ASPECTS of ALKALOID BIOSYNT7F,SIS a Thesis

ASPECTS OF ALKALOID BIOSYNT7F,SIS

a thesis subbitted by

ROBERT HENRY HESSE

in partial fulfilment of the requirements for the degree of

DOCTOR OF PHILOSOPHY of THE UNIVERSITY OF LONDON

Imperial College, London, S.W.7. December, 1964. (1)

ABSTRACT

Tracer experiments have shown that berberine is derived from reticuline, the N-methyl group of reticuline becoming the berberine ',bridge*. At least one step in this transformation is stereospecific. Reticuline has also been shown to be a precursor of protopine. An N-methyl cyclisation also takes place in this transformation and at least one step is stereospecific. Possible mechanisms for the N-methyl cyclisation are suggested and their implications in the biosynthesis of a variety of alkaloids are discussed. ACKNOWLEDGEMENTS

I am deeply grateful to Professor D.H.R. Barton for the privilege of research under his guidance. I wish to thank Dr. Gordon Kirby for his constant encouragement and assistance, and in particular for the many stimulating discussions we have had. I am indebted to many members of the Imperial College staff for their assistance; among them in particular: Dr. D. Turner, Mr. D. Aldrich, Mr. R. Young, and Mrs. L. Brown. I acknowledge with gratitude the financial support of the National Science Foundation (U.S.A.). Finally, to Dr. Maurice M. Pechet who provided the opportunity, and to my wife, Lucille, who has shared the burdens, I dedicate these efforts.

TABLE OF CONTENTS Eau_ Introduction 1 REVIE7 Synthesis of the Benzylisoquinoline Skeleton 5 Transformation of Larger Precursors 13 The Amaryllidaceae Alkaloids 16 Morphine 23 BERBER INE Introduction 28 One Carbon Metabolism 31 On the Origin of the Berberine Bridge 39 The Berberine Bridge in Otter Alkaloids 50 Protopine 54 OXIDATION OF AMINES Mechanistic Considerations 62 Alkaloids from Reticuline ... 75 SYNIfILSIS OF PRECURSORS 82 EXPERIMENTAL Precursors 100 Degradations 123 Tracer Experiments 126 REFERENCES.... 147 - 1 -

Although proposals for the biosynthesis of alkaloids and "biologically-patterned." synthesis have been with us for nearly fifty years,1'2 the knowledge of how Nature effects the elaboration of these diverse and often complex natural products is much more recent and still in a state of rapid extension. Recent advances are primarily a result of the application of tracer techniques, which have enabled us to observe the fate of a substance interacting with a complex living system. Isotopic tracers are so fundamental to the study of alkaloid biosynthesis that it is desirable to recognize the assumptions and limitations inherent in their use. The basic assumption is that isotopically- labelled molecules are subject to the same fate as molecules composed of only the natural isotopes. Although this idea is qualitatively sound, quantitative differences are the rule rather than the exception.3 For most isotopes these differences are small and not important; for others, particularly hydrogen isotopes, they may be significant. Although isotope effects might constitute a snare for the naive, they provide a powerful tool for the sophisticated investigator.4 As our knowledge of alkaloid biosynthesis is refined, these 2 ONO

subtle effects may well augment our understanding of the subject. Although we can label only individual atoms with isotopes, our interest for the most part is devoted to the transformation of molecules. In order to unequivocally follow these transformations, it is important that when we isolate a labelled substance, we provide evidence that it has been derived from its precursor in a specific fashion. This is a formidable problem. In the case of simple molecules degradation of products will often give evidence that the tracer atom has remained specifically attached to the various species involved in the transformation.5 In the case of complex substances, particularly those in which the tracer atom might be considered labile, it is often necessary to perform multiple-labelling experiments5,6 to insure that the appearance of radioactivity in the product is not the result of decomposition of the precursor followed by non-specific re-incorporation into the product. 'Multiple-labelling" experiments, as a rule, do not involve the preparation of a single molecular species containing all the requisite labels. These preparations are generally a mixture of singly- labelled species usually containing only small amounts of multiply-labelled species.7'8 The final distribution of labelling in the product is thus influenced by the nature of the isotopic species and by the rate at which each is incorporated. it is apparent, then, that this method is more susceptible to isotope effects than a method using a singly-labelled species. Observance of these criteria is basic to the proper pursuit of this method. Therefore, although details of degradations will not be presented in this review, it is to be understood that, withstanding evidence to the contrary, results are supported by proper degradations and, where applicable, multiple-labelling experiments. Since the use of tracers has become more widespread and since experience has inevitably led to a refinement of technique, the field of alkaloid biosynthesis is currently in a state of rapid flux. Because of the breadth of this topic and the rapid accrual of new information, any attempt by this author to exhaustively review this field would be presumptuous and of little value. A number of reviews have adequately covered various aspects of this subject. ,6,9,10 Our interest has for the most part been confined to the later 4

stages of biosynthesis, particularly those occurring in the benzylisoquinoline series, and an attempt has been made to introduce only material germane to this topic. REV IE7

Biosynthesis of the Benzylisoquinoline Skeleton

The relationship of certain phenethylamine alkaloids such as hordenine (1), mescaline (2), or ephedrine (3) to

the aromatic amino-acids, e.g.l phenylalanine (1k), tyrosine (5), and DOPA (6) was suggested more than fifty years ago.11

I I Ntle_ 2 OMe 1-

CO,4H

14 HO' NH H2 2 3 4 5

OH GO2 1-' I-13 2

HOB NH NH 2 2 Subsequent experiments have confirmed this relationship. Rordenine (1) has been shown to be derived from [2_14-1ujphenylalanine 12 or [2-14C]tyrosine12 as well as from [a-14C]tyramine.13 Similarly, mescaline (2) has been shown to arise from tyrosine.14 In experiments using 13N. as a tracer Shibata and Imaseki have demonstrated that the nitrogen of ephedrine (3) is derived exclusively from the nitrogen of phenylalanine.15 Since rL3- 14 Cjphenylalaninei is a precursor of the closely 16 related norpseudoephedrine (7), it appears that in this instance phenylalanine is a direct precursor and does not undergo conversion to an aldehyde or a-keto acid. That the more complex benzylisoquinoline skeleton, as typified by tetrahydropapaveroline (8), could in principle be derived from amino-acid units was suggested by Robinson12 and Winterstein and Trier.11 In general Robinson reasoned that if the reaction outlined below were to take place in the plant it could participate in the formation of a variety of alkaloids.

lz=r4 I I C C 0 NH C C —N I 1 - 7 -

Variations as this theme are legion. The nucleophilic carbon could arise from an It&ctivated" carbon or an aromatic ring, and the two components might be provided by a preformed Schiffsf base. The reaction of this sort utilizing a phenethylamine and an aldehyde has become familiar to chemists as the 18 Pietet-Spengler synthesis of isoquinolines.171 The first evidence that this hypothesis was sound was provided by Battersby and Harper who isolated radioactive papaverine (9) from poppies fed [2- 40]- tyrosine .19 Degradation showed the labelling pattern indicated in figure 1. More recent investigations have shown that the more complex benzylisoquinolines such as morphine20 (10), berberine21 (ii), and hydrastine21 (12) as well as benzphenanthridine alkaloid, chelidonine22 (13), are derived from two units of tyrosine. In each case degradation showed the pattern of labelling to be that anticipated if two aromatic amino-acids were first to condense to form a simple benzylisoquinoline which then underwent further transformation. Phenylalanine has been reported to be a much less efficient precursor than tyrosine for berberine,21 hydrastine,21 and morphine.23 This is not unexpected. B

Figure 1

I 1 HO H Me 0

3fric OH Otseie 8 9

H NH,

• OH Label from dopamine 4".

1 3 Label from tyrosine • 9

Although in mammalian systems tyrosine is formed by, the hydroxylation of phenylalanine,21 in E.Co1i 25 and in certain higher plants this reaction is not

important. 26,27 In these cases tyrosine is formed directly from prephenic acid28 (14). CO2H -0

CQH CO,H Calf

CO2H 2 Although it appears that a skeletal unit derived from two molecules of tyrosine is common to a number of the more complex alkaloids, the exact nature of the condensing units as well as the specific reactions involved in the formation of this skeleton remains unclear. It is apparent that the two amino-acid units from which the benzylisoquinoline skeleton is derived are probably not equivalent. The first evidence of this resulted from a study of the incorporation of tyrosine into hydrastine21'29 (12). In certain cases differential incorporation of tyrosine into the two - 10 - whalvesit of the molecule was observed. Although this effect was small, its presence strongly indicated that the apino-acid was being incorporated into the two halves via different paths. It now appears that one and only one of these paths involves dopamine. Recent investigations have shown that in the biosynthesis of morphine,3° berberine,31 hydrastine,32 and chelidonine33 only one unit of dopamine is incorporated. In each ease the position of labelling is consistent with the proposal that dopamine enters only the phenethylanine portion of the molecule and does not contribute to the formation of the C6-02 benzyl portion. On the basis of these data, it has been suggested that the basic benoylisoquinoline results from the condensation of a molecule of dopamine with a phenylaeetaldehyde or phenylpyruvie acid moiety as shown.31

••••••••••••••••••••••.> 0 —11 9H CO2 Although this scheme is compatible with the evidence, it is by no means proved. There is no evidence that the nitrogen of a benzylisoquinoline is in fact derived from the nitrogen of dopamine. In the absence of such data there is an equally plausible route which could involve the condensation of a molecule of dihydroxyphenylacetaldehyde with an amino-acid followed by the reactions shown.

N,7N,'"N

-5 ----)► H 0,C- HO2C

The phenylacetaldehyde could in principle be derived from dopamine itself as this type of oxidation in mammalian systems is well documented.34 The product of the action of monamine oxidase on dopamine has recently been shown to be tetrahydropapaveroline (8) which is formed from the intermediate reaction product dihydroxyphenylacetaldehyde.35 - 12 -

At present there is insufficient evidence to discriminate among the various proposals for the biosynthesis of the basic isoquinoline skeleton. Ue do not know, for instance, whether the benzyl group of the initial product bears one oxygen or two. Degradation of an alkaloid formed from labelled DOPA might settle that point since it has been reported that in certain plants DOPA is not converted into dopamine; the latter substance is derived from tyramine.36 It may be concluded that further study of the simple early stages of alkaloid synthesis will not be without merit. -13 -

Transformation of Larger Precursors

The most notable contributions to our understanding of how large precursors, those more complex than amino-acids, are transformed into the plant alkaloids have come from tho groups led by Battersby5 and Barton.6 Although experiments with labelled amino-acid derivatives have added much to our knowledge of alkaloid biosynthesis, vide supra, the results have not been for the most part sufficiently restrictive to confirm mechanistic proposals. A most illuminating use of complex precursors has been as a test of Barton's proposals on "phenol oxidatiod1P7 The mechanistic criteria on which these proposals rest place unusually severe restrictions on the predicted relationship of precursors and products. These criteria may be illustrated as follows: the oxidation of a phenol gives rise to a phenolate radical written as below. This is an ambident species; and if two radicals were to couple to give stable products 0* 0 o a variety of carbon-carbon or carbon-oxygen linkages could be produced. Since the nature of the mesomeric species above restricts coupling to the ortho or para positions, meta substituted products cannot be formed in this manner. A laboratory example of this type of reaction is provided by the oxidation of p- cresol to Fummerer's ketone30 which is now known to have the structure39 (2). Although the possibility that this reaction proceeds via a radical attack on a

2 ••••••••e phenolate anion has not been excluded, it has been shown40 that dimeric products are not formed by the attack of a phenolate radical on a phenol ether. Barton's proposals for phenol coupling via phenol oxidation thus require the presence of suitably placed hydroxyls in both reactants and predict the presence of a dienone intermediate such as (1). It was recognized that if such a process were to occur in the living plant, it could be utilized in the - 15 - elaboration of a large number of alkaloids.37 The specificity of these proposals has enabled then to be tested in several cases. Two of these have been ohonen for review! the Amaryllidaceae alkaloids, because these studies contain the first confirmation of the hypothesis; and morphine, because it is germane to our theme of isoquinoline biosynthesis and because it allows an introduction to a versatile precursor, reticuline. - 16 -

The Amaryllidaceae Alkaloids

The Amaryllidaceae alkaloids comprise a large group which are superficially of a widely divergent type.41 Closer inspection, however, indicates that most can be considered derivatives of three main types typified by galanthamine (7), haemanthamine (9), and lycorine (8). Barton proposed that each could be derived as indicated from a single precursor such as (5) by the process of phenol oxidation.37 (The fully methylated derivative of this compound was subsequently isolated from an kmaryllidomeae species as the alkaloid belladine42 (1).) Before discussing the evidence supporting these proposals, it should be pointed out that the researches into the incorporation of amino-acids into these alkaloids carried out by Battersby and 7ildman, 43,44,45 47 Jeffs, 46 and Barton have been quite fruitful. From these studies it appears that the Amaryllidaceae alkaloids are formed from a C6-C2 unit derived from tyrosine and a C6-C1 unit derived from phenylalanine, presumably by way of cinnamic acid. It is possible that two routes exist to these alkaloids from amino-acids.47 — 3.7 —

1. R1=R2=1 3=R4 Me 2. R1=R 4 =11, R2 = R3 =Pile 3. R1=R 2 =R4 =H, R3 =Me 4. R1=R 3= R R2=Me 5. R3.=R2=R3 4 „zii

6 . R1=Me, R2 =R3 =R 4 Me0 1 - 6 =.H

ome

Nme HO

Me0

9 10

4 OR - 18 -

The utility of the phenol oxidation hypothesis was first evidenced by its predictive powers. On the basis of this hypothesis the formulation (7) was suggested for galanthamine. An elegant laboratory synthesis of galanthamine from N10-dimethylnorbelladine48 (2), in addition to formulating a proof of structure, demonstrated the synthetic power of phenol oxidation. It also transpired that the postulated dienone intermediate (10) was identical to the natural product narwedine." ConfirnPtion of a more direct sort was subsequently achieved. It was found that intact plants could transform norbelladine (5), N-methylnorbelladine (3), and N,0-dimethylnorbelladine (2) into the alkaloid galanthamine .° Multiple-labelling experiments demonstrated that these precursors were incorporated without prior degradation. Surprisingly, it was found that 0-methylnorbelladine (4) was not incorporated into galanthamine.47 Although it is difficult to interpret a negative result, this fact suggests that in the biosynthesis of galanthamine a specific sequence of methylation is operative, i.e.9N-methylation precedes 0-methylatiou.47 A second type of coupling was demonstrated when it was shown that norbelladine (5) was an efficient - 19 -

precursor of haemanthamine (9).51 Surprisingly, 0-methylnorbelladine (4) was also incorporated into this alkaloid.52 The possibility that an 0-methyl might be converted to a methylenedioxy-group had been suggested by Sribney and Xirkwood,53 but there was no evidence to support the idea. 7.7hen the crucial experiment was performed with a multiply-labelled precursor containing 14C in the methoxyl group, it was found that the methoxyl group did in fact become the methylenedioxy-group of haemanthamine.52 These observations constituted the first evidence that such a pathway was actually existent. A final mode of coupling was proposed for the elaboration of lycorine (8). The proposal has been confirmed by the observation that norbelladine is an efficient precursor of lycorine.854 A subtle point exists in the formation of this class of alkaloids; for there are two possible pathways from the initial coupling product to the final product vide infra. These are outlined below.

- 20 -

14_

12 13 In the first case the nitrogen would directly attack the transient dienone of (11) thus forming the tetra- cyclic product (14) before tautomerism could occur. The second possible pathway involves rapid tautomerism of the dienone to a m-cyclophane (12). Additional steps such as hydroxylation, oxidation to a quinone (13) followed by nucleophilic attack, would be required to form the final product. 37Experiments involving a tritium label at the starred position have suggested that the latter process is, in fact, the actual pathway.55 It has been suggested that a fourth type of Amaryllidaceae alkaloid typified by homolycorine (15) could in principle be derived from a compound of the lycorine type as illustrated below. - 21 -

It has been reported that norbelladine is converted into homolycorine.56 Since definitive degradations have not been reported, this evidence is suggestive rather than conclusive. The foregoing data constitute compelling support for Barton's proposals37 regarding the biosynthesis of Amaryllidaceae alkaloids. Of particular importance is the fact that each reported precursor has borne two phenolic hydroxyls suitably placed for radical coupling. It is of considerable interest that the isomer of 0- methylnorbelladine (6), in which the phenolic hydroxyls are not suitably located for the proposed coupling, is metabolically inert.57 Experiments in this series have served to expand this hypothesis as well as to confirm it. It is - 22 - apparent that if a catechol is to undergo successful phenol coupling, the unused ortho hydroxyl must be masked. The mode of masking operative in vivo could not have been predicted a priori.37 The incorporation of 0-nethoxyphenols into the Amaryllidaceae alkaloids suggests that Nature may achieve this end by the use of methoxyl groups. This result is echoed by studies in morphine biosynthesis. -23 -

Morphine

Morphine (1) by virtue of its profound physiological effects and the richness of its chemistry has excited a considerable fascination in the organic chemist. In few cases has biogenetic speculation been so inextricably bound up with the chemistry of the substance. It is appropriate that the formulation proposed by Robinson in 1925P8 based on the brilliant conception that morphine was formed in vivo by cyclisation of a benzylisoquinoline, required nearly two decades for chemical confirmation." Robinson's reasoning was based on structural rather than mechanistic considerations; and a subsequent attempt along these lines to achieve biogenetic total synthesis of a morphine alkaloid failed.60 Since Robinson's proposals, numerous hypotheses regarding the biosynthesis of morphine have been forthcoming. One of them, however, is in every detail supported by experimental evidence.37 Early experiments with labelled amino-acids (see p. 7) confirmed the structural relationship between morphine and the simpler benzylisoquinolines proposed by Robinson. More recently, the mechanistic criteria - 2 4 - of phenol coupling has served as a guide for studies which have illuminated the details of morphine biosynthesis . These considerations led Barton to suggest that a suitably protected phenolic precursor such as (2) was converted by phenol coupling to a dienone (3)37 which was the direct precursor of the morphine alkaloids. A chain of evidence was begun when Battereby and his co- workers demonstrated that the fully demethylated compound ( 4 :) was an efficient precursor of morphine.63 Indirect evidence that this compound might be converted into morphine through the methylated derivative was provided by studies which showed that, in the poppy, thebaine (5) was converted into codeine (6) which was then converted into morptlne. 6162 kl)./ Later studies showed that the trimethyl compound (2; = Ye) was in fact an efficient precursor of thebaine and, therefore, of morphine. The most recent work has shown that this compound is converted into thebaine without demethylation.64 Further observations have been made regarding the 65 proposed dionone intermediate (3). Battersby and Ginsburg66 independently proposed that if the dienone were to exist in the %pen!' form, reduction and allylic elimination would provide an economical route to thebaine. - 25 -

This proposal was not in accord with behaviour of compounds of tho same type which have been shown to exist in the closed dibenzfuran form?9/ 48 Subsequently, however, the dienone was prepared from thebaine; and it was shown to exist in the open form (5a). Further- more, reduction gave isomeric dienols which were 67 converted into thebaine under mild conditions. The 67 Observation of two groups that the dienono as well as one of the dienols was a potent precursor of thebaine provided the final link of a substantive chain of evidence in support of the proposed biosynthesis of morphine.37 It is interesting to note that artifice has again preceded Nature, for only recently the natural product Salutaridine 68 was shown to be identical to the dienone (5a). As a postscript to this chapter, it is appropriate to mention that consideration of phenol coupling has led to a rational, if formal, biologically-patterned total synthesis of thebaine. It has been reported that the oxidation of the labelled-trimethyl precursor (2; R = Me) followed by dilution with inactive dienone, purification, and final conversion to thebaine resulted in a radiochemical yield of 0.0120.e 68A similar study69 using starting material of known absolute configuration - 2 6 -

ast 3h

HO Me

HOW me

4 5 - 27 has confirmed the stereochemistry previously assigned to the morphine alkaloids.70,71 It should be pointed out that although the substrate for this oxidation, the phenolic precursor. (2; R = Me) of the morphine alkaloids, was unknown when it was 72 first prepared in 1957, it has subsequently been isolated as a natural product: first from Anona reticulata73 as the (+)-enantiomer named reticuline, then from Phylica rogersii 74also as the (+)-form, and recently and appropriately from the opium poppy75 as a racemate. This compound may well be found to have an important role in the biosynthesis of mercy modified benzylisoquinolines. BE1ZBERINE - 28 -

Berberine W I a fine, bright yellow, crystalline alkaloid first isolated by Chevalier and Pelletan in 1826,76 has not quite the charm of morphine. Its chemistry is simpler; a correct formulation was proposed and confirmed as early as 1910.77 And its uses are not so dramatic. One use of this alkaloid is as a substitute for quinine in tonic water:78 It is, however, 2 ubiquitous; and it was recognized by Robinson that this compound might contain in its architecture an element germane to the biosynthesis of a wide variety of alkaloids. This element is the so-called "berberine bridge 11.2 It is clear that berberine can be represented as a benzylisoquinoline with a one-carbon link interposed between the nitrogen and the beuzyl group; and it is thus Robinson proposed that the alkaloid was formed - that is to say, by the reaction of benzylisoquinoline and a formaldehyde equivalent as illustrated.

-f- CH2 0 me

2 -29 -

Such a process is well known in the laboratory although, in most cases, the sense of ring closure is wrong leading to the oxidation pattern shown as (2). 79 A more elaborate mode of biosynthesis has been suggested involving a postulated-rearrangement product of prephenic acid,8° viz., CO 2 H

HO C. H 0,C 2 roe BEBRBID0F_REINE

OH OH

This latter scheme has been rendered unlikely as a result of experiments with labelled amino-acids (see p. 7). These data suggested that the most fruitful approach to the problem would follow the simple lines laid down by Robinson with the only ambiguity being the nature of the additional one-carbon fragment. Since the "formaldehyde equivalently has been a rather ill-defined concept in various. biogenetic speculations, it is rather fortunate that in recent years much evidence has been amassed regarding the manner in which the cell handles one-carbon units, and that the nature of these - 30 - various one-carbon units is becoming more clear. Before attempting a summary of current findings in the field, it must be pointed out that most of the evidence has been obtained in animal or bacterial systems, and the extension of these results to the higher plant is not well documented. It would, however, be surprising if the broad outlines of the scheme were not to obtain in the latter case. -31 -

One-Carboni_ 1et abolism

One-carbon units may enter metabolism at four oxidation levels: those of methanol, formaldehyde, formic acid, or carbon dioxide. (Although methane is formed by some bacteria,81 it is not of general metabolic importance.) Carbon dioxide, a major catabolic product and an important anabolic component of photosynthesis, plays a critical role in plant metabolism. However, the metabolism of carbonate is not pertinent to the type of carbon transfer observed during the later stages of alkaloid biosynthesis, and for that reason it is proposed to restrict this summary to the three lower- oxidation states. Our understanding of the processes involved in the transfer of 01-units at the level of methanol, i.e. , methyl groups attached to carbon, nitrogen, oxygen, or sulphur is fairly complete." It is known that with few exceptions, vide supra, such methyl groups arise by a process of transmethylation as written below.

2 -013 + R H ---> H -f R-CH3 -32 —

It has also been shown that in most cases the methyl donor in this reaction is the amino-acid methionine - CH3SCH2CH2CH(NE2)00217. Examples of methylation by methionine are numerous and have been collected in two reviews.82'33 Although it is difficult to establish the obligatory nature of a precursor in an intact living system, methionine has been shown by several investigators to provide 0-methyl,84 N-methyl,85 and methylenedioxy-groups53 added during alkaloid biosynthesis. Although it is possible that in several instances methionine itself serves as the "active" methyl donor,8 6 in most cases it is probable that methionine must first react with AT? to form S-Adenosylmethionine.87 60%Icyl mA1116:dz This is reasonable since .suirAler±mm is a much better ttiiol leaving group than sulphide..88 In Table 1 is listed a number of methylations known to proceed through the formation of S-Adenosylmethionine. There are few clear- cut examples of the intermediacy of this compound in alkaloid biosynthesis; but it does appear to be involved in the conversion of norbelladine to the 0-methyl derivative catalyzed by an enzyme system from Nerinii bulbs as shown below."

-33 --

S-Adenosyl- HO > methionine HO

The mode of formation of methionine is not as yet clearly defined. It appears, in fact, that it nay be formed in a variety of reactions.82 One sequence, mediated by a number of co-factors and enzymes, involves the transfer to homocysteine (7) of the methyl group of 5-N-methyltetrahydrofolic acid (8).94 (For the sake of brevity, the following abbreviations will be used: FA = folic acid, THFA = tetrahydrofolic acid, and the

C1-derivatives will be referred to as derivatives of THFA. Derivatives of tetrahydrofolic acid play a major

role in the transfer and transformation of C1-units. It is largely through the studies of this versatile compound that our knowledge of one-carbon units has been achieved. A summary of these reactions is presented in figure 1. Carbon can enter the cycle as formic acid which reacts with THFA (2) to form 10-formyl TETA (1).95 This compound may then cyclise to 5,10-methenyl Figure 1

0HC-. 14.-R NHR

I HCOe'

HCO NHR H

...... ••••••••••11i.

Ik0.011••••••=1.1•11111, 11Z••••••••• H N 2 3 4

1 1 C M NHR CO 2H T-TSCH2CH2OHYH2 CH3S CH2 CH2 CHIVH2 Ct. H 7 6 8

O2 H C 2 ONHCH(CH2)2CO2H - 35 -

THFA (4). 96Reversal of this process accompanied by rearrangement gives the 5-formyl-TUFA (3).97 Another mode of reaction available to the 5,10-methenyl THFA is reduction to the 5910-methylene-TaFA (5).98 It is this compound which brings about the transfer of carbon at the oxidation level of formaldehyde, e.g.,the donation of a hydroxymethylene-group to glycine to form serine,99 viz . 9

M2(NH2)002H + 5,10-methylene THFA H + THFA CH2 OHOH(NFT - 2 )002

Other examples of hydroxymethylations brought about by this compound are tabulated in Table 2. In two instances, viz. thymi idine 101and deoxycytidine,102 the resultant hydroxymethylene-group is reduced to methyl, thus constituting the rare examples of de novo methyl synthesis, vide supra. The reactions which 5,10-methylene THFA undergo certainly merit for it the appellation of "biological formaldehyde equivalent". In addition to its formation from 50.0-Mothenyi THFA, 5,10-methylene THFA is also formed by the

Table 1 Methylation by S-AdOnosyImethionine CH 89 CHgH NHOF.3 !j.1.-. 0HCH I\THCH 2 3

HC' HO OH OCH 3 go HO2OcH(NH2 )0112cH2soH3 ---.102COH(NH2 )CH2CH2S(CH3) 2 OlycH2 ) 5011=OH(oH2 )nCO2H-4.)0H3(oH0 ),oH--oHOH2 )n002H 91 n=7 or 9 'CH/2 HO CCH NH HO OCH NHCH 92 2 2 2 2 2 3

Table 2 10-methylene CH CH(NH )C0 H 5 > 0li30 ( 0112011)NH2CO211 2 2 3 THFA CH CH CHNH CO H > CH CH C( CH 0H )NH 3 2 2 2 3 2 2 2 C.!02H

CH(CH2OH)NH2CO2H ..." 0"J(CH2OH)2NH2002H - 37 - non-enzymatic reaction of formaldehyde and THFA.1" The latter reaction is of dubious biological significance since formaldehyde is highly toxic; formaldehyde poisoning is one of the risks ventured by drinkers of methylated spirits.'" Moreover, it has recently been shown that exogenous formaldehyde is metabolized to formic acid. In elegant experiments, Rachele and co- workers105 prepared fully deuterated [14C]formaldehyde. After administration to rats, the ratio of deuterium to carbon-14 was obtained for both methyl and hydroxymethyl groups. In each case the ratio indicated that all of the formaldehyde had passed through formate. A more interesting and important route to 5,10- methylene-THFA is provided by the oxj:dation of sarcosine. In the presence of THFA, sarcosine is enzymatically converted to serine. 06

THFA (0) CH NHCH CO H HOCH OHNH CO H 3 2 2 enzyme 2 2 2

The N-methyl group of sarcosine becomes the hydro ethylene of serine. In the absence of TETA, free formaldehyde appears in the reaotion medium. 107 Free formaldehyde - 38 -

is not involved in the reaction shown above, however, since it has been shown that exogenous formaldehyde does not provide the p-carbon of serine under these conditions.108 Furthermore, that the one-carbon unit transferred passes through the oxidation level of 109 formaldehyde is evidenced by multiple-labelling experiments. It thus appears that the N-methyl group of sarcosine is oxyidized to yield a "bound!' form of formaldehyde which reacts enzymatically with THEA to yield 5,10-methylene THFA. The latter may then react with glycine to form serine. This conversion of en IT-methyl group to a "formaldehyde equivalent" is certainly a provocative process in view of our interest in berberine. - 39 -

On the Origin of the Berberine Bridge

On the basis of their investigation of the biosynthesis of haemanthamine in which an 0-methyl group was shown to be converted into a methylenedioxy-group, Barton and Zirby suggested that the berberine bridge might arise via a similar cyclisation of an N-methyl group.110 The same scheme was independently proposed by Battersby.5 Consideration of the N-methyl group as

a formaldehyde equivalent rendered this an extremely attractive hypothesis, and we set about to test it. The plant chosen for these investigations was agrastis canadensis - Golden Seal - which produces major amounts of berberine (I) and hydrastine (2), as well as smaller amounts of canadine (3). Preliminary studies with DOPA showed this amino- acid to be an efficient precursor of both berberine and hydrastine, thus demonstrating the validity of our techniques of feeding and isolation. The choice of a suitable precursor to test our hypothesis was on the surface a complicated one. The presence of an N-methyl group was, of course, obligatory; and in order to facilitate the cyclisation, a free phenolic hydroxyl in position three prime was also desirable. Although the number of benzylisoquinolines which bear these features is formidable, one of them, reticuline (4), merited particular consideration. This compound has been demonstrated to be a precursor of the morphine alkaloids, vide supra. Since opium also contains alkaloids such as protopine (5) and narcotine (6) which are related to berberine and hydrastine, it was attractive to speculate that reticuline might be the precursor of these compounds. - 41 -

Accordingly, (±)-reticuline labelled with 140 in the N-methyl group was fed to several Hydrastis eanadensis plants. The berberine isolated was radioactive (0.7% incorporation). Reaction of the labelled berberine with phenyl Grignard reagent111 followed by oxidation of the resulting phenyldihydroberberine gave benzoic acid containing all of the radioactivity (106%). To confirm this result, a mixture of (±)-[6-methoxy1-14C]- reticuline (5.9%) and (±)-[N-nethyl-14C]reticuline (94.1%) was also fed. The resulting berberine was again radioactive (0.9% incorporation). Hydrolysis of the berberine with hot sulphuric acid112 gave formaldehyde as the dimedone derivative containing 5.7% of the original activity. Degradation as described above gave benzoic acid containing 91% of the activity. These results demonstrate that (±)-reticuline is converted into berberine without ftscrambling" of the label, and provide a second example of the conversion of a methoxyl group into a methylenedioxy-group. Independent evidence regarding the conversion of an N-methyl group into the berberine bridge has been forthcoming. Battersby has shown that the tetrahydric phenol Iaudanosoline (7) is converted into berberine in - 42 -

Berberis ;talkonica with the N-methyl again becoming the berberine bridge. 113Less direct evidence has been provided by Gupta and Spenser who have shown methionine to be an efficient precursor of the bridge carbon of berberine and of the carboxyl of hydrastine as well as the AI-methyl, methoxy, and methylenedioxy-groups of these alkaloids .11i Having achieved our first objective, the elucidation of the origin of the berberine bridge, we turned to other aspects of berberine biosynthesis. In particular we were curious about the specificity of the various steps. A benzylisoquinoline which meets our criteria, vide supra, for a berberine precursor is an isomer of reticuline, namely, protosinomenine (8). However,

CViN,N (D -N ,,N < if 0 Nkt,le 0 N me ome me

7 R=1-1 8 R =me when this compound, labelled with tritium by alkaline exchange, was administered to Hydrastis canadensis - 43 -

plants, there was a negligible incorporation of tritium into berberine. This demonstrates that certain step(s) in the biosynthesis of berberine from reticuline are highly specific. There are at least two explanations for this selectivity. If the cyclisation of the N-methyl is accompanied by an oxidative process, the presence of an unprotected hydroxyl at position six could complicate the course of the reaction, i.e.t

R +CH2O

Secondly, the construction of the 6,7-methylenedioxy- group is a process that might be specific. Since enzymatic reactions are generally stereospecific,116 a demonstration of stereo selectivity in the formation of berberine from reticuline would provide evidence that at least one of the steps involved was under specific enzymatic control. To achieve this end, (+) and (-)--reticuline, each generally labelled with tritium by acid exchange,117 were separately administered to matched Hydrastis canadensis plants. In this - 44 -

experiment, the (+)-enantiomer was incorporated with

13 times the efficiency of its antipode, 7.9% vs. 0.4%. Although this appears to be a clear-cut result, the possibility of variation between the two plants could not be ruled out. Confirmation was achieved as follows: a large mass of ()43_14c]..t reticuline mixed with a small mass of generally-tritiated (-)-reticuline was fed to several Eydrastis canadensis plants. The resulting berberine was purified as usual, converted into canadine, and assayed for 140 and 3H. It was obssrved in accord with the foregoing results that the 14 C label was incorporated with 7.5 times the efficiency of the 311 label. The apparent residual incorporation of the (-)-enantioner was also observed in the case of protopine, vide infra. Our criterion of optical purity for the two enantiomers is based on measurement of optical rotation, and it is quite possible that our (-)-reticuline was contaminated with sufficient of the (÷)-enantiomer to account for these results. Je cannot rule out the possibility that some racemization is taking place in the plant. However, the similarity of the results in two different systems tends to render this unlikely. Although berberine itself is symmetrical, these observations make it possible to correlate the configuration of intermediates in berberine biosynthesis with other benzylisoquinoline alkaloids. The absolute configurations of (+)-reticuline and (-)-reticuline are represented by the formulations (9) and (10) respectively.110 The absolute configuration of (-)- reticuline corresponds to that of the morphine alkaloids, e.g.,thebaine (11).71'72 The inter-relation of a number of protoberberine alkaloids has been reported by Carrodi and Eurdiggar.119 The (-)-protoberberines such as (-)-eanadine (12), (-)-stylopine (13), and (-)- caalrine (14) were shown to be of the sane configuration as (+)-laudanosine (15), and thus related to (+)- reticuline. Recently, hydrastinel20 (16) as well as narcotine121 (17) has been added to this group. On the basis of our evidence, we may conclude that certain of the intermediates in berberine biosynthesis in drastis canadensis also belong to this group. This is of interest because, although many of the protoberberines occur as both (+) and (-)-forms, it is usually those of the (-)-configuration which occur as companions to berberine.122 Meo".‹-N/N1 MeO Me HO

OH me 9 memeo it

N I 0/i; MeQ ,,_ I O ON-7N.,i MeO 1-1 Fie HOW Nr">A1 .‘ me oma %,,..,, 01 me 12 13 14

/ Off` N) <(-)s-J1 NM` NM H.' Me. 3Me Me 15 16 17

Encouraged by the selectivity observed in these studies, we decided to investigate the order of methylation in the steps leading to berberine. Since Battersby had reported the incorporation of the tetrahydric compound (7), vide supra, it appeared that the next biogenetic stage would be 0-methylation to produce either of the trihydric phenols (18) or (19).

18 19 20

Administration of a mixture of phenol (18), labelled with 140 in the N-methyl group, and of phenol (19), labelled with tritium by basic exchange, to several Hydrastis canadensis plants led to the formation of berberine labelled with both carbon-14 and tritium. Little selectivity was observed in this case. The ratio of 14C/3H in the berberine was only slightly different from that in the starting mixture. This - 48 -

result was not unexpected since the tetrahydroxy N-methyl compound (7) is converted into berberine and the dimethoxy N-i' compound (20) is converted into morphine p123 both presumably through reticuline. Although these data were obtained in different plants, it is possible that a variety of paths tothe reticuline-type precursor exists. This is certainly suggested by our results. Our final investigations were concerned with the transformation of the protoberberine compounds (t)- nandinine (21) and (±)-canadine to berberine. Although (±)-nandinine [labelled with 3a as shown (21)] was converted into berberine with an efficiency of only .06%, (±)-canadine [similarly labelled (22)] was incorporated to a much greater extent (8.9%). It is difficult to attach great biochemical significance to either of these results as both precursors are readily auto-oxidized. Therefore, although both are converted into berberine, we cannot be sure that the conversion is a specific enzymatic one. Experiments with optically active compounds might illuminate this matter. - 49 -

21 2-2 - 50 -

The Berberine Bridge in Other Alkaloids

Knowing that the berberine bridge is derived from an N-methyl group, it is tempting to speculate about the role that this reaction may play in the biosynthesis of other alkaloids. The extension of this reaction to the protoberberine alkaloids is obvious; and we have, in fact, isolated radioactive canadine (1) from Rydrastis canadensis after the administration of N-methyl-labelled reticuline. Unfortunately, the in of radioactivity was insufficient for proper degradation; and the result must be accepted as only indicative. Another class of alkaloids closely related to berberine is the phthalide-isoquinolines such as hydrastine (2) and narcotise (3).

/ N i ct:•-•Nme Me) me ome Mme Me Om e OM e 2 3

There is experimental evidence that these are derived from a benzylisoquinoline by the addition of a one- carbon unit,21'114,124 and it has been generally assumed that these alkaloids are derived from a berberine type of precursor.2125 however, although under our conditions of growth and feeding DOPA was an efficient precursor of both berberine and hydrastine, the incorporation of reticuline into hydrastine was negligible! Furthermore, protosinomenine (4) as well as the trihydric phenols (5) and (6) was also not incorporated into this alkaloid.

HO HO Me0 NMO MCO HO Me HC

4 .1111.10•••••

A similarly perplexing lack of incorporation has been observed in the case of the Amaryllidaceae alkaloid galanthine.126 Such a result is difficult to interpret. Although ono may invoke a number of explanations, it must be concluded that the biosynthesis of hydrastinc remains obscure.

-51a- INDOLI. ALKALOIDS Fiuure 1

Ajmaline Strychnine

MOO

Lburnamenine Quinine

MeO MSC?

OMC OM e

Pmetine - 52 -

A structural element similar to the berberine bridge is present in a wide variety of complex indole alkaloids. It is attractive to speculate that this element might also originate in an N-methyl group. Representative alkaloids are tabulated in figure 1, and the nberberinen type carbon is indicated. Although it is not an indole alkaloid, emetine has been included for Obvious reasons. Attempts to test these speculations have been discouraging. Independent work has shown that methionine is not an efficient precursor of C-21 of ajmaline (7).127,128 This result is complicated by the fact that the origin of the non-tryptophan derived portion of these alkaloids is obscure.12*,I29 A simple but interesting extension of AT-methyl oyclisation could be operative in the formation of the cactus alkaloids such as anhalinine (8). Cyclisation of an j-ethyl group would lead to the 1-methyl- isoquinolines such as pellotine (9). Thisspeculation is extremely attractive since the requisite AI-methyl and N-ethyl phenethylanine derivatives occur as companion substances in the plant. These speculations could - 53 -

1v1 e 0 H MG N Me. PHD MG 8

also be extended to the formation of the tetrahydro- carboline alkaloids such as harmane (10) or harmaline (11).

meo

A final extension of the berberine bridge concept is to the protopine alkaloids. This will be discussed in the next chapter. - 5 -

Protopine

Protopine (1) was first isolated by Rosso in 1871.130 Its correct formulation was expressed by Perkin.131,132 This alkaloid, like berberine, is quite ubiquitous;133 and its pharmacological properties are not such as to recommend its use in medical practice It is apparent that protopine may be regarded as derived from a protoberberine by oxygenation and methylation

In this scheme the 'berberine bridge!' is obvious. Perhaps less obvious is a second relationship to the N-methyl cyclisation reaction. In the formation of the berberine bridge, an intermediate oxidized species can be formulated as a carbinolamine or Schiff& base as below. ksimilar oxidation of a carbon linked to nitrogen could in principle -55-

OR I ÷ N=CH I Na-IOH 3 2

introduce the carbonyl function of protopine. Since it had been reported that [14C]methionine, in addition to providing the nethylenedioxy-group of protopine,54 was incorporated into the skeleton of the alkaloid, it was attractive to speculate that this activity might reside in the bridge carbon. Preliminary experiments indicated that tyrosine was an efficient precursor of the alkaloid(2.3% incorporation) in Dicentra spectabilis. Subsequently, administration of (±)-[N-methyl-14C]reticuline to a flowering Dicentra spectabilis plant resulted in the formation of radioactive protopine (1.0% incorporation). Zeisel determination indicated that there was negligible activity in the N-methyl group of the alkaloid. The protopine was, therefore, degraded according to the following scheme. Quarternization with dimethyl sulphate gave (2), which was reduced with Nailig to the aide base (3). -56-

MLA HOl mec 0 H 2

3 .1100111M.

Oxidation of the Emde base liberated the desired carbon as acetic acid. The degradation of acetic acid on a small scale and the derivatization of the products are very difficult.135 7e therefore sought a new and superior method. The following sequence was found to be efficient and applicable to a very small-scale procedure.

CO 2 CHO B Z 0 CH CO H H 3 2 4 aZO M e0 C H NH , meo 3 ge NCIBH4

The acetic acid was degraded according to the Schmidt 136 Procedure. The resulting nethylamine was treated with - 57 - benzylisovanillin, and the intermediate Schiffst base was reduced with HaBEI4 to give the benzylamine When this procedure was applied to the acetic acid derived from the labelled protopine, the benzylamine contained all of the radioactivity of the starting protopine, 96%. This clearly demonstrated that the bridge carbon was derived from the N-methyl group of reticuline. To provide confirmation, a mixture of (±)-[N-methyl- 14 C]reticuline (75%) and (±)-[3-14C]reticuline (25%) was administered to a flowering Dicentra spectabilis plant. The radioactive protopine (3.4% incorporation) was degraded as follows.

7 0 -58 -

Protopine was treated with FO013 to form isoprotopine chloride (6). Treatment of isoprotopine chloride with base gave the methine (7), ozonolysis of which gave formaldehyde isolated as the dimedone derivative. The formddehyde contained 23% of the radioactivity of the starting protopine. This is the same amount as that contained in the three position of reticuline; it confirms that reticuline is incorporated without scrambling of the label, the N-methyl carbon becoming the tibridgett carbon of protopine. When the dehydration was carried out with impure POC13, protopine hydrochloride (5), rather than isoprotopine chloride, was the produot. Since this compound has a carbinolamine structure, it follows that attack of nitrogen on the protopine carbonyl must precede or be concerted with the attack of the oxygen on phosphorous during the formation of isoprotopine chloride. The hydroxyl group of the hydrochloride (5) apparently is not sufficiently basic or is too hindered to react with PO013. Having demonstrated that the N-methyl cyclisation was also involved in the biosynthesis of protopine, we turned to other aspects of protopine biosynthesis. Administration of the enantiomers of reticuline labelled - 59 - with tritium to separate Dicentra plants, vide supra, produced a result which echoed those found in the case of berberine. The (+)-encntiomer was incorporated with much greater efficiency than the (-)-antipode (ratio 7.9% vs. 0.415%). Similarly, when both were administered in a mixture to Argemone plants (+)43H]reticuline was twice as efficient as (±)-[N-methyl _14-1ujreticuline as a precursor of protopine. Thus it appears that in Dicentra spectabilis and ArRemone intermediates in protopine biosynthesis are also related to the (-)-protoberberine alkaloids. Since in various Corydalis plants protopine often occurs as a companion to protoberberines of the (+)-configuration,122 it would be interesting to observe in these plants whether protopine biosynthesis followed the same stereochemical path as in the Dicentra and the Arplemone. • Our final experiment in the protopine series was designed to show that protopine is, in fact, derived from a protoberberine precursor. Administration of (±)- nandinine (9) labelled with tritium as indicated gave radioactive protopine (0.3% incorporation). Although the aldtloid was not degraded, steps were taken to insure radiochemical purity. Breakdown of the precursor seems -60 -

unlikely since the incorporation was good and since we have not, to date, observed non-specific incorporation of a complex precursor. On the basis of our data, we may propose a scheme for the biosynthesis of protopine.

m e 0 me() HO HO

0 C < NM C °'>„ c)

OMC -61 -

The data do not exclude alternative routes, and they do leave several points unanswered. It is not known whether the isoquinoline methylenedioxy-group is formed before or after ring closure, although we would prefer the latter; and it is not known whether oxygenation and nethylation precede or follow the formation of the beuzyl methylenedioxy-graup. However, the following points are well supported: protopine is formed from a protoberberine derived by N1-methyl eyclisation from reticuline, and at least one of the steps involved is stereo specific. OXIDATION OF AMINES - 62 -

Mechanistic Considerations

That the berberine bridge arises from an N-methyl group is well established. It is possible that this type of reaction, particularly if formulated in a more general sense, i.e.Las the two-electron oxidation of carbon linked to nitrogen, may play a wide role in alkaloid biosynthesis. Our knowledge of how this transformation night be accomplished is limited; but consideration of various related reactions which occur in vivo as well as in vitro may serve as a guide to further investigations. It is clear at the onset that the analogy between the formation of the berberine bridge and the formation of the methylenedioxy-group is a superficial one. Although carbon linked to nitrogen may undergo oxidation via a valence-expanded intermediate, viz.,

1 X 1 R I Ri R x+ R-CE R-CH=NC 2 a-CH2"Nh, 2 2 + R + R oxidation of carbon linked to oxygen usually involves direct hydrogen radical abstraction. Furthermore, the formation of a methylenedioxy-group from an 0-methoxy -63 -

phenol is a process for which few analogies exist. The formation of formaldehyde in the demethylation of catechol monomethyl ethers137 is the closest biochemical analogy, and little is known regarding the mechanism of this reaction. The formation of acetals in the auto- oxidation of ethers138 also bears a certain similarity, but the existence of such a radical chain process in the living cell is extremely unlikely. Hydrogen abstraction by an activated alcoholate radical139 may provide a model for the formation of a methylenedioxy- group. Formation of an nactivated” phenoxide radical and intramolecular hydrogen abstraction, followed by oxidation and nucleophilic attack, could provide such a route. Barton and his, collaborators have discussed

-4- CH 2

47 other possibilities, but at the moment it is not obvious how one could hope to test any of them. In contrast to the above, one is faced with numerous

- 641 -

analogies for our postulated amine-oxidation reaction. The oxidation of amines with reagents such as mercuric 140 acetate or iodine141 is a well known procedure of considerable utility. Although the overall course of the reaction depends on the nature of the substrate, one can often formulate the initial product as an imine or iminium ion; and in certain cases one nay view the reaction as proceeding through a transient valency- expanded intermediate.

+ Hg+2 Hg Hg+ Hg+2 RCH \ ROlif RCH,Y NCH= •; 14\

Although the biological oxidation of amines nay also proceed in a similar fashion, there are a number of reasons why these systems are unattractive as models for the reaction which interests us. The mechanism of these reactions probably varies, and the details are not well understood. Furthermore, as no stable intermediates exist, it would be difficult to verify the existence of such pathways in a biochemical system. The reactions of amine oxides provide a much more attractive model, and one which in many details is

- 65 analogous to biochemical processes. This has been pointed out by Battersby.5 Several reactions of mine oxides involve demethylation with the formation of 142 formaldehyde. One of these, the Polonovski reaction, outlined below has been studied in some detail by Oae, Kitao, and Kitaoka.143

0 .gi(CH + 3 2 A c20

+ CH 0 2 Although certain rearrangement products were shown to arise from a radical cage process, i.e.

NM e NMe2 m e 2 2 =0 i40 Me —30 "CA GE 1/ the authors were unable to exclude an ionic mechanism as below for the demethylation reaction.

AC0.5 0 AC I B + ItC H3 N= CHO CH CH 3 NCH 3 • 3 ----> -66-

The reaction is accomplished under non-physiological conditions by treatment with acetic anhydride, but it has recently been reported that a more physiological acyl phosphate can effect the same reaction.144 Another demethylation reaction brought about by iron and a reducing agent145 has recently been re-investigated by Ferris and Gerwe.146 On the basis of their data, these authors suggest the following reductive mechanism.

+ 4. (cTi y NOHt-+ +2 + 11-1. ----> (CH ) ON- + H2 - 3 3 3 3 + Fe+3 + + (CTJ 3) 3N° --' -. (CH3 )2NHCH2 + . + + (CH ) NHCH 3 2 2 + (CH3 )2NHCH2OH + (CH3 )3N4 (CH ) NOH 3 3

A slightly different mechanism had been suggested by earlier investigators.145 They postulated attack of the N-oxide on the vacant co-ordination sphere of a +3 chelated Fe species followed by heterolysis of the 0-N bond to give a radical cation as above. A proton is then transferred from the m-carbon to one of the oxy- ligands to give a neutral radical on the a-carbon. The oxidation level of the Fe then undergoes adjustment with -67 -

the extrusion from the chelate of a hydroxyl radical which then pairs with the electron on the m-carbon. This scheme requires that the hydrogen which is eventually replaced by hydroxyl must be cis to the oxygen of the amine oxide. This constraint has been used to ration- alize aspects of the reaction course of nicotine N-oxide.147 These two postulated mechanisms differ only in subtle points, and it is quite possible that Ferris and Gerwe who simplified the system were actually observing a different reaction. All of the evidence, however, clearly establishes that this type of reaction proceeds in two one-electron steps. These reactions proceed under very mild conditions; and since oxidizable metals are omnipresent in biochemical systems, several investigators consider them a likely model for biochemical N-demethylation reactions. Most interesting transformations can be effected by the treatment of an amine oxide with potassium chromate. Codeine N-oxide148 is demethylated to norcodeine in this way. But a more provocative transformation is the conversion of strychnine N-oxide (1) into pseudostrychnine (2).149 -68 -

This is completely analogous to the conversion of a protoberberine into a olatopine-type alkaloid. And, utilizing this reaction, Bentley and Murray150 have converted the protoberberine (3) into cryptopine (4).

MOO meo

There are several remarkable aspects of this conversion. The first is that the bridge-head hydrogen of strychnine is the one replaced by hydroxyl. This excludes any §_7a2 intermediates such as an /mine. Models - 69 - indicate that the bridge-head hydrogen is indeed cis co-planar with the oxide oxygen, but so is one of the allylic hydrogens: The reason why the allylic position is not attacked is not obvious. It is also of interest that the protoberberine is converted into a carbinolamine rather then a 1,2-dihydro compound. This is more evidence against an iminium intermediate, for 2,5- dihydroberberinium compounds rearrange to the 1,2- dihydro compounds rather than undergoing nucleophilic addition. In the light of these data, the most likely mechanism is one involving two one-electron transfers similar to the iron-catalyzed demethylation reaction vide sutra,. The two-electron oxidation of carbon linked to nitrogen is also extremely common in biochemical systems. One of the most widespread classes of enzymes accomplishing 151 such a reaction is the amino-acid oxidases. These enzymes catalize the conversion of an m-amino acid to the corresponding m-?veto acid, ammonia, and water as shown below. The electron acceptor is an oxidized pyridine nucleotide.

R0H(NH2 )002H 71-002H + Nh3 + H2O - 70 -

The mono and dianine oxidases152 form a second class of enzymes mediating a similar reaction. In this case the substrate is an alkylanine - primary, secondary, or tertiary - and the oxidant is molecular oxygen. The products of this reaction are en aldehyde, a derivative of ammonia, and hydrogen peroxide as below.

/R2 R2 R -CH N ------* R CHO + EN// 2 + H202 R3

Mono amine oxidases are of considerable physiological importance as they regulate the level of amines such as serotonin (5) and adrenaline (6) which affect the nervous system.

Furthermore, certain dietary items, particularly cheese, contain levels of toxic &lines which, in the absence of amine oxidase, would be lethal.153 As yet only a - 71 - little is known regarding the mode of action of these important enzymes. Recent studies 154 ' 155 suggest that copper as well as pyridoxal phosphate participates in these reactions. A reasonable mechanism might involve the formation of a transient t-amine followed by oxidation as below. The oxidation might proceed

0 OH // R - CH2 - N RCH211: RCH N

products through an amine oxide, and the peroxide may be formed as a by-product of the conversion of molecular oxygen to "cationic oxygen". This has been suggested in the 156 case of steroid hydroxylation. A third type of amine oxidace is involved in the demethylation of tertiary amines resulting in the 157 formation of a secondary puline and formaldehyde. In view of the known reactions of amine oxides vide supra and the presence of amine oxides in a number of living systems,158 information on the possible role of these compounds was sought. It was quickly found that N 9 N- dimethylaniline N-oxide was demethylated by this - 72 - enzyme,159 and more recently the presence of an amine oxide intermediate has been reported.160 Thus, not only do the reactions of amine oxides provide Pin intriguing model for the reactions which interest us, but there is also evidence that they are involved in the biological oxidation of carbon linked to nitrogen. Finally, amine oxides are particularly attractive because their role in the processes which interest us should be susceptible to test. - 73 -

Alkaloids from Reticuline

One observation of considerable interest to emerge from recent studies of alkaloid biosynthesis is that a variety of alkaloids of divergent structure and function may be derived from a common precursor. This has been clearly demonstrated in the case of the Anaryllidaceae alkaloids which are derived from the versatile norbelladine (1) by phenol oxidation.47 There is now evidence that reticuline (3.) may be an even more versatile precursor of modified beuzylisoquinoline alkaloids. The relationship of reticuline to the simple benzylisoquinolines such as laudanosine(ja) or Iftudaaine( b) is innediately obvious. By the processes of phenol oxidation, reticulino may be convertedinto a variety of more complex alkaloids. It has been shown 67 to be a precursor of the morphine alkaloids. A similar coupling could lead to sinomenine ( 'E) Coupling of a diradical derived from reticuline in another sense could lead to the aporphine alkaloids such as glaucine (5) or corytuberine (6). Since the potential of reticuline as a substrate for phenol oxidation has been discussed in considerable detail by Barton and Cohen, 37 me0/iNVN, MeO i meo Me

\4-NOH OR OH Ns z'OMe OM? OH 2 3a. R=H

MeO me HO Me

HO MCO OMe

4 6

OH OMe 7 -75-

this author will not reiterate these detailed suggestions but will instead discuss the possible role of amine oxidation in the conversion of reticuline into a variety of benzylisoquinoline alkaloids. It has become apparent to us that the conversion of an N-methyl group into the berberine bridge might be only one example of a group of transformations involving a two-electron oxidation of carbon linked to nitrogen. The potential of such a reaction is considerable as is illustrated below.

X lo TZ e` R-C=0 HN' R ----- /Y +// R - C = I \ri, 1 ''\ a ''''`A R + R ----->. A 2.1. C Y/\ I II ii12

The first intermediate may be written as an imine (9) which could undergo nucleophilic attack to yield a substituted product (10). Alternately, the imine could -76—

undergo rearrangement to an iemanine (11). This enemin4 upon electrophilic attack would yield substituted products of the type (12). Finally, the imine could be opened to yield an amine and a carbonyl compound. Although these processes as formulated involve two-electron transfer, the sane structures could be derived from a carbinolanine formed by two one- electron transfers. There is some evidence to support the latter type of mechanism, vide supra. Application of these reactions to reticuline results in the generation of a number of benzylisoquinoline structures. The most simple outcome of this reaction is dehydrogenation. Formation of an inmantihe: followed by a second oxidation step would yield a fully aromatic isoquinoline. An illustration of this would be the conversion of a protoberberine (7) into berberine (8). The same reaction sequence would result in the conversion of nor-reticuline (13) into papaverine (14). Nucleophilic attack on the mine could have a variety of outcomes. Attack by an flactivatcd" carbon such as an aromatic ring would yield a "berberine bridge'? cyclisation. Attack p to the hydroxyl would lead to the protoberberines of another oxygenation - 77 -

pattern such as norcoralydine (15). An interesting variation on this theme may be operative in the biosynthesis of argemonine (16). In this case the cyclisation would be onto the three position rather than the N-methyl group, i.e., OM e Me0 OH HQ

Recently, we have administered labelled reticuline to Argemone plants. Unfortunately, very little argemonine was isolated. Dilution of this and purification resulted in the isolation of inactive argemonine. In the absence of information regarding the incorporation of our precursors under our conditions, we must treat the results of this preliminary experiment with caution. Attack by hydroxide on an imine intermediate would provide a route to the protopine alkaloids. Such a reaction could be involved in the conversion of nandinine (7) into protopine (17). Leetel6l has suggested that the Corydalis alkaloids such as corycavine (20) are derived by methylation of a protopine type of precursor. A more economical route - 78 -

MeO 1%4e° N

OH OMe ame °Me _13 14

MeO OMB' MeO OMC

- 79 - would involve electrophilic attack on an enanini intermediate (18), followed by nucleophilic attack on the resulting imine (19). Reduction of the imine would lead to the thirteen substituted protoberberines such asophiocarpine (21) or corydaline (22). A final variation of the amine-oxidation reaction is the hydrolysis of an iminium intermediate to form a carbonyl and an amine. The utility of this is illustrated in the following postulated biosynthesis of chelidonine (25). Although Manske162 has stated that no obvious route to the benzphenanthridine alkaloids from the protoberberine alkaloids exists, such a route has been suggested in general terms by Woodward and Turner.165 On the basis of amine oxidation we can expand and justify this scheme as follows.

0 0 19 - GO -

Me0 me0

OMe OMe OMe' Ome

0 0 - 81 -

Oxidation of a protoberberine (derived via an N- methyl cyclisation) followed by methylation could give rise to the amino aldehyde (23). A second oxidation followed by rearrangement would give the aldehyde guaminG , (24). A conventional cyclination followed by reduction would lead to chelidonine (25). This scheme fixes the location of the hydroxyl group in the proper position, and is compatible with the results of amino-acid feeding 14c), experiments. Furthermore, we have fed (t)-N-methyl[ 1-[3H]reticuline to Chelidonium majus plants. The chelidonine isolated was radioactive. Furthermore, as expected the 311 label was lost during the transformation. Degradations were not performed, but the preliminary evidence is encouraging. Although these speculations have been confined to the isoquinoline alkaloids derivable in principle from reticuline, these principles may be extended to other classes of alkaloids. For example, cryptopine type alkaloids exist in the indole series.164 SYNTTiESIS OF PRECURSORS - 82 -

The labelled, simple benzylisoquinolines required for these studies were prepared by total synthesis. Carbon-14 was introduced during the course of the synthesis; and tritium was, in several instances, introduced at the end of the synthesis by suitable exchange procedures. Although these syntheses were exacting and time-consuming, they involved very little novel chemistry. For that reason they will not be discussed in great detail. The derivatives of laudanosoline were synthesized 165 by standard Bischler Napieralski methods. The required phenethylemine (3) as well as the phenylacetic acid equivalent watt prepared from suitable derivatives of protocatechuic aldehyde (1). The 0-methyl label was introduced at the earliest stage of the synthesis by alkylation of a monobenzyl protocatechuic aldehyde with [14C]methyl iodide. As the standard procedure

(HeI/K2CO3/acetone) was a sluggish reaction which resulted in a variable yield, it was necessary to seek a more suitable procedure. Although reaction of a phenoxide ion in dimethyl suiphoxide with stoichiometric amounts of methyl iodide gave a good yield of ether, consideration of they formation and methylating

- 83 -

NO2 1 R1 00 R RI 0 2 2 1 2 IS 0 R 0 s

R1 CH CI 0 2 R 0 R20 R20 4 . =Mc R -=Bz 5 .

3 R30 < 02H 44 0 OC RN 2 0 4 R 6 a. R3=111c, R4=Bz b. 713=R4=Bz

- 84 - RO 7 a, R1==Me,R 4 R.. 2=t =Bz 2 R O b. II1=R3=Me, R2 =R4=Bz c. 33.1=a4=Bz,. R2 =!,:t. =Me 1 2 - d. R =R = 3=Bz, R 4=Me e _I. =Me R2 =R 3 =R 4 =Bz

8 a-. R1=R4=1,1E.,,-, R2 =R3 =Bz b R1=R3=Me,,R2=R4=Bz c.1R =R 9- =Bz, =Me R,..=1 ,..2 =R-3 . =Bz, R4 Pie OR3

e. Ri =Me R2 =R3 =J-14 =BZ OR 4

RIOT 1 -4 2 3 9 a. =._i =Me, R =R. =Bz 2 1 3 2 R 0 me b. =_R R =R. =Bz

c . R1=R.4 =Bz, R2 =R3 =Me ' Nc) R3 . R1=13.2=-3 =Bz, LR=Me ) ''- 01;4 . R1=Me, R.2 =R3=R4=Bz 1 4 - 3 , 10 a. R. =R =Mr- ,IA 7-R =_OZ b. R1=R.3=Me, -a 2 -4.=Bz

C . R1=R4=Bz, R2=R3=Me d. iral=_2 =R- =Bz, R.4=Me R1=Me, R2=R3=13.4=Bz R1=R4 112 =R3 h. _R.1 R 3=H, R4 -]tile . 3 2 4, g • R' =R =Me . =tie, =R2 3 4 85

properties of the trimethylsulpivnium4m ion166 rendered this method unattractive for radiochemical synthesis. Subsequently, it was found that treatment of the requisite phenol with one equivalent of sodium hydride in dimethylformamide produced a phenolate ion which reacted rapidly and quantitatively with methyl iodide. This method was used for subsequent radiochemical preparations. Conversion of a protocatoehuic aldehyde derivative into a phenethylamine was accomplished in either of two ways. The method of choice for synthetic purposes involved the formation of the nitrostyrone (2) followed by reduction to the required amine. Treatment of the aldehyde with KOAc/MeNE 167 3ci in neat nitromethane has proved to be a superior method for the preparation of these nitrostyrenes. The lithium aluminium hydride reduction.; of these compounds has given variable results. The best yields have been obtained when pure nitrostyrene stabilized with a trace of acetic acid was slowly added to a very large excess of lithium aluminium hydride. The second route to the amine was utilized for the introduction of 14C into the three position of the isoquinoline nucleus. The benzylchloride (4) was treated with Na14ON in dimethyl sulphoxide168 to give

1•11111.1111•MIM••••111. 11•••••••••111011110= IETile are indebted to Dr. D.S. Bhakuni, who suggested this method. -86 -

the nitrile (5). For the success of this reaction it was necessary to use pure, dry dimethyl sulphoxide and a reaction temperature in excess of 100°. Reduction of the nitrile with lithium aluminium hydride had been reported to give a poor and variable yield of amine?'69 Sodium and alcohol reduction gave none of the desired compound. It was found, however, that reduction of the nitrile with lithium aluminium hydride-A1013 complexi" gave nearly quantitative yields of the desired phenethyl- Arline (3). Since stocks of 3-benzyloxy, 4-methoxy-phenylacetic acid were available to us, this material was, where suitable, converted into the acid chloride and used to acylate the appropriate amine. This method was used for the synthesis of the amide precursor (7) of reticuline and for that of 14(31 -benzyloxy, 4f-methoxy)benzyl]2- methyl, 6,7-dibenzyloxy, 1,2,3,4-tetrahydro-isoquinoline (7d). The appropriate phenylacetic acids were not available for the synthesis of isoquinolines which did not bear the 3'-benzyloxy, 41 -methoxy substituents. In these cases the appropriate derivative of protocatechuic acid was converted into diazoketonc (6) which was then - 87 -

rearranged in the presence of the suitable phenethylamine to give the required amide (7). The use of Ag20 to effect this rearrangement proved unreliable; and this hey reaction was, therefore, accomplished photochemically.171 Cyclodehydration of tho amides with POC13 in toluene proceeded smoothly in all cases. The N-methyl group was introduced by reaction of the 3,4-dihydro-isoquinoline (8) produced as above with methyl iodide. A [14c3N_ methyl could, of course, be introduced at this stage. The methylation was carried out at room temperature in benzene or in refluxing methanol. The first method, although sluggish, had the advantage that the product generally crystallized from the reaction mixture. The latter was faster and often gave a cleaner product. Furthermore, the required imine could be generated in situ by the addition of potassium t-butoxide to a solution of the hydrochloride. Finally, after concentration of the reaction mixture, the intermediate methiodide could be reduced vide infra without isolation. Complications were encountered during the reduction of the methiodides (9) with NaBELI. Under ordinary reaction conditions, from 10 to 40% of the starting - 88 -

material was converted into a non-basic, insoluble substance, presumably a borazine derivative .67 As it proved impossible to convert this material into the desired tetrahydrobonzylisoquinoline,172 its formation was a nuisance. Subsequently, it was found that

gradual addition of a moderate excess of 1 aBH4 to a suspension of iodide in iced methanol, followed by slow warming to room temperature, resulted in a good yield of the desired tetrahydro compound. Reduction of the methiodide with NaBH4 in pyridine was found to provide a useful alternative to the above procedure. Using this latter system we have observed the formation of a minor by-product, which is less polar on thin layer chromatography than the tetrahydro-isoquinoline. During the preparation of 14(31 ,41 -bisbenzyloxy)benzy132-methyl, 6-methoxy, 7-benzyloxy, 1,2,31 4-tetrahydro-isoquinoline (10e), we separated and isolated the by-product. This material gave a positive test with Dragendorfts reagent, and its properties were similar to those of the major and desired product. Bands at 2400 cm-1 in the tnfra red indicated the presence of B-E absorption;173 and the N.M.R. spectrum of this substance was almost identical to that of the major product. These properties exclude a borazine derivative. Mechanistic considerations

-89-

have led us to tentatively formulate this substance as the boron-containing compound (12). This could arise by conversion of the methiodide (9e) into the iso base, followed by electrophilic attack by BH3 on the ettamievi (11).

M e0 M BZO _es N M e B ZO Mc,

B H BH3 082 2 OBZ A•••••••••••••••••••* 082 OBZ

i t 12

The final step required to complete the synthesis of the simple benzylisoquinoline precursors was the removal of the protecting benzyl groups. Our results with the EC1 catalzed debenzylation were disappointing, particularly as the work-up was lengthy and the yields, on occasion, were nearly disastrous. Although hydrogenolysis was reported to produce erratic results,17 this method was investigated as an alternative. It was found that, by using a large excess of palladium on carbon, and by taking suitable precautions to insure the - 90 -

purity of the substrate, hydrogenolysis was completely reliable and gave a product of high quality. Although on occasions HC1 was added to increase the solubility of the substrate, the presence of acid was not necessary for the success of the reaction. On the one occasion that the reaction failed, removal of the catalyst followed by addition of fresh catalyst produced the desired result. Utilizing the reactions outlined above, we prepared (t)- 6-p-methyl[140] reticuline, (t)43-140]reticuline, (±)- N-methyl[140] reticuline, and 1-[(31 -hydroxy, 40-methoxy)benzyl]2-nethyl[140], 6,7-dihydroxy, 1,2,3,4-tetra hydroisoquinoline (10h). Another compound - 143'140- dihydroxy)benzyl]2-methyl, 6-methoxy, 7-hydroxy, 1,2,3,4- tetrahydro-isoquinoline (10i), obtained by total synthesis as above, was labelled by base (triethylamine) catal/zed exchanged with HTO in dimethylformamide. This type of exchange was formulated some time ago as an electrophilic attack on phenoxide by phenol175 and is specific for o and 2 positions. The enantiomers of reticuline, labelled by acid exchange,117 were provided by Dr. D.S. Bhakuni; and protosinomenine (10g), labelled by basic exchange, was provided by Mrs. A. Kirby. In - 91 - addition, two other isomers of reticuline (10b) and (10c) were prepared as dibenzyl ethers. These compounds were not labelled. Since it was felt that the total synthesis of labelled protoberberine compounds would involve a dis- proportionate amount of effort, attempts were made to label readily available protoberberines. It has been observed in these laboratories that in the presence of a moderate amount of H01, 0-methoxyphenols exchange aryl protons at positions o to methoxyl groups as well as those o to hydroxyls.117 Although berberine proved resistant to electrophilic attack, it was hoped that tetrahydroberberine would exchange its aryl protons under similar conditions. Unfortunately, tetrahydroberberine is not sufficiently soluble in strong mineral acids such as HCl, H2SO4, HC1041 and FTO3 for the exchange method to be feasible. Solution of tetrahydroberberine in warm D PO resultedin complete decomposition of the 3 substrate. The complex of and BF 176 has been H3PO4 3 reported to be a useful medium for the exchange of aryl protons.177 When this reagent prepared from D31,04 and BF unchanged 3 etherate was used the substrate was isolated. Preparation from D3PO4 and gaseous BF3 produced - 92 - a more vigorous reagent which rapidly destroyed the substrate. In view of these discouraging results, we sought a less direct route. When berberine chloride was roasted in a stream of CO2, methyl chloride was eliminated with the formation of a red phenol betaine, berbe3Tubine (13).178 This material, without further purification, was reduced with

Na ll4 in methanol to give tetrahydroberberkibine or (t)-nandinine (14). This sequence enabled us to

<0 O

OMC 0M0

13 14 introduce a "handle"; and subsequent alkaline exchange gave (t)-nandininelabelled with tritium p to the hydroxyl. Methylation with diazomethane completed the synthesis of (t) [3H]canadine. For our brief investigations of the biosynthesis of argemonine (16), we required a supply of this alkaloid. -93-

As only small amounts of plant material were available, isolation of the alkaloid was not practicable. We, therefore, prepared the racemic alkaloid (N-methylpavine) from papaverine as shown.179

M 00 MeO Me0 meo ome Me()

O 16 Me ScMC

The weak point of this synthesis lay in the preparation of 1,2-dihydro-N-methylpapaverine (15). The literature procedurel" involved the reduction of ally papaverine methiodide with lithium aluminium hydride in tetrahydrofuran. This procedure was tedious and the reported yield is only about 30%. We sought a simpler procedure with a mild work-up and a high yield. The generally accepted mechanism for the reduction of an isoquinolinivM compound to a tetrahydro compound with NaBELI181 is outlined below. - 94- H 4-

N d8H4

It was apparent that the use of NaBH4 in a basic, aprotic solvent might enable one to isolate the intermediate 1,2,-dihydrc-isoquinoline. In fact, when berberine

chloride was treated with NaBH4 in pyridine, a quantitative yield of 1,2-dihydroberberine was obtained. Admittedly, this represented a favorable case as the double bond of 1,2-dihydroberberine shows little tendency to migrate into the 2,3-position. However, when

papaverine methiodide was treated with NaBH4 in pyridine, 1,2-dihydro-N-methylpapaverine was obtained in 70% yield based on starting papaverine. It was also found that the 11 2-dihydro compound on treatment with NaBH4 in aqueous methanol was further reduced to laudanosine. The N.M.R. spectrum of 1,2-dihydro-N-methylpapaverine exhibits two unexpected features. The 3 and 4 protons produce the expected quartet, but the resonance of the 3 proton is split - J=ca. 1 cps. The absence of this fine splitting in the spectrum of the closely related -95-

compound, N-nethylisopapaverine (17) suggested that in the 1,2-dihydro-N-methylpapaverine, the 3 proton was coupled with the 1 proton. A subsequent spin de- coupling experiment confirmed this. Recent information suggests that such long range coupling is surprisingly common in dienones and dienols182 as well as in alicyclic 183 systems. To our knowledge, however, this is the first example of such long range coupling across a hetero atom. The steric requirements for this type of coupling differ from those of allylic coupling. The coupled protons must be equatorial, co-planar, and situated at the arms of a 171" as illustrated (18).

MeO me°

H Me©, OR Me0 17 18

The proton resonance of one of the methoxyls of 1,2- dihydro-N-mothylpapaverine occurs at 0.2 t higher than those of the other three. This was also unexpected, - 96 - but the examination of the N.M.R. spectra of a number of benzylisoquinolines proved instructive (Table 1). In each of these compounds, the resonance of the alkoxyl protons at position seven occurs at 0.2 - 0.5t higher field than the resonances of protons attached to similar alkoxyl groups. In all but one of these compounds, vide infra, the proton at position eight also resonates at higher field than similar aryl protons. These observations bring to mind the situation reported for the aporphine alkaloids1 184 and suggest that the 1-benzyl group is so oriented that the shielded protons lie within the shielding region of that group. Apart from enabling us to identify quickly the signal of an alkoxyl at position seven, this phenomenon provides an insight into the conformation of the molecule. Inspection of models indicates that the observed shielding, particularly that of the eight proton, is best explained by assuming a quasi-axial orientation of the benzyl group. Such a conformation does not appear unfavorable, and the long range coupling observed in 1,2-dihydro-N- methylpapaverine strongly suggests the presence of a quasi-axial benzyl in this compound at least. - 97- Table 1 Group Normal Compound at 7 Resonance Resonance 8-H ( ) ( rr ) (1w) Laudanosinc rye 0 6.4 6.1, 6.17 3.85 N-Methyl isopapavrine Me0 6.68 6.15,6.25 N-Methyl-1,2- -dihydropapaverino Mc° 6.41 6.15,6.25 4.07 Dibonzyl reticuline * Bz0 5.15 4.9 3.82 Dibenzyl protosinomenino * Mc° 6.40 6.15 3.92 10 b. Bz0 5.22 4.92 3.87 10 c. Mc° 6.45 6.19 3.9 10 c. Bz0 5.2 4.95 3.9

* We arc indebted to Yrs. A. Kirby who drew these examples to our attention. - 90 -

Since the 7 methoxyl is shielded in N-methylisopapaverine, it appears that the phenyl group is trans to the nitrogen. In this compound the benzylidine group is similar to an equatorial benzyl, and it is interesting to note that the eight proton does not appear to be shielded. -99-

EXPERIMITT.AL

All melting points, unless otherwise stated, were obtained using a micro ;oiler hotstage, and all are reported uncorrected. Micro-analyses were performed at Imperial College under the direction of Miss J. Cuckney. Organic extracts containing basic substances were dried over potassium carbonate. Those containing neutral or acidic substances were dried over sodium sulphate. Nuclear magnetic resonance spectra were run on a Varian A-60 spectrometer. Unless otherwise stated, these spectra were obtained on 10% solutions in chloroform. - 100 -

PRECURSORS

14 2=1,1911Sayl-r_a:A=122Eayloxybenzaldehyde-1 To a solution of 4-benzylprotocatechuic aldehyde (15 mg) in dimethylformamide (1 cc) was added 55% sodium hydride (3 mg). The mixture was warmed until hydrogen evolution, had ceased. The resulting yellow solution was frozen into an l'Amershamn break-seal ampule containing [14 03methyl iodide (2.9 ng, 0.1 mc). The ampule was evacuated and sealed. The break-seal was ruptured, and the methyl iodide was distilled into the frozen reaction mixture. After four days, inactive methyl iodide was added. The product was extracted from aqueous sodium hydroxide with ether and was crystallized to give material (16 mg) with m.p. 61-63° (0.07 mc).

3-Methoxy-4-benzyloxy-W-nitrostyrene (2a)(method of G. M. Thomas) A solution of 0-benzylvanillin (20 mg) in ethanol (0.6 cc) containing nitromethane (0.02 cc) was cooled to five degrees. A solution of 5% sodium hydroxide in ethanol (0.15 cc) was added. After a few minutes, - 101 - the reaction mixture was poured into iced 8N hydrochloric acid. The bright yellow product (16 mg) which crystallized was filtered off; m.p. 118-122° (lit.185 122-123°).

3-Benzyloxy-4-methoxy-W-nitrostyrene (2b) To a solution of 0-benzylisovanillin (2 g) in nitromethane (10 cc) was added methylamine hydrochloride (0.25 g) and potassium acetate (0.25 g). The mixture was shaken at room temperature overnight. Chloroform was added to dissolve the nitrostyrene, and the inorganic salts were filtered off. Most of the solvent was removed under vacuum, and the product was crystallized by adding hot ethanol (containing a trace of acetic acid). The product (2.4 g) melted at 126-120° (lit.187 128°).

3,4-Bisbenzyloxy-bi-nitrostyrene (2c) When dibenzylprotocatechuic aldehyde (2 g) was treated as above, 3,4-bisbenzyloxy-W-nitrostyrene (2.2 g) was obtained; m.p. 119-120° (lit.167 118-9°).

3 -Methov-4-benzyloxyphenethylamine (3a)186 A solution of 3 -methoxy-1f-benzyloxy-.LI-nitrostyrene (2a) (2g) in dry, distilled tetrahydrofuran (30 cc) was - 102 -

added slowly to a well-stirred, refluxing suspension of lithium aluminium hydride (5 g) in dry tetrahydrofuran (40 cc). After two hours, the mixture was treated with ethyl acetate and then with sodium hydroxide solution. The suspension was diluted with ether and filtered through celite. The filter cake was washed thoroughly with hot tetrahydrofuran and ether. The organic filtrates were concentrated and the residue was taken up in dilute hydrochloric acid. Non-basic material was removed by benzene extraction. A small amount of insoluble polymer separated at this stage and was discarded. The aqueous solution of amine hydrochloride was rendered basic and was exhausted with chloroform. The chloroform was dried and evaporated. The product was dissolved in a small volume of ethanol and treated with ethereal hydrochloric acid to give crystals (1.4 g), m.p. 176-178°.

5 -Benzyloxy-4-methoxyphenethylamine (3b) When 3.-benzyloxy-4-methoxy-W-nitrostyrene (2b) (2g) was treated as above, 3'-benzyloxy-4-methoxyphenethylamine hydrochloride (1.3 g) was obtained; m.p. 167° (lit.187 166°). - 103 -

5,4-Bisbenzyloxyphenethylamine (3c) When 3,4-bisbenzyloxy-td-nitrostyrene (2c) (2 g) was treated as above, 3,4-bisbenzyloxyphenethylanime hydrochloride (1.15 g) was obtained; m.p. 135° (lit.'" 132-133 °).

a372eiz-11etho-4-1Y 321 'lenlacetonitrile (5) To a solution of 3-methoxy-4-benzyloxybenzyl chloride (4) (630 mg) in dimethyl sulphoxide (2 cc) (freshly distilled from calcium hydride at 1 mm) was added sodium cyanide (180 mg) in dimethyl sulphoxide (1.5 cc). The mixture was heated at 95-100° for three hours, cooled, and treated with saturated aqueous sodium chloride. The product was extracted into ether (4X). The ether was washed with water (6X with 1/20 vol.), dried, and evaporated. Crystallization from ether/petrol gave the product (500 mg); m.p. 67° (lit.67 67-8°).

3-Methoxy-4-benzyloxyphen-ethylamine (3a) (Alternative procedure) To a solution of aluminium chloride (4 g) in dry ether (50 ce) was added lithium aluminium hydride (1 g). - 104 -

The solution was swirled for a few minutes, and then filtered. To the filtrate (20 cc) maintained under reflux was slowly added the nitrile (5) (370 mg, vide supra, in dry ether (25 cc). After thirty minutes under reflux, moist tetrahydrofuran (20 cc) was slowly added. After thirty minutes more, water was added to the iced mixture; and the reaction mixture was brought to pH < 2 with sulphuric acid. The ether was removed. The aqueous layer was taken to pH > 10 with potassium hydroxide and exhausted with ether. Evaporation of the ether gave the amine (320 mg); m.p. of the hydrochloride - 176-178°.

(3-Methoxy-4-benzyloaEldma22-121 14c anitrile (5) To Na[140]11 (1.5 mg, 0.5 mc) was added inactive sodium cyanide (6.7 mg) in dimethyl suiphoxide (0.3 cc). A solution of 3-methoxy-4-benzylozybenzyl chloride

(59 mg) dimethyl sulphoxide (0.5 cc) was added. The closed reaction vessel was maintained at 100-105° for three hours. The mixture was then cooled and diluted with saturated aqueous sodium chloride solution. The product was extracted into ether (4 x 5 cc) (carrier nitrile, 48 mg, was added to the last extract). - 105 -

The ether was washed with water (5 x 1.5 cc), dried and evaporated. The crude nitrile in ether (3 cc) was reduced as above using reagent solution (3 cc) to give 3-methoxy-4-benzyloxyphen[14C]ethylamine (0.35 mc, 70%).

3-Methoxy-4-benzylov-W-diazoacetophenone (6a) A suspension of 0-benzylvanillic acid (0.75 g) in dry benzene (50 cc) containing oxalyl chloride was warmed on the steam-bath for two hours. The solvent was then evaporated under reduced pressure. More benzene was added and evaporated. This was repeated several times. The resulting acid chloride, which spontaneously crystallized, was dissolved in ether (50 cc) and added slowly to a cold ethereal solution of diazomethane (from 5 g of nitrosomethylurea). The flask was stoppered. After storage overnight, the solution yielded 3-methoxy-4-benzyloxy-W-diazoacetonhenone (460 mg); m.p. 105-106° (lit.189'19° 109° or 98-99°). Concentration of the mother-liquors gave 180 mg more.

3,4-Bisbeny1oy-W-diazo-acetophenone (6b) The acid chloride derived as above from dibenzyl- protocatechuie acid (0.80 Op dissolved in a mixture - 106 - of ether (50 cc) and benzene (15 cc), was added to an ethereal solution of diazomethane as above. After storage overnight at 5°, the solution was concentrated. The product (0.720 g, m.p.100-102°) was crystallized from ether. The analytical specimen recrystallized from ether gave a m.p. of 103°. (Found: C, 73.48;

/11 5.13; N, 8.01. C2218 03N2 requires C, 73.73; HI 5.06; N, 7.82%.)

E:13-Methoxv-4-benzyloxy-phenethyl)-3-benzyloxy-4- methoxyphenylacetamide (7a) A solution of 3-benzybxy-4-methoxyphenylacetyl chloride (prepared from the phenylacetic acid, 30 mg, as above) in dry benzene (3 cc) was added to 3-methoxy- 4-benzyloxyphenethylamine (liberated from the oxylate, 20 mg) and potassium carbonate (7 rig). The suspension was stirred for two hours and boiled for ten minutes. The solvent was removed end the residue was briefly digested with aqueous sodium bicarbonate. The product was extracted into chloroform. The chloroform was washed, dried, and evaporated. Crystallization from ether/ethanol gave the amide (24 mg, 90% based on amine); m.p. 136-80 ( lt.67 135 _60; 139_410). - 107 -

phenylacetamide (7d) To a stirred solution of 3,k-bisbenzyloxyphenethylamine (liberated from the hydrochloride, 800 mg) in dry benzene was added magnesium oxide (1.5 equivalents), then a solution of 3-benzyloxy-4-methoxy-phenylacotyl chloride (650 mg). The stirred suspension was warmed for three hours and then worked up as above to give amide (1.1 g); m.p. 133-136° (lit.191 137°.).

N-(3-Methoxy-4-benzyloxy-phenW-3-methoxy-4-benzyloxy- phenylacetanide (7b) A solution of 3-methoxy-4-benzyloxyphenethylamine (liberated from the hydrochloride, 293 mg) and 3- methoxy-4-benzyloxy-00 -diazoaeetophenone (280 mg) in dry benzene (60 cc) was irradiated under nitrogen using a small mercury lamp. After eighteen hours, the diazoketone band was no longer visible in the infra red, and the reaction was terminated. The solvent was evaporated, the product (210 mg) was crystallized from ethanol/ether; p.p. 125-130°. Treatment of the mother-liquors with charcoal and concentration of these gave more product (120 mg; m.p. 125-128°). The - 108 - analytical specimen was recrystallized from ether; m.p. 129-130°(lit.192 1280). (Found: CP 75.46; E, 6.66. Calcid. for C 3)O5_ IT: 0, 75.12; H, 6 2 0 .50%).

N-(5-Methoxy-4-benzyloxy-phenothyl)-5,4-bisbenzyloxy- phonylaoetamicle (7e) A solution of 3-methoxy-4-benzyloxyohenethylamine (liberated from the hydrochloride, 290 mg) and 3,4- bisbonzyloxy-W-diazoacetophenone (360 mg) in dry benzene (60 cc) was irradiated as above. The usual work-up afforded two crops of crystalline material (405 mg; m.p. 128-132°, 75 mg; m.p. 125-129°. The analytical specimen was recrystallized from ethyl acetate/ ether; m.p.131°. (Found: 0, 77.52; HI 6.34;

14, 2.45. 0 0 11 requires: 0, 77.66; F19 6.35; 381137 5 N, 2.39%).

N-(3-Benzyloxy-4-methoxy-phenethyl)-3-methoxy-4-benzylwcy- phenylacetamide (7c) A solution of 3-benzyloxy-4-methoxyphenethylanine (liberated from the hydrochloride, 215 mg) and 3- methoxy-4-benzyloxy-W-diazoacetophenone (206 mg) in dry benzene (50 cc) was irradiated as above. The usual - 109 -

work-up gave c.v.:Ade (315 mg); m.p. 125-128°. The analytical specimen was recrystdilized from ethanol/other; m.p. 128°. (Found: C, 74.89; E, 6.62; N, 2.87. C32H3305N requires: C, 75.12; Et 6.50; N, 2.74%).

1=1(3T-Beazyloxy-41 -methoxy)-benzyl]-6-methoxy-7- benzy/o4Ey-3,4-dihydro-isoquinoline hydrochloride (8a) To the appropriate amide (7a) (1 g) in dry, refluxing toluene (16 cc) was added freshly-distilled POC13 (0.4 cc). After one hour, the solvent was removed in vacuo. The residue was triturated with petroleum ether and ether. The product (0.01 g) was crystallized from ethanol; m.p. 208-216° (lit.72 198-2000).

ILA-dihydroisoquinoline hydrochloride (8b) Toa suspension of the requisite amide (7b) (0.5 g) in dry, refluxing toluene (20 cc; was added freshly- distilled FOCI (0.5 cc). After tventy minutes, a 3 second portion of POC13 (0.5 cc) was added. After another twenty minutes, the reaction was worked up as above to sive the product (400 mg); m.p. 197-199° (lit.192 194-196°). - 110 -

1.715t-Benzyloxy-41 -methoxybenzy1)-6,7-bisbenzyloxy- 0-dihydroisoquinoline hydrochloride (8d) .inter treatment of the appropriate amide (7d) (0.51 g) as above, the product (447 mg) was obtained; 12.1). 175-1800 (lit.191 179-1810).

1-(51 ,4t-BisbenzyloxybenaV-6-methoxy-7-benzyloxy- 3,4-dihydroisoeuinoline hydrochloride (8e) After treatment of the appropriate amide (7e) (0.52 g) as above, the product (0.45 g) was obtained; m.p. '182-134°. The analytical specimen was recrystallized from ethanol/ether; m.p. 182-184°. (Found:

0, 75.41; El, 6.22. C38113604N:L1 requires: C9 75.30; H, 5.98%).

1-(31 -Methoxy-4t-benzyloxybenzy1)-6-benzyloxy-7-methoxy- 11 4-roiso ochloride (8c) After treatment of the appropriate amide (7c) (400 mg) as above, the product (380 mg) was obtained; m.p. 145-150°. This material showed a strong tendency to form solvates and, therefore, suitable analytical data were not obtained. It was, however, successfully 'converted into the known methyl tetrahydro derivative, (10 c), vide infra. 1=1,t-Benzyloxy.-41 -methoxybenayllza=2.anyltiam= 7-benzyloxy-5,4-dihydroisoouinolinium iodide (9a). This compound was prepared using Jain's method.72

1-(51 -Benzyloxy-4t-methoxybenzy1)-2-methyl[140]-6- methoxy-7-benaloxy-5,4-dihydroisoouinolinium iodide (9a) The free base (liberated from the hydrochloride, (8a), 68 mg) was dissolved in dry benzene (1 cc). Part of this solution (0.5 cc) was frozen into a reaction tube. Methyl[14C]iodide (0.9 mg, 0.1 mc) was distilled in from a break-seal via/. The break-seal was washed with methyl iodide (4 mg) in benzene (0.1 cc), and this was subsequently distilled into the reaction mixture. The mixture was sealed and stirred in the dark for seven days. The solid nethiodide was then removed and a fresh portion of the free base was added, with one drop of methyl iodide, to the mother-liquors. The seoond crop was collected after one day. The crops were combined and reduced with sodium borohydride, vide infra. - 112 -

1-(3!-Methoxy-4t-benzyloxybenzyl)-2-methyl-6-methoxY- 7-benzyloxy-3,4-dihydroisoquinolinium iodide (9b) A portion of aqueous sodium bicarbonate was added to a solution of dihydroisoquinoline hydrochloride (8b) (220 mg) in aqueous alcohol. The turbid mixture was exhausted with ether, and the ether was dried and evaporated in the cold. The crystalline free-base was dissolved in dry benzene (5 cc). The solution was flushed with nitrogen and treated with methyl iodide (0.3 cc). After storage for twenty hours in the dark, the solution yielded a crystalline material (180 mg), m.p. 170-1°, (lit.194 172.5-175.5°)p which was washed with ether and innediately reduced with sodium borohydride, vide infra.

1-(5t-Benzyloxy-41 -methoxybenzy1)-2-metLy1-6,7-bis- aDLY12a=,aAZWagE21s2 quino/inium iodide (9d) Men the isoquinoline hydrochloride (8d) (400 mg) was treated as above, the product (410 mg) was obtained; m.p. 178-180°. Recrystallization raised the melting point to 184-186° (lit.191 185-187o). - 113 -

1-(31 ,4t-Bisbenzyloxybenzy1)-2-methyl-6-methoxy-7- 22Ralay=221=2.1LEIElisoquinolinium iodide (9e) Application of the above procedure to the hydrochloride (8e) gave an oil. To a solution of hydrochloride (Se) (320 mg) in methanol (10 cc) was added potassium t-butoxide (61 mg) and then methyl iodide (0.4 cc). The suspension was filtered, and the filtrate was heated to reflux for fortyfive minutes. Concentration of the solution and addition of other gave methiodide (400 mg); m.p. 180°.

1-(31 -Mothoxy-41 -benzyloxy)-2-methyl-6-benzyloxy-7- methoxy-3,4-dihydroisoquinolinium iodide (9c) 'When the appropriate hydrochloride (8c) (0.3 g) was treated as above, mettiodide (390 mg) wc,s obtained; m.p. 143-146° (lit.193 144-145°).

1-(3!-3enzy1oxy-4t-methoxybenzy1)-2-metha:6-methoxy- 7-benzyloxy-1,2,3,4-tetrahydroisociuinoline (10a) (Method of D. S. Bhakuni): To a suspension of methiodide (9a) (25 rig) in iced methanol (0.4 cc) was added sodium borohydride (6 mg). The mixture was stirred and slowly allowed to come to - 114 - room temperature. After two hours,., water was added; and the solution was exhausted with ether. The ethereal extracts were chromatographed on alumina. The eluted dibenzyl-("±)-reticuline (10a) was cryStallized from ether/petroleum ether to give 15 ng; m.p. 87-89° (lit.72 890).

1-(31 -Benzyloxy-41-methoxybenzy1)-2-methyl-6 7-bisbenzyloxy-4204 4-tetrahydroisociuinoline (10d) The nethiodide (9d) (120 mg) was added to a suspension of sodium borohydride (18 mg) in pyridine (5 cc). The mixture was stirred for fifteen minutes, poured into water, and exhausted with ether. The ethereal extracts were dried and evaporated. Several portions of toluene were added to the residue and then evaporated to remove traces of pyridine. The resulting oil was dissolved in ether. Petroleum ether was added and, after refrigeration, the amine (90 mg) was obtained; 12.13. 83-85° (lit .151 86°).

1-(3T-Methoxy-41 -benzyloxybenzy1)-2-nethyl=k-methoxx= 7-benzyloxy-1,2,3,4-tetrahydroisoquinoline (lob) tihen the methiodide (9b) (so mg) was treated as above, the amine (60 ng) was obtained; m.p. 95-970 - 115 -

(lit.194 94-95°).

1-(31-Methoxy-41 -benzyloxybenzy1)-2-methy1-6- benzyloxy-7-methoxy-1,2,3,4-tetrahydroisoeuinoline (100 When the methiodide (90) (400 mg) was treated as above, the amine (290 mg) was obtained; m.p. 93-95° (lit.193 91-93°).

1-(31 ,4t-Bisbenzyloxybenzy1)-2-mothyl-6-methoxy-7- the Boron Compound (12) After treatment of the methiodide (NO (400 mg) as above, a mixture was obtained; p.p. 145-1520. Chromatography on alumina gave two fractions: a non- polar one (35 mg), m.p. 155-160°, with strong bands at 2400 cm-1; and a more polar one (221 mg), m.p. 62° (recrystallized from methanol). Both compounds gave a positive Dragendorfls test, and both had similar N.M.R. spectra. The more polar compound was the only product isolated when the methiodide (9e) was reduced with sodium borohydride in methanol. Analytical specimen (10e) crystallized from methanol; osp. 62°. - 116 -

Found: 0, 79.07, 79.29; H, 6.94, 6.87;. 0, 12.08,

N, 1.82, 2.04; Calculated for C39E3904N: C, 79.97; 6.71; N, 2.39; 0, 10.93. Calculated for C 11 NO ..iMe011: C, 78.87; H, 39 39 4 6.79; N, 2.33; 0, 11.94.

1-(31-1Benzyloxy-41-methoxy-benzy1)-2-methYlf14ci_ 6t7-bisbenv•loxy-112,3,4-tetrahydroisocuinoline (10d) The hydrochloride (8d) (60 mg) was dissolved in methanol (0.5 cc) and frozen. Sodium hydroxide (1N) in methanol (0.1 cc) was added to the frozen mixture. [14C)Methyl iodide (1.49 mg, .05 mc) was distilled into the mixture. This was followed by inactive methyl iodide (.0025 cc) in methanol (0.1 cc). The reaction vessel was sealed and the solution stirred in the dark for 4 days at 50°. The vessel was then opened and methyl iodide (lac) added. The solution was heated under reflux for one hour. The solvents were removed, methanol (1.5 cc) added to the residue, and the mixture cooled to 4°. Sodium borohydride (15 mg) was added and the mixture stirred for 3 hours. After the usual work- up, the crude product was chromatographed to give the amine m.p. 86°, (0.025 mc). - 117 -

1-(3t-Hydrozy-41 -methozybenzy1)-2-methyl-6-methoxy-7- hYdr°047-421.11.1=I2WWZ21222211 19111 122. The dibenzyl ether (10a) (18 rag) freshly chromatographed and recrystallized, was added to a suspension of pre-reduced 10% palladium on carbon (25 rag) in ethanol (2 cc). Upon shaking, there was an immediate uptake of hydrogen; it continued until two equivalents had been absorbed (about forty-five minutes). The shaking was continued for one hour during which little additional hydrogen was taken up. The catalyst was removed, and the ethanol was evaporated to give (.t) reticuline as a white foam.

1-(31 711ydroxy-411 -methoxybenzyl)-2-methyl-6,7-dihydroxy- 19295,4-tetrahydroisoquinoline (10h) When the tribenzyl ether (10d)(150 mg) was treated as above, there was no hydrogen uptake. A drop of concentrated hydrochloric acid was added to solubilize the starting material, and the catalyst was filtered off. Fresh catalyst (180 mg) was added. The reaction then proceeded as usual with three equivalents taken up in forty-five minutes. After two hours, the catalyst was removed. The ethanol was concentrated to a small - 118 -

volume and diluted with aqueous sodium bicarbonate. The suspension was exhausted with chloroform and ether. The organic solvents were evaporated to yield a yellow powder (73 mg); m.p. 119-23° (lit./91 120-2°). N.M.R. shoved the complete absence of benzyl groups. This compound was exceedingly unstable in the presence of air, acid, or base.

1-(3',41-Dihydroxybenzy1)-2-methyl-6-methoxy-7- _bmficy:-19 2.,4-tetrahydroisoquinoline (101) When the tribenzyl ether (10e) (300 mg) was treated as above (300 mg catalyst and 3 drops of acid), the usual work-up yielded a white solid (164 mg); N.M.R. in dinethylformamide showed the presence of N-methyl, 0-methyl, 5 aryl protons, and no benzyl groups.

CiT-Nandinine Berberine hydrochloride (500 mg) was roasted under carbon dioxide atmosphere at 185-205° for seventy-five minutes. The residue was suspended in water and exhausted with chloroform, and the chloroform extracts were evaporated. The blood-red residue was dissolved in aqueous methanol (20 cc, 90%) at 4° and treated with - 119 - sodium borohydride (150 mg). After thirty minutes, water was added; and the solution was brought to pH > 10 with sodium hydroxide. The solution was washed with ether, the pH adjusted to 8; and the solution was exhausted with chloroform. The chloroform was evaporated, and the crude product was chromatographed on alumina. Crystallization from methanol gave (±)-nandinine (185 mg); n.p. 170° (lit.'" 167°) . litl=2H3Nandinine After a mixture of (±)-nandinine hydrochloride (75 mg), potassium t-butoxide (75 mg), and deuterium oxide had been heated at 100° for 168 hours, it yielded a material which showed, in N.M.R. analysis, the expected loss of the proton 2 to the hydroxyl. To prepare the tritiated material (±)-nandinine (100 mg), potassium t-butoxide (72 ug), and tritiated water (0.5 cc, 0.1oni10 were heated at 100° for six clays. The reaction vessel was then opened, and 1 cc dimethylformamide was added. After four days more, the reaction was terminated. The (±)-PH]nandinine was chromatographed and crystallized; activity - 1.18 x 105 dps/mg. - 120 -

(±)-Canadine The (±)-Namainine (20 mg) dissolved in methanol (4 cc) was cooled in an ice-bath. Diazomethane in ether (standard preparation, 4 cc) was added. The flask was loosely stoppered, placed in a basin of ice, and stored in the dark overnight. Thin layer chromatography indicated nearly complete methylation by the next morning. After one more day the solvents were removed, and the residue was chromatographed to give the product (15 mg); u.p. 169-172°.

[8,2t,51 ,6?-3H]1-(350-dil y1-6- methoxy-7-hydromy-19 2,3,4-totrollydroisoquinoline To the phenol (101) (20 mg) in dimethylformamide (0.2 cc) was added tritiated water (0.5 cc, 0.1 curie) and triethylamine (0.2 cc). The solution was deoxygenated with dry, oxygen-free nitrogen, sealed under vacuum, and maintained at 100° for five days. The solvents were evaporated. Methanol was added to the residue and evaporated. This was repeated to remove labile tritium. The final product was extracted into chloroform/ether from aqueous sodium bicarbonate to give the material (5 mg); activity 2 x 107 dpm/mg. - 121 -

1,2-Dihydroberberine Berberine chloride (150 mg) was added to a suspension of sodium borohydride (30 mg) in pyridine (10 cc). After fifteen minutes, water was added; and the lemon- yellow crystals (105 mg) were filtered off; m.p. 168-9° (lit.'" 166-7 °).

1,2-Dihydro-N-methylpapaverine Papaverine methiodide (from papaverine, 0.5 g) was added to a suspension of sodium borohydride (60 mg) in pyridine (20 cc). After ten minutes, more sodium borohydride (40 mg) was added. The mixture was allowed to stand, with occasional agitation, for ten minutes. A large volume of ether was added, and the solution was washed with water. The ethereal solution was dried and concentrated in vacuo. The remaining pyridine was removed on a rotary evaporator attached to an oil pump. Addition and evaporation of toluene removed the last traces of pyridine. The product was crystallized from ether/petroleum ether to give a material (370 mg); m.p. 134-136° (lit.180 135°). - 122 -

N.M.R. Data

Tau values for the resonances are listed with the number of protons in parentheses.

1-(3t-methoxy-41 -benzyloxy-benzy1)-2-methyl-6-methoxy-7- benzyloxy-1,2,324-tetrahydro-isoquinoline (lob) 7.5(3); 6.25(3); 6.2(3); 5.22(2); 4.92 (2); 3.87(1); 3.3-3.5 (4).

1-(31 -methoxy-4I-benzyloxy-benzy1)-2-methyl-6-benzyloxy- 7-methoxy-1,2,314-tetrahydro-isoquinoline (10c) 7.44(3); 6.45 (3); 6.19(3); 4.91(2); 4.85(2); 3.91(1); 3.1-3.4(4).

1-(31 ,41 -bisbenzylozy-benzy1)-2-methy1-6-methoxy-7- benzyloxy-1,2,3,4-tetrahydro-isoquinoline (10t) 7.5(3); 6.17(3); 5.2(2); 4.95(4); 3.9(1); 3.1-3.6(4).

"Boron compound" (12) 7.38(3); 6.17(3); 5.45(2); 4.92(4); 4.4(1); 3.1-3.7(4).

N-methyl-i, 2-dihydropapaverine 7.1(3); 6.41(3); 6.25(3); 6.15(6); 4.75(doublet 1); 4.07(1); 3.95(doublet-1); 3.2-3.6(4). - 123 -

DEGRADATIONS

1-Phenyl-1,2,7dihydroberberine To a suspension of dried berberine chloride (63 mg) in dry ether (0.5 cc) was added IM ethereal phenyl magnesium bromide (1 cc). The suspension was stirred under reflux for 21 hours. Water was added, then hydrochloric acid. nScratching , produced a white powder which was removed by centrifugation. This solid was dissolved in hot water, and the free base was precipitated with ammonia. The free base was then dissolved in hot acetic acid and ethanol was added. On addition of ammonia, the product crystallized. It was washed with water and dried to give a crystalline solid (52 rig); m.p. 194-198° (lit.111 195o).

Emde base from protopine (3,p.55) To a solution of protopine methyl methano sulphate (2, p.55) in 5% aqueous sulphuric acid (5 cc) was slowly added freshly-prepared 4% sodium amalgam (20 g). The reaction was maintained at steam-bath temperature and kept strongly acid by the periodic addition of sulphuric acid. After the addition of the amalgam - 12' -

(about fifteen minutes), the mixture was poured into 4N sodium hydroxide. The resulting suspension was exhausted with ether, chloroform, and ethyl acetate. The combined organic extracts were dried and evaporated. The residue was crystallized from ether to give a solid (66 mg); m.p. 110-112°. The analytical specimen was recrystallized from ether; m.p. 109-111°. (Found: C, 68.01; H, 6.78. C21 2505Nrequires: C, 67.90; H, 6.78%).

Isoprotopine chloride (6, p.58) This compound was prepared according to Perkin's method.131 7Then POC13 with boiling point below 106° was used, the major product was protopine hydrochloride which was not converted with prolonged treatment with P0C1 into isoprotopine chloride. The desired 3 transformation was accomplished uhen POC1 b.p. 106-7°, 3 was used.

Anhydromethylprotopine (7, P.58) This compound was prepared according to Perkin.131 - 125 -

Ozonolysis of amhydromethylprotopine (7, p.58) A slow stream of ozone was bubbled through a solution of anhydromethylprotopine (40 ng) in ethyl chloride (20 cc) at -78°. After forty-five minutes, ozone appeared in the exit-gases. The solvent was allowed to evaporate. Zinc dust (0.8 g) and water (15 cc) were added to the residue. After thorough mixing, the suspension was allowed to stand one hour and then was steam-distilled. The distillate was collected in saturated aqueous dinedone solution. After storage of the solution overnight, it yielded formaldehyde dimedone; m.p. 190-191°.

Degradation of acetic acid Sodium acetate (5 mg) was subjected to the Schmidt Decarboxylation.136 The resulting methylamine was distilled into aqueous hydrochloric acid. The solution was evaporated and the residue was dissolved in methanol. 0-benzylisovanillin (25 mg) was added followed by one drop of sodium hydroxide solution. After one hour, sodium borohydride (30 mg) was added, and the solution was allowed to stand for another hour. The basic material was separated and isolated as the hydrochloride (12 mg); m.p. 212° (lit.48 212°). - 126 -

TRACER EXPERIMENTS

Counting techniques, Carbon-14-containing specimens were counted as thin films ( < 0.5 memi3 using either a windowless proportional counter or a thin-window low-background Geiger counter. All relevant samples were counted at the same time to obviate difficulties due to changing counter efficiency. All samples were counted in duplicate. Tritium was assayed by liquid-scintillation techniques. The sample (0.2 - 4.0 mg) was dissolved in dry dimethylformamide (four drops). Nuclear Enter- prises scintillator Ne.N.E.213 (1.2 cc) was then added. Samples were counted in duplicate; and any substantial quenching was corrected by extrapolating the log of specific activity-versus-concentration curve to zero 14-1 concentration. [11 25H]Hexadecane and [1_ ujHexadecane were used as standards. The ratios of carbon-14 to hydrogen-3 were determined by counting a scintillation sample containing both isotopes at two different EHT settings. Counting efficiencies for carbon-14 and hydrogen-3 at both - 127 - settings were determined at the same time. These values provided the coefficients of two simultaneous linear equations which were subsequently solved to obtain the desired information.

Method of feeding Unless otherwise stated, all feedings were by the wick method. The precursor (less than 20 mg) was dissolved in three cubic centimeters of water with the aid of a trace of acid. Phosphoric and acetic acids were the most useful for this purpose. An unmercerized cotton thread was run through the stem of the plant and then drawn back and forth to abra: de the tissues. The ends of the thread were then placed in the precursor solution contained in a small poly-topped vial. After all the solution had passed into the plant, water was added to the vial and allowed to pass via the wick into the plant. This resulted in a nearly quantitative transfer of the precursor.

Isolation of berberine, and hydrastine Eydrastis canadensis plants were disrupted in 50% aqueous methanol containing 5% acetic acid and a trace of hydrochloric acid. The resulting pulp was stirred - 128 -

at steam-bath temperature for several hours. Charcoal and celite were then added, and the pulp was filtered throtigh celite. The filter cake was repeatedly leached with hot aqueous methanol. The combined filtrates were concentrated to a small volume on a rotary evaporator. Ammonia was added, and the suspension was exhausted with chloroform. The aqueous layer was acidified, then neutralized with sodium bicarbonate, and exhausted with chloroform and ether. The organic extracts were combined and taken to dryness (at this point, if only a small amount of berberine was present, inactive berberine was added). The crude berberine was dissolved in aqueous acetic acid and treated with sulphuric acid. The berberine sulphate crystallized at this point and was removed. Inactive berberine sulphate was added to the mother-liquors and was removed after crystallization. The combined portions of berberine sulphate were recrystallized, dissolved in hot water) and treated with barium hydroxide. The barium sulphate was removed by centrifugation. The aqueous solution of berberine was treated with hydrochloric acid and concentrated, and the berberine chloride was crystallized. The barium sulphate was washed with ethanol. This was then added - 129 -

to the combined berberine mother-liquors. These combined fractions were made basic, diluted with water, and exhausted with chloroform. The chloroform was dried and evaporated. This residue contained hydrastine and canadine which could be separated by chromatography on alumina.

Isolation of protopine Dicentra spectabilis plants were macerated as above. The aqueous acidic filtrate was concentrated and washed with chloroform. The aqueous portion was then made basic and exhausted with chloroform. The chloroform was dried and evaporated. The residue crystallized on treatment with methanol. Further purification was achieved by dissolving the protopine in hot 5% acetic acid (10 cc/g) and treating the resulting solution with potassium nitrate (1 g/g). The crystalline nitrate was isolated and shaken with aqueous ammonia and chloroform. The chloroform was dried and evaporated to a small volume. Addition of hot methanol gave protopine of high purity.

Berberine from k-/4- )-LN-methyl-r 14 Cireticuline1 6 The (±)-reticuline (8.9 x 10 c/100 sec.) was - 130 - administered to three Hydrastis canadensis plants. After seven days, the plants were worked up, and the berberine fraction was diluted with inactive berberine (38 mg). Specific Activity Calculated (0/100 sec./mg) (c/100 sec./mg) Crude berberine sulphate 2140 Recrystallized berberine sulphate 1800 Mother-liquors 1720 Diluted and recrystallized 374 344 Mother-liquors 368 344 Converted to (±)-canadine 594 600 Recrystallized 609 600 Mother-liquors 625 600

The berberine sulphate was converted into the chloride as above and treated with phenyl magnesium bromide, vide supra. The phenyl dihydro compound was oxidized under Kuhn Roth conditions.195 from the phenyl dihydro compound (26 mg) was obtained benzoic acid (2 mg); m.p. 120-121°. Berberine chloride = 1.21 x 104 counts/100 sec./m.nole 4 Benzoic acid = 1.27 x 10 It Activity in the "bridge" carbon = 106% - 131 -

Berberine from (±)-[6r0-methyl-N-methy1-14C]reticuline

Doubly-labelled reticuline (2.28 x 107 0/100 sec.) was administered to three Hydrastis canadensis plants. After one week, the plants were worked up as usual. The berberine fraction was diluted with inactive berberine (42 mg), and the sulphate was prepared. The sulphate was crystallized to constant specific activity (2.1 x 106 c/100 sec./mmole, 0.9% incorporation). An aliquot of the berberine was diluted, converted into the chloride, and heated with 20% sulphuric acid.112 The formaldehyde was distilled into saturated aqueous dimedone. Berberine chloride (5 mg) gave formaldehyde dimedone (1.4 mg).

Berberine chloride 9.00 x 104 0/100 sec./mole Formaldehyde dimedone 5.07 x 103 tt Methylene di oxy 5.63% of total label.

An aliquot of the diluted dibeuzyl ether of the precursor was cleaved with Hi.195 The methyl iodide was trapped in triethylamine.

6 Dibenzyl ether 5.74 x 10 c/100 sec./mnole

Me-Et3 -N+-I- 1.7 x 104 It t+ 6-Methoxyl 5.93% of the total label. - 132 -

A second aliquot of berberine sulphate was diluted, converted into the chloride, and degraded via the phenyl dihydroberberino, vide summ.

Berberine chloride 1.13 x 104 0/100 sec./mmole Phenyldihydroberberine 1.13 x 104

Benzoic acid 1.03 x 104 It tt tiBridgen carbon 91% of total label. N-Methyl of precursor 94% of total label.

Hydrastine and canadine from (t)-reticuline The hydrastine fractions from the preceding feeding were chromatographod without dilution. It proved possible to isolate hydrastine (16 mg), characterized by m.p. 133-4°, mixed m.p.133-4°, and thin layer chromatography. After several recrystallizations, this material had only 16 c/100 sec./Mg (ca. 0.001% inc.) and had not yet reached constant activity. The canadine fractions (0.3 mg - estimated by U.V.) were diluted with (t)-canadine (20 mg). After rechromatography and several crystallizations, this material had a constant specific activity of 400 0/100 sec./mg, .035% incorporation. -133 -

Berberine from (+)-[3H]reticuline and 1:7)431]reticuline Two matched Hydrastis canadensis plants were chosen. To one was fed (+)-[31-I]reticuline (3 mg) (1.13 x 106 dps/mg). To the other was administered (-)-[31-i]reticuline (4.7 mg) (9.08 x 105 dps/mg). Both plants were sacrificed after eight days and worked up separately. These plants contained considerable berberine which was isolated as the sulphate without dilution. The mother- liquors were, however, diluted and a second crop was obtained.

Feeding (+)-reticuline (-)-reticuline

I3erberine isolated 34 ng 43 mg

Diluted with 50 mg 50 rag

The berberine sulphate was purified, converted into the chloride, and recrystallized several times. The berberine chloride was reduced to (±).canadine for counting purposes.

Feeding (+)-reticuline -) -reticuline (c/100 sec./Mg) 0/100 sec./mg) Canadine 5.2 x 104 4.1 x 103 Recrystallized, chromatographed and recrystallized 5.4 x 104 4.2 x 103

% Incorporation 7.89% 0.53% - 134 -

Feedina2111:1=141423xyliculine with 1=1=alreticuline A mixture of (±)-5414C]reticuline (ca. 17 mg, 1.88 x 105 dps) and (-)-[5H]reticuline (1.17 mg, 10.6 x 105 dps) was administered to a single Hydrastis canadensis plant. After seven days, the plant was worked up as usual. The mother-liquors from which berberine sulphate (23 mg) had crystallized were diluted with inactive berberine (90 mg) and a second crop was obtained. The berberine was purified as above, converted into canadine, and assayed for 14C aad 3H.

14C 14v3H (dps/mg) Ratio Can aline 25 Crystallized, chromatographed and recrystallized 24 1.45

iimmil..1•1•••••••••11. Incorporation 14C 3B. 1.5% .196%

Feeding of nor-reticulines (10h) and (100 A mixture of (±)-1-(31 -hydroxy-41 -methoxybenzy1)- 2-methyl-Lr 14 CJ-6,7-dihydroxy-112,3,4-tetrahydroisoquinoline (ca. 11 mg, 1.56 x 107 dpm) and (t)421 ,51 ,6',8-3H]-1- (31 ,41 -dihydroxybenzy1)-2-mothyl-6-methoxy-7-hydroxy- 1,2,3,4-tetrahydroisoquinoline (11 mg, 108 dpm) was fed

-135 -

to several Flydrastis canadensis plants. After one week, the plants were worked up as usual. The mother- liquors from which the berberine sulphate (67 mg) was crystallized were diluted with inactive berberine (50 ng) and a second crop was obtained. The berberine was purified as usual, converted into canadine, and assayed for 14C and 3H.

14c 14C/3H (dps/ng) Ratio Canadine 52 Recrystallized, chromatographed and recrystallized 54 0.5

Incorporation 14C 3E 2.5% 0.79%

The hydrastine fractions from this feeding were chromatographed to yield hydrastine (25 mg). This was combined with inactive alkaloid (50 mg). After two recrystallizations, it had lost all of its activity.

Berberine from (t)43Hlnandinine The (±)-nandinine (10 mg, 2.36 x 105 cps) was administered to a Hldrastis canadensis plant. After

-136 -

nine days, the plrAnt was worked up es usual. The mother- liquors from which berberine sulphate (64 mg) had been crystallized were diluted with inactive berberine (50 mg) and a second crop was obtained. The combined berberine fractions were purified as usual and converted into (t)- canadine for counting purposes.

Specific activity (c/100 sec./mg) Canadine 3.10 x lo3 Recrystallized 1.75 x 103 Chromatographed .18 x 103 Recrystallized .174 x 103 Yother-liquors .178 x lo3

11110=11=1=•••••••••

Incorporation .o64%

Berberine from (±)-PHicanadine The (t)-canadine (10 mg, 2.36 x 105 cps) was administered to a Hydrastis canadentliz; plant. After two days, the plant wilted badly (because of the acetic acid used to solubilize the canadine). After one week, the plant was harvested and worked up as usual. - 137 -

The berberine fraction was diluted with inactive berberine (100 mg) and the sulphate was isolated. The berberine was purified as usual to give berberine chloride (53 mg)•. The berberine chloride was diluted with inactive berberine chloride (40 mg) and converted into berberine acetone. This compound was crystallized twice to remove traces of canadine. The berberine acetone was then converted into berberine chloride which was recrystallized and converted into (±)-canadine for counting purposes.

Specific activity (000 sec./mg)

Canadine 1.4 x 104 Crystallized, chromatographed and recrystallized 1.37 x 104

Feeding of (t)4313]protosinomenine The (±)-[3H]protosinomenine (22 mg, 7.6 x 104 cps/mg) was fed to three Hjdrastis canadensis plants. After seven days, the plants were harvested and worked up as usual. The mother-liquors from which the berberine sulphate (50 mg) had been crystallized were diluted with inactive berberine (50 mg) and a second crop was obtained. The berberine was purified as usual and converted into

- 138 -

(±)-canadine for counting purposes.

Specific activity 1000 sec./mg)

Canadine 61 Recrystallized, chromatographed and recrystallized 148

Incorporation .0028%

The hydrastine fractions from the above feeding were chromatographed. The hydrastine isolated (18 mg) was diluted with inactive hydrastine (50 mg) and crystallized. After several recrystallizations, the hydrastine was inactive.

Feeding of DOPA to Uydrastis canadensis The (±)-2414C]dihydroxypheny1alanine (0.01 no) was administered to a young Hydrastis canadenit plant. After fourteen days, the plant was harvested and worked up as usual. The berberine fraction was diluted with inactive berberine (62 mg). The berberine sulphate was isolated and purified as usual to give radioactive berberine sulphate (1.77 x 103 elm/13g, 0.9% incorporation). - 139 -

The hydrastine fractions were diluted with inactive hydrastine (58 mg) and worked up as usual .

Specific activity (cpm/mg) Eydrastine 912 Mother-liquor 10,800 Recrystallized 804 Recrystallized 984 Mother-liquor 960

•••••11/111111•••••••=11.11111.1.•

Incorporation 0.62%

Protopine from (±N=LN-meIhy1=1Lix2.112uline The (±)-[N-methyl-14C]reticuline (7.94 x 106 c/100 sec.) was administered to a flowering Dicentra spectabilis plant. After one week, the plant was harvested and worked up as usual to give radioactive protopine (78 mg). Specific activity 1c/100 sec./mmole) First crystallization 3.60.103 Second crystallization 3.40 x 105 Purified as nitrate and crystallized 3.63 x 105 Mother-liquors 3.71 x 105 - 140 -

An aliquot of the radioactive protopine was diluted and demethylated with HI.

Pro to pine 1.8 x 105 c/100 sec./Mmole + - Me-Et -1J -I 4.6 x 103 It 3

Therefore, less than 2.3% of the activity resided in the N-methyl of protopine. An aliquot of the remaining radioactive protopine was diluted and converted into the Emde base. The Emde base (30 mg) was dissolved in 20% sulphuric acid (10 cc) and heated under reflux for fifteen minutes. Chromium trioxide (1.3 g) was added and the mixture was heated under reflux for ninety minutes. The resulting acetic acid was steam-distilled and neutralized. The water was evaporated and the sodium acetate was degraded, vide supra. Specific activity (c/100 sec./mnole)

Emde base 6.65 x 104 N-methy1-3-benzyloxy- 4-methoxybenzylamine 6.38 x 104

Activity in the Abridgers carbon = 96% - 141 -

Protopine from doubly-labelled precursor A mixture of (±)-3414C]reticuline (6.73 x 106 0/100 sec.) and (±)-[N-methyl-14C]reticuline (2.02 x 107 0/100 sec.) was administered to a flowering Dicentra spectabilis. After one week, the plant was sacrificed. The usual work-up gave protopine (540 mg).

Specific activity (c/100 sec./mg) Protopine 1.68 x 103 Converted to nitrate and recrystallized 1.71 x 103 Mother-liquors 1.63 x 103

Incorporation 3.4%

An aliquot of radioactive protopine was converted into anhydromethylprotopine (7, p.58). This methine (48 mg) was ozono4ized9 vide supra. The stew- distillation of the formaldehyde was spoiled by frothing; so, the total reaction mixture was mixed with saturated dimedone solution. The zinc was quickly filtered off, and the filtrate was stored for three days. The filtrate was then exhausted with benzene. The benzene - 142 -

extracts were chromatographed on thin layer plates. The formaldehyde dimedone zone was removed, and the product was isolated. After final purification on an alumina column, the formaldehyde dimedone was recrystallized from aqueous ethanol, m.p. 190-10, mixed m.p. 189°-91°.

Protopine 2.4 x 105 c/100 sec./Mmole Formaldehyde dimedone 5.42 x 104

The "chain" carbon therefore bore 23% of the total label.

Protopine -nandi n jLe. The (±)-nandinine (9 mg, 2.12 x 105 c.p.s.) was fed to a Dicentra speetabilis plant. After one week, the plant was harvested. The usual work-up gave protopine (286 mg). Specific activity (c/100 sec./mg) Protopine 1.06 x 103 Converted into nitrate and crystallized 0.42 x 103 Chromatographed 0.26 x 103 Treated with charcoal 0.28 x 103

- 143 -

Chromatographed - traction 1 0.25 x 103

ft 2 0.26 x 103 rr 3 0.26 x 103

ft 4 0,28 x 103

Incorporation = 0.3%

Protopine from (+)-13Elyeticuline The (+)43H]retiouline (2.1 mg, 1.13 x 106 dps/mg) was administered to a flowering Dicentra spectabilis plant. After one week, the plant was harvested and worked up to give protopine (163 mg).

Secific activity (c/100 sec./mg)

Protopine 1.44 x 10' Converted into nitrate and recrystallized 1.35 x 104

Incorporation 5.9%

Protopine from (-)-13Hlreticuline A parallel feeding with (-)-[3H]reticuline (2.2 ng, 9.08 x 105 dps/mg) gave protopine (126 mg).

- 144 -

Specific activity (c/100 sec./mg) Protopine 1.08 x 103 Converted into nitrate and recrystallized 1.3 x 103

Incorporation 0.45%

Experiments with Argemone plants A mixture of M.-IN- 14c]-1ujreticuline (ca. 12 mg, 5.12 x 105 dps) and (+)-[3H]reticuline (2.58 mg, 2.82 x 106 dps) was administered to a flowering Argemone hispida and to a flowering Argemone mexicana plant. After one week, the plants were macerated as usual. The aqueous acid extracts were made basic and were exhausted with chloroform and ether. The organic extracts were combined, dried, and evaporated. Thin layer chromatography (silica gel G, methanol) indicated the presence of a considerable amount of protopine, but only traces of argemonine. Inactive argemonine (20 mg) was added, and the alkaloids were separated preparatively on a silica gel M3“ plate. Argemonine and protopine were eluted and each was diluted with inactive alkaloid (80 mg each). - 143 -

Specific activity c/100 sec ./mg)

Argemonine 6.85 x 104 Chromatographed 0

Pro topine 2.24 x 103 Crystallized and converted into nitrate 8.45 x 102 Recrystallized 8.34 x 102 Chronat ographed 8.41 x 102

14C 14C/3E Ratio Protopine 3.2 dps/mg 1/10.9

Experiments with Chelidonium majus A mixture of (±)-[N-methyl-14C]reticuline (7 x 104 cps) and (±)-[1-31-1]reticuline (ratio 14C/3:1 = was administered by injection into the stalk of two Chelidonium majus plants. After one week, the plants were harvested and macerated as usual. The aqueous acid fraction was made alkaline and exhausted with chloroform and ether. These extracts were evaporated to dryness. The residue was leached with 10% aqueous acetic acid. This extract was made basic, and the - 146 - alkaloids were extracted into chloroform. The chloroform extract was evaporated, and the residue was chrmnatographed. Inactive chelidonine (5/ ng) was added to the pure chelidonine fractions, and the alkaloid was crystallized. Specific activity (000 sec./mg) Chelidonine 100 Mother-liquors 93 Recrystallized 95 Mother-liquors 94

.0•1•101•0•11•...•••••••

Incorporation .069%

Liquid scintillation counting indicated the absence of tritium. - 147 -

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