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Studies in natural products Part I. The biosynthesis of erythrina alkaloids Part II. An attempted in… Gervay, Joseph Edmund 1965

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The U n i v e r s i t y of B r i t i s h Columbia DEPARTMENT OF CHEMISTRY PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of JOSEPH E. GERVAY M.Sc, The Univ e r s i t y of B r i t i s h Columbia THURSDAY, SEPTEMBER 23, 1965 AT 10:30 A.M. IN ROOM 261, CHEMISTRY BUILDING 1963 COMMITTEE IN CHARGE Chairman: W. Hoar T. Money F. McCapra C. A. McDowell R. E'. Pincock A. Rosenthal G. M. Tener External Examiner: D. A. Nelson Department of Chemistry Un i v e r s i t y of Wyoming Laramie, Wyoming. STUDIES IN NATURAL PRODUCTS ABSTRACT Part I. THE BIOSYNTHESIS OF ERYTHRINA,ALKALOIDS Hypotheses for the biogenesis of Erythrina alk a l o i d s are discussed, Di - ( p-3, 4-dihydroxyphenyl)-ethylamine, the t h e o r e t i c a l precursor predicted by the biogenetic theory, was prepared and ring closure to the erythrinane ring system by oxidative coupling was attempted under various conditions, Consequently, the biogenesis of the Erythtrina a l k a l o i d s was re-examined and a new proposal i s advanced for the biosynthesis of these a l k a l o i d s , Synthetic routes to a hypothetical precursor;, proposed here for the f i r s t time as a potential, intermediate s, are described, The biogenetic-type synthesis of the spiro-amine rin g system present i n the Erythrina a l k a l o i d s was achieved by oxidative coupling of the blocked diphenolic precursor^ as predicted by the proposed biosyhthetic scheme, Oxidation of di-(/3-3-hydroxy-4-me.thoxyphenyl) -ethylamine by a l k a l i n e potassium ferr i c y a n i d e afforded 3 s 15-dimethoxy-16-hydroxy-2-oxoerythrina-1 (6), 3-diene i i i 1.5% y i e l d , Reduction of the l a t t e r by sodium borohydride gave 3, 15-dimethoxy-2,, 16^dihydroxyerythrina-l ( 6 ) 5 3-diene, Acetylation of the dienone yielded 3, 15-dimethoxy-1 6-acetoxy-2-oxoerythrina-l(6) j 3-diene. The t o t a l biogenetic-type synthesis of erysodine i s therefore but two steps from completion. The r e s u l t s as a whole confirm the hypothesis that Erythrina a l k a l o i d s are produced i n Nature by oxidative coupling of diphenols. They also demonstrate the d i r e c t i n g role of the protective groups in the phenolic precursor. The evidence allows a biosynthetic pathway for the aromatic Erythrina a l k a l o i d s to be considered s and the mechanism for the ring closure process i s discussed. The i s o t o p i c a l l y l a b e l l e d precursor 3-hydroxy-4-methoxy-N-(3-hydroxy-4-methoxyphen (1-^C) ethyl) -phenethylamine was prepared to test the biosynthetic hypothesis i n the plant. Feeding experiments are i n progress, . Part I I , AN ATTEMPTED IN VITRO DEMETHYLATION OF LANOSTEROL The biogenesis of ch o l e s t e r o l and methods for functional.ising inert methyl groups are reviewed 3 and a new t h e o r e t i c a l approach to removal of the 14 -methyl group from lano s t e r o l i s described. The removal of th i s methyl group i n v i t r o could not be achieved. but a series of i n t e r e s t i n g compounds was obtained, .• Evidence for the structures of these, compounds is presented, Thus ? photosensitized Oxygenation of dihydrolano-s t e r y l acetate i n the presence of para-nitrobenzene.su 1-phonyl chloride yielded 3|?-acetoxylanosta-7 ?9(11)-diene. 3.3-acetoxylanost-8-ene-7-one and 3f?-a,cetoxylanost-8-ene-7<*- hydroperoxide. In addition a compound having an ambiguous structure and designated as IP1 was obtained. The dibromo-derivative of the l a t t e r i s 3/3-acetoxy-7o<3 llo(-dibromolanostane-8o( ?9o(-epoxide,the structure of. which was determined by X-ray c r y s t a l l o g r a p h i c study, A working structure for compound IP1 based on the physical and chemical evidence i s discussed. GRADUATE STUDIES Topics i n Organic Chemistry Topics i n Inorganic Chemistry Topics i n Physical Chemistry Molecular Rearrangements Physical Organic Chemistry Seminar i n Organic Chemistry Organic Stereochemistry Recent Synthetic Methods Chemical Ki n e t i c s Natural Products Related Studies: Biochemistry W. J. Polglase S. H. Zbarsky G. M. Tener M. Darrach Computer Programming and R. Henderson Numerical Analysis PUBLICATION A. Rosenthal and J. Gervay: "Direct High Pressure Carbonylation of Aromatic N i t r i l e s with Dicobalt Octacarbonyl" chemistry and Industry, 1623 (1963) .( A. Rosenthal and J. Gervay: "Direct Conversion of Aromatic N i t r i l e s into Phthalimidines and Ureas Using Dicobalt Octacarbonyl" Canadian Journal of Chemistry, 42 1490 (1964). D. E. McGreer R.,S:E. I. Pincock J. P. Kutney W.-.R.. Cullen N. B a r t l e t t J. A. R. Coope R.• F. Snider A. Bree R. E . I . Pincock R. Stewart J. P. Kutney L- D- Hayward L. D. H a l l . D. E. McGreer G. B. Porter D. •, J. L. Jame s J. P. Kutney A. I. Scott P a r t I . P a r t I I . STUDIES- IN NATURAL PRODUCTS THE BIOSYNTHESIS OF ERYTHRINA A L K A L O I D S . AN ATTEMPTED IN VITRO DEMETHYLATION OF LANOSTEROL by JOSEPH EDMUND GERVAY B . S c . H o n o u r s , U n i v e r s i t y o f M o n t r e a l , L o y o l a C o l l e g e , 1961 M . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1963 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e D e p a r t m e n t o f C h e m i s t r y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d . THE UNIVERSITY OF BRITISH COLUMBIA S e p t e m b e r 1965 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f th e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r -m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s ^ I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i -c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Chemistry  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date September 24, 1965 ABSTRACT In Part I, hypotheses f o r the biogenesis of Erythrina a l k a l o i d s are discussed. Di-(B-3,4-dihydroxyphenyl)-ethylamine the t h e o r e t i c a l pre-cursor predicted by the biogenetic theory, was prepared and r i n g closure to the erythrinane r i n g system by oxidative coupling was attempted under various conditions. Consequently, the biogenesis of the Erythrina alk a l o i d s was re-examined and a new proposal i s advanced f o r the biosyn-thesis of these a l k a l o i d s . Synthetic routes to a hypothetical precursor, proposed here f o r the f i r s t time as a p o t e n t i a l intermediate, are described. The biogenetic-type synthesis of the spiro-amine r i n g system present i n the Erythrina a l k a l o i d s was achieved by oxidative coupling of the blocked diphenolic precursor, as predicted by the proposed biosynthetic scheme. Oxidation of di-(3-3-hydroxy-4-methoxyphenyl)-ethylamine by al k a l i n e potassium f e r r i c y a n i d e afforded 3,15-dimethoxy-16-hydroxy-2-oxo-erythrina-1(6),3-diene i n 15% y i e l d . Reduction of the l a t t e r by sodium borohydride gave 3,15-dimethoxy-2,16-dihydroxyerythrina-l(6),3-diene. A c e t y l a t i o n of the dienone y i e l d e d 3,15-dimethoxy-16-acetoxy-2-oxoerythrina-1(6),3-diene. . The t o t a l biogenetic-type synthesis of erysodine i s there-fore but two steps from completion. The r e s u l t s as a whole confirm the hypothesis that Erythrina alkaloids are produced i n Nature by oxidative coupling of diphenols. They also demon-st r a t e the d i r e c t i n g r o l e of the pr o t e c t i v e groups i n the phenolic pre-cursor. The evidence allows a b i o s y n t h e t i c pathway f o r the aromatic E r y t h r i n a a l k a l o i d s to be considered, and the mechanism f o r the r i n g closure process i s discussed. The i s o t o p i c a l l y l a b e l l e d precursor 3-hydroxy-4-methoxy-N-(3-hydroxy-4-methoxyphen[1- 1 4C]ethyl)-phenethylamine was prepared to t e s t the biosynthetic hypothesis i n the plant. Feeding experiments are i n progress. In Part I I , the biogenesis of cho l e s t e r o l and methods for f u n c t i o n a l -i s i n g i n e r t methyl groups are reviewed, and a new t h e o r e t i c a l approach to removal of the 14a-methyl group from lanosterol i s described. The removal of t h i s methyl group i n v i t r o could not be achieved, but a serie s of i n t e r e s t i n g compounds was obtained. Evidence f o r the structures of these compounds i s presented. Thus, photosensitized oxygenation of dihydrolanosteryl acetate i n the presence of para-nitrobenzenesulphonyl chloride y i e l d e d 33-acetoxylanosta-7,9(ll)-diene, 36-acetoxylanost-8-ene-7-one and 3$-acetoxylanost-8-ene-7a-hydroperoxide. In addition a compound having an ambiguous structure and designated as IP1 was obtained. The dibromo-derivative of the l a t t e r i s 33-acetoxy-7a,lla-dibromolanostane-8 a,9a-epoxide, the structure of which was determined by X-ray c r y s t a l l o g r a p h i c study. A working structure f o r compound IP1 based on the physical and chemical evidence i s discussed. if. TABLE OF CONTENTS PART I Page INTRODUCTION 1 DISCUSSION 21 EXPERIMENTAL 43 BIBLIOGRAPHY . . 63 L i s t of Figures 1. Simple dimerization of phenol r a d i c a l s 15 2. The Biosynthesis of Galanthamine 19 3. The Biosynthesis of Isothebaine 20 4. Hypothetical Biogenesis of the Erythrina a l k a l o i d s 25 5. Hypothetical Biogenesis of the Erythrina a l k a l o i d s v i a oxidative coupling of phenols 28 6. Reaction sequence, leading to di-(8-3,4-dihydroxyphenyl)-ethylamine 29 7. Reaction scheme f o r the preparation of 3-benzyloxy-4-methoxyphenethylamine and 3-benzyloxy-4-methoxyphenyl-ac e t i c a c i d 32 8. Reaction sequence, leading to di-(&-3-hydroxy-4-methoxyphenyl)-ethylamine 33 9. Biogenetic-type synthesis of the erythrinane s p i r o amine r i n g system 36 10. n.m.r. spectrum of 3,15-dimethoxy-16-hydroxy-2-oxoerythrina-1(6),3-diene 37 11. Proposed mechanistic scheme f or the formation of the spiro amine r i n g system v i a oxidative coupling 40 12. Reaction scheme f o r the preparation of 3-hydroxy-4-methoxy-N-(3-hydroxy -4-methoxyphen[l - !4c]ethyl)-phenethylamine 42 V. PART II page INTRODUCTION . . . 68 DISCUSSION 82 EXPERIMENTAL 93 BIBLIOGRAPHY . 98 L i s t of Figures 1. The Biosynthesis of Isopentenyl Pyrophosphate 71 2. Polymerization of Isopentenyl Pyrophosphate 72 3. Scheme f o r the formation of Cholesterol from Squalene 75 4. In v i t r o f u n c t i o n a l i s a t i o n of non-active methyl groups 78 5. Proposed scheme f o r the removal of the 14a-methyl group i n dihydrolanosterol 86 6. Photosensitized oxygenation of dihydrolanosteryl acetate, i n the presence of para-nitrobenzenesulphonyl chloride 88 7. Chart showing the reactions of compound IP1 with chromium t r i o x i d e , potassium iodide and pyridine perbromide 90 ACKNOWLEDGMENTS The w r i t e r wishes to express his thanks to Professor .A„ L.Scott and to Dr. Frank McCapra for t h e i r advice, patience and encouragement i n the d i r e c t i o n of t h i s research project. Thanks are also due to Dr. T. Money f o r h i s h e l p f u l c o l l a b o r a t i o n during the course of t h i s work. The wri t e r also expresses his indebtedness to Professor J . T r o t t e r and to Mr. J.K. Fawcett f o r the X-ray c r y s t a l l o g r a p h i c study. PART I THE BIOSYNTHESIS OF ERYTHRINA ALKALOIDS -1-INTRODUCTION The E r y t h r i n a Alkaloids The alkaloids found i n numerous species of the genus Erythrina have attracted general i n t e r e s t both because of t h e i r p h y s i o l o g i c a l a c t i v i t y , and because they contain a type of structure not previously encountered among the a l k a l o i d s . The occurrence of hypaphorine (1), an indole alkaloid,''" i n 2 the species of Er y t h r i n a has long been known and the presence of other uncharacterized amorphous alka l o i d s has been reported repeatedly.^>4 ,S ,6 H • C H 2 - C H - C O O ci) C H 3 X C H 3 An intensive search f o r alkaloids i n plants belonging to t h i s genus was star t e d some twenty f i v e years ago following the discovery of the curare-l i k e action of extracts of various species of Eryth r i n a , an action d e f i n i t e l y 6,7,8,9,10 not a t t r i b u t a b l e to hypaphorine. The f i r s t systematic examina-t i o n of some f i f t y - o n e species o f Ery t h r i n a showed that a l l contained 11 12 13 al k a l o i d s with paralyzing a c t i v i t y , the potency varying widely. ' ' P h y s i o l o g i c a l l y , the Ery t h r i n a a l k a l o i d s are c u r a r i z i n g agents of high potency, although t h e i r a c t i v i t y presents several unusual features. Unlike other known c u r a r i z i n g agents, both synthetic and na t u r a l l y - o c c u r r i n g , the Er y t h r i n a a l k a l o i d s are unique i n that they are t e r t i a r y bases with r e l a --2-t i v e l y high a c t i v i t y . Furthermore, . quaternization greatly diminishes the c u r a r i z i n g potency; t h i s also i s the only class of compounds i n which t h i s 7 14 i s true. ' They are e f f e c t i v e when administered o r a l l y and have been used c l i n i c a l l y with some success. The pioneering work on the i s o l a t i o n and characterization of a l k a l o i d s from various species of E r y t h r i n a was done by Folkers and h i s a s s o c i a t e s . ^ ' 12 13 ' The e l u c i d a t i o n of t h e i r c o n s t i t u t i o n has been achieved mainly by Prelog, ( who f i r s t suggested the nature of the r i n g system i n the bases, and by Boekelheide. ^ By 1960 the i s o l a t i o n and c h a r a c t e r i z a t i o n of the members of t h i s family of a l k a l o i d s was e s s e n t i a l l y complete, and excellent reviews of the h i s t o r y and chemistry of the E r y t h r i n a a l k a l o i d s have been presented i n 1 14 a u t h o r i t a t i v e manner. ' Besides hypaphorine, which occurs i n a number of E r y t h r i n a species, the bases found i n these plants f a l l i nto two groups. The bases of the f i r s t group or " f r e e " a l k a l o i d s , are named from the p r e f i x "erythr-", and these alk a l o i d s are i s o l a t e d d i r e c t l y from extracts of the plant without the necessity of previous h y d r o l y s i s . The second group or "combined" alkal o i d s occur i n the plant i n combination with some other moiety, 18 19 20 usually sulphoacetic a c i d ' or glucose and on hydrolysis y i e l d the " l i b e r a t e d " a l k a l o i d s . The stem "eryso-" i s used to i n d i c a t e the " l i b e r a t e d " a l k a l o i d s which do not apparently occur as such in the plant but are l i b e r a t e d i n the course of i s o l a t i o n by the h y d r o l y t i c action of d i l u t e mineral acids. ,The q u a n t i t i e s of l i b e r a t e d a l k a l o i d s obtained from the plants generally predominate and often greatly exceed those of the free a l k a l o i d a l f r a c t i o n s . - 3 -The known na t u r a l l y - o c c u r r i n g Erythrina alkaloids can be c l a s s i f i e d depending whether or not the a l k a l o i d s , as o r i g i n a l l y i s o l a t e d , contain an aromatic r i n g . There are seven members of the aromatic group, including the erysodine (2) and e r y t h r a l i n e (3) types; whereas the second group i n which an aromatic r i n g i s not present has only two members, a-erythroidine (4) and g-erythroidine (5). The two groups d i f f e r only i n that the e r y t h r o i -dines have a lactone r i n g where the benzjenoid r i n g occurs i n the aromatic E r y t h r i n a a l k a l o i d s . The p r i n c i p a l aromatic a l k a l o i d s d i f f e r only i n the nature of the oxygen function attached to the benzenoid r i n g or i n t h e i r degree of unsaturation. The numbering of the E r y t h r i n a a l k a l o i d s follows 2 that devised e a r l i e r as a common numbering f o r a l l ' the Ery t h r i n a a l k a l o i d s . (4) (5.) -4-One i n t e r e s t i n g feature of structures (2) - (5) i s the heteroannular diene system present i n rings A and B. A l l the Erythrina alkaloids r e a d i l y undergo c a t a l y t i c reduction to the corresponding d i - and tetrahydro d e r i -15 17 vates. ' Furthermore, when the o r i g i n a l alkaloids are treated with acid under mild conditions, loss of the a l i p h a t i c methoxyl group occurs as methanol and the diene system becomes lengthened to a conjugated t r i e n e 15,17,22,23 system,' (2). as shown f o r the desmethoxy der i v a t i v e (6) of erysodine MeO (2) (6) When the o r i g i n a l a l k a l o i d s or t h e i r desmethoxy derivatives are treated with acid under more severe conditions, i . e . b o i l i n g hydrobromic acid or polyphosphoric acid at 125°, a rearrangement with aromatization of r i n g A 23 15 17 24 occurs. ' ' ' This reaction, known as the "apo-rearrangement", leads to a dihydroindole d e r i v a t i v e as shown (8). By analogy with other known carbonium ion rearrangements, i t i s probable that an intermediate of the type shown by (7) i s involved. In the case of aromatic E r y t h r i n a a l k a l o i d s , the "apo-rearrangement" i s accompanied by cleavage of the aromatic ether linkages i n r i n g D so that the product i n each case i s apoerysopine (8). Also, since the "apo--5-rearrangement" r e s u l t s i n destruction of both asymmetric atoms (C-3 and C-5) apoerysopine and apo-B-erythroidine are o p t i c a l l y i n a c t i v e . The conversion of the dihydroindole de r i v a t i v e to a true indole structure has been accom-25 26 27 28 p l i s h e d i n both the aromatic ser i e s and with B-erythroidine. ' ' ' M e O (2) (7) (8) The Hofmann exhaustive methylation procedure has been used extensively 14 i n degrading the Erythrina a l k a l o i d s . With less highly hydrogenated derivatives the Hofmann reaction i s accompanied by aromatization of r i n g A and frequently elimination of functional groups may occur as w e l l . This 29 was f i r s t observed by Boekelheide and Agnello and l a t e r t h i s aromatiza-17 30 t i o n was studied extensively i n the case of dihydro-B-erythroidinol (9). ' The d i o l (9), formed by the l i t h i u m aluminium hydride reduction of dihydro-B-erythroidine, was subjected to the exhaustive methylation procedure. In t h i s case aromatization of r i n g A was accompanied by loss of methanol but the d i o l function remained i n t a c t as shown by (10). A s i m i l a r aromatization of r i n g A was demonstrated by Prelog with the 31 von Braun degradation of aromatic E r y t h r i n a a l k a l o i d s . From the degradative evidence i t can be seen that the Erythrina ' alk a l o i d s give two important ser i e s of products i n which r i n g A has become aromatized. As shown, the rupture of the carbon to nitrogen bond at -6-C-5-N-9 gives r i s e to the ortho d i s u b s t i t u t e d benzenoid system of structure (10), whereas, the migration of carbon atom 13 from C-5 to C-4 gives r i s e to the indoline-type structure (8). To explain the o r i g i n of these products, the s p i r o amine structure (2) (5) proposed for these al k a l o i d s appeared to be not only a reasonable one to co r r e l a t e and explain the degradative evidence, but a necessary requirement. (9) (10) A number of methods have been explored f o r the synthesis of molecules of t h i s type to obtain valuable evidence regarding the proposed structures, since the s p i r o amine system was f i r s t deduced as being common to a l l of 15 17 21 the E r y t h r i n a a l k a l o i d s . ' ' To obtain a c o r r e l a t i o n between synthetic and natural material, i t was necessary to accomplish the synthesis of a su i t a b l e d e r i v a t i v e of the Er y t h r i n a a l k a l o i d s i n which the spiro system remains i n t a c t . Most of the preliminary work on the synthesis of compounds s t r u c t u r a l l y r e l a t e d to the Ery t h r i n a a l k a l o i d s i s due to Wiesner and h i s 32 c o l l a b o r a t o r s . The f i r s t successful synthesis of a compound containing 33 the desired s p i r o amine system was that of Belleau. His elegant synthesis i s shown below f o r the aromatic d e r i v a t i v e (11) to which the t r i v i a l name erythrinane has been given. - 7 -(11) 34 Belleau repeated the synthesis using the corresponding dimethoxy de r i v a t i v e and obtained racemic 15,16-dimethoxyerythrinane (15). The i n f r a r e d spectrum of the p i c r a t e of t h i s racemic mixture proved to be superimpOsable on the spectrum of the p i c r a t e of natural 15,16-dimethoxy-erythrinane obtained from degradations.**' 35 Mondon has demonstrated that c y c l i z a t i o n to form the s p i r o amine system occurs even more r e a d i l y when the lactam carbonyl i s placed i n the f i v e membered r i n g instead of the s i x . When the ketal aci d (12) i s warmed with 3,4-dimethoxyphenethylamine (13) i n the presence of acid, the conden-s a t i o n - c y c l i z a t i o n reaction occurs i n excellent y i e l d to give the amide (14). This on reduction with l i t h i u m aluminium hydride, gives the same racemate of 15,16-dimethoxyerythrinane (15) obtained previously by Belleau. The r e s o l u t i o n of 15,16-dimethoxyerythrinane (15) was accomplished 36 37 independently by Belleau and Boekelheide, The levorotatory enantio-morph was shown to be i d e n t i c a l with the natural material. S i m i l a r l y , the i d e n t i t y o f synthetic and natural d e r i v a t i v e s established the presence of 38 the spi r o amine system i n g-erythroidine (5). a-Erythroidine d i f f e r s from -8-8-erythroidine i n having the a l i p h a t i c double bond l i n k i n g the 13-14 rather than the 12-13 p o s i t i o n s , but, since a-erythroidine has been converted to B-erythroidine the synthesis conclusively established the spiro amine 39 structure postulated f o r a-erythroidine and i t s d e r i v a t i v e s . Thus, the fact that the synthetic and natural isomers have the same mode of r i n g fusion i s beyond question. (12) (14) In the past few years important advances have been made i n elaborating the chemistry of the erythrina a l k a l o i d s . Other routes leading to the . . j j 38,46,47,48,75 , 40,41,42,43,44,49 spiro amine system have been devised. Mondon 45 has extended h i s previous synthetic studies and he also reported a new rearrangement of the erythrinane r i n g skeleton. The important question of stereochemistry of these alkaloids have been s e t t l e d quite recently. The aromatic E r y t h r i n a a l k a l o i d s with the exception of erythratine, have been i n t e r r e l a t e d and shown to have the same 14 configuration of the s p i r o carbon atom, C-5, From an X-ray c r y s t a l lo-^ 50 graphic study of e r y t h r a l i n e hydrobromide and from chemical studies of 47 erysodine and the proof of i t s structure through synthesis, the r e l a t i v e configurations at C-3 and C-5 are known f o r the aromatic e r y t h r i n a alkaloids -9-The methoxyl and s p i r o amine groups have a " c i s " r e l a t i o n s h i p as shown (16) C H O R C H C % ^ (16) (17) The conversion of a-erythroidine to 3-erythroidirie established that < 39 51 52 both have the same configuration at C-3 and C-5. Degradations ' and 53 X-ray study . allowed the complete assignment of r e l a t i v e and absolute configurations of the erythroidines as shown f o r (3R,5S,12S)-a-erythroidine (17). • 54 Boekelheide has shown, by the method of molecular r o t a t i o n d i f f e r -ences and by o p t i c a l rotatory dispersion measurements, that the aromatic er y t h r i n a a l k a l o i d s and the erythroidines have the same, configuration at the s p i r o carbon atom C-5. i n view of t h i s evidence, and of the determina-t i o n of absolute configuration of erythroidines, the absolute configuration of the aromatic s e r i e s can be given (16).^* The Biosynthesis of A l k a l o i d s . The structures of some 1700 n a t u r a l l y occurring a l k a l o i d s are known at present, and the processes by which a l k a l o i d s are synthesized i n plants •10-have long been the subject of study and speculation among organic chemists 55 56 57 58 and biochemists. ' ' ' A proper understanding of the pathways demands a knowledge of the substances which are involved as intermediates and also of the mechanisms by which the various transformations are c a r r i e d out. In the l a s t ten years, t h i s complex of problems, which previously was only the 59 object of speculations, has been attacked s u c c e s s f u l l y on a broad front„ ' 60,61,62,63 ^ e biogenetic studies with a l k a l o i d a l compounds are now at the very i n t e r e s t i n g stage of development where hypothesis and experiment can be combined, and at the moment the main i n t e r e s t l i e s i n the c l a r i f i c a -t i o n of the major formal r e l a t i o n s h i p s between alkaloids and t h e i r precursors. The d i f f i c u l t problems of the d e t a i l s of the reaction mechanisms now seem 64 to r e s t i n many cases on a secure t h e o r e t i c a l b a s i s . No simple synthesis has yet been devised for the a l k a l o i d s , and the multistep syntheses described i n the l a s t h a l f century seem unsatisfactory when compared to the paths used by nature. This problem has int r i g u e d many workers, and as early as 1917 S i r Robert Robinson^ had devised and executed the famous sythesis o f tropinone, patterned along l i n e s considered also to represent reasonable b i o s y n t h e t i c routes. He put forward many 5 8 important ideas on a l k a l o i d biosynthesis, and h i s ideas have been invalu-able i n guiding experimental work on l i v i n g plants. 59 The term "biogenetic-type" has been selected to describe an organic synthesis designed to follow, i n at l e a s t major aspects, biosynthetic pathways proved or presumed, to be used i n the natural construction of the end products. It implies that the r e l a t i o n s h i p of the laboratory synthesis to the biosynthesis i s not ne c e s s a r i l y very close and that the i n v i t r o route may be based on an i n vivo scheme which i s reasonable yet only -11-speculative, or f o r which only meager evidence may be a v a i l a b l e . The term i s meant to r e f e r to presumed intermediates and biosynthetic paths, and l i t t l e emphasis i s placed on reagents and conditions, In the laboratory d u p l i c a t i o n of the key b i o l o g i c a l step, any conditions or reagents may be used which are necessary for the completion of the reaction. The s t r i k i n g success of c e r t a i n "biogenetic-type" syntheses may depend upon u t i l i z a t i o n of reaction types which p a r a l l e l enzyme-promoted processes, and i n l i e u of the enzyme system, the organic chemist may need to resort to reagents and conditions not a v a i l a b l e to the l i v i n g system, i n order to follow the over-a l l b i o s y n t h e t i c route. The key intermediate may possess the exact struc-ture proposed, i n the b i o s y n t h e t i c scheme or i t may be a simple modification, to d i r e c t the intermediate along desired channels and preclude other re-action courses. Biogenetic-type synetheses often are neater, shorter, and more e f f i c i e n t than normal routes i n which no attention i s p a i d to natural processes. Sometimes, i t i s found that the only s a t i s f a c t o r y route to some natural product i s the biogenetic type. However, the success of a "bio-genetic-type" synthesis by i t s e l f does not constitute evidence f o r the operation of a p a r t i c u l a r chemical step i n nature, and the temptation i s great i n many cases to draw such a conclusion. Real progress i n the study of a l k a l o i d biosynthesis began when organic compounds l a b e l l e d with carbon-14 and with other isotopes became r e a d i l y a v a i l a b l e i n the early n i n e t e e n - f i f t i e s . Since then, many groups o f workers have tackled problems i n t h i s area and the information i s accumulating r a p i d l y , ^ > ^ 4 Isotopes have proved p a r t i c u l a r l y important i n c l a r i f y i n g the mutual r e l a t i o n s h i p s among the various alkaloids found i n any given plant at the same time. Many biosynthetic processes of fundamental importance can be studied by tr a c e r methods. Examples of such processes are condensation, r i n g closure, methylation and demethylation, several d i f f e r e n t oxidation reactions, dehydrogenation, and so on. By means of t h i s new experimental technique, i t was possible to e s t a b l i s h the biogenetic r e l a t i o n s h i p s between c e r t a i n "precursors" and the alkaloids within r e l a t i v e l y short p e r i o d s . ^ There have been a considerable number of postulated biogenetic routes, and looking back at these speculations one can see two main thought processes, which are often c l o s e l y a l l i e d . One approach has been the comparative anatomy method, in v o l v i n g inspection of the formulae of a l k a l o i d s to seek common s t r u c t u r a l units within a group of a l k a l o i d s , and suggesting possible r e l a t i o n -ship of these units to simpler natural products. Such deductions are p a r t i -c u l a r l y useful f o r c o r r e l a t i n g d i f f e r e n t groups of a l k a l o i d s and f o r p r e d i c t -ing the structures of new a l k a l o i d s . The simple units from which the alkaloids may a r i s e are the amino acids r e s u l t i n g from the decomposition of proteins. The recognition of the extremely close r e l a t i o n s h i p between a l k a l o i d s , simple plant bases, and basic amino acids led to important information concerning the synthesis of a l k a l o i d s . The s t r u c t u r a l examination based hypothesis can be supported by tools such as tracers or the i s o l a t i o n of enzymes which cata-lyse the successive steps of the biosynthesis, since both d i r e c t and i n d i r e c t approaches to biogenesis complement each other. The other approach has been to c o r r e l a t e a l k a l o i d a l structures on the basis of a u n i f y i n g reaction mechan-ism. The amino acid phenylalanine (18) can, by decarboxylation, give the amine (19) and, by oxidation, the aldehyde (20). In t h i s connection Robinson recognized that i f condensation of g-substituted ethylamines (19) with alde-hydes (20) could occur i n plants, then one could account f o r a wide v a r i e t y of a l k a l o i d a l structures, as t h i s reaction i s p a r t i c u l a r l y important f o r the -13-synthesis of N-heterocyclic systems. (19) + C H O (18) N-H (20) Other synthetic reactions which can be considered to be most important as keys to the synthesis of a l k a l o i d s are: the a l d o l condensation between aldehydes and B-keto acids and the s i m i l a r condensation of carbinolamines, -C(0H)-N- with the active methylene groups (-CH2-C=0) of ketones or 0-keto ... 66,61 acids. The b i o s y n t h e t i c s i g n i f i c a n c e of phenol oxidations has long been recognized, and the r o l e of oxidative condensations i n the biosynthesis of a l k a l o i d s have been discussed i n great d e t a i l i n excellent reviews.^4,67, ' Of the known alka l o i d s more than 10% can be derived, i n p r i n c i p l e , by coupling of appropriate phenolic precursors. The i n t e r p r e t a t i v e mechanistic approach to biosynthesis, of c o r r e l a t i n g the structures of natural products i n terms of oxidative coupling of phenolic precursors i n 64 biogenetic pathways, i s now c l a s s i f i e d as Biogenetic Analysis. It -14-i n c l u d e s i n i t s o p e r a t i o n t h o s e f e e d i n g e x p e r i m e n t s w i t h r a d i o c h e m i c a l l y l a b e l l e d s u b s t r a t e s w h i c h can be u s e d t o e v a l u a t e s u c h an a n a l y s i s . The o x i d a t i o n o f p h e n o l s o r o f p h e n o l • a n i o n s by o n e - e l e c t r o n t r a n s f e r o x i d i z i n g a g e n t s a f f o r d s mesomeric p h e n o l r a d i c a l s as shown (21). (21) T h e s e a r e s t a b l e r a d i c a l s , r e l a t i v e t o a l k y l r a d i c a l s , b e c a u s e o f t h e s p r e a d o f t h e odd e l e c t r o n by r e s o n a n c e o v e r t h e o r t h o and p a r a p o s i t i o n s o f t h e a r o m a t i c r i n g . The f r e e r a d i c a l s have b e e n d e t e c t e d b y m a g n e t i c s u s c e p t i -64 b i l i t y and e l e c t r o n s p i n r e s o n a n c e m e a s u r e m e n t s , and d e t a i l e d s t u d i e s h a v e shown t h a t t h e f r e e e l e c t r o n d e n s i t y i s g r e a t e r a t t h e para - , t h a n at t h e o r t h o - p o s i t i o n . The e x p e r i m e n t s a r e i n f u l l a c c o r d w i t h t h e v i e w t h a t t h e f i r s t s t e p i n t h e o x i d a t i o n o f a m o n o h y d r i c p h e n o l , by a one e l e c t r o n t r a n s f e r o x i d a n t i s t h e g e n e r a t i o n o f t h e p h e n o x y l r a d i c a l . Once p h e n o l r a d i c a l s h a v e b e e n g e n e r a t e d t h e y may be c o n v e r t e d t o s t a b l e m o l e c u l a r p r o d u c t s d e p e n d i n g on t h e s u b s t i t u t i o n p a t t e r n by s e v e r a l p r o c e s s e s . R e d u c t i o n g i v e s b a c k t h e p a r e n t p h e n o l , c o u p l i n g w i t h r e a c t i v e m o l e c u l e s , f o r example o x y g e n and h a l o g e n s a f f o r d n o n - r a d i c a l p r o d u c t s , s e l f c o u p l i n g f u r n i s h e s d i m e r s . Dimers c a n be f o r m e d by c a r b o n - c a r b o n , c a r b o n - o x y g e n o r o x y g e n - o x y g e n c o u p l i n g . S i m p l e d i m e r i z a t i o n o f t h e r a d i c a l s g i v e s r i s e t o d i p h e n y l s o r d i p h e n y l e t h e r s by o r t h o - o r p a r a - c o u p l i n g . (See F i g u r e 1). -15-Th e carbon-carbon coupling i s the most important and i t can be ortho-ortho, ortho-para or para-para. Figure 1. Simple dimerization of phenol r a d i c a l s . I f oxidative dimerization of the molecule i s assumed, one must d i s t i n g u i s h , i n p r i n c i p l e , between the r a d i c a l coupling process (homolytic coupling) and the s u b s t i t u t i o n of a phenol. r a d i c a l ( r a d i c a l i nsertion) i n t o a molecule of phenol .followed by furt h e r oxidation. Both mechanisms pre d i c t ortho-para type s u b s t i t u t i o n . Although r a d i c a l s u b s t i t u t i o n i n t o a phenol, : anion cannot be disregarded, the intervention of r a d i c a l i n s e r t i o n processes i n such phenol oxidations seems u n l i k e l y , and the r a d i c a l 68 coupling i s favoured . and accepted without further q u a l i f i c a t i o n . Evidence i s also lacking f o r the i n t r u s i o n of c a t i o n i c species (2-electron 64 oxidation) i n these oxidations. Phenols i n which one or preferably two reactive p o s i t i o n s are blocked by s u i t a b l e substituents, e.g. methoxyl, methyl or acetyl groups, give good y i e l d s of diphenyl d e r i v a t i v e s on oxidation. It i s c l e a r that 2,4,6-tri-substituted phenols not having a-CH i n the substituents produce stable -16-phenol r a d i c a l s , which are r e a d i l y detected not only by t h e i r chemical r e a c t i v i t y but also by t h e i r paramagnetism. This i s because phenol coupling i s prevented by s t e r i c reasons, and the absence of a-hydrogen i n the s u b s t i -6 7 tuents p r o h i b i t s the formation of a methylenequinone. Amino phenols and 64 amines also couple v i a mesomeric r a d i c a l s . Successful coupling reactions i l l u s t r a t i n g the formation of C-C, C-0 and C-N bonds have been c a r r i e d out i n the laboratory, however, the s e l e c t i o n of reagent and the experimental conditions f o r a given substrate are largely, empirical. A great v a r i e t y of ele c t r o n acceptors have been employed to ef f e c t the coupling of phenols e.g. f e r r i c y a n i d e s , f e r r i c c h l o r i d e , hydrogen peroxide, manganese dioxide, quinones, or enzyme preparations. The y i e l d s of the i s o l a b l e products of the reaction vary within wide l i m i t s and are greatly dependent upon the structure of the phenol, the s t a b i l i t y or solu-b i l i t y of the reaction products and the reaction conditions (pH). As a rule large amounts of amorphous by-products, generally i l l defined, are simul-taneously formed due to hydroxylations, r i n g f i s s i o n or polymerization. In the l i v i n g c e l l phenol oxidations occur i n a highly organized surrounding and i t i s probable that the reacting phenol molecules become s u i t a b l y oriented so as to make a d i r e c t e d coupling p o s s i b l e , thus minimizing the formation of by-products. This i s very d i f f i c u l t to imitate i n the labora-tory, and i t i s customary to work i n high d i l u t i o n to prevent polymeriza-t i o n . I f one assumes that r a d i c a l s such as (21) disappear by coupling i n pa i r s to fur n i s h molecular products, then c e r t a i n r e s t r i c t i o n s are imposed on the mode of coupling. I f such r a d i c a l s are also involved i n biogenetic pathways, then by blocking reactive p o s i t i o n s i n t e r e s t i n g r e s t r i c t i o n s are imposed on the precursors and products i n the biogenetic sequence. It was -17-6 7 f i r s t recognized by Barton and Cohen, that completely rigorous a p p l i -cation of the p r i n c i p l e of ortho- and para-C-C and C-0 coupling accounts f o r the s t r u c t u r a l features found i n many classes of a l k a l o i d s . They also postulated that the coupling step i s the r e s u l t of phenol oxidation., Nume-rous papers have appeared since concerning the use of oxidative procedures on phenolic and amino compounds and a va r i e t y of bios y n t h e t i c successes u u . • . 63,68,64 have been obtained. Amongst the many types of compounds which can, at le a s t formally, be derived by the coupling of phenoxide r a d i c a l s the a l k a l o i d galanthamine (25) present i n the Amaryllidaceae provides an outstanding example. Accord-ing to biogenetic theory the three main classes o f Amaryllidaceae a l k a l o i d s are a l l derived from a precursor (22) now known to be the a l k a l o i d norbella-dine, having two phenolic rings with the phenolic hydroxyl groups i n such 6 7 positio n s as to d i r e c t ortho- and/or para-coupling between the rings. The v a l i d i t y of t h i s scheme has been demonstrated by independent researches i n three laboratories.^3,68 Galanthamine was regarded as biosynthesised from norbelladine 0N-dimethyl ether (24) by oxidation to the dienone (26), r i n g closure to the enone (27), and reduction of the l a t t e r to the a l l y l i c alcohol galantha-mine (25) (see Figure 2). At the time when t h i s scheme was put forward the a l k a l o i d narwedine (27) had not been characterized, and the formula for galanthamine was uncertain. The correct formula (25) was, i n f a c t , chosen on the basis o f the proposed biogenesis. Biogenetic analysis has shown that not only was the correct formula (25) chosen f o r galanthamine but i t was proved that l a b e l l e d norbelladine ON-dimethyl ether (24) and 71 norbelladine (22) was incorporated i n t o galanthamine. In e a r l i e r -18-experiments, incorporation of [2-lkC] tyrosine (23) into galanthamine (25) was observed. The c o n s t i t u t i o n of galanthamine has been placedbeyond question by a t o t a l synthesis from norbelladine ON-dimethyl ether (24) based 70 on the biogenetic scheme. An important r e s u l t which bears on the phenol coupling theory f o r these alkaloids i s the evidence obtained for the pre-63 sence of phenolic norbelladine derivatives i n Amaryllidaceae plants* The aporphine a l k a l o i d isothebaine (29) has an unusual oxygenation pattern. It was suggested that bases of t h i s type are biosynthesized frqm the 1-benzyltetrahydroisoquinoline (28) by phenol oxidation to give the dienone (30) followed by reduction to the dienol (31) (see Figure 3). Migration of a bond by dienol benzene rearrangement leads d i r e c t l y to isothebaine (29). The f i r s t synthesis of isothebaine by t h i s sequence 72 has been achieved, and t r a c e r studies c a r r i e d out with o r i e n t a l poppies 73 showed that o r i e n t a l i n e (28) i s incorporated by plants into isothebaine. The influence of s e l e c t i v e p rotection of phenolic function i s n i c e l y i l l u s t r a t e d i n t h i s case, and i t seems probable that methylation controls the d i r e c t i o n of oxidative coupling i n the biosynthesis of a l k a l o i d s . 74 An extensive i n v e s t i g a t i o n of benzyltetrahydroisoquinolines has shown, that, provided they contain quaternary nitrogen, simple phenolic bases can undergo oxidative condensation under conditions s i m i l a r to those of biogenesis to form a l k a l o i d s of the isoquinoline s e r i e s with good y i e l d s . By means of such oxidative condensation, more than s i x t y alkaloids of various s t r u c t u r a l types have become more e a s i l y obtainable. Of the numerous pos s i b l e condensations of the intermediate mesomeric r a d i c a l s only those which lead to n a t u r a l l y occuring a l k a l o i d s give good y i e l d s and few by-products. These r e s u l t s suggest that oxidative condensations of -19-Figure 2. The Biosynthesis of Galanthamine-. (25). - 2 0 -Figure 3. The Biosynthesis of Isothebaine (29). quaternary bases may also be involved i n the biosynthesis of a l k a l o i d s i n the plant c e l l . There have been a number of speculations on the biogenesis of the 14 E r y t h r i n a a l k a l o i d s , however, no experimental evidence has been reported . so f a r with which to evaluate these proposals. The greater the importance of the complex natural products which can be i s o l a t e d and s t r u c t u r a l l y i d e n t i f i e d i n modern times, the more important does i t become to learn to synthesize them as simply and as r a p i d l y as i n the c e l l . It i s hoped that, by i m i t a t i n g such bi o s y n t h e t i c methods the increasing demand f o r p h y s i o l o g i c a l l y active b i o l o g i c a l products can be met more e f f i c i e n t l y than by the time-consuming extractions from the plant c e l l . -21-DISCUSSION The object of t h i s i n v e s t i g a t i o n was to obtain fundamental information concerning the biosynthesis of Erythrina a l k a l o i d s , and to achieve a model synthesis of the c h a r a c t e r i s t i c spiro amine structure along the l i n e s of a proposed biogenetic scheme. The problem arose as a d i r e c t consequence * , , 14,58,67,76,77,78 . c of much speculation concerning the formation of these alkaloids i n the plants, since the biogenesis of the Erythrina a l k a l o i d s had not been previously explored. The biogenesis of Er y t h r i n a a l k a l o i d s cannot r e a d i l y be r e l a t e d to any of the schemes proposed f o r other a l k a l o i d s . It appeared that a new approach i s required to explain the formation of the spiro amine system, and i t might be expected to ar i s e through a new type of v a r i a t i o n i n biogenesis. A study of the molecular structures of the Er y t h r i n a a l k a l o i d s , leads to c e r t a i n f i r m convictions as to the sort of precursors and processes involved i n t h e i r biosynthesis. The modes of possible biogenesis are set out as connected s e r i e s of reactions, however, a d e f i n i t e order f o r the various stages i s not assumed. 58 According to biogenetic theory the spiro amine system i s derived from a precursor (33), having two phenolic rings with phenolic hydroxyl groups at 3,4-positions, synthesised i n Nature from two C6-C2 units (Ar-C-C). The condensation presumably proceeds by a mechanism whereby one amino acid becomes rea c t i v e by decarboxylation to an amine and the other by oxidative deamination to an aldehyde or i t s equivalent. One s a t i s f a c -14 tory Scheme (see Figure 4) envisions the b u i l d i n g blocks to be two molecules of 3,4-dihydroxyphenylalanine (32), t h e i r union to give the precursor (33) and i t s subsequent oxidation to the orthoquinone (35). It -22-i s not known at what stage decarboxylation occurs. The r i n g closure to the di-hydroindole (34) and subsequently to the intermediate (37) i s a p l a u s i b l e one. S i m i l a r r i n g closures occur when 3,4-dihydroxyphenethylamines and 79 80 and 3,4-dihydroxyphenylalanine i t s e l f i s subjected to mild Oxidation. ' Methylation to give erysopine (38) may occur at some l a t e r stage. The out-standing feature of t h i s scheme i s that i t represents an overlap of the two great biogenetic pathways f o r forming indole and isoquinoline a l k a l o i d s , and i t explains the formation of the s p i r o amine system i n a very simple way. According to t h i s scheme erysopine (38) appears to be the key intermediate f o r the elaboration of the various aromatic alkaloids as well as for the erythroidines (4) (5). The non-aromatic alkaloids of t h i s family may be 17 derived by Woodward f i s s i o n of the dihydroxybenzenoid r i n g of erysopine (38), followed by appropriate modifications of the side-chains and ultimate lactortization (5). Since these further a l t e r a t i o n s do not a f f e c t the stereo-chemistry at C-3 and C-5 i t i s possible that erysopine (38) i s formed f i r s t i n the plant and then converted enzymatically to other a l k a l o i d s , i n c l u d i n g 6-erythroidine (5), the extent to which t h i s could occur would depend on the enzyme systems possessed by a p a r t i c u l a r plant species. It seems to be quite possible that the s t a r t i n g material f o r the biogenesis of the Ery-t h r i n a a l k a l o i d s i s 3,4-dihydroxyphenylalanine (32) , since an appropriate union of two molecules of t h i s amino acid can lead d i r e c t l y to a structure (38) representative of the ''aromatic" a l k a l o i d s . the r o l e that the coupling of phenoxide r a d i c a l s can play i n explain-64 ing the biosynthesis o f natural products i s by now well appreciated. The formation of a l l E r y t h r i n a a l k a l o i d s can be accommodated by t h i s e l e -67 gant and simple biogenetic hypothesis, that the carbon skeletons -23-are produced by oxidative phenolic coupling of a precursor of the type (33). The aromatic Erythrina a l k a l o i d s are p l a u s i b l y derived by oxidative coupling of t h i s intermediate (see Figure 5). Oxidation of the base (33), by some one-electron t r a n s f e r system to generate r a d i c a l s which, by coupling, would y i e l d the diphenoquinone (39). Addition of the amino group to the quinonoid system i n (39) leads to the dienone (37) and then subsequent unexceptional steps y i e l d the aromatic E r y t h r i n a a l k a l o i d s (40) (38). A v a r i a t i o n i n t h i s scheme i s the carbon-nitrogen coupling of the precursor (33) to give the hydroindole (34), f u r t h e r para-para carbon-carbon coupling w i l l furnish the same dienone (37) which was obtained from the other route. The non-aromatic members o f t h i s family can be formulated by oxidative f i s s i o n of the catechol r i n g as suggested above. Such processes as dehydration, hydro-genation and dehydrogenation may occur at any stage a f t e r the spir o amine system was formed. O-methylation can occur at some previous or subsequent stage, as i t i s not known yet whether the methylation pattern i s b u i l t i n from the ba s i c precursors or at the diphenol (33) l e v e l . However, since t h i s hypothetical precursor possesses the s p e c i f i c hydroxylation pattern from which a l l the known Er y t h r i n a a l k a l o i d s can be derived, i t i s strongly believed that methylation occurs at t h i s stage i n the biosynthesis. This hypothesis i s f i r m l y supported by the now well known " d i r e c t i n g " r o l e of 64 the p r o t e c t i v e groups i n both the synthesis and biosynthesis of a l k a l o i d s . ' 70 73 97 ' ' The di f f e r e n c e i n the methylation pattern of the phenolic rings demonstrates that biosynthesis i s di r e c t e d to d i f f e r e n t f i n a l skeletons, at l e a s t i n part, by O-methylation. 58 67 With the biogenetic theory ' as a background i n mind the programme of research was i n i t i a t e d , f i r s t to examine the i n v i t r o synthesis of the erythrinane r i n g system, followed by t r a c e r experiments to determine i n vivo -24-i n the plant how near to the truth the assumptions may be. Consequently, in order to prepare the base precursor (33) predicted by the biogenetic theory, i t was f i r s t necessary to develop a synthetic pathway to t h i s proposed key intermediate for the i n v i t r o synthesis (see Figure 6 ) . 3,4-Dimethoxyphenylacetic acid (42) was treated with t h i o n y l chloride and the resultant a c i d chloride was condensed with 3,4-dimethoxyphenethyl-amine (13) to give homoveratroyl-homoveratrylamine (43). The amide was reduced with l i t h i u m aluminium hydride i n ether to d i - ( 3 - 3 , 4-dimethoxy-phenyl)-ethylamine (45). This diamine was also prepared on a larger scale i n one step by the c a t a l y t i c reduction of 3,4-dimethoxyphenylacetonitrile 81 (44). Demethylation of t h i s product i n r e f l u x i n g concentrated hydrobro-mic acid then y i e l d e d di-(6-3,4-dihydroxyphenyl )-ethylamine hydrobromide (33) i d e n t i f i e d by chemical analysis. Its i n f r a r e d 3400 cm * (0-H) and u l t r a v i o l e t spectrum ^ m a x 284 my (e 7770)were i n agreement with the assigned structure. This hypothetical precursor (33) of the E r y t h r i n a alkaloids was then oxidized with a l k a l i n e potassium f e r r i c y a n i d e under various con-d i t i o n s . In 24 oxidation experiments i t was not possible to i s o l a t e any products or to obtain reproducible r e s u l t s . The major product i n a l l cases was an i n t r a c t a b l e chloroform-insoluble polymer. Even i n very d i l u t e s o l u t i o n extensive polymerization was observed. One crude product of minute amount had absorptions at 3450 (0-H) and 1720 cm 1 (C=0) i n the i n f r a r e d . The u l t r a v i o l e t spectrum showed X at 253 my and a shoulder r max at 284 my. S i m i l a r r e s u l t s were obtained when f e r r i c chloride was used as o x i d i z i n g agent. I t was c l e a r from these experiments that polymerization of the tetrahydroxy-precursor cannot be avoided under laboratory conditions and the proposed dienone (37), i f formed at a l l , could not be present i n - 2 5 -O H (34) O H (35) C H O 3 Figure 4. Hypothetical Biogenesis of the Erythrina alkaloids. -26-y i e l d s much greater than 1% -. Many combinations are possible i n the conden-sat i o n , since each phenolic hydroxyl group may produce two d i f f e r e n t r a d i c a l positions i n t h i s manner. In view of the above re s u l t s the biogenesis of the Erythrina al k a l o i d s was re-examined, and a new hypothesis was put forward. It was reasonable to suggest that blocking by methylation of the 4-hydroxyl group i n both 3,4-dihydroxybenzenoid rings of the diphenol (33) should reduce the number of r eactive s i t e s (both ortho and/or para) l i a b l e to oxidative coupling, and d i r e c t the reaction along the proposed route to the dienone (37) which i s , of course, the desired i n i t i a l product of phenol r a d i c a l coupling. It seemed quite probable that oxidation of the modified diphenol (58), proposed here f o r the f i r s t time as a p o t e n t i a l precursor of the spi r o amine r i n g system, would prove more succe s s f u l . In t h i s hypothetical precursor (58), where the subsequent coupling reaction i s d i r e c t e d only by the free phenolic groups, the phenoxide r a d i c a l s have a b e t t e r opport-unity to couple intramolecularly than before, f o r the very same reasons which were discussed e a r l i e r i n other s i m i l a r ortho-methoxyphenol systems. To test t h i s new proposal i n v i t r o , the chemical synthesis started from i s o v a n i l l i n (46), and the accomplished plan f o r t h i s new synthetic approach is o u t l i n e d i n Figures 7,8, and 9. I s o v a n i l l i n (46) was benzylated with benzyl c h l o r i d e , and i t s conden-sa t i o n with h i p p u r i c acid gave the oxazolone (49), the a l k a l i n e hydrolysis of which y i e l d e d a mixture of 3-benzyloxy-4-methoxyphenylpyruvic (52)and benzoic acid. These acids were separated a f t e r treatment of the mixture with a l k a l i n e hydrogen peroxide, r e s u l t i n g i n 3-benzyloxy-4-methoxyphenyl-85 a c e t i c acid'(55) separated by column chromatography. This acid was pre-pared i n much b e t t e r y i e l d s by the a l k a l i n e hydrolysis of 3-benzyloxy-4-- " 2 7 -methoxyphenylacetonitrile (54). A considerable quantity of 3-benzyloxy-4-methoxy-B-nitrostyrene (50) was desired f o r the preparation of 3-benzyloxy-4-methoxyphenethylamine (53). 82 Unsuccessful attempts were made to repeat an e a r l i e r procedure for the condensation of O - b e n z y l i s o v a n i l l i n (47) with nitromethane using methylamine as the condensing agent. The experiment was repeated s i x times but the pro-duct described was never obtained. Instead an unknown brown amorphous substance, melting above 200° was obtained and t h i s compound might be the polymer of the desired product. The nitrostyrene (50) was f i n a l l y prepared 38 by the method of Lange and Hambourger using aqueous sodium hydroxide instead of methylamine and carrying out the condensation reaction at 10°. 84 The ni t r o s t y r e n e was then reduced with l i t h i u m aluminium hydride m t e t r a -hydrofuran to y i e l d 3-benzyloxy-4-methoxyphenethylamine (53). This amine was also prepared by the reduction of 3-benzyloxy-4-methoxyphenylacetonitrile (54) with l i t h i u m aluminium hydride i n ether. The p h e n y l a c e t o n i t r i l e (54), which turned out to be an important i n t e r -mediate in the laboratory synthesis of the desired phenolic diamine precursor (58), was prepared as follows. O - b e n z y l i s o v a n i l l i n (47) from i s o v a n i l l i n (46) was reduced with sodium borohydride to the corresponding alcohol (48) which, with t h i o n y l c h l o r i d e y i e l d e d 3-benzyloxy-4-methoxybenzyl chloride (51). This underwent exchange with potassium cyanide i n dimethyl s u l -86 phoxide to y i e l d 3-benzyloxy-4-methoxyphenylacetonitrile (54). The acid c h l o r i d e of 3-benzyloxy-4-methoxyphenylacetic acid (55), f r e s h l y prepared, was treated at once with 3-benzyloxy-4-methoxyphenethyl-amine (53), y i e l d i n g 3-benzyloxy-N--(3-benzyloxy-4-methoxyphenethyl)-4-. methoxyphenylacetamide (56). The amide was best reduced by borane i n t e t r a ^ hydrofuran to give di-(B-3-benzyloxy-4-methoxyphenyl)-ethylamine (57) i n good y i e l d s , i d e n t i f i e d as the hydrochloride. This new compound analysed -28-Figure 5. Hypothetical biogenesis of the Erythrina alkaloids via oxidative coupling of phenols. -29-Figure 6. Reaction sequence, leading to di-(2 -3,4r4i-nydroxyphenyl)-ethylamine (33). ' -30-c o r r e c t l y f o r C 3 2H3 60L,NC1 . Its n.m.r. spectrum in deuterochloroform showed the expected aromatic resonance as a multiplet at x 2.65 and a s i n g l e t at T 3.24, a s i n g l e t f o r the methylene (-0-CH 2-) at x 4.94 and f o r the methyls (-0-CH 3) at x 6.2, and a broad s i n g l e t centered at x 6.88 f o r the protons of the phenethylamine side chain. The benzyl-groups of the amine (57) were removed by hydrogenolysis i n methanol containing concentrated hydrochloric acid over 10% p a l l a d i s e d charcoal; to give the desired di-(B-3-hydroxy-4-methoxyphenyl)-ethylamine (58). The fa c t that the righ t precursor was at hand was shown by i t s sub-sequent synthesis i n one step from 3-benzyloxy-4-methoxyphenylacetonitrile (54) by c a t a l y t i c reduction. Microelemental analysis was i n agreement with the proposed str u c t u r e , and i t s i n f r a r e d spectrum i n Nujol showed a strong absorption at 3550 cm * (0-H) c h a r a c t e r i s t i c of phenols substituted by ether groups i n the ortho p o s i t i o n , n.m.r. spectrum i n deuterochloroform showed the aromatic proton resonance as a multiplet centered at x 3.36, a s i n g l e t at x 4.47 f o r the phenolic protons which disappeared on addition of D2O (deuterium exchange), a s i n g l e t at x 6.2 for the methyls (-O-CH3), and a broad t r i p l e t centered at x 7.25 f o r the protons of the phenethylamine side chain. The previous oxidative condensations had given unsatisfactory r e s u l t s c h i e f l y because of side reactions. Thus, to achieve a biogenetic-type synthesis by oxidative condensation, i t appeared necessary to suppress unwanted phenol oxidation. This new approach has been very rewarding and with the modified diphenolic precursor (58), where the arrangement of the hydroxy groups i s such as to promote and d i r e c t the condensation i n the phenolic moiety, a simple synthesis of the spi r o amine system was achieved i n very reasonable y i e l d (see Figure 9). The exploratory experiments i n v e s t i g a t i n g the new p o t e n t i a l i t i e s of such phenol-coupling showed (U.V., I.R.) that these oxidations a c t u a l l y -31-proceed much bett e r than had been expected. F i r s t , at the most two equiva-lents of o x i d i z i n g agent were used and potassium f e r r i c y a n i d e appeared to be a convenient reagent. The oxidation proceeded best when a very d i l u t e s o l u t i o n of the diphenol was added slowly to a f o u r f o l d excess (by equivalent) of the f e r r i c y a n i d e containing potassium hydrogen carbonate (pH 8). By working up the product as d e t a i l e d i n the experimental section, c r y s t a l l i n e 3,15-dimethoxy-16-hydroxy-2-oxoerythrina-l(6),3-diene (59) was i s o l a t e d i n 9% y i e l d m.p. 224-227°. In more elaborate preparative experiments with f e r r i c y a n i d e as oxidant the y i e l d of dienone (59) a c t u a l l y i s o l a t e d was increased to 15%. There i s l i t t l e doubt that further improvement i n y i e l d could be achieved by increasing the d i l u t i o n of the reactants s t i l l more. Elemental analysis and s p e c t r a l data confirmed beyond doubt that the long sought dienone (59) had been obtained. T.L.C. of the dienone (59) gave one spot on s i l i c a g e l . The u l t r a v i o l e t spectrum i n ethanol showed X at r ° r max 238 (e 19370) and 283 (E 4150) my. The i n f r a r e d spectrum i n Nujol had absorptions at 3500 (0-H), 1690 (C=0) unsaturated s i x membered r i n g ketone), 1665 ( v i n y l ether), 1630 (C=C), 1595 (aromatic) and 915, 845 (substituted diene) cm The n.m.r. spectrum i n deuterochlorofrom (see Figure 10) showed the lone aromatic protons as s i n g l e t s at T 3.36 and T 3.62, the o l e f i n i c protons as a t r i p l e t centered at x 3.72 (Hi)(Ji7= 1.5 c.p.s) i n complete 87 agreement with the magnitude of long-range a l l y l i c spin-spin coupling and at T 3.99 (Hj+) . The phenolic proton appeared at x 3.79 and exchanged on addition of D 2 O , the expected methyl resonances as s i n g l e t s at x 6.28 and at x 6.38, and broad multiplets i n the region x 6.5-7.8 corresponding to the 8 a l i p h a t i c protons of r i n g B and C of the erythrinane r i n g system. The mass spectrum showed the molecular ion at m/e 313, and other s i g n i f i c a n t peaks at m/e 312 (M-l), 298 (M-15)(M-CH3), CHO C H = C - C = 0 I l N O C H C T ^ V s OCH-Ph i (49) 2 . Ph o CH-OCOJH 2 2 (52) OCH-Ph CHO OCH-Ph (47), CH=CH-NO„ OCHI Ph 2 (50) CH^CH-NH2 <-OCH OCH-Ph 2 CH-COH 2 2 OCH-Ph CH^OH OCH OCH-Ph 2 (48) c OCH. OCH-Ph 2 (51) CH^CN-OCH-Ph o c a (54) Figure 7, Reaction scheme for the preparation of 3-benzyloxy-4-methoxy-phenethylamine (53) and >-benzyloxy-4-methoxyphenylacetic acid (55). . -33-Figure 8. Reaction sequence, leading to di-(^ > -3>-hydroxy-4-methoxyphenyl)-ethylamine (58). -34-285 (M-28) (M-CO) (M-C2H1+) , 283 (M-30) (M-C2H6) , 282 (M-31)(M-1-C 2H 6). Additional evidence was gained by ac e t y l a t i o n of the dienone (59) with acetic anhydride and dry pyridine at room temperature to give 3,15-dimethoxy-16-acetoxy-2-oxoerythrina-l(6), 3-diene (60). T.L.C. of t h i s compound showed one spot on s i l i c a g e l . U l t r a v i o l e t spectrum i n ethanol showed X r ° r max at 214, 235 and 285 my. Infrared spectrum i n Nujol had absorptions at 1780 (phenol acetate), 1690 (unsaturated s i x membered r i n g ketone), 1665 (v i n y l ether), 1630 (C=C) and 1205 (phenol acetate) cm - 1. This also established that a t e r t i a r y nitrogen i s present i n the molecule, since a secondary nitrogen would acetylate r e a d i l y . Reduction of the dienone (59) by sodium borohydride i n ethanol at room temperature y i e l d e d 3,15-dimethoxy-2 (16-dihydroxyerythrina-l(6),3-diene (61). This compound was p u r i f i e d by chromatography on an alumina column, m.p. 166-169°. T.L.C. on s i l i c a gel showed one spot. Infrared spectrum i n Nujol had absorptions at 3510, 3450 (0-H), 1655 ( v i n y l ether), 1610 (C=C) and 1110, 1260 (secondary alcohol) cm"'1'. U l t r a - v i o l e t spectrum i n ethanol showed X m a x at 212, 240 (shoulder) and 287 my. The mass spectrum showed the molecular ion at m/e 315, and other s i g n i f i c a n t peaks at m/e 316 (M+l), 300 (M-15)(M-CH3), 297 (M-18)(M-H20), 287 (M-28){M-C 2W$ and 285 (M-30) (M-C 2H 6). The n.m.r. spectrum i n deuterochloroform showed the two lone aromatic protons as s i n g l e t s at x 3.43 and at x 3.68, a mult i p l e t at x 4.24 and a s i n g l e t at x 4.95 f o r the two o l e f i n i c protons, a broad s i n g l e t at x 4.72 which exchanged on addition of D 20 and was assigned to a hydroxyl proton, a mul t i p l e t centered at x 5.16 f o r the proton on the carbon atom bearing the hydroxyl group i n r i n g A, the methyl resonances (-O-CH3) at x6.28 and at r 6.47 and a complex pattern of l i n e s between x 6-8 corres--35 ponding to the aliphatic protons in rings B and C of the erythrinane ring system. Considering the mechanism of the cyclization reaction via oxidative coupling, there are two routes leading to the spiro amine ring system. One has as its first step the para-para coupling of the generated phenolic radicals to give a diphenoquinone type intermediate (39) (see Figure 5). The fourth ring may then be formed by the addition of the amino group to the quinonoid system to yield the dienone (37). There are two objections to this scheme. The major inherent objection to this route is the steric difficulty of attaching the nitrogen atom to the diphenoquinone system. A careful examination of the molecular model showed clearly that it is almost impossible to utilize the proposed diphenoquinone intermediate (39). The nitrogen atom is too far out and above the ring system and the site of its proposed attack, so that bond formation would be sterically difficult. The second objection is that i t has been reported that diphenoquinone does not 88 89 undergo addition reactions with amines. ' The second route (see Figure 11) can be visualized by the coupling of the phenolic radical with the un-paired electron on the nitrogen to give the phenethyl-indole intermediate (65). This can either, by para-para phenolic coupling (66), or by an in-doline type of ring closure (64), furnish (67) which, on aromatization of ring D will give rise to the dienone (59). This second mechanism is the favoured one and i t is proposed for the cyclization, strongly supported by 80 analogous products obtained enzymatically and by ferricyanide oxidation 90 of N-substituted 3,4-dihydroxyphenethylamines. The exact analogy for our case is, of course, oxidation of the methylester of 3-hydroxy-4-methoxy-90 phenylalanine to the corresponding indole by means of Fremy-salt. -36-Figure 9. Biogenetic-type synthesis of the erythrinane spiro-amine ring system. -38-This f i r s t laboratory r e a l i z a t i o n of such c y c l i z a t i o n , to provide the spiro amine skeleton present i n the E r y t h r i n a a l k a l o i d s by r a d i c a l coupling, has numerous i n t e r e s t i n g facets both from a biogenetic and a synthetic standpoint. It c e r t a i n l y renders strong support f o r the a t t r a c t i v e proposal that i n Nature these al k a l o i d s are formed by oxidative coupling, which pre-vi o u s l y has received no experimental v e r i f i c a t i o n . The r e s u l t s of t h i s i n v e s t i g a t i o n are consistent with the ideas presented e a r l i e r , but are not d e c i s i v e . However, they are s u f f i c i e n t to indicate i n broad outline the b i o s y n t h e t i c pathway to the spi r o amine system. The importance of choosing the correct p r o t e c t i o n pattern f o r the phenolic groups and also the proper state of the nitrogenous function i s well i l l u s t r a t e d by the synthesis. The most important conclusion to be drawn i s that i t seems very probable that methylation precedes and controls the d i r e c t i o n of oxidative coupling i n the biosynthesis of Erythrina a l k a l o i d s . The tetrahydroxy-diphenol (33) may well be the true precursor of these al k a l o i d s i n the plants, and the introduction of a l l y l i c hydroxyl or methoxyl at a l a t e r stage represents a minor divergence from our i n v i t r o scheme. Such a precursor would have the conceptual advantage of a s i n g l e oxygenation pattern i n the precursor f o r a l l the E r y t h r i n a a l k a l o i d s . The s t r u c t u r a l aspects of the theory are thus consistent with the r e s u l t s . Proof that the mechanism of coupling r e a l l y involves two phenolate r a d i c a l s i s d i f f i c u l t to secure by d i r e c t experiment, but so f a r as circumstantial evidence w i l l go the theory i s supported. Therefore, the case i s a very strong one i n favour of the biosynthesis of E r y t h r i n a a l k a l o i d s by coupling of the-diphenol (58). Although the r e s u l t of the feeding experiments are not yet a v a i l a b l e , i t seems quite c e r t a i n that the reaction c a r r i e d out i n the laboratory also -39-takes place in vivo. The evidence shows that such couplings under labora-tory conditions are facile, and the formation of the dienone (59) from the phenolic rings could be the key step in the biosynthesis of Erythrina alkaloids. The intermediates, therefore/must occupy attention. Isolation of intermediates from plants is quite difficult since they are present only in minute amounts. However, since i t was possible to prepare the dienone (59) , based on our present biosynthetic knowledge i t is possible and quite safe to predict that this hypothetical precursor is present in the Erythrina species. The validity of this proposal is supported by the examples of other similar alkaloidal structures occuring in plants, and known to ori-98 99 100 ginate from phenolic precursors. ' ' The hypothesis that methylene-dioxy-groups in alkaloids could be derived biogenetically by cyclization of 71 O-methoxyphenols is by now well established. Therefore i t seems reason-able to assume that the methylenedioxy-group present in erythraline (3) is formed by a radical cyclization mechanism from the dienone (59). There are five examples of derivation of this group from the O-methoxyphenol system in different alkaloids, and the generality of this step is in l i t -tie doubt. From the synthetic standpoint, the dienol (61) is a potential inter-mediate for a total biogenetic-type synthesis of erysodine (2) in view of its methylation pattern (see Figure 9). Elimination of the alcohol func-tion in (61) with the possibility of introducing the C-6 C-7 double bond and reduction at C-3 C-4 will lead directly to erysodine (2). Experiments in this direction are under.way in our laboratory. A final decision on the biosynthetic pathway can only be made by radiochemical labelling experiments. Therefore an extensive feeding pro-gram was initiated in our laboratory. First, in order to obtain an in - 4 0 -3 o (59) Figure 11. Proposed mechanistic scheme for the formation of the spiro-amine ring system via oxidative coupling. -41-vivo confirmation f o r our i n v i t r o laboratory synthesis the diphenol (58) 14 l a b e l l e d with C was prepared according to the scheme shown i n Figure 12. 3-Benzyloxy-4-methoxybenzyl chloride (51) was reacted with potassium 14 cyanide containing potassium [ C] cyanide i n dimethyl sulphoxide to y i e l d 14 3-benzyldxy-4-methoxyphenyl[1- C ] a c e t o n i t r i l e (68). The n i t r i l e was 14 hydrolysed to 3-benzyloxy-4rmethoxyphenyl[1- C]acetic acid (70) which on condensation with 3-benzyloxy-4-methoxyphenethylamine (53) gave 3-benzyloxy-N-(3-benzyloxy-4-methoxyphenethyl)-4-methoxyphenyl[carbonyl- 1 4C]acetamide (69), The amide was reduced by borane i n tetrahydrofuran (71) and debenzy-lated by hydrogenolysis to give the desired 3-hydroxy-4-methoxy-N-(3-hyd-14 roxy-4-methoxyphen[l- C]ethyl)—phenethylamine (72) shown i n r a d i o - i n a c t i v e runs to be i d e n t i c a l with an authentic sample. This l a b e l l e d substance, which i s assumed to be a precursor of the aromatic E r y t h r i n a a l k a l o i d s . and introduced into the plant's b i o s y n t h e t i c system, i s expected to produce on i s o l a t i o n radiochemically l a b e l l e d erysodine (2), erysopine (38) and e r y t h r a l i n e (3). Feeding experiments are i n progress, the r e s u l t s of which w i l l be of major i n t e r e s t , and they are expected to provide good support f o r the suggested mode of biosynthesis of the E r y t h r i n a a l k a l o i d s . ^ v C H 2 C I CH OCH Ph 2 ( 5 1 ) KCN OCH 2Ph (68) PhCH 2 0-i CH OCH 2Ph ( 7 0 ) OCH Ph 2 PhCH 2 0-( 7 1 ) O C ^ P h (53) OCH 2 Ph FieP.ire 1 2 •ation of 3-hydroxy-4-methoxy-1 ethyl)-phenexhylamine ( 7 2 ) . ' s marked, with, asterisk. -43-EXPERIMENTAL Melting points were determined on a Ko f l e r block and are uncorrected. U l t r a v i o l e t (U.V.) spectra were measured on a Cary 14 spectrophotometer and i n f r a r e d spectra (I.R.) were taken on a Perkin-Elmer model 137B spectro-photometer. Nuclear magnetic resonance (n.m.r.) spectra were recorded at 60 Mc/s on a Varian A60 instrument. The l i n e positions or centers of multi-plets are given i n the Ti e r s x scale with reference to tetramethylsilane as the i n t e r n a l standard, with the types of protons and integrated areas being indicated i n parentheses. S i l i c a gel G and alumina G (according to Stahl) plates were used f o r t h i n layer chromatography (T.L.C.) and were developed as given below. The alumina used for column chromatography was Shawinigan reagent, n e u t r a l i z e d with ethyl acetate, dried and deactivated with 60% of water. Every molecular weight quoted was determined mass spectrometrically. The mass spectrum was determined on a A.E.I. MS9 double focusing mass spectrometer. Elemental microanalyses were performed by Mrs. C. Jenkins of t h i s Department, and by Dr. A. Bernhardt and h i s associates of the Max Planck I n s t i t u t e , Mulheim, Ruhr, West Germany. The n.m.r. and mass spectrometric determinations were done by Mrs. A. Brewster and Mr. F. G. Bloss of t h i s Department r e s p e c t i v e l y . The r a d i o a c t i v i t y was determined with a Nuclear Chicago Model D47 gas flow detector operated as a Geiger counter and mounted i n a Model M-5 Semiautomatic Sample Changer, a l l i n conjunction with a Model 181B Decade Scalar. The a c t i v i t i e s were measured by depositing samples of 0.1 to 0.5 mg as t h i n films on standard 1.125 inch diameter aluminum planchets. The t o t a l a c t i v i t i e s of synthetic precursors are given i n m i l l i c u r i e s (mc), a -44-counter e f f i c i e n c y of 39.1% being assumed. Homoveratroyl-homoveratrylamine (43) 3,4-Dimethoxyphenylacetic acid (1 g) L. Light $ Co., England) and thionyl chloride (15 ml) were heated on a water-bath f o r 1 hour (45°) , and the excess of th i o n y l chloride was evaporated under reduced pressure. A so l u t i o n of the residue i n anhydrous ether (50 ml) was added to 3,4-dimethoxyphenethylamine (1.5 g) (Eastman Kodak) i n ether (20 ml). The amide which p r e c i p i t a t e d immediately was f i l t e r e d o f f and the crude product 92 r e c r y s t a l l i z e d from ethanol (1.5 g). m.p. 124° ( l i t e r a t u r e m.p. 124°). Anal. Found: C, 66,59%; H, 6.5%; N, 3.79%. Calc. f o r C 2oH 2 5N0 5: C, 66.9%; H, 6.97% N, 3.9%. Infrared spectrum i n chloroform: 3450 (s) (N-H), 3050 (s ) , 2990 ( s ) , 1680 (s ) , 1610 ( s ) , 1530 ( s ) , 1480 (s ) , 1430 ( s ) , 1270 (s) (broad), 1160 ( s ) , 1150 (s ) , 1035 ( s ) , 965 (w), 865 (s) and 815 (s) cm - 1. n.m.r. signals i n deuterochloroform, given i n T u n i t s : multiplet centered at 3.29 (aromatic H, area 6 H), multiplet centered at 6.15 (methyl H of 0 -0-CH3, area 12 H), mult i p l e t centered at 6.53 (methylene H of -CH2-c'-and methylene H of -N-CH2-, area 4H), t r i p l e t centred at 7.32 (S-methylene H of homoveratrylamine side chain, area 2H). Di-(J3-3,4-dimethoxyphenyl)-ethylamine (45) Homoveratroyl-homoveratrylamine.. (500 mg) was extracted (Soxhlet) into a r e f l u x i n g suspension of l i t h i u m aluminium hydride (600 mg) in dry ether (180 ml) (48 hours). The excess of reagent was decomposed with ethyl acetate (10 ml) and water was added (50 ml). The ether layer was separated -45-and the pasty aqueous layer further extracted with ether. Evaporation of the dried (anhydrous magnesium sulphate) ethereal solutions gave the o i l y amine (400 mg). The hydrochloride, prepared i n anhydrous ether, was recry-81 s t a l l i z e d from ethanol. m.p. 197° ( l i t e r a t u r e m.p. 195-196°). The amine 81 was also prepared on a l a r g e r scale by a previously described method as follows. A s o l u t i o n of 3,4-dimethoxyphenylacetonitrile (25 g) (K § K) i n ethanol (500. ml) was hydrogenated over activated Raney-Nickel c a t a l y s t at 1000 p . s . i . at 100° for 6 hours. A f t e r f i l t r a t i o n the solvent was removed under reduced pressure and then the amine was d i s t i l l e d o f f at 118-120°/1.5 mm. The r e s i d u a l o i l was dissolved i n dry ether (50 ml) and dry hydrogen chloride passed i n . The p r e c i p i t a t e d hydrochloride was c o l l e c t e d , recrys-t a l l i z e d from ethanol, giving di-(J3-3,4-dimethoxyphenyl)-ethylamine hydro-chloride (7 g) i d e n t i c a l with the product from the amide route (mixed m.p., I.R., n.m.r.). Anal.. Found: C, 62.98%; H, 7.23%; N, 3.75%. Calc. f o r C20 H28 N°4 C 1 : c > 62.8%, H, 7.32%; N, 3.67%. Infrared Spectrum i n Nujol: 2925 (s ) , 2500 (w), 1600 (m), 1525 ( s ) , 1460 ( s ) , 1380 ( s ) , 1340 (m), 1240 ( s ) , 1260 (s) 1165 ( s ) , 1030 ( s ) , 860 ( s ) , 815 ( s ) , 773 (w) and 725 (w) cm"1, n.m.r spectrum i n deuterochloroform, given in T u n i t s : doublet centered at 3.25 (aromatic K, area 6 H), s i n g l e t centered at 6.19 (methyl H of -0-CH3, area 12 H), broad s i n g l e t centered at 6.75 (H of phenethylamine side chain, area 8 H). Pi-(3-3,4-dihydroxyphehyl)-ethylamine Hydrobromide (33) Di-(3-3,4-dimethoxyphenyl)-ethylamine hydrochloride (1.3 g) was refluxed for 3 hours with hydrobromic acid (48%) (50 ml). The r e s u l t i n g s o l u t i o n was evaporated to dryness under reduced pressure on the steam-bath, and the -46-residual s o l i d r e c r y s t a l l i z e d from ethanol (charcoal) giving di-^-3,4-di-hydroxyphenyl)-ethylamine hydrobromide (1 g). It was necessary to dry the c r y s t a l s at 100° under vacuum for 5 hours, m.p. 151°. Anal. Found: C, 49.8%; H, 5.29%; N, 3.21%. Calc. f o r C 1 6H 2oN0 l tBrH 20: C, 49.6%; H, 5.15%; N, 3.61%. Infrared spectrum i n Nujol: 3400 (s) (0-H), 2995 ( s ) , 2750 (w), 1625 (m), 1545 (m), 1470 ( s ) , 1380 ( s ) , 1340 (w), 1295 ( s ) , 1270 (m), 1195 (m), 1155 (w), 1120 (m), 1070 (m), 1050 (m), 1039 (w), 950 (w), 888 (w), 825 (m) , 810 (w) , 783 ( s ) , and 725 (w) cm."1 U l t r a v i o l e t spectrum i n ethanol: X 284 my ( e 7770). r max v Oxidation of Pi-(g-3,4-dihydroxyphenyl)-ethylamine Hydrobromide with  Potassium Ferricyanide and F e r r i c Chloride In one t y p i c a l experiment 356 mg (1.08 mMole) of potassium f e r r i c y a n i d e i n 900 ml of d i s t i l l e d water containing 20 g of sodium bicarbonate was added through a f i n e c a p i l l a r y to a s t i r r e d s o l u t i o n of di-Q3-3,4-dihydroxyphenyl)-ethylamine hydrobromide (100 mg, 0.27 mMole) i n 340 ml of d i s t i l l e d water under nitrogen atmosphere during 6 hours. The reaction mixture was s t i r r e d f o r another 3 hours and f i l t e r e d to remove the large amount of polymer which separated. The s o l u t i o n was then extracted with ethylacetate and chloroform (4x200 ml r e s p e c t i v e l y ) . The combined and dried (anhydrous sod-ium sulphate) extracts were evaporated under reduced pressure to give a gummy material (18 mg). The i n f r a r e d spectrum (NaCl) of the crude product has absorptions at 3450 (OH) and 1720 cm"1 (C=0). U l t r a v i o l e t spectrum i n ethanol: X 253 my and a shoulder at 284 my. S i m i l a r r e s u l t s were max. obtained when f e r r i c c hloride was used as o x i d i z i n g agent. It was not possible to i s o l a t e any products or to obtain reproducible r e s u l t s i n these -47-oxidation experiments. O - B e n z y l i s o v a n i l l i n (47) A mixture of i s o v a n i l l i n (30 g) (K § K), benzyl chloride (37.8 g), f i n e l y powdered anhydrous potassium carbonate (15 g) and potassium iodide (7.5 g), and absolute methanol (100 ml) was refluxed f o r 15 hours. A f t e r f i l t r a t i o n from inorganic materials and concentration under reduce pressure, the product was steam d i s t i l l e d y i e l d i n g a gum-like residue which s o l i d i f i e d on standing at 0°. The residue was dissolved i n ether, washed f i r s t with d i l u t e sodium hydroxide s o l u t i o n then with water and dried (anhydrous magnesium sulphate). The brownish o i l , obtained by evaporation of the ether s o l u t i o n under vacuum, was r e c r y s t a l l i z e d twice from benzene-petroleum ether 93 (30-60°) to give c o l o r l e s s needles (32 g), m.p. 62° ( l i t e r a t u r e m.p. 62°). n.m.r s i g n a l s ; given i n T u n i t s , spectrum obtained i n deuterochloroform: s i n g l e t centered at 0.25 (aldehyde H, area = 1 H), multiplet centered at 2.7 (aromatic H, area = 8 H), s i n g l e t centered at 6.12 (methyl H of -0-CH3, area = 3 H) and a si n g l e centered at 4.91 (methylene of -0-CH 2-C 6H 5, area = 2 H). 3-Benzyloxy-4-methoxybenzyl Alcohol (48) A s t i r r e d s o l u t i o n of O - B e n z y l i s o v a n i l l i n (25 g), i n methanol (260 ml) was treated portionwise with sodium borohydride (3.5 g) over 1 V 2 hours. The s o l u t i o n was warmed at 40° f o r 1 hour, a c i d i f i e d with concentrated hydrochloric acid, and then b a s i f i e d with 2 N. sodium hydroxide. The organic solvent was evaporated under reduced pressure and the residue, an aqueous suspension, extracted thoroughly with chloroform. A f t e r drying -48-(anhydrous sodium sulphate) the chloroform s o l u t i o n was evaporated under vacuum, and the product r e c r y s t a l l i z e d from ether and l i g h t petroleum-ether (40-60°) to y i e l d the alcohol (24 g), m.p. 72° ( l i t e r a t u r e m.p. 7 2 - 7 3 ° ) . 8 6 3-Benzyloxy-4-methoxybenzyl chloride (51) Thionyl chloride (38 ml) was added dropwise during 30 minutes to a ra p i d l y s t i r r e d suspension of 3-benzyloxy-4-methoxybenzyl alcohol (21 g) in ether (150 ml). A f t e r a further 30 minutes, the c l e a r s o l u t i o n was evapo-rated under reduced pressure. The crude product was dissolved i n petroleum-ether (80-110°), p u r i f i e d (charcoal), and f i l t e r e d by gravity. On evapo-ra t i o n of the organic solvent 24 g of the required chloride was obtained, which was r e c r y s t a l l i z e d from petroleum-ether (80-110°) and ether, m.p. 72° ( l i t e r a t u r e m.p. 7 2 - 7 3 ° ) . 8 6 3-Benzyloxy-4-methoxyphenylacetonitrile (54) Potassium cyanide (15 g) was s t i r r e d f o r 15 minutes with dimethyl-sulphoxide (500 ml), and then 3-benzyloxy-4-methoxybenzyl chloride (40 g) was added to the s o l u t i o n . A f t e r s t i r r i n g the s o l u t i o n f or 6 hours at room temperature, 200 ml of water was added and the aqueous s o l u t i o n extracted s i x times with ether-petroleum-ether (80-110°) (1:1 by volume), washing each time with water (100 ml). Evaporation of the combined, drie d (anhydrous magnesium sulphate) extracts gave 28 gm of the n i t r i l e . It was r e c r y s t a l l i z e d from chloroform-petroleum ether (30-60°). m.p. 78° ( l i t e r a -ture m.p. 79.5-80.5 ). Anal. Found: C, 75.31%; H, 5.33%; N, 5.18^. Calc. f o r C i 6 H 1 5 N 0 2 : 75.8%; H, 5.93%; N, 5.51%. The i n f r a r e d spectrum i n -49-chloroform had the c h a r a c t e r i s t i c n i t r i l e absorption band at 2280 cm ^ (C=N st r e t c h i n g ) , 3-Benzyloxy-4-methoxy-8-nitrostyrene (50) 12 g of O - b e n z y l i s o v a n i l l i n was dissolved i n 400 ml of 95% ethanol at room temperature and the s o l u t i o n then cooled to 5-10°, a f t e r which 6 g of nitromethane was added. Then a s o l u t i o n of 5 g of sodium hydroxide dissolved i n the minimum amount of water i n 100 ml of ethanol, cooled to 5-10°, was added from a dropping funnel at a rate of 5 ml per minute. The s o l u t i o n of the nitromethane and O - b e n z y l i s o v a n i l l i n i n alcohol was vigorously s t i r r e d and kept below 15° during the addition of the a l c o h o l i c sodium hydroxide. As the reaction proceeded, the insoluble sodium s a l t of the condensation product p r e c i p i t a t e d . A f t e r a l l of the a l k a l i had been added and with the temperature kept below 15°, i c e water was slowly added u n t i l the p r e c i p i t a t e dissolved. The c l e a r cold s o l u t i o n was added i n a fi n e stream through a funnel to a s t i r r e d s o l u t i o n of 60 ml of con-centrated hydrochloric acid i n 90 ml of water. No attempt was made to control the temperature during the addition. .A f i n e , yellow p r e c i p i t a t e was immediately formed and a f t e r standing f o r V 2 hour was f i l t e r e d with suction and then washed with ethanol. 8 g of nitrostyrene was obtained, 82 m.p. 127°. ( l i t e r a t u r e m.p. 127-128°). The product thus formed was quite pure and was fwithout further p u r i f i c a t i o n by r e c r y s t a l l i z a t i o n ^ used i n the next step. 5-Benzyloxy-4-methoxyphenethylamine (53) A s o l u t i o n of 3-benzyloxy-4-methoxy-B-nitrostyrene (10.5 g) i n anhydrous tetrahydrofuran (200 ml) was added to a suspension of l i t h i u m -50-aluminium hydride (10 g) i n the same solvent (200 ml). A vigorous reaction was observed. A f t e r the mixture had been heated under r e f l u x f o r four days, ( i t was treated with i c e cold water (25 ml), s t i r r e d for 2 hours and f i l t e r e d , the f i l t e r - p a d being washed twice with ether. The combined organic solu-tions were evaporated, and the residue was dissolved i n 10% hydrochloric acid. The a c i d i c s o l u t i o n was extracted with ether, b a s i f i e d and extracted three times with chloroform (400 ml). Evaporation of the dried (anhydrous sodium sulphate) chloroform extracts l e f t a residue (5 g), which was d i s -solved i n anhydrous ether and treated with dry hydrogen chloride i n the same solvent. R e c r y s t a l l i z a t i o n of the bulky p r e c i p i t a t e from ethanol-ether yielded the required amine hydrochloride (5 g) m.p. 163-165° ( l i t -82 erature m.p. 162-166°). This amine was also prepared by another method, as follows. A s o l u t i o n of 3-benzyloxy-4-methoxyphenylacetonitrile (4 g) i n anhydrous ether (150 ml) was added to a s t i r r e d s o l u t i o n of lithium aluminium hydride (5 g) i n ether (100 ml). A f t e r the mixture had been heated under r e f l u x f o r 5 hours, i t was cooled, treated with i c e water, s t i r r e d f o r 1 hour and f i l t e r e d . The ethereal s o l u t i o n was evaporated under reduced pressure, and the residue was dissolved i n 10% hydrochloric acid. The a c i d i c s o l u t i o n was extracted with ether, b a s i f i e d and extracted three times with chloroform. Evapora-t i o n of the dry (anhydrous sodium sulphate) chloroform extracts y i e l d e d the amine (1.8 g). The hydrochloride was r e c r y s t a l l i z e d from ethanol-ether to give c r y s t a l s , i d e n t i c a l with the product from the other route, n.m.r. si g n a l s : given i n T u n i t s , spectrum obtained i n deuterochloroform: multi-p l e t centered at 2.67 (aromatic H of 0-CH2-C6H5, area 5 H), multiplet centered at 3.2 (aromatic H of t r i s u b s t i t u t e d benzene, area 3 H), s i n g l e t centered at 4.94 (methylene of -O-CT^-CgHs,area 2 H), s i n g l e t centered at -51-6.22 (methyl H of -0-CH3, area 3 H), multiplet centered at 6.95 (H of phenyl-ethylamine side chain, area 4H) . Infrared spectrum i n Nujol: 2990 (s), 1620 (m), 1530 ( s ) , 1460 (s), 1375 ( s ) , 1275 ( s ) , 1238 ( s ) , 1150 (s), 1030 ( s ) , 940 (m), 860 (m), 812 ( s ) , 750 (m) 735 (s) 706 (m) and 695 (m) cm - 1, 4-(3-Benzyloxy-4-methoxyben2ylidene)-2-phenyloxazolone (49) The oxazolone was obtained by heating O - b e n z y l i s o v a n i l l i n (20 g), h i p p u r i c acid (14.5 g) , anhydrous sodium acetate (A.R., 8.5 g) and acetic anhydride (50 ml) at 100° f o r 2 hours. The crude product was mixed with alcohol, and the yellow s o l i d f i l t e r e d by suction and washed with much b o i l i n g water (1 l i t e r ) . The oxazolone c r y s t a l l i z e d from a c e t i c acid i n 82 yellow prismatic needles (16 g). m.p. 154° ( l i t e r a t u r e m.p. 155°). The substance i s sparingly soluble i n hot alcohol. 3-Benzyloxy-4-methoxyphenylacetic Acid (55) A mixture of the oxazolone (35 g) and 10% sodium hydroxide (200 ml) was refluxed under nitrogen u n t i l evolution of ammonia ceased (8 hours), then saturated with carbon dioxide (pH 8-8.5) (4 hours), and cooled to 5°. Then 6% aqueous hydrogen peroxide was added (25 ml) at such a rate that the temperature of the reaction mixture d i d not r i s e 5° ( 1 hour). A f t e r storage at 0° f o r 24 hours the mixture was a c i d i f i e d with concentrated hydrochloric a c i d , and the p r e c i p i t a t e d gummy acids were exhaustively extracted with chloroform. The extracts, when washed, drie d (anhydrous sodium sulphate), and evaporated under, reduced pressure, afforded an o i l (34 g) which was chromatographed on s i l i c a gel (B.D.H.) (600 g). The s i z e -52-of the column was 27 x 5 cm diameter. The material was added to the top of the column by d i s s o l v i n g i t i n the minimum amount of benzene. On e l u t i o n the following f r a c t i o n s were obtained consecutively: (A) 3000 ml of benzene removed no material (B) 1000 ml of benzene-ether (10%) y i e l d e d nothing. (C) 1000 ml of benzene-ether (10%) gave a small amount of s o l i d material (D) 2000 ml of benzene-ether (10%) eluted benzoic acid, which gave a melting point of 122° undepressed on admixture with an authentic sample of benzoic acid. (E) 1000 ml of benzene-ether (10%) eluted o i l s i n two f r a c t i o n s . (F) 2500 ml of benzene-ether (10%) yielded 5 g of 3-benzyloxy-4-methoxyphenylacetic acid. The l a s t f r a c t i o n s eluted from the column were o i l s , and because of the large amount of solvent involved, further development of the chromato-graphic column was abandoned. The acid obtained was r e c r y s t a l l i z e d twice 82 from benzene, m.p. 122-124°. ( l i t e r a t u r e m.p. 125°), The required acid was prepared more r e a d i l y and i n b e t t e r y i e l d s , as follows. A s o l u t i o n of 3-benzyloxy-4-methoxyphenylacetonitrile (15 g) i n ethylene g l y c o l (300 ml) and water (80 ml) was heated under r e f l u x f or 13 hours with potassium hydroxide (8 g). A f t e r d i l u t i o n of the cooled s o l u t i o n with water (200 ml), i t was extracted twice with ether, then a c i d i f i e d with concentrated hydrochloric acid and extracted again with ether. The combined second set of extracts gave 3-benzyloxy-4-methoxy-phenylacetic acid (11 g) which was r e c r y s t a l l i z e d from benzene to give c r y s t a l s i d e n t i c a l with the product from the other route. Anal. Found: C, 71.06%; H, 5.96%. Calc. f o r C^H^O^: C, 70.70%; H, 5.83%. -53-Infrared spectrum of the c r y s t a l s showed the following major absorp-tions i n Nujol: 2990 (s) , 1720.(s), 1605 (w), 1530 (m) , 1460 (s ) , 1380 (s), 1260 (m), 1220 (m), 1160 (m), 1140 (m), 1010 (m), 860 (w), 815 (w), 780 (w), 745 (m), and 695 (w) cm The n.m.r. sig n a l s : given i n T u n i t s , spectrum obtained i n deuterochloroform: s i n g l e t centered at -1.4 (H of COOH), multi-p l e t centered at 2.65 (aromatic H of O-CH2C5H5, area 5 H), multiplet centered at 3.19 (aromatic H of t r i s u b s t i t u t e d benzene, area 3 H), s i n g l e t centered at 4.9 (methylene H of 0-CH 2-C eH 5, area 2 H), s i n g l e t centered at 6.2 (methyl H of 0-CH3, area 3 H), s i n g l e t centered at 6.5 (methylene H of -CH2-C00H, area 2 H). 3~Benzyloxy-N-(3-benzyloxy-4-methoxyphenethyl )-4-methoxyphenylacetamide  (56) 3-Benzyloxy-4-methoxyphenylacetic acid (600 mg) in dry chloroform (50 ml) was added portionwise to 3.5 ml of th i o n y l c h l o r i d e , and the re-action mixture was then allowed to stand on a water-bath (45°) f o r one hour. The solvent and the excess of th i o n y l chloride were evaporated under diminished pressure. The res i d u a l acid chloride s o l i d i f i e d and was used immediately. The acid chloride (from 600 mg of acid) i n dry tetrahydrofuran (40 ml) was added dropwise during 1 hour to a s t i r r e d s o l u t i o n of 3-benzyloxy-4-methoxyphenethylamine hydrochloride (800 mg) i n tetrahydrofuran (50 ml) and aqueous sodium hydroxide (0.25 g i n 1 ml). A f t e r an addit i o n a l V 2 hour the tetrahydrofuran was removed under reduced pressure, The residue was taken up i n chloroform, washed successively with d i l u t e hydrochloric acid, aqueous sodium bicarbonate and water, dried:(anhydrous magnesium -54-sulphate), and evaporated under vacuum to give an o i l which s o l i d i f i e d on standing at room temperature. R e c r y s t a l l i z a t i o n from ethyl acetate gave 82 the amide (900 mg) m.p. 116° ( l i t e r a t u r e m.p. 118°. Anal. Found: N, 2.73%, Calc. f o r C32H33NO5: N, 2.74%. Infrared spectrum i n Nujol: 3350 (m) (N-H), 2950 (s ) , 1650 (s), 1600 (m), 1525 (s ) , 1460 (s), 1380 (s ) , 1260 (s ) , 1240 (s), 1160 (w), 1140 ( s ) , 1080 (w), 1020 (s ) , 940 (w), 855 (w), 813 (m), 780 (w), 742 (m), 725 (m), and 700 (s) cm 1 . n.m.r. si g n a l s ; given i n x un i t s , spectrum obtained i n deuterochloroform: multiplet centered at 2.65 (aromatic H of -O-CH2-C5H5, area 10 H), s i n g l e t centered at 3.23 (aromatic H of t r i s u b s t i t u t e d benzene, area 6 H), s i n g l e t centered at 4.95 (methylene H of -0-CH 2-C 6H 5, area 4 H), s i n g l e t centered at 6.2 (methyl H of -O-CH3, are 6 H), multiplet centered at 6.5 (methylene H of CH2-C=0 and methylene H of -N-CH2-, area 4 H), t r i p l e t centered at 7.25 (6 methylene H of phenethyl-amine side chain, area 2 H). Pi-(B-3-benzyloxy-4-methoxyphenyl)-ethylamine (57) To a s o l u t i o n of 3-benzyloxy-N-(3-benzyloxy-4-methoxyphenethyl)-4-methoxyphenylacetamide (258 mg) i n dry tetrahydrofuran (50 ml) i n a 100 ml fla s k (nitrogen atmosphere) was added 10 ml of 1 M borane i n tetrahydro-94 furan over 20 minutes. The temperature was maintained at approximately 0° during the addition. A f t e r the reaction mixture had been heated under r e f l u x f o r 8 hours, i t was cooled to room temperature and 2 ml of d i l u t e hydrochloric acid was added. The tetrahydrofuran was removed by d i s t i l l a t i o n . Sodium hydroxide p e l l e t s were added to saturate the aqueous phase and the l a t t e r was extracted three times with a t o t a l of 100 ml of ether. The combined ether extracts were drie d (anhydrous sodium -55-sulphate) and evaporated. The residual amine was converted into i t s hydro-chloride (250 mg) with dry ethereal hydrogen chloride, m.p. 130°. The f i n a l product was obtained as the c r y s t a l l i n e amine hydrochloride by r e c r y s t a l l i -zation from ethanol-ether and drying at 100° under vacuum for f i v e hours, m.p. 164-167°. Anal. Found: C, 72.13%; H, 6.83%; N, 2.47%, Calc. for C 3 2 H 3 6 O 4 N C I : C, 72.1%; H, 6.76%; N, 2.63%. Infrared spectrum i n Nujol: 2950 ( s ) , 2480 (w) , 1600 (w) , 1530 (s) , 1460 (s) , 1380 (s) , 1285 (s) , 1240 (s) , 1150 ( s ) , 1085 (w), 1030 ( s ) , 990 (m), 880 (w), 858 (w), 814 (m), 775 (w), 752 (m), 735 (s) and 700 (m) cm - 1, n.m.r. s i g n a l s ; given i n T u n i t s , spectrum obtained i n deuterochloroform: mulitplet centered at 2.65 (aromatic H of - O - C H 2 - C 6 H 5 , area 10 H), s i n g l e t centered at 3.24 (aromatic H of t r i s u b s t i t u t e d benzene, area 6 H), s i n g l e t centered at 4.94 (methylene H of -0-CH 2-C 6H 5, area 4 H), s i n g l e t centered at 6.2 (methyl H of -0-CH3, area 6 H), s i n g l e t (broad) centered at 6.88 (H of phenylethylamine side chain, area 8 H). Pi-(g-3-hydroxy-4-methoxyphenyl)-ethylamine (58) The corresponding dibenzyl ether, Di-(3-3-benzyloxy-4-methoxyphenyl)-ethylamine hydrochloride (56 mg) was hydrogenolysed i n methanol (10 ml) containing concentrated hydrochloric acid (0.1 ml) and 10% p a l l a d i s e d charcoal (25 mg), the hydrogen uptake being complete i n 30 minutes. F i l t r a t i o n of the'solution and evaporation of the solvent gave di-(3-3-hydroxy-4-methoxyphenyl)ethylamine hydrochloride. It was r e c r y s t a l l i z e d from ethanol. m.p. 230°. The amine was also prepared on a larger scale by another method, as follows. A s o l u t i o n of 3-benzyloxy-4-methoxyphenylacetonitrile (3 g) i n -56-methanol (250 ml) was placed i n t o a high pressure bomb, with 1.5 g of fres h l y 95 prepared active Raney-Nickel c a t a l y s t . The hydrogen gas was passed into the bomb (1100 p.s.i.) and the mixture heated at 90° with s t i r r r i n g for 8 hours. The bomb was allowed to cool before i t was opened and then the contents were removed. The reaction product was then f i l t e r e d and the ammoniacal methanol s o l u t i o n evaporated. The residue was dissolved i n absolute ethanol and dry hydrochloric acid was added to the ethanolic s o l u t i o n i n the same solvent. The amine hydrochloride (lg) c r y s t a l l i z e d overnight at 0° and a f t e r r e c r y s t a l l i z a t i o n from absolute ethanol i t was found to be i d e n t i c a l (mixed m.p., I.R., n.m.r.) with that obtained from the amide route above. Anal. Found: C, 61.08%; H, 6.7%; N, 3.49%. Calc. for CigHji+O^NCl: C, 61.3%; H, 6.8%; N, 3.96%. Infrared spectrum i n Nujol: 3550 (m), 2950 (s ) , 2500 (w), 1600 (m) 1460 ( s ) , , 1380 ( s ) , 1340 (m), 1300 (m), 1265 (w), 1230 ( s ) , 1205 (w), 1158 (m), 1132 (m), 1025 ( s ) , 955 (w), 870 ( s ) , 812 ( s ) , 768 (w), 755 (w) and 725 (w) cm"1, n.m.r. s i g n a l s ; given i n T u n i t s , spectrum obtained i n deuterochloroform: multiplet centered at 3.36 (aromatic H, area 6 H), s i n g l e t centered at 4.47 (phenolic H, area 2 H), s i n g l e t centered at 6.2 (methyl H of -0-CH3, area 6 H), t r i p l e t (broad) centered at 7.25 (H of phenylethylamine side chain, area 8 H). 3,15-Dimethoxy-16-hydroxy-2-oxoerythrina-l(6),3-diene (59) A s o l u t i o n of di-(6-3-hydroxy-4-methoxyphenyl)-ethylamine hydrochloride (150 mg, 0.424 mMole) i n d i s t i l l e d water (900 ml) was added through a fin e c a p i l l a r y to a vigorously s t i r r e d s o l u t i o n of potassium f e r r i c y a n i d e (570 mg, 1.73 mMole) i n 100 ml of d i s t i l l e d water and 240 ml of 1 N. sodium bicarbonate, under nitrogen atmosphere, during 6 hours. The reaction -57-mixture was s t i r r e d f o r another three hours, then extracted with chloroform (6 x 200 ml). The combined extracts, a f t e r drying (anhydrous sodium sulphate), on evaporation of the solvent under reduced pressure yielded a brown gum (110 mg). The experiment was repeated ten times and the combined gummy residue (1.140 g) was chromatographed on alumina (30 g) (Shawinigan reagent, ne u t r a l i z e d with ethyl acetate, dried at 100° and then deactivated with~6% of water). The material was introduced i n 20 ml of chloroform into the column (size 13 x 2 cm diameter), and on e l u t i o n the following f r a c t i o n s were obtained consecutively: (A) 50 ml of chloroform removed 61 mg of colourless o i l . (B) 90 ml of chloroform gave 250 mg of c r y s t a l l i n e s o l i d . (C) 525 ml of chloroform y i e l d e d 165 mg of o i l . (D) 250 ml of chloroform-ethanol (4:1) eluted 145 mg of o i l material. The t o t a l recovery of organic material was 621 mg. The c r y s t a l s obtained from f r a c t i o n (B) were r e c r y s t a l l i z e d from, ethanol to give an a n a l y t i c a l sample, m.p. 224-229° with decomposition. One spot i n T.L.C. on s i l i c a gel (Stahl G) i n n-butanol-water-acetic acid (12:5:2) (R f=0.29). This compound was i d e n t i f i e d as 3,15-dimethoxy-16-hydroxy-2-oxoerythrina-l(6),3-diene (225 mg, 15%). Molecular weight 313 (determined by mass spectrometry). Anal. Found: C, 69.21%; H, 6.19%, N, 4.45%. Calc. f o r C 1 8H 1 9N0 H: C, 69.02%; H, 6.06%; N, 4.46%. U l t r a v i o l e t spectrum i n ethanol: ^ m a x 238 my (e 19370) and 283 my (e 4150). Infrared spectrum i n Nujol: 3500 (m), 1690 ( s ) , 1665 ( s ) , 1630 ( s ) , 1595 (m), 1505 ( s ) , 1460 ( s ) , 1380 ( s ) , 1320 (w), 1265 (m), 1200 ( s ) , 1175 ( s ) , 1130 (w), 1100 (w), 1070 (w), 1030 (m), 975 (w), 915 (w), 885 (m), -58-845 (m), 812 (w), 782 (m) and 723- (w) cm ^. n.m.r. si g n a l s ; given i n x un i t s , spectrum obtained i n deuterochloroform: s i n g l e t centered at 3.36 (aromatic H, area IH), s i n g l e t centered at 3.62 (aromatic H, area 1 H), t r i p l e t centered at 3.72 (J = 1.5 c.p.s) ( v i n y l H, area 1 H), broad s i n g l e t centered at 3.79 which disappeared on addition of D 20 (hydroxyl H, area 1 H), s i n g l e t centered at 3.99 (v i n y l H, area 1 H), s i n g l e t s centered at 6.28 and 6.38 (methyl H of -O-CH3, area 3 H respectively) and broad multiplets centered at 6.75 and 7.38 ( a l i p h a t i c protons of r i n g B and C of erythrinane skeleton, area 4 H r e s p e c t i v e l y ) . Mass spectrum showed s i g n i f i c a n t peaks at m/e = 313 (M +), 312 (M-l), 298 (M-15), 285 (M-28), 283 (M-30), 282 (M-31), 269, 254, 241, 238, 226, 210, 198, 176, and 170. 3,15-Dimethyoxy-16-acetoxy-2-oxoerythrina-l(6),3-diene (60) 6 mg of 3,15-dimethoxy-16-hydroxy-2-oxoerythrina-l(6),3-diene was acetylated, with 3 ml of ac e t i c anhydride (reagent) and 3 ml of dry pyridine (reagent) at room temperature overnight. The excess of reagent and the pyridine was removed under reduced pressure on the steam bath. The residue was dissolved i n 10 ml of chloroform, washed twice with water and dried (anhydrous sodium sulphate). Evaporation of the chloroform s o l u t i o n under reduced pressure gave 7 mg of an o i l y material which s o l i d i -f i e d on standing at room temperature. Infrared spectrum i n Nujol: 1780 (s), 1690 ( s ) , 1630 (s ) , 1600 (shoulder), 1510 ( s ) , 1465 ( s ) , 1380 (s ) , 1260 ( s ) , 1205 ( s ) , 1180 ( s ) , 1100 (m), 1080 (m), 980 (w) and 900 (w) cm U l t r a v i o l e t spectrum i n ethanol: A 214, 234, 285 mu. T.L.C. r max gave one spot on s i l i c a gel. Rf= 0.18 i n n-butanol-water-acetic acid -59-(12:5:2) . 3,15-Dimethoxy-2,16-dihydroxyerythrina-1(6),5-diene (61) To a s o l u t i o n of 3,15-dimethoxy-16-hydroxy-2-exoerythrina-l(6),3-diene (115 mg) i n ethanol (20 ml) was added sodium borohydride (70 mg), and the reaction mixture s t i r r e d f o r 6 hours at room temperature. The ethanolic s o l u t i o n was then concentrated under reduced pressure and 40 ml of water added. The aqueous s o l u t i o n was extracted three times with chloroform, and the combined extracts (150 ml) a f t e r drying (anhydrous sodium sulphate), on evaporation y i e l d e d 102 mg of o i l y material which was chromatographed on alumina (3 g). The s i z e of the column was 3.5 x 2 cm diameter. The material was introduced i n t o the column i n 4 ml of chloroform, and on e l u t i o n the following f r a c t i o n s were obtained consecutively: 180 ml of chloroform eluted a yellow o i l i n 3 f r a c t i o n s . The required alcohol was then eluted with 50 ml of chloroform (65 mg). R e c r y s t a l l i z a t i o n from ether-ethanol gave a m.p. 166-169°.. T.L.C. gave one spot. Rf= 0.19 i n n-butanol-water-acetic acid (12:5:2). Infrared spectrum i n Nujol: 3510 ( s ) , 3450 ( s ) , 1655 ( s ) , 1610 ( s ) , 1505 ( s ) , 1450 ( s ) , 1375 ( s ) , 1260 (s) , 1220 ( s ) , 1110 ( s ) , 1085 ( s ) , 1050 ( s ) , 1020 ( s ) , 985 (m), 920 (m), 870 (m), 840 (w), 806 (s) and 770 (m) cm - 1. U l t r a v i o l e t spectrum i n ethanol: X m 3.x 212, 240 (shoulder), 287 my. Mass spectrum showed s i g n i f i c a n t peaks at m/e = 315 (M +), 316 (M + 1), 300 (M-15), 297 (M-18), 287 (M-28), 285 (M-30), 279, 260, 259, 251, 242, 212, 199, 167, 149, 147, 129, 113 and 112. The n.m.r. spectrum i n deuterochloroform showed the expected methyl resonances (-OCH3) at T 6.28 and T 6.47, two s i n g l e t s at x 3.43 and x 3.68 f o r the aromatic protons, a multiplet centered at x 4.24 and a s i n g l e t at x 4.95 -60-f o r the two o l e f i n i c protons, a multiplet centered at x 5.16 for the lone proton on the carbon bearing the hydroxyl group i n r i n g A of the erythrinane r i n g system, a broad s i n g l e t at x 4.72 which disappeared on addition of D2O and was assigned to one hydroxyl proton, and multiplets x 6-8 corresponding to the a l i p h a t i c protons i n rings B and C. 14 3-Benzyloxy-4-methoxyphenyl[1- C]acetic Acid (70) Potassium cyanide (39.4 mg) was s t i r r e d f o r 10 minutes with dimethyl 14 sulphoxide (3 ml) and potassium[ C]cyanide (6.5 mg, 1.0 mc) was added and washed i n with dimethyl sulphoxide (4 ml). A f t e r 15 minutes, 3-benzyloxy-4-methoxybenzyl chloride (185.5 mg) was added, and the s o l u t i o n was s t i r r e d at room temperature f o r 6 hours. It was shaken with water (50 ml) and ether-petroleum ether (80-110°) (50 ml, 1:1 by volume) and the aqueous layer was fur t h e r extracted twice with the same solvent, washing each time with water (10 ml). Evaporation of the combined, drie d (anhydrous sodium s u l -14 phate) extracts l e f t 3-benzyloxy-4-methoxyphenyl[1- C ] a c e t o n i t r i l e (151 mg), shown i n r a d i o - i n a c t i v e runs to be i d e n t i c a l with authentic material (mixed m.p.). A l l the active sample was dissolved i n ethylene g l y c o l (3 ml) and water (1 ml) and heated with potassium hydroxide (0.5 g) under r e f l u x f o r 12 hours. The cooled s o l u t i o n was p a r t i t i o n e d between water and ether and the aqueous phase, a f t e r a c i d i f i c a t i o n , was extracted four times with 14 ether and dried to a f f o r d 3-benzyloxy-4-methoxyphenyl[1- C]acetic acid (137 mg; 0.8 mc), m.p. 123°. -61-3-Hydroxy-4-methoxy-N-(3-hydroxy-4-methoxyphen[l- C]ethyl)-phenethyl- amine (72) . 14 3-Benzyloxy-4-methoxyphenyl[1- C]acetic acid (137 mg) was warmed on a steam bath f o r 30 minutes with th i o n y l chloride (3 ml) and the excess o f reagent was, evaporated under reduced pressure. A s o l u t i o n of the residue i n anhydrous ether (10 ml) was added dropwise to a s t i r r e d s o l u t i o n of 3-benzyloxy-4-methoxyphenethylamine (417 mg) i n anhydrous ether 10 ml at 0°. The mixture was shaken with 2N hydrochloric acid (20 ml) and ethyl acetate (60 ml), and the aqueous layer was extracted twice with ethyl acetate. A f t e r the combined extracts had been shaken with an excess of aqueous potassium carbonate and water, they were drie d and evaporated to y i e l d 3-benzyloxy-N-(3-benzyloxy-4-methoxyphenethyl)-4-methoxyphenyl-14 [carbonyl- Cjacetamide as a s o l i d . R e c r y s t a l l i z a t i o n from ethyl acetate gave the amide (150 mg), m.p. 116°, s u i t a b l e f o r reduction. To a s o l u t i o n of the foregoing amide i n dry tetrahydrofuran (50 ml) i n a 100 ml f l a s k (nitrogen atmosphere) was added 10 ml of IM borane i n tetrahydrofuran over 20 minutes. The temperature was maintained at approximately 0° during the addition. A f t e r the reaction mixture had been heated under r e f l u x f o r 6 hours, i t was cooled to room temperature and 2 ml of d i l u t e hydrochloric acid was added. The tetrahydrofuran was removed by d i s t i l l a t i o n at atmospheric pressure (steam bath) as hydrogen was evolved from the hydro-l y s i s of excess reagent. Sodium hydroxide p e l l e t t s were added to saturate the aqueous phase and the l a t t e r was extracted three times with a t o t a l of 100 ml of ether. The combined ether extracts were dried (anhydrous sodium sulphate) and evaporated. The r e s i d u a l amine was debenzylated by hydro-genolysis i n methanol (10 ml) containing concentrated hydrochloric acid -62-(0.1 ml) and 10% p a l l a d i s e d charcoal (25 mg). The hydrogen uptake was complete i n 30 minutes. F i l t r a t i o n of the s o l u t i o n and evaporation of 14 1 the solvent gave 3-hydroxy-4-methoxy-N-(3-hydroxy-4-methoxyphen[l- C]ethyl-phenethyiamine hydrochloride which was r e c r y s t a l l i z e d from ethanol m.p. 230° (60 mg; 0.08 mc). It was shown i n r a d i o - i n a c t i v e runs to be i d e n t i c a l with an authentic sample (mixed m.p.). -63-BIBLIOGRAPHY 1. Manske, R. H. F. "The A l k a l o i d s " Vol. I I . Academic Press, New York. N.Y., 1952, Chapter 13. 2. Greshoff, M. Ber. 23, 3537 (1890). 3. Greshoff, M. Ber. deut. phar.. Ges. £, 215 (1899) 4. Chakravarti, S. N., Sitaraman, M. L. Venkatasubban, A. Chem. Abstracts 28, 1470 (1934). 5. Bochefontaine, A. and Rey, P. Gaz. Med. Paris (6), _3, 196 (1881). 6. Rey, P. J . Therapeutique 10, 843 (1883). 7. Manske, R. H. F. "The A l k a l o i d s " Vol. V. Academic Press, New York, N.Y. 1955, Chapter 46. 8 Lehman, A. J . J . Pharmacol. 60_, 69 (1937). 9. Cicardo, V. H. and Hug, E. Compt. rend. soc. b i o l . 126, 154 (1937). 10. Simon, I. Arch. Farmacol. sperim. 49_, 193 (1935). 11. Folkers, K. and Major, R. T. J . Am. Chem. Soc. 59_, 1580 (1937). 12. Folkers, K. J . Am. Pharm. Assoc. 27_, 689 (1938). 13. Folkers, K. and Unna, K. J . Am. Pharm. Assoc. 27_, 693 (1938); 28, 1019 (1939) . 14. Manske, R. H. F. "The A l k a l o i d s " Vol. VII. Academic Press, New York, N.Y., 1960, Chapter 11. 15. Carmack, M., McKusick, B. C. and Prelog, V. Helv. Chim. Acta 3£, 1601 (1951) . 16. Kenner, G. W., Khorana, H. G., Prelog, V. Helv. Chim. Acta 34, 1969 (1951) . 17. Boekelheide, V., Weinstock, J . , Grundon, M. F., Sauvage, G. L. and Agnello, E. J . J . Am. Chem. Soc. 75_, 2550 (1953). 18. Folkers, K. and Unna, K. J . Am. Pharm. Assoc. 62_, 1677 (1940). 19. Folkers, K., Koniuszky, F. and Shavel, J . J . Am. Pharm. Assoc. 66_, 1083 (1944). 20. Lapiere, C. "Contribution a l'Stude des alc a l o i d e s des Erythrine'es". Ligge, 1952. -64-21. Boekelheide, V. and Prelog, V. "Progress i n Organic Chemistry" Vol. I I I . Cook, J . W. Ed., Butterworths S c i e n t i f i c P u b l i c a t i o n , London, 1955, Chapter 5. 22. Koniuszky, F. and Folkers, K. J . Am. Chem. Soc. 72, 5579 (1950). 23. Sauvage, G. L. and Boekelheide, V. J . Am. Chem. Soc. 7_2> 2062 (1950). 24. Boekelheide, V. and Grundon, M. F. J . Am. Chem. Soc. 75, 2563 (1953). 25. Lapiere, C. and Robinson, R. Chem. and Ind. (London) 30_, 650 (1951). 26. Grundon, M. F. and Boekelheide, V. J . Am. Chem. Soc. 74, 2637 (1952). 27. Grundon, M. F. and Boekelheide, V. J . Am. Chem. Soc. 75_, 2537 (1953). 28. Grundon, M. F., Sauvage, G. L. and Boekelheide, V.. J . Am. Chem. Soc. 75, 2541 (1953). 29. Boekelheide, V. and Agnello, E. J . J . Am. Chem. Soc. 73, 2286 (1951). 30. Weinstock, J . and Boekelheide, V. J . Am. Chem. Soc. 75_, 2546 (1953). 31. Prelog, V., McKusick, B. C. Merchant, J . R., J u l i a , S. and Wilhelm, M. Helv. Chim. Acta 39, 498 (1956). 32. Manson, A. J . and Wiesner, K. Chem. and Ind. (London) 641 (1953). 33. Belleau, B. J . Am. Chem. Soc. 75.» 5 7 6 5 (1953). 34. Belleau, B. Chem. and Ind. (London) 410 (1956). 35. Mondon, A. Angew. Chem. 68, 578 (1956). 36. Belleau, B. Can. J . Chem. 35, 651, 663 (1957). 37. Boekelheide, V., Miiller, M., Jack, J . , Grossnickle, T. T. Chang, M. J. Am. Chem. Soc. 81_, 3955 (1959). 38. Miiller, M., Grossnickle, T. T. and Boekelheide, V. J . Am. Chem. Soc. 81_, 3959 (1959) . 39. Boekelheide, V. and Morrison, G. C. J . Am. Chem. Soc. 80_, 3905 (1958). 40. Mondon, A. Ber. 92, 1461 (1959). 41. Mondon, A., Hasselmeyer, G. and Zander, J . Ber. 92, 2543 (1959). 42. Mondon, A. Ann. 628, 123 (1959). 43. Mondon, A. and Hansen, K. F. Tetrahedron Letters 14, 5 (1960). -65-44. Mondon, A., Zander, J . Menz, Hans-Udo Ann. 667, 126 (1963). 45. Mondon, A. Tetrahedron 19, 911 (1963). 46. Mondon, A. Ber. 92, 1472 (1959). 47. Prelog, V., Langemann, A., Rodig, 0. and Ternbah, M. Helv. Chim. Acta 42, 1301 (1959). 48. Sugasava, S. and Yoshikawa, H. Chem. Pharm. B u l l . (Tokyo) 8_, 290 (1960). 49. Mondon, A. and Nestler, H. J . Angew Chem. 76_, 651 (1964). 50. ' Nowacki, W. and Bonsma, G. F. Z. Kryst. 110, 89 (1958). 51. Boekelheide, V. and Wenzinger, R. J . Org. Chem. 29, 1307 (1964). 52. H i l l , R. K. and Schearer, W. R. J . Org. Chem. 27_, 921 (1962). 53. Hanson, A. W. Proc. Chem. Soc. 52 (1963). 54. Boekelheide, V. and Chang, Mildred Y. J . Org. Chem. 29, 1303 (1964). 55. Gadamer, J . Arch. Pharm. 249, 680 (1911). 56. Gadamer, J . Arch. Pharm. 249, 498 (1911). 57. Schopf, C , T h i e r f e l d e r , K, Ann. 497, 22 (1932). 58. Robinson, R. "The S t r u c t u r a l Relationships of Natural Products" Clarendon Press, Oxford, 1955. 59. van Tamelen, E. E. Fortschr. Chem. Org. Naturstoffe 19_, 242 (1961). 60. Battersby, A. R. Quart. Rev. 15, 259 (1961). 61. Mothes, K. and Schiitte, H. R. Angewandte Chemie, International Ed. i n English 2_, 341 (1963) . 62. Mothes, K. and Schiitte, H. R. Angewandte Chemie Internation Ed. i n English 2, 441 (1963). 63. Battersby, A. R. Proc. Chem. Soc. 189 (1963). 64. Scott, A. I. Quart, Rev. 19_, 1 (1965). 65. Robinson, R. J . Chem. Soc. I l l , 762 (1917). 66. Schopf, C. Angew. Chem. 6j_, 31 (1949). 67. Barton, D. H. R. and Cohen, T. " F e s t s c h r i f t A. S t o l l " Birkhauser, Basle, 117 (1957). -66-68. Barton, D. H. R. Proc. Chem. Soc. 293 (1963). 69. Erdtman, H. and Wachtmeister, C. A. " F e s t s c h r i f t A. S t o l l " Birkhauser, Basle, 144 (1957). 70. Barton, D. H. R. and Kirby, G. W. J . Chem. Soc. 806 (1962). 71. Barton, D. H. R., Kirby, G. W., Taylor, J . B. and Thomas, G. M. J . Chem. Soc. 4545 (1963). 72. Battersby, A. R. and Brown, T. H. Proc. Chem. Soc. 85 (1964). 73. Battersby, A. R. and Brown, R. T., Clements, J . H. and Iverach, G. G. Chem. Communications, (London), No. 11 230 (1965). 74. Franck, B., Blaschke, G.. and S c h l i n g l o f f , G. Angewandte Chemie, International Ed. i n English, .3, 192 (1964). 75. Blake, J . , T r e t t e r , J . R. and Rapoport, H. J . Am. Chem. Soc. 87, 1398 (1965). 76. Witkop, B. and Goodwin, S. Experientia 377 (1952). 77. Wenkert, E. Experientia L5, 165 (1959). 78. Prelog, V. Angew. Chem. 69_, 33 (1957). 79. Bu'Lock J . D. and Harley-Mason, J . J . Chem. Soc. 2248 (1951). 80. Forbes, J . J . Chem. Soc. 513 (1956). 81 Cromartie, I. T., Harley-Mason, J . and Wannigama, D. J . P. J . Chem. Soc. 1938 (1958). 82. Robinson, R. and Sugasawa, S. J . Chem. Soc. 3163 (1931). 83. Lange, N. A. and Hambourger, W. E. J . Am. Chem. Soc. 53, 3865 (1931). 84. Gensler, W. J . and Samour, C. M. J . Am. Chem. Soc. 73_, 5555 (1951). 85. J a i n , M. K. J . Chem. Soc. 2203 (1962). 86. Battersby, A. R. Binks, R., Francis, R. J . , McCaldin, D. J . and Ramuz, H. J . Chem. Soc. 3600 (1964). 87. Bhacca, N. S. and Williams, D. H. "Applications of n.m.r. Spectroscopy i n Organic Chemistry" Holden-Day, 1964. 88. Fieser, L. F. J . Am. Chem. Soc. 52_, 5204 (1930). 89. Brown, B. R. and Todd, A. R. J . Chem. Soc. 1280 (1954). -67-90. Harley-Mason, J . J . Chem. soc. 200 (1953). 91. Wilcox, M. E., Wyler, H., Mabry, T. J . and Dreiding, A. S. Helv. Chim. Acta. 48, 252 (1965). 92. Shrinivasan, V. R. and Turba, F. Biochemische Z e i t s c h r i f t 327, 362 (1956). 93. Wong, E. J . Org. Chem. 2_8, 2336 (1963). 94. Brown, H. C. and Heim, P. J . Am. Chem. Soc. 86, 3566 (1964). 95. Vogel, A. I. " P r a c t i c a l Organic Chemistry" Longmans, Green and Co., London, (1957). 96. Battersby, A. R. Francis, R. J . , Ruveda, E. A. and Staunton, J . Chem. Communications, (London), No. 5 89 (1965). 97. Jackson, A. H. and Martin, J . A. Chem. Communications, (London), No. 8 142 (1965). 98. Battersby, A. R. and Herbert, R. B. Chem. Communications, (London), No. 11 228 (1965) . 99. Haynes, L.,J., Stuart, K. L., Barton, D. H. R. and Kirby, G. W. Proc. Chem. Soc. 280 (1963). 100. Barton, D. H. R., Kirby, A. J . (Mrs.) and Kirby G. W. Chem. Communications No.3 52 (1965). PART II AN ATTEMPTED IN VITRO DEMETHYLATION OF LANOSTEROL -68-INTRODUCTION The, i n v e s t i g a t i o n of the biogenesis of cholesterol (18) i s the most exhaustive and thorough of a l l the work which has been done on the bio-1 2 3 4 synthesis of s t e r o i d s , and the t o p i c has been well reviewed. ' ' ' The knowledge of t h i s c e n t r a l l y important precursor of other steroids i s v i t a l and i t s biogenesis i l l u s t r a t e s a "general" mechanism for s t e r o l formation. The f i r s t work which demonstrated the chemical nature of t h i s bio-5 synthesis began over 20 years ago when i t was found that s t e r o i d a l sub-stances are constructed from numerous small molecules, and that acetate i s 6 7 the source of the carbon atoms of s t e r o l s i n yeast and in animal t i s s u e . ^'^ B l o c h ' s ^ pioneering work showed that a c e t i c acid molecules are i n -corporated i n t o the structure of c h o l e s t e r o l according to a d e f i n i t e pattern. This work took on even greater i n t e r e s t with the r e a l i z a t i o n that the biogenesis of ch o l e s t e r o l and steroid s i s a small part of a vast biosynthetic panorama that now includes a l l terpenes and derived sub-stances. 4 Subsequent w o r k , 1 1 , 1 2 ' 1 3 ' 1 4 , 1 5 , 1 6 which stands as a b r i l l i a n t achievement, has located the o r i g i n of every carbon atom of cholesterol i n e i t h e r the carboxyl or methyl carbon of acetate, by su i t a b l e degrada-tions of the s t e r o l and measurement of the r e l a t i v e isotope incorporation into both nuclear and side-chain moieties. Thus, a l l of the carbon atoms in c h o l e s t e r o l can be derived from acetate. Besides acetate, many other low molecular weight compounds were tested f o r the p o s s i b i l i t y f o r t h e i r 17 incorporation i n t o c h o l e s t e r o l . It appeared that the e f f i c i e n c y of the many suggested precursors was proportional to t h e i r a b i l i t y to y i e l d the two carbon fragment, acetate. 18 19 20 The observations ' ' that hydroxymethylglutaryl CoA (3) i s -69-syrithesized from acetoacetyl CoA (2) and acetyl CoA (1) and could be 21 incorporated into cholesterol (18), r a i s e d the p o s s i b i l i t y that hydroxy-methylglutarate (3) might be a key intermediate. The search for intermediates i n the transformation of acetate into cholesterol met with l i t t l e success, u n t i l 1956 when the Merck group 22 23 24 i s o l a t e d mevalonic acid (4). ' ' The obvious s i m i l a r i t y to hydroxy-21 methylglutarate (3), which i s transformed into cholesterol only poorly, 25 prompted i n v e s t i g a t i o n of the possible r o l e of mevalonic acid (4) i n ch o l e s t e r o l biosynthesis. The r e s u l t s indicated that t h i s compound was capable of being incorporated into c h o l e s t e r o l in very high y i e l d . Sub-26 sequent study showed that a carboxyl group i s l o s t as carbon dioxide early i n the sequence of reactions and also conversion to squalene (11) 27 28 with high e f f i c i e n c y was observed under anaerobic conditions. ' This indicated that decarboxylation occurred, apparently at the s i x carbon 29 atom l e v e l , to give a f i v e carbon atom active intermediate. Ferguson has established that hydroxymethylglutaryl CoA (3) can be converted to 30 mevalonic acid (4) i n yeast and Knauss has recently demonstrated the formation of mevalonic acid (4) from acetate (1) i n l i v e r t i s s u e , so an excellent case f o r t h e i r intermediacy has now been made. 31 Tchen working with mevalonic acid (4) i n yeast preparations found an enzyme which transforms the acid to 5-phosphomevalonic acid (5). In addition he found that ATP was necessary to transform the 5-phosphomevalon-32 33 ate (5) to squalene (11). Lynen and Bloch have described the further reactions which convert 5-phosphomevalonate (5) to 5-pyrophosphomevalonate (6) and isopentenyl pyrophosphate (7), the i'soprenoid intermediate that evaded i s o l a t i o n f o r a long time. The condensation of d i m e t h y l a l l y l pyrophosphate (8), an isomerization product of isopentenyl pyrophosphate -70-34 (7), and isopentenyl pyrophosphate (7) has been reported by Lynen to r e s u l t i n geranyl pyrophosphate (9). This compound can then condense with an ad d i t i o n a l molecule of isopentenyl pyrophosphate (7) to y i e l d farnesyl pyrophosphate (10). The reductive dimerization of two molecules of farnesyl pyrophosphate (10), t a i l to t a i l , to y i e l d squalene (11) has 32 35 beendemdnstrated i n l i v e r and yeast. ' The incorporation of acetate into terpenes and t h e i r d e r i v a t i v e s , such as s t e r o i d s , d i f f e r s at an early stage from that f o r the biosynthesis of ^•cetogehins. 4 The acetogenins include compounds b i o g e n e t i c a l l y derivable by the acetate hypothesis and exclude the terpenes, which, although u l t i m a t e l y derived from acetate, are themselves a homogeneous family A r i s i n g from l i n e a r combination of ispprenoid u n i t s . Whereas the aceto-genins are formed by a l i n e a r l i n k i n g of acetate u n i t s , the terpenes are generated by conversion of acetate to a branched-chain intermediate, i s o -plentenyl pyrophosphate (7), the b i o l o g i c a l isqprene u n i t . The series of r'eactiohs as now postulated f o r the biogenesis of squalene (11) are given i n Figures 1 and 2 (P = P0 3 ). The intermediates i n t h i s b i o s y n t h e t i c sequence p r i o r to mevalonate (4) are capable of interconversion to many 4 other substances. However, the formation of mevalonate (4) i s an i r -r e v e r s i b l e process, and mevalonate once formed has e s s e n t i a l l y only one biochemical r o l e , the production of isoprenoid substances. Its discovery was, therefore, one of the important break-throughs i n terpene biosynthesis. As early as 1926 i t was suggested 3 5 simply on the bases of s t r u c t u r a l s i m i l a r i t i e s , that squalene (11) i s b i o g e n e t i c a l l y r e l a t e d to the s t e r o i d s . 36 The early experiments did in d i c a t e some involment of squalene (11) i n 37 chol e s t e r o l (18) biosynthesis, however , i t was not u n t i l 1953 that Bloch -71-CHCOSCoA Figure 1. The Biosynthesis of isopentenyl Pyrophosphate (7). Figure 2. Polymerization of Isopentenyl Pyrophosphate (7). -73-and h i s collaborators reinvestigated the matter using isotopes and were able to demonstrate that squalene (11) i s indeed converted into cholesterol (18). The c y c l i z a t i o n of squalene (11) to lanosterol (13) (see Figure 3) i s of p a r t i c u l a r . i n t e r e s t because of the intermediacy of the l a t t e r i n the pathway of s t e r o i d biogenesis. The r o l e of squalene as an obligatory pre-cursor of c h o l e s t e r o l , and the mechanism of i t s conversion to lanosterol proposed on purely s t r u c t u r a l and t h e o r e t i c a l grounds, now has firm experi-38 mental support. Woodward and Bloch i n t h e i r suggested mechanism implicated lanosterol (13) as an intermediate i n cholesterol biosynthesis, and t h e i r proposal i s consistent with a l l of the isotope d i s t r i b u t i o n data and enzy-4 mo l i g i c a l evide.nce that has been obtained so f a r . The conversions of squa-39 40 lene (11) to lanosterol (13) and of lanosterol (13) to ch o l e s t e r o l (18) haye been demonstrated. Very i n t e r e s t i n g l y , the formation of squalene from 41 i t s precursors does not require oxygen, the c y c l i z a t i o n of squalene to 42 lanoste r o l and cho l e s t e r o l does require oxygen.. Squalene (11) c y c l i s e s ' @ concertedly by a process i n i t i a t e d ; by atmospheric oxygen probably as the HO 42 43 cation on C-3 ' to give the hypothetical intermediate (12) , which can 44 45 undergo two 1,2-methyl s h i f t s ' to y i e l d l a n o s t e r o l (13). With the attainment of lanosterol (13), the biosynthesis of the Str u c t u r a l feature most c h a r a c t e r i s t i c of the s t e r o i d s , the cyclopentano-perhydrophenanthrene skeleton, has been achieved. For the f i n a l t ransfor-mation of lanost e r o l (13) to cho l e s t e r o l (18) there remains only the removal of three methyl groups and appropriate a l t e r a t i o n of the two o l e f i n i c centers :j.n the lanosterol (13). There are undoubtedly many enzymatic reactions involved i n these transformations, but to date only some of them have been elucidated i n d e t a i l . One of the two pathways by which the changes i n lano-s t e r o l (13) are eff e c t e d has as i t s terminal step the saturation of the -74-4 sidechain double bond (see Figure 3). The alternate route involves e a r l i e r reduction of the side-chain double bond. There i s of course the d i s t i n c t p o s s i b i l i t y of intermediates crossing from one of these pathways to the other. Some steps i n the conversion of lanosterol (13) to c h o l e s t e r o l (18) have recently been established. The conversion of desmosterol (17) to cholesterol 46 47 48 (18) has been shown by Stokes. Bloch and Schwenk have demonstrated the conversion of b i o s y n t h e t i c a l l y l a b e l l e d zymosterol (16) to cholesterol (18). The three methyl groups at C-4 and C-14 of lanosterol (13) are l o s t as carbon dioxide i n a sequence which requires oxygen and i s b e l i v e d to 49 50 51 involve oxidation of the methyl groups to carboxyl groups ' ' i n the f i n a l formation of cholesterol (18). Another intermediate has been i s o l a t e d i n the sequence between lanosterol (13) and c h o l e s t e r o l (18), and by a series 49 51 of ingenious biochemical and chemical methods i t has been shown to pos-sess the structure of 14-desmethyllanosterol (14) . It gives r i s e i n turn to c h o l e s t e r o l , thus, i t i s established that the 14a i s the f i r s t of the three extra methyi groups to be l o s t i n lan o s t e r o l (13). The exact order of events r e s u l t i n g in loss of the C-4 methyl groups i s not known with c e r t a i n t y , but there i s some argument f o r the existence of a hypothetical C-4-monomethyl intermediate compound (15), based upon the 4 recently i d e n t i f i e d 4-monomethyl steroids i n Nature. The scheme f o r the conversion of squalene (11) to cholesterol (18), as we now understand i t , i s shown i n Figure 3. It must be emphasized that the above pathways were the r e s u l t of an i n t r a c e l l u l a r " i n vivo" process. This process made extensive use of enzymes to synthesize the desired product. There i s an increasing amount of i n t e r e s t in the mechanism of these " i n vivo" reactions and, by u t i l i z i n g the same -75-Figure 3. Scheme for the formation of Cholesterol (18) from Squalene -76-intermediates as the c e l l , organic chemists now attempt to reproduce these reactions under, laboratory conditions 'in v i t r o ' without enzymes. This i s possible since the enzyme lowers the a c t i v a t i o n energy of the reaction by acting as a c a t a l y s t . In v i t r o , these reactions which occur " i n v i v o " should thus only require stronger conditions. A successful reaction of 52 t h i s type i s , of course, a "biogenetic type" synthesis. The loss of the methyl groups in the l a n o s t e r o l - c h o l e s t e r o l intercon-version has been of considerable i n t e r e s t . Since C-18 and C-19 hydroxylated s t e r o l s have been found i n n a t u r e * ^ ' ^ ' ^ and C-19 hydroxysteroids are considered to be l i k e l y precursors of r i n g A nor-steroids, i t i s reason-able to assume that the f i r s t step i n the demethylation may be hydroxylation of the methyl groups. The hydroxylation of saturated carbon atoms that are unactivated i n the c l a s s i c a l sense i s of great importance. .Investi-gation of b i o l o g i c a l hydroxylation of steroids has indicated that the hydroxylation occurs by replacement of hydrogen without inversion of 57 58 59 configuration, ' ' i . e . , the hydroxyl group occupies the same s t e r i c l o c a t i o n as the hydrogen which i t replaces. This stereochemical r e s u l t i s f i n d i n g increasing analogy i n studies of e l e c t r o p h i l i c s u b s t i t u t i o n reac-tions at saturated carbon. C o r e y ^ has suggested e l e c t r o p h i l i c oxygen as the active agent, comparing the enzyme-oxygen complex to a peroxide or peracid. Microrganisms which e f f e c t s t e r o i d hydroxylation at a s p e c i f i c satu-rated carbon atom often produce 1,2-epoxides 1 from steroids in which that carbon i s part of an o l e f i n i c linkage, and i t has been pointed out^ 1 that the same enzyme may be involved i n both processes. As a r e s u l t the enzy-matic reagent has been compared to a peroxide or peracid and has been con--77-sidered as an e l e c t r o p h i l i c , non-radical species. Furthermore, i t has been established that the hydroxylation process does not involve hydration of an o l e f i n i c intermediate, but di r e c t incorporation of molecular oxygen into the reagent and hence i n t o the s t e r o i d and that metal ions and TPNH are i n v o l -62 ved, providing a d d i t i o n a l evidence f o r a peroxidic intermediate. Another reaction of importance i s the introduction, removal, and re-arrangement , of double bonds. The f i r s t may involve p r i o r hydroxylation and subsequent loss of water. Rearrangement of double bonds may i n some cases occur as a r e s u l t of series of oxidations and reductions, as i n the case of the movement of C-8 C-9 unsaturation of zymosterol (16) to C-5 C-6 i n c h o l e s t e r o l (18). In v i t r o f u n c t i o n a l i s a t i o n of non-active methyl groups, by intramole-cular attack by groups near these groups i n space, has been achieved by a var i e t y of methods. The problem posed an i n t e r e s t i n g challenge i n synthetic chemistry, magnified by the widespread occurrence of such transformations i n nature under the influence of enzymes. The f i r s t reported f u n c t i o n a l i -sation of -inactive methyl groups was the use of the Lo e f f l e r - F r e y t a g r e a c t i o n * ' 3 ' ^ 4 , ^ to prepare conessine d e r i v a t i v e s as shown (19) - (21) (see Figure 4). The mechanism i s of the free r a d i c a l chain type,*' 5 and r a d i c a l abstraction by nitrogen i s favoured by the ju x t a p o s i t i o n of the two groups involved. The r a d i c a l decomposition of the i20-N-chloroamino s t e r o i d (19) i n a c i d s o l u t i o n l e d to a r i n g closure between C-18 and nitrogen. A s i m i l a r r a d i c a l a b s t r a c t i o n reaction i n v o l v i n g oxygen rather than nitrogen as the abstracting r a d i c a l , was the photolysis of C-20 ketosteroids which gave r i s e to cyclobutanol p r o d u c t s . ^ The p y r o l y s i s of 21-diazo-5a-pregnan-20-one (22) resulted i n the f o r --79-mation of a carbon-carbon bond between the C-18 methyl group and C-21 (24) by carbene (23) i n s e r t i o n as shown. Photolysis of azides w i l l cause nitrene i n s e r t i o n i n a s i m i l a r manner, and use has been made of t h i s i n Barton's conessine s y n t h e s i s ^ 8 and Edward's diterpene a l k a l o i d s y n t h e s i s . ^ The action of lead tetraacetate on alcohols leads d i r e c t l y to high y i e l d s of tetrahydrofuran d e r i v a t i v e s , without the formation of a free car-bonium ion, when a l l the p a r t c i p a t i n g centers are f i x e d i n the arrangement 70 which favours hydrogen abstraction. The treatment of s t e r o i d a l alcohols such as (25) with lead tetraacetate w i l l give r i s e to c y c l i c ethers (27), and the reaction i s thought to involve the t r a n s i t i o n state (26). The reaction has been used to f u n c t i o n a l i s e the C-18 methyl group i n an aldo-71 72 sterone synthesis. ' Hypochlorites can be photolysed to give f u n c t i o n a l i s a t i o n of s u i t a b l y 70 73 placed i n e r t methyl groups, ' and lead tetraacetate treatment of hypo-h a l i t e s gives s i m i l a r r e s u l t s . ^ 4 The photolysis of s u i t a b l y constituted organic n i t r i t e s provokes an intramolecular exchange of the NO of the n i t r i t e residue with a hydrogen atom attached to a carbon i n the y - p o s i t i o n . The C-nitroso compounds thus formed can be i s o l a t e d as the oximes. This reaction has been used to f u n c t i o n a l i s e both C-18 and C-19 methyl groups i n steroids by a mechanism 75 76 as shown (28) - (31). ' The whole process i s believed to take place within a solvent cage. The reaction has been used i n a p a r t i a l synthesis 77 of aldosterone. The methods of f u n c t i o n a l i s i n g methyl groups by intramolecular abstrac-t i o n of hydrogen, corresponds to the t r a n s f e r of a hydrogen atom to an attacking free r a d i c a l i n the same molecule, and hence to a hydrogen s h i f t . - 8 0 -Th e most f r e q u e n t l y o b s e r v e d i n t r a m o l e c u l a r h y d r o g e n t r a n s f e r s a r e 1 ,5 s h i f t s . The most f a v o u r a b l e t r a n s i t i o n s t a t e f o r t h e h y d r o g e n t r a n s f e r i s t h a t o f a 6-membered r i n g i n t h e c h a i r f o r m and t h e s t e r i c r e q u i r e m e n t s f o r t h e s e r e a c t i o n s a r e more i m p o r t a n t t h a n t h e e n e r g e t i c o n e s . H y d r o g e n s h i f t s c o r r e s p o n d i n g t o 1 , 2 - and 1 , 3 - h y d r i d e s h i f t s o b s e r v e d w i t h c a r b o n i u m and 78 oxonium i o n s do n o t o c c u r w i t h o x y - r a d i c a l s . The methods o f f u n c t i o n a l i s i n g m e t h y l g r o u p s , summarised a b o v e , have a l l i n v o l v e d some s o r t o f f r e e r a d i c a l r e a c t i o n , and a r e t h u s n o t good a n a l o g i e s f o r C o r e y ' s ^ ^ model f o r b i o l o g i c a l h y d r o x y l a t i o n w h i c h i s presumed t o i n v o l v e a t t a c k o f c a t i o n i c o x y g e n . A c l o s e r a n a l o g y t o t h i s m o d e l , o f non r a d i c a l d i s p l a c e m e n t o f a l k y l h y d r o g e n by o x y g e n w h i c h c a n be r e g a r d e d as e s p e c i a l l y s i m i l a r t o s t e r o i d h y d r o x y l a t i o n , i s t h e r e a c t i o n o f d e c a l i n w i t h o z o n e . B o t h c i s - and t r a n s - d e c a l i n w i t h ozone g i v e c i s - and t r a n s - 9 -h y d r o x y d e c a l i n , r e s p e c t i v e l y , a s t e r e o s p e c i f i c s u b s t i t u t i o n p r o c e e d i n g w i t h 79 r e t e n t i o n o f c o n f i g u r a t i o n and i n v o l v i n g e l e c t r o p h i l i c o x y g e n . A n o t h e r c l o s e c h e m i c a l a n a l o g f o r t h i s t y p e o f b i o l o g i c a l r e a c t i o n s o f s t e r o i d s i s t h e f o r m a t i o n o f a c y c l i c e t h e r (34) f r o m a h y d r o p e r o x i d e t o s y l a t e ( 3 2 ) , 80 85 w h i c h i n v o l v e s a t t a c k on a c a r b o n u n a c t i v a t e d i n any c l a s s i c a l f a s h i o n . ' (32) (33) (34) -81-Thus 1,3,3-trimethylcyclohexane hydroperoxide (32) reacted with para-nitro-benzenesulphonyl chloride i n cold pyridine arid methylene chl o r i d e . Since 0-0 cleavage of the para-nitrobenzensulphonyl d e r i v a t i v e of the hydroperoxide i s probably h e t e r o l y t i c under the conditions used the change from hydroper-oxide to b i c y c l i c ether (34) i s considered proceeding v i a the c a t i o n i c t r a n s i t i o n state such (33). The mechanism, i s an intermediate between the extremes f o r attack of c a t i o n i c oxygen, e x c l u s i v e l y on hydrogen or exclusively on carbon. The b i c y c l i c ether has not been detected under d i f f e r e n t reaction conditions which are known to proceed v i a a r a d i c a l mechanism. One important feature of the r e a c t i o n i n t h i s case i s i t s s t e r i c f a c i l i t a t i o n , owing to the proximity of the methyl group and hydroperoxide function. This reaction i s i n t e r e s t i n g as a chemical counterpart of enzymatic hydroxylation at a saturated carbon which also appears to involve e l e c t r o p h i l i c oxygen, and which proceeds by f r o n t a l displacement as implied by the intermediate (33). -82-DISCUSSION The f i r s t stage i n the conversion of lanosterol to c h o l e s t e r o l , " i n vivo", i s the removal of the 14a-methyl group. There are several p o s s i b i -l i t i e s f o r the removal of the extra methyl carbons: d i r e c t loss as a methyl group; preliminary oxidation to a hydroxy-methylene group and loss as form-aldehyde; furt h e r oxidation to a formyl group and loss as formic acid; or, f i n a l l y , complete oxidation and loss -as CO2. From the evidence p r e s e n t e d ^ the l a s t of these p o s s i b i l i t i e s seems a c t u a l l y to occur. The l o c a t i o n of the 8,9-double bond i n lanos t e r o l (13) _is a t t r a c t i v e as an a c t i v a t i n g fea-ture f o r the decarboxylation of the C-14 carboxylic a c i d (35). Such an activated decarboxylation usually r e s u l t s i n the migration of the double bond, which would i n t h i s case form the 8(14)-unsaturation (36). It i s possible that t h i s substance i s indeed an intermediate but that i t r a p i d l y rearranges to the thermodyriamically more stable isomer (37) or that:, i n the reaction, C-9 acquires an e l e c t r o p h i l e from an enzyme surface that i n i t i a t e s the loss of CO2 as shown. (35) (36) (37) -83-The enzyme-steroid complex i s then cleaved by attack of a proton at C-14 producing the i s o l a t e d intermediate (37). Such a mechanism w i l l lead to a product with the correct stereochemistry about the D/E r i n g juncture. To achieve " i n v i t r o " oxidation of the 14a-methyl, a s u i t a b l e group would have to be introduced i n a 1,3-diaxial r e l a t i o n s h i p to i t . The 8(9)-double bond makes the 9a and 7a positions p a r t i c u l a r l y l a b i l e , and these are, therefore, the best p o s i t i o n s f o r s u b s t i t u t i o n with a view to removal of the 14a-methyl group as they are both i n a 1,3-diaxial r e l a t i o n s h i p to 81 i t . Since molecular oxygen i s involved i n b i o l o g i c a l hydroxylation a reaction i n v o l v i n g molecular oxygen would be a c l o s e r " i n v i t r o " analogy.. 78 Autoxidation of o l e f i n s i s known to give a l l y l i c hydroperoxidation, 82 and lanosterol would be expected to oxidize i n the C-7 and C - l l positions, Autoxidation of lanosterol i s known, however, to give the 73 and 113 83 hydroperoxides, and t h i s would be useless as a means of obtaining the 7a-hydroperoxide. Photosensitized oxygenation of o l e f i n i c double bonds 84 gives a l l y l i c hydroperoxides i n which the double bond has rearranged. The i n i t i a l l y formed hydroperoxide usually survives the reaction conditions and can be i s o l a t e d and reduced to the a l l y l i c alcohol by any of a number of ways. When applied to o l e f i n s having nearby functional groups, t h i s oxygenation reaction has considerable p o t e n t i a l as a synthetic t o o l , and also y i e l d s information on various factors that might influence s e n s i t i z e d 84 87 84 photochemical processes. .' The proposed mechanism of t h i s reaction involves e x c i t a t i o n o f the s e n s i t i z i n g dye on absorption of l i g h t energy, and the formation of a l a b i l e photosensitizer-oxygen complex, which i n turn 86 oxidizes the substrate. The findings suggest a c y c l i c mechanism f o r the olefin-oxygen combination, a f t e r the system has been s u i t a b l y energized. -84-The sensitizer-oxygen complex breaks down to y i e l d excited oxygen (O2*) which w i l l react with the double bond (38) to give the rearranged a l l y l i c hydroperoxide (40)via the s i x membered t r a n s i t i o n state (39). (38) (39) (40) Several detailed variants for such a c y c l i c process can be envisaged according to whether i t i s concerted or not, and depending on the nature of the bonds i n the t r a n s i t i o n states or intermediates, and the extent ( i f any) of p a r t i c i p a t i o n by the s e n s i t i z e r when the oxygen attacks. This photosensitized oxygenation reaction i s also of special interest as a possible pathway for b i o l o g i c a l oxidations, and various steroidal 84 86 87 olefins have been hydroperoxidized i n t h i s manner. ' ' The attack has usually been from the less hindered a-face of the molecule and the bonds formed and broken (C-0 and C-H respectively) are c i s to each other, with no implication about the timing of the events or the extent of p a r t i -cipation by, the s e n s i t i z e r . The result with steroids and simpler olefins 87 indicated that the reaction i s subject to s t e r i c hindrance. In dihydro-lanosterol (41) the C-8 position i s more hindered than the C-9 position due to the proximity of the 14a-methyl group and thus 9a-hydroperoxylanost-7-ene-38-ol (42) would be the expected product of photosensitized oxidation. -85-When dihydrolanosterol was oxygenated i n the presence of l i g h t and haemato^ porphyrin,and the resultant product was reduced and acetylated, 36-acetoxy-88 lanosta-7,9(ll)-diene (48) was obtained as the only s o l i d product. This r e s u l t might indi c a t e that 9-hydroperoxidation had i n fac t occurred since the 9a-alcohpl would be r e a d i l y dehydrated under a c e t y l a t i o n conditions. However, the photo-oxygenation of dihydrolanosteryl acetate (47) gave the same r e s u l t , and attempts to i s o l a t e the 9a-hydroperoxide were unsuccessful. Dihydrolanosterol (41) could be used to f u n c t i o n a l i z e the C-14 methyl group i n s i t u under the influence of a powerful leaving group such as the para-nitrobenzenesulphonyl group, (see Figure 5). The C-9 C-14 methyl ether (43) would be the expected product by analogy to the Corey reaction (see Introduction)and t h i s ether could be oxidized to the unsaturated lactone (44) which would open to the acid (45) from which the C-14 group would be r e a d i l y l o s t by decarboxylation. In t h i s sequence, the C-14 methyl group would be removed, and the double bond would be s h i f t e d to the 7 p o s i t i o n . Both of these are steps i n the i n vivo conversion of lanosterol to c h o l e s t e r o l , and an,in v i t r o r e a l i z a t i o n of these steps v i a a serie s of oxidation reactions, would e s t a b l i s h a mile-stone i n an area t r a d i t i o n a l l y considered to be a sector of biochemistry, namely, the f i e l d of enzyme mechanism. Previous 88 attempts of C-14 methyl a c t i v a t i o n by photosensitized oxygenation were unsuccessful, as the expected C-9 C-14 c y c l i c ether could not be i s o l a t e d or detected i n the reaction product. When i n the hope of f u n c t i o n a l i z i n g the C-14 methyl group v i a the C-7 C-14 methyl ether 3B-acetoxylanost-8-ene-7a-hydroperoxide (50) was treated with para-nitrobenzensulphonyl chloride i n p y r i d i n e , no reaction occurred, the only compound i s o l a t e d was the s t a r t i n g material. The l a b i l t y or i n t r a c t a b i l i t y of any 9ot-hydroperoxide formed i n the -86-Figure 5. Proposed scheme for the removal of the 14o(-methyl group in dihydrolanosterol ( 4 l ) . -87-photo-oxygenation reaction might be overcome by carrying out the reaction i n the presence of para-nitrobenzensulphonyl chloride so that the peroxide might react with the 14o(-methyl group. Therefore the i n s i t u photo-oxygen-ation reaction was- repeated. Dihydrolanosteryl acetate (47) (see Figure 6) was oxygenated i n pyridine i n the presence of l i g h t , haematoporphyrin, and para-nitrobenzensulphonyl chloride. The reaction product was worked up the usual way, and a semi-solid was obtained which was chromatographed on an alumina colum. On developing the chromatographic column, f i r s t , petroleum ether eluted unchanged para-nitrobenzensulphonyl chloride followed by 33-acetoxy-lanosta-7,9(ll)-diene (48). Next petroleum ether' eluted a compound which was designated as IP1 m.p. 141-144°.. Its i n f r a r e d spectrum had peaks at 1730, 1240 (acetate) and 823 cm \ the u l t r a v i o l e t absorption was at 209 my ( e 8960). This compound was found to be i d e n t i c a l to that obtained, and not i d e n t i f i e d , i n a previous 88 study (Infrared, U l t r a v i o l e t spectrum and mixed m.p.). In subsequent f r a c t i o n s on e l u t i o n with petroleum ether and petroleum ether-benzene, 33-acetoxylanost-8-ene-7-one (49) and 33-acetoxylanost-8-ene-7a-hydroperoxide (50) were obtained r e s p e c t i v e l y . Since a l l y l i c hydroper-88 oxidation had yi e l d e d the 7a-hydroperoxide, i t was pos s i b l e that the l a t t e r compound had been formed by rearrangement of the f i r s t formed 9a-hydroperoxide. The rearrangement of t e r t i a r y to secondary hydroperoxides 89 90 had been reported, ' and the mechanism requires retention of configuration. The compound IP1 analyzed c o r r e c t l y f o r the required c y c l i c ether -(43), and the mass spectroscopic molecular weight of 484 i s i n agreement with the correct analysis f o r C32H52O3. Since i t had no carbonyl or hydroxyl + A c O A c O (48) 0 2 , h v sensitizer N O o <47) A c O ( + I P 1 ' C 3 2 H 5 2 ° 3 s o 2 c i O (49) + A c O ^ > Figure 6, Photosensitized oxygenation of d 1 ^ 0 ^ " ^ ^ ! ^ ^ 6 ( 4 ? ) ' " the presence of para-nitrobenzenesulphonyl c h l o n d e . in -89-88 functions, the compound was presumably an ether. An e a r l i e r study showed that treatment of t h i s compound with chromium t r i o x i d e i n a c e t i c acid gave 36-acetoxylanost—8-ene-7-one.(49), and :reduction with potassium iodide i n a c e t i c acid gave 3B-acetoxylanosta-7,9(ll)-diene (48) (see Figure 7). Oxidation under conditions which had y i e l d e d lactones from s t e r o i d a l c y c l i c ethers f a i l e d to give products with lactone.absorption i n the i n f r a r e d . On treatment with boron t r i f l u o r i d e etherate compound IP1 gave two products. The f i r s t one,of these was an.enone C 3 2 H 5 2 O 3 , supported by Infrared and U l t r a v i o l e t s p e c t r a l data. The n.m.r. showed no o l e f i n i c proton, so the enone double bond was t e t r a s u b s t i t u t e d . The second product from the boron t r i f l u o r i d e treatment of the ether IP1 was a k e t o l , substantiated by i t s i n f r a r e d spectrum. The U l t r a v i o l e t spectrum was transparent- The a l c o h o l i c grouping was considered to be secondary i n View of a proton at T 6.8 i n the n.m.r spectrum. This proton was coupled with two protons on an adja-cent carbon atom and was considered to be a x i a l since the quartet had s i m i l a r spin-spin coupling to the 3a a x i a l proton at x 5.51. Since t h i s proton i s a x i a l , the a l c o h o l i c group must be e q u a t o r i a l . At t h i s point none of the various hypothetical structures which are possible has f i t t e d a l l of the p h y s i c a l and chemical data a v a i l a b l e f o r compound IP1. Therefore, i t was decided to tackle the problem by X-ray methods. Since the compound i s presumably an ether but not the expected c y c l i c ether (43), i t was hoped that i t might.be a s u i t a b l e intermediate which can be f u n c t i o n a l i z e d i n subsequent steps f o r the removal of the 14a methyl group. The compound IP1 was r e a d i l y brominated by pyridine perbromide to give the dibromo-derivative which analyzed c o r r e c t l y f o r C32 H52°3 B r2• Its U l t r a v i o l e t spectrum was transparent, and the Infrared spectrum showed absorptions at 1745 and 1240 cm * (acetate). C r O I P 1 , C 3 2 H 5 2 0 3 A c O K I P y r - B r > A c O (51) igure 7. Chart showing the reactions of compound IP1 with chromium t r i o x i d e potassium iodide and pyridine perbromide. -91-The X.-ray c r y s t a l l o g r a p h i c study was c a r r i e d out i n t h i s Department. The c r y s t a l s of the dibromo-derivative are orthorhombic, a_ = 26.03,., b_ = 9 . 8 8 ^ , c = 12.26. A, Z = 4 space group P2 12 12 1. The i n t e n s i t i e s of about 2400 r e f l e c t i o n s were measured on a G.E. spectrogoniometer with s c i n t i l l a -t i o n counter, using CuKa r a d i a t i o n . The two bromine p o s i t i o n s were deter-mined by Patterson methods and a l l carbon atoms were located on three suc-cessive three-dimensional electron-density d i s t r i b u t i o n s . P o s i t i o n a l and i s o t r o p i c temperature parameters, were re f i n e d by four cycles of least squares; a further s i x cycles of a n i s o t r o p i c least squares completed the refinement g i v i n g a f i n a l R_ value of 13.3%. The absolute configuration was determined by the anomalous dispersion method. The dibromo-derivative.of compound IP1 i s 3g-acetoxy-7 a,llcrdibromo-lanostane-8a,9a-epoxide (51) . Steroid r i n g A i s in the normal c h a i r form and ring D has a h a l f - c h a i r conformation. ' The epoxide prevents rings B and C from adopting the chair form. The bond lengths and valency angles are normal, and the intermolecular separations correspond to Van der Waals i n t e r a c t i o n s . In summary, the- oxidation product IP1 i s an ether and we f e e l strongly that i t i s a d i s u b s t i t u t e d 1,2-epoxide i n view of the protons i n the 7x region of the n.m.r. spectrum. The formation of a ketone-secondary alcohol with boron t r i f l u o r i d e etherate would also indicate that the ether termini are very probably secondary, and the structure (52) can be assigned tenta-t i v e l y which would f i t a l l the phy s i c a l at hand. The n.m.r. spectrum i n deuterochloroform could not be evaluated with c e r t a i n t y . However, i n benzene s o l u t i o n , where a solvent s h i f t was observed, -92-a b e t t e r r e s o l u t i o n was obtained and assignments were r e a d i l y made as follows: The o l e f i n i c proton at C - l l was assigned to a quartet at T5.15 (area = 1 H). This proton i s coupled with the two proton, on C-12. The 3a-axial proton on the same carbon as the acetate function occurred at T5.48 with J . , . = 8 c.p.s. and J . , . .,= 3 c.p.s. as a x i a l - a x i a l r a x i a l - e q u a t o r i a l 1 expected (area = 1 H). The p a i r of doublets at x6.95 and at T7.15 were assigned to the two epoxide protons (area = 1 H and J = 3.5 c.p.s., respec-t i v e l y ) . A doublet at T7.55 ( J = 5 c.p.s., area = 1 H) was assigned to the proton at C-8, and the abnormal downfield s h i f t may be a t t r i b u t e d to the deshielding by the double bond. A complex pattern of l i n e s occurred between x7.6-9.5>where i n d i v i d u a l assignments are not p o s s i b l e . F i n a l l y , i t i s c l e a r from the above that a most unusual rearrangement, not encountered so f a r , must occur i n the bromination of compound IP1. It i s d i f f i c u l t to conceive of a d i s u b s t i t u t e d 1,2-epoxide i n the lano s t e r o l skeleton from which the dibromide (51) can be derived, and which also f i t s a l l the chemical and ph y s i c a l data a v a i l a b l e . -93-EXPERIMENTAL Melting points were determined on a Kofler block and are uncorrected. U l t r a v i o l e t spectra were measured on a Cary 14 spectrophotomer and i n f r a r e d spectra were taken on a,Perkin Elmer Model 137B spectrophotometer. Nuclear magnetic resonance (n.m.r.) spectra were recorded at 60 Mc/s on a Varian A60 instrument. The. l i n e positions or centers of m u l t i p l e t s . a r e given i n the Ti e r s T scale with reference to tetramethylsilane as the i n t e r n a l standard. Alumina G (according to Stahl) plates were used for t h i n layer chromatography ( T . L . C ) . The alumina used f o r column chromatography was Woelm neutral reagent, deactivated with 6% of water. The mass spectrum was determined on a A..E.I. MS9 Double Focusing Mass Spectrometer. Elemental microanalyses were performed by Mrs. C. Jenkins of t h i s Department, and by Dr. A. Bernhardt and h i s associates of the Max Planck I n s t i t u t e , Mulheim, Ruhr, West-Germany. The n.m.r. and mass spectrometric determinations were done by Mrs. A. Brewster and Mr. G. Bloss of t h i s Department r e s p e c t i v e l y . The X-ray c r y s t a l l o g r a p h i c study was c a r r i e d out by Dr. J . T r o t t e r and by Mr. J . K. Fawcett i n t h i s Department. Lanosteryl Acetate 30 g o f Lanosterol (K § K) was acetylated, with 50 ml of a c e t i c anhydride (reagent).and 80 ml of dry pyridine (reagent) at room temperature overnight. The excess of reagent was removed under reduced pressure on the steam bath and the residue was dissolved i n ether, washed with d i l u t e hydrochloric acid and then several times with water and dried (anhydrous -94-magnesium sulphate). Evaporation of the ethereal s o l u t i o n under reduced pressure gave 28.5 g of l a n o s t e r y l acetate which was r e c r y s t a l l i z e d from ethanol.. m.p. 123-124°. Infrared spectrum i n Nujol: 1739 (OAc) and 1240 (OAc) cm 1 . Dihydrolanosteryl Acetate (47) Lanosteryl acetate.(25 g) was hydrogenated i n ethyl acetate (200 ml) containing 1 g of platinum oxide, under hydrogen atmosphere with s t i r r i n g f o r 24 hours. A f t e r removal o.f\ the; c a t a l y s t by f i l t r a t i o n the dihydrolano-s t e r y l acetate, was r e c r y s t a l l i z e d to constant melting point from ethyl acetate, m.p. 119-120°, ( l i t e r a t u r e m . p . 120-121°). -Anal. Found: C, 81.5%; H, 11.3%. Calc. f o r C32H51+02.: C, 81.7%, H, 11.5%. T..L.C. one spot. = .87 i n benzene-chlorofrom (1:1). Infrared spectrum i n Nujol: 1750 ( s ) , 1242 ( s ) , 1035 ( s ) , 1010 (m), 905 (w) and 870 (w) cm"1. Oxygenation of Dihydrolanosteryl Acetate i n the presence  of Para-nitrobenzene Sulphonyl Chloride 10 g of dihydrolanosteryl acetate (3-/?-acetoxylanost-8-ene), 4.9 g of para-nitrobenzene sulphonyl chloride and 150 mg of haematoprophyrin were dissolved i n 260 ml of dry p y r i d i n e (reagent) contained i n a 500 ml. round-bottom f l a s k . The s o l u t i o n was i r r a d i a t e d with three G. E. 20 watt 250 v o l t fluorescent tubes f o r 8 days while oxygen was bubbled through the s o l u t i o n . The pyridine was removed under reduced pressure and the s o l i d residue was dissolved in.ether. The ether s o l u t i o n was subsequently -95--extracted with water, d i l u t e sulphuric acid and water. The ethereal s o l u t i o n was dried (anhydrous magnesium sulphate), and the solvent evapo-rated under reduced pressure to give 16 g of a semi-solid which was chromato-graphed on alumina (450 g). T.L.C. on the crude product showed 7 spots i n benzene-chloroform (1:1), and the product l i b e r a t e d iodine from sodium iodide s o l u t i o n . The organic material was added to the top of the column (22 x 2.5 cm diameter) by d i s s o l v i n g i t i n a mixture of petroleum ether (80-110°)-benzene (17:2). On e l u t i o n the following f r a c t i o n s were then obtained consecutively: E l u t i o n with 80 ml of petroleum ether (40-60°) gave unchanged para-nitrobenzene-sulphonyl chloride. E l u t i o n with 250 ml of petroleum ether (40-60°) gave.3^-acetoxylano-s t a - 7 , 9 ( l l ) - d i e n e which was c r y s t a l l i z e d from acetone as needles (658 mg). m.p. 161-164°. U l t r a v i o l e t spectrum i n ethanol:X 237, 246 and 255 m . r r max Infrared spectrum i n Nujol: 1745 (s),1240 ( s ) , 1030 (m), 980 (m), 905 (w), 875 (w) and 820 (w) cm This compound gave no depression i n melting point by admixing i t , and had an i d e n t i c a l i n f r a r e d spectrum with an authen-i 88 t i c sample. E l u t i o n with 500 ml of petroleum ether (40-60°) gave a s o l i d which c r y s t a l l i z e d from acetone as needles (95 mg). m.p. 141-144°. This compound was designated IP1, and was found to be i d e n t i c a l to that obtained e a r l i e r 88 by another group of workers (mixed m.p., Infrared and U l t r a v i o l e t Spectrum. [<*]D = +161 i n ethanol. U l t r a v i o l e t spectrum i n e t h a n o l : X m a x 209 (6 8960). Infrared spectrum i n Nujol: 1730 ( s ) , 1240 ( s ) , 1095 (w), 1070 (m), 1050 (w) 1030 (m), 1010 (w), 975 (w), 960 (w) and 823 (m) cm"1. T.L.C. showed one spot. R^= 0.61 i n benzene-chloroform (1:1). Mass spectrum showed s i g n i f i -cant peaks at m/e = 484 (M +), 483 (M-l), 469 (M-15), 441 (M-43), 424 (M-60), -96-371, 329 (M-155), 311, 289, 247, 231, 208, 207, 193 and 161. n.m.r. sign a l s ; given i n T u n i t s , spectrum obtained in benzene: mu l t i p l e t centered at 5.18, quartet centered at 5.4 correspondign to the 3a-axial proton on the same carbon as the acetate function, doublet centered at 6.96 (J = 3.5 c.p.s.), doublet centered at 7.15 (J = 3.5 c.p.s.) doublet centered at 7.52 (J = 5 c.p.s.), and a complex pattern of li n e s between 7 .8 -9 .5 . E l u t i o n with 1000 ml of petroleum ether ( 4 0 - 6 0 ° ) y i e l d e d nothing. E l u t i o n with 500 ml of petroleum ether ( 8 0 - 1 1 0 ° ) gave traces of c r y s t a l s . E l u t i o n with 1750 ml of petroleum ether ( 8 0 - 1 1 0 ° ) gave 3B-acetoxylan-ost -8-ene -7— one which c r y s t a l l i z e d from petroleum ether ( 4 0 - 6 0 ° ) as needles (1.32 g). m.p. 1 4 7 - 1 5 0 ° . U l t r a v i o l e t spectrum i n ethanol: X 254 my. Infrared spectrum i n Nujol: 1740 ( s ) , 1650 ( s ) , 1580 (s) , 1240 ( s ) , 1080 (w), 1030 (m), 1010 (m), 975 (m) and 900 (w) cm"1 T.L.C. showed one spot. Rp= 0.64 i n benzene-chloroform (1 :1 ) . This compound was found to be iden-88 t i c a l to that of an authentic sample (mixed m.p., i n f r a r e d spectrum). E l u t i o n with 1500 ml of petroleum ether ( 8 0 - 1 1 0 ° ) y i e l d e d 300 mg of o i l . E l u t i o n with 300 ml of petroleum ether ( 8 0 - 1 1 0 ° ) gave nothing. E l u t i o n with petroleum ether (80-110°)-benzene (2:1) gave 3/?-acetoxy-lanost-8-ene-7o<-hydroperoxide which c r y s t a l l i z e d from ethyl acetate as needles (310 mg). m.p. 1 7 4 - 1 7 5 ° . Infrared spectrum i n Nujol: 3400 ( s ) , 1730 ( s ) , 1275 ( s ) , 1040 (m) 1020 (m), 980 (m), 930 (w) and 860 (w) cm-1. T. L.C. showed one spot. R^ = 0.37 i n benzene-chloroform (1 :1 ) . The com-pound had only end absorption i n the u l t r a v i o l e t , and i t gave no depression -97-88 on a mixed melting point determination with an authentic sample. At t h i s point the development of the chromatographic columhwas discontinued. Bromination of Compound IP1 The compound IP1 (45 mg) was dissolved i n dioxan (8 ml) with pyridine perbromide (45 mg) and the s o l u t i o n was l e f t 4 hours at room temperature. The excess of bromine was removed by the addition of sodium thiosulphate s o l u t i o n . The aqueous suspension was extracted three times with ether and the combined ethereal extracts on evaporation, a f t e r drying (anhydrous magnesium sulphate), gave a s o l i d . The product was c r y s t a l l i z e d from acetone to give sturdy needles and dried, m.p. 199-200°. Anal. Found: C, 59.93%; H, 8.2%. Calc. f o r C 32H520 3Br2: C, 59.7%; H, 8.03%. Infrared spectrum i n Nujol: 1745 ( s ) , 1240 ( s ) , 1030 (m), and 905 (w) cm"1. The u l t r a v i o l e t spectrum was transparent. The dibromide of compound IP1 was i d e n t i f i e d by X-ray c r y s t a l l o g r a p h i c analysis as 3-B-acetoxy-7a,lla-dibromo-lanostane-8a,9a-epoxide. -98-BIBLIOGRAPHY 1. Popj&k, G. Ann. Rev. Biochem. 27_, 535 (1958). 2. Rieser, L. F. and Fiese r , M. "Steroids" Reinhold Publishing Corp., N.Y. 1959, 3. Wright, L. D. Ann. Rev. Biochem. 30, 525 (1961). 4. Richards, J . H. and Hendrickson, J . B. "The Biosynthesis of Steroids, Terpenes> and Acetogenins"'" W.A. Benjamin Inc.,, N.Y. 1964. 5. Rittfnberg, D. and Schoenheimer, R. J. B i o l . Chem. 121, 235 (1937). 6. Sonderhoff, R. and Thomas, H. Ann. 530, 195 (1937). 7. Bloch, K. and Rittenberg, D. J . B i o l . Chem. 145, 625 (1942). 8. Bloch, K. and Rittenberg, D. J . B i o l . Chem. 159, 45 (1945). 9. Bloch, K., Borek, E. and Rittenberg, D. J . B i o l . Chem. 162, 441 (1946) 10. L i t t l e , H. N. and Bolch, K. J . B i o l . Chem. 183, 33 (1950). .11. Wurschi J . , Huang, R. L. and Bloch, K. J . B i o l . Chem. 195, 439 (1952). 12. Cprnforthi J . W., Hunter, G. D. and Popjak, G. Biochem. J . 54_, 590 (1953). 13. Cprnforth, J . W. Hunter, G. D. and Popjak, G. Biochem. J . 54, 597 (1953). 14. Cprnforth, J . W., Gore, I. Y.and Popjak, G. Biochem. J . 65, 94 (1957). 15. Bloch, K. Helv. Chim. Acta. 36, 1611 (1953). 16. Daub en, W. G. and Takemura, K. H. J . Am. Chem. Soc. 75_, 6302 (1953). 17. BernfeldV P. "Biogenesis of Natural Compounds" Pergambri Press N.Y. 1963, Chapter 4. 18. Rabinowitz, J . L. and Gurin, S. J . B i o l . Chem. 208, 307 (1954). 19. Rudney, H. J . Am. Chem. Soc. 76, 2595 (1954).. 20. Rudney, H. Federation Proc. 15_, 342 (1956). 21. Bloch, K., Clark. L. C. and Harary, I. J . B i o l . Chem. 211, 687 (1954). 22. Skeggs, H. R., Wright, L. D., Cresson, E. L. MacRae, G. D. E. Hoffman, C. H., Wolf, D. E. and Folkers, K. J . B a c t e r i o l . 72, 519 (1956). -99-23. Wright, L. D., Cresson, E. L. Skeggs, H. R., MacRae, G. D. E., Hoffman, C. H., Wolf, D. E; and Folkers, K. J . Am. Chen. Soc. 78, 5273 (1956). 24. Wolf, D. E. Hoffman, C. H., A l d r i c h , P. E., Skeggs, H. R., Wright, L. D. and Folkers, K. J . Am. Chem. Soc. 78, 4499 (1956); 79, 1486 (1957). 25. Tavormina, P. A. Gibbs, M. H. and Huff, J . W. J . Am. Chem. Soc. 7£, 4498. (1956). 26. Tavormina, P, A. and Gibbs, M. H. J . Am. Chem. Soc. 78_, 6210 (1956) 27. Cornforth, J . W., Cornforth, R. H., Popjak, G. and Gore, I. Y. Biochem. J . 69^ , 146 (1958). 28. D i t u r i , F., Gurin, S. and Rabinowitz, J . L. J . Am. Chem. Soc. 79_, 2650 (1957). 29. Ferguson, J . , Durr, I. F. and Rudney, H. Proc. Natl. Acad. S c i . U. S. , 45_, 499 (1959) . 30. Knauss, H. J . , Porter, J . W. and Wasson, G. J . B i o l . Chem. 234, 2835 (1959). 31. Tchen, T. T. J . B i o l . (hem. ^ 33, 1100 (1958). 32. Lynen, F., Eggerer, H., Henning, U. and Kessel I. Angew. Chem. 70, 738 (1958). 33. Chaykin, S., Law, J . , P h i l l i p s , A. H., Tchen, T. T. and Bloch, K. Proc. Natl. Acad. S c i . U.S., 44, 998 (1958). 34. Lynen, F., Agranoff, B. W., Eggerer, H., Henning, U. and Moslein, E.M. Angew. Chem. 71_, 657 (1959) . 35. Heilbron, I. M., Kamm, E. D. and Owens, W. M. J . Chem. S o c , 1630 (1926). 36. Channon, H. J . Biochem, J . 20_, 400 (1926) . 37. Langdon, R. G. and Bloch, K. J . B i o l . Chem. Soc. 200,135 (1953). 38. Woodward, R. B. and Bloch, K. J . Am. Chem. Soc. 75_, 2023 (1953). 39. Tcheh, T,. T. and Bloch, K. J . Am. Chem. Soc. 77_, 6085 (1955). 40. Clayton, R. B. and Bloch, K. J . B i o l . Chem. 218, 319 (1956). 41. Bucher, N. L. R. and McGarrahan, K. J . B i o l . Chem. 222, 1 (1956). 42. Tchen, T, T. and Bloch, K. J . B i o l . Chem. 226, 921, 931, (1957). 43.. Eschenimoser, A., Ruzieka, L., Jeger, 0. and A r i g o n i , D. Helv. Chim. -100-Acta. 38, 1890 (1955). 44. Maudgal, R. K., Tchen, T. T. and Bloch, K. J . Am. Chem. Soc. 80_, 2589 (1958). 45. Cornforth, J . W., Cornforth, R. H., P e l t e r , A., Horning, M. G. and Popjak, G. Proc. Chem. Soc. 112 (1958). 46. Stokes, W. M. Hickey, F. C. and F i s h , W. A. J . B i o l . Chem. 232, 347 (1958). 47. Johnston,J. D. and Bloch, K. J . Am. Chem. Soc. 79, 1145 (1957). 48. Alexander, G. J . and Schwenk, E. Arch. Biochem. Biophys. 66_, 381 (1957). 49. Gautschi, F.. and Bloch, K. J.A.C.S. 79, 684 (1957). 50. Olson, J r . , J . A., Lindberg, M. and Bloch, K. J . B i o l . Chem. 226, 941 (1957). 51. Gautschi, F. and Bloch, K. J . B i o l . Chem. 233,1343 (1958). 52. van Tamelen, E. E. Fortschr. Chem. Org. Naturstoffe 19, 242 (1961). 53. Loke, K. H. , Watson, E. J . D. and Marrian, G. F. Biochim. Biophys. Acta. 26, 230 (1957). Biochem. J . 71_, 43 (1959). 54. Neher, R. and Wettstein, A. Helv. Chim. Acta. 3£, 2062 (1956). 55. von Euw, J . , Meystre, Ch., Neher, R., Reichstein, T. and Wettstein, A. Helv. Chim. Acta. 41_, 1516 (1958) . 56. Ehrenstein, M., Johnson, A. R., Olmsted, P. C , Vi v i a n , V. I., Wagner, M. A., Neumann, H. C. J . Org. Chem- 15., 264 (1950); 1_6, 335 (1951). 57. Hayano, M., Gut, M., Dorfman, R. I., Sebek, 0. K. and Peterson, D. H. J. Am. Chem. Soc. 80_, 2336 (1958). 58. Corey, E. J . , Gregoriou, G. A. and Peterson, D. H. J . Am. Chem. Soc. 80, 2338 (1958). 59. Bergstrom, S., Lindstredt, S., Samuelson, B., Corey, E. J . and Gregoriou, G. A. J . Am. Chem. Soc. 80, 2337 (1958). 60. Corey, E. J . and Gregoriou, G, A. J . Am. Chem. Soc. 81_, 3127 (1959). 61. Bloom, B. M. and S h u l l , G. M. J . Am. Chem. Soc. 77, 5767 (1955). 62. Talalay, P. Physiol. Rev. 37, 362 (1957). 63. Corey, E. J . and He r t l e r , W. R. J . Am. Chem. Soc. 80, 2903 (1958). -101-64. Buchschacher, P., Kalvoda, J., Arigoni, D. and Jeger, 0. J. Am. Chem. Soc. 80, 2905 (1958). 65. Wawzonek. S. and Thelen, P. J. J. Am. Chem. Soc. 72_, 2118 (1950). 66. Buchschacher, P., Cereghetti, M., Wehrli, H., Schaffner, K. and Jeger, 0. Helv. Chim. Acta. 42, 2122 (1959). 67. Greuter, F . , Kalvoda, J. and Jeger, 0. Proc. Chem. Soc. 349 (1958). 68. Barton, D. H. R. and Morgan, Jr., L. R. J. Chem. Soc. 622 (1962). 69. Apsimon, J. and Edwards, 0. E. Can. J. Chem. 40_, 896 (1962). 70. Heusler, K. and Kalvoda, J. Angew. Chem. International Ed. in English '3, 525 (1964). 71. Heusler, K. Kalvoda, J,, Meystre, Ch., Wieland, P., Anner, G., Wettstein, A., Cainelli, G., Arigoni, D. and Jeger, 0. Helv. Chim. Acta. 44, 502 (1961). 72. Cainelli, G. Mihailovic, M. Lj., Arigoni, D. and Jeger, 0. Helv. Chim. Acta. 42, 1124 (1959). 73. Ahtar, M. and Barton, D. H. R. J. Am. Chem. Soc. 8_3, 2213 (1961) . 74. Heusler, K., Kalvoda, J., Anner, G. and Wettstein, A. Helv. Chim. Acta. 46, 352, 618 (1963). 75. Barton, D. H. R., Beaton, J. M. Geller, L. E. and Pechet, M. M. J. Am. Chem. Soc. 83, 4076 (1961). 76. Nussbaum, A. L. and Robinson, C. H. Tetrahedron 17_, 35 (1962). 77. Barton, D. H. R. and Beaton, J. M. J. Am. Chem. Soc. 83, 4083 (1961). 78. Davies, A. G. "Organic Peroxides" Butterworths, London, 1961. 79. Durland, J. R. and Adkins, H. J. Am. Chem. Soc. 61_, 429 (1939). 80. Corey, E. J. and White, R.W. «J. Am. Chem. Soc. 80, 6686 (1958). 81. Hayano, M.and Dorfman, R* I. J. Biol. (hem. 211, 227 (1954). 82. Walling, C. "Free radicals in solution" Wiley, 1957. 83. Horn, D. H. S< and Use, D. J. Chem. Soc. 2280 (1957). 84. Schenck, G. 0. Angew. Chem. 69_, 579 (1957). 85. Sneen, R. A. and,Matheny, N . P. J. Am. Chem. Soc. 86, 3905 (1964). -102-86. Nickon, A. and Ba g l i , J . F. J . Am. Chem. Soc. 83, 1498 (1961). 87. Nickon, A. and Mendelson, W. L. Can. J . Chem. 43_, 1419 (1965); J. Am. Chem. Soc. 85, 1894 (1963). 88. Young, D. W. Ph.D. Thesis. The Univ e r s i t y of Glasgow 1963. The wri t e r i s indebted to Dr. D. W. Young f o r providing the authentic s t e r o i d samples. 89. Schenck, G. 0., Neumuller, 0. A. and E i s f e l d , W. Ann. 618, 202 (1958). 90. Lythgoe, B. and T r i p p e t t , S. J . Chem. Soc. 471. (1959). 91. The wr i t e r i s indebted to Professor J . T r o t t e r and to Mr. J . K; Fawcett f o r the X-ray c r y s t a l l o g r a p h i c study. 

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