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The chemistry of the Vinca alkaloids sitsirikine, catharanthine, and their derivatives Brown, Richard Talbot 1964

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THE CHEMISTRY OP THE VINOA ALKALOIDS ITSIRIKINE, CATHARANTHINE, AND THEIR DERIVATIV by Richard Talbot Brown B.A., Oxford U n i v e r s i t y , I960 B.Sc., Oxford Univ e r s i t y , 1961 A THESIS SUBMITTED IN PARTIAL FULFILMENT OP THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Chemistry We accept t h i s thesis as conforming to the required standard DEPARTMENT OF CHEMISTRY THE UNIVERSITY OF BRITISH COLUMBIA Jul y , 1964 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 of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the 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 reference and study* I f u r t h e r agree that per-m i s s i o n f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t . c o p y i n g or p u b l i -c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission-Department of The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada The University of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY RICHARD TALBOT BROWN B.A., University of Oxford, 1961 B.Sc, University of Oxford, 1961 MONDAY, AUGUST 10th, 1964, A T 2:00 P.M. IN ROOM 261, CHEMISTRY BUILDING of COMMITTEE IN CHARGE Chairman: I. McT. Cowan N. B a r t l e t t C. T. Beer G. G. S. Dutton J . P. Kutney C..A..McDowell R. E. I. Pincock External Examiner: Ernest Wenkert University of Indiana THE CHEMISTRY OF THE VINCA ALKALOIDS SITSIRIKINEy OATHARANTHINE> AND"THEIR DERIVATIVES ABSTRACT In part I of t h i s thesis are described the s t r u c t u r a l determinations of s i t s i r i k i n e , d i h y d r o s i t s i r i k i n e and i s o s i t s i r i k i n e , three new alk a l o i d s from Vinca rosea Linn. S i t s i r i k i n e , C 2 1 H 2 6 O 3 N 2 5 and dihydrositsirikine., ^21^28^3 N2» w e r e i s o l a t e d as an inseparable mixture, which was shown by hydrogenation studies to be com-prised of an o l e f i n and i t s dihydro d e r i v a t i v e . The formation of formaldehyde upon ozonisation of the mix-ture, and of propionic a c i d i n a modified Kuhn-Roth oxidation of d i h y d r o s i t s i r i k i n e demonstrated that, s i t -s i r i k i n e possessed a v i n y l group. Both s i t s i r i k i n e and d i h y d r o s i t s i r i k i n e gave mono-acetates, and the N.M.R. data indicated that primary hydroxyl groups were present i n the o r i g i n a l a l k a l o i d s . A methyl ester function suggested by spectral evidence Ttfas established by hydride reduction of d i h y d r o s i t s i r i -kine to a d i o l . Since the diol, y ielded an acetonide, i t was i n f e r r e d that d i h y d r o s i t s i r i k i n e possessed a hydroxy-ester u n i t . The U.V. spectrum of d i h y d r o s i t s i r i k i n e was character-i s t i c of an indole chromophore, which the mass spectrum showed to be part of a tetrahydro- (5-carboline system. Dehydrogenatioh afforded a compound with a fl a v o c o r y l i n e -type U.V. spectrum, and t h i s suggested that s i t s i r i k i n e was a r e l a t i v e of the t e t r a c y c l i c corynantheine class of al k a l o i d s . This was confirmed by conversion of dihydro-corynantheine i n t o d i h y d r o s i t s i r i k i n e . The structure of the r e l a t e d indole a l k a l o i d i s o s i t -s i r i k i n e , C21H26O3N2, was determined by a s i m i l a r series of reactions. Ozonolysis y i e l d e d acetaldehyde, which authenticated the ethylidene group indicated by the N.M.R. spectrum. A c e t y l a t i o n afforded a mono-acetate, whose N.M.R. spectrum suggested that i s o s i t s i r i k i n e had a primary hydroxyl function. A methyl ester was e s t a b l i -shed by hydride reduction to a d i o l , which formed an acetonide and hence showed the presence of a (^-hydroxy-ester unit i n the o r i g i n a l a l k a l o i d . Since dehydrogena-t i o n of d i h y d r o - i s o s i t s i r i k i n e yielded f l a v o c o r y l i n e , a t e t r a c y c l i c structure very s i m i l a r to that of s i t s i r i k i n e could be postulated f o r i s o s i t s i r i k i n e . Part II i s concerned with the chemistry of cleavamine, a s c i s s i o n product, of the Vinca a l k a l o i d catharanthine. Treatment of catharanthine with aqueous acid i n the pre-sence of a reducing agent, led to the i s o l a t i o n of descar-bomethoxycatharanthine, cleavamine and two epimeric dihydro-cleavamines. A t e n t a t i v e mechanism for the reaction i s proposed, which can account for the formation of these compounds .. Reduction of catharanthine i n g l a c i a l a c e t i c acid pro-vided carbomethoxy-dihydrocleavamine. Mercuric acetate oxidised t h i s compound to a mixture of two immonium ions, both of which underwent transannular c y c l i s a t i o n s . One of the ions gave the known Iboga a l k a l o i d s coronaridine and dihydrocathafanthine, whereas the other afforded pseudo-vincadifformine - a synthetic analogue of the known Vinca a l k a l o i d vincadifformine. The structure of pseudo-vincadifformine was determined by conversion into compounds which had U.V., I.R., N.M.R. and mass spectra completely analogous to the corresponding derivatives of vincadifformine. Similar transannular c y c l i s a t i o n s to.the above are pos-tulated i n the scheme advanced by-Wenkert for the biogenesis of Iboga and Aspidosperma a l k a l o i d s , and the s i g n i f i c a n c e of our r e s u l t s with regard to t h i s theory i s duscussed. The formation of coronaridine and dihydrocatharanthine i n the reaction constituted p a r t i a l syntheses of these alka-l o i d s , and the p o t e n t i a l use of transannular c y c l i s a t i o n s i n laboratory syntheses of Iboga and Aspidosperma a l k a l o i d s i s also considered. GRADUATE STUDIES F i e l d of Study: Chemistry Topics i n Organic Chemistry D.E. McGreer J.P. Kutney, R.E.I. Pincock Seminar i n Organic Chemistry J.P. Kutney Structure of Newer Natural Products J.P. Kutney Recent Synthetic Methods i n G.G.S. Dutton Organic Chemistry A. Rosenthal Related Studies: Topics i n Inorganic Chemistry N. B a r t l e t t W.R. Cullen Topics In Physical Chemistry J.A,R. Coope R.F. Snider, A.V. Bree PUBLICATIONS J.P. Kutney and R.I. Brown, The Structure of S i t s i r i k i n e - A new A l k a l o i d from Vinca rosea Linn, Tetrahedron L e t t e r s , No. 26, 1815 (1963) J.P. Kutney, R.T. Brown and E. Piers, The Synthesis of a Vincadifformine-Type Skelet v i a a Novel Transannular C y c l i z a t i o n Reaction, J . Am. Chem. S o c , 86, 2286 (1964) J.P. Kutney, R.T. Brown and E. Piers, The Synthesis of Iboga Al k a l o i d s v i a a Novel Transannular C y c l i z a t i o n Reaction, J . Am. Chem. S o c , 86,2287 (1964) A C KNOWLEDGEMENT S I w i s h t o e x p r e s s uiy g r a t i t u d e t o D r . J . P . K u t n e y f o r h i s c o n s t a n t h e l p a n d e n c o u r a g e m e n t d u r i n g t h e c o u r s e o f my r e s e a r c h . T h a n k s a r e due t o D r s . M. Gorman a n d N. E e u s s , L i l l y R e s e a r c h L a b o r a t o r i e s , f o r t h e g i f t o f t h e a l k a l o i d s w h i c h f o r m e d t h e b a s i s o f t h i s w o r k a nd f o r c o m m u n i c a t i o n o f t h e i r u n p u b l i s h e d r e s u l t s o n t h e c h e m i s t r y o f c a t h a r a n t h i n e . A l s o I w o u l d l i k e t o a c k n o w l e d g e w i t h t h a n k s t h e k i n d n e s s o f P r o f . C. D j e r a s s i , S t a n f o r d U n i v e r s i t y , a n d D r . P. K e b a r l e , U n i v e r s i t y o f A l b e r t a , i n r u n n i n g t h e mass s p e c t r a o f s e v e r a l c o mpounds. Abstract In part I of t h i s thesis are described the s t r u c t u r a l determinations of s i t s i r i k i n e , d i h y d r o s i t s i r i k i n e and i s o s i t s i -r i k i n e , three new a l k a l o i d s from Vinca rosea Linn. S i t s i r i k i n e , C21 H26°3 N2' a n d d i n y d r o s i t s i r i k i n e , C21 I i28°3 N2' w e r e i s o l a t e d as an inseparable mixture, which was shown by hydrogenation studies to be comprised of an ole-f i n and i t s dihydro d e r i v a t i v e . The formation of formaldehyde upon ozonisation of the mixture and of propionic acid i n a modified Kuhn-Roth oxidation of d i h y d r o s i t s i r i k i n e demonstrated that s i t s i r i k i n e possessed a v i n y l group. Both s i t s i r i k i n e and d i h y d r o s i t s i r i k i n e gave mono-acet-ates, and the N.M.R. data indicated that primary hydroxyl groups were present i n t h e o r i g i n a l a l k a l o i d s . A methyl ester function suggested by spe c t r a l evidence was established by hydride reduction of d i h y d r o s i t s i r i k i n e to a d i o l . Since the d i o l yielded an acetonide, i t was i n f e r r e d that d i h y d r o s i t s i r i k i n e possessed a {3-hydroxy-ester u n i t . The U.V. spectrum of d i h y d r o s i t s i r i k i n e was character-i s t i c of an indole chromophore, which the mass spectrum showed to be part of a tetrahydro-(2>-carboline system. Dehydrogena-t i o n afforded a compound with a flavocoryline-type U.V. spectrum,and t h i s suggested that s i t s i r i k i n e was a r e l a t i v e of the t e t r a c y l i c corynantheine class of a l k a l o i d s . This was confirmed by conversion of dihydrocorynantheine into dihydro-s i t s i r i k i n e . i v The structure of the r e l a t e d indole a l k a l o i d i s o s i t s i -r i k i n e , C21 H26°3 N2' w a s d e " t e r m i n e d a s i m i l a r series of reactions. Ozonolysis yielded acetaldehyde, which authenti-cated the ethylidene group indicated by the N.M.R. spectrum. A c e t y l a t i o n afforded a mono-acetate, whose N.M.R. spectrum suggested that i s o s i t s i r i k i n e had a primary hydroxyl function. A methyl ester was established by hydride reduction to a d i o l , which formed an acetonide and hence showed the presence of a ^-hydroxy-ester u n i t i n the o r i g i n a l a l k a l o i d . Since dehydro-genation of d i h y d r o - i s o s i t s i r i k i n e yielded f l a v o c o r y l i n e , a t e t r a c y c l i c structure very s i m i l a r to that of s i t s i r i k i n e could be postulated f o r i s o s i t s i r i k i n e . Part I I i s concerned with the chemistry of cleavamine, a s c i s s i o n product of the Vinca a l k a l o i d catharanthine. Treatment of catharanthine with aqueous acid i n the pre-sence of a reducing agent led to the i s o l a t i o n of descarbo-methoxycatharanthine, cleavamine and two epimeric dihydroclea-vamines. A tentative mechanism f o r the reaction i s proposed, v;hich can account for the formation of these compounds. Reduction of catharanthine i n g l a c i a l a c e t i c acid provided carbomethoxy-dihydrocleavamine. Mercuric acetate oxidised t h i s compound to a mixture of two immonium ions, both of which under-went transannular c y c l i s a t i o n s . One of the ions gave the known Iboga a l k a l o i d s coronaridine and dihydrocatharanthine, whereas the other afforded pseudo-vincadifformine — a synthe-t i c analogue of the known Vinca a l k a l o i d vincadifformine. V The structure of pseudo-viricadifformine was determined byconversion i n t o compounds which had U.V., I.R., N.M.R. and mass- spectra completely analogous to the corresponding d e r i -vatives of vincadifformine. S i m i l a r transannular c y c l i s a t i o n s to the above are postulated i n the scheme advanced by Wenkert for the biogene-s i s of Iboga and" Aspidosperma a l k a l o i d s , and the s i g n i f i c a n c e of our r e s u l t s with regard t o t h i s theory i s discussed. The formation of coronaridine and 'dihydrocatharanthine i n the r e a c t i o n constituted p a r t i a l syntheses of these a l k a l o i d s , and the p o t e n t i a l use of transannular c y c l i s a t i o n s i n labora-t o r y syntheses of Iboga and Aspidosperma a l k a l o i d s i s also considered. v i CONTENTS •Page GENERAL INTRODUCTION 1 PART I : The S t r u c t u r a l E l u c i d a t i o n of S i t s i r i k i n e , D i h y d r o s i t s i r i k i n e , and I s o s i t s i r i k i n e A. S i t s i r i k i n e and D i h y d r o s i t s i r i k i n e 11 B. I s o s i t s i r i k i n e 24 C. Other Work on S i t s i r i k i n e , D i h y d r o s i t s i r i k i n e and I s o s i t s i r i k i n e 32 D. The Biogenesis of Yohimbine, Corynantheine and Ajmaline Type A l k a l o i d s . 35 PART I I : Some Aspects of the Chemistry of Catharanthine and Cleavamine Introduction 44 A. The Catharanthine-Cleavamine Transformation 46 B. The Biogenesis of Strychnos, Iboga and Aspidosperma A l k a l o i d s 56 C. Transannular C y c l i s a t i o n s of Cleavamine Derivatives 66 ( i ) . Pseudo-vincadifformine and i t s Derivatives 69 ( i i ) Coronaridine and Dihydrocatharanthine 70 D. Discussion 81 EXPERIMENTAL 85 Part I Experimental Section I s o l a t i o n of S i t s i r i k i n e 85 S i t s i r i k i n e P i c r a t e 87 v i i Page S i t s i r i k i n e Acetate of D i h y d r o s i t s i r i k i n e oo D i h y d r o s i t s i r i k i n e P i c r a t e By D i h y d r o s i t s i r i k i n e Acetate 89 D i h y d r o s i t s i r i k i n e p-Bromobenaoate 69 Saponification o f D i h y d r o s i t s i r i k i n e 90 D i h y d r o s i t s i r i k i n e D i o l 93. Acetonide of D i h y d r o s i t s i r i k i n e D i o l y l Modified Kuhn-Roth O x i d a t i o n of D i b y d r o s . i t s i r i k i ne 92 Ozonisation o f S i t s i r i k i n e and I s o s i t s i r i k i n e 93 Lead Tetracetate Dehydrcgenation o f D i h y d r o s i u s i r i k i n e 9 4 Attempted Palladium Dehydrogenation of Tetradehydro-dihydrositsirikine Hydrochloride 95 P a l l a d i u m - c h a r c o a l Dehydrogenation o f Dihydro s i t s i r i k i n e 95 Palladium-charcoal Dehydrogenation o f D i h y d r o s i t s i r i k i n e Hydrobromide 97 Quinone Dehydrogenation of Compound B 98 D i h y d r o s i t s i r i k i n e O l e f i n i c Ester 99 Desoxy-dihydrositsirikine 99 Lithium Aluminium Hydride .Reduction of D i h y d r o s i t s i r i k i n e O l e f i n i c Ester 100 Dihydrocorynantheine 101 Desmethyl-dihydrocorynantheine 101 Synthesis of D i h y d r o s i t s i r i k i n e 102 I s o o i t s i r i k i n e 103 Acetonide of I s o s i t s i r i k i n e D i o l 104 D i h y d r o - i s o s i t s i r i k i n e 105 v i i i P a g e Lead Tetraeetate Oxidation of D i h y d r o s i t s i r i k i n e 106 Sodium Borohydride Seduction of Tetradehydro-d i h y d r o - i s o s i t s i r i k i n e 107 Palladium Dehydrogenation of I s o s i t s i r i k i n e Sulphate 107 Palladium Dehydrogenation of D i h y d r o - i s o s i t s i r i k i n e Hydrochloride 108 Part I I Experimental Section I s o l a t i o n of Cleavamine and Descarbornethoxy-catharanthine 110 4 "ot"-Dihydro cleavamine 111 Csrbomethoxy-4 , i^"-dihydrocleavamine 112 4"{5"-Dihydrocieavamine 113 Mercuric Acetate Oxidation of Carbomethoxy-4 "(3 "-dihydro cleavamine 114 Acid Hydrolysis of Pseudo-vincadifformine 116 Reduction of Pseudo-vincadifformine with Zinc and Sulphuric Acid 117 Epimerisation of Dihydro-pseudo-vincadifformine 119 REFERENCES 120 Figure 1 2 Following page 11 11 i x f i g ure Following page 5 13 6 16 7 19 8 19 9 25 10 69 11 f^ 12 70 GENERAL INTRODUCTION Interest i n the Apocynaceous plant Vinca rosea Linn. (Lochnera Reichb. or Catharanthus roseus G.Don) was stimulated by the observation that c e r t a i n preparations of the plant 1 2 exhibited a remarkable anti-tumour a c t i v i t y * . This discovery prompted a systematic f r a c t i o n a t i o n of extracts i n a search for the active p r i n c i p l e s free of extraneous substances. I n i -t i a l t e s t i n g indicated that the b i o l o g i c a l l y active e n t i t i e s were confined to the complex a l k a l o i d a l portion of the plant constituents, and a separation scheme was then devised which permitted the i s o l a t i o n of four active a l k a l o i d s — vincaleuko-b l a s t i n e (VLB) leurosine, l e u r o c r i s t i n e and leurosidine — i n "2. A addition to numerous other a l k a l o i d s of unknown structure ' . The experimental anti-tumour a c t i v i t y of these four a l k a l o i d s against the transplanted P-1534 leukemia i n mice has been 1 5 6 reported by the Canadian group of Noble, Beer and Cutts ' ' 7 8 and by workers at the L i l l y l a b o r atories ' . I t was also 1 5 found that VLB produced severe leukopenia i n rats ' , and, moreover, markedly i n h i b i t e d the growth of a transplanted Q human carcinoma i n the hamster cheek pouch . Reports on c l i n -i c a l t r i a l s of VLB and l e u r o c r i s t i n e have been presented by several g r o u p s 1 0 ' 1 1 ' 1 2 ' 1 3 . The e a r l i e s t chemical i n v e s t i g a t i o n of the plant was performed i n the l a t e 19th century by Greshoff 1^, who was only able to demonstrate the presence of a l k a l o i d a l material. - 2 -IK Cowley and Bennett "* i n 1928 succeeded i n i s o l a t i n g two c r y s t a l l i n e sulphates and a t a r t r a t e , but did not describe any chemical or physical properties, and i n 1953 French 16 workers reported an u n i d e n t i f i e d c r y s t a l l i n e a l k a l o i d . More recently, several groups have obtained known a l k a l o i d s : 1 7 1 7 18 1 7 ajmalicine , serpentine ' , akuammicine , tetrahydroalsto-18 1Q 20 nine , lochnerine , and reserpine „ In 1958 Kawat and co-2 1 workers i s o l a t e d two c r y s t a l l i n e and two amorphous alka l o i d s , and Noble, Beer and Cutts described VLB1. A major advance i n the phytochemistry of Vinca rosea Linn, was made when the L i l l y group3*'* devised an extraction scheme capable of separating the large number of a l k a l o i d s present i n the plant. The process consists e s s e n t i a l l y of separating the a l k a l o i d t a r t r a t e s which are soluble i n organic solvents from those which are in s o l u b l e ; a b r i e f outline of the procedure i s given i n Scheme 1. The constituents of each f r a c t i o n were further separated by chromatography on alumina and gradient pH extr a c t i o n , as shown i n Schemes 2 and 3. - 3 -Scheme 1 - Ext r a c t i o n Ground Whole Plant S k e l l y B Extract Defatted Drug 1) I) HC1(2N) Ammonia CHC15 1) 2) 2io T a r t a r i c Acid Benzene Sk e l l y Sol. (E) I "Acid" Benzene Extract 1) 2) Drug 2% T a r t a r i c Acid °2 H4 C 12 1) 2) Ammonia Benzene C 2H 4C1 2 S o l . U - ^ Acid Phase "A l k a l i n e " Benzene Extract i ! Ammonia CH 2H 4C1 2 1) 2) G 2H 4C1 2 Sol.(A) Drug 2% T a r t a r i c Acid C 2H 4C1 2 Marc EtOH Extract C 2 H 4 G 1 2 Sol.(B,) Phenolic A l k a l o i d s (C,D) Acid Phase 1) Ammonia 2) C 2 H 4 C 1 2 C 2H 4C1 2 Sol.(B) ] Aqueous Phase Sol. = Soluble A l k a l o i d s 1) NaOH (pH 11) 2) G2H 4 C 1 C2H * C 12 Sol.(P) - 4 -Scheme 2 - Fracti o n A [Fraction A Chromat, 011 CATHARANTHINE VINDOLININE (.2HC1) AJMALICINE VINDOLINE Sulphate Residues 1) 2) Free Bases Chromat. 0H-CHC1,(1:3) j CAROSIDINE Residues Gradient pH(4.4-5. VINCARODINE 0H-OHO15(1:1) LEUROSINE ISOLEUROSINE VLB(.H 2S0 4) 0H~CHC1,(1:1) i -> CAROSINE 0H-CHC15(1:3) Gradient pH Mother li q u o r s Chromat, (deact.) CHC1. PLEUROSINE CHC1. LOCHNERIDINE VIROSINE Residues Chromat. CHC1. Gradient pH(2.7-3.4) CATHARICINE pH 3.9-4.4 VINDOLINE Mother l i q u o r Chromat. CHC15-CH30H(99:1) NEOLEUROSIDINE pH 4.9-6.4 LEUROCRISTINE LEUROSIDINE Mother l i q u o r Gradient pH(3.4) NEOLEUROCRISTINE Chromat.= Chromatography on alumina deact. = deactivated (alumina.) 0H = Benzene - 5 -Scheme 3 - Fractions A,, (A+B), B, , B, F and E. Fractio n A-, Chromat. 0H 0fi - C H c l 5(3il) TETRAHYDROALSTONINE Vindoline Fraction B, Fractions A+B Modified Gradient pH(3.3) Amorphous Residues Chromat. Chromat. 0H-CHC13(3:1) 0H 0H-CHO15(3:1) 0H-CHC15(2:l) Ajmalicine Catharanthine Tetrahydro-serpentine Vindoline CATHARINE VINDOLICINE Fractio n B Chromat, g!!-CHCl, (3:1) 0H-CHC1- (1:1) \1 giaalicine LOCHNERINE PER] [VINE Mother l i q u o r H 2S0 4 SITSIRIKINE (.i-H2S04) Fractio n F CHOI. Chromat, CHC1, VINCAMICINE SERPENTINE ( .HNOj ) F r a c t i o n E r 0H Chromat, 0H-CHC13(3:1) LOCHNERICINE Tetrahydroalstonine Vindoline Table 1 Name Formula Fracti o n L i t . r e f . Akuammicine G20 H20°2 N2 B 17 ,29 Ajmalicine G21 H24°3 N2 17 Tetrahydroalstonine C21 H24°3 N2 A,E 18 Serpentine C21 H22°3 N2 F 17,18 Mit r a p h y l l i n e G21 H24°4 N2 A 29 Lochnerine C20 H24°2 N2 B 19 Ammocalline C19 H22 N2 B 29 Pe r i v i d i n e C20 H22°4 N2 A 29 Gavincine G20 H24°2 N2 A 29 Lochneridine C20 H24°3 N2 A 26 Perivine C20 H24°3 N2 B 3,23 Catharanthine G21 H24°2 N2 A 22,28 Lochnericine °21H24°2N2 E 22,24-Vindolinine °21H24°2N2 A 22 S i t s i r i k i n e G21 H26°3 N2 B 26 I s o s i t s i r i k i n e G21 H26°3 N2 B -Virosine G22 H26°4 N2 A 3,23 Vinosidine G22 H26°5 N2 A 29 Lochnerivine G24 H28°5 N2 A 29 Vindoline C25 H 3 2°6 N2 A,AX,E 22,27 Vin d o l i c i n e C25 H 3 2°6 N2 (A+B) 26 Leurosidine G41 H54°9 N4 A 29 Vincarodine C44 H52°10 N4 A 4 Catharine G46 H52°9 N4 (A+B) 4,26 Catharicine G46 H52°10 N4 A 4 Leurocristine G46 H54°10 N4 A 4,29 Table 1. (Cont.) Name Formula Fracti o n L i t . r e Pleurosine C46 H56 ° 1 0 N4 A 4 Neoleurocristine C46 H56 G 1 2 W4 A 4 Vincaleukoblastine C46 H58 ° 9 N4 A 1,25 Leurosine C46 H58°9 N4 A 25,25 Isoleurosine °46H60°9N4 A 26 Neoleuros±dine G48 H62°11 N4 A 4 Vindolidine C48 H64 ° 1 0 R4 A 4 Vincamicine B 26 Leurosidine A 4 Carosidine A 4 P e r i c a l l i n e B 29 Ammorosine A 29 Perosine B 29 Cavincidine B 29 Maandrosine B 29 Cathindine B 29 The a l k a l o i d s i s o l a t e d by the L i l l y group from various f r a c t i o n s are l i s t e d i n Table 1, which includes a l l those whose ch a r a c t e r i s a t i o n has been published up to A p r i l 1964. However, Dr.M.Gorman very recently indicated i n a private communication that over f i f t y a l k a l o i d s have now been i s o l a t e d from Vinca rosea Linn., so that even t h i s table i s already out of date. Of the a l k a l o i d s tabulated, the structures were already known f o r ajmalicine (1), t e t r a -hydroalstonine (1), serpentine (2), mitraphylline (3) and - 8 -akuammicine (4), and have since been determined for catha-r a n t h i n e 2 8 ^ ) , p e r i v i n e 3 0 ( 6 ) , v i n d o l i n e 2 ^ ( 7 ) , v i n d o l i c i n e 3 1 (8), v i n d o l i n i n e 3 2 ( 9 ) , l o c h n e r i n e 3 3 ( 1 0 ) , and l o c h n e r i d i n e 3 ^ (11). The structures of vincaleukoblastine (80) and leuro-68 c r i s t i n e ( v i n c r i s t i n e ) are discussed below. Me0 2C MeOj,C MeO^C (4) COnMe - 9 -(11) In Part I of t h i s thesis i s presented the evidence which led to the assignment of structures to s i t s i r i k i n e , d i h y d r o s i t s i r i k i n e and i s o s i t s i r i k i n e , three minor a l k a l o i d s i s o l a t e d from Vinca rosea Linn. Part I I discusses some aspects of the chemistry of cleavamine, an acid rearrange-ment product of the Vinca a l k a l o i d catharanthine, and the - 1 0 -use of cleavamine and i t s derivatives i n p a r t i a l synthese of Iboga and Aspidosperma-type a l k a l o i d s . - 11 -PART I The S t r u c t u r a l E l u c i d a t i o n of S i t s i r i k i n e ,  D i h y d r o s i t s i r k i n e and I s o s i t s i r i k i n e A. S i t s i r i k i n e and D i h y d r o s i t s i r i k i n e S i t s i r i k i n e was i s o l a t e d as a minor a l k a l o i d from Vinca Of. rosea Linn, by the L i l l y group . Chromatography of f r a c t i o n B (see Scheme 3) of the a l k a l o i d a l extract yielded several f r a c t i o n s from which perivine (6) was c r y s t a l l i s e d . The mother l i q u o r s and some of the following f r a c t i o n s were combined and converted to the sulphate s a l t s . A f t e r perivine sulphate had been removed by r e c r y s t a l l i s a t i o n from methanol, s i t s i r i k i n e sulphate was obtained as blades from ethanol, and analysed for. C 2-i^26 03 N2°^ H2 S 04' The free base was obtained only as an amorphous powder. On the basis of an in f r a - r e d comparison with -yohimbine, i t was suggested that 26 s i t s i r i k i n e might represent a new-yohimbine isomer Through the kind co-operation of Dr.M.Gorman, L i l l y Research Laboratories, Indianapolis, Indiana, U.S.A., we obtained a sample of s i t s i r i k i n e f o r further s t r u c t u r a l studies. Our preliminary work revealed that the o r i g i n a l a l k a l o i d was a mixture of at l e a s t three compounds, since on th i n - l a y e r chromatography a separation into three d i s t i n c t spots was observed. Several d i f f e r e n t p u r i f i c a t i o n techniques were applied without success — chromatography on alumina and s i l i c a g e l , sublimation, f r a c t i o n a l r e c r y s t a l l i s a t i o n of the s a l t s . F i n a l -l y , i t was found that a f t e r several r e c r y s t a l l i s a t i o n s of the base from acetone-petroleum ether, a material melting CM oo. -=^ —I-<0 o o o 10 Q O (/) UJ 00 Hen SITSIRIKINE ACETATE SOLVENT CD 3COCD 3 IH 4H 8 0 PPM (6) 4.7V 5.65 T 637f 9.02 f Figure 2 - 12 sharply at 181° was obtained. This showed only two spots on thi n - l a y e r chromatography and analysed f o r the acetone s o l -vate, C21 I I26°3 N2' G H3 C O CH 3. Attempts- to separate the two components were of no a v a i l , and indeed the mixture behaved as a homogeneous compound except on th i n - l a y e r chromato-graphy. The unsolvated a l k a l o i d was subsequently obtained from aqueous methanol as needles, m.p, 206-208° oC 2^-58° L -ID (MeOH), and analysed we l l f o r C2i H26°3 N2* T n i s molecular formula was supported by elemental analyses on the p i c r a t e , m.p. 226-228°(dec.) and f i n a l l y substantiated by a mass spectral molecular weight determination (354), The u l t r a - v i o l e t spectrum of s i t s i r i k i n e , with maxima at 226, 282 and 290 mjx indicated an unsubstituted indole chromophore. This was confirmed by signals i n the nuclear magnetic resonance (N.M.R.) spectrum (Figure 1) at 0.06T ( i n d o l i c NH) and i n the region 2.5-3,IT (four aromatic protons). A strong band i n the i n f r a - r e d spectrum at 1705 cm."1 was r e a d i l y a t t r i b u t e d to a carbonyl group and an ab-sorption at 3360 cm.""1 was compatible with the presence of NH and/or hydroxyl groups. In ad d i t i o n to a spike at 6.38T (CH^O), the N.M.R. spectrum of s i t s i r i k i n e displayed a two-proton m u l t i p l e t centred a t 6.IT that was possibly due to the methylene protons of a primary a l c o h o l i c function. The presence of a hydroxyl group was confirmed by the f o r -o r 126 © mation of a monoacetate, C^HggO^^, m.p. 198 , |^ oc.J -26 (MeOH), whose N.M.R. spectrum (Figure 2) was p a r t i c u l a r l y i n s t r u c t i v e . Apart from the expected signal at 8.02T due DIHYDROSITSIRIKINE SOLVENT CD 3 C0CD 3 3H 0 PPM(d) 289Y 6.2f 6.42V 9.07+ Figure 3 Figure 4 - 13 -to the a c e t y l group, the mu l t i p l e t present at 6 . I t i n the spectrum of the alcohol had s h i f t e d downfield and now appeared at 5.6T. This s h i f t of 0.5tupon a c e t y l a t i o n i s c h a r a c t e r i s t i c of primary alcohols, whereas the correspon-35 ding s h i f t f o r secondary alcohols i s about l t u n i t ^ . Besides the above-mentioned signals the N.M.R. spec-t r a of s i t s i r i k i n e and i t s acetate displayed a m u l t i p l e t centred at 4.7fdue to o l e f i n i c protons, which integrated for rather l e s s than two hydrogen atoms. A series of micro-hydrogenations was run on s i t s i r i k i n e and i t was found that only 0.6-0.7 mol. of hydrogen was taken up. Moreover, the product gave only one spot on t h i n - l a y e r chromatography whose Rf value corresponded to the smaller of the two spots exhibited by the o r i g i n a l a l k a l o i d . This evidence suggested that the two components of the mixture d i f f e r e d from each other merely by the presence of an o l e f i n i c bond i n one of the a l k a l o i d s . The unsaturated a l k a l o i d was named s i t s i r i -kine, whereas the corresponding dihydro d e r i v a t i v e w i l l be referred to as d i h y d r o s i t s i r i k i n e . This conclusion was fully-borne out by subsequent work. C a t a l y t i c hydrogenation on a lar g e r scale and r e c r y s t a l l i s a t i o n from acetone afforded solvated dihydro-s i t s i r i k i n e , m.p. 180°, which analysed f o r Cg-j^gO^^' CH^COCH^. Further r e c r y s t a l l i s a t i o n s from aqueous methanol gave the unsolvated a l k a l o i d , C ?, Hpft0,Np, m.p. 215 , |0(. -55^(MeOH). The N.M.R. spectrum of d i h y d r o s i t s i r i k i n e (Figure 3) showed a complete disappearance of the o l e f i n i c - 14 -proton absorption. A strong band at 1710 cm. i n the i n f r a -red spectrum of the reduced material excluded any conjuga-t i o n between the carbonyl group and the double bond i n s i t -s i r i k i n e , and the u l t r a - v i o l e t spectrum was unchanged with maxima at 226, 282 and 290 mji . Elemental analyses on the c r y s t a l l i n e p i c r a t e , m.p. 228-230°(dec.), acetate, m.p. 187°,foci 26-3i°(MeOH), and p-bromobenzoate, m.p. 174°, sup-D ported the formula assigned to d i h y d r o s i t s i r i k i n e , and f i n a l confirmation was obtained from a. mass spe c t r a l molecular weight determination (356). Evidence that the o l e f i n i c linkage i n s i t s i r i k i n e was i n fact a terminal double bond was provided by the appear-ance of a new C-methyl absorption at 9.07T i n the N.M.R. spectrum of the reduction product. This was corroborated when ozonolysis of the o r i g i n a l a l k a l o i d gave formaldehyde, i d e n t i f i e d by paper chromatography of i t s 2,4-dinitrophenyl-36 hydrazone^ . A conventional Kuhn-Roth determination on d i -h y d r o s i t s i r i k i n e 1 indicated 0.93fmol. C-methyl, while a 37 modified procedure yielded propionic acid and thus showed that the new C-methyl function was i n fact part of a C-ethyl group. These experiments established the presence of a v i n y l group i n s i t s i r i k i n e . However, i t was s t i l l necessary to explain why the o l e f i n i c proton absorption i n the N.M.R. spectrum of s i t -s i r i k i n e integrated f o r two rather than the three hydrogen atoms expected f o r a v i n y l group. The th i n - l a y e r chromato-graphy and microhydrogenation r e s u l t s had suggested that - 15 -the impurity present i n the o r i g i n a l a l k a l o i d was dihy-d r o s i t s i r i k i n e , and a close scrutiny of the N.M.R. spec-trum of the o r i g i n a l s i t s i r i k i n e (Figure 1) revealed a s l i g h t absorption at 9.02T i n t e g r a t i n g f o r about one hydrogen, whereas the mass spectrum indicated a small peak at m/e 356 i n addition to that at m/e 354. A Kuhn-Roth determination on the mixture showed 0.38 mol. C-methyl, and the modified method afforded propionic a c i d . From these r e s u l t s i t was deduced that the o r i g i n a l a l k a -l o i d was a mixture of s i t s i r i k i n e and d i h y d r o s i t s i r i k i n e i n an approximate r a t i o of 2 s i . Since d i h y d r o s i t s i r i k i n e was the only component which could be obtained pure, i t was used as the s t a r t i n g mate-r i a l i n a l l subsequent studies. Besides providing the information discussed above, the N.M.R. spectrum of d i h y d r o s i t s i r i k i n e (Figure 3) was very useful i n e s t a b l i s h i n g the-nature of the two oxygen functions present i n a d d i t i o n to the carbonyl group. In the region of 6.IX there was a two-proton absorption, a t -t r i b u t a b l e to hydrogen atoms attached to an oxygen-bearing carbon atom,which.upon a c e t y l a t i o n moved down to 5.6T« This p a r a l l e l e d the behaviour of the o r i g i n a l s i t s i r i k i n e and confirmed the presence of a primary a l c o h o l . The nature of the t h i r d oxygen atom was indicate d by a spike at 6.42X r e a d i l y assigned as before to the three protons of a methoxyl function. A Z e i s e l determination on di h y d r o s i t -s i r i k i n e showed the presence of one methoxyl group and gave - 16 -ample support to the N.M.R. designation. Since the u l t r a - v i o l e t and N.M.R. spectra excluded the p o s s i b i l i t y that the methoxyl was attached to the indole system, the presence of absorption bands at 1705 and 1165 cm.""1 i n the i n f r a - r e d region suggested that i t was part of a carbomethoxy group . An attempted sa p o n i f i c a -t i o n under mild conditions was unsuccessful, but a f t e r more d r a s t i c treatment an unsaturated acid was i s o l a t e d as the hydrochloride, m.p. 260-263°. The N.M.R. spectrum ( i n t r i f l u o r o a c e t i c acid) of t h i s substance showed loss of the methoxyl group. Reduction of d i h y d r o s i t s i r i k i n e with l i t h i u m alumi-nium hydride yielded a c r y s t a l l i n e d i o l , m.p. 203°, J 2^-3° (MeOH), which analysed for CgQHggOgNg. The in f r a - r e d spec-trum of the d i o l showed no carbonyl absorption, and the N.M.R.spectrum indicated a complete absence of the methoxyl s i g n a l . The presence of a carbomethoxy group i n di h y d r o s i t -s i r i k i n e was thus confirmed. Treatment of the d i o l with acetone containing p-toluenesulphonic acid afforded an acetonide, m.p. 105-109°, as shown by a six-proton N.M.R. si g n a l (gem-dimethyl) at 8.62f . Since both a l c o h o l i c groups were primary the formation of t h i s d e r i v a t i v e meant that the hydroxyl groups were i n a 1,3-relationship, and hence d i h y d r o s i t s i r i k i n e i t s e l f must contain a ^ -hydroxy-ester grouping. The N.M.R, spectrum of the acetonide was even more i n s t r u c t i v e i n that the s i g n a l at 6.25T , due to the methylene protons - 17 -on the two oxygen-bearing carbon atoms, was s p l i t into a doublet, thereby i n d i c a t i n g that there must be one proton on the carbon atom l i n k i n g the hydroxymethyl and carbomethoxy groups i n d i h y d r o s i t s i r i k i n e . At t h i s point i t had been established that s i t s i r i k i n e possessed a t e t r a c y c l i c skeleton and the following features; N H C: H C H . CH ^CH.OH During the course of the chemical i n v e s t i g a t i o n s , mass spectrometric analyses of s i t s i r i k i n e and various derivatives were undertaken. The mass spectrum of d i h y d r o s i t s i r i k i n e (Figure 8), run by the d i r e c t i n l e t procedure , was most help-f u l and i s discussed i n some d e t a i l . The molecular i o n peak at m/e 356 established the mole-cular formula assigned to d i h y d r o s i t s i r i k i n e , and fragments at m/e 338 (M-HpO) and 325 (M-CH20H) were consistent with the presence of a primary alcohol. A strong peak at m/e 253 was considered to a r i s e from loss of the entire oxygen-contai-ning portion of the molecule, i . e . M-CEK C H? 0 H. More important, NCO,Me however, were the ions at m/e 184, 170, 169 and 156. I t was immediately apparent from these four peaks that rings A,B, and C of the d i h y d r o s i t s i r i k i n e skeleton were of the type (12) encountered i n the yohimbine and r e l a t e d a l k a l o i d classes , - 18 -where these ions are also a t t r i b u t e d to the fragments shown i n Figure 8. The assignments made i n the case of yohimbine were quite rigorously established by deuterium l a b e l l i n g , as well as by studying the effe c t of functional substituents i n various positions of the molecule. Moreover, the occurrence of s i g n i f i c a n t peaks at m/e 169 and 170 and not at m/e 168 CO^Me 'CH z0H (12) and 169 excluded the type of pentacyclic r i n g system found i n polyneuridine (13)^°. Further information regarding the r i n g skeleton was ob-tained from semimicro dehydrogenation experiments. Treatment of d i h y d r o s i t s i r i k i n e with lead tetracetate afforded a product which exhibited u l t r a - v i o l e t spectra. (Amax. 253, 308 and 365 mix i n neutral or acid solution;Xmax. 284 and 328 I M A i n a l k a l i n e solution) i n good agreement with those of tetradehydroyohimbine and s i m i l a r compounds (14)'^1. Dehydrogenation of d i h y d r o s i t s i -r i k i n e with 10$ palladium-charcoal at 250° gave a mixture of (i5 ) Figure 6 - 19 -products which were separated by t h i n - l a y e r chromatography. The main product (compound A) displayed u l t r a - v i o l e t spectra (Figures 5 and 6) s i m i l a r to those of harman (15). I n neutral and a l k a l i n e media the spectra were the same, with maxima at 234, 250, 282, 288, 337 and 349 myuL , whereas i n acid s o l u t i o n there was a bathochromic s h i f t to 254, 303 and 372 m^u. These r e s u l t s were s u f f i c i e n t to confirm the tetrahydro-|^-carboline structure (12) indicated by the mass spectrum. D i h y d r o s i t s i r i k i n e hydrobromide was then subjected • o to palladium dehydrogenation at 280 , and the r e s u l t i n g mix-ture separated by t h i n - l a y e r chromatography. The u l t r a - v i o l e t absorption of one major f r a c t i o n (compound B) was i n close correspondence with that of 5,6~dihydroflavocoryline hydro-chloride (16)^ 3 with maxima at 221, 312, and 386 mjx . This provided the f i r s t piece of evidence f o r the entire r i n g system i n s i t s i r i k i n e . F i n a l confirmation was obtained when the dehydrogenation product was oxidised further with 2,3-dichloro-5,6-dicyano-p-benzoquinone to compound C, which possessed a completely aromatised r i n g system. The u l t r a - v i o l e t spectrum (Figure 7) with maxima at 237, 291, 345 and 385 IIUA. was i n good agreement QUINONE DEHYDROGENATION OF COMPOUND B (compound C, HCl salt) COMPARE FLAVOCORYUN HYDROCHLORIDE XMAX238, 291,346,385 m/,L XMIN 275, 305,374 m/L \T\fJL, Figure 7 DIHYDROSITSIRIKINE 120 150 200 250 300 350 m/e Figure 8 - 20 -with that reported f o r f l a v o c o r y l i n e (17, R = R^ = Et) but showed differences from other compounds (17)^ 3 with the same chromophore (see Table 2). Table 2 R Amax. ) H H 244 294 345 388 H. Et 235 295 350 390 E t Et 238 291 346 385 (0H 2)4 242 295 342 388 -Pr Et 240 292 346 386 An authentic sample of flavocoryline• hydrochloride was kindly provided by Dr.G.A. Swan, Chemistry Department, King's College, University of Durham, England, and the u l t r a - v i o l e t spectrum found to be superimposible on that of the compound derived from d i h y d r o s i t s i r i k i n e . Furthermore, the two materials had the same Rf value on paper chromatograms run i n several d i f f e r e n t solvent systems. Although the minute amounts of dehydrogenation products a v a i l a b l e prevented complete charac-t e r i s a t i o n , the u l t r a - v i o l e t s p e c t r a l data established the r i n g structure, and also suggested that s i t s i r i k i n e was a r e l a t i v e of the corynantheine (18a) class of a l k a l o i d s . A pr o v i s i o n a l structure such as (19) could thus be considered for d i h y d r o s i t s i r i k i n e . b; R = -CHgCH. The o r i e n t a t i o n of the hydrogen atom at C-3 i n (19) was indicated asOtby the presence o f Bohlmann bands^ at 2810 and 2760 cm."^ i n the i n f r a - r e d spectrum of d i h y d r o s i t s i r i k i n e . Furthermore, the hydrogen atom at 0=15 could be assumed to have the Ot-conf i g u r a t i o n since t h i s o r i e n t a t i o n has been found to be constant at the corresponding p o s i t i o n i n a l l related a l k a l o i d s . During t h e i r i n v e s t i g a t i o n s on corynantheine (18a) Karrer and co-workers^ 2 had reduced dihydrocorynantheine (18b) with l i t h i u m aluminium hydride and i s o l a t e d two isomeric al c o h o l s ; desmethoxy—dihydrocorynantheine alcohol(20) and iso-desmethoxy-dihydrocorynantheine alcohol (21). Since the configurations at C-3 and C-15 i n dihydrocorynantheine were the same as those projected f o r the corresponding positions i n d i h y d r o s i t s i r i k i n e , i t seemed f e a s i b l e to attempt a c o r r e l a t i o n between d i h y d r o s i t s i -- 22 -r i k i n e and dihydrocorynantheine. Dehydration of d i h y d r o s i t s i -r i k i n e to give the unsaturated ester (22), followed by hydride reduction should y i e l d the known a l k a l o i d (20), provided, of course, that the configuration at C-20 was also the same as i n corynantheine. Accordingly, d i h y d r o s i t s i r i k i n e was treated with sodium methoxide i n dry methanol to afford the 06(3-unsaturated ester (22), which r e c r y s t a l l i s e d from aqueous methanol as needles, m.p. 84-89° ,[bC[ "^+2° (MeOH), and analysed f o r the methanol solvate, Cg^ggOgNg'CHjOH. The presence of a terminal o l e f i n -1 was shown by a band i n the i n f r a - r e d spectrum at 1620 cm. and N.M.R, signals at 3.73 and 4.41T , each of which integrated f o r one proton. On hydrogenation one mol. of hydrogen was taken up to give desoxy-dihydrositsirikine (23) as a mixture of the two C-16 epimers, m.p. 172-177° . The in f r a - r e d and N.M.R. spectra showed the disappearance of the double bond, and the analysis was i n excellent agreement with the formula Cp.,HpoOpNp. D Hp (22) LiAl H . (20) + (21) - 23 -The o l e f i n i c ester was then reduced with l i t h i u m aluminium hydride, but the product contained r e l a t i v e l y l i t t l e terminal o l e f i n , as determined by the N.M.R. spectrum. By a combination of chromatographic and r e c r y s t a l l i s a t i o n techniques the major 0 component was obtained as l i g h t brown needles, m.p. 204 , foe]2^-24.3° (MeOH), which showed one spot on th i n - l a y e r chro-matography and analysed w e l l f o r OgQ^gONg. The melting point and s p e c i f i c r o t a t i o n were i n excellent agreement with the cor-responding constants — m.p. 204° » j^ 0 6 ]])^" 24.0° — quoted^ 2 for iso-desmethoxy-dihydrocorynantheinealcohol (21). This was good evidence f o r the structure (19) f o r d i h y d r o s i t s i r i k i n e but was not e n t i r e l y conclusive, since unfortunately no sample of the i s o - a l c o h o l could be obtained f o r d i r e c t comparison. However, the c o r r e l a t i o n between dihydrocorynantheine and d i h y d r o s i t s i r i k i n e was achieved by the fo l l o w i n g sequence of reactions. Mild acid hydrolysis converted dihydrocorynantheine (18b) to desmethyl-dihydrocorynantheine (24), which on reduction with sodium borohydride yielded a product (25) i d e n t i c a l i n every respect with d i h y d r o s i t s i r i k i n e . Having thus established the structure of d i h y d r o s i t s i r i k i n e , we could immediately assign structure (26) to g i t e i r i k i n e . - 24 -H H' (26) H' Me0 3C CH aOH B. I s o s i t s i r i k i n e Prom the amorphous post-perivine f r a c t i o n s of the chroma-tography of f r a c t i o n B (Scheme 3), the L i l l y group i s o l a t e d another a l k a l o i d which they also referred to as " s i t s i r i k i n e " but to which we have given the name " i s o s i t s i r i k i n e " since we have now shown that i t i s a new a l k a l o i d . Although the base, [a] 2 6-20°(CHCl 3), was an amorphous powder, i t was homogeneous by thin-layer chromatography and gave a sharp-melting c r y s t a l l i n e sulphate, m.p. 26.3.5°, and p i c r a t e , m.p. 216° . Analyses on i s o -s i t s i r i k i n e and i t s s a l t s indicated a formula of G21^26°3^2 f o r the base. This formula was established by a mass spectral mole-cular weight determination which showed a value of 354. Standard Kuhn-Roth and Z e i s e l determinations showed the presence of one C-methyl and one 0-methyl group resp e c t i v e l y . Maxima at 224, 283 and 291 m^ i i n the u l t r a - v i o l e t spectrum were charac-t e r i s t i c of an unsubstituted indole chromophore, and an absorp-t i o n band at 1720 cm.-1 i n the i n f r a - r e d region gave evidence for a carbonyl group. The N.M.R. spectrum of i s o s i t s i r i k i n e (Figure 4) confirmed that the indole system was unsubstituted, with signals at 1.33T(NH) and i n the region 2.4-3.It* (four - 25 -aromatic hydrogens), and a sharp three-proton s i n g l e t at 6.28T was r e a d i l y a t t r i b u t e d to the methoxyl group found i n the Z e i s e l determination. The presence of one o l e f i n i c hydrogen atom was shown by a quartet centred at 4.53T, whereas a doublet at 8.40t indicated that the methyl group was attached to an o l e f i n i c carbon atom. Since the coupling constants were the same (7 c/s) i n both cases, these signals almost c e r t a i n l y denoted an ethylidene group containing a t r i s u b s t i t u t e d double bond. C a t a l y t i c hydrogenation resulted- i n theuptake of one mol. of hydrogen to a f f o r d an amorphous mixture of two d i h y d r o - i s o s i t -s i r i k i n e s , Cg'jHggO^Ng, as shown by th i n - l a y e r chromatography. The major component (which was not the same as d i h y d r o s i t s i -r i k i n e ) was separated by chromatography and characterised as the c r y s t a l l i n e p i c r a t e , m.p. 187° . In the 1T.M.R. spectrum of the dihydro compound the signals d u e t o the ethylidene group had disappeared, and a new methyl absorption at 9.03T became evident. P i n a l confirmation of the ethylidene group was obtained when ozonolysis of i s o s i t s i r i k i n e yielded acet-aldehyde, i d e n t i f i e d by paper chromatography of i t s 2,4-dini-36 trophenylhydrazone^ . Carbonyl and methoxyl functions accounted f o r two of the oxygen atoms i n i s o s i t s i r i k i n e . The nature of the t h i r d oxygen atom was revealed-when a c e t y l a t i o n gave an amorphous acetate, C2 3H 2Q0 4N2, which displayed the appropriate bands i n the i n f r a - r e d region at 1730 (C=0) and 1235 (OAc) cm'.r^. The 26 N.M.R. spectrum of t h i s acetate was p a r t i c u l a r l y i n s t r u c t i v e . Apart from the expected new sharp si g n a l at 8.15£ due to the ace t y l group, a doublet i n t e g r a t i n g for two hydrogens now appeared at 6.05TJ , whereas a two-proton absorption present at 6.6"£in the spectrum of the alcohol had disappeared. The most obvious i n t e r p r e t a t i o n was that the doublet was due to the methylene protons of a primary hydroxyl group which had 35 undergone a downfield s h i f t of 0.5X u n i t on a c e t y l a t i o n " " . Moreover, the s p l i t t i n g of the si g n a l into a doublet suggested that the methylene, protons were part of an AgX system, and hence the hydroxymethyl group i n i s o s i t s i r i k i n e was probably l attached to a. carbon bearing one hydrogen atom, i . e . H-C-CHgOH. When i s o s i t s i r i k i n e was reduced with l i t h i u m aluminium hydride a d i o l was obtained, which showed neither a carbonyl absorption i n the in f r a - r e d regron nor a methoxyl s i g n a l i n the N.M.R. spectrum. This evidence demonstrated the presence of a carbomethoxy group i n I s o s i t s i r i k i n e . Treatment of the reduction product with acetone containing p-toluenesulphonic acid gave- an~ acetonide which c r y s t a l l i s e d and the N.M.R. spectrum c l e a r l y indicated a gem-dimethyl group with a pa i r of sharp signals at 8.63 and 8.68T. Since the d i o l had two primary alcohol functions, the formation of an isopropylidene derivative-meant that the hydroxyl groups were i n a 1,3 r e l a t i o n s h i p . Hence i s o s i t s i r i k i n e i t s e l f must - 27 -possess a {3-hydroxy-ester grouping s i m i l a r to that found i n s i t s i r i k i n e . At t h i s point the following features of the a l k a l o i d structure had been established: NC = C — CH 3 -CH / H \ CO RMe CHROH The nature of the r i n g system was revealed by dehydrogena-t i o n of a small amount of i s o s i t s i r i k i n e sulphate with palladium black at 280° . The r e s u l t i n g mixture was separated by t h i n - l a y e r chromatography, and two s i g n i f i c a n t f r a c t i o n s were obtained. One of the dehydrogenation products gave u l t r a - v i o l e t spectra of the harman(15) type (Figures 5 and 6 ) w i t h maxima at 232, 282, 289, 336 and 347 mjn, i n neutral s o l u t i o n , which s h i f t e d to 251, 302 and 374 rn^t upon a c i d i f i c a t i o n . Even more i n s t r u c -t i v e was the u l t r a - v i o l e t spectrum of the other f r a c t i o n , since i t was s i m i l a r to that of fl a v o c o r y l i n e hydrochloride (27) with maxima at 237, 291, 345 and 385 mjm (cf. Figure 7) and thus provided evidence f o r the entire t e t r a c y c l i c r i n g system of i s o s i t s i r i k i n e . I t was subsequently shown by paper chromatography, using an ethyl acetate-pyridine-water (8:2:1) system, that the l a t t e r dehydrogenationproduct was a c t u a l l y a mixture of two compounds. The major component (Rf 0.35) of t h i s mixture was not f l a v o c o r y l i n e (Rf 0.43) but the minor component - 28 -had the same Rf value as f l a v o c o r y l i n e , i n d i c a t i n g that some of t h i s known a l k a l o i d had presumably been obtained i n the de-hydrogenation. Since i t had been possible to degrade d i h y d r o s i t s i r i k i n e to f l a v o c o r y l i n e by a combination of palladium and quinone dehydrogenation reactions, a s i m i l a r procedure was followed with d i h y d r o - i s o s i t s i r i k i n e . The hydrogenation product of i s o s i t s i r i k i n e was conver-ted to the amorphous hydrochloride. The s a l t (without p u r i f i -cation) was then heated with palladium black at 280° , the residue taken up i n ac e t i c a c i d , and treated with 2,3-dichloro-5,6-dicyano-p-benzoquinone. From the reaction mixture was i s o l a t e d a c r y s t a l l i n e hydrochloride, m.p. 280-282° , which was i d e n t i c a l i n every respect with authentic f l a v o c o r y l i n e hydrochloride (27) s same melting point and undepressed mixed melting point; superimposible u l t r a - v i o l e t and i n f r a - r e d spectra; i d e n t i c a l Rf values on th i n - l a y e r and paper chromatography. From these r e s u l t s the gross structure (28) could be assigned with some cert a i n t y to i s o - s i t s i r i k i n e , since the al t e r n a t i v e structure with the positions of the ethylidene and ^-hydroxy-ester functions interchanged was considered very u n l i k e l y on biogenetic grounds. However, the structure (28) + CI M e O X C B ^ O H - 29 -contained three asymmetric centres at C-3, C-15, and G-16 whose configurations s t i l l remained to be determined. At f i r s t , the C-3 hydrogen atom was thought to have a (3-orientation, since the i n f r a - r e d spectrum of i s o s i t s i r i k i n e did not display Bohlmann bands^ i n the 2800 cm,"1 region. However, d i h y d r o - i s o s i t s i r i k i n e exhibited strong absorptions -1 at 2810 and 2760 cm. ,and thus presumably had the ex-configu-r a t i o n at C-3. This suggestion was substantiated when lead tetracetate oxidised d i h y d r o - i s o s i t s i r i k i n e (29) to the t e t r a -dehydro compound (30), which on subsequent reduction with sodium borohydride regenerated the s t a r t i n g material. Since t h i s sequence i s known to give the isomer with the C-3 hydrogen atom i n the OL-orientation , i t followed that d i h y d r o - i s o s i t s i -r i k i n e , and hence i s o s i t s i r i k i n e , must have t h i s configuration at C-3. M e O ^ C C H R O H H e O R C C H ^ O H The 0(-orientation of the hydrogen atom at C-15 could be 4 5 assumed on the basis of Wenkert's empirical rule , but there was no way of r e a d i l y f i n d i n g the configuration at C-16. There-fore the structure postulated f or i s o s i t s i r i k i n e was (31), and - 30 -i t i s noteworthy that t h i s a l k a l o i d bears a close r e l a t i o n s h i p to s i t s i r i k i n e (26). MeO^C CH^OH A f t e r the evidence indicated above for i s o s i t s i r i k i n e was already on hand, a mass spectrum (Figure 9) of t h i s a l k a l o i d was obtained. In addition to an accurate molecular weight, the mass spectrum provided valuable evidence about the structure, and thus supplemented the chemical i n v e s t i g a t i o n s . The peaks at m/e 336 (M-HgO) and 335 (M-l-HgO) were con-siderably stronger than the parent ions at m/e 354 (M +) and 353 ( M - l ) — a f a c i l e dehydration compatible with the presence of a l a b i l e proton at 0-16. A series of ions at m/e 184, 170, 169 and 156 corresponded to a s i m i l a r sequence displayed by d i h y d r o s i t s i r i k i n e , and were considered to be fragments derived P 40 -carboline system . However, the mass spectrum of i s o s i t s i r i k i n e (Figure 9) d i f f e r e d markedly from the spectrum of d i h y d r o s i t s i r i k i n e (Figure 8), inasmuch as a series of strong signals were obtained at m/e 275, 261, 247, 232 and 219 that were not found i n the l a t t e r spectrum. These peaks could be p l a u s i b l y a t t r i b u t e d to various r a d i c a l ions(32a,b,c,d, e) i n which the entire t e t r a c y c l i c r i n g structure of i s o s i t s i r i -- 31 kine had been aromatised. I t must be emphasised that the (32) as fi b: R = R x = Et: m/e 275 = Me, R x = Et; m/e 261 = fl, ^  = Et; m/e 247 = CH 2, R x = H; m/e 232 = R x = H; m/e 219 structures shown are only t e n t a t i v e , since no evidence i s av a i l a b l e to decide which among several a l t e r n a t i v e structures are correct. Since these ions are not produced i n the f r a g -mentation of d i h y d r o s i t s i r i k i n e (or s i t s i r i k i n e ) i t must be assumed that the presence of a double bond exo to the D-ring leads to i t s ready aromatisation, and subsequently to that of the C-ring, The difference i s most c l e a r l y demonstrated by the respective base peaks, both of which a r i s e by loss of the ^>-hydroxy-ester group. With d i h y d r o s i t s i r i k i n e t h i s ion (33) i s quite stable and i s registered at m/e 251, but i n the case of i s o s i t s i r i k i n e , dehydrogenation of the corres-ponding i o n (34) occurs to give the m/e 247 fragment f or which the structure (32c) i s suggested. - 32 -C. Other Work on S i t s i r i k i n e ; , D i h y d r o s i t s i r i k i n e and I s o s i t s i r i k i n e At about the same time as the p u b l i c a t i o n of our work on s i t s i r i k i n e and d i h y d r o s i t s i r i k i n e ^ , a r e p o r t ^ 8 appeared on the i s o l a t i o n of several related a l k a l o i d s from Aspidosperma  oblongum A.DC. S p i t e l l e r and Spiteller-Friedmann separated trace amounts of a l k a l o i d s by means of thin - l a y e r chromatography and postulated structures on the basis of mass spe c t r a l cracking patterns. Prom one f r a c t i o n was obtained an o l e f i n (M.W, 354), which on hydrogenation gave a dihydro compound (M.W. 356). A p a r t i a l structure containing a tetrahydro- (2> -carboline system was deduced when both compounds.displayed signals at m/e 184, 170, 169 and 156^*, whereas peaks-at M-31 and M-59 suggested the presence of primary alcohol and me t h y l e s t e r functions. Since both compounds showed a strong peak at M-103 i t was te n t a t i v e l y assumed that the oxygen-containing groups were j-jti nil present as a. ft-hydroxy-ester u n i t , -CH * . Reduction of the 1 "C0RMe o l e f i n with l i t h i u m aluminium hydride gave a compound of mole-cular weight 326, which confirmed the methyl ester. This pro-duct also displayed a peak at m/e 251, corresponding to loss CH OH of -CH. * , which substantiated the proposed a-hydroxy-ester CH^OH r group i n the o r i g i n a l a l k a l o i d . These deductions were supported by a p a r a l l e l series of reactions and mass spectra on an accompanying a l k a l o i d of mole-cular weight 384. This was considered to be merely a deri v a t i v e of the above a l k a l o i d (M.W. 354) with a methoxy substituent i n the aromatic r i n g , since the cracking pattern was s i m i l a r to the above except that the peaks were s h i f t e d upwards by 30 mass u n i t s . On the basis of these r e s u l t s the authors suggested the structures (35a) or (35b) f o r the a l k a l o i d of molecular weight 354, and (35c) for the dihydro d e r i v a t i v e (M.W. 356), Subsequent comparison by S p i t e l l e r of the mass spectrum of the dihydro compound with the mass spectrum of our dihydro-s i t s i r i k i n e indicated that they were p r a c t i c a l l y i d e n t i c a l . The small differences i n the spectra were considered, by S p i t e l l e r to be due to impurities i n t h e i r compound, since the minute amounts a v a i l a b l e i n t h e i r i n v e s t i g a t i o n prevented them from rigorously p u r i f y i n g t h e i r substance. Because the M-103 peak was lar g e r i n the o l e f i n than i n the dihydro compound, the i s o s i t s i r i k i n e structure (35a.) was favoured f or t h e i r parent a l k a l o i d (M.W. 354)? inasmuch as the cation (34) r e s u l t i n g from loss of the ^ >~hydroxy~ester unit would be s t a b i l i s e d by the a l l y l i c double bond. However, our mass spectrum of i s o s i t -s i r i k i n e was very d i f f e r e n t from that of S p i t e l l e r ' s a l k a l o i d , and consequently the above r a t i o n a l i s a t i o n Is open to question. _ 34 -Some months a f t e r our p u b l i c a t i o n , two Dutch workers 0 described the i s o l a t i o n of an a l k a l o i d , Cg-j^gO^^, m , P * ^16 » from P a u s i n y s t a l i a yohimbe P i e r r e . These authors indepen-50 dently derived a structure which corresponded to dihydro-s i t s i r i k i n e , and, indeed, the i n f r a - r e d spectrum of t h e i r a l k a l o i d was superlmposible on that of our/compound. The route by which t h e i r structure was determined was somewhat d i f f e r e n t from ours, and hence i s summarised below. Dehydrogenation with selenium afforded a l s t y r i n e (36), which established the r i n g system and the p o s i t i o n of s u b s t i -tuents. Kuhn-Roth oxidation indicated one C-methyl group, which was considered as part of an et h y l group i n view of the dehydro-genation r e s u l t s . The presence of a hydroxyl group was proven by formation of an acetate. From a Z e i s e l determination of one methoxyl group and a carbonyl band i n the i n f r a - r e d spectrum, a methyl ester was i n f e r r e d , and t h i s was confirmed by a s a p o n i f i c a t i o n - r e e ' s t e r i f i c a t i o n sequence. The, r e l a t i o n between the oxygen functions was elucidated by dehydration to an 0$~unsaturated ester (22) and hydrogenation to a mixture of the two deso x y - d i h y d r o s i t s i r i k i n e s > (23). A Kuhn-Roth oxidation revealed the presence cf an a d d i t i o n a l C-methyl group, and hence established a. (S-hydroxy-ester u n i t i n the o r i g i n a l a l k a l o i d . - 35 -M e O R C H O C H j (37) Prom these r e s u l t s a d i h y d r o s i t s i r i k i n e structure (25) was de-duced, and confirmed by a c o r r e l a t i o n with the known dihydro-corynantheine d e r i v a t i v e (37), This c o r r e l a t i o n was achieved by hydride reduction of (23) to a mixture of two alcohols, one of which was i s o l a t e d and found to be i d e n t i c a l with (37). D» The Biogenesis of Yohimbine, Corynantheine and A.jmaline Type A l k a l o i d s I t i s of i n t e r e s t to discuss b r i e f l y some of the bio-synthetic ideas pertaining to indole a l k a l o i d s , and more partic-u l a r l y to the corynantheine s e r i e s , i n order to consider the possible biogenetic r e l a t i o n s h i p of s i t s i r i k i n e and i t s re-l a t i v e s . F o r many years i t has been considered that indole a l k a l o i d s r e l a t e d to yohimbine (42) are derived i n part from tryptophan (38), a hypothesis that has been substantiated i n every instance where tracer experiments ••-have- been performed; 51 as, f o r example, with reserpine, ajmaline and serpentine . Hence the main i n t e r e s t at the present time i s i n the non-tryptophan portion of the molecules. 52 53 Thus i n the Robinson-Woodward ' theory, which i s based on an e a r l i e r scheme due to Barger and Hahn^, yohimbine (42) - 36 -i s produced v i a the intermediate (40) formed by condensation of a dihydroxyphenylalanine u n i t (39) and formaldehyde with tryptophan (38). Introduction of a carbomethoxy group into (40) and appropriate reduction steps are then postulated to 52 lead to yohimbine (42). Robinson's suggestion to account fo r the carbomethoxy group i s that the hydroxylated aromatic r i n g E i s expanded to a t r o p o l o n e ( 4 1 ) , which then crumples to a keto-acid. A cleavage of r i n g E along the dotted l i n e , 55 known as a "Woodward f i s s i o n " , i s invoked to account f o r a l k a l o i d s such as corynantheine (18a), and ajmalicine (1). Subsequent r i n g closures are required to affor d polyneuri-dine (13)^° and ajmaline" (43)-^, - 37 -H (18a) M e O R c \ MeO^C (13) CO^Me N Y ^CH*OH ( 43) An elegant a l t e r n a t i v e scheme in v o l v i n g prephenic acid (44) has been elaborated by Wenkert . Rearrangement of prephe-nic acid by a 1,2-shift of the pyruvate residue with retention of configuration, followed by hydration, affords a unit (45) re a d i l y d i s c e r n i b l e i n yohimbine (42). Condensation with a formaldehyde equivalent and retro-aldolisa11on then y i e l d s a jlseco, "prephenate-formaldehyde" (SPF) group (46), that can condense with tryptamine to y i e l d the ring-opened a l k a l o i d s t y p i f i e d by corynantheine (18a) and ajmalicine (1). - 38 OH One of the a t t r a c t i v e features of Wenkert's scheme i s that i t predicts the correct stereochemistry at the p o s i t i o n corresponding to C-15 of yohimbine (42) i n the various a l k a l o i d s . The hydrogen atom at t h i s p o s i t i o n i s found to have the <<x,-confi-guration i n a l l r e l a t e d indole a l k a l o i d s of known stereochem-4 5 5 7 i s t r y • , except for Y ~ a ^ a m m i c i n e a n d the Aspidosperma a l k a l o i d s discussed l a t e r i n t h i s t h e s i s . A group of cyclopentane glucosides, one example of which i s genipin ( 4 7 ) ^ 8 , has been found to have the same absolute configuration at the p o s i t i o n ^starred i n (47) ^| corresponding to C-15 i n (42). These compounds appear to have t h e i r structure based on the monoterpene unit (48), cleavage of which along the dotted l i n e would give a skeleton analogous to the SPP u n i t . - 39 -(47) (48) (49) Wenkert thus considered the hypothesis (previously suggested by Thomas^) that the indole a l k a l o i d s may have a mono t e r -penoid precursor derived from mevalonic acid (49), or equally, that the cyclopentano-monoterpenes evolve from prephenic acid. "' One can r e a d i l y v i s u a l i s e the r o l e of the SPP u n i t (46) i n the biosynthesis of corynantheine (18a) and rela t e d a l k a l o i d s . Formation of an SPP-tryptamine complex (51) v i a (50) i s followed by a Mannich-type condensation at the oL-position of the indole system to give (52), which can then undergo appropriate modi-f i c a t i o n s . S i t s i r i k i n e (26) and i t s r e l a t i v e s constitute an i n t e r -esting v a r i a t i o n of the corynantheine series which may l i e on one possible biogenetic pathway to pentacyclic a l k a l o i d s such as polyneuridine (13)^°. The intermediate (52) proposed by Wenkert can be v i s u a l i s e d as undergoing decarboxylation and oxidation fir) to the immonium i o n (53) , which then c y c l i s e s to (54). Addition of the aldehyde function to the ^ - p o s i t i o n of the indole can affor d a p l a u s i b l e precursor (55) of the ajmaline-61 type a l k a l o i d s such as vomeniline (58) . I f , however, reduction of the aldehyde group i n (52) occurs before decarboxylation, - 40 -- 41. -then a s i t s i r i k i n e type i s obtained. The corresponding immo-nium ion (56) can c y c l i s e only to a pentacyclic precursor (57) of polyneuridine (13), or i t s C-16 epimer, akuammidine. 6 2 6 3 Recently Leete ' has attempted to test the hypotheses described above by feeding l a b e l l e d compounds to Rauwolfia.  serpentina and degrading the ajmaline (43) obtained. In both the Robinson-Woodward and the Wenkert schemes C-21 of ajmaline derived from a formaldehyde equivalent, a theory which i s supported by incorporation of "^C-formate at t h i s p o s i t i o n ^ 2 . As phenylalanine i s a known precursor of dihydroxyphenyl-alanine, i t might be expected, on the basis of the Robinson-Woodward hypothesis, that administration of phenylalanine-2-^C would provide ajmaline-3-"^C, but the ajmaline extracted was 6 3 i n a c t i v e , as were the reserpine and serpentine, . I f Wenkert'a prephenic acid hypothesis were correct, then alanine-2-^C, which would l a b e l prephenic acid by way of pyruvate, should give a c t i v i t y at C-3 i n ajmaline. However, only 2fo of the r a d i o a c t i v i t y was a t t r i b u t a b l e to t h i s p o s i t i o n Ajmaline i s o l a t e d from a plant which had been fed mevalonate-2-^C, an established- precursor of terpenes, was 63 completely i n a c t i v e . This r e s u l t rendered u n l i k e l y another 63 - 42 -biosynthetic route i n which the non-tryptophan moiety was \56 59 supposed to be formed from a monoterpene unit (48)^ . A fourth hypothesis was then put forward by L e e t e ^ , on the basis offfia. degradation of ajmaline, l a b e l l e d by 1 4 - ' " ^ * a c e t a t e - l ~ G incorporation, which showed that C-3 and C-19 each contained a quarter of the t o t a l a c t i v i t y , whereas C-21 was i n a c t i v e . I f i t were assumed that the remaining ha l f of the a c t i v i t y was shared between C-15 and 0-17, then t h i s would support a theory, previously advanced by S c h l i t t l e r 65 and Taylor i n which the carbon chain 18-19-20-15-14-3 o r i -ginates by condensation of three molecules of acetyl-coenzyme A to a poly~[3 -keto fragment (59). Further condensations with a formaldehyde equivalent at C-20, and with the methylene 66 group of malonyl-coenzyme A (derived from acetyl-coenzyme A ) at C-15, were postulated to affor d an intermediate (60) very o 2 c ^ o C H 2 0 o 2 c ^ o ^ 2.0 18 ( 5 9 ) ^ iK^lK o O * / S C o A ° 2 C M #_ _ 0 C = X (60) s i m i l a r to Wenkert's SPF unit (46). I t should be emphasised that (60) would be expected to form a complex with tryptamine e s s e n t i a l l y i d e n t i c a l to the tryptamine-SPF complex (51), and hence the l a t t e r part of Wenkert's scheme i n which the various indole a l k a l o i d s are derived would s t i l l be v a l i d . - 43 -Unfortunately i n a r e p e t i t i o n of the work on R. serpentina, 6 7 Battersby and co-workers were unable to reproduce the above r e s u l t s , and i t was found that the radioactive l a b e l i n ajma-l i n e (43) from both acetate and formate was scattered. Hence, at the present time no one hypothesis has been established to the exclusion of others, and the origin" of the non-tryptophan portion of these indole a l k a l o i d s i s s t i l l a subject of contro-versy. - 44 -PART I I Some Aspects of the Chemistry of Catharanthine and Cleavamine' 112 ,69 Introduction During i n v e s t i g a t i o n s by the L i l l y g r oup y 3 on the dimeric Vinca a l k a l o i d s , vincaleukoblastine (VLB), leurosine, and leuro-c r i s t i n e , i t was found that each was cleaved by concentrated hydrochloric acid to an indole compound and a vindoline deriv-at i v e (61). In the instances of VLB and leurosine, the l a t t e r was desacetylvindoline (61a), whereas l e u r o c r i s t i n e gave des-N^-methyl-desacetyl vindoline•-('61b). Both VLB and leuro-c r i s t i n e afforded the same indole d e r i v a t i v e , velbanamine, C-^HggNgO, but the corresponding compound with leurosine was cleavamine, GigH24^2° Velbanamine was considered to be a hydroxy-dihydrocleavamine, sinee i t yielded some cleavamine on prolonged heating with acid 68 MeO' (61) a: R = Me bj R. = H C O ^ M e ,69 When catharanthine (5) was subjected to the same acid t r e a t -ment, one of the products was found to be cleavamine, which suggested that the dimeric a l k a l o i d s were constituted of vindo-l i n e and catharanthine-like moieties. Moreover, the i n f r a - r e d spectrum of VLB could be approximated by an equimolar mixture - 45 -of vindoline and catharanthine . When the structure of these on OQ a l k a l o i d s had been established ' , the L i l l y research group postulated the p a r t i a l structure (62a.) for VLB, the precise points of attachment of the vindoline unit and the p o s i t i o n of the hydroxyl group s t i l l remaining i n doubt. Leurosine was thought to be the anhydro-analogue, whereas l e u r o c r i s t i n e was probably des-N/N-methyl-N/ j-formyl VLB (62b). However, the i d e n t i t y of "the indole portion of VLB could not be established d i r e c t l y , since i t seemed that a rearrangement was taking place during the acid cleavage, and consequently the indole compound i s o l a t e d did not necessarily possess the skeleton present i n the o r i g i n a l a l k a l o i d . I t therefore became imperative to e s t a b l i s h the structure and mode of formation of cleavamine, and also to characterise the other products from the acid treatment of catharanthine. Establishment of the mechanism of the catharanthine-cleavamine transformation would f u r n i s h evidence for the structure of VLB, since catharan-thine constituted an excellent model f o r the postulated indole moiety. - 46 -Although the study of t h i s reaction was o r i g i n a l l y under-taken mainly i n connection with the structure of VLB, the u l t i -mate scope of the work went f a r beyond t h i s aspect. Conside-r a t i o n of l i k e l y intermediates from a mechanistic standpoint led to the use of cleavamine analogues i n the synthesis of immonium compounds, which i n turn were found to undergo trans-annular c y c l i s a t i o n s to Iboga (63) and Aspidosperma (64) - l i k e skeleta. (63) R = H or C02Me Hence the discussion of the work on cleavamine and i t s congeners can be divided i n t o two sections: the catharanthine -cleavamine transformation and a possible mechanism are presen-ted i n section A, whereas the synthetic use of the cleavamines and t h e i r biogenetic implications w i l l be discussed i n section C. A review of current bi©synthetic theories pertinent to the discussion on transannular c y c l i s a t i o n s i s given i n section B. A, The Catharanthine-Cleavamjne Transformation When catharanthine (5) was treated with concentrated hydrochloric acid i n the presence-of t i n and stannous ch l o r i d e , and the r e s u l t i n g m i x t u r e separated by chromatography, two of the products obtained were descarbomethoxycatharanthine (65) and cleavamine (66). The structure (66) of cleavamine had been - 47 -suggested mainly on the basis of a comparison of the mass spectral cracking patterns of cleavamine and dihydrocleavamine 7 2 with that of quebrachamine (67)? and was f i n a l l y established 73 by an X-ray analysis of cleavamine methiodide . H . Since the combined y i e l d of cleavamine and descarbometh-oxycatharanthine was only about 20$, i t was decided to examine some of the other components more c l o s e l y . In p a r t i c u l a r , i t was f e l t desirable to i s o l a t e other compounds which might provide information about the mechanism of t h i s i n t e r e s t i n g rearrangement. The cleavamine mother l i q u o r s and several cleava-mine-containing f r a c t i o n s were combined and subjected to a care-f u l column chromatography. Apart from the many f r a c t i o n s containing i n t r a c t a b l e gums and r e s i n s , one f r a c t i o n was obtained which could be w e l l characterised. I t i s appropriate to t discuss t h i s i n some d e t a i l , since a d d i t i o n a l evidence was furnished which was germane to any mechanistic i n t e r p r e t a t i o n . This f r a c t i o n , designated B9, was a mixture of two com-- 48 -pounds, as shown by t h i n - l a y e r chromatography. No o l e f i n i c protons were apparent i n the N.M.R. spectrum of B9, and the methyl t r i p l e t normally present at 8.96Yin cleavamine had shi f t e d to 9.13"E • The mass spectrum showed a molecular ion at m/e 282, and other s i g n i f i c a n t peaks at m/e 156, 143, 138, 124-could be a t t r i b u t e d to the following fragments, which are given by 4"oL"-dihydrocleavamine (68) : H m/e 156 m/e 143 m/e 124 m/e 138 In general the mass spectrum of B9 was p r a c t i c a l l y superimpo-s i b l e on that of 4"oc"-dihydrocleavamine, and also the i n f r a -red spectra were f a i r l y s i m i l a r . The leading spot of the mixture had the same Rf as 4"oc"-dihydrocleavamine on th i n - l a y e r chromatography and the second spot was thought to be due to a dihydrocleavamine epimeric at C-4, which we designated as 4"(3"-dihydrocleavamine. From other studies at the L i l l y labora-t o r i e s , a C-4 epimer of 4"Ot"-dihydroclea,varaine was i s o l a t e d , and a sample provided by Dr.M; Gorman, L i l l y Research Labora-t o r i e s f o r comparison purposes. I t was possible to demonstrate For the sake of c l a r i t y , the dihydrocleavamine (68) obtained by c a t a l y t i c hydrogenation of c l e a v a m i n e i s referred to as 4"oLl'-dihydroc] eavamine. This does not imply any d e f i n i t e s t e -reochemistry, out xs used merely to d i f f e r e n t i a t e t h i s com-pound from the corresponding C-4 epimer, 4"fi"-dihydrocleava-mine c - 49 -that our 4 , ,p"-dihydrocleavamine was i d e n t i c a l to the L i l l y sample. A synthetic mixture of the dihydrocleavamines duplica-ted the behaviour of f r a c t i o n B9 on thin-flayer chromatography and gave an i d e n t i c a l i n f r a - r e d spectrum. Two dihydrocleavamines epimeric at C-4 could be obtained i n t h i s r e a c t i o n ei t h e r from dihydrocatharanthine (present as an impurity or formed by reduction of catharanthine), or by reduction of cleavamine (or an equivalent r e a c t i o n intermediate). Since the catharanthine was-homogeneous, and i n any case dihydro-catharanthine (69) had been shown- to decarboxylate without • 1 1 3 formation of dihydrocleavamine ' , the former p o s s i b l i t y could be dismissed. We were thus l e f t with t h e l a t t e r a l t e r n a t i v e , that had to be incorporated i n t o a mechanism which would account for the formation of cleavamine (66), descarbomethoxycatharan-thine (65) and the two dihydrocleavamines (68). Some other re s u i t s pertinent to any proposed me onanism had been obtained by the L i l l y group^^^cDihydrocatharanthine (69) was decarboxylated r e a d i l y to epi-ibogamine(70) by heating with hydrazine i n ethanol^^, or by hydrolysis with ei t h e r aqueous potassium hydroxide or l i t h r u m iodide i n pyridine followed by 7 5 heating with d i l u t e mineral acid . These r e s u l t s f i t t e d the 7 5 proposed mechanism for the decarboxylation of Iboga al k a l o i d s s - 50 -However, none of these procedures was successful i n decarboxy-l a t i n g catharanthine (5), presumably because the corresponding intermediate (71) would be too strained to form"* . Therefore, the formation of descarbomethoxy-catharanthine ( 6 5 ) , a l b e i t in. poor y i e l d , upon treatment of catharanthine (5) with concentrated hydrochloric a c i d must involve some other mechanism. In order to explain the occurrence of cleava-mine ( 6 6 ) and the epimeric dihydrocleavamines ( 6 8 ) a r i n g -opened intermediate must be present at some stage, and more-over, a route has to be provided whereby the o l e f i n i c linkage present i n catharanthine can be reduced. With these considera-tions i n mind we postulate the speculative mechanism on p. 51 (66) (68) - 52 -for the reaction. The lone p a i r of electrons on the N^^-atom i n (5) can pa r t i c i p a t e i n a rearrangement to form an immonium io n , with concurrent r i n g cleavage and protonation at the ^ - p o s i t i o n of the indole. The r e s u l t i n g ring-opened intermediate (72) i s s t a b i l i s e d by two fa c t o r s : ( i ) the a l l y l i c nature of the immonium i o n , and ( i i ) the conjugation of the newly generated double bond between.C-17 and C-18 with both the ester and a n i -l i n o functions. A f t e r acid hydrolysis of the ester, decarboxy-l a t i o n may then occur v i a (73) i n a n analogous manner to the Iboga a l k a l o i d s . Absence of a G-5/018 bond renders the molecule more f l e x i b l e , and the decarboxylated product (74) i s obtained, whereas the corresponding intermediate (71) required i n the usual mechanism (see above, p. 50) cannot be formed. The c r u c i a l , intermediate ('74) may foll o w either of two reaction paths. I f the o r i g i n a l electron flow i s re'versed, then the C-5/C-18 bond:is regenerated and the product w i l l be des carbomethoxy catharanthine (65). But i f there i s merely an a l l y l i c s h i f t of a. proton (-perhaps because the immonium system has already been reduced) then the ring-opened t e t r a c y c l i c compounds must ul t i m a t e l y beformed. Assuming that (75) i s the actual intermediate, . 1.,2.-reduction of the immonium i o n w i l l give"cleavamine d i r e c t l y . On the other hand, 1,4-reduction can also occur to afford an eneamine (76), which rearranges 76 i n the well-known manner to the immonium compound (77) with subsequent reduction to (68). A mixture of 4"c0'- and 4"(2>"-di-53 -hydrocleavamines (68) i a obtained because the approach of the proton to C-4 i n . (76) can occur from above or below the plane of the r i n g with..essentially' equal f a c i l i t y . The reduction ...of catharanthine with zinc i n g l a c i a l ace-t i c acid to car..homethoxy-4"(o"-dihydrocieavamine ( 119) showed that the reduction, and decarbomethoxylatron were separate processes. Much...more, substantial support for the mechanism, however, was the transannular c y c l l s a t i o n of an immonium ion derived from ca.rbemethoxy--4l,p)"-dihydrocleavamine to an Iboga skeleton'(see secti.onC, p.79). This demonstrated that a s i m i -l a r c y c l l s a t i o n proposed for the formation of'descarbomethoxy-catharanthine (65) from the intermediate (74) was a c t u a l l y f e a s i b l e . In the absence of an inorganic reducing agent, the reduction step possibly takes place v i a van intramolecular redox reaction of two molecules of the intermediate(75) to y i e l d one molecule of cleavamine (66) and one' of a pyridinium com-pound (78). The increase i n y i e l d of cleavamine i n a reducing medium i s thus explicable on the grounds that (75) i s reduced d i r e c t l y to cleavamine and no pyridinium compound i s formed. Further i n t e r e s t i n the chemistry of cleavamine was - 54 -stimulated by the recent work of Buchi and co-workers o n voacamine (79) which indicated - that the o r i g i n a l structure (62a) proposed by the L i l l y group f o r VLB was probably wrong, and suggested that the indole moiety was a cleavamine (66) rather than a catharanthine (5) type. I t was noted that voacamine also represented a "dimeric" a l -k a l o i d constituted from two indole moieties. Even more impor-tant was the observation that voacamine could be cleaved into the respective monomeric units-by means of a c i d i c reagents under conditions s i m i l a r to those used for VLB. The carbon-carbon - bond l i n k i n g the two halves i s l a b i l e and i s ruptured during acid treatment. One of the weak features i n the L i l l y structure (62) for VLB had been the nature of the linkage•between the indole and dihydro-indole portions. As mentioned before, VLB i s cleaved by acid i n t o desacetylvindoline and hydroxy-dihydro-6Q cleavamine , but i t was d i f f i c u l t to r a t i o n a l i s e such a f r a c -ture on the basis of structure (62). One would not expect - 55 -a bond comprised of an aliphairic carbon on one hand and an aromatic carbon on the other to react i n t h i s manner. Consi-deration of the chemistry of voacamine led to a review of the structure f o r VLB.and further studies were undertaken. A high-r e s o l u t i o n mass spectrum gave a molecular :weight f or VLB of 810.4219, which showed that the correct formula was C^H^gOgN^ 7 8 and not C^gH^gOgN^ as previously thought . Thus VLB must contain a carbomethoxy-cleavamine rather than a catharanthine unit as the indole, portion of the molecule. On the basis of t h i s and other evidence, a revised structure (80) was very Co recently proposed f o r VLB (80) Me / 'OH C02Me Although the study of the catharanthine-cleavamine trans-formation was i n i t i a t e d to throw l i g h t upon a corresponding reaction thought to occur with VLB and i t s congeners, i t was also r e a l i z e d at an early stage of the i n v e s t i g a t i o n that the r e s u l t s were of wider p o t e n t i a l i n t e r e s t i n the areas of synthesis and biogenesis of indole a l k a l o i d s . These aspects were subsequently considered i n some d e t a i l , and are discussed i n the following sections. - 56 -B. The Biogenesis of Strychnos, Iboga and Aspidosperma Alkaloids In order to be i n a p o s i t i o n to discuss f u l l y the r e s u l t s of the transannular c y c l i s a t i o n s (section C), i t i s pertinent to review b r i e f l y the various theories advanced fo r the biosynthesis of a l k a l o i d s of the Strychnos, Iboga and Aspidosperma species, as t y p i f i e d by strychnine (82), coronaridine (86) and aspidospermine (87). These compounds are considered to be re l a t e d biogeneti-c a l l y (see below), and indeed have been found to occur t o -gether i n the same plants, as f o r example Vinca rosea Linn, (see i n t r o d u c t i o n to t h i s thesis) and Stemmadenia do n e l l -s m i t h i i (Rose) Woodson ' . I t has recently been demonstrated that l a b e l l e d tryptophan (38) i s incorporated into vindo-27 7 Q l i n e ( 7) and ibogaine (88) , and hence one may assume that the re l a t e d a l k a l o i d s are constructed i n part from t r y p t o -phan, " . Strychnine (82) may thus be derived, according to the Robinson-Woodward theory ' mentioned e a r l i e r (p.35), from a condensation of a dihydroxyphenylalanine u n i t (39) and formal-dehyde with tryptophan (38) to a f f o r d the intermediate (81), 55 which undergoes subsequent "Woodward f i s s i o n " of r i n g E (along the dotted l i n e ) and appropriate c y c l i s a t i o n s to pro-vide the Strychnos skeleton. In order to accommodate the Iboga a l k a l o i d s , a v a r i a t i o n of t h i s above scheme has been proposed 8 0 by Taylor , whereby condensation of tryptophan (38) and 3,4-dihydroxyphenylalanine (39) affords the a^-unsaturated acid (83). The l a t t e r intermediate by a Michael ad d i t i o n , and - 57 -(88) 58 -Woodward f i s s i o n of the aromatic r i n g , provides the i n t e r -mediate (84), which "then p a r t i c i p a t e s with formaldehyde i n a Mannich reaction to y i e l d the t e t r a c y c l i c compound (85). Subsequent a l d o l condensation, dehydration, and reduction lead to the Iboga skeleton (86). A more comprehensive scheme f o r the Strychnos and Iboga a l k a l o i d s , that has the a d d i t i o n a l merit of encompassing the Aspidosperma s e r i e s , i s furnished by Wenkert's prephenic acid hypothesis-^, which was discussed e a r l i e r (p.37) i n r e l a t i o n to the corynantheine-type bases. According to t h i s theory, the strychnine (82) group evolves from the tryptamine-SPP complex (51) by attack of the formyl acetate residue at the d e p o s i t i o n of the indole to give the immonium i o n (89), which bears on obvious resemblance to the known a l k a l o i d steimuade-nine ( 9 l ) 8 ^ . A transannular c y c l i s a t i o n then provides the strychnine precursor (90). (91) (90) - 59 -Derivation of the Iboga and Aspidosperma systems i s les s straightforward, since i t i s evident that they a r i s e from rearranged SPP u n i t s . The c r u c i a l rearrangement can be seen as proceeding v i a a retro-Michael reaction of the interme-diate (89), i n v o l v i n g an activated hydrogen atom on a carbon atom Oi to either the immonium system or to the a c e t y l group, with resultant cleavage of the SPP u n i t . I f the o r i g i n a l tryptamine-SPP complex (51) underwent t h i s reaction, the formyl acetate residue would be l o s t and a pathway to the fl a v o p e r e i r i n e (92) structure revealed. However, were the retro-Michael process to occur at a l a t e r stage, when the f o r -myl acetate moiety could not be l o s t because of i t s attach-ment to the indole system, as i n (89), then the cleavage pro-duct (95) could be modified by unexceptional reactions to give e i t h e r an Aspidosperma (94) or an Iboga (97) precursor. These compounds could then undergo a p a r a l l e l series of reactions: Michael additions to the ap-unsaturated acid systems? would a f f o r d the nine-membered r i n g compounds (95) and (98), i which could then, by transannular c y c l i s a t i o n s , y i e l d the Aspidosperma and Iboga skelata, (96) and (99) r e s p e c t i v e l y . No d i r e c t proof of Wenkert's hypothesis has been pub-- 61 -l i s h e d to date, but the l a t t e r portion of h i s proposal i s supported by a considerable weight of circumstantial evidence. For instance, the retro-Michael reaction of a strychnine-type a l k a l o i d i s exemplified by a cleavage that Smith and Edwards have found to occur with akuammicine (100). In order to explain the formation of the betaine (102) when akuammicine was heated i n methanol at 100° for three hours, the authors proposed a mechanism in v o l v i n g a retro-Michael cleavage of an intermediate such as (101). I t i s worthy of note that Wenkert predicted the occur-rence i n Nature of AspidoBperma a l k a l o i d s carboxylated as i n (96) and t h i s has since been v e r i f i e d by the i s o l a t i o n of - 62 -several a l k a l o i d s related to vincadifformine (103). Perhaps go the best example i s minovincine (104) , which possesses not only a carbomethoxy group i n the predicted p o s i t i o n but also the a c e t y l function. Counterparts o f the conjugated enone system i n the postulated Iboga-type precursor (99) have also been found: the double bond i n catharanthine (5), and the car-bonyl group i n voacryptine (105). Furthermore, the r i n g -opened precursor (95) i s represented i n Nature by vincadine (106a) 8^, and vincaminorine ( 1 0 6 b ) w h e r e a s the carbometh-H I (103) C O . M e (104) M e O (106) •\ r _ M a:H = H C0->Me * b:R = Me oxy-cleavamine portion of the VLB molecule (80) i s r e a d i l y derived from (98). A s i g n i f i c a n t point i n favour of the above scheme i s that i t predicts the correct absolute configuration at C-15 i n akuammicine (100), which has been found to be constant 89 i n t h i s and related Strychnos a l k a l o i d s .. The sole exception _ 63 i s the d , l mixture y-akuammicine, the formation of which can be a t t r i b u t e d to a r e v e r s i b l e transformation of (89) to (93) p r i o r to the complete evolution of the former to the Strychnos system. The occurrence i n Nature of racemic vinca-d i f f ormine (103)^''^ 2, the o p t i c a l antipodes of quebracha-mine ( 1 0 7 ) 8 5 , ( 1 0 8 ) 8 6 , and of vincadifformine ( 1 0 5 ) 8 2 ' 9 2 , and the enantiomeric a l k a l o i d s (— )-Q-methylaspidocarpine (113) and ( + ) - p y r i f o l i d i n e (114) can be interpreted on the basis of the above biosynthetic scheme. Since intervention of the non-asymmetric intermediate (93) i n Aspidosperma bio-synthesis destroys the f i x e d configuration of the starred po-s i t i o n i n (89), no o p t i c a l r e l a t i o n s h i p can e x i s t between the a l k a l o i d s of t h i s family and other indole bases. Randomisation of the absolute configuration of Aspidosperma a l k a l o i d s i s therefore considered by wenkert to be due to a lack of o p t i -c a l consistency i n the Michael r e a c t i o n (94) to (95). Evidence which tends to support the postulated trans-annular c y c l i s a t i o n of (95) to (96) has been accumulated i n studies of the r e l a t i v e configurations of various Aspidosper-ma a l k a l o i d s . Some of these have been correlated with (-)-aspidospermine (115), whose absolute configuration i s known 9 3 from the X-ray structure determination by M i l l s and Nyburg . (—)-Quebrachamine has been shown to have the same c o n f i -guration as aspidospermine at the asymmetric centre i n v o l -ving the ethyl group 9^, and hence has the structure (107); the epimeric (+)-quebrachamine must then be (108). - 64 -I t i s a t t r a c t i v e to speculate that the Aspidosperma a l k a l o i d s may be divided i n t o two stereochemical s e r i e s , one of which i s t h e o r e t i c a l l y derived by a transannular c y c l i -s ation of ( —)-quebrachamine (107), the other by c y c l i s a -t i o n of the (+)-enantiomer (108). The reverse processes can often be achieved i n the laboratory t In several cases the o r i e n t a t i o n of the ethyl equivalent of the a c e t y l group i n (95) and (96) seems to be c h a r a c t e r i s t i c of the a l k a l o i d s of a p a r t i c u l a r plant species. Thus (—)-quebrachamine (107) has been found i n conjunction with ( —)-aspidospermine (115) and (-O-pyrifolidine (115) £(— )-0-Methyl-aspidocarpineJ i n or Aspidosperma quebracho bianco , and with (+)-vincadifformine (109) and (+)-l,2-dehydroaspido3permidIne (111) i n Rhazya 92 s t r i c t a . On the other hand, (+)-quebrachamine occurs to-gether with (—)-tabersonine (110) £ (— )-6,7-dehydrovinca-difformine J i n Stemmadenia species 9^ 1, and with (-)-1,2-dehy-droaspidospermidine (112) i n Pleiocarpa t u b i c i n a 9 ^ . The stereo-chemistry seems to be determined by the o r i e n t a t i o n of the a c e t y l group i n the quebrachamine-like precursor (95) since the Mannich-type closure of the nine-membered r i n g requires formation of a cis-perfaydroquinoline system.in (96). Purther-56 more, Wenkert suggested that the c i s - a n t i - c i s backbone exhibited by the Aspidosperma. a l k a l o i d s may be the conse-quence of the Mannich condensation (and any subsequent r e -duction) following the path of l e a s t s t e r i c resistance. The i s o l a t i o n of stereochemically r e l a t e d a l k a l o i d s i n the same 66 -plant c e r t a i n l y supports t h i s suggestion, and furnishes circumstantial evidence f o r the occurrence i n Nature of a transannular c y c l l s a t i o n such as postulated i n Wenkert's hypothesis. Experimental evidence which demonstrates the f e a s i b i l i t y of such c y c l i s a t i o n s w i l l be presented i n sec-t i o n C. c- Transannular C y c l i s a t i o n s of Cleavamine Derivatives A consideration of the mechanistic aspects of the catharanthine (5)—cleavamine (66) transformation led us i n turn to examine the various biosynthetic hypotheses that have been outlined above. In p a r t i c u l a r , our i n t e r e s t was drawn to the part of Wenkert's scheme dealing with the Iboga and Aspidosperma a l k a l o i d s , which was found to have d i r e c t r e l e -vance to the work on cleavamine. F i r s t of a l l , when one con-sidered the conversion of intermediate (98) to the pentacyclic structure (99) of the Iboga a l k a l o i d s , i t was apparent that the transannular c y c l i s a t i o n of a cleavamine-like skeleton was involved. This was immediately reminiscent of a n i d e n t i c a l transannular c y c l i s a t i o n of (74) that had been proposed i n the mechanism (p.51) to explain the formation of descarbo-methpxycatharanthine (65). Secondly, the intermediate (95) - 67 -advanced by Wenkert as the d i r e c t precursor of the Aspido-sperma system (96) was a g a i n s i m i l a r to cleavamine ( 6 6 ) , e s s e n t i a l l y d i f f e r i n g only i n the p o s i t i o n of the ethyl group* I t was clear that cleavamine or one of i t s derivatives might be converted i n t o an-immonium intermediate of the type proposed by Wenkert, and thereby a f f o r d an excellent opportu-n i t y f or evaluating the f e a s i b i l i t y of such transannular c y c l i -s ation processes. Accordingly, oxidation of 4"a ! ,-dihydrocleavamine (68) with mercuric acetate gave an immonium ion (116)., which under-went transannular c y c l i s a t i o n to an Aspidosperma-like ske-l e t o n (117). This could not be i s o l a t e d as such, but the cor-responding dihydro-indole (118a) was obtained a f t e r reduction 9 7 with l i t h i u m aluminium hydride . (116) (117) CO^Me (118) a.;R = H bsR = Ac - 68 -This r e s u l t suggested that entry i n t o the v i n c a d i f f o r -mine (103) type of system could be r e a l i s e d by use of the 112 appropriate carbomethoxy-dihydrocleavamine (119; » Moreover, with t h i s cleavamine d e r i v a t i v e there was also a p o s s i b i l i t y of obtaining an Iboga a l k a l o i d system, since the C-18 hydrogen atom was rendered l a b i l e by the carbomethoxy group. Reaction with mercuric acetate "would be expected to generate an i n t e r -mediate with the immonium -system (^Ii=CC) -involving either C-19 or C-5. The intermediate (120) with the C-19 immonium group could, by the appropriate transannular c y c l i s a t i o n , afford a v i n c a d i f f o r m i n e - l i k e system (121), whereas that (122) with the >N=C-5 grouping could y i e l d an Iboga a l k a l o i d (86), We were able to demonstrate that i n fa c t both processes operate. Carbomethoxy-4"^"-dihydrocleavamine (119)^ was prepa^-red by reduction of catharanthine (5) with zinc and ac e t i c 98 acid . Acid hydrolysis and decarboxylation of t h i s product afforded 4"£>"-dihydrocleavamine which was not i d e n t i c a l to - 69 -that obtained by hydrogenation of cleavamine, and hence must have the ethyl group i n a d i f f e r e n t o r i e n t a t i o n . Oxidation of carbomethoxy-4"(5"-*idihydrocleavamine -with mercuric acetate i n a c e t i c acid afforded a complex mixture which was subjec-ted to chromatography on alumina. This procedure resulted i n the i s o l a t i o n of one major component and two other a l k a -l o i d s i n smaller amounts. The l a t t e r substances, which were 71 found to be the known a l k a l o i d s coronaridine' and dihydro-OQ catharanthine , w i l l be presented l a t e r , while the former i s discussed immediately below. ( i ) Pseudo-vincadifformine and i t s Derivatives The major product, which we have termed pseudo-vinca-d i f f ormine, was obtained from the i n i t i a l benzene f r a c t i o n s qq of the chromatography i n about 25> y i e l d . I t was a white amorphous powder,loci " -503 (EtOH), which analysed w e l l for L JD ^21^26^2^2' ^ l * 1 8 1 ! confirmation of the molecular formula was • obtained when a mass spectrometric molecular weight deter-mination showed a value of 338. Maxima i n the u l t r a - v i o l e t spectrum at 226, 298 and 326 mju_, and absorption bands i n the i n f r a - r e d region at 1675 and 1610 cm.-"*" c l e a r l y i n d i c a -ted an ot^-unsaturated ester function conjugated with the dihydro-indole system i n the same manner as i n v i n c a d i f f o r -mine (103). The N.M.R. spectrum exhibited a s i n g l e t at 1.05T (NH), a complex pattern i n the region 2.4-3.3t correspon-ding to four aromatic protons, and a spike at 6.23Tdueto the methoxyl group. A very strong s i g n a l at m/e 124 i n the mass spectrum (Figure 10) was i n d i c a t i v e of an Aspidosperma - 70 --type s k e l e t o n 1 0 0 (see l a t e r ) , and, indeed, the spectrum was very s i m i l a r to "that of v i n c a d i f f ormine. Chemical evidence i n support of "the conjugated ester system was provided by acid-catalysed hydrolysis and decarb-oxyl a t i o n of pseudo-vincadifformine (121) to y i e l d a gummy product (123) which showed the expected spectral properties of an indolenine s y s t e m 1 0 0 , 9 1 , X m o v 221, 227 ( i n f l e c t i o n ) . and 250 (broad) mjx; no carbonyl or NH absorption i n the i n f r a red r e g i o n S u b s e q u e n t reduction of the l a t t e r substance with l i t h i u m aluminium hydride afforded a c r y s t a l l i n e pro-duct, m.p. 89-90 ^OtJ^-eo (CHCl^), for which the structure (124) was deduced from the following evidence. Elemental ana-(123) lyses suggested a formula O-^HggNp^ which was confirmed by a mass spectrometric molecular weight (282). The reduc-t i o n of the indolenine system was c l e a r l y indicated by a t y p i c a l dihydro-indole u l t r a - v i o l e t spectrum ( X m Q V 243 and 295 myw,) and the appearance of a new absorption at 3230 cm."1 i n the i n f r a - r e d spectrum (NH). Moreover, a complex pattern of l i n e s i n the N.M.R. spectrum i n the re-gion 2 . 7 - 3 . 6 T , due to four aromatic protons, was i n complete I I I I I I — I — I — I — I — I <fr o o o o o o O CO ID <fr CM A|ISU9|UI 9AljD|8J - 71 -agreement with known Aspidosperma a l k a l o i d systems Invaluable information was provided by the mass spectrum (Figure 12) of the dihydro-indole (124), which showed s i g n i -f i c a n t peaks at m/e 282 (M +) and 254 (M-28), and a very strong s i g n a l at m/e 124. I t was recently observed by Biemann e t a L 1 0 0 that the appearance of M-28 and m/e 124 peaks may be consi-dered diagnostic of an Aspidosperma-type skeleton. In t h i s instance, a similar-fragmenta"fcion process leading to a m/e 124 ion may be postulated, whereby the molecular i o n (125) expels ethylene to y i e l d the m/e 254 fragment (126), which i s subse-quently cleaved to the m/e 124 i o n (127). This ion d i f f e r s only i n the p o s i t i o n of the eth y l group from that (128) pro-p o s e d 1 0 0 f o r the corresponding m/e 124 peak displayed by the 9° (129) m/e 124 Et - 72 -Aspidosperma a l k a l o i d s . Furthermore, the mass spectrum of (124) was superimposible on that of the s i m i l a r compound 97 (118a)previously synthesised from 4"(X."-dihydrocleavamine? . Further evidence f or the structure of (124) was ob-tained from the N-acetyl d e r i v a t i v e , m.p. 107.5-109°, C21^28°2iyf2* T t l e ul" t r a~ v i°l e" t spectrum of the l a t t e r displayed maxima at 253, 279 and 289 TDJUL , and the complex m u l t i p l e t i n the aromatic region of the N.M.R. spectrum of (124) had now collapsed into a broad three-proton peak centred at 2.85X and a s i g n a l at 1.87T due to one proton. These s p e c t r a l data were i n excellent agreement with the acetate (118b) previous-97 l y derived from 4"oL"-dihydrocleavamine^ and with the known Aspidosperma a l k a l o i d demethoxypalosine ( 1 2 9 ) 1 0 ^ . A d d i t i o n a l chemical proof of the conjugated ester system was provided by reduction of p B e u d o - v i n c a d i f f o r m i n e with zinc and sulphuric acid to y i e l d two isomeric dihydro d e r i v a t i v e s . The major product, dihydro-pseudovincadifformine, £oc]24--l6° (EtOH), analysed f o r C 2 1H 2 80 2N 2, a formula which was established by a mass spectrometric molecular weight of 340. The u l t r a - v i o l e t spectrum (X T O Q V 244 and 299 mil ) was c h a r a c t e r i s t i c of a dihydro-indole chromophore, and the ester carbonyl absorption i n the i n f r a - r e d had now moved to 1725 cm."1. A c e t y l a t i o n afforded a N-acetate, 0 2^H^ 00^N 2, 0 L j ^ r i 28°(EtOH), which showed u l t r a - v i o l e t maxima at 253, 282 and 291 iryu. , and the appropriate appearance of an amide band at 1660 cm."1 with concurrent loss of the NH peak i n the i n f r a - r e d spectrum. Besides the expected s i g n a l at - 73 -7.75T (CHjC=0) i n the N.M.R. spectrum, there was an un-expected u p f i e l d s h i f t of the methoxyl signal from 6.33 to 6.83T, which w i l l be discussed l a t e r . In general, the spectral properties of dihydro-pseudo-vincadifformine and i t s acetate were i n agreement with the structure (131), f o r which confirmation was found i n the mass spectral cracking pattern (Figure 11 )„ The base peak of the mass spectrum was m/e 124, whereas the second most intense peak was at m/e 254. A fragmentation process e n t i r e l y analogous to that discussed above (125) to (127) was obviously occurring, i n which the molecular ion (132) eliminated a molecule of methyl acrylate instead of ethylene to give an i d e n t i c a l m/e 254 fragment (126), which then cleaved as before to afford the m/e 124 i o n (127). The absence of a s i g n i f i c a n t m/e 254 peak i n the mass spectrum (Figure 10) of pseudo-vincadifformine (121) i t s e l f was due to the presence of a double bond, which prevented any e l i m i -nation of methyl acrylate (or i t s equivalent) ^ from the mole-cular ion (133). - 74 -(132) CO;>M<L H (133) C O a M e + -> (127) (134) m/e 124 C02Me + However, the rearrangement product (134) could s t i l l cleave to y i e l d the m/e 124 ion. F i n a l l y , a comparison of the mass spectra of pseudo-vincadifformine (121) and dihydro-pseudo-vincadifformine (131a) with those of authentic v i n c a d i f f o r -mine (103) and dihydrovincadifformine (130) revealed that both pairs of spectra were i d e n t i c a l . The minor product, [c*L 1 2 4~132° (EtOH), from the zinc /sulphuric acid reduction of pseudo-vincadifformine also analysed f o r 02^220^2, and spectral data indicated the presence of dihydro-indole and saturated ester functions. A c e t y l a t i o n afforded a N-acetate, C2^H^00^N2, j j x j 2 4+3° (EtOH), whose u l t r a - v i o l e t spectrum ( X m o v 250, 278 and 296 mjk) showed small but d i s t i n c t differences from the ace-- 75 -tate of the major dihydro compound. Moreover, the methoxyl absorption i n "the N.M.R. spectrum of the acetate was i n a more usual p o s i t i o n (6.42T). Vigorous treatment with sodium methoxide converted the major component in t o a substance which proved to be i d e n t i c a l with the minor product. I t was thus established that the minor component, which we r e f e r to as iso-dihydro-pseudo-vincadifformine, also had the gross structure (l31a)and that the compounds were i n f a c t stereoisomers, which d i f f e r e d only i n the configuration at the carbon atom (C-18) bearing the carbomethoxy group. The i s o l a t i o n and interconversion of the two dihydro compounds i n the reduction of pseudo-vincadifformine (121) could be r a t i o n a l i z e d by analogy with a s i m i l a r series of reactions performed by Smith and Edwards 1 0^ i n the akuammicine se r i e s . Reduction of dihydroakuammicine (135) with zinc and sulphuric acid afforded tetrahydroakuammicine (137), which was epimerised with sodium methoxide to iso-tetrahydroakuam-micine (138). The authors suggested that the f i r s t step i n the reduction was protonation of C-16 to give the immonium ion (136). The proton added to the {3-face i n order to allow the carbomethoxy group to take up the morestable equatorial o r i e n t a t i o n , with r i n g C i n the boat conformation. Reduction of the immonium system then proceeded with a d d i t i o n of hydro-gen at C-2, again from the (i-face, to give a compound (137) i n which the B/C r i n g junction was the more stable cis-form and rings C and D had chair conformations. This forced the - 76 -carbomethoxy group i n t o an unfavourable a x i a l o r i e n t a -t i o n . (135) (136) N. H (138) H CO*Me (137) Epimerisation of tetrahydroakuammicine (137) to the isomeric base (138) was then r e a d i l y understood as in v o l v i n g a change of o r i e n t a t i o n of the carbomethoxy group from a x i a l to equatorial. This mechanism enabled the authors to explain the hydrogen bonding of the carbonyl group indicated by the in f r a - r e d spectrum of(137), which did not occur i n the case of (138). Although the akuammicine derivatives (137) and (138) are not s t r i c t l y comparable to dihydro-pseudo-vincadiffor-mine (131a) and i t s epimer, i t i s nevertheless l i k e l y that the reduction of pseudo-vincadifformine (121) follows a s i m i l a r s t e r i c course. I f , for the sake of argument, the P-configuration i s assumed at C-9, then the predominant isomer would also be the k i n e t i c a l l y favoured one (139a), - 77 -which has the carbomethoxy group at C-B i n the axialCX-orien-t a t i o n . Treatment with base would then affo r d the thermodyna-mically more stable iso-dihydro compound (140a) with an equatorial carbomethoxy substituent i n the-(3-orientation. (139) a; R = H (140) a; R = H b: R = Ac b: R = Ac I t must be emphasised that, since the stereochemistry of pseudo-vincadifformine s t i l l remains to be established, the above arguments are not conclusive. I f one constructs models of the corresponding acetates (139b) and (140 b), the methoxyl group of the a x i a l carbo-methoxy function of (139b) i s found to come i n close p r o x i -mity to the benzene r i n g , whereas the methoxyl group i n (140b) cannot do so. Thus the high p o s i t i o n (6.83T) of the methoxyl proton signals i n the N.M.R.- spectrum of dihydro-pseudo-vincadifformine acetate (139b) may be due to diamag-n e t i c s h i e l d i n g by the benzene r i n g . The chemical and sp e c t r a l evidence c i t e d above estab-l i s h e d a vincadifformine-type structure (121) f o r pseudo-v i n c a d i f f ormine. I t should be mentioned at t h i s time that, i n f a c t , the mercuric acetate oxidation of carbomethoxy-4"(S , ,-dihydrocleavamine (119) can, and does, proceed i n two - 78 -di r e c t i o n s to provide immonium derivatives i n v o l v i n g either C-19 or C-5. At the outset i t was therefore necessary to consider the a l t e r n a t i v e c y c l i s a t i o n of (14-1) to (14-2). However, studies of models showed that s t e r i c repulsions made t h i s c y c l i s a t i o n extremely unfavourable, and the structure (142) was excluded even before experiments were run. Hence pseudo-vincadifformine (121) must be derived from the C-19 immonium i o n (120) as shown. ( i i ) Coronaridine and Dihydrocatharanthine The chromatography of the mixture r e s u l t i n g from mercuric acetate oxidation of carbomethoxy-4"(3"-dihydro-cleavamine (119) yielded, i n a d d i t i o n to pseudo-vincadiffor-105 mine, small amounts of two other a l k a l o i d s . Prom the l a t e r benzene f r a c t i o n s of the chromatography was i s o l a t e d an amorphous a l k a l o i d , which afforded a c r y s t a l l i n e hydro-- 79 -chloride, m.p. 221-223° . The u l t r a - v i o l e t spectrum of the base indicated an indole chromophore with maxima at 226, 285, and 293 inu. . The presence of an ester carbonyl absorption at 1705 cm."1, and the absence of the strong Bohlmann bands i n the region between 2700 and 2800 cm."1 displayed by the s t a r t i n g material, suggested that t h i s a l k a l o i d vas a member of the Iboga series (86). Indeed, comparison of our a l k a l o i d ( i n f r a - r e d spectra and t h i n - l a y e r chromatographic mobility) 71 with an authentic.sample of coronaridine (145) showed that they were', the same . Further comparison (mixed melting-point and i n f r a - r e d spectra) "of the c r y s t a l l i n e hydrochlorides completely established the i d e n t i t y . The other a l k a l o i d , eluted with benzene-ether ( l : l ) , was c r y s t a l l i n e , m.p. 143-145.5 ° ,[ot]26+49° (CHC1 3), and the spectral data showed the presence of an indole system and a saturated ester function. An authentic sample of dihydrocatha-ranthine was prepared by hydrogenation of catharanthine (5), and a d i r e c t comparison (mixed melting-point, i n f r a - r e d spec-t r a , t h i n - l a y e r chromatographic mobility) confirmed that our °8 107 product was a c t u a l l y dihydrocatharanthine (69)" ' Thus i t was established that the transannular c y c l i s a -t i o n of the other possible mercuric acetate oxidation pro-duct (122) with the >N=C-5 immonium system, l e d to the Iboga skeleton (86). 80 MeO^ C (122) M e 0 2 C (86) The i s o l a t i o n of both coronaridine (145) and dihydro-catharanthine (69 ) from t h i s r e action indicated that an isomerisation of the ethyl group at C-4 was taking place. (143) (144) MeQ,C (146) M e ° ^ C v (69) This was not unexpected, since the immonium i o n (144) or (146) formed by oxidation of carbomethoxy-4"(o"-dihydro-- 81 -cleavamine could r e a d i l y isomerise to the other epimer v i a the eneamine (143) before c y c l i s a t i o n . The m o b i l i t y of the 7 6 immonium-eneamine system i s w e l l known . D. Discussion The c y c l i s a t i o n to Iboga a l k a l o i d s provides somesupport fo r the mechanism postulated (p.5l) f o r the catharanthine -cleavamine transformation. An e s s e n t i a l part of the mechanism i s the transannular c y c l i s a t i o n of a cleavamine-like i n t e r -mediate (74) to descarbomethoxycatharanthine (6 5), and the above r e s u l t s demonstrate the f e a s i b i l i t y of t h i s process. Moreover, the immonium-eneamine tautomerism required to accommodate coronaridine and dihydrocatharanthihe i s also i n -volved to explain the formation of 4"CXI1- and 4"^"-dihydro-cleavamines (68) i n the cleavage of catharanthine (5) with a c i d i c reagents. I t i s apparent that the three products obtained i n our reaction prove that the kind of transannular c y c l i s a t i o n of immonium ions proposed i n Wenkert's biosynthetic scheme (p. 60) does take place quite r e a d i l y . Although these r e s u l t s do not prove that t h i s i s the actual biogenetic pathway, they cer-t a i n l y lend support to the l i k e l i h o o d of such reactions. I t appears that the Aspidosperma ( 64.) and Iboga (63) a l k a l o i d s may very w e l l evolve from a common biogenetic precursor [such as (147)], that can afford e i t h e r a quebrachamine (67)7 or cleavamine (66) skeleton, which subsequently undergoes - 82 -c y c l i s a t i o n i n the manner we have now r e a l i s e d i n the l a -boratory. The a l k a l o i d s of these types are t h e o r e t i c a l l y derivable from the same immonium i o n (147/, which involves the carbon atom between the nitrogen and ethyl-bearing carbon atoms. I t w i l l be of i n t e r e s t to see whether a l k a l o i d systems (147) ^ 0 ^ 1 H :. T (148) CO, 0^ (63) (150) such as (149) and (150), which could be formed from the a l t e r n a t i v e ion (148), w i l l be found i n Nature. This would then p a r a l l e l the r e l a t i o n s h i p between akuammicine (lOO) 1 0^" and condylocarpine (152) , which can be considered as a r i s i n g b i o g e n e t i c a l l y from a related p a i r of immonium ion precursors, (89) and (151) res p e c t i v e l y . Indeed, the Swiss 108 workers who recently i n t e r r e l a t e d akuammicine and condy-- 83 -locarpine proposed the transannular c y c l i s a t i o n of i o n i c intermediates s i m i l a r to (89) and (151). CO, Me ( 1 0 0 ) (151) C0 ,Me (152) The formation of coronaridine (145) and dihydrocatha-ranthine ( 69 ) i n the mercuric acetate reaction constitutes an a t t r a c t i v e entry into the Iboga a l k a l o i d s e r i e s . Since the removal of the carbomethoxy group i s r e a d i l y accomplished^ t h i s sequence obviously also provides p a r t i a l syntheses of ibogamine (153) and epi-ibogamine (70) . I t i s apparent that i f a synthesis of carbomethoxy-dihydrocleavamine ( 119) can be developed, t h i s would e f f e c t the t o t a l synthesis of the Iboga skeleton, which has not yet been accomplished. (153) - 84 -An I n t e r e s t i n g p o t e n t i a l route to the Aspidosperma system, p a r t i c u l a r l y of the v i n c a d i f f ormine (103) "type, i s revealed by the c y c l i s a t i o n of the immonium i o n (120) to pseudo-vinbadifformine (121). The obvious extension of t h i s r e a ction to an a l k a l o i d such as vincadine (106a) should pro-vide a synthesis of vincadifformine and i t s r e l a t i v e s . Since i t has recently proved possible to c y c l i s e (—)-quebracha-mine (107) to (+)-aspidospermidine (154) by means of mercuric 109 acetate oxidation and subsequent hydride reduction , the synthesis of vincadifforimine from vincadine i s now highly probable. (107) (154) EXPERIMENTAL Melting points were determined on a Kofler block and are uncorrected. - U l t r a - v i o l e t (U.V.) absorption curves were measured i n methanol s o l u t i o n on a Cary 14 spectrometer, and i n f r a - r e d (I.R.) spectra were taken on a, Perkin-Elmer Model 21 spectrophotometer. Nuclear magnetic resonance (N.M.R.) spectra were recorded at 60 megacycles/sec. on a Varian A60 instrument; the l i n e p ositions or centres of m u l t i p l e t s are given i n the Tiers X scale with reference to tetramethylsilane as the i n t e r n a l standard; the m u l t i p l i c i t y , and integrated area and type of protons are indicated i n parentheses. S i l i c a gel G plates were used f o r t h i n - l a y e r chromatography (T.L.C.) and were developed by ethyl acetate, ethyl acetate-chloro-form or ethyl acetate-ethanol mixtures as given below. The alumina used f o r column chromatography was Shawlnigan reagent grade, deactivated with 3$ of 10$ aqueous ac e t i c a c i d , unless otherwise stated. Analyses "were performed by Dr.A. Bernhardt and h i s associates, Mulheim (Ruhr), Germany and by the Micro-a n a l y t i c a l Laboratory, University of B r i t i s h Columbia. Every molecular weight (M.W.) quoted was determined mass spectro-m e t r i c a l l y . Part I Experimental Section I s o l a t i o n of S i t s i r i k i n e The crude sulphate (1 g.) provided by Dr.M. Gorman, - 86 -E l i L i l l y Research Laboratories, was dissolved i n methanol (20 ml.) and water (250 ml.), the s o l u t i o n cooled i n i c e and made basic with aqueous ammonia. The p r e c i p i t a t e was taken up i n ether (200 ml.) the layers separated, and the aqueous portion further extracted with ether (3 x 100 ml.). A f t e r drying over magnesium sulphate, the combined ethereal extracts were evaporated to give a powder, which showed three spots on T.L.C. (EtOAc). Several r e c r y s t a l l i s a t i o n s f r o m acetone-petroleum ether (b.p. 60-80°) afforded needles (320 mg.) m.p. 178-179°, which now displayed only two spots on T.L.C. Repeated r e c r y s t a l l i -sations f a i l e d to resolve the mixture, which behaved as a pure compound by a l l c r i t e r i a except T.L.C., and hence t h i s was c a l l e d s i t s i r i k i n e . The purest samples of the a l k a l o i d and i t s d e r i v a t i v e s are described below. S i t s i r i k i n e c r y s t a l l i s e d from acetone with one molecule of solvent as needles, m.p. 181° ,[oc126-520 (MeOH). Pound: i_ _ i D C, 69.52; H, 7.84. Calc. for C 2 1H 2 60 5N 2.Me 2C0: C, 69.88; H, 7.82. The unsolvated material was obtained from aqueous me-thanol as stout needles, m.p. 206-208°; foci 2 6-58° (MeOH); D \ n a x . ( l o g £ ) : 2 2 6 < 4'56), 282 (3.90), 290 (3.84) m/A. ; N > m a x < ( N u j o l ) : 3360 (NH and/or OH), 1705 (C=0), 740 (o-disub-s t i t u t e d benzene) cm."1. N.M.R. signals (CD^COCD^): 2.8 (multiplet* 4H, aromatic), 4.7 ( m u l t i p l e t , 1.8H, o l e f i n i c ) , 6.1 ( m u l t i p l e t , 2H, CH 20), 6.38 ( s i n g l e t , 3H, CH 30), 9.02 (1H)T . Pound: C, 71.15,71-43; H,7.51, 7.66; 0, 14.00; - 87 N, 7.89, 7.77; C-Me, 1.61; M.W. 354. Calc. f or C 21 H26°3 N2 : C, 71.16; H, 7.39; 0, 13.54; N, 7.90; (1) C-Me, 4.24; M.W. 354. S i t s i r i k i n e P i c r a t e A saturated a l c o h o l i c s o l u t i o n of p i c r i c acid (2 ml.) was added to s i t s i r i k i n e (50 mg„) i n ethanol (1 ml.) and the mixture heated to b o i l i n g . The p r e c i p i t a t e was r e c r y s t a l l i s e d from methanol io affo r d yellow hexagonal prisms (45 mg.), m.p. 226-228° ( d e c ) . Pound: C, 55.55, 55. 70; H, 5.46, 5.26; N, 12.10. Calc. f o r C 2 7 H 2 g 0 1 0 N 5 : C, 55.57; H, 5.01; N, 12.00. S i t s i r i k i n e Acetate S i t s i r i k i n e (110 mg.) was dissolved i n pyridine-acetic anhydride (1:1, 2 ml.) and l e f t overnight. The s o l u t i o n was poured i n t o ice-water (10 ml.), b a s i f i e d with ammonia, and the p r e c i p i t a t e taken up i n ether. A f t e r washing several times with water, the ethereal s o l u t i o n was dried over magnesium sulphate and evaporated. R e c r y s t a l l i s a t i o n from aqueous metha-nol afforded the acetate as needles (100 mg.), m.p. 198°; [c*-] 2 6-26° (MeOH); two spots on T.L.C. (EtOAc-CHCl 5, 1:1); ^ m a v (Nujol): 3340 (NH), 1735 (C=0), 1700 (0=0), 1240 (OAc) cm."*1; N.M.R. signals (CD^COCD^): 2.7 ( m u l t i p l e t , 4H, aroma-t i c ) , 4.7 ( m u l t i p l e t , 1.8H, o l e f i n i c ) , 5.6 ( m u l t i p l e t , 2H, CH 20Ac), 6.37 ( s i n g l e t , 3H, CH 30), 8.02 ( s i n g l e t , 3H, CH5C=0), 9.02 (1H)T. Pound: C, 69.65; ,H, 7.32; N, 7.01. Calc. f o r C 2 3 H 2 8 0 4 N 2 ! °' 6 9 ' 6 ? 5 H» 7.12; N , 7.07. - 88 -D i h y d r o s i t s i r i k i n e (25) S i t s i r i k i n e (450 mg.), i n methanol (10 ml.), was hydrogenated over palladium black (24 mg.). The hydrogen up-take ceased a f t e r 30 minutes when 0.65 mol. had been absorbed. Af t e r removal of the ca t a l y s t and solvent, the product was r e c r y s t a l l i s e d twice from acetone-petroleum ether (b.p. 60-80 ) to give d i h y d r o s i t s i r i k i n e (405 mg.), m.p. 177-179°. This compound displayed only one spot on T.L.C. (EtOAc) which corresponded to one of the two spots shown by s i t s i r i k i n e . Therefore the impurity which could not be removed from s i t s i -r i k i n e was i n fa c t the dihydro compound. D i h y d r o s i t s i r i k i n e c r y s t a l l i s e d from acetone, with one molecule of solvent, as needles, m.p. 180° . Pound: C, 69.42; H, 7.91; N, 6.97. Calc. f o r C 2 1H 2 80 3N 2*Me 2C0: C, 69.53; H, 8.27; N, 6.76. R e c r y s t a l l i s a t i o n from aqueous methanol afforded the unsolvated a l k a l o i d as prisms, m.p. 215° ;[oc]2^-55° (MeOH); L JD Xmax. ( l 0 S £ ) 5 2 2 6 (4.61), 282 (3-95), 290 (3.87) m u j A m i n > (log£): 247 (3.40), 287.5 (3.85) m/*. ; V f f i a x > ( C H C l 3 ) i 3480 (NH and OH), 2810 and 2760 (Bohlmann bands) 4 4, 1710 (0=0) cm."1; ^ m Q V (Nujol): 3400 (NH), 3200 (OH), 1710 (C=0)cm."1; N.M.R. signals (CDjCOCD^: 2.8 (m u l t i p l e t , 4H, aromatic) 6.1 ( m u l t i p l e t , 2H, CH 20), 6.42 ( s i n g l e t , 3H, CH 30), 9.07 (broad s i n g l e t , 3H, CH 3C)T. Pound: C, 70.80; H, 7.78; 0, 13.49; N, 7.77; O-Me, 9.01; C-Me, 3.95; M.W. 356. Calc. f o r C21 H28°3 N2 S C' 10'16'> H» 7 - 9 2 ; 0, 13.47, N, 7.86; ( l ) O-Me, 8.72; (1) C-Me, 4.22; M.W. 356. - 89 -D i h y d r o s i t s i r i k i n e Picrate D i h y d r o s i t s i r i k i n e (60 mg.) and a s o l u t i o n of p i c r i c a c i d were reacted i n the manner described above and the de r i vative r e c r y s t a l l i s e d from methanol to y i e l d amber prisms (55 mg.), m.p. 228-230° (dec.). Found: C, 55.30, 55.46; H, 5.55,5.71; N, 11.95. Calc. f o r C^H^O^^: C, 55.38; H, 5.34; N, 11.96. D i h y d r o s i t s i r i k i n e Acetate The acetate was prepared by treatment of d i h y d r o s i t s i -r i k i n e (200 mg.) with ac e t i c anhydride i n pyridine as above. The product was r e c r y s t a l l i s e d twice from acetone-petroleum ether (b.p. 60-80° ) to aff o r d needles (155 mg.), m.p. 187° one spot on T.L.C. (EtOAc-CHCl,, 1:1) ;M26-31° (MeOH); D V. M Y (Nujol): 3390 (NH), 1740 (0=0), 1705 (C=0), 1250 (OAc) cm."1; N.M.R. signals (CDClj):'1.34 ( s i n g l e t , IH, NH), 2.8 ( m u l t i p l e t , 4H, aromatic), 5.6 (m u l t i p l e t , 2H, CHgOAc), 6.38 ( s i n g l e t , 3H, CH 50), 7.96 ( s i n g l e t , 3H, CH5C=0), 9.02 (broad s i n g l e t , 3H, CE^G)X . Pound: C, 69.39, 68.98; H, 7.75 7.59; 0, 16.46; N, 6.85. Calc. f o r C ^ H ^ O ^ : C, 69.32; H, 7.69; 0, 16.06; N, 7.03. D i h y d r o s i t s i r i k i n e p-Bromobenzoate p-Bromobenzoyl chloride (130 mg.) was added to dihy-d r o s i t s i r i k i n e (65 mg.) i n dry pyridine (3 ml.). A f t e r standing overnight the s o l u t i o n was poured i n t o ice-water, made basic with aqueous ammonia and s t i r r e d f o r 10 minutes. - 90 -The p r e c i p i t a t e was taken up i n ether, the ethereal s o l u t i o n washed twice with water and dried over magnesium sulphate. Removal of the solvent gave a gum which was dissolved i n benzene and f i l t e r e d through alumina (5 g.). The benzene eluate was concentrated and petroleum-ether (b.p. 60-80° ) added dropwise to. the" b o i l i n g s o l u t i o n u n t i l a permanent t u r b i d i t y was obtained. The product was r e c r y s t a l l i s e d from acetone-petroleum ether (b.p. 60-80° ) to y i e l d the p-bromo-o benzoate as slender needles (60 mg.), m.p. 174 ; one spot on T.L.C.; \ ) m Q V (Nujol): 3370 (NH), 1725 (0=0), 1705 (C=0) cm."1; N.M.R. signals (CDCl^): 2.5 ( m u l t i p l e t , 6H, aromatic), 5.4 ( m u l t i p l e t , 5H, CH 20), 6.37 ( s i n g l e t , 3H, CH 50), 9.10 (broad s i n g l e t , 3H, CH3C)'T. Pound: C, 62.34; H, 5.70; N, 5.06. Calc. f o r CggH^O^Br: C, 62,33; H, 5.79; N, 5.19. Saponification of D i h y d r o s i t s i r i k i n e (25) D i h y d r o s i t s i r i k i n e (200 mg.) was heated under r e f l u x with 2 N methanolic sodium hydroxide (20 ml.) f o r 2 hours. A f t e r removal of the solvent, the residue was taken up i n water and extracted with methylene chloride to remove unsa-ponified material. The aqueous s o l u t i o n was a c i d i f i e d with hydrochloric acid and evaporated to dryness. The residue was leached with absolute ethanol and the s o l u t i o n f i l t e r e d from sodium chloride. Evaporation and r e c r y s t a l l i s a t i o n from aqueous alcohol gave an a(S-unsaturated acid hydrochloride, - 91 -m.p. 260-263° ; v m Q ^ (Nujol): 1695 (0=0), 1620 (0=0) cm."x; m a x * N.M.R. signals (CF^CO^): 3.2 (m u l t i p l e t , 4H, aromatic), 3.77 ( s i n g l e t , IH, o l e f i n i c ) and 4.20 ( s i n g l e t , IH, o l e f i n i c ) T . D i h y d r o s i t s i r i k i n e D i o l A s o l u t i o n of d i h y d r o s i t s i r i k i n e (250 mg.) i n t e t r a -hydrofuran (10 ml.) was run slowly into a s t i r r e d suspension of l i t h i u m aluminium hydride (200 mg.) i n tetrahydrofuran (10 ml.) and heated under r e f l u x for 3 hours. Afte r the mix-ture had stood overnight,the excess hydride was decomposed with saturated aqueous sodium sulphate s o l u t i o n (10 ml.), followed by water (20 ml.). The aqueous suspension was then extracted with methylene chloride (4 x 25 ml.), and the combi-ned extracts dried over sodium sulphate. Removal of the solvent and r e c r y s t a l l i s a t i o n from aqueous acetone gave the d i o l as needles (180 mg.), m.p. 203° ; one spot on T.L.C, (EtOAc-EtOH, 1:1); [ a ] 2 6-3° (MeOH); \ m a x < ( l o g O : 226 (4.55), 282 (3.85), 290 (3.78)mym ; v m a x < (Nujol): 3210 (NH and OH) cm."1; N.M.R. signals (CD^COCD^): 2.8 (m u l t i p l e t , 4H, aromatic), 6.4 ( m u l t i p l e t , 4H, 2 CHgO), 9.02 (broad s i n g l e t , 3H, CH 3C)r. Pound: C, 73.30; H, 8.46; N, 8.41. Calc. f o r C 2 0H 2g0 2N 2: C, 73.13; H, 8.59; N, 8.53. Acetonide of D i h y d r o s i t s i r i k i n e D i o l The above d i o l (150 mg.) was dissolved i n dry acetone (20 ml.) and p-toluenesulphonic acid (110 mg.) added. After - 92 -standing at room temperature for 48 hours, the s o l u t i o n was neutralised with aqueous ammonia and the acetone removed under vacuum. The product was i s o l a t e d with ether, the ethereal s o l u t i o n dried over magnesiumsulphate, and evaporated to leave a gum. On t r i t u r a t i o n with a l i t t i e anhydrous ether c r y s t a l s formed, which were f i l t e r e d o f f and found to be unreacted d i o l (95 mg.). The f i l t r a t e was evaporated, the residue taken up i n benzene and passed throught a column of alumina (3 g.). Removal o f the solvent gave the acetonide as an amorphous powder (30 mg.) which c r y s t a l l i s e d from methanol with one molecule of solvent, m.p. 105-109° ; one spot on T.L.C. (EtOAc-CHCl,, l:l);v„,QV (Nujol): 3200 (NH) cm.""1; N.M.R, signals (CDCl^): 1.61 ( s i n g l e t , IH, NH), 2.8 (m u l t i p l e t , 4H, aromatic), 6.25 (doublet, 4H, 2 CH 20), 8.62 ( s i n g l e t , -6H, (CH 3) 2C0 2), 9.06 (broad s i n g l e t , 3H, CH 3C)T. Found (powder): C, 74.56; H, 9.03; N, 7.31. Calc. f o r C2^202%2'. C, 74.96; H, 8.75; N, 7.60. Found (solvate): C, .72.21; H, 8.71. Calc. f o r C^H^O^.MeOH: C, 71.96; H, 9.06. Modified Kuhn-Roth Oxidation^ 7 of D i h y d r o s i t s i r i k i n e (25) D i h y d r o s i t s i r i k i n e (5 mg.) and 10$ chromic acid (2 ml.) were put i n a d i s t i l l a t i o n apparatus, d o u b l e - d i s t i l l e d water (2 ml.) added, and d i s t i l l a t i o n begun immediately. I t was continued with periodic a d d i t i o n of water u n t i l 30 ml. of d i s t i l l a t e had been c o l l e c t e d . This was neutralised with . 93 -2 N aqueous potassium hydroxide (pH meter), and evaporated to dryness. The residue was taken up i n pure water (0.3 ml.) and put on a small column of Dowex 50 acid r e s i n (2 x 0.5 cm.); the f l a s k was washed with water (2 x 0.5 ml.) and t h i s also added to the column. To the f i l t r a t e was added a few drops of 70$ aqueous ethylamine, and i t was then concentrated under vacuum at 30-40° down to 1-2 drops. This was spotted on Whatman No. 1 paper together with standard solutions of the ethylamine s a l t s of a c e t i c , propionic and butyric acids. The paper was developed by descending chromatography, using a 0.025 M ethylamine s o l u t i o n i n water-saturated n-butanol as the stationary phase, and water-saturated •57 n-butanol as the mobile phase^ . A f t e r 24 hours the paper was sprayed with a l c o h o l i c bromocresol green s o l u t i o n , when the acids became v i s i b l e as blue spots on a yellow background. D i h y d r o s i t s i r i k i n e gave a c e t i c and propionic a c i d s , and a blank oxidation showed only the barest trace of acetic a c i d . S i t s i r i k i n e under these conditions also gave a c e t i c and propionic acids, -while i s o s i t s i r i k i n e gave ac e t i c acid only. Ozonisation of S i t s i r i k i n e (26) and I s o s i t s i r i k i n e (31) S i t s i r i k i n e (5 mg.) i n g l a c i a l a c e t i c acid (1 ml.) was ozonised f o r 5 minutes, then transferred to a d i s t i l l a -t i o n apparatus containing 5$ aqueous ferrous sulphate (15 ml.). - 94 -After 30 minutes the mixture was steam d i s t i l l e d i n t o an aqueous s o l u t i o n of 2,4-dinitrophenylhydrazine sulphate u n t i l about 10 ml. of water had passed over. The-solution was ex-tracted several times with benzene, the combined extracts dried with magnesium sulphate and flushed through a column of Pisher acid-washed alumina (10 g.). A f t e r concentration of the benzene s o l u t i o n to about 0.5 ml., a few drops were spotted on Whatman No. 1 paper, together with standard solu-tions (5 mg./ml.) of the 2,4-dinitrophenylhydrazones of form-aldehyde , acetaldehyde and acetone. 36 The paper was developed by descending chromatography-^, using methanol-heptane as the stationary phase and heptane as the mobile phase. A f t e r 6 hours the paper was sprayed with 10$ aqueous sodium hydroxide s o l u t i o n . The 2,4-dinitro-phenylhydrazone of formaldehyde (red-brown spot, Rf 0.10) was c l e a r l y i n d i c a t e d , and a trace of acetone (dark-brown spot, Rf 0.30) was also present. Blank experiments gave no spots corresponding to formaldehyde or acetaldehyde, but always showed a trace of acetone. Repetition of the same procedure for i s o s i t s i r i k i n e (5 mg.) showed formation of acetaldehyde 2,4-dinitrophenyl-hydrazone (Rf 0.19). Lead Tetracetate Dehydrogenation of D i h y d r o s i t s i r i k i n e Lead tetracetate (400 mg.) was added i n small portions over a period of 10 minutes to a s o l u t i o n of d i h y d r o s i t s i -- 9 5 -r i k i n e (100 mg.) i n g l a c i a l a c e t i c acid (10 ml.). The mix-ture was kept at 50-60° f o r a further 20 minutes, then poured i n t o i c e - c o l d 50$ aqueous sodium hydroxide s o l u t i o n and extracted with chloroform. The chloroform extract was washed with a l i t t l e water, dried over sodium sulphate, and a c i -d i f i e d to Congo red with 8 M ethanolic hydrogen chloride. Evaporation of the solvent afforded tetradehydro-dihydrositsi-r i k i n e hydrochloride as a gum (70 mg.), which could not be induced to c r y s t a l l i s e ; X.,^- (acid and neutral s o l u t i o n ) : 253, 308, 365 mw.;X v ( a l k a l i n e s o l u t i o n ) : 284, 528 mu ; (CHOI,): 1700 (C=0), 1630 (aromatic) cm."1. Attempted Palladium Dehydrogenation of Tetradehydro - d i h y d r o s i t s i r i k i n e Hydrochloride The hydrochloride (55 T a g . ) from the previous reaction was mixed with palladium black (50 mg.) and heated at 250-270° under nitrogen f o r 7 minutes. The residue was leached with trot methanol, the s o l u t i o n f i l t e r e d , and the U.V. spectrum run d i r e c t l y on t h i s s o l u t i o n . I t was unchanged from that of the s t a r t i n g m a terial. The r e a c t i o n was repeated twice, heating a t 280° for 15 and 30 minutes; however, no change was observed i n the U.V. spectrum. Palladium-charcoal Dehydrogenation of D i h y d r o s i t s i r i k i n e (25) D i h y d r o s i t s i r i k i n e (50 mg.) was w e l l mixed with 10$ palladium-charcoal(250 mg.) and heated under nitrogen at 250° - 96 f o r 15 minutes. The residue was extracted with hot methanol and the U.V. spectrum r u n ; X m Q V ; 230, 290, 310, 385 mu. Aft e r removal of the methanol, the product was taken up i n ether-water, the ether la.yer separated and dried over sodium sulphate. Removal of the solvent gave a gum (30 m g -);X m a X o: 230, 288, 317 m^i . The aqueous portion was made strongly a l k a l i n e (pH>10) and extracted with chloroform. A f t e r drying, the chloroform s o l u t i o n was evaporated to afford a gum (5 nig.);"^... : 295, IH3 X « 313, 348, 389 mjx . The neutral extract was dissolved i n a few drops of methanol and spotted on a. preparative T.L.C. plate ( s i l i c a g e l , 0.5 mm. t h i c k ) . The plate was developed i n chloroform-ethyl acetate (3:1) f o r 45 minutes, dried, and then developed two more times. Under U.V, l i g h t four bands CDuld be seen, and each was cut out and extracted with methanol i n a Soxhlet apparatus f o r several hours. The extract from the main band (compound A) displayed U.V. spectra s i m i l a r to those of harman 4^ (Figures 5 and 6 ) ; \ m Q V (neutral and a l k a l i n e s o l u t i o n ) : 234, 250, 282, 288, 337, 349 mu. ;~k (acid ' max. so l u t i o n ) : 254, 303, 372 mjx . Evaporation of the methanol yielded a gum (10 mg.); V m Q V (CHC1,): 1710 (0=0), 1625 (0=0) „ m «i cm. . - 97 -Palladium-charcoal Dehydrogenation of D i h y d r o s i t s i r i k i n e Hydrobromide The hydrobromide (50 mg.) was well mixed with 10$ palladium-charcoal (200 mg.) and heated under nitrogen at 280° f o r 15 minutes. The residue was extracted with hot methanol, f i l t e r e d , and the U.V. spectra run; X„,_v (neutral and a c i d i c s o l u t i o n ) ; 221, 250, 308, 366, 380 mjx ; X m a X o ( a l k a l i n e solution)? 282, 310, 382 inu . The solvent was removed and the residue dissolved i n water, the s o l u t i o n made basic (pH 8) with ammonia and shaken with ether. A f t e r separation and drying the ether was removed to leave a gum (5 mg.); X m a x > • 290, 355 m^ . . 50$ Aqueous sodium hydroxide was added to the aqueous portion u n t i l i t was strongly a l k a l i n e (pH>10), and the s o l u t i o n was then extracted with chloroform. The red chloro-form extract was washed with water, dried, ana a c i d i f i e d with 8 W ethanolic hydrogen chloride (yellow s o l u t i o n ) . Eva-poration of the solvent afforded a gum (22 mg«)>X m a X i (acid and neutral s o l u t i o n ) : 222, 308, 383 myx ;Xfflax^ ( a l k a l i n e s o l u t i o n ) : 283, 315, 380 m/x. . This material was dissolved i n a l i t t l e methanol and spotted on a preparative T.L.C. plate ( s i l i c a g e l , 0.5 mm. t h i c k ) . This plate was run f o r 20 minutes i n ethyl acetate, and then twice i n ethanol-ethyl acetate (1:1) f o r 40 minutes. Under U.V. l i g h t a separation into two main bands was observed. These were cut out and extracted with methanol i n - 9 8 -a Soxhlet apparatus f o r several hours. One f r a c t i o n gave a U.V. spectrum analogous to that of tet r a d e h y d r o - d i h y d r o s i t s l r i k i n e h y d r o c h l o r i d e ; ^ m a x < ; 251, 307, 365 nyi . The other f r a c t i o n (compound B) gave a U.V. spectrum s i m i l a r to that of 5,6-dihydroflavocoryline h y d r o c h l o r i d e 4 3 ; A m a x >: 221, 312, 386 m^ ; X m i n > : 215, 277, 338 mjx . Quinone Dehydrogenation of Compound B The methanolic s o l u t i o n of compound B was evaporated to give a gum (2 mg.), which was dissolved i n g l a c i a l a c e tic acid (0.5 ml.). 2,3-Dichloro~5,6-dicyano-p~benzoquinone * o (10 mg.) was added, and the mixture heated at 80-90 for 6 hours. The s o l u t i o n was then d i l u t e d with water and extrac-ted several times with ether. A f t e r making strongly a l k a l i n e with 50$ aqueous sodium hydroxide, the aqueous s o l u t i o n was shaken with chloroform, the organic layer separated, washed with a l i t t l e water and dried over sodium sulphate. A c i d i -f i c a t i o n with 8 N ethanolic hydrogen chloride and removal of the solvent afforded a yellow gum (0.8 mg.). This (compound C) displayed a U.V* spectrum (Figure 7) very s i m i l a r to that of f l a v o c o r y l i n e h y d r o c h l o r i d e 4 5 ;X m a x < : 237, 291, 345, 385HUA The compound displayed the same Rf value (0.43) as an authentic sample of f l a v o c o r y l i n e on paper chromatography using an ethyl acetate-pyridine-water (8:2:1) system. - 99 -D i h y d r o s i t s i r i k i n e O l e f i n i c Ester (22) D i h y d r o s i t s i r i k i n e acetate (1.3 g.) was heated under r e f l u x with 0.1 N sodium methoxide i n dry methanol (60 ml.) for 45 minutes. S o l i d carbon dioxide was then added, the methanol removed under vacuum, and the residue taken up i n ether-water. The ethereal s o l u t i o n was dried and the solvent removed. Chromatography of the product on alumina (50 g.) a f f o r -ded the desired material (410 mg.) on e l u t i o n with benzene-ether (19*1)° Ether eluted d i h y d r o s i t s i r i k i n e (605 mg.). R e c r y s t a l l i s a t i o n from methanol afforded the solvated o l e f i n i c ester as needles (290 m g . ) , m.p. 84-89° ; one spot on T.L.C.(EtOAc-CHCl,, 1:1); Totl 2 6+2° (MeOH);V m Q V (Nujol): j L J nicix o 3160 (NH), 1708 (0=0), 1622 (C=C); N.M.R. signals (CDClj): 1.60 ( s i n g l e t , IH, NH), 2.8 (m u l t i p l e t , 4H, aromatic), 3.73 ( s i n g l e t , IH, o l e f i n i c ) , 4.41 ( s i n g l e t , IH, o l e f i n i c ) , 6,23 ( s i n g l e t , 3H, CH 30), 9.06 (broad s i n g l e t , 3H, C H j O f . Found: C, 71.41; H, 8.08; 0, 12.71; N, 7.12. Calc. f o r C 2 1H 2 60 2N 2.MeOH: C, 71.32; H, 8.16; 0, 12.96; N, 7.56. Desoxy-Dihydrositsirikine (23) The above o l e f i n i c ester (95 mg.) i n methanol was hydro-genated over palladium black (15 mg.). Uptake of hydrogen ceased a f t e r 3 hours when 1.04 mol. had been absorbed. The so l u t i o n was f i l t e r e d , heated to b o i l i n g , and water added drop-wise u n t i l a permanent t u r b i d i t y was observed. On cooling the saturated ester c r y s t a l l i s e d out as prisms (80 mg.), m.p. - 100 -172-177° ; two spots on T.L.C. (EtOAc-CHCl,, 1:1); (Nujol): 3370 (NH),1710 (C=0) cm."1. Pound: C, 74.04; H, 6.43; H , 8.36. Calc. f o r P 2 l H 2 8 0 2 N 2 : c» 74.08, H, 8.29; N, 8.23. Lithium Aluminium Hydride Reduction of D i h y d r o s i t s i r i k i n e • O l e f i n i c Ester (22) The o l e f i n i c ester (150 mg.) and l i t h i u m aluminium hydride (100 mg.) i n ether-tetrahydrofuran (1:1, 30 ml.) were heated under r e f l u x - f o r 2 hours. Excess hydride was decomposed with saturated aqueous sodium sulphate s o l u t i o n (20 ml.), the orga-nic l a y e r separated, and the aqueous suspension extracted with methylene chloride (3 x 10 ml.). The combined organic extracts were dried over magnesium sulphate and evaporated to leave a c r y s t a l l i n e s o l i d (140 mg.). This material had no carbonyl absorption i n the I.R. region, but showed three spots on T.L.C. The N.M.R. spectrum indicated that the mixture contained only 25$ of the expected terminal o l e f i n i c a l cohol. The product was taken up i n benzene and chromatographed on alumina (5 g., deactivated with 0.3$ of g l a c i a l a c e t i c a c i d ) . E t h y l acetate eluted the major f r a c t i o n (70 mg.), which was r e c r y s t a l l i s e d twice from acetone-petroleum ether (b.p. 60-80°) and once from aqueous methanol to afford l i g h t brown needles (23 mg.), m.p. 204° ; one spot on T.L.C. (EtOAc);foci 2 6-24.3° , L J D (MeOH); v m t l_ (Nujol): 3200 cm."1 (NH and OH); N.M.R. signals (CDCl^): 1.25 ( s i n g l e t , IH, NH), 2.8 (m u l t i p l e t , 4H, aromatic)T, no o l e f i n i c protons. Pound: C, 77.39; H, 8.49; N , 8.79. - 101 -Calc. f o r C 2 0H 2 60N 2: C, 77.38; H, 8.44; N , 9.03. The constants quoted i n the l i t e r a t u r e 4 2 for iso-des-methoxy-dihydrocorynantheine alcohol (21) are m.p. 204° , rocl 1 7-24.0° (MeOH). JD Dihydrocorynantheine (18h) Crude corynantheine 1" 1" 0 (700 mg., two spots on T.L.C.) was hydrogenated i n ethanol (5 ml.) over palladium black. Uptake of hydrogen ceased a f t e r 20 minutes when 0.6 mol. had been absorbed. A f t e r f i l t r a t i o n the s o l u t i o n was warmed to 65° and water added dropwise u n t i l the s o l u t i o n remained cloudy. On cooling dihydrocorynantheine was obtained as needles (610 mg.), m.p. 174-176°; one spot on T.L.C. (EtOAc); V ( C H C 1 , ) : 3420 XucLX a j (NH), 1690 (C=0), 1628 (C=C) cm.""1. N.M.R. signals (CD5COCD5)s 0.43 ( s i n g l e t , IH, NH), 2.8 ( m u l t i p l e t , 5H, aromatic and o l e -f i n i c ) , 6.27 ( s i n g l e t , 3H, CH^O), 6.40 ( s i n g l e t , 3H, CH^O), 9.1 (broad s i n g l e t , 3H, C H j O t . Pounds C, 71.78; H, 7.91; H, 7.56. Calc. f o r C 2 2H 2 80 3N 2: C, 71.71; H, 7.66; N, 7.60. I l l o The m.p. quoted f o r dihydrocorynantheine i s 173-174 . Desmethyl-dihydrocorynantheine (24) A s o l u t i o n of dihydrocorynantheine (500 mg.) i n acetone (50 ml.) was cooled i n ic e and dry hydrogen chloride passed i n f o r 15 minutes. A f t e r standing at 5° f o r 15 hours, the so l u t i o n was evaporated under vacuum to small bulk, d i l u t e d with water (100 ml.) and extracted with chloroform (10 x 50ml.). 102 The water layer was then made basic with aqueous ammonia, extracted with ether (3 x 50 ml.) and the combined ether extracts dried over magnesium sulphate. Removal of the ether afforded desmethyl-dihydrocorynantheine as an amorphous powder (240 mg.), which gave a p o s i t i v e f e r r i c chloride t e s t (purple); \> v (CHOI,): 3480 (NH) 1715 (0=0), 1658 (H0-C=C-C=0), 1607 (C=C) cm."1. N.M.R. signals (CD^COCDj): 1.01 ( s i n g l e t , IH, NH), 2.8 (m u l t i p l e t , 4H, aromatic), 6.28 and 6.52 (2 sing-l e t s , 3H, CH^O), 9.1 (broad s i n g l e t , 3H, CH 5C)T. Found: C, 71.22; H, 7.91; N, 8.21. Calc. f o r C21 H26°3 N2 ! C» 7 1 ° 1 6 » H> 7.39; N, 7.90. Desmethyl-dihydrocorynantheine (200 mg.), i n methanol (10 ml.), was reduced with sodium borohydride (500 mg.). A f t e r 1 hour the methanol was removed under vacuum, the residue t r e a -ted with water and then extracted with ether. The ethereal layer was separated, dried over magnesium sulphate and the s o l -vent evaporated. The r e s u l t i n g material was dissolved i n benzene and chromatographed on alumina (10 g.). E l u t i o n with ether yielded a s o l i d which was r e c r y s t a l l i s e d twice from pe-troleum ether (b.p. 60-80° ) to afford stout* needles (65 mg.), \) v (Nujol): 3400 (NH), 3200 (OH) 1710 (0=0) cm.". Found: I U c l X a C, 70.81; H, 7.94; N, 7.96. Calc. f o r C 2 1H 2 Q0 5N 2: C, 70.76; H 7.92; N, 7.86. m.p. 215° ; one spot on T.L.C. (MeOH); -1 ™_ - 103 -This compound was i d e n t i c a l with d i h y d r o s i t s i r i k i n e by a l l c r i t e r i a : m.p. and mixed m.p.; o p t i c a l r o t a t i o n ; Rf value on T.L.C; superimposible I.R. spectra. I s o s i t s i r i k i n e (31) I s o s i t s i r i k i n e was obtained from Dr.M. Gorman, E l i L i l l y Laboratories as the c r y s t a l l i n e sulphate, m.p. 263.5° . Pound: C, 62.70, H, 6.69; 0, 20.14; N, 6.93; S, 3-97; C-Me, 2.95; O-Me, 7.65. Calc. f or C^Hg^OjNg'iHgSO^: C, 62.52; H, 6.74; 0, 19.84; N, 7.00; S, 3-97; (1) C-Me, 3.72; (1) O-Me, 7.69. The free base was an amorphous powder, [cx.| 2^=20° (CHC1,); D ? one spot on T.L.C. (EtOAc); A. ( l o g € ) : 224 (4.55), 283 (3-92), in 3.x o 291 (3.84) ma ; V m o v (0HCl,)s 3400 (NH and OH), 1725 (C=0) cm."1, no Bohlmann bands 4 4; v m o v (Nujol): 3300 (NH and OH), 1720 (C=0), 740 (o-disubstituted benzene) cm,"1; N.M.R. signals (CDCl^): 1.33 ( s i n g l e t , IH, NH), 2.7 (m u l t i p l e t , 4H, aromatic), 4.53 (quartet, J = 7 c.p.s., IH, C=CH-CH5)9 6.28 ( s i n g l e t , 3H, CH 50), 8,40 (doublet, J = 7 c.p.s., 3H, CH 5-CH=C)t. Pound: C, 70.65; H, 7.75; 0, 14,15; N, 7.53; C-Me, 3.28; O-Me, 9.17; M.W. 354. Calc. for C 2 1H 2 60 5N 2: C, 71.16; H, 7,39; 0, 13.54; N, 7.90; (1) C-Me, 4.24; (1) O-Me, 8.76; M.W. 354, I s o s i t s i r i k i n e picrate was prepared i n the manner des-cribed f o r s i t s i r i k i n e and r e c r y s t a l l i s e d from methanol as yellow needles, m.p. 216° . Pounds C, 55.60, 55.69; H, 5.20, 5.38; 0, 27.29; N, 12.17, 11.92, Calc. f o r C 2 7 H 2 9 0 1 0 N 5 : - 104 -C, 55.57; H, 5.01; 0, 27.42; N, 12.00. Ac e t y l a t i o n of I s o s i t s i r i k i n e with a c e t i c anhydride i n pyridine gave an amorphous monoacetate which was homogeneous by T.L.C. (EtOAc-CHCl,, 1:1); v^,, (0C1.): 3380 (NH), 1730 (0=0), 1235 (OAc) cm."1; N.M.R. signals (CDClj): 1.40 ( s i n g l e t , IH, NH), 2.7 (m u l t i p l e t , 4H, aromatic), 4.39 (quartet, IH, C=CH-CH5), 6.05 (doublet, 2H, CH~CHo0Ac) 6.27 ( s i n g l e t , 3H, CH 50), 8.15 ( s i n g l e t , 3H, CH5C=0), 8.36 (doublet, 3H, CH3-CH=C)T. Found: C, 69.84; H, 7.78; N, 7.44. C a l c . for Co^H^O^Ng: C, 69.67, H5 7.12; N, 7.07. Acetonide of I s o s i t s i r i k i n e D i o l A s o l u t i o n of i s o s i t s i r i k i n e (300 mg.) i n tetrahydrofuran (10 ml,) was run i n t o a suspension of lithiumaluminium hydride (300 mg.) i n ether (5 ml.), and the mixture heated under r e f l u x f o r 3 hours. Saturated aqueous sodium sulphate (10 ml.) was added with s t i r r i n g and the organic l a y e r s e p a r a t e d . A f t e r d i -l u t i o n with water (20 ml.) the aqueous layer was extracted with methylene chloride (3 x 20 ml.), and the combined organic ex-tr a c t s were dried over sodium sulphate. Evaporation of the s o l u t i o n afforded a gum (240 mg.); v m a v : 3200 (OH and NH)cm."1, no carbonyl absorption; N.M.R. signals (CD^COCD^): 2.8 ( m u l t i -p l e t , 4H, aromatic), 4.53 (quartet, IH, C=CH-CH5), 8.37 (doublet, 3H, CH^-CH=C)T , no methoxyl absorption. Since i t could not be induced to c r y s t a l l i s e , the gum was taken up i n dry acetone (10 ml.), p-toluenesulphonic acid - 105 -(150 mg.) added, and the mixture heated under r e f l u x f o r 30 minutes. A f t e r standing overnight the s o l u t i o n was made basic with aqueous ammonia, and the acetone removed under vacuum. The residue was extracted with ether, the ethereal s o l u t i o n dried over sodium sulphateand evaporated to leave a gum. This was taken/up i n benzene and f i l t e r e d through alumina (5 g.). Removal of the benzene and r e c r y s t a l l i s a t i o n from methanol afforded i s o s i t s i r i k i n e acetonide as needles (42 mg.), m.p. 105-109° ; one spot on T.L.C. (EtOAc); f a 1 2 6-53° (CHOI,); D (Nujol): 3200 (NH) cm."1; N.M.R. signals (CDC1,): 1.87 ( s i n g l e t , IH, NH), 2.8 ( m u l t i p l e t , 4H, aromatic), 4.40 (quartet, IH, C=CH-CH5), 8.30 (doublet, 3H, CH5~CH=C), 8.63 ( s i n g l e t , 3H, (CH 5) 2C0 2), 8.68 ( s i n g l e t , 3H, (CH 5) 2C0 2)T. Found: C, 72.10,, 72.19; H, 8.39; 8.46; N, 7.20. Calc. f o r C 23 H30 02 N2* MeOH: C, 72.33, H, 8.60; N, 7.03. D i h y d r o - i s o s i t s i r i k i n e (29) I s o s i t s i r i k i n e base (200 mg.), i n methanol (5 ml.), was hydrogenated over palladium black (20 mg.). Uptake of hydrogen ceased a f t e r 5 hours, when 1.02 mol. had been absorbed. Removal of the c a t a l y s t and the solvent; yielded an amorphous product, which showed two spots on T.L.C. (EtOAc). The major component (120 mg.) was i s o l a t e d from the ether-benzene (1:3) eluate during chromatography on alumina (10 g.). D i h y d r o - i s o s i t s i r i k i n e was an amorphous powder which could not be induced to c r y s t a l l i s e , but was homogeneous on T.L.C. i\maXt (log£): 226 (4.57), 284 ( 3 . 9 2 ) , 291 (3.84) mju. ; - 106 -V m o v (CHOI,): 3480 (NH and OH), 2810 and 2760 (Bohlmann bands)'1 max o j 1720 (0=0) cm."1; N.M.R. signals (CDC1 5): 2.01 ( s i n g l e t , IH, NH), 2.8 (m u l t i p l e t , 4H, aromatic), 6.20 ( s i n g l e t , 3H, OH^O), 9.03 (broad s i n g l e t , 3H, CH 5C)X . Pounds 0, 70.30; H, 7.53; N , 8.13. Calc. f or C 2 1H 2 80 5N 2: C, 70.76; H, 7.92; N, 7.86. D i h y d r o - i s o s i t s i r i k i n e picrate was formed i n the usual manner and r e c r y s t a l l i s e d from aqueous methanol as yellow p l a t e l e t s , m.p. 187° . Pound: C, 54.65, 54.30; H, 5.68, 4.84; 0, 27.57; N, 11.89. Calc. f o r 0 27 H31°10 W5'^ H2 0 : G> 54.56; H, 5.43; 0, 28.23; N , 11.79. Lead Tetracetate, Oxidation of J P i h y d r o - i s o s l t s i r i k i n e (29) The dihydro compound (100 mg.) was dissolved i n acetic acid (10 ml.), lead tetracetate (400 mg.) added i n small portions, and the mixture heated at ca. 60° for 2 hours. A f t e r removal of the solvent the residue was taken up i n water (20 ml.), made strongly a l k a l i n e with 50$ aqueous potassium hydroxide and ex-tracted with chloroform ( 3 x 20 ml.). The combined extracts were dried over sodium sulphate, a c i d i f i e d with 8 N ethanolic hydrogen chloride and evaporated to dryness. Tetradehydro-dihydro-isositsirikine (30) hydrochlo-ride was thus obtained as a red glass (70 mg.);X m c i v : 253, 308, 360 mu;V m a x < (OHClj, free base): 1730 (0=0), 1615 cm."1; T.L.C. (EtOAc) showed no s t a r t i n g material. - 107 -Sodium Bprohydride Reduction of Tetradehydro-dihydro-i s o s i t s i r i k i n e (50) The above hydrochloride (60 mg.), i n methanol (5 ml.), was treated with sodium borphydride (200 mg.), and heated under r e f l u x f o r 1 hour. The solvent was removed'under vacuum, the residue taken up i n water (10 ml.), and extracted-with ether (4 x 10 ml.) A f t e r drying, the ethereal s o l u t i o n was evaporated, the product taken up i n benzene, and chromatographed on alumina( 3 g«). Benzene-ether (3^1) eluted a compound- (21 mg.) which was found to be i d e n t i c a l to d i h y d r o - i s o s i t s i r i k i n e (U.V. and I.R. spectra, T.L.C). Palladium Dehydrogenation of I s o s i t s i r i k i n e Sulphate I s o s i t s i r i k i n e sulphate (60 mg.) was intimately mixed with palladium black (60 mg.) and heated at ea. 270° under a nitrogen atmosphere f o r 10 minutes. The product was taken up i n hot methanol, the s o l u t i o n f i l t e r e d , evaporated down to a few drops and spotted on a preparative T.L.C. plate ( s i l i c a g e l , 0.5 mm. t h i c k ) . The plate was developed twice i n e t h a n o l - e t h y l acetate (1:1), then viewed underU.V. l i g h t and the fluorescent bands cut out. Each section was e x t r a c t e d - w i t h methanol i n a Soxhlet apparatus f o r several hours, and the U.V,spectrum run on the so l u t i o n . S i g n i f i c a n t spectra were shown by two f r a c t i o n s : ( i ) The f r a c t i o n with Rf 0.9 had a U.V. absorption s i m i l a r to that of harman 4 3 ( c f . Figures 5 and 6 ) ; \ (neutral solu-in 3.x • t i o n ) : 232, 282, 289, 336, 347 ;"K m i n <: 273, 305, 340 nyi ; - 108 -A m a x > (acid s o l u t i o n ) : 251, 302, 374 m/i ;X m i n > : 233, 279, 330 mjji . ( i i ) The f r a c t i o n with Rf 0.1 had a spectrum reminiscent of f l a v o c o r y l i n e 4 3 (neutral and acid s o l u t i o n ) : 237, 247, 291, 345, 385 m/x ; A m i n # : 245, 274, 304, 373 m^ t . The methanolic s o l u t i o n of ( i i ) was evaporated and the residue taken up i n a l i t t l e water. 50$ Aqueous potassium hy-droxide was then added u n t i l the s o l u t i o n was strongly* a l k a l i n e , and the mixture extracted with chloroform ( 3 x 10 ml.) A f t e r drying over potassium carbonate the chloroform extract was a c i d i f i e d with 8 N ethanolic hydrogen chloride and evaporated to leave a yellow gum (9 mg.). The I.R. spectrum was s i m i l a r but not i d e n t i c a l to that of f l a v o c o r y l i n e hydrochloride. Paper chromatography showed that most of the material obtained from i s o s i t s i r i k i n e was not f l a v o c o r y l i n e . Using an ethyl acetate-pyridine-water (8:2:1) system, the major component of f r a c t i o n ( i i ) had an Rf of 0.35, whereas f l a v o c o r y l i n e had a corresponding value of 0.43. Palladium Dehydrogenation of D i h y d r o - i s o s i t s i r i k i n e Hydrochloride D i h y d r o - i s o s i t s i r i k i n e (200 mg.) was converted to the amorphous hydrochloride s a l t , which was then intimately mixed with palladium black (200 mg.) and heated under a nitrogen atmosphere at ca. 280° f o r 10 minutes. The residue was taken up i n hot g l a c i a l a c e t i c acid (5 ml.), the s o l u t i o n f i l t e r e d - 109 -and 2,3-dichloro-5,6-dicya.no-p-benzoquinone (200 mg.) added. The mixture was then heated at 90-95° for 5 hours. A f t e r removal of the solvent under vacuum, d i l u t e aqueous ammonia (20 ml.) was added (pH 8), and the s o l u t i o n extracted several times with ether to remove weak bases. The aqueous s o l u t i o n was then made strongly a l k a l i n e (pH>10) with 50$ aqueous potassium hy-droxide and extracted with chloroform (3 x 20 ml.). When the chloroform extract had been dried over potassium carbonate, i t was a c i d i f i e d with 8 N e.thanolic hydrogen chloride and evapora-ted to y i e l d a yellow gum (40 mg.), which displayed a flavocory-line-type U.V. spectrum. Paper chromatography (as above) i n d i -cated that i t was a mixture of two compounds — the major c o n s t i -tuent having the same Rf value .as f l a v o c o r y l i n e , whereas the other corresponded to the compound obtained from i s o s i t s i r i k i n e . Three r e c r y s t a l l i s a t i o n s "from chloroform afforded the major component as yellow needles (6 mg.), m.p. 280-282° (block pre-heated to 150° );X-max# (log€): 238 (4.54), 247 (4.50), 290 (4.13), 345 (4.27), 384 (4.23) m/A : \ m i n > (log£): 210 (4.26), 244 (4.49), 273 (4.04), 303 (4.01), 373 (4.15) mu; v m a x > (Nujol): 3350 (NH), 1650, 1630, 1515 cm.""1; one spot on paper chromato-graphy. This compound was found to be i d e n t i c a l with authentic f l a v o c o r y l i n e hydrochloride i n a l l respects: m.p. and mixed m.p.; superimposible I.R. and U.V. spectra; same Rf values on paper chromatography. - n o -Part I I Experimental Section I s o l a t i o n of Cleavamine (66) and Descarboinethoxy-catharanthine (65) A mixture of catharanthine hydrochloride (40 g.) 1 0 1» stannous chloride (44 g.) and mossy t i n (4 g.) i n concentrated hydrochloric acid (520 ml.), was heated under r e f l u x i n a n i t r o -gen atmosphere for 75 minutes. By the end of t h i s time a red gum had formed. The a c i d i c s o l u t i o n was decanted from the gum and washed with methylene chloride (3 x 100 ml.). The washings were combined with the red gum, then methanol (50 ml.) and methylene chloride (100 ml.) were added so that a clear s o l u t i o n was obtained. This s o l u t i o n was shaken with 1 N aqueous sodium hydroxide (600 ml.), separated and washed with water (200 ml.); the sodium hydroxide s o l u t i o n was washed with ether (2 x 100 ml.) snd the ethereal extract added to the methylene chloride solu-t i o n . A f t e r drying over magnesium sulphate, the organic s o l u t i o n was -evaporated to leave a reddish o i l (32 g.), which was taken up i n benzene and chromatographed on alumina (1200 g.). 101 Cleavamine was eluted i n the i n i t i a l benzene-petroleum ether (b.p. 30-60° ) (1:1) f r a c t i o n s and r e c r y s t a l l i s e d from methanol to give needles (2.4 g.), m.p. 117-119° ; one spot on T.L.C. (EtOAc); [ c x ] 2 6+73° (CHClj) ;.\ m a x < ( log 6.): 225 (4.60), 285 (3.87), 292 (3.86) nyi ;V m a x > . ( N u j o l ) : 3420 (NH), 2000, 2740 and 2700 (Bohlmann bands) 4 4 cm."1, no carbonyl ab-sorption; N.M.R. signals (CD^COCD^): 0.87 ( s i n g l e t , IH, NH) 2.8 (m u l t i p l e t , 4H, aromatic), 4.72 (doublet, IH, o l e f i n i c ) , - I l l -8.96 ( t r i p l e t , 3H, CH 3CH 2)Y. Pound: C, 81.30; H, 8.54; N, 10.18; M.W, 280. Calc. f o r C i gH 2 4N 2: C, 81.38; H, 8.63; N, 9.99; M.W, 280. Benzene-chloroform (1:1) eluted s t a r t i n g material (~7 g.) i n the i n i t i a l f r a c t i o n s and descarhomethoxy-catharanthine (1.0 g.) i n the l a t e r f r a c t i o n s . The l a t t e r material was r e c r y s t a l l i s e d twice from ether to y i e l d needles, m.p. 103-104°, which were i d e n t i c a l with authentic descarbomethoxy-catharan-t h i n e 1 0 1 (I.R,, T.L.C.);V (Nujol): 3140 (NH) cm."1. Pound: C, 81.70; H, 8.12; N, 10.10. Calc.for C i gH" 2 2N 2: C, 81,97; H, 7.97; N, 10,06. 4"ol"-Dihydrocleavamine (68) Cleavamine (1.-5 g.), i n e t h y l acetate (20 ml.), was hydro-genated over Adam's catal y s t (150 mg.). Uptake of hydrogen ceased a f t e r 50 minutes when 1 mol. of hydrogen had been absorbed. F i l t r a t i o n and evaporation gave 4"OL"-dihydrocleavamine, which o r e c r y s t a l l i s e d from methanol as ;prisms (1.2 g.), m.p. 136-138 ; one spot on T.L.C. (EtOAc);V m o v (Nujol); 3410 (NH), 2790 and 2750 (Bohlmann bands) cm.*"1? N.M.R. signals (CD^COCI^): 0.88 ( s i n g l e t , IH, NH), 2.8 (multiplet,4H, aromatic), 9.17 ( t r i p l e t , 3H, CH 5CH 2)r. Pound: C, 81.02; H, 9.59; N, 9.88; M.W. 282. Calc. f or C i gH 2 6N 2: C, 80.80; H, 9.28; N, 9.92; M.W. 282. I s o l a t i o n of B9 The l a t e r benzene-petroleum ether (1:1) f r a c t i o n s from the above chromatography (p.110) displayed three spots on T.L.C. - 112 -(EtOAc) with Rf values of 0 . 77 , 0.53, and 0.27. The compound with a Rf value of 0 .77 was found to correspond to cleavamine. These f r a c t i o n s were combined (5.4 g.) and placed on alumina (450 g.). E l u t i o n was begun with benzene-petroleum ether (1:2), and the f i r s t - four f r a c t i o n s afforded cleavamine(I.4 g.) a f t e r r e c r y s t a l l i s a t i o n frommethanol. Later f r a c t i o n s contained progressively l e s s cleavamine and more of the other two c o n s t i -tuents. The la.st (39, 55 mg.) of the nine f r a c t i o n s obtained with t h i s eluentcontained no cleavamine, displaying only two spots on T.L.C. (Rf 0.53 and 0.27).. R e c r y s t a l l i s a t i o n from aqueous ethanol gave l i g h t brown prisms, m.p. 127-132° ;-"v_Q__ (Wujol): 3410 (NH),2790 and 2740 (Bohlmann bands) cm-."*1; N.M.R. signals (CDCl^): 2.05 ( s i n g l e t , IH, NH), 2.8 (m u l t i p l e t , 4H, aromatic), 9.13 (broad s i n g l e t , 3H, CH^C)T, no o l e f i n i c proton absorption. Pound: C, 80.56; H, 9.46; N, 10.04; M.W. 262. Calc.for C i gH 2 6M 2: C, 80.80; H, 9-28; N, 9.92; M.W. 282. Fraction B9 was shown to be a mixture of 4"cx"- and 4"p!I-dihydrocleavamine by comparison (T.L.C, I.E. spectra) with authentic samples. The 4"w.,,-dihydrocleavamlne was prepared by c a t a l y t i c hydrogenation of cleavamine (see above), and a sample of 4"^"-dihydrocleavamine was kindl y provided by Dr.M. Gorman, L i l l y Research Laboratories. Carbomethoxy-4"fi"-dihydrocleavamih-e (119) A mixture of catharanthine (5) (30 g.) and zinc dust (300 g.), i n g l a c i a l a c e t i c acid (750 ml.), was heated under - 113 -re f l u x i n a nitrogen atmosphere f o r 4 hours. The hot s o l u t i o n was decanted, most of the solvent removed under vacuum, and the residue taken up i n water (100 ml.). The s o l u t i o n was made basic with aqueous ammonia, extracted with ether (4 x 150 ml.), and the combined ether extracts dried over magnesium sulphate. Removal of the ether afforded an o i l which was taken up i n hot methanol (100 ml.). A c r y s t a l l i n e s o l i d (6.3 g.) was deposited on cooling. This material showed three spots on T.L.C. (EtOAc-CHCl^, 1:1) and consequently was chromatographed on alumina (200 g.). E l u t i o n with benzene-petroleum ether (b.p. 30-60° ) (1:1) provided carbomethoxy-4"(*>',-dihydroeleava-mine (4.8 g.), which was r e c r y s t a l l i s e d from methanol to affor d stout needles, m.p. 172° ; one spot on T.L.C. (EtOAc-CHCl^ 1:1); [oc]26+100° (CHC1 3); X m a X o (log-G): 227 (4.47), 286 (3.87), 293 (3.84) mu ; ^ O T . U u j o l ) : 3430 (NH), 2790 (Bohlmann band) 4 4, 1722 (C=0) cm."1; N.M.R. signals•(ODClj): 1.00 ( s i n g l e t , IH, NH), 2.7 (m u l t i p l e t , 4H, aromatic), 6.28 ( s i n g l e t , 3H, CH^O), 9.31 ( t r i p l e t , 3H, CH^CH^T, no o l e f i n i c proton absorption. Pound: C, 74.26; H, 8.35; N, 8.26. Calc. f o r C 2 1 H 2 Q 0 2 N 2 : C, 74.07; H, 8.29; N, 8.23. Neuss et a l . 6 8 quote m.p. 64-66°, [oc] 2 6+96° (CHC1,) for D ? carbomethoxy-dihydrocleavamine. 4 "fi'*-Dihydrocleavamine (68) Carbomethoxy-4 , ,p"-dihydrocleavamine (500 mg. ), i n 5N hydrochloric acid (30 ml.), was heated on the water-bath under - 114 -a nitrogen atmosphere f o r 8 hours. The s o l u t i o n was cooled i n i c e , made basic with, aqueous ammonia, and extracted with methylene chloride (3 x 50 ml.). The organic extract was dried, . concentrated to a small volume and f i l t e r e d through alumina (10 g.). Evaporation of the solvent gave an amorphous powder (340 mg.) which could not be induced to c r y s t a l l i s e ; one spot on T.L.C. (EtOAc);V (Nujol): 3350(HH), 2750 (Bohlmann band) cm."""1", no carbonyl absorption. This material was i d e n t i c a l (T.L.C., I.E. spectra) with an authentic sample of 4"|3"-dihydrocleavamine kindly supplied by Dr.M. Gorman, L i l l y Research Laboratories. Mercuric Acetate Oxidation of Carlomebhoxy^4"fi"-dihydrocleav-amine (119) Carbomethoxy-4"(2>l,-dihydrocleavamine (4.5 g.) and mercuric acetate (10.5 g. ), i n g l a c i a l a c e t i c acid (150 ml. ), were s t i r r e d under a nitrogen atmosphere f o r 40 hours. The s o l u t i o n was then, f i l t e r e d from the p r e c i p i t a t e d mercurous acetate (8.2 g, 90$) and heated under r e f l u x f o r 5 hours. The solvent was removed i as f a r as possible under vacuum, the residue made basic with d i l u t e ammonia (50 ml.) and extracted with methylene chloride (3 x 50 ml.). A f t e r drying over sodium sulphate the methylene chloride was removed, and the dark brown product chromatographed on a l u -mina (200 g.,deactivated with 0.6 ml. of g l a c i a l a c e t i c a c i d ) . The i n i t i a l benzene f r a c t i o n s afforded pseudo-vincadifformine - 115 -(121) as a white powder (1.15 g . ) ; O L 1 - 5 0 3 (EtOH); one spot D on T.L.C. (EtOAc-CHCl,, l : 9 ) ; 7 V m Q Y (log 6 ) : 226 (4.07), 298 (4.12), 326 (4.24) m/>L ; v m a 3 U (C01 4)s 3380 (NH), 2780 (Bohlmann band), 1675 (0=0), 1610 (0=0) cm."1; N.M.R signals• (ODClj): 1.05- ( s i n g l e t , IH, NH), 2.8 ( m u l t i p l e t , 4H, aromatic), 6.23 ( s i n g l e t , 3H, GH 50), 9.07 ( t r i p l e t , 3H, OHjCHgJT. Found: C, 74.48, 74.69; H, 7.80, 7.52; 0, 9.62; N, 8.27; M.W. 338. Calc. f o r C 2 1H 2 60 2N 2: .0, 74.52; H, 7.74; 0, 9.46; N, 8.28; M.W. 338. The l a t e r benzene f r a c t i o n s y i e l d e d an amorphous powder (105 mg.); one spot on T.L.C. (EtOAc-CHCl,, l s 9 ) ; X T r i Q V : 226, 285, 293 m / L ; V m a x > (CC1 4); 3400 (NH), 1705 (0=0) cm."1; N.M.R. signals (CDCl^): 2.01 ( s i n g l e t , IH, NH), 2.8 (m u l t i p l e t , 4H, aromatic), 6.30 ( s i n g l e t , 3H, CHjO), 9.10 ( t r i p l e t , 3H, CH 5CH 2)r. The Rf value on T.L.C. and the I.R. spectrum were i d e n t i c a l 71 to those of coronaridine (145) . The amorphous material was taken up i n anhydrous ether and the hydrochloride s a l t formed by passing i n dry hydrogen chloride. Two r e c r y s t a l l i s a t i o n s from acetone-ether afforded the hydrochloride as needles, m.p. 221-223 (dec.); \> (Nujol): 3160 (NH), 2530 (NH), 1715 (0=0) cm."1. An authentic 0 sample of coronaridine hydrochloride, m.p. 221-223 , pre-pared from a Bample of coronaridine k i n d l y provided by Dr.M. Gorman, L i l l y Research Laboratories, did not depress the melting point, andthe I.R. spectra were i d e n t i c a l . Benzene-ether (1 si) e l uted a compound (85 mg.), which was r e c r y s t a l l i s e d from petroleum ether (b.p. 60-80° ) to afford - 116 -prisms, m.p. 143-145.5°; [a] 2 3+49° ( C H G 1 3 ) ; X m a x < : 225, 286, 293 mu ;v „ Q V (KBr): 3350 (NH) 1700 (0=0) cm."1; N.M.R. signals (CDC1 5): 2,00 ( s i n g l e t , IH, NH), 2.8 ( m u l t i p l e t , 4H, aromatic), 1 0 7 6.38 ( s i n g l e t , 3H, CH 50), 9.04 ( t r i p l e t , 3H, CH 5CH 2)T. This material was i d e n t i f i e d as dihydro catharanthine (.'69.) by comparison (m.p., mixed m.p.,.T.L.C., I.R. spectra) with an authentic sample prepared by hydrogenation of catharanthine (5). Acid Hydrolysis of Pseudo-vincadifformine (121) Pseudo-vincadifformine (100 mg.) was heated with 2 N hydrochloric acid (3 ml.) i n a sealed tube a t 110° f o r 6 hous. The s o l u t i o n was made basic with aqueous ammonia and the p r e c i -p i t a t e taken up i n ether. Evaporation of the ethereal s o l u t i o n yielded a gummy product (123) which exhibited t h e s p e c t r a l properties of an i n d o l e n i n e : 221, 250 (broad) m ix ; » max. / v m a x (CCl^): 1605, 1575 cm."1, no NH absorption. The gum was dissolved i n tetrahydrofuran (5 ml.) and heated under r e f l u x with l i t h i u m aluminium hydride (100 mg.) for-3 hours. The excess of hydride was destroyed with saturated aqueous sodium sulphate (10 ml.) and the product i s o l a t e d with ether.Removal of the solvent afforded (124) as an o i l which c r y s t a l l i s e d from acetone as needles, m.p. 89-90° ;rcy."]2 -^.60° L JD (CHC1,); one spot on T.L.C. (EtOAc-CHCl,, 1:9);7v m Q V (log € ) : 243 (3.81), 295 (3.45) m/*.;vmax> (Nujol): 3230 (NH), 1600 (aromatic C=C) cm."1; N.M.R. signals (CDCl^): 3 . 1 ( m u l t i p l e t , 4H, aromatic) 9.10 ( t r i p l e t , 3 H , CH5CH2)*t . Pound: C, 81.00; -• 117 -H, 9.38: N, 9.86; M.W. 282. Calc. for C i qH 2 6N 2; C, 80.80, H, 9.28; N, 9.92; M.W. 282. A c e t y l a t i o n with a c e t i c anhydride i n pyridine afforded the N-acetate, which was r e c r y s t a l l i s e d from petroleum ether (b.p. 60-80° ), m.p. 107.5-109° ; one spot on T.L.C. (EtOAc-CHC13, l : 9 ) ; X m a X t (log£ ): 212 (4^35),253 (4.13), 279 (3.58), 289 (3.51)m^i ;V m a x < (KBr): 1655 (N-C=0), 1595 (aromatic C=C) cm."1; N.M.R. signals (CDC1 5): 1.87 (broad s i n g l e t , IH, aromatic), 2.85 ( m u l t i p l e t , 3H, aromatic), 7.78 ( s i n g l e t , 3H, CH3C=0), 9.10 ( t r i p l e t , 3H,CH 5CH 2 )r . Pound: C, 77.14; H, 8.64; 0, 5.07; N, 8.97. Calc. f o r C 2 4H 2 80N 2: C, 77.73; H, 8.70; 0, 4.93; N, 8.63. Reduction of Pseudo-vincadifformine (121) with Zinc and Sulphuric Acid Pseudo-vincadifformine(1.0 g.) and zinc dust (150 g.), i n 10$ methanolic sulphuric acid (500 ml.), were heated under r e f l u x f o r 30 minutes. The methanol was removed under vacuum, the acid neutralised with aqueous sodium carbonate, and the s o l u t i o n extracted with ether (3x 200 ml.). A f t e r drying,eva-poration of the solvent afforded a gum (0.8 g.) which showed two spots on T.L.C. (EtOAc-CHClj, 1:9). This material was then chromatographed on alumina (40 g.). The major product (640 mg.), dihydro-pseudo-vincadifformine (139&7* eluted with benzene-petroleum ether (b.p. 30-60° ) (l:l)was an amorphous powder (from ether); Tal 2 4-16 ° (EtOH); one spot on T.L.C. (EtOAc-CHC13, l : 9 ) ; X m a x < (loge.): 244 (3.82), 2 9 9 (3o45)m^; V m a X o - 118 -(CC1 4): 3 3 8 0 ' ( H H ) , 1720 (0=0), 1605 (aromatic C=C) cm."1; N.M.R. signals (CDCl^); 3.1 (m u l t i p l e t , 4H, aromatic), 6.33 ( s i n g l e t , 3H, CH 30), 9.12 ( t r i p l e t , 3H, CH 3CH 2)T. Found: C, 73.97; H, 8.13; N, 8.32; M.W. 340. Calc. f o r C 2 1H 2 80 2N 2: C, 74.08; H, 8.29; N, 8.23; M.W. 340. Acet y l a t i o n with a c e t i c anhydride i n pyridine gave an amorphous N-acetate, |od~j 2 3+28° (EtOH); one spot on T.L.C. (EtOAc-CHCl 3, l - 9 ) ; X m a x > (log 6 )'s 253 (4-01), 282 (3.49), 291 (3.45)myx ; " v m a X e (CC1 4): 1725 (C=0), 1660 (N-C=0), 1595 (aromatic C=C) cm."1; N.M.R. signals (CC1 4): 2.3 (broad s i n g l e t , IH, aromatic), 3.0 (m u l t i p l e t , 3H, aromatic), 6.83 ( s i n g l e t , 3H, CH 50), 7.75 ( s i n g l e t , 3H, CH3C=0), 9.08 ( t r i p l e t , 3H, CH 3CH 2)t. Found: C, 72.26; H, 8.24; N, 7.51. Calc. f o r C23 H30°3 N2 ! °» 7 2 ° 2 2 * H» 7.91; N, 7.32. The minor product (80 mg.), iso-dihydro-pseudo-vincadif-formine (I40a)(?was eluted with benzene-ether (1:1) and was also an amorphous powder,fod] 2 4-132° (EtOH); one spot on T.L.C. D (EtOAc-CHCl 3, l s 9 ) ; X m a x > ( l o g € ) : 244 (3.83), 297 (3.45) m^ ; ^ m o V ( C C l J ; 3380 (NH), 1720 (C=0), 1605 (aromatic C=C) cm."1; N.M.R. signals (CLCl 3)s 3.2 (m u l t i p l e t , 4H, aromatic), 6.32 (s i n g l e t , 3H, CH 30), 9.10 ( t r i p l e t , 3H, CH 3CH 2)T. Found: C, 74.38; H, 8.32; N, 8.29. Calc. f o r C 2 1H 2g0 2N 2: C, 74.08; H, 8.29; N, 8.23. Ace t y l a t i o n with acetate anhydride i n pyridine afforded an amorphous N-acetate, foil 2 4+3° (EtOH); one spot on T.L.C, L J D (EtOAc-CHCl,, l : 9 ) ; X T n Q Y (log£): 250 (4.09), 278 (3.47), 296 - 119 -(3.39)m^i ; " v m a x > (CC1 4): 1725 (0=0), 1660 (N-C=0), 1595 (aromatic C=C) cm.""1; N.M.R. signals (CCl^): 2.9 ( m u l t i p l e t , 4H, aroma-t i c ) , 6.42 ( s i n g l e t , 3H,CH 50), 7.86 ( s i n g l e t , 3H,'0H5C=0), 9.10 ( t r i p l e t , 3H, CH^CHgVT.. Found: C, 72.57; H, 8.10. Calc. f o r C 2 3H^0 5N 2: C, 72.22; H, 7.91. Epimerisation of Dihydro-pseudo-vincadifformine (150) A s o l u t i o n of dihydro-pseudo-vincadifformine (200 mg.) i n methanol (2 ml.) was sealed: i n a tube together with sodium methoxide (60 mg.) and saturated methanolic magnesium-methoxide so l u t i o n ( l ml.), and heated at 100° f o r 5 hours. The s o l u t i o n was then poured i n t o water (20 ml.) and extracted immediately with ether (4 x 20 ml.). A f t e r drying over sodium sulphate the ethereal extract was evaporated to give an amorphous powder (165 mg.), which was i d e n t i c a l with iso-dihydro-pseudovinca-difformine (140) (T.L.C, U.V. and I.R. spectra). - 120 -REFERENCES 1. R.L. Noble, C.T. Beer and J.H, Cutts, Ann. N.Y. Acad. S c i . , 16, 882 (1958); i b i d . . 16, 89?- (1958). 2. I.S. Johnson, H.F. Wright and G.H. Svoboda, J. Lab. C l i n . Med.. M, 850 (1959). 3. G.H. Svoboda, N. Neuss, M. Gorman, J. Am. Pharm. Assoc.  ( S c i . Ed.). 48, 659 (1959). 4. G.H. Svoboda, I.S. Johnson, M. Gorman and N. Neuss, J.. 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S p i t e l l e r , Tetrahedron L e t t e r s . 299 (1961) ; J . Am. Chem. Soc . M, 4578 (1962). 95. M. P l a t , J . Le Men, M.M. Janot, Tetrahedron L e t t e r s , 271 (1962) , 96. B.W. Bycroft, D. Schumann, M.B, Pa t e l and H. Schmid, Helv.  Chim. Acta . 47. i n press (1964). 1 2 8 97. J.P. Kutney and E. P i e r s , J , Am. Chem. Soc.. 86. 953 (1964). 98. V/e are g r a t e f u l to Drs, M. Gorman and N. Neuss, L i l l y Research Laboratories, for providing us with experi-mental d e t a i l s i n advance of t h e i r p u b l i c a t i o n ( r e f . 68). 99. J.P. Kutney, R.T. Brown and E. P i e r s , J . Am. Chem.Soc., 8£, 2286 (1964). 100. K. Biemann, M. Spiteller-Priedmarin and G„ S p i t e l l e r , Tetrahedron L e t t e r s , 485 (1961). 101. We wish to thank Dr. M. Gorman, L i l l y Research Laboratories, f o r supplying catharanthine hydrochloride and f o r providing comparison samples of cleavamine and descarbomethoxy-catharanthine. 102. C. D j e r a s s i , S.E. Plores, H. Budzikiewicz, J.M. Wilson, L.J. Durham, J . LeMen, M.M. Janot, M. P l a t , M. Gorman and N. Neuss, Proc. Natl.Acad. S c i . , 4.8, 113 (1962). 103. B. G i l b e r t , J.A. Br i s s o l e s e , J.M. Wilson, H. Budzikiewicz, L.J, Durham and C. D j e r a s s i , Chem. and Ind., 1949 (1962). 104. G.P. Smith and P.N. Edwards, J . Chem. S o c , 152 (1961). 105. J.P. Kutney, R.T. Brown and E. P i e r s , J . Am. Chem. Soc., 86, 2287 (1964). 106. We are g r a t e f u l to Dr. M. Gorman f o r supplying a sample of coronaridine. 107. We are unable to reconcile the differences between our m.p. and r o t a t i o n values and those reported f o r d i -- 129 -hydro catharanthine m.p. 63-65° , [<*-] \ +33° (CHC15) . All our data are consistent with those expected for dihydrooatharanthine. 108. D . Schumann and H. Schmid, Helv. Chim. Acta.. 46. 1996 (1963). 109. J . P . Kutney and B. Piers, unpublished results. 110. Our thanks are due to Dr. R. Goutarel, Institute de Chimie des Substances Naturelles, Gif sur Yvette, Prance, for supplying the corynantheine. 111. R. Goutarel, M.M. Janot, R. Mirza and V. Prelog, Helv.  Chim. Acta. j>6, 337 (1953); M.M. Janot and R. Goutarel, Compt. rend.. 234. 1562 (1952). 112. Por the sake of consistency, the conventional numbering of the Iboga alkaloids [see (5)] has been used for a l l cleavamine derivatives. 113. Some of these resultsLhave been.discussed very Li recently by Dr. M. Gorman at the Fifth Annual Meeting of the American Society of Pharmacognosy, University of Pittsburgh, June 22-25, 1964. 

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