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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 U n i v 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 t h e s i s as conforming to the r e q u i r e d standard  DEPARTMENT OF CHEMISTRY THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1964  In the  r e q u i r e m e n t s f o r an  British  mission  for reference  for extensive  p u r p o s e s may  be  cation  of  written  Department  of  and  by  the  study*  the  for  Library  the  I further  Head o f my  University  agree for  that  of  of  Department  shall  not  per-  scholarly or  t h a t . c o p y i n g or  f i n a n c i a l gain  Columbia,  fulfilment  s h a l l make i t f r e e l y  this thesis  permission-  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  in partial  degree at  I t i s understood  this thesis  w i t h o u t my  that  copying of  granted  representatives.  this thesis  advanced  Columbia, I agree  available  his  presenting  be  by publi-  allowed  The U n i v e r s i t y  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  of  RICHARD TALBOT BROWN  B.A., U n i v e r s i t y B.Sc,  University  o f Oxford, 1961 of Oxford, 1961  MONDAY, AUGUST 10th, 1964, A T 2:00 P.M. IN ROOM 261,  CHEMISTRY BUILDING  COMMITTEE IN CHARGE Chairman:  I . McT. Cowan  N. B a r t l e t t C. T. Beer G. G. S. Dutton External  Examiner:  University  J . P. Kutney C..A..McDowell R. E. I . P i n c o c k E r n e s t Wenkert of Indiana  THE  CHEMISTRY OF THE VINCA ALKALOIDS  SITSIRIKINEy OATHARANTHINE>  AND"THEIR DERIVATIVES  ABSTRACT In p a r t I o f t h i s t h e s i s are d e s c r i b e d the s t r u c t u r a l d e t e r m i n a t i o n s o f 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 , t h r e e new a l k a l o i d s from V i n c a 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 d i h y d r o s i t s i r i k i n e . , ^21^28^3 2» i s o l a t e d as an i n s e p a r a b l e mixture, which was shown by h y d r o g e n a t i o n s t u d i e s t o be comp r i s e d o f an o l e f i n and i t s d i h y d r o d e r i v a t i v e . The f o r m a t i o n o f formaldehyde upon o z o n i s a t i o n o f the mixture, and o f p r o p i o n i c a c i d i n a m o d i f i e d Kuhn-Roth o x i d a t i o n o f 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. N  w  e  r  e  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 monoa c e t a t e s , and the N.M.R. data i n d i c a t e d t h a t 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 e s t e r f u n c t i o n suggested by s p e c t r a l evidence Ttfas e s t a b l i s h e d by h y d r i d e r e d u c t i o n o f 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 i e l d e d an a c e t o n i d e , i t was i n f e r r e d t h a t 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 c h a r a c t e r i s t i c o f an i n d o l e chromophore, which the mass spectrum showed t o be p a r t o f a t e t r a h y d r o - ( 5 - c a r b o l i n e system. Dehydrogenatioh a f f o r d e d a compound w i t h a f l a v o c o r y l i n e type U.V. spectrum, and t h i s suggested t h a t s i t s i r i k i n e was a r e l a t i v e o f the t e t r a c y c l i c c o r y n a n t h e i n e c l a s s o f alkaloids. T h i s was confirmed by c o n v e r s i o n o f dihydrocorynantheine i n t o d i h y d r o s i t s i r i k i n e . The s t r u c t u r e o f the r e l a t e d i n d o l e 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 s e r i e s of r e a c t i o n s . O z o n o l y s i s y i e l d e d acetaldehyde, which a u t h e n t i c a t e d the e t h y l i d e n e group i n d i c a t e d by the N.M.R. spectrum. A c e t y l a t i o n a f f o r d e d a mono-acetate, whose N.M.R. spectrum suggested t h a t i s o s i t s i r i k i n e had a primary h y d r o x y l f u n c t i o n . A methyl e s t e r was e s t a b l i shed by h y d r i d e r e d u c t i o n t o a d i o l , which formed an a c e t o n i d e and hence showed the presence o f a (^-hydroxye s t e r u n i t i n the o r i g i n a l a l k a l o i d . Since dehydrogenat 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 s t r u c t u r e very s i m i l a r t o that of s i t s i r i k i n e  c o u l d be p o s t u l a t e d f o r i s o s i t s i r i k i n e . P a r t I I i s concerned w i t h the c h e m i s t r y of cleavamine, a s c i s s i o n product, of the V i n c a a l k a l o i d c a t h a r a n t h i n e . Treatment of c a t h a r a n t h i n e w i t h aqueous a c i d i n the presence of a r e d u c i n g agent, l e d to the i s o l a t i o n of descarbomethoxycatharanthine, cleavamine and two e p i m e r i c d i h y d r o cleavamines. A t e n t a t i v e mechanism f o r the r e a c t i o n i s proposed, which can account f o r the f o r m a t i o n of these compounds .. Reduction of c a t h a r a n t h i n e i n g l a c i a l a c e t i c a c i d prov i d e d carbomethoxy-dihydrocleavamine. M e r c u r i c a c e t a t e o x i d i s e d t h i s compound t o a mixture of two immonium i o n s , both of which underwent t r a n s a n n u l a r c y c l i s a t i o n s . One of the i o n s gave the known Iboga a l k a l o i d s c o r o n a r i d i n e and d i h y d r o c a t h a f a n t h i n e , whereas the other a f f o r d e d pseudov i n c a d i f f o r m i n e - a s y n t h e t i c analogue of the known V i n c a alkaloid vincadifformine. The s t r u c t u r e of p s e u d o - v i n c a d i f f o r m i n e was determined by c o n v e r s i o n i n t o compounds which had U.V., I.R., N.M.R. and mass s p e c t r a completely analogous to the c o r r e s p o n d i n g d e r i v a t i v e s of v i n c a d i f f o r m i n e . S i m i l a r t r a n s a n n u l a r c y c l i s a t i o n s t o . t h e above a r e post u l a t e d i n the scheme advanced by-Wenkert f o r the b i o g e n e 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 w i t h r e g a r d t o t h i s theory i s duscussed. The f o r m a t i o n of c o r o n a r i d i n e and d i h y d r o c a t h a r a n t h i n e i n the r e a c t i o n c o n s t i t u t e d 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 t r a n s a n n u l a r c y c l i s a t i o n s i n l a b o r a t o r y syntheses of Iboga and Aspidosperma a l k a l o i d s is also considered. GRADUATE STUDIES F i e l d of Study: Chemistry T o p i c s i n Organic Chemistry D.E. McGreer J.P. Kutney, R.E.I. Pincock Seminar i n Organic Chemistry J.P. Kutney S t r u c t u r e of Newer N a t u r a l Products J.P. Kutney Recent S y n t h e t i c Methods i n G.G.S. Dutton Organic Chemistry A. Rosenthal Related Studies: T o p i c s i n I n o r g a n i c Chemistry Topics In Physical Chemistry R.F.  N. B a r t l e t t W.R. Cullen J.A,R. Coope Snider, A.V. Bree  PUBLICATIONS  J.P.  Kutney and R.I. Brown,  The S t r u c t u r e o f S i t s i r i k i n e - A new A l k a l o i d from V i n c a r o s e a L i n n , Tetrahedron  Letters,  No. 26, 1815 (1963)  J.P. Kutney, R.T. Brown and E. P i e r s , The via  Synthesis of a Vincadifformine-Type Skelet a Novel T r a n s a n n u l a r C y c l i z a t i o n R e a c t i o n ,  J . Am. Chem. S o c , 86, 2286 (1964) J.P.  Kutney, R.T. Brown and E. P i e r s ,  The S y n t h e s i s of Iboga A l 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 wish his  constant  research.  to express help  Thanks  and encouragement  the basis  unpublished  f o r the gift  of this  results  t h e course  o f my  t o acknowledge  with  Prof.  C. D j e r a s s i , S t a n f o r d  thanks  of their  of catharanthine. the kindness  U n i v e r s i t y , a n d D r . P.  of A l b e r t a , i n running  Lilly  o f the a l k a l o i d s which  on t h e chemistry  like  compounds.  during  work and f o r communication  would  University  t o Dr. J.P. Kutney f o r  a r e d u e t o D r s . M. G o r m a n a n d N. E e u s s ,  Research Laboratories, formed  uiy g r a t i t u d e  t h e mass  spectra  Also  I  of Kebarle, of several  Abstract In p a r t I of t h i s t h e s i s 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 L i n n . S i t s i r i k i n e , 21 26°3 2' C  C  21 28°3 2' Ii  N  w e r e  H  N  a n d  d i n  y  d r o s i  tsirikine,  i s o l a t e d as an i n s e p a r a b l e mixture, which  was shown by hydrogenation s t u d i e s to be comprised  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 mixture and of p r o p i o n i c a c i d i n a modified Kuhn-Roth o x i d a t i o n of d i h y d r o s i t s i r i k i n e demonstrated t h a t 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 i n d i c a t e d that primary h y d r o x y l 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 e s t e r f u n c t i o n suggested by s p e c t r a l evidence was e s t a b l i s h e d by hydride r e d u c t i o n 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 y i e l d e d an acetonide, i t was i n f e r r e d t h a t d i h y d r o s i t s i r i k i n e possessed a {3-hydroxy-ester  unit.  The U.V. spectrum o f d i h y d r o s i t s i r i k i n e was c h a r a c t e r istic  of an i n d o l e chromophore, which the mass spectrum showed  to be p a r t of a tetrahydro-(2>-carboline system. Dehydrogenat i o n a f f o r d e d a compound w i t h a f l a v o c o r y l i n e - t y p e U.V. spectrum,and t h i s suggested t h a t 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 c l a s s of a l k a l o i d s . This was confirmed by conversion of dihydrocorynantheine i n t o dihydrositsirikine.  iv  The s t r u c t u r e o f the r e l a t e d i n d o l e a l k a l o i d r i k i n e , 21 26°3 2' C  H  N  w  a  s  d e  "  isositsi-  a s i m i l a r series of  t e r m i n e d  r e a c t i o n s . Ozonolysis y i e l d e d acetaldehyde, which a u t h e n t i cated the e t h y l i d e n e group i n d i c a t e d by the N.M.R. spectrum. A c e t y l a t i o n a f f o r d e d a mono-acetate, whose N.M.R. spectrum suggested  t h a t i s o s i t s i r i k i n e had a primary h y d r o x y l f u n c t i o n .  A methyl e s t e r was e s t a b l i s h e d by hydride r e d u c t i o n to a d i o l , which formed an acetonide and hence showed the presence o f 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 dehydrogenation o f d i h y d r o - i s o s i t s i r i k i n e y i e l d e d 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 s t r u c t u r e very s i m i l a r to t h a t of s i t s i r i k i n e could be p o s t u l a t e d f o r i s o s i t s i r i k i n e . P a r t I I i s concerned w i t h the chemistry o f cleavamine, a s c i s s i o n product o f the Vinca a l k a l o i d catharanthine. Treatment o f catharanthine w i t h aqueous a c i d i n the presence o f a reducing agent l e d t o the i s o l a t i o n of descarbomethoxycatharanthine,  cleavamine and two epimeric d i h y d r o c l e a -  vamines. A t e n t a t i v e mechanism f o r the r e a c t i o n i s proposed, v;hich can account f o r the formation o f these compounds. Reduction o f catharanthine i n g l a c i a l a c e t i c a c i d provided carbomethoxy-dihydrocleavamine.  Mercuric acetate o x i d i s e d t h i s  compound t o a mixture o f two immonium i o n s , both of which underwent t r a n s a n n u l a r c y c l i s a t i o n s . One o f the ions gave the known Iboga a l k a l o i d s c o r o n a r i d i n e and d i h y d r o c a t h a r a n t h i n e , whereas the other a f f o r d e d pseudo-vincadifformine — a synthet i c analogue o f the  known Vinca a l k a l o i d v i n c a d i f f o r m i n e .  V  The s t r u c t u r e of pseudo-viricadifformine was b y c o n v e r s i o n i n t o compounds which had U.V.,  I.R.,  determined N.M.R. and  mass s p e c t r a completely analogous to the corresponding  deri-  -  v a t i v e s of v i n c a d i f f o r m i n e . S i m i l a r t r a n s a n n u l a r c y c l i s a t i o n s to the above are p o s t u l a t e d i n the scheme advanced by Wenkert f o r 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 w i t h regard t o t h i s theory i s discussed.  The  formation of c o r o n a r i d i n e and 'dihydrocatharanthine i n the r e a c t i o n c o n s t i t u t e d 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 t r a n s a n n u l a r c y c l i s a t i o n s i n l a b o r a t o r y syntheses of Iboga and Aspidosperma a l k a l o i d s i s a l s o considered.  vi 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 D. The Biogenesis of Yohimbine, Corynantheine and Ajmaline Type A l k a l o i d s .  32 35  PART I I : Some Aspects o f the Chemistry o f Catharanthine and Cleavamine Introduction A. The Catharanthine-Cleavamine  44 Transformation  B. The Biogenesis of Strychnos, Iboga and Aspidosperma A l k a l o i d s  46 56  C. Transannular C y c l i s a t i o n s of Cleavamine D e r i v a t i v e s 66 ( i ) . Pseudo-vincadifformine and i t s D e r i v a t i v e s ( i i ) Coronaridine and Dihydrocatharanthine  69 70  D. D i s c u s s i o n  81  EXPERIMENTAL  85  P a r t I Experimental S e c t i o n 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 Picrate  87  vii Page of  S i t s i r i k i n e Acetate Dihydrositsirikine  oo  Dihydrositsirikine Picrate  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 Dihydrositsirikine  90  Dihydrositsirikine Diol  93.  Acetonide of D i h y d r o s i t s i r i k i n e Modified Kuhn-Roth  Oxidation  Diol  of D i b y d r o s . i t s i r i k i ne  O z o n i s a t i o n 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 Lead T e t r a c e t a t e Dehydrcgenation o f Dihydrosiusirikine Attempted P a l l a d i u m Dehydrogenation of T e t r a d e h y d r o - d i h y d r o s i t s i r i k i n e Hydrochloride  yl 92  93 94 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  P a l l a d i u m - c h a r c o a l 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  98  of Compound B  Dihydrositsirikine Olefinic Ester  99  Desoxy-dihydrositsirikine  99  L i t h i u m Aluminium Hydride .Reduction of Dihydrositsirikine Olefinic 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  Isooitsirikine  103  Acetonide of I s o s i t s i r i k i n e D i o l  104  Dihydro-isositsirikine  105  viii Page  Lead Tetraeetate O x i d a t i o n of Dihydrositsirikine  106  Sodium Borohydride Seduction of Tetradehydrodihydro-isositsirikine  107  P a l l a d i u m Dehydrogenation  of I s o s i t s i r i k i n e Sulphate 107  P a l l a d i u m 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  P a r t I I Experimental S e c t i o n  I s o l a t i o n of Cleavamine and  Descarbornethoxycatharanthine  110  4 "ot"-Dihydro cleavamine  111  Csrbomethoxy-4 ^"-dihydrocleavamine  112  4"{5"-Dihydrocieavamine  113  ,i  Mercuric Acetate O x i d a t i o n of Carbomethoxy4 "(3 "-dihydro cleavamine  114  A c i d H y d r o l y s i s of Pseudo-vincadifformine  116  Reduction of Pseudo-vincadifformine w i t h Zinc and S u l p h u r i c A c i d E p i m e r i s a t i o n of Dihydro-pseudo-vincadifformine  117 119  REFERENCES  120  Figure  F o l l o w i n g page  1  11  2  11  ix  figure  F o l l o w i n g page  5  13  6  16  7  19  8  19  9  25  10  69  11 12  f  ^ 70  GENERAL INTRODUCTION  I n t e r e s t i n the Apocynaceous p l a n t Vinca rosea L i n n . (Lochnera Reichb. or Catharanthus roseus G.Don) was s t i m u l a t e d by the o b s e r v a t i o n that c e r t a i n preparations of the p l a n t 1 2 e x h i b i t e d 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 e x t r a c t s i n a search f o r the a c t i v e p r i n c i p l e s f r e e of extraneous substances. I n i t i a l t e s t i n g i n d i c a t e d that the b i o l o g i c a l l y a c t i v e e n t i t i e s were confined to the complex a l k a l o i d a l p o r t i o n of the plant c o n s t i t u e n t s , and a s e p a r a t i o n scheme was then devised which permitted the i s o l a t i o n of f o u r a c t i v e a l k a l o i d s — v i n c a l e u k o b l a s t i n e (VLB) l e u r o s i n e , l e u r o c r i s t i n e and l e u r o s i d i n e — i n "2.  A  a d d i t i o n t o numerous other a l k a l o i d s of unknown s t r u c t u r e ' . The experimental  anti-tumour a c t i v i t y of these four a l k a l o i d s  a g a i n s t the t r a n s p l a n t e d P-1534 leukemia i n mice has been 15 6 reported by the Canadian group of Noble, Beer and Cutts ' ' 7 8  and by workers a t the L i l l y l a b o r a t o r i e s ' . I t was a l s o 1 5 found that VLB produced severe leukopenia i n r a t s ' , and, moreover, markedly i n h i b i t e d the growth of a t r a n s p l a n t e d 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  The  1 1  1 2  1 3  e a r l i e s t chemical i n v e s t i g a t i o n of the p l a n t was  performed i n the l a t e 19th century by G r e s h o f f ^ , who was only able t o demonstrate the presence of a l k a l o i d a l m a t e r i a l . 1  -  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 d i d not describe any chemical or p h y s i c a l p r o p e r t i e s , 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 r e c e n t l y , s e v e r a l groups have obtained known a l k a l o i d s : 1 7 17 18 1 7 ajmalicine , serpentine ' , akuammicine , t e t r a h y d r o a l s t o 18 1Q 20 nine , l o c h n e r i n e , and r e s e r p i n e „ I n 1958 Kawat and co21  workers  i s o l a t e d two c r y s t a l l i n e and two amorphous a l k a l o i d s ,  and Noble, Beer and Cutts described  VLB . 1  A major advance i n the phytochemistry of Vinca rosea L i n n , was made when the L i l l y group *'* devised an e x t r a c t i o n 3  scheme capable of separating the l a r g e number of a l k a l o i d s present i n the p l a n t . The process c o n s i s t s 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 s o l u b l e i n organic s o l v e n t s from those which are i n s o l u b l e ; a b r i e f o u t l i n e of the procedure i s given i n Scheme 1. The c o n s t i t u e n t s of each f r a c t i o n were f u r t h e r separated by chromatography on alumina and gradient pH e x t r a c t i o n , as shown i n Schemes 2 and 3.  - 3 -  Scheme 1 - E x t r a c t i o n Ground Whole P l a n t Skelly B Defatted  Extract HC1(2N) Ammonia CHC1  1)  I)  1) 2)  Drug  2io T a r t a r i c A c i d Benzene  5  Skelly S o l . (E) I  Drug  "Acid" Benzene E x t r a c t 1) 2)  C H C1 2  4  1) Ammonia 2) Benzene  2% T a r t a r i c A c i d °2 4 2 H  2  Sol.U-^  C1  Ammonia CH H C1  i!  2  G H C1 2  4  Drug  " A l k a l i n e " Benzene E x t r a c t  A c i d Phase 4  1) 2% T a r t a r i c A c i d 2) C H C 1 2  2  4  EtOH  2  2  Sol.(A)  Marc  Extract Phenolic Alkaloids (C,D)  2 4 2 Sol.(B,)  C  H  G 1  Acid Phase 1) 2)  C H C1 2  4  Ammonia 2 4 2 C  H  C 1  ]  2  Sol.(B)  Aqueous Phase 1) NaOH (pH 11) 2) G H 4 2  S o l . = Soluble A l k a l o i d s  CH* 2 Sol.(P) 2  C1  C 1  - 4 Scheme 2 - F r a c t i o n A [Fraction A Chromat, CHC1.  0H-OHO1 (1:1)  011  5  LEUROSINE ISOLEUROSINE  CATHARANTHINE VINDOLININE (.2HC1) AJMALICINE VINDOLINE  VLB(.H S0 ) 2  Sulphate Residues  4  0H~CHC1,(1:1) i -> CAROSINE  1) Free Bases 2) Chromat.  LOCHNERIDINE VIROSINE Mother liquors Chromat, (deact.)  Residues  Chromat.  CHC1. PLEUROSINE  0H-CHC1,(1:3)  j  CHC1.  0H-CHC1 (1:3)  CAROSIDINE  5  Gradient pH(2.7-3.4)  Residues Gradient pH  Gradient pH(4.4-5. VINCARODINE  CATHARICINE  pH 3.9-4.4  pH 4.9-6.4  VINDOLINE  LEUROCRISTINE LEUROSIDINE Mother liquor Chromat.  CHC1 -CH 0H(99:1) 5  Mother liquor Gradient pH(3.4)  NEOLEUROCRISTINE  3  NEOLEUROSIDINE  Chromat.= Chromatography on alumina deact. = d e a c t i v a t e d (alumina.) 0H = Benzene  - 5 Scheme 3 - F r a c t i o n s A,, (A+B), B, , B, F and E.  F r a c t i o n s A+B  F r a c t i o n A-,  Modified Gradient pH(3.3)  Chromat.  0H  0fi-CHcl (3il)  Amorphous Residues  5  TETRAHYDROALSTONINE  Vindoline  Chromat. F r a c t i o n B, Chromat. 0H-CHC1 (3:1) 3  Ajmalicine  0H  0H-CHO1 (3:1)  Catharanthine Tetrahydroserpentine  Vindoline  5  Fraction B  0H-CHC1 (2:l) 5  CATHARINE VINDOLICINE  Fraction F Chromat,  Chromat, g!!-CHCl, (3:1)  0H-CHC1- (1:1)  CHOI.  giaalicine  LOCHNERINE  VINCAMICINE  \1  CHC1, SERPENTINE ( . H N O j )  PER][VINE Mother liquor H S0 2  4  Fraction E  SITSIRIKINE (.i-H S0 ) 2  4  Chromat, r  0H LOCHNERICINE Tetrahydroalstonine  0H-CHC1 (3:1) 3  Vindoline  Table 1 Name  Formula  Akuammicine  G  20 20°2 2  G  21 24°3 2  C  21 24°3 2  Serpentine  C  21 22°3 2  Mitraphylline  G  21 24°4 2  C  20 24°2 2  Ammocalline  C  19 22 2  Perividine  C  20 22°4 2  G  20 24°2 2  C  Perivine Catharanthine  Ajmalicine  H  Fraction N  H  B  Lit.ref. 17,29  17  N  A,E  18  F  17,18  A  29  B  19  B  29  A  29  A  29  20 24°3 2  A  26  C  20 24°3 2  B  3,23  G  21 24°2 2  A  22,28  Lochnericine  °21 24°2 2  E  22,24-  Vindolinine  °21 24°2 2  A  22  G  21 26°3 2  B  26  G  21 26°3 2  B  -  Virosine  G  22 26°4 2  A  3,23  Vinosidine  G  22 26°5 2  A  29  Lochnerivine  G  24 28°5 2  A  29  Vindoline  C  25 32°6 2  A,A ,E  22,27  25 32°6 2  (A+B)  26  A  29  A  4  (A+B)  4,26  A  4  A  4,29  Tetrahydroalstonine  Lochnerine  Gavincine Lochneridine  Sitsirikine Isositsirikine  Vindolicine  H  N  H  N  H  N  H  N  H  N  H  N  H  N  H  N  H  N  H  N  H  H  H  C  H  H  H  H  H  H  N  N  N  N  N  N  N  N  N  Leurosidine  G  41 54°9 4  Vincarodine  C  44 52°10 4  Catharine  G  46 52°9 4  Catharicine  G  46 52°10 4  Leurocristine  G  46 54°10 4  H  N  H  H  H  H  N  N  N  N  X  Table 1. (Cont.) Fraction  Lit.re  46 56°10 4  A  4  46 56 12 4  A  4  46 58°9 4  A  1,25  A  25,25  A  26  A  4  A  4  Vincamicine  B  26  Leurosidine  A  4  Carosidine  A  4  Pericalline  B  29  Ammorosine  A  29  Perosine  B  29  Cavincidine  B  29  Maandrosine  B  29  Cathindine  B  29  Name  Formula  Pleurosine  C  Neoleurocristine  C  Vincaleukoblastine  C  Leurosine  C  Isoleurosine  H  N  H  H  W  G  N  46 58°9 4 H  N  °46 60°9 4 H  Neoleuros±dine  G  Vindolidine  C  N  48 62°11 4 H  N  48 64°10 4 H  R  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  v a r i o u s f r a c t i o n s are l i s t e d i n Table 1, which i n c l u d e s a l l those whose c h a r a c t e r i s a t i o n has been published A p r i l 1964. However, Dr.M.Gorman  up to  very r e c e n t l y i n d i c a t e d  i n a p r i v a t e 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 L i n n . , so that even t h i s t a b l e i s already  out of date. Of the a l k a l o i d s  tabulated,  the s t r u c t u r e s were already known f o r a j m a l i c i n e ( 1 ) , t e t r a hydroalstonine  ( 1 ) , serpentine  ( 2 ) , m i t r a p h y l l i n e (3) and  - 8-  akuammicine ( 4 ) , and have since been determined f o r cathar a n t h i n e ^ ) , perivine (6), vindoline ^(7), vindolicine 2 8  3 0  2  (8), v i n d o l i n i n e ( 9 ) , l o c h n e r i n e ( 1 0 ) , 3 2  3 3  3 1  and l o c h n e r i d i n e ^ 3  (11). The s t r u c t u r e s o f v i n c a l e u k o b l a s t i n e (80) and l e u r o 68 c r i s t i n e ( v i n c r i s t i n e ) are discussed below.  MeOj,C  Me0 C 2  MeO^C  COnMe  (4)  - 9 -  (11) In P a r t I of t h i s t h e s i s i s presented the evidence which l e d to the assignment of s t r u c t u r e s 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 V i n c a rosea L i n n . P a r t I I d i s c u s s e s some aspects of the chemistry of cleavamine, an a c i d rearrangement product of the Vinca a l k a l o i d c a t h a r a n t h i n e , and the  -10-  use of cleavamine and i t s d e r i v a t i v e s 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 L i n n , 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 e x t r a c t y i e l d e d s e v e r a l f r a c t i o n s from which p e r i v i n e (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 f o l l o w i n g 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 p e r i v i n e 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 -i^26 3 2°^ 2 4' The f r e e base was 0  N  H  S0  2  obtained only as an amorphous powder. On the b a s i s of an i n f r a - r e d comparison w i t h -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 L a b o r a t o r i e s , I n d i a n a p o l i s , Indiana, U.S.A., we obtained a sample of s i t s i r i k i n e f o r f u r t h e r s t r u c t u r a l s t u d i e s . Our p r e l i m i n a r y work revealed that the o r i g i n a l a l k a l o i d was a mixture of a t l e a s t three compounds, since on t h i n - l a y e r chromatography a s e p a r a t i o n i n t o three  distinct  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 a p p l i e d without success — chromatography on alumina and s i l i c a g e l , s u b l i m a t i o n , 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 s e v e r a l 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 m a t e r i a l m e l t i n g  CM  oo. -=^—I<0  o o o  10 Q O (/)  UJ  00  Hen  SITSIRIKINE ACETATE SOLVENT  CD COCD 3  3  IH  4H  0 PPM (6)  8  4.7V  5.65 T 637f Figure 2  9.02 f  - 12  sharply a t 181° was obtained. This showed only two spots on t h i n - l a y e r chromatography and analysed f o r the acetone s o l vate, 21 26°3 2' 3 C  II  N  GH  COC  H . Attempts- t o separate the two 3  components were o f no a v a i l , and indeed the mixture behaved as a homogeneous compound except on t h i n - l a y e r chromatography. The unsolvated a l k a l o i d was subsequently  obtained  from aqueous methanol as needles, m.p, 206-208° oC ^-58° 2  L  (MeOH), and analysed w e l l f o r 2i 26°3 2* C  H  N  T  n  i  s  -ID  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 s u b s t a n t i a t e d by a mass s p e c t r a l molecular weight d e t e r m i n a t i o n (354), The u l t r a - v i o l e t spectrum o f s i t s i r i k i n e , w i t h maxima at 226, 282 and 290 mjx i n d i c a t e d an u n s u b s t i t u t e d i n d o l e chromophore. This was confirmed by s i g n a l s i n the n u c l e a r magnetic resonance (N.M.R.) spectrum (Figure 1) a t 0.06T ( i n d o l i c NH) and i n the r e g i o n 2.5-3,IT ( f o u r aromatic p r o t o n s ) . A strong band i n the i n f r a - r e d spectrum a t 1705 cm." was r e a d i l y a t t r i b u t e d t o a carbonyl group and an ab1  s o r p t i o n a t 3360 cm."" was compatible w i t h the presence o f 1  NH and/or h y d r o x y l groups. I n a d d i t i o n to a spike a t 6.38T (CH^O), the N.M.R. spectrum o f s i t s i r i k i n e d i s p l a y e d a two-proton m u l t i p l e t centred a t 6.IT that was p o s s i b l y due to the methylene protons o f a primary a l c o h o l i c f u n c t i o n . The presence o f a h y d r o x y l group was confirmed by the f o r o r 126 © mation o f a monoacetate, C^HggO^^, m.p. 198 , |^oc.J  -26  (MeOH), whose N.M.R. spectrum ( F i g u r e 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 s i g n a l a t 8.02T due  DIHYDROSITSIRIKINE SOLVENT  CD C0CD 3  3H  3  0 PPM(d) 289Y  6.2f 6.42V Figure 3  9.07+  Figure  4  - 13 -  to the a c e t y l group, the m u l t i p l e t present a t 6 . I t i n the spectrum o f the a l c o h o l had s h i f t e d downfield and now appeared a t 5.6T. This s h i f t of 0 . 5 t u p o n 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 o f primary a l c o h o l s , whereas the correspon35 ding s h i f t f o r secondary a l c o h o l s i s about l t u n i t ^ . Besides the above-mentioned s i g n a l s the N.M.R. spect r a o f s i t s i r i k i n e and i t s a c e t a t e d i s p l a y e d a m u l t i p l e t centred a t 4 . 7 f d u e t o o l e f i n i c protons, which i n t e g r a t e d f o r r a t h e r l e s s than two hydrogen atoms. A s e r i e s o f microhydrogenations was r u n on s i t s i r i k i n e and i t was found that only 0.6-0.7 mol. o f hydrogen was taken up. Moreover, the product gave only one spot on t h i n - l a y e r chromatography whose R f value corresponded t o the s m a l l e r o f the two spots e x h i b i t e d 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 o f the mixture d i f f e r e d from each other merely by the presence o f an o l e f i n i c bond i n one o f 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 k i n e , whereas the corresponding dihydro d e r i v a t i v e w i l l be r e f e r r e d t o as d i h y d r o s i t s i r i k i n e . This c o n c l u s i o n was f u l l y - b o r n e out by subsequent work. C a t a l y t i c hydrogenation on a l a r g e r s c a l e and r e c r y s t a l l i s a t i o n from acetone a f f o r d e d s o l v a t e d dihydros i t s i r i k i n e , m.p. 180°, which analysed f o r Cg-j^gO^^' CH^COCH^. F u r t h e r 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 , Hp 0,Np, m.p. 215 , |0(. ?  ft  -55^(MeOH). The N.M.R. spectrum o f d i h y d r o s i t s i r i k i n e (Figure 3) showed a complete disappearance o f the o l e f i n i c  - 14 -  proton a b s o r p t i o n . A strong band a t 1710 cm.  i n the i n f r a -  red spectrum o f the reduced m a t e r i a l excluded any conjugat 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 w i t h maxima a t 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.), a c e t a t e , m.p. 187°,foci -3i°(MeOH), and p-bromobenzoate, m.p. 174°, supD 26  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  c o n f i r m a t i o n was obtained from a. mass s p e c t r a l molecular weight determination (356). Evidence  that the o l e f i n i c l i n k a g e i n s i t s i r i k i n e was  i n f a c t a t e r m i n a l double bond was provided by the appearance o f a new C-methyl a b s o r p t i o n a t 9.07T i n the N.M.R. spectrum o f the r e d u c t i o n product. This was corroborated when o z o n o l y s i s o f 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 o f i t s 2 , 4 - d i n i t r o p h e n y l 36 hydrazone^ . A conventional Kuhn-Roth determination on d i hydrositsirikine  1  i n d i c a t e d 0.93fmol. C-methyl, w h i l e a 37  modified procedure  y i e l d e d p r o p i o n i c a c i d and thus showed  that the new C-methyl f u n c t i o n was i n f a c t part o f a C-ethyl group. These experiments e s t a b l i s h e d t h e presence o f 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  t o e x p l a i n why the  o l e f i n i c proton a b s o r p t i o n i n t h e N.M.R. spectrum o f s i t s i r i k i n e i n t e g r a t e d f o r two r a t h e r than the three hydrogen atoms expected  f o r a v i n y l group. The t h i n - l a y e r chromato-  graphy and microhydrogenation  r e s u l t s had suggested  that  - 15 -  the i m p u r i t y present i n the o r i g i n a l a l k a l o i d was d i h y d r o s i t s i r i k i n e , and a c l o s e s c r u t i n y o f the N.M.R. spectrum o f 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 a b s o r p t i o n a t 9.02T i n t e g r a t i n g f o r about one hydrogen, whereas the mass spectrum i n d i c a t e d a s m a l l peak a t m/e 356 i n a d d i t i o n to t h a t a t m/e 354. A KuhnRoth d e t e r m i n a t i o n on the mixture showed 0.38 mol. Cmethyl, and the modified method a f f o r d e d p r o p i o n i c 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 loid  was a mixture o f 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 o f 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 mater i a l i n a l l subsequent s t u d i e s . Besides p r o v i d i n g the i n f o r m a t i o n discussed above, the N.M.R. spectrum o f d i h y d r o s i t s i r i k i n e ( F i g u r e 3) was very u s e f u l i n e s t a b l i s h i n g the-nature of the two oxygen f u n c t i o n s present i n a d d i t i o n t o the carbonyl group. I n the r e g i o n o f 6.IX there was a two-proton a b s o r p t i o n , a t t r i b u t a b l e to hydrogen atoms attached t o 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  sitsirikine  and confirmed the presence o f a primary a l c o h o l . The nature of the t h i r d oxygen atom was i n d i c a t e d by a s p i k e a t 6.42X r e a d i l y assigned as before t o t h e three protons o f a methoxyl f u n c t i o n . A Z e i s e l d e t e r m i n a t i o n on d i 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 t o the N.M.R. d e s i g n a t i o n . Since the u l t r a - v i o l e t and N.M.R. s p e c t r a  excluded  the p o s s i b i l i t y that the methoxyl was attached to the i n d o l e system, the presence o f a b s o r p t i o n bands a t 1705 and 1165  cm."" i n the i n f r a - r e d r e g i o n suggested that i t was 1  p a r t o f a carbomethoxy group  . An attempted s a p o n i f i c a -  t i o n under m i l d c o n d i t i o n s was u n s u c c e s s f u l , but a f t e r more d r a s t i c treatment an unsaturated  a c i d was i s o l a t e d as  the h y d r o c h l o r i d e , 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 a c i d ) o f t h i s substance showed l o s s o f 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 y i e l d e d a c r y s t a l l i n e d i o l , m.p. 203°, (MeOH), which analysed  J ^-3° 2  f o r CgQHggOgNg. The i n f r a - r e d spec-  trum of the d i o l showed no carbonyl a b s o r p t i o n , and the N.M.R.spectrum i n d i c a t e d a complete absence o f the methoxyl s i g n a l . The presence of a carbomethoxy group i n d i 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 w i t h acetone c o n t a i n i n g ptoluenesulphonic a c i d a f f o r d e d an acetonide, m.p. 105109°,  as shown by a s i x - p r o t o n N.M.R. s i 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 o f 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 - r e l a t i o n s h i p , 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 c o n t a i n a ^-hydroxy-ester  grouping.  The N.M.R,  spectrum o f the acetonide was even more i n s t r u c t i v e i n t h a t the s i g n a l a t 6.25T , due t o the methylene protons  - 17 -  on the two oxygen-bearing carbon atoms, was s p l i t i n t o 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 p o i n t i t had been e s t a b l i s h e d t h a t s i t s i r i k i n e possessed a t e t r a c y c l i c s k e l e t o n and the f o l l o w i n g f e a t u r e s ;  C:  N H  CH  CH.  H  ^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 v a r i o u s d e r i v a t i v e s 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 ) , r u n by the d i r e c t i n l e t procedure  , was most h e l p -  f u l and i s discussed i n some d e t a i l . The molecular i o n peak a t m/e 356 e s t a b l i s h e d the molec u l a r 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-CH 0H) were c o n s i s t e n t w i t h the 2  presence of a primary a l c o h o l . A strong peak a t m/e 253 was considered to a r i s e from l o s s of the e n t i r e oxygen-containing p o r t i o n of the molecule, i . e . M - C E K ? . More important, CH  N  0H  CO,Me  however, were the i o n s a t m/e 184, 170, 169 and 156. I t was immediately apparent from these f o u r peaks t h a t r i n g s A,B, and C of the d i h y d r o s i t s i r i k i n e s k e l e t o n 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 c l a s s e s  ,  - 18 -  where these ions are a l s o a t t r i b u t e d t o the fragments shown i n Figure 8. The assignments made i n the case of yohimbine were q u i t e r i g o r o u s l y e s t a b l i s h e d by deuterium l a b e l l i n g , as w e l l as by studying the e f f e c t of f u n c t i o n a l s u b s t i t u e n t s i n v a r i o u s p o s i t i o n s of the molecule. Moreover, the occurrence of s i g n i f i c a n t peaks a t m/e 169 and 170 and not a t m/e 168 CO^Me  'CH 0H z  (12) and 169 excluded the type of p e n t a c y c l i c r i n g system found i n polyneuridine  (13)^°.  Further i n f o r m a t i o n regarding  the r i n g s k e l e t o n was ob-  t a i n e d from semimicro dehydrogenation experiments. Treatment of d i h y d r o s i t s i r i k i n e w i t h lead t e t r a c e t a t e a f f o r d e d a product which e x h i b i t e d u l t r a - v i o l e t spectra. (Amax. 253, 308 and 365 mix i n n e u t r a l or a c i d s o l u t i o n ; X m a x . 284 and 328 IMA i n a l k a l i n e s o l u t i o n ) i n good agreement w i t h those of tetradehydroyohimbine and s i m i l a r compounds (14)'^ . Dehydrogenation of d i h y d r o s i t s i 1  r i k i n e w i t h 10$ palladium-charcoal  a t 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) d i s p l a y e d 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 n e u t r a l and a l k a l i n e media the s p e c t r a were the same, w i t h maxima at 234, 250, 282, 288, 337 and 349  myuL  , whereas i n a c i d s o l u t i o n  there was a bathochromic s h i f t to 254,  303 and 372  These r e s u l t s were s u f f i c i e n t to confirm the  m^u. tetrahydro-  | ^ - c a r b o l i n e s t r u c t u r e (12) i n d i c a t e d 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 ture separated  , and the r e s u l t i n g mix-  by t h i n - l a y e r chromatography. The  a b s o r p t i o n of one major f r a c t i o n (compound B) was  ultra-violet i n close  correspondence w i t h that of 5 , 6 ~ d i h y d r o f l a v o c o r y l i n e hydrochloride (16)^  3  w i t h maxima at 221, 312, and 386 mjx . This  provided the f i r s t piece of evidence f o r the e n t i r e r i n g system i n s i t s i r i k i n e .  F i n a l c o n f i r m a t i o n was product was  obtained when the dehydrogenation  oxidised further with  2,3-dichloro-5,6-dicyano-  p-benzoquinone to compound C, which possessed a aromatised  completely  r i n g system. The u l t r a - v i o l e t spectrum (Figure 7)  w i t h 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  X 238, 291,346,385 m/,L X 275, 305,374 m/L MAX  MIN  \T\fJL, Figure 7  DIHYDROSITSIRIKINE  120  150  200  250  m/e Figure 8  300  350  - 20 -  w i t h that r e p o r t e d  f o r f l a v o c o r y l i n e (17, R = R^ = E t )  but showed d i f f e r e n c e s from other compounds ( 1 7 ) ^  3  w i t h the  same chromophore (see Table 2 ) .  Table 2 R  Amax.  )  H  H  244  294  345  388  H.  Et  235  295  350  390  Et  Et  238  291  346  385  242  295  342  388  240  292  346  386  (0H )4 2  -Pr  Et  An a u t h e n t i c sample 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 was k i n d l y provided by Dr.G.A. Swan, Chemistry Department, King's C o l l e g e , U n i v e r s i t y 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 m a t e r i a l s had the same Rf value on paper chromatograms r u n i n s e v e r a l d i f f e r e n t s o l v e n t systems. Although the minute amounts of dehydrogenation products a v a i l a b l e prevented complete charact 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 e s t a b l i s h e d the r i n g s t r u c t u r e , and a l s o 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) c l a s s of a l k a l o i d s . A p r o v i s i o n a l s t r u c t u r e such as (19) could thus be considered for  dihydrositsirikine.  b; R = -CHgCH.  The o r i e n t a t i o n of the hydrogen atom a t C-3 i n (19) was i n d i c a t e d asOtby the presence o f Bohlmann b a n d s ^ a t 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 a t 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 a t the corresponding p o s i t i o n i n a l l r e l a t e d alkaloids  .  During t h e i r i n v e s t i g a t i o n s on corynantheine (18a) K a r r e r and co-workers^  2  had reduced dihydrocorynantheine (18b) w i t h  l i t h i u m aluminium hydride and i s o l a t e d two i s o m e r i c a l c o h o l s ; desmethoxy—dihydrocorynantheine  a l c o h o l ( 2 0 ) and iso-desmethoxy-  dihydrocorynantheine a l c o h o l (21). Since the c o n f i g u r a t i o n s a t C-3 and C-15 i n dihydrocorynantheine were the same as those p r o j e c t e d f o r the corresponding p o s i t i o n s 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 g i v e the unsaturated e s t e r (22), f o l l o w e d by hydride r e d u c t i o n should y i e l d the known a l k a l o i d (20), provided, o f course, t h a t the c o n f i g u r a t i o n a t C-20 was a l s o the same as i n corynantheine. A c c o r d i n g l y , d i h y d r o s i t s i r i k i n e was t r e a t e d w i t h sodium methoxide i n dry methanol t o a f f o r d the 06(3-unsaturated e s t e r (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 D s o l v a t e , Cg^ggOgNg'CHjOH. The presence o f a t e r m i n a l o l e f i n -1 was shown by a band i n the i n f r a - r e d spectrum a t 1620 cm. and N.M.R, s i g n a l s a t 3.73 and 4.41T , each of which i n t e g r a t e d f o r one proton. On hydrogenation  one mol. o f hydrogen was  taken up t o g i v e d e s o x y - d i h y d r o s i t s i r i k i n e (23) as a mixture of the two C-16 epimers, m.p. 172-177° . The i n f r a - r e d and N.M.R. s p e c t r a showed the disappearance  of the double bond, and the  a n a l y s i s was i n e x c e l l e n t agreement w i t h the formula Cp.,HpoOpNp.  Hp  (22) L i A l H .  (20) + (21)  - 23 -  The o l e f i n i c e s t e r was then reduced w i t h l i t h i u m  aluminium  h y d r i d e , but the product contained r e l a t i v e l y l i t t l e t e r m i n a l 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] ^-24.3° (MeOH), which showed one spot on t h i n - l a y e r chro2  matography and analysed w e l l f o r OgQ^gONg. The m e l t i n g p o i n t and s p e c i f i c r o t a t i o n were i n e x c e l l e n t agreement w i t h the corresponding constants — m.p. 204° » j^ ]])^" 24.0° — quoted^ f o r 2  06  iso-desmethoxy-dihydrocorynantheinealcohol  (21). This was  good evidence f o r the s t r u c t u r e (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 c o n c l u s i v e , s i n c e u n f o r t u n a t e l y 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 f o l l o w i n g sequence of r e a c t i o n s . M i l d a c i d h y d r o l y s i s converted dihydrocorynantheine (18b) to desmethyl-dihydrocorynantheine  (24), which on r e d u c t i o n  w i t h sodium borohydride y i e l d e d a product (25) i d e n t i c a l i n every respect w i t h d i h y d r o s i t s i r i k i n e . Having thus e s t a b l i s h e d the s t r u c t u r e of d i h y d r o s i t s i r i k i n e , we could immediately a s s i g n s t r u c t u r e (26) to g i t e i r i k i n e .  - 24 -  H  H'  H'  (26)  Me0 C 3  B.  CH OH a  Isositsirikine Prom the amorphous p o s t - p e r i v i n e 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 a l s o r e f e r r e d to as but  "sitsirikine"  to which we have given the name " i s o s i t s i r i k i n e " since  have now  shown that i t i s a new  [a] -20°(CHCl ), was 26  3  a l k a l o i d . Although the base,  an amorphous powder, i t was homogeneous  by t h i n - l a y e r chromatography and gave a sharp-melting sulphate, m.p.  we  26.3.5°, and p i c r a t e , m.p.  216°  crystalline  . 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 i n d i c a t e d a formula of 21^26°3^2 G  f o r  the base. This formula was  e s t a b l i s h e d by a mass s p e c t r a l mole-  c u l a r weight determination  which showed a value of  Standard Kuhn-Roth and Z e i s e l determinations  354.  showed the presence  of one C-methyl and one 0-methyl group r e s p 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 u n s u b s t i t u t e d i n d o l e chromophore, and an absorpt i o n band a t 1720  cm.  -1  i n the i n f r a - r e d r e g i o n gave evidence  f o r 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 i n d o l e system was  unsubstituted,  w i t h s i g n a l s at 1.33T(NH) and i n the r e g i o n 2.4-3.It* ( f o u r  - 25 -  aromatic hydrogens), and a sharp three-proton s i n g l e t a t 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 q u a r t e t centred a t 4.53T, whereas a doublet a t 8 . 4 0 t i n d i c a t e d t h a t the methyl group was attached to an o l e f i n i c carbon atom. Since the c o u p l i n g constants were the same (7 c/s) i n both cases, these s i g n a l s almost c e r t a i n l y denoted an e t h y l i d e n e group c o n t a i n i n g 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 t h e u p t a k e of one mol. of hydrogen t o 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 t h 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 c h a r a c t e r i s e d as the c r y s t a l l i n e p i c r a t e , m.p. 187° . I n the 1T.M.R. spectrum of the dihydro compound the s i g n a l s d u e t o  the e t h y l i d e n e  group had disappeared, and a new methyl a b s o r p t i o n a t 9.03T became e v i d e n t . P i n a l c o n f i r m a t i o n of the e t h y l i d e n e group was obtained when o z o n o l y s i s of i s o s i t s i r i k i n e y i e l d e d a c e t aldehyde, i d e n t i f i e d by paper chromatography of i t s 2 , 4 - d i n i 36  trophenylhydrazone^ . Carbonyl and methoxyl f u n c t i o n s 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  a c e t a t e , C 2 H Q 0 N 2 , which d i s p l a y e d the a p p r o p r i a t e bands i n 3  2  4  the i n f r a - r e d r e g i o n a t 1730 (C=0) and 1235 (OAc)  cm'. ^. r  The  26  N.M.R. spectrum of t h i s a c e t a t e 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 s i g n a l a t 8.15£ due to the a c e t y l group, a doublet i n t e g r a t i n g f o r two hydrogens now appeared a t 6.05TJ , whereas a two-proton a b s o r p t i o n present at 6.6"£in the spectrum o f the a l c o h o l had disappeared. The most obvious i n t e r p r e t a t i o n was t h a t the doublet was due t o the methylene protons o f a primary h y d r o x y l group which had 35  undergone a downfield s h i f t o f 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 o f the s i g n a l i n t o a doublet suggested that the methylene, protons were part o f an A g X 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 b e a r i n g 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 w i t h l i t h i u m aluminium hydride a d i o l was obtained, which showed n e i t h e r a c a r b o n y l a b s o r p t i o n i n the i n 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 o f the r e d u c t i o n product with acetone c o n t a i n i n g p-toluenesulphonic a c i d 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 i n d i c a t e d a gem-dimethyl group w i t h a p a i r o f sharp s i g n a l s a t 8.63 and 8.68T. Since the d i o l had two primary a l c o h o l f u n c t i o n s , the f o r m a t i o n o f an i s o p r o p y l i d e n e derivative-meant t h a t the h y d r o x y l 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 t o that found i n  sitsirikine. At t h i s p o i n t the f o l l o w i n g  f e a t u r e s of the  alkaloid  s t r u c t u r e had been e s t a b l i s h e d : CO Me R  N  /  C = C — CH H  -CH \  3  CH OH R  The nature of the r i n g system was revealed by dehydrogenat i o n o f a small amount of i s o s i t s i r i k i n e sulphate w i t h palladium black a t 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 s p e c t r a  of the harman(15) type (Figures 5 and 6 ) w i t h maxima a t 232, 282, 289, 336 and 347 mjn, i n n e u t r a l s o l u t i o n , which s h i f t e d to 251, 302 and 374rn^tupon 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 o f the other since i t was s i m i l a r to that of f l a v o c o r y l i n e  fraction,  hydrochloride  (27) w i t h maxima a t 237, 291, 345 and 385 mjm ( c f . F i g u r e 7) and thus provided evidence f o r the e n t i r e t e t r a c y c l i c  ring  system of i s o s i t s i r i k i n e . I t was subsequently  shown by paper chromatography,  u s i n g an e t h y l a c e t a t e - p y r i d i n e - w a t e r  (8:2:1) system, that  the l a t t e r d e h y d r o g e n a t i o n p r o d u c t was a c t u a l l y a mixture of two compounds. The major component (Rf 0.35) mixture was not f l a v o c o r y l i n e  (Rf 0.43)  of t h i s  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 dehydrogenation. Since i t had been p o s s i b l e 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 r e a c t i o n s , 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 t o the amorphous h y d r o c h l o r i d e . The s a l t (without  purifi-  c a t i o n ) was then heated w i t h palladium black a t 280° , the residue taken up i n a c e t i c a c i d , and t r e a t e d w i t h 2 , 3 - d i c h l o r o 5,6-dicyano-p-benzoquinone. From the r e a c t i o n mixture was i s o l a t e d a c r y s t a l l i n e h y d r o c h l o r i d e , m.p. 280-282° , which was i d e n t i c a l i n every respect w i t h a u t h e n t i c 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 (27) s same m e l t i n g point and undepressed mixed m e l t i n g p o i n t ; superimposible u l t r a - v i o l e t and i n f r a - r e d s p e c t r a ; i d e n t i c a l Rf values on t h i n - l a y e r and paper chromatography. From these r e s u l t s the gross s t r u c t u r e (28) could be assigned w i t h some c e r t a i n t y to i s o - s i t s i r i k i n e , s i n c e the a l t e r n a t i v e s t r u c t u r e w i t h the p o s i t i o n s of the e t h y l i d e n e and ^-hydroxy-ester  f u n c t i o n s interchanged was considered  very  u n l i k e l y on b i o g e n e t i c grounds. However, the s t r u c t u r e (28)  +  CI  MeOX  CB^OH  - 29 -  contained  three asymmetric centres at C-3, C-15, and  G-16  whose c o n f i g u r a t i o n s 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 - o r i e n t a t i o n , s i n c e 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 d i d not d i s p l a y Bohlmann b a n d s ^ i n the 2800 cm,"  1  However, d i h y d r o - i s o s i t s i r i k i n e e x h i b i t e d strong -1 at 2810 and 2760 cm.  region. absorptions  ,and thus presumably had the ex-configu-  r a t i o n at C-3. This suggestion was s u b s t a n t i a t e d when lead t e t r a c e t a t e o x i d i s e d 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 r e d u c t i o n w i t h sodium borohydride regenerated the s t a r t i n g m a t e r i a l . Since t h i s sequence i s known to give the isomer w i t h the C-3 hydrogen atom i n the OL-orientation  , i t f o l l o w e d 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 c o n f i g u r a t i o n at C-3.  MeO^C  CH OH R  HeO C  CH^OH  R  The 0 ( - o r i e n t a t i o n of the hydrogen atom a t C-15 could be 4 5  assumed on the b a s i s of Wenkert's e m p i r i c a l r u l e  , but there  was no way of r e a d i l y f i n d i n g the c o n f i g u r a t i o n at C-16. Theref o r e the s t r u c t u r e p o s t u l a t e d f o r 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 c l o s e 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 i n d i c a t e d above f o r i s o s i t s i r i k i n e was a l r e a d y on hand, a mass spectrum (Figure 9) of t h i s a l k a l o i d was obtained. I n a d d i t i o n t o an accurate molecular weight, the  mass spectrum provided v a l u a b l e evidence about the s t r u c t u r e ,  and thus supplemented the chemical i n v e s t i g a t i o n s . The peaks a t m/e 336 (M-HgO) and 335 (M-l-HgO) were cons i d e r a b l y stronger than the parent ions a t m/e 354 (M ) and +  353 ( M - l ) — a f a c i l e dehydration compatible w i t h the presence of a l a b i l e proton a t 0-16. A s e r i e s of i o n s a t m/e 184, 170, 169 and 156 corresponded to a s i m i l a r sequence d i s p l a y e d 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 - c a r b o l i n e system 40 . However, the mass  P  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  s e r i e s of strong s i g n a l s were obtained a t 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 t o v a r i o u s r a d i c a l ions(32a,b,c,d, e) i n which the e n t i r e t e t r a c y c l i c r i n g s t r u c t u r e of i s o s i t s i r i -  - 31  kine had been aromatised. I t must be emphasised t h a t the  (32) as fi = R b: R  = E t : m/e 275  x  = Me, R  = E t ; m/e 261  x  = fl, ^ = CH , R 2  = R  x  = E t ; m/e 247 x  = H; m/e 232  = H; m/e 219  s t r u c t u r e s shown are only t e n t a t i v e , since no evidence i s a v a i l a b l e to decide which among s e v e r a l a l t e r n a t i v e s t r u c t u r e s are c o r r e c t . 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 ( o r s i t s i r i k i n e ) i t must be assumed that the presence of a double bond exo t o the D-ring leads to i t s ready a r o m a t i s a t i o n , and subsequently to that of the C - r i n g , The d i f f e r e n c e i s most c l e a r l y  demonstrated  by the r e s p e c t i v e base peaks, both of which a r i s e by l o s s 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 q u i t e s t a b l e and i s r e g i s t e r e d a t 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 c o r r e s ponding i o n (34) occurs to give the m/e 247 fragment which the s t r u c t u r e (32c) i s suggested.  for  - 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 Isositsirikine 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 s e v e r a l r e l a t e d a l k a l o i d s from Aspidosperma oblongum A.DC. S p i t e l l e r and S p i t e l l e r - F r i e d m a n n  separated  t r a c e amounts of a l k a l o i d s by means of t h i n - l a y e r chromatography and p o s t u l a t e d s t r u c t u r e s on the b a s i s of mass s p e c t r a l c r a c k i n g p a t t e r n s . 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 s t r u c t u r e c o n t a i n i n g a t e t r a h y d r o - (2> - c a r b o l i n e system was  deduced when both compounds.displayed s i g n a l s a t m/e 184,  170, 169 and 156^*, whereas peaks-at M-31 and M-59 suggested the presence of primary a l c o h o l and m e t h y l e s t e r  functions.  Since both compounds showed a strong peak a t M-103 i t was t e n t a t i v e l y assumed t h a t the oxygen-containing groups were present as a.  ft-hydroxy-ester  j-jti nil  u n i t , -CH * . Reduction o f the "C0 Me o l e f i n w i t h l i t h i u m aluminium hydride gave a compound o f molec u l a r weight 326, which confirmed the methyl e s t e r . This product a l s o d i s p l a y e d a peak a t m/e 251, corresponding to l o s s CH OH of -CH. * , which s u b s t a n t i a t e d the proposed a-hydroxy-ester CH^OH group i n the o r i g i n a l a l k a l o i d . 1  R  r  These deductions were supported by a p a r a l l e l s e r i e s of r e a c t i o n s and mass spectra on an accompanying a l k a l o i d of molec u l a r weight 384.  This was considered  t o be merely a d e r i v a t i v e  of the above a l k a l o i d (M.W. 354) w i t h a methoxy s u b s t i t u e n t  i n the aromatic r i n g , s i n c e the c r a c k i n g p a t t e r n 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 b a s i s of these r e s u l t s the authors suggested the s t r u c t u r e s (35a) or (35b) f o r the a l k a l o i d of molecular weight 354,  and (35c) f o r 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 w i t h the mass spectrum of our dihydros 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 d i f f e r e n c e s i n the s p e c t r a were considered, by S p i t e l l e r to be due to i m p u r i t i e s i n t h e i r compound, s i n c e the minute amounts a v a i l a b l e rigorously  i n their investigation  purifying  prevented them from  t h e i r substance. Because the M-103 peak  was l a r g e r i n the o l e f i n than i n the dihydro compound, the isositsirikine  s t r u c t u r e (35a.) was favoured f o r t h e i r parent  a l k a l o i d (M.W.  354)? inasmuch as the c a t i o n (34) r e s u l t i n g  from l o s s of the ^>~hydroxy~ester u n i t 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 and  from that of S p i t e l l e r ' s  alkaloid,  consequently the above r a t i o n a l i s a t i o n I s 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 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*  0  ^16 »  from P a u s i n y s t a l i a yohimbe P i e r r e . These authors indepen50  d e n t l y derived a s t r u c t u r e  which corresponded to d i h y d r o -  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 s t r u c t u r e was determined was somewhat d i f f e r e n t from ours, and hence i s summarised  below.  Dehydrogenation w i t h selenium a f f o r d e d a l s t y r i n e ( 3 6 ) , which e s t a b l i s h e d 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 o x i d a t i o n i n d i c a t e d one C-methyl group, which was considered as p a r t of an e t h y l group i n view of the dehydrogenation r e s u l t s . The presence of a h y d r o x y l group was proven by f o r m a t i o n of an a c e t a t e . From a Z e i s e l d e t e r m i n a t i o n of one methoxyl group and a carbonyl band i n the i n f r a - r e d spectrum, a methyl e s t e r 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 f u n c t i o n s was e l u c i d a t e d by dehydration to an 0$~unsaturated e s t e r (22) and hydrogenation to a mixture of the two d e s o 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 o x i d a t i o n >  revealed the presence c f an a d d i t i o n a l C-methyl group, and hence e s t a b l i s h e d 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 -  (37) MeO C  HOCHj  R  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 s t r u c t u r e (25) was deduced, and confirmed by a c o r r e l a t i o n w i t h the known dihydrocorynantheine d e r i v a t i v e ( 3 7 ) , This c o r r e l a t i o n was achieved by hydride r e d u c t i o n of (23) to a mixture of two a l c o h o l s , 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 w i t h (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 d i s c u s s b r i e f l y some of the b i o s y n t h e t i c ideas p e r t a i n i n g to i n d o l e a l k a l o i d s , and more p a r t i c u l a r l y to the corynantheine s e r i e s , i n order to consider the p o s s i b l e b i o g e n e t i c 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 r e l a t i v e s . F o r many years i t has been considered t h a t i n d o l e a l k a l o i d s r e l a t e d to yohimbine (42) are d e r i v e d i n part from tryptophan ( 3 8 ) , a hypothesis that has been s u b s t a n t i a t e d i n every i n s t a n c e where t r a c e r experiments ••-have- been performed; 51 as, f o r example, w i t h r e s e r p i n e , ajmaline and serpentine  .  Hence the main i n t e r e s t a t the present time i s i n the nontryptophan p o r t i o n 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 H a h n ^ , yohimbine (42)  - 36 -  i s produced v i a the i n t e r m e d i a t e (40) formed by condensation of a dihydroxyphenylalanine u n i t (39) and formaldehyde w i t h tryptophan (38). I n t r o d u c t i o n of a carbomethoxy group i n t o (40) and a p p r o p r i a t e r e d u c t i o n steps are then p o s t u l a t e d to 52 lead to yohimbine (42). Robinson's suggestion  to account  f o r the carbomethoxy group i s t h a t 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 k e t o - a c i d . 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 a j m a l i c i n e ( 1 ) . Subsequent r i n g c l o s u r e s are r e q u i r e d t o a f f o r d p o l y n e u r i dine (13)^° and ajmaline" (43)-^,  - 37 -  H (18a)  MeO  R  MeO^C  c\  CO^Me NY  ^CH*OH  (13)  ( 43)  An elegant a l t e r n a t i v e scheme i n v o l v i n g prephenic a c i d (44) has been elaborated by Wenkert  . Rearrangement of prephe-  n i c a c i d by a 1 , 2 - s h i f t of the pyruvate residue w i t h r e t e n t i o n of c o n f i g u r a t i o n , followed by h y d r a t i o n , a f f o r d s a u n i t (45) r e a d i l y d i s c e r n i b l e i n yohimbine (42). Condensation w i t h a formaldehyde equivalent and r e t r o - a l d o l i s a 1 1 o n then y i e l d s a jlseco, "prephenate-formaldehyde" (SPF) group (46), that can condense w i t h 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 a j m a l i c i n e ( 1 ) .  - 38  OH  One of the a t t r a c t i v e f e a t u r e s of Wenkert's scheme i s t h a t i t p r e d i c t s the c o r r e c t stereochemistry corresponding  to C-15  at the p o s i t i o n  of yohimbine (42) i n the v a r i o u s a l k a l o i d s .  The hydrogen atom a t t h i s p o s i t i o n i s found to have the <<x,-config u r a t i o n i n a l l r e l a t e d i n d o l e a l k a l o i d s of known stereochem45  i s t r y • , except f o r Y ~ ^ a  a  m  m  ici  n  e  57  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 g l u c o s i d e s , one example of which i s g e n i p i n ( 4 7 ) ^ , has been found to have the same absolute 8  c o n f i g u r a t i o n a t the p o s i t i o n ^ s t a r r e d i n (47) ^| corresponding to C-15 i n (42). These compounds appear to have t h e i r s t r u c t u r e based on the monoterpene u n i t ( 4 8 ) , cleavage of which along the dotted l i n e would g i v e a s k e l e t o n analogous to the SPP u n i t .  - 39 -  (47)  (48)  (49)  Wenkert thus considered the h y p o t h e s i s ( p r e v i o u s l y suggested by Thomas^) t h a t the i n d o l e a l k a l o i d s may have a mono t e r penoid p r e c u r s o r derived from mevalonic a c i d ( 4 9 ) , or e q u a l l y , t h a t 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 b i o s y n t h e s i s of corynantheine (18a) and r e l a 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 a t the oL-position of the i n d o l e system to g i v e ( 5 2 ) , which can then undergo a p p r o p r i a t e modifications. 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 c o n s t i t u t e an i n t e r e s t i n g v a r i a t i o n of the corynantheine s e r i e s which may l i e on one p o s s i b l e b i o g e n e t i c pathway to p e n t a c y c l i c a l k a l o i d s such as p o l y n e u r i d i n e (13)^°. The i n t e r m e d i a t e (52) proposed by Wenkert can be v i s u a l i s e d as undergoing d e c a r b o x y l a t i o n and o x i d a t i o n fir)  to the immonium i o n (53)  , which then c y c l i s e s to (54).  A d d i t i o n of the aldehyde f u n c t i o n t o the ^ - p o s i t i o n of the i n d o l e can a f f o r d a p l a u s i b l e p r e c u r s o r (55) of the a j m a l i n e 61  type a l k a l o i d s such as vomeniline (58)  . I f , however, r e d u c t i o n  of the aldehyde group i n (52) occurs before d e c a r b o x y l a t i o n ,  - 40 -  - 41. -  then a s i t s i r i k i n e type i s obtained. The corresponding immonium i o n (56) can c y c l i s e only to a p e n t a c y c l i c precursor (57) of p o l y n e u r i d i n e (13), or i t s C-16 62  Recently Leete  epimer, akuammidine.  63  '  has attempted  to t e s t the hypotheses  described above by feeding l a b e l l e d compounds to Rauwolfia. s e r p e n t i n a and degrading the ajmaline (43) obtained. I n both the Robinson-Woodward and the Wenkert schemes C-21  of ajmaline  derived from a formaldehyde e q u i v a l e n t , a theory which i s supported by i n c o r p o r a t i o n 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 dihydroxyphenyla l a n i n e , i t might be expected, on the b a s i s of the Woodward h y p o t h e s i s , t h a t a d m i n i s t r a t i o n of  Robinson-  phenylalanine-2-^C  would provide ajmaline-3-"^C, but the ajmaline e x t r a c t e d was 63  i n a c t i v e , as were the r e s e r p i n e and serpentine, . I f Wenkert'a prephenic a c i d hypothesis were c o r r e c t , then a l a n i n e - 2 - ^ C , which would l a b e l prephenic a c i d by way of pyruvate, should give a c t i v i t y at C-3 i n a j m a l i n e . 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 63 Ajmaline i s o l a t e d from a p l a n t 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  - 42 -  b i o s y n t h e t i c route i n which the non-tryptophan moiety was \56 59  supposed to be formed from a monoterpene u n i t (48)^  .  A f o u r t h hypothesis was then put forward by L e e t e ^ , on the b a s i s of a. degradation of a j m a l i n e , l a b e l l e d by ffi  14-'"^*  acetate-l~  G i n c o r p o r a t i o n , 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 h a 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, p r e v i o u s l y advanced by S c h l i t t l e r 65  and T a y l o r  i n which the carbon c h a i n 18-19-20-15-14-3 o r i -  g i n a t e s by condensation of three molecules of acetyl-coenzyme A to a poly~[3 -keto fragment (59). F u r t h e r condensations w i t h a formaldehyde e q u i v a l e n t a t C-20, and w i t h the methylene 66 group of malonyl-coenzyme  A (derived from acetyl-coenzyme A  )  at C-15, were p^ o s2.0 t u l a t e18d to a f f o r d an intermediate (60) very o c ^ o o c ^ o  (59)^ iK^lK o O 2  C  H  2  0  2  */SCoA ° 2  C  M  0  #_  _  C= X  (60)  s i m i l a r to Wenkert's SPF u n i t (46). I t should be emphasised that (60) would be expected to form a complex w i t h tryptamine e s s e n t i a l l y i d e n t i c a l t o the tryptamine-SPF complex (51), and hence the l a t t e r part of Wenkert's scheme i n which the v a r i o u s i n d o l e a l k a l o i d s are derived would s t i l l be v a l i d .  - 43 -  U n f o r t u n a t e l y i n a r e p e t i t i o n of the work on R. s e r p e n t i n a , 67  Battersby and co-workers  were unable to reproduce the above  r e s u l t s , and i t was found t h a t the r a d i o a c t i v e l a b e l i n ajmal i n e (43) from both acetate and formate was s c a t t e r e d . Hence, a t the present time no one hypothesis has been e s t a b l i s h e d to the e x c l u s i o n of o t h e r s , and the o r i g i n " of the non-tryptophan p o r t i o n of these i n d o l e a l k a l o i d s i s s t i l l a subject of controversy.  - 44 PART I I Some Aspects of the Chemistry of Catharanthine 112 and Cleavamine' 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 o u ,69 p  y 3  on the  dimeric  Vinca a l k a l o i d s , v i n c a l e u k o b l a s t i n e (VLB), l e u r o s i n e , and leuroc r i s t i n e , i t was  found t h a t each was  cleaved by  concentrated  h y d r o c h l o r i c a c i d to an i n d o l e compound and a v i n d o l i n e d e r i v a t i v e (61). In the i n s t a n c e s of VLB and l e u r o s i n e , the was  latter  d e s a c e t y l v i n d o l i n e (61a), whereas l e u r o c r i s t i n e gave  d e s - N ^ - m e t h y l - d e s a c e t y l vindoline•-('61b). Both VLB and  leuro-  c r i s t i n e a f f o r d e d the same i n d o l e d e r i v a t i v e , velbanamine, C-^HggNgO, but the corresponding compound w i t h l e u r o s i n e was  cleavamine, ig 24^2° Velbanamine was G  H  considered  to be  a hydroxy-dihydrocleavamine, sinee i t y i e l d e d some cleavamine on prolonged h e a t i n g w i t h a c i d 68  (61)  a: R  = Me  bj R. = H  MeO' CO^Me  When catharanthine  (5),69 was  ment, one of the products was  subjected  to the same a c i d t r e a t -  found to be cleavamine, which  suggested t h a t the dimeric a l k a l o i d s were c o n s t i t u t e d of vindol i n e and c a t h a r a n t h i n e - l i k e m o i e t i e s . Moreover, the i n f r a - r e d spectrum of VLB  could be approximated by an equimolar mixture  - 45 -  of v i n d o l i n e and catharanthine  . When the s t r u c t u r e o f these on  a l k a l o i d s had been e s t a b l i s h e d  OQ  '  , the L i l l y research group  p o s t u l a t e d the p a r t i a l s t r u c t u r e (62a.) f o r VLB,  the p r e c i s e  p o i n t s of attachment of the v i n d o l i n e u n i t 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 t o 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 i n d o l e p o r t i o n o f VLB could not be e s t a b l i s h e d d i r e c t l y , since i t seemed that a rearrangement was  t a k i n g place during the a c i d cleavage, and consequently the  i n d o l e compound i s o l a t e d d i d not n e c e s s a r i l y possess the s k e l e t o n present i n the o r i g i n a l a l k a l o i d . I t t h e r e f o r e became imperative  to e s t a b l i s h the s t r u c t u r e and mode of formation  of cleavamine, and a l s o t o c h a r a c t e r i s e the other products from the a c i d treatment o f catharanthine.  Establishment of  the mechanism of the catharanthine-cleavamine  transformation  would f u r n i s h evidence f o r the s t r u c t u r e of VLB,  since catharan-  t h i n e c o n s t i t u t e d an e x c e l l e n t model f o r the postulated moiety.  indole  - 46 -  Although the study of t h i s r e a c t i o n was o r i g i n a l l y undertaken mainly i n connection w i t h the s t r u c t u r e o f 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 l e d to the use o f cleavamine analogues i n the s y n t h e s i s of immonium compounds, which i n t u r n were found to undergo t r a n s annular c y c l i s a t i o n s to Iboga ( 6 3 ) and Aspidosperma ( 6 4 ) - l i k e skeleta.  (63) R = H or C0 Me 2  Hence the d i s c u s s i o n of the work on cleavamine and i t s congeners can be d i v i d e d i n t o two sections: the catharanthine -cleavamine  t r a n s f o r m a t i o n and a p o s s i b l e mechanism are presen-  ted i n s e c t i o n A, whereas the s y n t h e t i c use of the cleavamines and t h e i r b i o g e n e t i c i m p l i c a t i o n s w i l l be discussed i n s e c t i o n C. A review of current bi©synthetic t h e o r i e s p e r t i n e n t to the d i s c u s s i o n on t r a n s a n n u l a r c y c l i s a t i o n s i s g i v e n i n s e c t i o n B.  A, The Catharanthine-Cleavamjne  Transformation  When catharanthine (5) was t r e a t e d w i t h concentrated h y d r o c h l o r i c a c i d i n the presence-of t i n and stannous c h 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 and cleavamine  (65)  (66). The s t r u c t u r e (66) of cleavamine had been  - 47 -  suggested mainly on the b a s i s of a comparison of the mass s p e c t r a l c r a c k i n g p a t t e r n s of cleavamine and w i t h that of quebrachamine  72  (67)? and was  dihydrocleavamine  f i n a l l y established  by an X-ray a n a l y s i s of cleavamine methiodide  73  .  H  . Since the combined y i e l d of cleavamine and descarbomethoxycatharanthine  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 d e s i r a b l e to i s o l a t e other compounds which might  provide i n f o r m a t i o n about the mechanism of t h i s i n t e r e s t i n g rearrangement. The mine-containing  cleavamine mother l i q u o r s and s e v e r a l c l e a v a -  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 c o n t a i n i n g 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 c h a r a c t e r i s e d . I t i s a p p r o p r i a t e t d i s c u s s 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  f u r n i s h e d 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-  to  - 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 a t 8 . 9 6 Y i n cleavamine had s h i f t e d to 9.13"E • The mass spectrum showed a molecular i o n a t m/e 282, and other s i g n i f i c a n t peaks a t m/e 156, 143, 138, 124could be a t t r i b u t e d to the f o l l o w i n g 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 a l s o the i n f r a -red  spectra were f a i r l y s i m i l a r . The l e a d i n g spot of the  mixture had the same Rf as 4"oc"-dihydrocleavamine on t h i n - l a y e r chromatography  and the second spot was thought to be due to  a dihydrocleavamine epimeric a t C-4, which we designated as 4"(3"-dihydrocleavamine. From other s t u d i e s a t 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 Laborat o r i e s f o r comparison purposes. I t was p o s s i b l e t o 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 r e f e r r e d t o 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 compound from the corresponding C-4 epimer, 4"fi"-dihydrocleavamine s  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 s y n t h e t i c mixture of the dihydrocleavamines d u p l i c a 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 e i t h e r from dihydrocatharanthine (present as an impurity or formed by r e d u c t i o n of c a t h a r a n t h i n e ) , or by r e d u c t i o n of cleavamine  (or an e q u i v a l e n t r e a c t i o n i n t e r m e d i a t e ) .  Since the catharanthine was-homogeneous, and i n any case dihydroc a t h a r a n t h i n e (69) had been shown- to decarboxylate without •  113  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 w i t h t h e l a t t e r a l t e r n a t i v e , that had to be i n c o r p o r a t e d i n t o a mechanism which would f o r the f o r m a t i o n of cleavamine  account  (66), descarbomethoxycatharan-  t h i n e (65) and the two dihydrocleavamines  (68).  Some other re s u i t s p e r t i n e n t to any proposed me onanism had been obtained by the L i l l y group^^^cDihydrocatharanthine (69) decarboxylated r e a d i l y to epi-ibogamine(70)  was  by h e a t i n g w i t h  hydrazine i n e t h a n o l ^ ^ , o r by h y d r o l y s i s w i t h e i t h e r aqueous potassium hydroxide or l i t h r u m i o d i d e i n p y r i d i n e f o l l o w e d by heating with d i l u t e mineral acid proposed mechanism  75  75  . These r e s u l t s f i t t e d  the  f o r the d e c a r b o x y l a t i o n of Iboga a l k a l o i d s s  - 50 -  However, none of these procedures was s u c c e s s f u l i n decarboxyl a t i n g catharanthine ( 5 ) , presumably because the corresponding intermediate (71) would be too s t r a i n e d t o form"* Therefore, the formation of (65),  .  descarbomethoxy-catharanthine  a l b e i t i n . poor y i e l d , upon treatment of catharanthine  (5) w i t h concentrated h y d r o c h l o r i c a c i d must i n v o l v e some other mechanism. I n order t o e x p l a i n the occurrence of c l e a v a mine ( 6 6 ) and the epimeric dihydrocleavamines  (68) a ring-  opened intermediate must be present a t some stage, and moreover, a route has to be provided whereby the o l e f i n i c l i n k a g e present i n catharanthine can be reduced. With these c o n s i d e r a t i o n s i n mind we p o s t u l a t e the s p e c u l a t i v e mechanism on p. 51  (66)  (68)  - 52 -  for  the r e a c t i o n . The lone p a i r of e l e c t r o n s on the N^^-atom  i n  (5) can  p a r t i c i p a t e i n a rearrangement to form an immonium i o n , w i t h concurrent r i n g cleavage and p r o t o n a t i o n a t the ^ - p o s i t i o n of the i n d o l e . 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 f a 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 c o n j u g a t i o n of the newly generated double bond between.C-17 and C-18 w i t h both the e s t e r and a n i l i n o f u n c t i o n s . A f t e r a c i d h y d r o l y s i s of the e s t e r ,  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) r e q u i r e d i n the u s u a l mechanism (see above, p. 50) cannot be formed. The c r u c i a l , intermediate ('74) may f o l l o w e i t h e r of two r e a c t i o n paths. I f the o r i g i n a l e l e c t r o n f l o w 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 t h e immonium system has a l r e a d y been reduced) then the ring-opened  tetracyclic  compounds must u l t i m a t e l y b e f o r m e d . Assuming t h a t (75) i s the a c t u a l i n t e r m e d i a t e , . 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 a l s o occur to a f f o r d an eneamine (76), which rearranges 76 i n t h e well-known manner  to t h e immonium compound (77) w i t h  subsequent r e d u c t i o n 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 r e d u c t i o n...of catharanthine w i t h z i n c i n g l a c i a l acet i c a c i d to car..homethoxy-4"(o"-dihydrocieavamine  (119)  showed  that the reduction, and decarbomethoxylatron were separate processes. Much...more, s u b s t a n t i a l support f o r the mechanism, however, was the t r a n s a n n u l a r c y c l l s a t i o n of an immonium i o n derived from ca.rbemethoxy--4 p)"-dihydrocleavamine to an Iboga l,  skeleton'(see s e c t i . o n C , 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 f o r the formation of'descarbomethoxycatharanthine (65) from the i n t e r m e d i a t e (74) was  actually  feasible. I n the absence of an i n o r g a n i c reducing agent, the r e d u c t i o n step p o s s i b l y takes place v i a van i n t r a m o l e c u l a r redox r e a c t i o n of two molecules of the i n t e r m e d i a t e ( 7 5 ) to y i e l d one molecule of cleavamine (66) and one' of a p y r i d i n i u m compound (78). The i n c r e a s e i n y i e l d of cleavamine i n a reducing medium i s thus e x p l i c a b l e on the grounds that (75) i s reduced d i r e c t l y to cleavamine and no p y r i d i n i u m compound i s formed.  F u r t h e r i n t e r e s t i n the chemistry of cleavamine was  - 54 -  s t i m u l a t e d by the recent work of Buchi and co-workers  o n  voacamine (79) which i n d i c a t e d that the o r i g i n a l s t r u c t u r e -  (62a) proposed by the L i l l y group f o r VLB was probably wrong, and suggested t h a t the i n d o l e moiety was a cleavamine (66) r a t h e r than a catharanthine  (5) type.  I t was noted t h a t voacamine a l s o represented  a "dimeric" a l -  k a l o i d c o n s t i t u t e d from two i n d o l e m o i e t i e s . Even more important was the o b s e r v a t i o n that voacamine could be cleaved  into  the r e s p e c t i v e monomeric u n i t s - b y means of a c i d i c reagents under c o n d i t i o n s s i m i l a r to those used f o r VLB. The carboncarbon bond l i n k i n g the two halves i s l a b i l e and i s ruptured -  during a c i d treatment. One of the weak f e a t u r e s i n the L i l l y s t r u c t u r e (62) for  VLB had been the nature of the linkage•between the i n d o l e  and d i h y d r o - i n d o l e  p o r t i o n s . As mentioned before, VLB i s  cleaved by a c i d i n t o d e s a c e t y l v i n d o l i n e and hydroxy-dihydro6Q 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 b a s i s of s t r u c t u r e (62). One would not expect  - 55 -  a bond comprised o f an a l i p h a i r i c carbon on one hand and an aromatic carbon on the other to r e a c t i n t h i s manner. C o n s i d e r a t i o n of the chemistry of voacamine l e d to a review of the s t r u c t u r e f o r VLB.and f u r t h e r s t u d i e s were undertaken. A h i g h r e s o l u t i o n mass spectrum gave a m o l e c u l a r w e i g h t f o r VLB of :  810.4219, which showed that the c o r r e c t formula was C^H^gOgN^ 78  and not C^gH^gOgN^ as p r e v i o u s l y thought c o n t a i n a carbomethoxy-cleavamine  . Thus VLB must  r a t h e r than a catharanthine  u n i t as the indole, p o r t i o n of the molecule. On the b a s i s of t h i s and other evidence, a r e v i s e d s t r u c t u r e (80) was very Co  r e c e n t l y proposed f o r VLB  (80)  Me  / 'OH  C0 Me 2  Although the study of the catharanthine-cleavamine transformation was i n i t i a t e d to throw l i g h t upon a corresponding r e a c t i o n thought to occur w i t h VLB and i t s congeners, i t was a l s o r e a l i z e d a t an e a r l y stage of the i n v e s t i g a t i o n t h a t 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 s y n t h e s i s and b i o g e n e s i s of i n d o l e 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 f o l l o w i n g s e c t i o n s .  - 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 d i s c u s s 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 ( s e c t i o n C), i t i s p e r t i n e n t to review b r i e f l y the v a r i o u s t h e o r i e s advanced f o r the b i o s y n t h e s i s of a l k a l o i d s of the Strychnos, Iboga and Aspidosperma s p e c i e s , as t y p i f i e d by s t r y c h n i n e (82), c o r o n a r i d i n e (86) and aspidospermine  (87).  These compounds are considered to be r e l a t e d b i o g e n e t i c a l l y (see below), and indeed have been found to occur t o gether i n the same p l a n t s , as f o r example Vinca rosea L i n n , (see i n t r o d u c t i o n to t h i s t h e s i s ) and Stemmadenia d o n e l l s m i t h i i (Rose) Woodson  '  . I t has r e c e n t l y been demonstrated  that l a b e l l e d tryptophan (38) i s i n c o r p o r a t e d i n t o v i n d o 27 7Q l i n e ( 7)  and ibogaine (88)  , and hence one may  assume  that the r e 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  Robinson-Woodward theory  thus be d e r i v e d , according to the '  mentioned e a r l i e r (p.35), from  a condensation of a dihydroxyphenylalanine u n i t (39) and f o r m a l dehyde w i t h 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 a p p r o p r i a t e c y c l i s a t i o n s to provide the Strychnos s k e l e t o n . 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 80  by T a y l o r  , whereby condensation of tryptophan (38) and  dihydroxyphenylalanine (39) a f f o r d s the a^-unsaturated  3,4-  acid  (83). The l a t t e r i n t e r m e d i a t e by a Michael a d 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 w i t h formaldehyde i n a Mannich r e a c t i o n t o y i e l d the t e t r a c y c l i c compound ( 8 5 ) . Subsequent a l d o l condensation, dehydration, and r e d u c t i o n lead to the Iboga s k e l e t o n ( 8 6 ) . 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 m e r i t of encompassing the Aspidosperma s e r i e s , i s f u r n i s h e d by Wenkert's prephenic  acid  h y p o t h e s i s - ^ , 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 t o t h i s theory,  the s t r y c h n i n e (82) group evolves from the tryptamine-SPP complex (51) by a t t a c k of the formyl acetate residue a t the deposition  of the i n d o l e t o g i v e the immonium i o n (89), which  bears on obvious resemblance to the known a l k a l o i d steimuadenine ( 9 l ) ^ . A t r a n s a n n u l a r c y c l i s a t i o n then provides the 8  s t r y c h n i n e precursor ( 9 0 ) .  (91)  (90)  - 59 -  D e r i v a t i o n of the Iboga and Aspidosperma systems i s l e s s s t r a i g h t f o r w a r d , 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 r e t r o - M i c h a e l r e a c t i o n of the interme-  d i a t e (89), i n v o l v i n g an a c t i v a t e d hydrogen atom on a carbon atom Oi to e i t h e r the immonium system or to the a c e t y l group, w i t h r e s u l t a n t 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 r e a c t i o n , the formyl acetate r e s i d u e would be l o s t and a pathway to the f l a v o p e r e i r i n e (92) s t r u c t u r e r e v e a l e d . However, were the r e t r o - M i c h a e l process to occur a t 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 a t t a c h ment to the i n d o l e system, as i n ( 8 9 ) , then the cleavage product (95) could be modified by u n e x c e p t i o n a l r e a c t i o n s 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 s e r i e s of r e a c t i o n s : M i c h a e l a d d i t i o n s to the ap-unsaturated a c i d 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 s k e l a t a , (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 p o r t i o n of h i s proposal i s supported by a considerable weight of c i r c u m s t a n t i a l  evidence.  For i n s t a n c e , the r e t r o - M i c h a e l r e a c t i o n of a s t r y c h n i n e type a l k a l o i d i s e x e m p l i f i e d by a cleavage t h a t Smith and Edwards  have found t o occur w i t h akuammicine (100). In order  to e x p l a i n the formation of the betaine (102) when akuammicine was heated i n methanol at 100°  f o r three hours, the authors  proposed a mechanism i n v o l v i n g a r e t r o - M i c h a e l cleavage of an intermediate such as  (101).  I t i s worthy of note t h a t Wenkert p r e d i c t e d 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 s i n c e been v e r i f i e d by the i s o l a t i o n of  - 62 -  s e v e r a l a l k a l o i d s r e l a t e d to v i n c a d i f f o r m i n e (103). Perhaps go  the best example i s minovincine (104)  , which possesses  not  only a carbomethoxy group i n the p r e d i c t e d p o s i t i o n but a l s o the a c e t y l f u n c t i o n . Counterparts o f the conjugated enone system i n the p o s t u l a t e d Iboga-type  precursor (99) have a l s o  been found: the double bond i n catharanthine ( 5 ) , and the carbonyl group i n voacryptine  (105). Furthermore,  the r i n g -  opened precursor (95) i s represented i n Nature by vincadine ( 1 0 6 a ) ^ , and 8  H  (103)  vincaminorine ( 1 0 6 b ) w h e r e a s the carbometh-  I  CO.Me  (104)  MeO  (106) •\  _ a:H = H C0->Me r  M  * oxy-cleavamine  b:R = Me  p o r t i o n 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 p o i n t i n favour of the above scheme i s that i t p r e d i c t s the c o r r e c t absolute c o n f i g u r a t i o n a t  C-15  i n akuammicine (100), which has been found to be constant 89 i n t h i s and r e l a t e d Strychnos a l k a l o i d s .. The s o l e 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 t r a n s f o r m a t i o n of (89) to (93) p r i o r to the complete e v o l u t i o n of the former to the Strychnos system. The occurrence i n Nature of racemic v i n c a d i f f ormine ( 1 0 3 ) ^ ' ' ^ , the o p t i c a l antipodes of quebracha2  mine ( 1 0 7 ) , ( 1 0 8 ) , and of v i n c a d i f f o r m i n e ( 1 0 5 ) ' , 8 5  8 6  8 2  and the enantiomeric a l k a l o i d s (113)  (—  and ( + ) - p y r i f o l i d i n e (114)  9 2  )-Q-methylaspidocarpine can be i n t e r p r e t e d on  the b a s i s of the above b i o s y n t h e t i c scheme. Since i n t e r v e n t i o n of the non-asymmetric intermediate (93) i n Aspidosperma b i o s y n t h e s i s destroys the f i x e d c o n f i g u r a t i o n of the s t a r r e d pos 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 f a m i l y and other i n d o l e bases.  Randomisation  of the absolute c o n f i g u r a t i o n of Aspidosperma a l k a l o i d s i s t h e r e f o r e considered by wenkert to be due to a l a c k of o p t i c a l c o n s i s t e n c y i n the Michael r e a c t i o n (94) to (95). Evidence which tends to support the p o s t u l a t e d t r a n s annular c y c l i s a t i o n of (95) to (96) has been accumulated i n s t u d i e s of the r e l a t i v e c o n f i g u r a t i o n s of v a r i o u s Aspidosperma a l k a l o i d s . Some of these have been c o r r e l a t e d w i t h (-)aspidospermine  (115), whose absolute c o n f i g u r a t i o n i s known 93  from the X-ray s t r u c t u r e d e t e r m i n a t i o n by M i l l s and Nyburg (—)-Quebrachamine has been shown to have the same c o n f i g u r a t i o n as aspidospermine  at the asymmetric centre i n v o l -  v i n g the e t h y l g r o u p ^ , and hence has the s t r u c t u r e (107); 9  the epimeric (+)-quebrachamine must then be (108).  .  - 64 -  I t i s a t t r a c t i v e to speculate t h a t the Aspidosperma a l k a l o i d s may be d i v i d e d 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 d e r i v e d by a t r a n s a n n u l a r c y c l i s a t i o n 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  o f t e n be a c h i e v e d i n the l a b o r a t o r y  t  In s e v e r a l cases  the o r i e n t a t i o n of the e t h y l e q u i v a l e n t of the a c e t y l group in  (95) and  (96) seems t o be c h a r a c t e r i s t i c of the  of a p a r t i c u l a r p l a n t s p e c i e s . Thus has been found and  i n conjunction with  ( - O - p y r i f o l i d i n e (115)  and  (—)-quebrachamine  (107)  ( —)-aspidospermine  (115)  £ ( — )-0-Methyl-aspidocarpineJ i n  Aspidosperma quebracho bianco (109)  alkaloids  or  , and w i t h ( + ) - v i n c a d i f f o r m i n e  (+)-l,2-dehydroaspido3permidIne  (111) i n Rhazya  92 stricta  . On the o t h e r hand, (+)-quebrachamine o c c u r s t o -  gether with (—)-tabersonine  (110)  £ (—  )-6,7-dehydrovinca-  d i f f o r m i n e J i n Stemmadenia s p e c i e s ^ , and w i t h  (-)-1,2-dehy-  droaspidospermidine  9  9  (112)  1  i n P l e i o c a r p a t u b i c i n a ^ . The  c h e m i s t r y seems to be determined  stereo-  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  p r e c u r s o r (95) s i n c e  the Mannich-type c l o s u r e of the nine-membered r i n g r e q u i r e s f o r m a t i o n of a c i s - p e r f a y d r o q u i n o l i n e system.in (96). P u r t h e r 56 more, Wenkert  suggested  t h a t the c i s - a n t i - c i s backbone  e x h i b i t e d by the Aspidosperma. a l k a l o i d s may  be the  quence of the Mannich c o n d e n s a t i o n  subsequent r e -  (and any  conse-  d u c t i o n ) f o l l o w i n g the p a t h of l e a s t s t e r i c r e s i s t a n c e .  The  i s o l a t i o n of s t e r e o c h e m i c a l l y r e l a t e d a l k a l o i d s i n the same  66 -  p l a n t c e r t a i n l y supports t h i s suggestion, and f u r n i s h e s c i r c u m s t a n t i a l 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 p o s t u l a t e d 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 sect i o n C. c  -  Transannular  C y c l i s a t i o n s of Cleavamine D e r i v a t i v e s  A c o n s i d e r a t i o n of the mechanistic aspects of the catharanthine ( 5 ) — c l e a v a m i n e  (66) t r a n s f o r m a t i o n l e d us i n  t u r n to examine the v a r i o u s b i o s y n t h e t i c hypotheses t h a t have been o u t l i n e d above. I n 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 d e a l i n g w i t h 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-  s i d e r e d the conversion of intermediate (98) to the p e n t a c y c l i c s t r u c t u r e (99) of the Iboga a l k a l o i d s , i t was apparent t h a t the transannular c y c l i s a t i o n of a c l e a v a m i n e - l i k e s k e l e t o n was i n v o l v e d . This was immediately reminiscent of a n i d e n t i c a l t r a n s a n n u l a r c y c l i s a t i o n of (74) t h a t had been proposed i n the mechanism (p.51) to e x p l a i n the formation of methpxycatharanthine  descarbo-  (65). Secondly, the i n t e r m e d i a t e  (95)  - 67  -  advanced by Wenkert as the d i r e c t precursor of the Aspidosperma system (96)  was a g a i n s i m i l a r to cleavamine  (66),  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 e t h y l group* I t was c l e a r t h a t cleavamine or one of i t s d e r i v a t i v e s might be converted i n t o an-immonium i n t e r m e d i a t e of the type proposed by Wenkert, and thereby a f f o r d an e x c e l l e n t opportun i t y f o r e v a l u a t i n g the f e a s i b i l i t y of such transannular c y c l i s a t i o n processes. A c c o r d i n g l y , o x i d a t i o n of 4"a -dihydrocleavamine  (68)  !,  w i t h mercuric acetate gave an immonium i o n (116)., which underwent 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 c o r responding d i h y d r o - i n d o l e (118a) was obtained a f t e r r e d u c t i o n w i t h l i t h i u m aluminium hydride  (116)  CO^Me  97  .  (117)  (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,  w i t h t h i s cleavamine d e r i v a t i v e there was a l s o a p o s s i b i l i t y of o b t a i n i n g an Iboga a l k a l o i d system, s i n c e the C-18 hydrogen atom was rendered l a b i l e by the carbomethoxy group. Reaction w i t h mercuric acetate "would be expected t o generate an i n t e r mediate w i t h the immonium -system (^Ii=CC) - i n v o l v i n g e i t h e r C-19 or C-5. The i n t e r m e d i a t e (120) w i t h the C-19 immonium group c o u l d , by the a p p r o p r i a t e transannular c y c l i s a t i o n , a f f o r d a v i n c a d i f f o r m i n e - l i k e system (121), whereas t h a t (122) w i t h 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 f a c t both processes operate.  Carbomethoxy-4"^"-dihydrocleavamine red by r e d u c t i o n of catharanthine 98 acid  (119)^  was prepa^-  (5) w i t h z i n c and a c e t i c  . A c i d h y d r o l y s i s and d e c a r b o x y l a t i o n of t h i s product  a f f o r d e d 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 e t h y l group i n a d i f f e r e n t o r i e n t a t i o n . O x i d a t i o n of carbomethoxy-4"(5"-* dihydrocleavamine i  -with mercuric acetate  i n a c e t i c a c i d a f f o r d e d a complex mixture which was ted to chromatography on alumina. This procedure  subjec-  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 s m a l l e r 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 c o r o n a r i d i n e ' and dihydrocatharanthine  OQ  , w i l l be presented l a t e r , w h i l e the former  i s discussed immediately below. ( i ) Pseudo-vincadifformine and i t s D e r i v a t i v e s 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 f o r D L  ^21^26^2^2' ^ l *  1 8 1  J  ! c o n f i r m a t i o n of the molecular formula  was  • obtained when a mass s p e c t r o m e t r i c molecular weight d e t e r m i n a t i o n showed a value of 338. Maxima i n the u l t r a - v i o l e t spectrum a t 226, 298 and 326 mju_, and a b s o r p t i o n bands i n the i n f r a - r e d r e g i o n a t 1675 and 1610 cm."*" c l e a r l y i n d i c a -  ted an ot^-unsaturated e s t e r f u n c t i o n conjugated w i t h the d i h y d r o - i n d o l e 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 e x h i b i t e d a s i n g l e t at  1.05T  (NH), a complex p a t t e r n i n the r e g i o n 2.4-3.3t corresponding to f o u r 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 ( F i g u r e 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 t o "that o f v i n c a d i f f ormine. Chemical evidence i n support o f "the conjugated e s t e r system was provided by a c i d - c a t a l y s e d h y d r o l y s i s and decarbo x y l a t i o n o f pseudo-vincadifformine (121) t o y i e l d a gummy product (123) which showed the expected s p e c t r a l p r o p e r t i e s of an i n d o l e n i n e 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 c a r b o n y l or NH a b s o r p t i o n 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 r e d u c t i o n o f the l a t t e r substance w i t h l i t h i u m aluminium hydride a f f o r d e d a c r y s t a l l i n e p r o duct, m.p. 89-90 ^ O t J ^ - e o  (CHCl^), f o r which the s t r u c t u r e  (124) was deduced from the f o l l o w i n g evidence. Elemental ana-  (123) l y s e s suggested a formula O-^HggNp^ which was confirmed by a mass spectrometric molecular weight (282). The reduct i o n o f the i n d o l e n i n e system was c l e a r l y i n d i c a t e d by a t y p i c a l d i h y d r o - i n d o l e u l t r a - v i o l e t spectrum ( X  m  Q  V  243  and 295 myw,) and the appearance o f a new a b s o r p t i o n a t 3230 cm." i n the i n f r a - r e d 1  spectrum (NH). Moreover, a  complex p a t t e r n o f l i n e s i n the N.M.R. spectrum i n the r e g i o n 2 . 7 - 3 . 6 T , due t o f o u r aromatic protons, was i n complete  I  o O  I  I  o  CO  I  I  o  ID  I — I — I — I — I — I  o  <fr  o  CM  A|ISU9|UI 9AljD|8J  o  <fr  - 71 -  agreement w i t h known Aspidosperma a l k a l o i d  systems  Invaluable i n f o r m a t i o n was provided by the mass spectrum (Figure 12) of the d i h y d r o - i n d o l e (124), which showed s i g n i f i c a n t peaks a t m/e 282 (M ) and 254 (M-28), and a v e r y strong +  s i g n a l a t m/e 124. I t was r e c e n t l y observed by Biemann e t a L  1 0 0  that the appearance of M-28 and m/e 124 peaks may be c o n s i dered d i a g n o s t i c of an Aspidosperma-type s k e l e t o n . I n t h i s i n s t a n c e , a similar-fragmenta"fcion process l e a d i n g to a m/e 124 i o n may be p o s t u l a t e d , whereby the molecular i o n (125) expels ethylene t o y i e l d the m/e 254 fragment (126), which i s subsequently cleaved to the m/e 124 i o n (127). This i o n d i f f e r s only i n the p o s i t i o n of the e t h y l group from t h a t (128) proposed  1 0 0  f o r the corresponding m/e 124 peak d i s p l a y e d by the  9° Et  (129)  m/e 124  - 72 -  Aspidosperma a l k a l o i d s . Furthermore, the mass spectrum o f (124) was superimposible  on t h a t o f the s i m i l a r compound 97 ?  (118a)previously synthesised from 4"(X."-dihydrocleavamine . F u r t h e r evidence f o r the s t r u c t u r e o f (124) was obt a i n e d from the N-acetyl d e r i v a t i v e , m.p. 107.5-109°, C  21^28°2 2* iyf  T t l e  u  l"  tra  ~ °l " vi  e  t  spectrum of the l a t t e r d i s p l a y e d  maxima a t 253, 279 and 289 TDJUL , and the complex m u l t i p l e t i n the aromatic r e g i o n of the N.M.R. spectrum of (124) had now c o l l a p s e d i n t o a broad three-proton peak centred a t 2.85X and a s i g n a l a t 1.87T due to one proton. These s p e c t r a l data were i n e x c e l l e n t agreement w i t h the acetate (118b) p r e v i o u s 97  l y d e r i v e d from 4"oL"-dihydrocleavamine^  and w i t h the known  Aspidosperma a l k a l o i d d e m e t h o x y p a l o s i n e ( 1 2 9 ) ^ . 10  A d d i t i o n a l chemical proof of the conjugated  ester  system was provided by r e d u c t i o n of p B e u d o - v i n c a d i f f o r m i n e w i t h z i n c and s u l p h u r i c a c i d t o y i e l d two i s o m e r i c d e r i v a t i v e s . The major product,  dihydro  dihydro-pseudovincadifformine,  £oc] --l6° (EtOH), analysed f o r C H 0 N , a formula which 24  2 1  2 8  2  2  was e s t a b l i s h e d by a mass spectrometric molecular weight of 340. The u l t r a - v i o l e t spectrum  (X  TOQV  244 and 299 mil ) was  c h a r a c t e r i s t i c of a d i h y d r o - i n d o l e chromophore, and the e s t e r carbonyl a b s o r p t i o n i n the i n f r a - r e d had now moved to 1725  cm." . A c e t y l a t i o n a f f o r d e d a N-acetate, 1  0 ^H^ 0^N , 2  0  2  28°(EtOH), which showed u l t r a - v i o l e t maxima a t 253, 282 and 291 iryu. , and the a p p r o p r i a t e appearance of an amide 0Lj^ i r  band a t 1660 cm." w i t h concurrent 1  l o s s 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 a t  - 73 -  7.75T (CHjC=0) i n the N.M.R. spectrum, t h e r e was an unexpected u p f i e l d s h i f t of the methoxyl s i g n a l from 6.33 to 6.83T, which w i l l be discussed l a t e r . I n g e n e r a l , the s p e c t r a l p r o p e r t i e s of dihydro-pseudo-vincadifformine and i t s a c e t a t e were i n agreement w i t h the s t r u c t u r e (131), f o r which c o n f i r m a t i o n was found i n the mass s p e c t r a l c r a c k i n g p a t t e r n (Figure 11 )„  The base peak of the mass spectrum was m/e 124, whereas the  second most i n t e n s e  peak was a t m/e 254. A fragmentation  process e n t i r e l y analogous to that discussed above (127)  (125) to  was o b v i o u s l y o c c u r r i n g , i n which the molecular i o n  (132) e l i m i n a t e d a molecule of methyl a c r y l a t e i n s t e a d of ethylene t o give an i d e n t i c a l m/e 254 fragment (126), which then cleaved as before to a f f o r d 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 n a t i o n of methyl a c r y l a t e ( o r i t s e q u i v a l e n t ) ^from the molec u l a r i o n (133).  - 74 -  (127)  m/e 124  CO;>M<L  (132)  +  +  -> H  C0 Me  CO Me a  2  (133)  (134)  However, the rearrangement product  (134) could s t i l l  cleave  to y i e l d the m/e 124 i o n . F i n a l l y , a comparison of the mass spectra of pseudo-vincadifformine  (121) and dihydro-pseudo-  v i n c a d i f f o r m i n e (131a) w i t h those of a u t h e n t i c v i n c a d i f f o r mine (103) and d i h y d r o v i n c a d i f f o r m i n e (130) revealed t h a t both p a i r s of s p e c t r a were i d e n t i c a l . The minor product, [c*L 1 ~132° (EtOH), from the z i n c 24  / s u l p h u r i c a c i d r e d u c t i o n of pseudo-vincadifformine  also  analysed f o r 0 2 ^ 2 2 0 ^ 2 , and s p e c t r a l data i n d i c a t e d the presence o f d i h y d r o - i n d o l e and saturated e s t e r f u n c t i o n s . A c e t y l a t i o n a f f o r d e d a N-acetate,  C2^H^ 0^N , j j x j  (EtOH), whose u l t r a - v i o l e t spectrum ( X  0  m o v  2  24  +3°  250, 278 and  296 mjk) showed s m a l l but d i s t i n c t d i f f e r e n c e s from the ace-  - 75  -  t a t e of the major dihydro compound. Moreover, the methoxyl a b s o r p t i o n i n "the N.M.R. spectrum of the acetate was i n a more u s u a l p o s i t i o n  (6.42T).  Vigorous treatment w i t h sodium methoxide converted the major component i n t o a substance which proved to be i d e n t i c a l w i t h the minor product. I t was thus e s t a b l i s h e d that the minor component, which we r e f e r to as i s o - d i h y d r o pseudo-vincadifformine, a l s o had the gross s t r u c t u r e (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 c o n f i g u r a t i o n at the carbon atom (C-18) bearing the carbomethoxy group. The i s o l a t i o n and i n t e r c o n v e r s i o n of the two  dihydro  compounds i n the r e d u c t i o n of pseudo-vincadifformine  (121)  could be r a t i o n a l i z e d by analogy w i t h a s i m i l a r s e r i e s of r e a c t i o n s performed by Smith and E d w a r d s ^ i n the akuammicine 10  s e r i e s . Reduction of dihydroakuammicine (135) w i t h z i n c and s u l p h u r i c a c i d a f f o r d e d tetrahydroakuammicine was epimerised w i t h sodium methoxide to micine (138). The authors suggested  (137), which  iso-tetrahydroakuam-  t h a t the f i r s t step i n  the r e d u c t i o n was p r o t o n a t i o n of C-16  to g i v e the immonium  i o n (136). The proton added to the {3-face i n order to a l l o w the carbomethoxy group to take up the m o r e s t a b l e e q u a t o r i a l o r i e n t a t i o n , w i t h r i n g C i n the boat conformation.  Reduction  of the immonium system then proceeded w i t h a d d i t i o n of hydrogen at C-2, a g a i n from the ( i - f a c e , to g i v e a compound  (137)  i n which the B/C r i n g j u n c t i o n was the more s t a b l e c i s - f o r m and r i n g s C and D had c h a i r 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 tion.  (136)  (135) N.  H (138)  H  CO*Me  (137)  E p i m e r i s a t i o n of tetrahydroakuammicine (137) to the isomeric base (138) was then r e a d i l y understood as i n 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 e q u a t o r i a l . This mechanism enabled the authors to e x p l a i n the hydrogen bonding of the carbonyl group i n d i c a t e d by the i n f r a - r e d spectrum o f ( 1 3 7 ) , which d i d not occur i n the case of (138). Although the akuammicine d e r i v a t i v e s (137) and (138) are not s t r i c t l y comparable to d i h y d r o - p s e u d o - v i n c a d i f f o r mine (131a) and i t s epimer, i t i s nevertheless l i k e l y  that  the r e d u c t i o n of pseudo-vincadifformine (121) f o l l o w s a s i m i l a r s t e r i c course. I f , f o r the sake of argument, the P - c o n f i g u r a t i o n i s assumed a t C-9,  then the predominant  isomer would a l s o be the k i n e t i c a l l y favoured one (139a),  - 77 -  which has the carbomethoxy group a t C-B i n the a x i a l C X - o r i e n t a t i o n . Treatment w i t h base would then a f f o r d the thermodynam i c a l l y more s t a b l e i s o - d i h y d r o compound (140a) w i t h an e q u a t o r i a l carbomethoxy s u b s t i t u e n t i n the-(3-orientation.  (139) a; R = H b: R = Ac  (140)  a; R = H b: R = Ac  I t must be emphasised t h a t , s i n c e the stereochemistry of pseudo-vincadifformine s t i l l remains t o be e s t a b l i s h e d , the above arguments are not c o n c l u s i v e . I f one c o n s t r u c t s models of the corresponding acetates (139b) and (140 b ) , the methoxyl group of the a x i a l carbomethoxy f u n c t i o n of (139b) i s found to come i n c l o s e 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 h i g h p o s i t i o n ( 6 . 8 3 T ) of the methoxyl proton s i g n a l s i n the N.M.R.- spectrum of dihydropseudo-vincadifformine a c e t a t e (139b) may be due to diamagn e t i c s h i e l d i n g by the benzene r i n g . The chemical and s p e c t r a l evidence c i t e d above e s t a b lished  a v i n c a d i f f o r m i n e - t y p e s t r u c t u r e (121) f o r pseudo-  v i n c a d i f f ormine. I t should be mentioned a t t h i s time t h a t , i n f a c t , the mercuric acetate o x i d a t i o n of carbomethoxy4"(S -dihydrocleavamine (119) can, and does, proceed i n two ,,  - 78 -  d i r e c t i o n s to provide immonium d e r i v a t i v e s i n v o l v i n g e i t h e r C-19 or C-5. At the outset i t was t h e r e f o r e 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, s t u d i e s of models showed t h a t s t e r i c r e p u l s i o n s made t h i s c y c l i s a t i o n extremely unfavourable, and the s t r u c t u r e (142) was excluded even before experiments were run. Hence pseudo-vincadifformine (121) must be d e r i v e d 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 a c e t a t e o x i d a t i o n of carbomethoxy-4"(3"-dihydrocleavamine  (119) y i e l d e d , i n a d d i t i o n to p s e u d o - v i n c a d i f f o r 105  mine, s m a l l amounts o f 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 a f f o r d e d a c r y s t a l l i n e  hydro-  - 79 -  c h l o r i d e , m.p. 221-223° . The u l t r a - v i o l e t spectrum of the base i n d i c a t e d an i n d o l e chromophore w i t h maxima a t 226, 285, and 293 inu. . The presence of an e s t e r carbonyl  absorption  at 1705 cm." , and the absence of the strong Bohlmann bands 1  i n the r e g i o n between 2700 and 2800 cm."  1  d i s p l a y e d by the  s t a r t i n g m a t e r i a l , suggested t h a t t h i s a l k a l o i d vas a member of the Iboga s e r i e s (86). Indeed, comparison of our a l k a l o i d ( i n f r a - r e d s p e c t r a and t h i n - l a y e r chromatographic m o b i l i t y ) 71  w i t h an authentic.sample of c o r o n a r i d i n e they were', the same  showed that  . Further comparison (mixed  and i n f r a - r e d spectra) "of the c r y s t a l l i n e completely  (145)  melting-point  hydrochlorides  e s t a b l i s h e d the i d e n t i t y .  The other a l k a l o i d , e l u t e d w i t h benzene-ether ( l : l ) , was c r y s t a l l i n e , m.p. 143-145.5 ° ,[ot] +49° (CHC1 ), and the 26  3  s p e c t r a l data showed the presence of an i n d o l e system and a saturated e s t e r f u n c t i o n . An authentic sample of dihydrocatharanthine was prepared by hydrogenation of catharanthine ( 5 ) , and a d i r e c t comparison (mixed m e l t i n g - p o i n t , i n f r a - r e d spect r a , t h i n - l a y e r chromatographic m o b i l i t y ) confirmed that our °8 107 product was a c t u a l l y dihydrocatharanthine  (69)" '  Thus i t was e s t a b l i s h e d that the transannular  cyclisa-  t i o n of the other p o s s i b l e mercuric acetate o x i d a t i o n product (122) w i t h the >N=C-5 immonium system, l e d to the Iboga skeleton (86).  80  MeO^C  Me0 C 2  (122)  (86)  The i s o l a t i o n of both coronaridine  (145) and dihydro-  catharanthine ( 6 9 ) from t h i s r e a c t i o n i n d i c a t e d that an i s o m e r i s a t i o n of the e t h y l group a t C-4 was t a k i n g 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 o x i d a t i o n 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 76  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  f o r the mechanism p o s t u l a t e d ( p . 5 l ) f o r the  catharanthine  -cleavamine t r a n s f o r m a t i o n . 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 c l e a v a m i n e - l i k e  inter-  mediate (74) to descarbomethoxycatharanthine (6 5), and 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  the  process.  Moreover, the immonium-eneamine tautomerism r e q u i r e d to accommodate c o r o n a r i d i n e and dihydrocatharanthihe volved to e x p l a i n the formation of 4"CXI- and 1  4"^"-dihydro-  cleavamines (68) i n the cleavage of catharanthine acidic  i s also i n -  (5) w i t h  reagents. I t i s apparent that the three products obtained i n our  r e a c t i o n 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 b i o s y n t h e t i c scheme (p. 60) does take place q u i t e r e a d i l y . Although these r e s u l t s do not prove t h a t t h i s i s the a c t u a l b i o g e n e t i c pathway, they c e r t a i n l y lend support to the l i k e l i h o o d of such r e a c t i o n s . I t appears that the Aspidosperma ( 64.) and Iboga ( 6 3 ) may  very w e l l evolve from a common b i o g e n e t i c  alkaloids  precursor  [such as ( 1 4 7 ) ] , that can a f f o r d e i t h e r a quebrachamine  (67)7  or cleavamine (66) s k e l e t o n , 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 d e r i v a b l e from the same immonium i o n (147/, which i n v o l v e s the carbon atom between the n i t r o g e n and e t h y l - b e a r i n g 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  1  H (147)  ^0^  (148)  :. T 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 i o n (148), w i l l be found i n Nature. T h i s 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) ^" 10  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 r e l a t e d p a i r of immonium i o n p r e c u r s o r s , (89) and (151) r e s p e c t i v e l y . Indeed, the Swiss 108  workers  who r e c e n t l y i n t e r r e l a t e d akuammicine  and condy-  - 83 -  l o c a r p i n e 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).  (151)  CO, Me  (100)  C0,Me  (152)  The f o r m a t i o n of c o r o n a r i d i n e (145) and dihydrocathar a n t h i n e ( 69 ) i n the mercuric a c e t a t e r e a c t i o n c o n s t i t u t e s an a t t r a c t i v e entry i n t o 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 a c c o m p l i s h e d ^ t h i s sequence o b v i o u s l y a l s o 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 s y n t h e s i s of carbomethoxy-dihydrocleavamine  (119)  can be developed, t h i s would e f f e c t the t o t a l s y n t h e s i s of the Iboga s k e l e t o n , 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 r e v e a l e d 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 e x t e n s i o n of t h i s r e a c t i o n to an a l k a l o i d such as v i n c a d i n e (106a) should provide a s y n t h e s i s of v i n c a d i f f o r m i n e and i t s r e l a t i v e s . Since i t has r e c e n t l y proved p o s s i b l e to c y c l i s e mine (107) to (+)-aspidospermidine  (—)-quebracha-  (154) by means of mercuric 109  acetate o x i d a t i o n and subsequent hydride r e d u c t i o n  , the  s y n t h e s i s of v i n c a d i f f o r i m i n e from v i n c a d i n e i s now  highly  probable.  (107)  (154)  EXPERIMENTAL  M e l t i n g p o i n t s were determined  on a K o f l e r block and  are uncorrected. - U l t r a - v i o l e t (U.V.) a b s o r p t i o n 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.) s p e c t r a were taken on a, Perkin-Elmer Model 21 spectrophotometer.  Nuclear magnetic resonance (N.M.R.)  spectra were recorded at 60 megacycles/sec.  on a V a r i a n A60  instrument; the l i n e p o s i t i o n s or centres of m u l t i p l e t s are g i v e n i n the T i e r s X s c a l e w i t h reference to t e t r a m e t h y l s i l a n e 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 i n t e g r a t e d area and type of protons are i n d i c a t e d i n parentheses.  Silica  g e l G p l a t e s were used f o r t h i n - l a y e r chromatography (T.L.C.) and were developed  by e t h y l a c e t a t e , e t h y l a c e t a t e - c h l o r o -  form or e t h y l a c e t a t e - e t h a n o l mixtures as g i v e n below. The alumina used f o r column chromatography was Shawlnigan reagent grade, d e a c t i v a t e d w i t h 3$ of 10$ aqueous a c e t i c a c i d , unless otherwise s t a t e d . Analyses "were performed by Dr.A.  Bernhardt  and h i s a s s o c i a t e s , Mulheim (Ruhr), Germany and by the M i c r o a n a l y t i c a l Laboratory, U n i v e r s i t y of B r i t i s h Columbia. Every molecular weight (M.W.) quoted was determined mass s p e c t r o metrically.  P a r t I Experimental S e c t i o n  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 L a b o r a t o r i e s , was d i s s o l v e d i n methanol (20 ml.) and water (250 m l . ) , the s o l u t i o n cooled i n i c e and made b a s i c w i t h aqueous ammonia. The p r e c i p i t a t e was taken up i n ether (200 ml.) the l a y e r s separated, and  the  aqueous p o r t i o n f u r t h e r e x t r a c t e d w i t h ether (3 x 100  ml.).  A f t e r d r y i n g over magnesium sulphate, the combined e t h e r e a l e x t r a c t s 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  acetone-petroleum ether  from  (b.p. 60-80°) a f f o r d e d needles (320 mg.) now  m.p.  178-179°, which  d i s p l a y e d only two spots on T.L.C. Repeated r e c r y s t a l l i -  s a t i o n s f a i l e d to r e s o l v e 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 w i t h one molecule of solvent as needles, m.p.  181° ,[oc1 -52 26  i_  C, 69.52; H, 7.84. H,  _ i  D  C a l c . f o r C H 0 N . M e C 0 : C, 69.88; 21  26  5  2  2  7.82. The unsolvated m a t e r i a l was  obtained from aqueous  thanol as stout needles, m.p. \nax. N>  (MeOH). Pound:  0  m a x <  ( l o g £ ) :  (Nujol):  2 2 6  me-  206-208°; foci -58° (MeOH); D < '56), 282 (3.90), 290 (3.84) m/A. ; 26  4  3360 (NH and/or OH),  1705  (C=0), 740  (o-disub-  s t i t u t e d benzene) cm." . N.M.R. s i g n a l s (CD^COCD^): 2.8 1  ( m u l t i p l e t * 4H, aromatic), 4.7 6.1  ( m u l t i p l e t , 2H, CH 0), 6.38 2  ( m u l t i p l e t , 1.8H,  olefinic),  ( s i n g l e t , 3H, CH 0),  (1H)T . Pound: C, 71.15,71-43; H,7.51, 7.66;  3  0, 14.00;  9.02  - 87  N, 7.89, 7.77; C-Me, 1.61; M.W.  354. C a l c . f o r C 1 2 6 ° 3 2 H  N  :  2  C, 71.16; H, 7.39; 0, 13.54; N, 7.90; (1) C-Me, 4.24;  M.W.  354.  Sitsirikine  Picrate  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 a c i d (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 t o 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 a f f o r d y e l l o w 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. C a l c . f o r C  2 7  H  2 g  0  1 0  N : C, 55.57; H, 5.01; N, 12.00. 5  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 d i s s o l v e d  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 m l . ) , b a s i f i e d w i t h 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 s e v e r a l times w i t h water, the e t h e r e a l s o l u t i o n was d r i e d over magnesium sulphate and evaporated. R e c r y s t a l l i s a t i o n from aqueous methan o l a f f o r d e d the acetate as needles (100 mg.), m.p.  198°;  [c*-] - 2 6 ° (MeOH); two spots on T.L.C. (EtOAc-CHCl , 1:1); 26  5  ^  ( N u j o l ) : 3340 (NH), 1735 (C=0), 1700 (0=0), 1240 (OAc)  m a v  cm."* ; N.M.R. s i g n a l s (CD^COCD^): 2.7 ( m u l t i p l e t , 4H, aroma1  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 0Ac), 6.37 ( s i n g l e t , 3H, CH 0), 8.02 ( s i n g l e t , 3H, CH C=0), 2  3  5  9.02 ( 1 H ) T . Pound: C, 69.65; ,H, 7.32; N, 7.01. C a l c . f o r C  23 28 4 2 H  0  N  !  °'  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) Sitsirikine  (450 mg.), i n methanol (10 m l . ) , was  hydrogenated over palladium b l a c k (24 mg.). The hydrogen uptake ceased a f t e r 30 minutes when 0.65 mol. had been absorbed. A f t e r removal of the c a t a l y s t and s o l v e n t , 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 g i v e d i h y d r o s i t s i r i k i n e (405 mg.), m.p. 177-179°. T h i s compound d i s p l a y e d 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 i m p u r i t y which could not be removed from s i t s i r i k i n e was i n f a 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, w i t h one molecule of s o l v e n t , as needles, m.p. 180° . Pound: C, 69.42; H, 7.91; N, 6.97. Calc. f o r C H 0 N * M e C 0 : C, 69.53; H, 8.27; N, 6.76. 21  28  3  2  2  R e c r y s t a l l i s a t i o n from aqueous methanol a f f o r d e d the unsolvated a l k a l o i d as prisms, m.p. 215° ;[oc] ^-55° D 2  L  X  max. (  l 0  S ) £  5  2  2  6  (MeOH);  J  (4.61), 282 (3-95), 290 (3.87) m u j A  (log£): 247 (3.40), 287.5 (3.85) m/*. ; V  m i n >  ( C H C l ) i 3480  ffiax>  3  (NH and OH), 2810 and 2760 (Bohlmann b a n d s ) , 1710 (0=0) cm." ; 4 4  ^  m Q V  ( N u j o l ) : 3400  N.M.R. s i g n a l s  1  (NH), 3200 (OH), 1710 (C=0)cm." ; 1  (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 0), 6.42 ( s i n g l e t , 3H, CH 0), 9.07 2  3  (broad s i n g l e t , 3H, C H C ) T . Pound: C, 70.80; H, 7.78; 0, 3  13.49; N, 7.77; O-Me, 9.01; C-Me, 3.95; M.W. 356. C a l c . f o r C  21 28°3 2 H  8.72;  N  S  C  ' ' '> » - 9 ; 0, 13.47, N, 7.86; ( l ) O-Me, 10  16  H  7  2  (1) C-Me, 4.22; M.W. 356.  - 89 -  Dihydrositsirikine  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 o f p i c r i c a c i d were r e a c t e d i n the manner d e s c r i b e d above and the d e r i v a t i v e 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.) w i t h a c e t i c anhydride i n p y r i d i n e 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° ) t o a f f o r d needles (155 mg.), m.p. 187° one spot on T.L.C. (EtOAc-CHCl,, 1:1) ;M -31° (MeOH); D 26  V.  M Y  ( N u j o l ) : 3390 (NH), 1740 (0=0), 1705 (C=0), 1250  (OAc) cm." ; N.M.R. s i g n a l s (CDClj):'1.34 ( s i n g l e t , IH, NH), 1  2.8 ( m u l t i p l e t , 4H, a r o m a t i c ) , 5.6 ( m u l t i p l e t , 2H, CHgOAc), 6.38 ( s i n g l e t , 3H, CH 0), 7.96 ( s i n g l e t , 3H, CH C=0), 9.02 5  (broad s i n g l e t , 3H, CE^G)X  5  . 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 c h l o r i d e  (130 mg.) was added t o d i h y -  d r o s i t s i r i k i n e (65 mg.) i n dry p y r i d i n e (3 m l . ) . A f t e r standing overnight the s o l u t i o n was poured i n t o i c e - w a t e r , made b a s i c w i t h 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 e t h e r e a l s o l u t i o n washed twice w i t h water and d r i e d over magnesium sulphate. Removal of the s o l v e n t gave a gum which was d i s s o l v e d 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  e t h e r (b.p. 60-80° ) t o y i e l d the p-bromoo  benzoate as s l e n d e r needles (60 mg.), on T.L.C.; \ ) (C=0)  Q  V  ( N u j o l ) : 3370 (NH), 1725  cm." ; N.M.R. s i g n a l s (CDCl^): 2.5 1  a r o m a t i c ) , 5.4 CH 0), 9.10 5  H, 5.70; N,  m  m.p.  ( m u l t i p l e t , 5H, CH 0), 6.37 2  174  ; one  spot  (0=0), 1705 ( m u l t i p l e t , 6H, ( s i n g l e t , 3H,  (broad s i n g l e t , 3H, CH C)'T. Pound: C, 62.34;  N, 5.06.  3  C a l c . f o r C g g H ^ O ^ B r : C, 62,33; H,  5.79;  5.19.  S a p o n i f i c a t i o n 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  w i t h 2 N methanolic sodium hydroxide (20 ml.) f o r 2 hours. A f t e r removal of the s o l v e n t , the r e s i d u e was taken up i n water and e x t r a c t e d w i t h methylene c h l o r i d e to remove unsap o n i f i e d m a t e r i a l . The aqueous s o l u t i o n was a c i d i f i e d w i t h h y d r o c h l o r i c a c i d and evaporated to dryness. The r e s i d u e was leached w i t h a b s o l u t e ethanol and the s o l u t i o n f i l t e r e d from sodium c h l o r i d e . Evaporation and r e c r y s t a l l i s a t i o n from aqueous a l c o h o l gave an a(S-unsaturated a c i d h y d r o c h l o r i d e ,  - 91 -  m.p. 260-263° ; v  m Q  ^  max *  ( N u j o l ) : 1695 (0=0), 1620 (0=0) cm." ; x  N.M.R. s i g n a l s (CF^CO^): 3.2 ( m u l t i p l e t , 4H, a r o m a t i c ) , 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 .  Dihydrositsirikine Diol A s o l u t i o n o f 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 r u n slowly i n t o a s t i r r e d suspension of l i t h i u m aluminium hydride (200 mg.) i n t e t r a h y d r o f u r a n (10 ml.) and heated under r e f l u x f o r 3 hours. A f t e r the mixture had stood o v e r n i g h t , t h e excess hydride was decomposed w i t h s a t u r a t e d aqueous sodium sulphate s o l u t i o n (10 ml.), f o l l o w e d by water (20 ml.). extracted ned  The aqueous suspension was then  w i t h methylene c h l o r i d e  (4 x 25 ml.),  and the combi-  e x t r a c t s d r i e d o v e r sodium sulphate. Removal o f 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, (EtOAcEtOH, 1:1); [ a ] - 3 ° (MeOH); \ 26  (3.85), 290 (3.78)mym ; v  m a x <  ( l o g O : 226 (4.55), 282  ( N u j o l ) : 3210 (NH and OH) cm." ; 1  m a x <  N.M.R. s i g n a l s (CD^COCD^): 2.8 ( m u l t i p l e t , 4H, a r o m a t i c ) , 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 C)r. 3  Pound: C, 73.30; H, 8.46; N, 8.41. Calc. f o r C H g 0 N : 2 0  2  2  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 d i s s o l v e d  i n dry acetone  (20 ml.) and p-toluenesulphonic a c i d (110 mg.) added. A f t e r  - 92 -  standing a t room temperature f o r 48 hours, the s o l u t i o n was neutralised  w i t h aqueous ammonia and the acetone removed under  vacuum. The product was i s o l a t e d w i t h ether, the e t h e r e a l s o l u t i o n d r i e d over magnesiumsulphate,  and evaporated t o  leave a gum. On t r i t u r a t i o n w i t h 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 t o 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 o f alumina (3 g.). Removal o f the s o l v e n t gave the acetonide as an amorphous powder (30 mg.) which c r y s t a l l i s e d from methanol w i t h one molecule of s o l v e n t , m.p. 105-109° ; one spot on T.L.C. (EtOAc-CHCl,, l:l);v„,  QV  signals  ( N u j o l ) : 3200 (NH) cm."" ; N.M.R, 1  (CDCl^): 1.61 ( s i n g l e t , IH, NH), 2.8 ( m u l t i p l e t ,  4H, a r o m a t i c ) , 6.25 (doublet, 4H, 2 CH 0), 8.62 ( s i n g l e t , 2  6H, ( C H ) C 0 ) , 9.06 (broad s i n g l e t , 3H, C H C ) T . Found 3  2  2  3  (powder): C, 74.56; H, 9.03; N, 7.31. C a l c . f o r  C ^ 0 % '. 2  2  2  2  C, 74.96; H, 8.75; N, 7.60. Found ( s o l v a t e ) : C, .72.21; H, 8.71. C a l c . f o r C^H^O^.MeOH: C, 71.96; H, 9.06. M o d i f i e d Kuhn-Roth O x i d a t i o n ^  7  o f 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 a c i d (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 w i t h p e r i o d i c a d d i t i o n o f water u n t i l 30 ml. o f d i s t i l l a t e had been c o l l e c t e d . This was n e u t r a l i s e d  with  . 93 -  2 N aqueous potassium hydroxide (pH meter), and evaporated to dryness. The r e s i d u e was taken up i n pure water (0.3 ml.) and put on a s m a l l column of Dowex 50  a c i d r e s i n (2 x 0.5 cm.);  the f l a s k was washed w i t h water (2 x 0.5 ml.) and t h i s a l s o added to the column. To t h e 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 a t 30-40° down to 1-2 drops. This was spotted on Whatman No. 1 paper t o g e t h e r w i t h standard s o l u t i o n s of the ethylamine s a l t s of a c e t i c , p r o p i o n i c and b u t y r i c a c i d s . The paper was developed by descending chromatography, u s i n g a 0.025 M ethylamine s o l u t i o n i n water-saturated n-butanol as the s t a t i o n a r y phase, and water-saturated •57  n-butanol as the mobile phase^ . A f t e r 24 hours the paper was sprayed w i t h a l c o h o l i c bromocresol green s o l u t i o n , when the a c i d s became v i s i b l e as blue spots on a y e l l o w 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 p r o p i o n i c a c i d s , and a blank o x i d a t i o n showed o n l y the barest t r a c e of a c e t i c acid. S i t s i r i k i n e under these c o n d i t i o n s a l s o gave a c e t i c and p r o p i o n i c a c i d s , -while i s o s i t s i r i k i n e gave a c e t i c a c i d only. O z o n i s a t i o n 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) Sitsirikine  (5 mg.) i n g l a c i a l a c e t i c a c i d (1 ml.)  was ozonised f o r 5 minutes, then t r a n s f e r r e d t o a d i s t i l l a t i o n apparatus c o n t a i n i n g 5$ aqueous f e r r o u s sulphate (15 ml.).  - 94 -  A f t e r 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. T h e - s o l u t i o n was ext r a c t e d s e v e r a l times w i t h benzene, the combined  extracts  d r i e d w i t h magnesium sulphate and flushed through a column of P i s h e r acid-washed alumina (10 g.). A f t e r c o n c e n t r a t i o n 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 w i t h standard s o l u t i o n s (5 mg./ml.) of the 2,4-dinitrophenylhydrazones of formaldehyde , acetaldehyde and acetone. 36  The paper was developed by descending chromatography-^, u s i n g methanol-heptane as the s t a t i o n a r y phase and heptane as the mobile phase. A f t e r 6 hours the paper was sprayed w i t h 10$ aqueous sodium hydroxide s o l u t i o n . The 2 , 4 - d i n i t r o 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 t r a c e of acetone (dark-brown s p o t , Rf 0.30) was a l s o present. Blank experiments gave no spots corresponding to formaldehyde or acetaldehyde, but always showed a t r a c e of acetone. R e p e t i t i o n of the same procedure f o r i s o s i t s i r i k i n e (5 mg.) showed f o r m a t i o n of acetaldehyde 2 , 4 - d i n i t r o p h e n y l hydrazone (Rf 0.19). Lead T e t r a c e t a t e Dehydrogenation of D i h y d r o s i t s i r i k i n e Lead t e t r a c e t a t e (400 mg.) was added i n s m a l l p o r t i o n s over a p e r i o d of 10 minutes t o a s o l u t i o n of d i h y d r o s i t s i -  -95  -  r i k i n e (100 mg.) i n g l a c i a l a c e t i c a c i d (10 m l . ) . The mixture was kept a t 50-60° f o r a f u r t h e r 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 e x t r a c t e d w i t h chloroform. The chloroform e x t r a c t was washed w i t h a l i t t l e water, d r i e d over sodium s u l p h a t e , and a c i d i f i e d t o Congo r e d w i t h 8 M e t h a n o l i c hydrogen c h l o r i d e . Evaporation of the s o l v e n t a f f o r d e d t e t r a d e h y d r o - d i h y d r o s i t s i r i k i n e h y d r o c h l o r i d e as a gum (70 mg.), which could not be induced to c r y s t a l l i s e ; X.,^253, 308, 365 mw.;X  v  ( a c i d and n e u t r a l s o l u t i o n ) :  ( 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 P a l l a d i u m Dehydrogenation of Tetradehydro -dihydrositsirikine  Hydrochloride  The h y d r o c h l o r i d e (55 T a g . ) from the p r e v i o u s r e a c t i o n was mixed w i t h palladium b l a c k (50 mg.) and heated a t 250-270° under n i t r o g e n f o r 7 minutes. The r e s i d u e was leached w i t h 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 t e r i a l . The r e a c t i o n was repeated t w i c e , h e a t i n g a t 280° f o r 15 and 30 minutes; however, no change was observed i n the U.V. spectrum. P a l l a d i u m - c h a r c o a l 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 w i t h 10$ palladium-charcoal(250  mg.) and heated under n i t r o g e n a t 250°  - 96  f o r 15 minutes. The r e s i d u e was e x t r a c t e d w i t h hot methanol and the U.V. spectrum r u n ; X  m Q V  ; 230, 290, 310, 385 mu.  A f t e r removal of the methanol, the product was taken up i n ether-water,  the ether la.yer separated and d r i e d  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 p o r t i o n was made s t r o n g l y a l k a l i n e (pH>10) and e x t r a c t e d w i t h chloroform. A f t e r d r y i n g , the chloroform s o l u t i o n was evaporated t o a f f o r d a gum (5 n i g . ) ; " ^ . . . : 295, IH3 X «  313, 348, 389  mjx  .  The n e u t r a l e x t r a c t was d i s s o l v e d i n a few drops o f methanol and spotted on a. p r e p a r a t i v e T.L.C. p l a t e ( s i l i c a g e l , 0.5 mm. t h i c k ) . The p l a t e was developed i n chloroforme t h y l acetate (3:1) f o r 45 minutes, d r i e d , and then developed two more times. Under U.V, l i g h t f o u r bands  CDuld  be seen,  and each was cut out and e x t r a c t e d w i t h methanol i n a Soxhlet apparatus f o r s e v e r a l hours. The e x t r a c t from the main band (compound A) d i s p l a y e d U.V. spectra s i m i l a r to those of harman ^ (Figures 5 and 6 ) ; \ 4  m Q V  ( n e u t r a l 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. s o l u t i o n ) : 254, 303, 372 mjx . Evaporation of the methanol y i e l d e d a gum (10 mg.); V „ «i cm. . m  m Q V  (CHC1,): 1710 (0=0), 1625 (0=0)  - 97 -  P a l l a d i u m - c h a r c o a l 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 w e l l mixed w i t h 10$ palladium-charcoal (200 mg.) and heated under n i t r o g e n a t 280° f o r 15 minutes. The residue was e x t r a c t e d w i t h hot methanol, f i l t e r e d , and the U.V. s p e c t r a run; X„,_  v  and  (neutral  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 s o l u t i o n ) ? 282, 310, 382 inu . The s o l v e n t was removed and the residue d i s s o l v e d i n water, the s o l u t i o n made b a s i c (pH 8) w i t h ammonia and shaken w i t h ether. A f t e r s e p a r a t i o n and d r y i n g the ether was removed to leave a gum (5 mg.);  X  m a x >  • 290, 355 m^. .  50$ Aqueous sodium hydroxide was added t o the aqueous p o r t i o n u n t i l i t was s t r o n g l y a l k a l i n e (pH>10), and the s o l u t i o n was then e x t r a c t e d w i t h chloroform. The red c h l o r o form e x t r a c t was washed w i t h water, d r i e d , ana a c i d i f i e d w i t h 8 W e t h a n o l i c hydrogen c h l o r i d e ( y e l l o w s o l u t i o n ) . Evap o r a t i o n o f the s o l v e n t a f f o r d e d a gum (22 and n e u t r a l s o l u t i o n ) : 222, 308, 383 myx  m  g«)>X  ;X  maXi  (acid  ^ (alkaline  fflax  s o l u t i o n ) : 283, 315, 380 m/x. . This m a t e r i a l was d i s s o l v e d i n a l i t t l e methanol and spotted on a p r e p a r a t i v e T.L.C. p l a t e ( s i l i c a g e l , 0.5 mm. t h i c k ) . This p l a t e was run f o r 20 minutes i n e t h y l a c e t a t e , and then twice i n e t h a n o l - e t h y l acetate (1:1) f o r 40 minutes. Under U.V.  l i g h t a s e p a r a t i o n i n t o two main bands was  observed. These were cut out and e x t r a c t e d w i t h methanol i n  -  98  -  a Soxhlet apparatus f o r s e v e r a l hours. One f r a c t i o n gave a U.V.  spectrum analogous to t h a t  of t e t 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 t h a t of 5 , 6 - d i h y d r o 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 ; A m a x > : 221, 312, 386 m^ ; X m i n > : 215, 277, 4 3  338 mjx .  Quinone Dehydrogenation of Compound B The methanolic s o l u t i o n of compound B was evaporated to g i v e a gum  (2 mg.), which was d i s s o l v e d i n g l a c i a l a c e t i c  a c i d (0.5 m l . ) . 2,3-Dichloro~5,6-dicyano-p~benzoquinone *  (10 mg.) was added, and the mixture heated at 80-90  o  for  6 hours. The s o l u t i o n was then d i l u t e d w i t h water and e x t r a c ted s e v e r a l times w i t h ether. A f t e r making s t r o n g l y a l k a l i n e w i t h 50$ aqueous sodium hydroxide, the aqueous s o l u t i o n was shaken w i t h chloroform, the organic l a y e r separated, washed w i t h a l i t t l e water and d r i e d over sodium sulphate. A c i d i f i c a t i o n w i t h 8 N e t h a n o l i c hydrogen c h l o r i d e and removal of the s o l v e n t a f f o r d e d a y e l l o w gum (0.8 mg.). This (compound C) d i s p l a y e d 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 ; X m a x < : 237, 291, 345, 4 5  385HUA  The compound d i s p l a y e d the same Rf value (0.43) as an a u t h e n t i c sample of f l a v o c o r y l i n e on paper  chromatography  u s i n g an e t h y l a c e t a t e - p y r i d i n e - w a t e r (8:2:1) system.  - 99 -  Dihydrositsirikine  O l e f i n i c E s t e r (22)  Dihydrositsirikine  acetate (1.3 g.) was heated under  r e f l u x w i t h 0.1 N sodium methoxide i n dry methanol (60 ml.) f o r 45 minutes. S o l i d carbon d i o x i d e was then added, the methanol removed under vacuum, and the residue taken up i n ether-water. The e t h e r e a l s o l u t i o n was d r i e d and the solvent removed. Chromatography of the product on alumina (50 g.) a f f o r ded the d e s i r e d m a t e r i a l (410 mg.) on e l u t i o n w i t h benzene-ether (19*1)° Ether e l u t e d 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 a f f o r d e d the solvated o l e f i n i c e s t e r as needles (290 m g . ) , m.p. 84-89° ; one spot on T.L.C.(EtOAc-CHCl,, 1:1); Totl + 2 ° ( M e O H ) ; V 26  j  L  mQV  (Nujol):  nicix o  J  3160 (NH), 1708 (0=0), 1622 (C=C); N.M.R. s i g n a l s ( C D C l j ) : 1.60 ( s i n g l e t , IH, NH), 2.8 ( m u l t i p l e t , 4H, a r o m a t i c ) , 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 0), 9.06 (broad s i n g l e t , 3H, C H j O f . Found: C, 71.41; 3  H, 8.08; 0, 12.71; N, 7.12. Calc. f o r C H 0 N .MeOH: C, 71.32; 21  26  2  2  H, 8.16; 0, 12.96; N, 7.56. D e s o x y - D i h y d r o s i t s i r i k i n e (23) The above o l e f i n i c e s t e r (95 mg.) i n methanol was hydrogenated over palladium black (15 mg.). Uptake of hydrogen ceased a f t e r 3 hours when 1.04 mol. had been absorbed. The s o 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 dropwise u n t i l a permanent t u r b i d i t y was observed. On c o o l i n g the saturated e s t e r 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); ( N u j o l ) : 3370 (NH),1710 (C=0) cm." . Pound: C, 74.04; H, 6.43; 1  H , 8.36.  Calc. f o r P l 2 8 2 2 H  0  N  2  :  c  » 4.08, H, 8.29;  N, 8.23.  7  L i t h i u m Aluminium Hydride Reduction o f 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 E s t e r (22) The o l e f i n i c e s t e r (150 mg.) and l i t h i u m aluminium hydride  (100 mg.) i n e t h e r - t e t r a h y d r o f u r a n (1:1, 30 ml.) were heated under r e f l u x f o r 2 hours. Excess hydride was decomposed w i t h -  saturated aqueous sodium sulphate s o l u t i o n (20 m l . ) , the organ i c l a y e r separated, and the aqueous suspension e x t r a c t e d w i t h methylene c h l o r i d e (3 x 10 ml.). The combined organic e x t r a c t s were d r i e d over magnesium sulphate and evaporated t o leave a crystalline  s o l i d (140 mg.).  This m a t e r i a l had no carbonyl  a b s o r p t i o n i n the I.R. r e g i o n , but showed three spots on T.L.C. The N.M.R. spectrum i n d i c a t e d t h a t the mixture contained only 25$ o f the expected t e r m i n a l o l e f i n i c a l c o h o l . The product was taken up i n benzene and chromatographed on alumina (5 g., d e a c t i v a t e d w i t h 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 a c e t a t e e l u t e d the major f r a c t i o n (70 mg.), recrystallised  t w i c e from acetone-petroleum  which was  ether (b.p. 60-80°)  and once from aqueous methanol t o a f f o r d l i g h t brown needles (23 mg.),  m.p. 204° ; one spot on T.L.C. (EtOAc);foci -24.3° , D (MeOH); v _ ( N u j o l ) : 3200 cm." (NH and OH); N.M.R. s i g n a l s (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, 26  L  J  1  m t l  no o l e f i n i c protons. Pound: C, 77.39; H, 8.49;  N , 8.79.  - 101 -  C a l c . f o r C H 0 N : C, 77.38; H, 8.44; N , 9.03. 2 0  2 6  2  The constants quoted i n the l i t e r a t u r e methoxy-dihydrocorynantheine  4 2  f o r iso-des-  a l c o h o l (21) are m.p. 204° ,  rocl -24.0° (MeOH). D 17  J  Dihydrocorynantheine (18h) Crude corynantheine " " 1  was hydrogenated  1  0  (700 mg., two spots on T.L.C.)  i n ethanol (5 ml.) over palladium b l a c k .  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 c o o l i n g 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."" . N.M.R. s i g n a l s (CD COCD )s 1  5  5  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. C a l c . f o r C H 0 N : C, 71.71; H, 7.66; N, 7.60. Ill o The m.p. quoted f o r dihydrocorynantheine i s 173-174 . 2 2  2 8  3  2  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 i c e and dry hydrogen c h l o r i d e passed i n f o r 15 minutes. A f t e r standing a t 5° f o r 15 hours, the s o l u t i o n was evaporated under vacuum t o s m a l l bulk, d i l u t e d w i t h water (100 ml.) and e x t r a c t e d w i t h chloroform (10 x 50ml.).  102  The water l a y e r was then made b a s i c w i t h aqueous ammonia, e x t r a c t e d w i t h ether (3 x 50 ml.) and the combined ether e x t r a c t s d r i e d over magnesium sulphate. Removal o f the ether a f f o r d e d 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 c h l o r i d e t e s t ( p u r p l e ) ; \>  v  (CHOI,): 3480 (NH) 1715 (0=0), 1658 (H0-C=C-C=0),  1607 (C=C) cm." . N.M.R. s i g n a l s (CD^COCDj): 1.01 ( s i n g l e t , 1  IH, NH), 2.8 ( m u l t i p l e t , 4H, a r o m a t i c ) , 6.28 and 6.52 (2 s i n g l e t s , 3H, CH^O), 9.1 (broad s i n g l e t , 3H, C H C ) T . Found: C, 5  71.22; H, 7.91; N, 8.21. C a l c . f o r C21 26°3 2 » H  N  !  C  7 1  °  1 6  » > H  7.39; N, 7.90.  Desmethyl-dihydrocorynantheine (200 mg.), i n methanol (10 ml.), was  reduced w i t h sodium borohydride (500 mg.).  After  1 hour the methanol was removed under vacuum, the r e s i d u e t r e a ted w i t h water and then e x t r a c t e d w i t h ether. The e t h e r e a l l a y e r was separated, d r i e d over magnesium sulphate and the s o l vent evaporated. The r e s u l t i n g m a t e r i a l was d i s s o l v e d i n benzene and chromatographed  on alumina (10 g.). E l u t i o n w i t h  ether y i e l d e d a s o l i d which was r e c r y s t a l l i s e d twice from petroleum ether (b.p. 60-80° ) t o a f f o r d stout* needles (65 (MeOH);  m.p. 215° ; one spot on T.L.C. \)  v  mg.),  -1 Found: ™_ ( N u j o l ) : 3400 (NH), 3200 (OH) 1710 (0=0) cm.".  IUclX a  C, 70.81; H, 7.94; N, 7.96. 7.92; N, 7.86.  C a l c . f o r C H 0 N : C, 70.76; H 2 1  2 Q  5  2  - 103 -  This compound was i d e n t i c a l w i t h 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. s p e c t r a .  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 L a b o r a t o r i e s 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. C a l c . f o r 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 f r e e base was an amorphous powder, [cx.| ^=20° (CHC1,); D one spot on T.L.C. (EtOAc); A. ( l o g € ) : 224 (4.55), 283 (3-92), 2  ?  in 3.x o  291 (3.84) ma ; V  (0HCl,)s 3400 (NH and OH), 1725 (C=0) cm." , 1  m o v  no Bohlmann b a n d s ; v  ( N u j o l ) : 3300 (NH and OH), 1720 (C=0),  44  m o v  740 ( o - d i s u b s t i t u t e d benzene) cm," ; N.M.R. s i g n a l s (CDCl^): 1.33 1  ( s i n g l e t , IH, NH), 2.7 ( m u l t i p l e t , 4H, a r o m a t i c ) , 4.53 ( q u a r t e t , J = 7 c.p.s., IH, C=CH-CH ) 6.28 ( s i n g l e t , 3H, CH 0), 8,40 5  9  5  (doublet, J = 7 c.p.s., 3H, CH -CH=C)t. Pound: C, 70.65; H, 5  7.75; 0, 14,15; N, 7.53; C-Me, 3.28; O-Me, 9.17; M.W. 354. Calc. f o r C H 0 N : C, 71.16; H, 7,39; 0, 13.54; N, 7.90; (1) C-Me, 2 1  2 6  5  2  4.24; (1) O-Me, 8.76; M.W. 354, Isositsirikine  p i c r a t e was prepared  i n the manner des-  c r i b e d 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, C a l c . f o r C H 0 N : 2 7  2 9  1 0  5  - 104 -  C, 55.57; H, 5.01;  0, 27.42; N, 12.00.  A c e t y l a t i o n of I s o s i t s i r i k i n e w i t h a c e t i c anhydride i n p y r i d i n e gave an amorphous monoacetate which was homogeneous by T.L.C. (EtOAc-CHCl,, 1:1); v ^ , , (0=0), 1235 (OAc) IH, NH),  (0C1.): 3380 (NH), 1730  cm." ; N.M.R. s i g n a l s ( C D C l j ) : 1.40 ( s i n g l e t , 1  2.7 ( m u l t i p l e t , 4H, a r o m a t i c ) , 4.39 ( q u a r t e t , I H ,  C=CH-CH ), 6.05 (doublet, 2H, CH~CH 0Ac) 6.27 ( s i n g l e t , 3H, 5  o  CH 0), 8.15 ( s i n g l e t , 3H, CH C=0), 8.36 (doublet, 3H, CH -CH=C)T. 5  5  Found: C, 69.84; H, 7.78; 69.67, H  Acetonide  5  7.12;  N, 7.44.  3  Calc.  f o r Co^H^O^Ng: C,  N, 7.07.  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 o f i s o s i t s i r i k i n e (300 mg.) i n t e t r a h y d r o f u r a n (10 ml,) was run i n t o a suspension  of lithiumaluminium  hydride  (300 mg.) i n ether (5 m l . ) , and the mixture heated under r e f l u x f o r 3 hours. Saturated aqueous sodium sulphate (10 ml.) was added w i t h s t i r r i n g and t h e 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 w i t h water (20 ml.) the aqueous l a y e r was e x t r a c t e d w i t h methylene c h l o r i d e (3 x 20 m l . ) , and the combined organic ext r a c t s were d r i e d over sodium sulphate. Evaporation of the s o l u t i o n a f f o r d e d a gum (240 mg.); v  : 3200 (OH and NH)cm." , 1  m  a  v  no carbonyl a b s o r p t i o n ; N.M.R. s i g n a l s (CD^COCD^): 2.8 ( m u l t i p l e t , 4H, a r o m a t i c ) , 4.53 ( q u a r t e t , IH, C=CH-CH ), 8.37 5  (doublet, 3H, CH^-CH=C)T , no methoxyl a b s o r p t i o n . Since i t could not be induced t o 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 s t a n d i n g overnight the s o l u t i o n was made b a s i c w i t h aqueous ammonia, and the acetone removed under vacuum. The r e s i d u e was e x t r a c t e d w i t h e t h e r , the e t h e r e a l  solution  d r i e d over sodium s u l p h a t e a n d 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 a f f o r d e d 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 - 5 3 ° (CHOI,); D 26  ( N u j o l ) : 3200 (NH) cm." ; N.M.R. s i g n a l s  (CDC1,):  1  ( s i n g l e t , IH, NH), 2.8 ( m u l t i p l e t , 4H, a r o m a t i c ) , 4.40 IH, C=CH-CH ), 8.30 5  ( d o u b l e t , 3H, CH ~CH=C), 8.63  5  2  ( s i n g l e t , 3H, ( C H ) C 0 ) T .  2  5  72.19; H, 8.39; 8.46; 72.33, H, 8.60; N,  2  (quartet,  (singlet,  5  3H, ( C H ) C 0 ) , 8.68  1.87  Found: C, 72.10,,  2  N, 7.20. C a l c . f o r C 3 3 0 2 2 * MeOH: C, H  0  N  2  7.03.  Dihydro-isositsirikine  (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 p a l l a d i u m b l a c k (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; y i e l d e d 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) e l u a t e  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 max o (CHOI,): 3480 (NH and OH), 2810 and 2760 (Bohlmann bands)' j  1  m o v  1720 (0=0) cm." ; N.M.R. s i g n a l s (CDC1 ): 2.01 ( s i n g l e t , IH, NH), 1  5  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 C)X . Pounds 0, 70.30; H, 7.53; N , 8.13. 5  Calc. f o r C H 0 N : C, 70.76; H, 7.92; N, 7.86. 2 1  2 8  5  2  D i h y d r o - i s o s i t s i r i k i n e p i c r a t e 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 7 3 1 ° 1 0 5 ' ^ 2 > 54.56; H, H  W  H  0:G  2  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 d i s s o l v e d i n a c e t i c a c i d (10 ml.), l e a d t e t r a c e t a t e (400 mg.) added i n small portions, and the mixture heated a t ca. 60° f o r 2 hours. A f t e r removal of the solvent the residue was taken up i n water (20 ml.), made s t r o n g l y a l k a l i n e w i t h 50$ aqueous potassium hydroxide and ext r a c t e d w i t h chloroform  ( 3 x 20 m l . ) .  The combined e x t r a c t s were d r i e d over sodium sulphate, a c i d i f i e d w i t h 8 N e t h a n o l i c hydrogen c h l o r i d e and evaporated to dryness. T e t r a d e h y d r o - d i h y d r o - i s o s i t s i r i k i n e (30) hydrochlor i d e was thus obtained 360 m u ; V  as a r e d g l a s s (70 m g . ) ; X  mciv  : 253, 308,  (OHClj, f r e e base): 1730 (0=0), 1615 cm." ; 1  m a x <  T.L.C. (EtOAc) showed no s t a r t i n g m a t e r i a l .  - 107 -  Sodium Bprohydride Reduction of Tetradehydro-dihydroisositsirikine  (50)  The above h y d r o c h l o r i d e (60 mg.), i n methanol  (5 ml.),  was t r e a t e d w i t h sodium borphydride (200 mg.), and heated under r e f l u x f o r 1 hour. The s o l v e n t was removed'under vacuum, t h e r e s i d u e taken up i n water (10 m l . ) , and e x t r a c t e d - w i t h ether (4 x 10 ml.) A f t e r d r y i n g , the e t h e r e a l s o l u t i o n was evaporated, the product taken up i n benzene, and on alumina( 3 g«). Benzene-ether  chromatographed  (3^1) e l u t e d 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. s p e c t r a ,  T.L.C).  P a l l a d i u m 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.) palladium black (60 mg.)  was i n t i m a t e l y mixed w i t h  and heated at ea. 270° under a n i t r o g e n  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 p r e p a r a t i v e T.L.C. p l a t e ( s i l i c a g e l , 0.5  mm.  t h i c k ) . The p l a t e 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 f l u o r e s c e n t bands cut out. Each s e c t i o n was e x t r a c t e d w i t h methanol i n a Soxhlet -  apparatus f o r s e v e r a l hours, and the U.V,spectrum r u n on the s o l u t i o n . S i g n i f i c a n t s p e c t r a were shown by two f r a c t i o n s : ( i ) The f r a c t i o n w i t h Rf 0.9 had a U.V. that of harman  43  a b s o r p t i o n s i m i l a r to  ( c f . F i g u r e s 5 and 6 ) ; \  t i o n ) : 232, 282, 289, 336, 347  ;"K  (neutral soluin 3.x •  min<  : 273, 305, 340 nyi ;  - 108 -  A  m a x >  ( a c i d 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 w i t h Rf 0.1 had a spectrum reminiscent of flavocoryline  ( n e u t r a l and a c i d s o l u t i o n ) : 237, 247,  4 3  291, 345, 385 m/x ; A The methanolic  m i n #  : 245, 274, 304, 373 m^t .  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 hydroxide 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 e x t r a c t e d w i t h chloroform ( 3 x 10 ml.) A f t e r d r y i n g over potassium carbonate the chloroform e x t r a c t was a c i d i f i e d w i t h 8 N e t h a n o l i c hydrogen c h l o r i d e and evaporated to leave a y e l l o w 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 h y d r o c h l o r i d e . Paper chromatography showed that most of the m a t e r i a l 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 e t h y l a c e t a t e - p y r i d i n e - w a t e r (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.  P a l l a d i u m 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 t o the amorphous h y d r o c h l o r i d e s a l t , which was then i n t i m a t e l y mixed w i t h palladium black (200 mg.) and heated under a n i t r o g e n atmosphere a t 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 a c i d (5 m l . ) , 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° f o r 5 hours. A f t e r removal of the s o l v e n t 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 e x t r a c t e d s e v e r a l times w i t h ether to remove weak bases. The aqueous s o l u t i o n was  then  made s t r o n g l y a l k a l i n e (pH>10) w i t h 50$ aqueous potassium  hy-  droxide and e x t r a c t e d w i t h chloroform (3 x 20 ml.). When the chloroform e x t r a c t had been d r i e d over potassium carbonate, i t was a c i d i f i e d w i t h 8 N e.thanolic hydrogen c h l o r i d e and ted  to y i e l d a y e l l o w gum  l i n e - t y p e U.V.  (40 mg.),  evapora-  which d i s p l a y e d a f l a v o c o r y -  spectrum. Paper chromatography (as above) i n d i -  cated t h a t 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 a f f o r d e d the major component as y e l l o w needles (6 mg.), heated to 150°  );X-  max#  m.p.  280-282° (block pre-  ( l o g € ) : 238 (4.54), 247 (4.50),  (4.13), 345 (4.27), 384 (4.23) m/A : \  m i n >  (log£): 210  244 (4.49), 273 (4.04), 303 (4.01), 373 (4.15) mu; v 3350 (NH), 1650, 1630, 1515  m a x >  290 (4.26), (Nujol):  cm."" ; one spot on paper chromato1  graphy. This compound was found to be i d e n t i c a l w i t h a u t h e n t i c 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 i n a l l r e s p e c t s : m.p. m.p.;  superimposible I.R. and U.V.  paper chromatography.  and mixed  s p e c t r a ; same Rf values on  - no  -  P a r t I I Experimental  Section  I s o l a t i o n of Cleavamine (66) and Descarboinethoxycatharanthine (65) A mixture of catharanthine h y d r o c h l o r i d e (40 g.) » 101  stannous c h l o r i d e (44 g.) and mossy t i n (4 g.) i n concentrated h y d r o c h l o r i c a c i d (520 ml.), was heated under r e f l u x i n a n i t r o gen atmosphere f o r 75 minutes. By the end of t h i s time a r e d 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 w i t h methylene c h l o r i d e (3 x 100 ml.). The washings were combined w i t h the r e d gum, then methanol (50 ml.) and methylene c h l o r i d e (100 ml.) were added so t h a t a c l e a r s o l u t i o n was obtained. This s o l u t i o n was shaken w i t h 1 N aqueous sodium hydroxide  (600 m l . ) , separated and washed w i t h water (200 ml.);  the sodium hydroxide s o l u t i o n was washed w i t h ether (2 x 100 ml.) snd the e t h e r e a l e x t r a c t added to the methylene c h l o r i d e  solu-  t i o n . A f t e r d r y i n g over magnesium sulphate, the organic s o l u t i o n was evaporated to leave a r e d d i s h o i l (32 g.), which was taken -  up i n benzene and chromatographed on alumina 101 Cleavamine  (1200 g . ) .  was e l u t e d 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 g i v e needles (2.4 g . ) , m.p. 117-119° ; one spot on T.L.C. (EtOAc); [ c x ] + 7 3 ° (CHClj) ; . \  ( l o g 6.): 225  26  (4.60), 285 (3.87), 292 (3.86) nyi ;V 2000, 2740 and 2700 (Bohlmann b a n d s )  m a x >  44  m a x <  . ( N u j o l ) : 3420 (NH), cm." , no carbonyl ab1  s o r p t i o n ; N.M.R. s i g n a l s (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, a r o m a t i c ) , 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, C H C H ) Y . Pound: C, 81.30; H, 8.54; N, 3  10.18; M.W, M.W,  2  280. Calc. f o r C H N : C, 81.38; H, 8.63; N, 9.99; i g  2 4  2  280. Benzene-chloroform (1:1) e l u t e d s t a r t i n g m a t e r i a l (~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 m a t e r i a l 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 w i t h a u t h e n t i c descarbomethoxy-catharanthine  1 0 1  (I.R,, T.L.C.);V  ( N u j o l ) : 3140 (NH) cm." . Pound: 1  C, 81.70; H, 8.12; N, 10.10. C a l c . f o r C H" N : C, 81,97; H, ig  22  2  7.97; N, 10,06.  4"ol"-Dihydrocleavamine (68) Cleavamine (1.-5 g.), i n e t h y l acetate (20 ml.), was hydrogenated over Adam's c a t a l 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 one spot on T.L.C. (EtOAc);V  m o v  (1.2 g . ) , m.p.  136-138 ;  ( N u j o l ) ; 3410 (NH), 2790 and  2750 (Bohlmann bands) cm.*"? N.M.R. s i g n a l s (CD^COCI^): 0.88 1  ( s i n g l e t , IH, NH), 2.8 ( m u l t i p l e t , 4 H , a r o m a t i c ) , 9.17 3H, C H C H ) r . Pound: C, 81.02; H, 9.59; N, 9.88; M.W. 5  2  C a l c . f o r C H N : C, 80.80; H, 9.28; N, 9.92; M.W. i g  2 6  2  (triplet, 282.  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) d i s p l a y e d three spots on T.L.C.  - 112 -  (EtOAc) w i t h Rf values o f 0 . 7 7 , 0.53, and 0.27.  The compound  w i t h a Rf value of 0 . 7 7 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 w i t h benzene-petroleum  ether (1:2),  and the f i r s t - f o u r f r a c t i o n s a f f o r d e d 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.  L a t e r f r a c t i o n s contained  p r o g r e s s i v e l y l e s s cleavamine and more o f the other two c o n s t i t u e n t s . The la.st (39, 55 mg.)  of the nine f r a c t i o n s obtained w i t h  t h i s e l u e n t c o n t a i n e d no cleavamine, d i s p l a y i n g 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-."*; N.M.R. s i g n a l s 1  (CDCl^): 2.05 ( s i n g l e t , IH, NH), 2.8 ( m u l t i p l e t , 4H, a r o m a t i c ) , 9.13  (broad s i n g l e t , 3H, CH^C)T, no o l e f i n i c proton a b s o r p t i o n .  Pound: C, 80.56; H, 9.46; N, 10.04; M.W. 262. C a l c . f o r C H M : i g  2 6  2  C, 80.80; H, 9-28; N, 9.92; M.W. 282. F r a c t i o n B9 was shown to be a mixture of 4"cx"- and 4"p !I  dihydrocleavamine by comparison ( T . L . C ,  I.E. spectra) w i t h  a u t h e n t i c 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 k i n d l y provided by Dr.M.  Gorman,  L i l l y Research L a b o r a t o r i e s .  Carbomethoxy-4"fi"-dihydrocleavamih-e  (119)  A mixture o f catharanthine (5) (30 g.) and z i n c dust (300 g.), i n g l a c i a l a c e t i c a c i d (750 ml.), was heated under  - 113 -  r e f l u x i n a n i t r o g e n atmosphere f o r 4 hours. The hot s o l u t i o n was decanted, most o f the s o l v e n t removed under vacuum, and the r e s i d u e taken up i n water (100 m l . ) . The s o l u t i o n was made b a s i c w i t h aqueous ammonia, e x t r a c t e d w i t h ether (4 x 150 m l . ) , and the combined ether e x t r a c t s d r i e d over magnesium sulphate. Removal of the ether a f f o r d e d 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 c o o l i n g . This m a t e r i a l 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 w i t h 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 a f f o r d stout needles, m.p. 172° ; one spot on T.L.C. (EtOAc-CHCl^ 1:1); [oc] +100° (CHC1 ); X 26  3  293 (3.84) mu ; ^  m a X o  ( l o g - G ) : 227 (4.47), 286 (3.87),  . U u j o l ) : 3430 (NH), 2790 (Bohlmann b a n d ) , 4 4  O  T  1722 (C=0) cm." ; N.M.R. s i g n a l s • ( O D C l j ) : 1.00 ( s i n g l e t , IH, 1  NH), 2.7 ( m u l t i p l e t , 4H, a r o m a t i c ) , 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 a b s o r p t i o n . Pound: C, 74.26; H, 8.35;  N,  8.26. C a l c . f o r C H Q 0 N : C, 2 1  2  2  2  74.07; H, 8.29; N, 8.23. Neuss et a l .  quote m.p. 64-66°, [oc] + 9 6 ° (CHC1,) f o r D carbomethoxy-dihydrocleavamine. 6 8  26  ?  4 "fi'*-Dihydrocleavamine (68) Carbomethoxy-4 p"-dihydrocleavamine (500 mg. ), i n 5N ,,  h y d r o c h l o r i c a c i d (30 ml.), was heated on the water-bath under  - 114 -  a n i t r o g e n atmosphere f o r 8 hours. The s o l u t i o n was cooled i n i c e , made b a s i c with, aqueous ammonia, and e x t r a c t e d w i t h methylene c h l o r i d e (3 x 50 ml.). The organic e x t r a c t was d r i e d , . concentrated  t o a s m a l l volume and f i l t e r e d through alumina  (10 g.). Evaporation of the s o l v e n t gave an amorphous powder (340 mg.) which could not be induced t o c r y s t a l l i s e ; one spot on T.L.C. (EtOAc);V  ( N u j o l ) : 3350(HH), 2750 (Bohlmann  band) cm."""", no carbonyl a b s o r p t i o n . 1  This m a t e r i a l was i d e n t i c a l  (T.L.C., I.E. spectra) w i t h  an a u t h e n t i c sample of 4"|3"-dihydrocleavamine k i n d l y s u p p l i e d by Dr.M. Gorman, L i l l y Research L a b o r a t o r i e s . Mercuric Acetate O x i d a t i o n of  Carlomebhoxy^4"fi"-dihydrocleav-  amine (119) Carbomethoxy-4"(2> -dihydrocleavamine (4.5 g.) and mercuric l,  acetate (10.5 g. ), i n g l a c i a l a c e t i c a c i d (150 ml. ), were s t i r r e d under a n i t r o g e n 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 s o l v e n t was removed i  as f a r as p o s s i b l e under vacuum, the r e s i d u e made b a s i c w i t h d i l u t e ammonia (50 ml.) and e x t r a c t e d w i t h methylene c h l o r i d e (3 x 50 ml.). A f t e r d r y i n g over sodium sulphate the methylene c h l o r i d e was removed, and the dark brown product chromatographed on a l u mina (200 g.,deactivated w i t h 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 a f f o r d e d  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 ( l o g 6 ) : 226 (4.07), 298 m Q Y  (4.12), 326 (4.24) m/>L ; v  m a 3 U  (C01 )s 3380 (NH), 2780 (Bohlmann 4  band), 1675 (0=0), 1610 (0=0) cm." ; N.M.R signals• (ODClj): 1  1.05- ( s i n g l e t , IH, NH), 2.8 ( m u l t i p l e t ,  4H, a r o m a t i c ) , 6.23  ( s i n g l e t , 3H, GH 0), 9.07 ( t r i p l e t , 3H, OHjCHgJT. Found: C, 5  74.48, 74.69; H, 7.80, 7.52; 0, 9.62; N, 8.27; M.W. 338. C a l c . f o r C H 0 N : .0, 74.52; H, 7.74; 0, 9.46; N, 8.28; M.W. 338. 2 1  2 6  2  2  The l a t e r benzene f r a c t i o n s  yielded  an amorphous powder  (105 mg.); one spot on T.L.C. (EtOAc-CHCl,, l s 9 ) ; X 285, 293 / L ; V m  signals  : 226,  T r i Q V  ( C C 1 ) ; 3400 (NH), 1705 (0=0) cm." ; N.M.R. 1  m a x >  4  (CDCl^): 2.01 ( s i n g l e t , IH, NH), 2.8 ( m u l t i p l e t , 4H,  a r o m a t i c ) , 6.30 ( s i n g l e t , 3H, CHjO), 9.10 ( t r i p l e t , 3H, CH CH )r. 5  2  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 o f c o r o n a r i d i n e (145)  .  The amorphous m a t e r i a l was taken up i n anhydrous e t h e r and the h y d r o c h l o r i d e 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  the h y d r o c h l o r i d e as needles, m.p. 221-223 (Nujol):  afforded  (dec.); \>  3160 (NH), 2530 (NH), 1715 (0=0) cm." . An a u t h e n t i c 1  0  sample o f c o r o n a r i d i n e h y d r o c h l o r i d e , m.p. 221-223  , pre-  pared from a Bample o f c o r o n a r i d i n e k i n d l y provided by Dr.M.  Gorman, L i l l y Research L a b o r a t o r i e s , d i d n o t depress  the m e l t i n g p o i n t , a n d t h e I.R. s p e c t r a were i d e n t i c a l . Benzene-ether (1 s i ) e l u t e d 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° ) t o a f f o r d  - 116 -  prisms, m.p. 143-145.5°; [a] +49° ( C H G 1 ) ; X  : 225, 286,  23  3  293 mu ;v „  Q V  (KBr): 3350 (NH) 1700 (0=0)  (CDC1 ): 2,00 ( s i n g l e t , IH, NH), 5  m a x <  cm." ; N.M.R. s i g n a l s 1  2.8 ( m u l t i p l e t , 4H, aromatic), 107  6.38  ( s i n g l e t , 3H, CH 0), 9.04 ( t r i p l e t , 3H, C H C H ) T . This 5  5  m a t e r i a l was i d e n t i f i e d as dihydro catharanthine  2  (.'69.)  by  comparison (m.p., mixed m.p.,.T.L.C., I.R. spectra) w i t h an a u t h e n t i c sample prepared by hydrogenation  o f catharanthine ( 5 ) .  A c i d H y d r o l y s i s o f Pseudo-vincadifformine  (121)  Pseudo-vincadifformine  (100 mg.) was heated w i t h 2 N  h y d r o c h l o r i c a c i d (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 b a s i c w i t h aqueous ammonia and the p r e c i p i t a t e taken up i n ether. Evaporation of the e t h e r e a l s o l u t i o n y i e l d e d a gummy product (123) which e x h i b i t e d t h e s p e c t r a l p r o p e r t i e s of an i n d o l e n i n e : 221, 250 (broad) m ix ; » max. / v  m  a  x  ( C C l ^ ) : 1605,  1575 cm." , no NH a b s o r p t i o n . 1  The gum was d i s s o l v e d i n t e t r a h y d r o f u r a n (5 ml.) and heated under r e f l u x w i t h l i t h i u m aluminium hydride (100 mg.) f o r - 3 hours. The excess of hydride was destroyed w i t h saturated aqueous sodium sulphate (10 ml.) and the product i s o l a t e d w i t h e t h e r . R e m o v a l of the solvent a f f o r d e d (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."]^-.60° D 2  L  (CHC1,); one spot on T.L.C. (EtOAc-CHCl,, 1 : 9 ) ; 7 v 243 (3.81), 295 (3.45) m/*.;v  max>  mQV  J  (log €):  ( N u j o l ) : 3230 (NH), 1600  (aromatic C=C) cm." ; N.M.R. s i g n a l s (CDCl^): 3 . 1 ( m u l t i p l e t , 1  4H, aromatic) 9.10 ( t r i p l e t , 3 H , CH CH )*t . Pound: C, 81.00; 5  2  -• 117 -  H, 9.38:  N, 9.86; M.W.  282. C a l c . f o r C H N ; C, 80.80,  H, 9.28;  N, 9.92;  282.  M.W.  i q  2 6  2  A c e t y l a t i o n w i t h a c e t i c anhydride i n p y r i d i n e a f f o r d e d 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. CHC1 , l : 9 ) ; X 3  C=C)  (log£ ): 212 (4^35),253 (4.13), 279  m a X t  289 (3.51)m^i ;V  107.5-109° ; one spot on T.L.C. (EtOAc-  m a x <  ( K B r ) : 1655  (N-C=0), 1595  cm." ; N.M.R. s i g n a l s (CDC1 ): 1.87  (aromatic  (broad s i n g l e t , IH,  1  5  a r o m a t i c ) , 2.85  (3.58),  ( m u l t i p l e t , 3H, a r o m a t i c ) , 7.78  (singlet,  3H,  CH C=0), 9.10 ( t r i p l e t , 3 H , C H C H ) r . Pound: C, 77.14; H, 3  0, 5.07;  N, 8.97.  0, 4.93; N,  C a l c . f o r C H 0 N : C, 77.73; H, 2 4  8.64;  2  5  2 8  2  8.70;  8.63.  Reduction of Pseudo-vincadifformine (121) w i t h Zinc and Sulphuric Acid P s e u d o - v i n c a d i f f o r m i n e ( 1 . 0 g.) and z i n c dust (150 g.), i n 10$ methanolic s u l p h u r i c a c i d (500 ml.), were heated under r e f l u x f o r 30 minutes. The methanol was removed under vacuum, the a c i d n e u t r a l i s e d w i t h aqueous sodium carbonate, and the s o l u t i o n e x t r a c t e d w i t h ether (3x 200 ml.). A f t e r drying,evap o r a t i o n of the s o l v e n t a f f o r d e d a gum  (0.8 g.) which showed  two spots on T.L.C. (EtOAc-CHClj, 1:9). This m a t e r i a l was  then  chromatographed on alumina (40 g.). The major product (640 dihydro-pseudo-vincadifformine (139&7* e l u t e d w i t h benzenepetroleum ether (b.p. 30-60° ) ( l : l ) w a s an amorphous powder (from e t h e r ) ; Tal CHC1 , l : 9 ) ; X 3  24  m a x <  - 1 6 ° (EtOH); one spot on T.L.C. (EtOAc-  (loge.):  244  (3.82),  299  (3o45)m^; V  m a X o  mg.),  - 118 -  (CC1 ): 3 3 8 0 ' ( H H ) , 1720 (0=0), 1605 (aromatic C=C) 4  cm." ; 1  N.M.R. s i g n a l s (CDCl^); 3.1 ( m u l t i p l e t , 4H, a r o m a t i c ) , 6.33  ( s i n g l e t , 3H, CH 0), 9.12 ( t r i p l e t , 3H, C H C H ) T . Found: 3  3  2  C, 73.97; H, 8.13;  N, 8.32;  M.W. 340. C a l c . f o r C H 0 N :  C, 74.08; H, 8.29;  N, 8.23;  M.W. 340.  2 1  A c e t y l a t i o n w i t h a c e t i c anhydride  2 8  2  2  i n p y r i d i n e gave an  amorphous N-acetate, |od~j + 2 8 ° (EtOH); one spot on T.L.C. 23  (EtOAc-CHCl , l - 9 ) ; X 3  291 (3.45)myx ; " v (aromatic C=C)  ( l o g 6 )'s 253 (4-01), 282 (3.49),  m a x >  ( C C 1 ) : 1725  m a X e  4  (C=0), 1660  (N-C=0), 1595  cm." ; N.M.R. s i g n a l s ( C C 1 ) : 2.3 (broad 1  4  singlet,  IH, a r o m a t i c ) , 3.0 ( m u l t i p l e t , 3H, a r o m a t i c ) , 6.83 ( s i n g l e t , 3H, CH 0), 7.75 ( s i n g l e t , 3H, CH C=0), 9.08 ( t r i p l e t , 3H, 5  3  C H C H ) t . Found: C, 72.26; H, 8.24; 3  C  2  23 30°3 2 H  N  °»  !  7 2  °  2 2  * » 7.91; H  The minor product formine  N, 7.51.  Calc. f o r  N, 7.32.  (80 mg.),  iso-dihydro-pseudo-vincadif-  (I40a) was e l u t e d w i t h benzene-ether (1:1) and was a l s o (?  an amorphous powder,fod] - 1 3 2 ° (EtOH); one spot on T.L.C. D 24  (EtOAc-CHCl , l s 9 ) ; X 3  ^mo  V  ( l o g € ) : 244 (3.83), 297 (3.45) m^ ;  m a x >  ( C C l J ; 3380 (NH), 1720  (C=0), 1605 (aromatic C=C) cm." ; 1  N.M.R. s i g n a l s ( C L C l ) s 3.2 ( m u l t i p l e t , 4H, a r o m a t i c ) , 6.32 3  ( s i n g l e t , 3H, CH 0), 9.10 ( t r i p l e t , 3H, C H C H ) T . Found: C, 3  74.38; H, 8.32;  3  N, 8.29.  2  C a l c . f o r C H g 0 N : C, 74.08; H, 2 1  8.29;  2  2  2  N, 8.23. A c e t y l a t i o n w i t h acetate anhydride i n p y r i d i n e a f f o r d e d an amorphous N-acetate, foil + 3 ° (EtOH); one spot on T.L.C, D (EtOAc-CHCl,, l : 9 ) ; X (log£): 250 (4.09), 278 (3.47), 296 2 4  L  T n Q Y  J  - 119 -  (3.39)m^i ; " v  m a x >  (CC1 ): 1725 (0=0), 1660 (N-C=0), 1595 (aromatic 4  C=C) cm."" ; N.M.R. s i g n a l s ( C C l ^ ) : 2.9 ( m u l t i p l e t , 4H, aroma1  t i c ) , 6.42 ( s i n g l e t , 3 H , C H 0 ) , 7.86 ( s i n g l e t , 3H,'0H C=0), 5  5  9.10 ( t r i p l e t , 3H, CH^CHgVT.. Found: C, 72.57; H, 8.10. C a l c . f o r C H ^ 0 N : C, 72.22; H, 7.91. 23  5  2  E p i m e r i s a t i o n 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 t o g e t h e r w i t h sodium methoxide (60 mg.) and s a t u r a t e d methanolic magnesium-methoxide s o l u t i o n ( l m l . ) , and heated a t 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 e x t r a c t e d immediately w i t h ether (4 x 20 ml.). A f t e r d r y i n g over sodium sulphate the e t h e r e a l e x t r a c t was evaporated to g i v e an amorphous powder (165 mg.), which was i d e n t i c a l w i t h iso-dihydro-pseudovincad i f f o r m i n e (140) ( T . L . C , U.V. and I.R. s p e c t r a ) .  - 120  -  REFERENCES  1. R.L. Noble, C.T. Beer and J.H, C u t t s , 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. M , 850 (1959).  Svoboda, J . Lab. 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D j e r a s s i , B. G i l b e r t , J.N. Shoolery, L.P. Johnson and K. Biemann, E x p e r i e n t i a . 17o 162 (1961). 89. D. Schumann and H. Schmid, Helv. Chim. A c t a , 46. 1996 (1963). 90. J . Trojanek, 0. S t r o u f , K. Blaha, L. D o l e j s and V. Hanus, A b s t r a c t s . IUPAC I n t e r n a t i o n a l Symposium on the Chemistry of N a t u r a l Products, Kyoto, Japan (1964), p. 100. . 91. C. D j e r a s s i , H. B u d z i k i e w i c z , J.M. Wilson, J . Gosset, J . Le Men, M.M. Janot, Tetrahedron L e t t e r s . 235 (1962). 92. G.F. Smith and M.A. Wahid, J . Chem. S o c , 4002 (1963). 93. J.F.D. M i l l s and S„C. Nyburg, Tetrahedron L e t t e r s , No. 11, 1 (1959); J . Chem. S o c . 1458 (I960) 94. K. Biemann and G. S p i t e l l e r , Tetrahedron L e t t e r s . 299 (1961) ; J . Am. Chem. S o c . 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. B y c r o f t , D. Schumann, M.B, P a 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 a r e g r a t e f u l to Drs, M. Gorman and N. Neuss, L i l l y Research L a b o r a t o r i e s , f o r p r o v i d i n g us w i t h e x p e r i 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. S p i t e l l e r - P r i e d m a r i n and G„ S p i t e l l e r , Tetrahedron L e t t e r s , 485 (1961). 101.  We wish t o thank Dr. M. Gorman, L i l l y Research L a b o r a t o r i e s , f o r s u p p l y i n g catharanthine h y d r o c h l o r i d e and f o r p r o v i d i n g comparison samples of cleavamine and descarbomethoxy-catharanthine.  102. C. D j e r a s s i , S.E. P l o r e s , H. B u d z i k i e w i c z , J.M. W i l s o n , 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. B r i s s o l e s e , J.M. Wilson, H. B u d z i k i e w i c z , 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 a r e g r a t e f u l t o Dr. M. Gorman f o r s u p p l y i n g a sample o f c o r o n a r i d i n e .  107.  We are unable t o r e c o n c i l e the d i f f e r e n c e s 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° (CHC1 ) . A l l our data are consistent with those expected for dihydrooatharanthine. 5  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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