UBC Theses and Dissertations

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UBC Theses and Dissertations

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

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The U n i v e r s i t y  of B r i t i s h  Columbia  DEPARTMENT OF CHEMISTRY  PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  of  JOSEPH E . GERVAY  M . S c , The U n i v e r s i t y  of B r i t i s h  Columbia  1963  THURSDAY, SEPTEMBER 23, 1965 AT 10:30 A.M. IN ROOM 261, CHEMISTRY BUILDING  COMMITTEE IN CHARGE Chairman:  W. Hoar R. E'. P i n c o c k A. R o s e n t h a l G. M. Tener  T. Money F. McCapra C. A. McDowell E x t e r n a l Examiner: Department University  D. A. Nelson  o f Chemistry o f Wyoming  Laramie, Wyoming.  STUDIES IN NATURAL PRODUCTS ABSTRACT  Part I.  THE  BIOSYNTHESIS OF  ERYTHRINA,ALKALOIDS  Hypotheses f o r the b i o g e n e s i s of E r y t h r i n a a l k a l o i d s are d i s c u s s e d , Di-(p-3, 4-dihydroxyphenyl)ethylamine, the t h e o r e t i c a l p r e c u r s o r p r e d i c t e d by the b i o g e n e t i c t h e o r y , was prepared and r i n g c l o s u r e to the e r y t h r i n a n e r i n g system by o x i d a t i v e c o u p l i n g was attempted under v a r i o u s c o n d i t i o n s , Consequently, the b i o g e n e s i s of the Erythtrina a l k a l o i d s was re-examined and a new p r o p o s a l i s advanced f o r the b i o s y n t h e s i s of these a l k a l o i d s , S y n t h e t i c r o u t e s to a h y p o t h e t i c a l precursor;, proposed here f o r the f i r s t time as a p o t e n t i a l , i n t e r m e d i a t e , are d e s c r i b e d , s  The b i o g e n e t i c - t y p e s y n t h e s i s of the spiro-amine r i n g system present i n the E r y t h r i n a a l k a l o i d s was a c h i e v e d by o x i d a t i v e c o u p l i n g of the blocked d i p h e n o l i c p r e c u r s o r ^ as p r e d i c t e d by the proposed b i o s y h t h e t i c scheme, O x i d a t i o n of di-(/3-3-hydroxy4-me.thoxyphenyl) -ethylamine by a l k a l i n e p o t a s s i u m ferricyanide afforded 3 15-dimethoxy-16-hydroxy-2o x o e r y t h r i n a - 1 ( 6 ) , 3-diene i i i 1.5% y i e l d , Reduction of the l a t t e r by sodium borohydride gave 3, 15dimethoxy-2,, 1 6 ^ d i h y d r o x y e r y t h r i n a - l ( 6 ) 3-diene, A c e t y l a t i o n of the dienone y i e l d e d 3, 15-dimethoxy16-acetoxy-2-oxoerythrina-l(6)j 3-diene. The t o t a l b i o g e n e t i c - t y p e s y n t h e s i s of e r y s o d i n e i s t h e r e f o r e but two steps from completion. s  5  The r e s u l t s as a whole c o n f i r m the h y p o t h e s i s that E r y t h r i n a a l k a l o i d s are produced i n Nature by o x i d a t i v e c o u p l i n g of d i p h e n o l s . They a l s o demonstrate the d i r e c t i n g r o l e of the p r o t e c t i v e groups i n the p h e n o l i c precursor. The evidence allows a b i o s y n t h e t i c pathway f o r the aromatic E r y t h r i n a a l k a l o i d s to be c o n s i d e r e d and the mechanism f o r the r i n g c l o s u r e process i s discussed. s  The i s o t o p i c a l l y l a b e l l e d p r e c u r s o r 3-hydroxy-4methoxy-N-(3-hydroxy-4-methoxyphen ( 1 - ^ C ) e t h y l ) -  phenethylamine was p r e p a r e d t o t e s t the b i o s y n t h e t i c h y p o t h e s i s i n the p l a n t . F e e d i n g experiments a r e i n progress, . Part I I ,  AN ATTEMPTED IN VITRO DEMETHYLATION LANOSTEROL  OF  The b i o g e n e s i s o f c h o l e s t e r o l and methods f o r f u n c t i o n a l . i s i n g i n e r t methyl groups a r e r e v i e w e d and a new t h e o r e t i c a l approach t o removal o f the 14 methyl group from l a n o s t e r o l i s d e s c r i b e d . The removal o f t h i s methyl group i n v i t r o c o u l d not be a c h i e v e d . but a s e r i e s o f i n t e r e s t i n g compounds was o b t a i n e d , .• E v i d e n c e f o r the s t r u c t u r e s of these, compounds i s p r e s e n t e d , 3  T h u s p h o t o s e n s i t i z e d Oxygenation of d i h y d r o l a n o s t e r y l a c e t a t e i n the presence o f para-nitrobenzene.su 1phonyl c h l o r i d e y i e l d e d 3|?-acetoxylanosta-7 9(11)-diene. 3.3-acetoxylanost-8-ene-7-one and 3f?-a,cetoxylanost-8-ene7<*- h y d r o p e r o x i d e . I n a d d i t i o n a compound h a v i n g an ambiguous s t r u c t u r e and d e s i g n a t e d as IP1 was o b t a i n e d . The d i b r o m o - d e r i v a t i v e of the l a t t e r i s 3/3-acetoxy-7o<3 llo(-dibromolanostane-8o( 9o(-epoxide,the s t r u c t u r e of. which was determined by X-ray c r y s t a l l o g r a p h i c study, A working s t r u c t u r e f o r compound IP1 based on the p h y s i c a l and c h e m i c a l e v i d e n c e i s d i s c u s s e d . ?  ?  ?  GRADUATE STUDIES T o p i c s i n Organic  Chemistry  D. E. McGreer R. :E. I. Pincock J . P. Kutney ,S  T o p i c s i n I n o r g a n i c Chemistry  Topics i n Physical  Chemistry  M o l e c u l a r Rearrangements P h y s i c a l Organic  Recent  Pincock  R.  Stewart  J . P. Kutney L-  S y n t h e t i c Methods  Natural  R. E . I .  Chemistry  Stereochemistry  Chemical  J . A. R. Coope R.• F. S n i d e r A. Bree  Chemistry  Seminar i n Organic Organic  W.-.R.. C u l l e n N. B a r t l e t t  D- Hayward L. D. H a l l  . D. E. McGreer G. B. P o r t e r D. •, J . L. Jame s  Kinetics  J . P. Kutney A. I . S c o t t  Products  Related Studies: Biochemistry  Computer Programming and Numerical A n a l y s i s  W. J . P o l g l a s e S. H. Zbarsky G. M. Tener M. Darrach R. Henderson  PUBLICATION A. R o s e n t h a l and J . Gervay: " D i r e c t High P r e s s u r e C a r b o n y l a t i o n of Aromatic N i t r i l e s w i t h D i c o b a l t O c t a c a r b o n y l " c h e m i s t r y and I n d u s t r y , 1623 (1963) . A. Rosenthal and J . Gervay: " D i r e c t C o n v e r s i o n of Aromatic N i t r i l e s i n t o P h t h a l i m i d i n e s and Ureas Using D i c o b a l t Octacarbonyl"  (  Canadian  J o u r n a l of Chemistry, 42  1490  (1964).  STUDIES- IN NATURAL PRODUCTS Part  I.  Part  II.  THE BIOSYNTHESIS  OF ERYTHRINA A L K A L O I D S .  AN ATTEMPTED IN VITRO DEMETHYLATION OF LANOSTEROL  by  JOSEPH B.Sc.  Honours,  University  M.Sc,  University  A THESIS  of  EDMUND GERVAY Montreal,  of  British  Loyola College, Columbia,  SUBMITTED IN P A R T I A L  THE REQUIREMENTS DOCTOR OF  in  1963  FULFILMENT  FOR THE DEGREE OF PHILOSOPHY  the  Department of  Chemistry  We a c c e p t t h i s required  thesis  as  conforming  to  the  standard.  THE U N I V E R S I T Y  OF B R I T I S H  September  1965  COLUMBIA  OF  1961  In p r e s e n t i n g the  this  thesis  Columbia,  I agree that  the Library  a v a i l a b l e f o r r e f e r e n c e and s t u d y . mission  f o rextensive  representatives^  cation  of this  w i t h o u t my w r i t t e n  forfinancial  permission.  D e p a r t m e n t o f Chemistry 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, C a n a d a  September 24,  thesis  per-  for scholarly  by t h e Head o f my D e p a r t m e n t o r by  It i s understood  thesis  s h a l l make i t f r e e l y  I f u r t h e r agree that  copying o f t h i s  p u r p o s e s may be g r a n t e d  Date  fulfilment of  r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f  British  his  in partial  1965  Columbia  that  gain  copying o r p u b l i -  shall  n o t be a l l o w e d  ABSTRACT  In P a r t I , hypotheses f o r t h e b i o g e n e s i s  o f E r y t h r i n a a l k a l o i d s are  discussed. Di-(B-3,4-dihydroxyphenyl)-ethylamine the t h e o r e t i c a l c u r s o r p r e d i c t e d by the b i o g e n e t i c t h e o r y , to t h e e r y t h r i n a n e  was prepared  pre-  and r i n g  closure  r i n g system by o x i d a t i v e c o u p l i n g was attempted under  various conditions.  Consequently, t h e b i o g e n e s i s  o f the E r y t h r i n a  a l k a l o i d s was re-examined and a new p r o p o s a l  i s advanced f o r t h e b i o s y n -  t h e s i s o f these a l k a l o i d s .  Synthetic routes  to a hypothetical precursor,  proposed here f o r the f i r s t  time as a p o t e n t i a l i n t e r m e d i a t e ,  The  biogenetic-type  s y n t h e s i s o f t h e spiro-amine  i n the E r y t h r i n a a l k a l o i d s was a c h i e v e d blocked scheme.  are described.  r i n g system  present  by o x i d a t i v e c o u p l i n g o f the  d i p h e n o l i c p r e c u r s o r , as p r e d i c t e d by the proposed b i o s y n t h e t i c O x i d a t i o n o f di-(3-3-hydroxy-4-methoxyphenyl)-ethylamine by  a l k a l i n e potassium f e r r i c y a n i d e a f f o r d e d 3,15-dimethoxy-16-hydroxy-2-oxoerythrina-1(6),3-diene borohydride  gave  i n 15% y i e l d .  Reduction  o f the l a t t e r by sodium  3,15-dimethoxy-2,16-dihydroxyerythrina-l(6),3-diene.  A c e t y l a t i o n o f t h e dienone y i e l d e d 3,15-dimethoxy-16-acetoxy-2-oxoerythrina1(6),3-diene. . The t o t a l b i o g e n e t i c - t y p e f o r e b u t two s t e p s The  from  synthesis o f erysodine  i s there-  completion.  r e s u l t s as a whole c o n f i r m t h e h y p o t h e s i s  that E r y t h r i n a a l k a l o i d s  are produced i n Nature by o x i d a t i v e c o u p l i n g o f d i p h e n o l s .  They a l s o demon-  s t r a t e t h e d i r e c t i n g r o l e o f t h e p r o t e c t i v e groups i n t h e p h e n o l i c cursor.  The evidence  allows a b i o s y n t h e t i c pathway f o r t h e aromatic  E r y t h r i n a a l k a l o i d s t o be c o n s i d e r e d , process  i s discussed.  pre-  and t h e mechanism f o r the r i n g c l o s u r e  The i s o t o p i c a l l y  labelled precursor  4-methoxyphen[1- C]ethyl)-phenethylamine 14  hypothesis i n the p l a n t .  3-hydroxy-4-methoxy-N-(3-hydroxywas p r e p a r e d t o t e s t the b i o s y n t h e t i c  F e e d i n g experiments a r e i n p r o g r e s s .  In P a r t I I , t h e b i o g e n e s i s o f c h o l e s t e r o l  and methods f o r f u n c t i o n a l -  i s i n g i n e r t methyl groups a r e reviewed, and a new t h e o r e t i c a l approach t o removal o f the 14a-methyl  group from l a n o s t e r o l i s d e s c r i b e d .  The removal  o f t h i s methyl group i n v i t r o c o u l d not be a c h i e v e d , but a s e r i e s o f i n t e r e s t i n g compounds was o b t a i n e d .  E v i d e n c e f o r the s t r u c t u r e s o f these  compounds i s p r e s e n t e d . Thus, p h o t o s e n s i t i z e d o x y g e n a t i o n o f d i h y d r o l a n o s t e r y l a c e t a t e i n t h e presence o f para-nitrobenzenesulphonyl c h l o r i d e y i e l d e d 33-acetoxylanosta7 , 9 ( l l ) - d i e n e , 36-acetoxylanost-8-ene-7-one and 3$-acetoxylanost-8-ene-7ahydroperoxide.  In a d d i t i o n a compound h a v i n g an ambiguous s t r u c t u r e and  d e s i g n a t e d as IP1 was o b t a i n e d . The d i b r o m o - d e r i v a t i v e o f the l a t t e r i s 3 3 - a c e t o x y - 7 a , l l a - d i b r o m o l a n o s t a n e - 8 a,9a-epoxide, t h e s t r u c t u r e o f which was determined by X-ray c r y s t a l l o g r a p h i c study. compound IP1 based on t h e p h y s i c a l  A working s t r u c t u r e f o r  and chemical e v i d e n c e i s d i s c u s s e d .  if. TABLE OF CONTENTS  PART I  Page  INTRODUCTION  1  DISCUSSION  21  EXPERIMENTAL  43  BIBLIOGRAPHY  .  .  63  List  o f Figures  1.  Simple d i m e r i z a t i o n o f phenol r a d i c a l s  15  2.  The B i o s y n t h e s i s o f Galanthamine  19  3.  The B i o s y n t h e s i s o f Isothebaine  20  4.  Hypothetical Biogenesis  25  5.  H y p o t h e t i c a l B i o g e n e s i s o f the E r y t h r i n a a l k a l o i d s v i a o x i d a t i v e c o u p l i n g o f phenols  28  R e a c t i o n sequence, l e a d i n g t o ethylamine  29  6.  7.  8.  9.  10.  11.  12.  o f the E r y t h r i n a a l k a l o i d s  di-(8-3,4-dihydroxyphenyl)-  R e a c t i o n scheme f o r the p r e p a r a t i o n o f 3-benzyloxy-4methoxyphenethylamine and 3-benzyloxy-4-methoxyphenylacetic acid  32  R e a c t i o n sequence, ethylamine  33  Biogenetic-type r i n g system  l e a d i n g t o di-(&-3-hydroxy-4-methoxyphenyl)-  s y n t h e s i s o f t h e e r y t h r i n a n e s p i r o amine 36  n.m.r. spectrum o f 3,15-dimethoxy-16-hydroxy-2-oxoerythrina1(6),3-diene  37  Proposed m e c h a n i s t i c scheme f o r the f o r m a t i o n amine r i n g system v i a o x i d a t i v e c o u p l i n g  40  o f the s p i r o  R e a c t i o n scheme f o r t h e p r e p a r a t i o n o f 3-hydroxy-4-methoxyN-(3-hydroxy-4-methoxyphen[l-!4c]ethyl)-phenethylamine  42  V.  PART I I  page  INTRODUCTION  ...  68  DISCUSSION  82  EXPERIMENTAL  93  BIBLIOGRAPHY  .  98  L i s t o f Figures 1.  The B i o s y n t h e s i s o f I s o p e n t e n y l Pyrophosphate  71  2.  P o l y m e r i z a t i o n o f I s o p e n t e n y l Pyrophosphate  72  3.  Scheme f o r t h e f o r m a t i o n o f C h o l e s t e r o l from Squalene  75  4.  In v i t r o f u n c t i o n a l i s a t i o n o f n o n - a c t i v e methyl groups  78  5.  Proposed scheme f o r t h e removal o f t h e 14a-methyl dihydrolanosterol  86  6.  7.  group i n  P h o t o s e n s i t i z e d oxygenation o f d i h y d r o l a n o s t e r y l acetate, i n the p r e s e n c e o f p a r a - n i t r o b e n z e n e s u l p h o n y l c h l o r i d e Chart showing t h e r e a c t i o n s o f compound IP1 w i t h chromium t r i o x i d e , p o t a s s i u m i o d i d e and p y r i d i n e perbromide  88  90  ACKNOWLEDGMENTS The  w r i t e r wishes t o express h i s thanks t o P r o f e s s o r .A„ L.Scott  and  t o Dr. Frank McCapra f o r t h e i r a d v i c e ,  the  d i r e c t i o n o f t h i s research  p a t i e n c e and encouragement i n  project.  Thanks a r e a l s o due t o Dr. T. Money f o r h i s h e l p f u l during  t h e course o f t h i s work. The  and  collaboration  w r i t e r a l s o expresses h i s indebtedness t o P r o f e s s o r  t o Mr. J.K. Fawcett f o r the X-ray c r y s t a l l o g r a p h i c  study.  J.  Trotter  PART I  THE BIOSYNTHESIS OF ERYTHRINA  ALKALOIDS  -1-  INTRODUCTION  The  Erythrina The  Alkaloids  a l k a l o i d s found i n numerous s p e c i e s  a t t r a c t e d general  i n t e r e s t both because o f t h e i r p h y s i o l o g i c a l a c t i v i t y , and  because they c o n t a i n the  alkaloids.  o f the genus E r y t h r i n a have  a type o f s t r u c t u r e n o t p r e v i o u s l y  The o c c u r r e n c e o f hypaphorine  ( 1 ) , an  encountered among i n d o l e alkaloid,''" i n  2 the  species  o f E r y t h r i n a has long been known  uncharacterized  and t h e p r e s e n c e o f o t h e r repeatedly.^>4,S,6  amorphous a l k a l o i d s has been r e p o r t e d  •CH -CH-COO 2  H  C H  X 3  C H  3  ci) An  i n t e n s i v e s e a r c h f o r a l k a l o i d s i n p l a n t s b e l o n g i n g t o t h i s genus was  s t a r t e d some twenty f i v e y e a r s ago f o l l o w i n g the d i s c o v e r y l i k e action o f extracts not  o f various  a t t r i b u t a b l e t o hypaphorine.  t i o n o f some f i f t y - o n e s p e c i e s  species  6,7,8,9,10  o f the c u r a r e -  o f E r y t h r i n a , an a c t i o n The  first  systematic  o f E r y t h r i n a showed t h a t  examina-  a l l contained 11  a l k a l o i d s with p a r a l y z i n g  definitely  a c t i v i t y , t h e potency v a r y i n g w i d e l y .  12 13 '  '  P h y s i o l o g i c a l l y , the E r y t h r i n a a l k a l o i d s a r e c u r a r i z i n g agents o f h i g h p o t e n c y , although t h e i r a c t i v i t y p r e s e n t s s e v e r a l unusual f e a t u r e s .  Unlike  o t h e r known c u r a r i z i n g agents, both s y n t h e t i c and n a t u r a l l y - o c c u r r i n g , the E r y t h r i n a a l k a l o i d s a r e unique i n t h a t they a r e t e r t i a r y bases w i t h  rela-  -2-  t i v e l y high a c t i v i t y .  Furthermore, . q u a t e r n i z a t i o n  c u r a r i z i n g potency; t h i s a l s o i s the 7 is  true.  only  greatly diminishes  the  c l a s s o f compounds i n which t h i s  14 '  They are  e f f e c t i v e when a d m i n i s t e r e d o r a l l y and have been  used c l i n i c a l l y w i t h some s u c c e s s . The  pioneering  from v a r i o u s 12  work on the  species  i s o l a t i o n and  o f E r y t h r i n a was  done by  characterization  of  Folkers  associates.^'  and h i s  alkaloids  13 '  The  Prelog, and  by  who  first  suggested the  Boekelheide.  By family  (  e l u c i d a t i o n o f t h e i r c o n s t i t u t i o n has  1960  the  r i n g system i n the  by  bases,  ^  i s o l a t i o n and  o f a l k a l o i d s was  h i s t o r y and  n a t u r e o f the  been a c h i e v e d mainly  c h a r a c t e r i z a t i o n o f the members o f t h i s  e s s e n t i a l l y complete, and  excellent  reviews o f  the  c h e m i s t r y o f the E r y t h r i n a a l k a l o i d s have been p r e s e n t e d i n 1 14  a u t h o r i t a t i v e manner. ' Besides hypaphorine, which occurs i n a number o f E r y t h r i n a the bases found i n t h e s e p l a n t s first  fall  i n t o two  groups.  The  species,  bases o f  the  group or " f r e e " a l k a l o i d s , are named from the p r e f i x " e r y t h r - " ,  these a l k a l o i d s are the n e c e s s i t y  i s o l a t e d d i r e c t l y from e x t r a c t s  of previous h y d r o l y s i s .  The  o f the p l a n t  second group o r  and  without  "combined"  a l k a l o i d s o c c u r i n the p l a n t i n combination w i t h some o t h e r moiety, 18 19 20 usually sulphoacetic  acid  "liberated" alkaloids. a l k a l o i d s which do not l i b e r a t e d i n the mineral acids. plants  alkaloidal  or g l u c o s e  and  on h y d r o l y s i s  yield  stem " e r y s o - " i s used t o i n d i c a t e the  a p p a r e n t l y o c c u r as such i n the p l a n t but  course o f i s o l a t i o n by  ,The  generally  The  '  "liberated" are  the h y d r o l y t i c a c t i o n o f d i l u t e  q u a n t i t i e s o f l i b e r a t e d a l k a l o i d s o b t a i n e d from  predominate and  fractions.  often  the  g r e a t l y exceed those o f the  the free  -3-  The  known n a t u r a l l y - o c c u r r i n g E r y t h r i n a a l k a l o i d s can be c l a s s i f i e d  depending whether o r n o t the a l k a l o i d s , as o r i g i n a l l y aromatic erysodine  ring.  There a r e seven members o f the aromatic  r i n g i s not p r e s e n t  g-erythroidine  (5).  has o n l y two members, a - e r y t h r o i d i n e (4)  The two groups d i f f e r o n l y i n t h a t the e r y t h r o i -  dines have a l a c t o n e r i n g where the benzjenoid Erythrina alkaloids. nature  group, i n c l u d i n g t h e  (2) and e r y t h r a l i n e (3) types; whereas the second group i n which  an aromatic and  i s o l a t e d , c o n t a i n an  The p r i n c i p a l a r o m a t i c  o f the oxygen f u n c t i o n a t t a c h e d  degree o f u n s a t u r a t i o n . that devised  r i n g occurs  i n the aromatic  a l k a l o i d s d i f f e r only i n the  t o the benzenoid r i n g or i n t h e i r  The numbering o f the E r y t h r i n a a l k a l o i d s f o l l o w s  e a r l i e r as a common numbering f o r a l l ' t h e E r y t h r i n a a l k a l o i d s .  (4)  (5.)  2  -4-  One i n t e r e s t i n g f e a t u r e o f s t r u c t u r e s diene system p r e s e n t i n r i n g s A and B.  (2) - (5) i s the h e t e r o a n n u l a r  A l l the E r y t h r i n a a l k a l o i d s  readily  undergo c a t a l y t i c r e d u c t i o n t o the c o r r e s p o n d i n g d i - and t e t r a h y d r o d e r i vates.  15 17 '  Furthermore,  when t h e o r i g i n a l  a l k a l o i d s are t r e a t e d with  acid  under m i l d c o n d i t i o n s , l o s s o f t h e a l i p h a t i c methoxyl group occurs as methanol and the diene system becomes lengthened t o a conjugated system,'  15,17,22,23  as shown f o r the desmethoxy d e r i v a t i v e  triene  (6) o f e r y s o d i n e  (2).  MeO  (2) When t h e o r i g i n a l  (6) a l k a l o i d s o r t h e i r desmethoxy d e r i v a t i v e s a r e t r e a t e d  with a c i d under more s e v e r e c o n d i t i o n s , i . e . b o i l i n g hydrobromic p o l y p h o s p h o r i c a c i d a t 125°, a rearrangement  acid or  with aromatization of r i n g A  23 15 17 24 occurs.  '  '  '  T h i s r e a c t i o n , known as the "apo-rearrangement", leads  t o a d i h y d r o i n d o l e d e r i v a t i v e as shown ( 8 ) . carbonium  i o n rearrangements,  By analogy with o t h e r known  i t i s p r o b a b l e t h a t an i n t e r m e d i a t e o f t h e  type shown by (7) i s i n v o l v e d . In the case o f aromatic E r y t h r i n a a l k a l o i d s , the "apo-rearrangement" i s accompanied by c l e a v a g e o f the aromatic e t h e r l i n k a g e s i n r i n g D so t h a t the p r o d u c t i n each case i s apoerysopine  ( 8 ) . A l s o , s i n c e the "apo-  -5-  rearrangement" r e s u l t s apoerysopine  i n d e s t r u c t i o n o f both asymmetric atoms (C-3 and  and a p o - B - e r y t h r o i d i n e are o p t i c a l l y  inactive.  The  C-5)  conversion  o f the d i h y d r o i n d o l e d e r i v a t i v e t o a t r u e i n d o l e s t r u c t u r e has been accom25 26 27 28 B-erythroidine. ' ' '  p l i s h e d i n b o t h the a r o m a t i c s e r i e s and with  MeO (2)  (8)  (7)  The Hofmann e x h a u s t i v e m e t h y l a t i o n procedure  has been used e x t e n s i v e l y  14 i n d e g r a d i n g the E r y t h r i n a a l k a l o i d s .  With l e s s h i g h l y  hydrogenated  d e r i v a t i v e s the Hofmann r e a c t i o n i s accompanied by a r o m a t i z a t i o n o f r i n g A and was  f r e q u e n t l y e l i m i n a t i o n o f f u n c t i o n a l groups may o c c u r as w e l l . This 29 f i r s t observed by Boekelheide and A g n e l l o and l a t e r t h i s aromatiza17  t i o n was  s t u d i e d e x t e n s i v e l y i n the case o f d i h y d r o - B - e r y t h r o i d i n o l ( 9 ) .  The  ( 9 ) , formed by the l i t h i u m aluminium 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 -  diol  B - e r y t h r o i d i n e , was  s u b j e c t e d t o the e x h a u s t i v e m e t h y l a t i o n procedure.  t h i s case a r o m a t i z a t i o n o f r i n g A was the d i o l  f u n c t i o n remained i n t a c t  accompanied by  as shown by  A s i m i l a r a r o m a t i z a t i o n o f r i n g A was  von  Braun d e g r a d a t i o n o f aromatic E r y t h r i n a From the d e g r a d a t i v e evidence  alkaloids  g i v e two  aromatized.  important  '  In  l o s s o f methanol but  (10).  demonstrated by P r e l o g w i t h 31  the  alkaloids.  i t can be seen t h a t the E r y t h r i n a '  s e r i e s of products  i n which r i n g A has become  As shown, the r u p t u r e o f the carbon t o n i t r o g e n bond at  30  -6C-5-N-9 gives  r i s e t o t h e o r t h o d i s u b s t i t u t e d benzenoid system o f s t r u c t u r e  (10), whereas, t h e m i g r a t i o n to the indoline-type  structure  the s p i r o amine s t r u c t u r e be n o t o n l y  o f carbon atom 13 from C-5 t o C-4 g i v e s (8).  To e x p l a i n the o r i g i n o f these p r o d u c t s ,  (2) (5) proposed f o r t h e s e a l k a l o i d s appeared t o  a r e a s o n a b l e one t o c o r r e l a t e and e x p l a i n t h e d e g r a d a t i v e  evidence, but a necessary  requirement.  (9)  (10)  A number o f methods have been e x p l o r e d o f t h i s type t o o b t a i n  valuable  15 the E r y t h r i n a a l k a l o i d s . natural material,  f o r the s y n t h e s i s  evidence regarding  s i n c e t h e s p i r o amine system was f i r s t  and  rise  o f molecules  the proposed  structures,  deduced as b e i n g common t o a l l o f  17 21 '  '  To o b t a i n  a c o r r e l a t i o n between  i t was n e c e s s a r y t o accomplish t h e s y n t h e s i s  synthetic of a  s u i t a b l e d e r i v a t i v e o f the E r y t h r i n a a l k a l o i d s i n which t h e s p i r o system remains i n t a c t .  Most o f the p r e l i m i n a r y  work on the s y n t h e s i s  o f compounds  s t r u c t u r a l l y r e l a t e d t o t h e E r y t h r i n a a l k a l o i d s i s due t o Wiesner and h i s 32 collaborators.  The f i r s t  successful  the d e s i r e d s p i r o amine system was t h a t is  synthesis  o f a compound 33  of Belleau.  containing  H i s elegant  shown below f o r t h e a r o m a t i c d e r i v a t i v e (11) t o which t h e t r i v i a l  erythrinane  has been  given.  synthesis name  -7-  (11) 34 Belleau  r e p e a t e d the s y n t h e s i s u s i n g the c o r r e s p o n d i n g dimethoxy  d e r i v a t i v e and o b t a i n e d racemic 15,16-dimethoxyerythrinane (15).  The  i n f r a r e d spectrum o f the p i c r a t e o f t h i s racemic m i x t u r e proved t o be superimpOsable on the spectrum o f the p i c r a t e o f n a t u r a l  15,16-dimethoxy-  e r y t h r i n a n e o b t a i n e d from degradations.**' 35 Mondon  has demonstrated t h a t c y c l i z a t i o n t o form the s p i r o amine  system occurs even more r e a d i l y when t h e l a c t a m c a r b o n y l i s p l a c e d i n the f i v e membered r i n g i n s t e a d o f the s i x . w i t h 3,4-dimethoxyphenethylamine  When the k e t a l a c i d  (12) i s warmed  (13) i n the p r e s e n c e o f a c i d , the  conden-  s a t i o n - c y c l i z a t i o n r e a c t i o n occurs i n e x c e l l e n t y i e l d t o g i v e the amide (14).  T h i s on r e d u c t i o n w i t h l i t h i u m aluminium h y d r i d e , g i v e s the same  racemate o f 15,16-dimethoxyerythrinane (15) o b t a i n e d p r e v i o u s l y by  Belleau.  The r e s o l u t i o n o f 15,16-dimethoxyerythrinane (15) was accomplished 36 37 i n d e p e n d e n t l y by B e l l e a u and B o e k e l h e i d e , The l e v o r o t a t o r y e n a n t i o morph was shown t o be i d e n t i c a l w i t h the n a t u r a l m a t e r i a l . S i m i l a r l y , the i d e n t i t y o f s y n t h e t i c and n a t u r a l d e r i v a t i v e s e s t a b l i s h e d the presence o f the s p i r o amine system i n g - e r y t h r o i d i n e  38 (5). a-Erythroidine d i f f e r s  from  -8-  8-erythroidine  i n h a v i n g the a l i p h a t i c double bond l i n k i n g the 13-14  than the 12-13 p o s i t i o n s , b u t , s i n c e a - e r y t h r o i d i n e B-erythroidine  the s y n t h e s i s  rather  has been c o n v e r t e d t o  c o n c l u s i v e l y e s t a b l i s h e d the s p i r o amine 39  structure postulated  f o r a-erythroidine  f a c t t h a t the s y n t h e t i c f u s i o n i s beyond  and n a t u r a l  the  Thus, the  isomers have the same mode o f r i n g  question.  (12)  In the p a s t  and i t s d e r i v a t i v e s .  (14)  few years important  advances have been made i n e l a b o r a t i n g  c h e m i s t r y o f the e r y t h r i n a a l k a l o i d s .  Other r o u t e s l e a d i n g t o the  . . j j 38,46,47,48,75 , 40,41,42,43,44,49 s p i r o amine system have been d e v i s e d . Mondon 45 has extended h i s p r e v i o u s s y n t h e t i c s t u d i e s rearrangement o f the e r y t h r i n a n e The important q u e s t i o n settled quite recently.  ring  and he a l s o r e p o r t e d  a new  skeleton.  of stereochemistry  o f these a l k a l o i d s have been  The aromatic E r y t h r i n a a l k a l o i d s  with the  exception  o f e r y t h r a t i n e , have been i n t e r r e l a t e d and shown t o have the same 14 c o n f i g u r a t i o n o f the s p i r o carbon atom, C-5, From an X-ray c r y s t a l lo-^ 50 g r a p h i c study o f e r y t h r a l i n e hydrobromide and from chemical s t u d i e s o f 47 e r y s o d i n e and the p r o o f o f i t s s t r u c t u r e through s y n t h e s i s , the r e l a t i v e configurations  at C-3 and C-5  are known f o r the aromatic e r y t h r i n a a l k a l o i d s  -9-  The  methoxyl and  s p i r o amine groups have a " c i s " r e l a t i o n s h i p as shown  C H  O  C H C % ^  R  (16)  The  conversion  (16)  (17)  of a-erythroidine  to 3 - e r y t h r o i d i r i e e s t a b l i s h e d  < both have the same c o n f i g u r a t i o n at C-3  and  C-5.  39  Degradations  51  '  that 52  and  53 X-ray study  . allowed the  configurations  complete assignment o f r e l a t i v e and  o f the e r y t h r o i d i n e s as shown f o r  absolute  (3R,5S,12S)-a-erythroidine  (17). •  54  Boekelheide ences and by  has  shown, by  o p t i c a l r o t a t o r y d i s p e r s i o n measurements, t h a t the  e r y t h r i n a a l k a l o i d s and  t i o n of absolute  The  i n view o f t h i s e v i d e n c e , and  c o n f i g u r a t i o n o f e r y t h r o i d i n e s , the  aromatic s e r i e s can be  Biosynthesis  The present,  given  o f the  absolute  determina-  configuration  (16).^*  of A l k a l o i d s .  s t r u c t u r e s o f some 1700 and  aromatic  the e r y t h r o i d i n e s have the same, c o n f i g u r a t i o n at  the s p i r o carbon atom C-5.  o f the  the method o f m o l e c u l a r r o t a t i o n d i f f e r -  n a t u r a l l y o c c u r r i n g a l k a l o i d s are known at  the p r o c e s s e s by which a l k a l o i d s are s y n t h e s i z e d  i n plants  •10-  have long been t h e s u b j e c t o f study and s p e c u l a t i o n among o r g a n i c 55 and b i o c h e m i s t s .  chemists  56 57 58 '  '  '  A p r o p e r u n d e r s t a n d i n g o f the pathways demands  a knowledge o f the substances which are i n v o l v e d as i n t e r m e d i a t e s o f the mechanisms by which the v a r i o u s the  l a s t ten years,  transformations  ^  e  a r e c a r r i e d out.  b i o g e n e t i c studies with  s u c c e s s f u l l y on a broad front„ alkaloidal  and experiment  can be combined, and at the moment the main i n t e r e s t l i e s t i o n o f the major formal  '  compounds are now at  the v e r y i n t e r e s t i n g s t a g e o f development where h y p o t h e s i s  The  In  t h i s complex o f problems, which p r e v i o u s l y was o n l y t h e 59  o b j e c t o f s p e c u l a t i o n s , has been a t t a c k e d 60,61,62,63  and a l s o  i n the c l a r i f i c a -  r e l a t i o n s h i p s between a l k a l o i d s and t h e i r  precursors.  d i f f i c u l t problems o f the d e t a i l s o f the r e a c t i o n mechanisms now seem 64  t o r e s t i n many cases on a secure No s i m p l e multistep  theoretical basis.  s y n t h e s i s has y e t been d e v i s e d  syntheses d e s c r i b e d  f o r t h e a l k a l o i d s , and t h e  i n the l a s t h a l f c e n t u r y  when compared t o the paths used by n a t u r e .  seem u n s a t i s f a c t o r y  T h i s problem has i n t r i g u e d  many workers, and as e a r l y as 1917 S i r Robert R o b i n s o n ^ had d e v i s e d and executed the famous s y t h e s i s o f t r o p i n o n e , also to represent  reasonable  patterned  b i o s y n t h e t i c routes. 58  along  lines  considered  He put forward many  important i d e a s on a l k a l o i d b i o s y n t h e s i s ,  and h i s i d e a s have been i n v a l u -  a b l e i n g u i d i n g experimental  plants.  The  work on l i v i n g 59  term " b i o g e n e t i c - t y p e "  has been s e l e c t e d t o d e s c r i b e  s y n t h e s i s d e s i g n e d t o f o l l o w , i n a t l e a s t major a s p e c t s ,  an o r g a n i c  biosynthetic  pathways p r o v e d o r presumed, t o be used i n t h e n a t u r a l c o n s t r u c t i o n o f t h e end p r o d u c t s .  I t implies that the r e l a t i o n s h i p o f the laboratory  to the b i o s y n t h e s i s  i s not n e c e s s a r i l y very  synthesis  c l o s e and t h a t the i n v i t r o  route may be based on an i n v i v o scheme which i s reasonable  yet only  -11-  s p e c u l a t i v e , o r f o r which o n l y meager evidence may  be a v a i l a b l e .  The  i s meant t o r e f e r t o presumed i n t e r m e d i a t e s and b i o s y n t h e t i c p a t h s , little  emphasis i s p l a c e d on reagents  and  conditions,  term  and  In the l a b o r a t o r y  d u p l i c a t i o n o f the key b i o l o g i c a l s t e p , any c o n d i t i o n s o r reagents may used which are n e c e s s a r y  f o r the completion  o f the r e a c t i o n .  success o f c e r t a i n " b i o g e n e t i c - t y p e " syntheses  may  The  be  striking  depend upon u t i l i z a t i o n  o f r e a c t i o n types which p a r a l l e l enzyme-promoted p r o c e s s e s , and i n l i e u the enzyme system, the o r g a n i c chemist  may  need t o r e s o r t t o reagents  c o n d i t i o n s not a v a i l a b l e t o the l i v i n g system, i n o r d e r t o f o l l o w the a l l b i o s y n t h e t i c route.  The  key i n t e r m e d i a t e may  t u r e proposed, i n the b i o s y n t h e t i c scheme o r i t may to d i r e c t the i n t e r m e d i a t e a l o n g d e s i r e d channels a c t i o n courses.  B i o g e n e t i c - t y p e synetheses  possess  the exact  of  and over-  struc-  be a simple m o d i f i c a t i o n , and p r e c l u d e o t h e r r e -  o f t e n are n e a t e r , s h o r t e r , and  more e f f i c i e n t than normal r o u t e s i n which no a t t e n t i o n i s p a i d t o n a t u r a l processes.  Sometimes, i t i s found t h a t the o n l y s a t i s f a c t o r y r o u t e t o some  n a t u r a l product  i s the b i o g e n e t i c t y p e .  However, the s u c c e s s o f a " b i o -  g e n e t i c - t y p e " s y n t h e s i s by i t s e l f does not  c o n s t i t u t e evidence  f o r the  o p e r a t i o n o f a p a r t i c u l a r chemical s t e p i n n a t u r e , and the t e m p t a t i o n i s g r e a t i n many cases t o draw such a c o n c l u s i o n . Real p r o g r e s s  i n the study o f a l k a l o i d b i o s y n t h e s i s began when  o r g a n i c compounds l a b e l l e d w i t h carbon-14 and w i t h o t h e r i s o t o p e s became r e a d i l y a v a i l a b l e i n the e a r l y n i n e t e e n - f i f t i e s .  S i n c e t h e n , many groups  o f workers have t a c k l e d problems i n t h i s a r e a and the i n f o r m a t i o n i s accumulating  rapidly,^>^  4  Isotopes have p r o v e d p a r t i c u l a r l y  important  i n c l a r i f y i n g the mutual r e l a t i o n s h i p s among the v a r i o u s a l k a l o i d s i n any g i v e n p l a n t at the same time. fundamental importance  Many b i o s y n t h e t i c p r o c e s s e s  can be s t u d i e d by t r a c e r methods.  found of  Examples o f  such p r o c e s s e s are several  c o n d e n s a t i o n , r i n g c l o s u r e , m e t h y l a t i o n and  different oxidation  o f t h i s new  r e a c t i o n s , dehydrogenation, and  e x p e r i m e n t a l t e c h n i q u e , i t was  By means  and  the  alkaloids within  biogenetic relatively  periods.^ There have been a c o n s i d e r a b l e  and  so on.  p o s s i b l e t o e s t a b l i s h the  r e l a t i o n s h i p s between c e r t a i n " p r e c u r s o r s " short  demethylation,  number o f p o s t u l a t e d  l o o k i n g back at these s p e c u l a t i o n s  which are o f t e n  closely allied.  One  method, i n v o l v i n g i n s p e c t i o n o f the structural units within  one  can  see  approach has  main thought p r o c e s s e s ,  been the  comparative anatomy  natural products.  suggesting possible r e l a t i o n Such d e d u c t i o n s are  c u l a r l y u s e f u l f o r c o r r e l a t i n g d i f f e r e n t groups o f a l k a l o i d s and i n g the s t r u c t u r e s  o f new  alkaloids.  The  parti-  for predict-  simple u n i t s from which the  a r i s e are the  The  r e c o g n i t i o n o f the extremely c l o s e r e l a t i o n s h i p between a l k a l o i d s , simple  the be  synthesis  amino a c i d s  of alkaloids.  s u p p o r t e d by  l y s e the  basic  r e s u l t i n g from the decomposition o f  alkaloids  may  p l a n t bases, and  amino a c i d s  routes,  formulae o f a l k a l o i d s t o seek common  a group o f a l k a l o i d s , and  s h i p o f these u n i t s to s i m p l e r  two  biogenetic  The  l e d to important i n f o r m a t i o n  i s o l a t i o n o f enzymes which  s t e p s o f the b i o s y n t h e s i s ,  approaches to b i o g e n e s i s  concerning  s t r u c t u r a l examination based h y p o t h e s i s  t o o l s such as t r a c e r s or the  successive  proteins.  can cata-  s i n c e both d i r e c t and i n d i r e c t  complement each o t h e r .  The  o t h e r approach has  been  to c o r r e l a t e a l k a l o i d a l s t r u c t u r e s  on the b a s i s o f a u n i f y i n g r e a c t i o n mechan-  ism.  (18)  The  amine (19)  amino a c i d p h e n y l a l a n i n e and,  recognized  that  hydes  could  (20)  by  oxidation,  the  can,  aldehyde  by d e c a r b o x y l a t i o n , (20).  i f condensation of g-substituted o c c u r i n p l a n t s , then one  could  give  the  In t h i s c o n n e c t i o n Robinson ethylamines  (19)  with  alde-  account f o r a wide v a r i e t y  o f a l k a l o i d a l s t r u c t u r e s , as t h i s r e a c t i o n i s p a r t i c u l a r l y important f o r  the  -13-  synthesis  of N-heterocyclic  systems.  (19)  +  CHO  N-H  (18)  (20)  Other s y n t h e t i c r e a c t i o n s which can be as keys to the s y n t h e s i s aldehydes and -C(0H)-N- w i t h  considered  t o be most important  o f a l k a l o i d s are: the a l d o l c o n d e n s a t i o n between  B-keto a c i d s and  the s i m i l a r c o n d e n s a t i o n o f  the a c t i v e methylene groups  carbinolamines,  (-CH -C=0) o f ketones or 0-keto 2  ... 66,61 acids. The  b i o s y n t h e t i c s i g n i f i c a n c e o f phenol o x i d a t i o n s has  recognized,  and the r o l e o f o x i d a t i v e condensations i n the b i o s y n t h e s i s  a l k a l o i d s have been d i s c u s s e d ' by  i n great d e t a i l  Of the known a l k a l o i d s more than 10%  in  derived, i n p r i n c i p l e ,  The i n t e r p r e t a t i v e  approach to b i o s y n t h e s i s , o f c o r r e l a t i n g the s t r u c t u r e s  of  n a t u r a l p r o d u c t s i n terms o f o x i d a t i v e c o u p l i n g o f p h e n o l i c p r e c u r s o r s b i o g e n e t i c pathways, i s now  of  e x c e l l e n t reviews.^4,67,  can be  coupling of appropriate phenolic precursors.  mechanistic  l o n g been  c l a s s i f i e d as B i o g e n e t i c A n a l y s i s .  64  It  in  -14-  includes  in its  operation  labelled  substrates  those  f e e d i n g experiments  w h i c h can be u s e d t o  The o x i d a t i o n o f p h e n o l s o r transfer  oxidizing  agents  evaluate  of phenol  affords  •  mesomeric  with radiochemically  such  an  anions by  analysis.  one-electron  phenol r a d i c a l s  as  shown  (21).  (21)  These of  are  the  stable  radicals, relative  odd e l e c t r o n  aromatic  ring.  by resonance  The f r e e  to  alkyl  over  radicals  the  radicals,  ortho  because  of  the  and p a r a p o s i t i o n s  have been d e t e c t e d  by magnetic  spread  of  the  suscepti-  64 bility have  and e l e c t r o n  shown t h a t  the  the  ortho-position.  the  first  step  spin free  electron  i n the  radicals  have been  products  d e p e n d i n g on t h e  for  example  furnishes  or  the  generation  generated  back  dimers.  oxygen-oxygen diphenyls  is  oxygen  density  oxidation of  oxidant  gives  measurements,  The e x p e r i m e n t s  transfer  Reduction  resonance  the  they  parent  Dimers can be  diphenyl  in  greater full  of  the  may b e  phenol,  the  b y a one  phenoxyl r a d i c a l . converted  to  electron  molecules,  self  coupling  carbon-oxygen  radicals  or para-coupling.  that  processes.  afford non-radical products,  by o r t h o -  view  at  molecular  coupling with reactive  formed by c a r b o n - c a r b o n ,  than  Once p h e n o l  stable  by s e v e r a l  studies  para-,  accord w i t h the  Simple d i m e r i z a t i o n o f the  ethers  at  a monohydric p h e n o l ,  substitution pattern  and h a l o g e n s  coupling.  are  is  and d e t a i l e d  gives  (See  rise  Figure  or to 1).  -15-  Th e carbon-carbon c o u p l i n g  i s the most important and i t can be  ortho-ortho,  ortho-para or para-para.  Figure  1.  Simple d i m e r i z a t i o n  I f oxidative dimerization  o f t h e molecule i s assumed, one must  d i s t i n g u i s h , i n p r i n c i p l e , between the r a d i c a l coupling)  o f phenol r a d i c a l s .  and the s u b s t i t u t i o n o f a phenol.  coupling process radical  (radical  i n t o a molecule o f phenol .followed by f u r t h e r o x i d a t i o n . p r e d i c t o r t h o - p a r a type s u b s t i t u t i o n . phenol, :  coupling  insertion)  Both mechanisms  Although r a d i c a l s u b s t i t u t i o n i n t o a  anion cannot be d i s r e g a r d e d ,  p r o c e s s e s i n such phenol o x i d a t i o n s  (homolytic  the i n t e r v e n t i o n o f r a d i c a l i n s e r t i o n  seems u n l i k e l y , and the r a d i c a l  i s f a v o u r e d . and a c c e p t e d w i t h o u t f u r t h e r  qualification.  Evidence i s a l s o l a c k i n g f o r the i n t r u s i o n o f c a t i o n i c s p e c i e s  68  (2-electron  64 oxidation)  i n these  oxidations.  Phenols i n which one o r p r e f e r a b l y two r e a c t i v e p o s i t i o n s are b l o c k e d by  s u i t a b l e s u b s t i t u e n t s , e.g. methoxyl, methyl o r a c e t y l groups, g i v e good  y i e l d s o f diphenyl  d e r i v a t i v e s on o x i d a t i o n .  I t i s c l e a r that  2,4,6-tri-  s u b s t i t u t e d phenols not h a v i n g a-CH i n the s u b s t i t u e n t s produce s t a b l e  -16-  phenol r a d i c a l s , which a r e r e a d i l y d e t e c t e d not o n l y by t h e i r r e a c t i v i t y b u t a l s o by t h e i r paramagnetism.  chemical  T h i s i s because phenol  coupling  i s p r e v e n t e d by s t e r i c reasons, and t h e absence o f a-hydrogen i n the s u b s t i tuents  p r o h i b i t s the f o r m a t i o n  o f a methylenequinone.  67  Amino phenols and  64 amines a l s o couple v i a mesomeric r a d i c a l s . Successful and  c o u p l i n g r e a c t i o n s i l l u s t r a t i n g the formation  o f C-C, C-0  C-N bonds have been c a r r i e d out i n t h e l a b o r a t o r y , however, the s e l e c t i o n  o f reagent and the experimental empirical. effect  conditions  f o r a given substrate  A great v a r i e t y o f e l e c t r o n a c c e p t o r s  have been employed t o  the c o u p l i n g o f phenols e.g. f e r r i c y a n i d e s , f e r r i c c h l o r i d e , hydrogen  peroxide,  manganese d i o x i d e , quinones, o r enzyme p r e p a r a t i o n s .  The y i e l d s  o f the i s o l a b l e p r o d u c t s o f the r e a c t i o n vary w i t h i n wide l i m i t s g r e a t l y dependent upon t h e s t r u c t u r e bility  are largely,  o f t h e p h e n o l , the s t a b i l i t y o r s o l u -  o f the r e a c t i o n p r o d u c t s and t h e r e a c t i o n c o n d i t i o n s  l a r g e amounts o f amorphous b y - p r o d u c t s , g e n e r a l l y i l l taneously  and a r e  formed due t o h y d r o x y l a t i o n s ,  (pH). As a r u l e  defined,  are s i m u l -  ring f i s s i o n or polymerization.  the  l i v i n g c e l l phenol o x i d a t i o n s  occur i n a h i g h l y organized  and  i t i s p r o b a b l e t h a t the r e a c t i n g phenol molecules become s u i t a b l y  In  surrounding  o r i e n t e d so as t o make a d i r e c t e d c o u p l i n g p o s s i b l e , thus m i n i m i z i n g the formation  o f by-products.  t o r y , and i t i s customary  T h i s i s very d i f f i c u l t  t o i m i t a t e i n the l a b o r a -  t o work i n h i g h d i l u t i o n t o p r e v e n t  polymeriza-  tion. I f one assumes t h a t r a d i c a l s such as (21) d i s a p p e a r p a i r s to f u r n i s h molecular on the mode o f c o u p l i n g .  products,  by c o u p l i n g i n  then c e r t a i n r e s t r i c t i o n s  a r e imposed  I f such r a d i c a l s a r e a l s o i n v o l v e d i n b i o g e n e t i c  pathways, then by b l o c k i n g r e a c t i v e p o s i t i o n s i n t e r e s t i n g r e s t r i c t i o n s are imposed on t h e p r e c u r s o r s  and p r o d u c t s i n the b i o g e n e t i c sequence.  I t was  -1767 first  recognized  by Barton and Cohen,  c a t i o n o f the p r i n c i p l e o f o r t h o f o r the s t r u c t u r a l features  t h a t completely  rigorous  and para-C-C and C-0 c o u p l i n g  appli-  accounts  found i n many c l a s s e s o f a l k a l o i d s .  They a l s o  p o s t u l a t e d t h a t t h e c o u p l i n g s t e p i s t h e r e s u l t o f phenol oxidation., rous papers have appeared s i n c e c o n c e r n i n g  Nume-  t h e use o f o x i d a t i v e procedures  on p h e n o l i c and amino compounds and a v a r i e t y o f b i o s y n t h e t i c u u . • . 63,68,64 have been o b t a i n e d . Amongst t h e many types o f compounds which can, a t l e a s t  successes  f o r m a l l y , be  d e r i v e d by the c o u p l i n g o f phenoxide r a d i c a l s the a l k a l o i d  galanthamine  (25)  example.  present  i n the A m a r y l l i d a c e a e  ing t o b i o g e n e t i c theory  two p h e n o l i c  p o s i t i o n s as t o d i r e c t The  an o u t s t a n d i n g  t h e t h r e e main c l a s s e s o f A m a r y l l i d a c e a e  are a l l d e r i v e d from a p r e c u r s o r dine, having  provides  the phenolic hydroxyl  and/or p a r a - c o u p l i n g  groups i n such 67 between the r i n g s .  v a l i d i t y o f t h i s scheme has been demonstrated by independent  i n three  researches  laboratories.^3,68  Galanthamine was r e g a r d e d as b i o s y n t h e s i s e d dimethyl  alkaloids  (22) now known t o be t h e a l k a l o i d n o r b e l l a -  rings with  ortho-  Accord-  ether  (24) by o x i d a t i o n t o t h e dienone  from n o r b e l l a d i n e 0N(26), r i n g c l o s u r e t o the  enone (27), and r e d u c t i o n o f the l a t t e r t o t h e a l l y l i c  alcohol  galantha-  mine (25) (see F i g u r e  2 ) . At t h e time when t h i s scheme was put forward  the a l k a l o i d narwedine  (27) had n o t been c h a r a c t e r i z e d , and t h e formula  f o r galanthamine was u n c e r t a i n .  The c o r r e c t f o r m u l a (25) was, i n f a c t ,  chosen on t h e b a s i s o f the proposed b i o g e n e s i s . shown t h a t n o t o n l y was t h e c o r r e c t f o r m u l a but  Biogenetic  a n a l y s i s has  (25) chosen f o r galanthamine  i t was proved t h a t l a b e l l e d n o r b e l l a d i n e ON-dimethyl e t h e r  norbelladine  (22) was i n c o r p o r a t e d  i n t o galanthamine.  71  (24) and  In e a r l i e r  -18-  [2- C]  experiments, i n c o r p o r a t i o n o f was  observed.  q u e s t i o n by  The  t y r o s i n e (23)  i n t o galanthamine  c o n s t i t u t i o n o f galanthamine has  been placedbeyond  lk  a t o t a l synthesis  on the b i o g e n e t i c scheme. coupling theory  70  f o r these  from n o r b e l l a d i n e ON-dimethyl e t h e r An  important  (25)  (24)  based  r e s u l t which bears on the phenol  a l k a l o i d s i s the evidence  obtained  f o r the  pre63  sence o f p h e n o l i c n o r b e l l a d i n e d e r i v a t i v e s i n A m a r y l l i d a c e a e The pattern.  aporphine a l k a l o i d i s o t h e b a i n e I t was  suggested  (30)  an unusual  t h a t bases o f t h i s type  frqm the 1 - b e n z y l t e t r a h y d r o i s o q u i n o l i n e the dienone  (29) has  f o l l o w e d by  plants* oxygenation  are b i o s y n t h e s i z e d  (28) by phenol o x i d a t i o n t o  r e d u c t i o n t o the d i e n o l (31)  (see F i g u r e  M i g r a t i o n o f a bond by d i e n o l benzene rearrangement leads d i r e c t l y isothebaine has  (29).  been a c h i e v e d ,  72  The  first  and  t r a c e r s t u d i e s c a r r i e d out w i t h  showed t h a t o r i e n t a l i n e (28) The  give  s y n t h e s i s o f i s o t h e b a i n e by  this  3).  to  sequence  oriental  poppies 73  i s i n c o r p o r a t e d by p l a n t s i n t o i s o t h e b a i n e .  i n f l u e n c e of s e l e c t i v e p r o t e c t i o n of phenolic f u n c t i o n i s n i c e l y  illustrated in this  case,  and  i t seems p r o b a b l e  that methylation  the d i r e c t i o n o f o x i d a t i v e c o u p l i n g i n the b i o s y n t h e s i s o f 74 An  extensive  investigation  shown, t h a t , p r o v i d e d they  yields.  alkaloids.  of benzyltetrahydroisoquinolines  contain quaternary  bases can undergo o x i d a t i v e condensation of biogenesis  controls  n i t r o g e n , simple  has  phenolic  under c o n d i t i o n s s i m i l a r t o  t o form a l k a l o i d s o f the i s o q u i n o l i n e s e r i e s w i t h  By means o f such o x i d a t i v e c o n d e n s a t i o n ,  those  good  more than s i x t y a l k a l o i d s  o f v a r i o u s s t r u c t u r a l types have become more e a s i l y o b t a i n a b l e . numerous p o s s i b l e condensations o f the i n t e r m e d i a t e  Of  the  mesomeric r a d i c a l s  o n l y those which l e a d t o n a t u r a l l y o c c u r i n g a l k a l o i d s g i v e good y i e l d s few  by-products.  These r e s u l t s  suggest t h a t o x i d a t i v e condensations  of  and  -19-  Figure 2.  The B i o s y n t h e s i s o f Galanthamine-. (25).  -20-  F i g u r e 3.  quaternary the p l a n t  bases may  The  Biosynthesis o f Isothebaine  a l s o be  (29).  i n v o l v e d i n the b i o s y n t h e s i s o f a l k a l o i d s i n  cell.  There have been a number o f s p e c u l a t i o n s on the b i o g e n e s i s  of  the  14 Erythrina alkaloids,  however, no  so f a r w i t h which t o e v a l u a t e The be  these  experimental  evidence  been r e p o r t e d .  proposals.  g r e a t e r the importance o f the complex n a t u r a l products  i s o l a t e d and s t r u c t u r a l l y  i d e n t i f i e d i n modern t i m e s ,  does i t become t o l e a r n to s y n t h e s i z e them as simply the c e l l .  has  I t i s hoped t h a t , by  and  which  the more  can  important  as r a p i d l y as i n  i m i t a t i n g such b i o s y n t h e t i c methods  i n c r e a s i n g demand f o r p h y s i o l o g i c a l l y a c t i v e b i o l o g i c a l products  the  can be  more e f f i c i e n t l y than by the time-consuming e x t r a c t i o n s from the p l a n t  met cell.  -21-  DISCUSSION The o b j e c t o f t h i s concerning  i n v e s t i g a t i o n was t o o b t a i n fundamental  information  the b i o s y n t h e s i s o f E r y t h r i n a a l k a l o i d s , and t o a c h i e v e  a model  s y n t h e s i s o f the c h a r a c t e r i s t i c s p i r o amine s t r u c t u r e along the l i n e s o f a proposed b i o g e n e t i c scheme.  The problem arose as a d i r e c t  * , , 14,58,67,76,77,78 o f much s p e c u l a t i o n  had not been p r e v i o u s l y The b i o g e n e s i s any  o f these  o f the E r y t h r i n a a l k a l o i d s  o f E r y t h r i n a a l k a l o i d s cannot r e a d i l y be r e l a t e d t o  o f the schemes proposed f o r o t h e r a l k a l o i d s .  I t appeared t h a t a new  o f the s p i r o amine system,  i t might be e x p e c t e d t o a r i s e through a new type o f v a r i a t i o n i n  biogenesis.  A study  o f the m o l e c u l a r  s t r u c t u r e s o f the E r y t h r i n a a l k a l o i d s ,  leads t o c e r t a i n f i r m c o n v i c t i o n s as t o the s o r t o f p r e c u r s o r s involved i n t h e i r biosynthesis. out  c  explored.  approach i s r e q u i r e d t o e x p l a i n the f o r m a t i o n and  . the f o r m a t i o n  concerning  a l k a l o i d s i n the p l a n t s , s i n c e the b i o g e n e s i s  consequence  The modes o f p o s s i b l e b i o g e n e s i s  as connected s e r i e s o f r e a c t i o n s , however, a d e f i n i t e o r d e r  v a r i o u s stages  and  processes are s e t  f o r the  i s n o t assumed. 58  According to biogenetic theory from a p r e c u r s o r  (33), having  groups at 3 , 4 - p o s i t i o n s , (Ar-C-C).  amine system i s d e r i v e d  two p h e n o l i c r i n g s with p h e n o l i c  hydroxyl  s y n t h e s i s e d i n Nature from two C6-C2 u n i t s  The condensation  amino a c i d becomes  the s p i r o  presumably proceeds by a mechanism whereby one  r e a c t i v e by d e c a r b o x y l a t i o n  t o an amine and the o t h e r  by o x i d a t i v e deamination t o an aldehyde o r i t s e q u i v a l e n t .  One  satisfac-  14 t o r y Scheme  (see F i g u r e 4) e n v i s i o n s the b u i l d i n g b l o c k s t o be two  molecules o f 3 , 4 - d i h y d r o x y p h e n y l a l a n i n e precursor  (32), t h e i r union  to  (33) and i t s subsequent o x i d a t i o n t o the orthoquinone  g i v e the (35). I t  -22-  i s not known at what stage hydroindole one.  (34)  and  decarboxylation  subsequently  to the  o c c u r s . The  intermediate  r i n g c l o s u r e t o the d i (37)  is a plausible  S i m i l a r r i n g c l o s u r e s o c c u r when 3,4-dihydroxyphenethylamines  and 79  and  3,4-dihydroxyphenylalanine  Methylation  to give erysopine  i t s e l f i s s u b j e c t e d to m i l d O x i d a t i o n . (38) may  occur  at some l a t e r s t a g e .  s t a n d i n g f e a t u r e o f t h i s scheme i s t h a t i t r e p r e s e n t s great b i o g e n e t i c pathways f o r forming it  e x p l a i n s the f o r m a t i o n  According  f o r the e l a b o r a t i o n o f the v a r i o u s (4)  (5).  The  lactortization chemistry  i n the p l a n t and 6-erythroidine  appears to be  aromatic  the key  and C-5  f u r t h e r a l t e r a t i o n s do not  i t i s p o s s i b l e that erysopine  be  erysopine ultimate  (38)  i s formed  stereofirst  enzymatically  to o t h e r a l k a l o i d s , i n c l u d i n g  ( 5 ) , the extent  t o which t h i s  c o u l d o c c u r would depend on  by  a p a r t i c u l a r plant species.  t h r i n a a l k a l o i d s i s 3,4-dihydroxyphenylalanine  (38)  way.  a f f e c t the  q u i t e p o s s i b l e t h a t the s t a r t i n g m a t e r i a l f o r the b i o g e n e s i s  o f two  and  then c o n v e r t e d  the enzyme systems possessed  union  two  a l k a l o i d s as w e l l as f o r the  o f the dihydroxybenzenoid r i n g o f  S i n c e these  out-  intermediate  a p p r o p r i a t e m o d i f i c a t i o n s o f the s i d e - c h a i n s and  (5).  at C-3  (38)  '  isoquinoline alkaloids,  non-aromatic a l k a l o i d s o f t h i s f a m i l y may 17  d e r i v e d by Woodward f i s s i o n (38), f o l l o w e d by  an o v e r l a p o f the  o f the s p i r o amine system i n a very simple  t o t h i s scheme e r y s o p i n e  erythroidines  i n d o l e and  The  80  (32) , s i n c e an  I t seems t o be o f the  Ery-  appropriate  m o l e c u l e s o f t h i s amino a c i d can l e a d d i r e c t l y t o a s t r u c t u r e  r e p r e s e n t a t i v e o f the ''aromatic"  alkaloids.  the r o l e t h a t the c o u p l i n g o f phenoxide r a d i c a l s  can p l a y i n e x p l a i n 64 i n g the b i o s y n t h e s i s o f n a t u r a l p r o d u c t s i s by now w e l l a p p r e c i a t e d . The f o r m a t i o n o f a l l E r y t h r i n a a l k a l o i d s can be accommodated by t h i s e l e 67 gant and simple b i o g e n e t i c h y p o t h e s i s , t h a t the carbon s k e l e t o n s  -23-  are The  produced by o x i d a t i v e p h e n o l i c c o u p l i n g o f a p r e c u r s o r o f the type ( 3 3 ) . aromatic  E r y t h r i n a a l k a l o i d s a r e p l a u s i b l y d e r i v e d by o x i d a t i v e c o u p l i n g  of t h i s intermediate one-electron  (see F i g u r e 5).  Oxidation  o f the base (33), by some  t r a n s f e r system t o generate r a d i c a l s which, by c o u p l i n g , would  y i e l d t h e diphenoquinone (39).  A d d i t i o n o f the amino group t o the q u i n o n o i d  system i n (39) leads t o t h e dienone (37) and then subsequent steps y i e l d t h e a r o m a t i c  E r y t h r i n a a l k a l o i d s (40) ( 3 8 ) . A v a r i a t i o n i n  t h i s scheme i s the c a r b o n - n i t r o g e n the h y d r o i n d o l e  coupling o f the precursor  (34), f u r t h e r p a r a - p a r a  the same dienone (37) which was o b t a i n e d aromatic  from t h e o t h e r r o u t e .  at any stage  O - m e t h y l a t i o n can o c c u r  as d e h y d r a t i o n ,  at some p r e v i o u s  o r at t h e d i p h e n o l  hydro-  a f t e r t h e s p i r o amine  stage, as i t i s n o t known y e t whether t h e m e t h y l a t i o n from t h e b a s i c p r e c u r s o r s  The non-  by o x i d a t i v e f i s s i o n o f  the c a t e c h o l r i n g as suggested above. Such p r o c e s s e s genation and d e h y d r o g e n a t i o n may occur  (33) t o g i v e  carbon-carbon c o u p l i n g w i l l f u r n i s h  members o f t h i s f a m i l y can be f o r m u l a t e d  system was formed.  unexceptional  o r subsequent  pattern i s b u i l t i n  (33) l e v e l .  However, s i n c e  t h i s h y p o t h e t i c a l p r e c u r s o r p o s s e s s e s the s p e c i f i c h y d r o x y l a t i o n  pattern  from which a l l the known E r y t h r i n a a l k a l o i d s can be d e r i v e d , i t i s s t r o n g l y b e l i e v e d that methylation hypothesis  occurs  i s f i r m l y supported  at t h i s stage  i n the b i o s y n t h e s i s .  This  by the now w e l l known " d i r e c t i n g " r o l e o f  the p r o t e c t i v e groups i n both t h e s y n t h e s i s and b i o s y n t h e s i s o f a l k a l o i d s . 70 73 97 '  '  The d i f f e r e n c e i n t h e m e t h y l a t i o n  pattern o f the phenolic rings  demonstrates t h a t b i o s y n t h e s i s i s d i r e c t e d t o d i f f e r e n t at l e a s t  64 '  final  skeletons,  i n p a r t , by O - m e t h y l a t i o n . 58 67  With t h e b i o g e n e t i c t h e o r y o f r e s e a r c h was i n i t i a t e d , erythrinane  first  '  as a background i n mind the programme  t o examine t h e i n v i t r o s y n t h e s i s o f t h e  r i n g system, f o l l o w e d by t r a c e r experiments t o determine i n v i v o  -24-  i n the p l a n t how  near t o the t r u t h the assumptions  may  be.  Consequently, i n  o r d e r t o p r e p a r e the base p r e c u r s o r (33) p r e d i c t e d by the b i o g e n e t i c t h e o r y , i t was  f i r s t n e c e s s a r y to develop a s y n t h e t i c pathway t o t h i s proposed 6).  i n t e r m e d i a t e f o r the i n v i t r o s y n t h e s i s (see F i g u r e  3,4-Dimethoxyphenylacetic  acid  and the r e s u l t a n t a c i d c h l o r i d e was  (42) was  treated with t h i o n y l  condensed  with  amine (13) t o g i v e homoveratroyl-homoveratrylamine  key  chloride  3,4-dimethoxyphenethyl(43).  The  amide  was  reduced w i t h l i t h i u m aluminium h y d r i d e i n e t h e r t o d i - ( 3 - 3 , 4-dimethoxyphenyl)-ethylamine  (45).  T h i s diamine was  a l s o p r e p a r e d on a l a r g e r  scale  i n one s t e p by the c a t a l y t i c r e d u c t i o n o f 3 , 4 - d i m e t h o x y p h e n y l a c e t o n i t r i l e 81 (44). mic  Demethylation o f t h i s p r o d u c t i n r e f l u x i n g c o n c e n t r a t e d hydrobroa c i d then y i e l d e d d i - ( 6 - 3 , 4 - d i h y d r o x y p h e n y l )-ethylamine  (33) i d e n t i f i e d by chemical a n a l y s i s . u l t r a v i o l e t spectrum structure. was  ^  m a x  284 my  I t s i n f r a r e d 3400 cm * (0-H)  and  (e 7770)were i n agreement w i t h the a s s i g n e d  T h i s h y p o t h e t i c a l p r e c u r s o r (33) o f the E r y t h r i n a  alkaloids  then o x i d i z e d w i t h a l k a l i n e p o t a s s i u m f e r r i c y a n i d e under v a r i o u s con-  ditions.  In 24 o x i d a t i o n experiments  i t was  products or to o b t a i n r e p r o d u c i b l e r e s u l t s . was  hydrobromide  not p o s s i b l e to i s o l a t e  The major product i n a l l cases  an i n t r a c t a b l e c h l o r o f o r m - i n s o l u b l e polymer.  s o l u t i o n e x t e n s i v e p o l y m e r i z a t i o n was  observed.  minute amount had a b s o r p t i o n s at 3450 (0-H) infrared.  at 284 my.  Even i n v e r y d i l u t e One  and 1720  The u l t r a v i o l e t spectrum showed X r  I t was  crude p r o d u c t o f cm  1  at 253 my  (C=0)  i n the  and a s h o u l d e r  max  S i m i l a r r e s u l t s were o b t a i n e d when f e r r i c  o x i d i z i n g agent.  any  c l e a r from these experiments  c h l o r i d e was  used as  that polymerization  o f the t e t r a h y d r o x y - p r e c u r s o r cannot be a v o i d e d under l a b o r a t o r y c o n d i t i o n s and the proposed  dienone  ( 3 7 ) , i f formed  at a l l ,  c o u l d not be p r e s e n t i n  -25-  OH  OH (34) (35)  C H O 3  Figure 4.  Hypothetical Biogenesis of the Erythrina alkaloids.  -26y i e l d s much g r e a t e r than 1% -.  Many combinations  s a t i o n , s i n c e each p h e n o l i c h y d r o x y l positions  group may  are p o s s i b l e i n the condenproduce two  different  radical  i n t h i s manner.  In view o f the above r e s u l t s the b i o g e n e s i s o f the E r y t h r i n a a l k a l o i d s was to  re-examined, and suggest  reactive sites  and d i r e c t is, It  h y p o t h e s i s was  put  forward.  I t was  t h a t b l o c k i n g by m e t h y l a t i o n o f the 4-hydroxyl  3,4-dihydroxybenzenoid of  a new  r i n g s o f the d i p h e n o l  reasonable  group i n both  (33) s h o u l d reduce the number  (both o r t h o and/or para) l i a b l e to o x i d a t i v e c o u p l i n g ,  the r e a c t i o n along the proposed route to the dienone  o f c o u r s e , the d e s i r e d i n i t i a l seemed q u i t e p r o b a b l e  proposed here  product  o f phenol  radical  (37)  coupling.  t h a t o x i d a t i o n o f the m o d i f i e d d i p h e n o l  f o r the f i r s t  time  (58),  as a p o t e n t i a l p r e c u r s o r o f the  amine r i n g system, would prove more s u c c e s s f u l .  which  spiro  In t h i s h y p o t h e t i c a l  p r e c u r s o r (58), where the subsequent c o u p l i n g r e a c t i o n i s d i r e c t e d o n l y by the f r e e p h e n o l i c groups, the phenoxide r a d i c a l s have a b e t t e r opportu n i t y to couple i n t r a m o l e c u l a r l y than b e f o r e , f o r the very same  reasons  which were d i s c u s s e d e a r l i e r i n o t h e r s i m i l a r ortho-methoxyphenol systems. To t e s t  t h i s new  isovanillin is  p r o p o s a l i n v i t r o , the chemical  (46), and the accomplished  o u t l i n e d i n F i g u r e s 7,8, Isovanillin  (46) was  and  p l a n f o r t h i s new  which y i e l d e d a mixture  benzoic  acid.  from  s y n t h e t i c approach  9.  b e n z y l a t e d w i t h b e n z y l c h l o r i d e , and i t s conden-  s a t i o n w i t h h i p p u r i c a c i d gave the oxazolone of  synthesis started  (49), the a l k a l i n e h y d r o l y s i s  o f 3-benzyloxy-4-methoxyphenylpyruvic  These a c i d s were s e p a r a t e d a f t e r treatment  o f the  (52)and mixture  w i t h a l k a l i n e hydrogen p e r o x i d e , r e s u l t i n g i n 3-benzyloxy-4-methoxyphenyl85 a c e t i c a c i d ' ( 5 5 ) s e p a r a t e d by pared  i n much b e t t e r y i e l d s by  column chromatography.  T h i s a c i d was  pre-  the a l k a l i n e h y d r o l y s i s o f 3-benzyloxy-4-  -  methoxyphenylacetonitrile  "27-  (54).  A c o n s i d e r a b l e q u a n t i t y o f 3-benzyloxy-4-methoxy-B-nitrostyrene was  (50)  d e s i r e d f o r the p r e p a r a t i o n o f 3-benzyloxy-4-methoxyphenethylamine  (53).  82 U n s u c c e s s f u l attempts  were made t o r e p e a t an e a r l i e r procedure  condensation of O - b e n z y l i s o v a n i l l i n as the condensing  agent.  duct d e s c r i b e d was  The  (47) with nitromethane  experiment  never o b t a i n e d .  substance, m e l t i n g above 200° was  was  f o r the  u s i n g methylamine  r e p e a t e d s i x times but the p r o -  I n s t e a d an unknown brown amorphous o b t a i n e d and t h i s  compound might be the  polymer o f the d e s i r e d p r o d u c t .  The n i t r o s t y r e n e (50) was f i n a l l y p r e p a r e d 38 by the method o f Lange and Hambourger u s i n g aqueous sodium h y d r o x i d e i n s t e a d o f methylamine and c a r r y i n g out the c o n d e n s a t i o n r e a c t i o n at 10°. 84 The n i t r o s t y r e n e was hydrofuran to y i e l d was  then reduced w i t h l i t h i u m aluminium 3-benzyloxy-4-methoxyphenethylamine  a l s o p r e p a r e d by the r e d u c t i o n o f  (54) w i t h l i t h i u m aluminium The p h e n y l a c e t o n i t r i l e  hydride (53).  mtetra-  T h i s amine  3-benzyloxy-4-methoxyphenylacetonitrile  hydride i n ether. (54), which t u r n e d out t o be an important  inter-  mediate i n the l a b o r a t o r y s y n t h e s i s o f the d e s i r e d p h e n o l i c diamine p r e c u r s o r (58), was (46) was  p r e p a r e d as f o l l o w s .  The  isovanillin  c h l o r i d e y i e l d e d 3-benzyloxy-4-methoxybenzyl  T h i s underwent exchange w i t h potassium  phoxide  (47) from  reduced w i t h sodium b o r o h y d r i d e t o the c o r r e s p o n d i n g a l c o h o l  which, w i t h t h i o n y l (51).  O-benzylisovanillin  to y i e l d  cyanide  (54).  a c i d c h l o r i d e o f 3-benzyloxy-4-methoxyphenylacetic t r e a t e d at once w i t h  chloride  i n dimethyl s u l 86  3-benzyloxy-4-methoxyphenylacetonitrile  f r e s h l y p r e p a r e d , was  (48)  acid  (55),  3-benzyloxy-4-methoxyphenethyl-  amine (53), y i e l d i n g  3-benzyloxy-N--(3-benzyloxy-4-methoxyphenethyl)-4-.  methoxyphenylacetamide  (56).  The  amide was  b e s t reduced by borane i n t e t r a ^  h y d r o f u r a n t o g i v e di-(B-3-benzyloxy-4-methoxyphenyl)-ethylamine good y i e l d s ,  identified  as the h y d r o c h l o r i d e .  T h i s new  (57) i n  compound a n a l y s e d  -28-  Figure 5.  Hypothetical biogenesis of the Erythrina alkaloids via oxidative coupling of phenols.  -29-  Figure 6.  Reaction sequence, leading to di-(2 -3,4r4i- ydroxyphenyl)ethylamine (33). ' n  -30c o r r e c t l y f o r C H 3 0 L , N C 1 . I t s n.m.r. spectrum  i n d e u t e r o c h l o r o f o r m showed  the expected  at x 2.65  32  at T 3.24,  6  aromatic resonance  a singlet  as a m u l t i p l e t  f o r the methylene  methyls  (-0-CH ) at x 6.2,  protons  o f the phenethylamine  and  3  The benzyl-groups  and a s i n g l e t  (-0-CH -) at x 4.94  and  2  c e n t e r e d at x 6.88  a broad s i n g l e t  (58).  o f the amine (57) were removed by h y d r o g e n o l y s i s i n  to g i v e the d e s i r e d  The  sequent  s y n t h e s i s i n one s t e p from  (54) by c a t a l y t i c r e d u c t i o n . s t r u c t u r e , and  a b s o r p t i o n at 3550 cm  at hand was  Microelemental  a n a l y s i s was  i t s i n f r a r e d spectrum  * (0-H)  c h a r a c t e r i s t i c o f phenols  showed the a r o m a t i c p r o t o n resonance  as a m u l t i p l e t  i n agreement with  substituted  by  i n deuterochloroform c e n t e r e d at x 3.36,  a  f o r the p h e n o l i c p r o t o n s which d i s a p p e a r e d on a d d i t i o n o f  D2O (deuterium exchange), a broad t r i p l e t  i t s sub-  i n N u j o l showed a s t r o n g  n.m.r. spectrum  at x 4.47  shown by  3-benzyloxy-4-methoxyphenylacetonitrile  e t h e r groups i n the o r t h o p o s i t i o n ,  singlet  palladised  di-(B-3-hydroxy-4-methoxyphenyl)-ethylamine  f a c t t h a t the r i g h t p r e c u r s o r was  the proposed  f o r the  side chain.  methanol c o n t a i n i n g c o n c e n t r a t e d h y d r o c h l o r i c a c i d over 10% charcoal;  f o r the  at x 6.2  a singlet  c e n t e r e d at x 7.25  f o r the methyls  f o r the p r o t o n s o f the  (-O-CH3), and  phenethylamine  side chain. The p r e v i o u s o x i d a t i v e condensations c h i e f l y because o f s i d e r e a c t i o n s .  had  given u n s a t i s f a c t o r y  Thus, t o a c h i e v e a b i o g e n e t i c - t y p e  s y n t h e s i s by o x i d a t i v e c o n d e n s a t i o n ,  i t appeared  unwanted phenol  approach  oxidation.  T h i s new  results  necessary to  suppress  has been v e r y rewarding  and  w i t h the m o d i f i e d d i p h e n o l i c p r e c u r s o r (58), where the arrangement o f the hydroxy  groups i s such  p h e n o l i c moiety,  as t o promote and d i r e c t  a simple s y n t h e s i s o f the s p i r o amine system was  i n very reasonable y i e l d The  the c o n d e n s a t i o n i n the achieved  (see F i g u r e 9 ) .  e x p l o r a t o r y experiments  i n v e s t i g a t i n g the new  such p h e n o l - c o u p l i n g showed (U.V., I.R.)  potentialities  of  t h a t these o x i d a t i o n s a c t u a l l y  -31-  proceed much b e t t e r than had been expected. lents  o f o x i d i z i n g agent were used and potassium  be a convenient reagent.  The  s o l u t i o n o f the d i p h e n o l was of  First,  o x i d a t i o n proceeded  at the most two  f e r r i c y a n i d e appeared b e s t when a v e r y  added s l o w l y t o a f o u r f o l d excess  the f e r r i c y a n i d e c o n t a i n i n g potassium hydrogen carbonate  working  3,15-dimethoxy-16-hydroxy-2-oxoerythrina-l(6),3-diene 224-227°.  There  (by e q u i v a l e n t )  is little  By  crystalline isolated in  In more e l a b o r a t e p r e p a r a t i v e experiments  f e r r i c y a n i d e as o x i d a n t the y i e l d o f dienone i n c r e a s e d t o 15%.  (59) was  to  dilute  (pH 8 ) .  up the product as d e t a i l e d i n the e x p e r i m e n t a l s e c t i o n ,  9% y i e l d m.p.  equiva-  with  (59) a c t u a l l y i s o l a t e d  was  doubt t h a t f u r t h e r improvement i n y i e l d  c o u l d be a c h i e v e d by i n c r e a s i n g the d i l u t i o n o f the r e a c t a n t s s t i l l  more.  E l e m e n t a l a n a l y s i s and s p e c t r a l d a t a c o n f i r m e d beyond doubt t h a t the long sought  dienone  spot on s i l i c a r  238  (59) had been o b t a i n e d . T.L.C. o f the dienone gel. °  (e 19370) and  The u l t r a v i o l e t  283  (E 4150)  a b s o r p t i o n s at 3500 (0-H), 1690 1665  (vinyl ether),  diene) cm  The  1630  my.  spectrum r  The  (C=0)  (C=C), 1595  n.m.r. spectrum  i n e t h a n o l showed X  i n f r a r e d spectrum  (aromatic) and 915,  i n deuterochlorofrom  c e n t e r e d at x 3.72  i n N u j o l had  845  ketone),  (substituted  (see F i g u r e 10)  and T 3.62,  ( H i ) ( J i 7 = 1.5  one  at  max  u n s a t u r a t e d s i x membered r i n g  the lone aromatic p r o t o n s as s i n g l e t s a t T 3.36 p r o t o n s as a t r i p l e t  (59) gave  showed  the  olefinic  c.p.s) i n  complete 87  agreement w i t h the magnitude o f long-range and at T 3.99  (Hj+) .  The p h e n o l i c p r o t o n appeared  on a d d i t i o n o f D 2 O , the expected methyl and at x 6.38, to  allylic  and broad m u l t i p l e t s  resonances  spin-spin coupling at x 3.79  and  exchanged  as s i n g l e t s at x  i n the r e g i o n x 6.5-7.8 c o r r e s p o n d i n g  the 8 a l i p h a t i c p r o t o n s o f r i n g B and C o f the e r y t h r i n a n e r i n g  The mass spectrum peaks at m/e  312  showed the m o l e c u l a r i o n at m/e ( M - l ) , 298  6.28  (M-15)(M-CH ), 3  313,  system.  and o t h e r s i g n i f i c a n t  CH^OH  CHO  CHO  OCH-Ph  OCH  OCH-Ph 2  (47),  (48)  CH=CH-NO„ CH=C-C=0 I l N O s  (49)  OCHI Ph 2  V  C H C T ^ OCH-Ph 2 .  i Ph  o  OCH-Ph 2 OCH.  (50)  CH^CH-NH  CH-OCOJH 2 2  OCH-Ph  c  (51)  2  CH^CN-  <OCH  OCH-Ph 2  (52)  OCH-Ph oca (54)  CH-COH 2 2  OCH-Ph  Figure 7,  Reaction scheme for the preparation of 3-benzyloxy-4-methoxyphenethylamine (53) and >-benzyloxy-4-methoxyphenylacetic acid (55). .  -33-  Figure 8. Reaction sequence, leading to di-(^> -3>-hydroxy-4methoxyphenyl)-ethylamine (58).  -34-  285  (M-28) (M-CO) (M-C H ) , 283 (M-30) (M-C H ) , 282 (M-31)(M-1-C H ). 2  1+  2  6  2  6  A d d i t i o n a l evidence was gained by a c e t y l a t i o n o f t h e dienone a c e t i c anhydride  and d r y p y r i d i n e a t room temperature  1 6 - a c e t o x y - 2 - o x o e r y t h r i n a - l ( 6 ) , 3-diene (60). showed one s p o t on s i l i c a r  at  214, 235 and 285 my.  gel. °  t o g i v e 3,15-dimethoxy-  T.L.C. o f t h i s compound  U l t r a v i o l e t spectrum  i n e t h a n o l showed X max  r  I n f r a r e d spectrum  (59) with  i n N u j o l had a b s o r p t i o n s at 1780  (phenol a c e t a t e ) , 1690 ( u n s a t u r a t e d s i x membered r i n g k e t o n e ) , 1665 ( v i n y l e t h e r ) , 1630 (C=C) and 1205 (phenol a c e t a t e ) c m . -1  This also established  t h a t a t e r t i a r y n i t r o g e n i s p r e s e n t i n t h e molecule,  s i n c e a secondary  n i t r o g e n would a c e t y l a t e r e a d i l y . R e d u c t i o n o f the dienone temperature  (59) by sodium b o r o h y d r i d e  i n e t h a n o l a t room  y i e l d e d 3,15-dimethoxy-2 16-dihydroxyerythrina-l(6),3-diene (  T h i s compound was p u r i f i e d by chromatography on an alumina 169°. had  T.L.C. on s i l i c a  g e l showed one s p o t .  (61).  column, m.p. 166-  I n f r a r e d spectrum  i n Nujol  a b s o r p t i o n s a t 3510, 3450 (0-H), 1655 ( v i n y l e t h e r ) , 1610 (C=C) and  1110,  1260 (secondary  showed X  m a x  a l c o h o l ) cm"''. 1  U l t r a - v i o l e t spectrum  at 212, 240 ( s h o u l d e r ) and 287 my.  i n ethanol  The mass spectrum  showed  the m o l e c u l a r i o n a t m/e 315, and o t h e r s i g n i f i c a n t peaks a t m/e 316 (M+l), 300  (M-15)(M-CH ), 297 (M-18)(M-H 0), 287 (M-28){M-C W$ and 285 (M-30) 3  ( M - C H ) . The 2  6  n.m.r. spectrum  2  2  i n d e u t e r o c h l o r o f o r m showed the two lone  a r o m a t i c p r o t o n s as s i n g l e t s a t x 3.43 and a t x 3.68, a m u l t i p l e t and x  a singlet  at x 4.24  at x 4.95 f o r t h e two o l e f i n i c p r o t o n s , a broad s i n g l e t at  4.72 which exchanged on a d d i t i o n o f D 0 and was a s s i g n e d t o a h y d r o x y l 2  p r o t o n , a m u l t i p l e t c e n t e r e d at x 5.16 f o r the p r o t o n on t h e carbon b e a r i n g the h y d r o x y l group i n r i n g A, the methyl x6.28 and  resonances  atom  (-O-CH3) at  a t r 6.47 and a complex p a t t e r n o f l i n e s between x 6-8 c o r r e s -  -35 ponding to the aliphatic protons in rings B and C of the erythrinane ring system. Considering the mechanism of the cyclization reaction via oxidative coupling, there are two routes leading to the spiro amine ring system.  One  has as i t s f i r s t step the para-para coupling of the generated phenolic radicals to give a diphenoquinone type intermediate (39) (see Figure 5). The fourth ring may then be formed by the addition of the amino group to the quinonoid system to yield the dienone (37). to this scheme.  There are two objections  The major inherent objection to this route is the steric  difficulty of attaching the nitrogen atom to the diphenoquinone system.  A  careful examination of the molecular model showed clearly that i t is almost impossible to u t i l i z e the proposed diphenoquinone intermediate (39).  The  nitrogen atom is too far out and above the ring system and the site of i t s proposed attack, so that bond formation would be sterically d i f f i c u l t .  The  second objection is that i t has been reported that diphenoquinone does not 88 89 undergo addition reactions with amines.  '  The second route (see Figure  11) can be visualized by the coupling of the phenolic radical with the unpaired electron on the nitrogen to give the phenethyl-indole (65).  intermediate  This can either, by para-para phenolic coupling (66), or by an in-  doline type of ring closure (64), furnish (67) which, on aromatization of ring D will give rise to the dienone (59).  This second mechanism is the  favoured one and i t is proposed for the cyclization, strongly supported by 80 analogous products obtained enzymatically and by ferricyanide oxidation 90 of N-substituted 3,4-dihydroxyphenethylamines. The exact analogy for our case i s , of course, oxidation of the methylester of 3-hydroxy-4-methoxy90 phenylalanine to the corresponding indole by means of Fremy-salt.  -36-  Figure 9.  Biogenetic-type synthesis of the erythrinane spiro-amine ring system.  -38-  This f i r s t  l a b o r a t o r y r e a l i z a t i o n o f such c y c l i z a t i o n , t o p r o v i d e the  s p i r o amine s k e l e t o n p r e s e n t i n the E r y t h r i n a a l k a l o i d s by r a d i c a l c o u p l i n g , has numerous i n t e r e s t i n g f a c e t s both standpoint.  I t c e r t a i n l y renders s t r o n g support f o r the a t t r a c t i v e p r o p o s a l  t h a t i n Nature these a l k a l o i d s v i o u s l y has  from a b i o g e n e t i c and a s y n t h e t i c  are formed by o x i d a t i v e c o u p l i n g , which p r e -  r e c e i v e d no e x p e r i m e n t a l v e r i f i c a t i o n .  The  results of this  i n v e s t i g a t i o n are c o n s i s t e n t with the i d e a s p r e s e n t e d e a r l i e r , but are not decisive.  However, they are s u f f i c i e n t t o i n d i c a t e i n broad  b i o s y n t h e t i c pathway t o the s p i r o amine system.  The  outline  importance  o f choosing  the c o r r e c t p r o t e c t i o n p a t t e r n f o r the p h e n o l i c groups and a l s o the s t a t e o f the n i t r o g e n o u s The  most important  probable  and c o n t r o l s the d i r e c t i o n o f o x i d a t i v e c o u p l i n g  i n the b i o s y n t h e s i s o f E r y t h r i n a a l k a l o i d s . may  proper  f u n c t i o n i s w e l l i l l u s t r a t e d by the s y n t h e s i s .  c o n c l u s i o n to be drawn i s t h a t i t seems v e r y  t h a t m e t h y l a t i o n precedes  the  The  w e l l be the t r u e p r e c u r s o r o f t h e s e a l k a l o i d s  tetrahydroxy-diphenol i n the p l a n t s , and  (33)  the  i n t r o d u c t i o n o f a l l y l i c h y d r o x y l o r methoxyl at a l a t e r stage r e p r e s e n t s a minor d i v e r g e n c e  from our i n v i t r o scheme.  Such a p r e c u r s o r would have  the c o n c e p t u a l advantage o f a s i n g l e oxygenation f o r a l l the E r y t h r i n a a l k a l o i d s . thus c o n s i s t e n t w i t h the r e s u l t s . r e a l l y i n v o l v e s two  phenolate  The  s t r u c t u r a l aspects o f the t h e o r y are  Proof  radicals  p a t t e r n i n the p r e c u r s o r  t h a t the mechanism o f c o u p l i n g  is difficult  t o s e c u r e by  experiment,  b u t so f a r as c i r c u m s t a n t i a l evidence w i l l  supported.  T h e r e f o r e , the case i s a v e r y s t r o n g one  go the t h e o r y i s  i n f a v o u r o f the  b i o s y n t h e s i s o f E r y t h r i n a a l k a l o i d s by c o u p l i n g o f t h e - d i p h e n o l Although i t seems  the r e s u l t  o f the f e e d i n g experiments  direct  are not y e t  (58). available,  q u i t e c e r t a i n t h a t the r e a c t i o n c a r r i e d out i n the l a b o r a t o r y a l s o  -39takes place in vivo.  The evidence shows that such couplings under labora-  tory conditions are facile, and the formation of the dienone (59) from the phenolic rings could be the key step in the biosynthesis of Erythrina alkaloids.  The intermediates, therefore/must occupy attention.  of intermediates  Isolation  from plants is quite difficult since they are present only  in minute amounts. However, since i t was possible to prepare the dienone (59) , based on our present biosynthetic knowledge i t is possible and quite safe to predict that this hypothetical precursor is present in the Erythrina species.  The validity of this proposal is supported by the examples of  other similar alkaloidal structures occuring in plants, and known to o r i ginate from phenolic precursors.  98 99 100 ' '  The hypothesis that methylene-  dioxy-groups in alkaloids could be derived biogenetically by cyclization of 71 O-methoxyphenols is by now well established.  Therefore i t seems reason-  able to assume that the methylenedioxy-group present in erythraline (3) is formed by a radical cyclization mechanism from the dienone (59).  There  are five examples of derivation of this group from the O-methoxyphenol system in different alkaloids, and the generality of this step is in l i t tie doubt. From the synthetic standpoint, the dienol (61) is a potential intermediate for a total biogenetic-type synthesis of erysodine its methylation pattern (see Figure 9).  (2) in view of  Elimination of the alcohol func-  tion in (61) with the possibility of introducing the C-6 C-7 double bond and reduction at C-3 C-4 will lead directly to erysodine  (2).  Experiments  in this direction are under.way in our laboratory. A final decision on the biosynthetic pathway can only be made by radiochemical labelling experiments. gram was initiated in our laboratory.  Therefore an extensive feeding proFirst, in  order to obtain an in  -40-  3  o  (59) Figure 11. Proposed mechanistic scheme for the formation of the spiro-amine ring system via oxidative coupling.  -41-  v i v o c o n f i r m a t i o n f o r our i n v i t r o  l a b o r a t o r y s y n t h e s i s the d i p h e n o l  (58)  14 l a b e l l e d with  C was  p r e p a r e d a c c o r d i n g t o the scheme shown i n F i g u r e 12.  3-Benzyloxy-4-methoxybenzyl  chloride  cyanide c o n t a i n i n g potassium 3-benzyldxy-4-methoxyphenyl[1-  (51) was  reacted with  potassium  14 [ C] cyanide i n d i m e t h y l s u l p h o x i d e t o y i e l d 14  C]acetonitrile  h y d r o l y s e d t o 3-benzyloxy-4rmethoxyphenyl[1-  14  (68).  The n i t r i l e  C]acetic  acid  was  (70) which on  c o n d e n s a t i o n w i t h 3-benzyloxy-4-methoxyphenethylamine (53) gave  3-benzyloxy-  N-(3-benzyloxy-4-methoxyphenethyl)-4-methoxyphenyl[carbonyl- C]acetamide 14  (69),  The  amide was  reduced by borane  i n t e t r a h y d r o f u r a n (71) and debenzy-  l a t e d by h y d r o g e n o l y s i s t o g i v e the d e s i r e d 3-hydroxy-4-methoxy-N-(3-hyd14 roxy-4-methoxyphen[l-  C]ethyl)—phenethylamine  runs t o be i d e n t i c a l w i t h an a u t h e n t i c sample.  (72) shown i n r a d i o - i n a c t i v e This labelled  substance,  which i s assumed t o be a p r e c u r s o r o f the aromatic E r y t h r i n a a l k a l o i d s and i n t r o d u c e d i n t o the p l a n t ' s b i o s y n t h e t i c system,  i s expected t o produce  on i s o l a t i o n r a d i o c h e m i c a l l y l a b e l l e d e r y s o d i n e ( 2 ) , e r y s o p i n e (38) erythraline  (3).  F e e d i n g experiments  .  are i n p r o g r e s s , the r e s u l t s  and of  which w i l l be o f major i n t e r e s t , and they are expected t o p r o v i d e good support f o r the suggested mode o f b i o s y n t h e s i s o f the E r y t h r i n a  alkaloids.  ^ v  C  H  C  I  2  KCN  CH OCH Ph  OCH Ph  2  2  (68)  (51)  PhCH 0-i  OCH Ph  2  2  CH  (70)  OCH Ph  2  OCH Ph 2  (53)  PhCH 02  OC^Ph (71)  FieP.ire 1 2  •ation o f 3-hydroxy-4-methoxy1 ethyl)-phenexhylamine ( 7 2 ) . ' s marked, with, a s t e r i s k .  -43-  EXPERIMENTAL  Melting Ultraviolet  p o i n t s were determined  on a K o f l e r b l o c k and are u n c o r r e c t e d .  (U.V.) s p e c t r a were measured on a Cary 14 spectrophotometer  i n f r a r e d s p e c t r a (I.R.) were taken on a P e r k i n - E l m e r model 137B photometer. 60 Mc/s plets  N u c l e a r magnetic  on a V a r i a n A60  resonance  instrument.  are g i v e n i n the T i e r s  spectro-  (n.m.r.) s p e c t r a were r e c o r d e d at  The  l i n e p o s i t i o n s or centers o f m u l t i -  x s c a l e w i t h r e f e r e n c e t o 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 s t a n d a r d , with the types o f protons i n d i c a t e d i n parentheses.  and  and i n t e g r a t e d areas b e i n g  S i l i c a g e l G and alumina G ( a c c o r d i n g t o S t a h l )  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  as g i v e n below.  The  alumina used f o r column chromatography was  developed Shawinigan  reagent, n e u t r a l i z e d w i t h e t h y l a c e t a t e , d r i e d and d e a c t i v a t e d with 60% water.  Every m o l e c u l a r weight  The mass spectrum  was  quoted was  determined  determined  on a A.E.I. MS9  of  mass s p e c t r o m e t r i c a l l y .  double  f o c u s i n g mass  spectrometer. E l e m e n t a l m i c r o a n a l y s e s were performed Department, and by Dr. A. Bernhardt  Department The  C. J e n k i n s o f t h i s  and h i s a s s o c i a t e s o f the Max  I n s t i t u t e , Mulheim, Ruhr, West Germany. d e t e r m i n a t i o n s were done by Mrs.  by Mrs.  Planck  The n.m.r. and mass s p e c t r o m e t r i c  A. Brewster  and Mr.  F. G. B l o s s o f t h i s  respectively. r a d i o a c t i v i t y was  determined  w i t h a N u c l e a r Chicago Model D47  flow d e t e c t o r o p e r a t e d as a G e i g e r counter and mounted i n a Model Semiautomatic Scalar. mg  The  as t h i n f i l m s on s t a n d a r d 1.125  total  M-5  Sample Changer, a l l i n c o n j u n c t i o n w i t h a Model 181B a c t i v i t i e s were measured by d e p o s i t i n g  activities  i n c h diameter  Decade  samples o f 0.1  aluminum p l a n c h e t s .  o f s y n t h e t i c p r e c u r s o r s are g i v e n i n m i l l i c u r i e s  gas  to  0.5 The  (mc),  a  -44counter  e f f i c i e n c y o f 39.1% b e i n g assumed.  Homoveratroyl-homoveratrylamine (43)  3,4-Dimethoxyphenylacetic thionyl  acid  c h l o r i d e (15 ml) were heated  the excess  (1 g) L. L i g h t $ Co., England) and on a water-bath f o r 1 hour (45°) , and  o f t h i o n y l c h l o r i d e was evaporated  s o l u t i o n o f the r e s i d u e i n anhydrous e t h e r  under reduced  pressure.  (50 ml) was added t o 3,4-  dimethoxyphenethylamine (1.5 g) (Eastman Kodak) i n e t h e r (20 m l ) . amide which p r e c i p i t a t e d immediately  A  The  was f i l t e r e d o f f and the crude  product 92  recrystallized  from e t h a n o l  (1.5 g ) . m.p. 124° ( l i t e r a t u r e m.p.  A n a l . Found: C, 66,59%; H, 6.5%; N, 3.79%. H, 6.97% N, 3.9%.  124°).  C a l c . f o r C o H N 0 : C, 66.9%; 2  I n f r a r e d spectrum i n c h l o r o f o r m :  2 5  5  3450 (s) (N-H), 3050  ( s ) , 2990 ( s ) , 1680 ( s ) , 1610 ( s ) , 1530 ( s ) , 1480 ( s ) , 1430 ( s ) , 1270 (s) ( b r o a d ) , 1160 ( s ) , 1150 ( s ) , 1035 ( s ) , 965 (w), 865 (s) and 815 (s) c m . -1  n.m.r. s i g n a l s at  i n deuterochloroform,  given i n T u n i t s : m u l t i p l e t  3.29 ( a r o m a t i c H, a r e a 6 H), m u l t i p l e t  centered  c e n t e r e d a t 6.15 (methyl H o f  0 -0-CH , a r e a 12 H), m u l t i p l e t 3  c e n t e r e d a t 6.53 (methylene H o f -CH -c'2  and methylene H o f -N-CH -, a r e a 4H), t r i p l e t c e n t r e d at 7.32 (S-methylene 2  H o f homoveratrylamine s i d e c h a i n , a r e a 2H). Di-(J3-3,4-dimethoxyphenyl)-ethylamine  (45)  Homoveratroyl-homoveratrylamine.. (500 mg) was e x t r a c t e d ( S o x h l e t ) i n t o a r e f l u x i n g suspension (180 ml) (48 h o u r s ) . acetate  o f l i t h i u m aluminium The excess  hydride  o f reagent  (10 ml) and water was added  (50 m l ) .  (600 mg) i n d r y e t h e r  was decomposed with  ethyl  The e t h e r l a y e r was s e p a r a t e d  -45-  and the p a s t y aqueous l a y e r f u r t h e r e x t r a c t e d w i t h e t h e r . the d r i e d  Evaporation of  (anhydrous magnesium s u l p h a t e ) e t h e r e a l s o l u t i o n s gave the  amine (400 mg). stallized  oily  The h y d r o c h l o r i d e , p r e p a r e d i n anhydrous e t h e r , was  from e t h a n o l . m.p.  197°  ( l i t e r a t u r e m.p.  195-196°).  81  recry-  The  amine 81  was  a l s o p r e p a r e d on a l a r g e r s c a l e by a p r e v i o u s l y d e s c r i b e d method  follows. ethanol 1000  A solution of 3,4-dimethoxyphenylacetonitrile (500. ml)  p.s.i.  was  at 100°  hydrogenated f o r 6 hours.  over a c t i v a t e d Raney-Nickel  The  r e s i d u a l o i l was  c h l o r i d e passed  in.  (K § K) i n c a t a l y s t at  A f t e r f i l t r a t i o n the s o l v e n t was  under reduced p r e s s u r e and then the amine was mm.  (25 g)  d i s s o l v e d i n dry e t h e r (50 ml)  The p r e c i p i t a t e d h y d r o c h l o r i d e was  and dry hydrogen collected,  from e t h a n o l , g i v i n g di-(J3-3,4-dimethoxyphenyl)-ethylamine  chloride  (7 g) i d e n t i c a l w i t h the p r o d u c t  n.m.r.).  C20 28 °4 H  N  C1: c  >  62.8%, H,  (s),  2500 (w), 1600  1260  (s) 1165  n.m.r 3.25  spectrum  from the amide r o u t e  A n a l . . Found: C, 62.98%; H,  (m),  ( s ) , 1030  7.32%; N, 1525  ( a r o m a t i c K,  a r e a 12 H), broad  a r e a 6 H), s i n g l e t singlet  ( s ) , 1380  ( s ) , 815  i n deuterochloroform,  7.23%; N,  recryshydro-  (mixed  m.p.,  3.75%. C a l c . f o r  3.67%. I n f r a r e d Spectrum i n N u j o l : 2925  ( s ) , 1460  ( s ) , 860  removed  d i s t i l l e d o f f at 118-120°/1.5  tallized  I.R.,  as  ( s ) , 773  ( s ) , 1340 (w)  (m),  and 725  1240  (w)  (s),  cm" , 1  g i v e n i n T u n i t s : d o u b l e t c e n t e r e d at c e n t e r e d at 6.19  c e n t e r e d at 6.75  (methyl H o f -0-CH , 3  (H o f phenethylamine s i d e c h a i n ,  a r e a 8 H).  Pi-(3-3,4-dihydroxyphehyl)-ethylamine  Hydrobromide  (33)  Di-(3-3,4-dimethoxyphenyl)-ethylamine h y d r o c h l o r i d e (1.3 g) was r e f l u x e d for  3 hours w i t h hydrobromic a c i d  was  evaporated  t o dryness  (48%)  under reduced  (50 m l ) .  The  resulting  solution  p r e s s u r e on the steam-bath, and  the  -46-  r e s i d u a l s o l i d r e c r y s t a l l i z e d from e t h a n o l ( c h a r c o a l ) g i v i n g h y d r o x y p h e n y l ) - e t h y l a m i n e hydrobromide  (1 g ) .  c r y s t a l s a t 100° under vacuum f o r 5 hours, m.p. H, 5.29%; N, 3.21%. I n f r a r e d spectrum  Calc. f o r C H oN0 BrH 0: 16  2  lt  2  di-^-3,4-di-  I t was n e c e s s a r y t o dry the 151°. A n a l . Found: C, 49.8%; C, 49.6%; H, 5.15%; N, 3.61%.  i n N u j o l : 3400 (s) (0-H), 2995 ( s ) , 2750 (w), 1625 (m),  1545 (m), 1470 ( s ) , 1380 ( s ) , 1340 (w), 1295 ( s ) , 1270 (m), 1195 (m), 1155 (w), 1120 (m), 1070 (m), 1050 (m), 1039 (w), 950 (w), 888 (w), 825 (m) , 810 (w) , 783 ( s ) , and 725 (w) Ultraviolet  spectrum r  cm."  1  i n ethanol: X 284 my max  v  ( e 7770).  O x i d a t i o n o f P i - ( g - 3 , 4 - d i h y d r o x y p h e n y l ) - e t h y l a m i n e Hydrobromide w i t h Potassium F e r r i c y a n i d e and F e r r i c C h l o r i d e  In one t y p i c a l  experiment  356 mg  (1.08 mMole) o f p o t a s s i u m  ferricyanide  i n 900 ml o f d i s t i l l e d water c o n t a i n i n g 20 g o f sodium b i c a r b o n a t e was added through  a fine c a p i l l a r y to a s t i r r e d solution of  ethylamine hydrobromide  (100 mg, 0.27 mMole) i n 340 ml o f d i s t i l l e d water  under n i t r o g e n atmosphere d u r i n g 6 hours. f o r another 3 hours which s e p a r a t e d .  di-Q3-3,4-dihydroxyphenyl)-  The r e a c t i o n mixture was  stirred  and f i l t e r e d t o remove the l a r g e amount o f polymer  The s o l u t i o n was then e x t r a c t e d with e t h y l a c e t a t e and  c h l o r o f o r m (4x200 ml r e s p e c t i v e l y ) .  The combined and d r i e d  (anhydrous  sod-  ium s u l p h a t e ) e x t r a c t s were evaporated under reduced p r e s s u r e t o g i v e a gummy m a t e r i a l  (18 mg).  The i n f r a r e d spectrum  has a b s o r p t i o n s a t 3450 (OH) and 1720 cm"  1  (NaCl) o f the crude product  (C=0).  in ethanol: X 253 my and a s h o u l d e r at 284 my. max.  Ultraviolet  spectrum  S i m i l a r r e s u l t s were  o b t a i n e d when f e r r i c c h l o r i d e was used as o x i d i z i n g agent.  I t was n o t  p o s s i b l e t o i s o l a t e any p r o d u c t s o r t o o b t a i n r e p r o d u c i b l e r e s u l t s i n these  -47-  oxidation  experiments.  O - B e n z y l i s o v a n i l l i n (47)  A mixture finely  of isovanillin  (30 g) (K § K ) , b e n z y l c h l o r i d e  powdered anhydrous potassium  (15 g) and p o t a s s i u m  iodide  (7.5 g ) , and a b s o l u t e methanol (100 ml) was r e f l u x e d f o r 15 hours.  After  filtration  carbonate  (37.8 g ) ,  from i n o r g a n i c m a t e r i a l s and c o n c e n t r a t i o n under reduce  the product was steam d i s t i l l e d y i e l d i n g a gum-like on s t a n d i n g a t 0 ° .  r e s i d u e which  pressure, solidified  The r e s i d u e was d i s s o l v e d i n e t h e r , washed f i r s t  d i l u t e sodium h y d r o x i d e magnesium s u l p h a t e ) .  s o l u t i o n then w i t h water and d r i e d  The brownish  with  (anhydrous  o i l , o b t a i n e d by e v a p o r a t i o n o f the e t h e r  s o l u t i o n under vacuum, was r e c r y s t a l l i z e d twice from benzene-petroleum e t h e r 93 (30-60°) t o g i v e c o l o r l e s s n e e d l e s  (32 g ) , m.p. 62° ( l i t e r a t u r e m.p. 6 2 ° ) .  n.m.r s i g n a l s ; g i v e n i n T u n i t s , spectrum  obtained i n deuterochloroform:  s i n g l e t c e n t e r e d at 0.25 (aldehyde H, a r e a = 1 H), m u l t i p l e t 2.7 ( a r o m a t i c H, a r e a = 8 H), s i n g l e t  c e n t e r e d at  c e n t e r e d a t 6.12 (methyl H o f -0-CH , 3  a r e a = 3 H) and a s i n g l e c e n t e r e d a t 4.91 (methylene  o f -0-CH -C H , 2  6  5  area =  2 H). 3-Benzyloxy-4-methoxybenzyl A l c o h o l (48) A stirred solution of O-Benzylisovanillin was t r e a t e d p o r t i o n w i s e w i t h sodium b o r o h y d r i d e The  (25 g ) , i n methanol (260 ml) (3.5 g) o v e r 1 V 2  s o l u t i o n was warmed at 40° f o r 1 hour, a c i d i f i e d w i t h  concentrated  h y d r o c h l o r i c a c i d , and then b a s i f i e d w i t h 2 N. sodium h y d r o x i d e . o r g a n i c s o l v e n t was evaporated aqueous s u s p e n s i o n ,  hours.  The  under reduced p r e s s u r e and t h e r e s i d u e , an  e x t r a c t e d thoroughly with chloroform.  A f t e r drying  -48-  (anhydrous  sodium s u l p h a t e ) the c h l o r o f o r m s o l u t i o n was  vacuum, and the product (40-60°) to y i e l d  r e c r y s t a l l i z e d from e t h e r and  the a l c o h o l  (24 g ) , m.p.  72°  3-Benzyloxy-4-methoxybenzyl c h l o r i d e  Thionyl  chloride  (38 ml)  was  evaporated under  light  petroleum-ether  ( l i t e r a t u r e m.p.  72-73°).  (51)  added dropwise  d u r i n g 30 minutes t o a  r a p i d l y s t i r r e d s u s p e n s i o n o f 3-benzyloxy-4-methoxybenzyl a l c o h o l e t h e r (150 m l ) .  A f t e r a f u r t h e r 30 minutes,  r a t e d under reduced p r e s s u r e .  The  r a t i o n o f the o r g a n i c s o l v e n t which was  24 g o f the r e q u i r e d  72-73°).  cyanide  On  c h l o r i d e was  evapo-  obtained,  (80-110°) and e t h e r , m.p.  (15 g) was  stirred  (54)  f o r 15 minutes with d i m e t h y l -  s u l p h o x i d e (500 ml), and then 3-benzyloxy-4-methoxybenzyl c h l o r i d e was  added t o the s o l u t i o n .  room temperature, extracted  (anhydrous  200 ml o f water was  time with water (100 ml).  magnesium s u l p h a t e ) e x t r a c t s  Calc.  79.5-80.5 ) .  for Ci H 6  1 5  Anal.  N 0 : 75.8%; H, 2  g)  at  added and the aqueous s o l u t i o n  r e c r y s t a l l i z e d from c h l o r o f o r m - p e t r o l e u m t u r e m.p.  (40  A f t e r s t i r r i n g the s o l u t i o n f o r 6 hours  s i x times with e t h e r - p e t r o l e u m - e t h e r  washing each  72°  8 6  3-Benzyloxy-4-methoxyphenylacetonitrile  Potassium  evapo-  d i s s o l v e d i n petroleum-  and f i l t e r e d by g r a v i t y .  r e c r y s t a l l i z e d from p e t r o l e u m - e t h e r  ( l i t e r a t u r e m.p.  (21 g) i n  the c l e a r s o l u t i o n was  crude product was  e t h e r (80-110°), p u r i f i e d ( c h a r c o a l ) ,  8 6  (80-110°) (1:1 by  volume),  E v a p o r a t i o n o f the combined, d r i e d gave 28 gm o f the n i t r i l e . e t h e r (30-60°). m.p.  Found: C, 75.31%; H, 5.93%; N, 5.51%.  The  78°  5.33%; N,  I t was (litera-  5.18^.  i n f r a r e d spectrum i n  -49-  c h l o r o f o r m had the c h a r a c t e r i s t i c n i t r i l e (C=N  a b s o r p t i o n band at 2280 cm ^  stretching),  3-Benzyloxy-4-methoxy-8-nitrostyrene  12 g o f O - b e n z y l i s o v a n i l l i n  was  (50)  dissolved  i n 400 ml o f 95% e t h a n o l at  room temperature  and the s o l u t i o n then c o o l e d t o 5-10°, a f t e r which 6 g o f  nitromethane was  added.  dissolved 5-10°, was  Then a s o l u t i o n o f 5 g o f sodium  i n the minimum amount o f water  i n 100 ml o f e t h a n o l , c o o l e d t o  added from a d r o p p i n g f u n n e l at a r a t e o f 5 ml p e r  The s o l u t i o n o f the nitromethane and O - b e n z y l i s o v a n i l l i n vigorously  hydroxide  stirred  i n alcohol  and kept below 15° d u r i n g the a d d i t i o n  sodium h y d r o x i d e .  As the r e a c t i o n proceeded,  o f the c o n d e n s a t i o n product p r e c i p i t a t e d .  minute.  o f the  the i n s o l u b l e  was  alcoholic  sodium  salt  A f t e r a l l o f the a l k a l i had been  added and w i t h the temperature kept below 15°, i c e water was s l o w l y added u n t i l the p r e c i p i t a t e d i s s o l v e d . The c l e a r c o l d s o l u t i o n was added i n a f i n e stream through a f u n n e l t o a s t i r r e d s o l u t i o n o f 60 ml o f concentrated hydrochloric c o n t r o l the temperature was  immediately  formed  a c i d i n 90 ml o f water. d u r i n g the a d d i t i o n .  No attempt was  made t o  .A f i n e , y e l l o w p r e c i p i t a t e  and a f t e r s t a n d i n g f o r V 2  hour was  f i l t e r e d with  suction m.p.  and then washed w i t h e t h a n o l . 8 g o f n i t r o s t y r e n e was o b t a i n e d , 82 127°. ( l i t e r a t u r e m.p. 127-128°). The p r o d u c t thus formed was  q u i t e pure and was without f  f u r t h e r p u r i f i c a t i o n by r e c r y s t a l l i z a t i o n ^  used i n the next s t e p . 5-Benzyloxy-4-methoxyphenethylamine  (53)  A s o l u t i o n o f 3-benzyloxy-4-methoxy-B-nitrostyrene anhydrous  t e t r a h y d r o f u r a n (200 ml) was  (10.5  g) i n  added t o a s u s p e n s i o n o f l i t h i u m  -50aluminium h y d r i d e was observed. (  (10 g) i n the same s o l v e n t (200 ml).  reaction  A f t e r t h e mixture had been heated under r e f l u x f o r f o u r days,  i t was t r e a t e d with i c e c o l d water (25 m l ) , s t i r r e d the f i l t e r - p a d b e i n g washed twice w i t h e t h e r . t i o n s were evaporated, acid.  A vigorous  f o r 2 hours and f i l t e r e d ,  The combined o r g a n i c s o l u -  and the r e s i d u e was d i s s o l v e d i n 10% h y d r o c h l o r i c  The a c i d i c s o l u t i o n was e x t r a c t e d with e t h e r , b a s i f i e d and e x t r a c t e d  t h r e e times with c h l o r o f o r m (400 m l ) .  E v a p o r a t i o n o f the d r i e d  sodium s u l p h a t e ) c h l o r o f o r m e x t r a c t s l e f t  (anhydrous  a r e s i d u e (5 g ) , which was d i s -  s o l v e d i n anhydrous e t h e r and t r e a t e d w i t h d r y hydrogen c h l o r i d e i n t h e same s o l v e n t .  R e c r y s t a l l i z a t i o n o f the b u l k y p r e c i p i t a t e  from e t h a n o l -  e t h e r y i e l d e d the r e q u i r e d amine h y d r o c h l o r i d e (5 g) m.p. 163-165°  (lit-  82 e r a t u r e m.p.  162-166°).  T h i s amine was a l s o p r e p a r e d by another method, as f o l l o w s . of 3-benzyloxy-4-methoxyphenylacetonitrile  (4 g) i n anhydrous e t h e r (150 ml)  was added t o a s t i r r e d s o l u t i o n o f l i t h i u m aluminium h y d r i d e (100 m l ) .  A solution  (5 g) i n e t h e r  A f t e r the mixture had been heated under r e f l u x f o r 5 hours, i t  was c o o l e d , t r e a t e d w i t h i c e water, s t i r r e d e t h e r e a l s o l u t i o n was evaporated  f o r 1 hour and f i l t e r e d .  The  under reduced p r e s s u r e , and t h e r e s i d u e  was d i s s o l v e d i n 10% h y d r o c h l o r i c a c i d .  The a c i d i c s o l u t i o n was e x t r a c t e d  with e t h e r , b a s i f i e d and e x t r a c t e d t h r e e times with c h l o r o f o r m .  Evapora-  t i o n o f t h e dry (anhydrous sodium s u l p h a t e ) c h l o r o f o r m e x t r a c t s y i e l d e d the amine (1.8 g ) . The h y d r o c h l o r i d e was r e c r y s t a l l i z e d from e t h a n o l - e t h e r t o g i v e c r y s t a l s , i d e n t i c a l w i t h t h e product s i g n a l s : g i v e n i n T u n i t s , spectrum plet  from t h e o t h e r r o u t e ,  n.m.r.  obtained i n deuterochloroform:  multi-  c e n t e r e d a t 2.67 (aromatic H o f 0-CH -C6H , a r e a 5 H), m u l t i p l e t 2  5  c e n t e r e d at 3.2 (aromatic H o f t r i s u b s t i t u t e d benzene, a r e a 3 H ) , s i n g l e t c e n t e r e d at 4.94 (methylene  o f -O-CT^-CgHs,area 2 H), s i n g l e t  c e n t e r e d at  -516.22  (methyl H o f -0-CH , a r e a 3 H), m u l t i p l e t c e n t e r e d at 6.95  ethylamine s i d e c h a i n , a r e a 4H) . (m), 940  (H o f phenyl-  3  1530  ( s ) , 1460  (m), 860  ( s ) , 1375  (m), 812  I n f r a r e d spectrum  ( s ) , 1275  ( s ) , 750  (m)  735  ( s ) , 1238 (s) 706  (m)  i n N u j o l : 2990 (s), 1620  ( s ) , 1150 and 695  ( s ) , 1030 ( s ) , (m)  4-(3-Benzyloxy-4-methoxyben2ylidene)-2-phenyloxazolone  The oxazolone was hippuric acid anhydride  (14.5  (50 ml)  cm , -1  (49)  o b t a i n e d by h e a t i n g O - b e n z y l i s o v a n i l l i n  g) , anhydrous sodium a c e t a t e (A.R., 8.5  at 100°  f o r 2 hours.  The  (20 g ) ,  g) and  crude product was  acetic  mixed with  a l c o h o l , and the y e l l o w s o l i d f i l t e r e d by s u c t i o n and washed w i t h much b o i l i n g water (1 l i t e r ) .  The oxazolone c r y s t a l l i z e d from a c e t i c a c i d i n 82  y e l l o w p r i s m a t i c needles  (16 g ) . m.p.  154°  The substance i s s p a r i n g l y s o l u b l e i n hot 3-Benzyloxy-4-methoxyphenylacetic  A mixture o f the oxazolone was  ( l i t e r a t u r e m.p.  155°).  alcohol.  Acid  (55)  (35 g) and 10% sodium h y d r o x i d e  r e f l u x e d under n i t r o g e n u n t i l  (200  ml)  e v o l u t i o n o f ammonia ceased (8 h o u r s ) ,  then s a t u r a t e d with carbon d i o x i d e (pH 8-8.5) (4 h o u r s ) , and c o o l e d to 5 ° . Then 6% aqueous hydrogen the temperature  p e r o x i d e was  added (25 ml) at such a r a t e t h a t  o f the r e a c t i o n mixture d i d not r i s e 5° ( 1 h o u r ) .  s t o r a g e a t 0° f o r 24 hours the mixture was  After  a c i d i f i e d with c o n c e n t r a t e d  h y d r o c h l o r i c a c i d , and the p r e c i p i t a t e d gummy a c i d s were e x h a u s t i v e l y e x t r a c t e d with  chloroform.  The e x t r a c t s , when washed, d r i e d  (anhydrous  sodium s u l p h a t e ) , and evaporated under, reduced p r e s s u r e , a f f o r d e d an o i l (34 g) which was  chromatographed on s i l i c a  g e l (B.D.H.) (600 g ) .  The  size  -52-  o f the column was  27 x 5 cm diameter.  The m a t e r i a l was  added t o the top  o f the column by d i s s o l v i n g i t i n the minimum amount o f benzene.  On  elution  the f o l l o w i n g f r a c t i o n s were o b t a i n e d c o n s e c u t i v e l y : (A)  3000 ml o f benzene removed no m a t e r i a l  (B)  1000  ml o f benzene-ether  (10%) y i e l d e d n o t h i n g .  (C)  1000  ml o f benzene-ether  (10%)  gave a s m a l l amount o f s o l i d  (10%)  e l u t e d b e n z o i c a c i d , which  material (D)  2000 ml o f benzene-ether  gave a m e l t i n g p o i n t o f 122° undepressed an a u t h e n t i c sample o f b e n z o i c  acid.  (E)  1000  eluted o i l s  (F)  2500 ml o f benzene-ether  ml o f benzene-ether  4-methoxyphenylacetic The  on admixture  last  (10%)  i n two  (10%) y i e l d e d 5 g o f  with  fractions.  3-benzyloxy-  acid.  f r a c t i o n s e l u t e d from the column were o i l s ,  and because o f  the l a r g e amount o f s o l v e n t i n v o l v e d , f u r t h e r development o f the chromatog r a p h i c column was  abandoned.  The  a c i d o b t a i n e d was  recrystallized  twice  82 from benzene, The follows.  m.p.  122-124°.  r e q u i r e d a c i d was  ( l i t e r a t u r e m.p.  125°),  p r e p a r e d more r e a d i l y and i n b e t t e r y i e l d s ,  A s o l u t i o n of 3-benzyloxy-4-methoxyphenylacetonitrile  ethylene g l y c o l  (300 ml)  13 hours w i t h potassium  and water (80 ml) was hydroxide  (8 g ) .  s o l u t i o n w i t h water (200 m l ) , i t was  (15 g) i n  heated under r e f l u x f o r  A f t e r d i l u t i o n o f the c o o l e d  e x t r a c t e d twice with e t h e r , then  a c i d i f i e d w i t h c o n c e n t r a t e d h y d r o c h l o r i c a c i d and e x t r a c t e d again with ether.  The  combined second  phenylacetic acid  s e t o f e x t r a c t s gave 3-benzyloxy-4-methoxy-  (11 g) which was  c r y s t a l s i d e n t i c a l w i t h the product C, 71.06%; H,  5.96%.  r e c r y s t a l l i z e d from benzene t o g i v e from the o t h e r r o u t e .  C a l c . f o r C ^ H ^ O ^ : C, 70.70%; H,  A n a l . Found:  5.83%.  as  -53-  I n f r a r e d spectrum o f the c r y s t a l s showed the f o l l o w i n g major t i o n s i n N u j o l : 2990 (s) , 1720.(s), 1260 745  absorp-  1605 (w), 1530 (m) , 1460 ( s ) , 1380 ( s ) ,  (m), 1220 (m), 1160 (m), 1140 (m), 1010 (m), 860 (w), 815 (w), 780 (w), (m), and 695 (w) cm  The n.m.r. s i g n a l s : g i v e n i n T u n i t s ,  obtained i n deuterochloroform: plet  singlet  spectrum  c e n t e r e d a t -1.4 (H o f COOH), m u l t i -  c e n t e r e d at 2.65 (aromatic H o f O-CH2C5H5, a r e a 5 H), m u l t i p l e t  c e n t e r e d at 3.19 (aromatic H o f t r i s u b s t i t u t e d benzene, a r e a 3 H), s i n g l e t c e n t e r e d at 4.9 (methylene H o f 0-CH -C H , area 2 H), s i n g l e t 2  e  5  at 6.2 (methyl H o f 0-CH , a r e a 3 H ) , s i n g l e t 3  centered  c e n t e r e d at 6.5 (methylene  H o f -CH -C00H, a r e a 2 H ) . 2  3~Benzyloxy-N-(3-benzyloxy-4-methoxyphenethyl )-4-methoxyphenylacetamide (56)  3-Benzyloxy-4-methoxyphenylacetic a c i d  (600 mg) i n d r y c h l o r o f o r m  (50 ml) was added p o r t i o n w i s e t o 3.5 ml o f t h i o n y l c h l o r i d e , and the r e a c t i o n mixture hour.  was then allowed t o s t a n d on a water-bath  The s o l v e n t and t h e excess  diminished pressure.  (45°) f o r one  o f t h i o n y l c h l o r i d e were evaporated  under  The r e s i d u a l a c i d c h l o r i d e s o l i d i f i e d and was used  immediately. The  a c i d c h l o r i d e (from 600 mg o f a c i d ) i n d r y t e t r a h y d r o f u r a n (40 ml)  was added dropwise d u r i n g 1 hour t o a s t i r r e d s o l u t i o n o f 3-benzyloxy-4methoxyphenethylamine h y d r o c h l o r i d e (800 mg) i n t e t r a h y d r o f u r a n (50 ml) and  aqueous sodium h y d r o x i d e  (0.25 g i n 1 ml).  A f t e r an a d d i t i o n a l  hour the t e t r a h y d r o f u r a n was removed under reduced  pressure,  V2  The r e s i d u e  was taken up i n c h l o r o f o r m , washed s u c c e s s i v e l y w i t h d i l u t e h y d r o c h l o r i c a c i d , aqueous sodium b i c a r b o n a t e  and water, d r i e d : ( a n h y d r o u s  magnesium  -54-  s u l p h a t e ) , and evaporated  under  s t a n d i n g at room temperature.  vacuum t o g i v e an o i l which s o l i d i f i e d on Recrystallization  from e t h y l a c e t a t e gave 82  the amide (900 mg) m.p. 116° ( l i t e r a t u r e m.p. 118°. C a l c . f o r C32H33NO5: N, 2.74%.  A n a l . Found: N, 2.73%,  I n f r a r e d spectrum i n N u j o l : 3350 (m) (N-H),  2950 ( s ) , 1650 ( s ) , 1600 (m), 1525 ( s ) , 1460 ( s ) , 1380 ( s ) , 1260 ( s ) , 1240 (s),  1160 (w), 1140 ( s ) , 1080 (w), 1020 ( s ) , 940 (w), 855 (w), 813 (m), 780  (w),  742 (m), 725 (m), and 700 (s) cm . 1  spectrum o b t a i n e d i n d e u t e r o c h l o r o f o r m :  n.m.r. s i g n a l s ; g i v e n i n x u n i t s , multiplet  (aromatic H o f -O-CH2-C5H5, a r e a 10 H), s i n g l e t  c e n t e r e d at 2.65  c e n t e r e d a t 3.23  (aromatic  H o f t r i s u b s t i t u t e d benzene, a r e a 6 H), s i n g l e t c e n t e r e d a t 4.95 (methylene H o f -0-CH -C H , a r e a 4 H), s i n g l e t 2  6  5  are 6 H ) , m u l t i p l e t  c e n t e r e d at 6.2 (methyl H o f -O-CH3,  c e n t e r e d a t 6.5 (methylene H o f CH -C=0 and methylene 2  H o f -N-CH -, area 4 H), t r i p l e t 2  c e n t e r e d a t 7.25 (6 methylene H o f p h e n e t h y l -  amine s i d e c h a i n , a r e a 2 H).  Pi-(B-3-benzyloxy-4-methoxyphenyl)-ethylamine  (57)  To a s o l u t i o n o f 3-benzyloxy-N-(3-benzyloxy-4-methoxyphenethyl)-4methoxyphenylacetamide (258 mg) i n d r y t e t r a h y d r o f u r a n (50 ml) i n a 100 ml flask  ( n i t r o g e n atmosphere) was added 10 ml o f 1 M borane i n t e t r a h y d r o 94  f u r a n over 20 minutes. 0° d u r i n g t h e a d d i t i o n . r e f l u x f o r 8 hours,  The temperature was m a i n t a i n e d  A f t e r the r e a c t i o n mixture had been heated  distillation.  under  i t was c o o l e d t o room temperature and 2 ml o f d i l u t e  h y d r o c h l o r i c a c i d was added. by  at approximately  The t e t r a h y d r o f u r a n  Sodium h y d r o x i d e  was removed  p e l l e t s were added t o s a t u r a t e  the aqueous phase and the l a t t e r was e x t r a c t e d t h r e e times with a t o t a l o f 100  ml o f e t h e r .  The combined e t h e r e x t r a c t s were d r i e d  (anhydrous sodium  -55-  s u l p h a t e ) and evaporated. chloride  (250 mg)  product was  The  residual  amine was  c o n v e r t e d i n t o i t s hydro-  with dry e t h e r e a l hydrogen c h l o r i d e , m.p.  130°.  o b t a i n e d as the c r y s t a l l i n e amine h y d r o c h l o r i d e by  The  recrystalli-  z a t i o n from e t h a n o l - e t h e r and d r y i n g at 100° under vacuum f o r f i v e m.p.  164-167°.  C32H36O4NCI:  A n a l . Found: C,  C,  72.1%; H,  72.13%; H,  6.76%; N,  2950 ( s ) , 2480 (w) , 1600  (w) , 1530  (s),  1030  1150  ( s ) , 1085 735  (w),  (s)  and 700  I n f r a r e d spectrum  (s) , 1460 (m),  cm ,  (s) , 1380 880  (m),  spectrum  obtained i n deuterochloroform: m u l i t p l e t  -1  i n Nujol:  (s) , 1285  (w), 858  (w), 752  (aromatic H o f - O - C H 2 - C 6 H 5 ,  (m)  c e n t e r e d at  c e n t e r e d at 4.94  5  a r e a 6 H), s i n g l e t  (broad)  (m),  775  2.65  H o f t r i s u b s t i t u t e d benzene, a r e a 6 H), s i n g l e t 6  1240  n.m.r. s i g n a l s ; g i v e n i n T u n i t s ,  c e n t e r e d at 3.24  2  (s) ,  (w), 814  a r e a 10 H), s i n g l e t  H o f -0-CH -C H , a r e a 4 H), s i n g l e t  hours,  6.83%; N, 2.47%, C a l c . f o r  2.63%.  ( s ) , 990  final  c e n t e r e d at 6.2  c e n t e r e d at 6.88  (aromatic (methylene  (methyl H o f -0-CH , 3  (H o f phenylethylamine  side  c h a i n , a r e a 8 H).  Pi-(g-3-hydroxy-4-methoxyphenyl)-ethylamine  The  corresponding dibenzyl ether,  ethylamine  h y d r o c h l o r i d e (56 mg)  was  (25 mg),  Filtration  Di-(3-3-benzyloxy-4-methoxyphenyl)-  hydrogenolysed  containing concentrated h y d r o c h l o r i c a c i d charcoal  (0.1 ml)  and  10%  (10  ml)  palladised minutes.  o f t h e ' s o l u t i o n and e v a p o r a t i o n o f the s o l v e n t gave d i - ( 3 - 3 -  from e t h a n o l . m.p.  follows.  i n methanol  the hydrogen uptake b e i n g complete i n 30  hydroxy-4-methoxyphenyl)ethylamine h y d r o c h l o r i d e .  The  (58)  amine was  I t was  recrystallized  230°. a l s o p r e p a r e d on a l a r g e r s c a l e by another method, as  A s o l u t i o n of 3-benzyloxy-4-methoxyphenylacetonitrile  (3 g) i n  -56-  methanol  (250 ml) was  p l a c e d i n t o a h i g h p r e s s u r e bomb, with 1.5  g of f r e s h l y  95 prepared  a c t i v e Raney-Nickel  the bomb (1100 8 hours.  catalyst.  The hydrogen gas was  p . s . i . ) and the mixture heated  The bomb was  contents were removed.  r e a c t i o n p r o d u c t was  ammoniacal methanol s o l u t i o n evaporated.  The  i n the same s o l v e n t .  The  opened and then  then f i l t e r e d  r e s i d u e was  a b s o l u t e e t h a n o l and dry h y d r o c h l o r i c a c i d was solution  into  at 90° w i t h s t i r r r i n g f o r  allowed t o c o o l b e f o r e i t was The  passed  and  the  the  dissolved i n  added t o the e t h a n o l i c  amine h y d r o c h l o r i d e ( l g ) c r y s t a l l i z e d  o v e r n i g h t at 0° and a f t e r r e c r y s t a l l i z a t i o n from a b s o l u t e e t h a n o l i t was found t o be i d e n t i c a l  (mixed m.p.,  the amide r o u t e above. f o r CigHji+O^NCl:  I.R.,  A n a l . Found: C, 61.08%; H,  C, 61.3%; H,  6.8%;  N,  3550 (m),  2950 ( s ) , 2500 (w), 1600  (m)  (m),  (w),  1158  870  1265  ( s ) , 812  1230  i n T u n i t s , spectrum 3.36  (aromatic H,  singlet  ( s ) , 1205  ( s ) , 768  (w), 755  (w), (w)  3.96%. 1460  6.7%;  I n f r a r e d spectrum  ( s ) , , 1380  (m),  and 725  N, 3.49%.  1132  (w)  ( s ) , 1340  (m),  cm" , 1  1025  a r e a 6 H), s i n g l e t  c e n t e r e d a t 4.47  mg,  1.73  (m),  1300 (w), given  c e n t e r e d at area 2 H),  (broad)  3  s i d e c h a i n , a r e a 8 H).  (59)  A s o l u t i o n o f di-(6-3-hydroxy-4-methoxyphenyl)-ethylamine  capillary  i n Nujol:  ( p h e n o l i c H,  3,15-Dimethoxy-16-hydroxy-2-oxoerythrina-l(6),3-diene  (150 mg,  Calc.  n.m.r. s i g n a l s ;  (methyl H o f -0-CH , a r e a 6 H), t r i p l e t  (H o f phenylethylamine  from  ( s ) , 955  obtained i n deuterochloroform: m u l t i p l e t  c e n t e r e d at 6.2  c e n t e r e d at 7.25  n.m.r.) w i t h t h a t o b t a i n e d  0.424 mMole) i n d i s t i l l e d water (900 ml) was  added through  t o a v i g o r o u s l y s t i r r e d s o l u t i o n o f potassium mMole) i n 100 ml o f d i s t i l l e d water and 240  hydrochloride  ferricyanide  ml o f 1 N.  b i c a r b o n a t e , under n i t r o g e n atmosphere, d u r i n g 6 h o u r s .  a fine  The  (570  sodium  reaction  -57-  mixture was (6 x 200  stirred  ml).  The  f o r another  t h r e e hours,  residue  The  experiment was  (1.140 g) was  p r e s s u r e y i e l d e d a brown  repeated t e n times  The  and  chromatographed on alumina  n e u t r a l i z e d with e t h y l a c e t a t e , d r i e d at 100° wa t e r ) .  chloroform  combined e x t r a c t s , a f t e r d r y i n g (anhydrous sodium s u l p h a t e ) ,  on e v a p o r a t i o n o f the s o l v e n t under reduced (110 mg).  then e x t r a c t e d with  m a t e r i a l was  the combined gummy  (30 g)  and  gum  (Shawinigan  reagent,  then d e a c t i v a t e d with~6%  i n t r o d u c e d i n 20 ml o f c h l o r o f o r m i n t o the column  ( s i z e 13 x 2 cm d i a m e t e r ) , and on e l u t i o n the f o l l o w i n g f r a c t i o n s were obtained consecutively: (A)  50 ml o f c h l o r o f o r m removed 61 mg  (B)  90 ml o f c h l o r o f o r m gave 250  (C)  525  ml o f c h l o r o f o r m y i e l d e d 165 mg  (D)  250  ml o f c h l o r o f o r m - e t h a n o l  mg  of colourless o i l .  of c r y s t a l l i n e  (4:1)  of  solid.  oil.  e l u t e d 145 mg  of o i l  material. The  total  r e c o v e r y o f o r g a n i c m a t e r i a l was  The  c r y s t a l s o b t a i n e d from f r a c t i o n  (12:5:2) (R =0.29).  g e l ( S t a h l G)  T h i s compound was  f  mg.  (B) were r e c r y s t a l l i z e d  e t h a n o l to g i v e an a n a l y t i c a l sample, m.p. spot i n T.L.C. on s i l i c a  621  224-229° with decomposition.  i n n-butanol-water-acetic  (225 mg,  (determined  A n a l . Found: C, 69.21%; H,  4.45%.  Calc. f o r C  1 8  H  1 9  N 0 : C, 69.02%; H, H  U l t r a v i o l e t spectrum i n e t h a n o l : ^ (e 4150). ( s ) , 1595 1175  m a x  15%).  238  my  6.19%, N,  (e 19370) and 1690  1505  ( s ) , 1460  ( s ) , 1380  ( s ) , 1320  (w),  ( s ) , 1130  (w),  1100  1070  1030  975  (m),  313  4.46%.  (m),  (w),  acid  M o l e c u l a r weight  6.06%; N,  I n f r a r e d spectrum i n N u j o l : 3500 (m),  (w),  One  i d e n t i f i e d as 3,15-dimethoxy-16-  hydroxy-2-oxoerythrina-l(6),3-diene by mass s p e c t r o m e t r y ) .  from,  283  ( s ) , 1665 1265  (s),  (m),  (w), 915  1200  my 1630 (s),  (w), 885  (m),  of  -58-  845  (m), 812 (w), 782  u n i t s , spectrum  (m) and 723- (w) cm ^.  obtained i n deuterochloroform:  (aromatic H, a r e a IH), s i n g l e t triplet  n.m.r. s i g n a l s ; g i v e n i n x singlet  c e n t e r e d at 3.36  c e n t e r e d at 3.62 (aromatic H, a r e a 1 H),  c e n t e r e d at 3.72 (J = 1.5 c.p.s) ( v i n y l H, a r e a 1 H), broad  c e n t e r e d at 3.79 which d i s a p p e a r e d on a d d i t i o n o f D 0 2  singlet  singlet  ( h y d r o x y l H, area 1 H),  c e n t e r e d at 3.99 ( v i n y l H, area 1 H), s i n g l e t s c e n t e r e d at 6.28  and 6.38 (methyl H o f -O-CH3, area 3 H r e s p e c t i v e l y ) and broad  multiplets  c e n t e r e d at 6.75 and 7.38 ( a l i p h a t i c p r o t o n s o f r i n g B and C o f e r y t h r i n a n e s k e l e t o n , area 4 H r e s p e c t i v e l y ) . Mass spectrum 298 210,  showed s i g n i f i c a n t peaks a t m/e = 313 ( M ) , 312 ( M - l ) , +  (M-15), 285 (M-28), 283 (M-30), 282 (M-31), 269, 254, 241, 238, 226, 198, 176, and 170.  3,15-Dimethyoxy-16-acetoxy-2-oxoerythrina-l(6),3-diene  (60)  6 mg o f 3,15-dimethoxy-16-hydroxy-2-oxoerythrina-l(6),3-diene a c e t y l a t e d , w i t h 3 ml o f a c e t i c anhydride pyridine  (reagent) a t room temperature  was  (reagent) and 3 ml o f d r y  overnight.  The excess o f reagent  and t h e p y r i d i n e was removed under reduced p r e s s u r e on the steam b a t h . The  r e s i d u e was d i s s o l v e d i n 10 ml o f c h l o r o f o r m , washed twice with water  and d r i e d  (anhydrous sodium s u l p h a t e ) .  E v a p o r a t i o n o f the c h l o r o f o r m  s o l u t i o n under reduced p r e s s u r e gave 7 mg o f an o i l y m a t e r i a l which f i e d on s t a n d i n g at room temperature.  I n f r a r e d spectrum  solidi-  i n N u j o l : 1780  (s),  1690 ( s ) , 1630 ( s ) , 1600 ( s h o u l d e r ) , 1510 ( s ) , 1465 ( s ) , 1380 ( s ) ,  1260  ( s ) , 1205 ( s ) , 1180 ( s ) , 1100 (m), 1080 (m), 980 (w)  cm  U l t r a v i o l e t spectrum r  gave one spot on s i l i c a  and 900 (w)  i n ethanol: A 214, 234, 285 mu. T.L.C. max g e l . R = 0.18 i n n - b u t a n o l - w a t e r - a c e t i c a c i d f  -59(12:5:2) .  3,15-Dimethoxy-2,16-dihydroxyerythrina-1(6),5-diene  To a s o l u t i o n o f (115 mg)  3,15-dimethoxy-16-hydroxy-2-exoerythrina-l(6),3-diene  i n e t h a n o l (20 ml) was  r e a c t i o n mixture s t i r r e d s o l u t i o n was added.  added sodium b o r o h y d r i d e (70 mg),  f o r 6 hours at room temperature.  The  and the  ethanolic  then c o n c e n t r a t e d under reduced p r e s s u r e and 40 ml o f water  The aqueous s o l u t i o n was  the combined e x t r a c t s  (150 ml)  on e v a p o r a t i o n y i e l d e d 102 mg on alumina  (61)  (3 g ) .  m a t e r i a l was  e x t r a c t e d t h r e e times w i t h c h l o r o f o r m , and  a f t e r drying  (anhydrous  sodium  o f o i l y m a t e r i a l which was  The s i z e o f the column was  3.5  sulphate),  chromatographed  x 2 cm diameter.  The  i n t r o d u c e d i n t o the column i n 4 ml o f c h l o r o f o r m , and on  e l u t i o n the f o l l o w i n g f r a c t i o n s were o b t a i n e d c o n s e c u t i v e l y : 180 ml o f chloroform eluted a yellow o i l i n 3 f r a c t i o n s . then e l u t e d w i t h 50 ml o f c h l o r o f o r m (65 mg). e t h a n o l gave  a m.p.  166-169°..  butanol-water-acetic acid 3450 ( s ) , 1655 ( s ) , 1110 840  ( s ) , 1610  ( s ) , 1085  (w), 806  (s) and 770  ( s ) , 1505  (m)  required alcohol  I n f r a r e d spectrum ( s ) , 1450  ( s ) , 1020 cm .  ( s ) , 1375  ( s ) , 985  R= f  0.19  i n n-  i n N u j o l : 3510 ( s ) , ( s ) , 1260  (m), 920  U l t r a v i o l e t spectrum  -1  was  R e c r y s t a l l i z a t i o n from e t h e r -  T.L.C. gave one s p o t .  (12:5:2).  ( s ) , 1050  The  (s) , 1220  (m), 870  (m),  i n ethanol: X m 3.x  212, m/e 279,  240  ( s h o u l d e r ) , 287 my.  = 315 260,  Mass spectrum showed s i g n i f i c a n t peaks at  ( M ) , 316  (M + 1 ) , 300  259,  242,  +  251,  212,  (M-15), 297  199,  167,  149,  (M-18), 287 147,  129,  (M-28), 285  113 and 112.  n.m.r. spectrum i n d e u t e r o c h l o r o f o r m showed the expected methyl (-OCH3)  at T  6.28  and T 6.47,  aromatic p r o t o n s , a m u l t i p l e t  two s i n g l e t s at x 3.43 c e n t e r e d at x 4.24  The  resonances  and x 3.68  and a s i n g l e t  (M-30),  f o r the  at x  4.95  -60o l e f i n i c p r o t o n s , a m u l t i p l e t c e n t e r e d at x 5.16  f o r the two  f o r the  lone  p r o t o n on the carbon b e a r i n g the h y d r o x y l group i n r i n g A o f the e r y t h r i n a n e r i n g system, a broad s i n g l e t and was  at x 4.72  which d i s a p p e a r e d on a d d i t i o n o f D2O  a s s i g n e d t o one h y d r o x y l p r o t o n , and m u l t i p l e t s  to the a l i p h a t i c protons  i n r i n g s B and  3-Benzyloxy-4-methoxyphenyl[1Potassium  cyanide  (39.4  mg)  14  was  x 6-8  corresponding  C.  C]acetic Acid stirred  (70)  f o r 10 minutes with  dimethyl  14 sulphoxide  (3 ml)  and potassium[  C]cyanide  washed i n w i t h d i m e t h y l s u l p h o x i d e 4-methoxybenzyl c h l o r i d e at room temperature ether-petroleum was  (4 m l ) .  (185.5 mg)  f o r 6 hours.  (6.5 mg,  1.0  mc)  was  A f t e r 15 minutes,  added  3-benzyloxy-  was  added, and the s o l u t i o n was  I t was  shaken w i t h water (50 ml)  e t h e r (80-110°) (50 ml,  1:1  by volume) and  stirred and  the aqueous  f u r t h e r e x t r a c t e d t w i c e with the same s o l v e n t , washing each time  water (10 m l ) .  E v a p o r a t i o n o f the combined, d r i e d  phate) e x t r a c t s l e f t  3-benzyloxy-4-methoxyphenyl[1-  water  A l l the a c t i v e sample was (1 ml)  C]acetonitrile  12 hours.  d i s s o l v e d i n ethylene g l y c o l  and heated w i t h potassium  The  c o o l e d s o l u t i o n was  hydroxide  0.8  mc),  m.p.  123°.  mg),  (mixed and  (0.5 g) under r e f l u x f o r  e x t r a c t e d f o u r times 14  e t h e r and d r i e d t o a f f o r d 3-benzyloxy-4-methoxyphenyl[1(137 mg;  with  (151  (3 ml)  p a r t i t i o n e d between water and  the aqueous phase, a f t e r a c i d i f i c a t i o n , was  layer  (anhydrous sodium s u l 14  shown i n r a d i o - i n a c t i v e runs t o be i d e n t i c a l w i t h a u t h e n t i c m a t e r i a l m.p.).  and  e t h e r and with  C]acetic  acid  -61-  3-Hydroxy-4-methoxy-N-(3-hydroxy-4-methoxyphen[l-  C]ethyl)-phenethyl-  amine (72) . 14 3-Benzyloxy-4-methoxyphenyl[1steam bath f o r 30 minutes  C]acetic acid  with t h i o n y l c h l o r i d e  reagent was, evaporated under reduced p r e s s u r e . i n anhydrous e t h e r (10 ml) was  The mixture was  acetate  and t h e excess o f  i n anhydrous e t h e r 10 ml at (20 ml)  and  ethyl  e x t r a c t e d t w i c e with e t h y l  A f t e r the combined e x t r a c t s had been shaken w i t h an excess o f  aqueous p o t a s s i u m carbonate and yield  warmed on a  A s o l u t i o n o f the r e s i d u e  shaken with 2N h y d r o c h l o r i c a c i d  (60 m l ) , and the aqueous l a y e r was  acetate.  (3 ml)  was  added dropwise t o a s t i r r e d s o l u t i o n o f  3-benzyloxy-4-methoxyphenethylamine (417 mg) 0°.  (137 mg)  water, they were d r i e d and evaporated t o  3-benzyloxy-N-(3-benzyloxy-4-methoxyphenethyl)-4-methoxyphenyl14  [carbonyl-  Cjacetamide  as a s o l i d .  gave the amide (150 mg),  m.p.  R e c r y s t a l l i z a t i o n from e t h y l a c e t a t e  116°, s u i t a b l e f o r r e d u c t i o n .  o f the f o r e g o i n g amide i n dry t e t r a h y d r o f u r a n (50 ml) ( n i t r o g e n atmosphere) was over 20 minutes. the a d d i t i o n .  temperature was  i n a 100 ml  flask  i n tetrahydrofuran  m a i n t a i n e d at a p p r o x i m a t e l y 0° d u r i n g  A f t e r the r e a c t i o n mixture had been h e a t e d under r e f l u x f o r  6 h o u r s , i t was a c i d was  The  added 10 ml o f IM borane  To a s o l u t i o n  added.  c o o l e d t o room temperature The t e t r a h y d r o f u r a n was  and 2 ml o f d i l u t e h y d r o c h l o r i c  removed by d i s t i l l a t i o n at  atmospheric p r e s s u r e (steam bath) as hydrogen l y s i s o f excess r e a g e n t .  e v o l v e d from the hydro-  Sodium h y d r o x i d e p e l l e t t s were added t o s a t u r a t e  the aqueous phase and the l a t t e r was 100 ml o f e t h e r .  was  e x t r a c t e d t h r e e times w i t h a t o t a l  The combined e t h e r e x t r a c t s were d r i e d  s u l p h a t e ) and evaporated.  The  g e n o l y s i s i n methanol (10 ml)  residual  amine was  (anhydrous  d e b e n z y l a t e d by  containing concentrated hydrochloric  of  sodium hydroacid  -62-  (0.1  ml) and 10% p a l l a d i s e d c h a r c o a l  was complete i n 30 minutes.  (25 mg).  The hydrogen uptake  F i l t r a t i o n o f the s o l u t i o n and e v a p o r a t i o n o f 14  the s o l v e n t gave 3-hydroxy-4-methoxy-N-(3-hydroxy-4-methoxyphen[lphenethyiamine h y d r o c h l o r i d e which was r e c r y s t a l l i z e d (60 mg; 0.08 mc). an a u t h e n t i c sample  1  C]ethyl-  from e t h a n o l m.p.  I t was shown i n r a d i o - i n a c t i v e runs t o be i d e n t i c a l (mixed m.p.).  230° with  -63-  BIBLIOGRAPHY  1.  Manske, R. H. F. "The A l k a l o i d s " V o l . I I . Academic P r e s s , New York. N.Y., 1952, Chapter 13.  2.  G r e s h o f f , M.  Ber. 23, 3537 (1890).  3.  G r e s h o f f , M.  Ber. deut. phar.. Ges. £ , 215 (1899)  4.  C h a k r a v a r t i , S. 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Communications No.3 52 (1965).  and Co.,  (London),  Chem.  PART I I  AN ATTEMPTED IN VITRO DEMETHYLATION OF LANOSTEROL  -68-  INTRODUCTION  The, i n v e s t i g a t i o n o f the b i o g e n e s i s o f c h o l e s t e r o l (18) i s the most exhaustive  and thorough o f a l l the work which has been done on the b i o 12  3 4  s y n t h e s i s o f s t e r o i d s , and the t o p i c has been w e l l reviewed. ' ' ' knowledge o f t h i s c e n t r a l l y important and  i t s biogenesis The  illustrates  The  precursor of other s t e r o i d s i s v i t a l  a " g e n e r a l " mechanism f o r s t e r o l  f i r s t work which demonstrated the chemical  nature  formation.  of this bio-  5  s y n t h e s i s began over 20 years stances  that s t e r o i d a l  a r e c o n s t r u c t e d from numerous s m a l l m o l e c u l e s , 6  the source ^'^  ago when i t was found  o f the carbon atoms o f s t e r o l s  Bloch's^  corporated pattern.  i n yeast  sub-  and t h a t a c e t a t e i s 7  and i n animal  p i o n e e r i n g work showed t h a t a c e t i c a c i d molecules  tissue.  are i n -  i n t o the s t r u c t u r e o f c h o l e s t e r o l a c c o r d i n g t o a d e f i n i t e T h i s work took on even g r e a t e r i n t e r e s t w i t h the r e a l i z a t i o n  t h a t the b i o g e n e s i s o f c h o l e s t e r o l and s t e r o i d s i s a s m a l l p a r t o f a v a s t b i o s y n t h e t i c panorama t h a t now i n c l u d e s a l l terpenes stances.  4  Subsequent w o r k ,  1 1 , 1 2  '  1 3  '  1 4 , 1 5 , 1 6  and d e r i v e d sub-  which stands  as a b r i l l i a n t  achievement, has l o c a t e d the o r i g i n o f every carbon atom o f c h o l e s t e r o l i n e i t h e r the c a r b o x y l o r methyl carbon o f a c e t a t e , by s u i t a b l e degradat i o n s o f the s t e r o l and measurement o f the r e l a t i v e i s o t o p e i n c o r p o r a t i o n i n t o both n u c l e a r and s i d e - c h a i n m o i e t i e s . i n c h o l e s t e r o l can be d e r i v e d from a c e t a t e . low m o l e c u l a r  Thus, a l l o f the carbon atoms Besides  a c e t a t e , many o t h e r  weight compounds were t e s t e d f o r the p o s s i b i l i t y 17  incorporation into cholesterol. two carbon fragment, a c e t a t e .  for their  I t appeared t h a t the e f f i c i e n c y o f the  20 p r o p o r t i o n a l t o t h e i r a b i l i t y t o y i e l d the many suggested p r e c u r s18 o r s19was The o b s e r v a t i o n s ' ' t h a t h y d r o x y m e t h y l g l u t a r y l CoA (3) i s  -69-  s y r i t h e s i z e d from a c e t o a c e t y l CoA  (2) and  a c e t y l CoA  (1) and  c o u l d be  21 incorporated into cholesterol methylglutarate  (18),  (3) might be a key  r a i s e d the p o s s i b i l i t y  t h a t hydroxy-  intermediate.  The s e a r c h f o r i n t e r m e d i a t e s i n the t r a n s f o r m a t i o n o f a c e t a t e c h o l e s t e r o l met  with  i s o l a t e d mevalonic methylglutarate  little  success, u n t i l 22 23 24 (4). ' ' The  acid  ( 3 ) , which i s transformed 25  prompted i n v e s t i g a t i o n  1956  when the Merck group  obvious  s i m i l a r i t y to hydroxy21 into c h o l e s t e r o l only poorly,  o f the p o s s i b l e r o l e o f mevalonic  cholesterol biosynthesis.  The  results  into  acid  (4) i n  i n d i c a t e d t h a t t h i s compound  was  capable o f b e i n g i n c o r p o r a t e d i n t o c h o l e s t e r o l i n v e r y h i g h y i e l d . Sub26 sequent study showed that a c a r b o x y l group i s l o s t as carbon d i o x i d e e a r l y i n the sequence o f r e a c t i o n s and a l s o c o n v e r s i o n t o squalene (11) 27 with h i g h e f f i c i e n c y was  observed  under a n a e r o b i c  conditions.  28 '  This  i n d i c a t e d t h a t d e c a r b o x y l a t i o n o c c u r r e d , a p p a r e n t l y at the s i x carbon 29 atom l e v e l , to g i v e a f i v e carbon atom a c t i v e i n t e r m e d i a t e . Ferguson has e s t a b l i s h e d t h a t h y d r o x y m e t h y l g l u t a r y l CoA (3) can be c o n v e r t e d t o mevalonic  acid  (4) i n y e a s t and  f o r m a t i o n o f mevalonic excellent  acid  Knauss  30  has  r e c e n t l y demonstrated  (4) from a c e t a t e  the  (1) i n l i v e r t i s s u e , so  case f o r t h e i r i n t e r m e d i a c y has now  an  been made.  31 Tchen  working w i t h mevalonic  acid  (4) i n y e a s t p r e p a r a t i o n s  an enzyme which t r a n s f o r m s  the a c i d to 5-phosphomevalonic a c i d  a d d i t i o n he  was  ate  found  t h a t ATP  (5) t o squalene  (11).  necessary 32  Lynen  and  found  (5).  In  to t r a n s f o r m the 5-phosphomevalon33  Bloch  have d e s c r i b e d the f u r t h e r  r e a c t i o n s which c o n v e r t 5-phosphomevalonate (5) to 5-pyrophosphomevalonate (6) and  i s o p e n t e n y l pyrophosphate  (7),  evaded i s o l a t i o n f o r a l o n g time.  The  the i'soprenoid i n t e r m e d i a t e t h a t condensation  pyrophosphate ( 8 ) , an i s o m e r i z a t i o n p r o d u c t  of d i m e t h y l a l l y l  o f i s o p e n t e n y l pyrophosphate  -7034 (7), and i s o p e n t e n y l pyrophosphate (7) has been r e p o r t e d by Lynen to r e s u l t i n g e r a n y l pyrophosphate ( 9 ) . T h i s compound can then condense with an a d d i t i o n a l molecule f a r n e s y l pyrophosphate of  o f i s o p e n t e n y l pyrophosphate  (10). The r e d u c t i v e d i m e r i z a t i o n  f a r n e s y l pyrophosphate  (10), t a i l  to t a i l , 32  beendemdnstrated i n l i v e r and y e a s t . The  (7) t o y i e l d o f two molecules  t o y i e l d squalene  (11) has  35  '  i n c o r p o r a t i o n o f a c e t a t e i n t o terpenes  and t h e i r d e r i v a t i v e s ,  such  as s t e r o i d s , d i f f e r s a t an e a r l y stage from t h a t f o r the b i o s y n t h e s i s o f ^•cetogehins.  4  The acetogenins  by the a c e t a t e h y p o t h e s i s  i n c l u d e compounds b i o g e n e t i c a l l y d e r i v a b l e  and exclude t h e t e r p e n e s , which,  although  u l t i m a t e l y d e r i v e d from a c e t a t e , a r e themselves a homogeneous f a m i l y A r i s i n g from l i n e a r combination genins  of ispprenoid units.  Whereas t h e a c e t o -  a r e formed by a l i n e a r l i n k i n g o f a c e t a t e u n i t s , t h e terpenes a r e  generated  by c o n v e r s i o n o f a c e t a t e t o a branched-chain  plentenyl pyrophosphate  intermediate, i s o -  ( 7 ) , the b i o l o g i c a l isqprene u n i t .  r'eactiohs as now p o s t u l a t e d f o r t h e b i o g e n e s i s o f squalene in  F i g u r e s 1 and 2 (P = P 0  3  sequence p r i o r t o mevalonate  ).  The s e r i e s o f (11) a r e g i v e n  The i n t e r m e d i a t e s i n t h i s b i o s y n t h e t i c  (4) a r e capable o f i n t e r c o n v e r s i o n t o many  4 other substances.  However, the f o r m a t i o n o f mevalonate (4) i s an i r -  r e v e r s i b l e p r o c e s s , and mevalonate once formed has e s s e n t i a l l y o n l y one biochemical r o l e , the production o f i s o p r e n o i d substances.  Its discovery  was, t h e r e f o r e , one o f t h e important break-throughs i n terpene b i o s y n t h e s i s . As e a r l y as 1926 i t was s u g g e s t e d simply on t h e bases o f s t r u c t u r a l 3 5  s i m i l a r i t i e s , t h a t squalene The  e a r l y experiments  cholesterol  36  (11) i s b i o g e n e t i c a l l y r e l a t e d t o the s t e r o i d s .  d i d i n d i c a t e some involment  o f squalene  (18) b i o s y n t h e s i s , however , i t was not u n t i l  (11)  1953 t h a t  in  Bloch  37  -71-  CHCOSCoA  Figure 1. The Biosynthesis of isopentenyl Pyrophosphate (7).  Figure 2. Polymerization of Isopentenyl Pyrophosphate (7).  -73-  and  h i s collaborators  reinvestigated  a b l e t o demonstrate t h a t (18).  squalene  t h e matter u s i n g i s o t o p e s  and were  (11) i s indeed c o n v e r t e d i n t o  The c y c l i z a t i o n o f squalene  (11) t o l a n o s t e r o l  cholesterol  (13) (see F i g u r e 3)  i s o f p a r t i c u l a r . i n t e r e s t because o f t h e i n t e r m e d i a c y o f t h e l a t t e r i n t h e pathway o f s t e r o i d b i o g e n e s i s .  The r o l e o f squalene as an o b l i g a t o r y  pre-  c u r s o r o f c h o l e s t e r o l , and t h e mechanism o f i t s c o n v e r s i o n t o l a n o s t e r o l proposed on p u r e l y  s t r u c t u r a l and t h e o r e t i c a l grounds, now has f i r m  experi-  38 mental s u p p o r t . lanosterol  Woodward and Bloch  i n t h e i r suggested mechanism  (13) as an i n t e r m e d i a t e i n c h o l e s t e r o l b i o s y n t h e s i s ,  proposal i s consistent  implicated  and t h e i r  with a l l o f the isotope  d i s t r i b u t i o n d a t a and enzy4 m o l i g i c a l evide.nce t h a t has been o b t a i n e d so f a r . The c o n v e r s i o n s o f squa39 40 lene (11) t o l a n o s t e r o l (13) and o f l a n o s t e r o l (13) t o c h o l e s t e r o l (18) haye been demonstrated.  Very i n t e r e s t i n g l y , the f o r m a t i o n o f squalene from  i t s p r e c u r s o r s does n o t r e q u i r e lanosterol  and c h o l e s t e r o l  oxygen,  41  the c y c l i z a t i o n o f squalene t o 42 oxygen.. Squalene (11) c y c l i s e s  does r e q u i r e  '  c o n c e r t e d l y by a p r o c e s s i n i t i a t e d ; by atmospheric oxygen p r o b a b l y as the HO c a t i o n on C-3  42 43 ' t o give the h y p o t h e t i c a l  intermediate  @  (12) , which can  44 45 undergo two 1,2-methyl s h i f t s  '  to y i e l d  With t h e attainment o f l a n o s t e r o l Structural  feature  l a n o s t e r o l (13).  (13), t h e b i o s y n t h e s i s  o f the  most c h a r a c t e r i s t i c o f t h e s t e r o i d s , the c y c l o p e n t a n o -  perhydrophenanthrene s k e l e t o n , mation o f l a n o s t e r o l  has been a c h i e v e d .  (13) t o c h o l e s t e r o l  For the f i n a l  transfor-  (18) t h e r e remains o n l y the removal  o f t h r e e methyl groups and a p p r o p r i a t e a l t e r a t i o n o f t h e two o l e f i n i c :j.n t h e l a n o s t e r o l involved  There a r e undoubtedly many enzymatic  reactions  i n these t r a n s f o r m a t i o n s , but t o date o n l y some o f them have been  elucidated sterol  (13).  centers  in detail.  One o f t h e two pathways by which t h e changes i n l a n o -  (13) are e f f e c t e d has as i t s t e r m i n a l  step the s a t u r a t i o n  o f the  -74-  s i d e c h a i n double bond  (see F i g u r e 3 ) .  The  r e d u c t i o n of the s i d e - c h a i n double bond. possibility  a l t e r n a t e route  Some steps i n the c o n v e r s i o n o f l a n o s t e r o l The  (18) has been shown by Stokes.  Bloch  47  (13) t o c h o l e s t e r o l  and Schwenk  48  the c o n v e r s i o n o f b i o s y n t h e t i c a l l y l a b e l l e d zymosterol (18).  The  as carbon  t h r e e methyl groups at C-4  and C-14  (17)  (16)  (13)  i n the sequence between l a n o s t e r o l 49 of ingenious biochemical  and  (13)  and c h o l e s t e r o l 51  (14) .  '  51 '  i n the isolated  (18), and by  a series  It gives r i s e i n turn  to c h o l e s t e r o l , t h u s , i t i s e s t a b l i s h e d t h a t the 14a  exact o r d e r o f events  lost  methods i t has been shown to pos-  sess the s t r u c t u r e o f 1 4 - d e s m e t h y l l a n o s t e r o l  The  50  Another i n t e r m e d i a t e has been  chemical  t h r e e e x t r a methyi groups t o be  are  i s b e l i v e d to  i n v o l v e o x i d a t i o n o f the methyl groups to c a r b o x y l groups (18).  to c h o l e s t e r o l  to c h o l e s t e r o l  of lanosterol  d i o x i d e i n a sequence which r e q u i r e s oxygen and  formation of c h o l e s t e r o l  (18) have  have demonstrated  49  final  distinct  o f t h e s e pathways t o the o t h e r .  c o n v e r s i o n o f desmosterol  46  involves e a r l i e r  There i s o f course the  o f i n t e r m e d i a t e s c r o s s i n g from one  r e c e n t l y been e s t a b l i s h e d .  4  lost in lanosterol  i s the f i r s t  o f the  (13).  r e s u l t i n g i n l o s s o f the C-4  methyl groups  i s not known w i t h c e r t a i n t y , but t h e r e i s some argument f o r the  existence  o f a h y p o t h e t i c a l C-4-monomethyl i n t e r m e d i a t e compound 4  (15), based upon the  r e c e n t l y i d e n t i f i e d 4-monomethyl s t e r o i d s i n Nature.  The  scheme f o r the  c o n v e r s i o n of squalene  now  understand i t ,  i s shown i n F i g u r e  (11) to c h o l e s t e r o l  (18), as we  3.  It must be emphasized t h a t the above pathways were the r e s u l t i n t r a c e l l u l a r " i n v i v o " process. t o s y n t h e s i z e the d e s i r e d p r o d u c t .  o f an  T h i s p r o c e s s made e x t e n s i v e use o f enzymes There i s an i n c r e a s i n g amount o f  i n the mechanism o f these " i n v i v o " r e a c t i o n s and,  interest  by u t i l i z i n g the same  -75-  Figure 3.  Scheme for the formation of Cholesterol (18) from Squalene  -76-  intermediates  as the c e l l ,  organic  chemists now  r e a c t i o n s under, l a b o r a t o r y c o n d i t i o n s p o s s i b l e s i n c e the enzyme lowers the a c t i n g as a c a t a l y s t . should  attempt t o reproduce these  ' i n v i t r o ' without enzymes.  This i s  a c t i v a t i o n energy o f the r e a c t i o n  by  In v i t r o , t h e s e r e a c t i o n s which o c c u r " i n v i v o "  thus o n l y r e q u i r e s t r o n g e r  conditions.  A successful reaction of 52  t h i s type i s , o f c o u r s e , a " b i o g e n e t i c The  type"  l o s s o f the methyl groups i n the  v e r s i o n has  been o f c o n s i d e r a b l e  synthesis.  lanosterol-cholesterol intercon-  interest.  Since  s t e r o l s have been found i n n a t u r e * ^ ' ^ ' ^ and considered  t o be  l i k e l y precursors  a b l e to assume t h a t the  first  of  The  the methyl groups.  are u n a c t i v a t e d  i n the  step  occurs by 57  configuration,  58 '  i n the  C-19  of saturated  o f s t e r o i d s has  hydroxylated  hydroxysteroids  d e m e t h y l a t i o n may  sense i s o f g r e a t  gation of b i o l o g i c a l hydroxylation hydroxylation  C-19  and  of ring A nor-steroids,  hydroxylation  classical  C-18  are  i t i s reasonbe  hydroxylation  carbon atoms t h a t  importance.  .Investi-  indicated that  replacement o f hydrogen w i t h o u t  the  i n v e r s i o n of  59 '  i . e . , the h y d r o x y l  group o c c u p i e s the  l o c a t i o n as the hydrogen which i t r e p l a c e s .  same s t e r i c  This stereochemical  result is  f i n d i n g i n c r e a s i n g analogy i n s t u d i e s o f e l e c t r o p h i l i c s u b s t i t u t i o n r e a c t i o n s at s a t u r a t e d the  carbon.  C o r e y ^ has  a c t i v e agent, comparing the  suggested e l e c t r o p h i l i c oxygen as  enzyme-oxygen complex t o a p e r o x i d e  or  peracid. Microrganisms which e f f e c t s t e r o i d h y d r o x y l a t i o n  at a s p e c i f i c  satu-  r a t e d carbon atom o f t e n produce 1,2-epoxides from s t e r o i d s i n which t h a t 1  carbon i s p a r t o f an o l e f i n i c the same enzyme may matic reagent has  be  l i n k a g e , and  involved  i t has  i n both p r o c e s s e s .  been p o i n t e d As  out^  a r e s u l t the  been compared t o a p e r o x i d e o r p e r a c i d and has  that  1  enzy-  been  con-  -77-  s i d e r e d as an e l e c t r o p h i l i c , n o n - r a d i c a l e s t a b l i s h e d that the hydroxylation o l e f i n i c intermediate,  species.  Furthermore, i t has been  p r o c e s s does n o t i n v o l v e h y d r a t i o n  o f an  but d i r e c t i n c o r p o r a t i o n o f m o l e c u l a r oxygen i n t o the  reagent and hence i n t o the s t e r o i d and t h a t metal ions  and TPNH a r e i n v o l -  62 ved,  providing  a d d i t i o n a l evidence f o r a p e r o x i d i c  intermediate.  Another r e a c t i o n o f importance i s t h e i n t r o d u c t i o n , removal, and r e arrangement , o f and  double bonds.  s u b s e q u e n t l o s s o f water.  The f i r s t may i n v o l v e p r i o r  Rearrangement o f double bonds may i n some  cases o c c u r as a r e s u l t o f s e r i e s o f o x i d a t i o n s case o f t h e movement o f C-8 C-9 u n s a t u r a t i o n in  hydroxylation  and r e d u c t i o n s ,  o f zymosterol  as i n the  (16) t o C-5 C-6  c h o l e s t e r o l (18). In v i t r o  f u n c t i o n a l i s a t i o n o f n o n - a c t i v e methyl groups, by  intramole-  c u l a r a t t a c k by groups n e a r these groups i n space, has been a c h i e v e d by a v a r i e t y o f methods.  The problem posed an i n t e r e s t i n g c h a l l e n g e  c h e m i s t r y , m a g n i f i e d by t h e widespread o c c u r r e n c e o f such i n n a t u r e under t h e i n f l u e n c e o f enzymes.  The f i r s t  i n synthetic  transformations  reported f u n c t i o n a l i -  s a t i o n o f - i n a c t i v e methyl groups was the use o f the L o e f f l e r - F r e y t a g reaction*' '^ 3  Figure  4).  4 ,  ^ t o p r e p a r e c o n e s s i n e d e r i v a t i v e s as shown (19) - (21)  The mechanism i s o f the f r e e r a d i c a l c h a i n  a b s t r a c t i o n by n i t r o g e n involved.  type,*'  5  (see  and r a d i c a l  i s f a v o u r e d by t h e j u x t a p o s i t i o n o f the two groups  The r a d i c a l d e c o m p o s i t i o n o f t h e 2 0 - N - c h l o r o a m i n o s t e r o i d (19) i  i n a c i d s o l u t i o n l e d t o a r i n g c l o s u r e between C-18 and n i t r o g e n .  A similar  r a d i c a l a b s t r a c t i o n r e a c t i o n i n v o l v i n g oxygen r a t h e r than n i t r o g e n  as t h e  a b s t r a c t i n g r a d i c a l , was t h e p h o t o l y s i s to c y c l o b u t a n o l The  o f C-20 k e t o s t e r o i d s  which gave r i s e  products.^  p y r o l y s i s o f 21-diazo-5a-pregnan-20-one (22) r e s u l t e d i n the f o r -  -79-  mation o f a carbon-carbon bond between the C-18 by  carbene (23)  i n s e r t i o n as shown.  i n s e r t i o n i n a s i m i l a r manner, and conessine s y n t h e s i s ^ The  8  and  Photolysis of azides w i l l use has  C-21  ion,  (24)  cause n i t r e n e  been made o f t h i s i n Barton's  Edward's d i t e r p e n e  alkaloid synthesis.^  a c t i o n o f l e a d t e t r a a c e t a t e on a l c o h o l s l e a d s d i r e c t l y t o  yields of tetrahydrofuran bonium  methyl group and  d e r i v a t i v e s , without the  when a l l the p a r t c i p a t i n g c e n t e r s  formation  high  of a free car-  are f i x e d i n the  arrangement  70 which favours such as and  (25)  hydrogen a b s t r a c t i o n .  The  with lead tetraacetate w i l l  treatment o f s t e r o i d a l a l c o h o l s  g i v e r i s e t o c y c l i c ethers  the r e a c t i o n i s thought t o i n v o l v e the t r a n s i t i o n s t a t e  r e a c t i o n has  (26).  (27), The  been  used to f u n c t i o n a l i s e the C-18 methyl group i n an a l d o 71 72 sterone s y n t h e s i s . ' H y p o c h l o r i t e s can be p h o t o l y s e d t o g i v e f u n c t i o n a l i s a t i o n o f s u i t a b l y 70 placed  i n e r t methyl groups,  halites The  73 '  gives s i m i l a r r e s u l t s . ^  and  l e a d t e t r a a c e t a t e treatment o f hypo-  4  p h o t o l y s i s of s u i t a b l y c o n s t i t u t e d organic  intramolecular  exchange of the NO  t o a carbon i n the y - p o s i t i o n .  formed can be  i s o l a t e d as the oximes. C-19  within a solvent  r e a c t i o n has  The  The  C-nitroso  T h i s r e a c t i o n has  f u n c t i o n a l i s e both C-18 and 75 76 as shown (28) - (31). ' cage.  an  o f the n i t r i t e r e s i d u e w i t h a hydrogen  atom a t t a c h e d  The  n i t r i t e s provokes  compounds thus  been used t o  methyl groups i n s t e r o i d s by  a mechanism  whole p r o c e s s i s b e l i e v e d t o take been used i n a p a r t i a l  place synthesis  77 of  aldosterone. The  tion  methods o f f u n c t i o n a l i s i n g methyl groups by  intramolecular  o f hydrogen, corresponds to the t r a n s f e r o f a hydrogen atom t o  a t t a c k i n g f r e e r a d i c a l i n the same m o l e c u l e , and hence t o a hydrogen  abstracan shift.  -80-  Th e most  frequently  shifts.  T h e most  that  of  these  observed  favourable  a 6-membered  reactions  corresponding  are  to  intramolecular  ring more  1,2-  transition  i n the  chair  important  state  for  the  form and t h e  than  and 1 , 3 - h y d r i d e  hydrogen t r a n s f e r s  the  1,5  hydrogen t r a n s f e r  steric  energetic  shifts  are  is  requirements  ones.  for  Hydrogen s h i f t s  o b s e r v e d w i t h c a r b o n i u m and  78 oxonium i o n s  do n o t  The methods all  for  as  ozone.  of  free  s i m i l a r to Both c i s -  hydroxydecalin,  oxy-radicals.  of  radical  groups,  reaction,  summarised above,  and a r e  thus  not  oxygen.  alkyl  steroid  A closer  to  this  hydroxylation,  is  the  w i t h ozone  a stereospecific  reaction  give  cis-  presumed  model,  h y d r o g e n by oxygen w h i c h can be  and t r a n s - d e c a l i n  respectively,  analogy  have  good  f o r b i o l o g i c a l h y d r o x y l a t i o n which i s  cationic  displacement  especially  with  of  C o r e y ' s ^ ^ model  involve attack  non r a d i c a l  with  f u n c t i o n a l i s i n g methyl  i n v o l v e d some s o r t  analogies to  of  occur  of  and  of  regarded decalin trans-9-  substitution proceeding  with  79 retention close the  of  configuration  chemical  analog  formation of  for  a cyclic  and i n v o l v i n g this ether  type (34)  electrophilic  oxygen.  of b i o l o g i c a l reactions from a h y d r o p e r o x i d e  of  Another  steroids  tosylate  is  (32), 80  which  involves  attack  (32)  on a c a r b o n u n a c t i v a t e d  (33)  i n any  classical  fashion.  (34)  85 '  -81-  Thus 1 , 3 , 3 - t r i m e t h y l c y c l o h e x a n e benzenesulphonyl  hydroperoxide  (32) r e a c t e d w i t h p a r a - n i t r o -  c h l o r i d e i n c o l d p y r i d i n e arid methylene c h l o r i d e .  c l e a v a g e o f the p a r a - n i t r o b e n z e n s u l p h o n y l d e r i v a t i v e o f the  o x i d e t o b i c y c l i c e t h e r (34) i s c o n s i d e r e d p r o c e e d i n g v i a the (33).  The  hydroper-  cationic  mechanism, i s an i n t e r m e d i a t e between the  extremes f o r a t t a c k o f c a t i o n i c oxygen, e x c l u s i v e l y on hydrogen o r on carbon.  The b i c y c l i c  e t h e r has not been d e t e c t e d under d i f f e r e n t  c o n d i t i o n s which are known t o proceed v i a a r a d i c a l mechanism.  One  f e a t u r e o f the r e a c t i o n i n t h i s case i s i t s s t e r i c f a c i l i t a t i o n , the p r o x i m i t y o f the methyl is  0-0  hydroperoxide  i s p r o b a b l y h e t e r o l y t i c under the c o n d i t i o n s used the change from  t r a n s i t i o n s t a t e such  Since  group and h y d r o p e r o x i d e  function.  exclusively reaction important  owing t o  This reaction  i n t e r e s t i n g as a c h e m i c a l c o u n t e r p a r t o f enzymatic h y d r o x y l a t i o n at a  s a t u r a t e d carbon which a l s o appears which proceeds  t o i n v o l v e e l e c t r o p h i l i c oxygen, and  by f r o n t a l displacement  as i m p l i e d by the i n t e r m e d i a t e (33).  -82-  DISCUSSION  The  first  stage i n the c o n v e r s i o n o f l a n o s t e r o l  to cholesterol, " i n  vivo",  i s the removal o f the 14a-methyl group.  lities  f o r t h e removal o f t h e e x t r a methyl carbons: d i r e c t l o s s as a methyl  group; p r e l i m i n a r y  oxidation  There a r e s e v e r a l  possibi-  t o a hydroxy-methylene group and l o s s as form-  aldehyde; f u r t h e r o x i d a t i o n  t o a formyl group and l o s s as f o r m i c a c i d ; o r ,  finally,  and l o s s -as CO2.  complete o x i d a t i o n  the  l a s t o f these p o s s i b i l i t i e s  the  8,9-double bond i n l a n o s t e r o l  ture  From t h e evidence p r e s e n t e d ^  seems a c t u a l l y t o o c c u r .  (13) _ i s a t t r a c t i v e as an a c t i v a t i n g  f o r t h e d e c a r b o x y l a t i o n o f the C-14 c a r b o x y l i c  activated  decarboxylation usually  The l o c a t i o n o f  acid  ( 3 5 ) . Such an  r e s u l t s i n t h e m i g r a t i o n o f t h e double  bond, which would i n t h i s case form t h e 8 ( 1 4 ) - u n s a t u r a t i o n possible  that  the  (36).  t h i s substance i s indeed an i n t e r m e d i a t e but t h a t  r e a r r a n g e s t o t h e thermodyriamically more s t a b l e reaction,  i t rapidly  that  l o s s o f CO2 as shown.  (36)  Iti s  isomer (37) o r that:, i n t h e  C-9 a c q u i r e s an e l e c t r o p h i l e from an enzyme s u r f a c e  (35)  fea-  (37)  initiates  -83-  The  enzyme-steroid complex i s then c l e a v e d by  producing product  the i s o l a t e d i n t e r m e d i a t e  with  To  (37).  the c o r r e c t s t e r e o c h e m i s t r y  achieve  a proton  Such a mechanism w i l l about the D/E  " i n v i t r o " o x i d a t i o n o f the  would have to be  attack of  ring  C-14  l e a d to a  juncture.  14a-methyl, a s u i t a b l e group  i n t r o d u c e d i n a 1 , 3 - d i a x i a l r e l a t i o n s h i p to i t . The  double bond makes the 9a and  7a p o s i t i o n s p a r t i c u l a r l y  l a b i l e , and  a r e , t h e r e f o r e , the b e s t p o s i t i o n s f o r s u b s t i t u t i o n w i t h o f the  at  14a-methyl group as they  8(9)-  these  a view to removal  are both i n a 1 , 3 - d i a x i a l r e l a t i o n s h i p to 81  it.  Since molecular  oxygen i s i n v o l v e d i n b i o l o g i c a l h y d r o x y l a t i o n  r e a c t i o n i n v o l v i n g molecular 82  Autoxidation and  of olefins  l a n o s t e r o l would be  oxygen would be  a  a c l o s e r " i n v i t r o " analogy..  i s known to g i v e a l l y l i c  hydroperoxidation,  expected t o o x i d i z e i n the C-7  A u t o x i d a t i o n o f l a n o s t e r o l i s known, however, t o g i v e the  78  and C - l l p o s i t i o n s , 73 and  113  83 hydroperoxides, hydroperoxide.  and  t h i s would be u s e l e s s as a means o f o b t a i n i n g the  Photosensitized  7a-  o x y g e n a t i o n o f o l e f i n i c double bonds 84  g i v e s a l l y l i c h y d r o p e r o x i d e s i n which the double bond has The  initially  and  can be  o f ways.  rearranged.  formed h y d r o p e r o x i d e u s u a l l y s u r v i v e s the r e a c t i o n c o n d i t i o n s  i s o l a t e d and  reduced to the a l l y l i c  a l c o h o l by  any  o f a number  When a p p l i e d to o l e f i n s h a v i n g nearby f u n c t i o n a l groups, t h i s  o x y g e n a t i o n r e a c t i o n has  c o n s i d e r a b l e p o t e n t i a l as  a synthetic tool,  and  a l s o y i e l d s i n f o r m a t i o n on v a r i o u s f a c t o r s t h a t might i n f l u e n c e s e n s i t i z e d 84 87 84 photochemical p r o c e s s e s . .' The proposed mechanism of this reaction i n v o l v e s e x c i t a t i o n o f the s e n s i t i z i n g dye on a b s o r p t i o n o f l i g h t energy, and  the f o r m a t i o n  of a l a b i l e photosensitizer-oxygen  o x i d i z e s the s u b s t r a t e . the o l e f i n - o x y g e n  The  findings  86  complex, which i n t u r n  suggest a c y c l i c mechanism f o r  combination, a f t e r the system has  been s u i t a b l y e n e r g i z e d .  -84The  sensitizer-oxygen complex breaks down to y i e l d excited oxygen (O2*)  which w i l l react with the double bond (38) to give the rearranged a l l y l i c hydroperoxide (40)via the s i x membered t r a n s i t i o n s t a t e (39).  (38)  (39)  (40)  Several d e t a i l e d v a r i a n t s f o r such a c y c l i c process can be envisaged according t o whether i t i s concerted or not, and depending on the nature of the bonds i n the t r a n s i t i o n states or intermediates, and the extent ( i f any) o f p a r t i c i p a t i o n by the s e n s i t i z e r when the oxygen attacks. This photosensitized oxygenation r e a c t i o n i s also of s p e c i a l i n t e r e s t as a possible pathway f o r b i o l o g i c a l o x i d a t i o n s , and various s t e r o i d a l 84 86 87 o l e f i n s have been hydroperoxidized  i n t h i s manner.  '  '  The attack  has u s u a l l y been from the less hindered a-face of t h e molecule and the bonds formed and broken (C-0 and C-H r e s p e c t i v e l y ) are c i s to each other, with no i m p l i c a t i o n about the timing of the events or the extent o f p a r t i c i p a t i o n by, the s e n s i t i z e r .  The r e s u l t with s t e r o i d s and simpler o l e f i n s 87  i n d i c a t e d that the r e a c t i o n i s subject to s t e r i c hindrance.  In dihydro-  l a n o s t e r o l (41) the C-8 p o s i t i o n i s more hindered than the C-9 p o s i t i o n due to the proximity o f the 14a-methyl group and thus 9a-hydroperoxylanost-7ene-38-ol (42) would be the expected product of photosensitized o x i d a t i o n .  -85-  When d i h y d r o l a n o s t e r o l was oxygenated i n t h e presence porphyrin,and  t h e r e s u l t a n t product was reduced  of light  and haemato^  and a c e t y l a t e d , 36-acetoxy88  lanosta-7,9(ll)-diene  (48) was o b t a i n e d as t h e o n l y s o l i d p r o d u c t .  r e s u l t might i n d i c a t e t h a t 9 - h y d r o p e r o x i d a t i o n the 9 a - a l c o h p l would be r e a d i l y dehydrated However, t h e photo-oxygenation  had i n f a c t o c c u r r e d s i n c e  under a c e t y l a t i o n c o n d i t i o n s .  o f dihydrolanosteryl acetate  same r e s u l t , and attempts t o i s o l a t e t h e 9a-hydroperoxide Dihydrolanosterol  This  (47) gave t h e  were u n s u c c e s s f u l .  (41) c o u l d be used t o f u n c t i o n a l i z e t h e C-14 methyl  group i n s i t u under t h e i n f l u e n c e o f a powerful para-nitrobenzenesulphonyl  group, (see F i g u r e 5 ) .  (43) would be t h e expected  product by analogy  l e a v i n g group such as t h e The C-9 C-14 methyl  t o t h e Corey r e a c t i o n (see  I n t r o d u c t i o n ) a n d t h i s e t h e r c o u l d be o x i d i z e d t o the u n s a t u r a t e d which would open t o t h e a c i d l o s t by d e c a r b o x y l a t i o n .  ether  l a c t o n e (44)  (45) from which t h e C-14 group would be r e a d i l y  In t h i s sequence, the C-14 methyl group would be  removed, and t h e double bond would be s h i f t e d t o t h e 7 p o s i t i o n .  Both o f  these a r e s t e p s i n t h e i n v i v o c o n v e r s i o n o f l a n o s t e r o l t o c h o l e s t e r o l , and a n , i n v i t r o r e a l i z a t i o n o f these s t e p s v i a a s e r i e s o f o x i d a t i o n r e a c t i o n s , would e s t a b l i s h a m i l e - s t o n e  i n an a r e a t r a d i t i o n a l l y c o n s i d e r e d t o be a  s e c t o r o f b i o c h e m i s t r y , namely, t h e f i e l d o f enzyme mechanism.  Previous  88 attempts  o f C-14 methyl a c t i v a t i o n by p h o t o s e n s i t i z e d oxygenation  u n s u c c e s s f u l , as t h e expected  C-9 C-14 c y c l i c e t h e r c o u l d n o t be i s o l a t e d  or detected i n the r e a c t i o n product.  When i n t h e hope o f f u n c t i o n a l i z i n g  the C-14 methyl group v i a the C-7 C-14 methyl e t h e r 7a-hydroperoxide  were  3B-acetoxylanost-8-ene-  (50) was t r e a t e d w i t h p a r a - n i t r o b e n z e n s u l p h o n y l  chloride  i n p y r i d i n e , no r e a c t i o n o c c u r r e d , t h e o n l y compound i s o l a t e d was t h e s t a r t The i n g mlaa tb ei rl ii ta yl . o r i n t r a c t a b i l i t y o f any 9ot-hydroperoxide  formed i n t h e  -86-  Figure 5.  Proposed scheme for the removal of the 14o(-methyl group i n dihydrolanosterol ( 4 l ) .  -87-  photo-oxygenation i n t h e presence  r e a c t i o n might be overcome by c a r r y i n g out t h e r e a c t i o n of para-nitrobenzensulphonyl  might r e a c t w i t h t h e 14o(-methyl group. a t i o n r e a c t i o n was- repeated.  T h e r e f o r e the i n s i t u photo-oxygen-  Dihydrolanosteryl acetate  was oxygenated i n p y r i d i n e i n the presence para-nitrobenzensulphonyl  c h l o r i d e so t h a t the p e r o x i d e  chloride.  (47) (see F i g u r e 6)  o f l i g h t , haematoporphyrin, and  The r e a c t i o n product was worked up the  u s u a l way, and a s e m i - s o l i d was o b t a i n e d which was chromatographed on an alumina  colum.  On d e v e l o p i n g the chromatographic  column, f i r s t , petroleum  e l u t e d unchanged p a r a - n i t r o b e n z e n s u l p h o n y l  ether  c h l o r i d e f o l l o w e d by 33-acetoxy-  l a n o s t a - 7 , 9 ( l l ) - d i e n e (48). Next petroleum m.p.  141-144°..  ether' e l u t e d a compound which was d e s i g n a t e d  as IP1  I t s i n f r a r e d spectrum had peaks a t 1730, 1240 ( a c e t a t e ) and  823 cm \  the u l t r a v i o l e t  a b s o r p t i o n was a t 209 my  ( e 8960).  T h i s compound  was found  t o be i d e n t i c a l t o t h a t o b t a i n e d , and not i d e n t i f i e d , i n a p r e v i o u s  88 study  ( I n f r a r e d , U l t r a v i o l e t spectrum and mixed m.p.). In subsequent f r a c t i o n s on e l u t i o n w i t h petroleum  ether-benzene, 33-acetoxylanost-8-ene-7-one (49) and 7a-hydroperoxide  (50) were o b t a i n e d r e s p e c t i v e l y .  e t h e r and petroleum  33-acetoxylanost-8-ene-  Since a l l y l i c  hydroper-  88 oxidation  had y i e l d e d the 7a-hydroperoxide,  i t was p o s s i b l e t h a t t h e  l a t t e r compound had been formed by rearrangement o f t h e f i r s t hydroperoxide.  The rearrangement o f t e r t i a r y t o secondary 89 90  had been r e p o r t e d , The (43),  '  formed 9a-  hydroperoxides  and t h e mechanism r e q u i r e s r e t e n t i o n o f c o n f i g u r a t i o n .  compound IP1 a n a l y z e d c o r r e c t l y f o r the r e q u i r e d c y c l i c e t h e r -  and t h e mass s p e c t r o s c o p i c m o l e c u l a r weight o f 484 i s i n agreement  w i t h t h e c o r r e c t a n a l y s i s f o r C32H52O3.  S i n c e i t had no c a r b o n y l o r h y d r o x y l  + A c O (48)  hv  0 , 2  +  (  sensitizer  A c O  NOo <47)  A c O  (49)  O  +  so ci 2  A c O ^ >  Figure  6,  P h o t o s e n s i t i z e d oxygenation o f d ^ ^ " ^ ^ ! ^ ^ " the presence o f p a r a - n i t r o b e n z e n e s u l p h o n y l c h l o n d e . 1  in  0  6  ( 4 ? )  '  I  P  1  '  C  3 2  H 5  2  ° 3  -89-  f u n c t i o n s , the compound was t h a t treatment  presumably an e t h e r .  An  e a r l i e r study  88  showed  o f t h i s compound w i t h chromium t r i o x i d e i n a c e t i c a c i d gave  36-acetoxylanost—8-ene-7-one.(49), and reduction :  a c e t i c a c i d gave 3 B - a c e t o x y l a n o s t a - 7 , 9 ( l l ) - d i e n e  w i t h potassium (48)  iodide i n  (see F i g u r e 7 ) .  O x i d a t i o n under c o n d i t i o n s which had y i e l d e d l a c t o n e s from s t e r o i d a l e t h e r s f a i l e d t o g i v e products  with  l a c t o n e . a b s o r p t i o n i n the i n f r a r e d .  treatment  w i t h boron t r i f l u o r i d e e t h e r a t e compound IP1  The  one,of these was  first  an.enone  U l t r a v i o l e t s p e c t r a l data.  The  C32H52O3,  t e t r a s u b s t i t u t e d . The  t r i f l u o r i d e treatment  o f the e t h e r IP1 was  grouping  was  the n.m.r  The  supported  by  products.  T h i s p r o t o n was  a k e t o l , s u b s t a n t i a t e d by i t s transparent-  coupled w i t h two  The  protons  at x 5.51.  alcoholic  T 6.8 on an  c o n s i d e r e d t o be a x i a l s i n c e the q u a r t e t  s i m i l a r s p i n - s p i n c o u p l i n g t o the 3a a x i a l p r o t o n  in  adja-  had  Since  this  i s a x i a l , the a l c o h o l i c group must be e q u a t o r i a l .  At t h i s p o i n t none o f the v a r i o u s h y p o t h e t i c a l s t r u c t u r e s which p o s s i b l e has compound IP1. methods.  f i t t e d a l l o f the p h y s i c a l and Therefore,  i t was  chemical  which can be  d e c i d e d t o t a c k l e the problem by  expected  hoped t h a t i t might.be a s u i t a b l e i n t e r m e d i a t e  The  compound IP1 was  r e a d i l y brominated by p y r i d i n e  perbromide to g i v e the d i b r o m o - d e r i v a t i v e which a n a l y z e d Br  X-ray  f u n c t i o n a l i z e d i n subsequent s t e p s f o r the removal o f the  14a methyl group.  C32 52°3 2•  are  data a v a i l a b l e f o r  S i n c e the compound i s presumably an e t h e r but not the  c y c l i c e t h e r (43), i t was  H  the  from the boron  c o n s i d e r e d t o be secondary i n View o f a p r o t o n at  spectrum.  I t s U l t r a v i o l e t spectrum was  spectrum showed a b s o r p t i o n s  at 1745  and  On  I n f r a r e d and  second product  U l t r a v i o l e t spectrum was  cent carbon atom and was  proton  gave two  n.m.r. showed no o l e f i n i c p r o t o n , so  enone double bond was  i n f r a r e d spectrum.  cyclic  correctly for  t r a n s p a r e n t , and the I n f r a r e d  1240  cm  * (acetate).  IP1,C  3 2  H  5 2  0  3  Pyr-Br>  CrO KI  A c O  (51)  A c O  i g u r e 7.  Chart showing the r e a c t i o n s o f compound IP1 with chromium potassium i o d i d e and p y r i d i n e perbromide.  trioxide  -91-  The The  X.-ray c r y s t a l l o g r a p h i c study was  c a r r i e d out i n t h i s  c r y s t a l s o f the d i b r o m o - d e r i v a t i v e are orthorhombic,  9.88^,  c = 12.26. A, Z = 4 space  group P 2 2 2 . 1  1  The  1  Department.  a_ = 26.03,., b_ =  i n t e n s i t i e s of  about  2400 r e f l e c t i o n s were measured on a G.E.  s p e c t r o g o n i o m e t e r with  t i o n c o u n t e r , u s i n g CuKa r a d i a t i o n .  two bromine p o s i t i o n s were d e t e r -  The  scintilla-  mined by P a t t e r s o n methods and a l l carbon atoms were l o c a t e d on t h r e e successive three-dimensional electron-density d i s t r i b u t i o n s . i s o t r o p i c temperature  Positional  and  parameters, were r e f i n e d by f o u r c y c l e s o f l e a s t  a f u r t h e r s i x c y c l e s o f a n i s o t r o p i c l e a s t squares g i v i n g a f i n a l R_ v a l u e o f 13.3%.  The  completed  the  a b s o l u t e c o n f i g u r a t i o n was  squares;  refinement determined  by the anomalous d i s p e r s i o n method. The  d i b r o m o - d e r i v a t i v e . o f compound IP1 i s 3 g - a c e t o x y - 7 , l l c r d i b r o m o a  lanostane-8a,9a-epoxide and r i n g D has  (51) .  a h a l f - c h a i r c o n f o r m a t i o n . ' The  C from a d o p t i n g the c h a i r form. normal,  S t e r o i d r i n g A i s i n the normal c h a i r  form  epoxide p r e v e n t s r i n g s B and  The bond l e n g t h s and v a l e n c y angles  are  and the i n t e r m o l e c u l a r s e p a r a t i o n s correspond t o Van d e r Waals  interactions. In summary, the- o x i d a t i o n p r o d u c t that i t i s a d i s u b s t i t u t e d  1,2-epoxide i n view o f the protons  r e g i o n o f the n.m.r. spectrum. w i t h boron  IP1 i s an e t h e r and we  The  feel  strongly  i n the  f o r m a t i o n o f a ketone-secondary  7x alcohol  t r i f l u o r i d e e t h e r a t e would a l s o i n d i c a t e t h a t the e t h e r t e r m i n i  are v e r y p r o b a b l y secondary,  and the s t r u c t u r e  (52) can be a s s i g n e d t e n t a -  t i v e l y which would f i t a l l the p h y s i c a l at hand. The n.m.r. spectrum certainty.  i n d e u t e r o c h l o r o f o r m c o u l d not be e v a l u a t e d with  However, i n benzene s o l u t i o n , where a s o l v e n t s h i f t was  observed,  -92-  a b e t t e r r e s o l u t i o n was follows:  The  o b t a i n e d and  o l e f i n i c proton  ( a r e a = 1 H).  at C - l l was  expected  a s s i g n e d t o a q u a r t e t at  T h i s p r o t o n i s coupled w i t h the two  3 a - a x i a l p r o t o n on the same carbon T5.48 w i t h J  assignments were r e a d i l y made as  . , . = 8 c.p.s. axial-axial  (area = 1 H).  The  1  p a i r o f d o u b l e t s at x6.95 and  epoxide  tively).  at T7.55 ( J = 5 c.p.s.,  proton  at C-8,  protons  so f a r , must o c c u r i n the b r o m i n a t i o n  to conceive of a d i s u b s t i t u t e d  s k e l e t o n from which the dibromide all  the c h e m i c a l and p h y s i c a l d a t a  (51)  c.p.s.,  respec-  a s s i g n e d t o the  occurred  possible.  i t i s c l e a r from the above t h a t a most unusual  not encountered is d i f f i c u l t  assignments are not  were  a t t r i b u t e d t o the  A complex p a t t e r n o f l i n e s  between x7.6-9.5>where i n d i v i d u a l Finally,  a r e a = 1 H) was be  as  at T7.15  ( a r e a = 1 H and J = 3.5  and the abnormal d o w n f i e l d s h i f t may  d e s h i e l d i n g by the double bond.  The  as the a c e t a t e f u n c t i o n o c c u r r e d at  a s s i g n e d t o the two A doublet  proton, on C-12.  and J . , . .,= 3 c.p.s. axial-equatorial  r  T5.15  rearrangement,  o f compound IP1.  1,2-epoxide i n the  lanosterol  can be d e r i v e d , and which a l s o  available.  It  fits  -93EXPERIMENTAL  M e l t i n g p o i n t s were determined  on a K o f l e r b l o c k and are u n c o r r e c t e d .  U l t r a v i o l e t s p e c t r a were measured on a Cary 14 spectrophotomer s p e c t r a were taken on a , P e r k i n Elmer Model 137B magnetic A60  resonance  instrument.  the T i e r s  and  infrared  spectrophotometer.  Nuclear  (n.m.r.) s p e c t r a were r e c o r d e d at 60 Mc/s  The. l i n e p o s i t i o n s o r c e n t e r s o f m u l t i p l e t s . a r e g i v e n i n  T s c a l e w i t h r e f e r e n c e t o t e t r a m e t h y l s i l a n e as the  standard.  Alumina  G ( a c c o r d i n g t o S t a h l ) p l a t e s were used  chromatography ( T . L . C ) .  The  determined  on a A..E.I. MS9  Double F o c u s i n g Mass  E l e m e n t a l m i c r o a n a l y s e s were performed Department, and by Dr. A. Bernhardt  d e t e r m i n a t i o n s were done by Mrs.  The  for thin  by Mrs.  The mass  layer was  spectrum  Spectrometer. C. J e n k i n s o f t h i s  and h i s a s s o c i a t e s o f the Max  I n s t i t u t e , Mulheim, Ruhr, West-Germany.  Department  internal  alumina used f o r column chromatography  Woelm n e u t r a l r e a g e n t , d e a c t i v a t e d w i t h 6% o f water. was  on a V a r i a n  Planck  The n.m.r. and mass s p e c t r o m e t r i c  A. Brewster  and Mr.  G.  Bloss of t h i s  respectively.  X-ray  c r y s t a l l o g r a p h i c study was  by Mr. J . K. Fawcett  i n this  c a r r i e d out by Dr. J . T r o t t e r  and  Department.  Lanosteryl Acetate  30 g o f L a n o s t e r o l (K § K) was anhydride overnight.  a c e t y l a t e d , w i t h 50 ml o f a c e t i c  ( r e a g e n t ) . a n d 80 ml o f d r y p y r i d i n e The  excess o f reagent was  steam b a t h and the r e s i d u e was  (reagent) at room  temperature  removed under reduced p r e s s u r e on the  d i s s o l v e d i n e t h e r , washed with  h y d r o c h l o r i c a c i d and then s e v e r a l times w i t h water and d r i e d  dilute (anhydrous  -94-  magnesium s u l p h a t e ) . p r e s s u r e gave 28.5 ethanol.. m.p. (OAc)  cm  1  E v a p o r a t i o n o f the e t h e r e a l s o l u t i o n under  g o f l a n o s t e r y l a c e t a t e which was  123-124°.  I n f r a r e d spectrum  reduced  recrystallized  i n N u j o l : 1739  (OAc)  and  (47)  L a n o s t e r y l a c e t a t e . ( 2 5 g) was  hydrogenated  i n e t h y l a c e t a t e (200  c o n t a i n i n g 1 g o f p l a t i n u m o x i d e , under hydrogen atmosphere with f o r 24 hours.  A f t e r removal  s t e r y l acetate, was a c e t a t e , m.p.  11.5%.  (w)  (literaturem.p.  C, 81.5%; H,  T..L.C. one s p o t .  I n f r a r e d spectrum and 870  (w)  ml)  stirring  o.f\ the; c a t a l y s t by f i l t r a t i o n the d i h y d r o l a n o -  r e c r y s t a l l i z e d t o c o n s t a n t m e l t i n g p o i n t from  119-120°,  A n a l . Found:  905  1240  .  Dihydrolanosteryl Acetate  H,  from  11.3%.  120-121°).  -  C a l c . f o r C32H51+02.:  = .87 i n benzene-chlorofrom  i n N u j o l : 1750  ( s ) , 1242  ethyl  ( s ) , 1035  C, 81.7%, (1:1).  ( s ) , 1010  (m),  cm" . 1  Oxygenation  o f D i h y d r o l a n o s t e r y l A c e t a t e i n the  presence  of Para-nitrobenzene Sulphonyl C h l o r i d e  10 g o f d i h y d r o l a n o s t e r y l a c e t a t e (3-/?-acetoxylanost-8-ene), 4.9 o f p a r a - n i t r o b e n z e n e s u l p h o n y l c h l o r i d e and d i s s o l v e d i n 260 ml o f dry p y r i d i n e round-bottom f l a s k . 250  volt  The  solution. r e s i d u e was  The p y r i d i n e was  o f haematoprophyrin  (reagent) c o n t a i n e d i n a 500  s o l u t i o n was  f l u o r e s c e n t tubes  150 mg  bubbled  e t h e r s o l u t i o n was  watt  through  removed under reduced p r e s s u r e and the The  were  ml.  i r r a d i a t e d w i t h t h r e e G. E. 20  f o r 8 days w h i l e oxygen was  dissolved in.ether.  g  the  solid  subsequently  -95--  e x t r a c t e d w i t h water, d i l u t e s u l p h u r i c a c i d and water. s o l u t i o n was  dried  The  ethereal  (anhydrous magnesium s u l p h a t e ) , and the s o l v e n t  evapo-  r a t e d under reduced p r e s s u r e t o g i v e 16 g o f a s e m i - s o l i d which was graphed on alumina (450 g ) .  T.L.C. on the crude p r o d u c t showed 7 s p o t s i n  b e n z e n e - c h l o r o f o r m (1:1), and the p r o d u c t l i b e r a t e d i o d i n e from iodide  added t o the top o f the column (22 x 2.5  diameter) by d i s s o l v i n g i t i n a m i x t u r e o f petroleum e t h e r (17:2).  nitrobenzene-sulphonyl  chloride.  s t a - 7 , 9 ( l l ) - d i e n e which was  r  I n f r a r e d spectrum i n N u j o l : 1745 (w) and 820  (w) cm  p o i n t by admixing i t ,  i  sample.  gave.3^-acetoxylano-  c r y s t a l l i z e d from acetone as n e e d l e s (658  U l t r a v i o l e t spectrum i n e t h a n o l : X  161-164°.  tic  (s),1240  max  ( s ) , 1030  237,  mg).  246 and 255 m .  (m), 980  (m), 905  (w),  T h i s compound gave no d e p r e s s i o n i n m e l t i n g and had an i d e n t i c a l  i n f r a r e d spectrum w i t h an authen-  88  E l u t i o n w i t h 500 ml o f p e t r o l e u m e t h e r (40-60°) crystallized was  (80-110°)-benzene  gave unchanged p a r a -  E l u t i o n w i t h 250 ml o f p e t r o l e u m e t h e r (40-60°)  875  cm  On e l u t i o n the f o l l o w i n g f r a c t i o n s were then o b t a i n e d c o n s e c u t i v e l y :  E l u t i o n w i t h 80 ml o f p e t r o l e u m e t h e r (40-60°)  r  sodium  solution.  The o r g a n i c m a t e r i a l was  m.p.  chromato-  from acetone as n e e d l e s (95 mg).  d e s i g n a t e d IP1, and was  gave a s o l i d  m.p.  141-144°.  which  T h i s compound  found t o be i d e n t i c a l t o t h a t o b t a i n e d e a r l i e r 88  by another group o f workers [<*] = +161 D  i n ethanol.  (mixed m.p.,  I n f r a r e d and U l t r a v i o l e t  U l t r a v i o l e t spectrum i n e t h a n o l : X  I n f r a r e d spectrum i n N u j o l : 1730  ( s ) , 1240  1030  (w) and 823  (m), 1010  spot. R^= cant peaks  0.61  (w), 975  (w), 960  ( s ) , 1095  i n b e n z e n e - c h l o r o f o r m (1:1).  at m/e  = 484  (M ), +  483  ( M - l ) , 469  m a x  (w), 1070  (m) cm" . 1  209  Spectrum. (6 8960).  (m), 1050  T.L.C. showed  Mass spectrum showed (M-15), 441  (w)  one  signifi-  (M-43), 424  (M-60),  -96-  371,  329 (M-155),  311, 289, 247, 231, 208, 207, 193 and 161.  n.m.r. s i g n a l s ;  g i v e n i n T u n i t s , spectrum o b t a i n e d i n benzene: m u l t i p l e t c e n t e r e d a t 5 . 1 8 , q u a r t e t c e n t e r e d a t 5.4 c o r r e s p o n d i g n carbon  t o the 3a-axial p r o t o n on the same  as t h e a c e t a t e f u n c t i o n , doublet  doublet  c e n t e r e d at 7.15  c e n t e r e d a t 6.96  ( J = 3.5 c . p . s . ) ,  ( J = 3.5 c.p.s.) doublet c e n t e r e d at 7.52  c . p . s . ) , and a complex p a t t e r n o f l i n e s between  E l u t i o n w i t h 1000 ml o f petroleum E l u t i o n w i t h 500 ml o f petroleum  ether  (J = 5  7.8-9.5.  (40-60°)  ether ( 8 0 - 1 1 0 ° )  y i e l d e d nothing. gave t r a c e s o f  crystals. E l u t i o n w i t h 1750 ml o f petroleum  ether ( 8 0 - 1 1 0 ° )  o s t - 8 - e n e - 7 — one which c r y s t a l l i z e d from petroleum (1.32 g ) . m.p. 1 4 7 - 1 5 0 ° .  Ultraviolet  gave  3B-acetoxylan-  ether ( 4 0 - 6 0 ° )  spectrum i n e t h a n o l : X  as needles  254 my.  I n f r a r e d spectrum i n N u j o l : 1740 ( s ) , 1650 ( s ) , 1580 (s) , 1240 ( s ) , 1080 1030 (m), 1010 (m), 975 (m) and 900 (w) cm"  (w), Rp=  0.64 i n benzene-chloroform  (1:1).  1  T.L.C. showed one spot.  T h i s compound was found t o be i d e n 88  tical  t o t h a t o f an a u t h e n t i c sample (mixed m.p., i n f r a r e d E l u t i o n w i t h 1500 ml o f petroleum  ether ( 8 0 - 1 1 0 ° )  spectrum).  y i e l d e d 300 mg o f  oil. E l u t i o n w i t h 300 ml o f petroleum E l u t i o n w i t h petroleum  ether ( 8 0 - 1 1 0 ° )  gave n o t h i n g .  e t h e r (80-110°)-benzene (2:1)  gave 3/?-acetoxy-  lanost-8-ene-7o<-hydroperoxide which c r y s t a l l i z e d from e t h y l a c e t a t e as needles  (310 mg).  m.p. 1 7 4 - 1 7 5 ° .  I n f r a r e d spectrum i n N u j o l : 3400 ( s ) ,  1730 ( s ) , 1275 ( s ) , 1040 (m) 1020 (m), T. L.C. showed one spot. R^ = 0.37  980 (m), 930 (w) and 860 (w) cm-1.  i n benzene-chloroform  (1:1).  The com-  pound had o n l y end a b s o r p t i o n i n the u l t r a v i o l e t , and i t gave no d e p r e s s i o n  -97-  on a mixed m e l t i n g p o i n t d e t e r m i n a t i o n w i t h an a u t h e n t i c sample. t h i s p o i n t t h e development  o f the chromatographic  88  At  columhwas d i s c o n t i n u e d .  B r o m i n a t i o n o f Compound IP1 The compound IP1 perbromide  (45 mg)  (45 mg)  the  d i s s o l v e d i n dioxan (8 ml) w i t h p y r i d i n e  and the s o l u t i o n was  The excess o f bromine was solution.  was  l e f t 4 hours at room temperature.  removed by the a d d i t i o n o f sodium  The aqueous s u s p e n s i o n was  e x t r a c t e d t h r e e times w i t h e t h e r and  combined e t h e r e a l e x t r a c t s on e v a p o r a t i o n , a f t e r d r y i n g  magnesium s u l p h a t e ) , gave a s o l i d .  The p r o d u c t was  acetone t o g i v e s t u r d y n e e d l e s and d r i e d , m.p. C, 59.93%; H, 8.2%. spectrum i n N u j o l : ultraviolet  1745  spectrum was  ( s ) , 1240  transparent.  (m), and 905  8.03%.  Infrared  (w) cm" . 1  The dibromide o f compound IP1  i d e n t i f i e d by X-ray c r y s t a l l o g r a p h i c a n a l y s i s as lanostane-8a,9a-epoxide.  from  199-200°. A n a l . 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