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Synthesis and biosynthesis of indole alkaloides. Hall, Ernest Stanley 1966

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SYNTHESIS AND BIOSYNTHESIS OF INDOLE ALKALOIDS by ERNEST STANLEY HALL B.Sc. Honours, The U n i v e r s i t y o f B r i t i s h Columbia, 1963.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Chemistry  We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1966  In presenting  t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements  f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y available f o r reference and study,  1 further agree that permission, f o r extensive copying of t h i s  thesis f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives.  I t i s understood that copying  or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission.  Department of  Cemistry  The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date  Aup;. 22.  1966  The U n i v e r s i t y of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of ERNEST STANLEY HALL B , S c , The U n i v e r s i t y of B r i t i s h Columbia, 1962 THURSDAY, AUGUST 18, 1966 at 3:30 P.M. IN ROOM 261, CHEMISTRY BUILDING COMMITTEE IN CHARGE Chairman:  P„ Ford  C„ T. Beer Fo McCapra C. A, McDowell External Examinerr  D. E. McGreer R. E„ Pincock M. Smith J . B. Hendrickson  Department of Chemistry BRANDEIS UNIVERSITY WALTHAM, MASSACHUSETTS Research Supervisor:  F. McCapra  SYNTHESIS AND BIO-SYNTHESIS OF INDOLE ALKALOIDS ABSTRACT In p a r t A a much sought s y n t h e s i s of the c a l y c a n t h a ceous a l k a l o i d s i s described.. O x i d a t i v e d i m e r i z a t i o n of N- m e t h y l t r y t a m i n e a f f o r d e d d l - chimonanthine and mesochimonanthine, and d l - c a l y c a n t h i n e was produced by subsequent a c i d - c a t a l y z e d rearrangement of the carbon s k e l e t o n through a tetraminodialdehyde,, As the suggested b i o s y n t h e s i s of t h e s e a l k a l o i d s i s r e p r e s e n t e d as o c c u r r i n g by an o x i d a t i v e d i m e r i z a t i o n of N- m e t h y l t r y p t a m i n e ( i t s e l f a n a t u r a l p r o d u c t ) the v e r y d i r e c t s y n t h e s i s d e s c r i b e d i s a b i o s y n t h e t i c model.. The d i s c o v e r y of meso-chimonanthine i n an e x t r a c t of Calycanthus f 1 o r i d u s i s a l s o r e p o r t e d and i s the f i r s t n a t u r a l calycanthaceous a l k a l o i d w i t h t h i s s t e r e o c h e m i s t r y t o be discovered,, As f o l i c a n t h i n e and c a l y c a n t h i d i n e a r e N-methyl chimonantbines and t h i s methyla t i o n has been r e p o r t e d , a s y n t h e s i s of chimonanthine a l s o r e p r e s e n t s a formal s y n t h e s i s of these a l k a l o i d s . , A proposal f o r t h e s t r u c t u r e of h o d g k i n s l n e , t h e remaining calycanthaceous a l k a l o i d ^ i s made„ A number of s y n t h e t i c by-products a r e a l s o described., 3  In p a r t B evidence f o r t h e mono-terpenoid o r i g i n of the n i n e or t e n carbon non-tryptophan d e r i v e d p o r t i o n of the i n d o l e a l k a l o i d s i s presented,, The monoterpene, g e r a n i o l - 2C, was a d m i n i s t e r e d t o V i n e a r o s e a c u t t i n g s and t h e Aspidosperma- type a l k a l o i d , v i n d o l i n e , was i s o l a t e d and shown by Kuhn-Roth d e g r a d a t i o n t o be l a b e l l e d at C-5 as p r e d i c t e d ^ y theory.. Feeding experiments w i t h mevalonic a c i d - 2C a r e a l s o described..  GRADUATE STUDIES Field  of Study:  Organic  Topics i n Physical  Chemistry Chemistry  J . A, E„ Coope W. C L i n J . P. Kutney Seminar Wc R o Cullen T o p i c s i n I n o r g a n i c Chemistry R . C„ Thompson N„ B a r t l e t t D . E., McGreer T o p i c s i n Organic Chemistry P„ Kutney F o McCapra L, D „ H a l l Organic S t e r e o c h e m i s t r y F o McCapra H e t e r o c y c l i c Chemistry J o P , Kutney A l k a l o i d Chemistry T„ Money I s o p r e n o i d Chemistry R . Stewart P h y s i c a l Organic Chemistry R E. P incock Organic Reaction Mechanisms J P , . Kutney S t r u c t u r e of Newer N a t u r a l Products A„ L S c o t t Recent S y n t h e t i c Methods i n Organic Chemistry  Do  E, McGreer  Biochemistry Modern B i o c h e m i s t r y  Go M.. Tenner W. T. Poglase Po H o J e l l i n c k S, H o Zbarsky V o J., O'Donnell  PUBLICATIONS A. I . Scott, F< McCapra, E. So H a l l , Chimonanthine, A One-Step Synthesis and Biosynthetic Model. J . Am. Chem Soc. 86, 302, (1964).  E. S. H a l l , F= McCapra, T, Money, K„ Fukamoto, J,. R„ Hanso^ Be S„ Mootoo, G„ T„ P h i l i p s and A, I,. Scott, Concerning the Terpenoid O r i g i n of Indole A l k a l o i d s . Chem. Comm. 348, (1966).  ABSTRACT  In part A, a much sought synthesis of the calycanthaceous alkaloids is described.  Oxidative dimerization of N-methyl-  tryptamine afforded dl-chimonanthine and meso-chimonanthine, and dl-calycanthine was produced by subsequent acid-catalyzed rearrangement of the carbon skeleton through a tetraminodialdehyde.  As the suggested biosynthesis of these alkaloids i s  represented as occurring by an oxidative dimerization of Nmethyltryptamine, i t s e l f a natural product, the very direct synthesis described is a biosynthetic model.  The discovery  of meso-chimonanthine in an extract of Calycanthus floridus is also reported and is the f i r s t natural calycanthaceous alkaloid  with this stereochemistry  to be discovered.  As  folicanthine and calycanthidine are N-methyl chimonanthines and this methylation has been reported, a synthesis of chimonanthine also represents a formal synthesis of these alkaloids A proposal for the structure of hodgkinsine, the remaining calycanthaceous alkaloid, is made.  A number of synthetic by-  products are also described. In part B evidence for the mono-terpenoid origin of the nine or ten carbon non-tryptophan derived portion of the indole alkaloids i s presented.  The monoterpene geraniol-2-  was ad  ministered to Vinca rosea cuttings and the Aspidosperma-type  iii  alkaloid,  v i n d o l i n e , was  degradation by  theory.  are a l s o  t o be  isolated  specifically  Feeding  described.  and  shown by  labelled  experiments  a t C-5  with mevalonic  Kuhn-Roth as p r e d i c t e d acid-2-l^C  iv  TABLE OF CONTENTS  ABSTRACT TABLE OF CONTENTS LIST OF FIGURES ACKNOWLEDGEMENTS INTRODUCTION  Page ii iv v viii 1  PART A Introduction  26  Discussion  46  Experimental  85  References  110  PART B Introduction  118  Discussion  140  Experimental  162  Appendix  195  References  196  V  LIST OF FIGURES PART A Figures  Page  1. A t y p i c a l A l k a l o i d s  2  2. B i o g e n e t i c - t y p e Synthesis of Tropinone  6  3. Amino Acids and A l k a l o i d B i o s y n t h e s i s  8  4. A l t e r n a t i v e Mechanisms and Evidence Against R a d i c a l I n s e r t i o n f o r P h e n o l i c O x i d a t i v e Coupling  10  5. C o u p l i n g of Mesomeric Phenol and Arylamine  11  Radicals  6. Morphine A l k a l o i d s , S y n t h e s i s and B i o s y n t h e s i s  13  7. Some Indole A l k a l o i d s  15  8. Tryptamine and Gramine from Tryptophan  17  9. Condensation  19  of Tryptamine and Aldehydes  10. B i o g e n e s i s of Ergot A l k a l o i d s  21  11. Pentose Shunt and the G l y c o l y t i c Pathway  24  12.  Shikimic-Prephenic  13. Degradation  A c i d Route to Aromatic  Amino Acids  of C a l y c a n t h i n e  29  14. E a r l y Proposals f o r the S t r u c t u r e of C a l y c a n t h i n e Calycanine 15. S y n t h e s i s of Calycanine 16. B i o g e n e s i s of Calycanthaceous 17. Condensation  25  and  30 31  Alkaloids  ofTetramino-dialdehyde  34 35  18. P r o t o n a t i o n of Tryptamine D e r i v a t i v e s  39  19.  Important Degradation  41  20.  Calycanthaceous  21.  S y n t h e s i s and Coupling of Oxytryptamine Urethan  Products of F o l i c a n t h i n e  A l k a l o i d s other than C a l y c a n t h i n e  44 48  vi  22. S y n t h e s i s o f F o l i c a n t h i n e  51  23. Kekule S t r u c t u r e s and E l e c t r o n Density o f Indole by Molecular O r b i t a l Approximations  53  24. Indole G r i g n a r d Reagent  54  25. P r e p a r a t i o n o f N -methyltryptamine  56  26. Fragmentation o f Nb-methyltryptamine  58  27. Mass Spectrum o f N -methyltryptamine  57  28. S y n t h e s i s o f Chimonanthine  60  b  b  and C a l y c a n t h i n e  29. Fragmentation o f Chimonanthine  62  30. Mass Spectrum o f N a t u r a l and S y n t h e t i c dl-Chimonanthine 63 31. Mass Spectrum o f N a t u r a l and S y n t h e t i c meso-Chimonanthine  64  32. N.M.R. Spectrum o f dl-chimonanthine  66  33. N.M.R. Spectrum o f meso-chimonanthine  67  34. Fragmentation o f C a l y c a n t h i n e  70  35. Mass Spectrum o f d l - c a l y c a n t h i n e  71  36. Mass Spectrum o f meso-calycanthine  72  37. N.M.R. Spectrum o f d l - c a l y c a n t h i n e  74  38. Mass Spectrum o f Dimer A  77  39. Mass Spectrum o f Dimer B  78  40. Suggested S t r u c t u r e s of Compounds A and B  79  41. Products from Attempted C o u p l i n g i n T e t r a h y d r o f u r a n  80  42. Mass Spectrum o f Compound D  81  43. Mass Spectrum o f Hodgkinsine  83  44. N.M.R. o f Hogkinsine  84  vii  PART B I'  Figure  Page  1. I n c o r p o r a t i o n of Tryptophan i n t o the Alkaloids 2.  Rauwolfia  I n c o r p o r a t i o n of Tryptophan i n t o Ibogaine Vindoline  119  and  121 123,124  3. Barger-Hahn-Woodward Theory  128  4. Wenkert-Bringi Hypothesis - S.P.F. Unit 5. Condensation of S.P.F. and 6.  In v i t r o Transannular  7. B i o g e n e s i s of Geranyl Wenkert Theory 8. Cyclopentanoid 9. 10.  129 ,130  Tryptamine  131  Cyclization pyrophosphate and  the Thomas-  134 135  Monoterpenes  Some Monoterpenes with the "Corynanthe" Carbon Skeleton  136  S c h l i t t l e r - T a y l o r - L e e t e Hypothesis  137  11. Delphinium A l k a l o i d s  142  12.  S t r u c t u r a l A n a l y s i s of Indole A l k a l o i d s  143  13.  E x o t i c Corynanthe Ring  144  14.  A l t e r n a t e Terpene  Precursor  148  15.  I n c o r p o r a t i o n of Mevalonic a c i d i n t o V i n d o l i n e  147  16. Feeding  Systems  of Mevalonate t o Vinca  Species  17.  Synthesis of 2 - C  18.  I n c o r p o r a t i o n of G e r a n i o l i n t o V i n d o l i n e  14  Geraniol  150 ,151 154 158  viii  ACKNOWLEDGEMENTS  I wish t o express my thanks t o P r o f e s s o r A . I . S c o t t , Dr. F.McCapra, and Dr.T.Money f o r the p r i v i l e g e  of working with  them and f o r t h e i r p a t i e n c e and e x c e l l e n t advice i n the d i r e c t i o n of t h i s  research.  Thanks are a l s o due t o Mr.P.Salisbury the capable  c u l t i v a t i o n of Vinca  f o r advice and f o r  rosea.  I am g r a t e f u l f o r having r e c e i v e d a N a t i o n a l Research C o u n c i l o f Canada s t u d e n t s h i p d u r i n g my s t u d i e s .  1  Introduction The  s t r u c t u r e s of some 1700  known at the present c o n t a i n i n g organic  time.*  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 are  A l k a l o i d s are b a s i c  nitrogen-  compounds u s u a l l y with the n i t r o g e n as part  of a h e t e r o c y c l i c system.  They are i n general  p r o d u c t s of higher p l a n t s .  . A few,  2  the  however, such as pyocyanin  (1) from the bacterium Pseudomonas aeruginosa  and  (2) the a c t i v e p r i n c i p l e of Mexican h a l u c i n o g e n i c of the genus P s i l o c y b e , occur i n d o l e a l k a l o i d , bufotenine  metabolic  psilocybine mushrooms  i n lower p l a n t forms.  (3), has  One  been i s o l a t e d from the venom  of the toad Bufo v u l g a r i s as w e l l as from p l a n t and sources.  some a l k a l o i d s occur  quaternary ammonium s a l t s or as t e r t i a r y amine The  taxonomic d i s t r i b u t i o n of a l k a l o i d s cannot be  c e r t a i n t y as the chemistry  of the  estimated estimate  fixed  f l o r a of only a few  m a j o r i t y of p l a n t s s t i l l . r e m a i n to be examined.  as  oxides.  of the world have been i n t e n s i v e l y s t u d i e d and the  with  regions  greater It has  been  that 10-20% of a l l p l a n t s c o n t a i n a l k a l o i d s . ^  This  i s l i m i t e d by a n a l y t i c a l methods which o f t e n f a i l  to  traces.^  A l k a l o i d s can occur of p l a n t s and  i n the r o o t , stem, l e a f , flower or seeds  accumulation occurs  juvenile tissues. and  not  C o l c h i c i n e (4) i s an example of an a l k a l o i d with a  n e u t r a l e x o c y c l i c amide n i t r o g e n while  detect  fungal  Some compounds g e n e r a l l y regarded as a l k a l o i d s are  basic.  any  simple  a l k a l o i d content  The and  d u r i n g the a c t i v e growth of  r e l a t i o n s h i p between the age  of the p l a n t  d i s t r i b u t i o n has been reviewed  by  2  Mothes. >  4  A maximum i n a l k a l o i d c o n t e n t o f t e n o c c u r s at o r about  the time o f f l o w e r i n g . seeds,  A l k a l o i d s then o f t e n accumulate i n the  A l t h o u g h the e f f e c t of growing c o n d i t i o n s on a l k a l o i d  c o n t e n t i s a complex m a t t e r , g e n e r a l l y c o n d i t i o n s which promote healthy  growth a r e b e s t f o r a l k a l o i d f o r m a t i o n .  F i g u r e 1.  Atypical Alkaloids.  Very l i t t l e i s known about the f u n c t i o n of a l k a l o i d s i n p l a n t s . Of s e v e r a l t h e o r i e s which have been advanced  none i s e n t i r e l y  3  satisfactory.  These t h e o r i e s i n c l u d e p r o t e c t i o n from i n s e c t s and  animals because of the t o x i c nature of the a l k a l o i d s and hence i n c r e a s e d chances f o r s u r v i v a l of the p l a n t , d e t o x i f i c a t i o n of p o t e n t i a l l y dangerous metabolic products by t r a n s f o r m a t i o n s i n t o a l k a l o i d s , and r e g u l a t i o n of metabolic p r o c e s s e s . ^  Objections  t o the f i r s t  theory i n c l u d e the o f t e n very s p e c i f i c a c t i o n of  alkaloids.  The a l k a l o i d s of Atropa b e l l a d o n a are extremely  t o x i c t o man yet c e r t a i n i n s e c t s eat the p l a n t with  impunity.  Ammonia i n high c o n c e n t r a t i o n s i s known t o be t o x i c t o c e l l s but glutamic and a s p a r t i c a c i d s have been r e c o g n i z e d as b e i n g e f f e c t i v e i n e l i m i n a t i n g f r e e ammonia.  The t o x i c i t y o f amino  a c i d s known t o be converted t o a l k a l o i d s has not been  demonstrated.  The argument a g a i n s t a r e g u l a t o r y r o l e f o r a l k a l o i d s i s the absence of a l k a l o i d s i n many p l a n t s .  A combination of these  three g e n e r a l r o l e s may e v e n t u a l l y be accepted as the r o l e of alkaloids i n plants.  In a number of cases p r o l i f e r a t i o n of  a l k a l o i d p r o d u c i n g p l a n t s has r e s u l t e d from t h e i r economic d e s i r a b i l i t y and r e s u l t a n t c u l t i v a t i o n by man.  Tea, c o f f e e ,  cocao, tobacco as w e l l as v a r i o u s n a r c o t i c s p e c i e s f a l l  into  this  category. While a l k a l o i d s are g e n e r a l l y harmless commercial on animals.  interest  t o p l a n t s , much of the  stems from t h e i r d i v e r s e p h y s i o l o g i c a l  effects  These compounds tend t o upset the balance o f  endogenous amines a s s o c i a t e d with the chemistry of the c e n t r a l nervous  system.^  The range of e f f e c t s produced  by a l k a l o i d s i s  d e t a i l e d i n p h a r m a c o l o g i c a l c o l l e c t i o n s ^ and many have been  4  medically useful. The  processes  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 p l a n t s  have l o n g been the s u b j e c t of study chemists and b i o c h e m i s t s . > ^ , 9 7  and  s p e c u l a t i o n among organic  A proper  understanding  of  the  pathways i n v o l v e d demands a knowledge of 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 various transformations study  and  a l s o of the mechanisms by which the  are c a r r i e d out.  Real progress  i n the  of a l k a l o i d b i o s y n t h e s i s began when organic compounds  l a b e l l e d with carbon-14 and  other 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 .  P r i o r to that time the  problems of b i o s y n t h e s i s were only the o b j e c t of s p e c u l a t i o n but are now  at the very  hypothesis  i n t e r e s t i n g stage  of development where  and experiment can be combined.  These problems have  been a t t a c k e d s u c c e s s f u l l y on a broad f r o n t and s e v e r a l e x c e l l e n t reviews have been w r i t t e n . » 1 0 - 1 5 2  secure  There now  seems t o be a  t h e o r e t i c a l b a s i s f o r the d e t a i l s of r e a c t i o n mechanisms  i n many i n s t a n c e s , and the formal r e l a t i o n s h i p between a l k a l o i d s and p r e c u r s o r s  i s becoming more c l e a r  Notable successes made i n the f i r s t  i n the s y n t h e s i s of n a t u r a l products  h a l f of t h i s century  were  but there i s g e n e r a l l y a  l a c k of s i m i l a r i t y between the s y n t h e t i c pathways used by  organic  chemists i n c o n s t r u c t i o n of the more i n t r i c a t e systems and  the  methods and r o u t e s presumably employed by nature.  this  During  time a s m a l l s c h o o l developed which was  i n t e r e s t e d i n the  s y n t h e s i s of n a t u r a l products  along l i n e s  a l s o t o represent  reasonable  patterned  biosynthetic routes.  considered Many  5  "biogenetic-type"  syntheses have been accomplished and these were  reviewed by van Tamelen i n 1961,10  I t seems p o s s i b l e that the  f u l l p o t e n t i a l i t i e s of b i o g e n e t i c a l l y p a t t e r n e d syntheses are only b e g i n n i n g t o be r e a l i z e d .  r e a c t i o n s and  Advantages a r i s e from  the r e a l i z a t i o n that even i n t r i c a t e molecules are c o n s t r u c t e d i n nature by a l i m i t e d number of simple, organic the  q u i t e understandable  r e a c t i o n s and that these r e a c t i o n s can be simulated i n  laboratory.  N a t u r a l products are u s u a l l y c o n s t r u c t e d i n  nature as a consequence of the chemistry of t h e i r Enzyme systems i n the c e l l  precursors.  have an a c t i v a t i n g e f f e c t , as w e l l as  s e r v i n g t o hold molecules i n a conformation f a v o u r a b l e  to  c e r t a i n r e a c t i o n s and can determine the s t e r e o c h e m i s t r y products.  The important c r i t e r i o n f o r a l a b o r a t o r y  i s the p r a c t i c a l one of whether or not the conceived  of  synthesis p l a n based  on suspected n a t u r a l p r o c e s s e s leads t o a new or improved l a b o r a t o r y method.  A knowledge of b i o g e n e t i c  a l s o i n v a l u a b l e i n the s t r u c t u r a l determination i s o l a t e d n a t u r a l products,  describe  an o r g a n i c  major a s p e c t s ,  of newly  as many p o s s i b l e s t r u c t u r e s on the  b a s i s of chemical evidence may The term " b i o g e n e t i c - t y p e "  principles i s  be u n l i k e l y i n terms of  synthesis  biogenesis.  has been s e l e c t e d t o  r e a c t i o n designed t o f o l l o w i n at l e a s t i t s  the b i o s y n t h e t i c pathways proved or presumed t o be  used i n the n a t u r a l c o n s t r u c t i o n of the end product.  Little  emphasis i s p l a c e d on reagents or c o n d i t i o n s - ^ and success  may  depend upon u t i l i z a t i o n of r e a c t i o n types which p a r a l l e l enzymepromoted p r o c e s s e s by u s i n g reagents and c o n d i t i o n s not a v a i l a b l e  6  to  the l i v i n g system.  A few " p h y s i o l o g i c a l - t y p e " syntheses have  a l s o been accomplished  i n which not only p l a u s i b l e b i o - o r g a n i c  s u b s t i t u t e s a r e used but a l s o s p e c i f i c c o n d i t i o n s of temperature, pH,  d i l u t i o n , e t c . , which supposedly compare t o those o b t a i n i n g  i n the l i v i n g c e l l .  Laboratory syntheses which proceed under ±n_ v i v o  these c o n d i t i o n s are l i k e l y t o correspond t o spontaneous syntheses, that i s syntheses not n e c e s s a r i l y The  first  enzyme-catalyzed.^  example of a b i o g e n e t i c - t y p e s y n t h e s i s was d e v i s e d  and executed by Robinson  as e a r l y - a s 1917.  Tropinone  (5) was  o b t a i n e d i n one stage from s u c c i n a l d e h y d e , methylamine and a c e t o n e d i c a r b o x y l i c a c i d which were regarded as reasonable b i o g e n e t i c p r e c u r s o r s ^ (Figure 2). 1  soon a f t e r  T h i s was accomplished  W i l l s t a t t e r ' s e x c e e d i n g l y lengthy f i r s t  synthesis of  the compound.  5  F i g u r e 2.  B i o g e n e t i c - t y p e S y n t h e s i s o f Tropinone.  Many p o s t u l a t e s have been made c o n c e r n i n g b i o g e n e t i c r o u t e s and these arose from two, o f t e n c l o s e l y a l l i e d , approaches. first  The  i n v o l v e d i n s p e c t i o n o f the s t r u c t u r e s of a l k a l o i d s or other  compounds, s e e k i n g common s t r u c t u r a l u n i t s and s u g g e s t i n g  7  p o s s i b l e r e l a t i o n s h i p s of these u n i t s to simpler  n a t u r a l products.  Such deductions have been u s e f u l i n c o r r e l a t i n g d i f f e r e n t groups of a l k a l o i d s and  f o r p r e d i c t i n g new  a b a s i s f o r t r a c e r experiments. a c i d s , e s p e c i a l l y l y s i n e (S), and  tryptophan  s t r u c t u r e s as w e l l as  The  r e c o g n i t i o n of the amino  ornithine  (7), p h e n y l a l a n i n e  (6) as w e l l as other simple p l a n t bases, as  u n i t s from which a l k a l o i d s c o u l d a r i s e , l e d to information The  simple  important  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  the b a s i s of a u n i f y i n g r e a c t i o n mechanism.  Robinson^  c o u l d account f o r a wide  v a r i e t y of a l k a l o i d a l s t r u c t u r e s with N - h e t e r o c y c l i c amino a c i d p h e n y l a l a n i n e  decarboxylation  and  (9) can  an aldehyde  systems.  give an amine (10)  (4) by o x i d a t i o n .  by  Other  s y n t h e t i c r e a c t i o n s which are important keys to the s y n t h e s i s  a c i d s , and  condensations of c a r b i n o l a m i n e s  a c t i v e methylene of ketones or biosynthetic  been r e c o g n i z e d reviewsl ' 5  1 8 - 2 0  syntheses make use of c o u p l i n g  (-C(OH)N-) w i t h  i s the  An  subject  of s e v e r a l  i n c r e a s i n g number of  of p h e n o l i c  the  -keto acids.17,13  s i g n i f i c a n c e of phenol o x i d a t i o n s  and  of  /3-keto  a l k a l o i d s are a l d o l condensations between aldehydes and  The  on  that i f condensation of ^ - s u b s t i t u t e d ethylamines with  aldehydes c o u l d occur i n p l a n t s then one  The  (9),  concerning a l k a l o i d synthesis.  second approach was  recognized  forming  has  long  excellent  biogenetic-type  oxidative coupling.  i s p a r t i c u l a r l y important i n the  T h i s mode  f i e l d of a l k a l o i d s  as more than 10% of the known a l k a l o i d s can be d e r i v e d by a p p l i c a t i o n of the p r i n c i p l e of ortho-  and  para-C-C and  C-0  coupling,  8  by c o u p l i n g o f the a p p r o p r i a t e p h e n o l i c p r e c u r s o r s . ^ ) ^ ) ^ 1  COOH  COOH  COOH  Hr NH  2  Hr  ^1 NHr  8  F i g u r e 3.  Amino A c i d s and A l k a l o i d B i o s y n t h e s i s .  1  9  In c o n s i d e r i n g the mechanism of o x i d a t i v e d i m e r i z a t i o n must d i s t i n g u i s h between homolytic r a d i c a l i n s e r t i o n ^ and  coupling  h e t e r o l y t i c coupling  o x i d a t i o n to a c a t i o n i c species)„ of a c a t i o n i c s p e c i e s capture  any  (radical coupling), (two  electron  Evidence f o r the i n c l u s i o n  i s l a c k i n g and  the i n a b i l i t y of A r O  t h i s two  electron oxidation.  Although r a d i c a l i n s e r t i o n cannot be d i s r e g a r d e d  p-cresol veratrole  to  +  n u c l e o p h i l e other than phenol anions o f f e r s some  c i r c u m s t a n t i a l evidence a g a i n s t  likely.  one  i t seems un-  Evidence i n c l u d e s the absence of c r o s s c o u p l i n g when (13)  i s o x i d i z e d i n the presence of a l a r g e excess of  (14), and  i n t e r n a l c o u p l i n g of the phenol  i t s monomethyl ether only d i m e r i z e s . ^  (15),  while  E l e c t r o n s p i n resonance  2  s p e c t r a of phenols undergoing o x i d a t i o n i n a l k a l i n e s o l u t i o n give d i r e c t evidence of r a d i c a l i n t e r m e d i a t e s .  The  o x i d a t i o n of  phenols or of phenol anions by reagents capable of r e d u c t i o n one-electron spreading  a f f o r d s mesomeric phenol r a d i c a l s s t a b i l i z e d  of the odd e l e c t r o n by resonance over the ortho  para p o s i t i o n s of the aromatic r i n g .  Coupling  i n the  or para p o s i t i o n or on the oxygen i s then p o s s i b l e . s t u d i e s by E.Muller hyperfine  and  his colleagues  by  by and  ortho  Detailed  on the a n a l y s i s of  s p l i t t i n g of e l e c t r o n s p i n resonance s p e c t r a of these  r a d i c a l s have shown that the f r e e e l e c t r o n d e n s i t y i s g r e a t e r at the para than at the ortho p o s i t i o n while shows a s m a l l but  non-zero d e n s i t y . ^ , 2 2  the meta p o s i t i o n  The  most v e r s a t i l e  reagents f o r o x i d a t i v e c o u p l i n g are a l k a l i n e potassium cyanide and  ferric chloride.  Other oxidants  ferri-  which have been  10  ArO"  + Fe(CN)  Homolytic  3-  ArO-  g  Coupling  2 ArO-  (ArO)  + Fe(CN) 2  Radical Insertion  ArO-  + ArO"—= *(ArO)  H e t e r o l y t i c Coupling  ArO  + »ArO  ArO  £  2  n  +  + ArO"  0CH  *(ArO)2  3  no c r o s s coupl i n g (same products as f o r oxidation of p-cresol) i n absence of veratrole  14  13  r=^  ^C__/^  H0  (CH2)4  46  (CH ) 2  ~^v__/^  0 R  15 when R =H, not when R = CHg  F i g u r e 4. A l t e r n a t i v e Mechanisms and Evidence I n s e r t i o n f o r P h e n o l i c O x i d a t i v e Coupling.  Against R a d i c a l  11  used i n C-C, C-0, and C-N f o r m a t i o n  a r e manganese and l e a d  d i o x i d e s , c e r i u m (IV) and vanadium (V) s a l t s , l e a d t e t r a a c e t a t e and Fenton's r e a g e n t . Aminophenols and amines a l s o c o u p l e v i a mesomeric Simple a r o m a t i c  amines ^  and o-aminophenols ^4  radicals.  give r i s e to  d i m e r i c p h e n a z i n e s (16) and phenoxazones r e s p e c t i v e l y . P a r t i c i p a t i o n of 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 of the morphine a l k a l o i d s has been demonstrated and a b i o g e n e t i c type s y n t h e s i s o f t h e b a i n e  (22) has been a c h i e v e d .  s e r v e s t o i l l u s t r a t e the u t i l i t y o f t h e p h e n o l i c  F i g u r e 5.  This coupling  C o u p l i n g o f Mesomeric P h e n o l and A r y l a m i n e R a d i c a l s ,  12  concept.  The  r e l a t i o n s h i p between b e n z y l i s o q u i n o l i n e s  and morphine (23) was  first  suggested by G u l l a n d and  and used t o deduce the c o r r e c t s t r u c t u r e f o r the  (12,17)  Robinson*  5  latter.  D e t a i l s of p o s s i b l e mechanisms f o r the c o u p l i n g were d i s c u s s e d by Barton and  Cohen*** and  d i p h e n o l i c base by ether  are i l l u s t r a t e d by c o n v e r s i o n  (17, R = Me)  formation  i n t o the dienone  (20) and a p p r o p r i a t e  of the proposed t r a n s f o r m a t i o n  of the dienone  "open" form and  The  (21)  dienone was  c o u l d be converted  (19)  Modifications into  f o l l o w e d by  found t o e x i s t  i n t o thebaine  the  followed  reduction.  morphine i n v o l v e r e d u c t i o n to the d i e n o l h y d r a t i v e rearrangement.  (19)  of  (22)  dei n the  under  very m i l d l a b o r a t o r y c o n d i t i o n s and  thence t o morphine  (23) i n  r e a l i z a t i o n of the l a t t e r scheme.  Barton and  t h e i r co-workers have independently  demonstrated the p a r t i c i p a t i o n  Battersby  and  of 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 of these alkaloids?-'*> 20,26  T  h  benzylisoquinoline precursor  e  (17, R = Me)  with carbon-14 and  t r i t i u m was  i n t o thebaine  i n Papaver somniferum.  (22)  of o x i d a t i v e c o u p l i n g was laudanosoline  l e s s e f f i c i e n t l y than the base (17, R =  (17, R = Me)  (18) was  not  u s i n g manganese d i o x i d e , as determined by 2 5  nor-  Me).  incorporated.  The  of t o t a l l y s y n t h e t i c o r i g i n  c o u l d be o x i d i z e d to the racemic dienone  dilution.  The p a r t i c i p a t i o n  i n c o r p o r a t e d more e f f i c i e n t l y  L a b e l l e d tetrahydropapavarine l a b e l l e d precursor  (0.14% i n c o r p o r a t i o n )  i n f e r r e d from experiments where  (17, R = H) was  than t y r o s i n e but  converted  labelled  (19)  i n 0.024% y i e l d  radiochemical  13  18  C  H  3 ° \  r  ^  OCH,  1  CH3O\J  CH o 3  20 CH30  CHO.  CH O^^X 3  21 F i g u r e 6.  22  thebaine  23  morphine  Morphine A l k a l o i d s , Synthesis and B i o s y n t h e s i s  14  Indole A l k a l o i d s A l a r g e number o f a l k a l o i d s o c c u r i n g i n nature aromatic  contain  r i n g s and of these a c o n s i d e r a b l e p o r t i o n c o n t a i n  the i n d o l e or d i h y d r o i n d o l e nucleus.  T h i s l a r g e f a m i l y of  i n d o l e a l k a l o i d s has been i s o l a t e d from more than twentyf i v e genera of p l a n t s and more than three-hundred s t r u c t u r e s have been e l u c i d a t e d , as l i s t e d by Hesse i n a very u s e f u l recent publication.27  No i n d o l e a l k a l o i d has a c q u i r e d the i l l i c i t  commercial n o t o r i e t y o f the p h e n y l a l a n i n e  d e r i v a t i v e morphine  (23) or the a v a i l a b i l i t y of i t s methyl e t h e r , codeine, i s so commonly used as an a n a l g e s i c .  However, some u s e f u l  and well-known drugs are i n d o l e a l k a l o i d s . has been used as a stimulant f o r vermin. Indians  which  Strychnine  (24)  f o r the heart and a l s o as a p o i s o n  Curare i s the name given by South American  i n the Amazon and Orinoco  v a l l e y s to concentrated  aqueous e x t r a c t s used as arrow poisons s k e l e t a l muscle p a r a l y s i s .  which produce c a r d i a c or  Over f o r t y a l k a l o i d s which bear  s t r i k i n g resemblances t o s t r y c h n i n e have been i s o l a t e d i n recent years  from the p a r t i c u l a r l y potent  (25) was employed as an Reserpine  Calabash c u r a r e .  Yohimbine  a p h r o d i s i a c i n v e t e r i n a r y medicine.  (26), which i s but one o f the a l k a l o i d s o f the Indian  Snakeroot, Rauwolfia  s e r p e n t i n a , was widely used i n n a t i v e  medicine, u s u a l l y as a s e d a t i v e . hypertension  I t i s u s e f u l i n treatment of  and of v a r i o u s mental d i s o r d e r s .  The a l k a l o i d s of  Ergot which i s a fungus p a r a s i t i c on c e r e a l grasses,  especially  r y e , have an o x y t o c i c e f f e c t u s e f u l i n c h i l d b i r t h .  These  15  25 yohimbine  F i g u r e 7.  Some I n d o l e  26  Alkaloids.  reserpine  16  a l k a l o i d s are l y s e r g i c a c i d amides. amides of l y s e r g i c a c i d amide which produces  Of the s e r i e s of s y n t h e t i c  (27) the most i n t e r e s t i n g i s the d i e t h y l -  symptoms l i k e those of s c h i z o p h r e n i a when  a d m i n i s t e r e d i n extremely s m a l l doses. **  Vinblastine ^  2  d i m e r i c a l k a l o i d produced clinically  by  2  (28) a  Vinca rosea L i n n i s used  as a potent a n t i - l e u k e m i c agent.  The  physiological  a c t i v i t y and d i v e r s i t y of i n t e r e s t i n g and e l a b o r a t e r i n g systems w i l l continue to provide i n t e r e s t  i n i n d o l e a l k a l o i d chemistry.  It has long been suspected that the i n d o l e a l k a l o i d s are d e r i v e d i n p a r t from tryptophan  (8) and t h i s has been confirmed  i n every case where t r a c e r experiments 30-33  have been c a r r i e d out. > 2  These a l k a l o i d s are formed i n nature from tryptophan i n  s e v e r a l ways. Although the i n d o l e a l k a l o i d s are c h a r a c t e r i z e d by e l a b o r a t e r i n g systems, Serotonin of  a few are simple d e r i v a t i v e s of tryptamine  (5-hydroxytryptamine)  i s a vascoconstrictive  (31).  principle  b l o o d , i s widely d i s t r i b u t e d i n animal t i s s u e and i s i n v o l v e d  i n the chemistry of the c e n t r a l nervous occurs i n p l a n t s .  Psilocybine  system.  5  (2) i s a r e l a t e d  It a l s o tryptamine  d e r i v a t i v e and r e q u i r e s no comment other than t o note o x i d a t i o n at  the 4 - p o s i t i o n of the i n d o l e nucleus, a p o s i t i o n which i s  important w i t h r e s p e c t t o s y n t h e s i s and b i o s y n t h e s i s of l y s e r g i c acid. was  Gramine (32) i s a d e g r a d a t i o n product of tryptophan  the s u b j e c t of some of the e a r l i e s t  tracer studies.^4  and The  most s i g n i f i c a n t r e s u l t of these s t u d i e s i s p r e s e n t e d i n a recent paper by 0'Donovan and Leete^5 i  n  which a mixture of  tryptophan-@-3H and DL-tryptophan-(J-l C was 4  DL-  fed to intact barley  17  32  gure 8.  gramine  Tryptamine and Gramine from Tryptophan.  18  seedlings.  It was  e s t a b l i s h e d that a l l of the a c t i v i t y  was  l o c a t e d i n the methylene group of the gramine s i d e c h a i n . biosynthetic  hypothesis c o n s i s t e n t  by Wenkert^® and phosphate  i s a t t r a c t i v e i n that  (29) which i s a l s o i n v o l v e d  decarboxylation base  i t involves  proposed  pyridoxal  i n transamination  of amino a c i d s through formation of the  and Schiff  (30).  Under p h y s i o l o g i c a l c o n d i t i o n s harman (35) tryptamine  (25°  C and  pH  5-6)  can be produced i n v i t r o i n good y i e l d (31)  and  acetaldehyde.  reasonable b i o s y n t h e s i s (36), harmine it  with t h i s r e s u l t was  A  (37)  and  f o r the  :.,  ^ 3 - c a r b o l i n e a l k a l o i d s harmaline  harman (38)  obtained  L a b e l l e d t r y p t o p h a n i s a proven p r e c u r s o r The  from Peganum harmala,  recognized  (33)  synthesis.  of the c a r b o l i n e s  compounds, f o r i n s t a n c e „-keto a c i d s ,  or i n d o l e n i n e  (34)  derivatives i s  as the well-known Mannich r e a c t i o n of organic  Yohimbine i s a good example of a more complex c a r b o l i n e strychnine The  has  ergot  carbon u n i t . Floss?**  an i n d o l e n i n e  a l k a l o i d s are  a c i d i s used not compounds but  skeleton.(Figures  only  the  7 and  first  has  chemistry. while  9).  formed from tryptophan and  Their biogenesis  Weygand was  of  condensation of tryptamine with  aldehydes or other c a r b o n y l to y i e l d carboline  from  As t h i s i s a l s o a  stands as an e a r l y example of a b i o g e n e t i c - t y p e  Peganum h a r m a l a . ^  tetrahydro-  a  five-  been reviewed by Weygand  to demonstrate that  f o r the s y n t h e s i s  a l s o f o r the b i o s y n t h e s i s  showed a s p e c i f i c r a t e of i n c o r p o r a t i o n  of t y p i c a l  and  mevalonic isoprenoid  of a l k a l o i d s when he of 16%  i n t o some ergot  19  35 t e t r a h y d r o h a r m a n  36 h a r m a l i n e  37 harmine  38 harman  F i g u r e 9.  Condensation of Tryptamine and Aldehydes.  20  alkaloids. ^  Furthermore mevalonic a c i d i s i n c o r p o r a t e d i n t o  4  ergot  a l k a l o i d s v i a i s o p e n t e n y l or d i m e t h y l a l l y l pyrophosphate  (39). *> 4  The  4 2  mechanism of condensation and c y c l i z a t i o n i s  a q u e s t i o n which has  not been answered completely.  Electro-  p h i l i c s u b s t i t u t i o n of the i n d o l e r i n g i s d i f f i c u l t four and  favoured  psilocybine  (2.)  that tryptophan and  in position five. 4 3  The  discovery  f o l l o w e d by t r a c e r s t u d i e s  can be h y d r o x y l a t e d  4 4  in position i n nature of  demonstrated  i n nature i n the  4-position  such a r e a c t i o n c o u l d p l a y a p a r t i n the b i o g e n e s i s  ergot  alkaloids.  of tryptophan  The  other p o s s i b i l i t y  i s direct  with dimethylallylpyrophosphate  simultaneous d e c a r b o x y l a t i o n  the  condensation  e i t h e r by  at the 4 - p o s i t i o n of the i n d o l e r i n g or by a t t a c k  of  attack  with  at the Ot^carbon of the  tryptophan  s i d e - c h a i n f o l l o w e d by c y c l i z a t i o n to p o s i t i o n four which i s favoured and  f o r stereochemical  seven are e l e c t r o n i c a l l y The  majority  reasons even though p o s i t i o n s f i v e favoured.  of i n d o l e a l k a l o i d s c o n s i s t of a tryptamine  u n i t p l u s a nine or ten carbon u n i t condensed w i t h the of the tryptamine s i d e c h a i n and one,  two,  any  combination of p o s i t i o n s  or three of the i n d o l e system.  of t h i s ten carbon u n i t i s arranged i n one  The  carbon s k e l e t o n  of three  each of which can condense with i t s e l f and with the u n i t to give r i s e t o the m u l t i t u d e  nitrogen  patterns, tryptamine  of e l a b o r a t e r i n g systems  which make the i n d o l e a l k a l o i d s so i n t e r e s t i n g .  Part B of  t h i s t h e s i s i s concerned with e l a b o r a t i o n of the b i o g e n e s i s the non-tryptophan d e r i v e d p o r t i o n of these  of  indole alkaloids.  21  F i g u r e 10.  B i o g e n e s i s o f Ergot  Alkaloids.  22  The  Calycanthaceous  a l k a l o i d s are a s m a l l but  interesting  group of i n d o l e a l k a l o i d s d e r i v e d from condensation of tryptamine u n i t s .  two  Part A of t h i s t h e s i s d e s c r i b e s the  s y n t h e s i s of these a l k a l o i d s i n a simple way c l o s e l y r e l a t e d t o the way  total  which i s probably  i n which they are s y n t h e s i z e d i n  nature. The b i o s y n t h e s i s of the aromatic amino a c i d s has been worked out u s i n g b i o c h e m i c a l techniques which i n c l u d e f e e d i n g of l a b e l l e d p r e c u r s o r s , growth r e q u i r e m e n t s  i  and i s o l a t i o n of  c e r t a i n i n t e r m e d i a t e s from mutant s t r a i n s of E . c o l i . acid-5-phosphate  (44) and prephenic a c i d  (45) are  Shikimic  important  i n t e r m e d i a t e s i n t h i s metabolic pathway t o aromatic compounds which has become known as the s h i k i m i c - p r e p h e n i c or the carbohydrate r o u t e .  Shikimic acid  (43) i s d e r i v e d from phospho-  enolpyruvic acid  (41) and erythrose-4-phosphate  (42), both b e i n g  d e r i v e d from glucose by g l y c o l y s i s and through the pentose respectively. 11 and 12.  shunt  These metabolic pathways are o u t l i n e d i n f i g u r e s The mechanism of a r o m a t i z a t i o n of s h i k i m i c a c i d -  5-phosphate (44) t o a n t h r a n i l i c a c i d  (46) has not been  completely e l u c i d a t e d but the amino group i s g l u t a m i n e - d e r i v e d . A n t h r a n i l i c a c i d i s converted t o i n d o l e - 3 - g l y c e r o l phosphate through i t s r i b o n u c l e o t i d e . i s then r e p l a c e d by s e r i n e Prephenic a c i d  The  g l y c e r o l phosphate s i d e c h a i n  (48) y i e l d i n g tryptophan (6).  (45) i s d e r i v e d from s h i k i m i c  and phosphoenolpyruvate.  (47)  acid-5-phosphate  A r o m a t i z a t i o n w i t h l o s s of carbon  d i o x i d e f o l l o w e d by amination of the  Ct-keto group y i e l d s  23  tyrosine.  A r o m a t i z a t i o n w i t h l o s s o f carbon d i o x i d e and water  g i v e s p h e n y l p y r u v i c a c i d and p h e n y l a l a n i n e by subsequent amination.  Reductive dehydration gives cinnamic acid, ** 4  24  CH2OH  CHO  I I I I  H,OH  I  HCOH  C =0 "1 HOCH  HOCH  HCOH  I  HCOH  HCOH  HCOH CH OP0 H glucose-6-phosphat 2  PENTOSE PHOSPHATE SHUNT  COOH  I  3  CH 0P0 H 2  2  3  2  fructose-6phosphate GLYCOLYSIS  HCOH  I  "transketolase thiamine pyrophosphate  HOCH  I  HCOH j HCOH  I  gluconic acid-6phosphate  I  HCOH  CH OP0 H 2  CH 0P0 H ^ — * CH20H C 0 C=0 2  3  2  I  CH20H t r a n s k e t o l - HCOH C=0 ase"  2  2  V  2  HCOH  HCOH  HCOH  I  HCOH  HCOH CH OP03H ribose-5phosphate 2  Figure  2  L OP03H 2  2  diydroxyacetone phosphate — ^ F i g u r e 12 CHO HCOH  HOCH  * I  A  C=0  I  —t— CH2OPO3H2 COH r * HCOH HOCH COH \ glyceraldehydeI 3-phosphate(40) HI \ HCOH CH OP03H CH2OPO3H2 \ ^ V r i b u l o s e - 5 - xylulose-5-phosphate phosphate CH 0H CHO C=0 I  3  CH2OH  CHO  2  'transketolase"  HCOH  I  transHCOH aldolase" |  > CH2OPO3H2  erythrose-4-phosphate (41)  HCOH  CH OP0 H 2  3  2  sedoheptulose7-phosphate  11. Pentose Shunt and the G l y c o l y t i c Pathway,  25  CHO  COOH  I  HCOH  >  I  COOH  I  I  HCOH CH2OPO3H2  5-dehydroquinic  I  I  HCOH  I  HOCH I HCOH  acid  CHO  HCOH <  2  Krebs cycle  2  COOH I c=o CH  +  'H phosphoenol pyruvic acid (41)  CH2OH  COOH  a c e t y l CoA  HC OPO3H2  HCOPO3H2  I  CH2OPO3H2 glyceraldehyde -3-phosphate (40)  COOH  CH OP0 H 2  3  2  erythrose-4phosphate (42)  I  HCOH  I  CH OP0 H 2  3  2  OOH  HOOC  CH-C-COOH 2  I'  tyrosine ^phenylala-  nine  H 0 P0 2  43 OH shikimic acid ("N" f r o m glutamine  3  cinnamic acid  OH  44 OH shikimic acid phosphate  / COOH  5-  4.5  0  H  prephenic  acid  HOOC OH .CHOH CHOH CH Q  CH OP0 H 2  3  s  NH 46  2  anthranilic acid  2  anthranilic nucleotide  ribo-  COOH  CHOH CHOH  CHNHr  CH OP0 H  CH OH  2  2  48  93 H  anthranilic 1-deoxyribonucleotide  3  2  47 indole-3-glycerol phosphate H  tryptophan Figure  12. S h i k i m i c - P r e p h e n i c  Acid  Route  t o A r o m a t i c Amino  Acids  PART A  Calycanthaceous A l k a l o i d s : A T o t a l S y n t h e s i s and B i o s y n t h e t i c  Model  26  Introduction The  d e t e r m i n a t i o n of the s t r u c t u r e of c a l y c a n t h i n e  (730Q ,  which i s the p r i n c i p a l a l k a l o i d of the b o t a n i c a l order Calycanthaceae  may be c o n s i d e r e d one of the c l a s s i c a l problems  of a l k a l o i d chemistry, while the deduction of the c o r r e c t s t r u c t u r e stands as a t r i b u t e t o the power of modern b i o g e n e t i c and m e c h a n i s t i c theory„  The seeds of Calycanthus  glaucus  W i l l d . , which i s a shrub n a t i v e t o Georgia, North C a r o l i n a and Tennessee, a t t r a c t e d a t t e n t i o n because of t h e i r nature.  poisonous  C a l y c a n t h i n e , the a c t i v e p r i n c i p l e , was f i r s t  from these seeds by G.R.Eccles i n 1 8 8 8 . ^  isolated  i t was not u n t i l  1952, however, that a deduction of the c o r r e c t s t r u c t u r e was first  made by R.B.Woodward  products at Harvard, calycanine i n 1960.49  48  i n an advanced course on n a t u r a l  a f t e r s y n t h e s i s of the d e g r a d a t i o n  (69) was accomplished  product  i n c o r r o b o r a t i o n and p u b l i s h e d  The same s u g g e s t i o n was made by R.Robinson and  H.J.Teuber i n 1 9 5 4 .  50  The chemical and s p e c t r a l  evidence  presented by Woodward i n 1960 f o r the s t r u c t u r e and c o n f i g u r a t i o n of c a l y c a n t h i n e was then confirmed by x-ray a n a l y s i s of the dihydrobromide  dihydrate.^l  The a b s o l u t e c o n f i g u r a t i o n has  a l s o been determined.^2 Before the advent  of modern p h y s i c a l t o o l s , s t r u c t u r e s of  o r g a n i c compounds were based weight,  on elemental a n a l y s i s ,  molecular  chemical t e s t s f o r f u n c t i o n a l groups and degradation  t o produce known compounds or at l e a s t s i m p l e r compounds, whose s t r u c t u r e or s t r u c t u r a l d e t e r m i n a t i o n would p r o v i d e c l u e s t o the  27  i n i t i a l molecular framework.  S t r u c t u r a l p r o p o s a l s based  t h i s evidence were then checked by s y n t h e s i s . of  The  c a l y c a n t h i n e became evident from the remarkable  nitrogenous h e t e r o c y c l e s produced  on degradation.  on  complexity v a r i e t y of The  f o r m a t i o n of both i n d o l e and q u i n o l i n e d e r i v a t i v e s c r e a t e d substantial d i f f i c u l t i e s  i n i n t e r p r e t a t i o n and  b i z a r r e s t r u c t u r a l p r o p o s a l s , eg. 59-65. d e g r a d a t i v e work are found i n "The It led 1905  l e d t o some  Reviews of t h i s  Alkaloids".  5 3  i s i n t e r e s t i n g t o examine some of the evidence which  t o the p r o p o s a l and r e j e c t i o n of these s t r u c t u r e s . Gordin  T h i s was i n 1939  5 4  a s s i g n e d the formula  later doubled  5 5  H  c o n s t i t u t i o n was  to calycanthine.  then f i n a l l y  t o i t s present C 2 2 2 6 4 N  5 6  In  T  r e v i s e d by Barger e_t a l . h  e  f  i  r  s  t  c l u e t o the  a f f o r d e d by the b e n z o y l a t i o n of c a l y c a n t h i n e  and o x i d a t i o n of the product w i t h potassium  permanganate,  57  y i e l d i n g a product shown to be i d e n t i c a l with s y n t h e t i c N-benzoyl-N-methyltryptamine  (49).  I t was  a l s o known by  formation of d i n i t r o s o d e r i v a t i v e s and Z e r e w i t i n o f f d e t e r m i n a t i o n of two a c t i v e hydrogens, n i t r o g e n atoms were secondary.  From t h i s the probable  i n the molecule of the grouping 59 was by the p r o d u c t i o n of ^ - c a r b o l i n e dehydrogenated  w i t h selenium.  that two of the f o u r  deduced and  presence  supported  (51) when the molecule i s I s o l a t i o n of i d e n t i c a l  when e i t h e r c a l y c a n t h i n e or tryptamine  (31) i s heated with  p h t h a l i c anhydride gave f u r t h e r support. lime on b e n z o y l c a l y c a n t h i n e produced  substances  The a c t i o n of soda  2-phenylindole  (52)  and  28  quinoline (50) and  (53) while c a l y c a n t h i n e y i e l d e d N^-methyltryptamine a s m a l l q u a n t i t y of a base C^2^10^2>  t o be 8-methyl-£-carboline56 but R e p e t i t i o n of t h i s degradation The  first  a t  l a t e r d i s p r o v e d by  assumed  synthesis.58  y i e l d e d mainly norharman (54). ® 5  weak base c a l y c a n i n e , C 2 2 1 0 2 (69) i s produced when H  N  c a l y c a n t h i n e i s p y r o l y z e d , or heated with l e a d oxide, oxide, sulfur, ** s e l e n i u m , 5  skatole  6 0  (55), 3 - e t h y l i n d o l e  or z i n c d u s t . (56) and  formed by the a c t i o n of selenium In 1939 was  a d i - i n d o l y l e n e (60)  (3-Carboline  lepidine ( 5 7 )  (Figure  Barger, M a d i n a v e i t i a  6 1  and  copper  6 1  13).  Streuli  5 6  assumed c a l y c a n i n e  ( c o n t a i n i n g ' a q u i n o l i n e nucleus) which,  t e n t a t i v e l y advanced to represent  calycanthine.  (61),  This  s t r u c t u r e had s e v e r a l p o i n t s a g a i n s t i t , i n c l u d i n g the r e a c t i o n of c a l y c a n t h i n e with E h r l i c h ' s reagent  only on h e a t i n g  that c a l y c a n t h i n e must be s u b s t i t u t e d i n the of the i n d o l e  c_ and  implying  ^positions  nucleus.  T h i s l e d to the p r o p o s a l of 63 as a more l i k e l y a l t e r n a t i v e although  the accompanying p r o p o s a l  for calycanine  (62) as a  l e p i d y l - / 3 - c a r b o l i n e r e q u i r e d an e m p i r i c a l formula  C21H15N3  s y n t h e s i s 62 proved to be q u i t e d i f f e r e n t  61  from c a l y c a n i n e .  On 62  i s i n t e n s e l y f l u o r e s c e n t i n n e u t r a l or a c i d s o l u t i o n s whereas c a l y c a n i n e i s only s l i g h t l y f l u o r e s c e n t and t h e r e f o r e not even a c a r b o l i n e d e r i v a t i v e .  6 0  are a l s o  with a methylamino s i d e c h a i n and a fused p i p e r i d i n e r i n g was  (51),  S t r u c t u r e 64,  probably  C15H10N2, was  then proposed f o r c a l y c a n i n e . Furthermore both of the s t r u c t u r a l p r o p o s a l s  for caly-  29  49  51  |3-carboline  53 q u i n o l i n e  F i g u r e 13.  Degradation  50  N^-methyltryptamine (dipterin)  52  2-phenylindole  54 norharman  of C a l y c a n t h i n e .  30  64 F i g u r e 14. and  65  E a r l y P r o p o s a l s f o r the S t r u c t u r e o f C a l y c a n t h i n e  Calycanine.  31  canthine m.p.  (61 and 63) were r e n d e r e d  115-6° C  6 2 w a s  u n l i k e l y when a base, C12H10N2,  o b t a i n e d by o x i d a t i o n of c a l y c a n t h i n e  s i l v e r a c e t a t e i n 1% a c e t i c a c i d s o l u t i o n s .  by  T h i s base which  f u r t h e r o x i d i z e d by a l k a l i n e p o t a s s i u m permanganate t o N - o x a l y l a n t h r a n i l i c a c i d and ammonia *^ has been shown by s y n t h e s i s 6  be 3 - ( N - m e t h y l ) - 4 - p y r r o q u i n o l i n e product  (58).  This  6 4  to  degradation  c o u l d o n l y be d e r i v e d from 61 or 63 by a s e r i e s of h i g h l y  unusual transformations.  More r e c e n t s p e c t r o s c o p i c p r o p e r t i e s  as w e l l as f u n c t i o n a l group a n a l y s i s and c o l o u r r e a c t i o n s a l s o exclude The  these  formulations.  c o r r e c t s t r u c t u r e of c a l y c a n t h i n e was  b i o g e n e t i c and m e c h a n i s t i c c a l y c a n i n e was  68 F i g u r e 15.  p r i n c i p l e s a f t e r the s t r u c t u r e of  f i n a l l y established.  S y n t h e s i s of  deduced on  69 Calycanine.  An x - r a y  calycanine  examination  6 5  32  of c a l y c a n i n e i n 1941 showed the molecule t o be c e n t r o symmetrical  and s t r u c t u r e 69 was f i n a l l y e s t a b l i s h e d by  s y n t h e s i s ® from l e u c o - i s o i n d i g o (60)®6(Figure 15).  Leuco-  4  i s o i n d i g o i s i s o m e r i z e d by h e a t i n g f o r f i v e hours i n 4 N h y d r o c h l o r i c a c i d t o the  6~-l "tam 67.  Reduction  ac  aluminum hydride i n t e t r a h y d r o f u r a n g i v e s the chrysene  with  lithium  hexahydrodiaza-  (68). Dehydrogenation with p a l l a d i u m c h l o r i d e i n  h y d r o c h l o r i c a c i d 67 hexahydroderivative  o  r  over m e t a l l i c palladium®** converted the  smoothly i n t o 6,12-diazachrysene  (69) which  was i d e n t i c a l i n a l l r e s p e c t s t o c a l y c a n i n e d e r i v e d from c a l y canthine.  Comparison o f the u l t r a v i o l e t  AjJJ|£  N  canthine;  252, 310  m/JL>  1,2,3,4-tetrahydroquinoline; 3.30 suggested  loge  XjJ°  H  m a x  spectrum o f c a l y -  . 4.26, 3.80 with that of  248, 299 m/x, l o g £  that the molecule c o n t a i n e d two aromatic  o r t h o - s u b s t i t u t e d with carbon and n i t r o g e n r e s i d u e s . s u b s t i t u t i o n p a t t e r n i s amply supported degradation  This  by c o n s i d e r a t i o n of the  b i o g e n e s i s was suggested  f o r c a l y c a n t h i n e by i t s  by Manske i n 1929 from the seeds of a t a x o n o m i c a l l y  d i s t a n t s p e c i e s , Meratia praecox, which i s a Compositae. f a c i l e formation of N -methyltryptamine D  formula,  rings  p r o d u c t s . ( F i g u r e 13).  A simple isolation  3.86,  m a x  The  (50) whose e m p i r i c a l  C11H14N2, i s one proton more than h a l f that of c a l y -  c a n t h i n e , as a degradation product occurrence  of c a l y c a n t h i n e , and i t s  as the n a t u r a l a l k a l o i d d i p t e r i n l e d t o the p r o p o s a l  of o x i d a t i v e c o u p l i n g o f two molecules as a reasonable  of Nb-methyltryptamine  b i o g e n e s i s f o r the a l k a l o i d . ® ' ^ 4  5  This  33  c o u p l i n g c o u l d be _<_' , OL(3 or @@' .  However on m e c h a n i s t i c  grounds e l e c t r o n s are p r i m a r i l y a v a i l a b l e f o r the o x i d a t i v e c o u p l i n g of two  i n d o l e s at the ( 3 - p o s i t i o n and the primary  product of such a c o u p l i n g would be 71. isomer may aldehyde  This calycanthine  be regarded as e q u i v a l e n t t o the t e t r a a m i n o d i -  72 v i a h y d r o l y s i s of the two  aminodialdehyde  imines.  This tetra-  i s capable of forming f i v e s t r u c t u r a l  isomers.  (Figure 17) through formation of i n t e r n a l N - a c e t a l s w i t h l o s s of two molecules of water.  The s t a b i l i t y of c a l y c a n t h i n e t o  a c i d suggested that i t has the c o n f i g u r a t i o n which i s most favoured on s t e r i c grounds.  Robinson  and T e u b e r  5 0  had  p r e f e r r e d s t r u c t u r e 73 8 while Harley-Mason i s r e p o r t e d t o have p r e f e r r e d 73(3.  51  As c a l y c a n t h i n e i s o p t i c a l l y a c t i v e the  forms Of-,(3 and 5must c o n t a i n a c i s - f u s i o n of the two r i n g s A and B. boat  In (3 and  six-membered  5 the r i n g s A and B must be i n the  form i n order t o allow the five-membered r i n g s t o reach  approximate  p l a n a r i t y whereas i n d  c h a i r s i s p o s s i b l e and hence was p r e f e r r e d s t r u c t u r e and  a conformation w i t h c i s - f u s e d  Woodward's c h o i c e f o r the  configuration. ^ 4  F u r t h e r chemical evidence f o r the s t r u c t u r e 73oL  was  p r o v i d e d by o x i d a t i o n of c a l y c a n t h i n e by mercuric a c e t a t e i n aqueous a c e t i c a c i d , y i e l d i n g dehydrocalycanthine with the l o s s of two hydrogen atoms.  Because dehydrocalycanthine was  hydrolyzed by a l c o h o l i c potassium an amide a l c o h o l i t was r a t h e r than an amidine.  smoothly  hydroxide t o methylamine and  deduced that i t was  an enamine  T h i s c o n c l u s i o n was  supported  (74) by  34  F i g u r e 16.  B i o g e n e s i s of C a l y c a n t h a c e o u s A l k a l o i d s .  i  35  36  c o n v e r s i o n of NN'-dimethylcalycanthine to a c l o s e l y Only i n the case of 73oc  dehydro-compound.  formation v i a o x i d a t i o n p r e c l u d e d by s t e r i c A comparison calycanthine  of the n u c l e a r magnetic  (40 megacycles)  a l k a l o i d physostigmine  analogous  i s amidine considerations.  resonance  spectrum of  w i t h that of the analogous  natural  (75) r e v e a l e d c e r t a i n d i f f e r e n c e s i n the  methylene resonances.  In the case of physostigmine the near  p l a n a r i t y of the five-membered r i n g makes geminal methylene hydrogens  approximately e q u i v a l e n t w i t h c o r r e s p o n d i n g s m a l l  H 74 c o u p l i n g c o n s t a n t s , and r e s u l t s i n a p a i r of d i s t o r t e d s t r u c t u r e s one of which resonance.  (-N-CH2-) i s overlapped by the N-methyl  The complexity of the f i n e s t r u c t u r e  w i t h the methylene groups  associated  of c a l y c a n t h i n e i s however more  t y p i c a l of methylene groups which are p a r t of a r i g i d puckered six-membered r i n g where the two hydrogen methylene are non-equivalent. implicated. in I960  5 1  The  and  atoms of each  S t r u c t u r e s 73OL or 8 are hence  s t r u c t u r e and c o n f i g u r a t i o n 73oi' was  by x-ray  triplet  confirmed  analysis.  The a b s o l u t e c o n f i g u r a t i o n of c a l y c a n t h i n e (730L')  was  37  a s s i g n e d by S . F . M a s o n circular  d i c h r o i s m curve  absorption ation  of It  will  band o f  Moffit's  the  be f o u n d  asymmetric  of  which i s  potentially mesomeric  striking  shown  the  isomers  (73).  other  nature  (ftxDn  coupling and t h i s  that  is,  there gives  of  are rise  structure  always  i s o m e r s must  be One o f  calycanthine  strongly  is  its  Natural chimonanthine  7 1  It  d-calycanthine the  two  exception  of  (CoGn = , - 3 2 9 ° ) .  these a l k a l o i d s belong to have t h e  72  are  be  optically active.  +684°).  =  that  they  or  natural  by i s o m e r i z a t i o n o f  4 8  isomer to  there  oxidative  isomers  and N - m e t h y l  d i s y m m e t r i c and hence  of  levorotatory  since  With the  racemic  properties  7 0  only other  one s e r i e s  consider-  calycanthine  intermediate  optically active  and t h e  stongly  the  must be n o t e d t h a t  of  theory. five  the  long wavelength  C h i m o n a n t h i n e (737)  structurally  dextrorotatory is  all  c e n t e r s p r o d u c e d by t h e  two s e r i e s  and s i g n s o f  a n i l i n e c h r o m o p h o r e and f r o m a  however r e p r e s e n t It  shape  w i t h the  coupled-oscillator  two d i a s t e r e o i s o m e r s  the  associated  i n nature.  identified.  73€.  from the  has b e e n p r e d i c t e d t h a t  chimonanthines  to  in 1962°^  has r e c e n t l y  into  been  1-chimonanthine  same e n a n t i o m o r p h i c  series,  same a b s o l u t e c o n f i g u r a t i o n .  Chimonanthine C h i m o n a n t h i n e was i s o l a t e d  i n 1960  from the  leaves  of 71  Chimonanthus This  fragrans  alkaloid,  metric  Lindle  C22 26^4J  titration  h  to  m  -P-  (Meratia praecox 188-189°  Rehd.  and W i l s . )  C was shown by p o t e n t i o -  be a weak d i a c i d i c b a s e  of  e q u i v a l e n t weight  38  173.  . The u l t r a v i o l e t spectrum  and underwent a hypsochromic (0.5 N HC1).  had a maxima at 246 and 304  shift  of about  T h i s type of spectrum  7 myu. i n d i l u t e  acid  had e a r l i e r been noted as  c h a r a c t e r i s t i c of a s o - c a l l e d Ph-N-C-N type of c h r o m o p h o r e and was  s u b s t a n t i a t e d by l a t e r work.  and analogues  such as e c h i t i n o l i d e >  calycanthine, c a l y c a n t h i d i n e  5 6  i n d o l i n e type u l t r a v i o l e t ca. 250 and 300 mjLL.  Physostigmine  7 4  coryminine,  and f o l i c a n t h i n e  t r i c y c l i c p y r r o l o i n d o l e system  m/u,.  7 6  7 5  (75)  as w e l l as  c o n t a i n i n g the  In d i l u t e a c i d i c s o l u t i o n , however, the  Hodgson and  undergo a  hypsochromic  Smit 72 attributed this  b  7 1  s p e c t r a i n n e u t r a l s o l u t i o n w i t h "X  h  t o p r o t o n a t i o n of N  72  (75) a l l showed m o d i f i e d  s p e c t r a of physostigmine and i t s congeners s h i f t of 8-10  m/x.  shift  (78), the p o s i t i v e charge on which i s then  s u f f i c i e n t l y close to p a r t i a l l y the lone p a i r of e l e c t r o n s on N  inhibit a  the d e r e a l i z a t i o n of  over the aromatic nucleus.  An  e x c e l l e n t study has r e c e n t l y been made on the p r o t o n a t i o n of tryptamine d e r i v a t i v e s i n a c i d i c media by Jackson and  A.E.Smith  77  which c o n f i r m s the e a r l i e r i n t e r p r e t a t i o n by G.F.Smith as t o the reasons f o r the s p e c t r a l s h i f t . important i n that i t was solution  (1 M HC1)  T h i s study i s a l s o very  noted that i n more s t r o n g l y  the s p e c t r a of these physostigmine  acidic derivatives  become very s i m i l a r t o those of tryptamines i n s t o n g l y a c i d i c s o l u t i o n , and of i n d o l e n i n e s .  T h i s was  clearly  consistent  w i t h the opening of r i n g C t o g i v e 3H-indolium s a l t s  (79).  T h i s a c i d induced r i n g opening i s e s s e n t i a l t o the b i o g e n e t i c argument f o r i n t e r c o n v e r s i o n of the calycanthaceous a l k a l o i d s .  39  Other s p e c t r o s c o p i c  s t u d i e s had a l s o shown that the p r o t o n a t i o n  of i n d o l e s i n s t r o n g l y a c i d i c media occurs e x c l u s i v e l y at the 3 - p o s i t i o n with the formation  o f t h e corresponding  3H-indolium  ( i n d o l e n i n e ) salt. ®* ** 7  7  R =R =CHo  75 p h y s o s t i g m i n e  78  2  1  79  R =OCONHMe 3  76 esermethole Ri=R =CH3 R3=OCH3 2  77  deoxynereseroline R =CH R!=R =H 2  3  3  F i g u r e 18.  Protonation  o f Tryptamine D e r i v a t i v e s .  Chimonanthine a l s o was s h o w n  71  t o c o n t a i n two N-methyl  groups and had a sharp band at 3440 c m  - 1  i n the i n f r a r e d ,  i n t e r p r e t e d as an aromatic N-H s t r e t c h i n g frequency.  Reduction  w i t h z i n c and h y d r o c h l o r i c a c i d gave a q u a n t i t a t i v e y i e l d o f an i n d o l i h e i d e n t i f i e d by comparison o f the i n f r a r e d and u l t r a v i o l e t s p e c t r a as  3-2'-methylaminoethylindoline, i n d i c a t i n g that  the s k e l e t o n o f chimonanthine was composed o f two tryptamine units.®  0  S t r u c t u r e s 73CL  o r 73 5 were proposed on the b a s i s o f  40  dehydrogenation with z i n c dust at 330° C. gave c a l y c a n i n e quinoline  from c a l y c a n t h i n e  skeleton,  and  conditions  with i t s preformed  quinolo-  but f a i l e d t o give more than a t r a c e with  e i t h e r chimonanthine or f o l i c a n t h i n e . presented that  These  Evidence was a l s o  f o l i c a n t h i n e was bis-N -methylchimonanthine a  the Hofmann degradation product of f o l i c a n t h i n e dimeth-  i o d i d e was assigned a s t r u c t u r e 80, based on i n d o l i n e than q u i n o l i n e ated  systems by s y n t h e s i s .  8 1  rather  T h i s evidence e l i m i n -  s t r u c t u r e s 73 £ and 73 (b . A d e t a i l e d x-ray a n a l y s i s o f chimonanthine dihydrobromide 82  e s t a b l i s h e d s t r u c t u r e 73 If as the c o r r e c t one f o r chimonanthine. F o l i c a n t h i n e (85) Folicanthine with a melting rotation  point  o f 118-119°  ttt-l = -365° ( r e v i s e d ) ^ was f i r s t 8  D  leaves  of Calycanthus f l o r i d u s L. i n 1 9 5 1  leaves  of Calycanthus o c c i d e n t a l i s .  after i n i t i a l  studies'  7 2  7 6  and a  i s o l a t e d from the and l a t e r from the  The s t r u c t u r e proposed  was based on an i n c o r r e c t molecular  0  formula and on four d e g r a d a t i o n products which were not definitely identified. three  Subsequent i n v e s t i g a t i o n  8  4  identified  o f these compounds as N ,Nk-dimethyltryptamine (81), a  N j N ^ - t r i m e t h y l t r y p t a m i n e and N-methylnorharman (54) and t h i s a  w i t h an i n d o l i n e type of u l t r a v i o l e t of the s t r u c t u r e . give an  spectrum l e d t o r e f o r m u l a t i o n  In l a t e r work f o l i c a n t h i n e was found t o  86% y i e l d o f N , N ^ - d i m e t h y l i n d o l i n e a  with zinc i n hydrochloric  acid.  7 0  (81) on r e d u c t i o n  The presence of a Ph-N-C-N  41  (0^)2^3  (CH ) 3  PH3  2  +  CH  ( c k  3  3  )  >£5  CH  2  3  CHQ  2  (CH )2 3  N-dimethyl 73 7 dimethiodide  N-dimethyl 73$ dimethiodide (CH )  3  ^(CH ) 3  N(CH ) 3  2  2  ^N(CH ) 3  CHQ  N ( C H ) ^ N(CH ) 3  2 r  3  2  80  folicanthine  Zn/HCJ. NHCH, CH81  F i g u r e 19.  Important Degradation Products of F o l i c a n t h i n e  2  42  system-was v e r i f i e d by u l t r a v i o l e t potentiometric supported  s p e c t r a l measurements, and  t i t r a t i o n with a molecular  a d i m e r i c s t r u c t u r e but  structural proposal.  weight  l e d t o another i n c o r r e c t  I d e n t i f i c a t i o n of the product  7 0  b a s i c treatment of f o l i c a n t h i n e d i m e t h i o d i d e finally  determination,  8 0  by  from  synthesis  8 1  l e d t o the p r o p o s a l of bis-N-methyl c a l y c a n t h i n e  s t r u c t u r e s 73 8 or T f o r f o l i c a n t h i n e .  An i n f r a r e d  f o r f o l i c a n t h i n e d i f f e r i n g only i n the NH and from that of chimonanthine but  quite different  spectrum  N-methyl r e g i o n s from c a l y -  c a n t h i n e , very s i m i l a r r o t a t i o n s and easy r e d u c t i o n of both by z i n c and a c i d to i n d o l i n e s while c a l y c a n t h i n e i s u n a f f e c t e d l e d t o the c o n v i c t i o n that f o l i c a n t h i n e was chimonanthine (737).  The  r a p i d formation  i o d i d e s i n c o n t r a s t to the complicated 49  bis-N -methyl a  of normal dimeth-  r e a c t i o n s of  calycanthine  8 '  and  the s i m i l a r i t y of the mass spectrum t o that of  chimonanthine one-half  86  showing f a c i l e cleavage  the molecular  weight supported  F i n a l l y the s u c c e s s f u l m e t h y l a t i o n to f o l i c a n t h i n e confirmed Calycanthidine  t o a s t r o n g fragment at this structure,  by G.F.Smith ' of chimonanthine 0  t h i s s t r u c t u r e (73 7,85).  (84)  C a l y c a n t h i d i n e , m e l t i n g p o i n t 142° C, was by Barger i n 1939 Calycanthus  isolated  as the minor a l k a l o i d from the seeds of  floridus L..  based on a molecular u n t i l recently.  first  5 6  formula  A hexahydro  (3-carboline s k e l e t o n  C13H16N2 was deduced and  In work p u b l i s h e d  in 1962  83  accepted  calycanthidine  43  was  shown t o have the formula C23H23N4, and two Ph-N-C-N  chromophores from the u l t r a v i o l e t t i t r a t i o n and was  spectrum  and by p o t e n t i o m e t r i c  thus a f o u r t h member of the d i m e r i z e d t r y p t -  amine group of a l k a l o i d s .  Comparison of the i n f r a r e d  w i t h those of the very s i m i l a r chimonanthine a similar specific rotation  (CO._ = -317) D  and  spectrum  folicanthine,  t o chimonanthine  and  f o l i c a n t h i n e , r a p i d f o r m a t i o n of a normal d i m e t h i o d i d e and smooth r e d u c t i o n by z i n c and h y d r o c h l o r i c a c i d t o a mixture of i n d o l i n e s , demonstrated like structure.  that c a l y c a n t h i d i n e had a  I s o l a t i o n of  chimonanthine-  3-2'-methylaminoethylindoline  and l-methyl-3-2'-methylaminoethylindoline i n d i c a t e d that  caly-  c a n t h i d i n e r e p r e s e n t e d the i n t e r m e d i a t e stage i n the m e t h y l a t i o n of chimonanthine  t o f o l i c a n t h i n e and t h i s i s supported  n u c l e a r magnetic  resonance  by  and mass s p e c t r a l evidence, e s p e c i a l l y  the f a c i l e cleavage of the molecular i o n .  The m e t h y l a t i o n of  chimonanthine, t o c a l y c a n t h i d i n e and then t o f o l i c a n t h i n e been  accomplished.  Hodgkinsine  87  (86)  Hodgkinsine  with a m e l t i n g p o i n t 128° C and a s p e c i f i c  r o t a t i o n of +60° was  i s o l a t e d i n 1960  A u s t r a l i a n shrub, Hodgkinsonia  from the l e a v e s of an  frutescens F . M u e l l . .  b a s i s of a n a l y s i s and molecular weight molecular formula C22 26 4^^ H  system  has  N  from i t s u l t r a v i o l e t  a n c  *  s  n  o  w  On the  assigned a  *° c o n t a i n the Ph-N-C-N  n  spectrum.  i t was  8 8  85  t o be a d i a c i d i c base w i t h e i t h e r o n e  8 8  I t was or t w o  8 5  also reported NH  stretching  44  CH  CH  3  CH  3  CH  ?  F i g u r e 20.  C,H  3  3  84 c a l y c a n t h i d i n e  86  3  hodgkinsine  85  folicanthine  (not d e f i n i t e l y established) C a l y c a n t h a c e o u s A l k a l o i d s o t h e r than C a l y c a n t h i n e .  45  r e g i o n s i n t h e i n f r a r e d and t o be i s o m e r i c w i t h c a l y c a n t h i n e . F a i l u r e t o produce c a l y c a n i n e on d e g r a d a t i o n w i t h z i n c dust i n d i c a t e d i t p r o b a b l y d i d not have a q u i n o l o q u i n o l i n e s k e l e t o n . T h i s was t h e e x t e n t o f knowledge about h o d g k i n s i n e when t h e p r e s e n t s t u d i e s began.  Subsequent s t u d i e s by G . F i S m i t h  i n d i c a t e d t h a t h o d g k i n s i n e c o u l d be a d e h y d r o c a l y c a n t h i n e C22 24 4 H  N  a  n  d  e v e n t u a l l y a mass s p e c t r a l d e t e r m i n a t i o n i n d i c a t e d  t h a t h o d g k i n s i n e was a t r y p t a m i n e t r i m e r .  This conclusion,  s u p p o r t e d by n u c l e a r magnetic resonance data, l e d P r o f e s s o r Smith t o propose s t r u c t u r e 86 f o r h o d g k i n s i n e . support i s c o n t a i n e d i n t h i s  thesis.  8 9  Parallel  46  Discussion The  o b j e c t of t h i s work was  t o achieve  a model s y n t h e s i s  of  the calycanthaceous a l k a l o i d s along the l i n e s of the proposed b i o g e n e t i c scheme.49-50  From the onset the problem was  r e a c t i o n c o n d i t i o n s s i n c e the p o s t u l a t e of tryptamine had  always seemed reasonable  t h i s c l a s s of dimeric  indole a l k a l o i d s .  /3/5'-coupling  of  of  f o r the b i o s y n t h e s i s of Experimental evidence  that tryptamine i s the b i o l o g i c a l p r e c u r s o r came with  one  of these compounds  f e e d i n g of t r y p t o p h a n - ^ - C to Calycanthus floridus® 14  a f t e r s i m u l a t i o n of the b i o s y n t h e s i s had been achieved  0  i n the  laboratory. Many attempts had been made by s e v e r a l workers t o c o u p l i n g of v a r i o u s N - s u b s t i t u t e d  achieve  N-methyltryptamines by  exposure t o such o x i d a t i v e media as f e r r i c , e e r i e , and manganous s o l u t i o n s under v a r i o u s c o n d i t i o n s known to promote o x i d a t i v e c o u p l i n g of p h e n o l i c e i t h e r recovered  systems.'  used an o x i n d o l e  i o d i n e under b a s i c c o n d i t i o n s .  The  (92)  secondary amine and was  The  first  found by Hendrickson ejt  2  involving oxidation  Coupling  of the urethan of  gave the urethan p r o t e c t e d b i s o x i n d o l e  urethan grouping was  was  c o u p l i n g r e a c t i o n f o l l o w i n g the  precedent of sodiomalonate coupling®  tryptamine  Starting material  or degraded t o i n t r a c t a b l e mixtures.  p r a c t i c a l s o l u t i o n t o the problem was a_1^91,48 who  one-electron  used t o prevent o x i d a t i o n of  with oxy-  (94). the  a n i c e c h o i c e as i t can be reduced to  the r e q u i s i t e N-methyl at the same time that the o x i n d o l e reduced t o the r e q u i r e d o x i d a t i o n s t a t e with  is  l i t h i u m aluminum  47  hydride.  C o u p l i n g of the o x i n d o l e p r e c l u d e d the p o s s i b i l i t y  of  c o u p l i n g i n the ( X - p o s i t i o n . Oxytryptamine h y d r o c h l o r i d e based on an e a r l i e r p r e p a r a t i o n i e n t procedure. isatin  (91) was 9 3  This p r e p a r a t i o n i n v o l v e d condensation  (87) and m e t h y l c y a n o a c e t a t e  (88).  by a method  and developed i n t o a convenof  i n p i p e r i d i n e f o l l o w e d by  z i n c dust r e d u c t i o n i n a c e t i c a c i d t o acetate  prepared  methyl-3-oxindolylcyano-  A f t e r s a p o n i f i c a t i o n i n 2 N potassium  the r e s u l t i n g 3 - o x i n d o l y l c y a n o a c e t i c a c i d (89) was  hydroxide  decarboxyl-  a t e d by a d d i t i o n t o hot e t h y l e n e g l y c o l , y i e l d i n g 3 - o x i n d o l y l acetonitrile  (90).  T h i s n i t r i l e was  then reduced w i t h  p l a t i n u m d i o x i d e and hydrogen i n e t h a n o l i c h y d r o c h l o r i c a c i d y i e l d i n g oxytryptamine a l l yield.  h y d r o c h l o r i d e i n t h i r t y per cent  over-  S h a k i n g an aqueous a l k a l i n e s o l u t i o n o f o x y t r y p t -  amine w i t h e t h y l c h l o r o f o r m a t e  smoothly a f f o r d e d the  urethan  (92). The  o x i d a t i v e c o u p l i n g of the u r e t h a n  u s i n g c u p r i c , f e r r i c and s i l v e r i o n s . b u t y l hydroperoxide starting material.  (92) was  attempted  E l e c t r o l y t i c and t -  o x i d a t i o n s were a l s o f u t i l e y i e l d i n g o n l y The  oxindole enolate  ( 9 3 ) , preformed by  a d d i t i o n of sodium h y d r i d e t o the u r e t h a n , was s u c c e s s f u l l y coupled added.  t o 94 when a s o l u t i o n of i o d i n e i n benzene was  slowly  In view of the d i f f i c u l t y of a c h i e v i n g t h i s c o u p l i n g  u s i n g normal one e l e c t r o n ,. o x i d i z i n g agents a r a d i c a l c o u p l i n g process  as e n v i s a g e d  i n v o l v e d here.  f o r the b i o s y n t h e s i s i s p r o b a b l y  not  A c o u p l i n g of one mole of o x i n d o l e e n o l a t e  (93)  48  ^  CN CH COOCH3 piper-  CN ^CHCOOCH,  CN ^CCOOCH3  2  0  hi  Zn dust  * 2 N KOH  N-^O I  H 87  88 CH CN  id.  2  P#2  H HC1, EtOH  N-^O H  2  90  89  NH3CI  91  O ll  O  *H  CICOEt ^ N 1^ O H  aqueous Na C03, CHCI3 2  II  NH-C-OEt  92  NaH tetrahydrofuran r  HC-OET"  -N ^sX> 1  NHCOOEt  H benzene H CK.N  EtOOCNH  L1AIH4  0  73 7  ^NHCOOEt  1  H F i g u r e 21  94 d i a s t e r e o i s o r a e r s A and B S y n t h e s i s and Coupling o f Oxytryptamine Urethan(92)  49  w i t h a mole of 3 - i o d o o x i n d o l e c o u l d be the mechanism. y  Reduction o f the two d i m e r i c d i a s t e r e o i s o m e r s  (94) w i t h  l i t h i u m aluminum h y d r i d e i n t e t r a h y d r o f u r a n y i e l d e d mixtures of b a s i c p r o d u c t s whose chromophores  were not d e s t r o y e d i n  a c i d but showed a hypsochromic s h i f t c h a r a c t e r i s t i c of the Ph-N-C-N system which i n d i c a t e d c y c l i z a t i o n had o c c u r r e d . chromophores  The  of any p r o d u c t s which might a r i s e from t o t a l r e -  d u c t i o n of the o x i n d o l e s t o i n d o l i n e s without c y c l i z a t i o n should be d e s t r o y e d i n a c i d by p r o t o n a t i o n o f the a n i l i n e nitrogen. Of the s i x compounds i s o l a t e d from the mixture o b t a i n e d from r e d u c t i o n o f one d i a s t e r e o i s o m e r  (94) the major  product had a m o l e c u l a r weight of 344 which i s two l e s s than c a l y c a n t h i n e . significantly different  The u l t r a v i o l e t  hydrogens  spectrum was  from that of c a l y c a n t h i n e .  also  It c o u l d  be transformed i n t o an isomer a l s o i s o l a b l e from the mixture by b o i l i n g i n aqueous a c i d .  These compounds were formulated  as monoamidines of 73(3 and  5 respectively.  (82) ( 3 % ) , d l - c a l y c a n t h i n e  (730/)  (0.2%),  dl-Chimonanthine Nb-methyltryptamine  and a t h i r d dehydrocompound were a l s o i s o l a t e d from the mixture. The c r y s t a l l i n e product (6%) i s o l a t e d from r e d u c t i o n of the second d i a s t e r e o i s o m e r  (94) was  a c a l y c a n t h i n e isomer by  a n a l y s i s and mass spectrum but the i n f r a r e d spectrum was nificantly different  from c a l y c a n t h i n e .  peak i n the mass spectrum at m/e of the m o l e c u l a r weight, was  sig-  The preponderant  172, which r e p r e s e n t e d o n e - h a l f  f o r t y - f i v e times more i n t e n s e than  the parent peak, an i n t e n s i t y d i f f e r e n c e which can only be  50  r a t i o n a l i z e d i n terms o f the c a l y c a n t h i n e isomer 73 't with two halves j o i n e d together by a s i n g l e bond.  T h i s compound was  i s o l a t e d and c h a r a c t e r i z e d b e f o r e Smith had announced the i s o l a t i o n o f n a t u r a l chimonanthine and showed that i t was i n f a c t the T isomer of c a l y c a n t h i n e .  S i m i l a r but not i d e n t i c a l  t h i n - l a y e r chromatographic behaviour t o the n a t u r a l chimonanthine l e d t o the c o n c l u s i o n that t h i s s y n t h e t i c compound was mesochimonanthine. Implicit  i n the chemistry  o f the c a l y c a n t h i n e isomers i s  the p o t e n t i a l f o r e q u i l i b r a t i o n o f the f i v e isomers i n a c i d i c medium v i a s p e c i e s such as 71 and 72 (Figure 16). d l Chimonanthine was heated with d i l u t e h y d r o c h l o r i c a c i d (0.01 N) and  the r e s u l t i n g bases, separated  on t h i n - l a y e r p l a t e s , produced  a p a t t e r n o f f i v e or s i x s p o t s .  The major ones corresponded t o  chimonanthine  (40%), and N -methyltryptamine  (5%).  (25%), c a l y c a n t h i n e  b  When c a l y c a n t h i n e was s u b j e c t e d t o the same treatment a  virtually  i d e n t i c a l t h i n - l a y e r p a t t e r n was produced which  demonstrated the e q u i l i b r a t i o n nature When n a t u r a l d - c a l y c a n t h i n e  o f the i n t e r c o n v e r s i o n .  was i s o m e r i z e d  the generated  chimonanthine was found t o be l e v o r o t a t o r y demonstrating that both a l k a l o i d s have the same a b s o l u t e s t e r e o c h e m i s t r y non-epimerizable  centers.  9 1  ?  at the two  4 8  Since chimonanthine and c a l y c a n t h i d i n e have both been methylated t o y i e l d f o l i c a n t h i n e and chimonanthine a l s o s e r v e s folicanthine.  8 9  the s y n t h e s i s o f c a l y c a n t h i n e  f o r m a l l y as a s y n t h e s i s o f  51  A t o t a l s y n t h e s i s o f racemic f o l i c a n t h i n e has a l s o beenachieved  by T.Hino i n Japan t h r o u g h the 3 , 3 ' - d i s u b s t i t u t e d  3,3'-bisoxindole  (95)  8 1  '  y 4  p r e p a r e d by c o n d e n s a t i o n o f N-methyl-  i s a t i n and N - m e t h y l o x i n d o l e i n a c i d f o l l o w e d by  cyanoethylation.  F o r m a t i o n o f t h e S c h i f f base 96 f o l l o w e d by t r e a t m e n t w i t h m e t h y l i o d i d e and a c i d h y d r o l y s i s y i e l d e d a  bisoxindole(97)which  a f f o r d e d d l - f o l i c a n t h i n e on r e d u c t i o n . The f a i l u r e o f v a r i o u s N - s u b s t i t u t e d N-methyl  tryptamines  t o c o u p l e when exposed t o v a r i o u s r e a g e n t s known t o promote onee l e c t r o n o x i d a t i v e c o u p l i n g i n p h e n o l i c systems (page 9) s u g g e s t e d t h e absence o f the ( 3 - r a d i c a l under t h e s e c o n d i t i o n s . As a u t o x i d a t i o n ( i . e . f o r m a t i o n  o f f r e e r a d i c a l s ) i s enhanced by  95  anion formation  i t was d e c i d e d  t o make use o f t h e s a l t - l i k e  p r o p e r t i e s a s s o c i a t e d w i t h i n d o l y l magnesium h a l i d e s .  F i g u r e 22.  97 Synthesis of F o l i c a n t h i n e .  52  As p r e v i o u s l y mentioned the s t r u c t u r a l determination calycanthine  was based i n p a r t on a mechanistic  of  preference f o r  c o u p l i n g of /3-radicals o f Nk-methyltryptamine as opposed t o c o u p l i n g i n the c£r-position.  The preference  for  (3-coupling  i s based on the e s t a b l i s h e d a v a i l a b i l i t y of e l e c t r o n s i n t h i s p o s i t i o n and the assumption that an odd e l e c t r o n would a l s o have i t s highest  d e n s i t y i n the (3-position.  The e l e c t r o n  d e n s i t i e s f o r i n d o l e have been c a l c u l a t e d by the method o f molecular orbitals,®® and have e s t a b l i s h e d the e l e c t r o n e g a t i v i t y of the  /3-position.  A c o n s i d e r a t i o n o f Kekule resonance  hybrids  leads t o the same conclusion.®  7  Those s t r u c t u r e s  c o n t a i n i n g an o r d i n a r y benzenoid r i n g would be expected t o be more s t a b l e and hence make more important c o n t r i b u t i o n s t o the h y b r i d but the only s t r u c t u r e c o n t r i b u t i n g a negative the  charge t o  06-position i s non-benzenoid while s e v e r a l c o n t r i b u t i n g t o  negative  charge i n the (3-position are benzenoid.  undergo 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 i n the facility. halogenation  Indoles  (3-position with  great  N i t r a t i o n , a l k y l a t i o n , a c y l a t i o n , Mannich r e a c t i o n , and d i a z o t i z a t i o n a l l take p l a c e i n the ( o - p o s i t i o n  i f s u f f i c i e n t l y m i l d c o n d i t i o n s are used t o prevent p o l y s u b s t i tution.  Recent s p e c t r o s c o p i c  s t u d i e s have shown that  protonation  of indoles i n strongly a c i d i c  s o l u t i o n occurs  e x c l u s i v e l y i n the (3-position even when the ^ - p o s i t i o n i s already  substituted. ® 7  53  1.009 1.015 riy  1.066  1.013  1.059  ^ 1.010  H  1.742  0 = 3  a =2 N = 1  H  Figure  23.  Kekule S t r u c t u r e s  by Molecular  The first  l  H  H  and E l e c t r o n Density  o f Indole  O r b i t a l Approximations.  utility  of G r i g n a r d  reagents d e r i v e d from i n d o l e was  i n v e s t i g a t e d by Oddo i n 1 9 1 1  been the o b j e c t  of s p e c u l a t i o n .  9 9  and t h e i r nature has s i n c e  Depending on r e a c t i o n  c o n d i t i o n s a l k y l a t i o n s , a c y l a t i o n , and c a r b o n a t i o n magnesium h a l i d e s y i e l d s N-, |3-, N,(J- and r a r e l y products. Grignard species.  1 0 0  '  1 0 1  of i n d o l y l a-substituted  These r e s u l t s l e d t o f o r m u l a t i o n  of the i n d o l e  reagent as e i t h e r N-MgX, C-MgX or e s s e n t i a l l y i o n i c Recent evidence i n favour  of i o n i c species  has been  54  obtained  from a study  o f the n u c l e a r magnetic resonance s p e c t r a  i n tetrahydrofuran.^2  of these reagents  f  h e  f i i a  u  r  an N-H i n the n u c l e a r magnetic resonance or i n f r a r e d and  the f a i l u r e of the (3-proton resonance t o s h i f t  f i e l d on p r e p a r i n g the Grignard formulations.  reagent  t o detect  e  spectra  t o higher  e l i m i n a t e C-MgX  The s t r i k i n g s i m i l a r i t y between the n.m.r.  s p e c t r a of indolylmagnesium bromide and i n d o l y l s o d i u m which presumably c o n t a i n s a l a r g e l y i o n i c N-N  a  bond excludes the  p o s s i b l i t y o f a c o v a l e n t N-Mg bond.  F i g u r e 24.  Indole G r i g n a r d  Reagent.  K o n d i r e f f and Fomin i n 1914 announced a method f o r prepa r a t i o n o f hydrocarbons based on the c o u p l i n g a c t i o n o f f e r r i c c h l o r i d e on organomanganese d e r i v a t i v e s . ^ 3  Perhaps the most  i n t e r e s t i n g consequence of t h i s r e a c t i o n was the d i s c o v e r y of ferrocene  i n 1951  1  0  4  when f e r r i c c h l o r i d e was added t o c y c l o p e n t a -  d i e n y l magnesium bromide.  A systematic  study  of the c o u p l i n g  of phenylmagnesium bromide with f e r r i c c h l o r i d e was made i n 1930  t o d e f i n e the m e c h a n i s m .  105  An i n t e r e s t i n g  stoichiometry  was observed by t i t r a t i o n , m e t a l l i c i r o n being one o f the products.  The c o u p l i n g c o u l d a l s o be i n i t i a t e d with  ferrous  55  chloride.  6 CgHgMgBr + 2 F e C l  »•  3  3 C H -C H 6  5  g  5  + 2 Fe + 3 MgBr + 3 MgCl  Nb-methyltryptamine 25)  (50)  ( d i p t e r i n ) , was  2  2  prepared  (Figure  u s i n g a method d e v i s e d by Dr. A.C.Day, U n i v e r s i t y of Oxford,  England, which i s i t s e l f a m o d i f i c a t i o n of an e a r l i e r a t i o n by Hoshiro  and K o b a y a s h i  prepar-  (An a l t e r n a t i v e p r e p a r a t i o n  1 0 6  has been d e s c r i b e d by Witkop which i n v o l v e s f o r m y l a t i o n of tryptamine  h y d r o c h l o r i d e f o l l o w e d by r e d u c t i o n w i t h l i t h i u m  aluminum h y d r i d e . was  prevented  1 0 7  )  Q u a t e r n i z a t i o n of the a l i p h a t i c  by N ^ - t o s y l a t i o n (99) before m e t h y l a t i o n  The p r o t e c t i v e t o s y l group was u s i n g the method of W i l k i n s o n  then c o n v e n i e n t l y  nitrogen (100).  eliminated  involving reductive displace-  1 0 8  ment by sodium i n l i q u i d ammonia.  The  N -methyltryptamine b  was  c h a r a c t e r i z e d by m e l t i n g p o i n t , i t s i n d o l e chromophore i n the u l t r a v i o l e t , n u c l e a r magnetic resonance and mass spectrum. the mass s p e c t r a of c o u p l i n g products d i s c u s s i o n of the fragmentation i s a l s o important. those w i t h m/e important  a  p a t t e r n of Nb-methyltryptamine  By f a r the most abundant fragments were  44 and 31.  primary process  It i s w e l l recognized  that the most  r e s u l t i n g from e l e c t r o n impact  a l i p h a t i c amines i s removal of one  on  of the lone p a i r e l e c t r o n s  o f the heteroatom f o l l o w e d by simple carbon bond adjacent  were very important  As  cleavage  t o the n i t r o g e n a t o m .  1 0 9  of the carbonT h i s would r e s u l t  56  50 "\  F i g u r e 25.  P r e p a r a t i o n of Nb-methyltryptamine.  i n an i o n w i t h m/e  44.  Simple cleavage of a C-N  g i v e a p o s i t i v e fragment of m/e h i g h e r mass has m/e  30.  130  c h a r a c t e r i s t i c fragments are m/e  i s also strong.  131.  dimethylamine The  other  144 c o r r e s p o n d i n g t o l o s s of  methylamine and the molecular i o n m/e abundance of m/e  The s t r o n g e s t peak at  131 c o r r e s p o n d i n g t o l o s s of  and a d d i t i o n of a p r o t o n ; m/e  bond should  (Figure 27).  174 w i t h 8% of the Fragmentation  patterns w i l l  be d i s c u s s e d i n terms of the c o n v e n t i o n i n t r o d u c e d by  Djerassi  1 0  RELATIVE  OQ  CO  o -r-  45>  o  o  -i  r  o  INTENSITY O  O  O  -r  -T-  00  vO O  ~T~  -T-  o  O O  p  CO CO  CO  •aa> o  31  rt  c 3  v. 44  O  H,  o  I S  a  rt r f >i <<  •8  m  3  ?  8"  ^-l30CM-44) 144 C M - 3 0  oi— o  174  (M+)  19  C X 3  '  6 : I  58  where a s i n g l e headed arrow r e p r e s e n t s t r a n s f e r of a s i n g l e electron  (m/e i s the r a t i o of mass t o charge). +  m/e Figure  26.  The  130 101  Fragmentation of N -Methyltryptamine. h  coupling  of N^-methyltryptamine was s u c c e s s f u l l y  achieved by o x i d a t i o n  of i t s magnesium i o d i d e s a l t w i t h  anhydrous f e r r i c c h l o r i d e .  Preparation  o f N^-methyItryptamine-  s-magnesium i o d i d e was c a r r i e d out under n i t r o g e n of an e t h e r e a l  by a d d i t i o n  s o l u t i o n of N^-methyltryptamine t o a s t i r r e d  s o l u t i o n o f methyl magnesium i o d i d e prepared i n the u s u a l way. Addition  of f e r r i c c h l o r i d e i n e t h e r f o l l o w e d  by m i l d  h y d r o l y s i s w i t h aqueous ammonium c h l o r i d e gave a mixture of p r o d u c t s which were then analyzed by t h i n - l a y e r chromatography and  ultraviolet  preparative  spectroscopy.  Several  f r a c t i o n s from  t h i n - l a y e r p l a t e s had u l t r a v i o l e t s p e c t r a  correspond'  59  i n g t o a Ph-N-C-N chromophore which i s c h a r a c t e r i s t i c o f c a l y c a n t h a c e o u s and p h y s o s t i g m i n e t y p e a l k a l o i d s chromophore w i t h a hypsochromic s h i f t i n d i l u t e  (an i n d o l i n e acid).  7 2  '  7 7  T h i s was t h e f i r s t i n d i c a t i o n t h a t t h e c o u p l i n g might have been successful.  On s e p a r a t i o n o f t h e p r o d u c t s by chromatography  a p a r t from unchanged Nb-methyltryptamine (30%) t h e p r i n c i p a l p r o d u c t s were d l - c h i m o n a n t h i n e (19%, based on 3 0 % r e c o v e r y o f s t a r t i n g m a t e r i a l ) , meso-chimonanthine  ( 7 % ) , two h i g h m e l t i n g  dimers named A (3.5%) and B ( 3 . 5 % ) , and some monomeric o x i d a t i o n p r o d u c t s which were not isolated i n c r y s t a l l i n e form.  The  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 from t h e c o u p l i n g r e a c t i o n ranged from 60 t o 80%.  C a l y c a n t h i n e was not d e t e c t e d i n t h e r e a c t i o n  m i x t u r e , and was not e x p e c t e d s i n c e c o n d i t i o n s do not f a v o u r e q u i l i b r a t i o n through the tetraminodialdehyde (72).  The  r e c o v e r y o f s t a r t i n g m a t e r i a l was poor u n l e s s t h e r e a c t i o n was c a r r i e d out under n i t r o g e n . The i d e n t i t y o f d l - c h i m o n a n t h i n e i s o l a t e d from t h e r e a c t i o n m i x t u r e was e s t a b l i s h e d beyond doubt by correspondence o f i t s i n f r a r e d , u l t r a v i o l e t , and mass s p e c t r a as w e l l as t h i n - l a y e r c h r o m a t o g r a p h i c b e h a v i o u r i n s e v e r a l s o l v e n t systems and i t s c o l o u r r e a c t i o n w i t h 1% e e r i e s u l p h a t e i n 3 5 % s u l p h u r i c spray, t o the n a t u r a l 1 - a l k a l o i d .  acid  There was no d e p r e s s i o n o f  m e l t i n g p o i n t on a d m i x t u r e w i t h a sample o f s y n t h e t i c r a c e m i c c h i m o n a n t h i n e p r e p a r e d by P r o f e s s o r  Hendrickson. > y l  4 8  60  F i g u r e 28.  S y n t h e s i s o f Chimonanthine and  Calycanthine.  61  P r e v i o u s l y o b s e r v e d d e p r e s s i o n o f m e l t i n g p o i n t on adm i x t u r e w i t h a sample o f n a t u r a l chimonanthine r e c e i v e d from P r o f e s s o r Smith o f Manchester l e d t o t h e d i s c o v e r y o f mesochimonanthine i n nature.  T h i s sample o f chimonanthine from  an e x t r a c t o f C a l y c a n t h u s f l o r i d u s was y e l l o w i n c o l o u r and was r e c e i v e d w i t h t h e i n s t r u c t i o n s t h a t i t c o u l d be p u r i f i e d by s i m p l e chromatography  on a l u m i n a .  benzene o f t h e f i r s t  A f t e r r e c r y s t a l l i z a t i o n from  f r a c t i o n s e l u t e d w i t h benzene-ether from an  a l u m i n a column t h e w h i t e c r y s t a l s w i t h m e l t i n g p o i n t 199-202° C were used f o r comparison w i t h t h e s y n t h e t i c p r o d u c t .  The  absence o f o p t i c a l a c t i v i t y , v e r y c l o s e resemblance o f u l t r a v i o l e t and s o l u t i o n i n f r a r e d s p e c t r a , a mass spectrum  showing  a m o l e c u l a r i o n a t m/e 346 w i t h f a c i l e c l e a v a g e t o a fragment m/e 172 and a l e s s i n t e n s e fragment a t m/e 130 e s t a b l i s h e d t h e i d e n t i t y o f t h i s compound as t h e mesomeric isomer o f chimonanthine.®  6  T h i s same compound was s u b s e q u e n t l y i s o l a t e d from t h e  r e a c t i o n m i x t u r e and was a l s o r e p o r t e d as a p r o d u c t from Hendrickson's s y n t h e s i s .  4 8  As t h e mass spectrum i s so i m p o r t a n t i n c h a r a c t e r i z a t i o n o f t h e c h i m o n a n t h i n e s i t s h o u l d be d i s c u s s e d i n d e t a i l . spectra of the calycanthaceous and d i s c u s s e d . , » 86  110  The mass  a l k a l o i d s have been p u b l i s h e d  Of t h e f i v e c a l y c a n t h i n e i s o m e r s (73)  o n l y c h i m o n a n t h i n e , j o i n e d by a s i n g l e bond would be e x p e c t e d t o undergo f a c i l e s y m m e t r i c a l c l e a v a g e .  S y m m e t r i c a l fragmen-  t a t i o n c a n y i e l d a s t a b i l i z e d b e n z y l c a t i o n (102) w h i c h may l o s e hydrogen t o form a fragment o f m/e 172 o r undergo  cyclic  62  CH  H  3  173  102 a  COT H  m/e  H  101  F i g u r e 29.  m/e  130  F r a g m e n t a t i o n o f Chimonanthine.  dH  172  3  RELATIVE -37 o  -r o  8  INTENSITY  -r o  O  o  O  CD o  "T~  O  o  o  o o  130  »  m  172 C M-174)  OO  OJ  OO  346 CM ) 4  89  CX  1,53  F i g u r e 31.  Mass Spectrum o f N a t u r a l and S y n t h e t i c meso-Chimonanthine.  65  c o l l a p s e as shown i n F i g u r e 29 t o a fragment  (101) of m/e 130.  ^-methylene  indolenine  The molecular i o n m/e 346 was found  t o be l e s s than 12% a s , i n t e n s e as the parent fragment  at m/e 172  f o r 1- and dl-chimonanthine and 7% f o r meso-chimonanthine. Previously published s p e c t r a  8 6  i n d i c a t e 3.5% abundance f o r the  molecular i o n , a d i f f e r e n c e r e a d i l y a t t r i b u t e d t o d i f f e r e n t experimental c o n d i t i o n s . The n u c l e a r magnetic resonance s p e c t r a are a l s o o f c o n s i d e r able i n t e r e s t . five-membered  Woodward  49  p r e d i c t e d that the n e a r l y p l a n a r  r i n g s o f 737, which was l a t e r d i s c o v e r e d i n nature  as chimonanthine  s h o u l d have more n e a r l y e q u i v a l e n t  protons than would be the case f o r c a l y c a n t h i n e 7306 other isomers w i t h puckered six-membered r i n g s . resonances o f racemic chimonanthines  (Figure 32) and mesomeric  or the  The methylene (Figure 33)  are not i d e n t i c a l but are s i m i l a r and c o n t r a s t  w i t h c a l y c a n t h i n e (Figure 37). shift  methylene  The d i f f e r e n c e i n chemical  of the N-CH3 resonances, 7.707", f o r racemic and 7.63T f o r  mesomeric chimonanthines,  i s consistent with d i f f e r e n c e s i n  s h i e l d i n g caused by d i f f e r e n t p o s i t i o n s r e l a t i v e t o the aromatic nucleus.  An n.m.r. spectrum of the n a t u r a l mixture from  which  meso-chimonanthine was i s o l a t e d had peaks o f very s i m i l a r i n t e n s i t y at 7.637* and 7.707"indicating an approximately 1:1 mixture o f the d i a s t e r e o i s o m e r s . t h i n - l a y e r chromatography alumina.  T h i s a n a l y s i s i s supported by  o f the mixture i n 2% methanol-ether on  The chemical s h i f t s o f NH and R3CH protons are a l s o  somewhat d i f f e r e n t .  The s p l i t t i n g o f the aromatic protons  68  c o u l d be a n a l y z e d i n the 100 Mc spectrum of racemic  chimonanthine  but not of the meso-isomer where s e v e r a l of these protons must be more n e a r l y e q u i v a l e n t .  The  (2.877) would be c o u p l e d t o H Proton H  b  low f i e l d aromatic p r o t o n  (3.407*, J  must a l s o be coupled t o H  b  a  b  -7.6  (3.07 7*,  c  J  b  H  Q  c.p.s.). c  = 7.6  c.p.s.)  i  which  i s para  than H .  Proton H  b  7.8  t o the n i t r o g e n and expected t o be more s h i e l d e d  c.p.s.).  c  must a l s o be coupled t o  c  (J  The  b  d  c (  j =  Long range c o u p l i n g e f f e c t s which are q u i t e  l a r g e through 7T e l e c t r o n s y s t e m s and H  (3.457", J  = 1.0  c.p.s., J  c  1 1 1  '  1 1 2  also s p l i t  protons  H  b  = 1.3).  a  i s o l a t i o n of meso-chimonanthine i n nature and the  i s o l a t i o n of calycanthaceous a l k a l o i d s from s e v e r a l o r d e r s of p l a n t s i s i n d i c a t i v e of a simple b i o g e n e s i s w i t h o n l y l o o s e enzyme c o n t r o l . The s y n t h e s i s o f c a l y c a n t h i n e was i s o m e r i z a t i o n o f racemic chimonanthine  completed  hours on the steam bath dl-chimonanthine was  Treat-  found t o be the  best means of a c c o m p l i s h i n g t h i s i s o m e r i z a t i o n .  mixture of chimonanthine  catalyzed  to calycanthine.  ment w i t h hot d i l u t e aqueous a c e t i c a c i d was  1:4  by a c i d  After  thirty  transformed i n t o a  and c a l y c a n t h i n e .  In c o n t r a s t  to  i s o m e r i z a t i o n s c a t a l y z e d by d i l u t e h y d r o c h l o r i c a c i d t h e r e were no minor byproducts or d e g r a d a t i o n t o N -methyltryptamine.  The  b  presence of o t h e r isomers to  separate  (73) c o u l d have been masked by  them from c a l y c a n t h i n e or chimonanthine  l a y e r chromatography.  by  failure thin-  I f they were present the q u a n t i t i e s  i n v o l v e d must have been s m a l l .  In view of the r e l a t i v e  stability  69  of c a l y c a n t h i n e and chimonanthine  i t seems u n l i k e l y that other  isomers w i l l be d e t e c t e d i n nature. d l - C a l y c a n t h i n e was chromatographic  i d e n t i f i e d on the b a s i s of t h i n - l a y e r  behaviour and the s u p e r i m p o s i b i l i t y of u l t r a -  v i o l e t and s o l u t i o n i n f r a r e d s p e c t r a w i t h those of the n a t u r a l alkaloid.  M e l t i n g p o i n t d e p r e s s i o n and d i f f e r e n c e s i n the  s o l i d s t a t e i n f r a r e d s p e c t r a were a t t r i b u t e d t o d i f f e r e n t l i n e forms f o r the r e s o l v e d and racemic a l k a l o i d . magnetic  resonance  Treatment  and mass s p e c t r a were a l s o  crystal-  Nuclear  identical.  of meso-chimonanthine w i t h d i l u t e aqueous a c e t i c  a c i d r e s u l t e d i n an e s s e n t i a l l y two component mixture of s t a r t i n g m a t e r i a l and a compound i d e n t i f i e d as (1:2) on the b a s i s of i t s mass spectrum, spectrum  meso-calycanthine an u l t r a v i o l e t  showing the Ph-N-C-N chromophore, and i t s m e l t i n g p o i n t  of 265-268° C which i s higher than d - c a l y c a n t h i n e as f o r a more symmetrical The mass spectrum molecular i o n m/e result  expected  molecule. of c a l y c a n t h i n e i s dominated by the  346 as the r u p t u r e of a s i n g l e bond cannot  i n fragmentation.  l o s s of the b r i d g e s .  8 6  Other peaks i n the spectrum Peaks at m/e  288  occur by  (17%) and 231  correspond t o l o s s of one and two b r i d g e s r e s p e c t i v e l y . a l s o occur at m/e  172 and 130.  component a f t e r treatment  The mass spectrum  (25%) Peaks  of the major  of meso-chimonanthine w i t h a c i d  was  s t r i k i n g l y s i m i l a r t o that of n a t u r a l or racemic c a l y c a n t h i n e showing o n l y s m a l l d i f f e r e n c e s i n abundance of some (notably m/e  fragments  172 which i s approximately twice as i n t e n s e  70  F i g u r e 34.  Fragmentation of  Calycanthine.  RELATIVE  CfQ  c o to  -1 O  INTENSITY  r  ro  o  .  OJ o  O  00  O  O  O  o  S CO CO CO 13 CD O c+  o. o  e a  o Hs  —  »  m  o p  I  3  cr H  1 3 0  72  ro  o o  231 C M - ||53  2 88  oo  3 0 2  C M - 5 8 ) C M - 4 4 )  3 4 6  IL  C M  +  )  ZL  73  ( F i g u r e s 3 5 and 3 6 ) . ) . The n u c l e a r magnetic  resonance spectrum o f n a t u r a l d - c a l y -  c a n t h i n e ( a n d the s y n t h e t i c racemate) has been p u b l i s h e d before ® 4  but i t i s o f i n t e r e s t  i n comparison  w i t h that of chimonanthine.  L i k e dl-chimonanthine the aromatic r e g i o n i n the 1 0 0 Mc spectrum  i s r e a d i l y analyzed and i s c o n s i s t e n t w i t h the known  structure.  The chemical s h i f t s and c o u p l i n g c o n s t a n t s a r e  shown i n f i g u r e 3 7 .  The methylene r e g i o n can be p a r t i a l l y  yzed even i n the 6 0 Mc spectrum. is split  The low f i e l d proton at 6 . 8 5 7"  i n t o a s e x t e t by c o u p l i n g w i t h a geminal proton ( J =  14 c . p . S j )  and two v i c i n a l  high f i e l d proton i s s p l i t coupling  anal-  ( J = 1 4 and 6 c.p.s.) p r o t o n s .  i n t o a doublet of q u a r t e t s by geminal  ( J = 1 4 c.p.s.) and c o u p l i n g w i t h two v i c i n a l  (J = 4 and 2 c . p . s . ) .  The  protons  From the i d e n t i c a l geminal c o u p l i n g  c o n s t a n t s i t i s probable that the high and low f i e l d methylene protons a r e geminal i n d i c a t i n g an u n u s u a l l y l a r g e chemical shift  ( 1 . 9 1 T ) between two protons on the same carbon.  s i t u a t i o n i s an i n t e r e s t i n g example o f a n i s o t r o p i c and d e s h i e l d i n g by aromatic  rings.  Such a  shielding  1 1 1 3  The two s y n t h e t i c byproducts A and B were o r i g i n a l l y thought t o be e i t h e r i s o m e r i c w i t h c a l y c a n t h i n e or t o be oxygenation p r o d u c t s .  The i n f r a r e d s p e c t r a were d i f f e r e n t  from those of the c a l y c a n t h i n e isomers, had a sharp NH band at about (see  3 4 4 0 cm"  1  and no c a r b o n y l bands.  The u l t r a v i o l e t  spectra  experimental) were not the same as those o f the c a l y c a n -  t h i n e isomers having an e x t r a peak about  2 8 0 mp. which showed a  75  hypsochromic  s h i f t and reduced i n t e n s i t y i n d i l u t e  acid.  Although the s p e c t r a o b t a i n e d by mixing N^-methyltryptamine calycanthine  and  (2:1) were q u i t e s i m i l a r , the i n d o l e chromophore  was r e l a t i v e l y u n a f f e c t e d by d i l u t e a c i d .  The i n f e r e n c e was  that A and B possessed an i n d o l i n e chromophore p l u s a second non-indole chromophore.  Treatment  o f these compounds w i t h  aqueous a c e t i c a c i d f a i l e d t o y i e l d any c a l y c a n t h i n e isomers. On the b a s i s of t h i n - l a y e r chromatographic  evidence compound  A was u n a f f e c t e d w h i l e compound B was p a r t i a l l y transformed A or a very s i m i l a r compound.  into  M i c r o - a n a l y s i s and mass  spectrometry e s t a b l i s h e d molecular weights of 344 w i t h C22H24N4 c o r r e s p o n d i n g t o d e h y d r o c a l y c a n t h i n e s .  formulae  The mass  s p e c t r a were very s i m i l a r t o c a l y c a n t h i n e w i t h the molecular i o n as the most abundant peak.  The minor peaks (with d i f f e r e n c e s  i n i n t e n s i t y ) a l s o corresponded spectrum  ( F i g u r e s 38, 39).  t o peaks i n the c a l y c a n t h i n e  The major d i f f e r e n c e i s the v i r t u a l  absence of m/e 231 a t t r i b u t e d t o a p r o t o n a t e d c a l y c a n i n e by l o s s of both ethanamine b r i d g e s . r e d u c t i o n byproducts  T h i s f e a t u r e was a l s o shared by  A - l and A-2 from the f i r s t  calycanthaceous a l k a l o i d s .  4 8  The u l t r a v i o l e t  synthesis of s p e c t r a of these  compounds were a l s o very s i m i l a r l e a d i n g t o the s u s p i c i o n that compound B w i t h m e l t i n g p o i n t 235-240° C might be i d e n t i c a l t o Hendrickson's  compound A - l w i t h a m e l t i n g p o i n t o f 238-242° C.  Compound A had a m e l t i n g p o i n t o f 274-275° C while A-2 was much lower, m e l t i n g at 204-205° C.  D i f f e r e n c e s were observed  however i n the n u c l e a r magnetic  resonance  spectra.  Compounds  76  A - l , A-2, A and B a l l had two  peaks i n c o n t r a s t t o one f o r  N-CH3  chimonanthine and c a l y c a n t h i n e e m p h a s i z i n g t h e asymmetric of t h e m o l e c u l e s a l r e a d y s u s p e c t e d from t h e u l t r a v i o l e t  nature  spectra.  Compound A - l has N-CH peaks a t 6.73 and 7.357" w h i l e A-2 has 3  peaks a t 6.73 and 7.76 7".  Compound B has N-CH peaks a t 6.78T 3  and 7.42 T w h i l e A has peaks a t 6.72 and 7.617".  In s p i t e of  d i f f e r e n c e s between t h e b y p r o d u c t s from i n d o l e and o x i n d o l e c o u p l i n g i t i s s t i l l a t t r a c t i v e t o f o r m u l a t e A and B as monoa m i d i n e s o f i s o m e r s 73, w h i c h c o u l d be formed by o v e r o x i d a t i o n during the coupling r e a c t i o n . S i n c e an amidine w i t h t h e c a l y c a n t h i n e s t r u c t u r e (73Ct) i s i m p o s s i b l e ^ and t h e mass spectrum r u l e s out 7 3 T t h e r e a r e three p o s s i b i l i t i e s .  Hendrickson ® e l i m i n a t e s 736. on t h e 4  grounds t h a t t h e d i f f e r e n c e i n c h e m i c a l s h i f t s t r o n g l y i m p l i e s t h a t one  N-CH3  i s an amidine m e t h y l but i n t h e case o f an £,  isomer t h e environments o f t h e two and s i m i l a r .  N-CH3  Compounds A - l and A - 2  4 8  groups a r e s a t u r a t e d  were i n t h e r a c e m i c  s e r i e s as b y p r o d u c t s from r e d u c t i o n t o d l - c h i m o n a n t h i n e . Compounds A and B c o u l d then be t h e mesomeric monoamidines o f 73 fi and 8 r e s p e c t i v e l y ( F i g u r e 4 0 ) .  Extra o x i d a t i o n i n the  meso s e r i e s would a l s o account f o r t h e lower y i e l d o f meso than of r a c e m i c c h i m o n a n t h i n e . Further evidence f o r t h i s f o r m u l a t i o n i n c l u d e s mono-acetyla t i o n o f A and c o n s i d e r a t i o n o f t h e n.m.r. spectrum o f B which has one a r o m a t i c p r o t o n (2.217") a t lower f i e l d t h a n t h e r e s t .  RELATIVE r——i  i  o  ro  "  —  o  OJ  0  o  1 j>  INTENSITY 1  o  <ji  o  1 o>  r  o  -si  1  o  go  o  1  O  1  §  ~  130  I 72  (23n  2 86  300 CM-4 4 ) 329 344 CM+)  LL  ^ — CX 1.33 * LX 1.4 ]  -100 -90 -80 - 70 -60 -50 -40  If)  -30 -20  OJ O ro —  oo r*—  oo 00  (\J OJ  ro  -4"  —  -10  oj  ill  I  200  100  M/E Figure 39.  i  Mass Spectrum o f Dimer B.  I*  j  lllll  300  79  T h i s i s probably the proton ortho t o the amidine n i t r o g e n . Compound B has only a s i n g l e NH and R CH by i n t e g r a t i o n and a 3  s i n g l e methylene proton at high f i e l d  (8.84D which i s  r e m i n i s c e n t o f c a l y c a n t h i n e with i t s six-membered r i n g s .  There  were e i g h t methylene protons by i n t e g r a t i o n , i n d i c a t i n g that the double bond c o u l d not be i n an ethanamine b r i d g e .  A F i g u r e 40.  73/3  B  5  Suggested S t r u c t u r e s of Compounds A and B.  Compound A was u n a f f e c t e d by attempted by attempted  73  hydrogenation and  r e d u c t i o n with l i t h i u m aluminum h y d r i d e .  O x i d a t i o n o f c a l y c a n t h i n e w i t h mercuric a c e t a t e and o f chimonant h i n e w i t h mercuric a c e t a t e or manganese d i o x i d e f a i l e d t o y i e l d spots on t h i n - l a y e r chromatography c o r r e s p o n d i n g t o compound A or B.  Treatment of these dehydrocompounds with d i l u t e  a c i d d i d not y i e l d compound A or B.  The u l t r a v i o l e t  acetic  spectrum  of t h e dehydrocompounds gave no evidence o f amidine formation. An attempt  was made t o couple  Ntj-methyltryptamine-N a  80  magnesium i o d i d e i n t e t r a h y d r o f u r a n i n the hope o f r a i s i n g the y i e l d , as i t was much more s o l u b l e i n t h i s s o l v e n t .  Only a  t r a c e amount of d i m e r i c product was o b t a i n e d as d e t e c t e d by mass spectrometry with  i n a p r e p a r a t i v e t h i n - l a y e r chromatographic  the same as chimonanthine.  reaction  The major product  (named C) was shown by a n a l y s i s and mass  fraction from  this  spectrometry  t o have a molecular weight o f 188 and a formula o f C i 2 l 6 2 H  The  i n f r a r e d had a sharp peak at 3500 cm"  2790 cm"  1  N  and peaks at 2840 and  a t t r i b u t a b l e t o the NH o f a s u b s t i t u t e d p y r r o l e and  1  -N(CH3)2>  A t y p i c a l i n d o l e chromophore appeared i n the  ultraviolet  spectrum.  The n.m.r. and mass s p e c t r a were c o n s i s t -  ent with f o r m u l a t i o n o f compound C as  N ,N -dimethyltryptamine. b  b  The m e l t i n g p o i n t , 45-49° C, a f t e r r e c r y s t a l l i z a t i o n from ether a l s o i s i n agreement w i t h t h i s s t r u c t u r e . The  second product, D, was shown by i t s i n d o l e type  v i o l e t spectrum, absence of an presence  ultra-  proton i n the n.m.r. spectrum,  o f two NH protons by exchange with D2O, a n a l y s i s  ^15^20^^'  a  n  d  m  a  s  s p e c t r o m e t r i c molecular weight 244, t o be  s  Nb-methyl-Ct-tetrahydrofuranyltryptamine.  T h i s compound  probably a r i s e s by c o u p l i n g o f the r e s p e c t i v e r a d i c a l s , the intermediate  06-indolyl r a d i c a l being l e s s commonly  found.  r f ^ l  N(CH ) 3  2  NHCH  H F i g u r e 41. Products  from Attempted Coupling i n T e t r a h y d r o f u r a n .  RELATIVE T — °  1  r\> o  1 CJ  o  IN T E N S I T Y  1  o  -t»  1  o  01  1  o  ci  1  o  244  T8  -M  1  o  oo  1 o  1  §  ' -  82  Hodgkinsine An 80 mg. sample o f hodgkinsine was r e c e i v e d from P r o f e s s o r T a y l o r , U n i v e r s i t y o f Queensland, when i t was thought isomer, o f c a l y c a n t h i n e o r a d e h y d r o c a l y c a n t h i n e . i s o m e r i z a t i o n o r hydrogenation  t o be an  Attempted  f o l l o w e d by i s o m e r i z a t i o n r e s u l t e d  i n no evidence f o r c a l y c a n t h i n e isomers.  Hodgkinsine  during  t h i s time was a l s o under study by P r o f e s s o r Smith o f Manchester who e s t a b l i s h e d that the compound was a t r i m e r o f N^-methyld i p t e r i n and suggested s t r u c t u r e 86. spectrum at  were determined  A mass spectrum  and a r e o f i n t e r e s t .  The base peak i s  m/e 172 w i t h secondary peaks at m/e 344 and 518.  p a t t e r n i n d i c a t e s f a c i l e cleavage of the molecule symmetrical  fragments.  The n.m.r. spectrum  peaks at 7.59, 7.65 and 7.717". for  and n.m.r.  This  i n t o three  has three NCH3  The aromatic r e g i o n i n t e g r a t e s  e l e v e n protons w i t h one at s l i g h t l y higher f i e l d  than the  r e s t and p r o v i d e s evidence f o r s u b s t i t u t i o n i n an aromatic  ring.  There a r e three NH, three R3CH protons and 21 (12 + 9) methylene p l u s N-methyl protons. to  The methylene protons do not extend  as high a f i e l d as does the high f i e l d proton o f c a l y c a n t h i n e  p r o v i d i n g evidence f o r t h r e e five-membered ethanamine r i n g s . A c o n s i d e r a t i o n o f the b i o g e n e s i s o f hodgkinsine as a probable o x i d a t i v e c o u p l i n g of chimonanthine  and  N -methyltryptamine b  makes s u b s t i t u t i o n on an aromatic r i n g para t o a n i t r o g e n the most probable. one.  S t r u c t u r e 86 f o r hodgkinsine i s a reasonable  85 Experimental M e l t i n g p o i n t s were determined scope 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  u s i n g a Cary 14 spectrophotometer Perkin-Elmer model 1376 stated.  Mrs.  A.Brewster  determined  and i n f r a r e d s p e c t r a u s i n g a  spectrophotometer unless otherwise  Nuclear magnetic  r e c o r d e d at 60 Mc/s  on a K o f l e r hot stage micro-  resonance  s p e c t r a (n.m.r.) were  on a V a r i a n A60 instrument by Mr.P.Horn or  and at 100 Mc/s  on a V a r i a n HA-100 instrument  by Mr.R.Burton of t h i s department.  The resonance  p o s i t i o n s are  given i n the T i e r s 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 an i n t e r n a l standard, w i t h types of protons, c o u p l i n g constants  (J) i n c y c l e s per second  areas i n parentheses. Mrs.C.Jenkins  ( c . p . s . ) , and the i n t e g r a t e d  Elemental analyses were performed  by  of t h i s department.  Mass s p e c t r o m e t r i c d e t e r m i n a t i o n s were done by Mr.G.Eigend o r f of t h i s department on an A t l a s CH4 s e r v i c e became a v a i l a b l e .  instrument a f t e r  Thanks are due t o  this  Dr.H.Budzikiewicz,  Stanford U n i v e r s i t y f o r the o r i g i n a l d e t e r m i n a t i o n of the mass spectrum.;of  s y n t h e t i c dl-chimonanthine  and t o D r . T a y l o r ,  U n i v e r s i t y of Queensland, f o r h i s d e t e r m i n a t i o n of the mass spectrum  of s y n t h e t i c meso-chimonanthine and f o r h i s supply of  hodgkinsine. We New  are g r a t e f u l t o Dr.A.Brossi of Hoffmann-LaRoche, Nutley,  Jersey f o r a l a v i s h g i f t  of tryptamine h y d r o c h l o r i d e .  thank Dr.G.F.Smith, Manchester U n i v e r s i t y , England, generous g i f t  of the crude sample of n a t u r a l  We  f o r a very  chimonanthine  86  which proved t o be so i n t e r e s t i n g and f o r a sample o f d - c a l y canthine.  Many thanks a r e a l s o due t o Dr.J.B.Hendrickson f o r  a sample o f h i s s y n t h e t i c d l - c h i m o n a n t h i n e and f o r a copy o f his manuscript before p u b l i c a t i o n . Alumina G ( a c c o r d i n g t o S t a h l ) was used f o r t h i n - l a y e r chromatography.  The alumina used f o r column chromatography  was Shawinigan r e a g e n t , n e u t r a l i z e d by treatment w i t h e t h y l acetate.  3,2'-Methylaminoethylindole  (N -methyltryptamine) (50). b  T h i s p r e p a r a t i o n was d e v i s e d by Dr.A.C.Day, U n i v e r s i t y of O x f o r d , England and i s a m o d i f i c a t i o n o f an e a r l i e r p r e p a r a t i o n by H o s h i r o and K o b a y a s h i .  1 0 6  3,2'-p-toluenesulphonylaminoethylindole Tryptamine  h y d r o c h l o r i d e ( 3 , 2 ' - a m i n o e t h y l i n d o l e ) (98)  (20 g., 0.102 moles) was suspended i n benzene (150 ml.) and treated wi^h p-toluenesulphonyl c h l o r i d e  (21.4 g., 0.112 moles,  10% e x c e s s ) f o l l o w e d by p o t a s s i u m h y d r o x i d e (17 g., 0.3 moles; l e s s when s t a r t i n g w i t h t r y p t a m i n e ) i n water  (150 m l . ) .  On  warming, a c l e a r two phase s o l u t i o n was o b t a i n e d and a l l o w e d t o cool with occasional shaking.  The w h i t e s o l i d ,  N^-tosyl-  t r y p t a m i n e , was f i l t e r e d o f f about o n e - h a l f hour a f t e r  acids'  i f i c a t i o n 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 and c h i l l i n g i n an i c e bath.  The s u p e r n a t a n t l i q u i d was a g a i n t r e a t e d w i t h t o s y l  c h l o r i d e a f t e r making b a s i c w i t h p o t a s s i u m h y d r o x i d e ( 7 g." e x c e s s )  87  and a few more grams of the t o s y l a t e  were o b t a i n e d .  e r a t u r e method i n v o l v e d r e c r y s t a l l i z a t i o n from but t h i s s o l v e n t p a i r which the product m.p.  114-115° ( l i t e r a t u r e  benzene-ethanol  soluble.  Y i e l d 30 g.,  N -tosyltryptamine b  (29.8  (50 ml.)  g., 0.095 moles)  and added to 50% w/w  g e n t l e warming.  moles, 24% excess) was erature overnight. two  (99)  (100) was aqeous sodium  (18 g. NaOH) whereby the compound went i n t o  on shaking and  after  90%;  115-116°).  3,2'-methyl-p-toluenesulphonylaminoethylindole  hydroxide  hours.  lit-  i s d e f i n i t e l y i n f e r i o r to hot e t h a n o l i n  i s extremely  suspended i n e t h a n o l  The  Methyliodide  added and the s o l u t i o n  (16.8  solution  g.,  0.118  kept at room temp-  Much c r y s t a l l i n e s o l i d had The white c r y s t a l l i n e product  precipitated was  filtered  o f f a f t e r c h i l l i n g i n an i c e bath, washed with a s m a l l amount of c o l d Dilute  e t h a n o l then r e c r y s t a l l i z e d sodium hydroxide  solution  was  from a minimum of e t h a n o l . added t o the f i l t r a t e  the s m a l l amount of o f f - w h i t e s o l i d p r e c i p i t a t e s t a r t i n g material»and t r e a t e d as b e f o r e . N -tosyltryptamine b  117°). ative  Although was  20.4  Y i e l d of N -methylb  118-119° ( l i t e r a t u r e  s t r o n g d e p r e s s i o n i s shown on  116-  mixing.  N -methyl-N -tosyltryptamine(100) b  b  N -methyl-Nb-tosyltryptamine D  m.p.  assumed t o be  the m e l t i n g p o i n t of the unmethylated d e r i v -  very s i m i l a r ,  D e t o s y l a t i o n of  g., 67%;  and  (20 g., 0.064 moles)  d i s s o l v e d i n t e t r a h y d r o f u r a n (350 ml.)  was  and added to l i q u i d  88  ammonia (1.5 1.)  w i t h s t i r r i n g i n a 3 l i t e r 3-necked  bottom f l a s k c o o l e d i n a d r y - i c e acetone bath.  round  Sodium  added i n s m a l l p i e c e s t o the s t i r r e d s o l u t i o n u n t i l  was  the  c h a r a c t e r i s t i c deep blue c o l o u r of s o l v a t e d e l e c t r o n s p e r s i s t e d f o r ten minutes.  Ammonium c h l o r i d e was  u n t i l the c o l o u r disappeared.  then added c a u t i o u s l y  Ammonia was  overnight at room temperature.  allowed t o  Water and 4 N sodium  evaporate hydroxide  were added and the o r g a n i c m a t e r i a l c o l l e c t e d with e t h e r 100 ml.).  The b a s i c product was  hydrochloric acid  (3 x  purified via solution in 1 N  (3 x 50 ml.), r e - e x t r a c t i o n w i t h ether  (3 x 50  ml.), washing u n t i l n e u t r a l w i t h water, d r y i n g over magnesium s u l p h a t e , and e v a p o r a t i o n t o give a brownish on c o o l i n g .  The  N -methyltryptamine b  l i z e d from ether but was  o i l which s o l i d i f i e d  (50) c o u l d be  recrystal-  very s u s c e p t i b l e t o o x i d a t i o n producing  a brown e t h e r - i n s o l u b l e gum,presumably through the 3-hydroperoxide and was  best c r y s t a l l i z e d under n i t r o g e n , m.p.  Crude y i e l d ; 9.8 274,  282,  g. , 93%.  291 mfx., 6  i n d o l e chromophore.  m a x  U l t r a v i o l e t spectrum; 7000, 7200, 6000.  (deuterochloroform) m u l t i p l e t s c e n t e r e d at 2.60 area 4.8  (ethanol)  ^-  spectrum; and 2.98T  H) , s i n g l e t at 3.28T (OL  p r o t o n , area 1 H), s i n g l e t at 7.12 T(methylene protons, area 4 H), s i n g l e t at 7.65T (N-methyl, area 3 H): resonance  from 3 - 4 T ( a r o m a t i c  (benzene) a broad  p l u s NH protons, area 6 H), a  broad s i n g l e t c e n t e r e d at 7.15T  (methylene  s i n g l e t at 7.77T (N-methyl, 3 H).  The  m a x  This i s a t y p i c a l  Nuclear magnetic resonance  (aromatic protons p l u s NH,  88-89° C.  p r o t o n s , 4 H), a  n.m.r. s p e c t r a are  89  c o n s i s t e n t w i t h the s t r u c t u r e o f Nb-methyltryptamine.  The  degeneracy o f the four methylene protons i n d e u t e r o c h l o r o f o r m was removed i n benzene.  The mass spectrum: m/e 174 (M , 8%), +  144 (M-31, 7 % ) , 131 (M-44+1, 100%), 44 (dimethylamine), 31 (methylamine).  C a l c . f o r C12H14N2;Molecular weight 174.  Coupling of D i p t e r i n  (N -methyltryptamine) (50) b  One gram o f d i p t e r i n was used f o r s e v e r a l t r i a l in  oxidations  order t o e s t a b l i s h reasonable c o n d i t i o n s f o r the o x i d a t i o n ,  work-up, and s e p a r a t i o n o f p r o d u c t s .  A c l e a n e r product i s  obtained when the c o u p l i n g i s c a r r i e d out under n i t r o g e n .  When  o n e - t h i r d o f the t h e o r e t i c a l amount o f f e r r i c c h l o r i d e i s used the  y i e l d o f chimonanthine i s decreased but the amount of  recoverable s t a r t i n g material increased.  As the i n d o l y l mag-  nesium i o d i d e was found t o be q u i t e i n s o l u b l e i n ether the c o u p l i n g r e a c t i o n was attempted i n t e t r a h y d r o f u r a n without success but w i t h an i n t e r e s t i n g byproduct.  The procedure  d e s c r i b e d i s f o r a s c a l e d up c o u p l i n g r e a c t i o n . Methyl magnesium i o d i d e was prepared i n the u s u a l manner from magnesium t u r n i n g s  (1.96 g., 0.805 mmoles) f r e s h l y crushed  i n a mortar and methyl i o d i d e  (11.4 g., 0.805 mmoles) i n a 3  l i t e r 3-necked f l a s k equipped w i t h a short condenser, a mecha n i c a l s t i r r e r w i t h a mercury and d r y i n g tubes.  gland, a 250 ml. dropping f u n n e l ,  The apparatus was flame d r i e d and the  r e a c t i o n c a r r i e d out under a p o s i t i v e p r e s s u r e of dry n i t r o g e n . A t o t a l volume o f 500 ml. of commercial anhydrous ether was used.  90  The d i p t e r i n  (14 g., 0.805 mmoles) used was  the crude m a t e r i a l  from d e t o s y l a t i o n of N -tosyl-Nb-methyltryptamine D  a z e o t r o p i n g w i t h benzene (3 x 50 ml.) ether was  (700 ml.).  d r i e d by  and d i s s o l v e d i n anhydrous  The s m a l l amount of e t h e r i n s o l u b l e i m p u r i t y  removed by f i l t r a t i o n through a pad of g l a s s wool as the  d i p t e r i n was  added dropwise t o the s t i r r e d  s o l u t i o n of methyl  magnesium i o d i d e over a p e r i o d of about one hour.  The  light  y e l l o w s o l i d indolylmagnesium i o d i d e , some i n suspension and some on the f l a s k w a l l s , was Anhydrous f e r r i c c h l o r i d e  s t i r r e d v i g o r o u s l y f o r two  hours.  (15 g., 0.920 mmoles, 15% excess  a l l o w i n g f o r i m p u r i t i e s ) was  d i s s o l v e d i n anhydrous  e t h e r (2 x  200 m l . ) , decanted from the rust-brown r e s i d u e , and added dropwise w i t h v i g o r o u s s t i r r i n g t o the indolylmagnesium suspension.  The b l u e - b l a c k r e a c t i o n mixture was  v i g o r o u s l y f o e i g h t e e n hours.  iodide  stirred  The G r i g n a r d reagent  decomposed with one and one-half l i t e r s of c o l d  was  saturated  ammonium c h l o r i d e w i t h s t i r r i n g f o r two hours l e a v i n g brown aqueous and e t h e r l a y e r s and a brown s o l i d . was  light  The  latter  removed by f i l t r a t i o n through a c e l i t e pad and washed with  s a t u r a t e d ammonium c h l o r i d e .  The ether l a y e r was washed with  b r i n e and d r i e d over magnesium s u l p h a t e . g. of a b l a c k s o l i d was mixture was  0.22  o b t a i n e d . " The composition of t h i s  not i n v e s t i g a t e d a f t e r i t was  chimonanthine  On e v a p o r a t i o n  found t o c o n t a i n no  by comparative t h i n - l a y e r chromatography.  aqueous l a y e r was  e x t r a c t e d w i t h c h l o r o f o r m (5 x 100 ml.)  enough 6 N sodium  hydroxide was  The then  added t o b r i n g the pH t o approx-  91  i m a t e l y ten,shaking a l l the w h i l e w i t h c h l o r o f o r m . c e l l u l o s e was  added t o absorb the green g e l a t i n o u s p r e c i p i t a t e  of f e r r o u s hydroxide which was r e p e a t e d l y , a-s was chloroform.  Powdered  then f i l t e r e d and  the o r i g i n a l brown s o l i d , w i t h b o i l i n g  The aqueous l a y e r was  with c h l o r o f o r m .  titurated  also repeatedly extracted  A l l c h l o r o f o r m e x t r a c t s which were red-brown  i n c o l o u r were combined, d r i e d over anhydrous  magnesium s u l p h a t e  and evaporated y i e l d i n g e l e v e n grams (80% r e c o v e r y ) of a product mixture.  A n a l y s i s by t h i n - l a y e r chromatography  methanol-ether^  (alumina G,  2%  e e r i e s u l p h a t e spray (1% e e r i e s u l p h a t e : 35%  s u l p h u r i c a c i d ) ) r e v e a l e d a mixture of f i v e major components i n c l u d i n g meso-chimonanthine ( R f 0.50, changing t o y e l l o w ) , dl-chimonanthine  r e d changing to blue-green ( R f 0.30,  blue changing t o yellow) and Nb-methyltryptamine yellow).  Two  r e d changing t o ( R f 0.10,  compounds more mobile i n t h i s system than chim-  onanthine were named compound A ( R f 0.68, and compound B ( R f 0.58,  purple, fading quickly)  brown a f t e r warming).  Of the s o l v e n t  systems t r i e d e t h e r , or e t h e r mixtures, were the o n l y ones found which would s e p a r a t e 2% Methanol-ether was ether  ( R f 0.80,  meso and racemic  chimonanthines.  the most g e n e r a l l y u s e f u l .  chimonanthine,  c a l y c a n t h i n e ) was  a n a l y s i s of monomeric components ( R f 0.30, A c i d i c e e r i e s u l p h a t e spray was  50%  Methanol-  useful for  Nb-methyltryptamine).  the most g e n e r a l l y u s e f u l f o r  d e t e c t i o n of compounds as i t not only gave b r i g h t l y c o l o u r e d spots but spots which changed i n c o l o u r with time i n a c h a r a c t e r i s t i c manner.  C h l o r o p l a t i n i c a c i d , Dragendorf r e a g e n t ^  4  and  92  i o d i n e vapour were a l s o u s e f u l .  C h l o r o p l a t i n i c a c i d a l s o gave  s p e c i f i c c o l o u r s and was more s e n s i t i v e i n the case of compounds A and B than was e e r i e s u l p h a t e .  Dragendorf reagent was p a r t -  i c u l a r l y s e n s i t i v e f o r a l l these compounds but showed uniform orange s p o t s .  Iodine vapour was very convenient but not very  specific.  Chromatographic  s e p a r a t i o n o f the p r o d u c t s .  The product mixture was d i s s o l v e d i n benzene and s u b j e c t e d t o c a r e f u l chromatography The chromatography and f r a c t i o n s  on n e u t r a l alumina  was f o l l o w e d by t h i n - l a y e r  (grade I, 400 g . ) . chromatography  (250 ml.) combined a c c o r d i n g l y .  A f t e r two l i t e r s  of benzene had passed through the column compound A was e l u t e d (500 ml., 0.25 g . ) .  E l u t i o n w i t h 2% ether-benzene  (1000 ml.)  produced a mixture of compounds A and B (0.26 g.) then w i t h 10% ether-benzene  (500 ml.) a mixture of meso-chimonanthine,  compound B and some of compound A (0.30 g . ) . ether-benzene  E l u t i o n w i t h 20%  (1000 ml.) y i e l d e d a mixture o f compounds A and B  w i t h both racemic and meso-chimonanthines (1.2 g . ) . onanthine w i t h some o f the meso isomer e l u t e d w i t h 50% ether-benzene  D  (1.05 g.) was then  (500 ml.) f o l l o w e d by a mixture o f  chimonanthine, u n i d e n t i f i e d m a t e r i a l N -methyltryptamine  dl-Chim-  ( i n d o l e chromophore) and  (1.22 g., 500 m l . ) .  The column was then  s t r i p p e d of o r g a n i c m a t e r i a l u s i n g e t h e r (250 m l . ) , 10% methanolether  (250 ml.) and f i n a l l y methanol  (1800 ml.).  N -methylD  tryptamine was the major component of t h i s dark brown gum  (5.2 g.)  93  and 30% of s t a r t i n g m a t e r i a l c o u l d be r e c o v e r e d by washing through  an alumina  column  chloroform-methanol, The  and  (grade  III) using chloroform,  finally  methanol.  other major components were f u r t h e r p u r i f i e d by r e -  peated chromatography then by c r y s t a l l i z a t i o n and y i e l d s  calc-  u l a t e d on the b a s i s of 30% r e c o v e r y of s t a r t i n g m a t e r i a l . Compound A ( y i e l d 3.5%) form or benzene and was  was  difficult  i n the c r y s t a l l i n e form. m.p. H, 7.44%; N, N,  16.5%.  15.78%.  276  Calc. for C  sh., 283,  Ultraviolet sh. , 264,  t o r e d i s s o l v e once o b t a i n e d  274-275° C. A n a l . Found: C, 76.26%;  Calc. for C 2 2  m o l e c u l a r weight 344. 249,  r e c r y s t a l l i z e d from c h l o r o -  H  2 4  2 2  H  C  4  sh. nyu., em  271  sh., 290  sh. mw.,  1590(s),  deuterochloroform:  7100,  ax  6  N,  16.25%;  1580(s).  The  at 7.61T"  (R N-CH 2  s i g n i f i c a n t peaks at m/e  (M + 2, 23%), 329  (4%).  239  m a x  singlets  and  at 5.34  and  area each 1 H), u n r e s o l v e d m u l t i p l e t s from  singlet  (M - 58, 4%), 209  A  u n r e s o l v e d m u l t i p l e t s c e n t e r e d at 2.95  (methylene p r o t o n s ) , s i n g l e t  spectrum had  x  n.m.r. spectrum i n  6.4  and  a  3440 (w), 2930, 2850,  max  R3CH,  to 8.IT  m  6000, 5300, 5000, 3800.  m a x  5.80T (NH or  130  7.0%;  7.5%;  7000, 7200, 4500.  3.45T (aromatic p r o t o n s , area 8 H), broad  10%),  H,  3  spectrum i n e t h a n o l i c h y d r o c h l o r i c a c i d :  2790, 1640(s),  346  - %5  spectrum i n e t h a n o l : X  I n f r a r e d spectrum i n c h l o r o f o r m : V >  area 3 H),  7 6  4  N : C, 76.7%; H,  Ultraviolet  304  N : >  2 g  (M - 15,  (M - 135,  344  12%), 300  5%), 197  at 6 . 7 2 T 3j  (R N-CH , 2  area 3 H). (M ), +  345  (M - 44,  (M - 147,  3  The mass  (M+1,  10%),  4%), 172  107%),  286 (M -  172,  94  Compound B ( y i e l d 3.5%) was d i f f i c u l t shown t o be a mixture same u l t r a v i o l e t  o f two isomers both of which have the  spectrum.  The p h y s i c a l data are f o r the isomer  which was s l i g h t l y more mobile e t h e r , m.p. 16.10%.  235-240° C.  Calc. f o r C  2 2  t o p u r i f y and was  H  molecular weight 344.  when developed  with  Anal. Found: C, 76.56%; H, 7.09%; N, 2 4  N : C, 76.7%; H, 7.0%; N, 16.25%; 4  spectrum i n e t h a n o l : A * max. 10080, 10100, 6500. Ultraviolet  Ultraviolet  &  274, 283, 310 sh. mju,, C  on alumina  m a x  spectrum i n e t h a n o l i c h y d r o c h l o r i c a c i d :  ~\  267, 276, 293  nicix, mix,  £  7600, 7600, 5900.  includes:  V  max.  1450(s) cm" . 1  doublet  I n f r a r e d spectrum i n c h l o r o f o r m  3440, 3200, 2950, 2830, 1630(s),  1580(s),  The n.m.r. spectrum i n d e u t e r o c h l o r o f o r m : a  ( J = 7.8 c.p.s.) c e n t e r e d at 2.21T  (aromatic  area 1 H), a wide m u l t i p l e t c e n t e r e d at 3.30T p r o t o n s , area 7 H), broad d o u b l e t s  proton,  (aromatic  ( J c a . 3 c.p.s.) c e n t e r e d  at 5.50 and 5.70T  (R3CH and R2NH, each area 1 H), a s e x t e t  from 6.1 t o 6.65 T  (methylene p r o t o n s , area 2 H), p a r t i a l l y  obscured m u l t i p l e t s from 7.0 t o 8.1 T  (methylene p r o t o n s ,  area  5 H), doublet of m u l t i p l e t s ( J = 14 c.p.s.) c e n t e r e d at 8.84T (methylene p r o t o n , area 1 H) , s i n g l e t at 6 . 7 8 T 3 H) and a s i n g l e t at 7.42T  (N-CH3,  spectrum had s i g n i f i c a n t peaks at m/e 346 (M + 2, 10%), 343 ( M - 1 ,  area 3 H).  (N-CH3,  area  The mass  344 ( M ) , 345 ( M + 1 , +  20%),  45%), 299 (M - 45, 22%), 288  (M - 56, 20%), 231 (M - 113, 4%), 172 (M - 172, 23%), 159 (16%), 143 (10%), 130 (16%). Meso-chimonanthine  ( y i e l d 7%) was r e c r y s t a l l i z e d  from  95  benzene,  m.p.  198-202° C.  There was  no d e p r e s s i o n of m e l t i n g  p o i n t on admixture w i t h n a t u r a l meso-chimonanthine erimental f o r i s o l a t i o n ) .  Anax  2  4  8  »  T * '  305  Ultraviolet ^max  1 3 0 0 0  ( t y p i c a l P h - N - C - N chromophore). (0.1 mg./ml. ) :  spectrum i n e t h a n o l : 4  900.  (0.1 N ) : A  in ethanolic hydrochloric acid  form  >  (see exp-  m a x  Ultraviolet  spectrum  239, 294 nyu,  I n f r a r e d spectrum i n c h l o r o -  3440(w), 2920(s), 2850(m), 2800 (m) ,  v  1615(s), 1480 ( s ) , 1460(s), 1395(w), 1340(m), 1310(m), 1240(w), 1150(m), 1120(m), 1022(m), 994(w), 935(w), 908(w) cm" .  The  n.m.r. spectrum i n d e u t e r o c h l o r o f o r m : m u l t i p l e t s from 2.7  to  7" (aromatic p r o t o n s , area 8 H) , broadened s i n g l e t  T  1  (R N-H, area 2 H), broadened  at 5.35  4.0  singlet  at 6.15T  ( R 3 C - H , area 2 H),  p a r t i a l l y masked m u l t i p l e t s from 7.0  to 8 . 3 T  (methylene p r o t o n s ,  2  area 8 H) , s i n g l e t  ( N - C H 3 , area 6 H) .  at 7.63 7"  spectrum had fragments at m/e 173  ( M - 173, 25%), 157 dl-Chimonanthine  benzene,  m.p.  ( M , 7%), 172  346  (2%) and 130  ( y i e l d 19%) was There was  p o i n t on admixture w i t h a sample  ( M - 174,  +  ( 2 % ) , 144  183-185° C.  The mass 100%),  (10%).  r e c r y s t a l l i z e d from no d e p r e s s i o n of m e l t i n g  of s y n t h e t i c dl-chimonanthine  k i n d l y s u p p l i e d by P r o f e s s o r J.B.Hendrickson and prepared by c o u p l i n g of o x i n d o l e s . 247, 303 mij., C  m  a  x  4 8  Ultraviolet  13600, 5600.  ethanolic hydrochloric acid: spectrum i n c h l o r o f o r m : V  A  m  spectrum i n e t h a n o l :  Ultraviolet a  x  spectrum i n  239, 294 myii.  Infrared  3440(w), 2920(s), 2850(m), 2800  (m), 1615(s), 1480(s), 1460(s), 1395(m), 1350(m), 1310(m), (w), 1150(m), 1120(m), 1090(w), 1055(w), 1022(m), 904(w)  1240 cm" . 1  96  The n.m.r. spectrum  i n deuterochloroform: m u l t i p l e t  analyzed i n the 100 Mc 2.7  t o 3.7T  spectrum  (can be  as a simple ABCD spectrum) from  (aromatic protons, area 8 H), broad s i n g l e t at 5.62T  (R3C-H, area 2 H), broader s i n g l e t at 5.75T(R N-H, area 2 H), 2  m u l t i p l e t s from 7.2  t o 8.2 7"  a s i n g l e t at 7.70  (N-CH3, area 6 H).  fragments at m/e 100%),  T  346(M ,  (methylene  p r o t o n s , area 8 H), The mass spectrum  <12%), 347(M + 1, 1%), 172(M  +  173 (M - 173, <33%), 157(2%),  144(2%) and  -  and had  174,  130(26%).  Attempted c o u p l i n g of d i p t e r i n ( 5 0 ) i n t e t r a h y d r o f u r a n Methyl magnesium i o d i d e was  prepared  i n tetrahydrofuran  ( d r i e d by r e f l u x i n g over l i t h i u m aluminum hydride)  from  magnesium (0.28  As not a l l  of  g.) and methyl i o d i d e (1.63  g.).  the magnesium d i s s o l v e d even a f t e r prolonged h e a t i n g the  reagent was reagent)  e s t i m a t e d by adding an a l i q u o t  to d i l u t e hydrochloric acid  titrating  (1 ml.,  (0.1 N, 3 ml.)  w i t h d i l u t e sodium carbonate  1/60  of  and back-  (0.0173 N, 7.9  ml.)  methyl r e d , as 75% of the t h e o r e t i c a l N^-methyltryptamine (1.5 g.)  d i s s o l v e d i n t e t r a h y d r o f u r a n was  stirring  and s t i r r i n g was  was  i n c r e a s e d (150 ml.)  c o n t i n u e d f o r two  hours.  The  volume  because the i n d o l y l G r i g n a r d reagent Anhydrous f e r r i c c h l o r i d e  i n t e t r a h y d r o f u r a n (50 ml.)  was  The workup was  (1.4  g.)  added s l o w l y g i v i n g a b l a c k  a p p a r e n t l y homogeneous s o l u t i o n which was  solvent  (50)  added dropwise w i t h  s e p a r a t e d as a brown gum.  overnight.  to  s t i r r e d under n i t r o g e n  i d e n t i c a l t o that u s i n g e t h e r as a  (see p r e v i o u s experiment) and recovery of o r g a n i c  97  m a t e r i a l was good (85%). The absence o f e i t h e r chimonanthine demonstrated  or c a l y c a n t h i n e was  by t h i n - l a y e r chromatography  eerie sulphate).  (alumina G, e t h e r ,  Besides s t a r t i n g m a t e r i a l , the two major  p r o d u c t s which were d e s i g n a t e d C (R^ 0.5, yellow, 15% o f product) and D (Rj 0.3, p u r p l e , 5% of product) were i s o l a t e d  .  by chromatography on alumina. Compound C was r e c r y s t a l l i z e d  from e t h e r and i d e n t i f i e d  from p h y s i c a l data as N , N - d i m e t h y l t r y p t a m i n e . b  m.p.  b  45-49° C, l i t . m . p . 49-50° C .  H, 8.72%; N, 14.26%. C a l c . f o r C N, 14.89%; m o l e c u l a r weight ethanol:  A  A  N  188.  1 1 3  ),  :  C  >  76.54%; H, 8.57%;  The u l t r a v i o l e t €  m  a  spectrum i n  6600, 7000, 6200,  x  T h i s i s a t y p i c a l i n d o l e chromo-  The i n f r a r e d spectrum  substituted p y r r o l e 1  16 2  277 sh. , 283, 292 mjUL,  m a x  u n a f f e c t e d by d i l u t e a c i d . phore.  Anal, found: C, 76.74%;  1 1 5  H 1 2  ( F i g u r e 41).  includes: V  mSLX  3500 (NH,  2950, 2870, 2840, 2790 cm"  1  (-N(CH ) ) 3  2  l O X  U  The n.m.r. spectrum  c e n t e r e d at 2.4 and 2.9 T a broadened doublet  i n deuterochloroform:  multiplets  (aromatic p r o t o n s , areas 1 and 3 H),  ( J = 1.8 c.p.s.) c e n t e r e d at 3.27 T (CV.  p r o t o n , area 1 H), m u l t i p l e t at 7 . 2 1 7 " (methylene 4 H) and a s i n g l e t at 7.61T spectrum  has m/e  85%), 115(40%),  (N(CH ) ),  188 (M+, 100%),  3  2  p r o t o n s , area  area 6 H).  The mass  143 (M - 45, 45%), 130 (M - 58  58 (M - 130), 44.  Compound D was r e c r y s t a l l i z e d from e t h e r , m.p.  134-147° C.  It was i d e n t i f i e d as a t e t r a h y d r o f u r a n y l t r y p t a m i n e d e r i v a t i v e . ( F i g u r e 41).  Anal. F o u n d ( s i n g l e d e t e r m i n a t i o n o n l y ) : C, 73.10%  98  H, 8.19%; N, 11.68%; O, 7.1% C  1 5  H  2 0  (by d i f f e r e n c e ) .  N 0 : C, 73.73%; H, 8.25%; N, 11.47%; 0 , 6.55%: molecular 2  weight 244.  The u l t r a v i o l e t  sh. , 283, 290 mu., C ' ' ' '  max.  acid.  The  spectrum  i n ethanol: ^  6700. 6900. 5200.  i n d o l e chromophore and was  includes: V  i n f r a r e d spectrum  2850, 2700 (N-CH ), 1630,  956 cm"" .  n.m.r. spectrum  The  1  s i n g l e t at 0.96 multiplet  m  (aromatic N-H,  from 2.3  to 3 , l T  a  3500  x  1010,  (M - 59, 144  had m/e  100%),  159  ( 6 % ) , and 130  area 0.7  exchanges w i t h D 0,  T  area  2  (-0-CH -, area 3 H) , 2  (-CH -CH -N-, area 4 H), 2  2  singlet  ( J = 22  (-CH-(CH ) -CH -, area 4 H). 2  2  ( M , 10%), 198  ( M - 59,  The  2  +  14%), 172  (M - 45,  1%),  (M - 72, 3%), 156  158 (5%),  of the mother l i q u o r s from which compound  e t h e r , alumina, c . f . chimonanthine R^ 0.25)  also evident.  was  346 which i s the  molecular weight of the c a l y c a n t h i n e isomers. 172 peak was  It was  The  corresponding  concluded that a s m a l l  amount of the c o u p l i n g r e a c t i o n took p l a c e i n t e t r a h y d r o f u r a n  (  >1%).  H),  (aromatic p r o t o n s , area 4 H) , broad-  c r y s t a l l i z e d showed a fragment at m/e  m/e  975,  (4%).  A mass spectrum C (Rj 0.30,  244  990,  2  at 7.44 7" (N-CH3, area 3 H) and a broadened doublet  mass spectrum  (NH,  exchanges w i t h D 0,  H) , m u l t i p l e t c e n t e r e d at 6.39 7  c.p.s.) c e n t e r e d at 8.22T  276  x  i n d e u t e r o c h l o r o f o r m : broadened  ened s i n g l e t 3.53 7" ( a l i p h a t i c N-H,  m u l t i p l e t c e n t e r e d at 7.12  a  This i s a t y p i c a l  1670,  3  T  m  e s s e n t i a l l y unchanged i n d i l u t e  p y r r o l e ) , 2910,  0.9  Calc. for  99  Discovery of meso-chimonanthine i n nature A sample of crude chimonanthine of  Calycanthus f l o r i d u s was  purposes  of comparison  m a t e r i a l was  (201 mg.)  from an e x t r a c t  r e c e i v e d from Dr.G.F.Smith f o r  w i t h the s y n t h e t i c product.  y e l l o w i n c o l o u r p a r t of i t (83 mg.)  As was  this  washed  through an alumina column w i t h benzene (50 m l . ) , 10% e t h e r benzene  (50 m l . ) , e t h e r , and f i n a l l y  benzene f r a c t i o n was white c r y s t a l s m.p.  7 1  m.p.  199-202° C. l i t . 1-chimonanthine  T h i s compound showed s t r o n g d e p r e s s i o n of  m e l t i n g p o i n t on admixture  w i t h the s y n t h e t i c  183-185° C).  chimonanthine  first  isolated  lO^Jjj  (chloroform) 0.0°, l e d t o i t s i d e n t i f i c a t i o n as meso-  chimonanthine  ( m.p.  The e t h e r -  r e c r y s t a l l i z e d twice from benzene y i e l d i n g  (20 mg.)  188-189° C .  chloroform.  and d i s c o v e r y of the same m a t e r i a l i n the Nb-methyl-  tryptamine c o u p l i n g product. data f o r the s y n t h e s i s . of  A check of r o t a t i o n ,  For p h y s i c a l data see experimental  There was  a complete  correspondence  i n f r a r e d , u l t r a v i o l e t , n.m.r., and mass s p e c t r a .  Behaviour  on t h i n - l a y e r chromatography i n s e v e r a l s o l v e n t systems i n c l u d i n g the c o l o u r r e a c t i o n with e e r i e s u l p h a t e was identical.  An n.m.r. spectrum  also  i n d e u t e r o c h l o r o f o r m of the crude  m a t e r i a l showed s i n g l e t s of equal i n t e n s i t y at 7.63 T  (-N-CH3 of  1-chimonanthine) and at 7.72 7" (-N-CH3 of meso-chimonanthine). T h i n - l a y e r chromatographic  analysis  (alumina G, 2% methanol-  e t h e r , 1% e e r i e s u l p h a t e : 3 5 % s u l p h u r i c a c i d ) r e v e a l e d a component  (approximately 1:1)  mixture.  two  100 t  P r e p a r a t i o n of d l - c a l y c a n t h i n e by i s o m e r i z a t i o n o f d l chimonanthine. T r i a l experiments were f i r s t  c a r r i e d out on samples o f  s y n t h e t i c dl-chimonanthine (5 mg.) i n d i l u t e h y d r o c h l o r i c  acid  (4 N, I N , 0.1 N and 0.01 N) (5 ml.) under n i t r o g e n at 20° and 100° C f o r times r a n g i n g from two hours t o twenty days.  After  a d d i t i o n o f potassium carbonate and e x t r a c t i o n w i t h c h l o r o f o r m the  a l k a l o i d mixture was examined by t h i n - l a y e r  (alumina, e t h e r , e e r i e s u l p h a t e ) . led  chromatography  The most d r a s t i c  conditions  t o r e c o v e r y o f only a s m a l l amount of a l k a l o i d and t h i s  was mainly N -methyltryptamine  while the m i l d e s t  b  produced no o b s e r v a b l e change.  conditions  Intermediate c o n d i t i o n s  produced s p o t s c o r r e s p o n d i n g t o N -methyltryptamine, d l - c h i m b  onanthine and c a l y c a n t h i n e  (R  f  0.80, r e d changing t o grey-green  changing t o mauve when sprayed w i t h 1% e e r i e s u l p h a t e : 35% s u l p h u r i c a c i d ) as w e l l as some very minor  spots.  A second s e r i e s o f experiments u s i n g a c e t i c a c i d , d l chimonanthine  (2 mg.) i n p r e b o i l e d d i s t i l l e d water  under n i t r o g e n was  j  (5 m l . ) ,  c a r r i e d out and found t o be s u p e r i o r i n  that e s s e n t i a l l y no N -methyltryptamine b  or minor products were  produced. dl-Chimonanthine containing acetic acid  (30 mg.) was d i s s o l v e d i n water  (5 ml.)  (5 drops) and heated on a steam bath  (30 hours) under n i t r o g e n .  The a l k a l o i d r e c o v e r e d (27 mg.) was  a mixture o f two components (1:4) c o r r e s p o n d i n g t o chimonanthine and c a l y c a n t h i n e , w i t h only a t r a c e o f N. -methyltryptamine.  101  The major component was on alumina,  i s o l a t e d by chromatography  (20  mg.)  r e c r y s t a l l i z e d from benzene and compared w i t h  natural d-calycanthine.  White c r y s t a l s :  m.p.244-247° C, which  showed d e p r e s s i o n of m e l t i n g p o i n t on admixture w i t h n a t u r a l d-calycanthine.  The  s o l i d s t a t e i n f r a r e d s p e c t r a (KBr d i s c s )  were not i d e n t i c a l but a l l other p h y s i c a l data, i n c l u d i n g s o l u t i o n i n f r a r e d , which were run on a P e r k i n Elmer 21 found t o be superimposable, The u l t r a v i o l e t C  max.  19000, 6300; '  298 mjLu.  The  and  were i d e n t i c a l .  spectrum i n e t h a n o l :  ^  m  a  250,  x  i n dilute hydrochloric acid; '  i n f r a r e d spectrum  (chloroform): V  1585(m), 1490(s),  m  }\ a  max.  x  1607(s),  1315(s),  1303(s),  1287(m), 1268(m), 1240(m), 1190(m),  958(w), 886(w), 865(w) c m .  The  -1  1040(s),  1450(m), 1378(m),  1025(s),  975(w),  t o 3.907", e a s i l y  (see F i g u r e 37)  p r o t o n s , area 8 H) , a broad s i n g l e t  1167(s),  n.m.r. spectrum i n deutero-  c h l o r o f o r m : complex of m u l t i p l e t s from 2.90 spectrum  240,  3480(m),  2820(s),  1157(m), 1118(w), 1107(m), 1067(s),  mfx,  309  2960(s),  a n a l y s a b l e i n a 100 Mc  the  (aromatic  at 5.527" (R2NH,  i n D2O, area 2 H), a broadened s i n g l e t at 5.72 T  exchanges  (N-CH-N, sharp  i n D2O, area 2 H), a s e x t e t c e n t e r e d at 6.85Twith c o u p l i n g cons t a n t s 14,  14 and 2 c.p.s.(methylene p r o t o n s , area 2 H),  m u l t i p l e t s from 7.2  t o 8.1T  4 H) and a s i n g l e t at 7 . 6 2 T spectrum was (M , +  (remaining methylene p r o t o n s , (NCH3, area 6 H).  The mass  i n sharp c o n t r a s t t o chimonanthine w i t h m/e  100%), 347(M + 1, 25%), 302(M - 44,  area  346  12%), 288(M - 58,  17%),  102  259(6%), 245(10%), 231(M - 115, 25%), 172(M - 174, 10%), and 130(6%) .  Isomerization of  meso-chimonanthine  Meso-chimonanthine acid  (10 mg„) was d i s s o l v e d i n d i l u t e  acetic  (10 drops i n 5 ml. water) and heated under n i t r o g e n on the  steam bath f o r 24 hours.  Examination by t h i n - l a y e r chromato-  graphy a f t e r work up r e v e a l e d a two component mixture (1:2) contaminated with a small amount o f N^-methyltryptamine. component  The  (major) other than s t a r t i n g m a t e r i a l had a decided  tendency t o t a i l and s t r e a k e d  from the o r i g i n t o R  f  0.8  (alumina,  e t h e r , mauve c o l o u r f a d i n g q u i c k l y with e e r i e  sulphate)  u n l e s s very l i g h t l y  spotted.  P a r t i a l s e p a r a t i o n was achieved by chromatography  on alumina  u s i n g benzene-ether mixtures and 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 from benzene. violet  White c r y s t a l s : m.p. 265-268° C.  spectrum i n e t h a n o l :  A  n  max.  245, 307 mu,, ' I  Ultra-  The mass  spectrum was s t r i k i n g l y s i m i l a r t o that o f d l - c a l y c a n t h i n e d i f f e r i n g only s l i g h t l y  i n the abundance o f some fragments:  m/e 346 (M+, 100%), 347 ( M + 1 ,  25%), 302 (M - 44, 7%), 288  (M - 58, 10%), 259 ( 5 % ) , 245 (11%), 231 (M - 115, 15%), 172 (26%) and 130 (16%). I s o m e r i z a t i o n of Compounds A and B Compounds A and B (10 mg.) were s u b j e c t e d t o the same i s o m e r i z a t i o n c o n d i t i o n s as was meso-chimonanthine  and examined  103  by t h i n - l a y e r chromatography.  A l a r g e p o r t i o n was not  recovered from aqueous s o l u t i o n but compound A was a p p a r e n t l y u n a f f e c t e d while compound B was p a r t i a l l y transformed T h i s was not confirmed by i s o l a t i o n .  i n t o A.  Neither calycanthine  nor chimonanthine was formed.  A c e t y l a t i o n o f compound A Compound A (20 mg.) was t r e a t e d with a c e t i c (0.5 ml.) and p y r i d i n e (0.5 ml.) f o r 15 minutes. (Rj  0.2, alumina,  anhydride The product  e t h e r ; orange, 1% e e r i e s u l p h a t e : 3 5 % s u l p h u r i c  a c i d ) was shown t o be the monoacetate by i n t e g r a t i o n of the a c e t a t e methyl  (8.01T) i n the n.m.r. spectrum.  m a t e r i a l was found i n the product. in ethanol:  A  m a x  No s t a r t i n g  The u l t r a v i o l e t  250 sh. , 273 sh. , 280 mjx, was very  spectrum similar  t o t h a t of compound A.  Attempted hydrogenation  o f compound A  Compound A (16 mg.) was shaken i n e t h y l a c e t a t e w i t h p r e hydrogenated p a l l a d i u m on c h a r c o a l (1:10) f o r two hours.  No  hydrogen was absorbed.  Attempted r e d u c t i o n of compound A Compound A (95 mg.) and l i t h i u m aluminum hydride  (60 mg.)  were r e f l u x e d overnight i n t e t r a h y d r o f u r a n (5 ml., d r i e d by r e f l u x i n g over L i A l H ^ ) . a d d i t i o n of e t h y l a c e t a t e .  Excess  hydride was destroyed by  A f t e r f i l t r a t i o n and e v a p o r a t i o n  104  t h i n - l a y e r chromatography showed v i r t u a l l y unchanged s t a r t i n g material.  The u l t r a v i o l e t  spectrum was  a l s o unchanged.  Hodgkinsine A sample of hodgkinsine Taylor i n Australia.  (80 mg.)  I t was  was  r e c e i v e d from  b e l i e v e d then t o be  i s o m e r i c with c a l y c a n t h i n e or to be a dehydro  Dr.  either  isomer.  Isomerization Hodgkinsine  (5 mg.)  was  dissolved in preboiled  water c o n t a i n i n g a c e t i c a c i d n i t r o g e n on the steam bath  distilled  (3 drops) and heated under  (15 hours).  The  sample  (4 mg.)  was  r e c o v e r e d by e x t r a c t i o n w i t h c h l o r o f o r m of the s o l u t i o n which had been made b a s i c with sodium b i c a r b o n a t e .  A n a l y s i s by  t h i n - l a y e r chromatography i n s e v e r a l s o l v e n t systems ( i . e . alumina, e t h e r , e e r i e sulphate) r e v e a l e d hodgkinsine red changing t o pink then t o y e l l o w ) , some o r i g i n , a major product and a minor product  (Rf 0.54,  (Rf 0.70,  (Rf  0.2,  m a t e r i a l at the  red changing t o red-brown)  r e d changing t o p i n k ) .  Neither  hodgkinsine nor i t s a c i d i s o m e r i z a t i o n products corresponded products or a c i d i s o m e r i z a t i o n products from the c o u p l i n g reaction.  Reduction Hodgkinsine  (9.8 mg.)  palladium-charcoal  was  shaken w i t h p r e s a t u r a t e d  (1:10) i n e t h a n o l f o r three hours.  After  to  105  90 minutes 0.70 e q u i v a l e n t s absorbed  (1.0 equiv.,  M.W.  (M.W. 344) of hydrogen had been 520) and a b s o r p t i o n o f hydrogen  proceeded only very slowly a f t e r t h i s time.  After  filtration  a n a l y s i s by t h i n - l a y e r chromatography showed some unchanged hodgkinsine  and a major spot at the o r i g i n  (Rf 0.0, alumina,  e t h e r , r e d changing t o orange changing t o dark brown; Rf 0.3, alumina, methanol-chloroform 1:10).  The chromatographic  behaviour of t h i s r e d u c t i o n product  was very s i m i l a r t o N^-  methyltryptamine but the c o l o u r r e a c t i o n s with sulphate were q u i t e d i f f e r e n t .  acid-ceric  dl-Chimonanthine  t r e a t e d i n the same way but no h y d r o g e n o l y s i s  (19.7 mg.) was  occurred.  Isomerization The  hodgkinsine  d i s t i l l e d water  r e d u c t i o n mixture  (3 mg.) was d i s s o l v e d i n  (5 ml.) c o n t a i n i n g a c e t i c a c i d (3 drops) and  heated under n i t r o g e n on the steam bath (18 hours). by t h i n - l a y e r chromatography f a i l e d isomers.  t o r e v e a l any c a l y c a n t h i n e  The p a t t e r n was that of a mixture of  r e d u c t i o n and a c i d i s o m e r i z a t i o n  hydride from  (5 mg.) was r e f l u x e d  (60 mg.) i n dry t e t r a h y d r o f u r a n  LIAIH4)  hodgkinsine  products.  Attempted r e d u c t i o n w i t h l i t h i u m aluminum Hodgkinsine  Analysis  hydride  with l i t h i u m aluminum ( d r i e d by  and worked up i n the u s u a l way.  l a y e r chromatography r e v e a l e d unchanged  distillation  A n a l y s i s by t h i n -  hodgkinsine.  106  Dehydrocalycanthine Calycanthine stirred in  under  dilute  nitrogen  acetic  dissolved  (30 mg.)  acid  in dilute  and m e r c u r i c a c e t a t e (58,1  a t room t e m p e r a t u r e (5%).  acetic  A few  acid  (30 min.)  t h e r e was was  no  The  further  then heated  precipitate workup  accompanied apparent  ®  involved  through the s o l u t i o n s u l p h i d e by  and  filtration  (2 h o u r s )  removing  o n l y about  precipitated  extraction  s e v e r a l minor  The  first  (R^ 0.65,  was  not  was  proven  mixture.  dissimilar  layer  components and  by c h r o m a t o g r a p h y (Rf 0.60,  not be  chromatography.  chromatography  solution  white  solution. sulphide  mercuric  basification  mg.).  washed w i t h d i l u t e (10 mg.)  and  Thin-layer  two  found  acetic to  chromatography  a mixture of major  with  caly-  components. edges)  t o the hodgkinsine i s o m e r i z a t i o n product  second  t o grey) c o u l d  a  but  purple changing to yellow with v i o l e t  different The  pad,  sulphate) revealed  canthine,  and  i n t o c h l o r o f o r m (16  25% o x i d a t i o n .  (alumina, e t h e r , e e r i e  yielding  b u b b l i n g hydrogen  ( 5 % ) , then w i t h acetone, weighed  represent  The  a lime-green  m e r c u r o u s a c e t a t e p r e c i p i t a t e was  acid  ing  (17 h o u r s ) .  through a c e l i t e  p o t a s s i u m c a r b o n a t e and The  change  filtration,  hours  c l o u d i n e s s soon  a yellow colouration  o f m e r c u r o u s a c e t a t e and 1 1  were  milligrams of calycanthine  A slight by  on a s t e a m b a t h  f o r seventeen  (5%) s e r v e d a s a b l a n k  y i e l d e d unchanged c a l y c a n t h i n e . appeared  mg.)  on a l u m i n a  of the a p p r o p r i a t e  reddish changing  t o mauve  s e p a r a t e d f r o m compound B by  This  but  compound was  isolated  thin-  (25%)  ( b e n z e n e - e t h e r m i x t u r e s ) and  chang-  by  recrystal  107  l i z e d from benzene, ethanol:  ^  m  a  x  m.p.  211-214° C.  238, 312 mfx, € <  U l t r a v i o l e t spectrum i n  4500, 1120:  max  2. 238, 295 m/u». max. ' / from that of compound B.  chloric acid: different  in dilute  T h i s spectrum was  hydro-  quite  Dehydrochimonanthine dl-Chimonanthine  (7 mg.)  and mercuric a c e t a t e (5% excess)  were s t i r r e d under n i t r o g e n on a steam bath f o r 2.5 dilute acetic acid canthine  (2 mg.  hours i n  (5%) and worked up i n the same way  recovered).  The r e c o v e r e d m a t e r i a l was a  mixture of c a l y c a n t h i n e , d e h y d r o c a l y c a n t h i n e and dl-Chimonanthine  as c a l y -  (16.5 mg.)  chimonanthine.  and manganese d i o x i d e  (35  were s t i r r e d under n i t r o g e n f o r two days i n benzene-ether  mg.) (1:1)  g i v i n g , b e s i d e s m a t e r i a l at the o r i g i n , a compound more mobile than chimonanthine ultraviolet  (R^ 0.8,  alumina, methanol-ether 1:20).  spectrum i n e t h a n o l :  -A  m a x  248, 305 m/X  The  indicated  no change i n the i n d o l i n e chromophore.  P h y s i c a l p r o p e r t i e s of hodgkinsine C o l o u r l e s s needles, m.p. ultraviolet  spectrum i n e t h a n o l :  13000, 6400. iplet  -A x  246, 306 nyx,  The  8 8  6  (aromatic p r o t o n s , area 10 H),  s i n g l e t c e n t e r e d at 4,12 T  m a x  broadened  (aromatic p r o t o n , area 1 H),  s i n g l e t s c e n t e r e d at 5.33  R2N-H, area 2.7  ma  128° C .  The n.m.r. spectrum i n d e u t e r o c h l o r o f o r m : mult-  from 2.8 t o 3.8 f  broadened  126-134° C. l i t .  and 5.88T  (N-CH-N and  and 2.9), a s e r i e s of m u l t i p l e t s from 7.0  to  108  8.3 T under three s i n g l e t s at 7.59, and R -N-CH 2  3  spectrum was 2%), 358 346  most i n s t r u c t i v e w i t h m/e  (M - 160,  (M - 172,  2%), 344  10%), 314  174 - 85, 7%), 245 (M - 346,  quoted may was  and 7.71T  protons, t o t a l area 21 H (12+9)).  (M - 174,  518  (M ,  7%), 519  +  23%), 345  (M - 174 - 73,  (M - 174 - 99, 4%), 186  100%), 143  (30%), 130  (methylene  The mass (M + 1,  (M - 173,  (M - 174 - 30, 3%), 302  7%), 287(M - 174 - 57, 3%), 271  172  7.63  10%),  (M - 174 5%), 259  ( M -  (M - 174 - 158,  (22%).  The  42,  8%),  abundances  not be a c c u r a t e as the i n t e n s i t y of the parent peak  not a c c u r a t e l y known.  A l k a l o i d s from the seeds of Calycanthus Calycanthus  floridus  f l o r i d u s L. seeds (50 g.) purchased  F.W.Schumacher Co.,  Sandwich, Mass., U.S.A., were ground i n a  Waring blender and t r i t u r a t e d w i t h l i g h t petroleum 200 ml.)  from the  ether  y i e l d i n g a q u a n t i t y of l i g h t yellow o i l (13.5  The remaining  s o l i d was  t r i t u r a t e d with c h l o r o f o r m  then e x t r a c t e d i n a s o x h l e t apparatus. c h l o r o f o r m e x t r a c t s a yellow o i l was examined by t h i n - l a y e r chromatography sulphate).  (3 x  g.,  27%).  (3 x 200  ml.)  On e v a p o r a t i o n of the  obtained  (2.2 g., 4.3%)  (ether, alumina,  and  eerie  Aside from the o i l at the s o l v e n t f r o n t , and a very  minor spot at the o r i g i n c a l y c a n t h i n e (major a l k a l o i d ) and calycanthidine  (R^ 0.35)  were d e t e c t e d .  other components c o r r e s p o n d i n g r e a c t i o n were d e t e c t e d . canthine c r y s t a l l i z e d  No chimonanthine or  t o minor products of the c o u p l i n g  A f t e r s t a n d i n g f o r s e v e r a l days c a l y -  from the o i l .  109  Attempted r e s o l u t i o n o f d l - c h i m o n a n t h i n e A l l a t t e m p t s t o r e s o l v e s y n t h e t i c d l - c h i m o n a n t h i n e by c r y s t a l l i z a t i o n o f t h e mono- o r d i - s a l t s o f d-10-camphors u l p h o n i c a c i d were u n s u c c e s s f u l .  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N.J.Leonard, J.Am.Chem.Soc., 77, 439(1955); 79, 5279(1957); 81, 5627(1959).  PART B  THE ORIGIN OF THE NON-TRYPTOPHAN DERIVED PORTION OF THE INDOLE ALKALOIDS  118  Introduction The  r e l a t i o n s h i p of tryptophan  t o the i n d o l e a l k a l o i d s  as d i s c u s s e d i n the general i n t r o d u c t i o n i s almost s e l f - e v i d e n t and has been c o n c l u s i v e l y demonstrated. T r y p t o p h a n - 2 - C (1) has been found  to be i n c o r p o r a t e d by  14  Rauwolfia  s e r p e n t i n a i n t o ajmaline  reserpine(4).  12 '  and  serpentine  C-5  as expected.  Systematic  converted  borohydride  degradation  9  of the  ajmaline^  showed that the r a d i o a c t i v i t y was  3  The  degradation  ed by f u s i o n with soda l i m e which was  (2), s e r p e n t i n e ( 3 ) , and  4  of ajmaline  confined to  (2) was  y i e l d i n g N -methylharman a  to i t s methiodide and reduced  t o a t e r t i a r y base (6).  Reduction  accomplish(5)  with sodium  (sodium i n  l i q u i d ammonia) of the methiodide of the t e r t i a r y base (6) gave a s i n g l e compound i n high y i e l d i d e n t i f i e d as 2 - e t h y l - l - m e t h y l N,N-dimethyltryptamine  (7).  Hofmann degradation  of  this  compound y i e l d e d a 3 - v i n y l i n d o l e (8) i n e x c e l l e n t y i e l d which was  converted  t o a d i o l with osmium t e t r o x i d e and c l e a v e d  p e r i o d a t e to y i e l d 2 - e t h y l - 3 - f o r m y l - l - m e t h y l i n d o l e  (9)  with  and  formaldehyde which was isolated as i t s dimedone d e r i v a t i v e . i n d o l e d e r i v a t i v e was d e r i v a t i v e and  i n a c t i v e but  formaldehyde dimedone  a l l other i n t e r m e d i a t e s i n the degradative  had e s s e n t i a l l y the same s p e c i f i c a c t i v i t y as the which i n d i c a t e d that the ajmaline was i4  scheme  ajmaline  l a b e l l e d s o l e l y at  D l - T r y p t o p h a n - 3 - C (10) has been i n c o r p o r a t e d ibogaine  The  C-5.  (6.8%) i n t o 5  (11) by f i v e month o l d Tabernanthe iboga p l a n t s .  l o c a t i o n of the a c t i v e carbon atom was  e s t a b l i s h e d as C-7  The by  119  F i g u r e 1. Alkaloids.  I n c o r p o r a t i o n o f Tryptophan i n t o the  Rauwolfia  120  degradation.  Fusion  of the a l k a l o i d w i t h a mixture of sodium  and potassium hydroxide y i e l d e d 1,2-dimethyl-3-ethyl-5-hydroxyindole  (12) which was  o x i d i z e d with aqueous 80% chromium  t r i o x i d e a f f o r d i n g a mixture of l a b e l l e d p r o p i o n i c labelled acetic acids. the p r o p i o n i c a c i d was  A f t e r s e p a r a t i o n by  and  chromatography  o x i d i z e d w i t h potassium dichromate i n  N s u l p h u r i c a c i d to a c e t i c a c i d which was  then s u b j e c t e d  Schmidt r e a c t i o n a f f o r d i n g i n a c t i v e methylamine and d i o x i d e w i t h 80% of the a c t i v i t y of Radioactive  un-  v i n d o l i n e has  18  to a  carbon  ibogaine.  r e c e n t l y been i s o l a t e d from Vinea  rosea p l a n t s which had been f e d D L - t r y p t o p h a n - 2 - C w i t h 0.32% 6 7 incorporation. ' A systematic d e g r a d a t i o n e s t a b l i s h e d that 7 e s s e n t i a l l y a l l of the a c t i v i t y was l o c a t e d at C-10. The 14  key  step of the d e g r a d a t i o n was  the  formation  of N -methylnora  , , 8 harmine (14) by soda lime d i s t i l l a t i o n of v i n d o l i n e . d e g r a d a t i o n was ajmaline  2  then i d e n t i c a l to that a l r e a d y  ( f i g u r e 1) and  dimedone d e r i v a t i v e had active vindoline The  for  the formaldehyde i s o l a t e d as i t s e s s e n t i a l l y the same a c t i v i t y as  the  of the non-tryptophan, nine or ten carbon,  i n d o l e a l k a l o i d s was  been the s u b j e c t  described  (13).  biogenesis  p o r t i o n of the  The  of s p e c u l a t i o n .  have been promulgated.  The  l e s s obvious and Several elaborate  has  long  theories  i n i t i a l t r a c e r s t u d i e s tended t o  confuse r a t h e r than c l a r i f y the matter. The  Barger-Hahn h y p o t h e s i s concerning  carbon s k e l e t o n of yohimbine  (19) was  the b i o g e n e s i s  of  advanced c o n c u r r e n t l y  the with  121  12  F i g u r e 2. Vindoline.  I n c o r p o r a t i o n o f Tryptophan i n t o I b o g a i n e and  122  the s t r u c t u r a l e l u c i d a t i o n of that a l k a l o i d . c o n s i d e r e d yohimbine  They  (19) t o be d e r i v e d from tryptamine (15),  t y r o s i n e as m-hydroxyphenylacetaldehyde e q u i v a l e n t and formaldehyde  (16, R = H) or i t s  or i t s one carbon e q u i v a l e n t .  This  p r o p o s a l was supported by condensation of tryptamine w i t h aldehydes and e n o l i c Ct-keto a c i d s t o y i e l d c a r b o l i n e d e r i v a t i v e s under p h y s i o l o g i c a l c o n d i t i o n s .  1 0  *  1 2  The aromatic analogue (18) 11  of yohimbine was a l s o s y n t h e s i z e d i n t h i s way. encountered  A difficulty  by t h i s s u g g e s t i o n i s that r i n g E would be aromatic  whereas i t i s a l i c y c l i c  i n yohimbine and most r e l a t e d  alkaloids.  The p r o p o s a l gained c o n s i d e r a b l e support, however, as a 13 consequence of the a t t r a c t i v e s u g g e s t i o n o f Woodward. r e l a t e d pathway i n v o l v e s condensation o f acetaldehyde of tryptamine give 25.  This  3,4-dihydroxyphenyl-  (16a, R = H) or i t s e q u i v a l e n t w i t h the (3-position (15), and r e a c t i o n w i t h a one carbon u n i t t o  A d d i t i o n of an a n i o n o i d C-2 t o the Opposition o f the  i n d o l i n e and f i s s i o n of the c a r b o c y c l i c r i n g between the h y d r o x y l b e a r i n g atom C-3 and C-4 w i t h i n c o r p o r a t i o n of an a c e t a t e u n i t (not n e c e s s a r i l y i n the order shown i n f i g u r e 3) c o u l d simply e x p l a i n the o r i g i n of s t r y c h n i n e (28).  T h i s type of r i n g  cleavage named a f t e r Woodward then p l a y e d a major r o l e i n t h e o r i e s d e a l i n g with the b i o g e n e s i s o f other a l k a l o i d s  includ-  i n g c i n c h o n i n e (23a), q u i n i n e (23b), ajmaline (2), e t c . .  1 4  '  1 5  Woodward r e c o g n i z e d that the carbon s k e l e t o n o f ajmaline (2) and c i n c h o n i n e (23a) c o u l d be d e r i v e d from a compound 20 by Woodward cleavage o f r i n g E of the aromatic analogue of  123  a.  Schematic.  b. B i o g e n e s i s o f Yohimbine,  F i g u r e 3.  A j m a l i n e and Q u i n i n e  B a r g e r - Hahn - Woodward Theory.  124  gure 3. c o n t i n u e d .  Suggested B i o g e n e s i s o f S t r y c h n i n e .  strychnine  125  yohimbine and  (18).  Ajmaline i s d e r i v e d by the  hydroxylation  condensation of r i n g E with the c a r b o l i n e r i n g as shown i n  f i g u r e 3.  I t was  f u r t h e r suggested that compound 20  a l s o undergo r i n g cleavage cinchonamine  r i n g c o u p l i n g to  yield  (21), cinchonamine, by a t h i r d cleavage  y i e l d i n g the c i n c h o n i n e , cinchonine  and  (23a)  and  quinine  quinine  (23) s k e l e t o n .  (23b)  could  and  coupling,  Although  are q u i n o l i n e a l k a l o i d s  14 tryptophan-2-  C (1) i s i n f a c t  Cinchona s u c c i r u b r a and was  found at C-6 Robinson  17  a f t e r degradation  as p r e d i c t e d by  elaborated  1 0  incorporated  i n t o quinine  a l l of the  theory.  by  radioactivity  14  the Barger-Hahn scheme to account f o r  the presence of the extraneous carbomethoxy group i n r i n g E of yohimbine. tropolone  He  suggested expansion of aromatic r i n g E t o a  which c o u l d then c o l l a p s e with e x t r u s i o n of the  carbo-  m et hoxy group. A study chemically  of the s t e r e o c h e m i s t r y  of the i n d o l e and  r e l a t e d a l k a l o i d s by Wenkert and  observation  that carbon-15 was  i//'-Akuammicine Wenkert and  la  was  Bringi  i n v a r i a b l y of one  the  u n t i l r e c e n t l y the only known  exception.  B r i n g i then drew a t t e n t i o n t o the d e f i c i e n c i e s of  The  the o l d e r theory the a b s o l u t e  led t o  configuration.  the Barger-Hahn b i o s y n t h e t i c model and proposed an alternative.  1 8  phyto-  three interdependent  are:  d i f f i c u l t i e s r a i s e d by  (a) the s t a t e of o x i d a t i o n of r i n g E,  c o n f i g u r a t i o n of C-15  carbomethoxy group.  elegant  and  (c) the o r i g i n of  With the e x c e p t i o n  of a l s t o n i n e  yohimbine type compounds possess aromatic r i n g E, and  (b)  the  no a l l have  126  asymmetric and  c e n t e r s a t t h e D/E r i n g  sempervirine.  Furthermore,  natural  systems  clarity  regarding the expansion  and  has never  the  ^3-  r a t h e r than  ester  group very  an  ester  a substitute  skeleton.  Davis  the aromatic  then  bases  had r e c e n t l y acids  amino a c i d s .  i s determined.  of  hydrated  shikimic itself  forms  t o prephenic  the direct  Rearrangement 1,2-shift  '  2 2  acid  A hydrated prephenic  2 1  i n t e r m e d i a t e from  However  Tiy-akuammmine  suggested  of prephenic  acid  (19).  be e x p e c t e d  (34)) enzymatic  products.  which  absolute  was  most config-  conversion of  that prephenic alkaloids.  acid  was  2 4  ( 4 5 , page 25) (29)  by a  r e s i d u e and condensation w i t h a C - l  While  a unit  (30) r e a d i l y  t h e rearrangement  to yield  intervention  be r e s p o n s i b l e f o r t h e s p e c i f i c natural  their  acid  the lack of intervention  progenitor of these  might  yohimbine  that shikimic (43,  i n the microbiological 23  i n yohimbine  acid  was then" s u g g e s t e d  ( 4 5 , page 25) were t h e p r o g e n i t o r s  f o l l o w e d by h y d r a t i o n a f f o r d s  discernible prephenic  1 8  of the pyruvate  equivalent  make t h e o r i g i n o f  moiety  demonstrated  were d e r i v e d and by which  uration such  of the latter  18  considered as the c r u c i a l  indole  the lack of  f o rphenylalanine as a precursor of the  page 25) and p r e p h e n i c of  by  E to a tropolone  of the contraction  A carboxysubstituted hydroaromatic as  Finally,  of phenolic ring  a£-conjugated  vague.  alstonine  reduction of a phenol  been observed.  the mechanistic unlikelihood  to a  juncture except  racemic  products ( i . e .  at this  point could  absolute configuration  Seco-prephenate-formaldehyde  of  i nthe  (S.P.F.) (31)  127  c o u l d then a r i s e by a r e t r o - a l d o l r e a c t i o n . tryptamine  through an i n t e r m e d i a t e  such as corynantheine and  ajmaline  skeleton  (36) c o u l d then y i e l d a l k a l o i d s  (32), a j m a l i c i n e (33), sarpagine  (2) as o u t l i n e d i n f i g u r e 5.  (e.g. 34)  group at the  f o l l o w e d by t r a n s a n n u l a r  (13, 61)  (36, 37) was  Iboga  tryptaminelater  These s t r u c t u r a l l y more complex systems r e q u i r e  2 4  v i s u a l i z e d as proceeding intermediate  T h i s rearrangement c o u l d  v i a a r e t r o - M i c h a e l r e a c t i o n of  of the i n d o l e  (38).  Michael  be  the  37 r e s u l t i n g i n a formyl a c e t a t e moiety attached  ft^-position  to  reactions after  a p p r o p r i a t e changes i n o x i d a t i o n l e v e l c o u l d r e s u l t  i n compounds  42 which are very s i m i l a r to the n a t u r a l a l k a l o i d s  quebrachamine (45) and cleavamine reactions  (46).  I n t e r n a l Mannich  (transannular c y c l i z a t i o n ) as shown i n f i g u r e 5 c o u l d  then y i e l d the c l o s e d Aspidosperma and (43 and  and  (37).  a l s o d e s c r i b e d by Wenkert i n the  rearrangement of the S.P.F. u n i t .  41 and  attack  indole  c y c l i z a t i o n t o the iminium i o n  a l k a l o i d s k e l e t o n s c o u l d be d e r i v e d from a  S.P.F. u n i t  the  strychnine  ( ^ - p o s i t i o n of the  A mechanism by which the Aspidosperma  paper.  The  (35),  c o u l d s i m i l a r l y be d e r i v e d from 36 by  of the f o r m y l a c e t a t e  (11, 62)  Condensation with  Iboga carbon  skeletons  44).  An i n t e r e s t i n g aspect r e a c t i o n s 41 t o 43 and  of the t r a n s a n n u l a r  42 t o 44 as p o s t u l a t e d by Wenkert f o r  f o r m u l a t i o n of Aspidosperma and  Iboga carbon s k e l e t o n s i n nature,  i s that model r e a c t i o n s have been accomplished. co-workers > 2 5  2 6  cyclization  Kutney  and  i n t h i s department e f f e c t e d an aspidosperma-  128  HOOC  (S.P.F.)  CH OH 2  COOCH,  34 akuammicine racemic) Figure  4.  35 sarpagine  Wenkert-Bringi Hypothesis-S.P.F.  Unit.  H  129  cf. 2 F i g u r e 5.  c f . 35  C o n d e n s a t i o n of S.P.F. and T r y p t a m i n e .  131  132  l i k e transannular mercuric  c y c l i z a t i o n on dihydrocleavamine  acetate i n a c e t i c a c i d .  50 and  (52) and  51 c y c l i z e s t o  a c e t a t e to a l t e r n a t i v e iminium  5-desethyl-7-ethylvincadifformine  t o the i b o g a i n e - l i k e bases c o r o n a r i d i n e  dihydrocatharanthine  with  Carbomethoxydihydrocleavamine  (49) when o x i d i z e d with mercuric salts  (47)  (53)  and  (54).  A t h i r d theory based on s t r u c t u r a l r e l a t i o n s h i p s was independently was  proposed by Thomas ^ and W e n k e r t 2  i n 1961.  24  suggested that the non-tryptophan moiety of the  a l k a l o i d s was  monoterpenoid i n o r i g i n .  Wenkert  s t r u c t u r a l i d e n t i t y of the carbon s k e l e t o n of the oleuropeine and  (77),  swertiamarin  unit  (31).  2 8  indole noted  24  2  3 1  the  glucosides  g e n t i o p i c r i n (78), ® bakankosin  (73)  It  (79),  3 0  with h i s seco-prephenate-formaldehyde  He a l s o noted that cleavage  of the  cyclopentane  r i n g of the monoterpenic g l u c o s i d e s , v e r b e n a l i n  (63), ^  (64),  at a s p e c i f i c  3 2  aucubin  (65),  3 3  and a s p e r u l o s i d e  (66)  3 4  3  bond would give an S.P.F. type of carbon s k e l e t o n with  genipin  the  r e q u i r e d a b s o l u t e c o n f i g u r a t i o n of the non-tryptophan p a r t of the i n d o l e a l k a l o i d s .  Hendrickson  3 5  when d i s c u s s i n g these  monoterpenes at a l a t e r date a s c r i b e d one f o r t h i s r i n g opening p r o c e s s . p o s s i b i l i t y that these  reasonable  (Figure 8; 71—••72) .  mechanism The  g l u c o s i d e s were prephenate d e r i v e d  a l s o suggested by Wenkert.  The  aromatic  was  r i n g E of yohimbine  (19) would be formed by r i n g c l o s u r e as opposed t o the preformed r i n g of the Barger-Hahn and prephenic proposed mode of formation  acid theories.  of Aspidosperma  (61) and  The Iboga  (62)  133  s k e l e t o n s would be the same as f o r the l a t e r stages o f the prephenic a c i d theory.  (Figure 5 ) .  27 Thomas (75),  3u  a l s o proposed that the b i o s y n t h e s i s o f g e n t i a n i n e  oleuropeine  (77) , swertiamarin  (73) and bakankosin (79)  was r e l a t e d t o t h a t of the non-tryptophan moiety of the i n d o l e a l k a l o i d s and p o i n t e d out t h a t t h i s concept  ( r e q u i r i n g the  combination o f tryptamine with a monoterpene) would be c o n s i s t e n t with the e s t a b l i s h e d mode o f b i o s y n t h e s i s o f the ergot a l k a l o i d s . Tryptophan and mevalonic a c i d were known t o be p r e c u r s o r s (see page 18) o f these mould a l k a l o i d s .  According  t o Thomas the  ten carbon u n i t would be d e r i v e d from two u n i t s of mevalonate (55) v i a a c y c l o p e n t a n o i d C-21 of yohimbine  monoterpene (59) i n such a way that  (19) would correspond t o C-5 of the p r e c u r s o r  (carbonyl carbon o f a c e t a t e ) .  2 7  The carbomethoxy group (C-22)  would be d e r i v e d from e i t h e r C-2 or C-3a of mevalonate carbon of a c e t a t e )  (methyl  and n e i t h e r of these r i n g E s u b s t i t u e n t s  would be d e r i v e d from one-carbon u n i t s as was r e q u i r e d by the other  hypothesis.  (Figure 7 ) .  Aromatic r i n g E would be formed  i n a manner analogous t o that i n v o l v e d i n the c y c l i z a t i o n o f swertiamarin and  (73) t o e r y t h r o c e n t a u r i n  Tomita.  3 1  (74) as observed by Kubo  Thomas a l s o r e c o g n i z e d  the c y c l o p e n t a n o i d  monoterpene s t r u c t u r e (59) i n the a l k a l o i d s a c t i n i d i n e skytanthine  (69),  3 8  (70),  3 7  and the r i n g opened s t r u c t u r e 60 i n the  Ipecacuanha a l k a l o i d emetine ( 7 6 ) . of a nine carbon monoterpene.  3 y  Aucubin  (65) i s an example  I t d i f f e r s from the s k e l e t o n of  genepin (64) i n that a carbomethoxy group i s m i s s i n g .  This  134  acetogenins  P 3 H  / N  2  XV  CH3COSC0A  -  0^T\  —  malonylCoA  CHH o - - - * C O S C o A  (acetate)  '  .  n < ! n  »H^CH  3  2 COOH COSCoA  CH OH  2  reduction  H 0 P 2  CH.  3  ^_  COOH  CH2 g ^ H  2  3-hydroxy-3-methylg l u t a r y l CbA  P2O6H3  mevalonic acid 55  '^2®^2 CH OP 2  2  f a t t y a c i d s by  acetoacetyl  O^CHg  /  CH COCH COCH COSCoA 3  a c e t y l CoA  9 3"  /  CH OP  2  2  higher t e r penes, s t e r o i d s  — * - other monoterpenes 2  dimethylallvl isopentenyl pyrophosphate pyrophosphate 56 57 7^  g e r a n y l pyrophosphate 58 Corynanthe 2 skeleton 3 (cf. S.P.F.)  3 \ 61 F i g u r e 7.  Biogenesis  Wenkert Theory.  Aspidosperma skeleton o f Geranyl  62  Iboga skeleton  pyrophosphate and the Thomas-  135  CH OH  CH2OH  CH.  H  H -OGluc H-  H' CH O CT ^ >  CH 02C'  3  3  verbenalin 63  2  .H  OH  Her'  H"'  0  2  -•H  aucubin 65  ,-CH,  OGluc  , OGluc  CHO CHO  asperuloside 66  6  2  genepin 64 CH OAc  ^  OGluc  iridodial 67  CH 0 Cf^! 3  2  plumieride 68  136  OGluc emuIsin  cf. ring E Yohimbine  HOOC swertiamarin .73  74 erythrocentaurin  NH OH 4  -NH  0  N  gentianin 75  CH3O OCH, OGluc  CH3OOC oleuropeine 77 Glue  OGluc  O gentiopicrin 78 F i g u r e 9. Skeleton.  bakankosin 79  Some Monoterpenes w i t h the "Corynanthe" Carbon  137  carbomethoxy present  group i s presumably  the same as that which i s o f t e n  i n the i n d o l e a l k a l o i d s .  The b i o s y n t h e s i s proposed f o r i r i d o d i a l c y c l i z a t i o n of c i t r o n e l l a l the  laboratory.  the b i o g e n e s i s plumieride  (67), i n v o l v i n g the  ( c f . 58—»-59) , has been simulated i n  Yeowell and Schmidt have r e c e n t l y i n v e s t i g a t e d  4 0  of the cyclopentane-monoterpene  (68)  4 1  glucoside  by f e e d i n g of m e v a l o n o l a c t o n e - 2 - C . 14  Radio-  a c t i v i t y was found at C-4 and C-7a (Figure 8) as p r e d i c t e d  from  the c y c l i z a t i o n o f geranyl pyrophosphate (58). A fourth p o s s i b i l i t y  f o r the o r i g i n o f the non-tryptophan  p o r t i o n o f the i n d o l e a l k a l o i d s was proposed by S c h l i t t l e r and Taylor  i n I960.  4 2  The suggestion  was that the r e l e v a n t  pre-  c u r s o r might be formed by condensation o f an open c h a i n s i x carbon a c e t a t e  u n i t , a one carbon u n i t and a three carbon u n i t .  (Figure 10).  alkaloids  HOOC^ ^COOH  Figure  10.  S c h l i t t l e r - T a y l o r - L e e t e Hypothesis.  138  A s e r i e s of f e e d i n g experiments u s i n g Rauwolfia c a r r i e d out by Leete prephenic  serpentina  seemed t o e l i m i n a t e the Barger-Hahn, the  a c i d , and the monoterpene t h e o r i e s f o r the b i o g e n e s i s  of the i n d o l e a l k a l o i d s .  These experiments give  results  c o n s i s t e n t only w i t h the S c h l i t t l e r - T a y l o r " a c e t a t e "  hypothesis.  When sodium f o r m a t e - C was f e d t o R.serpentina  i t was  i4  reported  4 3  that C-21 o f ajmaline  (2) became l a b e l l e d  (12% of  a c t i v i t y ) i n agreement with i t s d e r i v a t i o n from the one-carbon p o o l of the p l a n t s as p r e d i c t e d by a l l t h e o r i e s except the monoterpene theory. ajmaline  In a second p a p e r  4 4  i t was r e p o r t e d that  (2) i s o l a t e d from p l a n t s which had been f e d mevalonic  acid-2- C 1 4  (55) or p h e n y l a l a n i n e - 2 - C 14  T h i s was evidence schemes.  was completely  inactive.  a g a i n s t the monoterpene and the Barger-Hahn  A l a n i n e - 2 - C was f e d t o t e s t the prephenic 1 4  hypothesis  on the assumption that pyruvate formed from a l a n i n e  by t r a n s a m i n a t i o n prephenic  acid  would be i n c o r p o r a t e d i n t o the s i d e c h a i n o f  a c i d (29).  actual precursor  Phosphoenolpyruvate  however and i s apparently  (see page 25) i s the not r e a d i l y  formed  AC from p y r u v i c a c i d  , l J  hence low i n c o r p o r a t i o n probably  constitute a v a l i d objection.  Only 2% o f the a c t i v i t y was i n  the p o s i t i o n (C-3 of ajmaline) a c e t a t e - l - C was r e p o r t e d 1 4  4 4  doesn't  p r e d i c t e d by theory.  Sodium  as being i n c o r p o r a t e d i n t o ajmaline  (2) with 26% o f t o t a l a c t i v i t y at p o s i t i o n s 3 and 19, with p o s i t i o n s 14, 18 and 21 i n a c t i v e . w i t h the T a y l o r a c e t a t e hypothesis terpene  T h i s i s i n p e r f e c t agreement and i n c o n t r a s t t o the mono-  theory which p r e d i c t s that the l a b e l should  have been  139  i n p o s i t i o n s 21,  19, 16 and 14.  F u r t h e r support was  by i s o l a t i o n and degradation of s e r p e n t i n e (3). paper  4 6  malonic  a c i d - l , 3 - C was 1 4  provided  In a t h i r d  r e p o r t e d as being i n c o r p o r a t e d  i n t o s e r p e n t i n e (3) with 48% of the t o t a l a c t i v i t y at p o s i t i o n 22 and a l s o i n t o ajmaline with 74% of the a c t i v i t y at  C-17.  Both r e s u l t s are i n agreement w i t h the T a y l o r h y p o t h e s i s . (Figure  10). 47  L a t e r f e e d i n g experiments  by B a t t e r s b y  not only f a i l e d t o  c o n f i r m the Leete r e s u l t s but were i n sharp c o n t r a s t .  After  the f e e d i n g of sodium f o r m a t e - C and the i s o l a t i o n of ajmaline 14  from R.serpentina, i t was  found that the N-methyl group c a r r i e d  not l e s s than 25% of the a c t i v i t y and C-21 activity.  Cephaline  had l i t t l e  or no  (76, R = H) from C.ipecacuanha f e d with  sodium f o r m a t e - C had 67% of i t s a c t i v i t y i n the 0-methyl 14  groups whereas C-12  was  essentially inactive.  o x i d a t i o n showed a low s c a t t e r of a c t i v i t y skeleton. ajmaline  T h i s s c a t t e r of a c t i v i t y was  Kuhn-Roth  i n the  carbon  a l s o observed  with  (2) i s o l a t e d from R.serpentina p l a n t s f e d w i t h sodium  acetate-l- C. 1 4  A c e t i c and  propionic acids isolated  o x i d a t i o n had low and d i f f e r e n t  l e v e l s of a c t i v i t y .  r e s u l t s were s t r o n g evidence a g a i n s t the involvement carbon u n i t as p r e d i c t e d by s e v e r a l t h e o r i e s . a l evidence was  u r g e n t l y r e q u i r e d i n view of the  r e s u l t s obtained i n i n i t i a l t r a c e r  experiments.  Fresh  after These of a  one-  experiment-  conflicting  140  Discussion In s p i t e o f much s p e c u l a t i o n > > » > > > 1 4  experimentation ' ' ' ' 6  comprising  4 3  4 4  4 6  4 7  1 5  1 8  2 2  2 4  2 7  4 2  t h e o r i g i n o f t h e carbon s k e l e t o n  the non-tryptophan d e r i v e d p o r t i o n of the i n d o l e  a l k a l o i d s remained obscure and t h e r e s u l t s c o n f u s e d . hypothesis  and  had been u n e q u i v o c a l l y  The r e p e a t e d  claim that formation  No  s u p p o r t e d by f e e d i n g e x p e r i m e n t s . o f t h i s nine o r t e n c a r b o n  fragment i n v o l v e d one f o r m a t e , one malonate and t h r e e a c e t a t e 43,44,46 47 units had not been s u b s t a n t i a t e d by o t h e r workers who observed s c a t t e r i n g o f t h e l a b e l when a c e t a t e was f e d t o R.serpentina  and C.ipecacuanha.  t o be c o n f i r m e d  T h i s o b s e r v a t i o n was e v e n t u a l l y 7  by t h e o r i g i n a l workers.  Research was  i n i t i a t e d i n our l a b o r a t o r y t o t e s t what seemed t o us t h e most a t t r a c t i v e theory experimentally. 27 24 Thomas and Wenkert had proposed t h a t t h e n o n - t r y p t o p h a n p o r t i o n o f t h e i n d o l e a l k a l o i d s c o u l d be d e r i v e d from a t e n c a r b o n monoterpenoid u n i t .  I f t h i s p r o p o s a l were c o r r e c t t h e  u b i q u i t o u s t e r p e n e p r e c u r s o r , m e v a l o n i c a c i d ( 5 5 ) , s h o u l d be u t i l i z e d i n the biosynthesis of indole a l k a l o i d s .  A radio-  a c t i v e i s o t o p i c l a b e l i n m e v a l o n i c a c i d f e d t o a p l a n t s h o u l d be i n c o r p o r a t e d i n t o t h e i n d o l e a l k a l o i d s produced by t h a t p l a n t i n a s p e c i f i c manner. a c i d t o R.serpentina  L e e t e , however, had f e d a c t i v e m e v a l o n i c 44 and had f a i l e d t o observe any  i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o ajmaline of t h i s n e g a t i v e  (2).  On t h e b a s i s  r e s u l t and t h e r e p o r t e d but now d i s c r e d i t e d  s p e c i f i c i n c o r p o r a t i o n o f f o r m a t e , a c e t a t e and m a l o n a t e ' ' " 4 3  4 4  4  141  i n t o ajmaline  the monoterpene hypothesis  appeared untenable.  4 8  Kirby,  however, found that mevalonic a c i d was not  incorporated delpheline  by Delphinium elatum p l a n t s i n t o the a l k a l o i d  (79) which he recognized  as being c l e a r l y  i n o r i g i n and hence warned that negative i n t e r p r e t e d with c a r e .  terpenoid  experiments should be  The t e r p e n o i d o r i g i n o f d e l p h e l i n e  was based on a s t r u c t u r a l a n a l y s i s .  Postulates  concerning  b i o g e n e t i c routes o f t e n a r i s e from an i n s p e c t i o n o f s t r u c t u r e s seeking  common s t r u c t u r a l u n i t s and suggesting  s h i p s of these u n i t s t o s i m p l e r The  success  n a t u r a l products  (see page 8 ) .  o f t h i s approach has been p a r t i c u l a r l y s t r i k i n g i n  the l a r g e terpene f i e l d rearranged  possible relation-  3 5  where many complex and o f t e n h i g h l y  s t r u c t u r e s can always be r e l a t e d i n terms of reason-  able r e a c t i o n mechanisms t o ten, f i f t e e n , twenty or t h i r t y carbon atom u n c y c l i z e d p r e c u r s o r s . accordance with the b i o g e n e t i c  These p r e c u r s o r s isoprene  a r e formed i n  r u l e by condensation o f  the f i v e carbon u n i t s i s o p e n t e n y l pyrophosphate a l l y l pyrophosphate decarboxylation  (56) and dimethyl-  (57) which a r i s e by p h o s p h o r y l a t i o n  of mevalonic a c i d , i t s e l f d e r i v e d from  and three  a c e t a t e u n i t s . (Figure 7 ) . The  success  of f e e d i n g experiments depends, among other  f a c t o r s , upon the l a b e l l e d p r e c u r s o r  reaching  the s i t e o f  s y n t h e s i s at a time when a c t i v e s y n t h e s i s i s o c c u r r i n g . Success may thus depend on the choice of experimental age  o f the p l a n t and the method of f e e d i n g . 4 9  p l a n t , the  This p r i n c i p l e  was demonstrated by Benn and M a y " who observed the i n c o r p o r a t i o n  142  of mevalonic a c i d - 2 - C i n t o l y c o c t o n i n e  (81) and browniine (82)  i 4  when i n t a c t  D.brownii p l a n t s were f e d j u s t before  flowering. 48  These workers a t t r i b u t e d K i r b y ' s negative  results^*  0  when mevalon-  ate was f e d through the cut ends o f l e a f s t a l k s o f young D. elatum p l a n t s t o the p o s s i b l e confinement o f a l k a l o i d to the p l a n t  roots.  80 Figure  formation  81 82  RO R = H R = CH  3  11. Delphinium A l k a l o i d s .  S t r u c t u r a l a n a l y s i s r e v e a l s the a s t o n i s h i n g f a c t that i n s p i t e of the many and v a r i e d r i n g systems and the l a r g e number of i n d o l e a l k a l o i d s a l l of these a l k a l o i d s can be c l a s s i f i e d i n terms o f three b a s i c p a t t e r n s non-tryptophan p o r t i o n .  f o r the carbon s k e l e t o n of the  These three p a t t e r n s  have been named  a f t e r b o t a n i c a l f a m i l i e s i n which r e p r e s e n t a t i v e a l k a l o i d s were f i r s t  discovered.  In f i g u r e 12 a j m a l i c i n e  be r e p r e s e n t a t i v e of the Corynanthe s k e l e t o n of the Aspidosperma s k e l e t o n Iboga s k e l e t o n aranthine  (62).  are d e p i c t e d  (33) i s seen t o  (60), v i n d o l i n e (13)  (61) and c a t h a r a n t h i n e  (83) o f the  The s t r u c t u r e s of v i n d o l i n e and c a t h i n such a way as t o make t h i s r e l a t i o n s h i p  143  COOH  H  60  Figure  Corynanthe  12.  61  Structural Analysis  Aspidosperma  62  Iboga  of Indole A l k a l o i d s .  144  28  85  F i g u r e 13.  B r u c i n e R = OCH3 Strychnine R = H  Gelsemine  (skeletons only) E x o t i c Corynanthe R i n g Systems.  145  clear.  D i f f e r e n c e s i n oxygenation  the carbomethoxy group account  p a t t e r n and the absence of  f o r d i f f e r e n c e s of many i n d o l e  a l k a l o i d s from these three types.  Many other a l k a l o i d s have  q u i t e d i f f e r e n t r i n g systems r e s u l t i n g from condensation t h r e e b a s i c ten-carbon  s k e l e t o n s w i t h tryptophan  themselves i n a v a r i e t y of ways.  The  and  (3), ajmaline strychnine carbon  (2), corynantheine  (28) were encountered  with  Corynanthe s k e l e t o n  g i v e s r i s e t o the g r e a t e s t number of r i n g systems. these as r e p r e s e n t e d by yohimbine  A few  of  (19), r e s e r p i n e (4), s e r p e n t i n e (32), sarpagine  (35), and  i n the i n t r o d u c t i o n .  The  s k e l e t o n s of some more e x o t i c Corynanthe r i n g systems are  d e p i c t e d i n f i g u r e 13. drawn i n the u s u a l way  Echitamine  (84) and  gelsemine  and a l s o i n such a way  the Corynanthe carbon s k e l e t o n obvious.  Some of  tryptophan  are  these  5  have a p p a r e n t l y l o s t one  (85)  as t o make the  a l k a l o i d s such as a p p a r i c i n e , e l l i p t i c i n e ^ and u l e i n e  We  of the  or both of the carbon  (87)  atoms from the  side chain.  were f o r t u n a t e i n having c l o s e at hand workers  i e n c e d i n the growth, f e e d i n g and e x t r a c t i o n of the i n d o l e a l k a l o i d producing p l a n t Vinca rosea L i n n roseus G.Don) and are indebted to Dr. Beer of p l a n t c u t t i n g s .  exper-  prolific  (Catharanthus  f o r advice and  I n t e r e s t i n t h i s p l a n t and  gifts  i t s relatives  (the common p e r i w i n k l e ) has been c o n s i d e r a b l e s i n c e the d i s c o v e r y i n i t of a n t i l e u k e m i c a l k a l o i d s ' and has r e s u l t e d i n 5  52  60  e x t e n s i v e i n v e s t i g a t i o n of the a l k a l o i d a l c o n s t i t u e n t s . Of some t h r e e hundred a l k a l o i d s present the s t r u c t u r e s of more  146  than f i f t y Vindoline  a r e known and r e p r e s e n t many s t r u c t u r a l (13), > > 5 2  5 4  5 8  catharanthine  (83)  6 0  and a j m a l i c i n e  (33) a r e t h r e e o f the major a l k a l o i d s and possess sperma (61), Iboga  (62) and Corynanthe  types.  the Aspido-  (60) s k e l e t o n s r e s p e c t -  i v e l y making t h i s p l a n t an e x c e l l e n t c h o i c e f o r b i o g e n e t i c studies.  The, s t r u c t u r e of v i n c a l e u k o b l a s t i n e ( v i n b l a s t i n e )  61 (28)  which i s one o f the a n t i l e u k e m i c d i m e r i c a l k a l o i d s  i s o l a t e d from V.rosea  i s on page 15.  A d i s c u s s i o n o f the  a l k a l o i d s which are found i n Vinca s p e c i e s , while a t o p i c of c u r r e n t i n t e r e s t , i s beyond the scope o f t h i s In i n i t i a l mevalonic  experiments c a r r i e d out by Dr.I.G.Wright  acid-2- C 1 4  i n aqueous s o l u t i o n was a d m i n i s t e r e d t o  f r e s h l y cut shoots of Vinca rosea L i n n . . b i o g e n e s i s o f monoterpenes from mevalonic monocyclic of  thesis.  By the e s t a b l i s h e d acid  (55), the  monoterpene s k e l e t o n (59) can be d e r i v e d and cleavage  a s u i t a b l y f u n c t i o n a l i z e d d e r i v a t i v e a c c o r d i n g t o the Wenkert-  Thomas theory would y i e l d a fragment ( F i g u r e 15).  (60) l a b e l l e d as shown.  U t i l i z a t i o n o f t h i s fragment a c c o r d i n g t o the  Wenkert h y p o t h e s i s  2 4  would y i e l d v i n d o l i n e w i t h 50% o f the  a c t i v i t y at C-8 and 25% of the a c t i v i t y i n each o f p o s i t i o n s 4 and 22.  Seven days a f t e r a d m i n i s t r a t i o n o f 0.30 mc o f DL-  mevalonic  a c i d - 2 - l C r a d i o a c t i v e v i n d o l i n e w i t h 0.02% s p e c i f i c 4  i n c o r p o r a t i o n o f p r e c u r s o r was i s o l a t e d and r e c r y s t a l l i z e d t o constant a c t i v i t y as i t s h y d r o c h l o r i d e .  Desacetylvindoline  had e s s e n t i a l l y the same a c t i v i t y i n d i c a t i n g that mevalonic was not b e i n g degraded t o a c e t a t e and the l a b e l s c a t t e r e d .  acid  147  H  CH OP 0 H 2  ^0  2  6  3  CH 0P 0 H 2  2  6  2  O P  2°6 3 H  3  55 0.30 mc; 1.0 mc/mmole  HC1  desacetylvindoline 2 . 7 8 x l 0 c. /m./mmole 5  CH 0  OAc  3  'LiAlH,  COOCH  3  13  0.02%; 2 . 7 9 x l 0 c . /, m./mmole  5  CH 0 3  CH OH 2  88 F i g u r e 15.  2*  I n c o r p o r a t i o n o f mevalonic a c i d i n t o  0.55x10° c./ m./mmole 20% o f a c t i v ity Vindoline.  148  R e d u c t i o n o f d e s a c e t y l v i n d o l i n e t o v i n d o l i n o l (88) and c l e a v a g e of t h i s v i c i n a l g l y c o l w i t h p e r i o d i c a c i d produced formaldehyde which was i s o l a t e d as i t s dimedone d e r i v a t i v e and shown t o have 2 0 % ( a p p r o x i m a t e l y as e x p e c t e d . * 6 2  6 3  \) o f t h e a c t i v i t y o f t h e v i n d o l i n e ,  T h i s was e x c e l l e n t e v i d e n c e f o r a mono-  terpenoid o r i g i n f o r the non-tryptophan p o r t i o n o f the i n d o l e alkaloids.  An a l t e r n a t i v e mode o f c y c l i z a t i o n and c l e a v a g e 62  of monoterpene p r e c u r s o r s results.  ( F i g u r e 14).  A*3  '  was a l s o c o n s i s t e n t w i t h t h e s e  No mechanism was s u g g e s t e d .  a_ a F i g u r e 14.  2  A l t e r n a t e Terpene  Precursor CA  Soon a f t e r t h i s i n i t i a l experiment Goeggel and A r i g o n i reported  s i m i l a r r e s u l t s from f e e d i n g o f m e v a l o n i c a c i d - 2 - C 1 4  t o V i n c a r o s e a and V i n c a major L. f o l l o w e d by i s o l a t i o n o f v i n d o l i n e w i t h 0.12% i n c o r p o r a t i o n and r e s e r p i n i n e 0.01%  incorporation.  the a c e t a t e  (89) w i t h  Degradation of v i n d o l i n e e s t a b l i s h e d that  group, t h e 0-methyl, N-methyl and t h e e t h y l s i d e  149  c h a i n were i n a c t i v e w h i l e C-22 contained  22.5%  (the carbomethoxy carbon)  of the t o t a l a c t i v i t y .  r e s e r p i n i n e obtained  revealed  Degradation of  an a p p r e c i a b l e  the  amount of s c a t t e r  of l a b e l as each O-methyl accounted f o r about 7% of the (Figure 16).  These authors had p r e v i o u s l y e s t a b l i s h e d that  these methyls were d e r i v e d from m e t h i o n i n e . ^ carbon, C-22,  Battersby  a low  l e v e l of a c t i v i t y  also published  a c i d - 2 - C t o Vinca rosea  and  1 4  (13) w i t h 0.05%  d e g r a d a t i o n of C-21  (3) w i t h 0.02%  (87) w i t h 0.04% The had  24% and  incorporation  and  i n c o r p o r a t i o n were i s o l a t e d from  23% of the t o t a l a c t i v i t y .  catharanthine  implying  c a r r y 29% of the a c t i v i t y .  e s t a b l i s h e d that C-6,  l a b e l at C-5,  that C - l and The  a l t e r n a t i v e proposal  C-18  The  and  must by  25% at C-22.  had  difference requires These  p r e v i o u s l y mentioned  l a b e l at C-22  d e g r a d a t i o n of c a t h a r a n t h i n e  C-20  on r e d u c t i o n  f o r rearrangement of a terpene  however r e q u i r e s 50% of the  and  Kuhn-Roth  Thomas-Wenkert theory  25% at C - l , and  r e s u l t s were c l e a r l y c o n s i s t e n t .  ion.  vindoline  carbomethoxy groups (C-22) of a j m a l i c i n e  48% of the a c t i v i t y  The  feeding  (33) w i t h 0.003%  were i n a c t i v e w h i l e e t h y l p y r i d i n e obtained  50% of the  of  Radioactive  incorporation, ajmalicine  incorporation, serpentine  the  (0-3%).  Rhazia s t r i c t a and  1 4  catharanthine  carbomethoxy  the r e s u l t s of f e e d i n g mevalonic  mevalonic a c i d - 3 - C t o the l a t t e r . " * *  Vinca r o s e a .  The  5  accounted f o r 20% of the t o t a l a c t i v i t y w h i l e  e t h y l s i d e c h a i n had  catharanthine  activity.  of the  precursor  Iboga  skeleton.  hence e l i m i n a t e s t h i s suggest-  150 a.Arigoni  2 x 0CH  F i g u r e 16.  F e e d i n g o f Mevalonate t o V i n c a S p e c i e s .  3  -  7%  151 F i g u r e 16 c o n t i n u e d . c.Battersby.  Feeding o f Rhazia  stricta.  CH CH COOH  47%  * CH COOH  47%  3  CH NH + COo = C 2 o inactive 47% 3  2  2  3  152  A c t i v e 1,2-dehydroaspidospermine (90) was 0.15% C-5, C-8  isolated with  i n c o r p o r a t i o n of m e v a l o n a t e - 2 - C from Rhazia 14  C-20,  and C-21  stricta.  were shown t o be i n a c t i v e w h i l e i s o l a t i o n of  as formaldehyde a f t e r Emde and Hofmann d e g r a d a t i o n s e s t a b l i s h -  ed t h a t t h i s p o s i t i o n c a r r i e d 65% of the a c t i v i t y .  As  a l k a l o i d has no carbomethoxy group 67% i s r e q u i r e d by  this  theory.  Carbon-20 of the same a l k a l o i d l a b e l l e d by f e e d i n g of mevalonate3- C 14  and  was C-21  shown t o c a r r y 47% of the t o t a l a c t i v i t y w h i l e were i n a c t i v e as e x p e c t e d by  Geraniol-2- C 1 4  to Vinea rosea  was  precursor.  theory.  s y n t h e s i z e d f o r the purpose of  i n the hope of e s t a b l i s h i n g t h a t the  p o r t i o n of the i n d o l e a l k a l o i d s was  feeding  terpenoid  formed from a t e n c a r b o n  T h i s would emphasize the normal t e r p e n o i d  of the i n d o l e a l k a l o i d s .  C-5  nature  Successful i n c o r p o r a t i o n of g e r a n i o l  or g e r a n y l pyrophosphate would a l s o e s t a b l i s h t h i s compound as a useful precursor The  f o r o t h e r s t u d i e s i n the monoterpene  s u c c e s s f u l use of g e r a n i o l would a l s o l e a d t o use  field.  of  l a b e l l e d f a r n e s o l , the f i f t e e n c a r b o n u n i t from which the  sesqui-  t e r p e n e s are d e r i v e d , and p l a n s t o s y n t h e s i z e l a b e l l e d f a r n e s o l were a l s o made. concerning  In s p i t e of the v a s t amount of s p e c u l a t i o n  the b i o g e n e s i s of t e r p e n e s r e m a r k a b l y few t r a c e r  s t u d i e s have been completed.  A l t h o u g h g e r a n y l and 67  p y r o p h o s p h a t e s are proven s t e r o i d p r e c u r s o r s  farnesyl  i n no case have  t h e y been used f o r b i o g e n e t i c s t u d i e s i n the t e r p e n e f i e l d . far  As  as i s known a study of the b i o g e n e s i s of gibberellins® i n  which l a b e l l e d g e r a n y l g e r a n i o l was  8  not i n c o r p o r a t e d i s the  only  153  i n s t a n c e where t h i s compound has been used. There a r e s e v e r a l r e a s o n s why these p o t e n t i a l l y v e r y u s e f u l compounds had not been used f o r t r a c e r s t u d i e s .  They  are e x t r e m e l y i n s o l u b l e i n water and d i f f i c u l t i e s i n a d m i n i s t r a t i o n were e n v i s a g e d .  The phosphate o r pyrophosphate  i s the  m e t a b o l i t e u t i l i z e d by an o r g a n i c system and w h i l e t h e s e compounds c a n be made they would be i o n i c i n n a t u r e and were not e x p e c t e d t o be a b l e t o p e n e t r a t e c e l l membranes. r e a s o n was undoubtedly  A third  t h e f a c t t h a t l a b e l l e d g e r a n i o l i s not  commercially a v a i l a b l e . G e r a n i o l - 2 - C w i t h a s p e c i f i c a c t i v i t y o f 0.159 mc/mmole 1 4  was p r e p a r e d from one m i l l i c u r i e o f e t h y l b r o m o a c e t a t e - 2 - C . 14  The method employed i n v o l v e d a Reformatsky  condensation of e t h y l  b r o m o a c e t a t e - 2 - C (92) w i t h 6-methyl-5-hepten-2-one (91) t o 14  y i e l d e t h y l 3-hydroxy-3,7-dimethyl-6-octenoate  (93) i n a v e r y  s m a l l s c a l e m o d i f i c a t i o n o f a method d e s c r i b e d by R u z i c k a and S c h i n z . »' CT  1  D e h y d r a t i o n was a c c o m p l i s h e d by p y r o l y s i s o f  e t h y l 3-acetyl-3,7-dimethyl-6-octenoate ( 9 4 ) m i x t u r e o f c i s and t r a n s e t h y l 14  6 9  y i e l d i n g a 1:2  3,7-dimethyl-2,6-octadienoate-2-  C (95) w i t h o n l y a s m a l l amount o f o t h e r double bond i s o m e r s . T h i s e s t e r m i x t u r e was reduced t o a 1:2 m i x t u r e o f n e r o l (97) and g e r a n i o l - 2 - C (96) w i t h l i t h i u m aluminum h y d r i d e . ( F i g u r e 1 4  17).  A l l s t e p s i n t h e s y n t h e s i s were checked on an o r d i n a r y  s c a l e and compounds c h a r a c t e r i z e d by p h y s i c a l data (see experimental) . til  The r e a c t i o n s were then s c a l e d down and r e p e a t e d un-  t h e t e c h n i q u e s i n v o l v e d , e s p e c i a l l y d i s t i l l a t i o n , were  154  U^CH OH 2  LiAlH4  12  CH OH 2  96 geraniol  Figure  17.  Synthesis  of 2- C A4  97 nerol  Geraniol.  (2:1)  155  adequate t o g i v e r e a s o n a b l e The  y i e l d s i n the r a d i o a c t i v e s y n t h e s i s .  i n f r a - r e d and n.m.r. s p e c t r a o f p r o d u c t s  obtained i n the  r a d i o a c t i v e s y n t h e s i s were compared w i t h those o f p r o d u c t s i n the c o l d - r u n .  The 1:2  r a t i o o f c i s and t r a n s i s o m e r s was  e s t a b l i s h e d by vapor phase chromatography. g e r a n i o l are both n a t u r a l products  As n e r o l and  and p r o b a b l y i n t e r c o n v e r t i b l e  in vivo  no attempt was made t o s e p a r a t e  feeding.  The p r e p a r a t i o n o f a m i x t u r e  the mixture  o f g e r a n y l phosphate and  pyrophosphate by t h e method o f Cramer and B*6hm by Popjak and C o r n f o r t h  7 2 a  before  as m o d i f i e d  w a s also t r i e d i n a n t i c i p a t i o n of  d i f f i c u l t y i n the a d m i n i s t r a t i o n of g e r a n i o l t o the p l a n t . P l a n t t i s s u e i s permeable t o g e r a n i o l but i s d e s t r o y e d by high concentrations.  The problem o f a d m i n i s t r a t i o n was s i m p l y  one o f adequate d i s p e r s i o n .  S e v e r a l f e e d i n g e x p e r i m e n t s were  c a r r i e d out u s i n g c o l d g e r a n i o l u n t i l a method was found which d i d not d e s t r o y p l a n t t i s s u e .  I t was found t h a t when g e r a n i o l  was made s o l u b l e i n water w i t h  06-lecithin, a natural emulsify-  i n g agent from p l a n t s o u r c e s , o r w i t h t h e n o n - i o n i c Tween 20 i t c o u l d be a d m i n i s t e r e d without  detergent  t h r o u g h t h e c u t ends o f s h o o t s  o b v i o u s damage t o t h e p l a n t s .  A s m a l l amount o f g e r a n i o l - 2 - C 1 4  made s o l u b l e w i t h Tween  20 was f e d t o a s i n g l e V i n c a r o s e a c u t t i n g .  A f t e r one week  t h i s c u t t i n g was d r i e d between absorbant paper and a u t o r a d i o graphy p r e p a r e d by exposure o f a p i e c e o f x - r a y w i t h t h e l e a v e s and stem f o r seven days.  These  f i l m by c o n t a c t autoradiographs  showed t h a t r a d i o a c t i v i t y was d i s t r i b u t e d throughout t h e p l a n t .  156  The stem and l e a f v e i n s were a p p a r e n t l y t h e most a c t i v e but t h i s may be a consequence o f t h e b u l k o f these t i s s u e s .  The  topmost l e a v e s were a p p a r e n t l y as a c t i v e as t h e lower ones. G e r a n i o l - 2 - C was f e d t o t e n V i n c a r o s e a c u t t i n g s and t h e 1 4  a l k a l o i d s e x t r a c t e d a f t e r seven days:  2.4% o f t h e a c t i v i t y f e d  was p r e s e n t i n t h e crude a l k a l o i d a l f r a c t i o n .  Thin-layer  chromatography o f t h e a l k a l o i d m i x t u r e r e v e a l e d t h e well-known complexity of the mixture.  An a u t o r a d i o g r a p h o f a t h i n - l a y e r  p l a t e showed s p o t s c o r r e s p o n d i n g t o a l l o f t h e a l k a l o i d a l on t h e p l a t e and was good e v i d e n c e  spots  f o r incorporation of geraniol  i n t o many o f t h e a l k a l o i d s o f V i n c a r o s e a . V i n d o l i n e (13) was i s o l a t e d by p r e p a r a t i v e t h i n - l a y e r chromatography  54  and c r y s t a l l i z e d t o c o n s t a n t a c t i v i t y  d i l u t i o n with authentic material. t i o n o f about 0.005%.  after  I t had a r a t e o f i n c o r p o r a -  The s p e c i f i c i n c o r p o r a t i o n i s not known  as t h e amount o f v i n d o l i n e i s o l a t e d from t h e p l a n t i s unknown. V i n d o l i n e was p r e s e n t i n t h e p l a n t i n o n l y s m a l l amounts a t the time o f these f e e d i n g experiments  but was chosen f o r study  because o f e x p e r i e n c e w i t h t h i s a l k a l o i d and t h e a v a i l a b i l i t y o f r e a s o n a b l e amounts o f a u t h e n t i c v i n d o l i n e f o r purposes o f degradation. Two-hundred 10-14 i n c h c u t t i n g s o f one year o l d V i n c a r o s e a  14 p l a n t s were a d m i n i s t e r e d 0.282 mc o f g e r a n i o l - 2 specific  C with a  a c t i v i t y o f 0.159 mc/mmole made s o l u b l e i n 200 ml.  d i s t i l l e d water w i t h 8 drops o f Tween 20.  A f t e r one week t h e  crude a l k a l o i d e x t r a c t c o n t a i n e d 2.1% o f t h e a c t i v i t y  administered.  157  Vindoline  was  i s o l a t e d by chromatography and  l a y e r chromatography i n two  solvent  preparative  systems.  20 mg.  of  a l k a l o i d showing a s i n g l e spot on anautoradiograph had a c t i v i t y of 1350 a specific 0.011%.  c.p.m./mg. or 3.11  i n c o r p o r a t i o n of 0.37% The  x 10~ and  5  mc  corresponding to  a c t i v i t y changed very l i t t l e a f t e r s e v e r a l  regenerated a l k a l o i d was  v i n d o l i n e and  an  a r a t e of i n c o r p o r a t i o n  c r y s t a l l i z a t i o n s t o constant a c t i v i t y as the The  thin-  re-  dihydrochloride.  then d i l u t e d w i t h  r e c r y s t a l l i z e d from ether  of  authentic  t o p r o v i d e a sample  s u i t a b l e f o r degradation. I f the Thomas-Wenkert p r o p o s a l non-tryptophan p o r t i o n of the the  l a b e l of g e r a n i o l - 2 -  f o r the b i o g e n e s i s  i n d o l e a l k a l o i d s was  (Figure  18).  incorporated  P o s i t i o n 5 of  v i n d o l i n e i s a c c e s s i b l e by c a r e f u l Kuhn-Roth o x i d a t i o n as carbonyl  carbon of p r o p i o n i c  propionic  7 5  The  other two  A c e t i c a c i d would a l s o be d e r i v e d  group by h y d r o l y s i s d u r i n g  o x i d a t i o n and  separation  p-bromophenacyl e s t e r s  7 6  oxidation.  of p r o p i o n a t e and followed  s p e c i f i c a c t i v i t y of the two  carbons of  from  the same s p e c i f i c  Kuhn-Roth  acetate  as t h e i r  by d e t e r m i n a t i o n of  the  e s t e r s would unambiguously  a c t i v i t y as v i n d o l i n e and  a c i d was  inactive.  Any  activity  i n the a c e t a t e  c o u l d be  independently determined by  as  the  the a c t i v i t y of v i n d o l i n e as p o s i t i o n 5 i f the p r o p i o n i c had  the  a c i d from the e t h y l s i d e c h a i n should a l s o appear  acetic acid. acetate  acid.  the  correct  should be s p e c i f i c a l l y  at p o s i t i o n 5 of v i n d o l i n e .  of  locate acid  i f the a c e t i c of  h y d r o l y s i s to  vindoline desacetyl-  158  CHoOH  0.286 mc  21 OAc 5.1 x 1 0  0 H  COOCH3 a f t e r d i l u t i o n 5.54 x 10 - d./,m./mmole 0  HIO,  3  ?  %  4  CrO. 3 CHoI inactive  CH CH COOH + CH COOH p-bromophenacyl\ '. , bromide £^CH„6-0CH1-// V 3  2  3  Q  Br  5.50 x 10 d. /m./mmole 99.2% o f a c t i v i t y CH3C-OCH2 inactive F i g u r e 18.  5  Incorporation of Geraniol into Vindoline.  Br  159  vindoline.  The a c t i v i t y o f N-methyl and O-methyl groups i s  e a s i l y d e t e r m i n e d by v o l a t i l i z a t i o n as m e t h y l i o d i d e on r e a c t i o n 77  with hydriodic acid.  D e g r a d a t i o n o f t h e p r e c u r s o r and  i n c o r p o r a t i o n i n t o t h e one-carbon p o o l c a n thus be checked. A s e r i e s o f Kuhn-Roth o x i d a t i o n s were c a r r i e d out i n an e f f o r t t o e s t a b l i s h the best c o n d i t i o n s of p r o p i o n i c a c i d .  7 3  *  7 4  '  7 5  for  production  The y i e l d o f v o l a t i l e a c i d s was e s t a b l i s h e d  by t i t r a t i o n and a f t e r r e g e n e r a t i o n  o f t h e f r e e a c i d s by passage  t h r o u g h an i o n exchange column they were s e p a r a t e d and e s t i m a t e d as t h e i r e t h y l a m i n e s a l t s by paper c h r o m a t o g r a p h y .  74  The b e s t  c o n d i t i o n s were v e r y s i m i l a r t o c o n d i t i o n s p u b l i s h e d by Lemieux and  Purves.  7 3  Vindoline- C 1 4  (93.6  mg.) w i t h a s p e c i f i c a c t i v i t y o f 5.54  x 1 0 d./m./mmole was o x i d i z e d w i t h 3 0 % aqueous chromium t r i 4  o x i d e and 1.97 e q u i v a l e n t s o f v o l a t i l e a c i d s were o b t a i n e d as estimated  by t i t r a t i o n w i t h l i t h i u m h y d r o x i d e .  The s a l t s were  c o n v e r t e d t o t h e p-bromophenacyl e s t e r s and s e p a r a t e d by p r e p a r a t i v e t h i n - l a y e r chromatography on s i l i c a g e l . spot m a t e r i a l was o b t a i n e d  When s i n g l e  i t was 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 and s p e c i f i c a c t i v i t y d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n techniques.  The p-bromophenacylpropionate had  a s p e c i f i c a c t i v i t y o f 5.50 x 1 0 d./m./mmole (275 + 4.5 c./m. 7  4  f o r 2.01 mg. o f sample a t a c o u n t i n g  e f f i c i e n c y o f 64%) w h i c h  i s 99 + 2% o f t h e a c t i v i t y o f v i n d o l i n e .  The p-bromophenacyl-  a c e t a t e was i n a c t i v e w i t h i n t h e l i m i t s o f c o u n t i n g  error.  The  O-methyl and N-methyl groups were a l s o shown t o be i n a c t i v e w i t h -  160  i n the l i m i t of counting  error.  7 7  These r e s u l t s c o n s t i t u t e unambiguous e v i d e n c e f o r s p e c i f i c i n c o r p o r a t i o n o f C-2 o f g e r a n i o l i n t o C-5 o f v i n d o l i n e and e s t a b l i s h t h e monoterpenoid o r i g i n o f t h e n o n - t r y p t o p h a n p o r t i o n of the indole a l k a l o i d s .  Only some q u e s t i o n s remain  as t o t h e mechanism o f c o n d e n s a t i o n and c y c l i z a t i o n . Independent s t u d i e s by B a t t e r s b y ,  A r i g o n i , and S c o t t  7 8  were p u b l i s h e d  simultaneously  7 9  8 0  w i t h t h i s work and a l l r e s u l t s a r e  c o n s i s t e n t w i t h t h e Wenkert-Thomas t h e o r y .  A r i g o n i f e d geran-  i o l - 2 - C t o V i n c a r o s e a and i s o l a t e d v i n d o l i n e w i t h a l l i t s 1 4  a c t i v i t y a t p o s i t i o n 5. ajmalicine  M e v a l o n a t e - 3 - C was a l s o f e d and 14  (33) and v i n d o l i n e  (13) i s o l a t e d .  47% o f t h e a c t i v -  i t y o f v i n d o l i n e was a t C-20 as e x p e c t e d w h i l e C-5, C-21 and the carbomethoxy c a r b o n C-22, as w e l l as the($)- and N-methyls were i n a c t i v e .  I n t h e case o f a j m a l i c i n e C-19 c a r r i e d 4 0 % o f  the a c t i v i t y w h i l e C-18 was i n a c t i v e . Battersby  a l s o f e d m e v a l o n a t e - 3 - C t o V i n c a r o s e a and 14  showed t h a t 4 2 % o f t h e a c t i v i t y was a t C-19 as r e q u i r e d by theory. and  M e v a l o n a t e - 6 - C was i n c o r p o r a t e d 14  catharanthine  (87).  The p r o p i o n i c  i n t o v i n d o l i n e (13)  a c i d r e s i d u e was i n -  a c t i v e i n b o t h c a s e s as e x p e c t e d .  The mixed phosphate e s t e r s  of g e r a n i o l - 2 - C  r o s e a and a j m a l i c i n e ( 3 3 ) ,  1 4  serpentine  were f e d t o V i n c a  (3), catharanthine  (87) and v i n d o l i n e  Kuhn-Roth o x i d a t i o n on d e s a c e t y l v i n d o l i n e  (13) i s o l a t e d .  e s t a b l i s h e d t h a t 98%  of t h e a c t i v i t y was l o c a t e d a t C-5 as a l s o e s t a b l i s h e d by us and Arigoni.  Carbon 5 o f c a t h a r a n t h i n e  a l s o accounted f o r a l l o f  161  the a c t i v i t y i n t h a t m o l e c u l e .  I n t h e case o f a j m a l i c i n e  a c t i v i t y was l o c a t e d a t C-3, C-14, C-20 o r C-21 by t o a j m a l i c o l and Kuhn-Roth o x i d a t i o n .  degradatio  Theory r e q u i r e s  that  C-20 be l a b e l l e d . 80 Scott  f e d d e u t e r i u m l a b e l l e d mevalonate and g e r a n i o l t  V i n c a r o s e a and was a b l e t o observe enhancement o f r e l e v a n t fragments by mass s p e c t r o m e t r y o f i s o l a t e d v i n d o l i n e . t e c h n i q u e c o u l d prove t o be u s e f u l f o r b i o g e n e t i c provided  t h e i n c o r p o r a t i o n i s g r e a t e r than 0.2%.  This  studies  162  Experimental  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 uncorrected.  U l t r a v i o l e t s p e c t r a were measured on a Cary  spectrophotometer i n 95% e t h a n o l .  The  taken on a P e r k i n Elmer Model 137B meter.  The  or a Model 21  n u c l e a r magnetic resonance  instrument.  The  given i n T i e r s  spectrophoto-  (n.m.r.) s p e c t r a were  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 Alumina G p l a t e s ( a c c o r d i n g to S t a h l )  g e l G p l a t e s ( a c c o r d i n g to S t a h l ) were used f o r t h i n -  l a y e r chromatography ground was  (T.L.C.).  In more recent work the back-  made f l u o r e s c e n t t o long wave u l t r a v i o l e t  admixture of alumina or s i l i c a with 2% General P - l , Type 118-2-7 e l e c t r o n i c phosphor. determined u s i n g an A t l a s CH-4  Model D47  r a d i o a c t i v i t y was  mass  Mass s p e c t r a were  spectrometer. Chicago  as a Geiger counter  Decade S c a l e r .  were measured by d e p o s i t i n g samples of 0.2 1.125  The  and  t o 0.5  activities mg.  as  thin  i n c h diameter aluminum p l a n c h e t t e s .  t o t a l a c t i v i t i e s of s y n t h e t i c p r e c u r s o r s and of  m e t a b o l i t e s are given i n m i l l i c u r i e s e f f i c i e n c y of 39%.  by  Semiautomatic Sample Changer, a l l i n  c o n j u n c t i o n with a Model 181B  f i l m s on standard  light  E l e c t r i c Ratma  measured with a Nuclear  gas flow d e t e c t o r operated  mounted i n a Model M-5  The  60  l i n e p o s i t i o n s or c e n t e r s of m u l t i p l e t s are  the i n t e r n a l standard.  The  14  i n f r a r e d s p e c t r a were  measured at s i x t y megacycles per second on a V a r i a n A  or S i l i c a  are  A Nuclear  (mc)  assuming a  isolated counter  Chicago Model 180040 l i q u i d  163  s c i n t i l l a t i o n counter was of r a d i o a c t i v i t y  oxazole)  determinations  as r e q u i r e d f o r degradative work.  s c i n t i l l a t i o n mixture phenyloxazole  used f o r more accurate  c o n s i s t e d of toluene  (P.P.O.) (2 g.)  and  (P.O.P.O.P.) (25 m g . ) .  81  McGeer of the F a c u l t y of Medicine  The  (500 ml.),  2,5-di-  2-p-phenylenebis(5-phenylWe  are indebted  to  Dr.P.  and P r o f e s s o r G.H.N.Towers  of the Department of Botany f o r the use of t h e i r s c i n t i l l a t i o n apparatus. Institute  We  thank Dr.T.C.Beer of the Cancer Research  and a l s o the E l i L i l y Company f o r samples of  Vinca  alkaloids. I n c o r p o r a t i o n of a p r e c u r s o r i s r e p o r t e d i n two ways. r a t e of i n c o r p o r a t i o n i s d e f i n e d as the r a t i o of the a c t i v i t y of product one  hundred.  The  The  times  i n c o r p o r a t i o n i s the r a t i o of the  molar a c t i v i t y of the product p r e c u r s o r times one  total  to the t o t a l a c t i v i t y of p r e c u r s o r specific  The  to the molar a c t i v i t y of the  hundred.  r a d i o a c t i v e precursor  [JMQ -3 , 5-dihydroxy - 3-methyl-  pentanoic  lactone-2- C  purchased  from Merck Sharpe and Dohme of Canada L t d . i n e i t h e r  0.5  or 1.0  1 4  ( [DL]-mevalonolactone-2- C)  mc,quantities.  acetate-2- C 1 4  was  14  One  was  m i l l i c u r i e of e t h y l bromo-  o b t a i n e d from the same company and used f o r  the p r e p a r a t i o n of g e r a n i o l - 2 - C by standard 1 4  S y n t h e s i s of 3,7-dimethyl-2,6-oct.adien-l-ol  methods. '  7 0  (geraniol-2- C)  In each step of the s y n t h e s i s model r e a c t i o n s were out t o check r e a c t i o n c o n d i t i o n s , y i e l d s ,  6 9  1 4  carried  and t o serve as a  164  s o u r c e of a u t h e n t i c i n t e r m e d i a t e s f o r comparisons i n the r a d i o active series.  These r e a c t i o n s were c a r r i e d out f i r s t on an  o r d i n a r y s c a l e then on a s c a l e c l o s e t o t h a t e x p e c t e d t o be r e q u i r e d i n the r a d i o a c t i v e s y n t h e s i s . Normal p r e c a u t i o n s were o b s e r v e d w i t h r e s p e c t t o h a n d l i n g of  radioisotopes.  A l l work was c a r r i e d out on a m e t a l t r a y  (1 x 19 x 28 i n c h e s ) i n a fume hood l i n e d w i t h p o l y e t h y l e n e . A N u c l e a r Chicago Model 2650 G e i g e r c o u n t e r was used t o m o n i t o r any g r o s s c o n t a m i n a t i o n .  Ethyl 3-hydroxy-3 7-dimethyl-6-octenoate-2- C 14  >  (93)  6 9  The m i l l i c u r i e of e t h y l b r o m o a c e t a t e - 2 - C w i t h a s p e c i f i c 14  a c t i v i t y of 1.0 mc/mmole was p u r c h a s e d i n a b r e a k - s e a l tube.  The tube was m o d i f i e d by a d d i t i o n o f a B  1 0  sample  Quickfit  s o c k e t t h r o u g h w h i c h the b r e a k - s e a l c o u l d be broken by means of a g l a s s r o d and i n a c t i v e e t h y l bromoacetate added a f t e r l a t i o n f o r purposes of s c a v e n g i n g and of d i l u t i o n . set  1 4  C  A sidearm  a t f o r t y - f i v e degrees t o t h e tube and equipped w i t h a  Q u i c k f i t cone and vacuum t a k e o f f was a l s o added. the  distil-  B  1 4  After cooling  brown l i q u i d the s e a l was b r o k e n and e t h y l bromoacetate-2d i s t i l l e d under r e d u c e d p r e s s u r e (12 t o r ) i n t o a t a r e d , i c e -  c o o l e d , round bottom f l a s k .  A f t e r c o o l i n g the sample tube  i n a c t i v e e t h y l bromoacetate  (0.83 g.) was added and d i s t i l l e d  b e f o r e y i e l d i n g a sample o f s u f f i c i e n t volume (1.050 g.,  as  6.28  mmoles) f o r s y n t h e s i s w i t h a c a l c u l a t e d s p e c i f i c a c t i v i t y of 0.159  mc/mmole.  T h i s was mixed i n a dry 5 ml. d r o p p i n g f u n n e l  )  165  w i t h f r e s h l y d i s t i l l e d 6-methyl-5-hepten-2-one (0.794 g., mmoles) purchased  from A l d r i c h Chemical  r i n s e d w i t h one m i l l i l i t e r (2 x 0.5  Co..  ml.)  The  was  of benzene which had  been d r i e d by a z e o t r o p i c d i s t i l l a t i o n t o one  h a l f i t s volume  and the r i n s i n g s combined w i t h the reagents i n the funnel.  flask  6.28  dropping  The r e a c t i o n commenced a f t e r b r i e f warming w i t h an  o i l bath when about 10% of the reagent mixture  had been added  under anhydrous c o n d i t i o n s i n a n i t r o g e n atmosphere t o a  10%  excess of a c t i v a t e d g r a n u l a r z i n c  round  (0.452 g.)  i n a 10 ml.  bottom two-necked f l a s k equipped w i t h a condenser and magnetic stirring  flea.  The z i n c had been a c t i v a t e d by washing with  aqueous h y d r o c h l o r i c a c i d  ( 5 % ) , d i s t i l l e d water, e t h a n o l , a c e t -  one and f i n a l l y dry benzene, t r a c e s of which were removed i n vacuo b e f o r e weighing.  Dropwise a d d i t i o n was  at  as t o maintain r e f l u x i n g of the g e n t l y  such a r a t e (20 min.)  s t i r r e d mixture.  The r e a c t i o n mixture was  hour w i t h v i g o r o u s s t i r r i n g ,  then c o n t i n u e d  then r e f l u x e d f o r an  c o o l e d i n an i c e - b a t h and t r e a t e d  w i t h i c e - c o l d 10% s u l p h u r i c a c i d  (5 ml.).  After transfer to  a s e p a r a t o r y f u n n e l and r i n s i n g the r e a c t i o n f l a s k w i t h benzene (2 x 5 ml.)  the aqueous and benzene l a y e r s were separated.  benzene s o l u t i o n was  e x t r a c t e d w i t h c o l d 5% s u l p h u r i c a c i d  10 ml.), c o l d 10% sodium carbonate with water (2 x 5 ml.). extracted with ether sodium carbonate  and  which was  (2 x  f i n a l l y washed  The combined s o l u t i o n s were back  (2 x 5 ml.)  (5 ml.)  (5 ml.)  The  washed w i t h  and w i t h water (2 x 5 ml.).  10% The  combined o r g a n i c l a y e r s were d r i e d over anhydrous magnesium  166  sulphate and f i l t e r e d i n t o the s u b l i m a t i o n apparatus  which had  been m o d i f i e d f o r s m a l l s c a l e d i s t i l l a t i o n s by a d d i t i o n o f a cup  (2 ml.) t o the end of the c o l d f i n g e r .  was removed under a stream  The bulk of s o l v e n t  of warm,dry n i t r o g e n .  The r e s i d u a l  s o l v e n t and any low b o i l i n g i m p u r i t i e s were removed by d i s t i l l a t i o n of the m a g n e t i c a l l y s t i r r e d l i q u i d under reduced (12 t o r ) u s i n g a b o i l i n g water bath.  pressure  E t h y l 3,7-dimethyl-3-  h y d r o x y - 6 - o c t e n o a t e - 2 - C was then d i s t i l l e d 14  (11 t o r ) i n t o the  c l e a n e d d r i e d cup while the temperature of a m a g n e t i c a l l y  stir-  r e d g l y c e r i n bath was s l o w l y r a i s e d from 130 t o 150° C l e a v i n g a l i g h t brown r e s i d u e .  The product was c a r e f u l l y t r a n s f e r r e d  by p i p e t t e and c o l d f i n g e r cup and the d i s t i l l a t i o n j a c k e t r i n s e d with e t h e r .  A f t e r removal o f s o l v e n t and d i s t i l l a t i o n  as before a f u r t h e r 28 mg. o f the p r e c i o u s product (0.9784 g., 4.56 mmoles, 72.5%). 15 t o r . 1640(s),  Boiling point, l i t .  I n f r a r e d : ( l i q u i d f i l m ) 3540(s),  3000(s),  1460(ra), 1380(m), 1330(m), 1205(s),  840(w) c m . -1  was c o l l e c t e d 6  9  134°,  2940(s),  1040(m), 935(w),  T h i s spectrum i s i d e n t i c a l t o the one o b t a i n e d  f o r the compound i n c o l d runs as i s the n.m.r. spectrum which i s c o n s i s t e n t with the expected (deuterochloroform)  structure.  The n.m.r. spectrum:  broad t r i p l e t c e n t e r e d at 4.89 7" ( o l e f i n i c  proton;? area 1.0 H) , quartet c e n t e r e d at 5.89 T (methylene of  -OCH2CH3, area 2 H), s i n g l e t at 6.64 T (hydroxyl proton, area 1 H), s i n g l e t at 7.56 T (C-2 methylene, area 2 H), m u l t i p l e t c e n t e r e d at 7.99 T(allylic  methylene, area 2 H), s i n g l e t s at  8.33 and 8.39 T ( t e r m i n a l methyl protons, area 6 H), s i n g l e t  167  at  8.77  7* ( t e r t i a r y methyl p r o t o n s ) o v e r l a p p i n g a t r i p l e t  c e n t e r e d at 8.77  T (methyl o f -OCH CH  r e m a i n i n g methylene  2  3>  t o t a l a r e a 6 H).  p r o t o n s are obscured by methyl  Ethyl 3-acetyl-3,7-dimethyl-6-octenoate-2- C 14  7 0  (69) (0.978  14  mmoles) was r e f l u x e d f o r 6,5  hours w i t h a two  e x c e s s of a c e t i c a n h y d r i d e i n a 10 ml. round bottom  (5 m l . ) .  molar  flask.  Excess a c e t i c a n h y d r i d e i n the y e l l o w r e a c t i o n m i x t u r e c a r e f u l l y decomposed w i t h water  The  resonances.  E t h y l 3,7-dimethyl-3-hydroxy-6-octenoate-2- C g., 4.56  also  was  After separation i n  a f u n n e l the aqueous l a y e r was e x t r a c t e d w i t h e t h e r (2 x 5 ml.) which had j u s t been used t o r i n s e the r e a c t i o n f l a s k condenser.  and  The e t h e r washings were combined w i t h the o r g a n i c  l a y e r and c a r e f u l l y washed f r e e o f a c e t i c a c i d w i t h 10% carbonate  (1 x 5, 3 x 2.5 m l . ) .  sodium  The c a r b o n a t e e x t r a c t s were  t h e n back e x t r a c t e d w i t h e t h e r (1 x 5 m l . ) .  The  ethereal  s o l u t i o n was washed w i t h e t h e r ( 1 x 5, 1 x 2.5 m l , ) , t h e n w i t h brine  (1 x 4 ml.) and d r i e d over magnesium s u l p h a t e i n a C r a i g  f i l t r a t i o n apparatus.  Removal of s o l v e n t under a stream o f dry  n i t r o g e n a f t e r f i l t r a t i o n y i e l d e d a crude a c e t a t e (1.11 g., mmoles, 95%) whose i n f r a r e d spectrum was  4.34  i d e n t i c a l to that  o b t a i n e d f o r the same compound i n the i n a c t i v e s y n t h e s i s .  The  compound i s c h a r a c t e r i z e d by the absence of h y d r o x y l a b s o r p t i o n (3540 c m ) -1  infrared.  8 2  and by t y p i c a l bands at 1740 and 1245 c m  -1  i n the  168  Ethyl  3,7-dimethyl-2,6-octadienoate-2- C 1 4  P y r o l y s i s o f the a c e t a t e o f e t h y l 6 - o c t e n o a t e was  7 0  3-hydroxy-3,7-dimethyl-  shown by vapor phase chromatography  to y i e l d  two major p r o d u c t s and was t h e r e f o r e a s u p e r i o r method t o d e h y d r a t i o n w i t h phosphorus  oxychloride which y i e l d e d f o u r .  A  carbowax 20M (5 f e e t x 0 . 2 5 i n c h e s ) column at 1 4 0 ° C w i t h c o l l e c t o r , d e t e c t o r and i n j e c t o r t e m p e r a t u r e s o f  150,  250  C r e s p e c t i v e l y and a h e l i u m f l o w r a t e of 59 ml./min. was Injecting 2.0 / X L .  6-octenoate  used.  3,7-dimethy1-2-trans-6-  ( g e r a n i o l ) (44 min.), e t h y l  (67 min.)  oate ( 9 3 m i n . ) .  235°  o f sample r e t e n t i o n t i m e s were d e t e r m i n e d f o r  2 - m e t h y l - 2 - h e p t e n - 6 - o n e (5 m i n . ) , octadien-l-ol  and  3-hydroxy-3,7-dimethyl-  and e t h y l 3 - a c e t y l - 3 , 7 - d i m e t h y l - 6 - o c t e n -  The p r o d u c t s o f p y r o l y s i s of the a c e t a t e ,  i n t e g r a t i n g the a r e a s under the peaks, had r e t e n t i o n t i m e s o f 22 min.(33%) and 2 9 . 5  min.(67%).  The two components were  s e p a r a t e d u s i n g a carbowax 20M column ( 1 0 f e e t x 0 . 3 1 8  inches)  at an o p e r a t i n g temperature o f 1 8 0 ° C and a h e l i u m f l o w r a t e o f 100 ml./min.„ a t u r e s were  The c o l l e c t o r , d e t e c t o r , and i n j e c t o r temper-  155,  260,  and  240°  C  respectively.  Up t o  1 0 0 JJLL .  o f l i q u i d c o u l d be s e p a r a t e d under t h e s e c o n d i t i o n s . The more m o b i l e component i s e t h y l 3 , 7 - d i m e t h y l - 2 - c i s - 6 o c t a d i e n o a t e as deduced from i t s p h y s i c a l p r o p e r t i e s and by r e d u c t i o n t o n e r o l w i t h l i t h i u m aluminum h y d r i d e . i n d e x : Ln]  23 rk  Refractive „  =  1.4672.  U l t r a v i o l e t spectrum i n e t h a n o l :  JJ 2 1 5 mLL,  ^  m  a  x  11,400  I n f r a r e d spectrum  ( t y p i c a l of an Oi,@-unsaturated  (liquid film):  V  3002  „ max.  ester).  (s), 2925(s),  1723,  169  1715(s), 1650(s), 1450(m), 1245,  1215(m), 1160(s), 1099(w),  1065(w), 1039(w), 852(w), 821(w), 790(w) cm" .  The n.m.r.  1  spectrum  i n d e u t e r o c h l o r o f o r m which i m p l i e s that the  i s a mixture and  i s thereby d i f f i c u l t  at 4.87  t o analyze completely  T (newly o l e f i n i c proton 0.80  i n c l u d e s : broad s i n g l e t at 4.38 a broad t r i p l e t  T ( o l e f i n i c p r o t o n , area 1.0 T (methylene  quartet centered at 5.94  showed s i g n i f i c a n t peaks at m/e  151  (M - 69), 124  69. 43, 41, and 28.  (M - 72), 101  (M ), +  83,  column (5 f e e t  inches) operated at 180° C w i t h i n j e c t o r , d e t e c t o r and of 230,  flow r a t e of 91 ml./min.. reduced e s t e r  245,  One  from A l d r i c h Chemical  The  min.)  3,7-  purchased  Company as a mixture of the c i s ( n e r o l )  a l s o purchased  A sample of the t r a n s isomer from the same company.  l e s s mobile and major component of p y r o l y s i s i s e t h y l ^23  3 ,7-dimethyl-2-trans-6-octadienoate . = 1.4680.  U l t r a v i o l e t spectrum  H,200 ( t y p i c a l of an  spectrum  t o the un-  and the other t o  ( n e r o l ) which was  and t r a n s ( g e r a n i o l ) isomers. ( g e r a n i o l ) was  and 150° C with a helium  peak corresponded  ( r e t e n t i o n time 6.0  dimethyl-2-cis-6-octadien-l-ol  (liquid film):  V  R e f r a c t i v e index:  i n ethanol:  780 cm . -1  A  o v  215  0£,@-unsaturated e s t e r ) . 3001  The n.m.r. spectrum  1098,  1061,  [n] ^  mfJL  ,  Infrared  8 7  ( s ) , 2920(s), 1723,  1650(s), 1450(m), 1225(s), 1143(s), 1108, 815,  = 196  The r e d u c t i o n product showed two peaks on  c o l l e c t o r temperatures  ^max  H) and a  (M - 95), 86,  vapour phase chromatography u s i n g a'carbowax 20M x 0.25  H),  of -OCH2CH3, area 2 H).  The mass spectrum (M - 45), 128  fraction  1712(s),  1038.  i n deuterochloroform:  865,  170  broad s i n g l e t 4.37 7" ( o l e f i n i c proton on newly formed bbnd, area 1.0  H), broad t r i p l e t  o l e f i n i c p r o t o n , area 1.0 (methylene at  7.87  7" (methylene  finally  -OCH2CH3). = 196  H), quartet centered at 5.94  (M+),  and C-3  and 8.39  a triplet  T  methyl p r o t o n s , area 7 H), p a i r of  T ( t e r m i n a l methyl p r o t o n s , area 6 H),  c e n t e r e d at 8.80  The mass spectrum 151  (M - 45),  96), 82, 69, 41, and  129  T  (methyl protons of  showed s i g n i f i c a n t peaks at (M - 67),  124  (M - 72),  14  mmoles) was  100  m/e (M -  28.  Ethyl 3-acetyl-3,7-dimethyl-6-octenoate-2- C 4.35  (original  protons of -OCHgCHg, area 2 H), p o o r l y r e s o l v e d peak  s i n g l e t s at 8.33 and  c e n t e r e d at 4.90 T  double  very s l o w l y d i s t i l l e d  (1.11  at atmospheric  i n t o the c o l l e c t i o n cup of the m i c r o - d i s t i l l a t i o n  pressure  apparatus.  It had been found that r e f l u x i n g f o r ten minutes d i d not complete p y r o l y s i s .  Both d i s t i l l a t e  g.,  effect  and r e s i d u e were t r a n s -  f e r r e d t o a s e p a r a t o r y f u n n e l and washed f r e e of a c e t i c a c i d a 10% sodium carbonate w i t h water  solution  (3 x 3 ml.).  and removal of the ether under a  stream of n i t r o g e n the p y r o l y s a t e was  at  A f t e r washing  (4 x 3 ml.), b r i n e (2 ml.), d r y i n g over anhydrous  magnesium s u l p h a t e , f i l t r a t i o n  pressure  with  distilled  under  reduced  (12 t o r , 116-123° C) y i e l d i n g a mixture c o n s i s t i n g of  l e a s t 90% of c i s and t r a n s (1:3) isomers of e t h y l  2,6-octadienoate-2- C 14  composition  (0.70  g., 3.58  3,7-dimethyl-  mmoles, 83% y i e l d ) .  i s based on a comparison of the i n f r a r e d  w i t h that from a sample of the same compound which was by vapour phase chromatography and by i n t e r p r e t a t i o n  The  spectrum analyzed of the  171  n.m.r. spectrum.  The p y r o l y s i s of the a c e t a t e i s r e a d i l y  checked by disappearance of the a c e t a t e a b s o r p t i o n maxima at 1735  and 1250 c m  and appearance  -1  of a peak at 1650  a t t r i b u t e d to an asymmetrical double bond. ( no s o l v e n t ) w i t h a s i n g l e t at 4.38 newly formed double bond, area 0.90  cm"  1  The n.m.r. spectrum  T ( o l e f i n i c proton on H) i s a l s o i d e n t i c a l t o  that from the c o l d run.  3,7-dimethyl-2,6-octadien-l-ol-2- C (geraniol) ( 9 7 ) 14  Ethyl 3,7-dimethyl-2,6-octadienoate-2- C 14  mmoles) i n e t h e r (3 ml.) was  7 0 a  (0.70 g.,  3.58  added at such a r a t e as to m a i n t a i n  r e f l u x i n g to a magnetically s t i r r e d ethereal s o l u t i o n  (5 ml.)  of l i t h i u m aluminum hydride (100% excess) i n a 12 ml.  two-  necked round bottom  flask.  The mixture was  then r e f l u x e d f o r  f o r t y minutes and excess hydride destroyed by c a u t i o u s a d d i t i o n of wet e t h e r .  Dilute hydrochloric acid  (2 N, 3 ml.) was  added  t o d i s s o l v e aluminates and o r g a n i c m a t e r i a l e x t r a c t e d i n t o e t h e r (3 x 3 ml.).  The combined o r g a n i c l a y e r s were washed w i t h  water u n t i l n e u t r a l t o pH paper  (5 x 3 m l . ) , w i t h b r i n e  (2 m l . ) ,  d r i e d over magnesium s u l p h a t e , f i l t e r e d , the s o l v e n t removed under a stream of dry n i t r o g e n and the 3,7-dimethyl-2,6-octadienl - o l - 2 - i C d i s t i l l e d under reduced p r e s s u r e . 4  (0.3736 g., 2.46  mmoles, 68.6%  yield  from the e s t e r ) of the s y n t h e t i c  mixture of g e r a n i o l and n e r o l based on e t h y l i s 39.2%.  The  bromoacetate-2- C 14  The i n f r a r e d spectrum of t h i s mixture i s i d e n t i c a l  to that of p r e v i o u s s y n t h e t i c mixtures and d i f f e r s somewhat  172  from the spectrum of the t r a n s isomer  ( g e r a n i o l ) but only i n the  fingerprint region.  72a  3,7-dimethyl-2,6-octadien-l-phosphate Geraniol  (96)  chloroacetonitrile  (250 mg.,  1.62  (1.40 g., 9.75  and pyrophosphate  mmoles) was  mixed with  mmoles, 6 x)  i n a 100  three-necked round bottom f l a s k with s m a l l magnetic bar f i t t e d w i t h a condenser amine phosphate acetonitrile  (34 ml.) was  f u n n e l over a 3.5  a d d i t i o n then was  (20.6 g.)  Ditriethyl-  x) d i s s o l v e d i n  The r e a c t i o n mixture  was  and s t i r r e d c o n s t a n t l y d u r i n g the  allowed t o stand 2.5  in acetonitrile  hours.  (Ditriethylamine  (50 ml.)  evolved.  acid  then adding t r i e t h y l a m i n e  from a dropping f u n n e l t o the s t i r r e d  C o n s i d e r a b l e heat was  ml.  stirring  had been prepared by d i s s o l v i n g 85% phosphoric  (11.75 g.)  tri-  i n t r o d u c e d through the dropping  hour p e r i o d .  kept at room temperature  phosphate  and dropping f u n n e l .  (1.17 g., 3.9 mmoles, 2.4  a  solution.  A f t e r the f i r s t  mole of  tri-  ethylamine had been added the milky suspension became a c l e a r solution.  The product f a i l e d t o c r y s t a l l i z e out o v e r n i g h t but  c r y s t a l l i z e d b e a u t i f u l l y a f t e r a d d i t i o n of a few of acetone.  The phosphate  milliliters  i s h i g h l y d e l i q u e s c e n t and was  over a d e s i c c a n t a f t e r removal of s o l v e n t iri vacuo) . ammonia  (0.1 N, 50 ml.)  r e a c t i o n mixture.  kept  Dilute  and e t h e r (150 ml.) were added t o the  A f t e r s e p a r a t i o n the e t h e r l a y e r  e x t r a c t e d w i t h d i l u t e ammonia (0.1 N, 2 x 25 ml.)  was  and the  combined aqueous phase washed w i t h e t h e r (3 x 100 ml.).  The  173  aqueous phase was C) t o 20 ml..  then c o n c e n t r a t e d on a r o t a r y evaporator  A f t e r a d d i t i o n of cyclohexylamine  the c o n c e n t r a t i o n was s o l u t i o n was  (0.55  (60°  ml.)  continued u n t i l c r y s t a l s appeared  then the  allowed t o stand o v e r n i g h t at 0° C and the mono-  phosphate s a l t  f i l t e r e d and dried.(123  mg.).  The mother l i q u o r s were t r e a t e d with c o n c e n t r a t e d ammonia (1 ml.)  and e x t r a c t e d w i t h e t h e r t o remove  (2 x 15 ml.). was  cyclohexylamine  A s o l u t i o n of l i t h i u m c h l o r i d e  (IN, 5  ml.)  added t o the mother l i q u o r s which were then c o n c e n t r a t e d  u n t i l c r y s t a l s appeared, allowed t o stand overnight and f i l t e r e d .  The pyrophosphate  i n vacuo at 60°  C.  The  (85 mg.)  was  (0° C)  d r i e d overnight  homogeneity of the phosphate and pyrophosphate s a l t s  were t e s t e d by paper c h r o m a t o g r a p h y . chromatography was  83  Whatman #3 MM paper f o r  washed w i t h d i l u t e a c e t i c a c i d  A f t e r d r a i n i n g f o r f i v e minutes the paper was n e u t r a l w i t h d i s t i l l e d water.  (2 N, 250  washed u n t i l  Repeated r i n s i n g w i t h a t o t a l of  f i v e l i t e r s per sheet of paper was  necessary.  allowed t o dry at room temperature  overnight then was  w i t h the l i t h i u m s a l t  ml.).  The paper  was  spotted  of geranyl pyrophosphate and the c y c l o -  hexylamine s a l t of g e r a n y l phosphate.  The  r e s i d u e from  mother l i q u o r s , d i t r i e t h y l a m i n e phosphate and  dipotassium  phosphate were a l s o s p o t t e d on the paper which was i n t o a c y l i n d e r , s t a p l e d and developed  the  then  rolled  (25 hours) i n an i s o -  p r o p a n o l , ammonia, water (6:3:1) mixture u s i n g the  ascending  technique i n a chromatography tank which had been allowed  two  174  days t o e q u i l i b r a t e .  The paper was d r i e d by s t a n d i n g i n a  c u r r e n t o f warm a i r from a h a i r d r y e r f o r h a l f and hour then i n an oven f o r f i v e minutes  (80° C ) .  The paper was s p r a y e d ,  f i r s t w i t h a m i x t u r e o f 6 0 % w/w p e r c h l o r i c a c i d normal h y d r o c h l o r i c a c i d  (5 m l . ) ,  (10 ml.) and 4% w/v ammonium molybdate  (25 ml.) which had been d i l u t e d t o 100 m l . w i t h water. then d r i e d i n a c u r r e n t o f warm a i r (5 min.) then  I t was  momentarily  i n an oven (80° C ) . The paper was then s p r a y e d w i t h f r e s h l y p r e p a r e d 1% stannous c h l o r i d e i n 10% h y d r o c h l o r i c a c i d b l u e s p o t s o f a phosphomolybdate complex.  yielding  G e r a n y l phosphate  (R^ 0.6) showed up as a s i n g l e b l u e - g r e e n spot w i t h a v e r y s m a l l amount o f m a t e r i a l near t h e o r i g i n . pyrophosphate  The sample o f g e r a n y l  (R^ 0.5) c o n t a i n e d a l a r g e amount o f phosphate  (blue s p o t ) and a s i g n i f i c a n t amount o f i m p u r i t y near t h e o r i g i n . Orthophosphate  (Rf 1.3) was v i s i b l e as a y e l l o w spot b e f o r e  r e d u c t i o n w i t h stannous c h l o r i d e when i t t u r n e d b l u e - g r e e n . The r e s i d u e from t h e mother l i q u o r s c o n s i s t e d m a i n l y o f o r t h o phosphate w i t h a s m a l l amount o f g e r a n y l  phosphate.  A d m i n i s t r a t i o n of l a b e l l e d g e r a n i o l t o Vinca rosea Linn. (Catharanthus r o s e u s G.don) The p l a n t s (1.0 y e a r o l d ) were grown i n a bed i n an unh e a t e d , shaded greenhouse and about 5% were f l o w e r i n g .  These  p l a n t s had f l o w e r e d t h e p r e v i o u s summer and had had no f l o w e r s f o r t h r e e months p r i o r t o f e e d i n g .  The crude a l k a l o i d c o n t e n t  based on d r i e d weight o f stem and l e a f m a t e r i a l was 0.37% and  175  v i n d o l i n e was  shown t o be p r e s e n t  by t h i n - l a y e r chromatography  and by i t s c h a r a c t e r i s t i c c r i m s o n c o l o u r w i t h e e r i e  sulphate  s p r a y ( 1 % e e r i e s u l p h a t e , 35% s u l p h u r i c a c i d ) . Because t r o u b l e had been a n t i c i p a t e d i n a d m i n i s t e r i n g water i n s o l u b l e g e r a n i o l to plants experimental  d e t a i l s had been  worked out f o r p r e p a r a t i o n of the pyrophosphate. was  also considered  The  glucoside  as a s u i t a b l e water s o l u b l e d e r i v a t i v e .  B e f o r e p r e p a r i n g a d e r i v a t i v e a t t e m p t s were made t o s o l u b i l i z e geraniol Span 20  u s i n g e t h a n o l , d i m e t h y l s u l f o x i d e , soap, 0 6 - l e c i t h i n , ( s o r b i t a n l a u r a t e ) and Tween 20  laurate).  (polyoxyethylenesorbitan-  E t h a n o l and d i m e t h y l s u l f o x i d e k i l l e d the p l a n t s i n  concentrations  necessary f o r s o l u b i l i z a t i o n .  soap a l s o k i l l e d the p l a n t .  Ordinary  potassium  I n j e c t i o n of neat g e r a n i o l w i t h a  s y r i n g e i n t o the stem r e s u l t e d i n c o l l a p s e of the stem. ing  the c h e m i c a l  Paint-  or a d i l u t e s o l u t i o n i n p e t r o l e u m e t h e r on  the  l e a v e s r e s u l t e d i n , a b s o r p t i o n and d e s i c c a t i o n of c e l l s i n the p a i n t e d area w i t h c o l l a p s e of the v e i n s and e v e n t u a l stem c o l l a p s e as the m a t e r i a l was extent  transported.  Tween 20 and t o a l e s s e r  Span 20 and C t V l e c i t h i n were c a p a b l e of  g e r a n i o l so t h a t i t c o u l d be a d m i n i s t e r e d shoots i n s u f f i c i e n t concentrations any  solubilizing  t h r o u g h cut ends of  for tracer studies  without  o b v i o u s damage t o the p l a n t s . A s i x i n c h c u t t i n g (21 l e a v e s ) was  placed i n a  c o n t a i n i n g an aqueous s u s p e n s i o n (1 ml.) octadien-l-ol-2- C 1 4  mc/mmole).  The  (96,97) (1.67  s u s p e n s i o n was  mg.,  t e s t tube  of 3,7-dimethyl-2,6-  specific activity  p r e p a r e d by s h a k i n g the  0.159 labelled  176  p r e c u r s o r w i t h one m i l l i l i t e r  from a mixture of one drop of  Tween 20 i n 250 ml. of d i s t i l l e d water. w i t h i n f o u r hours and water was cut  end submerged.  The  absorbed  added as r e q u i r e d t o keep the  W i t h i n t h r e e days a c t i v i t y was d e t e c t a b l e  w i t h the r a d i a t i o n monitor i n even the topmost c u t t i n g which was  l i q u i d was  leaves.  The  kept a l i v e under continuous i l l u m i n a t i o n f o r  seven days, l o s t 5 l e a v e s i n t h i s time.  Leaves were removed  from o p p o s i t e s i d e s of the stem and p r e s s e d between t i s s u e paper between two books u n t i l dry. l e a v e s was left  also pressed.  The stem with remaining  Leaves and stem w i t h l e a v e s were then  f o r seven days i n c o n t a c t w i t h I l f o r d x-ray f i l m and the  r e s u l t i n g a u t o r a d i o g r a p h confirmed that a c t i v i t y was  distributed  throughout the p l a n t . T o x i c i t y of g e r a n i o l as a f u n c t i o n of c o n c e n t r a t i o n was checked i n a simple experiment  by hydrophonic a d m i n i s t r a t i o n t o  each of two c u t t i n g s o f one m i l l i l i t e r of s o l u t i o n ! the I.  containing  f o l l o w i n g c o n c e n t r a t i o n s of g e r a n i o l and Tween 20 r e s p e c t i v e l y : 1 mg.,  1/2500 drop; I I . 1 mg.,  drop; IV. 2 mg.,  1/10  1/10  drop; V, 5 mg.,  drop; I I I . 2 mg., 1/10  1/2500  drop; VI. a blank.  The ends of the c u t t i n g s were kept immersed i n water and a r e c o r d made of the water absorbed.  A f t e r t h r e e days the lower l e a v e s  s t a r t e d y e l l o w i n g i n a l l cases.  A f t e r f i v e days the lower  l e a v e s were p a r t i c u l a r l y dry and f a l l i n g , stem c o l o u r i n g disappeared, and the c u t t i n g stopped a b s o r b i n g water c o n d i t i o n V.  It was  rosea w i l l t o l e r a t e  had  under  concluded that h e a l t h y c u t t i n g s of Vinca one t e n t h drop of Tween 20 and two  milli-  177  grams o f g e r a n i o l but t h a t f i v e m i l l i g r a m s r e s u l t s i n o b s e r v a b l e damage.  P i l o t Run Feeding of g e r a n i o l - 2 - C t o Vinca rosea 1 4  When i t became o b v i o u s t h a t a l a r g e number o f c u t t i n g s would have t o be f e d i n o r d e r t o o b t a i n enough a c t i v e v i n d o l i n e f o r d e g r a d a t i o n an i l l u m i n a t i o n chamber was c o n s t r u c t e d . c o n s i s t e d o f a bank o f f o u r twenty-seven  This  watt, eighteen i n c h  f l u o r e s c e n t tubes s u p p o r t e d on a m e t a l frame s i x t e e n i n c h e s above a n i n e square f o o t a r e a c o v e r e d w i t h p o l y e t h y l e n e s h e e t i n g . E i g h t t w e n t y - f i v e i n c h t e s t tube b l o c k s were made each w i t h two s t a g g e r e d rows o f h o l e s t o f i t 12 x 75 mm. t e s t t u b e s .  Rows,  and h o l e s i n each row were spaced two i n c h e s a p a r t so each b l o c k c o n t a i n e d space f o r t w e n t y - f o u r t e s t tubes and a t o t a l o f one hundred and n i n e t y - t w o c u t t i n g s c o u l d be f e d and i l l u m i n a t e d at once.  P i l o t Run Ten s h o o t s o f year o l d greenhouse grown  Catharanthus  r o s e u s p l a n t s v a r y i n g from f i v e t o e l e v e n i n c h e s i n h e i g h t were c u t d i a g o n a l l y a c r o s s t h e stems. t a k e n from s i n g l e p l a n t s .  S e v e r a l c u t t i n g s were  Leaves were c a r e f u l l y removed so  t h a t t h e i n d i v i d u a l c u t t i n g s would s t a n d i n 12 x 75 mm. tubes w i t h o u t i n t e r f e r e n c e from t h e l e a v e s .  test  The c u t ends were  immersed i m m e d i a t e l y i n water i n o r d e r not t o break t h e c a p i l -  178  l a r i t y of the l i q u i d t r a n s p o r t system. mg.,  0.021  mc)  w i t h Tween 20  Geraniol-2- C  (20.1  1 4  e m u l s i f i e d i n ten m i l l i l i t e r s of d i s t i l l e d water (8 d r o p s / l i t e r ) was  absorbed  through the cut ends  of the ten shoots which were then kept a l i v e under constant i l l u m i n a t i o n t a k i n g care t o keep the cut ends immersed i n water f o r seven The  days. f r e s h p l a n t m a t e r i a l (51 g.) was  cut i n t o s m a l l e r  p i e c e s and macerated w i t h methanol-acetic ml.)  acid  (10:1, 3 x  200  i n a Waring b l e n d e r , f i l t e r i n g and washing each time  a Buchner f u n n e l u n t i l no more green c o l o u r remained. s o l v e n t was  on  The  removed on a r o t a r y evaporator and the r e s i d u e  p a r i t i t i o n e d between benzene (1 x 200 ml.,  1 x 50 ml.)  3 x 20 ml.).  dilute  hydrochloric acid  (2 N, 2 x 50 ml.,  aqueous l a y e r was  mixed w i t h an equal volume of c h l o r o f o r m ,  shaken,, and the o r g a n i c layers.combined aqueous l a y e r was  .  then washed w i t h c h l o r o f o r m  removed a s m a l l amount of yellow pigment. caused any l a y e r was prevent  The  The  and  combined  dark brown  (5 x 40 ml.)  Emulsions  had  which not  great amount of t r o u b l e to t h i s p o i n t but the aqueous f i l t e r e d through a l a r g e Whatman #1 paper t o help  future d i f f i c u l t i e s .  The aqueous l a y e r was  made b a s i c  w i t h ammonia (pH 10) and the a l k a l o i d s e x t r a c t e d i n t o c h l o r o form  (10 x 25 ml.)  y i e l d i n g a l i g h t y e l l o w e x t r a c t which a f t e r  washing w i t h water (4 x 20 ml.)  then b r i n e (1 x 30 ml.),  filter-  i n g , d r y i n g over anhydrous magnesium sulphate and e v a p o r a t i o n y i e l d e d the crude a l k a l o i d which was  (106 mg.,  0.2%  of f r e s h p l a n t m a t e r i a l ) ,  counted u s i n g the gas flow counter  (specific  activity  179  4070 c./m./mg.) and shown t o r e p r e s e n t 2.4%  o f the  activity  fed. The graphy"  crude a l k a l o i d was  w h i c h r e v e a l e d the now  m i x t u r e and suggested alumina  examined by t h i n - l a y e r chromatof a m i l i a r c o m p l e x i t y of the  a v e r y low v i n d o l i n e c o n t e n t .  G p l a t e (5 x 20 cm.)  w i t h t u n g s t e n phosphor was  w i t h the crude a l k a l o i d , a l k a l o i d from band V, and a u t h e n t i c v i n d o l i n e and was acetate (1:1).  developed  spotted  with  i n chloroform-ethyl-  This p l a t e a f t e r examination  l i g h t and exposure t o i o d i n e vapour was f i l m of C r a f t i n t Spray-Art  An  under u l t r a v i o l e t  sprayed w i t h a t h i n  F i x a t i v e t o p r e s e r v e the i o d i n e  c o l o u r a t i o n and t o f i x the alumina.  An  autoradiograph  o b t a i n e d by exposure of I l f o r d x-ray f i l m by c o n t a c t w i t h the p l a t e f o r seventeen days showed a s e r i e s o f s p o t s  corresponding  t o a l k a l o i d s but the band V m a t e r i a l i n t h i s system showed o n l y a s i n g l e r a d i o a c t i v e spot. A p r e p a r a t i v e t h i n - l a y e r chromatoplate Desaga apparatus  (blade s e t t i n g 0.8  grams o f alumina  G and one  mm.)  was  prepared  by s p r e a d i n g  hours.  c h l o r o f o r m s o l u t i o n was developed  plate.  d r i e d at room temperature i n a v e n t i l a t e d  f o r twenty-four  was  fifty  gram phosphor s l u r r i e d i n n i n e t y  m i l l i l i t e r s of water on a twenty by s i x t y c e n t i m e t e r The p l a t e was  using  The  crude a l k a l o i d i n a  s t r e a k e d a c r o s s one end.  area  concentrated The p l a t e  u s i n g a mixture of c h l o r o f o r m - e t h y l a c e t a t e  (1:1).  A l k a l o i d bands on the p l a t e were l o c a t e d by f l u o r e s c e n c e and quenching o f the f l u o r e s c e n t background.  E i g h t bands were  by  180  scraped from the p l a t e and packed  the alumina s l u r r i e d  i n g l a s s columns as one  graphy column.  The  alkaloid  i n chloroform  would normally pack a chromato-  was  then e l u t e d by  repeated  washing w i t h chloroform,weighed and  i t s a c t i v i t y determined by  counting  Band one  on aluminum p l a n c h e t t e s .  a complex of f l u o r e s c e n t and  (Rf 0-2.5)  was  quenching bands near to the  origin  5  (36 mg., II  (R  f  total activity 2.5-2.9) was  a c t i v i t y 3.8  x 10  l y quenching and  3  1.1  x 10  brightly  blue  c./m.).  by t h i n  counts per minute).  Band  fluorescent  total  Band I I I (R  f  (1.7 mg.,  2.9-3.7) was  l a y e r comparison with  the v i n d o l i n e c o n t a i n i n g band (1.7 mg.,  a c t i v i t y 4.9  x 10  s t r o n g l y quenching (R and were c o l o u r e d 2.9,  2.7  mg.,  f  Bands IV, V and  q u i c k l y and  Band IV had  intensely by 16.2,  nor very  total activity  s t o n g l y quenching 10  3  c./m.).  the s o l v e n t loaded was  x 10  front.  and  5.4  specific  x 10  activity neither  (Rf 4.9-5.4, 0.9  mg.,  p l a t e was  not  again  recovered.  The  Speed i s r e q u i r e d f o r s u c c e s s f u l  to l e t developing  fluormg.,  of the  of a l k a l o i d s from alumina as they tend to o x i d i z e . best  than  total activity  Only about f i f t y percent  (2.8, c./m.  3  no a c t i v i t y between band VIII  onto the p r e p a r a t i v e  not e x t r a c t e d .  i o d i n e vapour  c./m.) while band VIII was  (Rf 5.4-6.0, 1.6  There was  VI were very  Band VII was  s t r o n g l y quenching 1.8  5.6,  a higher  the v i n d o l i n e c o n t a i n i n g band. escent  total  3.7-4.1, 4.1-4.4, 4.4-4.9 r e s p e c t i v e l y )  total activity  respectively).  strong-  authentic  m a t e r i a l was  c./m.).  and  s o l v e n t evaporate e n t i r e l y  1.6  x  and alkaloid origin recovery It  was  from the  181  p l a t e b e f o r e bands were s c r a p e d o f f and s l u r r i e d w i t h c h l o r o f o r m . Band I I I was d i l u t e d w i t h f i v e m i l l i g r a m s o f a u t h e n t i c v i n d o l i n e as an a i d t o p u r i f i c a t i o n and c r y s t a l l i z e d from a v e r y s m a l l amount o f e t h e r a t low temperatures  (0° C) a f t e r  seeding.  V i n d o l i n e i s not an easy compound t o c r y s t a l l i z e i n s m a l l quantities.  Two l a r g e c o l o u r l e s s c r y s t a l s  (1.2 mg.) were  o b t a i n e d and counted as t h i n f i l m s d e p o s i t e d on t h r e e t a r e d p l a n c h e t t e s from e t h y l a c e t a t e u n t i l t h e s t a t i s t i c a l e r r o r was l e s s than f o u r p e r c e n t  counting  ( I . 0.286 mg., 149 + 6 counts  per minute p e r m i l l i g r a m (c./m./mg.), I I . 0.310 mg., 165 + 6 c . / m./mg., I I I . 0.279 mg., 147 + 6 c./m./mg.).  The v i n d o l i n e was  r e c o v e r e d from the p l a n c h e t t e s , c r y s t a l l i z e d one more time from e t h e r and counted as b e f o r e showing no d e t e c t a b l e l o s s o f a c t i v i t y . The  s p e c i f i c a c t i v i t y . w a s , 1 5 0 c./m./mg. o r 6.85 x 1 0 c,/m./ 4  mmole a f t e r d i l u t i o n w i t h 5 mg. v i n d o l i n e . Incorporation, 9 x _7 10 millicuries. Rate o f i n c o r p o r a t i o n , 0.005%. Preparative feeding of g e r a n i o l - 2 - C t o Vinca rosea Linn. 1 4  Geraniol-2- C 1 4  (0.272 mg.) s p e c i f i c a c t i v i t y 0.159 mc/mmole,  0.282 mc) was a d m i n i s t e r e d h y d r o p h o n i c a l l y t o one hundred and n i n e t y - t w o c u t t i n g s o f V i n c a r o s e a s o l u b i l i z e d w i t h Tween 20 (8 drops/200 ml.)  as p r e v i o u s l y d e s c r i b e d .  A f t e r seven days  the f r e s h p l a n t m a t e r i a l was e x t r a c t e d f o r a l k a l o i d . were macerated i n f i f t y gram l o t s » 1  (wet weight  The p l a n t s  835 g , ) , e x t r a c t s  \  combined, and volumes s c a l e d a p p r o p r i a t e l y y i e l d i n g 2.14 g. o f crude a l k a l o i d  (0.26%).  Rate o f i n c o r p o r a t i o n : 2.1%.  This  182  m a t e r i a l was  shown t o c o n t a i n some v i n d o l i n e by  chromatography  (Alumina G,  acetate-ether,  1:50,  The  a l k a l o i d was  ml.),  chloroform-ethylacetate,  t r i t u r a t e d w i t h chloroform-benzene shown to c o n t a i n  by t h i n - l a y e r chromatography, and  no was  the eluent 1:20,  200  ml.;  (3:100,  1:10,  200  ml;  1:4,  400  ml.;  1:1,  chromatographed. increasing  (3:100, 400 200  ml.)  a brown band s t a r t e d t o come o f f i n chloroform-benzene E l u t i o n was (1 x 100,  continued w i t h t h i s s o l v e n t  1 x 40,  2 x 200  contain vindoline. 200,  ml.)  E l u t i o n was  t h i c k - l a y e r chromatography. chloroform  (3:1).  continued with chloroform  some v i n d o l i n e .  f u r t h e r p u r i f i e d by The  column was  (1 x  being e l u t e d  f r a c t i o n s which r e p r e s e n t e d 0.59  combined but  until  which were a l l shown by T.L.C. to  t h i s a l k a l o i d a l mixture a l s o c o n t a i n e d  a l o i d were not  ml.;  collecting aliquots  1 x 500ml.) u n t i l very l i t t l e m a t e r i a l was  vindoline containing  100  loaded onto a  e l u t e d from the column while g r a d u a l l y  p o l a r i t y through chloroform-benzene  spray).  vindoline  grade I n e u t r a l alumina column (Woelm alumina) and No m a t e r i a l was  1:1,or e t h y l  w i t h i o d i n e vapour or e e r i e s u l p h a t e  the dark brown r e s i d u e  detectable  thin-layer  but  The g. of a l k -  preparative  washed w i t h methanol-  (1:20), methanol, then a c e t i c acid-methanol(1:10)  g i v i n g e s s e n t i a l l y q u a n t i t a t i v e recovery of a l k a l o i d . c h l o r o f o r m f r a c t i o n s were shown by T.L.C. to c o n t a i n Six t h i c k - l a y e r alumina p l a t e s w i t h f l u o r e s e c e n t were prepared as d e s c r i b e d  The no  post  vindoline.  background  i n the p r e v i o u s s e c t i o n .  A f t e r s t r e a k i n g approximately  100  mg.  of a l k a l o i d s i n a  t h i n band on each p l a t e they were developed i n c h l o r o f o r m -  133  e t h y l acetate  1:1 (8 hours) u n t i l t h e s o l v e n t  end  of the p l a t e  (R  0.3-0.4) were l o c a t e d by r e f e r e n c e  f  fluorescence  (60 cm.).  had reached t h e  The v i n d o l i n e c o n t a i n i n g bands t o t h i n - l a y e r p l a t e s by  and by quenching o f t h e f l u o r e s c e n t  background.  These bands were s c r a p e d from t h e p l a t e s as soon a f t e r d e v e l o p i n g as p o s s i b l e and t h e a l k a l o i d e l u t e d w i t h c h l o r o f o r m  f o l l o w e d by  a b i t o f methanol y i e l d i n g 73.6 mg. o f l i g h t brown s o l i d foam. T h i s m a t e r i a l was counted on a p l a n c h e t t e and  (4, 270 c./m./mg.)  found t o be more a c t i v e t h a n t h e t o t a l a l k a l o i d (3,500  c./m./mg.).  An alumina chromatograro s p o t t e d w i t h t h e t o t a l  a l k a l o i d , t h e s e m i - p u r i f i e d v i n d o l i n e , and a u t h e n t i c was d e v e l o p e d i n c h l o r o f o r m - e t h y l a c e t a t e  (1:1), spots  vindoline revealed  w i t h i o d i n e vapour and t h e p l a t e s p r a y e d w i t h C r a f t i n t SprayA r t F i x a t i v e t o h o l d t h e alumina i n p l a c e w h i l e an a u t o r a d i o g r a p h was exposed. and  A f t e r 30 days t h e a u t o r a d i o g r a p h was developed  showed a s e r i e s o f s p o t s c o r r e s p o n d i n g t o t h e a l k a l o i d  s p o t s made v i s i b l e w i t h i o d i n e and a s i n g l e spot c o r r e s p o n d i n g to the v i n d o l i n e f r a c t i o n . f r a c t i o n contained  In s p i t e of t h i s the v i n d o l i n e  l e s s than f i f t y percent  v i n d o l i n e as r e v e a l -  9^  ed by a n a l y s i s o f t h e u l t r a v i o l e t spectrum: T*  £  *ax.  8 0 0 0 j  6 0 0  °-  V i n d o l i n e  "  \ax.  7660, 5540 ( t y p i c a l i n d o l e chromophore). atography i n e t h y l a c e t a t e - e t h e r  2 5 2  mSiX  293, 247 s h .  > 304 m^, Thin-layer  £  m  a  x  chrom-  (1:50) demonstrated t h a t t h e  m a t e r i a l was a m i x t u r e o f t h r e e major components (R^ 0.5, 0.25 ( v i n d o l i n e ) , and 0.1) and s e v e r a l f l u o r e s c e n t V i n d o l i n e i s not f l u o r e s c e n t .  impurities.  The m i x t u r e was s e p a r a t e d by  184  preparative G  t h i c k - l a y e r (0.5 mm.)  (20 x 20 cm.)  plate.  quenching of background f o r two  fluorescent  The  chromatography on an alumina  vindoline f r a c t i o n located  fluorescence  i m p u r i t i e s not  s i n g l e spot m a t e r i a l  i n two  (20 mg.)  detectable  good s o l v e n t  s e v e r a l o t h e r s t r i e d which d i d not w i t h the o r i g i n a l mixtures.  The  was  now,  systems as w e l l  give as impressive  spectrum a l s o matched (ethanol) : A  c./m./mg., 20 mg.)  The  5 5  252,  303  i  at a c o u n t i n g e f f i c i e n c y of 39%  t o an i n c o r p o r a t i o n  of 3.11  mux,  corresponded  K  x 10  mc  or a r a t e of  incorporation (  As v i n d o l i n e i s d i f f i c u l t  The  to c r y s t a l l i z e as the  free a l k a l o i d  c r y s t a l l i z e d t o constant a c t i v i t y as i t s d i h y d r o c h l o r i d e .  v i n d o l i n e was  d i s s o l v e d i n ether,  f i l t e r e d , and  c h l o r i d e blown over the top of the s o l u t i o n . a t i o n the s o l u t i o n was  were then d e c a n t e d and amount of c o l d e t h e r . methanol  hydrogen  After centrifug-  again t r e a t e d w i t h hydrogen c h l o r i d e  u n t i l no more p r e c i p i t a t e was  obtained.  The  mother l i q u o r s  the white p r e c i p i t a t e washed w i t h a The  dihydrochloride  (2 drops) and warm e t h y l a c e t a t e  became t u r b i d .  C r y s t a l l i z a t i o n occurred  seed c r y s t a l .  The  on two  7600,  a  (1,350  of 0.011%.  i t was  colour  matched  mix ; €,  specific activity _  separations  ultraviolet  m£ix o  The  as  c h a r a c t e r i s t i c crimson  vindoline.  5500 ( t y p i c a l i n d o l i n e n e ) .  except  by other means, a  w i t h 1% e e r i e s u l p h a t e - 3 5 % s u l p h u r i c a c i d spray now p e r f e c t l y that of a u t h e n t i c  by  dihydrochloride  aluminum p l a n c h e t t e s  was  was  small  dissolved i n  added u n t i l the  solution  on s c r a t c h i n g w i t h a counted a f t e r  from c h l o r o f o r m .  deposition  Average s p e c i f i c  185  a c t i v i t y : 940 c./m./mg., 5.0 x 10° c./m./mmole.  The d i h y d r o -  c h l o r i d e was c r y s t a l l i z e d twice more, m.p. 150-152, from acetate-methanol  and again counted.  Average s p e c i f i c  960 c./m./mg., 5.1 x 1 0 c./m./mmole. 5  chloroform  (1 N) .  activity:  The f r e e a l k a l o i d was  regenerated by shaking a c h l o r o f o r m s o l u t i o n aqueous ammonia  ethyl  (4 ml.) with  The acjueous l a y e r was then washed w i t h  (1 ml.) and the o r g a n i c l a y e r d r i e d over anhydrous  magnesium s u l p h a t e , f i l t e r e d and taken t o dryness under a stream of dry n i t r o g e n , removing r e s i d u a l s o l v e n t i_n vacuo. vindoline  (6.27 mg.) now showed no f l u o r e s c e n t i m p u r i t i e s on  t h i n - l a y e r chromatography and had a s p e c i f i c 10  The r e c o v e r e d  c./m./mmole., 1120 d/'/m./mg. .  a c t i v i t y o f 5.1 x  Constant  a c t i v i t y had been  achieved w i t h i n the l i m i t s o f c o u n t i n g e r r o r which can be l a r g e if  great c a r e i s not taken weighing  samples as a uniform f i l m . w i t h i n three percent x 10  samples and d e p o s i t i n g  With care the count  (500 c./m.).  c./m./mmole corresponds  A specific  can be reproduced  a c t i v i t y o f 5.1  at a 39% c o u n t i n g e f f i c i e n c y t o  6 1.3 x 10 d i s i n t e g r a t i o n s p e r minute. As one m i l l i c u r i e 7 corresponds t o 3.700 x 10 d i s i n t e g r a t i o n s per second (2.22 x Q  10  d./m.) the s p e c i f i c  a c t i v i t y o f v i n d o l i n e can be expressed  -4 as 5.9 x 10 mc./mmole. 14 2-  As the s p e c i f i c  a c t i v i t y of g e r a n i o l -  C f e d t o Vinca rosea was 0.159 mc/mmole the s p e c i f i c  i n c o r p o r a t i o n o f the g e r a n i o l - n e r o l mixture 0.37%.  into vindoline i s  186  Degradation The  of V i n d o l i n e  a c t i v e v i n d o l i n e (6.27 mg„) o b t a i n e d by e x t r a c t i o n of  V i n c a rosea was crops  (first  d i l u t e d by r e c r y s t a l l i z a t i o n from e t h e r i n two  crop 89.4  mg)  with  Eli  L i l y Company.  mg.,  second crop 19.8  a u t h e n t i c v i n d o l i n e (111.6 mg.)  mg.,  total  o b t a i n e d from  T h i s m a t e r i a l and i t s d e g r a d a t i o n  109.2 the  products  81 were counted  i n the toluene s c i n t i l l a t i o n mixture  Nuclear Chicago Model 180040 L i q u i d S c i n t i l l a t i o n  using a Counter.  The c o u n t i n g e f f i c i e n c y was  e s t a b l i s h e d by the channel  method.  (a p l o t of c o u n t i n g e f f i c i e n c y  A quenching  curve  versus the r a t i o of c o u n t i n g r a t e i n two energy was  determined  u s i n g a s e r i e s of s i x acetone  channels(B/A)  quenched samples  w i t h a c t i v i t y of 210,200 d i s i n t e g r a t i o n s per minute. c o u n t i n g r a t e s i n channel A were r e s p e c t i v e l y  The  The  164,784(78.5%),  147,893(70.3%), 115,112(54.8%), 85,926(40.9%), 66,010 and 33,742(16.0%) counts per minute.  ratio  (31.4%),  counting rates i n  channel B were at the same time 55,787(B/A = 0.339), 74,595 (B/A = 0.506),  84,244(B/A = 0.731),  74,218(B/A = 0.86),  60,840XB/A = 0.922), 32,902(B/A = 0.975) counts per minute. C a l c u l a t i o n of the standard d e v i a t i o n i n the c o u n t i n g r a t e s then i n the r a t i o s  (Appendix  I) e s t a b l i s h e d that the standard  d e v i a t i o n s i n the r a t i o s were l e s s than 1%.  One  s h o u l d be a b l e  to use the curve p l o t t e d from the above data t o e s t a b l i s h i n g e f f i c i e n c y i n a r a d i o a c t i v e sample w i t h i n 1%,  subject to  the s t a t i s t i c a l e r r o r i n c o u n t i n g and i n the r a t i o B/A sample.  count-  f o r that  137  Oxidation  Conditions'' '' 3  '  1 J  A s e r i e s o f t h i r t e e n e x p e r i m e n t s were r e q u i r e d t o e s t a b l i s h the o x i d a t i o n c o n d i t i o n s under which a s u i t a b l e r a t i o o f p r o p i o n i c and a c e t i c a c i d s c o u l d be o b t a i n e d bromophenacyl  e s t e r s of t h e s e a c i d s c o u l d be p u r i f i e d and t h e i r  s p e c i f i c a c t i v i t y determined.  In t h e s e e x p e r i m e n t s t h r e e  d i f f e r e n t d e s i g n s o f a p p a r a t u s were used. modified  so t h a t the p-  Kjeldahl apparatus  8 4  One of t h e s e , a  involved bubbling  steam t h r o u g h  a s o l u t i o n o f v i n d o l i n e i n the o x i d i z i n g m i x t u r e .  The  second  c o n s i s t e d of an o x i d a t i o n chamber w i t h a vapour t r a p then a s p l a s h t r a p t o p r e v e n t n o n - v o l a t i l e a c i d s b e i n g c a r r i e d from the o x i d i z i n g s o l u t i o n during d i s t i l l a t i o n . for  A s i d e arm was  provided  a d d i t i o n o f water t o compensate f o r water removed as steam.  The t h i r d a p p a r a t u s was used i n the e v e n t .  I t was  from a f i f t y m i l l i l i t e r round bottom f l a s k w i t h B neck, a C l a i s e n head m o d i f i e d  1 4  constructed Quickfit  so t h a t the d i s t i l l a t i o n  flask  c o u l d be i n c l i n e d 30° from the normal v e r t i c a l p o s i t i o n , a d r o p p i n g f u n n e l , a L i e b i g condenser and f i n a l l y cylinder for collecting d i s t i l l a t e .  a graduated  Carborundum b o i l i n g s t o n e s  were used t o p r e v e n t bumping and i t was found t h a t the bent C l a i s e n head p r o v i d e d  remarkable p r o t e c t i o n against  The f i r s t seven o x i d a t i o n s were c a r r i e d out on 5 mg. the next f i v e on 10 mg.  splash. of v i n d o l i n e ,  and the t h i r t e e n t h w i t h 100 mg..  The  changes i n o x i d a t i o n c o n d i t i o n s i n v o l v e d changes i n c o n c e n t r a t i o n of chromium t r i o x i d e , c o n c e n t r a t i o n of  distillation.  o f s u l p h u r i c a c i d , and r a t e  183  A t y p i c a l o x i d a t i o n procedure i s as f o l l o w s :  Vindoline  (10 mg.) was washed i n t o t h e 50 m l . round bottom f l a s k w i t h d i s t i l l e d water marked w i t h  (5 m l . ) .  The 3 m l . and 5 m l . l e v e l s were  a grease p e n c i l on t h e f l a s k .  One m i l l i l i t e r o f  the o x i d i z i n g m i x t u r e (4 N chromic a c i d - s u l p h u r i c  acid-water  4:1:25) was added and t h e r e s u l t i n g m i x t u r e heated i m m e d i a t e l y to b o i l i n g w i t h a micro burner.  The chromic a c i d s o l u t i o n  (4 N)  was p r e p a r e d by d i s s o l v i n g chromium t r i o x i d e (67 g.) i n d i s t i l l e d water  (500 m l . ) , a l l o w i n g  i t t o stand overnight, f i l t e r i n g  through a s i n t e r e d glass f i l t e r , slowly sulphuric volatile  acid  (125 m l . ) , then d i s t i l l i n g t o remove steam  impurities.  when r e q u i r e d  adding concentrated  Distillation  was c o n t i n u e d a d d i n g water  from t h e d r o p p i n g f u n n e l  3-5 m l . o f l i q u i d  i n the f l a s k .  so t h a t t h e r e was always  Twenty-five m i l l i l i t e r s of  d i s t i l l a t e was c o l l e c t e d i n 20 minutes and then a f u r t h e r 25 m l . aliquot collected. point  Each a l i q u o t was brought t o t h e b o i l i n g  t o degas i t , a drop o f p h e n o l p h t h a l e i n s o l u t i o n added and  titrated  w i t h d i l u t e sodium h y d r o x i d e (0.01015 N).  A few  c r y s t a l s o f barium c h l o r i d e added t o a one ml. a l i q u o t b e f o r e titration  established  t h e absence o f s p l a s h i n g .  the f i r s t a l i q u o t was reduced i i i vacuo a t a b a t h of 55° C t o 0.5 m l . .  The volume o f temperature  T h i s s o l u t i o n was p a s s e d t h r o u g h a  column o f Dowex 50 ( a c i d form) (0.7 x 5 cm.) t o r e g e n e r a t e t h e free a c i d .  7 4  A 25% ethylamine s o l u t i o n  (0.2 ml.) was added t o  the e l u a n t and then t h e volume reduced i r i vacuo t o about 1 drop. The v o l a t i l e  a c i d m i x t u r e was t h e n a n a l y z e d by paper  chromatog-  189  raphy f o r a c e t i c and p r o p i o n i c a c i d s .  Whatman #3 a c i d washed  paper f o r chromatography was c u t i n s t r i p s  (6 i n c h e s wide) and  one end s e r r a t e d so s o l v e n t c o u l d d r i p from t h e paper. ethylamine  The  s a l t s o f t h e o x i d a t i o n m i x t u r e and a c e t i c , p r o p i o n i c  and f o r m i c s a l t s were s p o t t e d on t h e paper 6.5 i n c h e s from t h e end.  The paper was then f o l d e d f o r descending  chromatography.  Water s a t u r a t e d b u t a n o l s e r v e d as t h e m o b i l e phase w h i l e t h e chromatography tank was e q u i l i b r a t e d w i t h water s a t u r a t e d b u t a n o l , 0.025 N i n e t h y l a m i n e .  Papers were p l a c e d i n t h e  tank f o r one h a l f hour b e f o r e a d d i t i o n o f t h e m o b i l e phase, then developed  f o r 8 hours.  These s a l t s have a r e p r o d u c i b l e R^  ( p r o p i o n a t e 0.4, a c e t a t e 0.2, formate 0,15) when R from t h e c e n t e r o f g r a v i t y o f t h e s p o t .  f  i s measured  The f r o n t o f t h e spot  i s c o n c e n t r a t i o n dependent t o t h e e x t e n t t h a t i t was p o s s i b l e t o e s t i m a t e c o n c e n t r a t i o n from p o s i t i o n o f t h e f r o n t .  A one  t o one r a t i o o f a c e t a t e and p r o p i o n a t e was r e s o l v e d but o v e r l a p became s e r i o u s as t h e r a t i o o f p r o p i o n a t e t o a c e t a t e dropped. As t h e r e were two s o u r c e s o f a c e t a t e i n t h e m o l e c u l e ,  the a c e t y l  group and t h e e t h y l s i d e c h a i n , a r a t i o o f 1:3 f o r t h e e t h y l s i d e c h a i n r e s u l t s i n a 1:7 p r a c t i c a l r a t i o when h y d r o l y s i s i s considered.  P r o p i o n a t e was d e t e c t a b l e o n l y w i t h great  when t h e r a t i o became more than 1:20. d e s c r i b e d no p r o p i o n a t e was d e t e c t a b l e .  difficulty  I n t h e experiment The d e t e c t i o n o f  p r o p i o n a t e and a c e t a t e on paper i n t h e presence o f e t h y l a m i n e was not t o o easy but a s a t i s f a c t o r y m e t h o d  85  involved drying  the paper o v e r n i g h t o r i n a stream o f warm a i r from a h a i r  190  d r y e r then s p r a y i n g w i t h bromophenolblue  s o l u t i o n f o l l o w e d by  aqueous copper s u l p h a t e (2%) s o l u t i o n a f t e r d r y i n g . s p r a y gave b l u e s p o t s on a mauve background.  This  The bromophenol  b l u e s o l u t i o n was p r e p a r e d by d i s s o l v i n g bromophenolblue i n e t h a n o l (300 ml.) then a d d i n g 3 0 % aqueous sodium  (300 mg.)  hydroxide  (0.25 ml.) t o change t h e c o l o u r o f t h e s o l u t i o n from r e d t o b l u e .  O x i d a t i o n o f v i n d o l i n e - C (13) 1 4  V i n d o l i n e - C (93.6 mg., 0.205 mmoles) w i t h a s p e c i f i c 4 1 4  a c t i v i t y o f 5.54 x 10 chromium t r i o x i d e  d./m./mmole was added t o 3 0 % aqueous  (5 ml.) i n t h e 50 m l , round bottom f l a s k w i t h  m o d i f i e d C l a i s e n head a t room t e m p e r a t u r e . The o x i d a t i o n m i x t u r e (Lemieux)'° had j u s t p r e v i o u s l y been boiled  and 30 m l , o f water d i s t i l l e d from t h e reagent t o remove  any v o l a t i l e a c i d i m p u r i t i e s (blank t i t e r 0,25 m l . ) .  The reagent  was v e r y c a r e f u l l y heated as r a p i d e v o l u t i o n o f carbon d i o x i d e gave r i s e t o c o n s i d e r a b l e f r o t h i n g i n t h e f i r s t  few minutes.  The m i x t u r e was t h e n heated s t r o n g l y and f o u r 60 m l . f r a c t i o n s of  steam v o l a t i l e d i s t i l l a t e c o l l e c t e d b e i n g c a r e f u l t o keep t h e  volume o f t h e reagent m i x t u r e a t about 5 ml,. was d i s t i l l e d i n about f i f t e e n minutes.  Each f r a c t i o n  After bringing t o the  b o i l i n g p o i n t and c o o l i n g each f r a c t i o n was t i t r a t e d w i t h c a r b o n a t e f r e e l i t h i u m h y d r o x i d e (0.0128 N).  The c a r b o n a t e  f r e e base was p r e p a r e d by d i s s o l v i n g l i t h i u m h y d r o x i d e (7 g.) i n b o i l e d out d i s t i l l e d water  (40 ml.) and a l l o w e d t o s t a n d  u n d i s t u r b e d under a s o d a l i m e guard tube f o r two days w h i l e t h e  191  l i t h i u m c a r b o n a t e s e t t l e d out, s o l u t i o n was  A p o r t i o n (1.4 ml.)  d i l u t e d w i t h d i s t i l l e d water (500 ml.)  s o l u t i o n s t a n d a r d i z e d a g a i n s t 0.01 ml.)  2.45  ml.;  N h y d r o c h l o r i c a c i d (5.000  ml., 0.0313 mmoles; f r a c t i o n I I I 1.75  0.404 mmoles.  Reagent  m l . , 0.0224 mmoles;  ml., 0.0121 mmoles; t o t a l steam v o l a t i l e a c i d 1.97  e q u i v a l e n t s of v o l a t i l e a c i d were o b t a i n e d  from the r a d i o a c t i v e v i n d o l i n e . e v a p o r a t e d i n vacuo (55°  The  aqueous s o l u t i o n s were  C) t o about 2 ml. then t r a n s f e r r e d t o  a 25 ml. round bottom f l a s k .  A few drops of s o l u t i o n were  t r a n s f e r r e d t o a s c i n t i l l a t i o n c o u n t i n g v i a l and removed under a stream of n i t r o g e n . (1.67 mg.)  the  f r a c t i o n I 26.42 ml., 0.333 mmoles; f r a c t i o n I I  f r a c t i o n IV 0.93  salt  and  u s i n g a pH meter t o determine the e n d - p o i n t .  b l a n k 0.25  of t h i s  was  T h i s sample of  t o s e r v e as i n s u r a n c e  i s o l a t i o n of the e s t e r s .  I t was  solvent  i n case of  lithium unsuccessful  never counted.  P r e p a r a t i o n of p-bromophenacyl e s t e r s The  volume of s o l u t i o n was  f u r t h e r reduced (0.3 ml.)  the o r i g i n a l f l a s k r i n s e d w i t h a few m i l l i l i t e r s of b r i n g i n g the volume up t o 10 ml.. (125 mg.,  0.48  The  ethanol  p-Bromophenacylbromide  mmoles, 10% e x c e s s ) was  r e f l u x e d f o r 40 m i n u t e s .  added and the s o l u t i o n  r e s i d u e was  separated  by p r e p -  a r a t i v e t h i n l a y e r chromatography on a s i l i c a g e l G p l a t e mm.  x 20 x 20 cm.)  chloroform.  w i t h f l u o r e s c e n t background d e v e l o p i n g  S i x bands (A-H)  and  were s c r a p e d  from the p l a t e  oo eluted with ether i n a Craig f i l t r a t i o n apparatus.'  5  The  (0.5 with and  192  e l u t e d m a t e r i a l was t h e n a n a l y z e d by t h i n - l a y e r chromatography. Band A (Rf 0.0 - 0.12) c o n t a i n e d  a s m a l l amount o f an un-  i d e n t i f i e d i m p u r i t y which c o u l d have been p - b r o m o p h e n a c y l a l c o h o l . Band B (R^ 0.12 - 0.25, 40.33 mg) c o n s i s t e d o f p-bromophenacylacetate  ( R 0.37) w i t h two minor i m p u r i t i e s a t R f  (propionate). band.  0.3 and 0.53  Band C ( R 0.25 - 0.26) was a narrow y e l l o w f  Band D a t Rf 0.26 - 0.30 (4.01 mg.) was p-bromophenacyl-  propionate acetate  (Rf 0.53 on a t h i n p l a t e ) w i t h a s m a l l amount o f t h e  (Rf 0.37 on a t h i n p l a t e ) .  Band E was b l a n k .  (Rf 0.35 - 0.47) was p-bromophenacyl bromide. 0.54)  f  Band F  Band G ( R 0.47 f  was a s m a l l amount o f y e l l o w u n i d e n t i f i e d i m p u r i t y  band H (Rf 0.62) was a f l u o r e s c e n t i m p u r i t y . band was rechromatographed on a s t a n d a r d plate  while  The p r o p i o n a t e  s i l i c a gel thin layer  (0.1 mm. x 5 cm. x 20 cm.) and a f t e r e x t r a c t i o n w i t h  ether  p r o v e d t o be s i n g l e spot m a t e r i a l on t h i n - l a y e r chromatography. T h i s sample (0.4 mg.) was r e c r y s t a l l i z e d t h r e e t i m e s from l i g h t petroleum ether t o constant 86 m.p. 61-62° C mixture  m e l t i n g p o i n t , 59 - 60° C; l i t .  and counted i n t h e t o l u e n e  (2.01 mg., 275 + 4.5 c./m.).  scintillation  A channel r a t i o  0.607 c o r r e s p o n d s t o a c o u n t i n g e f f i c i e n c y o f 64%.  (B/A) o f  This implies  a s p e c i f i c a c t i v i t y o f 5.50 x 1 0 d./m./mmole which i m p l i e s t h a t 4  99.3  + 2% o f t h e a c t i v i t y o f t h e v i n d o l i n e was l o c a t e d i n t h e  propionic acid residue.  S i m i l a r l y t h e p-bromophenacylacetate  f r a c t i o n was rechromatographed and shown by t h i n - l a y e r chromatography t o c o n t a i n no p r o p i o n a t e . amount o f t h e slow r u n n i n g  impurity.  I t did s t i l l r e t a i n a small An attempt t o remove t h i s  193  i m p u r i t y by c r y s t a l l i z a t i o n  was not c o m p l e t e l y s u c c e s s f u l s o t h e  a c e t a t e was chromatographed one more time t h e n t h r e e t i m e s from l i g h t p e t r o l e u m e t h e r o 86 (lit.  84-85  C).  crystallized  (30-60°) m.p. 83-84° C  T h i s compound was counted as b e f o r e (5 mg.)  but no a c t i v i t y was d e t e c t a b l e :  background 2 4 + 1 c./m.,  p - b r o m o p h e n a c y l a c e t a t e 24 + 0.5 c./m..  This i n d i c a t e d that  none o f t h e a c t i v i t y o f v i n d o l i n e was l o c a t e d i n e i t h e r t h e e t h y l side chain or the a c e t y l function of vindoline(13). C o u n t i n g o f N-methyl and O-methyl groups The  determination  o f a l k o x y l groups i n the form o f e t h e r s  or e s t e r s i s c l a s s i c a l l y a c c o m p l i s h e d by t r e a t m e n t o f t h e o r g a n i c 77  compound w i t h b o i l i n g  hydriodic acid.  The l i b e r a t e d i o d i d e s  are f l u s h e d t h r o u g h a condenser and t r a p i n t o a s o l u t i o n o f bromine i n a c e t i c a c i d t h e n e s t i m a t e d i o d o m e t r i c a l l y . determination  For  o f t h e N - a l k y l group p y r o l y s i s o f t h e q u a t e r n a r y  ammonium compound i s u s u a l l y r e q u i r e d a f t e r • l i q u i d d i s t i l l e d from the r e s i d u e .  An a l k o x y l d e t e r m i n a t i o n  l y c a r r i e d out by Mr. P.Borda, t h e a n a l y s t In two d e t e r m i n a t i o n s of sodium t h i o s u l p h a t e  has been  vindoline  for this  was k i n d department.  (10.871 mg.) r e q u i r e d 42.24 m l .  (0.009678 N) and 6.146 mg. r e q u i r e d 12.24  ml.  o f sodium t h i o s u l p h a t e  2.87  and 2.86 e q u i v a l e n t s  (0.01967 N).  These f i g u r e s i m p l i e d  o f m e t h y l i o d i d e f o r one e q u i v a l e n t  of v i n d o l i n e w h i c h i n d i c a t e d t h a t t h e N-methyl, the m e t h o x y l and  the c a r b o m e t h o x y l m e t h y l s were a l l b e i n g v o l a t i l i z e d as  methyl i o d i d e .  Active vindoline  (4.197 mg, s p e c i f i c  activity  194  5.54  x 10  d./m./mmole) was t r e a t e d w i t h b o i l i n g h y d r i o d i c a c i d  and t h e methyl i o d i d e produced b u b b l e d , u s i n g t h e a n a l y t i c a l apparatus,  d i r e c t l y i n t o t h e s c i n t i l l a t i o n m i x t u r e (5 m l . ) .  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