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UBC Theses and Dissertations

Studies related to the synthesis of bisindole alkaloids of the vinblastine-Vincristine family Balsevich, J. John 1978

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STUDIES RELATED TO THE SYNTHESIS OF BISINDOLE ALKALOIDS OF THE VINBLASTINE-VINCRISTINE FAMILY by J . JOHN BALSEVICH B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1972 M.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Dept. o f C h e m i s t r y , Univ. o f B r i t i s h Columbia) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 19 7 8 © J . John B a l s e v i c h , 1978 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my written permission. Department of C h e m i s t r y The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date Ort. 4. 1978 • 6 A B S T R A C T The work done towards t h i s t h e s i s i s o u t l i n e d i n f o u r s e c t i o n s . The f i r s t s e c t i o n i n v o l v e d e v a l u a t i o n of the ' c h l o r o -i n d o l e n i n e c o u p l i n g ' of velbanamine d e r i v a t i v e s w i t h v i n d o l i n e (10). A c c o r d i n g l y , 18S-carbomethoxycleavamine ( 19 )/ chosen as s t a r t i n g m a t e r i a l , was c o n v e r t e d to 18S-carbomethoxy-3R-hydroxy-velbanamine c h l o r o i n d o l e n i n e (55) i n f i v e s t e o s . Attempted couu l i n g of t h i s d e r i v a t i v e w i t h v i n d o l i n e i n methanol c o n t a i n i n g hydrogen c h l o r i d e was u n s u c c e s s f u l . T h e r e f o r e , s t u d i e s were undertaken t o a s c e r t a i n a reason f o r the l a c k o f c o u p l i n g . C onsequently, i n t e r n a l q u a t e r n i z a t i o n of the t e r t i a r y n i t r o g e n was e s t a b l i s h e d as a c o m p e t i t i v e s i d e r e a c t i o n . Based on these r e s u l t s the c o n d i t i o n s o f the c o u p l i n g r e a c t i o n were m o d i f i e d t o e l i m i n a t e t h i s s i d e r e a c t i o n . C o u p l i n g o f v i n d o l i n e and 18S-carbomethoxy-3R-hydroxyvelbanamine c h l o r o i n d o l e n i n e was thus a t t a i n e d , however, the o n l y d i m e r i c p r o d u c t o b t a i n e d was 1 8 ' — e o i v i n c a d i o l i n e (74). The second s e c t i o n i n v o l v e d the study o f the f u n c t i o n a l -i z a t i o n o f 3 ' , 4 ' - a n h y d r o v i n b l a s t i n e (26) u s i n g a e r i a l o r t - b u t y l hydroperoxide o x i d a t i o n . Under the appropriate conditions the n a t u r a l l y o ccurring a l k a l o i d s l eurosine (3) and Catharine (76) could be obtained. A study was c a r r i e d out i n regards to the mechanism of the o x i d a t i o n with the r e s u l t being i m p l i c a t i o n of a r a d i c a l mechanism. The t h i r d s e c t i o n involved the study of the r e a c t i o n of 3',4'-anhydrovinblastine (and r e l a t e d d e r i v a t i v e s ) with potassium permanganate. From t h i s r e a c t i o n was obtained as the major product 3R-hydroxyvinamidine (8^3)—a d e r i v a t i v e of the n a t u r a l l y occurring a l k a l o i d vinamidine (82). Attempted deoxygenation of 3R-hydroxyvinamidine was unsuccessful. Therefore, vinamidine was prepared by potassium permanganate o x i d a t i o n of 4'-deoxy-l e u r o s i d i n e (93). The fourth s e c t i o n r e l a t e s to the preparation of v i n -b l a s t i n e - v i n c r i s t i n e analogs (for the purpose of e l u c i d a t i n g s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s ) . Thus, iodine/sodium bicarbon-ate o x i d a t i o n of leurosine (3), v i n b l a s t i n e (1), and l e u r o s i d i n e (JU provided access to the corresponding 19'-oxo d e r i v a t i v e s i n f a i r to moderate y i e l d s . Oxidation of 19'-oxoleurosine (91) with Jones reagent at low temperature afforded 19 1,22-dioxoleurosine (98). A l t e r n a t i v e l y 19 1,22-oxoleurosine could be obtained from leurosine i n b e t t e r y i e l d by f i r s t u t i l i z i n g Jones o x i d a t i o n to a f f o r d 22-oxoleurosine and then o x i d a t i o n of t h i s d e r i v a t i v e with iodine/sodium bicarbonate. t - B u t y l hydroperoxide o x i d a t i o n of 6 , 7-dihydro-3 1 , 4 '-anhydrovinblastine (100) afforded the novel analog 6,7-dihydroleurosine (101) , Jones o x i d a t i o n of which y i e l d e d 22-oxo-6 , 7-dihydroleurosine ( 1J02) . - i v -TABLE OF CONTENTS Page T i t l e page i A b s t r a c t i i T a b l e o f Contents i v L i s t o f F i g u r e s v L i s t o f T a b l e s i x Acknowledgements x I n t r o d u c t i o n 1 1.1 G e n e r a l Background 1 1.2 S t r u c t u r e E l u c i d a t i o n of V i n b l a s t i n e , V i n c r i s t i n e , L e u r o s i n e , and L e u r o s i -d i n e 6 1.3 S t u d i e s Aimed a t the S y n t h e s i s of the 'Dimeric' Catharanthus A l k a l o i d s — the ' C h l o r o i n d o l e n i n e Approach 1 14 1.4 S t u d i e s Aimed a t the S y n t h e s i s o f the D i m e r i c Catharanthus A l k a l o i d s the ' B i o g e n e t i c Approach' 20 1.5 V i n c r i s t i n e D e r i v a t i v e s 2 3 1.6 Scope of C u r r e n t Research 25 D i s c u s s i o n 28 2.1 F u r t h e r E v a l u a t i o n o f the C h l o r o i n d o l e -n i n e Approach 2 8 2.2 Chemistry of 3 ' , 4 ' - A n h y d r o v i n b l a s t i n e — S y n t h e s i s of L e u r o s i n e and C a t h a r i n e . 83 2.3 Chemistry of 3 1 , 4 ' - A n h y d r o v i n b l a s t i n e — S y n t h e s i s of V i n a m i d i n e ( C a t h a r i n i n e ) (82) and R e l a t e d D e r i v a t i v e s 9 3 2.4 P r e p a r a t i o n o f V i n b l a s t i n e - V i n c r i s t i n e Analogs 114 E x p e r i m e n t a l 125 B i b l i o g r a p h y 165 - v -LIST OF FIGURES Figure Page 1. Structure of some c l i n i c a l l y used a l k y l a t -ing agents 2 2. Some antimetabolite cancer drugs and t h e i r metabolite analogs 3 3. Structures of some c y t o t o x i c a n t i b i o t i c s .. 4 4. A s i g n i f i c a n t pathway f o r the fragmentation of le u r o s i n e i n the mass spectrometer 9 5. Outline of Kutney and Worth's s t r u c t u r e -proof of leurosine 11 6. P o t i e r and Langlois' structure-proof of l e u r o s i d i n e 13 7. Mechanism f o r the formation of 18S-carbo-methoxycleavamine from catharanthine 16 8. General o u t l i n e of the 'chloroindolenine approach 1 17 9. Condensation of the chloroindolenine of 4S-ethyl-18Scarbomethoxydihydrocleavamine with v i n d o l i n e 19 10. 'Biogenetic-type' coupling of catharanthine-N, -oxide (24) with v i n d o l i n e 22 b <— 11. Some 'dimeric' catharanthus a l k a l o i d s 26 12. P o s s i b l e mchanism f o r the formation of 4S,--18R-ether 37 from v i n b l a s t i n e 29 13. "*"Hmr spectrum of 16 ,18S-dicarbomethoxy-cleavamine (39) 33 14. Comparison of the u l t r a v i o l e t spectra of 18S-carbomethoxycleavamine (A)and 16,18S— dicarbomethoxycleavamine (B) 34 15. S p a t i a l r e l a t i o n s h i p of the 18-hydrogen to the N b e l e c t r o n p a i r i n 18-carbomethoxy-cleavamine 3 4 16. "^ Hmr spectrum of 16 ,18S-dicarbomethoxy— 3R,4S-epoxydihydrocleavamine (40) 36 - v i -F i g u r e Page 17. Sequence used f o r the d e t e r m i n a t i o n of the a b s o l u t e c o n f i g u r a t i o n of 16,18S-dicarbo-methoxy-3R,4S-epoxydihydrocleavamine (40) .. 39 18. "^Hmr spectrum o f f o r m y l ketone 45 42 19. I n f r a r e d spectrum o f f o r m y l ketone 45 43 20. I n f r a r e d spectrum of a l c o h o l 47 45 21. Mass s p e c t r a l f r a g m e n t a t i o n p a t t e r n of 16,-18S-dicarbomethoxy-3R-hydroxyvelbanamine (51) 49 22. "*"Hmr spectrum of 16 ,18S-dicarbomethoxy— 3R-hydroxyvelbanamine (51) 50 23. "^Hmr spectrum of 16,18S-dicarbomethoxy— 3S,4R-epoxydihydrocleavamine (52) 51 24. Mass s p e c t r a l f r a g m e n t a t i o n p a t t e r n o f 16,-18S-dicarbomethoxy-3S,4R-epoxydihydrocleav-amine (52) . . 52 25. U l t r a v i o l e t spectrum o f the c h l o r o i n d o l e n -i n e 55 55 26. Proposed g e n e r a l mechanism of c h l o r o i n d o l e n -i n e ' d i m e r i z a t i o n 1 56 27. Comparison of the u l t r a v i o l e t s p e c t r a o f a t y p i c a l i n d o l e d e r i v a t i v e ( A ) , d i h y d r o i n d o l e d e r i v a t i v e (B) , and 'dimer' (C) 62 28. I n f r a r e d spectrum of 16,18S-dicarbomethoxy— 3R,4S-epoxy-19-oxodihydrocleavamine (62) ... 64 13 29. Cmr spectrum of 16,18S-dicarbomethoxy-3R,-4S-epoxy-19-oxodihydrocleavamine (62) 65 13 30. Comparison o f the Cmr c h e m i c a l s h i f t s (<0 of hexahydroazepine (A) and c a p r o l a c t a m (B). 66 31. ^Hmr spectrum of 16,18S-dicarbomethoxy-3R,-4S-epoxy-19-oxodihydrocleavamine (62) 67 32. ^Hmr spectrum of 18S-carbomethoxy-3R,4S— epoxy-19-oxodihydrocleavamine (66) 69 - v i i -F i g u r e Page 33. O u t l i n e o f t h e r e a c t i o n o f 1 8 S - c a r b o m e t h o x y — 3 R , 4 S - e p o x y - 1 9 - o x o d i h y d r o c l e a v a m i n e (66) w i t h 1 - c h l o r o b e n y o t r i a z o l e 7". 70 34. ^Hmr s p e c t r u m o f 68 7 2 35. "4lmr s p e c t r u m o f 69 7 3 36. 1Hmr s p e c t r u m o f 1 8 1 - e p i - 1 9 1 - o x o l e u r o s i n e (70) 75 37. P r o p o s e d mechanism f o r c o u p l i n g o f c h l o r o i n -d o l e n i n e 61 w i t h v i n d o l i n e 77 38. "'"Hmr s p e c t r u m o f 1 8 - e p i l e u r o s i n e (73) 78 39. "'"Hmr s p e c t r u m o f 1 8 ' - e p i v i n c a d i o l i n e (74) ... 80 40. "^Hmr s p e c t r u m o f C a t h a r i n e (sample was ob-t a i n e d f r o m t h e - E l i L i l l y L a b o r a t o r i e s , I n d i a n a p o l i s ) 86 41. "^Hmr s p e c t r u m o f s y n t h e t i c C a t h a r i n e 87 42. P o s s i b l e mechanism f o r t h e f o r m a t i o n o f l e u r o s i n e f r o m 3 1 , 4 ' - a n h y d r o v i n b l a s t i n e 91 43. P o s s i b l e mechanism f o r t h e f o r m a t i o n o f C a t h a r i n e f r o m l e u r o s i n e 9 2 44. I n f r a r e d s p e c t r u m o f 3 R - h y d r o x y v i n a m i d i n e (83) 95 45. "^Hmr s p e c t r u m o f 3 R - h y d r o x y v i n a m i d i n e (83) .. 96 46. ^Hmr s p e c t r u m o f 3 R - a c e t o x y v i n a m i d i n e (84) .. 98 47. "^ Hmr s p e c t r u m o f t r i a c e t a t e 86 100 48. ''"Hmr s p e c t r u m o f t e t r a c e t a t e 87 101 49. "4lmr s p e c t r u m o f a l d e h y d e 90 103 50. I n f r a r e d s p e c t r u m o f 1 9 1 - o x o l e u r o s i n e (91) .. 105 51. "*"Hmr s o e c t r u m o f 1 9 ' - o x o l e u r o s i n e (91) 106 - v i i i -Figure Page 52. H^mr spectrum of vinamidine (sample was ob-tained from the E l i L i l l y Laboratories, Indianapolis) 10 8 53. "''Hmr spectrum of synthetic vinamidine 109 54. "*"Hmr spectrum of 19 1-oxo-4'-deoxyleurosi-dine (95) I l l 55. Possible mechanism for the formation of vinamidine from 4'-deoxyleurosidine 112 56. Possible mechanism for the formation of 3R-hydroxyvinamidine from 31,4'-anhydro-vinblastine 113 57. Possible mechanism for the formation of 3R-hydroxyvinamidine from leurosine 113 58. Infrared spectrum of the product obtained from the iodine/sodium bicarbonate oxida-tion of 4 1-deoxyleurosidine 116 59. '''Hmr spectrum of the product obtained from the iodine/sodium becarbonate oxidation of 4'-deoxyleurosidine 117 60. Comparison of u l t r a v i o l e t spectra of 19'— oxoleurosine (A) and 19',22-dioxoleurosine (B) . ... 119 61. 1Hmr spectrum of 19 1,22-dioxoleurosine (98). 120 62. Infrared spectrum of 19 1,22-dioxoleurosine (98) 121 63. ''"Hmr spectrum of N -desmethyl derivative 99 f 122 - ix -LIST OF TABLES Table Page 1. Comparison of some fragments exhibited i n the mass spectra of 16,18S-dicarbomethoxy-cleavamine (39), 16,18S-dicarbomethoxy-3R,— 4S-epoxydihydrocleavamine (40), and 16,18S— dicarbomethoxy-4S-dihydrocleavamine (41) .... 37 13 2. Comparison of Cmr chemical s h i f t s of leurosine, and the epoxides 40 and 42 38 3. Comparison of the chemical s h i f t of the C^g;— hydrogen of various 16,18S-dicarbomethoxy-cleavamine derivatives 59 4.' Study of the a e r i a l and t-butyl hydroperoxide oxidations of 3',4 1-anhydrovinblastine, leur-osine, and derivatives thereof 88 5. Comparison of some characterization data for 19'-oxoleurosine (91), 19'-oxovinblastine (96), and 19 '-oxoleurosidine(97) 115 - x -ACKNOWLEDGEMENTS I w i s h t o e x p r e s s ray a p p r e c i a t i o n t o P r o f e s s o r J . P . Kutn e y f o r h i s g u i d a n c e t h r o u g h o u t t h e c o u r s e o f t h i s r e s e a r c h . I w o u l d a l s o l i k e t o thank t h e v a r i o u s t e c h n i c i a n s and s t a f f who p r o v i d e d me w i t h t h e n e c e s s a r y s p e c t r o s c o p i c d a t a . F u r t h e r m o r e , I w o u l d l i k e t o e x p r e s s g r a t i t u d e t o t h e v a r i o u s p o s t - d o c t o r a l f e l l o w s who I h a d t h e p l e a s u r e o f w o r k i n g w i t h . I n p a r t i c u l a r , I w o u l d l i k e t o t h a n k B.R. Worth f o r h i s h e l p i n t h e p r e p a r a t i o n o f t h i s manu-s c r i p t as w e l l as i n c a r r y i n g o u t t h e r e s e a r c h . F i n a l l y , I w o u l d l i k e t o t h a n k my w i f e f o r h e r h e l p i n p r e p a r i n g t h i s m a n u s c r i p t as w e l l as f o r h e r g e n e r a l s u p p o r t . - 1 -INTRODUCTION 1.1 General Background Cancer Chemotherapy has over the past 30-40 years evolved into an e f f e c t i v e means of treatment; and although the somewhat limited number of cancers treatable by this modality has remained a major drawback, the further development of this 1-5 area continues with the promise of greater potential. H i s t o r i c a l l y , the evolution of antineoplastic chemo-therapy began with a series of independent observations. Thus, in 19 39, Loeser^ reported the use of androgens in the treatment of breast cancer, and in 19 41, Huggins and Hodges, 7 reported that estrogen was eff e c t i v e in the treatment of prostatic cancer. By 1946, Rhoads and many others had noted the anticancer 9 effects of nitrogen mustard. In 19 48, Farber and associates, discovered the potential of a n t i f o l i c acid compounds, and later,demonstrated the c l i n i c a l value of actinomycin D. 1 0 Then, in 1955, the leukopenic e f f e c t of the catharanthus alkaloids was noted by Beer and Noble. These miscellaneous results provided a s o l i d founda-tion, as well as much impetus for further development. Conse-quently, the observation that mustard g a s — b i s (2-chloroethyl) - 2 -s u l f i d e — h a d the a b i l i t y to produce leukopenia, led to the d i s -covery of the less toxic nitrogen mustard. A further consequence of this was the development of a series of analgous anticancer HN(CH2CH2CI)2 H 3C0 2 SOH 2 C(CH 2 ) 2 CH 2OS0 2 CH 3 Nitrogen mustard Busulfan 0 S P-N(CH2CH2CI)2 — P — N C J Cyclophosphamide N Thiotepa Fig. 1. Structure of some c l i n i c a l l y used alkylating agents. 12 compounds: the "alkylating agents' (Fig. 1). Farber's observation, that f o l i c acid treatment of anemic leukemic children resulted i n a worsened condition, eventually led to the development of methotrexate—a c l i n i c a l drug which s t i l l 13 finds wide usage. In turn, methotrexate has become one of 14 many antimetabolite drugs (Fig. 2 ) . Furthermore, the observa-tion, that actinomycin D possessed interesting anticancer pro-perties, i n i t i a t e d a search for other a n t i b i o t i c s which might possess similar potential. The result of this was the i s o l a t i o n - 3 -Drug M e t a b o l i t e N ^ J l / \ N C H N 2 1 M e t h o t r e x a t e , R=NH 2, R 1=CH 3 OH H 5 - F l u o r o u r a c i l SH N 6 - M e r c a p t o p u r i n e H.N 2 O NH I OH H y d r o x y u r e a i ' o l i c a c i d , R=OH, R 1=H OH O ^ N H U r a c i l NH. "CO A d e n i n e H N 2 1 NH. U r e a F i g . 2 . Some a n t i m e t a b o l i t e c a n c e r d r u g s and t h e i r m e t a b o l i t e a n a l o g s . - 4 -OC '— HC 9H(Me)2 -CH N Me i sarcosine i L-proline CHfMeL CH-i •CO—i NMe I sarcosine i L-proline Actinomycin D Kidamycin Fig. 3. Structures of some cytotoxic a n t i b i o t i c s . - 5 -and/or synthesis of several new and promising compounds belonging 15-17 to the a n t i b i o t i c group (Fig. 3). In a similar manner, the early work of Noble, Beer, 18 19 and Cutts ' stimulated the development of a new class of a n t i -cancer agents: the catharanthus alkaloids. These workers, while investigating the reputed hypoglycemic potential of extracts of Catharanthus roseus noted that injections of the extracts into rats produced a rapid onset of peripheral granulocytopenia and bone marrow depression. Eventually, this a c t i v i t y was loc a l i z e d to the alka l o i d fractions with the subsequent result being the is o l a t i o n of vinblastine (1). Meanwhile, at the L i l l y labora-20 21 tori e s , work carried out by Svoboda ' led to the i s o l a t i o n of the related cytotoxic alkaloids v i n c r i s t i n e (2), leurosine (3), and leurosidine (4). Within a decade of the o r i g i n a l discovery, vinblastine and v i n c r i s t i n e had advanced to c l i n i c a l 22 use; and as a result, the study of the catharanthus alkaloids quickly became an area of top i c a l interest to a multitude of researchers. 2 1, R=CH3 (Vinblastine) 2, R=CHO (Vincristine) - 6 -3 (Leurosine) 4 (Leurosidine) 1.2 Structure Elucidation of Vinblastine, V i n c r i s t i n e , Leurosine, and Leurosidine I n i t i a l l y , the chemistry of the catharanthus alkaloids consisted of work done towards the structure elucidation of the aforementioned 'dimeric'* alkaloids. Thus, by means of lithium 23 aluminum hydride reduction, Neuss et a l were able to convert *The term 'dimeric' alkaloids i s s t r i c t l y speaking a misnomer, however, i t has been popularly used in this area along with other terms such as bisindole alkaloids to refer to these compounds. - 7 -v i n b l a s t i n e and v i n c r i s t i n e to the pentahydroxy d e r i v a t i v e 5. Further c o r r e l a t i o n between v i n b l a s t i n e and v i n c r i s t i n e was obtained by the development of a cleavage r e a c t i o n . Hence, treatment of v i n b l a s t i n e or v i n c r i s t i n e i n hot concentrated hy d r o c h l o r i c a c i d containing stannous c h l o r i d e and m e t a l l i c t i n 23 24 le d to products a r i s i n g from cleavage of the C^g'-C^ bond. ' In the case of v i n b l a s t i n e these products were velbanamine (6), 5 and d e a c e t y l v i n d o l i n e (7), while i n the case of v i n c r i s t i n e , the products obtained were velbanamine and N^-desmethyldeacetylvin-dol i n e (8). Therefore, the d i f f e r e n c e between v i n b l a s t i n e and v i n c r i s t i n e was a t t r i b u t a b l e to a d i f f e r e n c e i n the dihydro-indole p o r t i o n of the 'dimers'. These r e s u l t s , coupled with a 25 d e t a i l e d mass s p e c t r a l study performed by Biemann et a l , led to reasonable s t r u c t u r a l assignments. However, i t was not u n t i l the 2 6 x-ray a n a l y s i s c a r r i e d out by Moncrief and Lipscomb i n 1965 that the absolute s t r u c t u r e of v i n c r i s t i n e (as i t s methiodide d e r i v -- 8 -ative) was conclusively determined. Since vinblastine and vin-c r i s t i n e had been chemically related, the determination of the absolute structure of v i n c r i s t i n e meant that the absolute structure of vinblastine was also established. Therefore, with the determination of the structures of vinblastine and v i n c r i s t i n e , greater emphasis was placed on the structure determination of leurosine and leurosidine. Accordingly, a comparison of spectral data indicated that v i n -blastine and leurosine were closely related. Reductive cleavage of leurosine with stannous chloride and metallic t i n i n hot concentrated hydrochloric acid led to the i s o l a t i o n of cleavamine 24 (9) and deacetylvindoline (7), therefore pinpointing the d i f f e r -ence between vinblastine and leurosine to the indole portion of 2 7 the 'dimers'. High resolution mass spectrometry established the molecular formula of leurosine as C.,H[_CN.C' — two hydrogens - 9 -less than that for vinblastine. Moreover, from the fragmenta-tion pattern exhibited i n the mass spectrum, the presence of an oxygen functionality i n the piperidine ring of the indole por-tion was indicated (Fig. 4). The conclusion that this oxygen 13 functionality was a 3',4'-epoxide was corroborated by a C-2 8 magnetic resonance study performed by Wenkert and associates However, the unambiguous assignment of the epoxide stereo-29 chemistry was determined only recently by Kutney and Worth , who using the recently developed method of coupling vindoline (10) Fig. 4. A s i g n i f i c a n t pathway for the fragmentation of leurosine in the mass spectrometer. - 10 -and catharanthine (11) to afford dimeric compounds of the vin-30-33 blastine-type*, synthesized 3R, 4S-epoxydihydrocatharanthine (12) and coupled this compound with vindoline to afford leuro-sine (Fig. 5). Since the absolute configuration of the sta r t i n g catharanthine derivative was known, the stereochemistry of the epoxide i n leurosine was established. The determination of the structure of leurosidine followed a similar path. A comparison of spectral data between leurosidine and vinblastine showed a close relationship between the two alkaloids. Reductive cleavage (stannous chloride and metallic t i n i n hot concentrated hydrochloric acid) yielded deacetylvindoline (7) and a new indole fragment vinrosamine (13), thus pinpointing the difference between the two 'dimeric' alka-loids to the indole portion. 13 High resolution mass spectrometry indicated that leurosidine was isomeric with vinblastine (C^gH^gN^Og), and o r i -g i n a l l y , based on these early results leurosidine and vinrosamine were incorrectly assigned as the secondary alcohols 14 and 15 *The coupling of vindoline and catharanthine to afford 'dimers' of the vinblastine-type constituted an important discovery and w i l l be discussed i n some d e t a i l i n section 1.4. - 11 -Fig. 5. Outline of Kutney and Worth's structure-proof of leurosine. - I r -respectively. However, these structures were later corrected (to the t e r t i a r y alcohols 4 and 13) on the basis of further 14 chemical evidence as well as a C- magnetic resonance study 28 performed by Wenkert et a l . More recently, Potier and 35 Langlois were able to confirm this assignment by means of an unambiguous synthesis.* Thus, vindoline (10) and dihydrocathar-anthine (16) were coupled to y i e l d the enamine 17. This enamine was not isolated, but was reacted with osmium tetroxide to afford the 4',5'-diol 18, which on treatment with sodium borohydride yielded leurosidine (Fig. 6). Since this reaction sequence could only have led to the C-41 alcohol, and since the configuration of the other centers was known, leurosidine 1s structure was established as the C-41 epimer of vinblastine. *This synthesis was an application of the 'biogenetic approach 1 to the synthesis of the 'dimeric' alkaloids — an approach which i s discussed in section 1.4. -13-Leurosidine Fig. 6. Potier'and Langlois 1 structure-proof of leurosidine. - 14 -Vinblastine, v i n c r i s t i n e , leurosine, and leurosidine represented some of the f i r s t alkaloids isolated from Catharanthus roseus. Further investigations in this area have continued and to date close to one hundred indole alkaloids of varying types have been isolated. Among these have been several new 'dimeric 1 36 — 3 8 alkaloids, some of which have shown cytotoxic properties. 1.3 Studies Aimed at the Synthesis of the 'Dimeric' Catharanthus Alkaloids — the 'Chloroindolenine Approach'. As the structures of various catharanthus alkaloids began to emerge, so too did interest i n their synthesis. In pa r t i c u l a r , the synthesis of the v i n b l a s t i n e - v i n c r i s t i n e type rapidly became a dominant concern, for not only were these novel compounds st r u c t u r a l l y complex and thus synthetically challenging, but also they were expensive and d i f f i c u l t to i s o l a t e and possessed tremendous anticancer potential — a l l of which com-bined to make them extremely attractive as synthetic targets. Consequently, two of the major alkaloids of C. roseus, vindoline (10) and catharanthine (11), became important as poten-t i a l synthons of the 'dimeric' alkaloids. Both of these compounds 10 (Vindoline) 11 (Catharanthine) were readily available (from plant material) and featured de-- 15 -sirable functionality as well as suitable carbon skeletons. Moreover, since both of these alkaloids had recently been syn-39-42 thesized, any natural products synthetically derived from them could formally be considered as t o t a l l y synthetic. The f i r s t synthetic effo r t s aimed at the dimeric alkaloids were based on the 'chloroindolenine approach'. 43 Kutney et a l had shown in 1970, that when a solution of catharanthine in hot g l a c i a l acetic acid was treated with sodium borohydride a high y i e l d of 18S-carbomethoxycleavamine (19) could be obtained (Fig. 7). This product possessed the required car-bon skeleton of the indole portion of the 'dimeric' alkaloids and required only appropriate functionalization of the 3, 4-double bond and subsequent condensation (via the chloroindole-nine) with vindoline to provide access to a number of dimeric alkaloids (Fig. 8). The f i r s t requirement, the functionalization of the 3,4-double bond, proved d i f f i c u l t . This d i f f i c u l t y was due to the competitive r e a c t i v i t i e s of the indole system and the basic nitrogen towards e l e c t r o p h i l i c reagents (e.g. many oxidizing agents). In general, attempted functionalization of the double bond either led to complex reaction mixtures or to products i n 4 4 which the double bond was s t i l l i ntact. However, two selective reactions of the 3,4-double bond were attained. One of these was hydrogenation using a platinum catalyst. The exclusive pro-duct obtained i n good y i e l d was 4S-ethyl-18S-carbomethoxydihydro-4 3 cleavamine (20). The other successful reaction achieved was hydroboration. Oxidation of the intermediate alkylborane with -16-i CO Me 19 (18S-Carbomethoxycleavamine) F i g . 7. Mechanism for the formation of 18S-carbomethoxy-cleavamine from catharanthine. - 17 -Fig. 8. General outline of the 'chloroindolenine approach'. - 18 -a l k a l i n e h y d r o g e n p e r o x i d e a f f o r d e d i n good y i e l d t h e 3S-hydroxy 44 d e r i v a t i v e 21 as a m i x t u r e o f C-18 e p i m e r s . T h e r e f o r e , a t 20 21 l e a s t t o a l i m i t e d e x t e n t , t h e f i r s t r e q u i r e m e n t was s a t i s f i e d . The s e c o n d r e q u i r e m e n t , t h e c o u p l i n g o f v i n d o l i n e and a c l e a v a m i n e d e r i v a t i v e , was s t u d i e d by a number o f r e s e a r c h -45-49 e r s . One o f t h e most t h o r o u g h i n v e s t i g a t i o n s i n t h i s a r e a 49 was c a r r i e d o u t by Kutney e t a l . These w o r k e r s by t r e a t i n g 4 S - e t h y l - 1 8 S - c a r b o m e t h o x y c l e a v a m i n e c h l o r o i n d o l e n i n e (22) w i t h a s o l u t i o n o f v i n d o l i n e i n 1.5% m e t h a n o l i c h y d r o g e n c h l o r i d e o b t a i n e d a s i n g l e d i m e r i c p r o d u c t ( F i g . 9 ) . M o r e o v e r , r e g a r d l e s s o f changes i n r e a c t i o n c o n d i t i o n s ( s o l v e n t , t e m p e r a t u r e , c o n c e n -t r a t i o n , t i m e , a c i d , and a d d i t i o n t e c h n i q u e ) no o t h e r d i m e r i c p r o d u c t s — e x c l u d i n g d e g r a d a t i o n p r o d u c t s o f t h e o r i g i n a l d i m e r — were p r o d u c e d . Hence, t h i s r e a c t i o n was c o m p l e t e l y s t e r e o -s e l e c t i v e . X - r a y a n a l y s i s o f t h i s p r o d u c t by C l a r d y e t a l ^ showed i t t o be 1 8 ' - e p i - 4 ' - d e o x y l e u r o s i d i n e ( 2 3 ) . Thus, a l t h o u g h v i n d o l i n e and t h e c l e a v a m i n e d e r i v a t i v e 22 were c o u p l e d a t t h e r e q u i r e d c e n t e r s , the c o n f i g u r a t i o n a t C-18 1 o f t h e c o u p l e d p r o d u c t 23 was o p p o s i t e t o t h a t o f t h e ' n a t u r a l d i m e r s ' . F u r -t h e r m o r e , e x t e n s i o n o f t h e c h l o r o i n d o l e n i n e method t o a number -19-Fig. 9. Condensation of the chloroindolenine of 4S-ethyl— 18S-carbomethoxydihydrocleavamine with vindoline. - 20 -of other cleavamine derivatives led to a variety of new 'dimeric' products, a l l of which possessed the 'unnatural' stereochemistry 49 at C-181. Therefore, as a general and ve r s a t i l e route to the synthesis of the 'dimeric' catharanthus alkaloids, the 'chloroin-dolenine approach' as outlined was a f a i l u r e . However, modifi-cations of t h i s approach may yet provide some measure of success, and further study towards this end continues. 1.4 Studies Aimed at the Synthesis of the 'Dimeric' Catharanthus Alkaloids — t h e 'Biogenetic Approach'.* The stereochemical outcome of the 'chloroindolenine approach' was unfortunately not predictable. A p r i o r i considera-tions required a conformational analysis of the nine-membered ring of carbomethoxycleavamine — a complex ring system with potential for conformational f l e x i b i l i t y , and thus not readily amenable to analysis. Conversely, the pentacyclic Iboga skeleton of catharanthine represented a r i g i d i n f l e x i b l e moiety; and i t was this fixed geometry which led to the development -of the 30 31 32 33 'biogenetic approach* by both Potier ' and Kutney ' Thus, treatment of catharanthine (11) with m-chloroperbenzoic acid in methylene chloride solution led to the formation of the N^-oxide 24. Treatment of this product with t r i f l u o r o a c e t i c anhydride (modified Polonovski r e a c t i o n 5 1 ) resulted i n the frag-mentation of the Ccj-C^g bond with concomitant generation of a *This approach has been termed 'biogenetic' on the basis of popular b e l i e f and circumstantial evidence, however the con-clusive proof of this conviction has yet to be accomplished. - 21 -c a t i o n i c c e n t e r a t C-18, and i n t h e p r e s e n c e o f v i n d o l i n e , u nder a p p r o p r i a t e c o n d i t i o n s , good y i e l d s (50-60%) o f 3 ' , 4 ' - a n h y d r o v i n -b l a s t i n e (26) c o u l d be o b t a i n e d ( F i g . 1 0 ) . The s u c c e s s o f t h i s a p p r o a c h was a t t r i b u t a b l e t o r e t e n t i o n o f 1 r e a c t a n t - l i k e ' g eometry d u r i n g t h e c o u p l i n g . M o l e c u l a r models had c l e a r l y e s t a b l i s h e d t h a t e i t h e r c o n c e r t e d attack, by v i n d o l i n e d u r i n g t h e f r a g m e n t a t i o n o r a s t e p - w i s e c o u p l i n g i n w h i c h t h e ' r e a c t a n t - l i k e ' c o n f o r m a t i o n o f t h e s e c o - c a t h a r a n t h i n e i n t e r m e d i a t e was f r o z e n w o u l d l e a d t o t h e r e -q u i r e d outcome. A c c o r d i n g l y , c a r r y i n g o u t t h e r e a c t i o n i n c o n c e n -t r a t e d s o l u t i o n a t low t e m p e r a t u r e s (ca -50°C) a f f o r d e d , a f t e r work-up, 3 ' , 4 ' - a n h y d r o v i n b l a s t i n e , w h i l e on t h e o t h e r hand, i n more d i l u t e s o l u t i o n a t h i g h e r t e m p e r a t u r e s ( c a 4 0 ° C ) , 1 8 ' - e p i - 3 ' , 4 ' - a n h y d r o v i n -b l a s t i n e was i s o l a t e d . The c o m p l e t e r e v e r s a l o f t h e s t e r e o c h e m i c a l outcome o f t h e r e a c t i o n c o u l d be r a t i o n a l i z e d by a s s u m i n g t h a t i n more d i l u t e s o l u t i o n s a t h i g h e r t e m p e r a t u r e s t h e r e a c t i v e i n t e r m e d -i a t e was a b l e t o a d o p t a more s t a b l e ' p r o d u c t - l i k e 1 c o n f o r m a t i o n (2 5a-»25b) p r i o r t o c o u p l i n g w i t h v i n d o l i n e . The s u c c e s s f u l r e a l i z a t i o n o f t h e ' b i o g e n e t i c a p p r o a c h 1 c o n s t i t u t e d a m a j o r b r e a k t h r o u g h i n t h e s y n t h e s i s o f t h e ' d i m e r i c ' c a t h a r a n t h u s a l k a l o i d s , and l a u n c h e d s e v e r a l i n v e s t i g a t i o n s i n t o t h e v e r s a t i l i t y o f t h e r e a c t i o n . I n g e n e r a l , i t was f o u n d t h a t s u b s t i -t u t i n g v a r i o u s n u c l e o p h i l i c a s p i d o s p e r m i n e d e r i v a t i v e s i n p l a c e o f 31 33 52 v i n d o l i n e was p o s s i b l e . ' ' The r e p l a c e m e n t o f c a t h a r a n t h i n e w i t h v a r i o u s f u n c t i o n a l i z e d d e r i v a t i v e s was n o t as s u c c e s s f u l . The 29 53 35 s y n t h e s e s o f l e u r o s i n e ' and l e u r o s i d i n e ( p r e v i o u s l y m e n t i o n e d i n c o n n e c t i o n w i t h t h e i r s t r u c t u r e e l u c i d a t i o n ) were a t t a i n e d by t h i s method, however t h e y i e l d s were d i s a p p o i n t i n g ( 5 - 3 0 % ) . F u r t h e r m o r e , - 22 -Me i C0 2Me 26 (34'-Anhydrovinblastine) 25b Fig. 10. 'Biogenetic-type' coupling of catharanthine-N^-oxide (24) with vindoline. - 23 -reaction of other catharanthine derivatives such as the epoxide 27 and the lactone 2 8 afforded only rearrangement products such as 29 and 30 respectively.^ 4 ^ As such, the u t i l i t y of the 'biogenetic approach' in regard to the use of functionalized catharanthine derivatives appeared limited. Consequently, the development of r e g i o s p e c i f i c reactions for the functionalization of 3',4'-anhydro-vinblastine has become an area of current interest. 7 30 1.5 V i n c r i s t i n e Derivatives Although the discussion of the chemistry relating to - 24 -the 'dimeric' alkaloids has so far been concerned with vinblas-t i n e - l i k e derivatives, the conversion of vinblastine to v i n c r i s -tine i s an important step and has been accomplished by Jovanovics 5 7 et a l . Thus, treatment of a solution of vinblastine sulfate i n acetone at -50°C with chromic acid afforded v i n c r i s t i n e . Extension of this reaction to leurosine was also successful and provided 5 8 the N -formyl derivative 31 i n good y i e l d . 31 This r e g iospecific oxidation represented a s i g n i f i c a n t development in the chemistry of the 'dimeric' catharanthus alkaloids since i t constituted the only method for the production of v i n c r i s t i n e analogs. Thus, although various aspidospermine derivatives had been successfully used i n the 'biogenetic approach' to y i e l d vinblastine analogs, the corresponding produc-31 tion of v i n c r i s t i n e analogs was not successful. This result was not surprising since in the successful coupling reactions the aspidospermine unit was either a nucleophilic methoxyaniline or an i l i n e system, while i n the unsuccessful case the electron don-- 25 -ating amino group was replaced by the electron withdrawing N a~formyl group. The resultant reduced r e a c t i v i t y towards e l e c t r o p h i l i c aromatic substitution of the N^-formyl derivative thus provided a rationale for the observed lack of reaction. Further significance of the regiospecific chromate o x i -dation was due to the d i f f e r e n t spectrum of a c t i v i t i e s exhibited by vinblastine and v i n c r i s t i n e . These two potent anticancer agents although s t r u c t u r a l l y very similar were activity-wise very 59 d i f f e r e n t . Moreover, the difference in b i o l o g i c a l a c t i v i t y r esulting from the modification of an N a-methyl to an N a-formyl group was further exemplified by 22-oxoleurosine (31), which 6 0 has advanced to c l i n i c a l t r i a l s i n Hungary. Leurosine on the other hand, has not been found c l i n i c a l l y useful. The extension of this reaction to available synthetic 'dimers' shows great potential for the production of new anticancer agents. 1.6 Scope of Current Research Thus, i t has been seen that the chemistry of the 'dimeric' catharanthus alkaloids has developed over the past twenty years to a point where the potential synthesis of a large number of analogs and natural products i s at hand. How-ever, the complete attainment of these goals s t i l l requires the further development of the chemistry of the indole portion of available synthetic 'dimers'. Indeed, extension of the previously mentioned 'biogenetic approach' by appropriate functionalizations of 3',4'-anhydrovinblastine (26) would lead to the synthesis of a number of natural 'dimers', some of which are i l l u s t r a t e d - 2 6 -V i n b l a s t i n e , R^OH, R 2=CH 2CH 3, R 3=R 4=H L e u r o s i d i n e , R - ^ C H ^ H ^ R 2=OH, R 3=R 4=H L e u r o s i n e , R 1=CH 2CH 3, ^ 2 R 4 = 0 ' R 3 = H D e o x y v i n b l a s t i n e , R 1=R 3=R 4=H, R 2=CH 2CH 3 V i n c a d i o l i n e , R =R.=OH, R =CH CH , R =H H3C02C Vind. C a t h a r i n e H3C02C Vind. V i n a m i d i n e ( C a t h a r i n i n e ) F i g . 11. Some ' d i m e r i c ' c a t h a r a n t h u s a l k a l o i d s . - 27 -i n Fig. 11. Furthermore, attainment of this result coupled with previously developed chemistry would provide access to a number of st r u c t u r a l l y diverse analogs — an important requirement for the production of new drugs as well as for the development of structure-activity relationships. Alternatively, another area of recent interest involves further evaluation of the 'chloroindolenine approach'. The continuing interest i n this approach revolves around the preparation of cleavamine derivatives whose geometry would be such that 'dimer-ization ' with vindoline could occur only from the oC-face to afford the 'natural' stereochemistry 'dimer'. Thus, the 4S,18R-ether 32 and the lactone 33 represent potential precursors of vinblastine in that their geometry should block access to the/3-facel 32 33 In summary, the further investigation of the 'chloro-indolenine approach', the development of the chemistry of 3',4'-anhydrovinblastine, and the production of vin b l a s t i n e - v i n c r i s t i n e analogs remain as current topics of research. Further discussion of these various aspects, combined with an outline of the work performed in attempting to achieve these ends, constitutes the next chapter of this thesis. - 28 -DISCUSSION 2.1 Further Evaluation of the Chloroindolenine Approach . Although previous attempts at coupling chloroindole-nines of several cleavamine derivatives with vindoline had re-sulted i n the formation of 'unnatural 1 stereochemistry 'dimers' reversed by u t i l i z a t i o n of other 'more appropriate' cleavamine de-ri v a t i v e s s t i l l remained as a v a l i d consideration. Indeed, there existed some j u s t i f i c a t i o n for the b e l i e f that derivatives of velbanamine (e.g. 34 and 35) might be such compounds. The basis for this conviction was a report that vinblastine could be cleaved i n hot aqueous 40% s u l f u r i c acid to y i e l d deacetyl-(at C-18') 49 the question of whether this outcome could be HO R R 34, R=H, R]_=C02Me 35, R=C02Me, R]_=H vindoline (7) and an indole fragment whose structure was assigned - 29 -Vinblastine Deacety1-vindoline (7) H02C 37 Fig. 12. Possible mechanism for the formation of 4S,18R-ether 37 from vinblastine. - 30 -as the 4S,18R-ether 37 (Fig. 12). The formation of this product could be explained by a sequence involving protonation of the electron-rich methoxyaniline system of vinblastine followed by indole-assisted cleavage of the C2.8'~C15 bon^* The resultant intermediate 36 could presumably be transformed into the 4S,18R-ether by intramolecular attack of the 4S-hy-droxyl group onto the C-18 position. Since the intermediate 36 should also be available from the chloroindolenine* 38/ i t therefore followed that 18— carbomethoxyvelbanamine (3_4 or 35) should via i t s chloroindolenine 37 be a precursor of the corresponding ether 32. Furthermore, MeO C 37, R=C02Me 38, R=C02H the geometry of this ether was such that formation of the 'natural' stereochemistry 'dimer' (on coupling with vindoline) would be expected. Therefore, coupling of 18-carbomethoxyvelban-amine chloroindoline (37) with vindoline should (assuming the *The chloroindolenine 'dimerization' involved the formation of an intermediate (analgous to 36) according to the mechanism proposed by A.M. Treasurywala.44 - 31 -intermediacy of 32) afford vinblastine. More generally, the synthesis of velbanamine derivatives* followed by coupling of their chloroindolenines with vindoline appeared a potentially viable route to vinblastine, v i n c r i s t i n e , vincadioline, and v a r i -ous novel analogs. Accordingly, exploration of this route was undertaken. 18S-Carbomethoxycleavamine (19), available from 43 catharanthine i n good y i e l d was chosen as the starting material. 19, R=H 39, R=C02Me Thus, successful u t i l i z a t i o n of this material required an over-a l l hydration of the 3,4-double bond. However, direct func-t i o n a l i z a t i o n of the 3,4-double bond of 19 had previously proven d i f f i c u l t with part of the problem being attributed to the com-44 p e t i t i v e r e a c t i v i t y of the indole chromophore. Elimination of this problem required deactivation (i.e. protection) of the *Buchi and co-workers have recently reported the synthesis of 18R-carbomethoxyvelbanamine. Their results in regard to dimer-ization with vindoline have not yet appeared. - 32 -indole chromophore and therefore 18S-carbomethoxycleavamine was treated with potassium hydride in tetrahydrofuran followed by methyl chloroformate to afford after p u r i f i c a t i o n , 16,18S— 6 2 6 3 dicarbomethoxycleavamine* (39) in 78% y i e l d . ' The identity of this compound was v e r i f i e d by i t s spectral properties. Thus, high resolution mass spectrometry confirmed the molecular f o r -mula as 023^2^0^. The proton magnetic resonance ("'"Hmr) spectrum (Fig. 13) lacked a signal for an N -hydrogen, while exhibiting the following: & 5.77 (IH, doublet, J=6 Hz, C^g-H), ^5.37 (IH, multiplet, C3-H)-, <$ 3 . 88 (3H, singlet, -0CH3) , 3 . 53 (3H, singlet, -OCH^). Further supportive evidence was gained from the u l t r a v i o l e t spectrum (Fig. 14) which was indicative of a modified indole chromophore. F i n a l l y , the assignment of the configuration at C-18 as S was based upon the low f i e l d position of the C^ g-hydrogen resonance (& 5.77) i n the "'"Hmr spectrum. Previously i t had been noted that the C^ g-hydrogen resonance of 18R-carbomethoxycleavamine derivatives occurred at ~<c£ 4, while that of the corresponding 18S-carbomethoxy 4 4 65 derivatives occurred below & 5 (i.e. to lower f i e l d ) . ' The reason for the observed difference was presumed due to the spa t i a l proximity of the 18S-hydrogen to the N b unshared pair of electrons (Fig. 15). *This compound had previously been prepared i n this laboratory I. Itoh and A.H. R a t c l i f f e using a diff e r e n t procedure. 3.88 - 34 -log <f log I 250 300 250 300 A. nm \ nm Fig. 14. Comparison of the u l t r a v i o l e t spectra of 18S-carbo-methoxycleavamine (A) and 16,18S-dicarbomethoxycleavamine (B). 18S configuration 18?. configuration Fig. 15. Spatial relationship of the 18-hydrogen to the Nfc electron pair i n 18-carbomethoxycleavamine. - 35 -With the protected derivative 39 available, the next requirement was oxygenation of the 3,4-double bond. E a r l i e r , i t had been observed that 39 readily underwent autoxidation* to afford 16 ,18S-dicarbomethoxy-3R, 4S-epoxydihydrocleavamine (4_0) as one of many products. The structure of this compound was de-duced from i t s spectral properties as well as via a correlation to a compound of known structure. Accordingly, the ^ Hmr spectrum 40 (Fig. 16) indicated that the o l e f i n i c proton was no longer present. High resolution mass spectrometry established the molecular formula as C 2 3H2 gN 20^. Comparison of the mass spectral fragmentation patterns of the o l e f i n 39, the dihydro derivative 41**,and 40 (Table 1) indicated the presence of an oxide func-t i o n a l i t y i n the piperidene ring. The fact that this func-t i o n a l i t y was a 3,4-epoxide was based on a comparison of the *This i n i t i a l observation was made by I. Itoh i n this laboratory. **41 was prepared by hydrogenation of 16,18S-dicarbomethoxy-cleavamine in the presence of a platinum catalyst. Fig. 16. Hmr spectrum of 16,18S-dicarbomethoxy-3R,4S-epoxydihydrocleavamine (40). - 37 -Cmpd. 39 40 41 MeO CHC m/e 208 (23%) C 1 2 H 1 8 N 0 2 Me02CHC m/e 224 (100%) C 1 2 H 1 8 N 0 3 MeC>2CHC m/e 210 (100%) C 1 2 H 2 0 N O 2 136 (43%) C H N 1 14 152 (15%) C 9 H 1 4 N O 138 (22%) C 9 H 1 6 N XT 122 (100%) C G H 1 2 N 138 (10%: C 8 H 1 2 N O 124 (13%) C 8 H 1 4 N Table 1. Comparison of some fragments exhibited i n the mass spectra of 16,18S-dicarbomethoxycleavamine (39), 16,18S-dicarbomethcxy-3R,4S-epoxydihydrocleavamine (40), and 16,18S-dicarbomethoxy-4S-dihydro-cleavamine (41). - 38 -13 2 8 C-magnetic resonance spectra of 40, leurosine (3) and the Me02C C0 2Me 41 2 8 epoxide 42 (Table 2) while the determination of the absolute 42 \ Compound Carbon^x 40 Leurosine 42* -CH2CH3 8.9 8.6 8.1 — CH 2CH ^  30.0 28.0 28.6 C-2 33.6 33.5 25.4 C-3 60.6 60.3 56.0 C-4 62.7 59.9 59.8 C-5 52.7 54.0 54.6 C-7 53. 4 49.6 51.3 C-19 50.6 42.3 45.5 Table 2. Comparison of 13rjmr chemical s h i f t s of leurosine and the epoxides 4_0 and 42. *The numbering system normally used i n cleavamine derivatives was also applied to the epoxide 42. - 39 -configuration of the epoxide was achieved by conversion of 40 to a derivative whose configuration at C-4 was known (Fig. 17). * In this regard, reduction of 40 with lithium aluminum hydride afforded the d i o l 43. 6 3 This d i o l (43) was also obtained by Fig. 17. Sequence used for the determination of the absolute configuration of 16,18S-dicarbomethoxy-3R,4S-epoxydihydrocleav-amine (40). 6 3 6 5 lithium aluminum hydride reduction of the known t r i o l 44. ' Since the configuration at C-4 of the t r i o l 44 (and hence of the d i o l 43) had been previously determined, the configuration at C-3 and C-4 in the epoxide 40 was established (3R,4S). * This sequence was performed by A. H. R a t c l i f f e i n this laboratory. - 40 -Inasmuch as the epoxide 40 represented an attractive intermediate for the synthesis of velbanamine derivatives, further study of the a e r i a l oxidation of the o l e f i n 39 was undertaken. Autoxidation of 39 was carried out i n peroxide-free tetrahydrofuran containing a small amount of aqueous 1% t r i f l u o r o -acetic acid.* Monitoring the course of the reaction v i a thin layer chromatography indicated that after three days at ambient temperature no reaction had occurred. After f i v e days, a s i g n i -ficant amount of epoxide 40 had formed, however the starting o l e f i n 39 s t i l l accounted for the majority of nitrogenous material present. After eight days most of the starting material had been consumed, however the epoxide 40 had also undergone further reaction. Thus, an i s o l a t i o n of the products, 40 was obtained i n 10% y i e l d while the major product (52% yield) was the formyl-ketone 45**. CHO 45 *These conditions had o r i g i n a l l y been developed by I. Itoh and A.H. R a t c l i f f e and found to afford r e l a t i v e l y uncomplicated product mixtures. **O r i g i n a l l y , based on the assumption that the cleavamine carbon skeleton was intact, this product was incorrectly assigned as the lactam 46.62,6 3 - 41 -The structural assignment of 45 was based on analysis of spectral properties coupled with a chemical transformation. Thus, the "*"Hmr spectrum (Fig. 18) exhibited the following signals*- i n accord with the proposed structure: ^8.13 (IH, singlet, Nb-CHO), c$5.23 (IH, broad singlet,Cg-H) , <£4.00 (3H, broad singlet,-OCH 3) , ^3.66 (3H,singlet,-OCH 3) , i l . 9 0 (2H, quartet,J=7 Hz, -CH_2CH3) , i0.79 (3H,triplet, J=7 Hz, -CH2CH_3) . The infrared spectrum (Fig. 19) exhibited strong absorptions at 1680 and 1650 cm - 1 which were i n agreement with the presence of an enamide func-t i o n a l i t y . High resolution mass spectrometry established the molecular formula as C„,H_,N~Cv while the presence of the ketone 2 3 2 6 2 6 C functionality was substantiated by sodium borohydride reduction of 45 enabling i s o l a t i o n of the alcohol 47. *The duplication of various resonances observed in this spectrum have also been noted i n a number of alkaloids which possess an N^-formyl group.66 3.66 l 1 1 1 1 1 r F i g . 18. Hmr spectrum of formyl ketone 45. Fig. 19. Infrared spectrum of formyl ketone 45. - 44 -CHO 47 The above information provided s u f f i c i e n t proof for structure 45. Accordingly/ the presence of the ketone functional-i t y was compatible only with a cleavage product and ruled out lactam 46. Furthermore, that the alcohol 47 displayed strong absorptions in i t s infrared spectrum at 1669 and 1651 cm 1 (Fig. 20) corroborated the enamide assignment i n 47 and 45 <~i •— while eliminating structures such as the <* ,/3 -unsaturated ketones 48 and 49. F i n a l l y , the alternative enamide 50 was ruled out CHO 48 49 due to incompatibility with the observed "^ Hmr spectrum in which the resonance due to the methylene hydrogens of the ethyl group was located at Si.90, i n good agreement with a methylene group 6 7 adjacent to a double bond (but not to a ketone). Fig. 20. Infrared spectrum of alcohol 47. - 46 -H a v i n g a s c e r t a i n e d t h e o v e r a l l c o u r s e o f t h i s a u t o x i -d a t i o n r e a c t i o n , i n t e r e s t was t u r n e d t o m o d i f i c a t i o n o f the r e a c t i o n c o n d i t i o n s . The p r e s e n c e o f an i n d u c t i o n p e r i o d s u g g e s t e d t h a t a c e r t a i n c o n c e n t r a t i o n o f p e r o x i d e m i g h t be n e c e s s a r y f o r r e a c t i o n t o o c c u r a t an a p p r e c i a b l e r a t e . * ' 8 ^° T h i s s u p -p o s i t i o n was t e s t e d by r e p e a t i n g t h e r e a c t i o n w i t h t e t r a h y d r o f u r a n , w h i c h had a l r e a d y undergone a e r i a l o x i d a t i o n * , as t h e s o l v e n t . U s i n g o t h e r w i s e s i m i l a r c o n d i t i o n s and m o n i t o r i n g o f the r e a c t i o n by t h i n l a y e r c h r o m a t o g r a p h y i n d i c a t e d t h a t a f t e r f o u r h o u r s t h e m a j o r i t y o f s t a r t i n g m a t e r i a l had been c o n v e r t e d t o t h e e p o x i d e 40. I s o l a t i o n o f t h e p r o d u c t s a f t e r twenty-two h o u r s a f f o r d e d 40 i n 32% y i e l d and t h e k e t o e n a m i d e 45 i n 25% y i e l d . I n o r d e r t o a t t a i n r e p r o d u c i b l e c o n d i t i o n s as w e l l as maximize the y i e l d o f e p o x i d e 40, o x i d a t i o n o f t h e o l e f i n 39 was p e r f o r m e d i n p e r o x i d e - f r e e t e t r a h y d r o f u r a n c o n t a i n i n g aqueous 1% t r i f l u o r o a c e t i c a c i d and a known amount o f t - b u t y l h y d r o p e r o x i d e * * . Thus i t was f o u n d t h a t under a p p r o p r i a t e c o n d i -*The c o n c e n t r a t i o n o f p e r o x i d e m a t e r i a l p r e s e n t was n o t measured. * * T h i s s t u d y was c a r r i e d o u t i n c o n j u n c t i o n w i t h T. H i b i n o . - 47 -tions 16,18S-dicarbomethoxy-3R,4S-epoxydihydrocleavamine (40) 6 2 63 could be obtained in 76% y i e l d . ' With the epoxide 40 now readily available, the next objective was acid catalysed hydrolysis to the trans d i a x i a l d i o l 51' ' — a compound possessing the required a x i a l C. hy-droxyl group. However, several attempts to e f f e c t hydration of HO the epoxide functionality led to disappointing results. In general, i t was observed that at ambient temperature, except in concentrated solutions of strong acids such as perchloric acid, no reaction occurred. On heating ( 50°C) the epoxide 40 readily underwent reaction i n various media yielding i n a l l cases complex product mixtures. Indeed, the best reaction con-ditions developed (aqueous 3 0% perchloric acid/tetrahydrofuran at 66°C for 24 h or aqueous 50% t r i f l u o r o a c e t i c acid at 65°C for 4 h) afforded the d i o l 51 in yields of only 17-25%. The identity of the d i o l 51 was confirmed by i t s spectral properties as well as by a chemical transformation. High resolution mass spectrometry established the molecular formula as C» ,H,nN.,Oc, while the mass spectral fragmentation - 48 -p a t t e r n v e r i f i e d t h e p r e s e n c e o f a d i o l f u n c t i o n a l i t y i n t h e p i p e r i d i n e r i n g ( F i g . 2 1 ) . The a s s i g n m e n t o f t h e 18S c o n f i g u r -a t i o n was b a s e d on t h e low f i e l d p o s i t i o n o f t h e C^g-hydrogen r e s o n a n c e (e$6.54) i n t h e "'"Hmr s p e c t r u m ( F i g . 22), w h i l e t h e 3R,4R c o n f i g u r a t i o n was p r o v e n by t r e a t m e n t o f 51 w i t h methane-s u l f o n y l c h l o r i d e i n p y r i d i n e . From t h i s r e a c t i o n were o b t a i n e d two p r o d u c t s . The m a j o r p r o d u c t (52% y i e l d ) was a s s i g n e d as th e 3S,4R-epoxide 52 on t h e b a s i s o f i t s s p e c t r a l p r o p e r t i e s . Thus, t h e "*"Hmr s p e c t r u m ( F i g . 23) was s i m i l a r t o (but d i f f e r e n t from) t h a t o f t h e 3R,4S-epoxide 40 ( F i g . 1 6 ) . H i g h r e s o l u t i o n mass s p e c t r o m e t r y e s t a b l i s h e d t h e m o l e c u l a r f o r m u l a as C_oH_flN_0 OH 52 53 w h i l e t h e p r e s e n c e o f t h e e p o x i d e f u n c t i o n a l i t y i n t h e p i p e r i d i n e r i n g was c o n f i r m e d by t h e mass s p e c t r a l f r a g m e n t a t i o n p a t t e r n ( F i g . 2 4 ) . The m i n o r p r o d u c t (29% y i e l d ) was a s s i g n e d as t h e 3R-mesyloxy d e r i v a t i v e 53 on t h e b a s i s o f i t s ^Hmr s p e c t r u m , w h i c h c o n t a i n e d t h e f o l l o w i n g s i g n a l s : ^ 4 . 6 0 ( I H , d o u b l e t , J = 2 . 5 Hz, C^-H) , c(3.03 ( 3 H , s i n g l e t ,-OS0 2CH 3) . T h i s compound was somewhat u n s t a b l e and on n o r m a l h a n d l i n g was p a r t i a l l y c o n v e r t e d t o t h e Fig. 21. Mass spectral fragmentation pattern of 16,18S-dicarbomethoxy— 3R-hydroxyvelbanamine (51). 3.97 3.96 F i g . 24. Mass s p e c t r a l f r a g m e n t a t i o n p a t t e r n o f 1 6 , 1 8 S - d i c a r b o m e t h o x y — 3 S , 4 R - d i h y d r o c l e a v a m i n e ( 5 2 ) . - 53 -3S,4R-epoxide 52*. The f a c i l e c o n v e r s i o n o f the d i o l 51 t o the 3S ,4R--i— epoxide 52 was c o n c l u s i v e p r o o f o f i t s 3R,4R c o n f i g u r a t i o n , as o n l y i n the case o f a t r a n s - d i a x i a l o r i e n t a t i o n o f the hydroxy and mesyloxy groups ( i n 53) c o u l d the c y c l i z a t i o n have o c c u r r e d 73 so r e a d i l y ; and though the p r e p a r a t i o n o f 51 was i n e f f i c i e n t , i t was now p o s s i b l e t o t e s t the e f f e c t of an a x i a l C ^ - h y d r o x y l group on the s t e r e o c h e m i c a l outcome of the c h l o r o i n d o l e n i n e ' d i m e r i z a t i o n ' . C onsequently, 51 was t r e a t e d w i t h a s o l u t i o n o f sodium methoxide i n methanol from which was o b t a i n e d the d i o l 54 i n q u a n t i t a t i v e y i e l d . The i d e n t i t y o f t h i s p r o d u c t was s u b s t a n t i a t e d HO i C02Me 54 by i t s u l t r a v i o l e t spectrum which was i n d i c a t i v e o f an i n d o l e chromophore. The f a c t t h a t the C-18 p o s i t i o n was not e p i m e r i z e d d u r i n g the r e a c t i o n was c o r r o b o r a t e d by the c h e m i c a l s h i f t ( i n the "'"Hmr spectrum) of the C^g-hydrogen («f5.45). *Although 53 was o r i g i n a l l y i s o l a t e d as a s i n g l e compound by t h i n l a y e r chromatography, a f t e r d e t e r m i n a t i o n of the iHmr spectrum the sample c o n t a i n e d c l o s e t o 50% of the epoxide 52. - 54 -Treatment of 54 with 1-chlorobenzotriazole in benzene afforded the chloroindolenine 55 i n 57% y i e l d . * The HO / i CO Me spectral properties of this product were in accord with the assigned structure. Thus, high resolution mass spectrometry established the molecular formula as C^^E^^2^' w h i l e the u l t r a v i o l e t spectrum (Fig. 25) was indicative of the new chromo-phore. The H^mr spectrum exhibited a signal due to the C^g— hydrogen (£5.79,lH,doublet of doublets,J=2,12 Hz) and lacked a signal for an N a-hydrogen. With the chloroindolenine 55 now available, the next step involved i t s coupling with vindoline under acidic conditions. Treatment of 55 with vindoline i n methanolic hydrogen chloride however afforded no i s o l a b l e 'dimeric' products.** Indeed *This compound tended to decompose during i s o l a t i o n and thus the low y i e l d of isolated product was not truly indicative of the chlorination reaction which by thin layer chromatographic analysis appeared to be quantitative. **This reaction was carried out by T. Hibino several times, using the various conditions which previously were successful for the coupling of 18S-carbomethoxy-4S-dihydrocleavamine chloro-indolenine with vindoline.44 - 55 -log £ 250 300 350 X nm Fig. 25. U l t r a v i o l e t spectrum of the chloroindolenine 55. greater than 90% of the vindoline could be recovered from the reaction mixture. This negative re s u l t cast some doubt on the o r i g i n a l premise, however due to the lack of any coupling reaction or other tangible consequences, no d e f i n i t i v e conclusions could be made. Therefore, studies were undertaken to ascertain why the 1dimerization 1 had f a i l e d and to determine whether the presence of an a x i a l C^-hydroxyl functionality could change the 'normal' stereochemical outcome of the chloroindolenine coupling reaction. Examination of the proposed general mechanism of the - 56 -Fig. 26. Proposed general mechanism of chloroindolenine 'dimerization'. - 57 -chloroindolenine 'dimerization 1 (Fig. 26) suggested that the lack of reaction between a chloroindolenine derivative and vindoline could be due to formation of the quaternary ammonium sa l t 57. This type of quaternary ammonium s a l t had previously 49 7 been prepared i n this laboratory ' and shown to be unreactive 49 towards vindoline. Thus, a rapid rate of internal quaterniza-tion r e l a t i v e to the rate of 'dimerization' would explain the lack of 'dimeric'product observed in the attempted coupling of 55 with vindoline. Support for this explanation was provided by examination of molecular models which showed that for internal quaternization to occur the N^-lone pair would have to occupy an a x i a l orientation r e l a t i v e to the piperidine ring, and as the quaternization reaction 'proceeded (i-»ii) there would be a strong s t e r i c interaction between C-18 and the C-4 axial (or pseudoaxial) substituent. It therefore followed that the greater Me the s t e r i c bulk of the C-4 substituent the less favorable would be internal quaternization. Accordingly, the effective coupling of 18S-carbomethoxy-4S-dihydrocleavamine chloroindolenine (22) with vindoline (to afford the dimer 23 in ~ 65% yield) could be - 58 -attributed to a slow rate of quaternization (relative to 'dimerization') which i n turn was due to the s t e r i c hindrance provided by the a x i a l C^-ethyl group. Conversely, the un-successful coupling of 18S-carbomethoxy-3R-hydroxyvelbanamine chloroindolenine (55) with vindoline could be attributed to an increased rate of internal quaternization (relative to 'dimerization') resulting from the diminished s t e r i c hindrance afforded by an a x i a l C^-hydroxyl group. Further substantiation of this rationale was furnished by the previously performed coupling of 18S-carbomethoxycleavamine chloroindolenine (58) with vindoline, from which 18'-epi-3',4'-anhydrovinblastine (59) was obtained i n ca 10% y i e l d . Since the s t e r i c impedance afforded by the v i n y l ethyl group* toward internal quaternization *The spacial relationship between C-18, the electron pair and the C-4 axial (or pseudoaxial) substituent is nicely demonstrated by a comparison of the chemical s h i f t of the C]_g-hydrogen in the 1Hmr spectra of various 18S-carbomethoxy-cleavamine derivatives (Table 3). Since the chemical s h i f t of the Cl8 -hydrogen depends upon i t s proximity to the electron pair, a comparison of this s h i f t as a function of the C o a x i a l (or pseudoaxial) substituent would r e f l e c t the 1,3-diaxial inter-action of the Nfc, electron pair with that substituent. Indeed, Table 3 shows a positive correlation between the C]_g-hydrogen' s chemical s h i f t and the s t e r i c bulk afforded by the C-4 func-t i o n a l i t y . Presumably, the greater the s t e r i c bulk, the greater the displacement of the N]-, electron pair away from C-18. Table 3 . Comparison of the chemical s h i f t s of the C l g — hydrogen of various 16,18S-dicarbomethoxycleavamine derivatives. - 60 should have been intermediate to that afforded by the ax i a l ethyl group in 22 and the axial hydroxyl group i n 55, the intermed-iate y i e l d of 59 (i.e. 10%) was reasonable. Another derivative available to further evaluate the 'standard chloroindolenine dimerization' was the epoxide 60 which was available from 40 in 50% y i e l d * by the action of sodium methoxide in methanol. In accord with the proposed structure, the u l t r a v i o l e t spectrum was indicative of the indole chromophore. High resolution mass spectrometry confirmed, the *This compound was found to be sensitive to acidic and basic media and the low y i e l d of isolated product obtained l i k e l y r e f l e c t s the occurrence of decomposition during the reaction and subsequent p u r i f i c a t i o n by chromatography on alumina. 4 0 , ***** 6 0 , R=C02Me R=H - 61 -molecular formula as C21 H26 N2°3' w^^ --'-e t l l e assignment of the 18S configuration was based upon the low f i e l d position of the C i g-hydrogen resonance i n the "'"Hmr spectrum (^5.19,doublet of doublets,J=10,5 Hz). Treatment of 60 with 1-chlorobenzotriazole afforded the chloroindolenine 61 i n 67% y i e l d . High resolution mass i C02Me 61 spectrometry confirmed the molecular formula as C 2^H 2 ,.N203C1, while the u l t r a v i o l e t spectrum was analgous to that obtained for 55. Treatment of 61 with a solution of vindoline i n methanolic hydrogen chloride afforded four 'dimeric' products in a combined y i e l d of ca 17%. Although structures were not assigned to these products the fact that they were 'dimers' was established by th e i r u l t r a v i o l e t spectra which were indica-tive of the superposition of an indole and dihydroindole chromophore (Fig. 27). The i s o l a t i o n of several dimers was l i k e l y due to the i n s t a b i l i t y of the epoxide functionality i n the strongly acidic reaction conditions employed. Of particu-lar interest however was the observed overall y i e l d of 'dimeric' - 62 -log £ 250 300 250 300 )\ nm X nm F i g . 27. Comparison of the u l t r a v i o l e t spectra of a t y p i c a l indole derivative (A), dihydroindole derivative (B), and 'dimer' (C). material which was similar to that obtained i n the 'dimerization' reaction of the o l e f i n 59. Since the s t e r i c requirements for the internal quaternization were similar in both cases the observed results were consistent. To this point the important role of internal quater-nization as a competing reaction i n the chloroindolenine ' d i -merization' had been strongly implied but not proven. Thus, this proof was undertaken. Oxidation of the 3R,4S-epoxide 40 77 with either chromium trioxide i n pyridine or mercuric acetate 7 8 in aqueous dioxane afforded the lactam epoxide 62. This de-- 63 -ri v a t i v e could not undergo internal quaternization as the electron pair was now involved i n resonance s t a b i l i z a t i o n of the adjacent carbonyl group. Me02C C02Me 62 The structural assignment of 62 was based on analysis of spectral data. Thus, the presence of the amide functionality was corroborated by a strong absorption at 164 2 cm ^ in the infrared spectrum (Fig. 28). High resolution mass spectrometry established the molecular formula as C»_.H_^N-0,. Furthermore, 2 3 26 2 6 that the lactam obtained was indeed the 19-oxo derivative 62 and not the 5- or 7-oxo derivatives 63 or 64 was deduced from 13 1 13 the Cmr and Hmr spectra. The Cmr spectrum (Fig. 29) exhibited 63 64 F i g . 28. Infrared spectrum of 16,18S-dicarbomethoxy-3R,4S-epoxy-19-oxodihydro-cleavamine (62). 171.9 lU 59.3 61.4 i / O ' w V ^ r v . TMS 1 3 Fig. 29. Cmr spectrum of 16,18S-dicarbomethoxy-3R,4S-epoxy-19-oxodihydrocleavamine (62). - 66 -s i g n a l s f o r C-3 and C-4 a t J61.4 and £59.3 r e s p e c t i v e l y . C o m p a r i s o n o f t h e s e s i g n a l s w i t h t h o s e o f t h e s t a r t i n g e p o x i d e 40 (£60.6 and £62.7) i n d i c a t e d t h a t C-3 had been s h i f t e d down-f i e l d by 0.8 ppm w h i l e C-4 had been s h i f t e d u p f i e l d by 3.4 ppm. S i n c e a c a r b o n y l g r o u p n o r m a l l y c a u s e s a s u b s t a n t i a l d o w n f i e l d 79 s h i f t o f t h e a d j a c e n t c a r b o n r e s o n a n c e ( c f F i g . 30), t h e o b s e r v e d p o s i t i o n o f t h e C-4 s i g n a l was i n c o m p a t i b l e w i t h s t r u c -t u r e 63. F i n a l l y , s t r u c t u r e 6 4 was deemed u n a c c e p t a b l e on t h e b a s i s o f t h e "'"Hmr s p e c t r u m . Thus, a 7-oxo d e r i v a t i v e w o u l d be A B 13 F x g . 30. Compari s o n o f t h e Cmr c h e m i c a l s h i f t s (S) o f h e x a h y d r o a z e p i n e 8 0 (A) and c a p r o l a c t a m 7 9 ( B ) . e x p e c t e d t o e x h i b i t an AB q u a r t e t due t o t h e C-8 h y d r o g e n s s u c h 81 1 as was o b s e r v e d f o r 65 (<$3.94, AB q u a r t e t ) . I n d e e d , t h e Hmr s p e c t r u m ( F i g . 31) l a c k e d such a s i g n a l (more e a s i l y seen i n t h e "'"Hmr s p e c t r u m — F i g . 3 2--of t h e N a - d e s c a r b o m e t h o x y d e r i v a t i v e 66) w h i l e e x h i b i t i n g r e s o n a n c e s i n a c c o r d w i t h s t r u c t u r e 62 U4.32, - 68 -OMe b 65 l H , m u l t i p l e t , C 2 - H * ; J4 .1, 1H,doublet ,J-6 Hz,C 1 8-H). With the i d e n t i t y of the lactam 6 2 secured the next step was regeneration of the i n d o l e chromophore. Treatment of 62 with sodium methoxide i n methanol a f f o r d e d 66 i n 95% y i e l d . Me02C 66 The presence of the i n d o l e chromophore was s u b s t a n t i a t e d by the u l t r a v i o l e t spectrum . Fu r t h e r c o r r o b o r a t i o n f o r s t r u c t u r e 66 was a v a i l a b l e from the "^ Hmr spectrum ( F i g . 32) which e x h i b i t e d the f o l l o w i n g s i g n a l s : <4 8.67 (lH,bs,N -H) ; & 4.33 (IH,doublet a . — of t r i p l e t s , J = 1 3 , 3 Hz,C 2~H), & 4.05 (IH,dd,J=10.5,1.5 Hz,C l g-H). *The low f i e l d p o s i t i o n of the C^-hydrogen resonance was a s c r i b e d to the f a c t t h a t i t was e c l i p s e d with the adjacent carbonyl group (at C-19) and thus d e s h i e l d e d s t r o n g l y by the a n i s o t r o p i c e f f e c t of the carbonyl. Some d e s h i e l d i n g could a l s o be a t t r i b u t e d to the s t e r i c a l l y crowded environment of the C2 _hydrogen. 6 3.71 - 70 -F i n a l l y , the 18S configuration was assigned on the basis of previous results where no epimerization of this center had been observed. Having 66 available, the next step attempted was chlorination of the indole. Treatment of 6 6 with 1-chloro-benzotriazole in benzene however did not y i e l d the expected chloroindolenine 67, instead two i n d o l i c products were isolated (Fig. 33). The major product (40% yield) was assigned as the CI Fig. 33. Outline of the reaction of 18S-carbomethoxy-3R,4S— epoxy-19-oxodihydrocleavamine (66) with 1-chlorobenyotriazole. - 71 -18R-benzotriazole adduct 68. In accord with t h i s s t r u c t u r e the "'"Hmr spectrum (Fig. 34) lacked a C^g-hydrogen resonance while e x h i b i t i n g s i g n a l s corresponding to four new aromatic protons. The chemical s h i f t s of these protons i n d i c a t e d that they were magnetically d i f f e r e n t from each other and thus l e d to the assignment of the linkage between C-18 and the 1-position of be n z o t r i a z o l e . The t e n t a t i v e assignment of the stereochemistry as 18R was based on the f a c t that i n previous 'couplings' the nucleophile ( i . e . v i n d o l i n e ) approached from the '/3-face 1. F i n a l l y , high r e s o l u t i o n mass spectrometry confirmed the molecu-l a r formula as C 2 7 H 2 7 N 5 0 4 . The minor product (27%) was assigned as the 18R-hy-droxy adduct 69. High r e s o l u t i o n mass spectrometry e s t a b l i s h e d the molecular formula as C2i H24 N2 <" )4 w*1-'--'-e ^ he ^ "Hmr spectrum (Fig. 35) lacked a C^-hydrogen resonance i n d i c a t i n g the hy-droxy 1 group at that p o s i t i o n . Although the chloroindolenine 6_7 was not obtained, the adducts 6 8 and 6 9 were as s u i t a b l e f o r the required purpose ( i . e . coupling with v i n d o l i n e ) , as under a c i d i c conditions the 44 e q u i l i b r i u m i i i ^ = s . i v would be at t a i n e d . Therefore, a s o l u -t i o n of 68 i n methylene c h l o r i d e was treated with v i n d o l i n e and a small amount of t r i f l u o r o a c e t i c a c i d . From t h i s r e a c t i o n a s i n g l e product was obtained (89% y i e l d ) which on the basis of i t s s p e c t r a l p r o p e r t i e s was assigned as 18'-epi-19'-oxoleuro-sine (70). High r e s o l u t i o n mass spectrometry e s t a b l i s h e d the molecular formula as C46 H54 N4° ] _ o ' T l l e ^ H m r spectrum ( F i g . 36) 3.83 i 1 r i 1 1 1 r Fig. 34. Hmr spectrum of 68. - 74 -contained singlets at i6.87 (C,.-H) and 5.9 8 (C,_-H) i n accord with a 15-substituted vindoline derivative. F i n a l l y , this 'dimer' was assigned the 'unnatural' stereochemistry at C-18' (i.e. 18R) on the basis of i t s c i r c u l a r dichroism (cd) curve which exhibited 8 2 8 strong absorptions at 208 nm (A£=+12.4) and 222 nm (A£=-41.3). ' Previously the cd curves of various natural and 'unnatural dimers' (at C-18') had been correlated and shown to be of interpretive value. Thus, the 'natural dimers' (i.e. 18'S configuration) exhibited a strong negative absorption at ca 208 nm and a strong positive one at ca 222 nm while the 'unnatural dimers' ( i.e. 18'R configuration) exhibited the reverse. 2 . 0 6 T 1 1 r F i g . 36. Hmr spectrum of 18'-epi-19'-oxoleurosine (70). - 76 -The excellent y i e l d of dimer obtained i n the coupling reaction of the lactam 68 with vindoline versus the poor y i e l d obtained i n the reaction of the amine 61 established the s i g n i - • ficance of internal quaternization as a side reaction during attempted 1dimerizations 1. Having established quaternization as a serious draw-back, a more eas i l y reversed method of blocking this path was sought. One p o s s i b i l i t y was formation of the N^-iminium species, which upon reduction with sodium borohydride would regenerate the parent amine. Therefore, 18S-carbomethoxy-3R,4S-epoxy-dihydrocleavamine chloroindoline (61) was converted to i t s N^-oxide 71 in the presence of vindoline (by the action of m— chloroperbenzoic acid) and this mixture was treated with t r i -fluoroacetic a c i d / t r i f l u o r o a c e t i c anhydride (presumably generating the iminium species 72*) (Fig. 37). Quenching of the reaction with sodium borohydride afforded after p u r i f i c a t i o n 18 1-epileurosine (73) i n 40% y i e l d . The identity of this product was in accord with i t s spectral properties. High resolution mass spectrometry confirmed the molecular formula as C^gH^gN^Og, while the cd curve established the configuration at C-18' as R. F i n a l l y , the "^ Hmr spectrum (Fig. 38) was similar to those of 44 various 'unnatural dimers' obtained e a r l i e r . *The formation of the iminium derivative i s an application of the Polonovski reaction.51 On the basis of geometric and electronic considerations i t was f e l t that the elimination process to form the iminium ion would occur rather than any C-C bond fragmentation process. - 77 -t r i f l u o r o -acetic acid t r i f l u o r o a c e t i c anhydride 72 Vindoline Fig. 37. Proposed mechanism for coupling of chloroindolenine 61 with vindoline. - 79 -T h e r e f o r e , h a v i n g m o d i f i e d the c o n d i t i o n s of the c h l o r o i n d o l e n i n e ' d i m e r i z a t i o n ' so as t o m i n i m i z e i n t e r n a l q u a t e r n i z a t i o n as w e l l as the d e c o m p o s i t i o n o f p r o d u c t s , the next aim was the c o u p l i n g o f 18S-carbomethoxy-3R-hydroxyvel-banamine c h l o r o i n d o l e n i n e (55) w i t h v i n d o l i n e . To t h i s end, 55 was t r e a t e d w i t h v i n d o l i n e u s i n g the m o d i f i e d r e a c t i o n c o n d i -t i o n s . I n v e s t i g a t i o n o f the p r o d u c t m i x t u r e a f f o r d e d a s i n g l e ' d i m e r i c ' p r o d u c t (19% y i e l d ) which on the b a s i s o f i t s s p e c t r a l p r o p e r t i e s was i d e n t i f i e d as 1 8 ' - e p i v i n c a d i o l i n e (74). High HO 74 r e s o l u t i o n mass sp e c t r o m e t r y e s t a b l i s h e d the m o l e c u l a r f o r m u l a as <-4gH5gN4<- )]_o' w n i l e t n e c& curve a f f i r m e d the 18R assignment. Furthermore, the ^Hmr spectrum ( F i g . 39) was s i m i l a r t o those 4 4 of p r e v i o u s l y p r e p a r e d ' u n n a t u r a l dimers'. I n summary, i t was shown t h a t i n t e r n a l q u a t e r n i z a t i o n c o u l d be a s e r i o u s s i d e r e a c t i o n . Hence, the c h l o r o i n d o l e n i n e ' d i m e r i z a t i o n ' was m o d i f i e d so as t o m i n i m i z e t h i s u n d e s i r a b l e r e a c t i o n . U s i n g the m o d i f i e d c o n d i t i o n s , 18S-carbomethoxy— 3R-hydroxyvelbanamine c h l o r o i n d o l e n i n e (55) c o u l d be co u p l e d w i t h v i n d o l i n e t o a f f o r d o n l y the ' u n n a t u r a l ' s t e r e o c h e m i s t r y 'dimer' ( i . e . 74). Fig. 39. Hmr spectrum of 18 1-epivincadioline (74). - 81 -In conclusion, the i n i t i a l premise, that the chloro-indolenine of 18-carbomethoxyvelbanamine would be a precursor of the 4S,18R-ether 32, was found to be incorrect. Rather i t was seen that derivatives possessing an axial C41-hydroxy1 functionality as well as a cationic center at C-18 readily underwent inte r n a l quaternization. Thus, the argument for the preparation and coupling (with vindoline) of derivatives po-ssessing an axial C^-hydroxyl group (i.e. various velbanamine derivatives) became f a l l a c i o u s . Furthermore, the structure 37, which was reported i n the l i t e r a t u r e as being the i n d o l i c product obtained from the acid catalyzed cleavage of vinblastine i n the absence of reducing agents, tended to contravene the re-sults obtained here. In view of these results i t was f e l t that a more plausible structure would be the ammonium s a l t 75, however this proposal must be considered purely speculative. F i n a l l y , although the synthesis and 'dimerization' (with vindoline) of cleavamine derivatives whose geometry precludes reaction with nucleophiles from the ' /9-face' (e.g. 32 and 33) s t i l l represents - 82 -a v a l i d goal, the d i f f i c u l t y encountered i n introducing an a x i a l C^-hydroxyl group into 18S-carbomethoxycleavamine (20) forced abandonment of the approach i n favor of other more pro-mising routes, such as the functionalization of 3',41-anhydro-vinblastine (26) (Sec. 2.2, 2.3).. - 83 -2.2 Chemistry o f 3 ' , 4 1 - A n h y d r o v i n b l a s t i n e — S y n t h e s i s of L e u r o s i n e and C a t h a r i n e . A l t h o u g h f u r t h e r e v a l u a t i o n o f the ' c h l o r o i n d o l e n i n e approach' d i d not l e a d t o the a n t i c i p a t e d outcome, the c h e m i s t r y t h a t was developed ( f o r the e l a b o r a t i o n o f 18S-carbomethoxy-cleavamine, 19) d i d p r o v i d e a f o u n d a t i o n f o r the i n i t i a l s t u d i e s aimed a t f u n c t i o n a l i z a t i o n o f 3 ' , 4 ' - a n h y d r o v i n b l a s t i n e (26). 19 26 Accordingly, the f i r s t modification of 26 attempted, was pro-tection of the indole chromophore. However, despite treatment of 26 with varying amounts of potassium hydride (2-5 equivalents) and methyl chloroformate i n tetrahydrofuran at various tempera-tures (0-50°C), only starting material or decomposition products could be isolated.* Indeed, no evidence for the formation of N ai-carbomethoxy derivatives could be obtained. The lack of reaction at the N a i position of the 'dimer' was presumed due to reduced a c c e s s i b i l i t y of this position as a result of s t e r i c hindrance afforded by the C-18' substituents (i.e. vindoline and *This study was carried out by T. Hibino. - 84 -the carbomethoxy group). Examination of molecular models i n d i -cated that the C-18' substituents would also afford s t e r i c im-pedance to approach of reagents at the ft-position of the indole chromophore (i.e. C-9), and thus i t was reasoned that protec-tion of the indole chromophore might not be necessary. Therefore, epoxidation of 3 1,4'-anhydrovinblastine using the previously developed conditions* (tetrahydrofuran, aqueous 1% t r i f l u o r o -acetic acid, t-butyl hydroperoxide) was performed. From this 62 63 reaction leurosine (3) was obtained i n 51% y i e l d . ' The identi t y of the synthetic leurosine was established v i a compari-son with an authentic sample. Further examination of the t-butyl hydroperoxide reaction i n -dicated that under prolonged reaction times leurosine would undergo further reaction** to y i e l d another naturally occurring *This reaction was i n i t i a l l y performed by T. Hibino. **Originally, the structure of the overoxidation product of leurosine was incorrectly assigned as the lactam 77.63/64 - 85 -catharanthus a l k a l o i d , Catharine (76). The s t r u c t u r e of 85 C a t h a r i n e h a d b e e n e l u c i d a t e d b y an x-ray a n a l y s i s , w h i l e 19 C H O 76 77 t h e i d e n t i t y of t h e s y n t h e t i c m a t e r i a l as C a t h a r i n e w a s e s -t a b l i s h e d by c o m p a r i s o n w i t h an a u t h e n t i c s a m p l e ( c f . F i g . 40 a n d 41) . The pr o d u c t i o n of two n a t u r a l products coupled with the unusual s t r u c t u r e of one of them prompted f u r t h e r examina-t i o n of t h e i r formation. Under the o r i g i n a l c o n d i t i o n s employed a r a d i c a l and/or a c i d c a t a l y s e d mechanism was p o s s i b l e . There-f o r e , a number of r e a c t i o n s were c a r r i e d out (Table 4) i n order to d i s t i n g u i s h between the p o s s i b i l i t i e s . A e r i a l or t - b u t y l hydroperoxide o x i d a t i o n of 26 i n tet r a h y d r o f u r a n c o n t a i n i n g aqueous 1% t r i f l u o r o a c e t i c a c i d gave r i s e to Catharine (ca 30% y i e l d ) a f t e r eleven and f i v e days r e s p e c t i v e l y (Rx #i t #2, #12} Monitor i n g the course of the r e a c t i o n v i a t h i n l a y e r chromatography i m p l i c a t e d l e u r o s i n e as the p r e c u r s o r of Catharine. A c c o r d i n g l y , a e r i a l or t - b u t y l h y d r o p e r o x i d e o x i d a t i o n of l e u r o s i n e a l s o a f f o r d e d C a t h a r i n e (Rx #10 and #11). A l t e r n a t i v e l y , the r e a c t i o n of 3',4'-anhydro-v i n b l a s t i n e or l e u r o s i n e with t - b u t y l hydroperoxide could be Fig. 40. Hmr spectrum of Catharine (sample was obtained from the E l i L i l l y Laboratories, Indeanapolis). - 88 -Table 4. Study of the a e r i a l and t-butyl hydroperoxide oxidations of 3 4 1 - a n h y d r o v i n b l a s t i n e , leurosine, and derivatives thereof. Rx# 1. 3. 3',4 1-anhydrovinblastine 3' , 4 1-anhydrovinblastine t-BuOOH THF,1%TFA(aq) 22 h t-BuOOH THF,1%TFA(aq) 5 days 3',4 1-anhydrovinblastine t-BuOOH 3 1,4'-anhydrovinblastine THF-H20 22 h t-BuOOH 3 ' , 4 ' - a n h y d r o v i n b l a s t i n e THF 22 h t-BuOOH THF,1%TFA(aq) MeOH 22 h 3 1,4'-anhydrovinblastine t-BuOOH THF,1%TFA(aq) rad. inhib.* rx carried out in dark 3 ' , 4'-anhydrovinblastine N bi-oxide 3',4'-anhydrovinblastine t-BuOOH THF,1%TFA(aq) 22 h l e u r o s i n e (+++) Catharine (±) 78 or 79 (±) l e u r o s i n e (+) Catharine (++] 78 o r 79 (+) ~ o t h e r s (+) l e u r o s i n e (+) C a t h a r i n e (+) 78 o r 79 (+)" " o t h e r ( + + + ) S.M. (±) leurosine (±) Catharine (++) 78 or 79 (±) Others (++) S.M. (++) leurosine (++] 78 or 79 (±) S.M. (+++) leurosine (+) 78 or 79(+) no rx t-BuOOH THF,5 %TFA (aq) 22 h no rx *The ra d i c a l i n h i b i t o r used was 3-t-butyl-4-hydroxy-5-methyl-phenyl s u l f i d e . - 89 -Table 4 con't Rx# 9. leurosine t-BuOOH THF,5 %/TFA (aq) 22 h no rx 10. leurosine t-BuOOH S.M. (++) THF,1%TFA(aq) catharine (+) 22 h 79 ( + ) 11. leurosine A i r = 2 * 12. or 3 1,4 1-anhydrovinblastine leurosine (+) THF,1%TFA(aq) catharine (++) 11 days others (+) 13. leurosine t-BuCOH others (+) c n 2 c i 2 catharine (+++) (48% yield) 14. leurosine t-BuOOH S.M. (+++) THF,1%TFA(aq) catharine (±) 22h, rad inh* 79 (±) & rx i n dark 1%TFA(aq) THF 15. leurosine t-BuOOH 79 (+++) 44h, rad inh*,** rx i n dark 16. leurosine-N, ,-oxide (PleurosineT t-BuOOH THF , 1%TFA (aq) 22 h no rx 17. leurosine A i r CH 2C1 2 22 h no rx *The radic a l i n h i b i t o r used was 3-t-butyl-4-hydroxy-5-methyl-phenyl su l f i d e . **The difference between this reaction and reaction #14 (aside from the length of time) was i n the amount of ra d i c a l i n h i b i t o r used (i.e. greater i n this case). - 90 -s i g n i f i c a n t l y i n h i b i t e d , o r t h e c o u r s e o f t h e r e a c t i o n changed, on a d d i t i o n o f a r a d i c a l i n h i b i t o r (Rx #6,#14,#15). The r o l e o f aqueous a c i d i n t h e o x i d a t i o n o f 3',4'— a n h y d r o v i n b l a s t i n e was n o t c l e a r . A l t h o u g h l e u r o s i n e was formed i n t h e ab s e n c e o f a c i d (Rx #3 and #4), t h e r e s u l t a n t p r o d u c t m i x t u r e s were complex and c o n t a i n e d a low p r o p o r t i o n o f l e u r o -s i n e . On t h e o t h e r hand C a t h a r i n e f o r m a t i o n d i d n o t r e q u i r e a c i d o r w a t e r (Rx #4 and #13). I n d e e d C a t h a r i n e c o u l d be o b t a i n e d from l e u r o s i n e i n 48% y i e l d by t h e a c t i o n o f t - b u t y l h y d r o -p e r o x i d e i n m e t h y l e n e c h l o r i d e . F i n a l l y , t h e i n t e r m e d i a c y o f t h e N b , - o x i d e s 78* and 86 79 was, un d e r t h e r e a c t i o n c o n d i t i o n s employed, a p o s s i b i l i t y . 78 79 T h e r e f o r e , 78 and 79 were t r e a t e d w i t h t - b u t y l h y d r o p e r o x i d e i n t e t r a h y d r o f u r a n c o n t a i n i n g aqueous 1% t r i f l u o r o a c e t i c a c i d (Rx #7 and #16). S i n c e no r e a c t i o n had o c c u r r e d a f t e r 22 h o u r s t h e i r i n t e r m e d i a c y was r u l e d o u t . I n summary, 3 1 , 4 1 - a n h y d r o v i n b l a s t i n e c o u l d be o x i d i z e d *3 ' , 4 1 —Anhydrovinblastine—Nj-)< — o x i d e (7,9) had p r e v i o u s l y been p r e p a r e d by G.H. Bokelman. 6 2,63 - 91 -to leurosine in the presence of peroxides presumably v i a a ra d i c a l mechanism. Aqueous acid was necessary for a syntheti-c a l l y useful conversion possibly due to i n h i b i t i o n of unwanted side reactions. Catharine was formed from leurosine via a rad-i c a l mechanism with neither acid nor water being required. The intermediacy of the N, .-oxides 7 8 and 79 was ruled out. b — ~~ In conclusion, the f i r s t syntheses of leurosine a n d C a t h a r i n e were a c h i e v e d from 3 1 , 4 ' - a n h y d r o v i n b l a s t i n e by u t i l i z i n g the appropriate conditions. Furthermore, based on the oxidation studies, possible mechanisms for the formation of leurosine and catharine could be put forth (Fig. 42 and 43). Fig. 42. Possible mechanism for the formation of leurosine from 3',4 1-anhydrovinblastine. - 92 -F i g . 43. P o s s i b l e mechanism f o r the formation of catharine from leu rosine-- 9 3 -2.3 Chemistry of 3',4 1-Anhydrovinblastine—Synthesis of Vinamidine* (Catharinine) (82) and Related Derivatives. Further investigations into the functionalization of 3 1,4 1-anhydrovinblastine (and leurosine) led to the discovery of the potassium permanganate oxidation as a propitious method. Thus, from the reaction of 3',4'-anhydrovinblastine with a solution of potassium permanganate in acetone was obtained two 8 T main products. The minor product (10%) was assigned as the 5' *Vinamidine o r i g i n a l l y was incorrectly assigned as the C]_gi-C2i cleavage derivative 80.3 6 Subsequently i t was found to be i d e n t i c a l with the alkaloid c a t h a r i n i n e 8 8 — a compound whose 19 CHO structure was assigned as the C 4 1 - C 5 1 , cleavage derivative 82 on the basis of an x-ray analysis of a degradation product. - 94 -lactam 81 based on a comparison with an authentic sample.* The major product (42%) was i d e n t i f i e d (ex post facto) as 3R-hydroxyvinamidine (83)—a novel derivative of the naturally 88 occurring alkaloid vinamidine (82). The identity of this product was established v i a several chemical transformations and exhibited spectral properties in agreement with the assign-ment. High resolution mass spectrometry confirmed the molecular formula as C^gH^gN^O^^, while the presence of the t-formyl group was indicated by the strong bond at 16 60 cm-''" in the infrared spectrum (Fig. 44). Further evidence for the i — formyl group was provided by the signal at 6 7.32 (IH,singlet, N^i-CHO) in the ^ Hmr spectrum (Fig. 45). The presence of a secondary alcohol was proven by acetylation of 83 using acetic anhydride/pyridine. From this reaction was obtained the ketoacetate 84 i n 65% y i e l d . In accord C H O Me02C Vind. 84 *Previously, this compound had been prepared by G.H. Bokelman in this laboratory from the reaction of 3 1,4'-anhydrovinblas-tine-N]-, i -oxide with one equivalent of osmium tetroxide. The structural assignment was based on analysis of spectral properties and should be considered tentative.6 2,6 3 Fig. 44. Infrared spectrum of 3R-hydroxyvinamidine (83). - 97 -with this structure, high resolution mass spectrometry gave the molecular formula as C4 8H5gN4°]_2' w n i i e t n e H^mr spectrum (Fig. 46) substantiated the presence of the new secondary ace-tate with signals at & 4.80 (IH,broad sing l e t / , -H) and cf 2.10 (3H,singlet,-OCOCH 3). The presence of a ketone functionality was proven by sodium borohydride reduction of 83 from which was obtained the t r i o l 85 i n 66% y i e l d . Accordingly, high resolution mass spec-trometry gave the molecular formula as C46 H5g N4°]_i' while the infrared spectrum confirmed the presence of several alcohol f u n c t i o n a l i t i e s with a moderately strong bond at 3585 cm i 1 1 r Fig. 46. Hmr spectrum of 3R-acetoxyvinamidine (8A-) . - 99 -Acetylation of the t r i o l 85 yielded two products. The major product (25% yield) was assigned as the triacetate 86. Confirming this assignment, high resolution mass spectrometry gave the molecular formula as C 5 o H 6 2 N 4 ° 1 3 ' w n :*- l e t h e "*"Hmr spectrum (Fig. 47) revealed the presence of two new secondary acetates with signals at<£4.89 (lH,multiplet,C 4 , -H) , & 4 . 41 (IH, t r i p l e t , J=6 Hz ,C3 ,-H) , & 2. 08 (3H, singlet,-OCOCH 3) , and «£ 1.97 (3H, singlet,_OCOCH 3). The minor product (24% yield) was assigned as the tetraacetate 87. Thus, high resolution mass spectrometry established the molecular formula as C c-H,.N.O.while the 52 64 4 14 H^mr spectrum (Fig. 48) exhibited signals corresponding to four acetates at £ 2.08 (6H,singlet,2X-OCOCH3) and £ 1.98 (6H,singlet, 2X-OCOCH3). Oxidation of 83 with cupric acetate i n hot methanol afforded the o<-diketone 88. Since this oxidation i s a s p e c i f i c MeCL C ... . 2 Vind. 88 89 method for the conversion of c<-ketols to o<.-diketones , the presence of this functionality (oC-ketol) i n 83 was established, In r e l a t i o n to characterization of the <X-diketone 88, high re-solution mass spectrometry gave the molecular formula as C46 H54 N4°11' wh:'--1-e t h e infrared spectrum provided further e v i -- 102 -dence with a strong absorption at 1713 cm in agreement with the presence of the diketone group. Elimination of the alternate cleavage structure '89 as a p o s s i b i l i t y for the potassium permanganate product was CHO Me0 2 G Vind. 89 accomplished by periodic acid oxidation of the ketol 83 or the d i o l 85. The exclusive nitrogenous product obtained from both of these reactions was the aldehyde 90. The "^Hmr spectrum 90 (Fig. 49) of 90 exhibited one methyl t r i p l e t at £ 0.74 r as well as singlets at &7.61 and &9.20 corresponding to the N^-formyl and aldehydic hydrogens respectively. High resolution mass spec-trometry established the molecular formula as C 4 3 H 5 o N 4 ° i o ' t h u s confirming the loss of a three carbon fragment.. Fig. 49. Hmr spectrum of aldehyde 90. - 104 -F i n a l l y , the stereochemistry at C-31 was assigned as R on the basis of the reaction of leurosine with potassium permanganate. From this reaction were obtained two products. The major product (27% yield) was found to be i d e n t i c a l with 83. Since the absolute configuration of leurosine was known, the assignment of the absolute configuration of 83 was based on the assumption that the stereochemical i n t e g r i t y of C-3' was main-tained. The minor product (19% yield) was assigned as 19'— oxoleurosine (91) on the evidence of i t s spectral properties. High resolution mass spectrometry affirmed the molecular formula as C.,Hr.N . 0 , w h i l e the infrared spectrum (Fig. 50) contained 46 54 4 10 ^ ^ a strong absorption at 1644 cm 1 i n accord with the presence of 13 an amide carbonyl. The Cmr spectrum exhibited signals at 61.6 and 59.8 corresponding to C-31 and C-4'. The lack of any si g n i f i c a n t change i n the position of these signals r e l a t i v e to the analgous signals i n leurosine (£60.3 and ^ 59.9) corroborated the l9'-oxo (rather the 5'-oxo) assignment. F i n a l l y , the "*"Hmr spectrum (Fig. 51) contained a signal at£4.76 (IH,multiplet) which was attributable to the C 2•-hydrogen. F i g . 51. Hmr spectrum of 19'-oxoleurosine (91). - 107 -Having 3R-hydroxvinamidine available, a synthesis of vinamidine (82) was sought. Since various methods for de-oxygenation of alcohols and their derivatives were available, this route was i n i t i a l l y investigated. However, attempted formation of the thiobenzoate 92—a derivative which normally CHO OC II Me0 2C Vind. 92 90 should be easily deoxygenated — r e s u l t e d only i n the formation of decomposition products, while the use of the acetate 84 as a substrate for deoxygenation reactions (such as zinc metal i n hot 91 92 acetic acid or lithium dimethyl cuprate i n tetrahydrofuran ) afforded starting material (84) and/or alcohol 83. Therefore, an alternative means of synthesizing vinamidine, v i a extension of the potassium permanganate reaction was undertaken. Treat-ment of 4 1-deoxyleurosidine (93) (available from 3',4'-anhydro-31 33 vinblastine by hydrogenation over a platinum catalyst ' ) afforded vinamidine in 22% y i e l d . The identity of the synthetic material was based on a comparison with an authentic sample (cf Fig. 52 and 53). Moreover, the o p t i c a l rotation of the sodium borohydride reduction product 94 was found to be i n agreement with the l i t e r -8 8 ature value. Fig. 53. Hmr spectrum of synthetic vinamidine. - n o -Also obtained from t h i s r e a c t i o n was the lactam 95 i n 11% y i e l d . In agreement with t h i s s t r u c t u r e high r e s o l u t i o n 9 5 mass spectrometry gave the molecular formula as C^gH^gN^Og while the "''Hmr spectrum ( F i g . 54) e x h i b i t e d a s i g n a l at £ 4.84 (IH, m u l t i p l e t ) a t t r i b u t a b l e to the ,-hydrogen. F i n a l l y , the i n f r a -red spectrum possessed an absorption at 1640 cm 1 corresponding to the presence of the amide f u n c t i o n a l i t y . In summary, potassium permanganate o x i d a t i o n of v a r i -9 3 ous a v a i l a b l e 'dimers 1 gave r i s e to 'normal 1 lactam products (81,91 and 95) as w e l l as to 'abnormal' C^,-C^, cleavage products (vinamidine and 3R-hydroxyvinamidine). The mechanism f o r f o r -mation of these cleavage products remains ambiguous, however, p o s s i b i l i t i e s have been suggested i n F i g . 55, 56 and 57. 3.82 - 112 -F i g . 55. P o s s i b l e mechanism f o r the f o r m a t i o n o f v i n a m i d i n e from 4 ' - d e o x y l e u r o s i d i n e . - 113 -OH F i g . 56. Possible mechanism for the formation of 3R-hydroxyvina-midine from 3',4 1-anhydrovinblastine. 3.:— Hy droxyvin am i d ine Fig. 57. Possible mechanism for the formation of 3R-hydroxyvina-midine from leurosine. - 114 -2.4 Preparation of V i n b l a s t i n e - V i n c r i s t i n e Analogs. Aside from the chemistry developed i n r e l a t i o n to the synthesis of n a t u r a l products, a number of reactions were c a r r i e d out i n order to obtain novel analogs of v i n b l a s t i n e and v i n -c r i s t i n e f o r the purpose of e l u c i d a t i n g s t r u c t u r e - a c t i v i t y r e -l a t i o n s h i p s . In general, the production of analogs required a good o v e r a l l conversion of r e a d i l y a v a i l a b l e m a t e r i a l s . Pre-v i o u s l y , i t was seen that potassium permanganate o x i d a t i o n afforded access to s e v e r a l i n t e r e s t i n g lactams. The low y i e l d s of lactams produced by t h i s method prompted use of other conditions to achieve improved r e s u l t s . Accordingly, i t was determined that o x i d a t i o n with i o d i n e , i n aqueous tetrahydrofuran containing 94 sodium bicarbonate c o n s t i t u t e d such an improvement. Thus, leur o s i n e could be converted to 19 1-oxoleurosine i n 56% y i e l d . 63,64 Extension of t h i s r e a c t i o n to v i n b l a s t i n e and l e u r o s i d i n e * afforded the corresponding lactams 96 and 97 i n y i e l d s of 33% and 62% r e s p e c t i v e l y . The s p e c t r a l p r o p e r t i e s associated with V i n b l a s t i n e and l e u r o s i d i n e were supplied by the E l i L i l l y l a b o r a t o r i e s , I n d i a n a p o l i s . - 115 -the lactams were in accord with the structural assignments and are partly summarized in Table 5. . , C=0 of Cp'-H in iHmr Molecular lactam in formula (from Compound i . r . soectrum spectrum high res. ms.) 91 16 44 cm 1 4. 76 C46 H54 N4°10 96 1640 cm"1 4.70 C46 H56 N4°10 97 1644 cm"1 4. 73 C46 H56 N4°10 Table 5. Comparison of some characterization data for 19'-oxo-leurosine (91), 19 1-oxovinblastine (96), and 19 1-oxoleurosidine (97) Attempted extension of the iodine/sodium bicarbonate oxidation to 4 1-deoxyleurosidine was not successful. Instead of the anticipated product (19'-oxo-4'-deoxyleurosidine) a new derivative of unknown structure was obtained. This derivative was not a lactam as evidenced by i t s infrared spectrum (Fig. 5 8). High resolution mass spectrometry gave the molecular formula as C^gH^N^Og while the ''"Hmr spectrum (Fig. 59) possessed a s i g -nal at c/8.78 (IH,singlet) which was s p e c i f i c to this 'dimer'. The structure of this product remains mysterious, however ef f o r t s aimed at i t s elucidation continue. Having available various analogs of vinblastine, atten-tion was turned to the preparation of analogs of v i n c r i s t i n e . Treatment of 19'-oxoleurosine in acetone at -7 8°C with Jones 95* 96 97 reagent led to the i s o l a t i o n of two products. ' The major *This procedure represents an adaptation of the chromic acid oxi-dation reported by Jovancvics et- 'al- -for the conversion of vinblas-tine to v i n c r i s t i n e . 5 ^ This adaptation was o r i g i n a l l y developed by T. Honda and P.H. Liao i n this laboratory. Fig. 58. Infrared spectrum of the product obtained from the iodine/sodium bicarbonate oxidation of 4'-deoxyleurosidine. 3.64 Fig. 59. Hmr spectrum of the product obtained from the iodine/ sodium bicarbonate oxidation of 4'-deoxyleurosidine. - 118 -p r o d u c t (29% y i e l d ) was a s s i g n e d as the N a ~ f o r m y l d e r i v a t i v e 98. A c c o r d i n g l y , the u l t r a v i o l e t spectrum o f t h i s d e r i v a t i v e ( F i g . 60) 4.5-4.0 H 4.5H 4.CH 250 300 250 I 300 \ nm A nm F i g . 60. Comparison of u l t r a v i o l e t s p e c t r a of 1 9 1 - o x o l e u r o s i n e (A) and 1 9 1 , 2 2 - d i o x o l e u r o s i n e (B). e s t a b l i s h e d the presence o f the new chromophore. High r e s o l u -t i o n mass sp e c t r o m e t r y gave the m o l e c u l a r f o r m u l a as C 4 g H 5 2 N 4 < ~ ) i i ' w h i l e the ^Hmr spectrum ( F i g . 61) l a c k e d a s i g n a l a t t r i b u t a b l e t o an N -methyl group. F i n a l l y , the i n f r a r e d spectrum ( F i g . 62) e x h i b i t e d a s t r o n g a b s o r p t i o n a t 16 7 8 cm ^ i n a c c o r d w i t h the presence o f the N - f o r m y l group. 3. The minor p r o d u c t (17% y i e l d ) was a s s i g n e d as the N a~desmethyl d e r i v a t i v e 99. A c c o r d i n g l y , h i g h r e s o l u t i o n mass sp e c t r o m e t r y v e r i f i e d the m o l e c u l a r f o r m u l a as C45 H52 N4°]_o' w n ;"- x e the Hmr spectrum ( F i g . 6 3) l a c k e d a s i g n a l f o r an N -methyl group, - 11-9 -91, R=Me 98, R=CHO 99, R=H A l t e r n a t i v e l y , a more e f f i c i e n t route f o r the preparation of 19 1,22-dioxoleurosine (98) was achieved by f i r s t converting leurosine to 22-oxoleurosine* (37) (75% y i e l d ) . The i d e n t i t y of t h i s product was corroborated by the u l t r a v i o l e t spectrum which was i n d i c a t i v e of the new chromophore as w e l l as by high r e s o l u t i o n mass spectrometry which confirmed *Jones reagent was again used to e f f e c t t h i s transformation rather than the conditions given by Jovanovics et a l " Fig. 62. Infrared spectrum of 19 1,22-dioxoleurosine (98). 3.81 - 123 -2 2 C H O C0 2 Me 37 the.molecular formula as C, CH C .N .0,n. Oxidation of 37 with 46 54 4 10 iodine i n aqueous tetrahydrofuran containing sodium bicarbonate yielded 19 1,22-dioxoleurosine (67% yield) i d e n t i c a l with the material obtained previously. F i n a l l y , u t i l i z a t i o n of 6,7-dihydro-3 1,4 1-anhydrovin-blastine (100) as a substrate gave rise to two more novel analogs Thus, epoxidation of 100 using the previously developed condition (t-butyl hydroperoxide, tetrahydrofuran, aqueous 1% t r i f l u o r o -acetic acid) afforded 6,7-dihydroleurosine (101) in 41% y i e l d . High resolution mass spectrometry affirmed the molecular formula as C4gH^gN4Og, while the ^ "Hmr spectrum lacked any resonances due to v i n y l hydrogens. - 124 -Oxidation of 101 with Jones reagent as before afforded 22-oxo-6,7-dihydroleurosine (102) i n 72% y i e l d . In accord with this structure, high resolution mass spectrometry gave the mole-cular formula as C46 H5g N4°io' w n ;"- l e t n e u l t r a v i o l e t spectrum sub-stantiated the presence of the new chromophore. F i n a l l y , the production of the various derivatives outlined represented a portion of an ongoing program. Preliminary b i o l o g i c a l results in re l a t i o n to the anticancer properties of several analogs have been obtained, however, the elaboration of this data was not possible at this time. - 125 -EXPERIMENTAL Melting points (mp) were determined on a K o f l e r block and are uncorrected. U l t r a v i o l e t (uv) spectra were recorded on a Cary 15 spectrophotometer and were obtained w i t h samples i n ethanol s o l u t i o n . The absorption maxima are reported i n nanometers (nm) with log <£ values i n parentheses. I n f r a r e d ( i r ) spectra were recorded on a Pe r k i n Elmer model 457 spectrophoto-meter and were obtained with samples i n chloroform s o l u t i o n . The absorption maxima are reported i n wavenumbers (cm - 1), and are c a l i b r a t e d with respect to the absorption band of polystyrene at 1601 cm 1. Proton magnetic resonance (''"Hmr) spectra were recorded on a Varian HA-10 0, XL-100, NTCFT-100, or Bruker-270 MHz spectrometer. Spectra were obtained with samples i n deutero-chloroform s o l u t i o n at ambient temperature.. Chemical s h i f t values are given i n the £ (ppm) scale r e l a t i v e to t e t r a m e t h y l s i l a n e which was used as the i n t e r n a l standard. The i n t e g r a t e d peak areas, s i g n a l m u l t i p l i c i t i e s , and proton assignments are given i n parentheses. Low r e s o l u t i o n mass spectra (ms) were determined on e i t h e r an AEI-MS-902 or an a t l a s CH-4B spectrometer. High r e s o l u t i o n mass spectra were measured on an AEI-MS-902 i n s t r u -ment. C i r c u l a r dichroism (cd) curves were recorded .on a Jasco - 126 -J-20 spectropolarimeter and were obtained w i t h samples i n methanol s o l u t i o n . The curves were c a l i b r a t e d with 10-camphor-s u l f o n i c a c i d and were obtained at 25.0±0.1°C. The wavelengths of absorption maxima are reported i n nanometers with A £ values 13 13 i n parentheses. C-magnetic resonance ( Cmr) spectra were obtained on a Varian CFT-20 spectrometer used i n the proton decouple mode. Samples were i n deuterochloroform s o l u t i o n and were measured at ambient temperature. Chemical s h i f t values are given i n the scale r e l a t i v e to t e t r a m e t h y l s i l a n e which was used as the i n t e r n a l standard. The carbon assignments are given i n parentheses. Microanalyses were c a r r i e d out by Mr. P. Borda of the M i c r o a n a l y t i c a l Laboratory, U n i v e r s i t y of B r i t i s h Columbia. Thin l a y e r chromatography ( t i c ) u t i l i z e d Merck s i l i c a g e l G (acc. to Stahl) containing 2-3% f l u o r e s c e n t i n d i c a t o r . For preparative l a y e r chromatography ( p i c ) , p l a t e s (20x20 cm) of one mm thickness were used. V i s u a l i z a t i o n was e f f e c t e d by viewing under u l t r a v i o l e t l i g h t and/or by c o l o r r e a c t i o n w i t h e e r i e s u l f a t e spray reagent. Column chromatography u t i l i z e d Merck s i l i c a g e l 60 (70-230 mesh) or Merck aluminum oxide 90 (neutral) . Portions of the f o l l o w i n g work have been published p r i o r to the completion of t h i s t h e s i s . However, f o r the sake of c o n t i n u i t y and convenience these experiments have been included. - 127 -1 6 , 1 8 S - D i c a r b o m e t h o x y c l e a v a m i n e (39) 43 A s o l u t i o n o f 1 8 S - c a r b o m e t h o x y c l e a v a m i n e (19) (502 mg, 1.5 mmole) i n anhydrous t e t r a h y d r o f u r a n (50 ml) under a n i t r o g e n atmosphere was t r e a t e d w i t h a s u s p e n s i o n o f p o t a s s i u m h y d r i d e i n m i n e r a l o i l (300 mg, 22.5% KH, 1.7 mmole). A f t e r s t i r r i n g f o r 10 min a t ambient t e m p e r a t u r e , t h e r e s u l t a n t m i x t u r e was t r e a t e d w i t h m e t h y l c h l o r o f o r m a t e (29 0 mg, 3.1 mmole) and s t i r r e d f o r a f u r t h e r 1 h. A c e t i c a c i d (0.5 ml) was,added and s t i r r i n g c o n t i n u e d f o r 5 min. The s o l v e n t was removed i n vacuo and t h e r e s i d u e was c h r o m a t o g r a p h e d on a l u m i n a (Act.. I l l , p e t . e t h e r - b e n z e n e ) t o a f f o r d 1 6 , 1 8 S - d i c a r b o m e t h o x y c l e a v a m i n e * (39) (461 mg, 7 8 % ) , mp 124-125°C ( a c e t o n e - e t h e r ) ; u v A : 294 ( 3 . 6 6 ) , ITLclX 283 sh ( 3 . 8 0 ) , 268 ( 4 . 0 9 ) , 262 ( 4 . 1 1 ) , 228 ( 4 . 3 7 ) ; i r V : 1725; iHmr 8.08 (IH, m, C 1 4~H) , 7.23 (3H, m, C ^ - C ^ - H ' s) , 5.77 (IH, d, J=6 Hz, C-^-H) , 5.37 (IH, m, C 3~H) , 3.88 (3H, s, -OCH 3) , 3.53 (3H, s, -OCH_ 3), 0.98 (3H, t , J=7 Hz, -CH2CH_3) ; ms: m/e 396 (M+) , 273 (base p e a k ) , 136, 124; 1 3 C m r : 173.7 (-C0 2CH 3), 152.0 (-C0 2CH 3), 141.1 (C^), 137.7 ( C 1 ? ) , 136.0 ( C 1 5 ) , 129.9 ( C 1 Q ) , 124.3 ( C 1 2 ) , 122.8 ( C ^ ) , 122.3 (C 3) , 119.5 ( C g ) , 118.5 ( C 1 3 ) , 115.8 ( C 1 4 ) , 54.5 ( C l g ) , 53.3 ( C 7 & -OCH 3), 52.5 (C 5) , 51.8 (-OCH3) , 38.6 ( C l g ) , 35.9 (Cg) , 34.8 (C- 2), 27.6 (-CH 2CH 3), 12.7 (-CH 2CH 3). H i g h r e s o l u t i o n m o l e c u l a r w e i g h t d e t e r m i n a t i o n c a l c d . f o r C 2 3 H 2 8 N 2 ° 4 : 3 9 6 - 2 0 4 8 ' " f o u n d : 396 .2070. * T h i s compound had p r e v i o u s l y been p r e p a r e d i n t h i s l a b o r a t o r y by I . I t o h and A.H. R a t c l i f f e u t i l i z i n g a d i f f e r e n t p r o c e d u r e . The c h a r a c t e r i z a t i o n d a t a f o r t h i s compound, w i t h t h e e x c e p -t i o n o f t h e 13cmr s p e c t r u m , was c o m p i l e d by t h e s e w o r k e r s . - 128 -Anal.calcd. for C 2 3 H 2 8 N 2 ° 4 : C 6 9 - 6 7 ; H 7- 1 2'* N 7 - 0 7 ? found: C 69.86; H 7.10; N 6.87. Autoxidation of 16,18S-dicarbomethoxycleavamine A solution of 16,18S-dicarbomethoxycleavamine ( 3 9 ) (400 mg, 1 mmole) in peroxide-free tetrahydrofuran (10 ml) con-taining aqueous 1% t r i f l u o r o a c e t i c acid (1 ml) was s t i r r e d at ambient temperature i n the presence of a i r for a period of 8 days. After drying (K^CO^), the solvent was removed i n vacuo. Chromatography of the residue on alumina (Act. I l l , benzene) afforded the epoxide 4 0 * (40 mg, 10%) and the ketoenamide 4 5 (230 mg, 54%). 16,18S-dicarbomethoxy-3R,4S-epoxydihydrocleavamine ( 4 0 ) , mp 131-132°C (ether); uv A m, v: 294 (3.66), 283 sh (3.76), 268 UIGLX (4.08), 262 (4.09), 227 (4.32); i r * : 1728; XHmr <£: 8.1 1113. X (IH, m, C 1 4-H) , 7.3 (3H, m, C^-C-^-H ' s) , 5.84 (IH, d, J=6 Hz, C 1 8-H) , 3.91 (3H, x, -OCH3) , 3.57 (3H, s, -OCH_3), 1.00 (3H, t, J=7.5 Hz, -GH2CH_3); ms: m/e 412 (M+) , 224 (base peak), 152, 138; 1 3Cmr S: 173.4 (-C02CH3), 152.0 (-C02CH3), 137.1 ( C 1 ? ) , 136.0 ( C 1 5 ) , 129.8 ( C 1 Q ) , 124.5 ( C ± 2 ) , 122.9 (C.^), 119.5 (C g), 118.2 ( C 1 3 ) , 115.9 ( C 1 4 ) , 62.7 (C 4), 60.6 (C 3), 53.4 (C ? & -OCH3), 52.7 (C 5), 51.9 (-OCH3), 50.6 ( C i g ) , 39.5 ( C l g ) , 33.6 (C g & C 2 ) , 30.0 (-CH2CH3) , 26.3 (C-^ , 8.9 (-CH2CH3) . High resolution mole-cular weight determination calcd. for C O ^ H O O N T 0 c : 412.1997; *This compound was o r i g i n a l l y isolated as a decomposition product of 3 9 by I. Itoh and A.H. R a t c l i f f e . The characterization data l i s t e d here was compiled by these workers. - 129 -found: 412.2027. A n a l . c a l c d . f o r C 2 3 H 2 8 N 2 ° 5 : C 6 6 - 9 7 ; H 6 - 8 4 ? N 6.79; found: C 66.81; H 6.87; N 6.71. Ketoenamide 45 (as a foam); uv * : 293 (3. 67), max ' 281 sh (3.78), 265 (4.05), 259 (4.06), 227 (4.38); i r V : rricLX 1732, 1680, 1650; 1Hmr <£: 8.13 (IH, s, N b-CHO), 8.10 (1H, m, C l 4 - H ) , 7.3 (3H, m, C ^ - C ^ - H ' s ) , 5.23 (IH, bs, Cg-H), 4.00 (3H, bs, -OCH_3) , 3.66 (3H, s, -OCH_3) , 1.90 (2H, q, J=7 Hz, -CH_2CH3) , 0.79 (3H, t, J=7 HZ, -CH2CH_3); ms: m/e 426 (M +, base peak), 394, 228; 1 3Cmr 188.8 (N bCHO), 163.0 ( - c p 2 C H 3 ) , 160.7 (-C02CH3) , .135.9 ( C 1 7 ) , 132.3 ( C 1 5 ) , 128.8 ( C 1 Q ) , 126.9 (Cg) , 125.1 ( C 1 2 ) , 123.3 ( C 1 1 ) , 122.7 (C^), 119.9 ( C g ) , 118.9 ( C 1 3 ) , 116.0 ( C 1 4 ) , 53.6 (-OCH3) , 52.5 (-OCH3) , 48.7 (C ?) , 44.9 ( C 3 ) , 44.3 ( C 2 ) , 40.0 ( C l g ) , 30.3 (Cg), 24.5 (_CH 2CH 3), 12.3 (-CH 2CH 3). High r e s o l u t i o n molecular weight determination c a l c d . f o r C 2 3 H 2 6 N 2 O g : 426.1791; found: 426.1795. A n a l . c a l c d . f o r C 2 3H 2 6N 20 6.liCH 3OH: C 63. 80; H 6.33; N 6.33; found: C 64.08; H 6.11; N 6.31. Oxidation of 16,18S-dicarbomethoxycleavamine (39) i n  1 p r e o x i d i z e d ' * t e t r a h y d r o f u r a n A s o l u t i o n of (39) (900 mg, 2.27 -mole) i n ' p r e -o x i d i z e d 1 t e t r a h y d r o f u r a n (20 ml) c o n t a i n i n g aqueous 1% t r i -f l u o r o a c e t i c a c i d (2 ml) was s t i r r e d at ambient temperature f o r *'Pr e o x i d i z e d ' t e t r a h y d r o f u r a n r e f e r s to tet r a h y d r o f u r a n which had undergone a e r i a l o x i d a t i o n and contained an u n s p e c i f i e d amount of peroxides. - 130 -20 hr. The reaction mixture was poured into a saturated solution of sodium bicarbonate (20 ml) and extracted with ethyl acetate (2x40 ml). The combined organic portion was washed with water (3x15 ml) and brine (1x20 ml). After drying (Na2SC>4) the solvent was removed i n vacuo and the residue was chromatographed on alumina (Act. I l l , benzene) to afford the epoxide (40) (306 mg, 33%) and the ketoenamide (45) (250 mg, 26%). These compounds were i d e n t i c a l to the products obtained above. Oxidation of 16,18S-dicarbomethoxycleavamine (39) using  t-butyl hydroperoxide A solution of 39 (1.76 g, 4.4 mmole) in freshly d i s t i l l e d tetrahydrofuran (50 ml) containing aqueous 1% t r i -fluoroacetic acid (10 ml) and t-buty hydroperoxide (9 ml) was s t i r r e d at ambient temperature for 21 h. The reaction mixture was poured into a saturated solution of sodium bicarbonate (40 ml) and extracted with ethyl acetate (2x30 ml). The combined organic portion was washed with 5% sodium hydroxide solution (1x20 ml), water (1x20 ml), and brine (1x20 ml). After drying (Na 2S0 4) the solvent was removed i n vacuo to give a viscous o i l . Chromatography on alumina (Act. I l l , benzene) afforded the epoxide 40 (1.22 g, 67%) i d e n t i c a l with that obtained before. - 131 -16,18S-Dicarbomethoxy-4S-dihydrocleavamine (41 ) To a solution of 16,18S-dicarbomethoxy cleavamine (39) (15 mg, 0.038 mmole) in 95% ethanol (2 ml) at 5°C was added Adam's catalyst (2 mg, 0.009 mmole). The reaction mixture was hydrogenated at atmospheric pressure for 1 h, at which point the solvent was removed i n vacuo and the residue was chromato-graphed on s i l i c a gel to y i e l d 16,18S-dicarbomethoxy-4S— dihydrocleavamine (41) (14 mg, 93%) as a colorless f i l m ; uv * m a x:293 (3.72), 283 (3.80), 262 (4.08), 226 (4.34); i r ^ m a x : 2962, 1732; 1Hmr i : 8.17 (IH, m, C^-H) , 7.6-7.2 (3H, m, C 1 1-C 1 4-H's) , 5.66 (IH, m, C^-H) , 3.96 (3H, s, -OCH_3) , 3.64 (3H, s, -OCH3) , 0.90 (3H, t, J=7 Hz, -CH 2CH 3); ms: m/e 398 (M +), 396, 210 (base peak), 114. High resolution molecular weight determination calcd. for C ^ H ^ N ^ : 398. 2206; found: 398.2231. Hydroxenamide 47 A solution of the ketoenamide 45 (10 mg, 0.023 mmole) in 95% ethanol (2 ml) was treated with sodium borohydride (4 mg, 0.1 mmole). The reaction mixture was s t i r r e d at ambient temperature for 20 min, taken up i n water (10 ml) and extracted with methylene chloride (3x5 ml). After drying (Na„S0.), the - 132 -solvent was removed in vacuo and the residue chromatographed on s i l i c a gel (ether) to afford the hydroxyenamide 47 (6 mg, 60%) as a film, uv A : 293 (3.50), 281 sh (3.68), 262 (4.03), ITISX 224 (4. 32); i r t> : 3530, 3420 , 1730 , 1669 , 1651; ^mr IU3.X 8.05 (IH, m, C 1 4-H), 8.02 (IH, s, N^CHO), 7.6-7.2 (3H, m, C 1 1-C 1 3-H's) , 5.48 (IH, bs, C5-H) , 4.00 (3H, s, -OCH_3), 3.66 (3H, s, -OCH_3) , 0.98 (3H, t, J=7.5 Hz, -CH2CH_3) ; ms: m/e 428 (M +), 315, 201, 126 (base peak). High resolution molecular weight determination calcd. for C23 H28 N2°6 : 428.1947; found: 428.1928. 16,18S-Dicarbomethoxy-3R-hydroxyvelbonamine (51) A. Using perchloric acid A solution of the epoxide 40 (25 mg, 0.6 mmole) in tetrahydrofuran (2 ml) containing aqueous 25% perchloric acid (0.25 ml) was heated at 66°C under a nitrogen atmosphere for 2 4 h. The reaction mixture was cooled to ambient temperature, cautiously added to cold concentrated ammonium hydroxide (10 ml), and extracted with methylene chloride (3x8 ml). The combined organic portion was washed with 5% sodium bicarbonate solution (1x8 ml) and dried (Na„SO.). Removal of the solvent i n vacuo - 133 -afforded a brown foam which was shown by t i c to consist of a rather complex mixture of products. Chromatography on alumina (Act. I l l , ether) afforded the trans-diol 51 (Rf 0.35) (4.5 mg, 17%), mp 153-157°C (ether); uv A : 294 (3.66), 282 sh (3.76), in 9.x 263 (4.09), 227 (4.37); i r : 3600, 3440, 1728; 1Hmr c£: 8.14 i n s x (IH, m, C 1 4-H) , 7.5-7.2 (3H, m, ^ - C ^ - H ' s ) , 6.54 (IH, d, J=8.5 Hz, C l g-H) , 3.97 (3H, S, -OCH_3) , 3.63 (IH, d, J-2 Hz, C3-H) , 3.59 (3H, s, -OCH_3) , 0.97 (3H, t, J=7.5 Hz, -CH2CH_3) ; ms: m/e 430 (M+) , 315, 242 (base peak),170, 158, 156. High resolution molecular weight determination calcd. for C„-,H-riN_0-: 23 30 2 6 430.2104; found:. 430.2112. B. Using t r i f l u o r o a c e t i c acid A solution of the epoxide 40 (108 mg, 0.262 mmole) in aqueous 52% t r i f l u o r o a c e t i c acid (10 ml) was heated at 6 5°C under a nitrogen atmosphere for 4 h. The reaction mixture was cooled to ambient temperature, cautiously added to cold concentrated ammonium hydroxide (50 ml), and extracted with methylene chloride (3x15 ml). The combined organic portion was dried (K^CC^) and the solvent was removed in vacuo• Chromatography of the residue on s i l i c a gel (ether) afforded the trans-diol 51 (28 mg, 25%) i d e n t i c a l with the product obtained above. 16,18S-Dicarbomethoxy-3S,4R-epoxydihydrocleavamine (52) A solution of the trans-diol 51 (13 mg, 0.03 mmole) in pyridine (2 ml) containing methanesulfonyl chloride (3 drops) - 134 -was s t i r r e d at ambient temperature under a nitrogen atmosphere for 2.5 h. The reaction mixture was poured into 5% sodium bicarbonate solution and extracted with methylene chloride (3x12 ml) . The combined organic portion was dried (K2CC>3) and the solvent was removed i n vacuo. Chromatography of the residue on s i l i c a gel (ether) afforded two products. The major product (Rf 0.8) was the 3S,4R-epoxide 52 (6.5 mg, 52%) while the minor product (R^ 0.6) was the unstable mesylate 53 (4.5 mg, 29%) which during normal handling was p a r t i a l l y converted (ca 50% by ^Hmr) to the 3S,4R epoxide 52. The i n s t a b i l i t y of the mesylate 5 3 resulted i n i t s incomplete characterization. 16,18S-Dicarbomethoxy-3S,4R-epoxydihydrocleavamine (52); uv> : 296 (3.67) 284 sh (3. 76), 266 (4.03), 262 (4.03), 227 (4.29); i r D : 1730; 1Hmr <£: 8.2 (IH, m, C..-H), 7.5-7.2 (3H, m, C 1 1-C 1 3-H's) , 6.24 (IH, d, J=7.5 Hz, C^-H) , 3.96 (3H, s, -OCH3) , 3.63 (3H, s, -OCH_3) , 0.97 (3H, t, J=7.5 Hz, -CH2CH3) ; ms: m/e 412 (M +), 383 (base peak), 353, 224, 152, 140, 110. High resolution molecular weight determination calcd. for C 2 3H 2gN 20 5: 412.1998; found: 412.2020. Mesylate 53; 1Hmr 8.18 (IH, m, C 1 4~H), 7.6-7.2 (3H, C 1 1-C 1 3-H*s), 6.37 (IH, d, J=8.5 Hz, C 1 8~H), 4.60 (IH, d, J=2.5 Hz, C 3-H), 3.97 (3H, s, - O C H 3 ) , 3.65 (3H, s, - O C H 3 ) , 3.03 (3H, s, -SOCH3), 0.98 (3H, t, J=7.5 Hz, -CH2CH_3) . - 135 -18S-Carbomethoxy-3R-hydroxyvelbanamine 5 4 A 1.5% solution of sodium methoxide in methanol (3 ml) under a nitrogen atmosphere was treated with a solution of 16,-18S-dicarbomethoxy-3R-hydroxyvelbanamine (51) (29 mg, 0.067 mmole) i n anhydrous methanol (0.5 ml). The reaction - mixture was s t i r r e d at ambient temperature for 4 h. Acetic acid (ca 10 drops) was added and the solvent was removed i n vacuo. The residue was taken up i n methylene chloride (5 ml) and the resultant sus-pension was f i l t e r e d through a short plug of alumina (Act. I l l , methylene chloride). Removal of the solvent i n vacuo afforded 18S-carbomethoxy-3R-hydroxyvelbanamine (54) (25 mg, 100%) as a white foam; uv ?\ : 292 (3.76), 283 (3. 79), 277 sh (3.75), ITtctX 224 (4.39); i r V : 3605, 3453, 1720; 1Hmr <4: 8.03 (IH, bs, ITldX NftH) , 7.60-7.06 (4H, m, C-^-C^-H ' s) , 5.45 (IH, dd, J=12 & 2 Hz, C l g-H) , 3.69 (3H, S, -OCH_3) , 3.50 (IH, d, J=2 Hz, C^-H) , 0.99 (3H, t, J=7 Hz, -CH2CH3) ; ms'.m/e 372 (M+) , 170 (base peak), 158. High resolution molecular weight determination calcd. for C 2 1 H 2 8 N 2 ° 4 : 3 7 2 - 2 0 4 9 ? found: 372.2049. 18S-Carbomethoxy-3R-hydroxyvelbanamine chloroindolenine (55) A solution of 18S-carbomethoxy-3R-hydroxyvelbanamine (54) (24 mg, 0.065 mmole) in benzene (3 ml) at 0°C under a nitrogen atmosphere was treated with 1-chlorobenzotriazole (11 mg, 0.072 mmole). The resultant solution was s t i r r e d for 15 min at which point i t was concentrated in vacuo. Chroma-tography of the residue on s i l i c a gel (ether) afforded the - 136 -chloroindolenine 55 (15 mg, 57%) as a yellow film; uv X : •z max 307 (3.28), 288-268 (3.43), 223 (4.19); i r D : 3602, 1730, rricix 1571; "''Hmr cf: 7.7-7.1 (4H, m, C^-C^-H's) , 5.79 (IH, dd, J=12 & 2 Hz, C18-H) , 3.65 (3H, S, -OCH_3) , 0.96 (3H, t, J=7.5 Hz, -CH2CH3) ; ms: m/e 408, 406 (M+) , 343, 311, 209 (base peak), 170. High resolution molecular weight determination calcd. for C 2 1H 2 7N 20 4 3 5C1: 406.1660; found: 406.1655. Attempted coupling of 18S-carbomethoxy-3R-hydroxyvelbanamine (54) with vindoline* A solution of 18S-carbomethoxy-3R-hydroxvelbanamine (54) (17 mg, 0.046 mmole) in methylene chloride (1.5 ml) at 0°C under a nitrogen atmosphere was treated with a 0.05 M solu-tion of t-butyl hypochlorite i n carbon tetrachloride (0.92 ml, 0.046 mmole). The reaction mixture was s t i r r e d for 10 min and checked by t i c to insure complete chloroindolenine formation. The solvent was removed in vacuo and the residue was treated with a solution of vindoline (20.8 mg, 0.046 mmole) in 3% methanolic hydrogen chloride (3 ml). The reaction mixture was s t i r r e d under a nitrogen atmosphere at ambient temperature for 4 days at which point i t was poured into cold concentrated ammonium hydroxide (10 ml) and extracted with methylene chloride (3x8 ml). The combined organic portion was dried (Na 2S0 4) and the solvent was removed in vacuo. Chromatography of the r e s i -due on s i l i c a gel (10% methanol/ethyl acetate) gave back vindoline (8 mg, 38%) and deacetylvindoline (10 mg, 53%). Also present were several minor more polar products (total *This reaction was carried^out by T. Hibino i n this laboratory, and is included here for the sake of completeness. - 137 -18S-Carbomethoxv-3R,4S-epoxycleavamine (60) To a 2% solution of sodium methoxide in methanol (25 ml) under a nitrogen atmosphere was added 16,18S-dicarbo-methoxy-3R,4S-epoxydihydrocleavamine (40) (500 mg, 0.824 mmole). The reaction mixture was s t i r r e d at ambient temperature for a period of 2.5 h at which point i t was made neutral by the addition of acetic acid. The resultant solution was concen-trated in vacuo and the residue was dissolved in ethyl acetate . (30 ml) and washed with 5% sodium bicarbonate solution (1x10 ml) and brine (1x10 ml). After drying (Na 2S0 4), the solvent was removed i n vacuo to y i e l d a gum which was chromatographed on alumina (Act. I l l , benzene/ethyl acetate) to give 18S-car-bomethoxy-3R,4S-epoxydihydrocleavamine (60) (146 mg, 50%) as a white foam; uv A : 292 (3.99), 283 (4.00), 276 sh (3.94), max 224 (4.58); i r V : 3458, 1721; 1Hmr J : 8.62 (IH, bs, NH), max — 7.56-7.08 (4H, m, C 1 ( )-C 1 4-H' s) , 5.19 (IH, dd, J=10 & 5 Hz, C1Q-H) , 3.71 (3H, s, OCH3) , 1.10 (3H, t, J=7.5 Hz,. - C H J C H J ) ; ms: m/e 354 (M+, base peak), 325, 152, 138. High resolution molecu-l a r weight determination calcd. for C21 H26 N2^3 : 354.1944;. found: 354.1939. Anal.calcd. for C-. H_ ,,N-0, • h CH-.CO-C-H,.: 21 26 2 3 3 2 2 5 C 69.35; H 7.54; N 7.04; found: C 69.13; H 7.45; N 7.15. 18S-Carbomethoxy-3R,4S-epoxydihydrocleavamine chloroindolenine (61) A solution of the epoxide 60 (48 mg, 0.136 mmole) in benzene (2 ml) at 0°C under a nitrogen atmosphere was treated with a solution of 1-chlorobenzotriazole (24 mg, 0.156 mmole) in - 138 -benzene (1.5 ml). The reaction mixture was s t i r r e d for a fur-ther 10 min at which point i t was concentrated in vacuo and the residue chromatographed on s i l i c a gel (benzene/10% ethyl ace-tate) to afford the chloroindolenine 61 , mp 149-150°C (ether) ; uv A : 303 (3.38), 288-272 (3.45), 225 (4.32); i r > : max max 1730; 1Hmr £: 7.6 (IH, m, C 1 4~H) , 7.4 (3H, m, C^-C^-H' s) , 4.48 (IH, d, J = l l Hz, C i g-H) , 3.70 (3H, s, -OCH_3) , 1.11 (3H, t, J=7.5 Hz, -CH 2CH 3); ms: m/e 390, 388 (M+, base peak), 353, 152. High resolution molecular weight determination calcd. for C 2 1 H 2 5 N 2 ° 3 3 5 c l : 3 8 8 - 1 5 5 4 ' ' found: 388.1567. Attempted dimerization of 18S-carbomethoxy-3R,4S-epoxydihydro- cleavamine ( 6 0 ) with vindoline i n methanolic hydrogen chloride A solution of the epoxide 6 0 (90 mg, 0.25 mmole) and triethylamine (0.05 ml) in methylene chloride (9 ml) at 0°C under a nitrogen atmosphere was treated with a 0.05 M solu-tion of t-butyl hypochlorite i n carbon tetrachloride (5.4 ml, 0.27 mmole). The reaction mixture was s t i r r e d at 0°C for a further 15 min and checked by t i c to ensure complete formation of the chloroindolenine 6 1 . The solvent was removed i n vacuo and the residue was treated with a solution of vindoline (10) (112 mg, 0.25 mmole) i n 1.8% methanolic hydrogen chloride (10 ml). The reaction mixture was s t i r r e d at ambient temper-ature under a nitrogen atmosphere for 48 h at which point i t was poured into cold ammonium hydroxide solution (30 ml) and extracted with methylene chloride (3x15 ml). The combined organ-i c portion was dried (Na2S04) and the solvent was removed - 139 -i n vacuo. The residue was chromatographed on s i l i c a gel to afford four dimeric products (total ~18 mg). These products were ascertained by their cd curves to be 'unnatural' stereo-chemistry 'dimers', however due to the low yields no further characterization was obtained. Preparation of 16,18S-dicarbomethoxy-19-oxo-3R,4S-epoxydihydro- cleavamine (62) A. Using Sarrett reagent To freshly prepared Sarrett reagent (75 mg CrO^ i n 2 ml pyridine) at 0°C under a nitrogen atmosphere was added a solution of 16,18S-dicarbomethoxy-3R,4S-epoxydihydrocleavamine 40 (71 mg, 0.172 mmole) in methylene chloride (1 ml). The reaction mixture was allowed to warm to ambient temperature and s t i r r e d for a further 17 h at which point i t was taken up in 10% sodium b i s u l f i t e solution (10 ml) and treated with acetic acid (0.5 ml). The resultant mixture was extracted with methylene chloride (2x15 ml) and the combined organic portion was dried (Na 2S0 4). Removal of the solvent in vacuo followed by chroma-tography of the residue on alumina (Act. I l l , ethyl acetate) afforded the lactam epoxide 62 (50 mg, 68%), mp 202-204°C (methanol); uv X : 293 (3.58), 282 (3.65), 262 (4.01), IUclX 227 (4.28); i r v : 1732 , 1642 ; 1Hmr <£: 8.25 (IH, m, C..-H), 7.5 (3H, m, C 1 1-C 1 3-H ,s) , 4.32 (IH, m, C2-H) , 3.93 (3H, s, -OCH_3) , 3.55 (3H, S, -OCH3), 1.00 (3H, t, J=7.5 Hz, -CH 2CH 3); ms: m/e 426 (M+), 394 (base peak); 13Cmr £: 171.9 (-C02CH3), 167.7 ( C 1 9 ) , 152.0 (-C02CH3) , 136.0 ( C 1 5 ) , 135.5 ( C 1 ? ) , 129.1 ( C 1 Q ) , 125.2 - 140 -( C 1 2 ) , 123.4 ( C n ) , 119.5 (C g), 118.4 (C 1 3) , 116.1 (C 1 4) , 61.4 (C3) , 59.3 (C 4) , 53.6 (-OCH3) , 52.4 (-OCH3), 47.7 (Cg) , 47.1 (C 38.6 (C 2) , 31.1 (C 1 8) , 30.6 (C g) , 24.3 (-CH2CH3), 22.1 (C^ , 9.4 (-CH2CH3). High resolution molecular weight determination calcd. for C~-,H0/.N-C>: 426. 1791; found: 426.1785. Anal.calcd. 2.6 26 2 6 for C23H2gN2Og.35CH3OH: C 63. 80; H 6.37; N 5.88; found: C 64.01; H 6.30; N 5.91. B . Using mercuric acetate A solution of the epoxide 40 (25 mg, 0.061 mmole) i n p-dioxane (2 ml) was treated with a solution of the disodium s a l t of ethylenediaminetetraacetic acid (65 mg, 0.232 mmole) and mercuric acetate (55 mg, 0.173 mmole) i n 1% aqueous acetic acid (2 ml). The reaction mixture was s t i r r e d at ambient temp-erature for 19 h, f i l t e r e d , and diluted with ethyl acetate (10 ml). The organic portion was washed with 5% hydrochloric acid (1x5 ml), 5% sodium bicarbonate solution (1x5 ml), and brine (1x5 ml). After drying (Na 2S0 4), the solvent was removed in vacuo and the residue was chromatograohed on s i l i c a gel (benzene/10% ethyl acetate) to afford the lactam epoxide 62 (13 mg, 50%) i d e n t i c a l to that obtained above. 18S-Carbomethoxy-19-oxo-3R,4S-epoxy-dihydrocleavamine (66) To a 1% solution of sodium methoxide in methanol (4 ml, 0.741 mmole) at ambient temperature under a nitrogen atmosphere was added the lactam epoxide 62 (45 mg, 0.106 mmole) The resultant suspension was s t i r r e d for 3 h at which point - 141 -a l l the s o l i d had dissolved. The reaction mixture was treated with acetic acid (5 drops) and the solvent was removed in vacuo. The residue was t r i t u r a t e d with methylene chloride (2 ml) and the resultant suspension was f i l t e r e d through c e l i t e . The f i l t r a t e was concentrated i n vacuo and chromatographed on s i l i c a gel (ether) to afford 18S-carbomethoxy-19-oxo-3R,4S-epoxy-dihydrocleavamine (66) (37 mg, 95%) as a white foam; uv A : max 292 (3.96), 283 (3.99), 276 (3.92), 224 (4.57); i r $ : 3452, III 3 .X 1728, 1648; -"-Hmr i : 8.67 (IH, bs, NH) , 7.56 (IH, m, C^-H) , 7.2 (3H, m, C 1 1-C 1 3-H's), 4.33 (IH, doublet of t r i p l e t s , J=3 & 13 Hz, C 2-H), 4.05 (IH, dd, J=10.5 & 1.5 Hz, C^-H) , 3.71 (3H, s, -OCH3) , 1.15 (3H, t, J=7.5 Hz, -CH2CH_3) ; ms: m/e 368 (M+) , 228 (base peak), 215, 183, 170, 168, 167. High resolution molecular weight determination calcd. for C2i H24 N2°4 : 368.1736; f D u n c j : 368.1736. Reaction of 18S-carbomethoxy-19-oxo-3R,4S-epoxydihydrocleavamine  (66) with 1-chlorobenzotriazole A solution of the lactam epoxide 66 (32 mg, 0.087 mmole) in benzene (2 ml) at ambient temperature under a nitrogen at-mosphere was treated with 1-chlorobenzotriazole (14.7 mg, 0.096 mmole). The reaction mixture was s t i r r e d for 10 min at which £>oint i t was chromatographed on s i l i c a gel (ether) to afford two products. The major and less polar (Rf 0.8) product was the benzotriazole derivative 68 (17 mg, 40%), while the minor product (Rf 0.7) was the hydroxy derivative 69 (9 mg, 27%). Both compounds were obtained as colorless films. - 142 -18-Benzotriazole derivative 68; uv A : 291 (3.87), ITtciX 283 (3.94), 277 sh (3.90), 258 (3.86), 221 (4.45); i r max 3442, 1732 , 1649; """Hmr «T: 9.39 (IH, bs, NH) , 8.03 (IH, dd, J=8 & 2 Hz, 1 benzotriazole H) , 7.6-7.0 (6H, m, c n " " c 1 4 ~ H ' s & 2 benzotriazole H's), 6.34 (IH, dd, J=8 & 2 Hz), 4.65 (IH, m, C 2~H), 3.83 (3H, s, -OCH3), 1.07 (3H, t, J=7.5 Hz, -CH 2CH 3); ms: m/e 485 (M +, base peak), 426 , 367, 228, 154 , 119 . High resolution molecular weight determination calcd. for C^H^NgO^: 485.2063; found: 485.2061. 18-hydroxv derivative 69, uv A : 292 (3.76), max 283 (3. 81), 276 sh (3. 78), 223 (4 . 48); ir S) : 3586, 3515, 3467, IIlclX 3442 , 1734 , 1641; 1Hmr J: 8.43 (IH, bs, NH) , 7.56 (IH, m, C 1 4-H) , 7.25 (3H, m, C 1 1^C ; L 3-H' s) , 4.40 (IH, doublet of t r i p l e t s , J=13 & 3 Hz, C2-H) , 3.94 (3H, s, -OCH_3), 2.02 (2H, q, J=7.5 Hz, -CH2CH3) ; ms: m/e 384 (M+, base peak), 325, 228, 215, 182, 164, 115. High resolution molecular weight determination calcd. for C 21 H24 N2°4 : 384.1685; found: 384.1689. 18'-epi-19'-oxoleurosine 70 A. solution of vindoline (12 mg, 0.026 mmole) and the benzotriazole derivative 68 (12 mg, 0.025 mmole) i n methylene chloride (2 ml) under a nitrogen atmosphere at ambient temper-ature was treated with t r i f l u o r o a c e t i c acid (5 drops). The reaction mixture was s t i r r e d for 1 h, diluted with methylene chloride (20 ml), washed with 5% sodium bicarbonate solution (5 ml), and dried (K 2C0 3). Removal of the solvent in vacuo followed by chromatography of the residue on s i l i c a gel (ethyl - 143 -acetate/10% methylene chloride, 2X) afforded 18'-epi-19' — oxoleurosine (70) (18 mg, 89%) as a white amorphous s o l i d ; uv ^ : 309 (3.94), 295 (4.07), 284 (4.04), 261 (4.08), ill 3.X 218 (4.73); i r T) : 3468, 1740, 1639, 1618; 1Hmr Si 9.16 (IH, bs, NH) , 7.5-7.0 (4H, m, C u , -C 1 4 , -H' s) , 6.87 (IH, s, C 1 4~H) , 5.98 (IH, s, C ?-H), 5.93 (IH, dd, J=10.5 & 4 Hz, C ?-H), 5.33 (IH, s, C.-H), 5.31 (IH, d, J=10.5 Hz, Cr-E), 4.71 (IH, m, 4 6 C 2,-H), 3.86 (3H, s, -OCH_3) , 3.82 (3H, s, -OCH_3) , 3.75 (3H, s, -OCH3) , 3.69 (Iff, s, C2-H) , 2.62 (3H, s, N-CH_3) , 2.06 (3H, s, -OCOCH3), 1.02 (3H, t, J=7 Hz, -CH2CH_3) , 0.59 (3H, t, 0.65, -CH2CH3) ; ms: m/e 822 (M+, base peak), 762, 663, 662, 661, 555, 135; cd A : 208 (+12.4), 222 (-41.3). High resolution molecu-max l a r weight determination calcd for C. <-H N .0 : 822. 3829; 46 54 4 10 found: 822.3856. 18' -Epileurosine (73) A solution of vindoline (10) (20 mg, 0.044 mmole) and the chloroindolenine 61 (18 mg, 0.046 mmole) in methylene chloride (1 ml) at ambient temperature under a nitrogen atmos-phere was treated with m-chloroperbenzoic acid (8 mg, 0.0 46 mmole). The reaction mixture was s t i r r e d for 5 min, treated with a 1:1 mixture (by volume) of t r i f l u o r o a c e t i c a c i d / t r i -fluoroacetic anhydride (6 drops) and s t i r r e d for a further 15 min. The resultant solution was cautiously added to a suspen-sion of sodium borohydride (100 mg, 2.63 mmole) in methanol (5 ml) at 0°C. This mixture was diluted with water (10 ml) and - 144 -extracted with methylene chloride (3x8 ml). The combined organic portion was dried (Na 2S0 4) and the solvent was removed in vacuo. Chromatography of the residue on s i l i c a gel (ethyl acetate/10% methanol) afforded 18'-epileurosine (15 mg, 40%) as a white amorphous s o l i d ; uv A : 307 (3.77), 290 (3.97), IT13.X 283 (3. 98), 262 (4.04), 217 (4. 63); i r : 3463 , 1739, 1616; in 3.x W 9.10 (IH, bs, NH) , 6.9-7.5 (4H,m, C n , -C 1 4 ,-H1 s) , 7.01 (IH, s , C 1 4 - H ) , 6.00 (IH, s, C^-H) , 5.91 (IH, dd, J=10 & 4 Hz, C 7-H), 5.38 (IH, s, C 4~H), 5.30 (IH, d, J=10 Hz, C g-H), 3.84 (3H, s, -OCH_3) , 3.77 (3H, s, -OCH_3) , 3.75 (3H, s, -OCH_3), 3.68 (IH, s, C2-H) , 2.61 (3H, s, NCH_3) , 2.06 (3H, s, -OCOCH_3) , 0.97 (3H, t, J=7 Hz, -CH2CH3) , 0.61 (3H, t, J=7 Hz, -CH2CH_3) ; ms m/e 822, 808 (M +), 610, 510, 282, 135, 121, 106 (base peak); cd : 207 (+21.8), 222 (-41.4). High resolution molecular max r weight determination calcd. for C4gH,-gN40g: 808.4046; found: 808.4036. 18'-epivincadioline (74) A solution of vindoline (10) (18 mg, 0.039 mmole) and the chloroindolenine 55 (24 mg, 0.059 mmole) in methylene chloride at 0°C under a nitrogen atmosphere was treated with m-chloroperbenzoic acid (9 mg, 0.052 mmole). The reaction mixture was s t i r r e d at 0°C for 30 min, treated with a 1:1 mixture (by volume) of t r i f l u o r o a c e t i c a c i d / t r i f l u o r o a c e t i c anhydride (8 drops), and s t i r r e d for a further 35 min at 0°C. The resultant solution was cautiously added to a suspension of - 145 -sodium borohydride (130 mg, 3.42 mmole) in methanol at 0°C. This solution was diluted with water (10 ml) and extracted with methylene chloride (3x8 ml). The combined organic portion was dried (Na 2S0 4) and the solvent was removed in vacuo. The residue was chromatographed on s i l i c a gel (76% ethyl acetate/ 12% methylene chloride/12% methanol) to y i e l d 18'-epivincadioline (9.5 mg, 19%) as a white amorphous s o l i d ; uv A : 30 8 (3.81), in 9.x 292 (3.97), 283 (3.95), 252 (4.10), 222 sh (4.58), 209 (4.66); i r V : 3438, 1737, 1616; 1Hmr <$: 9.00 (IH, bs, NH) , 7.5-7.0 (4H, m, C 1 1,-C 1 4,-H ,s) , 6.98 (IH, s, C 1 4-H),6.01 (1H, s, C-^-H) , 5.88 (IH, dd, J=10.5 & 4 Hz, C ?-H), 5.34 (IH, s, C 4~H), 5.30 (IH, d, J=10.5 Hz, C c-H), 3.87 (3H, s, -OCH.), 3.77 (3H, s, o — j -OCH_3) , 3.76 (3H, s, -OCHj), 3.68 (IH, s, Cj-H), 2.64 (3H, .s, NCH3), 2.09 (3H, s, -OCOCH^), 0.93 (3H, t, J=7 Hz, -CH 2CH 3), 0.66 (3H, t, J=7 Hz, -CH 2CH 3); ms: m/e 826 (M+) , 824, 344, 170, 169, 168, 135, 121, 115, 107 (base peak); cd A : 209 (+18.0), max 223 (-42.0). High resolution molecular weight determination calcd. for C . ,HcoN .0,.: 826.4152; found: 826.4129. 46 58 4 10 3',4'-Anhydrovinblastine (26) 3',4'-Anhydrovinblastine (26) was prepared using a 31 33 s l i g h t l y modified procedure to those previously published. ' A solution of catharanthine (11) (1.35 mg, 4 mmole) in methylene chloride (30 ml) at 0°C under a nitrogen atmosphere was treated with m-chloroperbenzoic acid (0.73 g, 4.2 mmole). The reaction mixture was s t i r r e d at 0°C for 5 min and checked - 146 -by t i c to ensure complete N-oxide formation. Vindoline (10) (1.2 g, 2.6 mmole) was added and the resultant solution was cooled to -50°C. T r i f l u o r o a c e t i c anhydride (2.4 ml, 12 mmole) was added and the reaction mixture was s t i r r e d for a further 3.5 h at -40°C. The resultant solution was cautiously added to a suspension of sodium borohydride (4 g, 0.105 mmole) in methanol (100 ml) at 0°C. This solution was diluted withwater (200 ml) and extracted with methylene chloride (3x60 ml). The combined organic portion was dried (Na 2S0 4) and the solvent was removed i n vacuo. C r y s t a l l i z a t i o n of the residue from methanol (ca 25 ml) afforded 3 1,4'-anhydrovinblastine (26) (1.55 g, 69% based on vindoline, 48% based on catharanthine). Leurosine ( 3 ) Leurosine ( 3 ) was prepared from 3',4 1-anhydrovin-blastine (26) v i a the method of T. Hibino u t i l i z i n g t-butyl hydroperoxide in tetrahydrofuran containing 1% aqueous t r i -fluoroacetic acid. However, the i s o l a t i o n procedure used was diffe r e n t from that published. Thus, upon completion of the reaction as indicated by t i c (usually 9 to 22 h ) the reaction mixture was treated with methanol (ca 10 ml/g of 3 1,4 1-anhydro-vinblastine used) and dried (K^CO^). The solvent was removed in vacuo and the residue was c r y s t a l l i z e d from methanol to afford leurosine ( 3 ) . - 147 -Catharine (76) A. A solution of leurosine (3) (30 mg, 0.037 mmole) in tetrahydrofuran (2 ml) containing aqueous 1% t r i f l u o r o a c e t i c acid (0.2 ml) was s t i r r e d i n the presence of a i r for 11 days. The reaction mixture was diluted with a saturated solution of sodium bicarbonate (5 ml) and extracted with methylene chloride (3x5 ml). The combined organic portion was dried (Na2SO^) and the solvent was removed i n vacuo. Chromatography of the residue on s i l i c a gel (ethyl acetate/12% methanol) afforded leurosine (4 mg) and catharine (4 mg, 15%). The synthetic catharine had mp 213-215°C (acetone) ( l i t . 213-215°C); mp 162-166°C 8 5 (ethanol) ( l i t . 171-175 C); undepressed on admixture with an authentic sample*; oC = -49° (C, 0.7, CHC13) ( l i t . 8 5 -51°). The ''"Hmr, uv, i r , and mass spectra were superimposable with those of authentic material. B. Oxidation of 3 1,4 1-anhydrovinblastine (26) as above afforded catharine in 34% y i e l d . C. A solution of leurosine (3) (45 mg, 0.56 mmole) in methylene chloride containing t-butyl hydroperoxide (0.06 ml) was s t i r r e d at ambient temperature for 24 h. The solvent was removed in vacuo and the residue chromatographed on s i l i c a gel (methylene chloride/5% methanol) to afford catharine (22 mg, 48%). *The authentic catharine was provided by the E. L i l l y Co., Indianapolis. - 148 -Comparative oxidations of 3',4'-anhydrovinblastine (26),  leurosine (3), and derivatives thereof u t i l i z i n g t-butyl  hydroperoxide Reactions were a l l carried out u t i l i z i n g 10 mg of substrate dissolved i n 0.5 ml tetrahydrofuran containing 0.05 ml of t-butyl hydroperoxide. To these solutions were added any further reagents used. The product composition of the reac-tions was ascertained v i a t i c . Authentic samples of 3',4'-anhydrovinblastine (26), leurosine (3), catharine (76), 86 63 pleurosine (79), and 3 1,4'-anhydrovinblastine-N^,-oxide (78) were used for comparison purposes. The solvent systems used for the chromatographic analysis were ethyl acetate/20% methanol and methylene chloride/6% methanol. Vi s u a l i z a t i o n was achieved by spraying with eerie sulfate spray reagent and heating at 10 0°C for 1 h. The r e l a t i v e amounts of products formed were estimated from the visualized chromatograms. Rx . Conditions Products (%) Substrate Time Additive 76 26 78 or 79 Others 26 22 h 0.05 ml 1% t r i -fluoroacetic acid 5 95 - 5 -26 5 days II 40 25 - 5 30 26 22 h 0.05 ml water 5 30 - 5 60 26 22 h - 30 10 5 10 45 26 22 h 0.05 ml 1% t r i -fluoroacetic acid 0.1 ml methanol — 50 45 5 — - 149 -cont' d Rx. Conditions Products (%) Substrate Time Additive 76 3 26 78 or 79 Others 26 22 h 0.05 ml 1% t r i -fluoroacetic acid 5 mg r a d i c a l i n -h i b i t o r * - 25 70 5 -78 22 h 0.05 ml 1% t r i -fluoroacetic acid - - 100 -26 22 h 0.05 ml 5% t r i -fluoroacetic acid - - 100 -3 22 h I I - 100 - -3 22 h 0.05 ml 1% t r i -fluoroacetic acid 20 65 15 -3 22 h 0.05 ml 1% t r i -fluoroacetic acid 5 mg r a d i c a l i n -h i b i t o r * 5 90 5 3 44 h 0.05 ml 1% t r i -fluoroacetic acid 10 mg radi c a l i n -h i b i t o r * 100 79 22 h 0.05 ml 1% t r i -fluoroacetic acid 100 * 3-t-Butyl-4-hydroxy-5-methylphenyl sulfide - 150 -Reaction of 3',4'-anhydrovinblastine (26),with potassium  permanganate A solution of 34'-anhydrovinblastine (26) (250 mg, 0.316 mmole) i n methylene chloride (2 ml) and acetone (5 ml) was treated at 0°C with a solution of potassium permanganate (105 mg, 0.665 mmole) i n acetone (5 ml). The reaction mixture was s t i r r e d at 0°C for 5 min and the solvent was removed i n vacuo. The residue was t r i t u r a t e d with methylene chloride (5 ml) and f i l t e r e d through s i l i c a gel (ethyl acetate/25% methanol). Removal of the solvent i n vacuo followed by chromatography of the residue on s i l i c a gel (ethyl acetate/15% methanol) afforded the ketol 83 (111 mg, 42%) as the major product (Rf 0.4). 19'-Oxo-3',4 1-anhydrovinblastine (81) (25 mg, 9.8%) i d e n t i c a l to an authentic sample ( t i c , ms, ^Hmr) was obtained as the minor product (R f 0.75). Ketol 83, mp 198-202°C (ethanol); uv : 310 (3.80), max 294 (4.08), 284 (4.14), 268 (4.19), 212 (4.72); i r : 3475, IuclX 1734, 1660, 1612; 1Hmr : 7.88 (IH, bs, NH), 7.51 (IH, m, C 1 4,-H), 7.32 (IH, s, NCHO) , 7.14 (3H, m, C 1 1 , -C13,-H's) , 6.71 (IH, s, C 1 4-H), 6.00 (IH, s , C 1 7 - H ) , 5.86 (IH, dd, J=10, 4 Hz, C 7-H), 5.49 (IH, s, C 4-H), 5.30 (IH, d, J=10 Hz, C g-H), 3.97 (IH, bs, C 3,-H), 3.79 (3H, s, -OCH_3) , 3.73 (3H, s, -OCH) , 3.51 (3H, s, ' -OCH3) , 2.69 (3H, s, -NCH_3) , 2.12 (3H, s, -OCOCH_3), 0.79 (3H, t, J=7 Hz, -CH2CH_3) , 0.70 (3H, t, J=7 Hz, -CH2CH_3); ms m/e 840 (M +), 781, 680, 573, 135 (base peak). High resolu-tion molecular weight determination calcd. for C . ,H_,N .0,,: ^ 46 56 4 11 840.3945; found: 840.3966. - 151 -Reaction of leurosine (3) with potassium permanganate A solution of leurosine (3) (105 mg, 0.13 mmole) in acetone (1 ml) and methylene chloride (0.5 ml) was treated with a solution of potassium permanganate (40 mg, 0.25 mmole) in acetone (4 ml). The reaction mixture was s t i r r e d at ambient temperature for 3 min and the solvent was removed i n vacuo. The residue was treated as above to afford the ketol 83 (30 mg, 27%) i d e n t i c a l with that obtained above, and 19'-oxoleurosine (91) (20 mg, 19%) as a colorless f i l m . 19'-Oxoleurosine (91), uv : 309sh (3.74), 294 max (4.00), 284 (4.05), 262 (4.13), 214 (4.66); i r : 3470, 1738, max 1644; •'"Hmr : 8.06 (IH, bs, NH) , 7.57 (IH, m, C 1 4,-H) f 7.18 (3H, m, C 1 1,-C 1 3,-H ,s), 6.65 (IH, s, C 1 4-H), 6.19 (IH, s, C 1 ?-H), 5.90 (IH, dd, J=10.5, 3.5 Hz,C7~H), 5.51 (IH, s, C 4~H), 5.33 (IH, d, J-10.5 Hz, C 6-H), 4.76 (IH, m, C 2,-H), 3.85 (3H, s, -OCH3) , 3.83 (3H, s, -OCH_3), 3.63 (3H, s, -OCH_3), 2.76 (3H, s, NCH3), 2.12 (3H, s, -OCOCH3), 1.01 (3H, t, J=7.5 Hz, -CH2 CH3) , 0.84 (3H, t, J=7 Hz, -CH2CH_3) ; ms m/e 822 (M +) , 763, 282, 135 (base peak); 1 3Cmr : 163.0 ( C 1 9 , ) , 61.6 (Cj,), 59.8 (C 4,), 8.9 (-CH 2£H 3), 8.5 (-CH2CH3). High resolution molecular weight determination calcd. for C46 H54 N4°io : 822.3839; found: 822.3806. Ketoacetate 84 A solution of the ketol 83 (22 mg, 0.026 mmole) i n pyridine (2 ml) at ambient temperature under a nitrogen atmos-phere was treated with acetic anhydride (4 drops). The reaction - 152 -mixture was s t i r r e d for 30 h. Methanol (1 ml) and toluene (10 ml) were added and the solvent was removed in vacuo. Chromatography of the residue on s i l i c a gel (ethyl acetate/10% methanol.) afforded the ketoacetate 84 (15 mg, 65%) as a white amorphous s o l i d , uv : 310 (3.81), 294 (4.10), 285 (4.17), n 3.x 270 (4.21), 213 (4.73); i r m : 3470, 1738, 1660, 1612; Iu3X •"•Hmr : 7.90 (IH, bs, NH) , 7.51 (IH, m, C 1 4,-H), 7.32 (IH, s, NCHO), 7.12 (3H, m, , -C13,-H's) , 6.64 (IH, s, C^-H) , 6.00 (IH, s, C 1 ?-H), 5.86 (IH, dd, J=10, 4 Hz, C?-H) , 5.48 (IH, s, C 4-H), 5.31 (IH, d, J=10 Hz, C g-H), 4.80 (IH, bs, C 3,-H), 3.79 (3H, s, -OCH3), 3.76 (3H, s, -OCH_3) , 3.72 (IH, s, C2~H) , 3.62 (3H, s, -OCH_3) , 2.69 (3H, s, NCH_3), 2.14 (3H, s, -OCOCH_3) , 2.10 (3H, s, -OCOCH3) , 0.77 (3H, t, J=7 Hz, -CH2CH_3), 0.70 (3H, t, J=7 Hz, -CH 2CH 3); ms m/e 882 (M +), 822, 762, 720, 613, 555, 354, 181, 169, 135, 131, 119 (base peak). High resolution molecular weight determination calcd. for C48 H58 N4°12 : 8 8 2 • 4 0 5 0 ; found: 882.4046. Diol 85 A solution of the ketol 8 3 (50 mg, 0.06 mmole) in 95% ethanol (3 ml) was treated with sodium borohydride ( 6 mg, 0.16 mmole). The reaction mixture was s t i r r e d at ambient temper-ature for 30 min and treated with acetone ( 1 ml). The solvent was removed in vacuo and the residue was tr i t u r a t e d with methylene chloride (20 ml) and f i l t e r e d through c e l i t e . Removal of the solvent in vacuo followed by chromatography of the residue on - 153 -s i l i c a gel (methylene chloride/5% methanol) afforded the d i o l 85 (33 mg, 66%) as a colorless f i l m , uv A : 310 (3.65), ITlcLX 295 (4.00), 284 (4.06), 267 (4.11), 212 (4.65); i r V : 3585, msx 3472, 1738,1660, 1613; 1Hmr &: 7.98 (IH, s, NH), 7.53 (IH, m, C 1 4,-H), 7.45 (IH, s, NCHO), 7.15 (3H, m, , -C 1 3 , -H 1 s) , 6.76 (IH, s, C 1 4-H), 6.12 (IH, s, C-^-H), 5.90 (IH, dd, J=10, 4 Hz, C 7-H), 5.46 (IH, s, C 4~H), 5.35 (IH, d, J-10 Hz, C g-H), 3.82 (3H, s, -OCH3) , 3.80 (3H, s, -OCH_3) , 3.76 (IH, s, C2-H) , 3.55 (3H, s, -OCH3), 2.71 (3H, s, NCH_3), 2.10 (3H, s, -OCOCH_3), 0.84 (3H, t, J=7.5 Hz, -CH2CH3) , 0.79 (3H, t, J=7 Hz, -CH2CH_3) ; ms: m/e 842 (M+) , 783, 681, 574, 516, 135 (base peak). High resolution molecular weight determination calcd. for C4gH^gN40^ 842.4102; found: 842.4060. Acetylation of diol85 A solution of the d i o l 8 5 (32 mg, 0.38 mmole) in pyridine (2 ml) at ambient temperature under a nitrogen atmos-phere was treated with acetic anhydride (0.1 ml). The reaction mixture was s t i r r e d for 22 h at which point methanol (0.5 ml) and toluene (10 ml) were added. The solvent was removed i n vacuo and the residue was chromatographed on s i l i c a gel (methy-lene chloride/5% methanol) to y i e l d the tetraacetate 8 7 (9 mg, 24%, Rf 0.4) and the triacetate 86 (9 mg, 26%, Rf 0.35). Both compounds were obtained as colorless films. Tetraacetate 8 7, uv A : 310 (3.73), 294 (4.06), max 284 (4.11), 267 (4.15), 212 (4.70); i r v : 3468, 1732, 1666, IT13.X 1618; 1Hmr <£: 8.06 (IH, bs, NH) , 7.50 (H, m, C,4,-H), 7.36 - 154 -(IH, s, NCHO), 7.14 (3H, m, ,-C 1 3, -H' s) , 6.53 (IH, s, C14~H) , 6.13 (IH, s, C 1 7-H) , 5.89 (IH, dd, J=10, 5 Hz, C ?-H), 5.52 (IH, s, C 4-H), 5.30 (IH, d, J=10 Hz, C g-H), 4.90 (IH, m, C 4,-H), 4.54 (IH, t, J=6 Hz, C 3,-H), 4.03 (IH, s, Cj-H), 4.82 (3H, s, -OCH3), 4.77 (3H, s, -OCH_3) , 4.59 (3H, s, -OCH3) , 2.85 (3H, s, NCH3), 2.08 (6H, s, 2X-OCOCH_3), 1.98 (6H, s, 2X-OCOCH_3), 0.75 (3H, t, J=7 Hz, -CH-CH ), 0.48 (3H, t, J=7 Hz, -CH.CH-); ms: m/e 968 (M +), 765, 659, 600, 135 (base peak). High resolu-tion molecular weight determination calcd. for ^ 52^64^4^14: 968.4419; found: 968.4395. Triacetate 86, uv A : 309 (3.70), 294 (4.03), max 284 (4.08), 269 (4.10), 212 (4.67); i r 0 : 3480, 1732, 1664, IHclX 1617; W 7.95 (IH, bs, NH) , 7.54 (IH, m, C^.-H), 7.38 (IH, s, NCHO), 7.15 (3H, m, C n , -C 1 3, -H' s) , 6.54 (IH, s, C 1 4~H) , 6.17 (IH, s, C 1 7-H) , 5.88 (IH, dd, J=10, 4 Hz, C?-H) , 5.55. (IH, s, C 4-H), 5.30 (IH, d, J=10 Hz, C g-H), 4.89 (IH, m, C 4,-H), 4.41 (IH, t, J=6 Hz, C 3,-H), 3.84 (3H, s, -OCH3), 3.82 (3H, s, -OCH3) , 3.75 (IH, s, C2-H) , 3.58 (3H, s, -OCH_3) , 2.73 (3H, s, NCH3) , 2.12 (3H, s, -OCOCH3) , 2.08 (3H, s, -OCOCH_3), 1.97 (3H, s, -OCOCH3), 0.76 (6H, t, J=7 Hz, 2X-CH2CH_3); ms: m/e 926 (M +), 867, 765, 659, 600, 135 (base peak). High resolution molecular weight determination calcd. for C 5 QHg 2N 40^ 3: 926.4313; found: 926.4331. Diketone 8 8 A solution of the ketol 83 (30 mg, 0.036 mmole) and cupric acetate (monohydrate) (20 mg, 0.11 mmole) in methanol - 155 -(3 ml) was heated at reflux for 25 min. The solvent was removed in vacuo and the residue was t r i t u r a t e d with methylene chloride (2 ml) and f i l t e r e d through c e l i t e . Removal of the solvent in vacuo followed by chromatography of the residue on s i l i c a gel (methylene chloride/5% methanol) afforded the dike-tone 88 (16 mg, 53%) as a pale yellow fi l m ; uv ?* m a x: 308 (3. 70), 293 (4.03), 277 (4.11), 260 ( 4.20), 207 (4.70); i r v : 3450, max 1739, 1713, 1662, 1615; 1Hmr<^: 7.94 (2H, bs, NH, NCHO), 7.52 (IH, m, C 1 4,-H), 7.18 (3H, m, C n , -C 1 3 , -H' s) , 6.77 (IH, s, C 1 4-H), 6.10 (IH, s, C 1 ?-H), 5.91 (IH, dd, J=10, 4 Hz, C ?-H), 5.51 (IH, s, C 4-H), 5.34 (IH, d, J=10 Hz, C g-H), 3.82 (3H, s, -OCH3) .3.74 (3H, bs, -OCH_3) , 3.62 (3H, s, -OCH.^ ) , 2.74 (3H, s, NCH 3), 2.12 (3H, s, -OCOCH_3), 1.04 (3H, t, J=7.5 Hz, -CH2 CH_3) , 0.79 (3H, t, J=7 Hz, -CH2CH_3); ms: m/e 838 (M+) , 779 , 678 (base peak), 570, 135. High resolution molecular weight determination calcd. for C . ,H_ .N.O., , : 838. 3789; found: 838. 3770. 46 54 4 11 Periodic acid cleavage of the d i o l 8 5 A solution of the d i o l 8 5 (25 mg, 0.030 mmole) in tetrahydrof.uran (2 ml) at ambient temperature under a nitrogen atmosphere was treated with a solution of periodic acid (8.5 mg, 0.066 mmole) in tetrahydrofuran (0.5 ml). The reaction mixture was s t i r r e d for 2 min and concentrated i n vacuo. Chromatography of the residue on s i l i c a gel (methylene chloride/ 5% methanol/0.1% ammonium hydroxide) afforded the N^-formyl-aldehyde 9 0 (15 mg, 65%) as a colorless film, uv h : 310 ^ max (3.67), 294 (4.00), 284 (4.08), 268 (4.15), 213 (4.66); - 156 -i r = 3464, 1732, 1661, 1612; "'•Hmr J : 9.20 (IH, s, -CHO), 7.96 (IH, bs, NH), 7.61 (IH, s, NCHO), 7.56 (IH, m, C 1 4,-H), 7.18 (3H, m, C 1 1 1 - C 1 3 , - H , s ) , 6.64 (IH, s, C 1 4-H), 6.11 (IH, s, C-^-H) , 5.88 (IH, dd, J=10,4 Hz, C ?-H), 5.49 (IH, s, C 4~H), 5.29 (IH, d, J=10 Hz, Cg-H) , 3.80 (6H, s, 2X-OCH_3) , 3.62 (3H, s, -OCH_3) , 3.50 (IH, s, C2-H) , 2.71 (3H, s, NCH_3), 2.11 (3H, s, -OCOCH3), 0.74 (3H, t, J=7 Hz, -CH2CH_3); ms'-m/e 782 (M+) , 723, 621 , 514, 135 (base peak). High resolution molecular weight determination calcd. for C 4 3H 5 0N 4O 1 Q: 782.3515; found: 782.3484. Periodic acid cleavage of the ketol 83 A solution of the ketol 83 (40 mg, 0.048 mmole) i n tetrahydrofuran (2 ml) at ambient temperature under a nitrogen atmosphere was treated with a solution of periodic acid (15 mg, 0.117 mmole) in tetrahydrofuran (1 ml). The reaction mixture was s t i r r e d for 4 h and the solvent was removed in vacuo. Chromatography of the residue on s i l i c a gel (methylene chloride/ 5% methanol/0.1% ammonium hydroxide) afforded the N^-formyl-aldehyde 90 (20 mg, 54%) i d e n t i c a l with that obtained above. Reaction of 4'-deoxyleurosidine (93) with potassium  permanganate A solution of 4 1-deoxyleurosidine (93) (260 mg, 0.33 mmole) i n acetone (5 ml) and methylene chloride (2 ml) at ambient temperature under a nitrogen atmosphere was treated with a solution of potassium permanganate (15 8 mg, 1 mmole) i n acetone (3 ml). The reaction mixture was s t i r r e d for 20 min 157 -and the solvent was removed in vacuo. The residue was t r i t u r -ated with methylene chloride (10 ml) and f i l t e r e d through s i l i c a gel (ethyl acetate/25% methylene chloride/15% methanol). Removal of the solvent i n vacuo followed by chromatography of the residue on s i l i c a gel (ethyl acetate/13% methanol) afforded vinamidine (82) (60 mg, 22%) and 19'-oxo-4 1-deoxyleurosidine (95) (30 mg, 11%). The synthetic vinamidine had oC= -35° 88 o 1 (C=l.l, CHC13) ( l i t -33 ). The Hmr and mass spectra as well as the t i c properties of the synthetic material were i n accord with those exhibited by an authentic sample.* 19'-Oxo-4 1-deoxyleurosidine (95), uv X : 311 (3.92), m 3.x 294 (4.03), 284 (4.08), 263 (4. 13), 212 (4.72); i r : 3476, 1736, 1640, 1615; 1Hmr <£: 8.05 (IH, bs, NH) , 7.58 (IH, m, C 1 4,-H), 7.17 (3H, m, C'11, -C 1 3 , -H' s) , 6.65 (IH, s, C^-H) , 6.16 (IH, s, C 1 ?-H), 5.88 (IH, dd, J=10.5, 4 Hz, C ?-H), 5.51 (IH, s, C 4-H), 5.31 (IH, d, J=10 Hz, C g-H), 4.84 (IH, m, C 2,-H), 3.82 (6H, s, 2X-OCH3), 3.78 (IH, s, C2~H) , 3.61 (3H, s, -OCH_3) , 2.74 (3H, s, NCH_3) , 2.12 (3H, s, -OC0CH_3), 0.92 (3H, t, J=7 Hz, -CH 2CH 3), 0.84 (3H, t, J=7 Hz, -CH2CH_3); ms:m/e 808 ( M + ) , 749, 690, 646 (base peak), 589, 540, 135. High resolution molecular weight determination calcd. for C. ,Hr/.N/,On: 808.4079; found: 46 56 4 9 808.4046. *The authentic sample was provided by the E. L i l l y Co., Indianapolis. - 158 -Catharininol (94) Catharininol was prepared from the synthetic (vinamidine) 88 catharimne v i a the l i t e r a t u r e procedure. This material had oC^-78 0 (C=0.42, CHC13) ( l i t -80°). 19 '-Oxoleurosine(91) A suspension of leurosine (3; (70 mg, 0.087 mmole) in aqueous 1.8% sodium bicarbonate solution (3.5 ml) was treated with a solution of iodine (45 mg, 0.35 mmole) in tetrahydro-furan (3 ml). The reaction mixture was s t i r r e d at ambient temperature for 10 min, diluted with 5% sodium bicarbonate solution (15 ml), and extracted with methylene chloride (3x15 ml). The combined organic portion was washed with 10% sodium b i s u l f i t e solution (1x15 ml) and brine (1x15 ml). After drying (Na 2S0 4), the solvent was removed i n vacuo and the residue chromatographed on s i l i c a gel (ethyl acetate/12% methylene chloride/12% methanol) to afford 19 1-oxoleurosine (91) (40 mg, 56%) i d e n t i c a l with the material prepared previously. 19'-Oxoleurosidine (96) Treatment of leurosidine (4) as above afforded 19'— oxoleurosidine (96) (62%) as an amorphous s o l i d , uv A : 309 max (3.74), 294 (4.00), 285 (4.04), 262 (4.11), 214 (4.67); i r ^ m a x : 3477, 1738, 1644, 1615; 1Hmr i : 8.07 (IH, bs, NE), 7.51 (IH, m, C 1 4,-H), 7.16 (3H, m, C^,-C 1 3 ,-H's) , 6.63 (IH, s, C 1 4-H), 6.16 (IH, s, C 1 7-H), 5.88 (IH, dd, J=10, 4 Hz, C ?-H), - 159 -5.50 (IH, s, C 4-H), 5.32 (IH, d, J=10 Hz, C g-H), 4.73 (IH, m, C 2,-H), 3.82 (6H, s, 2X-OCH_3) , 3.60 (3H, s, -OCH_3), 2.74 (3H, s, NCH_3), 2.11 (3H, s, -OCOCH_3), 0.96 (3H, t, J=7.5 Hz, -CH2CH_3) , 0.83 (3H, t, J=7 Hz, -CH2CH_3) ; ms: m/e 824 (M+) , 765, 665, 282, 135 (base peak). High resolution molecular weight determination calcd. for C^H^N^O., n : 824. 3996; found: 824. 3953 19'-Oxovinblastine (97) Treatment of vinblastine (1) as above afforded 19'— oxovinblastine (97) (33%) as an amorphous s o l i d , uv ?\ : 311 max (3.74), 294 (3.99), 285 (4.02), 259 (4.14), 213 (4.69); i r \l : 3475, 1740, 1640; 1Hmr i : 8.06 (IH, bs, NH) , 7.54 IUclX —• (IH, m, C 1 4,-H), 7.18 (3H, m, ,-C 1 3 ,-H' s) , 6.68 (IH, S, C 1 4-6.18 (IH, s, C 1 ?-H), 5.91 (IH, dd, J=10.5, 3.5 Hz, C ?-H), 5.54 (IH, s, C 4-H), 5.34 (IH, d, J=10.5 Hz, C g-H), 4.70 (IH, m, C 2,-H), 3.82 (3H, s, -OCH_3) , 3.80 (3H, s, -OCH_3) , 3.6 0 (3H, s, -OCH3) , 2.75 (3H, s, NCH_3) , 2.12 (3H, s, -OCOCH_3), 0.87 (6H, bt, J=7.5 Hz, 2X-CH2CH_3) ; ms: m/e 824 (M+) , 765, 665, 135 (base peak). High resolution molecular weight deter-mination calcd. for C. £.HccN.O. . : 824. 3996; found: 824. 3971. 46 56 4 10 - 160 -Reaction of 4'-deoxyleurosidine (93) with iodine/sodium  bicarbonate. Treatment of 4'-deoxyleurosidine (93) (40 mg, 0.05 mmole) as above afforded as the major product (28 mg, 69%) a compound possessing the following characteristics, i r ^ m a x : 3472, 1735, 1616; W i : 8.78 (IH, s), 8.10 (IH, bs, NH)f 7.53 (IH, m, C 1 4,-H), 7.16 (3H, m, , -C 1 3, -H' s) , 6.52 (IH, s, C14~H) , 6.13 (IH, s, C 1 ?-H), 5.88 (IH, dd, J=4.5,10 Hz, C ?-H), 5.46 (IH, s, C 4-H), 5.32 (IH, d, J=10 Hz, C g-H), 3.83 (3H, s, -OCH3), 3.81 (3H, s, -OCH3), 3.64 (3H, s, -OCH ), 2.72 (3H, s, NCH 3), 2.10 (3H, s, -OCOCH3), 0.89 (3H, t, J=7.5Hz, -CH 2CH 3), 0.79 (3H, t, J=7.5Hz, -CH 2CH 3); ms: m/e 808 ( M + ) , 806, 779, 749, 647, 448, 282, 135 (base peak). High resolution molecular weight determination calcd. for C . ,-H_^ N .0. : 808.4046; found: ^ 46 56 4 9 808.4048. Oxidation of 19'-oxoleurosine (91) with Jones reagent A solution of 19'-oxoleurosine (100 mg, 0.122 mmole) in acetone (6 ml) containing acetic anhydride (1.5 ml) at -78°C under a nitrogen atmosphere was treated with Jones reagent (0.4 ml, 1.06 mmole Cr0 3). The reaction mixture was s t i r r e d at -78°C for 35 min, treated with ammonium hydroxide solution - 161 -(10 ml), and allowed to warm to 0 C. The resultant solution was diluted with 1% sodium b i s u l f i t e solution (15 ml) and extracted with methylene chloride (3x15 ml). The combined organic portion was dried (Na 2S0 4) and the solvent was removed i n vacuo. Chromatography of the residue on s i l i c a gel (methy-lene chloride/5% methanol) afforded starting material (15 mg, Rp 0.7), N -desmethyl-19'-oxoleurosine (98) (17 mg, 17%, Rf 0.6) and the N A~formyl derivative 99 (30 mg, 29%, R f 0.5). The products were obtained as amorphous solid s . N -Desmethyl-19*-oxoleurosine, uv ?\ : 314 sh (3.75), A ITlclX 294 (4.07), 286 (4.11), 279 (4.09), 252 (4.10), 224 sh (4.58), 212 (4.70); i r 1 : 3476, 1735, 1645, 1620; W 8.02 max (IH, bs, Na,-H), 7.56 (1H-, m, C^.-H), 7.17 (3H, m, C-^.-C^,-H's), 6.66 (IH, s, C 1 4-H) , 6.30 (IH, s, C^-H) , 5.89 (IH, dd, 10, 3.5 Hz, C ?-H), 5.57 (IH, s, C 4~HJ, 5.34 (IH, d, J=10 Hz, C,-H), 4.78 (2H, m, C,,-H, N -H), 4.19 (IH, bs, C„-H), 3.81 (6H, b 2 a — A s, 2X-OCH3) , 3.62 (3H, s, -OCH_3), 2.14 (3H, s, -OCOCH_3) , 1. (3H, t, J=7.5 Hz, -CH2CH3) , 0.84 (3H, t, J=7 Hz, -CH2CH_3) ; 02 ms: m/e 808 (M') , 806, 749, 648, 296, 207, 174 (base peak). High resolution molecular weight determination calcd. for C45 H52 N4°io 808.3593; found: 808.3618. N -Formvl derivative 99, uv A • 296 (4.27), 288 (4.23), 280 (4.17),. 258 (4.24), 217 (4.78); i r i : 3478, ITlclX 1740, 1678, 1645, 1600; 1Hmr c£: 8.06 (IH, bs, NH) , 7.54 (IH, m, C 1 4,-H), 7.21 (3H, m, C n , -C 1 3, -H1 s) , 7.16 (IH, s, C 1 4~H) , 6.93 (IH, bs, C 1 ?-H), 5.97 (IH, dd, J=10, 4 Hz, C ?-H), 5.47 - 162 -(IH, d, J=10 Hz, Cg-H) , 5.28 (IH, s, C4~H) , 4.74 (2H, m, C2,-H, C2-H) , 3.95 (3H, s, -OCH_3) , 3.78 (3H, s, -OCH3> , 3.68 (3H, s, -OCH_3) , 2.10 (3H, s, -OCH_3) , 1.02 (3H, t, J=7 Hz, -CH2CH_3) , 0.87 (3H, t, J=7 Hz, -CH2CH_3) ; ms: m/e 836 (M+) , 834, 820, 777, 761, 677, 661, 636, 121 (base peak). High resolution molecu-l a r weight determination calcd. for C.^HroN.O,,: 836.3620; 4o OZ 4 JL± found: 836.3686. 22-Oxoleurosine (37) Oxidation of leurosine (3) as above afforded 22— oxoleurosine (75%), mp 211-216°C (methanol), uv A : 296 IuclX (4.22), 289 sh (4.18), 262 sh (4.15), 253 (4.20), 219 (4.68), i r ) : 3470, 1738, 1688; 1Hmr S: 8.80-8.20 (IH, m, NCHO), 8.01 (IH, bs, NH) , 7.53 (IH, m, C 1 4 I-H), 7.18 (3H, m, C^,-C 1 3 , - H , s ) / 6.88 (2H, bs, C-^-H, C-^-H), 5.94 (IH, dd, J=10, 4 Hz, C ?-H), 5.44 (IH, d, J=10 Hz, C g-H), 5.26 (IH, s, C 4~H), 4.66 (IH, m, C2-H) , 3.92 (3H, s, -OCH_3), 3.75 (3H, bs, -OCH_3) , 3.71 (3H, s, -OCH3), 2.09 (3H, s, -OCOCH3), 1.62 (2H, q, J=7.5 Hz, -CH 2CH 3), 0.97 (3H, t, J=7.5 Hz, -CH2CH_3), 0.84 (3H, t, J=7 Hz, -CH2CH_3) ; ms: m/e 822 (M+) , 820, 683 , 588, 121 (base peak), 106. High resolution molecular weight determination calcd. for C.,Hr.N.O,n: 822.3839; found: 822.3806. 46 54 4 10 Oxidation of 22-oxoleurosine (37) with iodine 22-Oxoleurosine (37) was oxidized with iodine in tetrahydrofuran as described previously in the preparation of 19'-oxoleurosine (91) from leurosine (3). 19',22-Dioxo-- 163 -leurosine (98), i d e n t i c a l with the product resulting from Jones oxidation of 19'-oxoleurosine, was obtained (67%). 6,7-Dihydroleurosine (101) A solution of 6,7-dihydro-3',4'-anhydrovinblastine (100) (60 mg, 0.076 mmole) in tetrahydrofuran (3 ml) containing 1% aqueous t r i f l u o r o a c e t i c acid (0.3 ml) and t-butyl hydro-peroxide (0.3 ml) was s t i r r e d at ambient temperature for 9 hr. The reaction mixture was diluted with a saturated solution of sodium bicarbonate (10 ml) and extracted with methylene chloride (3x6 ml) . The combined organic portion was dried (Na^O^) and the solvent was removed i n vacuo. Chromatography of the r e s i -due on s i l i c a gel (ethyl acetate/12% methanol) afforded 6,7— dihydroleurosine (101) (25 mg, 41%) as a glass, uvA : 310 max (3.75), 294 (4.00), 284 (4.03), 260 (4.12), 213 (4.68); i r L^ = ~ : 3478, 1738, 1612; 1Hmr <£: 8.03 (IH, bs, NH) , 7.54 III 3.X — (IH, m, C 1 4,-H), 7.18 (3H, m, C±1,-C13,-H's), 6.57 (IH, s, C 1 4-H), 6.13 (IH, s, C 1 7-H), 5.64 (IH, s, C 4~H), 3.84 (3H, s, -OCH_3), 3.82 (3H, s, -OCH_3), 3.73 (IH, s, C2~H) , 3.6 2 (3H, s, -OCH3), 2.68 (3H, s, NCH_3) , 2.12 (3H, s, -OCOCH_3) , 0.98 (3H, t, J=7.5 Hz, -CH2CH3) , 0.82 (3H, t, J=7 Hz, -CH2CH_3) ; ms: m/e 810 (M +), 808, 671, 453, 106 (base peak). High resolution molecular weight determination calcd. for C . ,-H,_nN .0„ : 810.4203; ' 46 58 4 9 found: 810.4203. 22-OXO-6,7-dihydroleurosine (102) Jones oxidation of 6,7-dihydroleurosine (101) as - 164 -described previously for 19'-oxoleurosine (91), afforded 22-OXO-6,7-dihydroleurosine (102) (72%), mp 201-205°C (methanol); uv A : 296 (4.22), 287 sh (4.18), 252 (4.20), 218 (4.68); i r i> : 3478, 1738, 1687; 1Hmr $: 8.00 (IH, bs, NH), 6.82 (IH, s, C 1 7-H), 5.40 (IH, s, C 4~H), 4.67 (IH, bs, C2-H) , 3.92 (3H, s, -OCH_3), 3.81 (3H, s, -OCH^), 3.66 (3H, s -OCH_3), 2.08 (3H, s, -OCOCH_3) , 0.99 (6H, t, J=7 Hz, 2X-CH2CH ms: m/e 824 (M +), 686, 658, 138, 124 (base peak), 106. High resolution molecular weight determination calcd. for C/l<.H1.cN.O. 824. 3995; found: 824.3955. 46 56 4 10 - 1 6 5 -BIBLIOGRAPHY 1. E.S. Greenwald, "Cancer Chemotherapy" (Medical Outline Series), 2nd ed., Medical Examination Publishing Co., Flushing N.Y., 1973. 2. "Cancer Chemotherapy I I " , edited by I. 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