UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies in natural products Inaba, Tadanobu 1967

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The U n i v e r s i t y o f B r i t i s h C o l u m b i a FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of TADANOBU INABA B.Eng., The U n i v e r s i t y o f Osaka P r e f . , 1962 M . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1964 THURSDAY, OCTOBER 26, 1967, AT 3:30 P.M. IN ROOM 225, CHEMISTRY BUILDING COMMITTEE IN CHARGE E x t e r n a l Examiner: P r o f e s s o r T.A. Giessman Department o f C h e m i s t r y U n i v e r s i t y o f C a l i f o r n i a Los A n g e l e s , C a l i f o r n i a Chairman: I . McT. Cowan B.A. Bohm J.P. Kutney E. P i e r s W.R. C u l l e n N.L. Paddock T. Money R e s e a r c h S u p e r v i s o r : J.P. Kutney STUDIES IN NATURAL PRODUCTS ABSTRACT In part I of t h i s t h e s i s i s described the s t r u c t u r determination of thamnosin, a minor component obtained from Thamnosma montana To r r . and Frem. Thamnosin, C„„H„ o0,, was shown by NMR and IR spect j U Zo o to posess a coumarin chromophore and the mass spectrum suggested that t h i s substance was cleaved under e l e c t r o impact i n t o two equal h a l v e s . C a t a l y t i c hydrogenation of thamnosin gave i t s d i h y d r o - d e r i v a t i v e and thereby i n d i c a t e d the presence of one e a s i l y reduced o l e f i n i c l i n k a g e . The UV spectrum of the l a t t e r suggested that two 6-alkyl-7-methoxycoumarin chromophores were present The cleavage of thamnosin i n t o lower molecular wei fragments was achieved by osmium t e t r o x i d e h y d r o x y l a t i c of the double bond followed by p e r i o d i c a c i d o x i d a t i o n of the r e s u l t i n g d i o l . The two aldehydic products (C,,H„0' and C.„H 0,) which were obtained from t h i s 11 8 4 19 20 4 r e a c t i o n were subsequently c h a r a c t e r i z e d . The smaller fragment was i d e n t i f i e d as 7-methoxycoumarin-6-aldehyde by d i r e c t comparison w i t h an authentic sample?. The oth fragment, C^9 H20^4' W a S ^^i-al-ly shown by s p e c t r o s c o p i evidence, to possess a 6- s u b s t i t u t e d 7-methoxycoumarin system. The nature of the s u b s t i t u e n t at C-6 was sub-sequently i d e n t i f i e d as cyclohexene d e r i v a t i v e bearing two methyl groups and a t e r t i a r y formyl func-t i o n s . Summation of the above and other evidence allowed a s t r u c t u r a l assignment to thamnosin,, I t i s seen that t h i s substance represents a novel system which has not been p r e v i o u s l y obtained i n nature. A d e t a i l e d d i s c u s s i o n of the mass sp e c t r a of thamnosin and i t s d e r i v a t i v e s i s presented. P a r t I I describes a p o s s i b l e s y n t h e t i c route to the vobasine- and sarpagine-type a l k a l o i d s . Three d i f f e r e n t approaches to the p r e p a r a t i o n of 5-dehydro s a l t s and to r i n g c l o s u r e - O f corynan-thenoid bases v i a transannular c y c l i z a t i o n s were attempted. F i r s t l y , o x i d a t i o n of s i t s i r i k i n e (121) and dihydrocorynantheic a c i d e t h y l e s t e r (126) by mercuric acetate predominantly gave 3-dehydro s a l t s (122) and (127), r e s p e c t i v e l y , while the formation of 5-dehydro s a l t s was not s i g n i f i c a n t to form the bridge between C-16 and C-5 v i a t r a n s -annular c y c l i z a t i o n . Secondly, mercuric acetate o x i d a t i o n s of 3-benzylderivatives of corynanthenoid bases followed by transannular c y c l i z a t i o n s were attempted. Preparation.of the isomeric 3o( - and 3(J - b e n z y l y o h i m b i n e s was a c c o m p l i s h e d by t h e r e a c t i o n o f b e n z y l magnesium bromide w i t h 3-dehydroyohimbine p e r c h l o r a t e . The s t e r e o c h e m i s t r y o f t h e s e compounds was e s t a b l i s h e d by NMR and mass s p e c t r a . A c c o r d i n g l y , 3c\- b e n z y l - d e r i v a t i v e s o f d i h y d r o c o r y n a n t h e i n e , d i h y d r o c o r y n a n t h e i c a c i d e t h y l and m e t h y l e s t e r and the t e t r a c y c l i c m e t h y l k e t o n e (157) were p r e p a r e d . O x i d a t i o n of t h e s e d e r i v a t i v e s by m e r c u r i c a c e t a t e p r o c e e d e d b u t t r a n s a n n u l a r c y c l i z a t i o n was not s u c c e s s f u l . T h i r d l y , o x i d a t i o n o f 3 , 4 - s e c o - c o r y n a n t h e i n o i d bases by m e r c u r i c a c e t a t e was a t t e m p t e d . D i h y d r o c o r y -' n a n t h e a l e t h y l e n e a e c t a l m e t h i o d i d e (173) was t r e a t e d w i t h sodium i n l i q u i d ammonia t o g i v e 3,4-seco-N^-m e t h y l d i h y d r o c o r y n a n t h e a l e t h y l e n e a c e t a l (174), w h i c h c o u l d be o x i d i z e d by m e r c u r i c a c e t a t e t o the dehydro s a l t . However subsequent h y d r o l y s i s o f the e t h y l e n e a c e t a l was not s u c c e s s f u l . GRADUATE STUDIES F i e l d of Study: O r g a n i c C h e m i s t r y H e t e r o c y c l i c C h e m i s t r y F.."McCapra A l k a l o i d C h e m i s t r y J.P. K u t n e y I s o p r e n o i d C h e m i s t r y T. Money P h y s i c a l O r g a n i c C h e m i s t r y R. S t e w a r t O r g a n i c R e a c t i o n Mechanism R.E. P i n c o c k PUBLICATIONS J.P. Kutney, A. By, T. I n a b a and S.Y. Leong. A T o t a l l y S y n t h e t i c E n t r y I n t o the V e r a t r u m . A l k a l o i d S k e l e t o n , T e t r a h e d r o n L e t t e r s , 2911 (1965) J.P. Kutney and T. Inaba - The S t r u c t u r e o f S u b m i t t e d f o r P u b l i c a t i o n Thamnosin STUDIES IN NATURAL PRODUCTS by TADANOBU INABA B . E n g . , The U n i v e r s i t y o f Osaka P r e f . , 1962 M . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f Chem i s t r y We a c c e p t t h i s t h e s i s as con fo rm ing to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September , 1967 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department or by hi's r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f C^^H ) ST/? ^ The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date 27 /7^7 11 A b s t r a c t In p a r t I o f t h i s t h e s i s i s d e s c r i b e d the s t r u c t u r a l d e t e r m i n a t i o n o f t hamnos in , a m inor component o b t a i n e d f rom Thamnosma montana T o r r . and Frem. Thamnos in , C ^ Q ^ g O g , was shown by NMR and IR s p e c t r a to posess a coumar in chromophore and the mass spec t rum sugges ted t h a t t h i s subs tance was c l e a v e d under e l e c t r o n impact i n t o two equa l h a l v e s . C a t a l y t i c h y d r o g e n a t i o n o f thamnos in gave i t s d i h y d r o - d e r i v a t i v e and t he r eby i n d i -c a t e d the p r e sence o f one e a s i l y r educed o l e f i n i c l i n k a g e . The UV spec t rum o f the l a t t e r sugges ted tha t two 6 - a l k y l -7-methoxycoumarin chromophores were p r e s e n t . The c l e a v a g e o f thamnos in i n t o lower m o l e c u l a r we ight f ragments was a c h i e v e d by osmium t e t r o x i d e h y d r o x y l a t i o n o f the doub l e bond f o l l o w e d by p e r i o d i c a c i d o x i d a t i o n o f the r e s u l t i n g d i o l . The two a l d e h y d i c p r o d u c t s (C^^HgO^ and C^gH2Q0^) wh ich were o b t a i n e d f rom t h i s r e a c t i o n were s u b s e q u e n t l y c h a r a c t e r i z e d . The s m a l l e r f ragment was i d e n t i f i e d as 7-methoxycoumar in-6-aldehyde by d i r e c t compar i son w i t h an a u t h e n t i c samp le . The o t h e r f r agmen t , ^19^20^4' w a s i 1 1 ^ * - 3 ! ^ shown by s p e c t r o s c o p i c e v i d e n c e , to posses s a 6 - s u b s t i t u t e d 7-methoxycoumarin s ys t em. The n a t u r e o f the s u b s t i t u e n t a t C-6 was s u b s e q u e n t l y i d e n t i -f i e d as c y c l ohexene d e r i v a t i v e b e a r i n g two methy l groups and a t e r t i a r y f o r m y l f u n c t i o n s . Summation o f the above and o t h e r e v i dence a l l o w e d a s t r u c t u r a l ass ignment to • • * 1 1 1 thamnosin. I t i s seen that t h i s substance represents a novel system which has not been previously obtained i n nature. A d e t a i l e d discussion of the mass spectra of thamnosin and i t s d e r i v a t i v e s i s presented. Part I I describes a possible synthetic route to the vobasine- and sarpagine-type a l k a l o i d s . Three d i f f e r e n t approaches to the preparation of 5-dehydro s a l t s and to ri n g closure of corynanthenoid bases v i a transannular c y c l i z a t i o n s were attempted. F i r s t l y , oxidation of s i t s i r i k i n e (121) and dihydro-corynantheic acid ethyl ester (126) by mercuric acetate predominantly gave 3-dehydro s a l t s (122) and (127), re-spec t i v e l y , while the formation of 5-dehydro s a l t s was not s i g n i f i c a n t to form the bridge between C-16 and C-5 v i a transannular c y c l i z a t i o n . Secondly, mercuric acetate, oxidations of 3-benzyl-der i v a t i v e s of corynanthenoid bases followed by trans-annular c y c l i z a t i o n s were attempted. Preparation of the isomeric 3ol- and 3p-benzylyohimbines was accomplished by the reaction of benzyl magnesium bromide with 3-dehydro-yohimbine perchlorate. The stereochemistry of these compounds was established by NtyR and mass spectra. Accordingly, 3o(-benzyl-derivattves of dihydrocorynantheine, dihydrocorynantheic acid ethyl and methyl ester and the t e t r a c y c l i c methyl ketone (157) were prepared. Oxidation of these derivatives by mercuric acetate proceeded but i v t r a n s a n n u l a r c y c l i z a t i o n was not s u c c e s s f u l . T h i r d l y , o x i d a t i o n o f 3 , 4 - s e c o - c o r y n a n t h e i n o i d bases by m e r c u r i c a c e t a t e was a t t e m p t e d . D i h y d r o c o r y n a n t h e a l e t h y l e n e a e c t a l m e t h i o d i d e (173) was t r e a t e d w i t h sod ium i n l i q u i d ammonia to g i v e 3 ,4-seco-N^-methy ld ihydro-c o r y n a n t h e a l e t h y l e n e a c e t a l ( 174 ) , wh ich c o u l d be o x i -d i z e d by m e r c u r i c a c e t a t e to the dehydro s a l t . However subsequent h y d r o l y s i s o f the e t h y l e n e a c e t a l was not s u c c e s s f u l . V T a b l e o f C o n t e n t s 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 Con ten t s v L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i Acknowledgements i x P a r t Is S t r u c t u r e E l u c i d a t i o n o f Thamnosin-A n o v e l Coumar in System I n t r o d u c t i o n 1 D i s c u s s i o n 15 I n t e r p r e t a t i o n o f Mass S p e c t r a 47 E x p e r i m e n t a l 66 Re f e r ences 81 P a r t I I : S t u d i e s i n I ndo l e A l k a l o i d s I n t r o d u c t i o n 86 D i s c u s s i o n 116 E x p e r i m e n t a l 161 Mass S p e c t r a l Da ta 195 Re f e r ences 203 v i L i s t of Tables Table Page 1 , 3 2 36 3 39 v i i L i s t of Figures Part I Figue Page Figure Page 22 51 1 6 23 53 2 .7 24 54 3 . 8 25 55 4 10 26 58 5 11 27 59 6 12 28 60 7 13 29 61 8 14 30 63 9 . .. . 16 31 64 10 18 11 . 20 Part II 12 21 1 87 13 23 2 88 14 27 3 89 15 28 4 91 16 35 5 92 17 41 6 93 18 44 7 95 19 48 8 96 20 49 9 97 21 50 10 99 v x i i L i s t of Figures (Cont'd) Figure Page 11 101 12 102 13 103 14 103 15 105 16 106 17 108 18 109 19 110 20 I l l 21 112 22 113 23 114 24 115 25 117 26 118 27 eooooeooooo 118 28 119 29 122 30 124 31 127 Figure 32 33 o » o o 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 o o o o o 0 0 0 * ooooooooo ooooooooo ooooooo ooooooooo OOOOOOOOO ooooooooo 0 0 0 0 OOOOOOOOO OOOOOOOO OOOOOOO 0 0 0 0 0 * 0 0 ooooooo ooooooo OOOOOOOO O O 0 o o o Page 128 130 132 133 134 136 137 139 142 143 146 147 149 151 152 153 155 156 157 158 160 i x Acknowledgements I w i s h to express my s i n c e r e thanks to D r . James P. Kutney f o r h i s c o n s t a n t h e l p and encouragement d u r i n g the cou r se o f my r e s e a r c h . Thanks a r e due to D r . John R. H a d f i e l d and D r . Edward P i e r s f o r t h e i r i n v a l u a b l e i n s t r u c t i o n s i n the e x p e r i m e n t a l a s p e c t s . PART I STRUCTURE ELUCIDATION OF THAMNOSIN-A NOVEL COUMARIN SYSTEM -1-INTRODUCTION One of the major problems i n organic chemistry has always been the structure e l u c i d a t i o n of organic molecules which were l a r g e l y supplied by natural sources. Interest-i n g l y the majority of natural products incorporate at lea s t one benzene r i n g . The plants of the Rutaceae family are well known to contain a large number of benzenoid compounds, coumarins, flavones and some quinoline a l k a l o i d s . The turpentine-broom, Thamnosma montana Torr. and Frem. (Rutaceae), i s found i n desert mesas and slopes and these shrubby plants were reported to have plant-growth-inhibitor 1 2 properties ' and to have been used by American Indians i n 3 f o l k medicine . Bennett and Bonner studied the t o x i c i t y of aqueous extracts of leaves of eleven desert plant species and found that Thamnosma montana Torr. and Frem. was the most t o x i c as judged by the response to tomato"''. The crude material caused death of young tomato plants at concentration of about 1 mg./ml. within seven days. Three c r y s t a l l i n e compounds were i s o l a t e d from Thamnosma montana and two of them were i d e n t i f i e d to be byakangelicin (1) and i s o p i m p i n e l l i n (2)^". The structure of the t h i r d compound was elucidated 4 by Dreyer and found to be the d i o l (3), 5-(3 1-methyl-2',3'-dihydroxybutanyl)-8-methoxypsoralen. Dreyer de-veloped a better extraction scheme f o r i s o l a t i n g not only -2-t h e t h r e e compounds o b t a i n e d by B e n n e t t and B o n n e r b u t was a b l e t o s e p a r a t e s i x o t h e r compounds. T h i s e x t r a c t i o n p r o c e d u r e i s s c h e m a t i c a l l y r e p r e s e n t e d b e l o w . E x t r a c t i o n Scheme g r o u n d whole a e r i a l p l a n t e x t r a c t e d w i t h a c e t o n e a c e t o n e e x t r a c t c o n c e n t r a t e d t o 1/3 volume d a r k h e a v y t a r a c e t o n e s o l u b l e hexane f a t s & waxes c h r o m a t o g r a p h y on a l u m i n a h e x a n e -b e n z e n e ( 1 : 1 ) benzene b e n z e n e -c h l o r . ( 1 : 1 ) I s o p i m p i n e l l i n T h a m n o s i n s o l u b l e i n 15% H C l c h l o r . c h l o r . -a c e t o n e ( 9 : 1 ) S k i m m i a n i n e X - F a g a r i n e ^ - S i t o s t e r o l A l l o i m p e r a t o r i n m e t h y l e t h e r N - M e t h y l a c r i d o n e Byakang e 1 i c i n The d i o l (3) -3-As shown i n the ext r a c t i o n scheme and i n Table 1, these s i x compounds were p-sitosterol, three known a l k a l o i d s , a known furocoumarin and an unknown compound, thamnosin. This i s the f i r s t report of N-methylacridone, the parent member of acridone a l k a l o i d s ^ , occurring as a natural product. Table 1 Name Formula m.p. Ref. ^ - S i t o s t e r o l (4) C 2 9 H 5 0 ° 137-9° Alloimperatorin methyl ether (5) C17 H16°4 108-10° 10 Isopimpinellin (2) C11 H10°5 148-9° 1,4 Thamnosin C25 H26°5 244-6° 4 N-Methylacridone (6) C 1 4 H n 0 N 202-3° 4,5 Skimmianine (7) C 1 4 H 1 3 ° 4 N 173-5° 7,8,9 y-Fagarine (8) C 1 3 H 1 1 0 3 N 140-2° 7,8,9 Byakangelicin (1) C17 H18°7 105-7° 1,4 The d i o l (3) C17 H18°6 174-6° 1,4 Dreyer's proposal f o r the structure of thamnosin was the following^. Thamnosin, m.p. 244-6°, was analyzed f o r ^25^26^5 a n c * ^"ts u l t r a v i ° l e t spectrum suggested that thamnosin might be a coumesterol d e r i v a t i v e with the long wavelength band s h i f t e d s l i g h t l y to higher energies. The presence of a lactone r i n g was shown by treatment with 5% sodium hydroxide i n aqueous methanol and regeneration o f t h e s t a r t i n g m a t e r i a l u p o n a c i d i f i c a t i o n . F u s i o n w i t h p o t a s s i u m h y d r o x i d e gave r e s o r c i n o l . The n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r u m showed t h e p r e s e n c e o f two m e t h y l g r o u p s two m e t h o x y l g r o u p s , one o l e f i n i c p r o t o n and a complex m u l t i p l e t w h i c h was a s s i g n e d t o n i n e a r o m a t i c p r o t o n s . D r e y e r s u g g e s t e d a t e n t a t i v e s t r u c t u r e f o r t h a m n o s i n as t h e f o l l o w i n g . T h i s s e c t i o n o f t h e t h e s i s w i l l be c o n c e r n e d w i t h t h e c o m p l e t e e l u c i d a t i o n o f t h e s t r u c t u r e o f t h a m n o s i n . -5-B i o s y n t h e s i s o f C o u m a r i n s B e f o r e d i s c u s s i n g t h e s t r u c t u r a l e l u c i d a t i o n o f t h a m n o s i n , I f e l t t h a t i t was a p p r o p r i a t e t o f i r s t p r o v i d e a b r i e f summary o f t h e i n v e s t i g a t i o n s d e a l i n g w i t h t h e b i o s y n t h e s i s o f c o u m a r i n s . B e f o r e t h e a p p l i c a t i o n o f r a d i o a c t i v e t r a c e r s became p o s s i b l e , a t t e n t i o n was f o c u s s e d on s t r u c t u r a l r e g u l a r i t i e s w i t h i n f a m i l i e s o f compounds and i n t h i s way a t t e m p t s were made t o p r e d i c t p l a u s i b l e b i o g e n e t i c p a t h w a y s . R u z i c k a ' s i s o p r e n e r u l e a r o s e f r o m s u c h c o n s i d e r a t i o n s . I n a s i m i l a r m a n n e r , R o b i n s o n r e g a r d e d f l a v o n e s , c h a l c o n e s , a n t h o c y a n i n s and r e l a t e d compounds as c o n t a i n i n g C ^ - C ^ - C ^ u n i t s ; c i n n a m i c a c i d s , c o u m a r i n s and some o t h e r g r o u p s o f n a t u r a l p r o d u c t s as c o n t a i n i n g C ^ - C ^ u n i t s ^ . I t a p p e a r s t h a t t h e l a r g e number o f b e n z e n o i d com-pounds i n n a t u r e d e r i v e t h e i r a r o m a t i c r i n g s by one o f two r o u t e s i (1) v i a s h i k i m i c a c i d o r (2) t h r o u g h p o l y a c e t y l 12 c h a i n c y c l i z a t i o n . The p h e n y l p r o p a n e s k e l e t o n ( C g - C ^ ) i s p r o b a b l y t h e b a s i s o f n a t u r a l c o u m a r i n s and i t has b e e n s u g g e s t e d t h a t t h e b e n z e n e r i n g s a r i s e most l i k e l y f r o m s h i k i m i c a c i d i n v i e w o f t h e o v e r w h e l m i n g p r e p o n d e r a n c e i n t h e Cg~Cg compounds o f t h e t y p i c a l s h i k i m i c - d e r i v e d o x i d a t i o n p a t t e r n s ( F i g u r e 1 ) . The f i r s t p r o p o s a l f o r c o u m a r i n b i o s y n t h e s i s f r o m 13 c i n n a m i c a c i d s was o f f e r e d by Haworth , who p o s t u l a t e d p a r a o x i d a t i o n o f a p - h y d r o x y - c i n n a m i c a c i d (9) t o y i e l d ( 1 1 ) , w h i c h c o u l d t h e n c y c l i z e by a M i c h a e l a d d i t i o n o f mo iHOH JHOH iH2op T e t r o s e CH 9 =C' z >COOH Phospho-eno l P y ruva t e (PEP) CooH P r e p h e n i c A c i d P h e n y l p y r u v i c A c i d COOH CO C H 2 CHOH CHOH $HOH CH 2OPP Ho C o o H C C o H per O H D e h y d r o q u i n i c S h i k i m i c A c i d A c i d P h e n y l a l a n i n e ccoH .CooH .CcoH CooH C innami c A c i d CocH T y r o s i n e F i g u r e 1 The S h i k i m i c A c i d Pathway to A roma t i c Compounds p-Coumar ic A c i d I -7-carboxyl, and f i n a l l y dehydrate to y i e l d a 7-hydroxy-coumarin (15), (Figure 2), I t should be noted that most of the known coumarins possess the 7-oxygen function. Grisebach and O l l i s " ^ suggested the d i r e c t oxidative coupling of the para-position with the cinnamoyl carboxyl, as i n (12), whereas Kenner et a l " ^ have favored i n i t i a l two-electron oxidation of the carboxyl, as i n (10),- so as not to have the process dependent on the p-hydroxyl function. (14) (15) Figure 2 The coumarins display an impressive v a r i a t i o n i n structure (Figure 3) and therefore provide ample -8-o p p o r t u n i t y f o r b i o s y n t h e t i c i n v e s t i g a t i o n s . O C H , C H S c o p o l e t i n X a n t h o x y l e t i n B e r g a m o t i n A n g e l i c i n F i g u r e 3 X a n t h y l e t i n The m e t h y l and i s o p e n t e n y l s u b s t i t u e n t s a r e a l w a y s f o u n d a t a r o m a t i c s i t e s c o n c o n a n t w i t h t h e mechanism o f 16 e n o l a l k y l a t i o n ( p p - p y r o p h o s p h a t e r e s i d u e ) The u l t i m a t e s o u r c e o f t h e s e i s o p r e n o i d u n i t s i s m e v a l o n i c a c i d , w h i c h e v e n t u a l l y g i v e s r i s e t o t h e a c t i v e s y n t h e t i c f r a g m e n t , i s o p e n t e n y l p y r o p h o s p h a t e . I t seems p r o b a b l e t h a t t h e c o u r s e o f b i o s y n t h e s i s d e s c r i b e d above r e p r e s e n t s t h e e s s e n t i a l n a t u r e o f t h e s e p r o c e s s e s b u t - 9 -o b v i o u s l y exper iments w i t h l a b e l l e d i n t e r m e d i a t e s a r e n e c e s s a r y to e s t a b l i s h o r exc lude them. Some o f the r e c e n t i n v e s t i g a t i o n s i n t h i s a r e a w i l l be d i s c u s s e d be low . E v i dence f rom f e e d i n g exper iments showed t h a t coumar in (21) was s y n t h e s i z e d f rom s h i k i m i c a c i d - d e r i v e d pheny lp ropane 17 18 19 p r e c u r s o r s (Cg-Cg) ' ' i n p r e f e r e n c e to a c e t a t e conden -s a t i o n . Brown, Towers and W r i g h t ^ s t u d i e d coumar in f o r m a t i o n w i t h i n the p e r e n n i a l g r a s s , H i e r o c h l o e o d o r a t a , and found t h a t the b e s t p r e c u r s o r s were o-coumar ic a c i d (18) and c i n n a m i c a c i d ; p h e n y l a l a n i n e (16) and s h i k i m i c a c i d were not as good , a c e t a t e and s a l i c y l i c a c i d v e r y p o o r . T h i s work i n d i c a t e d the same r e s u l t s as those o f K o s u g e ' s 18 f e e d i n g exper iment i n sweet c l o v e r , M e l i l o t u s a l b a . 20 The f o l l o w i n g r e s u l t s by Kosuge and Conn , by S toke r 21 33 and B e l l i s and by Gorz and H a s k i n s i n d e p e n d e n t l y showed t h a t the c o n v e r s i o n o f t r a n s - c i n n a m i c a c i d (17) to coumar in i n M e l i l o t u s a l b a o c c u r r e d by way o f o-coumar ic a c i d ( 1 8 ) , o-coumar ic a c i d ^ - g l u c o s i d e (19) and o-coumar i n i c a c i d (3>~glucoside ( 2 0 ) . T h i s p l a n t was a l s o shown to c o n t a i n 22 an t r a n s - c i s i somerase enzyme system (19-20) ( F i g u r e 4 ) . 23 Brown has shown t h a t p-coumar ic a c i d was seven ty t imes l e s s e f f e c t i v e than c i n n a m i c a c i d as a p r e c u r s o r of coumar in i n H i e r o c h l o e o d o r a t a , w h i l e t y r o s i n e was s i x t y t imes l e s s e f f e c t i v e than p h e n y l a l a n i n e ( 1 6 ) . I t was c l e a r t h a t a p re fo rmed p h e n o l i c n u c l e u s posed i n f a c t a d e c i d e d d i s a d v a n t a g e i n i t s use as a p r e c u r s o r . The above - 1 0 -(19) (20) (21) F i g u r e 4 B i o s y n t h e s i s o f c o u m a r i n ( 2 1 ) r e s u l t s s u p p o r t t h a t t h e o - h y d r o x y l a t i o n mechanism (17*18) 24 i s o p e r a t i n g i n t h e c o u r s e o f t h e b i o s y n t h e s i s o f c o u m a r i n 15 18 K e n n e r e t a l u s e d t y r o s i n e l a b e l l e d w i t h 0 i n t h e c a r b o x y l g r o u p t o p r o v e t h a t t h e oxygen atom a t p o s i t i o n 1 i n n o v o b i o c i n o r i g i n a t e d f r o m c a r b o x y l , i . e . t h a t t h e s i m p l e o x i d a t i v e c y c l i z a t i o n t h e y p o s t u l a t e c a n a l s o o p e r a t e . N o v o b i o c i n The b i o s y n t h e s i s o f t h e 7 - o x y g e n a t e d c o u m a r i n , u m b e l l i f e r o n e ( 2 4 ) , i n H y d r a n g e a m a c r o p h y l l a was s t u d i e d - 1 1 -by Brown, Towers and Chen • and by A u s t i n and Meyers and the f e e d i n g and t r a p p i n g expe r imen ts w i t h ^ C - l a b e l l e d compounds e s t a b l i s h e d the e x i s t e n c e o f the f o l l o w i n g pathway, I n t e r e s t i n g l y r a d i o a c t i v e c i s - p - h y d r o x y c i n n a m i c a c i d was i n c o r p o r a t e d about one-seven th as e f f i c i e n t l y as p-coumar ic a c i d ( 2 2 ) 2 6 ( F i g u r e 5 ) . (22) C o o H H O (23) C o o H Q U O COOH ^0 (24) F i g u r e 5 B i o s y n t h e s i s o f U m b e l l i f e r o n e ( 2 4 ) 27 28 A n a l y s e s o f l a v e n d e r p l a n t s by Brown ' w i t h c a r e f u l p r e c a u t i o n s to a v o i d enzymic h y d r o l y s i s , have shown t h a t h e r n i a r i n (27) e x i s t s i n the p l a n t a t l e a s t 99% i n a bound f o r m , presumably as c i s - 2 - g l u c o s y l o x y - 4 - m e t h o x y c i n n a m i c a c i d (c is-GMC) ( 2 6 ) . The use o f t r a p p i n g and f e e d i n g t e c h n i q u e s enab l ed Brown to p o s t u l a t e the f o l l o w i n g pathway f o r h e r n i a r i n (27) f o r m a t i o n ( F i g u r e 6 ) . B i o s y n t h e t i c i s o p r e n y l a t i o n o f coumar ins i s v e r y common, and these compounds o f t e n appear i n the fo rm o f 29 30 f u r a n s w i t h t h r e e m i s s i n g i s o p r e n o i d ca rbon atoms ' F l o s s and Mothes s t u d i e d the i n c o r p o r a t i o n o f r a d i o --12-c i s - G M C ( 2 6 ) (27) F i g u r e 6 B i o s y n t h e s i s o f H e r n i a r i n ( 2 7 ) a c t i v e c i n n a m i c a c i d and u m b e l l i f e r o n e i n t o f u r o c o u m a r i n s E v i d e n c e was p r e s e n t e d t h a t p a r a - h y d r o x y l a t i o n o f t h e c i n n a m i c a c i d p r e c u r s o r p r e c e d e d o r t h o - h y d r o x y l a t i o n , s i n c e u m b e l l i f e r o n e was a f a r b e t t e r p r e c u r s o r t h a n c i n n a m i c a c i d , whereas c o u m a r i n gave o n l y v e r y p o o r i n c o r p o r a t i o n . 32 More r e c e n t work by t h e same a u t h o r s d e m o n s t r a t e d t h e i n c o r p o r a t i o n o f r a d i o a c t i v e m e v a l o n i c a c i d i n t o 14 c o u m a r i n s . F e e d i n g e x p e r i m e n t s o f c i n n a m i c a c i d - ( C O O H - C) and m e v a l o n i c a c i d-(4-^Q) i n t o r o o t s o f P i m p i n e l l a magna and d e g r a d a t i o n o f t h e l a b e l l e d f u r o c o u m a r i n s , b e r g a p t e n and p i m p i n e l l i n , showed t h a t t h e c o u m a r i n p a r t o f t h e f u r o c o u m a r i n s k e l e t o n i s formed f r o m c i n n a m i c a c i d , whereas t h e two e x t r a - c a r b o n s o f t h e f u r a n r i n g o r i g i n a t e f r o m C-4 and C-5 o f m e v a l o n i c a c i d ( F i g u r e 7 ) . -13-B e r g a p t e n F i g u r e 7 T h i s r e s u l t r e f l e c t s t h e o n l y e x p e r i m e n t r e p o r t e d i n t h e l i t e r a t u r e w h i c h c a s t s some e v i d e n c e on t h e o r i g i n o f t h e two e x t r a c a r b o n atoms o f t h e f u r a n r i n g . G r i s e b a c h and B a r z ' i n v e s t i g a t e d t h e b i o s y n t h e s i s o f c o u m a r a n o - c o u m a r i n , c o u m e s t r o l , and showed t h a t t h i s compound i s a n i s o f l a v o n e , w i t h t h e b e n z e n o i d r i n g o f t h e c o u m a r i n n u c l e u s o r i g i n a t i n g f r o m a c e t a t e , and t h e r e m a i n -i n g n i n e c a r b o n s f r o m p h e n y l p r o p a n o i d p r e c u r s o r s ( C g - C g ) . C o u m e s t r o l - 1 4 -37 V e r y r e c e n t work by K u n e s c h and P o l o n s k y showed t h a t t h e s p e c i f i c i n c o r p o r a t i o n o f (-)-(3-^c] p h e n y l a l a n i n e ( a d e m o n s t r a t e d p r e c u r s o r o f c i n n a m i c a c i d ^ ) i n t o a 4 -p h e n y l c o u m a r i n ( n e o f l a v a n o i d ) , c a l o p h y l l o l i d e (28), s u p p o r t e d 38 t h e b i o g e n e t i c scheme s u g g e s t e d by S e s h a d r i and by O l l i s 3 9 ( F i g u r e 8). (28) F i g u r e 8 -15-DISCUSSION I would now l i k e to discuss the experimental r e s u l t s which allow us to postulate a structure f o r thamnosin, one 4 of the new constituents i s o l a t e d by Dreyer . As already mentioned i n the Introduction (see extraction scheme) thamnosin was obtained as a minor component from Thamnosma  montana Torr. and Frem. A sample of t h i s compound was obtained f o r further s t r u c t u r a l studies through the kind co-operation of Dr. D. L. Dreyer, F r u i t and Vegetable Laboratory, U.S. Department of A g r i c u l t u r e , Pasadena, C a l i f o r n i a . The molecular formula, ^25^26^6' n a < ^ D e e n previously assigned f o r thamnosin but the reexamination of t h i s substance by high r e s o l u t i o n nuclear magnetic resonance (NMR) and mass spectrometry i n our laboratory led to the correct molecular formula (^^Q^28^6' ^ OUN^S M.W. 484.188). A Z e i s e l determination on thamnosin showed the presence of two methoxyl groups. The strong and complex absorption (Amax 227, 256 and 333 mu) i n the u l t r a v i o l e t (UV) spectrum and i n the appropriate regions of the NMR spectrum suggested the presence of highly conjugated systems. Bands at 1725, 1610 and 1557 cm"1 i n the i n f r a r e d (IR) spectrum indicated the presence of oi-pyrone or coumarin 40 chromophores and c l e a r l y eliminated a tf-pyrone system from consideration. There were also absorption bands i n d i c a t i v e of t r i s u b s t i t u t e d and trans d i s u b s t i t u t e d double bonds at 820 and 980 cm"1, r e s p e c t i v e l y , but no ro CM P • H to O ON 0 0 U o <u a, m i n co PS vD 0) -17-bands c o r r e s p o n d i n g t o h y d r o x y l a b s o r p t i o n ( F i g u r e 9). The mass s p e c t r u m o f t h a m n o s i n was v e r y s t r i k i n g , s i n c e t h e r e were v i r t u a l l y no peaks between m/e 484 and m/e 243. The f a c t t h a t t h e b a s e peak a p p e a r e d a t m/e 242 and t h a t t h e m o l e c u l a r i o n peak was f a i r l y week (8%) s u g g e s t e d t h a t t h a m n o s i n was e a s i l y c l e a v e d u n d e r e l e c t r o n i m p a c t i n t o two e q u a l h a l v e s . T h i s s i t u a t i o n w o u l d be c o n s i s t e n t w i t h a n e s s e n t i a l l y s y m m e t r i c a l m o l e c u l e a n d , as w i l l be s e e n l a t e r , was a n i m p o r t a n t r e s u l t i n t h e s t r u c t u r a l e l u c i d a t i o n o f t h i s compound. The d e t a i l e d mass s p e c t r a o f t h a m n o s i n and i t s d e r i v a t i v e s w i l l be d i s c u s s e d l a t e r i n a s e p a r a t e s e c t i o n o f t h i s t h e s i s . The NMR s p e c t r u m o f t h a m n o s i n ( F i g u r e 10) i n d i c a t e d t h e p r e s e n c e o f a s i n g l e t m e t h y l ( p r o b a b l y a l l y l i c ) a t 7r8 . 78 , a v i n y l m e t h y l a t t 8 . 2 0 , two m e t h o x y l g r o u p s a t 1:6.29 and t6.27, a m u l t i p l e t c e n t e r e d a t 1:4.75 f o r a n o l e f i n i c p r o t o n , and a complex m u l t i p l e t i n t h e r e g i o n , T2 . 4 -3 .9 , w h i c h on t h e b a s i s o f i t s i n t e g r a t e d a r e a , c o u l d be a s s i g n e d t o t e n p r o t o n s . On t h e a s s u m p t i o n t h a t t h a m n o s i n c o n t a i n e d two m e t h o x y l g r o u p s , t h e t o t a l number o f p r o t o n s i n t h i s m o l e c u l e was twenty e i g h t . The e x -panded a r o m a t i c r e g i o n o f t h a m n o s i n , as shown i n F i g u r e 12, i n d i c a t e d t h e p r e s e n c e o f a n AB s y s t e m ( a t ^ 3 . 9 8 and t"3.82, J^g^lfc c p s ) , two s e t s o f d o u b l e t s a t t3.85 and t3.83 (J=9.5 c p s ) w h i c h c o u l d be t e n t a t i v e l y a s s i g n e d t o two p r o t o n s a t t h e C-3 p o s i t i o n s o f two c o u m a r i n systems and two s i n g l e t s a t ^3.39 and?:3o37 w h i c h c o u l d be a s s i g n e d -18 -19-to protons at the C-8 positions of these systems. In a s i m i l a r fashion, the two s i n g l e t s at'£'2.94 and r2.89 and two sets of doublets at ?r2.50 andt2.46 (J=9.5 cps) could be assigned to the C-5 and C-4 protons on the coumarin r i n g s . These assignments are c l e a r l y based on the as-sumption that thamnosin possessed two coumarin chromo-phores-a postulate which was supported by the UV spectral data (see below). It was c l e a r from the above molecular formula that seventeen degrees of unsaturation were present i n thamnosin and therefore the f i r s t selected r e a c t i o n was c a t a l y t i c hydrogenation to investigate the nature of any double bonds which may be present. Thamnosin i n the presence of 10% palladium on charcoal i n tetrahydrofuran smoothly absorbed one mole of hydrogen. The elemental analysis and the high r e s o l u t i o n mass spectrum confirmed that t h i s compound hereafter c a l l e d dihydro-thamnosin, m.p. 226-228°, had the molecular formula, C 3 0 H 3 0 ° 6 0 The IR spectrum of dihydro-thamnosin showed very s i m i l a r bands f o r the carbonyl and double bond stretching frequencies o r i g i n a l l y observed i n thamnosin but no bands were present at 980 cm"^ ". The complete disappearance of the AB system i n thamnosin (at t3„98 and?;3.82, J A B = 1 6 cps) as mentioned above was now observed i n the NMR spectrum of the dihydro-derivative (Figure 11). The above spectral data confirmed that a trans d i s u b s t i t u t e d double bond was -20-i n C N tn in • •a» O N 0 D -> -> t' vO O O N m C O • • • N N CM mm • • O N O N I 0 •n —> <r> i n CO CO 1 f /-v£ 1 1 ° i-l 1 • r-l B 0 »-><M oo •-> • • ro Dihydro-thamno s i n Thamnosin 1/ Figure 12 Expanded Aromatic Regions (lOOMc/s) -22-b e i n g r e d u c e d i n t h e m o l e c u l e . I t c o u l d now be f u r t h e r s u g g e s t e d f r o m t h e c h e m i c a l s h i f t and t h e m u l t i p l i c i t y -p a t t e r n o f t h i s p a r t i c u l a r o l e f i n i c s y s t e m t h a t i t was l i n k e d t o a n a r o m a t i c p o r t i o n on t h e one hand and a t e r t i a r y c a r b o n atom on t h e o t h e r . The c h r o m o p h o r i c change c r e a t e d by t h e h y d r o g e n a t i o n r e a c t i o n as shown i n t h e UV s p e c t r u m c o n f i r m e d t h e p r e s e n c e o f a d o u b l e b o n d c o n j u g a t e d t o a n a r o m a t i c s y s t e m , t h e l a t t e r most l i k e l y b e i n g a c o u m a r i n c h r o m o p h o r e . As shown i n F i g u r e 13, t h e UV s p e c t r u m o f d i h y d r o -t h a m n o s i n was f o u n d t o be a l m o s t s u p e r i m p o s a b l e on t h a t o f 41 s u b e r o s i n , 7 -methoxy-6 - isopent-2 1 - e n y l c o u m a r i n . I t s h o u l d be n o t e d t h a t t h e e x t i n c t i o n c o e f f i c i e n t s i n d i h y d r o -t h a m r i o s i n were a l m o s t t w i c e as l a r g e as t h o s e i n s u b e r o s i n and t h a t s u b e r o s i n h a d o n l y f o u r a r o m a t i c p r o t o n s . T h e r e f o r e t h e p r e s e n c e o f e i g h t a r o m a t i c p r o t o n s i n t h e NMR s p e c t r u m o f d i h y d r o - t h a m n o s i n c o u l d s u g g e s t t h a t t h a m n o s i n c o n s i s t e d o f two 7 - m e t h o x y c o u m a r i n m o i e t i e s ( C ^ Q x 2) and a C-^Q a l k y l r e s i d u e l i n k e d t o t h e 6 - p o s i t i o n o f t h e s e m o l e c u l e s . S u b e r o s i n The NMR s p e c t r u m o f d i h y d r o - t h a m n o s i n ( F i g u r e 11) showed s h a r p s i n g l e t s f o r a m e t h y l g r o u p a t t 8 . 9 7 and a - 2 3 -F i g u r e 13 UV S p e c t r a o f D i h y d r o - t h a m n o s i n and S u b e r o s -24-v i n y l methyl a t t 8 . 2 6 , two methoxyl resonances at T6.25 andT6.22, an o l e f i n i c proton at t4.83 and a series of signals f o r eight aromatic protons i n the r e g i o n , t 2 . 4 -3.9. The s i g n i f i c a n t u p f i e l d s h i f t of one methyl group si g n a l (8.78*8.97) i n converting thamnosin to i t s dihydro-d e r i v a t i v e suggested that the s h i f t e d methyl was situated on a carbon atom adjacent to the double bond which had been reduced. Futhermore i t s appearance as a s i n g l e t showed that t h i s methyl was attached to a t e r t i a r y carbon atom. On the basis of the above evidence i t was possible to postulate, as a working hypothesis, a p a r t i a l structure (I) f o r thamnosin. (I) The hydrogenation conditions used to obtain dihydro-thamnosin were known to be too mild to attack a coumarin system and t h i s was confirmed when an attempted reduction of the 3,4-double bond i n authentic 7-methoxycoumarin met with f a i l u r e . The presence of a v i n y l methyl and an o l e f i n i c proton shown by the NMR spectrum and a t r i s u b -s t i t u t e d double bond indicated by the IR spectrum (a band at 820 cm"^) i n dihydro-thamnosin was not s u r p r i s i n g since the mild reduction of thamnosin would not be-expected to a f f e c t the t r i s u b s t i t u t e d double bond (probably G H o - C = C H - ) . - 2 5 -In order to obtain lower molecular weight fragments which may be more e a s i l y compared with compounds of known structure, the cleavage of thamnosin was next considered. The trans di s u b s t i t u t e d double bond i n thamnosin was thought to be a convenient handle f o r t h i s purpose and therefore i t s conversion to a d i o l was attemped. The successful hydroxylation of thamnosin was ac-complished by t r e a t i n g i t with osmium tetroxide to give thamnosin-diol. C r y s t a l l i z a t i o n of the l a t t e r compound from ethanol provided two c r y s t a l l i n e modifications. One of these ( p l a t e s ) , turned out to be the free d i o l (elemtntal analysis) while the other modification (prisms) was shown by elemental analysis to be the d i o l bearing one molecule of ethanol of c r y s t a l l i z a t i o n . The l a t t e r could be con-verted to the former by merely grinding the c r y s t a l s and heating at 100° f o r three hours. I n t e r e s t i n g l y , the c r y s t a l l i z a t i o n of thamnosin-diol from methanol gave a single product which was analyzed as C^QH^QOg.CHgOH. The above three compounds showed i d e n t i c a l behavior on TLC with several solvent systems and the UV spectra were also i d e n t i c a l . On t h i s basis i t was concluded that thamnosin afforded only one d i o l i n the hydroxylation reaction. The UV spectrum of thamnosin-diol was almost super-imposable on that of dihydro-thamnosin and the IR spectrum showed no absorption at 980 cm~^. As expected, the hydroxylation and the c a t a l y t i c hydrogenation reactions were both proceeding on the same double bond of thamnosin. -26-The mass spectrum indicated a very weak molecular ion (1%) but the M-18 peak was s u f f i c i e n t l y strong to obtain an accurate mass measurement (found: 500.187). The IR spectrum showed very intense absorption f o r hydroxyl groups at 3480 cm"1, the usual bands f o r coumarin carbonyl functions and double bond absorptions at 1725, 1620 and 1565 cm"1 as well as the band at 820 cm"1. The NMR spectrum of thamnosin-diol (Figure 14 and 15) was very i n s t r u c t i v e and c l e a r l y indicated the presence of a l l thirty protons. The spectrum showed a three proton s i n g l e t at "2-8.69, a v i n y l methyl a t 8 . 2 1 , a one proton s i n g l e t f o r Hg at 7:7.03, (see below f o r p a r t i a l s t r u c t u r e s ) , a two proton m u l t i p l e t a t T 6 . 6 , a poorly defined one proton doublet a t t 6 . 2 8 (J=5 cps, H^) two methoxyl proton signals at t6.47 and T6.16, a broad s i g n a l centered at T4.98 (H A), an i l l - d i f i n e d one proton doublet at r 4 . 7 5 (J=5 cps, Hp) and complex peaks f o r eight aromatic protons i n the region, 7^2.3-4.0. H. B Addition of deuterium oxide to the NMR sample tube containing the d i o l sharpened the peaks at ^7.03 and T4.98 6.16 6.47 F i g u r e 15 NMR Spectruca .of T h a m n o s i n - d i c l (100 Mc/s) - 2 9 -and c a u s e d t h e two p r o t o n m u l t i p l e t a t 7 r 6 . 6 t o d i s a p p e a r . T h e r e f o r e t h e m u l t i p l e t a t T 6 . 6 was a s s i g n e d t o two h y d r o x y l i c p r o t o n s . V i r t u a l l y no c o u p l i n g s between p r o t o n s H A and Hg i n t h e d i o l were o b s e r v e d a f t e r d e u t e r i u m exchange and on t h i s b a s i s i t a p p e a r e d t h a t t h e d i h e d r a l a n g l e between H A and Hg was c l o s e t p 9 0 ° . A s t u d y o f m o l e c u l a r m o d e l s , u s i n g t h e a b o v e m e n t i o n e d p r o p o s a l s , s u g g e s t e d t h a t a c i s h y d r o x y l a t i o n o f t h e p o s t u l a t e d t r a n s d o u b l e bond w o u l d y i e l d a d i o l w i t h a d i h e d r a l a n g l e between H A and Hg o f a p p r o x i m a t e l y 6 0 ° . A c c o r d i n g t o K a r p l u s t h i s s h o u l d p r o v i d e a c o u p l i n g c o n s t a n t between t h e s e p r o t o n s o f a p p r o x i m a t e l y 2 c p s . B u t i t must be remembered t h a t s u b s t i t u e n t s w i t h h i g h e l e c t r o -n e g a t i v i t y a r e known t o r e d u c e t h e c o u p l i n g c o n s t a n t s o f v i c i n a l p r o t o n s . I n t h i s i n s t a n c e b o t h H A and Hg a r e a t t a c h e d t o c a r b o n atoms b e a r i n g h y d r o x y l g r o u p s . S p i n - d e c o u p l i n g e x p e r i m e n t s ( F i g u r e 15) d e m o n s t r a t e d t h a t t h e i r r a d i a t i o n a t t h e r e s o n a n c e f r e q u e n c y o f t h e o l e f i n i c p r o t o n (r4.75) a l l o w e d t h e d o u b l e t a t t 6 . 2 8 (J=5 c p s ) t o c o l l a p s e i n t o s i n g l e t . The p r o t o n (H^,) a t T - 6 . 2 8 w h i c h c o u p l e d o n l y w i t h t h e o l e f i n i c p r o t o n (H^) was, on t h e b a s i s o f i t s c h e m i c a l s h i f t , s i t u a t e d n e x t t o a n a r o m a t i c s y s t e m and t h e s i g n a l m u l t i p l i c i t y s u g g e s t e d t h a t t h e a d j a c e n t a l i p h a t i c c a r b o n atom must be f u l l y s u b s t i t u t e d . C o n s i d e r a t i o n o f t h e above s p e c t r a l e v i d e n c e a l l o w e d us t o expand t h e t e n t a t i v e s t r u c t u r e o f t h a m n o s i n t o I I -30-and the d i o l to I I I . It was now desirable to attempt a cleavage of thamnosin-d i o l into lower molecular weight fragments. For t h i s purpose, the d i o l was reacted with p e r i o d i c a c i d i n aqueous methanol at room temperature. Two aldehydic compounds de-signated as aldehyde-I and aldehyde-II were obtained from t h i s r e a c t i o n . Aldehyde-I was more polar than aldehyde-II on a s i l i c a gel G t h i n layer chromatogram (TLC). High r e s o l u t i o n mass spectra of these aldehydes deter-mined the molecular weights to be 204.042 and 312.138 f o r aldehyde-I and aldehyde-II, r e s p e c t i v e l y , where C-j^HgO^ requires 204.042 and ^i<^20^h requires 312.136. Further evidince f o r the correctness of the above formulae w i l l be presented below. It should be noted that the sum of these two formulae becomes C^Q^gOg and indicates conclusively that the p e r i o d i c a c i d r e a c t i o n merely cleaves the molecule -31-into these two fragments with no loss of any carbon atoms i . e . (C 1 0H 7O 3)—C^Cqj-CC-i^gH-j^gOg) Thamnosin 1) 0 S 0 4 2) HI0 4 v (C l 0H 70 3)-CHO OHC-(C 1 8H i g0 3) Aldehyde-I " Aldehyde-II Aldehyde-I, m.p. 242, showed an orange spot on a TLC plate a f t e r spraying with 2,4-dinitrophenylhydrazine reagent (2,4-DNP) and i t was suggested that t h i s compound may be an aromatic aldehyde. The UV spectrum showed a rather complex pattern with ^max at 255, 308 and 329 mu probably due to an extended conjugation of a coumarin system with the aldehyde group. When sodium borohydride was added to a methanolic s o l u t i o n of aldehyde-I and then the UV spectrum was recorded, the spectrum changed dramatically and was now almost superimposable on the spectra of dihydro-thamnosin arid suberosin. The hydroxymethyl group which would be derived from the aldehyde i n the hydride reduction would not be expected to contribute s i g n i f i c a n t l y to the UV spectrum and i t was not s u r p r i s i n g that a t y p i c a l 7-methoxy-coumarin chromophore was now i n hand. This r e s u l t immedi-a t e l y suggested that aldehyde-I could be 6-formyl-herniarin - 3 2 -( 7 - m e t h o x y c o u m a r i n - 6 - a l d e h y d e ) . The IR s p e c t r u m o f a l d e h y d e - I s t i l l showed t h e p r e s e n c e o f a n o(-pyrone s y s t e m . The NMR s p e c t r u m was o b t a i n e d a f t e r some d i f f i c u l t y due t o t h e h i g h l y i n s o l u b l e n a t u r e o f t h i s compound i n t h e common d e u t e r a t e d s o l v e n t s and a l s o due t o t h e s m a l l amount a v a i l a b l e . E v e n t u a l l y a l d e h y d e - I i n ( C F j C l ^ C C O D ^ p r o v i d e d a n NMR s p e c t r u m , f r o m w h i c h some e v i d e n c e o f t h e f u n c t i o n a l g r o u p s c o u l d be d e r i v e d . The p r e s e n c e o f one m e t h o x y l g r o u p and a s h a r p one p r o t o n s i n g l e t a t T - 0 . 2 3 f o r a n a l d e h y d i c p r o t o n were s u g g e s t e d f r o m t h e s p e c t r u m . On t h e b a s i s o f t h e above s p e c t r o s c o p i c e v i d e n c e t h e s t r u c t u r e o f a l d e h y d e - I was ' p r o p o s e d t o be 7 - m e t h o x y -c o u m a r i n — 6 - a l d e h y d e , T h i s l a t t e r s u b s t a n c e was r e p o r t e d 4 i n t h e l i t e r a t u r e as a d e g r a d a t i o n p r o d u c t f r o m o s t r u t h i n and f r o m s u b e r o s i n ^ 1 *****. An a u t h e n t i c sample o f t h i s compound was k i n d l y s u p p l i e d by P r o f e s s o r K i n g ^ 1 and a d i r e c t c o m p a r i s o n w i t h a l d e h y d e - I was made. TLC c o m p a r i s o n on s i l i c a g e l and a l u m i n a w i t h s e v e r a l s o l v e n t s y s t e m s , s u p e r i m p o s a b l e UV and IR s p e c t r a and a m i x e d m e l t i n g p o i n t d e t e r m i n a t i o n -33-Suberosin proved that aldehyde-I was 7-methoxycoumarin-6-aldehyde. On t h i s basis, a portion of the thamnosin structure was now established with c e r t a i n t y . The less polar aldehyde, aldehyde-II, showed a l i g h t yellow spot on a TLC plate when sprayed with 2,4-DNP and r e s i s t e d c r y s t a l l i z a t i o n . The UV spectrum of aldehyde-II was e s s e n t i a l l y superimposable on that of suberosin, which was again i n d i c a t i v e of the presence of a 7-methoxycoumarin system bearing a C-6 a l k y l side chain. The presence of a coumarin chromophore i n aldehyde-II confirmed the previous suggestion that the thamnosin molecule contained two coumarin chromophores most probably linked to a C^Q a l k y l chain. The NMR spectrum of aldehyde-II (Figure 16) was again -34-v e r y i n f o r m a t i v e . Two sharp s i n g l e t s a t T 8 . 8 2 and 7:8.21 c o n f i r m e d the p resence o f a s a t u r a t e d and a v i n y l methy l g r o u p . In a d d i t i o n a methoxy l s i g n a l a t 7 6 . 2 0 , a one-p r o t o n d o u b l e t a t 7r5 . 8 4 (J=5 c p s ) , a m u l t i p l e t a t 1 4 . 7 6 f o r one o l e f i n i c p r o t o n , a s e r i e s o f s i g n a l s f o r f o u r a r o m a t i c p r o t o n s and a one-p ro ton s i n g l e t a t t 0 . 7 3 f o r the s a t u r a t e d a ldehyde p r o t o n were the r ema in i ng s i g n i f i -can t s i g n a l s . The c h e m i c a l s h i f t o f one o f the methy l s i g n a l s ( t 8 . 8 2 ) and the p r e sence o f a s i n g l e t f o r the a l d e h y d i c p r o t o n gave some e v i dence t h a t b o t h o f t hese groups may be a t t a c h e d to the same f u l l y s u b s t i t u t e d c a rbon atom. T h i s s u g g e s t i o n was made e a r l i e r i n compar ing the NMR s p e c t r a o f thamnos in and d i h y d r o - t h a m n o s i n . A d d i t i o n a l i n f o r m a t i o n about the f o u r a r o m a t i c p r o t o n s o f a ldehyde- I I was o b t a i n e d when the s p l i t t i n g p a t t e r n s o f the a r o m a t i c p r o t o n s i g n a l s were examined i n the NMR s p e c t r a t aken a t 60 Mc/s and 100 M c / s . I t t u rned out t h a t t h i s r e g i o n c o n s i s t e d o f two s e t s o f d o u b l e t s a t 7 : 3 . 8 4 (J=9.5 cps ) a n d t 2 „ 4 5 (J=9.5 cps ) and two s i n g l e t s a t 7:3.31 and 7 :2 .85 f o r wh ich ass ignments c o u l d be r e a d i l y made. T a b l e 2 summarizes the p u b l i s h e d d a t a on s e v e r a l coumar in systems e x h i b i t i n g s i m i l a r a r o m a t i c r e g i o n s . As shown i n T a b l e 2 , the a r o m a t i c r e g i o n s o f o s t r u t h i n ^ , m a r m e s i n ^ and s u b e r o s i n ^ i n p a r t i c u l a r showed v i r t u a l l y the same p a t t e r n s and c h e m i c a l s h i f t s as those o f a l d e h y d e - I I . T h i s NMR ev idence showed w i thou t -36-Table 2 Chemical Shifts of Aromatic Protons7 of Coumarins at 60 Mc/s in Tiers tscale, J (cps), solventt CDCI3. H -C-3 a Hb-C-4 Hc-C-5 Hd-C-8 H -C-6 e i Ref. i) 3.75 d. Jab" 1 0 2.40 d. Jab- 1 0 2.95 s. and 2.80 s. 46 i i ) 3.92 s. 2.53 d. Jce" 8 3.27 d. Jde- 2 3.22 q. J-2,8 45 i i i ) 3.78 d. Jab- 1 0 2.10 d. Jab" 1 0 2.56 s. 3.26 8 . 47 iv) 3.77 d. Jab- 9- 5 2.37 d. Jab- 9' 5 2.81 s. 3.22 s. 48 v) 3.84 d. Jab- 9* 5 I 2.45 d. Jab- 9- 5 2.85 8 . 3.31 s. (Aldehyde-II) 0^0 i) Ostruthin i i ) i i i ) Marmesin iv) Suberosin - 3 7 -d o u b t , t h a t a l d e h y d e - I I c o n t a i n e d a 7 - m e t h o x y c o u m a r i n s y s t e m w i t h a n a l k y l s i d e c h a i n a t C - 6 . V e r y s t r o n g s u p p o r t f o r t h i s p r o p o s a l was a l s o a v a i l a b l e f r o m t h e UV s p e c t r u m o f t h i s compound, s i n c e t h e l a t t e r s p e c t r u m i s v e r y s e n s i -t i v e t o t h e r e l a t i v e s u b s t i t u e n t p o s i t i o n s on t h e c o u m a r i n c h r o m o p h o r e . A t h r e e p r o t o n s i n g l e t a t t 8 . 2 1 and a one p r o t o n m u l t i p l e t a t 7:4.76 f u r t h e r s u g g e s t e d t h e p r e s e n c e o f a C H 3 - C = C - H m o i e t y i n a l d e h y d e - I I . T h i s s u g g e s t i o n was l a t e r s u b s t a n t i a t e d b y s e l e c t i v e e p o x i d a t i o n o f t h i s d o u b l e b o n d . A t t h i s p o i n t i n t h e d i s c u s s i o n t h e p e r t i n e n t s t r u c t u r a l f e a t u r e s w h i c h a r e p r e s e n t i n a l d e h y d e - I I may be summarized as f o l l o w s : F u r t h e r i n f o r m a t i o n a b o u t t h e s t r u c t u r e o f a l d e h y d e -I I coxild now be o b t a i n e d f r o m c o m p a r i s o n o f t h e NMR s p e c t r u m o f t h i s compound w i t h t h o s e o f p r e v i o u s compounds a l r e a d y d i s c u s s e d . F o r example i t w i l l be n o t e d f r o m t h e NMR s p e c t r a o f t h a m n o s i n , d i h y d r o t h a m n o s i n and t h a m n o s i n -d i o l ( F i g u r e s 10, 11 and 15) t h a t a b r o a d , o n e - p r o t o n s i g n a l a l w a y s a p p e a r s b e n e a t h o r v e r y c l o s e t o t h e m e t h o x y l r e s o n a n c e s . T h i s p a r t i c u l a r p r o t o n ( d e s i g n a t e d as H r i n p a r t i a l s t r u c t u r e I I f o r t h a m n o s i n and on F i g u r e s -38-14 and 15) now appeared c l e a r l y as a doublet (J=5 cps see Figure 16) atT5.84. I t was also shown by spin decoupling (see Figure 15) that an o l e f i n i c proton (Hp, see p a r t i a l structure II) was coupled with t h i s p a r t i c u l a r proton ( H Q ) i n thamnosin-diol ( J ^ r j = ^ cps). The s i g n i f i c a n t downfield s h i f t of t h i s proton (H^ ,) i n aldehyde-II r e l a t i v e to thamnosin or i t s other derivatives must be due to i t s close proximity to the aldehyde group. In order to get some idea about the expected chemical s h i f t of proton H , a very approximate c a l c u l a t i o n was made u t i l i z i n g the s h i e l d -49 ing constants f o r various substituents as given by Shoolery and also mentioned by S i l v e r s t e i n et a l ~ ^ . For t h i s purpose, suberosin was selected as a t y p i c a l system i n which the methylene protons are desheilded by a 7-methoxycoumarin r i n g on the one hand and an o l e f i n i c linkage on the other. In t h i s way an approximate value f o r the s h i e l d i n g constant of the 7-methoxycoumarin system was obtained (see Table 3). A s u r p r i s i n g l y good agreement between the calcu l a t e d and found values was obtained. Therefore i f t h i s proton H^ possesses the suggested environment, the p a r t i a l structure f o r aldehyde-II may be represented as follows. Aldehyde-II -39-Table 3 Standard (CH,) t 9.77 A O Shielding constant C=C - 1.32 8.45 -CH2-C=C- 8.45 Obseved f o r suberosin - 6.74 1.71 Approximate s h i e l d i n g constant f o r 7-methoxycoumarin system i s 1.71. CH3-C-CHO 8.90 -C=C- - 1.32 7-methoxycoumarin - 1.71 Calc. f o r H c ^5.87 Found f o r H Q r5.84 I f we now consider that the molecular formula es-tablished f o r thamnosin required seventeen degrees of unsaturation, i t i s noted that we have up to t h i s point i n the disc u s s i o n accounted f o r sixteen degrees (fourteen ( 2 x 7 ) f o r two coumarin systems and two f o r o l e f i n i c l i nkages). One degree of unsaturation which s t i l l remains unaccounted can now be e a s i l y incorporated with the remain-ing C2H^ i n aldehyde-II to complete a cyclohexene r i n g . On th i s basis, aldehyde-II would have structure I I I and i n turn thamnosin must be assigned structure IV. The remain-ing p o r t i o n of the discussion w i l l be devoted to experi-ments which substantiate these proposals. In order to provide evidence f o r the presence of the t r i s u b s t i t u t e d double bond i n a cyclohexene system as -40-CH 3 ( I V ) p o s t u l a t e d i n I I I and I V , we c o n s i d e r e d s e v e r a l r e a c t i o n s on d i h y d r o - t h a m n o s i n i n wh ich the c o n j u g a t e d , d i s u b s t i t u t e d doub le bond had been removed. The e p o x i d a t i o n o f d i h y d r o - t h a m n o s i n by m-ch lo roper-b e n z o i c a c i d i n c h l o r o f o r m gave a s i n g l e p r o d u c t whose h i g h r e s o l u t i o n mass spec t rum and e l ementa ry a n a l y s e s were i n good agreement w i t h the m o l e c u l a r f o r m u l a o f a mono-epox ide , C ^ QH ^ QO ^ . T h i s epox ide e x h i b i t e d a U V spec t rum wh ich was supe r imposab l e on t h a t o f d i h y d r o -thamnos in , i n d i c a t i n g t h a t the coumar in systems were s t i l l i n t a c t . The I R spec t rum o f d i h yd ro- thamnos in-ox ide a l s o i n d i c a t e d the t h r e e c h a r a c t e r i s t i c coumar in bands a t 1725, 1612 and 1565 c m " 1 . The s i g n i f i c a n t f e a t u r e o f the N M R spec t rum ( F i g u r e 17) was t h a t one o f the two methy l peaks i n the s t a r t i n g m a t e r i a l had s h i f t e d s i g n i f i c a n t l y to h i g h e r f i e l d ( 8 . 2 6 » 8.59) and t h a t no o l e f i n i c p r o t o n was p r e s e n t i n the -41--42-m o l e c u l e . T h i s NMR e v i d e n c e now c o n f i r m e d t h e p r e s e n c e o f t h e m o i e t y , C H ^ - C ^ C H - , i n d i h y d r o - t h a m n o s i n . The NMR s p e c t r u m o f d i h y d r o - t h a m n o s i n - o x i d e s t i l l showed a s h a r p t h r e e p r o t o n s i n g l e t a t ^9.17, a s i n g l e peak f o r two m e t h o x y l g r o u p s a t t 6 . 1 6 , and e i g h t p r o t o n s i n t h e r e g i o n , t2.5-3.9. On t h e b a s i s o f t h e a b o v e , i t was c l e a r t h a t a s t r a i g h t f o r w a r d e p o x i d a t i o n o f t h e d o u b l e bond was o c c u r r -i n g , i . e . H c M 3 M — "A " " I t was h o p e d t h a t t h e e p o x i d e c o u l d s e r v e as a n i n t e r -m e d i a t e f o r s u b s e q u e n t d e g r a d a t i o n o f t h e m o l e c u l e . U n f o r t u n a t e l y a t t e m p t s t o c l e a v e t h e e p o x i d e r i n g u n d e r a v a r i e t y o f c o n d i t i o n s a l w a y s l e d t o a complex m i x t u r e o f p r o d u c t s . S i n c e we were r a p i d l y r u n n i n g s h o r t o f m a t e r i a l , we d e c i d e d t o abandon t h i s a p p r o a c h . C o u m a r i n s a r e known t o be s t a b l e towards a v e r y d i l u t e 51 52 s t r e a m o f ozone ' and i t was f e l t t h a t t h i s r e a c t i o n may y i e l d f r u i t f u l r e s u l t s . I n d e e d t h e c o n t r o l l e d o z o n o l y s i s o f d i h y d r o - t h a m n o s i n f o l l o w e d by c a t a l y t i c r e d u c t i o n o f t h e o z o n i d e gave a s i n g l e compound, d e s i g n a t e d as k e t o - a l d e h y d e -I I I . The mass s p e c t r u m o f t h i s s u b s t a n c e showed a v e r y weak m o l e c u l a r i o n peak b u t t h e M - l8 p e a k , w h i c h was more i n t e n s e , was s u b j e c t e d t o a n a c c u r a t e mass measurement. The UV and -43-IR s p e c t r a o f t h i s k e t o - a l d e h y d e - I I I s t i l l i n d i c a t e d t h e r e t e n t i o n o f t h e 6 - a l k y l - 7 - m e t h o x y c o u m a r i n c h r o m o p h o r e . The NMR s p e c t r u m o f k e t o - a l d e h y d e - I I I ( F i g u r e 18) i n d i c a t e d a t e r i a r y m e t h y l a t T 8 .91 , two m e t h o x y l g r o u p s a t 7:6.15 and e i g h t a r o m a t i c p r o t o n s f o r t h e two c o u m a r i n s y s t e m s . The p r e s e n c e o f a s h a r p , t h r e e p r o t o n s i n g l e t a t -7:7.87 ( C H 3 C O ) , a one p r o t o n d o u b l e t a t T0.02 ( - C H r C H O , J=2 c p s ) and a one p r o t o n d o u b l e t a t 7:5.76 (-CHRCHO, J=2 c p s ) c o n f i r m e d t h a t t h e o z o n o l y s i s h a d p r o c e e d e d i n t h e e x p e c t e d f a s h i o n . As s u g g e s t e d a b o v e , t h e a l d e h y d i c p r o t o n was i n d e e d f o u n d t o be c o u p l e d w i t h a s i n g l e p r o t o n (H^,) when a s p i n d e c o u p l i n g e x p e r i m e n t was c a r r i e d out ( s e e F i g u r e 1 8 ) . I t was a l s o c o n f i r m e d t h a t was c o u p l e d o n l y w i t h t h i s p r o t o n . T h e r e f o r e t h e c a r b o n atom b e a r i n g c o u l d o n l y be c o n n e c t e d t o a n a r o m a t i c s y s t e m , a t e t r a - s u b s t i t u t e d c a r b o n atom and a t r i - s u b s t i t u t e d d o u b l e bond whose o l e f i n i c p r o t o n was i n t u r n a l s o c o u p l e d w i t h H^.. T h e r e f o r e t h e i m m e d i a t e e n v i r o n m e n t o f H r must be e x p r e s s e d as f o l l o w s . I S p i n J L ( i r r - Decoup l ing a t z-5.76 0.02 (J-2) I II 6.15 2 8.91 F i g u r e 18 NMR Spectrum o f Keto-aldehyde-I I I (100 Mc/s) -45-When t h i s e v idence i s t aken i n c o n j u n c t i o n w i t h the p r e v i o u s r e s u l t s , i t i s c onc l uded t h a t ke to-a ldehyde- I I I had the i n d i c a t e d s t r u c t u r e . The above e p o x i d a t i o n and o z o n i z a t i o n p r o d u c t s o f d i h y d r o -thamnos in had now c o m p l e t e l y i d e n t i f i e d the n a t u r e o f the t r i - s u b s t i t u t e d doub le bond i n thamnos in . Some s u p p o r t i n g e v i dence f o r ke to-a ldehyde- I I I was o b t a i n e d when the l a t t e r was s u b j e c t e d to m i l d r e d u c t i o n w i t h sodium b o r o h y d r i d e . The p r o d u c t , a r b i t r a r i l y d e s i g -na t ed as a l c o h o l - I V , had some i n t e r e s t i n g s p e c t r a l c h a r -a c t e r i s t i c s , a l t h o u g h u n f o r t u n a t e l y a v e r y s m a l l q u a n t i t y was a v a i l a b l e and complete c h a r a c t e r i z a t i o n was not p o s s i b l e . C o n s e q u e n t l y the s p e c t r a l a ss ignments a r e no t e n t i r e l y d e f i n i t i v e . The mass spec t rum o f t h i s compound showed the p r e sence o f M-18 and M-36 peaks a l o n g w i t h a v e r y weak m o l e c u l a r i o n peak . The NMR spec t rum of a l c o h o l - I V showed a comple te d i s s a p e a r a n c e o f the me thy l s i g n a l a t f 7 . 8 7 , i n agreement w i t h the expec ted r e d u c t i o n o f the me thy l ke tone f u n c t i o n . T h i s d i s c u s s i o n now conc l udes our e x p e r i m e n t a l r e s u l t s on the s t r u c t u r a l e l u c i d a t i o n o f thamnos in . The e v i dence p r e s e n t e d a l l o w s us to a s s i g n s t r u c t u r e IV to t h i s n a t u r a l p r o d u c t . U n f o r t u n a t e l y , due to the i n a v a i l a --46-b i l i t y o f t hamnos in , no f u r t h e r d e g r a d a t i o n exper iments c o u l d be c a r r i e d out and we a r e unab l e to make any a s i g n -ment o f s t e r e o c h e m i s t r y a t the l one asymmetr i c c e n t r e . Thamnosin r e p r e s e n t s a n o v e l sys tem wh ich to the b e s t o f my knowledge , has no t been p r e v i o u s l y encoun te red i n any n a t u r a l s o u r c e . A b i o s y n t h e t i c s tudy on t h i s m o l e c u l e , u t i l i z i n g l a b e l l e d p r e c u r s o r s , c o u l d be v e r y i n t e r e s t i n g and i s a n t i c i p a t e d . -47-Mass S p e c t r a o f Thamnosin and i t s D e r i v a t i v e s Mass s p e c t r o m e t r y p l a y e d an impor tan t r o l e i n the s t r u c t u r e d e t e r m i n a t i o n o f thamnos in and i t was f e l t a p p r o p r i a t e to d i s c u s s the mass s p e c t r a l r e s u l t s i n a s e p a r a t e s e c t i o n o f t h i s t h e s i s . The a n a l y s i s o f these r e s u l t s was a i d e d by p r e v i o u s l y p u b l i s h e d i n v e s t i g a t i o n s as w e l l as by a d e t a i l e d s tudy on v a r i o u s coumar in systems i n our l a b o r a t o r y . A b r i e f d i s c u s s i o n o f some o f these r e s u l t s i s p r e s e n t e d f i r s t b e f o r e a n a l y s i s o f the thamnos in system i s c o n s i d e r e d . The mass s p e c t r o m e t r i c i n v e s t i g a t i o n s o f n a t u r a l l y o c c u r r i n g oxygen h e t e r o c y c l e s have been c a r r i e d out by s e v e r a l g r o u p s ^ ' ' ^ ' ^ ' ^ From these s t u d i e s i t i s appa ren t t h a t the coumar in r i n g sys tem, under e l e c t r o n i m p a c t , f ragments w i t h l o s s o f c a rbon monoxide to p r o v i d e r e l a t i v e l y s t a b l e i o n s . E l i m i n a t i o n o f a methy l r a d i c a l f rom an a r o m a t i c methoxy l f u n c t i o n and f a c i l e c l e a vage o f an a l k y l s i d e c h a i n (S t o the a r o m a t i c r i n g o f t e n o c c u r s as w e l l to g i v e r i s e to con juga t ed oxonium i ons and somet imes, the t r o p y l i u m i o n . F r agmen ta t i ons o f these m o l e c u l e s w i l l be shown 53 a c c o r d i n g to the D j e r a s s i c o n v e n t i o n i . e . - c—c - =. - c—c - > -c + #c-S V , V ^ X. The f r a g m e n t a t i o n s o f s imp l e coumar ins w i l l be d i s c u s s e d f i r s t and then these p o s t u l a t e s w i l l be extended - 4 8 -t o t h e t h a m n o s i n s e r i e s . I t must be e m p h a s i z e d t h a t t h e m e c h a n i s t i c i n t e r p r e t a t i o n s a r e m e r e l y r e a s o n a b l e r a t i o n -a l i z a t i o n t o e x p l a i n t h e a p p e a r a n c e o f v a r i o u s f r a g m e n t s i n t h e mass s p e c r o m e t e r . I n most i n s t a n c e s a good d e a l o f a d d i t i o n a l s t u d y i s n e c e s s a r y b e f o r e any d e f i n i t i v e comments a b o u t t h e s e i o n s c a n be made. The l o s s o f CO(M-28) and 2CO(M-56) f r o m t h e p y r o n e r i n g o f c o u m a r i n (21) and h e r n i a r i n (27) was o b s e r v e d u n d e r e l e c t r o n i m p a c t . H e r n i a r i n a l s o f r a g m e n t e d w i t h 54 f a c i l e l o s s o f a m e t h y l r a d i c a l as shown i n F i g u r e 19 m/e 146 (76%) m/e 118 (100%) m/e 90 (43%) C o u m a r i n (21) •o m/e 176 (100%) H e r n i a r i n (27) •cH 3 * m/e 148 (82%) cr 0' m/e 133 (83%) -co C 7 H 5 0 m/e 105 (12%) F i g u r e 19 -49-Barnes and Occolowitz"^ reported the i n t e r e s t i n g break-down of the prenylated 7-methoxycoumarin, osthol (29) and dihydroosthol (30), as shown i n Figures 20 and 21. m/e 159 (26%) m/e 131 (35%) Figure 20 The molecular ion of osthol l o s t a methyl r a d i c a l to give an ion at m/e 229 (92%) and i t was shown that t h i s methyl r a d i c a l was fragmented from the prenyl side chain to provide a highly conjugated ion (30). F i s s i o n of the side chain -50-t o t h e r i n g gave t h e t r o p y l i u r a - t y p e i o n (31) a t m/e 189 (70%) and l o s s o f t h e methoxy g r o u p w i t h o u t r e a r r a n g e m e n t gave t h e i o n (32) a t m/e 213 (42%). As shown i n F i g u r e 21, s t a b i l i z a t i o n o f t h e M-15 i o n c o u l d n o t o c c u r i n d i h y d r o o s t h o l (33) b e c a u s e o f t h e s a t u r a t e d s i d e c h a i n . L o s s o f a r a d i c a l from t h e m o l e c u l a r i o n a t m/e 246 (39%) y i e l d e d t h e b a s e peak a t m/e 189 w h i c h s u b s e q u e n t l y e l i m i n a t e d a m o l e c u l e o f f o r -m a l d e h y d e and c a r b o n monoxide t o g i v e t h e f r a g m e n t a t m/e 131 (36%). m/e 159 (6%) m/e 131 (36%) F i g u r e 21 S i m i l a r f r a g m e n t a t i o n s o f a l k y l s i d e c h a i n s o f f u r o c o u m a r i n s were r e c e n t l y o b s e r v e d by K u t n e y , E i g e n d o r f , D r e y e r and M i t s h e r " ^ . A t y p i c a l example i s p r o v i d e d by a l l o i m p e r a t o r i n m e t h y l e t h e r (34) ( F i g u r e 22). A l l o i m p e r a t o r i n m e t h y l e t h e r (34) and i t s d i o l (35) - 5 1 -(37) (38) Figure 22 -52-4 were i s o l a t e d from Thamnosma montana Torr. and Frem . and t h e i r mass spectra were obtained i n our laboratory. The d i f f e r e n c e i n the side chain of these two compounds made the r e l a t i v e i n t e n s i t i e s of peaks i n the mass spectra s i g n i f i c a n t l y d i f f e r e n t . The strong ion (36) at m/e 229 (97%) c h a r a c t e r i s t i c of furocoumarins was again recorded i n the mass spectrum of the d i o l ( 3 5 ^ . This ion (36) was also the base peak i n the mass spectra of compounds (37) f and ( 3 8 ) 5 7 . On the basis of the above fragmentations of the coumarin system and other considerations, the mass spectra of thamnosin and i t s d e r i v a t i v e s w i l l be discussed. It had been already mentioned i n the previous section of t h i s thesis that thamnosin and i t s derivatives showed several i n t e r e s t i n g features i n the mass spectra. Above a l l , the f a c i l e fragmentation of the molecule was noted by the weak molecular ion peak (m/e 484, 8%) and a base peak at m/e 242 (Figure 23). This f i s s i o n was so e f f i c i e n t that no fragments between these two peaks could be detected. This r e s u l t immediately suggested that a r e t r o D i e l s - A l d e r (D.A.) s c i s s i o n of a cyclohexene r i n g i n thamnosin may be occurring. This type of fragmentation i s well known from the mass sp e c t r a l studies on unsaturated terpenes and steroids i n p a r t i c u l a r . Furthermore i t must be recognized that t h i s process involves cleavage of the molecule into two fragments of equal mass and therfore the cyclohexene r i n g must be so RELATIVE INTENSITY RELATIVE INTENSITY - 5 4 -^ 0 T h a m n o s i n m/e 484 (8%) r e t r o D„A. v + m/e 242 (100%) m/e 211 (28%) F i g u r e 24 -55-Dihydro-thamnosin m/e 486 (1.4%) retro D.A, •0" T ) C H 5 m/e 242 (100%) m/e 189 (11%) cr o m/e 159 (2%) m/e 199 (2%) m/e 131 (3%) Figure 25 -56-oriented as to accomodate t h i s f a c t . Indeed t h i s r e s u l t provided the f i r s t suggestion that thamnosin consisted of two coumarin systems linked to a C^Q u n i t , with the l a t t e r bearing the cyclohexene r i n g . Figure 24 i l l u s t r a t e s the proposed fragmentation scheme f o r thamnosin. For the sake of c l a r i t y , the correct structure f o r t h i s substance i s u t i l i z e d . The fundamental ret r o D.A. f i s s i o n of the molecule i s followed by loss of C O , O C H g and CH3 - the l a t t e r fragmentations already noted i n the mass spectra of coumarins. Thamnosin C c m H 9 p O , M.W. 484 100) m/e %B • P. m/e %B 1F1 m/e %B. P. m/e %B.P. 484 8 227 74 199 16 141 5 244 6 214 6 189 6 131 5 243 58 213 8 185 5 128 7 242 100 212 9 183 6 115 5 241 10 211 28 171 7 228 12 210 7 155 9 In dihydro-thamnosin (Figure 25) the re t r o D.A. re a c t i o n also gave the base peak at m/e 242 due to the re t e n t i o n of a cyclohexene i n t h i s molecule. The double bond conjugated to a coumarin system i s not present i n dihydro-thamnosin and so the f i s s i o n to the coumarin could now give r i s e to a species at m/e 189 (11%), postu-lated as a tropylium-type ion. It should be noted that dihydro-thamnosin shows a weaker molecular ion at m/e 486 (1.4%) than that of thamnosin (Figure 23). -57-D ihydro-thamno s i n C30 H30°6 M.W. 486 (^100) m/e %B.P. m/e %B.P. m/e %B. P. m/e %B.P. 486 1.4 227 28.0 199 2.0 155 1.3 244 2.6 214 1.2 190 1.6 141 0.8 242 100.0 213 1.6 189 10.6 131 2.7 241 2.1 212 1.4 183 1.0 128 1.3 229 1.2 210 1.0 171 1.4 115 1.2 228 2.6 203 0.8 159 2.2 103 1.7 Thamnosin-dio1, obtained from thamnosin by treatment with osmium tetroxide, has two hydroxyl groups one of which i s on a be n z y l i c carbon and i t was expected that the molecular ion might be very weak. Indeed immediate loss of was observed to give a f a i r l y stable ion at m/e 500 (9%) (Figure 26). As shown i n Figure 27, further f i s s i o n o( to a carbonyl group and also (3 to the coumarin system now gave r i s e to the base peak at m/e 189. The ret r o D.A. react i o n of the cyclohexene r i n g i n t h i s molecule gave a very strog ion at m/e 242 (77%), which was found to be the base peak i n the mass spectra of thamnosin and dihydro-thamnosin. Thamnosin-diol C30 H30°8 M.W. 518 (> 100) m/ e %B.P. m/e %B.P. m/e %B 0 P. m/e %B. P. 518 0.1 227 39 161 10 113 12 516 0.5 211 15 159 23 111 16 500 9 205 10 149 15 110 14 364 16 204 16 142 10 109 14 363 47 203 19 139 21 108 40 283 15 199 10 131 24 107 16 243 16 190 18 129 11 105 16 242 77 189 100 128 18 103 15 241 15 185 11 127 14 229 13 175 12 115 16 -8S--59-0^0 ^0 Tharanosin-diol m/e 518 (M-18) + m/e 500 (9%) retr o D.A. 0 - . A . ' V U L M J m/e 242 (77%) V C14 H11°3 m/e 227 (39%) v-*ocH3 C14 H11°2 m/e 211 (15%) C H 3 0 ^ ^ ^ 0 ^ 0 m/e 189 (100%) -t-C10 H7°2 m/e 159 (23%) — C O C13 H11°2 m/e 199 (10%) - C o C g H 7 0 T m/e 131 (24%) Figure 27 -60-Chemical degradation of thamnosin-diol into two units was accomplished by the a c t i o n of p e r i o d i c a c i d to give two aldehydes. The structure of aldehyde-I was i d e n t i f i e d to be 7-methoxy-coumarin-6-aldehyde by d i r e c t comparison with an authentic sample and t h i s highly conjugated molecule, as a n t i c i p a t e d , showed a molecular ion at m/e 204 which also was the base peak. Loss of H and CO gave highly conjugated ions at m/e 203 and m/e 175, successively, (Figure 28) Aldehyde-I C 1 1 H g 0 4 M.W. 204 (^100) m/e %B.P. m/e %B.P. m/e %B.P. m/e %B.P. 205 21 159 14 127 18 113 19 204 100 158 14 126 14 112 19 203 33 149 17 125 21 111 33 187 23 139 12 123 15 110 15 175 15 137 12 119 19 109 21 OWc Aldehyde-I m/e 204 (100%) 0 CH,0 m/e 175 (15%) m/e 203 (33%) Figure 28 -61-Aldehyde-II m/e %B.P. 312 45 243 21 242 100 227 49 211 21 189 22 C19 H20°4 M ' W -m/e %B.P. 167 16 155 17 149 29 140 16 138 17 136 16 312 (>100) m/e %B.P. 127 25 126 19 125 29 124 17 123 21 119 16 m/e %B.P. 113 29 112 30 111 49 110 21 109 29 105 16 Aldehyde-II m/e 312 (45%) retr o D.A. C14 H11°3 m/e 227 (49%) C14 H11°2 m/e 211 (21%) Figure 29 -62-A n o t h e r d e g r a d a t i o n p r o d u c t o f t h a m n o s i n - d i o l , a l d e h y d e - I I , showed a s t a b l e m o l e c u l a r i o n a t m/e 312 (45%) and t h e f r a g m e n t a t i o n p a t t e r n i n t h e l o w e r mass r e g i o n was somewhat s i m i l a r t o t h a t o f d i h y d r o - t h a m n o s i n ( F i g u r e 26). The b a s e peak a t m/e 242 must a r i s e f r o m t h e r e t r o D . A . r e a c t i o n o f t h e c y c l o h e x e n e r i n g . As shown i n F i g u r e 9, a n a l d e h y d i c h y d r o g e n c o u l d m i g r a t e t o f o r m a k e t e n e i n t e r m e d i a t e and s u b s e q u e n t f i s s i o n (?> t o t h e c o u m a r i n s y s t e m c o u l d p r o v i d e a n t r o p y l i u m t y p e i o n a t m/e 189 (22%). I n c o n t r a s t t o t h e c h a r a c t e r i s t i c f r a g m e n t a t i o n p a t t e r n s o f t h a m n o s i n and i t s d e r i v a t i v e s d i s c u s s e d a b o v e , t h e d i h y d r o - t h a m n o s i n - o x i d e showed a f e a t u r e l e s s mass s p e c t r u m ( F i g u r e 30) e x c e p t f o r t h e f a i r l y s t a b l e m o l e c u l a r i o n a t m/e 502 (23%) and t h e b a s e peak a t m/e 189. The a b s e n c e o f a c y c l o b e x e n e r i n g i n t h i s m o l e c u l e and t h e s t a b i l i t y o f a n e p o x i d e r i n g u n d e r e l e c t r o n i m p a c t w o u l d be e x p e c t e d t o l e a d t o t h i s t y p e o f f r a g m e n t a t i o n . D i h y d r o - t h a m n o s i n - o x i d e C ? nrU n0 A M.W. 502 (>100) m/e % B . P . m/ e % B . P . m/ e %B , P , m/ e % B . P . 502 23 242 31 185 19 141 22 313 10 229 11 163 12 139 62 282 20 227 10 159 17 131 19 281 13 203 14 158 20 113 13 280 12 190 25 156 55 111 36 245 32 189 100 149 14 103 13 F i n a l l y , a mass s p e c t r u m o f k e t o - a l d e h y d e - I I I ( F i g u r e 30) was o b t a i n e d . T h i s compound was o b t a i n e d by - 6 4 -K e t o - a l d e h y d e - I I I m/e 518 m/e 227 (10%) m/e 242 (14%) F i g u r e 31 -65-c l e a v a g e o f a c y c l o h e x e n e r i n g i n d i h y d r o - t h a m n o s i n by t r e a t m e n t w i t h o z o n e . T h i s m o l e c u l e , w i t h l a b i l e f u n c t i o n a l g r o u p s , was e x p e c t e d t o g i v e a c h a r a c t e r i s t i c and c o m p l e x f r a g m e n t a t i o n u n d e r e l e c t r o n i m p a c t ( F i g u r e 3 1 ) . K e t o - a l d e h y d e - I I I C 3 0 H 3 0 ° 8 M.W. 518 (%, 100) m/e % B . P . m/e % B . P o m/e %B o P . m/e % B . P . 518 0.1 242 14 177 28 149 23 500 34 229 40 175 15 135 15 311 29 203 87 163 20 133 18 269 27 191 74 161 21 131 59 256 34 190 87 160 21 115 21 255 91 189 100 159 59 103 38 As summarized i n F i g u r e 31 , t h e s t r o n g peaks a t m/e 189 (100%) , m/e 255 (91%) and m/e 203 (87%) c o u l d come from t h r e e ways o f f i s s i o n ( a , b and c ) o f K e t o - a l d e h y d e - I I I . S u b s e q u e n t l o s s o f CO f r o m e a c h f r a g m e n t c o u l d be e x p l a i n e d by t h e f a c t t h a t e a c h f r a g m e n t c o n t a i n e d a c o u m a r i n s y s t e m . I n c o n c l u s i o n , t h e mass s p e c t r a o f t h a m n o s i n and i t s d e r i v a t i v e s showed c h a r a c t e r i s t i c f r a g m e n t a t i o n p a t t e r n s and a l l t h e d a t a were i n good a c c o r d w i t h t h e p r o p o s e d s t r u c t u r e s d e s c r i b e d i n the p r e v i o u s s e c t i o n o f t h i s t h e s i s . - 6 6 -EXPERIMENTAL M e l t i n g p o i n t s were d e t e r m i n e d on a K o f l e r b l o c k and a r e u n c o r r e c t e d . The u l t r a v i o l e t (UV) s p e c t r a were r e c o r d -ed i n m e t h a n o l s o l u t i o n on a C a r y 14 s p e c t r o p h o t o m e t e r , and t h e i n f r a r e d ( I R ) s p e c t r a were t a k e n on P e r k i n - E l m e r M o d e l 21 and M o d e l 137 s p e c t r o p h o t o m e t e r s . N u c l e a r m a g n e t i c r e s o n a n c e (NMR) s p e c t r a were r e c o r d e d i n d e u t e r i o c h o r o f o r m ( u n l e s s o t h e r w i s e i n d i c a t e d ) a t 100 M c / s on a V a r i a n HA100 i n s t r u m e n t . I n a l l i n s t a n c e s , s p e c t r a were a l s o r e c o r d e d a t 60 M c / s on a V a r i a n A60 i n s t r u m e n t b u t t h e s e a r e n o t q u o t e d h e r e . ' T h e c h e m i c a l s h i f t s a r e g i v e n i n t h e T i e r s ?~scale w i t h r e f e r e n c e t o t e t r a m e t h y l s i l a n e as t h e i n t e r n a l s t a n d a r d . Mass s p e c t r a were r e c o r d e d on a n A t l a s CH-4 mass s p e c t r o m e t e r and h i g h r e s o l u t i o n d e t e r m i n a t i o n s were c a r r i e d o u t on a n A E I MS-9 mass s p e c t r o m e t e r . A n a l y s e s were p e r -formed by D r . A . B e r n h a r d t , M u l h e i m ( R u h r ) , Germany and M r . P . B o r d a o f t h e m i c r o a n a l y t i c a l l a b o r a t o r y , U n i v e r s i t y o f B r i t i s h C o l u m b i a . S i l i c a g e l G and Woelm n e u t r a l a l u m i n a c o n t a i n i n g e l e c t r o n i c p h o s p h o r were u s e d f o r t h i n -l a y e r c h r o m a t o g r a p h y ( T L C ) and Woelm n e u t r a l a l u m i n a ( a c t i v i t y I ) was u s e d f o r c o l u m n c h r o m a t o g r a p h y . T h a m n o s i n The c r u d e t h a m n o s i n , p r o v i d e d by D r . D . L . D r e y e r , F r u i t and V e g e t a b l e L a b o r a t o r y , U . S . D e p a r t m e n t o f A g r i c u l t u r e , P a s a d e n a , C a l i f o r n i a , was r e c r y s t a l l i z e d t h r e e t i m e s f r o m b e n z e n e - d i c h l o r o m e t h a n e t o p r o v i d e t h e a n a l y t i c a l -67-sample (as prisms), m.p, 244-247°; one bright fluorescent spot on TLC ( s i l i c a gel G, CHCI3J EtOAc ( l s l ) ) . ORDs [^<^>~JYP° » [^400 ' P^a^-n p o s i t i v e curve. IR (KBr)s 1725, 1610, 1557 (o(-pyrone), 980 (trans d i s u b s t i -tuted double bond), 820 ( t r i s u b s t i t u t e d double bond) cm"1. UV,^max ( e ) : 227 (30,000), 256 (23,100), 298 (Sh, 14,800) 333 m|x(22,900); A m i n (e) s 243 (20,600), 282 (12,100). NMR signals (100 Mc/s)sT 2.46 (IH, doublet, J=9.5 cps, H-C^ coumarin), 2.50 (IH, doublet, J=9.5 cps, H-C^ coumarin), 2.89 (IH, s i n g l e t , H-C5 coumarin), 2.94 (lH, s i n g l e t , H-C5 coumarin), 3.37 (lH, s i n g l e t , H-Cg coumarin), 3.39 (lH, s i n g l e t , H-Cg coumarin), 3.82 (IH, doublet, J=16 cps, Hg-C=C-), 3.83 (IH, doublet, J=9.5 cps, H-C3 coumarin), 3.85 (IH, doublet,, J=9.5 cps, H-C3 coumarin), 3.98 (lH, doublet, J=16 cps, HA-C=C-), 4.75 (lH, m u l t i p l e t , HD-C=C-), 6.18 (IH, m u l t i p l e t , H c-C-), 6.27 (3H, s i n g l e t , CH30-Cy coumarin), 6.29 (3H, s i n g l e t , CH 30-C 7 coumarin), 8.20 (3H, s i n g l e t , CH3-C=C-), 8.78 (3H, s i n g l e t , CH3-C-C=C-). Anal. Found: C, 74.26; H, 5.74; 0, 20.08; O-Me, 12.87. Calc. f o r C 3 0H 2g0 6: C, 74.36; H, 5.82; 0, 19.81; (2) O-Me, 12.7. Molecular wt.: 484.188 (Calc. 484.189). Attempted Ozonization of Thamnosin Thamnosin (M.W. 484, 90 mg.) was dissolved i n dichloromethane (20 ml„), cooled to -78° and a slow stream of ozone was passed through f o r 15 minutes. Acetic a c i d (25 ml.) was added to the mixture and a s o l u t i o n of ferrous -68 -s u l f a t e (1 g.) i n water (10 ml.) was added. The mixture was s t i r r e d f o r 30 minutes, heated f o r 15 minutes and im-mediately steam d i s t i l l e d into a s o l u t i o n of 2,4-dinitro-phenylhydrazine (0.6 g.) i n water (40 ml.) and concentrated s u l f u r i c a c i d (10 ml.). Very slow p r e c i p i t a t i o n was observed a f t e r 30 minutes and 250 ml. of d i s t i l l a t e was c o l l e c t e d . The orange p r e c i p i t a t e was separated and chromatographed on ac i d washed alumina (Shawinigan, pH 3). E l u t i o n with benzene-ethyl acetate gave a reddish brown s o l i d (5 mg„), crude m.p. 250° (decomp.)o Attempted r e c r y s t a l l i z a t i o n f a i l e d . UV, Amaxs 224 and 355 nyx. ( i n ether). 2.4-dinitrophenylhydrazine showed ^max at 258 and 342 vajx i n ether. D ihydro-thamno s i n Thamnos i n (238 mg.), i n absolute tetrahydrofuran (40 ml.), was hydrogenated over 10% palladium on charcoal (220 mg.). The hydrogen uptake ceased a f t e r 25 minutes when 1 mol. had been absorbed„ A f t e r removal of the cata-l y s t and solvent, the product was r e c r y s t a l l i z e d from benzene-petroleum ether to give dihydro-thamnosin (174 mg.), m.p. 226-228°. This compound displayed one d u l l fluorescent spot on TLC ( s i l i c a g e l , CHCI3S EtOAC ( I s 1) ) whose R f value was the same as that of thamnosin„ IR (KBr): 1728, 1618, 1563 (o(-pyrone), 820 ( t r i s u b s t i t u t e d double bond) cm"^0 UV, ^max ( e ) : 224 (36,300), 246 (Sh, 13,300), 254 (12,000), 300 (Sh, 14,500), 330 mjL (27,200) 5 A m i n (e)s 266 (5,900). -69-NMR singnals (100 Mc/s)s 2.47 (lH, doublet, J=9.5 cps, H-C^ coumarin), 2.53 (lH, doublet, J=9.5 cps, H-C^ coumarin) 2.93 (lH, s i n g l e t , H-C5 coumarin), 3.07 (lH, s i n g l e t , H-C5 coumarin), 3.28 (lH, s i n g l e t , H-Cg coumarin), 3.33 (IH, s i n g l e t , H-Cg coumarin), 3.86 (2H, two doublets, J=9.5 cps, H-C3 coumarin), 4.83 (lH, m u l t i p l e t , HD~C=C-), 6.33 (lH, doublet, J=3.5 cps, H C » C - ) , 6.22 (3H, s i n g l e t , CH 30-C 7 coumarin), 6.25 (3H, s i n g l e t , CH 30-C 7 coumarin), 8.26 (3H, s i n g l e t , CH3-C=C-), 8.97 (3H, s i n g l e t , CH 3-C-). Anal. Found; C, 73.47; H, 6.43; 0, 20.25; O-Me, 12.87. Calc. f o r C 3 0 H 3 0 0 6 : C, 74.07; H, 6.23; 0, 19.73; (2) O-Me, 12.8. Molecular wt.: 486.204 (Calc. 486.204). Hydrogenation of 7-Methoxycoumarin A sample of 7-methoxycoumarin (5 mg.) i n absolute ethanol (2 ml.) was hydrogenated over 10% palladium on charcoal (5 mg.). No uptake of hydrogen was detected a f t e r s t i r r i n g f o r 2 hours. However 7-methoxycoumarin (5 mg.) i n absolute tetrahydrofuran (2 ml.) was hydrogenated over 5% rhodium on alumina (5 mg.) i n a micro-hydrogenator, and a rapid uptake of 1 mol. was observed. At t h i s point the absorption ceased. The c a t a l y s t was f i l t e r e d o f f and evaporation of the solvent gave as an o i l , 3,4-dihydro-7-methoxycoumarin (5 mg.). UV,Amaxs end absorption (220 m^ .), 278, 284 nyi. IR (CHClo): 1750 (-C0-0-C=C-), 1626, 1590 (aromatic r i n g ) . -70-T h a m n o s i n - d i o l Thamnosin (223 mg.) was d i s s o l v e d i n a b s o l u t e t e t r a -h y d r o f u r a n (40 m l . ) and osmium t e t r o x i d e (140 m g . , 1.2 m o l . ) was added to the s o l u t i o n . The m i x t u r e was a l l o w e d to s tand f o r 3 days a t room tempera tu re and then methano l (100 m l . ) was added . Dry hydrogen s u l f i d e was passed th rough the m i x t u r e f o r 20 m i n u t e s . The s u l f i d e was f i l t e r e d o f f to g i v e a p a l e y e l l o w s o l u t i o n . E v a p o r a t i o n o f the s o l v e n t gave c r y s t a l l i n e t h a m n o s i n - d i o l ( 1 5 0 m g . ) . T h a m n o s i n - d i o l c r y s t a l l i z e d as p r i sms f rom m e t h a n o l , w i t h one m o l e c u l e o f s o l v e n t , ( a ) , m.p. 2 7 3 - 2 7 6 ° . A n a l . Found : C , 6 7 . 9 9 ; H, 6 . 4 0 . C a l c . f o r £30^30 ° 8 * CH3OH: C , 6 7 . 6 1 ; H, 6 . 2 3 . These p r i sms were ground and d r i e d i n the d r y i n g p i s t o l f o r 3 hours a t 100° to a f f o r d u n s o l v a t e d t h a m n o s i n - d i o l ( b ) , Hup. 2 4 3 - 2 4 8 ° , r e f o r m i n g p l a t e s , m.p. 2 6 7 - 2 7 2 ° . A n a l . Found : C , 6 9 . 0 1 ; H, 6 . 3 3 . C a l c . f o r C-jQHgoOg: C , 6 9 . 4 9 ; H, 5 . 79 . R e c r y s t a l l i z a t i o n f rom e t h a n o l a f f o r d e d the u n s o l v a t e d t h a m n o s i n - d i o l ( c ) as p l a t e s , m 0 p . 2 4 3 - 2 4 8 ° , r e f o r m i n g p l a t e s , m.p. 269-272° mixed m e l t i n g p o i n t w i t h thamnos in-d i o l (b) showed no d e p r e s s i o n . A n a l . Found : C , 6 9 . 7 8 ; H, 5 . 9 1 ; 0 , 2 4 . 5 1 ; 0-Me, 1 1 . 7 1 . C a l c . f o r C 3 0 H 3 0 0 8 s C , 6 9 . 4 9 ; H, 5 .83 ; 0 , 2 4 . 6 8 ; (2) 0-Me, 1 2 . 0 . In a d d i t i o n to the above u n s o l v a t e d p l a t e s ( c ) , thamnos in-d i o l c r y s t a l l i z e d , w i t h one m o l e c u l e o f s o l v e n t , as p r i sms -71-(d), m.p. 267-272°. Anal. Found: C, 68.01; H, 6.13; 0, 25.72. Calc. f o r C 3 0H 3 00 8.C 2H 5OH: C, 68.06; H, 6.43; 0, 25.51. These prisms (d) were ground and dried at 100° i n the drying p i s t o l f o r 6 hours to y i e l d unsolvated thamnosin-diol (e), m.p. 243-247°, reforming p l a t e s , m.p. 266-272°, whose mixed melting point with thamnosin-diol (c) showed no depression. The above thamnosin-diol (a, b, c, d and e) showed one spot, r e s p e c t i v e l y , on TLC with the i d e n t i c a l R^ valves (alumina and s i l i c a g e l , benzene-EtOAc, CHCl 3, CHCl 3-Et0Ac, EtOAc). IR (KBr): 3480 (hydroxyl), 1725, 1620, 1565 (coumarin), 820 ( t r i s u b s t i t u t e d double bond) cm"1. UVj^max (e): 223 (36,800), 251 (Sh, 12,900), 300 (Sh, 18,200), 328 (29,700),A m i n . (e)s 267 (6,400). NMR signals (100 Mc/s) ^ 2 . 4 3 (lH, doublet, J=9.5 cps, H-C^ coumarin), 2.49 (lH, doublet, J=9.5 cps, H-C^ coumarin) 2.61 (IH, s i n g l e t , H-C5 coumarin), 2.80 (lH, s i n g l e t , H-C5 coumarin), 3.25 (lH, s i n g l e t , H-Cg coumarin), 3.49 (lH, s i n g l e t , H-Cg coumarin), 3.82 (lH, doublet, J=9.5 cps, H-C3 coumarin), 3.92 (lH, doublet, J=9.5 cps, H-C3 coumarin) 4.75 (lH, doublet, J=5 cps, H D»C=C-), 4.98 (lH, m u l t i p l e t , H A-C-0-), 6.16 (3H, s i n g l e t , C H 30-C 7 coumarin), 6.28 (lH, doublet, J=5 cps, H c-C-), 6.47 (3H, s i n g l e t , C H 30-C ? coumarin), 6.6 (2H, mutiplet, 2H0-), 7.03 (lH, s i n g l e t , H - D - C - O-), 7.8 and 8.3 (4H, two sets of doublets, J=7 cps, -C- C H 2 - C H 2 - C - ) , 8.21 (3H, s i n g l e t , CH 3"C=C-), 8.69 (3H, s i n g l e t , CHo-C-). -72-NMR signals (+D20, 100 Mc/s): 4.98 (IH, sharp s i n g l e t , HA-C-OD), 5.30 ( s i n g l e t , HOD), no peaks at 6.6 (2DO-), 7.03 (IH, sharp s i n g l e t , Hg-C-OD), the re s t of the peaks remained the same. High r e s o l u t i o n mass determination, m/e 500 (CgQHgQOg-^O)* peak: 500.187 (Calc. 500.184). Periodate Cleavage of Thamnosin-diol To a s o l u t i o n of thamnosin-diol (M.W. 518, 200 mg.), i n methanol (220 ml.), was added an aqueous s o l u t i o n of pe r i o d i c a c i d (1.5 mol.^) and the reac t i o n mixture was allow-ed to stand f o r 24 hours. The solvent was evaporated i n  vacuo and the r e s i d u a l material was extracted with d i c h l o r o -methane. The organic layer was washed with water, aqueous sodium bicarbonate and water and dried over sodium s u l f a t e . Removal of the sovent gave a white s o l i d . The white ma-t e r i a l was p u r i f i e d by preparative TLC on s i l i c a gel (CHCl^: EtOAc (1 s 1) ). Af t e r the plate was developed, a small p o r t i o n of the pla t e was sprayed with 2,4-DNP reagent to give two d i s t i n c t bands. The more polar compound (aldehyde-I) showed an orange color while the less polar compound (aldehyde-II) was yellow i n c o l o r . E x t r a c t i o n of these two f r a c t i o n s with methano]/chloroform y i e l d e d aldehyde-I (49 mg.) and aldehyde-II (25 mg.). Aldehyde-I was c r y s t a l l i z e d as prisms from methanol, m.p. 242-246°. IR (KBr): 1735, 1670, 1610 (ol-pyrone, aldehyde). UV,Amax: 255, 308, 329, 342 (Sh.) mu, ^ min: 237, 278, -73-318 mu. UV (+NaBH4), ^max: 222, 251 (Sh.), 295 (Sh.), 327 mu, min: 261 mu. NMR signals (100 Mc/s i n ( C F 2 C l ) 2 C ( O D ) 2 ) : -0.23 (IH, s i n g l e t , -CHO), 1.90 (IH, s i n g l e t , H-C5 coumarin), 2.09 (IH, doublet, J=9.5 cps, H-C4 coumarin), 3.00 (IH, s i n g l e t , H-Cg coumarin), 3.75 (IH, doublet, J=9.5 cps, H-C^ coumarin) 6.01 (3H, s i n g l e t , CH3O-C7 coumarin). Molecular wt.: 204.042 (Calc. f o r C n H g 0 4 : 204.042). An authentic sample of 7-methoxycoumarin-6-aldehyde, m.p. 248-251°, was obtained from Dr. F. E. King, Forest Products Laboratories, Ayresburg, Bucks, England, (King et a l 4 1 give m.p. 252-253°). IR (KBr): 1735, 1670, 1610 cm"1. UV, Xmax: 255, 308, 328, 342 (Sh.) mu, A m i n . : 237, 277, 316 mu. Mixed m.p. with aldehyde-I: m.p. 243-246°. Aldehyde-I was i d e n t i c a l with 7-methoxycoumarin-6-aldehyde by a l l c r i t e r i a : mixed m.p.5 R^ values on TLC ( s i l i c a gel and alumina, CHCI3, CHClg-EtOAc, EtOAc, benzene-EtOAc); superimposable UV and IR spectra. Aldehyde-II r e s i s t e d c r y s t a l l i z a t i o n but data was obtained on TLC pure m a t e r i a l . IR (CHCI3): 1720, 1615 cm 1 (o(-pyrone, saturated aldehyde). UV,>max: 229, 254 (Sh.), 296 (Sh.), 328 mu, Amin: 261 mu. NMR signals (100 Mc/s): 2.45 (IH, doublet, J=9.5 cps, H-C4 coumarin), 2.85 (IH, s i n g l e t , H-Cr coumarin), 3.31 (IH, -74-s i n g l e t , H-Cg coumarin), 3.84 (lH, doublet, J=9.5 cps, H-C3 coumarin), 4.76 (IH, m u l t i p l e t , HD-C=C-), 5.84 (lH, doublet, H C~C-), 6.20 (3H, s i n g l e t , CH^O-^ coumarin), 8.21 (3H, s i n g l e t , CH3-C=C-), 8.82 (3H, s i n g l e t , CH3-C-). Molecular wt. ; 312.138 (Calc. f o r C ^ ^ Q O ^ 312.136). Dihydro-thamnosin-oxide Dihydro-thamnosin (M.W. 486, 220 mg.), i n chloroform (50 ml.), was treated with m-chloroperbenzoic a c i d (1.5 mol.) and the s o l u t i o n was maintained at room temperature f o r 36 hours. The s o l u t i o n was then washed with aqueous sodium bicarbonate s o l u t i o n and dried over sodium s u l f a t e . Removal of the sovent gave c r y s t a l l i n e material (200 mg.). R e c r y s t a l l i z a t i o n from d i i s o p r o p y l ether afforded dihydro-thamnosin-oxide as plates, m.p. 243-246°. IR (KBr)5 1725, 1612, 1565 cm"1 (coumarin). U V , X m a x ( e ) s 223 (39,700), 243 (Sh. 12,800), 253 (Sh. 9,700), 298 (Sh. 17,300), 329 mu (27,400), A m i n ( € - ) : 263 (3,900). NMR signals (100 Mc/s)i 2.47 (lH, doublet, J=9.5 cps, H-C^ coumarin), 2.51 (lH, doublet, J=9.5 cps, H-C^ coumarin) 3.00 (IH, s i n g l e t , H-C5 coumarin), 3.05 (lH, s i n g l e t , H-C5 coumarin),3.26 (IH, s i n g l e t , H-Cg coumarin), 3.28 (lH, s i n g l e t , H-Cg coumarin), 3.85 (2H, doublets, J=9.5 cps, 2H-C3 c o u m a r i n ) , 6 . 1 6 (6H, s i n g l e t s , 2CH3O-C7 coumarin), 6.4 (IH, m u l t i p l e t , H C~C-), 7.11 (lH, broad s i n g l e t , H D-C-C-), 8.59 (CH 3, s i n g l e t , CH 3 - ( A > ) , 9.17 (3H, s i n g l e t , CH o — C — ) . -75-Anal. Found; C, 72,00; H, 5.77; 0, 22.29; 0-Me, 12.15. Calc. f o r C 3 0H 3 0O 7: C, 71.69; H, 6.02; 0, 22.28; (2) 0-Me, 12.3. Molecular wt. : 502.202 (Calc. 502.199). Attempted Epoxide Opening on Dihydro-thamnosin-oxide Dihydro-thamnosin-oxide (M.W. 502, 10 mg.) was added to a b o i l i n g 5% aqueous o x a l i c a c i d s o l u t i o n (3 ml.) and r e f l u x i n g continued f o r 30 minutes. The s o l u t i o n was cooled and extracted with dichloromethane. The extracts were washed with 5% aqueous sodium bicarbonate and dried over sodium s u l f a t e . The solvent was removed to give a white residue (8 mg.). This residue was i d e n t i f i e d to be the unreacted s t a r t i n g oxide by TLC, UV and IR spectra. Under more f o r c i n g conditions ( f o r example r e f l u x i n g i n dioxane f o r 2 hours) the oxide gave i n t r a c t a b l e mixtures. Attempted Hydroxyation of Dihydro-thamnosin Dihydro-thamnosin (50 mg.), i n absolute tetrahydro-furan (2 ml.), was treated with osmium tetroxide (31 mg., 1.2 mol.). The re a c t i o n mixture was allowed to stand at room temperature f o r 5 days, and then was s t i r r e d with a s o l u t i o n of sodium b i s u l f i t e (100 mg.) i n water (5 ml.) and methanol (10 ml.) f o r 20 hours. The s o l u t i o n was separated, a c i d i f i e d with a few drops of a c e t i c a c i d , concentrated to a small volume and extracted with chloroform. The organic layer was separated and dried over sodium s u l f a t e . -76-Evaporation of the solvent afforded a grayish brown s o l i d (23 mg.)- TLC on s i l i c a gel (CHClg-EtOAc) indicated that the major component i n t h i s mixture was unreacted s t a r t i n g m a t e r i a l . Preparative TLC on s i l i c a gel (with very poor recovery) showed that the s t a r t i n g material represented 60% while a few more polar compounds represented the remain-ing 40% of the r e a c t i o n mixture. Controlled Ozonization of Osthol Osthol (8-isopent-2 1-eny1-7-methoxycoumarin, 5 mg.) was dissolved i n dichloromethane (40 ml.) and cooled to -78°. A slow stream of ozone was passed through the s o l u t i o n f o r 50 minutes u n t i l the excess ozone was detected with aqueous potassium iodide-boric acid at the o u t l e t . The s o l u t i o n was poured into aqueous ferrous s u l f a t e (100 mg.) and the s t i r r i n g was continuted f o r 10 minutes. The organic layer was separated and dried over sodium s u l f a t e . Removal of the solvent gave a pale yellow material (4 mg.). This material showed one spot on TLC ( s i l i c a g e l , EtOAc-CHClg) and i t s R^ value was smaller than that of osthol. On spraying with 2,4-DNP reagent the ozonization product showed a d i s t i n c t yellow spot on a TLC p l a t e . Under these conditions, osthol showed no c o l o r . The UV spectrum of the ozonization product was the same as that of the s t a r t -ing m a t erial. I t was apparent that the c o n t r o l l e d ozoni-zation of osthol was most l i k e l y y i e l d i n g an aldehydic product. - 7 7 -C o n t r o l l e d O z o n i z a t i o n o f D i h y d r o - t h a m n o s i n D i h y d r o - t h a m n o s i n (100 m g . ) , i n d i c h l o r o m e t h a n e , was o z o n i z e d f o r 1 h o u r u n d e r t h e same c o n d i t i o n s as p r e v i o u s l y d e s c r i b e d f o r o s t h o l . The c r u d e p r o d u c t (90 m g . ) was p u r i f i e d by p r e p a r a t i v e TLC ( s i l i c a g e l , E t 0 A c - C H C l 3 ) and t h e a l d e h y d i c band was d e t e c t e d by 2,4~DNP as s p r a y r e -a g e n t . E x t r a c t i o n o f t h e a l d e h y d e by c h l o r o f o r m - m e t h a n o l a f f o r d e d a n amorphous s o l i d (36 m g . ) , m . p . 1 3 5 - 1 4 0 ° , d e -s i g n a t e d as k e t o - a l d e h y d e - I I I . I R ( C H C l 3 ) : 1721, 1616 c m " 1 ( c o u m a r i n , a l d e h y d e , m e t h y l k e t o n e ) . U V , A m a x ( f ) : 223 ( 4 2 , 9 0 0 ) , 254 ( S h . 1 2 , 0 0 0 ) , 296 ( S h . 15, 5 0 0 ) , 329 mu (2 9 , 9 0 0 ) , - A m i n ( e ) : 265 mu ( 4 , 8 0 0 ) . NMR s i g n a l s (100 M c / s ) ; 0 . 0 2 ( l H , d o u b l e t , J=2 c p s , - C H O ) , 2 .39 ( l H , d o u b l e t , J = 9 . 5 c p s , H - C 4 c o u m a r i n ) , 2 . 4 4 ( l H , d o u b l e t , J= s9.5 c p s , H - C ^ c o u m a r i n ) , 2.68 ( l H , s i n g l e t , H-Cg c o u m a r i n ) , 2 . 8 6 ( I H , s i n g l e t , H - C ^ c o u m a r i n ) , 3.18 ( l H , s i n g l e t , H-Cg c o u m a r i n ) , 3.27 ( l H , s i n g l e t , H-Cg c o u m a r i n ) , 3.77 ( l H , d o u b l e t , J = 9 . 5 c p s , H - C 3 c o u m a r i n ) , 3.81 ( I H , d o u b l e t , J = 9 . 5 c p s , H - C 3 c o u m a r i n ) , 5.76 ( l H , d o u b l e t , J=2 c p s , H ^ - C - C H O ) , 6.15 ( 6 H , s i n g l e t s , 2 C H 3 0 - C 7 c o u m a r i n ) , 7.87 ( 3 H , s i n g l e t , C H 3 - C 0 - ) , 8.91 ( 3 H , s i n g l e t , C H 3 ~ C - ) . H i g h r e s o l u t i o n mass d e t e r m i n a t i o n , m/e 500 ( C 3 0 H 3 0 ° 8 " H 2 ° ) + P e a k s 500.184 ( C a l c . 5 0 0 . 1 8 4 ) . Sodium B o r o h y d r i d e R e d u c t i o n o f K e t o - a l d e h y d e - I I I K e t o - a l d e h y d e - I I I (16 m g . ) , i n i s o - p r o p a n o l (2 m l . ) -78-and c h l o r o f o r m ( i n i l . ) , was r e d u c e d w i t h s o d i u m b o r o h y d r i d e (8 m g . ) ' A f t e r t h e m i x t u r e was a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 55 m i n . , t h e s o l v e n t was removed i n v a c u o . The r e s u l t i n g r e s i d u e was e x t r a c t e d w i t h c h l o r o f o r m . E v a p o r a t i o n o f t h e c h l o r o f o r m gave a n amorphous s o l i d (8 m g . ) . IR (CHCI3): 3436 ( h y d r o x y l ) , 1718, 1613, 1560 ( c o u m a r i n ) c m " 1 . UV, Amaxj 225, 254, 298 ( S h . ) , 329 m p . NMR s i g n a l s (100 M c / s ) s 3.43 (2H, d o u b l e t , J=9.5 c p s , H-C4 c o u m a r i n ) , 3.66 (IH, s i n g l e t , H-C5 c o u m a r i n ) , 3.84 (IH, s i n g l e t , H-Cg c o u m a r i n ) , 3.29 (2H, s i n g l e t , 2H-Cg c o u m a r i n ) , 3.85 (IH, d o u b l e t , J=9.5 c p s , H-C3 c o u m a r i n ) , 3.89 (IH, d o u b l e t , J=9.5 c p s , H-Cg c o u m a r i n ) , 6.15 (6H, s i n g l e t , 2 CHgO-C7), 5.8-6.4 (6H, m u l t i p l e t ) , 8.82 (3H, m u l t i p l e t , CHj-CH-OH), 9.09 (3H, s i n g l e t , CHg-C-). S u b e r o s i n D e m e t h y l s u b e r o s i n (6 - i s o p e n t-2 * - e n y l u m b e l l i f e r o n e ) was k i n d l y s u p p l i e d by D r . F . E . K i n g , F o r e s t P r o d u c t s R e s e a r c h L a b o r a t o r i e s , A y r e s b u r g , B u c k s , E n g l a n d . D e m e t h y l s u b e r o s i n was m e t h y l a t e d by r e f l u x i n g w i t h m e t h y l -i o d i d e and p o t a s s i u m c a r b o n a t e i n a c e t o n e f o r 10 h o u r s t o a f f o r d s u b e r o s i n . R e c r y s t a l l i z a t i o n f r o m d i i s o p r o p y l e t h e r gave s u b e r o s i n (7 - m e t h o x y-6 - i s o p e n t-2 ' - e n y l c o u m a r i n ) as p r i s m s , m . p . 82-87° ( K i n g e t a l 4 1 g i v e m . p . 87-88°). IR (CHClo): 1724, 1621, 1563 ( c o u m a r i n , o ( - p y r o n e ) c m " 1 . -79-UV,>max: 223, 253, 297 (Sh.), 330 mu. NMR signals (100 Mc/s): 2.47 (lH, doublet, J=9.5 cps, H-C4 coumarin), 2.90 (lH, s i n g l e t , H-Cej coumarin), 3.30 (lH, s i n g l e t , H-Cg coumarin), 3.87 (lH, doublet, J=9.5 cps, H-C3 coumarin), 4.78 (lH, broad t r i p l e t , J=7 cps, H-C2'), 6.18 (3H, s i n g l e t , CH3O-C7 coumarin), 6.74 (2H, doublet, J=7 cps, 211-C^), 8.27 (3H, s i n g l e t CH3-C=C-), 8.33 (3H, s i n g l e t , CH3-C=C-). T e t rahyd r o-1hamriosin Dihydrd-thamnosin (M.W. 486, 50 mg.), i n a c e t i c a c i d (25 ml.), was hydrogenated over 10% palladium on charcoal (100 mg.). The hydrogenation was interrupted when 1 mol. of hydrogen was absorbed. The c a t a l y s t was f i l t e r e d o f f and removal of the solvent gave an amorophous s o l i d (40 mg.), t e trahydro-thamno s i n . IR (CHC1 3): 1721, 1623, 1560 (coumarin) cm"1. UV, ^ maxs end absorption (220 mu), 254 (Sh.), 300 (Sh.), 332 mu, Xmin: 266 mu. NMR signals (100 Mc/s): 2.46 (lH, doublet, J=9.5 cps, H-C4 coumarin), 2.50 (lH, doublet, J=9.5 cps, H-C4 coumarin), 2.92 (IH, s i n g l e t , H-C5 coumarin), 2.97 (IH, s i n g l e t , H-C5 coumarin), 3.26 (2H, s i n g l e t s , 2H-Cg coumarin), 3.82 (IH, doublet, J=9.5 cps, H-C3 coumarin), 3.86 (lH, doublet, J=9.5 cps, H-C3 coumarin), 6.16 (3H, s i n g l e t , CU^O-Cy coumarin), 6.18 (3H, s i n g l e t , CH.3O-C7 coumarin), 8.96 (3H, doublet, J=4 cps, CH 3-C-H), 9.16 (3H, s i n g l e t , CHo-C-). -80-Octahydro-thamno s i n Dihydro-thamnosin (49 mg.), i n dichloromethane-methanol (10 ml./10 ml.), was hydrogenated over 10% palladium on charcoal. The hydrogen uptake was ceased a f t e r 3 mol. and the c a t a l y s t was f i l t e r e d o f f . Removal of the solvent gave an amorphous s o l i d (46 mg.), octahydro-thamnosin. IR (CHCI3): 1761, 1618 cm"1 (C=0, aromatic). UV,^maxs end absorption (220 mu) , 285 mji, A-mins 253 mu. NMR signals (60 Mc/s): 3.25 (2H, broad s i n g l e t s , 2H-C5), 3.55 (2H, broad s i n g l e t s , 2H-Cg), 6.28 (3H, s i n g l e t , CHgO-C^, 6.32 (3H, s i n g l e t , CH 30-C 7), 9.08 (3H, m u l t i p l e t , CHo-CH), 9.23 (3H, s i n g l e t , CHo-C-). -81-REFERENCES 1. E . L . B e n n e t t and J . B o n n e r , Am. J . B o t a n y , 40 , 29 (1953 ) . 2. W. H . M u l l e r and H . M u l l e r , Am. J . B o t a n y , 4 3 , 354 (1956 ) . 3. T . H . K e a r n e y and R. H . P e e b l e s , A r i z o n a F l o r a , U n i v e r s i t y o f C a l i f o r n i a P r e s s , B e r k e l e y and L o s A n g e l e s , C a l i f . (1960) p . 4 9 4 . 4 . D. L . D r e y e r , T e t r a h e d r o n , 22 , 2923 (1966 ) . 5. J . R. P r i c e , The A l k a l o i d s , V o l . I I , ( E d . ) R . H . F . Manske a n d H . L . H o l m e s , A c a d i m i c P r e s s , New Y o r k (1952) p . 3 5 3 . 6. P r i v a t e c o m m u n i c a t i o n f r o m D r . D . L . D r e y e r . 7. H . T . 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Stainton, A.P. Johnson and M. Barber, J . Heterocyclic Chem., 2, 256 (1965). 56. R.A.W. Johnstone, B.J. M i l l a r d , F.M. Dean and A.W. H i l l , J . Chem. Soc., 1712 (1966). 57. J.P. Kutney, G. Eigendorf, D.L. Dreyer and L.A. Mitscher, submitted f o r p u b l i c a t i o n . 58. N.S. Wulfson, V.I. Z a r e t s k i i and V.G. Zyakoon, Is v. Akad. Nauk SSR, Ser. Khim., 2215 (1963). i PART II STUDIES IN INDOLE ALKALOIDS - 8 6 -INTRODUCTION R e s e a r c h on a l k a l o i d s i n t e r e s t s c h e m i s t s b e c a u s e o f t h e complex s t r u c t u r e s , s p e c i a l b i o l o g i c a l a c t i v i t i e s and p h y t o c h e m i c a l i m p o r t a n c e . B e c a u s e a l k a l o i d s g e n e r a l l y a r e c o m p l e x m o l e c u l e s , c l a s s i c a l s t r u c t u r a l e l u c i d a t i o n methods r e q u i r e l a r g e amounts o f sample t o deduce t h e o r i g i n a l s t r u c t u r e f r o m t h e d e g r a d a t i o n p r o d u c t s and t h i s i n c o n -v e n i e n c e has b e e n t h e m a i n d i f f i c u l t y i n a l k a l o i d c h e m i s t r y . The p r o b l e m has b e e n l a r g e l y overcome by t h e e x t e n s i v e u s e o f p h y s i c a l m e t h o d s , e s p e c i a l l y n u c l e a r m a g n e t i c r e s o n a n c e and mass s p e c t r o s c o p y , w h i c h r e q u i r e o n l y s m a l l q u a n t i t i e s o f m a t e r i a l . I n a d d i t i o n t o t h e s t r u c t u r a l e l u c i d a t i o n o f a l k a l o i d s , a c o n s i d e r a b l e e f f o r t has b e e n made towards t h e s y n t h e s i s o f t h e s e s u b s t a n c e s . I n many i n s t a n c e s , l a b o r a -t o r y c o n s t r u c t i o n o f complex o r g a n i c systems has b e e n s t u d i e d by " b i o g e n e t i c - t y p e " s y n t h e s i s . S t u d i e s o f t h e b i o s y n t h e s i s o f a l k a l o i d s became f e a s i b l e w i t h t h e a d v e n t o f r a d i o a c t i v e compounds. F o r e x a m p l e , i n c o r p o r a t i o n e x p e r i m e n t s w i t h r a d i o a c t i v e t r y p t o p h a n i n t o g r a m i n e ( 1 ) , a j m a l i n e ( 2 ) , s e r p e n t i n e ( 3 ) , v i n d o l i n e (4) , c a t h a r a n t h i n e (5) and r e s e r p i n e ( 6 ) , h a v e now shown c o n c l u s i v e l y t h a t t h e i n d o l e p o r t i o n o f t h e i n d o l e a l k a l o i d s i s d e r i v e d f r o m t r y p t o p h a n o r i t s b i o -1 2 l o g i c a l e q u i v a l e n t ' . I n c o n t r a s t t o t h e " t r y p t o p h a n " p o r t i o n o f t h e i n d o l e a l k a l o i d s , v a r i o u s h y p o t h e s e s ( d i s c u s s e d b e l o w ) c o n c e r n e d -87-w i t h t h e b i o s y n t h e s i s o f t h e " n o n - t r y p t o p h a n " o r Cg_^Q u n i t have b e e n p u t f o r w a r d . (6) OMe F i g u r e 1 The S h i k i m i c - P r e p h e n i c A c i d H y p o t h e s i s A b i o g e n e t i c scheme i n v o l v i n g p r e p h e n i c a c i d as a p r e c u r s o r was p r o p o s e d by W e n k e r t ^ f o r t h e b i o s y n t h e s i s - 8 8 -(13) (14) (12) Figure 2 -89-Figure 3 -90-o f t h e " n o n - t r y p t o p h a n " p o r t i o n o f i n d o l e a l k a l o i d s . I n t h i s scheme, as shown i n F i g u r e 2, p r e p h e n i c a c i d ( 7 ) , known as a p r e c u r s o r o f n a t u r a l l y o c c u r r i n g b e n z e n o i d compounds, i s r e a r r a n g e d and d e h y d r a t e d t o g i v e t h e i n t e r m e d i a t e ( 8 ) , whose r o l e i n t h e b i o s y n t h e s i s o f y o h i m b i n e (10) c o u l d be v i s u a l i z e d as f o l l o w s . C o n d e n s a t i o n o f f o r m a l d e h y d e w i t h t h e i n t e r m e d i a t e (8) f o l l o w e d by a r e t r o - a l d o l i z a t i o n l e a d s t o a " s e c o - p r e p h e n a t e - f o r m a l d e h y d e " ( S P F ) u n i t (9) w h i c h c o u l d be i n c o r p o r a t e d i n t o c o r y n a n t h e i n e (11) and a j m a l i c i n e ( 1 2 ) , The o c c u r r e n c e o f n e u t r a l p l a n t c o n s t i t u e n t s w i t h a common SPF n u c l e u s ( e . g . g e n i t i o p i c r i n (13) and b a k a n k o s i n (14) ) seemed t o l e n d s u p p o r t f o r t h e i n t e r m e d i a c y o f t h e SPF u n i t . An i m p o r t n a t f e a t u r e o f t h e p r e p h e n i c a c i d t h e o r y was t h a t i t a c c o u n t e d f o r t h e a l m o s t c o n s i s t a n t l y o b s e r v e d c o n f i g u r a t i o n a t C-15 i n t h e i n d o l e a l k a l i d s . W e n k e r t ' s t h e o r y a l s o c o v e r s t h e b i o g e n e s i s o f t h e A s p i d o s p e r m a and I b o g a g r o u p s o f b a s e s ' * . C o n d e n s a t i o n o f t h e SPF u n i t w i t h t r y p t a m i n e f o l l o w e d by a r e t r o - M i c h a e l r e a c t i o n p r o v i d e s t h e k e y p r o d u c t ( 1 5 ) . As shown i n F i g u r e 3 , t h i s c l e a v a g e p r o d u c t (15) l e a d s t o b o t h t h e A s p i d o s p e r m a (17) and I b o g a (19) a l k a l o i d s , T r a n s a n n u l a r c y c l i z a t i o n r e a c t i o n s s i m i l a r t o t h e p o s t u l a t e d c o n v e r s i o n s 25 (16-»17 and 18-»19) h a v e b e e n p e r f o r m e d i n t h e l a b o r a t o r y ' 2 6 , 2 7 , 2 8 The p r e p h e n i c a c i d t h e o r y has a l s o b e e n e x t e n d e d t o 21 c o v e r t h e b i o g e n e s i s o f Akuamma and S t r y c h n o s a l k a l o i d s - 9 1 -The new s t r u c t u r a l p a t t e r n s f o u n d i n p l e i o c a r p a m i n e (21) 20 and p i c r a l i n e (22) o f f e r a p o s s i b l e s o l u t i o n t o t h e b i o g e n e s i s o f t h e Akuamma (24) and o f t h e S t r y c h n o s (26) b a s e s , as i l l u s t r a t e d i n F i g u r e 5. (21) (22) (27) (28) F i g u r e 4 A l t h o u g h t h e r e has b e e n no d i r e c t p r o o f o f W e n k e r t ' s p r e p h e n i c a c i d t h e o r y , i t s h o u l d be n o t e d t h a t t h e c a r b o n s k e l e t o n o f t h e SPF u n i t (9) i s i d e n t i c a l , e x c e p t f o r one c a r b o n a t o m , w i t h t h a t p r o p o s e d i n t h e m o n o t e r p e n e h y p o t h e s i s (32) o u t l i n e d b e l o w . -92-(26) Figure 5 -93-The M o n o t e r p e n e H y p o t h e s i s T h o m a s 4 and W e n k e r t ^ n o t e d t h e s t r u c t u r a l s i m i l a r i t y between t h e " n o n - t r y p t o p h a n " p o r t i o n o f i n d o l e a l k a l o i d s and m o n o t e r p e n i c g l u c o s i d e s , e . g . v e r b e n a l i n (27 )"*4 and 53 g e n i p i n (28) and t h e r e f o r e p o s t u l a t e d a m o n o t e r p e n e o r i g i n f o r t h e " n o n - t r y p t o p h a n " p o r t i o n o f i n d o l e a l k a l o i d s . As shown i n F i g u r e 6, t h e C^Q monoterpene u n i t (31) c l e a v e d a l o n g t h e d o t t e d l i n e p r o v i d e s a c a r b o n s k e l e t o n v e r y s i m i l a r t o t h e SPF u n i t (9 ) . T h i s s k e l e t o n (32) and two o t h e r s o b t a i n e d by r e a r r a n g e m e n t s (33) and (34) a r e shown by t h e t h i c k e n e d bonds i n a j m a l i c i n e (12) , c a t h a r a t h i n e (5) and v i n d o l i n e (4 ) , r e s p e c t i v e l y . (33) (34) F i g u r e 6 -94-The t r a n s f o r m a t i o n r e a c t i o n s r e q u i r e d to produce these r e a r r a n g e d s k e l e t o n s and the f i n a l a l k a l o i d s a r e s i m i l a r to those e l a b o r a t e d i n the p r e p h e n i c a c i d h y p o t h e s i s . The e s s e n t i a l d i f f e r e n c e between the two t h e o r i e s b e i n g i n the o r i g i n o f the i n i t i a l C^Q u n i t , and i n e x p l a i n i n g the d e r i v a t i o n o f yoh imb ine- t ype a l k a l o i d s . In the p r e p h e n i c a c i d t heo r y t hese appear a t the v e r y b e g i n n i n g whereas i n the monoterpene h y p o t h e s i s they a r e a t the v e r y end o f b i o g e n e t i c deve lopment . B i o s y n t h e s i s o f the "Non-Tryp tophan " P o r t i o n o f I ndo l e A l k a l o i d s (Yohimbe-, Co r ynan the- , Asp idope rma- , Iboga-type ) Recent t r a c e r exper iments by s e v e r a l groups have p r o v i d e d v e r y s t r o n g e v i dence f o r a monoterpene o r i g i n . Meva lona te ( 3 0 ) , a known p r e c u r s o r o f t e r p e n e s , has been shown to be i n c o r p o r a t e d i n t o the " n o p - t r y p t o p h a n " p o r t i o n o f v i n d o l i n e ( 4 ) 6 ' 7 ' 8 , s e r p e n t i n e ( 3 ) 6 ' 1 0 , c a t h a r a n t h i n e ( 5 ) 6 ' 1 , 2-dehyd roasp idospe rm id ine ( 3 5 ) 6 , a j m a l i c i n e ( 1 2 ) 6 ' 9 g and r e se rpene (6) . A monoterpene , g e r a n i o l ( 2 9 ) , was shown to be a p r e c u r s o r o f r e p r e s e n t a t i v e examples o f the Co r ynan the , Iboga and Asp idosperma groups o f b a s e s 9 ' ^ ' 1 1 . B a t t e r s b y and c o - w o r k e r s 1 ^ r e p o r t e d the i n c o r p o r a t i o n o f [2- 1^c) g e r a n i o l i n t o a j m a l i c i n e ( 12 ) , s e r p e n t i n e ( 3 ) , c a t h a r a n t h i n e 12 ( 5 ) , v i n d o l i n e (4), and r a d i o a c t i v e l o g a n i n (37) i n t o c a t h a r a n t h i n e ( 5 ) , v i n d o l i n e (4), p e r i v i n e ( 36 ) , s e r p e n t i n e (3) and a j m a l i c i n e (12) i n V i n c a r o s e a p l a n t s . -95-(37) Figure 7 Independently, geraniol (39) was reported to be incorpo-rated into the "non-tryptophan" portion of vindoline (4) by Scott and co-workers 1 1 and also by Arigoni and co-q workers i n Vinca.rosea plants. Degradation of the radio active a l k a l o i d s f u l l y supported the biogenetic scheme shown i n Figure 6. Biogenetic-type Reactions Leading to Iboga- and  Aspidosperma-alkaloids The i n v i t r o conversion of 9-membered r i n g i n t e r -mediates to the Iboga and Aspidosperma skeletons (see compounds (16) and (18) i n Figure 3) by transannular c y c l i z a t i o n reaction was demonstrated by Kiitney and co-workers 2 5 » 2 6 ' 2 7 ' 2 8 . As Figure 8 i l l u s t r a t e s , the Aspidosperma skeleton present i n both 7-ethyl-5-desethyl-F i g u r e 8 - 9 7 -Figure 9 -98-2 5 26 a s p i d o s p e r m i d i n e (39r J and (+) - a s p i d o s p e r m i d i n e (41) was c o n s t r u c t e d f rom d i h y d r o c l e a v a m i n e (38) and (-)-quebrachamine ( 4 0 ) , r e s p e c t i v e l y . Carbomethoxyd ihydro c l eavamine (42) was a l s o i nduced to c y c l i z e to y i e l d 27 28 b o t h the Asp idosperma- and Iboga-type a l k a l o i d s ( F i g u r e 9 ) . M e r c u r i c a c e t a t e o x i d a t i o n o f carbomethoxy-d i h y d r o c l e a v a m i n e (42) to a m i x t u r e of im in ium s a l t s (43) and ( 44 ) , f o l l o w e d by t r a n s a n n u l a r c y c l i z a t i o n , p r o v i d e d p s e u d o - v i n c a d i f f o r m i n e (45) and the Iboga d e r i v a t i v e s ( 4 6 ) . Bo th c o r o n a r i d i n e (46 , @> -Et a t C-4) and d i h y d r o c a t h a r a n t h i n e (46 , o(-Et a t C-4) were i s o l a t e d s i n c e i s o m e r i z a t i o n a t C-4 v i a the enamine (47) o c c u r s d u r i n g the r e a c t i o n . T h i s t r a n s a n n u l a r c y c l i z a t i o n was thus shown to be a v e r s a t i l e s y n t h e t i c e n t r y to the Asp idosperma and Iboga a l k a l o i d s . A B i o g e n e t i c Theory o f V o b a s i n e - , S a rpag ine- and A j m a l i n e - type Bases I t i s q u i t e obv ious f rom s t r u c t u r a l s i m i l a r i t i e s , t ha t the c o r y n a n t h e n o i d s k e l e t o n c o u l d be r e l a t e d to p e n t a c y c l i c a l k a l o i d s such as s a r p a g i n e (48) and then to h e x a c y c l i c bases such as a j m a l i n e ( 2 ) . I n t e r c o n v e r s i o n between s a r p a g i n e - and a j m a l i n e - l i k e bases seemed to be p o s s i b l e i n the p l a n t and t h i s i n t e r c o n v e r s i o n was s i m u l a t e d i n a l a b o r a t o r y as w i l l be d i s c u s s e d l a t e r . However i t can r e a d i l y be seen t he r e e x i s t a t l e a s t two p o s s i b l e r o u t e s f o r the b i o g e n e s i s o f s a r p a g i n e - l i k e bases f rom a c o r y n a n t h e i n e - t y p e m o l e c u l e „ .99 = Figure 10 -100-As shown i n F i g u r e 10, i s o s i t s i r i k i n e ( 4 9 ) 1 7 c o u l d be a p p r o p r i a t e l y o x i d i z e d to the im in ium s a l t ( 5 0 ) and the subsequent a t t a c k o f an a n i o n a t C-16 v i a t r a n s a n n u l a r c y c l i z a t i o n would l e a d to akuammidine ( 5 1 ) . Q u a t e r n i z a t i o n 13 o f akuammidine (51) , f o l l o w e d by o x i d a t i o n a t C-3 and r i n g open ing c o u l d g i v e r i s e to a 2 - a c y l i n d o l e a l k a l o i d , v i n c a d i f f i n e ( 5 4 ) 1 5 . A l t e r n a t i v e l y , b i o g e n e s i s o f a 2 - a c y l i n d o l e base f o l l o w e d by r i n g c l o s u r e to a s a r p a g i n e - t y p e base can be v i s u a l i z e d . The f o r m a t i o n o f 3 ,4-seco base (52) f rom i s o s i t s i r i k i n e (49) f o l l o w e d by a t r a n s a n n u l a r c y c l i z a t i o n o f the im in ium s a l t (53) c o u l d y i e l d v i n c a d i f f i n e ( 54 ) , w h i c h , upon r i n g c l o s u r e , c o u l d f u r n i s h a s a r p a g i n e - t y p e base ( 5 1 ) . The d i r e c t b i o g e n e t i c r e l a t i o n s h i p between these c l a s s e s o f a l k a l o i d s draws suppor t f rom the obse r ved c o - o c c u r r e n c e o f the s a r p a g i n e - t y p e ( i . e . a f f i n i s i n e (55) ) and o f the v o b a s i n e - t y p e ( i . e . a f f i n i n e (56) ) a l k a l o i d s i n the same p l a n t , as r e p o r t e d by Cava e t a l H C HiO H HoHtc H As f a r as the f e e d i n g exper iments a r e c o n c e r n e d , r a d i o a c t i v e l o g a n i n (37) was r e p o r t e d bo be s p e c i f i c l y i n c o r p o r a t e d i n t o p e r i v i n e (36) by B a t t e r s b y and co-workers 12 - 1 0 1 -Some R e a c t i o n s o f B i o g e n e t i c I n t e r e s t I n S a r p a g i n e - , V o b a s i n e - and A j m a l i n e - t y p e B a s e s A c o n s i d e r a b l e amount o f work has b e e n done on l a b o r a -t o r y s y n t h e s i s o f t h e s e t y p e s o f i n d o l e a l k a l o i d s . C o r r e l a t i o n s between s a r p a g i n e - and a j m a l i n e - t y p e b a s e s 22 have b e e n t r i e d by B a r t l e t t e t a l , who r e p o r t e d t h a t d e o x y a j m a l a l - A ( 5 7 ) , u p o n t r e a t m e n t w i t h s t r o n g a c i d f o l l o w e d by r e d u c t i o n , gave d e o x y a j m a l i n e ( 5 8 ) . T h i s r i n g c l o s u r e was a l s o s u p p o r t e d by t h e c o n v e r s i o n o f t o s y l a t e d d e o x y -a j m a l o l - A (60) t o 2 - h y d r o x y d i d e o x y a j m a l i n e (61) a t room t e m p e r a t u r e ( F i g u r e 1 1 ) . F i g u r e 11 - 1 0 2 -23 A s i m i l a r r i n g c l o s u r e was r e p o r t e d by M a r t i n e t a l i n t h e c a s e o f v o a c h a l o t i n e ( 6 2 ) . M e t h a n e s u l f o n y l c h l o r i d e , p - t o l u e n e s u l f o n y l c h l o r i d e and t h i o n y l c h l o r i d e r e a c t e d w i t h v o a c h a l o t i n e (62) t o g i v e , i n e a c h c a s e , t h e same c y c l i z e d d i h y d r o i n d o l e compound: 2 - h y d r o x y - 1 7 - d e o x y -v i n c a m a j i n e (63) ( F i g u r e 1 2 ) . ( 6 2 ) , R=H R = M e s , T s , C l S O (63) F i g u r e 12 The r e v e r s e r e a c t i o n i s a l s o p o s s i b l e and has b e e n u t i l i z e d foV«*fche d e g r a d a t i o n o f a j m a l i n e - l i k e a l k a l o i d s . As shown i n F i g u r e 13, o x i d a t i o n o f v i n c a m a j i n e w i t h chromium t r i o x i d e o r l e a d t e t r a a c e t a t e i n p y r i d i n e gave a n i n d o l e a l d e h y d e ( 6 6 ) , w h i c h * o n r e d u c t i o n f u r n i s h e d t h e h y d r o x y e s t e r , v o a c h a l o t i n e (62) A t t e m p t s have a l s o b e e n made t o c o r r e l a t e d i r e c t l y between t h e p o t e n t i a l l y r e l a t e d p e n t a c y c l i c s a r p a g i n e - and t e t r a c y c l i c 2 - a c y l i n d o l e t y p e s . F o r e x a m p l e , t h e c o n v e r s i o n o f p e r i v i n e (36) i n t o n o r m a c u s i n e B (69) was r e p o r t e d by 31 Gorman e t a l ( F i g u r e 1 4 ) . - 1 0 3 -F i g u r e 13 P e r i c y c l i v i n e was i s o l a t e d d u r i n g t h e p h y t o c h e m i c a l 32 i n v e s t i g a t i o n s o f G a b u n i a o d o r a t i s s i m a and C a t h a r a n t h u s  l a n c e u s and f o u n d t o have t h e s t r u c t u r e ( 6 7 ) , a compound p r e v i o u s l y o b t a i n e d f r o m p e r i v i n e ( 3 6 ) , F i g u r e 14 -104-A s i m i l a r r i n g c l o s u r e was a c c o m p l i s h e d i n the case o f p i c r a p h y l l i n e , a t e t r a c y c l i c 2-acy i ndo l e compound which was i s o l a t e d f rom P i c r a l i m a n i t i d a . The c o n v e r s i o n o f t h i s a l k a l o i d to t e t r a h y d r o a l s t o n i n e c o n f i r m e d the s t r u c t u r e and 35 36 s t e r o c h e m i s t r y o f the fo rmer ' . R e d u c t i o n o f p i c r a p h y l l i n e (71) w i t h p o t a s s i u m b o r o h y d r i d e p roduces an equ imo l a r m i x t u r e o f e p i m e r i c p i c r a p h y l l i n o l s whose N^-methoch lo r ides were p r epa red i n the u s u a l manner. P y r o l y s i s o f the m e t h o c h l o r i d e a t 280° y i e l d e d a s i n g l e compound i d e n t i f i e d as t e t r a h y d r o -a l s t o n i n e ( 72 ) . I t i s o f i n t e r e s t t h a t N ^ - m e t h y l t e r t r a h y d r o a l s t o n i n e 37 ( m e l i n o n i n e A ) was a l s o i s o l a t e d f rom t h i s p l a n t spec imen . As d i s c u s s e d p r e v i o u s l y (see F i g u r e 10, 4 9 * 5 2 ) , the r e v e r s e r e a c t i o n ( 72*71 ) , c o u l d a l s o be o f s i g n i f i c a n c e f o r the b i o g e n e s i s o f 2 - a c y l i n d o l e a l k a l o i d s (70) and ( 71 ) . 39 Do lby and Saka i s u c c e s s f u l l y demonst ra ted the c o n v e r s i o n o f d i h y d r o c o r y n a n t h e i n e (73) to a t r i c y c l i c 2 - a c y l i n d o l e , - 1 0 5 -d i h y d r o b u r n a m i c i n e ( 7 7 ) . As shown i n F i g u r e 15, d i h y d r o -c o r y n a n t h e i n e (73) was c o n v e r t e d t o 7 - a c e t o x y - 7 H - d i h y d r o -c o r y n a n t h e i n e by t h e a c t i o n o f l e a d t e t r a a c e t a t e . The m e t h i o d i d e (74) o f t h e a c e t o x y i n d o l e n i n e was t r e a t e d w i t h b o i l i n g a c e t i c a c i d c o n t a i n i n g s o d i u m a c e t a t e . E x t r a c t i o n f r o m s t r o n g l y a l k a l i n e s o l u t i o n gave 3 - k e t o - 3 , 4 - s e c o - N ^ -m e t h y l - d i h y d r o c o r y n a n t h e i n e ( 7 5 ) , w h i c h u p o n h y d r o l y s i s f o l l o w e d by d e c a r b o x y l a t i o n gave t h e k e t o a l d e h y d e ( 7 6 ) . F i g u r e 15 R e d u c t i o n w i t h s o d i u m b o r o h y d r i d e i n aqueous m e t h a n o l y i e l d e d d i h y d r o b u r n a m i c i n e ( 7 7 ) . T h i s s y n t h e s i s by D o l b y and S a k a i was t h e f i r s t r e p o r t e d a t t e m p t t o o b t a i n t h e -106-(83) Figure 16 - 1 0 7 -39 2-acy l mo ie t y i n i n d o l e a l k a l o i d s . The same au tho r s a l s o r e p o r t e d the f o l l o w i n g sequence summarized i n F i g u r e 16 . Compounds (78a) and (78b ) , d e r i v e d f rom n a t u r a l d i h y d r o - c o r y n a n t h e i n e , on t r ea tment w i t h a c e t i c a n h y d r i d e -sodium a c e t a t e y i e l d e d the c o r r e s p o n d i n g a c e t o x y l a c t a m s (79a) and ( 79b ) . S a p o n i f i c a t i o n o f (79a) y i e l d e d the a l c o h o l ( 8 0 ) , wh i ch r e v e r t e d to (79a) upon a c e t y l a t i o n o r was c o n v e r t e d to the 2 - a c y l i n d o l e (81) on o x i d a t i o n w i t h manganese d i o x i d e . At tempted p r e p a r a t i o n o f a t r i c y c l i c base by h y d r o l y s i s o f the l a c t am (81) was not s u c c e s s f u l . Ve ry r e c e n t l y , Do lby and G r i b b l e r e p o r t e d o t h e r ways 52 o f s y n t h e s i z i n g 2 - a c y l i n d o l e s . They have s t u d i e d the c o n v e r s i o n o f the t e t r a c y c l i c base (82) i n t o the t r i c y c l i c ke tone (83) by two independent r o u t e s . One r o u t e i s s i m i l a r to the sequence l e a d i n g to the s y n t h e s i s o f d i h y d r o -39 bu rnam i c i ne (77) and the o t h e r i n c l u d e s the d i r e c t o x i -d a t i o n o f a 10-membered r i n g ( 3 ,4-seco base ), The key p r o d u c t o f the f i r s t sequence i s an e p i m e r i c m i x t u r e o f @ - c h l o r o i n d o l e n i n e s wh ich were a l k y l a t e d w i t h methy l i o d i d e to form m e t h i o d i d e s . The m e t h i o d i d e m i x t u r e was t r e a t e d w i t h sodium a c e t a t e i n aqueous e t h a n o l and then b a s i f i e d w i t h sodium h y d r o x i d e to g i v e the d e s i r e d t r i c y c l i c ke tone ( 83 ) . The second r o u t e i n c l u d e d B i r c h r e d u c t i o n on the m e t h i o d i d e o f t e t r a c y c l i c base (82) to f u r n i s h 3 ,4-seco base wh ich was then s u b j e c t e d to p e r i o d i c a c i d o x i d a t i o n to g i v e the t r i c y c l i c ke tone ( 83 ) . T h i s o x i d a t i o n i s a t t r a c t i v e f rom the s y n t h e t i c p o i n t o f v i ew , s i n c e the 108-i n t r o d u c t i o n of a r e a c t i v e ketone f u n c t i o n can be achieved i n the l a t e r stage of the s y n t h e s i s of 2 - a c y l i n d o l e a l k a l o i d s , 40 Recently Harley-Mason et a l r e p o r t e d b r i e f l y a r e -a c t i o n which introduced an a c e t o x y l at C-3 of the hexa-- h y d r o i n d o l o p y r r o c o l i n e (84). As shown i n F i g u r e 17, the i a c e t o x y l was converted to a ketone b y j m i l d a l k a l i n e h y d r o l y s i s f o l l o w e d by manganese d i o x i d e o x i d a t i o n . F i g u r e 17 A s i m i l a r cleavage by a c e t i c anhydride was a p p l i e d to the p r e p a r a t i o n of some indolobenzazonines (85, 86) by F r e t e r et a l , a f t e r they observed t h a t treatment of l - p h e n y l - 2 - m e t h y l t e t r a h y d r o - - c a r b o l i n e (87) w i t h a c e t i c 41 anhydride gave the 1,2-seco compound (88) ( F i g u r e 18). In a d d i t i o n to these syntheses of 3-keto-3,4-seco bases, p r e p a r a t i o n s of 9- and 10-membered r i n g compounds by a cleavage of the C/D r i n g are of p a r t i c u l a r i n t e r e s t from a s y n t h e t i c p o i n t of view. Hence, the methods now F i g u r e 18 a v a i a b l e a r e r e v i e w e d b e l o w , Wenkert and c o - w o r k e r s 4 " ^ d e v e l o p e d a u n i q u e s y n t h e s i s o f a q u e b r a c h a m i n e model (90) by B i r c h r e d u c t i o n o f t h e i n d o l o p y r r o c o l i n e s y s t e m ( 8 9 ) . - 1 1 0 -LL. / NH3 (89) (90) D o l b y and B o o t h ^ r e p o r t e d t h e C/D r i n g c l e a v a g e o f t h e o c t a h y d r o - 2 - h y d r o x y i n d o l o p y r i d o c o l i n e (91) by l i t h i u m aluminum h y d r i d e r e d u c t i o n o r by B i r c h r e d u c t i o n t o f u r n i s h a 10-membered r i n g compound ( 9 2 ) . ( K (92) H e r c h e l S m i t h and co-workers^"* i n d e p e n d e n t l y s t u d i e d t h e m e t a l - a m m o n i a r e d u c t i o n o f h e x a h y d r o - 4 - m e t h y l i n d o l o -i n d o l i z i n i u m i o d i d e (93) and o c t a h y d r o - 5 - m e t h y l i n d o l o -q u i n o l i z i n i u m i o d i d e (94) i n g r e a t d e t a i l . Optimum y i e l d s were o b t a i n e d by u s i n g s l i g h t l y more t h a n 2 e q u i v . o f l i t h i u m and 1 mole o f l - m e t h o x y - 2 - p r o p a n o l i n l i q u i d ammonia ( F i g u r e 1 9 ) . (93) , n = l (R=H,Me) (94) ,n=2 (R=H,Me) L i /HM 3 O H F i g u r e 19 N /He "I - I l l -Figure 20 -112-The a p p l i c a t i o n o f t h e m e t a l - a m m o n i a r e d u c t i o n s m e n t i o n e d above was t h e i m p o r t a n t k e y t o t h e t o t a l s y n t h e s e s 29 o f s e v e r a l a l k a l o i d s , q u e b r a c h a m i n e ( 3 9 ) , d i h y d r o -c l e a v a m i n e 3 0 , 4 6 (^28) and c a r b o m e t h o x y d i h y d r o c l e a v a m i n e ^ ' 4 ^ (42) ( F i g u r e 2 0 ) . I n t r o d u c t i o n o f a carbomethoxy g r o u p a t C - 3 was a c h i e v e d by t r e a t m e n t o f t h e q u a t e r n a r y m e s y l a t e (95) w i t h p o t a s s i u m c y a n i d e f o l l o w e d by h y d r o l y s i s o f t h e n i t r i l e w i t h 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 t o g i v e ( 4 2 ) . O t h e r p o s s i b l e ways o f p r e p a r i n g 3 , 4 - s e c o - and 3 -k e t o - 3 , 4 - s e c o - c o r y n a n t h e n o i d b a s e s c o u l d come from a p p l i -c a t i o n s o f c a r b o n - n i t r o g e n h y d r o g e n o l y s i s o f a l l y l i c 48 q u a t e r n a r y s a l t s r e p o r t e d by H a r l e y - M a s o n e t a l o r f r o m a p p l i c a t i o n o f a r e a c t i o n s i m i l a r t o t h e r i n g o p e n i n g o f a 1 - a z a b i c y c l o - a l k a n e t o a 9-membered r i n g amine as d e m o n s t r a t e d by R e i n e c k e e t a l 5 ^ . r>J Me-(97) ( 9 6 ) , R = H , 0 H = / \ \ (98) F i g u r e 21 - 1 1 3 -48 H a r l e y - M a s o n e t a l r e p o r t e d t h a t t h e h y d r o g e n a t i o n o f a g r o c l a v i n e m e t h i o d i d e ( 9 6 , R s H ) and e l y m o c l a v i n e m e t h i o d i d e ( 8 8 , R s O H ) o v e r Adam's c a t a l y s t gave t h e s e c o -compound ( 9 7 , R=H and R r O H , r e s p e c t i v e l y ) a f t e r two m o l e c u l e s o f h y d r o g e n had b e e n s m o o t h l y a b s o r b e d . As shown i n F i g u r e 21, n o t o n l y t h e r e d u c t i o n o f t h e d o u b l e bond b u t a l s o a l l y l i c c a r b o n - n i t r o g e n h y d r o g e n o l y s i s h a d o c c u r r e d . However, a n Emde r e d u c t i o n o f a g r o c l a v i n e and e l y m o c l a v i n e m e t h i o d i d e s w i t h s o d i u m and l i q u i d ammonia t o f u r n i s h ( 9 8 , R r H and R s O H , r e s p e c t i v e l y ) was r e p o r t e d by 49 B i r c h and c o - w o r k e r s I t s h o u l d be n o t e d t h a t h y d r o g e n o l y s i s o f N - b e n z y l d e r i v a t i v e s t o g e n e r a t e t h e c o r r e s p o n d i n g b a s e s (NH) i s w e l l known. The c a t a l y t i c r e d u c t i o n r e p o r t e d by F r e t e r e t a l 4 1 ( F i g u r e 22) a p p e a r s t o be t h e f i r s t example where t h e n i t r o g e n atom and t h e b e n z y l i c bond a r e i n t h e same 6-membered r i n g . F i g u r e 22 T h i s h y d r o g e n o l y s i s c o u l d be a p p l i e d t o t h e p r e p a r a t i o n o f 3 , 4 - s e c o compounds i n a p p r o p r i a t e i n d o l e a l k a l o i d s . -114-R e i n e c k e e t a l i n v e s t i g a t e d t h e s y n t h e s i s o f t h e 9-membered r i n g a m i n o k e t o n e ( 1 0 6 ) . (101) (102) (103) (104) (105) (106) F i g u r e 23 As shown i n F i g u r e 2 3 , r e a c t i o n o f b e n z y l magnesium b r o m i d e w i t h t h e i m i n i u m s a l t ( 1 0 2 ) , o b t a i n e d by m e r c u r i c a c e t a t e o x i d a t i o n o f t h e 1 - a z a b i c y c l o a l k a n e ( 1 0 1 ) , l e d t o t h e t e r t i a r y amine (103) whose q u a t e r n a r y s a l t (104) underwent a n i n t e r n a l e l i m i n a t i o n t o g i v e t h e 9-membered r i n g amine ( 1 0 5 ) . The o x i d a t i o n (OsO^-NalO^) o f t h e amine (105) gave b e n z a l d e h y d e and a n a m i n o k e t o n e ( 1 0 6 ) . T h i s G r i g n a r d r e a c t i o n has b e e n u s e d by Z i n n e s e t al"* ( F i g u r e 24) t o p r e p a r e 3 - b e n z y l y o h i m b a n e ( 1 0 8 ) . M e r c u r i c a c e t a t e o x i d a t i o n o f yohimbane y i e l d e d t h e e x p e c t e d 3 -d e h y d r o y o h i m b a n e s a l t ( 1 0 7 ) . R e a c t i o n o f t h e l a t t e r w i t h b e n z y l m a g n e s i u m b r o m i d e gave 3 - b e n z y l y o h i m b a n e (108) as t h e m a j o r p r o d u c t . The m e t h i o d i d e o f 3 - b e n z y l y o h i m b a n e would be expected to give a 10-membered r i n g upon t r e a t -ment with base i n analogy with Reinecke's sequence and subsequent oxidation (OsO^-NalO^) could f u r n i s h 3-keto-3,4-seco-yohimbane. Figure 24 It can be seen that although several groups of workers have investigated some of the reactions described i n Figure 10, no reports concerned with bridge formations between C-15 and C-5 v i a transannular c y c l i z a t i o n s have as yet appeared. Therefore i t was of great i n t e r e s t to investigate the p o s s i b i l i t y of transannular c y c l i z a t i o n s on appropriate corynanthenoid-type bases including the 3,4-seco d e r i v a t i v e s . These reactions would be of i n t e r e s t not only as biosynthetic models but also as a part of possible synthetic approaches to sarpagine- and vobasine-type a l k a l o i d s . This part of the thesis w i l l mainly be concerned with the synthesis of c o r y n a n t h e i n e - l i k e bases, the generation of iminium double bonds between and C-5 (intermediates (50) and (53) i n Figure 10) and with some attempted trasannular c y c l i z a t i o n s . -116-DISCUSSION The successful transannular c y c l i z a t i o n s c a r r i e d out 25 26 27 28 by Kutney and co-workers ' ' ' providing a general synthetic entry into Iboga and Aspidosperma a l k a l o i d s , prompted us to attempt chemical transformations of cory-nanthenoid - l i k e a l k a l o i d s to sarpagine- and vobasine-type bases. This part of the thesis w i l l be concerned with three d i f f e r e n t approaches to the r i n g closure of cory-nanthenoid- l i k e a l k a l o i d s and these are summarized b r i e f l y below. a) Oxidation of corynanthenoid bases by mercuric acetate i s known to give predominantly the corresponding 3-dehydro-iminium s a l t (109), which might be i n equilibrium with the 5- dehydro- (110) and the 21-dehydroiminium s a l t (111). I f a properly generated anion at C-16 would attack the iminium s a l t to accomplish a r i n g closure v i a a transannular c y c l i z a t i o n , bond formation between C-16 and C-5 would r e s u l t , g i v i n g the base (112). As shown i n Figure 25, r other possible bond formations (109 and 111) would y i e l d 4-membered rings , which would be more strained than the 6- membered r i n g (112). b) A second approach (Figure 26) to the synthesis of a properly oxidized compound was to block C-3 with a benzyl group i n order to prevent the formation of a 3-dehydro -117-d e r i v a t i v e , wh i ch i s known to be the major p r o d u c t i n the case of ( a ) . The b e n z y l group a l s o might p r o v i d e a c o n -v e n i e n t e n t r y i n t o the 2-acy l i n d o l e f a m i l y as d e s c r i b e d p r e v i o u s l y ( F i g u r e 2 3 ) . F i g u r e 25 c ) The t h i r d app roach i n v o l v e d the C/D r i n g c l e a v a g e o f a c o r y n a n t h e n o i d base under B i r c h r e d u c t i o n c o n d i t i o n s to g i v e the c o r r e s p o n d i n g 3 ,4-seco b a s e , wh ich c o u l d be o x i -d i z e d , by m e r c u r i c a c e t a t e , to im in ium s a l t s (119) and ( 120 ) . Subsequent a t t a c k o f an a n i o n a t C-16 o f the fo rmer (119} would g i v e r i s e to a b r i d g e d compound whose s k e l e t o n i s the same as t h a t o f a t y p i c a l v o b a s i n e - t y p e a l k a l o i d ( F i g u r e 2 7 ) . -118-6. Figure 27 -119-(a) Attempted Transannular C y c l i z a t i o n s of Corynantheine-l i k e Bases The f i r s t attempt of t h i s type was made on s i t s i r i k i n e 17 62 (121) by G. Eigendorf . As summarized i n Figure 28, mercuric acetate oxidation of s i t s i r i k i n e (121) i n a c e t i c acid gave predominantly a 3-dehydro d e r i v a t i v e (122) as 62 shown by the UV spectrum (125) Figure 28 - 1 2 0 -Prolonged s t i r r i n g of the mixture and subsequent heat-ing f o r 6 hours gave i n t r a c t a b l e mixtures ( i . e . a t a r under fo r c i n g conditions). I t was concluded that the mercuric acetate oxidation gave mainly the expected iminium s a l t (122) and that the r i n g closure beween C-3 and C-16 ( r e s u l t -ing i n a 4-membered ring) was not favorable. The extent of formation of the iminium s a l t s (123) and (124) e i t h e r by d i r e c t oxidation or by e q u l i b r a t i o n with the 3-dehydro -121-derivative (122) was i n s u f f i c i e n t to detect any of the c y c l i z e d base (125). In the case of s i t s i r i k i n e (121), the t e r t i a r y proton on C-16 might not be e a s i l y abstracted and t h i s way provide an a l t e r n a t i v e explanation for the f a i l u r e of the r i n g closure reaction. Dihydrocorynantheic acid ethyl ester (126) was also investigated with regard to the above reaction. When t h i s compound was subjected to mercuric acetate oxidation, the desired r i n g closure was again not successful. In a t y p i c a l experiment which involved s t i r r i n g the reactants at room temperature f o r 75 hours, a complex mixture of more than 14 compounds (as shown by TLC) was obtained. (b) Attempted Ring Closures of Corynanthenoid Bases with  4 Substituent at C-3 As discussed previously, introduction of a benzyl group at C-3 of yohimbane was achieved by Zinnes et a l " * 1 , although no NMR or mass spectral data on the 3-benzyl d e r i v a t i v e were reported. Since yohimbine was r e a d i l y a v a i l a b l e i n quantity, t h i s compound was used to investigate i n d e t a i l , the introduction of a benzyl group into the 3-position. Preparation of 3-dehydroyohimbine perchlorate had been 58 reported previously by Weisenborn and D i a s s i and t h i s reaction was r e a d i l y repeated to give a yellow c r y s t a l l i n e s a l t (131) i n good y i e l d (Figure 29). Treatment of 3-dehydroyohimbine perchlorate (131) with a large excess of benzyl magnesium bromide i n ether, as -122-reported by Zinness et a l " * 1 , gave e s s e n t i a l l y a single 3-benzyl d e r i v a t i v e i n 40% y i e l d a f t e r chromatographic separation. The UV spectrum of the Grignard product showed an indole chromophore and the IR spectrum indicated the bands f o r hydroxyl and methyl ester groups at 3333 and 1709 cm"1, re s p e c t i v e l y . I t was therefore immediately obvious that the introduction of a 3=benzyl group could be accomplished without destroying the methyl ester function at C-16. Yohimbine(10) Figure 29 -123-The mass s p e c t r u m showed a weak m o l e c u l a r i o n a t m/e 444 (1.9%) and a b a s e peak a t m/e 353, p r o b a b l y due t o l o s s o f t h e b e n z y l g r o u p (M-91). The NMR s p e c t r u m ( F i g u r e 30) c o n f i r m e d t h e p r e s e n c e o f a carbomethoxy g r o u p (?~6.25), i n d o l e NH (^3.46) and n i n e a r o m a t i c p r o t o n s i n t h e r e g i o n , £2.4-3.2. A d e u t e r i u m exchange e x p e r i m e n t d e m o n s t r a t e d a s l o w exchange o f t h e b r o a d s i n g l e t a t 7:3.46 (30 m i n . t o c o m p l e t i o n ) . The c h e m i c a l s h i f t o f t h e i n d o l e NH i n t h i s compound when compared t o t h e i n d o l e NH r e s o n a n c e i n y o h i m b i n e ( a t 7:2.21) must be due t o t h e s h i e l d i n g e f f e c t o f t h e a r o m a t i c r i n g o f t h e 3 - b e n z y l s u b s t i t u e n t . A l l t h e p h y s i c a l d a t a s u p p o r t e d t h e s u c c e s s f u l i n t r o -d u c t i o n o f a 3 - b e n z y l g r o u p i n t o y o h i m b i n e , e i t h e r f r o m t h e at- o r f^>-side o f t h e m o l e c u l e . Z i n n e s e t a l 5 1 s u g g e s t e d i n c o n c l u s i v e l y , o n t h e b a s i s o f ORD and IR s t u d i e s , t h a t 3 - b e n z y l y o h i m b a n e (108) p o s s e s s e d a 3 oU b e n z y l g r o u p . I n t h e c a s e o f 3 - b e n z y l y o h i m b i n e , t h e NMR s p e c t r a l d a t a s u g g e s t s t h a t t h e 3 - b e n z y l g r o u p i s i n t h e o r i e n t a t i o n (132). M o l e c u l a r m o d e l s o f 3d- and 3@<=benzylyohimbine i n d i c a t e d t h a t o n l y t h e 3d - b e n z y l g r o u p c o u l d e x t e n s i v e l y s h i e l d t h e i n d o l e NH as was s u g g e s t e d f r o m i t s NMR s p e c t r u m . I t was a l s o s e e n t h a t t h e 3o(-benzyl g r o u p was i n a t r a n s d i a x i a l o r i e n t a t i o n t o t h e l o n e p a i r o f e l e c t r o n s on t h e n i t r o g e n (N^) when t h e m o l e c u l e e x i s t e d i n t h e most f a v o r a b l e c o n f o r m a t i o n . A p a r t f r o m t h e above compound, a s l i g h t l y more p o l a r s p o t w i t h a s i m i l a r c o l o r was d e t e c t e d on TLC (3^ - b e n z y l -125-d r i v a t i v e ? ) , but i n s u f f i c i e n t quantities were a v a i l a b l e f o r complete c h a r a c t e r i z a t i o n . This more polar component was obtained as one of the major products when the Grignard reaction was done i n tetrahydrofuran, as w i l l be discussed below. The y i e l d of 3-benzylyohimbine from the Grignard reaction i n ether as solvent was low, possibly because the intermediate complex forms an insoluble layer around the yohimbine perchlorate and prevents further reaction. The use of a more polar solvent such as tetrahydrofuran would a l l e v i a t e t h i s d i f f i c u l t y since i t i s known that most Grignard intermediate complexes are soluble i n t h i s solvent. Indeed when f i n e l y powdered 3-dehydroyohimbine perchlorate (131) was added to benzyl magnesium bromide i n tetrahydro-furan, s o l u t i o n occurred immediately. Since the perchlorate (131) i s not soluble i n tetrahydrofuran, i t was apparent that reaction must have taken place i n s t a n t l y . The UV spectrum of the crude Grignard products was recorded and indicated the presence of not only an indole chromophore (Amaxs 283, 291 mu) but also a considerable amount of a 3-dehydro base (^maxs 320 mu). After a d d i t i o n of one drop of concentrated hydrochloric acid to the UV c e l l , the absorption at 320 mu was l o s t and a new peak at 352 mu 52 appeared. This dramatic bathochromic s h i f t (320-^352 mu) i s t y p i c a l of a 3,14-dehydro base (130) being isomerized to a 3-dehydro iminium s a l t (131). These UV spectral studies c l e a r l y show that the Grignard reagent acts as -126-a base to p u l l o f f a proton at C-14 as well as a nucleophile i n attacking the iminium bond at O3o The e x t i n c t i o n co-e f f i c i e n t s of both the indole chromophore (yohimbine) and the 3-dehydro s a l t are known and, therefore, the approximate r a t i o of these components i n the mixture could be c a l c u l a t e d . In t h i s way, the r a t i o of saturated indole d e r i v a t i v e s to the 3-dehydro s a l t was found to be 100 s 11. The crude Grignard product mixture was p u r i f i e d by column chromatography. E l u t i o n with benzene-ether gave two products i n o v e r a l l 60% y i e l d . The r a t i o of the less polar to the more polar material was found, by preparative TLC' on s i l i c a g e l , to be 2 s 1. The less polar component was found to be i d e n t i c a l with the 3c(-benzylyohimbine obtained previously as the major product by the Grignard r e a c t i o n i n ether. The more polar material was i d e n t i c a l , by TLC, with the minor component obtained i n the ether r e a c t i o n . This material showed an indole chromophore i n the UV spectrum and, i n t e r e s t i n g l y , the mass spectra showed a r e l a t i v e l y strong molecular ion peak at m/e 444 (14%) and a base peak at m/e 353 (M-91). The NMR spectrum of the more polar compound (Figure 31) showed a s i m i l a r pattern to that of the less polar material except f o r the chemical s h i f t of the indole NH proton. There was a s i n g l e t f o r the methyl protons of the carbomethoxy group att 6.17, and a ten proton m u l t i p l e t i n the region, 7:2.3-3.2. I t should be noted that i n t h i s -127-RELATIVE INTENSITY RELATIVE INTENSITY 831 -129-compound the chemical s h i f t of the indole NH became i n d i s -tinguishable from that of the expected nine aromatic protons. Therefore, i t appeared that the benzyl group was not exert-ing a sh i e l d i n g e f f e c t on the indole NH as i n the less polar compound. Thus, i t could be concluded from the NMR spectra of these two 3-benzyl derivatives that the less polar material bearing the 3c(-benzyl group shields the indole NH (t3.46) whereas the more polar compound with a 3^-benzyl group has l i t t l e influence. Further evidence to support these proposals could be obtained from the mass spectra of these two isomers. I t i s very w e l l known, from mass spectrometric measure-ments, that yohimbine-, ajmalicine- and corynantheine-type a l k a l o i d s (134) give r i s e to a strong ( M - l ) + peak under electron impact and i t i s speculated that t h i s ( M - l ) + peak 66 i s due to loss of the proton at C-3 For example, the mass spectrum of yohimbine (M.W. 354) showed a large (M-l) peak at m/e 353 . I t i s not sur-p r i s i n g , therfore, that the mass spectra of both 3<^ - (132) and 3(*>-benzylyohimbine (133) showed a base peak at m/e 353 undoubtedly due to loss of the benzyl group as shown i n Figure 33. The benzyl group and the lone p a i r of electrons -130-m/e 353 (100%) F i g u r e 33 on n i t r o g e n (N^) a r e c o n s i d e r e d t o be t r a n s d i a x i a l i n t h e c a s e o f 3o(-benzylyohimbine (132) whereas i n t h e (B>isomer (133) d i h e d r a l a n g l e o f a p p r o x i m a t e l y 60° e x i s t s between them. I t w o u l d t h e r e f o r e be e x p e c t e d t h a t 3o(-benzylyohimbine (132) w o u l d l o s e t h e b e n z y l g r o u p more e a s i l y , u n d e r e l e c t r o n i m p a c t j F i g u r e 33 b e a r s o u t t h i s s u g g e s t i o n . The m o l e c u l a r i o n peak o f 3^ - b e n z y l y o h i m b i n e (132) was o b s e r v e d a t m/e 444 (1.9%) and was f o u n d t o be weaker t h a n t h a t o f 3(3 - b e n z y l --131-y o h i m b i n e (133) (14%). T h e s e o b s e r v a t i o n s a r e i n good a c c o r d w i t h t h e p r e v i o u s s t r u c t u r a l a s s i g n m e n t s . F u r t h e r e l u t i o n o f t h e G r i g n a r d p r o d u c t m i x t u r e w i t h m e t h a n o l gave t h e 3-dehydro b a s e (130) w h i c h was c h a r a c -t e r i z e d as i t s p e r c h l o r a t e s a l t . A l t h o u g h t h e p e n t a c y c l i c a l k a l o i d , y o h i m b i n e , gave two i s o m e r i c 3 -benzyl d e r i v a t i v e s , i t does n o t n e c e s s a r i l y f o l l o w t h a t t e t r a c y c l i c c o r y n a n t h e n o i d - t y p e b a s e s w o u l d * a l s o g i v e two i s o m e r i c 3 -benzyl compounds. However, t h e above s t r u c t u r a l a s s i g n m e n t s i n t h e y o h i m b i n e s e r i e s , b a s e d on NMR and mass s p e c t r a , m i g h t p r o v e t o be u s e f u l f o r s i m i l a r s t r u c t u r a l s t u d i e s on c p r y n a n t h e n o i d d e r i v a t i v e s . A n o t h e r p o s s i b l e b l o c k i n g g r o u p ( a t C-3) w h i c h was c o n s i d e r e d was t h e n i t r i l e g r o u p , s i n c e t h i s f u n c t i o n c o u l d be e a s i l y e l i m i n a t e d by h y d r o l y s i s f o l l o w e d by d e c a r b o x y -l a t i o n . However, 3 -cyanoyohimbine (122) p r e p a r e d a c c o r d -i n g t o t h e p r o c e d u r e r e p o r t e d by Z i h n e s e t a l 5 1 was f o u n d t o be v e r y u n s t a b l e i n s o l u t i o n . L o s s o f h y d r o g e n c y a n i d e o c c u r r e d e v e n i n " s p e c t r o g r a d e " m e t h a n o l w h i l e t h e UV s p e c t r u m was b e i n g r e c o r d e d . P u r i f i c a t i o n o f 3-c y a n o y o h i m b i n e was i m p o s s i b l e due t o t h e u n s t a b l e n a t u r e o f t h i s m a t e r i a l i n s o l u t i o n and t h e NMR s p e c t r u m was a l s o u n o b t a i n a b l e . A l t h o u g h t h e s t e r e o c h e m i s t r y o f 3-cyano-y o h i m b i n e was c o n s i d e r e d t o be s i m i l a r t o t h a t o f t h e 3-b e n z y l d e r i v a t i v e s ( i . e . p r e d o m i n a n t l y 3d-cyano) no p h y s i c a l d a t a was a v a i l a b l e t o s u p p o r t t h i s a s s i g n m e n t . However, t r e a t m e n t o f t h e 3 -cyanoyohimbine (137) w i t h b e n z y l -132-Figure 34 magnesium bromide i n tetrahydrofuran furnished predomi-nantly 3(3—benzylyohimbine, together with a minor amount of the 3d isomer. This r e s u l t suggests that the o r i g i n a l 3-cyanoyohimbine was predominantly the 3ck d e r i v a t i v e since the Grignard reaction occurs by Sn 2 displacement of the cyano group. Before the introduction of the 3-benzyl group into a corynanthenoid-type base was attempted, the r e a c t i v i t y of tetradehydroyohimbine perchlorate (140) with benzyl magnesium bromide was examined (Figure 35). The enamine compound (141) which may be formed i n t h i s reaction could - 1 3 3 -F i g u r e 35 p r o v i d e , u p o n t r e a t m e n t w i t h a c i d , t h e i m i n i u m s a l t ( 1 4 2 ) , a s y s t e m w h i c h w o u l d be v e r y d e s i r a b l e i n t h e c o r y n a n t h e i n e 59 f a m i l y . However, t e t r a d e h y d r o y o h i m b i n e p e r c h l o r a t e (140) d i d n o t r e a c t w i t h b e n z y l magnesium b r o m i d e e v e n u n d e r r e f l u x i n g c o n d i t i o n s i n t e t r a h y d r o f u r a n as s o l v e n t . S i n c e t h e s u c c e s s f u l p r e p a r a t i o n o f 3 - b e n z y l d e r i v a -t i v e s had now b e e n c a r r i e d out on model compounds, t h e i n t r o d u c t i o n o f a b e n z y l g r o u p a t C - 3 o f d i h y d r o c o r y n a n t h e i n e was a t t e m p t e d ( F i g u r e 3 6 ) . The p r e p a r a t i o n o f 3 - d e h y d r o -d i h y d r o c o r y n a n t h e i n e p e r c h l o r a t e (143) was a c h i e v e d by - 1 3 4 -m e r c u r i c a c e t a t e o x i d a t i o n f o l l o w e d by t r e a t m e n t w i t h p e r c h l o r i c a c i d . The 3 - d e h y d r o s a l t (143) r e a c t e d i n s t a n t -l y w i t h b e n z y l magnesium b r o m i d e i n t e t r a h y d r o f u r a n and a f t e r s t i r r i n g was c o n t i n u e d f o r 10 m i n u t e s , t h e u s u a l w o r k - u p o f t h e r e a c t i o n m i x t u r e gave a s i n g l e p r o d u c t , as shown by T L C . The UV s p e c t r u m o f t h e c r u d e p r o d u c t i n d i c a t e d t h e p r e s e n c e o f a 3 - d e h y d r o b a s e (17%). F i g u r e 36 -135-The IR spectrum of the main product ( a f t e r chromatography) showed the retention of the carbomethoxy (1692 cm"1) and the enol ether (1631 cm"1) groups. The mass spectrum showed a weak molecular ion at m/e 458 (1%) and a base peak at m/e 367 (M-91), thereby confirming the successful introduction of a benzyl group at C-3. The NMR spectrum (Figure 37) showed s i n g l e t s at 7:6.27 and 2:6.35 f o r the methyl protons of the cabomethoxy and methyl ether groups, and a one proton m u l t i p l e t at 7:1.40. Upon addition of deuterium oxide, the m u l t i p l e t at 7H.40 disappeared immediately and a slow exchange of the s i n g l e t at£3.40 (30 min. to completion) was observed. The one proton m u l t i p l e t at ^1.40 could be assigned to the o l e f i n i c proton at C-17, being deshielded by the phenyl nucleus of the benzyl group (the o l e f i n i c 6 3 s i g n a l of dihydrocorynantheine appears at 7:2.60 ). The s i n g l e t at 7-3.40 could be assigned to the indole NH, and slow exchange of the indole NH may be due to s t e r i c hindrance from the benzyl substituent. The chemical s h i f t suggests that the o r i e n t a t i o n of the benzyl group i s (A at C-3. The presence of a weak molecular ion (1%) i n the mass spectrum also supported t h i s assignment. I t was concluded, therefore, that the reaction of benzyl magnesium bromide with 3-dehydrodihydrocorynantheine perchlorate gives e x c l u s i v e l y 3o(-benzyl- and no 3^-benzyldihydrocory-nantheine. I t was also c l e a r , from molecular models, that the absence of r i n g E i n dihydrocorynantheine made the molecule less r i g i d and that the stereospecific attack of -137 = t h e G r i g n a r d r e a g e n t f r o m t h e ol - s i d e o f t h e m o l e c u l e i s much more f a v o r a b l e . I n o r d e r t o s t u d y t h e t r a n s a n n u l a r c y c l i z a t i o n o f 3(k-b e n z y l d i h y d r o c o r y n a n t h e i n e ( 1 4 4 ) , i t was n e c e s s a r y t o p r e p a r e a n i m i n i u m s y s t e m i n v o l v i n g C-5 (146) and t o g e n e r a t e a p o t e n t i a l a n i o n a t C - 1 6 . F i r s t , m e r c u r i c a c e t a t e o x i d a t i o n o f t h e 3cUbenzyl d e r i v a t i v e (144) was a t t e m p t e d and t h e i s o l a t i o n o f m e r c u r o u s a c e t a t e (77% o f t h e t h e o r e t i c a l amount) and TLC p r o p e r t i e s s u p p o r t e d t h e f o r m a t i o n o f a n i m i n i u m s a l t i n -v o l v i n g e i t h e r C-5 o r C - 2 1 . However, a s u b s e q u e n t a t t e m p t a t h y d r o l y s i s o f t h e enol e t h e r t o t h e a l d e h y d i c f u n c t i o n i n o r d e r t o a c t i v a t e C - 1 6 , u n d e r c o n d i t i o n s w h i c h were e f f e c t i v e i n t h e c a s e o f d i h y d r o c o r y n a n t h e i n e i t s e l f , gave a n i n t r a c t a b l e m i x t u r e . As summarized i n F i g u r e 3 8 , a n a l t e r n a t i v e way, i n v o l v -i n g d e m e t h y l a t i o n o f t h e 3 - b e n z y l d e r i v a t i v e (144) and s u b -s e q u e n t o x i d a t i o n t o g e n e r a t e t h e i m i n i u m s y s t e m (147) ( F i g u r e 38), was c o n s i d e r e d and a t t e m p t e d . (144) —>fe > -> (146) (147) M e o o C C H O H F i g u r e 38 - 1 3 8 -As d e t a i l e d i n t h e e x p e r i m e n t a l s e c t i o n , s i x d i f f e r e n t e x p e r i m e n t s i n v o l v i n g v a r i o u s c o n d i t i o n s ( h y d r o g e n c h l o r i d e i n a c e t o n e , b o r o n t r i f l u o r i d e e t h e r a t e , h y d r o g e n b r o m i d e i n m e t h y l a c e t a t e , h y d r o g e n b r o m i d e i n a c e t o n e , b o r o n t r i -b r o m i d e i n d i c h l o r o m e t h a n e , and b o r o n t r i c h l o r i d e i n d i -c h l o r o m e t h a n e ) a f f o r d e d no s u c c e s s i n t h e i s o l a t i o n o f t h e d e m e t h y l d e r i v a t i v e ( 1 4 7 ) . T h e r e f o r e , s i n c e t h e e n o l e t h e r s i d e c h a i n o f d i h y d r o c o r y n a n t h e i n e e v i d e n t l y c a u s e d d i f f i -c u l t y i n a c h i e v i n g s u c c e s s f r o m t h i s s e q u e n c e , t h e p r e p a -r a t i o n o f a 3 - b e n z y l d e r i v a t i v e w i t h a p r i m a r y e s t e r a t C-15 was s t u d i e d f o r p o s s i b l e t r a n s a n n u l a r c y c l i z a t i o n . The s y n t h e s i s o f t h e s e 3 - b e n z y l d e r i v a t i v e s was c a r r i e d o u t u n d e r t h e same c o n d i t i o n s as d i s c u s s e d p r e v i o u s l y . M e r c u r i c a c e t a t e o x i d a t i o n o f d l - d i h y d r o c o r y n a n t h e i c a c i d m e t h y l e s t e r and e t h y l e s t e r gave t h e c o r r e s p o n d i n g 3 -d e h y d r o d e r i v a t i v e s (149) and ( 1 5 0 ) , r e s p e c t i v e l y . The 3 -d e h y d r o m e t h y l e s t e r ( 1 4 9 ) , was t r e a t e d w i t h b e n z y l magnesium b r o m i d e i n t e t r a h y d r o f u r a n and gave a s i n g l e 3 -b e n z y l compound. The UV s p e c t r u m o f t h e c r u d e p r o d u c t showed t h e p r e s e n c e o f a s a t u r a t e d i n d o l e chromophore (97%) and 3 - d e h y d r o b a s e ( 3 % ) . The mass s p e c t r u m o f t h e m a i n p r o d u c t showed a weak m o l e c u l a r i o n a t m/e 416 (0.3%) and a b a s e peak a t m/e 325 ( M - 9 1 ) , i n d i c a t i n g t h a t t h e b e n z y l g r o u p had b e e n s u c c e s s f u l l y i n t r o d u c e d a t C - 3 p r o b a b l y from t h e o ( - s i d e . The NMR s p e c t r u m showed a s i n g l e t f o r t h e i n d o l e NH a t 7 r 3 . 3 5 a g a i n i n d i c a t i n g t h e s h i e l d i n g o f t h e p h e n y l n u c l e u s o f t h e 3 J l - b e n z y l g r o u p . A d d i t i o n a l l y , t h e - 1 3 9 -(155) F i g u r e 39 s p e c t r u m showed n i n e a r o m a t i c p r o t o n s i n t h e r e g i o n ^ 2 . 4 -3 . 2 , a s i n g l e t f o r a c a r b o m e t h o x y g r o u p a t 7 r 6 . 2 9 and a b r o a d s i n g l e t f o r t h e b e n z y l i c p r o t o n s o f t h e 3 - b e n z y l g r o u p a t t 7 . 1 8 . A l l t h e d a t a s u p p o r t e d t h e s u c c e s s f u l p r e p a r a t i o n o f t h e 3o(-benzyl d e r i v a t i v e ( 1 5 1 ) . -140-I n t r o d u c t i o n o f a 3c\-benzyl g r o u p was a n a l o g o u s l y a c c o m p l i s h e d i n t h e c a s e o f t h e e t h y l e s t e r d e r i v a t i v e t o a f f o r d d l-3c ( - b e n z y l d i h y d r o c o r y n a n t h e i c a c i d e t h y l e s t e r (152). M e r c u r i c a c e t a t e o x i d a t i o n o f t h e 3c/,-benzyl m e t h y l e s t e r (151) was a t t e m p t e d and 65% o f t h e t h e o r e t i c a l amount o f m e r c u r o u s a c e t a t e was i s o l a t e d . However, t r e a t m e n t o f t h e r e a c t i o n m i x t u r e w i t h s o d i u m b o r o h y d r i d e gave b a c k o n l y s t a r t i n g m a t e r i a l . O b v i o u s l y , t h e c o n v e r s i o n (153* 155) h a d n o t o c c u r r e d , s i n c e t h e c y c l i z e d compound (155) c o u l d n o t be a t t a c k e d by s o d i u m b o r o h y d r i d e t o g i v e b a c k s t a r t i n g m a t e r i a l . T r e a t m e n t o f t h e 3c(-benzyl m e t h y l e s t e r (151) w i t h m e r c u r i c a c e t a t e u n d e r more f o r c i n g c o n d i t i o n s r e s u l t e d i n t h e l o s s o f a b e n z y l g r o u p t o y i e l d a 3 -dehydro d e r i v a t i v e . The g e n e r a t i o n o f a n a n i o n a t C-16 i n t h i s m o l e c u l e m i g h t n o t be v e r y f a v o r a b l e i n a c i d i c m e d i a . An a l t e r n a t i v e compound c o u l d be t h e m e t h y l k e t o n e d e r i v a t i v e (158) i n s t e a d o f t h e m e t h y l e s t e r (151). The m e t h y l k e t o n e w o u l d be e x p e c t e d t o e n o l i z e towards t h e s e c o n d a r y c a r b o n atom r a t h e r t h a n t h e p r i m a r y c a r b o n a t o m , t o g e n e r a t e a p o t e n t i a l a n i o n a t C-16. An e f f i c i e n t way o f c o n v e r t i n g a n e s t e r g r o u p t o a c o r r e s p o n d i n g methyl k e t o n e was r e c e n t l y d e v e l o p e d by C o r e y and C h a y k o v s k y 5 ^ . A l t h o u g h t h i s method has n e v e r b e e n a p p l i e d t o a n i n d o l e c o n t a i n i n g e s t e r , d l - d i h y d r o -c o r y n a n t h e i c a c i d m e t h y l e s t e r (156) was s u b j e c t e d t o t h i s r e a c t i o n . Thus r e a c t i o n o f m e t h y l s u l f i n y l c a r b a n i o n w i t h -141-the methyl ester (156) gave the corresponding (3-ketosulf-oxide, which was c r y s t a l l i z e d from ethyl acetate. Reduction of the ketosulfoxide with aluminum amalgam provided the corresponding methyl ketone (157). The successful conversion was supported by the following physical data. The NMR spectrum showed a s i n g l e t f o r the methyl ketone a t t 7 . 8 4 and no signals between^6.7 and t 3 . 1 . The mass spectrum confirmed the presence of the molecular ion peak at m/e 310 (100%). In a s i m i l a r manner (Figure 40), dl-3o(-benzyldihydro-corynantheic acid methyl ester (151) was converted to the corresponding methyl ketone (158). The success of t h i s transformation was indicated by the following physical data. The NMR spectrum of the product showed a sharp s i n g l e t f o r a methyl ketone a t t 7 . 8 8 , no signals between £6.5 and 7:3.6, a one proton s i n g l e t f or the indole NH at t3.58 and nine aromatic protons i n the r e g i o n , t 2.4-3.2. The chemical s h i f t of the indole NH showed that the configuration at C-3 was retained. The mass spectrum showed a molecular ion at m/e 400 (1.4%) and a base peak at m/e 309 (M-91). The structure of the methyl ketone (158) was thus confirmed. Mercuric acetate oxidation of the 3o(-benzyl methyl ketone (158) was c a r r i e d out and mercurous acetate (96%) was i s o l a t e d . A f t e r the work-up and chromatography, the product was i s o l a t e d as the perchlorate s a l t (32%), m.p. 236-241°. The melting point of t h i s s a l t was quite -142-Figure 40 -143-Figure 41 -144-s i m i l a r to that of the s t a r t i n g material (m.p. 232-237°) but a mixture melting point showed a depression (m.p. 212-215°), i n d i c a t i n g that the two compounds were not i d e n t i c a l . This oxidation product (as the free base) was shown to contain an indole chromophore i n the UV spectrum and an indole NH and a carbonyl absorption at 3289 and 1709 cm"1, re s p e c t i v e l y , i n the IR spectrum. The NMR spectrum s t i l l showed nine aromatic protons i n the r e g i o n , 2 . 5 - 3 . 3 , an indole NH af£3.45 and a methyl ketone at 7:7.90. The chemical s h i f t s of the aromatic, indole NH and methyl ketone signals were quite s i m i l a r to those of the s t a r t i n g methyl ketone (140). The mass spectrum of the product showed a very weak molecular ion at m/e 400 (0.4%, while the s t a r t i n g material had a molecular ion at m/e 400, 1.4%) and a ( M - l ) + ion at m/e 399 (1.1%). The ion at m/e 355 (4.7%) appeared i n the s t a r t i n g material was no longer present i n the mass spectrum of the product. In addition to the base peaks at m/e 309 i n the mass spectra of both the s t a r t i n g material and the products, the rest of the fragmentation patterns were almost superimposable. The c y c l i z e d compounds (161) and (162) must have a molecular weight of 398 and the base peaks i n the mass spectra could be expected to be at m/e 307. The oxidation product, therefore, cannot have structures (161) or (162). The enamine compounds (163) and (164) and the iminium s a l t s (159) and (160) f o r the structure of the oxidation product are also excluded by the mass spectrometry measure--145-ments. These physical data show that the product i s isomeric with the s t a r t i n g 3o(-benzyl methyl ketone (158). One p o s s i b i l i t y i s that t h i s product i s the C-20 ethyl 56 epimer (166) formed by a redox reaction as outlined i n Figure 41. In the redox reaction, the iminium s a l t (165) can be reduced to the ($-ethyl epimer (166) with the enamine (163) being oxidized to the highly conjugated (^-carboline d e r i v a t i v e (165). In conclusion, none of the corynanthenoid-type bases with the 3-benzyl blocking group gave the desired bridged compounds (between C-5 and C-16) under the conditions examined. I t i s of i n t e r e s t to summarize the mass spectra of the 3-benzyl derivatives i n the corynanthenoid s e r i e s , since four t e t r a c y c l i c bases with various substituents at C-15 were a v a i l a b l e . In a l l instances, (M-91) +, r e s u l t i n g from easy loss of the 3-benzyl group, was the base peak. I t should be noted that a l l of these compounds (144), (151), (152) and (158) possess carbonyl functions at C-16, e i t h e r as an ester or a methyl ketone. When a hydrogenYto a carbonyl group i s a v a i l a b l e to form a 6-membered r i n g 1 t r a n s i t i o n state involving the carbonyl bond (see fragment a i n Figure 43), i t i s known that the carbon-carbon bond Pj to the carbonyl group cleaves under electron impact. Therefore i t was expected that an ion at m/e 251 (fragment b) should be common to the spectra of bases (144), (151), (152) and (158). In f a c t , the mass spectrum of 3-benzyl-HELATivC INTENSITY o 8 * 8 8 S ? (D - 2 » t fc 8-8 RELATIVE NTENSITY O 3 8 S 8 8 NELATIVC INTENSITY 3 8 S 8 8 Ul s i fttlATlVt i s H H & T i r 3 S 8 8 8 - 1 4 7 -a m/e 367 (100%) f m/e 167 F i g u r e 43 -148-dihydrocorynantheine showed a f a i r l y strong peak at m/e 251 (14%) and also a peak at m/e 221 (c, 12%) probably due to loss of ethane to form the highly conjugated ion (fragment c ) . As shown i n Figure 42, the occurrence of these peaks at m/e 251 and m/e 221 could also be observed i n the mass spectra of the 3-benzyl derivatives (151), (152) and (158). There were also common fragments at m/e 169, m/e 168 and m/e 167 i n a l l the four spectra and these ions are known to occur i n yohimbine-like bases. I t i s reasonable to spectulate that ions at m/e 169, m/e 168 and m/e 167 are 66 due to (5-car bo l i n e fragments d, e and f, respectively Although these assignments f o r ion fragments i n the mass spectra were not based on d i r e c t experimental evidence, the above speculations appear to be very reasonable f o r the s t r u c t u a l l y s i m i l a r compounds as discussed. c) Attemped Transannular C y c l i z a t i o n s of 3,4-seco-Corynan-thenoid Alkaloids (Figure 27) When the preparation of 3,4-seco derivatives was attempted, the only procedure a v a i l a b l e from the l i t e r a t u r e was the Bi r c h reduction or l i t h i u m aluminum hydride cleavage of the methiodide of the t e t r a c y c l i c base (91) described by Dolby and Booth 4 4. Hence, the methiodides of corynantheic acid ethyl ester (126) were prepared and a sodium i n l i q u i d ammonia reduction was attempted. The only products i s o l a t e d were the recovered s t a r t i n g material and a compound which was probably the 3,4-seco-alcohol (167) -149-by analogy with the r e s u l t s of Dolby and B o o t h ^ (Figure 44). The i d e n t i t y of the t r i c y c l i c alcohol (167) was established by the following physical data. The NMR spectrum showed a s i n g l e t f o r a N^-methyl group a t ? 7 . 6 9 , only three protons above^9, and the absence of an ester group. The IR spectrum indicated the presence of a hydroxyl and the absence of a carbonyl group. Figure 44 The mass spectrum confirmed the molecular ion at m/e 314 (47%). The poor y i e l d from t h i s reduction was thought to be due to the low s o l u b i l i t y of the methiodide of the s t a r t i n g material (126) i n l i q u i d ammonia. To overcome t h i s problem the B i r c h reduction of the methiodide of the -150-alcohol (168) was attempted, however, the y i e l d of the 3,4-seco-alcohol (167) was not improved. Since a carbonyl-containing group i s necessary to acti v a t e a proton at C-16 fo r the transannular c y c l i z a t i o n (Figure 27), attempts were made to oxidize the alcohol (167) to the aldehyde (169). Oxidat ion of the alcohol (167) with 6 5 dicyclohexylcarbodiimide i n dimethylsulfoxide lead to only a poor conversion to the corresponding aldehyde (169). Since t h i s reaction sequence provides the aldehyde (169) i n very poor y i e l d , the a l t e r n a t i v e sequence (Figure 45) s t a r t i n g from the commercially a v a i l a b l e dihydrocorynantheine (170) was investigated. Base catalyzed hydrolysis of dihydrocorynantheine (170) followed by treatment with aqueous acid gave dihydrocorynan-68 theal (171) i n good y i e l d . This substance was converted to the ace t a l (172) and then to the methiodide by the usual methods as outlined i n Figure 45. Bi r c h reduction of the methiodide (173) gave some s t a r t i n g material (40%) back and a mixture of 3,4-seco-acetal (174) and dihydrocorynantheal ethylene a c e t a l (172). By re c y c l i n g the recovered s t a r t i n g material (172 and 173) the 3,4-seco-acetal (174) could be obtained i n 70% o v e r a l l y i e l d . The structure of the 3,4-seco-acetal (174) was f u l l y supported by the mass spectrum which indicated a very stable molecular ion at m/e 356 (100%). The NMR spectrum (Figure 46) was very i n s t r u c t i v e and showed the presence of N^-CHg at7-7.83 and ethylene acetal at^6.26 ( s i n g l e t , 4H) and atT5.30 ( t r i p l e t , J=5 - 1 5 1 -F i g u r e 45 153 -154-cps, H-Cj^). Treatment of the 3,4-seco-acetal (174) with r e f l u x i n g hydrochloric acid for 5 minutes gave the desired 3,4-seco-aldehyde (175) as shown by the IR spectrum (2835 and -1\ 1707 cm ) and the NMR spectrum (sing l e proton m u l t i p l e t at '£"0.56). This proposal was confirmed by the mass spectrum which showed a molecular ion peak at m/e 312 (91%) compared to that of dihydrocorynantheal at m/e 296 (3%). The t r i c y -c l i c aldehyde (169) i s now a suitable compound for trans-annular c y c l i z a t i o n studies i f oxidation to the iminium s a l t (176) can be achivedo Attempted mercuric acetate oxidation of the 3,4-seco-aldehyde (169) under various conditions was not very successful, only 30% of the theo-r e t i c a l amount of mercurous acetate p r e c i p i t a t e d and pro-longed s t i r r i n g at room temperature did not promote any further oxidation. Thin layer chromatographic examination of the reaction mixture showed only the s t a r t i n g material (169) and very polar material (possibly various iminium s a l t s ) . No trace of the t e t r a c y c l i c compound (178), which would not be expected to stay at the base l i n e , could be found. 1 Since the oxidation of the aldehyde (169) was not very successful, a t t e n t i o n was turned to oxidation of the corresponding acetal (174). Mercuric acetate oxidation of the 3,4-seco-acetal (174) proceeded i n good y i e l d (85% of the t h e o r e t i c a l amount of mercurous acetate was p r e c i p i -tated), however, hydrolysis of the acetal to the desired -155-aldehyde was a d i f f i c u l t step. Hydrolysis by sodium acetate i n a c e t i c acid at room temperature was found to be too mild whereas under r e f l u x only an i n t r a c t a b l e t a r was obtained. Figure 48 Since most of the d i f f i c u l t i e s i n the proposed synthesis appear to be associated with the aldehydic function, attempts were made to convert t h i s to an ester function (Figure 49). Oxidation of the aldehyde (169) with Jones' reagent and Fisher e s t e r i f i c a t i o n of the crude oxidation product did not y i e l d any of the desired 3,4-seco methyl ester (179). Oxidation with s i l v e r oxide and -156-e s t e r i f i c a t i o n with diazomethane again was unsuccessful. Since these i n i t i a l experiments did not y i e l d the desired ester (179), the more r e a d i l y a v a i l a b l e dihydrocorynantheal (171) was used as a model compound to study these reactions. Figure 49 Fourteen d i f f e r e n t conditions with s i l v e r oxide, Jones' or potassium permanganate oxidation and Fisher or d i a -zomethane e s t e r i f i c a t i o n conditions were t r i e d , but only the s t a r t i n g material or an i n t r a c t a b l e t a r was obtained. Although the desired ester (156) i s a known compound and the reaction could be followed by TLC, no trace of the RELATIVE INTENSITY RELATIVE INTENSITY 3 ro -159 = e s t e r (156) c o u l d be f o u n d i n t h e p r o d u c t m i x t u r e f r o m t h e v a r i o u s r e a c t i o n s . P o s s i b l y t h e i n d o l e chromophore h a d b e e n d e s t r o y e d by t h e o x i d a t i o n m i x t u r e s . The mass s p e c t r a o f 3 , 4 - s e c o - c o r y n a n t h e n o i d b a s e s have n o t y e t a p p e a r e d i n t h e l i t e r a t u r e . C o r y n a n t h e n o i d a l k a l o i d s a r e known t o g i v e a s t r o n g ( M - l ) + peak due t o t h e i o n h ( s e e b e l o w ) and ( 3 - c a r b o l i n e - t y p e f r a g m e n t s a t m/e 170 ( g ) , m/e 169 and m/e 168 ( i o n s d and e i n F i g u r e 4 3 , r e s p e c t i v e l y ) u n d e r e l e c t r o n i m p a c t . The mass s p e c t r a o f t h e t r i c y c l i c 3 , 4 - s e c o - b a s e s ( F i g u r e s 50 and 51) show v e r y weak ( M - l ) + peaks as w o u l d be e x p e c t e d , s i n c e t h e y c a n n o t f o r m a v e r y s t a b l e i o n o f t h e t y p e h . However t h e t h r e e t r i c y c l i c b a s e s p r e p a r e d d i d n o t g i v e v e r y c h a r a c t e r i s t i c f r a g m e n t a t i o n p a t t e r n s p o s s i b l y due t o t h e u n s t a b l e s i d e c h a i n s a t C - 1 5 . None o f t h e t h r e e a p p r o a c h e s t o s t u d y t r a n s a n n u l a r c y c l i z a t i o n d i s c u s s e d above were s u c c e s s f u l , a l t h o u g h more d e t a i l e d i n v e s t i g a t i o n s were n e c e s s a r y i n most c a s e s . U n f o r t u n a t e l y , a f i r e i n o u r l a b o r a t o r y d e s t r o y e d a l l t h e i m p o r t a n t i n t e r m e d i a t e s n e c e s s a r y f o r f u r t h e r work a t t h i s t i m e . Work i s now i n p r o g r e s s on a d i f f e r e n t sequence o u t l i n e d i n F i g u r e 52. - 1 6 0 -l ) O s O * > *) Me X /Me 3 ) ^ CooMe Figure 52 (Proposed Scheme) -161-EXPERIMENTAL M e l t i n g p o i n t s were d e t e r m i n e d on a K o f l e r b l o c k and a r e u n c o r r e c t e d . The u l t r a v i o l e t (UV) s p e c t r a were r e -c o r d e d i n m e t h a n o l s o l u t i o n on a C a r y 14 s p e c t r o p h o t o m e t e r , and t h e i n f r a r e d ( I R ) s p e c t r a were t a k e n on P e r k i n - E l m e r M o d e l 21 and M o d e l 137 s p e c t r o p h o t o m e t e r s . N u c l e a r m a g n e t i c r e s o n a n c e (NMR) s p e c t r a were r e c o r d e d i n d e u t e r i o c h l r o f o r m a t 60 Mc/s on a V a r i a n A60 o r a t 100 M c / s on a V a r i a n HA100 i n s t r u m e n t . The c h e m i c a l s h i f t s a r e g i v e n i n t h e T i e r s f s c a l e w i t h r e f e r e n c e t o t e t r a m e t h y l s i l a n e as t h e i n t e r n a l s t a n d a r d . Mass s p e c t r a were r e c o r d e d on a n A t l a s CH-4 mass s p e c t r o m e t e r o r on a n AEI MS-9 mass s p e c t r o m e t e r . A n a l y s e s were p e r f o r m e d by D r . A . B e r n h a r d t , M u l h e i m ( R u h r ) , Germany and M r . P. B o r d a o f t h e m i c r o a n a l y t i c a l l a b o r a t o r y , U n i v e r s i t y o f B r i t i s h C o l u m b i a . S i l i c a g e l G and Woelm n e u t r a l a l u m i n a c o n t a i n i n g e l e c t r o n i c p h o s p h o r were u s e d f o r t h i n l a y e r c h r o m a t o g r a p h y ( T L C ) and Woelm n e u t r a l a l u m i n a ( a c t i v i t y I ) was u s e d f o r column c h r o m a -t o g r a p h y . 3 - D e h y d r o y o h i m b i n e P e r c h l o r a t e (131) 58 The method d e s c r i b e d by W e i s e n b o r n and D i a s s i was e m p l o y e d . Y o h i m b i n e ( 7 . 6 g . ) was o x i d i z e d by m e r c u r i c a c e t a t e ( 7 . 0 g . ) i n m e t h a n o l (50 m l . ) a t 64° f o r 30 m i n u t e s . = 162-M e r c u r o u s a c e t a t e was f i l t e r e d o f f and h y d r o g e n s u l f i d e was b u b b l e d t h r o u g h t h e f i l t r a t e . The p r e c i p i t a t e d s u l f i d e was s e p a r a t e d by f i l t r a t i o n . The f i l t r a t e was r e d u c e d t o a volume o f 20 m l . u n d e r r e d u c e d p r e s s u r e and was t r e a t e d w i t h a n e q u i m o l a r amount o f p e r c h l o r i c a c i d t o a f f o r d c r y s t a l l i n e 3 - d e h y d r o y o h i m b i n e p e r c h l o r a t e ( 6 . 0 g . ) , m . p . 2 0 0 - 2 0 5 ° . UVj-^max: 246 and 352 mu. ( L i t . 5 8 m . p . 2 0 5 - 2 0 6 ° , UV, A m a x : 246 and 352 mu) R e a c t i o n o f B e n z y l Magnesium B r o m i d e w i t h 3 - D e h y d r o - y o h i m b i n e P e r c h l o r a t e a) I n D i e t h y l E t h e r A s u s p e n s i o n o f 3 - d e h y d r o y o h i m b i n e p e r c h l o r a t e (1 g . ) i n e t h e r was added t o t h e G r i g n a r d r e a g e n t ( f r o m magnesium ( 0 . 8 g . ) and b e n z y l b r o m i d e ( 3 . 9 6 m l . ) ) i n e t h e r . The m i x t u r e was s t i r r e d f p r 7 h o u r s u n d e r r e f l u x . The c o o l e d r e a c t i o n m i x t u r e was p o u r e d i n t o aqueous ammonium c h l o r i d e , t h e o r g a n i c l a y e r s e p a r a t e d , d r i e d o v e r magnesium s u l f a t e and t h e s o l v e n t e v a p o r a t e d . C h r o m a t o g r a p h y on a l u m i n a and e l u t i o n w i t h b e n z e n e gave 1 , 2 - d i p h e n y l e t h a n e . E l u t i o n w i t h e t h e r p r o v i d e d 3 ^ - b e n z y l y o h i m b i n e (400 m g . ) , w h i c h c r y s t a l l i z e d f r o m d i i s o p r o p y l e t h e r as l a r g e p r i s m s , m . p . 1 5 5 - 1 6 0 ° . I R , " P m a x ( n u j o l ) ; 3333, 1709 c m " 1 . UV, A m a x ; end a b s o r p t i o n ( a t 230 m p ) , 2 8 3 , 291 mji. NMR s i g n a l s (60 M c / s ) : T 2 . 4 - 3 . 2 ( 9 H , m u l t i p l e t , a r o m a t i c ) , -163-3,46 (1H, s i n g l e t , indole NH), 6,25 (3H, s i n g l e t , CH3COO-), 7,16 (2H, s i n g l e t , CH 2-C 3). Mass spectrumj m/e 444 (M + Calc, f o r C28**32°3 N2 ! 4 4 4 ) b) In Tetrahydrofuran 3-Dehydroyohimbine perchlorate (500 mg,) was ground to a f i n e powder and added to the Grignard reagent which was made from magnesium (200 mg,) and benzyl bromide (0.99 ml.) i n tetrahydrofuran (100 ml.). A f t e r s t i r r i n g f o r 2 min. at room temperature, the s o l u t i o n was poured into saturated aqueous ammonium chloride (200 ml.). The organic layer was separated, dried over sodium s u l f a t e and the solvent evaporated leaving white s e m i - c r y s t a l l i n e material, which contained 10% of 3-dehydro base as shown by the UV spectrum. (UV,^max (neutral) % 291, 320 mu; Amax ( a c i d i c ) ; 291 (O.D. 0.47), 353 mp (O.D. 0.19) ) The c r y s t a l l i n e material was chromatographed on alumina (30 g.). E l u t i o n with benzene gave 1,2-diphenylethane, further e l u t i o n with benzene-ether ( 9 % 1) gave a mixture of 3d-and 3(^-benzylyohimbine. Further p u r i f i c a t i o n by preparative s i l i c a gel TLC (ethyl acetata) provided the less polar product (220 mg„), 3c(-benzylyohimbine (132), m.p. 155-160° and the more polar product (99 mg.), 3(2-benzylyohimbine (133), m.p. 110-115°. The 3o(~benzyl-yohimbine (132) was found to be i d e n t i c a l with that from the Grignard reaction i n ether, by TLC comparison with several solvent systems. 3£>-Benzylyohimbine (133) - 1 6 4 -showed t h e t y p i c a l i n d o l e chromophore (Amaxs end a b s o r p t i o n ( a t 230 mp)5 2 8 3 , 291 m u ) . IR,5>max ( n u j o l ) : 3333, 1709 c m " 1 . NMR s i g n a l s (60 M c / s ) : t 2 . 3 - 3 . 2 (10H, m u l t i p l e t , a r o m a t i c + i n d o l e N H ) , 6.17 (3H, s i n g l e t , CH3COO-), 7.42 ( 2 H , b r o a d s i n g l e t , ^ C H 2 - ) • Mass s p e c t r u m : m/e 444 ( M + C a l c . f o r C 2 8 H 3 2 ^ 3 N 2 5 4 4 4 ) . 3 - C y a n o y o h i m b i n e 5 1 The p r o c e d u r e d e s c r i b e d by Z i n n e s e t a l 5 1 was e m p l a y e d . 3 - D e h y d r o y o h i m b i n e p e r c h l o r a t e (215 mg.) was d i s s o l v e d i n m e t h a n o l (20 m l . ) and w a t e r (15 m l . ) and p o t a s s i u m c y a n i d e (93 mg.) a d d e d . The s o l u t i o n was s t i r r e d f o r 10 m i n . a t room t e m p e r a t u r e and t h e c r y s t a l l i n e p r e c i p i t a t e o f 3 -c y a n o y o h i m b i n e (143 mg.) c o l l e c t e d by f i l t r a t i o n , m . p . 1 7 5 ° (decomp. s o f t e n i n g a t 1 5 0 ° ) . I R , ^ max'.2230 ( - C N ) , 1735 (-COOMe) c m " 1 . U V , A m a x : 233 ( O . D . 0 . 2 9 ) , 283 ( 0 . 4 3 ) , 292 ( 0 . 4 2 ) , 322 ( 0 . 1 8 ) mu. The s o l u t i o n i n t h e UV c e l l was a l l o w e d t o s t a n d f o r 1 h o u r a t room t e m p e r a t u r e and t h e UV s p e c t r u m was r e c o r d e d , A m a x : 230 ( O . D . 0 . 1 7 ) , 253 ( 0 . 1 6 ) , 283 ( 0 . 2 2 ) , 292 ( 0 . 2 1 ) , 323 ( 0 . 1 6 ) , 352 ( 0 . 2 6 ) mp. Amax ( a c i d i c ) : 252 ( O . D . 0 . 2 2 ) , 356 ( 0 . 5 2 ) mji. Z i n n e s e t a l 5 1 r e p o r t e d t h e f o l l o w i n g p h y s i c a l c o n s t a n t s f o r 3 - c y a n o y o h i m b i n e ( m . p . 1 7 5 - 1 8 5 ° , d a r k e n s a t 1 5 2 ° , IR ( n u j o l ) : 3440, 3240, 2300 and 1732 c m " 1 . UV (95% E t 0 H ) , > m a x : 2 2 8 , 257 ( S h ) , 295 ( S h ) , 307 and 318 m u ) . -165-Attempted Dehydrogenation of 3-Dehydroyohimbine  Perchlorate with 2,3-Dichloro-5,6-dicyanobenzoquinone (DDQ) A mixture of 3-dehydroyohimbine perchlorate (54 mg.) and DDQ (31 mg., 1.1 mole) i n g l a c i a l a c e t i c acid (150 ml.) was refluxed f o r 6 hours. Evaporation of the solvent l e f t a dark brown residue, ^taax at 255 (Sh, DDQ), 365 mu (3-dehydroyohimbine) and there was no i n d i c a t i o n of aromatization. Tetradehydroyohimbine Perchlorate (140) A s o l u t i o n of lead tetraacetate (2.6 g.) i n a c e t i c acid (120 ml.) was added dropwise over a 2 hour period to a s t i r r e d s o l u t i o n of yohimbine (1 g.) i n a c e t i c acid (25 ml.) at 60°. Most of the a c e t i c acid was removed under reduced pressure, chloroform (150 ml.) and water (25 ml.) were added, and the mixture was made a l k a l i n e (pH9) by the slow ad d i t i o n of 50% potassium hydroxide. The chloroform s o l u t i o n was dried over sodium su l f a t e and a c i d i f i e d with methanolic p e r c h l o r i c a c i d . Upon evapo-r a t i o n the tetradehydroyohimbine perchlorate (0.6 g=) was obtained as needles, m^p. 175-178° „ TLC (alumina) showed the presence of a small amount of 3-dehydroyohimbine perchlorate. P u r i f i c a t i o n by column chromatography (alumina) provided the pure tetradehydro s a l t . IR, l^max ( n u j o l ) : 3540, 3190, 1720, 1635, 1570 cm"1. UV,^maxs 253, 308, 361 mu. Anal. Found: C, 54.0: H, 5.6. -166-Calc. for C 2 1H 230 3N2.C10 4.CH 30HJ C, 54.6; H, 5.6. 59 Wenkert and Roychaudhri reported the m.p. 200-201° f o r tetradehydroyohimbine perchlorate. S c h l i t t l e r 6 0 et a l obtained tetradehydro-c(-yohimbine chloride by lead tetraacetate oxidation of 3-epil-c^-yohimbine and the UV spectrum of tetradehydro-oUyohimbine chloride showed ^max (EtOH) at 253, 308 and 361 mu. Attempted Grignard Reaction on Tetradehydroyohimbine  Perchlorate (140) Tetradehydroyohimbine perchlorate (50 mg.) was ground to a f i n e powder and added to the Grignard reagent which was prepared from magnesium (75 mg.) and benzyl bromide (0.4 ml.) i n tetrahydrofuran (95 ml,). Afte r s t i r r i n g f o r 2 hours under r e f l u x , the insoluble material (30 mgl) was f i l t e r e d o f f . This material was found to be the recovered s t a r t i n g material by the UV spectrum. The f i l t r a t e was poured into saturated ammonium chloride (100 ml.). The organic layer was separated and dried over sodium s u l f a t e . Evaporation of the solvent gave a brown o i l (5 mg.), which was i d e n t i c a l with the s t a r t i n g material by TLC and by the UV spectral examination. 3-Dehydrodihydrocorynantheine Perchlorate (143) A s o l u t i o n of dihydrocorynantheine hydrochloride (2.0 g.), sodium acetate (404 mg.) and mercuric acetate (6.28 g.) i n methanol (100 ml.) was s t i r r e d f o r 2 hours -167-a t 4 0 ° . The p r e c i p i t a t e o f m e r c u r o u s a c e t a t e ( 2 .4 g . ) was removed by f i l t r a t i o n and t h e f i l t r a t e was t r e a t e d w i t h h y d r o g e n s u l f i d e . The s u l f i d e was f i l t e r e d o f f . The f i l t r a t e was r e d u c e d t o a volume o f 10 m l . and 60% p e r c h l o r i c a c i d (1 m l . ) was added t o g i v e t h e c r y s t a l l i n e p e r c h l o r a t e ( 1 .9 g . ) . R e c r y s t a l l i z a t i o n f r o m m e t h a n o l gave y e l l o w n e e d l e s , 3 - d e h y d r o d i h y d r o c o r y n a n t h e i n e p e r c h l o r a t e , m . p . 2 6 1 - 2 6 4 ° . IR, ^max ( K B r ) ; 3260, 1698 (COOMe), 1636 (- C=N1T ), 1620 O C = C - 0 - ) , 1575, 1558 c m " 1 . UV, ^rnaxj 24 0, 352 mu. UV ( b a s i c ) , ^ m a x : end a b s o r p t i o n ( a t 2 50 m u ) , 292, 362 m y . A n a l . F o u n d : C , 5 6 . 6 1 ; h , 6 . 0 0 ; N, 6 . 2 0 . C a l c . f o r C 2 2 H 2 7 ° 3 N 2 , C 1 0 4 : C ' 5 6 o 6 ; H ' 5 o 8 ; N » 6 , 0 ° 3 p ( - B e n z y l d i h y d r o c o r y n a n t h e i n e (144) B e n z y l magnesium b r o m i d e was p r e p a r e d by d r o p w i s e a d d i t i o n o f a s o l u t i o n o f b e n z y l b r o m i d e (5 .6 m l . ) i n d r y t e t r a h y d r o f u r a n (100 m l . ) t o a s t i r r e d s u s p e n s i o n o f magnesium (1 .2 g . ) i n t e t r a h y d r o f u r a n (10 m l . ) o v e r a p e r i o d o f 30 m i n . When a d d i t i o n was c o m p l e t e d t h e m i x t u r e was s t i r r e d a t room t e m p e r a t u r e f o r 2 h o u r s . To t h e G r i g n a r d r e a g e n t was added 3 - d e h y d r o d i h y d r o c o r y n a n t h e i n e p e r c h l o r a t e ( 1 .9 g . ) w i t h v i g o r o u s s t i r r i n g . The r e a c t i o n m i x t u r e was s t i r r e d a t room t e m p e r a t u r e f o r 10 m i n . and t h e n p o u r e d i n t o s a t u r a t e d aqueous ammonium c h l o r i d e s o l u t i o n (200 m l . ) . A f t e r s e p a r a t i o n o f t h e l a y e r s , t h e - 1 6 8 -aqueous l a y e r was e x t r a c t e d w i t h s e v e r a l p o r t i o n s o f e t h e r , t h e combined e x t r a c t s were d r i e d o v e r s o d i u m s u l f a t e , and were e v a p o r a t e d t o d r y n e s s . The UV s p e c t r u m o f t h e r e s i d u e i n d i c a t e d t h e p r e s e n c e o f 3 -dehydro b a s e (17%) ( A max ( a c i d i c ) : 291 ( O . D . 0 . 2 5 ) , 352 (0 o D. 0.19) m u ) . The 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 (100 g . ) . E l u t i o n w i t h b e n z e n e y i e l d e d 1,2 - d i p h e n y l e t h a n e . E l u t i o n w i t h b e n z e n e - e t h e r (10 : 1) gave t h e d e s i r e d 3of-benzyl d i h y d r o -c o r y n a n t h e i n e (144) as amorphous s o l i d . C r y s t a l l i z a t i o n f r o m d i i s o p r o p y l e t h e r - c h l o r o f o r m gave p r i s m s , m . p . 95-100°. I R , >*max (CHCI3): 3356 ( i n d o l e N H ) , 1692 ( e s t e r ) , 1631 (C=C) c m " 1 . U V , ^ m a x ( O . D ) : 226 (1 . 4 1 ) , 283 (0 . 2 4 ) , 291 (0.21) mu. NMR s i g n a l s (100 Hc/s):T 1.40 ( I H , m u l t i p l e t , -C=CH0Me), 2,5-3.2 (9H, m u l t i p l e t , a r o m a t i c ) , 3.40 ( l H , b r o a d s i n g l e t , i n d o l e N H ) , 6.27 and 6.35 (6H, two s i n g l e t s , -COOCH3 and C H 30 - C = C ) . Mass s p e c t r u m : m/e 458 ( M + C a l c . f o r C29**34^3^2J 4 5 8 ) . The m e t h i o d i d e was p r e p a r e d i n t h e u s u a l m a n n e r . R e c r y s t a l l i z a t i o n f r o m d i i s o p r o p y l e t h e r - m e t h a n o l gave p r i s m s , m . p . 2 2 5 - 2 3 1 ° . A n a l . F o u n d : C , 6 0 . 2 8 ; H , 6.45; 0, 8.20; N , 4 .70. C a l c . f o r 03^3703^1: C , 59.99; H , 6.21; 0, 7.99; N , 4.66. - 1 6 9 -M e r c u r i c A c e t a t e O x i d a t i o n o f 3 o ( - B e n z y l d i h y d r o -c o r y n a n t h e i n e (144) f o l l o w e d by a t t e m p t e d D e m e t h y l a t i o n - B e n z y l d i h y d r o c o r y n a n t h e i n e (84 mg.) was added t o a s o l u t i o n o f m e r c u r i c a c e t a t e (238 mg.) i n m e t h a n o l (15 m l . ) c o n t a i n i n g a c e t i c a c i d (10 d r o p s ) and t h e m i x t u r e was s t i r r e d a t room t e m p e r a t u r e f o r 22 h o u r s . M e r c u r o u s a c e t a t e (74 m g . , 77%) was s e p a r a t e d . The UV s p e c t r u m showed a n i n d o l e chromophone (^max, end a b s o r p t i o n ( a t 230 m u ) , 285, 292 m u ) . I R , •) max ( n u j o l ) : 1692 ( C 0 0 C H 3 ) , 1631 (Me0-C=C-) c m " 1 . TLC e x a m i n a t i o n o f t h e p r o d u c t showed no s t a r t i n g m a t e r i a l ( s i l i c a g e l G ) . The f i l t r a t e was t r e a t e d w i t h h y d r o g e n s u l f i d e and t h e s u l f i d e was f i l t e r e d o f f . E v a p o r a t i o n o f t h e f i l t r a t e gave a l i g h t brown r e s i d u e , w h i c h was d e m e t h y l a t e d w i t h h y d r o g e n b r o m i d e i n m e t h y l a c e t a t e a t room temperature f o r 30 m i n . and t h e s o l v e n t was e v a p o r a t e d o f f t o y i e l d a brown i n t r a c t a b l e m i x t u r e o f more t h a n 9 compounds. A t t e m p t e d D e m e t h y l a t i o n o f 3 c ^ - B e n z y l d i h y d r o -c o r y n a n t h e i n e (144) a ) H y d r o c h l o r i c A c i d i n aqueous M e t h a n o l 3 o ( - B e n z y l d i h y d r o c o r y n a n t h e i n e ( 7 . 5 mg.) i n 50% aqueous m e t h a n o l (2 m l . ) was t r e a t e d w i t h c o n c e n t r a t e d h y d r o c h l o r i c a c i d ( 0 . 1 m l . ) . The m i x t u r e was a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 1 h o u r . TLC ( s i l i c a g e l G) e x a m i n a t i o n o f t h e r e a c t i o n m i x t u r e showed o n l y t h e s t a r t i n g m a t e r i a l . -170-The mixture was refluxed for 30 min., but no reaction took place as shown by TLC b) Hydrogen Chloride i n Acetone 3c/-Benzyldihydrocorynantheine (50 mg.) i n acetone (5 ml.) saturated with hydrogen chloride was allowed to stand f o r 25 hours at 0 ° . TLC showed no s t a r t i n g material and none of the spots (streak) on TLC exhibited a p o s i t i v e f e r r i c chloride reaction. c) Boron T r i f l u o r i d e Etherate 3d-Benzyl-dihydrocoynantheine (61 mg.) with BF^. etherate (1 ml.) was heated at 90 ° f o r 30 min. and no s t a r t i n g was shown on TLC ( s i l i c a gel G, EtOAc). The mixture was poured into water and b a s i f i e d with aqueous ammonia. The aqueous s o l u t i o n was extracted with ether and the organic layer was dried over sodium s u l f a t e . Evaporation of the solvent gave an amorphous yellow s o l i d . TLC showed the presence of more than 7 compounds one of which gave a p o s i t i v e f e r r i c chloride t e s t , but further p u r i f i c a t i o n by preparative TLC f a i l e d due to decomposition of the demethyl d e r i v a t i v e . d) Hydrogen Bromide i n Methyl Acetate 3o(-Benzyldihydrocorynantheine (50 mg.) was treated with methyl acetate (10 ml.) saturated with hydrogen bromide. The mixture was allowed to stand at room -171= t e m p e r a t u r e f o r 44 h o u r s . TLC ( s i l i c a g e l G) showed a t r a c e o f s t a r t i n g m a t e r i a l and s e v e n more compounds. F u r t h e r p u r i f i c a t i o n by p r e p a r a t i v e TLC f a i l e d due t o d e c o m p o s i t i o n o f t h e p r o d u c t s . e) H y d r o g e n B r o m i d e i n A c e t o n e 3o ( - B e n z y l d i h y d r o c o r y n a n t h e i n e (50 mg.) was d i s s o l v e d i n a c e t o n e (15 m l . ) and h y d r o g e n b r o m i d e was b u b b l e d t h r o u g h t h e m i x t u r e f o r 30 m i n . a t 0°. The m i x t u r e was a l l o w e d t o s t a n d o v e r n i g h t a t 0°. TLC showed no s t a r t i n g m a t e r i a l ( p o s i t i v e f e r r i c c h l o r i d e t e s t ) , h o w e v e r , p u r i -f i c a t i o n by column c h r o m a t o g r a p h y ( a l u m i n a ) was n o t s u c c e s s f u l . f ) B o r o n T r i b r o m i d e i n D i c h l o r o m e t h a n e 3o! - B e n z y l d i h y d r o c o r y n a n t h e i n e (15 mg.) was d i s s o l v e d i n d i c h l o r o m e t h a n e (7 m l . ) and b o r o n t r i b r o m i d e (0.1 m l . ) was a d d e d . The r e d d i s h m i x t u r e was s t i r r e d a t 0° f o r 2 h o u r s u n t i l no s t a r t i n g m a t e r i a l was d e t e c t e d . No i n d o l e chromophore was p r e s e n t as shown by t h e UV s p e c t r u m ( W ; 229, 304 mu) . g) B o r o n T r i c h l o r i d e i n D i c h l o r o m e t h a n e 3d - B e n z y l d i h y d r o c o r y n a n t h e i n e (20 mg.) was d i s s o l v e d i n d i c h l o r o m e t h a n e (15 m l . ) and b o r o n t r i c h l o r i d e was b u b b l e d t h r o u g h t h e m i x t u r e a t 0° f o r 20 m i n . O n l y t h e s t a r t i n g m e t e r i a l was d e t e c t e d by TLC ( s i l i c a g e l G ) . - 1 7 2 -The r e a c t i o n m i x t u r e was a l l o w e d t o s t a n d a t room t e m p e r -a t u r e f o r f u r t h e r 2 h o u r s , and TLC showed no s t a r t i n g m a t e r i a l . The b a s i f i e d m i x t u r e ( w i t h aqueous ammonia) showed no c a r b o n y l a b s o r p t i o n s i n t h e IR s p e c t r u m and no i n d o l e chromophore i n t h e UV s p e c t r u m . d l - 3 - D e h y d r o d i h y d r o c o r y n a n t h e i c A c i d M e t h y l E s t e r  P e r c h l o r a t e (149) 61 d l - D i h y d r o c o r y n a n t h e i c a c i d m e t h y l e s t e r (1 g . ) , k i n d l y s u p p l i e d by D r . B . D o u g l a s , S m i t h K l i n e and F r e n c h L a b o r a t o r i e s , P h i l a d e l p h i a , was d i s s o l v e d i n m e t h a n o l (50 m l . ) and t r e a t e d w i t h a s o l u t i o n o f m e r c u r i c a c e t a t e ( 4 . 1 g . , 4 mole) i n m e t h a n o l (100 m l . ) . The m i x t u r e was r e f l u x e d f o r 1 h o u r , c o o l e d t o room t e m p e r a -t u r e and t h e p r e c i p i t a t e o f m e r c u r o u s a c e t a t e f i l t e r e d o f f . H y d r o g e n s u l f i d e was p a s s e d t h r o u g h t h e f i l t r a t e f o r 15 m i n . and t h e r e s u l t i n g s u l f i d e f i l t e r e d o f f . The f i l t r a t e was r e d u c e d t o a volume o f 10 m l . and p e r c h l o r i c a c i d (60%, 0 . 4 m l . ) was added t o g i v e a y e l l o w p r e c i p i t a t e o f 3 - d e h y d r o p e r c h l o r a t e ( 1 . 5 g . ) . R e c r y s , t l l i z a t i o n from m e t h a n o l a f f o r d e d a n a n a l y t i c a l sample o f d l - 3 - d e h y d r o -d i h y d r o c o r y n a n t h e i c a c i d m e t h y l e s t e r p e r c h l o r a t e ( 1 4 9 ) , m . p . 2 0 9 - 2 1 2 ° . I R , ^ max ( K B r ) : 3220, 1727 (C=0), 1643 ( C = N + ) , 1568, 1550 c m " 1 . U V , A m a x ( 6 ) : .247 ( 1 6 , 2 0 0 ) , 352 ( 2 1 , 3 0 0 ) mu, ^mim 231 ( 1 4 , 5 0 0 ) , 280 ( 6 , 0 0 0 ) mu. - 1 7 3 -A n a l . F o u n d : C , 5 6 . 6 ; H , 6 . 1 . C a l c . f o r C2QH25N2O2.ClO^; C , 5 6 . 5 3 ; H , 5 . 9 3 . d l - 3 - D e h y d r o d i h y d r o c o r y n a n t h e i c A c i d E t h y l E s t e r  P e r c h l o r a t e (150) d l - D i h y d r o c o r y n a n t h e i c a c i d e t h y l e s t e r ( 1 . 6 g . ) was o x i d i z e d by m e r c u r i c a c e t a t e ( 6 . 5 g . , 4 m o l . ) as p r e v i o u s l y d e t a i l e d f o r t h e m e t h y l e s t e r . The c r u d e y e l l o w p e r c h l o r a t e ( 1 . 7 g . ) was r e c r y s t a l l i z e d from e t h a n o l t o g i v e p r i s m s , m . p . 2 1 5 - 2 1 8 ° . UV, ^ m a x : 2 4 6 , 352 mu. UV ( b a s i c ) , ^ m a x : end a b s o r p t i o n ( a t 230 m u ) , 292, 362 mp. A n a l . F o u n d : C , 5 7 . 6 2 ; H , 5 . 9 8 ; N , 6.10. C a l c . f o r C 2 1H2 7 0 2N2.C10 4: C , 5 7 . 4 5 ; H , 6.20; N , 6 . 3 8 . A t t e m p t e d S y n t h e s i s o f d l - 3 - C y a n o d i h y d r o c o r y n a n t h e i c  A c i d M e t h y l E s t e r d l - 3 - D e h y d r o d i h y d r o c o r y n a n t h e i c a c i d m e t h y l e s t e r (25 m g . ) i n 50% aqueous m e t h a n o l (30 m l . ) was t r e a t e d w i t h p o t a s s i u m c y a n i d e (11 m g . ) . No p r e c i p i t a t e s had formed a f t e r 10 m i n . , b u t t h e UV s p e c t r u m o f t h e r e a c t i o n m i x t u r e ( A m a x ( O . D . ) : 235 ( 0 . 3 3 ) , 284 ( 0 . 5 8 ) , 292 ( 0 . 5 9 ) , 307 ( 0 . 4 4 ) , 319 ( 0 . 4 2 ) mu) d i d n o t c o r r e s p o n d t o t h a t o f t h e s t a r t i n g m a t e r i a l ( 1 4 3 ) . The s o l v e n t was e v a p o r a t e d o f f u n d e r r e d u c e d p r e s s u r e and f u r t h e r p u r i f i c a t i o n by c h r o m a t o g r a p h y f a i l e d t o g i v e any p u r e compounds. However t h e peaks a t 284 and 292 mu a r e p r o b a b l y due t o t h e d e s i r e d - 1 7 4 -3 - c y a n o - e s t e r and t h e peaks a t 307 and 319 mu due t o 3 -d e h y d r o b a s e a f t e r l o s s o f HCN 0 d l - 3 o ( - B e n z y l d i h y d r o c o r y n a n t h e i c a c i d m e t h y l e s t e r (151) d l - 3 - D e h y d r o d i h y d r o c o r y n a n t h e i c a c i d m e t h y l e s t e r p e r c h l o r a t e (1 .15 g . ) was a d d e d , w i t h v i g o r o u s s t i r r i n g , t o t h e G r i g n a r d r e a g e n t p r e p a r e d f r o m magnesium ( 0 . 6 g . ) and b e n z y l b r o m i d e (3 m l . ) i n t e t r a h y d r o f u r a n (130 m l . ) as p r e v i o u s l y d e s c r i b e d . The m i x t u r e was s t i r r e d f o r 1 m i n . a t room t e m p e r a t u r e and t h e n p o u r e d i n t o s a t u r a t e d aqueous ammonium c h l o r i d e (200 m l . ) . A u s u a l w o r k - u p gave a b r o w n i s h o i l . A c c o r d i n g t o t h e UV s p e c t r u m o f t h i s o i l , a p p r o x i m a t e l y 3% o f 3 - d e h y d r o b a s e was p r e s e n t ( ^ m a x ( a c i d i c ) : 291 ( O . D . 0 . 7 0 ) , 352 ( O . D . 0 . 0 9 ) m u ) . The o i l was c h r o m a t o g r a p h e d on a l u m i n a and e l u t i o n w i t h b e n z e n e -e t h e r (5 : 1) y i e l d e d t h e d e s i r e d 3 - b e n z y l d e r i v a t i v e . R e c r y s t a l l i z a t i o n f r o m p e t r o l e u m e t h e r ( 3 0 - 6 0 ° ) gave n e e d l e s (485 m g . ) , 3 A - b e n z y l d i h y d r o c o r y n a n t h e i c a c i d m e t h y l e s t e r ( 1 5 1 ) , m . p . 1 4 1 - 1 4 4 ° . ( p e r c h l o r a t e , m . p . 2 2 5 - 2 3 3 ° ) IR ( K B r ) : 3440 ( i n d o l e N H ) , 1730 (COOMe), 1600 ( p h e n y l ) c m - 1 . U V , > m a x ( £ ) : 225 ( 3 8 , 5 0 0 ) , 275 ( S h , 7 , 0 0 0 ) , 283 ( 8 , 3 0 0 ) , 291 ( 6 , 8 0 0 ) mp, ^ m i n ( 6 ) : 252 ( 2 , 4 0 0 ) , 288 ( 6 , 0 0 0 ) mp. NMR s i g n a l s (60 M c / s ) : ^ 2 . 4 - 3 . 2 ( 9 H , m u l t i p l e t , a r o m a t i c ) , 3 .35 ( I H , b r o a d s i n g l e t , i n d o l e N H ) , 6 .29 ( 3 H , s i n g l e t , COOMe), 7.18 ( 2 H , b r o a d s i n g l e t , (f> - C H 2 - C - 3 ) . A n a l . F o u n d : C , 7 7 . 1 9 ; H , 7 . 6 5 ; N , 7 . 1 2 . C a l c . f o r C 9 7 H o 9 0 9 N 9 : C , 7 7 . 6 7 ; H , 7 . 7 4 ; N , 6 . 7 . -175-Mass s p e c t r u m : m/e 416 ( M + C a l c . f o r C27H32O2N2! 416). The m e t h i o d i d e was p r e p a r e d i n a u s u a l m a n n e r , m . p . 194-199° (MeOH). A n a l . F o u n d : C,58.90; H , 6.66; N, 4.94. C a l c . f o r C 2 7 H 3 2 ° 2 N 2 , M e I ' M e O H ; C ' 5 8 - 5 * H> 6 ' 6 6 ? N» 4.75. d l-3p ( - B e n z y l d i h y d r o c o r y n a n t h e i c A c i d E t h y l E s t e r d l - 3 - D e h y d r o d i h y d r o c o r y n a n t h e i c a c i d e t h y l e s t e r p e r c h l o r a t e (1.6 g . ) was t r e a t e d w i t h t h e G r i g n a r d r e a g e n t p r e p a r e d f r o m magnesium (1.0 g . ) and b e n z y l b r o m i d e (4.8 m l . ) i n a b s o l u t e t e t r a h y d r o f u r a n (125 m l . ) as p r e v i -o u s l y d e s c r i b e d . R e c r y s t a l l i z a t i o n o f t h e c r u d e m a t e r i a l f r o m p e t r o l e u m e t h e r (30-60°) gave 720 mg. o f 3o(-benzyl-d e r i v a t i v e (152) as p l a t e s , m . p . 121-125°. ( p e r c h l o r a t e , m . p . 216-222°). U V , ^ m a x : 225, 275 ( S h ) , 283, 291 mu. A n a l . F o u n d : C , 77.86; H , 7.77; N, 6.53. C a l c . f o r C 2 3 H 3 4 0 2 N 2 S C , 78.09; H , 7.96; N, 6.51. Mass s p e c t r u m : m/e 430 ( M + C a l c . f o r C28H34^2N2S 430). M e r c u r i c A c e t a t e O x i d a t i o n o f d l-3 ( / - B e n z y l d i h y d r o c o r y n a n t h e i c  A c i d M e t h y l E s t e r (151) A m i x t u r e o f t h e 3xj\-benzyl d e r i v a t i v e (151) (16.4 mg.) and m e r c u r i c a c e t a t e (31 m g . ) i n a c e t i c a c i d was h e a t e d a t 90° f o r 2 h o u r s and t h e r e s u l t i n g m e r c u r o u s a c e t a t e (13.1 m g . , 65%) was f i l t e r e d o f f . The r e a c t i o n m i x t u r e was t r e a t e d w i t h s o d i u m b o r o h y d r i d e and worked up as u s u a l . - 1 7 6 -TLC on s i l i c a g e l G showed a t l e a s t s i x s p o t s and t h e m a j o r s p o t c o r r e s p o n d e d t o t h e s t a r t i n g m a t e r i a l . F u r t h e r p u r i f i c a t i o n was n o t a t t e m p t e d . P r e p a r a t i o n o f M e t h y l s u l f i n y l C a r b a n i o n The p r o c e d u r e d e s c r i b e d by C o r e y and C h a y k o v s k y 5 7 w a s e m p l o y e d . Sodium h y d r i d e ( 3 . 0 g . , 50% m i n e r a l o i l d i s -p e r s i o n ; M e t a l h y d r i d e s , I n c . , ) was p l a c e d i n a t w o -n e c k e d 50 m l . f l a s k and washed t h r e e t i m e s w i t h p e t r o l e u m e t h e r , by s w i r l i n g , a l l o w i n g t h e h y d r i d e t o s e t t l e , and d e c a n t i n g t h e l i q u i d p o r t i o n i n o r d e r t o remove t h e m i n e r a l o i l . The f l a s k was i m m e d i a t e l y f i t t e d w i t h a n i n l e t o f n i t r o g e n gas and a r u b b e r cap t h r o u g h w h i c h r e a g e n t s c a n be i n t r o d u c e d v i a h y p o d e r m i c s y r i n g e . A t h r e e - w a y s t o p c o c k , c o n n e c t e d t o t h e f l a s k , was c o n n e c t e d t o a w a t e r a s p i r a t o r and a s o u r c e o f d r y n e t r o g e n . The s y s t e m was e v a c u a t e d u n t i l t h e l a s t t r a c e s o f p e t r o l e u m e t h e r were removed f r o m t h e s o d i u m h y d r i d e and was t h e n r e p l a c e d u n d e r n i t r o g e n by e v a c u a t i n g and f i l l i n g w i t h n i t r o g e n s e v e r a l t i m e s . D i m e t h y l s u l f o x i d e (20 m l . , d i s t i l l e d f r o m c a l c i u m h y d r i d e , b . p . 76° a t 3 . 5 mm Hg) was i n t r o d u c e d and t h e m i x t u r e was h e a t e d w i t h s t i r r i n g t o 7 0 - 7 5 ° u n t i l t h e e v o l u t i o n o f h y d r o g e n c e a s e d ( a p p r o x i m a t e l y 60 m i n . ) . A somewhat c l o u d y , p a l e y e l l o w - g r e y s o l u t i o n c o n t a i n e d 3 m o l e s / l i t e r o f s o d i u m s a l t . -177 = R e a c t i o n o f d l - D i h y d r o c o r y n a n t h e i c A c i d M e t h y l E s t e r (156) w i t h M e t h y l s u l f i n y l C a r b a n i o n and s u b s e q u e n t l y w i t h  Aluminum Amalgam d l - D i h y d r o c o r y n a n t h e i c a c i d m e t h y l e s t e r (62 mg.) was d i s s o l v e d i n d r y t e t r a h y d r o f u r a n (5 m l . ) w i t h a t r a c e o f t r i p h e n y l m e t h a n e . The s t a n d a r d s o l u t i o n o f m e t h y l s u l f i n y l c a r b a n i o n s o d i u m s a l t as d e s c r i b e d above (1 .1 m l . , 16.5 m o l e ) was i n t r o d u c e d v i a h y p o d e r m i c s y r i n g e o v e r a p e r i o d o f 15 m i n . a t 0° and t h e r e s u l t i n g m i x t u r e was s t i r r e d f o r a f u r t h e r 20 m i n . a t room t e m p e r a t u r e . The r e a c t i o n m i x t u r e was t h e n p o u r e d i n t o w a t e r and a c i d i f i e d w i t h d i l u t e h y d r o c h l o r i c a c i d . The a c i d i c s o l u t i o n was b r o u g h t t o pH 8 by aqueous s o d i u m b i c a r b o n a t e and e x t r a c t e d w i t h c h l o r o f o r m . The e x t r a c t s were d r i e d o v e r s o d i u m s u l f a t e and e v a p o r a t e d t o d r y n e s s . The o i l y r e s i d u e was c r y s t a l l i z e d f r o m e t h y l a c e t a t e t o e l i m i n a t e a s m a l l amount o f t h e s a t r t i n g m a t e r i a l ( c h e c k e d by s i l i c a g e l G TLC p l a t e , 20% EtOAc i n b e n z e n e ) . The p u r i f i e d (3-keto s u l f o x i d e , i n 10% aqueous t e t r a -h y d r o f u r a n (20 m l . ) , was r e a c t e d w i t h aluminum amalgam p r e p a r e d as f o l l o w s . Alminum f o i l (54 mg.) was c u t i n t o s t r i p s a p p r o x i m a t e l y 10 cm. x 1 cm. and i m m e r s e d , a l l a t o n c e , i n t o a 2% aqueous m e r c u r i c c h l o r i d e f o r 15 s e c o n d s . The s t r i p s were r i n s e d w i t h a b s o l u t e e t h a n o l and t h e n w i t h e t h e r and c u t i m m e d i a t e l y w i t h s c i s s o r s , i n t o p i e c e s 1 cm. s q u a r e , d i r e c t l y i n t o t h e r e a c t i o n v e s s e l . The r e a c t i o n m i x t u r e was h e a t e d a t 65° f o r 40 m i n . and t h e w h i t e s o l i d - 1 7 8 -was f i l t e r e d o f f . The f i l t r a t e was c o n c e n t r a t e d t o remove most o f t h e t e t r a h y d r o f u r a n , c h l o r o f o r m was added and t h e c h l o r o f o r m phase was s e p a r a t e d f r o m t h e w a t e r , d r i e d o v e r s o d i u m s u l f a t e , and e v a p o r a t e d t o l e a v e t h e o i l y m e t h y l k e t o n e (50 m g . ) . C r y s t a l l i z a t i o n from p e t r o l e u m e t h e r ( 3 0 - 6 0 ° ) - e t h e r y i e l d e d t h e m e t h y l k e t o n e (157) as p r i s m s , m . p . 1 6 7 - 1 7 2 ° . I R , pmax (CHC 1 3 )J 3370 ( i n d o l e N H ) , 1695 (C=0) c m " 1 . U V , A m a x ( O : 225 ( 3 9 , 5 0 0 ) , 274 ( S h , 8 , 1 5 0 ) , 282 ( 8 , 4 0 0 ) , 290 ( 7 , 1 0 0 ) mu, ^ m i n ( 6 ) : 247 ( 4 , 2 0 0 ) , 288 ( 6 , 8 5 0 ) mp. NMR s i g n a l s (60 M c / s ) s t 2.4-3.0 ( 5 H , m u l t i p l e t , a r o m a t i c + i n d o l e N H ) , 7.8 ( S h , s i n g l e t , C H 3 C 0 - ) , 9 . 0 3 ( 3 H , t r i p l e t , CH 3 CH2 ~ ) • Mass s p e c t r u m : m/e 310 ( M + C a l c . f o r C2o H 2 6 O N 2 s 3 1 0 ) . R e a c t i o n o f d l - 3 o ( - B e n z y l d i h y d r o c o r y n a n t h e i c A c i d M e t h y l  E s t e r (151) w i t h M e t h y l s u l f i n y l C a r b a n i o n and w i t h  Aluminum Amalgam d l - 3 o ( - B e n z y l d i h y d r o c o r y n a n t h e i c a c i d m e t h y l e s t e r (200 m g . ) , i n t e t r a h y d r o f u r a n (15 m l . ) , was t r e a t e d w i t h 3 . 4 m l . o f t h e s t a n d a r d s o l u t i o n o f m e t h y l s u l f i n y l i o n s o d i u m s a l t p r e p a r e d as p r e v i o u s l y d e s c r i b e d (3 m o l e s / l i t e r s o l u t i o n ) . The r e s u l t i n g (i - k e t o s u l f o x i d e was c r y s t a l l i z e d f r o m e t h y l a c e t a t e t o y i e l d 143 mg. o f w h i t e n e e d l e s . R e d u c t i o n o f t h e (B> - k e t o s u l f o x i d e (124 mg.) w i t h t h e amalgam p r e p a r e d f r o m aluminum (75 mg.) gave t h e d e s i r e d m e t h y i k e t o n e (158) (104 m g . ) . R e c r y s t a l l i z a t i o n -179= f r o m d i i s o p r o p y l e t h e r gave 3o(-benzyl m e t h y l k e t o n e (158) as n e e d l e s , m . p . 1 1 5 - 1 2 0 ° . I R , >^ max ( n u j o l ) : 3484 ( N H ) , 1718 (C=0), 1592 ( p h e n y l ) c m " 1 . U V , ^ m a x : 225, 275 ( S h ) , 2 8 3 , 291 mu. NMR s i g n a l s (60 M c / s ) : t 2 . 4 - 3 . 2 ( 9 H , m u l t i p l e t , a r o m a t i c ) , 3.58 ( I H , b r o a d s i n g l e t , i n d o l e N H ) , 7 .20 ( 2 H , b r o a d s i n g l e t , <fi - C H 2 - ) , 7.88 ( 3 H , s i n g l e t , C H g C O - ) . Mass s p e c t r u m : m/e 400 ( M + C a l c . f o r Q,^c\^r^M2' 4 0 0 ) . M e r c u r i c A c e t a t e O x i d a t i o n o f 3p(-Benzyl M e t h y l K e t o n e (158) A m i x t u r e o f 3 d ~ b e n z y l m e t h y l k e t o n e (140) (63 mg.) and m e r c u r i c a c e t a t e (210 mg.) i n m e t h a n o l (25 m l . ) c o n t a i n -i n g a c e t i c a c i d (15 d r o p s ) was s t i r r e d a t room t e m p e r a t u r e f o r 26 h o u r s . M e r c u r o u s a c e t a t e p r e c i p i t a t e d was s e p a r a t e d by f i l t r a t i o n and w e i g h e d (79 m g . , 9 6 % ) . H y d r o g e n s u l f i d e was b u b b l e d t h r o u g h t h e f i l t r a t e and t h e s u l f i d e was f i l t e r e d o f f . The s o l v e n t was e v a p o r a t e d t o d r y n e s s and t h e r e s i d u e was d i s t r i b u t e d between d i c h l o r o m e t h a n e and aqueous s o d i u m b i c a r b o n a t e . The o r g a n i c l a y e r was s e p a r a t e d and d r i e d o v e r s o d i u m s u l f a t e . E v a p o r a t i o n o f t h e s o l v e n t a f f o r d e d a l i g h t brown r e s i d u e w h i c h was c h r o m a t o g r a p h e d on a l u m i n a (10 g . ) . E l u t i o n w i t h d i c h l o r o m e t h a n e y i e l d e d a n o i l ( c o n t a i n i n g an i n d o l e chromophore c h e c k e d by t h e UV s p e c t r u m ) , w h i c h was t r e a t e d w i t h m e t h a n o l i c p e r c h l o r i c a c i d t o a f f o r d t h e c r y s t a l l i n e p e r c h l o r a t e (25 m g . ) , m . p . -2 3 6 - 2 4 1 ° . The s t a r t i n g m a t e r i a l gave t h e p e r c h l o r a t e , m . p . 2 3 2 - 2 3 7 ° , on t r e a t m e n t w i t h m e t h a n o l i c p e r c h l o r i c a c i d . - 1 8 0 -A m i x e d m . p . o f t h e above two p e r c h l o r a t e s showed a s i g n i f i c a n t d e p r e s s i o n , m . p . 2 1 2 - 2 1 5 ° . I R , y>max ( f i l m ) : 3289 ( N H ) , 1709 (C=0) c m " 1 . U V , A m a x : 226, 2 8 3 , 290 mu. NMR (100 M c / s ) : t 2 . 5 - 3 . 3 ( 9 H , m u l t i p l e t , a r o m a t i c ) , 3 .45 ( I H , s i n g l e t , i n d o l e N H ) , 7 .90 ( 3 H , s i n g l e t , C H 3 C 0 - ) . Mass s p e c t r u m : m/e 400 ( M + C a l c . f o r C 2 7 H 3 2 O N 2 s 4 0 0 ) . 71 72 S y n t h e s i s o f d l - D i h y d r o c o r y n a n t h e o l (168) ' d l - D i h y d r o c o r y n a n t h e i c a c i d e t h y l e s t e r (200 mg.) was r e d u c e d w i t h l i t h i u m alminum h y d r i d e (400 mg.) i n t e t r a -h y d r o f u r a n (25 m l . ) by r e f l u x i n g f o r 3 h o u r s . The u s u a l w o r k - u p y i e l d e d a w h i t e s o l i d (168 m g . , 96%), w h i c h was c r y s t a l l i z e d t w i c e f r o m c h l o r o f o r m t o g i v e a n a n a l y t i c a l s a m p l e , d l - d i h y d r o c o r y n a n t h e o l , m . p . 1 8 5 - 1 8 8 ° . I R , l ) m a x ( C C l 4 f i l m ) : 3155 c m " 1 ( h y d r o x y l ) . UV, ^raax ( O : 226 ( 2 0 , 0 0 0 ) , 274 ( S h , 6 , 0 8 0 ) , 279 ( S h , 6 , 3 3 0 ) , 290 ( 5 , 3 0 0 ) , ^ m i n ( ^ ) ; 247 ( 1 , 8 0 0 ) , 288 mu ( 5 , 1 0 0 ) . NMR s i g n a l s (100 M c / s ) s t 1.92 ' ( l H , s i n g l e t , i n d o l e N H ) , 2 . 5 - 3 . 0 ( 4 H , m u l t i p l e t , a r o m a t i c ) , 6.32 ( 2 H , t r i p l e t , J=6 c p s , - C H Q C H Q - O H ) . A n a l . F o u n d : C , 7 6 . 4 4 : H , 8 . 7 0 ; 0 , 5 . 2 8 ; N , 9 . 6 0 . C a l c . f o r C i g H 2 6 O N 2 s C , 7 6 . 4 7 ; H , 8 . 7 8 ; 0 , 5 . 3 6 ; N , 9 . 3 9 . Mass s p e c t r u m : m/e 298 ( M + C a l c . f o r C i q H 9 f i 0 N 9 s 2 9 8 ) . Sodium B o r o h y d r i d e R e d u c t i o n o f D i h y d r o c o r y n a n t h e a l (171) D i h y d r o c o r y n a n t h e a l (5 m g . ) , p r e p a r e d f r o m n a t u r a l l y - 1 8 1 -o c c u r r i n g c o r y n a n t h e i n e as d e s c r i b e d l a t e r , was t r e a t e d w i t h s o d i u m b o r o h y d r i d e (5 mg.) i n m e t h a n o l (2 m l . ) a t room t e m p e r a t u r e f o r 10 m i n . The m e t h a n o l was e v a p o r a t e d and t h e w h i t e r e s i d u e was e x t r a c t e d w i t h c h l o r o f o r m . E v a p o r a t i o n o f t h e s o l v e n t gave a n o i l (5 m g . ) ; one s p o t on TLC ( a l u m i n a , C H C l g ) . T h i s o i l had i d e n t i c a l R£ w i t h d l - d i h y d r o c o r y n a n t h e o l (167) by TLC ( s i l i c a g e l , a l u m i n a ) . B i r c h R e d u c t i o n o f d l - d i h y d r o c o r y n a n t h e o l M e t h i o d i d e d l - D i h y d r o c o r y n a n t h e o l (168) (150 mg.) was added t o m e t h y l i o d i d e (2 m l . ) and m e t h a n o l (5 m l . ) , t h e m i x t u r e was k e p t a t room t e m p e r a t u r e f o r 12 h o u r s , and t h e e x c e s s r e a g e n t was removed u n d e r r e d u c e d p r e s s u r e . The r e s i d u e , i n l i q u i d ammonia (50 m l . ) , was t r e a t e d w i t h s o d i u m (280 m g . ) , w h i c h was added i n s m a l l p i c e s w i t h s t i r r i n g d u r i n g 30 m i n . , t h e b l u e c o l o r b e i n g a l l o w e d t o d i s c h a r g e between e a c h a d d i t i o n . On c o m p l e t i o n o f t h e r e a c t i o n , ammonium c h l o r i d e was added and ammonia was e v a p o r a t e d o f f by p a s s i n g a stream o f n i t r o g e n t h r o u g h . The r e s i d u e was e x t r a c t e d w i t h c h l o r o f o r m and e v a p o r a t i o n o f t h e s o l v e n t gave a n amorphous s o l i d (155 mg.) w h i c h was c h r o m a t o g r a p h e d on a l u m i n a . E l u t i o n w i t h c h l o r o f o r m gave d l - N ^ - m e t h y l -3 , 4 - s e c o - d i h y d r o c o r y n a n t h e o l (167) (53 m g . ) . IR, 9 max ( C H C I 3 ) : 3200 (OH) c m " 1 . UV, A m a x ; 226 ( O . D . 1 . 3 4 ) , 284 ( 0 . 3 1 ) , 291 ( 0 . 2 7 ) . NMR s i g n a l s (60 M c / s ) : 1 2 . 4 - 3 . 1 ( 5 H , a r o m a t i c p r o t o n s and - 1 8 2 -i n d o l e N H ) , 7 .69 ( 3 H , s i n g l e t , C H 3 ~ N b ) . Mass s p e c t r u m : m/e 314 ( M + C a l c . f o r C20H3ON2: 3 1 4 ) . F u r t h e r e l u t i o n w i t h m e t h a n o l gave t h e r e c o v e r e d m e t h i o d i d e (89 mg.) i d e n t i f i e d by s p e c t r a l and TLC c o m p a r i s o n s w i t h a n a u t h e n t i c s a m p l e . B i r c h R e d u c t i o n o f d l - D i h y d r o c o r y n a n t h e i c A c i d E t h y l E s t e r  M e t h i o d i d e The m e t h i o d i d e (95 m g . ) , p r e p a r e d f r o m d l - d i h y d r o -c o r y n a n t h e i c a c i d e t h y l e s t e r (126) i n t h e u s u a l manner, was added t o a b l u e s o l u t i o n o f s o d i u m (130 mg.) i n l i q u i d ammonia (11 m l . ) w i t h t - b u t a n o l (1 m l . ) . The m i x t u r e was s t i r r e d f o r 2 m i n . , t h e n ammonium c h l o r i d e was added t o d e s t r o y e x c e s s s o d i u m . Ammonia was a l l o w e d t o e v a p o r a t e and w a t e r and c h l o r o f o r m were a d d e d . E v a p o r a t i o n o f t h e c h l o r o f o r m l a y e r y i e l d e d a w h i t e s o l i d (22 m g . ) , w h i c h was i d e n t i f i e d as d l - N ^ - m e t h y l - 3 , 4 - s e c o - d i h y d r o c o r y n a n t h e o l (167) by d i r e c t c o m p a r i s o n w i t h t h e B i r c h r e d u c t i o n p r o d u c t o f d l - d i h y d r o c o r y n a n t h e o l m e t h i o d i d e ( T L C , UV, I R ) . The aqueous l a y e r was e v a p o r a t e d t o g i v e a s o l i d , w h i c h was e x t r a c t e d w i t h c h l o r o f o r m . E v a p o r a t i o n o f t h e c h l o r o f o r m y i e l d e d t h e s t a r t i n g m a t e r i a l (55 mg.) ( U V , ^ m a x ; 220, 272, 282, 289 m u ) . A t t e m p t e d C o n v e r s i o n o f d l - 3 , 4 - s e c o - N | J - M a t h y l d i h y d r o - c o r y n a n t h e o l (167) i n t o d l - 3 , 4 - s e c o - N ^ " Me t h y l d i h y d r o -c o r y n a n t h e a l (169) - 1 8 3 -The t r i c y c l i c a l c o h o l (167) (33 mg.) i n d r y d i m e t h y l -s u l f o x i d e (2 m l . ) was t r e a t e d w i t h p h o s p h o r i c a c i d (15 mg.) f o l l o w e d by d i c y c l o h e x y l c a r b o d i i m i d e (80 mg.)^. 5 The m i x t u r e was a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 24 h o u r s and p o u r e d i n t o aqueous s o d i u m b i c a r b o n a t e . The m i x t u r e was e x t r a c t e d w i t h e t h e r and t h e e t h e r l a y e r was s e p a r a t e d . E v a p o r a t i o n o f t h e s o l v e n t gave s e m i - c r y s t a l l i n e m a t e r i a l (24 m g . ) . TLC ( a l u m i n a ) i n d i c a t e d t h a t t h i s m a t e r i a l c o n t a i n e d m o s t l y t h e s t a r t i n g m a t e r i a l and a v e r y s m a l l s p o t c o r r e s p o n d i n g t o t h e t r i c y c l i c a l d e h y d e (169) c o u l d be d e t e c t e d . P r e p a r a t i o n o f D i h y d r o c o r y n a n t h e i n e (170) C o r y n a n t h e i n e t a r t r a t e ( 1 . 4 g . ) , k i n d l y s u p p l i e d by P r o f e s s o r R. G o u t a r e l o f C . N . R . S . , I n s t i t u t de C h i m i e des S u b s t a n c e s N a t u r a l l e s de G i f - s u r - Y v e t t e , F r a n c e , was t r e a t e d w i t h aqueous s o d i u m b i c a r b o n a t e t o g i v e a f r e e b a s e c o r y n a n t h e i n e (995 m g . ) . C o r y n a n t h e i n e (995 m g . , M.W. 3 6 6 ) , i n e t h a n o l (100 m l . ) , was h y d r o g e n a t e d o v e r 10% p a l l a d i u m on c h a r c o a l and 1 mole o f h y d r o g e n was a b s o r b e d . The c a t a l y s t was f i l t e r e d o f f and e v a p o r a t i o n o f t h e s o l v e n t y i e l d e d d i h y d r o c o r y n a n t h e i n e (990 m g . ) , w h i c h showed one s p o t on TLC ( s i l i c a g e l G , E t O A c ) and 67 was i d e n t i c a l w i t h t h e sample p r e p a r e d by R. T . Brown P r e p a r a t i o n o f D i h y d r o c o r y n a n t h e a l (171) A m i x t u r e o f d i h y d r o c o r y n a n t h e i n e (325 m g . ) , p o t a s s i u m -184-hydroxide (128 mg.), methanol (5 ml.) and water (1.5 ml.) was refluxed f o r 7 hours under an atmosphere of nitrogen. The cooled reaction mixture was poured into water (85 ml.) containing concentrated hydrochloric acid (7,3 ml.). The r e s u l t i n g s o l u t i o n was refluxed for 4 hours, then cooled i n an ice-bath, made basic by addition of concentrated ammonia water (10 ml.) and extracted thoroughly with chloroform. The combined extracts were washed with a small amount of water and dried over anhydrous magnesium su l f a t e . Removal of the solvent afforded dihydrocorynantheal (171) as a brownish s o l i d (277 mg.). IR,?)max (CHCl 3)s 3390 cm _ 1(NH), 2703 (-CH0), 1718 (OO). UV, xmax: 226, 283, 290 mp. Mass spectrum: m/e 296 (M + Calc. f o r C^gr^O^: 296). Dihydrocorynantheal Ethylene Acetal A mixture of crude dihydrocorynantheal (700 mg.) and p-toluenesulphonic acid (820 mg.) i n ethylene g l y c o l (20 ml.) was heated with s t i r r i n g i n a small vacuum d i s t i l l a t i o n apparatus at 110° and 24 mm Hg pressure f o r 3 hours. Approximately 3 ml. of d i s t i l l a t e was obtained. The cooled reaction mixture was then poured into 100 ml. of ice cold 2N sodium bicarbonate s o l u t i o n and the re s u l t i n g mixture was extracted with chloroform. The extracts were washed with a small amount of water and then dried over anhydrous sodium s u l f a t e . Removal of the solvent gave a crude brownish material (820 mg.) -185-whlch was chromatographed on alumina (40 g.). E l u t i o n with benzene/ethyl ether (3 : 1) gave dihydrocorynantheal ethylene a c e t a l (172) (405 mg.), m.p. 219-223° (needles). IR,^max ( C H C I 3 ) : 3380 (NH) cm"1, no C=0 absorption. UV,Amax; 226, 283, 290 mu. NMR signals (60 Mc/s);t2.1-3.1 ( 5 H , m u l t i p l e t , aromatic H and indole NH), 5.30 (IH, t r i p l e t , J=5 cps, -CHCQ" ), 6.26 ( 4 H , s i n g l e t , -0-CH 2-CH 2-0-), 7.83 ( 3 H , s i n g l e t , CH 3-N b). Mass spectrum: m/e 340 (M + Calc. f o r C 2^H 2g0 2N 2: 340). Bi r c h Reduction of Dihydrocorynantheal Ethylene Acetal  Methiodide (173) Dihydrocorynantheal ethylene ac e t a l (412 mg.) was added to a s o l u t i o n of methyl iodide (2 ml.) i n methanol (50 ml.) and the mixture was allowed to stand at room temperature f o r 18 hours. Evaporation of the solvent gave the yellow methiodide (583 mg.) which showed no spot corresponding to the s t a r t i n g material on TLC. The methiodide (173), without further p u r i f i c a t i o n , was subjected to a B i r c h reduction. The methiodide (173) (101 mg.) was suspended i n l i q u i d ammonia (25 ml.) and absolute ethanol (0.7 ml.) was added. Sodium (280 mg.) was added i n small pieces over a period of 20 min. with continuous s t i r r i n g . Ammonium chloride was then added u n t i l blue color d i s -appeared. Ammonia was allowed to evaporate with a =186-stre^m o f n i t r o g e n t o y i e l d a w h i t e r e s i d u e , w h i c h was e x t r a c t e d w i t h e t h e r f i r s t and s u b s e q u e n t l y w i t h c h l o r o -f o r m . E v a p o r a t i o n o f e t h e r and c h l o r o f o r m y i e l d e d a w h i t e s o l i d (55 m g . ) and a y e l l o w r e s i d u e (44 m g . ) , r e s p e c t i v e l y . The e t h e r e x t r a c t showed two s p o t s on TLC ( a l u m i n a , c h l o r o f o r m ) and was p u r i f i e d by p r e p a r a t i v e T L C . The more p o l a r compound (10 m g . ) , m . p . 2 2 0 - 2 2 3 ° , was i d e n t i c a l w i t h d i h y d r o c o r y n a n t h e a l e t h y l e n e a c e t a l (172) ( T L C , UV, m i x e d m . p . ) . The l e s s p o l a r compound (32 mg.) was t h e d e s i r e d 3 , 4 - s e c o - N ^ - m e t h y l - d i h y d r o c o r y n a n t h e a l e t h y l e n e a c e t a l ( 1 7 4 ) , m - p . 1 7 3 - 1 7 6 ° ( p l a t e s ) . I R , $ max (CHCI3): 3413 c m " 1 ( N H ) . UV, ^ m a x ; 225, 282, 290 mu. NMR s i g n a l s (60 M c / s ) : ^ 2 . 2 9 ( I H , s i n g l e t , i n d o l e N H ) , 2.4-3.1 (4H, m u l t i p l e t , a r o m a t i c ) , 5 . 3 0 ( I H , t r i p l e t , J=5 c p s , -CHCQI), 6 .29 (4H, s i n g l e t , - 0 - C H 2 C H 2 - 0 - ) , 7 .86 (3H, s i n g l e t , C H 3 " N b ) , 9.12 (3H, t r i p l e t , C H g - C H j - ) . A n a l . F o u n d : C , 74 . 0 9 ; H , 8 . 8 7 ; N , 7 . 8 5 ; 0 , 8 . 8 7 . C a l c . f o r C 2 2 H 3 2 0 2 N 2 : C , 74 .12; H , 9 . 0 5 ; N , 7 . 8 6 ; 0 , 8 . 9 8 . Mass s p e c t r u m : m/e 356 ( M + C a l c . f o r C 2 2 H 3 2 0 2 N 2 : 3 5 6 ) . 3,4 - s e c o - N ^ - M e t h y l d i h y d r o c o r y n a n t h e a l (169) 3 , 4 - s e c o - N ^ - M s t h y l d i h y d r o c o r y n a n t h e a l e t h y l e n e a c e t a l (174) (40 mg.) i n 2N h y d r o c h l o r i c a c i d (5 m l . ) was r e f l u x e d f o r 7 m i n . The c o o l e d r e a c t i o n m i x t u r e was n e u t r a l i z e d w i t h aqueous s o d i u m b i c a r b o n a t e and e x t r a c t e d w i t h c h l o r o -f o r m . The e x t r a c t s were d r i e d o v e r s o d i u m s u l f a t e and - 1 8 7 -e v a p o r a t i o n o f t h e s o l v e n t gave a b r o w n i s h s o l i d (38 m g . ) . C r y s t a l l i z a t i o n from e t h e r - h e x a n e a f f o r d e d a n a n a l y t i c a l sample o f t h e a l d e h y d e ( 1 6 9 ) , m . p . 1 4 9 . 5 - 1 5 2 . 5 . I R , v'max ( K B r ) : 3420 ( N H ) , 2835 ( - C H 0 ) , 1707 (C=0) c m " 1 . U V , A m a x ( € ) : 226 ( 2 4 , 3 0 0 ) , 283 ( 5 , 5 7 0 ) , 291 ( 5 , 0 1 0 ) , > m i n (6).- 250 ( 2 , 0 1 0 ) , 288 ( 4 , 8 6 0 ) mp. NMR s i g n a l s (60 M c / s ) : T 0 . 5 6 ( l H , m u l t i p l e t , - C H O ) , 2 . 1 - 3 . 1 ( 5 H , m u l t i p l e t , . a r o m a t i c H and i n d o l e N H ) , 7.85 ( 3 H , s i n g l e t , N b - C H 3 ) , 9 . 0 8 ( 3 H , t r i p l e t , J=3 c p s , C H _ 3 C H 2 - ) . A n a l . F o u n d : C , 7 6 . 5 9 , H , 9 . 1 55 N , 8 . 8 4 ; 0 , 5 . 0 1 . C a l c . f o r C 2 o H 2 8 O N 2 s C> 7 6 - 8 8 » H , 9 . 0 3 ; N , 8 . 9 7 ; 0 , 5 . 1 2 . Mass s p e c t r u m : m/e 312 ( M + C a l c . f o r C 2 Q H 2 g 0 N 2 : 3 1 2 ) . M e r c u r i c A c e t a t e O x i d a t i o n o f 3 , 4 - s e c o - N - L j - M e t h y l d i h y d r o -c o r y n a n t h e a l E t h y l e n e A c e t a l (174) A m i x t u r e o f t h e t r i c y c l i c a c e t a l (174) (16 m g . ) , m e r c u r i c a c e t a t e (120 m g . , 2.1 m o l e ) and e t h a n o l (17 m l . ) was s t i r r e d a t room t e m p e r a t u r e f o r 17 h o u r s and m e r c u r o u s a c e t a t e (80 m g . , 85%) was i s o l a t e d by f i l t r a t i o n . A s m a l l p o r t i o n o f f i l t r a t e s p o t t e d on a s i l i c a g e l TLC p l a t e and d e v e l o p e d w i t h e t h y l a c e t a t e gave no movable s p o t s . The p r o d u c t was t r e a t e d w i t h s o d i u m a c e t a t e (100 m g . ) i n a c e t i c a c i d (5 m l . ) a t 100° f o r 20 h o u r s . The r e a c t i o n m i x t u r e was p o u r e d i n t o s a t u r a t e d s o d i u m b i c a r b o n a t e s o l u t i o n and e x t r a c t e d w i t h c h l o r o f r o m . E v a p o r a t i o n o f t h e s o l v e n t gave a b l a c k t a r . -188-M e r c u r i c A c e t a t e O x i d a t i o n o f 3 , 4 - s e c o - N ^ - M e t h y l d i h y d r o -c o r y n a n t h e a l (169) A m i x t u r e o f 3 , 4 - s e c o a l d e h y d e (169) (17 m g . ) , m e r c u r i c a c e t a t e (34 m g . , 2 m o l e ) , e t h a n o l (10 m l . ) and a c e t i c a c i d (5 m l . ) was s t i r r e d a t room t e m p e r a t u r e f o r 72 h o u r s . The p r e c i p i t a t e d m e r c u r o u s a c e t a t e was s e p a r a t e d by f i l t r a t i o n (8 m g . , 30%) and t h e f i l t r a t e was e v a p o r a t e d t o d r y n e s s . The IR s p e c t r u m o f t h e brown r e s i d u e showed no c a r b o n y l bands b u t a b r o a d band a t 1557 cm ^. The UV s p e c t r u m i n d i c a t e d t h e a b s e n c e o f a n i n d o l e c h r o m o p h o r e . A t t e m p t e d C o n v e r s i o n o f 3 , 4 - s e c o - N - ^ - M e t h y d i h y d r o -c o r y n a n t h e a l (169) i n t o a n E s t e r a ) J o n e s ' O x i d a t i o n and F i s h e r E s t e r i f i c a t i o n The a l d e h y d e (169) (82 mg.) was d i s s o l v e d i n a c e t o n e (15 m l . ) and 8N s t a n d a r d J o n e s ' r e a g e n t ( 1 . 5 mN) was added t o t h e m i x t u r e . The r e a c t i o n m i x t u r e was s t i r r e d a t room t e m p e r a t u r e f o r 20 m i n . and a c e t o n e was e v a p o r a t e d u n d e r r e d u c e d p r e s s u r e . A n h y d r o u s m e t h a n o l (15 m l . ) was added t o t h e r e s i d u e and t h e m i x t u r e was r e f l u x e d f o r 1 h o u r . The whole m i x t u r e was p o u r e d i n t o aqueous s o d i u m b i c a r b o n a t e and was e x t r a c t e d w i t h c h l o r o f o r m . The o r g a n i c l a y e r was d r i e d o v e r s o d i u m s u l f a t e and e v a p o r a t e d t o g i v e a d a r k brown r e s i d u e (62 m g . ) . The major p r o d u c t (10 mg.) s e p a r a t e d by p r e p a r a t i v e TLC ( a l u m i n a , C H C l ^ ) showed a n i n d o l e chromophore i n t h e UV s p e c t r u m b u t no - 1 8 9 -c a r b o n y l a b s o r p t i o n i n t h e IR s p e c t r u m . b) S i l v e r O x i d e O x i d a t i o n A m i x t u r e o f 3 , 4 - s e c o - a l d e h y d e (169) (25 m g . ) , f r e s h l y p r e p a r e d s i l v e r o x i d e (1 mM) and m e t h a n o l (10 m l . ) was s t i r r e d a t room t e m p e r a t u r e f o r 1 h o u r . The m i x t u r e was f i l t e r e d and t h e f i l t r a t e e v a p o r a t e d t o y i e l d a brown r e s i d u e . The IR s p e c t r u m showed a v e r y weak c a r b o n y l a b s o r p t i o n and t h e UV s p e c t r u m showed a n i n d o l e c h r o m o p h o r e . A t t e m p t e d C o n v e r s i o n o f D i h y d r o c o r y n a n t h e a l (171) i n t o  t h e c o r r e s p o n d i n g E s t e r (156) a ) P o t a s s i u m Permanganate O x i d a t i o n .(1 m o l e ) D i h y d r o c o r y n a n t h e a l (30 m g . ) , i n a c e t o n e (25 m l . ) was t r e a t e d w i t h 0.1 m o l a r p o s t a s s i u m permanganate s o l u t i o n (1 m l . ) a t 0° and t h e m i x t u r e was s t i r r e d f o r 30 m i n u t e s . The r e s u l t i n g brown r e s i d u e was f i l t e r e d o f f and t h e f i l t r a t e was e v a p o r a t e d t o g i v e a brown r e s i d u e (30 m g . ) . T h i s r e s i d u e was i d e n t i f i e d as t h e s t a r t i n g m a t e r i a l by TLC and IR s p e c t r a . b) P o t a s s i u m Permanganate O x i d a t i o n ( 2 . 0 m o l e ) D i h y d r o c o r y n a n t h e a l (10 m g . ) , i n a c e t o n e (10 m l . ) was t r e a t e d w i t h 0 . 1 m o l a r p o t a s s i u m permanganate s o l u t i o n ( 0 . 6 7 m l . ) . The m i x t u r e was s t i r r e d f o r 30 m i n u t e s a t 0° and t h e s o l v e n t was e v a p o r a t e d t o g i v e a d a r k brown r e s i d u e . T h i s r e s i d u e c o n t a i n e d no i n d o l e chromophore as - 1 9 0 -shown by t h e UV s p e c t r u m . c ) Chromium T r i o x i d e i n A c e t i c A c i d and F i s h e r  E s t e r i f i c a t i o n D i h y d r o c o r y n a n t h e a l (15 mg.) was d i s s o l v e d i n 90% a c e t i c a c i d ( 1 . 5 m l . ) and chromium t r i o x i d e (5 mg.) was a d d e d . The r e a c t i o n m i x t u r e was s t i r r e d a t room t e m p e r a -t u r e f o r 12 h o u r s . The s o l v e n t was e v a p o r a t e d o f f and t h e r e s u l t i n g brown o i l was d i s s o l v e d i n m e t h a n o l (3 m l . ) w i t h a few d r o p s o f c o n c e n t r a t e d s u l f u r i c a c i d . The m i x t u r e was r e f l u x e d f o r 30 m i n u t e s and c o o l e d t o 0 ° . The m i x t u r e was t r e a t e d w i t h s a t u r a t e d s o d i u m b i c a r b o n a t e and c h l o r o -f o r m . The c h l o r o f o r m l a y e r was s e p a r a t e d and d r i e d o v e r s o d i u m s u l f a t e . E v a p o r a t i o n o f t h e s o l v e n t gave a d a r k brown r e s i d u e , w h i c h was p u r i f i e d by p r e p a r a t i v e TLC ( a l u m i n a ) . The compound w i t h t h e same v a l u e as t h a t o f d i h y d r o c o r y n a n t h e i c a c i d m e t h y l e s t e r (156) was s e p a r a t e d , b u t t h e c h a r a c t e r i z a t i o n o f t h i s compound was n o t p o s s i b l e due t o t h e p o o r y i e l d ( 0 . 5 m g , ) . d) J o n e s ' O x i d a t i o n and E s t e r i f i c a t i o n w i t h D i a z o m e t h a n e D i h y d r o c o r y n a n t h e a l (20 mg.) was t r e a t e d w i t h J o n e s * reagent ( 0 . 3 2 mN) i n a c e t o n e (2 m l . ) and t h e m i x t u r e was s t i r r e d a t room t e m p e r a t u r e f o r 20 m i n u t e s . E v a p o r a t i o n o f t h e s o l v e n t gave a brown r e s i d u e , w h i c h was t h e n d i s s o l v e d i n m e t h a n o l (2 m l . ) . A l a r g e e x c e s s o f d i a z o m e t h a n e i n e t h e r was added t o t h e m i x t u r e and t h e - 1 9 1 -whole m i x t u r e was a l l o w e d t o s t a n d f o r 1 h o u r . The s o l v e n t was e v a p o r a t e d and t h e r e s u l t i n g r e s i d u e was p u r i f i e d by p r e p a r a t i v e TLC ( a l u m i n a ) . The compound w h i c h showed i d e n t i c a l b e h a v i o r w i t h d i h y d r o c o r y n a n t h e i c a c i d m e t h y l e s t e r (156) was s e p a r a t e d ( y i e l d : 2 m g . ) . U V ^ m a x : 284, 290 mp ( i n d o l e ) . IR,2^max ( C H C l 3 ) : 1705 c m " 1 (C=0). P r e p a r a t i o n o f N - F o r m y l t r y p t a m i n e 64 The p r o c e d u r e d e s c r i b e d by S z a n t a y e t a l was e m p l o y e d . T r y p t a m i n e f o r m a t e (20 g . , m . p . 1 6 7 ° ) , p r e p a r e d f r o m t r y p t a m i n e and 90% f o r m i c a c i d i n m e t h a n o l , was h e a t e d a t 180° f o r 45 m i n u t e s . The r e a c t i o n was m o n i t o r e d by TLC ( s i l i c a g e l , E t O A c ) . On c o o l i n g , c r y s t a l s formed s l o w l y , m . p . 7 5 - 7 7 ° ( L i t . 6 4 m . p . 7 5 - 7 6 ° ) . IR, ^max ( C H C 1 3 ) : 3401 ( N H ) , 1689 (Amide I band) c m " 1 . The c r u d e N - f o r m y l t r y p t a m i n e was u s e d f o r t h e n e x t r e a c t i o n w i t h o u t f u r t h e r p u r i f i c a t i o n . 64 P r e p a r a t i o n o f 3 , 4 - D i h y d r o - ( B - c a r b o l i n e The c r u d e N - f o r m y l t r y p t a m i n e (20 g . ) p r e p a r e d above was b o i l e d w i t h a n h y d r o u s b e n z e n e (400 m l . ) u n t i l t h e major p a r t o f t h e s u b s t a n c e d i s s o l v e d . T h e n the m i x t u r e was c o o l e d t o 7 5 ° and p h o s p h o r o u s o x y c h l o r i d e (60 m l . ) was added o v e r a p e r i o d o f 10 m i n u t e s . The m i x t u r e was r e f l u x e d f o r 30 m i n u t e s . The s o l v e n t was e v a p o r a t e d u n d e r r e d u c e d p r e s s u r e a t 4 0 ° . The d a r k brown r e s i d u e was v i g o r o u s l y s h a k e n w i t h a c e t i c a c i d (100 m l . ) . The a c i d i c = 192-s o l u t i o n was s e p a r a t e d f r o m b l a c k t a r and was d i l u t e d w i t h w a t e r (900 m l . ) . The r e s u l t i n g brown amorphous s o l i d was s e p a r a t e d by f i l t r a t i o n and t h e f i l t r a t e was b a s i f i e d a t 0° w i t h 50% p o t a s s i u m h y d r o x i d e s o l u t i o n t o pH 10. The l i g h t y e l l o w p r e c i p i t a t e o f 3 , 4 - d i h y d r o - ( 3 - c a r b o l i n e was d r i e d " - u n d e r r e d u c e d p r e s s u r e a t 56° f o r 5 h o u r s ( y i e l d 18.6 g). U V , ^ m a x ( n e u t r a l ) ; 236, 242, 318 mu; -^max: (+HC1): 246, 69 359 mu. ( F l e m i n g and H a r l e y - M a s o n r e p o r t e d ^ m a x ( n e u t r a l ) : 236, 242, 318 mu; 3-max (+HC1); 246, 359 m u ) . P r e p a r a t i o n o f d l - 3 - E t h y l - l , 2 , 3 , 4 , 6 , 7 , 1 2 , 1 2 b - o c t a h y d r o -2 - k e t o i n d o l o ( 2 , 3 - a ) q u i n o l i z i n e _ (181) The p r o c e d u r e o f Openshaw and W h i t t a k e r 7 ^ was f o l l o w e d . A s o l u t i o n o f 3 , 4 - d i h y d r o - p > - c a r b o l i n e ( 1 8 . 6 g . , 0.11 M) i n a b s o l u t e a l c o h o l (190 m l . ) was h e a t e d w i t h 3 - d i m e t h y l a m i n o - m e t h y l p e n t a n - 2 - o n e m e t h i o d i d e ( 3 2 . 7 g . , 0.11 M) u n d e r r e f l u x f o r 2 h o u r s . The c o o l e d m i x t u r e was d i l u t e d w i t h 5% p o t a s s i u m h y d r o x i d e s o l u t i o n (500 m l . ) and e x t r a c t e d w i t h c h l o r o f o r m (800 m l . ) . The e x t r a c t s were washed w i t h w a t e r and d r i e d o v e r s o d i u m s u l f a t e . E v a p o r a t i o n o f t h e s o l v e n t gave a d a r k brown r e s i d u e . The r e s i d u e was e x t r a c t e d w i t h h o t benzene (700 m l . ) and t h e b e n z e n e s o l u t i o n was f i l t e r e d from amorphous m a t e r i a l . E v a p o r a t i o n o f t h e s o l v e n t gave a l i g h t brown r e s i d u e , w h i c h was c r y s t a l l i z e d from a l c o h o l t o g i v e c r u d e t e t r a c y c l i c k e t o n e (181) ( 7 . 5 g . ) , m . p . 1 9 9 - 2 0 2 ° . R e c r y s t a l l i z a t i o n - 1 9 3 -70 f r o m a l c o h o l gave p r i s m s , m . p . 2 0 5 - 2 0 9 ° . ( L i t . m . p , 2 0 5 -2 0 6 ° ) . I R , ^ m a x (CHClo): 3401 ( i n d o l e NH), 1715 (C=0) c m " 1 . A s o l u t i o n o f t r i m e t h y l p h o s p h o n o a c e t a t e (12 m l . , 60 mM) i n d r y t e t r a h y d r o f u r a n (100 m l . ) was added t o s o l i d p o t a s s i u m t - b u t o x i d e ( p r e p a r e d f r o m 2.0 g . o f p o t a s s i u m and t - b u t a n o l ) . The m i x t u r e was c o o l e d t o 0°, and a s o l u t i o n o f t h e t e t r a c y c l i c k e t o n e (181) (5.0 g . ) i n d r y t e t r a h y d r o f u r a n (250 m l . ) was a d d e d . The s o l u t i o n was a l l o w e d t o warm t o room t e m p e r a t u r e , s t i r r e d f o r 2 h o u r s and d i l u t e d w i t h 2% p o t a s s i u m h y d r o x i d e (1000 m l . ) and d i c h l o r o m e t h a n e (500 m l . ) . The o r g a n i c l a y e r was s e p a r a t e d , washed w i t h s a t u r a t e d s o d i u m b i s u l f i t e , d r i e d o v e r s o d i u m s u l f a t e and e v a p o r a t e d t o d r y n e s s t o g i v e a y e l l o w r e s i n o u s p r o d u c t (4.5 g . ) . TLC ( a l u m i n a , C H C l ^ ) showed one s p o t and t h e p r o d u c t was l e s s p o l a r t h a n t h e s t a r t i n g m a t e r i a l . T h i s ^ ( ^ - u n s a t u r a t e d e s t e r (182) had t h e f o l l o w i n g p h y s i c a l d a t a . IR,7^max (CHCI3): 1698, 1637 (o< ^ - u n s a t u r a t e d e s t e r ) c m " 1 . NMR s i g n a l s (100 M c / s ) : ? - 1 . 5 9 ( l H , s i n g l e t , i n d o l e N H ) , 2 . 5 - 3 . 1 ( 4 H , m u l t i p l e t , a r o m a t i c ) , 4 . 3 3 ( l H , s i n g l e t , v i n y l i c ) , 6 . 3 0 ( 3 H , s i n g l e t , COOMe), 9 . 0 4 ( 3 H , t r i p l e t , W i t t i g R e a c t i o n on t h e T e t r a c y c l i c K e t o n e (181) and  s u b s e q u e n t H y d r o g e n a t i o n - 1 9 4 -J=7 c p s , C H 3 C H 2 - ) ( L i t . ^ 4 . 3 0 ( v i n y l i c , s i n g l e t ) a t 60 M c / s ) A s m a l l p o r t i o n of d ^ - u n s a t u r a t e d e s t e r was d i s s o l v e d i n m e t h a n o l and a few d r o p s o f c o n c e n t r a t e d p e r c h l o r i c a c i d was a d d e d . C r y s t a l s d e v e l o p e d o v e r n i g h t and had m . p . 2 4 5 - 2 5 0 ° . The W i t t i g p r o d u c t (182) ( 4 . 4 g . ) , i n m e t h a n o l (250 m l . ) , was h y d r o g e n a t e d o v e r 10% p a l l a d i u m on c h a r c o a l (3 g . ) and h y d r o g e n u p - t a k e (350 m l . ) c e a s e d a f t e r 2 h o u r s . The c a t a l y s t was f i l t e r e d o f f and t h e f i l t r a t e was r e d u c e d t o a volume o f 100 m l . u n d e r r e d u c e d p r e s s u r e . The s o l u t i o n was t r e a t e d w i t h c o n c e n t r a t e d p e r c h l o r i c a c i d (2 m l . ) and t h e r e s u l t i n g y e l l o w p r i s m s ( 3 . 5 g . ) were s e p a r a t e d by f i l t r a t i o n , m . p . 2 6 0 - 2 6 5 ° . T r e a t m e n t o f t h e p e r c h l o r a t e w i t h aqueous s o d i u m b i c a r b o n a t e gave t h e f r e e b a s e , d l - d i h y d r o c o r y n a n t h e i c a c i d m e t h y l e s t e r ( 1 5 6 ) , w h i c h was c r y s t a l l i z e d f r o m n - h e p t a n e - b e n z e n e t o g i v e n e e d l e s , m . p . 1 4 2 - 1 4 4 ° . D i r e c t c o m p a r i s o n o f t h i s m e t h y l 61 e s t e r (156) w i t h a n a n t h e n t i c s p e c i m e n , k i n d l y s u p p l i e d by D r . B . D o u g l a s , S m i t h K l i n e and F r e n c h L a b o r a t o r i e s , P h i l a d e l p h i a , P a . , showed t h e two t o be i d e n t i c a l ( m i x e d m . p . , NMR, T L C , I R ) . I R , l)max ( C H C I 3 ) : 3530 ( N H ) , 1725 (C=0) c m " 1 . NMR s i g n a l s (100 M c / s ) : t l . 8 4 ( I H , s i n g l e t , i n d o l e N H ) , 2 . 5 - 3 . 1 ( 4 H , m u l t i p l e t , a r o m a t i c ) , 6.33 ( 3 H , s i n g l e t , COOMe), 9.12 ( 3 H , t r i p l e t , J=7 c p s , C H o C H ~ - ) . Mass Spectral Data - 1 9 5 -34-5enzylyohlmbine (132) C23 H32°3 K2 M.W. 444 9 0 ) m/e %B.P. m/e %B.P. 444 355 354 353 352 351 293 277 235 1.9 3.5 23.0 100.0 4.5 2.6 2.2 4.1 2.4 223 222 221 219 209 207 206 198 197 3.2 2.2 1°.0 2.0 3.0 2.7 2.1 4.3 13.0 m/e %B.P. m/e ?6B.P. 195 184 183 182 169 168 167 156 155 2.0 2.0 4.0 2.9 3.3 4.5 3.5 3.0 3.4 154 144 143 142 130 92 91 3.2 3.1 2.1 2.0 2.1 2.5 5.0 3^-Benzylyohimbine ( 1 3 3 ) C28H32°3N2 M.W. 444 -''90) m/e 444 443 413 355 354 353 352 351 %B.P. m/e %B.P. m/e %B.P. m/e 14 335 13 197 14 155 5 6 293 7 184 14 154 5 .-5 278 5 183 5 153 5 20 277 20 182 6 144 6 39 235 5 170 .5 130 5 100 221 12 169 7 97 7 19 209 5 168 9 92 7 14 198 6 167 6 91 16 -196-3<^-Benzyldihydro- corynantheine C29 H34°3 N2 M.W. 458 100) (144) m/e #B.P. 458 457 369 3 6 8 367 366 365 351 335 m/e %B.P. 1 252 5 2 251 14 10 249 7 . 67 223 5 100 221 12 10 209 5 10 208 5 5 207 5 5 198 6 m/e %B.P. m/e %B.P. 197 23 155 5 195 5 154 8 183 5 144 5 182 5 142 6 180 5 130 5 169 14 115 6 168 16 106 6 167 10 105 8 156 6 3c<-Benzyldihydro-'  corynantheine methiodide c30 H37°3 N a I M.W. 600 ( £ 9 0 ) m/e %B.P. 472 3 368 23 367 79 295 5 251 10 249 7 m"/e %B.P. 221 6 197 13 196 6 169 6 168 9 167 5 m/e %B.P. 154 5 143 16 142 100 141 82 140 68 139 72 m/e %B.P. 128 54 127 93 92 9 91 32 -197-3^-Benzyldihydro- C.Prynantheic acid methyl ester M.W. 416 ( ?y 100) m/e 70B.P. m/e %B.P. 416 415 385 325 324 0.3 0 .9 • 1.2 3 . 0 100.0 3.1 323 252 251 249 237 223 2 .3 1.3 7 . 0 0 .7 1.1 1.2 m/e %B.P. 222 221 209 197 183 182 1.1 4.1 0 .9 1.1 0 .9 0 . 8 m/e %B.P. 169 168 167 135 154 1.0 1.5 1.1 0 . 7 1.0 3 i i - Benzyldihydro-corynantheic acid ethyl ester M.W. 430 ( 9 0 ) £\0 0C' m/e #B.P. m/e %B.P. m/e %B.P. 430 0.3 252 2 . 5 197 3.1 429 0.7 251 11.8 195 2 . 0 341 3 . 7 237 3.1 I84 3.1 340 24.1 223 3.1 183 3.1 339 100.0 222 182 2 . 5 338 2 .6 221 8 . 6 169 3.1 337 2.3 219 2 . 0 168 4 .9 311 3.4 209 3 .0 167 4 .2 m/e %B.P. 156 155 154 144 143 115 92 91 2 . 0 2 .9 4 . 0 2 . 0 2.1 2 . 7 2 . 5 15.6 -198-Tetracycllc methyl ketone (157) C20H260N2 y,.w. 3 1 0 100) m/e SSB.P. ;.1 1 3 1 0 309 303 281 253 20 100 66 4 9 19 m/e $B.P« 252 251 225 224 223 221 9 26 13 4 11 4 m/e %B.P. m/e %B.P« 184 170 169 168 156 155 5 11 11 5 8 5 149 144 143 111 109 11 4 4 5 5 ^ - B e n z y l t e t r a c y c l i c  methyl ketone (158) C27H32°N2 M.W. 400 (^ 90) m/e %B.P. m/e %B. P. m/e &B.P. m/e %B.P. 400 1.4 265 1.5 221 11 .0 182 1.5 355 4.7 252 4.3 219 1.5 169 1.9 311 5.4 251 22.0 209 1.5 168 3.0 310 43.0 250 1.8 207 1.5 167 2.4 309 100.0 249 2.9 206 1.4 155 1.6 308 5.5 223 2.0 197 1.7 154 2.1 307 3.4 222 3.3 183 1.6 91 3.7 -199-.IgCOAc)^ oxidation product CLom_ 3j-benzyl t e t r a c y c l i c diethyl ketone (158) C2?*320N2 H.W. /+00 ( , 90) m/e %B.P. ^00 399 311 310 309 305 307 252 0.4 1 .1 3.0 21 .4 100.0 2.9 2.4 5.3 m/e 251 250 249 223 222 221 220 219 %B.P. m/e %B.P. m/e 26.2 209 1 .9 168 2.0 207 1.9 167 3-2 206 1.7 155 2.3 197 1.8 154 4.3 195 1.7 91 13.7 183 1.8 1.5 182 1.8 1.9 169 1.7 %B.P. 2.1 1.7 1.5 1 . 6 2.4 d1-Dihydrocorynantheol (168) C 1 Q.4 2 60N 2 M.W, 298 ( ^100) (168) HO, m/e %B.P. 299 298 297 296 269 253 20.0 100/0 90.5 7.5 5.1 9.1 m/e %B.P. m/e %B.P. m/e %B.P. 225 197 184 170 169 168 14.0 4.8 5.3 12.0 10.8 6.0 156 154 149 144 143 119 8.7 4.0 7.6 4.4 4.0 7.9 i P17 111 110 109 8.8 5.4 5.4 4.2 di . 3 , i r-seco - K D-Methyl-dihydrocorynantheol (167) C 2 0 H 3 0 0 N 2 M.W. 3 1 4 ( £ 1 0 0 ) m/e %B.P. m/e ?6B.P. 3 1 5 3H 3 1 3 271 2 7 0 2 6 9 1 4 4 7 9 1 2 1 2 1 0 2 5 8 2 5 6 2 2 7 2 1 4 2 0 3 2 0 2 1 0 1 6 1 4 1 0 9 3 0 -200-(167) m/e %B.P. 2 0 1 2 0 0 1 9 9 1 8 6 1 7 2 1 7 1 1 0 2 1 9 1 2 1 8 1 0 0 m/e %B.P. 1 5 9 1 5 8 1 5 7 1 4 6 1 4 5 1 4 4 23 3 2 4 5 1 0 1 4 4 2 Dihydrocorynantheal (171) C 1 9 H 2 4 0 N 2 M.W. 2 9 6 1 0 0 ) m/e %B.P. m/e %B.P, 296 2 6 8 2 6 7 2 5 3 2 5 1 2 2 5 3 . 0 4 . 0 1 6 1 8 1 8 1 8 3 8 2 2 3 1 8 4 1 8 3 1 7 1 1 7 0 169 1 6 8 1 7 3 0 16 26 100 86 3 9 C-7Wh m/e #B.P. m/e %B.P. 167 1 5 7 1 5 6 1 5 5 1 5 4 1 4 4 1 4 3 2 3 1 9 71 2 4 3 2 4 5 5 2 1 4 2 1 3 0 1 2 9 1 2 8 1 2 7 1 2 6 1 1 5 3 6 3 4 3 2 ft 3 2 4 2 -201 Dihydroc orynantheal  ethylene acetal (172) C 2 1 S 2 8 ° 2 N 2 M.W. 3 4 0 (i< 1 0 0 ) (172) H w* m/e %B.P. 3 4 0 3 3 9 2 6 8 2 6 7 2 5 3 2 5 1 2 3 9 2 2 5 8 51 ' 4 9 1 4 2 7 1 8 4 4 2 2 3 2 2 1 2 1 1 1 9 7 1 8 4 1 8 3 1 7 1 1 7 0 %B.P. m/e %B.P. 1 6 1 6 9 1 0 0 1 7 1 6 8 4 0 2 0 1 6 7 2 4 1 5 1 5 7 1 7 3 2 1 5 6 65 1 9 1 5 5 2 3 2 8 1 5 4 2 7 8 8 1 4 4 3 7 3,4-seco-N b-Methyldihydro- corynantheal ethylene acetal M.W. 3 5 6 O M 0 0 ) m/e %B.P. 3 5 7 2 5 3 5 6 . 1 0 0 3 5 5 11 3 2 5 11 2 9 8 11 m/e %B.P. 2 1 3 1 3 2 1 2 5 5 2 0 1 46 2 0 0 1 2 1 8 5 1 0 (174) m/e %B.P. 1 7 0 1 2 1 5 8 11 1 5 7 1 5 1 5 6 1 8 1 4 4 2 1 -202-3,4-sec o- Nb-Met hy 1-dihydrocorynantheal (169) C 2 0 * 2 8 0 N 2 M.W. 3 1 2 (>< ioo) m/e %B.P. m/e %B.P. m/e 5&B.P. m/e 3 1 3 2 1 2 0 1 1 9 1 5 8 1 3 1 4 4 4 0 3 1 2 91 1 7 0 1 4 1 5 7 2 1 1 4 3 3 9 269 5 5 1 6 9 1 5 1 5 6 4 4 140 1 3 2 2 7 * 2 2 1 6 8 6 9 1 5 4 16 1 3 7 1 4 2 2 6 100 167 1 5 1 4 9 1 9 1 3 0 1 5 -203= REFERENCES 1. 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