UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies on mycophenolic acid and lignan chemistry Knittel, Peter 1971

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STUDIES ON MYCOPHENOLIC ACID AND LIGNAN CHEMISTRY by PETER KNITTEL B . S c , 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 , 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department o f C h e m i s t r y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1971 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall'make, jt .freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of • CHEMISTRY The University of British Columbia Vancouver 8. Canada Date APPTT. ?Dr 1Q71 - i i -ABSTRACT P r e v i o u s work on the b i o s y n t h e s i s o f mycophenolic a c i d i s d i s c u s s e d . [ 1 1 - 1 4 C ] - O r s e l l i n i c a c i d (22) and [ 1 ' - 1 4 C ] - 2 , 4 -d i h y d r o x y - 5 , 6 - d i m e t h y l b e n z o i c a c i d (23) were s y n t h e s i z e d and a d m i n i s t e r e d t o P e n i c i l l i u m brevi-compactum. The purpose of f e e d i n g these two a c i d s was t o determine the o r d e r o r sequence o f the r e a c t i o n s i n v o l v e d i n the b i o s y n t h e s i s o f mycophenolic a c i d . I t was found t h a t a c i d (22) was not i n c o r p o r a t e d i n t o mycophenolic a c i d but t h a t a c i d (23) was i n c o r p o r a t e d s p e c i f i c a l l y t o the e x t e n t o f 11.5%. D e g r a d a t i o n of the mycophenolic a c i d from the f e e d i n g o f (23) showed t h a t a co m p l e t e l y s p e c i f i c i n c o r p o r a t i o n had o c c u r r e d and thus supported the r o l e o f t h i s 14 a c i d (23) as a p r e c u r s o r o f mycophenolic a c i d . [1- C ] - g e r a n i o l (24) was a l s o f e d i n an attempt t o determine the o r i g i n o f the Cy s i d e c h a i n o f mycophenolic a c i d . (24) was i n c o r p o r a t e d but d e g r a d a t i o n o f the mycophenolic a c i d showed t h a t the g e r a n i o l was decomposed t o a c e t a t e b e f o r e i n c o r p o r a t i o n , l e a v i n g the p r e c u r s o r s t a t u s i n doubt. Three attempts were made t o s y n t h e s i z e mycophenolic a c i d u s i n g suspected b i o l o g i c a l i n t e r m e d i a t e s w i t h no s u c c e s s . T h i s was presumably due t o the d i f f i c u l t y encountered i n t r y i n g t o a l k y l a t e the aromatic s k e l e t o n o f mycophenolic a c i d . A s y n t h e s i s o f l i g n a n s i n v o l v i n g i n t r a m o l e c u l a r p h e n o l i c o x i d a t i v e c o u p l i n g was attempted w i t h l i t t l e s u c c e s s , due t o the i n a b i l i t y t o o b t a i n the e s t e r r e q u i r e d t o couple t o g i v e the l i g n a n s k e l e t o n . - i i i -T A B L E OF CONTENTS P A R T I M Y C O P H E N O L I C A C I D I N T R O D U C T I O N 1 D I S C U S S I O N 14 E X P E R I M E N T A L 34 P A R T I I L I G N A N S Y N T H E S I S I N T R O D U C T I O N 5 6 D I S C U S S I O N 5 9 E X P E R I M E N T A L 6 9 B I B L I O G R A P H Y 7 9 - i v -ACKNOWLEDGEMENT I would l i k e t o thank Dr. T. Money f o r h i s a s s i s t a n c e and guidance throughout the three years i t took t o complete the work presented here. I would a l s o l i k e t o thank Mr. P. S a l i s b u r y f o r h i s expert t e c h n i c a l a s s i s t a n c e i n the growing of the c u l t u r e s of P e n i c i l l i u m brevi-compactum. PART I MYCOPHENOLIC ACID INTRODUCTION I ISOLATION AND STRUCTURE Mycophenolic acid (1) i s perhaps one of the most i n t e r e s t -ing natural metabolites known and i t s biosynthesis and b i o l o g -i c a l a c t i v i t y have been the subject of considerable i n v e s t i g a t i o n . The compound was f i r s t discovered by Gossio''" i n 1896. He i s o l a t e d i t from a s t r a i n of P e n i c i l l i u m mould named P e n i c i l l i u m glaucum, which i s probably a member of the P e n i c i l l i u m b r e v i -compactum s e r i e s . Naming of the compound was l e f t to Alsberg 2 and Black , who i n 1913 i s o l a t e d i t from a s t r a i n of P e n i c i l l i u m stoloniferum. These workers assigned the molecular formula C H 0 to mycophenolic a c i d . Further studies on the metabolite 17 20 6 . * * 3-7 were c a r r i e d out from 1932 to 1952 by Birkinshaw and co-workers As a r e s u l t of an extensive chemical and s t r u c t u r a l i n v e s t i g a t i o n of mycophenolic acid i s o l a t e d from P e n i c i l l i u m brevi-compactum, they a r r i v e d at structure (1) f o r the metabolite. HOOC CH 30 II BIOSYNTHESIS (1) The biosynthesis of mycophenolic acid was f i r s t studied 8 — 12 by Bir c h and his co-workers from 1957 to 1964 and then by - 2 -13 Le d e r e r and co-workers i n 1964. Very r e c e n t l y , i n 1970, more 14 work has been r e p o r t e d by Canonica and co-workers B i r c h proposed the the o r y t h a t the s k e l e t o n of mycophenolic a c i d i s d e r i v e d from a head t o t a i l c o n d e n s a t i o n of a c e t i c a c i d u n i t s i n the n a t u r a l system. T h i s b i o g e n e t i c scheme has been extended t o o t h e r n a t u r a l p r o d u c t s and i s now p o p u l a r l y known as the a c e t a t e h y p o t h e s i s . The o r i g i n s o f t h i s h y p o t h e s i s can •I C "I c be a t t r i b u t e d t o work c a r r i e d out by C o l l i e ' from 1893 t o 1907. He made some i n t e r e s t i n g o b s e r v a t i o n s on the base c a t a l -yzed t r a n s f o r m a t i o n s o f p o l y a c e t y l compounds, e s p e c i a l l y t h a t o f d e h y d r a c e t i c a c i d (2) t o o r s e l l i n i c a c i d (3) a known n a t u r a l p r o d u c t . He reasoned t h a t p o l y a c e t y l compounds might undergo s i m i l a r t r a n s f o r m a t i o n s t o aromatic systems i n n a t u r e . (2) (3) C o l l i e ' s t h e o r y was i g n o r e d u n t i l the e a r l y 1950's, p r e -sumably because t h e r e was no known p r a c t i c a l way t o s u b s t a n t i a t e i t . A l s o t h e r e e x i s t e d a g e n e r a l b e l i e f t h a t u n i t s were so s m a l l t h a t they c o u l d be the b u i l d i n g b l o c k s f o r p r a c t i c a l l y a n y t h i n g . However, w i t h the advent of r a d i o a c t i v e i s o t o p e s , t h e r e was a f l u r r y o f e x p e r i m e n t a t i o n t o see i f a c e t a t e d i d indeed condense w i t h i t s e l f and c y c l i z e t o b u i l d up the s k e l e t o n s o f n a t u r a l p r o d u c t s . By f e e d i n g carbon-14 l a b e l l e d a c e t a t e t o - 3 -v a r i o u s n a t u r a l systems, r e c o v e r i n g and degrading the v a r i o u s m e t a b o l i t e s i t soon became apparent t h a t the a c e t a t e h y p o t h e s i s was v a l i d . Most o f the work was done by B i r c h and h i s co-workers and 17 i n a review i n 1957 he e n u n c i a t e d a modern view o f the a c e t a t e h y p o t h e s i s . T h i s view i s t h a t a l i n e a r polyketomethylene c h a i n (4) formed by head t o t a i l c o n d e n s a t i o n o f a c e t a t e u n i t s c o u l d c y c l i z e , v i a a l d o l or C l a i s e n c o n d e n s a t i o n (see FIG. 1 ) , t o y i e l d a remarkable a r r a y o f complex s t r u c t u r e s . Using a 3 - t r i -k eto a c i d i n t e r m e d i a t e , two major groups of n a t u r a l p r o d u c t s can be o b t a i n e d : the a c y p h l o r o g l u c i n o l s (A) and the o r s e l l i n i c a c i d d e r i v a t i v e s (B), (see FIG. 1 ) . Due t o the oxygen sub-s t i t u e n t p a t t e r n of mycophenolic a c i d , i t would seem most pr o b a b l e t h a t i t a r i s e s v i a the a l d o l c o n d e n s a t i o n p a t h . 0 0 0 (4) 0 0 0 0 II R-C :OOH HO iH (A) FIG. 1 (B) - 4 -In 1958 Birch"1"^ confirmed that the aromatic skeleton of mycophenolic acid does derive from the condensation of a c e t i c acid u n i t s . By feeding [ 1 - 1 4 C ] - and [2-"*"4C] - a c e t i c acid to P e n i c i l l i u m brevi-compactum and degrading the i s o l a t e d myco-phenolic a c i d , he showed that the carbon-14 l a b e l was present i n the alternate l a b e l l i n g pattern expected i f mycophenolic acid was derived from a head to t a i l condensation of a c e t i c acid u n i t s , (see FIG. 2). FIG. 2 The r a d i o a c t i v e acetate work of Birch also indicated that the side chain of mycophenolic acid i s derived from acetate. However, Birc h suggested 1^ that the side chain was formed independently from the aromatic nucleus. To t e s t t h i s theory, B i r c h c a r r i e d out a time study on the rate of incorporation of 14 the l a b e l from [1- C]-acetate. He showed that the l a b e l i s more ra p i d l y introduced i n t o the aromatic nucleus than i n t o the side chain. This can be a t t r i b u t e d to the f a c t that i n the l a t t e r case the a c e t y l coenzyme A i s thought to go v i a a more involved route to isopentenyl pyrophosphate (5). If t h i s were true then i t would seem that the side chain i s of terpenoid o r i g i n . Since i t was known that mevalonic acid (6) can be derived - 5 -18 from a c e t a t e i n the n a t u r a l system and s i n c e Lynen demon-s t r a t e d t h a t i s o p e n t e n y l pyrophosphate can be o b t a i n e d from mevalonic a c i d , t h i s seemed t o be the compound t o f e e d i n order t o t e s t the independent t e r p e n o i d o r i g i n of the C_ s i d e c h a i n . (5) I f the C_ s i d e c h a i n and the aromatic nucleus were formed 7 s e p a r a t e l y then on f e e d i n g r a d i o a c t i v e mevalonic a c i d , no l a b e l s h ould o c c u r i n the aromatic n u c l e u s , u n l e s s the mevalonic a c i d i s degraded t o a c e t a t e p r i o r t o i n c o r p o r a t i o n . A l s o , whether o r not the m u t i l a t e d t e r p e n o i d C_ s i d e c h a i n does a r i s e from C - a l k y l a t i o n w i t h g e r a n y l pyrophosphate ( 7 ) ( d e r i v e d from meval-o n i c a c i d v i a i s o p e n t e n y l pyrophosphate) would be s o l v e d by f e e d i n g r a d i o a c t i v e mevalonic a c i d . (6) (7) B i r c h and c o - w o r k e r s 1 ^ f e d [ 2 - 1 4 C ] - m e v a l o n i c a c i d and degraded the i s o l a t e d mycophenolic a c i d . They found the complete absence of carbon-14 i n the aromatic nucleus and one l a b e l on the expected p o s i t i o n o f the C s i d e c h a i n , (see FIG. 3 ) . - 6 -These f i n d i n g s support the independent f o r m a t i o n of the C-, s i d e c h a i n v i a C - a l k y l a t i o n w i t h g e r a n y l pyrophosphate, (see F I G . 4 ) . HOOC *= 1 4C F I G . 3 I f t h e C._ s i d e c h a i n arose from the condensation of two mevalonic a c i d u n i t s t o form g e r a n y l pyrophosphate (7), which then a l k y l a t e d the aromatic n u c l e u s , then o x i d a t i v e cleavage would be n e c e s s a r y t o reduce the c h a i n t o a C_ c h a i n and a fragment, acetone (see F I G . 4 ) . The acetone so formed should have t h e same p r o p o r t i o n of l a b e l as the mycophenolic a c i d (see F I G . 4 ) . T h i s was shown t o be the case by steam d i s -t i l l i n g r a d i o a c t i v e acetone out of the n a t u r a l system. Thus i t seems l i k e l y , t h a t the p r e c u r s o r o f the C_, s i d e c h a i n was a C fragment, presumably g e r a n y l pyrophosphate. An e x p l a n a t i o n 14 of the l a b e l l i n g p a t t e r n and the proposed r o u t e from [2- C ] -mevalonic a c i d t o mycophenolic a c i d and acetone i s shown below i n F I G . 4. To c o n f i r m t h a t the C_ u n i t i s d e r i v e d from two mevalonic a c i d u n i t s . B i r c h 1 1 f e d [ 4 - 1 4 C ] - m e v a l o n i c a c i d . Based on the 14 e x p l a n a t i o n produced i n F I G . 4, i n c o r p o r a t i o n of [4- C ] -mevalonic a c i d s hould r e s u l t i n the l a b e l l i n g p a t t e r n shown i n F I G . 5 below. T h i s was indeed found t o be the case and pro-v i d e d e x c e l l e n t support f o r the t e r p e n o i d o r i g i n of the C_ s i d e c h a i n . In summary i t can be s a i d t h a t the above experiments of B i r c h imply t h a t the i s o p r e n o i d s i d e chain of mycophenolic a c i d could a r i s e b i o s y n t h e t i c a l l y by condensation of two meva-l o n i c a c i d u n i t s to form a u n i t (presumably geranyl pyrophosphate) which i s i n v o l v e d i n the c o n s t r u c t i o n of the a c i d i c s i d e c h a i n . Subsequent o x i d a t i v e cleavage of the C i n u n i t could then produce the C_ si d e chain and acetone. *= C FIG. 4 - 8 -Whether or not the o x i d a t i v e cleavage occurs before or a f t e r a l k y l a t i o n of the aromatic r i n g remains obscure. A l s o the r e s u l t s o u t l i n e d above do not n e c e s s a r i l y support the i n v o l v -ment of a C a l k y l a t i n g agent or a C a l k y l a t e d i n t e r m e d i a t e . *= C FIG. 5 Another c o n t r i b u t i o n of B i r c h ' towards the establishment of the b i o s y n t h e s i s of mycophenolic a c i d was the di s c o v e r y of the b i o g e n e t i c o r i g i n of the nuclear C- and O-methyl groups. Again by r a d i o a c t i v e t r a c e r experiments he showed t h a t methio-- 9 -nine ( 8 ) , a known n a t u r a l m e t h y l a t i n g agent, was i m p l i c a t e d i n the i n t r o d u c t i o n of the nuclear C- and O-methyl groups i n t o mycophenolic a c i d . Feeding of [methyl- 1 4C]-methionine and subsequent degradation of the recovered mycophenolic a c i d showed an i n c o r p o r a t i o n of about 7 5 per cent of the carbon-14 i n t o the nuclear C- and O-methyl groups. More d e t a i l e d degradations ^^x^^V^^OOH NH 2 (8) showed t h a t more a c t i v i t y was a s s o c i a t e d w i t h the O-methyl group than w i t h the C-methyl group. This would seem to i n d i c a t e t h a t C-and O-methylation occur at d i f f e r e n t times and t h a t C-meth y l a t i o n occurs before O-methylation. This work was confirmed by experiments c a r r i e d out by 13 Lederer and co-workers , who fed deuterium l a b e l l e d methionine and showed by degradation t h a t the deuterium was present i n the nuclear, methyl group and methoxy group. Thus having e s t a b l i s h e d an o v e r a l l g e n e r a l pathway from 11 12 acetate to mycophenolic a c i d , B i r c h ' then turned h i s a t t e n -t i o n to determining the s p e c i f i c steps i n v o l v e d . A p o s s i b l e i n t e r m e d i a t e was o r s e l l i n i c a c i d (3) si n c e t h i s compound had the general s t r u c t u r a l f e a t u r e s of mycophenolic a c i d and was a 14 known n a t u r a l product. B i r c h thus fed [5- C ] - o r s e l l i n i c a c i d to P e n i c i l l i u m brevi-compactum w i t h d i s s a p p o i n t i n g r e s u l t s . The r e s u l t s showed t h a t a very poor i n c o r p o r a t i o n had occurred. The carbon-14 l a b e l appeared i n the expected p o s i t i o n of myco-phe n o l i c a c i d to about one qu a r t e r of what i t should have been i n d i c a t i n g p r i o r degradation of o r s e l l i n i c a c i d to acetate by the mould. This work was meant to solv e the p u z z l e of whether or not mycophenolic a c i d arose from acetate v i a o r s e l l i n i c a c i d type compounds (from a C p o l y k e t i d e chain) or compounds of the typ 8 (9) (from a p o l y k e t i d e c h a i n ) . However, the i n c o n c l u s i v e COOH OH (R = H or OH) R (9) nature of the r e s u l t s d i d nothing to solv e the controversy of mycophenolic a c i d a r i s i n g from a C Q or C polyketomethylene o 10 p r e c u r s o r . Thus B i r c h had demonstrated the acetate o r i g i n of the aromatic r i n g , the t e r p e n o i d o r i g i n of the C_, s i d e chain and the methionine o r i g i n of the nu c l e a r methyl groups. From t h i s he p o s t u l a t e d the set of r e a c t i o n s shown i n FIG. 6 as being i n v o l v e d i n . t h e t o t a l b i o s y n t h e s i s of mycophenolic a c i d . The only unknown f a c t o r i s the sequence of r e a c t i o n s which lead from acetate t o mycophenolic a c i d . Since the t e r m i n a t i o n of the work presented i n . t h i s t h e s i f u r t h e r r e s u l t s on the b i o s y n t h e s i s of mycophenolic a c i d have 14 very r e c e n t l y been reporte d by Canonica and h i s co-workers - 11 -Separate feeding experiments u s i n g p h t h a l i d e s (10), (11), (12) and (13) have produced r e s u l t s which show c o n c l u s i v e l y t h a t 1 4 only [7- C]-5,7-dihydroxy-4-methylphthalide (10) xs a t r u e precursor of mycophenolic a c i d . As a r e s u l t of these f i n d i n g s OH (R = CH 3 or CH(OH)-CH(OH)-CH3) 0 F I G . 6 - 12 -Canonica and h i s colleagues c l a i m to have e s t a b l i s h e d the p a i n t at which; 1) the methyl group at C-4 and the i s o p r e n o i d s i d e c h a i n are int r o d u c e d , 2) the lact o n e r i n g forms and 3) the stage at which m e t h y l a t i o n of the p h e n o l i c hydroxyls occurs. (12) (13) However, t h e i r experimental evidence cannot be used t o determine when C-methylation occurs. The f a c t t h a t (10) i s and (11) i s not i n c o r p o r a t e d does not show t h a t C-methylation occurs before a r o m a t i z a t i o n as they c l a i m . I t i s q u i t e p o s s i b l e t h a t aroraat-i z a t i o n to o r s e l l i n i c a c i d can occur, then m e t h y l a t i o n and then p h t h a l i d e r i n g formation t o y i e l d (10). I l l BIOLOGICAL IMPORTANCE Although mycophenolic a c i d has been e x t e n s i v e l y s t u d i e d - 13 -because of i t s i n t e r e s t i n g b i o g e n e s i s , r e c e n t l y i t s b i o l o g i c a l a c t i v i t y has aroused much i n t e r e s t . Known f o r sometime as an a n t i b i o t i c , more r e c e n t l y much work has been done on i t s a n t i -cancer a c t i v i t y . 19 20 In two papers i n 1969 F r a n k l i n and co-workers ' repor t e d an e x t e n s i v e i n v e s t i g a t i o n of the i n h i b i t i o n of n u c l e i c a c i d s y n t h e s i s by mycophenolic a c i d , thereby i n h i b i t i n g cancerous growth. Apparently the i n h i b i t i o n i s due t o the a b i l i t y of mycophenolic a c i d to i n h i b i t the enzyme IMP dehydrogenase and i n t e r f e r e w i t h the b i o s y n t h e s i s of guanine n u c l e o t i d e s . The study showed t h a t mycophenolic a c i d has a potent a n t i m i t o t i c e f f e c t on mammalian c e l l s and t h a t i t i s an e f f e c t i v e antitumor agent w i t h a marked a c t i v i t y a g a i n s t a range of tumors i n mice and r a t s . The compound has unusual p r o p e r t i e s i n t h a t the i n h i b i t i o n of DNA s y n t h e s i s can be reversed by guanine, the s i d e e f f e c t s are n e g l i g i b l e , i t i s r e l a t i v e l y nontoxic and the e f f e c t s of overdosage dissappear on d i s c o n t i n u i n g the drug. Thus from the experimental i n v e s t i g a t i o n s of F r a n k l i n , i t seems p o s s i b l e t h a t mycophenolic a c i d may i n f u t u r e serve as a cure f o r some types of cancer. DISCUSSION I BIOSYNTHESIS 68 At the outset of our i n v e s t i g a t i o n s we were interested i n the order or sequence i n v o l v e d i n the b i o s y n t h e s i s of mycophenolic a c i d . Our work was i n i t i a l l y d i v i d e d i n t o two phases. In the f i r s t phase we were i n t e r e s t e d i n determining the nature of the l e a s t complex aromatic intermediate on the b i o s y n t h e t i c route. For t h i s purpose we decided t o t e s t the precursor a c t i v i t y of [ l ' - 1 4 C ] - o r s e l l i n i c a c i d (22) and [1 1- 1 4C]-2,4-dihydroxy-5,6-dimethylbenzoic a c i d (23). (22) (23) By f e e d i n g these two compounds, s e v e r a l p o s s i b l e c o n c l u -s i o n s c o u l d be reached: 1) i f (22) and/or (23) were in c o r p o r a t e d s p e c i f i c a l l y then the C p o l y k e t i d e o r i g i n of mycophenolic a c i d o would be e s t a b l i s h e d . 2) i f both (22) and (23) were i n c o r p o r a t e d then (22) would be a precursor of (23) which subsequently goes on t o mycophenolic a c i d . 3) i f only (22) were in c o r p o r a t e d then presumably C-methylation occurred a f t e r p h t h a l i d e formation. 4) i f only (23) were i n c o r p o r a t e d then t h i s would i n d i c a t e t h a t C-methylation occurred before a r o m a t i z a t i o n . 5) i f n e i t h e r (22) or (23) was i n c o r p o r a t e d then a new search f o r the b a s i c aroma-t i c p r e c u r s o r would be i n i t i a t e d . - 15 -In order to t e s t whether or not (22) and/or (23) were precur s o r s of mycophenolic a c i d , i t became necessary to synthe-s i z e them w i t h r a d i o a c t i v i t y at a p a r t i c u l a r s i t e i n t h e i r s t r u c t u r e . The s y n t h e t i c routes used are shown i n f l o w sheet I wi t h sequences A and B f o r (22) and (23) r e s p e c t i v e l y . The s y n t h e s i s of (22) was c a r r i e d out i n a manner s i m i l a r to t h a t d e s c r i b e d by Thomas^ . Treatment of o r c i n o l (25) w i t h [•'•^C]-sodium cyanide, z i n c cyanide and hydrogen c h l o r i d e i n e t h e r 2 ^ (Gatterman r e a c t i o n ) provided [1'-1 4C]-orcylaldehyde (26) i n 84% y i e l d . This was then converted t o [ 1 ' - 1 4 C ] - o r s e l -l i n i c a c i d (22) by o x i d a t i o n of the 2,4-0-dicarbethoxy d e r i v a -t i v e (27) w i t h potassium permanganate t o ( 2 8 ) 2 4 f o l l o w e d by s a p o n i f i c a t i o n . P r i o r conversion of the p h e n o l i c hydroxyls to the carbethoxy d e r i v a t i v e s was necessary s i n c e i t was found t h a t d i r e c t o x i d a t i o n of (26) to (22) proceeded i n extremely low y i e l d . The r e l u c t a n c e of the hydroxy aldehyde (26) t o o x i d i z e may be due to the strong i n t r a m o l e c u l a r hydrogen bond present i n the molecule as shown i n FIG. 8. FIG. 8 Removal of the b l o c k i n g groups a f t e r o x i d a t i o n proved somewhat d i f f i c u l t due t o the ease w i t h which o r s e l l i n i c a c i d FLOW SHEET I (R-COOCHoCHo) (*=14C SEQUENCE A J OH - 17 -d e c a r b o x y l a t e s . However, d e c a r b o x y l a t i o n was k e p t a t a minimum by u s i n g m e t h a n o l as s o l v e n t f o r t h e s a p o n i f i c a t i o n and k e e p i n g o t h e r e a c t i o n a t 0 C and under n i t r o g e n a t a l l t i m e s , e s p e c i a l l y when t h e s o l u t i o n was a c i d i c . Thus (22) was o b t a i n e d i n good y i e l d and a f t e r p u r i f i c a t i o n t o c o n s t a n t r a d i o a c t i v i t y was a d m i n i s t e r e d t o P e n i c i l l i u m brevi-compactum. The s y n t h e s i s o f (23) was a c c o m p l i s h e d u s i n g t h e method o f P e t t e r s o n ^ 1 . T h i s method i s i d e n t i c a l i n a l l r e s p e c t s t o t h a t f o r t h e s y n t h e s i s o f (22) e x c e p t t h a t 3 , 5 - d i h y d r o x y - o - x y l e n e (29) i s used i n t h e i n i t i a l r e a c t i o n i n s t e a d o f o r c i n o l ( 2 5 ) . Reduc-t i o n o f o r c y l a l d e h y d e (45) w i t h z i n c amalgam i n h y d r o c h l o r i c a c i d (Clemmenson r e d u c t i o n ) p r o c e e d e d s m o o t h l y and gave (29) i n al m o s t q u a n t i t a t i v e y i e l d . A g a i n some d i f f i c u l t y was e x p e r i -enced i n t h e . f i n a l s a p o n i f i c a t i o n t o th e h y d r o x y a c i d ( 2 3 ) . I t was f o u n d t h a t t h i s a c i d was much more prone t o d e c a r b o x y l a t i o n t h a n (22) . Thus where i t was p o s s i b l e t o use aqueous sodium h y d r o x i d e i n t h e c a s e o f ( 2 2 ) , we found t h a t f o r s a p o n i f i c a t i o n t o (23) i t was n e c e s s a r y t o use 20% a l c o h o l i c p o t a s s i u m h y d r o x -i d e . The a c i d (23) was o b t a i n e d i n l o w e r y i e l d t h a n (22) and a l s o p r o v e d more d i f f i c u l t t o p u r i f y . A f t e r , p u r i f i c a t i o n t o c o n s t a n t s p e c i f i c a c t i v i t y ( e s t a b -l i s h e d by l i q u i d s c i n t i l l a t i o n c o u n t i n g ) , t h e a c i d s (22) (2.5 x 10 J mc/mm) and (23) (5.4 x 10 mc/mm) were d i s s o l v e d i n e t h a n o l and a d m i n i s t e r e d s e p a r a t e l y t o shake c u l t u r e s o f P e n i -c i l l i u m brevi-compactum (grown on Raulin-Thom medium) when t h e pH o f th e b r o t h was 5.2 t o 5.6. When pH 7 was r e a c h e d (14 t o 20 d a y s ) , m y c o p h e n o l i c a c i d was i s o l a t e d from t h e p a r a l l e l e x p e r i m e n t s by e x t r a c t i n g b o t h t h e b r o t h and m ycelium w i t h e t h e r . - 18 -P u r i f i c a t i o n t o constant r a d i o a c t i v i t y was achieved by t r i t u r -a t i o n w i t h ether and c r y s t a l l i z a t i o n from e t h y l a c e t a t e . I t was found t h a t when the P e n i c i l l i u m brevi-compactum was grown on Czapek-Dox medium, the c o r r e c t pH 1s were reached i n s h o r t e r times but the recovered mycophenolic a c i d was much more impure and more d i f f i c u l t to p u r i f y . Mycophenolic a c i d from the o r s e l l i n i c a c i d (22) experiment was r a d i o i n a c t i v e w h i l e t h a t from the experiment using the a c i d (23) as precursor had a s p e c i f i c a c t i v i t y of 6.3 x 10 -^ mc/mm (an i n c o r p o r a t i o n of 11.5%). To determine how the carbon-14 was i n c o r p o r a t e d (randomly or s p e c i f i c a l l y ) , a degradation of mycophenolic a c i d was c a r r i e d out. I f (23) were i n i t i a l l y degraded to acetate by the mould before i n c o r p o r a t i o n . t h e n one would expect a random d i s t r i b u -t i o n of the l a b e l throughout mycophenolic a c i d . Other workerslO had found t h a t o z o n o l y s i s of the C 7 s i d e c hain was s u f f i c i e n t to determine i f randomization had occurred, s i n c e both the l e v u l i n i c a c i d (33) and the aldehyde (34) would be r a d i o a c t i v e . However, i f i n c o r p o r a t i o n were s p e c i f i c then only the aldehyde (34) would be r a d i o a c t i v e ( i e . have the same r a d i o a c t i v i t y as the o r i g i n a l mycophenolic a c i d ) . We found t h a t o z o n o l y s i s of mycophenolic a c i d i n methanol at -78°C f o l l o w e d by decomposition of the ozonide w i t h e i t h e r dimethyl s u l f i d e ^ or water7 y i e l d e d only a crude y e l l o w o i l . S e v e r a l attempts t o p u r i f y t h i s o i l and i s o l a t e l e v u l i n i c a c i d (33) and the aldehyde (34) r e s u l t e d i n the i s o l a t i o n of only a s m a l l amount of l e v u l i n i c a c i d (33) , too impure to count r a d i o -a c t i v i t y . We concluded t h a t the i n a b i l i t y to i s o l a t e the - 119- -aldehyde (34) was due t o i t s i n s t a b i l i t y or r e a c t i v i t y , s i n c e other w o r k e r s 2 8 i n our l a b o r a t o r y had found t h a t even a pure sample of (34) decomposes on standing to g i v e s e v e r a l products. I t i s p o s s i b l e t h a t i n t e r a c t i o n between the p h e n o l i c h y d r o x y l and the aldehydic f u n c t i o n i s o c c u r r i n g . Thus we decided to convert the f r e e hydroxyl of mycophenolic a c i d t o i t s methoxy d e r i v a t i v e w i t h diazomethane, g i v i n g the methyl ether methyl e s t e r of mycophenolic a c i d (35). This was then ozonized i n the same manner as above. Again the only i s o l a b l e product was methyl l e v u l i n a t e (36) and the aldehyde (37) was not d e t e c t a b l e . HOOC An attempt t o convert methyl l e v u l i n a t e t o i t s semicarbazone d e r i v a t i v e , i n order to o b t a i n a pure sample f o r l i q u i d s c i n -t i l l a t i o n counting was a l s o u n s u c c e s s f u l s i n c e the d e r i v a t i v e formed very s l o w l y and i n very poor y i e l d . (35) (36) (37) Thus i t became apparent t h a t we had t o cleave the ozonide of e i t h e r mycophenolic a c i d (1) or i t s methyl ether methyl e s t e r - 20 -FLOW S H E E T I I (41a) (41b) _ 21 _ (35) i n such a way as to y i e l d some other f u n c t i o n a l group than the aldehyde. One p o s s i b i l i t y was r e d u c t i v e cleavage w i t h l i t h -ium aluminum h y d r i d e 2 ^ , which should y i e l d the a l c o h o l (38) and y - v a l e r o l a c t o n e (39). However, the only product i s o l a t e d was y - v a l e r o l a c t o n e (39), w i t h no t r a c e of the a l c o h o l (38). ROOC The other p o s s i b i l i t y was o x i d a t i v e cleavage, s i n c e the a c i d (40) had been reported as a s t a b l e c r y s t a l l i n e s o l i d . Thus we ozonized the methyl ether methyl e s t e r of mycophenolic a c i d (35) i n methanol at -78°C. F o l l o w i n g the procedure of Perry-^O, the methanol was removed and the ozonide t r e a t e d w i t h 30% hydrogen peroxide and formic a c i d at 100°C f o r one hour. The r e s u l t was the comparatively easy i s o l a t i o n of the dimethoxy a c i d (4 0) which on p u r i f i c a t i o n was found t o have t o t a l r e t e n -t i o n of r a d i o a c t i v i t y (1.77 x 10"^ mc/mm) from the o r i g i n a l mycophenolic a c i d . See flow sheet I I . (page 20) Thus we had e s t a b l i s h e d t h a t i n c o r p o r a t i o n was nonrandom, s i n c e a l l the a c t i v i t y was i n the aromatic nucleus of mycophenolic a c i d . I f i n c o r p o r a t i o n was s p e c i f i c , then the carbon-14 l a b e l should be l o c a t e d e n t i r e l y on one carbon as shown i n FIG. 9. (page 22) To confirm t h a t t h i s was indeed the.case, we employed the method of B i r k i n s h a w 7 to decarboxylate the dimethoxy a c i d (40) t o the coumaran-2-one (41a) and/or (41b)' and carbon d i o x i d e . I f s p e c i f i c - 22 -i n c o r p o r a t i o n had occurred, then the carbon d i o x i d e should have n e a r l y 100% r e t e n t i o n of the r a d i o a c t i v i t y of (40). Thus (40) was t r e a t e d w i t h red phosphorus and h y d r i o d i c a c i d under r e f l u x t o y i e l d (41a) and/or (41b), which was r a d i o i n a c t i v e , and carbon d i o x i d e ( i s o l a t e d as barium carbonate and counted i n suspension-^) c o n t a i n i n g 96% of the o r i g i n a l r a d i o a c t i v i t y of the dimethoxy a c i d (40) . Although Birkinshaw^ r e p o r t e d t h a t the product of the d e c a r b o x y l a t i o n of (40) was the coumaran-2-one (41a) and/or (41b), the only proof he had f o r t h i s assumption was a m e l t i n g p o i n t , a mass a n a l y s i s , i t s s o l u b i l i t y i n sodium carbonate and a y e l l o w f e r r i c c h l o r i d e t e s t . We have i s o l a t e d (41a) and/or (41b) and shown i t to have the same m e l t i n g p o i n t and f e r r i c c h l o r i d e t e s t as t h a t reported by Birkinshaw. To provide f u r -t h e r proof f o r the s t r u c t u r e we have taken i n f r a r e d , u l t r a v i o l e t , n u clear magnetic resonance and mass s p e c t r a l data. These data confirm t h a t the product of the d e c a r b o x y l a t i o n of (40) i s indeed the coumaran-2-one (41a) and/or (41b). (40) (41a) (41b) These r e s u l t s show t h a t s p e c i f i c i n c o r p o r a t i o n of r a d i o -a c t i v i t y from (23) i n t o mycophenolic a c i d had occurred and support the r o l e of t h i s hydroxy a c i d as a pre c u r s o r i n the b i o s y n t h e t i c sequence. From t h i s i t can be seen t h a t mycophen-o l i c a c i d i s of Cg p o l y k e t i d e o r i g i n and t h a t C-methylation occurs before a r o m a t i z a t i o n . Thus other secondary transforma-t i o n s (0-methylation, p h t h a l i d e r i n g formation, and c o n s t r u c t i o n of the C 7 s i d e chain) are o c c u r r i n g a f t e r a r o m a t i z a t i o n . The next step i n the b i o s y n t h e t i c sequence seems t o be the formation of the p h t h a l i d e r i n g , s i n c e Canonica and h i s c o l l e a -gues-*-4 have reported the high s p e c i f i c i n c o r p o r a t i o n of 5,7-dihydroxy-4-methylphthalide (10) i n t o mycophenolic a c i d . A revised- t e n t a t i v e scheme f o r the b i o s y n t h e s i s of mycophenolic a c i d can be considered as shown i n FIG. 10. FIG. 10 - 24 -The second phase of our work was a s s o c i a t e d w i t h attempts t o confirm or r e f u t e the suggestion t h a t the C 7 moiety i s d e r i v e d from g e r a n i o l . To t h i s end we fed [ 1 - 1 4 C ] - g e r a n i o l (24) t o P e n i c i l l i u m brevi-compactum. Previous work-*2'-^ i n our l a b o r a t o r y had shown t h a t admin-i s t r a t i o n of t r i t i u m l a b e l l e d g e r a n i o l (42) and geranyl pyro-phosphate (43) to P e n i c i l l i u m brevi-compactum y i e l d e d r a d i o -a c t i v e mycophenolic. a c i d . However, the r e s u l t s were i n c o n c l u s i v e s i n c e degradation of the recovered mycophenolic a c i d showed th a t c o n s i d e r a b l e randomization of the l a b e l had occurred. I t was thought t h a t t h i s randomization might be due to the redox system i n nature removing and r e p l a c i n g t r i t i u m w i t h hydrogen. However, i t was a l s o p o s s i b l e t h a t g e r a n i o l o r v g e r a n y l pyrophosphate were degraded to acetate before i n c o r p o r a t i o n and t h i s would a l s o e x p l a i n randomization of the l a b e l . (42) (43) To c l a r i f y t h i s r e s u l t , we decided to feed [ 1 - 1 4 C ] - g e r a n i o l (24) s i n c e w i t h t h i s molecule randomized i n c o r p o r a t i o n can occur only i f p r i o r degradation of the carbon s k e l e t o n occurs. The r a d i o a c t i v e g e r a n i o l was prepared by Dr. J . F a i r l i e i n our l a b -o r a t o r y and was fed t o P e n i c i l l i u m brevi-compactum i n a water s o l u t i o n c o n t a i n i n g tween-20, an " e m u l s i f i e r . Growth time, recovery, p u r i f i c a t i o n and degradation of mycophenolic a c i d were e x a c t l y as des c r i b e d before (see a l s o e x p e r i m e n t a l ) . We found t h a t the l a b e l was d i s t r i b u t e d i n e x a c t l y the same propor-t i o n s as B i r c h ^ ' l O found when he fed [ l - ^ - ^ c ] - a c e t i c a c i d . This would suggest t h a t the [1- 1 4C] - g e r a n i o l was degraded to acetate and then i n c o r p o r a t e d . This does not n e c e s s a r i l y r u l e out a g e r a n i o l type precursor of the C-j s i d e chain s i n c e the g e r a n i o l may be degraded before i t can reach the s i t e of synthe-s i s i n the n a t u r a l system. A summary of the r a d i o a c t i v e work i s given i n Table 1. Table 1. ( a l l f i g u r e s are s p e c i f i c a c t i v i t i e s i n u n i t s of mc/mm) Compound fed (22) (23 ) (24) Mycophenolic a c i d recovered 0 6 .318 X 10" 5 6 .070 X IO" 4 Mycophenolic a c i d d i l u t e d 0 . 1 .782 X 10~ 5 2 5 .685 .332 X X IO""4* 10 _ 5+ Mycophenolic a c i d ether e s t e r (35) 0 1 .782 X 10~ 5 2 5 . 685 .332 X X -4 10 I* 10 _ 5+ Ozonolysis product (40) 0 1 .777 X 10" 5 1 .985 X l O " 4 Decarboxylation product (41) 0 0 3 .162 X I O - 5 Barium carbonate 0 1 .714 X 10" 5 7 .778 X 10~ 6 [co 2 ] *-used f o r the o z o n l y s i s r e a c t i o n . +-used f o r the d e c a r b o x y l a t i o n r e a c t i o n . - 26 -I I ATTEMPTED SYNTHESIS Having e s t a b l i s h e d a t e n t a t i v e sequence for. the biosynthe-s i s of mycophenolic a c i d , we thought i t would be i n t e r e s t i n g t o attempt a l a b o r a t o r y s y n t h e s i s using some of the suspected n a t u r a l p r e c u r s o r s . Three d i f f e r e n t s y n t h e t i c routes were t r i e d using o r c i n o l (25) and 3,5-dimethoxybenzoic a c i d (44) as s t a r t -i ng m a t e r i a l s , s i n c e these were the aromatic compounds a v a i l a b l e w i t h the b a s i c s k e l e t o n of mycophenolic a c i d . The f i r s t route (see flow sheet I I I ) i n v o l v e d the use of 2.4- dihydroxy-5,6-dimethylbenzoic a c i d (49) as an in t e r m e d i a t e . O r c i n o l (25) was converted t o orcylaldehyde (45) using the method of Adams and L e v i n e ^ , which i n v o l v e d r e a c t i o n of (25) w i t h hydrogen cyanide and hydrogen c h l o r i d e t o giv e the imide h y d r o c h l o r i d e which was subsequently hydrolyzed to (45). Re-d u c t i o n of (45) v i a Clemmenson r e d u c t i o n c o n d i t i o n s gave 3.5- dihydroxy-o-xylene (29) i n good y i e l d . Treatment of (29) under the m o d i f i e d Gatterman c o n d i t i o n s of Adams and Levine23 gave the aldehyde (46), which was converted t o the a c i d (49) by using the method of Hoesch 2 4. This i n v o l v e d b l o c k i n g the p h e n o l i c hydroxyls as dicarbethoxy d e r i v a t i v e s before o x i d a t i o n s i n c e the hydrogen bond (see FIG. 8) prevents o x i d a t i o n from o c c u r r i n g . The dicarbethoxy aldehyde (47) was formed by t r e a t -ment of (46) w i t h e t h y l chloroformate i n the presence of base and was subsequently o x i d i z e d t o the corresponding a c i d (48) using potassium permanganate. S a p o n i f i c a t i o n of (48) w i t h 20% a l c o h o l i c potassium hydroxide at 0°C under n i t r o g e n proceeds smoothly and on a c i d i f i c a t i o n g i v e s the a c i d (49) i n f a i r y i e l d . Some d e c a r b o x y l a t i o n occurs but i s reduced to a minimum i f the - 28 -r e a c t i o n i s kept at 0°C and under n i t r o g e n at a l l times. Treatment of the hydroxy a c i d (49) w i t h diazomethane i n ether gave the monomethyl ether methyl e s t e r (50). I r r a d i a t i o n of (50) w i t h bromine i n carbon t e t r a c h l o r i d e s o l u t i o n - ^ produced the dibromomethyl compound (51). Formation of the p h t h a l i d e (52) was accomplished by r e f l u x i n g (51) i n dioxan-water f o r 24 hours-^S. P u r i f i c a t i o n of (52) was found to be exceedingly d i f f i c u l t and i t was thought t h i s might be due t o the presence of the bromomethyl group. Thus we decided t h a t hydrogenation of (52) to (53) might a i d i n p u r i f i c a t i o n . However, hydrogen-a t i o n over p a l l a d i u m on c h a r c o a l f a i l e d t o y i e l d (53) g i v i n g only a black gummy t a r . On the b a s i s of these r e s u l t s we decided t h a t i f the methyl group at C-4 were introduced a f t e r bromination and p h t h a l i d e formation we would have a b e t t e r chance of success. The second s y n t h e t i c sequence (see f l o w sheet IV) i n v o l v e d o r s e l l i n i c a c i d (3) as an i n t e r m e d i a t e . O r c i n o l (25) was con-v e r t e d t o orcylaldehyde (45) as d e s c r i b e d above (page 24). Conversion t o the dicarbethoxy d e r i v a t i v e (55) f o l l o w e d by o x i -d a t i o n y i e l d e d the a c i d (56) which was hydrolyzed t o o r s e l l i n i c a c i d (3). The o r s e l l i n i c a c id.so obtained.was methylated w i t h diazomethane to g i v e (57) and t h i s was brominated i n an i d e n t -i c a l manner to (50) to g i v e the bromomethyl compound (58). The p h t h a l i d e (59) formed smoothly and i n good y i e l d by r e f l u x -ing (58) i n dioxan-water f o r 24 h o u r s ^ . Thus we had an intermediate (59) which a f t e r two simple a l k y l a t i o n s and s e l e c t i v e o z o n o l y s i s might be expected to give mycophenolic a c i d . Treatment of (59) under F r i e d e l - C r a f t s c o n d i t i o n s w i t h methyl c h l o r i d e and aluminum c h l o r i d e was - 29 -FLOW SHEET IV (R-COOCH2CH3) - 30 -un s u c c e s s f u l i n producing the d e s i r e d C-methylation at p o s i t i o n 4. S i m i l a r r e s u l t s were obtained when g e r a n y l a t i o n w i t h g e r a n y l h a l i d e s w a s attempted. Treatment of (59) w i t h e i t h e r g e r a n y l c h l o r i d e or bromide and n - b u t y l l i t h i u m using the method of C a r d i l l o " ^ f a i l e d to induce g e r a n y l a t i o n at e i t h e r p o s i t i o n 6 or 4.. A l s o t r i e d without success was the method of Mechoulam37, which i n v o l v e d s t i r r i n g (59) w i t h g e r a n i o l and p-toluene s u l f o n i c a c i d i n methylene c h l o r i d e at room temperature f o r 2 4 hours. Since i t seemed impossible to a l k y l a t e (59) t o gi v e e i t h e r (60) or (61) we devised a t h i r d s y n t h e t i c approach (see flow sheet V). We considered t h a t the a l k y l a t i o n s had f a i l e d due t o the d e a c t i v a t i n g e f f e c t of the p h t h a l i d e carbonyl on the aromatic system. Mechoulam37 reported the g e r a n y l a t i o n of o l i v e t o l (62) and s i n c e t h i s i s an analog of o r c i n o l (25) we attempted to ger a n y l a t e o r c i n o l (25) i n the same manner using g e r a n i o l and p-toluene s u l f o n i c a c i d i n methylene c h l o r i d e . The attempt was s u c c e s s f u l g i v i n g the product (63). (62) (63) Thus we concluded t h a t g e r a n y l a t i o n had t o be accomplished before i n t r o d u c t i o n of the carbonyl on the aromatic r i n g . How-ever, t h i s meant t h a t a l l r e a c t i o n s a f t e r g e r a n y l a t i o n must not a f f e c t the ge r a n y l group. I t was found t h a t the aromatic methyl - 32 -group had t o be f u n c t i o n a l i z e d t o the bromide or a l c o h o l before g e r a n y l a t i o n s i n c e the f r e e r a d i c a l bromination c o n d i t i o n s would a l s o a t t a c k the g e r a n y l group. A l l the other subsequent r e a c t i o n s (see flow sheet V) could be m o d i f i e d so as to leave the g e r a n y l group i n t a c t . Thus the next step was t o prepare the bromomethyl d e r i v -a t i v e (64) of o r c i n o l (25). However, on attempting the bromin-a t i o n w i t h bromine i n carbon t e t r a c h l o r i d e under i r r a d i a t i o n and w i t h N-bromosuccinimide under v a r y i n g c o n d i t i o n s , we were never able t o prepare (64). The products i s o l a t e d i n a l l cases had been brominated on the aromatic r i n g . This was v e r i -f i e d by the abscence of the aromatic proton resonance i n the n u c l e a r magnetic resonance s p e c t r a . To overcome t h i s o b s t a c l e we set out t o prepare t h e . a l c o h o l (65) s i n c e i n the p h t h a l i d e formation step the bromide i s probably hydrolyzed t o the a l c o -h o l which then l a c t o n i z e s t o form the p h t h a l i d e . OH OH (64) (65) A l i t e r a t u r e survey revealed t h a t (65) was exceedingly u n s t a b l e . However, we a l s o found t h a t the dimethoxy d e r i v a t i v e (66) of (65) was a s t a b l e s o l i d and s i n c e 3,5-dimethoxybenzoic a c i d (44) was a v a i l a b l e , we reduced t h i s w i t h l i t h i u m aluminum hydride t o g i v e the alcohol.(66) i n good y i e l d . Since the a c t i v a t i n g powers of h y d r o x y l and methoxyl are s i m i l a r , - 33 -g e r a n y l a t i o n (using Mechoulam's method^ 7) should a l s o occur on (66) . Conversion of the a l c o h o l (66) to the benzyl, ether d e r i v a t i v e ( 6 7 ) ^ w a s f o l l o w e d by treatment w i t h g e r a n i o l and p-toluene s u l f o n i c acid-* 7 i n methylene c h l o r i d e s o l u t i o n . The r e s u l t was negative. Compound (68) could not be detected a f t e r 48 hours of r e a c t i o n . From the above work i t can be seen t h a t a l k y l a t i o n of the aromatic s k e l e t o n of mycophenolic a c i d i s exceedingly d i f f i c u l t and overcoming t h i s i s the key t o a s u c c e s s f u l s y n t h e s i s of t h i s i n t e r e s t i n g n a t u r a l product.using suspected b i o s y n t h e t i c i n t e r -mediates. Only one s y n t h e s i s of mycophenolic a c i d has been 3 8 reported and t h i s appeared w h i l e our own i n v e s t i g a t i o n s were i n progress. This s y n t h e s i s i s not analogous to the bio s y n t h e -t i c route and at no time i s a complete g e r a n y l group introduced i n t o the aromatic r i n g . In a very recent paper, a f t e r termin-a t i o n of t h i s work, C a n o n i c a 1 4 claims to have introduced a 6-ge r a n y l group i n t o the p h t h a l i d e (59) using s i l v e r oxide as a c a t a l y s t w i t h g e r a n y l bromide. EXPERIMENTAL The U l t r a v i o l e t s p e c t r a were r e c o r d e d i n methanol on a UNICAM SP800 s p e c t r o p h o t o m e t e r . The I n f r a r e d s p e c t r a were r e c o r d e d on a P e r k i n Elmer 137 s p e c t r o p h o t o m e t e r w i t h sodium c h l o r i d e c e l l s u s i n g e i t h e r n u j o l m u l l s o r c h l o r o f o r m s o l u t i o n s . The N u c l e a r M a g n e t i c Resonance s p e c t r a were r e c o r d e d on T-60, A-60 and C-60 s p e c t r o p h o t o m e t e r s w i t h 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 and t h e s o l v e n t s as i n d i c a t e d i n t h e t e x t . M e l t i n g p o i n t s were r e c o r d e d on a K o f l e r M i c r o H e a t i n g Stage ( R e i c h e r t ) and a r e u n c o r r e c t e d . The c h e m i c a l s used came l a r g e l y from F i s c h e r , A l d r i c h , Eastman O r g a n i c and B r i t i s h Drug Houses. The r a d i o a c t i v e compounds were o b t a i n e d from New E n g l a n d N u c l e a r . The r a d i o a c t i v e s c i n t i l l a t i o n c o u n t i n g was c a r r i e d o u t i n s c i n -t i l l a t i o n v i a l s by d i s s o l v i n g t h e sample i n one m i l l i l i t r e o f methanol and m i x i n g w i t h f o u r t e e n m i l l i l i t r e s o f L i q u i f l u o r ( N E N ) - t o l u e n e , t h e s c i n t i l l a t i n g a gent. The s c i n t i l l a t i o n c o u n t e r used was a MARK I l i q u i d s c i n t i l l a t i o n s y stem made by C h i c a g o N u c l e a r . The r a d i o a c t i v e p r o d u c t s were i d e n t i f i e d by c o m p a r i s o n o f t h e i r m e l t i n g p o i n t s , u l t r a v i o l e t and i n f r a r e d s p e c t r a t o t h o s e o f t h e c o m p l e t e l y c h a r a c t e r i z e d n o n r a d i o a c t i v e compounds. T h i s was n e c e s s a r y due t o t h e s m a l l amount o f r a d i o a c t i v e m a t e r i a l a v a i l a b l e as w e l l as i t s v a l u e . - 3 5 -PREPARATION OF [1'- 1 4C]-ORCYLALDEHYDE(26) 22 The method used was as d e s c r i b e d by Thomas . . Anhydrous o r c i n o l (25) (1.0 g, 0.008 mol) was d i s s o l v e d i n anhydrous e t h e r (30 ml) i n a 50 ml 3-necked f l a s k e q u i p p e d w i t h a m e c h a n i c a l s t i r r e r , c o n denser and gas i n l e t t u b e . Then z i n c 14 c y a n i d e (1.5 g) and C-sodium c y a n i d e (3.1 mg) were added. The m i x t u r e was s t i r r e d v i g o r o u s l y and anhydrous hydrogen c h l o r i d e gas was p a s s e d i n r a p i d l y . The r e s u l t was t h e s e p a r a -t i o n o f a red-brown o i l w h i c h t u r n e d t o a p a l e orange s o l i d a f t e r 1/2 h r . The r e a c t i o n was st o p p e d a f t e r 1 1/2 h r and t h e e t h e r was d e c a n t e d l e a v i n g a gummy orange s o l i d . Water (30 ml) was added s l o w l y r e s u l t i n g i n a v i g o r o u s r e a c t i o n . T h i s was r e f l u x e d f o r 2-3 min and th e n c o o l e d t o 0°C r e s u l t i n g i n an orange p r e c i p i t a t e . F i l t r a t i o n and d r y i n g y i e l d e d ' [ l ' - 1 4 C ] -o r c y l a l d e h y d e (26) (1.03 g, 84%) m.p. 182-185°C. 14 PREPARATION OF [!'- C ] - 2 , 4-O-DICARBETHOXY- 5 -METHYJJBENZ ALDEHYDE (27) 22 The method used was as d e s c r i b e d by Thomas Crude [ 1 ' - 1 4 C ] - o r c y l a l d e h y d e ( 2 6 ) (1.03 g, 0.007 mol) was d i s -s o l v e d i n a c e t o n e (5 ml) and e t h y l c h l o r o f o r m a t e (0.63 ml) was added. The s o l u t i o n was s t i r r e d a t 0°C and IN sodium h y d r o x i d e (6.5 ml) was s l o w l y added. A f t e r p a r t i a l s e p a r a t i o n o f a gummy s o l i d , more ac e t o n e (7 ml) was added and t h e s o l u t i o n t r e a t e d t w i c e more w i t h e t h y l c h l o r o f o r m a t e (0.63 ml) and IN sodium h y d r o x i d e (6.5 m l ) . A f t e r s t i r r i n g a t 0°C f o r 1/2 h r , t h e s o l u t i o n was d i l u t e d w i t h w a t e r (50 ml) and l e f t a t 0°C o v e r -n i g h t . F i l t r a t i o n , d r y i n g and s u b s e q u e n t ' r e c r y s t a l l i z a t i o n o f - 36 -the r e s u l t i n g y e l l o w p r e c i p i t a t e f r om p e t e t h e r ( 3 0 - 6 0 ) gave -[ l ' - 1 4 C ] - 2 , 4 - 0 - d i c a r b e t h o x y - 5 - m e t h y l b e n z a l d e h y d e ( 2 7 ) (1.82 g, 92%) m.p. 60-60.5°C as w h i t e f l a k e s . PREPARATION OF [1'- 1 4C]-2,4-0-DICARBETHOXY-5-METHYLBENZOIC  ACID(28) 22 The method used was as d e s c r i b e d by Thomas [ 1 1 - 1 4 C ] - 2 , 4 - 0 - D i c a r b e t h o x y - 5 - m e t h y l b e n z a l d e h y d e ( 2 7 ) (1.82 g, 0.006 mol) was d i s s o l v e d i n ac e t o n e (20 ml) and p o t a s s i u m permanganate (1.82 g) i n w a t e r (30 ml) a t 40°C was added t o t h e s t i r r e d s o l u t i o n . S t i r r i n g was c o n t i n u e d a t room temper-a t u r e f o r 1 h r and t h e n s u l f u r d i o x i d e was b u b b l e d t h r o u g h t h e i c e - c o o l e d s o l u t i o n t o remove t h e formed manganese d i o x i d e . A c l e a r o i l s e p a r a t e d w h i c h s o l i d i f i e d on s t a n d i n g a t 0°C. F i l t r a t i o n , d r y i n g and subsequent r e c r y s t a l l i z a t i o n o f t h i s s o l i d from e t h y l a c e t a t e - p e t e t h e r ( 6 0 - 8 0 ) y i e l d e d [ 1 * - 1 4 C ] -2 , 4 - 0 - d i c a r b e t h o x y - 5 - m e t h y l b e n z o i c a c i d ( 2 8 ) (1.53 g, 80 %) m.p. 116-117°C as c o l o r l e s s c r y s t a l s . PREPARATION OF [1'- 1 4C]-ORSELLINIC ACID(22) 22 The method used was as d e s c r i b e d by Thomas [ 1 ' - 1 4 C ] - 2 , 4 - 0 - D i c a r b e t h o x y - 5 - m e t h y l b e n z o i c a c i d ( 2 8 ) (1.53 g, 0.005 mol) was d i s s o l v e d i n methanol (15 ml) and IN sodium h y d r o x i d e (49 ml) was added d r o p w i s e w i t h s t i r r i n g a t 0°C under a n i t r o g e n atmosphere. Upon complete a d d i t i o n t h e mix-t u r e was s t i r r e d f o r 2 h r and t h e n w a t e r (10 ml) was added and t h e me t h a n o l removed under vacuum. The s o l u t i o n was a g a i n s t i r r e d a t 0°C under n i t r o g e n and 3N h y d r o c h l o r i c a c i d was added d r o p w i s e u n t i l t h e s o l u t i o n was s l i g h t l y a c i d i c . On s t a n d i n g o v e r n i g h t a t 0°C, a w h i t e p r e c i p i t a t e formed and was f i l t e r e d and d r i e d . R e c r y s t a l l i z a t i o n from w a t e r - e t h a n o l and s u b l i m a t i o n a t 120°C i n a h i g h vacuum y i e l d e d [ 1 ' - 1 4 C ] - o r s e l -l i n i c a c i d (22) (0.75 g, 92%) m.p. 175-177°C w i t h a c o n s t a n t -3 s p e c x f x c a c t i v i t y o f 2.505 x 10 mc/mm. PREPARATION OF [1'- 1 4C]-2,4-DTHYDROXY-5,6-DIMETHYLBENZALDEHYDE  (30) 25 The method used was as d e s c r i b e d by P e t t e r s o n Anhydrous 3 , 5 - d i h y d r o x y - o - x y l e n e ( 2 9 ) (1.0 g, 0.007 mol) was d i s s o l v e d i n anhydrous e t h e r (30 ml) and r e a c t e d w i t h z i n c 14 c y a n i d e (1.5 g) and C-sodium c y a n i d e (3.1 mg). The p r o c e d u r e was i d e n t i c a l t o t h a t f o r t h e p r e p a r a t i o n o f [ l 1 - C ] - o r c y l a l -14 dehyde(26) and gave [ l 1 - C ] - 2 , 4 - d i h y d r o x y - 5 , 6 - d i m e t h y l b e n z a l -dehyde(30) (1.08 g, 89%) m.p. 190-195°C as a y e l l o w g r a n u l a r s o l i d . PREPARATION OF [1'- 1 4C]-2,4-0-DTCARBETHOXY-5,6-DIMETHYL  BENZALDEHYDE(31) 25 The method used was as d e s c r i b e d by P e t t e r s o n 14 Crude [ 1 ' - C ] - 2 , 4 - d i h y d r o x y - 5 , 6 - d i m e t h y l b e n z a l d e h y d e ( 3 0 ) (1.08 g, 0.006 mol) was d i s s o l v e d i n a c e t o n e (10 ml) and e t h y l c h l o r o f o r m a t e (0.65 ml) was added. The s o l u t i o n was s t i r -r e d a t 0°C and IN sodium h y d r o x i d e (10 ml) was s l o w l y added. A f t e r p a r t i a l s e p a r a t i o n o f a gummy s o l i d , more acetone (18 ml) - 38 -was added and t h e s o l u t i o n t r e a t e d t w i c e more w i t h e t h y l c h l o r o f o r m a t e (0.65 ml) and IN sodium h y d r o x i d e (10 m l ) . The s o l u t i o n was l e f t a t 0°C o v e r n i g h t and t h e n was a c i d i f i e d . The a c e t o n e was removed under vacuum and t h e r e s u l t i n g aqueous s o l u t i o n was e x t r a c t e d w i t h e t h e r . The e t h e r was d r i e d w i t h sodium s u l f a t e and removed under vacuum t o g i v e a brown o i l . T h i s o i l was e x t r a c t e d w i t h warm p e t e t h e r ( 3 0 - 6 0 ) w h i c h on subsequent c o o l i n g y i e l d e d y e l l o w c r y s t a l s . R e c r y s t a l l i z a t i o n 14 from p e t e t h e r ( 3 0 - 6 0 ) gave [ 1 ' - C ] - 2 , 4 - 0 - d i c a r b e t h o x y - 5 , 6 -d i m e t h y l b e n z a l d e h y d e ( 3 1 ) (1.42 g, 71%) m.p. 60-61°C as s l i g h t l y y e l l o w f l a k e s . PREPARATION OF [1'- 1 4C]-2,4-0-DICARBETHOXY-5,6-DIMETHYLBENZOIC  ACID(32) 25 The method used was as d e s c r i b e d by P e t t e r s o n [ 1 ' - 1 4 C ] - 2 , 4 - 0 - D i c a r b e t h o x y - 5 , 6 - d i m e t h y l b e n z a l d e h y d e ( 3 1 ) (1.42 g, 0.005 mol) was d i s s o l v e d i n a c e t o n e (50 ml) and p o t a s -sium permanganate (1.42 g) i n w a t e r (50 ml) a t 40°C was added t o t h e s t i r r e d s o l u t i o n . S t i r r i n g was c o n t i n u e d a t room temp-e r a t u r e f o r 4 h r and t h e n s u l f u r d i o x i d e was b u b b l e d t h r o u g h t h e i c e - c o o l e d s o l u t i o n t o remove t h e formed manganese d i o x i d e . The o i l w h i c h formed would n o t c r y s t a l l i z e and t h e 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 a f t e r r e m o v a l o f t h e a c e t o n e under vacuum. The e t h e r s o l u t i o n was e x t r a c t e d w i t h sodium b i c a r b o n a t e s o l u -t i o n and a f t e r a c i d i f i c a t i o n t h i s s o l u t i o n was e x t r a c t e d w i t h e t h e r . D r y i n g and r e m o v a l o f t h e e t h e r under vacuum r e s u l t e d i n a y e l l o w gummy s o l i d . R e c r y s t a l l i z a t i o n from e t h y l a c e t a t e -14 p e t e t h e r ( 6 0 - 8 0 ) y i e l d e d [!'- C ] - 2 , 4 - 0 - d i c a r b e t h o x y - 5 , 6 -d i m e t h y l b e n z o i c a c i d ( 3 2 ) (0.80 g, 53%)' m.p. 112-113°C as c o l o r l e s s c r y s t a l s . PREPARATION OF [!'- C]-2,4-DTHYDROXY-5,6-DTMETHYLBENZOIC  ACID (23) 14 [ 1 ' - C ] - 2 , 4 - 0 - D i c a r b e t h o x y - 5 , 6 - d i m e t h y l b e n z o i c a c i d (32) (0.80 g, 0.003 mol) was d i s s o l v e d i n e t h a n o l (10 ml) and 20% a l c o h o l i c p o t a s s i u m h y d r o x i d e (4.7 ml) was added d r o p w i s e w i t h s t i r r i n g a t 0°C under a n i t r o g e n atmosphere. Upon complete a d d i t i o n t h e s o l u t i o n was s t i r r e d f o r a n o t h e r h r and t h e n was d i l u t e d w i t h an e x c e s s o f w a t e r , a l l o w i n g t h e e t h a n o l t o be removed under vacuum. Then t h e s o l u t i o n was a g a i n s t i r r e d a t 0°C under n i t r o g e n and 3N h y d r o c h l o r i c a c i d was added d r o p w i s e u n t i l t h e s o l u t i o n was s l i g h t l y a c i d i c . On s t a n d i n g o v e r n i g h t a t 0°C a y e l l o w p r e c i p i t a t e formed w h i c h was f i l t e r e d and d r i e d . R e c r y s t a l l i z a t i o n from w a t e r - e t h a n o l and s u b l i m a t i o n a t 100°C i n a h i g h vacuum gave [ 1 ' - ^ 4 C ] - 2 , 4 - d i h y d r o x y - 5 , 6 -d i m e t h y l b e n z o i c a c i d ( 2 3 ) (0.37 g, 82%) m.p. 160-162°C as a s l i g h t l y y e l l o w s o l i d w i t h a c o n s t a n t s p e c i f i c a c t i v i t y o f -4 5.465 x 10 mc/mm. ADMINISTRATION OF RADIOACTIVE COMPOUNDS TO PENICILLIUM BREVI- COMPACTUM C u l t u r e s o f P e n i c i l l i u m brevi-compactum were grown on Raulin-Thom medium w i t h s h a k i n g u n t i l t h e pH o f t h e b r o t h was between 5.2 and 5.6. Then s o l u t i o n s o f t h e r a d i o a c t i v e com-pounds i n e t h a n o l o r w a t e r ( e m u l s i f i e d w i t h tween 20) were added d i r e c t l y t o t h e b r o t h . The c u l t u r e s were t h e n a l l o w e d to grow u n t i l the pH of the b r o t h reached 7.0 (14-20 days). The mycelium was then f i l t e r e d o f f and both the mycelium and the b r o t h were e x t r a c t e d w i t h l a r g e amounts of ether. Drying and removal of the ether under vacuum y i e l d e d gummy ye l l o w r e s i d u e s which c o n s i s t e d mostly of mycophenolic a c i d . P u r i -f i c a t i o n was accomplished by t r i t u r a t i o n w i t h ether and e t h y l acetate and f i n a l l y by r e c r y s t a l l i z a t i o n from e t h y l acetate to constant s p e c i f i c a c t i v i t y , m.p. 136-138°C; mixed m.p. 137-139°C. DEGRADATION OF THE RADIOACTIVE MYCOPHENOLIC ACID The r a d i o a c t i v e mycophenolic a c i d was d i l u t e d w i t h c o l d mycophenolic a c i d by mixing and r e c r y s t a l l i z a t i o n together from e t h y l a c e t a t e . This was done to i n c r e a s e the amount of m a t e r i a l thereby making subsequent r e a c t i o n s e a s i e r to accomplish. 30 A) QZONOLYSIS - The method used was t h a t of Perry R a d i o a c t i v e mycophenolic a c i d (75 mg, 0.23 mmol) was t r e a t e d w i t h e t h e r e a l diazomethane to give the methyl ether methyl e s t e r of mycophenolic a c i d (35). This crude product was then d i s s o l v e d i n methanol (10 ml) and cooled t o -78°C. Ozone was passed through the s o l u t i o n u n t i l i t turned b l u e . Nitrogen was then bubbled through to remove the excess ozone and the methanol was removed under vacuum w i t h very g e n t l e h e a t i n g . To the r e s i d u a l pungent y e l l o w o i l were added formic a c i d (0.38 ml) and 30% hydrogen peroxide (0.26 m l ) . This s o l u t i o n was heated at 100°C f o r 1/2 hr and then the formic a c i d was removed under vacuum. Saturated sodium bicarbonate - 41 -was added to the r e s i d u e and t h i s was then e x t r a c t e d w i t h e t h e r , and the ether s o l u t i o n d i s c a r d e d . The bicarbonate s o l u t i o n was a c i d i f i e d and again e x t r a c t e d w i t h ether. The ether was d r i e d and removed under vacuum y i e l d i n g a y e l l o w o i l y s o l i d . T r i t u r -a t i o n w i t h ether and r e c r y s t a l l i z a t i o n from ether-pet ether(30-60) gave 2-(4,6-dimethoxy-7-methyl-3-oxo-5-phthalanyl)-ethanoic acid(40) m.p. 150-152°C(lit.7 m.p. 150-151°C) as white needles. v (nujol) 3300-2600, 1760, 1705, 1601 cm - 1; X 293, 247 mu; max max T(CDC1 3) 1.59(s, IH), 4.83(s, 2H), 5.90(s, 3H), 6.81(d, 5H), 7.82(s, 3H) . 7 B) DE C ARB OX Y LAT I ON _ The method used was t h a t of Birkmshaw . 2-(4,6-Dimethoxy-7-methyl-3-oxo-5-phthalanyl)-ethanoic acid(40) (100 mg, 0.4 mmol) and red phosphorus (0.3 g) were combined i n h y d r i o d i c a c i d (3 ml) and t h i s was r e f l u x e d i n an atmosphere of carbon d i o x i d e f r e e n i t r o g e n . The r e s u l t i n g carbon d i o x i d e was trapped as barium carbonate by bubbling through a barium hydroxide s o l u t i o n . The r e a c t i o n was termin-ated a f t e r 19 hr and the phosphorus f i l t e r e d o f f . The f i l t r a t e was e x t r a c t e d w i t h e t h y l acetate (4 x 15 ml) and the e t h y l acetate s o l u t i o n was then washed w i t h water and aqueous sodium t h i o s u l f a t e . Drying and removal of the e t h y l acetate y i e l d e d a gummy pungent y e l l o w s o l i d . S u blimation at 120°C at 0.05 mm Hg and subsequent r e c r y s t a l l i z a t i o n from benzene gave the coumaran-2-one(41a) and/or (41b) as white needles, m.p. 179-183°C(lit.7 m.p. 184-185°C). v (CHC10) 3200-3500, 1801, 1745, max 3 1650 cm - 1; X 283, 222 my; T (CDC1 0) 3.60(s, IH), 6.32(s, 2H), max o 7.78(s, 3H) , 7.86(s, 3H) ; m/e 178. PREPARATION OF ORCYLALDEHYDE(45) 23 The method used was th a t of Adams and Levine Anhydrous o r c i n o l (25) (1.0 g, 0.008 mol) was d i s s o l v e d i n anhydrous ether (25 ml) and z i n c cyanide (1.5 g) was added. The mixture was s t i r r e d v i g o r o u s l y and anhydrous hydrogen c h l o r i d e gas was bubbled through r a p i d l y . The apparatus c o n s i s t e d of a 3-necked f l a s k equipped w i t h a s t i r r e r , condenser and gas i n l e t tube and connected t o a s e r i e s of traps designed t o remove any excess hydrogen cyanide or hydrogen c h l o r i d e present. A f t e r 1/2 hr a t h i c k o i l separated and t h i s e v e n t u a l l y s o l i d i f i e d . A f t e r 1 hr the hydrogen c h l o r i d e a d d i t i o n was stopped and the ether was decanted. The r e s u l t i n g gummy residue was b o i l e d 2 to 3 min w i t h water and then l e f t to c o o l to 0°C. The r e s u l t i n g orange s o l i d was r e c r y s t a l l i z e d from d i l u t e ethanol g i v i n g orcylaldehyde(45) (0.93 g, 76%) m.p. 179-180°C ( l i t . 2 3 m.p. 178-180°C) as orange needles, v (nujol) 3100, 1620 cm - 1; A 290, 234, max max 220 my(e 10500, 5850, 7520); T (CF 3C00H) -0.42 (S, IH) , 3.49 (s, 2H), 7.40(s, 3H). PREPARATION OF 2,4-0-DICARBETHOXY-6-METHYLBENZALDEHYDE(55) 24 The method used was s i m i l a r t o t h a t of Hoesch Orcylaldehyde (45) (0.93 g, 0.007 mol) was d i s s o l v e d i n acetone (5 ml) and e t h y l chloroformate (0.63 ml) was added. This was cooled to 0°C and IN sodium hydroxide (6.5 ml) was added w i t h s t i r r i n g . A f t e r the se p a r a t i o n of a gummy s o l i d , more acetone (7 ml) was added and the s o l u t i o n was t r e a t e d twice more w i t h - 43 -the above amounts o f e t h y l c h l o r o f o r m a t e and sodium h y d r o x i d e . I n about 1/2 h r a t h i c k o i l s e p a r a t e d and t h i s s o l i d i f i e d a f t e r a s h o r t w h i l e . F i l t r a t i o n and subsequent r e c r y s t a l -l i z a t i o n from p e t e t h e r (30-60) gave 2 , 4 - 0 - d i c a r b e t h o x y - 6 -m e t h y l b e n z a l d e h y d e ( 5 5 ) (1.4 g, 78%) m.p. 58-59°C ( l i t . 2 4 m.p. 60°C) as w h i t e f l u f f y f l a k e s . v ( n u j o l ) 1750, 1675 c m - 1 ; 1 max ' ' A 256 my (e 10,000); x(CDCl-) - 0 . 3 1 ( s , IH) , 2.95(s, IH) , in 9.x o 5.63(q, 4H) , 7.35(s, 3H) , 8 . 6 0 ( t , 6H). PREPARATION OF 2,4-0-DICARBETHOXY-6-METHYLBENZOIC ACID(56) 24 The method used v/as s i m i l a r t o t h a t o f Hoesch 2 , 4 - 0 - D i c a r b e t h o x y - 6 - m e t h y l b e n z a l d e h y d e ( 5 5 ) (1.4 g, 0.005 mol) was d i s s o l v e d i n ac e t o n e (15 ml) and p o t a s s i u m perman-ganate (1.4 g) i n w a t e r (25 ml) a t 40°C was added w i t h s t i r r i n g . T h i s was l e f t f o r 1 h r and t h e n t h e formed man-ganese d i o x i d e was removed by b u b b l i n g s u l f u r d i o x i d e t h r o u g h t h e i c e c o o l e d s o l u t i o n . The r e s u l t was t h e p r e c i p -i t a t i o n o f a w h i t e s o l i d . R e c r y s t a l l i z a t i o n from e t h y l a c e t a t e - p e t e t h e r (60-80) gave 2 , 4 - 0 - d i c a r b e t h o x y - 6 - m e t h y l b e n z o i c a c i d ( 5 6 ) (1.18 g, 79%) m.p. 111-112°C ( l i t . 2 4 m.p. 112°C) as c o l o r l e s s c r y s t a l s . v ( n u j o l ) 3200-2550, 1760, 1 max J 1690, 1625 cm" 1; A 305, 270 my(e 280, 580); T(CDC1-.) -0.52 ITlclX j ( s , IH) , 2.99(s, 2H) , 5.66(q, 4H) , 7.45(s, 3H) , 8 . 6 1 ( t , 6H) . PREPARATION OF ORSELLINIC A C I D ( 3 ) 24 The method used was s i m i l a r t o t h a t o f Hoesch 2 , 4 - 0 - D i c a r b e t h o x y - 6 - m e t h y l b e n z o i c a c i d (56) (1.18 g, 0.004 mol) - 44 -was d i s s o l v e d i n methanol (10 ml) and s t i r r e d a t 0°C under n i t r o g e n . Then IN sodium hydroxide (37.8 ml) was added dropwise. Upon complete a d d i t i o n the s o l u t i o n was s t i r r e d f o r 2 hr and then the methanol was removed under vacuum. Again w i t h s t i r r i n g a t 0°C and under n i t r o g e n , h y d r o c h l o r i c -a c i d (6N) was added u n t i l the s o l u t i o n was a c i d i c . The r e s u l t was a white p r e c i p i t a t e which on r e c r y s t a l l i z a t i o n from water-e t h a n o l gave o r s e l l i n i c a c i d ( 3 ) (0.46 g, 72%) m.p. 1 7 4 - 1 7 6 ° C 24 n ( l i t . m.p. 1 7 6 ° C ) as white n e e d l e s . v (nujol) 3600-2400, ^ max J ' 1640 cm - 1; X 300, 262 mu(e 3300, 8 6 5 0 ) ; T (a c e t o n e - d 6 ) 3.73 max ' (s, 2H) , 7.46(s, 3H) . PREPARATION OF METHYL 2-HYDROXY-4-METHOXY-6-METHYLBENZOATE(57) O r s e l l i n i c acid.( 3 ) (5.0 g, 0.03 mol) was r e a c t e d w i t h excess e t h e r e a l diazomethane and the s o l u t i o n was al l o w e d t o stand a t room temperature f o r 1 hr. The e t h e r was removed and the r e s u l t i n g y e l l o w s o l i d was chromatographed on a column of s i l i c a g e l f i n e r than 200 mesh, e l u t i n g w i t h c h l o r o f o r m under p r e s s u r e . The f i r s t f r a c t i o n c o n t a i n e d the d e s i r e d p roduct and the second f r a c t i o n c o n t a i n e d methyl 2,4-dihydroxy-6-methylbenzoate. R e c r y s t a l l i z a t i o n from aqueous e t h a n o l gave methyl 2-hydroxy-4-methoxy-6-methylbenzoate(57) (4.4 g, 76%) m.p. 6 5 . 5 - 6 6 ° C as white f l a k e s . v (nujol) 1640, 1620, c max J 1570 cm" 1; 301, 262 mp; T ( C D C 1 _ ) -2.05 (s, IH) , 3.70 (s, 2H) , 6.07(s, 3H) , 6.20(s, 3H) , 7.50(s, 3H) . - 45 -PREPARATION OF METHYL 6-BROMOMETHYL-2—HYDRQXY-4-METHO.XY  BENZOATE (58) The method used was s i m i l a r t o t h a t o f A l l i s o n and 34 Newbold M e t h y l 2 - h y d r o x y - 4 - m e t h o x y - 6 - m e t h y l b e n z o a t e ( 5 7 ) (3.1 g, 0.016 mol) was d i s s o l v e d i n c a r b o n t e t r a c h l o r i d e (45 ml) and t h i s was i r r a d i a t e d and r e f l u x e d w i t h a 250 w a t t lamp. Bromine (2.53 g) i n c a r b o n t e t r a c h l o r i d e (15 ml) was added d r o p w i s e o v e r 45 min and t h e s o l u t i o n was r e f l u x e d f o r an a d d i t i o n a l 15 min a f t e r c o m p l e t e a d d i t i o n . Removal o f t h e s o l v e n t under vacuum and chromatography o f t h e r e s u l t i n g cream c o l o r e d s o l i d on a column o f s i l i c a g e l f i n e r t h a n 200 mesh, e l u t i n g w i t h c h l o r o f o r m under p r e s s u r e y i e l d e d a w h i t e s o l i d i n t h e f i r s t f r a c t i o n . R e c r y s t a l l i z a t i o n from p e t e t h e r (60-80) gave m e t h y l 6-bromomethyl-2-hydroxy-4-methoxybenzoate(58) (3.0 g, 70%) m.p. 1 0 2 - 1 0 5 ° C as a f l u f f y w h i t e s o l i d . V T 1 1 = V (CHC1.,) IUclX J 1650, 1610, 1570 cm" 1; A. 309, 263, 220 my; T ( C D C I ^ ) -2.05 ( s , IH) , 3.58(m, 2H) , 5 . 2 5 ( s , 2H) , 6 . 0 3 ( s , 3H) , 6 . 2 3 ( s , 3H) . PREPARATION OF 7-HYDROXY-5-METHOXYPHTHALTDE(59) The method used was s i m i l a r t o t h a t o f A l l i s o n and 34 Newbold M e t h y l 6-bromomethyl-2-hydroxy-4-methoxybenzoate(58) (2.1 g, 0.008 mol) was d i s s o l v e d i n a m i x t u r e o f d i o x a n (40 ml) and w a t e r (10 m l ) . T h i s s o l u t i o n was r e f l u x e d f o r 24 h r and t h e n c o n c e n t r a t e d t o 10 ml under vacuum. The r e s u l t i n g y e l l o w s o l i d was r e c r y s t a l l i z e d from a c e t o n e - p e t e t h e r (60-80) and - 46 -s u b l i m e d a t 110°C a t 0.05 mm Hg t o g i v e 7-hydroxy-5-methoxy p h t h a l i d e ( 5 9 ) (1.22 g, 89%) m.p.. 183-185°C ( l i t . 3 4 m.p. 186-188°C) as a w h i t e f l a k e y s o l i d . v ( n u j o l ) 3250, 1740, 1603 cm J max ' ' ' \ 298, 257, 219 my; T(CDC1,) 2.23(s, IH) , 3.45(s, 2H) , 4.67 ( s , 2H) , 6.17(s, 3H) . ATTEMPTED PREPARATION OF 7-HYDROXY-5-METHOXY-4-METHYL  PHTHALIDE(60) 7- H y d r o x y - 5 - m e t h o x y p h t h a l i d e ( 5 9 ) (100 mg, 0.0006 mol) was d i s s o l v e d i n n i t r o b e n z e n e (15 ml) and aluminum c h l o r i d e (100 mg) was added. T h i s 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 and m e t h y l c h l o r i d e was b u b b l e d t h r o u g h . A f t e r 1 h r t h e s o l u t i o n was h e a t e d f o r 2 h r a t 100°C. The m i x t u r e was t h e n d i l u t e d w i t h an e x c e s s o f w a t e r and e x t r a c t e d w i t h e t h e r . The e t h e r was removed under vacuum and t h e n i t r o b e n z e n e steam d i s t i l l e d l e a v i n g a brown s o l i d . P u r i f i c a t i o n y i e l d e d o n l y t h e s t a r t i n g p h t h a l i d e ( 5 9 ) . ATTEMPTED PREPARATION OF 6-GERANYL-7-HYDROXY-5-METHOXY  PHTHALIDE(61) 36 A) The method used was s i m i l a r t o t h a t o f C a r d i l l o e t a l 7 - H y d r o x y - 5 - m e t h o x y p h t h a l i d e ( 5 9 ) (100 mg, 0.0006 mol) was d i s s o l v e d i n d r y benzene (5 ml) and n - b u t y l l i t h i u m (0.07 g) was added d r o p w i s e . S t i r r i n g a t room t e m p e r a t u r e f o r 2 h r gave an orange s o l u t i o n . G e r a n y l c h l o r i d e (0.12 ml) was added and t h e m i x t u r e r e f l u x e d o v e r n i g h t . The e x c e s s n - b u t y l l i t h i u m was d e s t r o y e d w i t h some e t h a n o l and t h e m i x t u r e was a c i d i f i e d - 47 -and e x t r a c t e d w i t h e t h e r . Removal o f t h e e t h e r and e x c e s s g e r a n y l c h l o r i d e under vacuum y i e l d e d a s o l i d w h i c h on examin-a t i o n p r o v e d t o be t h e s t a r t i n g p h t h a l i d e ( 5 9 ) . 37 B) The method used was s i m i l a r t o t h a t o f Mechoulam 7- H y d r o x y - 5 - m e t h o x y p h t h a l i d e ( 5 9 ) (100 mg, 0.0006 mol) and g e r a n i o l (90 mg) were d i s s o l v e d i n m e t hylene c h l o r i d e (1.8 m l ) . To t h i s s t i r r e d s o l u t i o n was added a s u s p e n s i o n o f p - t o l u e n e s u l f o n i c a c i d (0.01 g) i n m e t h y l e n e c h l o r i d e (1.4 m l ) . The r e a c t i o n 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 1/2 h r , f i l t e r e d , washed w i t h b r i n e s o l u t i o n , d r i e d w i t h sodium s u l f a t e and t h e m e t h ylene c h l o r i d e removed under vacuum. The e x c e s s g e r a n i o l was d i s t i l l e d o f f under h i g h vacuum l e a v i n g a w h i t e s o l i d . T h i s s o l i d upon e x a m i n a t i o n a g a i n p r o v e d t o be t h e s t a r t i n g p h t h a l i d e ( 5 9 ) . C) 7 - H y d r o x y - 5 - m e t h o x y p h t h a l i d e ( 5 9 ) (0.5 g, 0.003 mol) and aluminum c h l o r i d e (0.5 g) were combined i n n i t r o b e n z e n e (25 m l ) . Then g e r a n o y l c h l o r i d e (2 ml) was added d r o p w i s e r e s u l t i n g i n a b l a c k s o l u t i o n and t h e e v o l u t i o n o f hydrogen c h l o r i d e . T h i s was h e a t e d a t 100°C u n t i l t h e e v o l u t i o n o f hydrogen c h l o r i d e ceased ( a p p r o x i m a t e l y 8 h r ) . The m i x t u r e was t h e n poured i n t o a m i x t u r e o f c r u s h e d i c e (7.5 g) and 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 (7.5 ml) and s t i r r e d o v e r n i g h t u n t i l s o l u t i o n was e f f e c t e d . The r e s u l t i n g b l a c k s o l u t i o n 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 was d r i e d w i t h sodium s u l f a t e and removed under vacuum. The n i t r o b e n z e n e was steam d i s t i l l e d g i v i n g a b l a c k s o l i d , w h i c h on t r i t u r a t i o n w i t h e t h e r y i e l d e d a w h i t e s o l i d . Upon e x a m i n a t i o n t h i s s o l i d a g a i n p r o v e d t o be t h e s t a r t i n g p h t h a l i d e ( 5 9 ) - 48 -PREPARATION OF 2,4-DIHYDROXY-6-METHYLTOLUENE(29) 2 6 The method used was t h a t o f R o b e r t s o n and W h a l l e y Z i n c (23.8 g ) , m e r c u r i c c h l o r i d e (1.8 g ) , c o n c e n t r a t e d h y d r o -c h l o r i c a c i d (7.3 ml) and w a t e r (183 ml) were combined and shaken t o g e t h e r f o r 15 min. The l i q u i d was t h e n d e c a n t e d from t h e formed z i n c amalgam and 6N h y d r o c h l o r i c a c i d (93 ml) was added. T h i s was t h e n r e f l u x e d and o r c y l a l d e h y d e (45) (5.5 g, 0.036 mol) i n e t h a n o l (100 ml) was added d r o p w i s e . Upon complete a d d i t i o n t h e m i x t u r e was r e f l u x e d f o r a f u r t h e r 2 h r and t h e n t h e s o l u t i o n was d e c a n t e d from t h e r e s i d u a l z i n c amalgam. Removal o f t h e e t h a n o l under vacuum and c o o l i n g t o 0°C r e s u l t e d i n t h e d e p o s i t i o n o f a p i n k i s h s o l i d . R e c r y s t a l -l i z a t i o n from benzene gave anhydrous 2 , 4 - d i h y d r o x y - 6 - m e t h y l t o l u e n e ( 2 9 ) (3.9 g, 78%) m.p. 132-133°C ( l i t . 6 0 m.p. 135-137°C) as s l i g h t l y p i n k f l a k e s . v ( n u j o l ) 3310, 1615, 1520 cm "*"; X o v 283 my(e 1840); T (CF-COOH) 3.49 ( s , 2H), 7.71 ( s , 3H), 7.85 ItlclX O ( s , 3H). PREPARATION OF 2,4-DIHYDROXY-5,6-DIMETHYLBENZALDEHYDE(46) 2 , 4 - D i h y d r o x y - 6 - m e t h y l t o l u e n e ( 2 9 ) (1.0 g, 0.007 mol) was r e a c t e d w i t h z i n c c y a n i d e (1.5 g) i n anhydrous e t h e r (25 ml) i n e x a c t l y t h e same manner as o r c i n o l (25). R e c r y s t a l l i z a t i o n from d i l u t e e t h a n o l gave 2 , 4 - d i h y d r o x y - 5 , 6 - d i m e t h y l b e n z a l d e -hyde(46) (1.07 g, 89%) m.p. 195-196°C ( l i t . 2 6 m.p. 196°C) as y e l l o w n e e d l e s . v ( n u j o l ) 3500-2500, 1660, 1600 cm" 1; •* max J ' ' ' A 292, 238, 222 my (e 12450, 7720, 8960); T (CF_C00H) -0.42 ITlclX ( s , IH) , 3.45(s, IH) , 7.42(s, 3H) , 7.80(s, 3H) . - 49 -PREPARATION OF 2,4-0-DICARBETH0XY-56-DIMETHYLBENZALDEHYDE(47) 2,4-Dihydroxy-5,6-diraethylbenzaldehyde (4 6) (1.07 g, 0.006 mol) was r e a c t e d w i t h e t h y l c h l o r o f o r m a t e (0.65 ml) and sodium hydroxide (10 ml) i n e x a c t l y the same manner as o r c y l a l d e h y d e ( ). A t h i c k o i l formed but i t d i d not s o l i d i f y and the s o l u t i o n was e x t r a c t e d w i t h e t h e r which was subsequently d r i e d and removed under vacuum. The r e s u l t i n g y e l l o w gummy s o l i d was r e c r y s t a l -l i z e d from p e t ether(30-60) t o g i v e 2,4-0-dicarbethoxy-5,6-dimethylbenzaldehyde (47) (1.15 g, 58%) m.p. 60-61°C as white n e e d l e s , v (nujol) 1750, 1690, 1590 cm - 1; A 300, 255 mn max J ' ' max v (e 1780, 10000); T (CF3COOH) -0.48 (S, IH) , 2.90 (s, IH) , 5.50 (q, 4H) , 7.30(s, 3H) , 7.72(s, 3H) , 8.55(t, 6H) . PREPARATION OF 2,4-0-DTCARBETHOXY-5,6-DIMETHYLBENZ0IC ACID(48) 2,4-0-Dicarbethoxy-5,6-dimethylbenzaldehyde(47) (1.15 g, 0.004 mol) was r e a c t e d w i t h potassium permanganate (1.15 g) i n e x a c t l y the same manner as 2,4-0-dicarbethoxy-6-methyl benzaldehyde (55). The product o i l e d out a f t e r the treatment w i t h s u l f u r d i o x i d e and the s o l u t i o n was e x t r a c t e d w i t h e t h e r . The e t h e r was d r i e d and removed under vacuum and the pro d u c t was r e c r y s t a l l i z e d from e t h y l a c e t a t e - p e t e t h e r (60-80) t o g i v e 2,4-0-dicarbethoxy-5,6-dimethylbenzoic acid(48) (0.76 g, 63%) m.p. 115-117°C as c o l o r l e s s c r y s t a l s . v (nujol) 3000-2500, * 2 max J 1775, 1700, 1600 cm" 1; A ^ 273 my (e 670); x(CDCl-) 2.95(s, IH), 5.61(q, 4H) , 7.54(s, 3H) , 7.79(s, 3H) , 8.56(t, 6H) . - 50 -PREPARATION OF 2,4-DIHYDROXY-5,6-DTMETHYLBENZOIC ACID(49) 2,4-0-Dicarbethoxy-5,6-dimethylbenzoic acid(48) (0.76 g, 0.004 mol) was d i s s o l v e d i n e t h a n o l (7 ml) and c o o l e d t o 0°C under a n i t r o g e n atmosphere. Then 20% a l c o h o l i c potassium hydroxide (4.6 ml) was added dropwise and the s o l u t i o n was s t i r r e d a t room temperature f o r 1 hr a f t e r complete a d d i t i o n . The s o l u t i o n was d i l u t e d w i t h an excess of water and the e t h a n o l was removed under vacuum. Again w i t h s t i r r i n g a t 0°C and under n i t r o g e n , 3N h y d r o c h l o r i c a c i d was added dropwise u n t i l the s o l u t i o n was a c i d i c . The r e s u l t was a cream c o l o r e d s o l i d which on r e c r y s t a l l i z a t i o n from w a t e r - e t h a n o l gave 2,4-dihydroxy-5,6-dimethylbenzoic acid(49) (0.29 g, 67%) m.p. 160-161°C(lit. m.p. 163°C) as white n e e d l e s . v_, (nujol) 3300, 3150-2450, 1600 cm" 1; X 309, 264 my (e 4000, 9460); max T (acetone-d 6) 3.63 (s, IH), 7.48(s, 3H), 7.89(s, 3H). PREPARATION OF METHYL 5,6-DIMETHYL-2-HYDROXY-4-METHOXY  BENZOATE(50) 2,4-Dihydroxy-5,6-dimethylbenzoic a c i d (49) (1.8 g, 0.01 mol) was t r e a t e d w i t h excess e t h e r e a l diazomethane. A f t e r s t a n d i n g a t room temperature f o r 1 hr the e t h e r was removed under vacuum l e a v i n g a y e l l o w s o l i d . T h i s was chromatographed on a column of s i l i c a g e l f i n e r than 200 mesh, e l u t i n g w i t h c h l o r o f o r m under p r e s s u r e . The f i r s t f r a c t i o n c o n t a i n e d the d e s i r e d product and the second f r a c t i o n c o n t a i n e d methyl 2,4-dihydroxy-5,6-dimethylbenzoate. R e c r y s t a l l i z a t i o n o f the f i r s t f r a c t i o n p roduct from aqueous e t h a n o l gave methyl 5,6-- 51 -d i m e t h y l - 2 - h y d r o x y - 4 - m e t h o x y b e n z o a t e ( 5 0 ) (1.47 g, 71%) m.p. 8 3 - 8 5 ° C as w h i t e n e e d l e s . v (CHC1.) 3020-2960, 1725, 1650, max 3 1600 cm" 1; A 307, 263 my; T(CDC1.) -1.25 (S, I H ) , 3.64 ( s , I H ) , 6 . 1 0 ( s , 3H) , 6 . 1 8 ( s , 3H) , 7 . 5 4 ( s , 3H) , 7 . 9 6 ( s , 3H) . PREPARATION OF METHYL 5,6-DTBR0M0METHYL-2-HYDROXY-4-METHOXY  BENZOATE(51) M e t h y l 5 , 6-dimethyl-2-hydroxy-4-methoxybenzoate(50) (1.4 7 g, 0.007 mol) was d i s s o l v e d i n c a r b o n t e t r a c h l o r i d e (20 ml) and t r e a t e d w i t h bromine (2.15 g) i n c a r b o n t e t r a c h l o r i d e (10 ml) i n e x a c t l y t h e same manner as m e t h y l 2-hydroxy-4-methoxy-6-m e t h y l b e n z o a t e ( 5 7 ) . The r e s u l t i n g y e l l o w s o l i d was r e c r y s t a l -l i z e d from p e t e t h e r (30-6 0) t o g i v e m e t h y l 5,6-dibromomethyl-2-' hydroxy-4-methoxybenzoate(51) (1.0 g, 41%) m.p. 132-134°C as s m a l l w h i t e n e e d l e s . v (CHC1.) 3020-2965, 1655, 1601, 1590 ITlciX 3 c m - 1 ; A 313, 261, 228 my; T(CDC1_) - 1 . 7 3 ( S , IH) , 3.50(s, IH) , IU3.X J 5.00(s, 2H) , 5.31(s, 2H) , 5.96(s, 3H), 6.08(s, 3H) . ATTEMPTED PREPARATION OF 4-BR0M0METHYL-7-HYDR0XY-5-METH0XY  PHTHALIDE (52) M e t h y l 5,6-dibromomethyl-2-hydroxy-4-methoxybenzoate (51) (1.0 g, 0.00 3 mol) was r e a c t e d i n a m i x t u r e o f d i o x a n (20 ml) and w a t e r (5 ml) i n e x a c t l y t h e same manner as m e t h y l 6-bromo-me t h y l - 2 - h y d r o x y - 4 - m e t h o x y b e n z o a t e ( 5 8 ) . Upon e t h e r e x t r a c t i o n , t h e r e s u l t was a r e d o i l w h i c h had t h e u l t r a v i o l e t s p e c t r u m o f th e d e s i r e d p h t h a l i d e ( 5 2 ) b u t w h i c h p r o v e d i m p o s s i b l e t o p u r i f y . A 291, 256, 219 my ( c f . 7 - h y d r o x y - 5 - m e t h o x y p h t h a l i d e ( 5 9 ) ) • m.ctx - 52 -PREPARATION OF 3 , 5-DIHYDROXY-4-GERANYLTOLU.ENE (63) 37 The method used was s i m i l a r t o t h a t o f Mechoulam O r c i n o l (25) (5.0 g, 0.04 mol) and g e r a n i o l (6.5 g) were d i s s o l v e d i n methylene c h l o r i d e (80 m l ) . To t h i s was added a s u s p e n s i o n o f p - t o l u e n e s u l f o n i c a c i d (0.6 g) i n methylene c h l o r i d e (70 ml) and t h e m i x t u r e was s t i r r e d a t room tempera-t u r e f o r 2 h r . Then th e s o l u t i o n was washed w i t h b r i n e and d r i e d . The methylene c h l o r i d e was removed under vacuum and t h e r e s i d u a l o i l was chromatographed on a column o f f l o r i s i l , e l u t i n g w i t h 95:5 p e t e t h e r ( 6 0 - 8 0 ) - e t h e r . The p r o d u c t was r e c o v e r e d i n t h e second f r a c t i o n and removal o f t h e s o l v e n t y i e l d e d a c l e a r t h i c k o i l . v ( f i l m ) 3460, 1635, 1600, 1520 max cm" 1; x ( C D C l 3 ) 3.81(s, 2H) , 5.00 ( s , 3H) , 7.50 ( t , 2H), 7.80 ( s , 3H), 7.70-8.70(m, 13H). ATTEMPTED PREPARATION OF l-BROMOMETHYL-3,5-DIHYDROXYBENZENE(64) 34 The method used was t h a t o f A l l i s o n and Newbold O r c i n o l (25) (8.0 g, 0.064 mol) was d i s s o l v e d i n c a r b o n t e t r a -c h l o r i d e (120 ml) and r e f l u x e d w i t h a 250 w a t t lamp. Then bromine (10.3 g) i n c a r b o n t e t r a c h l o r i d e (40 ml) was added d r o p w i s e o v e r t h r e e h o u r s . Upon complete a d d i t i o n , t h e s o l u t i o n was r e f l u x e d f o r a f u r t h e r 10 min, f i l t e r e d and c o n c e n t r a t e d t o 100 m l . On c o o l i n g t h i s d e p o s i t e d p a l e w h i t e n e e d l e s (8.3 g ) . T h i s p r o d u c t was shov/n t o be r i n g b r o m i n a t e d p r o d u c t by 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 i n c e t h e a r o m a t i c p r o t o n r e s o n a n c e was d i m i n i s h e d compared t o t h a t o f o r c i n o l (25) and t h e r e was no bromomethylene p r o t o n r e s o n a n c e . - 53 -O r c i n o l ( 2 5 ) (5.0 g, 0.04 mol) and N-b r o m o s u c c i n i m i d e (6.3 g) were combined i n c a r b o n t e t r a c h l o r i d e (200 ml) and t h i s was i r r a d i a t e d w i t h a 250 w a t t lamp f o r 1 1/2 h r . The r e s u l t was a c l e a r s o l u t i o n w i t h a f l o a t i n g s o l i d , s u c c i n i m i d e . F i l t r a t i o n , c o n c e n t r a t i o n t o 75 ml and c o o l i n g t o 0°C y i e l d e d i v o r y c r y s t a l s , w h i c h were shown t o be t h e r i n g b r o m i n a t i o n p r o d u c t s as above. When t h i s r e a c t i o n was. c a r r i e d o u t i n t h e p r e s c e n c e o f 2 , 2 ' - a z o b i s ( 2 - m e t h y l p r o p r i o n i t r i l e ) t h e same r e s u l t was o b t a i n e d as above. PREPARATION OF 3,5-DTMETHOXYBENZYL ALCOHOL(66) 3,5-Dimethoxybenzoic a c i d ( 4 4 ) (25 g, 0.14 mol) was c o n v e r t e d t o t h e m e t h y l e s t e r ( oo) v i - a t h e F i s c h e r - S p e i e r e s t e r -i f i c a t i o n g i v i n g m e t h y l 3,5-dimethoxybenzoate(00) (25.5 g, 98%) m.p. 34-36°C. The crude m e t h y l e s t e r (00) was t h e n d i s s o l v e d i n anhydrous e t h e r (250 ml) and l i t h i u m aluminum h y d r i d e (5.1 g) was added s l o w l y . The m i x t u r e was r e f l u x e d f o r 1 h r and t h e n t h e e x c e s s l i t h i u m aluminum h y d r i d e was d e s t r o y e d w i t h w a t e r and aqueous t a r t a r i c a c i d . The e t h e r l a y e r was s e p a r a t e d , d r i e d and e v a p o r a t e d t o y i e l d a w h i t e s o l i d . R e c r y s t a l l i z a t i o n from p e t e t h e r ( 3 0 - 6 0 ) gave 3,5-dimethoxy b e n z y l a l c o h o l ( 6 6 ) (18.4 g, 86%) m.p. 44-45.5°C as w h i t e n e e d l e s . v ( n u j o l ) 3500-3200, 2900, 1600, cm" 1; X 277 my; max J ' max T ( C C 1 4 ) 3.75(m, 3H), 5.59(s, 2H), 6.30(s, 6H) 6.62(s, I H ) . PREPARATION OF 1-(3,5-DIMETHOXY)-BENZYL BENZYL ETHER(67) 59 The method used was s i m i l a r t o t h a t o f McCloskey - 54 -3,5-Dimethoxybenzyl alcohol (66) (15.2 g, 0.09 mol) was dissolved i n excess benzyl chloride and powdered sodium hydroxide (8 g) was added. This mixture was s t i r r e d at 100°C for 4 hr and then at room temperature overnight. Water was added to remove the sodium hydroxide and t h i s was then extracted with ether. The • ether solution was dried and the ether removed under vacuum. The r e s u l t i n g l i q u i d residue was d i s t i l l e d at 178-180°C at 0.35 mm Hg giving 1-(3,5-dimethoxy)-benzyl benzyl ether(67) (14.1 g, 61%) as a clear viscous l i q u i d . v m a x ( f i l m ) 2950, 1600 cm"1; x(CCl 4) 2.76(s, 5H), 3.65(m, 3H) , 5.56(s, 2H) , 5.63(s, 2H) , 6.38(s, 6H); A 277, 224 my. max ATTEMPTED PREPARATION OF 1-(3,5-DIMETHOXY-4-GERANYL)-BENZYL  BENZYL ETHER(68) 3 6 A) The method used was s i m i l a r to that of C a r d i l l o et a l 1-(3,5-Dimethoxy)-benzyl benzyl ether(67) (2.0 g, 0.008 mol) was dissolved i n anhyrous benzene (50 ml) and n-butyl l i t h i u m (1.0 g) was added dropwise with s t i r r i n g . After s t i r r i n g at room temperature for 1 1/2 hr, geranyl chloride (3.0 g) was added and the mixture was refluxed for 24 hr. A few drops of ethanol were added to destroy any excess n-butyl l i t h i u m and the mixture was a c i d i f i e d . Extraction with ether, drying and removal of the ether under vacuum yielded a yellow o i l . Vacuum d i s t i l l a t i o n yielded only geranyl chloride and the s t a r t i n g ether ( 6 7 ) . The use of methyl l i t h i u m and geranyl bromide gave the same r e s u l t . - 55 -37 B) The method used was t h a t o f .Mechoulam 1 - ( 3 , 5 - D i m e t h o x y ) - b e n z y l b e n z y l e t h e r (67) (2.0 g, 0.008 mol) and g e r a n i o l (2.0 g) were d i s s o l v e d i n methylene c h l o r i d e (40 ml) and p - t o l u e n e s u l f o n i c a c i d (0.1 g) was added. The mix-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 24 h r and t h e n i t was washed w i t h b r i n e . Removal o f t h e s o l v e n t a f t e r d r y i n g and subsequent r e m o v a l o f any e x c e s s g e r a n i o l by vacuum d i s t i l l a t i o n l e f t o n l y t h e s t a r t i n g e t h e r ( 6 7 ) . 3 6 C) The method used v/as t h a t o f C a r d i l l o e t a l 1 - ( 3 , 5 - D i m e t h o x y ) - b e n z y l b e n z y l e t h e r (67) (2.0 g, 0.008 mol) and g e r a n i o l (2.0 g) were combined i n d e c a l i n (25 ml) and t h e s o l u t i o n was r e f l u x e d f o r 24 h r . Vacuum d i s t i l l a t i o n a f t e r t h i s t i m e y i e l d e d o n l y d e c a l i n , g e r a n i o l and t h e s t a r t i n g e t h e r ( 6 7 ) . PART I I LIGNAN SYNTHESIS INTRODUCTION The term " l i g n a n s " r e f e r s t o a group o f n a t u r a l l y o c c u r r i n g p h e n o l s d e r i v e d from two p h e n y l p r o p a n e u n i t s (C -C ) l i n k e d by 6 3 th e c e n t r a l c a r b o n atom o f t h e propane s i d e c h a i n ( 1 4 ) . The l i n k a g e i n n a t u r e p r o b a b l y o c c u r s t h r o u g h o x i d a t i v e c o u p l i n g o f two p h e n y l p r o p a n e u n i t s . (14) There a r e two g e n e r a l c l a s s e s o f l i g n a n s ; t h e a c t u a l l i g n a n s e x e m p l i f i e d i n s t r u c t u r e s (15) and (16) and t h e c y c l o -l i g n a n s ( 1 7 ) . The l i g n a n s can be f u r t h e r s u b d i v i d e d i n t o f o u r s t r u c t u r a l subgroups; t h e l i g n a n s ( 1 8 ) , t h e l i g a n o l i d e s ( 1 9 ) , the monoepoxylignans (20) and t h e b i s e p o x y l i g n a n s ( 2 1 ) . S e v e r a l examples o f each t y p e have been i s o l a t e d from v a r i o u s n a t u r a l s o u r c e s . (15) (16) (17) - 57 -The s y n t h e s i s of l i g n a n s i n v i t r o by o x i d a t i v e coupling-has been accomplished. O r i g i n a l l y i t i n v o l v e d the use of an enzyme mixture c o n t a i n i n g l a c c a s e as the dehydrogenating agent. 21 However, i t was then found.by Freudenberg t h a t the r e a c t i o n was n o n s p e c i f i c and thus l a r g e l y independent of the dehydrog-enating agent. He showed t h a t the a c t i v i t y of the enzyme was l i m i t e d to the dehydrogenation and the succeeding r e a c t i o n s proceeded a u t o m a t i c a l l y . R (R = <$>, R' = H) (20) (21) This then allowed the.use of various dehydrogenating agents, the most popular now i n use being peroxidase i n the prescence of hydrogen peroxide. Since then various copper, iron and manganese s a l t s have also been used. The coupling reaction seems to occur with the formation of the phenoxy r a d i c a l s which can then form several resonance structures and the product i s the r e s u l t of coupling of the most stable r a d i c a l s (see FIG. 7 ) . Although the product usually r e s u l t s from the most stable r a d i c a l s , other r a d i c a l s can react giving various undesired side products. In the labora-tory t h i s procedure leads to racemic products whereas the natur-a l l y occurring compounds are o p t i c a l l y a c t i v e . Thus i t would seem that asymmetric induction must be involved i n the natural process. R R R further reactions FIG. 7 DISCUSSION Our i n t e r e s t i n l i g n a n s centered around f i n d i n g a d i f f e r e n t , more e f f i c i e n t s y n t h e s i s i n v o l v i n g p h e n o l i c o x i d a t i v e c o u p l i n g . The method we chose would i n v o l v e i n t r a m o l e c u l a r c o u p l i n g as opposed t o i n t e r m o l e c u l a r c o u p l i n g which had p r e v i o u s l y been used . A f t e r s e v e r a l t r i a l s , we chose t o s y n t h e s i z e the hydroxy e s t e r (69) s i n c e o x i d a t i v e p h e n o l i c c o u p l i n g of t h i s compound would gi v e the b a s i c s k e l e t o n of the l i g a n o l i d e s (70). Due t o the comparative r i g i d i t y of t h i s molecule, i t seemed reasonable to expect more s p e c i f i c c o u p l i n g t o (70) w i t h l e s s s i d e products being formed. OH OH (69) (70) Since f r e e p h e n o l i c hydroxyls are very r e a c t i v e , i t was necessary t o p r o t e c t them by conversion to an ether before proceeding w i t h the s y n t h e s i s of (69). Our f i r s t s y n t h e t i c approach (see flow sheet VI) i n v o l v e d the use of the methyl ethers as p r o t e c t i n g groups. They were chosen s i n c e they are very s t a b l e and u n r e a c t i v e but can be cleaved under reasonable c o n d i t i o n s . - 60 -p-Anisaldehyde (71) on heating w i t h malonic a c i d , p y r i d i n e o q and p i p e r i d i n e gave p-methoxycinnamic a c i d (72) i n high y i e l d . Conversion of (72) t o the a c i d c h l o r i d e (73) was accomplished by s t i r r i n g w i t h an excess of o x a l y l c h l o r i d e at room tempera-t u r e . P a r t of (73) was then e s t e r i f i e d by r e f l u x i n g i n methanol to g i v e (74). Reduction of (74) w i t h l i t h i u m aluminum hydride gave the a l c o h o l (7 5) i n almost q u a n t i t a t i v e y i e l d . R e f l u x i n g (75) w i t h sodium hydride i n benzene gave the sodium s a l t (76), which we considered t o be a b e t t e r n u c l e o p h i l e than the c o r r e s -ponding a l c o h o l (75). E s t e r i f i c a t i o n to (77) was achieved by combining the a c i d c h l o r i d e (73) w i t h the sodium s a l t (76) and r e f l u x i n g i n benzene. C r y s t a l l i z a t i o n from e t h y l acetate gave (77) i n e x c e l l e n t y i e l d . I t remained only t o cleave the methyl ethers and we would have the d e s i r e d p h e n o l i c e s t e r (69) . However, s e v e r a l attempts at cleavage f a i l e d . In each case cleavage of the ether was accompanied by cleavage of the e s t e r . The methods t r i e d i n c l u d e d treatment of (77) w i t h boron t r i b r o m i d e at -78°C 4 1 and 0°C 4 0; r e f l u x i n g w i t h aluminum c h l o r i d e i n benzene 4 2 or s t i r r i n g at room t e m p e r a t u r e 4 3 ; r e f l u x i n g w i t h p-toluene s u l f o n i c a c i d i n s o l v e n t s ranging from dimethyl s u l f o x i d e to benzene 4 4; r e f l u x i n g w i t h p y r i d i n e h y d r o c h l o r i d e 4 5 . I t became apparent t h a t another p r o t e c t i n g group was needed. P r e f e r r a b l y i t should be a c i d l a b i l e but s t a b l e under the reac-t i o n s used. A f t e r some study we decided t o t r y the t e t r a h y d r o -p y r a n y l ethers 46/47 (see flow sheet V I I ) . We found t h a t p-hydroxy cinnamic a c i d (78) was a v a i l a b l e and we converted t h i s t o the methyl e s t e r (79) by r e f l u x i n g w i t h methanol and concentrated - 61 -FLOW SHEET VI - 62 -s u l f u r i c a c i d o v e r n i g h t . P r i o r e s t e r i f i c a t i o n was necessary, s i n c e i n the p y r a n y l a t i o n step the a c i d would form the t e t r a -hydropyranyl e s t e r . The t e t r a h y d r o p y r a n y l ether (80) of (79) was formed by shaking (7 9) w i t h an excess of dihydropyran c o n t a i n i n g a few drops of concentrated h y d r o c h l o r i c a c i d . P a r t of (80) was then reduced w i t h l i t h i u m aluminum hydride t o gi v e the a l c o h o l (82) which was subsequently converted to the sodium s a l t (83) w i t h sodium hydride i n benzene. To o b t a i n the a c i d c h l o r i d e (84) we h y d r o l i z e d (80) to the a c i d (81) by r e f l u x i n g i n a l c o h o l i c base and then t r e a t e d (81) w i t h excess o x a l y l c h l o r i d e . U n f o r t u n a t e l y , one of the byproducts of t h i s r e a c t i o n i s hydrogen c h l o r i d e , and although.we thought the t e t r a h y d r o -p y r a n y l ether would be s t a b l e t o the dry gas, i t became obvious t h a t the ether was cleaved under these c o n d i t i o n s . Thus i n s t e a d of o b t a i n i n g (85), we obtained the polymeric s e l f - c o n d e n s a t i o n product (86) of the phenol w i t h the a c i d c h l o r i d e . 0 ^0 (86) Since i t seemed t h a t use of an a c i d l a b i l e p r o t e c t i n g group and the e s t e r i f i c a t i o n method used i n sequences VI and V I I above were not compatible, we set out t o f i n d another method of FLOW - 63 -SHEET V I I - 64 -e s t e r i f i c a t i o n . A f t e r some l i t e r a t u r e r e s e a r c h we d e c i d e d t o 4 8 t r y e s t e r i f i c a t i o n u s i n g d i c y c l o h e x y l c a r b o d i i m i d e (DCC) (87) I t had been r e p o r t e d 4 ^ - 5 ^ t h a t when a c a r b o x y l i c a c i d and an a l c o h o l a r e combined i n t h e p r e s e n c e o f DCC t h e c o r r e s p o n d i n g e s t e r i s formed v i a t h e mechanism Shown i n FIG. 11. The DCC (87) i s r e v e r s i b l y p r o t o n a t e d and t h e n a t t a c k e d by t h e c a r b o x y l a n i o n y i e l d i n g t h e O - a c y l i s o u r e a i n t e r m e d i a t e w h i c h can t h e n r e v e r s i b l y p r o t o n a t e s e t t i n g i t up f o r a t t a c k by an a l c o h o l t o form t h e e s t e r (path a) and d i c y c l o h e x y l u r e a . A l t e r n a t i v e l y , i t can r e a r r a n g e t o t h e N - a c y l u r e a ( p a t h b) w h i c h does n o t r e a c t f u r t h e r . Thus i t can be seen t h a t i f t h e a l c o h o l p r e s e n t i s n o t a s t r o n g enough n u c l e o p h i l e t o t r a p t h e i n t e r m e d i a t e O - a c y l i s o u r e a i t w i l l r e a r r a n g e t o g i v e t h e n o n - r e a c t i v e N - a c y l u r e a and e s t e r i f i c a t i o n w i l l n o t o c c u r . R - N = C = N - R — R - N H R - N H C =f N H C I f 0 - R - R' - R 0 1 (b) (a) R - N - C - N - R H 0 R' - C - R* R' OH fl C - R' II 0 OR" + R - N H C - N " H 0 H - R FIG. 11 (R = C6E1±) - 65 -When methanol was used as the a l c o h o l , e s t e r i f i c a t i o n of p-methoxycinnamic a c i d (72) t o the methyl e s t e r (74) proceeded w i t h ease (see flow sheet V I I I ) . However, using p-O-tetrahydro-pyranylcinnamyl a l c o h o l (82) and p-O-tetrahydropyranylcinnamic a c i d (81) w i t h DCC r e s u l t e d i n i s o l a t i o n of only the N-acyl urea (88) produced by rearrangement of the O-acyl i s o u r e a (89) and recovery of the a l c o h o l (82) (see flow sheet V I I I ) . This would seem to i n d i c a t e t h a t (82) i s not a strong enough nucleo-p h i l e f o r t h i s e s t e r i f i c a t i o n . OTHP (88) OTHP (89) The l i t e r a t u r e ^ 4 - 5 8 contains r e p o r t s of other workers who have encountered t h i s problem and i t has been solved by develop-i n g a r e a c t i o n whereby the a l c o h o l i s f i r s t reacted w i t h DCC, using copper c h l o r i d e as a c a t a l y s t , to give the O - a l k y l i s o u r e a (90). This i s then attacked by the a c i d anion r e s u l t i n g i n the e s t e r and d i c y c l o h e x y l u r e a (see FIG. 12). The advantage of t h i s method i s t h a t the intermediate formed i s not prone to rearrange-ment and the c a r b o x y l i c a c i d i s r e a c t i v e enough t o atta c k i t i n most cases. We combined p-O-tetrahydropyranylcinnamyl a l c o h o l (82) w i t h - 6 6 -FLOW SHEET V I I I ( 8 2 ) - 6 7 -DCC and copper c h l o r i d e i n d i m e t h y l formamide and 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 4 hours (see f l o w s h e e t I X ) . p-Hydroxy c i n n a m i c a c i d (78) was t h e n added and workup y i e l d e d a sweet s m e l l i n g y e l l o w o i l w h i c h had t h e u l t r a v i o l e t c h a r a c t e r i s t i c s o f t h e e x p e c t e d e s t e r (91). P u r i f i c a t i o n was found t o be v e r y d i f f i c u l t and t h e a t t e m p t was abandoned. I t i s p o s s i b l e t h a t use o f an a c i d w i t h a p r o t e c t e d p h e n o l i c h y d r o x y l m i g h t r e s u l t i n a c l e a n e r p r o d u c t . Had t h e e s t e r been o b t a i n e d , v a r i o u s o x i d a t i v e methods would have been t r i e d t o c o n v e r t i t t o t h e l i g a n o l i d e s k e l e t o n (70). R - N = C = N - R + R' OH Cu C l R - N - C = H | OR1 N - R R N - C = & H K H O 1 R - R R"-COO R - N - C -H N - R H + R"COOR (R = C 6H l ; L) FIG. 12 - 68 -FLOW SHEET IX THPO COOH OH EXPERIMENTAL The U l t r a v i o l e t s p e c t r a were r e c o r d e d i n m e t hanol on a UNICAM SP800 s p e c t r o p h o t o m e t e r . The I n f r a r e d s p e c t r a were r e c o r d e d on a P e r k i n Elmer 137 s p e c t r o p h o t o m e t e r w i t h sodium c h l o r i d e c e l l s u s i n g e i t h e r n u j o l m u l l s o r c h l o r o f o r m s o l u t i o n s . The N u c l e a r M a g n e t i c Resonance s p e c t r a were r e c o r d e d on T-60, A-60 and C-60 s p e c t r o p h o t o m e t e r s w i t h 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 and t h e s o l v e n t s as i n d i c a t e d i n t h e t e x t . The Mass s p e c t r a were r e c o r d e d on a MS-9 mass s p e c t r o p h o t o m e t e r . M e l t i n g p o i n t s were r e c o r d e d on a K o f l e r M i c r o H e a t i n g Stage ( R e i c h e r t ) and a r e u n c o r r e c t e d . The c h e m i c a l s used came l a r g e l y from F i s c h e r , A l d r i c h , Eastman O r g a n i c and B r i t i s h Drug Houses. - 70 -PREPARATION OF p-METHOXY CINNAMIC ACID(72) The method used was t h a t o f D u t t 3 9 . p - A n i s a l d e h y d e ( 7 1 ) (44.8 m l , 0.37 m o l ) , m a l o n i c a c i d (37 g ) , p y r i d i n e (2.5 ml) and p i p e r i d i n e (2.5 ml) were combined i n a 250 ml f l a s k and h e a t e d a t 120°C f o r 1/2 h r . The r e s u l t was a s o l i d mass w h i c h on r e c r y s t a l l i z a t i o n from e t h a n o l gave p-methoxy c i n n a m i c a c i d (72) (59 g, 92%) m.p. 173-174.5°C(lit. m.p. 173°C) as w h i t e n e e d l e s . v ( n u j o l ) 2900-2400, 1690, c max 1601 cm" 1; X 313, 299, 292, 226, 210 my; x (CDCl ) 2.50-3.83 IU3.X _3 (m, 6H), 6.16(s, 3H). PREPARATION OF METHYL p-METHOXYCTNNAMATE(74) p-Methoxy c i n n a m i c a c i d (7 2) (18 g, 0.1 mol) was s t i r r e d a t room t e m p e r a t u r e w i t h e x c e s s o x a l y l c h l o r i d e f o r 1/2 h r . The e x c e s s o x a l y l c h l o r i d e was t h e n removed under vacuum by c h a s i n g w i t h benzene. The r e s u l t i n g y e l l o w s o l i d was d i s s o l v e d i n an e x c e s s o f methanol and t h i s s o l u t i o n was r e f l u x e d f o r 1 1/2 h r . C o n c e n t r a t i o n o f t h e methanol and c o o l i n g t o 0°C r e s u l t e d i n a w h i t e p r e c i p i t a t e . R e c r y s t a l l i z a t i o n from methanol gave m e t h y l p-methoxycinnamate (7 4 ) (19.3 g, 98%) m.p. 91.5-92°C as w h i t e c u b i c c r y s t a l s . v ( n u j o l ) 1705, 1640, 1601 cm 1 ; 2 max i t 1 X 318, 300, 293, 227, 211 my; T (CDCl-)• 2.32-3.95 (m, 6H) , nicix -_J 6.21 ( s , 3H), 6. 29 ( s , 3H). PREPARATION OF p-METHOXY CINNAMYL ALCOHOL(75) M e t h y l p-methoxycinnamate (7 4) (19.3 g, 0.1 mol) v/as d i s s o l v e d i n anhydrous e t h e r (2 5 0 ml) and l i t h i u m aluminum - 71 -h y d r i d e (3.0 g) was added s l o w l y . The m i x t u r e was r e f l u x e d • f o r 3 h r and t h e n t h e e x c e s s l i t h i u m aluminum h y d r i d e was d e s t r o y e d w i t h w a t e r and aqueous t a r t a r i c a c i d . E x t r a c t i o n w i t h e t h e r and subsequent d r y i n g and r e m o v a l o f t h e e t h e r under vacuum y i e l d e d a y e l l o w o i l w h i c h s o l i d i f i e d on s t a n d i n g . R e c r y s t a l l i z a t i o n from e t h e r gave p-methoxycinnamyl a l c o h o l ( 7 5 ) (10 g, 61%) m.p. 79-80°C as w h i t e f l a k e s . v ( n u j o l ) 3300-IU3.X 3200, 1601, 1501 c m - 1 ; X 261, 207 my; T ( a c e t i c a c i d - d 4 ) max 2.60-4.10(m, 6H) , 5.78(d, 2H) , 6.23(s, 3H) , 7.82 ( s , IH) . PREPARATION OF p-METHOXYCINNAMYL p-METHOXYCINNAMATE(77) p-Methoxycinnamic a c i d ( 7 2 ) (5.0 g, 0.03 mol) was s t i r r e d w i t h e x c e s s o x a l y l c h l o r i d e f o r 1/2 h r and t h e n t h e e x c e s s o x a l y l c h l o r i d e was removed under vacuum by c h a s i n g w i t h benzene. The r e s u l t i n g y e l l o w s o l i d was d i s s o l v e d i n anhydrous benzene (100 ml) and t h i s was s l o w l y added t o a benzene s u s p e n s i o n o f t h e sodium s a l t o f p-methoxycinnamyl a l c o h o l ( 7 6 ) , formed by r e f l u x i n g p-methoxycinnamyl a l c o h o l ( 7 5) (5.0 g, 0.03 mol) w i t h sodium h y d r i d e (1.6 g) i n benzene (25 m l ) . The r e s u l t was a v i g o r o u s r e a c t i o n i n a c l o u d y s o l u t i o n . The m i x t u r e was r e f l u x e d f o r 1 h r and t h e n f i l t e r e d h o t . Removal o f t h e benzene under vacuum y i e l d e d an amorphous s o l i d . R e c r y s t a l l i z a t i o n from e t h y l a c e t a t e gave p-methoxycinnamyl p-methoxycinnamate(77) (8.3 g, 83%) m.p. 96-97°C as w h i t e f l u f f y n e e d l e s . v (CHC1 ) . max 3 3000, 2950, 2850, 1730, 1640, 1603 c m - 1 ; X 306, 295, 270, max 229 my; T(CDG1 3) 2.10-3.85(m, 12H) , 5.15(d, 2H) , 6.18(s, 3H) , 6.20(s, 3H); C a l c u l a t e d C-74.07% found C-74.34% H-6.17% H-6.31%. - 72 -ATTEMPTED PREPARATION OF p-HYDROXYCINNAMYL p-HYDROXYCINNAMATE( 69) A) The method used was s i m i l a r t o t h a t o f Benton and D i l l o n 4 0 . p - Methoxycinnamyl p-methoxycinnamate(77) (1.0 g, 0.003 mol) was d i s s o l v e d i n methylene c h l o r i d e (50 ml) and c o o l e d t o 0°C. Then boron t r i b r o m i d e (1.6 ml) was s l o w l y added and t h e m i x t u r e was a l l o w e d t o come t o room t e m p e r a t u r e o v e r n i g h t . An e q u a l amount o f w a t e r was added and t h e m i x t u r e was shaken and l e f t a t room t e m p e r a t u r e f o r 3 h r . E x t r a c t i o n w i t h e t h e r y i e l d e d an o i l w h i c h on a n a l y s i s was found t o c o n t a i n o n l y p r o d u c t s i n w h i c h t h e r e was no e s t e r f u n c t i o n . Thus e s t e r c l e a v a g e had o c c u r r e d as w e l l as e t h e r c l e a v a g e . B) The method used was s i m i l a r t o t h a t o f S c h a f e r and F r a n c k 4 1 . p -Methoxycinnamyl p-methoxycinnamate(77) (1.0 g, 0.003 mol) was d i s s o l v e d i n methylene c h l o r i d e and c o o l e d t o -78°C. To t h i s s t i r r e d s o l u t i o n b o ron t r i b r o m i d e (0.5 ml) was added and t h e r e d s o l u t i o n was a l l o w e d t o come t o room t e m p e r a t u r e o v e r 4 h r . The methylene c h l o r i d e was removed under vacuum and t h e r e s i d u e was t a k e n up i n e t h a n o l (30 ml) and w a t e r (3 ml) and t h i s was r e f l u x e d f o r 4 h r . Removal o f t h e e t h a n o l under vacuum and e x t r a c t i o n w i t h e t h e r y i e l d e d a m i x t u r e o f p r o d u c t s due t o e s t e r c l e a v a g e as w e l l as e t h e r c l e a v a g e . C) The method used was s i m i l a r t o t h a t o f L e d n i c e r and G r o s t i c p - Methoxycinnamyl p-methoxycinnamate(77) (0.5 g, 0.0015 mol) was d i s s o l v e d i n anhydrous benzene and aluminum c h l o r i d e (0.21 g) was added w i t h s t i r r i n g . The mixture was r e f l u x e d f o r 1/2 hf and then the benzene was removed under vacuum. The residue was cooled i n an i c e - s a l t bath and 3N h y d r o c h l o r i c a c i d (20 ml) was s l o w l y added. E x t r a c t i o n w i t h e t h y l acetate y i e l d e d s e v e r a l products, a l l of which were e s t e r cleavage fragments. D) The method used was s i m i l a r t o t h a t of Klamann and 44 W e y e r s t a l l p-Methoxycinnamyl p-methoxycinnamate(77) (0.5 g, 0.0015 mol) was d i s s o l v e d i n d i m e t h y l s u l f o x i d e (20 ml) and p-toluene s u l f o n i c a c i d (0.13 g) was added. This s o l u t i o n was r e f l u x e d f o r 3 hr and then the d i m e t h y l s u l f o x i d e was removed by vacuum d i s t i l l a t i o n . The r esidue was taken up i n water and t h i s was then e x t r a c t e d w i t h ether. Examination of the r e s u l t a n t brown o i l showed t h a t e s t e r cleavage had occurred as w e l l as ether cleavage. The experiment was repeated using water and benzene as s o l v e n t s w i t h s i m i l a r r e s u l t s . 43 E) The method used was s i m i l a r to t h a t of O l l i s e t a l p-Methoxycinnamyl p-methoxycinnamate(77) (0.5 g, 0.0015 mol) was d i s s o l v e d i n anhydrous benzene (10 ml) and aluminum c h l o r i d e (0.21 g) was added. This mixture was s t i r r e d at room temperature f o r 2 hr and then the benzene was removed under vacuum. H y d r o c h l o r i c a c i d (3N) (20 ml) was added to the residue and t h i s v/as shaken u n t i l s o l u t i o n was e f f e c t e d . E x t r a c t i o n w i t h ether revealed t h a t e s t e r cleavage had again occurred as w e l l as ether cleavage. 45 F) The m thod used was s i m i l a r to t h a t of Curphey et a l p-Methoxycinnamyl p-methoxycinnamate(77) (1.0 g, 0.003 mol) - 74 -was added t o a m i x t u r e o f p y r i d i n e (5.2 ml) and 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 (5.7 ml) w h i c h had been h e a t e d t o 210°C w h i l e d i s t i l l i n g o f f t h e wa t e r and t h e n c o o l e d t o 140°C. The s o l u t i o n was r e f l u x e d under a n i t r o g e n atmosphere f o r 3 h r and t h e n was d i l u t e d w i t h w a t e r . E x t r a c t i o n w i t h e t h e r y i e l d e d a b l a c k t a r , e x a m i n a t i o n o f w h i c h a g a i n r e v e a l e d t h a t e s t e r c l e a v a g e had t a k e n p l a c e . PREPARATION OF METHYL p-HYDROXYCINNAMATE(7 9) p-Hydroxy c i n n a m i c a c i d (78) (10 g, 0.06 mol) was d i s s o l v e d i n d r y methanol (850 ml) and c o n c e n t r a t e d s u l f u r i c a c i d (25 ml) was added s l o w l y . The s o l u t i o n was r e f l u x e d o v e r n i g h t and th e n n e u t r a l i z e d w i t h s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n . Most o f t h e methanol was removed under vacuum and c o o l i n g o f th e r e s i d u a l s o l u t i o n r e s u l t e d I n a p i n k i s h p r e c i p i t a t e . R e c r y s -t a l l i z a t i o n from e t h a n o l gave m e t h y l p - h y d r o x y c i n n a m a t e ( 7 9 ) (8.3 g, 76%) m.p. 140-141°C as w h i t e f l a k e s . v ( n u j o l ) 3400, ^ ' r , m a x \ J > r 1695, 1640, 1603, 1590 cm" 1; A 312, 300, 294, 228, 211 my; IT13.X T ( C D C 1 3 ) 2.20-3.90(m, 6H), 6.20(s, 3H), 8.18(s, I H ) . PREPARATION OF METHYL p-O-TETRAHYDROPYRANYLCINNAMATE(80) 46 47 The method used was s i m i l a r t o t h a t o f Parham ' M e t h y l p - h y d r o x y c i n n a m a t e ( 7 9 ) (9.8 g, 0.055 mol) was added s l o w l y t o d i h y d r o p y r a n (13.3 ml) c o n t a i n i n g 2 drops o f concen-t r a t e d h y d r o c h l o r i c a c i d . The s o l u t i o n was shaken o v e r n i g h t on a m e c h a n i c a l s h a k e r r e s u l t i n g i n a w h i t e s o l i d mass. T h i s was d i s s o l v e d i n e t h e r and t h e e t h e r s o l u t i o n was washed w i t h 2N sodium h y d r o x i d e t o remove t h e c a t a l y s t and any s t a r t i n g - 75 -m a t e r i a l s t i l l p r e s e n t . D r y i n g and removal of the e t h e r under vacuum gave a y e l l o w o i l which c r y s t a l l i z e d on s t a n d i n g . R e c r y s t a l l i z a t i o n from e t h a n o l gave methyl p - O - t e t r a h y d r o p y r a n y l cinnamate (80) (12.2 g, 85%) m.p. 65-67°C as white f l a k e s . v (CHC1-.) 3005, 2930, 2860, 1695, 1630, 1603 cm" 1; A 306, max 3 ' ' ' ' max ' 299, 293, 225, 209 m ; T ( C C 1 4 ) 2.35-4.00 (m, 6H) , 4.60(s, IH) , 6.30(s, 3H) , 8.00-8.70 (m, 8H) . PREPARATION OF p - 0 - T E T RAHYDROPYRANYLC ITSf NAM IC ACID (81) Methyl p-O-tetrahydropyranylcinnamate(80) (13.0 g, 0.05 mol) was d i s s o l v e d i n methanol (200 ml) and 2N a l c o h o l i c sodium hydroxide (100 ml) was added. The mixture was s t i r r e d and r e f l u x e d f o r 1/2 hr and then the methanol was removed under vacuum. The water s o l u t i o n was a c i d i f i e d t o a pH of 6.8 and immediately e x t r a c t e d w i t h e t h e r . D r y i n g and removal o f the e t h e r under vacuum y i e l d e d a y e l l o w s o l i d . T r i t u r a t i o n w i t h e t h e r and r e c r y s t a l l i z a t i o n from c h l o r o f o r m gave p - O - t e t r a -h y d r o p y r a n y l c i n n a m i c a c i d (81) (11.0 g, 90%) m.p. 158-160°C as white n e e d l e s . v (CHC1-) 3500-2400, 1690, 1640, 1603 cm" 1; nicix o A 305, 298, 291, 224, 209 my; x(CDCl^) 2.00-3.80(m, 6H), nicix o 2.48(s, IH) , 6.00-6.40 (m, 2 H ) , 7 . 8 - 8 . 6 ( m , 6 H ) . PREPARATION OF p-O-TETRAHYDROPYRANYL'CTNNAMYL ALCOHOL (82) Methyl p-O-tetrahydropyranylcinnamate(80) (10.0 g, 0.04 mol) was d i s s o l v e d i n anhydrous e t h e r (50 ml) and l i t h i u m aluminum h y d r i d e (1.2 g) i n anhydrous e t h e r (50 ml) v/as added s l o w l y . The mixture was r e f l u x e d f o r 1 hr and then the excess - 76 -l i t h i u m aluminum h y d r i d e was d e s t r o y e d w i t h w a t e r and aqueous' t a r t a r i c a c i d . 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 was d r i e d and removed under vacuum t o g i v e p - 0 - t e t r a -h y d r o p y r a n y l c i n n a m y l a l c o h o l ( 8 2 ) (9.0 g, 98%) as a l i g h t y e l l o w o i l . v ( f i l m ) 3400, 2940, 2860, 1605 cm" 1; X 260 my; max ' ' ' ' max H T ( C D C 1 3 ) 2.50-4.00(m, 6H), 4.55(s, I H ) , 5.70(d, 2H), 6.00-6.60 (m, 2H) , 8.00-8.60 (m, 6H) . ATTEMPTED PREPARATION OF p-O-TETRAHYDROPYRANYLCINNAMYL p-O- TETRAHYDROPYRANYLCINNAMATE(85) p - O - T e t r a h y d r o p y r a n y l c i n n a m i c a c i d (81) (11.9 g, 0.05 mol) was d i s s o l v e d i n anhydrous benzene (50 ml) and sodium h y d r i d e (2.3 g) was added s l o w l y . T h i s m i x t u r e was r e f l u x e d f o r 3 h r and on c o o l i n g , o x a l y l c h l o r i d e (10 ml) was added and t h e mix-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 1/2 h r . The e x c e s s o x a l y l c h l o r i d e was removed under vacuum and t h e r e s i d -u a l y e l l o w - g r e e n s o l i d was d i s s o l v e d i n benzene. p - O - T e t r a -h y d r o p y r a n y l c i n n a m y l a l c o h o l ( 8 2 ) (9.0 g, 0.04 mol) was d i s s o l v e d i n anhydrous benzene (50 ml) and sodium h y d r i d e (3.0 g) was added. T h i s m i x t u r e was r e f l u x e d f o r 15 min and was t h e n added t o t h e benzene s o l u t i o n o f t h e y e l l o w - g r e e n p r o d u c t o b t a i n e d above. T h i s c o m b i n a t i o n was r e f l u x e d f o r 1 h r , f i l t e r e d h o t and t h e benzene removed under vacuum t o g i v e a y e l l o w o i l . S p e c t r a l e x a m i n a t i o n o f t h i s o i l showed t h a t i t was n o t t h e d e s i r e d e s t e r b u t r a t h e r a p o l y m e r i c s p e c i e s i n d i c a t i n g t h a t the t e t r a h y d r o p y r a n y l group had been removed i n t h e r e a c t i o n . - 77 -ATTEMPTED PREPARATION OF p-O-TETRAHYDROPYRANYLCINNAMYL p-O- TETRAHYDROPYRANYLCINNAMATE(8 5) 52 The method used was s i m i l a r t o t h a t o f Khorana p - O - T e t r a h y d r o p y r a n y l c i n n a m y l a l c o h o l ( 8 2 ) (0.5 g, 0.002 mol) and p - O - t e t r a h y d r o p y r a n y l c i n n a m i c a c i d ( 8 1 ) (0.5 g, 0.002 mol) were combined w i t h d i c y c l o h e x y l - c a r b o d i i m i d e (0.8 g) i n e t h e r (10 m l ) . T h i s s o l u t i o n was s t i r r e d o v e r n i g h t a t room temper-a t u r e r e s u l t i n g i n a w h i t e p r e c i p i t a t e and a c l e a r e t h e r s o l u -t i o n . The w h i t e s o l i d was s p e c t r a l l y a n a l y z e d and shown t o be the N - ( p - O - t e t r a h y d r o p y r a n y l c i n n a m o y l ) - u r e a ( 8 8 ) . Removal o f th e e t h e r under vacuum from t h e f i l t r a t e y i e l d e d an o i l w h i c h was shown t o be p - O - t e t r a h y d r o p y r a n y l c i n n a m y l a l c o h o l ( ) by co m p a r i s o n w i t h an a u t h e n i c sample. No t r a c e o f t h e e x p e c t e d e s t e r was f o u n d . ATTEMPTED PREPARATION OF p-O-TETRAHYDROPYRANYLCINNAMYL p- HYDROXYCINNAMATE(91) 56 The method used was t h a t o f Schmidt and Mo o s m t i l l e r p - O - T e t r a h y d r o p y r a n y l c i n n a m y l a l c o h o l ( 8 2 ) (8.8 g, 0.04 m o l ) , d i c y c l o h e x y l - c a r b o d i i m i d e (7.8 g) and copper c h l o r i d e (4 mg) were combined i n anhydrous d i m e t h y l f o r m a m i d e (30 ml) and t h e r e s u l t i n g g r e e n s o l u t i o n 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 24 h r . p - H y d r o x y c i n n a m i c a c i d ( 7 8 ) (4.4 g, 0.03 mol) was added r e s u l t i n g i n a v e r y b r i g h t g r e e n s o l u t i o n . T h i s s o l u -t i o n was s t i r r e d f o r a f u r t h e r 2 h r a t room t e m p e r a t u r e and th e r e s u l t i n g d i c y c l o h e x y l u r e a was f i l t e r e d . The f i l t r a t e was d i l u t e d w i t h t w i c e i t s volume o f wat e r and e x t r a c t e d w i t h ether. 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