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Studies related to natural products : biosynthesis of coumarins Collier, Peter Lawrence 1971

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STUDIES RELATED TO NATURAL PRODUCTS: BIOSYNTHESIS OF COUMARINS  BY  PETER LAWRENCE COLLIER B.Sc. Honours, University of Alberta, 1967  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE i n the DEPARTMENT OF CHEMISTRY  We accept this thesis as conforming  to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA May, 1971  In p r e s e n t i n g t h i s an the  thesis  advanced degree at Library  in p a r t i a l f u l f i l m e n t o f  the U n i v e r s i t y  of  the  requirements  B r i t i s h Columbia, I agree  s h a l l make i t f r e e l y a v a i l a b l e  for  r e f e r e n c e and  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f f o r s c h o l a r l y purposes may by  his  of  this  written  representatives.  be g r a n t e d by  the Head of my  It i s understood that  t h e s i s f o r f i n a n c i a l gain s h a l l not permission.  Depa rtment The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Columbia  be  that  study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  - i i-  ABSTRACT  This thesis describes a biosynthetic i n v e s t i g a t i o n of coumarins i n turpentine-broom, Thamnosma montana  Torr. and Frem.  In contrast to simple coumarins, the biosynthetic pathways leading to the furanocoumarins were found to be i n a state of confusion as indicated by e x i s t i n g published  data.  None of the r e s u l t s were i n t e r n a l l y  consistent with any general postulate and there was question  considerable  concerning the actual meaning of a s u b s t a n t i a l portion of the  experimental data. Studies as described here were performed i n several d i f f e r e n t areas. In the i n i t i a l i n v e s t i g a t i o n s , a d e t a i l e d study of i s o l a t i o n procedures for the many coumarins present i n the plant was necessary. appropriate  Subsequently,  chemical degradative pathways were developed f o r umbelliprenin  (11), i s o p i m p i n e l l i n (2), and alloimperatorin methyl ether i s o l a t i o n of relevant carbon atoms i n these coumarins.  (8) to allow  Finally,  3 incorporation studies with DL-mevalonic-5 H acid were conducted and -  subsequent degradative reactions were performed on umbelliprenin and alloimperatorin methyl ether preliminary  (11)  (8). The implications of these  experiments i n terms of biosynthetic pathways are presented.  - iii -  TABLE OF CONTENTS Page TITLE PAGE  i  ABSTRACT  i i  TABLE OF CONTENTS LIST OF FIGURES  i i i .'  iv  ACKNOWLEDGEMENTS  vi  STUDIES RELATED TO THE BIOSYNTHESIS OF COUMARINS INTRODUCTION  i  2  DISCUSSION  41  EXPERIMENTAL  60  BIBLIOGRAPHY  85  - iv -  LIST OF FIGURES Figure  Page  1  Some representative n a t u r a l l y occurring coumarins...  7  2  The shikimic acid pathway to aromatic compounds ....  9  3  Radioactive compounds i s o l a t e d from H. odorata  13  4  Proposed scheme for coumarin biosynthesis i n Melilotus and H_. odorata  16  Proposed scheme f o r biosynthesis of umbelliferone i n Eydrangea  18  6  Proposed scheme f o r h e r n i a r i n biosynthesis  21  7  Possible stages of O-methylation i n h e r n i a r i n biosynthesis  22  8  Modified scheme f o r h e r n i a r i n biosynthesis  22  9  Involvement of a spirolactone i n coumarin biosynthesis  24  10  Suggested biosynthetic scheme f o r c a l o p h y l l o l i d e ...  26  11  Degradation of sphondin  28  12  Degradation of pimpinellin  29  13  Proposed scheme f o r the biosynthesis of furanocoumarins  32  Proposed.alternative biosynthesis  35  5  14 15  pathway of furanocoumarin  Furanocoumarins recovered from Ruta graveolens and Heracleum lanatum  38  16  Proposed scheme f o r the biosynthesis of marmesin....  39  17  T y p i c a l p u r i f i c a t i o n sequence of components from Thamnosma montana, Torr. and Frem  44  A t y p i c a l thin layer chromatoplate of i s o l a t e d fractions obtained from the chloroform extract  45  A thin layer chromatoplate of some authentic samples of compounds i n Thamnosma montana Torr. and Frem. ..  46  P u r i f i c a t i o n of f r a c t i o n s G, H and I , J and K, and L of the i n i t i a l column chromatography  47  18  19  20  - v Figure  Page  21  Conversion of umbe11iprenin to umbelliferone  48  22  Degradations  of i s o p i m p i n e l l i n  49  23  Degradations  of, alloimperatorin  24  R e c r y s t a l l i z a t i o n of various coumarins to constant radioactivity  54  R e c r y s t a l l i z a t i o n of umbelliferone to constant radioactivity  56  R e c r y s t a l l i z a t i o n of alloimperatorin d i o l to constant r a d i o a c t i v i t y  57  25  26  27  methyl ether .........  51  methyl ether  R e c r y s t a l l i z a t i o n of the alcohol, compound (48), to constant r a d i o a c t i v i t y  57  - vi-  ACKNOWLEDGEMENTS  I wish to express my gratitude to Professor James P. Kutney f o r his  encouragement and guidance throughout the course of this  research. I am also extremely g r a t e f u l to R.N. Young and A.K. Verma f o r t h e i r collaboration and patience with me i n t h i s research and f o r many h e l p f u l suggestions. Special thanks i s due also to Miss Diane Johnson f o r typing the manuscript.  - 1-  BIOSYNTHESIS OF COUMARINS  - 2 -  INTRODUCTION  The turpentine-broom, Thamnosma montana Torr. and Frem. (Rutaceae), i s found i n desert mesas and slopes.  These shrubby plants were 12  reported to have plant-growth-inhibitor properties. '  In addition,  they were reported to have been used by American Indians i n f o l k medicine.^ Bennett and Bonner studied the t o x i c i t y of aqueous extracts of leaves of eleven desert plant species and found that Thamnosma montana Torr. and Frem. was the most toxic as judged by the response to tomato.^ The crude material caused the death of young tomato plants at a concent r a t i o n of about 1 mg/ml within seven days. The plants of the Rutaceae family are w e l l known to contain a large number of benzenoid compounds, coumarins, flavones and some quinoline alkaloids.  Bennett and Bonner i s o l a t e d three c r y s t a l l i n e compounds  from Thamnosma montana and i d e n t i f i e d two of them as byakangelicin(1) and i s o p i m p i n e l l i n (2), respectively.^"  - 3 -  The s t r u c t u r e o f the t h i r d  compound was e l u c i d a t e d by Dreyer  and found t o be a l l o i m p e r a t o r i n methyl e t h e r d i o l  (3) [ 5 - ( 3 ' - m e t h y l -  2',3'-dihydroxybutanyl)-8-methoxypsoralen].  OCH Dreyer developed a b e t t e r e x t r a c t i o n scheme f o r i s o l a t i n g n o t o n l y t h e t h r e e compounds o b t a i n e d by Bennett to s e p a r a t e s i x o t h e r compounds.  and Bonner, but was a b l e  These s i x compounds were  (A); t h r e e known a l k a l o i d s , N - m e t h y l a c r i d o n e  g-sitosterol  ( 5 ) , skimmianine ( 6 ) ,  and y ~ f a g a r i n e ( 7 ) ; a known furanocoumarin  a l l o i m p e r a t o r i n methyl  ( 8 ) ; and an unknown compound, thamnosin.  T h i s was the f i r s t  of N - m e t h y l a c r i d o n e ,  report  t h e p a r e n t member o f a c r i d o n e a l k a l o i d s , ~*  o c c u r r i n g as a n a t u r a l p r o d u c t .  ether  However, recent work i n our laboratories by T. Inaba has proved t h i s tentative structure to be i n c o r r e c t .  Furthermore, Inaba^ as a r e s u l t  of a complete e l u c i d a t i o n of the structure of thamnosin, assigned the following structure to the compound.  - 5 -  H  (10)  As a r e s u l t o f f u r t h e r r e c e n t  work i n our l a b o r a t o r i e s on  Thamnosma montana, e i g h t o t h e r compounds were s e p a r a t e d and i n a d d i t i o n t o the n i n e p r e v i o u s l y above.  i s o l a t e d by the workers mentioned  These compounds were u m b e l l i p r e n i n  a l l o i m p e r a t o r i n methyl ether  epoxide  (15), x a n t h o t o x i n ( 1 6 ) , p s o r a l e n e  characterized  (11), i s o i m p e r a t o r i n (12),  ( 1 3 ) , thamnosmin ( 1 4 ) , b e r g a p t e n  ( 1 7 ) , and p h e l l o . p t e r i n ( 1 8 ) .  - 6-  Following  the i s o l a t i o n and i d e n t i f i c a t i o n o f t h e numerous  compounds i n the p l a n t , i t was d e c i d e d work on some o f the coumarins. with  t o conduct some b i o s y n t h e t i c  T h i s s e c t i o n o f the t h e s i s i s concerned  some o f t h e b i o s y n t h e t i c work done on some o f the coumarins i n  Thamnosma montana.  I t i s therefore appropriate  of b i o s y n t h e s i s i n t h i s  Biosynthesis The  to discuss the status  area.  o f Coumarins  coumarins form a d i v e r s i f i e d  and q u i t e w i d e l y d i s t r i b u t e d  c l a s s o f n a t u r a l l y o c c u r r i n g a r o m a t i c compounds.  Coumarin i t s e l f i s  the s i m p l e s t member o f t h i s c l a s s .  Other compounds i n t h i s c l a s s have  substituents of varying  ranging  complexity,  methoxyl groups to i s o p r e n o i d s i d e c h a i n s  from simple  hydroxyl or  and i s o p r e n o i d - d e r i v e d  A s t r i k i n g p o i n t about t h e s u b s t i t u t e d coumarins i s t h a t w i t h very  few known e x c e p t i o n s ,  i s oxygenated  para  only a  the benzene r i n g o f the coumarin n u c l e u s  to the p o s i t i o n of the side chain  t h a t i s , a t p o s i t i o n 7.  rings.  Figure  1 illustrates  n a t u r a l l y o c c u r r i n g coumarins, b e a r i n g  attachment,  some r e p r e s e n t a t i v e  a v a r i e t y of d i f f e r e n t  functions  -  7 -  and s u b s t i t u t i o n patterns.  Figure 1.  Some representative n a t u r a l l y occurring coumarins.  Before commencing with the biosynthesis of coumarins, a discussion of the shikimic acid pathway to aromatic compounds •  -  appropriate.  8  (Figure 2) i s  - 8 -  COOH 0 I sT\ / C—0-p—0  (Glycolysispyruvic acid pathway)  COOH C=0  - ^ 1 1  I  H C\OH  0  2  H,  2  -HPO,  D-Glucose  HO-C-H H 0=CH  (Pentose phosphate pathway)  COOH  DPN. Co  H-C-OH  OH  H-C-OH CH OP  H-C-OH  2  AH 0P 2  D-Erythrose-4-phosphate  u  COOH  H0-  3-Deoxy-Darabino-heptulosonic acid-7-phosphate (DAHP)  OOOH  T° DPNH  I C=0 I C-OH  HO-  HQ *''/  COOH Dehydration (5-dehydroquin4se)  Cyclization^  j-H  H-C-OH  O  OH  | C-0 1 CH. 3  n  CH„  OH 5-Dehydroquinic acid  COOH  COOH  COOH OH  OH 5-Dehydroshikimic  acid  Shikimic acid  »  OH  Phosphoenol pyruvate 5-Phosphoshikimic acid  - 9 -  0  COOH  0  II  COOH  H ~\OjT '^' ^ C  H 0  per  t:;H  J  CH--  COOH  ^COOH  OH  HO  3-Enolpyruvyl shikimic acid-5-phosphate  Chorismic  |*V OH  2  C  0 0 0 1 1  Prephenic acid  acid  COOH CH  \  2  CH  COOH  COOH  DPN  NH„  H  Transamination Cinnamic acid  Phenylalanine  Phenylpyruvic acid  COOH  p-Coumaric acid  Tyrosine  Figure 2.  p-Hydroxyphenylpyruvic acid  The shikimic acid pathway to aromatic  compounds.  - 10 -  Phosphoenolpyruvic acid and D-erythrose-4-phosphate intermediates i n the metabolic pathways of D-glucose.  are independent  The reaction of  the two can be formulated as a concerted reaction between two phosphate esters leading to the formation of orthophosphate and 3-deoxy-D-arabinoheptulosonic acid-7-phosphate  (DAHP).  The D-arabino configuration i s  favored since, i n the assumed c y c l i z a t i o n of DAHP to 5-dehydroquinic acid, the hydroxyl groups on C-4 and C-5 of DAHP would correspond to the i d e n t i c a l configuration of 0-3 and C-4 of 5-dehydroquinic a c i d . The enzymic condensation of phosphoenolpyruvic acid and D-erythrose4-phosphate i s formulated as an attack by a n u c l e o p h i l i c group of the enzyme, symbolized here by OH , on the phosphoenolpyruvic acid.  This  process i s concerted with attack by carbon 3 of the phosphoenolpyruvic acid on the e l e c t r o p h i l i c carbon atom of the aldehyde and protonation of the carbonyl oxygen atom by an a c i d i c group.  The o v e r a l l r e s u l t i s  the release of orthophosphate or of a transient, phosphorylated enzyme and the open chain form of DAHP. In the limited sense that t h i s reaction may be viewed as an attack by a n u c l e o p h i l i c species on an aldehyde, i t resembles the chemical a l d o l condensation and the enzymic  aldolase  condensation. In order to convert DAHP to 5-dehydroquinic acid, the hydroxyl group on C-5 of DAHP i s f i r s t oxidized by diphosphopyridine nucleotide CDPN), to f a c i l i t a t e the elimination of phosphate  i n the next step.  The carbonyl group on C-5 i s then reduced by reduced DPN formed i n the f i r s t reaction to a hydroxyl group having the o r i g i n a l configuration. F i n a l l y , the r e s u l t i n g 2,6-diketone i s c y c l i z e d to 5-dehydroquinic acid. A l l these reactions are catalyzed by one enzyme or enzyme complex and  -  11 -  are probably concerted. To convert 5-dehydroquinic acid to shikiraic acid, the former compound i s f i r s t dehydrated to 5-dehydroshikimic acid. responsible f o r this was 5-dehydroquinase for  which was highly s p e c i f i c  t h i s reaction as i t was unable to dehydrate the closely related  quinic acid to shikimic acid. to  The enzyme  The reduction of 5-dehydroshikimic acid  shikimic acid by p a r t i a l l y p u r i f i e d 5-dehydroshikimate  reductase  has also been studied.' A cofactor requirement f o r the reduction i s triphosphopyridine nucleotide (TPNH) . To convert shikimic acid to prephenic acid,  5-phosphoshikimic  acid i s f i r s t formed by phosphate transfer from adenosine-5-triphosphate (ATP) to shikimic a c i d .  This then reacts with enol pyruvate phosphate  to give the 3-enol pyruvate ether of phosphoshikimic acid.  The  prephenic acid i s formed by the attack of n u c l e o p h i l i c enolpyruvate on C-l of the ring associated with the phosphate leaving group i n a trans  manner.  With external n u c l e o p h i l i c reagents, such reactions  are known to require  c i s stereochemistry.  No exact analogies appear  to be known f o r the type of i n t e r n a l rearrangement  postulated f o r the  conversion of enol pyruvate to prephenic acid. Aromatic compounds are formed from prephenic acid by decarboxylat i o n and reductive decarboxylation which y i e l d s the immediate precursors of tyrosine and phenylalanine, r e s p e c t i v e l y .  The l a t t e r  two are f i n a l l y formed by transamination and can undergo conversion to cinnamic acid and p-coumaric  a c i d , respectively.  Both the acid  catalyzed and the enzymic aromatization of prephenic acid may be v i s u a l i z e d as i n i t i a t e d by e l e c t r o p h i l i c attack on the hydroxyl group. Prephenic acid has also been shown to be converted by enzymes to  - 12 -  p-hydroxyphenylpyruvic acid.  Diphosphopyridine nucleotide (DPN)  is  required for t h i s r e a c t i o n , suggesting oxidation of C-4 followed by decarboxylation.  Biosynthesis of Coumarin: Evidence from feeding experiments showed that coumarin was synthesized from shikimic acid-derived phenylpropane precursors i n * preference  . acetate , - condensation. J' • 9,10,11 to 9  Brown, Towers, and Wright  studied the biosynthesis of coumarin  i n Hierochloe odorata and M e l i l o t u s o f f i c i n a l i s . precursors were fed.  A number of possible  From this the best precursors of coumarin were  found to be o-coumaric acid and cinnamic acid while shikimic acid and L-phenylalanine were found to be s l i g h t l y l e s s e f f i c i e n t as whereas a c e t i c a c i d , s a l i c y l i c  precursors  a c i d , o-tyrosine» f e r u l i c a c i d , and  m e l i l o t i c acid were found to be very poor precursors.  This work  indicated approximately the same r e s u l t s as Kosuge and Conn's feeding experiments i n sweet clover, M e l i l o t u s alba  The r e s u l t s obtained  by Brown, Towers, and Wright suggested that the shikimic acid-phenylpropanoid acid pathway predominates i n coumarin biosynthesis i n H.  odorata.  Furthermore, the r e l a t i v e e f f i c i e n t u t i l i z a t i o n of cinnamic acid and o-coumaric acid implied that the former undergoes hydroxylation ortho  to the side chain during the The e f f e c t of varying metabolic  process. periods on the r a d i o a c t i v i t y of  several phenolic constituents of H. odorata s t r u c t u r a l l y related compounds was Wright  9  including coumarin and  also studied by Brown, Towers, and 14 after the administration of cinnamic a c i d - 3 - C. The radio-  - 13 -  a c t i v i t y of the following compounds i s o l a t e d from the plant was p a r t i c u l a r i n t e r e s t (Figure 3).  o-Coumaric acid  of  Changes i n s p e c i f i c a c t i v i t y of coumarin  o-Coumaryl glucoside  Coumarin  COOH  p-Coumaric acid  Figure 3.  Ferulic c i d  M e l i l o t i c acid  a  Radioactive compounds i s o l a t e d from H. odorata.  14 with, time a f t e r feeding cinnamic acid-3-  C to Hierochloe odorata  indicated a d e f i n i t e lag phase p e r s i s t i n g u n t i l 8 hours from the complete absorption of the precursor. hours, there was  From that time u n t i l about 24  a l i n e a r increase i n s p e c i f i c a c t i v i t y .  Changes of  s p e c i f i c a c t i v i t y of o-coumaric acid, p-coumaric acid, f e r u l i c acid, m e l i l o t i c acid, and experiment indicated  o-coumaryl glucoside with time from the same that a l l four free acids exhibited  s p e c i f i c a c t i v i t y at or before 16 hours, and  a maximum i n  that a l l but m e l i l o t i c acid  attained  a r e l a t i v e l y high s p e c i f i c a c t i v i t y i n 0 to 4 hours. 14  slow and  s l i g h t accummulation of  C i  n  The  m e l i l o t i c acid again suggested  i t to be a secondary metabolic product of cinnamic acid.  - 14 -  A clearer picture of the d i s t r i b u t i o n of the active carbon was obtained from an examination of the t o t a l a c t i v i t y i n the various constituents throughout the period of the experiment.  From t h i s , i t  was clear that the largest reservoir of o-hydroxylated phenyl propanoid material throughout the experiment was o-coumaryl glucoside.  I t was  14 found that the  C content of the glucoside was even higher shortly  a f t e r feeding than at 4 hours, i n d i c a t i n g that cinnamic acid administered to H. odorata undergoes rapid  ortho  hydroxylation and that  glucoside formation also occurs r a p i d l y , with a large accumulation of 14 C i n o-coumaryl glucoside even while absorption of the presursor i s i n progress. In order to determine whether o-coumaryl glucoside i s an i n t e r mediate i n coumarin biosynthesis a sample of radioactive o-coumaryl 14 14 glucoside and cinnamic acid-3- C were fed to H. odorata. The C d i l u t i o n during conversion of cinnamic acid to coumarin was 315 and that of o-coumaryl glucoside was 435, i n d i c a t i n g the two compounds to be comparable i n e f f i c i e n c y as precursors of coumarin. 12 Brown  has also shown that p-coumaric acid was seventy times  less e f f e c t i v e than cinnamic acid as a precursor of coumarin i n Hierochloe odorata, while tyrosine was s i x t y times less e f f e c t i v e than phenylalanine.  From this  i t was clear that a preformed phenolic  nucleus posed a decided disadvantage i n i t s use as a precursor. In l a t e r studies on the biosynthesis of coumarin i n H. odorata, 13 14 Brown fed CO^ and showed that coumarin exists i n plant c e l l s as a glucoside of o-coumarinic acid (19) and can be recovered by emulsin 14 hydrolysis. A study of the incorporation of C from CC^ into coumarin  - 15 -  and the aglycone of o-coumaryl glucoside with time was subsequently made.  The results indicated that the t o t a l a c t i v i t y of coumarin showed  (19)  a slow, almost uninterrupted increase u n t i l 17 days after which the total  declined markedly.  The maximum a c t i v i t y of the aglycone,  however, occurred at about 4 days and was followed by a marked decrease i n a c t i v i t y . In addition to showing the presence of the cis-glucoside of coumarinic acid i n H. odorata, the r e s u l t s of Brown also suggested that o-coumaryl glucoside was a metabolic intermediate rather than an end product, since the peak i n i t s t o t a l a c t i v i t y i s reached e a r l i e r than that of coumarin.  The demonstration of the presence of coumarinic  acid glucoside suggested that glucoside formation was necessary to e f f e c t trans-cis inversion which must precede the formation of the coumarin lactone ring. such a pathway.  The cis-glucoside would be an intermediate i n  Also the incorporation of l a b e l l e d o-coumaryl gluco-  side to coumarin, supported the b e l i e f that o-hydroxylation was an e s s e n t i a l feature of coumarin biosynthesis.  Furthermore,  coumarin  l i k e o-coumaryl glucoside was not an end product, but underwent r e l a t i v e l y 14 slow metabolism experiments.  as revealed by the eventual decline i n  C i n the  - 16 -  Therefore,  Brown  14  proposed the following scheme f o r coumarin  biosynthesis i n Melilotus and H. odorata (Figure 4).  Figure 4.  Proposed scheme f o r coumarin biosynthesis irt Melilotus and H. odorata.  Results by Kosuge and Conn  15  , Stoker and B e l l i s  16  , and Gorz  and Haskins"^ also independently showed that the conversion  of trans-  cinnamic acid to coumarin i n Melilotus alba occurred by a s i m i l a r scheme.  Furthermore, t h i s plant was shown to contain a t r a n s - c i s  _ 18 isomerase enzyme system  Biosynthesis of 7-0xygenated Coumarins: A.  Umbelliferone (7-hydroxycoumarin)  (20)  The formation of umbelliferone (20) has been studied by Brown, Towers, and Chen  19  and by Austin and Meyers  20 21 ' using Hydrangea  macrophylla . The l a t t e r workers have reported that umbelliferone exists as the free coumarin only to a very small extent i f at a l l , and have i d e n t i f i e d two bound forms i n Hydrangea (21 and 22).  The  COOH  Glu 7-3-D-Glucosyloxycoumarin or skimmin (21)  Glu  Glu  cis-2,4-di-g-D-Glucosyloxycinnamic acid (22) 14  f i r s t compound was predominant.  Experiments with  C - l a b e l l e d compounds,  by the two groups have been i n substantial accord and led to the following biosynthetic route being proposed for umbelliferone i n Hydrangea (Figure 5).  - 18 -  Glu (21)  Figure 5.  (20)  Proposed scheme for biosynthesis of umbelliferone i n Hydrangea.  B.  Herniarin  (7-methoxycoumarin)  (23) Lavender (Lavandula o f f i c i n a l i s Chaix) i s one of the few species which elaborate both coumarin and a 7-hydroxylated coumarin, i n t h i s 22 case h e r n i a r i n (23).  Therefore, i t was chosen by Brown  as a con-  venient species i n which to compare the biosynthesis of coumarin and a  - 19 -  7-hydroxylated  coumarin.  Like coumarin, h e r n i a r i n ' a l s o occurred i n the bound state as a glucoside.  I t seemed quite c e r t a i n that this glucoside was  syloxy-4-methoxy-cis-cinnamic  acid (cis-GMC) (24).  2-gluco-  The presence of  t h i s compound implied the formation, at some stage, of an orthohydroxylated precursor and indicated that the lactone ring of 7hydroxylated coumarins also can be synthesised v i a ortho-hydroxylation. Brown  22  compared i n studies on lavender a number of d i f f e r e n t  14  C  l a b e l l e d compounds as precursors of h e r n i a r i n , and i n some cases coumarin.  L-phenylalanine was  incorporated with moderate d i l u t i o n of  14 C into h e r n i a r i n as well as coumarin.  Glucose was used with  e f f i c i e n c y as a precursor of both coumarins.  lower  However,o-coumaric  and p-coumaric acids were u t i l i z e d with a high degree of s e l e c t i v i t y . The former was used for the synthesis of coumarin some 150-200 times as e f f i c i e n t l y as i t was used for h e r n i a r i n synthesis, and the l a t t e r was  s e l e c t i v e l y u t i l i z e d for herniarin,synthesis by a s l i g h t l y lesser  factor.  Furthermpre cinnamic  both coumarins.  }  acid was  found to be a precursor of  1  These findings showed that h e r n i a r i n , unlike coumarin, was synthesized by way  not  of the o-coumaric acid - o-coumaryl glucoside pathway.  They confirmed the theory that coumarin and herniarin a r i s e v i a orthoand para-hydroxylation, respectively, of a common precursor, probably some form of cinnamic  acid. 14  The low d i l u t i o n of was  C i n trans-GMC suggested  the intermediate precursor of cis-GMC.  that this compound  The trans-cis inversion  necessary for this step would be analogous to that postulated i n the  - 20  formation of coumarinyl glucoside. of p-coumaric acid to cis-GMC,an  In a d d i t i o n , during the conversion ortho-hydroxylation glucoside formation  and an 0-methylation of the para-hydroxyl group must occur.  Glucoside  formation must obviously follow the o-hydroxylation, but the order of the other steps remained i n question. In an attempt to elucidate the above problem Brown  compared p-  14 methoxycinnamic acid-a- C and 2,4-dihydroxy-trans-cinnamic  acid-1-  (umbellic acid) with p-coumaric acid as precursors of h e r n i a r i n .  14  C  The  r e s u l t s c l e a r l y demonstrated the very high e f f i c i e n c y with which pmethoxycinnamic acid was converted to h e r n i a r i n .  Umbellic acid and  umbelliferone, while moderately well utilized,were both poorer precursors than p-coumaric acid acid.  and 25-50 times poorer than p-methoxycinnamic  Thus., these experiments revealed the following order of precursor  e f f i c i e n c i e s for h e r n i a r i n synthesis:  trans-GMC > p-methoxycinnamic  acid > p-coumaric acid > umbellic acid _ umbelliferone > glucose. Therefore, the scheme for the h e r n i a r i n biosynthesis proposed by 22 Brown  at the time can be represented by the following scheme  (Figure 6). The stage at which O-methylation h e r n i a r i n remained uncertain, however.  occurred i n the biosynthesis of The two p o s s i b i l i t i e s shown  below CFigure 7) seemed the most probable, and as has already been noted a comparison of the two l a b e l l e d intermediates showed that 22 p-methoxycinnamic acid was much the better h e r n i a r i n precursor. 23 On the basis of e x i s t i n g evidence, Brown  recently proposed the  following scheme (Figure 8) f o r h e r n i a r i n biosynthesis. acid i s converted to an intermediate "X",  which may  Here p-coumaric  be an enzyme-substrate  COOH  COOH  C  6 11°5 H  trans-GMC  2-Hydroxy-4-methoxy-transcinnamic a c i d 1  0  CH 0 3  COOH V  C  CH 0  6 11°5 H  (24)  ure 6.  Proposed scheme for herniarin  (23)  biosynthesis.  - 22 -  COOH  Umbellic a c i d  Figure 7.  Possible stages of O-methylation i n h e r n i a r i n biosynthesis.  1  i  trans-GHC  Figure 8.  trans-GMC  Modified scheme f o r h e r n i a r i n biosynthesis.  (24)  - 23 -  complex.  To overcome the d i f f i c u l t y posed by the poor e f f i c i e n c y of  umbellic acid as an intermediate,  i t was  suggested that  o-hydroxylation  and glucosylation occurred without the substrate leaving the enzyme surface, or whatever other complex may  be concerned.  The product, trans-  2-glucosyloxy-4-hydroxy-cinnamic acid (trans-GHC), could then undergo O-methylation to trans-GMC, a known intermediate.  However, trans-GHC,  an unknown compound, has not yet been tested as a h e r n i a r i n precursor.  C.  Novobiocin  HN 2  OCH„ 3  2A 25  1  Kenner and h i s co-workers  '  have shown that the lactone ring  i n the coumarin residue of the a n t i b i o t i c novobiocin  (25),  formed by a Streptomyces, originates i n a d i f f e r e n t way.  By the use of  14 C  they showed that the coumarin residue was formed from tyrosine, 18 and further work with 0 yielded good evidence that the ring oxygen originated from the carboxyl oxygens of tyrosine. They postulated an oxidative c y c l i z a t i o n of the amino acid to explain their r e s u l t s . 26 Others  27 '  have raised the question whether a s i m i l a r mechanism  may 27  not also operate i n higher plants, and Scott, Meyers, and  co-workers  have suggested the involvement of a spirolactone as shown (Figure 9 ).  - 24 -  (20)  Figure 9.  6-Hydroxycoumarin  Involvement of a spirolactone i n coumarin biosynthesis.  27 The model reaction shown was demonstrated _in v i t r o ,  but  subsequent ^n vivo work by Austin and Meyers ^ with a " ^ C - l a b e l l e d 2  spirolactone did not bear out the theory.  The fact that the coumarins  i n question a c t u a l l y exist i n the c e l l as glucosides of coumarinic acids argued strongly that at least i n plants an  o-hydroxylation 28  mechanism, rather than oxidative c y c l i z a t i o n i s the favored pathway.  D,  Coumestrol OH  (26)  - 25 -  The investigations of Grisebach and Barz  29  '  30  on the coumarano-  coumarin, coumestrol (26), have shown that this compound i s biosynthetic a l l y an isoflavone with the benzenoid r i n g of the coumarin nucleus o r i g i n a t i n g from acetate, and the remaining nine carbons from phenyl26 propanoid precursors. aryl-migration was an intermediate. may  As i n the formation  of isoflavones,  involved, and a chalcone glucoside was  an apparently  These findings demonstrated that i d e n t i c a l structures  have e n t i r e l y d i f f e r e n t biosynthetic o r i g i n s , even i n species of  the.same family. E.  Calophyllolide  (27)  31 Recent work by Kunesch and Polbnsky showed that the s p e c i f i c 14 incorporation of (-)-phenylalanine-3C into a 4-phenylcoumarin (neoflavanoid), c a l o p h y l l o l i d e (27), supported the biogenetic scheme (Figure 10) suggested by Seshadri Edwards and Stoker  32  and  Ollis.  33  presented a d d i t i o n a l information  concerning  the isomerization of o-coumaroyl glucoside to coumarinoyl glucoside  34 (19) i n the biosynthesis of coumarin  and the isomerization of 2-  glucosyloxy-4-methoxy-trans-cinnamic acid (trans-GMC) to 2-glucosyloxy4-methoxy-cis-cinnamic acid (cis-GMC) (24) i n the biosynthesis of , . . 35 herniarin.  - 26 -  Figure 10.  Suggested biosynthetic scheme f o r c a l o p h y l l o l i d e .  The r e s u l t s of Edwards and Stoker indicated that an enzyme i s not involved i n the isomerization of o-coumaroyl glucoside to coumarinoyl glucoside i n M. o f f i c i n a l i s , but that the reaction i s catalyzed by light.  Furthermore, their r e s u l t s also indicated that the isomerization  - 27 stage i n the biosynthesis of h e r n i a r i n i s a photochemical step, no isomerase enzyme being involved.  Consequently, t h i s isomerization  stage has now been shown to be photochemical both for coumarin i t s e l f and for a t y p i c a l 7-oxygenated coumarin.  It i s probable,  therefore,  that the isomerization step i n the biosynthesis of a l l plant coumarins i s e n t i r e l y photochemical.  Biosynthesis of Furanocoumarins: In one of the i n i t i a l studies on the biosynthesis of  furano-  36 coumarins, Caporale and h i s co-workers reported the incorporation of 3 14 3 r a d i o a c t i v i t y from acetate-2- H, tyrosine-2- C and -U- H, mevalonic acid-2-  14  3 C, and succinic acid-2,3- Hinto bergapten (15) and  (17) by leaves  psoralen  of Fiscus c a r i c a . OCH  (17)  (15)  37 Floss and Mothes  presented  evidence that the coumarin skeleton  of furanocoumarins i n Pimpinella magna (Umbelliferae) was  formed from  cinnamic acid and also that para-hydroxylation  ortho-hydroxyla-  t i o n , since umbelliferone was  preceded  a far better precursor than cinnamic  acid whereas coumarin gave only poor incorporation, i n d i c a t i n g that umbelliferone but not coumarin was  an intermediate  Pimpinella furanocoumarins from cinnamic acid.  i n the formation of  The co-occurrence of  - 28 -  simple furanocoumarins, isopropylfuranocoumarinsand isoprenylated furanocoumarins suggested the p o s s i b i l i t y that the furan ring originated from carbons 1 and 2 of the isoprene residue or from carbons 4 and 5 of mevalonic, acid, respectively.  38  39 Floss and Mothes reported r e s u l t s  which confirmed the incorporation of cinnamic acid and apparently c l a r i f i e d the o r i g i n of the two extra  carbon atom's of the furan r i n g ,  and hence allowed some conclusions to be made regarding the pathway of furanocoumarin formation. Labelled cinnamic acid-(  14  COOH) and DL-mevalonic acid-4-  14  C were  fed to Pimpinella magna and various furanocoumarins present were isolated.  In order to e s t a b l i s h the d i r e c t conversion of cinnamic  acid to furanocoumarins, a degradation, using sphondin (28), was performed (Figure 11).  The CO^ obtained from C-2 of sphondin had about  Cu Powder >  C0_(C-2)  COOH 5'  4' (28)  Figure 11.  Degradation of sphondin.  90% of the s p e c i f i c a c t i v i t y of the s t a r t i n g material.  Thus, most of  the a c t i v i t y was located at C-2 of the furanocoumarin, i n d i c a t i n g that cinnamic acid was indeed incorporated into the furanocoumarins. 14 Mevalonic acid-4-  C was also incorporated into the furanocoumarins.  To determine the l o c a t i o n of the isotope i n the furanocoumarins i s o l a t e d ,  a degradation was attempted.  A l l the  position 5', i f the furan ring was mevalonic acid.  C would be expected to be i n  indeed formed from C-4 and C-5 of  Labelled p i m p i n e l l i n (29) was degraded by ozonization  (Figure 12) and the product was i d e n t i f i e d as  2,4-dihydroxy-5,6-  dimethoxy-m-phthaldehyde (30).  (29)  Figure 12.  (30)  Degradation of p i m p i n e l l i n .  The ozonization reaction involved removal of carbon atoms 2, 3, and 5'.  Since p i m p i n e l l i n had a constant s p e c i f i c a c t i v i t y and the  dialdehyde had only 11% of the o r i g i n a l s p e c i f i c a c t i v i t y , most of the must have been located i n C-2, C-3 and/or C-5'.' As shown before, the coumarin portion of the molecule, which includes C-2 and originated from cinnamic a c i d .  C-3,  A s p e c i f i c incorporation of C-4 of  mevalonic acid was highly u n l i k e l y . mevalonic acid indeed l a b e l l e d C-5'  Thus, i t confirmed that C-4 of furanocoumarins. 39  Therefore,  the r e s u l t s of Floss and Mothes  apparently  the o r i g i n of the carbon skeleton of the furanocoumarins.  clarified  Thus, the  coumarin portion i s formed from cinnamic acid, while the furan ring originated from an isoprenoid residue, probably v i a isoprenylation of  - 30 the aromatic ring followed by c y c l i z a t i o n and loss of a 3-carbon isopropyl side chain. The general, mechanism of furanocoumarin biosynthesis e s p e c i a l l y the sequence of the i n d i v i d u a l steps was  also considered by Floss and  39 Mothes.  With very few exceptions  a l l furanocoumarins have the furan  ring attached to p o s i t i o n 6 and 7 or 7 and 8 of the coumarin system, the oxygen being i n p o s i t i o n 7.  This would be explained most e a s i l y  by the assumption that umbelliferone i s the compound which i s isoprenylated, and that further hydroxylations and a l k y l a t i o n s take place a f t e r this step.  However, the r e s u l t s of the cinnamic acid and mevalonic acid  feeding experiments c l e a r l y showed that t h i s was  not the case.  If  isoprenylation of umbelliferone preceded further hydroxylations, i t was  expected that the r e l a t i v e s p e c i f i c a c t i v i t i e s of the various  furanocoumarins i s o l a t e d would be the same i n the cinnamic acid as i n the mevalonic acid experiment, since a l l the reactions which create the difference between the products would occur a f t e r the introduction of the isotope i n both cases.  However, the r e l a t i v e s p e c i f i c a c t i v i t i e s  of the various furanocoumarins were not the same i n the cinnamic acid and mevalonic acid experiments. The r e s u l t s pointed to the a l t e r n a t i v e whereby the hydroxylation and methylation pattern was  established f i r s t and isoprenylation then  occurred as the l a s t step.  Since, however, the 7-hydroxy group must  be protected from methylation i n order to allow furan ring i t was  l i k e l y that umbelliferone was  and this compound was methylations.  f i r s t converted  formation,  to the glucoside  the substrate for further hydroxylations  and  The sugar could then be removed at a l a t t e r stage to  allow furan ring formation to take place.  39 Floss and Mothes  also found that, the hydroxylation pattern of the  -  furanocoumarins  31  -  i n Pimpinella could be derived by a combination of  three types of reactions: (i)  a hydroxylation of umbelliferone and  para  ortho  to the hydroxyl group  to the lactone oxygen,  ( i i ) a hydroxylation i n p o s i t i o n 5 ("second ortho hydroxylation"), ( i i i ) opening of the lactone ring and r e c y c l i z a t i o n i n the opposite direction. An observation i n favor of t h i s scheme was that xanthotoxin (16), the only member of a l l the possible mono- and dimethoxylated furanocoumarins of this type which could not be formed by these reactions, was not found i n Pimpinella.  , (16) The data also suggested that reaction ( i ) occurred more r e a d i l y than reaction ( i i ) , since i n the cinnamic acid experiment,  sphondin  (28) had a much higher s p e c i f i c a c t i v i t y than bergapten (15) and isobergapten (31).  As expected bergapten and isobergapten had the  same s p e c i f i c a c t i v i t y i n the cinnamic acid experiment, whereas the value f o r pimpinellin (29) and i s o p i m p i n e l l i n (2 ) were lower.  Since  the value of p i m p i n e l l i n was lower than that of i t s isomer i t appeared as i f i t was obligatory f o r reaction ( i i i ) to occur together with reaction  (ii).  - 32 -  The data from the mevalonic acid experiments also indicated that isoprenylation at p o s i t i o n 8 was preferred as a l l angular furanocoumarins had a higher s p e c i f i c a c t i v i t y than the l i n e a r ones.  This  was more pronounced with the pair bergapten and isobergapten, where there was a true competition f o r the same precursor. 39 On the basis of the r e s u l t s Floss and Mothes  proposed the  following scheme f o r the biosynthesis of furanocoumarins (Figure 13).  COOH  Figure 13.  COOH  Proposed scheme f o r the biosynthesis of furanocoumarins.  - 33 Recently, Floss and Paikert The evidence obtained  40  examined the scheme experimentally.  d i d not favour the proposed biogenetic scheme  and indicated that an a l t e r n a t i v e pathway, involving isoprenylation at the umbelliferone stage followed by further modification of the benzenbid r i n g , must be considered. These workers attempted to determine whether the ortho- or the para-hydroxyl group was introduced into the cinnamic acid  molecule.  The fact that coumarin was not u t i l i z e d i n the biosynthesis d i d not rule out o-coumaric acid as an intermediate.  Both o-coumaric acid  14 and p-coumaric acid with a Pimpinella magna.  C l a b e l i n the carboxyl group were fed to  However, the r e s u l t s together with previous work by  Brown and h i s co-workers on the biosynthesis of umbelliferone i n Hydrangea macrophylla  suggested  that o-coumaric acid was not a natural  intermediate i n the synthesis, but that p-coumaric acid was the l i k e l y precursor.  40 Floss and Paikert  also studied the incorporation of umbelli-  ferone and i t s 7-glucoside into furanocoumarins.  I t was found that the  glucoside was incorporated s l i g h t l y less e f f i c i e n t l y than the aglycone  41 Furthermore, e a r l i e r experiments  had shown very rapid g l y c o s y l a t i o n  of added umbelliferone, and thus, they examined the metabolic fate of umbelliferone i n P. magna. plant.  Umbelliferone-S-^H  was root fed to the  A f t e r work up of the plants and subsequent enzymatic hydrolysis  of the coumarins to y i e l d the aglycones, i t was noted that umbelliferone was the only l a b e l l e d material, that i s , there was no detectable conversion of umbelliferone glucoside to other coumarin glucosides. If the hypothetical pathway of furanocoumarin biosynthesis, as  - 34 -  postulated by Floss and Mothes, was correct, i t was expected that scopoletin (7-hydroxy-6-methoxycoumarin)  (32) would be a precursor of  sphondin and p i m p i n e l l i n , possibly also of i s o p i m p i n e l l i n , but not of bergapten and isobergapten. administered  Therefore, scopoletin-methyl-^  was  to the roots of P. magna and, as a reference, i t s isomer  (6-hydroxy-7-methoxyc6umarin-methyl-^H) (33) was fed i n a p a r a l l e l experiment.  (32)  (33)  It was found that scopoletin-me  was not incorporated  preferentially  into any one furanocoumarin as would be expected i f isoprenylation were a l a t e step i n the biogenetic pathway.  Furthermore, i t l a b e l l e d  the furanocoumarins i n about the same manner of magnitude as i t s 3 isomer 6-hydroxy-7-methoxycoumarin-methyt- H which could not be an intermediate i n the formation of the furanocoumarins of Pimpinella. This indicated that the scopoletin molecule was not transformed into furanocoumarins as a u n i t , but that the incorporation of r a d i o a c t i v i t y was possibly only due to demethylation and p a r t i a l r e u t i l i z a t i o n of the l a b e l l e d  fragment.  This was also suggested by the rather low  rate of incorporation of scopoletin as compared to umbelliferone. Therefore, i t seemed very l i k e l y that scopoletin was not a d i r e c t precursor of any of the furanocoumarins of P. magna.  35  Therefore,  the r e s u l t s  presented by Floss and Paikert  substantiate the assumption that isoprenylation occurred  40  did not  as a l a t e  reaction i n furanocoumarin biosynthesis and that the hydroxylations and methylations of the coumarins portion occurred stage.  at the glucoside  An a l t e r n a t i v e pathway of furanocoumarin biosynthesis had to be  considered,  involving isoprenylation of umbelliferone  (20) followed by  further modification of the r e s u l t i n g 6- and 8-dimethylallylumbelliferone (Figure 14).  Such a pathway was a t t r a c t i v e from a phytochemical point  (31) R^H, R =OCH , 2  3  (28) R^OCH^ R =H 2  (29) R  X  = R  2  = 0CH  3  (15) R =0CH , R 1  3  2  = H  = 0CH„  Figure 14.  Proposed a l t e r n a t i v e pathway of furanocoumarin biosynthesis.  of view since i t could explain the almost exclusive occurrence, i n nature, of 6,7- and 7,8-furanocoumarins. 42 Floss, Guenther, and Hadwiger  recently presented work on the  biosynthesis of furanocoumarins i n diseased celery.  Upon i n f e c t i o n  with the fungus S c l e r o t i n i a sclerotiorum,celery was known to develop a condition c a l l e d "pink r o t " which has been correlated with the s k i n i r r i t a t i n g properties of diseased celery.  Two phototoxic psoralen  derivatives have been i s o l a t e d from diseased celery t i s s u e , xanthotoxin (8-methoxypsoralen)  (16) and 4,5',8-trimethylpsoralen (34).  OCH (16)  3  CH  3  (34)  These workers conducted some feeding experiments to determine whether the two psoralen derivatives originated from the same biosynthetic precursors as other furanocoumarins found i n higher plants, namely cinnamic acid and mevalonic acid with the O-methyl groups presumably coming from the  pool (formate or the methyl group of methionine).  Xanthotoxin has the same s t r u c t u r a l pattern as other higher plant furanocoumarins and would thus be expected to be derived from the same biogenetic precursors.  On the other hand, the psoralen derivative (34)  has some rather unusual features i n that i t c a r r i e s three carbon bound methyl groups.  I f , as i n other furanocoumarins, the ring skeleton of  this compound i s derived from cinnamate and mevalonate, the three methyl  - 37 -  groups, or at least the two attached to the coumarin portion, would be expected  to originate from the  Cinnamic acid-1-  14  pool by  C-methylation.  C, methionine-methyl-  fed to celery plants after i n f e c t i o n .  14  C, and formate-  As expected,  a considerably  i n t e r e s t i n g l y , however,  none of the three substrates l a b e l l e d 4,5',8-trimethylpsoralen s i g n i f i c a n t extent.  I t was  C were  a l l three compounds  were incorporated into xanthotoxin, although formate was less e f f i c i e n t methyl donor than methionine,  14  to any  conceivable that the two products were  possibly synthesized at d i f f e r e n t s i t e s , that i s , xanthotoxin may  have  been formed by the plant and trimethylpsoralen by the fungal c e l l s . As an a l t e r n a t i v e explanation of the r e s u l t s of t h i s study, one  had,  however, to consider the p o s s i b i l i t y that the two furanocoumarins found i n diseased celery were formed by two e n t i r e l y d i f f e r e n t b i o synthetic pathways. 43 Steck, El-Dakhakhny and Brown  recently presented material  strongly i n d i c a t i n g the p a r t i c i p a t i o n of two  a-hydroxyisopropyldi-  hydrofuranocoumarins, marmesin (35) and columbianetin  (36) as natural  intermediates i n the elaboration of l i n e a r and angular furanocoumarins, respectively.  To date, this was  the f i r s t time intermediates i n the  reaction sequence from umbelliferone to furanocoumarins had been identified.  - 38 -  Trapping experiments were conducted i n which umbelliferone-2was  1A  C  administered to shoots of Ruta graveolens together with non14  l a b e l l e d marmesin, and skimmin-2non-labelled columbianetin.  C to Heracleum lanatum together with  In addition to marmesin, the l i n e a r furano-  coumarins psoralen (17), bergapten from Ruta. sphondin  (15), and xanthotoxin (16) , were recovered  In addition to columbianetin, a n g e l i c i n (37) , isobergapten (31),  (28), and pimpinellin (29) were recovered from Heracleum ( F i g . 15).  R  2  (17) (Rj_ = R (15) (16)  2  (37) ( R x = R  = H)  (R = OCH , R x  3  ( R 1 = H, R  2  2  = H)  (31)  = OCH ) 3  = H)  2  (R^ = H, R  (28) (R^ = OCH (29)  3>  (R = R x  2  R  2  £  = OCH ) 3  = H)  = OCH ) 3  Figure 15. Furanocoumarins recovered from Ruta graveolens. and Heracleum lanatum.  From the r e s u l t s , i t was clear that i n each species umbelliferone had been converted to the dihydrofuranocoumarin.  Furthermore, the  lower s p e c i f i c a c t i v i t i e s of the furanocoumarins were consistent with their formation from marmesin or columbianetin. 43 Steck, El-Dakhakhny and Brown  also conducted  d i r e c t feedings  of t r i t i a t e d marmesin and columbianetin to confirm the roles of these compounds i n furanocoumarin  biosynthesis.  In each case  there was  good conversion to the analogous unsubstituted furanocoumarin and a lower degree of tritium incorporation into the oxygenated  furanocoumarins.  Umbelliferone was known to occur i n Ruta graveolens and i n Heracleum lanatum.  Therefore, i t can be regarded as a natural i n t e r -  mediate i n furanocoumarin biosynthes i s i n these species.  The  formative route to marmesin proposed by Steck, El-Dakhakhny and Brown can be envisaged as the following (Figure 16).  ?  (35)  i  Figure 16.  Proposed scheme f o r the biosynthesis of marmesin.  There was an analogous sequence leading to columbianetin, but there was no firm experimental evidence to support this hypothesis.  The  nature of the reactions leading on to psoralen and a n g e l i c i n and ultimately to the substituted furanocoumarins, however, remained unclear. As the above discussion reveals, the biosynthetic pathways leading  -  40  -  to the furanocoumarins, i n contrast to those of simple coumarins are indeed i n a state of confusion.  None of the r e s u l t s are i n t e r n a l l y  consistent with any general postulate and there was question i n our minds about the r e a l meaning of a s u b s t a n t i a l portion of the experimental data.  For this reason we decided to investigate the biosynthesis  of several furanocoumarins i s o l a t e d from Thamnosma montana. Furthermore, the eventual goal of studying the biosynthesis of the novel dimeric thamnosin system required an i n i t i a l ,  careful,  investigation'of the appropriate monomeric units which would be v i s u a l i z e d as the biosynthetic templates  f o r the dimeric n a t u r a l product.  - 41 -  DISCUSSION  The discussion within the Introduction section of this thesis has revealed that a considerable amount of work has been done on biosynthesis of some of the simpler monomeric coumarins.  the  Unfortunately,  c a r e f u l analysis of a large portion of the published data leads to conclude that much s t i l l remains to be done i n t h i s area. instances the incorporations reported are very low, to the i s o l a t i o n of natural products possessing  one  In many  thereby leading  low l e v e l s of a c t i v i t y .  Furthermore, the eventual characterization'and, i n p a r t i c u l a r , s u f f i c i e n t degradation to i s o l a t e the relevant carbon atoms i s incomplete i n a number of instances. confusions  It was  our f e e l i n g that some of the  already mentioned i n the Introduction are simply due to the  incompleteness of the i n v e s t i g a t i o n s .  In order to t r y and e s t a b l i s h  more f i r m l y the various postulates i n the l i t e r a t u r e and also to provide an i n t e r n a l l y consistent picture of the various coumarins i  within the same plant system, we decided turpentine-broom, Thamnosma montana  to i n i t i a t e investigations i n  Torr. and Frem. As  already  mentioned, t h i s plant possesses a wonderful array of monomeric and dimeric coumarins. In e f f e c t these' aims required investigations i n several d i f f e r e n t areas.  These are the following:  -  (a)  42  -  Detailed i n v e s t i g a t i o n of s u i t a b l e i s o l a t i o n procedures to  allow i s o l a t i o n and characterization of small quantities of the natural coumarins, (b)  Development df appropriate chemical degradation  pathways  which allow i s o l a t i o n of relevant carbon atoms i n the coumarins, 14 Cc)  Incorporation studies with appropriately l a b e l l e d (  3 C and  H)  precursors. For this purpose, R.N. involved i n separate;but now  Young, A.K.  Verma, and myself became  complementary areas of the research.  discuss those areas with which I was  I will  d i r e c t l y involved but wish  to emphasize that a great deal of r e l a t e d work which  strengthens  some of the conclusions made l a t e r i s available from the e f f o r t s of my collaborators. For the sake of c l a r i t y , I propose to discuss my work i n the order of research areas (a-c). mentioned above. Old l i v i n g plants of' the turpentine-broom, Thamnosma montana  Torr.  and Frem., containing rather thick woody roots, were obtained i n the desert region near Morongo Valley, C a l i f o r n i a with the help of Dreyer and his associates at the F r u i t and Vegetable Chemistry Pasadena, C a l i f o r n i a .  Laboratory,  From the whole a e r i a l part of the plant  including the crown as well as the roots, i t was  considered desirable  to i s o l a t e a l l the compounds previously found i n the plant as w e l l as any new  ones which might be present.  In addition to the plant material,  Dreyer and his associates also provided us with an acetone extract of Thamnosma montana, Torr. and Frem. a s u i t a b l e i s o l a t i o n procedure.  Both of these were used to develop  -  (a)  43  -  I s o l a t i o n of Natural Products from Thamnosma montana  Torr. and Frem.  The plants were a i r dried thoroughly and ground to a course powder i n a high speed Waring blender. the pulp was  extracted with acetone.  and the clear acetone s o l u t i o n was tar.  The l a t t e r was now  evaporated  Using a Soxhlet extractor, The r e s u l t i n g s o l u t i o n was  evaporated  filtered  to provide a dark heavy  extracted with chloroform, f i l t e r e d ,  and  as before leaving a dark heavy o i l y residue.  The crude extract was  separated into numerous f r a c t i o n s using  column chromatography on deactivated alumina.  A t y p i c a l i s o l a t i o n scheme  i s portrayed i n Figure 17 with a t y p i c a l t h i n layer s i l i c a g e l chromatoplate of the i s o l a t e d fractions i l l u s t r a t e d i n Figure 18. For purposes of comparison  the appropriate colour c h a r a c t e r i s t i c s  under u l t r a v i o l e t l i g h t of previously characterized compounds found i n Thamnosma montana chromatoplate  Torr. and Frem. are shown on the s i l i c a gel  i n Figure 19.  The compounds of p a r t i c u l a r i n t e r e s t for our i n i t i a l biosynthetic work were umbelliprenin, alloimperatorin methyl ether, and It was of  isopimpinellin.  desirable, therefore, to i n i t i a l l y accummulate s u f f i c i e n t quantities  the above mentioned compounds along with alloimperatorin methyl  ether d i o l for purposes, of subsequent d i l u t i o n and degradative work i n the biosynthetic i n v e s t i g a t i o n s . . For this purpose the various f r a c t i o n s from the i n i t i a l column chromatography were combined i n an appropriate manner and subjected to a d d i t i o n a l column chromatography, thin layer chromatography, and f i n a l l y c r y s t a l l i z a t i o n to y i e l d the desired products as i l l u s t r a t e d i n Figure 20.  - 44 Ground  Plants Soxhlet Extraction (acetone)  Crude Extract Extraction (chloroform) Column Chromatography Fraction  Solvent petroleum  ether (30-60)  petroleum  ether (30-60):benzene (3:1) ether (30-60):benzene (2:1) ether (30-60):benzene (1:1)  petroleum petroleum  Compounds u n i d e n t i f i e d non-polar components such as hydrocarbons and waxes  benzene benzene  H  benzene:chloroform (1:1)  p r i m a r i l y umbelliprenin  chloroform  umbelliprenin, isoimperat o r i n , alloimperatorin methyl ether thamnosmin, and i s o p i m p i n e l l i n  chloroform  chloroform K  M  chloroform  i s o p i m p i n e l l i n , N-methylacridone, and alloimperat o r i n methyl ether epoxide.  chloroform  N-methylacridone, and alloimperatorin methyl ether d i o l  chloroform  primarily unidentified polar components  acetone acetone methanol Figure 17. T y p i c a l p u r i f i c a t i o n sequence of components from Thamnosma montana Torr. and Frem.  - 45 -  Fraction A B  C D  E F  G  H  I  1  J  1  K  L  M  N O  1 0 3f  0  0  »  Q i 0 ft o ' l o o  o  5  0 . o  0  0 0 0 0 0  W  5  Figure 18.  A t y p i c a l thin layer chromatoplate  of i s o l a t e d f r a c t i o n s  obtained from the chloroform extract. observed on the chromatoplate presented:  C h a r a c t e r i s t i c colours  under u l t r a v i o l e t l i g h t are  (1) purple; (2) blue; (3) yellowish-brown;  (4) brown; (5) yellowish-green.  - 46 -  Figure 19.  A thin layer chromatoplate of some authentic samples of compounds i n Thamnosma montana  Torr. and Frem.  The  legend i n d i c a t i n g the names of the compounds and t h e i r t y p i c a l colour under u l t r a v i o l e t l i g h t follows: (1)  umbelliprenin - blue; (2) isoimperatorin - yellowish-brown;  (3)  alloimperatorin methyl ether - yellowish-brown;  (4)  thamnosmin - purple; (5) i s o p i m p i n e l l i n - brown;  (6)  N-methylacridone  - blue; (7) alloimperatorin methyl  ether epoxide - yellowish-green; ether d i o l - yellowish-brown.  (8) alloimperatorin methyl  -  47  -  Fraction  Fraction H and I  and K  Column Chromatography and/or Thin Layer Chromatography and Crystallization  7  umbelliprenin  Figure 20.  J  1  umbelliprenin, isoimperatorin, alloimperatorin methyl ether, thamnosmin, and isopimpinellin  isopimpinellin, N-methylacridone and alloimperat o r i n methyl ether d i o l  N-methylacridone an'd alloimperat o r i n methyl ether d i o l  P u r i f i c a t i o n of Fractions G, H. and I, J and K,  and L of  the i n i t i a l column chromatography..  (b)  Development of Appropriate Chemical Degradation Pathways. The coumarins of i n t e r e s t p r i m a r i l y for the development of  degradative reactions were umbelliprenin (11), i s o p i m p i n e l l i n ( 2 ) , and alloimperatorin methyl ether ( 8 ) .  From these reactions  i t was  thought possible to i s o l a t e relevant carbon atoms i n the above mentioned coumarins.  Before  these degradative reactions could be performed  on any active material, i s o l a t e d from the incorporation experiments, i t was  necessary  to perfect them on the i n a c t i v e compounds previously  i s o l a t e d from the plant.  R.N.  Young, A.K.  Verma and myself,  consequently,  became involved i n the rather extensive research on the chemistry of the appropriate coumarins.  For the sake of c l a r i t y I w i l l summarize a l l  - 48 -  of the pertinent reactions which were studied.  However, experimental  d e t a i l s of only those reactions which were performed by me are recorded i n the thesis. (i)  Degradation  of Umbelliprenin  The degradative reaction employed for umbelliprenin involved 44 a c i d i c cleavage of the farnesol side chain to y i e l d umbelliferone (Figure 2 l ) . Umbelliprenin  (11) was  (20)  treated with g l a c i a l acetic acid to  which a c a t a l y t i c amount of concentrated s u l f u r i c  acid had been  added.  (11)  Figure 21.  (20)  Conversion of umbelliprenin to umbelliferone.  By the above degradative procedure  the relevant carbon atoms i n the  c y c l i c portion of umbelliprenin could be separated from the i s o prenoid side chain i n the ( i i ) Degradation  molecule.  of Isopimpinellin  There were two degradative sequences developed i s o p i m p i n e l l i n (2) (Figure 22).  i  n  i n connection with  one sequence i s o p i m p i n e l l i n  was  demethylated by heating i t with anhydrous aluminum chloride to y i e l d 5,8-dihydroxypsoralene  (38) which i n turn was  i t with acetic anhydride (39).  and pyridine to y i e l d  acetylated by treating 5,8-diacetylpsoralene  This l a t t e r substance was more e a s i l y handled  product.  In the other sequence  than the former  i s o p i m p i n e l l i n underwent ozonolysis i n  - 49 OCH  0  0 C H  OCH,  3  OCH,  (2) "  (40)  A1C1' ,A 3  \\  .CH,  (CH C0) 0 3  0'  C  5 5 H  2  N  (38)  Figure 22.  (39)  (f  ^CH  Degradations of i s o p i m p i n e l l i n .  g l a c i a l acetic acid solution.  The r e s u l t i n g ozonide was then reduced  by using zinc dust to y i e l d 5,8-dimethoxy-6-formylumbelliferone  (40).  Another degradation attempted with i s o p i m p i n e l l i n was to convert i t to  5,8-dimethOxyumbelliferone-6-carboxylic  0  (41)  OCH  acid (41). I t was f e l t  -50 -  that t h i s compound could i n turn be decarboxylated and hence this would provide a technique of i s o l a t i n g the carbon atom attached to p o s i t i o n 7 of the coumarin system. with d i l u t e s u l f u r i c  In one experiment i s o p i m p i n e l l i n was heated  acid and potassium dichromate to t r y and obtain  45,46 •, desired product. In the other case i s o p i m p i n e l l i n was subjected to 1  the  ozonolysis i n a g l a c i a l a c e t i c acid s o l u t i o n , followed by an oxidative work-up using hydrogen peroxide.  However, neither procedure produced  s u b s t a n t i a l amounts of the desired carboxylic acid and further attempts to obtain i t were abandoned. ( i i i ) Degradations of Alloimperatorin Methyl Ether There were two degradative sequences developed i n connection with alloimperatorin methyl ether (8) (Figure 23). Alloimperatorin methyl ether i n both cases was converted f i r s t to alloimperatorin methyl ether d i o l [5-C3'-methyl-2',3'-dihydroxybutanyl)-8-methoxypsoralen] (3) v i a the  epoxide (13). This l a t t e r compound was obtained by f i r s t  treating  the  methyl ether with m-chlorcperbenzoic acid i n chloroform at 0°C and then'  heating the r e s u l t i n g alloimperatorin methyl ether epoxide with d i l u t e o x a l i c acid. In one sequence alloimperatorin methyl ether d i o l was f i r s t with acetic anhydride i n pyridine to y i e l d  treated  5-(3 -methyl-3'-hydroxy-2',  acetoxybutanyl)-8-methoxypsoralene (42) which i n turn was ozonized and reduced, respectively,  using ozone i n g l a c i a l a c e t i c acid followed by  reduction with zinc dust to y i e l d  5-(3'-methyl-3'-hydroxy-2'-acetoxy-  i  butanyl)-6-al-8-methoxyumbelliferone (43). In the other sequence alloimperatorin methyl ether d i o l was treated with periodic acid i n a mixture of acetone free methanol and  Figure 23.  Degradations of alloimperatorin methyl ether.  - 52 -  water to y i e l d The  the aldehyde, compound  acetone was d i s t i l l e d into  present  (44), and acetone (45).  p-bromobenzenesulfonhydrazide (46),  i n a mixture of g l a c i a l a c e t i c acid and water, to y i e l d  acetone p-bromobenzenesulfonhydrazone ( 4 7 ) . ^ The aldehyde, compound (44)»was treated with sodium borohydride i n a mixture of methanol and chloroform,  followed by hydrolysis with water to y i e l d the alcohol,  compound (48).  (c)  Biosynthetic Investigations with Radioactive Mevalonic Acid 3 DL-Mevalonic-5- H acid as i t s dibenzylethylenediamine  s a l t (49)  was u t i l i z e d i n the various experiments discussed i n t h i s t h e s i s .  The  OH 3 • I HOC H_-CH -C-CH C00H(C H CH NHCH CHNHCH C>H_), Z Z \ Z o J Z Z Z Z o o 1/ Z CH(49) o  o  £  c  o  o  0  0  3  free a c i d i s unstable  and r e a d i l y l a c t o n i z e s , therefore, the DBED  (dibenzylethylenediamine) for the plant feedings  s a l t i s normally employed.  In preparation  the methanol s o l u t i o n of the radioactive compound  was d i l u t e d to a known volume (25 ml) and aliquots of t h i s s o l u t i o n were then used i n the experiments described. Similar experiments were conducted by R.N. Young and A.K. Verma 14 using the following possible biosynthetic precursors: glycine-1- C, 14 14 14 glycine-2- C, DL-mevalonic acid-2- C, DL-mevalonic acid-4- C, 3 ' 3 DL-mevalonic acid-2- H, and DL-mevalonic acid-4- H. In the area of research with which I was d i r e c t l y involved, that 3 i s with the biosynthetic investigations of DL-mevalonic-5- H acid as i t s dibenzylethylenediamine  salt  (49), Thamnosma montana plants of  about 19 to 22 months of age were u t i l i z e d f o r the various  feedings.  - 53 -  In a t y p i c a l biosynthetic feeding  an aliquot of the previously mentioned  solution of the radioactive compound (49) was  fed to a small number  of the young plants (6 to 8) for a t o t a l exposure time of about ten days.  The whole plant was  used for the feeding experiments conducted,  the radioactive compound being incorporated through the roots with the feeding being hydroponic i n nature. After the biosynthetic feeding was  completed umbelliprenin,  alloimperatorin methyl ether, thamnosmin, i s o p i m p i n e l l i n , and  allo-  imperatorin methyl ether epoxide were i s o l a t e d by the procedure discussed under section (a) of the Discussion and summarized b r i e f l y i n Figures 17 and 20.  The a c t i v i t y of the combined r e s i d u a l aqueous  solutions i n which the plants were fed was  determined as was  the a c t i v i t y  of the acetone and chloroform extracts. Before c r y s t a l l i z a t i o n was each of the isolated coumarins, the amount obtained was  u t i l i z e d on  determined and then  d i l u t e d with a weighed amount of cold material previously i s o l a t e d from the plant. Following t h i s procedure, attempts were made to obtain, by crystallization techniques, constant r a d i o a c t i v i t y .counts for each coumarin (Figure 24). Subsequent degradation of these compounds provides an i n d i c a t i o n of the l o c a t i o n of the l a b e l , and, hence, allows some conclusions to be drawn concerning (i)  the biosynthesis of these compounds.  Degradation of Radioactive  Umbelliprenin  After radioactive umbelliprenin (11) had been r e c r y s t a l l i z e d to constant r a d i o a c t i v i t y (Figure 24), the compound was umbelliferone  degraded to  (20) as described under Section (b) ( i ) of the Discussion  for i n a c t i v e umbelliprenin.  The umbelliferone obtained  was  r e c r y s t a l l i z e d to constant r a d i o a c t i v i t y from e t h y l acetate as shown i n Figure 25  after p a r t i a l p u r i f i c a t i o n by preparative thin layer chromato-  Figure 24.  R e c r y s t a l l i z a t i o n of various coumarins to constant r a d i o a c t i v i t y .  Coumarin Isolated  umbelliprenin  Weight Isolated (mg)  24.6  Weight after Dilution (mg.) 40.8  (Rediluted to 64.2)  alloimperatorin methyl ether  18.1  40.1  Recrystallization Number  Weight a f t e r Recrystallization (mg.)  Radioactive Counts after Recrystallization (dpm/mg)  Counting Time (minutes)  40x3(no blank) 40x3(no blank) 40x3(no blank) 40x3 40x3 40x3 40x3 100x3  1 2 3 4 5 .6 7 8  28.6 24.5 22.2 20.6 18.7 .17.7 14.2 61.1  50,900 19,610 31,750 26,790 35,400 25,700 26,650 3,710  9 10  55.8 53.5  3,445 3,335  1 2 3 4  29.3 24.7 22.2 19.1  24,000 5,770 1,152 343  40x3(no blank) 40x3(no blank) 40x3(no blank) 40x3  1 2 3 4 5  18.7 16.1 11.5. 10.2 6.7  21,600 13,450 1,542 1,155 368  40x3(no blank) 40x3(no blank) 40x3(no blank) 40x3 40x3  100x3 100x3  (Rediluted to 68.8)  thamnosmin  21.3  31.9  Figure  24.(Continued)  Coumarin Isolated  isopimpenellin  Weight Isolated (mg)  Weight after Dilution (mg)  32.8  49.5  (Rediluted to 50.4)  alloimperatorin methyl ether epoxide  17.8  34.7  Weight a f t e r Recrystallization (mg)  Radioactive Counts a f t e r Recrystallization (dpm/mg)  1 2 3 4 5 6 7 8  36.3 30.2 28.5 27.6 19.3 16.8 15.6 45.4  1,130 650 596 349 186 175 171 37.3  9 10  43.3 38.7  23.3 13.4  1 2 3 4  29.2 22.4 19.4 14.6  Recrystallization Number  1,812 2,035 1,405 266  Counting Time (minutes)  40x3(no blank) 40x3(no blank) 40x3(no blank) 40x3 40x3 40x3 40x3 40x3 40x3 40x3  40x3(no blank) 40x3(no blank) 40x3(no blank) 40x3  Ln Ln  - 56 -  graphy.  R e c r y s t a l l i z a t i o n Number  Radioactive Counts a f t e r Recrystallization (dpm/mg)  1 2 3  Figure 25.  Counting Time (minutes)  122 100 113  100x3 100x3 100x3  R e c r y s t a l l i z a t i o n of umbelliferone to constant radioactivity.  (ii)  Degradation  of Radioactive Isopimpinellin  Radioactive i s o p i m p i n e l l i n (2) was not subjected to degradative reactions due to the fact that r e c r y s t a l l i z a t i o n to constant radio3 a c t i v i t y was very d i f f i c u l t and that DL-mevalonic-5- H acid was incorporated apparently only to a very l i m i t e d extent i n t o this furanocoumarin (Figure 24). ( i i i ) Degradations  of Radioactive Alloimperatorin Methyl Ether  Since d i f f i c u l t i e s were encountered i n attempting  to r e c r y s t a l l i z e  alloimperatorin methyl ether (8) to constant r a d i o a c t i v i t y  (Figure  24), t h i s furanocoumarin was f i r s t p a r t i a l l y degraded to alloimperatorin methyl ether d i o l  [5-(3'-methyl-2',3'-dihydroxybutanyl)-8-methoxy-  psoralen] (3) as described under section (b) ( i i i ) of the Discussion. It was hoped that t h i s l a t t e r compound could be r e c r y s t a l l i z e d to constant r a d i o a c t i v i t y more e a s i l y than alloimperatorin methyl ether (8).  After i s o l a t i o n the d i o l was then r e c r y s t a l l i z e d to constant  r a d i o a c t i v i t y from ethyl acetate as shown i n Figure 26  after p a r t i a l  Recrystallization Number  Weight After Recrystallization (mg )  Radioactive Counts After R e c r y s t a l l i z a t i o n (dpm/mg)  1 2 3 4  37.0 33.0 25,4 22.6  32.8 18.95 19.85 17.30  Figure 26.  Counting Time (minutes)  100x3 100x3 100x3 100x3  R e c r y s t a l l i z a t i o n of alloimperatorin  methyl ether d i o l to  constant r a d i o a c t i v i t y .  p u r i f i c a t i o n by preparative thin layer chromatoraphy. Alloimperatorin to  methyl ether d i o l (3) was subjected to degradation  the aldehyde, compound  were converted to  the alcohol,  (44),and acetone (45) which i n turn compound  (48), and acetone p-bromo-  benzenesulfonhydrazone (47), respectively, as described under (b)  ( i i i ) of the Discussion.  section  Both compounds were p a r t i a l l y p u r i f i e d  by preparative thin layer chromatography and then r e c r y s t a l l i z e d to constant a c t i v i t y .  An i n s i g n i f i c a n t amount of r a d i o a c t i v i t y was  obtained i n the acetone derivative.  The alcohol,  compound (48),  however, had s i g n i f i c a n t r a d i o a c t i v i t y (Figure 27).  Recrystallization Number 1 2 3  Weight After Recrystallization (mg )  Radioactive Counts After R e c r y s t a l l i z a t i o n (dpm/mg)  8.9 7.3 6.8  15.4 12.6 12.9  Figure 27. R e c r y s t a l l i z a t i o n of the alcohol, compound constant r a d i o a c t i v i t y .  Counting Time (minutes)  100x3 100x3 100x3  (48), to  - 58 -  Conclusions From the degradation of radioactive umbelliprenin (11) to umbelliferone  (20) as described under section (c) ( i ) of the Discussion,  i t i s worthy to note that most of the r a d i o a c t i v i t y present i n umbelliprenin (> 95%) was  lost.  This indicated i n turn that most of  the o r i g i n a l r a d i o a c t i v i t y present i n umbelliprenin must have been present i n the f a r n e s y l side chain.  Therefore t h i s information would  suggest that mevalonic acid i s not a d i r e c t precursor of the coumarin nucleus or aromatic portion of umbelliprenin i n Thamnosma montana Torr. and Frem.  However^as expected, the r e s u l t s do i n d i c a t e that  mevalonic acid i s the precursor of the f a r n e s y l u n i t .  The w e l l known  conversion of mevalonic acid into isopentenyl pyrophosphate and subsequently  i n t o If jtf-dimethylallyl pyrophosphate, followed by head-  t o - t a i l condensation  of three of these 5-carbon u n i t s ^ i s obviously the  pathway involved. From the degradation of radioactive alloimperatorin methyl ether C8) to alloimperatorin methyl ether d i o l (3) and subsequent and conversion to  the a l c o h o l , compound  degradation  (48), and acetone p-bromo-  benzenesulfonhydrazone (47), r e s p e c t i v e l y , as described under s e c t i o n Cc) C i i i ) of the Discussion, i t i s worthy to note that most of the r a d i o a c t i v i t y present i n compound (3) (> 70%) was (48).  retained i n compound  On the other hand, only a trace of r a d i o a c t i v i t y was  compound (47).  This r e s u l t was  i n accordance with other  found i n  experimental  data by other research workers and by Verma and Young i n our l a b o r a t o r i e s . That i s , i n alloi.mperatorin methyl ether, i t was  believed that the  two  carbon atoms (4" and 5') i n the furan ring were derived from carbon atoms 4 and 5 of mevalonic acid and that the 5-carbon side chain at  - 59 p o s i t i o n 5 was derived also from mevalonic acid by condensation with carbon atom 5, of mevalonic acid (Figure 23). Consequently, i f DL3 mevalonic acid-5- H i s incorporated into alloimperatorin methyl ether as described, no appreciable loss i n r a d i o a c t i v i t y should occur i n the conversion to compound (48) from compound (3) as the o r i g i n a l carbon 5 of mevalonic acid i s not l o s t i n such a sequence.  The data thus  described under Section (c) ( i i i ) supports this theory.  - 60 -  EXPERIMENTAL  Throughout  t h i s work column chromatography  on Woelm neutral alumina.  was usually performed  This adsorbent was deactivated to a c t i v i t y IV  by the addition of water as directed by the manufacturers.  On occasion  s i l i c a gel for chromatographic adsorption (B.D.H. reagent) was for  column chromatography.  In column chromatography  used  of the crude  extract where large quantities of adsorbent were used, the adsorbent used was Shawinigan Aluminum oxide, deactivated by the addition of 6% of a 10% a c e t i c a c i d s o l u t i o n . the  solvents were d i s t i l l e d For  Except i n large scale column  chromatography,  before use.  a n a l y t i c a l and preparative t h i n layer chromatography  (TLC),  Woelm neutral alumina and s i l i c a g e l G (according to S t a h l ) , containing 2% by weight of General E l e c t r i c Retma p-1, Type 188-2-7 e l e c t r o n i c phosphor,were used.  The chromatograms, 0.3 mm  i n thickness, were a i r  dried and activated i n an oven at 100°C for three hours. t h i n layer chromatography used.  a thicker layer (0.5 mm)  For preparative  of adsorbent was  Detection of bands i n both cases was possible with u l t r a v i o l e t  light. Melting points were determined on a Kofler block and are uncorrected. Radioactivity was measured with a Nuclear-Chicago Mark 1 Model 6860 L i q u i d S c i n t i l l a t i o n counter i n counts per minute a c t i v i t y of a sample i n disintegrations per minute  (cpm).  The radio-  (dpm) was  calculated  - 61 using the counting e f f i c i e n c y which was determined  f o r each sample by the  external standard technique*^ u t i l i z i n g the b u i l t — i n barium 133 gamma source.  The r a d i o a c t i v i t y of the sample was determined using an organic  s c i n t i l l a t o r s o l u t i o n made up of the following components: (1 l i t e r ) ; 2,5-diphenyloxazole  toluene  (4 gm ); and 1,4-bis[2-(5-phenyloxazolyl)J  benzene (50 mg ) or an aqueous s c i n t i l l a t o r s o l u t i o n made up of the following components: ml); naphthalene  toluene (385 ml); dioxane  (80 gm ); 2,5-diphenyloxazole  (5-phenyloxazolyl)]benzene was  (385 ml)j methanol (230  (5 gm ); and l , 4 - b i s [ 2 -  (62.5 mg ). In p r a c t i c e an organic sample  dissolved i n benzene (1 ml) i n a counting v i a l or, i n the case of a  water soluble sample, the sample was dissolved i n methanol (1 ml) i n a counting v i a l .  Then, i n both cases, the volume was made up to 15 mis  with the above organic and aqueous s c i n t i l l a t o r solutions, r e s p e c t i v e l y . For each sample counted the background a c t i v i t y was determined  f o r the  counting v i a l to be used by f i l l i n g the v i a l with one of the above s c i n t i l l a t o r solutions and counting to determine  the background i n cpm.  The counting v i a l was emptied, r e f i l l e d with sample to be counted and the s c i n t i l l a t o r s o l u t i o n , and counted again.  The difference i n cpm  between the background count and the sample count was used f o r subsequent calculations.  (a)  I s o l a t i o n of Natural Products from Thamnosma montana  (i)  Extraction of Thamnosma montana  Torr. and Frem.  Torr. and Frem.  The plants for t h i s study were obtained i n the desert region near Morongo Valley, C a l i f o r n i a with the help of Dreyer and h i s associates at the F r u i t and Vegetable Chemistry Laboratory, Pasadena C a l i f o r n i a .  - 62 -  An acetone extract of the plant was  also obtained from the same source.  The plants were a i r dried and ground to a powder i n a Waring blender. The a i r dr:ied ground powder 6 1/2  C393 gm. ) was extracted with acetone for  hours i n a large glass Soxhlet extractor.  F i l t r a t i o n and evapora-  tion of the solvent gave a crude extract (26.3 gm ) as a dark heavy t a r . Extraction of the residue with chloroform and subsequent f i l t r a t i o n and evaporation gave a dark heavy o i l y residue (23.8 gm ).  (ii)  Column Chromatography of Chloroform The crude extract (67 gm ) was  Extract  applied i n chloroform to the top  of a column of deactivated Shawinigan .alumina (2,000 gm  , deactivated  by the addition of 6% by weight (120 ml ) of 10% a c e t i c a c i d ) . of the column was Fraction  performed with the various solvents as  Solvent  (volume, ml )  wt  (gm )  A  petroleum ether (2,000)  0.28  B  25% benzene i n petroleum ether (1,000)  0.02  33 1/3% benzene i n petroleum ether (1,000)  0.10  D  50% benzene i n petroleum ether (1,000)  0.37  E  benzene (1,000)  0.30  F  benzene (2,000)  0.86  Elution  illustrated.  Compounds u n i d e n t i f i e d non-polar components such as hydrocarbon or waxes  50% chloroform i n benzene (1,750)  1.82  p r i m a r i l y umbelliprenin  H  chloroform  0.92  I  chloroform  umbelliprenin, i s o imperatorin, alloimperat o r i n methyl ether, thamnosmin, and i s o pimpinellin  (750) (1,125)  1.76  - 63 Fraction  Solvent (volume, ml )  J  chloroform (875)  6.42  K  chloroform (500)  6.93  L  chloroform (500)  2.45  M  chloroform (1,000)  1.40  N  acetone (1,000)  0.77  0  acetone (1,000)  3.43  P  methanol  0.24  (1,000)  Compounds  wt (gm )  i s o p i m p i n e l l i n , Nmethylacridone and alloimperatorin methyl ether epoxide  N-methylacridone and alloimperatorin methyl ether d i o l primarily u n i d e n t i f i e d polar components  The numerous f r a c t i o n s obtained from column chromatography were examined under u l t r a v i o l e t l i g h t on a t h i n layer s i l i c a g e l G chromatoplate.  Authentic samples of umbelliprenin (11), isbimperatorin  (12),  alloimperatorin methyl ether (8), thamnosmin (14), i s o p i m p i n e l l i n (2), N-methylacridone  (5), alloimperatorin methyl ether epoxide (13), and  alloimperatorin methyl ether d i o l (3) were examined i n a s i m i l a r manner for purposes of comparison.  The chromatograms were developed i n an  ether-hexane mixture (1:1). Fractions A to F: A portion of f r a c t i o n s A to F when examined by TLC showed only the presence of u n i d e n t i f i e d non-polar components such as hydrocarbons and waxes.  Since these f r a c t i o n s had none of the desirable  components, further examination was not conducted.  coumarin  - 64 -  Fraction G: A portion of f r a c t i o n G when examined by TLC showed the presence of at l e a s t f i v e compounds with one component being predominant.  When  compared with authentic samples, this compound proved to be umbelliprenin (R  f  0.88).  Fractions H. and I: A portion of f r a c t i o n s H and I when examined by TLC indicated the presence of at l e a s t seven compounds. samples, umbelliprenin  When compared with  (R^ 0.88), isoimperatorin  authentic  (R^ 0.85), a l l o -  imperatorin methyl ether (R^ 0.75), thamnosmin (R^ 0.67), and i s o p i m p i n e l l i n (R^ 0.50) were found  present.  Fractions J and K: A' portion of f r a c t i o n s J and K when examined by TLC indicated the presence of at l e a s t eight compounds.  When compared with authentic  samples, i s o p i m p i n e l l i n (R^ 0.50), N-methylacridone (R^ 0.45), and alloimperatorin methyl ether epoxide (R^ 0.44) were present. Fraction L: A portion of f r a c t i o n L when examined by TLC indicated the presence of at l e a s t three compounds.  When compared with authentic samples, N-  methylacridone (R^ 0.45) and alloimperatorin methyl ether d i o l (R^ 0.37)  were present.  Fractions M to P: A portion of f r a c t i o n s M to P when examined by TLC showed primarily u n i d e n t i f i e d polar components and these were not examined further i n this study. The  compounds of p a r t i c u l a r i n t e r e s t , namely,umbelliprenin (11),  - 65 -  alloimperatorin methyl ether (8), i s o p i m p i n e l l i n (2), and alloimperatorin methyl ether d i o l (3) were i s o l a t e d from the various f r a c t i o n s of the column chromatography by a d d i t i o n a l chromatographic p u r i f i c a t i o n and/or crystallization.  (iii)  I s o l a t i o n of Umbelliprenin from Fraction G Umbelliprenin  (11), was  obtained from f r a c t i o n G (1.82 gm. ) p r i m a r i l y  by d i r e c t c r y s t a l l i z a t i o n from hexane.  Further amounts were obtained  by preparative t h i n layer chromatography on the mother l i q u o r s , using t h i n layer s i l i c a g e l G chromatopates developed i n an ether-hexane mixture.  The combined t o t a l was  (1:1)  then r e c r y s t a l l i z e d several times from  hexane to y i e l d a pure white product  (0.210 gm ) which was  found to be  umbelliprenin when compared with an authentic sample previously i s o l a t e d and characterized i n our laboratories by R.N.  (iv)  Young.  I s o l a t i o n of Coumarins from Fractions H and I Fractions H and I (2.68 gm ) were combined and adsorbed on about  10 grams of alumina.  This portion was  then added to a column of alumina  ( a c t i v i t y IV, 100 gm ) and eluted with various solvents as i l l u s t r a t e d . Fraction  Solvent  (volume, ml ) (150)  Wt  (mg )  Compounds u n i d e n t i f i e d non-polar components  A  petroleum ether  B  20% benzene i n petroleum ether (150)  249  p r i m a r i l y umbelliprenin  C  25% benzene i n petroleum ether (150)  564  umbelliprenin, and isoimperatorin  D  33 1/3% benzene i n petroleum ether (80)  339  umbelliprenin, isoimperat o r i n , alloimperatorin methyl ether, and thamnosmin  36  - 66 -  Fraction  Solvent  (volume, ml )  Compounds  Wt (mg )  50% benzene i n petroleum ether (100)  447  isoimperatorin, a l l o imperatorin methyl ether, thamnosmin, and i s o pimpinellin  F  benzene  315  isoimperatorin, a l l o imperatorin methyl ether, thamnosmin, and i s o pimpinellin  G  chloroform  (140)  315  isoimperatorin, a l l o imperatorin methyl ether, thamnosmin, and i s o pimpinellin  E  chloroform  (45)  27  I  chloroform  (200)  48  (80)  primarily unidentified polar components  The fractions from the column chromatography were examined  under  u l t r a v i o l e t l i g h t on a thin layer s i l i c a g e l G chromatoplate, developed i n an ether-hexane (1:1) mixture.  Preparative thin layer  chromato-  graphy, using s i m i l a r plates and solvent mixture, was used to obtain the desirable coumarins from f r a c t i o n s B to G, i n c l u s i v e .  Further amounts  of umbelliprenin (0.323 gm ) were obtained from f r a c t i o n s B, C, and D by preparative thin layer chromatography, followed by c r y s t a l l i z a t i o n from hexane.  Preparative t h i n layer chromatography on f r a c t i o n s C to G,  i n c l u s i v e , yielded isoimperatorin (0.258 gm ) which was from a mixture of hexane and ethyl acetate.  crystallized  Preparative thin layer  chromatography on fractions D to G, i n c l u s i v e , y i e l d e d alloimperatorin methyl ether (0.238 gm ) and thamnosmin (0.158 gm ) which were both c r y s t a l l i z e d from a mixture of hexane and ethyl acetate.  Preparative  - 67 t h i n layer chromatography on f r a c t i o n s E, F, and G yielded i s o p i m p i n e l l i n (0.203 gm ) which was c r y s t a l l i z e d from a mixture of hexane and ethyl acetate.  The coumarins i s o l a t e d were i d e n t i f i e d by comparison with  authentic samples previously i s o l a t e d and characterized i n our laboratories by R.N. Young.  (v)  I s o l a t i o n of Coumarins from Fractions J and K Fractions J and K (~»10 gm. ) were combined and adsorbed on about  40 gm  of alumina.  This portion was then added to a column of alumina  ( A c t i v i t y IV, 400 gm ) and eluted with various solvents as i l l u s t r a t e d . Fraction  Solvent (volume, ml )  wt  (mg )  Compounds  A  33 1/3% benzene i n petroleum ether  B .  50% benzene i n petroleum ether  C  33 1/3% petroleum ether i n benzene  D  benzene (300)  288  E  benzene (200)  577  F  benzene (125)  519  G  benzene (300)  2,035  i s o p i m p i n e l l i n and Nmethylacridohe  H  benzene (250)  2,690  p r i m a r i l y N-methylacridone  I  chloroform (250)  211  primarily N-methylacridone but some alloimperatorin ' methyl ether epoxide  J  chloroform (1,000)  264  K  acetone (1,000)  98  u n i d e n t i f i e d non-polar components  N-methylacridone and u n i d e n t i f i e d polar components  - 68 -  The  f r a c t i o n s from the column chromatography  ultraviolet  light  on a t h i n l a y e r s i l i c a  were examined  under  g e l G c h r o m a t o p l a t e , developed  s  in  an ether-hexane  (1:1) m i x t u r e .  I s o p i m p i n e l l i n was  crystallized  d i r e c t l y out of f r a c t i o n G and a l l o i m p e r a t o r i n m e t h y l e t h e r epoxide  was  c r y s t a l l i z e d d i r e c t l y out o f f r a c t i o n I , u s i n g a h e x a n e - e t h y l a c e t a t e solvent mixture i n both cases.  F u r t h e r amounts of the two  o b t a i n e d by p r e p a r a t i v e t h i n l a y e r chromatography of  the r e s p e c t i v e c r y s t a l l i z a t i o n s .  was  then r e c r y s t a l l i z e d  isopimpinellin (0.057 gm samples R.N. (vi)  ).  (0.692 gm The  The  compounds were  on the mother  liquors  combined t o t a l o f each compound  from an ethyl a c e t a t e - h e x a n e m i x t u r e t o y i e l d  pure  ) and pure a l l o i m p e r a t o r i n methyl e t h e r epoxide  coumarins were i d e n t i f i e d by comparison w i t h a u t h e n t i c  p r e v i o u s l y i s o l a t e d and c h a r a c t e r i z e d i n our l a b o r a t o r i e s  by  Young. I s o l a t i o n o f A l l o i m p e r a t o r i n M e t h y l E t h e r D i o l from F r a c t i o n L A l l o i m p e r a t o r i n methyl ether d i o l  (2.45 gm  ) p r i m a r i l y by d i r e c t  (3) was  o b t a i n e d from f r a c t i o n L  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 .  F u r t h e r amounts were o b t a i n e d by p r e p a r a t i v e t h i n l a y e r on the mother l i q u o r s , u s i n g s i l i c a an ether-hexane  (1:1) m i x t u r e .  from ethyl .acetate t o y i e l d  chromatography  g e l G chromatoplates developed i n  The combined t o t a l was  a pale yellow product  then  (0.772 gm  recrystallized ) which  was  found t o be a l l o i m p e r a t o r i n m e t h y l e t h e r d i o l when compared w i t h an a u t h e n t i c sample p r e v i o u s l y i s o l a t e d  and c h a r a c t e r i z e d by D.L.  Dreyer.  4  - 69 -  (b)  Development of Appropriate Chemical Degradation Pathways  (i)  Degradation of Umbelliprenin (11) Umbelliprenin (49.8 mg ) was dissolved i n g l a c i a l a c e t i c acid  (1.5 ml ) and concentrated s u l f u r i c acid (4 drops). mixture was s t i r r e d at room temperature f o r 50 hours.  The reaction The mixture  was treated with sodium hydroxide (5%, 50 ml ) and extracted with ether to remove unreacted material.  The aqueous solution was  acidified  with hydrochloric acid (20%, 50 ml ) and extracted with ether.  The  ether extract was dried over anhydrous sodium s u l f a t e , f i l t e r e d , and evaporated to dryness.  Preparative t h i n layer chromatography, using  a s i l i c a gel G chromatoplate developed i n an ether-hexane mixture (3:1), was used to obtain the product which appeared as a bright blue band near the baseline. to y i e l d  18.6 mg  The compound was c r y s t a l l i z e d from ethyl acetate of the desired product.  Comparison of the l a t t e r  by mixed melting point and TLC with a commercially available authentic sample of umbelliferone (20), m.p.  230-231°C, indicated the i d e n t i t y  of these compounds. ( i i ) Degradations of Isopimpinellin (2) Degradation of Isopimpinellin (2) to  5,8-Dimethoxy-6-formylumbelliferone(40)  An ozonolysis of i s o p i m p i n e l l i n (2) was  conducted, using  an ozonolysis medium prepared by passing ozone from a Welsbach . Ozonator through acetic acid (170 ml ) f o r 11/4 mmole)  hours.  Isopimpinellin (120 mg  , 0.5  was dissolved i n a small amount of acetic acid and added to the  s o l u t i o n containing ozone (0.91mmole).The reaction mixture was for 3 3/4 hours followed by reduction with zinc dust (492 mg for several hours.  stirred  , 7.6 mmole)  The s o l u t i o n was f i l t e r e d , evaporated to small volume  - 70  -  (*-25 ml ) and f i n a l l y taken to dryness i n vacuo.  The residue (131 mg )  was dissolved i n chloroform and subjected to column chromatography, using s i l i c a g e l (25 gm ) and the various solvents as i l l u s t r a t e d . Fraction  Solvent (volume, ml )  A  benzene (125)  B  33 1/3% chloroform i n benzene (125)  wt (mg )  Compounds  10  u n i d e n t i f i e d nonpolar components  primarily 5,8dimethoxy-6-formylumbelliferone  50% chloroform i n benzene (125) 33 1/3% benzene i n chloroform (125)  E  chloroform (125)  25  F  chloroform (125)  34  G  chloroform (125)  10  H  chloroform (125)  12  I  acetone (125)  15  acetone (125)  17  J  •  u n i d e n t i f i e d polar components and possibly some 5,8dimethoxyumbelliferone-6-carboxylic acid  i  K  acetone (125)  L  methanol (250)  5 30  Fractions C to K, i n c l u s i v e , from the column chromatography were examined under u l t r a v i o l e t l i g h t on a thin layer s i l i c a gel G chromatop l a t e , developed i n an ethyl acetate-chloroform (2:1) mixture. Preparative t h i n layer chromatography on f r a c t i o n s E and F yielded a l i g h t yellow product (24.8 mg ). a n a l y t i c a l sample, m.p.  C r y s t a l l i z a t i o n from acetone gave an  214-216°C, which was i d e n t i f i e d as 5,8-dimethoxy-  - 71 -  6-formylumbelliferone  (40) when compared with an authentic  previously i s o l a t e d and characterized Young.  IR (KBr)  1758,  sample  i n our laboratories by R.N.  1730 (-C0-0-C=C-, aldehyde carbonyl); 1640, MPOH  1593  (aromatic r i n g ) . M e 0 H  e  4.43); x max  UV X  226 my (sh, log e 4.02), 275 my (log  IU3.X  (NaOH added) 238 my (log e 4.26), 269 my (log  e  4.20),  MPOH  299 my (log  E  4.08), 360 my (log E 4.16); X  (HC1 added) 226 my  (sh, log E 4.17), 263 my (log e 4.08), 320 my (log E 4.16).  NMR (100  MHz)  -2.03 (IH, s i n g l e t , phenolic OH),  -0.23 (IH, s i n g l e t , -CHO),  2.15  (IH, doublet, J = 9.5 Hz, H-C cbumarin), 3.72 (IH, doublet, J = 4  9.5 Hz, H-C coumarin), 6.00 (3H, OMe) 3  Anal. Calcd. for C, H,.0,:  C, 57.60; H, 4.00.  0  lz  s i n g l e t , OMe), and 6.02 (3H, s i n g l e t , Found:  C, 57.38;  10 b  H, 4.07.  Degradations of Isopimpinellin  (2) to 5,8-Dimethoxy belliferone-6um  carboxylic Acid (41) Isopimpinellin  (56 mg ) was reacted with s u l f u r i c acid  (3%,  10 ml ) and potassium dichromate (169 mg ) dissolved i n water (2 ml ) for 6 hours under r e f l u x .  The reaction mixture was extracted with a  wide v a r i e t y of solvents, but no stable product was obtained from these or the r e s i d u a l aqueous reaction mixture. An ozonolysis  of i s o p i m p i n e l l i n was attempted,using an  ozonolysis medium prepared by passing ozone through a c e t i c acid (140 ml ) for 1 hour.  Isopimpinellin  (120 mg , 0.5 mmole) was dissolved i n  a small amount of acetic acid and added to the s o l u t i o n  containing  ozone (0.75mmole). The reaction mixture was s t i r r e d for 4 3/4 hours  - 72 followed by oxidation with 30% hydrogen peroxide (0.17 gm , 1.5 mmole) for 1 hour.  The solvent was removed i n vacuo and the residue (139 mg )  dissolved i n chloroform and subjected to column chromatography, using s i l i c a g e l (12 gm ) and the various solvents as i l l u s t r a t e d . Fraction  Solvent (volume, ml )  wt (mg )  Compounds  13  u n i d e n t i f i e d nonpolar components  chloroform (25)  29  p r i m a r i l y 5,8dimethoxy-6-formylumbelliferone  G  chloroform (25)  15  H  chloroform (50)  21  5,8-dimethoxy-6formylumbelliferone and possibly some 5,8-dimethoxyumbelliferone-6carboxylic acid  50% acetone i n chloroform (25)  21  possibly 5,8-dimethoxyumbellif erone-6carboxylic acid  J  acetone (125)  30  u n i d e n t i f i e d polar components  K  acetone (125)  13  L  methanol' (250)  31  A  benzene (100)  B  33 1/3% chloroform i n benzene (50) 50% chloroform i n benzene (50)  D  33 1/3% benzene i n chloroform (50) chloroform (15)  - 73 -  The f r a c t i o n s from the column chromatography were examined under u l t r a v i o l e t l i g h t on a thin layer s i l i c a g e l G chromatoplate, developed  i n an ethyl acetate-chloroform (1:1) mixture.  Preparative  thin layer chromatography on f r a c t i o n s F, G, and H yielded some of the aldehyde  (27.6 mg ), 5,8-dimethoxy-6-formylumbelliferone  no s i g n i f i c a n t amounts of the carboxylic acid,  (40). However,  5,8-dimethoxyumbelli-  ferone-6-carboxylic acid (41) was obtained either by preparative TLC on f r a c t i o n s G and H followed by sublimation or by d i r e c t sublimation of f r a c t i o n I.  (iii)  Degradations of Alloimperatorin Methyl Ether (8)  Conversion of Alloimperatorin Methyl Ether (8) to Alloimperatorin Methyl Ether D i o l (3): Alloimperatorin methyl ether (67.8 mg ., 0.239 mmole) and m-chloroperbenzoic acid (48.8 mg, ,0.284mmole) were dissolved i n separate portions (6 1/2 ml ) of chloroform.  The solutions were cooled to 0°C,  mixed, and s t i r r e d f o r 11 hours at i c e bath temperature. mixture was washed with 5% sodium bicarbonate  The reaction  and water, respectively.  Evaporation to dryness and c r y s t a l l i z a t i o n from an ethyl  acetate-hexane  mixture yielded a pale yellow s o l i d i d e n t i f i e d as alloimperatorin methyl ether epoxide  (13), m.p. 103-104°C, when compared with an authentic  sample previously i s o l a t e d and characterized i n our laboratories by R.N. Young.  IR (KBr) 1717 (C=0), 1588 (C=C).  UV x  MPOH  max  221 my (log  e 4.40), 244 my (sh, log e 4.30), 251.5 my (log e 4.36), 266 my (log e 4.31), 305 my (log e 4.11).  NMR (100 MHz) i n CDC1 , TMS lock, 1.82  (IH, doublet, J = 10 Hz, H-C^ furanocoumarin), 2Hz, H-C  7  furanocoumarin),  3  2.33 (IH, doublet, J =  3.11 (IH, doublet, J = 2 Hz, H-C, furano-  - 74 coumarin), 3.64 (IH, doublet, J = 10 Hz, H-C^ furanocoumarin), 5.78 (3H, s i n g l e t , OMe), 6.5-7.2 (3H; ABC m u l t i p l e t ; JAB  = 14.5 Hz, JAQ  =  3 Hz, J,,., = 7.5 Hz; benzylic methylene and epoxide protons), 8.51 and BC o 8.70  (6H, two s i n g l e t s , ^ C — C ( C H ) ) . 3  2  Mass spectrum  (m/e). 300(M) , 285  (M-15), 271(M-29), 257(M-43), 229(M-71), 201, 199, 186, 171, and 158. Anal. Calcd. f o r C ^ H ^ O ^  C, 67.99; H, 5.37. Found:  C, 67.91;  H, 5.98.  Alloimperatorin methyl ether epoxide was treated with o x a l i c acid (5%, 25 ml ) f o r 2 hours under r e f l u x .  The reaction mixture was  extracted with chloroform which was washed with 5% sodium carbonate and water, respectively.  The chloroform solution was dried with anhydrous  magnesium s u l f a t e and evaporated to dryness to y i e l d a pale yellow solid  C75.5 mg ).  Preparative thin layer chromatography,  using s i l i c a  gel G chromatoplates developed i n a chloroform-ethyl-acetate (1:1) mixture yielded a pale yellow s o l i d (65.8 mg ). C r y s t a l l i z a t i o n from ethyl acetate gave an a n a l y t i c a l sample, m.p. 174-176°C, i d e n t i f i e d as alloimperatorin methyl ether d i o l (40.7 mg ) when compared with characteriza4 tion data previously acquired f o r the compound by D.L. Dreyer. [a]^ °-30.6 3  (95% EtOH).  o  IR (nujol) 3410 (hydroxyl), 1713 (C=0), 1592  Ft" OH  (C=C).  UV x  220 my (log e 4.46),^244 my (log e 4.19), 251 my (log  H13.X  e 4.23), 266 my (log e 4.19), 309 my (log e 4.06).  NMR (60 MHz) i n CDC1 3  deuteriodimethylsulfoxide, TMS lock, 1.75 (IH, doublet, J = 10 Hz, H-C^ furanocoumarin), 2.25 (IH, doublet, J = 2Hz, H-C^ furanocoumarin), 3.00 (IH, doublet, J = 2Hz, H-C, furanocoumarin),  3.73 (IH, doublet, J = 10 Hz,  b H-C furanocoumarin),  5.83 (3H, singlet, OMe),  H-C,,, furanocoumarin),  6.87 (2H, doublet, J = 7 Hz, b e n z y l i c protons),  3  6.53 (IH, t r i p l e t , J = 7Hz,  - 75 -  8.70  (6H, s i n g l e t , C-methyls).  Degradation of Alloimperatorin Methyl Ether D i o l (3): Acetone free chloroform was prepared by passing chloroform through a c e l i t e column impregnated with. 2,4-dinitrophenylhydrazine and d i s t i l l i n g the eluant.  Acetone free methanol was prepared by the addition of  iodine (25 gm ) to methanol (1,000 ml ), followed by addition of the mixture to sodium hydroxide (500 ml  , 1 N) with constant s t i r r i n g .  Water (150 ml.) was added to p r e c i p i t a t e iodoform.  The mixture was  f i l t e r e d , refluxed to remove the iodoform odours, and f i n a l l y d i s t i l l e d . Alloimperatorin methyl ether d i o l  (21.3 mg  }  0.0671 mmole) was  dissolved i n acetone free methanol (3 ml ) and periodic acid (47.8 mg 0.215mmole) was dissolved i n water (3 ml stirred  for'*-'24 hours.  ). The solutions were mixed and  Nitrogen was flushed slowly through the reaction  mixture, which was maintained at'*' 50°C, i n order to d i s t i l l over any acetone formed during the course of the reaction. c o l l e c t e d as a derivative (47) by d i s t i l l a t i o n into  The acetone  p-bromobenzenesulfon-  hydrazide (150 mg ),dissolved i n a mixture of water (5 ml )and acetic acid (5 ml ). P r i o r to' t h i s d i s t i l l a t i o n a blank was into the reagent i n a s i m i l a r manner.  was  Both the blank and  glacial  distilled the  reaction mixture were extracted with acetone free chloroform.  The  extracts were washed with water, dried with anhydrous sodium s u l f a t e , f i l t e r e d , and evaporated to dryness.  Preparative t h i n layer chromatoi  graphy, using Woelm neutral alumina chromatoplates developed i n a  - 76 -  chloroform-methanol  (20:1) mixture was used to i s o l a t e the d e r i v a t i v e ,  acetone p-bromobenzenesulfonhydrazone  (R^ 0.62), separate from the  reagent, p-bromobenzenesulfonhydrazide  (R^ 0.76).  The blank was  discarded as no s i g n i f i c a n t amount of d e r i v a t i v e was apparent when the chromatoplate was analyzed under u l t r a v i o l e t l i g h t .  Crystallization  from chloroform yielded a white a n a l y t i c a l sample (10.A mg), m.p. 148°C, i d e n t i f i e d as the d e r i v a t i v e , acetone hydrazone  146-  p-bromobenzenesulfon-  (47) when compared with an authentic sample previously i s o l a t e d  arid characterized i n our laboratories by R.N. Young.  IR (KBr) 3220  MPOH  ( ^N-H)J 1343, 1180  (-S0„).  UV X  235 my  MHz)  i n CDC1 , TMS  3.24  (IH, broad s i n g l e t , ^N-H), 8.12 and 8.24  3  -N=C-(CH ) ). 3  lock, 2.1-2.6 (4H, A B 2  Mass spectrum (m/e)  2  NMR  (100  m u l t i p l e t , aromatic protons),  292 and 290  Anal. Calcd. f o r C g H ^ ^ S B r : C, 36,86; H, 3.88; N,  2  (log e 4.15).  (6H, two s i n g l e t s , (M).  C, 37.18; H, 3.81; N, 9.62.  Found:  9.40.  The aqueous non-volatile portion of the reaction mixture was . extracted with chloroform.  The extract, was washed with water,  dried with anhydrous sodium s u l f a t e , and evaporated to dryness to y i e l d an aldehyde, compound (44), as a crude residue, i d e n t i f i e d by TLC comparison to a sample previously i s o l a t e d and p a r t i a l l y characterized i n our laboratories by R.N. Young. (IH, s i n g l e t , -CHO), 1.83 2.02  NMR  (100 MHz)  ?  furanocoumarin), 3.65  coumarin), 5.62 Mass spectrum  3  (IH, doublet, J = 10 Hz, H-C^  (IH, doublet, J = 2 Hz, H-C  2 Hz, H-Cg  i n CDC1 , TMS  lock,  furanocoumarin),  furanocoumarin), 2.87  (IH, doublet, J =  (IH, doublet, J = 10 Hz, H-C  (2H, s i n g l e t , benzylic protons), 5.82  0.19  3  furano-  (3H, s i n g l e t ,  (m/e) 258(M), 229(M-29), 214(M-44), 201,, 186, and  158.  OMe).  The residue was dissolved i n a mixture of chloroform and methanol (1:1) and cooled to 0°C.  Sodium borohydride (41.6 mg ) was  dissolved  i n methanol (*»*1 ml) and added to the solution of the aldehyde.  The i  reduction was conducted f o r i hour with s t i r r i n g followed by hydrolysis with water (~10 ml ) f o r 1 hour with s t i r r i n g . was extracted with chloroform.  The reaction mixture  The extract was washed with water,  dried with anhydrous sodium s u l f a t e , f i l t e r e d , and evaporated to dryness to y i e l d the crude product.  Preparative thin layer chromatography,  using s i l i c a gel G chromatoplates developed i n an ethyl acetate-chloroform (2:1) mixture yielded the desired product.  C r y s t a l l i z a t i o n from e t h y l  acetate gave a pale yellow a n a l y t i c a l sample (5.6 mg), m.p.  167-167.5°C,  i d e n t i f i e d as the alcohol, compound (48) when compared with an 9  authentic sample previously i s o l a t e d and characterized i n our laboratories by R.N.  Young.  IR (KBr) 3450 (OH), 1705-1690 (C=0), 1585  (C=C).  Me OH  UV  220 my  x  (log e 4.39), 251 my  (log e 4.32), 265 my  (log e 4.24),  TT13X  306 my  (log e 4.11).  NMR  (100 MHz)  doublet, J = 10 Hz, H-C^  furanocoumarin), 3.12  3.66  (IH, doublet, J = 10 Hz, H-C^ 6.08  2  (IH, doublet, J = 2 Hz, H-C^  Mass spectrum  (m/e)  (IH,  furanocoumarin),  furanocoumarin), 5.78 2  201, 186, and  lock, 1.92  (IH, doublet, J = 2 Hz,  (2H, t r i p l e t , J = 6 Hz, -CH_0H) , 6.74  benzylic protons).  (3H, s i n g l e ^  (3H, t r i p l e t , J = 6 Hz,  260(M), 229(M-31), 214(M-46),  158.  Anal. Calcd. f o r H, 4.61.  3  furanocoumarin), 2.32  H-C^  OMe),  i n CDC1 -D 0, TMS  C 1  4  H 1 2  °5  :  C  '  6  4  ,  6  2  Mol. Wt. Calcd. f o r C H 0 :  mass spectrometry):  1 4  260.070.  1 2  5  5  H  >  4.62.  260.068.  Found:  C, 64.66;  Found (high resolution  - 78 (c)  Biosynthetic Investigations with Radioactive  Mevalonic Acid  3 DL-Mevalonic-5- H acid as i t s dibenzylethylenediamine s a l t was  obtained from the New  England Nuclear Corporation  (49)  for the purpose  of conducting the biosynthetic i n v e s t i g a t i o n . The r a d i o a c t i v i t y of the 3 9 DL-mevalonic-5- H acid (DBED s a l t ) was 1.0 m i l l i c u r i e s ( 2 . 2 x 10 dpm) and  the s p e c i f i c a c t i v i t y was  active compound (1.21 mg )was In preparation  221 millicuries per m i l l i m o l e .  The  radio-  obtained dissolved i n methanol (1.0  ml).  for the biosynthetic feeding the methanol s o l u t i o n of the  radioactive compound'was d i l u t e d with d i s t i l l e d water to a known volume (25 ml ) i n a volumetric  (i)  flask.  3 Feeding of DL-Mevalonic-5- H Acid In a t y p i c a l biosynthetic feeding  2.2 x 10  9  dpm  compound was  = 5.64  x 10  8  dpm)  (DBED Salt) an aliquot (6.4060 gm ,6.4060/25 x  of the s o l u t i o n of the radioactive  fed to six young Thamnosma montana plants  for a t o t a l exposure time of about 10 days.  ('"'19 months old)  The aliquot was  divided  up approximately into three equal portions, each portion to be fed to two plants contained feeding which was being incorporated  in  a test tube.  The whole plant was  used for the  hydroponic i n nature with the radioactive compound through the roots.  After the plants had  taken up  the  i n i t i a l radioactive s o l u t i o n , the test tubes were maintained f u l l with d i s t i l l e d water to a l e v e l j u s t near the top of the roots.  ( i i ) I s o l a t i o n of Coumarins from the Biosynthetic Feeding The  s i x plants from the biosynthetic feeding were ground to a powder  i n a Waring blender.  The ground powder («11 gm ) was  extracted with  acetone (750 m l ) for 9 3/4 hours i n a Soxhlet extractor.  Filtration  and evaporation of the solvent yielded a dark residue (779.4 mg ). Extraction of the residue with chloroform and subsequent f i l t r a t i o n and evaporation yielded a further dark residue (549.3 mg  ). The  total  r a d i o a c t i v i t y of the combined r e s i d u a l aqueous solutions i n which the plants were fed was  determined (7.41 x 10^ dpm)  as was  r a d i o a c t i v i t y of the acetone extract (23.2 x 10^ dpm) extract (14.6 x 10^ dpm).  the t o t a l  and the  chloroform  From the t o t a l r a d i o a c t i v i t y recovered from  outside the plants, the corrected r a d i o a c t i v i t y fed was established (5.64 x 10  8  dpm-0.0741 x 10  8  dpm  = 5.57  The crude extract (549.3 mg ) was alumina.  This portion was  x 10  8  dpm).  adsorbed on about 3 grams of  then added to a column of alumina ( A c t i v i t y  IV, 30 gm )and eluted with the various solvents as i l l u s t r a t e d . Fraction  Solvent  (volume, ml  )  Wt  (mg  )  Compounds u n i d e n t i f i e d nonpolar compounds such as hydrocarbons and waxes  A  petroleum ether (125)  63  B  petroleum ether (125)  11  C  25% benzene i n petroleum ether (125)  24  33% benzene i n petroleum ether (125)  56  50% benzene i n petroleum ether (125)  26  benzene  (75)  16  umbelliprenin, a l l o imperatorin methyl ether, thamnosmin, and i s o p i m p i n e l l i n  G  benzene  (50)  15  i s o p i m p i n e l l i n and N-methylacridone  H  benzene  (50)  9  umbelliprenin, a l l o imperatorin methyl ether, and thamnosmin  - 80 Fraction  Solvent  I  benzene  J K L  ,  (volume, ml )  (125)  Wt  (mg )  Compounds  28  chlorophylls, isopimpinellin, a l l o imperatorin methyl ether epoxide, and N-methylacridone  chloroform (125)  16  primarily unidentified polar components  chloroform (125)  19  chloroform  11  (125)  The f r a c t i o n s from the column chromatography were examined  under  u l t r a v i o l e t l i g h t on a thin layer s i l i c a g e l G chromatoplate, developed i n an ether-hexane (1:1) mixture.  Authentic samples of umbelliprenin  (11), isoimperatorin (12), alloimperatorin methyl ether (8), thamnosmin t  (14), i s o p i m p i n e l l i n (2), N-methylacridone (5), alloimperatorin methyl ether epoxide (13), and alloimperatorin methyl ether d i o l (3) were examined i n a s i m i l a r manner f o r purposes of comparison.  Preparative  thin layer chromatography was used to obtain umbelliprenin (24.6 mg ), alloimperatorin methyl ether (18.1 mg), thamnosmin (21.3 mg), (32.8 mg)  t  and alloimperatorin methyl ether epoxide (17.8 mg),  isopimpinellin identified  by comparison to authentic samples previously i s o l a t e d and characterized i n our l a b o r a t o r i e s .  '  In preparation f o r degradative work to be performed on some of the coumarins i s o l a t e d , each coumarin was d i l u t e d with a weighed amount of cold material previously i s o l a t e d from the plant.  Attempts were  made to obtain by c r y s t a l l i z a t i o n techniques, constant counts for each coumarin as i l l u s t r a t e d .  radioactivity  Coumarin Isolated  Weight Isolated (mg)  Weight After Dilution (mg)  umbelliprenin  24.6  40.8 (first dilution) 64.2 (second dilution)  alloimperatorin methyl ether  18.1  40.1  thamnosmin  21.3  isopimpinellin  alloimperatorin methyl ether epoxide  Recrystallizing Solvent  hexane  Weight After Final Recrystallization (mg)  Radioactive Radioactive counts After status of F i n a l Pecryst- Coumarin allization (dpm/mg)  53.5  3,335  hexane and ethyl, acetate  19.1  343  not constant  31.9  hexane and ethyl acetate  6.7  368  not constant  32.8  49.5 (first dilution) 50.4 (second dilution)  hexane and ethyl acetate  38.7  13.4  not constant  17.8  34.7  hexane and ethyl acetate  14.6  266  not constant  constant (incorporation 0.032%)  - 82 ( i i i ) Degradation  of Radioactive Umbelliprenin  Radioactive umbelliprenin (53.5 mg ,3.335 dpm/mg) was dissolved i n g l a c i a l acetic acid (2 ml )and concentrated s u l f u r i c acid (5 drops). The reaction mixture was treated with sodium hydroxide extracted with ether.  (5%, 50 ml )and  The aqueous s o l u t i o n was a c i d i f i e d with hydrochloric  acid (20%, 50 ml )and extracted with ether.  The ether extract was  dried over anhydrous sodium s u l f a t e , f i l t e r e d , and evaporated to dryness.  Preparative thin layer chromatography, using a s i l i c a g e l G  chromatoplate  developed  obtain the product  i n an ether-hexane (3:1) mixture was used to  (21.5 mg ) , i d e n t i f i e d as umbelliferone  comparison with a commercially  a v a i l a b l e authentic sample.  (20) by Umbelliferone  was r e c r y s t a l l i z e d several times from ethyl acetate to constant radioa c t i v i t y (14.0 mg ,122 dpm/mg; 12.2 mg ,100 dpm/mg; 10.4 mg ,113 dpm/mg).  (iv)  Degradations  of Radioactive Alloimperatorin Methyl Ether  Since radioactive alloimperatorin methyl ether (19.1 mg , 343 dpm/mg) was not r e c r y s t a l l i z e d to constant r a d i o a c t i v i t y e f f i c i e n t l y , i t was d i l u t e d again (to 68.6 mg ) with cold material and converted to alloimperatorin methyl ether d i o l which was r e c r y s t a l l i z e d to constant  radioactivity.  Conversion of Radioactive Alloimperatorin Methyl Ether (8) to Radioactive Alloimperatorin Methyl Ether D i o l (3): Radioactive alloimperatorin methyl ether (68.8 mg ,0.243 mmole) and m-chloroperbenzoic  acid (47.6 mg ,0.278mmdle) were dissolved i n separate  portions (6 1/2 ml )of chloroform.  The solutions were cooled to 0°C,  - 83 mixed, and s t i r r e d for 12 1/2 hours at i c e bath temperature. The reaction mixture was washed with 1% sodium bicarbonate and water, respectively.  The chloroform extract was dried with anhydrous  sodium s u l f a t e , f i l t e r e d , and evaporated  to dryness  to y i e l d crude  radioactive alloimperatorin methyl ether epoxide (13), i d e n t i f i e d by TLC comparison to an authentic sample. Radioactive alloimperatorin methyl ether epoxide was treated with o x a l i c acid (5%, 26 ml ) f o r 3 hours under r e f l u x . mixture was extracted with chloroform. washed with 5% sodium carbonate  The reaction  The chloroform extract was  and water, r e s p e c t i v e l y , followed by  drying with anhydrous sodium s u l f a t e and evaporation to dryness to y i e l d the crude product  (62.8 mg ). Preparative t h i n layer chromato-  graphy, using s i l i c a g e l G chromatoplates  developed  i n a chloroform-  ethyl acetate (1:1) mixture yielded a pale yellow s o l i d  (46.1 mg ),  i d e n t i f i e d as alloimperatorin methyl ether d i o l (3) by TLC comparison to an authentic sample.  Alloimperatorin methyl ether d i o l was  r e c r y s t a l l i z e d several times from ethyl acetate to constant  radioactivity  (37.0 mg,32.8 dpm/mg; 33.0 mg ,18.95 dpm/mg; 25.4 mg ,19.85 dpm/mg; 22.6 mg ,17.30 dpm/mg). This represented an incorporation of 0.00007% into the o r i g i n a l alloimperatorin methyl ether. Degradation  of Radioactive Alloimperatorin Methyl Ether D i o l (3):  Radioactive alloimperatorin methyl ether d i o l (22.3 mg ,0.0704 mm) was dissolved i n acetone free methanol (3 ml )and p e r i o d i c acid (47.4 mg ,0.213mmole)was dissolved i n water (3 ml ). The solutions were mixed and s t i r r e d for/~'24 hours.  Acetone, formed during the course of the  reaction was i s o l a t e d as a d e r i v a t i v e (47) i n a s i m i l a r manner as i t  - 84 was during  the reaction previously conducted on cold material.  C r y s t a l l i z a t i o n from chloroform yielded the p u r i f i e d d e r i v a t i v e (8.0 mg ), i d e n t i f i e d as acetone p-bromobenzenesulfonhydrazone (47) by TLC comparison to an authentic sample.  The d e r i v a t i v e when counted,  however, displayed i n s i g n i f i c a n t r a d i o a c t i v i t y . The aqueous non-volatile portion of the reaction mixture was worked up as i n the reaction previously conducted on cold material to y i e l d crude aldehyde, compound (44), i d e n t i f i e d by TLC comparison with an authentic  sample.  Reduction with sodium borohydride (42.5 mg ) and  subsequent hydrolysis and workup as before yielded crude alcohol (9.1 mg), compound (48), i d e n t i f i e d by TLC comparison with an authentic sample.  This degradation product was r e c r y s t a l l i z e d several times from  ethylacetate 12.6  to constant r a d i o a c t i v i t y (8.9 mg ,15.4 dpm/mg; 7.3 mg  dpm/mg; 6.8 mg ,12.9 dpm/mg).  t  - 85 -  BIBLIOGRAPHY  1.  E.L. Bennett and J . Bonner, Am. J . Botany, 40, 29 (1953).  2.  W.H. Muller and H. Muller, Am. J . Botany, 43, 354 (1956).  3.  T.H. Kearney and R.H. Peebles, "Arizona F l o r a " , p. 494, University of; C a l i f o r n i a Press, Berkeley and Los Angeles, C a l i f o r n i a , 1960. i D.L. Dreyer, Tetrahedron, 22, 2923 (1966).  4. 5.  J.R. P r i c e , "The A l k a l o i d s " , V o l . I I , p. 353, R.H.F. Manske and H..L. Holmes, Ed., Academic Press, New York, 1952.  6.  D.L. Dreyer, private  7.  T. Inaba, Ph.D. Thesis, p. 40, The University of B r i t i s h Columbia, 1967.  8.  D.B. Sprinson, Advan. i n Carbohyd. Chem. , 15_, 235 (1960).  9.  S.A. Brown, G.H.N. Towers, and D. Wright, Can. J . Biochem. and P h y s i o l . , 38, 143 (1960).  10.  communication.  T. Kosuge and E.E. Conn, J . B i o l . Chem., 234, 2133 (1959).  11.. F. Weygand and H. Wendt, Z. Naturf., 14b, 421 (1959). 12.  S.A. Brown, Z. Naturf., 15b, 768 (1960).  13.  S.A. Brown, Can. J . Biochem. and P h y s i o l . , 40, 607 (1962).  14.  S.A. Brown, "Biosynthesis of Aromatic Compounds", p. 15, G. B i l l e k , Ed., Pergamon Press, London, 1966.  15.  T. Kosuge and E.E. Conn, J . B i o l . Chem., 236, 1617 (1961).  16.  J.R. Stoker and D.M. B e l l i s , J . B i o l . Chem., 237, 2303 (1962).  17.  H.J. Gorz and F.A. Haskins, Crop S c i . , 4_, 193 (1964).  18.  J.R. Stoker, Biochem. Biophys. Res. Comm.,14, 17 (1964).  19.  S.A. Brown, G.H.N. Towers, and D. Chen, Phytochemistry, 3>> 469 (1964),  20.  D.J. Austin and M.B. Meyers, Phytochemistry, 4_, 245 (1965).  21.  D.J. Austin and M.B. Meyers, Phytochemistry, 4_, 255 (1965).  22.  S.A. Brown, Phytochemistry, _2, 137 (1963).  - 86 -  23.  S.A. Brown, Can. J . Biochem. , 43_, 199 (1965).  24.  K. Chambers, G.W. Kenner, M.J.T. Robinson, and B.R. Webster, Proc. Chem. S o c , 291 (1960).  25.  C A . Bunton, G.W. Kenner, M.J.T. Robinson, and B.R. Webster, Tetrahedron, 19, 1001 (1963).  26.  E. Grisebach and W.D.  27.  A . I . Scott, P.A. Dodson, F. McCapra, and M.B. Meyers, J . Amer. Chem. Soc. , 85, 3702 (1963).  28.  S.A. Brown, Lloydia, 26, 211 (1963).  29.  H. Grisebach andW. Barz, Z. Naturf., 18b, 466 (1963).  30.  H. Grisebach and W. Barz, Z. Naturf. , 19b, 569 (1964).  31.  G. Kunesch and J . Polonsky, Chem. Comm., 317 (1967).  32.  T.R. Seshadri, Tetrahedron, 6^, 173 (1959).  33.  W.D.  34.  K.G. Edwards and J.R. Stoker, Phytochemistry, 6^, 655 (1967).  35.  K.G. Edwards and J.R. Stoker, Phytochemistry, ]_, 73 (1968).  36.  G. Caporale, A. Breccia, and G. Rodighiero, "Prepn. Bio-Med. Appl. Labeled Mol., Proc. Symp", p. 103, Venice, 1964.  37.  K.G. Floss and U. Mothes, Z. Naturf., 19b, 770 (1963).  38.  R.' Aneja, S.K. Mukerjee, and T.R. Seshadri, Tetrahedron, 4_, 256 (1958).  39.  H.G. Floss and U. Mothes, Phytochemistry, _5» 161 (1966).  40.  H.G. Floss and H. Paikert, Phytochemistry, 8^, 589 (1969).  41.  F. Weygand,.H. Simon, H.G. F l o s s , and U. Mothes, Z. Naturf. 15b, 765 (1960).  42.  H.G. Floss, H. Guenther, and L.A. Hadwiger, Phytochemistry, .8, 585 (1969).  43.  W; Steck, M. El-Dakhakhny, and S.A. Brown, Tetrahedron Letters, 54, 4805 (1969).  O l l i s , Experientia, 17, 4 (1961).  O l l i s , Experientia, 22_, 777 (1966).  - 87 -  44.  E. Spath, Ber. dtsch. Chem. Ges., 71, 1667 (1938).  45.  E.A. Abu-Mustafa and M.B.E. Fayez, J . Org. Chem., 26_, 161 (1961).  46.  A. Chatterjee and S.S. Mitra, J . Amer. Chem. S o c , 73^, 606 (1949).  47.  R.J.W. Cremlyn, J . Chem. Soc. (C), 1229 (1966).  48.  "Mark 1 Liquid S c i n t i l l a t i o n Systems Manual", Nuclear Chicago Company, p. 18, Section I CL966).  

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