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Studies related to bark extractives of some fir and spruce species, and synthesis and biosynthesis of… Westcott, Neil Douglas 1970

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STUDIES RELATED TO: BARK EXTRACTIVES OF SOME FIR AND SPRUCE SPECIES; AND SYNTHESIS AND BIOSYNTHESIS OF INDOLE ALKALOIDS  by  NEIL DOUGLAS WESTCOTT B.Sc. Honours, U n i v e r s i t y of A l b e r t a , 1966  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the Department of  Chemistry  We accept t h i s t h e s i s as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1970  In p r e s e n t i n g  this  thesis  an a d v a n c e d d e g r e e a t the L i b r a r y I  further  for  agree  scholarly  by h i s of  shall  this  written  the U n i v e r s i t y  make  it  freely  that permission  for  It  financial  of  Columbia,  British for  gain  Columbia  the  requirements  reference copying o f  I agree and this  shall  that  not  copying or  for  that  study. thesis  by t h e Head o f my D e p a r t m e n t  is understood  Depa r t m e n t  Date  of  for extensive  permission.  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  fulfilment  available  p u r p o s e s may be g r a n t e d  representatives. thesis  in p a r t i a l  or  publication  be a l l o w e d w i t h o u t my  ABSTRACT P a r t I of the thesis describes four i n v e s t i g a t i o n s of some of the n e u t r a l components of bark e x t r a c t i v e s . The petroleum ether e x t r a c t of grand f i r [Abies grandis (Dougl.) L i n d l . ] was found to contain two t r i t e r p e n e lactones.  The f i r s t compound, c y c l o -  g r a n d i s o l i d e , was shown by chemical and spectroscopic considerations and confirmed by X-ray a n a l y s i s to be (2 3R)-3a-methoxy-9,19-cyclo-93-lanost-24ene-26 ,23-lactone (38) . The second component, e p i - c y c l o g r a n d i s o l i d e , was isomeric with the f i r s t and x^as assigned as (23S)-3ct-methoxy-9,19-cyclo-93~ lanost-24-ene-26,23-lactone (43). In the second i n v e s t i g a t i o n , three t r i t e r p e n e s of the chloroform e x t r a c t of P a c i f i c s i l v e r f i r [A. amabilis (Dougl.) Forbes] were examined. The main  methoxylanosta-9(11),24—diene-26,23-lactone ( 3 0 ) . Chemical and spectroscopic evidence i s considered which i n d i c a t e s that assignment to be i n c o r r e c t and abieslactone i s t e n t a t i v e l y re-assigned as (23R)-3a-methoxy-93-lanosta-7,24diene-26,23-lactone (81). A minor component, AA^ was assigned on the basis of methylation studies as 3-desmethylabieslactone or (23R)-3a-hydroxy-9glanosta-7,24-diene-26,23-lactone (83). Oxidation of AA^ gave a ketone i d e n t i c a l to the second minor component, AA^, which i s then (23R)-3-oxo93-lanosta-7,24-diene-26,23-lactone (82). The t h i r d i n v e s t i g a t i o n concerns the s t r u c t u r e of W^, a t r i t e r p e n e ketone from the petroleum ether e x t r a c t of Western white spruce [Picea glauca (Moench) Voss. var. a l b e r t i a n a (S. Brown) Sarg.].  The s t r u c t u r e  t e n t a t i v e l y assigned on the b a s i s of spectroscopic evidence i s 33-methoxy8a-serrat-13-en-21-one (91).  iii  The fourth i n v e s t i g a t i o n was a chemosystematic study of the petroleum ether extract of Engelmann spruce [P.. engelmannii Parry].  The presence of  methoxyserratene derivatives known to be present i n other members of the same genus were not detected i n the present investigation. Part I I of the thesis describes synthetic endeavors leading to possible bio-intermediates  of indole alkaloids and the biosynthetic evaluation of one  synthetic compound. Condensation of 3-ethylpyridine with (60) followed by reduction gave ethy1-1,2,5,6-tetrahydropyridine  2-carboethoxy-3(8-chloroethyl)indole  N-[${3(2-hydroxymethylindolyl)}ethyl]-3(64). The benzoxymethyl derivative 65 of  compound 64 was treated with potassium cyanide to give the cyanomethyl derivative 66 which could be.hydroxyzed to N-[3{3(2-carbomethoxymethylindolyl)}  with methyl formate followed by reduction of the resulating enol, gave 16,17-dihydrosecodin~17-ol (69). This compound was shown to be not, or very s l i g h t l y , incorporated into the alkaloids of Vinca rosea L. plants. to oxidize the tetrahydropyridine 64 with mercuric  acetate under various  conditions f a i l e d to give detectable amounts of the corresponding salt.  Attempts  pyridinium  , In another synthetic sequence, condensation of the tryptophyl derivative  60 with 3-acetylpyridine ethylene k e t a l followed by the same sequence of reduction and homologation as employed before gave N-[3{3(2-carbomethoxymethylindolyl)}ethyl]-3-acetyl-l,2,5,6-tetrahydropyridine  (82). Attempts  to oxidize 82 with mercurous acetate followed by hydrogenation f a i l e d to give the desired  N-[g{3(2-carbomethoxymethylindolyl)}ethyl]-3-acetyl-l,4,5,6-  tetrahydropyridine (83). In a second attempt to synthesize 83, the pyridinium chloride s a l t  iv  84 from the condensation 60, was  of 3~acetylpyridine with the tryptophyl derivative  hydrogenated to N-f (3{3(2-carboethoxyindolyl) }ethyl]-3-acetyl-l,4,5,  6-tetrahydropyridine gave major amounts of  (85).  Reduction  of 85 under a variety of conditions  N-[3{3(2-hydroxymethylindolyl)}ethyl]-3-acetylpiperi-  dine (86) with only trace amounts of N-[B{3(2-hydroxymethylindoiy 1)}ethy1]3-acety1-1,4,5,6-tetrahydropyridine  (87) containing the necessary  vinylogous  amide chromophore. In a t h i r d approach to the synthesis of 83, methyl indole-2-carboxylate (88) was (92).  reduced and homologated as before to give methyl indole-2-acetate  Treatment of 92 with ethylene oxide and stannic.chloride gave methyl  3(B.-hydroxyethyl)indole-2-acetate  (93).  Treatment of the tryptophyl  bromide derivative 94, produced by the action of phosphorous tribromide on  hydrogenated to the vinylogous  amide 83.  More conveniently, treatment of  93 i n 3-acetylpyridine with phosphorous tribromide and immediate hydrogenation gave 83 i n better y i e l d .  V  TABLE OF CONTENTS Page T i t l e page  i  Abstract  i i  Table of Contents  v  L i s t of Tables  vi  Lis t of Figures  vii  Acknowledgements Part I  x  Studies Related to Bark Extractives of Some F i r and- Spruce Species  Introduction (a)  2  S t r u c t u r a l Studies on Triterpenes from Grand F i r Dis cuss ion Experimental...  (b)  15 '  48  Investigations Concerning the Structure of Abieslactone Discussion  59  Experimental... (c)  101  S t r u c t u r a l Studies on W^ from Western White Spruce Discussion Experimental  (d)  110 '  133  Chemosystematic Studies on Engelmann Spruce Discussion  142  Experimental  151  Bibliography  158  Part II Studies Related to Synthesis and Biosynthesis of Indole Alkaloids Introduction  163  Discussion  183  Experimental.  204  Bibliography  231  vi  LIST OF TABLES Page Part I Table I II III  Hydrocarbons o f A b i e s c o r t i c a l p l e o r e s i n s P o s i t i o n o f m e t h y l groups i n T v a l u e s ( i 0.03T)  V VI  28  C o n t r i b u t i o n o f f u n c t i o n a l groups t o the c h e m i c a l s h i f t change (AT)  IV  6  o f m e t h y l groups .  Observed and c a l c u l a t e d c h e m i c a l s h i f t s  . .. . o f m e t h y l group  resonances i n t y p i c a l l a n o s t - 9 ( 1 1 ) - e n e s . .  29  Resonance f r e q u e n c i e s of m e t h y l groups o f lanost-9(11)-enes...  30  Mass s p e c t r a l comparison of c y c l o a r t e n y l a c e t a t e and rvr 1 n aran Hi q n 1 i Hp  VII VIII  28  36  Range of resonance f r e q u e n c i e s of t h e C-methyl groups Comparison  72  of resonance f r e q u e n c i e s of o l e f i n i c p r o t o n i n  a b i e s l a c t o n e s e r i e s w i t h some C(9) - C ( l l ) u n s a t u r a t e d triterpenes  74  Part I I Table I  R e s u l t s of i n c o r p o r a t i o n of s y n t h e t i c i n t e r m e d i a t e s V_. r o s e a L. p l a n t s  into 189  vii  LIST OF FIGURES Page Part I Figure 1.  Postulated biosynthesis  of serratenediol  9  2.  Typical p u r i f i c a t i o n sequence of components from grand f i r bark  16  3.  P u r i f i c a t i o n of Fraction G. . .  4.  P u r i f i c a t i o n of Fraction M.. .  5.  NMR spectrum of cyclograndisolide  (x 4 - 10 region).........  21  5a.  NMR spectrum of cyclograndisolide  (T 2 - 9 region)  22  6.  ORD curve of cyclograndisolide  7  P.T\  .  n;j  r-\7P<  rt-f  r\T  r  T_n  a  r* z?n  . ..  17 18  23 24  A n c r> T _ i r ? o , .  8.  NMR spectrum of grandisolide  26  9.  Mass spectrum of cycloartenyl acetate...  34  9a.  Mass s p e c t r a l fragmentation of cycloartenyl acetate  35  10.  Mass spectrum of cyclograndisolide  37  11.  NMR spectrum of epi-cyclograndisolide  (x 4 - 1 0  11a.  NMR spectrum of epi-cyclograndisolide  (T 2 - 9 region)  region)  42 43  12.  ORD curve of epi-cyclograndisolide  44  13.  CD curve of epi-cyclograndisolide  44  14.  Mass spectrum of epi-cyclograndisolide  46  Fragmentation pattern of epi-cyclograndisolide  47  15.  Degradation sequence of abieslactone  62  16.  NMR spectrum of abieslactone  69  17.  ORD curve of abieslactone...  70  14a.  viii  Figure  Page  18.  CD curve of abieslactone  70  19.  Mass spectrum of dihydroparkeyl acetate  77  20.  Mass spectrum of g r a n d i s o l i d e .  79  21.  Mass spectrum of abies lactone............. .  81  22.  ORD curve of AA  8 3  23.  CD curve of AA  24.  ORD curves of lanost-9(11)-en-3-one, AA , and dihydro  25.  Degradation sequence of abieslactone  26.  T y p i c a l p u r i f i c a t i o n sequence of components from western  2  .•  2  84 2  AA2....  91  white spruce bark  H2  27.  P u r i f i c a t i o n of F r a c t i o n 2 [Western white spruce]....  0  n  0  • c±  —  —  -  X  T  86  *  I-J  29.  NMR spectrum of W  30.  ORD curve of W.  —  114 11^  r  4  ... 116  4  31.  Fragmentation pattern of 33-methoxyserrat-13-en-21a-ol  125  32.  Mass spectrum of 33-methoxyserrat-13-en-21a-ol  33.  Mass spectrum of W^ a l c o h o l  34.  P o s t u l a t e f o r the biosynthesis of 8a- and 83-serrat-13-ene  "  126 128  • derivatives  131  35.  Separation sequence of Engelmann spruce bark  143  36.  P u r i f i c a t i o n of F r a c t i o n 2 [Engelmann spruce]  37.  P u r i f i c a t i o n of p r e c i p i t a t e from F r a c t i o n 3  148  38.  P u r i f i c a t i o n of mother l i q u o r s of F r a c t i o n 3  149  '  145  ix  Part I I Figure 1.  Page Wenkert's proposal f o r the biosynthesis of Aspidosperma and Iboga a l k a l o i d s  .  168  2.  Formal transformation of the monoterpene u n i t . . . . .  170  3.  Proposals  177  4.  Postulated biosynthesis of tabersonine  5.  Postulated biosynthesis of catharanthine  6.  Summary of pathway from loganin to i n d o l e a l k a l o i d s  182  7.  Synthesis of 16 ,17-dihydrosecodin-17-ol (57)  185  8.  Attempted synthesis of vinylogous  197  9.  Synthesis of vinylogous amide 83.  f o r the biosynthesis of pre-akuammicine (45).... (4)..  amide 83  179 179  202  ACKNOWLEDGEMENTS  I w i s h t o e x p r e s s my g r a t i t u d e t o P r o f e s s o r James P. Kutney f o r h i s encouragement and guidance throughout t h e c o u r s e o f my research. I w i s h t o e x p r e s s my i n d e b t e d n e s s t o Dr. I.H. Rogers f o r the g i f t o f e x t r a c t s o f Grand f i r b a r k and f o r h i s a b l e  assistance.  G i f t s of a u t h e n t i c t r i t e r p e n e samples from P r o f e s s o r s D.H.R. B a r t o n Uyeo, and G.- O u r i s s o n  and Dr. J.W. Rowe  were  of valuable  assistance  I w i s h t o thank my many h e l p f u l c o l l e a g u e s , p a r t i c u l a r l y D r s . R.B.  S w i n g l e and G.D. Knowles f o r t h e i r a s s i s t a n c e i n the mass  spectrometric i n v e s t i g a t i o n s . P i r i ^ir>.  <~'i  n  1  .^p. p, i  1'  . i n r^p  f rnm  th  1 0  N n i " n n n n |[  ^'^S'PHrch  C o u n c i l of  Canada and the U n i t e d S t a t e s Department o f A g r i c u l t u r e , F o r e s t r y S e r v i c e i s g r a t e f u l l y acknowledged.  I was f o r t u n a t e i n b e i n g  awarded a Dr. F . J . N i c h o l s o n S c h o l a r s h i p , a U n i v e r s i t y Graduate F e l l o w s h i p , a N a t i o n a l Research C o u n c i l o f Canada b u r s a r y , and a H.R. M a c M i l l a n  Family  F e l l o w s h i p d u r i n g the course o f my s t u d i e s .  PART STUDIES  RELATED  I  TO BARK  OF SOME F I R AND  SPRUCE  EXTRACTIVES SPECIES  Introduction  The mystery of man's natural world can be found i n the e a r l i e s t of records.  I t i s probable that the u t i l i z a t i o n of substances now known as  natural products started before the time of recorded h i s t o r y .  Extracts of  plants gave the ancient world indigo and a l i z a r i n used f o r dyeing. Aromatic plants afforded perfumes.  Other naturally occurring materials  were, and are s t i l l used for healing or k i l l i n g . Early investigations were deeply involved with the chemistry associated with these molecules.  Many researchers were severely hampered by the volume  of work needed to make the s l i g h t e s t progress.  As chemical theory evolved  two main areas or research proved to be scumbling blocks; p u r i f i c a t i o n of products and physical measurements on these products were inadequate.  The  past few decades have seen remarkable advances i n both these areas. The progress made i n biosynthetic theory has also played a role i n natural product i n v e s t i g a t i o n .  Natural products are often c l a s s i f i e d i n  families, thus biogenetic considerations determined  for one member are  usually applicable to other members of the family.  These considerations  have l e d to predictions concerning the occurrence of new and, at the time, unknown structures and even to suggest that some structures already assigned should be re-investigated since they did not. f i t the e x i s t i n g biosynthetic patterns. While much of the world i s forested, s u r p r i s i n g l y l i t t l e i s known about the chemical composition of the plant l i f e .  What i s known i s often  3  fragmented and incomplete making systematic studies nearly impossible. Despite the economic importance of the forestry industry the extractables found i n the trees are poorly understood. The pulp and paper industry has know of the existence of extractives but has often viewed them as a nuisance to be removed.  Industrial ingenuity  has accomplished their removal and i n the kraft process two by-products, sulfate turpentine and t a l l o i l , are of economic importance. Sulfate turpentine consists of the volatile terpenes condensed from the relief gases.  The production of synthetic pine o i l which i n turn i s  used for conversion to terpin hydrate and other chemicals, as well as a solvent and i n ore flotation, uses a large portion of the turpentine. Paints, lacquers, synthetic resin, and the perfumery industry use smaller ....  i  The composition of t a l l o i l is variable depending on the kind of wood and the pulping and recovery processes.  According to Browning the range 2  of composition may be from 35% to 55% resin acid, 35% to 60% fatty acid, and 10% to 20% unsaponifiable material.  The unsaponifiable matter  includes sterols, higher alcohols, hydrocarbons, and even some sterol esters of fatty acids which are d i f f i c u l t to saponify.  Industrial uses  of t a l l o i l are i n the manufacture of adhesives, binders, drying o i l s , soaps, printing ink, and varnishes.  Esters of t a l l o i l are used i n drying  o i l s , alkyd resins, plasticizers, and lubricant derivatives. The resin ducts of certain trees when wounded secrete a viscous o i l known as oleoresin.  It consists essentially of a resin i n a volatile o i l .  In the United States, oleoresin is collected from deliberately wounded  4  l o n g l e a f and s l a s h p i n e . the remaining much l i k e t a l l  The v o l a t i l e  "wood t u r p e n t i n e " i s removed and  r e s i n , c o n s i s t i n g m a i n l y o f r e s i n and f a t t y a c i d , i s used oil.  About 10% o f t h e o l e o r e s i n i s n e u t r a l o r u n s a p o n i f i a b l e  m a t e r i a l c o n t a i n i n g 3 - s i t o s t e r o l (1) and o t h e r a l c o h o l s ; two d i t e r p e n e (3);  s t e r o l s ; long chain  aldehydes, dextropimarinal  t r i c y c l i c diterpenes; diterpene  1  2,  (2) and i s o d e x t r o p i m a r i n a l  a l c o h o l s ; and 3,5  P. =  fatty  dimethoxystilbene.  3.,  CHO  2b, R = COOH  P = CHO  3b, R = COOH  The r e s i n a c i d f r a c t i o n o f t a l l o i l o r p i n e o l e o r e s i n c o n s i s t s m a i n l y o f diterpene  acids with  the abietane  r e s i n acids are dextropimaric palustric  4  skeleton.  T y p i c a l examples o f t h e  (2b) , i s o d e x t r o p i m a r i c  ( 5 ) , and l e v o p i m a r i c ( 6 ) .  5  6  (3b) , a b i e t i c ( 4 ) ,  3  5  The are  volatile  responsible  components, whether  f o r the c h a r a c t e r i s t i c  Some t r a i n e d o b s e r v e r s  same  odors  wood, b a r k  associated, with  c a n d i s t i n g u i s h c e r t a i n woods on t h i s  a p p r o a c h when p e r f o r m e d differentiating  from  they be  on a more s c i e n t i f i c  or characterizing different  or leaves, some  trees.  basis.  This  b a s i s may b e u s e f u l f o r species  or v a r i a t i o n s of the  species. The  heartwood  extracts  Erdtman * and t a x o n o m i c  species noted.  a n d C u p r e s s a c e a e h a v e b e e n made. has been  studied  and t h e p r e s e n c e  The genus P i n u s  5  monoterpenes [Plc6<a.  c o n i f e r s have been examined by  c o r r e l a t i o n s on t h e g e n e r i c  1  Pinaceae  o f many  found  K.la.ui;<j.  has been  i n t h e gum  (T'lociiuli)  Vuss]  7  remarkably  consistent  of several diterpenoids  and d i s t i n c t i v e  who  6  The l e a f  He c o n c l u d e d  8  of several  distribution  has been  o i l of white  that both  Abies  i n v e s t i g a t e d the  sptuce [?• a a i i a n a  e.uu b i a e k  were examined by von R u d l o f f . '  i n the f a m i l i e s  The heartwood  studied by M i r o v  turpentine.  level  spruce  v K i i x ) BSr. j species  pattern  have  a  of leaf o i l  terpenes. The  s i g n i f i c a n c e of these  chemosystematic x mariana]  in  white  others.  reflecting A been  to be a h y b r i d  Rosendahl  or black  the hybrid  s i m i l a r study  spruce  spruce  The a n a l y s i s  i s best  by von R u d l o f f . ^  i s considered  Morphologically like  studies  studies  of the l e a f  1 0  of white  »  1 1  »  1 2  i n some  o i l showed  by t h e  spruce  and b l a c k  and c o m p l e t e l y  of the Rosendahl  of the c o r t i c a l  conducted by Z a v a r i n .  The R o s e n d a h l  i s intermediate  i n others,  origin  illustrated  [P. g l a u c a  spruce.  characteristics,  different  from  a similar pattern  thus  spruce.  o l e o r e s i n from North American Nine  either  distinct  Abies  species are  f i r has  6  r e c o g n i z e d n o r t h o f Mexico and seven o f these may be grouped l a r g e r s p e c i e s complexes.  i n three  A. m a g n i f i c a A. Murr and A<> p r o c e r a Rehd.  are i n one complex; A. c o n c o l o r (Gord. & Glend.) L i n d l . and A. g r a n d i s (Dougl.) L i n d l . i n another A. balsamea  (L.) M i l l . ,  Both A. a m a b i l i s (Dougl.)  complex; w i t h A., l a s i o c a r p a  (Hook.) N u t t . ,  and A., f r a s e r l (Pursh) P o i r . as t h e t h i r d  complex.  Forbes and A. b r a c t e a t a D. Don a r e s e p a r a t e and  a r e n o t a s s o c i a t e d w i t h any l a r g e r complex.  The a n a l y s i s o f t h e mono-  t e r p e n o i d s and t h e s e s q u i t e r p e n o i d hydrocarbons  revealed chemical  d i f f e r e n c e s between many s p e c i e s and v a r i e t i e s . Of the n i n e s p e c i e s , grand [A. .amabilis] a r e o f i n t e r e s t  f i r [A. g r a n d i s ] and P a c i f i c s i l v e r f i r  i n c o n n e c t i o n w i t h the p r e s e n t work.  The  major d i f f e r e n c e s i n chemic-al c o m p o s i t i o n o f t h e o l e o r e s i n s may be seen -ticric  (a) and (b) o f T a b l e  Compound (a)  Sesquiterpenes 3-Bisabolene  grandis  1%  amabilis  30%  a-Cubebene  24  trace  a-Copaene  18  1  3-Copaene  trace  17  (b) Monoterpenes 3-Phellandrene  Table I .  T  15  Camphene  46  3-Carene  1  Hydrocarbons o f A b i e s c o r t i c a l o l e o r e s i n s  46  20  7  As The  can be seen, t h e r e a r e major d i f f e r e n c e s between these two s p e c i e s . d i f f e r e n c e s extend t o the t r i t e r p e n e s i s o l a t e d from the b a r k o f these  species  and w i l l be d i s c u s s e d  later i n this thesis.  At p r e s e n t , b a r k i s a major waste p r o d u c t i n t h e p u l p and paper industry.  Not o n l y  provides a d i f f i c u l t  does i t p o s s e s s l i t t l e d i s p o s a l problem.  commercial v a l u e b u t i t a l s o  Bark r e p r e s e n t s  10% t o 15% o f t h e  t o t a l w e i g h t o f t h e wood t r e a t e d and, i n t h e l o n g r u n , i t i s disadvantageous from the economic p o i n t The  o f view t o use i t o n l y as f u e l .  p h y s i c a l components o f b a r k , c o r k , f i b e r , and b a r k powder, have  been examined f o r p o s s i b l e u s e s . as a d d i t i o n s  1 3  F i b e r s may be used i n f i b e r b o a r d and  t o f i l l i n g s and i n s u l a t i o n m a t e r i a l s .  Bark powder has found  some use as a c a r r i e r f o r i n s e c t i c i d e s and i n s o i l improvement.  Fractions  Bark c h e m i c a l s may. be i s o l a t e d e i t h e r by p y r o l y s i s or e x t r a c t i o n . A l k a l o i d s such as q u i n i n e  (7) and s t r y c h n i n e  (8)  a r e i s o l a t e d from b a r k .  A f l a v a n o i d , q u e r c i t i n ( 9 ) , and some o f i t s d e r i v a t i v e s have been i s o l a t e d from many b a r k s .  They have a n t i o x i d a n t  because o f p o s s i b l e uses i n food  and a r e o f some m e d i c i n a l  treatment o f c a p i l l a r y b l o o d v e s s e l  7  p r o p e r t i e s which a r e o f i n t e r e s t  disorders.  8  l t f  v a l u e i n the  8  OH /  Extracts of western hemlock f i n d use as s o i l s t a b i l i z e r s , cold-setting waterproof adhesives, and o i l well d r i l l i n g A study was made by Chang and M i t c h e l l  fluids. 1 6  1 5  on the amount of extractable  ™tpri?l - i " the bark of some comr.cn North American pulpvcod spscies.  They  successively extracted the bark with benzene, ethanol, hot water, and  one  per cent aqueous sodium hydroxide s o l u t i o n .  The values obtained f o r  Engelmann spruce [Picea engelmannii Parry] were 5.2% benzene and ethanol soluble. respectively. 3.3%  14.6%  The only f i r mentioned, balsam f i r , had values of 13.2%  respectively.  varied from 28.7% 2.1%  The values f o r black spruce were 5.0% and  25.9%  Rowe  17  and  found that the benzene extract of pine bark  f o r lodgepole pine [Pinus contorta Dougl.] to a low of  f o r sugar pine [P. lambertianna Dougl.] The composition of the extract obtained by Chang and M i t c h e l l was  investigated f u l l y .  not  Rowe's study was more thorough and l i s t e d several  s t e r o l s which he i s o l a t e d .  More i n t e r e s t i n g was  the finding of a methoxy  t r i t e r p e n o l i n jack pine [P_. bank s i ana Lamb.] and a triterpene d i o l , which he c a l l e d pinusenediol, from l o b l o l l y pine [P. taeda L.] and jack pine.  9  The  s t r u c t u r e o f p i n u s e n e d i o l was shown by R o w e  10  t o be i d e n t i c a l  w i t h t h a t o f s e r r a t e n e d i o l (10) i s o l a t e d by Inubushi from a c l u b moss, Lycopodium serratum Thumb, v a r . Thumbergie M a l c i n o .  occurring  from a - o n o c e r i n  19  (11a) w i t h i n c o r p o r a t i o n o f one of the v i n y l  groups i n t o r i n g C ( F i g u r e 1 ) .  Figure  1.  Postulated  b i o s y n t h e s i s of s e r r a t e n e d i o l .  10  The r e s u l t of the incorporation of one of the methylene groups into the ring system.leaves  seven angular methyl groups rather than the usual  eight for other pentacyclic triterpenes. Evidence for this biosynthesis i s speculative since no l a b e l l i n g studies have been reported.  Also neither a-onocerin nor i t s derivatives  has been found to co-occur with the serratene derivatives i n trees.  A  club moss, Lycopodium clavatum, was found to contain a-onocerin and also diepiserratenediol ( 1 2 ) .  19  OH  H0-" "  12  The isomerization of a-onocerin (11) by mineral acid into 3- (13) and y-onocerin (14) i s known.  21  Inubushi  20  was able to confirm the e a r l i e r work  by the isomerization of a-onocerin diacetate ( l i b ) into 3- and y-onocerin diacetate (13b) and (14b) with the use of mineral acid.  With a bulkier  Lewis acid, boron t r i f l u o r i d e , Inubushi was able to i s o l a t e serratenediol diacetate (10b) and y o n o c e r i n diacetate.  S i m i l a r l y , s t a r t i n g with the  corresponding diketone, serratenedione (15) was i s o l a t e d .  The conversion  of. a-onocerin i n t o serratenediol represented a t o t a l synthesis of that compound since a-onocerin had been synthesized by S t o r k .  2 2  The position  of the double bond i n serratenediol and the position and configuration  11  14. R = H 14b, R = Ac  15  of the methyl group a r i s i n g from the v i n y l group of a-onocerin were determined by degradation.  HO 16 In a l a t e r paper Rowe  Zd  reported the i s o l a t i o n and structure of s i x  serratenediol derivatives from pine bark.  Of the s i x compounds only one,  21-eplserratenediol (16), had been previously characterized. The remaining f i v e were novel and three of them contained methoxy groups.  The three  12  methoxy compounds were sho WIT. to be 33»21ct—dimethoxyserrat-14-ene (17); 33-methoxyserrat-l4-en-21a-ol (IS); and 33-methoxyserrat-14-en-21-one (19). The other two compounds were serrat-14-en-21-on-33~ol (20) and serrat-14en-3a,213-diol (21). Further work on jack pine revealed 213-methoxyserrat14-en-3-one (22) and 21a-methoxyserrat-14-en-33-ol (23). The methylene chloride extract of the bark of Scots pine [P. sylvestus] contained 33-methoxyserrat-14-en-21-one 14-ene (17).  (19) and 33,21a-dimethoxyserrat-  2h  In a study of the bark of Sitka spruce [Picea  sitchensi3  (Bong.) Carr.]  conducted in our own laboratories and at the Forest Products Laboratory, Vancouver, several serratene derivatives were i s o l a t e d .  2 5 , 2 5  The two  in greatest abundance were 33-methoxyserrat-14-en-213-ol (24) and 3amethoxyserrat-I4-sn-2ip-oI (7.5) . T h e f i r s t naturally occurring serratene compound with a 13-ene system, 3a-methoxyserrat-13-en-213-ol (28), was also characterized.  Among the minor components were the following  serrat-14-enes: 33,213-diol (16); 33,21a-diol (10a); 21-on-33-ol (20); 3a-methoxy-21-one (26); 33-methoxy-21-one (19); and 3ct,213-dimethoxy (27). The diterpene alcohol 13-epimanool (29) was characterized as i t s 3,5dinitrobenzoate derivative.  28  29  13  17,  R  l  R. = H, R„ = R = 4 2 3  18,  R  l  R  4  = H, R  2  = OMe,  R  l  R  3  = H, R  2  = OMe,  R  l  H,  R  = H, R  19, 20,  I. H. R o g e r s of some spruces.  27  OMe  0  21, R  2  R  22, R  3  H, R  3  2  4  = OH,  R  = R  = OMe,  = R  3  4  =  R  OH  = 0  4  = 0  4  = OH  R-j = R  2  = 0  23,  R  l  R. = H, R_ = OMe, 4 3  R  24,  R  l  R  3  = H, R  2  = OMe,  R  4  = OH  25,  R  2  R  3  = H, R  1  = OMe,  R  4  = OH  = R  4  = 0  26, R  2  27, R  2  H,  R  R  = H, R  3  Q  ±  = OMe, T  R  3  = R. = 1 4  2  = OH  OMe  has compiled a review on the wood and bark extractives  Other than the above work on Sitka spruce most of the  work on spruce has centered on the phenolics or r e s i n acid and as such i s not d i r e c t l y applicable to this t h e s i s . The occurrence of serratene derivatives i n the family Pinaceae i n the genus Pinus and l a t e r i n the genus Picea i s now w e l l established.  A  methoxylated triterpene, abieslactone (30), has been reported from the genus Abies of the same f a m i l y .  2 8  This compound, however, i s not a  serratene d e r i v a t i v e but possesses rather a lanosterol skeleton. Abieslactone was i n i t i a l l y i s o l a t e d i n 1938  from the bark and leaves of Abies m a r i e s s i i  Masters, ^ a f i r tree of northern Japan. 2  i s o l a t e d by H e r g e r t  30  The same compound had been  from the North American P a c i f i c s i l v e r f i r [A. amabilis  (Dougl.) Forbes] and Noble f i r [A., procera Rehd.] and named by him as  14  methoxyabiesadienolide.  30 On that The As  the  basis  abieslactone structure be  will  some o f  relate  to  must  contain  absolute  discussion  the  first of  dehydrogenation the  skeleton  configuration  i t was  of  of  the  present  provides  portion  of  compounds  this found  suggested  1964  in  trimethylsteroids.  suggested  i l i . i s CoiuyuU'uu uucupietj a  above d i s c u s s i o n  characterization trees.  selenium  secu. l a t c l  the  The  and  of  3 1  for abieslactone  is  taelusr c e n t r a l p o s i t i o n  30 in  work.  a b r i e f summary o f thesis  concerning  i n extracts  of  investigations the  isolation  some s p r u c e  and  which and  f i r  15  Structural Studies on Triterpenes from Grand F i r  Discussion  As part of a long range study aimed at the eventual u t i l i z a t i o n of chemicals found i n the bark of coniferous species, I.H. Rogers, Forest Products Laboratory,  i n i t i a t e d an examination of the extractives of grand  f i r bark [Abies grandis  (Dougl.) L i n d l . ] .  The bark f o r this study was obtained  3 2  from a hundred year o l d tree  growing on the University of B r i t i s h Columbia campus.  The bark was a i r  dried and ground to a coarse powder before being extracted i n a large Soxhlet extractor using petroleum ether as solvent.  Upon evaporation  of the solvent the crude extract s o l i d i f i e d to give a brown wax. on the weight of a i r dried bark, the crude extract represented  Based  a petroleum  ether extractable content of 0.8%. The crude extract was separated  into numerous fractions using  successive column chromatography on alumina.  A t y p i c a l separation  emphasizing the fractions relevant to this discussion i s shown i n Figures• 2-4.  The i n i t i a l f r a c t i o n of i n t e r e s t i n the present work i s G  (Figure 2) containing a mixture of fatty alcohols, lactones, and epimanool. Further p u r i f i c a t i o n provided  three new f r a c t i o n s , M, N, and 0 . *  * The receipt of Fractions M, N, and 0 from I.H. R o g e r s acknowledged.  32  i s gratefully  16  Bark Petroleum Soxhlet Crude Extract  Column Chromatography  Solvent  Fraction  Figure 2.  Compound(s)  petroleum  ether  hydrocarbons  petroleum  ether  hydrocarbons, s t e r o l esters  petroleum  ether  s t e r o l esters  petroleum  ether  s t e r o l and wax esters  petroleum  ether  wax ester, 2 unknowns  benzene:pet. ether (1:9) benzene:pet. ether (1:4)  unidentified  benzene:pet. ether (3:7) benzene:pet. ether (1:1)  fatty alcohol, lactones epi-manool  benzene:pet. ether (3:2)  fatty alcohol, s i t o s t e r o l  benzene:pet. ether (3:2)  s i t o s t e r o l , f e r u l i c ester  benzene  s i t o s t e r o l , f e r u l i c ester 2 unknowns  d i e t h y l ether me thanol  f e r u l i c ester  Typical purification  sequence of components from grand f i r bark.  17  Fraction G  Column Chromatography  Solvent  Fraction M  Figure 3.  • •-• Compounds  petroleum etheribenzene (4:1)  epimanool, f a t t y a l c o h o l lactones  petroleum ether:benzene (1:1)  lactones fatty alcohol  benzene  lactones fatty alcohol  Purification of Fraction G.  Thin layer chromatography (TLC) of M, N, and 0 showed that a l l three fractions were mixtures.  Fraction ri contained at least four components:  epimanool, rapidly recognized by comparison with an available authentic sample (see later); two components revealing lactonic absorption in the infrared; and fatty alcohols.  Fractions N and 0 contained the same two  lactones and fatty alcohol with the fatty alcohol being the main component. On this basis i t appeared that Fraction M would be the most profitable to examine i n i t i a l l y .  Chromatography of Fraction M on a column of alumina  gave separation into two further fractions P and Q (Figure 4 ) . had TLC and nuclear magnetic resonance (NMR)  Fraction P  properties which appeared  consistent with manool (31) or epimanool (29).  These two alcohols may  be  conveniently distinguished from each other as their 3,5-dinitrobenzoate derivative.  The 3,5-dinitrobenzoate of Fraction P had a m.p.  of 115 -  117°C  in  agreement w i t h the r e p o r t e d  this  basis  the s t r u c t u r e  3 3  ascribed  v a l u e o f 116 was  that  -  118°C  of epimanool  OH.  Column  31  M  Chromatography  4.  Compound  Solvent  Fraction  Figure  (29).  OH.  29  Fraction  f o r epimanool.  petroleum  ether:benzene  (4:1)  epimanool  petroleum  ether:benzene  (3:2)  lactones fatty alcohol  Purification  of Fraction  M.  19 Fraction Q contained at least two compounds.' The NMR  of this fraction  had resonances at x 3.0, 5.0, and 6.7 similar to those reported for abieslactone (30) ,  2 9  In addition, a vinylic methyl at T 8.1 was  distinguishable, a signal also found i n abieslactone. A signal at T 8.7 which was also present was assigned to the methylenes of the long chain fatty alcohol, one of the components In Fraction Q.  MeO-'  The necessity to obtain material free of the fatty alcohol was obvious.  Rowe in his work with pine bark extracts had some success i n 33  removing this type of material with an urea channel complex.  Consequently  a portion of Fraction M was taken up in a warm alcoholic solution of urea and, after standing overnight, the crystalline complex containing the fatty alcohol component was removed by f i l t r a t i o n .  Thin layer chromato-  graphy of the mother liquors revealed a mixture of two compounds. These were conveniently separated on s i l i c a gel preparative layer plates impregnated with Rhodamine 6G.  3tf  The top band was the greater of the two and could be eluted from the s i l i c a gel with chloroform.  Evaporation of the chloroform left an  20  orange-red  solid  as some of the dye had  through a s h o r t . c o l u m n The  o f d e a c t i v a t e d alumina  compound so o b t a i n e d was  named c y c l o g r a n d i s o l i d e . mixture of  o f the second  a l s o been e l u t e d .  second band was  component, AG^,  and  subsequently  s i m i l a r l y treated to give a  some AG^„  Additional  quantities  above.  For a n a l y t i c a l purposes  c y c l o g r a n d i s o l i d e so o b t a i n e d was  from methanol t o g i v e a w h i t e s o l i d , m.p.  191  f o r m u l a by h i g h r e s o l u t i o n mass s p e c t r o m e t r y found 1745  to be C^^H^-gO^. cm  and  -1  suggested  The  infrared  the u l t r a v i o l e t  The  2 8  two h i g h f i e l d  NMR  spectrum  this CDC1  a t x 7.2;  a one  at 60 MHz and  9.7,  a t T.8.1;  had  t o be  t h i s i n t e r f e r e d w i t h the h i g h f i e l d the h i g h f i e l d  p r o t o n a t x 3.0 CHC1  .  a  x  209 mu  was  (log E  4.33)  o b t a i n e d i n CDCl^ r e v e a l e d  s u g g e s t i n g the presence - 9.15)  a t T 5.0;  a  was  was  doublets.  used  A t r a c e o f TMS  was  portions.  proton but i n  With  f o r the l o c k s i g n a l but  U s i n g CHCl^ f o r the l o c k  d o u b l e t s c o u l d be r e c o r d e d but  unobservable.  five  diffused  and an o l e f i n i c  o b t a i n e d i n two  of a  c o r r e s p o n d i n g to  More a c c u r a t e c h e m i c a l s h i f t s were o b t a i n e d at 100 MHz  as s o l v e n t , t e t r a m e t h y l s i l a n e (TMS) \  signal  m  an 0-methyl a t T 6.7;  proton m u l t i p l e t  i n s t a n c e the spectrum 3  molecular  elemental a n a l y s i s  with X  a C-methyl r e g i o n (T 8.95  C-methyls; a v i n y l i c m e t h y l  x 3.0.  and  The  (IR) spectrum w i t h a s t r o n g band at  (UV) spectrum  d o u b l e t s a t T 9.5  cyclopropane r i n g ;  triplet  - 193°C.  crystallized  a u u , p — u n s a t u r a t e d — 7 — l a c t o n e , the same chromophore found i n  abieslactone.  in  color.  t h i s m i x t u r e were o b t a i n e d from F r a c t i o n s N and 0 i n the manner  described  at  flush  removed most o f the  i n i t i a l l y coded as A.G^ and  The  A quick  the low  field  olefinic  Thus F i g u r e 5 shows the r e g i o n T 4 -  added to mark x 10.0.  F i g u r e 5a i s the  10  low  21  N> M  Figure  5a.  NMR  spectrum  of  cyclograndisolide  (x  2 -  9  region).  23  f i e l d . r e g i o n of cyclograndisolide i n CDCl^ r e l a t i v e to TMS.  The cyclopropane  methylene absorbs at T 9.50 and 9.68 ( J = 4 Hz); C-methyl groups absorb at T 8.98 - 9.14; the v i n y l i c methyl at T 8.10 i s an apparent t r i p l e t ; the one proton t r i p l e t at T 7.20 ( J = 1.8 Hz) i s assigned as an equatorial hydrogen geminal to the a x i a l methyl ether at T 6.72.  The one proton multiplet at  x 5.05 was assigned to the proton which i s geminal to the ring oxygen of the lactone and the apparent t r i p l e t at T 3.02 was assigned to the o l e f i n i c proton on the lactone ring.  The o p t i c a l rotatory dispersion (ORD) curve  (Figure 6) of cyclograndisolide (in dioxane) had a trough at 225 my C E ^]225  =  ~26,700°).  The c i r c u l a r dichroism (CD) curve (Figure 7) had a  weak p o s i t i v e peak, [01250  =  +370°, and a strong negative value with no  minimum observed above 215-mp ( [ Q ^ - ^  0-  »  220  260 -J  L  =  300  -44,000°).  340  J  I  o X  I—I  -e-  -2 -  -3H  Figure 6-  ORD curve of cyclograndisolide.  These measurements  380  i  L  X(mp)  24  are of the same sign and of s i m i l a r magnitude to those obtained f o r abieslactone  2 8  and would suggest the R. configuration about the lactone.  A(mp)  Figure 7 .  CD curve of cyclograndisolide.  C  ' The p o s i t i o n of the cyclopropane ring i n the above substance could not  be determined from the spectral evidence but the known triterpenes  35  and the cyclopropanoid  Buxus a l k a l o i d s  3 8  cyclopropanoid are a l l based on  cycloartenol (32), providing some suggestions. The  chemistry of cycloartenol has been w e l l s t u d i e d . ' 3 7  3 8  I t was  known that treatment of cycloartanyl acetate with gaseous hydrogen chloride  25 21  31  ^  20  22  30  32 in chloroform resulted i n a mixture of o l e f i n s .  The major isomer was the  9(11)-ene with minor amounts of the 7-ene and 8-ene isomers.  This  reaction was extremely convenient f o r our purposes since opening of the cyclopropane i n cyclograndisolide should give abieslactone (30) as the  However, when gaseous HC1 was bubbled into a chloroform solution of cyclograndisolide, the product i s o l a t e d was not the expected abieslactone. In the NMR spectrum of abieslactone, the o l e f i n i c proton absorbs at T 4.48 while the o l e f i n i c proton of the new reaction product, i n i t i a l l y  called  iso-AG^ and subsequently called grandisolide, absorbed at x 4.80 (Figure 8). This l a t t e r value was i n much better agreement with the r e p o r t e d  39  value  of x 4.75 f o r the C ( l l ) o l e f i n i c proton of ester (33), a result which seemed at variance with that reported f o r abieslactone.  In order to obtain  further evidence on the expected chemical s h i f t f o r the o l e f i n i c proton i n a 9(ll)-ene system, I examined dihydroparkeyl acetate (34) and observed a value of x 4.81. The other i n t r i g u i n g aspect of the NMR spectrum of grandisolide was the C-methyl group region which exhibited a series cf signals i n the  27  OOMe  AcO  AcO  33  34  range T 8.95 - 9.36. The C-methyl group region of abieslactone covered the range x 8.90 - 9.08.  This data provided a strong i n d i c a t i o n that  although abieslactone may indeed be a member of the t e t r a c y c l i c lanostane  suggested i n the structure  30  2  8  may be open to question.  For this reason  I w i l l discuss i n some d e t a i l the chemistry of abieslactone i n a l a t e r section of this thesis while at this time i t i s convenient to present the remaining data which provides  the completion of the structure of  cyclograndisolide. Considerable  systematic  data * * ' * 39  1  0  1  1  , l + 2  has been c o l l e c t e d on the  p o s i t i o n of NMR signals due to methyl groups of triterpenes and complete assignments of these signals have become possible for c e r t a i n triterpene families. The facts available to this point suggested that cyclograndisolide was a member of the cycloartane family.  Hence grandisolide would be a  member of the lanostane family (35) for which a considerable body of MR data was a v a i l a b l e .  In p a r t i c u l a r the r e l a t i v e positions of the methyl  28  21  22  -'2 4 '•  26  35  groups had been extensively studied and the pertinent values for the lano.stane family are found i n Table I I . Table I I I contains the changes in the chemical shifts (AT)- of the methyl group resonances produced by ^,  _  jr  x-  -  i  i-  _ -1„  -i  .  ~i  Compound  lanostane lanostan-3a-ol lanostan-3a-acetate lanostan-33-ol lanostan-313-acetate lanos tan-3-one II.  3Q  Methyl Group 30  31  9.17 9.13 9.09 9.20 9.13 8.94  9.17 9.07 9.17 9.02 9.16 8.94  18  19  9.22 9.08 9.07 9.23 • 9.23 9.09 9.20 9.09 9.08 9.23 9.22 8.94  32 9.20 9.19 9.17 9.20 9.21 9.22  Position of methyl groups in T values (±0.03 x).  Function  Methyl Group 30  8-ene 9-ene 8-ene-7,11-diketo Table I I I .  •  -0.01 -0.02 -0.08  31 -0.04 -0.05 -0.05  19 -0.13 -0.19 -0.44  18  32  +0.08 +0.11 -0.00  -0.07 +0.05 -0.34  Contribution of functional groups to the chemical shift change (Ax) of methyl groups.  29  Tables II and III in combination with each other may serve for the calculation of chemical shift values for other compounds i n this series whether or not they are listed.  34  As 3n example, the calculated values of  '  36  parkeyl acetate (36) and dihydroparkeyl acetate (34) are given with the observed  v a l u e s *  iii T&oxe  iv ,  Methyl Group  parkeyl acetate calculated observed dihydroparkeyl acetate calculated observed Table IV.  30  31  19  18  32  9.11 9.14  9.11 9.14  8.90 8.94  9.34 9.38  9.26 9.28  9.11 9.13  9.11 9.13  8.90 8.95  9.34 9.36  9.26 9.25  Observed and calculated chemical shifts of methyl group resonances in typical lanost-9(11)-enes.  It is noted that the observed and calculated values are i n good agreement.  Attention is directed to the calculated and observed values  for the 18, 19, and 32 methyl groups.  The 19 methyl group is both  observed and calculated to be in the region T 8.90 to T 8.95, an expected result since this methyl group is a l l y l i e to the 9(11)-ene system.  30  The 32 methyl group has a range .x 9.25 - 9.28, while the 18 methyl group is consistently at the highest f i e l d with a range of x 9.34 to 9.36. Hence a typical lanost-9(11)-erie derivative should have the 19 methyl group resonating near x 8.95 with the 18 and 32 methyl groups near x 9.35 and x 9.25 respectively. If one makes the assumption that grandisolide is a member of the lanostane series, the most likely position for the methoxyl function would be C(3) while the a, 0-unsaturated--y-lactone would foriu part of the side chain.  An examination of Table II shows the only functionality at C(3)  which significantly affects the resonance frequencies of the 18, 19, and 32 methyl groups i s the 3-ketone.  Hence i f the study i s focussed on these  three methyl groups in compounds with functionality at C(3) other than a kstor.s, the valuas p r c s s n t G d in Tablo II should, be applicable* Methyl Groups  lanost-9(11)-en-3a-ol calculated grandisolide observed  30  31  19  18  32  9.11  9.02  8.89  9.34  9.24  9.12  9.02  8.95  9.36  9.29  Table V . Resonance frequencies of methyl groups of lanost-9(11)-enes The effect of the lactone ring on the resonance frequencies of these methyl groups is unknown but i t would be surprising i f i t was appreciable. A comparison of the calculated values for lanost-9 (11)- en-3a-ol (37) with the observed data for grandisolide i s made i n Table V, and indeed the similarities are striking.  This result provided further evidence in  support of the general structural features present in grandisolide and,  31  MeO '  in turn, i n cyclograndisolide. At this stage i t appeared desirable to e s t a b l i s h the r e l a t i o n s h i p , i f any, between grandisolide and the only known t e t r a c y c l i c triterpene which contained  an unsaturated y-lactone  i n the side chain, abieslactone (30).  Indeed when abieslactone was treated with gaseous HC1 i n the manner already described spects  f o r cyclograndisolide, a reaction product i d e n t i c a l i n a l l r e -  (m.p., mixed m.p., IR, and TLC) with, grandisolide was obtained.  This result established the suspected relationship between the two series and strengthened the argument that grandisolide possesses a t e t r a c y c l i c system c h a r a c t e r i s t i c of the lanostane family.  On this basis, the most  l i k e l y structure for grandisolide would be 30.  In turn,  cyclograndisolide  could be either 38 or 39 since both cyclopropane rings could lead to the 9(11)-ene shown i n 30.  The alternative 38 was somewhat more favored  since  the cycloartenol series discussed previously was already w e l l established.'  MeO' 38  39  32  The quantities of cyclograndisolide were so small that further chemical investigations were net possible at this time. decided that an extensive mass spectrometric evidence i n support of 38 or 39.  study may  I t was  therefore  reveal further  For this purpose the known cyclopropane  system present i n the cycloartenol family was  f i r s t investigated.  The  mass spectrum of cycloartenol and some of i t s derivatives have been determined by several workers. ' ^ 1+3  The presence of the 9,19  cyclopropane  ring i s manifested i n the mass spectra by the appearance of an ion peak having an even mass number.  The p o s i t i o n of the peak i s unaffected by  s u b s t i t u t i o n pattern at C(4)  or by the oxygen function at C(3).  however, s h i f t e d by varying  It i s ,  the s u b s t i t u t i o n i n the side chain.  proposals for the o r i g i n of this fragment have been advanced. envisagf-S .1 ns.s o f ring A including the C(19)  carbon (nath i ) . ^  Two One  3  envisages loss of ring A with i n c l u s i o n of C(6) but not with the carbon (path i i ) . ^  4  Without proper l a b e l l i n g studies these two  cannot be distinguished.  proposal  The  other  C(19) paths  Regardless of the path followed, the r e s u l t i n g  ion fragment for cycloartenol or cycloartenyl acetate has a value of m/e  the  286.  \ i.  33  In order to compare our subsequent results under closely i d e n t i c a l conditions, the spectrum of cycloartenyl acetate was run on our instrument and i t i s reproduced i n Figure 9.  The observed fragmentation pattern  agrees well with the published data.  A comparison of cycloartenyl acetate  and cyclograndisolide (Figure 10) i s now i n order. Cycloartenyl acetate (Figures 9 and 9a) exhibits a molecular ion at m/e 468.  Loss of methyl from the molecular ion gives a M-15 i o n at  m/e 453 and a metastable at m/e 438.1.  Acetic acid i s also l o s t from the  molecular ion to give i o n a (m/e 408) and a metastable at m/e 356.2. Ion b at m/e 393 arises by loss of a c e t i c acid from the M-15 i o n and by loss of methyl from ion a as evidenced by metastables at m/e 341.1 and m/e 378.8. Ion a loses C^Hy* to give ion c at m/e 367 and a metastable at m/e  m/e 281.8. An ion e at m/e 286 corresponds to loss of ring A from the molecular ion.  Although another group of workers reported the observation of a  metastable ion from a s i m i l a r fragmentation of cycloartenol, no metastable t r a n s i t i o n was observed i n this instance.  The ion e can also lose methyl  to give ion f at m/e 271 with a metastable at m/e 256.9.  A metastable  ion at m/e 164.8 corresponds to loss of C^Hg• from i o n e to give an i o n g at m/e 217. The entire side chain could cleave with a loss of 111 mass units from ion f to give ion h at m/e 175.  Ion a could cleave through  ring C to give ions h and i at m/e 203 and m/e 205; metastable ions at 101.0 and m/e 102.9 may arise by this process.  The intense ion at  m/e 69 l i k e l y a r i s e s , i n part, from fragmentation of the side chain.  34  00 rH  LO-  CD  sr  o oo oI -<f  CD . CD  sr  rQ O N -  r--  O i D "  ro  CD o-v  x l roro  CD r d — CD CH Q)  00 .  CM  rs  CD  LU  .in  CD  o •rH  ro O rM  CD  rH  4-1  u rd O  rH  CD  -in  o  >1 o  < 4 H  o 0 p 4J  o  .CD  ON .  O (U  Q<  trfln CO  a)  rM  00 J  i S/_  Ob U I S N 3 1 N I  3AIldTJa  -CD  in  60 •rH  35  -CH, M-15 m/e 453 AcO _ l M m/e 468 -HOAc  -HOAc  V -CH,  ion a m/e 408  ion b m/e 393  "37 C  H  ion d m/e 339  ion c m/e 367  ion f m/e 271  " 11 18°2 C  H  ion e m/e 286  C  5 9 s. —?>  C  8 15  H  i  § m/e 217  AcO e  or e  H  ion h m/e 175 f8 15 H  ion a ion j m/e 20.5 ion i m/e 203 Figure 9a. Mass s p e c t r a l fragmentation  of cycloartenyl acetate.  o  n  36  Ion  Cycloartenyl  acetate  Cyclograndisolide  E l e m e n t a l Compi  M  468  468  C  31 44°3  M-15  453*  453*  C  30 45°3  a  408*  436*  C  30 44°2  b  393*  421*  C  29 41°2  c  365*  393*  C  27 37°2  d  339*  367  C  25 35°2  e  286  314  C  31 30°2  f  271*  299  C  20 27°2  h  175  175  j  205*  233  C  15 21°2  H  H  H  H  H  H  H  H  H  met as t a b l e i o n o b s e r v e d f o r p r o c e s s d e s c r i b e d i n t e x t -.,„, 1-.-: „ u .-. ~ it--: ,;n mass s p e c t r o m e t r y c Table V I .  Mass s p e c t r a l c o m p a r i s o n o f c y c l o a r t e n y l cyclograndisolide.  When t h e d i f f e r e n c e  a c e t a t e and  i n f u n c t i o n a l i t y a t C(3) a n d i n t h e s i d e  is  t a k e n i n t o a c c o u n t , t h e mass s p e c t r u m o f c y c l o g r a n d i s o l i d e  is  remarkably s i m i l a r t o c y c l o a r t e n y l acetate  in Table,VI. ( C H . 0 ). 0 1  31  HO  (Figure  The m o l e c u l a r i o n o f c y c l o g r a n d i s o l i d e T h e M-15  i o n f o r loss  (Figure  10)  as can be seen  i s a t m/e 468  o f m e t h y l i s a t m/e 453 w i t h  a  J  m e t a s t a b l e a t m/e  438.1.  c o m p o s i t i o n C^QH^^^. lose methyl to give  Loss of methanol gives  i o n a (m/e 436) o f  As .was s e e n i n c y c l o a r t e n y l  i o n b (m/e 421) o r l o s e  acetate,  £3^7' to give  both fragmentations being supported by metastable ions m/e 354.2 r e s p e c t i v e l y . is  9)  chain  s e e n a t m/e  Ion d corresponding  367 (^25^35^2^ *  this  i o n may  i o n c (m/e 393)  a t m/e 406.4 a n d  t o l o s s o f C^H • f r o m i o n b Q  M-32 436 b 421  c 393  M-15 453  314 f 299  300  F i g u r e 10.  Mass spectrum of  cyclograndisolide.  d 367  350  400  *i—r  450  M 468  ~i—i—i—r  38  Ion e at m/e 314 (C2.l 30°2^ ^ H  f at m/e 299  I^Q^^C^)  s  °b  s e r v e a  along with the s a t e l l i t e ions  a r i s i n g by loss of methyl and i o n h at m/e 175  arising' by loss of the e n t i r e side chain. A fragment corresponding to ion j at m/e 233 i s observed with composition Ci5 21°2*' ^ H  n e v e n  i o n , k,. at m/e 272 with composition  m a s s  ^18^24^2 "*" °^ unknown o r i g i n . s  The s t r i k i n g s i m i l a r i t i e s of the mass spectra of c y c l o a r t e n y l acetate and c y c l o g r a n d i s o l i d e coupled w i t h the other chemical and s p e c t r a l evidence presents a convincing argument f o r the presence of a 9,19 cyclopropane system.  On the b a s i s of a l l the evidence presented the s t r u c t u r e 38 can  be assigned f o r c y c l o g r a n d i s o l i d e . H  38  Since rather heavy reliance  on spectral data was necessary i n  deducing this structure, i t was decided to submit a derivative molecule for X-ray  of this  analysis.  To this end cyclograndisolide was reduced under mild conditions to give dihydrocyclograndisolide (40) whose NMR spectrum s t i l l contained the  39  MeO-  40  resonances f o r the cyclopropane protons.  The mass s p e c t r a l fragmentation  of dihydrocyclograndisolide was l i k e that of cyclograndisolide with ions containing the lactone ring now being observed at two mass units higher than i n the cyclograndisolide. Dihydrocyclograndisolide could be reduced with lithium aluminum  4 1  the cyclopropane ring was s t i l l i n t a c t and i t was clear that the d i o l s t i l l contained a l l the asymmetric centers of the o r i g i n a l natural product. Treatment  of the d i o l with p-bromobenzoyl chloride i n pyridine gave  the bis-p-bromobenzoate (42).  The NMR spectrum of this derivative contained  the resonances f o r the cyclopropane ring and f o r eight aromatic protons.  40  MeO' "  42 In addition, elemental analysis and molecular ions at m/e 842  838,  840,  and  F. H. A l l e n of this department performed an X-ray analysis' >  on  confirmed that the bis derivative had been obtained. 15  75  space group P2^2-^2^, with a. = 6.635, b_ = 20.919, c_ = 30.530 A, and units of C^fjHkQOcjB^  P  e r  unit c e l l .  The  four  i n t e n s i t i e s of the 2528 independ-  ent r e f l e c t i o n s with 265100° were measured on a Datex-automated GE XRD6 d i f f T a c t o m e t e r using N i - f i l t e r e d CuKa r a d i a t i o n . using Patterson  The  the X-ray fluorescence  the configuration at C(23)  has  of 0.096.  The  absolute  technique.  as shown i n structure 42.  depicted for 42 i s also correct.  the structure and absolute stereochemistry  configuration  solved leastwas  7G  analysis confirms the presence of the 9,19  stereochemistry  structure was  and Fourier techniques and refined by block-diagonal  squares methods to an R-factor determined by  The  cyclopropane ring and The  absolute  Hence, cyclograndisolide  as shown i n structure  and i s (23R)-3a-methoxy-9 ,19-cyclo-9f3-lanost-24-ene-26 ,23-lactone.  38  41  MeO  With the structure of cyclograndioslide established, grandisolide i s (23R)-3a-methoxylanosta-9(11),24-diene-26,23-lactone  (30) as suggested e a r l i e r .  As mentioned i n the early portion of the present discussion, a second component containing a lactonic absorption had been i s o l a t e d from Fraction M.  This material, i n i t i a l l y code named AG  2  and subsequently called e p i -  cyclograndisolide, was p u r i f i e d by preparative layer chromatography. n  J- _ i -.  —  U X J C t - U X X X l ^ a i  — L  xr U  U  X  I.  „ ^ i - i  T  UfO  X  g(XV»-  _ .  -.„-!-:.?  CX  ^>V^J.J-VX  WX.X-WV—  „ ,  111 . £S •  ino _  i n /. ° n  J  J _ ^ - T  ' ^1  »  Elemental analysis and high resolution mass spectrometry gave a molecular formula of ^ 3 ^ - 4 3 ^ 3 '  ^  e  ^  a n <  ^ ^  spectral properties were s i m i l a r to  those of cyclograndisolide and suggested an a,(3-unsaturated-Y lactone. _  The NMR spectrum  (100 MHz, Figures 11 and 11a) obtained i n the manner  used f o r cyclograndisolide exhibited, among other s i g n a l s , cyclopropane protons at T 9.50 and 9 . 6 8 (J = 4 Hz); the v i n y l i c methyl at T 8.12 as an apparent t r i p l e t (J = 1.7 Hz); the proton at x 7.20 (J = 1.8 Hz) f o r an equatorial proton geminal to an 0-methyl (x 6.72); a one proton multiplet at x 5.10 f o r a proton geminal to the lactone ring oxygen; and a one proton t r i p l e t at x 2 . 9 8 f o r the o l e f i n i c proton of the lactone ring.  The  differences of note from cyclograndisolide are the downfield s h i f t of the o l e f i n i c proton of the lactone, the u p f i e l d s h i f t of the proton geminal to  Figure 11.  NMR  spectrum of epi-cyclograndisolide (T 4 - 10 region).  I  T  6  Figure 11a.  NMR  spectrum of epi-cyclograndisolide ( T 2 - 9 region)  44  the lactonic oxygen, and a s l i g h t u p f i e l d s h i f t of the v i n y l i c methyl The ORD  curve (Figure 12) was  with a peak at [*]225 ([6J25Q  =  =  group.  of opposite sign to that of cyclograndisolide  22,640°.  The CD curve (Figure 13) had a shoulder  1708°) with no maximum being observed above 220 mp  ([6] = 39,140°)  Ji  o  1  220 Figure 12.  ORD  260  300  340  1  P  420  380  curve of epi-cyclograndisolide,  4H  i  o  A(mp)  X(mp)  45  The p o s i t i v e nature of both these measurements suggests that the configuration about the lactone, namely C(23), i s opposite isolide.  to that of cyclogrand-  In other words, the S configuration i s tentatively assigned as  shown i n structure 43.  H.  MeO''  43  further i l l u s t r a t e d when i t was seen that the mass spectrum of e p i cyclograndisolide (Figure 14) was nearly i d e n t i c a l to cyclograndisolide (Figure IQ). In f a c t , the fragmentation pattern substances  is. the same.  (Figure 14a) of both  The molecular ion i s seen at m/e 468.  As i n  cyclograndisolide the M-15 ion f o r loss of methyl i s at m/e 453 and ion a for loss of methanol i s at m/e 436.  Ion a may.lose methyl to give ion b  (m/e 421) or the M-15 ion may.lose methanol to give the same ion.  Ion a  may lose C-Jl^' to give i o n c (m/e 393) or C^Hg• to give ion d (m/e 367). Ion e (m/e 314) corresponds to loss of ring A from the molecular ion. Loss of methyl from i o n e results i n i o n f (m/e 299) and loss of the entire side chain gives ion h (m/e 175). observed at m/e 233. origin.  A fragment corresponding to ion j i s  As i n cyclograndisolide i o n k (m/e 272) i s of unknown  46 00 m cn  CD  m  i o-  i—i  sr  CM KO co cn I -3-  ^3 CM  cn  cn  o  .ID  m  CM CM CM  O CO •A  TJ d cd r-l  o  .CD  60  O rH  o O  I •A  P-,  cu CD  .1X1  o 0  3 •  M  4J  o  CD .CD  OJ &. • CO CO to cd  rH  0) M  - CD  001  ID  A1I9N3J.NI  3AIia"13a  60 •A  47 ion f m/e 299  ion h m /e 175-<  M-15 m/e 453 ion c m/e 39 3  -CH OH 3  V ion d m/e 36 7  Figure 14a.  ion a m/e 436  Fragmentation pattern  ion b m/e 421  of"epi-cyclograndisolide.  Because of the limited amount of epi-cyclograndisolide a v a i l a b l e , no chemical work was p r a c t i c a l at this time.  On the basis of the s p e c t r a l  evidence obtained f o r epi-cyclograndisolide, the structure suggested i s (23S)-3a-methoxy-9,19-cyclo-9e-lanost-24-ene-26,23-lactone (43).  H  MeO' 43  48  Experimental Throughout this work Merck s i l i c a gel G with added fluorescent indicator was used as adsorbent  i n thin layer chromatography (TLC).  The chromatograms,  0.3 mm. i n thickness, were a i r dried and activated i n an oven at 100°C f o r three hours.  The chromatograms were developed  i n chloroform and sprayed  with antimony pentachloride i n carbon tetrachloride (1:2) unless  otherwise  noted. For preparative layer chromatography a thicker layer (0.5 mm.) of adsorbent was u t i l i z e d , with 0.01% Rhodamine 6G added as indicator.  3Lf  Spraying with antimony pentachloride was done only along one edge or not at a l l as detection of bands was possible with u l t r a v i o l e t  .light: .In most  instances. Column chromatography was performed on either Woelm s i l i c a gel or neutral alumina. III)  The preferred adsorbent was deactivated alumina ( A c t i v i t y  prepared by the addition of water as directed by the manufacturers.  Except i n large scale work, the solvents were d i s t i l l e d before use. The nuclear magnetic resonance (NMR) spectra were measured i n chloroform or deuterochloroform  at room temperature.  The NMR spectra were  obtained at either 60 MHz using a Jelco C-60, Varian A-60, or a Varian T-60 instrument  or at 100 MHz using a Varian HA-100 instrument.  The positions  of a l l NMR resonances are given i n the Tiers x scale with tetramethylsilane as i n t e r n a l standard set at 10.0 units.  For multiplets the x values given  49  represent the center of the s i g n a l . Mass spectra were measured on an Associated E l e c t r i c a l Industries MS 9 high resolution mass spectrometer or, where noted, on an Atlas CH 4 spectrometer.  High resolution molecular weight determinations were deter-  mined on the MS 9 spectrometer. Infrared (IR) spectra were measured on Perkin Elmer model 21, 137, or 457 instrument.  The samples were usually measured as KBr  however, some were measured i n chloroform or neat. absorption maxima are quoted i n wave numbers Ultraviolet  pellets,  The positions of  (cm ). -1  (UV) absorptions were measured i n methanol or ethanol on  a Cary model 11 or model 15 spectrophotometer. •A Jasco model UV/ORD/Cp 5 spectropolarimeter was used to measure the c i r c u l a r dichroism  (CD)  and  optical rotatory dispersion  (ORD)  curves u s i n g  methanol or dioxane as solvent. Melting points were determined on a Kofler block and are uncorrected. Elemental analyses were performed by Mr. P. Borda, University of British  Columbia.  Extraction of grand f i r b a r k  3 2  Bark was obtained from a 100 year old grand f i r tree growing on the campus of the University of B r i t i s h Columbia.  The bark was a i r dried and  ground i n a Wiley m i l l to pass through a 3 mm.  sieve.  The ground bark was  extracted for 24 hours i n a large glass Soxhlet extractor. was  The extract  taken to dryness to provide a crude extract i n a y i e l d of 0.8% based  on the a i r dried weight of bark extracted.  50  Chromatography of crude e x t r a c t  3 2  The crude extract (26.7 gms.) was chromatographed on alumina (450 gms.) into a number of fractions as shown below.  The compounds  subsequently  i s o l a t e d from each f r a c t i o n are shown f o r c l a r i t y . Fraction  Solvent (volume, mis.)  Weight, mgs.  Compounds  A  petroleum ether (200)  690  hydrocarbons  B  petroleum  ether (100)  22 8  hydrocarbons, s t e r o l ester  C  petroleum  ether (200)  263  s t e r o l ester  D  petroleum  ether (200)  450  s t e r o l and wax ester  E  petroleum  ether (500)  994  wax ester, 2 unknowns  F  10% benzene i n pet. ether (500) 20% benzene i n pet. ether (400)  826  unidentified  307 ij.9-.-.ue^e l " e^her (400) 50% benzene i n pet. ether (300)  l :': :  60% benzene i n pet. ether (400)  909  f a t t y alcohol, 8-sitosterol  60% benzene i n pet. ether (300)  1016  8-sitosterol, f e r u l i c ester  benzene (400)  /  /  5004  T o t a l recovery  ~ 1 „,.1-. ~ 1  epimanool, lactones  •- 1925  ether (500) methanol (500)  £ „ *. « - „  8-sitosterol, ferulic ester, 2 unknowns f e r u l i c ester  13.75 gms.*  * r e s i n and fatty acids present i n crude extract were i r r e v e r s i b l y adsorbed on the alumina  51  Chromatography of Fraction G Fraction G (2.77 gins.) was chromatographed on alumina (200 gms.). E l u t i o n with 20% benzene i n petroleum ether (400 mis.) gave Fraction M (770 mgs.) subsequently shown to contain epimanool, fatty alcohol, and lactones.  Further elution with 50% benzene i n petroleum ether (400 mis.)  gave Fraction N (860 mgs.) subsequently shown to contain fatty alcohol. (950 mgs.)  lactones  and  F i n a l l y e l u t i o n with benzene (600 mis.). gave Fraction 0  containing  fatty alcohol and lactones.  Chromatography of Fraction M Fraction M (500 mgs.) was chromatographed on alumina (50 gms.). E l u t i o n with 20% benzene i n petroleum ether (250 mis.) gave Fraction P (80 mgs.).  Further elution with 40% benzene i n petroleum ether (500 mis.)  Fraction P Fraction P was a pale yellow o i l .  IR (neat) .3300 (OH), 3065 ( v i n y l ) ,  1635 (C=C), 1410, 990, 915 ( v i n y l ) , 878 (terminal methylene), 1383 and 1365 ( g e m i n a r d i m e t h y l ) .  NMR  (60 MHz) 4.15 (IH, quartet, J = 17.5 Hz,  J = 10.5 Hz), 4.88 (IH, quartet, J = 17.5 Hz, J = 1.5 Hz), 5.05 (IH, quartet, J = 10.5 Hz, J = 1.5 Hz), 5.26 and 5.55 methylene), 8.79  (2H, m u l t i p l e t s , exocyclic  (CH-j-C-OH), and 9.17, 9.23, and 9.36  (angular methyl).  Fraction P (80 mgs.) was treated with freshly prepared benzoyl chloride. (80 mgs.) i n pyridine temperature.  3,5-dinitro-  (2 mis.) f o r three days at room  The pyridine was removed i n vacuo.  The residue was  dissolved  in methylene chloride, washed with water, dried over sodium s u l f a t e , and evaporated.  C r y s t a l l i z a t i o n of this residue  from methylene chloride -  52  methanol gave needles m.p.  116 - 118°C, mixed m.p. with authentic epimanoyl-  3,5-dir.itrobenzoate 116 - 118°C. Fraction Q Fraction Q (400 mgs.) was obtained from Fraction M as a white low melting waxy s o l i d . R  f  TLC showed the presence of at least three compounds  0.35, 0.25, 0.17).  NMR  (60 MHz) had resonances at 3.0, 5.0, 6.7, and  8.1 s i m i l a r to a b i e s l a c t o n e  28  i n addition to broadened s i n g l e t at 8.7  assigned to methylene protons of a long chain fatty alcohol. Removal of fatty alcohol from Fraction Q A methanolic solution (4 mis.) of Fraction Q (200 mgs.) was heated to reflux and urea (2 gms.) was added followed by 1 ml. of benzene.  The  solution was allowed to cool slowly and was l e f t standing f o r 2 days before  with chloroform (2 mis.) and the combined f i l t r a t e was taken to dryness. The f i l t r a t e  residue was p a r t i t i o n e d between water and methylene chloride  and the organic layer separated, washed with water, and dried over sodium sulfate.  Evaporation of the solvent gave a white s o l i d (85 mgs.) which  contained by TLC two compounds, AG-^ and AG2 (R^ 0.35 and 0.25). The urea complex was dissolved i n water and extraction with methylene chloride gave, upon drying and evaporation, the fatty alcohol as a low melting wax  (R^ 0.17).  P u r i f i c a t i o n of Fraction N Fraction N (500 mgs.) was dissolved i n hot methanol and l e f t to cool for  four hours after which time a white solid,(120 mgs.) was removed by  filtration.  This s o l i d was found to be f a t t y alcohol and not further  53  examined. and  The f i l t r a t e was concentrated to 4 mis. and urea (2 gms.) added  the solution was warmed to r e f l u x to dissolve the urea; benzene (1 ml.)  was added and the solution l e f t to c r y s t a l l i z e f o r 2 days. urea complex by f i l t r a t i o n  Removal of the  and evaporation of the f i l t r a t e gave a residue  which was p a r t i t i o n e d between water and.methylene chloride.  The organic  layer was washed with water, dried over sodium s u l f a t e , and evaporated to give a residue  of AG-^ and AG  2  (250 mgs.) .  P u r i f i c a t i o n of Fraction 0 Fraction 0 (950 mgs.) was dissolved i n hot methanol and l e f t to cool for  four hours after which time fatty alcohol (505 mgs.) was removed by  filtration.  The f i l t r a t e was concentrated to 4 mis. and urea (2 gms.) was  added and dissolved at r e f l u x , benzene (1 ml.) was added. 4-v.o T.^-^«  „,™„-i„,r  v-"3 remove1  by  1  *"ion  After 2 days  and the f i l t r a t e evaporated tc  give a residue which was p a r t i t i o n e d between water and methylene chloride. The methylene chloride layer was washed with water, dried (sodium s u l f a t e ) , and evaporated to give a residue of AG^ and AG Preparative  layer chromatography of AG^ and AG  The mixture containing  AG^ and AG  2  (0.3 mis.) and applied to a preparative  (185 mgs.).  2  2  (85 mgs.) was dissolved i n chloroform layer chromatogram (20x60 cm.).  The  plate was developed i n chloroform and three bands were v i s i b l e when the chromatogram was examined under UV l i g h t . plate and extracted with chloroform. amount (2 mgs.) and was not examined.  The bands were scraped o f f the  The top band (R^ 0.6 7) was of small The second band (R^ 0.40) was the  major band (35 mgs.) and was one compound (AG^) when examined by TLC. The t h i r d band (R  r  0.25) overlapped the second band and contained both  54  AG^ and AG  (22 mgs.)-  2  Re-chromatography on preparative layer  as before gave pure AG-^ (3 mgs.) and pure AG  chromatograms  (15 mgs.).  2  AG^ or cyclograndisolide (38)' The AG-^ from the preparative layer chromatography was flushed through a short column of alumina with benzene to remove most of the orange color which came from the Rhodamine 6G dye. a white s o l i d m.p. 191 - 193°C. [<J>]  250  - 4,67.0° , [<}>]  225  dioxane) [ e ] [e]  2 1 5  2 7 0  ORD (c, 0.0368 i n dioxane) [<b]  - 26,700°, [ * ] o - 18,000°. 2 5 0  + 370°, [ 6 ]  0°, [ 6 ]  2 3 5  2 2 5  - 12,600°,  IR (KBr) 1745 (lactone carbonyl), 1665 (C=C).  Me0H 209 my (log e 4.33). max  A  ~ 890°,  30Q  CD (c, 0.0368 i n  2 2  + 120°, [ 6 ]  - 44,000°.  C r y s t a l l i z a t i o n from methanol gave  NMR (100 MHz) i n CDC1,, TMS lock o  UV  3.02 (IH,  .  apparent t r i p l e t , J = 1.7 Hz, H-C=C-C=0), 5.05 (IH, m u l t i p l e t , H-C-0), 6.72 (3H, s i n g l e t . OMe). 7.20 (IH. t r i p l e t , J = 1.8 Hz. equ.itnria.1 H-C-OMe), 8.10 (3H, apparent t r i p l e t , J = 1.7 Hz, v i n y l i c methyl), 8.98, 9.02, 9.07, 9.14 (5 C-methyls); in-CHCI3,CHC1  3  lock  3.02 unobservable,  5.05 - 9.14 region as before, 9.50 and 9.68 (2H, p a i r of doublets, J = 4 Hz, cyclopropane protons). 421 (M-47), and 314.  Mass spectrum  m/e 468 (M), 453 (M-15), 436 (M-32),  (Found: C, 79.43; H, 10.17; C H g 0 3 1  4  3  requires C,  79.44; H, 10.32%); high r e s o l u t i o n : 468 .365  C  31 48°3  436 .334  C  30 44°2  393 .278  C  2 7 37°2  339 .229  C  23 31°2  299 .204  C  20 27°2  233 .154  C  15 21°2  H  H  H  H  H  H  requires  468 .360,  453. 333  C  30 45°3  requires  436 .334,  421. 311  C  29 41°2  requires  39 3 .279 ,  367. 265  C  25 35°2  requires  339 .232,  314. 227  C  21 30°2  requires  299 .201,  272. 179  C  18 24°2  requires  233.154.  H  H  H  H  H  requires  453. 336,  requires  421. 311,  requires  367. 264,  requires  314. 224,  requires  272. 178,  55  AG^ or epi-cyclograndisolide (43) The AG  from preparative layer chromatography was flushed through a  2  short column of alumina with benzene to remove most of the orange color which came from the Rhodamine 6G dye. a white solid'm.p. 193 - 194°C.  U]  3 0 0  + 2,370°,  [<j,]  2 5 0  [6] 1660  2 5 0  + 1700°, [ 0 ]  lock 2.98 6.72  UV x  (C=C).  MPDH  2 7 0  22Q  NMR  '  5.10 - 9.14  "  -inram.uit-h  '  + 1424°, (carbonyl),  i n CDC1- TMS J  (IH, multiplet, H-C-0),  (IH, t r i p l e t , J = 1.7 Hz, e q u i t o r i a l ,  (3H, t r i p l e t , J =1.7  c-mpt-hvlsl •  "  2 5 5  IR (KBr) 1740 (100 MHz)  + 53°,  4 5 0  10,780°.  +  26  + 39,140°.  2 1 5  [<j>]  +  [ 6 ] o 1030°, [ 6 ]  (log e 4.15).  (3H, s i n g l e t , O-Me), 7.20  C5  + 22,640°, W  225  + 462°,  + 1850°, [ e ] 210 my  max  (c, 0.0434 i n dioxane)  (IH, apparent t r i p l e t , J = 1.7 Hz)', 5.10  H-C-OMe), 8.12 9.14  2 4 5  ORD  + 6,420°, W  CD (c, 0.0434 i n dioxane) [ 6 ]  C r y s t a l l i z a t i o n from methanol gave  j  region as before, 9.50  Hz, v i n y l i c methyl), 8.98, 9.02, mm  _ i^.-v ?  OH  ^ =..™-,™.->o a  „ „  n  i ,  5  n  9.07,  r „ n i , i «  '"2 -  and 9.68  '  (2H, p a i r of doublets,  J = 4 Hz, cyclopropane protons).  Mass spectrum m/e  468 (M), 453 (M-15),  436 (M-32) , 421 (M-47) and 314.  (Found C, 79.32 ; H, 10.20; C-j-jH^gO-j  requires C, 79.44; H, 10.32%; high resolution 468.363  21^H5°3  C  r e c  l  u : i  -  r e s  468.360). Grandisolide (30) Cyclograndisolide (33 mgs.)  was dissolved i n dry chloroform (5 mis.)  and hydrogen chloride, dried by passing throught concentrated sulphuric acid, was bubbled through for 2 hours.  Evaporation of the chloroform  gave grandisolide which was one spot on TLC.  C r y s t a l l i z a t i o n from  acetone gave a white s o l i d m.p.  IR (KBr) 1740  carbonyl), 1665  (C=C).  NMR  212 - 214°C.  (CDClo, 100 MHz)  3.02  (lactone  (IH, apparent t r i p l e t ,  56  J = 1.7 Hz, H-C=C-C=0), 4.80 H-C-O) , 6.72  (3H, s i n g l e t , OMe) , 7.20  H-C-OMe), 8.10 9.12,  9.29,  (IH, m u l t i p l e t , R-C=C), 5.05  (IH, m u l t i p l e t ,  (III, t r i p l e t , J = 1.7 Hz,  (3H, t r i p l e t , J = 1.7 Hz, v i n y l i c methyl),  9.36  (6 C-methyls).  (Found C, 79.34; H, 10.36; C  3 1  Mass spectrum (m/e)  H  4 8  0  8.95,  468,  453,  equitorial, 9.02,  436,  421.  requires C, 79.44; H, 10.32%).  3  Acid catalyzed isomerization of abieslactone Abieslactone  (35 mgs.)  dry hydrogen chloride was Evaporation m.p.  195 - 207°C.  H-C-O), 6.72  9.30,  1740  NMR  T  (100 MHz)  and 9.34  3.02  1  7.20  ,-.tTl~  ~7 Ur,  (18H,  C-methyls).  212 - 214°C, mixed m.p.  (lactone carbonyl)  Parkeyl acetate  1665  m  9.38  Q  Ql.  o  no  TLC,  Hz,  (IH, m u l t i p l e t ,  o  m  O  8.10  10  Q  1 /,  Repeated c r y s t a l l i z a t i o n s from acetone with grandisolide 212 - 214°C.  IR  (KBr)  (C=C) ; superimposable with IR of grandisolide.  (36) received from Professors D.H.R. Barton  (Imperial College, London) and G. Ourisson 4.80  one spot on  (IH, t r i p l e t , J = 1.7 Hz),  „).l,„1^  A sample of parkeyl acetate was  (100 MHz)  and  (IH, apparent t r i p l e t , J = 1.7  (non-integral, m u l t i p l e t , H-C=C), 5.05  —  (5 mis.)  bubbled through the solution for 2 hours.  (3H, s i n g l e t , OMe),  i-^^Ut-  gave m.p.  dissolved i n dry chloroform  of the chloroform gave a white s o l i d which was  H-C=C-C=0), 4.80  l"iU  was  - 5.00  (C.N.R.S., Strasbourg).  (2H, m u l t i p l e t s , o l e f i n i c protons), 8.94,  9.14,  NMR 9.28,  (6 C-methyls).  Dihydroparkeyl  acetate  Parkeyl acetate  (34)  (15 mgs.)  was  dissolved i n tetrahydrofuran  and  hydrogenated for 2 hours over 10% palladium on charcoal (20 mgs.). catalyst was  removed by f i l t r a t i o n and the f i l t r a t e evaporated and  The  57  c r y s t a l l i z e d from e t h y l acetate .m.p. 172°C * ). 1  NMR  9  (100 MHz)  (8 C-methyls).  (M-75).  4.81  173 - 174°C ' ( l i t e r a t u r e value 171 -  (1H, m u l t i p l e t , H-C=C) , 8.95,  Mass spectrum (m/e)  (Found C, 81.59; H,  470  11.53;-C  (M), 455  3 2  H  5 4  0  9.13,  (M-15), 410  9.25,  9.36  (M-60), 395  requires C, 81.64; H, 11.56%).  2  Cycloartenyl acetate An authentic sample of cycloartenyl acetate was (Forest Products Laboratory, Madison) m.p.  121 - 122°C ). 1+7  H-C=C), 5.51  117 - 119°C  (IH, quartet, J = 5 Hz and J = 10 Hz, H-C-OAc), 8.02 9.15,  9.19  (M), 453  (M-15), 408  Tjlhy u L'ucy'Clug  protons).  (M-60), 39 3 (M-65) and  catalyst was  455 C  31 50°3 H  NMR  (60 MHz)  5.50  (2H, p a i r of doublets, J = 4 Hz).  r e  <J  u i r e s  >  7 9  -  1 0  ; > H  IR  (KBr)  Mass spectrum (m/e)  470  (M),  (Found C, 79.00; H, 10.81;  10.71%).  Dihydrocyclograndisolide d i o l  (41)  Dihydrocyclograndisolide (30 mgs.) s t i r r e d with l i t h i u m aluminum hydride amount of water was  198 - 199°C.  (Hi, m u l t i p l e t , H-C-0), 9.50  (M-32), 423 (M-47), and 316. c  hydro-  removed by f i l t r a t i o n and the f i l t r a t e  (lactone carbonyl).  (M-15), 438  (10 mis.) was  at room temperature for  evaporated and c r y s t a l l i z e d from e t h y l acetate m.p.  and 9.68  (m/e)  286.  i n tetrahydrofuran  genated over 10% palladium on charcoal (50 mgs.)  1770  Mass spectrum  (40)  J." a i i d x S u i l u t :  Cyclograndisolide (50 mgs.)  The  (3H,  (5 C-methyls), 9.47 and 9.70 (2H,  pair, of doublets, J = 4 Hz, cyclopropane  2 hours.  ( l i t e r a t u r e value  (100 MHz) i n CHCI3, CHCI3 lock 4.97 (IH, multiplet,  NMR  s i n g l e t , 00CCH3), 9.05,  468  received from Dr. Rowe  i n tetrahydrofuran (10 mgs.)  added and the solvent was  (10 mis.)  for 18 hours. evaporated.  The  was  A small residue  58  was c a r e f u l l y a c i d i f i e d with d i l u t e hydrochloric acid and extracted with ether.  The extract was washed with water, dried (sodium s u l f a t e ) , and  evaporated  to give a white residue.  Column chromatography on alumina  (1 gm.) of the residue and elution with ether gave the d i o l , from petroleum  crystallization  ether - ether gave a white s o l i d m.p. 133 - 134°C.  IR (KBr)  3540, 3350 (OH). NMR (60 MHz) 6.2 - 6.6 (3H, overlapping m u l t i p l e t s , H-C-OH, CH -0H) 8.97, 9.03, 9.06, 9.10 (6 C-methyls) 9.50 and 9.68 (2H, 2  p a i r of doublets, J = 4 Hz, cyclopropane 474  protons).  Mass spectrum (m/e)  (M), 459 (M-15), 456 (M-18), 442 (M-32), 438 (M-36).  (Found C, 78.58;  H, 11.35; C ,H 0^ requires C, 78.43; H, 11.46%). 31 54 3 Bis-p-bromobenzoate of dihydrocyclograndisolide d i o l o  c/  Dihydrocyclograndisolide d i o l (41) (10 mgs.) and freshly  at room temperature for 2 days.  crystallized  The pyridine was removed i n vacuo and  the residue dissolved i n methylene chloride and washed with water, 5% aqueous sodium bicarbonate s o l u t i o n , water and dried over sodium s u l f a t e . Evaporation of the solvent and c r y s t a l l i z a t i o n from petroleum bis-p-bromobenzoate (42) as a white s o l i d m.p. 154 - 156°C. 1720  (ester carbonyl).  ether the IR (KBr)  NMR (60 MHz) 1.9 - 2.4 (8H, aromatic protons),  9.50 and 9.68 (2H, p a i r of doublets, J = 4 Hz, cyclopropane  protons).  Mass spectrum (m/e) 838, 840, 842 (M). (Found C, 64.10; H, 7.32; C,,H, 0,-Br requires C, 64.-29; H, 7.17%). n  o  59  Investigations concerning the structure of abieslactone Discussion  Abieslactone i s a triterpene occurring i n the bark and leaves of Abies m a r i e s i i Masters, a f i r tree of northern Japan, and also i n the North American P a c i f i c s i l v e r f i r [A. amabilis (Dougl.) Forbes] Noble f i r [A. procera Rehd.]. investigators  2 8  and  The arguments presented by the o r i g i n a l  f o r the structure of abieslactone as 30 are summarized  in this section.  For purposes of c l a r i t y i n the present discussion, the  published structure f o r abieslactone and i t s derivatives w i l l be u t i l i z e d i n i t i a l l y while alternative structures are postulated subsequently wherever possible.  MeO-  The molecular formula for abieslactone (30) established by analysis and mass spectrometry was  C^i^^gOy  ^  t n e  t  n  r  e  e  elemental  oxygen atom  present, one could be assigned to a methyl ether group from a Z e i z e l determination and a three proton s i n g l e t at x 6.73 The presence of an a,$-unsaturated-Y-lactone absorption at 1745  and 1660  cm  -1  was  i n the NMR  spectrum.  indicated by the IR  and the UV absorption maximum at  60  207.5 mu  (log e 4.30  The NMR (T 8.98  i n EtOH).  spectrum e x h i b i t e d s i g n a l s a t t r i b u t e d to s i x C-methyl groups  - 9.08), a v i n y l i c methyl as a t r i p l e t at x 8.10,  at x 6.73, at x 5.05  a narrow d i f f u s e d t r i p l e t at x 7.20, and x 4.48,  x 3.00  Spin decoupling  coupled to both the o l e f i n i c proton at  and the m u l t i p l e t at x 5.05.  The m u l t i p l e t at x 4.48 was  with protons i n the methylene envelope (x 7.61 d i f f u s e d t r i p l e t was  two one proton m u l t i p l e t s  and a one proton quintet at x 3.00.  showed that the v i n y l i c methyl was  a methyl ether  and 7.81).  coupled  The narrow  assigned as an e q u a t o r i a l proton geminal to the methyl  ether. M i l d hydrogenation  of abieslactone i n tetrahydrofuran with Pd/C  l y s t afforded dihydroabieslactone (44).  carbonyl at 1770 x 3.00,  5.03,  cm . 1  and 8.10  The NMR  The IR spectrum revealed a lactone  spectrum was  a l t e r e d i n that s i g n a l s at  had disappeared while a new  x 5.53  and a new  x 4.48  s i g n a l remained unchanged.  cata-  three proton doublet at x 8.73 now  one proton, m u l t i p l e t at appeared.  Prolonged hydrogenation  The  over Adams  c a t a l y s t i n a c e t i c a c i d - e t h y l acetate r e s u l t e d i n tetrahydroabieslactone (45) w i t h loss of the x 4.48  signal.  61  Oxidation of abieslactone with potassium permanganate a c e t i c a c i d afforded a trisnor-hydroxy acid (46) as a r e s u l t of cleavage of the unsaturated  lactone r i n g .  The c o n f i g u r a t i o n at C(23) i n the t r i s n o r -  hydroxy a c i d , and hence i n abieslactone, was  e s t a b l i s h e d as R by measure-  ment of the c i r c u l a r dichroism curves f o r the trisnor-hydroxy a c i d .  H  angelicalactone ( 4 7 ) T 3.00,  5.03,  7  and 8.10  , a keto a c i d (48).  The NMR  spectrum now  resonances but retained the x 4.48  a c i d function was e s t e r i f i e d with diazomethane; the NMR ed the x 4.48 ester.  resonance plus a new  lacked the  resonance.  The  spectrum s t i l l r e t a i n -  three proton s i n g l e t at x 6.30  f o r a methyl  Treatment of the keto e s t e r 49 w i t h boron t r i f l u o r i d e etherate  and ethane d i t h i o l followed by d e s u l f u r i z a t i o n with Raney n i c k e l , gave deoxy e s t e r (51). at x 4.78.  The x 4.48  resonance was now s h i f t e d to a new p o s i t i o n  The r e s u l t i n g deoxy e s t e r was  hydride to give an a l c o h o l (52) which was  treated with l i t h i u m aluminum converted  to the t o s y l a t e (53).  Reduction of the t o s y l a t e with l i t h i u m aluminum hydride gave a compound (54) i n which the oxygen functions of the side chain of abieslactone  62  63  were completely removed. region x 8.9 3 to 9.35,  The NMR  spectrum showed 8 C-methyl groups i n the  one 0-methyl s i n g l e t at x 6.70,  the one proton  diffused t r i p l e t at x 7.20, and the o l e f i n i c proton at x  4.75.  Hydrogenation of 54 gave a dihydro derivative, 55, whose NMR was devoid of the o l e f i n i c proton s i g n a l .  spectrum  Demethylation of the dihydro  derivative was accomplished by hydrogen bromide i n b o i l i n g acetic anhydride - acetic acid.  The r e s u l t i n g compound, 56, was  o l but when 56 was It was now  d i f f e r e n t from lanostan-3$-  oxidized i t was i d e n t i c a l with lanostan-3-one (57).  clear that 56 was  the C(3) epimer, lanostan-3a-ol. In turn this  result also revealed that the various degradation products and abieslactone i t s e l f would possess the t e t r a c y c l i c lanostane skeleton. i s v a l i d only i f no major s k e l e t a l rearrangements  This assumption •  occur during the degra-  be made l a t e r . The position of the double bond was  the one•remaining uncertainty.  Abieslactone could be oxidized with chromium trioxide i n acetic acid to an ene-dione system (58).  The UV spectrum (^jjj!^*  274, log e 3.84)  was  64  the 8 - e n e - 7 , 1 1 - d i k e t o  c h a r a c t e r i s t i c of s t e r o i d s or t r i t e r p e n e s p o s s e s s i n g chroraophore.  1+6  The o l e f i n i c p r o t o n a t x 4 . 4 8 i n the NMR s p e c t r u m o f  a b i e s l a c t o n e was no l o n g e r p r e s e n t  suggesting  t h a t t h e d o u b l e bond i n t h e - C(9)  s k e l e t o n o f a b i e s l a c t o n e had s h i f t e d t o t h e C(8) c o u r s e o f the o x i d a t i o n .  Uyeo w r i t e s  2 8  p o s i t i o n i n the  , "Of the two p o s s i b i l i t i e s  [7-ene o r 9 ( l l ) - e n e ] , t h e p o s i t i o n o f t h e d o u b l e bond between C ( 7 )  -  C(8)  c o u l d be r u l e d out because o f the f a c t t h a t a b i e s l a c t o n e and i t s d e r i v a t i v e [54]  showed no f a c i l e s h i f t o f the double bond i n the r i n g s y s t e m on  t r e a t m e n t w i t h m i n e r a l a c i d s , analogous t o l a n o s t - 9 ( 1 1 ) - e n - 3 ( 3 - y l [ d i h y d r o p a r k e y l a c e t a t e , 34 ]. 3g-yl acetate  [59]  5 0  3g-yl acetate  [61]  5 2  ,  »  1+7  >  1+8  >  1+9  Compounds such as  euph-7-en-3f3-yl acetate  [60]  5 1  ,  and t i r u c a l l - 7 - e n and C(8)  c o n v e r t e d w i t h a c i d s i n t o compounds h a v i n g t h e  - C(9).  F u r t h e r , the f a c t that the i s o l a t e d  bond can be h y d r o g e n a t e d under c a t a l y t i c c o n d i t i o n s p a r a l l e l s of l a n o s t - 9 ( l l ) - e n - 3 B - o l , w h i l e l a n o s t - 7 - e n - 3 | 3 - o l  34  lanost-7-en-  t h a t c o n t a i n t h e double bond between C ( 7 )  are known t o be r e a d i l y d o u b l e bond a t C(8)  146  acetate  59  double  the b e h a v i o r  i s resistant."  65  An  observation  t h a t s u p p o r t e d the view t h a t  the n u c l e a r  double bond  must be i n the C(9) - C ( l l ) p o s i t i o n was s e c u r e d by o x i d a t i o n of the 3a-methoxylanostene afforded  (54)'with  chromium t r i o x i d e i n a c e t i c a c i d which  two p r o d u c t s a f t e r , chromatography.  product W A R  The minor and l e s s p o l a r  an p.ne-rK n n p . (f>2) whnsf TIVfir>ecfri>inwas s i m i l a r t o the  chromium t r i o x i d e p r o d u c t of a b i e s l a c t o n e . compound was an a , 8 - u n s a t u r a t e d ketone  The major and more p o l a r  (6 3) whose UV s p e c t r a l p r o p e r t i e s  MeO ' '  62  63  (A  max  275 my,  log e 4.10)  were l i k e those of 38-acetoxylanost-9(ll)-en.  12-one ( 6 4 ) , 3a-acetoxyarbor-9(ll)-en-12-one 1+7  detodavallate (66) .  The ORD  5i+  9(ll)-en-12-one  (65)  5 3  curve was  ( 6 5 ) , and methyl 125 3  l i k e that of 3a-acetoxyarbor-  and 33-acetoxy-18a-olean-9(11)-en-12-one  (67) . 5 5  MeOOC 66  67  The mass spectrum of the 3a-methoxylanostene (54) was s i m i l a r cracking pattern to that of arborene ( 6 8 ) double bond at C(9) - C ( l l ) .  5 3  reported to show  which contains a  67  On the basis of the above data the structure for abieslactone suggested by Uyeo and co-workers  28  i s shown i n 30 and the systematic name  given i s '3a--methoxylanos ta-9 (11) ,24-dien—-27 ,23R-o.lide  [ (23R)-3a-methoxy-  lanosta-9(11),24-diene-26,23-lactone].  MeO''  It w i l l be remembered that 30 i s the structure proposed for grandis-  was  not i d e n t i c a l with abieslactone c a r e f u l re-evaluation of the  of abieslactone was  chemistry  necessary.  This called f o r , i n part, a larger amount of abieslactone than had been o r i g i n a l l y received from Professor Uyeo.  Hergert  30  had reported i t s  presence i n the bark of s i l v e r f i r [Abies amabilis (Dougl.) Forbes]. supply of that bark was Laboratory  obtained through the kindness of the Forest  A Products  i n Vancouver from the University of B r i t i s h Columbia Research  Forest near Haney, B r i t i s h Columbia. Extraction of the ground, a i r dried bark with chloroform or d i e t h y l ether and evaporation of the^solvent gave a brown residue. was  This residue  dissolved i n hot ether and l e f t to c r y s t a l l i z e overnight at room  temperature.  The p r e c i p i t a t e was  f i l t e r e d , washed with cold ether,  and,  68  after drying, provided  a l i g h t brown powder.  showed one main .compound corresponding lactone obtained  TLC of this brown powder  to an authentic sample of abies-  from Professor Uyeo, plus two minor spots which were  both more polar than abieslactone.  R e c r y s t a l l i z a t i o n of this brown powder  from e t h y l acetate indicated by TLC  that very l i t t l e enrichment of abies-  lactone had  occurred.  It was  found that abieslactone could be p u r i f i e d by chromatography on-  neutral deactivated alumina.  E l u t i o n with petroleum ether - benzene gave  abieslactone as a white s o l i d , while further e l u t i o n with benzene methylene chloride gave the second compound i n i t i a l l y coded as AA,,.F i n a l l y e l u t i o n with methylene chloride gave the t h i r d compound, Compounds AA j - u c  and AA^ w i l l be discussed  2  d u x e s  J . ( 3 C L . U U C  u u t - d x u c u  x i i  L U I O  later. uicuil.iir; x  w ci.T>  U i y a  ethyl acetate to give a white c r y s t a l l i n e s o l i d m.p. value 251 - 2 5 3 ° C ) .  28  abieslactone showed no melting point depression.  a three proton s i n g l e t at x 6.73;  This spectrum was  J.J.UUt  251 - 253°C ( l i t e r a t u r e authentic  The NMR  spectrum of  the three low f i e l d signals at T 3.00,  the v i n y l i c methyl at x 8.10; groups'.  u w x c e  In addition, the TLC of  both specimens'were the same as were the IR spectra.  and 5.05;  L c t J . l X 6 c u  A mixed melting point determination with  our product (Figure 16) had  AA^.  4.48,  the diffused t r i p l e t at T  and signals at x 8.98  - 9.08  7.20;  for s i x methyl  i n agreement with the reported NMR  spectrum of  abieslactone. The [<j>]  224  ORD  curve (Figure 17) of abieslactone (in dioxane) had a trough  - 49,000°.  The CD curve (Figure 18) of abieslactone (in dioxane)  had a weak p o s i t i v e peak [9125Q  +  ^50°, and a strong negative value with  Figure  18.  CD  curve  of  abieslactone.  71  no minimum observed below 220 my (1^^220  ~  ^5,050°).  As a further check that the i s o l a t e d abieslactone was indeed to the reported material, a derivative was prepared.  identical  The derivative (58),  the 8-ene-7,11-diketone obtained by chromium trioxide oxidation, was selected f o r a number of reasons.  The chromophore produced had character-  i s t i c s p e c t r a l properties and i t s physical properties were .known as i t had been prepared  i n the o r i g i n a l s t r u c t u r a l elucidation of abieslactone.  The derivative (58) was prepared expected UV absorption (X^|°  H  and the product exhibited the  274 log e 3.85), IR absorption (1735, 1670  cm ), and a melting point of 217 - 219°C i n agreement with the r e p o r t e d -1  28  value of 215 - 21.6°C. In addition to the above d e r i v a t i v e , dihydroabieslactone  (44), t e t r a -  reported . 28  OH  MeO' 69  On the basis of the above work, i t was concluded  that the abieslactone  i s o l a t e d from _A. amabilis was indeed i d e n t i c a l with the reported substance obtained from A_. m a r i e s i i . The re-evaluation of the data f o r abieslactone started with the NMR  72  spectral results.  In the.last section i t was  demonstrated that the p o s i t i o n  of C-methyl groups i n the triterpene series could be predicted with reasonable accuracy  using certain empirical calculations .  3 9  >^ >  An examination  1+1  of Table II (page 28) shows that i t i s only the 3-keto group which affects the signals of the C(18), C(19), and C(32) methyl groups. a -3a-methoxyl substituent was  assumed to be n e g l i g i b l e i n the  study i f the 3a-hydroxyl group was  used as a model.  that the lactone ring would have n e g l i g i b l e e f f e c t . assumptions was  The effect of previous  Also i t was  assumed  The v a l i d i t y of these  eventually established when our previous work coupled  with  X-ray analysis l e f t no doubt about the structures of cyclograndisolide and grandisolide. One  of the anomalies associated with the NMR  methyl groups.  spectrum of abieslactone  This point can be best i l l u s t r a t e d by considering several  examples from the p u b l i s h e d  28  chemistry  of abieslactone and the present  s tudy. Resonance Frequency (x units)  Compound abieslactone (30) dihydroabieslactone keto acid (48) keto ester (49) d i o l (69) grandisolide Table VII.  (44)  8.98 8.96 8.73 8.76 8.97 8.95  9 .00 9 .00 8 .84 8 .87 9 .00 9 .02  ;  9.06 9.06 8.95 8.95 9.05 9.07  9 .08 9 .08 9 .00 9 .03 9 .08 9 .12  9 .05 9 .07  9.09  9 .29  9.36  Range of resonance frequencies of the C-methyl groups.  Table VII i l l u s t r a t e s that the lactone ring has l i t t l e e f f e c t on the positions of the methyl s i g n a l s .  I f the lactone ring was  causing  the  C-methyls to absorb over a narrow range i n abieslactone, this range would  73  t  S t r u c t u r e 30 i s the s t r u c t u r e a s s i g n e d t o a b i e s l a c t o n e by U y e o ; a d i f f e r e n t compound, g r a n d i s o l i d e , was shown t o have t h i s structurei n the p r e s e n t s t u d y . 2 8  74  be  expected  t o b r o a d e n as  dihydroabieslactone to s l i g h t l y assigned  or beta the  requires the  are  i n the k e t o the  f r o m an  a c i d and  resonance frequency  of  keto  oxygenated carbon.  e s t e r can  feature  the m e t h y l  1 pt^d. and  fo  the  -  ^t nfll ^ ip  b*  3  first  obtained  by  Of  resonance frequency p.c]art^np .  lower than i n the  removal of  Compound abieslactone (30) dihydroabieslactone (44) keto acid (48) keto ester (49) diol (69) deoxy e s t e r (51) grandisolide dihydroparkeyl acetate (34) arborene ( 6 8 ) 5 3  Table V I I I .  the  attached  to  the  upper, l i m i t -  29)  or  grandisolide lowers  groups.  f u r t h e r i n t e r e s t i s the  reductive  signals be  abieslactone molecule  of  the  ^IIX  Tc*ble  C(9)  -  olefinic that  olefinic  there  the  proton  Resonance Frequency  (x  now  a-  postu-  abieslactone signal  triter-  deoxy e s t e r  f u n c t i o n i n 49  i s an  proton  C ( l l ) unsaturated  f a c t that  carbonyl  spectra  PVIAT.TO  f o u r d e r i v a t i v e s , m e n t i o n e d p o s s e s s , an  which i s considerably penes noted.  the  two  w i t h known C(9)  IV, page  I n a d d i t i o n t o t h e m e n t i o n e d a n o m a l i e s i n t h e NMR nomaly a s s o c i a t e d w i t h  The  of  Even i n t h e s e d e r i v a t i v e s  these values  i n the  range  likely  i s anomalous i n t h a t  d e r i v a t i v e s (Table  t h a t some s t r u c t u r a l  The  unchanged.  s i d e c h a i n w h i c h a r e now  Comparison of a l l of  lanostene  is altered.  essentially  resonance frequencies  i s never over T 9.08. unsaturated  functionality  diol  t o m e t h y l g r o u p s on  range of  C(ll)  the  lower f i e l d  carbons alpha the  and  this  (51)  shows  units)  4.48. 4.47 4.48 4.45 4.47 4.78 4.80 4.81 4.73  Comparison of r e s o n a n c e f r e q u e n c i e s of o l e f i n i c p r o t o n i n a b i e s l a c t o n e s e r i e s w i t h some C(9) - C ( l l ) u n s a t u r a t e d triterpenes.  75  absorption i n a region consistent with a C ( l l ) o l e f i n i c proton.  One  cannot  help but wonder whether "the. conversion, 49->51, i s not associated with double bond migration. In view of the previously successful comparison of cyclograndisolide and. cycloartenyl acetate, a mass spectrometric comparison of dihydroparkeyl acetate and grandisolide, both known members of the lanostane family possessing C(9) - C ( l l ) unsaturation, with abieslactone was The elemental composition resolution mass  of ions where given was  undertaken.  determined by high  spectrometry.  The fragmentation  of 9(ll)-ene triterpenes i s not w e l l studied, the  p r i n c i p l e examples being arborene ( 6 8 )  5 3  and arborenone ( 6 8 a ) . In both 58  cases the spectra are characterized by strong loss of methyl and a base  68, R = 68a, peak corresponding  R  H  2  =o  to fragmentation mode n with smaller ions due to  fragments from f i s s i o n s v i a pathways o and p.  However, triterpenes with  double bonds i n positions other than C(9) - C ( l l ) also exhibited some of the same fragments  56  so care must be exercised i n the i n t e r p r e t a t i o n .  Furthermore, a p p l i c a t i o n of these fragmentation pathways to the  76  present study i s of doubtful v a l i d i t y . involve cleavages i n rings C and D.  Fragmentations n, o, and p a l l  Arborene represents a pentacyclic  triterpene which may fragment rather d i f f e r e n t l y from the t e t r a c y c l i c system portrayed i n abieslactone.The mass spectrum of dihydroparkeyl acetate (34) (Figure 19) shows a molecular ion at m/e 470. A loss of methyl gives a M-15 ion at m/e 455, which i s the base peak, plus a metastable  ion at m/e 440.5.  A loss of  acetic acid from the molecular ion gives ion a (m/e 410) plus a metastable ion at m/e 357.9.  An ion b at m/e 395 can come from either the M-15 ion  ion e m/e 288  M-15 m/e 455 AcO M -HOAc  m/e 470 -HOAc  V  V. -CH,  M-60 m/e 410  ion c m/e 36 7  ion d m/e 341  ion b m/e 395  77  o  •s7  a o  vO O I rH •  a  -a-  ON •  ro  a vo ro a . tn cn  T3 <t  ro  CD CD  cn 00 0)00'-^  CM  CD  LU  . in  o rS >•>  a) ,M CD . CD  l-i <T)  p. o  u  •H 4-1  CD  o M +J O CU ft  to to to  _  CD CD  a  U 00  •H IX)  001  DS  UISN31NI  3AIldl3d  78  by loss of a c e t i c acid as seen by a metastable ion at m/e 343.1 or from ion a by loss of methyl as seen by a metastable ion at m/e 380.6. loses  C-^ij-  to give ion c at m/e 367 and a metastable at m/e  A peak at m/e  341 could correspond to loss of  Ion a  328.8.  from ion a.  CrAi^'  An ion, e (m/e 288), could correspond to the loss of ring A as observed f o r cycloartenyl acetate and cyclograndisolide. approximately one f i f t h as intense i n dihydroparkeyl  This ion i s  acetate as i t was i n  either of the cyclopropanoid triterpenes. Grandisolide  (30) obtained from cyclograndisolide was next compared  in the mass spectrometer since, i n addition to the 9,11 double bond, i t possesses the unsaturated abieslactone.  lactone side chain, the same as reported for  The spectrum (Figure 20) shows a molecular ^™  /~  A„  JJI  ion (m/e 468), i  J  i  ion e m/e 314  ^  M-15 m/e 453  MeO'  -Me OH ion b m/e 421  ion a m/e 436  ion c m/e 39 3  ion d m/e 36 7  79  CO -3-  m co t-H in -  CD  i -<r  a XI  CM  CO  •3 vD  I CO •  CM -  CD . CD  CO  sr  ro  ro CD . ID  01  CD . CD  cn  . C D ^ !  . i n \  CD . CD  a; Tj •rl  rH O  00  o .in  4H O  In  .4-)  o  QJ  ft  CO  CD  . a  CO  a  o CM ai  u . CD  001  S/-  05  UISN31NI 3AUU13d  0  tn  00 •rl  80  ion a can lose methyl (ion b, m/e 421), (ion d, m/e 367). Ion e corresponding  (ion c, m/e 393), and C<^Q'  to loss of ring A i s seen at  m/e 314, j u s t s l i g h t l y more intense, than i o n e i n dihydroparkeyl  acetate.  Comparison of the mass spectra of dihydroparkeyl acetate and grandi s o l i d e reveals that the major difference i s the r e l a t i v e i n t e n s i t i e s of the M-15 fragment (m/e 455 i n the former and m/e 453 i n the l a t t e r ) . lactone side chain does not otherwise a l t e r appreciably.the of the lanostane  The  fragmentation  skeleton.  The mass spectrum of abieslactone ion at m/e 468 with composition  (Figure 21) exhibits a molecular  ^^jj^-^^^'  ^  n e  ^-15 ion i s seen at m/e 453  (C^gH^^O^) but, as i n grandisolide, i t s r e l a t i v e i n t e n s i t y i s 20% of the corresponding ! Dnn  421  ! sr  i o n i n dihydroparkeyl  TOT ; -  " i c opon  (^28^37^2^ S l a  a  n  oo  -I r\r\  o of-  acetate.  TV,  /, T f t  tr  Loss of methanol from the vi  r\  \  r.-rV,•: T ;  „, _ ... v ~ 2 9 " 4 1 ~ 2 ~ ~  K  o f  m  arises v i a loss of methanol from the M-15 ion or  v i a loss of methyl from i o n a.. Furthermore, the ion a may lose C^H-,"  to give ion c at m/e 393 (C27H.J7O2).  7?  -CH0H_ 3  xon a . m/e 436 CH3  ion c m/e 393  7  ion e m/e 314 M-15 . m/e 453  Abies lactone M m/e 468  V  /  V  -CH, -7"  -CH OH 3  ion b m/e 421  „  81 CO  LO CO r H LO  O .iii  sr  CM VD CO CO  ,n  CM •  CJ  CD . CD  co  sr  ON>  ro  o .in  Q) r H CO  CD . CD  OS LW 0 \ CM  cn  CM CM  CD  .tn  LU  CO •r-)CO CM  Q)  d  CD . CD  cd rH CO QJ  LO  rd  o o  4-)  •A  •8  r-. CD  MH O  .m  n o . QJ  P.  CO CO  _  CD CD  •a rH CM  cu  u  3  001  n S'_  1  1  05 A1ISN31NI  3AIiai3U  . CD  in  60 •H  fn  82  Ion e (m/e 314), with elemental composition ^'21^30^2  ,  ^  S  a  PP  r o x i m a t e  ly'  three f o l d more intense i n abieslactone than i n grandisolide. Ion f (m/e 299, ^20^27^2^ (m/e 175, CijHig)  m a  a  r  i  s  e  s  from loss of methyl from i o n e while ion h  y represent fragmentation of the side chain from i o n f.  Ion k (m/e 272, C-^gR^O;?) i s seen i n the mass spectrum  of .abieslactone  along with i o n j (m/e 233, ^15^21^2^ corresponding to cleavage  through  ring D.  ion j m/e 233  Although i t i s clear that d e f i n i t e conclusions concerning the struct u r a l differences between grandisolide and abieslactone cannot be made from the consideration of the mass s p e c t r a l data, i t i s s i g n i f i c a n t that the region below m/e 300 i n abieslactone i s appreciably altered when compared with that of grandisolide. In p a r t i c u l a r , ions k and j noted i n abieslactone suggest a d i f f e r e n t orientation f o r the double bond i n this substance. In order to provide additional data concerning the structure of abieslactone i t i s necessary to consider f i r s t some results on two additional components i s o l a t e d from P a c i f i c s i l v e r  fir.  83  The f i r s t component coded as AA , m.p. 236 - 238°C, had formula ^30^44 - 3" <  >  2  The  IR spectrum  had two carbonyl absorptions, the a,3-unsaturated-y-  lactone (1745 cm ) and a ketone (1705 c m ) . -1  -1  The NMR spectrum had a quartet  at T 7.45 and this was assigned to protons adjacent to the ketone.  There  was no absorption for a methoxyl group while the C-methyl region contained signals at T 8.90, 8.98, and 9.18 integrating f o r s i x methyl groups. x 9.18 s i g n a l was a s i n g l e t and integrated for one methyl group.  The  Signals  at T 3.00, 5.02, and 8.10 were assigned to the same protons at C(24), C(23), and C(26) as i n abieslactone.  The o l e f i n i c proton absorbed at x 4.40.  The ORD curve (Figure 22) had a peak, [ 4> ] 317 + 1485°,' and a trough at lower wavelengths, [<r>]224 ~~ 25,400°. ^•'294  +  3,160° and a trough.at lower wavelengths, [9]215 ~ 35,510°.  J-  300  220  t  o  •o-  The CD curve (Figure 23) had a peak,  -1—  -2-  -3—  Figure 22.  ORD curve of AA  2<  340  380  A(mu)  84  ~]  1  260  -T o  A(my)  320  -1  -2  —  Figure 23. The  300  280  CD curve of  mass snectrum had  at m/e  314 was  AA^•  the m o l e c u l a r i o n at m/p.  also present as w e l l as the m/e  452  (C...V... .0.1.  A nftak  299 ion peak, both of which  were of lesser r e l a t i v e i n t e n s i t y than i n abieslactone. The other minor component, AA^ most polar of a l l three products absorption (3520 cm ) -1  (m.p.  249 - 250°C, C^QH^O^) , was  from s i l v e r f i r .  plus the lactone carbonyl.  the  The IR spectrum had The NMR  hydroxyl  spectrum had  signals at T 3.02, 5.02, and 8.10 c h a r a c t e r i s t i c of the lactone system; a broadened s i g n a l at x 6.6 assigned to a proton geminal to the hydroxyl; and C-methyl signals at x 8.98, 9.00, 9.03, and 9.06 o l e f i n i c proton was a molecular  at x 4.48 as i n abieslactone.  ion at m/e  454 (C^QH^O^) .  for s i x methyls.  The  The mass spectrum had  The ion peaks at m/e  314 and  m/e  299 were both present and were of greater r e l a t i v e i n t e n s i t y than i n AA but s l i g h t l y smaller than i n abieslactone.  2  85  Oxidation of AA^ with chromium, trioxide i n pyridine gave AA , suggest2  ing that they d i f f e r e d only i n oxidation l e v e l at one center.  AA3 upon  methylation gave a product whose TLC, m.p., mixed m.p., and IR were i d e n t i c a l to abieslactone.  Thus AA^ i s 3-desmethylabieslactone  and AA  2  i s the 3-keto  derivative of abieslactone. C a t a l y t i c reduction, of AA  2  i n tetrahydrofuran with palladium on char-  coal as catalyst gave dihydro AA-,.. The NMR signals formerly associated with the lactone had disappeared with a new one proton multiplet at T 5.5 now v i s i b l e . 9.20;  The C-methyls absorbed at x 8.96, 9.00, 9.02, and  the higher f i e l d resonance was s t i l l a three proton s i n g l e t .  curve of dihydro AA  had a peak [^]3x5  2  3055° and a trough [4*3275 ~ 3921°  +  associated with the ketone plus a peak at 220 mu ([<j>] with the lactone r i n e .  The ORD  + 220  '  o l l 0  ° ) associated  The CD curve had a mavirmi-m f o r th?. ketone,  [ 8 ] g ^ + 3170°, and the lactone had a p o s i t i v e curve with no maximum 2  observed above 220 mu ([G] 20  +  2  As was shown, AA  12,960°).  d i f f e r s from abieslactone only i n the nature of the  2  f u n c t i o n a l i t y at C(3), with the nuclear double bond and the lactone i n the same positions. lactone  2 8  I f AA  2  i s a 9(ll)-ene system as i s reported f o r abies-  then the ORD curve associated with the ketone function of AA  and dihydro AA  2  should be s i m i l a r to other 3-keto-9(ll)-ene triterpenes.  The ORD curves of AA ORD curve of  2  and dihydro AA  2  are shown i n Figure 24 along with the  lanost-9(11)-en-3-one. > 28  53  From Figure 24 i t can be seen that lanost-9(11)-en-3-one miniumum i n the 320 my region. this region.  2  AA  2  and dihydro AA  2  has a  have a maximum i n  The data f o r the abieslactone series i s at variance with the  86  Figure 24.  ORD . curves, of lanos.t—9 (11)-en—3-one , AA , and dihydro 2  reported results for known 3-keto-9(11)-ene triterpene systems.  AA . 2  On this  basis the p o s i t i o n of the double bond i n AA , AA-j, and, i n turn, i n 2  abieslactone cannot be at C(9) - C ( l l ) . The above discussion reveals that the NMR, ORD, and mass spectrometric studies of abieslactone and i t s derivatives present several anomalies. I t i s recognized that a l l three techniques  are s e n s i t i v e to the p o s i t i o n of  any o l e f i n i c linkages present i n the system.  In f a c t , i t was the i n t r o -  duction of an o l e f i n i c linkage v i a the cyclopropane  ring opening reaction  which caused doubt to be cast on the structure of abieslactone. For these reasons an examination of the chemistry  of abieslactone was  undertaken v^ith p a r t i c u l a r reference to the assignment of the p o s i t i o n of the nuclear double bond.  87  F i v e f a c t o r s t h a t were c o n s i d e r e d by the p r e v i o u s  authors^  i n the -  0  p l a c i n g of the double bond i n the C(9) - C ( l l ) p o s i t i o n a r e : (a) a b i e s l a c t o n e i s r e d u c i b l e by c a t a l y t i c methods; (b). a b i e s l a c t o n e i s s t a b l e t o m i n e r a l a c i d ; (c) a b i e s l a c t o n e upon chromium t r i o x i d e o x i d a t i o n gave a diketone  8-ene-7,ll-  (58);  (d) a 3a-methoxylanostene o b t a i n e d  from d e g r a d a t i o n  of a b i e s l a c t o n e gave,  upon chromium t r i o x i d e o x i d a t i o n , a 9(11)-ene-12-ketone (e) a d e g r a d a t i o n  product  (63);  of a b i e s l a c t o n e was compared t o an a u t h e n t i c  lanost-9(11)-ene d e r i v a t i v e . The double bond was thought t o be p a r t of t h e t r i t e r p e n e n u c l e u s s i n c e i t was n o t h y d r o g e n a t e d under m i l d c o n d i t i o n s .  I t c o u l d be  p o s i t i o n o f a t r i s u b s t i t u t e d n u c l e a r double bond i n a t r i t e r p e n e i s between C(7) and C(8), a l t h o u g h  double bonds between C(9) and C ( l l ) a r e  known.. The f a c t t h a t the i s o l a t e d double bond can be reduced p a r a l l e l s t h e r e p o r t e d b e h a v i o r  catalytically  of l a n o s t - 9 (11) -en-3r3-ol, w h i l e  l a n o s t - 7 - e n - 3 B - o l i s r e s i s t a n t even under r i g o r o u s c o n d i t i o n s . The o r i g i n i a l i n v e s t i g a t o r s a l s o r e p o r t t h a t a b i e s l a c t o n e i s s t a b l e to m i n e r a l a c i d .  I t i s known t h a t compounds such as l a n o s t - 7 - e n - 3 8 - y l  a c e t a t e , euph-7-en-38-yl a c e t a t e , and t i r u c a l l - 7 - e n - 3 B - y l a c e t a t e 5 0  5 1  t h a t c o n t a i n the double bond between C(7) and C(8) a r e r e a d i l y i n t o compounds h a v i n g  a double bond at C(8) - C(9).  5 2  converted  On the o t h e r hand,  compounds l i k e p a r k e y l a c e t a t e w i t h a C(9) - C ( l l ) double bond a r e s t a b l e to a c i d .  88  Furthermore,  abieslactone was  oxidized with chromium trioxide i n  aqueous acetic acid to a 8-ene~7,11-diketo derivative.  This suggested to  the o r i g i n a l investigators that the C(9) - C ( l l ) double bond was  probable.  Before continuing with other evidence present i n the o r i g i n a l struct u r a l determination, some observations from- the present study are i n order. It was  found i n our work that under mild hydrogenation conditions abies-  lactone gave a dihydro derivative, and that under more rigorous conditions a tetrahydro derivative was  formed.  The oxidation of abieslactone with  chromium t r i o x i d e gave a compound with the same properties as the 8-ene7,11  diketone reported by the o r i g i n a l i n v e s t i g a t o r s . 28  However, s i g n i f -  i c a n t l y d i f f e r e n t results were obtained upon treatment of abieslactone with acid. T T-~ J  ^ ~-  .1 -  ,~  ~  ,1^  4— - J  -U  ~  w.iu^j. uiitt^ uj.uuu k.t/uu4.^j.k/uu ,  uj  ULUg^U  1  • J _  -~  - T _ T  r  _• _  .- _.. .  \-LJ-J-^i-J-VJCi J-LL ^ilXf i. UJ. UliU J-O U1UC J_  ized abieslactone into a mixture of new products.  The NMR  spectrum  of the  product mixture had signals for the protons of the lactone ring indicating that the isomerization was not about that f u n c t i o n a l i t y . was  A new  signal  observed f o r an o l e f i n i c proton at T 4.8 with complete absence of the  former o l e f i n i c proton signal at x 4.4.  In addition to the o l e f i n i c  s i g n a l differences, the C-methyl groups now to 9.34.  The above NMR  resonated over the range x  8.94  data i s i n good agreement with the values obtained  for grandisolide (30) as mentioned previously.. Indeed, c r y s t a l l i z a t i o n of the above product gave a white s o l i d , 212 - 214°C, which was with grandisolide (30).  i d e n t i c a l i n every respect (mixed m.p., It was now  TLC,  m.p.  IR)  certain that abieslactone can be  isomerized with hydrogen chloride i n chloroform into a C(9) - C ( l l ) o l e f i n .  89  0  MeO"  30 If abieslactone i s dissolved i n acetic acid, i t may be recovered unchanged with no changes i n the NMR data of melting point being  observed.  However, dissolving abieslactone i n one percent concentrated hydrochloric acid i n acetic acid affected an isomerization but gave a d i f f e r e n t isomer t i o fh.an befores i n g l e t s at x 9.30 and 9.34; i n this case only one s i n g l e t at x 9.30 was of appreciable i n t e n s i t y . integral.  As before the o l e f i n i c proton s i g n a l was non-  The NMR spectrum of the l a t t e r agrees with the c a l c u l a t e d  3 9 - 1  *  1  NMR spectrum f o r a mixture of lanost-7-en-3a-ol and lanost-8-en-3a-ol. Obtaining d i f f e r e n t isomer ratios with d i f f e r e n t acid i s not unusual. In the Introduction i t was seen that treatment acids gave g- and y - o n o c e r i n .  21  of a-onocerin with p r o t i c  Treatment of a-onocerin with a Lewis acid  such as boron t r i f l u o r i d e gives y o n o c e r i n and s e r r a t e n e s . A cursory examination  of the oxidation reaction of abieslactone with  chromium t r i o x i d e seems to reveal no abnormalities. examination  20  However, on closer  this reaction i s found to be a t y p i c a l of C(9) - C ( l l ) t r i t e r -  pene o l e f i n s .  Oxidation of dihydroparkeyl a c e t a t e  49  or arborene  53  gives  90  the 9(11)-ene-12-keto derivative, none of the 8-ene-7,11-diketo derivative being reported.  Compounds such as lanost-7-ene, lanost-8-ene, or lanosta-  7,9(ll)-diene can be o x i d i z e d ' 5 7  5 8  to the 8-ene-7,11-diketo derivative,  but.these systems were rejected by the o r i g i n a l authors on the basis of previous arguments. I f one returns to the o r i g i n a l arguments for the assignment of the double bond p o s i t i o n , one must consider the degradation.sequence earlier.  It i s re-presented i n tabular form i n Figure 25.  25 the appropriate NMR  on cliromium t r i o x i d e oxidation, gave two products.  minor"  rivodnct  Also i n Figure  data i s l i s t e d and this w i l l be discussed shortly.  In the degradation sequence a 3a-methoxylanostene was  i s o l a t e d was  described  obtained which,  The major product  reported as a 9(11)-ene-12-keto derivative (63), while the was  ?  R-pnp-7  11— diketo derivative  (62).  It  will  that abieslactone gave only the 8-ene-7,11-diketo derivative.  be  The  recalled obser-  vation that the methoxylanostene gives two products i s inconsistent with the previous r e s u l t s .  I t also contradicts the precedents established for  either 7-ene or 9(11)-ene systems. The f i n a l piece of evidence presented f o r the C(9) - C ( l l ) double bond i s that the lanost-9(11)-en-3-one  and lanost-9(11)-en-38-yl acetate  obtained from the degradation sequence were compared to authentic samples of those materials and found to be i d e n t i c a l . This result seems s u r p r i s i n g since compounds l i k e abieslactone and 3-keto abieslactone had s p e c t r a l properties which were d i f f e r e n t from the observed or calculated values for C(9) - C ( l l ) o l e f i n i c systems. The NMR  data for the compounds of the degradative sequence i s presente  Figure  25.  Degradation  sequence  of  abieslactone.  Figure  25.  Degradation  sequence  of abieslactone  (cont'd.).  93  94  i n Figure 25.  In the previous discussion the NMR  data of abieslactone  and keto acid (48), keto ester (49), and d i o l (69) has been described as anomalous f o r a 9(ll)~ene system.  The NMR  data as presently reported f o r  the t h i o k e t a l (50) i s i n agreement with the "expected" NMR values of 9(ll)-enes.  The o l e f i n i c proton now  absorbs at T 4.78  the p o s i t i o n i n other C(9) - C ( l l ) o l e f i n s . absorption has an upper l i m i t of x 9.31, C(9) - C ( l l ) o l e f i n s .  i n agreement with  The C-methyl groups range of  again i n agreement with other  The NMR values for a l l products obtained l a t e r in  the degradation sequence are also i n agreement with the "expected"  NMR  results. The dramatic changes observed i n the NMR  spectra of the t h i o k e t a l and  the subsequent compounds were not commented upon by the o r i g i n a l i n v e s t i sators.  The channes are defi.nif.p. 1 v siioo-estive of a rearrangement reaction  occurring during the preparation of the thioketal derivative to give the C(9) - C ( l l ) o l e f i n as the major isomer.  This behavior p a r a l l e l s the  observation reported e a r l i e r that hydrogen chloride i n chloroform can convert abieslactone into a mixture of isomers.  Indeed, i f such a rear-  rangement did occur under the a c i d i c conditions employed, the subsequent comparison of the degradation products with authentic samples of lanostene derivatives would not give d i r e c t evidence for the structure of abieslactone With t h i s rearrangement i n mind i t i s possible to explain the  two  products obtained i n the oxidation of the methoxylanostene (54). Since the methoxylanostene i s obtained i n the l a t e r stages of the degradation sequence, that i s , after the t h i o k e t a l ( 5 0 ) , i t i s l i k e l y a mixture of olefins.  This mixture could e a s i l y explanin the oxidation products obtained  95  Lack of s u f f i c i e n t q u a n t i t i e s of a b i e s l a c t o n e p r e v e n t s  further  c h e m i c a l work at t h i s time on the s t r u c t u r e of a b i e s l a c t o n e .  With the  d a t a p r e s e n t l y a v a i l a b l e p o s s i b l e s t r u c t u r e s f o r a b i e s l a c t o n e may be discussed. S e v e r a l f a c t o r s must be t a k e n i n t o account f o r any proposed s t r u c ture of a b i e s l a c t o n e .  The n u c l e a r double bond must be t r i s u b s t i t u t e d .  F u r t h e r , c l e a v a g e of the double bond w i t h osmium  tetroxide/periodate  g i v e s a p r o d u c t c o n t a i n i n g l a c t o n e and aldehydo c a r b o n y l groups as w e l l as a k e t o n i c c a r b o n y l on an a l i c y c l i c system o r a s i x member, o r l a r g e r , r i n g . T h i s r e q u i r e s t h a t the double bond must n o t be e x o c y c l i c t o a c y c l o p e n t a n e ring. I t i s n e c e s s a r y t h a t the p r o p o s e d s t r u c t u r e f o r a b i e s l a c t o n e be a b l e  '  ~~ — -.-~-- ~ 0  0  — .~ —  - \-*-— /  ' — — ~~ ~ ' ~* " ^ '  r e p o r t e d comparison w i t h a u t h e n t i c l a n o s t e n e  derivatives.  ~ I n keeping with  o t h e r o b s e r v e d rearrangements o f t r i t e r p e n e s o r s t e r o i d s , the m i g r a t i n g groups a r e u s u a l l y a x i a l and i n a t r a n s c o p l a n a r  relationship.  these r e q u i r e m e n t s i n mind, s e v e r a l p o s s i b i l i t i e s may be  Keeping  presented.  W h i l e i t i s p o s s i b l e t o p l a c e the double bond a t C ( l ) - C(10)  as i n  p a r t i a l s t r u c t u r e 73, t h i s f e a t u r e i s u n l i k e l y i n view of the mass s p e c t r a l  73  96  fragmentation.  Ion e, mentioned previously, corresponding to loss of ring  A would not be as feasible and, i f i t did occur, several migrations are needed before fragmentation to give an ion of correct elemental  composition.  It i s possible to place the double bond i n ring D as i n structures 74 and 75.  Neither of these two structures- can explain the f a c i l e loss of  ring A i n the mass spectrum of abieslactone unless rearrangement occurs before fragmentation. for  Structure 74 does not allow a r a t i o n a l explanation  the anomalies noted i n the C-methyl group region of the NMR  of abieslactone.  spectrum  Structure 75 on the other hand, has both the C(18)  C(32) methyl groups i n a a l l y l i c p o s i t i o n which may  and  cause them to resonate  at the lower f i e l d as observed. The Cotton e f f e c t associated with the 3-ketone of 74 and 75 would be expected to be negative using the onocerin part structure 76 as the model.  97  The double bond could be placed i n ring C at C(9) - C ( l l ) i f the C(8) hydrogen i s i n the abnormal alpha configuration (structure 77).  I f C(8)  MeO"  7 7  was beta, a lanost-9(11)-ene type system could be the r e s u l t .  It i s known  that such systems are stable to a c i d i c reagents, that i s , no double bond ;vr.ri/r>T* r> n rnpf-H-ul  arnim  mjorafinpc  n^ntir  ^_2_SO  ^ l ^ e C^t^'OP  e^feC*"  associated  with the 3-ketone of 77 would be expected to be negative based on the octant r u l e  6 0  and molecular models.  The Cotton e f f e c t for the 3-keto  derivative of abieslactone measured i n this study i s p o s i t i v e , so structure 77 i s not l i k e l y on this basis. There are two positions i n ring B for the double bond.  I t i s possible  to place i t between C(5) - C(6) (structure 78) as i s found i n bryogenin (79).  78  79  80  98  The Cotton e f f e c t associated with p a r t i a l structure 80 i s negative. S i m i l a r l y , the Cotton e f f e c t associated with the 3-keto derivative of 79 i s negative, opposite to the observed Cotton e f f e c t f o r the 3-keto derivative of abieslactone. . On this basis, structure 78 i s an u n l i k e l y candidate.  MeO' '  The remaining p o s s i b i l i t y , 81, has a C(7) - C(8) double bond with the C(9) 8 hydrogen configuration.  This l a t t e r assignment  i s necessary  for two reasons: (a) i f the C(9) a configuration prevailed, 81 would reveal chemical and physical properties c h a r a c t e r i s t i c of the lanost-7-ene system which i s not the case with abieslactone;  (b) i n the isomerization  from the  7-ene shown i n 81 to the lanost-9(11)-ene system,.therC(9) hydrogen i n migrating to C(8) must remain on the B face of the molecule thereby requiring that the C(9) configuration i s 3 i n the o r i g i n a l structure. Structure  81 can also account f o r ion e i n the mass spectrum of abies-  lactone since a retro Diels-Alder collapse of ring B would give an ion of correct composition without any further migrations being necessary.  99  Using molecular models and the octant rule, the 3-keto derivative of 81 should give a p o s i t i v e Cotton effect as i s observed f o r the 3-keto derivative of abieslactone. The observed NMR data of abieslactone i s not as e a s i l y explained. The C(32) methyl group i s a l l y l i c to the double bond and could be expected to resonate at lower f i e l d , however, this explanation does not account f o r the C(18) methyl s i g n a l unless i t s resonance frequency i s altered by the C(9) g configuration proposed i n 81. the  ring junction may a f f e c t the resonance frequency of the angular methyl  on the adjacenb c a r b o n . the  I t i s known that change of configuration at  The actual e f f e c t of the C(9) r3 configuration on  63  resonance frequencies i s not known.  Cycloartenyl acetate with the C(9)  g configuration had the resonance frequencies of the C(18)/C(32) methyl pmunR  assi  cmed  tr*  RT c r n a l . c  T  Q,in/Q Q3 ;  6  4  So^ie of this effect may be the  r e s u l t of deshielding by the cyclopropane but this again i s not known. Chemically structure 81 i s also favored, as oxidation with chromium t r i o x i d e could give the observed 8-ene-7,11-diketo  system without rearrange-  ment p r i o r to oxidation. X-ray analysis w i l l be conducted on this molecule i n order to s e t t l e the  complete structure.  At this time structure 81 would appear to be the  best postulate f o r abieslactone which i s then (23R)-3 oi —methoxy—9g—lanos ta— .. 7,24-diene-26,23-lactone.  MeO  81  100  With the assignment of structure 81 to abieslactone, the structures of the two minor components i s o l a t e d from P a c i f i c s i l v e r f i r may now be assigned.  Compound AA^, previously shown to be the 3-keto derivative of  abieslactone, would be (82).  (23R)-3-oxo-98-lanosta-7,24-diene-26,23-lactone  The other component, AA^, was shown to be  3-desmethylabieslactone  which may now be assigned as (23R)-3a-hydroxy-9pl-lanosta-7,24-diene26,23-lactone (83).  82  83  101  Experimental Throughout this work Merck s i l i c a gel G with added fluorescent indicator was used as adsorbent  i n thin layer chromatography (TLC).  The chromatograms,  0.3 mm. i n thickness, were a i r dried and activated i n an oven at 100°C f o r three hours.  The chromatograms were developed  i n chloroform and sprayed  with antimony pentachloride i n carbon t e t r a c h l o r i d e (1:2) unless  otherwise  noted. For preparative layer chromatography a thicker layer (0.5 mm.) of adsorbent was u t i l i z e d , with 0.01% Rhodamine 6G added as i n d i c a t o r . ^ 3  Spraying with antimony pentachloride was done only along one edge or not at a l l —.—  pic —  . - 1 o r ^ r - r - i f-\r-i ,  ,  —  4- K o n A o  T.T o c -. —  -r-. r . o o - i K i ^ r  r.Ti r n ..  n l f r ^ m ' A ~] e\ iI -i nl-i +• — - —-o-*- - —  mr\c r — ~  instances. •••• Column chromatography was performed on either Woelm s i l i c a gel or neutral alumina. III)  The preferred adsorbent was deactivated alumina ( A c t i v i t y  prepared by the addition of water as directed by the manufacturers.  Except i n larger scale work the solvents were d i s t i l l e d before use. The nuclear magnetic resonance (NMR) spectra were measured i n deuterochloroform at room temperature.  The NMR spectra were obtained at either  60 MHz using a J e l c o C-60, Varian A-60, or a Varian T-60 instrument 100 MHz using a Varian HA-10Q instrument.  or at  The positions of a l l NMR  resonances are given i n the Tiers x scale with tetramethylsilane as i n t e r n a l standard set at 10.0 units.  For multiplets the x values given represent  102  X  the center of the s i g n a l .  Mass spectra were measured on an Associated E l e c t r i c a l Industries MS 9 high resolution mass spectrometer or, where noted, on an Atlas CH 4 ' spectrometer.  High resolution molecular weight determinations were deter-  mined on the MS 9 spectrometer. Infrared (IR) spectra were measured on Perkin Elmer model 21, 137, or 457 instrument.  The samples were usually measured as KBr p e l l e t s ,  however, some were measured i n chloroform or neat.  The positions of  absorption maxima are quoted i n wave numbers (cm - -). -  1  U l t r a v i o l e t (UV) absorptions were measured i n methanol or ethanol on a Cary model 11 or model 15 spectrophotometer. A Jasco model UV/ORD/CD 5 spectropolarimeter was used to measure the c i r c u l a r dichroism (CD) and o p t i c a l rotatory dispersion (CRD) curves using dioxane as solvent. Melting points were determined on a Kofler block and are uncorrected. Elemental analyses were performed by Mr. P. Borda, University of B r i t i s h Columbia. I s o l a t i o n of triterpenes from P a c i f i c s i l v e r  fir  The bark of a P a c i f i c s i l v e r f i r growing i n the University of B r i t i s h Columbia forest preserve near Haney, B r i t i s h Columbia was removed from the log and a i r dried. through a 3 mm.  The dried bark was ground i n a Wiley m i l l to pass  sieve.  The ground bark was extracted with chloroform f o r  18 hours i n a large a l l glass Soxhlet extractor.  The chloroform was  evaporated to give a crude extract i n a y i e l d of 5.6% based on the weight of a i r dried bark extracted.  103  Ninety granis of crude extract were dissolved i n 300 mis. of hot ether and l e f t to cool whereupon 2.1 gms. by f i l t r a t i o n .  of l i g h t brown powder could be collected  TLC of the l i g h t brown powder showed the presence of three  compounds, the major one corresponded to an authentic sample of abieslactone received from Professor Uyeo. C r y s t a l l i z a t i o n of triterpene mixture The l i g h t brown powder (4.4 gms.) was dissolved i n the minimum amount of refluxing ethyl acetate. filtration.  The p r e c i p i t a t e (2.2 gms.) was  collected by  Examination of the p r e c i p i t a t e and the mother liquor by TLC  showed very l i t t l e , i f any, enrichment of the desired abieslactone. Column chromatography  of triterpene mixture  The p r e c i p i t a t e (2.2 gms.) was  chromatographed  on alumina (220 gms.).  Elution with 50% benzene in n p f rol p.imi e'fhpr (&S00 m i s . 1 oaye abi es lacto "!'? 1  (1.5 gms.).  Further elution with 50% methylene chloride i n benzene (400 mis.)  gave a compound coded AA  2  (75 mgs.).  E l u t i o n with methylene  (800 mis.) gave a compound coded AA^  chloride  (45 mgs.). •  Abies lactone Abieslactone obtained from the column was c r y s t a l l i z e d twice from e t h y l acetate to give a white s o l i d m.p. 2 5 3 ° C ) ; mixed m.p.  251 - 253°C ( l i t e r a t u r e m.p.  with authentic sample obtained from Professor Uyeo  28  (Kyoto University, Kyoto) was 251 - 253°C.  [<J,]  589  - 520°, [<fr]  [<j>]  - 26,600°.  [e]  + 290°, [ 9 ]  216  2 4 Q  carbonyl), 1660 (IH,  251 -  300  - 4,000°,  U]  2 5 0  CD (c, 0.0422) [ 6 ] 2 3 0  (C=C).  - 6,430°, [ & ) UV A^2  H  209  2 2 0  ORD  (c, 0.0422)  - 12,900°, [ $ ] 2 6 5  0°,  [e]  2 6 0  - 45,050°.  (log e 4.30).  apparent t r i p l e t , J = 1.7 Hz, H-C=C-C=0), 4.48  2 2 Q  [<f>] o ~ 412°, 70  - 45,500°,  + 225°, [ 6 ]  2 5 0  IR (KBr) 1745 NMR  (100 MHz)  + 450°,  (lactone 3.02  (IH, m u l t i p l e t , H-C=C) ,  104  5.05 (IH, multiplet, H-C-O), 6.72 (3H, s i n g l e t , OMe), 7.20 (IH, triplet., J = 1.8 Hz, e q u i t o r i a l H-C-OMe), 8.10 (3H, t r i p l e t , J = 1.7 Hz, v i n y l i c methyl), and 8.90, 9.00, 9.06, 9.08 (6 C-methyls). Mass spectrum (m/e) 468 (M), 453 (M-15), 436 (M--32), 421 (M-47), and. 314. H, 10.20  ;  C  31 48°3 H  468 .357  C  31 48°3  436 .330  C  36 44°2  393 .278  C  2 7 39°2  314 .224  C  21 30°2  272 .177  C  18 24°2  175 .149  C  13 19  H  H  H  H  H  H  (Found C, 79,.50;  requires C, 79.44; H, 10.32%) ; high resolution: requires  468.360,  453.333  C  30 45°3  requires  436.334,  421.310  C  29 41°2  requires  393.279,  339.232  C  23 31°2  requires  314.224,  299.203  C  20 27°2  requires  272.178,  233.155  C  15 21°2  requires  175.149.  H  H  H  H  H  requires  453. 336,  requires  421. 311,  requires  339. 232,  requires  299. 201,  requires  233.154,  I s o l a t i o n of AA The f r a c t i o n containing AA-, from chromatography was c r y s t a l l i z e d from e t h y l acetate to give a white s o l i d m.p. 236 - 238°C.  ORD (c, 0.0210)  [<j>]  589  + 150°, [<H 4 0 0 + 262°, [<J>] 5 + 348°, [<j>]330 + 701°, [<fr]  [<t»]  300  0°, [ < H 2 8 0 - - 3,230°, [<f)]260 - 4,950°, [<f>]224 - 25,400°, U ]  3  18,950°.  [e]  2 9 4  0  CD (c, 0.0210) [ e ] 3 3 0 0°, [ 6 ]  3 2 5  + 106°, [ 0 ]  3 1 0  317  + 1485°, 2 2 0  + 1,640°,  + 3,160°, [ e ] 2 6 0 + 710°, [ e ] 2 5 0 + 781°, [ e ] 2 3 0 - 5,750°, [ e ] 2 1 5  35,510°, [ 6 ] carbonyl).  2 1 2  NMR  - 30,190°. (60 MHz)  -  -  IR (KBr) 1745 (lactone carbonyl), 1705 (ketone  3.00 (IH, t r i p l e t , J =1.7  Hz, H-C=C-C0),  4.40 (IH, m u l t i p l e t , H-C=C), 5.02 (IH, m u l t i p l e t , H-C-O), 7.45 (2H, quartet, -CH -C0), 8.10 (3H, t r i p l e t , J = 1.7 Hz, v i n y l i c methyl), 8.90, 8.98, 9.18 2  (6 C-methyls). Mass spectrum (m/e) 452 (M), 437 (M-15), and 314. C, 79.52; H, 9.70; C  3 Q  H  4 4  0  3  requires C, 79.64; H, 9.73%).  (Found  105  I s o l a t i o n of AA  3  The f r a c t i o n containing AA^ from chromatography was from e t h y l acetate to give a white s o l i d m.p.  crystallized  249 - 250°C.  IR (KBr)  3520 (OH), 1760,1745 (lactone carbonyl); (CHC1 ) 1755 (lactone carbonyl). 3  NMR  (60 MHz)  3.02  (IH, t r i p l e t ) , 4.48  6.6  (IH, multiplet, H-COH), 8.10  (IH, m u l t i p l e t ) , 5.02  (IH, m u l t i p l e t ) ,  (3H, t r i p l e t ) , 8.98, 9.00, 9.03,  9.06  (6 C-methyls). Mass spectrum (m/e) 454 (M), 439 (M-15), 436 (M-18), and 314.  (Found C, 79.11; H, 10.07; C  3 Q  H  4 6  0  3  requires C, 79.25; H, 10.20%).  8-ene-7,11-diketo derivative of abieslactone Chromium t r i o x i d e (230 mgs.)  i n 90% acetic acid (10 mis.) was added  slowly i n t o a solution of abieslactone (258 mgs.) mis.) at 55°C.  i n hot a c e t i c acid (40  S t i r r i n g was continued at 60°C f o r 4.5 hours and then the T V . ,-.  „ -:  .1  washed with aqueous sodium carbonate and water, and dried over sodium sulfate.  Evaporation of the ether gave a yellow s o l i d  was chromatographed on s i l i c a g e l (15 gms.). a yellow s o l i d (200 mgs.). s o l i d m.p.  210 - 215°C.  (300 mgs.)  that  E l u t i o n with chloroform gave  C r y s t a l l i z a t i o n from methanol gave a yellow  Re-chromatography of the c r y s t a l l i n e product on  s i l i c a g e l (10 gms.) gave the 8-ene-7,11-diketo derivative (58) as a yellow s o l i d which was twice c r y s t a l l i z e d from methanol, m.p. ture m.p. carbonyl).  215 - 2 1 6 ° C ) . 28  Mp  (IH, m u l t i p l e t ) , 6.72  NMR  (60 MHz)  3.02  (IH, t r i p l e t ,  (3H, s i n g l e t , OMe), 8.10  t r i p l e t , J = 1.7 Hz, v i n y l i c methyl), and 8.69, 8.83, 9.05, 9.18 methyls).  (litera-  IR (KBr) 1740 (lactone carbonyl), 1678 (ketone  UV A -0H 274 my (log e 3.85).  J = 1.7 Hz), 5.05  217 - 219°C  (3H, (6 C-  106  Dihydroabieslactone Abieslactone  (44)  (50 mgs.)  i n tetrahydrofuran  over 10% palladium on charcoal (50 mgs.)  (15 mis.) was  hydrogenated  at room temperature for 2 hours.  The catalyst was removed by f i l t r a t i o n and the f i l t r a t e evaporated to give a white s o l i d . m.p.  C r y s t a l l i z a t i o n from e t h y l acetate gave a white s o l i d  216 - 218°C ( l i t e r a t u r e m.p.  219 - 2 2 1 ° C ) .  IR (KBr) 1770  28  carbonyl); (CHC1 ) 1765  (lactone carbonyl).  m u l t i p l e t , H-C=C), 5.53  (IH, m u l t i p l e t , H-C-O), 6.72  3  7.20  NMR  (60 MHz).  Hz, CH -CH=C0), and 8.96, 9.00, 3  9.06,  9.08  4.47 (IH,  (3H, s i n g l e t ,  (IH, t r i p l e t , J = 1.7 Hz, equatorial H-C-OMe), 8.74  J =6.3  (lactone  OMe),  (3H, doublet,  (6 C-methyls).  Mass  spectrum (m/e) 470 (M), 455 (M-15), 438 (M-32), 423 (M-47) and 316. (Found C, 78.95; H.-10.66; J —  —  — ——~  Abieslactone  "~  H 3 1  0 5 0  3 requires C, 79.10; H, 10.71%).  \ '• ^ /  (50 mgs.)  Adams catalyst (15 mgs.) was  C  i n acetic acid (30 mis.) was hydrogenated over  at room temperature for 40 hours.  The catalyst  f i l t e r e d o f f and the solvent evaporated to give a white s o l i d .  C r y s t a l l i z a t i o n from methylene chloride - methanol then hexane gave a white s o l i d m.p.  226 - 228°C ( l i t e r a t u r e m.p.  1770  (lactone carbonyl).  NMR  6.72  (3H, s i n g l e t , OMe),  7.20  8.98,  9.03, 9.10,  9.15  5.50  28  IR (KBr)  (IH, m u l t i p l e t , H-C-O),  (IH, t r i p l e t , J = 1.7 Hz, H-C-OMe), and  (6 C-methyls).  (M-15), 440-(M-32), 425 requires C, 78.76; H,  (60 MHz)  230 - 2 3 1 ° C ) .  (M-47).  Mass spectrum (m/e)  (Found C, 78.84; H, 11.18;  472 (M), 457 ^^^-^  11.09%).  Lithium aluminum hydride reduction of dihydroabieslactone Dihydroabieslactone  (25 mgs.)  i n tetrahydrofuran (10 mis.) was  stirred  107  with lithium aluminum hydride (10 mgs.) for 18 hours. water was added and the solvent was evaporated.  A small amount of  The residue was c a r e f u l l y  a c i d i f i e d with d i l u t e hydrochloric acid and extracted with ether. The extract was washed with water, dried (sodium sulfate) and evaporated to give a white residue.  Column chromatography on alumina  (1 gm.) of the  residue and e l u t i o n with ether gave the d i o l (69) ; c r y s t a l l i z a t i o n  from  petroleum ether - ether gave white needles m.p. 161 - 162°C ( l i t e r a t u r e m.p. 161 - 1 6 3 ° C ) . 28  IR (KBr) 3350 (OH).  NMR (60 MHz)  H-C=C), and 8.97, 9.00, 9.05, 9.08 (6 C-methyls). 474  4.47 (IH, m u l t i p l e t , Mass spectrum (m/e)  (M), 459 (M-15), 456 (M-18), 442 (M-32), 438 (M-36).  H, 11.36; C  3 1  H  5 4  0  3  Methylation of AA A_A .  fa ™r.s \  (Found C, 78.36;  requires C, 78.43; H, 11.46%). .  3  TT;,,,  rUfCAi^o.-!  thoroughly i n an i c e bath.  »tifh„ior,o  chloride (5 rr.lc.) and cooled  A few mis. of ethereal diazomethane was added  followed by a c a t a l y t i c amount of dry aluminum chloride.  Fresh diazo-  methane s o l u t i o n was added over a period of 3 hours to maintain a yellow color i n the solution.  The excess diazomethane was destroyed with a drop  of d i l u t e acetic acid.  The solution was f i l t e r e d and the f i l t r a t e washed  with water and dried (sodium s u l f a t e ) ; evaporation gave a s o l i d which was p u r i f i e d by preparative layer chromatography. R^ 0.40 was c o l l e c t e d .  (15 mgs.)  The band with  NMR of this band showed methoxy (6.72) and o l e f i n i c  proton (4.48) as i n abieslactone.  C r y s t a l l i z a t i o n from ethyl acetate gave  a white s o l i d m.p. 249 - 250°C, mixed m.p. with abieslactone 249 - 250°C. IR was superimposable  with that of abieslactone.  108  Oxidation of AA^ AA^  \  (20 mgs.)  i n dry pyridine (3 mis.) and chromium trioxide (15  were s t i r r e d at room temperature f o r 3 days.  mgs.)  The solvent was removed  in vacuo and the residue dissolved i n methylene chloride and washed with water, dried (sodium s u l f a t e ) , and evaporated. acetate gave a white s o l i d m.p.  C r y s t a l l i z a t i o n from ethyl  236 - 238°C, mixed m.p. with AA  2 38°C.  IR and TLC were i d e n t i c a l with those .of AA .  Dihydro  AA  AA  2  236 -  2  2  2  (30 mgs.) was dissolved i n tetrahydrofuran (10 mis.) and hydro-  genated over 10% palladium on charcoal (15 mgs.)  for 2 hours.  The catalyst  was removed by f i l t r a t i o n and f i l t r a t e evaporated.  C r y s t a l l i z a t i o n from  ethyl acetate gave a white.solid m.p.  ORD  Ul~„„ + 283°. m , ~ „ - ' - /UU  U]  2 ? 5  [G]  3 3 0  '  -  0°, [ G ]  + 388°. r<bl„„ + 1.440°. rcb"I  • - J O J  - 392°, [<j,]  265  - • - O J U  0°, [ * ]  1760 (lactone carbonyl). m u l t i p l e t ) , 8.73  NMR  •  '  + 360°, [ 0 ]  2 5 0  (60 MHz)  4.40  + 3.055°. [41..... 0°. " Z O O  218  2 2 Q  + 4,360°.  3 Q  H  4 6  0  3  CD (c, 0.0208)  + 12,960°.  IR (CHC1 ) 3  (IH, m u l t i p l e t ) , 5.50 (IH,  (3H, doublet), and 8.96, 9.00, 9.02,  (Found C, 79.04; H, 10.32; C  (c, 0.0208)  *  + 6,110°, [<f,]  2 2 0  + 3,170°, [ 6 ]  2 9 4  223 - 225°C.  9.20  (7 C-methyls).  requires C, 79.25; H, 10.20%).  Treatment of abieslactone with acetic acid Abieslactone (25 mgs.) was dissolved i n acetic acid (20 mis.) and heated at 50°C f o r 4 hours.  The solvent was evaporated and the residue  dissolved i n methylene chloride and washed with water, sodium bicarbonate solution and dried (sodium s u l f a t e ) . a white s o l i d m.p. The NMR  Evaporation and c r y s t a l l i z a t i o n gave  250 - 252°C, mixed m.p. with abieslactone 250 - 252°C.  spectrum was the same as that of abieslactone.  109  Treatment: of abieslactone with 1% concentrated hydrochloric acid i n acetic acid Abieslactone (20 Bigs.) was dissolved i n 1% concentrated hydrochloric acid i n acetic acid (by volume) (15 mis.) and heated at 50°C for 2 hours. The solvent was evaporated and the residue dissolved i n methylene chloride and washed with water, sodium bicarbonate s o l u t i o n and dried sulfate).  Evaporation gave a white s o l i d .  NMR (60 MHz)  (sodium  3.02 (IH, t r i p l e t ,  J = 1.7 Hz), 4.81 (non-integral, m u l t i p l e t , H-C=C), 5.05 (IH, multiplet, H-C-0) , 6.72 (3H, s i n g l e t , OMe), 7.20 (111, t r i p l e t , J = 1.7 Hz, e q u i t o r i a l H-C-OMe), 8.10 (3H, t r i p l e t , v i n y l i c methyl), and 8.94, 9.00, 9.05, 9.07, 9.12,  9.30 (6 C-methyls).  Cleavage of double bond i n dihydroabieslactone Dihydroabieslactone (30 mgs.) was dissolved i n ether (5 mis.) containJ.'  / n  1  _i„  \  ...„.3  ...„„,-,.„,  „ „ „  s o l u t i o n was l e f t f o r 10 days at room temperature.  "\  .-.AA^A  'TT-! o  The solution was then  saturated with hydrogen s u l f i d e gas and the black solution f i l t e r e d c e l i t e with thorough washing of the f i l t e r cake with chloroform.  through  The  f i l t r a t e was evaporated f i r s t on a rotatory evaporator under reduced pressure and l a t e r with a mechanical pump.  The residue was dissolved i n  ether (5 mis.) and periodic acid (30 mgs.) was added.  The solution was  s t i r r e d f o r 18 hours before being extracted with water and aqueous sodium bicarbonate s o l u t i o n .  The ethereal layer was dried over sodium s u l f a t e  and evaporated to dryness..  IR (CHCl^) 1760 (lactone carbonyl) , 1720  (aldehydo carbonyl), 1705 (ketone carbonyl).  110  S t r u c t u r a l Studies on W  A  from Western White Spruce  Discussion  It was known from e a r l i e r work i n our l a b o r a t o r i e s ' 2 5  2 5  that  triterpenes of the serratane family (84) were constituents i n at least one species of the genus Picea (spruce).  The presence or absence of  these novel triterpenes i n other species of the same genus would be of chemotaxonomic i n t e r e s t .  84 The two major triterpenes which had been i s o l a t e d from Sitka spruce [Picea sitchensis ]  2  5  were 3r3-methoxyserrat~14-en-21g-ol (24) and the  corresponding 3a-methoxy isomer (25). included the f i r s t r e p o r t e d  25  The minor constituents of the bark  i s o l a t i o n of the double bond isomer, Sa-  me thoxyserrat-13-en-21g-ol (28).  With authentic samples of these and  other serratenes available for comparison, the study of Western white spruce [P_- glauca (Moench) Voss. var. albertiana (S. Brown) Sarg.] and Engelmann spruce [P_. engelmannii Parry] was  undertaken.  Ill  28  26  E a r l i e r i n v e s t i g a t i o n of the bark extracts  of Western white spruce had  been conducted by Drs. Gletsos and Gladstone i n these l a b o r a t o r i e s . By a combination of column and thin layer chromatography,  65  •  they had succeeded  in i s o l a t i n g three triterpenes, a l l found previously i n Sitka spruce.  The  f i r s t compound was found to be i d e n t i c a l to 3a-methoxyserrat-14-en-218-ol (25).  The second compound was shown to be i d e n t i c a l to the corresponding  ketone, 3a-methoxyserrat-l4-en-21-one  (26).  The t h i r d compound was  epimeric with the f i r s t and was shown to be 33-methoxyserrat-14-en-218-ol (24).  112  A fourth triterpene code named W^,  a ketone, was" i s o l a t e d but was of  undetermined structure. The present work describes the i s o l a t i o n and s t r u c t u r a l studies of this new  triterpene.  The ground, a i r dried bark of Western white spruce was extracted i n a Soxhlet extractor with petroleum ether.  Evaporation of the petroleum  ether solution l e f t a crude extract as a brown gummy s o l i d . The crude extract was (Figure 26).  chromatographed  The f i r s t f r a c t i o n eluted was  on a large column of alumina evaporated to give a yellowish,  Bark  i—I—  Soxhlet extraction petroleum ether  Crude Extract  Chromatography on alumina A c t i v i t y I I I  Fraction  Figure 26.  Solvent  Compounds  petroleum ether  hydrocarbons., s t e r o l and wax esters  benzene  3a-methoxyserrat— 14-en—21(3—ol, 3a-methoxyserrat— 14-en—21-one, W^, and epimanool  benzene - chloroform  fatty alcohol, fatty ester  chloroform  33-methoxys.errat—14-en-21B-ol and B - s i t o s t e r o l  chloroform — methanol  unidentified polar components  T y p i c a l p u r i f i c a t i o n sequence of components from Western white spruce bark.  113  low melting wax. was  The TLC properties of this f r a c t i o n suggested that i t  non-polar and did not contain any of the desired The second f r a c t i o n was  gave a gummy s o l i d . plus two  W^.  eluted with benzene and removal of solvent  TLC analysis showed that this f r a c t i o n  contained  of the other known triterpenes, 3a-methoxyserrat-14-en-21(3-ol  and 3cx-methoxyserrat-14-en-3-one (26) . present whose TLC behavior was  29  In addition a fourth compound  (25) was  l i k e that of epimanool (29) or manool (31).  31  The t h i r d f r a c t i o n was  eluted with benzene - chloroform.  This  fraction  appeared to be mainly fatty ester, f a t t y alcohol. The tion.  fourth f r a c t i o n eluted with chloroform gave a s o l i d upon evapora-  TLC investigation of this f r a c t i o n using two d i f f e r e n t  systems revealed the presence of 3 - s i t o s t e r o l  (1) and  eh-218-ol (24) by comparison with authentic samples.  1  solvent  3B-methoxyserrat-14-  114  The l a s t f r a c t i o n consisted of chloroform - methanol and methanol washings of the column.  TLC investigation showed few d i s t i n c t spots but  showed that the f r a c t i o n contained mainly polar compounds. Fraction 2 from the above chromatography was chromatographed on A c t i v i t y I alumina (Figure 27). containing  E l u t i o n with benzene gave, a f r a c t i o n 6  and the known 3a-methoxyserrat-14-en-21-one  (26).  Further  e l u t i o n with chloroform (Fraction 7) gave manool or epimanool plus Same thoxys err at- 14-en-21|3-ol.  Fraction 2  Column chromatography on alumina A c t i v i t y I  Fraction  Solvent benzene  Compounds 3a-methoxyserrat-14-en-21-one, w  chloroform  Figure 27.  4  3a-methoxyserrat-14-en-21B-ol, epimanool  P u r i f i c a t i o n of Fraction 2.  The ketone f r a c t i o n (6) was chromatographed on alumina (Figure 28). I n i t i a l e-lution with petroleum ether gave a f r a c t i o n (8) which had TLC and NMR properties c h a r a c t e r i s t i c of a f a t t y ester.  Further elution  with petroleum ether - benzene gave Fraction 9 whose TLC showed the presence of 3a-methoxyserrat-14-en-21-one by comparison with an authentic sample.  Continued e l u t i o n with the same solvent gave Fraction 10  115  containing an unidentified o i l y component, which'on TLC analysis was of the desired  free  W/,.  Fraction 6  Column Chromatography on'alumina A c t i v i t y I I I  Compound  Solvent  Fraction  petroleum ether  fatty ester  pet.  ether.benzene (19:1)  3a-methoxyserrat-14-en-21-one  10  pet.  ether:benzene (9:1)  unidentified  11  pet.  ether:benzene (9:1)  W  i i?I  Figure 2 8.  4  m o f-H ar» r\ 1  P u r i f i c a t i o n of Fraction 6.  Continued e l u t i o n with petroleum ether:benzene (19:1) gave Fraction 11 containing W^.  A methanol wash of the column gave only base l i n e material  (Fraction 12) when examined by TLC. The i n i t i a l NMR  spectrum of Fraction 11 (m.p. 190 - 225°C) containing.  WY showed the resonance f o r an 0-methyl group at x 6.65 but i t had a s l i g h t shoulder to i t .  When this region was examined on an expanded scale  one large 0-methyl resonance was seen plus a small s i g n a l . recrystallizations  Several  of a portion of this f r a c t i o n provided a c r y s t a l l i n e  product (m.p. 200 - 230°C) which, upon sublimation, provided further p u r i f i c a t i o n (m.p. 215 - 225°C).  Finally, additional crystallizations  116  from ethanol and then sublimation gave an a n a l y t i c a l sample as a white s o l i d , m.p. 227 - 229°C.  Elemental analysis and high resolution mass  spectrometry of this material established the molecular formula ^3^50^2" The NMR spectrum  (Figure 29) of this compound had resonances  f o r an  0-methyl group (x 6.65); a one proton quartet (x 7.30, J = 5 and 11 Hz); a two proton quartet, (x 7.55, J = 6 and 8 Hz); and signals at x 8.92, 8.99 , 9.02, 9.12, 9.16, 9.17, and 9.23 integrating for. seven methyl  groups.  The x 7.30 quartet was assigned to an a x i a l proton geminal to the 0-methyl group.  This assignment  and Jae = 5 Hz.  was based on the coupling constants Jaa = 11 Hz  Similar coupling was observed i n  33-methoxyserratene  derivatives previously i s o l a t e d i n our laboratories. 25 The IR spectrum of fQsCA  had absorption at 1700 cm  anr! 1100 e r f (O-Me) 1  1  (C=0), 1665 cm  The ORD curve (Figure 30) showed  306 mu ([<j>] = +8800°) and a trough at 276 my ([<(.] = +2160°).  O rH  2 —  240  Figure 30.  280 ORD curve of W^.  320  360  400  A (my)  V  1  r- f-  118  The mass spectrum showed a molecular ion at m/e ion peaks at m/e  454.  439, 422, and 407 were assigned to loss of methyl, methanol,  and both methyl and methanol from the molecular ion. found at m/e  Other strong  Weaker ions were  221, 203, and 189.  A p r i o r i t h i s s p e c t r a l data suggested-a possible structure for W^. The occurrence of only seven C-inethyl groups i n the NMR serratane family of triterpenes which was  i s t y p i c a l of the  known to occur i n the same genus.  The degree of unsaturation i s seven, f i v e i n the pe.ntacyclic skeleton, one i n the carbonyl; the remaining degree of unsaturation would then be present as a tetrasubstituted double bond since no o l e f i n i c protons were observed i n the NMR  spectrum.  The natural occurrence of another serrat-13-ene  derivative had been reported from our laboratories e a r l i e r , b<or>d w.-ip. t nt"-?tiv^] p  ORD  u  2 5  so the double  ace;! o-npd tfhi^ ^ o s i t i o r i i n a serr2.tei.e skeleton.  The  c h a r a c t e r i s t i c s of both serrat-13-en-3-one and serrat-13-en-21-one  were known  19  and the ORD  curve r e p o r t e d function was  19  curve of W^ was  of the same shape as the  f o r a serrat—13-en-21-one derivative.  on a secondary carbon atom since there was  geminal to i t and i t s most probable location was  ORD  The 0-methyl ether only one proton  at C(3).  With these  results i n hand, the postulated structure of W^ would then be 3B-methoxyserrat-13-en-21-one  (85). This compound had not been previously reported  119  but was  accessible from known serrat-14-ene derivatives.  When I n u b u s h i  19  was  i n v e s t i g a t i n g the chemistry of the serrat-14-enes,  he found that by treatment with acid the corresponding serrat-13-enes were produced.  In the case of the 21-ketones,  13- enes was very nearly quantitative.  the formation of the serrat-  The desired compound for isomer-  i z a t i o n , 3$-methoxyserrat-14-en-21-one, had been i s o l a t e d  i n small  2 5  amounts but the corresponding 21g-hydroxy compound was more abundant both in this study and i n previous work on Sitka s p r u c e .  25  Thus the ketone obtained from the Jones oxidation of 14- en-218-ol was  subjected to treatment by a mixture of sulphuric acid and  a c e t i c acid at room temperature The NMR  33-methoxyserrat-  to give a new ketone, m.p.  19  241 - 243°C.  spectrum of the new product showed an 0-methyl s i g n a l at x  and seven C-methyl groups fx 8.90. There was no NMR  8.96,  9.05.  9.07,  9.16,  9.23,  9.26).  s i g n a l for the o l e f i n i c proton suggesting complete  i z a t i o n of the o l e f i n i c linkage to the tetrasubstituted p o s i t i o n . spectrum had bands at 1700  cm  curve had a peak at 304 my  ([cj>] = +6230°) and a trough at 270 my  +2035°).  6.67  -1  (C=0)  and 1100  cm  -1  ORD  ([cj>] =  454 with peaks of  439, 422, and 407 f o r loss of methyl, methanol, and  both methyl and methanol from the molecular ion. were also seen at m/e  The IR  (OMe), while the  The mass spectrum had a molecular ion at m/e  low i n t e n s i t y at m/e  isomer-  221, 203, and  Reasonably  intense peaks  189.  The above data was e n t i r e l y consistent with the expected oxidation and isomerization product, 38-methoxyserrat-I3-en-21-one (85). remembered that the data for W^  I t w i l l be  also suggested that structure.  However,  when both sets of data are compared s i g n i f i c a n t differences e x i s t .  The  120  positions of the C-methyl groups i n the NMR spectrum of each compound were different.  The ORD curve of each compound, while of the same shape, always  had W^ with greater i n t e n s i t y .  In addition, the IR spectrum of W^ had a  C=C stretching frequency at 1665 cm have this absorption.  -1  while the synthetic compound did not  The mass spectra had s i m i l a r fragmentations but  differed significantly i n intensities. With two d i f f e r e n t compounds, both of which could be assigned the same structure on evidence obtained so f a r , the problem became more difficult.  Skeletal rearrangements or methyl migrations, both w e l l known  processes i n triterpene chemistry, could not be readily excluded i n the double bond isomerization reaction.  In f a c t , i t was possible that W^ had '  the proposed structure while the acid isomerized product had suffered more o  ~w~-  ~ -  ~ -^J-^-v-w^.^ .  The most conclusive way to determine each structure would be by X-ray analysis.  Since each was a ketone, the derivative selected was a  p-bromophenylhydrazone.  Each ketone was i n d i v i d u a l l y reacted with p-  bromophenylhydrazine hydrochloride i n ethanol containing acetic acid. The synthetic ketone was reacted f i r s t and the derivative was c r y s t a l l i z e d from ethanol as pale yellow needles, m.p. 204 - 207°C ( d e c ) .  Ultraviolet  absorption at 233, 293, and 302 my suggested that the expected hydrazone had reacted further to give a bromoindole derivative.  The elemental formula  C2yH^2N0Br'C2H^0II, determined by elemental analysis indicated that one molecule of ethanol was associated with each molecule of the bromoindole derivative i n the c r y s t a l . The X-ray analysis of the c r y s t a l l i n e bromoindole derivative was  121  performed by Dr. F . H . Allen o f , t h i s department.  bb  The crystals were  o  orthorhombic, ja = 10.20, b_ = 11.12, c. = 31.23 A, space group P2^2^2^, with four formula units of Cg-zH^NOBr• ^ H t j O H i n the unit c e l l .  The i n t e n s i t i e s  of 2105 r e f l e c t i o n s with 262.100° were measured on a Datex-automated diffractometer using CuKa radiation and a 6 - 2 9  scan.  GE XRD6  The structure 86  was determined f o r the bromoindole using Patterson and Fourier techniques together with c a r e f u l restrained least-squares refinement of portions of the structure as they emerged from the electron density map, the f i n a l R being 0.086 f o r 1459 observed r e f l e c t i o n s .  The absolute configuration  depicted i n 86 was determined by the X-ray fluorescence technique' ^. 7  u  MeO  86  The X-ray analysis showed that the bromoindole had been formed i n the reaction.  More important to the present study was the fact that the  parent ketone was indeed the expected 3B-methoxyserrat-13-en-21-one  (85)  and no s k e l e t a l rearrangements had occurred. In the c r y s t a l l i n e state the X-ray analysis showed that the seven  122  member r i n g C i n 86 adopts a chair conformation w i t h some out-of-plane d i s t o r t i o n s about the C(13) - C(14) o l e f i n i c bond.  The plane through the  terpenoid nucleus i s s l i g h t l y concave to minimize the methyl - methyl i n t e r a c t i o n s , a common feature i n s t e r o i d a l s y s t e m s . 67  When W^ was reacted with p-bromophenylhydrazine i n the same manner as before, the product, a dark s o l i d , was obtained i n low y i e l d .  Attempts to  c r y s t a l l i z e the product from ethanol gave a dark s o l i d that d i d not have a sharp melting point but rather decomposed at temperatures above 300°C. This s o l i d when examined was not s u i t a b l e f o r X-ray a n a l y s i s . The d i f f i c u l t y i n obtaining a s u i t a b l e c r y s t a l l i n e d e r i v a t i v e f o r X-ray analysis from W^ prompted another approach.  A p o r t i o n of W^ was  reduced with sodium borohydride i n methanol to give a mixture of alcohols T  - ^-ic^* T  v.TOT-iti  nnr^f^o^  I^T7 T ~ . n o v ~ i v c  layer chromatography.  The NMR. spectrum  of the major a l c o h o l had s i g n a l s f o r the 0-methyl (T 6.66), a proton geminal to the hydroxyl (T 6.84, J = 7 and 9 Hz, q u a r t e t ) , a proton geminal to the methoxyl (x 7.32, J = 4 and 10 Hz, q u a r t e t ) , and C-methyl groups (x 9.02, . 9.04, 9.12, 9.18, 9.24, seven methyls). The I R spectrum had l o s t the carbonyl band found i n the ketone and now had a new absorption at 3495 cm f o r the OH s t r e t c h i n g frequency. A small p o r t i o n of the major reduction product r e f e r r e d to as W^ a l c o h o l was treated with bromoacetyl c h l o r i d e and sodium bromoacetate i n benzene to give the bromoacetate d e r i v a t i v e of W^. submitted f o r X-ray a n a l y s i s .  This d e r i v a t i v e was  These c r y s t a l s seemed s u i t a b l e f o r X-ray  a n a l y s i s and much data was gathered on t h i s m a t e r i a l .  However, s e v e r a l  months were spent on t h i s problem before i t was decided that the data  -1  123  collected could not be solved with methods currently available. While the above X-ray analyses were being done, the mass spectra of W^ alcohol and 33-methoxyserrat-13-en-21a-ol (87) were examined.  The mass  MeO'  87 spectra of several serratenes had been previously studied i n our laborato-  examined i n the mass spectrometer and the data had provided s t r u c t u r a l information.  59  The mass spectrum of 3a-methoxyserrat-13-en-21B-ol and had been p a r t i a l l y a n a l y z e d .  68  the present study are located at m/e that the ion at m/e  (28) was  available  The major fragment ions of interest to 221, 203, and 189.  I t was suggested  221 originates from rings A and B as a fragment q.  ion r m/e 189  ion q m/e 221  124  Ion r (m/e 189) i s produced by loss of methanol from the m/e 221 i o n . A metastable peak observed at m/e 161.6 tends to confirm this process. Ion s (m/e 203, ^-[5^23^  w  a  s  Prominent in both 3a-methoxyserrat-13-  en-21-one and 3a-methoxyserrat-13-218-ol.  68  This i o n i s not as prominent  i n serrat-14-ene derivatives and f o r this reason i t was f e l t  6 8  that i t  i s diagnostic of serrat-13-ene derivatives; however, the o r i g i n of this ion was not postulated. The mass spectra of 38-methoxyserrat-13-en-2ia-ol and  alcohol  were determined and are presented i n Figures 32 and 33 respectively.  The  elemental composition of ions where given were determined by high resolution mass spectrometry. As expected the mass spectrum of 3B-methoxyserrat-13-en-21a-ol ^/jo«">si~o  t-u^t-  87 i s seen at m/e 456. Figure 31.  fi-^Q^^'S"rrat~]  3 cn—213-cl. —  (87)  The molecular ion of  A p a r t i a l fragmentation pattern i s given i n  The molecular ion may lose methyl (M-15, m/e 441), water (M-18,  m/e 438), and methanol  (M-32, m/e 424)..  Ion t (m/e 423) would correspond  to loss of methyl and water from the molecular ion.  Loss of both methyl  and methanol from the molecular ion would account f o r ion u (m/e 409). Ion v (m/e 391) would correspond to loss of methyl, water, and methanol from the molecular ion. As i n the r e p o r t e d  68  spectrum of 3ct-methoxyserrat-13-en-21B-ol,  ions q, s, and r at m/e 221, 203, and 189 respectively are again prominent. A metastable ion at m/e 161.6 suggests that i o n q (m/e 221, ^ 1 5 ^ 2 5 ^  l°  methanol to give ion r (m/e 189, ^14^21^" It was found i n this study that a decomposition of ion x (m/e 235,  s e s  125  ion v m/e 391 ion u m/e 409  ion t m/e 423  M-15 m/e 439  M-18 m/e 436  M-32 m/e 424  ion s m/e 203  ion r m/e 189  ion r m/e 189  m/e Figure 31.  207  Fragmentation pattern of 33-methoxyserrat-13-en-21a-ol.  126  VO  o - in sr CO oo  co I <*  r-i  CD > OS CO  CD  CD . CD cn  O  I  S  rH CM  r\i: X co  I  CO rH  I  CM  •U  r-l  cd  CTCM •  u u  CM CO CO O CM  5  <u —  U  CD . CD  • crs  co  % o  rC  00.  4-1 0)  0 I  ca CD . IT)  CO  O  u  4J  U 0)  o, CO  CD . CD  CO CO  s CM CO  OJ  5-1  3  001  n Sc-  . CD  i  1  OS  Sr!  U I S N 3 1 N I  3AIltn3S  Ul  00  •H  Pn  12 7  C H 0) by loss of methanol to give ion s (m/e 203, C H ) could account 16 2 7 15 2 3 r  for a metastable ion at m/e 175.4.  Previously  68  an observed metastable  ion at m/e 175.4 was assigned to the m/e 203 ion losing 14 mass units to give the m/e 189 i o n . A loss Of 14 mass units i s not a favored process. An ion y (m/e 20 7, C-^H^^O) "'* st n  o u  §  n t  t  o  contain rings D and E as  there i s a metastable ion at m/e 172.8 f o r the loss of water to give ion r (m/e 189) . The high mass region of the mass spectrum of W^ alcohol (Figure 33) has many of the same peaks as the mass spectrum of 33-methoxyserrat-13en-21cx-ol (87).  In fact the general fragmentation pattern given i n  Figure 31 f o r 87 applies equally w e l l to W^ alcohol.  The molecular ion  i s seen at m/e 456; a M-15 ion at m/e 441, a M-18 ion at m/e 438, and a M—32 inn  m/e 424  ai*e  a l s o n re.sent -  of water and methyl as before.  T.nn t (m/e. 423^ raav a r i s e by 1 nsfi  Ion u at m/e 409 would arise by loss of  both methyl and methanol from the molecular ion and ion v (m/e 391) by loss of methyl, water, and methanol from the molecular ion. While this region of the spectra i s comparable, the r e l a t i v e i n t e n s i t y of several peaks i s very much d i f f e r e n t . more intense than,in 87.  The M-15 i o n of W^ alcohol i s  Loss of methanol gives a M-32 ion which i s four  times more intense i n W^ alcohol than i n 87.  Furthermore, loss of both  methyl and methanol gives ion u (m/e 409) which i s s i x times more intense i n W, alcohol than i n 87. 4 A f a c i l e loss of a methyl group i s often suggestive of an a l l y l i e methyl group.  In this case, however, 36-methoxyserrat-13-en-21a-ol (87)  would contain a methyl group i n the same p o s i t i o n as W^ alcohol i f the l a t t e r i s a serrat-13-ene derivative as thought.  The reason, although not  RELATIVE INTENSITY 0  5D  25  ro M^Q ^ ro oo X  .  75  '  I DO  ro  o ><;  IS ru LP '  LO CD • CD  LO  err  CD  LO  VO <  —-O  tort oo  "  M  T  l o  ro  •o c VO  •e- I C O I - *  0000  •S I  LJl'  o  2ZT  U i  •t- u i  S  129  known, may be accounted f o r i f there was greater s t e r i c crowding about the methyl group(s) lost i n  alcohol as opposed to 87.  The differences observed i n the loss of methanol i s puzzling.  On the  basis of NMR spectra the methoxyl groups i n both compounds should have the same 3 orientation.  A more q u a l i t a t i v e observation i s that the TLC proper-  ties of these compounds are the same, either as the parent ketone or as the alcohol.  From previous work i n our laboratories i t was found that the  3a-methoxyserratene derivatives had d i f f e r e n t TLC properties from the 33-methoxyserratene d e r i v a t i v e s .  70  I t would seem f o r both of these reasons  that both compounds have a 33 methoxyl substituent. E a r l i e r i n this thesis the mass spectra of several lanosterol derivatives were presented. ^-^.nnni  ar, A  I r\^r  r*  I t can be seen from these spectra that configura-  on f o rm ?  OP  ^1  ^Vioprroc  n_n  f"^^ molecule  f a c i l i t y of the loss of the C(3) f u n c t i o n a l i t y .  C E " affect  the  This e f f e c t has also been  7 1  reported i n the case of 5a- and 53~steroids. Returning to the spectra of  1  alcohol, attention i s directed to the  ions at m/e 235 (x), 221 (q), 207 ( y ) , 203 ( s ) , and 189 ( r ) .  A l l these  i •  ions were seen i n the spectrum of 87. As before ion x (m/e 235, C^gH^O) can lose methanol to give ion s (m/e 203, O-^H^) and a metastable ion at m/e 175.4.  Both the ions at m/e 235 and 203 are approximately the same  r e a l t i v e i n t e n s i t y as they were i n 33-methoxyserrat-13-en-21a-ol (87). As i n the spectrum of 87, ion q (m/e 221, C-15H25O)  c  to give ion r (m/e 189, C-^H^) •  a  n  lose methanol  In comparison with the previous spectrum  ion q i s about a factor of f i v e less intense than before. In contrast to t h i s observation, ion y (m/e 207, C..-H,„0) i s now a  130  factor of f i v e more intense than i n the previous spectrum.  As before,  ion y can lose water to give ion r (m/e 189) and a metastable ion at m/e 172.7. The o r i g i n of the m/e 221 ion has been p o s t u l a t e d . t o involve a 68  hydrogen transfer from the C(26) methyl group to C ( l l ) with rupture of the C(9) - C ( l l ) bond.  For this type of fragmentation to occur i t i s  necessary f o r the methyl group to be s t e r i c a l l y close to C ( l l ) .  The  postulated o r i g i n of ion y (m/e 207), on the other hand, requires a hydrogen transfer to C(13) from the C(26) methyl group and, as could be expected, the proper s p a t i a l arrangement i s r e q u i s i t e again. In the normal serratene skeleton the C(26) methyl group i s B-oriented which, on the basis of molecular models, would seem to place i t closer  (m/e 221) could be expected to be more favored than fragmentation leading to i o n y (m/e 207) .. I f the C(26) methyl group i s a-oriented, a change i n conformation of r i n g C occurs.  Now the C(26) methyl group i s closer to C(13) and  farther away from C ( l l ) than i t was i n the normal serratenes.  The expected  r e s u l t of this s i t u a t i o n would be an increase i n the m/e 207 ion at the r e l a t i v e expense of the m/e 221 ion. Although the B-orientation of the C(26) methyl group p r e v a i l s . i n the normal serratane family, the a isomer could be expected on biogenetic grounds.  The biosynthesis of the serratenes has not been studied but i s  thought to occur v i a the o n o c e r i n s .  20  Protonation of C(26) i n  131  a-onocerin  (11) followed by ring closure and loss of a proton from C(13)  could give serrat-13-ene-3B,21a-diol (88) d i r e c t l y  88  Figure 34.  (Figure 34).  89  Postulate f o r the biosynthesis of 8a- and 8t3-serrat-13-ene derivatives.  However, protonation at C(26) makes C(8) planar and subsequent completion of the reaction may lead to the a-orientation f o r the C(26) methyl group as i n 8a-serrat-13-ene-3B.,21a-diol (89) , thereby allowing structure 90 to be postulated f o r  alcohol.  This postulate f o r the structure of  skeleton i s compatible with a l l evidence so far obtained.  The ORD  curve of W. suggests that rings D and E are the same as found i n  132  serrat~13-en~21-one derivatives.  The NMR  spectrum suggests seven C-methyl  groups and a 3 methoxyl group on a secondary carbon atom. ions at m/e  221 and m/e  by this system.  The formation of  207 i n the mass, spectrum would also be  explained  Furthermore, the conformational and configurational  changes may be the factor responsible ions observed f o r W.  for the more intense M-15  and  M-32  alcohol.  90  91  The above evidence does, not prove the structure of W. alcohol, but 4  would suggest that  alcohol i s 38-meth.oxy-8a-serrat-13-en-21a-ol  In turn the parent ketone, W^, (91)..  would be  (90) .  3B-methoxy-8a-serrat-13-en-21-one  133  Experimental  Throughout t h i s work Merck s i l i c a g e l G w i t h  added f l u o r e s c e n t  i n d i c a t o r was' used as adsorbent i n t h i n l a y e r chromatography chromatograms, 0.3 mm.  form u n l e s s s t a t e d o t h e r w i s e . antimony p e n t a c h l o r i d e  noted.  The  i n t h i c k n e s s , were a i r d r i e d and a c t i v a t e d i n an  oven at 100°C f o r t h r e e h o u r s .  with  (TLC).  The chromatograms were developed i n c h l o r o Detection  of compounds was done by s p r a y i n g  i n carbon t e t r a c h l o r i d e (1:2) unless  otherwise  .  F o r p r e p a r a t i v e l a y e r chromatography a t h i c k e r l a y e r of adsorbent . (0.5 mm.) S p " 5 v i n ( >  was u t i l i z e d w i t h wi  th  gn,timonv  0.01% Rhodamine 6G added as i n d i c a t o r  np,nt.arhl.nri  Hp  s n l iiti  nn  w-?s  donp only  3 4  .  along one edge  or n o t a t a l l as d e t e c t i o n o f bands was p o s s i b l e w i t h u l t r a v i o l e t  light i n  most i n s t a n c e s . Column chromatography was u s u a l l y performed on Woelm n e u t r a l alumina. The  p r e f e r r e d adsorbent was d e a c t i v a t e d alumina ( A c t i v i t y I I I ) p r e p a r e d by  the a d d i t i o n of water as d i r e c t e d by the m a n u f a c t u r e r s .  I n column chromato-  graphy o f the crude e x t r a c t where l a r g e q u a n t i t i e s of adsorbent were used, Shawinigan alumina was d e a c t i v a t e d by a d d i t i o n o f 3% o f a 10% a c e t i c a c i d solution.  Except i n l a r g e s c a l e column chromatography, the s o l v e n t s were  d i s t i l l e d b e f o r e use. The  n u c l e a r magnetic resonance  chloroform  a t room temperature.  (NMR) s p e c t r a were measured i n d e u t e r o -  The NMR s p e c t r a were o b t a i n e d  at e i t h e r  60 MHz u s i n g a J e l c o C-60, V a r i a n A-60, or a V a r i a n T-60 i n s t r u m e n t  or a t  100 MHz u s i n g a V a r i a n HA-100 i n s t r u m e n t .  reso-  The p o s i t i o n s o f a l l NMR  134  nances are given i n the Tiers T scale with tetramethylsilane as i n t e r n a l standard set at 10.0 units.  For multiplets the x values given represent  the center of the s i g n a l . Mass spectra were measured on an Associated E l e c t r i c a l Industries MS 9 high resolution mass spectrometer or, where noted, on an Atlas CH 4 spectrometer.  High resolution molecular weight determinations wer deter-  mined on the MS 9 spectrometer. Infrared (IR) spectra were measured on Perkin Elmer model 21, 137, or 457 instrument.  The samples were usually measured as KBr p e l l e t s , however,  some were measured i n chloroform or carbon tetrachloride or neat. positions of absorption maxima are quoted i n wave numbers  The  (cm ). -1  A Jasco model UV/ORD/CD 5 spectropolarimeter was used to measure the o n t i c a l rotatorv d i s D e r s i o n (ORD) curves using methanol as solvent. Melting points were determined on a Kofler block and are uncorrected. Elemental analyses were performed by Mr. P. Borda, University of B r i t i s h Columbia. Extraction of Western white spruce The bark f o r this study was obtained from a Western white spruce tree growing i n the Prince George region of B r i t i s h Columbia.  The bark was  a i r dried and ground i n a Wiley m i l l to pass through a 3 mm. sieve. A i r dried bark (1,850 gms.) was extracted with petroleum ether for 18 hours i n a large glass Soxhlet extractor.  Evaporation of the solvent gave a crude  extract (40 gms.) as a thick, brown, gummy wax. Column chromatography of crude extract Crude extract (80 gms.) was applied i n petroleum ether (2 1.) to the top of a column prepared from deactivated Shawinigan  alumina (5 l b s . ) .  135  E l u t i o n of the column was with various solvents as below. Fraction  Solvent (volume, 1.)  Wt.(gms.)  Compounds  1  petroleum ether (14)  8.0  hydrocarbons, s t e r o l and wax esters  2  benzene (13)  5.6  3a-methoxyserrat-14-en213-ol, 3a-methoxyserrat14-en-21-one, W^, ( e p i ) manool  3  20% chloroform i n benzene (8)  2.9  f a t t y e s t e r , f a t t y alcoho  4  chloroform (5)  4.2  3r3-methoxyserrat-14-en21g-ol, g - s i t o s t e r o l  13.0  u n i d e n t i f i e d components  5  50% methanol i n chloroform (2) methanol (4)  Fraction 1 A p o r t i o n of F r a c t i o n 1. when examined by TLC showed the presence of at .^ t-'h-.-oo ^ Q r n p ^ . ^ ^ o / ' p . Q . 5 7 0 . 6 7 , ard 0 . 7 2 ) . This f r a : }  components with chromatographic properties l i k e W  4  and was not further  examined. Fraction 2 A p o r t i o n of F r a c t i o n 2 when examined by TLC showed the presence of at l e a s t three compounds: (epi)manool (R^ 0.29), 3a-methoxyserrat-14-en-2ip-ol (R^ 0.36), and  (R^ 0.42) when compared with authentic  samples.  Fraction 3 A p o r t i o n of Fraction 3 was compared with authentic l i g n o c e r o l a l c o h o l (C H OH) 2 4  4 g  (60 MHz)  and showed the same R  f  of 0.25. IR ( f i l m ) 3300 (OH).  8.75 (broadened s i n g l e t ) .  NMR  F r a c t i o n 3 seemed to be mainly f a t t y  a l c o h o l and was not f u r t h e r examined. Fraction 4 A p o r t i o n of F r a c t i o n 4 when examined by TLC showed the presence of at  136  least  two compounds, 38-methoxyserrat-14-en-213-ol (R^ 0.17) and 8 - s i t o s t e r o l  (Rr. 0.22), when compared  t o a u t h e n t i c samples.  Fraction 5 A p o r t i o n o f F r a c t i o n 5 when examined by TLC showed o n l y p o l a r and was n o t examined  further i n - t h i s study.  Column chromatography  of F r a c t i o n 2  F r a c t i o n 2 (10.6 gms.) was d i s s o l v e d (100 mis.) and a p p l i e d I , 600 gms.).  i n 50% benzene  i n petroleum ether  to the top of a column p r e p a r e d from alumina  E l u t i o n w i t h benzene  (1.5 1.) gave a y e l l o w o i l y  Further  (3.5 1.) gave F r a c t i o n 6 (3.9 gms.) c o n t a i n i n g W^ and  3a-methoxyserrat-14-en-21-one gave F r a c t i o n  (Activity  material  which was examined by TLC and found to be f r e e o f the d e s i r e d W^. e l u t i o n w i t h benzene  compounds  by TLC e x a m i n a t i o n .  rr-.-ntr'ini.p.*?  ~i ^6 : 5  ( e p i ) m a n o o l by TCL e x a m i n a t i o n .  E l u t i o n with  chloroform  3^ ~r .p-th x^ 9 rr?t"~l^ ~pp— 2!R~ >l n d J  Washing  n  ri  7  r:>  L  r  a  the column w i t h methanol gave, upon  e v a p o r a t i o n , a brown r e s i d u e which was seen t o be p o l a r m a t e r i a l by TLC e x a m i n a t i o n and was n o t f u r t h e r  investigated.  I s o l a t i o n o f W^ Fraction 300 gms.).  6 (3.9 gms.) was chromatographed on alumina ( A c t i v i t y I I I ,  E l u t i o n with petroleum ether  as a y e l l o w i s h waxy s o l i d NMR  (60 MHz)  (R  f  0.76).  IR  (3.5 1.) gave F r a c t i o n 8 (800 mgs.) (CHCI3)  carbonyl).  8.75 (broadened s i n g l e t ) .  E l u t i o n w i t h 5% benzene i n p e t r o l e u m e t h e r (415 mgs.) c o n t a i n i n g authentic  1725 ( e s t e r  (1.5 1.) gave F r a c t i o n 9  3a-methoxyserrat-14-en-21-one  by TLC comparison w i t h  sample.  E l u t i o n with  10% benzene  i n petroleum ether  (2 1.) gave an u n i d e n t i f i e d  137  o i l (Fraction 10) which was not further examined i n this study. Continuing to elute with 10% benzene i n petroleum ether gave the desired  (1,100 mgs.) i n Fraction 11 when examined by TLC.  Eluting with methanol (1 1.) gave only polar or baseline material when examined by TLC. Properties o f A portion of Fraction 11 (600 mgs.) was c r y s t a l l i z e d from ethyl acetate to give a white s o l i d m.p.  190 - 225°C.  s l i g h t shoulder at 6.67, OMe).  NMR  (60 MHz)  6.65  (3H, s i n g l e t with  Several r e - c r y s t a l l i z a t i o n s from ethyl  acetate provided a c r y s t a l l i n e product m.p. tion provided further p u r i f i c a t i o n , m.p.  200 - 230°C which, upon sublima-  215 - 225°C.  Additional c r y s t a l -  l i z a t i o n s from ethanol and the sublimation gave an a n a l y t i c a l sample 227  -  [<j>]  3  229°C.  5  0  ORT)  0.02011  f . c  + 3,790°, [ cj)]  3 3 0  + 8,800°, [ * ]  frf> 1 ,  + 4,870°,  + 7,860°, [<f>]  :  3 0 0  +  280  [<j>]3  677°.  2 0  rcb 1  + 6,500° ,  + 2,430°, W  276  +  677°.  [<j>]  3  1  (3H, s i n g l e t , OMe), 7.30  +  + 8,266° ,  + 2,160°, [ $ }  IR (KBr) 1700 (ketone carbonyl), 1665 (C=C), 1100 (OMe). 6.65  0  r<b 1 . —  NMR  2 5 Q  H  r  e  c  l  u  i  r  e  3  0  6  + 5,690°.  9.12,  (7 C-methyls). Mass spectrum (m/e) 454 (M), 439 (M-15),  422 (M-32), 407 (M-47), 221, 203, and 189. 31 50°2  [<j>]  (IH, quartet, J = 5 and 11 Hz, a x i a l H-C-OMe), 2  C  2.300°.  (100 MHz)  7.55 (2H, quartet, J = 6 and 8 Hz, -CH C0-), and 8.92, 8.99, 9.02, 9.16, 9.17, 9.23  m.p.  s  c  >  (Found C, 81.74; H, 11.23;  81.88; H, 11.08%; high resolution 454.379 3 C  H 1  O 5 0  2  requires 454.381; 4 3 9 . 3 5 6 _ C H 0 ( M - 1 5 ) requires 439.357. 2  3B-methoxyserrat-13-en-21~one (85) Jones reagent was prepared by dissolving chromium t r i o x i d e (2.668 i n concentrated s u l f u r i c acid (2.13 mis.) and d i l u t i n g to 10 mis. with  gms.)  138  water i n a volumetric flask. 38-methoxyserrat-14-en-218-ol  (24) (140 mgs.)  was dissolved i n  acetone (50 mis.) and was oxidized with Jones reagent (0.2 mis.).  After •  30 minutes the solution was f i l t e r e d and the f i l t r a t e evaporated to dryness. This s o l i d was dissolved i n a mixture of acetic acid (15 mis.) and concentrated s u l f u r i c acid (1 ml.) and l e f t for 18 hours.  The acid mixture was  poured onto crushed i c e ; a f t e r the i c e had melted the white s o l i d was extracted with methylene chloride.  The methylene chloride extract was  washed with water, 5% sodium bicarbonate s o l u t i o n , saturated s a l t solution and dried over sodium s u l f a t e . of yellowish s o l i d . 10 gms.).  Evaporation of the solvent gave 120  This s o l i d was chromatographed on alumina ( A c t i v i t y I,  Prolonged e l u t i o n with petroleum ether gave a few mgs.  vellow o i l .  (c, 0.0201) [<fr]  W  2,843°, [<j)]  290  of pale  Elution with benzene gave a white soHH; crystal!) i zstH °r> from  ethyl acetate and sublimation gave a n a l y t i c a l sample m.p. ORD  mgs.  33Q  650  + 670°, [<j>]  + 3,795°, [<f>]  3 2 Q  + 3,650°, [«f>]70 2  carbonyl), 1100  589  (OMe).  +  2  >  0 3 5  NMR  + 670°, [<j>]  400  + 4,740° , [<j>]  °>  3 1 0  [*]  2 5 0  (100 MHz)  + 5,014°. 6.67  241 - 243°C.  + 1,762°, [<fr]  + 5 ,967° , [*>]  3  + 6 ,230° ,  7.30 (IH,  (2H, quartet, J = 6 and 8  Hz, -CH -C0-), and 8.90, 8.96, 9.05, 9.07, 9.16, 9.23, 9.26 2  4  +  IR (KBr) 1700 (ketone  (3H, s i n g l e t , OMe),  quartet, J = 5 and 11 Hz, a x i a l H-C-OMe), 7.55  Q  350  (7 C-methyls).  Mass spectrum (m/e) 454 (M), 439 (M-15), 422 (M-32), 407 (M-47), 221, 203, and 189.  (Found C, 81.65; H, 11.14; C  H 3 1  5 0  0  2  requires C, 81.88; H, 11.08%).  Bromoindole derivative (86) 33-methoxyserrat-13-en-21-one  (25 mgs.)  was dissolved i n warm ethanol  (5 mis.) containing a few drops of acetic acid; p-bromophenylhydrazine  139  hydrochloride (100 mgs.) was added and the s o l u t i o n r e f l u x e d f o r 10 hours and then l e f t at room temperature f o r a f u r t h e r 10 hours.  The e t h a n o l i c  s o l u t i o n was poured i n t o water and the p r e c i p i t a t e c o l l e c t e d by f i l t r a t i o n The p r e c i p i t a t e was c r y s t a l l i z e d twice from ethanol to give 86 as yellow needles m.p. 204 - 207°C ( d e c ) . 1  IR (KBr) 3280 (NH) .  UV X max  (log e 3.64), 29 3 my. ( l o g e 2.81), 302 my (log e 2.69). H, 9.09; B r , 12.38; C^H^NOBr• C ^ O H  M e 0 H  233 my  (Found C, 71.30;  requires C, 71.24.; H, 9.06; B r ,  12.50%). Reaction of  with p-bromophenylhydrazine  (25 mgs.) was d i s s o l v e d i n warm ethanol (5 mis.) containing a few drops of a c e t i c a c i d ; p-bromophenylhydrazine hydrochloride (100 mgs.) was added and the s o l u t i o n r e f l u x e d f o r 10 hours and l e f t at room temperas ti.ir<? f o 3p.0th.er .10 hoi."*Rr  The P t h ^ n o L i o sni.ut 'op 1  and the p r e c i p i t a t e c o l l e c t e d by f i l t r a t i o n .  VMR  noy^cl i n t o water  The p r e c i p i t a t e was  c r y s t a l l i z e d from ethanol to give a s m a l l amount of dark s o l i d which decomposed on heating over 300°C with no sharp melting p o i n t , alcohol  -  (100 mgs.) was d i s s o l v e d i n methanol (30 mis.) and was reduced with sodium borohydride (300 mgs.) over a period of 2 hours.  Excess sodium  borohydride was destroyed with a few drops of d i l u t e h y d r o c h l o r i c a c i d and the solvent was evaporated to leave a white paste.  This paste was d i s - .  solved i n water and chloroform and the chloroform l a y e r was washed with water and d r i e d over sodium s u l f a t e .  Evaporation of the solvent gave  110 mgs. of white s o l i d which was applied to a preparative l a y e r chromatogram  and developed twice i n chloroform.  The top band when extracted  140 from the adsorbent gave 15 mgs. of orange-red s o l i d ; TLC showed t h i s to be a mixture of at l e a s t two compounds. the  The lower band when extracted from  adsorbent gave 80 mgs. of orange-red s o l i d .  The 80 mgs. of s o l i d was  flushed through a short column of alumina (5 gms.) using benzene as the eluant.  Evaporation of the benzene gave a white s o l i d (65 mgs.).  Crys-  t a l l i z a t i o n from ethanol gave an a n a l y t i c a l sample m.p. 299 - 300°C. IR  (KBr) 3495 (OH). NMR (100 MHz)  6.66 (3H, s i n g l e t , OMe), 6.84 (IH,  quartet, J = 7 and 9 Hz, a x i a l H-C-OH), 7.32 (IH, quartet, J = 4 and 10 Hz, a x i a l H-C-OMe), and 8.95, 9.02, 9.04, 9.12, 9.18, 9.24 (7 C-methyls). Mass spectrum (m/e) 456 (M), 441 (M-15), 438 (M-18), 424 (M-32), 423 (M-33), 409 (M-47), 391 (M-65), 235, 221, 207, 203, and 189. H, 11.16; 031^52^2  rec  l ^ u  res  -  (Found C, 81.65;  C, 81.52; H, 11.48%); high r e s o l u t i o n  456.396  C-,H_-0.  renin'res  456.397.  235.206  CH.....0  renuires  235.206.  221.190  C H 0  requires  221.191,  207.174  C'^H^O  requires  207.175,  203.178  C H„„ 15 ^3  requires  203.180,  189.164  C, H 14 21  requires  129.164.  Bromoacetate d e r i v a t i v e of  alcohol  1 5  2 5  1C  a l c o h o l (15 mgs.) was d i s s o l v e d i n benzene (10 mis.) and sodium bromoacetate (50 mgs.) was added and s t i r r i n g was s t a r t e d .  Bromoacetyl  c h l o r i d e (30 mgs.) was d i l u t e d with benzene (0.5 mis.) and added dropwise. The f l a s k was f i r m l y stoppered and s t i r r i n g was continued f o r 2 days. The benzene was washed with water, 5% sodium bicarbonate s o l u t i o n , water and  d r i e d over sodium s u l f a t e .  Evaporation of the benzene l e f t a white  s o l i d which was c r y s t a l l i z e d from methylene c h l o r i d e - hexane to give white c r y s t a l s m.p. 245 - 246°C. (2H,  IR (KBr) 1725 (C=0).  s i n g l e t , 0CCH Br), 6.63 (3H, s i n g l e t , OMe). ?  NMR (60 MHz) 6.13  Mass spectrum (m/e)  141  578 and 576 (M). 3g-methoxyserrat-13-en-21a-ol (87) 3g-methoxyserrat-13-en-21-one  (35 mgs.) was dissolved i n methanol  (20 mis.) and reduced over a period of 2 hours with sodium borohydride (50 mgs.).  Excess sodium borohydride was destroyed with a few drops of  d i l u t e hydrochloric acid and the s o l u t i o n evaporated to dryness to give a white paste.  The paste was dissolved i n water and chloroform and the .  chloroform solution washed with water and dried over sodium s u l f a t e . Evaporation gave a white s o l i d which was p u r i f i e d by preparative layer chromatography.  The main band of material was extracted with chloroform to  give an orange-red s o l i d which was flushed through a short alumina column with benzene as the eluant.to give a white s o l i d .  An a n a l y t i c a l sample was  obtained b^ cr^^st^lli'^atio -  — 9fift n.  r  1  3450 (OH).  1  NMR (60 MHz)  fr ? . T p p * b a ' ' l 0  11  -  m-?: 1  nr  0  TT^  ^I^R^^  6.66 (3H, s i n g l e t , OMe), and 8.95, 9.01, 9.09,  9.13, 9.17 (7 C-methyls). Mass spectrum (m/e) 456 (M), 441 (M-15), 438 (M-18), 424 (M-32), 423 (M-33), 409 (M-4 7), 391 (M-65), 235, 221, 207, 203, and 189.  (Found C, 81.46; H, 11.84;  H, 11.48%); high resolution  456.397  C  C  5  H 3 1  2  H 3 1  0 5 2  2  0  r 2  e  q  u  i  r  e  requires  s  c  » 81.52; 456.397,  235.205  C H 0  requires  235.206,  221.190  C^H^O  requires  221.191,  207.174  C H 0  requires  207.175,  203.179  C^H^  requires  203.180,  189.165  l 5  2 7  1 Z  C  2 3  H 1  4  2  1  requires  189.164.  142  Chemosys tenia t i c studies on Engelmann spruce  Discussion  Investigations of the bark extractives of Sitka and Western white spruce had revealed the presence of readily i s o l a b l e amounts of methoxyserratene derivatives.  I t was of taxonomic interest to see i f the  occurrence of methoxyserratenes was a chemosystematic feature of the genus Picea.  For this reason a t h i r d member of the genus was examined.  Engelmann spruce [Picea engelmannii Parry] i s common throughout the i n t e r i o r mountain region of southern and central B r i t i s h Columbia. I t often forms hybrids with white spruce i n B r i t i s h Columbia but grows i n pure-stands i n Colorado.  The bark of Engelmann spruce for this study was  obtained from a region near Fort C o l l i n s , Colorado. The bark as obtained was a i r dried and ground so i t would pass through a 3 mm. sieve.  A portion of the ground bark was continuously extracted i n  a large Soxhlet apparatus with petroleum ether.  Upon evaporation of the  solvent a brown gummy crude extract was obtained i n 3% y i e l d based on the weight of the a i r dried bark- extracted. Following the petroleum ether extract, the bark was extracted with benzene to give, upon evaporation, a dark residue amounting weight of the o r i g i n a l a i r dried bark.  to 1% of the  F i n a l l y , the bark was extracted  with methanol to give a syrupy residue corresponding to 21% of the weight of the o r i g i n a l a i r dried bark.  The benzene and methanol extracts were  not further examined i n this study. A portion of the petroleum ether extract was chromatographed activated alumina (Figure 35).  The f i r s t f r a c t i o n was eluted with  on de-  143  Bark  petroleum ether Soxhlet extraction Crude Extract  Column Chromatography on alumina A c t i v i t y III 1  Fraction  Solvent  petroleum ether pet. ether-benzene  (4:1)  benzene-chloroform  (4:1)  hydrocarbons, s t e r o l and wax  esters  wax ester, (epi)manool, abienol  chloroform  wax ester, f a t t y alcohol, (epi)manool, unidentified  chloroform  8-sitosterol  chloroform-methanol  Figure 35.  Compound(s)  (1:1)  u n i d e n t i f i e d polar components  Separation sequence of components of Engelmann spruce bark.  144  petroleum ether and petroleum ether - benzene to give only o i l y material. The TLC of this .fraction revealed very non-polar materials i n the nature of hydrocarbons and wax or s t e r o l esters. The second f r a c t i o n was eluted with benzene - chloroform to give a syrupy f r a c t i o n .  Thin layer chromatography showed at least three compounds  (wax ester, (epi)manool, and abienol) were present. The t h i r d f r a c t i o n was eluted with chloroform to give a waxy s o l i d . The TLC of this f r a c t i o n showed the presence  of several components much  l i k e the components of the second f r a c t i o n . The fourth f r a c t i o n eluted with chloroform gave a white s o l i d . Comparison of this material with authentic g - s i t o s t e r o l (1) by TLC using two d i f f e r e n t solvent systems suggested W n i t e  OUJ.IU  they were i d e n t i c a l .  m . p .  J - J - J  —  X H K I  Crystallization  v.i ( p - s j . L u s L e i u i  at 139 - 1 4 0 ° C ) ; a mixed melting point of 139 - 140°C was observed 72  proving their i d e n t i t y .  1  uiexCS  145  The f i f t h f r a c t i o n was the column washings material.  and contained the polar  Examination of this f r a c t i o n by TLC f a i l e d to detect any com-  pounds of s i m i l a r chromatographic properties to the sought a f t e r methoxyserratene d e r i v a t i v e s . Both Fractions 2 and 3 had TLC properties s i m i l a r to the methoxyserratene derivatives i s o l a t e d from other spruce species. A portion of Fraction 2 (Figure 36) was d i s t i l l e d i n vacuum to give  Fraction 2 distillation Distillate  iCnlumn  Ch  T  nm a t nor-a  r\hv  Fraction  Solvent  Compounds  petroleum ether  hydrocarbons  pet. ether:benzene (19:1)  (epi)manool, abienol  pet. ether:benzene (19:1)  (epi)manool, abienol  Figure 36. P u r i f i c a t i o n of Fraction 2.  a clear yellow d i s t i l l a t e .  The residue was dark brown and contained  only base l i n e materials on a TLC chromatogram and was not examined further. The d i s t i l l a t e was chromatographed  on alumina to afford a p a r t i a l  146  separation. compound.  Petroleum ether eluted Fraction 6 containing the least polar Evaporation  of the solvent gave a waxy semi-solid.  spectrum had no peaks for hydroxyl or carbonyl functions.  The  IR  The NMR  spectrum  had none of the distinguishing features of the methoxyserratene derivatives i s o l a t e d i n other spruce species. Further e l u t i o n with petroleum ether - benzene gave mixtures of the two more polar compounds i n the d i s t i l l a t e .  The faster running  of the  compounds had TLC properties l i k e manool (31) or epimanool (29). slower running  The  compound had TLC properties l i k e abienol (92).  29  31  Fraction 7 was  92  enriched i n the f a s t e r running  8 was.enriched i n the slower running  component.  component, while Fraction  A NMR  spectrum of Fraction  7 showed signals at x 3.15. (quartet, J = 17 and 11 Hz); x 4.10 J = 17 and 11 Hz); x 4.5 1 Hz); x 4.93  two  - 5.10  singlets at x 8.76,  ( t r i p l e t , J = 7 Hz); x 4.85  ( m u l t i p l e t ) ; x 5.22, 9.15,  9.22,  and 9.34.  5.55  (quartet,  (quartet, J = 17  (broadened s i n g l e t s ) ;  Signals and couplings at  and and  147  x  4.10, 4.85, 4.93 - 5.10, 5.22, 8.76, 9.15, 9.22,  agreement with the reported NMR  and 9.34 are a l l i n  spectrum of manool  other signals mentioned were of lesser i n t e n s i t y .  73  or epimanool.  The  Signals at x 3.15,  4.5,  and 4.93 - 5.10 were assigned to the. o l e f i n i c protons of abienol on the basis of the reported spectrum . 74  same signals as the NMR  The NMR  spectrum of Fraction 8 had the  spectrum of Fraction 7 i n the o l e f i n i c region  with C-methyl groups at x 8.84, 9.14, 9.18, and 9.22. these methyl groups i n the NMR  The p o s i t i o n of  spectrum i s i n agreement with the reported  spectrum of a b i e n o l . 7 4  Vapor phase chromatography  of the above mixture gave peaks with  retention time of 13.5 and 15.2 minutes.  Injection of manorol or e p i -  manool gave a peak with a retention time of 13.5 minutes; abienol gave a neak with  retention  ri'rnp n f  1.5-2  mi,nv.t?S .  The mixture was not further p u r i f i e d as i t appeared clear that neithe of these compounds was  the sought a f t e r methoxyserratene derivative.  The t h i r d f r a c t i o n was dissolved i n warm acetone for c r y s t a l l i z a t i o n (Figure 37).  The p r e c i p i t a t e was c o l l e c t e d and chromatographed  on alumina  The i n i t i a l f r a c t i o n (9) gave an o i l y wax which exhibited no absorption for either hydroxyl or carbonyl.in the infrared spectrum. gave a low melting waxy substance. carbonyl absorption at 1725 cm . -1  Fraction 10  The IR spectrum of this f r a c t i o n had The NMR  spectrum had a dominant peak  at x 8.7 as a broadened s i n g l e t and a s i g n a l of small i n t e n s i t y at x 9.1. Upon amplification, a x 7.7 s i g n a l was seen as a t r i p l e t a x 5.9 s i g n a l was seen as a t r i p l e t  (J = 6 Hz).  (J = 6 Hz) and  I t i s not thought that  these two t r i p l e t s are coupled to each other but rather represent  148  Fraction 3 c r y s t a l l i z a t i o n from acetone Mother Liquors Precipitate  Column Chromatography  Solvent  Compound  petroleum ether  hydrocarbons  10  petroleum ether pet. ether-benzene (3:1)  wax ester  11  pet. ether-benzene (1:1)  fatty alcohol  Fraction  •• oo u r s  T?1  ^ *7 -•  P u r l f l r ^ f " ! rin -  i-i-p T->*v~or'-ir\-if-r->r'<ri r r .-— f  ~...17^ -.^ ^^^ t .4^ . ^-!i ^-.^ .. Q  v r t m  methylenes adjacent to the ester function of a wax ester. had a weak hydroxyl absorption  at 3570 cm  -1  Fraction 11  i n the IR spectrum.  The NMR  spectrum again had the dominating s i g n a l at T 8.7; a weak resonance at T 9.1; and, upon amplification, weak signals as t r i p l e t s at x 5.9, 6.4, and 7.7.  TLC of the material showed that the main component had the same  properties as an authentic fatty alcohol, i n this case l i g n o c e r o l [CH.j-(CH.2) 22-CH2OH] •  The minor component had properties l i k e the wax  ester of Fraction 10. The mother liquors of Fraction 3 were separated by chromatography on alumina (Figure 38).  Fraction 12 gave a compound that had the same  properties as manool or epimanool.  Further e l u t i o n gave Fraction 13  149  Mother Liquors of Fraction 3  Column Chromatography  Fraction  Solvent  Compound  12  pet.  ether-benzene (9:1)  (epi)manool  13  pet.  ether-benzene  unidentified  Figure 38.  (9:1)  P u r i f i c a t i o n of mother liquors of Fraction 3. .  which was o i l y and contained at least two major and two minor components. Separation of the mixture on preparative TLC gave four bands (Rr 0.70, 0.55, 0..45, and 0.37). mi,  _  J.J1C  at  T.TT,TT\  The- f i r s t —  - _  '  two bands were present i n small amounts -  lWLJ-i.V D p C UL J. Cllli  f  \.l 1.  -  LLJC  1-1  r\  AV.p  W.  —i r\  i  -  i  U &XL U  -u  i  11 ci.U  i „  .  ?  „ •  .  "  fi Ll LJilLl- LlclLl t- [-»CttS-  x 8.7 with a small s i g n a l centered at x 9.1, as found i n early fractions  containing fatty ester or alcohol.  The NMR spectrum of the R  r  0.55 band  again had the x 8.7 peak t y p i c a l of the fatty esters or alcohols and was not further examined.  The NMR spectrum of the R^ 0.45 and 0.37 bands  were reminiscent of the NMR spectrum of the mixture of (epi)manool and abienol i s o l a t e d e a r l i e r .  On the basis of t h e i r NMR spectra and physical  c h a r a c t e r i s t i c s , i t was f e l t that these compounds were not the sought after methoxyserratene  derivatives.  While both Sitka  and Western white spruce had contained e a s i l y  i s o l a b l e quantities of methoxyserratene the above cursory examination.  derivatives, none were detected i n  Repeating the examination on a fresh  portion of crude petroleum ether extract f a i l e d again to detect any of  150  the sought f o r methoxyserratene d e r i v a t i v e s . I.H. Rogers of the Forest Products Laboratory, who had done much of the e a r l i e r work on S i t k a s p r u c e ,  25  examined the petroleum ether extract  and various chromatographic f r a c t i o n s .  This examination also f a i l e d to  detect any of the serratenes which were i n r e l a t i v e abundance i n the other spruces examined. Two possible reasons may be advanced f o r the apparent absence of serratenes i n Engelmann spruce.  I t may be that Engelmann spruce, because  of i t s phytochemical background, does not synthesize these t r i t e r p e n e s . The other possible reason involves the anatomical s t r u c t u r e of the bark. In the o r i g i n a l s t u d y  2 5  of S i t k a spruce the crude plant m a t e r i a l was  hand picked to c o l l e c t the pork layer from the bark of overmature trees. This portion of the bark apparently has an enrichment of serratenes. although serratenes have been i s o l a t e d from whole'bark of S i t k a and Western white spruce i n our l a b o r a t o r i e s . quite t h i n . ?  8 :  The bark of Engelmann spruce i s generally  In t h i s study the bark had been removed from the log and  broken i n t o s m a l l chunks p r i o r to being received.  The hand s o r t i n g of  t h i s bark was not p o s s i b l e nor was i t f e l t necessary before grinding and extraction. In summary, i t may be stated that t h i s survey d i d not detect any of the serratenes i n Engelmann spruce which were previously i s o l a t e d from S i t k a or Western white spruce..  151  Experimental Throughout this work Merck s i l i c a gel G with added fluorescent i n d i c a t o r was used as adsorbent i n thin layer chromatography (TLC).  The  chromatograms, 0.3 mm. i n thickness, were a i r ' d r i e d and activated i n an oven at 100°C f o r three hours.  The chromatograms were developed i n chloro-  form unless stated otherwise. with antimony pentachloride  Detection  of compounds was done by spraying  i n carbon tetrachloride (1:2) unless  otherwise  noted. For preparative  layer chromatography a thicker layer (0.5 mm.) of  adsorbent was u t i l i z e d with 0.01% Rhodamine 6G added as i n d i c a t o r . 3 4  S n r a u j n o  u i H i ari t  irnrmv  r> p  n  r .q rh.  1  n r \ H p  s n l n f i  n n  w a s  doce  O n l y  along on? cdg?  or not at a l l as detection of bands was possible with u l t r a v i o l e t l i g h t i n most instances. Column chromatography was usually performed on Woelm neutral alumina. The preferred adsorbent was deactivated  alumina ( A c t i v i t y III) prepared by  the addition of water as directed by the manufacturers.  In column chromato  graphy of the crude extract where large quantities of adsorbent were used, Shawinigan alumina was deactivated by addition of 3% of a 10% acetic acid solution.  Except i n large scale column chromatography the solvents were  d i s t i l l e d before use. The nuclear magnetic resonance (NMR) spectra were measured i n deuterochloroform at room temperature.  The NMR spectra were obtained at either  60 MHz using a Jelco C-60 or Varian A-60 instrument or at 100 MHz using a Varian HA-100 instrument.  The positions of a l l NMR resonances are given  152  in the Tiers T scale with tetramethj^lsilane as i n t e r n a l standard set at 10.0  units.  For multiplets the T values given represent  the center of the  signal. Infrared (IR) spectra were measured on Perkin Elmer model 21 or instrument.  137  Samples were measured i n KBr p e l l e t s , i n chloroform or carbon  tetrachloride s o l u t i o n or neat.  The p o s i t i o n of absorption maxima are  given i n wave numbers (cm ). -1  Melting points were determined on a Kofler block and are uncorrected. Extraction of Engelmann spruce bark The bark for this study was  obtained from an Engelmann spruce tree  growing i n the Fort C o l l i n s region of Colorado.  The bark was  and ground i n a Wiley m i l l to pass through a 3 mm. ( "\ RAA V  —  >  frmc O — ~  *\ ' '  V . T O O  "  ~ ~  ov  +- v i  - - - - - - -  -t-o A •  glass Soxhlet extractor.  T.T-if-V. " -  +- -v- y. 1 —  hours. (21.2  ~ ~ — —  o * i">  w  4-V, ^ v  £ ~  ^ *^  IQ  A i r dried bark —  ..^^.^^.-^.L  ~  ^  1 ~ ~ ~ ~ J..-*..  Evaporation of the petroleum ether gave a crude  petroleum ether extract (6 7.1 gms.) bark was  -  sieve.  a i r dried  as a brown, gummy, semi-solid.  The  l e f t i n the extraction thimble and extracted with benzene for 18 The benzene extract was  gms.).  evaporated to give a crude benzene extract  F i n a l l y the bark was  extracted with methanol for 24 hours.  Evaporation of the methanol gave a crude methanol extract (375 gms.).  The  benzene and methanol extracts were not further examined in this study. Column chromatography of crude extract Crude petroleum ether extract (43 gms.) ether  was  dissolved i n petroleum  (2 1.) and applied to the top of a column prepared from Shawinigan  alumina (5 lbs..) deactivated by addition of a 3% (67.5 mis-) aqueous acetic acid s o l u t i o n .  E l u t i o n with, petroleum. ether  of a 10% (6 1.,)  and  153  20% benzene i n petroleum ether (15 1.) gave Fraction 1 (4.5 gms.).  TLC  suggested that Fraction 1 was non-polar material i n the nature of hydrocarbons and s t e r o l or wax esters. Further elution with 20% chloroform i n petroleum ether (6 1.) gave Fraction 2 (1.9 gms.) whose TLC properties suggested abienol, (epi)manool, and wax ester. E l u t i o n with chloroform (8 1.) gave Fraction 3 (1.8 gms.) whose TLC properties suggested  (epi)manool, fatty alcohol, and two other components.  Elution with chloroform (8 1.) gave Fraction 4 (3.8 gms.) containing 3 - s i t o s t e r o l by TLC  comparison.  F i n a l l y , elution with 50% methanol i n chloroform (3 1.) gave polar compounds (5.1 gms.) as Fraction 5. th i  s  Fraction 5 was not further examined i n  s t~ i.i rl v .  D i s t i l l a t i o n of Fraction 2 Fraction 2 (550 mgs.) was placed i n a bulb f o r hot box d i s t i l l a t i o n at a temperature  of 140°C and a pressure of 0.07 mm.  of mercury.  A pale  yellow o i l was collected (416 mgs.) as the d i s t i l l a t e ; the residue was a brown s o l i d (125 mgs.). The d i s t i l l a t e contained 3 components when examined by TLC (R^ 0.79, 0.39, and 0.32).  The residue was mainly polar materials on the base l i n e  of the chromatogram with a trace of the d i s t i l l a t e s t i l l present. Chromatography of the d i s t i l l a t e of Fraction 2 The d i s t i l l a t e of Fraction 2 (400 mgs.) was chromatographed on alumina  (40 gms.).  Elution with petroleum ether (300 mis.) gave a waxy  semi-solid (50 mgs.) as Fraction 6.  Elution with 5% benzene i n petroleum  154  ether (300 mis.)  gave F r a c t i o n 7 (223 mgs.) as an o i l .  with the same solvent  (200 mis.)  Continuing to elute  gave F r a c t i o n 8 (.118 mgs.) as an o i l .  P r o p e r t i e s of F r a c t i o n 6 Fraction 6 was a waxy semi-solid which contained a major component (Rj 0.79) and a trace of a second component (R^ 0.65) when examined by TLC. IR (neat) 2950 (CH) , 1440 (CH ) , 1380 (CH ), and 970 ( o l e f i n i c C-H). 2  (60 MHz) 9.12,  3  NMR  8.24 (3H, s i n g l e t ) , 8.44 (3H, s i n g l e t ) , 8.52 (3H, s i n g l e t ) , and  9.16, 9.22 (3 C-methyls).  P r o p e r t i e s of Fraction 7 F r a c t i o n 7 was an o i l which contained a major component at R^ 0.39 and a second component at R^. 0..32 when examined by TLC. The f a s t e r running component had the same TLC properties as (epi)manool (R^ 0.39),  IR ( f i l m ) 3330 (OH). NMR (100 MHz) showed s i g n a l s a t t r i b u t a b l e to ( e p i ) • •' . manool at: 4.12 (IH, quartet, J = 11 Hz -and 17 Hz, Hx i n system  Hx  \  w  Ha — ) > lb  4.85 (IH, quartet, J = 17 Hz and 1 Hz, Hb), 5.05 (IH, quartet, J = 11 Hz and 1 Hz, Ha), 5.22, 5.51 (2H, p a i r of broadened s i n g l e t s , e x o c y c l i c methylene), 8.76 (3H, s i n g l e t , a l l y l i c methyl), and 9.13, 9.21, 9.34 (3 C-methyls). a d d i t i o n , s i g n a l s a t t r i b u t a b l e to abienol were seen at: 3.15 (IH, Hy Hx 1 I ,/Ha  J = 11 Hz and 17 Hz, Hx i n system  ^ ^ ^ ^ c ^  Me  In  quartet,  ) , 4.54 (IH,  " Hb  t r i p l e t , J = 7 Hz, Hy), 4.93 - 5.10 (2H, m u l t i p l e t , Ha and Hb), 8.21 (3H, broadened s i n g l e t , v i n y l i c methyl), 8.84 (3H, s i n g l e t , CH^-COH),  155  and 9.14, 9.18, 9.22 (3 C-methyls).  Vapor phase chromatography of Fraction  7 on a 3% SE 30 column (8 f t . ) at 205°C gave -peaks with retention time of 13.5 and 15.2 minutes. time of 13.5 minutes;  Injection of (epi)manool gave a peak with retention abienol had a retention time of 15.2 minutes.  Fraction 8 Fraction 8 was .an o i l which contained a major component at  0.32  and a second component at Rj 0.39 when examined by TLC. The major component had TLC properties l i k e abienol (Rj 0.32); the minor component had properties l i k e  (epi)manool (R 0.39). f  IR (film) 3335 (OH). NMR (100 MHz)  showed signals a t t r i b u t a b l e to abienol and (epi)manool as i n the NMR spectrum of Fraction 7.  Vapor phase chromatography on a 3% SE 60 column  (8 f t . ) at 205°C gave peaks with retention time 13.5 and 15.2 minutes; under the same conditions (epi)manool and abienol had retention times of 13.5 and 15.2 minutes respectively. C r y s t a l l i z a t i o n of Fraction 3 Fraction 3 (1.8 gms.) was taken up i n warm acetone (35 mis.) and l e f t to c r y s t a l l i z e  f o r one day. The p r e c i p i t a t e was f i l t e r e d , washed with cold  acetone and dried to give a yellowish wax (307 gms.).  The f i l t r a t e was  evaporated to give 1.4 gms. of gummy o i l . Chromatography of p r e c i p i t a t e from Fraction 3 The p r e c i p i t a t e (307 mgs.) was chromatographed on alumina (30 gms.). Elution with petroleum ether (50 mis.) gave Fraction 9 as an o i l y wax. IR had no absorption f o r either hydroxyl or carbonyl functions.  The  Elution  with petroleum ether (50 mis.) and 25% benzene i n petroleum ether (600 mis.) gave Fraction 10 as a low melting waxy substance.  IR (film) 1725.  NMR  156  (60 MHz) 5.9 ( t r i p l e t , J = 6 Hz, -CH -OCOR), 7.7 ( t r i p l e t , J = 6 Hz, -0C0CH ), 8.7 (broad s i n g l e t , -CH  , 9.1 (multiplet, -CH ).  2  3  On this  basis Fraction 10 was thought' to be fatty ester and not further examined. Fraction 11 was eluted with 50% benzene i n petroleum ether (360 mis.) to give a low melting wax. R  TLC revealed  0.14 and a minor component at R  f  f  two components, a major component at  0.48.  IR (film) 3570.  NMR  (60 MHz)  5.9 ( t r i p l e t , J = 6 Hz, -CH -0C0R), 6.4  ( t r i p l e t , J = 6 Hz, -CH -0H),  7.7 ( t r i p l e t , J = 6 Hz, -0C0-CH -), 8.7  (broad s i n g l e t , -CH -), 9.1  2  2  (multiplet, -CH ). 3  2  2  The s p e c t r a l data suggested the Fraction 11 was mainly  fatty alcohol with some fatty ester from Fraction 10 as a contaminant. Chromatography of mother liquors from Fraction 3  • • •- •  A portion of the mother liquors from Fraction 3 (0.4 mgs.) was chrnmatocranhp.d  nn  a J i . i n i i r i ft  C125  cms . 1 .  F.lnfi.nn  v i th  10%  ben?-<at>e i n pet-  roleum ether (1,000 mis.) gave Fraction 12 (102 mgs.) 'as an o i l . e l u t i o n with the same solvent  Further  (1,500 mis.) gave Fraction 13 (170 mgs.) as  an o i l . Properties  of Fraction 12  Fraction 12 was a gummy o i l that contained two components, by TLC (R  f  0.39 and 0.32).  Comparison with, (epi)manool (R  f  0...39) and abienol  (R^ 0.32) suggested i d e n t i t y . Preparative  layer chromatography of Fraction 13  A portion of Fraction 13 (.90 mgs...) was applied to a preparative  layer  ch.romatogram and developed i n chloroform to reveal four bands. (R^ Q..70, 0.55, 0.45, and 0.37)., i n small amounts.  The f i r s t two bands ( R 0.70 and 0.55) were present f  Their NMR spectra were dominated by a broadened s i n g l e t  15 7  at 8.7 suggestive of the f a t t y ester or f a t t y alcohol components i n early fractions. The band with R (60 MHz)  3.00  a viscous o i l .  IR (film) 3425 (OH).  NMR  (unresolved m u l t i p l e t ) ,  4.95  (unresolved m u l t i p l e t ) , and 8.98,  The band with R (60 MHz)  0.45 was  (unresolved m u l t i p l e t ) , 4.20  ( s i n g l e t ) , 5.15  NMR  f  4.50  f  0.37 was  9.10,  also a viscous o i l .  (unresolved m u l t i p l e t ) , 4.95  9.15  (singlets).  IR (film) 3390 (OH).  ( s i n g l e t ) , 8.72  (singlet)  P u r i f i c a t i o n of Fraction 4 A portion of Fraction 4 (500 mgs.)  was  c r y s t a l l i z e d from ethanol to  give a white s o l i d m.p.  139 - 140°C, mixed m.p.  140°C, ( l i t e r a t u r e m.p.  139 - 1 4 0 ° C ) .  was  collected m.p.  137 - 139°C.  72  with 8 - s i t o s t e r o l 139 -  A second crop of white crystals  TLC on s i l i c a gel with chloroform as  solvent showed onlv 1 compound with Rr 0.14  (8-sitosterol. R  X  c  0.14);  X  with 25% e t h y l acetate i n benzene as developing solvent the white s o l i d and 8 - s i t o s t e r o l both had i d e n t i c a l properties (R 4.66  (IH, m u l t i p l e t , H-C=C), 6.50  f  0.30).  NMR  (IH, m u l t i p l e t , H-C-OIL), 8.35  s i n g l e t , H-0-C-; exchangable with D.O),  8.98  - 9.27  (60  MHz)  (IH,  (6 C-methyls).  158 Bibliography  1.  M.A. Buchanan, "The Chemistry of Wood", p.358, B.L. Browning, Ed., Interscience Publishers, New York, 1963.  2.  B.L. Browning, "Methods of Wood Chemistry", Vol. 1, p.189, Interscience Publishers, New York, 1967.  3.  D.B. Mutton, "The Chemistry of Wood Extractives", p.348, W.E. H i l l i s , Ed., Academic Press, New York, 1962.  4.  H. Erdtman, Pure Appl. Chem., 6_, 679 (1963).  5.  E.P. Swan, Can. J . Chem., 45, 1588 (1967).  6.  N.T. Mirov, Lloydia, 26_, 117 (1963).  7.  E. von Rudloff, Can. J . Bot., 45, 891 (1967).  8.  E. von Rudloff, Can. J . Bot., 45, 1703 (1967).  9.  E. von Rudloff, Can. J . Bot., 46, 1 (1968).  10.  L.A. Smedman, K. Snajberk, and E. Zavarin, Phytochem., _8, 1471 (1969).  11.  L.A. Smedman, E. Zavarin, and R. Teranishi, Phytochem. , -3, 1457 (1969).  12.  E. Zavarin, Phytochem., ]_, 92 (1968).  13.  W. Jensen, K.E. Fremer, P. S i e r i l a , and V. Wartiovaara, "The Chemistry of Wood", p.649, B.L. Browning, Ed., Interscience Publishers, New York, 1963. • ' M.A. Buchanan, "The Chemistry of Wood", p.361, B.L. Browning,- Ed., Interscience Publishers, New York, 1963.  14.  15.  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(B).  PART L I STUDIES RELATED TO  SYNTHESIS AND  OF INDOLE  ALKALOIDS  BIOSYNTHESIS  163  Introduction  In the l a s t century Serturner recognized morphine and referred to i t as a vegetable the term a l k a l o i d f o r such vegetable alkaloids were the indole a l k a l o i d s .  the basic character of  alkali.  alkalis.  Meisner l a t e r proposed  Among the e a r l i e s t known  In a recent compilation by Hesse  some f i v e hundred of these bases have been reported from about three hundred p l a n t s .  1  The study of the biosynthesis of the indole alkaloids has intrigued and interested workers i n this f i e l d f o r many years.  Early studies on the  biosynthesis were based on natural compounds with s t r u c t u r a l s i m i l a r i t i e s tc the prepceed intermediate  c"d c i " ^ c r i c a l  "n" wh 'ch "ere thought -1  to be of biogenetic s i g n i f i c a n c e . With the a v a i l a b i l i t y of radioactive isotopes these hypotheses could be tested. A common s t r u c t u r a l feature of many indole a l k a l o i d s , the |3-(2-aminoethyl)-indole moiety suggested the intermediacy tryptamine (2).  of tryptophan (1) or  In fact radioactive l a b e l l e d tryptophan has been shown to  be incorporated into a number of indole alkaloids including vindoline ( 3 ) » > \ catharanthine 2  3  (A) * *, ajmalicine ( 5 ) \ vincaminoreine ( 6 ) , 2  1  5  vincamine ( 7 ) , and minovine ( 8 ) . 6  1  5  2  164  In the  c o n t r a s t ' to origin  of  the the  discussion with  acceptance  of  the  origin  n o n - t r y p t o p h a n p o r t i o n was  a number  of  theories  of  the  the  emerging.  "tryptophan"  subject  of  portion,  considerable  165  The e a r l i e s t theory due to-Barger - Hahn^^-Robinson^-Woodward 7  11  >  12  suggested the intermediacy of a dihydroxyphenylacetaldehyde (9), or equivalent, plus two yohimbine  units.  This theory predicted the biosynthesis of  (10) as shown:  10  That theory had a number of deficiencies and prompted W e n k e r t > 13  to suggest a new p o s s i b i l i t y .  14  His i n i t i a l postulate involved the  intermediacy of a hydrated prephenic acid (11). . L a t e r so prephenic acid i t s e l f was u t i l i z e d .  1 5  this was altered  Condensation with a one carbon  unit and various rearrangements would afford the seco-prephenate-formaldehyde (SPF) unit (12). This unit would then condense i n a subsequent step to give corynantheine (13). COOH  HOOC \ = 0  COQH  CHO  COOH  CHO  1  0  J  J  H.  HOOC OH  11  HOOC  CHO 12  13  MeOOC  OMe  166  A third t h e o r y acetate background.  1 6 - 1 9  proposed that the non-tryptophan portion had an  Here three acetate units, a malonate unit, and a one  carbon fragment would condense to give the proposed intermediate 14. However, this hypothesis could not be substantiated experimentally and was subsequently withdrawn.  Yet another proposal was advanced by wenkert based on s t r u c t u r a l relationships.  1  and Thomas  /u  They suggested that the non-tryptophan  portion of the indole alkaloids was monoterpenoid i n o r i g i n . suggestion followed the discovery of several monoterpenes,  This  for example  g e n t i o p i c r i n (15), bakankosin (16), swertiamarin (17), and genipin (18) which a l l have a s l i g h t l y masked form of the seco-prephenate-formaldehyde unit.  15  16  167  •OH HO ._OH  ,-OGlu  r 0  MeOOC'  ri  0 18  17  When Wenkert suggested  advanced h i s monoterpenoid hypothesis, he also  a sequence for the formation of the Aspidosperma and Iboga  families of indole a l k a l o i d s .  Condensation of tryptophan with the SPF  unit followed by the appropriate reactions as outlined i n Figure 1 could ieau Lo Lnese two i. ami x A ess. Attention should be drawn to some ideas presented proposal.  I t can be seen that this proposal suggests  i n the Wenkert that more than one  family can arise from the same precursor, for example 19. The a c r y l i c acid intermediates 20 and 23 are important mentioned l a t e r i n this thesis.  intermediates and w i l l be  The t h i r d feature i s the transannular  c y c l i z a t i o n of 21 to 22 and 24 to 25. The transannular c y c l i z a t i o n reaction i s a f a c i l e reaction i n v i t r o  and, on the basis of Wenkert's  2 1  postulate, should occur i n vivo as well.. experiments were performed no evidence 5  found.  However, when the appropriate  for an i n vivo c y c l i z a t i o n was  Also the reverse ring opening process i s known i n v i t r o but  does not appear to be operational i n the b i o s y n t h e s i s .  5  5  This  discrepancy does not, however, seriously affect the o v e r a l l proposal.  Figure  1.  Wenkert's p r o p o s a l f o r the b i o s y n t h e s i s and I b o g a a l k a l o i d s .  of  Aspidosperma  169  If the monoterpene hypothesis i s correct the established precursor terpenes, mevalonic acid (26), would be expected to be incorporated. experiments In 1965  were unable to detect  18  Scott and co-workers  mevalonic acid into vindoline (3). groups  the successful incorporation of  Subsequent publications by several  showed that the non-tryptophan portion of indole alkaloids was  2 3 - 2 6  derived from s p e c i f i c a l l y l a b e l l e d mevalonic acid. thesis was  further corroborated  The monoterpene hypo-  when geraniol (27) was  porated into vindoline, catharanthine, plants.  Early  the incorporation of mevalonic acid.  reported  22  of  and ajmalicine i n Vinca  These three alkaloids are representative  2 7 - 3 0  shown to be incorrosea'  of the three major  families of indole a l k a l o i d s : Aspidosperma, Iboga, and Corynanthe families respectively. Further s n p p n r t i n o evidence for the monoterpene hypothesis provided  by B a t t e r s b y  31  who  vindoline, catharanthine, reports  3 2 - 3 4  serpentia  33  the incorporation of loganin (28) into  and ajmalicine i n Vinca rosea plants.  Subsequent  confirmed these results and extended them to Rauwolfia plants.  transformations  Degradation of the alkaloids suggested the formal  as i l l u s t r a t e d i n Figure 2.  from Vinca rosea p l a n t s , ) 3 1  26  reported  was  3 5  Loganin has  also been i s o l a t e d  s a t i s f y i n g a requirement for a true  27  28  precursor  170  Iboga  Figure 2 .  Corynanthe  Aspidosperma  Formal t r a n s f o r m a t i o n of the monoterpene u n i t .  In an e x t e n s i o n of the i n c o r p o r a t i o n s t u d i e s , B a t t e r s b y vivo experiments l o g a n i n and  3 5  which impose s t r i c t  requirements  of doubly  l a b e l l e d specimens he  s t e r e o s p e c i f i c i t y e s t a b l i s h e d f o r the f o r m a t i o n bonds i n o t h e r b i o l o g i c a l s y s t e m s the c o n f i g u r a t i o n of C ( 7 ) of the c o r r e s p o n d i n g Corynanthe and  of  a l s o holds  t h a t : (a) the  of the two  g e r a n i o l double  good i n V i n c a r o s e a ;  i n loganin i s determining  f o r the  (b)  stereochemistry  compounds; (c) the s t e r e o c h e m i c a l c o r r e l a t i o n s  the c o r r e s p o n d i n g  l o s s of a p r o t o n  enamine i n t e r m e d i a t e .  3 7  concluded  c e n t e r i n a j m a l i c i n e and by e x t e n s i o n f o r o t h e r  Strychnos  i n loganin with  the observed  on the f o r m a t i o n  i t s c o n v e r s i o n i n t o the t h r e e major c l a s s e s of i n d o l e a l k a l o i d s .  From the study  C(2)  reported i n  of  center i n ajmalicine i s f o r t u i t o u s ,  from t h i s p o s i t i o n s u p p o r t s  the i d e a of an  171  The discovery that loganin i s a precursor of the indole alkaloids prompted detailed study of the l a t e r stages of the biosynthesis. Wenkert  15  and Thomas  20  Both  suggested that the indole alkaloids are derived from  a cyclopentane monoterpene by some process  involving cleavage of the  cyclopentane ring.  that a cleavage compound, seco-  loganin (29) , was  Battersby  33  recognized  found i n a masked form i n menthiafolin  (30).  Thus mild  OE  0 oo  in  a l k a l i n e hydrolysis of menthiafolin, followed by careful a c i d i f i c a t i o n and e s t e r i f i c a t i o n by diazomethane afforded seco-loganin. loganin available B a t t e r s b y  38  was  With seco-  able to prove that i t i s : a constituent  of Vinca rosea plants; biosynthesized from loganin; a s p e c i f i c  precursor  of representative examples of the Iboga, Corynanthe, and Aspidosperma families. The incorporation of sweroside (31) into vindoline has been  .OGlu  31  172  demonstrated pathway  by  n i t r o g e n  rosea and  Vinca  rosea."  b i o l o g i c a l  containing  discovery Rhazya  i n  of  p l a n t s  4  1  4  by  (32)  presence  d i l u t i o n  l i k e l y  i n t o  intermediate  I t s  0  is  conversion  s t r i c t o s i d i n e  s p e c i e s .  I t  i n  that  sweroside  seco-ioganin.  the  was  not  subsequently  analysis  Evidence  biosynthesis  (stereochemistry  s t a r t i n g  was  two  i n  bases,  f o r  an  obtained  demonstrated  w i t h  the  early the  i n  i n  r a d i o a c t i v e  d i r e c t  w i t h  established)  loganin. The  enters  Vinca  tryptophan  *' v i t r o  condensation  vincoside  (33)  and  of  tryptamine  isovincoside  and  s e c o - l o g a n i n  (34),  32,  OGlu  which  have  4  produced  1  the  same  gross  s t r i c t o s i d i n e  33,  C^H a :  34,  C^HB:  35,  C Ha, 5  vinc  oslae  isovincoside C(3)  -  C(4)  double  bond  reduced: dihydrovincoside  s t r u c t u r e  as  s t r i c t o s i d i n e .  I t  i d e n t i c a l  to  s t r i c t o s i d i n e .  While  been of  i s o l a t e d  v i n d o l i n e  from ( 3 )  4  1  Vinca >  4  2  ,  rosea,  is  l i k e l y both  only  catharanthine  that  one  vincoside  of and  these  is  ( 4 )  ajmalicine  1  >  4  2  ,  bases  isovincoside  vincoside  4  two  involved  i n ( 5 )  have  the  4  1  »  4  is  biosynthesis  2  ,  and  173  36  5  akuammicine (36) , the l a t t e r being an example of a Strychnos a l k a l o i d . 1+2  Dihydrovincoside  (35) produced by reduction of the ethylenic side chain  was not incorporated into the three representative alkaloids usually examined.  41  The b i o l o g i c a l conversion' of vincoaide into the Aspidospenua and Iboga bases necessitates rearrangement of the skeleton. that this was an intramolecular process.  E a r l i e r work proved  Cleavage of the glucose moiety  of vincoside results i n an aglucone which should be i n equilibrium with, or convertible to, the aldehyde (37).  Ring closure could lead to corynan-  theine aldehyde (38) and/or geissoschizine (39).  39, 19(20)-ene, geissoschizine  174  Early experiments  36  >  43  with mature Vinca rosea plants had shown no  incorporation of corynantheine aldehyde into the alkaloids examined. Geissoschizine, however, was and also was  shown to be present i n Vinca rosea plants  incorporated into ajmalicine (5), vindoline (3), catharan-  thine (4), and akuammicine ( 3 6 ) . was  4 2  In Vinca minor L shoots geissoschizine  incorporated into vincamine (7) and minovine  8  (8).  4 4  7  A d i f f e r e n t approach to the study of the biosynthesis was Scott.  4 3  J  4 5  J  4 6  It was  adopted by  known that seeds of Vinca rosea were e s s e n t i a l l y  devoid of a l k a l o i d a l content.  Thus i n growing Vinca rosea plants from  seeds i t should be possible to observe the sequential formation of a l k a l o i d a l material.. Using t h i s t e c h n i q u e , vincoside (33) was 46  f i r s t alkaloids i s o l a t e d at 26 hours a f t e r germination. hours f i v e alkaloids were i s o l a t e d : corynantheine  among the  Between 28 and  40  aldehyde (38), geisso-  schizine (39), a g-hydroxy indolenine (40), a d i o l (41), and geissoschizine oxindole (42).  While corynantheine  mature Vinca rosea plants, i t , into seedlings.  aldehyde was not incorporated i n  along with geissoschizine, was incorporated  The s i g n i f i c a n c e of the l a s t three alkaloids i s o l a t e d i n  the 28 to 40 hour time period w i l l be discussed l a t e r .  175  S t i l l working loids  isolated  43  on s e q u e n t i a l s t u d i e s , S c o t t . found t h a t 4 1  (40 - 50 hours) were pre*akuammicine  36  the next  alka-  (43) , akuammicine  (36) ,  176  44  45  stemmadenine ( 4 4 ) , and tabersonine  ( 4 5 ) . The occurrence  of pre-akuammicine  i s a s i g n i f i c a n t development since i t represents an intermediate between geissoschizine (Corynanthe), (Corynanthe-Strychnos).  akuammicine (Strychnos), and stemmadenine  Pre-akuammicine i s a key member since i t retains  a l l ten carbons of geraniol, yet can also s u f f e r loss of a single carDon atom necessary  for Strychnos  alkaloids and, by rearrangement, generates  Aspidosperma or Iboga a l k a l o i d s .  •  Three processes have been suggested for the biosynthesis of preakuammicine (Figure 3 ) .  Wenkert ' 15  47  suggested a one electron oxidative  coupling to give a strictamine type d e r i v a t i v e , 4 6 .  Precedent for the  rearrangement of compounds such as 4 7 to the Strychnos akuammicine ( 3 6 ) i s a v a i l a b l e  4 8  representative  so that the indoline ( 4 8 ) or i t s reduced  form, pre-akuammicine ( 4 3 ) , could be reached by such a mechanism.  An  alternative to this process, also with i n v i t r o analogy, i s a protonation. of the indole nucleus Figure 3 .  5  0  49  as shown i n  A t h i r d p o s s i b i l i t y i s oxidation of geissoschizine to a 8 -  hydroxyindolenine (42).  followed by a to 8 rearrangement  ( 4 5 ) followed by formation of geissoschizine oxindole  This l a t t e r could react v i a the imino ether  (49)  to give  177  Figure  3.  Proposals  f o r the b i o s y n t h e s i s  of  pre-akuammicine.  178  pre-akuammicine (43).  As mentioned previously, S c o t t  4 6  found the 8-  hydroxyindolenine and geissoschizine oxindole i n his sequence studies and showed that Vinca rosea seedlings were able to incorporate the oxindole (42) into akuammicine and vindoline.  Thus the oxindole hypothesis has  been shown to be a viable process i n Vinca rosea. The occurrence of pre-akuammicine, stemmadenine, and tabersonine at approximately  the same time i s i n t e r e s t i n g .  I t can be seen formally that  reduction of pre-akuammicine can lead to stemmadenine.  Scott  5 0  found,  in v i t r o , sodium borohydride reduction of pre-akuammicine afforded akuammicine (36) and stemmadenine (44).  In fact, stemmadenine appears to  be the next intermediate as i t i s incorporated into catharanthine, vindol i n e , and tabersonine i n Vinca rosea s e e d l i n g s .  50  In Vinca minor  vincamine and minovine are l a b e l l e d s t a r t i n g from l a b e l l e d stemmadenine."* The conversion of stemmadenine to tabersonine i s s t i l l speculative. It i s thought give a new (Figure 4).  5  that the exocyclic double bond of stemmadenine migrates to  compound, iso-stemmadenine (50), with an endocyclic double bond Iso-stemmadenine can, with the aid of the lone pair of  electrons on nitrogen, expel the hydroxide group to give an a c r y l i c ester derivative (51).  The iminium system can, by loss of a proton and ring  closure, give tabersonine (45); or, i t i s also possible that a c r y l i c ester (51) may  lose a proton without c y c l i z a t i o n to give a new dihydropyridine  a c r y l i c ester (52) (Figure 5).  This derivative may  then close i n a D i e l s -  Alder fashion to give tabersonine (45) or, by another Diels-Alder reaction using both double bonds i n the dihydropyridine, to produce catharanthine (4).  179  F i g u r e 5.  P o s t u l a t e d b i o s y n t h e s i s of c a t h a r a n t h i n e  (4).  180  Tabersonine has been incorporated by Vinca rosea p l a n t s lings  4 3  into both vindoline and catharanthine.  4  and  seed-  The conversion of taber-  sonine into catharanthine i s i n t e r e s t i n g since i t formally requires a p a r t i a l reversal to a c r y l i c ester (52).  In Vinca minor, tabersonine i s  incorporated into vincamine (7) and minovine  8  (8).  5 2  7  The detection of 11-methoxytabersonine (53)  as a time intermediate  between the occurrence of tabersonine and vindoline i s expected since that methyl ether grouping i s necessary f o r vindoline ( 3 ) . The a c r y l i c ester derivatives 51 or 52 remain s t i l l to be detected i n the b i o l o g i c a l system although derivatives of them have been detected. derivatives tetrahydrosecodine (54) and dihydrosecodine  53  (55) have been  The  181  detected i n Rhazya s t r i c t a  5 j  and tetrahydrosecodiri-17-ol (56) has been  detected i n Rhazya o r i e n t a l i s . 5 3  17-ol (57) i n Vinca rosea plants.  Battersby  54  has detected dihydrosecodin-  Both B a t t e r s b y  55  and K u t n e y  57  have  found that dihydrosecodin-17-ol (57) was not a precursor i n Vinca rosea and Vinca minor.  Work on this aspect i s continuing and synthetic steps  leading to a c r y l i c ester derivatives w i l l be described i n this  thesis.  A summary of the biosynthetic sequence i s found i n Figure 6.  182  Figure  6.  Summary  o f pathway  from  loganin  to i n d o l e  alkaloids.  183  Discussion  A c r y l i c ester - dihydropyridine  d e r i v a t i v e s were p o s t u l a t e d  intermediates  i n indole alkaloid biosynthesis.  intermediates  of that  studies  synthesis  dihydropyridine or imagined. portion.  to synthesize  or d e r i v a t i v e s t h e r e o f , p l u s  of a p o s s i b l e p r e c u r s o r w i l l be  The  some b i o s y n t h e t i c  discussed.  of a d e r i v a t i v e c o n t a i n i n g b o t h a c r y l i c e s t e r and  functionalities  The f i r s t  i s fraught.with  d i f f i c u l t y was f o r e s e e n  difficulties  either  i n the a c r y l i c  real  ester  A c r y l i c a c i d polymers were p r e p a r e d i n the e a r l y 1870's w i t h  polymerization j u t i u c i.  type,  Efforts  as b i o -  „ uiivr.  of e s t e r s noted s h o r t l y a f t e r .  -r„.ci-.-.j-uj. j - u c u C c  these r e s u l t s  - sz l — J— ucavj  KJX  i t was f e l t  i  J_  . L X ^ U L  S  U  A  V  , - .gcu .  KJ J.  A c r y l i c esters  polymerized  . ... ? .1 - pcLUAJ-UCD .  ..i.i ,•^ . r Ulltr. l> cl£> -LI? U.L  U1.I  t h a t the a c r y l i c e s t e r f u n c t i o n s h o u l d  r a t e d a t a l a t e s t a g e i n the s y n t h e s i s .  I t was a l s o u n d e s i r a b l e  be  elabo-  to have the  r e a c t i v e a c r y l i c e s t e r moeity undergo r e a c t i o n when i t was s u b m i t t e d t o precursor  studies  i n the p l a n t system.  To circumvent t h i s p o s s i b i l i t y  h y d r a t e d form o f the a c r y l i c e s t e r was the s y n t h e t i c g o a l . allows  f o r a p o s s i b l e chemical dehydration  studies The  o r In v i v o by an enzymatic other  difficulty  pyridine systems.  5 7  choice  e i t h e r p r i o r t o the b i o s y n t h e t i c  process.  t o be c o n s i d e r e d  was the i n s t a b i l i t y  of d i h y d r o -  They a r e known t o o x i d i z e t o the c o r r e s p o n d i n g  p y r i d i n e r e a d i l y , c o n t a c t w i t h atmospheric oxygen w i l l sufficient.  This  a .  Some d i h y d r o p y r i d i n e s  f r e q u e n t l y be  which have s u b s t i t u e n t s  capable of  conjugation  w i t h the u n s a t u r a t e d system o f the d i h y d r o p y r i d i n e  ly stable.  The reduced form of n i c o t i n a m i d e  are r e l a t i v e -  adenine d i n u c l e o t i d e  (NADH, 58)  184  OH  may  OH  be cited as such a case.  58  Two  choices were a v a i l a b l e : the use of an  acetyl group at the C(3) p o s i t i o n of the dihydropyridine; or the use of an ethyl group at this p o s i t i o n but i n a tetrahydropyridine system. p o s s i b i l i t y , while i t may  The  first  give a r e l a t i v e l y stable dihydropyridine, would  i n the alkaloids being examined.  The second p o s s i b i l i t y , using the e t h y l  side chain, circumvents that drawback but gives an undesired oxidation l e v e l i n the h e t e r o c y c l i c ring. ed with the understanding  The second choice was  that an i n vivo oxidation may  The o v e r a l l sequence i s outlined i n Figure 7. 2-carboethoxy-3-(8-chloroethyl)-indole  subsequently be  necessary.  The synthesis of  (60) had been previously  designed  i n our laboratories but was modified s l i g h t l y i n the present case. sodium s a l t of d i e t h y l malonate was  select-  The  treated with l-chloro-3-bromopropane  to give d i e t h y l y-chloropropylmalonate  (59).  In the previous synthetic  scheme, dry powdered benzenediazonium chloride was  added to the sodium  s a l t of 59 and the r e s u l t i n g adduct refluxed i n a c i d i c ethanol to produce the indole derivative 60.  In this work, the benzenediazonium chloride  185  COOEt  l)NaOEt  COOEt  X  l)NaOEt  CQOMe Figure 7.  Synthesis of 16,17-dihydrosecodin-17-ol (57).  186  was  replaced with the corresponding  fluoroborate s a l t which i s l e s s s e n s i -  t i v e to shock and thermally more s t a b l e . t h i s way was  The indole d e r i v a t i v e prepared i n  i d e n t i c a l to the product i s o l a t e d previously and i t was  obtain-  ed i n higher o v e r a l l y i e l d . The required 3-ethylpyridine (61) obtained by Wolff-Kishner of 3 - a c e t y l p y r i d i n e , was  reduction  condensed with the indole d e r i v a t i v e 60 to give  N-[B{3(2-carboethoxyindolyl)  } e t h y l ] - 3 - e t h y l p y r i d i n i u m c h l o r i d e (62) as a  white c r y s t a l l i n e s o l i d . Reduction of the pyridinium c h l o r i d e (62) i n cold methanol with sodium borohydride gave  N-[3(3(2-carboethoxyindolyl)}ethyl]-3-ethyl-l,2,5,6-  tetrahydropyridine (63).  Lithium aluminum hydride reduction i n r e f l u x i n g  tetrahydrof uran gave, a f t e r chromatography, N-[(3{ 3(2-hydroxymethylindolyl) } 1  O  ~J-1-.~1  1  O  C  C  T  ^  —  J-?  /  I, \  ml,,.  XTWTA  ..  .  .  1.^1  broadened s i n g l e t f o r one o l e f i n i c proton (x 4.45); a s i n g l e t f o r the hydroxymethylene (x 5.19); a w e l l r e s o l v e d q u a r t e t 1  of the e t h y l side chain (x 8.02,  f o r the methylene group  J = 6.5 Hz); and a t r i p l e t f o r the methyl  group of the e t h y l side-chain (x 8.98,  J = 6.5  Hz); a l l i n good agreement  with s t r u c t u r e 64. The a l c o h o l (64) could be benzoylated  i n dry p y r i d i n e w i t h benzoyl  c h l o r i d e to give N-[g{3(2-benzoxymethylindolyl)}ethyl]-3-ethy1-1,2,5,6tetrahydropyridine (65).  This d e r i v a t i v e so obtained could be used  d i r e c t l y i n the next r e a c t i o n .  A s o l u t i o n of the benzoate (65) i n dimethyl-  formamide containing a large excess of potassium cyanide was slowly heated to a maximum of 120°C f o r two hours and t h i s mixture upon work up gave N-[8{3(2-cyanomethylindolyl)}ethyl]-3-ethyl-l,2,5,6-tetrahydropyridine  (66)  187  in varying y i e l d s .  The NMR spectrum of the n i t r i l e had a two proton  s i n g l e t f o r the methylene carrying the n i t r i l e absorbing at x 6.12. The n i t r i l e  (66) was readily hydrolyzed to N-[8{3(2-carbomethoxymethy.l  indolyl)}ethyl]-3-ethy1-1,2,5,6-tetrahydropyridine concentrated hydrochloric acid.  (67) i n methanol with  A less s a t i s f a c t o r y procedure was a l k a l i n e  hydrolysis of the n i t r i l e (66) and subsequent e s t e r i f i c a t i o n . gave the same ester (67).  Both methods  The ester (67) could be alkylated i n benzene  using methyl formate and sodium hydride as the base.  The r e s u l t i n g enol  (68) was not i s o l a t e d but was reduced d i r e c t l y with sodium borohydride i n methanol under c a r e f u l l y controlled conditions  to give 16,17-dihydrosecodin  17-ol (57). The NMR spectrum of 57 exhibited signals f o r the o l e f i n i c proton ( L 4.61), the hydroxyinethyI group (x 6.02), che methyl ester (x 6.38), and the methylene group (x 8.10) and methyl group (x 9.04) of the ethyl side chain.  This data was i n f u l l agreement  f o r 16,17-dihydrosecodin-17-ol (57)  With a possible precursor now a v a i l a b l e , attention was focused on providing a radioactive substance necessary for biosynthetic investigations A method had been developed i n our laboratories for exchanging the aromatic protons of the indole system f o r t r i t i u m atoms. transferring t r i t i a t e d  The method involves  t r i f l u o r o a c e t i c acid, prepared from equal molar  quantities of t r i f l u o r o a c e t i c anhydride and t r i t i a t e d system to the a l k a l o i d a l material.  water, i n a vacuum  After a period of one or two days the  acid i s removed i n a vacuum system and the l a b e l l e d a l k a l o i d neutralized and i s o l a t e d . The above procedure had been used many times to prepare l a b e l l e d  188  derivatives with no d i f f i c u l t i e s being encountered. DL-tryptophan was l a b e l l e d i n this manner. secodin-17-ol (57) was subjected  this treatment.  However when 16,17-dihydro-  to these conditions none of the l a b e l l e d  alcohol (57) could be detected by TLC. posed during  In the present case  The alcohol had evidently decom-  Successful l a b e l l i n g could, however, be  carried out on the ester (67) p r i o r to a l k y l a t i o n .  The l a b e l l e d ester  thus obtained could then be alkylated, and reduced as before to give a product i d e n t i c a l to 5 7 except f o r the enrichment of t r i t i u m i n the aromatic portion. E a r l i e r investigators i n our laboratories had developed a method f o r the incorporation of various substances i n t o Vinca rosea L. plants a cotton wick i n the stem of the l i v i n g plant. e x p e n r r i e r i t a t e c h n j . G u e was  s a t i s f a c t o r y artCi to  using  In order to ensure that the matve  s u i e  Elicit  iiuc  dge  o<'  the plant was suitable f o r biosynthetic i n v e s t i g a t i o n s , the t r i t i a t e d DLtryptophan prepared previously was fed. After nine days, the optimum conditions as determined by e a r l i e r investigations, the plants were macerated and extracted f o r a l k a l o i d s .  Chromatography of the crude a l k a l o i d a l  extract on alumina gave, among other a l k a l o i d s , vindoline (3), catharanthine (4), and ajmalicine  (5). The l a b e l l e d alkaloids were d i l u t e d with cold  4  189  MeOOC  or unlabelled alkaloids and were repeatedly  c r y s t a l l i z e d either as the  free base or as the hydrochloride s a l t to constant a c t i v i t y . age-incorporations  observed for this experiment are shown i n Table I.  Compound Fed  Percent cathar an thine  [ar- H]-DL- tryptophan  0.9 80  [ar- H]-alcohol (57)  0.0007  3  3  Table I.  The percent-  '  Incorporation vindoline  ajmalicine  0.155  0.312  inactive  0.0004  Results of incorporation of synthetic intermediates _V. rosea L. plants.  into  The successful incorporation of tryptophan indicated that the experimental method was s a t i s f a c t o r y and that the age of the plants selected was suitable for biosynthetic i n v e s t i g a t i o n . The synthetic alcohol (57) was tested i n the same manner. after work up, chromatography, and c r y s t a l l i z a t i o n to constant  The results a c t i v i t y are  given above i n Table I. Although a very small incorporation was observed, i t was impossible to t e l l i f this was evidence of true incorporation or i f i t was simply the  190  result of an aberrant biosynthetic pathway. Battersby  reported  54  Several months l a t e r Professor  that h i s laboratories had found evidence f o r the  alcohol ( 5 7 ) i n V. rosea plants.  They too had tested the compound as a  precursor and had found very s l i g h t or no i n c o r p o r a t i o n .  55  At the same time as alcohol ( 5 7 ) was being tested i n V. rosea L. other members of our laboratories were testing the compound i n V. minor L. The  test period i n V_. minor i s two days and i n the time • i t took to obtain  results i n V.. rosea detailed observations  several testings could be done on V. minor.  The  w i l l be reported elsewhere, but e s s e n t i a l l y the  i s o l a t e d alkaloids were i n a c t i v e or p o s s i b i l i t y with a trace of a c t i v i t y . With the apparent f a i l u r e of the alcohol ( 5 7 ) to act as an e f f i c i e n t ' precursor  f o r the indole a l k a l o i d s , i t was necessary to r e d i r e c t our  ftv n Liifc". Lie  c i .L'OJL LS .  It w i l l be r e c a l l e d that the desired system was an a c r y l i c ester dihydropyridine  derivative.  The compound j u s t tested d i f f e r e d i n oxidation  l e v e l i n both the a c r y l i c ester and the h e t e r o c y c l i c portions of the molecule.  A l t e r a t i o n of the oxidation l e v e l i n the ester portion was  investigated by other members of our laboratories and w i l l be reported elsewhere. Although the postulated intermediate was a dihydropyridine d e r i v a t i v e , i t was s t i l l considered biosynthetic evaluation.  to be too l a b i l e to be i s o l a t e d and submitted f o r In this s i t u a t i o n i t seemed that the corresponding  pyridinium s a l t may provide  the s o l u t i o n .  Pyridinium s a l t s are found i n vivo and are known to be reduced enzymatically  to dihydropyridines.  Two w e l l known examples of pyridinium  191  s a l t s which may  be reduced are the co-enzymes nicotinamide  dinucleotide (NAD,  69) and the phosphorylated  +I  adenine  form (NADP, 70).  -CONHo  0  69, R = H 70, R = P0oH OH  2  OR  . A pyridinium s a l t had been prepared  i n the above synthetic sequence  but i t s oxidation l e v e l could not be preserved i n l a t e r synthetic steps, j- i " .  » d i iie;cc!t»««ry  4.H.  u i a L  .i.tj& L a u u e  i.O . ( c u u C e  Lut;  Lo an  e s ttr't' f u i i C L x O u  under conditions which also would reduce the pyridinium s a l t .  axcohoj.  Even i f the  pyridinium s a l t had been preserved at this step, i t would not l i k e l y survive either the n i t r i l e formation or a l k y l a t i o n  steps.  The  tetrahydro-  pyridine system seemed to overcome these d i f f i c u l t i e s as this was  the  oxidation l e v e l which had been used previously and found stable.  What was  necessary  then was  to f i n d conditions for oxidation of the tetrahydropyridine  (57) to pyridinium s a l t (71).  X  CH 0H  CH 0H  2  2  OOMe 57  COOMe 71  192  To study this sequence a model pyridinium s a l t N - [ 3 ( 3 - i n d o l y l ) e t h y l ] 3-ethylpyridinium bromide (72) was phyl bromide and 3-ethyl pyridine.  prepared by the condensation of tryptoThe pyridinium bromide was  reduced with  sodium borohydride as before to give N[3(3-indolyl)ethyl]-3-ethyl-l,2,5,6tetrahydropyridine  (73).  Cl  11  Ii  72  73  Mercuric acetate i s a common oxidant i n a l k a l o i d chemistry the reagent selected for these oxidations.  Thus .compound 73 was  a s o l u t i o n of 2% aqueous acetic acid containing mercuric disodium s a l t of ethylenediaminetetraacetic  with methylene chloride.  was  added to  acetate and  acid ( E D T A ) ' 58  ed at a temperature of 100°C for one and a h a l f hours. p r e c i p i t a t e d mercurous acetate was  and  59  and  the  maintain-  A f t e r cooling the  f i l t e r e d off and the s o l u t i o n extracted  The resultant product, obtained as a brown  residue, consisted of several components and i t s weight corresponded to 70% o v e r a l l recovery  of organic material.  The major component was  separated  by preparative layer chromatography and gave a t y p i c a l i n d o l i c u l t r a v i o l e t absorption spectrum (<^  max  294,  283, 274,  and 230 my).  The NMR  spectrum  had, among other s i g n a l s , the NH resonance ( T 2.21), four aromatic protons  193  (T  2.45  -  3.05),  and one o l e f i n i c proton (r  spectrometry revealed a molecular ion at m/e by this data i s 7 4 .  4.38).  Low resolution mass  2 5 2 . The structure suggested  Another s t r u c t u r a l p o s s i b i l i t y possessing this mole-  cular weight could be the dihydropyridine derivative 7 5 .  However, the  s t a b i l i t y of the product i n a i r , the lack of s u f f i c i e n t aromatic and o l e f i n i c protons i n the NMR  spectrum, and the t y p i c a l i n d o l i c UV  spectrum  would suggest that this alternative ( 7 5 ) i s u n l i k e l y .  75  74  The aqueous portion of the above oxidation reaction mixture was treated with hydrogen s u l f i d e gas to p r e c i p i t a t e the excess mercuric s a l t used as an oxidant.  The p r e c i p i t a t e was  removed by f i l t r a t i o n  through  C e l i t e and the f i l t r a t e evaporated to dryness to give a yellow s o l i d .  This  s o l i d was mainly the added EDTA s a l t plus some organic compound which could not be separated. The next mercuric acetate oxidation experiment was ways.  F i r s t , the model compound was  changed i n several  changed to the previously prepared  2,3-disubstituted indole derivative 63 i n an e f f o r t to minimize  cyclization  194  onto the indole ring.  Also, the EDTA s a l t was  eliminated i n this case.  I t was w e l l known that alkaloids had been oxidized with mercuric acetate without  the addition of EDTA s a l t although  i t s addition was  decrease the p o s s i b i l i t y of carbon - carbon bond f i s s i o n . temperature was  shown to Further, the  5 9  changed to room temperature and the solvent changed from  aqueous acid to ethanol i n an e f f o r t to perform  the reaction under the  mildest conditions possible. The sought for pyridinium s a l t should e x i s t i n the oxidation solution as the acetate 76.  As an aid to recognition  ethyl]-3-ethylpyridinium acetate (76) was  N-[g{3(2-carboethoxyindolyl)}  prepared by treatment  of a  solution of the pyridinium chloride 62 with a solution .of s i l v e r acetate. The r e s u l t i n g s i l v e r chloride p r e c i p i t a t e d and was  removed by f i l t r a t i o n to  give crude pyridinium acetate 76 upon evaporation.  62, X = C l ~ 76, X =  The tetrahydropyridine derivative 63 was  OAc"  oxidized with mercuric  acetate i n ethanol at room temperature for four days.  Removal of the  mercurous acetate and treatment with hydrogen s u l f i d e as before gave, a f t e r f i l t r a t i o n , a yellow solution.  Concentration of this solution  195  and examination by TLC showed some s t a r t i n g materia], and both faster and slower running  compounds, plus some material s t i l l at the o r i g i n of the  chromatogram.  None of the compounds had s i m i l a r TLC properties to the  desired pyridinium acetate.  Separation' of the mixture on preparative  layer chromatography and examination of each f r a c t i o n , including the mater i a l at the o r i g i n , by UV showed that none.of the fractions had absorption of the pyridinium system 76.  the  Repeating the oxidation reaction  using the same conditions f a i l e d to produce the desired pyridinium Since there had been a lack of success  i n the previous  salt.  mercuric  acetate oxidations, the conditions were varied i n a further study.  In  these cases, the temperature and period of reaction were both varied. one  case oxidation was KD°r  was  -p^v  -F^,,-.- >>^.,-.-o  allowed TT-,  to proceed at 35°C for 18 hours, i n another erime ^te the y i e l d cf mercurcus acetate -  K^t-v.  about 90% of the t h e o r e t i c a l amount, yet i t was  any pyridinium acetate 76.  In  not possible to detect  Other experiments i n g l a c i a l acetic acid and  in 10% aqueous acetic acid, l e f t for three days at room temperature, gave approximately 60% of the t h e o r e t i c a l amount of mercurous acetate but none of the desired material was Concurrently with synthetic work was  found.  the mercuric  being done.  acetate oxidation experiments other  The p o s s i b i l i t y of using a 3-acetylpyridine  as one of the condensing units was  i n t e r e s t i n g . The a c e t y l side chain  s t a b i l i z e a dihydropyridine intermediate and subsequent biosynthetic studies.  The  may  s u f f i c i e n t l y to allow i s o l a t i o n other i n t e r e s t i n g point i s that  compounds with an a c e t y l side chain had been proposed by Wenkert possible intermediates  again  15  as  and would be accessible for biosynthetic evaluation  196  i f 3-acetylpyridine were used i n the i n i t i a l condensation. Condensation  of 2-carboethoxy-3(B-chloroethy.l)indole (60) prepared  e a r l i e r with the ethylene k e t a l of 3-acetylpyridine, gave N-[8{3(2-carboethoxyindoly 1)}ethyl]-3-acetylpyridinium chloride ethylene k e t a l (77) as a l i g h t gray powder (Figure 8). Sodium borohydride reduction of pyridinium chloride 77 i n methanol at 0°C followed by careful work up gave N-[g{3(2-carboethoxyindolyl)}ethyl]-3acetyl-1,2,5,6-tetrahydropyridine ethylene k e t a l (78) as a white solid. for  crystalline  The NMR spectrum of 78 contained a four proton multiplet (x6.14)  the protons of the ethylene k e t a l protecting group. The carboethoxy  desired  derivative 78 was reduced i n tetrahydrofuran and the  N-[8{3(2-hydroxymethylindolyl)}ethyl]-3-acety1-1,2,5,6-tetrahydro-  py ridiiie e t h y l e n e ke Lai  (79) p u r i f i e d by column ch roitta L Og x'aphy On alumina..  The k e t a l alcohol- 79 was then subjected to the same homologation steps as performed previously. Treatment of the k e t a l alcohol with benzoyl chloride gave N-[8{3(2-benzoxymethylindolyl)lethyl]-3-acety1-. 1,2,5,6-tetrahydropyridine ethylene k e t a l (80). The benzoate group i n compound 80 was then displaced by cyanide ion to give  N-[g{3(2-cyanomethylindolyl)}ethyl]-3-acety1-1,2,5,6-tetrahydro-  pyridine ethylene k e t a l (81).  This compound was recognizable by the  c h a r a c t e r i s t i c n i t r i l e absorption i n the IR spectrum at 2255 Hydrolysis of the k e t a l n i t r i l e 81 was accomplished  cm . -1  i n a methanolic  s o l u t i o n saturated with hydrogen chloride gas. P u r i f i c a t i o n by column chromatography on alumina afforded  N-[B{3(2-carbomethoxymethylindolyl)}  ethyl]-3-acetyl-l,2,5,6-tetrahydropyridine (82). The IR spectrum  of this  197  / \ 0  /6  0  /9  COOMe  COOMe 83  Figure  8.  Attempted  synthesis  of vinylogous  amide 83.  198  compound had two carbonyl absorptions (1727 and 1652 cm function and an' a,3-unsaturated ketone.  The NMR  ) for an ester  spectrum had  resonances  for one o l e f i n i c proton ( T 3.17), a methyl group of the ester function (x 6.33), and a methyl group of a ketone  (x 7.75)  i n f u l l agreement with  structure 82. When this synthetic sequence was  conceived, i t was intended to  oxidize the unsaturated ketone 82 to the corresponding pyridinium system and then c a t a l y t i c a l l y r e d u c e  47  the l a t t e r to the vinylogous amide 83.  Hopeful that this might s t i l l be the case, even though the concurrently conducted oxidation reactions i n the ethyl series were unsuccessful, the keto ester 82 was subjected to the oxidation reaction. . The oxidation mixture, worked up as before', was immediately subjected to c a t a l y t i c reduction.  The IR spectrum of the crude product obtained from the l a t t e r  reaction was not i n accord with the expected d a t a attempts  4 7  for the amide while  to purify the products resulted i n great' losses of material.  The o v e r a l l lack of success with the oxidation reactions necessitated yet another approach.  Condensation of 2-carboethoxy-3-(6-chloroethy1)-  indole (60) with 3-acetylpyridine gave, i n early preparations, a very dark s o l i d which tended to become gummy upon standing i n a i r . Later condensations performed  at a lower temperature gave lower y i e l d s of  N-[3(3(2-carboethoxy-  indolyl)}ethyl]-3-acetylpyridinium chloride (84) but the product now an orange - red c r y s t a l l i n e s o l i d .  Hydrogenation  was  of the s a l t with palladium  catalyst gave N-[3(3(2-carboethoxyindolyl)}ethyl]-3-acetyl-l,4,5,6-tetrahdyropyridine (85) quickly recognized by i t s c h a r a c t e r i s t i c spectroscopic properties.  Thus the next step i n the sequence required reduction of the  199  e s t e r w h i l e l e a v i n g the amide i n t a c t .  84  85  Use of l i t h i u m aluminum h y d r i d e i n t e t r a h y d r o f u r a n at 0°C as the r e d u c i n g agent gave 15 minutes.  complete d i s a p p e a r a n c e of s t a r t i n g m a t e r i a l w i t h i n  Work up and chromatography  on alumina gave one main compound.  j.he ilv spectrum Iiad one c a r b o n y l a b s o r p t i o n at 1/ID s a t u r a t e d ketone.  The NMR  spectrum had l o s t  cm  A  suggesting a  the s i g n a l s f o r the o l e f i n i c  and m e t h y l group p r o t o n s of the amide chromophore and had new x 5.32  f o r the hydroxymethyl group and a s i n g l e t  at x 7.90  signals at  f o r the s a t u r a t e d  ketone  thereby suggesting N[S{3(2-hydroxymethylindolyl)}ethyl]-3-  a c e t y l p i p e r i d i n e (86).  86  87  200  If the lithium aluminum-hydride  reduction was started at -30°C and the  temperature slowly raised to 0°C over a few hours some N-[8{3(2-hydroxymethylindolyl)}ethyl]-3-acety1-1,4,5,6-tetrahydropyridine (87) could be i s o l a t e d i n about 10% y i e l d .  In this instance some s t a r t i n g material as  w e l l as keto alcohol 86 were also obtained.  The presence of 87 indicated  that the ester group was being reduced p r e f e r e n t i a l l y to the vinylogous amide. When the reduction was done with lithium borohydride i n tetrahydrofuran at 0°C no reaction was observed after three hours.  Allowing the solution  to warm to room temperature f o r one and a half hours seemed to be without effect.  However, at reflux temperature the major product was again the keto  alcohol 86 and not the desired amide alcohol 87. Sodium borohydride w i l l nor. normally reduce the ester carbonyl although there are reports of esters being reduced with this r e a g e n t .  60  Thus to a  cold (0°C) methanol solution of the amide ester 85 sodium borohydride was added and the temperature slowly raised to r e f l u x over a two hour period. However no reduction products could be detected.  Repeating the reaction  but maintaining the temperature at 0°C f o r one hour before warming again f a i l e d to give any i n d i c a t i o n of reduction. Although the amide alcohol 87 could be obtained from some of the reductions,, the small amounts i s o l a t e d precluded the usefulness of this synthetic approach. The next approach has, of yet, not given the product desired f o r biosynthetic investigations but has allowed synthesis to proceed closer to that objective than has other syntheses reported thus f a r . The  201  synthetic approach i s outlined i n Figure 9. Methyl indole-2-carboxylate (88) was reduced with lithium aluminum hydride to give 2-hydroxymethylindole crystallization  (89) .  After chromatography and  the alcohol was obtained as a white s o l i d i n 70% y i e l d .  Use of the same homologation  sequence as before allowed extension of  the C(2) substituent by one carbon.  Thus treatment of 2-hydroxymethyl-  indole with benzoyl chloride gave, i n near q u a n t i t a t i v e ' y i e l d s , 2benzoxymethylindole  (90).  Displacement  of the benzoate group by cyanide  ion resulted i n the formation of 2-cyanomethylindole crystalline  (91) as a white  solid.  • Formation of the desired carbomethoxy function was accomplished by hydrolysis of the n i t r i l e 91 i n methanol saturated with hydrogen chloride gas.  The resulting' methyl mdoie-2-acetate  IR absorption at 1719 cm  -1  (92) was recognizable by i t c  f o r the ester carbonyl.  The NMR spectrum had  the C(3) proton of the indole moiety at x 3.68 and the methyl ester group at x 6.32. The necessary ethyl bridge at C(3) of the indole nucleus could be inserted under r e l a t i v e l y mild conditions using ethylene oxide and stannic chloride.  6 1  The y i e l d of methyl 3(3-hydroxyethyl)indole-2-acetate (93)  was only moderate but some s t a r t i n g material (92) could be recovered and re-used i n subsequent reactions. The NMR spectrum had new signals at x 6.25 and 7.12 f o r the two methylene groups of the hydroxyethyl side chain. Significantly  the signals formerly assigned to the C(3) proton of the  indole nucleus was now absent, suggesting s u b s t i t u t i o n had occurred at C(3) as indicated.  202  COOMe 93 Figure 9.  COOMe 83  Syntheses of vinylogous amide. 83.  203  Treatment of 93 with phosphorous tribromide gave methyl 3(8-bromoethyl)indoie-2-acetate (94) as an o i l which decomposed on storage and hence was condensed with 3-acetylpyridine immediately..  The r e s u l t i n g  pyridinium bromide 95 was formed but was contaminated with the excess of 3-acetylpyridine.  The pyridinium bromide 95 could be c a t a l y t i c a l l y  reduced to give the desired  N-[3{3(2-carbomethoxymethylindolyl)}ethyl]-  3-acety1-1,4,5,6-tetrahydropyridine (83). . A more s a t i s f a c t o r y procedure f o r the preparation of 83 was achieved with phosphorous tribormide i n 3-acetylpyridine. The product of this reaction was not i s o l a t e d but immediately  subjected to hydrogenation to  y i e l d the same vinylogous amide 83 as before but i n much better y i e l d . The vinylogous amide 83 obtained by either procedure had absorption in the IR spectrum f o r the ester carbonyl (1720 cm ) and the amide carbonyl -1  (1610  cm ). -1  The NMR spectrum had a s i n g l e t f o r one o l e f i n i c proton  ( T 3.15), a three proton s i n g l e t f o r the carbomethoxy group (T 6.38), and a three proton s i n g l e t f o r the methyl of the a c e t y l group (x 8.20). With the synthesis of the vinylogous amide now established, other . members of the laboratory are i n v e s t i g a t i n g methods f o r the synthesizing of the possible bio-intermediate 96 i n indole a l k a l o i d biosynthesis.  0  COOMe 96  \  204  Experimental  Throughout this work Woelm neutral alumina or Merck s i l i c a gel G with added fluorescent indicator were used.as adsorbent graphy (TLC)..  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. chromatography a thicker layer (0.5 mm.) tograms were developed  for thin layer chromato-  For preparative layer  of adsorbent was  i n a variety of solvents.  used.  The chroma-  Compounds were detected  using antimony pentachloride i n carbon t e t r a c h l o r i d e (1:2). ' Column chromatography was  usually performed on Woelm neutral alumina  deactivated to A c t i v i t y III according to the manufacturers'  direction.  Infrared ( I R ) spectra were measured on a Perkin Elmer model 21, or 457 instrument. chloroform s o l u t i o n .  137,  Samples were measured either as KBr p e l l e t s or in The p o s i t i o n of absorption maxima are given i n  wave numbers ( c m ) . -1  U l t r a v i o l e t (UV) spectra were measured i n methanol or ethanol on a Cary model 11 or model 15 instrument. (X _„) m  The position of absorption maxima  are given i n millimicrons (mu).  Nuclear magnetic resonance (NMR)  spectra were measured i n deutero-  chloroform, unless otherwise noted, at e i t h e r 60 MHz a Varian A-60, instrument.  or a Varian T-60  or at 100 MHz  using a Jelco  C-60,  using,a Varian HA-100  The chemical s h i f t s are given i n the Tiers T scale with  reference to tetramethylsilane as i n t e r n a l standard set at 10.0  units.  Mass spectra were measured on an Associated E l e c t r i c a l Industries MS 9 high resolution mass spectrometer  or on an Atlas CH 4  spectrometer.  205  High resolution molecular weight determinations were determined on the MS 9 spectrometer. Melting points were determined on a Kofler block and are uncorrected. Elemental analyses were performed by Mr. P. Borda, University of British  Columbia.  Radioactivity was measured with a Nuclear-Chicago Mark 1 Model 6860 Liquid S c i n t i l l a t i o n  counter i n counts per minute (cpm).  The r a d i o a c t i v i t y  of a sample i n disintegrations per minute (dpm) was calculated using the counting e f f i c i e n c y which was determined for each sample by the external standard technique  62  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 a s c i n t i l l a t o r solution made up of the following components: toluene (1 l i t r e ) ;  2,5-diphenyloxazole  (4 gms.); and 1,4-bis[2-(5-phenyloxazolyl)jbenzene (50 mgs.). .In practice a sample of an a l k a l o i d as the free base was dissolved i n benzene (1 ml.) i n a counting v i a l or, i n the case of an a l k a l o i d a l s a l t , 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 s c i n t i l l a t o r solution. For each sample counted the background was determined for 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 and counting to determine the background cpm.  solutions  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 solution-, and counted.  Hie difference i n cpm between the background count and the  sample count was used for subsequent  calculations.  Diethyl y-chloropropylmalonate A solution of diethylmalonate (160 gms.) and 1,3-chlorobromopropane  206  (160  gms.)  i n anhydrous ether (200 mis.)  was  added i n one portion to a  solution of sodium ethoxide prepared by dissolving sodium (24 gms.) dry ethanol (350 mis.). and  The  reaction was. maintained at 35°C for 4 hours  then allowed to stand at room temperature for 24 hours.  mixture was  poured i n water (1,000 mis.)  ether extract was  dried over sodium s u l f a t e , and  liquid  (120  J = 7  Hz,  2  triplet, J = 4 (6H,  IR (film) 1730  -CO0CH CH ), 6.56 Hz,  triplet, J = 7  aenz ene a i a z oni um  (2H,  3  2  The  NMR  (60 MHz)  5.75  (4H,  quartet,  -CH C1), 6.75  (IH,  2  (4H, multiplet, _C1CH CH CH ), 2  2  8.80  2  -COOCH CH_ ) . 2  3  f iuo rob o rate  and concentrated hydrochloric  (69 gms.)  sodium chloride solution,  t r i p l e t , J = 4 Hz,  A n i l i n e hydrochloride (180 gms.)  solution was  The  to give a clear, colorless  (COOEt).  -CH(C00Et) ), 8.10 Hz,  reaction  concentrated under reduced pressure.  d i s t i l l e d (110°C at 1 mm.)  gms.).  The  and extracted with ether.  washed with water, saturated  r e s u l t i n g o i l was  in  was  dissolved i n water (275  acid (140 mis.)  mis.)  i n a 3 l i t r e beaker.  This  cooled to —5°C i n an ice - s a l t bath and sodium n i t r i t e  i n water (150 mis.)  remained below 5°C.  was  added dropwise so that the temperature  No more sodium n i t r i t e solution was  added when a  drop of the a n i l i n e solution gave a blue coloration when tested on potassium iodide - starch paper. cooled to 0°C was  A s o l u t i o n of 48% fluoroboric acid (183 mis.)  and added slowly to the diazonium s a l t solution.  immediate but s t i r r i n g was  was  Precipitation  maintained for 10 minutes after a l l the  fluoroboric acid had been added.  About one h a l f of the p r e c i p i t a t e  was  transferred to a sintered glas.s funnel and  sucked as dry as possible with  the aid of a rubber sheet.  removed and  The vacuum was  the p r e c i p i t a t e  207  was  washed thoroughly with ice cold water (50 mis.)  before.  In a s i m i l a r manner the p r e c i p i t a t e was  methanol (25 mis.) transferred  and,  The  The  2-carboethoxy-3(3-chloroethyl)indole  126  Combined weight  (60) gms.)  and ychloropropylmalonate (147  was  dissolved  gms.)  was  in  added  the mixture s t i r r e d for 30 minutes at room temperature under a s l i g h t  p o s i t i v e pressure of nitrogen. gms.)  The was  solution was  diazonium chloride  (126  Elemmyer flask and  then added slowly and  transferred  cooled to -10°C. Benzene-  i n portions (-20  cautiously  below -5°C.  The  to an  solution  After a l l the benzenediazonium fluoroborate had been added,  the solution was hours at  gms.)  through a short length  of Gooch tubing to the s t i r r e d ethanolic solution' keeping the  s t i r r e d for 2 more hours at -5°C  and  then stored  for  18  -5°C. solution was  separated. and  the  gms.  In a 5 l i t r e , 3 neck flask sodium metal (14.7 dry ethanol (1,000 mis.)  was  containing  remaining half of  collected and stored i n a s i m i l a r manner.  of the benzenediazonium fluoroborate was  and  precipitate  dried i n a vacuum desiccator  anhydrous calcium s u l f a t e for 18 hours.  as  washed with i c e cold  f i n a l l y , ether (50 mis.).  to a tared beaker and  p r e c i p i t a t e was  and sucked dry  The  then poured into water (1,500 mis.)  aqueous solution was  and  the red o i l  thoroughly extracted with chloroform  the chloroform washings combined with the red o i l . This chloroform  solution was  washed thoroughly with water, saturated s a l t solution,  dried  over sodium s u l f a t e , and concentrated to a dark red o i l . The  dark red o i l was  trated s u l f u r i c acid (200  dissolved i n ethanol (1,000 mis.) mis.)  was  and  added slowly with s t i r r i n g .  concenThe  208  mixture was  refluxed for 12 hours then cooled and poured onto i c e and  neutralized.  This mixture was extracted with chloroform thoroughly and  the chloroform extract washed with water, saturated s a l t solution, dried over sodium s u l f a t e , and concentrated under reduced pressure to give a brown s o l i d  (140 gms.).  This brown s o l i d was Shawinigan alumina  dissolved i n benzene and applied to a column of  (3 legs.) deactivated by the addition of 3% (90 mis.) of  a 10% acetic acid solution.  E l u t i o n with benzene gave i n i t i a l l y a red o i l  and f i n a l l y the desired product as a c r y s t a l l i n e s o l i d .  Further elution  with chloroform eluted more of the desired product.  crystalline  The  fractions were combined and c r y s t a l l i z e d from benzene -. petroleum ether to give 29 gms. m.p.  1 J U  -  of white c r y s t a l l i n e 2-carboethoxy-3(B-chloroethyl)indole (60)  i j Z  3-ethylpyridine  L.  (61)  A mixture of 3-acetylpyridine (60 gms.), 85%'hydrazine hydrate (90 mis.), potassium hydroxide (50 gms.), and triethylene g l y c o l (400 was heated under nitrogen for 2 hours at a bath temperature The s o l u t i o n was  mis.)  of 130°C.  cooled and gradually reheated with a take off condensor  to a bath temperature was  .  of 200°C.  During this time the d i s t i l l a t e  collected. - The d i s t i l l a t e was  (175  mis.)  dissolved i n ethyl ether and washed with  water, dried over sodium s u l f a t e , and evaporated to a colorless l i q u i d . D i s t i l l a t i o n of this l i q u i d and c o l l e c t i o n of the f r a c t i o n b o i l i n g at 60°C at 17 mm. band.  gave 3-ethylpyridine as a colorless l i q u i d .  NMR  (60 MHz)  aromatic), 7.38 7 Hz, -CH CH ) 2  3  1.58  IR (film) no carbonyl  (2H, m u l t i p l e t , aromatic), 2.70  (2H, quartet, J = 7 Hz, -CH CH ), 8.82 2  3  (2H, multiplet,  (3H, t r i p l e t , J =  209  N-[S{3 (2-carboethoxyindolyl) }ethyl]-3-ethylpyrldini.um chloride (62) 2-carboethoxy-3(8-chloroethyl)indole (12 mis.) was hours.  (2.9 gms.)  i n 3-ethylpyridine  heated under nitrogen i n a Carius. tube at 120°C for 18  The reaction mixture was  cooled and t i t u r a t e d with ether and  pyridinium chloride 62 c o l l e c t e d by f i l t r a t i o n (3.7 gms.) IR (KBr) 1680  (COOEt)..  UV 233, 297..  J = 7 Hz, CH -CH -0-), 7.34 3  NMR  (CD 0D)  5.6.3  3  m.p.  87 - 89°C.  (2H, quartet,  (2H, quartet, J = 7.5 Hz, CH CH ~C),  2  3  (3H, t r i p l e t , J = 7 Hz, CH_ -CH 0) , and 8.96 3  2  the  2  (3H, t r i p l e t , J = 7.5  8.60 Hz,  CH CH C). 3  2  N-[8(3(2-carboethoxyindolyl)}ethyl]-3-ethyl-l,2,5,6-tetrahydropyridine The pyridinium chloride 62 (4.1 gms.) mis.)  and triethylamine (3 mis.).  tions to keep the temperature, below 0°C.  was  added (-50  mixture was  mis.)  acidic.  sodium bicarbonate  dissolved i n methanol  The s o l u t i o n was  s a l t bath to -5°C and sodium borohydride  the sodium borohydride,  was  the s o l u t i o n was  (7 gms.)  (63) (300  cooled i n an i c e —  was. added i n small por-  Four hours after the addition of evaporated  to near dryness, water  and d i l u t e hydrochloric acid was  added u n t i l  A f t e r 15 minutes the s o l u t i o n was and extracted with chloroform.  the  b a s i f i e d with  The chloroform extract  was washed with water, dried over sodium s u l f a t e , and evaporated  to give .  63 as a s l i g h t l y yellow o i l (4 gms.)  IR  (CHC1 ) 1680 3  which was  one spot on TLC.  (COOEt).  N-[g{ 3(2-hydroxymethylind'olyl) }ethyl]-3-ethyl-l,2 ,5 ,6-tetrahydropyridine The  carboethoxy d e r i v a t i v e 63 (4 gms.), dissolved i n tetrahydrofuran  (20 mis.), was hydride  (4 gms.)  added over 30 minutes to a suspension i n tetrahydrofuran  (250 mis.).  of lithium aluminum  The mixture was  refluxed  (64)  210  under nitrogen for two hours then cooled i n an i c e water bath. mis.) was  Water (4  added' dropwise, followed by 15% sodium hydroxide solution (4 mis.),  and f i n a l l y water (12 mis.) was  added.  The p r e c i p i t a t e was  and washed and the f i l t r a t e evaporated.  f i l t e r e d off  The residue was re-dissolved i n  chloroform and the chloroform s o l u t i o n washed with water, dried over sodium s u l f a t e , and evaporated. alumina (50 gms.) 64 (2.5 gms.)  This extract was  chromatographed  on  and elution with benzene then chloroform gave the alcohol  m.p.  (3.81), 292 (3.73).  108 - 110°C. NMR  IR (KBr) 3340, 3180.  (100 MHz)  4.45  5.19  (2H, s i n g l e t , CH_-0H) , 8.02  8.98  (3H, t r i p l e t , J = 6 Hz, CH_ CH ).  UV (log e) 284  (IH, broad s i n g l e t , H-C=C),  (2H, quartet, J = 6 Hz, CH -CH ~) , and  2  3  3  2  (high resolution found 284.184  2  C_ H N 0 requires 284.188). 18 24 2 0  N-[pl3(2-benzoxyraethylindolyi)Jethylj-3-ethyl-l,2,5,6-tetrahydropyridine (65) The alcohol 64 (2.1 gms.)  was dissolved in dry pyridine (25 mis.),  cooled to 0°C, and the benzoyl chloride (7 mis.) added dropwise.  After  3 hours the reaction mixture was d i l u t e d with water and b a s i f i e d with sodium bicarbonate. The benzoate 65 was extracted with chloroform and the  solution washed with water, dried over sodium s u l f a t e , and evaporated.  Chromatography of the crude benzoate (2.5 gms.)  on alumina and  crystalliza-  tion from methylene chloride — petroleum ether gave 65 as a white m.p.  110 - 112°C.  IR (KBr) 1720  (-CH0C0cj>) .  274 (3.94), 284 (3.96), 293 (3.84)..  2  NMR  (100 MHz)  NH), 2.1 - 3.1 (9H, aromatic protons), 4.6 -CH -0) , 7.40 2  CH.CH„-). —3 z  UV  (log e) 224 (4.60), 1.4  (IH, broad s i n g l e t ,  (3H, m u l t i p l e t , H-C=C and  (2H, quartet, J = 6 Hz, CH CH_ -) , 9.00 3  solid  2  (3H, t r i p l e t , J = 6 Hz,  (Found C, 77.05; H, 7.29; N, 7.05; C..HN 0 requires C, 77.27; Z H z o 2. Z  211  H, 7.28; N, 7.21%; high resolution 388.215 C H N 0 2 4  2 g  2  2  requires 388.216).  N-[3{3(2-cyanomethylindolyl)}ethyl]-3-ethy1-1,2,5,6-tetrahydropyridine (66) The benzoate 65 (1.1 gms.) was dissolved i n N,N-dimethylformamide; potassium cyanide (5 gms.) was added. temperature for  The mixture was s t i r r e d at room  f o r 45 minutes and the temperature was slowly raised to 120°C  3 hours.  After cooling and addition of water the solution was extracted  with methylene chloride.  The methylene chloride, was washed with water,  dried over sodium s u l f a t e , and evaporated to a thick o i l .  Chromatography  on alumina (50 gms.) and elution with benzene and then with 25% methylene chloride i n benzene gave n i t r i l e 66 (460 gms.).  C r y s t a l l i z a t i o n from  methylene chloride - petroleum ether gave an a n a l y t i c a l sample m.p. 135 137°C.  IR (CHC1 ) 2210 (CN)'. 3  (3.85), 291 (3.77). 6.12  UV (log e) 221 (4.69), 274 (3.84), 281  NMR (60 MHz)  4.55 (Hi, broad s i n g l e t , H-C=C),  (2H, s i n g l e t , CH_CN) , 8.97 (3H, t r i p l e t , J = 7 Hz, CH_ CH -). 2  3  (Found C, 77.65; H, 7.86; N, 14.16; C N, 14.36%; high resolution 293.186  C  i g  H  H 1 9  N 2 3  2 3  N  3  requires C, 77.75; H, 7.92;  3  r e  2  q  u i  res  293.189).  N-[B{3(2-carbomethoxymethylindolyl)}ethyl]-3-ethy1-1,2,5,6-tetrahydropyridine (67) The n i t r i l e 66 (360 mgs.) was dissolved i n methanol (35 mis.) and concentrated hydrochloric  acid (35 mis.) was added.  After 3 days the  solution was concentrated, neutralized with d i l u t e ammonium hydroxide, and extracted with methylene chloride.  The methylene chloride extract was  washed with water, dried over sodium s u l f a t e , and evaporated. on alumina (107 mgs.).  Chromatography  (20 gms.) and e l u t i o n with chloroform gave ester 67 as an o i l IR (CHC1 ) 1720 (COOMe). 3  UV (log e) 224 (4.42), 274 (3.87),  212  284 (3.91), 292 (3.83). 6.22  NMR  (100 MHz)  (2H, s i n g l e t , CH COOMe), 6.30 2  t r i p l e t , J = 7 Hz, CH_ CH -). 3  2  4.55  (IH, broad s i n g l e t , H-OC) ,  (3H, s i n g l e t , C00CH ), 8.98  (3H,  3  (high resolution found 326.202  c  i 26 2°2 H  2  N  requires 326.199). 16,17-dihydrosecodin-17-ol  (69)  The ester 67 (450 mgs.)  was  dissolved i n dry benzene (15 mis.)  dried by d i s t i l l a t i o n of benzene (3 mis.).  and  Sodium hydride (500 mgs.  of  suspension) was washed with benzene (2x10 mis.) suspended i n benzene (5 mis.), and methyl formate  (500 mgs.)  was  d i s t i l l e d from phosphorous pent-  oxide d i r e c t l y i n t o the sodium hydride suspension; then the benzene solution containing the ester 67 was  added dropwise.  heated to 35°C f o r 90 minutes then cooled to 0°C. was  The mixture was  Methanol (3 mis.)  added roliowed by i c e cold water and the mixture a c i d i f i e d with 2N  hydrochloric acid and b a s i f i e d with sodium bicarbonate solution.  The  reaction product was extracted with chloroform and the chloroform extract washed with water, dried over sodium s u l f a t e , and evaporated to give crude enol 68 (450 mgs.). The enol 68 (190 mgs.)  was  dissolved i n methanol (15 mis.), cooled  to -10°C i n an i c e - s a l t bath, and sodium borohydride (200 mgs.)  was  added.  After 1 hour at -10°C the methanol was evaporated and water  added.  The mixture was extracted with chloroform and the chloroform  extract washed with water,' dried over sodium s u l f a t e , and evaporated. The crude product was  chromatographed on alumina (5 gms.)  ether gave 16,17-dihydrosecodin-17-ol IR (KBr) 1725  (ester).  UV (log e) 292  (57) (150 mgs.), m.p.  and elution with 131 - 132°C.  (3.86), 284 (3.93), 274  (3.88), and  213  223  (4.49).  NMR (100 MHz) 1.22-(IH, broad s i n g l e t , NH), 2.4 - 3.1  (4H, aromatic protons), 4.6 3 (IH, broad s i n g l e t , H-C=C), 6.02 (2H, s i n g l e t , CH 0H), 6.38 (3H, s i n g l e t , COOMe), 8.10 (2H, quartet, J = 6 Hz, 2  CH CH ), and 9.04 (3H, t r i p l e t , J = 6 Hz, CH CH ). 2  3  2  3  (high resolution  found 356.207 C , H 0 N „ requires 356.209). o  o o  o  o  Trifluoroacetic acid- H T r i f luoroace t i c anhydride (1.17 gms.., 5.55 mmole) was added to w a t e r - % (0.10  g., 5.55 mmole, 100 mcurie/g) using a vacuum transfer system. The  resulting t r i f l u o r o a c e t i c a c i d — % (1.27 gms., 0.9 mc/mmole) was stored under an atmosphere of nitrogen at —10°C u n t i l required. Extraction of alkaloids from Vinca rosea Linn The  following procedure was developed i n order to extract and p u r i f y  the alkaloids of Vinca rosea Linn plants.  This procedure was used for a l l  extractions of V_. rosea L. plants and was scaled according to the wet weight of plants used. V. rosea L. plants were obtained of B r i t i s h Columbia.  The plants (30 gms., wet weight) were mascerated with  methanol i n a Waring Blender, was  from the greenhouse at the University  f i l t e r e d and re-mascerated u n t i l the f i l t r a t e  c o l o r l e s s . This green f i l t r a t e (combined volume was 300 mis.) was  evaporated to dryness, the residue taken up i n 2N hydrochloric acid (200 mis.)  and washed with benzene (3 x 100 mis.).  The combined benzene extracts  were back extracted with 2N hydrochloric acid (2 x 50 mis.).  The combined  aqueous phases were made basic with 15N ammonium hydroxide and extracted with chloroform  (3 x 100 mis.).  The combined chloroform extracts were  washed with water (100 mis.), dried over sodium s u l f a t e and evaporated to give a brown o i l (100 mgs.).  214  The o i l was dissolved i n benzene — methylene chloride (1:1, 1 ml.) and chromatographed on alumina (10 gms.).  The column was eluted succes-  s i v e l y with petroleum ether, benzene, chloroform, and methanol; of 10 mis. were taken.  fractions  The l a t e r benzene - petroleum ether (1:1)  fractions were combined and c r y s t a l l i z e d from methanol affording catharanthine (5 mgs.), the benzene fractions were combined and c r y s t a l l i z e d from methanol affording ajmalicine (2.5 mgs.), and the i n i t i a l benzene chloroform (4:1) fractions were combined and c r y s t a l l i z e d from ether giving vindoline (2.9 mgs.).  When required, the hydrochloride s a l t of  catharanthine and vindoline was formed by blowing hydrogen  chloride gas  on the surface of an ethereal s o l u t i o n of the a l k a l o i d ; catharanthine hydrochloride was c r y s t a l l i z e d from methanol, whereas vindoline hydrochloride was c r y s t a l l i z e d from acetone.  The hydrochloride s a l t of  ajmalicine was prepared by adding concentrated hydrochloric acid (1 drop) to a concentrated methanolic solution of the a l k a l o i d and ajmalicine hydrochloride was c r y s t a l l i z e d from methanol. Tritium l a b e l l e d radioactive alkaloids f o r biosynthesis studies The following procedure i s t y p i c a l for the formation of a l l the radioactive precursors u t i l i z i n g tritium i n the aromatic portion of the a l k a l o i d molecule. Trifluoroacetic a c i d — % tophan  (0.5 g., 0.9 mc/mmole) was added to DL-tryp-  (40 mgs.) using a vacuum transfer system.  The solution was  allowed to stand under an atmosphere of nitrogen at room temperature f o r 24 hours. system.  The t r i f l u o r o a c e t i c a c i d - 3  H  was removed using a vacuum transfer  Concentrated ammonium hydroxide solution (10 mis.) was c a r e f u l l y  215  added to the residue and the organic components extracted with dichloromethane (10 x 15 mis.)-  The organic extract was washed with water (10 mis.),  dried over sodium s u l f a t e , and concentrated to dryness under reduced pressure to afford [ar-%]-DL-tryptophan  (31 mgs., 7.6 x 10  s  dpm/mg.).  Feeding of [ar-%]-PL-tryptophan [ar- H]-DL-tryptophan  (11.387 mgs., 8.63 x 10 dpm) was dissolved i n  3  7  0.1N acetic acid and the solution administered to V. rosea plants (wet weight 48.3 gms.) by the cotton wick method.  After 9 days under i n t e r -  mittent fluorescent lamp i l l u m i n a t i o n , the alkaloids were i s o l a t e d and p u r i f i e d by chromatography to give catharanthine (7.00 mgs.), ajmalicine (5.68 mgs.), and vindoline (5.03 mgs.).  After dilution, with cold alkaloids  and c r y s t a l l i z a t i o n to constant a c t i v i t y the following incorporations were obtained: catharanthine (0.980%), ajmalicine (0.312%), and vindoline (0.155%). [ar- H]-16,17-dihydrosecodin-17-ol 3  (a)  16,17-dihydrosecodin-17-ol  with t r i f l u o r o a c e t i c acid- H.  (57) (50 mgs.) was treated .as before  Work up as before revealed decomposition  J  of 57. (b)  Carbomethoxy derivative 67 (150 mgs.) was.treated with t r i f l u o r o -  a c e t i c acid- H as before. 3  Work up gave [ar- H]-carbomethoxy derivative 67 3  which could be converted into [ar-%]-16,17-dihydrosecodin-17-ol by the described procedure.  '  Feeding of [ar-%]-16,17-dihydrosecodin-17-ol (57) [ar- H]-16,17-Dihydrosecodin-17-ol 3  (57) (8.0 mgs., 1.97 x 10  was fed to V. rosea plants (wet weight 75 gms.) as before.  7  dpm)  A f t e r chroma-  216  tography, d i l u t i o n with cold a l k a l o i d s , and c r y s t a l l i z a t i o n to constant a c t i v i t y the following incorporations were obtained: catharanthine  (0.0007%),  ajmalicine (0.0004%), and vindoline ( i n a c t i v e ) . Tryptophyl bromide A solution of phosphorus tribromide (0.44 mis.) was  i n ether (10  added dropwise to an ice cold solution of tryptophol (2 gms.)  i n ether (200 mis.). for the f i r s t 6 hours.  The reaction was  mis.) dissolved  s t i r r e d for 16 hours with i c e cooling  The supernatant was  decanted, x^ashed with sodium  bicarbonate solution, water, and dried over sodium s u l f a t e .  Removal of  the solvent yielded the product as white c r y s t a l s (2.46 gms.), m.p. 102°C ( l i t e r a t u r e m.p.  100 -  90 - 9 5 ° C ) . 63  N-[g(3-indolyl)ethyl]-3-ethylpyridinium bromide Tryptophyl bromide (2.46 gms.)  was  (72)  heated under nitrogen at 80°C  for 16 hours with 3-ethylpyridine (8 mis.).  The supernatant was  decanted  and the s o l i d t i t u r a t e d with ether and suction dried to give 72 as a yellow s o l i d  (3.6 gms.).  UV 290, 282  (shoulder), 267.  N-[B(3-indolyl)ethyl]-3-ethy1—1,2,5,6-tetrahydropyridine Pyridinium bromide 72 (2 gms.) and triethylamine (2 mis.) was and sodium borohydride temperature below 5°C. and evaporated  (7 gms.)  was  added. was  (73)  dissolved i n methanol (75 The solution was  mis.)  cooled to 0°C  added i n portions i n order to keep the  After 2 hours the solution was  to a s t i c k y paste which was  d i l u t e d with water  a c i d i f i e d with d i l u t e hydro-  c h l o r i c acid, b a s i f i e d with sodium bicarbonate, and extracted with chloroform.  The chloroform extract was washed with water, dried over sodium  s u l f a t e , and chromatographed on alumina to give 73 as an o i l (1.3 gms.).  217  NMR  (60 MHz)  2.23  - 3.13  J = 2 Hz, H-C=C), 8.98  (511, aromatic protons), 4.51  (IH, t r i p l e t ,  (3H, t r i p l e t , J = 6.5 Hz, CH CH ~). 3  2  Mercuric acetate oxidation of 73 Tetrahydropyridine 73 (300 mgs.), ethylenediaminetetraacetic acid (EDTA, 800 mgs.), sodium hydroxide mgs.)  (200 mgs.), and mercuric acetate  were dissolved i n 2% acetic acid (45 mis.).  maintained  The solution  (213 mgs.).  A portion of this o i l was  (100 MHz)  2.21  aromatic protons), 4.38 J = 7 Hz, CH-jC^-) .  to give a brown o i l  p u r i f i e d by preparative layer  chromatography to give 74 as a brown - orange s o l i d . NMR  The  extracted with methylene- chloride and the extract was washed  with water, dried over sodium s u l f a t e , and evaporated  294.  was  at a temperature of 100°C f o r one and a quarter hours.  solution was  (800  (IH, broad s i n g l e t , NH),  1>V 230, 274,  2.45  - 3.05  (IH, m u l t i p l e t , H-C=C), and 8.83  Mass spectrum  m/e  (3H,  283,  (4H, triplet,  252.  The aqueous layers of the extract were combined and saturated with hydrogen s u l f i d e gas.  The solution was  tration of the f i l t r a t e to near dryness which was  filtered off.  Concen-  resulted i n p r e c i p i t a t i o n of EDTA  Further concentration resulted i n more EDTA  being p r e c i p i t a t e d and f i l t e r e d o f f . dryness  f i l t e r e d through C e l i t e .  to give a yellow s o l i d .  The f i l t r a t e was  evaporated  to  Attempts to wash the organic products  out of this s o l i d with methanol were unsuccessful. N-[g{3(2-carboethoxyindolyl)}ethyl]-3-ethylpyridinium Pyridinium chloride 62 (50 mgs.)  was  (76)  dissolved i n ethanol (5 mis.)  and an ethanolic solution of s i l v e r acetate was drop f a i l e d to produce fresh p r e c i p i t a t e .  acetate  added dropwise u n t i l a  The p r e c i p i t a t e was  filtered  218  off and the f i l t r a t e evaporated which l a t e r s o l i d i f i e d . multiplets,  N-H);  2.05  NMR  (60 MHz)  - 3.01  J = 7 Hz, -CH -0C0-) ; 7.42 2  to dryness  to give 76 as a brown o i l  0.65  (1H, NH);  1.06,  (6H, aromatic protons); 5.70  1.40  (2H,  (2H, quartet,  (2H, quartet, J = 7 Hz, CH^H^-C) ; 8.63  t r i p l e t , J = 7 Hz, CH^CI-^-O) ; 9.01  (3H,  (3H, t r i p l e t , J = 7 Hz, CH^CH^C).  Oxidation of 63 i n ethanol at room temperature Tetrahydropyridine 63 (71 mgs.)  and mercuric acetate (310  were dissolved i n ethanol (25 mgs.). . S t i r r i n g was under nitrogen.  The mercurous acetate (140 mgs.)  mgs.)  continued for 4 days was  f i l t e r e d o f f , the  f i l t r a t e saturated with hydrogen s u l f i d e gas, and the solution f i l t e r e d through C e l i t e .  The f i l t r a t e was  evaporated  desired pyridinium acetate 7'6 on TLC.  (75 mgs.)  .and compared to the  No spot corresponding  to 76 could  be detected. A portion of the f i l t r a t e was  separated on a preparative layer chroma-  togram (alumina, CHCl^/MeOH) and four bands of material were examined. The f i r s t band (R^ 0.65)  was  less polar than 76 and i t s UV revealed  tion at 290 my and a shoulder at 334 my.  The second band (R^ 0.50)  less polar than 76 and had UV absorption maxima at 315 my. corresponded  to s t a r t i n g material.  absorpwas  The t h i r d band  The fourth band examined remained at  the o r i g i n of the chromatogram and had a maxima at 320 my i n the UV spectrum. Oxidation of 63 at 35°C i n ethanol Tetrahydropyridine 63 (100 mgs.)  and mercuric acetate (350 mgs.)  ethanol (10 mis.) were heated at 35°C for 18 hours under nitrogen. curous acetate (245 mgs.) worked up as before.  was  f i l t e r e d off and the reaction mixture  TLC f a i l e d to detect any of the desired 76.  in  Mer-  219  Oxidation of 63 at 50°C i n ethanol  •.  Tetrahydropyridine 63 (100 mgs.)  and mercuric acetate (350 mgs.)  ethanol were heated at 50°C for 4 hours under nitrogen. (233 mgs.)  was  in  Mercurous acetate  f i l t e r e d off and the reaction worked up as before.  TLC  f a i l e d to detect any of the desired pyridinium acetate 76. Oxidation of 6 3 i n acetic acid Tetrahydropyridine 6 3 (100 mgs.) were dissolved i n g l a c i a l  and mercuric acetate (350  acetic acid (10 mis.)  for 3 days at room temperature.  and s t i r r e d  mgs.)  under nitrogen  The mercurous acetate (130 mgs.)  f i l t e r e d off and the reaction worked up as before.  was  TLC f a i l e d to detect  any-of the desired pyridinium acetate 76. Oxidation of 63 i n 10% acetic acid Tetrahydropyridine 53 (93 mgs.)  and mercuric acetate (325 mgs.)  10% acetic acid (10 mis.) were s t i r r e d temperature.  under nitrogen for 3 days at room  Mercurous acetate (157 mgs.)  reaction worked up as before.  in  TLC f a i l e d  was  f i l t e r e d off and the  to detect any of the desired  pyridinium acetate 76. 3-acetylpyridine ethylene k e t a l  1  A s o l u t i o n of 3-acetylpyridine (104 gms.), ethylene g l y c o l (80 m i s . ) and p-toluene s u l f o n i c acid hydrate  (175 gms.)  i n benzene (400 mis.)  was  refluxed for 18 hours with a Dean - Stark apparatus to remove water.  The  mixture was poured into excess sodium bicarbonate s o l u t i o n , the layers separated, and the aqueous phase extracted with benzene.  The combined  benzene layers were washed with sodium bicarbonate s o l u t i o n , water, dried over sodium s u l f a t e , and evaporated  to give 150 gms.  of o i l .  This o i l  x  220  when d i s t i l l e d through a short Vigeraux column at 120°C at 20 mm. gave 3-acetylpyridine ethylene k e t a l (120 gms.).  NMR (60 MHz)  1.23 (IH,  doublet, J = 2 Hz, C -H), 1.47 (IH, quartet, J = 4 and 2 Hz, C ~H), 2  2.25  6  (IH, m u l t i p l e t ) , 2.77 (IH, m u l t i p l e t ) , 6.15 (4H, multiplet, k e t a l ) ,  8.37 (3H, s i n g l e t , CH ). 3  N-[g{3(2-carboethoxyindolyl)}ethyl]-3-acetylpyridinium chloride ethylene k e t a l (77) In a Carius tube 2-carboethoxy-3(g-chloroethyl)indole (60) (5.2 gms.) and 3-acetylpyridine ethylene k e t a l (15 mis.) was heated f o r 16 hours at 125°C.  The solution was cooled, dissolved i n methanol, f i l t e r e d , and  evaporated.  T i t u r a t i o n with ether produced  m.p. 232 - 233°C.  a gray p r e c i p i t a t e (7.5 gms.),  IR (KBr) 1703 (COOEt).  UV (log e) 296 (4.16), 272 sh  (3.85), 265 sh. (3.80), 227 (4.38), 222 (4.32). multiplet, k e t a l ) , 8.62 (3H, s i n g l e t , C0CH ) . 3  NMR (CD 0D) 3  6.15 (4H,  (Found C, 63.50; Ii, 6.15;  N, 6.52; C H N 0 C 1 requires C, 63.38; H, 6.04;'N, 6.72%). 22  25  2  4  N-[8{3(2-carboethoxyindolyl)}ethy1]-3-acety1-1,2,5,6-tetrahydropyridine ethylene k e t a l (78) The pyridinium chloride 77 (5.6 gms.) i n methanol (300 mis.) with added triethylamine (3 mis.) was cooled to 0°C i n an i c e - s a l t bath under nitrogen.  Sodium borohydride (18 gms.) was added i n small portions  such that the temperature  remained at 0°C.  After the addition was complete  the s o l u t i o n was s t i r r e d f o r 2 hours and the methanol then slowly evaporated under reduced pressure with, a bath temperature  of 25°C.  Addition of  water to this paste followed by extraction with methylene chloride, washing with water, drying over potassium carbonate, and evaporating, gave a  221  yellow o i l (5 gms.)  which was  c r y s t a l l i z e d from petroleum ether - methylene  chloride to give 78 as a white s o l i d , m.p. 1678  (COOEt).  UV (log e) 290  0.78  (IH, NH),  4.08  8.50  (3H, s i n g l e t , CH^CO) ..  129 - 131°C.  (4.47), 253 (.4.59), 210  (IH, broad s i n g l e t , H-C=C), 6.14  IR (KBr) 3320 (NH),  (4.15).  NMR  (60  MHz)  (4H, multiplet, k e t a l ) ,  N-[3{3(2-hydroxymethylindolyl)}ethyl]-3~acetyl-l,2,5,6-tetrahydropyridine ethylene k e t a l  (79)  The carboethoxy was  derivative 78 (4i8 gms.)  i n tetrahydrofuran (50 mis.)  added dropwise to a s t i r r e d suspension of lithium aluminum hydride i n  tetrahydrof uran (300 mis..) at 0°C.  The solution was  refluxed for 2 hours,  cooled to 0°C, and the excess hydride destroyed by dropwise addition of saturated potassium carbonate solution.  The r e s u l t i n g s o l i d s were separa-  ted by f i l t r a t i o n and washed with chloroform. potassium  carbonate and evaporated.  chromatography on alumina  (250 gms.)  i n chloroform, and chloroform.  The f i l t r a t e was  The alcohol 78 was p u r i f i e d by by e l u t i o n with benzene, 50% benzene  C r y s t a l l i z a t i o n from petroleum ether -  methylene chloride gave 79 as a white s o l i d m.p. 3200 (OH).  UV  (log e) 294  212 sh (4.45).  NMR  s i n g l e t , k e t a l ) , 8.60 N, 8.05;  C  20 26 2°3 H  N  342.193 C H N 0 2 0  2 6  2  3  (3.95), 268  (60 MHz)  r  e  4.15  l  u  :  L  r  e  s C  >  7  0  •  114 - 116°C.  (IH, multiplet, H-C=C), 6.20  1  5  5 ' H  IR  (4.02), 255 sh (3.98), 248  (3H, s i n g l e t , CH3-C). c  dried over  7  -  6  5  5  >  (4.66),  (4H,  (Found C, 70.21; H, N  (KBr)  7.64;  8.18%; high resolution  requires 342.194).  N-[B{3(2—benzoxymethylindolyl)}ethyl]-3-acetyl-l,2,5,6-tetrahydropyridine ethylene k e t a l  (80)  The alcohol 79 (2.8 gms.) amine (2.28 mis.) was  i n tetrahydrofuran (20 mis.) with t r i e t h y l -  cooled to 0°C and benzoyl chloride (1.14 mis.)  was  222  added dropwise. was  After 3 hours at 0°C saturated sodium carbonate solution  added dropwise and the tetrahydrofuran decanted, dried over sodium  carbonate, and evaporated to give crude benzoate (3.6 gms.). by column chromatography on alumina  (150 gms.)  and e l u t i o n with methylene  chloride followed by c r y s t a l l i z a t i o n gave benzoate 80, m.p. IR (KBr) 1713. MHz)  1.35  UV  (log e) 285  (4.53), 260  (IH, broad s i n g l e t , NH),  (4.66), 224  1.9 - 2.9  protons), 4.12  (IH, broad s i n g l e t , H-C=C), 6.16  protons), 8.54  (3H, s i n g l e t , C H 3 - C ) .  C  27 30 2°4 H  N  r  e  c  l  u  i  r  e  s  c  >  7 2  ' ; 6  H  ' -5 6  8  N  >  Purification  147 - 148°C.  (5.33).  NMR  (60  (9H, multiplets, aromatic (4H, m u l t i p l e t , k e t a l  (Found C, 72.4; H, 6.6; N,  6.2;  6.3%).  N-[g{3(2-cyanomethylindolyl)}ethyl]-3-acety1-1,2,5,6-tetrahydropyridine ethylene k e t a l  (81)  The benzoate 80 (2.4 gms.) potassium cyanide (4 gms.) the temperature was that temperature  was  i n N,N-dimethylformamide (75 mis.) with s t i r r e d at room temperature  for 1 hour;  raised to 110°C over 45 minutes and maintained at  for a further 45 minutes.  The reaction was  d i l u t e d with water, and extracted with chloroform.  cooled,  The organic layer  was washed with water (3 times), saturated potassium carbonate, and dried over potassium carbonate. (200 gms.)  with methylene chloride as eluant gave n i t r i l e 81 (1.6 gms.).  IR (CHC1 ) 2255 (CN). 3  6.12  Evaporation and chormatography on alumina  NMR  (60 MHz)  4.12  (4H, m u l t i p l e t , k e t a l protons), 6.23  (IH, broad s i n g l e t , H-C=C), (2H, s i n g l e t , CH CN), 8.52 2  (3H,  s i n g l e t , CH -C). 3  N-[8{3(2-carboethoxymethylindolyl)}ethyl]-3-acetyl-1,2,5,6-tetrahydropyridine  (82) The n i t r i l e 81 (750 mgs.)  was  dissolved i n absolute methanol (20 mis.)  223  and water  (0.2 mis.) and dry hydrogen chloride gas was bubbled into the  solution for 45 minutes.  After 48 hours at room temperature, the methanol  was evaporated, the residue neutralized with saturated sodium carbonate solution  and extracted with methylene dichloride.  The organic phase was  washed with water, dried over sodium s u l f a t e , and evaporated to give crude ester (660 mgs.). e l u t i o n with methylene  Column chromatography  chloride gave keto ester 82 (230 mgs.)  IR (film) 3400 (NH), 1727 (IH,  on alumina (50 gms.)  (COOMe), and 1652  broad s i n g l e t , NH), 3.17  (^CO).  NMR  as an o i l .  (60 MHz)  (IH, broad s i n g l e t , H-C=C), 6.29  s i n g l e t , CH C00), 6.33.(311, s i n g l e t , C00CH ) , 7.75 2  3  and  1.50 (2H,  (3H, s i n g l e t , CH C0) . 3  Mercuric acetate oxidation of keto ester 82 Keto ester 82 (52 mgs.)' and mercuric acetate (200 mgs.)  i n methanol  (15 mis.) were s t i r r e d under nitrogen for 36 hours at room temperature. Mercurous  acetate (103 mgs.)  was f i l t e r e d  o f f and hydrogen s u l f i d e gas  bubbled into the f i l t r a t e .  This f i l t r a t e was r e - f i l t e r e d through C e l i t e  and evaporated to dryness.  Fresh methanol  (10 mis.), triethylamine (0.5  mis.), and 10% palladium on charcoal (25 mgs.)  were hydrogenated at  atmospheric pressure and room temperature f o r 48 hours. filtered  The catalyst was  o f f and the f i l t r a t e evaporated to give an o i l (35 mgs.).  IR (CHC1 ) 1754, 1724, and 1681. 3  Chromatography on alumina (5 gms.)  e l u t i o n with chloroform and the methanol gave an o i l (5 mgs.). 1750, 1722, and  and  IR (CHC1 ) 3  1685.  N-[B{3(2-carboethoxyindolyl)lethyl]-3-acetylpyridinium chloride (84) 2-carboethoxy-3(8-chloroethyl)indole 60 (2.15 gms.)  i n 3-acetylpyridine  (6 mis.) was heated under nitrogen i n a Carius tube at 110°C for 18 hours  224  after which time the reaction mixture was  t i t u r a t e d with ether and  pyridinium chloride collected by f i l t r a t i o n as an orange powder. r e - c r y s t a l l i z a t i o n from methanol - ether gave 84, m.p. IR (KBr) 1702 (3.98), 227  (ester), 1691  (4.55), 221  s i n g l e t , C i H ) ; 0.97  3.09  C,iH), 1.90 4  (4.54).  NMR  (100.MHz, DMSO - d )  3  174°C.  0.61  &  r  2  Careful  (4.36), 268 sh  (IH, quartet, J = 8 and 6 Hz, C ,E); 5  Hz, 0-CH_ CH ); 6.29  CH C0) ; 8.70  (log e) 295  &1  (4H, aromatic protons); 4.97  J = 6.5  UV  (HI, doublet, J = 6 Hz, C H ) ; 1.12  2  8.Hz,  (ketone).  172.-  the  (2H, m u l t i p l e t , CH^N)  (IH, broad  (IH, doublet, J =  2.52,  2.60,  ; 5.78  (2H, m u l t i p l e t , CH -Ind.); 7.44 2  2.80,  (2H, quartet,  (3H, s i n g l e t ,  (3H, t r i p l e t , J = 6.5 Hz, 0-CH -CK_ ) .  3  2  3  N-[g{3(2-carboethoxyindolyl)}ethyl]-3-acetyl-l,4,5,6-tetrahydropyridine The pyridinium chloride 84 (500 mgs.)  was  hydrogenated at room temper-  ature and atmospheric pressure i n ethanol (35 mis.) with 10% palladium on charcoal (100 mgs.) The catalyst was residue was mis.)  filtered  (85)  and triethylamine (1 ml.)  u n t i l uptake ceased (48 hours).  o f f and the f i l t r a t e evaporated to dryness.  The  dissolved i n chloroform and extracted with pH3 buffer (3 x 50  and pH2  acid (0.01N HC1,  3 x 25 mis.), dried over sodium s u l f a t e , and  evaporated to give crude amide 85.  Chromatography on alumina (25  gms.)  and e l u t i o n with chloroform gave amide 85 as a yellow o i l (215 mgs.). IR (CHC1 ) 1706  (ester); 1626,  (4.52), and 211  (4.47).  3  NMR  1553  (amide).  (60 MHz)  (IH, m u l t i p l e t , aromatic protons), 2.80  0.47  UV  (log e) 305  (4.63),  (IH, broad s i n g l e t , NH),  (3H, m u l t i p l e t , aromatic  2.42  protons),  3.02  (IH, s i n g l e t , H-C=C), 5.65  (2H, quartet, J = 7.1 Hz, -OCH CH ),  7.80  (3H, s i n g l e t , COCH^ , 8.63  (3H, t r i p l e t , J = 7.1 Hz, OCH^H^).  2  257  3  225  N~[B{3(2-hydroxymethylindolyl)}ethyl]-3-acetylpiperidine The amide 85 (40 mgs.)  (86)  i n tetrahydrofuran (10 mis.) was  0°C and lithium aluminum hydride (5 mgs.)  was  TLC showed absence of s t a r t i n g material.  A f t e r addition of a few drops  of saturated potassium  carbonate  the solution was  dried over sodium s u l f a t e and evaporated. (5 gms.) 5.32  gave 86 as an o i l .  3  2  A f t e r 15 minutes  f i l t e r e d and the f i l t r a t e  Chromatography on alumina  IR (CHC1 ) 1710  (2H, s i n g l e t , CH_ 0H), 7.90  added.  cooled to  (ketone).  NMR  (60  MHz)  (3H, s i n g l e t , CH C0) . 3  N-[p{3(2-hydroxymethylindolyl)}ethy1]-3-acety1-1,4,5,6-tetrahydropyridine The amide 85 (105 mgs.)  i n tetrahydrofuran (15 mis.) was  (87)  cooled to  -30°C i n a dry i c e - carbon t e t r a c h l o r i d e bath and lithium aluminum hydride (10 mgs.)  was  added.  After 1 hour at -30°C the temperature was  raised to 0°C and the reaction worked up as beforealumina (10 gms.) mgs.).  Reduction  Chromatography on  and elution with chloroform gave amide alcohol 87  IR (CHC1 ) 1625, 3  H-C=C), 5.33  slowly  1550  (amide).  NMR  (2H, s i n g l e t , CH 0H), and 7.80 2  (60 MHz)  3.05  (11  (IH, s i n g l e t ,  (3H, s i n g l e t , C0CH ). 3  of 85 with lithium borohydride  The amide 85 (40 mgs.) lithium borohydride  (5 mgs.)  only s t a r t i n g material was  i n tetrahydrofuran was added. present.  cooled to 0°C  and  After 3 hours at 0°C TLC showed that Allowing the solution to warm to  room temperature for one and a h a l f hours seemed to be without  effect.  The solution was  TLC  refluxed for 1 hour and worked up as before.  showed that the major product was keto alcohol 86 with a trace amount of amide alcohol 87 present.  226  Reduction of 85 with sodium borohydride The amide 85 (45 mgs.)  i n methanol (10 mis.) was  and sodium borohydride (10 mgs.)  was  cooled to 0°C  added and the s o l u t i o n slowly warmed  and f i n a l l y refluxed for 2 hours.  TLC examination showed that no reduction  had taken place.  The s o l u t i o n was  cooled to 0°C and fresh sodium boro-  hydride (10 mgs.)  was  added.  The temperature was maintained at 0°C for  l.hour before warming to reflux.  TLC examination revealed the presence  of only s t a r t i n g material. 2-hydroxymet.hylindole  (89)  Methyl indole-2-carboxylate (88) (10 gms.)  i n tetrahydrofuran was  added dropwise to a s t i r r e d suspension of lithium aluminum hydride (4 in tetrahydrofuran (250 mis.) at 0°C.  gms.)  After addition the solution was  refluxed for 2 hours, cooled, and excess hydride decomposed by addition of saturated potassium carbonate solution.  -The-solids were removed by  f i l t r a t i o n and washed with methylene d i c h l o r i d e . 'The organic solution was washed with saturated potassium carbonate s o l u t i o n , dried over sodium s u l f a t e , and evaporated to y i e l d crude alcohol 89. alumina (250 gms.)  and e l u t i o n with chloroform gave, after c r y s t a l l i z a t i o n  from benzene, the alcohol 89 (5.8 gms.), m.p. 3380 (OH). NMR  (60 MHz)  C H), 3  5.48  Chromatography on  73 - 74°C.  IR (KBr)  UV (log e) 290 (3.81), 281 (3.97), 271 (3.98), 218 1.66  (IH, broad s i n g l e t , NH),  (2H, s i n g l e t , CH_0H) , 7.04  2-benzoxymethylindole  2  (IH, doublet, J = 3 Hz,  (IH, s i n g l e t ,  OH).  (90)  The alcohol 89 (5 gms.) amine (9.3 mis.) was  3.77  (4.64).  i n tetrahydrofuran (100 mis.) and t r i e t h y l -  cooled to 0°C and benzoyl chloride (4.8 mis.)  was  22 7  added dropwise.  After 3 hours at 0°C saturated potassium carbonate was  added, followed by methylene chloride. washed with methylene dichloride.  The organic layers were combined, dried  over sodium s u l f a t e , and evaporated. (250 gms.)  The water layer was separated and  Column chromatography on alumina  and e l u t i o n with benzene gave benzoate 90 (8.4 gms.).  l i z a t i o n from benzene gave a white s o l i d m.p. 3355 (NH), 1700 (ester). 217 (4.79).  NMR  128 - 129°C.  Crystal-  IR (KBr)  UV (log e) 290 (3.90), 282 (4! 14), 270 (4.19),  (60 MHz)  1.01 (IH, broad s i n g l e t , NH), 1.95  (2H, -  m u l t i p l e t , aromatic protons), 2.3 - 3.0 (7H, multiplets, aromatic protons), 3.40  (IH, doublet, J = 3 Hz, C H) , 4.52 3  (2H, s i n g l e t , CH_ 0). 2  2-cyanomethylindole (91) The benzoate 91 (5.7 gms.) was dissolved i n N,N-dimethylformamide (130 mis.) and potassium cyanide (7.5 gms.).  After s t i r r i n g at room temper-  ature f o r 1 hour the temperature was slowly raised to 80°C over a 1 hour period and maintained at that temperature for 3 hours.  After cooling to  room temperature methylene d i c h l o r i d e and water were added.  The layers  were separated and the water layer washed with fresh methylene d i c h l o r i d e . The organic layers were combined, washed with water, dried over sodium s u l f a t e , and evaporated.. The residual N,N— dime thy If ormamide was  removed  by freeze drying and the s o l i d residue chromatographed on alumina. with benzene gave n i t r i l e 91 (2.8 gms.). gave a white s o l i d m.p. 2245 (CN). NMR  (60 MHz)  102 - 103°C.  Elution  C r y s t a l l i z a t i o n from benzene  IR (KBr) 3370, 3320 (NH); 2270,  UV (log e) 298 (3.94), 277 (4.09), 265 (4.15), 217 (4.83). 1.82  (IH, broad s i n g l e t , NH), 2.4 - 3.0  aromatic protons), 3.56  (4H, m u l t i p l e t ,  (IH, doublet, J = 3 Hz, C„H), 6.10  (2H, s i n g l e t ,  228  CH CN).  (Found C, 76.83; H, 5.00;  H, 5.12;  N, 17.95%).  2  N, 17.94; C ^ H ^  requires C, 76.92  Methyl indole-2-acetate (92) The n i t r i l e 91 (2.1 gms.) water added) was  i n methanol (200 mis. absolute with 1%  treated with hydrogen chloride gas and allowed to stand  at room temperature  for 48 hours.  sodium bicarbonate solution was chloroform.  After evaporation of the methanol,  added and the mixture extracted with  The chloroform was washed with water, dried over sodium  s u l f a t e , and evaporated.  Chromatography on alumina  (100 gms.)  e l u t i o n with benzene gave the acetate 92 (2.1 gms.).  Crystallization  from benzene - petroleum ether gave a white s o l i d m.p. IR (KBr) 3350 (NH), 1719  (ester).  270  NMR  (4.04), 217  (4.69).  UV  (60 MHz)  (log e) 288 1.43  3.68  (IH, doublet, J = 3 Hz, C^U) , 6.29  6.32  (3H, s i n g l e t , C00CH ).  C  3  11 11 2 H  N 0  r  e  c  l  u  i  r  e  s  c  71 - 72°C.  (3.92), 279  (4.01),  (IH, broad s i n g l e t ,  NH),  (2H, s i n g l e t , CH C00) , and 2  (Found C, 69.55'; H, 5.80;  » 69.83; H, 5.86;  and  N,  7.42;  N, 7.40%).  Methyl 3(B-hydroxyethyl)indole-2-acetate (93) The acetate 92 (1.0 gm.)  i n carbon tetrachloride (100 mis.)  cooled to 0°C and ethylene oxide (0.4 mis.) added.  The solution  was was  cooled to -15°C and stannic chloride (0.65 mis.) i n carbon tetrachloride (20 mis.) was was  added dropwise.  After the addition was  complete s t i r r i n g  continued for 20 minutes keeping the temperature below 0°C.  form (35 mis.) and saturated sodium carbonate rapidly keeping the temperature  below 10°C.  Chloro-  (16 mis.) were added The organic layer was  rated and the aqueous layer washed with ether.  sepa-  The combined organic  229  layers were dried and evaporated. on alumina (50 gms.). (460 mgs.) mgs.)  and elution with chloroform - methanol gave alcohol 93 (465  as a brown o i l .  IR (CHClg) 3603 (OH), 3453 (NH), 1732 NMR  (60 MHz)  (2H, t r i p l e t , J = 6.7 Hz, CH_OH) , 6.32 2  (3H, s i n g l e t , C00CH ), 7.12 3  7.40  chromatographed  Elution with benzene gave s t a r t i n g acetate 92  UV 292, 284, 276 sh, and 224. 6.25  The r e s u l t i n g o i l was  1.20  (ester).  (IH, broad s i n g l e t , NH),  (2H, s i n g l e t , CH C00) , 6.40 2  (2H, t r i p l e t , J = 6.7 Hz, Ind.-CH ~), and 2  (IH, broad s i n g l e t , OH).  Methyl 3(g-bromoethyl)indole-2-acetate (94) To the alcohol 93 (340 mgs.)  i n ether (30 mis.) at 0°C,  tribromide (60 uls) i n ether (10 mis.) was added dropwise. was  The mixture  l e f t overnight at 0°C and then poured into saturated sodium carbonate  solution. ether.  The layers were separated and the aqueous layer extracted with  The combined ether extracts were washed with water, dried over  sodium s u l f a t e , and evaporated. on alumina (10 gms.) (190 mgs.). and 213.  NMR  CH C00), 6.60 2  phosphorous  The crude bromide 94 was  chromatographed  and elution with benzene gave 94 as a yellow o i l  IR (CHC1 ) 3450 (NH), 1721 (ester). 3  (60 MHz)  1.40  (4H, multiplet,  UV 325, 292, 284, 272 sh,  (IH, broad s i n g l e t , NH), 6.26 Ind.-CH CH Br), 6.30 2  2  (2H, s i n g l e t ,  (3H, s i n g l e t , C00CH ).. 3  N-[g{3(2-carbomethoxymethylindolyl)}ethyl]-3-acetylpyridinium bromide (95) The tryptophyl bromide derivative 94 (190 mgs.) was heated to 100°C f o r 3 hours.  i n 3-acetylpyridine  T i t u r a t i o n with ether produced the  pyridinium bromide 95 as a yellow s o l i d  (205 mgs.).  UV 289, 267, and 220.  2 30  N-[B{3(2-carbomethoxymethylindolyl)}ethyl]-3-acetyl-l,4,5,6-tetrahydropyridine (a)  (83)  Pyridinium bromide 95 (175 mgs.)  i n ethanol (50 mis.) .containing  triethylamine (0.5 mis.) was hydrogenated over 10% palladium on charcoal (80 mgs.)  f o r 48 hours.  evaporated,  The catalyst w a s . f i l t e r e d o f f , the ethanol  and the residues dissoved i n chloroform.  extracted wtih pH3 b u f f e r (3 x 25 mis.) and pH2 drying over sodium s u l f a t e the chloroform was on alumina  (10 gms.).  3  NMR  s i n g l e t , H-C=C), 6.35  (ester), and 1610  (100 MHz)  acid (2 x 25 mis.); a f t e r  evaporated and chromatographed  0.98  (amide), 1560  2  (3H, s i n g l e t , C0CH ).  (b)  To the alcohol 93 (20 mgs.)  (C=C).  (IH, broad s i n g l e t , NH),  (2H, s i n g l e t , CH_ C00) , 6.38  8.20  UV 294 sh,  3.15  (IH,  (3H, s i n g l e t , C00CH ) , 3  3  i n 3-acetylpyridine (5 mis.) at 0°C,  phosphorous tribromide (37 jiis.) was was  was  Elution with chloroform gave the amide 83 (34 mgs.).  IR (CHC1 ) 3450 (NH), 1720 286 sh, and 223.  The chloroform  raised to 85°C and maintained  added.  After addition the temperature  there for 6 hours.  t i t u r a t i n g with ether, the brown s o l i d s (600 mgs.) dried.  After cooling and  were f i l t e r e d off and  The s o l i d s were dissoved i n ethanol (100 mis.), f i l t e r e d ,  hydrogenated over 10% palladium on charcoal (100 mgs.) The catalyst was  and  for 48 hours.  f i l t e r e d o f f , the ethanol evaporated, and the residues  dissolved i n chloroform.  The chloroform solution was extracted with  b u f f e r (3 x 50 mis.), pH2  solution (2 x 50 mis.), dried over sodium s u l f a t e ,  and evaporated.  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