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Wood extractives : the structure and chemistry of some triterpenes isolated from the bark of Sitka spruce… Roger, Ian Henry 1967

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The Uni v e r s i t y of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of IAN HENRY ROGERS' B.Sc, Queen's University, B e l f a s t , 1954 M.Sc, The Un i v e r s i t y of B r i t i s h Columbia, 1959 WEDNESDAY, MAY 24th,' 1967, AT'3:30 P.M.: IN ROOM 261, CHEMISTRY BUILDING COMMITTEE IN CHARGE Chairman: I.McT.- Cowan. J.A.F. Gardner. E. Piers J.P. Kutney R.E. Pincock C A . McDowell G.B. Porter External Examiner: John W. Rowe D i v i s i o n of Wood Chemistry Forest Products Laboratory U.S. Department of Agri c u l t u r e • Madison, Wisconsin Research Supervisor: J. P. Kutney WOOD EXTRACTIVES: THE STRUCTURE AND CHEMISTRY OF SOME TRITERPENES ISOLATED FROM THE BARK OF SITKA SPRUCE (PICEA SITCHENSIS) ABSTRACT The neutral f r a c t i o n of the petroleum ether extract of Sitka spruce (Picea sitchensis) bark was shown to be r i c h i n compounds of the t r i t e r p e n e c l a s s . In particu-l a r three monomethyl ether derivatives of triterpene d i o l s were i s o l a t e d (compounds A, B and C). The structure of the main component, compound A, was proved by methylation studies to be 3^3-methoxy-2b3-hydroxy-^•A-serratene (LXXI) and the parent d i o l , 21-episerra-tenediol (LIV), and. r e l a t e d ketone, 3/3-methoxy-21 keto-^•^-serratene-(LVIIa) were also i s o l a t e d and i d e n t i f i e d . The other major component, compound B,was shown to have the structure 3oC-methoxy-21^S-hydroxy-^-^-serratene (LXXX) by appropriate c o r r e l a t i o n with known compounds of the serratene s e r i e s . Compound C i s isomeric with A and B but the double bond ex i s t s in the f u l l y s u b s t i t u t e d ^ ^ p o s i t i o n . Th< most l i k e l y structure for t h i s compound i s 3oC-methoxy-2LS-hydroxy-^-3-serratene (LXXXI) as shown by the corre-l a t i o n of i t s acetate with iso compound B acetate. Two other triterpenes, a k e t o l , C3oH4g02, and a d i -a x i a l d i o l , C 3 0 H 5 0 O 2 , were also- i s o l a t e d but t h e i r structures were- not c o n c l u s i v e l y established. A d i t e r pene alcohol, C 2 0 H 3.4-0, was separated and had s p e c t r a l properties suggesting i t s ' i d e n t i t y with the known compound manool. The extract also contained f a t t y acid esters of^S -s i t o s t e r o l and presumably of g l y c e r o l . The i d e n t i t y o, the acids was not investigated. A low melting wax est> was recovered but was not examined in d e t a i l . I t i s interesting, to note that the fundamental triterpene skeleton of the above compounds i s one in which ri n g C Is seven-membered and only-recently have such compounds been obtained i n Nature. They undoubt-edly present an i n t e r e s t i n g v a r i a t i o n i n triterpene bio-synthesis. GRADUATE STUDIES F i e l d of Study: Chemistry of Natural Products Organic Stereochemistry L.D. Hayward Heterocyclic Compounds F. McCapra A l k a l o i d Chemistry J.P. Kutney Isoprenoid Compounds T. Money Organic Reaction Mechanisms R.E. Pincock Structure of Newer Natural Products T. Money PUBLICATION G.G.S. Dutton and I.H. Rogers, Occurrence of 2-0-(4-0-Me D-glucopyranosiduronic acid)-D-xylose i n jute hemicellu-lose. J. Am. Chem. Soc., 81, 2413 (1959). WOOD EXTRACTIVES: THE STRUCTURE AND CHEMISTRY OF SOME TRITERPENES ISOLATED FROM THE BARK OF SITKA SPRUCE (PICEA SITCHENSIS) by IAN HENRY ROGERS B. S c , Queen's U n i v e r s i t y , B e l f a s t , 1954 M.Sc., The U n i v e r s i t y of B r i t i s h Columbia, 1959 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 req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1967 In p re sen t i ng t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permiss ion f o r ex ten s i ve copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r ep re sen t a t i v e s , It i s understood that copying o r p u b l i c a t i o n of t h i s t he s i s f o r f i n a n c i a l gain s h a l l not be a l lowed wi thout my w r i t t e n pe rm i s s i on . Depa rtment The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8 , Canada i i ABSTRACT The neutral fraction of the petroleum ether extract of Sitka spruce (Picea sitchensis) bark was shown.to be,rich in compounds of the triterpene class. In particular three monomethy1 ether derivatives of triterpene diols were isolated (compounds A, B and C). The structure of the main component, compound A, was proved by methylation studies to be 3g-methoxy-216-hydroxy-A1 4-serratene (LXXI) and the parent diol , 21-episerratenediol (LIV), and related ketone, 3B-methoxyT21 keto-A11+-serratene (LVIIa) were also isolated and identified. The other major component, compound B, was shown to have the structure 3a-methoxy-213-hydroxy-A1^-serratene (LXXX) by appropriate correlation with known compounds of the serratene series. Compound C is isomeric with A and B but the,double bond -exists in the fully substituted A 1 3 position.The most likely structure for this compound is 3a-methoxy-r21g-hydroxy-A13-serratene (LXXXI) as shown,by the correlation of its acetate with iso compound B acetate. Two other triterpenes, a ketol, C 3 o H i + 8 0 2 > a n < i a diaxial diol, C 3 0 H 5 Q O 2 , were also isolated but their structures were not conclusively established. A diterpefie alcohol, 0 2 0 ^ 3 1 + 0 , was separated and had spectral properties suggesting its identity with the known compound manool. The extract also contained fatty acid esters of g-sitosterol and; presumably of glycerol. The identity of the acids was not investigated•. A low melting wax ester was recovered .but was not examined in detail. It is interesting to note that the fundamental triterpene skeleton ofthe above compounds is one in which ring C is seven-membered and only recently have such compounds been obtained in Nature. They undoubtedly present an interesting variation in triterpene biosynthesis. i i i TABLE OF CONTENTS Page T i t l e Page i Ab s t r a c t i i Table o f Contents i i i L i s t of Tables i v L i s t o f Figures v Acknowledgement v i I n t r o d u c t i o n 1 Dis c u s s i o n 31 Experimental . . 117 Bi b l i o g r a p h y 178 iv LIST OF TABLES Page 1. Pine Bark Sterols 8 2. Values of Components on a Sil ica Plate Developed with Chloroform and Sprayed with Antimony Pentachloride . . 32 3. Methyl Group Integrals from N.M.R. Spectra of A and B Derivatives 53 4. Effects of Changes in Functionality on Mass Spectral Fragmentation in A and B Series . . 59 5. Polar Components Isolated from Sitka Spruce Bark Extract . 70 6. Separation of Olefin Mixture on a Silver Nitrate/Silica Column 98 7. Influence of Functional Group Configuration on T.L.C. Properties 102 V LIST OF FIGURES Figure No. Page 1. Separation of Triterpenes on a Sil ica Thin Layer . . 31 2. Infrared Spectrum of Compound A . . 34 3. N.M.R. Spectrum of Compound B. 35 4. N.M.R. Spectrum of Compound A 36 5. Mass Spectral Fragmentation of Compound A . . . . . 38 6. Mass Spectral Fragmentation- of Compound B . . . . . 39 7. ORD Curves of Compounds A and B Ketones 43 8. Mass Spectral Fragmentation of Compound A Ketone Ethylene Ketal 49 9. Mass Spectra of Sawamilletin and Desoxy Compound A . 55 10. N.M.R. Spectrum (100 Mc/s), of Compound A Acetate . . 61 11. N.M.R. Spectrum (100 Mc/s) of Compound B Acetate . . 62 12. ORD Curves of Compound A Ketone and"Pine Bark Ketone, 69 13. N.M.R. Spectrum (100 Mc/s) of 3a,216-DihydroxyTA l l +-Serratene 89 14. Decoupling Experiments on A2-6-Serratane in Benzene 100 15. Infrared Spectrum of Compound P I l l 16. N.M.R. Spectrum (100 Mc/s) of Compound P 112 v i ACKNOWLEDGEMENT The author wishes to express h i s indebtedness to Dr. H.L. Hergert and t o Rayonier Incorporated f o r the g i f t of e x t r a c t s from S i t k a spruce bark on which t h i s study is.based. He i s a l s o deeply indebted t o . P r o f e s s o r Y. Inubushi, to Dr. J.W. Rowe^ and t o . P r o f e s s o r B e r t i f o r the g i f t of aut h e n t i c samples of compounds r e l a t e d to A 1 ^ - s e r r a t e n e . F i n a n c i a l a s s i s t a n c e from the N a t i o n a l Research C o u n c i l o f Canada i s g r a t e f u l l y acknowledged. The author was fortunate i n being awarded a Cominco Graduate Fellowship f o r two years during h i s p e r i o d of study and t h i s was of great b e n e f i t to him. I t has been a most worthwhile experience to work with Dr. Kutney and h i s group during the past two and one h a l f years. His wide v a r i e t y of i n t e r e s t s have been both s t i m u l a t i n g and e n r i c h i n g and I record my a p p r e c i a t i o n of h i s guidance. 1 Wood Extractives: The Structure and Chemistry of Some  Triterpenes Isolated from Si t k a Spruce Bark INTRODUCTION The past ten years has seen a great development i n our knowledge of the chemistry of natural products. This advance has been spectacular because of the more or less simultaneous a v a i l a b i l i t y of improved methods of separa-ti o n and p u r i f i c a t i o n together with powerful physical techniques which have greatly a s s i s t e d i n s t r u c t u r a l e l u c i d a t i o n . Not only have these techniques speeded up the processes leading to the assignment of s t r u c t u r a l formulae, but they have also frequently led to the s o l u t i o n of d i f f i c u l t problems which might otherwise have been insoluble by s t r i c t l y chemical techniques. Perhaps even more remarkable has been the progress i n the c l o s e l y r e l a t e d f i e l d of biochemistry i n which the mechanisms by which enzymatic reactions are c a r r i e d out i n vivo are now becoming much better understood. One of the r e s u l t s of t h i s advance i n our knowledge has been the development of theories to explain the biogenesis of the quite remarkable range of organic compounds e x i s t i n g i n the plant and animal kingdoms. These theories have been supported by t r a c e r techniques involving the use of l a b e l l e d com-pounds which are considered as being intermediates i n the biogenetic pathways. The r e s u l t of t h i s work has been a r e a l i z a t i o n that nature uses only a very few biogenetic pathways to synthesize t h i s vast array of organic material and i n consequence there has been generated a f e e l i n g that these widely diverse chemical compounds f a l l i n t o quite well-defined patterns. Use of this has i n fact been already made not only to c o r r e c t l y predict the occurrence of new and, at the time, unknown structures but even to predict that c e r t a i n structures already assigned were inco r r e c t because they obviously did not f i t into the e x i s t i n g biosynthetic patterns. 2 The forests of the world provide a most intriguing hunting ground for the natural products chemist eager to discover compounds with novel structures or for the pharmacist who wishes to examine the use of these extractives in the combat of disease. Canada, and particularly Britsh Columbia, is well endowed in this respect. Our country is indeed well forested,it is vast in area, the species of indigenous trees are numerous, and forest products play a large part in our economy. Nevertheless we remain in a state of ignorance of the structure and chemistry of very many of the extractives present in our woods, not to mention the potential uses which these compounds might have. In fact, a survey of the scientific literature shows that we really are only beginning to apply the powerful new techniques now available to determine the composition of the minor extraneous components of our North American softwood species. The pulp and paper industry has, of course, long been aware of the occurrence in trees of minor quantities of extraneous materials soluble in organic solvents and that these materials have undesirable effects i f not thoroughly removed during the pulp manufacturing operations. The presence of more than trace amounts of extractives, especially polyphenolic materials, is manifested by poor colour, brightness and light ageing properties in the finished pulps or papers. These undesirable effects have caused the industry in the past to.regard extractives as an annoyance and as something to be eliminated during the production process. This they have done and in the kraft process the two by-products, sulfate turpentine and ta l l oil, are recovered and are nowadays extremely valuable raw materials for the naval stores industry in the United States, besides which they earn a lot of money for the pulp manufacturers. 3 Some of the main modern uses for sulfate turpentine include the manu-facture of synthetic pine oil used in detergents, ore flotation, household cleansing fluids, bactericides, etc., the synthesis of _1-menthol used as a 53 tobacco additive in cigarette manufacturing and as a perfume , and the synthesis of a whole range of alicyclic monoterpenes widely used in the perfumery industry5"*. Tall oil is a mixture containing about 90% of resin and fatty acids in roughly equal proportions. The remainder is a complex mixture of fatty acid esters, sterols, higher alcohols and hydrocarbons in which most of the sterol fraction is esterified with fatty acids. According to Stevens* some of the main industrial uses of tall o il and its derivatives are in the manufacture of adhesives, binders, drying oils, soaps, printing inks, varnishes, etc. Esters of t a l l oil acids are used in drying oils, alkyd resins, plasticizers and lubricant additives. When coniferous trees are wounded, they slowly secrete a sticky terpenoid substance which may be collected and is known as "oleoresin". In areas of Florida and Georgia this process is carried out deliberately until the tree reaches maturity and the collected oleoresin is steam distilled to remove the "gum turpentine" after which the remaining fatty and resin acid esters are used by the naval stores industry. The species which are treated in this way are usually longleaf pine (Pinus palustris Mill.) and slash pine 2 (P. e l i o t t i i ) . According to Mutton , about 10% of the weight of the oleoresin is neutral material and the unsaponifiable fraction contains B-sitosterol (I) (24a-ethyl cholesterol) and other sterols, long chain fatty alcohols such as lignoceryl alcohol, CH3(CH2)22GH2OH, two diterpene aldehydes, dextropimarinal (Iia) and isodextropimarinal (Ilia), tricyclic diterpenes, diterpene alcohols and 3,5-dimethoxystilbene. II b R = COOH 2 Besides oleoresin, Mutton ' mentions the occurrence of "fatty-resin" in the parenchyma c e l l walls of the wood. This resin, as the name implies, is very rich in fatty acid esters but the unsaponifiable fraction has not been extensively investigated. . The sterols however form a major com-ponent and, once again, B-sitosterol (I) is the one usually reported. The resin acids present in the oleoresin of Pinus species and in the t a l l o i l derived from them in the kraft pulp process are diterpene acids "•• • typified by dextropimaric ( l i b ) , isodextropimaric ( I l l b ) , abietic (IV), palustric (V) and levopimaric acids (VI). In most cases the structures of these compounds were established many years ago. a R = CHO III b R = COOH 5 The p u l p and paper i n d u s t r y removes the bark from i t s logs p r i o r t o t h e i r c o n v e r s i o n i n t o p u l p . Most o f the bark i s used as f u e l but some i s e x t r a c t e d t o remove p o l y p h e n o l i c m a t e r i a l s . These e x t r a c t s , p a r t i c u l a r l y i n the case o f western hemlock (Tsuga h e t e r o p h y l l a ) f i n d important markets i n s o i l s t a b i l i z a t i o n , c o l d - s e t t i n g w a t e r p r o o f a d h e s i v e s , and o i l w e l l d r i l l i n g f l u i d s 5 . I t i s noteworthy t h a t western hemlock bark i s r i c h i n p o l y p h e n o l i c m a t e r i a l s but does not c o n t a i n s i g n i f i c a n t amounts o f h i g h e r t e r p e n o i d s and o n l y a s m a l l q u a n t i t y o f e s t e r i f i e d p h y t o s t e r o l s . A u s e f u l s tudy o f the amount o f e x t r a c t a b l e m a t e r i a l i n the barks o f twenty f o u r common North American pulpwood s p e c i e s was c a r r i e d put at the 3 F o r e s t Products L a b o r a t o r y , M a d i s o n , W i s c o n s i n by Chang and M i t c h e l l u s i n g s u c c e s s i v e l y benzene, e t h a n o l , hot water and one per cent aqueous sodium h y d r o x i d e s o l u t i o n . The va lues found f o r Engelmann spruce were 5.2 % benzene s o l u b l e and 25.9 % e t h a n o l s o l u b l e m a t e r i a l based on the weight o f the dry b a r k . For b l a c k spruce the va lues were 5 .0 % and 14.6 % r e s p e c t i v e l y . U n f o r t u n a t e l y S i t k a spruce was not among the s p e c i e s s t u d i e d and the i n v e s t i -g a t i o n o f what compounds were present i n the v a r i o u s e x t r a c t s was very super-f i c i a l . 4 A recent and more thorough study was the work o f Rowe and Scroggins who examined the benzene s o l u b l e e x t r a c t i v e s o f lodgepole p i n e (Pinus c o n t o r t a D o u g l . ) . The bark o f t h i s t r e e i s unusual i n that i t c o n t a i n s up to 30 % benzene s o l u b l e m a t e r i a l a l though t h i s v a r i e s w i d e l y from one l o c a t i o n t o another . The f r e e and combined a c i d s were removed from the benzene e x t r a c t by the u s u a l procedures o f washing w i t h a l k a l i and s a p o n i f i c a t i o n . The com-b i n e d a c i d s were predominant ly o f the u n s a t u r a t e d t y p e , e s p e c i a l l y o l e i c , l i n o l e i c and 5 , 9 , 1 2 - o c t a d e c a t r i e n o i c a c i d . The normal a l i p h a t i c f r a c t i o n was separated from the u n s a p o n i f i a b l e f r a c t i o n by f o r m a t i o n o f the u r e a c h a n n e l ' 6 i n c l u s i o n complex and chromatography separated t h i s m i x t u r e i n t o s m a l l amounts o f normal p a r a f f i n s and a major amount o f wax a l c o h o l s . Gas chromato-graphy r e v e a l e d the presence o f a m i x t u r e o f homologues i n both f r a c t i o n s w i t h the C2o> C22 and C 2 i + compounds b e i n g most p l e n t i f u l f o r the wax a l c o h o l s and the C 2 i and C23 compounds f o r the normal p a r a f f i n s . The s t e r o l f r a c t i o n o f lodgepole p i n e bark was separated from the u n s a p o n i f i a b l e f r a c t i o n by p r e c i p i t a t i o n as the d i g i t o n i d e s . T h i s f r a c t i o n c o n s i s t e d main ly o f ^ - s i t o s t e r o l (I) accompanied by 10 % campesterol (24a-methyl c h o l e s t e r o l ) (VII) and two ' i s t e r o l " a u t o x i d a t i o r i productsv a j - s i t o s t e r o l ( V I I I ) was not present•; V I I S ** V I I I A f t e r removal o f minor q u a n t i t i e s o f sesqui terpenes by steam d i s t i l -l a t i o n o f the remainder o f the u n s a p o n i f i a b l e f r a c t i o n , the o i l y r e s i d u e , s t i l l a c c o u n t i n g f o r 38 % o f the benzene e x t r a c t , was separated by column chromatography t o y i e l d f o u r new d i t e r p e n e compounds. These compounds were shown t o be 13-epimanool (IXa) ( A 8 ( 2 ° ) > 1 ^ - l a b d a d i e n - l S a - o l ) , c o n t o r t o l a l (Xa) ( 1 5 - h y d r o x y - A 8 ( 2 0 ) , 1 3 - l a b d a d i e n - 1 8 - a l ) , c o n t o r t a d i o l (Xb) ( A 8 ( 2 0 ) , 1 3 -l a b d a d i e n - 1 5 , 1 9 - d i o l ) and 18-hydroxy-13-epimannool(IXb) ( A 8 ( 2 0 ) , 1 ^ - l a b d a d i e n -1 3 a , 1 8 - d i o l ) . 7 OH In a l a t e r paper Rowe has d e s c r i b e d the s t e r o l s o f p i n e b a r k . The s p e c i e s examined were jack p i n e (Pinus b a n k s i a n a Lamb.) sugar p i n e (Pinus i a m b e r t i a h a D o u g l . ) , l o b l o l l y p i n e (P. taeda L . ) and lodgepole p i n e (P. c o n t o r t a Dougl . ) t o which r e f e r e n c e has a l r e a d y been made. The s t e r o l s were p r e c i p i t a t e d from the u n s a p o n i f i a b l e f r a c t i o n s o f the benzene e x t r a c t s w i t h d i g i t o n i n and were then separated from each o t h e r by c a r e f u l column chromato-graphy. The major component was, as expected , 6 - s i t o s t e r o l (I) which was 7 a l r e a d y w e l l known t o occur i n t h i s genus, i n t a l l o i l and i n h i g h e r p l a n t s 8 9 i n genera l ' . The analyses o f the s t e r o l f r a c t i o n s from the f o u r p i n e s p e c i e s are g i v e n i n Table 1. The " t o t a l o x i d i z e d 6 - s i t o s t e r o l " column was broken down by Rowe i n t o t h r e e components, 7 - k e t o - B - s i t o s t e r o l , A 3 > 5 - s t i g m a -s t a d i e n-7 - o n e , and A^-st igmasten-S-one . The f i r s t pure sample o f 6 - s i t o s t e r o l was o b t a i n e d by S t e e l e and M o s e t t i g 1 ^ o n l y i n 1963. A l l p r e v i o u s l y i s o l a t e d m a t e r i a l a p p a r e n t l y was contaminated w i t h campesterol V I I which can be removed o n l y by gas chromato-graphy. T h i s m a t e r i a l was found t o be present i n a l l the p i n e s t e r o l f r a c t i o n s . Another s t e r o l shown t o be present by gas chromatography i s a-s i t o s t e r o l V I I ) (4a-methyl -A 7 > 2 1 * ^ 2 8 ^ - s t i g m a s t a d i e n - 3 g - o l ) which i s b e l i e v e d TABLE 1 PINE BARK STEROLS % S t e r o l s % Benzene e x t r a c t i n benzene e x t r a c t 6 - s i t o s t e r o l campesterol a ( - s i t o s t e r o l T o t a l o x i d i z e d 6 - s i t o s t e r o l Other Lodgepole p i n e (28.7) 3.9 73 5 4 2.4 -L o b l o l l y p i n e ( 4.6) 2.8 68 7 4 4 .8 12 P i n u s e n e d i o l 1 C h o l e s t e r o l Sugar p i n e ( 2.1) 5.0 63 9 - 0.2 • -Jack p i n e ( 4.2) 2.0 60 6 4 2.1 5 P i n u s e n e d i o l 1 C h o l e s t e r o l 5 d i h y d r o s t e r o l s 0 .5 methoxy-t r i t e r p e n o l 9 to be an i n t e r m e d i a t e i n the b i o s y n t h e t i c pathway between l a n o s t e r o l and 6 - s i t o s t e r o l . From the p o i n t o f view o f t h i s t h e s i s , the most i n t e r e s t i n g f a c t about Rowe's paper on the p i n e bark s t e r o l s i s h i s d i s c o v e r y o f the presence o f p i n u s e n e d i o l i n l o b l o l l y and j a c k p i n e . 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 12 shown by Rowe to 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 i s o l a t e d by Inubushi and c o - w o r k e r s 1 1 from Lycopodium serratum Thumb, v a r . T h u n b e r g i i Makino, a type o f Japanese c l u b moss. S i n c e the paper on the s t r u c t u r e o f s e r r a t e n e d i o l appeared f i r s t i n the l i t e r a t u r e , the name s e r r a t e n e d i o l takes precedence over t h a t o f p i n u s e n e d i o l , and the l a t t e r w i l l not be used again i n the p r e s e n t c o n t e x t . A l s o o f e s p e c i a l note i s Rowe's r e f e r e n c e t o the occurrence o f a methoxylated t r i t e r p e n o l i n j a c k p i n e bark a l though the q u a n t i t y present was a p p a r e n t l y minute . He s p e c u l a t e d t h a t t h i s compound might be a monomethyl e t h e r o f s e r r a t e n e d i o l . In a footnote t o h i s paper Rowe mentioned the i s o l a t i o n by H e r g e r t o f a s m a l l amount o f a t r i t e r p e n e d i o l from the bark o f l o n g l e a f p i n e (Pinus p a l u s t r i s M i l l . ) . T h i s m a t e r i a l was shown to be a r a t h e r impure sample o f s e r r a t e n e d i o l on the b a s i s o f t h i n l a y e r and gas chromatographic comparisons w i t h an a u t h e n t i c s t a n d a r d . A search o f the l i t e r a t u r e shows t h a t much l e s s work has been done on the e x t r a c t i v e s o c c u r r i n g i n the spruces (genus P i c e a ) than on those from the p ines (genus P i n u s ) . Thus the compounds p a l u s t r i c ( V ) , l e v o p i m a r i c (VI) and a b i e t i c a c i d (IV) were shown t o be the main c o n s i t u e n t s o f the r e s i n a c i d 13 f r a c t i o n o f European spruce ( P i c e a a b i e s ) . Some Japanese workers i n v e s t i -g a t i n g P. j e z o e n s i s have r e p o r t e d the occurrence o f l e v o p i m a r i c , a b i e t i c , 14 i s o d e x t r o p i m a r i c and d i h y d r o a b i e t i c a c i d s . Another group, working on the same s p e c i e s , has r e p o r t e d the i s o l a t i o n o f l i g n o c e r i c a c i d CH 3(CH 2)22COOH, l i g n o c e r y l a l c o h o l CH 3(CH 2)22CH2OH and p h y t o s t e r o l s 1 5 . I n s o m e e a r l y w o r k a t 10 M c G i l l U n i v e r s i t y , Purves and co-workers e x t r a c t e d white spruce bark (Picea glauca) with methanol on a p i l o t p l a n t s c a l e . The ether s o l u b l e p o r t i o n of t h i s e x t r a c t contained about 50 % r e s i n acids w i t h the remainder being a mixture o f f a t t y acids and e s t e r s . L i g n o c e r i c a c i d , behenic a c i d CH3(CH2)20COOH and l i g n o c e r y l a l c o h o l were i s o l a t e d from a wax f r a c t i o n and the non-saponifiable p o r t i o n was s t a t e d t o contain 5 % c r y s t a l l i n e p l a n t s t e r o l s and ah u n i d e n t i f i e d hydroxy diterpene, C 2 0 H 3 4 O . More r e c e n t l y 17 Hergert examined the chemical composition o f the monomeric polyphenols and polymeric polyphenols (tannins) of twenty four species i n c l u d i n g S i t k a spruce (P. s i t c h e n s i s ) and Engelmann spruce (P. engelmannii). The v o l a t i l e o i l from the l e a f needles o f these two species was i n v e s t i g a t e d by Von 18 R u d l o f f using vapour phase chromatography . He found the main components to be d-myrcene, 1-piperitone, and d-camphor while a- and 8-pinene were present i n only t r a c e amount. Other monoterpenes present i n c l u d e d 1,8 c i n e o l e , 1-8-phellandrene, p-cymene, d-bornyl acetate, d-borneol and d-t e r p i n e n - 4 - o l . In a recent paper by A s s a r s s o n ^ , the e f f e c t o f wood seasoning on the chemical composition of the e x t r a c t i v e s i n European spruce (P. abies Karst.) was examined with p a r t i c u l a r reference to the f a t t y and r e s i n a c i d s . The present study i s concerned w i t h the chemical s t r u c t u r e of a s e r i e s of n e u t r a l t r i t e r p e n e s present i n the n o n - v o l a t i l e p o r t i o n of the benzene e x t r a c t from S i t k a spruce bark. The crude m a t e r i a l was a g i f t from Dr. H. L. Hergert of the Olympic Research D i v i s i o n of Rayonier, Incorp., Sheltonj Washington with whom the w r i t e r was p r i v i l e g e d t o work i n the pe r i o d 1961-64. Dr. Hergert has had wide experience i n the chemistry of wood.and bark e x t r a c t i v e s and, during the course of one of h i s i n v e s t i g a t i o n s , he had i s o l a t e d 26 gm. of a white s o l i d m a t e r i a l from the cork l a y e r of S i t k a 11 spruce b a r k . To prepare t h i s e x t r a c t the bark from the b u t t logs o f mature S i t k a spruce t r e e s was f i r s t removed by mechanical s o r t i n g and the ground bark was then e x t r a c t e d w i t h petro leum e t h e r . The t o t a l petro leum e t h e r s o l u b l e m a t e r i a l , which was a brown wax, was then d i s s o l v e d i n warm acetone, e t h e r or benzene and a l l o w e d t o s t a n d o v e r n i g h t . A w h i t e c r y s t a l l i n e d e p o s i t was formed and removed by f i l t r a t i o n . The same m a t e r i a l i s r e c o v e r a b l e from whole S i t k a spruce bark but i n lower y i e l d . D r . Hergert a l s o p r o v i d e d a b o t t l e c o n t a i n i n g about 100 gm. o f a brown waxy m a t e r i a l r e p r e s e n t i n g the recovered r e s i d u e from the acetone mother l i q u o r . From i n f r a r e d s p e c t r a l measurements on these two crude m a t e r i a l s , H e r g e r t was aware t h a t they c o n t a i n e d methyl e t h e r groups and t h a t they p r o b a b l y c o n t a i n e d n e u t r a l t r i t e r p e n e s . I t i s o f c o n s i d e r a b l e i n t e r e s t , t o note t h a t t r i t e r p e n e s o f known s t r u c t u r e c o n t a i n i n g methyl e t h e r groups are indeed q u i t e r a r e . The f i r s t example was the compound s a w a m i l l e t i n (XI) i s o l a t e d .from sawa m i l l e t g r a i n 19 o i l . This compound was c h a r a c t e r i z e d by Abe and Obara and shown t o be i d e n t i c a l w i t h the methyl e t h e r o f the known compound t a r a x e r o l ( X I I ) . 3B-h y d r o x y - A 1 ^ - o l e a n e n e ) . The second t r i t e r p e n e b e a r i n g a methyl e t h e r f u n c t i o n was the com-pound m i l i a c i n ( X I I I ) which was i s o l a t e d from the u n s a p o n i f i a b l e f r a c t i o n o f m i l l e t seed o i l (Panicum m i l i a c e u m L . ) 20 by Abe and co-workers . The s t r u c t u r e o f t h i s m a t e r i a l was proven t o be 3 6 - m e t h o x y - A 1 8 - o l e a n e n e by i n t e r - r e l a t i n g i t s s t r u c t u r e w i t h t h a t o f s a w a m i l l e t i n by s u i t a b l e double bond XI R = C H 3 X I I R = +i 12 X I I I a R = C H 3 XIV b R = H rearrangement r e a c t i o n s under a c i d i c 21 22 c o n d i t i o n s ' Next t o be announced was the compound g-amyrin methyl e t h e r . ( X l V a ) i s o l a t e d by E g l i n t o n and co-workers from New Zealand t o e - t o e grass 23 (Arundo conspicua) . This compound was i d e n t i c a l w i t h the compound i s o s a w a m i l l e t i n prepared by Abe and co-workers from s a w a m i l l e t i n by r e -arrangement o f the double bond under 22 a c i d i c c o n d i t i o n s . However, the major component from Arundo conspicua was a new compound a r u n d o i n , the s t r u c t u r e o f which proved t o be q u i t e d i f f i c u l t t o e l u c i d a t e . In f a c t E g l i n t o n and h i s group proposed the s t r u c t u r e XV which was l a t e r shown by 24 Nishimoto et a l to be erroneous The Japanese work was undertaken f o r a r a t h e r i n t e r e s t i n g reason. In Chinese medic ine the rhizome o f the p l a n t Imperata c y l i n d r i c a P. Beauv. v a r . media Hubbard was used as a d i u r e t i c . The presence o f two t r i t e r p e n o i d s i n 25 t h i s drug was r e p o r t e d i n 1961 by Ohno, Komatsu and Ohmoto . These were separated by Nishimoto and h i s c o l l a b o r a t o r s and shown t o be methyl e t h e r s . The more s o l u b l e compound proved t o be arundoin and the o t h e r was a new com-26 pound named c y l i n d r i n . The l a t t e r compound was shown t o be the methyl e t h e r o f i s o a r b o r i n o l , the s t r u c t u r e o f which was o n l y p a r t l y worked out at t h a t 13 t i m e 4 . With the very recent s o l u t i o n o f . t h e problem o f the s t r u c t u r e o f 28 a r b o r i n o l XVI by X - r a y d i f f r a c t i o n , the s t r u c t u r e o f c y l i n d r i n i s ,thus X V I I . XVI XVII 24 The c o r r e c t e d s t r u c t u r e d e r i v e d by Nish imoto f o r the compound arundoin i s X V I I I ( 3 8 - m e t h o x y f e r n - 9 ( 1 1 ) - e n e ) . He has r e c e n t l y shown t h a t the secondary a l c o h o l s c o r r e s p o n d i n g to c y l i n d r i n s> and a r u n d o i n , v i z . i s o a r b o r i n o l and f e r n e n o l c o - o c c u r i n the same p l a n t 29 source XVIII The f i r s t example o f a 3a-methoxylated t r i t e r p e n e i s a b i e s l a c t o n e XIX which i s a l s o the f i r s t known n a t u r a l l y o c c u r r i n g methyl e t h e r o f a t e t r a c y c l i c t r i t e r p e n e . T h i s compound occurs i n the bark and leaves o f the. Japanese : f i r ( A b i e s m a r i e s i i Masters] and 30 was i s o l a t e d and c h a r a c t e r i z e d by Uyeo and h i s c o l l a b o r a t o r s . This compound turned out t o be i d e n t i c a l w i t h a white c r y s t a l l i n e compound i s o l a t e d by Hergert from P a c i f i c s i l v e r f i r . (Abies a m a b i l i s (Dougl . ) Forbes)and a l s o from^Abies p r o c e r a Rehd.^and named by him as m e t h o x y a b i e s a d i e n e o l i d e . 14 Ap art from the methyl e t h e r d e r i v a t i e s o f the s e r r a t e n e d i o l t y p e , which occur i n p i n e s p e c i e s and which w i l l be d i s c u s s e d l a t e r , t h e r e are a p p a r e n t l y no o t h e r f u l l y c h a r a c t e r i z e d n a t u r a l l y o c c u r r i n g t r i t e r p e n e methyl e thers known. Three methoxyl c o n t a i n i n g t r i t e r p e n e s o f unknown s t r u c t u r e have however, been i s o l a t e d from n a t u r a l s o u r c e s . One o f these 31 i s found i n the r o o t s o f the yew t r e e (Taxus baccata) and c o n t a i n s an epoxide and a lac tone r i n g system. Another i s known as a c e t y l c o l l e t o -32 t r i c h i n and i s found i n the pathogenic p l a n t fungus C p l l e t o t r i c h u m C a p s i c i . T h i s compound i s s a i d to c o n t a i n o n l y twenty e i g h t carbon atoms. and has h y d r o x y l and u n s a t u r a t e d ketone groups. F i n a l l y the t r i t e r p e n e s o f the genus Commiphora are known t o c o n t a i n a s e r i e s o f compounds known as the commie a c i d s . One o f t h e s e , commie a c i d A c o n t a i n s a methoxyl 33 34 group, a secondary a l c o h o l and a methyl e s t e r grouping ' F i n a l l y i t may be mentioned t h a t some methyl e t h e r s , which are not n a t u r a l l y o c c u r r i n g , have been s y n t h e s i z e d d u r i n g s t r u c t u r a l e l u c i d a t i o n work on t r i t e r p e n e s c o n t a i n i n g p r i m a r y o f secondary a l c o h o l g r o u p i n g s . 35 36 These c o n s i s t o f the methyl e thers o f a-amyrin XX , 6-amyrin XXI , a s i a t i c a c i d X X I I " ^ and c i n c h o l i c a c i d X X I I I ^ 8 . XX XXI 15 Two t r i t e r p e n o i d compounds containing seven membered a l i c y c l i c r i n g systems i n t h e i r skeletons have recently been described. One of these i s the t r i t e r p e n e a l k a l o i d Buxenine-G XXIV i s o l a t e d by Kupchan and Asbun from 52 the tree species Buxus sempervirens L. This compound i s i n t e r e s t i n g from a biosynthetic standpoint as the seven membered r i n g B i s considered to ar i s e by cyclopropane r i n g cleavage of a cycloartane d e r i v a t i v e . Various, cycloartane based al k a l o i d s of the type now known as the "Buxus a l k a l o i d s " co-occur with t h i s compound. A t y p i c a l example i s cyclobuxine-D XXV. A t r i t e r p e n e d i o l with a most i n t e r e s t i n g novel r i n g system was discovered by Inubushi and co-workers 1 1 i n the Japanese club moss Lycopodium serratum as has already been mentioned. This compound, serratenediol XXVI i s the f i r s t known triterpene containing a seven membered r i n g C i n i t s . 39 structure and i t s biosynthesis has been postulated by Inubushi as occurring 16 from a - o n o c e r i n XXVII w i t h i n c o r p o r a t i o n o f one o f the methylene groups i n t o the r i n g system a c c o r d i n g t o Scheme 1. The i n t e r e s t i n g r e s u l t o f t h i s methylene i n c o r p o r a t i o n i s t h a t s e r r a t e n e d i o l c o n t a i n s o n l y seven a n g u l a r methyl groups i n s t e a d o f the normal e i g h t and i n t h i s respect i t i s unique among the p e n t a c y c l i c t r i t e r p e n e s . I t s h o u l d be observed t h a t , a l though the above theory e x p l a i n s the b i o s y n t h e s i s very n e a t l y , i t i s not as yet supported by any l a b e l l i n g s t u d i e s and t o date n e i t h e r a - o n o c e r i n n o r any o f i t s d e r i -v a t i v e s has been shown t o co-occur w i t h s e r r a t e n e d i o l i n t r e e s . However, another spec ies o f c lub moss, Lycopodium c l a v a t u m , was found t o c o n t a i n 39 major q u a n t i t i e s o f a - o n o c e r i n and has very r e c e n t l y been shown to c o n t a i n d i e p i s e r r a t e n e d i o l ( i n which the c o n f i g u r a t i o n o f both h y d r o x y l groups are 64 the r e v e r s e o f XXVI) and a r e l a t e d t r i o l named l y c o c l a v a n o l S e r r a t e n e d i o l i s a white c r y s t a l l i n e s o l i d , m.p. 300° a n a l y s i n g f o r C 3 0 H 5 0 O 2 . The compound c o n t a i n s two secondary h y d r o x y l groups as i t forms a d i a c e t a t e and a d i k e t o n e by s tandard r e a c t i o n procedures . W o l f f - K i s h n e r 17 reduction of the diketone gives the unsaturated hydrocarbon serratene, C 3 0 H 5 0 , XXVIII, m.p. 237-9° which can be hydrogenated over Adam's catalyst to give a mixture of two stereo-isomeric hydrocarbons C 3 Q H 5 2 , a and 6 serratane. The NMR spectrum of serratene-diol diacetate shows a signal for an XXVIII olefinic proton at T 4.69 and three signals for methyl groups at x 9.21, 9.16, and 9.11 in the rat io . l :5: l with two acetate methyls at x 7.97. A l l the methyl groups are quaternary as there is no coupling observed. The structure of the two terminal rings was deduced from the series of reactions shown in Scheme 2. Treatment of serratanediol XXIX by ring con-traction to produce the bis-isopropylidene derivative XXX. This compound shows two peaks at x 8.42 and 8.29 each integrating for six protons arising from two isopropylidene groupings. The three remaining methyl groups give signals at x 9.46, 9.40 and 9.11 in the ratio 1:1:1. Scheme 2 XXXI XXXII 18 The compound XXX can be i s o m e r i z e d when heated w i t h 3 % a l c o h o l i c hydrogen c h l o r i d e t o y i e l d compound X X X I , C^oH^s i n which the reappearance o f seven methyl groups above T 8.90 i n the NMR spectrum i n d i c a t e s the s h i f t o f the double bonds from the i s o p r o p y l i d e n e groups back i n t o the r i n g systems. Treatment o f compound XXX w i t h osmium t e t r o x i d e f o l l o w e d by l e a d t e t r a a c e t a t e cleavage y i e l d s acetone and the 0,2k d iketone X X X I I , 0 2 ^ 3 5 0 2 . T h i s compound shows a s i n g l e c a r b o n y l a b s o r p t i o n at 1736 c m - 1 (KBr) i n d i -c a t i n g the presence o f the c a r b o n y l groups i n two f i v e membered r i n g s . The t e r m i n a l r i n g s o f s e r r a t e n e d i o l must t h e r e f o r e be s i x membered and c o n t a i n the normal geminal d i m e t h y l groups i n the p o s i t i o n s v i c i n a l t o the secondary a l c o h o l f u n c t i o n s as i n g-amyr in , XVIb. When the r e a c t i o n s shown i n Scheme 2 are performed on the correspond-i n g u n s a t u r a t e d s e r r a t e n e d e r i v a t i v e s , n e i t h e r the b i s i s o p r o p y l i d e n e d e r i -v a t i v e nor i t s a c i d i s o m e r i z a t i o n product shows i n f r a - r e d o r u l t r a - v i o l e t a b s o r p t i o n s p e c t r a t y p i c a l o f a conjugated system. The double bond i n s e r r a t e n e d i o l must t h e r e f o r e be w e l l removed from the t e r m i n a l r i n g s . The p o s i t i o n o f the double bond i n the molecule was e s t a b l i s h e d by means o f the s e r i e s o f r e a c t i o n s shown i n Scheme 3. H y d r o b o r a t i o n o f s e r r a t e n e f o l l o w e d by p e r a c i d o x i d a t i o n y i e l d s an a l c o h o l X X X I I I , C 3QH520, m.p. 173-175°. O x i d a t i o n o f t h i s a l c o h o l by means o f chromic o x i d e / p y r i d i n e reagent y i e l d s the corresponding ketone , serratanone XXXIV, C 3 0 H 5 0 O , m.p. 202-4°. Reduct ion o f serratanone by the W o l f f - K i s h n e r process f u r n i s h e s a - s e r r a t a n e showing t h a t no s k e l e t a l change has o c c u r r e d . I t i s c l e a r t h a t the r i n g c o n t a i n i n g the c a r b o n y l group i n serratanone i s s i x membered o r l a r g e r s i n c e the c a r b o n y l a b s o r p t i o n i s found at 1692 c m " 1 . The NMR spectrum o f t h i s compound shows methyl group a b s o r p t i o n s at T 9.33, 9.20, 9.14 and 9.10 i n the r a t i o 1:2:1:3. 19 N i t r i c a c i d o x i d a t i o n o f serratanone f o l l o w e d by m e t h y l a t i o n y i e l d s the d i m e t h y l e s t e r o f a d i c a r b o x y l i c a c i d XXXV. This proves the presence o f the - C O - C H 2 - grouping i n s e r r a t a n o n e . The r e a c t i o n which p r o v i d e s the key t o the s t r u c t u r e o f r i n g D i s the o x i d a t i o n o f serratanone w i t h se lenium d i o x i d e i n a c e t i c a c i d when t h r e e products can be i s o l a t e d by c a r e f u l chromatography over a lumina and s i l i c a . The f i r s t o f these i s the compound XXXVI, C 3 0 H 4 8 O , m.p. 247-249° 20 isolated in 16 % yield. This compound is a conjugated ketone (I.R. and U.V) but the double bond must be in a tetrasubstituted position since no olefinic proton appears in the NMR spectrum. The methyl group signals are observed as singlets at T 8.97, 9.11 (6H),'9.15, 9.22, 9.26 and 9.35. This compound also arises when serratanone is first brominated and then dehydrobrominated. The second compound XXXVII, CaoHi+eO, m.p. 228-229°, is isolated in 46 % yield and shows a maximum absorption in the U.V. spectrum at 256 mu with a shoulder at 282mu (log. e 4.13 and 3.67). The I.R. spectrum exhibits three strong absorptions.at 1647, 1623 and 1613 cm - 1 and the NMR spectrum shows the presence of one olefinic proton. The compound is therefore con-sidered to be a dienone arising by further oxidation of the previously des-cribed compound XXXVI. This is borne-out in that further oxidation of XXXVI yields,XXXVII and no other product. XXXVII XXXIX When the dienone XXXVII is heated with zinc dust in acetic acid, a methyl group is spontaneously eliminated from the system with the formation of a tetrasubstituted phenol XXXIX, CagH^O, m.p. 238-239°. This compound has absorption maxima in the U.V. spectrum at 285 and 291 my (log.e 3.45 and 3.49) and the I.R. spectrum shows absorption bands at 3484 cm - 1 due to the hydroxyl function and at 1600 and 1582 cm-1 due to the presence of an aromatic system. The NMR spectrum shows a sharp singlet at T 3.44 for one aromatic proton and a broad singlet at.T 5.62 disappearing on treatment with 2 1 D20 due to the -OH group. Sinee this aromatization reaction is well known 4 0 in steroid and lanosterol chemistry , it establishes the structure of ring D and also the presence of a methyl group on the D/E ring juncture. The NMR spectrum of the dienone XXXVII shows three methyl peaks in the region below x 9 . 0 0 , namely at x 8 . 7 3 ( 6 H ) and 8 . 7 7 whereas the phenol XXXIX shows two at x 8 . 7 5 and 8 . 7 8 . On the other hand al l the methyl groups in serratanone XXXIV absorb above x 9 . 1 0 . This low field shift of the methyl groups sug-gests that the dienone or aromatic system is situated in the a-position to a gem-dimethyl group in the terminal ring E. To confirm this supposition the compound, serratadienonediol, XL was prepared from serratenediol diacetate by means of reactions already described followed by hydrolysis of the acetate groupings. When the dehydration reaction involving the use of phosphorous, pentachloride in benzene is now applied to this compound the bis-isopro-pylidene derivative XLI, C ^ H i ^ O , m.p. 2 2 5 - 2 2 8 is obtained. One of the double bonds formed in this reaction is evidently conjugated to the dienone system as shown by a marked bathochromic shift in the ultra-violet absorption spectrum which shows maxima at 2 7 6 and 3 2 0 my (log. e 4 . 0 4 and 4 . 0 0 ) . The I.R. spectrum shows three peaks at 1 6 4 7 , 1 6 1 8 and 1 5 9 7 cm - 1 while the NMR spectrum shows a singlet for one olefinic proton at x 3 . 7 8 arid methyl groups at x 8 . 1 4 ( 6 H ) ; 8 . 4 3 and 8 . 2 6 (from C = C - C H 3 at C \ ) ; 8 . 8 8 (from C = C - C - C H 3 at ( C 1 8 ) ; and at 9 . 3 4 and 9 . 4 8 ( - C - C H 3 at C 8 and C 1 0 ) . 22 The t h i r d product from the se lenium d i o x i d e o x i d a t i o n o f s e r r a t a n o n e , XXXVII m.p. 189-191° i s o b t a i n e d i n o n l y 3 % y i e l d and analyzes f o r C3oH^6°- I t ; i s t h e r e f o r e i s o m e r i c w i t h the dienone XXXVII which i s the second p r o d u c t . However, i t d i s p l a y s a U . V . spectrum w i t h a maximum a b s o r p t i o n at 266 mu ( l o g . e 4.11) and the I . R . spectrum shows.two s t r o n g a b s o r p t i o n s at 1650 and 1597 c m - 1 . The NMR spectrum i n the o l e f i n i c p r o t o n r e g i o n shows a sharp s i n g l e t at T 3.89 and a c l e a n d o u b l e t at T 3.35 (J = 2.5 c . p . s . ) , both s i g n a l s c o r r e s p o n d i n g t o one p r o t o n . The l a t t e r 41 s i g n a l i s c o n s i d e r e d t o e x h i b i t t y p i c a l a l l y l i c c o u p l i n g r a t h e r than c o u p l i n g w i t h an adjacent hydrogen. The methyl group r e g i o n o f the NMR spectrum shows three s p i k e s at h i g h f i e l d (T 9 . 2 0 , 9.16 and 9.14) and f o u r at low f i e l d (x 8 .97 , 8 . 9 0 , 8.83 and 8 . 7 9 ) . The s h i f t o f a f o u r t h methyl group to low f i e l d , when compared w i t h the dienone X X X V I I , h a v i n g o n l y t h r e e so l o c a t e d , i n d i c a t e s t h a t t h i s a l s o i s a t o a double bond l e a v i n g o n l y the three methyl groups on r i n g A appear ing at h i g h f i e l d . The formula XXXVIII i s i n harmony w i t h these e x p e r i m e n t a l r e s u l t s . In support o f t h i s assignment i t i s p o i n t e d out t h a t c l e a n doublets i n the compounds XXXVI and XXXVII appear ing at T 7.25 and 7.01 r e s p e c t i v e l y and w i t h c o u p l i n g constants o f 13 c . p . s . are complete ly absent i n the spectrum o f X X X V I I I . These s i g n a l s are a t t r i b u t e d t o one component o f the AB system o f the C 2 7 methylene group. When the phenol XXXIX undergoes v i g o r o u s , o z o n i z a t i o n f o l l o w e d by p e r a c i d o x i d a t i o n as i n Scheme 4, a d i c a r b o x y l i c a c i d i s formed from which by p y r o l y s i s the ketone X L I I r e s u l t s . This compound analyzes f o r C i s H 3 o O , m.p. 156-158° and shows i n i t s ORD curve a peak at 314 my ( [ a ] + 1700) t y p i c a l o f a s a t u r a t e d ketone system. The I . R . spectrum shows a c a r b o n y l a b s o r p t i o n b a n d . a t 1721 c m - 1 (KBr d i s c ) c o n f i r m i n g t h a t the system i s t h a t o f a cyclohexanone r a t h e r than o f a cyc lopentanone. The NMR spectrum shows 23 Scheme 4 1. CT a. 2 . P e r a c i d ' O x i d n . X L I I XXXIX two sharp peaks c o r r e s p o n d i n g to f o u r methyl groups at T 9.15 (6H) and 9.12 (6H). The s t r u c t u r e X L I I t h e r e f o r e r e p r e s e n t s the ketone and the o r i g i n a l r i n g C i n s e r r a t e n e -d i o l - must be seven membered. On the b a s i s o f the evidence here d e s c r i b e d , Inubushi et a l a s c r i b e d the s t r u c t u r e XXVI t o s e r r a t e n e d i o l , apart from the s t e r e o c h e m i s t r y which was d e f i n e d l a t e r . They then proceeded t o c o n f i r m t h i s s t r u c t u r e by c o r r e -l a t i n g s e r r a t e n e d i o l w i t h the o n o c e r i n s e r i e s and thereby a l s o o b t a i n i n g the 39 c o n f i g u r a t i o n o f the molecule I t i s w e l l known t h a t a - o n o c e r i n i s i s o m e r i z e d by the a c t i o n o f m i n e r a l a c i d i n t o 6- and y o n o c e r i n , the s t r u c t u r e s o f which were deduced 42 by Barton and Overton . Double p r o t o n a t i o n t o each double bond i n a - o n o c e r i n leads t o B-onocer in w h i c h , by f u r t h e r s i n g l e p r o t o n a t i o n y i e l d s y o n o c e r i n . The p r o c e s s , as c a r r i e d out by I n u b u s h i , i s shown i n Scheme 5. The s t a r t i n g m a t e r i a l , a - o n o c e r a d i e n e d i o l d i a c e t a t e , X L I I I was used i n c o n f i r m i n g the f i n d i n g s o f e a r l i e r workers by i s o l a t i n g 6- and y - o n o c e r a d i e n e d i o l d i a c e t a t e s 24 X L I I I XLIV XLV XLIV and XLV. However,the Japanese group argued t h a t i t might be p o s s i b l e t o e f f e c t s i n g l e p r o t o n a t i o n o f a - o n o c e r a d i e n e d i o l d i a c e t a t e a c c o r d i n g t o Scheme 1 by the use o f a b u l k y Lewis a c i d such as boron t r i f l u o r i d e . They succeeded i n a c h i e v i n g t h e i r g o a l by treatment w i t h boron t r i f l u o r i d e i n c h l o r o f o r m d u r i n g which the two products c o r r e s p o n d i n g t o s e r r a t e n e d i o l d i a c e t a t e and y - o n o c e r e n e d i o l d i a c e t a t e XLV were o b t a i n e d i n the r a t i o o f 1:2 r e s p e c t i v e l y . T h i s r e a c t i o n sequence was then repeated u s i n g a-onocera-dienedione XLVI as s t a r t i n g m a t e r i a l w i t h the subsequent f o r m a t i o n o f s e r r a -tenedione X L V I I . The l a t t e r compound can be reduced to s e r r a t e n e d i o l by means o f sodium and n - p r o p a n o l , and, s i n c e a - o n o c e r i n had a l r e a d y been s y n t h e s i s e d by S t o r k ^ and c o - w o r k e r s , t h i s completed the t o t a l s y n t h e s i s o f s e r r a t e n e d i o l . Moreover the s t e r e o c h e m i s t r y o f s e r r a t e n e d i o l was proven by t h i s s y n t h e s i s w i t h the e x c e p t i o n o f the c o n f i g u r a t i o n o f the methyl group at 25 CQ. The hydroxyl groups must be equatorial since they can be formed from serratenedione by reduction with sodium and n-propanol. The fact that the ORD curves of synthetic and natural serratenedione are superimposable shows that serratenediol has the same absolute configuration as that of a-.44 onocenn Occurring along with serratenediol in its natural source is a mono-acetate derivative C32H52O3, m.p. 319-320° to which Inubushi assigned the structure XLVIII. Oxidation of this compound gives serrateneolone acetate XLIX, C32H50O3, m.p. 305-307°. Application of the Wolff-Kishner reduction to this compound gives a mono-ol,serrateneol, which may be oxidised to a ketone C^oH^O* m.p. 204-206°, L. The ketonic function in this compound must be situated on the opposite terminal ring to that in the case of serra-teneolone acetate XLIX. It was shown by Inubushi that the ORD curve of serrateneolone acetate gives a negative Cotton effect with molecular amplitude a = -31 which is 7 45 similar to the curves obtained from the compounds A'-lanosten-3-one LI , 46 or bauerenone, LII . Thus the assignment of the acetate group- to the" C3 position in the naturally occurring compound XLVIII was justified and the 26 c a r b o n y l group i n XLIX i s at p o s i t i o n C 2 1 . On the o t h e r hand the ketone L g ives a p o s i t i v e Cot ton e f f e c t 47 (a = +30) s i m i l a r t o t h a t found i n normal t r i t e r p e n e C3 ketones . The ketone i s t h e r e f o r e A l l * - s e r r a t e n - 3 - o n e and the p o s i t i v e Cotton e f f e c t suggests t h a t r i n g B/C i s t r a n s fused and the Cs methyl group i s 8 o r i e n t e d s i n c e t r i t e r p e n e C 3 ketones o f t h i s s t r u c t u r a l type have always been found t o g i v e a p o s i t i v e Cot ton e f f e c t ^ ' ^ 8 . In c o n f i r m a t i o n o f t h i s c o n c l u s i o n , the C28 ketone X L I I i s found t o g i v e a p o s i t i v e Cot ton e f f e c t (a = +55) i n e x c e l l e n t agreement w i t h measure-49 ments on analogous compounds L I I I o f known c o n f i g u r a t i o n . (R = H , a = +48; R = CN, a = +65; R = OH, a = +76; R = C 0 2 C H 3 , a = +80; R = C H 2 C 0 2 C H 3 , a = +32). This evidence completes the s t r u c t u r a l p r o o f o f s e r r a t e n e d i o l L I U which i s t h e r e f o r e r e p r e s e n t e d by the a b s o l u t e s t e r e o - s t r u c t u r e XXVI. Another compound which co-occurs w i t h s e r r a t e n e d i o l i n Lycopodium serratum was shown by Inubushi t o be 2 1 - e p i s e r r a t e n e d i o l L I V 5 ^ . Comparison o f the NMR spectrum o f the d i a c e t a t e d e r i v a t i v e w i t h t h a t o f s e r r a t e n e d i o l d i a c e t a t e showed t h a t the C-methyl reg ions are almost i d e n t i c a l . However, i n 27 LIV s e r r a t e n e d i o l d i a c e t a t e o n l y one s i g n a l appears f o r the a c e t a t e methyl groups at T 7.97 whereas i n the d i a c e t a t e o f LIV there are two s i g n a l s at T 7.91 and 7.95 each c o r r e s p o n d i n g t o one methyl group. Moreover, i n t h i s case , a broad m u l t i p l e t at x 5.52 (IH) and a sharp t r i p l e t at x 5.31 (IH) c o u l d be a s c r i b e d t o two protons geminal t o a c e t a t e groups w i t h the former p r o t o n b e i n g a x i a l and the l a t t e r e q u a t o r i a l . O x i d a t i o n o f the d i o l y i e l d e d s e r r a t e n e d i o n e X L V I I . P a r t i a l acety-l a t i o n w i t h a c e t i c anhydride and p y r i d i n e y i e l d e d a monoacetate which was d i f f e r e n t from the known s e r r a t e n e d i o l 3-acetate X L V I I I . This a c e t a t e showed a broad m u l t i p l e t at x 5.55 (IH) s u g g e s t i n g the presence o f an a x i a l p r o t o n geminal t o an a c e t a t e group and a sharp t r i p l e t at x 6.55 (IH) f o r an equato-r i a l p r o t o n geminal t o a h y d r o x y l group. O x i d a t i o n o f the monoacetate y i e l d e d the known compound s e r r a t e n e o l o n e a c e t a t e XLIX so t h a t the d i o l was confirmed t o be 2 1 - e p i s e r r a t e n e d i o l LIV o r A 1 4 - s e r r a t e n e - 3 8 , 2 1 8 - d i o l . 64 Very r e c e n t l y Inubushi et a l have d e s c r i b e d the i s o l a t i o n and s t r u c -t u r a l e l u c i d a t i o n o f the two t r i o l s l y c o c l a v a n o l LV and s e r r a t r i o l LVI b e l o n g -i n g t o the s e r r a t e n e c l a s s . The l a t t e r compound i s found i n Lycopodium serratum but the former occurs o n l y i n the r e l a t e d s p e c i e s L. c lavatum. OH 28 As has a l r e a d y been mentioned Rowe had i s o l a t e d p i n u s e n e d i o l from the barks o f jack p i n e (Pinus b a n k s i a n a Lamb.) , sugar p i n e (P. l a m b e r t i a n a D o u g l . ) , l o b l o l l y p i n e (P. taeda L . ) and l o n g l e a f p i n e (P. p a l u s t r i s M i l l . ) . Subsequent t o the p u b l i c a t i o n o f I n u b u s h i ' s f i r s t p a p e r ^ on s e r r a t e n e d i o l , he was able t o c o r r e l a t e h i s m a t e r i a l w i t h that o b a t i n e d from Lycopodium serratum. From the p o i n t o f view o f e l u c i d a t i o n o f the c h e m i s t r y o f the s e r r a t e n e system t h i s paper had l i t t l e t o add t o the work o f Inubushi a l r e a d y d e s c r i b e d above and w i l l not be d i s c u s s e d i n d e t a i l h e r e . S m a l l amounts o f two r e l a t e d d i o l s were i s o l a t e d from p i n e barks by Rowe and two methoxyl c o n t a i n i n g d e r i v a t i v e s were a l s o s e p a r a t e d . The s t r u c t u r e s o f these com-pounds were not r i g i d l y e s t a b l i s h e d . In a l a t e r paper Rowe and Bower 5 ^ d e s c r i b e d the i s o l a t i o n and i d e n t i -f i c a t i o n o f s i x s e r r a t e n e d i o l d e r i v a t i v e s from p i n e b a r k . One o f these i s i d e n t i c a l w i t h 2 1 - e p i s e r r a t e n e d i o l LIV which had a l r e a d y been c h a r a c t e r i z e d by I n u b u s h i 5 ^ . The o t h e r f i v e compounds are n o v e l and t h r e e o f them c o n t a i n methoxyl groups. One o f the methoxyl c o n t a i n i n g compounds was proved t o be i d e n t i c a l w i t h the d i m e t h y l e t h e r d e r i v a t i v e o f s e r r a t e n e d i o l by a p p r o p r i a t e comparisons. A monomethyl e t h e r o f a d i o l was a l s o d e s c r i b e d w h i c h , upon m e t h y l a t i o n y i e l d s s e r r a t e n e d i o l d i m e t h y l e t h e r . O x i d a t i o n w i t h chromic a c i d g ives r i s e to the methoxyketone L V I I , the s t r u c t u r e o f which f o l l o w s from the n e g a t i v e Cotton e f f e c t curve o b t a i n e d i n the ORD measurement. The m o l e c u l a r ampli tude o f t h i s compound (-27) i s i n agreement w i t h a 21-keto group as 39 shown by Inubushi so t h a t the o r i g i n a l compound i s o l a t e d from the bark was s e r r a t e n e d i o l 3-monomethyl e t h e r (3g-Methoxy-21a-Hydroxy-A 1 ? l t - . • .,. s e r r a t e n e ) . The t h i r d methoxyl c o n t a i n i n g compound i s a methoxy ketone which was proved i d e n t i c a l w i t h L V I I a and thus has the s t r u c t u r e 38-Methoxy-29 21 keto-A14-serratene. Of the two remaining compounds one has spectral properties in agree-ment with a keto-alcohol. The NMR spectrum shows a broad multiplet at T 6.8 corresponding to a proton geminal to an equatorial hydroxyl group. The ORD curve shows a single negative Cotton effect with a molecular amplitude of -36 suggesting a C 2 i keto function. The structure LVIIb in keeping with this evidence was confirmed by comparison with an authentic sample prepared from serratenediol 3-monoacetate by oxidation followed by saponification. This compound therefore has the structure 38-Hydroxy-21 keto-a R = CH3 LVII b R = H serratene. Finally a diol was isolated whose diacetate derivative displays two sharp overlapping triplets at T 5.38 in the NMR spectrum correspond-ing to two protons geminal to two axial acetoxy groups. Oxidation of the diol Mo" JX^ ^ yields serratenedione which was identi-L V i i l fied by comparison with an authentic standard. The structure of this com-pound is therefore 3a,213-Dihydroxy-All*-serratene LVIII (Diepiserratenediol). The above discussion has provided a summary of the published investi-gations which relate directly to my own research problem. The work now to be described involves the isolation and characterization of a number of serratene 30 derivatives from the benzene soluble extractives of Sitka spruce bark. It must however be mentioned that Kohlbrenner and Schuerch*^ have examined the benzene-alcohol soluble extract of Sitka spruce woodmeal. They isolated the phenolic compounds acetovanillone, vanill in, and vanillyl alcohol and the non-volatile neutral components B-sitosterol, 1-2-octyl-B-sitosterol phthalate. They also mentioned the presence of an unsaturated alcohol and a conjugated monoterpene diene in the neutral fraction. 31 DISCUSSION As has already been mentioned, the starting material in this project consisted of a cream-coloured powder (26 gm.) which had been obtained by Dr. Hergert as a precipitate when the petroleum ether soluble fraction of Sitka spruce bark was dissolved in acetone and allowed to stand for twenty four hours. Although the yield of this solid was reasonably high (sometimes as much as 30 % by weight of the petroleum ether extract) and the procedure for obtaining i t fairly straightforward, there is nevertheless a difficulty in obtaining an unlimited supply. The problem lies in being able to separate out the purple coloured cork from the whole bark. Dr. Hergert hand picked his samples from the butt logs of over-mature spruce trees grown in the Gray's Harbour area of the State of Washington. In normal Sitka spruce logs the bark is quite thin and it is not possible to remove the cork. Solvent front Start Figure 1, A preliminary examination of the powder showed it to have a melting range of 250-254°C accompanied by some "sweating" below the melting point. There was a marked tendency for subli-mation to occur above 220°C. The material was spotted on 5 x 20 cm. alumina and s i l ica thin layer chromatoplates which were developed in chloroform and sprayed with antimony pentachloride reagent. It was at once clear that the extract was a mixture . composed mainly of two compounds with 32 traces of several minor components being also present. The separation of the spots on alumina was poor but excellent results were obtained using s i l ica as is shown in the sketch in Figure 1. The compounds were named by letters of the alphabet as shown according to their relative abundances and the values and colours of the freshly sprayed spots are listed in Table 2. It was observed that developed but unsprayed plates did not show any fluore-scent spots when examined with a U.V. scanning lamp. TABLE 2 J*£ Values of Components on a Silica Plate  Developed with Chloroform and Sprayed with  Antimony Pentachloride Identification of Compound R f Value Colour C .44 golden brown B .37 ginger E .26 pink A .14 ginger F .08 pink D .05 golden brown The excellent separation of compounds A and B, the major components of the mixture, at once suggested the possibility of purification by prepara-tive scale thin layer chromatography on s i l ica gel. Accordingly plates were 33 prepared c o n t a i n i n g a f l u o r e s c e n t i n d i c a t o r and about 110-120 mg. q u a n t i t i e s o f the m i x t u r e a p p l i e d t o each one. D i f f i c u l t y was exper ienced i n t h a t as the c h l o r o f o r m s o l v e n t evaporated , the s i l i c a coated w i t h the a p p l i e d m a t e r i a l showed a tendency t o p e e l o f f from the l a y e r but t h i s problem was reduced by u s i n g more d i l u t e s o l u t i o n s o f the m a t e r i a l t o be a p p l i e d . The developed p l a t e s were examined under U . V . l i g h t and s t r i p s were a l s o sprayed w i t h antimony p e n t a c h l o r i d e t o a i d i n the l o c a t i o n o f the bands. A f t e r s c r a p i n g o f f the a p p r o p r i a t e s e c t i o n s from the p l a t e s , the compounds were recovered by e x t r a c t i o n o f the s i l i c a w i t h warm c h l o r o f o r m and m e t h a n o l . . In t h i s way a sample o f c h r o m a t o g r a p h i c a l l y pure compound A weighing 184 mg. and one o f compound B weighing 125 mg. was f a i r l y r e a d i l y o b t a i n e d from 580 mg. o f crude powder. The l a t t e r compound was not however, o b t a i n e d c h r o m a t o g r a p h i c a l l y pure as i t was not p o s s i b l e t o separate compound B from compound C by t h i s p r o c e d u r e . The t o t a l recovery o f compounds A and B from the p r e p a r a t i v e p l a t e s was o n l y 54 % by weight o f the m a t e r i a l a p p l i e d . R e c r y s t a l l i z a t i o n o f compound A from 95 % e t h a n o l y i e l d e d s m a l l white c r y s t a l s , m.p. 305-6°C. F r a c t i o n a l c r y s t a l l i z a t i o n o f compound B from normal hexane y i e l d e d a TLC pure product as s m a l l whi te p r i s m s , m.p."275-7°C. Compound A possesses a s p e c i f i c r o t a t i o n va lue [a]^ o f +2.8° and B o f - 5 5 . 4 ° , both measured i n c h l o r o f o r m s o l u t i o n . The i n f r a - r e d s p e c t r a o f compounds A and B were, determined and t o i l l u s t r a t e some c h a r a c t e r i s t i c a b s o r p t i o n s the spectrum o f compound A i s i l l u s t r a t e d i n F i g u r e 2. The most noteworthy f e a t u r e s o f t h i s spectrum a r e - t h e s t r o n g and f a i r l y broad h y d r o x y l s t r e t c h i n g a b s o r p t i o n at 3550 c m - 1 , the pronounced peak at 1100 c m - 1 caused by the carbon-oxygen s t r e t c h i n g v i b r a t i o n o f an e t h e r f u n c t i o n and the absence o f c a r b o n y l c o n t a i n i n g s u b s t i t u t e n t s . The spectrum 34 1/1 4 11 '1 ifCOO ".3ooo 2ooo •p"75o /5&0 /OOO ~75o Figure 2. Infra-Red Spectrum (KBr) of Compound A of compound B was quite similar to the above except that the hydroxyl stretching absorption was very weak and centred in this case at 3650 cm"1 Neither compounds A nor B exhibited characteristic ultra violet absorption spectra. Elemental analysis indicated that both compounds contained only carbon, hydrogen and oxygen. Furthermore the analytical results were con-sistent with both compounds having the empirical formula C 3 1 H 5 2 O 2 indicating that A and B are isomeric. The mass spectra of the compounds, which will be discussed later, indicated in both cases a parent ion peak at m/e 456. Finally the molecular weights and empirical formulae were rigidly established by high resolution mass spectrometry. The parent ion peaks from both com-pounds were measured at m/e 456.3976 while the theoretical value for a com-pound of empirical formula C 3 1 H 5 2 O 2 is 456.3967. Methoxyl analysis by the Zeisel method indicated that both compounds possess one methyl ether function. 35 Samples of both compounds were dissolved in deuterochloroform and submitted for NMR spectral measurements on the Varian A 60 instrument. In the case of compound B the solubility of the material was sufficiently high so that a 15 % solution could be prepared at room temperature1 and tetramethyl silane (TMS) used as an internal standard. However, in the case of compound A, the solubility was much lower and finally a spectrum was obtained at a temperature of 65°C with an external TMS standard. The spectra of compounds B and A are shown in Figures 3 and 4 respectively. A. £ 8 t f o _! i I l I ! ( Figure 3. Compound B. From the integration of the spectrum based on the total integral corresponding to fifty two protons, i t is clear that the signals at x 4.70, 6.57 and 7.25 correspond to single protons in each case whereas the sharp signal at T 6.72 arises from the three protons of a methoxyl group. From the chemical shift data i t was concluded that the signal at x 4.70 arises from the olefinic proton of a trisubstituted double bond, that at x 6.57 is 36 p r o b a b l y a p r o t o n geminal t o a secondary a l c o h o l f u n c t i o n , w h i l e the s i g n a l at x 7.25 corresponds t o a p r o t o n geminal t o a secondary methoxyl group. The h i g h f i e l d r e g i o n (T 9 . 0 - 9 . 3 5 ) o f the spectrum showed f o u r sharp s i g n a l s o f unequal h e i g h t which were not w e l l r e s o l v e d from one another . The t o t a l i n t e g r a l o f these s i g n a l s taken from p o i n t a on the i n t e g r a t i o n curve c o r r e s -ponds t o 21.0 protons which would seem to i n d i c a t e the presence o f seven 4- 6 8 r io I ! 1 I 9 I I F i g u r e 4. N.M.R. Spectrum o f Compound A . The NMR spectrum o f compound A was i n many r e s p e c t s s i m i l a r t o t h a t o f compound B, However, one important d i f f e r e n c e observed was the l a c k o f a s i g n a l at T 7.25 corresponding t o a p r o t o n geminal t o the methoxyl group. The angular methyl group r e g i o n was a l s o b e t t e r r e s o l v e d w i t h s i x s i g n a l s showing i n the r e g i o n T 9 .00 t o 9 . 2 6 . In r e l a t i o n s h i p t o F i g u r e 3 the whole spectrum appeared t o have been s h i f t e d about x 0 .1 d o w n f i e l d and t h i s i s p o s s i b l y a t t r i b u t a b l e t o the use o f an e x t e r n a l s t a n d a r d i n the present 37 i n s t a n c e . In t h i s case the a n g u l a r methyl group i n t e g r a l taken at p o i n t a corresponded t o 23.8 H a p p a r e n t l y i n d i c a t i n g e i g h t such groupings i n the m o l e c u l e . The next l o g i c a l s tep i n the s t r u c t u r a l e l u c i d a t i o n was to determine the degree o f u n s a t u r a t i o n i n the m o l e c u l e . Both compounds were shown t o g ive a y e l l o w c o l o u r w i t h t e t r a n i t r o m e t h a n e reagent , which i s i n support o f the presence o f double bonds a l r e a d y noted from the NMR s p e c t r a . The l a t t e r p o i n t was c o n v e n i e n t l y i n v e s t i g a t e d by microhydrogenat ion s t u d i e s u s i n g p l a t i n u m oxide c a t a l y s t . The microhydrogenat ion apparatus was f i r s t c a l i b r a t e d u s i n g c a r e f u l l y p u r i f i e d m a l e i c a c i d i n a s e r i e s o f seven d e t e r m i n a t i o n s on samples o f v a r y i n g w e i g h t s . No r e d u c t i o n took p l a c e when an attempt was made t o hydrogenate compounds A or B i n the presence o f e t h a n o l as s o l v e n t . However, when g l a c i a l a c e t i c a c i d was used i n s t e a d , uptake o f one mole o f hydrogen p e r mole o f s u b s t r a t e was achieved i n each case. The r e a c t i o n was complete a f t e r t h r e e hours and no f u r t h e r r e d u c t i o n c o u l d be observed. That the r e d u c t i o n d i d not proceed i n the presence o f e t h a n o l as s o l v e n t was a t t r i b u t e d to the double bonds i n these molecules b e i n g q u i t e s t e r i c a l l y h i n d e r e d , which i s not s u r p r i s i n g c o n s i d e r i n g t h a t they are known t o be t r i s u b s t i t u t e d . The hydrogenat ion r e s u l t s t h e r e f o r e e s t a b l i s h e d the presence o f one r e d u c i b l e double bond. When t h i s d a t a was c o n s i d e r e d w i t h the m o l e c u l a r formulae g iven above i t was c l e a r t h a t these substances are most l i k e l y members o f the p e n t a c y c l i c t r i t e r p e n e s e r i e s . I t was o b v i o u s l y p o s s i b l e t h a t t e t r a s u b s t i t u t e d double bonds might be present i n these compounds s i n c e such o l e f i n i c l i n k a g e s are n o t o r i o u s l y 38 difficult to hydrogenate and normally require much more drastic reduction conditions. It may be concluded however, that the presence of such functions in A and B is rather unlikely in that their dihydro derivatives failed to give a positive colour test for unsaturation with tetranitromethane. It is now opportune to discuss the mass spectral fragmentation patterns of compounds A and B. The results, represented schematically in Figures 5 and 6, were obtained using the AEI-MS9 double focussing mass spectrometer. COMPOUND A 200 Figure 5 Once again a comparison of the two spectra leads one to speculate that there is a close structural relationship between A and B. Almost a l l the major fragments in either spectrum are faithfully reproduced in the other. One may note however, the presence of fragments at m/e 284 and 285 in B and their absence in A. On the other hand the M+-15 peak caused by loss of a methyl group in A is not observed in B and this difference may relate to the presence of a significant fragment at m/e 284 in the latter (284 = 269 + 15) 39 F i g u r e 6 The l a r g e gap i n the 320-420 r e g i o n where no fragments are observed i m p l i e s t h a t the molecules are fragmented i n t o s m a l l e r u n i t s somewhere near the middle i . e . perhaps i n r i n g C o f a t r i t e r p e n e system. This s i t u a t i o n may p r e v a i l due to the presence o f a double bond o r i t might be due to some o t h e r f e a t u r e o f the c a r b o c y c l i c s k e l e t o n . The fragments o c c u r r i n g at M + - 3 2 , M + -18 and M + -15 are most e a s i l y r a t i o n a l i z e d by the e l i m i n a t i o n o f methanol , water and a l a b i l e methyl group from the s k e l e t o n . T h i s r e s u l t would support the presence o f a methyl e t h e r and an a l c o h o l f u n c t i o n i n both m o l e c u l e s . The abundant fragments at m/e 220 and 221 which represent approximate ly one-h a l f the t o t a l mass o f these compounds are dependent on changes i n the f u n c t i o n a l i t y o f the parent molecules and t h i s important f e a t u r e w i l l be d i s c u s s e d l a t e r . B e a r i n g i n mind the need f o r f u r t h e r c h a r a c t e r i z a t i o n o f these com-pounds through the p r e p a r a t i o n o f s u i t a b l e d e r i v a t i v e s , i t became necessary to o b t a i n l a r g e r s u p p l i e s o f the pure m a t e r i a l s . T h i s problem was s o l v e d by 40 column chromatography o f p o r t i o n s o f the o r i g i n a l m i x t u r e on n e u t r a l s i l i c a g e l , a procedure which o b v i o u s l y lends i t s e l f b e t t e r t o l a r g e s c a l e work than does p r e p a r a t i v e t h i n l a y e r chromatography. S e p a r a t i o n o f the v a r i o u s components i n the m i x t u r e c o u l d be achieved by a m i x t u r e o f benzene and c h l o r o f o r m as e l u a n t s . The f r a c t i o n c o n t a i n i n g compounds B and C was e l u t e d w i t h benzene-chloroform (1:1) w h i l e the f r a c t i o n c o n t a i n i n g E + A was o b t a i n e d w i t h benzene-chloroform (3.*7). Pure compound B was f i n a l l y o b t a i n e d by f r a c t i o n a l r e c r y s t a l l i z a t i o n o f the e l u t e d m a t e r i a l i n 95 % e t h a n o l . The f r a c t i o n con-t a i n i n g A as the major component was always contaminated w i t h a s m a l l amount o f B and a f u r t h e r p u r i f i c a t i o n was n e c e s s a r y . The components which were more p o l a r than A were recovered as a m i x t u r e c o n t a i n i n g some A and kept f o r l a t e r p u r i f i c a t i o n . Repeated chromatography on s i l i c a g e l columns f i n a l l y p r o v i d e d pure A. The compound des ignated as E c o u l d be separated from A only by f r a c t i o n a l r e c r y s t a l l i z a t i o n and i n t h i s case c h l o r o f o r m was found to be the b e s t s o l v e n t . A c e t y l a t i o n o f the secondary a l c o h o l f u n c t i o n s i n A and B proceeded r e a d i l y and q u a n t i t a t i v e l y i n a c e t i c a n h y d r i d e / p y r i d i n e mixtures at room temperature.. The acetates were w h i t e , b e a u t i f u l l y c r y s t a l l i n e m a t e r i a l s and were markedly more s o l u b l e i n o r g a n i c s o l v e n t s than were the parent compounds. Compound A acetate , m.p. 199-201°C, [ a ] D + 4 .5° was o b t a i n e d a n a l y t i c a l l y pure by r e c r y s t a l l i z a t i o n i n 95 % e t h y l a l c o h o l . In a s i m i l a r f a s h i o n , compound B a c e t a t e , m.p. 205.5-207°C, [ a ] ^ -64 .6° was o b t a i n e d i n pure form. Both acetates on a n a l y s i s y i e l d e d r e s u l t s compatible w i t h the e m p i r i c a l formula C 3 3 H 5i t 0 3 and the mass s p e c t r a showed the parent i o n peaks to occur at m/e 498 i n accordance w i t h the presence o f one a c e t a t e group. The i n f r a - r e d s p e c t r a o f both compounds measured i n KBr d i s c s , showed the presence of acetate (1730 and 1250 c m - 1 ) and methoxyl (1105 c m - 1 ) 41 f u n c t i o n s . The NMR s p e c t r a o f the acetates were measured i n d e u t e r o c h l o r o f o r m s o l u t i o n and i t was now p o s s i b l e t o use an i n t e r n a l TMS s t a n d a r d i n both cases . The expected chemical s h i f t s i n the low f i e l d s i g n a l s o r i g i n a l l y observed at x 6.50 and 6.57 i n the parent a l c o h o l s (F igures 3 and 4) had now o c c u r r e d and s i g n a l s at x 5.28 were present i n both cases . T h i s data con-f i r m e d the p r e v i o u s assignment o f these s i g n a l s as a r i s i n g from protons geminal t o secondary a l c o h o l groups. New s i g n a l s were a l s o observed at x 7.93 and 7.94 i n the A and B s e r i e s r e s p e c t i v e l y c o r r e s p o n d i n g to acetate methyl p r o t o n s . F i v e peaks c o u l d be seen i n the methyl group r e g i o n (x 9.02-9.25) o f compound A a c e t a t e which i n t e g r a t e d f o r 23.0 protons and o n l y t h r e e peaks were d i s c e r n i b l e i n the c o r r e s p o n d i n g r e g i o n o f B a c e t a t e (x 9.06-9.30) which had a t o t a l methyl group i n t e g r a l corresponding t o 20.3 p r o t o n s . Once again no p r o t o n geminal t o the methoxyl group c o u l d be d i s t i n g u i s h e d i n the A s e r i e s . O x i d a t i o n o f compounds A and B w i t h chromic o x i d e / p y r i d i n e reagent 57 a c c o r d i n g to the method o f S a r e t t and co-workers proceeded smoothly . The pure ketones were o b t a i n e d i n 70-85 % y i e l d a f t e r chromatography o f the crude o x i d a t i o n products on h i g h a c t i v i t y n e u t r a l a l u m i n a . R e c r y s t a l l i z a t i o n from e t h a n o l p r o v i d e d the pure compounds, A k e t o n e , m.p. 262-3°C, [ a ] ^ -0 .1° and B k e t o n e , m.p. 24q-2°C, [ a ] ' - 7 1 . 5 ° . Elementa l analyses on these compounds gave r e s u l t s c o n s i s t e n t w i t h the e m p i r i c a l formula C31H5QO2 and t h i s was supported by mass s p e c t r a l fragmenta-t i o n s t u d i e s which showed the parent ions o c c u r r i n g at m/e 454. Both com-pounds showed weak a b s o r p t i o n s i n the u l t r a - v i o l e t s p e c t r a which were r e a d i l y a s c r i b e d t o the presence o f the s a t u r a t e d carbonyl chromophore (^max 272 my, e =. 137 and 90). I n f r a - r e d s p e c t r a l measurements (KBr) i n d i c a t e d c a r b o n y l 42 a b s o r p t i o n bands at 1712 c m - 1 f o r A ketone and at 1707 c m - 1 f o r the B analogue thus i n d i c a t i n g t h a t the r i n g s b e a r i n g the c a r b o n y l f u n c t i o n s c o n t a i n s i x o r more carbon atoms. The NMR s p e c t r a o f the two ketones were aga in taken i n d u e t e r c h l o r o -form s o l u t i o n . The low f i e l d protons o c c u r r i n g at x 6.50 and 6.57 i n the parent compounds A and B and a s c r i b e d t o protons geminal t o secondary a l c o h o l groups were no longer p r e s e n t . The p o s i t i o n s o f the o t h e r s i g n a l s were very s i m i l a r to those o f the parent compounds A and B. Compound A ketone showed seven s i g n a l s i n the methyl group r e g i o n (x 8 .91-9.25) w i t h a t o t a l i n t e g r a l c o r r e s p o n d i n g t o 24.1 p r o t o n s . I t s compound B c o u n t e r p a r t showed f o u r peaks (x 8.91-9.17) e q u i v a l e n t t o 21.7 p r o t o n s . The o p t i c a l r o t a t o r y d i s p e r s i o n curves o f these ketones were measured i n methanol s o l u t i o n s on the JASCO 0RD/UV5 r e c o r d i n g s p e c t r o p o l a r i -meter. Compound A ketone e x h i b i t e d a n e g a t i v e Cot ton e f f e c t curve c e n t r e d at 290 my w i t h [<j>] . at 310 my (-1652°) and [<f>] at 271 my (+1789°) . The 1 J m i n v J L T J m a x ^ J m o l e c u l a r ampli tude was determined t o have the v a l u e - 3 4 . 4 . Compound B ketone a l s o showed a n e g a t i v e Cot ton e f f e c t curve c e n t r e d at 293 my w i t h [<t>] . at 310 my (-3332°) and [<f>] at 276 my ( - 2 2 7 ° ) . The m o l e c u l a r L J m m v 1 L Jmax v J ampli tude was c a l c u l a t e d t o be - 3 1 . 0 . These curves are shown i n F i g u r e 7. The shapes o f the ORD curves shown i n F i g u r e 7 are o f the same type as the curves g i v e n by compounds such as A 7 - l a n o s t e n - 3 - o n e (LI) and baurenone ( L I I ) which have been d i s c u s s e d by D j e r a s s i ^ ' ^ and C r a b b e ^ i n t e x t books d e a l i n g w i t h the a p p l i c a t i o n o f o p t i c a l r o t a t o r y d i s p e r s i o n measurement t o s t r u c t u r a l problems. Indeed, as has been mentioned i n the i n t r o d u c t i o n ^ Inubushi made r e f e r e n c e to these model substances i n p r o v i n g the s t e r e o -c h e m i s t r y at CQ i n the s e r r a t e n e d i o l system. He showed t h a t 38-acetoxy-2 1 - k e t o - A 1 1 * - s e r r a t e n e (XLIX) e x h i b i t s a s i n g l e n e g a t i v e Cot ton e f f e c t curve 43 w i t h a m o l e c u l a r ampli tude o f -31 whereas 3 - k e t o - A 1 ^ - s e r r a t e n e (L) shows a s i n g l e p o s i t i v e Cot ton e f f e c t curve w i t h a m o l e c u l a r ampl i tude o f +30. On t h i s b a s i s i t i s very l i k e l y t h a t A and B ketones both possess r i n g s D and E s i m i l a r t o the c o r r e s p o n d i n g r i n g s i n S B - a c e t o x y - Z l - k e t o - A 1 ^ - s e r r a t e n e (XLIX) and t h a t i n f a c t the o r i g i n a l secondary a l c o h o l groups are s i t u a t e d at the C 2 1 p o s i t i o n . F i g u r e 7. ORD curves o f Compounds A and B k e t o n e s . Important i n f o r m a t i o n as t o the s t e r i c environment around the c a r b o n y l chromophore may be o b t a i n e d by a study o f the r a t e o f h e m i k e t a l f o r m a t i o n i n a c i d i f e d methanol s o l u t i o n a c c o r d i n g t o the e q u a t i o n ,] 0 OH II H + I R-C-R' + CH3OH — • R-C-R* OCH3 The method was d e v i s e d by D j e r a s s i , M i t s c h e r and M i t s c h e r ^ who s t u d i e d a large number o f s t e r o i d and t r i t e r p e n e ketones as w e l l as s i m p l e r systems. The procedure i n v o l v e d i s very s i m p l e . F i r s t the ORD curve f o r the compound 44 under study i s measured i n methanol s o l u t i o n i n the u s u a l way. Then the s o l u t i o n i s t r a n s f e r r e d to a v o l u m e t r i c f l a s k c o n t a i n i n g a s m a l l amount o f h y d r o c h l o r i c a c i d and mixed t h o r o u g h l y . The f i n a l c o n c e n t r a t i o n o f the h y d r o c h l o r i c a c i d i n methanol should be i n the r e g i o n 0.05 t o 0.10 N . As r a p i d l y as p o s s i b l e the a c i d i f i e d s o l u t i o n i s t r a n s f e r r e d back i n t o the sample c e l l and the r o t a t i o n observed at the wavelength o f one o f the extrema i n the ORD curve . Hemiketa l f o r m a t i o n i s f o l l o w e d by the r a t e o f quenching o f the r o t a t i o n at t h i s extremum and measurements are taken at i n t e r v a l s o f time u n t i l an e q u i l i b r i u m s t a t e i s achieved as mani fes ted by a constant r e a d i n g . In the case o f a normal unhindered c a r b o n y l group t h i s e q u i l i b r i u m w i l l be f a r to the r i g h t i n the above e q u a t i o n w i t h consequent almost complete quenching o f the ORD curve whereas, i n the case where the chromophore i s s t r o n g l y h i n d e r e d s t e r i c a l l y , the e q u i l i b r i u m w i l l be f a r t o the l e f t and the ORD curve w i l l remain almost unchanged. In the examples o f normal unhindered s t e r o i d ketones s t u d i e d by D j e r a s s i and h i s co-workers the quenching o f the ORD curve was observed to be very r a p i d and e q u i l i b r i u m was reached i n l e s s than ten minutes a f t e r a d d i t i o n o f the a c i d . In cases where a geminal d i m e t h y l group o c c u r r e d v i c i n a l t o the carbon atom o f the c a r b o n y l chromophore the change i n r o t a t i o n was complete a f t e r about one h o u r . When o n l y one a l k y l s u b s t i t u e n t was present i n the v i c i n a l p o s i t i o n , an i n t e r -mediate s t a t e o f a f f a i r s was observed w i t h quenching p r o c e e d i n g about 50 t o 60 % t o complet ion a f t e r f i f t e e n t o twenty f i v e minutes . A p p l i c a t i o n o f the above method t o A and B ketones showed a n e g l i g i b l e degree o f quenching o f the r o t a t i o n n i n e t y minutes a f t e r the a d d i t i o n . o f the h y d r o c h l o r i c a c i d . S i m i l a r r e s u l t s were achieved at two d i f f e r e n t a c i d c o n c e n t r a t i o n s so t h a t i t was concluded t h a t i n both molecules the c a r b o n y l group i s present v i c i n a l t o a geminal d i m e t h y l group as i n the p a r t i a l s t r u c t u r e L I X . 45 S i m i l a r i n f o r m a t i o n t o t h a t o b t a i n e d above may be deduced from the study o f the mass s p e c t r a l f ragmentat ion p a t t e r n s o f the e thy lene k e t a l d e r i v a t i v e s o f s t e r o i d or t r i t e r p e n e k e t o n e s . Such s t u d i e s have been c a r r i e d 62 61 out i n d e p e n d e n t l y by the goups headed by F e t i z o n and D j e r a s s i who d i s -covered t h a t t h i s type o f k e t a l f u n c t i o n s t r o n g l y and p r e d i c t a b l y d i r e c t s bond f i s s i o n , thus p e r m i t t i n g the l o c a t i o n o f f u n c t i o n a l groups and o t h e r n u c l e a r s u b s t i t u t e n t s . The reason suggested f o r the very powerfu l e f f e c t o f the e t h y l e n e k e t a l f u n c t i o n i s i n d i c a t e d i n the sequence below. Homolys is o f a carbon-carbon bond i n the m o l e c u l a r i o n adjacent t o the s i t e o f the o r i g i n a l c a r b o n y l f u n c t i o n can g i v e r i s e t o resonance s t a b i l i z a t i o n o f the r e s u l t i n g i o n r a d i c a l by u t i l i z i n g the two adjacent heteroatoms t o c a r r y the charge. The work o f the D j e r a s s i group was concerned i n l a r g e measure w i t h the b e h a v i o u r o f v a r i o u s l y s u b s t i t u t e d 3-keto s t e r o i d e thy lene k e t a l s and t h r e e d i f f e r e n t types o f f r a g m e n t a t i o n were e l u c i d a t e d w i t h the mechanisms proposed b e i n g supported by deuterium l a b e l i n g s t u d i e s . These mechanisms are shown i n Scheme 6 w i t h r e f e r e n c e to the c leavage o f 5a-androstan-3-one ( L X ) . In the a c t u a l spectrum o f 5a-androstan-3-one e thy lene k e t a l these fragments 46 are the most abundant i n the e n t i r e spectrum w i t h that at m/e 99 b e i n g much more p l e n t i f u l than those at m/e 112 o r 125. m/e 112 Scheme 6. From the cleavage mechanism i l l u s t r a t e d above i t can be seen t h a t d i s u b s t i t u t i o n by a l k y l groups at e i t h e r the C2 or C4 p o s i t i o n s should have an important e f f e c t on the f ragmentat ion p a t t e r n s i n c e some o f the p o s s i b i l i -t i e s o f hydrogen t r a n s f e r would-be b l o c k e d o u t . Thus d i s u b s t i t u t i o n at C2 would e l i m i n a t e the f o r m a t i o n o f the m/e 99 fragment w i t h o u t g r e a t l y a f f e c t i n g the f o r m a t i o n o f the o t h e r fragments at m/e 112 o r 125. T h i s expected r e s u l t was conf irmed when the spectrum o f 2 , 2 - d i m e t h y l - 5 a - a n d r o s t a n - 1 7 g - o l - 3 - o n e 47 e thy lene k e t a l (LXI) was determined. The m/e 99 peak was complete ly m i s s i n g w i t h the m/e 125 peak b e i n g more i n t e n s e than t h a t at m/e 112. I f on the o t h e r hand two methyl groups are i n t r o d u c e d i n t o the C4 p o s i t i o n , then the pathways l e a d i n g t o the f o r m a t i o n o f the fragments at m/e 112 and m/e 125 ought t o be b l o c k e d so t h a t on ly the fragment at m/e 99 s h o u l d remain. T h i s was conf irmed by o b t a i n i n g the s p e c t r a o f the c o r r e s -ponding d e r i v a t i v e s o f the t r i t e r p e n e ketones a-amyrone L X I I and lupanone L X I I I . By f a r the most abundant peaks i n the s p e c t r a were those at m/e 99. The work o f the French s c h o o l has been very e x t e n s i v e i n examining the f ragmentat ion p a t t e r n s o f the e thy lene k e t a l d e r i v a t i v e s o f v a r i o u s l y s u b s t i t u t e d s t e r o i d a l ketone systems. However, i t does not add a n y t h i n g t o the arguments a l r e a d y advanced above and need not be f u r t h e r d i s c u s s e d i n the present c o n t e x t . Compounds A and B ketones were r e a c t e d w i t h an excess o f e thy lene g l y c o l i n dry benzene and t o l u e n e s u l p h o n i c a c i d as c a t a l y s t . The water 48 formed in the course of the reaction was removed continuously in a Dean-Stark trap to push the equilibrium to the right according to the equation The reaction was discontinued after five hours under reflux and the ketal derivatives recovered from the unreacted ketones by preparative TLC in the case of Compound A and by column chromatography on alumina in the case of B. In the former case the yield was only 50 % but in the latter it was almost quantitative. The A derivative was obtained in the form of colourless platelike crystals decomposing at 317-319°C after recrystallization from petroleum ether. The B derivative came out of solution in benzene as colourless rod-like crystals decomposing at 317-8°C. Both compounds yielded analytical values in agreement with the empirical formulae 0 3 3 1 1 5 ^ 0 3 and their mass spectra showed the molecular ion peaks at 498 as expected. absorption bands at 1107 cm - 1 in the region where the methoxyl group absorbs. A number of weak bands were also present between 900 and 1200 cm - 1 but were not helpful in the structural assignment. The NMR spectra, measured in deuterochloroform solution showed the usual signals mentioned previously plus sharp singlets at x 6.02 correspond-ing to four protons and arising from the methylene groups of the ketal rings. The IR spectra, measured on KBr discs, showed unusually strong 49 The most interesting feature of these derivatives in the present context is their mass spectra which, once again, resembled each other closely. The spectrum of the compound A derivative is illustrated in Figure 8. 100 % 8 0 S 6 0 •I 4 0 | 2 0 0 9 9 C O M P O U N D A K E T O N E • to ' Elhytana H«ta l |yiii,4 1 0 0 s .1.11. 3 0 0 _ ! I I I I 4 0 0 -j C-i i i _ 5 0 0 Figure 8. The first noteworthy feature of the fragmentation pattern is the tremendous abundance of the m/e 99 fragment which is more than ten times as intense as any other fragment represented. There is no trace of any signi-ficant quantity of fragments occurring at m/e 112 or 125 so that the con-clusion that the triterpenes A and B possess secondary alcohol functions vicinal to dimethyl substituted ring carbon atoms is inescapable. Of the other peaks in Figure 8 those occurring at m/e 189 and 221 are typical of the A and B series in general as will be discussed later. On the other hand the second most plentiful peak in the spectrum is that at m/e 399 which represents the remainder of the molecule after loss of the m/e 99 fragment. Because known methyl ether derivatives of triterpene alcohols are relatively rare, i t was obviously desirable to cleave the ether groups with 50 the aim of isolating known triterpene compounds such as alcohols, olefins, or acetates. Since methyl ethers attached to saturated carbon atoms are normally somewhat difficult to cleave and require fairly drastic acidic con-ditions, i t was considered advisable to first remove the free secondary alcohol function in ring E. Although this reaction is normally accomplished by carrying out a Wolff-Kishner reduction of the corresponding ketone, i t was considered more attractive to avoid the strongly basic conditions used in this procedure and employ a recently published method. The procedure adopted has been described by Cagliotti and Grasselli^^ who demonstrated its ut i l i ty in reducing a number of steroidal ketones to the parent hydrocarbons. Their method consists in reducing the tosylhydra-zone derivatives of the ketones with either lithium aluminium hydride or sodium borohydride using either methanol or dioxane as solvent. In a l l the cases described, in which steroid derivatives were employed, yields of 60 % or better of the desired hydrocarbons were achieved. The authors preferred the use of sodium borohydride as the reaction products were in this case not contaminated by small amounts of olefinic materials formed in side reactions. The mechanism and stereochemical course of this reduction was investi-gated by Djerassi's group^. Through the use of lithium aluminum deuteride and sodium borodeuteride they obtained monodeuterated hydrocarbons from the steroidal tosylhydrazones studied. The dependence of the competing olefin formation on a-substituents and on the reaction conditions was also elucidated. The tosylhydrazones of A and B ketones were prepared by reacting these compounds with an excess of tosylhydrazine reagent in dry benzene under reflux for seven hours. The water formed in the course of the reaction was removed continuously in a Dean-Stark trap as in the preparation of the ketal derivatives. After removal of most of the benzene the reaction mixture was 51 a l l o w e d t o s tand f o r a few hours whereupon whi te c r y s t a l l i n e products were o b t a i n e d . These compounds were r e c r y s t a l l i z e d from e t h a n o l i n which the unreacted t o s y l h y d r a z i n e reagent i s . - q u i t e s o l u b l e . The d e r i v a t i v e i n the A s e r i e s mel ted w i t h decomposit ion at 234-7°C and t h a t i n the B s e r i e s at 257-8°C. Both products gave a n a l y t i c a l r e s u l t s i n s a t i s f a c t o r y agreement w i t h the e m p i r i c a l formula C38H58N2O3S. The. NMR spectrum o f the compound A d e r i v a t i v e was determined i n d e u t e r o c h l o r o f o r m s o l u t i o n and showed the presence o f f o u r aromat ic protons i n . the r e g i o n T 2.00-2-72 and an aromatic methyl group at T 7.55 bes ides the u s u a l methoxyl and o t h e r s i g n a l s from the t r i t e r p e n e moiety . The IR spectrum o f t h i s compound (KBr) showed a sharp band at 3210 cm" 1 a t t r i b u t e d t o N-H s t r e t c h i n g , another at 1600 c m - 1 due t o the presence o f the t o s y l h y d r a z o n e moiety . The desoxy compounds A and B were r e a d i l y prepared by r e d u c t i o n o f the t o s y l h y d r a z o n e s w i t h sodium b o r o h y d r i d e i n r e f l u x i n g methanol f o r twelve h o u r s . Chromatography o f the crude r e d u c t i o n products on alumina columns y i e l d e d the d e s i r e d desoxy compounds i n 50-55 % y i e l d based on the weight Of the ketones . Both were b e a u t i f u l l y c r y s t a l l i n e m a t e r i a l s ^ m . p . 267-9° i n the A^and ,250-251° i n the B s e r i e s . The a n a l y t i c a l r e s u l t s were i n . b o t h cases i n agreement w i t h the e m p i r i c a l formulae C31H51O and the mass s p e c t r a y i e l d e d . m o l e c u l a r i o n peaks at m/e 440 i n c o n f i r m a t i o n o f t h e t m o l e c u l a r w e i g h t s . The IR s p e c t r a o f the desoxy compounds showed the customary absorp-t i o n s a l r e a d y mentioned p r e v i o u s l y . The NMR spectrum o f desoxy A showed s i g n a l s at x 4.60 ( o l e f i n i c H, IH), 6.60 (OCH3) , a s u g g e s t i o n o f a. wide m u l t i p l e t i n the r e g i o n x 7.15 t o 7.5 and f o u r peaks i n the s a t u r a t e d methyl group r e g i o n at x 9 . 0 1 , 9 .11 , 9.23 and 9 . 3 0 . The m u l t i p l e t i n the r e g i o n x 7.15 t o 7.5 c o u l d be r e s u l t i n g 52 from an axial proton geminal to an equatorial methoxyl group in which case it would take the form of a quartet. This was the first indication that the methoxyl group in the compound A series might be secondary in character. Another interesting feature of this spectrum was the total integral of 21.5 protons observed for the angular methyl group region, which in a l l the com-pound A derivatives so far examined appeared to indicate the presence of eight methyl groups but in this case appeared to indicate only seven. The corresponding spectrum for desoxy compound B showed analogous signals at T 4.70 (1H), 6.71 (3H), 7.25 (1H), and three signals from methyl groups at T 9.09, 9.16, and 9.34 with a total methyl group integral of 20.2 protons. The signal at 7.25, attributed to the proton geminal to the methoxyl group, was quite clear and much narrower than in the spectrum of desoxy A. Further derivatives which were prepared from compounds A and B were the dihydro products obtained by catalytic hydrogenation studies. These experiments were merely an extension of. the reaction conditions already described for the catalytic micro-hydrogenation studies using Adams' catalyst (Pt02) in glacial acetic acid. Thus dihydro compound A yielded white fernlike crystals from dioxane, m.p. 288-290°C, whereas dihydro B separated in the form of white needles from dioxane, m.p. 211-216°C. Both compounds gave analytical results in agreement with the empirical formula CsiHsi+C^ and exhibited molecular ion peaks at m/e 458 in the mass spectra. The I.R. spectra (KBr) showed hydroxyl absorption at 3550 and 3500 cm-1 for A and B respectively and C-0 stretching bands for methyl ether functions at 1082 and 1088 cnr1 respectively. The weak bands formerly present at 795 cm - 1 and attributed to the tertiary substituted ethylenic bonds were no longer observed. 53 The NMR s p e c t r a o f these compounds were measured i n d e u t e r o c h l o r o f o r m s o l u t i o n and both showed the absence o f the s i g n a l n o r m a l l y present at x 4.65 a r i s i n g from the o l e f i n i c p r o t o n . Dihydro compound A showed s i g n a l s at x 6.59 (1 H) from the p r o t o n geminal t o the h y d r o x y l f u n c t i o n , 6.64 (3 H) from the methoxyl group and seven methyl group s i g n a l s i n the r e g i o n x 9 . 0 2 - 9 . 2 6 i n t e g r a t i n g f o r 24.8 p r o t o n s . Dihydro compound B showed s i m i l a r s i g n a l s at x 6.10 (1 H ) , 6.70 (3 H ) , 7.25 (1 m u l t i p l e t ) f o r a p r o t o n geminal t o the methoxyl group, and f i v e s i g n a l s i n the methyl group r e g i o n (x 9 . 10-9 .24) i n t e g r a t i n g f o r 21.2 p r o t o n s . So f a r the d i s c u s s i o n has c e n t r e d o n . t h e p r e p a r a t i o n and s p e c t r a l f e a t u r e s o f d e r i v a t i v e s o f compounds A and B and the evidence d e s c r i b e d to date shows a very c l o s e r e l a t i o n s h i p between these two substances . However one d i f f e r e n c e which was apparent c o u l d be seen i n comparing the t o t a l methyl group i n t e g r a l s from the NMR s p e c t r a f o r compounds i n the two s e r i e s and these values are recorded i n Table 3. TABLE 3 M e t h y l Group I n t e g r a l s from NMR S p e c t r a o f A and B D e r i v a t i v e s D e r i v a t i v e A S e r i e s (H) B S e r i e s (H) O r i g i n a l compounds 23.8 21.0 Acetate 23.0 20.3 Ketone 24.1 21.7 Dihydro 24.8 21.0 Des.oxy 21.5 20.2 Ethylene K e t a l 22.8 21.3 54 Cl e a r l y i n a l l cases except i n the desoxy analogue the compound A series appeared to possess eight methyl groups whereas the B series indicated only seven. On t h i s basis compound A might be a "normal type" triterpene system whereas the B series could belong to the unique serratenediol family. The l a t t e r compounds, as Inubushi points out**, are the only triterpenes known to contain only seven angular methyl groups apart from those which possess primary alcohol or carboxylate groupings derived from methyl groups by enzymatic oxidation processes. The l a t t e r features are not present i n the S i t k a spruce compounds. Bearing i n mind the ORD r e s u l t s on compound A ketone i n which i t was concluded that the carbonyl function i s located at the C 21 p o s i t i o n , i t seemed very reasonable to postulate from biosynthetic considerations that the methoxyl group was located at the C 3 p o s i t i o n . That being the case and considering the ORD evidence i n support of the presence of the double bond at the A 1 4 p o s i t i o n , it\appeared that desoxy compound A might be i d e n t i c a l with the taraxerene d e r i v a t i v e sawamilletin XI which has already been mentioned i n the introduction. The equatorial methoxyl group at C 3 shown i n XI would be revealed i n the NMR spectrum by the presence of a quartet corresponding to the C 3 proton which would of course be i n an a x i a l o r i e n t a t i o n . 55 Professor Obara at Tokyo Kyoiku University very kindly provided us with a sample of the naturally occurring sawamilletin, m.p. 278°, [a]p +8°. Comparison with desoxy compound A by mixed melting point, thin layer chromato-graphy and quantitative I.R. spectroscopy (KBr) showed conclusively that these compounds are non-identical. The structural difference is also well illustrated by examining the mass spectra of these substances as presented in.Figure 9. 100 1 60 c „ 4 0 2 20 V IX 0 D E S O X Y C O M P O U N D A 2 0 0 •too 100 >e° £ 6 0 Uo a: 0 S A W A M I L L E T I N 100 . 2 0 0 m/e 3 0 0 M-47 4 0 0 ' ' ' ' i 0 0 Figure 9. Mass Spectra of Sawamilletin and Desoxy Compound A 56 Before examining these s p e c t r a i n d e t a i l i t : i s necessary at t h i s 67 stage to d i s c u s s some r e s u l t s from an important paper by D j e r a s s i et a l d e s c r i b i n g the a p p l i c a t i o n o f mass spectrometry i n s t r u c t u r a l e l u c i d a t i o n o f s a t u r a t e d and u n s a t u r a t e d p e n t a c y c l i c t r i t e r p e n e s . Most commonly, the u n s a t u r a t e d p e n t a c y c l i c compounds possess a A 1 2 double bond and be long t o the a- o r 6-amyrin c l a s s e s . However the s m a l l s u b - c l a s s o f the taraxerenes possesses a A 1 1 * double bond and the s p e c t r a o f the compounds taraxerone LXIV, t a r a x e r o l , LXV a n d m y r i c a d i o l d i a c e t a t e LXVI were d i s c u s s e d . One o f the tWo most important types o f f ragmentat ion i n t h i s r i n g system i s the r e t r o D i e l s - A l d e r process which r e s u l t s i n the cleavage o f r i n g D as shown above. In a d d i t i o n t o the i o n s shown, s a t e l l i t e peaks were observed caused by l o s s o f methyl and o t h e r groups from the main cleavage p r o d u c t s . In a d d i t i o n t o the r e t r o D i e l s - A l d e r p r o c e s s , another type o f 57 fragmentation gives rise to the most intense peaks at m/e 204 in the spectra of taraxerone and taraxerol. The cleavage giving rise to these peaks occurs in ring C at the position al ly l ic to the double bond and the fragment giving the peak at 204 comprises rings D and E. In the case of myricadiol diacetate the corresponding fragment at m/e 262 is rather weak because this species readily eliminates acetic acid to give an intense fragment at m/e 202. The mechanism suggested for this cleavage process is indicated in Scheme 7. X^ X X Scheme 7 The fragment (a) occurs at m/e 204 in cases where R2 is a methyl group. Subsequent loss of this group yields another abundant ion at m/e 189. If we now consider the spectrum of sawamilletin in Figure 9, the intense peak at m/e 204 is immediately evident( as being the most intense in the spectrum and the satellite peak at m/e 189 is also apparent. The retro Diels-Alder cleavage product appears at m/e 316 being shifted fourteen mass units higher than in taraxerol by the presence of the methoxyl function at C 3. The peaks at m/e 301, 284 and 269 are a l l satellites of the retro Diels-Alder cleavage product and may be explained as arising by loss of a . methyl group, loss of methanol, and loss of both methyl and methanol respectively. The fragments occurring at M-15 and M-47 may also be rational-ized as arising by loss of a methyl group and of methanol plus a methyl group respectively. 58 When the molecule o f s a w a m i l l e t i n i s c leaved i n the a l l y l i c p o s i t i o n , as i n Scheme 8, the l o s s o f i o n (a) leaves a r e s i d u e which should appear at m/e 236 i f i t were t o become p o s i t i v e l y charged. In f a c t no fragment having t h i s mass i s observed but by l o s s o f a methyl group t h i s s p e c i e s can g ive r i s e t o a s a t e l l i t e peak at m/e 221 which i s present though r a t h e r weak i n the case o f s a w a m i l l e t i n . The f ragmentat ion o f desoxy compound A i s on the whole q u i t e s i m i l a r t o t h a t o f s a w a m i l l e t i n at l e a s t i n so f a r as the masses o f the main f r a g -ments are concerned. Thus peaks are observed at m/e 316, 284 and 269 c o r r e s -ponding t o the r e t r o D i e l s - A l d e r cleavage process and at 204 and 189 from the f ragmentat ion i n r i n g C at the p o s i t i o n a l l y l i c t o the double bond which must be i n the A 1 1 + p o s i t i o n . The mass s p e c t r a l f ragmentat ion evidence t h e r e -f o r e c o r r o b o r a t e s the p o s t u l a t e d e r i v e d from a study o f the ORD curves o f compounds A and B k e t o n e s . The fragment at m/e 221 a r i s i n g from the a l l y l i c cleavage i n desoxy compound A i s much more abundant than i n the case o f s a w a m i l l e t i n and t h a t at m/e 204 much l e s s i n t e n s e . The very i n t e n s e m o l e c u l a r i o n peak i n desoxy compound A i s a l s o worthy o f n o t e . F u r t h e r evidence i n support o f a A 1 4 double bond a r i s e s i n an examinat ion o f , t h e mass s p e c t r a l f ragmentat ion o f o t h e r compounds i n both the A and B s e r i e s . The p o s i t i o n s o f the main r e t r o D i e l s - A l d e r and a l l y l i c c leavage fragments are summarised i n Table 4. The r e t r o D i e l s - A l d e r cleavage product at m/e 316 d i d not appear i n the spectrum o f e i t h e r A o r B ketones . However systems c o n t a i n i n g a A 1 4 double bond were p r e s e n t . U n f o r t u n a t e l y , because o f the methoxyl groups b e l i e v e d t o be l o c a t e d at the C 3 p o s i t i o n s , i t was not p o s s i b l e to vary the f u n c t i o n a l i t y at t h a t end o f the molecule so the m/e 221 peak remained unchanged throughout , as d i d a l s o the main r e t r o D i e l s - A l d e r peak. 59 TABLE 4 E f f e c t s o f Changes i n F u n c t i o n a l i t y on Mass S p e c t r a l  Fragmentat ion i n A and B S e r i e s D e r i v a t i v e r e t r o D i e l s - A l d e r A A l l y l i c A B B O r i g i n a l a l c o h o l 316. 316 220 220 221 221 Ketone - _ 218 218 221 221 A c e t a t e 316 316 262 262 221 221 Desoxy 316 316 204 204 221 . 221 Al though no complete spectrum o f s e r r a t e n e d i o l nor o f any o f i t s 11 12 d e r i v a t i v e s has been p u b l i s h e d t o d a t e , both Inubushi and Rowe have commented upon the observed masses o f the p r i n c i p a l fragments. The l a t t e r author i n d i c a t e d the most important fragments i n the spectrum o f s e r r a t e n e at m/e 191, 204, and 218 as a r i s i n g from cleavage through r i n g C. Replace-ment o f a methoxyl group by hydrogen i n s e r i e s A and B would lower the m/e 221 peak by 30 mass u n i t s t o m/e 191 and the o t h e r p o r t i o n o f the molecule remaining from t h i s f ragmentat ion s h o u l d appear at m/e 204 as i s observed i n desoxy A and B. The fragment at m/e 218 i n the spectrum o f s e r r a t e n e must a r i s e by f i s s i o n o f the 9,11 and 14,27 carbon-carbon bonds as i n LXVII i n s t e a d o f the more common 11,12 and 14,27 bond f i s s i o n which r e s u l t s i n the peak observed at m/e 204. In f a c t analogous, though r a t h e r weak, fragments are observed i n the A and B s e r i e s and appear at m/e 234 i n the s p e c t r a o f the 60 parent a l c o h o l s (F igures 5 and 6 ) . Rowe a l s o mentioned the expected r e t r o D i e l s - A l d e r fragments as o c c u r r i n g i n the f ragmentat ion p a t t e r n o f s e r r a t e n e at m/e 286 and 271. A g a i n , the i n t r o -LXVII C 3 p o s i t i o n i n s e r r a t e n e ought to r a i s e the p o s i t i o n o f the main r e t r o d u c t i o n o f a methoxyl f u n c t i o n at the D i e l s - A l d e r fragment by 30 mass u n i t s t o 316 as i s observed i n a l l the com-pounds o f the A and B s e r i e s w i t h the e x c e p t i o n o f the ketones . The i n t e n s i t y o f the m/e 316 peaks i s however q u i t e low so t h a t the r e s u l t i n g s a t e l l i t e peaks at m/e 301, a r i s i n g by l o s s o f a methyl group, are not shown i n the f o r m a l i s e d diagrams. The mass s p e c t r a l f ragmentat ion p a t t e r n s o f the compounds thus f a r d i s c u s s e d i n the A and B s e r i e s are e n t i r e l y c o n s i s t e n t w i t h the l o c a t i o n o f the double bond at the A 1 4 p o s i t i o n and w i t h the presence o f a s e r r a t e n e r i n g s k e l e t o n . When the work had reached t h i s stage i n i t s development, a new V a r i a n HA100 h i g h r e s o l u t i o n NMR spectrometer became a v a i l a b l e and i t was now p o s s i b l e t o d e r i v e a d d i t i o n a l s p e c t r a l i n f o r m a t i o n . Compounds A and B acetates were submit ted and the s p e c t r a shown i n F i g u r e s 10 and 11 were o b t a i n e d . The s i g n a l at T 4 .68 from the o l e f i n i c p r o t o n i n compound A acetate was an u n r e s o l v e d m u l t i p l e t w h i l e t h a t at x 5.31 a r i s i n g from the p r o t o n geminal t o the acetate group was a narrow t r i p l e t (J 2.5 c . p . s . ) . The methoxyl group s i g n a l was observed at x 6.68 and t h a t o f the acetate methyl group at x 7 .95. A r a t h e r wide m u l t i p l e t observed at x 7 . 4 2 , 61 corresponding to a single proton, upon examination at high resolution was seen to be a quartet (J^g 11 c.p.s., J ^ c 3.5 c.p.s.). This signal was assigned to the proton geminal to the methoxyl goup and was never distinguish-able in NMR spectra run on the A60 instrument with the possible exception of desoxycompound A. Finally seven distinct spikes could be seen in the saturated methyl group region of the spectrum (T 9.05-9.31) and these groups were apparently not coupled. COMPOUND A ACETATE 531T 7.37 T t ' '• 6 ' 7 ' 8 ' 9 ' rTiO Figure 10. N.M.R. Spectrum (100 Mc/s) of Compound A Acetate. It is well known that protons attached to ring carbon atoms bearing functional groups and having two adjacent methylene protons in the a position will appear either as narrow triplets or as wider quartets. When the signal is a triplet the proton is equatorial because only equatorial-equatorial or equatorial-axial coupling can occur and in both cases the coupling constant J is small (1 to 5 c.p.s.). On the other hand axial protons can undergo 62 a x i a l - a x i a l c o u p l i n g as w e l l and here the J v a l u e i s much l a r g e r (8-14 c . p . s . ) so t h a t the a x i a l - e q u a t o r i a l c o u p l i n g causes the s i g n a l t o appear as a q u a r t e t . From t h i s i n f o r m a t i o n i t was q u i t e c l e a r t h a t compound A acetate c o n t a i n s an e q u a t o r i a l methoxyl group and an a x i a l a c e t a t e f u n c t i o n . COMPOUND B ACETATE F i g u r e 11. N.M.R. Spectrum (100 Mc/s) o f Compound B A c e t a t e . In the spectrum o f compound B acetate (F igure 11) the o l e f i n i c p r o t o n s i g n a l at T 4.70 was aga in an u n r e s o l v e d m u l t i p l e t and the p r o t o n geminal t o the acetate group appeared as a narrow t r i p l e t (J 2.5 c . p . s . ) c e n t r e d at T 5 . 3 3 . The methoxyl group i n t h i s case absorbed at h i g h e r f i e l d (x 6.75) than i n the case o f A a c e t a t e (T 6.68) and the methyl group protons o f the acetate f u n c t i o n were observed at x 7 .98. However the most important f e a t u r e o f the two s p e c t r a l i e s i n the q u i t e d i f f e r e n t s i g n a l s a r i s i n g from the protons geminal to the. two methoxyl groups. In t h i s case a narrow t r i p l e t was observed (J 2.5 c . p . s . ) corresponding t o an e q u a t o r i a l p r o t o n geminal t o 63 an a x i a l methoxyl whereas i n the case of A acetate t h i s proton appeared as a quartet i n d i c a t i n g the methoxyl f u n c t i o n to be e q u a t o r i a l . F i n a l l y only four spikes could be seen i n the s a t u r a t e d methyl group region (x 9.10-9.33) of the spectrum of B acetate. I t was concluded that B acetate possesses an a x i a l methoxyl group and an a x i a l acetate f u n c t i o n . The d i s c u s s i o n up to t h i s point has considered the chemical and s p e c t r a l p r o p e r t i e s of the A and B s e r i e s i n p a r a l l e l to d e l i b e r a t e l y b r i n g i n t o focus the very close analogy between these compounds. However at t h i s stage i t i s necessary to depart from t h i s approach and to deal s e p a r a t e l y with the two s e r i e s i n i n d i c a t i n g how the s t r u c t u r e s were f i n a l l y proven. An attempt was made to remove the methyl ether f u n c t i o n from com-pound A acetate, through the use of a Lewis a c i d . Following the procedure of 68 Narayanan and Iyer , the compound was d i s s o l v e d i n a c e t i c anhydride and t r e a t e d overnight at 0°C with.boron t r i f l u o r i d e etherate. The crude product of t h i s r e a c t i o n contained at l e a s t s i x components but the major one was i s o l a t e d by p r e p a r a t i v e t h i n l a y e r chromatography. This cleavage product was a glassy substance which could not be induced to c r y s t a l l i z e . The NMR spectrum was determined i n deuterochloroform s o l u t i o n and showed the complete removal of the methyl ether, f u n c t i o n . The o l e f i n i c proton s i g n a l was s t i l l observed at x 4.63, apparently i n d i c a t i n g that the A 1^ double bond had not isomerized. The usual sijgnal was"present f o r a proton geminal to an acetate residue (x 5.31) and the methyl group of the acetate f u n c t i o n which appeared as a very strong sharp s i g n a l at x 7.93 apparently corresponded to only one acetate group. No other l o w f i e l d protons were observed but f i v e spikes were present i n the s a t u r a t e d methyl group region (x 8.98-9.29). No mass s p e c t r a l data was obtained on t h i s m a t e r i a l but from the f a c t s a v a i l a b l e i t was p o s s i b l e to speculate that the s t r u c t u r e of t h i s cleavage product might be 64 as shown in LXVIII. In this postulate the loss of an equatorial functional group at C 3 has been followed by ring contraction in ring A with the formation of an isopropyl side chain -a reaction well known in the chemistry of triterpenes. A considerable amount of LXVIII effort was expended in trying to cleave the methyl ether function in compound A acetate and in desoxy A using 36 % HBr in glacial acetic acid mixed with an equal volume of acetic anhydride. This reagent was used with success by Uyeo et al in the proof of structure of abieslactone XIX iii which both the unsaturated methyl ether LXIX and its dihydro derivative were cleaved to yield acetates which were then saponified and oxidised with chromic oxide/pyridine to produce ketones of known structure. As this work was presented in the form of a communi-cation, temperature conditions in the cleavage reaction were not specified. Part of the work in the cleavage reactions, now to be described, was performed, by Dr. W. A. F. Gladstone, a post-doctoral fellow in Dr. Kutney's group. Two cleavage experiments were carried out on A acetate, one under reflux conditions for 90 minutes, the other at 65°C for one hour. Apparently the two experiments gave similar products but the milder conditions gave a much better yield (80 %) of a white LXIX crystalline substance m.p. 175-202° 65 (from e t h a n o l ) . A sample o f t h i s m a t e r i a l d i s s o l v e d i n d e u t e r c h l o r o f o r m p r o v i d e d the f o l l o w i n g NMR spectrum (60 M c / s ) ; T 5.31 (1 H) ( t r i p l e t ) from a p r o t o n geminal t o an a x i a l a c e t a t e f u n c t i o n , T 7.93 (3 H) from the methyl o f an acetate group, and f o u r s p i k e s i n the s a t u r a t e d methyl group r e g i o n (x 8.95-9.18) but no o l e f i n i c p r o t o n from the A 1 4 double bond. An IR spectrum o f t h i s compound conf irmed the absence o f the methoxyl group and showed the presence o f a c e t a t e (1730 and 1250 c m " 1 ) . The mass spectrum showed a m o l e c u l a r i o n peak at m/e 466 c o r r e s p o n d i n g t o the expected va lue f o r a d i o l e f i n a c e t a t e . A h i g h r e s o l u t i o n NMR spectrum o b t a i n e d at 100 Mc/s r e v e a l e d , i n a d d i t i o n t o the s i g n a l s a l r e a d y d e s c r i b e d , a septet c e n t r e d at x 7.42 which might p o s s i b l y be a s s i g n e d t o the t e r t i a r y p r o t o n o f an i s o p r o p y l group. This s i g n a l was not present i n the 100 Mc/s spectrum o f desoxy, compound A which was run as a s t a n d a r d . The evidence t h e r e f o r e , though not c o n c l u s i v e , , i s i n favour o f the s t r u c t u r e LXX f o r the major product o f the cleavage r e i a c t i o n . j ^ o A c J LXX A s i m i l a r c leavage r e a c t i o n was c a r r i e d out on desoxy compound A but at room temperature f o r s i x t e e n h o u r s . From 511 mg. o f s t a r t i n g m a t e r i a l was recovered 362 mg. o f " r i n g c o n t r a c t i o n p r o d u c t " (76 % y i e l d ) , 36 mg. o f a compound t e n t a t i v e l y p o s t u l a t e d t o be A 1 3 desoxy compound A , and 61 mg. o f a complex m i x t u r e o f g l a s s y s o l i d s c o n t a i n i n g a c e t a t e and p o s s i b l y a l s o bromide f u n c t i o n s . The major product c r y s t a l l i z e d i n c o l o u r l e s s p l a t e l e t s , m.p. 160-165°C 66 and a n a l y s i s showed i t to possess an e m p i r i c a l f o r m u l a , C S Q H ^ S - Gas chromato-graphy on t h i s m a t e r i a l r e v e a l e d the presence o f two compounds i n a r a t i o o f 7:1. The NMR spectrum showed the absence o f low f i e l d protons but once aga in a wide m u l t i p l e t c e n t r e d at x 7.4 (probably a s e p t e t ) was p r e s e n t . The two isomers c o u l d not be separated on a s i l i c a g e l TLC p l a t e . S e p a r a t i o n by column chromatography on s i l v e r n i t r a t e impregnated s i l i c a g e l might prove s u c c e s s f u l .although t h i s technique was not attempted. C l e a r l y the data a v a i l a b l e p r e c l u d e s any d e f i n i t e s t r u c t u r a l assignment but i t i s f e l t t h a t the major product may possess a s t r u c t u r e s i m i l a r t o LXX but wi thout the C 2 1 a c e t a t e f u n c t i o n . The compound t e n t a t i v e l y d e s c r i b e d as A 1 3 desoxy compound A , m.p. 242-7°C analysed f o r C31H52O. The mass spectrum had a m o l e c u l a r i o n peak at 440 but no r e t r o D i e l s - A l d e r fragments were observed. The IR spectrum showed the u s u a l e t h e r f u n c t i o n s i g n a l at 1100 c m - 1 but the s i g n a l n o r m a l l y present at 795 c m - 1 i n the A s e r i e s and a t t r i b u t a b l e to the t r i s u b s t i t u t e d double bond was now absent . I n s u f f i c i e n t amounts o f m a t e r i a l prevented f u r t h e r i n v e s t i g a t i o n s on t h i s compound so t h a t no d e f i n i t e s t r u c t u r a l assignment can be made. The m i x t u r e o f p o l a r c leavage products was a l s o examined. An NMR spectrum o f t h i s f r a c t i o n (100 Mc/s) showed weak low f i e l d p r o t o n m u l t i p l e t s i g n a l s c e n t r e d at x 4 . 7 0 , 5 . 3 5 , 5.55 and 6 .10 . A r a t h e r broad a c e t a t e methyl s i g n a l was a l s o present at x 7.95 and the presence o f an acetate f u n c t i o n was a l s o apparent from the IR spectrum (1725 and 1250). This s p e c t r a l i n f o r m a t i o n may be e x p l a i n e d i f i t i s supposed t h a t the m i x t u r e o f p o l a r compounds i n c l u d e s A 1 3 and A 1 4 a x i a l and e q u a t o r i a l a c e t a t e s . The m u l t i p l e t c e n t r e d at x 6.10 c o u l d perhaps be due t o a p r o t o n geminal t o a bromine atom. In f a c t an examinat ion o f t h i s m i x t u r e on a t h i n l a y e r s i l i c a g e l chromatoplate showed 67 two spots of very s i m i l a r p o l a r i t y , one g i v i n g a brown colour and the. other pink. A f t e r d e a c e t y l a t i o n by r e a c t i o n with l i t h i u m aluminium hydride, the product was a yellow o i l y l i q u i d (31 mg.). An examination of t h i s mixture by TLC showed t h a t the compound g i v i n g r i s e t o the pink spot was apparently unchanged whereas the former brown spot, b e l i e v e d to have been caused by a mixture of acetates, was now replaced by two brown spots running c l o s e r to the base l i n e and of f a i r l y s i m i l a r p o l a r i t y . These might have been caused by a p a i r o f epimeric a l c o h o l s . Because so l i t t l e m a t e r i a l was a v a i l a b l e t h i s i n v e s t i g a t i o n was not pursued. I t was c l e a r from the above r e s u l t s that ether cleavage r e a c t i o n s were u n a t t r a c t i v e i n the A s e r i e s i n that they proceeded to give high y i e l d s of r i n g contracted products which could not conveniently be r e l a t e d to any compound of known s t r u c t u r e i n the s e r r a t e n e d i o l f a m i l y . The minor products of the cleavage experiments appeared t o be complex mixtures of acetates and brominated compounds which were d i f f i c u l t t o separate and were not c r y s t a l s l i n e . About t h i s time i t became necessary to o b t a i n samples of s e r r a t e n e d i o l and other c l o s e l y r e l a t e d compounds f o r the purpose of c o r r e l a t i o n w i t h the t r i t e r p e n e s i s o l a t e d from S i t k a spruce. P r o f e s s o r Inubushi at the F a c u l t y of Pharmacy, Osaka U n i v e r s i t y , Japan, k i n d l y sent us samples of s e r r a t e n e d i o l , serratene, serratenediol-3-monoacetate, a- and 8-serratane. Dr. Rowe at the Forest Products Laboratory, U.S. Department of A g r i c u l t u r e , Madison, Wisconsin, a l s o sent us generous samples of s e r r a t e n e d i o l and 21-episer.ratenediol. , Dr. Rowe, as i t t r a n s p i r e e d , had r e c e i v e d from Dr. Hergert a few years p r e v i o u s l y a sample of the crude t r i t e r p e n e mixture from S i t k a spruce i n connection with some IR s t u d i e s and, unknown to Dr. Hergert, he had separated the mixture by column chromatography on d e a c t i v a t e d alumina t o i s o l a t e f i v e components. 68 He had a l s o obtained some IR and NMR s p e c t r a l data on the two major com-ponents, which were i d e n t i c a l to compounds A and B although,due t o i n s u f f i -c i e n t q u a n t i t i e s , he was unable t o conduct any extensive i n v e s t i g a t i o n s . When he learned o f our work on the S i t k a spruce t r i t e r p e n e s , Dr. Rowe g r a c i o u s l y agreed to leave the problem i n our hands and has been most.help-f u l i n supplying us with samples (250 mg) of s e r r a t e n e d i o l and serratenedione which were needed i n work, on the B. s e r i e s . In c o l l a b o r a t i o n with our work Dr. Rowe re-examined h i s compounds from the S i t k a spruce bark e x t r a c t and c o n c l u s i v e l y showed one of the minor compounds t o be i d e n t i c a l with 2 1 - e p i s e r r a t e n e d i o l which he had p r e v i o u s l y i s o l a t e d from pine bark."' 1 With the presence of 2 1 - e p i s e r r a t e n e d i o l i n the o r i g i n a l t r i t e r p e n e mixture now known, i t was obvious from the mass s p e c t r a l and high r e s o l u t i o n NMR data that our compound A was probably i t s 3-monomethyl ether d e r i v a t i v e . Dr. Rowe sent us a sample of a n a t u r a l l y o c c u r r i n g ketone from pine bark to which he had assigned,the s t r u c t u r e 3B-methoxy-21-keto-A l l t-serratene^''' LVIIa. We compared t h i s ketone with out compound A ketone by melting p o i n t , mixed mel t i n g p o i n t , TLC i n two d i f f e r e n t solvent systems, mass spectrometry, ORD and q u a n t i t a t i v e IR spectroscopy (KBr). I t was apparent that these compounds were i d e n t i c a l . They d i d not however have the same s p e c i f i c r o t a t i o n values. Our present sample of compound A ketone a f t e r vacuum sublimation (240-50°C/0.07 mm. Hg) had m.p. 270-270.5°C. This m a t e r i a l 69 showed [a] 2 0 -0 .1° . (C. 1.03 in CHC13). The pine bark ketone sample provided by Dr. Rowe was sublimed twice but the highest melting point which could be obtained was 266.5-267°C. Rowe had previously shown this compound to have the rotation value, [a]2)3 -29° . No spot corresponding to an impurity could be seen in either case when these samples were examined by TLC. The ORD curves of the two ketones were identical in shape but one was displaced below the other as illustrated in Figure 12. Because of these discrepancies it seemed prudent to seek additional evidence to establish the correlation. Figure 12. ORD curves of Compound A ketone and Pine Bark ketone. From previous separations of the crude triterpene mixture on s i l ica columns, a fraction (1-730 gm.) enriched in the minor components more polar 70 than compound A was available. Column chromatography of this extract on deactivated alumina yielded the six compounds shown,in Table 5. The quanti-ties isolated after further purification and the colours given when chromato-plates were freshly sprayed with antimony pentachloride are included. The R f values quoted refer to the solvent system ethyl acetate/benzene (1:3). TABLE 5 Polar Components Isolated from Sitka.Spruce Bark Extract Compound Yield, mg. R f Colour with SbCl5 B 164 , 0.64 ginger brown A 641, 0.57 ginger brown (At)F 125 0.45 ginger brown D 131 0.39 orange pink G 209 0.30 brown H 59 0.21 brown Compound G was a white crystalline material, m.p. 289-291° • [a]20 -12 .3° ( C H C 1 3 ) . Elemental analysis showed i t to possess the empirical formula, C30H50O2, which was confirmed by the presence of a molecular ion peak at m/e 442 in the mass spectrum. The IR spectrum (KBr) showed a strong oxygen-hydrogen stretching absorption at 3500 cm-1 and weak bands around 1650 cm"1 and at 795 cm - 1 attributable to the presence of a trisub-stituted double bond. The NMR spectrum showed signals at x 4.65 (1 H, multiplet) for an olefinic proton, a triplet centred at 6.55 (1 H) corres-ponding to the proton geminal to an axial hydroxyl group, a broad multiplet (6.80, 1 H) corresponding to the proton geminal to an equatorial hydroxyl 71 group and seven sharp s p i k e s i n the s a t u r a t e d methyl group r e g i o n (T 9 . 0 4 -9 . 3 2 ) . The compound was a p p a r e n t l y a d i o l o f the s e r r a t e n e type and f u r t h e r i n f o r m a t i o n i n support o f t h i s c o n c l u s i o n was forthcoming from the mass s p e c t r a l f ragmentat ion p a t t e r n . The l a t t e r spectrum showed the expected r e t r o D i e l s - A l d e r fragment o c c u r r i n g at m/e 302 w i t h a s a t e l l i t e peak at m/e 284 a r i s i n g p r o b a b l y by l o s s o f water and a second s a t e l l i t e peak at m/e 269 i n d i c a t i n g the f u r t h e r l o s s o f a methyl group. The main a l l y l i c c leavage mexhanism was r e p r e s e n t e d by s t r o n g peaks at m/e 220 and 207 w i t h s a t e l l i t e peaks at 205, 202, and 189. Compound G y i e l d e d a d i a c e t a t e d e r i v a t i v e when a s m a l l sample was a c e t y l a t e d but t h i s compound was however not o b t a i n e d i n an a n a l y t i c a l l y pure form. The white c r y s t a l l i n e compound, m.p. 2 1 0 - 2 1 7 ° , showed the f o l l o w i n g s i g n a l s i n the NMR spectrum: T 4.65 (1 H) a m u l t i p l e t f o r an o l e f i n i c p r o t o n , a m u l t i p l e t at T 5.30 f o r one p r o t o n geminal t o a c e t a t e , a wide m u l t i p l e t at 5.47 f o r another p r o t o n geminal t o a c e t a t e , two s i n g l e t s a t 7.94 and 7.98 f o r the methyl groups o f the two a c e t a t e f u n c t i o n s , and o n l y t h r e e s p i k e s i n the s a t u r a t e d methyl group r e g i o n (T 9 . 0 7 - 9 . 3 1 ) . Apparent ly ; however one methyl group had been d e s h i e l d e d f o r a s i g n a l i n t e -g r a t i n g f o r t h r e e protons was now observed a t x 8 .75 . These r e s u l t s were conf irmed by r u n n i n g an NMR spectrum at 100 Mc/s which showed the m u l t i p l e t c e n t r e d at, x 5.30 to be a t r i p l e t (J^g 2.5 c . p . s . ) and t h a t c e n t r e d at x 5.47 t o be a q u a r t e t ^ c - P - s - > 5.2 c . p . s . ) the mass spectrum o f the d i a c e t a t e showed the parent i o n peak at m/e 526 w i t h s t r o n g s a t e l l i t e peaks at m/e 466 (M + -60) arid m/e 406 (M + -120) c o r r e s p o n d i n g t o the l o s s o f one and two molecules o f a c e t i c a c i d r e s p e c t i v e l y . The r e t r o D i e l s - A l d e r cleavage fragment d i d not appear but i t s s a t e l l i t e peaks were present at m/e 284 and 269. The expected fragments at m/e 262 and 249 from the a l l y l i c c leavage 72 mechanism were present t o g e t h e r w i t h the u s u a l s a t e l l i t e peaks at m/e 202, 189 and 187. The c h a r a c t e r i z a t i o n o f compound G as 2 1 - e p i s e r r a t e n e d i o l LIV was completed by comparison w i t h an a u t h e n t i c sample o b t a i n e d from Dr . Rowe. The compounds were i d e n t i c a l on the b a s i s o f m e l t i n g p o i n t , mixed m e l t i n g p o i n t , TLC b e h a v i o u r , mass s p e c t r a , NMR s p e c t r a and q u a n t i t a t i v e IR (KBr) comparisons. M e t h y l a t i o n o f a sample (100 mg.) o f 2 1 - e p i s e r r a t e n e d i o l was achieved u s i n g potass ium metal i n r e f l u x i n g to luene under an i n e r t atmosphere i n a f l a s k equipped w i t h a h i g h speed s t i r r e r . Chromatography o f the product on d e a c t i v a t e d a lumina y i e l d e d the d i m e t h y l e t h e r (18 mg.) a monomethyl e t h e r (7 mg.) another monomethyl e t h e r (43 mg.) and unreacted s t a r t i n g m a t e r i a l (8 mg.) . The more abundant o f the two monomethyl e thers was shown t o be i d e n t i c a l w i t h compound A on the b a s i s o f i t s m e l t i n g p o i n t and mixed m e l t i n g p o i n t , mass s p e c t r o m e t r y , NMR, TLC behaviour and q u a n t i t a t i v e IR (KBr) comparison. Compound A t h e r e f o r e has the s t r u c t u r e 36-methoxy-218-h y d r o x y - A 1 ^ - s e r r a t e n e , LXXI ( 2 1 - e p i s e r r a t e n e d i o l - 3 - m o n o m e t h y l e t h e r ) . Compound A i t s e l f was methylated under the same c o n d i t i o n s as above but w i t h somewhat g r e a t e r d i f f i c u l t y . From a sample o f the a l c o h o l (352 mg.) the methyl e t h e r d e r i v a t i v e (191 mg.) was o b t a i n e d and some unreacted a l c o h o l (100 mg.) was a l s o recovered from the column. This compound was a 73 white crystalline solid, m.p. 272-3°C, [a] 2 0 -16.4° (CHC13) which yielded analytical data in agreement with the empirical formula C32H54O2. No hydroxyl absorption band was evident in the IR spectrum but a strong ether band was present at 1100 cm-1 and the usual weak bands at 1650 and 795 cm"1 indicated the presence of the trisubstituted double bond. The NMR spectrum showed a multiplet at T 4.70 (1 H) for an olefinic proton, singlets at 6.68 and 6.73 for two methoxyl groups, a multiplet centred at 7.23 and a wide multiplet centred at 7.42 for the two protons geminal to methoxyl groups, one deshielded methyl group at 8.78 and six saturated methyl group spikes (9.08-9.37). A spectrum determined at 100 Mc/s showed the multiplet centred at T 7.23 to be a triplet ( J A g = 2.6 c.p.^.) and that at\* 7.42 to be a quartet (J,A X = 11.6 c.p.s., J g x = 4.0 c.p.s.). The mass spectrum of the ether derivative showed a molecular ion peak at m/e 470 and a retro Diels-Alder cleavage fragment at m/e 316 with satellite peaks at m/e 284 and 269. The main fragments from the al ly l ic clea.vage were present at m/e 234 and 221 with satellites at m/e 219, 189 and 187. A comparison was made between the dimethyl ether obtained by methylation of 21-episerratenediol and the methyl ether derivative of compound A. These compounds were found to be identical from evidence similar to that obtained in the case of the monomethyl ether derivatives. Previous attempts to separate compounds A and B on s i l ica gel columns had never been successful in yielding any fractions rich in compound E which runs just in front of A on a s i l ica gel thin layer chromatoplate. However it was discovered that column chromatography on high activity alumina yielded pure compound E although the compounds A and B were not separated from each other by this procedure. The crystalline material, m.p. 262-3° isolated in this manner was. optically active, [°0n° -16° (CHCI3) 74 and y i e l d e d the e m p i r i c a l formula C 3 1 H 5 0 O 2 on a n a l y s i s . I t showed a s t r o n g carbonyl a b s o r p t i o n at 1715 c m - 1 i n the IR spectrum i n d i c a t i n g the presence o f a ketone f u n c t i o n i n a s i x o r h i g h e r membered c a r b o c y c l i c r i n g . A l s o present was the u s u s a l band at 1105 c m - 1 a t t r i b u t a b l e to the presence o f ah e t h e r f u n c t i o n and a weak band at 795 cm" 1 i n d i c a t i n g the presence o f a t r i s u b s t i t u t e d double bond. T h i s compound was shown t o be compound A ketone by mixed m e l t i n g p o i n t , , TLC, q u a n t i t a t i v e IR spectroscopy and o p t i c a l r o t a t i o n measurements. The work which has j u s t been d i s c u s s e d completes the s t r u c t u r a l e l u c i d a t i o n o f compounds i s o l a t e d from S i t k a spruce b a r k . a n d b e l o n g i n g to the compound A s e r i e s . , I t i s now necessary t o r e t u r n t o the compound B s e r i e s and t o develop the arguments l e a d i n g t o s t r u c t u r a l assignments i n t h i s r e l a t e d f a m i l y o f compounds. The n e c e s s i t y o f c a r r y i n g out e t h e r c leavage r e a c t i o n s w i t h a view to the i s l o a t i o n o f i d e n t i f i a b l e products o f the s e r r a t e n e type has a l r e a d y been r e f e r r e d t o and i t was p o i n t e d out t h a t removal o f the secondary h y d r o x y l f u n c t i o n t o y i e l d the desoxy compound would p r o v i d e a g o o d . s t a r t i n g p o i n t f o r t h i s r e a c t i o n . I t would be even more p r e f e r a b l e i f the double bond were a l s o removed i n the s t a r t i n g m a t e r i a l so t h a t any u n d e s i r a b l e s i d e r e a c t i o n s a s s o c i a t e d w i t h double bond m i g r a t i o n s or s k e l e t a l rearrangements c o u l d be e l i m i n a t e d . Dihydro compound B was o x i d i s e d w i t h Jones reagent ^9>70 ^ chromic oxide i n aqueous s u l f u r i c a c i d ) and gave an almost q u a n t i t a t i v e y i e l d of the corresponding ketone. The white c r y s t a l l i n e m a t e r i a l , m.p. 167-9° was submit ted f o r a n a l y s i s and the a n a l y t i c a l data i n d i c a t e d the e m p i r i c a l formula C31H52O2. Th is r e s u l t was i n agreement w i t h the va lue m/e 456 o b t a i n e d f o r the m o l e c u l a r i o n peak i n the mass spectrum. The IR spectrum 75 showed a b s o r p t i o n bands at 1705 c m - 1 due t o the carbonyl chromophore and at 1090 c m - 1 from the methyl e t h e r f u n c t i o n . The NMR spectrum showed the f o l l o w i n g s i g n a l s ; a s i n g l e t at x 6.66 f o r the methoxyl group, a m u l t i p l e t c e n t r e d at 7.20 f o r one p r o t o n geminal t o a methoxyl group, an apparent q u a r t e t c e n t r e d at x 7.64 (2 H) ass igned to a p a i r o f methylene protons v i c i n a l to the c a r b o n y l group,and f o u r s p i k e s i n the methyl group r e g i o n (x 8 . 9 5 - 9 . 2 2 ) . The mass spectrum showed a s t r o n g M + -32 peak corresponding to l o s s o f methanol and another weaker peak at M + -47 i n d i c a t i n g the f u r t h e r l o s s , o f a methyl group. As expected there was no r e t r o D i e l s - A l d e r cleavage fragment and the whole nature o f the spectrum had changed s i n c e t h e r e was now no s t r o n g f ragmentat ion at the p o s i t i o n a l l y l i c t o a double bond. Prominent peaks were observed at m/e 261, 219, 189, i 5 0 and 136 w i t h the l a t t e r b e i n g the base peak i n the spectrum. D i h y r o compound B ketone was reduced to desoxy d i h y d r o compound B by means o f the W o l f f - K i s h n e r r e d u c t i o n i n which the Huang-Minion m o d i f i -c a t i o n was used.] A white c r y s t a l l i n e product was i s o l a t e d i n 85 % y i e l d . However t h i s m a t e r i a l a f t e r s e v e r a l r e c r y s t a l l i z a t i o n s from dioxane s t i l l possessed a broad m e l t i n g p o i n t (m.p. 171-193°) and was o b v i o u s l y a m i x t u r e . The r e d u c t i o n was repeated t w i c e and the product p u r i f i e d by p r e p a r a t i v e t h i n l a y e r chromatography on s i l i c a g e l . E v e n t u a l l y i t was observed t h a t r e c r y s -t a l l i z a t i o n from e t h y l acetate y i e l d e d two types o f c r y s t a l s - p l a t e l e t s (m.p. 185-192°) and needles (m.p. 215-223°C). S i n c e i t was p o s s i b l e t h a t the s t r o n g l y b a s i c c o n d i t i o n s o f . t h e W o l f f - K i s h n e r r e d u c t i o n might have caused some rearrangement r e a c t i o n t o take p l a c e i t was dec ided t o prepare the above compound i n a d i f f e r e n t manner. Dihydro compound B ketone was converted t o i t s t o s y l h y d r a z o n e d e r i v a t i v e and the l a t t e r t r e a t e d w i t h sodium b o r o h y d r i d e i n methanol . This r e a c t i o n 76 sequence p r o v i d e d an e x c e l l e n t y i e l d (>80 %) o f a desoxy d i h y d r o compound but the product was s t i l l a m i x t u r e as i n d i c a t e d above. The r e d u c t i o n products prepared by both methods were examined by vapour phase chromatography (VPC) and i t was shown t h a t both were mixtures of two compounds i n the r a t i o o f 4 : 1 . U s i n g hand p i c k e d c r y s t a l s i t was p o s s i b l e t o demonstrate t h a t the compound y i e l d i n g the p l a t e l e t type c r y s t a l s was the predominant component. Moreover a comparison o f the VPC c h a r t s showed the r e t e n t i o n times o f the p a i r o f peaks from the W o l f f -K i s h n e r m a t e r i a l t o be the same as those i s o l a t e d from the t o s y l h y d r a z o n e r e d u c t i o n r e a c t i o n . A q u a n t i t a t i v e IR s p e c t r a l comparison o f the p l a t e l e t type c r y s t a l s i n c h l o r o f o r m s o l u t i o n e s t a b l i s h e d t h e i r i d e n t i t y . A s t r o n g a b s o r p t i o n band at 1090 c m - 1 was a t t r i b u t e d t o the methoxyl group. Because the same p a i r s o f i s o m e r i c compounds were o b t a i n e d by both r e d u c t i o n processes i t was c l e a r t h a t the g e n e r a t i o n o f the two forms must have o c c u r r e d i n the sequence before the d i h y d r o compound B ketone s t a g e . O b v i o u s l y the c a t a l y t i c r e d u c t i o n o f the o l e f i n i c l i n k a g e i t s e l f must have y i e l d e d a and 8 forms a r i s i n g by a t t a c k o f the c a t a l y s t from above o r below the r i n g system a t . t h e p o s i t i o n o f the o r i g i n a l t e r t i a r y double bond. This type o f behav iour was observed by I n u b u s h i 1 1 who i s o l a t e d a and 8-serratane by the c a t a l y t i c hydrogenat ion o f s e r r a t e n e . A sample o f the mixed isomers gave a n a l y t i c a l data i n agreement w i t h the e m p i r i c a l formula CsiHsi+O and. the mass spectrum showed a m o l e c u l a r i o n peak at m/e 442 i n agreement w i t h t h i s a n a l y s i s . The mass spectrum a l s o showed fragments at M + - 1 5 , M + -32 and M + - 4 7 corresponding to l o s s o f a methyl group, l o s s o f methanol and l o s s o f both methyl and methanol r e s p e c t i v e l y . The base peak i n the spectrum again o c c u r r e d at m/e 136 and prominent peaks 77 were observed at m/e 177, 189, 190 and 221. The NMR spectrum showed s i g n a l s at x 6.68 f o r the methoxyl group, a m u l t i p l e t centred at 7.22 f o r one proton geminal to methoxyl and four spikes ( T 9.10-9.25) i n the satura t e d methyl group region. Some p r e l i m i n a r y r e a c t i o n s were c a r r i e d out t o t r y to f i n d a 71 s u i t a b l e reagent f o r cleavage of the methoxyl group. Youssefyeh and Mazur have obtained 90 % y i e l d s of 3a and 3 B-chlorocholestane by the cleavage of 3a - and 38-methoxychlolestane with boron t r i c h l o r i d e . I t seemed reasonable to use t h i s procedure on the s a t u r a t e d t r i t e r p e n e system. A mixture of the above desoxy dihydro compounds (122 mg.) was reacted with boron t r i c h l o r i d e at -89°C i n dichloromethane s o l u t i o n . . The temperature was allowed to r i s e s l o w l y t o 20° and the r e a c t i o n was l e f t standing overnight. A brown syrupy l i q u i d ( i n mg.) was recovered and t h i s was separated by p r e p a r a t i v e TLC on s i l i c a g e l t o give two non-polar yellow l i q u i d s (55 mg. and,17 mg.). An IR spectrum i n chloroform on the more p l e n t i f u l r e a c t i o n product i n d i c a t e d the complete removal o f the methoxyl group and an NMR spectrum showed only two broad s i g n a l s centred at x 8.75 and 9.15 corresponding to methyl group resonances. I t was thought that t h i s m a t e r i a l might be a mixture of c h l o r i n a t e d hydrocarbons and an attempt was made to,dehydrochlorinate the major (55 mg.) r e a c t i o n product by treatment of the l a t t e r with a s o l u t i o n of potassium hydroxide i n a mixture of d i e t h y l e n e , g l y c o l and diethylene g l y c o l dimethyl ether. When the mixture was heated on the steam bath a white s o l i d separated and was shown t o be potassium c h l o r i d e . Recovery of the r e a c t i o n product proved d i f f i c u l t and only 10.5 mg. of a c o l o u r l e s s l i q u i d was r e t r i e v e d . This product appeared to be a pure compound when examined by TLC. An NMR spectrum was run i n a micro c e l l and again only methyl group s i g n a l s were observed, at x 8.71 and : 78 9 .12 . However two weak o l e f i n i c p r o t o n peaks were a l s o present at T . 4 . 3 7 and 4.60 so t h a t a p p a r e n t l y d e h y d r o c h l o r i n a t i o n had o c c u r r e d t o some e x t e n t . Another s m a l l s c a l e c leavage r e a c t i o n was c a r r i e d o u t . u s i n g boron t r i b r o m i d e as the Lewis a c i d . This reagent was more convenient t o handle and the r e a c t i o n was a l l o w e d t o proceed f o r f o u r hours at room temperature . A f t e r f l u s h i n g the r e a c t i o n product through a s i l i c a g e l column a h i g h y i e l d o f a c o l o u r l e s s o i l was o b t a i n e d . The IR and NMR s p e c t r a l data on t h i s m a t e r i a l were e x a c t l y the same as observed on the boron t r i c h l o r i d e t r e a t e d sample. V a r i o u s attempts t o dehydrobrominate t h i s product were a l l unsuc-c e s s f u l and no o l e f i n i c . p r o t o n s were apparent i n the NMR s p e c t r a o f the r e a c t i o n p r o d u c t s . No f u r t h e r e t h e r c leavage experiments were attempted f o r a p e r i o d 30 of some months and then the p u b l i c a t i o n o f Uyeo's paper i n which he s u c c e s s f u l l y used 36 % HBr i n a c e t i c a c i d / a c e t i c anhydride s o l u t i o n to c leave the methyl e t h e r o f a t e t r a c y c l i c t r i t e r p e n e prompted us to t r y t o c leave the methoxyl group i n compound B acetate under these c o n d i t i o n s . A sample o f compound B a c e t a t e (312 mg.) was d i s s o l v e d i n a c e t i c anhydride (50 ml) and an equal volume g l a c i a l a c e t i c a c i d c o n t a i n i n g , 3 6 % HBr was added. The r e a c t i o n m i x t u r e was r e f l u x e d f o r one hour and a g l a s s y s o l i d was recovered (316 m g . ) . This product was shown,by TLC to be a m i x t u r e o f at l e a s t t h r e e compounds (R^ 0 . 7 4 , 0.53 and 0.23 w i t h the centre component b e i n g the main p r o d u c t ) . By column chromatography on h i g h a c t i v i t y a lumina these t h r e e f r a c t i o n s were r e a d i l y , s e p a r a t e d . The f i r s t two components were o b t a i n e d i n a pure s t a t e (55 mg. and 173 mg. r e s p e c t i v e l y ) but the t h i r d one (87 mg.) was contaminated w i t h c o l o u r e d i m p u r i t i e s . The t h r e e f r a c t i o n s were examined s e p a r a t e l y and s p e c t r a l data were o b t a i n e d . The f i r s t f r a c t i o n was' a syrupy l i q u i d which gave a m o l e c u l a r i o n 79 peak at m/e 4 0 6 - i n the mass spectrum i n d i c a t i n g t h a t i t might be t r i o l e f i n . An NMR spectrum showed the f o l l o w i n g s i g n a l s : - m u l t i p l e t s at T 4.55 and 4.62 which t o g e t h e r i n t e g r a t e d f o r only one proton^strong methyl group peaks at T 8.75 and i n the r e g i o n 9 . 0 2 - 9 . 1 8 . The l a c k o f o t h e r o l e f i n i c protons i n t h i s spectrum was p u z z l i n g and no l i k e l y s t r u c t u r e was ev ident from the d a t a . The second and l a r g e s t f r a c t i o n appeared t o c o n t a i n some c r y s t a l l i n e m a t e r i a l and when t r e a t e d w i t h a few drops o f c o l d e t h a n o l i t y i e l d e d 40 mg. o f whi te needle l i k e c r y s t a l s , m.p. 200-220°C. The m e l t i n g p o i n t c o u l d not be improved e i t h e r upon r e c r y s t a l l i z a t i o n o r vacuum s u b l i m a t i o n . The mother l i q u o r was a g l a s s y s o l i d which gave a m o l e c u l a r i o n peak at m/e 466 i n the mass spectrum and t h i s r e s u l t i n d i c a t e d t h a t t h i s m a t e r i a l may be a d i o l e f i n a c e t a t e . The NMR spectrum o f t h i s f r a c t i o n showed a m u l t i p l e t at x 4.60 corresponding t o one o l e f i n i c p r o t o n , a m u l t i p l e t at 5.30 f o r a p r o t o n geminal t o a c e t a t e and a s i n g l e t at 7.92 f o r an a c e t a t e methyl group. Weak s i g n a l s a t t r i b u t e d to two other a c e t a t e methyl groups at ,x 7.98 and 8.02 and a v e r y p o o r l y d e f i n e d methyl group r e g i o n i n d i c a t e d beyond,doubt t h a t t h i s f r a c t i o n was a m i x t u r e o f s e v e r a l compounds. The t h i r d and most p o l a r f r a c t i o n i s o l a t e d from the c leavage r e a c t i o n was aga in a g l a s s y s o l i d and showed a m o l e c u l a r i o n peak at m/e 526 i n the mass spectrum c o r r e s p o n d i n g to the expected v a l u e f o r a d i a c e t a t e . . The NMR spectrum showed s i g n a l s as f o l l o w s : - a m u l t i p l e t at T 4.60 f o r an o l e f i n i c p r o t o n , a m u l t i p l e t at 5.33 f o r two protons geminal to two a c e t a t e groups and a broad a c e t a t e methyl group s i g n a l at x 7 .95 . The c r y s t a l l i n e m a t e r i a l i s o l a t e d from the main f r a c t i o n a l s o gave a m o l e c u l a r i o n peak at m/e 466 i n the mass spectrum. The NMR spectrum o f t h i s substance was i d e n t i c a l to t h a t o f i t s mother l i q u o r a l r e a d y mentioned above except t h a t the methyl group s i g n a l was somewhat b e t t e r r e s o l v e d . 80 From more recent exper ience i n the chromatographic s e p a r a t i o n o f t r i t e r p e n e acetates o f the s e r r a t e n e t y p e , i t i s not d i f f i c u l t to understand t h a t these mono- and d i a c e t a t e f r a c t i o n s were complex m i x t u r e s . In f a c t the acetate d e r i v a t i v e s o f r e l a t e d d i o l s tend t o c o i n c i d e n on a TLC p l a t e whereas the parent d i o l s u s u a l l y separate very w e l l on s i l i c a g e l . I t was not c l e a r at a l l however i n t h i s c leavage experiment t o what extent double bond m i g r a t i o n t o the A 1 3 p o s i t i o n was t a k i n g p l a c e and whether the o l e f i n i c s i g n a l s observed were p a r t l y o r complete ly due t o e l i m i n a t i o n r e a c t i o n s o c c u r r i n g at the s i t e s o f both the methoxyl and the acetate f u n c t i o n s . The f i r s t compound prepared i n the A 1 3 ( i s o ) compound B s e r i e s was i s o l a t e d by a c c i d e n t i n another ;cleavage r e a c t i o n on compound B -acetate . The r e a c t i o n c o n d i t i o n s i n v o l v e d treatment o f t h i s compound f o r one and a h a l f hours w i t h 36 % H B r / a c e t i c a c i d reagent at 60°C but the a d d i t i o n o f the equal volume o f a c e t i c anhydride was o v e r l o o k e d . The white c r y s t a l l i n e ; m a t e r i a l i s o l a t e d as one o f the major components o f the r e a c t i o n product was r e c r y s t a l l i z e d from e t h a n o l (m.p. 222-235°C) and was c l e a r l y d i f f e r e n t from B acetate (m.p. 205-7°C). T h i s compound gave a n a l y t i c a l data i n accordance w i t h the e m p i r i c a l formula 033^1+03 and the mass spectrum had a m o l e c u l a r i o n peak at m/e 498. The IR spectrum c l e a r l y showed the presence o f acetate (1735 and 1255 cm" 1 ) and methoxyl (1104 c m - 1 ) f u n c t i o n s . That the methoxyl f u n c t i o n had not been removed was f u r t h e r conf irmed i n the NMR spectrum. There was however no s i g n a l at T 4.65 c o r r e s p o n d i n g t o an o l e f i n i c p r o t o n on a t r i s u b -s t i t u t e d double bond. The presence o f the secondary methoxyl group was i n d i -cated by the u s u a l s i n g l e t at T 6.70 and a m u l t i p l e t at 7.23 (1 H) f o r the p r o t o n adjacent t o the methoxyl group. The s i g n a l s a r i s i n g from the secondary acetate group were a l s o observed i n t h e i r normal l o c a t i o n s (T 7 .95 , '3 H) s i n g l e t and 5.33 (1 H m u l t i p l e t ) . T h i s evidence i s c o n s i s t e n t w i t h the A 1 1 + 81 double bond h a v i n g migrated t o the A 1 3 t e t r a s u b s t i t u t e d p o s i t i o n . I t was concluded from the f o r e g o i n g r e s u l t s t h a t 36 % H B r / a c e t i c a c i d reagent was p r o b a b l y too d r a s t i c f o r the cleavage o f compounds o f t h i s type c o n t a i n i n g the A 1 1 + double bond. The evidence i n d i c a t e d a r a t h e r complex m i x t u r e o f . p r o d u c t s some o f which may a r i s e through m o l e c u l a r rearrangements as a r e s u l t o f a c i d c a t a l y s e d r e a c t i o n s presumably i n v o l v i n g carbonium i o n i n t e r m e d i a t e s . A d i f f e r e n t approach to the problem was suggested by Dr . Rowe i n which an S N 2 d isplacement r e a c t i o n by methoxide i o n on a 33, 21a d i t o s y l d e r i v a t i v e o f s e r r a t e n e d i o l LXXII was e n v i s a g e d . A s u c c e s s f u l c o n v e r s i o n would y i e l d the d i m e t h y l e t h e r d e r i v a t i v e o f 3a , 2 1 3 - A 1 ^ - s e r r a t e n e d i o l L X X I I I i n one s t e p . The b a s i s f o r t h i s p r o p o s a l was some work c a r r i e d out on LXXII L X X I I I s t e r o i d systems by Nace and by Henbest arid h i s a s s o c i a t e s . The former worker had shown t h a t the methanolys is o f 5 a - c h o l e s t a n - 3 8 - y l t o s y l a t e LXXIV u s i n g dry methanol y i e l d s 25 % o f the e l i m i n a t i o n product LXXV and 75 % o f the e t h e r d e r i v a t i v e LXXVI i n which the c o n f i g u r a t i o n o f the s u b s t i t u e n t at the 3 p o s i t i o n has been i n v e r t e d . The Henbest group conf irmed t h i s r e s u l t and showed t h a t when a 5 - h y d r o x y l group i s p r e s e n t i n the system the y i e l d o f the corresponding 82 LXXIV LXXV LXXVI o l e f i n i s 30 % and o f the e t h e r o n l y 29 %. No example was g i v e n by e i t h e r group i n which a geminal 4 , 4 - d i m e t h y l system was present but i t c o u l d be a n t i c i p a t e d t h a t such a system would not be f a v o r a b l e t o S N 2 d isplacement s i n c e C3 i s e f f e c t i v e l y , a neopenty l p o s i t i o n . Al though low y i e l d s might be expected i n such a r e a c t i o n i t was decided t o i n v e s t i g a t e t h i s p o s s i b i l i t y s i n c e i t would a l l o w d i r e c t c o r r e l a t i o n w i t h the known s e r r a t e n e d i o l f a m i l y . From p r e v i o u s exper ience w i t h a l k a l o i d s i n D r . K u t n e y ' s group, i t was known t h a t mesylates are e a s i e r to prepare than the corresponding t o s y l a t e s . Moreover, i n the present i n s t a n c e , the presence o f the two adjacent geminal d i m e t h y l f u n c t i o n s i n the system gave r i s e to concern t h a t the d i t o s y l a t e might be d i f f i c u l t t o prepare because o f the s t e r i c h i n d r a n c e t h a t these groups would c r e a t e . I t t h e r e f o r e appeared more l i k e l y t h a t the s m a l l e r mesyl groups c o u l d be i n t r o d u c e d w i t h l e s s d i f f i c u l t y and t h e i r a b i l i t y t o undergo displacement r e a c t i o n s ought to be analogous t o the corresponding t o s y l a t e s . S e r r a t e n e d i o l d imesy la te was prepared by the method i n which the d i o l and methanesul fonyl c h l o r i d e were a l l o w e d t o r e a c t i n p y r i d i n e s o l u t i o n at -15°C f o r t h r e e h o u r s . The i s o l a t e d d imesy la te was examined by TLC, and was found t o be almost p u r e . An NMR spectrum o f t h i s compound showed o v e r l a p p i n g m u l t i p l e t s i n the r e g i o n T 5 . 5 0 - 5 . 8 5 corresponding t o two protons geminal t o mesylate groups. A sharp s p i k e at x 7.00 was a t t r i b u t e d t o methyl groups 83 from two mesylate f u n c t i o n s and the o t h e r s i g n a l s present were as expected f o r one o l e f i n i c p r o t o n and seven methyl groups. Before a t t e m p t i n g the methanolys is r e a c t i o n on the d imesy late i t was necessary t o prepare the methyl e t h e r d e r i v a t i v e o f compound B and t h i s was accomplished by the method a l r e a d y d e s c r i b e d f o r the m e t h y l a t i o n o f 21-e p i s e r r a t e n e d i o l . Thus 278 mg. o f compound B a f t e r one m e t h y l a t i o n and chromatographic s e p a r a t i o n y i e l d e d 128 mg. o f pure methyl e t h e r and 139 mg. o f unreacted a l c o h o l . This white c r y s t a l l i n e s o l i d , m.p. 243-5°C, [a] 2 ) 0 -76.4 (CHCI3) gave a n a l y t i c a l data i n agreement w i t h the e m p i r i c a l formula C'32^5|+<-)2 a n d the m o l e c u l a r i o n peak was measured at m/e 470 i n the mass spectrum. The NMR spectrum showed s i g n a l s as f o l l o w s : a s i x p r o t o n s i n g l e t at T 6.70 f o r the two methoxyl groups, o v e r l a p p i n g m u l t i p l e t s i n the r e g i o n x 7.16-7.23 f o r two protons geminal t o a x i a l methoxyl groups and the u s u a l s i g n a l s f o r methyl groups and o n e . o l e f i n i c p r o t o n . That the o v e r l a p p i n g m u l t i p l e t s were i n f a c t t r i p l e t s was conf irmed by r u n n i n g an NMR spectrum, at 100 Mc/s at 50°C. Both t r i p l e t s had c o u p l i n g constants J = 3.0 c . p . s . and the centres o f the two t r i p l e t s were separated from each o t h e r by 6.5 c . p . s . The mass spectrum o f t h i s compound showed s i g n i f i c a n t fragments at m/e 316, 284, 234, 221, 190, 189, 167 and 135. The methanolys is o f s e r r a t e n e d i o l d imesy la te was conducted under an i n e r t , anhydrous atmosphere.and i n r e f l u x i n g methanol f o r two days. The product was separated i n t o t h r e e f r a c t i o n s by column chromatography and examinat ion by TLC on a s i l i c a . p l a t e showed the complete absence o f any compound analogous to compound B methyl e t h e r . Examinat ion o f a l l t h r e e f r a c t i o n s by IR and NMR spectroscopy i n d i c a t e d t h a t they were mixtures o f compounds but no s i g n o f any s i g n a l s i n d i c a t i n g the presence o f methoxyl groups c o u l d be d e t e c t e d . Mass s p e c t r a were a l s o measured on the three 84 f r a c t i o n s and i t appeared that the most p o l a r product was s u b s t a n t i a l l y unreacted dimesylate, the middle f r a c t i o n ( a c t u a l l y two d i s t i n c t spots on a TLC p l a t e ) a mixture of monomesylates i n which one methanesulfonyl group had been e l i m i n a t e d from e i t h e r the C3 or C 2 i p o s i t i o n s , and the l e a s t p o l a r f r a c t i o n a t r i o l e f i n formed by e l i m i n a t i o n o f both methanesulfonyl groups. As already mentioned b r i e f l y above i t does not seem s u r p r i s i n g that t h i s r e a c t i o n d i d not y i e l d the d e s i r e d dimethyl ether. The presence of the func t i o n s must c e r t a i n l y i n t e r f e r e w i t h attack by any,incoming methoxide anion approaching from the d i r e c t i o n necessary to cause SN 2 displacement t o occur as i n d i c a t e d i n the p a r t i a l s t r u c t u r e LXXVII. Moreover the s u b s t i t u e n t on the C3 p o s i t i o n i s analogous,to a halogen.atom i n a neopentyl h a l i d e which, as i s very w e l l known, does not undergo the SN 2 type displacement r e a c t i o n An a l t e r n a t i v e approach to LXXVII the c o r r e l a t i o n of Compound B with OCM, the s e r r a t e n e d i o l system was to methylate the d i a x i a l a l c o h o l , d i e p i s e r r a -t e n e d i o l , and compare the 3-monomethyl ether. Although Dr. Rowe had reported 12 having i s o l a t e d t h i s compound from pine bark, i t was present i n very small traces and he could not provide us with a sample f o r methylation s t u d i e s . Instead he suggested using the Meerwein-Ponndorf r e d u c t i o n procedure on serratenedione of which he was able to send us a generous sample (250 mg.). Although normally r e d u c t i o n of c y c l i c ketones by metal hydrides y i e l d s predominantly the e q u a t o r i a l a l c o h o l s , there was precedent f o r t h i s suggestion 76 i n the form of a paper by Huneck i n which oleanonic a c i d methyl e s t e r LXXVIII was reduced i n 55 % y i e l d t o the corresponding a x i a l a l c o h o l by the Meerwein-85 Ponndorf t e c h n i q u e . To t e s t the y i e l d of* a x i a l a l c o h o l formed i n our system; we f i r s t a p p l i e d the Meerwein-Ponndorf r e d u c t i o n procedure t o compound B ketone and a f t e r a chromatographic s e p a r a t i o n on d e a c t i v a t e d a lumina were encouraged by the i s o l a t i o n o f a 45 % y i e l d o f compound B and a 30 % y i e l d o f an u n f a m i l i a r and more p o l a r compound which was assumed t o be the corresponding C-21 epimer. T h i s substance , a f t e r p u r i f i c a t i o n by vacuum s u b l i m a t i o n , had m.p. 271.5-272°C whereas the recovered compound B had m.p. 280-281°C a f t e r s i m i l a r p u r i f i c a t i o n . The IR spectrum o f the e p i m e r i c compound showed a s t r o n g 0-H s t r e t c h i n g a b s o r p t i o n at 3450 c m - 1 i n c o n t r a s t t o compound B i n which t h i s a b s o r p t i o n band i s very weak. The u s u a l a b s o r p t i o n bands were observed f o r the methoxyl group (1100 c m - 1 ) and the t r i s u b s t i t u t e d double bond (795 cm * ) . The mass spectrum had a m o l e c u l a r i o n peak at m/e 456 and the u s u a l r e t r o D i e l s - A l d e r fragments (m/e 316, 286, 269) and fragments a r i s i n g from cleavage a l l y l i c t o the double bond (m/e 221, 220, 190, 189) were observed. The NMR spectrum o f t h i s compound at 100 Mc/s i n d i c a t e d the u s u a l s i g n a l s f o r the p r o t o n on a t r i s u b s t i t u t e d double bond (x 4 . 7 2 , 1 H ) , a methoxyl group (x 6 .74 , 3 H ) , a p r o t o n g e m i n a l . t o an a x i a l methoxyl group, (x 7 .26, 1 H , t r i p l e t , J = 2.5 c . p . s . ) and sharp s p i k e s f o r s a t u r a t e d methyl groups. The p r o t o n geminal t o the h y d r o x y l group i n t h i s spectrum was observed as a q u a r t e t c e n t r e d at x 6 . 8 0 . On t h i s b a s i s the c o n f i g u r a t i o n 86 of the hydroxyl group atC 21 was concluded to be equatorial. As the reduction had apparently proceeded so favourably in the case of B ketone, i t was now applied on a small scale to serratenedione (55 mg.). When examined on a s i l ica gel TLC plate, developed in ethyl acetate/benzene, the reduction product indicated the presence of at least three components (R^ 0.35, 0.45 and 0.59) and comparison with authentic standards on the same plate showed the slowest of these fractions to be similar in polarity to serratenediol and the middle one to 21-episerratenediol. By the same token no sign could be seen of the presence of unreacted serratenedione or of ketols arising by partial reduction. Separation of the mixture by column chromatography on deactivated alumina was successful in yielding TLC pure fractions of a l l three components as well as a mixed fraction of the two more polar compounds. The most plentiful compound was the leastpolar material of which a 19 mg. sample had been prepared (yield 35 %) and this, being the only product not identifiable by TLC was thought to be the desired diaxial A 1 4 diol. Unfortunately this fraction had a wide melting range (183-193°) indicating that it was probably impure. Moreover it was discovered that, when a TLC plate spotted with this fraction was developed three times in ethyl acetate/benzene solvent and then sprayed, the original "spot" was actually resolved into two spots indicating the presence of.two components of very similar polarity but with the faster running spot being two to three times the more intense. The reason for the contamination of the desired diaxial alcohol fraction was not at first clear and i t was considered ' that in fact the fourth possible reduction product of serratenedione, namely 3-episerratene-diol, might possess similar TLC properties and be almost inseparable from the desired product. This supposition was eliminated when NMR spectra were 87 determined at 100 Mc/s on the two most n o n - p o l a r f r a c t i o n s i s o l a t e d from the crude r e d u c t i o n p r o d u c t . The major p r o d u c t , b e l i e v e d t o c o n t a i n the d i a x i a l a l c o h o l , d i d indeed i n d i c a t e a m u l t i p l e t c e n t r e d at T 6.65 shown upon m a g n i f i c a t i o n t o be due t o a p a i r o f o v e r l a p p i n g t r i p l e t s (J = 3.0 c . p . s . ) . No s i g n was observed o f a q u a r t e t c e n t r e d at T 6.80 which would a r i s e from a p r o t o n geminal t o the e q u a t o r i a l h y d r o x y l group i n 3 - e p i s e r r a t e n e d i o l and so t h i s compound c o u l d not have been present i n t h i s f r a c t i o n to any s i g n i -f i c a n t e x t e n t . However the NMR evidence d i d suggest an a l t e r n a t i v e e x p l a n a -t i o n f o r the presence o f two components i n the d i a x i a l a l c o h o l f r a c t i o n . In f a c t the o l e f i n i c p r o t o n s i g n a l at x 4.72 was much too weak r e l a t i v e to the o t h e r l o w f i e l d p r o t o n s i g n a l at T 6.65 and i n t e g r a t i o n o f the spectrum showed t h a t we were p r o b a b l y d e a l i n g w i t h a m i x t u r e c o n t a i n i n g 30 % o f the d e s i r e d d i a x i a l A 1 4 d i o l and 70 % o f the c o r r e s p o n d i n g A 1 3 i somer . T h i s would t h e r e f o r e i n d i c a t e t h a t the f a s t e r r u n n i n g component observed on the TLC p l a t e was the u n d e s i r e d A 1 3 i somer . Examinat ion o f the second (middle) f r a c t i o n : i s o l a t e d from the r e -d u c t i o n product p r o v i d e d evidence i n agreement w i t h these c o n c l u s i o n s . The NMR spectrum o b t a i n e d from t h i s m a t e r i a l c o n t a i n e d both e q u a t o r i a l (x 6.60) and a x i a l (x 6.85) protons geminal t o a x i a l and e q u a t o r i a l h y d r o x y l groups r e s p e c t i v e l y . . However these s i g n a l s c o u l d not be p r o p e r l y r e s o l v e d and appeared t o a r i s e from the s u p e r p o s i t i o n o f d u p l i c a t e q u a r t e t a n d . t r i p l e t m u l t i p l e t s . Once aga in the o l e f i n i c p r o t o n s i g n a l at x 4.70 was too weak when compared w i t h the i n t e n s i t i e s o f the o ther l o w f i e l d protons and the i n t e g r a l va lues recorded c o u l d be e x p l a i n e d by p o s t u l a t i n g the presence o f the r e l a t e d A 1 3 compound. I t was a l s o b e l i e v e d t h a t t h i s f r a c t i o n was a m i x t u r e o f the two p o s s i b l e a x i a l , e q u a t o r i a l d i o l s , namely the 3- and 21-e p i s e r r a t e n e d i o l s . 88 When c a r r y i n g out the Meerwein-Ponndorf r e d u c t i o n s on compound B ketone and on s e r r a t e n e d i o n e , no care had been taken t o l i m i t the q u a n t i t y o f hydro-c h l o r i c a c i d used i n the decomposi t ion o f the complex formed between the aluminum i s o p r o p o x i d e and the r e d u c t i o n p r o d u c t . In both cases an excess o f a c i d was employed. C l e a r l y t h i s had caused double bond m i g r a t i o n t o occur i n the case o f s e r r a t e n e d i o n e but n o t , f o r some r e a s o n , i n the o r i g i n a l r e d u c t i o n o f B ketone. The Meerwein-Ponndorf r e d u c t i o n r e a c t i o n was now repeated on a sample (59 mg.) o f s e r r a t e n e d i o n e and the amount o f h y d r o c h l o r i c a c i d used t o decompose the complex w i t h aluminum i s o p r o p o x i d e was l i m i t e d by employing a pH meter and thereby not p e r m i t t i n g the pH t o drop below 3 . 5 . R e p e t i t i o n o f the p r e v i o u s l y d e s c r i b e d chromatographic s e p a r a t i o n y i e l d e d 18 mg. o f the pure d i a x i a l a l c o h o l and a h i g h r e s o l u t i o n NMR spectrum on t h i s m a t e r i a l showed a s i g n a l at T 4.73 i n t e g r a t i n g f o r e x a c t l y one o l e f i n i c p r o t o n . T h i s substance was a whi te s o l i d which c o u l d be p u r i f i e d r e a d i l y by vacuum sub-l i m a t i o n and melted at 273-5°C. The mass spectrum i n d i c a t e d a m o l e c u l a r i o n peak at m/e 442 and the expected r e t r o D i e l s - A l d e r (m/e 302, 284, 269) and a l l y l i c c leavage fragments (m/e 220, 207, 190, 189) were a l l observed. R e p e t i t i o n o f the r e d u c t i o n on a f u r t h e r sample o f s e r r a t e n e d i o n e (116 mg.) y i e l d e d a f u r t h e r 44 mg. sample o f the d e s i r e d . d i a x i a l A 1 4 d i o l making a t o t a l o f 62 mg. a v a i l a b l e f o r m e t h y l a t i o n s t u d i e s . The NMR spectrum o f - t h e pure A 1 4 d i a x i a l a l c o h o l i s shown below i n F i g u r e 13. An examinat ion by TLC o f a t o t a l e t h e r s o l u b l e e x t r a c t o f S i t k a spruce bark p r o v i d e d by Dr . Hergert showed t h a t t h i s substance p r o b a b l y does not occur n a t u r a l l y i n the t r e e and t h i s was l a t e r confirmed when the e t h e r e x t r a c t e d t r i t e r p e n e f r a c t i o n was chromatographed on d e a c t i v a t e d a l u m i n a . U n f o r t u n a t e l y , before i t was r e a l i z e d t h a t the double bond i n the 89 d i a x i a l A 1 4 , a l c o h o l was b e i n g i s o m e r i z e d i n t o the A 1 3 p o s i t i o n by the excess h y d r o c h l o r i c a c i d , a second and r a t h e r l a r g e sample o f s e r r a t e n e d i o n e (200 mg.) was reduced by the Meerwein-Ponndorf method w i t h o u t p r e c a u t i o n s b e i n g F i g u r e 13. N . M . R . Spectrum (100 Mc/s) o f 3a-hydroxy-21B-hydroxy A l l 4 - s e r r a t e n e . taken t o use o n l y the p r o p e r amount o f a c i d t o decompose the aluminum complex. A f t e r chromatography on d e a c t i v a t e d a lumina t h i s r e a c t i o n y i e l d e d 92 mg. o f the d i a x i a l a l c o h o l which c o u l d be shown by TLC on a s i l i c a g e l p l a t e t o c o n t a i n r o u g h l y equal q u a n t i t i e s o f the A 1 3 and A 1 1 * i s o m e r s . The f i r s t two f r a c t i o n s recovered from the alumina column were found to c o n t a i n an almost pure sample o f the A 1 3 d i o l (31 m g . ) . The NMR spectrum o f t h i s f r a c t i o n taken at 100 Mc/s showed e s s e n t i a l l y no o l e f i n i c p r o t o n s i g n a l at x 4.65 and a , p a i r o f o v e r l a p p i n g t r i p l e t s c e n t r e d at x 6.64 as a l r e a d y observed i n the case o f i t s A 1 4 i somer . 90 The m e t h y l a t i o n technique p r e v i o u s l y u t i l i s e d i n the p r e p a r a t i o n o f compounds A and B methyl e thers and i n the s y n t h e s i s o f the c o r r e s p o n d i n g d e r i v a t i v e s o f 2 1 - e p i s e r r a t e n e d i o l was now a p p l i e d t o a sample (31 mg.) o f the s y n t h e s i z e d 3a, 2 1 3 - d i h y d r o x y - A 1 1 + - s e r r a t e n e . As b e f o r e ; the r e a c t i o n was conducted f o r two hours i n a n i t r o g e n atmosphere i n hot, dry to luene as s o l v e n t u s i n g a l a r g e excess o f potass ium metal (0.15 to 0.25 gm.) . The r e a c t i o n mixture was then a l l o w e d to c o o l , excess dry methyl i o d i d e (2 m l . ) was added and h e a t i n g and s t i r r i n g were, c o n t i n u e d f o r a f u r t h e r t h r e e h o u r s . The r e a c t i o n product was i s o l a t e d i n the u s u a l way and a f t e r two such m e t h y l a t i o n sequences, an examinat ion o f the product by T . L . C . r e v e a l e d , the presence o f a r a t h e r s m a l l q u a n t i t y o f two compounds h a v i n g p r o p e r t i e s s i m i l a r t o the compound B s t a n d a r d . In an e f f o r t to i n c r e a s e the y i e l d o f t h e s e , m a t e r i a l s , a t h i r d m e t h y l a t i o n r e a c t i o n was performed o n , t h e sample only t h i s t ime u n d e r , m o r e . d r a s t i c c o n d i t i o n s . Thus the r e a c t i o n w i t h potass ium metal was a l l o w e d t o proceed f o r f o u r hours i n to luene under r e f l u x and t h e , f i n a l r e a c t i o n w i t h methyl i o d i d e was a l l o w e d f i v e hours f o r c o m p l e t i o n . The product i s o l a t e d from t h i s r e a c t i o n was a y e l l o w o i l (50 m g . ) . S e p a r a t i o n by column chormatography on d e a c t i v a t e d a lumina y i e l d e d main ly a c o l o u r l e s s o i l (28 mg.) and t h r e e s m a l l f r a c t i o n s c o n t a i n i n g t r a c e s o f two i n c o m p l e t e l y separated monomethyl e t h e r s . The f a s t e r r u n n i n g o f the two monomethyl e thers showed T . L . C . behaviour analogous to t h a t o f compound B (R^ 0 . 4 3 , CHC]^). S u b l i m a t i o n o f the p u r e s t f r a c t i o n o f t h i s compound gave m a t e r i a l w i t h a wide m e l t i n g range (200-240°C). The c o l o u r l e s s o i l o b t a i n e d as the major product i n the m e t h y l a t i o n r e a c t i o n was d i s t i l l e d i n vacuo l e a v i n g a s m a l l amount o f white s o l i d which was subl imed t w i c e i n the u s u a l way. This m a t e r i a l was a l s o impure but had T . L . C . p r o p e r t i e s analogous to those o f a s t a n d a r d sample o f compound B 91 methyl e t h e r ( R f 0 , 8 4 , C H C I 3 ) . The o i l y d i s t i l l a t e was s t u d i e d by NMR spectroscopy and showed a s t r o n g s i g n a l i n the aromatic p r o t o n r e g i o n . There was a l s o a s i g n a l w i t h a chemical s h i f t c o r r e s p o n d i n g to t h a t o f an aromat ic methyl group. The o i l c o n t a i n e d at l e a s t t h r e e components as seen when s p o t t e d on a s i l v e r n i t r a t e impregnated s i l i c a t h i n l a y e r p l a t e and developed i n petro leum e t h e r . The nature o f the r e a c t i o n g i v i n g r i s e t o such a product was not understood. In a second experiment the r e m a i n i n g d i a x i a l A 1 4 a l c o h o l (30 mg.) was methylated u s i n g i n t h i s case r e f l u x i n g benzene, p r e v i o u s l y d r i e d by d i s t i l l a t i o n . f r o m l i t h i u m aluminum h y d r i d e . Two m e t h y l a t i o n s were performed as before u s i n g s i m i l a r q u a n t i t i e s o f potass ium metal and methyl i o d i d e . At t h i s stage the product c o n t a i n e d l a r g e l y unreacted s t a r t i n g m a t e r i a l p l u s t r a c e amounts o f the two monomethyl e thers but no d i m e t h y l e t h e r . A t h i r d m e t h y l a t i o n was conducted i n p r e c i s e l y the same manner as the f i r s t two except t h a t the r a t e o f s t i r r i n g w a s , i n c r e a s e d c o n s i d e r a b l y . A f t e r t h i s r e a c t i o n , .an examinat ion o f the o i l y product by T . L . C . showed t h a t a l l the s t a r t i n g m a t e r i a l had been u t i l s e d but , once a g a i n , t h e main f r a c t i o n i s o l a t e d was the " a r o m a t i c " o i l w i t h o n l y t r a c e amounts o f the two monomethyl e thers b e i n g p r e s e n t . I t was decided to abandon the m e t h y l a t i o n approach to the problem because o f l a c k o f s u f f i c i e n t l y l a r g e q u a n t i t i e s o f the d i a x i a l A 1 4 d i o l t o permit i s o l a t i o n o f the d e s i r e d methyl e t h e r d e r i v a t i v e s . C o n s i d e r a t i o n o f the s t a t u s o f the compound B s t r u c t u r a l problem at t h i s s tage. f o r c e d a r e t u r n to the e t h e r c leavage r e a c t i o n approach as b e i n g the o n l y obvious,method l i k e l y to y i e l d compounds o f known s t r u c t u r e which might be c o r r e l a t e d w i t h a u t h e n t i c samples. I t was decided to examine the use o f boron t r i f l u o r i d e e t h e r a t e . r e a g e n t at low temperature on compound B a c e t a t e . A c c o r d i n g l y a sample o f compound B a c e t a t e (240 m g . ) , d i s s o l v e d 92 i n a mixture o f e t h e r and a c e t i c a n h y d r i d e , was t r e a t e d w i t h boron t r i -f l u o r i d e e t h e r a t e at 0°C f o r f o r t y f i v e minutes . The r e a c t i o n was t e r m i n a t e d by p o u r i n g the m i x t u r e on to crushed i c e even though a l a r g e p r o p o r t i o n o f the o r i g i n a l compound had not d i s s o l v e d • Workup o f the r e a c t i o n by the u s u a l procedure y i e l d e d a crude product which was then r e a c t e d w i t h an excess o f l i t h i u m aluminum h y d r i d e to remove the acetate . group. F i l t r a t i o n and e v a p o r a t i o n o f the s o l v e n t r e s u l t e d i n the recovery o f a m i x t u r e o f s e v e r a l a l c o h o l s (225 m g . ) . The l a t t e r m i x t u r e was then o x i d i s e d i n the u s u a l way w i t h chromic o x i d e / p y r i d i n e reagent and the crude ketone mix t u r e thus o b t a i n e d (224 mg.) was separated by column chromato-graphy on d e a c t i v a t e d a l u m i n a . The main products i s o l a t e d were compound B ketone and a s l i g h t l y l e s s p o l a r compound c o n s i d e r e d t o be i s o compound B ketone. A T . L . C . pure sample o f a s lower r u n n i n g r e a c t i o n product (20 mg. F r a c t i o n I) was a l s o i s o l a t e d . This f r a c t i o n d i d not have p r o p e r t i e s s i m i l a r t o those o f s e r r a t e n e d i o n e i n t h a t i t ran f a s t e r and gave a d i f f e r e n t c o l o u r e d spot when sprayed w i t h antimony p e n t a c h l o r i d e . Moreover, no t r a c e whatever o f any s e r r a t e n e d i o n e was detected i n any o f the f r a c t i o n s recovered from the chromatography. The i n f r a r e d spectrum o f the unknown m a t e r i a l i n . F r a c t i o n I i n d i c a t e d the presence o f h y d r o x y l (3400 c m - 1 ) , c a r b o n y l (1695) and the o r i g i n a l t r i s u b s t i t u t e d double bond (795). However the mass spectrum o f • the m a t e r i a l at onc'e i n d i c a t e d i t t o be a m i x t u r e o f s e v e r a l components. Thus peaks were observed at m/e 421, 422, .436, 438, 439, 440, 452, 464 and 470. The m o l e c u l a r i o n peak f o r s e r r a t e n e d i o n e occurs at m/e 438 so t h a t the presence o f peaks at h i g h e r mass c a n . o n l y mean ; the i n t r o d u c t i o n o f f u r t h e r oxygen i n t o the system. Thus, f o r example, the i n t r o d u c t i o n o f h y d r o x y l at a p o s i t i o n a l l y l i c to the 14, 15 double bond would r a i s e the m o l e c u l a r i o n peak to m/e 454.and i n t r o d u c t i o n o f a second 93 hydroxyl at the Alternative al ly l ic position in the molecule would account for the observed peak at m/e 470. The process would be further complicated, by the likelihood of elimination reactions occurring during the ether cleavage process. Clearly therefore this result established that ether cleavage reactions would not be likely to give interpretable results so long, as the original double bond in the molecule remained intact. For this reason it was decided to conduct further ether cleavage experiments in the dihydro series. The preparation of desoxy dihydro compound B has already been discussed. A sample of this material (370 mg.) was cleaved at.room, temperature for 20 hours using a mixture of almost equal proportions of acetic anhydride and 44% HBr in glacial acetic acid. The resulting brown semi-solid product which was recovered was separated by column chromatography on deactivated alumina into two fractions 1 and 2. Fraction 1 (85 mg.) was considered to be olefinic in nature because of its T . L . C . properties and lack of either acetate or methyl ether, bands in the infrared spectrum. The NMR data confirmed the absence of these groups and indicated the presence of rather weak olefinic proton multiplets at T 4.64 and,4.80. That this fraction was a mixture of several compounds was clear from T . L . C i . using silver nitrate impregnated s i l ica thin layer plates. Development with petroleum ether showed the presence of two spots. Further,investigation of the olefin fraction by VPC on an S.E. 30 silicone gum rubber loaded column (20% liquid phase) showed the situation to be even more complex and no less than four peaks were observed. No further work was performed on this fraction. Fraction 2 (137 mg.) was a white, low melting solid whose infrared spectrum clearly showed the presence of acetate (1730 and 1230) and the absence of a methoxy.group. The NMR spectrum of- this material showed 94 a one proton triplet centred at T 5.36 and a three proton singlet at T 7.92 confirming the presence of. an acetate group. Reduction of Fraction 2 was accomplished by reaction with excess lithium aluminum hydride in refluxing tetrahydrofuran. The isolated product was examined by T.L.C. (silica, CHC13) and showed the presence of three compounds (R^ 0.55, 0.48 and 0.28) with the latter being present as l i t t l e more than a trace. Separation of this mixture by column chromatography on deactivated alumina resulted in the recovery of a fraction containing a mixture of the first two compounds (50 mg.) and a second fraction (59 mg.) consisting essentially of only the middle component. A small fraction 8 mg.) containing the most polar component was also isolated. Further chromatographic separations on the two main fractions resulted in the isolation of a small pure sample (4 mg.) of the least polar alcohol (a) and the purification of the main fraction of the second alcohol (6). The alcohol 6 so isolated was a white crystalline solid, m.p. 219-221°C. The NMR spectrum (100 mc/s) showed the presence of a triplet centred at T 6.66 for the proton geminal to an axial secondary hydroxyl group and the presence of the hydroxyl group was also indicated in the infrared spectrum (3560). The. empirical formula, C30H52O, was established by means of high resolution mass measurements on the jmolecular ion peak. The fact that this compound is an axial alcohol is doubly significant. In the first place i t indicates retention of configuration during the ether cleavage reaction. Secondly i t makes very unlikely the possibility that skeletal rearrangement may have occurred during this process. A sample of the pure alcohol (50 mg.) was oxidised at room temperature with freshly prepared Jones reagent. The crude ketone was purified by column chromatography on deactivated alumina and was' obtained as 95 a white c r y s t a l l i n e s o l i d (44 m g . ) , m.p. 185-193°C w i t h o u t r e c r y s t a l l i z a t i o n . I n t e r e s t i n g evidence concerning the s i z e o f r i n g A , the l o c a t i o n o f the o r i g i n a l methoxyl group, and the n a t u r e o f the A/B r i n g j u n c t u r e was d e r i v e d through the study o f the s p e c t r a l c h a r a c t e r i s t i c s o f t h i s compoud. Thus the i n f r a r e d spectrum d i s p l a y e d a c a r b o n y l a b s o r p t i o n band (1695 cm~l) i n d i c a t i n g the l o c a t i o n o f t h i s chromophore i n a r i n g o f s i x or more carbon atoms. The ORD curve showed a p o s i t i v e C o t t o n e f f e c t t y p i c a l f o r a C-3 ketone i n a t r i t e r p e n e h a v i n g a t r a n s A/B r i n g j u n c t u r e as 58 d i s c u s s e d by D j e r a s s i . F u r t h e r support f o r t h i s c o n c l u s i o n arose from a study o f the quenching o f the C o t t o n e f f e c t a f t e r the a d d i t i o n o f h y d r o -c h l o r i c a c i d t o the methanol s o l u t i o n o f the k e t o n e . In f a c t the k e t a l i -z a t i o n o f the c a r b o n y l was n e g l i g i b l e and i n d i c a t e d the presence o f a s t e r i c a l l y h i n d e r e d ketone f u n c t i o n as expected f o r a C-3 c a r b o n y l v i c i n a l t o a 4;4 geminal d i m e t h y l g r o u p i n g . The ampli tude o f the Cot ton e f f e c t curve was +53.7, perhaps somewhat h i g h e r than might have been expected from p r e v i o u s l y d i s c u s s e d measurements such as the +30.0 v a l u e quoted by 39 i L Inubushi f o r the u n s a t u r a t e d compound, 3 k e t o - A 1 - s e r r a t e n e L or f o r o t h e r 47 48 t r i t e r p e n e C-3 ketones . ' The NMR spectrum o f t h i s compound r e v e a l e d only the absence o f protons a b s o r b i n g at low f i e l d . A m u l t i p l e t c e n t r e d at x 7.57 was however a t t r i b u t a b l e to the C 2 protons b e i n g d e s h i e l d e d by the v i c i n a l c a r b o n y l group. The e m p i r i c a l . f o r m u l a , C30H50O, o f ketone 3 was e s t a b l i s h e d by a h i g h r e s o l u t i o n mass measurement on the m o l e c u l a r i o n peak. The removal o f the c a r b o n y l grouping i n the s a t u r a t e d ketone was accomplished by means o f the t o s y l h y d r a z o n e route used a l r e a d y so many times i n t h i s s tudy . Thus the tosy lhydrazone d e r i v a t i v e was r e a d i l y prepared by r e a c t i o n of the ketone w i t h p - t o l u e n e s u l f o n h y d r a z i d e reagent i n r e f l u x i n g 96 methanol w i t h a c e t y l c h l o r i d e as c a t a l y s t . The white c r y s t a l l i n e t o s y l -hydrazone, m.p. 202-4°C, o b t a i n e d from t h i s r e a c t i o n showed i n the i n f r a r e d spectrum the presence o f the u s u a l t y p i c a l a b s o r p t i o n bands. Reduct ion o f the t o s y l h y d r a z o n e was c a r r i e d out i n r e f l u x i n g s p e c t r a l grade dioxane u s i n g a l a r g e excess o f sodium b o r o h y d r i d e (from a f r e s h l y opened b o t t l e ) . An e x a m i n i a t i o n o f the r e d u c t i o n product by T . L . C . ( s i l i c a , petro leum ether) showed the presence o f a n o n - p o l a r compound (R^ 0 . 7 0 , g inger) and almost no unreacted t o s y l h y d r a z o n e . The pure s a t u r a t e d hydrocarbon was i s o l a t e d by column chromatography on n e u t r a l ' a l u m i n a . The white c r y s t a l l i n e s o l i d (8 mg.) so recovered was r e C r y s t a l l i z e d from s p e c t r a l grade acetone and gave f e r n l i k e c r y s t a l s , m.p. 184-6°C. This m a t e r i a l was compared w i t h an a u t h e n t i c sample o f g - s e r r a t a n e , m.p. 184-7°C, p r o v i d e d by P r o f e s s o r I n u b u s h i . The mixed m e l t i n g p o i n t was 184-6°C and the compounds showed s i m i l a r behaviour on both s i l i c a and a lumina t h i n l a y e r p l a t e s developed i n petroleum e t h e r . The e m p i r i c a l formula o f the hydrocarbon was e s t a b l i s h e d as C30H52 by means o f h i g h r e s o l u t i o n mass measurements on the m o l e c u l a r i o n peak and the mass s p e c t r a l f ragmentat ion p a t t e r n o f the hydrocarbon was very s i m i l a r t o t h a t o f the a u t h e n t i c sample o f g - s e r r a t a n e . Having now at l a s t o b t a i n e d a known compound v i a e t h e r cleavage i n the compound B s e r i e s i t seemed a p p r o p r i a t e t o study the cleavage r e a c t i o n , i n more d e t a i l , p a r t l y w i t h a view t o examining the o l e f i n i c f r a c t i o n more t h o r o u g h l y and p a r t l y t o o b t a i n l a r g e r q u a n t i t i e s o f the v a r i o u s compounds o b t a i n e d i n t h i s r e a c t i o n . Desoxy compound B was c o n s i d e r e d t o be a s u i t a b l e s t a r t i n g p o i n t i n p r e p a r i n g more desoxy d i h y d r o compound B but the c o n d i t i o n s f o r the hydrogenat ion o f t h i s m a t e r i a l were unknown. P r e l i m i n a r y attempts t o hydrogenate desoxy compound B a t atmospheric pressure u s i n g Adam's c a t a l y s t were u n s u c c e s s f u l p r o b a b l y because t h i s 97 material was very insoluble in glacial acetic acid. The reaction could not be made to proceed in mixtures of acetic acid and tetrahydrofuran (THF). Attempts to conduct the reaction in a Parr hydrogenator in mixtures of THF and acetic acid were also unsuccessful even at a temperature of 60°C. However the hydrogenation did proceed in good yield when a solvent mixture of equal volumes of acetic acid and chloroform was used. The product recovered from the hydrogenation was purified by column chromatography on alumina followed by crystallization from n-heptane^m.p. 195-8°C^white prisms. This material was later shown by VPC analysis to be almost a pure form of the dihydro compound. The other form, generated by the formation of a new asymmetric centre at C 14 during the hydrogenation, remained mainly in the mother liquor. The ether cleavage reaction was again performed on a sample (590 mg.) of the dihydro desoxy compound. On this occasion a mixture of equal volumes of acetic anhydride and 36% HBr in glacial acetic acid was used as the reagent. Isolation of the product in the usual way after allowing the reaction to proceed at room temperature for twenty hours showed that in fact no cleavage at a l l had occurred. The experiment was therefore repeated for 2 1/2 hours in a water bath at a temperature in the range 70-80°C. The product from this reaction was examined on a s i l ica coated thin layer plate (CHCI3) and two fractions (R^ 0.80 and 0.49) were seen to be present. Separation of these fractions was accomplished by column chromatography on alumina giving rise to a semi-crystalline olefin mixture (457 mg., 84% yield), a fraction (34 mg.) containing a mixture of olefin and unreacted starting material, and an impure fraction, containing an ,acetate group (97 mg., 15% yield). No further Work was performed on the latter two fractions. The olefin fraction as isolated from the column showed a single 98 spot when examined on a s i l ica thin layer plate developed in chloroform. Use of a silver nitrate impregnated s i l ica thin layer plate in the same solvent system resolved the mixture into two spots which ran quite close together. However when the olefin mixture was spotted on a.silver nitrate/sil ica plate developed in petroleum ether we were considerably dismayed to see no less than six spots. (Rf 0.67, 0.61, 0.55, 0.47, 0.42 and 0.33). The separation so achieved was however reproducible on a column of silver nitrate impregnated s i l ica (20% Ag NO3) using petroleum:ether as the eluting solvent. Thus in thirteen fractions (25 c c . ) a l l the material applied was success-fully recovered with the separation illustrated in Table 6. ,, TABLE 6 Separation of Olefin Mixture on a  Silver Nitrate/Silica Column Fraction No. Weight mg. Rf of Components 3 18 0.69 4 121 0.69, 0.63 5 35 0.63> 0.58 6 8 0.51 7 ' 68 0.42 8 100 0.39 9 37 0.39 10 10 0.39 The material recovered in fractions 8 to 10 was a white crystalline substance. This material was separated again on a small silver nitrate/ s i l ica column and was then recrystallized several times from acetone. The 99 rodlike crystals so obtained melted at 188-191°C. The empirical formula, C30H50, was established for the olefin by.both elemental analysis and high resolution mass measurements. The infrared spectrum of this compound provided useful information as to its structure. Thus the presence of a weak band at 1650 cm - 1 was attributed to a non-conjugated double bond and two strong sharp signals at 730 and 720 cm-1 may be.assigned with some caution to the out of plane deformation modes of cis olefinic hydrogen atoms. This assignment is 80 supported however by a recent paper by Meakins et al on the preparation and spectra of steroid olefins. These workers indicated the presence of absorption bands at 730 and 713 cm - 1 in the infrared spectrum of the triterpene: lup-2-ene LXXIX. ,The NMR spectrum was also imformative. Thus a pair of olefinic proton signals of very similar chemical shift was observed in deuterochloroform soluti The signal at T 4.63 was apparently a singlet and the other at T 4.60 appeared to be a narrow triplet (J 1.0 c.p.s.). It was demonstrated LXXIX iii Jdecoupling experiments (Figure 14) that these olefinic protons are coupled to the pair of Cl a l ly l ic protons which :appear as multiplets at T 7 and 8.04.The spectrum was however top complex to allow a complete analysis. 80 Meakins et al have also commented on the N.M.R. spectrum oflup-2-ene and have indicated the presence of a pair of olefinic proton signals at x 4.56 and 4.59, the former being only half as high as the latter. These authors commented on the surprisingly weak coupling constants observed in the A 2 compounds and speculated on the simplicity of the spectra having a 100 c o m p l i c a t e d o r i g i n . - L 4 - 5 7 1 F i g u r e 14. Decoupl ing Experiments on A 2 - g - s e r r a t a n e i n benzene. The i d e n t i t y o f the above o l e f i n was proven by hydrogenat ion t o be A 2 - 3 - s e r r a t a n e . Thus the o l e f i n (38 mg.) r e a d i l y absorbed hydrogen when r e a c t e d i n e t h y l a c e t a t e s o l u t i o n i n the presence o f Adam's c a t a l y s t . From t h i s r e a c t i o n t h e r e was i s o l a t e d a f t e r column chromatography on d e a c t i v a t e d a lumina a white c r y s t a l l i n e s o l i d (26 mg.) and r e c r y s t a l l i z a t i o n from acetone gave whi te n e e d l e s , m.p. 172-7°C. The m e l t i n g p o i n t o f t h i s m a t e r i a l was e l e v a t e d by vacuum s u b l i m a t i o n t o 181-4°C. The 8 - s e r r a t a n e p r e v i o u s l y i s o l a t e d from the displacement r e a c t i o n sequence had m.p. 182-5°C, mixed m.p. 181-4°C. Comparison o f these two samples on a q u a n t i t a t i v e b a s i s showed t h e i r i n f r a r e d s p e c t r a t o be superimposable and t h e i r T . L . C . p r o p e r t i e s ( s i l i c a , petro leum ether) were a l s o i d e n t i c a l . 101 I One further proof was desired of the identity of the two 3-serratane samples from the ether cleavage experiments. There was not sufficient material remaining of the original sample of B-serratane provided by Professor Inubushi to permit;a quantitative infrared spectral comparison with these 81 compounds. Fortuitously at this time a paper was published by Berti et al describing the isolation of serratene from the fern Polypodium vulgare. These authors kindly provided us with a sample (34 mg.) of this hydrocarbon which we hydrogenated at atmospheric pressure in acetic acid solution using Adam's catalyst. The white crystalline product (m.p. 157-160°C) was a mixture of the a and 6 forms of serratane and Inubushi, who separated these compounds, has reported the ratio as 20 to 1 in favour of the 6 form. We were unable to raise the melting point of the mixture by fractional crystallization from acetone or by vacuum sublimation. However use of the mixed solvent system ethyl acetate/methanol, permitted the recovery of some rieedlelike crystals; m.pi 180-184°C. The infrared spectrum of this material was compared on a,quantitative basis with the .corresponding spectra obtained, from the two ether cleavage reaction sequences. Al l three spectra were superimposable. On the basis of the experimental evidence which has been described to date we can now ascribe to compound B the structure 3a-methoxy-216-hydroxy-A1 k-serratene LXXX. 102 Before l e a v i n g the compound B s e r i e s t h e r e i s one f u r t h e r comment which i s worthy o f n o t e . This concerns the r a t h e r remarkable d i f f e r e n c e s i n T . L . C . p r o p e r t i e s between c o r r e s p o n d i n g d e r i v a t i v e s i n the A and B s e r i e s as i l l u s t r a t e d i n Table 7. Thus f o r example one can note the marked d i f f e r e n c e i n R^ v a l u e between A and B acetates which d i f f e r o n l y i n the c o n f i g u r a t i o n o f the C 3 methoxyl group. A g a i n s t t h i s one a l s o notes the i n a b i l i t y o f the t h i n l a y e r technique .to d i s t i n g u i s h between e q u a t o r i a l methoxyl and acetate f u n c t i o n s i n A a c e t a t e and 2 1 - e p i s e r r a t e n e d i o l d i a c e t a t e . Even more'remarkable: i s the i n a b i l i t y o f t h i n l a y e r t o separate the m i x t u r e o f acetates o f the compounds A , F, D and H i n which one i s a k e t o a c e t a t e , one a m e t h p x y : a c e t a t e and the o t h e r two are p r o b a b l y d i a c e t a t e s . A f u r t h e r example o f the d i s t i n c t i o n between the T . L . C . b e h a v i o u r , o f the A and B s e r i e s i s p r o v i d e d by the c o r r e s p o n d i n g desoxy compounds. , Thus the d i f f e r e n c e i n c o n f i g u r a t i o n o f the C 3 methoxyl group appears to. have a r a t h e r profound and unexpectedly l a r g e i n f l u e n c e on the T . L . C . p r o p e r t i e s o f these compounds. TABLE 7 Inf luence o f F u n c t i o n a l Group C o n f i g u r a t i o n on T . L . C . P r o p e r t i e s Compound R^ va lue A acetate 0.24 2 1 - e p i s e r r a t e n e d i o l d i a c e t a t e 0.24 0 24 Mixed acetates (A, F , D, H) B a c e t a t e 0.47 C a c e t a t e 0.47 Desoxy A 0.61 Desoxy B 0.81 103 The recovery of a brown resinous neutral extract Z. from Sitka spruce bark was mentioned early in this section. The separation of this mixture into crude fractions was accomplished by means of column chromatography on Activity III alumina using mixtures of the solvents petroleum ether, benzene and ether. This work is described in detail in the experimental section. Al l the fractions contained yellow oily residues consisting of fatty acid esters of phytosterols and (presumably) glycerol. The phytosterol esters were apparently less polar than the fats and tended to be removed with the earlier fractions. Among the new compounds isolated and not previously discussed were the triterpene C and the compounds X and P-which were of a different type. Compound C, as has earier been mentioned, was a minor component of the original triterpene extract Y. However, because of its similar value to compound B, with which i t co-occurs, this compound was most difficult to separate in a pure form. Fortunately the compound has a greater solubility in non-polar organic solvents than has B-and so extract Z was found to be considerably more rich in this substance than extract Y. A fraction (520 mg.) of chromatographycally pure C was for the first time obtained during a column chromatographic separation of the crude acetone soluble extract (including acids and phenolic compounds) of Sitka spruce bark. Recrystallization of this material from ethanol gave white needles, m.p. 179-182°C. Further recrystallization from acetone gave material, m.p. 180-185°C, which obviously was s t i l l impure, The infrared spectrum of this compound showed the presence of methoxyl (1100 cm-l) and hydroxyl (3400) substituents and the absence of the usual trisubstituted double bond. This conclusion was supported by an examination of the NMR spectrum which disclosed the absence of the usual olefinic proton signal at T 4.65. Moreover the one 104 p r o t o n m u l t i p l e t at x 7.22 and the sharp three p r o t o n s i n g l e t at x 6.67 were o b v i o u s l y caused by the presence o f a secondary methoxyl group. A one p r o t o n m u l t i p l e t a t T 6.54 was a t t r i b u t e d t o the p r o t o n geminal t o a secondary h y d r o x y l group. The nature o f the m u l t i p l e t s at x 6.54 and 7.22 was f u r t h e r e x p l o r e d when another NMR spectrum was o b t a i n e d a t 100 Mc/s . Both m u l t i p l e t s were t r i p l e t s (J = 2.5 c . p . s . ) i n d i c a t i n g t h a t t h i s compound possesses a x i a l methoxyl and h y d r o x y l f u n c t i o n s . The mass spectrum o f compound C. showed the m o l e c u l a r weight t o be 456 and t h i s compound i s t h e r e -f o r e i s o m e r i c w i t h compounds A and B. This c o n c l u s i o n was conf irmed when h i g h r e s o l u t i o n mass measurements were made on the m o l e c u l a r i o n and compound C was shown t o have the e m p i r i c a l , f o r m u l a C31H52O2. The most reasonable e x p l a n a t i o n o f these r e s u l t s would appear t o be t h a t t h i s compound i s d e r i v e d from compound B by m i g r a t i o n o f the double bond from the A 1 4 i n t o the t e t r a s u b s t i t u t e d A 1 3 p o s i t i o n . I f t h i s be so then the double bond s h o u l d remain i n t a c t when a m i x t u r e c o n t a i n i n g compound B and C i s hydrogenated i n g l a c i a l a c e t i c a c i d u s i n g Adam's c a t a l y s t . This r e a c t i o n was i n f a c t performed on such a m i x t u r e and the d i h y d r o compound B, which has a somewhat lower R^ va lue than compound B i t s e l f , was now f a i r l y r e a d i l y removed from compound C by column chromatography on d e a c t i v a t e d a l u m i n a . The object o f t h i s experiment was to i s o l a t e a pure sample o f compound C h a v i n g a sharp m e l t i n g p o i n t . In t h i s we were d i s a p p o i n t e d as the bes t sample, a f t e r r e c r y s t a l l i z a t i o n from n-heptane, had a m e l t i n g range.180-186°C and gave u n s a t i s f a c t o r y r e s u l t s from e lementa l a n a l y s i s as a l s o d i d the sample i s o l a t e d p r e v i o u s l y . A sample o f compound C was a c e t y l a t e d i n a m i x t u r e o f dry p y r i d i n e and a c e t i c anhydride at room temperature as i n the p r e p a r a t i o n o f B acetate." The a c e t a t e was p u r i f i e d by f l u s h i n g through a s h o r t column o f a lumina and 105 by s e v e r a l r e c r y s t a l l i z a t i o n s from petro leum e t h e r . C o l o u r l e s s r o d l i k e c r y s t a l s , m.p. 222-225°C were thus o b t a i n e d . The NMR spectrum o f t h i s compound showed a one p r o t o n m u l t i p l e t at T 5.31 and-a t h r e e p r o t o n s i n g l e t at T 7.95 c o r r e s p o n d i n g to a secondary a c e t a t e group and the p o s i t i o n s o f the s i g n a l s a s s o c i a t e d w i t h the methoxyl group remained v i r t u a l l y unchanged. The mass spectrum e x h i b i t e d a m o l e c u l a r i o n peak at m/e 498 and an i n t e n s e fragment at m/e 438 c o r r e s p o n d i n g to the l o s s o f a c e t i c a c i d , A q u a n t i t a t i v e comparison o f t h i s m a t e r i a l w i t h i s o compound B a c e t a t e from an e t h e r c leavage r e a c t i o n as p r e v i o u s l y d e s c r i b e d showed t h a t the i n f r a r e d s p e c t r a o f the two compounds were super imposable . The compounds were a l s o c o n s i d e r e d t o be i d e n t i c a l on the b a s i s o f t h e i r T . L . C , N . M . R . and mass s p e c t r a l p r o p e r t i e s . On the b a s i s o f the a v a i l a b l e evidence we suggest t h a t the most l i k e l y s t r u c t u r e f o r compound C i s t h a t o f 3 a - m e t h o x y - 2 1 $ - h y d r o x y - A 1 3 -s e r r a t e n e LXXXI. Because the compound was not i s o l a t e d i n a pure form and because the d i r e c t i s o m e r i z a t i o n o f compound B to C by treatment w i t h a c i d has not been performed we cannot c l a i m the s t r u c t u r e o f t h i s compound as c o n c l u s i v e l y p r o v e n . LXXXI Two o t h e r t r i t e r p e n e s were i s o l a t e d as minor components o f the o r i g i n a l e x t r a c t Y but i n n e i t h e r case c o u l d pure samples o f these m a t e r i a l s 106 be o b t a i n e d . Al though i n both cases the i s o l a t e d f r a c t i o n s showed o n l y one spot when examined on s i l i c a t h i n l a y e r p l a t e s , n e v e r t h e l e s s the wide m e l t i n g ranges and poor e lementa l a n a l y s i s r e s u l t s i n d i c a t e d t h a t the compounds were s t i l l impure. The f i r s t o f these mater ia ls ,compound D, m.p. 245-251°C, showed the presence o f the c a r b o n y l chromophore (1707 cm ) and a h y d r o x y l f u n c t i o n (3500) i n the i n f r a r e d spectrum. The NMR spectrum (100 Mc/s) showed the customary o l e f i n i c p r o t o n s i g n a l at T 4.70 i n d i c a t i n g the presence o f a j t r i s u b s t i t u t e d double bond. A l s o appearing i n the r e g i o n T 6 . 6 - 7 . 0 was a m u l t i p l e t c o r r e s p o n d i n g t o the p r o t o n geminal t o a h y d r o x y l f u n c t i o n but i t was i m p o s s i b l e to t e l l whether t h i s was a t r i p l e t o r .a q u a r t e t because t h e r e was a l s o present i n t h i s r e g i o n a sharp s i n g l e t at T 6;72 p o s s i b l y a r i s i n g from a methoxyl group a l though the l a t t e r was not seen i n the i n f r a r e d spectrum. The i n t e n s i t y o f the methoxyl s i g n a l wks q u i t e low however and suggested t h a t the i m p u r i t y o f the sample was due to the presence o f an unknown t r i t e r p e n e methyl e t h e r . The methyl group r e g i o n o f the NMR spectrum a l s o showed evidence o f the i m p u r i t y o f the specimen i n t h a t n i n e peaks o f a p p r o x i m a t e l y equal i n t e n s i t y were observed. F u r t h e r evidence as t o the l o c a t i o n o f the c a r b o n y l group was f o r t h -coming when the ORD curve o f the k e t o l was determined. T h i s e x h i b i t e d a n e g a t i v e Cotton e f f e c t w i t h trough and peak at 314 and 276 my t y p i c a l o f an i s o l a t e d c a r b o n y l chromophore i n a s a t u r a t e d environment. The measured 39 m o l e c u l a r ampli tude was - 2 6 . 2 . w h i c h , by analogy w i t h I n u b u s h i ' s r e s u l t s , and assuming t h a t the compound does have a s e r r a t e n e s k e l e t o n , would i n d i -cate the l o c a t i o n o f the chromophore at the C 21 p o s i t i o n . T h i s c o n c l u s i o n was a l s o supported by the mass s p e c t r a l f ragmentat ion p a t t e r n which showed the l o c a t i o n o f the pr imary a l l y l i c cleavage fragments at m/e 218 and 207. 107 By analogy w i t h the cleavage processes a l r e a d y d i s c u s s e d (see Table 4) t h i s i n d i c a t e s the l o c a t i o n o f the c a r b o n y l group at the C 21 p o s i t i o n as i n A or B ketones . Had the c a r b o n y l been present at C 3 the corresponding a l l y l i c c leavage fragements would have been observed at m/e 220 and 205. High r e s o l u t i o n mass measurements on the m o l e c u l a r i o n peak e s t a b l i s h e d the e m p i r i c a l formula C^oH^C^ f o r the k e t o l . U n f o r t u n a t e l y no d e f i n i t e evidence was o b t a i n e d to show t h a t compound D.has a s e r r a t e n e s k e l e t o n . A crude mother l i q u o r f r a c t i o n of , the m a t e r i a l was o x i d i s e d w i t h chromic oxide i n p y r i d i n e and an impure sample (36 mg.) o f a compound h a v i n g T . L . C . p r o p e r t i e s s i m i l a r t o an a u t h e n t i c sample o f A 1 4 s e r r a t e n e d i o n e was indeed i s o l a t e d by column chromatography. However t h i s compound may have been p a r t l y i s o m e r i s e d t o the A 1 3 analogue by contact w i t h h y d r o c h l o r i c a c i d d u r i n g the workup process and no d e f i n i t e statement may be made as to i t s i d e n t i t y . A c c o r d i n g l y we are content to s a y . t h a t compound D i s a t r i t e r p e n e k e t o l w i t h the e m p i r i c a l formula C 3 0 H 4 Q O 2 which may belong t o the s e r r a t e n e s e r i e s . To separate t h i s compound from i t s t r i t e r p e n e methyl e t h e r con-taminant f o r any subsequent i n v e s t i g a t i o n , i t would be bes t t o prepare the t o s y l h y d r a z o n e d e r i v a t i v e which c o u l d then be recovered undamaged by column chromatography on alumina o f s u f f i c i e n t l y low a c t i v i t y (perhaps a c t i v i t y I V ) . The pure t o s y l h y d r a z o n e c o u l d then be o x i d i s e d w i t h l e a d t e t r a a c e t a t e as 82 suggested by B h a t t i t o r e c o v e r the pure k e t o l . Al though t h i s a u t h o r ' s examples d i d not i n c l u d e the a p p l i c a t i o n o f the r e a c t i o n to any t r i t e r p e n e systems, p r e l i m i n a r y r e s u l t s o b t a i n e d by Dr. Gladstone i n t h i s l a b o r a t o r y suggest t h a t the method works very w e l l f o r such systems. The other t r i t e r p e n e minor component, code-named H , was a white c r y s t a l l i n e s o l i d , m.p. 256-270°C and the e m p i r i c a l formula C30H50O2 was 108 e s t a b l i s h e d by both e lementa l a n a l y s i s and h i g h r e s o l u t i o n mass measurements. The i n f r a r e d spectrum o f t h i s compound c o n t a i n e d a s t r o n g h y d r o x y l a b s o r p t i o n band (3400-3300 c m - 1 ) and a weak band (795) a t t r i b u t a b l e to a t r i s u b s t i t u t e d double bond. The NMR spectrum (100 Mc/s) i n d i c a t e d a one p r o t o n m u l t i p l e t at x 4.72 f o r the o l e f i n i c hydrogen o f a t r i s u b s t i t u t e d double bond and two o v e r l a p p i n g t r i p l e t s ( J 5.0 c . p . s . ) c e n t r e d at x 6.79 and 6 . 9 0 . These were ass igned to the protons geminal t o a p a i r o f secondary a x i a l h y d r o x y l groups. The mass spectrum showed s a t e l l i t e peaks correspond-i n g to the e l i m i n a t i o n o f a methyl group and o f two molecules o f water from the m o l e c u l a r i o n . Weak r e t r o D i e l s - A l d e r fragments may o r may not have been p r e s e n t . Thus s m a l l peaks were observed at m/e 302 and 269 but v a r i o u s fragments of low i n t e n s i t y were a l s o p r e s e n t i n the r e g i o n m/e 300 t o 380 and were p r o b a b l y caused by the unknown i m p u r i t y i n the sample. The major a l l y l i c c leavage fragments were seen at m/e 220 and 207 as i s a l s o observed f o r example i n the spectrum o f a u t h e n t i c 3 a - h y d r o x y — 2 1 6 - . h y d r o x y - A l l + - s e r r a t e n e . That compound H i s not i n f a c t an impure sample o f t h i s d i a x i a l d i o l was concluded from examinat ion o f d i f f e r e n c e s i n some o f the minor fragments i n the two s p e c t r a . For example, the s t a n d a r d compound o f known s t r u c t u r e c l e a r l y d i s p l a y s r e t r o D i e l s - A l d e r fragments at m/e 302, 284, and 269 o f r e l a t i v e i n t e n s i t y 7-10% and a l s o s a t e l l i t e peaks corresponding to the l o s s o f 51, 57, 74 and 75 mass u n i t s from the m o l e c u l a r i o n . The r e t r o D i e l s - A l d e r peaks i n the unknown compound, as a l r e a d y n o t e d , are very weak ( l e s s than 3%) and the m o l e c u l a r i o n s a t e l l i t e peaks noted above are c o m p l e t e l y absent . I n s t e a d , o ther s a t e l l i t e peaks were observed c o r r e s p o n d i n g to the l o s s o f 61, 63, 76, 77 and 79 mass u n i t s from the m o l e c u l a r i o n . Other d i f f e r e n c e s were noted i n the methyl group r e g i o n o f the c o r r e s p o n d i n g NMR s p e c t r a o f these compounds. Thus the s tandard d i a x i a l d i o l showed f i v e peaks l o c a t e d at x 9 . 0 9 , 9^14, 9 .19 , 9.21 109 and 9 . 3 4 . Compound H on the o ther hand showed peaks at the p o s i t i o n s T8.75, 9 . 0 5 , 9 .18 , 9.24 and 9 . 3 3 . Most c o n v i n c i n g o f a l l was the T . L . C . ev idence . Thus on a s i l i c a t h i n l a y e r p l a t e developed i n e t h y l acetate/benzene ( 1 : 3 ) , the s tandard d i a x i a l d i o l has 0.59 and compound H 0 . 3 7 . In summary one can say t h a t compound H was found t o be a t r i t e r p e n e d i a x i a l d i o l c o n t a i n i n g a t r i s u b s t i t u t e d double bond. Although the e m p i r i c a l formula C30H50O2 shows t h i s compound t o be i s o m e r i c w i t h 3a-hydroxy-21B-h y d r o x y - A 1 1 + - s e r r a t e n e , these compounds were n o n - i d e n t i c a l . I n s u f f i c i e n t q u a n t i t i e s were a v a i l a b l e f o r the p r e p a r a t i o n o f s u i t a b l e d e r i v a t i v e s and so we must conclude t h a t e i t h e r the compound possesses a s k e l e t o n which i s not t h a t o f s e r r a t e n e or e l s e one or o ther o f the h y d r o x y l f u n c t i o n s must be l o c a t e d on carbon atoms other than 3 and 21. In our o p i n i o n however t h i s compound p r o b a b l y possesses a s k e l e t o n c l o s e l y r e l a t e d t o but not i d e n t i c a l w i t h A 1 " 4 - s e r r a t e n e . Such a compound might w e l l a r i s e by a d i f f e r e n t type o f r i n g c l o s u r e d u r i n g the b i o s y n t h e s i s from a - o n o c e r i n . This concludes the d i s c u s s i o n o f the s t r u c t u r a l e l u c i d a t i o n work p e r -formed on the t r i t e r p e n e s i s o l a t e d from S i t k a spruce b a r k . There remain to be d i s c u s s e d the two compounds X and P and the f a t t y a c i d e s t e r f r a c t i o n s . Compound X was a white waxy s o l i d o f low m e l t i n g p o i n t , 69-70°C. The i n f r a r e d spectrum showed the presence o f a s t r o n g c a r b o n y l a b s o r p t i o n band (1725 c m - 1 ) and a p a i r o f s t r o n g . s h a r p s i g n a l s at 730 and 720 w h i c h , 83 a c c o r d i n g t o Be l lamy, a r i s e from the r o c k i n g v i b r a t i o n o f methylene groups i n polymethylene chains (-CH2-) n i n which n r e p r e s e n t s a number equal to or g r e a t e r than f o u r . A s t r o n g band a t 1178 i s i n d i c a t i v e o f 84 an e s t e r o f a l o n g , c h a i n f a t t y a c i d . The NMR spectrum (100 Mc/s) of t h i s compound showed a t r i p l e t ( J 6.5 c . p . s . ) at x 6.01 ass igned to the protons on the a l p h a methylene group o f the a l c o h o l p o r t i o n i n a c a r b o x y l i c 110 a c i d e s t e r . Another t r i p l e t (J = 7.5 c . p . s . ) was a l s o observed at x 7.77 and was a s s i g n e d t o the protons on the a l p h a methylene group o f the c a r b o x y l i c a c i d p o r t i o n , o f the e s t e r . In a d e c o u p l i n g experiment i t was demonstrated t h a t the protons i n both m u l t i p l e t s are coupled to methylene protons a b s o r b i n g around x 8.25. A l s o i n d i c a t e d i n the NMR spectrum was a very i n t e n s e s i g n a l at x 8.76 a r i s i n g from long c h a i n methylene and a weak methyl group s i g n a l at x 9.15. I n t e g r a t i o n o f the NMR spectrum, assuming the s i g n a l at x 6.01 t o be due to two p r o t o n s , gave the t o t a l number o f protons i n the molecule as b e i n g o f the o r d e r o f 110. Th is f i g u r e may very w e l l be m i s l e a d i n g but i t does at l e a s t u n d e r l i n e the f a c t t h a t long chains are i n v o l v e d i n both the a l c o h o l and a c i d m o i e t i e s o f t h i s e s t e r . Because o f the low m e l t i n g p o i n t o f the compound and problems w i t h o c c l u s i o n o f s o l v e n t , i t was not p o s s i b l e t o o b t a i n a s a t i s f a c t o r y e lementa l a n a l y s i s . Moreover the mass spectrum o f the m a t e r i a l was not h e l p f u l as no m o l e c u l a r i o n peak was observed. No s a p o n i f i c a t i o n s t u d i e s have been performed and no f u r t h e r i n f o r m a t i o n i s a v a i l a b l e concerning the nature o f compound X. The l a s t o f the n o n - e s t e r i f i e d compounds i s o l a t e d from S i t k a spruce bark i n the present study has been d e s c r i b e d as compound P. T h i s compound was v e r y s o l u b l e i n c o l d petroleum e t h e r , acetone and o t h e r s o l v e n t s and so was present o n l y i n e x t r a c t Z . Dur ing the chromatographic s e p a r a t i o n of t h i s e x t r a c t , f r a c t i o n s c o n t a i n i n g the t r i t e r p e n e s B and C t o g e t h e r w i t h an unknown m a t e r i a l were i s o l a t e d . I t was found t h a t the unknown m a t e r i a l c o u l d be removed by d i s t i l l a t i o n i n vacuo (130-135°C a t 0.07 mm. Hg) . The compound i s o l a t e d was a v i s c o u s l i q u i d w i t h a p a l e y e l l o w - g r e e n c o l o u r and f a i n t sweet odour. Examinat ion o f a sample o f the d i s t i l l a t e on a s i l i c a t h i n l a y e r p l a t e (chloroform) showed the i s o l a t e d m a t e r i a l t o be a p p a r e n t l y I l l chromatographically pure. Elemental analysis of the distillate was in keeping with the empirical formula C20H34O. When the mass spectrum was examined however no parent ion peak was present (m/e 290). The highest observed fragment at m/e 272 was therefore studied and high resolution mass measurements indicated this species to have the empirical formula C2oH32-Quite clearly then the molecule was losing the elements of water in the mass spectrometer. Compound P was found to have a weak ultraviolet maximum at 228 my (e 190) with a shoulder at 276 my. Such behaviour was believed to arise from j trace Contaminants .; •. ..v.'.. The infrared spectrum was however very much more informative and is shown in Figure 15. Figure 15. Infrared Spectrum of Compound P. 112 The first and most obvious feature of the infrared spectrum of compound P is the strong hydroxyl 0-H stretching absorption at 3400 cm"1. Secondly there is the fairly intense signal at 1635 arising from the carbon, carbon stretching frequency of an isolated olefinic double bond. Consul-85 tation of the text by Bellamy indicates the presence of two types of double bond in this system. Thus the weak signal at 3060 could arise either from a vinyl group or from a terminal methylene. The absorption bands at 1410, 990, and 915 are a l l attributable to a vinyl double bond whereas the signal at 887 must arise from a terminal methylene group. Finally the doublet at 1365 and 1383 must be associated with a gem dimethyl function. This spectrum is in fair agreement with the published spectrum of the 88 diterpene alcohol manool which was run as a nujol mull. The NMR spectrum of compound P was also very informative and is shown in Figure 16. The low field protons in this spectrum^include an ABX : type •system associated with a Vinyl group and two wide multiplets-arising from the protons of an exocyclic methylene. Figure 16. NMR Spectrum (100 Mc/s) of Compound P. 113 The observed coupling constants,in the ABX system were J t r a n s 17.5 c.p.s., J . 10.5 c.p.s., and J 1.5 c.p.s. On this basis the multiplet cis gem . c centred at x 4.15 was assigned to H in structure LXXXII, that centred at x 4.88 to H c, and the third centred at 5.05 to H^. The wide signals at x 5.26 and 5.55 were not displaced when the solution temperature was raised from 30° to 50°C and were assigned to an exocyclic methylene group. The change in temperature did however He Hb R H r move the signal at x 8.58 by 8 c.p.s. to higher field and this signal was therefore assigned to the hydroxyl function. The weak multiplets LXXXII observed at 6.45, 7.60 and 7.73 were considered to indicate the sample as being impure. Three methyl group signals were clearly seen in the 9.18 to 9.36 region and the intense signal at 8.78 .was considered to be caused partly by methylene protons and partly by another methyl group deshielded by proximity to the vinyl double bond or the tertiary hydroxyl group. The N.M.R. spectral evidence on compound P is in reasonable agreement with the identification of this compound as the diterpene alcohol manool LXXXIII^ although an authentic sample of the latterwasnot compared with the above. The spectrum of this alcohol (60 Mc/s) has been published by Varian Associates8.^ The only difference O H LXXXIII observed in the published spectrum is that the multiplets assigned to H £ and H^ are better separated and do not quite overlap. The mass spectrum of compound P was measured and 114 prominent fragments were observed at m/e 272 (M-18), 257 (M-33), 204 (M-86), 189 (M-101), 161 and 137. The suggestion that the isolated diterpene alcohol is impure, as seen from the presence of the multiplets at x 7.61 and 7.73, each corres-ponding to less than one proton in the integral, was confirmed when the material was spotted on a silver nitrate impregnated s i l ica thin layer plate developed in chloroform. Two components were seen with the main compound barely moving from the origin (grey colour, SbCls) and the contaminant, present in about 10% concentration, having 0.30 (pink). These compounds were not separated further but similar mixtures have been separated by 87 4 other authors ' by column chromatography of the 3,5-dinitrobenzoates on silver nitrate impregnated s i l ica . Finally brief mention must be made of the fatty acid containing components isolated from extract Z. A large fraction of a yellow viscous liquid was recovered from the column chromatographic separation of this extract after.all the triterpenes had already been removed. Saponification of this fraction by means of the potassium hydroxide in diethylene glycol treatment afforded a large amount of free fatty acid material and a smaller fraction of a dark coloured resinous pitch. The latter was examined by T.L.C. and showed the presence of traces of triterpene diols such as 21-epi-serratenediol but no 3-sitosterol. It was concluded that the fatty acids were present as mixed glycerides.in the original extract and this point was not further investigated. The chromatographic fractions containing the triterpenes were also contaminated with yellow oily liquids. This material was recovered after precipitation of the crystalline triterpenes and saponification in a solution of Sodium in aqueous ethanol gave rise to another fraction of free 115 f a t t y a c i d s and a l s o to a white c r y s t a l l i n e s o l i d , m.p. 140-141°C which was concluded t o be 8 - s i t o s t e r o l . T h i s substance was shown t o have the e m p i r i c a l formula C29H50O by h i g h r e s o l u t i o n mass measurements on the m o l e c u l a r i o n peak. Attempts to o b t a i n a s a t i s f a c t o r y e lementa l a n a l y s i s were u n s u c c e s s f u l p r o b a b l y because the m a t e r i a l had been r e c r y s t a l l i z e d from e t h a n o l which had caused s o l v a t i o n o f the p r o d u c t . The i n f r a r e d spectrum o f t h i s compound showed the presence o f h y d r o x y l (3400 c m - 1 ) and a non-conjugated t r i s u b -s t i t u t e d double bond (1635 and 797). The absence o f the f a m i l i a r gem d i m e t h y l doublet was a l s o n o t e d . The NMR spectrum (100 Mc/s) showed a one p r o t o n m u l t i p l e t at x 4.74 a r i s i n g from the o l e f i n i c p r o t o n o f a t r i s u b -s t i t u t e d double bond. A l s o present was a p o o r l y r e s o l v e d ; m u l t i p l e t at 6.63 and a sharp s i n g l e t at 7^44 r i s i n g out o f the methylene envelope r e g i o n may have been due to the h y d r o x y l group; The methyl group r e g i o n ( 9 . 0 - 9 . 3 ) was complex and i n c o m p l e t e l y r e s o l v e d . The mass spectrum o f the 6 - s i t o s t e r o l f r a c t i o n , showed a .prominent fragment at m/e 400 (M-14). This was taken as c l e a r evidence o f the presence o f the r e l a t e d compound, campesterol (VII) i n the sample. This co-occurrence i s a f a m i l i a r phenomenon, as has a l r e a d y been mentioned i n the i n t r o d u c t i o n . Other prominent fragments were observed at m/e 396 (M-18), 381 (M-33), 329 (M-85) , 315 (M-99), 272 (M-212) and 255 (M-139). These peaks i n d i c a t e d cleavages o c c u r r i n g i n the s i d e c h a i n and a l s o e l i m i n a t i o n o f water and o f a l a b i l e methyl group from the m o l e c u l e . This b r i n g s to a c o n c l u s i o n the d i s c u s s i o n o f the s t r u c t u r a l e l u c i d a t i o n work c a r r i e d out on the v a r i o u s compounds i s o l a t e d from S i t k a spruce b a r k . F u r t h e r work on the f a t t y a c i d f r a c t i o n s and on the compounds X and P would improve the scope o f the s t u d y . The t r i t e r p e n e s D and H are present to such a minor extent and are so d i f f i c u l t t o p u r i f i y t h a t not much 116 more is likely to be learned about them. There was probably at least one other diterpene alcohol present in the extract as a minor constituent. 117 EXPERIMENTAL General Throughout this work Merck s i l ica gel G was used as adsorbent in thin layer chromatography (TLC). The chromatoplates, 0.25 mm. in thickness, were air dried and activated in an oven at 100°C for three hours. In pre-parative scale TLC a thicker film (0;5 mm.) was utilised. The plates were developed in either chloroform or a mixture of benzene:ethyl acetate (3:1) and were then sprayed with a solution of antimony pentachloride in carbon tetrachloride (1:2). It was not necessary to heat the plates after spraying with this reagent. In a few instances s i l ica gel plates impregnated with silver nitrate were prepared for the separation of mixtures of olefinic substances. These plates were made by slurrying s i l ica gel (30 gm.) in a 7 % aqueous silver nitrate solution (60 ml.) and drying in the oven as above. For preparative scale TLC it was advantageous to use s i l ica gel containing 2 % by weight of a fluorescent indicator. Even in these instances the bands were so difficult to detect that it was necessary to also spray strips to determine their location. In general the recovery of material by preparative TLC was no better than 60 % so that i t was preferable to separate mixtures by column chromatography whenever possible. Column chromatography was performed using either Woelm grade s i l ica gel or neutral alumina. By far the best general purpose adsorbent for the separation of these triterpene mixtures was deactivated alumina (Activity III) prepared by the addition of the correct quantity of water to the adsorbent as directed on the container. The solvents used were petroleum ether (b.p. 65-110°) , benzene, and ether. Except in large-scale work these 118 s o l v e n t s were d i s t i l l e d before use . NMR s p e c t r a were measured i n d e u t e r o c h l o r o f o r m s o l u t i o n at room. temperature u n l e s s otherwise s p e c i f i e d . Most o f the NMR s p e c t r a l evidence was o b t a i n e d at 60 Mc/s u s i n g a V a r i a n A 60 instrument and, u n l e s s s t a t e d t o the c o n t r a r y , i t i s t o be understood t h a t s p e c t r a quoted i n t h i s s e c t i o n were run at t h i s frequency. In the few cases where a d d i t i o n a l r e s o l u t i o n was r e q u i r e d , the NMR s p e c t r a were, o b t a i n e d at 100 Mc/s u s i n g the V a r i a n HA 100 instrument and t h i s i s i n d i c a t e d i n the t e x t . The p o s i t i o n s o f a l l NMR 77 a b s o r p t i o n s i g n a l s are g iven i n the T i e r s T s c a l e w i t h r e f e r e n c e t o t e t r a m e t h y l s i l a n e as the i n t e r n a l s t a n d a r d set at T 10.0 u n i t s . For m u l t i -p l e t s the x va lues g iven r e p r e s e n t the centre o f the s i g n a l . Mass s p e c t r a were measured e i t h e r on an A t l a s CH 4 instrument , o r e l s e on an A s s o c i a t e d E l e c t r i c a l I n d u s t r i e s MS 9 h i g h r e s o l u t i o n mass spectrometer . The m o l e c u l a r formulae were determined by h i g h r e s o l u t i o n mass spectrometry on the MS 9 i n s t r u m e n t . S u i t a b l e standards o f known m o l e c u l a r weight were employed f o r t h i s purpose . I n f r a r e d s p e c t r a o f s o l i d samples were measured as KBr p e l l e t s on the P e r k i n Elmer 21 double beam spectrophotometer or e l s e i n c h l o r o f o r m s o l u t i o n on the P e r k i n Elmer 137 i n s t r u m e n t . The p o s i t i o n s o f a b s o r p t i o n maxima are quoted i n wave numbers ( c m - 1 ) . M e l t i n g p o i n t s were determined,on a K o f l e r b l o c k and are u n c o r r e c t e d . S p e c i f i c r o t a t i o n va lues were determined i n c h l o r o f o r m s o l u t i o n at 1 % con-c e n t r a t i o n and ORD curves were o b t a i n e d on a JASCO Model ORD/UV 5 s p e c t r o -p o l a r i m e t e r at 1 % c o n c e n t r a t i o n i n e i t h e r methanol o r dioxane s o l u t i o n . The analyses were performed by Dr . A . Bernhardt and h i s a s s o c i a t e s , Mulheim (Ruhr) Germany, and by Mr. P. Borda, U n i v e r s i t y o f B r i t i s h Columbia. 119 I s o l a t i o n o f S i t k a Spruce Bark T r i t e r p e n e s Bark was o b t a i n e d from the b u t t s o f mature S i t k a spruce t r e e s . ( P i c e a s i t c h e n s i s ) , Grays Harbor County, Washington S t a t e and m e c h a n i c a l l y separated i n t o a c o r k - r i c h f r a c t i o n by hand s o r t i n g . The bark was a i r - d r i e d and ground i n a hammermill t o pass a 20-mesh s i e v e . The bark was then e x t r a c t e d i n a b o r o s i l i c a t e g l a s s Sohxlet e x t r a c t o r f o r 18 hours w i t h petroleum e t h e r ( b . p . 40-60°C). The e x t r a c t was taken t o dryness t o p r o v i d e a crude m i x t u r e i n a y i e l d o f 4 .3 % based on the oven dry weight o f e x t r a c t e d b a r k . The e x t r a c t was d i s s o l v e d i n hot acetone (50 gm. e x t r a c t i n 500 m l . acetone) and a l l o w e d t o s tand, two days at ambient temperature whereupon white c r y s t a l s were d e p o s i t e d . The crude c r y s t a l l i n e d e p o s i t was f i l t e r e d o f f and r e c r y s t a l l i z e d . o n c e from hot acetone t o g ive a crude whi te t r i t e r -pene m i x t u r e , m.p. 250°C i n a y i e l d o f 15 % o f the s t a r t i n g e x t r a c t . For the sake o f convenience t h i s m i x t u r e w i l l h e r e a f t e r be r e f e r r e d t o as " e x t r a c t Y " . The f i l t r a t e s were combined and evaporated to dryness to g i v e a y e l l o w , t a c k y wax, m.p. 40°C. This wax (81.3 gm.) was d i s s o l v e d i n 1 l i t r e o f benzene and e x t r a c t e d t w i c e (600 and 150 m l . ) w i t h IN sodium hydroxide s o l u t i o n . The combined a l k a l i n e e x t r a c t s were r e - e x t r a c t e d w i t h benzene (2 x 300 m l . ) and the com-b i n e d benzene e x t r a c t s were washed w i t h water (3 x 500 m l . ) , d r i e d over magnesium s u l f a t e and evaporated t o r e c o v e r a p a l e g r e e n i s h - y e l l o w s e m i r s o l i d r e s i d u e (27.7 gm.) c o n t a i n i n g the n e u t r a l c o n s t i t u e n t s . T h i s f r a c t i o n w i l l be r e f e r r e d to l a t e r as " e x t r a c t Z " . The a l k a l i n e s o l u t i o n c o n t a i n i n g a c i d i c and p h e n o l i c m a t e r i a l s was a c i d i f i e d t o pH 3.5 by the slow a d d i t i o n o f con-c e n t r a t e d h y d r o c h l o r i c a c i d and. the f r e e a c i d s and phenols were then. .recovered by e x t r a c t i o n w i t h benzene ( 3 x 400 m l . ) . The combined benzene e x t r a c t I 120 was washed w i t h water (3 x 500 m l . ) d r i e d as above and evaporated t o y i e l d a dark brown tacky r e s i n (42.0 gm.) . T h i s f r a c t i o n was not f u r t h e r i n v e s t i g a t e d . S e p a r a t i o n o f Crude M i x t u r e s o f N e u t r a l T r i t e r p e n e s A. P r e p a r a t i v e T h i n Layer Chromatography In the e a r l y p a r t o f t h i s work s m a l l samples o f (200-300 mg.) compounds A and B were o b t a i n e d i n a pure s t a t e v i a p r e p a r a t i v e s c a l e TLC f o l l o w e d by r e c r y s t a l l i z a t i o n . Some d i f f i c u l t y was e x p e r i e n c e d w i t h p e e l i n g o f the adsorbent from the p l a t e a f t e r e v a p o r a t i o n o f the s o l v e n t used i n a p p l y i n g the m i x t u r e . This c o u l d be l a r g e l y overcome through the use o f more d i l u t e s o l u t i o n s . The p l a t e s used measured 60 x 20 cm. and were coated w i t h a l a y e r (0.5 mm. t h i c k ) o f s i l i c a g e l . Thus i n a t y p i c a l experiment 5 x 125 mg. o f e x t r a c t Y was a p p l i e d as a s o l u t i o n i n c h l o r o f o r m (5 x 8 m l . ) t o f i v e such p l a t e s . A f t e r d e v e l o p i n g once i n c h l o r o f o r m , s t r i p s on both s i d e s and i n the centre o f the p l a t e were sprayed w i t h antimony p e n t a c h l o r i d e . Examinat ion under a UV lamp then p e r m i t t e d l o c a t i o n o f the bands c o n t a i n i n g compounds A ( R f 0.18) and B ( R f 0.45) and these bands were scraped o f f the p l a t e s . The compounds were recovered by e x t r a c -t i o n w i t h warm c h l o r o f o r m (5 x 100 m l ) . In t h i s way 122 mg. o f crude com-pound B was recovered and 231 mg. o f compound A. ( T o t a l recovery. 56 % of s t a r t i n g m a t e r i a l ) . The compound B f r a c t i o n so recovered was examined by TLC on a s i l i c a chromatoplate developed i n c h l o r o f o r m and was shown to be contaminated w i t h a t r a c e o f compound C ( R f 0 . 5 2 ) . This was removed a f t e r two r e c r y s t a l l i z a t i o n s from n-hexane. The t o t a l y i e l d o f pure whi te c r y s t a l l i n e B, m.p. 275-7° was 92 mg. The compound A f r a c t i o n c o n t a i n e d a t r a c e o f the f a s t e r r u n n i n g compound E ( R f 0.36) which was removed by two 121 r e c r y s t a l l i z a t i o n s from benzene:hexane ( 2 : 3 ) . The y i e l d o f white c r y s t a l l i n e A , m.p. 296-8° was 170 mg. B. Column Chromatography on S i l i c a A sample o f e x t r a c t Y (4.985 gm.) was a p p l i e d as a s l u r r y i n the e l u t i n g s o l v e n t chloroform:benzene (9:11) on a column o f n e u t r a l s i l i c a (250 gm.; 4 .0 x 40 c m . ) . A f t e r c o l l e c t i o n o f 500 ml e l u a t e , the m a t e r i a l a p p l i e d t o the head o f the column had a l l d i s s o l v e d and been t r a n s p o r t e d i n t o the s i l i c a . The f i r s t l i t r e o f e l u a t e y i e l d e d a y e l l o w o i l (40 mg.) E l u t i o n w i t h chloroform:benzene (1:1,1000 ml) y i e l d e d s i m i l a r m a t e r i a l (35 mg.) w h i l e e l u t i o n w i t h chloroform:benzene (11:9, '3700 ml) e l u t e d compound B (1.720 gm) . F u r t h e r e l u t i o n w i t h t h i s s o l v e n t (1400 ml) then y i e l d e d a mixed f r a c t i o n c o n t a i n i n g compounds A , B and E (1.832 gm.) . F i n a l l y a m i x t u r e o f compound A w i t h more p o l a r minor components was removed (1.237 gm.) upon s t i l l f u r t h e r e l u t i o n (1000 m l ) . S t r i p p i n g the column w i t h pure c h l o r o f o r m (600 ml) y i e l d e d a mixed f r a c t i o n (85 m g . ) . The t o t a l recovery was 4.789 gm. Pure compound B.was o b t a i n e d by two r e c r y s t a l l i z a t i o n s o f the above m a t e r i a l from 95 % e t h a n o l ( y i e l d 1.200 gm., m.p. 276-7°C) . Attempts t o p u r i f y the f r a c t i o n s c o n t a i n i n g compound A by f r a c t i o n a l r e c r y s t a l l i z a t i o n from e t h a n o l o r petro leum e t h e r were u n s u c c e s s f u l . The two f r a c t i o n s r i c h i n compound A were combined and the chromato-graphy repeated as above except t h a t the m a t e r i a l was f i r s t d i s s o l v e d i n hot c h l o r o f o r m (50 m l . ) and t r a n s f e r r e d t o the column. E l u t i o n w i t h c h l o r o f o r m : benzene (11:9, 1800 m l ) p r o v i d e d i n i t i a l l y more compounds (0.163 gm.), then a m i x t u r e o f A and B (0.118 gm.) upon f u r t h e r e l u t i o n (300 m l ) , a f r a c t i o n o f almost pure A (1.190 gm.^ a d d i t i o n a l 800 ml o f s o l v e n t ) , and a m i x t u r e o f 122 A with more polar compounds (1.440 gm., 1000 ml of solvent). The present fraction of compound A so obtained s t i l l contained traces of B and E as impurities. Two r e c r y s t a l l i z a t i o n s from chloroform yielded TLC pure compound A (650 mg.) m.p. 307.5-308°. C. Column Chromatography on Alumina A sample of extract Y (2.014 gm.) was dissolved in the minimum quantity of chloroform. To the solution was added a few grams of neutral alumina (Activity III) and the chloroform was evaporated off from the slurry. The alumina coated with the extract was then transferred to the top of a column f i l led with deactivated alumina (100 gm.) in petroleum ether. Elution with successive amounts of petroleum ether-benzene (as shown) provided the various compounds. Elution with petroleum ether:benzene (4'1,300 ml) yielded a mixture of compounds B and C in roughly equal proportions (62 mg.) followed by a fraction containing pure compound B (322 mg., 200 ml.). The next fraction eluted contained a mixture of compounds B and E (297 mg., 400 ml). Use of petroleum ether:benzene (3:2) as solvent provided a fraction containing a mixture of compounds B, E and A (109 mg. 100 ml) followed by the main compound A fraction (1084 mg., 800 ml of the above solvent followed by elution with petroleum ether:benzene 2:3). Elution with pure benzene and with benzene:ethyl ether (9:1) yielded an impure fraction of compound D (35 mg. 300 ml) while further elution with this solvent system removed a mixed fraction containing compounds G and H (52 mg., 300 ml). The values of these compounds were measured on a s i l ica coated chromatoplate (20 x 20 cm) developed with ethyl acetate:benzene (1:3) and are as indicated: Compound B (0.64), A (0.57), F (0.45), D (0.39), G (0.30) and H (0.21). 123 D. Purification of Compound E by Column Chromatography on Alumina The mixed fraction containing compounds B and E (297 mg.) mentioned above was applied to the surface of alumina (Activity I) as previously described. A chromatographic separation was then carried out on a column of alumina (25 gm. , Activity I). Elution with benzene:ethyl ether (19:1, 125 ml) yielded pure compound E (203 mg.) and the column was then stripped with ethyl ether to recover the compound B (97 mg.). Composition of Extract Y On the basis of the above chromatographic separations the approxi-mate composition of extract Y was thus found to be as below:-Compound Weight Recovered (mg.) % of Extract from 2.014 gm. of Y C 30 1.5 B 440 22.0 E 240 12.0 A 1100 55.0 F weight unknown since never isolated in pure form D 30 1.5 G 35 1.7 H 20 1.0 Compound A White crystals from chloroform m.p. 307.5-308° C, [a]2,0 +2 .8° , ORD (C, 0.02; dioxane), 21°C; [<J>] 7 0 0 0 ° , [<j>] 5 8 9 +46°, [<f>] 5 0 0 +91°, [<\>]h00 +137°, [<J>] 3 0 0 +274°. Infrared (KBr): 3550 (hydroxyl), 1100 (methoxyl) and 795 (trisubstituted double bond). Ultraviolet, no absorption. NMR signals at 124 65°G w i t h e x t e r n a l TMS; 4.61 (1 H , m u l t i p l e t , o l e f i n i c H ) , 6.46 (1 H , m u l t i -p l e t , H-C-OH), 6.58 (3 H, s i n g l e t , m e t h o x y l ) , and 9 . 0 0 - 9 . 2 7 (23.8 H , s i x s p i k e s , angular m e t h y l ) . Mass spectrum (MS 9 ) ; fragments at m/e 441 (M-15), 438 (M-18) and 423 (M-33); 316 and 269 ( r e t r o D i e l s - A l d e r ) ; 221, 220, 189 ( a l l y l i c c l e a v a g e ) ; o ther i n t e n s e fragments at m/e 187, 147 and 135. Found: C, 81.52; H , 11.48; 0 , 7 .01; M.W. (M.S. 9) 456. C a l c . f o r C 3 1 H 5 2 0 2 : C, 8 1 . 0 8 ; H, 11.37; 0 , 7 .51; M.W. 456. High R e s o l u t i o n mass measurement 456.398 (standard 464); C a l c . f o r C 3 1 H 5 2 0 2 456. 397. Methoxyl ( Z e i s e l ) 7'.01; C a l c . f o r 1 0 C H 3 6 . 8 0 . Compound B White c r y s t a l s from e t h a n o l , m.p. 2 7 6 - 2 7 7 . 5 ° , [ a ] 2 0 - 5 5 . 4 . ORD (C 0 . 0 2 , CH3OH), 21°C; [<j>]700 - 1 2 8 ° , [<f>] 5 8 9 -260°, [<fr] 5 0 0 - 3 4 7 ° , [<(,]1 + 0 0 -433° , [<j>]3oo - 1290°. I n f r a r e d ( K B r ) : 3650 ( h y d r o x y l , weak), 1103 (methoxyl) , and 795 ( t r i s u b s t i t u t e d double bond) . U l t r a v i o l e t , no a b s o r p t i o n . NMR l s i g n a l s : 4.71 (1 H , m u l t i p l e t , o l e f i n i c H ) , 6.57 (1 H , m u l t i p l e t , H-C-OH), 6.72 (3 H, s i n g l e t , m e t h o x y l ) , 7.25 (1 H , m u l t i p l e t , H - C - 0 C H 3 ) , and 9.10-9.34 (21.0 H, angular m e t h y l ) . NMR (100 M c / s ) : 4.71 (unreso lved m u l t i p l e t ) , 6.57 ( t r i p l e t , J = 2.5 c . p . s . ) and 7.25 ( t r i p l e t , J = 2.5 c . p . s . ) Mass spectrum (MS 9 ) ; fragments at m/e 454 ( M - 2 ) , 438. (M-18), 424 (M-32); 316, 284, 269 ( r e t r o D i e l s - A l d e r ) J 221, 220, 190, 189 ( a l l y l i c c l e a v a g e ) ; o t h e r i n t e n s e f r a g -ments at m/e 187, 147 and 135. Found : C , 81 .04; H, 11.37; 0 , 7 .64; M.W. (MS 9) 456. C a l c . f o r C 3 i H 5 2 0 2 : C, 8 1 . 0 8 ; H, 11 .37;0, 7 .51; M.W. 456. High r e s o l u t i o n mass measurement" 456.398 (s tandard 464); C a l c . f o r C 3 i H 5 2 0 2 , 456.397. Methoxyl ( Z e i s e l ) 6 .97; C a l c . f o r 1 0CH 3 6 . 8 0 . ' 125 Compound B A c e t a t e Compound B (520 mg.) was t r e a t e d w i t h a m i x t u r e o f d r y , r e d i s t i l l e d p y r i d i n e (10 ml) and a c e t i c anhydride (10 ml) at room temperature f o r f o u r days. The r e a c t i o n m i x t u r e was poured on to crushed i c e and a l l o w e d t o s tand f o r t h r e e h o u r s . The crude a c e t a t e was e x t r a c t e d i n t o c h l o r o f o r m (3 x 75 ml) and the combined c h l o r o f o r m e x t r a c t s washed w i t h 5 % h y d r o c h l o r i c a c i d , 5 % sodium b i c a r b o n a t e and water . The e x t r a c t was d r i e d over anhydrous magnesium s u l f a t e , f i l t e r e d and the s o l v e n t evaporated t o y i e l d the crude a c e t a t e (520 mg)- T h i s compound was p u r i f i e d by column chromatography on d e a c t i v a t e d a lumina ( A c t i v i t y I I I , 50 gm.) . E l u t i o n w i t h petro leum e t h e r : benzene (17*.3, 500 ml) y i e l d e d the TLC pure a c e t a t e (510 mg. , 90 % y i e l d , R f CHC1 3 0 . 4 5 ) . R e c r y s t a l l i z a t i o n o f the a c e t a t e from ethanol p r o v i d e d whi te c r y s t a l s , m.p. 205.5-207° , [ a ] 2 0 - 6 4 . 6 ° . ORD (C, 0 . 0 2 , C H 3 0 H ) , 21°C; [<f>] 7 0 0 "100° , [>] 5 8 9 - | 5 5 ° , [4>]5oo " 1 5 2 ° , [.frhoo - 2 2 8 °> M 300 -1066°. I n f r a r e d ( K B r ) : 1733 and 1245 ( a c e t a t e ) , 1103 (methoxyl ) , and 792 ( t r i s u b s t i t u t e d double bond) . U l t r a v i o l e t , no a b s o r p t i o n . NMR s i g n a l s : 4.65 (1 H, m u l t i p l e t , l o l e f i n i c H ) , 5.28 (1 H , m u l t i p l e t , H-C-OAc), 6.70 (3 H , s i n g l e t , m e t h o x y l ) , I 7.23 (1 H , m u l t i p l e t , H-(j"-0CH 3), 7.94 (3 H, s i n g l e t , a c e t a t e ) , 9 . 0 7 - 9 . 3 0 (20.3 H , angular m e t h y l ) . NMR s i g n a l s (100 mc/s): 4.65 (unreso lved m u l t i p l e t ) , 5.28 ( t r i p l e t , J = 2.5 c . p . s ) , 7.23 ( t r i p l e t , J = 2.5 c . p . s . ) . Mass spectrum (MS 9 ) ; fragments at m/e 483 (M-15), 466 (M-32), 451 (M-47), 438 (M-60) , 423 (M-75); 316, 284, 269 ( r e t r o D i e l s - A l d e r ) ; 262, 221, 203, 202, 190, 189, ( a l l y l i c c l e a v a g e ) ; o ther i n t e n s e fragments at m/e 187 and 135. Found: C, 79.55; H, 10.70; 0 , 9 . 8 0 ; M.W. (MS 9) 498. C a l c . f o r C 3 3 H 5 t + 0 3 : C, 79 .46; H, 10.91; 0 , 9 . 6 2 ; M.W. 498. 126 Compound A A c e t a t e Compound A (630 mg.) was r e a c t e d i n a m i x t u r e o f a c e t i c anhydride (15 ml) and dry p y r i d i n e (15 ml) f o r f o u r days at room temperature . The s o l u t i o n c o n t a i n i n g the d i s s o v e d a c e t a t e was worked up as d e s c r i b e d above t o y i e l d crude compound A acetate (715 mg.) Examinat ion o f t h i s m a t e r i a l by TLC on s i l i c a showed the absence o f unreacted s t a r t i n g m a t e r i a l . R e c r y s t a l -l i z a t i o n from e t h a n o l y i e l d e d the a c e t a t e as w h i t e , n e e d l e - l i k e c r y s t a l s (570 mg. , y i e l d 83 %, R £ CHC1 3 0 . 2 1 ) , m.p. 199-201° , [ a ] 2 0 + 4 . 5 ° . ORD ( 0 . 0 2 , C H 3 0 H ) , 21°C; [<j,] 7 0 0 +97°, [ 4 > ] 5 8 9 +97°, [<t>] 5 0 0 +97°, [<frK0  +194°, [<f>] 300 +438°. I n f r a r e d ( K B r ) ; 1730 and 1250 ( a c e t a t e ) , 1105 (methoxyl) , and 795 ( t r i s u b s t i t u t e d double bond) . U l t r a v i o l e t , no a b s o r p t i o n . NMR I s i g n a l s : 4.64 (1 H, m u l t i p l e t , o l e f i n i c H ) , 5.28 (1 H, m u l t i p l e t , H-C-OAc), 6.63 (3 H, s i n g l e t , m e t h o x y l ) , 7.93 (3 H , s i n g l e t , a c e t a t e ) , and 9 . 0 2 - 9 . 2 8 (23.0 H, angular m e t h y l ) . NMR s i g n a l s (100 M c / s ) ; 4.64 (unreso lved m u l t i p l e t ) , 5.28 ( t r i p l e t J = 2.5 c . p . s . ) , 7.45 ( q u a r t e t , J A g = 11.0 c . p . s . , J A C = 4 . 0 c . p . s . ) . Mass spectrum (MS 9 ) ; fragments at m/e 483 (M-15), 438 (M-60), and 423 (M-75); 316, 284 and 269 ( r e t r o D i e l s - A l d e r ) ; 262, 221, 204, 203, 190, 189 ( a l l y l i c c l e a v a g e ) ; o t h e r i n t e n s e fragments at m/e 187 and 135. Found: C, 79 .43; H, 10.82; 0 , 9 . 5 5 ; M.W. (MS 9) 498. C a l c . f o r C 3 3 H 5 t t 0 3 ; C, 79.46; H , 10.92; 0 , 9 . 6 3 ; M.W. 498. Compound B Ketone Compound B (1.000 gm.) was d i s s o l v e d i n d r y , r e d i s t i l l e d p y r i d i n e (25 m l ) . T h i s s o l u t i o n was added s l o w l y t o a s l u r r y o f chromic oxide (975 mg.) i n dry p y r i d i n e (10 ml) and the r e a c t i o n mixture was s t i r r e d m a g n e t i c a l l y f o r 60 hours at room temperature . The s o l u t i o n was poured i n t o c o l d water (400 m l ) , a l l o w e d to s t a n d f o r h a l f an h o u r , and f i l t e r e d under s u c t i o n . The dark 127 brown precipitate on the suction funnel was washed thoroughly with cold water. The crude ketone was then recovered by extraction with warm chloroform (600 ml) while the inorganic residues remained on the suction funnel. The chloroform extract was washed with 5 % aqueous hydrochloric acid, water,dried over anhydrous magnesium sulfate, and filtered. Evaporation of the solvent yielded a brown coloured crude ketone (R^ CHCI3 0,45). This material was purified by column chromatography on deactivated alumina (Activity III, 50 gm.). Elution with benzene:petroleum ether (3:17, 1000 ml) yielded white crystalline material (892 mg., yield 85 %). Recrystallization from ethanol gave white flakes, m.p. 240-2°, [a] 2 0 - 7 1 . 5 ° . ORD (C, 0.02; CH30H), 21°C; W 700 -137°, [4] 5 8 9 -228 0 , [*hi2 -3332°, [ 4 > ] 2 7 6 -227°, [<J>]21+0 -4106; molecular amplitude -31.0. Addition of hydrochloric acid to the methanol solution of the ketone (0.02 N) had almost no effect on the amplitude of the Cotton effect after ninety minutes. Infrared (KBr): 1707 (carbonyl), 1100 (methoxyl), 795 (trisubstituted double bond). NMR signals: 4.63 (1 H, multiplet, olefinic H), 6.71 (3 H, singlet, methoxyl), 7.23 (1 H, multiplet H-C-OCH3), 8.91-9.17 (21.7 H, angular methyl). Mass spectrum (CH 4); fragments at m/e 422 (M-32) and 407 (M-47); 284 and 269 (retro Diels-Alder); 221, 218, 204, 203, 190, 189 (allylic cleavage); other intense fragments at m/e 147 and 135. Found: C, 81.58; H, 11.23; 0, 7.18; M.W. (CH 4) 454. Calc. for C 3 1 H 5 0 O 2 : C, 81.88; H, 11.08; 0 ,^7.04; M.W. 454. Compound A Ketone Compound A (1.133 gm.) dissolved in dry redistilled pyridine was added to a slurry of chromic oxide (1.015 gm.) in dry pyridine (20 ml) and the reaction mixture stirred magnetically at room temperature for 70 hours. Workup of this reaction as described above yielded the crude brown ketone (1.174 gm.). This material was purified by column chromatography on alumina (Activity I, 128 100 gm.)- E l u t i o n w i t h b e n z e n e : e t h y l e t h e r (19:1) y i e l d e d TLC pure A ketone (740 mg. , 65 % y i e l d , R f CHC1 3 0 . 4 1 ) . F u r t h e r e l u t i o n w i t h b e n z e n e : e t h y l e t h e r (4:1) y i e l d e d a f r a c t i o n (300 m g . J R f CHC1 3 0.23) c o n t a i n i n g l a r g e l y unreacted compound A. The pure ketone was o b t a i n e d as white f l a k e s from e t h a n o l , m.p. 262-3°. Vacuum subl imed m a t e r i a l (250-60°C/0•07) has m.p. 270-270.5° , [ a ] J 0 - 0 . 1 ° . ORD (C, 0 . 0 2 ; C H 3 0 H ) , 21°C; [<t>]70o + 36° , [<j>] 5 8 9 0f> O] 3 1 0 -2002°, [<|>]273 +1680°, [<f>]2-+0 -929° , m o l e c u l a r ampli tude - 3 6 . 8 . A d d i t i o n o f h y d r o c h l o r i c a c i d to the methanol s o l u t i o n o f the ketone (0.02 and 0.06 N) had v i r t u a l l y no e f f e c t on the ampli tude o f the C o t t o n e f f e c t a f t e r n i n e t y minutes . I n f r a r e d ( K B r ) ; 1712 ( c a r b o n y l ) , 1105 (methoxy l ) , and 795 ( t r i s u b s t i t u t e d double bond) . NMR s i g n a l s : 4.62 (1 H , m u l t i p l e t , o l e f i n i c H ) , 6.65 (3 H, s i n g l e t , m e t h o x y l ) , 8 .91-9.25 (24.1 H , angular m e t h y l ) . Mass spectrum (CH 4 ) ; fragments at m/e 439 (M-15), 422 (M-32), 407 (M-47); 284 and 269 ( r e t r o D i e l s - A l d e r ) ; 221, 218, 203, 190 and 189 ( a l l y l i c c leavage); o t h e r i n t e n s e fragments at m/e 147 and 135. Found: C, 8 2 . 0 3 , ; H , 11.24, 0 , 6 . 9 0 ; M.W. (CH 4) 454. C a l c . f o r C 3 1 H 5 0 0 2 : C, 81 .88, H,. 11.08; 0 , 7 .04; M.W. 454. Compound B Ketone Ethy lene K e t a l Compound B ketone (250 mg.) was r e a c t e d w i t h e thy lene g l y c o l (1.5 ml) i n the presence o f p - t o l u e n e s u l f o n i c a c i d hydrate (40 mg.) as c a t a l y s t . The r e a c t i o n was c a r r i e d out f o r f i v e hours i n r e f l u x i n g benzene p r e v i o u s l y d r i e d by d i s t i l l a t i o n from l i t h i u m aluminum h y d r i d e . The water formed d u r i n g the condensat ion was removed c o n t i n u o u s l y by means o f a Dean-Stark t r a p . The benzene s o l u t i o n was washed w i t h water , d r i e d over anhydrous magnesium s u l f a t e , and f i l t e r e d . E v a p o r a t i o n o f the s o l v e n t y i e l d e d the crude k e t a l which was p u r i f i e d by column chromatography on d e a c t i v a t e d alumina ( A c t i v i t y I I I , 20 gm.). E l u t i o n w i t h benzene:petroleum e t h e r (19:1 , 200 ml) y i e l d e d the TLC pure 129 ethylene ketal derivative (250 mg., yield 92 %) . White rod-like crystals from benzene, m.p. 317-8° (decomposition), [a]2.0 - 5 6 . 5 ° . Infrared (KBr); angular methyl). Mass spectrum (MS 9); fragments at m/e 399, 221, and 99 (very intense). Found: C, 79.26; H, 11.01; 0, 9.61; M.W. (MS 9) 498. Calc. for C33H54O3: C, 79.52; H, 10.84; 0, .9.64;. M.W. 498. Compound A Ketone Ethylene Ketal Compound A ketone (245 mg.) was reacted for six hours with ethylene glycol (0.75 ml) in dry refluxing benzene in the presence of p-toluene-sulfonic acid hydrate (70 mg.) as catalyst. The water formed in the reaction was removed by means of a Dean-Stark trap. Workup of the reaction as described above yielded the crude ketal (278 mg.). This was purified by. column chromatography on neutral alumina (Activity I, 30 gm.). Elution with benzene yielded the TLC pure ethylene ketal derivative (180 mg., 67 % yield). Recrystallization from petroleum ether (b.p. 65-110°) gave colourless plates, m.p. 317-9° (decomposit ion),[a]^° - 4 . 4 ° . Infrared (KBf); 1105 (methoxyl), and 795 (trisubstituted double bond). NMR signals: 4.63 (1 H, multiplet, olefinic H), 6.02 (4 H, singlet, -q-G^-CH^-O-), 6.61 (3 H, singlet, methoxyl), 9.00-9.27 (22.8 H, angular methyl). Mass spectrum (MS 9): fragments at m/e 399, 221, 189 and 99 (very intense). Found: C, 79.33; H, 10.94; 0, 9.91; M.W. (MS 9) 498. Calc. for C 3 3 H 5 1 + 0 3 : C, 79.52; H, 10.84; 0, 9.64; M.W. 498. Desoxy Compound B. Compound B ketone tosylhydrazone was prepared by the method described by Djerassi et. al . The ketone (300 mg.) and p-toluenesulfonhydrazide 1110 (methoxyl), and 795 (trisubstituted NMR signals: 4.65 130 reagent (310 mg.) were reacted together in refluxing methanol (10 ml) in the presence of three drops of acetyl chloride as catalyst. After half an hour heating was terminated, the reaction flask and contents allowed to cool to room temperature, and the white crystalline product removed by filtration. This material was shown by TLC on a s i l ica chromatoplate to be chromato-graphically pure and to contain no unreacted ketone. M.p. 257-8° (decomp.). Infrared (KBr); 3180 (NH), 1635 ( C=N-), 1592 and 1490 (aromatic), 1165 (-S02-), and 1095 (methoxyl). Found: C, 73.81; H, 10.00; N, 4.54; S, 4.96; Calc. for C38H58N203S: C, 73.26, H, 9.38, N, 4.50, S, 5.14. ! The tosylhydrazone derivative (220 mg.) was reduced by means of sodium borohydride (500 mg.) in moist refluxing dioxane according to the method of Cagliotti and G r a s e l l i A f t e r five hours heating was terminated, the dioxane was removed by evaporation under reduced pressure and the product was partitioned between benzene (150 ml) and water. The benzene layer was removed and washed with water, dried over anhydrous magnesium sulfate, filtered and evaporated to yield the crude desoxy compound. This substance was purified by column chromatography on deactivated alumina (Activity III, 20 gm.). Elution with petroleum ether (200 ml) yielded the TLC pure desoxy compound (115 mg., 73 % yield based on the ketone, R £ CHCI3 0.71). Crystal-lization from ethanol provided white crystals, m.p. 249.5-251°, [a]2)0 - 4 9 . 1 ° . Infrared (KBr): 1100 (methoxyl), and 797 (trisubstituted double bond). NMR signals: 4.70 (1 H, multiplet, olefinic H), 6.71 (3 H, singlet, methoxyl), 7.25 (1 H, multiplet, H-C-OCH3), and 9.09-9.34 (20.2 H, angular methyl). Mass spectrum (CH 4); fragments at m/e 425 (M-15), 408 (M-32), and 393 (M-47); 316, 284, and 269 (retro Diels-Alder); 221, 204, 190, 189 (allylic cleavage); other intense fragments at m/e 187 and 135. Found: C, 84.82; H, 11.94; 0, 3.71; M.W. (CH 4) 440. Calc. for C 3 1 H 5 2 0 : C, 84.54; H, 11.82; 0, 3.65; 131 M.W. 440. Desoxy Compound A Compound A ketone (330 mg.) and p-toluenesulfonhydrazide reagent (330 mg.) were reacted together in refluxing benzene for seven hours and most of the solvent removed by evaporation. The tosylhydrazone derivative which crystallized was filtered off (and recrystallized from ethanol, m.p. 234-7° (decomposition).. Infrared (KBr): 3210 (NH), 1635 ( C=N-), 1165 (-S02-), and 1107 (methoxyl). NMR signals: 2.00-2-72 (4 H, complex, aromatic), 6.63 (3 H, singlet, methoxyl), 7.55 (3 H, singlet, aromatic methyl), and 8.96-9.25 (angular methyl). Found: C, 73.19; H, 9.54; N, 4.61; S, 5.20. Calc. for C38H58N2S03: C, 73.26; H, 9.38; N, 4.50; S, 5.14. The tosylhydrazone (390 mg.) was reacted with sodium borohydride (500 mg.) for 8 hours in refluxing dioxane. Workup of the reaction product as described above .yielded the crude desoxy compound (277 mg.). This was purified by column chromatography on alumina (Activity I, 30 gm.). Elution with benzene yielded the desoxy compound (158 mg., 55 % yield based on the ketone). Examination by TLC (silica, CHCI3) showed the product to be pure (R^ CHCI3 0.50) and recrystallization from ethyl acetate provided needle-like crystals, m.p. 267-9°, [a] 2 0 +33.8°. ORD (C 0.02, dioxane), 21°C; [$ ] 7 0 0 +164°, [$] 583 +245°, [<t>]i+G0 +368°, [<J>]3oo +409°. Infrared (KBr): 1107 (methoxyl), and 795 (trisubstituted double bond). Ultraviolet, no absorption. NMR signals: 4.60 (1 H, multiplet, olefinic H), 6.60 (3 H, singlet, methoxyl), 7.15-7.50 (1 H,multiplet, H-C-0CH3), and 9.01-9.30 (21.5 H, angular methyl). NMR (100 Mc/s): 7.36•(quartet, J A g = 11.3 c.p.s, J A C = 4.0 c.p.s.). Mass spectrum (CH 4): fragments at m/e 425 (M-15), 388 (M-52); 316, 284, 269 (retro Diels-Alder); 221, 204, 191, and 189 (allylic cleavage); other intense frag-132 ments at m/e 184 and 135. Found: C, 84.68; H, 11.99; 0, 3.61; M.W. (CH 4) 440. ! Calc. for C 3 iH 5 2 0: C, 84.54; H, 11.82; 0, 3.64; M.W. 440. Dihydro Compound B Compound B (10.25 mg., 22.5 micromoles) was hydrogenated in a micro-hydrogenation apparatus previously calibrated against pure maleic acid (7 determinations). Using a suspension of Adam's catalyst.(Pt0 2, 5.17 mg.) in glacial acetic acid, a smooth uptake of hydrogen (25.0 micromoles) was observed. On a larger scale compound B (360 mg.) was hydrogenated under atmospheric pressure in glacial acetic acid solution using Adam's catalyst (100 mg.). The uptake of hydrogen over a period of 8 hours was 17.2 c c . (calc. 17.7 c c ) . Examination on a s i l ica chromatoplate ( C H C I 3 ) showed the presence of a trace of unreacted compound B. After the addition of some fresh catalyst (50 mg.) the hydrogenation was repeated. The acetic acid was removed by evaporation under reduced pressure, the product dissolved in chloroform, washed with 5 % sodium bicarbonate solution and water. After drying over anhydrous magnesium sulfate, filtration and evaporation, the TLC pure product was recovered (366 mg., C H C I 3 0.30). White needle-like crystals from petroleum ether, m.p. 211-216°. ORD (C 0.02, C H 3 O H ) , 21°C; ->] 7 0 0 -92° , [ < f > ] 5 8 9 -132° , [*] 5 0 0 - W> '[+] 1+ 0 0-366°, [cj>] 3 0 0 -864 o . Infrared (KBr): 3500 (hydroxyl), 1088 (methoxyl) and no absorption at 795 (trisub-stituted double bond). Ultraviolet, no absorption. NMR signals: no olefinic I H, 6.10 (1 H, multiplet, H-C-OH)6.70 (3 H, singlet, methoxyl), 7.25 (1 H, . I multiplet, H-C-OCH3), and 9.10-9.24 (21.0 H, angular methyl). Mass spectrum (MS 9): fragments at m/e 456 (M-2), 440 (M-18), 426 (M-32), 411 (M-47), 393 (M-65), and 369 (M-89); no retro Diels-Alder; other fragments at m/e 221, 133 190, 189, 163, 150, 149, and 136. Found: C, 81.41; H, 11.48; 0, 7.15; M.W. (MS 9) 458. Calc. for C 3 1 H 5 l t 0 2 : C , 81.16, H, 11.86; 0, 6.97; M.W. 458. Dihydro Compound A Compound A (10.54 mg., 23.1 micromoles) was hydrogenated in a micro-hydrogenation apparatus at atmospheric pressure using Adam's catalyst (5.56 mg.). No hydrogen uptake was observed using ethanol as solvent. Use of glacial acetic acid instead resulted in the absorption of 22.3 micromoles of hydrogen. ORD (C 0.02, dioxane), 21°C; [<j>]70o +139°, [<(>] 5 8 9 +139°, [^ 1 500 + 1 3 9 ° , [4>]tt00 +185°, [^>] 300 + 2 7 8 ° . In a second experiment the hydrogenation was repeated on a larger scale. Compound A (200 mg.) was hydrogenated in glacial acetic acid solution using Adams catalyst (95 mg.). After fifteen hours the observed hydrogen uptake was 11.6 ml (Calc. 9.8) and the reaction product was isolated as previously described. The crude dihydro compound (205 mg.) was examined on a s i l ica chromatoplate (R£ CHCI3 0.20) and apparently did not contain unreacted starting material. Crystallization from dioxane provided white crystals, m.p. 288-293°. Infrared (KBr): 3550 (hydroxyl), 1082 (methoxyl) and no absorption at 795 (trisubstituted double bond). NMR signals: no olefinic proton, 6.59 (1 H, multiplet, H-C-OH), 6.64 (3 H, singlet, methoxyl), 9.02-9.26 (24.8 H, angular methyl). Mass spectrum (MS 9); fragments at m/e 440 (M-18), 426 (M-32), 385 (M-73), and 369 (M-89); no retro Die l s-Alder; other fragments at m/e 245, 221, 189, 177,. 176, 149, and 136. Found: C, 81.29; H, 12.00; 0, 6.66; M.W. (MS 9) 458. Calc. for C 3 1 H 5 8 ° 2 : c > 81.16; H, 11.87; 0, 6.98; M.W. 458. Dihydro Compound B Ketone Dihydro compound B was oxidized with Jones' reagent as described by 70 Henbest et al . The reagent was prepared by dissolving reagent grade chromic 134 oxide (2.668 gm.) i n c o n c e n t r a t e d s u l f u r i c a c i d (2.13 ml) and d i l u t i n g t o 10 ml w i t h d i s t i l l e d water i n a v o l u m e t r i c f l a s k . The d i h y d r o compound (155 mg.) was d i s s o l v e d i n warm acetone (30 ml) and p l a c e d i n a 100 ml f l a s k equipped w i t h magnetic s t i r r e r and c a l c i u m c h l o r i d e d r y i n g tube. Jones reagent (0.14 ml) was added and r e a c t i o n a l l o w e d t o proceed f o r 20 m i n u t e s , whereupon the excess Jones reagent was destroyed by the a d d i t i o n o f an excess o f d r y , reagent grade methanol . The s o l v e n t was removed by e v a p o r a t i o n and the product p a r t i t i o n e d between benzene and water . The benzene l a y e r was washed w i t h 5 % sodium carbonate s o l u t i o n , water , and d r i e d over anhydrous magnesium s u l f a t e . A f t e r f i l t e r i n g o f f the d r y i n g agent, the benzene s o l u t i o n was run through a one i n c h bed o f n e u t r a l s i l i c a to remove t r a c e s o f chromous s a l t . E v a p o r a t i o n o f s o l v e n t y i e l d e d the pure ketone (152 mg. , R f CHC1 3 0 . 6 2 ) . White p l a t e l e t s from e t h a n o l , m.p. 1 6 7 - 9 ° . I n f r a r e d (CHCI3): 1705 ( c a r b o n y l ) , and 1090 (methoxyl) . NMR s i g n a l s : 6.66 (3 H, I s i n g l e t , m e t h o x y l ) , 7.20 (1 H , m u l t i p l e t H-C-OCH3), 7.64 ( m u l t i p l e t , - C H 2 - C 0 - ) , . and 8 . 9 3 - 9 . 2 3 (22.4 H, angular m e t h y l ) . Mass spectrum (MS 9 ) ; fragments at m/e 441 (M-15), 424 (M-32), 409 (M-47), 385 (M-71), and 383 (M-73); prominent fragments at m/e 219, 189, 150, 149, and 136. Found: C, 81.47; H, 11.23; 0 , 7 .25; M.W. (MS 9) 456. C a l c . f o r C 3 1 H 5 2 0 2 : C, 81 .52; H , 11.48; 0 , 7 .01; M.W. 456. D esoxy Dihydro Compound B This compound was prepared by two d i f f e r e n t methods. A. W o l f f - K i s h n e r Reduct ion Dihydro B ketone (150 mg.) was suspended i n e t h a n o l (15 ml) c o n t a i n i n g d i e t h y l e n e g l y c o l (1.5 ml) and r e a c t e d i n a 25 ml f l a s k under r e f l u x w i t h h y d r a z i n e h y d r a t e (0.75 ml) and potass ium h y d r o x i d e (0.14 gm.) . A f t e r 30 135 minutes the reagents had d i s s o l v e d and the e t h a n o l and excess h y d r a z i n e were then d i s t i l l e d o f f u s i n g an o i l b a t h at 150°C. D i e t h y l e n e g l y c o l (2 ml) was added t o the r e a c t i o n f l a s k and the o i l bath temperature r a i s e d to 190°G. A f t e r 4 hours under r e f l u x the s o l u t i o n was cooled, , poured i n t o water (20 ml) and e x t r a c t e d w i t h benzene (4 x 10 m l ) . A f t e r washing w i t h water the benzene s o l u t i o n was d r i e d over anhydrous magnesium s u l f a t e , f i l t e r e d and the s o l v e n t evaporated t o r e c o v e r a white c r y s t a l l i n e s o l i d (123 m g . , 85 % y i e l d ) . T h i s m a t e r i a l was examined on a s i l i c a chromatoplate and found t o be pure ( R f CHC1 3 0 . 6 8 ) . A second and l a r g e r sample (350 mg.) o f the crude desoxy d i h y d r o compound was prepared i n the same way and p u r i f i e d by chromatography on a column o f n e u t r a l s i l i c a (17-5 gm.) U s i n g benzene;petroleum e t h e r (3:17) as e l u a n t the pure m a t e r i a l was o b t a i n e d (154 mg.)- F u r t h e r e l u t i o n w i t h c h l o r o f o r m y i e l d e d a m i x t u r e o f unreacted s t a r t i n g m a t e r i a l and an unknown p o l a r by-product (140 m g . ) . B. Borohydride Reduct ion o f Dihydro B Ketone Tosylhydrazone, Dihydro B ketone (96 mg.) was r e a c t e d w i t h p - t o l u e n e s u l f o n h y d r a z i d e reagent (90 mg.) i n dry r e f l u x i n g benzene and the water formed was removed by a z e o t r o p i c d i s t i l l a t i o n . A f t e r t h r e e hours the r e a c t i o n was stopped and the product p u r i f i e d by chromatography on a n e u t r a l s i l i c a column (15 gm.) . E l u t i o n w i t h benzene y i e l d e d a m i x t u r e o f unreacted ketone and tosy lhydrazone (45 m g . ) . F u r t h e r e l u t i o n w i t h c h l o r o f o r m y i e l d e d almost pure t o s y l h y d r a z o n e (113 mg.) which was leached w i t h a l i t t l e c o l d methanol and d r i e d i n vacuo m.p. 211-2° ( d e c o m p o s i t i o n ) . I n f r a r e d (CHC1 3 ) : 3300 (NH), 1605 ( a r o m a t i c ) , and 1085 (methoxyl) . NMR s i g n a l s : 2 .10-2.75 (4 H , complex, a r o m a t i c ) , 6.70 I (3 H, s i n g l e t , m e t h o x y l ) , 7.22 (1 H , m u l t i p l e t , ti-(j-0CH3), 7.58 (3 H , s i n g l e t , aromat ic m e t h y l ) , 9 . 0 8 - 9 . 2 8 ( o v e r l a p p i n g s i g n a l s , angular m e t h y l ) . 136 In a second experiment dihydro B ketone (600 mg.) was reacted as above with p-toluenesulfonhydrazide reagent (600 mg.) for four hours in benzene under reflux. After removal of the benzene an examination by TLC on a s i l ica chromatoplate showed the product to be almost exclusively the tosylhydrazone. This product, without.further purification, was reduced with sodium borohydride (1.70 gm.) in dioxane (55 ,ml). After ten hours under reflux the solvent was removed in vacuo and the product recovered as pre-viously described. This material was purified by column chromatography on neutral s i l ica (65 gm.). Elution with petroleum ether yielded the pure desoxy dihydro compound (380 mg., 66 % yield). The desoxy dihydro compound B samples prepared by these two experi-ments, when recrystallized from ethyl acetate, could both be mechanically separated by hand picking into platelet type crystals m.p. 188-194° and needle-like crystals m.p. 215-223°. The platelet crystals, which pre-dominated in each of the two reduction experiments^were quantitatively compared by the running of infrared solution spectra (13.9 mg/ml in CHC13) and were shown to be identical. Examination by VPC using a 20 % silicone gum rubber (S.E. 30) column in an Aerograph instrument showed both samples to be mixtures of two components in the ratio 4:1 with the corresponding pairs having identical retention times on the column. The mixture of the two compounds, as prepared, gave the following data, Infrared (CHC13): 1090 (methoxyl). NMR signals: 6.68 (3 H, singlet, I methoxyl), 7.22 (1 H, multiplet, H-C-OCH3), 9.08-9.25 (22.8 H, angular methyl). Mass spectrum (CH 4); fragments at m/e 440 (M-2), 427 (M-15), 410 (M-32), and 395 (M-47); other intense fragments at 272, 221, 190, 189, 177, 163, 150, 149 and 136. Found:: C, 83.96 H, 12.22 0, 3.68 M.W. (CH 4) 442. Calc. for C S T H S ^ O : C, 84.16; H, 12.22; 0,3.62; M.W. 442. 137 Ether Cleavage of Dihydro Desoxy B with Boron Trichloride 71 The procedure adopted was that of Youssefyeh and Mazur. Dihydro desoxy B (123 mg.) dissolved in dichloromethane (5 ml) and added to liquid boron trichloride (approx. 2 ml) at -80°C. After maintaining at -80° for thirty minutes, the solution was allowed to warm to room temperature and left overnight. The solvent was removed in vacuo and the reaction product twice treated with 5 ml portions of methanol.and evaporated. The residue was taken, up in chloroform,washed well with water, dried and evaporated to yield a brown glassy solid (100 mg). Infrared (CHCI3): No absorption at 1090 (methoxyl).This product was separated on a preparative scale s i l ica coated chromatoplate (CHCI3) into two fractions, a yellow viscous liquid (55 mg., 0.76) and a dark yellow viscous liquid (17 mg.) R^  0.22). NMR signals (main product): no protons absorbing at low field, 8.75 and 9-9.3 (angular methyl). It was assumed that the major product described above was a chlorinated hydrocarbon by analogy.with Youssefyeh and Mazur's results on the cleavage of steroidal ethers. The main ether cleavage product (55 mg.) was warmed on the steam bath with IN potassium hydroxide solution (3 ml) in diethylene glycol to which was added diglyme (2 ml) to dissolve the oily hydrocarbon. After a few minutes a white precipitate of potassium chloride was.obseryed. After warming for two hours the reaction mixture was filtered, diluted with water and extracted with benzene (5 x .25 ml). This solution was washed with water, dried and evaporated to yield a product s t i l l containing residual high boiling solvent. When the latter was dissolved in n-hexane (50 ml), washed with water (4 x 50 ml), dried and evaporated, a colourless oily liquid was recovered (10 mg.) which showed only one spot on a.si l ica TLC plate. NMR signals: 4.37 and 4.60 (olefinic H), 8.71 and 9.12 (angular 138 methyl, very poorly resolved). The signals arising from the olefinic protons were very weak and i t was apparent that any olefin which was present was pre-sent only as a minor component. Ether Cleavage of Dihydro Desoxy B with Boron Tribromide Desoxy dihydro B (77 mg.) was dissolved in dichloromethane (10 ml) and reacted with boron tribromide (1 ml) at room temperature for four hours. Excess reagent was decomposed by addition of water. The reaction product was dissolved in petroleum ether, the latter extract washed four times with water, dried and flushed through a short bed of neutral s i l ica . A colourless oily liquid (78 mg.), with TLC properties similar to that of the main pro-duct from the boron trichloride cleavage, was recovered. Infrared (CHCI3): no absorption at 1090 (methoxyl). NMR signals: No protons absorbing at low field, 8.72 and 9.11 (angular methyl groups,very poorly resolved). This cleavage product (25 mg.) was reacted in 0.5 N ethanolic potassium hydroxide solution (15 ml) under reflux for five hours. The ethanol was removed in vacuo, replaced with water and the alkali carefully neutralized by the addition of dilute hydrochloric acid. The reaction pro-duct was extracted into petroleum ether, the extract washed twice with water and dried. The pale yellow oi l recovered from this solution was purified by column chromatography on alumina(Activity I, 10 gm.). Elution with benzene: petroleum ether (1:4) yielded a colourless oi l (10.7 mg.) and further elution with chloroform gave a pale yellow oi l (16.3 mg.). An NMR spectrum of the former showed only a very faint trace of olefinic proton at 4.6 and examin-ation by TLC appeared to indicate that the reaction product was mainly unreacted starting material. In a second experiment a sample of the BBr3 cleavage product (40 mg) 139 was reacted in a refluxing solution of 0.5 N potassium hydroxide in n-butanol (20 ml) for two days. The alcohol was evaporated in vacuo, the residue taken up in chloroform and the solution washed three times with IN hydrochloric acid, twice with water, dried and evaporated. The yellow oily liquid (34 mg.) so obtained did not show any indication of olefinic protons in the NMR spectrum. Ether Cleavage Reaction on Compound A Acetate f\ ft The procedure adopted was that described by Narayanan and Iyer. Compound A acetate (117 mg.) was dissolved in a mixture of acetic anhydride (6 ml) and ether (2 ml) and boron trifluoride etherate (1.2 ml) was added. The reaction was allowed to proceed for eighteen hours at 0°C and was then poured on to crushed ice and allowed to stand for three hours. The product was extracted into chloroform,the extract was washed with 5 % sodium bicarbonate solution, water, dried and the solvent evaporated. Chromato-graphy of the crude product on a column of neutral s i l ica (10 gm.) provided after elution with benzene:chloroform (1:1) a yellow syrup (110 mg.). This substance was further separated by preparative TLC on s i l ica with chloroform as developing solvent. Extraction of the major component with hot methanol and chloroform yielded a colourless viscous liquid (42 mg, R^  0.73, compare R^  A acetate 0.45). Traces of at least five other more polar compounds were seen on the TLC plate but were not recovered. NMR signals: 4.63 (1 H, multiplet, I olefinic H), 5.31 (1 H, multiplet, H-C-OAc), 7.93 (3 H, singlet, acetate methyl), 8.98-9.29 (angular methyl). No trace of a methoxyl signal was present. Attempted Correlation of Desoxy A with Sawamilletin A sample of sawamilletin was obtained from Professor Obara in Tokyo. 140 T h i s m a t e r i a l has a r e p o r t e d m e l t i n g p o i n t o f 278°. However our sample had a s t r o n g tendency t o sublime and no t r u e m e l t i n g o c c u r r e d u n t i l 288° was reached. A mixed m e l t i n g p o i n t d e t e r m i n a t i o n w i t h desoxy compound A (m.p. 267-9°) was 234-260°. The samples were s p o t t e d on a s i l i c a TLC p l a t e and developed i n c h l o r o f o r m . The compounds had i d e n t i c a l va lues 0 . 5 0 ; however s a w a m i l l e t i n gave a p u r p l e c o l o u r e d spot and desoxy A an o l i v e green spot when sprayed w i t h antimony p e n t a c h l o r i d e . A q u a n t i t a t i v e i n f r a r e d s p e c t r a l comparison o f these compounds i n potass ium bromide p e l l e t s at a r a t i o o f 300 p a r t s KBr t o one p a r t o f sample gave n o n - i d e n t i c a l s p e c t r a . The mass s p e c t r a a l s o showed marked d i f f e r e n c e s . S a w a m i l l e t i n (MS 9) showed fragments at m/e 425 (M-15) and 393 (M-47); 316, 301 and 269 ( r e t r o D i e l s - A l d e r ) ; 204, 191 and 189 ( a l l y l i c c l e a v a g e ) ; o t h e r fragments at m/e 147 and 135. E t h e r Cleavage React ions on A A c e t a t e w i t h 36 % H B r / A c e t i c A c i d A s o l u t i o n o f hydrogen bromide (36 %) i n g l a c i a l a c e t i c a c i d was prepared by p a s s i n g the dry gaseous HBr i n t o the i c e - c o o l e d a c e t i c a c i d and measuring the weight i n c r e a s e . Compound A a c e t a t e (660 mg.) was t r e a t e d unde r e f l u x c o n d i t i o n s w i t h a m i x t u r e o f equal volumes (30 ml) o f t h i s reagent and a c e t i c anhydride f o r 90 minutes . The c o o l e d r e a c t i o n m i x t u r e was poured on t crushed i c e and a l l o w e d t o s tand two h o u r s . The product was then e x t r a c t e d i n t o c h l o r o f o r m , washed w i t h 5 % potass ium b i c a r b o n a t e , w i t h water and d r i e d over anhydrous magnesium s u l f a t e . F i l t r a t i o n f o l l o w e d by e v a p o r a t i o n o f the s o l v e n t y i e l d e d a v i s c o u s l i q u i d (550 mg.) which p a r t l y c r y s t a l l i z e d on s t a n d i n g . The product was d i s s o l v e d i n the minimum q u a n t i t y o f warm 95 % e t h a n o l . a n d a l l o w e d t o c r y s t a l l i z e . The white c r y s t a l l i n e product (95 mg.) had m.p. 186-195° , not a f f e c t e d by f u r t h e r r e c r y s t a l l i z a t i o n from e t h a n o l . 141 Infrared (nujol): 1730 and 1250 (acetate), no absorption at 1100 (methoxyl). I NMR signals: no olefinic proton 5.28 (1 H, multiplet, H-C-OAc), 7.92 (3 H, singlet, acetate), and 8.95-9.18 (21.9 H, angular methyl). M.W. (CH 4) 466 (corresponding to a diolefin acetate). The main fraction from the ether cleavage reaction (450 mg.) was a non-crystalline glass which gave an infrared spectrum similar to that from the crystalline fraction. Treatment of this fraction with ethanolic potassium hydroxide solution under reflux failed to remove the acetate group. Treatment with lithium aluminum hydride in benzene followed by separation of the product on a column of alumina yielded some non-crystalline fractions which were.not futher investigated. In a second experiment compound A acetate (360 mg.) was treated with a mixture of equal volumes (25 ml) acetic anhydride and 36 % HBr/acetic acid at 65-68° in a water bath for one hour. The acetate was completely dissolved within five minutes under these conditions. Isolation of the product as before yielded a partly crystalline material (360 mg.). Examin-ation of this product on a s i l ica chromatoplate (CHCI3) showed a product (Rf 0.47, violet colour) and some unreacted starting material (R^ 0.19, yellow). Also present were a trace of a non-polar material (R^ 0.74) and some highly polar material not moved by this solvent system. The crude material was separated by column chromatography on alumina (Activity I, 22 gm.). Elution with benzene:petroleum ether (1:1) yielded a non-polar viscous liquid (10 mg.) not further investigated. Elution with benzene yielded a white crystalline product (200 mg.) and with diethyl ether:benzene (1:9) a pale yellow syrup (110 mg.) was removed. The latter was a mixture containing mainly unreacted starting material and some more polar compounds. The crystalline fraction thus recovered had m.p. 175-200° from ethanol, 142 u n a f f e c t e d by vacuum s u b l i m a t i o n . The NMR, i n f r a r e d and mass s p e c t r a l data on t h i s compound were complete ly i d e n t i c a l to the data o b t a i n e d on the d i o l e f i n i c a c e t a t e produced i n the p r e c e e d i n g experiment . NMR (100 Mc/s) showed a one p r o t o n s e p t e t c e n t r e d at 7.39 (J 6.5 c . p . s ) . Attempts t o decouple t h i s s i g n a l were u n s u c c e s s f u l . E t h e r Cleavage R e a c t i o n on Desoxy Compound A w i t h 36 % H B r / A c e t i c A c i d Desoxy A (510 mg.) was r e a c t e d o v e r n i g h t at room temperature w i t h a m i x t u r e o f equal volumes (25 ml) o f 36 % H B r / a c e t i c a c i d and a c e t i c anhydride The crude product was i s o l a t e d i n the u s u a l way and separated by column chromatography on d e a c t i v a t e d a lumina ( A c t i v i t y I I , 20 gm.) . E l u t i o n w i t h petro leum e t h e r y i e l d e d f i r s t a white c r y s t a l l i n e product (360 m g . , 76 % y i e l d ) and then some a p p a r e n t l y unchanged s t a r t i n g m a t e r i a l (36 mg.) E l u t i o n w i t h benzene:petroleum e t h e r (7:3) y i e l d e d an impure f r a c t i o n (87 mg.) c o n t a i n i n g more p o l a r c o n s t i t u e n t s . The main product had m.p. 160-165° from petro leum e t h e r . NMR s i g n a l s : no o l e f i n i c p r o t o n , 7.40 (1 H , m u l t i p l e t , - C H ( C H 3 ) 2 ) , 8 .97-9 .20 (20.8 H, angular m e t h y l ) . Examinat ion by V . P . C . on a column packed w i t h s i l i c o n e gum rubber (S.E 30, 20 % ) showed t h i s f r a c t i o n t o be a m i x t u r e o f two compounds i n the r a t i o 7:1. Found: C, 8 8 . 3 5 ; H, 12.08. C a l c . f o r CZ0HhB; C, 88.16; H, 11.94. The f r a c t i o n c o n t a i n i n g the p o l a r by-products o f the e t h e r cleavage r e a c t i o n was re-chromatographed on d e a c t i v a t e d a lumina ( A c t i v i t y H I , 20 gm.) E l u t i o n w i t h petro leum e t h e r (100 ml) y i e l d e d a f r a c t i o n (61 mg.) w h i c h , when examined on a s i l i c a chromatoplate (CHCI3), gave r i s e t o two spots o f very s i m i l a r R £ v a l u e , one p i n k i n c o l o u r and the o t h e r y e l l o w - b r o w n . I n f r a r e d (CHCI3); 1725 and 1250 ( a c e t a t e ) . NMR s i g n a l s : no o l e f i n i c p r o t o n , 143 7.95 (acetate, rather broad), 8.75-9.17 (angular methyl). NMR (100 Mc/s); weak multipletswere observed at 4.70, 5.35, 5.55 and 6.10. The recovered "starting material" from the ether cleavage reaction was also studied. Colourless needlike crystals were obtained from ethanol, m.p. 242-7°. Infrared (KBr); 1100 (methoxyl), no band at 795 (trisubstituted double bond). Mass spectrum (CH 4); fragments at m/e 425 (M-15) and 408 (M-32); other intense fragments at m/e 221, 205, 204, 191, 189, 149 and 135. Found: C, 84.25; H, 12.01; M.W. (CH 4) 440. Calc. for C 3 1 H 5 2 0 : C, 84.54; H, 11.81; M.W. 440. Correlation of Compound A Ketone with 38-Methoxy-21- Keto-A14-Serratene The authentic sample, isolated from pine bark, was provided by Dr. Rowe. This had m.p. 243-9°, elevated to 266.5-267° after two vacuum sublimations (240-250°C bath temperature/0.05 -mm.). Compound A ketonehad m.p. 245-260°, elevated to 270-270.5° by one vacuum sublimation. Mixed m.p. 265 .5-266.5° . The compounds ran identically on s i l ica chromatoplates in two solvent systems, [a]^0 -0 .1° for A ketone (Rowe has reported -29° for the pine bark ketone). ORD (C 0.02, CH30H), 16°C; [o>] 7 0 0 -87° , [<t>]589 -130°, [ > ] 3 i 2 -2269°, M 2 7 5 +1440°, [<t>]2ito -1833°; molecular amplitude -37.1. This curve was exactly the same shape as that obtained from A ketone but was somewhat more negative across the wavelength region scanned. Infrared (authentic sample, KBr); 1712 (carbonyl), 1105 (methoxyl), and 795 (trisubstituted double bond). A quantitative comparison of this compound (300 mg. . KBr per mg. sample) with compound A ketone gave completely super- ... impdsable spectra. Mass spectrum pine bark ketone (CH 4); fragments at m/e 439 (M-15), 422 (M-32) and 407 (M-47); 221, 218, 204, 203, 190, 189 (allylic cleavage); other intense fragments at m/e 170, 147 and 135. This spectrum was.very similar to that of A ketone. 144 Compound G White crystals from ethanol, m.p. 289-91°, |p<.f* - 1 2 . 3 ° . Low solubility in common solvents. Infrared (KBr); 3500 (-0H), and 795 (tri-substituted double bond). NMR signals: 4.65 (1 H, multiplet, olefinic H), 6.55 (1 H, triplet, H-C-OH), 6.80 (1 H, multiplet, H-C-OH), and 9.04-9.32 (23.0 H, angular methyl). Mass spectrum (CH 4); fragments at m/e 440 (M-2), 427 (M-15), 424 (M-18), and 409 (M-33); 302, 284, and 269 (retro Diels-Alder); 221, 220, 207, 203, 190, and 189 (allylic cleavage); other intense fragments at m/e 234, 187, 149 and 135. Found: C, 81.17; H, 11.26, O, 7.35; M.W. (CH 4) 442. Calc. for C 3 0 H5 0 O 2 :C, 81.45; H, 11.31, 0, 7.24; M.W. 442. Compound G Diacetate A crude fraction of compound G obtained from the mother liquors during crystallization (75 mg.) was acetylated as previously described using a mixture of equal volumes (10 ml) of acetic anhydride and dry pyridine at room temperature. Isolation of the crude acetate in the usual way was followed by chromatographic separation on a column of deactivated alumina (Activity III, 10 gm.). Elution with benzene: petroleum ether (2:3) yielded a white crystalline diacetate (45 mg.). Examination on a s i l ica chromatoplate (ethyl acetate:benzene, 1:4) showed the product to be pure diacetate (R^ 0.75, Compound G R £ 0.41). A pure sample was obtained by recrystallization from ethanol, m.p. 210-7°C. NMR signals; 4.65 (1 H, ' I multiplet, olefinic H), 5.30 (1 H, multiplet, H-C-OAc), 5.47 (1 H, multiplet, I H-C-OAc), 7.94 and 7.98 (6 H, two singlets, acetate), and 8.75-9.31 (19.6 H, angular methyl). NMR (100 Mc/s); 4.65 (unresolved multiplet), 5.30 (triplet J = 2.5 c.p.s.), and 5.47 (quartet, = 10.0 c.p.s., = 5.2 c.p.s.). Mass spectrum (MS 9); fragments at m/e 511 (M-15), 466 (M-60), 451 (M-75), 145 423 (M-103), 406 (M-120), 391 (M-135), and 363 (M-163); 203, 202, 190, and 189 (allylic cleavage); and other fragments at m/e 187, 147 and 135. Found: M.W (MS 9) 526. Calc. for 0 3 ^ 5 ^ ; M.W. 526. Correlation of Compound G with 21-Episerratenediol An authentic sample, isolated from pine bark, was sent to us by Dr. Rowe. White crystals, m.p. 284-5° (Literature value 289-290°, [a] 2 0 - 1 9 ° ) . This compound behaved in an identical fashion to compound G when examined on s i l ica chromatoplates in two solvent systems. The quantitative infrared spectra (KBr) were completely superimposable. The NMR spectrum, as measured by Dr. Rowe, was very similar to that obtained by us with com-pound G and the mass spectra of the samples were also very similar. Methylation of Compound A A three-necked 100 ml flask was fitted with a high speed stirrer^ a nitrogen gas inlet tube, a condenser with a calcium chloride drying tube and a dropping funnel. Full precautions were taken to exclude moisture from the walls of the apparatus. Compound A (175 mg.) was dissolved in warm, dry toluene (10 ml) and admitted to the reaction vessel via the dropping funnel. The apparatus was then thoroughly purged by passing purified grade nitrogen gas through it for twenty minutes and freshly cut potassium metal (300 mg.) was added. The flask was warmed with a heating mantle until the potassium was molten and stirring was then commenced. The reaction was allowed to proceed for two hours under a slow stream of nitrogen. At this stage a yellow powder was present together with finely divided particles of potassium metal. The reaction vessel was allowed to cool and an excess of pure dry methyl iodide (5 ml) was added from the dropping funnel. Stirring and heating were recommenced and the reaction was continued for a further 146 three hours. The contents of the f l a s k at t h i s stage were a b r i l l i a n t purple colour. The reaction was f i n a l l y stopped by cooling the fla s k and destroying the excess potassium by the addition of an excess of methanol. The solvent was then removed on a rotary evaporator and the reaction products p a r t i t i o n e d between chloroform and water. The combined chloroform extracts were washed with water, dried over anhydrous magnesium s u l f a t e , f i l t e r e d and evaporated i n vacuo to y i e l d the crude methyl ether. This product was p u r i f i e d by chromatography on a column of deactivated alumina ( A c t i v i t y I I I , 18 gm.). E l u t i o n with petroleum ether y i e l d e d f i r s t an o i l y l i q u i d f r a c t i o n (35 mg.) and then the pure methyl ether (95 mg.). E l u t i o n with benzene removed unreacted compound A (50 mg.). The methyl ether, a f t e r r e c r y s t a l l i z a t i o n from petroleum ether (b.p. 65-110°) was obtained as a white c r y s t a l l i n e s o l i d , m.p. 272-3°; [ a ] ^ 0 -16.4°. Infrared (KBr); no absorption at 3500 (-OH), 1100 (methoxyl), and 793 ( t r i s u b s t i t u t e d double bond). NMR s i g n a l s : 4.70 (1 H, m u l t i p l e t , o l e f i n i c H), 6.67 and 6.70 (6 H, I two s i n g l e t s , methoxyl), 7*20 (1 H, m u l t i p l e t , H-C - 0 C H 3 ) , 7.45 (1 H, m u l t i p l e t , H-C - O C H 3 ) , 9.05-9.33 (21.2 H, angular methyl). Mass spectrum (CH 4); fragments at m/e 455 (M-15), 438 (M-32) and 423 (M-47); 316, 284, and 269 (retro Diels-Alder).; 221, 189 ( a l l y l i c cleavage ); other fragments at m/e 279, 187, 167, and 149. Found: C, 81.68; H, 11.45; 0 , 6.95; M.W. (CH 4) 470. Calc. f o r C 3 2 H 5 1 t 0 2 ; C, 81.70; H, 11.49; 0 , 6.81; M.W. 470. Methylation of 21-Episerratenediol and C o r r e l a t i o n with Compound A A sample of 21-episerratenediol (102 mg.) was methylated according to the procedure outlined i n the preceeding section using again potassium metal (247 mg.) and methyl iodide (5 ml). The crude product was separated on a column of deactivated alumina ( A c t i v i t y I I I , 20 gm.). E l u t i o n with petroleum ether y i e l d e d an u n i d e n t i f i e d o i l y l i q u i d (45 mg.). Further 147 e l u t i o n w i t h benzene:petroleum e t h e r (1:9) removed the d i m e t h y l e t h e r (18 mg.) and the use o f benzene:petroleum e t h e r (4:1) y i e l d e d f i r s t a f r a c t i o n of a monomethyl e t h e r (8 mg.) and then a l a r g e r f r a c t i o n o f another mono-methyl e t h e r (43 mg.) . F i n a l l y e l u t i o n w i t h benzene removed some unreacted 2 1 - e p i s e r r a t e n e d i o l (8 m g . ) . The more p l e n t i f u l monomethyl e t h e r was p u r i f i e d by vacuum s u b l i -mation at 250-260°/0.05 mm. t o y i e l d a white c r y s t a l l i n e s o l i d , m.p. 305-6° (Compound A , 3 0 4 - 5 ° ) , mixed m.p. 306-7°. The two compounds gave i d e n t i c a l behaviour when examined on s i l i c a chromatopiates i n two d i f f e r e n t s o l v e n t systems and the q u a n t i t a t i v e i n f r a r e d s p e c t r a l comparison (KBr) y i e l d e d superimposable s p e c t r a . The NMR and mass s p e c t r a o f t h i s monomethyl e t h e r d e r i v a t i v e o f 2 1 - e p i s e r r a t e n e d i o l were a l s o shown to be very s i m i l a r t o the c o r r e s p o n d i n g s p e c t r a o b t a i n e d from compound A. The d imethyl e t h e r d e r i v a t i v e was a l s o compared w i t h compound A methyl e t h e r . A f t e r vacuum s u b l i m a t i o n t h i s compound had m.p. 277-8° (Compound A methyl e t h e r 2 7 2 - 3 ° ) , mixed m.p. 272-5° . The compounds aga in showed i d e n t i c a l TLC b e h a v i o u r , gave superimposable i n f r a r e d s p e c t r a (KBr) and the mass and NMR s p e c t r a were very s i m i l a r . NMR 100 M/cs; 7*22 ( t r i p l e t , J = 2.5 c . p . s . ) , 742 ( q u a r t e t , J ^ B = 11.6 c . p . s . , = 4 . 0 c . p . s . ) . C o r r e l a t i o n o f Compound E w i t h Compound A Ketone The i s o l a t i o n o f t h i s compound has a l r e a d y been d e s c r i b e d . The m a t e r i a l was p u r i f i e d by three r e c r y s t a l l i z a t i o n s from e t h a n o l which p r o -v i d e d white r o d - l i k e c r y s t a l s , m.p. 262-3°, [a]p° - 1 6 . 0 ° . Vacuum s u b l i -mation r a i s e d the m e l t i n g p o i n t to 270-271° . I n f r a r e d (KBr) : 1710 ( h y d r o x y l ) , 1105 (methoxyl) , and 795 ( t r i s u b s t i t u t e d double bond) . NMR s i g n a l s (100 M / c s ) ; 4.68 (1 H, m u l t i p l e t , o l e f i n i c H ) , 6.73 (3 H, 148 singlet, methoxyl), 7.34 (partly hidden multiplet, H-C-OCH3), and 8.94-9.28 (24.3 H, angular methyl). Found C, 81.69; H, 11.78; 0, 6.32; M.W. (MS 9) 454. Calc. for C 3 1 H 5 0 O 2 : C, 81.88; H, 11.08; 0, 7.04; M.W. 454. High resolution mass measurement 454.379 (standard 464). Calc. for C3 1 H 5 0 O 2 454.381. This compound was compared with compound A ketone. Compound A ketone, m.p. 270^-270.5°, mixed m.p. 270-271°. The compounds showed identical behaviour on s i l i ca chromatoplates developed in two solvent systems. The quantitative infrared spectral comparison (KBr) showed superimposable spectra. Ether Cleavage Reactions on B Acetate with 36 % HBr/Acetic Acid A. Under Reflux Compound B acetate (310 mg.) was suspended in a mixture of equal volumes (50 ml) acetic anhydride and 36 % HBr/acetic acid reagent. Reaction was allowed to continue for one hour under reflux and the cooled reaction mixture was poured into cold water. The precipitated material was filtered off, washed well with cold water, air dried and dissolved in benzene (200 ml). The benzene layer was washed with 1 % sodium carbonate and water. After drying over anhydrous magnesium sulfate, followed.by filtration, the solvent was removed under reduced pressure to give a pale brown glass (316 mg.) A TLC examination on a s i l ica chromatoplate (CHCI3) showed the presence of three fractions (R f 0.74, 0.53, and 0.23; R f B acetate 0.45). This mixture was separated by column chromatography on alumina (Activity I, 50 gm.) Elution with petroleum ether:benzene (1:1) yielded the least polar fraction (55 mg.) in a TLC pure state. Further elution with benzene:diethyl ether (19:1) removed the main fraction (173 mg.) also in a TLC pure condition. Finally elution with benzene:diethyl ether (1:1) resulted in the recovery 149 o f the most p o l a r f r a c t i o n (87 mg.) which c o n t a i n e d c o l o u r e d i m p u r i t i e s . The main f r a c t i o n when d i s s o l v e d i n the minimum q u a n t i t y o f warm e t h a n o l y i e l d e d a white c r y s t a l l i n e compound (40 m g . ) , m.p. 200-220° which was unchanged a f t e r f u r t h e r r e c r y s t a l l i z a t i o n o r vacuum s u b l i m a t i o n . The l e a s t p o l a r f r a c t i o n was a s y r u p . NMR s i g n a l s : 2.65 (about 6 H, aromat ic H, p o s s i b l y an a r t i f a c t ) , 4.55 and 4.62 ( o v e r l a p p i n g m u l t i p l e t s , o l e f i n i c H , r a t h e r weak), 8.75 and 9 . 0 2 - 9 . 1 8 (angular m e t h y l ) . M.W. (CH 4) 406. C a l c . f o r a t r i o l e f i n C 3 0 H 4 6 , M.W. 406. The c r y s t a l s i s o l a t e d from the main f r a c t i o n were next examined. I^ MR s i g n a l s : 4.60 (1 H, m u l t i p l e t , o l e f i n i c H ) , 5.26 (1 H , m u l t i p l e t , 1 H-C-OAc), 7.90 (3 H, s i n g l e t , a c e t a t e ) , and 9.08 (20.0 H, u n r e s o l v e d , angular m e t h y l ) . MW. 466. C a l c . f o r a d i o l e f i n acetate ^ 2 ^ 1 + 0 2 , M.W. 466. The n o n - c r y s t a l l i n e mother l i q u o r gave almost i d e n t i c a l s p e c t r a except t h a t weak s i g n a l s from two o t h e r a c e t a t e groups were seen i n the NMR spectrum (7.95 and 8 . 0 0 ) . The p o l a r f r a c t i o n was a p p a r e n t l y a m i x t u r e . NMR s i g n a l s : 2.65 (about 1 H, aromat ic H, p o s s i b l y an a r t i f a c t ) , 4.60 (0.5 H , wide m u l t i p l e t , i o l e f i n i c H ) , 5.33 (1 H, m u l t i p l e t , H-C-OAc), 7.95 (3 H, r a t h e r wide s i n g l e t , a c e t a t e ) , 8.78 and 9.15 (angular m e t h y l ) . M.W. (CH 4) 526. C a l c . f o r an o l e f i n d i a c e t a t e 03^51+0^, M.W. 526. B. At Room Temperature Compound B a c e t a t e (360 mg.) was suspended i n a m i x t u r e o f equal volumes (30 ml) o f 36 % H B r / a c e t i c a c i d reagent and a c e t i c anhydride f o r 20 hours at room temperature . The product was poured i n t o c o l d water and e x t r a c t e d t w i c e w i t h benzene. The e x t r a c t was washed w i t h 1 % sodium carbonate , d r i e d over anhydrous c a l c i u m c h l o r i d e and evaporated t o r e c o v e r a whi te c r y s t a l l i n e s o l i d (360 mg.) m.p. 212-220°C. A TLC examinat ion 150 o f t h i s product showed o n l y one spot i d e n t i c a l w i t h B a c e t a t e . T h i s m a t e r i a l was c l e a v e d f o r a second time as above but the r e a c t i o n temperature had t o be g r a d u a l l y r a i s e d t o 47°C to f o r c e the cleavage r e a c t i o n t o take p l a c e . A f t e r i s o l a t i o n o f the product as before a brown gum (404 mg.) was r e c o v e r e d . An examinat ion by TLC showed main ly o l e f i n i c m a t e r i a l p r e s e n t , some unchanged s t a r t i n g m a t e r i a l , and a more p o l a r f r a c t i o n . A chromato-g r a p h i c s e p a r a t i o n was performed on a column o f d e a c t i v a t e d a lumina ( A c t i v i t y I I I , 60 gm.). E l u t i o n w i t h petro leum e t h e r y i e l d e d a p a l e y e l l o w gum (130 mg.) w h i l e f u r t h e r e l u t i o n w i t h b e n z e n e : c h l o r o f o r m (19:1) y i e l d e d unreacted s t a r t i n g m a t e r i a l (70 m g . ) . F i n a l l y the column was s t r i p p e d w i t h benzene:chloroform (4:1) and t h i s e l u t i o n removed a y e l l o w syrup (100 mg.) . Treatment o f t h i s l a s t f r a c t i o n w i t h 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 gave i n low y i e l d a complex m i x t u r e o f p r o d u c t s . E t h e r Cleavage R e a c t i o n on B A c e t a t e w i t h 36 % H B r / A c e t i c A c i d Compound B acetate (590 mg.) was t r e a t e d w i t h 36 % H B r / a c e t i c a c i d reagent (45 ml) f o r 90 minutes at 60-62°C. Al though the r e a c t i o n m i x t u r e was s t i r r e d m a g n e t i c a l l y , some o f the s t a r t i n g m a t e r i a l was a p p a r e n t l y unchanged and d i d not d i s s o l v e . The " s o l u b l e " and ' " i n s o l u b l e " f r a c t i o n s were recovered s e p a r a t e l y by the normal workup procedure p r e v i o u s l y d e s c r i b e d . A TLC examinat ion on a s i l i c a chromatoplate (CHCI3) i n d i c a t e d the s o l u b l e p o r t i o n t o c o n t a i n one f r a c t i o n (R^ 0.76) and the i n s o l u b l e p o r t i o n two f r a c t i o n s ( R £ 0.76 and 0 . 3 3 ; R £ B a c e t a t e 0 . 3 4 ) . The r e a c t i o n products were recombined and separated by column chromatography on alumina ( A c t i v i t y I , 30 gm.) . E l u t i o n w i t h petro leum e t h e r y i e l d e d a brown syrup ( R £ 0 . 7 6 , 173 mg.) . F u r t h e r e l u t i o n w i t h b e n z e n e : d i e t h y l e t h e r (19:1) removed a p a l e 151 y e l l o w s o l i d (180 mg. R f 0.33) and f i n a l l y use o f b e n z e n e : d i e t h y l e t h e r (1:1) r e s u l t e d i n the recovery o f a dark y e l l o w syrup (130 m g . ) . The most non-polar , f r a c t i o n was examined by TLC on s i l v e r n i t r a t e impregnated s i l i c a coated chromatoplates . Use o f benzene as d e v e l o p i n g s o l v e n t showed t h i s f r a c t i o n t o c o n t a i n at l e a s t two compounds i n the r a t i o 4 :1 . Comparison w i t h the most n o n - p o l a r product from the B a c e t a t e e t h e r c leavage r e a c t i o n i n which a c e t i c anhydride was used ( r e f l u x c o n d i t i o n s ) showed these two f r a c t i o n s t o have d i f f e r e n t TLC p r o p e r t i e s . An attempt t o separate the m a t e r i a l from the present experiment i n t o i t s components by column chromatography on a s i l i c a column impregnated w i t h s i l v e r n i t r a t e (25 % Ag NO3, 20 gm.) was a complete f a i l u r e . The most p o l a r f r a c t i o n was a l s o separated by column chromatography on d e a c t i v a t e d alumina ( A c t i v i t y I I , 20 gm.) . Ten f r a c t i o n s were r e c o v e r e d , a l l y e l l o w c o l o u r e d gums and none h a v i n g TLC p r o p e r t i e s corresponding to a d i a c e t a t e . R e c r y s t a l l i z a t i o n o f the middle f r a c t i o n from e t h a n o l y i e l d e d white r o d - l i k e c r y s t a l s , m.p. 222-235° (B a c e t a t e m.p. 205-7°C). I n f r a r e d ( K B r ) ; 1735 and 1255 ( a c e t a t e ) , 1104 (methoxyl) and no s i g n a l at 795 ( t r i s u b -s t i t u t e d double bond). NMR s i g n a l s : no o l e f i n i c H, 5.33 (1 H , m u l t i p l e t , 1 l H-C-OAc), 6.70 (3 H, s i n g l e t , m e t h o x y l ) , 7.23 (1 H , m u l t i p l e t , J-C-OCH3), 7.95 (3 H, s i n g l e t , acetate), 8.66 and 9.13-9.17 (19.2 H , angular m e t h y l ) . Mass spectrum (CH 4 ) ; fragments at m/e 483 (M-15), 438 (M-60) , 423 (M-75) and 391 (M-107); no r e t r o D i e l s - A l d e r ; o t h e r i n t e n s e fragments at m/e 221, 203, 190, 189, 187, 149, 138, and 135. Found: C, 79 .51 ; H. 10.84; 0 , 9 . 8 9 ; M.W. (CH 4) 498. C a l c . f o r C 3 3 H 5 1 + 0 3 : C, 79 .52; H, 10.84; 0 , 9 . 6 4 ; M.W. 498. 152 Serratenediol Dimesylate This compound was prepared according to the procedure described by 75 Bobbitt et al . Serratenediol (48 mg.) was dissolved in dry pyridine (2 ml) and cooled to -15°C in an ice-salt bath. Methanesulfonyl chloride reagent (0.5 ml) was added from a pipette and the reaction mixture allowed to warm slowly to 0°C over a period of three hours. The mixture was again cooled to -12 °C and water added until a solid precipitated. Further addition of water dissolved this solid but a second heavy white precipitate formed. This precipitate was filtered off and washed thoroughly with cold water. The material was almost pure as evidenced by its TLC behaviour on a s i l ica chromatoplate. Attempts to recrystallize the compound from benzene or petroleum ether were unsuccessful. NMR signals: 4.68 (1 H, I multiplet, olefinic H), 5.50-5.85 (2 H, overlapping multiplets, H-C-OMes), 7.00 (6 H, singlet, 2 methane-sulfonyl groups), 9.00-9.30 (22.3 H, angular methyl). Methanolysis of Serratenediol Dimesylate Serratenediol dimesylate (62 mg.) was suspended in dry methanol (25 ml) in a dry 50 ml round bottom flask equipped with heating mantle, upright condenser and calcium chloride drying tube. Reaction was allowed to proceed for 48 hours under reflux. A sample was removed from the flask, evaporated to dryness and examined by TLC on a s i l ica chromatoplate (CHCI3). Three purple spots were observed (R^ . 0.73, 0.58 and 0.50; R^  starting material 0.26). On a second plate the mixture of reaction products was compared with compound B methyl ether as standard (R^ 0.66, brown spot). There did not appear to be any trace of this compound in the reaction product. The mixture was separated by column chromatography on deactivated alumina 153 ( A c t i v i t y I I I , 10 gm.)- E l u t i o n w i t h petroleum e t h e r y i e l d e d the l e a s t p o l a r component (6.5 mg.) and f u r t h e r e l u t i o n w i t h the same s o l v e n t removed the middle f r a c t i o n (10.0 mg.) . Use o f benzene:petroleum e t h e r (3:17) recovered the t h i r d component (9.0 m g . ) . A l l three f r a c t i o n s were non-c r y s t a l l i n e m a t e r i a l s and t h e i r TLC p r o p e r t i e s d i d not suggest the presence i n them o f any methyl e t h e r d e r i v a t i v e s . I n f r a r e d (CHC1 3 ) ; no a b s o r p t i o n band at 1100 i n any o f the f r a c t i o n s . NMR s i g n a l s (100 Mc/s^ m i c r o c e l l ) ; [ f i r s t f r a c t i o n ] , 4.66 ( o l e f i n i c H ) , 8.80 and 8 .92-9.14 (angular m e t h y l ) ; [second f r a c t i o n ] , 4.66 ( o l e f i n i c H, 6.88 ( s i n g l e t , m e t h a n e s u l f o n a t e ) , 8.78 and 8 . 9 2 - 9 . 2 0 (angular m e t h y l ) ; [ t h i r d f r a c t i o n ] , 4.69 ( o l e f i n i c H ) , 5.83 ( m u l t i p l e t , H - i-OMes) , 7.06 ( s i n g l e t , m e t h a n e s u l f o n a t e ) , 8.79 and 9 . 0 3 - 9 . 3 2 (angular m e t h y l ) . An attempt t o measure the. m o l e c u l a r weights o f the components i n the three f r a c t i o n s gave i n d e f i n i t e r e s u l t s . A l l o f these substances s t i l l seemed t o be m i x t u r e s . Compound B Methyl E t h e r Compound B (115 mg.) was methylated as a l r e a d y d e s c r i b e d f o r the p r e p a r a t i o n o f compound A methyl e t h e r i n which potass ium metal (0.29 gm.) and methyl i o d i d e (5 ml) were u t i l i z e d i n hot to luene as the r e a c t i o n medium. Workup by the u s u a l procedure gave the crude methyl e t h e r (151 m g . ) . Examinat ion by TLC (CHCI3) showed m e t h y l a t i o n to be o n l y p a r t i a l l y complete (Rf compound B 0 . 3 6 , R^ methyl e t h e r 0 . 6 5 , brown s p o t s ) . The r e a c t i o n was repeated u s i n g compound B (164 mg.) and the crude methyl e t h e r (183 mg.) was recovered as b e f o r e . These two f r a c t i o n s were combined and p u r i f i e d by column chromatography from d e a c t i v a t e d a lumina ( A c t i v i t y I I I , 25 gm.) . E l u t i o n w i t h petroleum e t h e r y i e l d e d the pure methyl e t h e r (128 mg. , y i e l d 44 %). F u r t h e r e l u t i o n w i t h benzene:petroleum e t h e r (1:1) recovered 154 unreacted compound B (139 mg.). Crystallization of the methyl ether from petroleum ether yielded white crystals, m.p. 243-5°, [a]2,0 - 7 6 . 4 ° . Infrared (KBr); no absorption at 3500 (hydroxyl),1100 (methoxyl), and 790 (trisubstituted double bond). NMR signals: 4.67 (1 H, multiplet, olefinic H), 6.70 (6 H, singlet, methoxyl), i 7.16 and 7.23 (2 H, overlapping multiplets, H-C-OCH3); 9.10-9.33 (18.1 H, angular methyl). NMR (100 Mc/s); 7.16 and 7.23 (overlapping triplets, J = 3.0 c.p.s.)- Mass spectrum (CH 4); fragments at m/e 455 (M-15), 438 (M-32), and 423 (M-47); 316, 284, and 269 (retro Diels-Alder); 234, 221, 190, and 189 (allylic cleavage); other intense fragments at m/e 279, 187, 167, 149, 137, and 135. Found: C, 81.78; H, 11.56; 0, 7.01; M.W. (CH 4), 470. Calc. for C32^5k02: C, 81.70; H, 11.49; 0,6.81; M.W. 470. Meerwein-Ponndorf Reduction of B Ketone Isopropanol was dried by distillation from calcium oxide, previously ignited at 700-900°C for five hours. A one molar solution of aluminum iso-propoxide in dry isopropanol was prepared by a standard procedure. The apparatus for the reduction consisted of a 50 ml pear-shaped flask with fractionating side-arm attachment equipped for distil lation. Compound B ketone (270 mg.) was reacted with 1 M aluminum isopropoxide in isopropanol (5 ml) in a hot oi l bath. The temperature at the head of the column varied in the range 60-80°C, and isopropanol was added to the reaction flask from a dropping funnel so as to maintain a fairly constant volume. The presence of acetone in the distillate was demonstrated using 2,4-dinitrophenylhydra-zine reagent. After five hours heating was discontinued and the isopropanol removed in vacuo. The crude product was dissolved in ether, the ether extract washed with 5 % hydrochloric acid, then with water and was dried 155 over anhydrous magnesium sulfate. The product was recovered as a yellow solid (270 mg.) after filtration and evaporation of the solvent. An examin-ation by TLC (silica, CHCI3) showed the formation of two products (Rf 0.36 and 0.19) in about the ratio 3:2. (Rf B ketone 0.46, compound B 0.36). Separation was accomplished by column chromatography on deactivated alumina (Activity III, 25 gm.). Elution with benzene'.petroleum ether (3:7) yielded compound B (120 mg.^ 45 % yield). Further elution with this solvent mixture yielded epi compound B (80 mg., 30 % yield). Compound B fraction, m.p. 280-281°C after vacuum sublimation. NMR signals (100 Mc/s); 4.74 (1 H, multiplet, olefinic H), 6.60 (1 H, triplet, 1 J = 2.5 c.p.s., H-C-OH), 6.75 (3 H, singlet, methoxyl), 7.29 (1 H, triplet, J = 2.5 c.p.s, H-C-OCH3), and 9.10-9.36 (angular methyl). Epi compound B fraction, m.p. 271.5-272°C after vacuum sublimation. Infrared (KBr);3450 (hydroxyl), 1100 (methoxyl), and 795 (trisubstituted double bond). NMR signals (100 Mc/s); 4.72 (1 H, multiplet, olefinic H), 1 6.74 (3 H, singlet, methoxyl), 6.80 (1 H, partly hidden multiplet, jj-C-OH), 7.26 (1 H, triplet, J = 2.5 c.p.s., H-C-OCH3), and 9.04-9.34 (angular methyl). Mass spectrum (MS9); fragments at m/e 441 (M-15), 438 (M-18), 424 (M-32), 409 (M-47), and 391 (M-65); 316, 301, 284, 269 (retro Diels-Alder); 220, 221, 203, 190 and 189 (allylic cleavage); other fragments at m/e 175, 161, 147 and 135. Found: M.W. (MS9) 456.3972 (standard 464). Calc. for C31H52O2 456.3967. Meerwein-Ponndorf Reduction of Serratenedione A sample of serratenedione (55 mg.) was reacted as previously described with a molar solution of aluminum isopropoxide in isopropanol (5 ml.). After five hours the reaction was stopped and the solvent evaporated 156 under reduced p r e s s u r e . The product was d i s s o l v e d i n e t h e r , washed w i t h aqueous h y d r o c h l o r i c a c i d , then w i t h water and was d r i e d over anhydrous magnesium s u l f a t e . A f t e r f i l t r a t i o n and e v a p o r a t i o n the crude r e a c t i o n product was r e c o v e r e d . A TLC examinat ion ( e t h y l acetate:benzene 1:3) showed the presence o f at l e a s t t h r e e products ( R £ 0 . 5 9 , 0.45 and 0 . 3 5 , grey c o l o u r ) . Two o f these spots were i d e n t i c a l i n p o s i t i o n w i t h a u t h e n t i c samples o f 2 1 - e p i s e r r a t e n e d i o l ( R £ 0.44) and s e r r a t e n e d i o l ( R £ 0 . 3 6 ) . The t h i r d and most p l e n t i f u l product was not f a m i l i a r and was not unreacted s e r r a t e n e d i o n e (Rf 0 . 7 0 , orange c o l o u r ) . The m i x t u r e was separated by column chromatography on d e a c t i v a t e d a lumina ( A c t i v i t y I I I , 10 gm.) . E l u t i o n w i t h petro leum ether:benzene (1:1) y i e l d e d the major component (19 mg. , 34 % y i e l d , F r a c t i o n 1), F u r t h e r e l u t i o n w i t h benzene:ether (9:1) removed the second component (14 mg. , 25 % y i e l d , F r a c t i o n 2 ) . F i n a l l y e l u t i o n w i t h benzene:ether (4:1) recovered the most p o l a r component (6 mg. , 11 % y i e l d , F r a c t i o n 3 ) . The three f r a c t i o n s , as r e c o v e r e d , possessed wide m e l t i n g ranges (183-193° , 215-230°, and 215-245° r e s p e c t i v e l y ) . When F r a c t i o n 1 was examined c a r e f u l l y on a s i l i c a chromatoplate (CHCI3) t h e r e was a s u g g e s t i o n o f two components b e i n g p r e s e n t . T h i s was conf irmed when a p l a t e was developed three times i n c h l o r o f o r m before s p r a y i n g . The spots were then b e t t e r d i f f e r e n t i a t e d . I n f r a r e d (CHCI3): no a b s o r p t i o n at 1700 showing t h a t no k e t o l was present as an i m p u r i t y . NMR s i g n a l s (100 M c / s ) : F r a c t i o n 1; 4.72 (weak m u l t i p l e t , o l e f i n i c H ) , 6.65 (two p a r t i a l l y o v e r l a p p i n g I t r i p l e t s , J = 2.5 c . p . s . , H-C-OH). F r a c t i o n 2; 4.70 (weak m u l t i p l e t , I o l e f i n i c H ) , 6.60 (unreso lved m u l t i p l e t , ^ H - C - O H ) , and 6.85 (unresolved 1 m u l t i p l e t , H-C-Oti). A second p o r t i o n o f s e r r a t e n e d i o n e (200 mg.) was reduced and separated i n the same way. The r e s u l t was i d e n t i c a l t o the above and 157 Fractions 1, 2 and 3 weighed 92, 57, and 59 mg. respectively. However, in this instance the third fraction was a mixture of the second and third components. Once again Fraction 1 was seen to contain two components, this time in roughly equal proportions. On this occasion a sample of the pure rearranged diol (31 mg.) was eluted first in the chromatographic separation of the reaction product. NMR signals (100 Mc/s); no absorption at4.70, 6.64 l (2 H, partly overlapping triplets,_H-C-OH), 9.04-9.20, angular methyl). The reduction of serratenedione (59 mg.) was again performed using one molar aluminum isopropoxide solution (3 ml.). After evaporation of the solvent^ water was added and the pH lowered to 3.5 by potentiometric tritration with dilute hydrochloric acid. The product was extracted into ether and separated as before to yield Fraction 1.(18 mg.) which now conclusively showed only one spot when examined by TLC. Reduction of another quantity of serratenedione (116 mg.) followed by the modified workup procedure yielded a further quantity (44 mg.) of pure Fraction 1. Vacuum sublimation gave white crystals, m.p. 289°C, [a] 2 0 - 3 5 . 8 ° . Infrared (KBr); 3400-3300 (hydroxyl) and 787 (trisubstituted double bond). NMR signals (100 Mc/s; 4.73 (1 H, multiplet, olefinic H), 6.56-6.72 (2 H, partially overlapping i triplets, J = 2.5 c.p.s., H-C-OH) and 9.09-9.24 (20.0 H, angular methyl). Mass spectrum (MS 9J; fragments at m/e 427 (M-15), 424 (M-18), 409 (M-33), 406 (M-36), 391 (M-51), 385 (M-57), and 367 (M-75); 302, 284 and 269 (retro Diels-Alder); 220, 207, 202, 190 and 189 (allylic cleavage); other intense fragments at m/e 187, 161, 147 and 135. Found: M.W.(MS9) 442.3837 (standard 464). Calc. for C 3 oH 5 0 0 2 ; M.W. 442.3810. Methylation of 3a, 21g-dihydroxy-Altt-serratene A sample of 3a, 21g-dihydroxy-Altf-serratene (31 mg.) was twice methylated with potassium metal (150-250 mg.) and methyl iodide (2 ml.) in 158 hot toluene (area 80°C) as previously.described for the preparation of, compound B methyl ether. After the usual workup procedure an examination of .the product by T.L.C. (CHC13 , silica) showed the presence of small; amounts of two compounds behaving similarly to compound B but no trace was seen of any compound with value comparable to that of compound B tiethyl ether* In a third attempt the reaction with potassium metal was conducted under reflux conditions•for four hours and, after the addition of.the methyl iodide, reaction was allowed to take place for a futther five hours< Isolation of the reaction product in the usual way yielded a yellow oi l (50 mg.) which was separated by column chromatography on.deactivated alumina (Activity III, 10 gm.). Elution with petroleum ether (b.p. 65-110°C) yielded a colourless oily liquid (28 mg.) containing a trace of ;a compound with T.L.C. behaviour similar to that of compound B methyl ether. Further elution with petroleum;ether:benzene. (4:1) yielded trace amounts of two compounds with T.L.C. behaviour,similar to compound B and stripping the column with petroleum ether:benzene (3:7) removed an impure fraction containing some unreacted starting material. An attempt was made to further purify the above.fractions as fallows. The main fraction was distilled (150-160°G at 0.07 mm.) using a glass tube placed.in a;vacuum sublimation .apparatus. A.colourless liquid was removed and a small amount of a white solid residue.remained in the tube. This was 11 sublimed under the usual conditions to give a solid apparently contaminated with traces, of an o i l . The material was compared with compound Bmethyl ether on a s i l ica .chromatoplate (CHC13; ginger colour, R^  0.84; compound B methyl ether, ginger, R^  0.84) but was impure. The liquid distillate gave the following NMR signals: 2.75 [strong, complex aromatic], 7.06 [aromatic CH 3], 8.75 and 9.12 [angular CH 3]. 100 Mc/s; additional weak signals at 159 4.56 and 4.84 [olefinic proton], 6.14, 6.76 and 6.95. Examination by T.L.C. (silica/AgNO3 } petroleum ether) showed the presence of at least three components.. A fraction containing mainly a compound with T.L.C. behaviour similar to compound B was vacuum sublimed (220-240°C at 0.07 mm.) The sublimate had m i p . 200-240°C andwas examined on a s i l ica chromatoplate (CHC13, ginger, R £ 0.43; compound B, ginger, R £ 0.43). A second component; was also present (ginger, R £ 0.39). The methylation experiment was repeated twice on a second sample (30 mg.) of ,3a, 213-dihydroxy-All4-serratene.. This time dry benzene was used in place of.toluene and the reaction was carried out under reflux conditions. Again very l i t t le 'of the dimethyl ether derivative appeared to be present. In a third methylation the rate, of stirring was sharply increased whereupon most of the diol reacted. Separation of the reaction product by column chromatography as previously described gave analogous results with the desired methyl ether being present in very low yield. Ether Cleavage of Compound B;.acetate with Boron Trifluoride Etherate A sample of compound B acetate (239 mg.) was dissolved in a mixture of acetic anhydride (8 ml.) and anhydrous ether (2 ml.). The solution was cooled to 0°C in the refrigerator and borontrif luoride etherate (1.5" ml.) added; The temperature was maintained at,0°C for forty five minutes and was poured on:to crushed ice even,though not, a l l of the compound had; dissolved. After leaving for two hours the acetic acid was neutralized by the addition of solid potassium bicarbonate and the reaction product partitioned between chloroform.and water. The chloroform extract was dried over anhydrous magnesium sulfate; filtered and evaporated to yield the crude reaction product (269 mg.) An examination by T.L.C. (si l ica, chloroform) 160 showed most of the compound B acetate apparently unreacted (olive green, Rf 0.64) with two other fractions also present (chocolate brown, Rf 0;68 and 0.44). The presence of uncleaved material was seen from the NMR spectrum; signals at 6.68 (singlet, methoxyl) and 7.22 (multiplet, H-CJ-OCH3): The crude.reaction; product was reacted with lithium aluminum hydride (500 mg.) in dry refluxing benzene for five hours. After cooling to room, temperature the excess reducing agent was destroyed by the controlled addition of moist tetrahydrofuran. The inorganic.residues were removed by filtration and the solvent evaporated to yield a white solid (225 mg.). Examination by T.LC. (sil ica, chloroform) showed,the complete absence of compound B acetate. The major product was apparently compound B and four more polar compounds, possibly diols, were noted. The crude product so obtained was dissolved in dry,pyridine (2 ml.) and poured into a stirred suspension of dry chromic oxide (197 mg.) in pyridine (5 ml.). After allowing to react at room temperature for twenty hours the reaction mixture was poured into cold water and the product isolated as described for the preparation of compound A ketone. The crude oxidation product (224 mg.) was separated into its components by column chromatography on deactivated alumina (Activity III, 25 gm.). Elution with petroleum ether:benzene (37:3) yielded a mixture (128 mg.) of compound B ketone,(Rf 0.52, brown) with a slightly less polar material (Rf 0.57, brown). Elution with petroleum ether:benzene (7:3) yielded,a T.L.C. pure fraction (Fraction A, 20 mg.) of an unknown material which might have been a dione (Rf 0.32, copper colour). Further elution With petroleum,ether: benzene (1:1) yielded a mixture (10,mg.) of this compound with two more polar compounds and stripping of the column with ether gave a final fraction (16 mg.) containing at least six components. A comparison of these fractions 161 with authentic A*^-serratenedione on a silica, T,L.C. plate (CHCI3) showed the complete absence of this compound (R£ 0.23, orange) in a l l the above fractions. "Fraction A" as isolated above was further examined. Infrared (KBr); 3400 (hydroxyl, fairly broad), 1695. (carbonyl, rather broad), no methoxyl group (1100), and 795 (trisubstituted double bond). The mass spectrum (MS 9) was clearly that of a-mixture. Peaks.were observed at m/e 421, 422, 436, 438, 439, 440, 452, 464, 465 and 470. Ether Cleavage of Desoxy Dihydro Compound B. (44% HBr/Acetic Acid) Desoxy dihydro compound B (370 mg.) was reacted at room temperature for twenty hours in a mixture of saturated HBr in acetic acid (44%, 50 ml.) and acetic anhydride (40 ml.). A l l the material Had dissolved before the reaction was terminated, by pouring on to crushed ice. After standing for two hours the product was extracted into chloroform washed with 5% potassium bicarbonate solution, then with water and was dried over anhydrous magnesium sulfate. Filtration and evaporation of the solvent yielded a brown syrup containing some semi-crystalline material. This product Was separated into two main fractions-by column chromatography.on deactivated alumina (Activity III, 36 gm.). Elution with petroleum ether yielded a colourless syrup (158 mg/) which was apparently olefinic and showed two components when examined by.T.LC. (silver nitrate/sil ica, petroleum ether). Further elution with petroleum ether:chloroform•(9:1) yielded a white crystalline solid (137 mg.) Infrared (CHCI3): 1730 and 1230 (acetate), absence of methoxyl (1100). NMR Signals: 5.36 (1 H, multiplet, H_-C-0Ac), 7.92 (3 H, singlet, acetate), and 9.10-9.21 (angular methyl). Further information was obtained on the olefin: fraction as follows. N.M.R. signals: 4.64 and 4.80 (weak multiplets, olefinic protons), 8.76 and 9.14-9.18 (angular methyl). An examination by VPC using as liquid phase 162 S.E. 30 gum rubber (20%) indicated the presence of at least four components. The acetate fraction was treated with lithium aluminum hydride (500 mg.) in dry refluxing tetrahydrofuran for four hours and the excess reducing agent was decomposed by the controlled addition of wet tetrahydrofuran. Filtration and evaporation?of solvent yielded a white crystalline product which upon examination by T.L.C. (silica, CHC13) was observed to contain three components (R^ . 0.55, 0.48 and 0.28, yellow). The slowest spot was present only as a minor component. The mixture was separated by column chromatography on deactivated alumina (Activity III, 20 gm.). Elution with petroleum ether:benzene (17:3) yielded first a mixture of the two less polar alcohols (50,mg.) and then a pure fraction of the second alcohol (59 mg., alcohol 6).; Further elution with petroleum ether:benzene (1:1) yielded the third component (8 mg.) which upon examination by T.L.C. (si l ica, CHCI3) was seen to contain two compounds in the approximate.ratio 1:4. An attempt to separate the mixed i fraction containing the first pair of alcohols by further column chromatography on deactivated alumina (Activity III) was largely unsuccessful. However a pure fraction (4 mg.) of the faster running alcohol a was recovered by elution with petroleum ether:benzene (19:1). The pure alcohol 3 , m.p. 219-221°C was further examined as follows. Infrared (KBr); 3560 (hydroxyl) . NMR signals (100 Mc/s); 6.66 (1 H, triplet 1 J = 3.0 c.p.s, H-C-OH) and 9.02-9.24 (23.7 H, angular methyl). Mass spectrum (MS 9); fragments at m/e 413 (M-15), 410 (M-18), and 395 (M-33); most intense fragments above m/e 100 occurred in.the order,121, 136, 123, 109, 107, 137, and 149; other significant peaks were observed at 189, 190, 207, 231 and;259. Found* M.W. 428.4003 (Standard m/e 464) Calc. for C30H52O: M. W. 428.4017. 163 Jones Oxidation of Alcohol B Alcohol 3.(50 mg.) was dissolved in warm spectral grade acetone,(20 ml.) in a flask equipped with a magnetic stirrer. Freshly prepared J,ones reagent (0.3 ml.) was added and the reaction was terminated after twenty minutes by the addition of reagent grade methanol (5 ml.). After stirring for a further ten minutes the solvent was evaporated and the product partitioned between chlorofrom and water. The chloroform layer was washed with water, dried over anhydrous magnesium sulfate, filtered and evaporated. The product was purified by column chromatography on deactivated alumina (Activity III, 10 gm.). Yield 44,mg. white crystals, m.p* 185-193°C without recrystallization. ORD (C, 0.02, CH30H), 21°C, O J 7 0 0 0°> [*] 589 -40° , [<t>] 305 +2867°, [<|>]266 -25099, _ [«J>]2itO "1396. Molecular amplitude +53.7. Addition of hydrochloric acid to the methanol solution of the ketone (0.05 N) had almost no effect on the amplitude of the Cotton effect after ninety minutes. Infrared (KBr): no hydroxyl absorption, 1695 (carbonyl). NMR signals (100 Mc/s);,7,52 (2 H, multiplet, -CH_2-C0-) , and.8.89-9.13 (23.7 H, angular methyl). Mass spectrum (CH 4); fragment at m/e 411 (M-15), other important;fragments at 274, 232, 205; 138 and 123. Found: M.W. 426.3909 (standard m/e 464). Calc. for C 3 0 H 5 0 O ; M.W. 426.3861. Borohydride Reduction of Ketone B Tosylhydrazone A sample of ketone,B (30 mg.) was reacted with tosylhydrazine reagent (31 mg.) in methanol (3 ml.) in a 25 ml. flask equipped with reflux condenser. Acetyl chloride (3 drops) was used as catalyst and reaction was allowed to take place for 45 minutes under reflux. The white crystalline tosylhydrazone derivative Was filtered off from the methanol after cooling. The air dried material had m.p. 202-4°C. I.R. (KBr).; 3200 (NH), 1635 ( C = N-), 1590 (aromatic), and 1162 (-S02-) . 164 The tosylhydrazone was transferred to a 50 ml. round bottom flask equipped with reflux condenser. Sodium borohydride (55 mg.) and spectral grade dioxane (15.ml.) were added and reduction was allowed to proceed for ten hours under reflux. The solvent was removed in vacuo and the reaction product taken up in chloroform solution. The chloroform.extract was washed with water, dried over anhydrous magnesium sulfate, filtered and evaporated to yield a white crystalline solid (23 mg.). Examination by T.L.C. (silica, petroleum ether) showed mainly one component present. This was recovered by column chromatography from alumina (Activity I, 10 gm.). Elution with petroleum ether yielded a white crystalline solid (8 mg.). This compound was recrystallized from spectral grade acetone. White leaflike crystals, m.p. 184-6°C. Authentic 6-serratane (Professor Inubushi) m.p. 184-7°C. Mixed m.p. 184-6°C. The compounds showed identical T.L.C. behaviour on both s i l ica (R£ 0.70, ginger) and alumina plates developed with petroleum ether. However a-serratane could not be distinguished from 8-serratane by T.L;C. Mass spectrum (CH 4); fragment at m/e 397 (M-15); major fragments at 231, 192, 191, 163, 137, and 123- Found: M.W. 412.4078 (standard m/e 414). Calcd. for C30H52: M.W. 412.4068 Hydrogenation of Desoxy Compound B Attempts to hydrogenate desoxy compound B with Adam's catalyst at atmospheric pressure in acetic acid or acetic acid : tetrahydrofuran (1:1) solution were unsuccessful. The hydrogenation was successfully accomplished in a Parr bomb at 50 p . s . i . hydrogen pressure using a solution of desoxy compound B (708 mg.) in acetic acid: chloroform (1:1, 140 ml.) with Adam's catalyst (320 mg.). After three hours the catalyst was removed by filtration and the product recovered by evaporation of the solvent under reduced pressure. Examination by T.L.C. (sil ica, CHCI3) showed that hydrogenation 165 had occurred (yellow spot, starting material brown, similar R £ value). The reaction product was purified by column chromatography on alumina (Activity I, 50 gm.). Elution with petroleum ether (b.p. 65-110°C) removed-dihydro desoxy compound B (687 mg.) 97% yield). White prisms from petroleum ether, m.p. 195-8°C. Infrared (KBr); 1100 (methoxyl), no absorption at.795 (trisubstituted double bond). N.M.R. signals; no olefinic proton, 6.67 (3 H, singlet, methoxyl), 7.20 (1 H, multiplet, H-C-0CH3) and 9.06-9.21 (angular methyl). Ether Cleavage Reaction of Desoxy Dihydro Compound B (36% HBr/Acetic Acid) Isolation of A2-8-Serratane. Dihydro desoxy compound B (590 mg.) was suspended in a mixture of freshly prepared 36% HBr in glacial acetic acid (50 ml.) and acetic:.. anhydride (50 ml.). After stirring for 19 hours at room temperature the reaction mixture was poured on,to crushed ice, allowed to stand for-two. . hours and extracted into petroleum ether (b.p. 30-60°C). The solution was washed twice with water, dried over anhydrous potassium carbonate, filtered and evaporated to recover a white solid. This was seen by T.L.C. examination to be unreacted starting material. Infrared (CHCI3); no acetate group (1730 and 1250)^ , 1085 (methoxyl) . The experiment was repeated for 2 1/2 hours at 70-80°C in a water bath. Recovery of the product as above.yielded a semi-crystalline material with a foul odour. Examination by,T.L.C. (silica, CHCl^ ) showed the presence of two fractions(R£ 0.80, brown; 0.49, brown). The.mixture was separated by column chromatography on alumina (Activity I, 25 gm..). Elution with petroleum ether removed an olefinic,fraction (457 mg.). Further elution with benzene removed a mixed fraction (34 mg.) containing olefinic and unreacted starting material. Finally an acetate fraction (97 mg.) was recovered by elution with benzene:ether (4:1). Infrared 166 (CHC13); 1710 and 1250 (acetate), no methoxyl (1100). This fraction was seen to contain polar impurities when examined by T.L.C. and was not further studied. The olefin fraction showed only one spot on a s i l ica plate when developed in chloroform. On a silver nitrate impregnated s i l i ca plate developed in this solvent two narrowly separated spots were seen. However, when petroleum ether was utilised as developing solvent^use of the silver . nitrate impregnated layer showed the presence of six spots (R^ . 0.67, 0.61, 0.55, 0.47, 0.42 and 0.33). The latter spot was the most,intense. The olefin mixture was separated on a column packed with silver nitrate impregnated s i l ica (20% AgN03, 50 gm.). Elution with petroleum ether (b.p. 65-110°, 25 ml. fractions) removed al l the material applied in thirteen fractions according to the table below. A white crystalline material was isolated in fractions 8 to 10. Fraction No Weight mg. R^ Value of Components 3 18 0.69 4 121 0.69 and 0.63 5 35 0.63 and 0.58 6 8 0.51 7- 68 0.42 8 100 0.39 9 32 0.39 10 10 0.39 Fractions 8-10 were bulked and rechromatographed on silver nitrate impregnated s i l ica (15 gm.). The main fraction from the second chromato-graphy (67 mg.) was recrystallized three times from spectral grade acetone. 167 White needles, m.p. 188-191°C. Infrared (KBr); 1650, 730 and 720 (cis disubstituted double bond), no acetate or methoxyl. NMR signals (100 Mc/s, benzene); 4.57 and 4.60 (2 H, complex multiplet, olefinic H), 7.85 (1 H, triplet, J = 2.0 c.p.s., a l ly l ic proton), 8.04 (IH, multiplet, a l lyl ic proton), and 9.01-9.13 (21.4 H, angular methyl). Irradiation at x 8.04 caused the alteration of this region to two rather wide peaks of equal height. Alternately irradiation at x 4.58 caused a simplification of the signals at 7.90 and 8.04 attributed to the al lyl ic protons. The spectrum was also run in deuterochloroform solution and this permitted slightly better resolution of the olefinic proton region which now showed a narrow triplet (J = 1.0 c.p.s.) centred at 4.62 and an apparent singlet at 4.65. Mass spectrum (CH 4); fragments observed at m/e 395 (M-15), 355 (M-55) and 328 (M-82); other prominent fragments at m/e 191, 189, 150, 137 and 123. Found: C, 87.90, H, 12.43; M.W. 410.3919 (standard m/e 414). Calculated for C30H52; C, 87.80, H,.12.20; M.W. 410.3912. Hydrogenation Of A2-g-Serratane ~ A sample of Az-B-serratane (38 mg.) was hydrogenated in ethyl acetate solution (30 ml.) in the presence of Adam's catalyst (17 mg.) for two hours. The catalyst was removed by filtration and the solvent evaporated. The product was purified by chromatography on a short column of deactivated alumina (Activity III, 10 gm.). Elution with petroleum ether yielded a white solid (26 mg.) which after recrystallization from acetone yielded needlelike crystals, m.p. 175-180°C. Vacuum sublimation raised the melting point to 181-4°C. The g-serratane obtained from the previous ether cleavage reaction sequence (ex acetate) had m.p. 182-5°C. Mixed m.p. 181-4°C. The two samples had identical T.L.C. properties (silica, petroleum ether) and gave superimposable quantitative infrared spectra (KBr). NMR signals: no low field protons, 9.10-9.24 (22.5 H, angular methyl) Mass spectrum 168 (CH 4); fragment at m/e 397 (M-15); intense fragments at m/e 231, 202, 200, 191, 137 and 123. (Standard m/e 414). Found: C, 87.20, H, 12.65; M.W. 412.4066. Calculated for C 3 o H 5 2 ; C, 87.37, H, 12.62; M.W. 412.4068. Hydrogenation of Sample of A ^ -Serratene A sample of serratene (32 mg.) was received from Professor Berti. This material, m.p. 237-9°C, was hydrogenated at atmospheric pressure in glacial acetic acid solution (50 ml.) in the presence of Adam's catalyst (21 mg.) for sixteen:hours. The catalyst was removed by filtration and the solvent evaporated. After chromatography on a short1 column of deactivated alumina (Activity III, 10 gm.) a white crystalline material (34 mg.), m.p. 157-160°C, was recovered. Attempts to elevate the melting point by recrystallization from acetone and by vacuum sublimation were unsuccessful. Finally recrystallizationfrom ethyl acetate:methanol yielded some needlelike crystals, m.p. 180-184°C. NMR signals (100 Mc/s); no low field protons, 9.13-9.26 (25.3 H, angular methyl). The quantitative infrared spectrum (KBr) of this material was compared with similar spectra obtained from the B-serratane samples derived from the ether cleavage reaction sequences on desoxy dihydro compound B. Al l three spectra were superimposable. Separation of Extract Z from Sitka Spruce Bark Extract Z (27.7 gm.) containing only neutral constituents was first widely separated by column chromatography on deactivated alumina (Activity III, 1000 gm.). Thus elution with petroleum ether (b.p. 65-110°C) yielded first a pale yellow oil (I, 0.366 gm.) having astrong blue fluorescence in U.V. light. This was followed by a paleyellow wax (II, 1.491 gm.) and then by a crystalline fraction (III, 7.066 gm.) containing mainly the 169 triterpenoid compounds B and C plus a third, unknown component. Further elution of the column with petroleum ether:benzene (7:3) removed another crystalline residue (IV, 1.796 gm.) containing also the triterpenes B and C but without the third component. Further elution with this solvent system and with petroleum ether:benzene (1:1) removed a fraction (V, 0.776 gm.) containing' two other unknown materials with R £ values falling between those of compound B and A on s i l ica plates. Use of the solvent system petroleum ether:benzene (3:7) resulted in the recovery of a fraction (VI, 2.327 gm.) containing mainly compound A and further elution with this solvent removed a green wax (VIII, 0.979;gm.) containing a mixture of compounds A and F with a third more polar constituent. The remainder of the material (VIII, 10.194 gm.) was recovered from the column by stripping with benzene and finally with ether. This fraction, a sticky yellow syrup, did not contain appreciable quantities of compounds showing discrete spots when examined by T.L.C. (silica, ethyl acetate/benzene, SbCls). The separation is summarised in the table below. Fraction II was dissolved in Column Chromatographic Separation of Extract Z  Fraction No. Solvent Compounds present Weight gm. I pet. ether Unknown 0.366 II " X etc. 1.491 III " C + B + P 7.066 IV pet.ether/benzene C + B 1.796 7:3 V pet. ether/benzene B + A + 2 unknowns 0.776 1:1 VI pet. ether/benzene . A 2.327 3:7 VII " A + F + unknown. 0.979 VIII ether stripped fats 10.194 Total 24.995 170 the minimum quantity of warm acetone and allowed to stand whereupon a white waxy solid "X" was precipitated. This was filtered off and dried. Yield; 317 mg. m.p. 69-70°C. Fractions III and IV were bulked'together and treated three times succesively with warm petroleum ether. After each treatment: the solvent was allowed to cool and was filtered off,from the 'undissolved crystalline residue. In this way was recovered a fraction (3.403 gm.) of almost pure compound B containing a trace of compound Ci The soluble. portion (5.459 gm.) was a yellow syrup with a sweet smell. From this was distil led (up to 220°C at 0.2 mm. Hg.) a yellow5 oi l (1.930 gm.) containing mainly an unknown compound P and traces of compounds B and C. Further treatment of the pot residue with cold petroleum ether resulted in the removal of a white solid containing compounds B and C (0.868 gm.). The remaining residue was a yellow syrup (2.749 gm.) containing traces of compounds C and B. This residue exhibited a carbonyl absorption band (1700) in the I.R. Spectrum (CHCl^ ) and was believed to contain mainly, fatty acid ester components •. Treatment of fraction V in the usual way with petroleum ether resulted in the recovery of a white solid (0.152 gm.) containing compounds A and B. The residue showed a.carbonyl absorption band (1700) in the I*R. spectrum (CHC1 ). Fraction VI also was treated three times with petroleum ether to recover a T.L.C. pure fraction (1;280 gm.) of compound A. The remaining yellow liquid also showed the presence of the carbonyl grouping. Isolation of Compound C Attempts to isolate pure compound C from mixtures with B by means of preparative scale T.L.'C. on s i l ica were rather unrewarding because of the very similar R,, values. An almost pure sample (38 mg.) was however 171 obtained by this method from 250 mg. of such a mixture. A sample of the compound (526 mg.) was obtained by column chromo-tography on deactivated alumina (Activity III, 1000 gm.) of a sample (20.638 gm.) of the total acetone soluble material (including acidic,and phenolic components) removed, from the original petroleum ether bark extract. The eluting solvent used was petroleum ether:benzene (4:1). Recrystallization from acetone gave white needlelike crystals; m.p. 180-185°C. The compound Was also isolated by a modified procedure. Thus the mixture of compounds B and C (868 mg.) isolated from Fractions III and IV of extract Z was hydrogenated in glacial acetic acid solution with Adam's catalyst as described under the preparation of dihydro compound B. The latter has a somewhat lower R £ value than compound B and compound C did not hydrogenate so that the mixture became easier to separate. Thus the hydrogenated mixture was separated by column chromatography on deactivated alumina (Activity III, 25 gm.) Elution with petroleum.ether yielded 195 gm. of T.L.C. pure compound C. Recrystallization of this material from n-heptane gave long,white needles, m.p. 180-186°C. [a]2)0 +23.1° . Infrared (KBr): 3400 (hydroxyl, strong), 1100 (methoxyl) and no.trisubstituted double l bond (795). NMR signals; no olefinic proton, 6.54.(1 H, multiplet, _H-C-OH), 6.67 (3H, singlet, methoxyl), 7.22 (IH, multiplet,. H-C-0CH3), and 9.04-9.24 (angular methyl). NMR signals (100 Mc/s): 6.54 (triplet, J = 2.5 c.p.s.), 7.22 (triplet, J = 2.5 c.p.s.), Mass•spectrum (CH 4);.fragments at m/e 454 (M-2), 441 (M-15), 438 (M-18), 424 (M-32), 409 (M-47) and 391 (M-65); no retro Diels-Alder; prominent fragments at m/e 236, 221, 203, 190, 189, 187, 147 and 135. Found: M.W. 456.3954 (standard m/e 464). Calc. for C31H52O2: MW. 456.3977. 172 Acetylation of Compound C Compound C (108 mg.) was acetylated at room temperature for three days in a mixture of dry pyridine (5 ml.) and acetic anhydride (5 ml.). The solution was poured into cold water, the crude acetate filtered off and recovered as previously described for the preparation of;compound A acetate, The crude acetate (109 mg.) was examined by T . L . C . ( s i l i c a , CHC13, R^  0.53; compare compound C,0.40). The product was chromatographed on deactivated alumina (Activity III, 20 gm.). Elution with petroleum ether:benzene (17:3) yielded the pure . acetate (70 mg.). Recrystallization from petroleum ether gave colourless rods,m.p. 222-5°C Infrared (KBr); 1725 and 1250 (acetate), 1100 (methoxyl) and no trisubstituted doubie bond (795). NMRjsignals: 5.31 (1 H, multiplet, H-C-OAc), 6.70 (3 H, singlet, methoxyl), 7.25 (1 H, multiplet, H-^-0CH3), 7.95 (3 H, singlet, acetate), and 8.67-9.18 (angular methyl). Mass spectrum (MS 9); fragments at m/e 483 (M-15), 466 (M-32), 452 (M-47), 438 (M-60), 432 (M-75), 406 (M-92) and 391 (M-107); no retro Diels-Alder; prominent fragments at m/e 255, 221, 203, 190, 189, 187, 147 and 135. Found : M.W. (MS 9) 498. Calc. for C 3 3 H 5 1 t 0 3 ; M.W. -498. Correlation of Compound C Acetate with Iso Compound B Acetate The isolation of iso compound B acetate from the ether cleavage reaction (36% HBr:acetic acid) on compound B acetate has already been described. These compounds were shown to be identical on the basis of a quantitative I.R. spectral comparison, identical T.L.C. behaviour (silica, CHC13) and similar NMR and mass spectra. Because of the rather wide melting point ranges of the two compounds, a mixed melting point comparison was not attempted. 173 Properties of Compound D This product was isolated in the chromatographic separation of Extract Y but a l l attempts to purify the material by further column chromato-graphy were unavailing. Crystallization from ethanol yielded white crystals m.p. 245-252°C. ORD (C, 0.02, CH30H), 21°C, |>] 7 0 0 - 2 3 1 ° , [<f>] 5 8 9 -231°, [<J>] 31U -2168°, [4>]276 +461°, [<j>]2i+o "2906°. Molecular amplitude -26.2. Infrared (KBr); .3500 (hydroxyl), 1707 (carbonyl), 1387.and 1370 (geminal dimethyl) and 797 (trisubstituted double bond). •NMR signals: 4.65 (1 H, I multiplet, olefinic H), 6.84 (1 H, multiplet, H-C-OH), 8.94-9.25 (22.6.H, angular methyl). Mass spectrum (CH 4): fragments observed at m/e 425 (M-15), 422 (M-18), and 407 (M-33); no retro Diels-Alder; 218, 207, 203, and 189 (allylic cleavage); other prominent fragments at 177, 149, 147, 138 and 135. Found: M.W. 440.3692 (standard m/e 464) . Calc. for C 3 0 H l + 8 O 2 ; M.W. 440.3654. Properties of Compound H This product was also a minor component of Extract Y and was not obtained in a pure state. The crude fraction (152 mg.) as previously isolated was chromatographed a second time on a column of deactivated alumina (Activity III). The recovered material (59 mg.) was recrystallized from ethanol, m.p. 256-270°C. Infrared (KBr); 3400-3300 (hydroxyl), 1387 and 1365 (fgeminal dimethyl) and 795 (trisubstituted double bond) . NMR signals (100 Mc/s); 4.72 (1 H, multiplet, olefinic H), 6.79 and 6.90 (2 H, overlapping triplets, J = 5.0 c.p.s., H-C-OH), 8.78 and 9.06-9.35 (angular methyl). Mass spectrum (MS 9); fragmentsat-m/e 427 (M-15), 424 (M-18), 409 (M-33), 406 (M-36), and 391 (M-51); 220, 207, and 189 (allylic cleavage); other prominent fragments at 187, 147, 135, 127 and 121. Found: C, 80.66, H, 12.03, 0, 7.50; M.W. 442.380 (standard m/e 464). Calc. for C30H50O2: 174 C, 81.45, H, 11.31, 0, 7.24; M.W. 442.381. Properties of Compound X This compound was isolated from fraction II of Extract Y as already described. The white waxy solid was recrystallized from acetone, m.p. 69-70°C. Infrared (KBr); 1725 (carbonyl), 730 and 720 (-(CH2) - where n > 4). NMR signals (100 Mc/s); -6.02 (triplet, J 6.5 c.p.s., -CHji-0-CO-) , 7.76 (triplet, J 7.5 c.p.s,, CH^-CO-O-), 8.50 (-CH2->- very intense), and 9.16 (methyl). Irradiation at T 8.38 (+ 0.05) caused the collapse of the triplet at,6.02 to a sharp singlet and that at 7.76 to a pseudo-singlet. The compound did not show a molecular ion peak in the mass spectrum (highest observed fragment at m/e 408). Major cleavage:fragments were observed at m/e 173 and 207. It was not possible to obtain a satisfactory elemental analysis because of problems arising from the low melting point and from occlusion of solvent. Properties of Compound P. This compound was isolated from fractions III andlV of Extract Y. as previously described. A sample of the crude extract Was distilled at, 160-165°C at 0.3 mm. Hg. using a micro cold finger apparatus cooled by running water. Thus in two batches was removed a sample (370 mg.) of a pale green viscous liquid. Ultraviolet (n-heptane); ^ m a x 228, shoulder 276 (€ 190 and 60). Infrared (liquid film); 3300 (hydroxyl), 3065 (vinyl or terminal methylene), 1635 (non-conjugated double bond),.1410, 990 and 915 (vinyl), 878 (terminal methylene) and 1383 and 1365 (geminal dimethyl). NMR signals (100 mc/s); ABX system from vinyl groups 4.15 (1 H, quartet, Jtrans 1 7 ' 5 c°P's-> J c i s 1 0 , 5 C , p ' S ' H a i n s y s t e m ^ C = G X )» 4.88 (1 H, H a Hb 175 R H c quartet, J. 17.5 c.p.s., J m 1.5 c.p.s., H in system C=(/ ), 5.05 trans gem ^ c ^ \ ^ (1 H, quartet, 10.5 c.p.s., J g g m 1.5 c.p.s., H b in system / C = \ )» 5.26 and 5.55 (2 H, pair of wide multiplets, exocyclic methylene),7.60 and 7.73 (pair of multiplets believed due to impurities), 8.57 (singlet,.-OH moved to 8.65 when, temperature raised from 30° to 50° ) , 8.79 (CH3-COH) and 9.17, 9.23 and 9.36 (angular methyl). Mass spectrum (MS 9);. highest observed peak at m/e 272; intense fragments at 257, 204, 189,.161, 137 and 135. Found:.C, 82.20, H, 12.19, 0, 5.62. Calc. for C 2 fjH 3 t t0: .C,.,-82.69, H, 11.80, 0, 5.51. The mass spectrum did not reveal a molecular ion peak. However high resolution mass measurement on the m/e 272 peak gave the value 272.2511 (standard m/e 314). C a l c for C 2 0 H 3 2 ; M.W. 272.2504. This peak, therefore corresponds to M+ -H 20. Saponification of Fatty Acid Esters The fat fraction (VIII, 10.194 gm.) isolated in the column chromato-graphic separation of extract Z was saponified according to a method 78 discussed by Vogel. The fat was treated for four hours in an oi l bath at 130°C in the presence of 50 ml. IN potassium hydroxide in diethylene; glycol. After cooling the diethylene glycol was removed by distillation in vacuo (b.p. 90°C at 0.1 mm. Hg.). • The residue was distributed between chloroform and water, the chloroform extracts combined, washed with water and dried over anhydrous magnesium sulfate. Filtration and distillation of the solvent yielded a dark coloured resinous solid (3.75 gm.). Examination of this fraction by T.L.C. showed the absence of phytosterols. Present were traces,of 21-episerratenediol and compound H besides unknown coloured materials. No further work was performed on this material. The aqueous phase from the chloroform extraction was retained and 176 a c i d i f i e d t o pH 3.by the a d d i t i o n o f h y d r o c h l o r i c a c i d . The brown o i l so r e l e a s e d was e x t r a c t e d i n t o c h l o r o f o r m ; the e x t r a c t washed w i t h water and d r i e d over anhydrous magnesium s u l f a t e . F i l t r a t i o n and e v a p o r a t i o n o f ; s o l v e n t y i e l d e d the crude f a t t y a c i d f r a c t i o n (6.91 gm.) as a brown semi-s o l i d m a t e r i a l , t h i s f r a c t i o n was not f u r t h e r i n v e s t i g a t e d . The crude f r a c t i o n s I I I to V I I o b t a i n e d i n the chromatography o f . e x t r a c t Z a l l c o n t a i n e d y e l l o w o i l y r e s i d u e s which c o u l d be recovered a f t e r the p r e c i p i t a t i o n o f c r y s t a l l i n e t r i t e r p e n e c o n s t i t u e n t s from acetone s o l u t i o n . A sample (5.2 gm.) of t h i s o i l was s a p o n i f i e d w i t h sodium (2-gm.) 79 i n aqueous e t h a n o l (20 m l . ) as d e s c r i b e d by V o g e l . A f t e r t h r e e hours under r e f l u x the f r e e f a t t y a c i d s were f i l t e r e d o f f as t h e i r i n s o l u b l e sodium s a l t s . This m a t e r i a l was t r e a t e d w i t h d i l u t e h y d r o c h l o r i c a c i d and e x t r a c t e d i n t o c h l o r o f o r m , the e x t r a c t washed w i t h water , d r i e d over anhydrous magnesium s u l f a t e and f i l t e r e d . E v a p o r a t i o n o f the s o l v e n t y i e l d e d a p a l e y e l l o w s e m i - s o l i d f r a c t i o n (665 mg.) o f f r e e f a t t y a c i d s not f u r t h e r s t u d i e d . The f i l t r a t e from the s a p o n i f i c a t i o n r e a c t i o n was evaporated to dryness i n vacuo and was taken up i n c h l o r o f o r m s o l u t i o n . A f t e r washing w i t h d i l u t e h y d r o c h l o r i c a c i d and water the e x t r a c t was d r i e d over anhydrous magnesium s u l f a t e and f i l t e r e d . E v a p o r a t i o n o f . t h e s o l v e n t y i e l d e d a y e l l o w o i l c o n t a i n i n g t r a c e s o f s o l i d m a t e r i a l . When t h i s was d i s s o l v e d i n acetone and a l l o w e d to s tand o v e r n i g h t a white c r y s t a l l i n e p r e c i p i t a t e appeared. R e p e t i t i o n o f t h i s procedure produced a second crop o f . w h i t e c r y s t a l s ( t o t a l y i e l d 182 m g . ) . The y e l l o w o i l y mother l i q u o r (3.7 gm.) was a p p a r e n t l y unhydrolysed e s t e r m a t e r i a l . I n f r a r e d (CHC1 3 ) ; 3500 and 3350 ( h y d r o x y l ) , 1710 (carbonyl) and 1600 (double bond) , The f i r s t crop o f c r y s t a l s when examined by T . L . C . was seen t o 177 contain two fractions with the main one showing properties similar to.an authentic sample of 6-sitosterol; The second crop of crystals apparently contained only 3-sitosterol. Properties of g-Sitosterol Recrystallization from ethanol yielded flat swordlike crystals, m.p. 141-2°G. Infrared (KBr); 3400-3300 (hydroxyl), 1635 (non-conjugated. double bond), and 795 (trisubstituted double bond). NMR signals (100.Mc/s), 4.74 (1 H, multiplet, olefin H), 6.63 (1 H, multiplet,. H-C-OH), 7.64.: i i (CH3-C-C=C), and 9.0-9.3 (complex, methyl, ethyl and isopropyl). Mass spectrum (CH-4); the sample was impure and contained campesterol.(24a- . methyl cholesterol) as seen by the appearance of an intense peak at m/e 400 (M-14); and satellites at 385, .382 and 367; fragments were observed at m/e 399 (M-15), 396 (M-18) and 381 (M-33); cleavages in the side.chain gave rise to peaks at 329 (M-85, cleavage between C 23 a n d ^ i j j 315 (M-99i cleavage between C 2 2 and C 2 3 ) , 273 (M-141, loss of side chain) and 255 (M-159, loss of side chain and water); other intense fragments at 303, 289, 231, 213, 159, 149, 145 and 133.; Found: M.W. 414.3814 (standard m/e 426)., Calc. for C 2 9 H 5 0 O ; M.W. 414.3861. 178 BIBLIOGRAPHY 1. R. H. Stevens, Encyclopedia of Chemical Technology, edited by R. E. Kirk and D. E. Othmer, Volume 13, p. 572, Interscience Encyclopedia, New York, (1954). 2. D. B. Mutton, the Chemistry of Wood Extractives, p. 348, edited by W. E. Hi l l i s , Academic Press, New York, (1962). 3. Y. P. Chang and R. L. 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