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Studies related to natural products : part I selected electrophilic substitution reactions of 3-methylfuran… Hanssen, Harald Wilhelm 1970

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STUDIES RELATED TO NATURAL PRODUCTS: PART I SELECTED ELECTROPHILIC SUBSTITUTION REACTIONS OF 3-METHYLFURAN PART I I STRUCTURAL ELUCIDATION OF OXYGEN HETEROCYCLES FROM ZEYHERA TUBERCULOSA BY HARALD WILHELM HANSSEN B.Sc. Honours, University of B r i t i s h Columbia, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the DEPARTMENT OF CHEMISTRY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1970 In p resenting 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 t h a t permission for e x t e n s i v e 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 e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date 7 ^ 7 - i i -ABSTRACT Part I describes an i n v e s t i g a t i o n of the following e l e c t r o p h i l i c s u b s t i t u t i o n reactions of 3-methylfuran. Acylation under F r i e d e l -Crafts conditions yielded 3-methyl-2-acetylfuran (8) and 3-methyl-5-acetylfuran (9) i n the r a t i o 65:35. Vilsmeier and Gatterman formylation procedures yielded 3-methylfurfural (10) and 3-methyl-5-furfural (11) i n the same r a t i o , 93.5:6.5. Mercuration of 3-methylfuran yielded only the 2-mercuro d e r i v a t i v e . Part II describes 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 of two major oxygen h e t e r o c y c l i c compounds C and D occurring i n the leaves of Zeyhera tuberculosa Bur. et Verlot (Bignoniaceae). Compound C i s assigned the structure 5,6,7,8-tetramethoxyflavone (25) on the basis of spectroscopic evidence. Compound D i s shown to possess the structure 5,6,7-trimethoxyflavone (35). In t h i s l a t t e r instance spectroscopic and chemical evidence could be obtained i n support of the s t r u c t u r a l assignment. In p a r t i c u l a r , a l k a l i n e hydrolysis of D yielded 2,3,4-trimethoxy-6-hydroxyacetophenone (34), the i d e n t i t y of which was established by an unambiguous synthesis. In the synthesis of the acetophenone d e r i v a t i v e (34), the key intermediate i s 2,6-dimethoxyhydroquinone diacetate (31) which was subjected to F r i e s rearrangement conditions, followed by acid h y d r o l y s i s , to give 2,4-dimethoxy-3,6-dihydroxyacetophenone (33). P a r t i a l methylation of (33) yielded (34). TABLE OF CONTENTS Page TITLE PAGE 1 ABSTRACT 1 1 TABLE OF CONTENTS 1 1 1 LIST OF FIGURES i v _ v LIST OF TABLES V 1 ACKNOWLEDGEMENTS .' . . •". V 1 1 PART I . . . ; 1 INTRODUCTION 2. DISCUSSION ,. 9 PYDPDIMENTA-L . . .' ^ BIBLIOGRAPHY • • • • 3 6 PART II 3 8 INTRODUCTION 3 9 DISCUSSION . ' 4 8 EXPERIMENTAL 7 5 BIBLIOGRAPHY ., • - 8 8 - i v -LIST OF FIGURES PART I Figure Page 1 Mechanism for the e l e c t r o p h i l i c substitution of furan 6 2 Energy diagram for the e l e c t r o p h i l i c substitution of furan at the 2- and 3-position 6 3 E l e c t r o p h i l i c substitution reactions of furans with an electron-withdrawing group at C-3 7 4 E l e c t r o p h i l i c substitution reactions of furans with an electron-donating group at C-3 8 5 Synthesis of 3-methylfuran . 10 6 Friedel-Crafts acetylation of 3-methylfuran 17 7 Vilsmeier formylation of 3-methylfuran 17 8 Gatterman formylation of 3-methylfuran 18 9 Nuclear magnetic resonance spectrum of 3-methyl-2-acetylfuran . . 23 10 Decoupling studies of 3-methyl-2-acetylfuran 24 11 Nuclear magnetic resonance spectrum of 3-methyl-5-acetylfuran 25 12 Decoupling studies of 3-methyl-5-acetylfuran 26 - v -LIST OF FIGURES PART I I Figure Page 1 Flavones reported i n Bignoniaceae 41 2 Flavones reported i n Bignoniaceae 42 3 Anthocyanins reported i n Bignoniaceae 43 4 3-Deoxyanthocyanins reported i n Bignoniaceae 44 5 A possible biogenetic relationship of isoprenoid quinones reported i n Bignoniaceae 45 6 Is o l a t i o n procedure for the constituents of Z^. tuberculosa 49 7 Nuclear magnetic resonance spectrum of compound C... 57 8 Mass spectrum of compound C 58 9 P r i n c i p l e mass spectrometric fragmentations of compound C 59 10 Nuclear magnetic resonance spectrum of compound D .. 62 11 Mass spectrum of compound D • 63 12 P r i n c i p l e mass spectrometric fragmentations of compound D 64 13 The t o t a l synthesis of compound D hydrolysis product 67 14 Nuclear magnetic resonance spectrum of 2,6-dimethoxy- . hydroquinone diacetate (31) 69. 15 Nuclear magnetic resonance of 2,4-dimethoxy-3-acetoxy-6-hydroxyacetophenone (32) 70 16 Nuclear magnetic resonance spectrum of 2,5-dihydroxy-4,6-dimethoxyacetophenone (33) 72 17 Nuclear magnetic resonance spectrum of 2,3,4-trimethoxy-6-hydroxyacetophenone (34) 73 18 Infrared spectra of natural and synthetic 1,2,3-trimethoxy-5-hydroxyacetophenone (34) 74 - v i -LIST OF TABLES PART I Table Page I Coupling constants of 3-methylfuran derivatives .... 21 II Chemical s h i f t s of protons i n some 3-methylfuran derivatives 22 PART II Fractions from the ethanolic extracts of Zeyhera tuberculosa 50 neutrals f r a c t i o n . 51 - V l l -ACKNOWLEDGEMENTS The author i s grateful to Dr. James P. Kutney for giving him the opportunity to do research i n several interesting, areas of natural products chemistry and also for. his encouragement and i n s p i r i n g supervision. Many helpful discussions with many helpful colleagues are gr a t e f u l l y acknowledged. Special thanks "are due to Dr. Robert Swingle for proof-reading and Miss Diane.Johnson for typing the manuscript. - v i i i -To my Parents - i x --Quota pars-operis -tanti nobis .-.committitur? - Seneca PART I SELECTED ELECTROPHILIC SUBSTITUTION REACTIONS OF 3-METHYLFURAN INTRODUCTION The chemistry of furan derivatives has been of great interest to organic chemists since the discovery of furoic acid i n 1780, and the accidental discovery of f u r f u r a l f i f t y years l a t e r . However, i t was not u n t i l the early 1920's that the economic and medicinal value of these compounds was appreciated. Soon, however, f u r f u r a l became available " i n drums and tank cars"''" as a res u l t of oat husk research, and a whqie new chemical industry e v o l v e d . Today f u r f u r a l i t s e l f i s converted into a d i p o n i t r i l e for the production of nylon. It i s also used as a highly selective solvent i n the p u r i f i c a t i o n of wood resins, i n the r e f i n i n g of petroleum o i l s , vegetable o i l s and animal o i l s . Furfural and f u r f u r y l alcohol are both used as solvents for i n d u s t r i a l coatings, dyes, sludge and varnish removers, and a wide variety of adhesives. Derivatives of furan have also become important as chemical intermediates i n the synthesis of pharmaceutical products such as the n i t r o - and mercurial furans which have bacteriocidal properties. Other derivatives are used as food preservatives, fungicides, i n s e c t i c i d e s , and herbicides. The rapid growth of furan chemistry has been documented i n a c l a s s i c monograph by Dunlop and Peters''' which was published i n 1953. - 3 -2 This work has since been extended by Bosshard and Eugster who have surveyed the chemistry of simple furan systems developed from 1952 to 1963. It i s the purpose of the present thesis (Part I) to attempt to further the understanding of this very important area of heterocyclic chemistry. The structure of furan i t s e l f was f i r s t proposed by Markwald i n 1888 to be the oxygen analogue of cyclopentadiene. Today the furan nucleus i s best described as a resonance hybrid as shown i n the structures l a - l g . The atomic o r b i t a l s of furan consist of a 1 l a «« + lb 0 » + l c ^ 0 . + Id + le 7) . 0 > I f 2 pentagonal frame work of a-bonds made with sp -hybridized ring carbon atoms plus four carbon p-orbitals with one ir-electron, each overlapping with a doubly f i l l e d p - o r b i t a l of the oxygen heteroatom. This d e r e a l i z a -t i o n of 6 TT-electrons gives furan and i t s nitrogen and sulphur analogues, pyrrole and thiophene, a certain s t a b i l i z a t i o n energy, or aromatic 3 character, found to be 23, 31, and 31 kcal/mole, respectively. The extent of resonance increases with decrease i n the electro-negativity of the heteroatom. The very electronegative oxygen has less tendency to assume the positive charge accompanying structures lb to l e , than has nitrogen or sulphur i n pyrrole and thiophene. Measurements of bond lengths i n furan, pyrrole and thiophene suggest that there i s 23 percent t o t a l contribution of structures l b - l e for furan, 24 percent 3 for pyrrole, and 28 percent for thiophene. Thus, furan i s the least aromatic compound of t h i s series and this fact i s reflected i n i t s chemistry. The most important resonance structures of furan are expected to be lb and l c since the energy required to separate charges w i l l be lower, than that of the other charge separated structures, as the distance involved i s less. Experimental support of t h i s conclusion i s seen i n the behavior of furan derivatives i n e l e c t r o p h i l i c substitu-t i o n reactions. The proposed mechanism of e l e c t r o p h i l i c substitution i s i l l u s t r a t e d i n Figure 1. The rate of substitution at either the 2- or 3-position i s dependent on the energy difference between the ground state of the reactants and the p a r t i c u l a r t r a n s i t i o n state involved. The reaction path through the most stable t r a n s i t i o n state w i l l be favoured (Figure 2) Therefore, an incoming group i s oriented almost exclusively to the 2-position. Examples of 3-substitution when there i s a vacant 2-position are very rare. Only one such example has been reported to date.^ - 5 -i s o - C . ^ iso-C H Cl —-> CHO A 1 C 1 3 ^ 0^ "CHO A set of general rules of e l e c t r o p h i l i c substitution have been 2 proposed regarding the orientation of substitution of furan derivatives. (i) substitution occurs p r e f e r e n t i a l l y at the 2-position, and i s independent of the nature of the group at 5-position. ( i i ) substituents at 3-position which are electron-withdrawing i . e . , meta -directing, direct substitution to 5-position, and those which are electron-donating, or ortho-para d i r e c t i n g , direct substitution to the 2-position. ( i i i ) i f both 2- and 5-positions are substituted, the substituent with the stronger electron-donating effect w i l l direct the substitution to the v i c i n a l position. A recent survey of the literature of furan chemistry indicates that there are no exceptions to these rules, other than the a l k y l a t i o n mentioned above which i s contrary to rule ( i ) . Recently i n our 6 laboratories, however, the second part of rule ( i i ) was shown to be i n error. It was discovered that i n the acylation of 3-methylfuran using propionic anhydride, two compounds were formed i n the r a t i o 70:30. These were proven to be 2-propionyl-3-methylfuran and 2-propionyl-4-methylfuran, respectively. This example appears to be the f i r s t exception to rule ( i i ) i n furan chemistry, and thus warrants further v 6 T H H -i n ^ 0 ' Figure 1. Mechanism for the e l e c t r o p h i l i c substitution of furan. Reaction pathway Figure 2. Energy diagram for the e l e c t r o p h i l i c substitution of 4 furan at the 2- and 3-position. - 7 -investigation. The f i r s t part of rule ( i i ) was formulated using only the following furan systems (Figure 3). ref CHO UNO, 0 Ac 20 X NO. C02H 7,8 CO„H Br, IT \ 0 ^ CHC1, Br 0 X. C02H CO.R z Ac 20 C02R BF. C02R C02R DMF P0C1. 10 CHO Figure 3. E l e c t r o p h i l i c substitution reactions of furans with an electron-withdrawing group at C-3. - 8 -At present there appears to be no evidence contrary to the f i r s t part of rule ( i i ) . Preliminary evidence from our laboratories, however, has shown that the second part of t h i s r u l e , based on the following reactions i s incomplete (Figure 4). For t h i s reason further investigation was under-taken on these types of reactions. ref 1 1 7 1 2 Figure 4. E l e c t r o p h i l i c substitution reactions of furans with an electron-donating group at C-3. DISCUSSION The p o s s i b i l i t y of e l e c t r o p h i l i c substitution at the 5-position i n a 3-alkylfuran i n which the 2-position was vacant was f i r s t proven 6 -i n our laboratories. 3-Methylfuran was acylated under Friedel-Crafts conditions with propionic anhydride, using orthophosphoric acid as the catalyst. Formed i n th i s reaction were 2-propionyl-3-methylfuran (3) and 2-propionyl-4-methylfuran (4) i n the r a t i o 7:3. I t then became apparent that a re-investigation of some e l e c t r o p h i l i c substitution reactions of 3-methylfuran could y i e l d new and interesting results for the synthesis of furan derivatives. The synthetic route for the preparation of 3-rnethylfuran that was chosen i s shown i n Figure 5. The reaction of 4,4-dimethoxy-2-butanone with a-chloroacetate i n the presence of sodium methoxide afforded the epoxyester (5) without d i f f i c u l t y and i n nearly quantitative y i e l d . - 10 -Without further p u r i f i c a t i o n this compound was pyrolysed at 120°. The methanol produced was removed during the course of the reaction, and the residue was subsequently d i s t i l l e d at reduced pressure to y i e l d a 13 white c r y s t a l l i n e material. Spectral data on th i s compound revealed the. Cl-CH -C02CH + CHr V /°C H3 C-CH.-CH // 2 \ 0 OCH, CH. \ 3 0. • 0CH_ / 3 C-CH„-CH 2 \ OCH ' CI I I CO CH (5) 12 0° Cuc 200-230' 0 (2) (7) Figure 5. Synthesis of 3-methylfuran. C02H NaOH 0^ C02CH (6) presence of a furan system (A 252 nm; v(chloroform) 1725, 885 cm . max Subsequent characterization confirmed t h i s material to be methyl-3-methyl furoate (6). Hydrolysis with aqueous sodium hydroxide converted the methyl ester to 3-methylfuroic acid (7), which upon decarboxylation by means of powdered copper i n quinoline provided 3-methylfuran (2) i n good y i e l d . Analysis of this product by gas-liquid chromatography and nuclear magnetic resonance spectroscopy revealed that the 3-methylfuran prepared i n this way was very pure, and could therefore be used d i r e c t l y i n subsequent experiments. Since 3-methylfuran decomposes on stand-ing, i t was advantageous to prepare this compound shortly before each reaction. Our f i r s t consideration was to establish a method by which substitution patterns i n furan derivatives could e a s i l y be established. The technique chosen was nuclear magnetic resonance (n.m.r.) spectro-scopy. Examination of the n.m.r. spectrum of 3-methylfuran suggested that evidence for the position of substitution could be obtained from the chemical s h i f t s of protons, as well as the coupling constants between various protons on the furan ri n g . Therefore, a detailed study of the n.m.r. spectra of 3-methyl-2-furoic acid and 3-methylfuran was.carried out. multiplets at x 2.48 and 3.60 i n the low f i e l d region. Those signals 14 can readily be assigned to protons at C-5 and C-4, respectively. The 3-methyl protons absorb as a multiplet at x 7.60. A double resonance experiment i n which this signal was irr a d i a t e d revealed that both low f i e l d m u l t i p l e t s could be decoupled to give doublets, thereby establishing the coupling constant = 1.8 Hz for the adjacent protons on the furan ring. To establish the long range coupling constants between the ring protons and the 3-methyl protons each of the low f i e l d multiplets were ir r a d i a t e d . I r r a d i a t i o n at x 2.48 reduced the C-3 methyl protons signal to a doublet, thus revealing ^4 Me = K z* S i m i l a r l y , i r r a d i a t i o n of the C-4 proton at x 3.60 reduced the C-3 methyl signal to a doublet with J r = 0.5 Hz. J to 5,Me Examination of the nuclear magnetic resonance spectrum of - 12 -3-methylfuran showed that, corresponding coupling constants were found to have, similar values as-those i n 3-methyl-2-furoic acid. Double i r r a d i a t i o n studies revealed an additional coupling constant ^ e = 1.1 Hz. The coupling constants thus measured for the 3-methylfuran nucleus are summarized i n Table I. In Table I I , the chemical s h i f t s obtained for some derivatives of 3-methylfuran are presented. Our f i r s t step i n the re-investigation of orientation i n electro-p h i l i c substitution reactions of 3-alkylfurans was acylation under Friedel-Crafts conditions. This type of reaction i s believed to involve attack on an acylium ion, followed by alkali n e hydrolysis of the intermediate (Figure 6). 3-Methylfuran was treated with an equimolar amount of acetic anhydride at 0° and a c a t a l y t i c amount of 85% ortho-p I i u s p i i O L i u ctciil a.a Lius c o n d e n s i n g a g e r i L . Almost: i n m n e d i a u e i y a brown coloration occurred due probably to f a c i l e r e s i n i f i c a t i o n of the furan. Workup of t h i s reaction, followed by d i s t i l l a t i o n , yielded a fragrant colorless o i l . Examination of this product by gas l i q u i d chromatography indicated that two major components were present i n the r a t i o 65:35. These two components were then separated by preparative gas-liquid chromatography. The nuclear magnetic resonance spectrum (Figure 9) of the major component shows one-proton multiplets at T 2.55 and 3.55. These signals can readily be assigned to protons at carbons 5 and 4 respectively of structure (8), i n Figure 6. Further evidence for this s t r u c t u r a l assignment was provided by double resonance experiments. The C-3 methyl protons absorb as a multiplet at T 7.55. I r r a d i a t i o n of t h i s signal reduced the low f i e l d multiplets to doublets with a coupling - 13 -constant of 1.8 Hz. I t could also be shown that i r r a d i a t i o n of the C-5 proton signal at x 2.55 reduced the 3-methyl protons multiplet to a doublet with a J-value of 1.0 Hz. Also, i r r a d i a t i o n at x 3.55 revealed another coupling to the C-3 methyl protons of 0.5 Hz. This data i s i n complete agreement with the assignments made for the substitution pattern i n 3-methyl-2-furoic acid (7). Figure 10 shows an example of a double resonance study of the major component from t h i s reaction. Oxidation of this acylated material with sodium hypochlorite''"*' yielded a colorless c r y s t a l l i n e compound with spectroscopic properties i d e n t i c a l with those of the previously prepared 3-methyl-2-furoic acid (7). I t can therefore be concluded that the major component of t h i s reaction i s me expected ^-acetyl-J-metnylturan. The second acetylated component was assigned structure (9). The nuclear magnetic resonance spectrum of this.compound(Figure 11) indicates one-proton multiplets at x 2.60 and x 2.91. These signals were assigned to protons at C-4 and C-2, respectively. The C-3 methyl group absorbs as an i l l - d e f i n e d quartet at x 7.90. The fact that this signal appears at higher f i e l d than the corresponding methyl signal i n 2-acetyl-3-methylfuran i s ra t i o n a l i z e d on the basis that i t i s not i n proximity to the paramagnetic shielding of the carbonyl oxygen. I r r a d i a t i o n at the 3-methyl protons absorption frequency reduced both low f i e l d signals to doublets, with a J-value of 1.0 Hz, which further suggests that these signals must be assigned to protons at positions 2 and 4. I r r a d i a t i o n of the multiplet at x 2.60 reduced the C-3 methyl protons - 14 -absorption signal to a doublet with a J-value of 1.0 Hz, while a coupling constant of 1.1 Hz was obtained when the multiplet at T 2.90 was i r r a d i a t e d . This data can only be interpreted i n terms of substitution at C-5 of the 3-methylfuran nucleus. I t can therefore be concluded that Friedel-Crafts acetylation of 3-methylfuran catalyzed by orthophosphoric acid yields two isomers as a r e s u l t of substitution at position 5 as well as p o s i t i o n 2. A s i m i l a r Friedel-Crafts acylation reaction was investigated by Eugsten and Waser^ (see Introduction). They used tri f l u o r o - b o r o -etherate to catalyze the acetylation of 3-furoic acid and methyl-3-furoate, but only the expected 5-substituted isomer i s reported. I t should also be noted here that Hartough and Kosak^acetylated 3-methylthiophene with acetic anhydride and orthophosphoric acid to obtain 2-acetyl-3-methylthiophene and 5-acetyl-3-methylthiophene i n the'ratio 8:2, respectively. The next type of e l e c t r o p h i l i c substitution reaction that was considered was that of formylation. The e a r l i e s t method of introduc-ing a formyl group on a furan nucleus was the Gatterman reaction which involves attack of hydrogen cyanide (Figure 8 ). A l i t e r a t u r e survey revealed that formylation of a 3-alkylfuran under Gatterman conditions has not been published since the procedure reported by Reichstein, i n 1931 (see Introduction). A more modern method of introducing a formyl group on an aromatic nucleus i s the so-called Vilsmeier formylation using dimethylformamide and phosphorus oxychloride. This reaction i s believed to involve attack on the chloro-iminium s a l t of dimethyl formamide to give a Schiff's base intermediate. Hydrolysis - 15 -of this intermediate by potassium carbonate yields the aldehydic products (Figure 7). Since this procedure i s reputed to give better o v e r a l l yields and 17 also to obey the orientation rules "without exception", i t seemed prudent to investigate this.method of'formylation before repeating the Gatterman formylation procedure. 3-Methylfuran was added to an equimolar mixture of dimethylformamide and phosphorus oxychloride according to the general procedure reported 18 by Traynelis and coworkers. After hydrolysis of the Schiff's base intermediate using potassium carbonate, an excellent y i e l d of a s l i g h t l y yellow o i l was obtained. Analysis of this material by gas. l i q u i d chromatography revealed that only, two components were present, preparative g.l.c. The nuclear magnetic resonance spectra of these components when compared with the Friedel-Crafts acetylation products c l e a r l y revealed the substitution patterns i n f u r f u r a l products. The n.m.r. of the major product show one-proton multiplets at x 2.43 and 3.58. The C-3 methyl protons absorption appears at x 7.60. Again • double i r r a d i a t i o n studies were used to reveal coupling constants. In summary, the spectral data obtained was i n accord with the structure (10) for the major product. The major Vilsmeier product was oxidized with s i l v e r oxide i n water to give a good y i e l d of a colorless c r y s t a l l i n e material whose spectral properties were i d e n t i c a l with those of 3-methylfuroic acid (7). This experiment established the Vilsmeier - 16 -formylation provided 3-methylfurfural (10) as the major product. The minor component i n t h i s reaction shows one-proton multiplets at x 2.52 and 2.93 which i s i n accord with the expected absorption frequencies of protons on C-4 and C-2, respectively, of structure (11). These assignments are further supported by double i r r a d i a t i o n studies that revealed coupling constants consistent with the substitution pattern i n 3-methyl-5-furfural ( l l ) . I t can therefore be concluded that the Vilsmeier formylation of 3-methylfuran yields a s i g n i f i c a n t amount of the 5-substituted isomer, contrary to the accepted orientation rule. We chose now to re-investigate the Gatterman formylation of 3-metbylfuran according -to Reichstein's procedure. A n h y d r o u s hydrogen cyanide was B e v i c j _ c i L e d i n a dropping funnel under ethyl ether by treating zinc cyanide with hydrogen chloride gas. After two hours the hydrogen cyanide, now saturated with hydrogen chloride, was dropped into a solution of 3-methylfuran i n ether. Subsequent hydrolysis and steam d i s t i l l a t i o n yielded a small amount of a yellow o i l which was found to contain several components by gas l i q u i d chromatography. The major component had spectral properties i d e n t i c a l to that of 3-methylfurfural. 3-Methy1-5-furfural was also believed to be present i n trace amounts. This p o s s i b i l i t y was indicated by close examination of the g.l.c. tracing as well as the n.m.r. spectrum of the crude product. I t seemed reasonable that 3-methyl-5-furfural was being formed i n s i g n i f i c a n t amounts, but being a less stable compound than 3-methyl-2-furfural, the former decomposed at a faster rate. I t i s expected that the 5-substituted isomer i s r e l a t i v e l y less stable to the prolonged acid conditions of this - 17 -(CH CO) 0 + -> CH3C02H + CH3CO (CH3CO)20 H + „„ H + S C ^0 ii + + 0 (8) (9) Figure 6. Friedel-Crafts acetylation of 3-methylfuran, P0C1. 0P0C1, .Cl + Cl H P0C1 ^ 0 ^ DMI: 3 -—> NJ - >>/ - H + :N S \ CH, CH H /fCl + Cl 0 ^ + 3 3 CH„ CH„ + N(CH 3) 2 +N(CH ) 3'2 K 2C0 3 0 CHO (10) Figure 7. Vilsmeier formylation of 3-methylfuran. CHO i H \ o ( 1 1 ) - 18 -reaction due to the fact that, in this molecule the position most susceptible to protonation i s s t i l l vacant, thus more f a c i l e r e s i n i -f i c a t i o n could occur. In order to test t h i s p o s s i b i l i t y , Reichstein's formylation procedure was repeated using a reaction time of s i x hours rather than the overnight time span called for o r i g i n a l l y . This experiment yielded as major components 3-methylfurfural and 3-methyl-5-furfura.l i n the ra t i o 93.5:6.5, respectively. + + HC1 + HCN > [HC=NH <-—3> HC=NH]Cl" • n - <J (2) HCN. L CH=NH CH=NH CH=NH + CH=NH H20 J 0 CHO (10) CHO ( I D Figure 8. Gatterman formylation of 3-methylfuran. - 19 -A t h i r d type of e l e c t r o p h i l i c substitution reaction that was considered for reinvestigation was metalation with mercuric chloride. Mercuration, followed by replacement reactions, has been a useful method i n rendering available furan derivatives which would otherwise have been inaccessible. A l i t e r a t u r e survey, however, revealed that 12 the only mercuration reaction of a 3-alkylfuran was done by Gilman. He reported the formation of the expected 3-methyl-2-furylmercuric chloride as the sole product. It was therefore decided that t h i s reaction should also be reinvestigated. 3-Methylfuran was added to an aqueous solution of mercuric chloride to y i e l d a colorless c r y s t a l l i n e p r e c i p i t a t e . The nuclear magnetic resonance spectrum of t h i s material indicated one-proton multiplets at x 2.35 and 3.58, as well, as a three-proton mn.l t i p ! et p + T ">. «?• J'*-can therefore be concluded that i n th i s reaction only 3-methyl-2-furfurylmercuric chloride was formed, with no evidence of other isomers. The last, e l e c t r o p h i l i c substitution reaction considered was that of bromination. Gilman has reported the bromination of 3-furoic acid, but there are no reports i n the l i t e r a t u r e of direct halogenation of 3-methylfuran. In the present study, bromination of 3-methylfuran using dibromodioxane was attempted. Removal of the solvent from the reaction product, however, resulted i n what appeared to be a rapid polymerization to give a brown, p l a s t i c - l i k e material. No further work was done on thi s reaction. In summation, the preceding work has shown that 3-methylfuran undergoes e l e c t r o p h i l i c substitution at position.5, as well as the expected position 2 i n some reactions. This fact has been established i n a Friedel-Crafts acyla.t:i.on, a Vilsmeier formylation, and also i n the - 20 -c l a s s i c Gatterman formylation reaction. Mercuration, however, appeared to give only the 2-substituted isomer. In the l i g h t of these r e s u l t s , i t may be concluded that the presently accepted orientation rule ( i i ) , as discussed i n the Introduction, i s i n error. A previous attempt to test rule ( i i ) has been reported by Finan 22 . and F o t h e r g i l l . They treated 3-methylfuran with acetic anhydride and excess of trifluoro-boro-etherate and obtained, i n low y i e l d , only the 2-acetyl compound. In a s i m i l a r experiment using i s o v a l e r i c anhydride only* the 2-substituted 3-methylfuran was obtained. Because' these workers were also searching for the 5-substituted isomers, this report i s of p a r t i c u l a r i n t e r e s t . The low yields and the severe reaction conditions described leaves open the p o s s i b i l i t y that the 5-substituted Z S C i a d i T S C O u l d h a V C f OiliiteCi. C l ^ i r X I i ^ uile: I'caCU j-<_»Vi D U L D C J . I I ^ iuG'.Le ctCi-Li xctuXJLt: were subject to more rapid decomposition to give results s i m i l a r to those previously described from the re-investigation of the Gatterman formylation. • • \ I t i s clear that orientation i n e l e c t r o p h i l i c substitution reactions of 3-alkylfurans i s not completely understood. The work described i n this part may support the suggestion by Paquette that product 'distribution i s dependent not only on r e a c t i v i t i e s but also on such secondary effects such as " r e l a t i v e s t e r i c bulk of the 3-alkyl group and/or the entering 23 e l e c t r o p h i l e . " Table I. Coupling constants of 3-methylfuran derivatives Positions of Protons Coupling Constant (Hz) 4,5 7 A 4, C-3 methyl 5, C-3 methyl 2, C-3 methyl 1.8 i n 1.0 0.5 1.1 - 22 -Table I I . Chemical s h i f t s , of protons i n some 3-methylfuran derivatives. Derivative C-2 C-4 C-5 C-3 Methyl 4 3 1 2.80 3.85 2.70 8.05 3.60 2.44 7.60 -0 3.55 2.55 7.55 0 ^  CHO 3.58 2.43 7.60 O HgCl 3.58 2.33 7.86 0 2.60 2.91 7.90 CHO 2.52 2.93 7.88 Figure 9- Nuclear magnetic resonance spectrum of 3-methyl-2-acetylfuran (8). Figure 11. Nuclear magnetic resonarce spectrum of 3-methyl-5-acetylfuran (9). I r r a d i a t i o n at T 7.90 K3 ON x 2.60 x 2.91 x 7.-90 C-2 proton , C-4 proton . C-3 methyl proton ;ure 12. Decoupling studies of 3-methyl-5-acetylfuran (60 MHz, 25 Hz scan width). EXPERIMENTAL Gas l i q u i d chromatography (g.l.c.) was performed on a Varian model A-700 instrument, using helium as a c a r r i e r gas at a flow rate of 80-85 ml/min. For routine analysis a column 1/4 inch x 10 feet consisted of 20% FFAP on 60/80 mesh Chromsorb W support packing was used. A similar column (3/8 inch, 30% FFAP) was used i n preparative chromatography. A l l work was done using a column temperature of 172-J L ' / D " . U l t r a v i o l e t spectra were recorded i n methanol on a Cary 11 spectrophotometer. Absorption values (\ ) are given i n nanometers max-(nm). Infrared spectra were measured on a Perkin-Elmer model 137 double beam instrument, and absorption bands (v ) are quoted i n max wavenumbers (cm •"*) . Nuclear magnetic resonance (n.m.r.) spectra were recorded i n deuterochloroform at room temperature. Generally, a Varian T-60 spectrometer was used to record routine spectra at 60. Megahertz (MHz) and for double resonance studies. Where additional resolution was required to confirm assignments, spectra were recorded at 100 MHz on a Varian HA-100 spectrometer. The positions of n.m.r. absorption signals are given i n the Tier's T scale using tetramethylsilane as the i n t e r n a l standard at T lO.OOt The x-value given for multiplet signals - 28 -represent the center of the multiplet. Coupling constants (J) are given i n Hertz (Hz). • Mass spectra were recorded at 70 eV on an Associated E l e c t r i c a l Industries MS-9 double-focussing mass spectrometer. Fragmentation data i s given i n mass to charge r a t i o s (m/e) followed by percent r e l a t i v e abundance. Melting points were determined on a Kofler block and are uncorrec Elemental analyses were performed by Mr. P. Borda of the Microanalytic; Laboratory, University of B r i t i s h Columbia, Vancouver, B.C. Methyl-5,5-dimethoxy-3-methyl-2,3-epoxy-pentanoate (5) 4,4- Dimethoxy-2-butanone (120 g, 1.0 moles) was added to methyl 3 l i t r e 3-necked round bottom f l a s k . This mixture was then cooled to -10° with an i c e - s a l t bath, and the system was flushed with dry nitrogen. Freshly prepared dry sodium methoxide (86 g, 1.6 moles) was added gradually with rigorous s t i r r i n g , at such a rate that the temperature of the mixture did not exceed 0° (about 2 hrs); This mixture was then s t i r r e d for an additional 2 hrs and allowed to come to room temperature overnight. It was again cooled to -10° and made s l i g h t l y a c i d i c by the addition of d i l u t e acetic acid (15%, 10 ml). Th organic layer was decanted o f f and the residue was extracted with several portions of ether. The combined organic layers were then washe with saturated sodium bicarbonate solution followed by saturated sodium chloride solution. After drying over sodium sulphate and removal of solvent under reduced pressure, a near quantitative y i e l d of the g l y c i ester (190 g) was obtained. This material was e s s e n t i a l l y pure by - 29 -g.l.c. and could be used i n the following experiment without further p u r i f i c a t i o n . Methyl-3-methyl-2-furoate (6) The crude g l y c i d i c ester (90 g) prepared as above was placed i n a 250 ml round-bottom flask f i t t e d with a 30 cm Vigreux column, d i s t i l l a t i o n head and condenser. It was then heated i n an o i l bath to 120°. By t h i s time methanol began to d i s t i l off. Heating was continued u n t i l d i s t i l l a t i o n of methanol ceased. The pressure i n the system was lowered to 6 mm and crude methyl-3-methyl-2-furoate was d i s t i l l e d at bp 68-75°. The d i s t i l l a t e (41 g) was collected i n an ice cooled receiver as a colorless c r y s t a l l i n e material, m.p. 33-35°; i o r n „ . /" _1_ i .c - ^ . -iT->r r> r> r\ — 1 . „ _ J , T _ . _ o r *~> max max ' ' • (IH, m ultiplet, J = 1.8, 0.5), 3.62 (IH, multiplet, J = 1.8, 1.0), 6.13 (3H, singlet),. 7.65 (3H, multiplet, J = 1.0, 0.5). 19 Anal. Calculated for C_Ho0 : C, 60.00; H, 5.75. Found: C, 60.10; l o o H, 5.85. 3-Methyl-2-furoic acid (7) A solution of methyl-3-methyl-2-furoate (20 g) i n aqueous sodium hydroxide (10%, 200.^ml) was refluxed for 1.2 hr. This mixture was then cooled and a c i d i f i e d with hydrochloric acid (6 N). A brown granular material separated out and was collected by f i l t r a t i o n . After washing with cold water and drying ir\_ vacuo, the product (13 g) was r e c r y s t a l l -ized from methylene chloride to give colourless c r y s t a l s , m.p. 139-140° ( l i t . m.p. 140°), v m a x (chloroform): 2700-2500, 1765 cm'1; n.m.r. - 30 -signals: T 2.44 (IH, multiplet, J = 1.8, 0.5), 3.60 (IH, multiplet, J = 1.8, 1.0), 7.60 (3H, m u l t i p l e t ) ; mass spectrum: 126(52), 109(45), 81(10), 80(17). Anal. Calculated for C,H.O,: C, 65.45; H, 5.49. Found: C, 65.44; b o o H, 5.47. 3-Methylfuran (2) Preceding each remaining experiment i n t h i s investigation 3-methylfuran was freshly prepared by the following procedure: 3-Methyl-2-furoic acid (2.3 g) was added to a mixture of powdered copper (1.0 g) and anhydrous quinoline (10 ml) i n a 25 ml round-bottom fla s k f i t t e d with a short-path d i s t i l l a t i o n head and a cooled receiver. Tiic Sv.-iLcin nds cvctC.Uis.tcu cti'iu i l u S h d u S c v c i a.1 uxi'nes i v i i n u i y fix L x u g c i i . The mixture was then slowly heated i n a sand bath to 200°, at which time the evolution of carbon dioxide was evident. Heating was continued to 240° u n t i l no more gas evolved and d i s t i l l a t i o n of 3-methylfuran ceased (about 2 hrs). The clear.colorless product (1.3 g, 79-83%) thus obtained was found to be homogeneous by g . l . c ; b.p. 65-66°; v (chloroform): 1500, 1200, 890 cm"1; n.m.r. signals: T 2.70 (IH, ITlclX multiplet, J = 1.8, 1.0, 0.5), 2.80 (IH, mul t i p l e t , J = 1.1, 1.0), 3.85 (IH, multiplet, J = 1.8, 1.1, 1.0), 8.05 (3H, multiplet, J = 1.1, I. 0, 0.5). Anal. Calculated for C^O: C, 73.15; H, 9.15. Found: C, 73.10; H, 9.13. - 31 -Acetylation of 3-methylfuran 3-Methylfuran (1 g) was added to anhydrous acetic anhydride (1.0 g) at 0°. Two drops of orthophosphoric acid (85%) was added with vigorous s t i r r i n g . The resulting brown mixture was warmed to room temperature and s t i r r e d for 1 hr. I t was then heated b r i e f l y to 50°, cooled, and water (2 ml) was added. After further s t i r r i n g (0.5 hr) a saturated sodium bicarbonate solution (8 ml) was added and the mixture was s t i r r e d overnight at room temperature. The brown organic layer was taken up i n ether and washed with water. After drying over sodium sulphate, the solvent was removed under reduced pressure and the brown o i l so obtained was also d i s t i l l e d at reduced pressure i n a Kugel'rohr hot-box. The colorless fragrant l i q u i d (0.5 g) was examined by g.l.c. and founa to ue a mixcurt o± -cwu cumuuuuua j.u Lhe i a L i u 65; 35, with retention times of 9.0 and 13.5 minutes, respectively. These components were separated by preparative g.l.c. The major component was characterized as 3-methyl-2-acetylfuran (13); X : 279, 230 nm; v ( f i l m ) : 1670, J J max max 1505, 880 cm _ 1; n.m.r. signals: T 2.55 (IH, m u l t i p l e t , J = 1.8, 1.0), 3.55 (IH, m u l t i p l e t , J = 1.8, 0.5), 7.52 (IH, s i n g l e t ) , 7.60 (IH, mu l t i p l e t , J = 1.0, 0.5); mass spectrum: 124(44), 109(100), 81(10). Anal. Calculated for C_,Ho0o: C, 67.73; H, 6.50. Found: C, 67.71; H, 6.50. The second component was characterised as 3-methyl-5-acetylfuran (14) , X : 280, 230 nm; v ( f i l m ) : 1670, 1505, 880 cm"1; n.m.r. signals: x 2.60 max ' max (IH, m u l t i p l e t , J = 1.1, 1.0), 2.91 (IH, m u l t i p l e t , J = 1.1, 1.0), 7.54 (iH, s i n g l e t ) , 7.90 (3H, m u l t i p l e t , J = 1.1,1.0); mass spectrum 124(40), - 32 -109(100), 80(10). Anal. Calculated for C_Ho0_: C, 67.73; H, 6.50. Found: C, 67.70; H, 6.48. Oxidation of the major acetylation product The major product (0.1 g) obtained above was added drop-wise to an aqueous sodium hypochlorite (5%, 4 ml), which had been preheated to 60°. This mixture was then s t i r r e d at 60-65° for f i f t e e n minutes, after which time sodium bisulphite solution was added to destroy the unreacted hypochlorite. The reaction mixture was then cooled to ice-bath temperature, a c i d i f i e d with d i l u t e hydrochloric acid, and extracted several times with ethyl ether. The organic. of the solvent yielded a s l i g h t l y brown material (0.6 g). R e c r y s t a l l i z a -t i o n of t h i s product from ethanol yielded colorless c r y s t a l s , m.p. 138.5-139.5°, which had spectral properties i d e n t i c a l to those of / 3-methyl-2-furoic acid (7). Vilsmeier formylation of 3-methylfuran Phosphorous oxychloride (2.1 g, 0.014 moles) was added with constant s t i r r i n g to dimethylformamide (1.0 g, 0.014 moles) i n a round-bottom flask f i t t e d with a condenser, dropping funnel and a source of dry nitrogen. The addition, carried out at ice-bath temperature, required t h i r t y minutes and the mixture was then kept cold for a further t h i r t y minutes. 3-Methylfuran (1.1 g, 0.015 moles) was then added to the mixture over f i f t e e n minutes. S t i r r i n g was maintained for - 33 - -one hour at 0° and a further hour at room temperature. The reaction mixture was then poured into cracked ice and water (10 g), neutralized with potassium carbonate, and allowed to s t i r overnight. The organic layer was separated and the aqueous layer was extracted several times with ethyl ether. The organic layer and extracts were combined and dried over sodium sulphate. Removal of the ether yielded a s l i g h t l y yellow o i l (1.2 g) which was examined by g.l.c. and found to contain two components i n the r a t i o 93.5:6.5, having retention times of 8 min. and 10 min, respectively. The major component was characterized as 3-methylfurfural (13); v (f i l m ) : 1680, 890 cm - 1; n.m.r. signals: x 2.43 (IH, multiplet, max ' • ° ' v ' J = 1.8, 0.5), 3.58 (IH, mul t i p l e t , J = 1.8, 1.1), 7.60 (3H, multiplet, Anal. Calculated for C&H 0: C, 65.45; H, 5.49. Found: C, 65.34; H, 5.55. The minor component was characterized as 3-methyl-5-furfural (14); vmax 1 6 7 5 ' 8 8 8 c m " 1 ;  n- m-l- signals: x 2.52 (IH, mul t i p l e t , J = 1.1, I. 0), 2.93 (IH, mul t i p l e t , J = 1.1, 1.0), 7.88 (3H, multiplet, J =1.1, 1.0); mass spectrum: 110(98), 109(100), 81(30). Anal. Calculated for CHO: C, 65.45; H, 5.49. Found: C, 65.67; H, 5.75. Gatterman formylation of 3-methylfuran Anhydrous hydrogen cyanide was generated, i n a dropping funnel, from zinc cyanide (2.7 g, 0.03 moles) i n ether (20 ml) by bubbling anhydrous hydrogen chloride through the mixture f o r two hours. The l i g h t brown two-phase system was then added with vigorous s t i r r i n g to - 34 -a cooled (-10°) 100 ml round bottom flask equipped with a solution of 3-methylfuran (1.1 g, 0.015 moles) i n ether (20 ml). After s t i r r i n g for one hour, the stoppered flask was l e t warm to room temperature and s t i r r e d overnight. The ethereal layer was then decanted and the residue was washed with a small amount of ether. Water (50 ml) was then added to the residue, and the mixture was steam d i s t i l l e d . The aqueous d i s t i l l a t e was extracted several times with ether and the combined organic extracts were dried over sodium sulphate. Removal of the solvent yielded a small amount of yellow o i l . Examination of t h i s material by g.l.c. revealed the presence of several components. The major component (retention time 8 min) was isolated by preparative g.l.c. and was found to have spectral properties i d e n t i c a l to those n f 3— m p f h v l f u r f u r a l (~\ '•i'l . This reaction was repeated under the conditions described above using a shorter reaction time of s i x hours. In this case, the product obtained was found to contain an additional minor component with a retention time of 10 min. Subsequent i s o l a t i o n and characterization of this material proved that i t had spectroscopic properties i d e n t i c a l with those of 3-methyl-5-furfural (14). The two f u r f u r a l isomers occurred i n this reaction i n the r a t i o , 93.5:6.5. Oxidation of major Vilsmeier product The major Vilsmeier formylation product (0.1 g) was added a mixture of s i l v e r oxide (0.3 g) i n aqueous sodium hydroxide solution (10%, 2 ml) and s t i r r e d for t h i r t y minutes at room temperature. The reaction mixture was then f i l t e r e d cooled to ice-bath temperature, and - 35 -neutralized with d i l u t e hydrochloric acid. This mixture v/as then extracted several times with ethyl ether. The extracts were dried over-sodium sulphate and the solvent was removed to y i e l d colorless c r y s t a l -l i n e material (0.09 g), m.p. 139-140°. Characterization of th i s product revealed that i t had spectral properties i d e n t i c a l to those of 3-methyl-2-furoic acid (7). 3-Methyl-2-mercurylfuran chloride 3-Methylfuran (1.6 g, 0.02 moles) i n methanol (1 ml) was added to a s t i r r i n g solution of mercuric chloride (5.0 g, 0.02 moles) i n water (20 ml) at room temperature. Almost immediately a white material.... . precipitated. After s t i r r i n g for one-half hour the reaction mixture was f i l t e r e d . The precipitate was washed with water and a i r dried. (6.1 g); m.p. 138-139°, 237 nm; (nujol): 140, 1060, 885 cm"1; fflcLX iHclX n.m.r. signals: 2.33 (1H, multiplet, J = 1.8, 0.5), 3.58 (IH, multiplet, J = 1.8, 1.0), 7.86 (3H, multiplet, J = 1.0, 0.5). Bromination of 3-methylfuran Anhydrous dibromodioxane was prepared according to the procedure 20 by B i l l i m o r i a and Maclogan. This material (2.5 g) was dissolved i n anhydrous ethyl ether (20 ml) and added to a solution of 3-methylfuran (1.6 g) i n ether (10 ml) previously cooled to -10°. The addition was carried out over f i f t e e n minutes and the mixture was s t i r r e d for a 9 I further f i v e minutes. Water (10 ml) was then added and the mixture was extracted with methylene chloride. After drying over sodium sulphate the solvent was removed to y i e l d a black o i l (1.1 g), which s o l i d i f i e d on standing. No spectroscopic data was obtained on this mate-- 36 -BIBLIOGRAPHY 1. A.P. Dunlop and F.N. Peters, "The Furans", P.V. Reinhold, New York, 1953. 2. P. Basshard and C.N. Eugster i n "Advances i n Heterocyclic Chemistry" (A.R. Katritsky and A.J. Boulton ed.) p. 377, Academic Press, New York, 1966. 3. L. Pauling, "The Nature of the Chemical Bond" 3rd ed. p. 303, Cornell University Press, New York, 1960. 4. L. Paquette, "Principles of Modern Heterocyclic Chemistry", p. 117, W.A. Benjamin, New York (1968). 5. H. Gilman, M. McCorkle, and N.O. Calloway, J. Amer. Chem. Soc. 56, 745 (1934). 6. G.V. Nair, "The Total Synthesis of Veratrum Alkaloids", p. 34, . Ph.D. Desertation, University of B r i t i s h Columbia, Vancouver, B.C. 7. Rinkes, Rec. Trans. Chim. 5_0, 981 (1931). 8. H. Gilman and R. Burtner, J. Amer. Chem. Soc. 55, 2903 (1933). 9. CH. Eugster and P.G. Waser, Helv. Chim. Acta 40, 888 (1957). 10. G. Zwicky, C. Eugster and P.G. Waser, Helv. Chim. Acta 42, 1177, (1957). 11. T. Reichstein, H. Zschokke, and A. Goerg, Helv. Chim. Acta 14, 1277 (1931). 12. H. Gilman and R. Burtner, J. Amer. Chem. Soc. 55.,' 559 (1933). 13. D.M. Burness, i n "Organic Synthesis" _39, (M. Tishler ed.), John Wiley and Sons, Inc., New York, 1959. 14. R.J. Abrahams and H.J. Bernstein, Can. J. Chem. 39_, 905 (1961). 15. M.W. Farrar and R. Levine, J . Am. Chem. Soc. 71, 1496 (1949). - 37 -16. H.D. Hartough and A.I. Kosak, J. Amer. Chem. Soc. j39, 3093 (1947). 17. P. Bosshard and C.N. Eugster, Ibid. p. 399, (1966). 18. V.J. Traynelis, J . J . Miskel, and J.R. Sowd, J. Org. Chem. 22_, 1269 (1957). 19. G.H. Stout, "Composition Tables", W.A. Benjamin, Inc, New York, 1963. 20. J.D. B i l l i m o r i a and N.F. Maclogan, J . Chem. Soc. 3257 (1954). "21. A. Terentev, L. Belenkiz and L. Yanovskoya, Zh. Obschch. Khim. 24, 1265 (1955); see Chem. Abstr. 49, 12327 (1955). 22. P.A. Finan and G.A. F o t h e r g i l l , J. Chem. Soc. 2723 (1963). 23. L. Paquette, Ibid. p. 125 (1968). PART I I STRUCTURAL ELUCIDATION OF OXYGEN HETEROCYCLES FROM ZEYHERA TUBERCULOSA INTRODUCTION Zeyhera tuberculosa Bur. ex. Verlot i s a species of arboraceous plants which are native to B r a z i l . I t belongs to the dicotyledonous family Bignoniaceae of the Scrophulariales order. The present thesis w i l l describe the f i r s t work concerning the chemical constituents of t h i s species. The Bignoniaceae family (commonly cal l e d the trumpet vine family) i s a large one, consisting of some s i x hundred and f i f t y species from one hundred genera of mainly climbing plants. The woody vines are found mainly i n the forest vegetation of South America, however, of the •better known genera, f i v e are native to North America. These include the huge Catalpa trees, the climbing vines of Arrabideae, the conspicous flowers of Tecoma, the cala-bash trees of Crescentia, and desert willows of Chilopsis. Due to the r e l a t i v e i n a c c e s s i b i l i t y of fresh plant material, phytochemical studies of the t r o p i c a l Bignoniaceae family have been lim i t e d , but work has been done i n two main areas of interest. One such area concerns a search for l i g h t o i l s which could be of i n d u s t r i a l value. A variety of unsaturated f a t t y acids has been reported i n the seeds of several species.''' The main area of i n t e r e s t , however, has been concerned with the flavonoid content, of these plants, with emphasis - 40 -on chemotaxonomy. Tbe present sui-vey w i l l deal with these compounds. Flavonoids, the largest group of naturally occurring phenolic compounds are a series of pigments which occur i n almost every plant species. The structures of these compounds i s based on the flavone skeleton (1) which consists of two benzene rings (A and B) joined by a three-carbon chain that i s usually cyclized into a Y ~ P v r o n e r i n g - These compounds are always hydroxylated at various positions, and are usually found as methyl ether derivatives, as O-glycosides, and sometimes as C-glycosides. (1). Several flavones that are hydroxylated only i n ring A have been 2 isolat e d from the Bignoniaceae plant family. Bose and Bhattacharyya found chrysin (2), b a i c a l e i n (3) and the 6-methyl ether of baicalein 3 (called oroxylin-A) i n the species Oroxylum indicum. Harbourne has' reported a s t r u c t u r a l isomer of chrysin, c a l l e d apegenin (4) i n the leaves of Chilopsis saligna Don. Luteolin (5) which contains one more hydroxyl group than apegenin, i s a very common constituent of Bignoniaceae 3 and has been found i n seven out of sixteen species in one survey. In Campensis radicans Seem, and Catalpa bignoides Walt., l u t e o l i n was isola t e d as the 7-glucoside. In four species, Catalpa bignoides Walt, - 41 -C. bungei Mey, C. speciosa Ward, and Tecoma au s t r a l i s R. Br., l u t e o l i n was found to co-exist with a new flavone, 6-hydroxyluteolin (6). Recently, the f i r s t discovery of a flavanone (or 2,3-dihydroflavone) 4 i n Bignoniaceae was made i n our laboratories. The major constituent of the f r u i t of Sparattosperma vernicosum was found to be the 76-neo-hesperidoside of pinocembrin (7). (2) OH 0 (5) OH HO ( 6 ) ° OH 0 Figure 1. Flavones reported i n Bignoniaceae (7) - 42 -Flavones which are hydroxylated at the 3-position are ca l l e d flavonols. The flavonol quercetin (8) i s the most widespread of a l l flavonoids. Bate-Smith'*' f i r s t reported quercetin i n f i v e Bignoniaceae 3 species, and Harbourne l a t e r found t h i s pigment i n the leaves of three more species. Kaempherol (9) i s also a common constituent of dicotyledons and i s most often found as the 3- or 7-glycoside. However, i n Bignoniaceae, i t has been found free and co-occurring with quercetin i n I n c o r v i l l e a marrei and Tecoma stans Juss. (8) •• . (9) Figure 2. Flavonols reported i n Bignoniaceae Those compounds which are generally responsible for the most v i v i d colors i n the plant kingdom are the 4-desoxyflavonols, commonly cal l e d anthocyanins. These flavylium sal t s are most often found as 3rglycosides, In an e a r l i e r survey, anthocyanins have been found in-forty-nine percent of some eight hundred dicotyledonous species. The most common of these pigments i s cyanidin (10). In the - 43 -7 Bignoniaceae family, Forsyth and Simmonds reported cyanidin i n eight 3 species. More recently, Harbourne* reported cyanidin 3-rutinoside i n the petals of four more species. A methylated derivative of cyanidin, c a l l e d paeonidin (11) has been found i n only one Bignoniaceae family, Tecomaria a u s t r a l i s R. Br. Forsyth also found delphinidin (12) i n Jacarunda caerulea and pelargonidin (13) i n two Spathodea species, S. campanulata and S. n i l o t i c a . OH OH (12) (13) Figure 3. Anthocyanins reported i n Bignoniaceae Anthocyanins which lack the 3-hydroxyl are of considerable phylogenic interest because they appear to be rare i n dicotyledonous - 44 -g plant families. The Bignoniaceae i s the only family of the Scrophulariales order from which such pigments have been isolated. In 1927, Chapman, 9 Perkin and Robinson extracted the leaves of Arrabicleae chica and obtained carajurin (14) and carajurone (15). This i s the only report of these two pigments to date. For the purpose of the present discussion these compounds are best represented as the oxonium cations shown i n Figure 4, but they are also often represented as quinones. (14) ' ' (15) Figure 4. 3-Desoxyanthocyanins reported i n Bignoniaceae The type of plant pigments that are classed as quinones, unlike the flavonoids, are found i n lower plants as well as the higher evolved species. In the order Scrophulariales, a great variety of quinones have been found, however, only four such compounds have been isolated from the Bignoniaceae family. Lapachol (16) was f i r s t reported by Lee 1^ i n 1901 i n the sawdust of Tecoma trees. Later, 8-lapachone (17) and dehydrolapachone (18) have been isolat e d from Tabebuia and - 45 -Stereospermum species.''"'1" With the i s o l a t i o n of the previously known techtoquinone (19) from Tecoma. species, a possible biogenetic r e l a t i o n -ship between these four isoprenoid quinones could be suggested, 12 (Figure 5) . Figure 5. A possible biogenetic relationship of isoprenoid quinones reported i n Bignoniceae. - 46 -Another class of compounds isolated from Bignoniaceae are the monoterpenoid cyclopentanoid lactones, or irridoids . Representative . of t h i s class i s catapoie (20a) which was isolated by Bobbitt and 13 -co-workers . along with i t s p_-hydroxybenzoyl ester (20b) from Catalpa ovata. Although i r r i d o i d s are fairly' widespread i n dicotyledenous plant f a m i l i e s , t h i s i s the only report of th e i r existence i n Bignoniaceae. (20a) R = H ronK-v R = n-HOC ,H,-CO > 6 4 At t h i s point i t i s clear that only a limited variety of chemical constituents have been elucidated i n the Bignoniaceae plant family. For chemotaxonomic purposes the work to date i s i n s u f f i c i e n t . It i s apparent that 6-hydroxylation i s a common str u c t u r a l feature of Bignoniaceae flavonoids.. At th i s w r i t i n g , 6-hydroxylation i s a feature of s i x compounds isolated from eight species. Such flavonoids have also been found i n three related f a m i l i e s , Labiateae, Scrophu-3 lariaceae and Verbenanceae. Another feature which may be an important taxonomic marker i s the notable lack of hydroxylation i n ring-B of Bignoniaceae flavones. S i m i l a r l y , O-methylation at the 4'-position i s an unusual feature that i s - 47 -present only i n the 3-desoxyanthocyanins. O-Methylation i n ring-A i s also a rare feature of these pigments. 3-Desoxyanthocyanins characterize the sub-family Gesherioideae of the Gesneriaceae family but have been reported i n only one species of the closely related Bignoniaceae. Ir r i d o i d s may also prove to be of considerable taxonomic importance to the Bignoniaceae family, as they have been frequently reported i n some closely related families such as Scrophulariaceae, Plantiginaceae . 14 and Sympetaleae. In addition to phytochemical investigations i n the Bignoniaceae plant family, some species have been investigated for certain therapeutic value. Kerharo and Bouquet1*' have described the u t i l i z a t i o n of f i v e South African species by the natives of the Gold Coast. Potions made from f r u i t s , leaves and cortex have been used i n the treatment of dysentry, snake-bite, fungal infections, and epilepsy as well as anti-inflammatory and haemeostatic agents. The fact that only limited work has been done on chemical constituents coupled with the p o s s i b i l i t y that some Bignoniaceae species possess p r i n c i p a l s of therapeutic value has encouraged us to begin investigations i n th i s area. Our work begins with the species Zehyera tuberculosa Bur. ex. Verlot, the extracts of which are reputed to have some anti-tumour properties. 1"^ No other work has been reported on th i s species to date. DISCUSSION As there have been no reported studies of the Zeyhera tuberculosa 17 since i t s c l a s s i f i c a t i o n i n 1895, an investigation of the chemical constituents of t h i s plant have begun i n our laboratories. An extract of leaves was prepared i n Rio de Janeiro. The leaves were f i r s t crushed and the pulp was extracted with hot ethanol. Removal of the solvent l e f t a dark green residue with a strong odor, ... .,„ . .5 .. 1 .....!. . .1 C .., .- -., 4-U ~ »-«-<-;+im«tr >-i -P J I 1 C p i U C C ! U . i r; ctuvj.1 LI^VJ. .I.O.L uli^ ^ w u a.j. ai. O n ^ . w - — ~-.--i-— . this extract i s shown i n Figure 6. The f i r s t step involved a separation into sub-extracts according to s o l u b i l i t y in organic solvents. In the second step, each sub-extract was divided into a c i d i c , basic and neutral fractions. It would appear from the u l t r a v i o l e t spectra that s i g n i f i c a n t separation between the sub-extracts was achieved (see Table I ) . The largest fraction was the benzene-neutrals f r a c t i o n , and therefore i t was decided that investigations should begin on t h i s group of constituents. Thin layer chromatography of the benzene-neutrals f r a c t i o n indicated that four major components were present. These components were labelled compounds A, B, C, and D in order of increasing p o l a r i t y on s i l i c a gel chromatoplates (Table I I ) . Ethanolic Extract Benzene Chloroform H 20 Ether ~ 1 Water Acids Bases Neutrals Acids Bases Neutrals 'Acids Bases Neutrals -C-Compound A  Compound B  Compound C Compound D Figure 6. Isolation procedure for constituents of Z. tuberculosa. - 50 -TABLE I Fractions from the Ethanolic Extract of Zeyh.era tuberculosa (25 g). Fraction Yield (g) , MeOH A ' •. max Benzene Acidic 4.60 304,270,213 Basic 0.03 303,269,212 Neutral 14.00 306,269,210 Chloroform Acidic • 0.05 317,282,216 Basic 0.03 320,280,237,212 Neutral 0.17 320 (sh),278,216 Ethyl Ether Acidic 0.14 318,270,332(sh),212 Basic 0.04 320(sh),276,213 Neutral 2.14 310,267,243,211 Water soluble 0.30 - 51 -TABLE II T.l.c; Properties of Components i n the Benzene-Neutrals Fraction Component • * Rf. A 0.95 B 0.90 C 0.35 D 0.30 * Values refer to s i l i c a gel chromatoplates developed with chloroform. - 52 -It i s s i g n i f i c a n t that none of these components could be detected on chromatoplates by application of common spray reagents. 2,4-Dinitro-phenylhydrazine f a i l e d to demonstrate the presence of simple aldehydes and ketones. V a n i l l i n - s u l p h u r i c acid spray showed that terpenoids were probably not present. S i m i l a r l y , the application of antimony t r i c h l o r i d e and pentachloride i n chloroform f a i l e d to indicate the presence of 18 terpenoids, steroids, or a l i p h a t i c l i p i d s . V i s u a l i z a t i o n was best achieved by u l t r a v i o l e t scanning. These preliminary experiments suggest that the major components of the benzene-neutrals f r a c t i o n are probably unsaturated and/or aromatic i n nature. The separation of compounds A, B, C, and D were best achieved by iKintr f w n m 1 limn nhrrnnaf n n r p n h i n nrnrprlnrpc TVIA n n r n n c p r*-£ 1~hp> -Fi r c ; t chromatography on s i l i c a gel was to separate out woody suspensions present i n the extract and also to i n i t i a l l y separate the polar constituents from the non-polar constituents. The f i r s t chromatographic fractions contained compound A, a slightly- yellow o i l which appeared by u l t r a v i o l e t scanning as a single blue spot on the chromatoplate. The u l t r a v i o l e t spectrum of t h i s compound (275,282 nm) indicates that a benzenoid system i s present. Further evidence for this i s indicated by a multiplet absorption at T 2.42 i n the nuclear magnetic resonance spectrum. An absorption at 1725 wavenumbers (cm *) i n the infrared spectrum of compound A suggests the presence of a carbonyl group. No further work was done on t h i s compound at t h i s time. Component B also appeared on s i l i c a gel chromatoplates.as a blue spot under u l t r a v i o l e t scanning. It could be seen, however, that several minor compounds were also present i n these chromatographic fractions. Therefore no further p u r i f i c a t i o n of this compound was attempted. Those chromatographic fractions that contained the polar components C and D were pooled and rechromatographed on alumina. Good separation of these components was achieved. Compound C was r e c r y s t a l l i z e d from ethyl ether to give colorless needle-like c r y s t a l s , m.p. 116-116.5°. The mass spectrum of this material revealed the molecular ion to have an m/e value of 342. High resolution measurement of th i s peak showed the molecular formula to -iy-'i8'"'6' -•-"•^  -• • ••  " • analysis. The u l t r a v i o l e t spectrum of t h i s compound shows absorption bands at 272 nm (Band I) and 304 nm (Band I I ) . This data suggests the p o s s i b i l i t y of aromatic chromophores of the type found i n anthra-• 19 quinones,coumarins, chromones and several types of flavonoids. The spectrum did not change appreciably on addition of a l k a l i , acid or aluminum chloride to the sample c e l l . These results mean that there are no free hydroxyl groups or enolizable carbonyls associated with the chxomophore of .compound C. (It i s known that the formation of tire e n o l - l i k e cinnamoyl grouping (21) of simple flavones results i n 20 a bathochromic s h i f t of Band I, usually with a change i n intensity.) - 54 -0 (21) The most s i g n i f i c a n t feature i n the infrared spectrum of compound C i s a strong absorption at 1655 cm 1. The most probable assignment for t h i s band i s to the stretching carbonyl frequency of an aryl 21 y-pyrone of the type occurring i n chromones (22) . 0 (22) Absorption bands ch a r a c t e r i s t i c of aromatic methyl ethers are also, evident i n this infrared spectrum. C-H symmetrical stretching frequencies appear at 2995> 2940 and 2850 cm 1, while methyl rocking frequencies appear at 1195 and 1120 cm"1. A strong C-O-C stretching band at 1040 cm 1 supports previous assignment of a y-pyrone as well as being additional evidence for the presence of aromatic methyl 22 ethers i n compound C. In the i o w frequency region of the spectrum there are bands at 880, 780, and 700 cm 1 which are ch a r a c t e r i s t i c of C-H out-of-plane - 55 -bending frequencies in. highly unsubstituted benzenoid systems. The nuclear magnetic resonance spectrum of compound C (Figure 7) gives clear evidence for the presence of four aromatic methyl ether groups. Signals at x 5.'90 and T 5.96 each integrate for three protons. A six-proton singlet at T 6.03 may be assigned to two magnetically equivalent aromatic methoxyl groups. In the low-field region of the n.m.r. spectrum, there are two multiplet signals appearing at T 2.1 and x 2.5, which integrate for two and three protons, respectively. A double resonance experiment f a i l e d to completely decouple these signals, but did demonstrate the li k e l i h o o d of mutual coupling. Signals i n th i s region of the n.m.r. spectrum are usually assigned to protons on aromatic systems, or •j^^^Cn.^' M H J . V H 1^ .-1- w UXJ. k-iy V> w j W l - V W <_ U J . W I I I U U J . V - I 1 U V . J . V - J . . 1 i 1 i»» j ^ t i x I. of the spectrum strongly suggests that a mono-substituted benzene ring i s present i n the structure of compound C. The physical data thus far presented strongly suggests that compound C i s a flavonoid which contains four methoxyl groups, but has an unsubstituted ring B. Evidence as to the type of flavonoid i s revealed by a one-proton singlet absorption at x 3.30. This signal could represent the proton on a t r i - s u b s t i t u t e d double bond of a highly conjugated system. The proton at the three-position of flavones (23) generally absorbs i n the region x 3.6 to. 3.7, while the proton at the two-position of iso-flavones (24) usually appears i n the region ; T 2.28 to 2.36. 2 3 (23) (24) Therefore, the most probable structure for compound C i s 5,6,7,8-tetramethoxyflavone (25). Further evidence to support t h i s OCH, OCH7 ^ OCH OCH3 0 (25) assignment was revealed i n the mass spectrum of th i s material (Figure 8). The most obvious feature i n the mass spectrum of compound C (Figure 8) i s the f a c i l e loss of a methyl r a d i c a l from the parent ion to give the base peak at m/e 327. Therefore, i t i s expected that subsequent major fragmentations occur from t h i s ion rather than the parent ion CFigu^e 9). Loss of carbon monoxide from the m/e 327 ion gives the next major ion at m/e 299. The l a t t e r then undergoes 24 cleavage which i s t y p i c a l of flavones. Phenyl acetylene ion i s produced to give a prominent peak at m/e 102, while the other part of Figure 7. Nuclear magnetic resonance spectrum of compound C. - 58 -LZE-esz-o _ a CJ Q ZD ED CU «>_ cu CJ t>SS-£22 • L6T 2BT a .in cn UJ . in ra ro .tn a 007 SL DS • ee o JULJQN31NJ 3AJldl3cJ . a in - 59 -m/e 197 m/e 102 Figure 9. Pr i n c i p l e mass spectrometric fragmentations of compound C. (It i s not intended to infer that the structures presented actually occur i n the instrument.) - 60 -the ion-becomes a fragment giving r i s e to m/e 197. This cleavage i s further evidence that ring B i s not substituted i n the proposed parent molecule i n that an unsubstituted phenyl acetylene ion appears to be produced. Another important fragmentation pathway occurs by the cleavage of the ion m/e 327 to give phenyl acetylene ion d i r e c t l y and also an ion giving r i s e to a s i g n i f i c a n t peak at m/e 225. The l a t t e r then loses carbon monoxide to give the ion at m/e 197. A t h i r d pathway, possibly of lesser importance, occurs from the loss of a methyl r a d i c a l from the ion m/e 299 to give a prominent peak at m/e 284. This ion then cleaves to give the phenyl acetylene ion and an ion which gives a prominent peak at m/e 182. Comnound I) the most, n n l a r r n n v n n n n r l -F-rrvm thi* r h r n m n t n g r a p V i - i t~ c Apar a t j r m was r e c r y s t a l l i z e d from chloroform/n-hexane to give small, colorless c r y s t a l s , m.p. 172-172.5°. The mass spectrum of this compound showed a molecular ion at m/e 312. High resolution mass spectrometric measurement and elemental analysis were i n good agreement with the molecular formula C.oH...0,-. 16 lo D The u l t r a v i o l e t spectrum of compound D was superimposable on that of compound C, thus suggesting a direct relationship between these components. Again there was no change on addition of a l k a l i to the sample c e l l , thereby excluding the p o s s i b i l i t y of enolic or phenolic hydroxyl groups being present i n th i s material. The infrared spectrum further supported a relationship between these compounds. A strong absorption which appears at 1625 cm 1 can be assigned to a y-lactone carbonyl of the type occurring i n chromones. - 61 -In addition, there i s again clear evidence of methoxyl C-H stretching bands at 3000, 2940 and 2850 cm"1. At 1200 and 1120 cm - 1 methyl rocking frequencies are evident. Strong bands at 820, 770 and 690 cm 1 are i n d i c a t i v e of C-H out-of-plane bending frequencies i n aromatic systems. The nuclear magnetic resonance spectrum of compound D (Figure 10) also' reveals a s i g n i f i c a n t relationship to compound C. In the low-f i e l d region there are two multiplets at x 2.22 and x 2.59 integrating for two and three protons, respectively. The most probable assignment for these signals i s two protons of a mono-substituted benzenoid system s i m i l a r to that of compound C. At s l i g h t l y higher f i e l d , there are two one-proton singlet absorptions at x 3.28 and x 3.42. These rlrrnnlr- -C ~ U . - J „ ~ - 1- . - J —4 1- - - 1--.... -•-fe"'-A -' •-'-•"-•- - - - - - -V w" ~ ---.fe ....-w.. i . C ^ p a r t l y substituted by electron-donating substituents ( i . e . methoxyls), or from a proton on the 3-position of a flavone system (23). Two signals at x 6.09 and x 6.15 are singlets that integrate for six and three protons respectively. These can readily be assigned to three aromatic methoxyl groups. Thus, i t would appear that compound D i s of the same type as compound C, but d i f f e r i n g i n structure by one less methoxyl group. The mass spectrum of compound D (Figure 11) reveals a fragmentation pattern s i m i l a r to that of the tetramethoxy compound. The most prominent cleavage i s the loss of a methyl r a d i c a l from the parent ion at m/e 312 to give an ion which gives r i s e to the base peak at m/e 297 (Figure 12). The l a t t e r then loses carbon monoxide to give the next prominent peak COMPOUND D 04 1 1 1 r — 1 — 1 1—1—1 1 r 250 300 350 400 -1 1 r 50 100 to in -1 1 1 1 1 r 150 200 . M/E Figure 11. Mass spectrum of compound D. - 64 -'Figure 12. P r i n c i p l e mass spectre-metric fragmentations of compound D. 65 -at m/e 269. It appears that the ion m/e 269 then cleaves to produce the phenyl acetylene ion at m/e 102 and a fragment giving r i s e to the prominent peak at m/e 167. Another fragmentation pathway i s evident. The ion m/e 269 also appears to undergo another cleavage to lose a methyl r a d i c a l to give ion m/e 254. The l a t t e r then cleaves to give the phenyl acetylene ion plus a prominent ion at m/e 152. This mass spectrum thus supports the previous proposal that compound D i s a flavone which appears to be substituted only i n ring A with three methoxyl groups. To further elucidate the structure of compound D i t was decided to submit a small quantity of t h i s material to a l k a l i n e hydrolysis. TVi <a m c i - i n - p r p a r t i n n r . - r n r l i i r ' f - w a c -i crt 1 3 1-£>rl Irt^r - n - r & r \ a - r a t i -\rr> th i n— 1 zt-\rf*-r " • - ' J ~ ^ - . . •.• -• . . . j i. j. • .* chromatography and was found to be a yellow o i l . The infrared spectrum of t h i s material indicated that an aromatic ketone was present (1610 cm . The nuclear magnetic resonance spectrum c l e a r l y shows three methoxyl signals at x 6.00, x 6.10 and x 6.25. Another three-proton singlet appears at x 7.35 which can be assigned to the methyl group of an acetophenone-type structure. The one-proton single t appearing at x 3.75 occurs i n the region usually associated with the absorption of protons on highly substituted benzenoid systems. At x 3.5 a sharp singlet i s in d i c a t i v e of the presence of an i n t e r n a l l y hydrogen-bonded phenolic group. The parent ion of t h i s material appears i n the mass spectrum at m/e 226.. Although t h i s ion undergoes very f a c i l e loss of a methyl r a d i c a l to give the base peak at m/e 211, i t also undergoes cleavage • 66 -cha r a c t e r i s t i c of aryl methyl ketones. The loss of 43 mass units from the parent ion gives r i s e to a prominent peak at m/e 183. At t h i s point i t can be tentatively concluded that the major.,1'.3. . •.•.-••<x a l k a l i n e hydrolysis fragment of compound D i s an a-hydroxyacetophenone which contains three methoxyl groups! Since i t i s known that flavones give a-hydroxyacetophenones on alkali n e hydrolysis i t would appear that compound D i s a flavone which contains three methoxyl groups i n ring A. The next aspect of the structural elucidation of compound D was the determination of the substitution .pattern. A review of the l i t e r a t u r e revealed that i n s u f f i c i e n t data on trimethoxyflavones was. available for conclusive i d e n t i f i c a t i o n of compound D. It was therefore decided to synthesize what would appear to be the most l i k e l y candidate for the acetophenone hydrolysis product. A review of the flavones previously isolated from the Bignoniaceae plant family (see Introduction) revealed that two t r i s u b s t i t u t e d flavones. have been i d e n t i f i e d . These are baicalein (5,6,7-trihydroxyflavone) and i t s 6--methyl ether. I t , therefore, seemed reasonable from a biogenetic point of view, that f u l l y methylated bacalein could exist and might prove to be compound D. The remainder of the present work describes the t o t a l synthesis of the product resulting from the al k a l i n e hydrolysis of D. The chosen synthetic pathway to the compounds i s shown i n Figure 13. Pyrogallol (27) was methylated without d i f f i c u l t y . A small quantity '26 of sodium hydrosulphite ensured a clean c r y s t a l l i n e product. Without further p u r i f i c a t i o n the trimethyl p y r r o g a l l o l (28) was - 67 --Figure 13. The t o t a l synthesis of compound D hydrolysis product. - 68 -27 28 oxidized to 2,6-dimethoxybenzoquinone (29) with n i t r i c acid. * A good y i e l d i n t h i s reaction could only be achieved i f the reaction tempera-ture was s t r i c t l y controlled. The bright yellow product showed t y p i c a l quinone absorptions i n the infrared near 1700, 1640 and 1600 cm \ The benzoquinone was reduced to the corresponding hydroquinone 27 28 (30) i n good y i e l d with sodium hydrosulphite. ' The diacetate of t h i s compound was prepared by treatment of (30) with excess acetyl chloride to give compound 31 which showed bands i n the infrared near 1750 and 1610 cm \ indicating the presence of an a r y l acetate system. The n.m.r. spectrum of this compound (Figure 14) revealed two three-proton singlets at T 7.68 and 7.72 to further establish the presence of two acetate groups. The methoxy (six-proton s i n g l e t , T 6.22) and •_ .-1 - . _• - J - _ .- - - • - 1 _ / _ 1 _ O £ C \ _ 1 ~ 4- « L U C l - W O a i . U l H d U . J . 1 - " A. O L - W L l O J - g H U X O V.'-* J - f l g J - ^ - 1-5 I -j • \Jj ». ^- w j - j . i w w * . » j ^ - i - C - w ^ -accord with structure 31. Acylation of compound 31 could now be done Via a Fries rearrange-ment. The conditions chosen for t h i s reaction were modified from the 29 method developed by Reynolds and co-workers. The acetophenone (32) was obtained i n good y i e l d without d i f f i c u l t y . Absorptions i n the infrared at 3550, 1765 and 1620 cm 1 c l e a r l y indicated that t h i s product possessed a phenolic group, an a r y l acetate and an a r y l ketone. The n.m.r. spectrum (Figure 15) was p a r t i c u l a r l y informative. In the o f f s e t , a one-proton singlet at x 3.45 indicated the presence of an intramolecular hydrogen-bonded phenolic proton. In-the high f i e l d region of the spectrum a three-proton singl e t at x 7.32 confirmed the presence of an a r y l methyl ketone. A three-proton singlet at x 7.66 showed that one acetate group was present i n the product. Thus, i t i s 8.0 7.0 6.0 5.0 PPM(°» 4.0 3.0 2.0 1.0 0 Figure 14. Nuclear magnetic resonance spectrum of 2,6-rdimetnoxyhydroquinone dxacetate C31).. - 71 -clear that acylation of compound 31 had occurred with loss of one acetate group to give 2,4-dimethoxy-3-acetoxy-6-hydroxyacetophenone (32). Deacetylation of th i s compound under acid conditions yielded the corresponding hydroquinone (33). The l a t t e r was evident by a strong infrared absorption near 3600 cm ^ and the absence of an acetate proton absorption i n the n.m.r. spectrum (Figure 16). ' P a r t i a l methylation of 33 under the mild conditions reported by Seshadri^^'^"'" yielded 2,3,4-trimethoxy-6-hydroxyacetophenone (34). The one-proton singlet absorption at x-3.50 i n the n.m.r. spectrum of thi s material (Figure 17) i s of special i n t e r e s t . This signal can be assigned to an intramolecularily hydrogen-bonded phenolic proton that corresponds to the sim i l a r system found i n the acetate 32. In the the 3-hydroxyl may be ra t i o n a l i z e d on the basis of some deactivation of the 6-hydroxyl by the acetyl group at position 1 through hydrogen bonding, there-by leaving the 3-hydroxyl r e l a t i v e l y more nucleophilic. The methylation producti(34) was isolated as a yellow o i l by preparative th i n layer chroma-tography and found to have i d e n t i c a l spectral properties to the product which was isolated from the alkalin e hydrolysis of compound D (Figure 18). The fact that the acetophenone (34) was obtained on alkalin e hydrolysis establishes the i d e n t i t y of compound D as 5,6,7-trimethoxy-flavone (35). 1 1 1 1 1 1 1 1 1 1 I I I ! 1 .1 1 , 1 1 1 I 1 1 1 1 1 1 1 1 1 I i i S 1 1 1 ' 500 I 1 | I I ' 1 400 l 1 I. 1 1 I l 1 3( I ' 1 1 1 )0 1 : , 1 1 200 1 1 | 1 1 ' I N ' 100 I i I 1 i I 1 I 1 () Hz >-H> 3.o a Figure 16. Nuclear magnetic resonance sy.ectrum of 2,5-dihydroxy-4,6-dimethoxyacetophenone (33). Figure 17. Nuclear magnetic resonance spectrum of 2,3,4-trimethoxy-6-hydroxyacetophenone (34). Figure 18. The infrared spectra of (a) natural and (b) synthetic 1,2,3-trimethoxy-5-hydroxyacetophenone (34). - 75 -(35) (34) 31 This flavone has previously been synthesized by Seshadri i n his studies of b a i c a l e i n . In summation, the investigation of a leaf extract from the plant Zeyhera tuberculosa has revealed the presence of a major component (compound C) which on spectroscopic data was t e n t a t i v e l y i d e n t i f i e d as 5,6,7,8-tetramethoxyflavone (25). Another component (compound D) was i d e n t i f i e d as the trimethyl ether of baicalein on the basis of spectroscopic data as well as the synthesis of a major degradation product. To the best of our knowledge neither of these compounds have previously been found i n nature. The presence of these flavones i n a species from the Bignoniaceae plant family i s of p a r t i c u l a r taxonomic interest as they possess certain features which highlight a trend within t h i s family. These features are ( i ) hydroxylation i n the 6-position, ( i i ) lack of hydroxylation i n ring B; and, ( i i i ) incidence of O-methylation. EXPERIMENTAL Thin layer chromatographic studies were carried out using either Merck s i l i c a gel or Woelm neutral alumina as the absorbents. The chromatoplates, 0.3 mm i n thickness, were a i r dried and activated i n an oven at 100°C for about three hours. In preparative scale th i n layer chromatography a thicker layer of 0.5 mm was used. In a l l cases, electronic phosphor (about 2% by weight) was added to the absorbent ?.S fluorescent ir\Ai ratnr The rhrnmatrm 1 ates were developed i n either chloroform, or a mixture of chloroform plus 2% methanol, and examined under a short and long wavelength u l t r a v i o l e t scanning lamp. Column chromatography was performed using Woelm s i l i c a g el, generally deactivated by the addition of 10% water. The dimensions of the columns were generally maintained at the accepted optimum r a t i o of diameter to height as 1:10. Throughout t h i s work, the solvents were d i s t i l l e d before use. U l t r a v i o l e t spectra were recorded i n methanol on a Cary 11 recording spectrophotometer. Infrared spectra were obtained on a Perkin-Elmer model 21 double-beam spectrophotometer. Solid samples were measured i n KBr p e l l e t s . Samples i n chloroform solution were measured on a Perkin-Elmer model 137 double-beam instrument. The positions of absorption maxima are quoted i n wave numbers (cm ^ ) . - 77 -Nuclear magnetic resonance (n.m.r.) spectra were measured i n deuterochloroform at room temperature. These were measured at 60 MHz on either a Varian A-60 or a Varian T-60 spectrometer. Where additional resolution or double resonance studies were required, the spectra were measured at 100 MHz using a Varian HA-100 instrument. The positions of a l l n.m.r. absorption signals are given i n the Tiers x scale with tetramethylsilane as the internal standard at x 10.00. For multiplet signals x-values given represent the center of the signal. Mass spectra were measured on an Associated E l e c t r i c a l Industries MS-9 double-focusing mass spectrometer. Fragmentation data i s given i n mass to charge r a t i o s (m/e) followed by percent r e l a t i v e abundance. High resolution measurements were also determined on t h i s instrument u s i n g sui'Lctbie SL.aiiua.rub uf known m u l e c u i a r weigh L. Melting points were determined on a Kofler block and are uncorrected. Elemental analyses were performed by Mr. P. Borda of the Microanalytical Laboratory, University of B r i t i s h Columbia, Vancouver. Fraction of the Ethanolic Extract of Z. tuberculosa Leaves of"Zeyhera tuberculosa were picked i n the v i c i n i t y of Poo de Janeiro, B r a z i l . After crushing, they were extracted with hot ethanol. Removal of most of the solvent l e f t a dark green residue. This extract (25 g) was dissolved i n warm water (500 ml) and extracted three times with benzene (1 l i t r e t o t a l ) using separatory funnel. Removal of benzene i n vacuo yielded a dark green gum (18.6 g). The aqueous layer was then extracted three times with chloroform (1 l i t r e ) to give a brown gum (2.7 g). F i n a l l y , the aqueous layer was extracted with ethyl ether (1 l i t r e ) to give another brown gum (0.4 g). Rotovaporation of the remaining aqueous layer yielded a brown, semi-c r y s t a l l i n e residue (0.3 g) with which no further work was done. Each of these sub-extracts was dissolved i n chloroform and treated i n the following manner: F i r s t the extract was washed several times with aqueous ammonium hydroxide (10%). This aqueous layer was then a c i d i f i e d with acetic acid (10%) and extracted with chloroform. Rotovaporation yielded a residue which was called acid material. The organic layer (which had been washed with ammonium hydroxide) was then washed several times with acetic acid (10%). The aqueous layer was again extracted with chloroform, and rotovaporation yielded a residue called basic material.. Rotovaporation of the o r i g i n a l organic layer Treated i n this way the benzene sub-extract yielded: Benzene-acids (4.6 g); A ' : 213, 270, 304 nm; v(chloroform): 1695, 1639 cm - 1. nicLX Benzene-bases (0.03 g); A : 212, 269, 303, 343 nm; v(chloroform): fficlX 1724, 1639, 1587 cm - 1. Benzene-neutrals (14.0 g); A 210, 269, 306 nm; v(chloroform): 1724, 1639 cm 1. The chlorform sub-extract yielded the following materials: Chloroform-acids (0.05 g); A : 216, 282, 317 nm; v(chloroform): 1695, nicix 1613 cm"1. Chloroform-bases (0.03 g); A : 212, 237, 280, 320 nm; ^ 6 ' max . v(chloroform): 3333, 1695, 1587 cm 1. Chloroform-neutrals (0.17 g); A : 216, 278, 320 (sh) nm; v(chloroform): 3571, 1754, 1613 cm"1, in 3.x F i n a l l y , the ether sub-extract treated i n the above manner gave the following materials: Ether-acids (0.14 g); A : 212, 232 (sh), e- v. b J , m a x , 270, 318 nm; v(chloroform): 1675, 1613 cm" . Ether-bases (0.04 g); A : 213, 276, 320 (sh) nm; v(chloroform): 3333, 1724, 1639 cm"1, max ' ' \ • J > \ J > > Ether-neutrals (2.14 g); A : 216, 278, 320 (sh) nm; v(chloroform): nicix 2857, 1667 cm"1. Thin Layer Chromatography of the Benzene-neutrals Fraction A sample of the benzene-neutrals f r a c t i o n from the above separation procedure was chromatographed on neutral alumina, using chloroform as the developer. Considerable d i f f i c u l t y was experienced i n v i s u a l i z a -t i o n . Several common spray reagents such as antimony t r i c h l o r i d e , antimony pentachloride, v a n i l l i n , eerie sulphate and 2,4-dinitrophenyl-hydrazine was t r i e d with l i t t l e success. The best v i s u a l aid was the alumina were less than well-defined. Chromatography on s i l i c a gel with chloroform plus 1% methanol as developer gave better r e s u l t s . Under u l t r a v i o l e t l i g h t i t could be seen that t h i s f r a c t i o n consisted mainly of two closely running compounds (dark blue spot, = 0.35, and yellow spot, R^  = 0.30), and also considerable front running material. Subsequent, chromatography of the l a t t e r on s i l i c a gel using benzene as developer showed this to consist of two compounds. Thus, these four compounds were labelled i n order of increasing p o l a r i t y as compounds A, B, C, and D. Separation of Components of the Benzene-neutrals Fraction (a) Column Chromatography on S i l i c a Gel A t y p i c a l separation procedure was carried out as follows: A sample of benzene-neutrals extract (5.0 g) was dissolved i n a minimum - 80 -amount of benzene and applied to the head of a column of s i l i c a gel (500 g, 10% water). .Elution with benzene (700 ml) yielded a s l i g h t l y yellow o i l (0.34 g) which was named Compound A. Further elution with benzene (750 ml) gave an orange and p a r t l y - c r y s t a l l i n e gum (0.4 g), cal l e d Compound B. The column was then stripped with ether (1 l i t r e ) to give a green gum (3.2 g) consisting mainly of Compounds C and D. (b) Column Chromatography,on Alumina The gum (3.2 g) obtained from the i n i t i a l chromatography on s i l i c a gel was dissolved i n benzene and applied to a column of alumina (150 g + 3% water). Elution with benzene (1 l i t r e ) plus benzene with 10-20% ether (1 l i t r e ) gave a white c r y s t a l l i n e compound C i n a green gum (z. 0 g) . rui'Liicr c i u L x u u j j n v c anotiic-i' white c r y s t a l l i n e ccrr.pcur.d D i n a green gum (0.4 g). Properties of Compound A This material appeared as a single blue spot on a s i l i c a gel chromatoplate under u l t r a v i o l e t scanning. The following data was obtained without further p u r i f i c a t i o n : X /. 275, 282 nm; v(neat): 3119. X 2900, 1725, 1250, 1110 cm - 1; n.m.r. signals: x 2.40 (multiplet), 5.75 (multiplet), 8.65 (multiplet), 9.1 (multiplet). Properties of Compound B Several attempts to c r y s t a l l i z e t h i s material from common solvents f a i l e d . Data was obtained on crude material: X 225, 274 nm; max v(chloroform): 2941, 1724, 1449 cm - 1; n.m.r. signals: x 2.4 (multiplet, - 81 -J = 5 Hz), 4.7 (multiplet), 5.75 (doublet, J = 5 Hz), 5.99 (doublet, J = 7 Hz), 6.32 ( s i n g l e t ) , 7.8 (mul t i p l e t ) , 8.35 (doublet, J = 5 Hz), 8.7 ( s i n g l e t ) , 9.1 (multiplet). Properties of Compound C This material (2.0 g) was r e c r y s t a l l i z e d from ethyl ether to give long colorless needle-like crystals (1.9 g), m.p. 116.5-117.0°, ^ m a x : 212, 271, 304 nm (no s i g n i f i c a n t change i n 0.002 M NaOCH^ or on addition of aluminum ch l o r i d e ) , v(KBr): 2995, 2940, 2850, 1655, 1587, 1195, 1120, 1040, 880, 780, 700 cm"1; n.m.r. signals: T 2.1 (2H, m u l t i p l e t ) , 2.5 (3H, m u l t i p l e t ) , 3.30 (IH, s i n g l e t ) , 5.96 (3H, s i n g l e t ) , 6.03 (6H, s i n g l e t ) ; mass spectrum: 342(35), 328(25), 327(100), 299(15), 284(25), ?67(.1.6) ,. 19"/ (20) , iH'^O.'y) . 102 0.6).-Anal. Calculated for C 1 9 H 1 8 ° 6 " c, 66.66; H, 5.30; M.W. 342.110; Found: C, 66.49; H, 5.35; M.W. 342.110 (high resolution mass spectro-metry) . Properties of Compound D Re c r y s t a l l i z a t i o n of th i s material was achieved by dissolving the crude compound (0.4 g) i n a minimum amount of hot chloroform and then adding i n one portion a large volume of n-hexane. This method yielded short colorless crystals (0.16 g after three r e c r y s t a l l i z a t i o n s ) , m.p. 172.0-172.5°; X : 212, 272, 304 nm; v(KBr): 3000, 2940, 2850, 1625, max 1595, 1350, 1200, 1120, 820, 770, 690 cm"1; n.m.r. signals: T 2.22 (2H, m u l t i p l e t ) , 2.59 (3H, mul t i p l e t , 3.28 (IH, s i n g l e t ) , 3.42 (IH, s i n g l e t ) , 6.09 (6H, s i n g l e t ) , 6.15 (3H, s i n g l e t ) ; mass spectrum: 312(25), 297(100), 295(7), 271(7), 269(13), 254(15), 167(12), 128(12). - 82 -Anal. Calculated for C 1 oH n,0 r: C, 69.22; H, 5.16; M.W. 312.100. 18 16 5 Found: C, 69.50; H, 4.80; M.W. 312.110. Hydrolysis of Compound D Compoudn D (70 mg) was added to a methanolic solution of potassium hydroxide (10 ml, 50%). The mixture was refluxed for s i x hours. The reaction mixture was then cooled and neutralized with d i l u t e hydro-c h l o r i c acid. Extraction with methylene chloride yielded a yellow gum (72 mg). Preparative thin-layer chromatography on s i l i c a gel (chloroform plus 1% methanol) made possible the i s o l a t i o n of a yellow o i l (24 mg) which was the least polar component. The following spectral data was obtained: A 281. 333 nm: vt'neat): 2940. 1610. 1110. 590 cm "*": max ' n.m.r. signals: x 3.50 (IH, s i n g l e t ) , 3.75 (IH, s i n g l e t ) , 6.0 (3H, s i n g l e t ) , 6.10 (311, s i n g l e t ) , 6.25 (3H, s i n g l e t ) , 7.35 (3H, s i n g l e t ) ; Mass spectrum: 226 (72), 211 (100), 195 (11), 193 (20), 186 (55), 165 C 4 9 ) , 151 (41). Anal. Calculated for C 1 1 H 1 4 0 5 : c> 58.40; H, 6.24. Found: C, 58.60; H, 6.14. 1,2,3-Trimethoxybertzene (28) 1,2,3-Trihydroxybenzene (100 g) was added to a solution of ethanol (300 ml, 95%), dimethylsulphate (355 ml), and sodium hydro-sulphite (5 g) i n a 2 l i t r e , three-necked round bottom fla s k . The l a t t e r was equipped with a thermometer, a pressure-equalizing dropping funnel, and a source of nitrogen. This apparatus was then flushed with nitrogen and cooled i n an ice-water bath. The dropping funnel was charged with a solution of sodium hydroxide (150 g) i n water - 83 -(350 ml). This solution was dropped into the reaction mixture (over 2 hours) with s t i r r i n g . The dropping rate was adjusted to prevent the temperature of the reaction mixture from exceeding 25°. S t i r r i n g at room temperature was continued for a further 4 hours. The reaction mixture was then poured into ice (800 g) to complete c r y s t a l l i z a t i o n of the product. F i l t r a t i o n yielded a s l i g h t l y pink c r y s t a l l i n e material (125 g). A small portion of th i s material was sublimed (100-110°/15 mm) to give colorless long needle-like c r y s t a l s , m-F- 47°,(lit.m,p. 47°) , X 267,277(sh) nm; v(nujol): 1600,1450,775,735, TTlelX 695 cm"1; mass spectrum: 168 (100), 153 (81), 125 (49), 110 (62), 95 (47). Anal. Calculated for CgH^O,^ C, 64.27; H, 7.19. Found: C, r>4-. i u : n . 7 . 21 . 2,6-Dimethoxybenzoquinone (29) 1,2,3-Trimethoxybenzene (120 g) was dissolved i n ethanol (550 ml, 95%) i n a 3 - l i t r e , 3-necked round bottom flask which was equipped with a r e f l u x condenser, dropping funnel, a thermometer, and an e f f i c i e n t magnetic s t i r r e r . This reaction mixture was then warmed to 50°. N i t r i c acid (200 ml, density 1.42) was dropped into the mixture at such a rate that the reaction temperature did not exceed 55°, about 2 hours (occasionally i t became necessary to cool the mixture rapidly with a dry ice-acetone bath, as i t was very important to control the tempera-ture). S t i r r i n g at 50° was continued for a further 2 hours. The reaction mixture was then cooled i n ice and f i l t e r e d . The lemon-yellow product was washed thoroughly with cold water, and then a i r dried to y i e l d 90 g of material. R e c r y s t a l l i z a t i o n of a small portion of t h i s compound from methanol yielded bright yellow needle-like c r y s t a l s , m.p. 248.5-249.0°(lit. m.p.249°) 2 7, X r 285nm; v(chloroform): 290,1700,1650, 1600 cm "*•; n.m.r. signals: T 6.15 (2H, s i n g l e t ) , 6.75 (6H, s i n g l e t ) ; ' mass spectrum: 168 (34), 138 (15), 125 (10), 87 (11), 80 (27), 69 (100). Anal. Calculated for C gH g0 4: C, 57.14; H, 4.80. Found: C, 57.11; H, 4.91. 2,6-Dimethoxyhydroquinone (30) 2,6-Dimethoxybenzoquinone (55 g) was mixed with sodium hydrosulphite (120 g). Boiling water (550 ml) was added to the dry mixture i n one portion. The reaction mixture was then swirled for 2 minutes and off , washed with cold water and dried, to give colorless c r y s t a l s , m.p. 158-160°Clit. m.p.158°) , \ 285 nm; v(nujol): 3300,1460,1120 cm"1; n.m.r. signals: T 2.55 (2H, s i n g l e t ) , 6.06 (6H, s i n g l e t ) , mass spectrum: 170 (55)', 168 (18), 155 (54), 127 (100), 112 (42), 109 (18), 84 (18). 2,6-Dimethoxyhydroquinone diacetate (31) Acetyl chloride (150 ml) was added to dry 2,6-dimethoxyhydroquinone (50 g). After flushing the apparatus with nitrogen, the reaction mixture was refluxed for 1 hour. The solution was then cooled and cautiously poured onto ice (200 g). The product was then f i l t e r e d , washed with water, and dried to y i e l d s l i g h t l y yellow c r y s t a l l i n e material (41 g). A small portion of th i s material was r e c r y s t a l l i z e d f r om methanol to give colorless c r y s t a l s , m. p. 123-124° ( l i t .m. p. 123°) 2 7 , X 267,275(sli - 85 -v(chloroform): 1750; 1125 cm - 1; n.m.r. signals: T 3.62 (2H, s i n g l e t ) , 6.20 (6H, s i n g l e t ) , 7.68 (3H, s i n g l e t ) , 7.72 (3H, s i n g l e t ) ; mass spectrum: 254 (7), 212 (25), 170 (100), 155 (21). Anal. Calculated for C 1 2 H 1 4 0 6 : c> 56.69; H, 5.55. Found: C, 56.40; H, 5.44. Fries Rearrangement of 2,6-Dimethoxyhydroquinone Diacetate 2,6-Dimethoxyhydroquinone diacetate (2 g) was ground to a powder and mixed thoroughly with anhydrous aluminum chloride (2 g). About one t h i r d of t h i s mixture was added to a thick-walled flat-bottom flask which had been suspended i n an oil-bath maintained at 120-125°. After t h i s i n i t i a l reaction, had begun as indicated by the evolution of hvrlrncrp/n PVI 1 O T*i d p. na? f a f t e r ? - 4 mi iintp.s; 1 t h e m i y t n r f i wa<; n t . i r r p . i i vigorously with a glass rod u n t i l the reaction had subsided. A second portion was added and rapid s t i r r i n g was resumed. After the last portion was added the reaction mixture was s t i r r e d for about 10 minutes. Heating with periodic s t i r r i n g was continued u n t i l the evolution of hydrogen chloride was no longer evident (about 30 minutes). The reaction mixture was then cooled and ground i n a mortor and then added, with s t i r r i n g , to a mixture of ice (2 g) and concentrated hydrochloric acid (_1 ml). The res u l t i n g s l u r r y was s t i r r e d for one-half hour, after which time the s o l i d was f i l t e r e d and washed with cold water. After thorough drying i n vacuo t h i s yellow material (2 g) was examined by th i n layer chromatography ( s i l i c a g e l , chloroform) and was found to consist predominantly of 2,4-dimethoxy-3-acetoxy-6-hydroxyacetophenone (32) with some of the corresponding deacetylated compound (33). - 86 -A small portion of t h i s mixture was separated by preparative thin layer chromatography. The major product was r e c r y s t a l l i z e d from methanol to y i e l d colorless c r y s t a l s , m.p. 109-110°, X : 278,317 nm; v: mux 3520, 1760, -1620 cm"1; nmr signals: -3.5 (IH, s i n g l e t ) , 3.72 (IH, s i n g l e t ) , 6.10 (3H, s i n g l e t ) , 6.15 (3H, s i n g l e t ) , 7.32 (3H, s i n g l e t ) , 7.66 (3H, s i n g l e t ) ; mass spectrum: 254(5), 212(100), 197(80),194(15),151(17). Anal. Calculated for C 1 oFL.0,: C, 56.69; H,-5.55. Found: C, 12 14 6 56.61; H, 5.60. 2,5-Dihydroxy-4,6-dimethoxyacetophenone (33) The crude Fries rearrangement mixture was thoroughly dried, and then refluxed with methanolic hydrogen chloride (5%) for one hour^ a i l c r wuxCii L x i i i e die s u i v e n l was r c i i i u v c u , cAciiiixiia l i o n ox l i i i S p i u O u c L by t h i n layer chromatography ( s i l i c a gel, chloroform) revealed the presence of one major component. Preparative th i n layer chromatography on a small portion of crude product yielded yellow c r y s t a l l i n e material, m.p. 162-163°; X 242, 283 nm; v (chloroform): 3600, 1640 cm - 1; n.m.r. signals: T 3.70 (IH, s i n g l e t ) , 6.00 (3H, s i n g l e t ) , 6.02 (3H, s i n g l e t ) , 7.30 (3H, si n g l e t ; mass spectrum: 212(90), 197(100), 182(38), 179(20), 169 (25), 151(50). Anal. calculated for C I Q H 1 2 0 S : C> 5 6 - 6 0 " H» 5.70. Found: C, 56.53; H, 5.68. ' - 87 -2,3,4-Trimethoxy-6-hydroxyacetophenone (34) A mixture of the above dihydroxyacetophenone (33, 1 g), dry acetone (5 ml), dry benzene .(50 ml), anhydrous potassium carbonate (3 g) and acid-free dimethylsulphate (0.5 ml) was refluxed for 12 hours. The inorganic s a l t s were then f i l t e r e d o f f and washed with a small quantity of hot benzene. The benzene f i l t r a t e was washed twice with water and then extracted with sodium hydroxide (5%). After a c i d i f i c a -t i o n with d i l u t e hydrochloric acid at 0°, the mixture was extracted with several portions of methylene chloride. This organic extract was then washed with water and dried over sodium sulphate. Removal of the solvent yielded a brown amorphous material which was found to contain several components on t h i n layer chromatography ( s i l i c a g e l , nT-i 1 ri;rr\-pQ-rTr)"\ P r p n a v a t i y c tViip "layer r - h r r n p n t n r r r f i p h v n f th'i'; n r n r i l l C i ' . yielded a small amount of yellow o i l as the least polar component. This o i l was found to have spectroscopic properties i d e n t i c a l with those of the hydrolysis product of compound D. - 88 -BIBLIOGRAPHY 1. (a) F. E a r l , J. Am. O i l Chemists Soc. 37_, 440 (1960); (b) M.J. Chisholm and C.Y. Hopkins, Can. J. Chem. 43, 2566 (1965). 2. P.K. Bose and S.N. Bhattacharyya, J. Indian Chem. Soc. 15, 311 (1938). 3. J.B. Harbourne, Phytochemistry 6_, 1643 (1967). 4. J.P. Kutney, W.D.C. Warnock, and B. G i l b e r t , Phytochemistry, i n press. 5. E.C. Bate-Smith, J. Linn Soc. (Botany) 58, 39 (1962). 6. ' G.H. Beale, J.R. Price and V.C. Sturgess, Proc. Roy. Soc. B130, 113 (1941). 7. W.G.C. Forsyth and N.W, Simmonds. Proc. Roy. Soc, B142, 549 (195A). 8. J.B. Harbourne, i n "Comparative Phytochemistry" (T.W. Swain, ed.) .p. 278, Academic Press, New York (1966). 9. E. Chapman, A.G. Perkin and R. Robinson, J. Chem. Soc. 3015 (1927). 10. C. Mathis, i n "Comparative Phytochemistry" (T. Swain, ed.), p. 267, Academic Press, New York (1966). 11. R.H. Thompson, i n "Chemistry and Biochemistry of Plant Pigments" (T.W. Goodwin ed.) p. 328, Academic Press, New York (1965). 12. R.H. Thompson, "Naturally Occurring Quinones", p. 59, Butterworths S c i e n t i f i c Publications, London (1957). 13. J.M. Bobbit, D.W. Spiggle, S. Mahboob, W. Philipsborn and H. Schmid, Tetrahedron Letters, 321 (1962). 14. E.C. Bate-Smith and T. Swain, i n "Comparative Phytochemistry", (T.W. Swain ed.) P. 170, New York (1966). 15. J . Kerharo and A. Bouquet, B u l l . Soc. Bot. France 94 (7), 251 (1947) - 89 -o ^  T T T i,' — J 11 n - - i ~ ~ ^ . — ~.c T-1 ^ ^i-«^ r> T,UT^ r n4-^ 16. B. G i l b e r t , Private Communication. 17. C.F.P. de Martius, Flora B r a s i l i e n s i s , Vol. I l l , Weldon and Wesley Ltd., Codicote, Herts, England, p. 354. 18. Bobbit "Thin-Layer Chromatography',' Reinhold Publishing Corporation, New York, 1963, p. 88. 19. L. Jurd, i n "The Chemistry of Flavonoid Compounds", T.A. ..Geissman (ed.) p. 107, Macmillan Co. New York (1962). 20. K. Venkataramen i n "The Chemistry of Flavonoid Compounds". (T.A. Geissman (ed.),p. 83, Macmillan Co. New York (1962). 21. K. Nakanishi, "Infrared Absorption Spectroscopy" p. 43-.Holden-Day, Inc. San Francisco, 1962. 22. K. Nakanishi, Ibid. p. 36, (1962). _~ j b l i c a t i o n no. 6418 (1964). 24. H. Budzikiewicz, C. Djerassi, and D. William."Structural Elucidation of Natural Products by Mass Spectrometry I I " , p. 262 Holden-Day, Inc., San Francisco (1964). 25. F.M. Dean "Naturally Occurring Oxygen Ring Compounds". Butterworths, London (1963) p. 291. . 26. E. Chapman, A.G. Perkin and R. Robinson, J . Chem. Soc.3028 (1927). 27. F. Manthner, J. Prak Chem. 287 (1936). 28. W. Baker, J. Chem. Soc. 662 (1941). 29. D. Reynolds, J. Cathcart and J. Williams, J. ORg. Chem. 18_, 1709 (1953) 30. V.D. Nageswara S a s t r i and T.R. Seshadri, Proc. Ind. Acad. S c i . A . 253 (1946). 31. V.D. Nageswara S a s t r i and T.R. Seshadri, Ibid. 262, (1946). 

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