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Synthesis of potential intermediates in the biosynthesis of flavoglaucin Avelino, Norman G. 1983

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SYNTHESIS OF POTENTIAL INTERMEDIATES IN THE BIOSYNTHESIS OF FLAVOGLAUCIN by NORMAN G. AVELINO c , The U n i v e r s i t y of B r i t i s h Columbia, 19 A THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 19B3 (T) Norman G. A v e l i n o , 19B3 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of V-The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 5 5 DE-6 (3/81) i i Abstract Previous work on the biosynthesis of compounds derived by the acyl-polymalonate route i s discussed. Flavoglaucin(84), a pigment produced by Aspergillus glaucus, i s derived by the acyl-polymalonate route. Feeding experiments using labelled acetate and malonate on flavoglaucin (84), have proven i t s origin as being from this route, however, no feeding experiments using potential intermediates have been reported. Thus, the i n i t i a l objective of our research was to synthesize deuterium-labelled compounds which could be regarded as probable intermediates in the biosyn-thetic route. Subsequent incorporation experiments using cultures of A. glaucus could then provide information which would assist in the eluc i -dation of the biosynthetic sequence leading to flavoglaucin(84). 6-n-heptyl-o<-[deuterio]-2-hydroxybenzaldehyde(92a) and i t s correspond-ing acid(92b) are potential intermediates in the biosynthesis of flavo-glaucin. Compound(92a) was synthesized from furan(157) and dimethylace-tylene dicarboxylate(158) in eleven steps. Various attempts to synthesize the corresponding acid (92b) f rom N,N-diethyl-6-n-heptyl-2-methoxybenzairu.de (97) , 3-n-hexyl-7-methoxyphthalide (109) , and methyl-6-n-heptyl- ©<.- [oxo] -2-methoxybenzoate(133) were unsuccessful. The syntheses of 6-n-heptyl-c* -[deuterio] -2-benzyloxybenzaldehyde (165) and 6-n-heptyl-o< - [deuterio] -2-methoxybenzaldehyde were successful but various attempts using several different oxidizing agents fai l e d to oxidize either aldehyde to their corresponding acids. i i i Table of Contents A b s t r a c t Page i i Table of Contents 1 1 1 L i s t of Schemes l v Table v i i i L i s t of A b b r e v i a t i o n s • • • i x Acknowledgements x A. I n t r o d u c t i o n 1 B. R e s u l t s and D i s c u s s i o n 22 C. Experimental 4 7 D. B i b l i o g r a p h y 6 9 E. S p e c t r a l Index 7 2 iv List of Schemes Page Figure . • 2 1 . . . 3 2 . . 3 3 . . 4 4 . . . 5 5 . . . 6 6 . . . 11 7 .... 1 5 8 17 9 . . . . 18 10 V List of Schemes (conf d) Page Figure — 11 . . . 21 12 . . . . 23 13 . . . . 25 14 . . . 26 15 . . . 27 16 . . 28 17 . . 29 18 . . . 30 19 . . . 31 20 . . . . 31 21 v i List of Schemes(contvd) Page Figure . . . 32 22 . . 32 23 . . . 33 24 . . . 34 25 . . . 35 26 . . . 36 27 . . . 37 28 . . 38 29 . . . 39 30 . . . 39 31 . . . . 40 32 v i i L i s t of Schemes(cont'd) Page Figure  41 33 42 34 . . 43 35 . . 44 36 v i i i Table Table Page 1 13 ix L i s t of A b b r e v i a t i o n s s i n g l e t broad s i n g l e t doublet t r i p l e t broad t r i p l e t q u a r t e t broad q u a r t e t n u l t i p l e t broad n u l t i p l e t tetrahydrofuran N,N-dimethyIformamide X Acknowledgements I would l i k e to thank my s u p e r v i s o r , Dr. Thomas Money, f o r -h i s i n v a l u a b l e help throughout the p e r i o d of time the research f o r and p r e p a r a t i o n of t h i s t h e s i s were undertaken. I w o u l d a l s o l i k e to thank Dr. Michael Dadson f o r h i s h e l p f u l s u g g e s -t i o n s . -1-INTRODUCTION Early explanations for the biosynthesis of natural products were largely based on the recognition of structural s i m i l a r i t i e s among various secondary metabolites. Of particular Importance was the recognition of a common structural sub-unit i n many compounds and this led to a c l a s s i f i c a t i o n of natural products based on their proposed biosynthesis. For example, amino acid residues were recognizable structural sub-units i n alkaloids while iso-prene units were discernible in the structures of terpenoids. For many naturally occuring phenolic compounds, the i n i t i a l biosynthetic proposals were made by J.N. C o l l i e 1 ' 2 i n 1907. Col l i e postulated a relationship be-tween certain laboratory and biosynthetic processes on the assumption that, chemical mechanisms should be recognizable i n biosynthetic processes even though enzymes and co-enzymes are clearly important. For example, he sug-gested that o r s e l l i n i c acid (5), a common lichen constituent, came from two molecules of acetoacetic acid. This was based on his observation in the laboratory that two molecules of ethyl acetoacetate condensed to form de-hydracetic acid(l) which upon treatment with a l k a l i gave o r s e l l i n i c acid (5) (Scheme 1). SCHEME 1 Collie's theory was largely forgotten presumably because isotopically labelled precursors were not available at that time to test his hypothesis. In 1951, A.J. Birc h 3 revived the interest i n polyketide biosynthesis. While Collie's theory stemmed from the chemical reactivity of diketonates, Birch's theory was an extrapolation of the known importance of acetic acid (6) i n fatty acid and terpenoid biosynthesis. As a modification of fatty acid biosynthesis, Birch's theory was i n i t i a l l y an inquiry as to what could result (on paper) from chemically acceptable cyclizations of poly-^ -ketomethylene compounds(8) derived by the condensation of acetic acid molecules (Scheme 2) . - 3 -2 CH 3C0 2H 6 CH3COCH2C02H n CH^CO?H CH3CO(CH2CO)nCH2C02H 8 SCHEME 2 Development o f h i s t h e o r y was f u r t h e r encouraged by the p o s s i b i l i t y t ha t such o r i g i n s , i . e . o r i g i n a t i n g from a c e t i c a c i d , might be r e v e a l e d by "marker" oxygen atoms (Scheme 3) i n n a t u r a l l y o c e u r i n g p h e n o l i c compounds 2 . B i r c h found t h a t i t was p o s s i b l e i n many i n s t a n c e s t o d i s t i n g u i s h the b i o -s y n t h e t i c u n i t s by i n s p e c t i o n o f the formulae o f the f i n a l p r o d u c t s : p o l y -k e t i d e s are marked by the f requent occurences o f oxygen atoms a t t ached i n p o s i t i o n s (3 t o each o the r and a l s o 0 t o p o s i t i o n s o f r i n g c l o s u r e (Scheme 3 ) . PRECURSOR NATURAL PRODUCT 0 0 0 HO-" v ^0H ORSEtUNlC ACID 5 ALTENAR I0. :3 O H u ENDOCROCIN 15 SCHEME 3 -4-The involvement of malonate in fatty acid biosynthesis^ and phenolic degradation products^>6,8 i e ( j Bu'lock and H.M. Smalley to prove that malonate was also involved in phenolic biosynthesis 7. This led to a more sophisticated general statement of phenolic biosynthesis which is now called the acyl-polymalonate route. The acyl-polymalonate route to phenolic compounds and derivatives in-volves condensation of an enzyme-bound acyl-starter unit (RCO-s-Enzyme), usually acetate, with a variable number of malonate units as their co-enzyme A esters (H02CCH2COSCoA) to produce a poly-^-keto enzyme-bound thiol ester intermediate^. Subsequent cyclization of this intermediate can then produce phenolic compounds of widely varying structures (Scheme 3 ) . The term poly-ketide has been coined to describe phenolic compounds derived in this way (Scheme 4 ) . RCOSEnzyme • n HCuCCH-COSCoA > 2 2 -n CoASH -n C 0 2 RCO(CH 2 CO) n 0 CH 2 COSEnzyme C y c l i z o t i o n > PHENOLIC COMPOUNDS SCHEME 4 Polyketides are produced mainly by fungi and to a lesser extent by higher organisms and often have no obvious function. Several compounds have been classified as antibiotics and-plant pigments. A flow chart for the elaboration of a polyketide precursor is shown in Scheme 5. The chain-initiating unit can vary from acetic acid to long-chain fatty acids and aromatic acids 2 ' 5 . However, as in fatty acid bio-synthesis, malonate is the chain-propagating unit. - 5 -Gcetote oleate cinnamate benzoate RC0 2 H RCOSCoA EnzSH RCOSEnz (chain-initiating unit) CHLCO,H CoASH CH 3COSCoA 'b io t in/Mg 2 * A T P / H C O : v 3 HO2CCH2COSCOA EnzSH H02CCH 2COSEnz (chain-propagating unit) RCOCH 2COSEnz -n CO RCO(CH 2 CO) n . 1 CH 2 C0SEnz 1 Intramolecular cyclization 2 Secondary transformations PHENOLIC COMPOUNDS 8, DERIVATIVES SCHEME 5 - 6 -A proposed mechanism for the assembly process (Scheme 6) involves an enzyme or enzymes containing two sulfhydryl groups. ( ^ ^ S H = Enzyme- (SH ) . "SH / ^ V 5 H RCOSCoA \ * _ ^ chain-initiating unit aSCOR HO^CCH^COSCoA ^ chain-propagating unit S— COR 5C0CH 2 —C0 2 " H0 2CCH 2C0SCoA SC0CH 2 C0R >->fSC0CH 2 7 C0 2 - > ^ S C 0 C H 2 C 0 C H 2 C 0 R ^ 5 - C 0 C H 2 C 0 R - C 0 2 ^ S * n H0 2 CCH 2 C05CoA - n C O , ^ ^ C 0 C H 2 (COCH 2 ) n _ 1 COR 1 intramolecular cyclizat.on ^ , C 0 M p 0 U N D S 2 Secondary transformations ^ DERIVATIVES SCHEME 6 - 7 -The variety of phenolic products and other non-phenolic products obtained by the polyketide route i s made possible by the manipulation of several variables, v i z . , (a) the nature of the chain-initiating unit (usually acetate but other p o s s i b i l i t i e s could be certain fatty acids, propionate, cinnamate, or benzoate). (b) the number of chain-propagating units i.e. the length of the polyketide intermediate. (c) the different modes of cyclization and aromatiza-tion processes (e.g.aldol, Claisen condensations, and lactonization). (d) secondary transformation (e.g. methylation, prenylation, reduction, and oxidation) which can occur before or after cyclization and aromatization processes and i n some complex cases these additional reactions may even obscure the fact that the compound i s of polyketide origin. (A) Different Modes of Cyclizations. There are two major types of cyclization reactions: intra-molecular aldol and Claisen condensations z. (A.l.) Aldol Condensation 16 17 18 OH 5 - 8 -(A.2.) Claisen Condensation <0) •EnzSH B. Secondary Transformations Often, after the i n i t i a l investigation using labelled acetate and/or malonate has shown that a particular compound i s of polyketide origin, further inquiries usually are concerned with determining the biosynthetic sequence i.e. determining when secondary transformations such as a l k y l a t i o n reduction, or oxidation occur. In such cases, the best way to approach the problem i s to synthesize labelled compounds suspected of being advanced biosynthetic precursors of the particular polyketide and then test their precursor activity by feeding them to the natural system. (B.l.) Alkylation Reactions There are two problems posed to the investigator regarding alkylations i n biosynthetic problems. They are: (i) the nature of the alkylating agent ( i i ) the stage i n the biosynthetic sequence of reactions when alkylation occurs (e.g. before or after aromatization). - 9 -The two most common alkylating agents 9 are: (i) s-adenosylmethionine(23) in methylation reactions. (i i ) Dimethylallylpyrophosphate(24) in prenylation reactions. 23 0 „ II II O - p - 0 - P -I OH OH OH 24 - 1 0 -(B.l.a.) Methylation Reactions (before or after Aromatization) ^ L ^ J X c O S E n z C--methylation before dramatization j ^ X ^ Y ^ C C S E n z CH, 25 26 ALDOL ALDOL 1/ C-methylation ofter 0—H oromatizatior. H-OH COSEnz 27 C 0 2 H 2B (B .l.b.) Prenylation Reactions (before or after Aromatization) COSEnz C-prgnylotion> before aromatization COSEnz O P 2 ° 6 H 3 ALDO. C-prenylation after * " aromctization -11-(B.2.) Reduction Reactions 6-Methylsalicylic acid(36) i s a good example of a fungal metabolite of polyketide origin where reduction followed by dehydration occurs not only before cyclization and aromatization but also before the complete poly-ketide chain has been assembled. Numerous investigations involving enzym-i c studies and the use of labelled acetate and malonate have proven that reduction of the appropriate carbonyl group followed by dehydration occurs before cyclization and aromatization 1 0. The sequence shown in Scheme 7 was deduced from the results of these investigations. CH3COSC0A Acetyl coenzyme A H02CCH2COSCoA E n z S H > Malonyl coenzyme A X J ^COSEnz N A D P H / H ^ COSEnz 32 33 -H20 COSEnz HO?CCH?COSCoA 1 ALDOL 2 AROMATIZATION •COSEnz 3 HYDROLYSIS C02H OH 6-Methylsalicylic acid 36 SCHEME 7 -12-(C) Classification of Polyketides As indicated previously, phenolic compounds produced by operation of the acyl-polymalonate route can be classified as triketides, tetraketides, pentakedtides, etc. and a brief discussion of the triketide and tetraketide groups i s provided below. (C.l.) Triketides (Compounds derived from RCCCH2COCH2COSEnz) RC0SC6A • 2 H02CCH2COSCoA chain-initiating unit chain-propagating unit -> •SEnz EnzSH in the triketide category, 2-pyrone systems rather than phenolic com-pounds are produced. And as mentioned earlier, the chain-initiating units can vary quite considerably (Table 1 ) . -13-TABLE 1 TRIKETIDES CHAIN-INITIATING UNITS TRIKETIDES (os thiol esters of coenzyme A) -14-Compound(40) above has two added f e a t u r e s . The f i r s t b e i n g a s imple O - m e t h y l a t i o n o f the pyrone h y d r o x y l group and the second b e i n g an o x i d a -t i v e c y c l i z a t i o n o f a ae thoxyphenol p r e c u r s o r . The o r i g i n o f bo th methyl and methylene e the r s is from S-adenosy lme th ion ine . (C.2.) Tetraketides (Compounds derived from RCO(CH2CO)2CH2COSEnz) EnzSH _ • 3 H0 2 CCH 2 COSCoA RCOSCoA choin-initialing unit choin-propagating units OSEnz 43 Examination of compounds derived from the tetraketides have shown that this group of polyketide intermediates can cyclize in three ways, viz., loctonizctior Enz 43 oldol COSEnz 0 43 a 2-pyrone R derivatives 44 OH orsellmx ocici & derivatives 45 >7X 0 SEnz 43 Claisen 0 OH acyl-phloroglucinol I derivatives 46 -15-(C.2.1.) 2-Pyrones and Derivatives The different possible chain-initiating units can give a wide variety of 2-pyrones belonging to the tetraketide group. In addition, the basic system, compound(44) can be subjected to secondary transformations giving an even wider variety of compounds, viz; ACQ 47 48 Secondary transformation such as reduction, dehydration, and oxygenation can be invoked to explain the biosynthesis of asperline(47) and desacetyl-6,7-deoxyasperline (48). The biosynthesis of radicinin(49) can be explained by C-alkylation using a crotonyl derivative(51), cyclization, and hydroxy-lation. The proposed sequence for the biosynthesis of radicinin i s i l l u s -trated i n Scheme 8. 50 ,C0SEnz A A 1 49 SCHEME 8 - 1 6 -(C.2.2. ) Tetraketides Derived by Intramolecular Aldol Condensation A large number of polyketide derived compounds arise from this type of cyclization. The simplest tetraketide formed by aldol condensation i s o r s e l l i n i c acid ( 5 ) . 'COSEnz 0 55 Aside from the different possible chain-initiating units and several secondary transformations that can occur before or after cyclization/ aromatization that produces a wide variety of compounds from this sub-group, o r s e l l i n i c acid (5) i t s e l f has been proposed and i n some cases proven to be an intermediate i n the biosynthesis of a variety of fungal metabolites a l -though the sequence of reactions from o r s e l l i n i c acid to the derived meta-bolite remains to be completely elucidated 6' 7' 9*!!. This aspect further adds another variation to the class of compounds belonging to this sub-group. Shown below are three examples of compounds derived from o r s e l l i n i c acid ( 5 ) . H0> OH fumigatin 59 spinulosin 61 penicillic acid 67 Scheme 9 shows the proposed sequence of reactions leading to fumigat (59) and spinulosin(61). COoH OH orsellinic acid 5 hydroxylation HO' OH 56 OH 57 0-methylation oxidation OH 58 H 0 ^ f f i f / C H 3 0 ^ V 0 fumigat in 59 hydroxylation 60 H C K ^ ^ T / oxidation ^ ^ " ^ O H CH H° TT PA^OH Spinulosin 61 SCHEME 9 The unusual structure of p e n i c i l l i c acid(67) provides an excellent example of the way i n which secondary transformations can obscure the origin of the metabolite. I t was K. Mosbach6 i n 1960 who fed labelled o r s e l l i n i c acid to " Penicillium baamese and found that the label had been carried through to p e n i c i l l i c acid(67). Further investigations led to findings that compounds (62) - (64) were also involved 1 2 i n p e n i c i l l i c acid biosynthesis (Scheme 10). SCHEME 10 Like o r s e l l i n i c acid, many fungal metabolites are derived by further modifications of 6-methylsalicylic acid(36). Patulin (74), lik e p e n i c i l l i c acid (67) i s another example of how secondary transformations can obscure the relationship between the biosynthesis and origin of a metabolite. The established biosynthetic relationship between patulin(74) and 6-methylsali-c y l i c acid(36) along with compounds(69) and (70) i s shown below (Scheme 11) ] SCHEME 11 -20-Mycophenolic acid(63) i s a good example of a polyketide-derived compound where C-methylation is occuring at a pre-aromatic l e v e l 1 ^ . The sequence of reactions involved in the biosynthesis of mycophenolic acid, an anti-cancer agent produced by Penicillium brevicompactum, has been investigated by several research groups 1^. I n i t i a l investigations showed that the basic carbocyclic system was derived from four acetate units (presumably one acetate and three malonate units) and that the 0 and C-methyl groups (^0*3) attached to the aromatic ring were derived from S-adenosylmethionine. In addition, the acidic side-chain was shown to be derived from two molecules of mevalonic acid(79). Later studies established the interroediacy of com-pounds (77), (78), (81), and (82) i n the biosynthetic route. This informa-tion, coupled with the fact that o r s e l l i n i c acid(5) was not a specific pre-cursor of mycophenolic acid led to the suggestion that C-methylation was occuring prior to cyclization/aromatization of the (3-triketoester intermed-iate (75) A summary of the various types of secondary transformations which occur in the biosynthetic route i s shown in Scheme 12. SCHEME 1? 22-DISCUSSION in 1934, Raistrick and Could" iBolated yellow and orange pigments, flavoglaucin(84) and auroglaucin(85) respectively from the mycelia of several fungal species belonging to the Aspergillus olaucus series. In 1965, A.J. Birch and co-workersl 7 used carbon-14 labelled acetate and mevalonate i n feeding experiments to establish the biosynthetic origin of auroglaucin(85). In these experiments i t was found that mevalonate was incorporated into the dimethylallyl moiety of the pigment while a l l other carbons originated from acetate. This was i n agreement with what was pre-dicted from the polyketide hypothesis. More recently, Barrow and co-workerslS used carbon-13 labelled acetate and mevalonate to confirm what Birch had concluded from the carbon-14 labelling studies. The experimental evidence obtained by Birch and Barrow indicated that the main carbon framework of these compounds i s derived by the acyl-polymal-onate route (cf.Scheme 5). However, the sequence of steps or nature of intermediates involved i n the biosynthesis of auroglaucin or flavoglaucin are unknown and the sequence shown i n Scheme 13 i s one of several equally reasonable p o s s i b i l i t i e s . For example, introduction of the dimethylallyl unit could occur before or after aromatization and the possible involvement of acetate-derived starter units such as octanoic acid or 3,5,7-octatrienoic acid i s also consistent with the experimental evidence. In addition, the - 2 3 -the aldehyde group could be formed at an early stage by reduction of an enzyme-bound intermediate or at a later stage by reduction of a carboxylic acid intermediate. C o A S H > 7 CH3COSC0A b' 0 t i n / M g 2' > 6 H02CCH2COSCoA 7 CH3CO2H ATP/HCO3 0 Q EnzSH ^ Y ^ C 0 S E N Z R = C7H15 89a R.C7H9 8£b R=C7Hi5 84 HQ R = C7H9 85 SCHEME 13 -24-Thus, the i n i t i a l objective of our research was to synthesize deuterium-labelled compounds which could be regarded as probable intermediates in the biosynthetic route. Subsequent incorporation experiments using cultures of A.glaucus could then provide information which would assist in the elucida-tion of the biosynthetic sequence leading to auroglaucin and flavoglaucin. Up to the present date however, no information regarding the identities of any advanced precursors for either of these pigments have been estab-lished. The specific compounds we chose to synthesize as potential inter-mediates were the deuterium-labelled 6-n-heptyl-2-hydroxybenzaldehyde(92a) and i t s corresponding acid(92b). Following this, we had hoped to synthesize the deuterium-labelled 2,5-dihydroxy-6-n-heptylbenzaldehyde(93a) and i t s corresponding acid(93b). OH 93 Q OH 93b -25-5 i s Q f 6-n-Heptyl-2-hvdroxv-oc-fdeuterioWbenzoic acid (92b) 97 X = H or D 98 92b SCHEME U In the i n i t i a l attempt to synthesize the deuterium-labelled acid(92b), 2-methoxybenzoic acid(94) was treated with oxalyl chloride i n benzene t o furnish the crude acid chloride(95) which was immediately transformed into the diethylamide(96) by adding i t dropwise to a diethylaroine and triethylamine solution in benzene. Ortho-lithiation of (96) according to the method of Snieckus 1^ followed by condensation with iodoheptane provided compound(97). Attempted hydrolysis of amide (97) by refluxing in 10% aqueous perchloric acid for 24 hours, or base h y d r o l y s i s 2 0 using a slurry of potassium tertiary butoxide in ether and water at room tempera-ture or at reflux a l l f a i l e d to provide the required carboxylic acid(99). At this point, further investigation of this synthetic route was aban-doned. It was also found that the hydrolysis conditions l i s t e d above fa i l e d to convert the simple amide(96) to i t s corresponding acid(94). At this stage, i t was noticed that literature reportsl9»21 describing the successful hydrolysis of aromatic tertiary amides could be explained by invoking a neighboring group effect. Thus, i n the examples shown in Scheme 15, the ortho -C-OH group participates in the hydrolysis of the tertiary amide group. SCHEME 15 On the basis of these observations, acid(92b) was constructed (Scheme 16). an alternate synthetic approach SCHEME 16 - 2 8 -As shown in Scheme 16, 7-methoxyphthalide(101) was synthesized from amide(96) by a sequence of reactions developed by Snieckus and co-workers 1 9. Treatment of phthalide(101) with lithium diisopropylamine(LDA) followed by addition of iodohexane provided 3-n-hexyl-7-methoxyphthalide (109). However, the poor yield in the condensation reaction led us to devise another method (Scheme 17)for preparing compound(109). CHO CONEt-1 n-C 7H nMgBi j p \ 2H * / H,0 p-TsOH/CH C^frHr it CH,0 92 b SCHEME 17 In this alternate process, compound(106) was condensed with n-hexyl-magnesium bromide to provide hydroxy-amide(111). Acid-catalyzed cycliza-tion of (111) occured i n good yie l d to furnish 3-n-hexyl-7-methoxyphthal-ide(109). Unfortunately, a l l attempts to cleave the lactone ring of (109) f a i l e d . For example, treatment of alkylphthalide(109) with trimethylsilyl iodide in quinoline for 2 minutes at 175°C according to the method reported by T r o s t 2 2 provided unchanged starting material. Varying the length of the reaction time met with no success and extending the reaction time to 1.0 hour resulted i n extensive decomposition. It i s possible that the reported 2 2 successful ring-opening of phthalide(112) (Scheme 18) i s due to the fact that the intermediate iodo-acid(113) readily undergoes dehydrohalogenation to furnish the -unsaturated acid(114). SCHEME 16 Other methods23 which fai l e d to cleave the lactone ring of compound (109) are il l u s t r a t e d in Scheme 19. -30-Reflux/24 hr 1 Nal/HMPA/j00_C/72_hr_ 2 H7H2O CH-6 3 Vi6 24 hr CH,0 117 HI/Red Phosphorous ^ Reflux/40hr SCHEME 19 I t has been reported24 t h a t trimethylsilyl iodide f a i l s to ring-open phthalide(119) but that some success (30% yield) can be achieved when trimethylsilyl bromide i s used (Scheme 20). -31-SCHEME 20 An equilibrium i s achieved between phthalide(119) and bromosilyl ester (120) but the low yield of the bromosilyl ester (120) discouraged us from trying this method on our 3-n-hexyl-7-methoxyphthalide(109). The p o s s i b i l i t y of a similar equilibrium existing for compound(109) in the reactions illustrated in Scheme 19 could account for the failure of the attempted ring-opening reactions. Thus, i t i s possible that the alkyliodo-acids(115) or (116) could readily cyclize back to starting material (Scheme 21). -HI ^ S o O H R0 122 SCHEME 21 Similarly, when methanol and sulfuric acid were used in an effort to ring-open phthalide(109), a similar unfavorable equilibrium could have been i n effect preventing production of significant amounts of hydroxy-ester(117) (Scheme 22) . -32-SCHEME 22 Thus, a l l attempts to obtain a substituted alkylbenzoic acid derivative from alkylphthalide(109) were f u t i l e . However, reduction with lithium aluminum hydride did furnish the alkylhydroxy-benzyl alcohol(126). The proposed mechanism for this reaction i s illustrated in Scheme 23. CH?o H" HVH20 , ( ^ ^ 0 H CH3O OH 126 SCHEME 23 - 3 3 -The attempted oxidation of diol{126) to the keto-acid(127) and/or the corresponding lactol (hydroxyphthalide) (128) with pyridinium dichromate in N,N-dimethylformamide (DMF) afforded not the desired keto-acid(127) but the original alkylphthalide(109) as the major product (Scheme 24). SCHEME 24 The mechanism proposed to account for these results i s depicted in Scheme 25. -34-SCHEME 25 -35-The equilibrium favoring the lactol(131) over the hydroxy-aldehyde(129) could explain why phthalide(109) was produced as the major product. In li e u of these results, another method was devised for synthesizing hydroxyphthal-ide(128) (Scheme 26). .OH £2b SCHEME 26 Oxidation of hydroxy-amide(111) to keto-amide(132) proceeded smoothly with pyridinium chlorochromate. Acid hydrolysis of the keto-amide(132) afforded the hydroxyphthalide (128) i n 80% yield based upon recovered starting material. Esterification of hydroxyphthalide(128) with methyl-iodide and sodium bicarbonate provided the keto-ester(133). It i s inter-esting to note that esterification conditions using methanol and sulfuric acid yielded the hydroxyphthalide methyl ether(138) (Scheme 27). CH3OH CH30H/H2504 CHoO SCHEME 27 - 3 7 -The different products formed by two different esterification methods i s consistent with recent s t u d i e s 2 5 which demonstrate that under neutral or acidic conditions, hydroxyphthalide(141) was favored over keto-acid(142) while under alkaline conditions, the equilibrium favored the keto-acid anion(144) (Scheme 28). N0HCC>3 KC02H U2 SCHEME 28 Having successfully produced keto-ester (133), we now attempted to trans-form this compound to the desired 6-n-heptylsalicylic acid(926). In essence, this involved reduction of the acyl group to the corresponding alkyl group. The proposed synthetic method i s shown i n Scheme 29. -38-OH 92 b SCHEME 29 However, the attempted reduction of keto-ester(133) with sodium boro-hydride f a i l e d to give the desired hydroxy-ester(145) and instead furnished the alkylphthalide(109). The proposed mechanism to account for this result i s i l l u s t r a t e d i n Scheme 30. -39-0 C H 3 -CH3O-S C H E M E 30 Another method for reducing the ketone functionality of keto-ester(133) was then considered. This involved conversion of the ketone moiety to i t s tosylhydrazone derivative(150) followed by sodium borohydride reduction (Scheme 31). 0 NNHS02CftU./CH3  R J R ' H 3 C C 6 H , S 0 2 N H N H 2 ^ R J R , NoBH^ ^ R C H 2 K U9 150 151 S C H E M E 31 This reduction method, however, furnished a product that i s suspected to be a phthalazine derivative(153). The proposed mechanism for the form-ation of (153) i s shown in Scheme 32. -40-N/^S0 2C eH.CH 3 -0 H3CC6HAS02NHNH2 J ^ H ^ C02CH3 X V CH30 V ) 152 N0EH4 N N^S02C6H4C^; CH30 1E3 SCHEME 32 Having found these ester route was also abandoned and a new (92b) was investigated (Scheme 3 3 ) . last two methods of reduction to be f u t i l e , the keto-route to 6-n-heptylsalicylic acid - 4 1 --42-In this route, 3-hydroxydimethylphthalate(157) was prepared^" by a Diels-Alder reaction between furan(154) and dimethylacetylene dicarboxylate (155) followed by aromatization of the adduct(156) with boron trifluoride in methylene chloride. An alternate literature method 2 7 for obtaining hydroxy-diester(157) was also investigated but found not to be as satis-factory (Scheme 34). f) Q -> S C H E M E 34 Reduction of hydrox-diester(157) with lithium aluminum hydride yielded triol(158) which was converted to the acetonide(159) by reacting i t with 2,2-dimethoxypropane in benzene. Oxidation of (159) with pyridinium chloro-chromate furnished the aldehydo-ketal(160) which was reacted with n-hexyl-magnesium bromide to yield hydroxy-ketal(161). Compound(161) was trans-formed to the deuterio-ketal(162) by treatment with mesyl chloride, t r i e -thylamine, and 4-N,N-dimethylaminopyridine in the THF to yield an o i l which was immediately reduced with lithium triethylborodeuteride. Deketalizatien of (162) with p-dioxane, water, and a trace of sulfuric acid provided the deuterio-diol (163) which was converted to the benzyl ether-benzyl alcohol (164) by treatment with benzyl bromide and pottasium carbonate in acetone. -43-Protection of the aromatic hydroxyl group of diol(163) was necessary, as noted i n preliminary investigations, to prevent undesirable decomposition i n subsequent steps in the synthesis. Oxidation of compound (164) with pyridinium dichromate in DMF28 fa i l e d to give the desired carboxylic acid (166) but did furnish the aldehyde(165).' Other oxidation methods such as treatment of (165) with tetrabutylammonium permanganate in pyr i d i n e 2 9 , or treatment with s i l v e r oxide^O failed to give the desired acid(166). The p o s s i b i l i t y that the benzyl ether was too bulky a protecting group and that along with the side-chain, each being ortho to the aldehyde func-tionality, that steric effects prevented oxidation was considered. Thus, there was a po s s i b i l i t y that substituting a methyl group for the benzyl protecting group might relieve some of the steric hindrance. The benzyl ether-benzaldehyde(165) was then debenzylated with trimethylsilyl chloride and sodium iodide in acetonitrile to furnish the salicylaldehyde derivative (92a) which incidentally, was one of our synthetic objectives. Treatment of (92a) with dimethyl sulfate and potassium carbonate in acetone provided the corresponding methyl ether (171) (Scheme 35). CeH^HjO (MehSiCl/No.1 CH3CN /Reflux ocetone 92o SCHEME 35 -44-An alternate route to the methyl ether(171) was also devised (Scheme 36). This involved treatment of diol(163) with methyl iodide and potassium carbonate in acetone to furnish methyl ether(170) which was then oxidized to aldehyde(171) with pyridinium dichromate in DMF. CH30 OH 170 CH36 171 92 b SCHEME 36 Having achieved this, various oxidation methods were then tried on aldehyde (171) in the hope of producing the corresponding carboxylic acid (172). Oxidation with tetrabutylammonium permanganate29 and also with si l v e r o x i d e 3 0 provided only unchanged aldehyde. Oxidation with aqueous potassium permanganate in acetone 3 1 or by bubbling oxygen to an ethanolic solution of the aldehyde both produced numerous side-products that a l l - 4 5 -attempts at isolating any of the products were f u t i l e . However, oxidation of the model compound, 2-methoxybenzaldehyde with either aqueous potassium permanganate in acetone-**, tetrabutylammonium permanganate^9 or s i l v e r oxide^O a l l furnished the corresponding acid, 2-methoxybenzoic acid (94). Thus, a l l attempts to oxidize the methyl ether-benzaldehyde(171) fai l e d and any further attempts were abandoned. It i s interesting to note that the methyl ether-benzyl alcohol(170) was readily oxidized to the corresponding benzaldehyde(171) with pyridinium dichromate and yet this aldehyde could not be oxidized to i t s corresponding acid(172). The mechanism for the oxidation of the benzyl alcohol with pyridinium dichromate involves formation of a chromate ester which involves reaction of the oxygen moiety of the alcohol with the oxidizing agent. However, the mechanism for the oxidation of an aldehyde to i t s corresponding acid involves reaction of the carbonyl carbon i t s e l f with the oxidizing agent. The results indicate that steric effects from the side-chain prevent oxida-tion when reaction takes place at a center that i s one carbon bond-length from the aromatic ring and ortho to the side-chain as in aldehyde (171) but that no steric effects are experienced when oxidation occurs at a center that i s two bond-lengths away from the ring as in the benzyl alcohol (170). Conclusion: It i s interesting to note that the natural products themselves, flavo-glaucin(84) and auroglaucin(85) exist as aldehydes. Having been established however, as being of polyketide o r i g i n 1 7 , 1 8 , i t was more logical to i n i t i a l -ly assume that the precursors of these metabolites were carboxylic acid -46-derivatives existing i n nature as coenzyme A esters or enzyme-bound thiol esters. However, in view of the fact that the 2-methoxy-benzaldehyde is quite resistant to further oxidation i t i s not surprising that nature chose these metabolites to exist as aldehydes. Thus, i t is reasonable to assume that 2-hydroxybenzaldehyde(92a) could be an intermediate in the bio-synthesis of flavoglaucin(84) and auroglaucin(85). The methodology for the synthesis of 6-n-heptyl-<*-{deuterio]-2-hydroxybenzaldehyde (92a) is sound (Schemes 33 and 35) and incorporation experiments using this aldehyde as precursor could be done in the near future. -47-Experimental General Unless otherwise stated, the following are implied. Melting points (m.p.) were determined on a Kofler micro-heating stage and are uncorrected. Gas-liquid chromatography (g.l.c.) was performed on a Hewlett-Packard model 5831 A using 6' x 1/8" columns and nitrogen as carrier gas. The column used was 3% OV-17 as the stationary phase with a chromosorb support (80/100 mesh). Carrier-gas flow rate was about 35 ml/min. Nuclear mag-netic resonance (n.m.r.) was performed on the following instruments: 60 MHz spectra on a Varian Associates model T-60 and on a model EM 360 L, 100 MHz (CW or FT) spectra on a Varian Associates model HA-100 or XL-100, 80 MHz (FT) spectra on a Bruker model WP-80, 400 MHz (FT) spectra on a Bruker model WA-400, and 270 MHz (FT) spectra on a 270 MHz instrument con-structed from a Bruker consul, a Nicolet computer model 293 A with a Diablo disc and a 270 MHz super-conducting magnet supplied by Oxford Instruments. Signal positions are given in the delta scale (£ ) with tetramethylsilane as an internal reference (J"-0.00). Signal multiplicity, coupling constants (when necessary), integrated area, and proton assignments are indicated in parenthesis. Infra-red spectra (i.r.) were recorded on a Perkin-Elmer spectrophotometer model 710 B. Solution spectra were performed using sodium chloride c e l l s of 0.2mm. thickness. Absorption positions (V max) are given in cm-1 units and are calibrated by means of the 1601 cm-1 band of polysty-rene. Low resolution mass spectra were recorded on a Varian MAT model CH4B spectrophotometer. High resolution mass spectra were determined on a KRATOS-AE1 model MS50 instrument. Micro-analyses were performed by Mr. P. - 4 8 -Borda, Micro-analytical Laboratory, University of British Columbia, Van-couver. A l l solvents used for n.m.r. and i . r . were of spectral grade. Solvents and reagents used were either Reagent grade, Certified grade, or Spectral grade. Solvents were d i s t i l l e d before use. The term "pet. ether 35-60°" refers to the low-boiling fraction of Reagent grade petroleum d i s t i l l a t e (b.p.ca. 35-60°C). Dry solvents or reagents where indicated were prepared as follows: diethyl ether (ether) and tetrahydrofuran (THF) by refluxing over sodium wire or lithium aluminum hydride followed by dis-t i l l a t i o n ; N.N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), die-thylamine, triethylamine, benzene, toluene, and 2,2-dimethoxypropane by d i s t i l l a t i o n from calcium hydride; and methylene chloride by d i s t i l l a t i o n from phosphorous pentoxide. Silica-gel used for column chromatography was Woelm s i l i c a 100-200, aktiv. (70-150 mesh) purchased from ICN Pharmaceuti-cals, Inc. as the activity grade I material and was deactivated to various grades with d i s t i l l e d water according to instructions from the manufacturer. F l o r i s i l ( R ) was purchased from the Floridin Company. Analytical thin-layer chromatography were purchased from J.T. Baker Chemical Co. 2-Methoxybenzoylchloride(95) Into a 250 ml round-bottomed flask equipped with an air-condenser, drying tube, and a st i r r i n g suspension of o-anisic acid(94)(5 gm;0.033 mol.) in dry benzene (85 ml) cooled in an ice bath, was added in one portion, oxalyl chloride (29 ml, 0.33 mol.). The reaction was stirred and allowed to warm-up to room temperature. After 3.0 hours, benzene and oxalyl chlor-ide were carefully removed on the rotary evaporator providing a brown o i l . This crude acid chloride was used immediately for the next reaction. -49-N,N-diethyl-2-methoxybenzamide(96) Into a pressure equalized addition funnel was added a solution of the crude acid chloride(95) in dry benzene (13 ml). This was slowly added drop-wise into a 3-necked 250 ml round-bottomed flask while sti r r i n g a solution of diethylamine (13.6 ml, 0.132 mol.), triethylamine (4.6 ml, 0.033 mol.) in dry benzene (13 ml) which had been cooled to 0°C and put under an atmos-phere of nitrogen. The reaction mixture was allowed to warm-up to room temperature overnight. The reaction mixture was suction f i l t e r e d and the f i l t r a t e diluted with more benzene (75 ml), washed with water, IN HC1, and saturated brine. It was then dried (MgSO^ j) and the benzene removed on the rotary evaporator to afford a yellow o i l which after d i s t i l l a t i o n (112-113°C/ 0.01 mm.) provided a viscious clear o i l (3.95 gm, 0.0192 mol., 58%). S (60MHz, CDC12) 0.99 (t, 3H,CH3- of N-CH2CH3a), 1.18(t, 3H, CH3- of N-CH2CH3b), 3.04-3.40 (two superimposed q, 4H, both -CH2- of N(CH2CH3)2), 3.70 (S, 3H, -OCH3), 6.59-7.30 (m, 4H, ArH's). i . r . (CHCI3): V max 1618 (-C0NEt2)cm-l. High resolution mass spectrum: m/e (rel.intensity) 207 (18)M*t, 206(29), 135(100), 77(14). Mol.wt. calc'd for C 1 2H 1 7N0 2: 207.1255. Found: 207.1253. N,N-diethyl-6-n-heptyl-2-jnethoxybenzamide(97) Into a 500-ml round-bottomed flask equipped with a magnetic stir r e r and a nitrogen i n l e t tube containing the benzamide(96) (4.26 gm, 0.021 mol.) st i r r i n g in dry THF (200 ml) was added N,N,N',N'-tetramethylethylendediamine (TMEDA) (3.1 ml, 0.021 mol.), cooled down to -78°C and placed under an atmosphere of nitrogen. Then sec-butyllithium (17.2 ml, 0.021 mol.) was added. After 1.5 hours at -78°C, iodoheptane (6.75 ml, 0.042 mol.) was slowly injected into the system. The reaction mixture was allowed to warm - 5 0 -up to room temperature overnight. The usual work-up provided a yellow o i l which after f i l t r a t i o n through s i l i c a - g e l (activity grade XIX) with ether/ pet. ether 35-60° (40/60) provided a clear o i l (2 . 6 gm, 0.0084 mol., 40%). S(100 MHz, CDC13) 0.72-1.09 (two superimposed t, 6 H , CH3- of side chain and CH3- of N-CH2CH3a), 1.09-1.40 (br.m, 11 H, -CH2- of side chain and CH3> of N-CH 2CH3b), 1.40-1.76 (br.m, 2H, ArCH2-CH2-), 2.38 (t, 2H, Ar-CH 2-), 3.0 (q, 2H, -CH2- of N-CH 2CH 3«), 3.10-3.44 (m, 1H, H from -CH2- of N-CH2CH3b), 3.46-3.69 (m, 1H, H from - C H 2 - of N -CH2CH3b) 3.70 (£, 3H, - O C H 3 ) 6.44-7.24 (m, 3H, ArH's). High resolution mass spectrum: m/e (rel.inten-sity) 305(10.5)M*, 233(100), 206(44.7). Mol.wt. calc'd for C 1 9H 3 1N0 2: 305.2347. Found: 305.2359. Attempted hydrolysis of compound(97) Method A: The alkylated benzamide(97) (0.166 gm, 0.00055 mol.) was re-fluxed in 10% aqueous perchloric acid (52 ml)) for 60 hours and then con-tinuously extracted with ether for 24 hours. Drying over MgSO^ and evapor-ation providing only unchanged starting material. Method B: The procedure used was the sane as in method B except that the benzamide(97) was refluxed for 24 hours in 50% aqueous perchloric acid. After work-up and ether evaporation only starting material was present. Method C: Into a 100 ml round-bottomed flask was placed the benzamide(97) (2.06 gm, 010067 mol.) i n ether (25 ml) followed by potassium tertiary butoxide (4.96 gm, 0.044 mol.) and water (0.24 ml). The slurry was re-fluxed for 24 hours. The reaction mixture was cooled i n i c e . Then ice and Iti HC1 were carefully added to the reaction mixture before extracting with ether. Drying (MgS04) and ether evaporation provided only starting mater-i a l . -51-Method D; The procedure used was the same as for method C except that THF was used instead of ether. After work-up and ether evaporation, only starting material was present. N,N-diethyl-6-n-heptyl-o(-{hydroxy}-2-methoxybenzamide (111) Into a dry 500 ml 3-necked round-bottomed flask equipped with a magnetic stirr e r and a reflux condenser was added powdered magnesium (14.6 gm, 0.609 mol.). Dry THF (40 ml) and 1,2-dibromoethane (0.1 ml as a catalyst) were then added to the system. The system was then put under an atmosphere of nitrogen followed by dropwise addition of n-bromohexane (77.6 ml, 0.553 mol.) in THF (60 ml) via a pressure-equalized addition funnel. A vigorous exothermic reaction ensured and the Grignard reagent prepared in this way was allowed to cool down to room temperature before using. A solution of the aldehydo-amide (106) 1 9 (13 gm, 0.055 mol.) in dry THF (150 ml) was then added dropwise to the Grignard reagent via a pressure-equalized addition-funnel and the reaction l e f t to s t i r at room temperature for 4.0 hours. Careful acidification with saturated ammonium chloride, ether extraction, washing the ether extract with saturated brine, drying the ether solution (MgSO,}) and ether evaporation l e f t a brown o i l which crystallized upon standing for a few hours. Recrystallization (ethyl ace-tate/pet . ether 35-60°) afforded white flakes (5.8 gm, 0.182 mol., 33%). m.p.=99-101°C. <T(400 MHz, CDC13) 0.87 (br.t, 3H, CH3- of side-chain), 1.09 (br.t, 3H, CH3- of N-CH2CH3a), 1.18-1.42(br.m, 11H, -CHj- of side-chain and CH3- of N-CH2CH3b), 1.75(br.m, 2H, ArCH (OH)-CH2-), 2.77(br.S, IH, exch. with D20, -OH), 3.18(br.m, 2H, -CH2- of N-CH2CH3a), 3.50 (br.m, 2H, -CH2-of N-CH2CH3b), 3.82 (S, 3H, -0CH3), 4.55(br.m, IH, ArCH-), 6.75-7.45(m, 3H, ArCH's). i.r.(CHC1 3): V max 3600 (free -OH), 3100-3550 (hydrogen-bonded -52--OH), 1618{-CONEt2Jem"1. High resolution mass spectrum: m/e(rel.intensity) 321(10)M*, 303(11), 249(18), 236 (93). Mol.wt. calc'd for C 1 9 H 3 1 N 0 3 : 321.2296. Found: 321.2306. Elemental analysis calc'd for C l 9H 3 lN0 3: C, 70.99; H, 9.72; N,4.36. Found: C, 70.92; H, 9.B1; K, 4.33. 3-Hexyl-7-methoxyphthalide (109) Into a 50 ml round-bottomed flask equipped with a magnetic s t i r r e r and a reflux condenser was added the hydroxy-amide(111) (0.9 gm, 0.002B mol.), p-toluene-sulfonic acid monohydrate (0.59 gm, 0.0031 mol.) and dry toluene (IB ml). The reaction mixture was heated to a bath temperature of about 65o C for 18 hours. The toluene was then removed on the rotary evaporator leaving a brown o i l which was taken up i n ether and the ether solution washed with water, saturated sodium bicarbonate, saturated brine, dried (MgS0 4) and then the ether evaporated leaving a light brown o i l (crude wt. 0.68 gm). g.l.c. (col.temp.: 200°C), retention time: 7.91 min., % purity: 9B. / (60 MHz, CDC13) 0.74-1.0(m, 3H, CH3- of side-chain), 1.0-1.60(br.m, 8H, -CH2- of side-chain), 1.6-2.28(br.m, 2H, -CH2- next to ben-z y l i c position),3.9 (S, 3H, -0CH3), 5.17-5.53 (br.m, 1H, ArCH-), 6.75-7.79 (m, 3H, ArH's). i.r.(CHC1 3): )/max 1759 (C»0cm-1). High resolution mass spectrum: m/e(rel.intensity) 24B(33)M*, 164(14)163(100). Mol.wt.calc'd for C 1 5H 2 003: 248.1407. Found: 248.1399. Attempted ring-opening reactions on phthalide(109) Method A: Into a 50 ml round-bottomed flask was added phthalide(109) (0.51 gm, 0.0020 mol.) in dry quinoline (10 ml) and then s t i r r e d at room temperature for a few minutes. Then trimethylsilyl iodide (1.26 ml, 0 . 0 0 8 ° mol.) was injected a l l at once and the reaction apparatus immediately -53-f i t t e d with a reflux condenser. It was then immersed in an o i l bath pre-heated to 175°C and stirred at this temperature for 2.0 minutes. The reac-tion mixture was then cooled i n a water bath and diluted with ethyl acetate (150 ml) followed by 20 ml of IN HC1. The reaction mixture was stirred for 5 minutes and then extracted with ethyl acetate. The ethyl acetate extract was then dried (MgS04), decolorized with Norite and f i l t e r e d through a bed of Celite. Evaporation of the solvent yielded an o i l which after a mixed g.l.c. showed i t to be starting material. An n.m.r. and i . r . also confirmed this. Method B; Into a 250 ml round-bottomed flask was added a solution of phthalide(109) (0.58 gm, 0.0023 mol.), sodium iodide (2.8 gm, 0.187 mol.) in dry hexamethyl phosphoramide (HMPA) (70 ml). The reaction mixture was stirred and heated at about 100°C for 68 hours. The reaction mixture was then cooled, diluted with water (50 ml) and acidified with IN HC1 before being extracted with ethyl acetate. The ethyl acetate extract was then washed with 10% aqueous sodium b i s u l f i t e and saturated brine. Drying (MgS04) and solvent evaporation yielded only starting material as verified by n.m.r. and i . r . Method C: Into a 50 ml round-bottomed flask equipped with a reflux condens-er was added the phthalide (109) (2.20 gm, 0.0089 mol.) followed by sodium iodide (6.7 gm. 0.045 mol.) and dry acetonitrile (20 ml). The reaction mixture was put under an atmosphere of nitrogen before trimehtylchloride (5.7 ml, 0.045 mol.) was injected i n . The resulting mixture was then stirred and heated to about 83°C for 21 hours. After cooling to room temp-erature i t was diluted with water (40 ml) and extracted with ethyl acetate, The ethyl acetate extract was washed with water, 10% aqueous sodium b i s u l -54-f i t e , and saturated brine. Drying and evaporation yielded an o i l which spectral data suggested was only starting material. Method D: Into a 50 ml round-bottomed flask was added phthalide(109) (1.04 gm, 0.0042 mol.) in spectrograde methanol (30 ml) followed by 20 drops of concentrated sulfuric acid. The reaction mixture was stirred at about 74°C for 19 hours. After cooling to room temperature the reaction mixture was diluted with ethyl acetate (50 ml) and washed with water, sat-urated sodium bicarbonate, saturated brine and water again. After drying (MgSO^ ) and solvent evaporation an o i l was l e f t which spectral data re-vealed to be only starting material. Method E: Into a 25 ml round-bottomed flask was added the phthalide(109) (0.35 gm, 0.0014 mol.) and 47% hydriodic acid (7 ml) followed by red phos-phorous (0.13 gm). The reaction mixture was then refluxed for 48 hours. The reaction mixture was cooled and extracted with ether. The ether extract was then washed with 10% aqueous sodium thiosulfate followed by ammonium hydroxide. The aqueous fraction was then acidified and then re-extracted with ether. The ether extract was then washed with water and saturated brine. Drying and evaporation yielded a solid (0.030 gm, 0.0012 mol., 85%). Spectral analysis suggested i t to be the demethylated phthalide(118). J(60 MHz, CDC13) 0.8-1.06(m, 3H, CH3- of side-chain), 1.06-1.60(br.m, 8H, -CH2- of side-chain), 1.7-2.4(br.m, 2H, -CH2~ next to benzylic position), 5.25-5.79(br.m, IH, ArCH-), 6.72-7.88(m, 3H, ArH's). i.r.(CHCl 3): Vmax 3300-3600 (hydrogen-bonded-OH), 1740 (C»0)cm"l. Low resolution mass spec-trum: m/e: 234M*, 149, 121. Reduction of phthalide (109)with lithium aluminum hydride To a s t i r r i n g suspension of lithium aluminum hydride (0.096 gm, 0.00253 -55-xnol.) in dry THF (20 ml) i n a 3-necked round-bottomed flask equipped with a reflux condenser was added dropwise via a pressure-equalized addition-funnel a solution of phthalide(109) (0.52 gm, 0.0021 mol.) i n THF (15 ml). After s t i r r i n g at room temperature for 7.0 hours, 3N HC1 was carefully added, It was then gravity f i l t e r e d and washed with ether. The f i l t r a t e was d i -luted with more ether, transferred into a separatory funnel and washed with saturated sodium bicarbonate and brine. Drying (MgS04) and solvent evap-oration provided a pale o i l . Spectral analysis indicate i t to be diol(126). ^(60 MHz, CDCI3) 0.79-1.02(br.m, 3H, CH3- of side-chain),1.02-1.43(br.m, 8H, -CH2- of side-chain), 1.43-1.99(br.m, 2H, ArCH(OH)-CH2-), 3.77 (S, 3H, -OCH3) , 4 . 3-5 . 0 (br .m, 3H, 1H f o r ArC — H and 2H f o r ArCH 20), ^ 0 6 . 42-7. 38 (m,3H,ArH's) . i . r . ( C H C l ): max 3624 ( free-OH) , 3150-3600 (hydrogen-bonded -OH)cr 3 -1 Attempted oxidation of diol(126) to hydroxyphthalide(128) Into a 100 ml round-bottomed flask containing a s t i r r i n g suspension of pyridinium dichromate (6.8 gm, 0.018 mol.) in dry DMF (10 ml) was added a solution of the d i o l (126) (0.456 gm, 0.0018 mol.) in DMF (10 ml). The reaction mixture was stirred at room temperature for 4.5 hours. This was then poured into a separating funnel containing 250 ml water and extracted with ether. The ether extract was then washed with IN HC1 and water. Drying (MgS04) and ether evaporation yielded an o i l which n.m.r. and i . r . suggest to be mostly phthalide(109). The crude product was taken up in ether and washed with saturated sodium bicarbonate. Neutralization of the bicarbonate wash with 3N HC1, re-extraction with ether, drying (mgS04), and ether evaporation yielded very few crystals whose n.m.r. and i . r . sug-gest i t to be the hydroxyphthalide (128). (cf .p. 56) . - 5 6 -N,N-diethyl-6-n-heptyl-o< -[oxc}-2-methoxybenzamide (132) To a st i r r i n g suspension of pyridinium chlorochromate (8.2 gm, 0.038 mol.) in dry methylene chloride (100 ml) in a 250 ml round-bottomed flask was added in several portions a solution of the hydroxy-amide(111) (3.5 gm, 0.011 mol.) in methylene chloride (100 ml). The reaction mixture was stirred at room temperature for 3.0 hours. F i l t r a t i o n through a bed of F l o r i s i l (25 gm, 100-200 mesh) provided a pale o i l (3.3 gm, 0.0104 mol., 96%). Micro-distillation (bath temp=141°C/0.005 mm.) provided a clear o i l . ^(400 MHz CDC13) 0.88(br.t, 3H, CH3- of side-chain), 1.04(t, 3H, CH3-of N-CH2CH3a), 1.16-1.42 (br.m, 11H, -CH2- of side-chain and CH3- of N-CH2CH3b), 1.67(br.quintet, 2H, ArC(0)CH2~CH2-), 2.88(br.m, 2H, ArC(0)-CH2~) 3.12(q, 2H, -CH2- of N-CH2CH3a), 3.51 (sextet, J=8Hz, IH, H from -CH2- of N-CH2CH3b), 3.71 (sextet, J=8Hz, IH, H from -CH2~ of N-CH2CH3b), 3.86 (S, 3H, -OCH3), 7.02-7.44 (m, 3H, ArH's). i . r . (CHCI3): V max 1690 (C=0), 1640(-C0NEt2)cm_1. High resolution mass spectrum: m/e(rel.intensity) 319(3)Mf, 248(37), 247(95). Mol.wt. calc'd for C 1 9H 2 9N0 3: 319.2140. Found: 319.2148. 3-Hexyl-3-hydroxy-7-methoxyphthalide(128) A suspension of the keto-amide(132) (2.93 gm, 0.0092 mol.) was refluxed i n 3N HC1(200 ml) for 22 hours. Ether extraction, washing the ether extract with saturated sodium bicarbonate, neutralization of the wash with cone. HC1 and re-extraction of the neutralized aqueous wash with ether afforded a crude white solid. Recrystallization (ethyl acetate/pet.ether 35-60°) provided small white crystals (0.56 gm, 0.00214 mol. 80% based on recovered starting material.). / (80 MHz, CDCI3) 0.87(br.t, 3H, CH3- of side-chain), 1.0-1.50(br.m, 8H, -CH2- of side-chain), 1.50-2.38(br.m, 3H, Ar-C-CH2- f -57--OH which exch. with D 20), 3.90 (S, 3H, -OCH3), 6.9-7.7(m, 3H, ArH's). i . r . ( C H C I 3 ) : Vniax 3560 (free-OH) , 3100-3500 (hydrogen-bonded -OH), 1759 (C=0) cm~l. High resolution mass spectrum: m/e(rel.intensity) 246(6)M*, 179(100). MOl.wt. calc'd for C 1 5H 2 00 4: 264.1356. Found: 264.1363. Methyl-6-heptyl-p( -[oxo}-2-methoxybenzoate (133) Into a 250 ml round-bottomed flask was added the hydroxyphthalide(128) (1.75 gm, 0.066 mol.) in dry N,N-dimethylacetamide (150 ml) followed by sodium bicarbonate (1.4 gm, 0.0165 mol.) and then methyl iodide (4.9 ml, 0. 079 mol.). The reaction mixture was stirred overnight. The usual work-up provided a light brown o i l (crude weight: 1.6 gm). S (60MHz, CDCl^) (br.m, 3H, CH3- of side-chain), 1.0-1.45 (br.m, 6H, -CH2- of side-chain), 1.45-1.9(br.m, 2K, ArC(0)CH2-CH2), 2.6-3.0 (br.t, 2K, ArC(0)-CH 2-), 3.79 (S, 3H, -OCH3 of ester), 3.82 (S, 3H, A r - 0 C H 3 ) , 6.8-7.42(br.m, 3H, ArH's). 1. r. ( C H C I 3 ) : V max 1690 (C=0 of ketone), 1730(C=0 of ester)cm -1. Attempted reduction of keto-ester(133) with sodium borohydride The keto-ester (133) (0.508 gm, 0.00183 mol.) in reagent grade ethanol (10 ml) was stirred in a 25 ml round-bottomed flask. To this was added sodium borohydride (0.104 gm, 0.00274 mol.). The resulting reaction mix-ture was then stirred at room temperature for 9.0 hours. Then i t was care-f u l l y acidified with 3N HC1 and diluted with water (50 ml). Extraction with ether, washing the extract with water and brine, drying (MgS04), and solvent evaporation yielded a clear o i l which spectral analyses (n.m.r. and i . r . ) indicate to be phthalide(109). The experiment was repeated at a lower temperature (ice/water bath) and only for 10 minutes. However, the results were the same. -58-Attempted deoxygenation of the ketone functionality of keto-ester(133) Into a dry 25 ml round-bottomed flask equipped with a reflux condenser and a drying tube was placed the keto-ester(133) (0.515 gm, 0.00185 mol.), dry ethanol (10 ml) and tosylhydrazide (0.413 gm, 0.00222 mol.). The re-action mixture was refluxed for 2.0 hours and then cooled. The ethanol was removed on the rotary evaporator leaving a thick paste which failed to crystallize even after prolonged cooling and trituration. An i . r . of the crude product suggested i t to be the tosylhydrazone. i.r.(CHCl3): V max 3625 (free-NH), 3350-3550 (hydrogen-bonded -NH), 1730 (C=0, ester), 1170 and 1380 (sym.and asym.stretches of -S0 2~)cm~l. The crude product was then suspended in glacial acetic acid (6.5 ml) and treated with several small portions of sodium borohydride over a per-iod of 20 minutes. Slow addition was necessary so that foaming did not become an inconvenience. The reaction mixture was then stirred at room temperature for 1.0 hour then at about 80°C for 4.0 hours. It was then poured into a beaker of crushed ice (50 gm) and treated with 2N NaOH until, an alkaline pH was reached. At this point, a tarry gum had precipitated which was insoluble in both ether and water. The aqueous portion was ex-tracted with ether and dried (MgS04). Evaporation of the ether afforded fine white crystals whose n.m.r., i . r . , and mass spectrum suggested that i t was neither the desired product nor starting material. The spectral data suggests that i t could be a phthalazine derivative(153). <J (400 MHz, CDCI3) 0.87-0.96(br.t, 3H, CH3- of side-chain), 1.26-1.56(br.m, 6H, -CH2-of side-chain), 1.69-1.85 (br. quintet, 2H, -CH2- £to C=Il) , 2.4 (S, 3H, CH3-of Ar0CH3), 2.87-3.0 (br.t, 2H, -CH 2-*to C=N), 3.95 (S, 3H, -OCH3), 7.11-7.2 (d, 1H, H from Ar0CH3), 7.24-7.41 (t as two overlapping d, 3H, H from Ar0CH3 and 2H each proton being ortho to the methyl group of -O2S-C6H4-CH3) 0 -59-7.68-7.82(br.t, IH, H from ArOCH3), 8.05-8.19(d, 2H, both protons ortho to -S0 2- i n -0 2S-C 6H 4-CH 3). i . r . (CHCI3) : Vmax 1680-1690(C=0),1180 and 1380 (sym.and asym. stretches of -S0 2-)cm _ 1. Low resolution mass spectrum: m/e 414M*,350, 259, 189. 2-Hydroxy-6-hydroxymethylbenzyl alcohol(158) A 3-necked 500 ml round-bottomed flask f i t t e d with a reflux condenser and containing a stirring suspension of lithium aluminum hydride (8.4 gm, 0.22 mol.) i n dry THF was put under an atmosphere of nitrogen. To this was added dropwise via a pressure equalized addition funnel, a solution of the crude hydroxy-diester (157)26 (23 gm) in dry THF (100 ml). After st i r r i n g at room temperature for 6.0 hours, the reaction mixture was carefully treated with 6N HC1. The acidified reaction mixture was extracted with ether and the ether extract was washed with water and brine. Drying (MgS04) and ether evaporation yielded a dark brown o i l which crystallized upon standing for about 0.5 hour. Recrystallization from ethyl acetate/pet. ether 35°-60° provided light brown crystals (8.3 gm, 0.054 mol.49%). m.p.110-112°C. S ( 4 0 0 MH2/ Acetone-d6) 3.08 (br.s, 2H, exch.with D20, 2X - OH), 3.90(S, IH, exch.with D20, OH), 4.74 (S, 2H, ArCH2~), 4.98 (S, 2H, ArCH2~), 6.83-7.30(m, 3H, ArH's). Low resolution mass spectrum: m/e 154M*, 136. Elemental analysis calc'd for C 8H 1 003: C,62.33; H,6.54. Found: C,61.97; H,6.35. 9,10-Benzo-2,2-dimethyl-5-hydroxymethyl-l,3-dioxane(159) Into a 100 ml round-bottomed flask f i t t e d with a short-path d i s t i l l a t i o n apparatus was added the triol(158) (6.13 gm, 0.04 mol.), dry 2,2-dimethoxy-propane (5.4 ml, 0.044 mol.), dry benzene (70 ml) and about 4 small crystals of p-toluene sulfonic acid monohydrate. The reaction mixture was stirred -60-and slowly heated to the azeotropic boiling-point of methanol/benzene (55oc). Slow careful heating was necessary to avoid the undesirable azeotrope form-ation between 2,2-dimethoxypropane and methanol (61°C). The usual work-up provided a light brown o i l (6.B gm, 88% crude yiel d ) . A micro-analytical sample was prepared by bulb-to-bulb d i s t i l l a t i o n of the crude product (173OC/0.01 mm) to furnish the desired product as a clear o i l which crystal-lized upon standing, m.p.: 49-51°C. cf (400 MHz, C D C I 3 ) 1.57 (S, 6H, iso-propylidene CH 3's), 1.77(br.s, IH, exch.with D20, -OH), 4.57 (S, 2H, ArCH 20-), 4.93 (S, 2H, ArCK2-0R), 6.75-7.24(m, 3H, ArH's). Low resolution mass spec-trum: m/e 194M*, 136. Elmental analysis calc'd for CuH 1 403: C,68.02; H,7.27. Found: C,67.83; H,7.39. 9,10-Benzo-2,2-dimethyl-5-formyl-1,3-dioxane(160) To a s t i r r i n g suspension of pyridinium chlorochromate (15.1 gm, 0.070nol.) in dry methylene chloride (250 ml) in a 500 ml round-bottomed flask was added a solution of the alcohol (159) (6.8 gm, 0.035 mol.) in methylene chlor-ide (25 ml) via a pressure-equalized addition-funnel. After s t i r r i n g at room temperature for 3.0 hours the dark brown reaction mixture was suction f i l t e r e d through a bed of F l o r i s i l and the f i l t r a t e transferred to a sep-aratory funnel. The f i l t r a t e was then washed with 1W HC1 and saturated brine. Drying (MgS04) and solvent evaporation yielded a light brown o i l (5.4 gm, 77% crude y i e l d ) . & (400 MHz, CDC13)1.55 (S, 6H, isopropylidene CH 3's), 5.24 (S, 2H, ArCH20), 7.05-7.45(m, 3H ArH's), 10.0(S, IH, -CHO). High resolution mass spectrum: m/e (rel.intensity) 192 (73)M*, 164 (12), 134(57), 106(100). Mol.wt.calc'd for C^H^C^: 192.0783. Found: 192.0787. -61-, "x:"&enzo-2,2-dimethyl-5-n-heptyl-0<-(hydroxy)-!, 3-dioxane (161) The procedure used to prepare the Grignard reagent, n-hexylmagnesium bromide, was identical to the procedure for making the same Grignard reagent in the synthesis of N,N-diethyl-2-methoxy-6-n-heptyl-^-hydroxy-benzamide (111). A solution of the aldehyde-ketal(160) $5.14 gm, 0.0268 mol.) in dry THF (100 ml) was added dropwise via a pressure-equalized addition-funnel to the Grignard reagent prepared above. The reaction mixture was stirred at room temperature for 5.0 hours. Careful acidification with saturated aqueous ammonium chloride, ether extraction, washing the ether extract with satur-ated brine, drying (MgSO,;) and ether evaporation provided a brown o i l . Chromatography on s i l i c a - g e l (activity grade III) with gradient elution from pet.ether 35°-60oc. to pet.ether 35°-60°/ether (20/80) provided a clear o i l (6.3 gm, 0.023 mol. 85%). 4 (400 MHz, C D C 1 3 ) 0.87 (br.t, 3H, CH3- of side-chain), 1.12-1.39 (br.m, 8H, -CH2- of side-chain), 1.53 (S, 3H, isoprop-ylidene CH3-), 1.57 (S, 3H, isopropylidene CH3-), 1.60-1.82(br.m, 2H, ArCH (OH) -CH2-), 1.85(br.s, IH, exch.with D20, -OH), 4.62(br.t, IH, ArCH-), 4.92 (AB q, J*16 Hz, IH, H from -CH2- of ArCH2_0R), 4.98(AB q, J=16 Hz, IH, H from -CH2- of ArCH2-0R), 6.72-7.28(m, 3H, ArH's). Low resolution mass spectrum: m/e 278M*, 260, 202. Elemental analysis calc'd for C17 H26°3 : C,73.35; H,9.41. Found: C,73.40; H,9.33. 9,10-Benzo-2,2-dimethyl-5-n-heptyl-oC-[deuteriol-I, 3-dioxane (162) A s t i r r i n g solution of the alcohol(161) (0.109 gm, 0.00040 mol.) in dry THF (15 ml) i n a 50 ml round-bottomed flask was put under an atmosphere of nitrogen and then lowered into an ice-water bath before triethylamine (0.14 ml, 0.001 mol.), 4-N,N-dimethylaminopyridine (0.122 gm, 0.001 mol.) and mesyl chloride (0.077 ml, 0.001 mol.) were added successively. Form--62-ation of a precipitate was immediate and the reaction mixture was l e f t to warm-up to room temperature overnight. The reaction mixture was then f i l t e r e d and washed several times with ether. The f i l t r a t e was transferred to a separatory funnel and washed successively with IN HC1, saturated sodium bicarbonate and water. Drying (MgSC^) and solvent evaporation provided a light yellow o i l which was immediately reduced with lithium triethylboro-deuteride (0.72 ml, 0.00072 mol., 1.0M in THF) in dry THF (3.0 ml) under an atmosphere of nitrogen for 6.0 hours. The reaction mixture was then cooled in a water bath before water (1.5 ml), 2N NaOH (0.5 ml), and 3% aqueous hydrogen peroxide (0.5 ml) were carefully added. The heterogeneous reac-tion mixture was 'then refluxed for 1.0 hour. Then i t was cooled, diluted with ether (50 ml) and washed successively with water, saturated aqueous sodium b i s u l f i t e , IN HC1, saturated sodium bicarbonate and water. Drying (MgS04) and ether evaporation provided a clear o i l (0.084 gm, 0.00032 mol., 80%). A micro-analytical sample was prepared by f i l t r a t i o n through s i l i c a -gel (activity grade III) by eluting with pet.ether 35°-60°C. J" (400 MHz, C D C I 3 ) (br.t, 3H, CH3- of side-chain), 1.17-1.42(br.m, 8H, -CH2- of side-chain), 1.54 (S, 6H, isopropylidene CH 3*s), 1.55(br.m, 2H, ArCH(D)-CH2-), 2.40(br.t,J-8Hz, 1H, ArCH-), 4.83 (S, 2H, ArCH2-0R), 6.63-7.14(m, 3H, ArH's). High resolution mass spectrum: m/e(rel.intensity) 263 (40)M*, 205(46), 162(143), 123(100). Mol.wt. calc'd for C 1 7 H 2 5 D 0 2 : 263.1989. Found: 263.1996. Elemental analysis for C 17H 2 6D0 2 calc'd as Ci7H 2 60 2: C,77.82; H,9.98. Found: C,77.75; H,10.10. 6-n-heptyl-gC-deuterio-2-hydroxybenzyl alcohol (163) Into a 250 ml round-bottomed flask was placed the deuterio-ketal (162) (2.4 gm, 0.0091 mol.) followed by p-dioxane (180 ml), water (37 ml), and -63-conc. sulfuric acid (10 drops). This mixture was stirred at room tempera-ture for 96 hours. After this time, the reaction mixture was neutralized (pH7-8) with sodium bicarbonate. Ether extraction, washing the ether - extract with brine, drying (MgS04) and ether evaporation provided a light green o i l (2.23 gm crude weight). £{270 MHz, C D C I 3 ) 0.85 ( b r . t , 3H, CH 3" side-chain), 1.01-1.40(br.m, 8H, -CH2- of side-chain), 1.40-1.63(br.m, 2H, ArCH(D)-CH2->,2.53(br.t, 1H, ArCH-), 4.90(S, 2H, ArCH20-), 6.63-7.24 (m, 3H, ArH's), 7.90(br.s, 1H, exch.with D20,-0H). High resolution mass spectrum: m/e(rel.intensity) 273(27)M*, 205(14), 162(9), 148(26), 123(100), 92(45). Mol.wt. calc'd for C 1 4H 2 1D0 2: 223.1677. Found: 223.1685. 2-Benzyloxy-6-n-heptyl-oC-deuterio-benzyl alcohol(164) To a s t i r r i n g solution of diol(163) (1.60 gm, 0.0072 mol.) in acetone (125 ml) was added anhydrous pottasium carbonate (1.1 gm, 0.0079 mol.) and benzyl bromide (0.94 ml, 0.0079 mol.). The flask was f i t t e d with a reflux condenser and refluxed for 10 hours. The reaction mixture was cooled, suction f i l t e r e d , and washed several times with ether. The f i l t r a t e was transferred into a separatory funnel and washed with IN HC1, saturated brine and water. Drying (MgS04) and ether evaporation yielded a pale o i l (crude wt. 2.34 gm). 2-Benzyloxy-6-n-heptyl-o(-deuterio-benzaldehyde(165) To a s t i r r i n g suspension of pyridinium dichromate (3.94 gm, 0.0105 mol.) in dry DMF (15 ml) was added in one portion a solution of the crude alcohol (164) (0.819 gm, 0.00262 mol.) in dry DMF (15 ml). After st i r r i n g at room temperature overnight i t was poured into a separatory funnel containing water (300 ml) and extracted with ether. The ether extract was washed w i t h IN HC1 and water. Drying (MgS04) and ether evaporation provided a light yellow o i l (0.74 gm crude weight). Spectral analysis of the crude product -64-suggest i t to be the benzyloxy-benzaldehyde(165) with a trace of starting alcohol(164). $ (60 MHz, CDC13) 0.78-1.0 (m, 3H, CH3-) of side-chain), 1.0-1.79(br.m, 10H,-CH2- of side-chain), 2.62-3.10 (br.m, 1H, ArCH-), 5.0KS, 2H, ArCH20-Ar), 6.48-7.58(m, 8H, both ArH's), 10.60(S, 1H,-CH0). i.r.(CHCI3): y max 1695(C=0 of aldehyde)cm - 1. Attempted oxidation of the benzyloxy-benzaldehyde(165) Method A: To a s t i r r i n g solution of aldehyde(165) (0.224 gm, 0.00072 mol.) in dry pyridine (10 ml) was added dropwise via a pressure-equalized addition-funnel a solution of tetrabutylammonium permanganate (0.35 gm, b.0010 mol.) in pyridine (25 ml) while being chilled at ice-water temperatures. The reaction mixture was stirred under these conditions for 6.0 hours. The reaction mixture was poured into a beaker containing a mixture of water, IN HC1, and saturated sodium b i s u l f i t e (150 ml, 1/1/1). It was then extract-ed with ether and the ether extract washed several times with saturated sodium bicarbonate. Drying (MgSO^ and ether evaporation yielded starting aldehyde (0.134 gm, 60% recovery). Neutralization of the bicarbonate washings with cone. HC1 and re-extraction with ether afforded, after drying (MgS04) and ether evaporation a brown o i l (0.064 gm) whose n.m.r. did not have any aromatic protons and therefore was not analyzed any further. Method B: Into a 50 ml round-bottomed flask containing a solution of the aldehyde(165) (0.56 gm, 0.00182 mol.) in ethanol (12 ml) was added with s t i r r i n g a solution of s i l v e r nitrate (0.46 gm, 0.00424 mol.) in d i s t i l l e d water (1.0 ml). Then dropwise by pipette was added a solution of powdered potassium hydroxide (1.28 gm, 0.0023 mol.) in d i s t i l l e d water (3.0 ml). The reaction mixture was stirred at room temperature for 3.5 hours. The reaction mixture was then suction f i l t e r e d and washed with water and acidi--65-fied with IN HC1. The f i l t r a t e was then extracted with ether. Drying (MgSOij) and ether evaporation yielded 9 5 % of starting aldehyde. 6-n-Heptyl-o£-deuterio-2-hydroxybenzaldehyde(92a) A solution of the benzyloxy-benzaldehyde(165) (0.68 gm, 0.0022 mol.) in dry acetonitrile (30 ml) and sodium iodide (1.5 gm, 0.0098 mol.) was equipped with a reflux condenser. The reaction mixture was stirred, put under an atmosphere of nitrogen and then lowered into an oil-bath pre-heated to 80°C. Then trimethylsilychloride was slowly injected into the reaction mixture and stirred under these conditions for 3.0 hours. After this time, the reaction mixture was poured into a separatory funnel containing water (50 ml) and extracted with ether. The ether extract was then washed succes-sively with 1 0 % sodiurnthiosulfate and saturated brine. Drying (MgS04) and evaporation of the ether provided a light brown o i l (0.35 gm crude weight). Fi l t r a t i o n through s i l i c a - g e l (activity grade III) by eluting with pet.ether 35°-60oc provided a pale o i l (0.23 gm, 0.00105 mol., 48%) cf(60 MHz, C D C I 3 ) 0.60-1.0 (m, 3H, CH3- of side-chain), 1.0-1.99(br.m, 10H, -CH2- of side-chain), 2.52-3.15(br.t, 1H, ArCH-), 6.40-7.58(m, 3H, ArH's), 10.10(S, 1H, -CHO), 11.82 (S, 1H, exch.with D2O, -OH). 6-h-Heptyl-cC-deuterio-2-methoxybenzyl alcohol(170) Into a 1 0 0 ml round-bottomed flask equipped with a reflux condenser was placed diol(163) (2.2 gm, 0.0010 mol.), anhydrous pottasium carbonate (2.7 gm, 0.020 mol.) and dry acetone (60 ml). The reaction mixture was stirred for a few minutes at room temperature before methyl iodide (2.5 ml, 0.040 mol.) was added. It was then refluxed for 8 hours. After this time, the reaction mixture was cooled, f i l t e r e d , and washed with ether. The com-bined acetone and ether f i l t r a t e s were washed with water and brine. Drying -66-(MgS04) and ether evaporation provided a dark yellow o i l . F i l t r a t i o n on s i l i c a - g e l (activity grade III) by eluting with pet.ether 35-60°/ether (9.5/0.5) provided an analytically pure sample (1.6 gm, 0.0068 mol., 68%). ^(400 MHz, C D C I 3 ) 0.87 (br.t, 3H, CH3- of side-chain), 1.13-1.42 (br.m, 8H, -CH2- of side-chain), 1.55(br.q,J-8Hz, 2H, ArCH(D) - C H 2 - ) , 2.51 (br.s, IH, exch.with D20, -OH), 2.65 (br.t,J=8Hz, IH, ArCH-), 3.85 (S, 3H, - O C H 3 ) , 4.74(br.d, 2H, ArCH20-), 6.72-7.23(m, 3H, ArH's). Low resolution mass spectrum: ja/e 237M?, 219, 153. Elemental analysis for C15H23DO2 calc'd as C15 H24°2 : C,76.23; H,10.24. Found: C,76.06; H,10,34. 6-n-Heptyl-o^-fdeuterioj-2-methoxybenzaldehyde (171) Method A: To a st i r r i n g suspension of pyridinium dichromate (0.22 gm, 0.00058 gm) in dry DMF (12 ml) in a 25 ml round-bottomed flask was added the alcohol(170) (0.069 gm, 0.00030 mol.) as a solution in DMF (3.0 ml). The reaction mixture was stirred at room temperature for 2.5 hours. Then, the reaction mixture was poured in a separatory funnel containing water (100 ml) and extracted with ether. The ether extract was washed with IN HC1 and saturated brine. Drying (MgS04) and ether evaporation provided a yellow o i l (0.059 gm). F i l t r a t i o n on s i l i c a - g e l (activity grade III) using pet.ether 35-60°C provided a clear o i l (0.046 gm, 0.00020 mol., 67%) $ (400 MHz, C D C I 3 ) 0.87 (br.t, 3H, CH3~ of side-chain), 1.18-1.42(br.m, 8H, -CH2- of side-chain), 1.54 (br.q,J=7.5 Hz, ArCH(D)-CH2-), 2.90 (br.t,J=7.5 Hz, IH, ArCH-), 3.90(S, 3H, - O C H 3 ) , 6.78-7.46(m, 3H, ArH's), 10.62 (S, IH, -CHO). i . r . ( C H C I 3 ) : V max 1695(-CH0)cm"1. High resolution mass spectrum: m/e (rel.intensity) 235(100)M*, 234(58), 164(99). Mol.wt. calc'd for Ci5H 2iD0 2: 235.1677. Found: 234.1606. Method B: A mixture of the hydroxybenzaldehyde(92a) (0.146 gm, 0.00066 mol.), -67-anhydrous eotassium carbonate (0.10 gm, 0.00073 mol.), and dimethyl sulfate (0.07 ml, 0.00073 mol.) i n dry acetone was refluxed under an atmosphere of -nitrogen for 20 hours. Then the reaction mixture was cooled, f i l t e r e d , and washed with ether. The combined f i l t r a t e s were washed with water and 2N sodium hydroxide. Drying (MgSO^ and evaporation of the ether provided a yellow o i l (0.156 gm). F i l t r a t i o n through s i l i c a - g e l (activity grade III) with pet.ether 35-60°C provided a clear o i l . Spectral data was the same as that for method A. Attempted oxidation of aldehyde(171) Method A: Into a 50 ml round-bottomed flask containing a solution of alde-hyde (171) (0.32 gm, 0.00136 mol.) in ethanol (10 ml) was added with s t i r r i n g , a solution of silve r nitrate in water (1.0 ml). Then dropwise by pipette was added a solution of powdered pottasium hydroxide (0.95 gm, 0.0170 mol.) in water (3.0 ml). The reaction mixture was then stirred in an oil-bath pre-heated to 50°C for 3.0 hours. The reaction mixture was then suction f i l t e r e d and washed with water. The combined f i l t r a t e s were then extracted with ether and the aqueous portion neutralized with cone. HC1 and then re-extracted with ethyl acetate. The ether solution was dried (MgS04) and evaporated leaving an o i l which n.m.r. suggested was only starting material (0.305 gm, 95% recovery). Method B; The procedure for the attempted oxidation of aldehyde(171) was similar to that for the attempted oxidation of aldehyde(165) with tetrabutyl-ammonium permanganate on p(64). The results were also similar. Method C; Into a 50 ml round-bottomed flask containing a s t i r r i n g solution of aldehyde (171) (0.0915 gm, 0.00039 mol.) in acetone (5 ml) was added, by pipet, a solution of potassium permanganate (0.111 gm, 0.00070 mol.) in -68-water (15 ml). The resulting mixture was stirred at room temperature for 1.0 hour. Sodium b i s u l f i t e was then added u n t i l the brown reaction mixture turned clear. Then i t was suction f i l t e r e d , acidified with 6N HC1 and extracted with ethyl acetate and the ethyl acetate extract washed with saturated brine. Drying (MgSO^ and solvent evaporation provided an o i l which analytical thin-layer chromatography showed was mostly starting alde-hyde. The reaction was thus repeated and monitored by t . l . c . u n t i l the alde-hyde was consumed (approx. 4 hours). However, after this time, there were a total of 6 spots detected by thin-layer chromatography. Repeated column chromatography of the crude mixture (0.116 gm) with Kieselgel 60 (230-400 mesh ASTM) by gradient elution from pet.ether 35°-60°/ethyl acetate 9/1 to 5/5 were f u t i l e and none of the products were successfully isolated. Method D; Oxygen was slowly bubbled into a solution of aldehyde(171) (0.024 gm, 0.00010 mol.) in ethanol (5 ml) in a 25 ml round-bottomed flask with an exit valve for excess oxygen. Only starting aldehyde was observed by thin-layer chromatography (pet.ether 35-60°/ethyl acetate, 7/3) of aliquots taken for the f i r s t 3 days. On the fourth day thin-layer chroma-tography revealed several spots. None of the products were successfully isolated by column chromatography using Kieselgel 60 (230-400 mesh ASTM) by gradient elution using pet.ether 35-60o/ethyl acetate 9/1 to 4/6. -69-BIBLIOGRAPHY (1) J.N. Collie, J.Chem. Soc, 1907, 91, 1806. (2) A.J. Birch, Forstchr Chem. Org. Naturst, 1957, 14, 186. (3) A.J. Birch, J. Chem. Soc, 1951, 30266. (4) S.J. Wakil, J.W. Porter, and D.M. Gibson, Biochim. et Biophys. Acta., 1957, 2_4, 453. (5) A.J. Birch, Ann.Rev. Plant Physiol., 1968, _19, 321. (6) E.W. Basset and S.W. Tannenbaum, Experientia, 1958, 14_, 37. (7) K. Mosbach, Acta. Chem. Scand., 1960, 14_, 457. (6) J.D. Bu'Lock and H.K. Smalley, Proc Chem. Soc, 195E, 45B2. (9) T.A. Geissman, and D.H.G. Crout, 'Organic Chemistry of Secondary Fiar.t Metabolism', Freeman-Cooper, San Francisco, 1969. (10) (a) P. Dimroth, H. Walter, and F. Lynen, European J. Biochem., 1970, 13, 98. (b) A.I. Scott, G.T. Ph i l l i p s and U. Kircheiss, Bioorganic Chem., •1971, _1, 380. (11) J.H. Richards and J.B. Hendrickson, 'The Biosynthesis of Steroids, Terpenes, and Acetogenins', W.H. Benjamin, Inc., New York, 1964. (12) K. Axberg and S. Gatenbeck, Acta. Chem. Scand., 1975, B29, 749. (13) (a) P.I. Forrester and G.M. Gaucher, Biochemistry, 1972, 11_, 1102. (b) G. Murphy and F. Lynen, European J. Biochem., 1975, 58, 467. -70-(14) C.T. Bedford, P. Knittel, T. Money, G.T. P h i l l i p s , and P. Salisbury, Can.J.Chem., 1973, SI, 694. (15) L. Canonica, W. Krozczynski, B.M. Ranzi, B. Rindone, E. Santianello, and C. Scolastico, J.C.S. Perkin I., 1972, 2639. (16) S. Gould and H. Raistrick, Biochem. Journal, 1934, 26, 1640. (17) A.J. Birch, A.J. Ryan, J. Schoefield, and H. Smith, J. Cher;. Sec, 1965, 1231. (18) J.K. Allen, K.D. Barrow, A.J. Jones, and P. Hannisch, J.C.S. Che~. Comm., 1978, 152. (19) 0. de'Silva, J.N. Reed, and V. Snieckus, Tetrahedron Lett., 1978, 5099. (20) P.G. Gassman, P.K.G. Hodgson, and R.J. Balchunis, J. Aner. Cheir:. Soc, 1976, 98, 1276. (21) O. de Silva and V. Snieckus, Tet. Lett., 197B, 5103. (22) B.M. Trost, G.T. Rivers, and J.M. Gould, J. Org. Cher.., 1980, 45, 1635. (23) (a) A.A. Durrani and J.H.P. Tyman, J.C.S. Perkin I, 1979, 2069. (b) G. A. Olah, S.C. Harang, B.G. Balaram Gupta, and R. Malhotra, J. Org. Chem., 1979, 44_, 1247. (24) H.R. Kricheldorf, Angew. Chem. Int. Ed. Engl., 1979, JLS, 689. (25) J.P. Tyman and A.A. Najam, Spectrochimica, 1977, 33A, 479. (26) A.W. McCulloch, B. Stanovnik, D.G. Smith, and A.G. Mclnnes, Can.J.Chem., 1969, 47, 4139. (27) E. E l i e l , A.W. Burgstahler, D.E. Rivard, and L. Hawfele, J. Aner. Chem. Soc, 1955, 7_7, 5092. (28) E.J. Corey, and G. Schmidt, Tetrahedron Lett., 1979, 339. -71-(29) T. Sala and M.V. Sargent, J.C.S. Chen. Comm., 1978, 253. (30) M. Shamma and H.R. Rodrigues, Tetrahedron, 1968, 6583. (31) P. Knittel, M.Sc. Thesis (University of British Columbia), 1971, 49. - 7 2 -60 M H z n.m.r. -73--74-60 M H z n.m.r. -77--78-400 M H z n.m.r. -79-- 8 0 --81-83-OH OH 158 ACETONE-(dt) 1 r- T 4 0 0 M H z n.m.r. 4 0 0 MHz n.m.r. - 8 5 -r—T s—1  400 M H z n.m.r. 270 M H z n.m.r. - 8 7 -0 0 : :- = • m *m tm >m tm *m LL. ; 1 , 1 IB f{ 1 , 1 c 7 S'2.40 _ - ; 1 4 0 0 M H z n.m.r. -88-270 M H z n.m.r. -B9-- 9 0 --92-

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