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Branched-chain sugar nucleosides. Synthesis of structural analogues of puromycin Baker, Donald Arthur 1972

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BRANCHED-CHAIN SUGAR NUCLEOSIDES. SYNTHESIS OF STRUCTURAL ANALOGUES OF PUROMYCIN BY DONALD ARTHUR BAKER B . S c , The Un i v e r s i t y of B r i t i s h Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of CHEMISTRY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1972 In present ing t h i s thes is in p a r t i a l f u l f i l m e n t o f the requirements fo r an advanced degree at the Un ivers 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 for reference and study. I fu r the r agree that permission for extensive copying of th i s t h e s i s fo 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 rep resenta t i ves . It i s understood that copying or p u b l i c a t i o n of th i s t h e s i s f o r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion . Department of The Un i ve rs i t y of B r i t i s h Columbia Vancouver 8, Canada - i -ABSTRACT Several new routes to nitrogenous branched-chain sugars have been investigated and the preparation of several novel branched-chain sugar nucleosides having a structural relationship to puromycin has been described. The cyanomethyl branched-chain sugars 3-C_-cyanomethyl^3-deoxy-1,2:5,6-di-0_-isopropylidene-ct-D-allof uranose [LXXXVI], 3-C-cyanomethy1-3-deoxy-l,2:5,6-di-0-isopropylidene-a-D-gulofuranose [LXVII], and 5-0_-benzyl-3-C_-cyanomethyl-3-deoxy-l,2-0-isopropylidene-a-p-ribofuranose [LXXVIII] were prepared by condensation of diethyl cyanomethylphosphonate with 1,2 :5,6-di-0_-isopropylidene-a-D-r_ibo-hexofuranos-3-ulose [XVIII], 1,2;5,6-di-0-isopropylidene-q-D-xylo-hexofuranos-3-ulose [LXVII], and 5-0-benzyl-l,2-0-isopropylidene-q-g-erythro-pentofuranos-3-ulose [LXVIII], respectively, followed by stereoselective hydrogenation over palladium-on-charcoal of the intermediate unsaturated sugars. Reduction of the n i t r i l e group of LXXXVI and LXXXVIII gave the D-amino sugars, isolated as their acetamido derivatives, 3-C_-(2'-acetamidoethyl)-3-deoxy-l,2:5,6-di-O-isopropylidene-a-D-allofuranose [XCII] and 3-£-(2'-acetamidoethyl)-5-0-benzyl-3-deoxy-l,2-0_-isopropylidene-a-D-ribofuranose [XCIII]. Selective hydrolysis of the 5,6-0_-isopropylidene ketal of LXXXVII followed by sodium periodate degradation and sodium borohydride reduction afforded the L-cyanomethyl branched-chain sugar 3-C_-cyanomethyl-3-deoxy-l,2-0_-isopropylidene-6-L-lyxofuranose [XCVI]. Reduction of the n i t r i l e group of this compound gave the L-amino sugar characterized - i i -as i t s acetamido derivative 3-C_-(2'-acetamidoethyl)-3-deoxy-l,2-0_-isopropylidene-8-L-lyxofuranose [XCVI]. The carbamoylmethyl branched-chain sugar 3-C_-carbamoylmethyl-3-deoxy-l,2:5,6-di-0_-isopropylidene-a-D-allofuranose [C] was prepared via three different routes. Hydrolysis of LXXXVI using alkaline hydrogen peroxide afforded C in 70 % yield. The same compound was also obtained by ammonolysis of 3-C-carbomethoxymethyl-3-deoxy-l,2:5,6-di-O-isopropylidene-a-D-allofuranose [XXXIX] using liquid ammonia and ammonium chloride and by the stereoselective photoaddition of formamide to the methylene branched-chain sugar 1,2:5,6-di-0-isopropylidene-3-C-methylene-q-D-ribo-hexofuranose [XX]. A nitromethyl branched-chain sugar l,2:5,6-di-0-isopropylidene-3-C-nitromethyl-a-D-glucofuranose [CV] was also prepared by condensing XVIII with nitromethane. The cyanomethyl branched-chain sugar LXXXVI was the key intermediate in the synthesis of the branched-chain sugar nucleosides. Selective hydrolysis of LXXXVI to the 1,2-0-isopropylidene compound followed by benzoylation, hydrolysis of the 1,2-isopropylidene ketal and acetylation yielded 1,2-di-0_-acetyl-5,6-di-0-benzoyl-3-C-cyanomethyl-3-deoxy-B-D-allofuranose [CX]. Fusion of CX with 6-chloropurine followed by reaction with methanolic-aqueous dimethyl amine gave the branched-chain sugar nucleoside 6-N,N-dimethylamino-9-(3'-C-N,N-dimethylcarbamoylmethy1-31-deoxy-g-D-allofuranosyl)-purine [CXXI]. Sodium metaperiodate oxidation of CXXI followed by sodium borohydride reduction gave the corresponding ribo nucleoside 6-N,N_-drmethylamino-9- (3' -C_-J},N-dimethylcarbaraoylmethyl-3'-deoxy-8-D~ - i i i -ribofuranosyl)-purine [CXXII]. In a separate procedure CXXII was obtained by fusion of 6-chloropurine with l,2-di-0-acetyl-5-0-benzoyl-3-C_-cyanomethyl-3-deoxy-g-D-ribofuranose [CXIII] prepared from LXXXVI by selective hydrolysis of the 5,6-isopropylidene group followed by sodium periodate degradation, sodium borohydride reduction of the aldehydo intermediate, benzoylation, hydrolysis of the 1,2-isopropylidene group and acetylation), followed by reaction with methanolic aqueous dimethylamine. The corresponding unblocked cyanomethyl branched-chain ribo sugar nucleoside 6-N ,N-dimethylamino-9- (3' -C_-cyanomethy 1-31 -deoxy-g-D-ribofuranosyl)-purine [CXXXI] was obtained by fusion of CXIII with 6-chloropurine followed by reaction of the blocked nucleoside with anhydrous dimethylamine. Pyrolysis of the N.,N-dimethylcarbamoylmethyl ribonucleoside CXXII gave the novel lactone nucleoside 6-N,N_-dimethylamino-9-(3'-C-carboxymethyl-2',3'-Y~lactone-3-deoxy-g-p-ribofuranosyl)-purine [CXXVIII]. Condensation of this compound with ammonia afforded 6-N,N-dimethylamino-9-( 3' -C_-carbamoylmethyl-3-deoxy-g-D-ribof uranosyl) -purine [CXXIX] and condensation of CVIII with ethyl glycinate gave the peptide nucleoside 6-N,N-dimethylamino-9-(3'-C-carbamoylmethyl-N-glycine ethyl ester-3'-deoxy-g-D-ribofuranosyl)-purine [CXXX]. Reduction of cyanomethyl branched-chain ribo-nucleoside CXXXI afforded an amino branched-chain sugar nucleoside which was characterized as i t s N-acetyl derivative 6-^,N_-dimethylamino-9-(3,-C-(2"-acetamido-ethyl)-3'-deoxy-g-D-ribofuranosyl)-purine [CXXXIV]. - i v -Compounds CX and CXIII were also converted into the corresponding blocked adenyl nucleosides 6-benzamido-9-(2'-0-acetyl-5',6 1-di-O-benzoyl-3'-C-cyanomethyl-3 1-deoxy-g-D-allofuranosyl)-purine [CXXXVI] and 6-benzamido-9-(2'-O-acety1-5'-0-benzoyl-3'-C-cyanomethyl-3'-deoxy-3-D-ribofuranosyl)-purine [CXXXXVII] by reaction with hydrogen bromide followed by condensation with chloromercuri-6-benzamido purine. -v-TABLE OF CONTENTS ABSTRACT i TABLE OF CONTENTS v LIST OF FIGURES x i ACKNOWLEDGEMENTS x i i I. OBJECTIVE 1 II. INTRODUCTION 4 1. Branched-chain sugars 4 1.1 Synthesis of branched-chain sugars 6 2. Oxidations of secondary carbohydrate hydroxyl groups 8 3. The Wittig reaction 10 3.1 Application of the Wittig reaction to carbo-hydrates 11 3.2 The phosphonate modification of the Wittig reaction 14 3.3 Mechanism and stereochemistry of the phosphonate modification of the Wittig reaction 15 3.4 The modified Wittig reaction in carbohydrate chemistry 17 4. Nitroparaffin addition to carbohydrates 19 5. Photo-addition of formamide to olefins 23 5.1 Photo-additions to carbohydrates 25 6. Nucleosides 27 6.1 Nucleoside synthesis 28 6.2 Synthesis of purine nucleosides 28 6.3 Branched-chain sugar nucleosides 32 6.4 Biological activity of branched-chain sugar nucleosides 32 - v i -l l i . RESULTS AND DISCUSSION 34 1. Synthesis of branched-chain cyanomethyl sugars by a Wittig reaction 34 1.1 1,2:5,6-Di-O-isopropylidene-q-D-ribo-hexofuranos-3-ulose [XVIII] 35 1.2 1,2:5,6-Di-0-isopropylidene-q-D-xylo-hexof uranos-3-ulose [LXVII] 36 1.3 5-0-Benzyl-l,2-0-isopropylidene-a-Il-erythro-pentofuranos-3-ulose [LXVIII] 38 1.4 3-C-Cyanomethyl-3-deoxy-l ,2:5, 6-di-0-isopro-pylidene-a-E-allofuranose [LXXXVI], 3-C-cyanomethyl-3-deoxy-l ,2:5, 6-di-0_-isopropyli-dene-a-g-gulofuranose [LXXXVII] and 5-0-benzyl-3-C-cyanomethyl-3-deoxy-l, 2-0-isopro-pylidene-q-g-ribofuranose [LXXXVIII] 40 2. Synthesis of branched-chain amino sugars by re-duction of branched-chain cyano sugars 48 2.1 3-C-(2'-Acetamidoethyl)-3-deoxy-l,2:5,6-di-0-isopropylidene-a-g-allofuranose [XCI] 49 2.2 3-C-(2'-Acetamidoethyl>5-0-benzyl-3-deoxy-l,2-O-isopropylidene-a-Pj-ribofuranose [XCIII]... 51 2.3 3-C-Cyanomethyl-3-deoxy-l,2-0-isopropylidene-B-L-lyxofuranose [XCV] and 3-C_-(2'-acetamido-ethyl)-3-deoxy-l,2-0-isopropylidene-B-^-lyxo-furanose [XCVI] 7 52 3. Synthesis of branched-chain carbamoylmethyl sugars 55 3.1 3-C-Carbamoylmethyl-3-deoxy-l,2:5,6-di-0-isopropylidene-a-D-allofuranose [C] 56 4. Synthesis of nitrogenous branched-chain sugars having a single carbon in the branched-chain 60 4.1 Photoamidation of 4,6-di-0-acetyl-2,3-dideoxy-ct-D- ery thro-hex-2- enopyranos id e [CII] 60 4.2 Addition of nitromethane to 1,2:5,6-di-0-isopropylidene-q-D-ribo-hexofuranos-3-ulose [X\'III] 7 63 5. Nucleoside synthesis 65 - v i i -5.1 Attempted acetolysis of 3-C-(2'-acetamidoethyl) -5-0-benzyl-3-deoxy-l, 2-£-isopropylidene-a-D-ribofuranose [XCIII] 66 5.2 Conversion of 3-C_-cyanomethyl-3-deoxy-l,2: 5,6-di-O-isopropylidene-a-D-allofuranose [LXXXVI] into 1,2-di-0-acetyl-5,6-di-0-benzoyl -3-£-cyanomethyl-3-deoxy-g-D-allofuranose [CX] and 1,2-di-£-acetyl-5-p_-benzoyl-3-C-cyano-methyl-3-deoxy-B-D^-ribof uranose [CXIII] 69 5.3 6-Chloro-9-(2'-0-acetyl-5',6'-di-0-benzoyl-3' -C-cyanomethyl-3 '-deoxy-g-p_-allof uranosyl) -purine [CXVI] and 6-chloro-9-(2'-0-acetyl-5' -0-benzoyl-3' -C-cyanomethyl-3' -deoxy-g-D-ribofuranosyl)-purine [CXVIII] 74 5.4 6-N_,N_-Dimethylamino-9- (3' -C-N ,N-dimethylcarba-moylmethyl-3'-deoxy-g-D-allofuranosyl)-purine [CXXI] and 6-N,N-dimethylamino-9-(3'-C-N,N-diraethylamino-9-(3 '-C_-N_,N-dimethylcarbamoyl-methyl-3 '-deoxy-g-D_-ribof uranosyl)-purine [CXXII] 77 5.5 6-N.,N-Dimethylamino-9-(3'-C_-carboxymethyl-2' , 3'-y-lactone-3-deoxy-g-D-ribofuranosyl)-purine [CXXVIII] 86 5.6 Conversion of 6-N_,N_-dimethylamino-9-(31 -C-carboxymethyl-2',3'-y-lactone-3-deoxy-g-D-ribofuranosyl)-purine [CXXVIII] to 6-N,N-dimethylamino-9- (3' -C_-N.,N_-dimethylcarbamoyl-methyl-3'-deoxy-g-D-ribofuranosyl)-purine [CXXII], and e-J^Nj-dimethylamino-g-(3'-C-carbamoylmethyl-3' -deoxy-g-Dj-ribof uranosyl) -purine [CXXIX] and 6-N,JS[-dimethylamino-9-(3'-£-carbamoylmethyl-N-glycine ethyl ester-3'-deoxy-g-D-ribofuranosyl)-purine [CXXX]... 88 5.7 6-N,N-Dimethylamino-9-(3'-C-cyanomethyl-3'-deoxy-g-p =-ribofuranosyl)-purine [CXXXI] 91 5.8 6-N,N.-Dimethylamino-9- (3' -C- (2"-acetamidoethyl) -3'-deoxy-g-D_-ribofuranosyl)-purine [CXXXIV] 92 5.9 6-Benzamido-9-(2'-0-acetyl-5',6'-di-0-benzoyl-31-C-cyanomethyl-3'-deoxy-g-D-allofuranosyl)-purine [CXXXVI] and 6-benzamido-9-(2'-O-acetyl -5' -()-benzoyl-31 -£-cyanomethyl-3' -deoxy-g-D-ribof uranosyl)-purine [CXXXVI I] 94 5.10 9-(3/-£-Aminoethyl-3*-deoxy-g-D-allofuranosyl}-adenine [CXXXIX] 97 - v i i i -6. Biological activity evaluation of branched-chain sugar nucleosides 98 IV. EXPERIMENTAL 99 1. General methods 99 2. Chromatography 99 2.1 Column 99 2.2 Thin layer chromatography 100 2.3 Paper chromatography 100 2.4 Gas liquid chromatography 100 3. Photolysis reactions 101 1,2:5,6-Di-0_-isopropylidene-a-g=-glucofuranose [LXIX] . 101 5-0-Benzyl-l, 2-C^-isopropylidene-a-Dj-xylof uranose [LXXVII] 102 1, 2: 5, 6-Di-O-isopropylidene-ct-gj-gulofuranose [LXXIa] . . 103 1,2:5, 6-Di-O-isopropylidene-a-Dj-ribof uranos-3-ulose [XVIII] 103 1,2:5,6-Di-0-isopropylidene-a-D-xylo-furanos-3-ulose [LXVII] 104 5-0-Benzyl-l, 2-0-isopropylidene-ct-P_-erythro-pentofur-anos-3-ulose [LXVIII] 105 3-C_-Cyanomethyl-3-deoxy-l ,2:5, 6-di-0-isopropylidene-a-g-allof uranose [LXXXVI] 107 3-C_-Cyanomethyl-3-deoxy-l, 2: 5, 6-di-0-isopropylidene-a-D-gulofuranose [LXXXVII] 108 5-£-Benzyl-3-C_-cyanomethyl-3-deoxy-l, 2-0-isopropyli-dene-a-D-ribofuranose [LXXXVIII] 109 3-C-(2'-Acetamidoethyl)-3-deoxy-l,2:5,6-d i-O-isopropy-lidene-a-D-allofuranose [XCII] 110 3-C_-(2'-Acetamidoethyl)-5-0-benzyl-3-deoxy-l, 2-0-iso-propylidene-a-D-ribof uranose [XCIII] I l l - i x -Acetolysis of 3-C_-(2'-acetamidoethyl)-5-0_-benzyl-3-deoxy-1.2- 0_-isopropylidene-a-D-ribof uranose [ X C I I I ] 112 3-C-Cyanomethyl-3-deoxy-l, 2-0-isopropylidene-B-L_-lyxo-furanose [XCV] 112 3-C-(2'-Acetamidoethyl)-3-deoxy-l, 2-0-isopropylidene-B-L-lyxof uranose [XCVI] 113 3-C-Carbamoylmethyl-3-deoxy-l,2:5,6-di-0_-isopropyli-dene-a-H-allofuranose [C] 114 Ethyl 4,6-di-0-acetyl-2,3-dideoxy-ct-D-erythro-hex-2-enopyranoside [CII] 115 Photo-addition of formamide to ethyl 4,6-di-0-acetyl-2.3- d ideoxy-ct-D-ery thro-hex-2-enopyranos id e [ C I I ] . . . . 116 1,2:5, 6-Di=0_-isopropylidene-3-C_-nitromethyl-a-D-glucof uranose [CV] 117 3-C-Cyanomethyl-3-deoxy-l, 2-()-isopropylidene-a-p=-allofuranose [CVIII] 118 5, 6-Di-0-benzoyl-3-C_-cyanomethyl-3-deoxy-l, 2-0-isopro-pylidene-a-D^allof uranose [CIX] 119 5- C)-Benzoyl-3-C-cyanomethyl-3-deoxy-l, 2-0-isopropyli-dene-cc-D-ribofuranose [CXII] 119 1,2-Di-0-acetyl-5, 6-di-0-benzoyl-3-C_-cyanomethyl-3-deoxy-B-D-allof uranose [CX] 120 3-C-Cyanomethyl-3-deoxy-l, 2-0_-isopropylidene-ct-D-ribofuranose [CXI] 121 1,2-Di-£-acetyl-5-0-benzoyl-3-C-cyanomethyl-3-deoxy-B-D-ribofuranose [CXIII] 122 6- Chloro-9-(2'-O-acetyl-5',6'-di-0-benzoyl-3'-C-cyanomethyl-3'-deoxy-B-D-allofuranosyl)-purine [CXVI] 123 6-Chloro-9-(2'-0-acetyl-5'-0-benzoyl-3'-C-cyanomethyl-3'-deoxy-3-D-ribofuranosyl)-purine [CXVIII] 124 6-HsN.-Dimethylamino-9- (3' -C_-N,^-dimethylcarbamoylmethyl -3'-deoxy-8-g-allofuranosyl)-purine [CXXI] 124 6-N., N-Dimethylamino-9- (2 1 ,5' -di-0-acetyl-3 '-C-N.N-dimethylcarbamoylmethyl-3'-deoxy-B-D-ribofuranosyl)-purine 125 -x-6-N_,_N-Di!i!ethylamino-9- (3' -£-N ,tI-dimethylcarbamoylmethyl -3'-deoxy-S-g-ribofuranosyl)-purine [CXXII] 126 Preparation of 6-N_,_N-dimethylamino-9-(3-£-N_,N_-dimethyl-carbamoylmethyl-3' -deoxy-S-D^-r ibof uranosyl) -purine [CXXII] from CXXXI 127 6-N,^-Dimethylamino-9-(3'-C_-carboxyraethyl-2t-3l-Y-lactone-3-deoxy-S-D-ribofuranosyl)-purine [CXXVIII].. 128 6-N_,N_-Dimethylamino-9- (3' -C-carbamoylmethyl-31 -deoxy-e-D-ribofuranosyl)-purine [CXXIX] 128 6-JN,N-Dimethylamino-9-(31-C-carbamoylmethyl-N-glycine ethyl ester-3'-deoxy -B-D-ribofuranosyl)-purine [CXXX] 129 6--N,_N_-Dimethyl;imino-9- (3' -£-cyanomethyl-3' -deoxy-B-D-ribofuranosyl)-purine [CXXXI] 130 6-_N,N_-Dimethylamino-9-(3'-(2"-acetamidoethyl)-31 -deoxy -g-D-ribofuranosyl)-purine [CXXXIV] 130 Chloromercuri-6-benzamidopurine 131 6-Benzamido-9-(2'-£-acetyl-5' , 6'-di-£-benzoyl-3 '-£-cyanomethyl-3'-deoxy-B-D=-allofuranosyl)-purine [CXXXVI] 132 6-Benzamido-9-(2*-£-acetyl--5'-0-benzoyl-3' -C-cyano-methyl-3'-deoxy-B-D-ribofuranosyl)-purine [CXXXVII].. 134 9-(3'-£-Aminoethyl-3'-deoxy-g-D-allofuranosyl)-adenine [CXXXIX] 134 ADDENDA 136 - x i -LIST OF FIGURES FIGURE 1. Proton magnetic resonance spectrum at 100 MHz in deuteriochloroform of 3-C-cyanomethyl-3-deoxy-l,2: 5,6-di-O-isopropylidene-a-Dj-allofuranose [LXXXVI] 47 2. Proton magnetic resonance spectrum at 100 MHz in dimethyl sulfoxide-d^ of 6-_N,N-dimethylamino-9-(3 '-C_-li,N-dimethylcarbamoylmethyl-3' -deoxy-g-D-allofuranosyl)-purine [CXXI] 79 - x i i -ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude to Professor A. Rosenthal who suggested this problem and provided s k i l l f u l guidance throughout the course of this work. Professor G.G.S. Dutton and Dr. P. Legzdins are to be thanked for reading and commenting on this manuscript. The encouragement and assistance of my wife during the prepara-tion of this thesis was greatly appreciated. Finally, the financial support of the H. R. MacMillan Family Fellowship (1971 - 1972) and the F. J. Nicholson Scholarship Fund (1969 - 1971) are gratefully acknowledged. I. OBJECTIVE: In recent years a wide v a r i e t y of unusual nitrogen containing carbohydrate d e r i v a t i v e s has been i s o l a t e d from a n t i b i o t i c s (1). This has l e d to a great i n t e r e s t i n the synthesis of compounds of t h i s type as not only has there been the motivation of preparing materials having i n t e r e s t i n g b i o l o g i c a l a c t i v i t i e s but a l s o , because of the high density of diverse f u n c t i o n a l groups present, t h e i r preparation has presented to the synthetic organic chemist i n t r i g u i n g challenges. The f i r s t o b j e c t i v e of t h i s work was to explore ways of prepar-ing carbohydrate d e r i v a t i v e s containing deoxy-nitrogenous branched-chains. Although no carbohydrates having a nitrogenous branched-chain have as yet been encountered i n nature, one n i t r o (10) and two amino (9,11) branched-chain sugars have been found to be components of a n t i b i o t i c s . To a t t a i n the above obje c t i v e three new methods of introducing nitrogenous branched-chains, the a d d i t i o n of the W i t t i g reagent d i e t h y l cyanomethylphosphonate to several 3-ketoses, the condensation of nitromethane with ketoses and the photo-amidation of unsaturated sugars, were examined. During the completion of t h i s work the con-densation of nitromethane with carbohydrates to give branched-chain n i t r o methyl and amino methyl sugars was reported by several other groups (20,79,80). -2-The second o b j e c t i v e of t h i s work was to convert some of the 3-deoxy branched-chain sugars prepared v i a the above W i t t i g r e a c t i o n i n t o hexo- and, i n p a r t i c u l a r , p e n to-furanosyl branched-chain sugar n u c l e o s i d e s . The C-3' m o d i f i e d n u c l e o s i d e s prepared were mainly d i m e t h y l -aminopurine d e r i v a t i v e s . Dimethylaminopurine was chosen to be the h e t e r o c y c l i c base i n these compounds i n order that these branched-c h a i n sugar n u c l e o s i d e s would be s t r u c t u r a l analogs of the a n t i b i o t i c puromycin [I] (2a). The b i o l o g i c a l a c t i v i t y of puromycin and i t s analogs i s known to be very dependent on the C-3' s u b s t i t u e n t . For example, the dimethylaminopurine n u c l e o s i d e I I , having a h y d r o x y l group at the 3' p o s i t i o n , has been shown to be completely i n a c t i v e (2b) w h i l e the corresponding £-31-amino-C-3-deoxy-dimethylaminopurine nu c l e o s i d e III (3) and v a r i o u s C-3' secondary amino nucl e o s i d e s IV (4) show a range of a n t i m e t a b o l i c a c t i v i t y (3,4). T h i s suggests that dimethylaminopurine nucleosides having C-3' branched-chains be a r i n g n i t r o g e n s u b s t i t u e n t s could be p o t e n t i a l t h e r a p e u t i c agents. [I] -3-In order to provide a perspective for subsequent di s c u s s i o n , methods of synthesis of branched-chain sugars, W i t t i g r e a c t i o n s , n i t r o p a r a f f i n condensations, photo-additions of formamide to o l e f i n s and photo-additions to carbohydrates w i l l a l l be b r i e f l y reviewed. In a d d i t i o n , some comments on methods of nucleoside synthesis and the b i o l o g i c a l a c t i v i t i e s of branched-chain sugar nucleosides w i l l be made. I I . INTRODUCTION: 1. Branched-chain sugars A branched-chain sugar i s a carbohydrate i n which a hydrogen or hydroxyl group i s replaced by a carbon so as to lead to branching of the carbon skeleton. Over the years these modified sugars have been i s o l a t e d from a number of natural products ( 5 ) . However, i t has been the r e l a t i v e l y recent discovery that these unusual sugars are components of some important a n t i b i o t i c s that has led to a heightened i n t e r e s t i n the preparation and properties of these compounds. In Table I are shown some representative branched-chain sugars i s o l a t e d from n a t u r a l l y occurring a n t i b i o t i c s . I t can be seen that there e x i s t s a v a r i e t y of branched-chains and unusual sugars. Of p a r t i c u l a r i n t e r e s t here are the branched-chain sugars garosamine [VIII] and evernitrose [IX] as these compounds are examples of nitrogen containing branched-chain sugars. Very r e c e n t l y another amino branched-chain sugar, sibirosamine (11), so f a r only i d e n t i f i e d as a 4,6-dideoxy-3-C-methyl-4-methylaminohexo-pyranose, has been i s o l a t e d from the a n t i b i o t i c sibiromycin. Grisebach and Schmid (12) have l a t e l y reviewed the chemistry and biochemistry of these unusual sugars. -5-TABLE I Branched-chain Sugars Isolated from A n t i b i o t i c s Structure Reference Structure Reference OH OH [V] 6 HO/ 0 LlMe V °H Me" / OH [VIII] 9 7 10 CH 3 [VI] Me [IX] Me MeO A \ OH [VII] 8 -6-1.1 Synthesis of branched-chain sugars There are two po s s i b l e ways branched-chain sugars can be prepared. One can e i t h e r construct the desired sugar i n stages by condensing together small non-carbohydrate u n i t s , or one can introduce a branch-chain into an already pre-formed carbohydrate. Because i n the former method racemic mixtures r e s u l t whenever asymmetric centers are produced, and carbohydrates generally have a high density of asymmetric centers, t h i s has not been a popular approach f o r the preparation of branched-chain sugars. One notable exception, however, was i n the synthesis of mycarose [VI] by Lemal and coworkers (13). As at the time there was no c l e a r evidence f o r the r e l a t i v e c o n f i g u r a t i o n at C-3, C-4 and C-5 they devised a scheme whereby the four p o s s i b l e racemic mycarose isomers could be prepared. This was done by condensing the keto a c e t a l X with the Grignard reagent 1-propynylmagnesium bromide followed by p a r t i a l hydrogenation and c i s hydroxylation. This gave a mixture of t r i o l s which were c y c l i z e d and separated as t h e i r methyl glycosides. In t h i s way they were able to synthesize racemic mycarose and i t s 3-epimer. CH, 3 [VI] [X] -7-Methods used to date to introduce branching into the carbon skeleton of a pre-formed sugar are summarized i n Table I I . Although a wide v a r i e t y of reactions has been employed i n the majority of cases the branched-chains obtained are formally produced by TABLE I I Methods f o r Introduction of Branched--chains into Sugars Method Reference 1. From Carbohydrate Ketoses (a) A c e t o n i t r i l e a d d i t i o n 14 (b) Cyanohydrin r e a c t i o n 15 (c) Diazomethane a d d i t i o n 16 (d) Enamine a l k y l a t i o n 17 (e) Grignard a d d i t i o n 18 (f) Methyl l i t h i u m a d d i t i o n 19 (g) Nitromethane a d d i t i o n 20 (h) W i t t i g r e a c t i o n 21, 22 2. From Carbohydrate Epoxides (a) Ad d i t i o n of carbanions 23 (b) Methyl l i t h i u m a d d i t i o n 24 3. From Unsaturated Carbohydrates (a) Oxo r e a c t i o n 25 (b) Photoamidation 26 4. Miscellaneous Approaches (a) Dimerization to branched-chain sugars 27 (b) Nitromethane addition to sugar dialdehydes 28 (c) Reduction of a lactone to a branched-chain sugar 29 (d) Ring c o n t r a c t i o n to a branched--chain sugar 30 -8-s u b s t i t u t i n g a branch-chain for a hydrogen atom i n the carbon skeleton of the sugar. The other type of branched-chain sugars (deoxy branched-chain sugars), where a hydroxyl group i s replaced by a branch-chain, are l e s s r e a d i l y a v a i l a b l e . Three reactions: W i t t i g a d d i t i o n s , n i t r o p a r a f f i n additions and photo-additions, which do lead to deoxy branched-chain sugars, w i l l be discussed i n greater d e t a i l . But f i r s t , because the synthesis of many branched-chain sugars i s dependent on the preparation of carbohydrate ketoses (see Table I I ) , methods of ox i d a t i o n of secondary hydroxyl groups of sugars w i l l be reviewed. 2. Oxidations of secondary carbohydrate hydroxyl groups P r i o r to about 1963 the means generally a v a i l a b l e f o r o x i d a t i o n of secondary carbohydrate hydroxyl groups were: platinum oxide and oxygen (31), lead t e t r a c e t a t e - p y r i d i n e (32) and chromium t r i o x i d e -p y r i d i n e (33). Y i e l d s of ketones i n blocked d e r i v a t i v e s were of t e n low and i n some cases the r e a c t i o n f a i l e d completely (34). Since then however, the a d d i t i o n of two new o x i d i z i n g agents, dimethyl s u l f o x i d e (DMSO) and ruthenium tetro x i d e (RuO^) and improvements i n the older procedures have made a v a i l a b l e many new carbonyl carbohydrates. The a p p l i c a t i o n of a l l these reagents to carbohydrates has been reviewed (35) and only those reagents employed i n t h i s work (DMSO and RuO.) w i l l be considered f u r t h e r . 4 Dimethyl sulf o x i d e has proved to be a very powerful o x i d i z i n g agent for carbohydrate hydroxyl groups (36). Using t h i s reagent a secondary a l c o h o l i s oxidized to a carbonyl and the DMSO i s reduced -9-to dimethyl s u l f i d e . Although some alcohols have been s u c c e s s f u l l y oxidized using DMSO alone .(37) , f o r sugar d e r i v a t i v e s the best y i e l d s are obtained by using combinations of DMSO and some " a c t i v a t -i n g " agent such as N, N-dicyclohexylcarbodiimide (38), a c e t i c anhydride (39) or phosphorus pentoxide (40). These e l e c t r o p h i l i c " a c t i v a t i n g " agents (E) (Equation 1) react with DMSO (36) to form an intermediate which i s subsequently attacked by the al c o h o l r e s u l t i n g i n d i s p l a c e -ment of the " a c t i v a t i n g " agent and formation of a dimethylalkoxy-sulfonium s a l t . Reaction with base followed by intramolecular hydrogen tr a n s f e r (41) then gives the carbonyl product and dimethyl s u l f i d e (DMS). The most common by-products of t h i s oxidation are methylthio-methyl ethers (42, 43). + (CH ) S=0 + E (CH ) S-O-E + R-CH-R' J OH (1) P , + R DMS + 0=0^, • * 2 2 S - (CH_)„S-0-CH R.' Oxidation with ruthenium tetroxide i s a method whereby secondary carbohydrate hydroxyl groups can be oxidized to ketones under r e l a t i v e l y mild conditions. This reagent can eith e r be prepared separately and added to a s o l u t i o n of the al c o h o l to be oxidized (44) (at l e a s t one equivalent of ruthenium tetroxide f o r each hydroxyl group to be ox i d i z e d ) , or generated jLn s i t u from ruthenium dioxide and sodium or potassium periodate (45). The i n s i t u preparation of -10-ruthenium tetroxide i s generally preferred as i t i s simpler and only trace amounts ( about-20 mg per g substrate) of c o s t l y ruthenium dioxide are used. The source of supply of ruthenium dioxide used i n t h i s oxidation i s very important (46). Commercial ruthenium dioxide i s prepared i n one of two ways, e i t h e r by d i r e c t combination of ruthenium with molecular oxygen, or from ruthenium t r i c h l o r i d e v i a the p r e c i p i t a -t i o n process (47). Using aqueous periodate solutions i t i s only p o s s i b l e to prepare ruthenium tetroxide from ruthenium dioxide prepared v i a the p r e c i p i t a t i o n process. Side products i n ruthenium tetro x i d e oxidations u s u a l l y r e s u l t from over-oxidation (48) r e s u l t i n g i n lactone formation. 3. The W i t t i g r e a c t i o n The W i t t i g r e a c t i o n (49) i s a method for preparing o l e f i n s from aldehydes or ketones. This r e a c t i o n involves a condensation e l i m i n -a t i o n between a phosphonium y l i d and the carboxyl group of an aldehyde or ketone to form an o l e f i n and a phosphine oxide (Equation 2). + - 9 (R) 3P-CHY + R -d-R »> R j R ^ C H Y + (R) 3P0 (2) Y=H or e l e c t r o n withdrawing group As t h i s r e a c t i o n i s widely used by organic chemists, general reviews (50) and d e t a i l e d discussions of the mechanism and stereo-chemistry (51) are a v a i l a b l e i n the l i t e r a t u r e and therefore these features w i l l not be reviewed here. The a p p l i c a t i o n of t h i s r e a c t i o n -11-to carbohydrates w i l l be examined, however. 3.1 Application of the Wittig reaction to carbohydrates The f i r s t application of the Wittig reaction in carbohydrate chemistry was reported in 1963 by Kochetkov et a l . (52). These workers had undertaken to develop a general route to higher aldoses. The unsaturated aldonic esters obtained via a Wittig reaction (Equation 3) were key intermediates in their program. By means of CO. Et I 2 CHO CH I II (CHOAc)n + (C H c) oPCHC0 oEt CH (3) | 6 5 3 2 i CH2OAc (CHOAc)n CH2OAc different Wittig reagents others have used the same strategy to obtain unsaturated higher ketoses (53), aldonic acids (54), deoxy sugars (55) and C_ glycosides (56) . The Wittig reaction has also been used to extend the carbon skeletons at the opposite end of the sugar chain. Condensation of the aldehyde XI (57) with either n-pentadecyltriphenyl phosphonium bromide or ethoxycarbonylmethylene triphenylphosphorane gave the expected unsaturated compounds XII and XIII respectively. R=CH OCH 1 J [XI] R = 0 i Ph [XII] R = CH(CH 2) nCH 3 [XIII] R = CHC02Et -12-Nucleosides having a 5' aldehydo group have also been condensed with W i t t i g reagents. When pyrimidine nucleoside XIV (58) was reacted with the phosphorane y l i d , generated i n s i t u by the a c t i o n of sodium ethoxide on (ethoxycarbonylmethyl) triphenylphosphonium bromide, a rapid r e a c t i o n ensued which gave f i v e products. These were subsequently i d e n t i f i e d as u r a c i l , unsaturated acids XV and XVI and the e t h y l esters of these acids. B B R=CH-[XIV] R r= 0 [XVI] R = CHC02H [XV] R -• CHCQ2H B = u r a c i l In t h i s laboratory the W i t t i g r e a c t i o n has been employed to prepare deoxy branched-chain sugars. Thus condensation of the basic W i t t i g reagent methyltriphenylphosphonium bromide with carbohydrate ketoses XVII and XVIII has been used by Rosenthal and S p r i n z l to obtain the 2-C-2-deoxy- XIX (59) and 3-C-3-deoxy- XX (21) ex o c y c l i c methylene branched chain sugars. Compound XX has also been prepared under s l i g h t l y differe..t conditions by Jones et a l . (60). Other branched-chain sugars prepared v i a a W i t t i g r e a c t i o n have included the unsaturated cyano sugars XXI (61). These compounds were prepared by condensation of ketose XX with cyanomethylene-t r i p h e n y l phosphorane. -13-0. OMe Me' Me 0 •Me 0 o - j — Me Me Me Me [XVII] R = 0 [XIX] R = CH 2 [XVIII] R = 0 [XX] R = CH 2 [XXI] In a l l of the above examples the carbohydrates have served only as the carbonyl components of the Wittig reaction. However, Zhdanov and Polenov (62) have reversed this approach and prepared a Wittig reagent from a carbohydrate. This carbohydrate phosphorane XXII was found to react with activated aldehydes to give both cis and trans o l e f i n i c ketones. For example, reaction with p-nitrobenzaldehyde gave the c i s and trans isomers of ketone XXIII. R=CH Me Me [XXII] R = P(Ph) 3 [XXIII] R = CH(C6H4)N02-p -14-3.2 The phosphonate mo d i f i c a t i o n of the W i t t i g r e a c t i o n As mentioned previously the W i t t i g r e a c t i o n involves a condensa-t i o n e l i m i n a t i o n between a phosphonium y l i d and an aldehyde or ketone. I t has been shown (63) that y l i d s can be obtained from any phosphorus system having a hydrogen atom adjacent to a phosphorus atom bearing a reasonable degree of p o s i t i v e charge. One phosphorus system which meets the above c r i t e r i a i s the phosphonates XXIV and the condensation of y l i d s prepared from these compounds with aldehydes and ketones i s r e f e r r e d to as the phosphonate mo d i f i c a t i o n of the W i t t i g r e a c t i o n . 0 II (RO)2P-CHR'R" [XXIV] The f i r s t reported r e a c t i o n of t h i s type was due to Horner and coworkers (64) who obtained triphenylethylene i n q u a n t i t a t i v e y i e l d by condensing benzophenone with the y l i d prepared from d i e t h y l b e n z y l -phosphonate. The use of phosphonates i n Wittig-type reactions was thoroughly investigated by Wadsworth and Emmons (65) who found that i n general compared to phosphonium y l i d s , the phosphonates were more r e a c t i v e , and gave better y i e l d s of o l e f i n . In a d d i t i o n , phosphonates are cheaper than phosphonium s a l t s . A l s o , the phosphorus by-product from phosphonates i s a water soluble phosphate ester which i s easier to separate from the o l e f i n than triphenylphosphine oxide, the usual by-product from the standard W i t t i g r e a c t i o n . -15-3.3 Mechanism and stereochemistry of the phosphonate modification  of the Wittig reaction The Wittig olefin synthesis is usually considered to proceed as shown below (Equation 4) (51) with the erythro-betaine XXVI leading to the cis o l e f i n and the threo-betaine XXVII leading to the trans. For stabilized ylids (XXV, R = electron withdrawing group) reaction with aldehydes gave predominately trans olefins while non-stabilized ylids (XXV, R = alkyl) gave the cis isomers, particularly in s a l t -free non-polar solvents (66). 0 (Ph) 0P 1 /\R' H R H R R' \ / - C / \ H H (Ph)o p 0 <$r erythro [XXVI] Jl_ II — CIS R H . R' H (Ph) 0P L R H ^ 3| C \ / [XXV] [XXVIII] C ' \ 1 / \ , *" C = C / \ H R' / \ R H H R* threo [XXVII] trans The reaction of phosphonate carbanions with aldehydes and ketones is also presumed to proceed as shown above for the phosphoranes. Although the stereochemistry of the products has not been as extensive-ly studied as the products from the standard Wittig reaction, i t would appear that in most instances both stabilized and non-stabilized anions give a predominance of the trans o l e f i n (67). Fortuitously, the condensation of diethyl cyanomethylphosphonate anion with aldehydes and ketones has been the subject of two recent studies (68, 69). This is the modified Wittig reaction used in this work. ^16-In the e a r l i e r i n v e s t i g a t i o n (68) the products a r i s i n g from the r e a c t i o n of alkylphenyl ketones with the carbanion generated from d i e t h y l cyanomethylphosphonate and sodium hydride (Equation 5) were examined. I t was found that when the phenyl group (R^) was unsubstitu-ted i n the or t h o - p o s i t i o n and the a l k y l group (R 2) w a s unbranched, a predominance of trans o l e f i n ( c i s / t r a n s r a t i o 0.1 - 0.2) r e s u l t e d . However, i f the a l k y l group was secondary, s u b s t a n t i a l amounts of c i s isomer were formed ( c i s / t r a n s r a t i o 0.5 - 0.7), and i f w a s a t e r t i a r y a l k y l group the c i s o l e f i n was the major product. Ortho s u b s t i t u t i o n i n the phenyl r i n g also increased the proportion of c i s isomer, whereas meta and para s u b s t i t u t i o n d i d not appreciably a f f e c t the product composition. 0 I-.-C=0 + (EtO)_P-CHCN / R2 [XXIX] [XXX] N C ^ P ( 0 ) ( 0 E t ) 2  erythro [XXXI] R, = Ro = H U P ( 0 ) ( 0 E t ) 2  threo [XXXII] substituted or unsubstituted phenyl a l k y l or H R- CN ^ / C=C / \ R 2 H c i s [XXXIII] (5) > = c x R 2 CN trans [XXXIV] -17-In the l a t e r study (69) the erythro XXXV and threo XXXVI diethyl-l-cyano-2-hydroxy-2-phenylethylphosphonates were prepared and separated. By studying the decomposition of these compounds i n b a sic media, (sodium hydride i n tetrahydrofuran), with and without the presence of competing aldehyde, the following conclusions were reached: (a) In t h i s basic medium anions XXXI and XXXII (R^ = phenyl, R 2 = H) p a r t l y reverted to benzaldehyde and anion XXX and p a r t l y interconverted d i r e c t l y ( i e . XXXI XXXII) without the formation of intermediates XXIX and XXX. (b) The c i s - t r a n s r a t i o of cinnamonitriles ( c i s / t r a n s about 0.18) ( c i s XXXIII, trans XXXIV, R^^ = phenyl, R 2 = H) was very nearly the same no matter i f the r e a c t i o n was c a r r i e d out using XXXV or XXXVI, with or without competing aldehyde, or d i r e c t l y using benzaldehyde and anion XXX. This indicated that the c i s - t r a n s r a t i o depended mainly on the r e l a t i v e rates of o l e f i n formation from oxyanions XXXI and XXXII. The f i r s t a p p l i c a t i o n of the phosphonate m o d i f i c a t i o n of the W i t t i g r e a c t i o n to a carbohydrate ketose was reported by Rosenthal and Nguyen (22) i n 1967. Here the ketose XVIII was condensed with the carbanion prepared by the a c t i o n of potassium t-butoxide on [XXXV] [XXXVI] 3.4 The modified W i t t i g r e a c t i o n i n carbohydrate chemistry -18-trimethylphosphonoacetate. This procedure gave as the major prod-ucts a mixture of cis and trans unsaturated esters (XXXVII and XXXVIII). Hydrogenation of either isomer gave as the only product the same 3-deoxy ali o branched-chain sugar XXXIX. Application of the same reaction to the 2-deoxy keto sugar XL afforded, after stereoselective hydrogenation, the 2,3-dideoxy branched-chain ribo sugar XLI (70). [XVIII] R = 0 H [XXXIX] [XXXVII] R = X C 0 2 C H 3 [XL] [XLI] -19-P r o t e c t e d 5'-aldehyde- n u c l e o s i d e s X L I I and X L I I I have a l s o been condensed w i t h phosphonate W i t t i g reagents (71) . Thus X L I I when condensed w i t h the carbanion from d i p h e n y l t r i p h e n y l p h o s p h o r -onylidine-methylphosphonate [XLIV] gave the 5*-deoxy-5'-(dihydroxy-p h o s p h i n y l m e t h y l ) - n u c l e o s i d e XLV. Nucleoside X L I I I condensed w i t h the same reagent gave the corresponding u r i d i n e compound XLVI. [XLII] B = adenine R = 0 [XLV] B = adenine R = CHPO(OPh) 2 [ X L I I I ] B = u r a c i l R = 0 [XLVI] B = u r a c i l R = CHPO(OPh) 2 4 N i t r o p a r a f f i n a d d i t i o n to carbohydrates N i t r o p a r a f f i n condensation r e a c t i o n s w i t h carbohydrates have been e x t e n s i v e l y s t u d i e d (72). H i s t o r i c a l l y these condensations were used as a r o u t e to h i g h e r carbon aldoses or ketoses (Equation 6 ) , the i n i t i a l a l k y l n i t r o condensation products being converted to aldehydes or ketones by the Nef r e a c t i o n (73). A m o d i f i c a t i o n of t h i s r e a c t i o n (Equation 7) has provided a general r o u t e to the 2-deoxy a l d o s e s . Thus a c e t y l a t i o n of the i n i t i a l nitromethane-a l d o s e condensation products f o l l o w e d by a Schmidt - Rutz r e a c t i o n (74) y i e l d s unsaturated n i t r o carbohydrates. Reduction of these compounds f o l l o w e d by a Nef r e a c t i o n on the n i t r o - s u g a r y i e l d s the 2-deoxy a l d o s e s . -20-HC=0 I R CH.OH I 2 CH NO CHJDH I 2 CHNO„ I 2 CHOH I R CH 3N0 2 1. OH 2. H 30 CH 2N0 2 CHOH I R CH„OH I 2 c=o I CHOH I R 1. OH 2. H 30 + HC=0 I CH„OH I 2 R (6) CH 2N0 2 CHOH I R 1. (Ac) 20 2. NaHCO„ CHNO. I 2 CH I R 1. H r 2. OH 3. H 30 HC=0 CH 2 (7) R As w e l l as these above reactions which serve to extend the sugar chain, n i t r o p a r a f f i n condensations have been used to prepare deoxyamino sugars (75), branched-chain deoxynitro sugars (28, 76) and dideoxy branched-chain d i n i t r o sugar d e r i v a t i v e s (77). The majority of these reactions were developed by H. H. Baer and coworkers at the U n i v e r s i t y of Ottawa. The condensation of sugar dialdehydes with nitromethane (75) (Equation 8) i s p a r t i c u l a r l y i n t e r e s t i n g . Under the r e a c t i o n conditions c y c l i z a t i o n occurred and reduction of the a c i n i t r o compound afforded deoxyamino sugars. By s u b s t i t u t i n g nitroethane for nitromethane i n t h i s r e a c t i o n deoxynitro branched-chain sugars have been prepared (28, 76). 0 0CH„ CH 3N0 2 NaOCH ' 0. 0CH„ KHSO OCH, NH 2 OH -21-Despite this considerable body of work done on the addition of nitroparaffins to carbohydrates, u n t i l very recently no attempt had been made to condense nitromethane with carbohydrate ketoses in order to obtain branched-chain sugars. This situation changed, however, in 1969 when two groups (78, 79) independently reported the addition of nitromethane to keto sugars. In the report from our laboratory (78) the 2-deoxy-3-keto-hexo-pyranose XLVII was condensed with nitromethane to afford the 2-deoxy-3-C-nitromethyl-D-rlbo-hexopyranose XLVIII. [XLVII] [XLVIII] The second study dealt with the addition of nitromethane to the 3-keto furanoses XLIX (79) . By this means the 3-C-nitromethyl-D-ribofuranose branched-chain sugar L was obtained. Me Me [XLIX] [L] R = t r i t y l or tosylate -22-Shortly after these i n i t i a l investigations appeared, Overend et a l . (20) published an extensive study on the addition of nitromethane to methyl glycopyranosiduloses, and Albrecht and Moffat (80) announced the condensation of nitromethane with 3-keto-furanose XVIII to obtain the 3-Cj-nitromethyl-E-ribofuranose LI. This compound was converted by a Schmidt-Rutz reaction (74) followed by reduction to the deoxy nitromethyl branched-chain sugar LII from which were obtained 3-C_-nitromethyl LUIa and 3-C-amino methyl LUIb branched-chain sugar nucleosides. Me. / 0 \ Mev ,0>^ Me Me [XVIII] [LI] [LII] [LUIa] R = N02 [LUIb] R = NH2 -23-5. Photo-addition of formamide to olefins The photo-addition of formamide (81-84) to olefins was devel-oped by Elad and Rokach as a general process for converting olefins to amides. It was found that in the absence of ketones the photo-addition of formamide required long irradiation periods and gave low yields. However, in the presence of ketones such as acetone, irradiation (X > 300) of formamide solutions of olefins gave good (50-80%) yields of amides. For terminal olefins (81) the photo-products were found to be mainly the 1:1 adducts resulting from anti-Markovnikov addition of formamide to the double bond (Equation 9). Minor side products RCH=CH 2 + HC0NH2 — — - RCH2CH2CONH2 (9) included non-terminal 1:1 formamide olefin adducts, acetone addition products and teleomeric material. Non-terminal olefins in this reaction were found to give primarily a mixture of 1:1 formamide olefin adducts. Reaction of a,B-unsaturated esters afforded mainly products where formamide addition had taken place at the B carbon (84). Of particular interest in this reaction is the sensitivity of the addition to steric features of the olefin. As well as the nearly exclusive anti-Markovnikov addition discussed above for terminal olefins, i t was found that the photo-condensation of formamide with norbornene [LIV] (82) gave only the single amide, norbornane-2-exo-carboxyamide [LV]. -24-CONH, [LIV] [LV] The mechanism postulated by Elad and Rokach (82) for the acetone initiated photo-addition of formamide was as follows (Equation 10). They hypothesized that the carbamoyl radicals are generated either from the collapse of the photo-activated formamide molecule or through hydrogen atom abstraction from formamide by the excited t r i p l e t of acetone. The olefin then serves as a scavenger for the carbamoyl radicals thereby forming the formamide addition products. Chain propagation can continue as shown in equation 10 d-f. Termination acetone hv [acetone] (no-*Tr*) HC0NH2 + [acetone] ( n ^ * ) RCH= CH2 + • CONH, RCHCH2CONH2 + HC0NH2 RCHCH2C0NH2 + RCH=CH 2 •CONH, RCHCH2C0NH2 RCH„CH„C0NHo + «C0NH, 1 1 2 i RCHCH„C0NHo I 2 2 CH -CHR (a) (b) (c) (d) (10) (e) RCHCH-CONH. + HC0NH„ , 2 2 2 CH2-CHR RCHCHoC0NH„ + CH2CH2R •CONH, (f) RCH = CH2 + •CONH, RCHCH2CONH2 + 'C0NH2 RCHCONH2CH2« RCHCHoC0NHo I 2 2 C0NH„ (g) (h) -25-of the chain may occur in many different ways, as for example in equation lOh. Products resulting from the side reactions (10 e-h) were isolated in some instances (82,83). The exact role of acetone in this reaction was recently c l a r i f i e d (85). For a direct energy transfer between the n "•IT* t r i p l e t of acetone and the n-nr* f i r s t t r i p l e t of formamide to occur, the t r i p l e t energy of acetone (3.5 ev) (86) would have to be higher than that of formamide. When the energy of the n "Hr* f i r s t t r i p l e t of formamide was calculated i t was found to be 4.2 ev. Therefore direct energy transfer to formamide from an acetone t r i p l e t is not possible and consequently the carbamoyl radicals in this photolysis must be produced by extraction of a formyl hydrogen from ground state formamide by a photo-activated acetone molecule. 5.1 Photo-additions to carbohydrates Although photo-degradation reactions of sugars have been well studied (87), there is only a limited number of instances of photo-additions to carbohydrates reported in the literature. In 1966 Horton and Turner (88), while studying carbohydrates having heteroatoms other than oxygen in the ring, prepared the thioacetate LVI by photo-addition of thioacetic acid to the unsatur-ated sugar LVII. Other sulfur-containing sugar derivatives have been prepared (89) by the addition of thioacetic acid and benzyl mercaptan to the unsaturated sugar LVIII. Various other reagents photo-condensed with unsaturated sugar LVII have included phosphine and phenylphosphine (90) and 1,3 dioxalan (91). With the exocyclic "ene" sugar XX photo-addition of -26-•C7 0 0 OMe 0 Me 0 •Me Me Me Me-^ ^Me [LVI] [LVII] [LVIII] dioxalan afforded the 3-deoxy-allo-branched-chain sugar LIX as the only product. More recently reports of photo-condensations of acetone with triacetyl-D-glucal [LX] (92), and 2,3 dimethylbut-2-ene with hexenopyranoses (93) have appeared. In this laboratory photo-amidation has been examined as a route to deoxy branched-chain amido and amino sugars. It was found that the acetone sensitized photo-addition of formamide to triacetyl-g-glucal [LX] (26) gave the mixture of amides depicted in equation 11 as well as trace amounts of acetone addition products. The same reaction applied to compound [LXI] (94) afforded the carbamoyl [XX] [LIX] -27-[LXII] I X — CK XMe [LXIII] branched-chain sugars LXII and LXIII along with some products result-ing from the photo-addition of acetone. 6. Nucleosides The term nucleoside is used to denote compounds containing a nitrogen heterocycle (purine or pyrimidine and their close analogs) in glycosidic linkage with a carbohydrate moiety. For naturally -28-occurring nucleosides the heterocyclic bases are attached via 8 glycosyl linkages, with the most commonly occuring bases being the purines adenine and guanine and the pyrimidines cytosine, uracil and thymine. The carbohydrate portion i s usually D-ribose or "2-deoxy-D-ribose" in the furanose form. 6.1 Nucleoside synthesis Rapid advances in nucleic acid chemistry have resulted in the development of many new methods of nucleoside syntheses. A complete review of the available methods is beyond the scope of this thesis; therefore only those methods of purine nucleoside synthesis used in this work w i l l be discussed. For more complete surveys of the synthetic methods available see references (95) and (96). 6.2 Synthesis of purine nucleosides Fischer and Helferich prepared the f i r s t purine glycosides (purine nucleosides) by condensing silver salts of some purine derivatives with acetylated glycosyl halides (97). Subsequent modifications of this procedure resulted in the replacement of the purine silver salts with chloromercuri derivatives (98) and the in  situ generation of the glycosyl halide from the ester with titanium tetrachloride (99) [Equation 12]. For sugars having at C-2 an ester hydroxyl protecting group, the anomeric (C-l') configurations of the nucleosides obtained by the above condensations are predicted by Baker's trans rule (100): "condensations of a heavy metal salt of a purine or pyrimidine with an acylated glycosyl halide w i l l form a nucleoside with a C-l', C-2' trans -29-configuration in the sugar moiety regardless of the original configura-tion of C-l, C-2." The mechanistic considerations underlying this observation have been reviewed (101) and although some exceptions are known (102), in the vast majority of cases the C-l', C-2' cis configuration is not observed, or observed only in minor proportions. Experimentally the anomeric configuration of glycofuranosyl purine nucleosides have been determined from the sign of their Cotton effect (103); the 9-B-P_-glycofuranosyl derivatives give negative Cotton effects (104) and the 9-a-Dj-compounds show positive Cotton effects. RO OR Although in equation 12 the N-9 substituted nucleoside is depicted as being the only product formed, as i t is in most instances, in some cases the N-7 isomer is produced, occasionally to the complete -30-exclusion of the N-9 form. A case in point occurred in the preparation of the puromycin analog LXIV. When ct-bromoacetoglucose was condensed with the chloromercuri derivative of 6-dimethylamino purine (105) only the N-7 nucleoside LXIV was isolated. Fortunately, in the case of puromycin derivatives, the N-7 and N-9 isomers are easily differentiated on the basis of their ultraviolet spectra, N-9 isomers at pH-7 having a X maximum at about 275 nm and N-7 isomers having a X maximum at about 295 nm (105). At present there i s s t i l l no completely satisfactory rule for predicting whether the N-9 or N-7 isomer w i l l be formed in this reaction. A more recently developed method for purine nucleoside synthesis is the fusion procedure (106,107). Here the acetylated sugar, with or without an acid catalyst, i s simply fused under reduced pressure with a purine derivative. The resultant nucleosides are usually the N-9 substituted isomers having anomeric configurations consistent with that which would be predicted by Baker's rule. Aside from simplicity, the fusion procedure has the additional advantages that the relatively unstable glycosyl halide is not necessary as an intermediate and that the purine may be substituted with amino, oxo, or thio functionalities which need not be protected AcO 2 OAc [LXIV] -31-during the reaction. Also this procedure results in nucleosides free of mercury contamination which is sometimes not possible using the above halo-mercuri method. This i s important in cases where the biological activity of a nucleoside is to be evaluated as i t has been —8 shown (108) that mercuric ion concentration as low as 10 molar can lead to erroneous interpretations of biological activity. 6.3 Branched-chain sugar nucleosides As branched-chain sugars had been isolated from a number of important antibiotics, in the middle years of the last decade a number of research groups independently began programs leading to the synthesis of nucleosides containing branched-chain sugars instead of the normal D_-ribo furnoses in order that their potential as therapeutic agents could be evaluated. The f i r s t report of a synthesis of a branched-chain sugar nucleoside came in 1966 from Walton et a l . (109) working in the research laboratories of Merck, Sharp and Dohme. These workers reported the preparation of the 2'-C-methyl-Dj-ribofuranose LXV and 3'-C-methyl-D-ribofuranose LXVI analogs of adenosine. NH [LXV] R = H R' = CH3 [LXVI] R = CH3 R' = H OH OH -32-Since then, in addition to the 3'-deoxy-3'-C-hydroxyethyl (110) , 3'-deoxy-3'-C-methyl (22) and 3'-deoxy-3'-C_-hydroxymethyl (111) ribo and alio furanosyl adenine nucleosides prepared in this laboratory, numerous other branched-chain nucleosides (Table III) have been synthesized. The continuing interest of both academic and industrial research groups in the chemistry of these compounds has made this a very active area of research. 6.4 Biological activity of branched-chain sugar nucleosides Although no studies have been published concerning the biological activity of a l l these modified nucleosides in a single system, the fragmentary reports which do exist in the literature indicate that some of these compounds might be developed into useful thera-peutic agents. For example, the methyl branched-chain sugar nucleo-sides have been shown to inhibit the growth of KB cells in culture (112) and to be effective anti-neurovaccinia agents in mice (113). In addition, the 3'-amino-3'-hydroxymethyl derivative of adenosine has been shown to exhibit weak inhibition against vaccinia Dairen (118). It should be noted that where compounds have been assayed biologically those having branches at the 3' position (eg. LXVI) showed the greatest ac t i v i t y . -33-TABLE III Synthesis of Branched-chain Sugar Nucleosides HOCH2 C or FC HOCH2 Q > C or FC (114) (114) (115) HOCH2 Q > DMP CH2R OH R = N0 2 or NH 2 (80) R = H or Me (116) HOCH NH 2 OH B = A, DMP, G, C, U (117) A = adenine; C = cytosine; CP = 6-chloropurine; DMP = 6-dimethylaminopurine FC = 5 - f l u o r o u r a c i l ; G = guanine; P = purine. The numbers i n parentheses are the references f o r these compounds. III. RESULTS AND DISCUSSION 1. Synthesis of branched-chain cyanomethyl sugars by a  Wittig reaction In the objective i t was indicated that the f i r s t goal of the work described here was to explore various means for introducing deoxy-nitrogenous branched-chains into carbohydrates. As a modified Wittig reaction had been shown i n this laboratory to be a useful way of preparing carbomethoxymethyl branched-chain sugars (22), i t was decided to attempt the condensation of the phosphonate Wittig reagent diethyl cyanomethylphosphonate (68,69) with the carbohydrate 3-keto furanoses 1,2:5,6-di-O-isopropylidene-q-D-ribo-hexofuranos-3-ulose [XVIII], 1,2:5,6-di-0-isopropylidene-D-q-xylo-hexofuranos-3-ulose [LXVII] and 5-0-benzyl-l,2-0-isopropylidene-q-D-erythro-pentofuranos-3-ulose [LXVIII]. This particular Wittig reagent was chosen because i t was f e l t that after reduction of the i n i t i a l cyanovinyl addition products to cyanomethyl deoxy branched-chain sugars, these cyanomethyl compounds could be converted into various other nitrogenous branched-chain sug'ars such as aminomethyl and car bamoy line thy 1 derivatives. As the second objective was to use these branched-chain compounds to prepare branched-chain sugar nucleosides and to examine the biological activity -35-of these modified nucleosides, i t was decided to use 3-keto-furanoses as the carbonyl component of this Wittig reaction. In this way the branched-chain sugars prepared would be in the furanose con-figuration (the carbohydrate configuration present in most naturally occurring nucleosides) and the branched-chain nucleosides prepared from these sugars would have 3'-branched-chains. As was pointed out i n the Introduction (p.32 ) branching at this position appears to confer the greatest degree of biological a c t i v i t y . Mev ,0 Me 0 .CK. PhCH2OCH2 ^ Q [XVIII] [LXVIII] 1.1 1,2:5,6-Di-O-isopropyl idene-ct-D-ribo-hexofuranos-3-ulose [XVIII] This compound was prepared from D-glucose by known procedures. Condensation of acetone with D-glucose i n the presence of an acid catalyst (118) afforded 1,2:5,6-di-O-isopropylidene-a-D-glucofuranose - 3 6 -[LXIX]. The secondary hydroxyl group of this compound was then oxidized to the hydrated 3-keto compound LXX (Equation 13) using sodium periodate and a "cataly tic'-' amount of ruthenium dioxide (119) . The water of hydration was removed from LXX by azeotroping with toluene to afford ketose XVIII. A point of technique frequently omitted in the discussion of the above oxidation i s the neccessity for carefully controlled addition of the periodate solution, particularly at the beginning of the reaction. Too rapid addition of periodate generally results i n precipitation of the ruthenium catalyst as an insoluble complex on the walls of the reaction flask. The best yields of ketose were obtained by i n i t i a t i n g the reaction by adding only a few drops of periodate solution. After several small additions of oxidant, i t was added in larger portions (1-2 ml) a n d the colour changes i n the reaction mixture were observed. The presence of the actual oxidant, ruthenium tetroxide was indicated by the reaction mixture taking on a green-black colour; when only ruthenium dioxide was present the solution appeared black. Additions of periodate were made only when a l l the ruthenium tetroxide generated by the previous addition of periodate had been consumed. 1.2 1,2:5,6-Di-O-isopropylidene-q-D-xylo-hexofuranos-3-ulose [LXVII] This ketose was prepared from hydrated ketose LXX following the procedure of Slessor and Tracey (119) (Equation 13). Thus compound LXX was acetylated to afford the enol acetate LXXI which after hydro-genation over palladium-on-charcoal followed by removal of the -37--38-3-acetate using sodium methoxide and oxidation (with ruthenium tetroxide as before) gave the required 3-keto compound LXVII. A point to note here for future discussion i s the s t e r i c c o n t r o l exerted by the 1,2-0-isopropylidene group of enol acetate LXXI. Because of the d i r e c t i v e e f f e c t exhibited by t h i s group, hydrogenation of LXXI was s t e r e o s e l e c t i v e and re s u l t e d i n formation of only one product [LXXIa]. 1.3 5-0-Benzyl-l,2-0-isopropylidene-q-D-erythro-pentofuranos-3-ulose [LXVIIIJ This compound was obtained by a rather lengthy procedure (Equation 14) s t a r t i n g from D-xylose [LXXIII]. The di-O-isopropylidene compound LXXIV was prepared by the a c i d catalyzed condensation of acetone with D-xylose (120). The 3,5-isopropylidene group of LXXIV was then s e l e c t i v e l y cleaved with d i l u t e acid to a f f o r d the mono-isopropylidene compound LXXV (120). T o s y l a t i o n of the 5-hydroxyl group of LXXV (121) followed by treatment of the t o s y l a t e with sodium methoxide, gave the 3,5-anhydro sugar LXXVI (121). Opening of the 3,5-anhydro r i n g with benzyl a l c o h o l and sodium afforded 5-0_-benzyl-l,2-0_-isopropylidene-a-D-xylo-furanose [LXXVII] (122). Preparation of t h i s compound by d i r e c t monobenzylation of LXXV was not su c c e s s f u l . Two methods f o r the oxidation of the secondary hydroxyl group of alc o h o l LXXVII were examined. In the f i r s t method the oxidant was dimethyl s u l f o x i d e with phosphorus pentoxide serving as the " a c t i v a t i n g " agent (40). This procedure afforded a 65% y i e l d of 5—0—benzyl—1,2—0— isopropylidene-q-D-erythro-pentofuranos-3-ulose [LXVIII] as a homogeneous -39-OCH ^ , acetone Dr x y l o s e • *- Me H 30 HOCH H 30 2/° OH Me -Me [LXXIV] Me [LXXV] Me •Me 1. TsCl 2. Na/MeOH ROCH 2 ^ 0 0 0 ROCH. RuO. I 2 4 4 6 DMSO/P 20 5 •Me CH ROH 2 ^ 0 • Me •Me Me Me Me [LXVIII] [LXXVII] [LXXVI] R = benzyl syrup (R^ 0.76, benzene:methanol 4:1) having no hyd r o x y l a b s o r p t i o n and a s t r o n g carbonyl a b s o r p t i o n at 1760 cm Attempted chromatography of t h i s ketose (on s i l i c a g e l ) l e d to decomposition so i t was t h e r e f o r e c h a r a c t e r i z e d as i t s 2,4-dinitrophenylhydrazone d e r i v a t i v e . In the second method the o x i d a t i o n of LXXVII was accomplished using ruthenium t e t r o x i d e generated as i n the previous two o x i d a t i o n s from ruthenium d i o x i d e and sodium p e r i o d a t e (119). This procedure gave very good y i e l d s (ca. 90%) of ketose LXVIII i d e n t i c a l by t i c , i r and nmr w i t h the product from the DMSO o x i d a t i o n . Although t h i s o x i d a t i o n -40-required about twenty hours to complete, the yi e l d of ketose was very good and none of the ring insertion lactone product was detected. In the oxidation of the related compound LXXIX with ruthenium tetroxide (123) (the ruthenium tetroxide being generated externally and added to a solution of alcohol LXXIX ) the 3-ketose LXXX was isolated i n about a 50% yield after a three hour reaction time. Longer reaction periods were found to give substantial amounts of the lactone side product LXXXI. [LXXIX] [LXXX] [LXXXI] 1.4 3-C-Cyanomethyl-3-deoxy-l, 2:5,6-di-0_-isopropylidene-a-B-allof uranose [LXXXVI], 3-C-cyanomethyl-3-deoxy-l,2:5 ,6-di-0-isopropylidene-ct-D-gulofuranose [LXXXVII] and 5-0_-benzyl-3-C_-cyanomethyl-3-deoxy-1,2-0-isopropylidene-a-D-ribofuranose [LXXXVIII] Having obtained the 3-ketoses just described the next step was to condense these compounds with the carbanion prepared from diethyl cyanomethylphosphonate and sodium hydride. The method followed here was essentially that u t i l i z e d by Jones and Maisey (68) i n the preparation of a,B-unsaturated n i t r i l e s from alkyl phenyl ketones. The only modifications to their procedure were that the reaction mixture was held -41-at 0° during addition of the ketose to the solution (this was found to eliminate the formation of side products) and that the i n i t i a l l y produced a,^-unsaturated n i t r i l e s were hydrogenated (at atmospheric pressure using palladium on charcoal) without prior purification to afford the 3-C_-cyanomethyl-3-deoxy branched-chain sugars. Thus, the above reactions applied to l,2:5,6-di-0-isopropylidene-ct-D-ribo-hexof uranos-3-ulose [XVTII] afforded 3-C-cyanomethyl-3-deoxy-1,2:5,6-di-0-isopropylidene-ct-D-allofuranose [LXVII] in 78% yield; 1,2:5,6-di-0-isopropylidene-q-D-xylo-hexofuranos-3-ulose [LXVIII] afforded 3-C-cyanomethyl-3-deoxy-l,2:5,6-di-0-isopropylidene-a-I)-gulofuranose [LXXXVI] in 79% yield, and 5-0-benzyl-l,2-0-isopropylidene-ot-g-erythro-pentofuranos-3-ulose [LXXXVII] afforded 5-0-benzyl-3-C-cyanomethyl-3-deoxy-a-D-ribofuranose [LXXXVIII] i n 93% yield (Equation 15). Although the intermediate a, 3-unsaturated n i t r i l e sugars XXI, LXXXIV and LXXXV were not characterized, i t i s presumed these were the i n i t i a l condensation products as they are the expected reaction products and hydrogenation of these compounds gave the cyanomethyl branched-chain sugars as would be expected. Furthermore, in each case the i r spectra contained characteristic stretching absorptions for carbon-nitrogen tr i p l e bonds (ca. 2250 cm ^) and the nmr spectra showed the presence of an o l e f i n i c proton (chemical shift about T 4.1). The palladium-on-charcoal atmospheric pressure hydrogenation of the o l e f i n i c bond proceeded smoothly in each instance, the uptake of hydrogen stopping spontaneously after absorption of about one equivalent. The s t a b i l i t y of the 5-0-benzyl group of compound LXXXVIII in this [LXVIII] -43-hydrogenation is somewhat surprising as these hydroxyl protecting groups are known to be hydrogenolyzed under mild conditions (124a); however, cases are known where hydrogenolysis of this group has required both heat and pressure (124b). That this series of reactions did indeed lead to the cyanomethyl branched-chain sugars was clearly shown by the i r and nmr spectra. The presence of the n i t r i l e group was confirmed by the characteristic C=N stretching absorption (ca. 2250 cm "*") in the i r spectra and the presence of the methylene protons adjacent to the cyano group was confirmed by finding a two proton multiplet in the region 7.0-7.5 T. In each instance the product after hydrogenation was judged homogeneous (by t i c and nmr). No trace of isomeric compounds having a C-3 cyanomethyl configuration epimeric with those shown in equation 15 was ever detected. The configuration at C-3 of these cyanomethyl branched-chain sugars was determined by nmr spectroscopy in the following manner. It has been shown by Hall and coworkers (125) that for 1,2-0-isopropylidene-a-D-glucofuranose and of 1,2-0-isopropylidene-g-L-idofuranose compound in a l l cases the twist conformation LXXXIX i s adopted. That i s , C-2 lie s below and C-3 above the plane formed by C-l, 0 and C-4. Assuming this conformation was adopted by the above 3-C-cyanomethyl branched-chain sugars and assuming a f i r s t order Karplus relationship (126), holds for H-l, H-2 and H-3, i t i s possible to make the following predictions: (a) If the C-3 cyanomethyl substituent projects above the plane (opposite to the configuration shown in equation 15) the H-2 nmr signal -44-Me [LXXXIX] should appear as a doublet (J^ ^  ~ 3-4 ^z, 3 < 0.5 Hz). (b) If the C-3 substituent is as shown in equation 15 the H-2 resonance should appear as a t r i p l e t or quartet (J- _ - 3-4 Hz, J2 3 = 3 - 6 H z ) * In Table IV are l i s t e d the H-2 chemical shifts and coupling constants for compounds LXXXVI, LXXXVII and LXXXVIII plus the same values for some representative 1,2-0-isopropylidene-furanoses. As can be seen from this Table, the H-2, H-3 coupling constant values indicate that the cyanomethyl branched-chain sugars have assumed the configurations depicted (i.e. the cyanomethyl branched chain is cis to the 1,2-isopropylidene group). -45-TABLE IV H-2 Chemical S h i f t s and Coupling Constants f o r Some 1,2-0_-Isopropylidene~furanoses Compound H-2 Chemical S h i f t J l , 2 J2,3 [LXXXVI] 5.23 x C D C 1 3 3.6 3.6 [LXXXVII] 5.27 x C D C 1 3 4.0 5.0 [LXXXVIII] , CDC1. 5.34 x 3 3.6 3.9 [LXIX] 5.60 x x4 (146) 3.9 =0 [LXIXa] r n 5.53 T 4 (146) 3.9 5.0 [LXXXVII] [LXXXVIII] -46-To i l l u s t r a t e the typical H-l, H-2 coupling pattern of these 3-C-cyanomethyl-3-deoxy-l,2-0-isopropylidene branched-chain sugars the 100 MHz spectrum of compound LXXXVI is reproduced in Figure I. The hydrogen assignments for LXXXVI were made in the following way: 1. irradiation of the doublet at 4.13 T (H-l) collapsed the t r i p l e t at 5.23 T to a doublet indicating this was the H-2 resonance, 2. irradiation of the t r i p l e t at 5.23 r collapsed the doublet at 4.13 T to a singlet, confirming the previous assignment and altered the multiplet 7.6-7.8 x indicating this was H-3 resonance. Another factor corroborating the C-3 configurational assignment i s the known directive effect of the 1,2-0-isopropylidene furanoses. As was previously noted (p. 38 ), the hydrogenation of enol acetate LXXI gave only one product LXXII. There i s a generally observed trend that for compounds of this sort the bulky isopropylidene group blocking the C-l, C-2 hydroxyls interferes with the approach of reagents from the underside (cis to the 1,2-0-isopropylidene group) of the ring (80,123). Therefore, i t was to be expected that catalytic hydrogenation of the unsaturated bond, took place via cis addition from the topside of the ring, thereby resulting in compounds having the proposed C-3 configuration. Some time after our i n i t i a l report on the preparation of cyanomethyl branched-chain sugars via the above modified Wittig reaction (127), Tronchet et a l . (61) reported the synthesis of branched-chain unsaturated cyano sugars by reaction of the Wittig reagent cyanomethylene triphenyl-phosphorane with keto sugars. The unsaturated cyano sugars were c i s -dihydroxylated (KMnO^) to yield aldehydo branched-chain sugars (e.g. equation 16). This procedure gave compounds having the same branched-chain r i i I i i i i I I I ' l l l l' T T I I 'I I I > I I ' I I I II I I I I I I l 1 T TV I | I I I I' l l l "I I T T T TV I I I r i' i i i i i T T T T 7 I I I I I 9 II I I | M I 1 0 Figure 1. Proton magnetic resonance spectrum at 100 MHz in deuteriochloroform of 3-C-cyanomethyl-3-deoxy-l,2:5,6-di-0-isopropylidene-a-D-allofuranose [LXXXVI]. -48-OH OH [V] which occurs i n the branched-chain sugar streptose [V]. 2. Synthesis of branched-chain amino sugars by reduction of branched- chain cyano sugars As i t was desired to prepare branched-chain sugars having a variety of nitrogenous functionalities, the reduction of the previously described branched-chain cyanomethyl compounds to the corresponding aminoethyl branched-chain sugars. Although alkyl cyanides provide useful intermediates for the synthesis of alkyl amines, the cyano group being reduced by metal hydrides (128), by catalytic hydrogenation (129), or by diborane (130); there have been very few reports of successful conversion of carbohydrate cyanides into carbohydrate amines. Coxon and Fletcher (131) have reported the lithium aluminum hydride reduction of a galactopyranosyl cyanide to an amino heptitol and interestingly, the branched-chain cyano sugar XC (132) has reportedly been hydrogenated (no details given) to the corresponding amino sugar isolated as the tri-acetal derivative XCI. -49-2.1 3-C_-(2'- Acetamidoethyl)-3-deoxy-l, 2:5 ,6-di-O-isopropylidene-a-D— allofuranose [XCI] As a successful catalytic reduction of a n i t r i l e branched-chain sugar had been reported (see above) i t was decided to attempt the reduction of 3-C- cyanomethyl-3-deoxy-l,2:5,6-di-j3-isopropylidene-a-D-allofuranose [LXXXVI] by catalytic hydrogenation. Catalytic hydro-genation of n i t r i l e s to amines has been assumed to proceed through an imine intermediate (128) (Equation 17a). H H R-CHN -=—*• RCH=NH — > RCH2NH2 (a) RCH=NH + RCH2NH2 RCH (NH2)NHCH2R (b) H RCH(NH2)NHCH2R • (RCH^NH + NH^ (c) (17) RCH=NH + RCH2NH2 RCH=NCH2R + NH^ (d) RCH=NCH R + H • (RCH ) NH (e) -50-Complications in this reaction occur when the primary amine couples with the intermediate imine, (Equation 17b) giving a product from which a secondary amine may be formed by hydrogenolysis (Equation 17c) or by elimination of ammonia (Equation 17d) followed by hydrogenation (Equation 17e) of the resultant imine. Variations on this general scheme have been used to account for the formation of other observed side products (128). The above coupling reactions have been prevented by either forming a derivative of the primary amine as soon as i t was produced, this being done by hydrogenation in the presence of mineral acid (133) or acetic anhydride (134), or by hydrogenation in an ammonia saturated solution (135) which reverses the equilibrium in equation 17d. As n i t r i l e branched-chain sugar LXXXVI contains the very acid labile 5,6-O-isopropylidene group, i t was decided to hydrogenate LXXXVI in ethanol saturated with ammonia. Accordingly LXXXVI in ethanol saturated with ammonia at 0° was hydrogenated at 60 psi for twenty hours at room temperature over 5% rhodium-on-alumina. Because of the presence of ammonia i t was impossible to monitor the hydrogen uptake; however, the reaction was continued u n t i l no starting material remained (as evidenced by t i c ) . This procedure gave the expected aminoethyl branched-chain sugar characterized as i t s N-acetyl derivative 3-C-(2'-acetamidoethyl)-3-deoxy-l,2:5,6-di-0-isopropylidene-ct-D-allofuranose [XCII] in 80% yield. The i r spectrum of this compound contained no C=N absorption but did show an N-H stretch at 3300 cm"1 and a carbonyl absorption at 1640 cm"1. The nmr spectrum -51-showed the presence of the N-H proton as a broad t r i p l e t at 3.27 x and the N-acetate as a 3 proton singlet at 8.03 x. 2.2 3-C-(21-Ace tamidoe thy l)-5-0j-benzyl-3-deoxy-l,2-0-isop ropy lidene-a-D-ribofuranose [XCIII] To reduce the n i t r i l e group of the 5-0-benzyl cyanomethyl branched-chain sugar LXXXVIII catalytic hydrogenation procedures were judged to be inapplicable as the rather vigorous conditions involved would in a l l likelihood have hydrogenolyzed the 5-0-benzyl hydroxyl protecting group. Had this happened i t would have then been necessary to reblock the 5-hydroxyl group to use this compound in nucleoside syntheses. In view of this i t was decided to attempt the reduction of the cyano group of LXXXVIII using lithium aluminum hydride in ether. Reduction to the pentose amino sugar proceeded smoothly with the product being characterized as before as the N-acetyl derivative 3-C-(2'--52-acetamidoe thy l)-5-0_-benzyl-3-deoxy-l,2-0-isop ropy lidene-a-D-ribof uranose [XCIII]. That the product of t h i s reduction was the expected acetamido-e t h y l branched-chain sugar was confirmed by the NH and carbonyl absorptions found i n the i r spectrum at 3300 cm ^ and 1650 cm ^ r e s p e c t i v e l y and by the broad NH s i g n a l and the N-acetyl s i g n a l found i n the nmr spectrum at 4-4.4 T and 8.1 T r e s p e c t i v e l y . PhCH 2OCH 2 0 PhCHOCH | H2 CN -Me Me f H2 HNAc •Me Me [LXXXVIII] [XCIII] 2.3 3-C_-Cyanomethyl-3-deoxy-l,2-0_-isopropylidene-g-L-lyxof uranose [XCV] and 3-C-(2'-acetamidoethyl)-3-deoxy-l,2-0-isopropylidene-B-L-lyxofuranose [XCVI]. As various L-amino sugars are known to e x i s t i n Nature (136) i t was decided to undertake the synthesis of the above cyanomethyl and acetamidoethyl branched-chain sugars. The manner i n which t h i s was done i s i l l u s t r a t e d i n equation 18. -53-(18) [XCVI] [XCV] The f i r s t step in this sequence i s the selective hydrolysis of the 5,6-isopropylidene group of LXXXVII. It has been widely observed that for 1,2:5,6-di-0-isopropylidene furanose derivatives the 5,6-isopropylidene ketal i s hydrolyzed much more rapidly than the 1,2 isopropylidene moiety (137). The hydrolysis of 1,2,5:6-di-O-isopropylidene-a-D-glucofuranose [LXIX] provides an excellent example of the selectivity of this reaction. In this instance the 5,6-isopropylidene group i s cleaved with dilute hydrochloric acid -54-some eighty times faster than the 1,2-isopropylidene (138). The 5,6-isopropylidene group has been selectively hydrolyzed using a variety of acidic conditions (138,139,140). In the present work this group was removed using an aqueous methanol solution containing sulfuric acid as in the case of the preparation of 1,2-0-isopropylidene glucose (140). The hydrolysis was conducted at room temperature for 7 hours to afford the mono-isopropylidene compound XCIV as a syrup in 88% yield. The nmr spectrum of this compound showed the presence of two hydroxyl groups and only two methyl groups (at 8.40 and 8.64 T) belonging to the 1,2-isopropylidene group. The L sugar 3-C_-cyanomethyl-3-deoxy-l,2-0_-isopropylidene-(3-L-lyxofuranose [XCVI] was obtained from mono-isopropylidene compound XCIV by sodium periodate oxidative cleavage to the 5-aldehydo compound followed by sodium borohydride reduction of the 5-aldehydo group (21). This series of reactions i s very frequently used in carbohydrate chemistry to prepare sugars having one carbon less than the starting compound. The best yields are usually obtained by reducing the intermediate aldehydo compound without isolation, as was done in this instance. That the cyanomethyl branched-chain of XCVI had survived the above operations was confirmed by the i r spectrum (C=N 2245 cm "*"). Reduction of the cyanomethyl branched-chain sugar XCV to the corresponding acetamidoethyl compound XCVI was accomplished by catalytic hydrogenation. As hydrogenation is technically simpler than lithium aluminum hydride reduction, where possible i t is the method of choice for reduction of the cyano moiety. In this instance the hydrogenation medium chosen was an acetic anhydride-ethanol 1:1 mixture. -55-As the 1,2-isopropylidene group i s comparatively acid stable, there was no danger of hydrolysis in this weakly acid medium. In the presence of acetic anhydride the N-acetyl derivative i s formed in situ preventing the previously discussed coupling reactions. Accordingly XCV was hydrogenated at 60 psi over platinum oxide for four and a half hours. A t i c examination of the reaction mixture after this time indicated that no starting material remained and showed the presence of only one product. Spectral data (nmr 3.8-4.3 T, broad N-H, 8.02 T singlet, N acetyl) confirmed the reduction had taken the expected course to afford crystalline 3-C-(2'-acetamidoethyl)-3-deoxy-l,2-0-isopropylidene-g-L-lyxofuranose [XCVI] in 93% yield. 3. Synthesis of branched-chain carbamoylmethyl sugars In order that the variety of nitrogenous deoxy branched-chain sugars available might be increased, the preparation of carbamoyl branched-chain sugars was also examined. Although no branched-chain carbamoyl sugars have been reported as yet, the nucleoside antibiotic gougerotin fXCVII] (141a) i s known to contain a C-6' carbamoyl group and 5'-carboxyamide adenosine analogs are used in the treatment of circulation disorders (141b). [XCVI] -56-3.1 3-C-Carbamoylmethyl-3-deoxy-l ,2:5, 6-di-O-isopropylidene-a-D-allofuranose [C] One method which has been u t i l i z e d in carbohydrate chemistry for the preparation of amides involves ammonolysis of an ester by reacting i t with l i q u i d ammonia in the presence of ammonium chloride. Heynes and Baltes (142) had used this procedure to convert the C-6 methyl ester of compound XCVIII to the C-6 amide XCIX. Fortunately a 3-C-carbomethoxymethyl sugar XXXIX (22) had already been prepared in this laboratory (p. 18 ) so therefore i t was only necessary to apply the above ammonolysis to this compound to obtain the desired carbamoylmethyl branched-chain sugar 3-C_-carbamoylmethyl-3-deoxy-l,2:5,6-di-CJ-isopropylidene-a-35-allofuranose [C] . Surprisingly under the conditions used by Heynes and Baltes (heat-ing i n a sealed tube at 50° for 6 hr in liquid ammonia containing ammonium chloride) only about 10% conversion (as evidenced by t i c ) of XXXIX to C occurred. It was found that to obtain satisfactory yields i t was necessary to allow the reaction to proceed at 60° for 24 hr. Under these conditions XXXIX was converted to C in 76% yield (Equation 19). As an alternate route to carbamoylmethyl branched-chain sugars MeO MeO 0 •Me 0 Me Me Me [XCVIII] [XCIX] -57--58-the base catalyzed reaction of cyanomethyl branched-chain sugar LXXXVI with hydrogen peroxide was examined. Although this reaction has been known for sometime (143) as a means of converting n i t r i l e s to the corresponding amides, to the best of our knowledge this reaction has not been previously applied to a carbohydrate n i t r i l e . When an ethanol solution of n i t r i l e LXXXVI was reacted with hydrogen peroxide and 6 N sodium hydroxide at 50° for 6 hr, i t was smoothly hydrolyzed to the carbamoylmethyl sugar C (70% yield) (Equation 19) identical (by nmr, i r , mixed m.p.) to the compound prepared via ammonolysis of ester XXXIX. As well as providing two routes to the carbamoyl methyl sugars, the above procedures provide a means of interrelating the products of two different Wittig reactions (Equation 19). As both LXXVI and XXXIX give the same amide both these compounds must have the same relative configuration. Since in connection with another problem an x-ray study of a derivative of compound XXXIX is underway, i t was desireable to have a way of chemically relating the two Wittig products LXXXVII and XXXIX As a third route to these carbamoylmethyl branched-chain sugars the photoaddition of formamide to the exocyclic unsaturated sugar XX was undertaken. The preparation of compound XX and the mechanism of this photoamidation have already been dealt with in the Introduction (p. 12 and p. 24 respectively). When compound XX was irradiated (X > 300) for seven hours in an oxygen-free mixture of formamide, tertiary butanol and acetone products were isolated from the reaction mixture (Equation 20). The major product (50% yield) proved to be the carbamoylmethyl branched-chain -59-[CI] sugar C i d e n t i c a l ( i r , nmr, mixed m.p.) to the product i s o l a t e d from the l a s t two r e a c t i o n s . In view of the f a c t that photoaddition of formamide to terminal o l e f i n s i s known to take place i n an a n t i -Markovnikov manner (81), and as t h i s r e a c t i o n i s known to be influenced by s t e r i c features of the o l e f i n (82) (p. 23 ), i t i s not too s u r p r i s i n g that the a d d i t i o n of formamide to XX takes place i n a s t e r e o s e l e c t i v e manner to give only the a l i o carbamoylmethyl a d d i t i o n product of XX. - 6 0 -Th e minor product CI (11%) was not characterized but was tentatively assigned the structure CI on the basis of nmr evidence: H-2 appears as a t r i p l e t indicating an alio configuration, there is one exchangeable proton present in the molecule indicating the l i k e l y presence of a hydroxyl group, and there are six methyl signals, four belonging to the isopropylidene groups and presumably two for the methyl groups in the branched-chain. The formation of acetone addition in this reaction has been noted previously (81). 4. Synthesis of nitrogenous branched-chain sugars having a single  carbon in the branched-chain Concommitant with the program to develop routes to nitrogenous branched-chain sugars having two carbons i n the branched-chain, the preparation of analogous compounds having only a single carbon in the branched-chain was attempted. 4.1 Photoamidation of 4,6-di-0-acetyl-2,3-dideoxy-a-D-erythro-hex-2-enopyranoside [CII] As the photoaddition of formamide to the exocyclic methylene sugar C just described had been relatively successful, i t was decided to apply the same reaction to the unsaturated sugar 4,6-di-0_-acetyl-2,3-dideoxy-ct-D-erythro-hex-2-enopyranoside [CII] . This compound was prepared from triacetyl-D-glucal by the method of Ferrier and Prasad (144). It was anticipated that the photoaddition of formamide to this compound would result in 2- and/or 3-C-carbamoyl-2,3-dideoxy pyranosides -61-(Equation 21) which could hopefully be differentiated on the basis of their nmr spectra. Accordingly olefin CII dissolved in a de-oxygenated (21) mixture of formamide, t_-butanol and acetone was irradiated (X > 300) for 9 hr. After this time t i c examination showed that no starting material remained and that there were two product spots. These two components were separated by chromatography on s i l i c a - g e l and examined. The minor component (about 12% of the total product) was judged on the basis of i t s nmr spectra to be a mixture of acetone addition products and was not further investigated. The major component, a syrup amounting to about 88% of the total product mixture had the following characteristics: (a) The i r spectrum contained in addition to the usual C-H absorptions an absorption at 3400 cm 1 and a strong absorption at 1660 cm"1. (b) The nmr spectrum showed a broad low f i e l d 2 proton absorption at 3.47 T and a total of 21 protons present. -62-(c) The elemental a n a l y s i s was consistent with that expected f o r the a d d i t i o n of the elements of formamide to o l e f i n CII. From the above data i t was concluded that t h i s product was a 1:1 formamide:olefin adduct. However, as the nmr spectrum showed s e v e r a l superimposed t r i p l e t s f o r the methyl peaks of the e t h y l glycoside, i t was assumed that t h i s product was a mixture of isomers. That t h i s product was a mixture was f u r t h e r substantiated by f i n d i n g that the de-acetylated (methanolic sodium methoxide) t r i m e t h y l s i l y l a t e d d e r i v a t i v e s (145) showed the presence of four components i n about equal amounts when examined by gas l i q u i d chromatography. Despite repeated chromatography no s i n g l e isomer could be separated i n a pure form. From the above i t would appear that the major product of the photo-amidation of CII i s a mixture of a l l four p o s s i b l e formamide a d d i t i o n products C i l i a , CHIb, CIVa, and CIVb. As i t was apparent that i t R' [ C i l i a ] R = CONH2, R' = H [CHIb] R' = H, R = CONH2 [CIVa] R = CONH2, R' = H [CIVb] R' = H, R = CONH2 -63-would be very d i f f i c u l t to prepare useful amounts of a single pure carbamoyl branched-chain sugar by this method, work along these lines was discontinued. In contrast to these results as indicated in the Introduction (p. 21 ) other workers in this laboratory have been able to apply this reaction with some success to the unsaturated sugars LX LXI. It i s evident that photoaddition of formamide to carbohydrate Me [LX] [LXI] olefins i s most useful when steric features of the olefin result in the preferential formation of a single product as was the case with compound XX. 4.2 Addition of nitromethane to 1,2;5,6-di-0-isopropylidene-ct-g-ribo-hexofuranos-3-ulose [XVIII] A second route investigated leading to nitrogenous branched-chain sugars having a single carbon in the branched-chain was the addition of nitromethane to carbohydrate ketoses. As was mentioned in the Introduction (p. 21 ) although the addition of nitroparaffins to -64-carbohydrates had been extensively i n v e s t i g a t e d , when t h i s work was begun there had been only two reports (78,79) (one of which (78) was from t h i s laboratory) on the a d d i t i o n of nitromethane to carbohydrate ketoses to a f f o r d branched-chain sugars. The manner i n which t h i s r e a c t i o n was used i s i l l u s t r a t e d i n equation 22. Condensation of ketose XVIII with the carbanion prepared R = N0 o or NH -65-from nitromethane and sodium methoxide afforded the branched-chain nitro sugar 1,2:5 ,6-di-C_-isopropylidene-3-C_-nitromethyl-a-D-glucof uranose [CV]. It was planned to use this compound to prepare deoxy nitromethyl (by acetylation of the 3° hydroxyl group followed by a Schmidt-Rutz reaction (74) and reduction of the nitro-olefin double bond) and deoxy aminomethyl branched-chain sugars (Equation 23). However, shortly after the preliminary results concerning the addition of nitromethane to XVTII were published (148), a brief communication from Albrecht and Moffatt (80) reported their results on condensing nitromethane with the same ketose XVIII, the conversion of the i n i t i a l nitromethane condensation product into deoxy nitromethyl and aminomethyl branched-chain sugars, and the conversion of these compounds into branched-chain sugar nucleosides. In view of this, further work here along these lines was discontinued and no further attempts were made to prepare branched-chain sugars of this type. 5. Nucleoside synthesis Having synthesized the 3-deoxy, two carbon nitrogenous branched-chain sugars just described, the next step was to use these compounds to prepare the corresponding branched-chain sugar nucleoside derivatives. In order to u t i l i z e these compounds in standard nucleoside syntheses, i t was necessary to convert them f i r s t into their l,2-di-0-acetyl derivatives. As the procedures for preparation of amino sugar nucleosides had been extensively investigated (149), i t was decided to attempt f i r s t the preparation of an amino branched-chain sugar nucleoside using the acetamidoethyl branched-chain sugar XCIII. - 6 6 -5.1 Attempted acetolysis of 3-£-(2'-acetamidoethyl)-5-0-benzyl-3-deoxy-l,2-0_-isopropylidene-a-D-ribofuranose [XCIII] The preparation of the blocked branched-chain sugar 3-C-(2'-acetamidoethyl)-l,2-di-0_-acetyl-5-0-benzyl-3-deoxy-ribofuranose [CVI] was f i r s t attempted. It was hoped that acetolysis by the normal procedure (150) using acetic acid, acetic anhydride, and sulfuric acid, would convert the 1,2-isopropylidene compound XCIII into the correspond-ing 1,2-di-acetyl derivative CVI (Equation 24). (24) [XCIII] [CVI] Unfortunately, under these acetolysis conditions the C-5 benzyl ether group was cleaved. This was evidenced by the nmr spectrum of the major product which indicated that no aromatic protons were present in the molecule. Cleavage of the benzyl ether moiety was not desirable as this would allow the branched-chain sugar to revert to the unwanted but more stable pyranose configuration. That the benzyl ether was unstable to these acetolysis conditions was not entirely unexpected in view of the findings of Allerton and Fletcher (151) that -67-benzyl ethers are r e a d i l y removed i n a c e t o l y s i s media. A c e t o l y s i s t r i a l s , on the model compound LXXVII, i n which the percentage of s u l f u r i c a c i d i n the r e a c t i o n medium was v a r i e d , revealed that the C-5 primary benzyl group of LXXVII was acetolyzed at about the same rate as the 1,2-isopropylidene k e t a l . In view of t h i s i t was decided to prepare CVI by a two-step procedure. F i r s t XCIII was reacted with 90% t r i f l u o r o a c e t i c a c i d to hydrolyze the isopropylidene k e t a l (152), and then the r e a c t i o n product was a c e t y l a t e d with a c e t i c anhydride and p y r i d i n e (Equation 25). This procedure r e s u l t e d i n the formation of a complex product mixture. A major component of t h i s mixture was t e n t a t i v e l y i d e n t i f i e d as the n i t r o g e n heterocycle CVII. This conclusion was based on the observations that the nmr spectrum of t h i s compound contained s i g n a l s corresponding to one benzyl and four acetate groups and that the i r spectrum showed no NH absorption, and a low wavelength carbonyl absorption (1640 cm ^) t y p i c a l of 3° amides. The nmr spectrum of the desired 1,2-0-acetyl product CVI would be expected to show the presence of only three a c e t y l groups. -68-The formation of the nitrogen heterocycle CVIII was rationalized by postulating that after hydrolysis an equilibrium was set up between [CVII] the oxygen and the nitrogen heterocyclic compounds; subsequent acetylation of this mixture lead to compounds having both nitrogen and oxygen as the ring heteroatom (Equation 25). The rearrangement of C-4 and C-5 amino and amido monosaccharides to nitrogen hetero-cycles has been well studied (153). Normally where there i s a competi-tion for ring formation between a hydroxyl group and an acetamido group the oxygen heterocycle is formed predominantely even i f i t has the -69-thermodynamically less favoured five membered ring structure (153). Thus i t had been anticipated that CVII would not be a major constituent of the product mixture. Since this was not so, further attempts to prepare CVI were abandoned. 5.2 Conversion of 3-C_-cyanomethyl-3-deoxy-l,2:5,6-di-0_-isopropylidene-a-g-allofuranose [LXXXVI] into l,2-di-0_-acetyl-5,6-di-0-benzoyl-3-C_-cyanomethyl-3-deoxy-8-D-allofuranose [CX] and 1,2-di-O-acetyl-5-0-benzoyl-3-C-cyanomethyl-3-deoxy-8-D-ribofuranose [CXIII] Since the preparation of amino branched-chain sugar derivatives suitable for nucleoside synthesis had been unsuccessful, i t was decided to prepare an appropriate derivative from the cyanomethyl branched-chain sugar LXXXVI. To prepare the hexose derivative required the following steps: (1) selective hydrolysis of the 5,6-isopropylidene group; (2) blocking of the free 5,6-hydroxyl groups as the benzoate esters; (3) hydrolysis of the 1,2-isopropylidene group; (4) acetylation of the 1,2-hydroxyl groups. To prepare the pentose derivative required a modifiction of this procedure in that after step (1) a sodium periodate oxidative cleavage followed by sodium borohydride reduction was carried out to remove the C-6 hydroxymethyl group. The resultant pentose compound was then benzoylated and subjected to steps (3) and (4). The reaction scheme representing these steps is shown in equation 26. -70-1. 10. 4 [CXI] [CXII] [CXIII] -71-The selective hydrolysis of a 5,6 isopropylidene ketal and the removal of a C-6 hydroxymethyl group by oxidative cleavage, followed by reduction, were discussed previously in the preparation of compound XCVI and w i l l not be considered in detail again. Suffice i t to say that treatment of LXXXVI with an aqueous methanol solution containing a small amount of sulfuric acid selectively hydrolyzed the 5,6-isopropylidene group of LXXXVI to afford CVIII as a syrup in nearly quantitative yield. Compound CXI was obtained as a crystalline solid in 90% yield by cleavage of the 5,6-hydroxyl groups of CVIII with sodium periodate followed by reduction of the 5-aldehydo group with sodium borohydride (21). The hydroxyl groups of compounds CVIII and CXI were benzoylated using the method of Molau (154). In this procedure the compound to be benzoylated i s dissolved in anhydrous benzene to which i s added only a slight excess of the amount of benzoyl chloride necessary for esterification and two equivalents of pyridine. Work up consists of f i l t e r i n g the reaction mixture through a short column of grade II alumina (a ratio of about 5:1 alumina to compound was used), evaporation of the f i l t r a t e and removal of traces of pyridine by azeotroping with toluene. Applying this procedure to compound CVIII afforded a 90% yield of crystalline benzoate CIX and a 93% yield of crystalline benzoate CXII from CXI. The chief advantage of this procedure l i e s in the fact that no opportunity for benzoic anhydride contamination of the product arises. Standard benzoylation procedures in carbohydrate chemistry sometimes result in the contamination of the benzoylated product with benzoic anhydride produced during the addition of water during work up (155). -72-Attempted acetolysis (150) of the 1,2-isopropylidene ketal of CIX using a mixture of acetic acid, acetic anhydride and sulfuric acid for twenty four hours at room temperature led to a complex mixture of products. The major component of this mixture (about 30% based on starting material) was isolated by column chromatography on s i l i c a . Elemental analysis of this material showed that i t contained no nitrogen, indicating that the n i t r i l e group on the branched-chain was apparently unstable to these conditions. It was therefore decided to proceed as before and use a two-step procedure; f i r s t hydrolysis of the isopropylidene ketal with t r i f l u o r o -acetic acid followed by acetylation with acetic anhydride in pyridine. Several exploratory runs using different acid concentrations were performed i n order that the optimal conditions for hydrolysis might be found. For compound CIX reaction with 80% aqueous trifluoroacetic acid for 45 minutes at room temperature was found to give the best yield; for compound CXII i t was found to be more advantageous to use a greater percentage of acid (90%) and run the hydrolysis for a shorter time (22 minutes). Acetylation, using acetic anhydride and pyridine, of the hydrolysis product from CIX gave after chromatography on s i l i c a gel l,2-di-0-acetyl-5,6-di-0-benzoy1-3-C-cyanomethy1-3-deoxy-g-D-allofuranose [CX] as a crystalline solid in 69% yield. The anomeric (H-l) hydrogen of this compound appeared in the nmr spectrum as a singlet at 3.77-r . As there was no measurable coupling between H-l and H-2 i t was concluded that a trans relationship existed between these two protons and that therefore CX had a 8 anomeric -73-configuration. Because of the conformational mobility of furanose systems (125a) i t i s not generally possible to definitely assign anomeric configurations on the basis of H-l, H-2 coupling constants alone. However, in instances where there is no appreciable coupling between two neighbouring protons i t can be f a i r l y safely assumed that a trans relationship exists between them (156). Acetylation of the hydrolysis product from CXII, using the same conditions as above, gave after column chromatography on s i l i c a gel two components. The major component isolated as a crystalline solid in 69% yield proved to be the expected acetylated cyanomethyl branched-chain sugar 1,2-di-0-ace tyl-5-0-benzoyl-3-C_-cyanome thy 1-3-deoxy-g-g-ribof uranose [CXIII], The g-configuration was tentatively assigned as before from the nmr spectrum (H-l appeared as a singlet at 3.80 T ) . The minor product, isolated in about 5% yield as a crystalline solid, gave the following data upon examination: (1) the nmr spectrum indicated that only one benzoate and one acetate ester were present; (2) the infrared spectrum showed no n i t r i l e absorption and three carbonyl absorptions. From the above i t was concluded that this compound was the lactone l-0-acetyl-5-0_-benzoyl-3-C_-carboxymethyl-2,3-Y-lactone-3-deoxy-g-D-ribofuranose [CXV]. The elemental analysis of this compound was found to be in agreement with the proposed structure. The B-anomeric configuration was again assigned on the basis of the nmr spectrum (H-l was observed as a singlet at 3.6 T ) . -74-It i s probable that the above side product arose as is i l l u s t r a t e d in equation 27. During the trifluoroacetic acid hydrolysis of compound CXII the n i t r i l e group in the branched-chain underwent p a r t i a l hydrolysis to the carboxylic acid which lactonized to afford compound CXIV. Acetylation of this material would then lead to CXV. 0 0 [CXII] [CXIV] [CXV] 5.3 6-Chloro-9-(2'-0-acetyl-5', 6'-di-O-benzoyl-3'-C-cyanomethyl-31-deoxy -B-D-allofuranosyl)-purine [CXVI] and 6-chloro-9-(2'-0-acetyl-5'-0-benzoy1-3 1-C-cyanomethyl-3'-deoxy-B-D-ribofuranosyl)-purine [CXVIII] As i t was desired to obtain 6-^,N_-dimethylaminopurine nucleoside derivatives of the previously described cyanomethyl branched-chain sugars, the methods used for the preparation of dimethylaminopurine nucleosides were examined. B.R. Baker and coworkers (157), in their classic synthesis of puromycin, formed the carbon nitrogen glycosidic bond by condensation of the titanium-amino sugar complex with -75-chloromercuri-2-methylmercapto-6-dimethylaminopurine. Raney nickel desulfurization of the purine then gave the 6-dimethylaminopurine nucleoside (Equation 28). While a 6-dimethylaminopurine nucleoside was obtained by this method several steps were required and the yield in the desulfurization was only f a i r . AcNH OBz AcNH OBz An alternate route to these nucleosides has been devised by R.K. Robins (158). .Here a 6-mercaptopurine nucleoside (159) was f i r s t prepared and then this compound was reacted with dry chlorine gas to afford the 6-chloropurine nucleoside. This chloropurine nucleoside was then converted to the 6-dimethylaminopurine nucleoside with aqueous dimethylamine. The chief disadvantage of this procedure is the number of manipulations of the base required to obtain the -76-6-dimethylaminopurine compound. This disadvantage has been overcome by the relatively recently developed fusion procedure (106). Using this method the 6-chloro-purine nucleosides were prepared directly by fusion of a C-l acetylated sugar with 6-chloropurine. The 6-dimethylamino functionality was then introduced as above, by reacting the 6-chloropurine nucleoside with aqueous dimethylamine. In view of i t s simplicity, and the fact that the nucleosides prepared are free of mercury contamination, this procedure was selected for the preparation of dimethylaminopurine nucleosides from the cyanomethyl branched-chain sugars. Accordingly CX was fused with 6-chloropurine at 155-160° under reduced pressure for 45 minutes. Chromatography on s i l i c a gel afforded as the only nucleoside product 6-chloro-9-(2 1 -0_-acetyl-5' ,6'-di-0_-benzoyl-3' -C-cyanomethyl-3' -deoxy-g-D-allofuranosyl) -purine [CXVI] in 69% yield. Similarly fusion of CXIII with 6-chloropurine under the same conditions gave 6-chloro-9-(2'-0_-acetyl-5'-0_-benzoyl-3'-C-cyanomethy1-3'-deoxy-g-D-ribofuranosyl)-purine [CXVIII] in 66% yield. The 0-anomeric configuration was tentatively assigned to these nucleosides on the basis of their small H-l', H-2' coupling constants (CXVI J , , = 2 Hz; CXVIII J 1 , , =1 Hz). This assignment was later confirmed by the circular dichroism (cd) spectra of the unblocked nucleosides. A thorough examination of a l l the reaction products did not reveal the presence of any other nucleoside indicating as expected that there was no appreciable formation of the a-anomer. -77-Cl [CXIII] R = H [CXVIII] R = H 5.4 6-N, N-Dimethylamino-9-(3' -C-N,_N-dimethylcarbamoylmethyl-3' -deoxy-g-D-allof uranosyl)-purine [CXXI] and 6-N^,N-dimethylamino-9-(3!-£-N,N-dimethylamino-9-(3,-£-N,N-dimethylcarbamoylmethyl-3'-deoxy-g-D-ribofuranosyl)-purine [CXXII] The second step in the preparation of the 6-dimethylaminopurine nucleosides was the reaction of the above 6-chloropurine blocked nucleosides with aqueous dimethylamine. This procedure was intended to remove the ester hydroxyl protecting groups and replace the 6-chloro group by a dimethylamino functionality thereby resulting in the formation of the cyanomethyl branched-chain nucleosides CXIX and CXX. When the chloropurine nucleoside CXVI was reacted for four hours with an aqueous methanol solution of dimethylamine and the products were separated by chromatography on s i l i c a gel a single crystalline nucleoside was isolated in 78% yield. -78-Th e nmr spectrum In dimethyl sulfoxide-d^ of this nucleoside is reproduced in Figure 2. Some features of this spectrum were readily interpreted: the two low f i e l d singlets at 1.69 and 1.88 T were assigned to the H-2 and H-8 protons of the heterocyclic base; the doublet at A.08 T is the H-l' signal; the two doublets at A.28 and A.6A T which disappeared on addition of deuterium oxide were assigned to the secondary hydroxyl groups of C-2' and C-5', not necessarily respectively; a third hydroxyl group i s superimposed on the C-2' signal at 5.A8 x; the large singlet at 6.7A x was assigned to the six protons of the N,N-dimethyl group of the heterocyclic base, a small water peak was superimposed on this signal. However, the two singlets at 7.02 x and 7.20 x which integrate for three protons each were not consistent with the expected nmr spectrum of the cyanomethyl branched-chain sugar nucleoside CXIX. N(Me) CH„ OH I 2 CN [CXIX ] R = CH20H [CXX] R = H T T 2 I i ' I I I i I i i I I I i I I I I 1 I H-2 H-8 i i i I i i i i I i i i i I i i i i I i i i i I i i i i | i i i i | i i i i | i i i , i I i i 11 r ; i i i' i r i i i ii r i i i II i i I I i n i i ' i i, N(Me), HO—CH purine -N(Me), [CXXI] NMe H-l' I I I I I I" NMe DMSO \J \*J Vu» I ' 1 ' ' 1 i I ' l l ! I 1 1 I I i i i i i I i i i i i i i i i I ' i i i i i ! i i I i t i i I i i I i i l . 1 : : 1 i 1 i ' i I i i ' i l : i i i I I ' l l ! I Figure 2. Proton magnetic resonance spectrum at 100 MHz in dimethyl sulfoxide-d 6 of 6-N,N-dimethylaniino-9_(3'_C-N,N-climethylcarbamoylmethyl-3'-deoxy-3-D-allofuranosyl)-purine [CXXI]. -80-A point to note here was the method of assigning the hydroxyl signals in this spectrum. In dimethyl sulfoxide i t is possible to differentiate between primary, secondary and tertiary hydroxyl groups on the basis of their couplings with adjacent protons. Primary hydroxyl groups generally appear as a t r i p l e t because of coupling to the two adjacently methylene protons; secondary hydroxyl groups appear as a doublet, and tertiary hydroxyl groups appear as a singlet. This simple method of differentiating hydroxyl groups was frequently used in this work. Further investigation of the above nucleoside provided the following information: the ultraviolet absorption (A = 275 nm) indicated v max that the position of attachment of the base to the sugar was at N-9 (105); the circular dichroism (cd) spectrum showed a negative Cotton effect confirming that this nucleoside had the expected g-anomeric configuration (104); the i r spectrum contained no n i t r i l e absorption but did show an unexpected carbonyl absorption (1610 cm ; the nmr spectrum in deuterochloroform showed the molecule contained about 26 hydrogens; the mass spectrum gave a value of m/e = 394 for the highest non-isotopic fragment, and no fragment at m/e = 348, which would correspond to the molecular ion of CXIXa was found. On the basis of the above data i t was concluded that this compound was the dimethyl-carbamoylmethyl branched-chain sugar nucleoside 6-N,N_-dimethylamino-9-(3' -C_-N,N_-dime thylcarbamoylme thy 1-3 '-deoxy-g-D-allof uranosyl) -purine [CXXI]. That this structure for CXXI was in agreement with the observed data is readily apparent. The two singlets at 6.78 and 6.86 T were assigned to the N,N-dimethyl moiety in the branched-chain; the carbonyl absorption -81-at 1610 cm is typical of tertiary amides and the mass spectral m/e value of 394 was consistent with the molecular formula C.-,H. ,N,0,_ 17 26 6 5 obtained from this structure. Reaction of the pentose chloropurine nucleoside CXVIII with the same methanol aqueous dimethylamine mixture afforded the analogous pentose nucleoside 6-N,N-dimethylamino-9-(3'-C_-N,N-dimethylcarbamoyl methyl-3'-deoxy-i3-D-ribofuranosyl)-purine as a syrup in 72% yie l d after chromatography on s i l i c a gel. Although CXXII appeared homogeneous by nmr, t i c and paper chromatography, i t could not be obtained crystalline, nor could a satisfactory elemental analysis be obtained. Consequently this compound was characterized as i t s 2',5'-di-O-acetyl derivative. To further verify the structure of nucleoside CXXII, the previously described sodium periodate, oxidative cleavage sodium borohydride reduction (59) was used to remove the C-6'-hydroxymethyl group from [CXXI] R = CH20H [CXXII] R = H -82-nucleoside CXXI. This procedure afforded a homogeneous syrup i n 69% y i e l d i d e n t i c a l by i r and nmr with the product obtained from the r e a c t i o n of aqueous dimethylamine with chloropurine nucleoside CXVTII. To account f o r the formation of the above nucleosides i t was i n i t i a l l y postulated that i n the b a s i c aqueous dimethylamine medium the n i t r i l e group on the branched-chain underwent hy d r o l y s i s to give the carboxylic a c i d , followed by l a c t o n i z a t i o n and addi t i o n of dimethylamine to the lactone (Equation 29). The hy d r o l y s i s of n i t r i l e s ? H2 C=N f *0H OH VH2 C0 2H OH -H20 (29) f«2 0=C \ N(Me), OH ||*-NH(Me), 0 to carboxylic acids i s known to be catalyzed by both acids and bases; however, as a ru l e h y d r o l y s i s proceeds f a s t e r i n a c i d i c media (160). It should be r e c a l l e d here that i n the t r i f l u o r o a c e t i c a c i d h y d r o l y s i s of the 1,2-isopropylidene k e t a l of CXII, that a f t e r a c e t y l a t i o n a small amount of lactone CXV was recovered. -83-BzOCH BzOCH 0 OAc Me 0 Me / C II 0 [CXII] [CXV] In order to examine the reactivity of the n i t r i l e group in these branched-chain sugars towards aqueous dimethylamine, compounds LXXXVI CVIII and XCIV were subjected to the same hydrolysis conditions as the above chloro nucleosides. In each instance there was no detectable hydrolysis (even after twenty-four hours) and the starting materials were recovered unchanged. Furthermore, i t was found that the n i t r i l e group of the chloro-nucleoside CXXIII (161) under the same conditions was not hydrolyzed and only the branched-chain cyanomethyl nucleoside CXXIV was obtained. Obviously, therefore, the hydrolysis of the n i t r i l e moiety in branched-chain nucleosides CXXI and CXXII was unusually f a c i l e . Although the alkaline hydrolysis of n i t r i l e s having adjacent hydroxyl groups has not been extensively studied some example which could have a bearing on these results were found in the literature. For example, the addition of hydrogen cyanide (usually via aqueous sodium cyanide) to reducing sugars (Kiliani syntheses (162), equation 30) is a classical method for extending sugar chains. After addition of the -84-hydrogen cyanide the alkaline solution of n i t r i l e was heated to 60-100° to effect hydrolysis. It has been found, however, that for some sugars (e.g. 2-deoxy-ribose and ribose (163) ) hydrolysis of the n i t r i l e proceeded spontaneously under very mild conditions (room temperature in a carbonate buffered solution). -85-H 0 \ ' I R HCN C=N l CHOH R OH C02H CHOH I R (30) Another pertinent instance of n i t r i l e hydrolysis was found in the addition of cyanide to epoxide CXXV (164). When this epoxide was heated to 100° in aqueous potassium cyanide solution no cyano addition products (e.g. CXXVI) were isolated, but rather only the lactone CXXVII. In rationalizing this result the authors postulated that the intermediate n i t r i l e addition product CXXVI underwent intramolecular hydrolysis by attack of the C-5 alkoxide anion on the C-3 cyano group (Equation 31). [CXXV] [CXXVI] [CXXVII] If i t i s assumed that intramolecular hydroxyl group participation can aid in the hydrolysis of a n i t r i l e group i t is possible to rationalize the facil e hydrolysis of the n i t r i l e group in nucleosides CXVI and CXVIII as the C-2 hydroxyl ion formed after acetate cleavage, is in a position to participate in hydrolysis of the n i t r i l e (Equation 32). However, this theory does not account for the -86-resistance of the n i t r i l e group compounds XCIV and CXXIV to hydrolysis, as i t would be expected that intramolecular hydroxyl group participation leading to hydrolysis of the n i t r i l e moiety would be possible with these compounds also. Apparently there are other factors involved here and the f u l l explanation awaits further investigation. [CXVI] R - CH2OBz [CXVIII] R = H 5.5 6-N,N-Dimethylamino-9-(3'-C-carboxymethyl-21,3'-y-lactone-3-deoxy-B-D.-ribofuranosyl)-purine [CXXVIII] In an attempted purification of the branched-chain nucleoside CXXII i t was sublimed at 200-205° and 0.1 mm. This procedure afforded a crystalline nucleoside in 73% yield. It was immediately obvious, however, that during sublimation nucleoside CXXII had undergone decomposition as the i r spectrum of the sublimed nucleoside had a -87-distinctly different carbonyl absorption (1770 vs. 1610 cm for CXXII) and the nmr spectrum in DMSO-d^  indicated that there was now only a single (primary) hydroxyl group present and that the molecule no longer contained the N.,N_-dimethyl group on the branched-chain. That nucleoside CXXII had undergone deamination was further substantiated by the finding that the highest nonisotopic fragment in the mass spectrum occurred at m/e 319. This implied that the new nucleoside had a molecular weight 45 units less than CXXII corresponding to the loss of dimethylamine from CXXII. On the basis of the above information i t was determined that this new compound was the novel lactone nucleoside 6-.N ,N-dimethylamino-9- (3' -C_-carboxymethyl-2 ', 3'-y-lactone-3-deoxy-g-D-ribofuranosyl)-purine [CXXVIII]. The deamination of CXXII under these conditions was not surprising, as amides having a neighboring hydroxyl group which can participate in the displacement of an amine from an amide have been shown to be easily deaminated (165) in acidic, neutral, or basic media. Presumably, the C-2' hydroxyl group of branched-chain tertiary amide nucleoside CXXVIII (Equation 33) assists in displacement of dimethylamine to give lactone CXXVIII. It is also possible that this reaction was further f a c i l i t a t e d by intermolecular catalysis by the heterocyclic base of the nucleoside. -88-5.6 Conversion of 6-N,N-dimethylamino-9-(3'-C-carboxymethyl-21,3'-y-lactone-3-deoxy -B-D-ribofuranosyl)-purine [CXXVIH] to 6-N,N-dimethylamino-9- (3* -C_-21,N-dimethylcarbamoylmethyl-3 '-deoxy-B-D-ribof uranosyl)-purine [CXXII], and 6-N,N_-dimethylamino-9- (3'-C-carbamoylmethyl-3'-deoxy-B-D-ribofuranosyl)-purine [CXXIX] and 6-N,N-dimethylamino-9-(31-C_-carbamoylmethyl-N-glycine ethyl ester-3'-deoxy-g-D-ribofuranosyl)-purine [CXXX] The lactone nucleoside CXXVIII proved to be a very useful compound for preparing amido branched-chain sugar nucleosides. The reaction of this nucleoside with a variety of amines is illustrated in equation 34. -89-(34) CH 2 OH 0=C %NHCH CO Et DMP = 6-N,N-dimethylaminopurine [CXXX] Reaction of CXXVIII with dimethylamine for four hours at zero degrees centigrade afforded the N,N-dimethylcarbamoylmethyl branched-chain nucleoside CXXII as a syrup i n quantitative yield. This compound was identical by nmr and i r with the compound prepared by reaction of dimethylamine with chloropurine nucleoside CXVIII. Using ammonia (166) in place of dimethylamine afforded the carbamoylmethyl nucleoside CXXIX as a crystalline solid i n 95% yield. As the above condensation of lactone nucleoside CXXVIII with ammonia -90-and dimethylamine were successful i t was decided to undertake the preparation of a peptide nucleoside using this compound. Lately interest has increased in nucleosides containing non-hydroxyl linked peptides. This has come about partly because commercially important antibiotics such as the polyoxins (167), gougerotin (141), blasticidin S (168), and puromycin (2a) have been shown to be nucleoside peptide derivatives with the aminoacyl moiety attached through an amino group of the sugar, and partly because amino acids which are not removed by the usual deproteinization procedures have been found in highly purified samples of ribonucleic acid (169) and deoxy ribonucleic acid (170) . The group of investigators led by R.K. Robins at the ICN Nucleic Acid Research Institute have been at the forefront in the preparation of these nucleoside peptides. These workers have made use of the active ester (171) and N_,N'-dicyclohexylcarbodiimide (172) methods of peptide synthesis to prepare various 5'-N-aminoacyl-5'-amino-ribofuranosyl purine nucleosides (173). The method chosen here to prepare a peptide nucleoside, analogous with the above reactions of the lactone nucleoside with amines, was to simply condense glycine ethyl ester (174) with lactone nucleoside CXXVIII (Equation 34). Thus a mixture of glycine ethyl ester and nucleoside CXXVIII in dimethylformamide were stirred at room temperature for 30 hours. After removal of the solvent and chromatography on s i l i c a gel the peptide nucleoside 6-N_,N-dimethylamino-9-(3'-C-carbamoylmethyl-N-glycine ethyl ester-3 1-deoxy-g-D-ribofuranosyl)-purine [CXXX] was isolated as a crystalline solid in 72% yield. That the condensation had taken place as expected was easily verified by the -91-i r spectrum which showed the characteristic amide carbonyl absorption at 1650 cm 1 as well as an, ester carbonyl absorption at 1780 cm \ 5.7 6-N,N-Dimethylamino-9- (3' -C_-cyanomethyl-3'-deoxy-g-D-ribof uranosyl)-purine [CXXXI] In order to prepare the cyanomethyl branched-chain nucleoside CXXXI i t was necessary to prevent the previously discussed hydrolysis of the n i t r i l e moiety in the branched-chain which occurred during reaction of the blocked chloropurine nucleosides with aqueous dimethylamine. Although the fine points of the mechanism for the above hydrolysis had not been definitely elucidated, by simply comparing the reactants and products i t was apparent that in anhydrous dimethylamine conversion of the n i t r i l e functionality to the tertiary amide was not possible. Consequently the chloropurine branched-chain sugar nucleoside CXVIII was dissolved in anhydrous dimethylamine and allowed to stand at -10° for twenty days. Upon removal of the solvent and trituration of the reaction mixture with ether, a portion of the desired cyanomethyl branched-chain sugar nucleoside CXXXI crystallized out. Chromatography of the remaining material gave a further portion of nucleoside CXXXI (total yield 78%). That this compound was the desired nucleoside 6-N,N-dimethylamino-9-(3'-C-cyanomethyl-31-deoxy-g-D-ribofuranosyl)-purine [CXXXI] was confirmed by spectral data. The i r spectrum contained hydroxyl and n i t r i l e absorptions at 3200-3400 cm 1 and 2230 cm \ respectively, and no carbonyl absorptions, indicating that no hydrolysis of the n i t r i l e had taken place and that the ester hydroxyl protecting groups had been completely removed. The nmr -92-CH_ OH 0=CN(Me)2 [CXXII] spectrum and elemental composition were also consistent with the above structure. It i s interesting to note that when this compound was dissolved in an aqueous dimethylamine-methanol mixture hydrolysis to the amide branched-chain sugar nucleoside CXXII took place but proceeded at a much slower rate (12 hr for complete hydrolysis) than the hydrolysis of chloropurine nucleoside CXVIII to CXXII. 5.8 6-N,N-Dimethylamino-9-(3*-C-(2"-acetamidoethy1)-3'-deoxy-g-D-ribof uranosyl) -purine [CXXXIV] The preparation of an aminoethyl branched-chain sugar nucleoside was i n i t i a l l y attempted by reduction of the amido branched-chain nucleoside CXXIX with lithium aluminum hydride in pyridine (175). Pyridine was chosen as the reaction solvent because of the negligible -93-solu b i l i t y of nucleoside CXXI in ethers. Unfortunately using these conditions no appreciable reduction took place and the starting material was recovered unchanged. An alternate approach to the desired aminoethyl nucleoside through reduction of the n i t r i l e moiety of nucleoside CXXXI was then investigated. Hydrogenation at room temperature and 60 psi of this compound over platinum oxide in a 1:1 mixture of acetic anhydride and ethanol gave after four hours two products (R^ 0.18 and 0.10 on s i l i c a gel with dichloromethane:ethyl acetate:ethanol as developer). These were separated by column chromatography on s i l i c a gel and their nmr spectra in dimethyl sulfoxide-d^ were examined. The spectra of both compounds exhibited a single low f i e l d broad t r i p l e t (2.14 x for the faster moving component and 2.22 x for the slower moving one) characteristic of a N-H acetamido proton. This indicated that reduction of the n i t r i l e had taken place. However, surprisingly the faster moving component CXXXII had no hydroxyl absorptions and 3 methyl singlets (7.85 x, 8.04 x, 8.20 x); the slower moving component CXXIII on the otherhand, showed one hydroxyl absorption (a doublet at 4.18 x indicating a secondary hydroxyl group) and two methyl singlets (7.98 x and 8.17 x). From this i t was concluded that as well as reduction of the n i t r i l e group acetylation of some of the hydroxyl groups had taken place to give as the reduction products an approximately 50:50 mixture of compounds CXXXII and CXXXIII (Equation 35). Presumably i t is the heterocyclic base of the nucleoside which catalyzes the acetylation of the hydroxyl groups in the nucleoside. This is somewhat remarkable in view of the low concentration of base present in the reaction mixture. -94-HOCH 2 ^0 DMP (Ac) 20 EtOH R'OCH DMP (35) [CXXXI] DMP = // N(Me), [CXXXII] R = R' = Ac [CXXXIII] R* = Ac, R [CXXXIV] R = R' = H = H These two compounds were de-O-acetylated by reaction with aqueous dimethylamine to give the same acetamidoethyl branched-chain sugar nucleoside 6-N,N-dimethylamino-9-(3'-C-(2"-acetamidoethyl)-3'-deoxy-g-D-ribof uranosyl) -purine [CXXXIV] as a crystalline solid. 5.9 6-Benzamido-9-(21-0-acetyl-5',6'-di-O-benzoyl-3'-C-cyanomethyl-3'-deoxy-g-D-allofuranosyl)-purine [CXXXVI] and 6-benzamido-9-(2'-0-acetyl-5 '-0-benzoyl-3'-C-cyanomethyl-3'-deoxy-g-D-ribofuranosyl)-purine [CXXXVII] In order to extend the u t i l i t y of cyanomethyl branched-chain sugars in nucleoside synthesis the preparation of nucleosides using the standard glycosyl halide, chloromercuri purine method (98,99) was br i e f l y examined. When the titanium tetrachloride, chloromercuri-6-benzamido-purine method (176) was used with CX (Equation 36) no appreciable yield -95-( 3 6 ) CH- OAc I 2 NHBz CN CMP = [CXXXVI] of nucleoside was obtained. The main product of this reaction appeared from spectral data to be the C-l hydrolysis product CXXXV, indicating that the glycosyl halide had been formed but that i t had not undergone condensation with the base, but rather had been hydrolysized to CXXXV probably during workup. In view of the above result i t was decided to prepare the more reactive glycosyl bromo derivative and to condense this compound with chloromercuri-6-benzamido purine. Thus the glycosyl bromide of CX was synthesized by reacting this compound with a saturated solution of -96-hydrogen bromide i n dichloromethane (177). The unstable bromo-g l y c o s i d e obtained a f t e r e v a p o r a t i o n of the s o l v e n t was immediately added to a suspension of c h l o r o m e r c u r i 6-benzamido purine i n toluene at 65° (176). A f t e r the u s u a l work up and chromatography on s i l i c a g e l t h i s procedure a f f o r d e d the b l o c k e d adenyl n u c l e o s i d e 6-benzamido-9-(2 1-0-acety-5 1,6 r-di-0-benzoyl-3'-C-cyanomethyl-3'-deoxy-B-D-a l l o f u r a n o s y l ) - p u r i n e [CXXXVI] as a syrup i n 60% y i e l d . The assignment of the g - c o n f i g u r a t i o n to t h i s compound was based on Baker's trans r u l e (100) and the s m a l l H - l ' , H-2' c o u p l i n g constant ( J ^ , 2 ' = 1 H z ^ • A p p l i c a t i o n of the above procedure to CXIII gave the corresponding pentose n u c l e o s i d e 6-benzamido-9- (2' -0-acetyl-5'-0-benzoyl-3'-C_-cyanomethyl-3'-deoxy-B-D-ribofuranosyl)-purine [CXXXVII]. The anomeric c o n f i g u r a t i o n was deduced as before from Baker's r u l e and the f a c t t h a t the anomeric proton appeared as a s i n g l e t at 3.95 T. -97-5.10 9-(3*-C-Aminoethyl-3'-deoxy-B-D-allofuranosyl)-adenine [CXXXIX] In order to reduce the n i t r i l e group in the branched-chain of the blocked adenyl nucleoside CXXXVI catalytic hydrogenation at 60 psi in acetic anhydride with platinum oxide catalyst was i n i t i a l l y attempted. However under these conditions no detectable reduction took place after 24 hours at room temperature. It was therefore decided to reduce this compound using lithium aluminum hydride in tetrahydrofuran (131). Using this procedure the desired aminoethyl nucleoside CXXXIX was obtained as a crystalline solid albeit i n low (20%) yie l d . Although the compound was purified by passage through an ion exchange resin (Dowex 50W-X2 (NH^+ form)) and crystallized several times from methanol, the product always remained contaminated with a trace of inorganic material. This tendency of nucleosides to complex with metals was noted before (108). Wherever possible, reaction conditions which could introduce such contamination should be avoided, especially i f the compounds are to undergo biological testing. NHBz [CXXXVII] [CXXXIX] -98-6. Biological activity evaluation of branched-chain sugar nucleosides A l l the nucleosides whose preparation i s described herein (with the exception of the last mentioned compound CXXXIX) are currently undergoing biological testing at the United States National Cancer Institute, Bethesda, Maryland. The means being used to evaluate the activity of these nucleosides is the Leukemia L 1210 system, as this type of compound generally shows i t s greatest activity in this system (178). IV. EXPERIMENTAL: 1. General Methods Unless otherwise specified a l l solvent evaporations were done in vacuo at prevailing aspirator pressure and a bath temperature less than 50°. Circular dichroism (cd) spectra were recorded on a Jasco ORD/UV-5 spectropolarimeter or a Jasco J-20 automatic recording spectropolarimeter. Optical rotations were measured with a Perkin Elmer model 141 polarimeter. Infrared (ir) spectra were recorded on a Perkin Elmer model 137 spectrophotometer. Sixty MHz nuclear magnetic resonance (nmr) spectra were measured on a Varian T-60 spectrometer; 100 MHz spectra were recorded on a Varian HA-100 or XL-100 spectrometer. Absorptions are given in x units with tetramethylsilane as internal standard ( set at x 10). The following abbreviations are used: (b) = broad, (d) = doublet(s), (s) = singlet(s), (t) = t r i p l e t ( s ) , (p) = proton(s). Mass spectra were recorded on an A. E. I. MS 9 spectrometer. Elemental analyses were performed by Mr. Peter Borda at the University of British Columbia. 2. Chromatography 2.1 Column S i l i c a gel column chromatography was accomplished using either s i l i c a gel 60-200 mesh, Davidson commercial grade H, indicated in the -100-experimental as " s i l i c a g e l " , or s i l i c a gel for t i c D 0 Mondray Ltd., indicated as " t i c s i l i c a g e l . " For s i l i c a gel column chroma-tography the r a t i o of material to absorbent was about 1 to 70. Grade II a c t i v i t y i n d i c a t e s that 10% of water has been added to the absorbent. For t i c s i l i c a gel column chromatography the r a t i o of material to absorbent was about 1 to 200 and columns were run under a p o s i t i v e pressure of 2 to 7 p s i . Alumina column chromatography was done using aluminum oxide Woelm ne u t r a l , the desired a c t i v i t y grade being pre-pared according to the d i r e c t i o n s on the container. 2.2 Thin Layer Chromatography A l l t h i n layer chromatography ( t i c ) was performed using s i l i c a gel f o r t i c D 5 Mondray Ltd. containing 1% e l e c t r o n i c phosphor. Compounds were detected e i t h e r by u l t r a v i o l e t absorbtion or by spraying with ca. 20% s u l f u r i c a c i d followed by heating on a hot pl a t e . 2.3 Paper Chromatography Paper chromatograms were developed on Whatman No. 1 paper. Nucleosides were detected with u l t r a v i o l e t l i g h t . 2.4 Gas L i q u i d Chromatography Gas l i q u i d p a r t i t i o n chromatographic separations (glc) were performed using a Varian aerograph model 1525 with the following columns: column A i s a s t a i n l e s s s t e e l column (10' x 3/8") packed with 5% butane d i o l succinate on Chromosorb W-AW-DMCS 60-80 mesh; column B i s a s t a i n l e s s s t e e l column (8' x 1/4") packed with 8.5% -101-SF 96 on Chromosorb W. 3. P h o t o l y s i s Reactions The l i g h t source i n these r e a c t i o n s was a Hanovia 450 w type L lamp. Large s c a l e ( i n t e r n a l ) photolyses were c a r r i e d out by p l a c i n g the lamp, and f i l t e r i f r e q u i r e d , i n s i d e a water cooled quartz immersion w e l l apparatus which was placed i n s i d e a 3-necked pyrex v e s s e l ( c a p a c i t y w i t h lamp about 300 ml ). Small s c a l e ( e x t e r n a l ) photolyses were performed by p l a c i n g the s o l u t i o n to be photolysed i n a pyrex tube ( c a p a c i t y about 80 ml ) and clamping t h i s tube to the ou t s i d e of the quartz immersion w e l l . The immersion w e l l and p h o t o l y s i s tube then were placed i n a water bath i n order that the s o l u t i o n being photolysed would remain a t room temperature. I n both of the above procedures i n order to prevent a c c i d e n t a l exposure to u l t r a v i o l e t r a d i a t i o n and to make the most e f f i c i e n t use of the r a d i a t i o n source , the whole p h o t o l y s i s apparatus was wrapped i n aluminum f o i l . A l l p h o t o l y s i s s o l v e n t s were reagent grade, d i s t i l l e d and d r i e d before use. P h o t o l y s i s s o l u t i o n s were deoxygenated w i t h Matheson p r e p u r i f i e d n i t r o g e n . 1,2:5, 6-Di-0_-isopropylidene-a-D-glucofuranose [LXIX] To an e f f i c i e n t l y s t i r r e d suspension of a-D-glucose (300 g) i n absolute acetone (21) was added p u l v e r i z e d anhydrous z i n c c h l o r i d e (280 g) and 85 % phosphoric a c i d ( 15 g ) . The mixture was allowed to shake at room temperature f o r two days. The unreacted sugar (108 g) was removed by f i l t r a t i o n and the f i l t r a t e was made s l i g h t l y a l k a l i n e -102-with sodium hydroxide (170 g in 170 ml of water). The insoluble inorganic material was removed by f i l t r a t i o n and washed with acetone. The f i l t r a t e and washings were concentrated under reduced pressure. The residue was dissolved in water (300 ml) and extracted with chloroform (300 ml x 3). The combined chloroform extracts were washed again with water, then dried over sodium sulfate. Evaporation of the solvent yielded a solid residue, which was recrystallized from cyclohexane to afford crystalling LXIX (220 g, 80% yield based on ID-glucose consumed), m.p. 109°. Reported (179): m.p. 110-111°. 5-0-Benzyl-l,2-0-isopropylidene-a-D-xylofuranose [LXXVII] This compound was synthesized following known procedures. Starting with 100 g of D-xylose [LXXIII], diacetone xylose [LXXIV] was prepared following a procedure given by Baker and Schaub (120); yield 104 g (73%) b.p. 97 - 98° (0.25 mm). Reported (120): 90 - 92° (0.2 mm). The diacetone xylose (104 g) was then hydrolyzed to monoacetone xylose (120) by dilute sulfuric acid; yield 95 g (95%). The monoacetone xylose [LXXIV] (75 g) was converted to 1,2-0-isopropylidene-5-0_-tosyl-a-D xylofuranose; yield 71 g (52%), m.p. 135 - 136°. Reported (121): m.p. 133 - 134°. Treatment of the tosylate (60 g) with sodium methoxide converted i t to 1,2-0-isopropylidene-3,5 anhydro-a-D-xylofuranose [LXXVI]; yield 24.5 g (83%) b.p. 48 - 50° (about 0.05 mm). Reported (121): 63 - 65° (0.1 mm). Finally the anhydro sugar LXXVI (23 g) was allowed to react with benzyl alcohol and sodium (122) to afford 5-0-benzyl--103-1,2-0-isopropylidene-ct-D-xylof uranose [LXVIII] (32.5 g, 87%), m.p. 63-64°. Reported (122): m.p. 63-65°. 1,2:5,6-Di-0-isopropylidene-a-D-gulofuranose [LXXIa] This compound was prepared by known procedures (119) from the hydrate of 1,2:5,6-di-0-isopropylidene-q-g-ribo-hexofuranos-3-ulose [LXX]. The hydrate of LXX (6.5 g) was reacted with acetic anhydride and pyridine to afford 3-0-acetyl-1,2:5,6-di-0-isopropylidene-ct-D-erythro-hex-3-enofuranose [LXXI] (3.1 g) , m.p. 56-57°. Reported (119): m.p. 62°. Hydrogenation of LXXI (3 g) over 5% palladium-on-charcoal (1 g) gave 3-0-acetyl-l,2:5,6-di-0-isopropylidene-ct-D-gulofuranose [LXXII] (2.4 g). De-acetylation of this material with methanolic sodium methoxide afforded the t i t l e compound (2.01 g, 33% yield based on LXX), m.p. 105°. Reported (119): 105-106°. 1,2:5,6-Di-0-isopropylidene-a-D-ribofuranos-3-ulose [XVIII] To a solution of 1,2:5,6-di-0-isopropylidene-a-D-glucofuranose [LXIX] (5 g) in carbon tetrachloride (80 ml) was added water (15 ml), sodium bicarbonate (1 g) and finely powdered ruthenium dioxide (80 mg). this solution was added with vigorous s t i r r i n g a few drops of 10% sodium periodate solution. After approximately 5 min another small addition of periodate was made. This process was then repeated several times while gradually increasing the volume of periodate solution added. Addition was discontinued when the solution appeared a greenish-black, which indicated the presence of ruthenium tetroxide, and was -104-restarted when the solution appeared black, which indicated only ruthenium dioxide was present. The reaction was stopped when t i c examination indicated that no more starting material remained (the total volume of periodate solution added was about 50 ml). Any residual ruthenium tetroxide was then destroyed by addition of isopropyl alcohol (0.5 ml) and the ruthenium dioxide removed by f i l t r a t i o n . The carbon tetrachloride layer was separated and the water layer extracted with chloroform (10 x 20 ml). The combined organic extracts were dried over sodium sulfate and the solvent evaporated to yield the ketose hydrate LXX (4.8 g, 97%) m.p. 109-110°. Reported (119): m.p. 109-111°. The hydrate was suspended in dry toluene (200 ml) and 50 ml was d i s t i l l e d off at atmospheric pressure.' The remaining toluene was then removed by flash evaporation using a rotary evaporator connected to an o i l pump. The crude ketose was then d i s t i l l e d , inan apparatus having a very short d i s t i l l a t i o n path (bulb-to-bulb), (150°, 0.1 mm) to afford XVIII (4.5 g, 90%) as a syrup. The i r spectrum showed a carbonyl absorption at 1760 cm 1 and no hydroxyl absorption. 1,2:5,6-Di-O-isopropylidene-q-g-xylo-furanos-3-ulose [LXVII] 1,2 :5,6-Di-O-isopropylidene-a-D-gulofuranose (5 g) was oxidized with sodium periodate and ruthenium dioxide as previously described for compound [XVIII]. Only five extractions with chloroform were necessary to remove the ketose from the water layer. The product crystallized from petroleum ether (65-110°) to give the ketose LXVII (3.9 g, 78%), m.p. 75°. Reported (119): m.p. 76-77°. -105-5-O-Benzyl-l,2-0-isopropylidene-g-D-erythro-pentofuranos-3-ulose [LXVIII] Method A: A solution of 5-0-benzy 1-1,2-0-isopropylidene-ct-D-xylofuranose [LXXVII] (10 g in anhydrous dimethyl sulfoxide (60 ml)) was cooled in an ice bath u n t i l frozen. Phosphorus pentoxide (4 g) was then added and after one hour at 0° the mixture was slowly allowed to come to room temperature. After twenty-four hours t i c examination of the reaction mixture indicated that a l l the starting material had been consumed (Rf LXVIII 0.76; Rf LXXVII 0.47, benzene methanol (4:1)). The reaction mixture was then added with s t i r r i n g to a cold saturated sodium bicarbonate solution (100 ml) and after f i l t r a t i o n , the f i l t r a t e was extracted with chloroform (7 x 50 ml). The chloroform extract was washed once with sodium bicarbonate solution (10 ml) and once with water and dried over magnesium sulfate. The majority of the solvent was then removed by evaporation and the residue dried by dissolving i t in anhydrous benzene (50 ml) and d i s t i l l i n g off 25 ml at atmospheric pressure. The remainder of the benzene and residual DMSO was removed by evaporation on a rotary evaporator connected to a vacuum pump (pressure about 1 mm) leaving 6.5 g of ketose LXVIII as a viscous yellow o i l . The i r spectrum of this material showed no hydroxyl absorbtion and a strong carbonyl absorbtion at 1760 cm \ This compound was characterized as i t s 2,4-dinitrophenylhydrazone derivative 22 which was crystallized from acetone water, m.p. 143-144°, [a]^ +140° (c 2, i n chloroform). -106-Anal. Calcd. for C o 1H n o0 oN.: C, 55.02; H, 4.84; N, 12.22. Found: C, 55.25; H, 5.03; N, 12.08. Method B: To a solution of 5-0-benzyl-l,2-0_-isopropylidene-a-D-xylofuranose [LXXVII] (5 g) in carbon tetrachloride (80 ml) was added ruthenium dioxide (80 mg), water (10 ml), and sodium bicarbonate (1 g). While the mixture was being vigorously stirred about 0.25 ml of 10% sodium metaperiodate solution was added dropwise. After 5 minutes another 0.5 ml of 10% sodium metaperiodate solution was added and after a further 5 minutes more periodate solution was added unt i l the solution was observed to turn a green-black colour (indicating the presence of ruthenium tetroxide). Addition of periodate solution was then continued at intervals whenever the solution turned black (indicating only ruthenium dioxide was present). When t i c examination indicated that a l l the starting material had been oxidized (this required about 1.3 equivalents of sodium periodate) a few drops of isopropyl alcohol were added to the reaction mixture to decompose any unreacted ruthenium tetroxide and the ruthenium dioxide was fi l t e r e d off. The carbon tetrachloride layer was then separated and the aqueous layer extracted with chloroform (3 x 75 ml). The combined organic extracts were then dried over magnesium sulfate and the solvent evaporated. After drying with benzene as previously described there remained 4.8 g of ketose LXVII (90% of theoretical) identical by nmr and i r to the product from the DMSO oxidation. -107-3-C-Cyanomethyl-3-deoxy-l, 2:5,6-di-0-isopropylidene-a-D-allof uranose [LXXXVI] 1,2:5,6-Di-0-isopropylidene-a-D-ribofuranos-3-ulose [XVIII] (14.7 g, 0.057 mole) dissolved in 1,2-dimethoxy ethane (DME) (250 ml) was added dropwise with s t i r r i n g to a solution (kept at 0°) of diethyl cyanomethylphosphonate carbanion (prepared as in the synthesis of LXXXVIII from sodium hydride (1.64 g, 0.0685 mole) and diethyl cyanomethylphosphonate (12.1 g, 0.0685 mole)) in DME (50 ml). After addition was complete the reaction mixture was allowed to come to room temperature and after 4 hr i t was diluted with ice water (100 ml) and the product extracted with ether (3 x 200 ml). The combined ether extracts were washed with water (3 x 20 ml), dried (sodium sulfate) and evaporated. The residue after evaporation of the solvent was bulb-to-bulb d i s t i l l e d (190°, 0.1 mm) to afford 13.6 g of a colourless syrup (Rf 0.68, benzene:methanol (19:1)); i r (film) 2250 cm - 1 ( C E N ) ; CDC1 T 3 3.9-4.1 (m, 2p, H-l and ol e f i n i c proton). Hydrogenation of this material in ethanol (150 ml) at ambient pressure and temperature over 5% palladium-on-charcoal (4 g) (1.01 equivalents of hydrogen absorbed) gave, after removal of the catalyst by f i l t r a t i o n and evaporation of the solvent, 13.6 g of syrup. Crystallization of this material from ether-petroleum ether 30-60°, afforded the branched-chain sugar LXXXVI (12.6 g, 78%), m.p. 109°, 2? —1 r n n [a] +91° (c 2, in chloroform); i r (nujol) 2270 cm (C=N); T 3 4.18 (d, lp, H-l, J± 2 = 3.6 Hz), 5.23 (t, lp, H-2, ^ 2 3 = 3.6 Hz). Irradiation of LXXXVI at the H-l signal collapsed H-2 into a doublet. -108-Anal. Calcd. for C^H $N 0 5: C, 59.3; H, 7.47; N, 4.94. Found: C, 59.26; H, 7.35; N, 4.81. 3-C-Cyanomethyl-3-deoxy-l ,2:5,6-di-0_-isopropylidene-a-D-gulof uranose [LXXXVII] 1,2: 5 ,6-Di-0_-isopropylidene-a-D-xylohexafuranos-3-ulose [LXVII] (290 mg) dissolved in DME (20 ml) was added dropwise with s t i r r i n g to a solution (kept at 0°) of diethyl cyanomethylphosphonate carbanion (prepared as in the synthesis of LXXXVIII from sodium hydride (30 mg)) and diethylcyanomethyl phosphonate (220 mg) in DME (15 ml). After addition was complete the reaction mixture was allowed to come to room temperature and after 4 hours i t was diluted with ice water (20 ml) and the product was extracted with ether (3 x 25 ml) as previously described. Crystallization from ether-pet. ether 30-60° afforded 260 mg (80%) of the unsaturated cyano branched-chain sugars 3-C-cyanovinyl-3-deoxy-l,2:5,6-di-0-isopropylidene-a-D-xylofuranose - l r n n [LXXXIV], m.p. 98°; i r (nujol) 2260 cm (C=N);• T 3 4.06-4.33 (m, 2p, H-l and ol e f i n i c proton). Hydrogenation of LXXXIV (260 mg in 10 ml ethanol) at ambient pressure and temperature over 5% palladium-on-charcoal (100 mg) (1 equivalent of hydrogen absorbed) gave the t i t l e branched-chain sugar LXXXVII (260 mg, 79%) which was crystallized from ether-petroleum ether 30-60°, m.p. 112°, [a]*5 -28.6° (c 2.3, in chloroform); i r (nujol) — l r nn 2280 cm (C=N); T 3 4.17 (d, lp, H-l, J = 4.0 Hz), 5.27 (broad t, lp, H-2, J _ = 5.0 Hz). Irradiation of H-l collapsed H-2 to a doublet. -109-Anal. Calcd. for C 1 4 H 2 1 N ° 5 : c> 59.35; H, 7.47; N, 4.94. Found: C, 59.33; H, 7.63; N, 4.69. 5-0-Benzyl-3-C_-cyanomethyl-3-deoxy-l, 2-0-isopropylidene-a-D-ribofuranose [LXXXVIII] To a suspension of sodium hydride (NaH) (0.36 g, 15 mg) in anhydrous 1,2-dimethoxyethane (DME) (20 ml) was added a solution of diethyl cyanomethylphosphonate (2.7 g, 15 mmole) in DME (20 ml). When evolution of hydrogen had ceased the mixture was f i l t e r e d ( a l l the above operations were performed in a dry box under nitrogen atmosphere) and the solution of phosphonate carbanion cooled to 0°. To this solution was then added dropwise 5-0_-benzyl-l,2-0-isopropylidene-a-D-erythro-pentofuranos-3-ulose [LXVIII] (2.8 g, 10.1 mmole) in DME (60 ml). When the addition was complete the reaction mixture was allowed to come to room temperature and after four hours the solution was diluted with ice water (75 ml) and extracted with ether (3 x 50 ml), The combined ether extracts were washed with water ( 3 x 5 ml), dried (magnesium sulfate) and evaporated. The remaining residue was dissolved in benzene (50 ml) and decolourized with charcoal. Evaporation of the solvent gave 3 g of syrup. Hydrogenation of this syrup in ethanol (25 ml) at ambient pressure and temperature over 10% palladium-on-charcoal (1 g) (1.05 equivalents of hydrogen absorbed) gave after removal of the catalyst and evaporation of the solvent 23 the t i t l e compound LXXXVIII as a homogeneous syrup (3 g, 93%); [a]p -1 CT)C\ +50° (c_ 3, in chloroform); i r (nujol) 2270 cm (C=N) ; T 3 4.2 (d, lp, H-l, J = 3.6 Hz), 5.34 (t, lp, H-2, J = 3.9 Hz). -110-Irradiation of the H-l signal of LXXXVIII collapsed H-2 into a doublet. Anal. Calcd. for C^H^NO^: C, 67.31; H, 6.98; N, 4.62. Found: C, 67.45; H, 7.20; N, 4.78. 3-C-(2'-Acetamidoethyl)-3-deoxy-l,2:5,6-di-O-isopropylidene-a-D-allofuranose [XCII] The 3-C_-cyanomethyl-3-deoxy-l, 2:5 ,6-di-0_-isopropylidene-a-D-allofuranose [LXXXV] (1 g) dissolved in absolute ethanol (70 ml) saturated with ammonia was hydrogenated over 5% rhodium-on-alumina (200 mg) at room temperature and 60 psi for 20 hr. The catalyst was then removed by f i l t r a t i o n and the solvent evaporated. The resulting syrup was acetylated with a mixture of acetic anhydride (3.5 ml) and pyridine (3.5 ml) for 24 hr at room temperature. The mixture was then diluted with ice water (20 ml) and the product extracted with dichloromethane (3 x 20 ml), washed with water ( 2 x 5 ml) and dried over sodium sulfate. Evaporation of the solvent afforded 0.92 g (80%) 23 of the above amide XCII as a syrup; [a]^ +41° (c 1, in chloroform); —1 - 1 m n i r (film) 3300 cm (N-H), 1640 cm (N-C=0); x 3 3.27 (broad t, lp, H-N ), 4.30 (d, lp, H-l J± 2 = 4.0 Hz), 5.30 (t, lp, H-2), 8.03 (s, 3p, Ac). Anal. Calcd. for C^H.^N.O,: C, 58.34; H, 8.20; N, 4.25. Found: 16 II l b C, 58.27; H, 8.44; N, 4.00. -111-3-C- (2' -Acetamidoethyl)- 5-0-benzyl-3-deoxy-l, 2-0-isopropylidene-a-D-ribofuranose [XCIII] A solution of 5-0_-benzyl-3-C-cyanomethyl-3-deoxy-l,2-0-isopropylidene-a-D-ribofuranose [LXXXVIII] (7 g in anhydrous ether (50 ml)) was added dropwise to a suspension of lithium aluminum hydride (LAH) (2.03 g) in anhydrous ether (100 ml). After two hours unreacted LAH was decomposed by the slow addition of ethyl acetate (35 ml) i n ether (50 ml) followed by water (2 ml). The solution was then f i l t e r e d and the f i l t r a t e was evaporated. The residue was taken up in chloroform (100 ml) and the chloroform solution was washed with water (3 x 10 ml), dried (sodium sulfate) and evaporated. The remaining material was acetylated by treatment with a mixture of acetic anhydride (10 ml) and anhydrous methanol (19 ml) for 3 hours. The mixture was then poured into ice water (50 ml) and the product extracted with chloroform (3 x 75 ml). The combined chloroform extracts were washed with 5% sodium bicarbonate solution (2 x 10 ml) and water (2 x 10 ml) and dried (sodium sulfate). Evaporation of the f i l t r a t e gave 6.6 g of syrup which was chromatographed on s i l i c a gel using benzene:ethyl acetate (2:1) as developer to afford amide XCIII as a syrup (5.5 g, 68.5% from LXXXVIII]; [a] 2, 2 +39° (c 3, in chloroform); 0 D - i n r n n i r (film) 3300 (N-H), 1650 cm (-C-N); x 3 4.2 (d, lp, H-l), 4-4.4 (b, lp, N-H), 8.1 (s, 3p, N-Ac). Anal. Calcd. for C^H^N^O : C, 65.31; H, 7.79; N, 4.01. Found: C, 65.60; H, 8.02; N, 3.87. -112-Acetolysis of 3-C-(2'-acetamidoethyl) -5-0-benzyl-3-deoxy-l, 2-0-isopropylidene-a-D-ribofuranose [XCIII] Concentrated sulfuric acid (0.25 ml) was added dropwise to a cooled (0°) solution of 3-C-(2'-acetamidoethyl)-3-deoxy-l,2-0-isopropylidene-ct-D-ribofuranose (500 mg) in acetic anhydride (0.5 ml) and gla c i a l acetic acid (5 ml). After addition was complete the reaction mixture was allowed to come to room temperature and let stand for one day. Workup was accomplished by pouring the reaction mixture into ice water (30 ml) and extracting the product with chloroform (3 x 25 ml). A t i c examination of the chloroform extract showed the presence of five products (R^ 0.0, 0.1, 0.45, 0.61 and 0.75, benzenermethanol (9:1)) in about equal amounts. 3-£-Cyanomethyl-3-deoxy-l,2-£-isopropylidene-8-L-lyxofuranose [XCV] To a solution of 3-C-cyanomethyl-3-deoxy-l,2:5,6-di-0-isopropylidene-a-D-gulofuranose [LXXXVII] (160 mg in 9 ml methanol) was added 0.7 M sulfuric acid (1.5 ml) and the mixture l e f t to stand for 7 hours. The hydrolysis mixture was then neutralized with solid sodium bicarbonate and extracted with chloroform (4 x 15 ml). The combined chloroform extracts were dried (sodium sulfate) and evaporated to afford 3-C-cyanomethyl-3-deoxy-l,2-0_-isopropylidene-a-D-gulofuranose [XCIV] (121 mg, 88%) as a syrup; x 3 4.07 (d, lp, H-l), 5.23 (broad t, lp H-2), 7.0-7.5 (m, 5p, CH2C N, H-3, C-5 OH, C-6 OH), 8.40, 8.64 (2s, 6p, l p ) . Upon addition of D20 two absorbtions in the region x 7.0-7.5 disappeared. The above diol XCIV (121 mg) was reacted with sodium periodate and sodium borohydride as described for the preparation -113-of XCI to afford the t i t l e branched-chain sugar XCV (105 mg, 87% 24 based on LXXXVII) which was crystallized from ether, m.p. 81°, [a]^ +10.4° (c_1.6, in chloroform); i r (nujol) 3500 (OH, 2245 cm"1 (C E N ) ; r n n T 3 4.10 (d, lp, H-l, J 1 2 = 4 Hz), 5.23 (t, lp, H-2, J 2 3 = 4.5 Hz), 8.43, 8.67 (2 s, 6p, lp). Anal. Calcd. for C,rtH, ..N-O. : C, 56.33; H, 7.09; N , 6.57. Found: 10 15 1 4 C, 56.22; H, 7.05; N , 6.50. 3-C-(2'-Acetamidoethyl)-3-deoxy-l,2-O-isopropylidene-B-L-lyxofuranose [XCVI] A solution of 3-C_-cyanomethyl-3-deoxy-l, 2-0-isopropylidene -B-L-lyxofuranose [XCV] (18 mg) dissolved in acetic anhydride (2 ml) and ethanol (2 ml) and containing platinum oxide (19 mg) was hydrogenated at room temperature and 60 psi for 4.5 hrs. A t i c examination at this time indicated that the reaction was complete (R^ XCV 0.47, R^  XCVI 0.05, dichloromethane:ethyl acetate:ethanol (5:5:1)). The catalyst was then removed by f i l t r a t i o n and the solvent evaporated to afford the t i t l e compound XCVI (20 mg) (92%) which crystallized on standing (R, 0.59 dichloromethane:methanol 9:1); m.p. 133° [a] 2, 5 +1.50° (c_ t 0 D - l n r n n 1.6, in chloroform); i r (KBr) 1630 cm (C-N); T 3 3.8-4.3 (b, lp, N-H), 4.17 (d, lp, H-l, J 1 2 = 4 Hz), 5.35 (t, lp, H-2, J 2 3 = 5 Hz), 8.0 (s, 3p, N-Ac). Anal. Calcd. for C 1 2 H 2 1 N 1 ° 5 : C ' 5 5 , 5 8 » H» 8- 1 65 N> 5.40. Found: C, 55.72; H, 8.27; N, 5.10. -114-3-C_-Carbamoylmethyl-3-deoxy-l ,2:5,6-di-O-isopropylidene-a-D-allofuranose [C] To a solution of 3-C_-cyanomethyl-3-deoxy-l,2:5,6-di-0-isopropylidene-a-D-allofuranose [LXXXVI] (0.566 g in 6 ml ethanol) was added 30% hydrogen peroxide (0.8 ml) and 6 N sodium hydroxide. (0.8 ml). The mixture was then stirred at 50° for 6 hrs. Any unreacted hydrogen peroxide was then decomposed by the addition of a few milligrams of platinum oxide, and the solution was f i l t e r e d and evaporated to dryness. The residue after evaporation, was extracted with dichloromethane (40 ml) and the dichloromethane extract washed with water (5 ml) and dried over sodium sulfate. Evaporation of the solvent gave a solid which was recrystallized from ether to afford the t i t l e compound C (0.40 g, 70%), m.p. 138°; [ a ] J 4 +81° (c_2, in chloroform); i r (nujol) 3490, 3250 (NH), —1 f n n 1650 cm (C=0); T 3 3.8-4.4 (b, 2p, NH2). Anal. Calcd. for O, .H„-N-0,: C, 55.8: H, 7.69; N, 4.47. Found: 14 23 1 6 C, 55.7; H, 7.91; N, 4.57. Preparation of C from XXIX 3-C_- (Carbomethoxymethyl) -3-deoxy-l ,2:5,6-di-0-isopropy lidene-a-D-allofuranose [XXIX] (150 mg) and ammonium chloride (15 mg) were dissolved in liquid ammonia (3 ml) and heated in a sealed stainless steel tube at 60° for 24 hrs. The ammonia was then evaporated and the residue dissolved in ether. Insoluble material was removed by f i l t r a t i o n and the f i l t r a t e was evaporated. The remaining material was taken up in a minimum amount of ether and stored at 0° for 24 hrs. A portion of the t i t l e amide C (90 mg) crystallized from this solution and was removed by -115-f i l t r a t i o n ; a further 18 mg of C (total yield 76%) was obtained by concentrating the mother liquor and allowing i t to stand at 0° for a further 24 hrs. The amide prepared by this procedure was identical ( i r , nmr, melting point, and mixed melting point) with the amide prepared from LXXXVI. Preparation of C from XX A de-oxygenated solution of l,2:5,6-di-0_-isopropylidene-3-C-methylene-q-g-ribo-hexofuranose [XX] in formamide (40 ml), t-butanol (20 ml) and acetone (5 ml) was photolyzed externally i n a pyrex vessel for 7 hrs. The vo l a t i l e solvents were then evaporated and the remaining solution diluted with saturated sodium chloride solution. Extraction of this solution with dichloromethane (3 x 35 ml) afforded (after drying over sodium sulfate and evaporation of the solvent) 300 mg syrup. Column chromatography of this material on t i c s i l i c a gel using benzene:ethyl acetate:ethanol (5:5:1) as developer gave amide C (152 mg, 50%) identical (by i r , nmr, and mixed m.p.) to the product prepared from LXXXVI and some uncharacterized acetone addition product (35 mg, 11%). The nmr spectrum of the acetone addition product contained the r n n following signals: x 3 4.23 (d, lp, H-l), 5.27 (t, lp, H-2), 8.0 (s, lp, OH), 8.5-8.8 (m, 18p, 6Me). Upon addition of D20 the singlet at x 8.0 was removed. Ethyl 4,6-di-0-acetyl-2,3-dideoxy-a-D-erythro-hex-2-enopyranoside [CII](144) Tri-O-acetyl-D-glucal (5 g) was dissolved in anhydrous benzene (20 ml, dried over molecular sieves) and d i s t i l l e d anhydrous ethanol -116-(1.8 ml). Boron trifluoride-ether (1 ml) was added to the mixture under anhydrous conditions. Vigorous s t i r r i n g was maintained at room temperature for twenty-five minutes, after which time anhydrous sodium carbonate (5 g) was quickly added. Stirring was then continued for a further fifteen minutes so as to ensure the complete neutralization of any excess boron trifluoride. The solid sodium carbonate was removed by f i l t r a t i o n . Upon evaporation of the solvents, the resulting syrupy residue crystallized spontaneously. Recrystallization from ether-petroleum ether afforded CII (4 g, 80%), m.p. 78-79°. Reported (144): m . p . 78-79°. Photo-addition of formamide to ethyl-4, 6-di-0-acetyl-2,3-dideoxy-ct-Dj-erythro-hex-2-enopyranoside [CII] Ethyl 4,6-di-0-acetyl-2,3-dideoxy-a-p-erythro-hex-2-enopyranoside [CII] (0.500 g) was dissolved in a mixture of freshly d i s t i l l e d formamide (190 ml), t-butanol (70 ml) and acetone (17 ml) and the solution purged with oxygen-free nitrogen for ten hrs. Irradiation (pyrex f i l t e r A >300) was then commenced. After 1 1/2 hr a further 2.5 g of CII in oxygen-free t-butanol (20 ml) and acetone (3 ml) was added dropwise over a three hour period. Examination of the reaction mixture by t i c (95:5 benzene :methanol R^  CII 0.50, R^. products -0 and 0.1) indicated that after 9 hrs no more starting material remained. The volatile solventswere then removed by evaporation and the remainder of the solution diluted with saturated sodium chloride solution (200 ml) and extracted with chloroform (4 x 100 ml). The combined chloroform -117-extracts were washed with water (3 x 20 ml), dried over sodium sulfate and evaporated to yield 3.1 g of syrup. Column chromatography of this material on s i l i c a gel grade II (benzene:ethyl acetate:ethanol 10:10:1) gave two components: 0.33 g of the f i r s t eluted component and 2.1 g of the second eluted component. The f i r s t eluted component proved to be a mixture of acetone r n n addition products: x 3 7.66 (s, lp, OH), 7.94 (s, 6p, 2Ac), 8.80 (t, 3p, CH^ of ethyl glycoside) 8.80 (s, 6p, 2 methyl peaks of acetone addition branched-chain). Glc column A: 2 unresolved peaks, retention time 24-26 min at 200°. Column B: 2 unresolved peaks, retention time 15 1/2-18 1/2 min at 210°. Anal. Calcd. for C.cH„-0-,: C, 56.59: H, 8.23. Found: C, 56.31; lb / H, 8.00. The second component proved to be the amide addition product 0 - l - i - i ii r n n i r (film) 3400 cm (NH.), 1740 cm (C=0), 1660 cm (C-N; x 3 0 1 3.47 (b, 2p, C-NH2), 7.90 (s, 6p, Ac), 8.74 (t, 3p, methyl peak of ethyl glycoside). Anal. Calcd. for c 1 3 H 2 i N i ° 7 : c» 51.47; H, 6.97; N, 4.61. Found: C, 51.93; H, 6.67; N, 4.20. I, 2:5,6-Di-0-isopropylidene-3-£-nitromethy1-a-D-glucofuranose [CV] A solution of one M sodium methoxide in methanol (1.95 ml, 1.95 mmoles) was added dropwise with s t i r r i n g to a solution of 1,2:5,6-di-O-isopropylidene-q-D-ribo-hexofuranos-3-ulose [XVIII] (0.5 g, 1.95 mmoles) in 5 ml of nitromethane. The reaction mixture was stirred for 16 hr at room temperature and then deionized, and the f i l t r a t e then -118-evaporated to a syrup. Crystallization from petroleum ether (b.p. 60-110°) gave 0.430 g (71% of pure, crystalline nitro derivative CV, m.p. 138-140°, [ a ] 2 2 +31° (c 2, chloroform); i r (CC14) 3650 (s) (OH) -1 r n n 1560 cm (N0 2); x 3 4.05 (d, H-l, 2 3.5 Hz), 5.13 (an AB system, J , 12.5 Hz, methylene protons a and b on C-l'), 5.38 (d, 3. y D H-2, 31 2 3.5 Hz), 5.5-6.3 (m), 6.50 (OH), 8.40, 8.55 (2s, 6p, lp) 8.62 and 8.66 (2s, 6p, lp). Anal. Calcd. for C 1 0H„.N0 0: C, 48.89; H, 6.63; N, 4.39. Found: C, 48.73; H, 6.49; N, 4.54. 3-C-Cyanomethyl-3-deoxy-l,2-0-isopropylidene-a-D-allofuranose [CVIII] To a solution of 3-C_-cyanomethyl-3-deoxy-l,2:5,6-di-0_-isopropylidene-ct-D-allofuranose [LXXXVI] (6.5 g) in methanol (300 ml) was added 1 N sulfuric acid (30 ml). The hydrolysis mixture was l e f t to stand at room temperature u n t i l t i c indicated that a l l the starting material was gone (about 4 hr), then neutralized with solid sodium bicarbonate and extracted with chloroform (3 x 200 ml). The combined chloroform extracts after drying (over sodium sulfate) were evaporated to afford 25 compound CVIII (5.5 g) as a syrup in nearly quantitative yield: [ a l n +99.4° (£1.67, in chloroform); i r (film) 3500 cm"1 (OH), 2280 cm"1 ( C H N ) ; x C D C 1 3 8.17, 8.33 (2s, 6p,Ip). Anal. Calcd. for C ^ ^ N ^ : C, 54.31; H, 7.04; N, 5.76. Found: C, 54.01; H, 7.21; N, 5.56. -119-5,6-Di-0_-benzoyl-3-C_-cyanomethyl-3-deoxy-l,2-0_-isopropylidene-a-D-allofuranose [CIX] To a solution of 3-Cj-cyanomethyl-3-deoxy-l, 2-0-isopropylidene-a-D-allofuranose [CVIII] (6.0 g in anhydrous benzene (30 ml)) was added dropwise a mixture of benzoyl chloride (3.2 ml) and pyridine (4.5 ml). After 14 hrs at room temperature the reaction mixture was fi l t e r e d through a short column of grade II alumina (25 g) and the column washed with benzene (150 ml). Evaporation of the combined eluents gave the t i t l e ester CIX which was crystallized from ether-petroleum ether 30-60° to give 10.0 g (90%) of product; m.p. 71-72°, [ a ] 2 4 +48.2° (c 1.3, in chloroform). Anal. Calcd. for C ^ H ^ N ^ : C, 66.6; H, 5.57; N, 3.10. Found: C, 66.33; H, 5.54; N, 2.95. 5-0-Benzoyl-3-C_-cyanomethyl-3-deoxy-l, 2-0-isopropylidene- o-D-ribo-furanose [CXII] To a solution of 3-C_-cyanomethyl-3-deoxy-l,2-0-isopropylidene-a-D-ribofuranose [CXI] (4.75 g, 22.3 mmole in anhydrous benzene (25 ml)) was added dropwise a mixture of benzoyl chloride (2.9 ml, 24.8 mmole) and pyridine (4 ml). After 20 hr at room temperature the reaction mixture was f i l t e r e d through a short column of grade II alumina (20 g) and the column was washed with benzene (100 ml). Evaporation of the solvent from the eluent gave the t i t l e ester CXII which was crystallized from ether-petroleum-ether 30-60°: (6.55 g, 93%), m.p. 110°, [ a ] 2 2 +59° (c_ 1.8, in chloroform). -120-Anal. Calcd. for C H ^ N ^ : C, 64.34; H, 6.03; N, 4.45. Found: C, 61.11, H, 5.93; N, 4.31. Attempted acetolysis of CIX 5,6-Di-0-benzoyl-3-C-cyanomethyl-3-deoxy-l,2-0-isopropylidene-a-D-allofuranose CXII (350 mg) was dissolved in a mixture of acetic acid (4 ml) and acetic anhydride (0.4 ml). To this solution was added dropwise concentrated sulfuric acid (0.4 ml). After 24 hr the reaction mixture was diluted with ice water (20 ml) and extracted with chloroform (3 x 15 -ml). Tic examination of the chloroform extract showed three products; (R^ 0.65 major, 0.3, and 0.1, benzene:methanol (9:1)). The chloroform extract was dried (over sodium sulfate) and evaporated to a syrup which was chromatographed on a column of grade II s i l i c a gel using benzene:ethyl acetate (4:1) as developer. The major component was recovered (100 mg). Elemental analysis of this product showed that i t contained no nitrogen. Found: C, 64.40; H, 5.03. 1,2-Di-0_-acetyl-5 ,6-di-0_-benzoyl-3-£-cyanomethyl-3-deoxy-g-D-allof uranose [CX] 5,6-Di-0_-benzoyl-3-C_-cyanomethyl-3-deoxy-l, 2-0-isopropylidene-a-D-allof uranose [CIX] (4.5 g) was allowed to react with an 80% solution of trifluoroacetic acid (60 ml) at room temperature for 45 minutes. The reaction mixture was then neutralized with solid sodium bicarbonate, f i l t e r e d , and the f i l t r a t e was extracted with methylene chloride (6 x 50 ml). Evaporation of the combined methylene chloride extracts -121-afforded a syrup (4.1 g) which was acetylated with acetic anhydride (15 ml) and pyridine (15 ml). After 20 hrs the reaction mixture was poured into ice water (100 ml) and worked up in the usual way to obtain 4.4 g of syrup. Column chromatography of this material on grade II s i l i c a gel using benzene-ethyl acetate (3:1) as developer yielded after crystallization from ether 3.3 g (69%) of acetate CX, m.p. 110°, [ a ] 2 3 -31° (c 2, in chloroform); i r (KBr) 2230 cm"1 (C=N); TC D C 1 3 1.8-2.8 (m, 10p, 2 Bz), 3.77 (s, lp, H-l), 7.87, 7.97 (2s,6p, 2 Ac). Anal. Calcd. for C o,H- cN n0„: C, 63.00: H, 5.08; N, 2.81. Found: zo zi> i y C, 63.00; H, 4.97; N, 2.65. 3-C-Cyanomethyl-3-deoxy-l,2-0_-isopropylidene-a-D-ribofuranose [CXI] To a solution of 3-C-cyanomethyl-3-deoxy-l,2-0-isopropylidene-a-JD-allof uranose [CVIII] (1.5 g, 6.2 mmole, in ethanol (40 ml)) was added with s t i r r i n g saturated sodium bicarbonate solution (2 ml) and sodium periodate (1.32 g, 6.2 mmole, dissolved in water (70 ml)). After the solution was l e f t stand for 3 hr in the dark at room temperature a few drops of ethylene glycol were added to destroy any unreacted periodate. Sodium borohydride (120 mg) was then added followed after 4 hr by acetone (0.5 ml) and the mixture stirred for an additional 0.5 hr. After f i l t r a t i o n the solution was extracted with methylene chloride (4 x 100 ml) and-; the organic extracts were combined and dried over sodium sulfate. Evaporation of the solvent gave a syrup which was crystallized from ether to afford CXI (lg, 90%), m.p. 70°, [a] 2) 3 +97° (c 1.1, in chloroform); i r (nujol) 3500 (OH), 2250 cm"1 (C=N). -122-Anal. Calcd. for C 1 0 H 1 5 N 1 ° 4 : c> 56.4; H, 7.05; N, 6.57. Found: C, 56.6; H, 6.99; N, 6.67. 1,2-Di-0-acetyl-5-0_-benzoyl-3-C-cyanome thy 1-3-deoxy-S-D-ribof uranose [CXIII] 5-0_-Benzoyl-3-C-cyanomethyl-3-deoxy-l,2-0-isopropylidene-a-D-ribofuranose [CXII] (7 g) was dissolved in 90% trifluoroacetic acid (42 ml) and let stand at room temperature for 22 minutes. The reaction mixture was then diluted with toluene (100 ml) and the solvent evaporated under vacuum (about 1 mm). The last traces of acid were removed by a second d i s t i l l a t i o n of toluene from the product and the remaining material (6.1 g) was then acetylated with acetic anhydride (20 ml) and pyridine (20 ml) for 24 hr at room temperature. The acetylation mixture was poured into ice water and worked up as described for compound CX. The syrupy product (7 g) was dissolved in ethanol and allowed to stand at 0° overnight during which time some of the t i t l e acetate CXIII (3.5 g, 44% based on CXII) crystallized. The mother liquor was concentratedto a syrup and chromatographed on a s i l i c a gel column using benzene:ethyl acetate (3:1) as developer to afford an additional 2 g of acetate CXII (25%) and a slightly faster moving component (0.4 g). The main component was crystallized from ethanol to afford 1,2-di-0_-acetyl-5-£-benzoyl-3-C-cyanomethyl-3-deoxy-24 g-D-ribofuranose [CXII], m.p. 117°; [a] -21.9° (c_1.5, in chloroform); i r (nujol) 2260 cm (C=N) ; T^±3 1.8-2.7 (m, 5p, Bz), 3.80 (s, lp, H-l), 4.66 (d, lp, H-2), 5.46 (d, 2p, C-5 CH ), 5.6-6.0 (m, lp, H-4), 7.2-8 (m, 3p, CH2-C^N and H-3), 8.43, 8.63 (2s, 6p, 2 Ac). -123-Anal. Calcd. for C ^ H ^ N ^ : C, 59.82; H, 5.31; N, 3.81. Found: C, 59.56; H, 5.17; N, 3.53. The minor component was crystallized from ethanol to give 1-0-acetyl-5-0_-benzoyl-3-C_-carboxymethyl-2 ,3-Y-lactone-3-deoxy-3-D-2A ribofuranose [CXV], m.p. 137°, [cJ^ -95.7° (c 1.6, in chloroform); - i r n n i r (nujol) 1700-1780 cm (C=0); x 3 1.9-2.7 (m, 5p, Bz), 3.6 (s, lp, H-l), 5.0 (d, lp, H-2), 5.5-5.9 (m, 3p, C-5CH2 and H-4), 6.7-7.5 (m, -CH2-C-0 and H-3), 8.0 (s, 3p, Ac). Anal. Calcd. for C.^ H.,,0-,: C, 60.00; H, 5.04. Found: C, 59.80; I D I D / H, 5.18. 6-Chloro-9-(2'-0-acetyl-5',6'-di-0-benzoyl-3'-C-cyanomethyl-3'-deoxy-g-D-allof uranosyl)-purine [CXVI] A thoroughly dried, finely powdered intimate mixture of 1,2-di-0-acety1-5,6-di-0-benzoyl-3-C-cyanomethy1-3-deoxy-g-D-allofuranose [CX] (1 g) and 6-chloropurine (350 mg) was heated in an o i l bath at 160° and 30 mm pressure for 5 minutes, followed by further heating at 160° and 0.1 mm for an additional 40 minutes. The melt was then cooled to room temperature and extracted with dichloromethane (50 ml). Fi l t r a t i o n and evaporation of the dichloromethane extract gave a yellow foam which was chromatographed on a column of grade II s i l i c a gel using benzene:ethyl acetate (1:1) as developer to afford two fractions. The f i r s t eluted component proved to be unreacted starting material (150 mg) and the second component was the t i t l e nucleoside CXVI (700 mg, 69% yield). This nucleoside remained as an amorphous 22 foam and could not be crystallized: [ ° t ] n -13° (c 1.7, in chloroform); -124-- i r n n i r (film) 2230 cm (C=N); T^U<J±3 1.42, 1.74 (2s, H-2, H-8), 3.9 (d, lp, H - l 1 , J l t 2, = 2 Hz), 7.2 (d, 2p, CH2C=N). Anal. Calcd. for C o nH 0.C1 1N C0^: C, 59.19; H, 4.10; N, 11.87. 29 24 1 5 7 Found : C, 59.46; H, 4.35; N, 11.47. 6-Chloro-9-(2'-0_-acetyl-5,-0-benzoyl-3'-C_-cyanomethyl-3'-deoxy-6-D-ribofuranosyl)-purine [CXVIII] A thoroughly dried mixture of 1, 2-di-0J-acetyl-5-0-benzoyl-3-C-cyanomethyl-3-deoxy-B-p-ribof uranose [CXIII] (722 mg) and 6-chloropurine (325 rag) was fused as described for compound CXVI. Chromatography of the material isolated after fusion on a column of t i c s i l i c a gel using benzene:ethyl acetate:ethanol (10:10:1) as developer afforded the t i t l e nucleoside CXVIII (600 mg, 66%) after crystallization from 23 ethanol, m.p. 136.5-137°; [ c t ] D +15.5° (c 1.5, in chloroform); i r - i r n n (nujol) 2250 cm x (C=N); T 3 1.5, 1.74 (2s, 2p, H-2, H-8), 3.96 (d, lp, H-l', J l t 2 , = 1 Hz), 7.2 (d, 2p, C H 2 C H N ) , 7.78 (s, 3p, Ac). Anal. Calcd. for O N c 0 c C l : C, 55.33; H, 3.98; N , 15.35. Found: C, 55.00; H, 3.6; N , 15.14. 6-N,N-Dimethylamino-9-(3'-C-N,N-dimethylcarbamoylmethyl-3'-deoxy-B-D-allofuranosyl)-purine [CXXI] To a solution of 6-chloro-9'- (2' -0-acetyl-5' , 6' -di-0-benzoyl-3'-C-cyanomethyl-3'-deoxy -B-p _allofuranosyl)-purine [CXVI] (450 mg in 20 ml methanol) was added dropwise 25% aqueous dimethylamine solution (10 ml) and the mixture l e f t to stand at room temperature for four hrs. After -125-evaporation of the solvent the remaining syrup was chromatographed on a column of t i c s i l i c a gel using dichloromethanemethanol (93:7) as developer to afford the t i t l e nucleoside (240 mg, 78% yield) which was crystallized from a methanol-ether mixture, m.p. 184-185°, 23 [a]_ -66° (c 1.8, in methanol); uv X 275 nm (e 20,000 in methanol); D — * max cd X 275 nm (6 -11,000, c 0.0047, in methanol); i r (KBr) 1630 cm"1 max ' ' ' ' r n n (0=0); T 3 2.0, 2.17 (2s, 2p, H-2, H-8), 6.57 (s, 6p, N(Me)2), 6.93, 7.04 (2s, 6 p, 0CN(Me)2); T D M S 0 _ d 6 4.28, 4.64 (2d, 2p, C-2'0H, C-5'0H). Molecular weight Calcd: 394. Found by mass spectrometry: 394. Anal. Calcd. for C.-,H_,0CN,: C, 51.79; H, 6.64; N, 21.31. Found: 17 26 5 6 C, 51.69; H, 6.71; N, 21.28. 6-N,N-Dimethylamino-9- (3' -C_-N,N-dimethylcarbamoylmethyl-3' -deoxy- g-D-ribofuranosyl)-purine [CXXII] To a solution of 6-chloro-9-(2'-0-acetyl-5'-O-benzoyl-3'-C-cyanomethyl-3'-deoxy-g-D-ribofuranosyl)-purine [CXVIII] (102 mg in methanol (7 ml)) was added dropwise a 25% aqueous solution of dimethyl-amine (2 ml). After 4 hr the solvent was evaporated and the residue chromatographed on a column of t i c s i l i c a gel using dichloromethane: methanol (93:7) as developer to afford the t i t l e amide nucleoside CXXII (64 mg, 72% yield) as a syrup. This compound was homogeneous by chromatography on paper (R^ 0.68 butanol:ethanol:water, 40:19:11), CDC1 and on s i l i c a gel (R^ 0.42 dichloromethane:methanol 9:1); x 1.80 (s, 2p, H-2, and H-8), 6.10 (d, lp, H-l'), 6.43 (b, 2p, C-5'0H and C-2'0H), 5.2-6.5 (m, 4p, H-2, H-4, and C-5'CH2), 6.53 (s, 6p, N(Me2)), -126-0 (i 6.97, 7.08 (2s, 6p, CN^le^). This compound could not be induced to 25 crystallize; [a]„ -31.2° (c 1.37, in water); uv X 275 nm (e 14,300, D — max in water) i r (film) 3200-3500 cm"1 (OH), 1640 cm"1 (C=0). Anal. Calcd. for C-.H-.N-O.: C, 52.74; H, 6.65; N, 23.06. Found: 16 24 6 4 C, 50.86; H, 6.43; N, 22.40 6-N,N-Dimethylamino-9-(2* ,5 '-di-0--acetyl-3'-Cj-N,N-dimethylcarbamoylmethyl-3'-deoxy-g-D-ribofuranosyl)-purine A solution of CXXII (50 mg) in pyridine (0.5 ml) and acetic anhydride (0.5 ml) was stored at room temperature for 20 hrs. After this time the reaction mixture was diluted with ice water (10 ml) and extracted with chloroform (3 x 20 ml). The chloroform extracts were dried over sodium sulfate and evaporated. The material remaining after evaporation was chromatographed on a column of t i c s i l i c a to 25 yield 55 mg (40%) of the t i t l e nucleoside as a syrup; f a ] D -25.2° (c 1, in chloroform). Anal. Calcd. for c 2 o H 2 8 N 6 ° 6 : C ' 5 3 - 6 4 ; H> 6- 2 95 N» 18.74. Found: C, 53.90; H, 6.31; N, 18.65. Preparation of 6-N,N-Dimethylamino-9-(3'-C_-N,N_-dimethylcarbamoylmethyl-3'-deoxy-g-D-ribofuranosyl)-purine [CXXII] from CXXVIII 6-N, .N-Dimethylamino-9-(3'-C-carboxymethy1-2',3'-y-lactone-3'-deoxy-g-D-ribofuranosyl)-purine [CXXVIII] (30 mg) was dissolved in dimethylamine (3 ml) and allowed to stand at 0° for 4 hr. After evaporation of the dimethylamine from the reaction mixture, the branched-chain N_,N_-dimethylcarbamoylmethyl nucleoside CXXII (34 mg, -127-quantitative yield) was recovered, having i r and nmr spectra identical to those of the product obtained by treatment of CXVIII with aqueous dime thylamine. Preparation of CXXII from CXXI Sodium periodate (152 mg) was added to a solution of 6-N,N-dimethylamino-9-(3'-C_-N,N-dimethylcarbamoylmethyl-3'-deoxy-g-D-al l o f uranosyl) -purine [CXXI] (275 mg in 21 ml water, 14 ml ethanol and 0.5 ml saturated sodium bicarbonate solution) and the mixture was stirred in the dark for 2.5 hrs. Sodium borohydride (212 mg) was then added and the reaction mixture was stirred for an additional 3 hrs. Unreacted borohydride was destroyed by the addition of a few drops of gla c i a l acetic acid and the solvent was then evaporated. The residue was taken up in methanol, refluxed for five minutes and the methanol evaporated. The remaining material was dissolved in dichloromethane and inorganic material removed by f i l t r a t i o n . The material remaining after evaporation of the f i l t r a t e was chromatographed on a column of t i c s i l i c a gel using dichloromethane:methanol (93:7) as developer to afford the pentose amide nucleoside CXXI (170 mg, 68% yield) as a syrup identical by nmr and i r with the product obtained by treatment of CXVIII with dimethylamine. Preparation of 6-N,N-dimethylamino-9-(31 -C_-N,N-dimethylcarbamoy Diethyl-s'-deoxy-g-D-ribof uranosyl)-purine [CXXII] from CXXXI 6-N,N-Dimethylamino-9-(3'-C-cyanome thy1-3'-deoxy-g-D-ribofuranosyl)-purine [CXXXI] (20 mg) was dissolved in a mixture of methanol (4 ml) -128-and 25% aqueous dimethylamine (2 ml). After the reaction mixture had stood at room temperature for 12 hrs the solvent was evaporated to yield CXXII (23 mg, quantitative yield) as a syrup identical by i r and nmr with the product obtained by treatment of CXVIII with aqueous dimethylamine. 6-N,N-Dimethylamino-9-(3'-C_-carboxymethyl-2',3'-y-lactone-3-deoxy-B-D-ribofuranosyl)-purine [CXXVIII] Sublimation of 6-N,N_-dimethylamino-9-(3'-C_-N,N-dimethylcarbamoyl-methyl-3'-deoxy-B-D-ribofuranosyl)-purine [CXXII] (30 mg) at 210° and 0.1 mm afforded, after crystallization from ethyl acetate, the t i t l e lactone nucleoside CXXVIII (19 mg, 73%); m.p. 198-199° (with 22 sublimation); [a]^ -57.5° (c 1.1, in chloroform); uv X 274 nm D — max (e 14,500, in methanol); cd X 274 nm (e -10,000, c 0.004 in methanol); max i r (KBr) 1770 cm (C=0) ; TK'uy'x3 1. 73, 2.23 (2s, 2p, H-2, H-8), 6.48 (s, 6p, N(Me) 2); T D M S 0 _ d 6 4.93 ( t, lp, C-5'OH). The hydroxyl absorption disappeared on addition of B^O. Molecular weight Calcd: 319. Found by mass spectrometry: 319. Anal. Calcd. for C, .H., _,NC0. : C, 52.65; H, 5.37; N, 21.93. Found: 14 17 5 4 C, 52.43; H, 5.54; N, 21.83. 6-N_,N_-Dimethylamino-9- (3* -C-carbamoylmethyl-31 -deoxy-g-D-ribof uranosyl) -purine [CXXIX] 6-N,N-Dimethylamino-9-(3'-C-carboxymethyl-2',3'-y-lactone-3-deoxy-g-D-ribofuranosyl)-purine [CXXVIII] (30 mg) was dissolved in liquid ammonia (3 ml) and the ammonia was allowed to evaporated slowly during -129-a period of six hrs. The resultant residue was crystallized from ethanol to afford the t i t l e nucleoside CXXIX (30 mg, 95%), m.p. 207°, [ a ] 2 3 -29.9° (c 0.5, in water); i r (nujol) 1650 cm"1 (C=0); T D M S 0 - d 6 D — 0 II 2.60, 3.13 (b, 2p, -C-NH2), 4.08 (d, lp, C-2'0H), 4.83 (t, lp, C-5'0H); uv X 275 nm (e 14,100 in water); cd X 275 nm (6 -6,000, max max c 0.0057, in water). Anal. Calcd. for C1.HonN,0.: C, 49.99; H, 5.99; N, 24.98. Found: 14 20 6 4 C, 49.59; H, 5.94; N, 24.72. 6-N,N-Dimethylamino-9-(3'-C-carbamoylmethy1-N-glycine ethyl ester-3'-deoxy-8-D-ribofuranosyl)-purine CXXX] 6-N, N-Dimethylamino-9-(3 '-C_-carboxymethyl-2 1 ,3' -y-lactone-3-deoxy-8-D-ribofuranosyl)-purine [CXXVII] (40.mg) was dissolved in a mixture of N»N_-dimethylformamide (0.75 ml) and ethyl glycinate (0.25 ml) and stirred at room temperature for 30 hr. Volatile material was removed by d i s t i l l a t i o n (50°, 0.1 mm) and the remaining residue column chromatographed on t i c s i l i c a gel using dichloromethane:methanol (9:1) as developer to afford, after crystallization from ethyl acetate, the t i t l e nucleoside CXXX (38 mg, 72%), m.p. 155-7°, [ c t ] 2 5 -48.8° (c 1.3, in chloroform); uv X 275 nm (e 14,600, in water); cd X — ' max ' ' max 275 nm (6 -8,550, c 0.0043, in water); i r (KBr) 1730 (C=0 ester), - i r n n 1650 cm (C=0 amide); x 3 1.83, 2.00 (2s, 2p, H-2, H-8); 1.8 (b, lp, NH), 4.10 (d, lp, H-l), 8.70 (t, 3p, CH3 of ethyl ester). Anal. Calcd. for C. oHo£N,0-: C, 51.1; H, 6.21; N, 19.89. lO ZD 0 O Found: C, 50.92; H, 6.19; N, 19.61. -130-6-f£,N-DInethylamino-9-(3 '-C-cyanomethyl-3' -deoxj'-g-D-ribof uranosyl) -purine fCXXXI] 6-Chloro-9-(2'-0-acetyl-5 '-O-benzoyl-31 -C-cyanomethyl-3' -deoxy-8-D-ribofuranosyl)-purine [CXVIII] (268 mg) was dissolved in anhydrous dimethylamine (30 ml) and stored at -10° for twenty days. The dimethylamine was then evaporated and the residue triturated with ether (5 ml). The material remaining after the ether was decanted was taken up in ethanol and allowed to stand at 0° for twenty four hrs. A portion of the t i t l e nucleoside (94 mg) crystallized directly out of this solution and a further 60 mg (total yield 78%) was obtained by chromatography of the mother liquor on a column of t i c s i l i c a gel using dichloromethane:ethanol (93:7) as developer. Nucleoside CXXXI was crystallized from ethanol, m.p. 206° with 25 sublimation, [a]_ -39.4° (c 0.6, in ethanol); uv X 275 nm (e 15,800, D max in water); cd X 275 nm (6 -6,100, c 0.0048, in water); i r (KBr) max 2230 cm"1 ( C E N ) ; T D M S 0 ~ d 6 1.70, 1.76 (2s, sp, H-2, H-8), 3.98 (d, lp, H-l'), 6.80 (s, 6p, N(Me)£). Anal. Calcd. for C, ,H., oN,0o: C, 52.82; H, 5.70; N, 26.40. 14 lo o j Found: C, 52.64; H, 5.64; N, 26.42. 6-N,N-Dimethylamino-9-(3'-(2"-acetamidoethyl)-3'-deoxy-g-D-ribofuranosyl)-purine [CXXXIV] 6-N,Nj-Dimethylamino-9-(3'-C-cyanomethy1-3'-deoxy-g-D-ribofuranosyl)-purine [CXXXI] (32 mg) was dissolved in a mixture of acetic anhydride (2 ml) and absolute ethanol (2 ml) and hydrogenated over platinum oxide -131-(20 mg) at 60 psi for four hrs. The catalyst was then removed by f i l t r a t i o n and the solvent evaporated to afford 40 mg of syrup. Examination of this product by t i c showed that i t contained two components (Rf 0.18 and 0.10, Rf CXXI 0.62 in dichloromethane:ethyl acetate:ethanol 5:5:1). These-two components were separated by column chromatography on t i c s i l i c a gel using the above developer, to afford 17 mg of the faster component, X D M ^ ° d6 2.14 (broad t, lp, N-H), 7.85, 8.04, 8.20 (3s, 9p, 3Ac), no hydroxyl signals and 19 mg of the slower component, T D M S 0 _ d 6 2.22 (broad t, lp, N-H), 4.18 (d, lp, C-2'0H), 7.98, 8.17 (2s, 6p, 2Ac). The slower moving component was dissolved in 25% aqueous dimethyl-amine solution for 3 hrs. After evaporation of the solvent the remaining material crystallized on trituration with dichloromethane. Reaction of the faster moving component under the same conditions afforded the identical product. The above two products were combined and recrystallized from an isopropanol water mixture to yield CXXIV (23 mg, 63%) which crystallized as the hemi-hydrate, m.p. 193-194°, 25 [aU 1.0 (c 0.9, in ethanol); uv \ 274 nm (e =23,900 water); D — max x D M S 0 " d 6 1.56, 1.76 (2s, 2p, H-2, H-8), 2.19 (t, lp, N-H), 4.0 (s, lp, H-l'), 8.23 (s, 3p, N-Ac). Anal. Calcd. for C. ,H-.N.0 •1/2H.0: C, 51.47; H, 6.74; N, 22.47. 16 24 6 4 2 Found: C, 51.38; H, 6.39; N, 22.07. Chloromercuri-6-benzamidopurine To a stirred solution of 7.8 g (0.028 mole) of mercuric chloride in 100 ml of 50% aqueous ethanol was added 6.8 g (0.028 mole) of 6-benzamidopurine. To the resulting suspension, 10.3 ml of 10% aqueous -132-sodium hydroxide (0.028 mole) was added dropwise with s t i r r i n g . The yellow mixture was stirred 1 hr and then allowed to stand at room temperature for a period of 20 hr. The white solid was f i l t e r e d , washed with 25 ml of cold 50% aqueous ethanol and dried in vacuo over phosphorus pentoxide: yield, 13g (96%). Reported (176): yield 96%. 6-Benzamido-9-(2'-0-acetyl-51,6'-di-0-benzoyl-3'-C-cyanomethyl-3'-deoxy -B-D-allofuranosyl)-purine [CXXXVT] Hydrogen bromide was bubbled into a solution of l,2'di-0-acetyl-5,6-di-0_-benzoyl-3-C_-cyanomethy 1-3-deoxy-g-D-allofuranose [CX] (1 g, 2.02 mmole) in anhydrous dichloromethane (50 ml) at 0° for fifteen minutes. After the reaction mixture was l e f t to stand at 0° for 1 hr and then at room temperature for 15 minutes, the solution was evaporated to a syrup and the last traces of hydrogen bromide were removed by co-evaporation with dry toluene. The syrup, dissolved in dry toluene (40 ml), was immediately added to a suspension of chloromercuri-6-benzamidopurine (960 mg, 2.02 mmole) and Celite (300 mg) in dry toluene (30 ml). After refluxing for 45 minutes the hot solution was f i l t e r e d and evaporated. The residue after evaporation was taken up in dichloromethane (120 ml) and washed successively with aqueous potassium iodide (30%, 2 x 20 ml) and water (2 x 20 ml). The dried (sodium sulfate) solution was evaporated and the resultant material chromatographed on s i l i c a gel (60 g, benzene:ethyl acetate (1:1) as developer) to give the t i t l e nucleoside CXXXVI (900 mg, 60% 22 yield) as aa amorphous foam; [ a ] n -37° (c 1.5, in chloroform); i r film - i r n n 2230 cm (CEN); x 3 0.8 (b, lp, NH), 1.38 (s, lp, H-2), 1.9-2.8 -133-(m, 16p, 3 Bz and H-8), 7.25 (d,2p, CH^C^N) . Anal. Calcd. for C„,H„ nN,0 o: C, 64.07; H, 4.45; N, 12.47. Found: JO J U D O C, 63.76; H, 4.72; N, 12.08. Attempted preparation of CXXXVI using titanium tetrachloride chloromercuri-6-benzamido-purine method (176) A mixture of CX (100 mg), chloromercuri-6-benzamidopurine (105 mg), Celite (100 mg) and anhydrous xylene (15 ml) was dried by d i s t i l l i n g off the xylene under reduced pressure (about 50 mm)• To the resulting residue was added anhydrous ethylene chloride (25 ml) and 15 ml of the solvent was d i s t i l l e d . The mixture was then cooled to 30° and titanium tetrachloride was (30 yl) added and the reaction mixture was heated under reflux for 16 hrs. The cooled reaction mixture was poured into saturated sodium bicarbonate solution and stirred vigorously for 30 min. and then f i l t e r e d . The f i l t e r cake was washed withdichloromethane (10 ml) and the combined organic extracts were washed with 30% aqueous potassium iodide solution (5 ml) and water ( 3 x 5 ml). The organic extract was then dried over sodium sulfate and the solvent evaporated. The remaining residue was chromatographed on a column of s i l i c a gel to yield 50 mg of a homogeneous syrup; CDC1 T 3 1.8-2.8 (m, lOp, 2 Bz), 7.80 (s, 3p, Ac). The compound did not contain a purine moiety. -134-6-Benzamido-9- (2' -O-acetyl-5 '-O-benzoyl-31 -C-cyanomethyl-3' -deoxy-g-D-ribof uranosyl) -purine [CXXXVII] Hydrogen bromide was bubbled into a solution of 1,2-di-0-acetyl-5-0_-benzoyl-3-£-cyanomethyl-3-deoxy-g-D-ribofuranose [CXIII] (500 mg) in anhydrous dichloromethane (25 ml) at 0° for 15 minutes. After being kept at 0° for 1 hr and at room temperature for 15 minutes, the solution was evaporated to a syrup and the last traces of hydrogen bromide were removed by co-evaporation with dry toluene. The resultant syrup was redissolved in toluene (10 ml) and added to a suspension of chloromercuri-6-benzamidopurine (658 mg) and Celite (500 mg) in toluene (50 ml) at 65°. (The above Celite, chloromercuri-6-benzamido-purine mixture had been previously dried by d i s t i l l i n g off 20 ml of toluene from the mixture.) When addition was completed the mixture was refluxed for one hr and then worked up as previously described for compound CXXXVII. The material resulting from this procedure (508 mg) was chromatographed on s i l i c a gel using benzene:ethyl acetate:ethanol (5:5:1) as developer to afford nucleoside CXXXVII 25 (298 mg, 40% yield) as an amorphous foam; [ a ] n +3.1° (c 1.2, in -1 CT)C~\ chloroform); i r (film) 2250 cm (C=N); x 3 0.75-1.00 (b, l p , N H ) 1.46 (s, lp, H-2 or H-8), 7.26 (d, 2p, - C H 2 ~ C E N ) , 7.83 (s,3p, Ac). Anal. Calcd. for C„ oH O / N,0,: C, 62.22; H, 4.48; N , 15.55. Found: C , 61.99; H, 4.80; N , 15.50. 9-(3'-C-Aminoethy1-3'-deoxy-g-D-allofuranosyl)-adenine [CXXXIX] To a suspension of LAH (210 mg, 5.5 mmole) in tetrahydrofuran (150 ml) was added dropwise a solution of 6-benzamido-9-(2' -O-acetyl--135-5',6'-di-O-benzoy1-3'-C-cyanomethy1-3'-deoxy-g-D-allofuranosyl)-purine [CXXXVI] (826 mg, 1.23 mmole) in THF. After 0.5 hr at room temperature and 2 hr reflux the excess reagent was destroyed by slow addition of water (10 ml), ethanol (10 ml), and 5 N NH^ OH (10 ml). The resulting precipitate was removed by f i l t r a t i o n and washed with ethanol (50 ml). The residue obtained by evaporation of the combined f i l t r a t e and washings was partitioned between dichloromethane (10 ml) and water (75 ml). Examination of the dichloromethane extract showed that i t contained no nucleoside, nor any material giving a positive test with ninhydrin. The water extract was evaporated to dryness and the remaining material (700 mg)taken up in ethanol and l e f t at 0° overnight. From this solution was obtained 200 mg of crystalline product having an ultraviolet spectrum similar to that of adenosine. The ultraviolet spectrum of the mother liquor indicated that i t contained a negligible amount of nucleoside. The above crystalline material was dissolved in 2% acetic acid (2 ml) and chromatographed on 5 ml of Dowex 50 W-X2 (NH^+ form) resin. The column was f i r s t washed with 100 ml water and then with 5% ammonium hydroxide to afford after crystallization of the main component from methanol nucleoside CXXXIX (80 mg, 20%), m.p. 170-171°, 25 [a]_ -59.1° (c 1.2, in water); uv X , 261 nm (e 15,000, in water); u in 3.x D^MSO-dg 1 % 6 6 t 1 > 8 2 ( 2 S j 2p, H-2, H-8), 2.70 (b, 2p, NH ), 4.10 (d, lp, H-l'), 4.2-4.6 (b, 2p, NH ) , 5.28 (t, lp, H-2'). Anal. Calcd. for C1oHOAN,0,: C, 48.14; H, 6.62; N, 25.91. Found: 13 20 6 4 C, 44.45; H, 5.41; N, 21.69. The sample contained some inorganic material which could not be removed. -136-ADDENDA An interesting rearrangement of cyanovinyl sugar LXXXIV was discovered too late to be included in the body of this thesis and is therefore added as a brief note here. When a sample of LXXXIV (1 g, Rf 0.49 benzene:methanol 9:1) was hydrogenated at ambient pressure and temperature over 5% palladium-on-charcoal (500 mg), i t was observed that reduction was very slow (0.19 equivalents of hydrogen absorbed in 24 hours). Apparently in this case the catalyst had been inadvertently poisoned as normally this hydrogenation was completed in about two hours. A t i c examination of the hydrogenation mixture after 24 hours indicated the presence of three components (R^ 0.57 major, R^  0.49 and R^  0.35 benzene:methanol 9:1). The catalyst was then removed by f i l t r a t i o n , the solvent was evaporated and the remaining material was chromatographed on tic s i l i c a gel (with benzene:ethyl acetate 4:1 as developer). This procedure afforded three compounds. Two of these products were readily identified, one being the unreduced starting material LXXXIV (170 mg, 0.49) and the other being the expected reduction product LXXXVII (140 mg, R^  0.35). Surprisingly the third component was identified on the basis of CDC1 nmr evidence as the unsaturated sugar CXL (x 3, 3.97 (d, lp, H-l, J „ = 5 Hz), 4.71 (broad d, lp, H-2), 5.28 (broad t, lp, H-5), 5.6 - 6.2 (m, 2p, C-6 methylene), 6.55 (d, 2p, CH2CN). These nmr values should be compared with those obtained from enol acetate LXXI r n n (x 3 3.97 (d, lp, H-l), 4.60 (d, lp, H-2), 5.30 (t, lp, H-5), -137-5.97 (d, 2p, C-6 methylene). Hydrogenation at ambient pressure and temperature of CXL over active 5% palladium-on-charcoal afforded as the only product the cyanomethyl sugar LXXXVII. A palladium catalyzed rearrangement of a double bond has been noted by Slessor and Tracy (119) in the hydrogenation of enol acetate LXXI. Further investigation of the palladium catalyzed rearrangements of these branched-chain sugars is underway. Me 0 Me' •Me Me Me [LXXXIV] [CXL] Me Pd/C [LXXXVII] -138-REFERENCES 1. a) J. S. Brimacombe. Angew. Chem. Internat. Ed. 10, 236 (1971). b) J. D. Dutcher. Adv. Carbohydr. Chem. 18, 259 (1963). 2. a) G. W. Waller, P. W. Fryth, B. L. Hutchings, and J. H. Williams. J. Am. Chem. Soc. _75, 2025 (1953). b) H. M. Kissman, C. Pidacks, and B. R. Baker. J. Am. Chem. Soc. T7_y 18 (1955). 3. a) R. I. Hewitt, A. R. Gumble, W. S. Wallace, and J. H. Williams. Antibiotic and Chemotherapy. 4_, 1222 (1954). b) P. L. Bennett, 5. L. Halliday, J. J. Oleson, and J. H. Williams. Antibiotics Annual (1954-55). 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