Patent Application: US-91029197-A

Abstract:
a class of substituted and unsubstituted nucleo - base analogs and related azoles , designated as &# 34 ; phosphazoles ,&# 34 ; is disclosed , certain preferred embodiments having the basic structure of ## str1 ## also disclosed are methods of making and using the new compounds .

Description:
the new class of compounds of the present invention , called &# 34 ; phosphazoles &# 34 ; are substituted pyrimidine fused derivatives of three 5 - member heterocyclic rings ( compounds 5 , 6 and 7 ), wherein ## str6 ## trivalent phosphorus ( p ) is an integral part of the heterocycle . among the preferred embodiments of the present invention are compounds having p introduced into a nucleo - base or related heterocycle and the corresponding nucleoside , nucleotide or 3 &# 39 ;, 5 &# 39 ;- cyclic nucleotide , or analogs thereof . phosphazoles can be used in pharmaceuticals or in industrial or agricultural applications , similar to other nucleo - base analogs , with some embodiments of this class being equivalent or superior to known nucleo - base derivatives or related compounds . the following examples are offered by way of illustration only and are not intended to limit the scope of the invention in any manner . the methods of the invention being as readily applicable to any substitution of a heterocycle containing a phosphorus atom in the ring system . the fundamental 1 , 3 - azaphospholine ring was formed by substituting phosphorus for the oxygen atom in a suitable starting material such as 5 - aminooxazole - 4 - carbonitrile ( compound 8 ). the 5 - aminooxazole - 4 - carbonitrile ( compound 8 ), was prepared according to the method by ferris and orgel ( 50 ). treatment of 5 - aminooxazole - 4 - carbonitrile ( compound 8 ) with tris ( trimethyl - silyl )- phosphine ( ttp ) ( 51 ) in the presence of naked fluoride ion , such as a mixture of 18 - crown - 6 / kf ( commercially available from aldrich chemical co ., milwaukee , wis . 53233 ) in anhydrous toluene at reflux temperature gave the key intermediate , 4 - amino - 1h - 1 , 3 - azaphospholine - 5 - carbonitrile ( compound 9 ) in a modest yield . the preparation of compound 9 is described in more detail as follows : dry argon was bubbled for 5 min into a suspension of 18 - crown - 6 ( 50 mg ) and dry potassium fluoride ( 3 . 5 g , 60 mmol ) in dry toluene ( 110 ml ). to this suspension , tris ( trimethylsilyl ) phosphine ( 51 ) ( 21 . 0 ml , 72 . 3 mmol ) and 5 - amino - oxazole - 4 - carbonitrile ( 50 ) ( compound 8 , 6 g , 61 . 8 mmol ) were added and the mixture was heated under reflux for 14 h under an argon atmosphere . the reaction mixture was allowed to cool to room temperature and methanol ( 75 ml ) was added under an argon atmosphere . after stirring for 2 h , the ph of the solution was adjusted to 5 . silica gel ( 50 g ) was added and after stirring for 2 h , the solvents were evaporated to dryness under reduced pressure . the dry powder was placed on a silica gel column ( 4 × 10 cm ). the column was flash eluted with hexane containing 0 - 20 % ethyl acetate . eluate containing the desired product was evaporated to yield 1 . 75 g ( 23 %) of white powder . ir ( kbr ): v 640 (═ p - c ), 1200 (- p ═ ch ), 2205 ( cn ), and3280 - 3440 ( nh , nh 2 ) cm - 1 . 31 p nmr ( cdcl 3 ): δ 72 . 17 . 1 h nmr ( dmso - d 6 ): δ 5 . 75 ( br , 2 h , nh 2 ), 8 . 00 ( dd , 1 h , c 2 h ), and 12 . 25 ( br s , 1 h , nh ). anal . calcd . for c 4 h 4 n 3 p : c , 38 . 41 ; h , 3 . 22 ; n , 33 . 60 ; p , 24 . 76 . found : c , 38 . 73 ; h , 3 . 19 ; n , 33 . 27 ; p , 24 . 51 . the phosphorus ( p ) atom was introduced into the heterocycle in place of the oxygen ( o ) by a ring - opening and recyclization mechanism . this ring - opening and recyclization reaction is believed to be facilitated by the presence of a nitrile function at the meta position to the ring oxygen , as indicated in the steps shown below . ## str7 ## the ring - opening involves the initial formation of the oxazolium zwitterion ( a ) by the reaction of compound 8 and the naked fluoride ion , generated by 18 - crown - 6 / kf . nucleophillic attack on the tertiary carbon atom of the oxazolium zwitterion ( a ) by the tris ( trimethylsilyl ) phosphine gives the intermediate ( b ) . this should be followed by a ring - opening to form the unstable phosphonium salt ( c ), which readily recyclizes into compound 9 with the separation of hexamethyldisiloxane . the compound , 4 - amino - 1h - 1 , 3 - azaphospholine - 5 - carbonitrile ( compound 9 ), is obtained as a stable , light yellow amorphous powder . from this key intermediate compound numerous compounds are synthesized , the preferred embodiments of which are described below . 4 - amino - 1h - 1 , 3 - azaphospholine - 5 - carbonitrile from example 1 is reacted with formamidine acetate in ethanol at reflux temperature to form the ring annulated product 7 - amino - 1h - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( the adenine congener , compound 10 ): ## str8 ## preparation of 7 - amino - 1h - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 10 ) to a solution of 4 - amino - 1h - 1 , 3 - azaphospholine - 5 - carbonitrile ( compound 9 , 0 . 7 g , 5 . 6 mmol ) in absolute ethanol ( 50 ml ) was added formamidine acetate ( 1 . 16 g , 11 . 2 mmol ) and the mixture was heated under reflux for 1 . 5 h . the product that precipitated after cooling was collected by filtration . the filtrate was evaporated under reduced pressure , the residual solid was dissolved in methanol ( 10 ml ) and impregnated onto silica gel ( 10 g ). the solvent was evaporated and the dry powder was loaded on a silica gel column ( 2 × 10 cm ). the column was flash eluted with dichloromethane containing 0 - 8 % methanol . eluate containing the desired product was evaporated and the residue was combined with the solid obtained previously . crystallization of the combined solid from aqueous ethanol gave 0 . 72 g ( 84 . 5 %) of the product , mp & gt ; 300 ° c . ; ir ( kbr ): ν720 (═ p - c ), 1215 (- p ═ ch ), and 3100 - 3440 ( nh , nh 2 ) cm - 1 . uv ( h 2 o ): λmax244 nm ( ε27 , 500 ), and 304 ( 6900 ); 31 p nmr ( dmso - d 6 ): δ74 . 52 . 1 h nmr ( dmso - d 6 ): δ 7 . 13 ( s , 2 h , nh 2 ), 8 . 19 ( s , 1 h , c 5 h ), 8 . 91 ( d , 1 h , c 2 h ), and 12 . 50 ( br s , 1 h , nh ). anal . calcd . for c 5 h 5 n 4 p : c , 39 . 48 ; h , 3 . 31 ; n , 36 . 84 ; p , 20 . 36 . found : c , 39 . 86 ; h , 3 . 25 ; n , 36 . 53 ; p , 20 . 32 . hydrolysis of the nitrile function of 4 - amino - 1h - 1 , 3 - azaphospholine - 5 - carbonitrile ( compound 9 ), from example 1 , is carried out by treatment with 0 . 1n aqueous naoh at gentle reflux for 5 hours to obtain 4 - amino - 1h - 1 , 3 - azaphospholine - 5 - carboxamide ( compound 11 ). the procedure is described in more detail , as follows : a suspension of 4 - amino - 1h - 1 , 3 - azaphospholine - 5 - carbonitrile ( compound 9 , 0 . 5 g , 4 mmol ) in aqueous sodium hydroxide solution ( 28 ml , 0 . 11 n ) was heated at 110 ° c . ( oil bath temp ) for 6 h . the reaction mixture was allowed to cool to room temperature and then cooled in an ice bath . the ph of the solution was adjusted to 6 . 8 ( using 2 n hydrochloric acid ) during which time some product precipitated . the flask was kept in the refrigerator overnight and the solid product was collected by filtration and dried to yield 0 . 4 g ( 70 %) of the product , mp 184 - 186 ° c . ; ir ( kbr ): ν3185 - 3445 ( nh , nh 2 ), and 1720 ( c ═ o ) cm - 1 . 1 h nmr ( dmso - d 6 ): δ 6 . 25 ( br s , 2 h , nh 2 ), 6 . 94 ( s , 2 h , conh 2 ), 7 . 85 ( dd , 1 h , c 2 h ), and 11 . 51 ( br s , 1 h , nh ). anal . calcd . for c 4 h 6 n 3 op : c , 33 . 57 ; h , 4 . 23 ; n , 29 . 37 ; p , 21 . 65 . found : c , 33 . 68 ; h , 4 . 05 ; n , 28 . 83 ; p , 21 . 38 . ## str9 ## compound 11 is then fused with formamide to form the hypoxanthine analog , 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidin - 7 ( 1h , 6h )- one ( compound 12 ) using the following procedure . a mixture of 4 - amino - 1h - 1 , 3 - azaphospholine - 5 - carboxamide ( compound 11 , 0 . 36 g , 2 . 5 mmol ) and formamide ( 0 . 5 ml , 12 . 5 mmol ) in a 5 ml pear shaped flask was placed in a preheated oil bath at 185 ° c . for 45 min . the flask was allowed to cool to room temperature . the solid thus obtained was triturated with cold water ( 5 ml ). the solid was collected by filtration and crystallized from aqueous ethanol to yield 0 . 3 g ( 78 %) of the title compound , mp & gt ; 300 ° c . ir ( kbr ): ν710 (═ p - c ), 1170 and 1200 (- p ═ ch ), 1650 ( c ═ o ), and 2860 - 3080 ( nh , nh 2 ) cm - 1 . uv ( meoh ): λmax244 nm ( ε43 , 300 ), and 274 ( 6200 ). 31 p nmr ( dmso - d 6 ): δ 85 . 54 ; 1 h nmr ( dmso - d 6 ): δ 7 . 90 ( s , 1 h , c 5 h ), 8 . 66 ( dd , 1 h , c 2 h ), 12 . 22 ( br s , 1 h , nh ), and 13 . 32 ( br s , 1 h , nh ). anal . calcd . for c 5 h 4 n 3 op : c , 39 . 23 ; h , 2 . 63 ; n , 27 . 45 ; p , 20 . 23 . found : c , 39 . 39 ; h , 2 . 75 ; n , 27 . 08 ; p , 20 . 02 . this hypoxanthine analog ( compound 12 ) was found to be identical to the one obtained by deamination of adenine analog ( compound 10 ) with aqueous nitrous acid . 4 - amino - 1h - 1 , 3 - azaphospholine - 5 - carboxamide ( compound 11 ), from example 3 , is fused with urea at 160 ° c . for 45 min to obtain the xanthine analog 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine - 5 , 7 ( 1h , 4h , 6h )- dione ( compound 13 ), by the following procedure : a finely powdered mixture of 4 - amino - 1h - 1 , 3 - azaphospholine - 5 - carboxamide ( compound 11 , 2 . 14 g , 14 . 96 mmol ) and urea ( 4 . 29 g , 71 . 43 mmol ) was placed in an open round bottom flask . the flask was immersed up to the neck in a preheated oil bath at 160 ° c . with efficient stirring . heating was continued until the starting material ( compound 11 ) was consumed ( tlc on silica gel plate using ch 2 cl 2 : meoh , 85 : 15 , as developing solvent ). the reaction mixture was cooled to 25 ° c . and sonicated with cold water ( 25 ml ) for 1 h . the precipitate was collected by filtration , washed thoroughly with cold water ( 3 × 10 ml ). the solid was suspended in hot ( 80 ° c .) water ( 25 ml ) and 0 . 1 n naoh was added dropwise until a clear solution was obtained . the solution was filtered and the ph of the filtrate was adjusted to 6 with acetic acid . the solid that separated was collected by filtration , washed with ice cold water ( 5 × 15 ml ), followed by ethanol ( 3 × 15 ml ) and dried at 100 ° c . under vacuum for 15 h to yield 2 . 16 g ( 85 %) of the title compound , mp & gt ; 300 ° c . uv ( meoh ): λmax242 nm ( ε39 , 300 ), 276 ( 9200 ), and 300 sh ( 4000 ). 1 h nmr ( dmso - d 6 ): δ 8 . 43 ( d , 1 h , j = 43 . 5 hz , c 2 h ), 11 . 07 and 11 . 21 ( 2s , 2 h , 2nh ). anal . calcd . for c 5 h 4 n 3 o 2 p . 0 . 5h 2 o : c , 33 . 72 ; h , 2 . 83 ; n , 23 . 59 ; p , 17 . 39 . found : c , 33 . 72 ; h , 2 . 62 ; n , 23 . 50 ; p , 17 . 61 . ## str10 ## the hypoxanthine analog from example 3 is reacted with pocl 3 at reflux temperature for 50 min to form 7 - chloro - 1h - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 14 ). reaction of compound 14 with thiourea in boiling ethanol produced the 6 - thiopurine analog 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidin - 7 ( 1h , 6h )- thione ( compound 15 ). compounds 14 and 15 are prepared as follows : a mixture of 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidin - 7 ( 1h , 6h )- one ( compound 12 , 5 . 0 g , 32 . 66 mmol ) and phosphorus oxychloride ( 125 ml ) was heated under reflux with stirring for 50 min under an argon atmosphere . the reaction mixture was cooled to room temperature and unreacted phosphorus oxychloride was removed under reduced pressure at 25 ° c . the residual syrup was cooled to 0 ° c . and carefully neutralized with 2 n aqueous ammonium hydroxide . the precipitate formed was collected by filtration , washed with cold water ( 2 × 25 ml ) and dried over p 2 o 5 under vacuum , first at 25 ° c . and then at 100 ° c . the dry solid was crystallized from acetonitrile to yield 2 . 73 g ( 49 %) of the title compound , mp & gt ; 300 ° c . ( became black at 190 ° c .). 1 h nmr ( dmso - d 6 ): δ 8 . 81 ( s , 1 h , c 5 h ), 9 . 36 ( dd , 1 h , j c2h , nh = 4 . 8 hz , j c2h , p = 39 . 4 hz , c 2 h ), and 13 . 68 ( br s , 1 h , nh ). anal . calcd . for c 5 h 3 cln 3 p : c , 35 . 02 ; h , 1 . 76 ; cl , 20 . 67 ; n , 24 . 50 ; p , 18 . 05 . found : c , 35 . 36 ; h , 2 . 08 ; cl , 21 . 10 ; n , 24 . 21 ; p , 17 . 78 . a mixture of 7 - chloro - 1h - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 14 , 0 . 23 g , 1 . 33 mmol ), thiourea ( 0 . 15 g , 2 . 0 mmol ) and absolute ethanol ( 10 ml ) was heated under reflux for 90 min with stirring under anhydrous conditions . the reaction mixture was cooled to 25 ° c . and then the solvent was evaporated in vacuo . the residue was sonicated with ice - cold water ( 10 ml ) and the precipitate was collected by filtration , washed with cold water ( 3 × 5 ml ) and dried over p 2 o 5 in vacuo first at 25 ° c . and then at 100 ° c . to yield 0 . 12 g ( 55 %) of the title compound , mp & gt ; 300 ° c . ir ( kbr ): ν710 (═ p - c ), 1170 and 1200 (- p ═ ch ), 1580 ( c ═ s ), and 3280 ( nh ) cm - 1 . uv ( meoh ): λmax230 nm ( ε19 , 400 ), 276 ( 9400 ), and 338 ( 17 , 200 ). 1 h nmr ( dmso - d 6 ): 8 . 11 ( s , 1 h , c 5 h ), 8 . 93 ( dd , 1 h , j c2h , nh = 5 . 4 hz , j c2h , p = 39 . 6 hz , c 2 h ), δ 13 . 15 ( s , 1 h , nh ), and 13 . 82 ( s , 1 h , nh ). anal . calcd . for c 5 h 4 n 3 ps . h 2 o : c , 32 . 08 ; h , 3 . 23 ; n , 22 . 45 ; s , 17 . 13 . found : c , 32 . 39 ; h , 3 . 61 ; n , 22 . 54 ; s , 17 . 40 . ## str11 ## the guanine analog 5 - amino - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidin - 7 ( 1h , 6h )- one ( compound 18 ) is obtained from the amino - carboxamide analog of example 3 by reacting with benzoylisothiocyanate to form 4 -( n &# 39 ;- benzoylthiocarbonyl ) amino - 1h - 1 , 3 - azaphospholine - 5 - carbox - amide ( 16 ), which is then reacted with ch 3 i in 0 . 1n naoh solution to give 4 -( n &# 39 ;- benzoyl - s - methylisothiocarbamoyl ) amino - 1h - 1 , 3 - azaphospholin - 5 - carboxamide ( 17 ), which in the presence of ammonia , forms the guanine analog ( compound 18 ). the preparation of compounds 16 - 18 are described in more detail as follows : 4 - amino - 1h - 1 , 3 - azaphospholine - 5 - carboxamide ( compound 11 , 0 . 95 g , 6 . 64 mmol ) was dissolved in a mixture of ethanol ( 15 ml ) and water ( 7 . 5 ml ) at 75 ° c . the resulting solution was cooled to 35 ° c . benzoylisothiocyanate ( 1 . 79 g , 10 . 97 mmol ) was added dropwise under an argon atmosphere with stirring . the mixture was stirred at room temperature for 2 h and the precipitate was collected by filtration , washed with water ( 3 × 15 ml ) and then with cold ethanol ( 10 ml ). the solid was dried over p 2 o 5 in vacuo , first at 25 ° c . ( 8 h ) and then at 80 ° c . ( 24 h ) to yield 2 . 01 g ( 99 %) of the title compound , mp & gt ; 300 ° c . 1 h nmr ( dmso - d 6 ): δ 7 . 50 - 7 . 66 ( m , 5 h , m , p - protons of benzoyl and conh 2 ), 7 . 97 ( d , 2 h , o - protons of benzoyl ), 8 . 15 ( dd , 1 h , j c2h , nh = 5 . 4 hz , j c2h , p = 39 . 0 hz , c 2 h ), 11 . 31 ( s , 1 h , nh ), 12 . 57 ( s , 1 h , nh ), and 14 . 55 ( s , 1 h , nh ). to a solution of 4 -( n &# 39 ;- benzoylthiocarbamoyl ) amino - 1h - 1 , 3 - azaphospho - line - 5 - carboxamide ( compound 16 , 0 . 918 g , 3 . 0 mmol ) in 0 . 1 n sodium hydroxide ( 115 ml ) was added iodomethane ( 0 . 205 ml , 3 . 30 mmol ) dropwise at 25 ° c . the mixture was stirred at 25 ° c . for 3 h , then cooled to 0 ° c . and acidified to ph 6 with acetic acid . the mixture was extracted with ethyl acetate ( 5 × 75 ml ). the combined ethyl acetate extracts were dried over anhydrous sodium sulfate and concentrated to 25 ml under reduced pressure and allowed to stand at 25 ° c . for crystallization to occur . the crystalline material was collected by filtration and dried to give 0 . 21 g ( 22 %) of the title compound , mp & gt ; 250 ° c . ( dec ). 1 h nmr ( dmso - d 6 ): δ 2 . 62 ( s , 3 h , sch 3 ), 7 . 47 - 7 . 64 ( m , 5 h , benzoyl ), 7 . 84 ( s , 2 h , conh 2 ), 8 . 39 ( m , 1 h , c 2 h ), 12 . 44 ( d , 1 h , nh ), and 12 . 70 ( br s , 1 h , nh ). a mixture of 4 -( n &# 39 ;- benzoyl - s - methylisothiocarbamoyl ) amino - 1h - 1 , 3 - azaphospholine - 5 - carboxamide ( compound 17 , 2 . 0 g , 6 . 24 mmol ) and 2 % ammonia in dimethylformamide ( 60 ml ) was placed in a closed steel reaction vessel and heated at 130 ° c . with stirring for 16 h . the mixture was cooled to 25 ° c ., diluted with methanol ( 50 ml ) and stirred in an open flask for 1 day . a white solid that separated from the reaction mixture was collected by filtration , washed with cold methanol ( 3 × 5 ml ) and dried in vacuo at 100 ° c . the dry solid was dissolved in 2 n naoh , filtered and the filtrate was acidified to ph 6 with glacial acetic acid to give a white precipitate . the precipitate was collected by filtration , washed with cold water ( 3 × 15 ml ), followed by acetone ( 2 × 5 ml ) and dried in vacuo at 100 ° c . to yield 0 . 50 g ( 48 %) of the title compound , mp & gt ; 300 ° c . ir ( kbr ): ν690 (═ p - c ), 1170 and 1230 (- p ═ ch ), 1690 ( c ═ o ), and 3170 - 3320 ( nh 2 ) cm - 1 . uv ( meoh ): λmax240 nm ( ε26 , 900 ) and 276 ( 6850 ). 1 h nmr ( dmso - d 6 ): δ 6 . 15 ( br s , 2 h , nh 2 ), 8 . 38 ( dd , 1 h , c 2 h ), 10 . 95 ( br s , 1 h , conh ), and 12 . 76 ( br s , 1 h , nh ). anal . calcd . for c 5 h 5 n 4 op . 0 . 5h 2 o : c , 33 . 90 ; h , 3 . 41 ; n , 31 . 63 ; p , 17 . 49 . found : c , 34 . 07 ; h , 3 . 34 ; n , 31 . 31 ; p , 17 . 07 . ## str12 ## the guanine analog from example 6 is heated under reflux with pocl 3 at 140 ° c . for 50 min to form 5 - amino - 7 - chloro - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 19a ), which on reaction with thiourea in boiling ethanol produced the 6 - thioguanine analog 5 - amino - 1 , 3 - azaphospholo [ 4 , 5 - d ]- pyrimidin - 7 ( 1h , 6h ) - thione ( compound 19b ). ## str13 ## a suspension of 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine - 5 , 7 ( 1h , 4h , 6h ) - dione ( compound 13 , 0 . 2 g , 1 . 18 mmol ) in dmf ( 5 ml ) and n , n - dimethylformamide dimethyl acetal ( 15 ml ) was heated at 80 ° c . for 55 h under anhydrous conditions . after cooling to room temperature the insoluble material was removed by filtration and the filtrate was evaporated in vacuo . the residue was coevaporated with toluene ( 10 ml ) and purified by chromatography over a silica gel column ( 0 . 5 × 5 cm ). the column was flash eluted with dichloromethane containing 1 % methanol . the appropriate fractions containing the desired product were collected and evaporated to give 0 . 13 g ( 52 %) of the title compound as a colorless powder , mp 166 - 168 ° c . uv ( meoh ): λmax240 nm ( ε29 , 350 ), 282 ( 7000 ), and 298 sh ( 4500 ). 31 p nmr ( dmso - d 6 ): δ 48 . 48 . 1 h nmr ( dmso - d 6 ): δ 3 . 20 ( s , 3 h , ch 3 ), 3 . 39 ( s , 3 h , ch 3 ), 4 . 10 ( s , 3 h , ch 3 ), and 8 . 46 ( d , 1 h , j = 43 . 5 hz , c 2 h ). anal . calcd . for c 8 h 10 n 3 o 2 p : c , 45 . 50 ; h , 4 . 77 ; n , 19 . 90 . found : c , 45 . 77 ; h , 4 . 72 ; n , 19 . 87 . ## str14 ## -- ch 2 ch ═ chch 2 ch 3 ( cis and trans ) ## str16 ## -- ch 2 ch ═ c ( ch 3 ) 2 --( ch 2 ) 3 ch 2 oh ## str17 ## the synthesis of compounds corresponding to structure 21 was accomplished by the direct alkylation of the adenine analog 7 - amino - 1h - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( example 2 , compound 10 ) with requisite alkyl halides or properly protected glycosyl ( carbohydrate ) halides . in the case of glycosyl derivatives , subsequent deprotection of the condensed product under alkaline conditions is required . the procedure is described more specifically as follows : preparation of 7 - amino - 1 -( n - pentyl )- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine [ compound 21 , r =( ch 2 ) 4 ch 3 ] 7 - amino - 1h - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 10 , 0 . 25 g , 1 . 64 mmol , dried by coevaporation with dry dmf , 15 ml ) was suspended in dry dmf to which sodium hydride ( 80 mg , 2 mmol , 60 % dispersion in mineral oil ) was added under argon atmosphere . the mixture was stirred at room temperature for 40 min and 1 - bromopentane ( 0 . 25 ml , 2 mmol ) was added . the reaction was continued for 3 h and the solvent was evaporated . the residue was coevaporated with toluene ( 25 ml ). the solid thus obtained was dissolved in a mixture of dichloromethane and methanol ( 1 : 1 , 10 ml ) and impregnated onto silica gel ( 10 g ). the solvents were evaporated under reduced pressure and the dry powder was loaded on a silica gel column ( 2 × 10 cm ). the column was flash eluted with dichloromethane containing 0 - 5 % methanol to yield 0 . 26 g ( 71 %) of the title compound , mp 196 - 198 ° c . ir ( kbr ): ν690 (═ p - c ), 1220 (- p ═ ch ), and 3110 and 3280 ( nh 2 ) cm - 1 . uv ( meoh ): λmax266 nm ( ε23 , 300 ). 31 p nmr ( dmso - d 6 ): δ 62 . 57 . 1 h nmr ( dmso - d 6 ): δ 0 . 83 ( t , 3 h , ch 3 ), 1 . 25 ( m , 4 h , 2ch 2 ), 1 . 92 ( m , 2 h , ch 2 ), 4 . 27 ( t , 2 h , nch 2 ), 8 . 37 and 8 . 67 ( 2s , 2 h , nh 2 ), 8 . 60 ( d , 1 h , c 5 h ), and 9 . 03 ( d , 1 h , j = 54 hz , c 2 h ). anal . calcd . for c 10 h 15 n 4 p . 0 . 5 ch 2 cl 2 : c , 47 . 63 ; h , 6 . 09 ; n , 21 . 17 . found : c , 47 . 47 ; h , 6 . 09 ; n , 21 . 86 . preparation of 7 - amino - 1 -( n - hexyl )- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine [ compound 21 , r =( ch 2 ) 5 ch 3 ] 7 - amino - 1h - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 10 , 0 . 5 g , 3 . 29 mmol ) was suspended in dry dmf to which sodium hydride ( 160 mg , 4 mmol , 60 % dispersion in mineral oil ) was added under an argon atmosphere . the mixture was stirred at room temperature for 40 min and 1 - bromohexane ( 0 . 56 ml , 4 mmol ) was added . the reaction was continued for 3 h and the solvent was evaporated . the residue was dissolved in dichloromethane ( 150 ml ) and washed with water ( 25 ml ). organic layer was separated and the aqueous layer was extracted with dichloromethane ( 2 × 50 ml ). the combined organic layer was dried ( na 2 so 4 ) and evaporated . the residue was purified by silica gel column ( 2 × 15 cm ) chromatography eluting with dichloromethane containing 0 - 4 % methanol . the appropriate fractions containing the desired product were evaporated to yield 0 . 53 g ( 68 %), mp 220 - 222 ° c . ir ( kbr ): ν690 (═ p - c ), 1220 (- p ═ ch ), and 3295 ( nh 2 ) cm - 1 . uv ( ph 1 ): λmax 254 nm ( ε23 , 700 ), 282 ( 11 , 500 ), and 314 ( 5200 ); ( meoh ): λmax 266 ( ε31 , 600 ); ( ph 11 ): λmax 266 nm ( ε33 , 300 ), and 330 ( 2200 ). 31 p nmr ( dmso - d 6 ): 63 . 21 . 1 h nmr ( dmso - d 6 ): δ 0 . 80 ( t , 3 h , ch 3 ), 1 . 23 ( br s , 6 h , 3ch 2 ), 1 . 92 ( t , 2 h , ch 2 ), 4 . 24 ( t , 2 h , nch 2 ), 8 . 15 and 8 . 23 ( 2s , 2 h , nh 2 ), 8 . 44 ( d , 1 h , c 5 h ), and 8 . 86 ( d , 1 h , j = 58 . 5 hz , c 2 h ). anal . calcd . for c 11 h 17 n 4 p : c , 55 . 92 ; h , 7 . 25 ; n , 23 . 72 . found : c , 55 . 49 ; h , 7 . 31 ; n , 23 . 28 . preparation of 7 - amino - 1 -( 4 - acetoxybutyl )- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine [ compound 21 , r =( ch 2 ) 3 ch 2 oac ] to a suspension of 7 - amino - 1h - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 10 , 0 . 3 g , 2 mmol ) in dry dmf ( 20 ml ) was added sodium hydride ( 96 mg , 2 . 4 mmol ) under an argon atmosphere . the mixture was stirred at ambient temperature for 30 min and 4 - bromobutyl acetate ( 0 . 35 ml , 2 . 4 mmol ) was added . after stirring for 3 h , dmf was evaporated and the residue was purified by chromatography over a silica gel column ( 2 × 15 cm ). the column was flash eluted with dichloromethane containing 0 - 5 % methanol . the homogeneous fractions containing the desired product were collected and evaporated under reduced pressure to yield 0 . 34 g ( 64 %) of the title compound , mp 168 - 170 ° c . 31 p nmr ( dmso - d 6 ): δ 62 . 34 . 1 h nmr ( dmso - d 6 ): δ 1 . 56 ( m , 2 h , ch 2 ), 1 . 90 ( m , 5 h , ch 2 , ch 3 ), 4 . 00 ( t , 2 h , nch 2 ), 4 . 30 ( t , 2 h , ch 2 ), 8 . 27 and 8 . 38 ( 2s , 2 h , nh 2 ), 8 . 50 ( d , 1 h , c 5 h ), and 8 . 92 ( d , 1 h , j = 56 . 4 hz , c 2 h ). anal . calcd . for c 11 h 15 n 4 o 2 p : c , 46 . 99 ; h , 5 . 43 ; n , 19 . 49 . found : c , 46 . 69 ; h , 5 . 44 ; n , 19 . 37 . preparation of 7 - amino - 1 -( 4 - hydroxybutyl )- 1 , 3 - amaphospholo -[ 4 , 5 - d ] pyrimidine [ compound 21 , r =( ch 2 ) 3 ch 2 oh ] a mixture of 7 - amino - 1 -( 4 - acetoxybutyl )- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine [ compound 21 , r =( ch 2 ) 3 ch 2 oac ], 0 . 3 g , 1 . 13 mmol ) and methanolic ammonia ( 100 ml , saturated at 0 ° c .) was stirred overnight and the methanolic ammonia was evaporated in vacuo . the residue was purified by silica gel column chromatography . the column was flash eluted with dichloromethane containing 0 - 8 % methanol to yield 0 . 2 g ( 79 %) of the title compound , mp 164 - 166 ° c . ir ( kbr ): ν700 (═ p - c ), 1220 (- p ═ ch ), and 2940 , 3060 and 3260 ( nh 2 , oh ) cm - 1 . uv ( ph 1 ): λmax254 4m ( ε26 , 000 ), 282 ( 13 , 100 ), and 314 ( 5900 ); ( meoh ): λmax266 nm ( ε35 , 900 ), and 330 ( 3100 ); ( ph 11 ): 266 nm ( ε38 , 500 ), and 334 ( 3300 ). 31 p nmr ( dmso - d 6 ) 63 . 64 . 1 h nmr ( dmso - d 6 ): δ 1 . 41 ( m , 2 h , ch 2 ), 1 . 97 ( m , 2 h , ch 2 ), 3 . 40 ( t , 2 h , ch 2 ), 4 . 34 ( t , 2 h , ch 2 ), 4 . 47 ( br s , 1 h , oh ), 8 . 28 and 8 . 39 ( 2s , 2 h , nh 2 ), 8 . 53 ( d , 1 h , c 5 h ), and 8 . 96 ( d , 1 h , j = 56 . 4 hz , c 2 h ). anal . calcd . for c 9 h 13 n 4 op . 0 . 25 ch 2 cl 2 : c , 45 . 26 ; h , 5 . 54 ; n , 22 . 83 . found : c , 45 . 05 ; h , 5 . 49 ; n , 22 . 73 . preparation of 7 - amino - 1 -[( 2 - acetoxyethoxy ) methyl ]- 1 , 3 - azaphospholo -[ 4 , 5 - d ] pyrimidine [ compound 21 , r = ch 2 och 2 ch 2 oac ] to a suspension of 7 - amino - 1h - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 10 , 0 . 5 g , 3 . 29 mmol ) in dmf ( 20 ml ) was added nah ( 0 . 16 g , 4 mmol ) under an argon atmosphere . after stirring the mixture at room temperature for 40 min , the reaction flask was cooled to - 50 ° c . and a solution of 2 -( acetoxyethoxy ) methyl bromide ( 108 ) ( 0 . 78 g , 3 . 96 mmol ) in dmf ( 5 ml ) was added . the reaction flask was allowed to warm to room temperature in 1 . 5 h and the reaction was continued for further 2 h . the solvent was evaporated in vacuo and the residue was coevaporated with toluene ( 10 ml ). the residue was dissolved in a mixture of dichloromethane and methanol ( 1 : 1 , 10 ml ) and impregnated onto silica gel ( 10 g ). the sovents were evaporated and the dry powder was loaded on a silica gel column ( 2 × 15 cm ). the column was flash eluted with dichloromethane containing 0 - 7 % methanol . the appropriate fractions containing the desired product were evaporated to yield 0 . 62 g ( 70 %) of the title compound , mp 134 - 135 ° c . 1 h nmr ( dmso - d 6 ): δ 1 . 89 ( s , 3 h , ch 3 ), 3 . 69 ( dd , 2 h , ch 2 ), 4 . 05 ( t , 2 h , ch 2 ), 5 . 67 ( s , 2 h , ch 2 ), 8 . 57 and 8 . 88 ( 2s , 2 h , nh 2 ), 8 . 70 ( d , 1 h , c5h ), and 9 . 00 ( d , 1 h , j = 51 . 9 hz , c 2 h ). anal . calcd . for c 10 h 13 n 4 o 3 p . 0 . 4 ch 2 cl 2 : c , 39 . 74 ; h , 4 . 60 ; n , 18 . 54 . found : c , 40 . 04 ; h , 4 . 20 ; n , 18 . 38 . preparation of 7 - amino - 1 -[( 2 - hydroxyethoxy ) methyl ]- 1 , 3 - azaphospholo -[ 4 , 5 - d ] pyrimidine [ compound 21 , r = ch 2 och 2 ch 2 oh ] a mixture of 7 - amino - 1 -[( 2 - acetoxyethoxy ) methyl ]- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 21 , r = ch 2 och 2 ch 2 oac , 0 . 45 g , 1 . 68 mmol ) and methanolic ammonia ( 100 ml , saturated at 0 ° c .) was stirred at room temperature for 18 h . methanolic ammonia was evaporated in vacuo and the residue was purified by silica gel column ( 2 × 8 cm ) chromatography . the column was flash eluted with dichloromethane containing 0 - 12 % methanol . the homogeneous fractions were pooled and evaporated to yield 0 . 35 g ( 92 %) of the title compound , mp 165 - 166 ° c . ir ( kbr ): ν705 (═ p - c ), 1210 (- p ═ ch ), 2930 , 3050 , and 3270 ( nh 2 , oh ) cm - 1 . uv ( ph 1 ): λmax254 nm ( ε24 , 200 ), 282 ( 15 , 200 ), and 312 ( 5000 ); ( meoh ): λmax266 nm ( ε28 , 900 ), and 340 ( 2300 ); ( ph 11 ): 266 nm ( ε34 , 700 ), and 340 ( 1900 ); 31 p nmr ( dmso - d 6 ): δ 65 . 85 ; 1 h nmr ( dmso - d 6 ): δ 3 . 46 ( m , 4 h , 2ch 2 ), 4 . 70 ( br s , 1 h , oh ), 5 . 70 ( s , 2 h , ch 2 ), 8 . 52 and 8 . 69 ( 2s , 2 h , nh 2 ), 8 . 65 ( d , 1 h , c 5 h ), and 8 . 96 ( d , 1 h , j = 54 hz , c 2 h ). anal . calcd . for c 8 h 11 n 4 o 2 p . 0 . 35 ch 2 cl 2 : c , 37 . 54 ; h , 4 . 61 ; n , 21 . 89 . found : c , 37 . 43 ; h , 4 . 13 ; n , 21 . 53 . preparation of 7 - amino - 1 -[( 1 , 3 - dibenzoyloxy - 2 - propoxy ) methyl ]- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine [ compound 21 , r = ch 2 och ( ch 2 obz ) 2 ] to a suspension of 7 - amino - 1h - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 10 , 0 . 5 g , 3 . 29 mmol ) in dmf ( 20 ml ) was added nah ( 0 . 16 g , 4 mmol ) under an argon atmosphere . after stirring the mixture at room temperature for 40 min , ( 1 , 3 - dibenzoyloxy - 2 - propoxy ) methyl bromide ( 109 ) ( 1 . 38 g , 3 . 5 mmol ) was added and the reaction was continued for 18 h . the solvent was evaporated in vacuo . the residue was dissolved in dichloromethane ( 150 ml ) and the organic solution was washed with water ( 30 ml ). aqueous layer was extracted with dichloromethane ( 2 × 100 ml ) and the combined organic extracts were dried ( na 2 so 4 ) and evaporated . the residue was purified by silica gel column ( 2 × 15 cm ) chromatography . the column was flash eluted with dichloromethane containing 0 - 2 . 5 % methanol . the appropriate fractions containing the desired product were evaporated to yield 0 . 68 g ( 44 %) of the title compound , mp 148 - 150 ° c . 1 h nmr ( dmso - d 6 ): δ 4 . 35 ( m , 3 h , ch , ch 2 ), 4 . 52 ( m , 2 h , ch 2 ), 5 . 79 ( s , 2 h , ch 2 ), 7 . 45 - 7 . 75 ( m , 10 h , 2 bz ), 8 . 33 ( s , 2 h , nh 2 ), 8 . 66 ( d , 1 h , c 5 h ), and 8 . 81 ( d , 1 h , j = 57 . 9 hz , c 2 h ). anal . calcd . for c 23 h 21 n 4 o 5 p : c , 59 . 48 ; h , 4 . 56 ; n , 12 . 07 . found : c , 59 . 08 ; h , 4 . 56 ; n , 12 . 02 . preparation of 7 - amino - 1 -[( 1 , 3 - dihydroxy - 2 - propoxy ) methyl ]- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine [ compound 21 , r = ch 2 och ( ch 2 oh ) 2 ] a mixture of 7 - amino - 1 -[( 1 , 3 - dibenzoyloxy - 2 - propoxy ) methyl ]- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 21 , r = ch 2 och ( ch 2 obz ) 2 , 0 . 65 g , 1 . 4 mmol ) and methanolic ammonia ( 125 ml , saturated at 0 ° c .) was stirred at room temperature for 20 h . the solvent was evaporated under reduced pressure . the solid thus obtained was triturated with cold methanol ( 5 ml ) and the product was collected by filtration . to the filtrate silica gel was added and the solvent was evaporated . the dry powder was loaded on top of a silica gel column ( 2 × 8 cm ). the column was flash eluted with dichloromethane containing 0 - 25 % methanol to give 0 . 28 g ( 78 %) of the title compound , mp 180 - 182 ° c . ir ( kbr ): ν695 (═ p - c ), 1215 (- p ═ ch ), and 3060 , 3300 , 3380 and 3500 ( nh 2 , oh ) cm - 1 . uv ( ph 1 ): λmax254 nm ( ε19 , 000 ), 282 ( 12 , 100 ), and 314 ( 4000 ); ( meoh ): λmax266 nm ( ε24 , 700 ), and 336 ( 24 , 000 ); ( ph 11 ): 266 nm ( ε27 , 650 ), and 340 ( 1900 ). 31 p nmr ( dmso - d 6 ): δ 65 . 02 . 1 h nmr ( dmso - d 6 ): δ 3 . 43 ( m , 3 h , ch , ch 2 ), 3 . 53 ( m , 2 h , ch 2 ), 4 . 63 ( br s , 2 h , 2oh ), 5 . 68 ( s , 2 h , ch 2 ), 8 . 23 ( s , 2 h , nh 2 ), 8 . 53 ( d , i h , c 5 h ), and 8 . 82 ( d , 1 h , j = 58 . 5 hz , c 2 h ). anal . calcd . for c 9 h 13 n 4 o 3 p : c , 42 . 19 ; h , 5 . 11 ; n , 21 . 87 . found : c , 42 . 37 ; h , 5 . 12 ; n , 21 . 66 . a mixture of 7 - amino - 1h - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 10 , 0 . 3 g , 1 . 97 mmol ) in 1 , 1 , 1 , 3 , 3 , 3 - hexamethyldisilazane ( hmds , 5 ml ), pyridine ( 5 ml ) and ammonium sulfate ( 50 mg ) was heated under reflux for 18 h . the reaction mixture was allowed to cool to room temperature and the excess of hmds and pyridine were evaporated in vacuo . the silyl derivative of compound 10 was dried under high vacuum for 3 h . the dried material was dissolved in 1 , 2 - dichloroethane ( 30 ml ) to which 1 - o - acetyl - 2 , 3 , 5 - tri - o - benzoyl - d - ribofuranose ( 1 . 28 g , 2 . 4 mmol ) was added . the mixture was cooled in an ice bath . a solution of sncl 4 in dichloromethane ( 1 m , 2 . 8 ml , 2 . 8 mmol ) was added keeping the flask under an argon atmosphere . after stirring the mixture in the ice bath for 4 h , the ice bath was removed and the reaction was continued at ambient temperature for 18 h . methanol ( 10 ml ) was added and after 10 min it was diluted with dichloromethane ( 100 mll ). the organic layer was washed with saturated sodium hydrogen carbonate solution ( 50 ml ) and the resultant emulsion was filtered through a celite pad . organic layer was separated and the aqueous layer was extracted with dichloromethane ( 2 × 50 ml ). the combined organic layer was dried ( na 2 so 4 ) and evaporated . the solid thus obtained was crystallized from a mixture of ethyl acetate and methanol to give 0 . 65 g ( 56 %) of the title compound , mp 248 - 250 ° c . ( dec ). 1 h nmr ( dmso - d 6 ): δ 4 . 85 ( m , 2 t c 5 h 2 ), 4 . 96 ( m , 1 h , c 4 &# 39 ; h ), 6 . 01 ( m , 1 h , c 3 &# 39 ; h ), 6 . 19 ( m , 1 h , c 2 &# 39 ; h ), 6 . 51 ( d , 1 h , c 1 &# 39 ; h ), 7 . 44 - 8 . 05 ( m , 15 h , 3bz ), 8 . 44 and 8 . 47 ( 2s , 2 h , nh 2 ), 8 . 75 ( d , 1 h , c 5 h ), and 8 . 95 ( d , 1 h , j = 57 hz , c 2 h ). anal . calcd . for c 31 h 25 n 4 o 7 p . 0 . 5 h 2 o : c , 61 . 49 ; h , 4 . 33 ; n , 9 . 25 . found : c , 61 . 61 ; h , 3 . 88 ; n , 9 . 21 . a mixture of 7 - amino - 1 -( 2 , 3 , 5 - tri - o - benzoyl - β - d - ribofuranosyl )- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 21 , r = 2 , 3 , 5 - tri - o - benzoyl - β - ribofuranose , 0 . 45 g , 0 . 75 mmol ) and methanolic ammonia ( 100 ml , saturated at 0 ° c .) was stirred at room temperature for 20 h and the methanolic ammonia was evaporated under reduced pressure . the residue was triturated with methanol ( 10 ml ) and the product was collected by filtration . the filtrate was evaporated and the residue was purified by chromatography over a silica gel column . the column was flash eluted with dichloromethane containing 0 - 20 % methanol . the appropriate fractions containing the pure product were collected and evaporated to give a total yield of 0 . 18 g ( 84 %) of the title compound , mp 220 - 222 ° c . ir ( kbr ): ν700 (═ p - c ), 1205 (- p ═ ch ), and 2930 - 3470 ( nh 2 , oh ) cm - 1 . uv ( ph 1 ): λmax254 nm ( ε23 , 400 ), 284 ( 15 , 600 ), and 314 ( 5600 ); ( meoh ): λmax266 nm ( ε32 , 900 ), and 340 ( 2100 ); ( ph 11 ): 266 nm ( ε36 , 050 ), and 338 ( 3250 ). 31 p nmr ( dmso - d 6 ): δ 62 . 50 ; 1 h nmr ( dmso - d 6 ): δ3 . 73 ( dd , 2h , c 5 &# 39 ; h 2 ), 3 . 98 ( m , 1 h , c 4 &# 39 ; h ), 4 . 10 ( br s , 1 h , c 3 &# 39 ; h ), 4 . 28 ( m , 1 h , c 2 &# 39 ; h ), 5 . 24 ( br s , 2 h , 2oh ), 5 . 63 ( br s , 1 h , oh ), 5 . 70 ( d , 1 h , c 1 &# 39 ; h ), 8 . 24 ( s , 2 h , nh 2 ), 8 . 86 ( d , 1 h , c 5 h ), and 8 . 83 ( d , 1 h , j = 58 . 8 hz , c 2 h ). anal . calcd . for c 10 h 13 n 4 o 4 p . 0 . 25 ch 2 cl 2 : c , 40 . 30 ; h , 4 . 45 ; n , 18 . 35 . found : c , 40 . 40 ; h , 4 . 15 ; n , 18 . 54 . 7 - amino - 1h - 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 10 , 0 . 5 g , 3 . 29 mmol , dried by coevaporation with dry dmf , 35 ml ) was suspended in dry acetonitrile ( 30 ml ) to which sodium hydride ( 160 mg , 4 mmol , 60 % dispersion in mineral oil ) was added under an argon atmosphere . the mixture was stirred at room temperature for 40 min and 1 - chloro - 2 - deoxy - 3 , 5 - di - o - p - toluoyl - α - d - erythro - pentofuranose ( 110 ) ( 1 . 55 g , 4 mmol ) was added . the reaction mixture was stirred for 2 . 5 h and the solvent was evaporated . the residue was partitioned between dichloromethane ( 150 ml ) and water ( 50 ml ). aqueous layer was separated and extracted with dichloromethane ( 2 × 100 ml ). the combined organic extracts were dried ( na 2 so 4 ) and evaporated . the residue was purified by chromatography over a silica gel column ( 2 × 20 cm ). the column was flash eluted with dichloromethane containing 0 - 2 % methanol . the fractions containing the desired product were collected and evaporated to yield 0 . 8 g ( 48 %), mp 128 - 130 ° c . 1 h nmr ( dmso - d 6 ): δ2 . 33and 237 ( 2s , 6 h , 2ch 3 ), 2 . 90 ( m , 1 h , c 2 &# 39 ; h ), 3 . 03 ( m , 1 h , c 2 &# 34 ; h ), 4 . 70 ( m , 3 h , c 4 &# 39 ; h and c 5 &# 39 ; h 2 ), 5 . 64 ( m , 1 h , c 3 &# 39 ; h ), 6 . 39 ( t , 1 h , c 1 &# 39 ; h ), 7 . 24 and 7 . 34 ( 2d , 4 h . tol ), 7 . 80 and 7 . 90 ( 2d , 4 h , tol ), 8 . 33 ( s , 2 h , nh 2 ), 8 . 33 ( d , 1 h , c 5 h ), and 8 . 85 ( d , 1 h , j = 59 hz , c 2 h ). anal . calcd . for c 26 h 25 n 4 o 5 p : c , 61 . 90 ; h , 5 . 00 ; n , 11 . 11 . found : c , 61 . 57 ; h , 5 . 21 ; n , 10 . 76 . a mixture of 7 - amino - 1 -( 2 - deoxy - 3 , 5 - di - o - p - toluoyl - β - d - erythro - pentofuranosyl )- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 21 , r = 2 - deoxy - 3 , 5 - di - o - p - toluoyl - β - d - erythro - pentofuranose , 0 . 6 g , 1 . 19 mmol ) and methanolic ammonia ( 100 ml , saturated at 0 ° c .) was stirred at room temperature for 20 h in a pressure bottle . the reaction flask was cooled in an ice bath , carefully opened and evaporated to dryness . the residue was dissolved in methanol ( 10 ml ) and impregnated onto silica gel ( 10 g ). the solvent was evaporated and the dry powder was loaded on a silica gel column ( 2 × 10 cm ). the column was flash eluted with dichloromethane containing 0 - 12 % methanol to yield 0 . 24 g ( 75 %) of the title compound , mp 132 ° c . ( foams ). ir ( kbr ): ν700 (═ p - c ), 1210 (- p ═ ch ), and 2930 - 3320 ( nh 2 , oh ) cm - 1 . uv ( ph 1 ): λmax254 nm ( ε45 , 238 ), 282 ( 25 , 800 ), and 314 ( 10 , 100 ); ( meoh ): λmax266 nm ( ε59 , 900 ), and 330 ( 5050 ); ( ph 11 ): 266 nm ( ε65 , 150 ), and 334 ( 5400 ). 31 p nmr ( dmso - d 6 ): δ 62 . 19 . 1 h nmr ( dmso - d 6 ): δ 2 . 40 ( m , 2 h , c 2 &# 39 ; h and c 2 &# 39 ; h ), 3 . 65 ( m , 2 h , c 5 &# 39 ; h 2 ), 3 . 96 ( m , 1 h , c 4 &# 39 ; h ), 4 . 32 ( br s , 1 h , c 3 &# 39 ; h ), 5 . 16 ( br s , 1 h , oh ), 5 . 40 ( br s , 1 h , oh ), 6 . 14 ( t , 1 h , c 1 &# 39 ; h ), 8 . 35 ( s , 2 h , nh 2 ), 8 . 81 ( d , 1 h , c 5 h ), and 8 . 88 ( d , 1 h , j = 57 . 3 hz , c 2 h ). anal . calcd . for c 10 h 13 n 4 o 3 p . 0 . 25 ch 2 cl 2 : c , 42 . 53 ; h , 4 . 70 ; n , 19 . 36 ; p , 10 . 70 . found : c , 42 . 08 ; h , 4 . 75 ; n , 19 . 11 ; p , 10 . 28 . similar to the 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine chemistry as outlined in examples 1 - 9 , 1 , 3 - azaphospholo [ 5 , 4 - d ] pyrimidines are made from properly functionalized oxazoles . the key starting material for this purpose is ethyl oxazole - 4 - carboxylate ( compound 22 ), which is prepared as reported ( 52 ). bromination of compound 22 with one equivalent of n - bromosuccinimide in the presence of 1 , 1 &# 39 ;- azobis ( cyclohexanecarbonitrile ) in ccl 4 gives the corresponding 5 - bromo derivative ( compound 23 ). it has been found ( 53 - 55 ) that the nuclear bromination of oxazoles either with bromine or with n - bromosuccinimide occurs preferentially at c - 5 ; if this position is occupied , then at c - 4 , but not at c - 2 . selective ammonolysis of compound 23 with methanolic ammonia ( saturated at 0 ° c .) at controlled temperature yields 5 - bromo - 1 , 3 - oxazole - 4 - carboxamide ( compound 24 ). ## str18 ## dehydration of compound 24 with phosgene provides 5 - bromooxazole - 4 - carbonitrile ( compound 25 ), which on treatment with ttp in the presence of naked fluoride ion ( 18 - crown - 6 / kf ) in anhydrous toluene at reflux temperature provides 4 - bromo - 1h - 1 , 3 - azaphospholine - 5 - carbonitrile ( compound 26 ). ## str19 ## this o → p conversion also undergoes a ring - opening and recyclization mechanism as described in example 1 ( compound 9 ). hydrolysis of compound 26 with aqueous naoh at room temperature gives 4 - bromo - 1h - 1 , 3 - azaphospholine - 5 - carboxamide ( 27a ). the c 4 - br of 27a may not be quite reactive toward conventional nucleophilic displacement reactions . however , the use of copper compounds as catalysts in nucleophilic aromatic ( 56 , 57 ) and heteroaromatic ( 58 ) substitution reactions are known . thus , treatment of compound 27a with cucn in the presence of copper powder or cucl at elevated temperature gives the cyano compound 27b . decarbonylation of compound 27b under hofman conditions ( naobr ) provides 5 - amino - 1h - 1 , 3 - azaphospholine - 4 - carbonitrile ( compound 28 ). ## str20 ## 5 - amino - 1h - 1 , 3 - azaphospholine - 4 - carbonitrile ( compound 28 ) is reacted with formamidine acetate in ethanol at reflux temperature to form the ring annulated product 4 - amino - 1h - 1 , 3 - azaphospholo [ 5 , 4 - d ] pyrimidine ( the adenine congener , compound 29 ). ## str21 ## a further embodiment includes phosphazole compounds having the structure : ## str22 ## wherein r is --( ch 2 ) 4 ch 3 the synthesis of the compounds in example 14 proceeds by the direct alkylation of the adenine analog ( compound 29 ) with requisite alkyl halides or properly protected glycosyl halides in appropriate solvents . in the case of glycosyl derivatives , subsequent deprotection of the glycosylated product under alkaline conditions is required . phosphorylation of the unprotected adenosine analog with pocl 3 in trimethylphosphate , according to the general procedure of yoshikawa et al . ( 59 ) gives the corresponding 5 &# 39 ;- monophosphate , which when reacted with n , n &# 39 ;- dicyclohexylcarbodiimide ( dcc ) in anhydrous pyridine in the presence of 4 - morpholino - n , n &# 39 ;- dicyclohexylcarboxamidine under high - dilution conditions ( 60 ) provides the camp analog 7 - amino - 3 -( β - d - ribofuranosyl )- 1 , 3 - azaphospholo [ 5 , 4 - d ] pyrimidine 3 &# 39 ;, 5 &# 39 ;- cyclic phosphate . hydrolysis of the nitrile function of 5 - amino - 1h - 1 , 3 - azaphospholine - 4 - carbonitrile ( compound 28 ), from example 12 , is carried out by treatment with 0 . 1n aqueous naoh at gentle reflux for 5 hours to obtain 5 - amino - 1h - 1 , 3 - azaphospholine - 4 - carboxamide ( compound 30 ), which when fused with formamidine forms the hypoxanthine analog , compound 31 . ## str24 ## 5 - amino - 1h - 1 , 3 - azaphospholine - 4 - carboxamide ( compound 30 from example 15 ) is fused with urea at 160 ° c . for 45 min to obtain the xanthine analog 32 . ## str25 ## the hypoxanthine analog ( compound 31 from example 15 ) is reacted with pocl 3 in the presence of diethylaniline at reflux temperature for 1 hour to form 4 - chloro - 1h - 1 , 3 - azaphospholo [ 5 , 4 - d ] pyrimidine ( compound 33 ), which on reaction with thiourea in boiling ethanol produces the 6 - thiopurine analog ( compound 34 ). ## str26 ## the guanine analog ( compound 37 ) is obtained from compound 30 ( from example 15 ) by reacting with benzoylisothiocyanate to form 5 -( n &# 39 ;- benzoylthiocarbonyl ) amino - 1h - 1 , 3 - azaphospholine - 4 - carboxamide ( compound 35 ), which when reacted with ch 3 i in 0 . 1 n naoh will afford 5 -( n &# 39 ;- benzoyl - s - methylisothiocarbamoyl ) amino - 1h - 1 , 3 - azaphospholine - 4 - carboxamide ( compound 36 ), which , in the presence of ammonia , produces the guanine congener ( compound 37 ). ## str27 ## the guanine congener from example 18 ( compound 37 ) on heating under 5 reflux with pocl 3 in the presence of diethylaniline provides 6 - amino - 4 - chloro - 1h - 1 , 3 - azaphospholo [ 5 , 4 - d ] pyrimidine ( compound 38a ), which on reaction with thiourea in boiling ethanol produces the 6 - thioguanine anlaog 6 - amino - 1 , 3 - azaphospholo [ 5 , 4 - d ] pyrimidine - 4 ( 1h , 5h )- thione ( compound 38b ). ## str28 ## a further embodiment includes phosphazole compounds having the structure : ## str29 ## wherein r is --( ch 2 ) 4 ch 3 -- ch 2 ch ═ chch 2 ch 3 ## str30 ## --( ch 2 ) 3 ch 2 oh -- ch 2 ch ═ chch 2 oh ## str31 ## the synthesis of the compounds in example 20 proceeds by the direct alkylation of the guanine analog ( compound 37 ) with requisite alkyl halides or properly protected glycosyl halides in appropriate organic solvents . in the case of glycosyl derivatives , subsequent deprotection of the protected glycosylated product under alkaline conditions is required . phosphorylation of the unprotected guanosine analog with pocl 3 in trimethylphosphate ( 59 ) produces the corresponding 5 &# 39 ;- monophosphate , which when dehydrated with dcc in anhydrous pyridine under high - dilution conditions ( 60 ) provides the cgmp analog . a further embodiment includes phosphazole compounds having the structure : ## str32 ## wherein r is -- ch 2 ch ═ ch 2 -- ch 2 ch ═ chch 2 ch 3 ## str33 ## the synthesis of the compounds in example 21 proceeds by the direct alkylation of the adenine analog ( example 2 , compound 10 ) with requisite alkyl halides or properly protected glycosyl halides in the presence of the phase - transfer catalyst cryptand tris -[ 2 -( 2 - methoxyethoxy ) ethyl ] amine ( tda - 1 ) and solid kox in acetonitrile . for the synthesis of the target adenosine analog ( compound 44 ), compound 9 serves as a useful starting material . the protection of the amino group is effected by the treatment of compound 9 with diethoxymethylacetate in acetonitrile to give 4 - ethoxymethyleneamino - 1 , 3 - azaphospholine - 5 - carbonitrile ( compound 41 ). coupling of compound 41 with 2 , 3 , 5 - tri - o - benzoyl - d - ribofuranosyl bromide ( compound 42 ) ( 61 ) provides the corresponding p - nucleoside ( compound 43 ). compound 43 is also prepared by alkylation of compound 41 with compound 42 in the presence of lithium dusopropylimide ( lda ) in a nonpolar solvent at - 70 ° c . this synthetic route is similar to the one used for p - alkylation of 1 , 3 - azaphospholines ( 62 ). compound 43 on reaction with methanolics ammonia ( saturated at 0 ° c .) at room temperature for 24 hours , deprotection of the sugar moiety with concomitant ring cyclization occurs to give the adenosine analog 7 - amino - 3 -( β - d - ribofuranosyl )- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine ( compound 44 ). compound 44 is also prepared by the direct glycosylation of the sodium salt of compound 14 ( generated in situ by the treatment with nah in ch 3 cn ) with 2 , 3 - o - isopropylidene - 5 - o - tert - butyldimethylsilyl )- α - d - ribofuranosyl chloride ( 63 ) in acetonitrile at room temperature to produce the p - glycosylated product ( along with n - glycosylated product ), which on separation by flash silica gel column chromatography and deisopropylidenation with aqueous trifluoroacetic acid followed by amination provides compound 44 . p - nucleosides of example 23 ( compound 47 ) are readily accessible by substituting the substrate 42 with the α - halogenoses 45 and 46 in the glycosylation of compound 10 . both compounds 45 and 46 are prepared by the literature procedures ( 64 , 65 ). structural manipulation of the protected azaphosphole nucleosides thus obtained provides the target nucleosides 47 . 5 &# 39 ;- monophosphate ( compound 48 ) and 3 &# 39 ;, 5 &# 39 ;- cyclic phosphate ( compound 49 ) of adenosine analog , compound 44 ## str36 ## the direct utilization of certain cyclic nucleotide derivatives as medicinal agents is well documented ( 27 ). with a view of gaining greater tissue specificity and longer lasting potency over camp , the 3 &# 39 ;, 5 &# 39 ;- cyclic phosphate of compound 44 will be prepared via the intermediate compound 48 . phosphorylation of unprotected adenosine analog 44 with pocl 3 in trimethylphosphate , according to the general procedure of yoshikawa and coworkers ( 59 ) will provide the corresponding 5 &# 39 ;- monophosphate ( compound 48 ). compound 48 is reacted with n , n &# 39 ;- dicyclohexylcarbodiimide ( dcc ) in anhydrous pyridine in the presence of 4 - morpholino - n , n &# 39 ;- dicyclohexylcarboxamidine under high - dilution conditions ( 60 ) to obtain 7 - amino - 3 -( β - d - ribofuranosyl )- 1 , 3 - azaphospholo [ 4 , 5 - d ]- pyrimidine 3 &# 39 ;, 5 &# 39 ;- cyclic phosphate ( compound 49 ). concomitant hydrolysis of the nitrile function and the sugar protecting groups of compound 41 is carried out by the treatment with 0 . 1n naoh solution to obtain 4 - amino - 3 -( β - d - ribofuranosyl )- 1 , 3 - azaphospholine - 5 - carboxamide ( compound 50 ). compound 50 is ring closed with formamide or with triethyl orthoformate to obtain the inosine analog , compound s1 ( x ═ o ), which is also prepared by deamination of the adenosine analog , compound 44 , with aqueous nitrous acid . acetylation of the sugar hydroxyls with acetic anhydride / 4 -( dimethylamino ) pyridine , followed by thiation with phosphorus pentasulfide and subsequent deacetylation provides the 6 - thioinosine analog 3 -( β - d - ribofuranosyl )- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine - 7 ( 6h )- thione ( compound 51 , x ═ s ). in a similar manner as described for the preparation of the guanine analog ( compound 37 , example 18 ), nucleoside 50 is ring - closed to the guanosine analog 5 - amino - 3 -( β - d - ribofuranosyl )- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidin - 7 ( 6h ) - one ( compound 54 ), via the 4 - thioureido intermediate ( 52 ). 5 &# 39 ;- monophosphate ( compound 55 ) and 3 &# 39 ;, 5 &# 39 ;- cyclic phosphate ( compound 56 ) of inosine analog ( compound 51 , x ═ o ) ## str39 ## in a similar manner as described for the preparation of the camp analog ( example 24 , compound 49 ), phosphorylation of compound 51 ( x ═ o ) with pocl 3 in trimethylphosphate at 0 - 5 ° c . provides the corresponding 5 &# 39 ;- monophosphate ( compound 55 ), which on subsequent ring closure by the treatment with dcc in pyridine results in the formation of the 3 &# 39 ;, 5 &# 39 ;- cyclic phosphate ( compound 56 ). 5 &# 39 ;- monophosphate ( compound 57 ) and 3 &# 39 ;, 5 &# 39 ;- cyclic phosphate ( compound 58 ) of guanosine analog ( compound 54 ) ## str40 ## in a similar manner as described for the preparation of the camp analog ( example 24 , compound 49 ), phosphorylation of the guanosine analog ( compound 54 ) provides the corresponding 5 &# 39 ;- monophosphate ( compound 57 ), which on ring closure with dcc / pyridine results in the cgmf analog ( compound 58 ). the chemical synthesis of compound 61 is accomplished by the following sequence of reactions . treatment of the guanosine analog ( compound 54 ) with α - acetoxyisobutyryl bromide in moist acetonitrile ( 66 , 67 ) results in the formation of a mixture of 2 &# 39 ;( 3 &# 39 ;)- bromo - 3 &# 39 ;( 2 &# 39 ;)- o - acetyl nucleosides ( compounds 59 ). treatment of compounds 59 with a mixture of zinc - copper in dmf ( 67 ), followed by deprotection gives 5 - amino - 3 -( 2 , 3 - dideoxy - β - d - glycero - pent - 2 - enofuranosyl )- 1 , 3 - azaphospholo [ 4 , 5 - d ] pyrimidine - 7 ( 6h ) - one ( compound 60 ). catalytic hydrogenation ( pd / c , h 2 ) of compound 60 will afford compound 61 . since the isolation of the antibiotics tubercidin , toyocamycin and sangivamycin in the early 1960 &# 39 ; s and subsequent structural elucidation of these antibiotics as pyrrolo [ 2 , 3 - d ] pyrimidine nucleosides , the developments in 7 - deazapurine nucleosides are phenomenal ( 68 ). isolation of additional 7 - deazapurine nucleosides from natural sources in recent years , such as nucleoside q ( 69 - 72 ), cadeguomycin ( 73 - 77 ), antibiotic ab - 116 ( 78 , 79 ), dapiramicin ( 80 - 82 ), 5 - iodo - 5 &# 39 ;- deoxytubercidin ( 83 ), kanagawamicin ( 79 ), and mycalisines a and b ( 84 , 85 ) has generated tremendous interest in the synthesis of deazapurine nucleosides . a large number of these nucleosides exhibited significant antiviral and antitumor activities ( 68 , 86 , 87 ) in vitro as well as in vivo . therefore , the synthesis of the phosphorus congeners of certain of these antibiotics was undertaken . the synthesis of these compounds in this totally unexplored area uses essentially the same synthetic sequences that were used for the preparation of 1 , 3 - azaphospholo [ 4 , 5 - d ] primidines . the rudimentary 5 - member ring having only phosphorus as a heteroatom is prepared from 2 - aminofuran - 3 - carbonitrile ( compound 62 ). condensation of malononitrile with chloroacetaldehyde in the presence of triethylamine in anhydrous tetrahydrofuran furnishes 2 - aminofuran - 3 - carbonitrile ( compound 62 ). the proposed reaction mechanism is : reaction of malononitrile with chloroacetaldehyde yields the intermediate a , which then cyclizes to give the compound 62 via the intermediate b . similar cyclizations using a substituted halo - aldehyde and malononitrile are reported in the literature ( 88 , 89 ). treatment of compound 62 with tris ( trimethylsilyl ) phosphine gives the versatile starting material 2 - aminophospholine - 3 - carbonitrile ( compound 63 ). synthesis of the tubercidin analog 4 - amino - 7 -( β - d - ribofuranosyl ) phospholo -[ 2 , 3 - d ] pyrimidine ( compound 67 ). ## str43 ## protection of the amino group of compound 63 is effected by the fusion with phthalimide to give compound 64 , which on glycosylation with 2 , 3 - o - isopropylidene - 5 - o -( tert - butyldimethylsilyl )- α - d - ribofuranosyl chloride ( compound 65 ) ( 63 ), under sodium salt ( 90 ) or phase - transfer ( 91 ) glycosylation condition gives the corresponding protected nucleoside ( compound 66 ). compound 66 , on successive treatment with 0 . 1 n aqueous naoh ( to cleave the phthalimido group ), fusion with formamidine acetate ( to ring close ) and aqueous trifluoroacetic acid ( to cleve the tbdms group and the isopropylidene group ) provides the tubercidin congener 4 - amino - 7 -( β - d - ribofuranosyl ) phospholo [ 2 , 3 - d ] pyrimidine ( compound 67 ). alkaline hydrolysis of compound 66 under nonoxidative conditions provides the amino - carboxamide derviative ( compound 68 ), which is then cyclized with treithyl orthoformate , followed by deacetonation under acidic conditions to furnish the 7 - deazainosine analog ( compound 69 ). compound 69 is also prepared by deamination of compound 67 ( the adenosine analog ) with aqueous nitrous acid . ## str44 ## synthesis of 7 - deazaguanosine analog 2 - amino - 7 -( β - d - ribofuranosyl )- phospholo [ 2 , 3 - d ] pyrimidin - 4 ( 3h - one ( compound 72 ) ## str45 ## in a similar manner to that set forth in example 6 , compound 68 is converted to the 7 - deazaguanosine analog ( compound 72 ) by a conventional four - step reaction sequence via the thioureido intermediate , compound 70a . other nucleoside derivatives of adenine and guanine analogs ( compound 74 and compound 77 ) ## str46 ## the nucleoside analogs include those species which contain modifications of the carbohydrate moiety , for example , wherein one or more of the hydroxyl groups are replaced with a hydrogen , halogen , a heteroatom , amines , thiols , and the like . in certain embodiments of the new compounds , the pentose moiety is replaced by a hexose and the stereochemistry of the carbohydrate carbons can be other than that of d - ribose . nucleosides as defined herein are prepared as described in examples 9 and 14 . sangivamycin analog of p - substituted deazaphosphole ( compound 84 ) ## str47 ## synthesis of sangivamycin analog in which phosphorus is a component of the pyrrole moiety is accomplished using appropriate furan . based on chloroacetaldehyde precedent ( 92 ), a 2 , 3 , 4 - trisubstituted furan ( compound 79 ) is generated . the key substrate that is used for the synthesis of compound 79 is methyl chloroformylacetate ( compound 78 ) and is prepared as previously described ( 93 , 94 ). treatment of malononitrile with compound 78 in aqueous solution containing naoac at reflux temperature ( 94 ) to give methyl 2 - amino - 3 - cyanofuran - 4 - carboxylate ( compound 79 ). o - p conversion of compound 64 to obtain compound 80 is effected with tris - ( trimethylsily ) phosphine ( ttp ) in the presence of naked fluoride ion in boiling toluene . the amino group of the tri - substituted phospholine ( compound 80 ) is protected with phthalimide group to obtain compound 81 , which on glycosylation with 2 , 3 - o - isopropylidene - 5 - o -( tert - butyldimethylsilyl )- α - d - ribofuranosyl chloride , under the stereospecific sodium salt glycosylation conditions ( 90 ) yields the key intermediate compound 82 . a removal of the blocking groups by the treatment with a weak base provides methyl 2 - amino - 3 - cyano - 1 -( 2 , 3 - o - isopropylidene - β - d - ribofuranosyl ) phospholine - 4 - carboxylate ( compound 83 ). ring closure of this o - aminonitrile ( compound 83 ) by the conventional procedure provides the sangivamycin analog compound 84 . toyocamycin analog of p - substituted deazaphosphole ( compound 96 ) ## str48 ## selective acetylation ( 96 ) of sugar hydroxyls of compound 84 by the treatment with acetic anhydride in the presence of 4 -( dimethylamino ) pyridine ( dmap ) at - 25 ° c . and subsequent dehydration of the carbamoyl group with phosgene ( 95 ) gives compound 85 . careful deacetylation of compound 85 with methanolic ammonia at 0 ° c . or with aqueous sodium bicarbonate ( 97 ) affords the toyocamycin analog of p - substituted deazaphosphole ( compound 86 ). thiosangivamycin and related analogs of p - substituted deazaphosphole ( compound 87 ) ## str49 ## treatment of toyocamycin analog ( compound 86 ) with hydrogen sulfide gas in dry pyridine in the presence of triethylamine at room temperature provides the thiosangivamycin analog ( compound 87 , x ═ s ). similarly , compound 86 is converted into the corresponding carboxamidine ( compound 87 , x ═ nh ) and carboxamidoxime ( compound 87 , x ═ noh ) by the treatment with liquid anmmonia / ammonium chloride and hydroxylamine , respectively . the carbonitrile function of compound 86 is also available for further transformation reactions . p - substituted deazaphosphole analogs related to 7 - deazainosine ( compound 89 ). ## str50 ## intermediate compound 4 - amino - 7 -( 2 , 3 , 5 - tri - o - acetyl - β - d - ribofuranosyl ) phospholo -[ 2 , 3 - d ] pyrimidine - 5 - carbonitrile ( compound 85 ) from example 36 may be deaminated with aqueous nitrous acid ( 98 ) to form the intermediate compound 7 -( 2 , 3 , 5 - tri - o - acetyl - β - d - ribofuranosyl )- 4 ( 3h )- phospholo [ 2 , 3 - d ] pyrimidine - 5 - carbonitrile ( compound 88 ). since the carbonitrile function of compound 88 is highly reactive toward nucleophilic substitution displacement reactions ( 99 ), a variety of 5 - substituted - 4 ( 3h ) - phospholo [ 2 , 3 - d ] pyrimidine nucleosides is prepared . thus , oxidative hydrolysis of compound 88 by the treatment with nh 4 oh / h 2 o 2 at room temperature ( 97 ) forms 7 -( β - d - ribofuranosyl )- 4 ( 3h )- phospholo [ 2 , 3 - d ] pyrimidine - 5 - carboxamide ( compound 89a ). the corresponding 5 - thiocarboxamide ( compound 89b ) is obtained by treatment of compound 88 with h 2 s in dry pyridine in the presence of triethylamine . a facile conversion of the nitrile function of compound 88 is also accomplished using the procedures previously employed in our laboratory ( 100 ) to obtain compounds 89c ( by the treatment with nh 3 / nh 4 cl ) and 89d ( by the treatment with free nh 2 oh in etoh ). other nucleoside derivatives of toyocamycin and sangivamyein analogs of p - substituted deazaphospholes ( compound 90 ) ## str51 ## the fraudulent nucleosides , 2 &# 39 ;- deoxysangivamycin , ara - toyocamycin , arafluoro - tubercidin and the like have shown selective activity against hcmv . therefore , the analogs of the above nucleosides in the p - substituted deazaphospholes ( compound 90 ) are prepared analogously . the nucleoside analogs include those species which contain modifications of the carbohydrate moiety , for example , wherein one or more of the hydroxyl groups are replaced with a hydrogen , halogen , a heteroatom ( e . g . azido ), amines , thiols , and the like . the pentose moiety is replaced by a hexose . the stereochemistry of the carbohydrate carbons can be other than that of d - ribose . nucleosides as defined herein are prepared as described in examples 9 and 14 . synthesis of 2 - azaphosphazoles ( 1 , 3 - azaphospholo [ 4 , 5 - d ]- v - triazines and their nucleosides ) ## str52 ## the synthesis of the compounds in example 40 proceeds by the direct alkylation of the phosphazoline compound ( 28 , from example 15 ) with requisite alkyl halides or properly protected glycosyl halides in appropriate solvents . in the case of glycosyl derivatives , subsequent deprotection of the glycosylated product under alkaline conditions is required . the alkylated compound 28 on further treatment with liquid amonia in the presence of nh 4 cl at elevated temperature and pressure affords the corresponding 4 - carboxamidines ( compound 91 ), which on treatment with sodium nitrite in aqueous acetic acid furnishs various 2 - azaphosphazoles ( compound 92 ). synthesis of 2 - azaphosphazolones related to 2 - azainosine ## str53 ## compounds 92 ( from example 40 ) on deamination with aqueous nitrous acid produces 2 - azainosine analogs ( compound 94 ), which can also be prepared by ring - annulation employing amino - carboxamide compound 93 under stronger acidic conditions ( 6n hcl ) and low temperature (- 25 ° c .) ( 101 ). the isolation of pathocidin ( 8 - azaguanine ) from natural sources ( 102 , 103 ) together with the observed array of biological activities ( 68 , 104 ) generated tremendous interest in the synthesis of 8 - azapurine nucleosides . the synthesis of 8 - azaphosphazole nucleosides is of biological interest . synthesis of 8 - azaadenosine analog 4 - amino - 1 -( β - d - ribofuranosyl )- 1 , 3 , 2 - diazaphospholo [ 4 , 5 - d ] pyrimidine ( compound 100 ) ## str54 ## reaction of diaminomaleonitrile with hexamethylphosphorus triamide in anhydrous acetonitrile gives the key starting material 4 , 5 - dicyano - 1 , 3 , 2 - diazaphospholine ( compound 95 ) ( 105 ). glycosylation of the sodium salt of 95 with the α - halogenose 2 , 3 - o - isopropylidene - 5 - o -( tert - butyldimethylsilyl )- α - d - ribofuranosyl chloride ( 63 ) in acetonitrile furnishs the protected nucleoside , compound 96 , as the only product , since n - 1 and n - 3 being symmetrical . deprotection of the carbohydrate moiety with aqueous trifluoroacetic acid gives 1 -( β - d - ribofuranosyl )- 1 , 3 , 2 - diazaphospholin - 4 , 5 - dicarbonitrile ( compound 97 ). treatment of compound 97 with 1 &# 39 ; equivalent of sodium methoxide in methanol at room temperature results in the formation of the imidate ester , compound 98 , regiospecifically . similar regiospecific formation of the imidate ester from 4 , 5 - dicyano - 1 -( 2 , 3 , 5 - tri - o - benzoyl - β - d - ribofuranosyl ) imidazole has been documented ( 106 ). in analogy to the imidazole chemistry ( 106 ), when compound 98 is treated with 5 % sodium hypochlorite solution , 5 - amino - 1 -( β - d - ribofuranosyl )- 1 , 3 , 2 - diazaphospholine - 4 - carbonitrile ( compound 99 ) is produced . this reaction may proceed by the elimination of ch 3 cl from the initially formed n - chloro derivative of compound 98 . such beckman type rearrangement of an n - chloroimidate has been documented ( 107 ). ring closure of compound 99 with diethoxymethyl acetate in the presence of ammonia gives the 8 - azaadenosine analog , 4 - amino - 1 -( β - d - ribofuranosyl )- 1 , 3 , 2 - diazaphospholo [ 4 , 5 - d ] pyrimidine ( compound 100 ). synthesis of 8 - phosphainosine , 1 -( β - d - ribofuranosyl )- 1 , 3 , 2 - diazaphospholo [ 4 , 5 - d ] pyrimidine - 4 ( 5h ) - one ( compound 102 ) ## str55 ## hydrolysis of compound 99 with aqueous naoh at room temperature produces 5 - amino - 1 -( β - d - ribofuranosyl )- 1 , 3 , 2 - diazaphospholine - 4 - carboxamide ( compound 101 ), which on further treatment with diethoxymethyl acetate in the presence of ammonia gives 8 - phosphainosine ( compound 102 ). compound 102 can also be prepared by the deamination of the adenosine analog , compound 100 , with aqueous nitrous acid . synthesis of 8 - phosphaguanosine , 6 - amino - 1 -( β - d - ribofuranosyl )- 1 , 3 , 2 - diazaphospholo [ 4 , 5 - d ] pyrimidin - 4 ( 5h )- one ( compound 105 ) ## str56 ## isopropylidenation of compound 101 , followed by conventional four - step reaction sequence via the 5 - thioureido intermediate ( compound 103 ), as described in example 26 , yields the 8 - phosphaguanosine analog ( compound 105 ) other nucleoside derivatives of 8 - phosphaadenine and 8 - phosphaguanine ( compound 107 and compound 109 ). ## str57 ## the nucleoside analogs include those species that contain modifications of the carbohydrate ( sugar ) moiety , for example , wherein one or more of the hydroxyl groups are replaced with a hydrogen , halogen , a heteroatom ( e . g . azido ), amines , thiols , and the like . the pentose moiety can be replaced by a hexose . the stereochemistry of the carbohydrate carbons can be other than that of d - ribose . nucleosides as defined herein are prepared as described in examples 9 and 14 . table 1 provides a list of preferred embodiments of the phosphazoles of the present invention that are suitable for use as pharmaceutical agents . the number in parentheses beside each compound indicates the example number where its manner of synthesis is described . the &# 34 ; t &# 34 ; numbers , such as t70241 , t70242 , t70245 , t70254 , t70256 , t70262 , t70268 , etc . indicate proprietary reference numbers of preferred embodiments for which physical characteristics are denoted in the corresponding experiments . efficacy and non - toxicity of representative compounds of the present invention are shown in the following examples , and in tables 2 and 3 and fig1 - 9 . the adenine analog phosphazole ( compound 10 , t70241 ) was assayed for activity against the cytokines tnf ( x and il - 1β . the effect of t70241 on lps - stimulated tnfα production was assessed in both thp - 1 cells and human pbmc cells in culture . the results of these assays are presented in the following examples and in the figures referred to therein . cell line . thp - 1 cells ( human monocyte ) were maintained in rpmi 1640 medium containing 25 mm hepes buffer , 2 mm l - glutamine , 50 units of penicillin per ml , 50 μg of streptomycin sulfate per ml , and 10 % heat - inactivated fetal bovine serum ( fbs ) in a humidified incubator containing 5 % co 2 . the cells were split 1 : 4 weekly as recommended by the american type culture collection ( atcc ). tnfα assay . varying concentrations of the test compound or control compound were added to thp - 1 cells plated at a density of 1 × 10 6 cells / ml in 600 μl of 10 % rpmi in a 48 well plate . after one hour the cells were stimulated with 0 . 5 μg of lipopolysaccharide ( lps ; sigma , lot number 43h40491 ) per ml . six hours post - stimulation supernatants were collected and assayed for tnfα or il - 1β production using a commercially available cytokine detection elisa ( enzyme linked immunosorbent assay ) kit according to the suppliers instructions . a suitable assay kit may be obtained from biosource , or r and d systems . a novel xanthine analog , hwa - 3138 , having the structure ( as shown below ) was used as the control compound ( 30 ) in this study to monitor the response of the thp - l cells . xanthine analogs are known inhibitors of tnfα but not il - 1β . in the following experiments compound hwa - 3138 was used as a positive control . ## str58 ## cytotoxicity analysis . the cytotoxicity of the compounds were assayed using the celltiter 96 ™ aqueous non - radioactive cell proliferation ( mts ) assay kit obtained from promega ( madison , wis .) following the instructions provided by the supplier . the viable cell number was determined by trypan blue staining and cells ( thp - 1 ) were resuspended in prmi supplemented with 10 % fbs ( gibco ). eighty microliters of cell suspension ( 1 . 7 × 10 4 cells / well ) was dispensed onto a 96 - well microtiter plates . at this time 20 μl of drug ( or control ) was added to appropriate wells . each concentration was assayed in quadruplicate . the plates were incubated at 37 ° c . in a humidified 5 % co 2 atmosphere for 4 days and mts assay sample was graphed for each concentration and the data obtained was used to calculate tc 50 ( the concentration of the compound necessary to inhibit the cell growth by 50 %) for each compound . fig1 a shows the toxicity of compound t70241 in thp - 1 cells . in this figure , the concentration of compound t70241 added is plotted against the observed percent of thp - 1 cells surviving after 4 days incubation . even the highest concentration of compound t70241 tested , 1 mg / ml , had no toxic effect on the thp - 1 cells . in fact , not only is t70241 non - toxic to thp - 1 cells , it stimulates thp - 1 growth at higher concentrations , as shown in fig1 a . these results demonstrate that the biological activity of compound t70241 obtained in the subsequent experiments are not influenced by toxicity . fig1 b is a graph showing the percent inhibition of tnfα production obtained at each concentration of compound t70241 . from fig1 b , 50 % inhibitory concentration ( ic 50 ) of t70241 was calculated to be about 23 . 9 μg / ml . referring now to fig2 a dose response graph is provided showing the anti - tnfα activity of compound t70241 compared to a control , the hwa - 3138 xanthine analog , and to another phosphazole compound t70254 . certain xanthine analogs , such as hwa - 3138 , are known inhibitors of tnfol and are currently used as the standard compounds to measure the effectiveness of any given anti - tnfα compound . the data plotted are the results obtained by monitoring the level of tnfα in culture medium 6 hours post - stimulation of thp - 1 cells with lps . at concentrations as little as 10 μg / ml , t70241 inhibits tnfα production in these cells better than the control xanthine compound . the control compound , a xanthine analog hwa - 3138 , provided no inhibition at concentrations of 10 and 100 μg / ml , whereas the phosphazole compound t70241 yielded about 25 % and 90 % inhibition at the same concentrations . time of addition . varying concentrations of test or control compounds were added to thp - 1 cells ( 1 × 10 6 cells / ml ) at different time points post - stimulation using 0 . 5 μg of lps ( sigma , lot number 43h40491 ) per ml in 300 μl of 10 % rpmi in a 48 well plate . six hours post - stimulation supernatants were collected and assayed for tnfα or il - 1β production using a biosource cytokine detection elisa ( enzyme linked immunosorbent assay ) kit according to the supplier &# 39 ; s instructions . as shown in fig3 even if compound t70241 is added to cultures 5 hours post - lps stimulation , the phosphazole analog is still able to significantly reduce the level of tnfα in the culture medium one hour later , i . e ., at 6 hours post - lps stimulation . although this property is not unique to compound t70241 , it clearly shows that t70241 does not interfere with lps . the membrane bound tnfα was separated from the free tnfα in the culture medium at 6 hours post - lps stimulation as follows : the corresponding cells were centrifuged at 3500 rpm (˜ 1 , 000 × g ) for 6 min . the supernatants were removed and the cell pellets were then resuspended in 200 μl of 0 . 25 mm tris - hcl ( ph 7 . 4 ). the cell associated tnfα was prepared by freezing the cell suspension in dry ice / ethanol and thawing at 37 ° c . three times with vortexing in - between freeze - thaw cycles . each cycle was 4 - 5 min apart . fig4 shows the level of membrane bound ( intracellular ) and free ( extracellular ) tnfα present in cells or in the culture medium 6 hours post - lps induction for cultures treated with compound t70241 . as seen from the data , compound t70241 is able to reduce the levels of both cell associated and free tnfα . at the lowest concentration tested , tnfα is reduced by about 50 % and 30 %, respectively , in the extracellular and membrane - bound fractions . although this finding is not unique to compound t70241 , it suggests that compound t70241 is acting inside the cell and the observed reduction in tnfα production in supernatants represents the actual inhibition of tnfα expression . to determine the long - term effects of phosphazole compound t70241 on tnfα production after a single treatment , thp - 1 cells were pre - incubated with t70241 , washed and resuspended in drug free medium . the xanthine analog hwa - 3138 was used as a control . the results of this study are presented in fig5 . compound t70241 was able to inhibit tnf ( x production in a dose dependent fashion for 72 hours after the drug removal as shown in fig5 a . the xanthine analog hwa - 3138 produced a similar effect but at much higher drug concentration as shown in fig5 b . thp - 1 cells were cultured and il - 1β inhibition assays were conducted as described for tnfα in example 46 , except a commercially available il - 1β assay kit , obtained from r and d systems , minneapolis , minn ., was used instead of the tnfα kit . fig6 shows the dose response profile obtained for the phosphazole compound t70241 when tested for its ability to inhibit il - 1β production . the t70241 compound is graphed with comparable data for the t70254 phosphazole and the xanthine analog hwa - 3138 control . the control compound hwa - 3138 does not inhibit il - 1β production at the concentrations tested , i . e ., 10 - 250 μg / ml . as seen in fig6 the adenine analog phosphazole ( t70241 ) is able to inhibit il - 1β production at concentrations in the range of 50 to 100 μg / ml . the ic 50 for t70241 in this experiment was calculated to be 38 . 76 μg / ml . these results suggest that compound t70241 and the xanthine analog hwa - 3138 exhibit different mechanisms of action . in &# 34 ; time of addition &# 34 ; studies , shown in fig7 it can be seen that t70241 must be added within 1 to 2 hours of lps induction of il - 1β in order to produce a dramatic reduction in il - 1β production at 6 hours post stimulation , which is different from the results obtained for tnfα inhibition by t70241 . in those studies , the phosphazole compound t70241 could be added as late as 5 hours post - induction of tnfα and still sharply inhibit the amount of cytokine in the culture at 6 hours post induction ( as shown in fig3 ). with reference now to fig8 for each concentration of t70241 tested , the membrane bound il - 1β was separated from the il - 1β found free in the culture medium at 6 hours post - lps stimulation as described in example 46 for tnfα , and the percent inhibition of each fraction was graphed . compound t70241 was found to reduce both the cell - associated ( intracelluar ) and the free ( extracellular ) il - 1β , similar to the results shown for inhibition of tnfα ( shown in fig4 ). the phosphazole t70241 was further evaluated for its activity against il - 1β in pbmcs stumulated with lps and aliquotes collected after 4 , 24 and 48 hour post - stimulation . the results of this study are compiled in table 3 . compound t70241 inhibited il - 1β up to 48 hour in a dose dependent fashion ( table 3 ). in the same experiment we show that cell viability was not affected after 48 hours of exposure to compound t70241 . the adenine analog phosphazole ( compound t70241 ) was assayed for antiviral activity using a hcmv plaque reduction assay , in accordance with known methods such as that described in u . s . pat . no . 5 , 446 , 045 . cell culture . vero ( african green monkey kidney cells ) and mrc - 5 ( diploid human embryonic lung fibroblasts ) cells were obtained from the american type culture collection ( atcc ) and were grown in minimal essential medium ( mem ) with earl &# 39 ; s salts and glutamine ( gibco brl , life technologies , inc .) with 10 % heat inactivated fetal bovine serum ( gibco brl ) and penicillin ( 100 μg / ml )/ streptomycin ( 100 μg / ml ). cmv cytopathic effect ( cpe ) assay . assays were performed essentially as described by lewis et al . ( 111 ), which report is incorporated herein by reference . mrc - 5 cells ( 100 , 000 cells / well ) were seeded in 24 - well plates overnight . medium was then removed and wells were rinsed once with 250 μl of 2 % fcs - mem before adding 40 - 50 pfu of hcmv ( town strain ) to each well . after 2 hours of virus adsorption , the medium was removed and the cells were rinsed once with 2 % fcs - mem . varying concentrations of the phosphazole compound in the culture medium ( 1 ml ) was then added to appropriate wells and plates were incubated for 5 - 6 days at 37 ° c . in a humidified 5 % co 2 atmosphere . each concentration was tested in duplicate wells . the extent of the plaque reduction was quantitated by removing the media and adding 200 μl of 0 . 1 % crystal violet in 20 % methanol to all wells . the crystal violet was then removed after 10 min staining at room temperature by rinsing in the sink with tap water and the plates dried overnight . the plaques were then counted using a nikon inverted microscope . the results of the hcmv assays are given in table 4 and fig9 b , showing a nearly logrithmic increase in inhibition of virus production over a 0 . 01 - 10 μg / ml range of t70241 phosphazole . cytotoxicity analysis . the cytotoxicity of the compound was assayed using few the celltiter 96 ™ aqueous non - radioactive cell proliferation ( mts ) assay ( promega ). briefly , viable cell number was determined by trypan blue staining and cells ( mrc - 5 and vero ) were resuspended in minimal essential medium ( mem ) supplemented with 10 % fbs ( gibco ). eighty microliters of cell suspension ( 1 . 7 × 10 4 cells / well ) was dispensed onto a 96 - well microtiter plates . at this time 20 μl of drug ( or control ) was added to appropriate wells . each concentration was assayed in quadruplicate . the plates were incubated at 37 ° c . in a humidified 5 % co 2 atmosphere for 4 days and mts assay was performed according to the manufacturer &# 39 ; s instructions . the average absorbence of the sample was graphed for each concentration and the data obtained was used to calculate tc 50 ( the concentration necessary to give one - half maximum growth ) for each compound . fig9 a is a plot of phosphazole ( t70241 ) concentration versus percent mrc - 5 cells surviving . the data shows that compound t70241 has a slight toxic effect on growing mrc - 5 cells over a four day period , with a tc 50 & gt ; 50 μg / ml . as noted above , at the same time compound t70241 is able to inhibit the number of hcmv induced plaques in static mrc - 5 cultures over a 5 to 6 day assay ( fig9 b ) with a 50 % inhibitory concentration of ( ic 50 ) of approximately 8 . 25 μg / ml . this selective activity is very encouraging for the application of phosphazole derivative candidate drugs for the treatment of hcmv . cell lines . the routine growth and passage of vero cells were performed in monoloyer cultures using minimal essential medium ( mem ) with either hanks [ mem ( h )] or earle [ mem ( e )] salts supplemented with 10 % calf serum , 100 u / ml penicillin g , and 100 μg / ml streptocycin . cells were passaged at 1 : 2 to 1 : 10 dilutions by using 0 . 05 % trypsin plus 0 . 02 % edta in a hepes buffered salt ( hbs ) solution . cpe assays . vero cells were plated at 4 × 10 4 cells / well in a 96 - well microtiter dish in 0 . 1 ml of culture medium 24 hours before infection with virus at an moi of 0 . 001 . the virus was allowed to adsorb to the cells for 10 min at 37 ° c . the virus - containing medium was then removed , and the cells were rinsed three times with fresh medium . finally , 100 - μl aliquots of fresh medium containing the various dilutions of test compounds were added to each well . plaques were observed 24 hour post - infection , and the degree of cpe was scored 40 to 48 hour post - infection . acv ( acyclovir ) was used as a standard in all hsv assays . several of the phosphazole compounds were assayed for their ability to inhibit hsv - 1 and hsv - 2 production in vitro only compound t70269 was active against hsv - 1 and hsv - 2 with ic 50 values of 2 . 75 and 27 . 75 μg / ml , respectively ( table 4 ). all the compounds tested were relatively nontoxic and ganciclovir ( dhpg ) and acyclovir ( acv ) were used as controls in these assays . adenosine deaminase ( ada , adenosine aminohydrolase , ec 3 . 5 . 4 . 4 , sigma ) from calf intestinal mucosa was prepared to give a stock of 1 mg / ml ( 250 - 280 units / ml ) in ph 7 . 4 , 0 . 01 m phosphate buffer . the relative rates of hydrolysis of selected compounds by ada and characterization of the products was carried as follows . briefly , 0 . 05 - 0 . 1 units of ada was added to 1 . 0 ml of a 50 μm solution of a given compound in ph 7 . 4 , 0 . 01 m phosphate buffer and incubated at 37 ° c . fifty - microliter aliquots were taken at time intervals and hydrolysis was quenched by mixing with 0 . 45 ml of distilled water and then the diluted sample was heated at 95 ° c . for 6 min to ensure enxyme deactivation . the decrease in substrate concentration and the formation of the products was followed over time by uv - spectroscopic analysis . adenosine was used as a control in this experiment and one unit is defined as the amount of ada that hydrolyzes 1 . 0 μmol of adenosine / min at 25 ° c . adenosine deaminase ( ada ) is a ubiquitous catabolic enzyme present in many animal and human tissues ( 112 ). in addition to converting adenosine to inosine , this enzyme catalyzes the hydrolysis of numerous 6 - substituted purine nucleosides to inosine and guanosine analogs ( 113 ) and most adenosine analogs active in vitro usually become inactive in vivo due to ada hydrolysis . based on these observation we subjected compound t70241 ( the adenine analog ) to ada hydrolysis in vitro using commercially available , purified anzyme . uv spectra of t70241 remained unchanged up to 5 hours in the presence of ada . at the same time adenosine , used as control in this experiment , was hydrolyzed at the rate of 1 . 0 pmole / unit per min at 25 ° c . these results suggest that compound t70241 is not a substrate for ada . primary cell assays . primary human peripheral blood mononuclear cells ( pbmcs ) were isolated as described by ojwang et al . ( 114 ). isolated monocytes were added to 24 - well cluster plates ( costar , cambridge , mass .) at a concentration of 1 × 10 5 viable cells per well . culture medium or test material dilutions in culture medium were added to the wells , the plates were then incubated at 37 ° c . for one hour . tnfα production was subsequently induced by stimulation with 100 μg / ml ( final concentration ) of lps ( lot number 20126f ) obtained from sigma , st . louis , mo . the plates were incubated an additional 4 , 24 or 48 hours . the supernatant fluids were then harvested and centrifuged to pellet cells or cellular debris , and the supernatant fluids were stored at - 70 ° c . until they were assayed for the presence of tnfα using a commercially available elisa kit , such as that described in example 46 . the effect of compound t70241 on lps - induced tnfα production in primary human blood cells ( pbmcs ) is shown in table 3 . pbmcs are a mixed population of cells which are considered to be involved in the inflammatory responses due to high levels of tnfα or il - 1β at the inflammation sites . experiments performed in pbmcs closely mimic the in vivo situation and based on this observation we evaluated compound t70241 in this system . these data show that compound t70241 is able to significantly reduce the levels of tnfα production in the pbmcs even 48 hours post - lps induction . the level of measurable toxicity in these cells is minimal at the 48 hour time point . also shown is the percent viable cells after 48 hours of exposure to t70241 in the medium . the fact that compound t70241 inhibited both tnfα and il - 1β in pbmcs is very encouraging to pursue the development of such novel class of compounds . thp - 1 cells were grown and the t70254 phosphazole compound ( n - 1 pentyl derivative of t70241 ) was assayed for its effect on tnfα production in the same manner as described in example 46 for the t70241 compound . the results of the tnfα assay is presented in fig2 along with the comparable results for the xanthine analog ( hwa - 3138 ) and the t70241 compound . the n - pentyl substituted phosphazole ( t70254 ) significantly inhibits tnfα production in thp - 1 cells when introduced 6 hours after induction with lps , showing approximately 30 % inhibition at 10 μg / ml and approximately 55 % at 100 μg / ml . certain embodiments of the present invention provide pharmaceutical compositions in which the active ingredient comprises an effective amount of one or more of the phosphazoles disclosed herein , or a suitable salt thereof , along with a pharmaceutically acceptable carrier and any other compatible therapeutic ingredients . in practical use , the inventive pharmaceutical compositions are prepared according to conventional pharmaceutical compounding techniques and the carrier may take a wide variety of forms depending on the form desired for administration , e . g ., oral or parenteral . in preparing the compositions for oral dosage form , any of the usual pharmaceutical media may be employed , such as water , glycols , oils , alcohols , lipids , flavoring agents , preservatives , coloring agents , and the like in the case of oral liquid preparations . in the case of aerosols , surfactants for delivery through mucosal membranes . in the case of oral solid preparations , carriers such as starches , sugars , microcrystalline cellulose , diluents , granulating agents , lubricants , binders , disintegrating agents , and the like may be employed . rectal preparations may include carbowax . additionally , the compounds of the present invention are suitable for encapsulation in liposomes or for crosslinking with protein carriers and the like . the pharmaceutical compositions of the present invention may be administered by conventional methods such as are usually employed with known nucleo - base , nucleoside or nucleotide analogs . certain embodiments of the present invention include methods of treating a broad spectrum of viral infections in humans and animals , including birds , comprising administering to the subject in need of treatment or prevention of a viral infection an effective amount of one of the phosphazole compounds or compositions of the present invention . other embodiments of the present invention encompass methods of treating a wide variety of cancerous tumors in animals and plants . the optimum dosages , manner of administration and other variables routinely determined when optimizing a treatment regime may be readily determined by the clinician as is usually done with other imidizole , azole , deazole or triazole based compounds . a new class of compounds is disclosed herein , along with the manner of synthesizing representative examples . exemplary compounds have been tested for activity as antiviral and antitumor agents by determining their ability to inhibit tnfα production or il - 1β production in cells in culture , and to reduce the number of hcmv plaques in in vitro culture . agents that can inhibit the production or maturation of tnfα and il - 1β in these different indications are expected to have excellent therapeutic potential . it is also expected that where existing heterocyclic compounds and / or nucleosides / nucleotides are known to have a certain function ( for example , as a component of a therapeutic composition , a pesticide used in agriculture or an industrial reagent ), nitrogen or oxygen atoms within that heterocycle will be exchanged with phosphorus , using the methods of the present invention , to generate a phosphazole analog . routine testing will thereafter establish the relative activity of the phosphazole analog compared to the progenitor heterocycle or corresponding nucleoside / nucleotide . a phosphazole analog having the desired level of activity may be substituted for the conventional compound to provide an alternative reagent or component with equivalent or superior results . 1 . m . honjo , t . maruyama , m . horikawa , j . balzarini and e . de clercq , chem . pharm . bull . tokyo , 35 , 3227 ( 1987 ). 2 . t . maruyama and m . honjo , nucleosides nucleotides , 7 , 203 ( 1988 ). 3 . k . issleib , h . oehme , h . schmidt and g .- r . vollmer , in : phosphorus chemistry . proceedings of the 1981 international conference . acs symposium as series 171 , am . chem . soc ., washington , d . c . 1981 , pp 405 - 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smith , t . l . wallace , e . de clercq , m . s . mcgrath and r . f . rando , antimicrob . agents chemother ., 39 , 2426 ( 1995 ). 115 . riley , t . a ., s . b . larson , t . l . avery , r . a . finch and r . k . robins , j . med . chem ., 33 , 572 - 6 ( 1990 ) all patents and publications mentioned in this specification are indicative of the level of skill of those of knowledge in the art to which the invention pertains . all patents and publications referred to in this application are incorporated herein by reference to the same extent as if each was specifically indicated as being incorporated by reference , to the extent that they provide materials and methods not specifically shown . table 1 shows certain phosphazoles arranged by type of heterocycle and by type of substituent , correlated with their compound numbers used in the examples . table 2 shows the inhibition of tnfα and il - β production in thp - 1 cells stimulated with lipopolysaccaride ( lps ) by certain phosphazoles , as well as the cytotoxicity of the corresponding compounds on thp - 1 cells . table 3 shows the inhibition of tnfα and il - 1β production in pbycs stimulated with lps by the adenine analog , compound 10 ( t70241 ), as well as the viability of the cells in the presence of t70241 . table 4 shows anti - hcmv , hsv - 1 and hsv - 2 activity as well as the cytotoxicity in vitro of certain phosphazoles . table 1__________________________________________________________________________representative phosphazoles for use as pharmaceutical agents nucleosides / nucleotides ( ribo , deoxyribo , 3 &# 39 ;, 5 &# 39 ;- cyclic , etc .) miscellaneous phosphazolesheterocycles n - nucleosides p - nucleosides n and / or p substituted__________________________________________________________________________adenine ( 10 ), t70241 soado ( 21 ), r = ribo , t70264 denosine ( 44 ) cyclovir of ade ( 21 ), 70268hypoxanthine ( 12 ), t70256 sodado ( 21 ), r = dribo , t70265 nosine ( 51 ), x = 0 anciclovir of ade ( 21 ), 70269xanthine ( 13 ), t70270 denosine ( 30 ), r = ribo hioinosine ( 51 ), x = 0 lkyl of ade ( 21 ), r = pentyl , 6 - chlompurine ( 14 ) nosine ( 31 ), r = ribo uanosine ( 54 ) t702546 - mercaptopurine ( 15 ), uanosine ( 39 ), r = ribo dg ( 61 ) klenyl of ade ( 21 ), t70245 = pentenyl , guanine ( 18 ), t70242azaado ( 92 ), r = ribo mp ( 48 ) t70262 , t702632 - nh . sub . 2 - 6 - ci - purine ( 19a ) azaino ( 94 ), r = ribo amp ( 49 ) pmpa ( 30 ), r = hpmp linkagel - thioguanine ( 19b ) phosphaado ( 100 ) gmp ( 58 ) cyclovir ( 39 ), r = ch . sub . 2 och . sub . 2 ch . sub . 2caffeine ( 20 ), t70271phosphalno ( 102 ) ubercidin ( 67 ) anciclovir ( 39 ), phosphaguo ( 105 ) deazalno ( 69 ) r = ch . sub . 2 och ( ch . sub . 2 oh ). sub . 2deazaguo ( 77 ), r = ribo angivamycin ( 84 ) oyocamycin ( 86 ) hiosangivamycin ( 87 ), x = s__________________________________________________________________________ numbers in parentheses refer to certain target compound nos . in the reaction schemes described in the examples . &# 34 ; t &# 34 ; numbers such as t7024 i , 70242 , 70245 , 70254 , 70256 , 70262 , 70263 , 70268 , 70269 , 70270 , etc . indicate proprietary reference nos . of the compounds . table 2______________________________________inhibition of tnfα and il - 1β inthp - 1 cells by representative phosphazoles ic . sub . 50 ( μg / ml ). sup . a tc . sub . 50 ( μg / ml ). sup . bcompd no . tnfα il - 1β growing thp - 1______________________________________t70241 23 . 9 16 . 10 & gt ; 1000t70270 238 . 85 & gt ; 250 ndt70256 168 . 5 170 . 4 ndt70271 37 . 33 & gt ; 250 ndt70264 126 . 27 & gt ; 100 117 . 51t70254 19 . 4 152 . 5 14 . 72t70260 & gt ; 250 nd 18 . 9t70262 & lt ; 10 & gt ; 10 3 . 9t70261 & gt ; 250 nd & gt ; 250t70268 & gt ; 100 & gt ; 100 ndt70269 & gt ; 100 & gt ; 100 ndt70265 26 . 17 & gt ; 100 104 . 52adenine & gt ; 250 & gt ; 250 ndadenosine 100 & gt ; 250 ndhwa - 3138 254 . 6 & gt ; 500 316 . 02______________________________________ . sup . a ic . sub . 50 = inhibitory concentration at 50 % level ; . sup . b drug dose required to inhibit logphase growing cells by 50 %; nd , not determined . * hwa3138 is a xanthine analog reported by semmler et al ., 1993 , immunolog 78 : 520 . table 3______________________________________inhibition of tnfα and il - 1β in pbmcs by compound t70241 % inhibition % inhibition % viable of tnfα * of il - 1β * cellstreatment of pbmcs 4 h 24 48 h 4 h 24 h 48 h 48 h______________________________________ + lps + 0 0 0 0 0 0 1050 μg / ml of t70241 + lps + 0 - 3 - 9 18 6 4 10410 μg / ml of t70241 + lps + 50 58 51 86 24 36 8350 μg / ml of t70241 + lps + 76 81 79 98 78 70 94100 μg / ml of t70241 + lps + 76 84 82 99 . 7 86 75 88250 μg / ml of t70241______________________________________ * percent inhibition of tnfα and il1β are presented relative to the control (+ lps + 0 μg / ml of t70241 ) lps = lipopolysaccharide table 4__________________________________________________________________________anti - hcmv activity of representative phosphazoles tc . sub . 50 ( μg / ml ). sup . bic . sub . 50 ( μg / ml ). sup . a growing static growing staticcompd hcmv hsv - i hsv - 2 mrc - 5 nirc - 5 vero vero__________________________________________________________________________t70241 8 . 08 & gt ; 50 & gt ; 50 & gt ; 50 & gt ; 50 & gt ; 50 & gt ; 50t20254 & gt ; 50 & gt ; 50 & gt ; 50 17 . 95 9 . 25 8 . 18 18 . 17t70268 & gt ; 50 & gt ; 50 & gt ; 50 & gt ; 50 & gt ; 50 & gt ; 50 & gt ; 50t70269 26 . 1 2 . 75 27 . 75 & gt ; 50 & gt ; 50 41 . 41 & gt ; 50acv nd 0 . 1 0 . 74 & gt ; 250 & gt ; 250 & gt ; 250 & gt ; 250dhpg 3 . 5 nd nd & gt ; 250 & gt ; 250 & gt ; 250 & gt ; 250__________________________________________________________________________ . sup . a ic . sub . 50 = inhibitory concentration at 50 % level . sup . b drug dose required to inhibit logphase growing cells by 50 % or concentrations resulting in 50 % cell death ( static cells ); nd = not determined .