the present invention relates to the use of pyrrolo [ 2 , 3 - d ] pyrimidine nucleosides in the treatment of viral infections , and , more specifically , to compounds of the following general formula and pharmaceutically acceptable salts thereof : ## str3 ## where r 1 is nh 2 or oh ; r 2 is h , cn , ## str4 ## and r 3 is 2 &# 39 ;, 3 &# 39 ;- dideoxy - 2 &# 39 ;, 3 &# 39 ;- didehydroribofuranose or 2 &# 39 ;, 3 &# 39 ;- dideoxyribofuranose . the compounds of the present invention exhibit antiviral activity with acceptable levels of cytotoxicity and can thus be used in the treatment of viral infections . in particular , as shown in table 1 , these compounds are effective against human immunodeficiency virus type 1 ( hiv - 1 ), human cytomegalovirus ( hcmv ) and herpes simplex virus type 1 ( hsv - 1 ). additional viruses contemplated to be within the broad scope of treatment of the present invention include , but are not limited to , the following : human b lymphotropic virus , herpes simplex virus type 2 , varicella - zoster virus , epstein - barr virus , necrotic rhinitis , malignant catarrh , allerton virus , equine herpesvirus -- 1 , equine herpesvirus -- 2 , equine herpesvirus -- 3 , neurolymphomatosis , influenza viruses , a , b and c , parainfluenza viruses -- 1 , 2 , 3 and 4 , adenovirus , rheovirus , respiratory syncytial virus , rhinoviruses , coxsackie virus , echo viruses , epidemic gastroenteritis virus , rubeola virus , hepatitis a and b viruses , and papovavirus . the compounds of the present invention can be used in a therapeutically effective amount to treat viral infections in vivo in accordance with conventional procedures , for example , as an active ingredient in pharmaceutical compositions . the pharmaceutical compositions may take the form of tablets , lozenges , granules , capsules , pills , ampoules or suppositories . they may also take the form of ointments , gels , pastes , creams , sprays , lotions , suspensions , solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents , syrups , granulates or powders . moreover , pharmaceutical compositions containing a compound of the present invention , can also contain other pharmaceutically active compounds or a plurality of compounds of the invention to maximize their therapeutic effectiveness . the compounds of the present invention can be synthesized by the general procedure illustrated in figs . i - iv . referring now to fig . i , the parent pyrrolo [ 2 , 3 - d ] pyrimidine ribofuranosyl compound tubercidin ( 1 ) was dissolved in acetonitrile and reacted with 2 - methyl - 2 - acetoxypropionyl bromide to introduce the bromine moiety in a trans orientation relative to the 2 &# 39 ;- acetoxy moiety . the compound was then reacted with pyridine and acetic anhydride to acetylate any free hydroxyl moieties . the resultant intermediate compound ( 2 ) was then treated with zinc - copper couple and dimethylformamide to form a second intermediate compound ( 3 ), then with methanolic ammonia to yield 2 &# 39 ; 3 &# 39 ;- dideoxy - 2 &# 39 ;, 3 &# 39 ;- didehydrotubercidin ( 4 ). a similar scheme of synthesis was followed with the parent compounds toyocamycin ( 6 ), as illustrated in fig . ii , and sangivamycin ( 11 ), as illustrated in fig . iii . as shown in figs . ii and iii , the only additional step in the synthesis of their 2 &# 39 ;, 3 &# 39 ;- dideoxy - 2 &# 39 ;, 3 &# 39 ;- didehydro derivatives was the addition of methanol after reaction with the bromide compound to remove the 5 &# 39 ; blocking moiety . referring now to fig . iv , conversion of the 2 &# 39 ; 3 &# 39 ;- dideoxy - 2 &# 39 ;, 3 &# 39 ;- didehydroribofuranosyl compounds ( 4 , 9 , 14 ) of figs . i , ii and iii to their respective 2 &# 39 ; 3 &# 39 ;- dideoxyribofuranosyl compounds ( 5 , 10 , 15 ) was achieved by hydrogenating in a parr apparatus the 2 &# 39 ;, 3 &# 39 ;- dideoxy - 2 &# 39 ;, 3 &# 39 ;- didehydro compound dissolved in alcohol with palladium on charcoal . the specific procedure followed for the synthesis of each compound depicted in figs . i - iv is set out below in the specific examples . 2 - methyl - 2 - acetoxypropionyl bromide ( 7 . 07 g , 33 . 9 mmole ) was added , with vigorous stirring to a suspension of finely ground tubercidin ( 3 . 0 g . 11 . 3 mmole ) in acetonitrile ( 100 ml , dried over 3 å sieves ) and the mixture then stirred at room temperature for 1 . 5 hr . the resulting solution was partitioned between ice - cold 50 % saturated sodium bicarbonate ( 300 ml ) and ethyl acetate ( 300 ml ). the organic layer was washed with brine ( 100 ml ), dried over sodium sulphate , filtered , and the filtrate was evaporated to afford a crisp white foam ( 5 . 8 g ). this foam was dissolved in pyridine ( 100 ml ), 4 - dimethylaminopyridine was added ( 40 mg , 0 . 4 mmole ), and the solution was treated with acetic anhydride ( 4 . 0 ml , 43 mmole ). the mixture was stirred at room temperature for 1 hr . and then evaporated to a thick oil . this oil was partitioned between ethyl acetate ( 300 ml ) and water ( 300 ml ). the organic layer was then washed with brine ( 100 ml ), dried over sodium sulphate , filtered , and the filtrate evaporated to a yellow foam which was kept in vacuo for 72 hr . this foam was dissolved in dimethylformamide ( 150 ml ) and treated with zinc - copper couple ( 20 g ). the mixture was stirred for 1 . 5 hr . at 80 °, then filtered through celite . the celite bed was washed with dimethylformamide ( 50 ml ) and the combined filtrates were evaporated at 50 °- 60 ° to afford a syrup which was partitioned between ethyl acetate ( 100 ml ) and water ( 300 ml ). the water layer was then extracted with ethyl acetate ( 2 × 100 ml ) and the combined organic fractions were washed with brine ( 200 ml ), dried over magnesium sulphate , filtered and the filtrate was evaporated to a thick oil . this oil was further evaporated in vacuo to afford a soft foam . this foam was dissolved in methanol ( 35 ml ), methanolic ammonia ( 35 ml , saturated at 0 °) was added , and the solution was sealed in a pressure bottle and stirred at room temperature for 12 hr . the solution was evaporated to a thick oil and then further evaporated in vacuo at 50 °- 60 ° to afford a very thick oil . this oil was dissolved in chloroform ( 10 ml ) and the solution was applied to a bed of silica ( 2 cm deep , 3 cm wide ) slurry packed in a fritted disc funnel . this bed was eluted with solvent system a ( 300 ml ). the first 100 ml of eluant was discarded , and the next 200 ml of eluant was collected and evaporated at 50 °- 60 ° to afford a thick oil . this oil was triturated with ether ( 5 × 20 ml ), chloroform ( 15 ml ) was added to the remaining residue and this mixture then heated at reflux . ether was then slowly added ( 30 ml ) and the mixture solidified upon cooling to room temperature . additional ether was added ( 50 ml ), the solid was collected by filtration and air dried to yield a tan solid ( 1 . 63 g ). the mother liquor was combined with the ethereal extracts , the resulting solution was evaporated and then triturated with chloroform / ether ( 1 : 2 , 30 ml ) to afford an additional 0 . 4 g of solid . the solids were combined and suspended in boiling ethyl acetate ( 200 ml ). isopropanol ( 30 ml ) was added , and the boiling mixture was stirred until all solid material had dissolved . norit ( 200 mg ) was added , and the mixture was filtered through celite . the celite bed was washed with ethyl acetate ( 20 ml ). the combined filtrates were reduced in volume to approximately 50 ml by boiling . the solution was allowed to stand at room temperature for 4 hr . the solid was collected by filtration and air dried to afford 1 . 3 g of a tan solid . the material was further purified by dissolving 1 . 2 g in boiling ethanol ( 150 ml ) and reducing the volume of the solution to 50 ml by boiling . the solution was allowed to cool to room temperature and then allowed to stand at 4 ° for 16 hr . the solid was collected by filtration to yield a solid ( 0 . 93 g ), after drying at 80 ° under reduced pressure over phosphorous pentoxide . some additional product ( 0 . 32 g ) could be obtained by evaporating the ethyl acetate and ethanol mother liquors and crystallizing the resulting residue from ethanol ( 25 ml ) for a total yield of 1 . 25 g ( 48 %). mp 204 °- 205 °; 1 h nmr ( dmso - d 6 ): δ , 8 . 07 ( s , 1h , h - 2 ), 7 . 15 ( d , 1h , h - 6 , j . sub . ( 5 , 6 ) = 3 . 71 hz ), 7 . 12 ( bs , 1 h , h - 1 &# 39 ;), 7 . 02 ( bs , 2h , nh 2 ), 6 . 56 ( d , 1h , h - 5 ), 6 . 42 ( ddd , 1h , h - 3 &# 39 ;, j . sub . ( 2 &# 39 ;, 3 &# 39 ;) = 6 . 01 hz , j . sub . ( 3 &# 39 ;, 4 &# 39 ;) = j . sub . ( 3 &# 39 ;, 1 &# 39 ;) = 1 . 62 hz ), 6 . 02 ( ddd , 1h , h - 2 &# 39 ;), 4 . 96 ( t , 1h , 5 &# 39 ;- oh ), 4 . 79 ( m , 1h , h - 4 &# 39 ;), 3 . 50 ( m , 2h , h - 5 &# 39 ; a , b ); uv : λ max ( nm ) ( log epsilon ): meoh , 271 ( 4 . 11 ); ph 1 , 272 ( 4 . 04 ), 226 ( 4 . 36 ); ph 11 , 271 ( 4 . 04 ), 224 ( 3 . 82 ); tlc : solvent a , r f = 0 . 21 ; anal calcd . for c 11 h 12 n 4 o 2 : c , 56 . 89 : h , 5 . 20 ; n , 24 . 13 . found : c , 56 . 88 ; h , 5 . 22 ; n , 24 . 09 . a solution of compound 4 ( 0 . 5 g , 2 . 14 mmole ) in absolute ethanol ( 75 ml ), containing 10 % palladium on charcoal ( 0 . 04 g ) was hydrogenated for 7 hr . on a parr hydrogenation apparatus at a pressure of 50 psi . the catalyst was removed by filtration through celite . the celite bed was washed with ethanol ( 10 ml ) and the filtrates were evaporated to afford an oil . this oil was dissolved in chloroform ( 30 ml ) and the solution was evaporated to afford a foam . after being kept in vacuo for 16 hr ., this foam was dissolved in chloroform / methanol ( 6 : 1 , v : v , 3 ml ) and the solution was applied to a silica column ( 300 mm × 22 mm ). the column was eluted at a flowrate of 17 ml / min ., with chloroform / methanol ( 6 : 1 , v : v ). the elution was followed by observing the absorbtion at 275 nm . the major fraction ( eluting between 7 . 2 and 16 . 4 min .) was evaporated to dryness and kept in vacuo for 48 hr . to afford 380 mg of a crisp foam ( 75 %): 1 h nmr ( dmso - d 6 ): δ , 8 . 04 ( s , 1h , h - 2 ), 7 . 33 ( d , 1h , h - 6 , j . sub . ( 5 , 6 ) = 3 . 6 hz ), 6 . 99 ( bs , 2h nh 2 ), 6 . 55 ( d , 1h , h - 5 ), 6 . 33 ( dd , 1h , h - 1 &# 39 ; j . sub . ( 1 &# 39 ;, 2 &# 39 ; a ) = j . sub . ( 1 &# 39 ;, 2 &# 39 ; b ) = 6 . 8 hz ), 4 . 99 ( bs , 1h , 5 &# 39 ;- oh ), 4 . 03 ( m , 1h , h - 4 &# 39 ;), 3 . 55 and 3 . 46 ( 2m , 2h , h - 3 &# 39 ; ab ); uv λ max ( nm ) ( log epsilon ): meoh , 273 ( 3 . 99 ), 228 ( 4 . 35 ); ph 1 , 273 ( 4 . 02 ), 277 ( 4 . 35 ); ph 11 , 271 ( 4 . 07 ), 225 ( 3 . 95 ); tlc ; solvent a , r f = 0 . 24 : anal . calcd . for c 11 h 14 n 4 o 2 ( 1 / 4 h 2 o ): c , 55 . 34 ; h , 6 . 12 ; n , 23 . 47 . found : c , 55 . 25 ; h , 6 . 29 ; n , 23 . 27 . a vigorously stirred suspension of finely ground toyocamycin ( 5 . 8 g , 20 mmole ) in acetonitrile ( 125 ml , dried over 3 å was chilled with an ice bath to ca . 5 °. to this suspension was added 2 - methyl - 2 - acetoxypropionyl bromide ( 11 . 7 g , 56 mmol ) over a 5 min . period via a syringe , with continuous stirring and cooling . after the addition was complete , the ice bath was removed and stirring was continued for an additional 1 hr . after this time , tlc analysis of the mixture revealed two bands ( r f = 0 . 60 , 0 . 30 ; solvent system a ; plate exposed to conc . ammonia vapor for ca 1 sec before developing ). the solution was then treated with methanol ( 5 ml ) and stirring was continued for an additional 15 min . after this time , tlc analysis showed an absence of the faster running band . the solution was then evaporated to afford a sticky foam which was kept in vacuo for 0 . 5 hr . and then dissolved in pyridine ( 150 ml ). acetic anhydride ( 12 ml , 127 mmole ) was then added to this solution and stirring was continued for an additional 0 . 5 hr . at this time , tlc showed one major band ( r f = 0 . 6 ). the solution was then evaporated in vacuo to a thick oil which was partitioned between ethyl acetate ( 300 ml ) and water ( 600 ml ). the organic layer was washed with water ( 2 × 600 ml ) and brine ( 300 ml ), dried over magnesium sulphate , filtered , and evaporated to a foam which was treated with methanol ( 100 ml ) and again evaporated to a foam which was then kept in vacuo for 0 . 5 hr . this foam was dissolved in hot methanol ( 125 ml ) and the solution was allowed to cool to room temperature . the solution was allowed to stand at room temperature for 2 hr . then kept at ca . 5 ° for 1 hr . the crystalline product was collected by filtration , washed with cold methanol ( 30 ml ) and dried at 80 ° under reduced pressure over phosphorous pentoxide for 16 hr . to afford 4 . 2 g of a white solid . additional material could be obtained by evaporating the mother liquor , triturating the residue with hot ligroin ( 3 × 75 ml ), co - evaporating the residue with methanol ( 2 × 50 ml ), and finally crystallizing the resulting foam from hot methanol ( 30 ml ). drying the solid as above , yielded 1 . 2 g of a hard glass which was identical by tlc ( r f = 0 . 6 , solvent system a ) to the first crop . to a solution of the material obtained as described above ( 7 . 5 g , 17 mmole ), in dimethylformamide ( 25 ml ), was added acetic acid ( 0 . 25 ml , 2 . 6 mmole ) and zinc - copper couple ( 7 . 5 g ). after stirring for 10 min ., the mixture became warm and tlc analysis ( solvent system a ) shows the absence of starting material ( r f = 0 . 63 , chars yellow ) and the presence of a new spot ( r f = 0 . 57 , chars blue ). the mixture was treated with water ( 10 ml ) and filtered through celite . the celite bed was washed with dimethylformamide ( 20 ml ) and the combined filtrates diluted to a volume of 200 ml wth water . this solution was extracted with ethyl acetate ( 5 × 40 ml ) and the combined extracts were washed with water ( 40 ml ) and then brine ( 3 × 40 ml ). the organic layer was then dried over magnesium sulphate , filtered , and evaporated to afford a sticky foam which was co - evaporated with methanol ( 2 × 40 ml ). this foam was then dissolved in hot methanol ( 20 ml ), filtered , and allowed to crystallize for 2 hr . at room temperature . the product was collected by filtration , and dried as described above to afford 2 . 7 g ( 52 %) of the 5 &# 39 ;- o - acetate of the title compound : mp 148 °- 149 °; 1 h nmr ( dmso - d 6 ): δ8 . 25 ( s , 1h , h - 2 ), 8 . 09 ( s , 1h , h - 6 ), 7 . 13 ( bs , h , h - 1 &# 39 ;), 6 . 88 ( bs , 2h , nh 2 ), 6 . 51 ( d , 1h , h - 3 &# 39 ;, j . sub . ( 2 &# 39 ;, 3 &# 39 ;) = 6 hz ), 6 . 17 ( d , 1h , h - 2 &# 39 ;), 5 . 06 ( bs , 1h , h - 4 &# 39 ;), 4 . 16 ( m , 2h , h - 5 &# 39 ; ab ), 2 . 01 ( s , 3h , 5 &# 39 ; ch 3 co ). a suspension of the 5 &# 39 ;- o - acetate ( 2 . 0 g 6 . 3 mmole ) in methanolic ammonia ( 75 ml , saturated at 0 °) was sealed in a pressure bottle and stirred at room temperature for 6 hr . the resulting yellow solution was reduced in volume by boiling on a steam bath to approximately 20 ml . the solution was then allowed to crystallize at room temperature . the solid was collected by filtration , washed with methanol ( 5 ml ) and air dried to afford 1 . 2 g of a white solid ( 22 % overall from toyocamycin ). a sample ( 1 . 0 g ) of this material was recrystallized by dissolving it in methanol ( 40 ml ), diluting the solution with water to a final volume of 100 ml , and allowing the solution to stand overnight at room temperature . the solid was collected by filtration and washed with water ( 10 ml ) to yield 0 . 56 g of material after air drying . a sample of this material ( 0 . 2 g ) was dissolved in solvent system a ( 10 ml ) and the solution was chromatographed on a silica column ( 22 mm × 300 mm ) using solvent system a at a flowrate of 17 ml / min . the elusion was followed by observing the absorbtion at 280 nm . the desired fraction ( eluting between 4 . 2 and 10 . 0 min .) was immediately evaporated under water aspirator pressure at room temperature . the residue was suspended in methanol ( 5 ml ) and filtered . the product cake was washed with methanol ( 5 ml ) and ether ( 5 ml ) to afford 0 . 12 g of a white powder , after air drying : mp 201 °- 202 °; 1 h nmr ( dmso - d 6 ): δ , 8 . 25 ( s , 1h , h - 2 ), 8 . 22 ( s , 1h , h - 6 ), 7 . 13 ( bs , 1h , h - 1 &# 39 ;), 6 . 87 ( bs , 2h , nh 2 ), 6 . 48 ( d , 1h , h - 3 &# 39 ;. j . sub . ( 2 &# 39 ;, 3 &# 39 ;)= 6 . 05 hz ), 6 . 07 ( d , 1h , h - 2 &# 39 ;), 5 . 00 ( t , 1h , 5 &# 39 ;- oh ), 4 . 87 ( m , 1h , h - 4 &# 39 ;), 3 . 58 ( m , 2h , h - 5 &# 39 ; ab ); uv λ max ( nm ) ( log epsilon ): [ meoh , 279 ( 4 . 31 ), 229 ( 4 . 17 ); ph 1 , 273 ( 4 . 26 ), 233 ( 4 . 39 ); ph 11 , 278 ( 4 . 33 ), 231 ( 4 . 20 );] tlc : solvent system a , r f = 0 . 38 ; ir ( kbr ) 2220 cm - 1 ( cyano ); anal . calcd . for c 12 h 11 n 5 o 2 : c , 56 . 03 ; h , 4 . 31 : n , 27 . 23 . found : c , 56 . 08 ; h , 4 . 36 ; n , 27 . 16 . a solution of compound 9 ( 0 . 4 g , 1 . 7 mmole ) in ethanol / water ( 90 : 10 , v : v , 30 ml ) containing 5 % palladium on charcoal ( 0 . 05 g ) was hydrogenated on a parr apparatus for 1 hr . at a pressure of 50 psi . an additional portion ( 0 . 1 g ) of the catalyst was then added , and the hydrogenation was continued for an additional 2 hr . the catalyst was removed by filtration through celite , and the celite bed was then washed with ethanol ( 20 ml ). the combined filtrates were evaporated under aspirator pressure at 45 °- 50 °, and the residue was co - evaporated with ethanol ( 2 × 20 ml ). the residue was crystallized from isopropanol ( 20 ml ), and washed with ether ( 20 ml ) to yield 0 . 23 g ( 57 %) of a white solid , after air drying . a sample ( 0 . 1 g ) of this material was dissolved in dmso ( 2 ml ) and the solution was chromatographed in two portions on a c - 18 column ( 22 mm × 250 mm , 50 mm guard column ) using water / methanol ( 70 : 30 , v : v ) at a flowrate of 12 ml / min . the elution was monitored by observing the absorbtion at 280 nm . the desired fraction eluted between 23 and 42 min . this fraction was combined with the corresponding fraction from a duplicate run and evaporated under water aspirator pressure at 35 ° to a volume of 8 ml . the resulting suspension was heated to dissolve the solids and the solution was then allowed to stand at room temperature for 3 hr . the resulting solid was collected by filtration , washed with water ( 10 ml ) and air dried to afford 0 . 053 g of a white solid : mp 197 °- 197 . 5 °; 1 h nmr ( dmso - d 6 ): δ8 . 44 ( s , 1h , h - 2 ), 8 . 20 ( s , 1h , h - 6 ), 6 . 83 ( bs , 2h , nh . sub . 2 ), 6 . 36 ( dd , 1h , h - 1 &# 39 ;, j = 3 . 13 , 6 . 84 hz ), 5 . 02 ( t , 1h , 5 &# 39 ;- oh ), 4 . 08 ( m , 1h , h - 4 &# 39 ;), 3 . 62 and 3 . 54 ( 2m , 2h , h - 5 &# 39 ; ab ), 2 . 41 and 2 . 22 ( 2m , 2h , h - 2 &# 39 ; ab ), 1 . 97 ( m , 2h , h - 3 &# 39 ; ab ); uv λ max ( nm ) ( log epsilon ): meoh , 280 ( 4 . 05 ), 231 ( 3 . 93 ); ph 1 , 274 ( 3 . 92 ), 235 ( 4 . 08 ); ph 11 , 279 ( 4 . 03 ), 234 ( 3 . 89 ); tlc ; solvent system a , r f = 0 . 40 , chars yellow ; ir ( kbr ) 2220 cm - 1 ( cyano ); anal . calcd . for c 12 h 13 n 5 o 2 : c , 55 . 59 ; h , 5 . 05 ; n , 27 . 02 . found : c , 55 . 68 ; h , 5 . 08 ; n , 26 . 91 . a rapidly stirred suspension of sangivamycin ( 6 . 18 g , 20 mmole ) in acetonitrile ( 125 ml , dried over 3 å sieves ) was treated with 2 - methyl - 2 - acetoxypropionyl bromide ( 10 . 4 g , 50 mmole ) and the stirring was continued for 1 . 5 hr . an additional portion of the bromo compound was added ( 1 g , 5 mmole ) and stirring was continued for another 5 min . methanol was then added ( 5 ml ) and the mixture was stirred for 10 min . the solvent was removed by evaporation . pyridine ( 100 ml ) and acetic anhydride ( 10 ml 0 . 1 mmole ) were added to the residue and the mixture was shaken vigorously for 5 min . in order to cause dissolution of the gummy residue . after an additional 15 min . of stirring , tlc analysis of the mixture shows one major product ( r f = 0 . 51 , solvent system a ). the solution was evaporated , and the resulting gummy residue was treated with ethanol / water ( 1 : 1 , v : v , 100 ml ) and the solution was once again evaporated to afford a gummy residue . this gum was treated with isopropanol ( 200 ml ) and the mixture was heated to reflux temperature . the mixture was allowed to cool to room temperature , the solid was collected by filtration ( 6 . 8 g ) and combined with the solid obtained ( 1 . 2 g ) by concentration of the mother liquor and trituration of the residue with isopropanol ( 100 ml ). a sample ( 7 . 5 g ) of this material was dissolved in hot isopropanol / methanol ( 4 : 2 , v : v , 600 ml ) and the volume of the solution was reduced to 300 ml by boiling on a hot plate . the solid , which had precipitated on cooling to room temperature , was collected by filtration , washed with isopropanol ( 20 ml ) and ether ( 50 ml ) to afford 5 . 2 g of a white powder . this powder ( 4 . 0 g ) was dissolved in dimethylformamide ( 70 ml ) and the solution was treated with zinc - copper couple ( 1 . 5 g ). after stirring for 2 hr ., tlc ( solvent system a ) shows the absence of starting material ( r f = 0 . 51 and the presence of product ( r f = 0 . 31 ). the spent zinc - copper couple was removed by filtration through celite , and the celite bed was washed with dimethylformamide ( 3 × 15 ml ). the combined filtrates were evaporated to afford an oil which solidified upon treatment with ethanol ( 50 ml ). the solid was collected by filtration and washed with ethanol ( 10 ml ) to afford 3 . 1 g of the crude acetate of the title compound . a suspension of the acetate ( 2 . 0 g ) in methanolic ammonia ( 40 ml , saturated at 0 °) was stirred at room temperature in a pressure bottle for 24 hr . and then kept at - 20 ° for 24 hr . the solid was removed by filtration and washed with methanol ( 5 ml ) and ether ( 10 ml ). the combined filtrates were evaporated to dryness and the residue was triturated with hot ethanol ( 30 ml ). the solid material was collected by filtration , after cooling the suspension to room temperature , washed with ethanol ( 10 ml ) and ether ( 10 ml ) to afford 0 . 76 g of the crude title compound ( 29 % overall from sangivamycin ). a sample ( 0 . 5 g ) of this material was dissolved in water ( 10 ml ) and the solution was applied to a sephadex lh - 20 column ( 2 . 5 cm × 20 cm ). the column was eluted with water while the elution was monitored by tlc ( solvent system a ). the first product containing fraction ( r f = 0 . 14 , 10 ml ) was discarded with the next 100 ml of eluent being collected and then evaporated to dryness . the residue was triturated with ethanol ( 20 ml ), the solid was collected by filtration , washed with ethanol ( 5 ml ) and ether ( 10 ml ) to afford 0 . 29 g of a white powder , after air drying . a sample ( 0 . 1 g ) of this material was dissolved in chloroform / methanol / water ( 80 : 10 : 2 , v : v : v , 3 ml ) and the solution was chromatographed on a silica column ( 20 mm × 100 mm , 10 micron silica ) using chloroform / methanol ( 8 : 1 , v : v ) at a flowrate of 15 ml / min . the elution was followed by observing the absorbtion at 280 nm and the fraction containing the product ( eluting between 4 and 12 min .) was collected and evaporated . the residue was triturated with methanol ( 3 ml ) and the solid was collected by filtration , washed with methanol ( 2 ml ) and ether ( 5 ml ) to afford 0 . 068 g of a white solid , after air drying : mp 240 ° dec ., 1 h nmr ( dmso - d 6 ): δ , 8 . 10 ( 2 , 1h , h - 2 ), 8 . 00 and 7 . 32 ( 2bs , 2h , nh 2 ), 7 . 95 ( s , 1h , h - 6 ), 7 . 15 ( bs , 1h , h - 1 &# 39 ;), 6 . 54 ( d , 1h , h - 3 &# 39 ;, j . sub . ( 2 &# 39 ;, 3 &# 39 ;)= 5 . 98 hz ), 6 . 08 ( d , 1h , h - 2 &# 39 ;), 4 . 92 ( t , 1h , 5 &# 39 ;- oh ), 4 . 81 ( bs , 1h , h - 4 &# 39 ;), 3 . 53 ( m , 2h h - 5 &# 39 ; ab ); uv λ max ( nm ) ( log epsilon ): meoh , 281 ( 4 . 12 ), 230 ( 3 . 97 ); ph 1 , 275 ( 4 . 02 ), 226 ( 4 . 10 ); ph 11 , 280 ( 4 . 08 ), 233 ( 3 . 87 ); tlc : solvent system a , r f = 0 . 14 ; anal . calcd , for c 12 h 13 n 5 o 3 : c , 52 . 36 ; h , 4 . 76 ; n , 25 . 44 . found : c , 52 . 16 ; h , 4 . 57 ; n , 25 . 28 . a suspension of compound 14 ( 0 . 15 g , 0 . 54 mmol ) in methanol ( 50 ml ) containing 5 % palladium on charcoal ( 0 . 05 g ) was hydrogenated on a parr apparatus for 8 hr . at 35 psi . the catalyst was removed by filtration through celite and the celite bed was washed with methanol ( 2 × 10 ml ). the combined filtrates were evaporated to a thick oil which was then co - evaporated with ethanol ( 2 × 10 ml ) and isopropanol ( 10 ml ). the resulting foam was then treated with ether ( 30 ml ), and the resulting solid was collected by filtration , washed with ether ( 10 ml ) and air dried to afford 0 . 12 g of a white solid . a sample ( 0 . 09 g ) was dissolved in chloroform / methanol ( 8 : 1 , v : v , 5 ml ) and the solution was chromatographed on a silica column ( 20 mm × 100 mm , 10 micron silica ) using chloroform / methanol ( 8 : 1 , v : v ) at a flowrate of 15 ml / min . the elution was monitored by observing the absorbtion at 280 nm and the fraction eluting between 5 min . and 10 min . was collected and evaporated . the residue was triturated with methanol ( 3 ml ). the solid was collected by filtration , washed with methanol ( 3 ml ) and ether ( 5 ml ), then allowed to air dry to afford 0 . 071 g of a white solid ( 47 %): mp 207 °- 208 °. 1 h nmr ( dmso - d 6 ): δ , 8 . 08 ( s , 1h , h - 2 ), 8 . 05 ( s , 1h , h - 6 ), 7 . 94 , 7 . 32 ( 2bs , 2h , nh 2 ), 6 . 38 ( q , 1h , h - 1 &# 39 ;), 4 . 86 ( t , 1h , 5 &# 39 ;- oh ); 4 . 06 ( q , 1h , h - 4 &# 39 ;), 3 . 54 ( m , 2h , h - 5 &# 39 ; ab ), 262 ( m , 1h h - 2 &# 39 ; ab ), 2 . 20 9m , 1h , h - 2 &# 39 ; b ), 2 . 05 ( m , 2h , h - 3 &# 39 ; ab ); uv λ max ( nm ) ( log epsilon ): meoh , 282 ( 4 . 08 ); ph 1 , 232 ( 4 . 15 ), 276 ( 4 . 09 ); ph 11 , 236 ( 3 . 93 ), 245 ( 3 . 89 ), 281 ( 4 . 13 ); tlc : solvent system a r f = 0 . 14 ; anal . calcd . for c 12 h 15 n 5 o 3 1 / 4 h 2 o : c , 51 . 15 ; h , 5 . 54 ; n , 24 . 86 . found : c , 51 . 43 ; h , 5 . 50 ; n , 24 . 67 . the following procedures were followed to test the compounds of the specific examples : assays with herpes virus type 1 ( hsv - 1 ) and human cytomegalovirus ( hcmv ) kb cells , an established human cell line derived from an epidermoid oral carcinoma , were routinely grown in minimal essential medium ( mem ) with hanks salts [ mem ( h )] supplemented with 5 % fetal bovine serum . african green monkey kidney ( bsc - 1 ) cells and diploid human foreskin fibroblasts ( hff cells ) were grown in mem with earle &# 39 ; s salts [ mem ( e )] supplemented with 10 % fetal bovine serum . cells were passaged according to conventional procedures as described in shipman , c ., jr . ; smith , s . h . ; carlson , r . h . ; drach , j . c . antimicrob . agents chemother ., 1976 , 9 , 120 . the plaque - purified isolate , p o , of the towne strain of hcmv was used in all experiments and was a gift of dr . mark stinski , university of iowa . the s - 148 strain of hsv - 1 was provided by dr . t . w . schafer of schering corp . stock preparations of hcmv and hsv - 1 were prepared and titered as described in turk , s . r . ; shipman , c ., jr . ; nassari , r . ; genzlinger , g . ; krawczyk , s . h . ; townsend , l . b . ; drach , j . c . antimicrob . agents chemother ., 1987 , 31 , 544 . hcmv plaque reduction experiments were performed using monolayer cultures of hff cells by a procedure similar to that referenced above for titration of hcmv , with the exceptions that the virus inoculum ( 0 . 2 ml ) contained approximately 50 pfu of hcmv and the compounds to be assayed were dissolved in the overlay medium . hsv - 1 plaque reduction experiments were performed using monolayer cultures of bsc - 1 cells . the assay was performed exactly as referenced above for hsv - 1 titration assays except that the 0 . 2 ml of virus suspension contained approximately 100 pfu of hsv - 1 and the compounds to be tested were dissolved in the overlay medium . two basic tests for cellular cytotoxicity were routinely employed for compounds examined in antiviral assays . cytotoxicity produced in hff and bsc - 1 cells was estimated by visual scoring of cells not affected by virus infection in the plaque reduction assays described above . cytopathology was estimated at 35 - and 60 - fold magnification and scored on a zero to four plus basis on the day of staining for plaque enumeration . cytotoxicity in kb cells was determined by measuring the effects of compounds on the incorporation of radioactive precursors into dna , rna and protein as described in turk , s . r . ; shipman , c ., jr . ; nassari , r . ; genzlinger , g . ; krawczyk , s . h . ; townsend , l . b . ; drach , j . c . antimicrob . agents chemother ., 1987 , 31 , 544 . the human okt4 + t - cell clone , ath8 , was obtained from dr . samuel broder &# 39 ; s laboratory ( nci ) through dr . hiroaki mitsuya . these cells are propagated in rpmi 1640 medium supplemented with 4 mm glutamine , 15 % heat - inactivated fetal bovine serum , antibiotics ( 50 units of penicillin and 50 μg of streptomycin per ml ), and 50 units of recombinant - derived human interleukin - 2 ( ala - 125 ; amgen biologicals ) per ml . the clone h9 cell lines , an okt4 + human t - cell line which is permissive for hiv replication but largely resistant to virus - induced cpe , h9 cells productively infected with the htlv - iii b strain of hiv , and h9 cells productively infected with the rf - ii haitian variant of hiv have been obtained from dr . robert gallo &# 39 ; s laboratory ( nci ) through dr . howard streicher . these cell lines are propagated in rpmi 1640 medium supplemented with 4 mm glutamine , 20 % heat - inactivated fetal bovine serum , and antibiotics ( 50 units of penicillin and 50 g of streptomycin per ml ). for infectious virus , undiluted culture supernate from ho / htlv - iii b producer cells is used . the testing of compounds for antiviral activity employed the modified cpe - inhibition assay described in mitsuya , h . ; weinhold , k . j . ; furman , p . a . ; st . clair , m . h . ; lehrman , s . n . ; gallo , r . c . ; bolognesi , d . ; barry , d . w . ; broder , s . proc . natl . acad . sci . usa , ( 1985 ) 82 , 7096 and mitsuya , h . ; broder , s . proc . natl . acad . sci . usa , 1986 , 83 1911 . the assay is based on the ability of uninfected aht8 cells to grow and form a pellet at the bottom of a culture tube . starting about 4 days after hiv addition , infected ath8 cells begin to die and the pellet starts to break up . the cell pellet is completely destroyed within 10 days . the protective effect of test compounds was assessed by adding them at varying concentrations to the cultured cells at the time of virus infection , then monitoring the status of the cell pellet . ath8 cells were used as the primary targeted cells in the hiv - induced cpe - inhibition assay . cells were treated with polybrene ( 2 μg / ml in growth medium ) for 30 minutes at 37 ° c ., then collected by gentle centrifugation ( 40 × g for 15 minutes at room temperature ) and resuspended in clarified ( 8000 × g for 15 minutes at 4 ° c .) supernate freshly harvested from 48 hour post - passage h9htlv - iii b cells . following a 60 minute absorption period at 37 ° c ., the cells were dispensed into the u - bottom wells of 96 - well trays ( 2 × 10 4 cells in 0 . 1 ml per well ). an equal volume ( 0 . 1 ml ) of supplemented romi 1640 medium containing test compound and twice the normal concentration of interleukin - 2 were added to each well . these compounds were evaluated at seven half - log dilutions . triplicate virus - infected cultures and one uninfected compound cytotoxicity control culture were included at each dosage level . cultures were incubated at 37 ° c . in a humidified atmosphere of 5 % co 2 in air . on day 10 post - infection aliquots were taken from individual wells and the total cell number and cell viability ( based on trypan - blue dye exclusion ) determined . dideoxycytidine was assayed in parallel as a positive control . dose - response relationships were constructed by linearly regressing the percent inhibition of parameters derived in the preceding sections against log drug concentrations . 50 % inhibitory ( i 50 ) concentrations were calculated from the regression lines . the three i 50 concentrations for inhibition of dna , rna and protein synthesis were averaged to give the values reported in table i for kb cell cytotoxicity . samples containing positive controls ( acyclovir , ganciclovir , or vidarabine ) were used in all assays . results from sets of assays were rejected if inhibition by the positive control deviated from its mean response by more than 1 . 5 standard deviations . table1__________________________________________________________________________antiviral activity and cytotoxicity of 4 , 5 - substituted 7 -( β - d - pentofuranosyl ) pyrrolo [ 2 , 3 - d ] pyrimidines . 50 % inhibitory concentration ( μm ) virus tested cytotoxicitycompound no . hiv hcmv hsv - 1 . sup . a ath8 hff . sup . b bsc . sup . b kb . sup . c__________________________________________________________________________dideoxytubercidin 5 -- & gt ; 100 & gt ; 100 -- & gt ; 100 & gt ; 100 -- dideoxydidehydrotubercidin 4 -- & gt ; 100 & gt ; 100 -- & gt ; 100 & gt ; 100 -- dideoxytoyocamycin 14 3 18 21 100 & gt ; 100 & gt ; 100 54dideoxydidehydrotoyocamycin 9 -- 5 . 1 41 -- & gt ; 100 & gt ; 100 & gt ; 100dideosysangivamycin 15 3 & gt ; 100 & gt ; 100 100 & gt ; 100 & gt ; 100 & gt ; 100dideoxydidehydrosangivamycin 13 -- & gt ; 100 & gt ; 100 -- & gt ; 100 & gt ; 100 54__________________________________________________________________________ . sup . a in experiments with hsv1 both the hf and 148 strains were used . . sup . b visual cytotoxicity scored on uninfected hff or bsc cells at time of hcmv or hsv1 plaque determination . . sup . c average percent inhibition of dna , rna and protein synthesis determined in kb cells as described in the text .
this invention relates to the use of certain substituted pyrrolo - pyrimidine nucleosides in the treatment of viral infections , in particular those caused by human immunodeficiency virus type 1 , herpes simplex virus type 1 and human cytomegalovirus . these substituted compounds retain antiviral properties present in their parent compounds yet exhibit significantly decreased levels of cytotoxicity .