the present invention relates to the use of 8 - azaprostaglandin analogs as ocular hypotensives . the compounds used in accordance with the present invention are encompassed by the following structural formula i : the preferred group of the compounds of the present invention includes compounds that have the following structural formula ii . in the above formulae , the substituents and symbols are as hereinabove defined . preferably d represents a covalent bond or is ch 2 ; more preferably d is ch 2 . preferably r is h or c 1 - c 5 lower alkyl . preferably r 3 is selected from the group consisting of phenyl and monosubstituted derivatives thereof , e . g . chloro and trifluoromethyl phenyl , i . e . m - chlorophenyl . preferably x is co 2 r and more preferably r is selected from the group consisting of h and methyl . the above compounds of the present invention may be prepared by methods that are known in the art or according to the working examples below . the compounds , below , are especially preferred representative , of the compounds of the present invention . pharmaceutical compositions may be prepared by combining a therapeutically effective amount of at least one compound according to the present invention , or a pharmaceutically acceptable acid addition salt thereof , as an active ingredient , with conventional ophthalmically acceptable pharmaceutical excipients , and by preparation of unit dosage forms suitable for topical ocular use . the therapeutically efficient amount typically is between about 0 . 0001 and about 5 % ( w / v ), preferably about 0 . 001 to about 1 . 0 % ( w / v ) in liquid formulations . for ophthalmic application , preferably solutions are prepared using a physiological saline solution as a major vehicle . the ph of such ophthalmic solutions should preferably be maintained between 6 . 5 and 7 . 2 with an appropriate buffer system . the formulations may also contain conventional , pharmaceutically acceptable preservatives , stabilizers and surfactants . preferred preservatives that may be used in the pharmaceutical compositions of the present invention include , but are not limited to , benzalkonium chloride , chlorobutanol , thimerosal , phenylmercuric acetate and phenylmercuric nitrate . a preferred surfactant is , for example , tween 80 . likewise , various preferred vehicles may be used in the ophthalmic preparations of the present invention . these vehicles include , but are not limited to , polyvinyl alcohol , povidone , hydroxypropyl methyl cellulose , poloxamers , carboxymethyl cellulose , hydroxyethyl cellulose and purified water . tonicity adjustors may be added as needed or convenient . they include , but are not limited to , salts , particularly sodium chloride , potassium chloride , mannitol and glycerin , or any other suitable ophthalmically acceptable tonicity adjustor . various buffers and means for adjusting ph may be used so long as the resulting preparation is ophthalmically acceptable . accordingly , buffers include acetate buffers , citrate buffers , phosphate buffers and borate buffers . acids or bases may be used to adjust the ph of these formulations as needed . in a similar vein , an ophthalmically acceptable antioxidant for use in the present invention includes , but is not limited to , sodium metabisulfite , sodium thiosulfate , acetylcysteine , butylated hydroxyanisole and butylated hydroxytoluene . other excipient components which may be included in the ophthalmic preparations are chelating agents . the preferred chelating agent is edentate disodium , although other chelating agents may also be used in place or in conjunction with it . the actual dose of the active compounds of the present invention depends on the specific compound , and on the condition to be treated ; the selection of the appropriate dose is well within the knowledge of the skilled artisan . the ophthalmic formulations of the present invention are conveniently packaged in forms suitable for metered application , such as in containers equipped with a dropper , to facilitate the application to the eye . containers suitable for dropwise application are usually made of suitable inert , non - toxic plastic material , and generally contain between about 0 . 5 and about 15 ml solution . sodium hydride ( 29 mg , 60 % dispersion in oil , 0 . 73 mmol ) was added to a solution of [ 3 -( 3 - chlorophenyl )- 2 - oxopropyl ]- phosphonic acid dimethyl ester ( 180 mg , 0 . 65 mmol ) in thf ( 5 ml ) at 0 ° c . the mixture was aged at rt for 40 min then recooled to 0 ° c . a solution of [ 4 -( r )-( 2 - formyl - 5 - oxo - pyrrolidin - 1 - yl )- butoxy ]- acetic acid methyl ester ( 0 . 72 mmol , crude from procedure 1 ) in thf ( 2 ml ) was added via cannula . the reaction was allowed to warm to rt and aged for 20 h . acetic acid and water ( 1 : 1 , 20 ml ) was then added and the mixture was extracted with etoac ( 3 × 20 ml ). the combined organic phase was washed with saturated aqueous nahco 3 ( 3 × 15 ml ) and brine ( 30 ml ) then dried ( na 2 so 4 ), filtered and concentrated in vacuo . the residue was purified by flash column chromatography ( 0 %→ 2 % meoh / ch 2 cl 2 ) and then by preparative thin layer chromatography ( 5 % meoh / ch 2 cl 2 ) to afford 106 mg ( 40 %) of ( 4 -{( r )- 2 -[( e )- 4 -( 3 - chlorophenyl )- 3 - oxo - but - 1 - enyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid methyl ester . a mixture of ( 4 -{( r )- 2 -[( e )- 4 -( 3 - chlorophenyl )- 3 - oxo - but - 1 - enyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid methyl ester ( 2 . 4 mg , 0 . 006 mmol ), rabbit liver esterase ( 1 mg , 134 units / mg ), ph 7 . 2 phosphate buffer ( 2 . 5 ml ) and acetonitrile ( 0 . 1 ml ) was stirred together at rt for 18 h . acetonitrile ( 5 ml ) was then added and the mixture was concentrated in vacuo to dryness . the residue was purified by flash column chromatography ( 0 %→ 10 % meoh / ch 2 cl 2 ) to afford 1 . 1 mg ( 47 %) of ( 4 -{( r )- 2 -[( e )- 4 -( 3 - chlorophenyl )- 3 - oxo - but - 1 - enyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid . pd / c ( 6 mg , 10 wt %) was added to a solution of ( 4 -{( r )- 2 -[( e )- 4 -( 3 - chlorophenyl )- 3 - oxo - but - 1 - enyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid methyl ester ( 59 mg , 0 . 14 mmol ) in methanol ( 2 ml ). the reaction mixture was evacuated and refilled with hydrogen ( 3 ×) then stirred at rt under a balloon of hydrogen for 3 . 5 h . the reaction mixture was filtered through celite , washing with methanol ( 5 ml ). the filtrate was concentrated in vacuo to afford 59 mg ( 99 %) of ( 4 -{( r )- 2 -[ 4 -( 3 - chlorophenyl )- 3 - oxo - butyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid methyl ester . a mixture of ( 4 -{( r )- 2 -[ 4 -( 3 - chlorophenyl )- 3 - oxo - butyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid methyl ester ( 8 . 1 mg , 0 . 02 mmol ), rabbit liver esterase ( 1 mg , 134 units / mg ), ph 7 . 2 phosphate buffer ( 3 ml ) and acetonitrile ( 0 . 1 ml ) was stirred together at rt for 16 . 5 h . aqueous hcl ( 0 . 5 m , 5 ml ) was added and the mixture was extracted with ch 2 cl 2 ( 3 × 5 ml ). the combined organic phase was dried ( na 2 so 4 ), filtered and concentrated in vacuo to afford 1 . 6 mg ( 20 %) of ( 4 -{( r )- 2 -[( e )- 4 -( 3 - chlorophenyl )- 3 - oxo - but - 1 - enyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid . sodium borohydride ( 4 . 1 mg , 0 . 11 mmol ) was added to a solution of ( 4 {( r )- 2 -[ 4 -( 3 - chlorophenyl )- 3 - oxo - butyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid methyl ester ( 44 mg , 0 . 11 mmol ) in ch 2 cl 2 ( 1 ml ) at rt . methanol ( 3 drops ) was added and the reaction was stirred at rt for 4 . 5 h . aqueous hcl ( 0 . 5 m , 5 ml ) was added and the mixture was extracted with ch 2 cl 2 ( 2 × 8 ml ). the combined organic phase was dried ( na 2 so 4 ), filtered and concentrated in vacuo . the residue was purified by flash column chromatography ( 0 %→ 3 % meoh / ch 2 cl 2 ) to afford 9 . 1 mg ( 21 %) of ( 4 -{( r )- 2 -[ 4 -( 3 - chlorophenyl )- 3 - hydroxy - butyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid methyl ester . aqueous lithium hydroxide ( 1 . 0 n , 0 . 04 ml ) was added to a solution of ( 4 -{( r )- 2 -[ 4 -( 3 - chlorophenyl )- 3 - hydroxy - butyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid methyl ester ( 15 mg , 0 . 036 mmol ) in thf ( 0 . 5 ml ) at rt . after 1 h , aqueous hcl ( 1 . 0 n , 3 ml ) was added and the mixture was extracted with ch 2 cl 2 ( 3 × 5 ml ). the combined organic phase was dried ( na 2 so 4 ), filtered and concentrated in vacuo . the residue was purified by flash column chromatography ( 0 %→ 3 % meoh / ch 2 cl 2 ) to afford 6 . 3 mg ( 43 %) of ( 4 -{( r )- 2 -[ 4 -( 3 - chlorophenyl )- 3 - hydroxy - butyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid . sodium borohydride ( 4 . 3 mg , 0 . 11 mmol ) was added to a solution of ( 4 -{( r )- 2 -[( e )- 4 -( 3 - chlorophenyl )- 3 - oxo - but - 1 - enyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid methyl ester ( 42 mg , 0 . 10 mmol ) in ch 2 cl 2 ( 1 ml ) at 0 ° c . methanol ( 3 drops ) was added and the reaction was stirred at rt for 3 h . aqueous hcl ( 0 . 5 m , 5 ml ) was added and the mixture was extracted with ch 2 cl 2 ( 3 × 5 ml ). the combined organic phase was dried ( na 2 so 4 ), filtered and concentrated in vacuo . the residue was purified by flash column chromatography ( 0 %→ 5 % meoh / ch 2 cl 2 ) to afford 31 mg ( 73 %) of ( 4 -{( r )- 2 -[( e )- 4 -( 3 - chlorophenyl )- 3 - hydroxy - but - 1 - enyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid methyl ester . aqueous lithium hydroxide ( 1 . 0 n , 0 . 04 ml ) was added to a solution of ( 4 -{( r )- 2 -[( e )- 4 -( 3 - chlorophenyl )- 3 - hydroxy - but - 1 - enyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid methyl ester ( 16 mg , 0 . 039 mmol ) in thf ( 0 . 25 ml ) at rt . after 1 h , aqueous hcl ( 0 . 5 n , 3 ml ) was added and the mixture was extracted with etoac ( 2 × 5 ml ). the combined organic phase was washed with brine ( 10 ml ), dried ( na 2 so 4 ), filtered and concentrated in vacuo to afford 15 mg ( 97 %) of ( 4 -{( r )- 2 -[( e )- 4 -( 3 - chlorophenyl )- 3 - hydroxy - but - 1 - enyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid . sodium hydride ( 4 . 44 g , 60 % dispersion in oil , 111 mmol ) was added to a solution of 1 , 4 - butanediol ( 10 . 0 g , 111 mmol ) in thf ( 200 ml ). after stirring 1 h at rt , the mixture was cooled to 0 ° c . and methyl bromomethylacetate ( 10 . 82 ml , 114 mmol ) was added and the reaction was allowed to warm to room temperature . after 16 h , water ( 100 ml ) was added and the mixture was extracted with etoac ( 3 × 100 ml ). the combined organic phase was washed with brine ( 2 × 100 ml ), dried ( na 2 so 4 ), filtered and concentrated in vacuo . the residue was purified by flash column chromatography ( 20 %→ 30 % etoac / hexane ) to afford 2 . 88 g ( 16 %) of ( 4 - hydroxy - butoxy )- acetic acid methyl ester as a colorless oil . triphenylphosphine ( 5 . 59 g , 21 . 3 mmol ), bromine ( 1 . 1 ml , 21 . 5 mmol ) and imidazole ( 1 . 41 g , 20 . 7 mmol ) were sequentially added to a solution of ( 4 - hydroxy - butoxy )- acetic acid methyl ester ( 2 . 88 g , 17 . 8 mmol ) in ch 2 cl 2 ( 15 ml ) at 0 ° c . under nitrogen . after 1 h , the mixture was filtered through basic alumina , rinsing with 5 % etoac / hexane ( 40 ml ). the filtrate was concentrated and the residue was purified by flash column chromatography ( 0 %→ 20 % etoac / hexane ) to afford 2 . 02 g ( 51 %) of ( 4 - bromo - butoxy )- acetic acid methyl ester as a colorless oil . sodium hydride ( 192 mg , 60 % dispersion in oil , 4 . 8 mmol ) was added to a solution of 5 -( tert - butyldimethylsilanyloxymethyl )- pyrrolidin - 2 - one ( 1 . 0 g , 4 . 4 mmol ) in dmf ( 8 ml ). after stirring for 1 h at rt , a solution of ( 4 - bromo - butoxy )- acetic acid methyl ester ( 1 . 08 g , 4 . 8 mmol ) in dmf ( 4 ml ) was added . the resulting mixture was heated at 90 ° c . for 18 h . the reaction was cooled to rt then water ( 75 ml ) was added and the mixture was extracted with etoac ( 3 × 50 ml ). the combined organic phase was washed with brine ( 100 ml ), dried ( na 2 so 4 ), filtered and concentrated in vacuo . the residue was purified by flash column chromatography ( 0 %→ 20 % etoac / ch 2 cl 2 ) to afford 758 mg of a mixture of starting material and { 4 -[( r )- 2 -( tert - butyldimethylsilanyloxymethyl )- 5 - oxo - pyrrolidin - 1 - yl ]- butoxy }- acetic acid methyl ester , which was taken on in the next step without further purification . tetrabutylammonium fluoride ( 3 . 0 ml , 1 . 0 m in thf , 3 . 0 mmol ) was added to a solution of impure { 4 -[( r )- 2 -( tert - butyldimethylsilanyloxymethyl )- 5 - oxo - pyrrolidin - 1 - yl ]- butoxy }- acetic acid methyl ester ( 758 mg , ˜ 1 . 98 mmol ) in thf ( 3 ml ) at 0 ° c . under nitrogen . the reaction was allowed to warm to rt and was aged for 18 h . thf was removed in vacuo , saturated aqueous nahco 3 ( 50 ml ) was added and the mixture was extracted with chcl 3 ( 2 × 30 ml ). the combined organic phase was dried ( na 2 so 4 ), filtered and concentrated in vacuo . the residue was purified by flash column chromatography ( 0 %→ 3 % meoh / ch 2 cl 2 ) to afford 188 mg ( 17 % over two steps ) of [ 4 -( r )- 2 - hydroxymethyl - 5 - oxo - pyrrolidin - 1 - yl )- butoxy ]- acetic acid methyl ester . 1 -( 3 - dimethylaminopropyl )- 3 - ethylcarbodiimide hydrochloride ( 412 mg , 2 . 15 mmol ) and dmso ( 0 . 20 ml , 2 . 82 mmol ) were added to a solution of [ 4 -( r )- 2 - hydroxymethyl - 5 - oxo - pyrrolidin - 1 - yl )- butoxy ]- acetic acid methyl ester ( 185 mg , 0 . 72 mmol ) in benzene ( 5 ml ). the mixture was cooled to 0 ° c . then pyridinium trifluoroacetate ( 153 mg , 0 . 79 mmol ) was added . the reaction was aged at 0 ° c . for 15 min , then at rt for 2 h . the solution was decanted from the oil and the oil residue was washed with benzene ( 3 × 4 ml ). the combined benzene phase was concentrated in vacuo to afford the title compound which was used without further purification . these compounds are tested for in vitro activity as described below and the results given in the tables . human recombinant ep 1 , ep 2 , ep 3 , ep 4 , fp , tp , ip and dp receptors : stable transfectants plasmids encoding the human ep 1 , ep 2 , ep 3 , ep 4 , fp , tp , ip and dp receptors were prepared by cloning the respective coding sequences into the eukaryotic expression vector pcep4 ( invitrogen ). the pcep4 vector contains an epstein barr virus ( ebv ) origin of replication , which permits episomal replication in primate cell lines expressing ebv nuclear antigen ( ebna - 1 ). it also contains a hygromycin resistance gene that is used for eukaryotic selection . the cells employed for stable transfection were human embryonic kidney cells ( hek - 293 ) that were transfected with and express the ebna - 1 protein . these hek - 293 - ebna cells ( invitrogen ) were grown in medium containing geneticin ( g418 ) to maintain expression of the ebna - 1 protein . hek - 293 cells were grown in dmem with 10 % fetal bovine serum ( fbs ), 250 μg ml − 1 g418 ( life technologies ) and 200 μg ml − 1 gentamicin or penicillin / streptomycin . selection of stable transfectants was achieved with 200 μg ml − 1 hygromycin , the optimal concentration being determined by previous hygromycin kill curve studies . for transfection , the cells were grown to 50 - 60 % confluency on 10 cm plates . the plasmid pcep4 incorporating cdna inserts for the respective human prostanoid receptor ( 20 μg ) was added to 500 μl of 250 mm cacl 2 . hepes buffered saline × 2 ( 2 × hbs , 280 mm nacl , 20 mm hepes acid , 1 . 5 mm na 2 hpo 4 , ph 7 . 05 - 7 . 12 ) was then added dropwise to a total of 500 μl , with continuous vortexing at room temperature . after 30 min , 9 ml dmem were added to the mixture . the dna / dmem / calcium phosphate mixture was then added to the cells , which had been previously rinsed with 10 ml pbs . the cells were then incubated for 5 hr at 37 ° c . in humidified 95 % air / 5 % co 2 . the calcium phosphate solution was then removed and the cells were treated with 10 % glycerol in dmem for 2 min . the glycerol solution was then replaced by dmem with 10 % fbs . the cells were incubated overnight and the medium was replaced by dmem / 10 % fbs containing 250 μg ml − 1 g418 and penicillin / streptomycin . the following day hygromycin b was added to a final concentration of 200 μg ml − 1 . ten days after transfection , hygromycin b resistant clones were individually selected and transferred to a separate well on a 24 well plate . at confluence each clone was transferred to one well of a 6 well plate , and then expanded in a 10 cm dish . cells were maintained under continuous hygromycin selection until use . radioligand binding studies on plasma membrane fractions prepared for cells stably transfected with the cat or human receptor were performed as follows . cells washed with tme buffer were scraped from the bottom of the flasks and homogenized for 30 sec using a brinkman pt 10 / 35 polytron . tme buffer was added as necessary to achieve a 40 ml volume in the centrifuge tubes . tme is comprised of 50 mm tris base , 10 mm mgcl 2 , 1 mm edta ; ph 7 . 4 is achieved by adding 1 n hcl . the cell homogenate was centrifuged at 19 , 000 rpm for 20 - 25 min at 4 ° c . using a beckman ti - 60 or tt - 70 rotor . the pellet was then resuspended in tme buffer to provide a final protein concentration of 1 mg / ml , as determined by bio - rad assay . radioligand binding assays were performed in a 100 μl or 200 μl volume . the binding of [ 3 h ]( n ) pge 2 ( specific activity 165 ci / mmol ) was determined in duplicate and in at least 3 separate experiments . incubations were for 60 min at 25 ° c . and were terminated by the addition of 4 ml of ice - cold 50 mm tris - hcl followed by rapid filtration through whatman gf / b filters and three additional 4 ml washes in a cell harvester ( brandel ). competition studies were performed using a final concentration of 2 . 5 or 5 nm [ 3 h ]( n ) pge 2 and non - specific binding was determined with 10 − 5 m unlabelled pge 2 . for radioligand binding on the transient transfectants , plasma membrane fraction preparation was as follows . cos - 7 cells were washed with tme buffer , scraped from the bottom of the flasks , and homogenized for 30 sec using a brinkman pt 10 / 35 polytron . tme buffer was added to achieve a final 40 ml volume in the centrifuge tubes . the composition of tme is 100 mm tris base , 20 mm mgcl 2 , 2m edta ; 10n hcl is added to achieve a ph of 7 . 4 . the cell homogenate was centrifuged at 19000 rpm for 20 min at 4 ° c . using a beckman ti - 60 rotor . the resultant pellet was resuspended in tme buffer to give a final 1 mg / ml protein concentration , as determined by biorad assay . radioligand binding assays were performed in a 200 μl volume . the binding of [ 3 h ] pge 2 ( specific activity 165 ci or mmol − 1 ) at ep 3d , receptors and [ 3 h ]- sq29548 ( specific activity 41 . 5 ci mmol − 1 ) at tp receptors were determined in duplicate in at least three separate experiments . radiolabeled pge 2 was purchased from amersham , radiolabeled sq29548 was purchased from new england nuclear . incubations were for 60 min at 25 ° c . and were terminated by the addition of 4 ml of ice - cold 50 mm tris - hcl , followed by rapid filtration through whatman gf / b filters and three additional 4 ml washes in a cell harvester ( brandel ). competition studies were performed using a final concentration of 2 . 5 or 5 nm [ 3 h ]- pge 2 , or 10 nm [ 3 h ]- sq 29548 and non - specific binding determined with 10 μm of the respective unlabeled prostanoid . for all radioligand binding studies , the criteria for inclusion were & gt ; 50 % specific binding and between 500 and 1000 displaceable counts or better . the effects of the compounds of this invention on intraocular pressure may be measured as follows . the compounds are prepared at the desired concentrations in a vehicle comprising 0 . 1 % polysorbate 80 and 10 mm tris base . dogs are treated by administering 25 μl to the ocular surface , the contralateral eye receives vehicle as a control . intraocular pressure is measured by applanation pneumatonometry . a topical once daily dosing of ( 4 -{( r )- 2 -[( e )- 4 -( 3 - chlorophenyl )- 3 - hydroxy - but - 1 - enyl ]- 5 - oxo - pyrrolidin - 1 - yl }- butoxy )- acetic acid [ example 8 ] ( 0 . 1 %) gave a maximum iop decrease from baseline of 4 . 9 mmhg ( 26 . 6 %) at 52 h in normotensive dogs ( n = 8 ). the maximum ocular surface hyperemia score was 1 . 5 ( 1 . 5 at 26 , 50 and 52 h ). certain of the compounds of this invention are useful in lowering elevated intraocular pressure in mammals , e . g . humans , and in treating other diseases and conditions which are responsive to prostaglandin analogues , e . g . glaucoma ; cardiovascular ; e . g . acute myocardial infarction , vascular thrombosis , hypertension , pulmonary hypertension , ischemic heart disease , congestive heart failure , and angina pectoris ; pulmonary - respiratory ; gastrointestinal ; reproductive and allergic diseases ; osteoporosis and shock . the foregoing description details specific methods and compositions that can be employed to practice the present invention , and represents the best mode contemplated . however , it is apparent for one of ordinary skill in the art that further compounds with the desired pharmacological properties can be prepared in an analogous manner , and that the disclosed compounds can also be obtained from different starting compounds via different chemical reactions . similarly , different pharmaceutical compositions may be prepared and used with substantially the same result . thus , however detailed the foregoing may appear in text , it should not be construed as limiting the overall scope hereof ; rather , the ambit of the present invention is to be governed only by the lawful construction of the appended claims .
the present invention provides a method of treating ocular hypertension or glaucoma which comprises administering to an animal having ocular hypertension or glaucoma therapeutically effective amount of a compound represented by the general formula i ; wherein x , y , z , d and r 3 are as defined in the specification .