Patent Application: US-44898108-A

Abstract:
a catecholamine or related compound of formula having - configuration at β - carbon and having a lipophilicity greater than - noradrenalme , a physiologically tolerated salt thereof , a prodrug thereof , a physiologically functional derivative thereof or any mixture thereof is useful as an anti - angiogemc agent a catecholamine or related compound of formula in which a β - hydroxy group has been modified is also anti - angiogemc

Description:
chemicals : ( s )- isoproterenol l - bitartrate , ( s )- noradrenaline l - bitartrate , d - mannitol , benzalkonium chloride , pivaloyl chloride ( trimethylacetyl chloride ), iodoethane , 1 - iodopropane , 1 - iodobutane , sodium borohydride , methyl trifluoromethanesulfonate , 2 - chloro - 3 , 4 - dihydroxyacetophenone , 1 , 4 - dioxane , p - toluoyl chloride , disodium sulfate , chlorobutanol , aminocaproic acid , sodium perchlorate , [ glu 1 ]- fibrinopeptide b , ( r )- isoproterenol hydrochloride , isoetharine mesylate , di - tert - butyl dicarbonate , triethylamine , acetyl chloride , 2 - propanol , dimethylformamide , tert - butylamine , isobutyryl chloride , benzoyl chloride , cetylpyridinium chloride and povidone ( k30 ) were obtained from sigma - aldrich ( oakville , ontario , canada ). acetone , methylene chloride , ethyl acetate , glacial acetic acid , disodium carbonate , sodium chloride and naoh were from emd science ( gibbstown , n . j ., usa ). disodium edetate , trifluoroacetic acid ( tfa ), and water were purchased from j . t . baker ( phillipsburg , n . j ., usa ). 1 . 0 m hcl was obtained from vwr ( montreal , quebec , canada ). water for mass spectrometry was purchased from anachemia ( lachine , qc , canada ). formic acid was purchased from riedel de haën ( oakville , ontario , canada ). acetonitrile and hexane were from fisher scientific ( nepean , ontario , canada ). ag ® 4 - x4 resin , 100 - 200 mesh , free base form was purchased from bio - rad laboratories , inc ( hercules , calif ., usa ). all the chemicals were used without further purification . cetylpyridinium trifluoroacetate was prepared from cetylpyridinium chloride . to a solution of sodium trifluoroacetate in methanol was added trifluoroacetic acid . the resulting methanolic solution of tfa - na was then added dropwisely to the solution of cetylpyridinium chloride in ethanol with stirring . after 30 min , precipitated nacl was filtered off and evaporated with rotary evaporator (& lt ; 40 ° c .). the residue was dissolved in dichloromethane and insoluble components were filtered off . after evaporation , tert - butanol was added and lyophilized to obtain as a white crystal . cetylpyridinium acetate was prepared from cetylpyridinium chloride . cetylpyridinium chloride was dissolved in methanol , and acetic acid and sodium acetate were added . after evaporating the solvent , the residue was dissolved in methylene chloride . cetylpyridinium acetate was soluble in methylene chloride , whereas sodium chloride was precipitated and removed by filtration . the solvent was evaporated and the absence of chloride ion was confirmed as no precipitate was formed when silver nitrate solution was added to the product . this compound is used in the synthesis of ( s )- n - ethylnoradrenaline hydrochloride , ( s )- n - propylnoradrenaline hydrochloride , and ( s )- n - butylnoradrenaline hydrochloride . to a solution of ( s )- noradrenaline bitartrate ( 3 . 83 g , 12 mmol ) in 100 ml of tetrahydrofuran ( thf )- water ( 1 : 1 v / v ) was added sodium bicarbonate ( 3 . 3 g , 40 mmol ), and stirred vigorously . di - tert - butyl dicarbonate ( 2 . 9 g , 13 . 2 mmol ) in thf ( 10 ml ) was then added dropwise at room temperature . the reaction mixture was stirred for 3 hrs . the product ( s )- n - boc - noradrenaline was extracted with 50 ml of dichloromethane ( dcm ) three times . the combined extract was washed with water ( 50 ml ) and brine ( 50 ml ). the product solution was dried over sodium sulfate , filtered and then evaporated to dryness , yielding the desired product . the product showed over 95 % purity based on the hplc profile ( an analytical waters hplc ( waters symmetryshield ™ 3 . 5 μm ; 4 . 6 × 50 mm c18 - reverse phase column ). a gradient 10 - 90 % acetonitrile in water , 0 . 1 % trifluoroacetic acid ( tfa ), for 9 min at a flow rate of 2 ml / min ( system a ) was used . the product was further used without purification . c 13 h 19 no 6 ; white solid ; rt = 4 . 9 min ( system a ). hrms : calcd . for [ m + h + ]= 270 . 1341 , found = 270 . 1342 . esi - ms / ms ( collision energy ( ce )= 2 ): m / z (% relative intensity ): 270 ( m + h + , 94 ), 252 ( 100 ), 214 ( 1 . 3 ), 196 ( 51 ). nmr : 1h nmr ( 500 mhz , cd 3 od ): δ 6 . 80 ( s , 1h ), 6 . 73 ( d , j = 8 hz , 1h ), 6 . 67 ( d , j = 7 hz , 1h ), 4 . 54 ( dd ( br ), j = 5 , 7 hz , 1h ), 3 . 24 ( dd , j = 5 , 13 hz , 1h ), 3 . 16 ( dd , j = 7 , 14 hz , 1h ), 1 . 43 ( s , 9h ). to a mixture of ( s )- n - boc - noradrenaline ( 3 . 0 g , 11 mmol ) and triethylamine ( 5 ml , 36 mmol ) in dcm ( 100 ml ) was slowly added pivaloyl chloride ( 2 . 9 ml , 24 mmol ) at 0 ° c . under nitrogen . the reaction mixture was stirred for 15 min at 0 ° c ., and then for 2 hrs at room temperature . the product solution was washed with water ( 50 ml ), dried over sodium sulfate and concentrated . silica gel column chromatography ( using a gradient of 20 - 40 % ethyl acetate / hexane ) afforded pure product ( 2 . 40 g , 46 % yield from ( s )- noradrenaline bitartrate ) as a viscous liquid . c 23 h 35 no 7 ; viscous liquid ; rt = 9 . 0 min ( system a ). hrms : calcd . for [ m + h + ]= 438 . 2492 , found = 438 . 2482 . esi - ms / ms ( ce = 2 ): m / z (% relative intensity ): 438 ( m + h + , 100 ), 420 ( 2 . 3 ), 382 ( 86 ). nmr : 1h nmr ( 500 mhz , cd 3 od ): δ 7 . 29 ( d , j = 7 hz , 1h ), 7 . 17 ( s , 1h ), 7 . 14 ( d , j = 9 hz , 1h ), 4 . 74 ( t ( br ), j = 8 hz , 1h ), 3 . 28 ( dd , 1h ), 3 . 22 ( dd , j = 8 , 15 hz , 1h ), 1 . 44 ( s , 9h ), 1 . 36 ( s , 9h ), 1 . 34 ( s , 9h ). to a solution of ( s )- n - boc - noradrenaline dipivalate ( 2 . 4 g , 5 . 5 mmol ) in dry methanol ( 100 ml ) was added dropwise acetyl chloride ( 8 ml , 110 mmol ) at 0 ° c . the reaction mixture was stirred for 15 min at 0 ° c . and then at room temperature . the reaction was monitored by tlc . after completion of the reaction ( 3 hrs ), the solvent was evaporated under reduced pressure and the product was purified by high performance displacement chromatography ( hpdc ) ( 0 . 1 % tfa / water , 4 g / l of cetylpyridinium trifluoroacetate as eluent ). the product was further used in the preparations of ( s )- n - ethylnoradrenaline dipivalate hydrochloride , ( s )- n - propyrnoradrenaline dipivalate hydrochloride , and ( s )- n - butylnoradrenaline dipivalate hydrochloride as described below . c 18 h 28 no 5 cl ; white solid ; rt = 6 . 0 min ( system a ). hrms : calcd . for [ c18h27no5 + h + ]= 338 . 1967 , found = 338 . 1983 . esi - ms / ms ( ce = 13 ): m / z (% relative intensity ): 338 ( m + h + , 5 . 9 ), 320 ( 100 ), 236 ( 26 ). nmr : 1h nmr ( 500 mhz , cd 3 od ): δ 7 . 36 ( dd , j = 2 , 8 hz , 1h ), 7 . 27 ( d , j = 2 hz , 1h ), 7 . 20 ( d , j = 8 hz , 1h ), 4 . 94 ( dd , j = 4 , 9 hz , 1h ), 3 . 20 ( dd , j = 4 , 13 hz , 1h ), 3 . 04 ( dd , j = 9 , 13 hz , 1h ), 1 . 36 ( s , 9h ), 1 . 35 ( s , 9h ). to a solution of ( s )- noradrenaline dipivalate hydrochloride ( 0 . 37 g , 1 . 0 mmol ) and triethylamine ( 0 . 4 ml , 3 . 0 mmol ) in 10 ml of dry dimethylformamide ( dmf ) was added iodoethane ( 0 . 12 ml , 1 . 5 mmol ). the reaction mixture was stirred for 24 hrs at room temperature under nitrogen and lyophilized . the mixture was taken up in ethyl acetate ( 30 ml ) and extracted with nah 2 po 3 buffer ( 4 × 20 ml , ph 3 ). the organic layer was extracted again with water ( 3 × 20 ml ). combined buffer extracts and water extracts were adjusted to ph 11 using 1 . 0 n naoh and extracted with ethyl acetate ( 3 × 20 ml ). the separated organic phase was further washed with water ( 20 ml ) and brine ( 20 ml ), dried over sodium sulfate and concentrated . crude mixture was purified by hpdc ( 0 . 1 % tfa / water , 4 g / l of cetylpyridinium trifluoroacetate as eluent ). the isolated compound was treated with excess of 1 . 0 n hcl and lyophilized to give a solid mono - ethylation product ( 0 . 11 g , 27 % yield ). c 20 h 32 no 5 cl ( mono - ethylation ); white solid ; rt = 6 . 2 min ( system a ). hrms : calcd . for [ c 20 h 31 no 6 + h + ]= 366 . 2280 , found = 366 . 2268 . esi - ms / ms ( ce = 15 ): m / z (% relative intensity ): 366 ( m + h + , 55 ), 348 ( 100 ), 264 ( 24 ). nmr : 1h nmr ( 500 mhz , cd 3 od ): δ 7 . 37 ( dd , j = 2 , 8 hz , 1h ), 7 . 27 ( d , j = 2 hz , 1h ), 7 . 20 ( d , j = 8 hz , 1h ), 4 . 98 ( dd , j = 3 , 10 hz , 1h ), 3 . 24 ( dd , j = 3 , 13 hz , 1h ), 3 . 12 ( m , 3h ), 1 . 36 ( s , 9h ), 1 . 35 ( s , 9h ), 1 . 34 ( t , j = 7hz , 3h ). to a solution of ( s )- n - ethylnoradrenaline dipivalate hydrochloride ( 50 mg , 125 μmol ) in dry methanol ( 3 ml ) was added excess solid sodium borohydride and the reaction mixture was monitored by hplc until no unreacted starting material remained . the solvent was evaporated to dryness giving white solid . crude mixture was purified by preparative hplc using 0 . 1 % tfa / water as eluent . the residue was treated with excess 1 . 0 n hcl and lyophilized to give desired product as a white solid ( 22 mg , 76 % yield ). c 10 h 16 no 3 cl ; white solid ; rt = 0 . 53 min ( system a ). hrms : calcd . for [ c 10 h 15 no 3 + h + ]= 198 . 1130 , found = 198 . 1128 . esi - ms / ms ( ce = 8 ): m / z (% relative intensity ): 198 ( m + h + , 48 ), 180 ( 100 ). nmr : 1h nmr ( 500 mhz , dmso - d6 ): δ 8 . 99 ( s ( br ), 1h ), 8 . 96 ( s ( br ), 1h ), 6 . 79 ( s , 1h ), 6 . 72 ( d , j = 8 hz , 1h ), 6 . 61 ( d , j = 9 hz , 1h ), 5 . 92 ( s ( br ), 1h ), 4 . 77 ( d , j = 10 hz , 1h ), 2 . 94 ( m ( br ), 3h ), 2 . 87 ( t , j = 12 hz , 1h ), 1 . 21 ( t , j = 7 hz , 3h ). to a mixture of ( s )- noradrenaline dipivalate hydrochloride ( 0 . 37 g , 1 . 0 mmol ) and triethylamine ( 0 . 4 ml , 3 . 0 mmol ) in dry dmf ( 10 ml ) was added iodopropane ( 0 . 15 ml , 1 . 5 mmol ) and stirred for 24 hrs at room temperature under nitrogen . the resulting mixture was lyophilized . the mixture was taken up in ethyl acetate ( 30 ml ) and extracted with nah 2 po 3 buffer ( 4 × 20 ml , ph 3 ). the organic layer was extracted again with water ( 3 × 20 ml ). combined buffer extracts and water extracts were adjusted to ph 11 using 1 . 0 n naoh and extracted with ethyl acetate ( 3 × 20 ml ). the separated organic phase was further washed with water ( 20 ml ) and brine ( 20 ml ), dried over sodium sulfate and concentrated . crude mixture was purified by hpdc ( 0 . 1 % tfa / water , 4 g / l of cetylpyridinium trifluoroacetate as eluent ). the isolated compound was treated with excess of 1 . 0 n hcl and lyophilized to give a solid product ( 0 . 09 g , 22 % yield ). c 21 h 34 no 5 cl ; white solid ; rt = 6 . 4 min ( system a ). hrms : calcd . for [ c 21 h 33 no 5 + h + ]= 380 . 2437 , found = 380 . 2421 . esi - ms / ms ( ce = 15 ): m / z (% relative intensity ): 380 ( m + h + , 78 ), 362 ( 100 ), 278 ( 20 ), 250 ( 0 . 9 ). nmr : 1h nmr ( 500 mhz , cd 3 od ): δ 7 . 38 ( d , j = 8 hz , 1h ), 7 . 28 ( s , 1h ), 7 . 20 ( d , j = 8 hz , 1h ), 5 . 01 ( dd , j = 3 , 10 hz , 1h ), 3 . 26 ( d , j = 13 hz , 1h ), 3 . 14 ( t , j = 10 hz , 1h ), 3 . 03 ( t , j = 8 hz , 2h ), 1 . 76 ( m ( br ), 2h ), 1 . 36 ( s , 9h ), 1 . 35 ( s , 9h ), 1 . 04 ( t , j = 7 hz , 3h ). to a solution of ( s )- n - propylnoradrenaline dipivalate hydrochloride ( 40 mg , 96 μmol ) in dry methanol ( 3 ml ) was added excess solid sodium borohydride and the reaction mixture was monitored by hplc until no unreacted starting material remained . the solvent was evaporated to dryness giving white solid . crude mixture was purified by preparative hplc using 0 . 1 % tfa / water as eluent . the residue was treated with excess 1 . 0 n hcl and lyophilized to give desired product as a white solid ( 15 mg , 63 % yield ). c 11 h 18 no 3 cl ; white solid ; rt = 0 . 57 min ( system a ). hrms : calcd . for [ c 11 h 17 no 3 + h + ]= 212 . 1286 , found = 212 . 1280 . esi - ms / ms ( ce = 8 ): m / z (% relative intensity ): 212 ( m + h + , 63 ), 194 ( 100 ). nmr : 1h nmr ( 500 mhz , dmso - d6 ): δ 8 . 96 ( s ( br ), 1h ), 8 . 93 ( s ( br ), 1h ), 6 . 78 ( s , 1h ), 6 . 72 ( d , j = 8 hz , 1h ), 6 . 62 ( d , j = 9 hz , 1h ), 5 . 93 ( d ( br ), j = 3 hz , 1h ), 4 . 76 ( d , j = 9 hz , 1h ), 2 . 99 ( m ( br ), 1h ), 2 . 87 ( m ( br ), 2h ), 2 . 72 ( m ( br ), 1h ), 1 . 65 ( m , 2h ), 0 . 89 ( m , 3h ). to a mixture of ( s )- noradrenaline dipivalate hydrochloride ( 0 . 56 g , 1 . 5 mmol ) and triethylamine ( 0 . 6 ml , 4 . 5 mmol ) in dry dmf ( 15 ml ) was added iodobutane ( 0 . 26 ml , 2 . 25 mmol ) and stirred at room temperature under nitrogen for 24 hrs . the resulting mixture was lyophilized . the mixture was taken up in ethyl acetate ( 40 ml ) and extracted with nah 2 po 3 buffer ( 4 × 20 ml , ph 3 ). the organic layer was extracted again with water ( 3 × 20 ml ). combined buffer extracts and water extracts were adjusted ph to 11 using 1 . 0 n naoh and extracted with ethyl acetate ( 3 × 20 ml ). the separated organic phase was further washed with water ( 20 ml ) and brine ( 20 ml ), dried over anhydrous sodium sulfate and concentrated . crude mixture was purified by hpdc ( 0 . 1 % tfa / water , 4 g / l of cetylpyridinium trifluoroacetate as eluent ). the isolated compound was treated with excess of 1 . 0 n hcl and lyophilized to give a solid product ( 0 . 22 g , 34 % yield ). c 22 h 38 no 5 cl ; white solid ; rt = 6 . 7 min ( system a ). hrms : calcd . for [ c 22 h 35 no 6 + h + ]= 394 . 2593 , found = 394 . 2582 . esi - ms / ms ( ce = 15 ): m / z (% relative intensity ): 394 ( m + h + , 100 ), 376 ( 34 ), 292 ( 4 ). nmr : 1h nmr ( 500 mhz , cd 3 od ): δ 7 . 37 ( dd , j = 2 , 8 hz , 1h ), 7 . 28 ( d , j = 2 hz , 1h ), 7 . 21 ( d , j = 8 hz , 1h ), 5 . 00 ( dd , j = 3 , 10 hz , 1h ), 3 . 25 ( dd , j = 3 , 12 hz , 1h ), 3 . 14 ( dd , j = 10 , 12 hz , 1h ), 3 . 06 ( t , j = 8 hz , 2h ), 1 . 71 ( m ( br ), 2h ), 1 . 46 ( m , 2h ), 1 . 36 ( s , 9h ), 1 . 35 ( s , 9h ), 1 . 01 ( t , j = 7 hz , 3h ). to a solution of ( s )- n - butylnoradrenaline dipivalate hydrochloride ( 50 mg , 120 μmol ) in dry methanol ( 3 ml ) was added excess solid sodium borohydride and the reaction mixture was monitored by . hplc until no unreacted starting material remained . the solvent was evaporated to dryness giving white solid . thf ( 10 ml ) and saturated nacl solution ( 10 ml ) were added to the solid and extracted with thf ( 4 × 15 ml ). combined organic extracts were dried over anhydrous sodium sulfate , filtered , dried in vacuo . the residue was treated with excess 1 . 0 n hcl and lyophilized to give desired product as a white solid ( 25 mg , 80 % yield ). c 12 h 20 no 3 cl ; white solid ; rt = 0 . 84 min ( system a ). hrms : calcd . for [ c 12 h 19 no 3 + h + ]= 226 , 1443 , found = 226 . 1437 . esi - ms / ms ( ce = 8 ): m / z (% relative intensity ): 226 ( m + h + , 78 ), 208 ( 100 ). nmr : 1h nmr ( 500 mhz , dmso - d6 ): δ 8 . 95 ( s ( br ), 1h ), 8 . 93 ( s ( br ), 1h ), 6 . 78 ( d , j = 2 hz , 1h ), 6 . 72 ( d , j = 9 hz , 1h ), 6 . 62 ( dd , j = 2 , 9 hz , 1h ), 5 . 93 ( d ( br ), j = 3 hz , 1h ), 4 . 75 ( d , j = 9 hz , 1h ), 2 . 99 ( dd , j = 3 , 13 hz , 1h ), 2 . 89 ( m , 3h ), 1 . 61 ( m , 2h ), 1 . 31 ( m , 2h ), 0 . 89 ( t , j = 8 hz , 3h ). to a solution of ( r )- isoproterenol hydrochloride ( 123 . 6 mg , 0 . 5 mmol ) in methanol ( 5 ml ) was added methyl trifluoromethanesulfonate ( 220 μl , 2 . 0 mmol ) at room temperature , and stirred overnight . after adding another methyl trifluoromethanesulfonate ( 220 μl , 2 . 0 mmol ), the solution was stirred again overnight . the solvent was removed using evaporator . then the residue was purified by preparative hplc ( vydac c18 , 5 × 25 cm column , isocratic solvent ( 0 . 1 % tfa in 5 % acetonitrile / water ), flow rate 20 ml / min ). the purified product was treated with 0 . 05 n hcl ( 10 ml ), and lyophilized to give ( r )- o - methylisoproterenol hydrochloride ( 62 . 3 mg 0 . 24 mmol , 48 % yield ) as a pale brown solid . c 12 h 20 no 3 cl ; pale brown solid ; rt = 4 . 6 min ( system b ). hrms : calcd . for [ c 12 h 19 no 3 + h + ]= 226 . 1443 , found = 226 . 1440 . esi - ms / ms ( ce = 10 ): m / z (% relative intensity ): 226 ( m + h + , 23 ), 194 ( 100 ). nmr : 1h nmr ( 500 mhz , cd 3 od ): δ 6 . 79 ( m , 2h ), 6 . 69 ( dd , j = 3 , 8 hz , 1h ), 4 . 34 ( dd ( br ), j = 3 , 10 hz , 1h ), 3 . 40 ( sept , j = 6 hz , 1h ), 3 . 23 ( s , 3h ), 3 . 04 - 3 . 16 ( m , 2h ), 1 . 34 ( d , j = 6 hz , 3h ), 1 . 32 ( d , j = 6 hz , 3h ). to a solution of ( s )- isoproterenol bitartrate ( 361 mg , 1 . 0 mmol ) in methanol ( 1 ml ), was slowly added methyl trifluoromethanesulfonate ( 1 ml , 8 . 0 mmol ) at room temperature , and stirred for 4 hrs . the solvent was rapidly removed using evaporator and the product was then purified by preparative hplc ( vydac c18 , 5 × 25 cm column , gradient elution ( 0 - 40 %, acetonitrile / water ( 0 . 1 % tfa ) with flow rate 20 ml / min ). the purified compound was treated with 0 . 1 n hcl ( 10 ml ) and lyophilized to give ( s )- o - methylisoproterenol hydrochloride as a white solid . the product was analyzed by an analytical waters hplc ( waters symmetryshield ™ 3 . 5 μm ; 4 . 6 × 50 mm c18 - reverse phase column ). a gradient 0 - 90 % acetonitrile in water , 0 . 1 % trifluoroacetic acid ( tfa ), for 12 min at a flow rate of 2 ml / min ( system b ) was used . c 12 h 20 no 3 cl ; white solid ; rt = 4 . 6 min ( system b ). hrms : calcd . for [ c 12 h 19 no 3 + h + ]= 226 . 1443 , found = 226 . 1432 . esi - ms / ms ( ce = 10 ): m / z (% relative intensity ): 226 ( m + h + , 17 ), 194 ( 100 ). nmr : 1h nmr ( 500 mhz , cd 3 od ): δ 6 . 75 ( m ( br ), 2h ), 6 . 64 ( d ( br ), j = 6 hz , 1h ), 4 . 33 ( d ( br ), j = 8 hz , 1h ), 3 . 36 ( m ( br ), 1h ), 3 . 19 ( s , 3h ), 3 . 00 - 3 . 12 ( m , 2h ), 1 . 29 ( t ( br ), j = 7 hz , 6h ). to a solution of ω - chloro - 3 , 4 - dihydroxyacetophenone ( 5 g , 26 . 8 mmol ) in dioxane ( 10 ml ), was added tent - butylamine ( 8 . 4 ml , 80 mmole ), and stirred for 3 hrs at 70 - 80 ° c . the reaction was monitored by hplc until no unreacted starting material remained . to the product amino ketone base was added 10 % hcl . after cooling overnight at room temperature , the solid was filtered , washed with acetone , and dried to give amino ketone hydrochloride ( 5 . 62 g , 81 % yield ; m . p . 233 - 235 ° c .). to a stirred solution of ω - tert - butylamino - 3 , 4 - dihydroxyacetophenone hydrochloride ( 900 mg , 3 . 5 mmol ) in methanol ( 20 ml ), was slowly added nabh 4 ( 266 mg , 7 mmol ) at room temperature . the reaction was monitored by hplc until no unreacted starting material remained ( 4 hrs ). the solvent was evaporated and the product was further lyophilized , giving a crude free base ( 1 . 32 g ). the crude free base ( 500 mg ) was dissolved in 2 . 5 ml of water and adjusted to ph 9 - 10 with 10 % hcl . after filtration , the resulting brown solution was eluted with 40 % methanol / water on rp - 18 column chromatography . the collected fractions were combined and dried in vacuo afforded a brown product , which was subjected to ag ® 4 - x4 resin ( 100 - 200 mesh ) column chromatography using pure water as eluent . after lyophilization , pure n - tert - butyl - noradrenaline was obtained as a yellowish crystal . c 12 h 19 no 3 ; yellowish crystal ; rt = 4 . 5 min ( system b ). hrms : calcd . for [ c 12 h 19 no 3 + h + ]= 226 . 1443 , found = 226 . 1432 . esi - ms / ms ( ce = 10 ): m / z (% relative intensity ): 226 ( m + h + , 100 ), 208 ( 60 ), 152 ( 9 ). nmr : 1h nmr ( 500 mhz , cd 3 od ): δ 6 . 63 ( d , j = 2 hz , 1h ), 6 . 50 - 6 . 60 ( m , 2h ), 4 . 66 ( t , j = 7 hz , 1h ), 3 . 00 ( d , j = 7 hz , 2h ), 1 . 33 ( s , 9h ). to a solution of ( s )- isoproterenol bitartrate ( 1 . 0 g , 3 mmol ) in dmf ( 15 ml ), were added triethylamine ( 1 . 25 ml , 9 mmol ) and boc 2 o ( 0 . 67 g , 3 mmol ) at room temperature . the mixture was vigorously stirred overnight and the reaction was monitored by hplc . the crude material was extracted with dcm ( 3 × 50 ml ). the combined extract was washed with water ( 3 × 50 ml ), and brine ( 50 ml ) and dried over anhydrous sodium sulfate . the resulting residue was evaporated to dryness to obtain 0 . 96 g of pale brown solid . the product was used further without purification . to a mixture of ( s )- n - boc - isoproterenol ( 0 . 96 g , 3 mmol ) and triethylamine ( 0 . 98 ml , 7 mmol ) in dcm ( 10 ml ) was dropwisely added isobutyryl chloride ( 0 . 65 ml , 6 mmol ) over 10 min at 0 ° c . under nitrogen and stirred for 2 hrs at room temperature . the crude material was extracted with dcm ( 3 × 50 ml ), washed with water ( 3 × 50 ml ) and brine ( 50 ml ), and dried over anhydrous sodium sulfate . the resulting solution was evaporated to dryness to provide ( s )- n - boc - isoproterenol diisobutyrate as a white crystal ( 1 . 2 g ), which was used further without purification . to a solution of ( s )- n - boc - isoproterenol diisobutyrate ( 1 . 2 g , 2 . 6 mmol ) in isopropanol ( 10 ml ) was added acetyl chloride ( 1 . 88 ml , 26 mmol ) at 0 ° c . and stirred for 1 . 5 hrs at room temperature . the reaction was monitored by hplc . after removing the solvent , the product ( s )- isoproterenol diisobutyrate was purified by hpdc ( 0 . 1 % tfa / water , 4 g / l of cetylpyridinium trifluoroacetate as eluent ) to afford 319 mg ( 32 % overall yield ). c 19 h 30 no 5 cl : white solid : rt = 5 . 8 min ( system a ). hrms : calc . for [ c 19 h 29 no 5 + h + ]= 352 . 2124 , found = 352 . 2119 . nmr : 1h nmr ( cd 3 od ): δ 7 . 40 ( d , j = 8 hz , 1h ), 7 . 34 ( brs , 1h ), 7 . 24 ( d , j = 8 hz , 1h ), 5 . 02 ( dd , j = 3 , 11 hz , 1h ), 3 . 47 ( m , 1h ), 3 . 25 ( dd , j = 3 , 12 hz , 1h ), 3 . 13 ( dd , j = 10 , 12 hz , 1h ), 2 . 83 ( m , 2h ), 1 . 37 ( d , j = 7 hz , 6h ), 1 . 30 ( d , j = 3 hz , 12 h ). to a solution of ( s )- isoproterenol bitartrate ( 1 . 0 g , 3 mmol ) in dmf 15 ml , were added triethylamine ( 1 . 25 ml , 9 mmol ) and boc 2 o ( 0 . 67 g , 3 mmol ) at room temperature . the mixture was vigorously stirred overnight and the reaction was monitored by hplc . the product was extracted with dcm ( 3 × 50 ml ), washed with water ( 3 × 50 ml ) and brine ( 50 ml ), and dried over anhydrous sodium sulfate . the residue was further evaporated to dryness to obtain 0 . 85 g of pale brown solid . the product was used further without purification . to a mixture of ( s )- n - boc - isoproterenol ( 0 . 85 g , 2 . 7 mmol ) in dcm ( 10 ml ) and triethylamine ( 0 . 89 ml , 6 . 4 mmol ) was dropwisely added benzoyl chloride ( 0 . 63 ml , 5 . 4 mmol ) over 10 min at 0 ° c . under nitrogen and stirred for 2 hrs at room temperature . the product was extracted with dcm ( 3 × 50 ml ), washed with water ( 3 × 50 ml ) and brine ( 50 ml ), and dried over anhydrous sodium sulfate . the resulting solution was evaporated to dryness , providing ( s )- n - boc - isoproterenol dibenzoylate as a white crystal ( 1 . 43 g ). to a solution of ( s )- n - boc - isoproterenol dibenzoylate ( 1 . 43 g ) in isopropanol ( 10 ml ) was added acetyl chloride ( 2 . 01 ml , 28 mmol ) at 0 ° c . and stirred for 1 . 5 hrs at room temperature . after removal the solvent , the desired ( s )- isoproterenol dibenzoylate was purified by hpdc ( 0 . 1 % tfa / h 2 o , 4 g / l of cetylpyridinium trifluoroacetate as eluent ) to yield 806 mg ( 64 % overall yield ). c 25 h 26 no 5 cl : white solid : rt = 6 . 3 min ( system a ). hrms : calc . for [ c 25 h 25 no 5 + h + ]= 420 . 1811 , found = 420 . 1815 . nmr : 1h nmr ( cd 3 od ): δ 8 . 03 ( m , 4h ), 7 . 63 ( m , 2h ), 7 . 60 ( d , j = 6 . 6 hz , 1h ), 7 . 49 ( d , j = 7 . 8 hz , 1h ), 7 . 49 ( brs , 1h ), 7 . 44 ( m , 4h ), 5 . 14 ( dd , j = 3 , 10 hz , 1h ), 3 . 53 ( m , 1h ), 3 . 33 ( dd , j = 16 . 6 hz , 1h ), 3 . 21 ( dd , j = 10 , 12 hz , 1h ), 1 . 41 ( d , j = 6 . 7 hz , 6h ). to a solution of ( s )- isoproterenol bitartrate ( 1 . 0 g , 3 mmol ) in dmf 15 ml , were added triethylamine ( 1 . 25 ml , 9 mmol ) and boc 2 o ( 0 . 67 g , 3 mmol ) at room temperature . the mixture was vigorously stirred overnight , and the reaction was monitored by hplc . ( s )- n - boc - isoproterenol was extracted with dcm ( 3 × 50 ml ), washed with water ( 3 × 50 ml ) and brine ( 50 ml ), and dried over anhydrous sodium sulfate . the solvent was evaporated to dryness to obtain 0 . 91 g of pale brown solid . to a mixture of ( s )- n - boc - isoproterenol ( 0 . 91 g , 2 . 9 mmol ) in dcm ( 10 ml ) and triethylamine ( 0 . 94 ml , 6 . 8 mmol ) was dropwisely added toluoyl chloride ( 0 . 77 ml , 5 . 4 mmol ) over 10 min at 0 ° c . under nitrogen and stirred for 2 hrs at room temperature . the product was extracted with dcm ( 3 × 50 ml ), washed with water ( 3 × 50 ml ) and brine ( 50 ml ), and dried over anhydrous sodium sulfate . the solution was evaporated to dryness to provide ( s )- n - boc - isoproterenol ditoluoylate as a white crystal ( 1 . 52 g ). to a solution of ( s )- n - boc - isoproterenol ditoluoylate ( 1 . 52 g ) in isopropanol ( 10 ml ) was added acetyl chloride ( 2 . 04 ml , 28 mmol ) at 0 ° c . and stirred for 1 . 5 hrs at room temperature . after removal the solvent , the desired ( s )- isoproterenol ditoluoylate was purified by hpdc ( 0 . 1 % tfa / h 2 o , 4 g / l of cetylpyridinium trifluoroacetate as eluent ) to afford 715 mg ( 55 % overall yield ). c 27 h 30 no 5 cl : white solid : rt = 6 . 8 min ( system a ). hrms : calc . for [ c 27 h 29 no 5 + h + ]= 448 . 2124 , found = 448 . 2109 . nmr : 1h nmr ( cd 3 od ): δ 7 . 89 ( d , hz , 4h ), 7 . 53 ( d , j = 8 hz , 1h ), 7 . 50 ( d , j = 8 hz , 1h ), 7 . 45 ( brs , 1h ), 7 . 24 ( d , j = 7 hz , 4h ), 5 . 06 ( dd , j = 3 , 10 hz , 1h ), 3 . 49 ( m , 1h ), 3 . 31 ( dd , j = 10 hz , 1h ), 3 . 19 ( dd , j = 10 , 13 hz , 1h ), 2 . 38 ( s , 6h ), 1 . 38 ( d , j = 7 hz , 6h ). to a solution of isoetharine mesylate ( 1 . 68 g , 5 mmol ) in acetone ( 25 ml ) was added 1 . 0 n naoh ( 25 ml , 25 mmol ) at room temperature and stirred for 10 sec , followed by addition of pivaloyl chloride ( 2 . 47 ml , 20 mmol ) in one portion . the reaction solution was stirred for additional 15 sec . and then quenched with 1 . 0 n hcl ( 20 ml ). the product was extracted with dcm ( 3 × 100 ml ), washed with 10 % sodium bicarbonate ( 100 ml ) and brine ( 50 ml ), dried over anhydrous sodium sulfate , filtered and concentrated in vacuo . crude product was purified by hpdc ( 0 . 1 % tfa / water , 4 g / l of cetylpyridinium trifluoroacetate as eluent ) yielding white solid ( 1 . 0 g , 45 % yield ). c 23 h 38 no 5 cl ; white solid ; rt = 6 . 6 min ( system a ). hrms : calcd . for [ c 23 h 37 no 5 + h + ]= 408 . 2750 , found = 408 . 2732 . esi - ms / ms ( ce = 15 ): m / z (% relative intensity ): 408 ( m + h + , 100 ), 390 ( 16 ), 348 ( 0 . 8 ), 306 ( 2 . 3 ). nmr : 1h nmr ( 500 mhz , cd 3 od ): δ 7 . 39 ( dd , j = 2 , 9 hz , 1h ), 7 . 32 ( d , j = 2 hz , 1h ), 7 . 21 ( d , j = 9 hz , 1h ), 5 . 17 ( d , j = 4 hz , 1h ), 3 . 60 ( sept , j = 6 hz , 1h ), 3 . 42 ( m , 1h ), 1 . 69 ( m , 1h ), 1 . 59 ( m , 1h ), 1 . 44 ( s , 3h ), 1 . 43 ( s , 3h ), 1 . 36 ( s , 9h ), 1 . 35 ( s , 9h ), 0 . 87 ( t , j = 7 hz , 3h ). the purity of ( s )- isoproterenol dipivalate hydrochloride was examined by waters analytical hplc system ( 600 - ms controller , 600e pump , 717 autosampler , 996 photodiode array detector ). the optical purity of ( s )- isoproterenol bitartrate and ( s )- isoproterenol dipivalate hydrochloride was examined by using another waters hplc system ( 600 controller , 600e pump , 717 autosampler , an d2996 photodiode array detector ). high performance displacement chromatography ( hpdc ) was also carried out by using the latter system . nmr spectra were measured by bruker avance 500 mhz nmr . high - resolution mass spectra were measured by micromass waters q - t of ultima ™ global mass spectrometer ( mississauga , ont , canada ) with nanolockspray ([ glu 1 ]- fibrinopeptide b as a reference compound ). huvecs were purchased from cedarlane laboratories ( burlington , canada ) and maintained in endothelial cell basal medium - 2 ( ebm - 2 ) supplemented with egm - 2 growth factor mixture ( clonetics ) and 2 % foetal bovine serum . the cells were cultured at 37 ° c . under a humidified 95 %/ 5 % ( v / v ) mixture of air and co 2 . matrigel endothelial cell tube formation assays were performed using bd matrigel ™ ( becton , dickinson ). bd matrigel ™ was thawed at 4 ° c ., and 150 μl were quickly added to each well of a 48 - well plate and allowed to solidify for 60 min at 37 ° c . huvecs ( 7 × 10 4 cells / ml ) in ec basal medium - 2 ( ebm - 2 ) were seeded 250 μl per well onto the surface of the solid bd matrigel ™ in the presence of the compounds to be tested . the cells were incubated for 22 h at 3 ° c . in a 5 % co 2 incubator . tube formation was monitored by using a leitz labovert ( leitz , wetzlar , germany ) inverted microscope . two randomly selected two microscopic fields were photographed with a digital camera ( nikon , coolpicks 995 ). the total length of tube structures in each photograph was measured using imagej ™ free software ( http :// rsb . info . nih . gov / ij /) and was normalized to that of control with no test compound . each reported value represents the average of total six photographs from three independent experiments . invasion of endothelial cells was measured using bd biocoat ™ growth factor reduced matrigel ™ invasion chambers ( beckton - dickinson ; 8 mm pore size ). ebm - 2 medium ( 500 μl each ) was placed into the lower wells . huvecs ( 2 . 5 × 10 4 ) in ebm - 2 serum - free medium ( 500 μl ) were seeded into each of the upper wells and incubated in the presence of a test compound . huvecs were allowed to migrate for 36 h at 37 ° c . in an atmosphere of 95 % air / 5 % co 2 . huvecs that remained on the upper surface of the filter were removed using a cotton swab . huvecs that had migrated to the lower surface of the filters were fixed with methanol , stained with 0 . 1 % crystal violet / 20 % ( v / v ) methanol and examined using a leitz labovert ( leitz , wetzlar , germany ) inverted microscope after mounting on a slide . three randomly selected microscopic fields were digitally captured using a nikon coolpicks ™ 995 digital camera and the number of huvecs in each photograph were directly counted . each reported value represents the average and standard deviation of a total four photographs from two independent experiments . ( s )- isoproterenol dipivalate hydrochloride used in the animal study was synthesized from ( s )- isoproterenol bitartrate . pivaloyl chloride ( trimethylacetyl chloride ) ( 4 . 1 mmol , 500 ml ) was added to a solution of ( s )- isoproterenol bitartrate ( 1 . 0 mmol , 361 . 3 mg ) in 50 % 1 . 0 n naoh aq / acetone ( 5 . 5 ml / 5 . 5 ml ). the mixture was allowed to react at room temperature for 1 h . the solution was acidified to ph 3 - 5 using 1 . 0 n hcl . after washing with n - hexane ( fisher ; nepean , ontario , canada ), the solution was extracted with dcm . the organic layer was washed with 10 % na 2 co 3 aqueous solution , dried over anhydrous sodium sulfate , and concentrated under reduced pressure . the residue was purified by high performance displacement chromatography ( column ; shiseido capcell ™ pak c18 aq 5 μm ; 250 × 4 . 6 mm ; 4 . 0 mg / ml cetylpyridinium acetate 0 . 1 % acetic acid in water , flow rate ; 1 . 0 ml / min ). the product was eluted out by a displacer , 4 . 0 mg / ml cetylpyridinium acetate 0 . 1 % acetic acid in water . after salt exchange using 0 . 1 n hcl and lyophilization , ( s )- isoproterenol dipivalate hydrochloride was obtained in 32 ± 4 % yield and 97 . 2 ± 0 . 7 % purity based on quantification of impurities described below . c 21 h 34 no 5 cl ; white solid ; rt = 6 . 6 min ( system a ). hrms : calcd . for [ c 21 h 33 no 5 + h + ]= 380 . 2437 , found = 380 . 2426 . 1h nmr ( 500 mhz , cd 3 od ) δ 7 . 33 ( dd , 7 . 2 , 1 . 9 hz , 1h ), 7 . 24 ( d , 1 . 9 hz , 1h ), 7 . 15 ( d , 7 . 2 hz , 1h ), 4 . 96 ( dd , 9 . 9 , 3 . 1 hz , 1h ), 3 . 40 ( m , 1h ), 3 . 18 ( dd , 12 . 3 , 3 . 1 hz , 1h ), 3 . 07 ( dd , 12 . 62 , 9 . 9 hz , 1h ), 1 . 32 ( d , 7 . 0 hz , 6h ), 1 . 30 ( s , 9h ), 1 . 29 ( s , 9h ). the optical isomers of ( s )- isoproterenol bitartrate and ( s )- isoproterenol dipivalate hydrochloride were separated by hplc using shiseido chiral cd - ph column ( 250 × 4 . 6 mm ; 5 μm ; isocratic 60 : 40 of 0 . 5 m sodium perchlorate / water and acetonitrile ; flow rate , 1 . 0 ml / min ). the elution profile was monitored by the absorption at 223 nm for isoproterenol bitartrate and 264 nm for isoproterenol dipivalate hydrochloride . the optical impurities were quantitated by the absorbance at 223 nm for isoproterenol bitartrate and 264 nm for isoproterenol dipivalate hydrochloride and by using a curve fitting software tablecurve2d ( systat ). the impurities of ( r )- isoproterenol bitartrate and ( r )- isoproterenol dipivalate hydrochloride were estimated as 2 . 0 ± 0 . 3 % and 3 . 3 ± 0 . 2 %, respectively . thus , the racemization induced during synthesis and purification was minimal , if it occurred . ( s )- isoproterenol hydrochloride and ( s )- isoproterenol dipivalate hydrochloride used in in vitro studies have 99 . 9 % or higher optical purity . in vivo studies : ( s )- isoproterenol dipivalate hydrochloride was used to study diabetic retinopathy in a rat model . another advantage of adrenalines is the formulation , i . e ., commercial eye drop dipivefrin is a prodrug of ( r , s )- adremaline . it is more lipophilic than adrenaline , is still water soluble and stable in eye drop solution . adrenaline is release when it passes through cornea , and pivalic acid , cleaved form of the blocking group , has a wide margin of safety , even at large oral administration . dipivefrin enhances the ocular absorption 17 time better than adrenaline , allowing one to reduce the amount of the dose and the potential side effects ( mandell et al ., 1978 ). the same formulation of prodrug was successfully applied to isoproterenol ( hussain & amp ; truelove , 1975 ). thus , the effect of the formulated ( s )- isoproterenol dipivalate hydrochloride on diabetic retinopathy with rat model was examined . active ingredient in the eye drop is 0 . 10 % ( w / v ) ( s )- isoproterenol dipivalate hydrochloride , and inactive ingredients are 1 . 84 % ( m / v ) d - mannitol , 0 . 005 % ( w / v ) disodium edetate , 0 . 10 % ( w / v ) chlorobutanol , 0 . 16 % ( w / v ) ε - aminocaproic add , 0 . 5 % ( w / v ) sodium chloride , 0 . 003 % ( w / v ) benzalkonium chloride , and 0 . 20 % ( w / v ) povidone . the ph of the eye drop was adjusted to 5 . 5 with 1 . 0 n hcl . the control eye drop has the same inactive ingredients , but lacks the active ingredient . the eye drop was freshly prepared every month and was stored at 4 ° c . no degradation of the active and inactive gradients was detected based on their hplc profiles after one month of storage at 4 ° c . the volume of each eye drop was 50 μl . prevention of diabetic retinopathy by ( s )- isoproterenol dipivalate eye drop was studied using a rat model . two - month - old male sprague - dawley rats were purchased from charles river , canada . they were housed in the biotechnology research institute ( bri )- animal facility . housing and all experimental manipulations were approved by the bri animal care committee that functions under the guidelines of the canadian council of animal care . diabetes was induced in male sprague - dawley rats weighing approximately 200 to 250 g by a single intraperitoneal injection of the beta - cell toxin , streptozotocin ( stz ) ( sigma , st . louis , mich . ), at a dose of 60 mg / kg body weight in 0 . 1m citrate buffer ph 4 . 5 . non - diabetic control rats received citrate buffer only . one week following induction of diabetes , glucose levels were determined in the blood sampled from the tail vein using a blood glucose monitoring system ( ascensia elite blood glucose meter , bayer inc , toronto , on , canada ). since the limit of detection of the blood glucose meter was 33 mm , any value above that has been assigned a maximum value of 35 mm . only animals with blood glucose levels higher than 15 mm were retained in the study . animals were thus allocated into one of four groups : group i ( n ˜ 20 ): non - diabetic rats - receiving vehicle ; group ii ( n ˜ 20 ): non - diabetic rats receiving eye drops containing ( s )- isoproterenol dipivalate ; group iii ( n ˜ 20 ): diabetic rats - receiving vehicle ; group iv ( n ˜ 20 ): diabetic rats - receiving eye drops containing ( s )- isoproterenol dipivalate . eye drops or vehicle were administered twice a day , seven days a week , on the cornea of different groups of rats , with a minimum interval of 7 h between the two treatments . to promote weight gain and limit hyperglycemia , diabetic rats were injected sub - cutaneously with 2 iu ultralente insulin ( humulin , eli lilly , toronto , on , canada ) three times a week . animal weights were monitored every week . the effects of ( s )- isoproterenol on diabetic retinopathy were studied by staining retinal capillaries with antibodies against lectin . the capillary density at the retinal center , corresponding to the macular of the human eye , was analyzed . diabetes induces glycation in retina . since ( s )- isoproterenol is a potent anti - glycation agent , penetration of ( s )- isoproterenol reduces glycation in diabetic retinal capillaries . thus , the retinas , which were stained with antibodies against lectin , were also stained with antibodies against glycated bovine serum albumin , and the degree of glycation of retinal capillaries was analyzed . anti - angiogenic activity of catecholamines and related compounds was measured by inhibiting capillary tube formation of ( huvecs in matrigel assay ( fig1 ). the potency of ( s )- isoproterenol is around 10 μm under the angiogenic assay condition used in fig1 . as dopamine showed comparable potency in the same assay ( fig2 a ) and showed effective anti - angiogenic activity at 1 μm ( basu et al ., 2001 ), the in vivo potency of ( s )- isoproterenol could be lower than 10 μm . the anti - angiogenicity of some anti - angiogenic agents of the present invention is listed in table 1a . table 1b lists dopamine , a known anti - angiogenic compound , and some catecholamines that do not show anti - angiogenic effects . fig2 a is the quantitative expression of the anti - angiogenicity of these compounds and fig2 b are some of the photographs used in the analysis . dopamine is an anti - angiogenic agent through dopamine d2 receptor ( basu et al ., 2001 ), whereas ( r )- adrenalines ( belonging to the catecholamine class of compounds ) are angiogenic through β2 - adrenergic receptor ( thake et al ., 2006 ). it was surprising and unexpected that ( s )- noradrenaline , which is structurally homologous to dopamine , was not anti - angiogenic , whereas ( s )- isoproterenol , which is less homologous to dopamine than ( s )- noradrenaline , was anti - angiogenic . moreover , even less homologous ( r , s )- n - tert - butylnoradrenaline was anti - angiogenic , despite ( r )- n - tert - butylnoradrenaline being an agonist of β2 - adrenergic receptor ( walker et al ., 1985 ). consequently , anti - angiogenic activity of the compounds listed in table 1a is unexpected . ( r )- isoproterenol is known to be angiogenic and competes with the anti - angiogenic activity of ( s )- isoproterenol as racemic mixture ( r , s )- isoproterenol did not show anti - angiogenic activity . thus , the optical purity of ( s )- isoproterenol required to express anti - angiogenic activity was examined with various optical purity of ( s )- isoproterenol ( fig3 ). anti - angiogenic activity was observed with 97 % w / w or higher optical purity of ( s )- isoproterenol , while 95 % w / w optically pure ( s )- isoproterenol was in the transition from non - antianglogenic to anti - angiogenic . consequently , at least 97 % w / w optically purity of ( s )- isoproterenol is preferred , more preferably at least 99 . 9 % w / w , even more preferably at least 99 . 99 % w / w , because the in vivo expression of β2 - adrenergic receptor in the targeted tissue could be higher than that in huvecs used in the assay , requiring further elimination of ( r )- isoproterenol in the drug . some ( s )- adrenalines listed in tables 1a and 1b are ( s )- noradrenaline , ( r , s )- adrenaline , ( s )- n - ethylnoradrenaline , ( s )- n - propylnoradrenaline , ( s )- n - butylnoradrenaline , ( r , s )- n - tert - butylnoradrenaline ( colterol ), and isoetharine . the least lipophilic ( s )- noradrenaline was not anti - angiogenic , whereas more lipophilic ( s )- n - ethylnoradrenaline , ( s )- n - propylnoradrenaline , ( s )- isoproterenol , ( s )- n - butylnoradrenaline showed anti - angiogenic activity . in racemic mixture , relatively lipophilic ( r , s )- adrenaline and ( r , s )- isoproterenol showed no anti - angiogenic activity . however , more lipophilic ( r , s )- n - tert - butylnoradrenaline and isoetharine showed anti - angiogenic activity . since ( r )- isoform of these adrenalines is angiogenic through their β2 - adrenergic receptor agonist activity , anti - angiogenic activity of ( s )- isoform of these adrenalines might be enhanced as their lipophilicity is increased . examples of some other ( s )- adrenalines useful in the present invention are listed in fig7 . since ( s )- isoproterenol and its analogs are anti - glycation agents ( yeboah et al ., 2005 ) and we have now found that they are anti - angiogenic , these activities may show synergy effects when both glycation and angiogenesis are contributing to the disease , for example diabetic retinopathy . for such applications , the compounds and their prodrugs with anti - glycation activity and anti - angiogenic activity , but lacking adrenergic activity are preferred . more specifically , ( s )- isoproterenol and its prodrugs are in particular interest . some prodrugs listed in fig9 are ( s )- n - ethyladrenaline dipivalate , ( s )- n - propylnoradrenaline dipivalate , ( s )- n - n - butylnoradrenaline dipivalate , ( s )- isoproterenol dipivalate , ( s )- isoproterenol diisobutyrate , ( s )- isoproterenol dibenzoylate , ( s )- isoproterenol ditoluoylate , and isoetharine dipivalate . anti - angiogenic activity was measured on some prodrugs , resulting in no or weak anti - angiogenic activity as shown in fig1 . considering that a part of some prodrugs may be hydrolyzed to anti - angiogenic ( s )- isoproterenol during 22 hrs of the incubation period , the prodrugs in fig1 may be non - anti - angiogenic or very weak anti - angiogenic agents . tumor cell invasion is a process involved in the late stage of cancer development where tumor migrates from one tissue to other tissues . the present invention provides a novel activity of ( s )- isoproterenol to prevent tumor cell invasion . fig3 shows that ( s )- isoproterenol ( 20 μm ) reduced the invasion of huvecs in matrigel by 60 %. in contrast , its optical isomer ( r )- isoproterenol did not show any anti - invasion activity . together with anti - angiogenic activity , ( s )- isoproterenol has dual activities to treat tumor metastasis . the present invention excludes the use of the compounds with β2 - adrenergic agonist activity such as ( r )- isoproterenol as β2 - adrenergic agonists are angiogenic ( thaker et al ., 2006 ), unless anti - angiogenic activity of the ingredients exceeds the angiogenic activity of the β2 - adrenergic agonist component ( s ) and the β2 - adrenergic activity does not interfere the therapeutic effects of the present invention and does not provide adrenergic adverse effects . the present invention includes the use of prodrugs of ( s )- isoproterenol and its analogs , where the aromatic hydroxyl group ( s ) are modified . the prodrugs may enhance and accelerate the tissue absorption and penetration due to its high lipophilicity . the prodrugs also protect the aromatic hydroxyl group ( s ) from chemical reactions such as oxidation during storage . the prodrugs are also less uv - light sensitive compared with the corresponding drugs . in case that a high optical purity is desired for anti - angiogenic activity and for minimizing adverse adrenergic effects , prodrugs reduce the racemization during storage . table 2 describes the optical purities of ( s )- isoproterenol and ( s )- isoproterenol dipivalate after incubation in various eye drop formulations for 10 days at 55 ° c . and compared with those before incubation . the incubation for 10 days at 55 ° c . corresponds to the incubation for 167 days at 22 ° c . the average optical purity of ( s )- isoproterenol after incubating 10 days at 55 ° c . was 97 . 35 ± 0 . 52 %, resulting in conversion of 2 . 55 % of ( s )- isoproterenol to ( r )- isoproterenol . this corresponds to a reduction of optical purity of ( s )- isoproterenol from 99 . 90 % to 90 % after 655 days of storage at room temperature . on the contrary , the average optical purity of ( s )- isoproterenol dipivalate after incubating 10 days at 55 ° c . was approximately 99 . 85 ± 0 . 08 %, resulting in 0 . 02 % of ( s )- isoproterenol dipivalate conversion to the corresponding ( r )- isoform . this implies that the optical purity of ( s )- isoproterenol dipivalate is reduced from 99 . 87 % to 99 . 78 % after 2 years of storage at room temperature . since a high optical purity of ( s )- isoproterenol is greatly desired for anti - angiogenic activity , prodrug formulation is preferred . prodrug formulation is not limited to pivalate esters . examples of some protecting groups of the aromatic hydroxyl groups are listed in fig8 . streptozotocin was used to induce diabetes in rats . the blood glucose levels were monitored once a week over a 27 week period for non - diabetic and diabetic rats . control , group i ( receiving vehicle ) and group ii ( receiving prodrug ) of non - diabetic animals ( filled diamond ) have a steady blood glucose level of 5 . 1 ± 0 . 4 and 5 . 1 ± 0 . 4 mm , respectively ). an increase in blood glucose levels was noted for group iii ( receiving vehicle ) and group iv ( receiving prodrug ) diabetic rats , during the first 2 weeks of diabetes induction . the glucose levels then stabilized at 28 ± 4 and 27 ± 5 mm , respectively . as the glucose level at around 5 mm is considered normal , rats in group i and group ii are non - diabetic . rats are considered diabetic when the blood glucose level exceeds 15 mm . thus , all of the rats in group iii and group iv are diabetic . the consistency of the blood glucose level between groups i and ii and between groups iii and iv shows that ( s )- isoproterenol does not affect the blood glucose level and diabetes . another potential adverse effect of ( s )- isoproterenol on body weight loss / gain was monitored . the weight gain during the experiments is essentially the same between group i ( non - diabetic rats receiving vehicle ) in fig5 a and group ii ( non - diabetic rats receiving prodrug ) in fig5 b , suggesting that no effect of ( s )- isoproterenol in weight gain / loss of non - diabetic rats . the weight gain is much less in the diabetic rats compared to those of non - diabetic rats ( chen et al ., 2004 ); however , the weight gain of group iii ( diabetic rats receiving vehicle ) in fig5 a is essentially the same as that of group iv ( diabetic rats receiving prodrug ) in fig5 b , showing no effect of ( s )- isoproterenol in weight gain / loss of diabetic rats . while rats are not an appropriate model organism for studying anti - angiogenicity directly , they may be used to study the ability of a drug to penetrate to the retina of an eye . it is known that ( s )- isoproterenol is a potent anti - glycation agent ( yeboah et al ., 2005 ). the delivery of ( s )- isoproterenol into the retina was demonstrated by inhibiting retinal glycation with ( s )- isoproterenol dipivalate eye drops . antibodies against glycated bovine serum albumin stained glycated retinal proteins in red fluorescence . the glycation of retinal capillaries was evaluated by superimposing the red fluorescence of anti - glycation antibodies with the above green fluorescence of anti - lectin antibodies , resulting green to light orange fluorescence of healthy retinal capillaries with less glycation and red fluorescence of highly glycated retinal capillaries . the retinal capillaries of non - diabetic rat eyes with least glycation showed green to light orange fluorescence . similar healthy capillaries were observed in diabetic rat eye with ( s )- isoproterenol dipivalate . the retinal capillaries in diabetic rat eye with vehicle were stained in red fluorescence and were also damaged as less number of capillaries were stained . fig6 quantifies the effect by normalizing the red fluorescence intensity of ages by the retinal capillary density , resulting 0 . 43 ± 0 . 15 , 0 . 44 ± 0 . 17 , and 0 . 76 ± 0 . 14 for non - diabetic rats , diabetic rats with ( s )- isoproterenol dipivalate treatment , and diabetic rats with vehicle , respectively . the p values are 0 . 052 , 0 . 068 , and 0 . 971 for non - diabetic rats vs . diabetic rats with vehicle , diabetic rats with ( s )- isoproterenol dipivalate vs . diabetic rats with vehicle , and non - diabetic rats vs . diabetic rats with ( s )- isoproterenol dipivalate , respectively . it should be noticed that acellular capillaries , which are enriched in diabetic retina with vehicle , are expected to be highly glycated such that the degree of glycation for diabetic rats with vehicle would be greater than 0 . 76 ± 0 . 14 in fig6 , demonstrating that ( s )- isoproterenol dipivalate effectively delivered ( s )- isoproterenol into retina and significantly reduced the glycation in retinal capillaries . since ( s )- isoproterenol can be effectively delivered to the retina of an eye where it can exert its anti - angiogenic effect , it is expected to be useful in the treatment and / or prevention of diabetic retinopathy . in one preferred embodiment , the present invention provides a novel use of ( s )- isoforms of isoproterenol and its analogs , for preventing and / or treating diseases related to angiogenesis and / or invasion . these compounds satisfy several criteria important for this application . first of all , the anti - angiogenic activity of ( s )- isoproterenol and its analogs is high . the anti - angiogenic activity at cellular level must be observable at or below 1 mm , preferably at or below 100 μm , more preferably at or below 20 μm . secondly , the adrenergic activity , which is characteristic of the ( r )- isoform is insignificant for the ( s )- isoform . the adrenergic activities of the ( s )- isoform of adrenalines are much lower that that of the corresponding ( r )- isoform ( patil et al ., 1974 ). in particular , topical administration of up to 20 % ( s )- isoproterenol hydrochloride did not show any indication to lower intra - ocular pressure in the human eye ( kass et al ., 1976 ). the ( s )- isoform of isoproterenol and its analogs are considered to be adrenergically inert and be safe for topical and systemic administration . the evidence that most commercial drugs of adrenergic agonists are racemic mixtures provides strong practical reason for the inertness of the ( s )- isomers ( boulton & amp ; fawcett , 2002 ). preparations according to one preferred embodiment of the present invention contain only the ( s )- isoform of isoproterenol and its analogs in order to reduce potential adverse effects through stimulation of adrenergic receptors . isoproterenol is known to have a duration long enough for a reasonable frequency of administration such as a twice - 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