we have synthesized 5 - benzylamino salicylic acid ( bas ) and its derivatives and demonstrated that these synthetic compounds have multiple neuroprotective action . first , bas and its derivatives attenuate nmda neurotoxicity at doses of 100 - 1 , 000 um . second , bas and its derivatives are antioxidants and block free radical neurotoxicity at doses of 1 - 300 um . finally , bas and its derivatives attenuate zn 2 + neurotoxicity . these novel and multiple neuroprotective effects of bas and its derivatives are merited to treat stroke , traumatic brain and spinal cord injury , epilepsy , and neurodegenerative diseases that are accompanied by excitotoxicity , zn 2 + neurotoxicity , and free radical neurotoxicity . the bas and its derivatives may be synthesized from 5 - aminosalicylic acid by reacting it with an appropriate compound . the following reaction schemes illustrate the synthesis of bas and its derivatives . reagents ( a ): benzyl bromide , 4 - nitrobenzyl bromide , 4 - chlorobenzyl chloride , 4 -( trifluoro methyl ) benzyl chloride , 4 - fluorobenzyl bromide , 4 - methoxybenzyl chloride , n , n - dimethyl foramide ( dmf ), triethylamine , room temperature , 4 hr . a preferred class of compounds within formula ( i ) comprises those compounds wherein x is co , so 2 or ( ch 2 ) n ( where n is an integer of 1 - 5 , inclusive ); r 1 is hydrogen , c 1 - c 5 alkyl or c 2 - c 5 alkanoyl ; r 2 is hydrogen or c 1 - c 5 alkyl ; r 3 is hydrogen or an acetoxy group ; and r 4 is phenyl group which is unsubstituted or substituted with one or more selected from the group consisting of nitro , halogen , haloalkyl , and c 1 - c 5 alkoxy ; or a pharmaceutically - acceptable salt thereof . reagents ( a ) etoh , h 2 so 4 , reflux , 6 hr . ( c ) acetic anhydride , h 2 so 4 , 0 ° c ., 30 min . a more preferred class of compounds within formula ( i ) encompasses those compounds wherein x is co , so 2 or ( ch 2 ) n ( where n = 1 , 2 , 3 ); r 1 is hydrogen , c 1 - c 3 alkyl or c 2 - c 3 alkanoyl ; r 2 is hydrogen or c 1 - c 3 alkyl ; r 3 is hydrogen or an acetoxy group ; r 4 is phenyl group which is unsubstituted or substituted with one or more selected from the group consisting of nitro , halogen , halo ( c 1 - c 3 ) alkyl and c 1 - c 3 alkoxy ; or a pharmaceutically - acceptable salt thereof . the term “ pharmaceutically - acceptable salts ” embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases . the nature of the salt is not critical , provided that it is pharmaceutically acceptable , and acids or bases which may be employed to form such salts are , of course , well known to those skilled in the art . examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid , sulfuric acid and phosphoric acid and such organic acids as maleic acid , succinic acid and citric acid . other salts include salts with alkali metals or alkaline earth metals , such as sodium , potassium , calcium and magnesium , or with organic bases , such as dicyclohexylamine . all of these salts may be prepared by conventional means from the corresponding compound of formula ( i ) by reacting , for example , the appropriate acid or base with the compound of formula ( i ). the synthesis examples show the exemplary method for the preparation of the representative compounds ( i ). to a solution of 5 - aminosalicylic acid ( 2 . 0 g , 13 mmole , purchased from aldrich chemical company , usa ) and triethylamine in dried dmf ( 25 ml ) was added benzyl bromide ( 2 . 68 g , 1 . 90 ml , 15 . 6 mmole ) at room temperature under a nitrogen atmosphere . the reaction mixture was stirred for 4 hr at room temperature . ice chips were added to the reaction mixture and then solvent was removed in vacuo . the reaction mixture was diluted with water and then extracted with ethyl acetate . the organic layer was washed with water and brine , and then dried over anhydrous mgso 4 . after evaporation of the solvent , the residue was purified by column chromatography and recrystallized from methanol / ethyl acetate / hexane ( 1 : 3 : 1 ) to give 3 . 6 g ( 73 % yield ) of 5 - benzylaminosalylic acid as a white solid . : mp 173 . 5 - 174 . 5 ° c . ( decompose ). % c % h % n calculated 69 . 12 5 . 39 5 . 76 found 69 . 30 5 . 18 5 . 63 by following the similar procedure in synthesis example 1 by using 5 - aminosalicylic acid ( 2 . 00 g , 13 . 0 mmole ) and 4 - nitrobenzyl bromide ( 3 . 38 g , 15 . 6 mmole ), 2 . 90 g ( 79 % yield ) of 5 -( 4 - nitrobenzyl ) aminosalicylic acid was obtained as a pale yellow solid . : mp 211 - 212 ° c . elemental analysis for c 14 h 13 n 2 o 5 . % c % h % n calculated 58 . 33 4 . 20 9 . 72 found 58 . 38 4 . 21 9 . 71 by following the similar procedure in synthesis example 1 by using 5 - aminosalicylic acid ( 500 mg , 3 . 26 mmole ) and 4 - chlorobenzyl chloride ( 630 mg , 3 . 91 mmole ), 480 mg ( 53 % yield ) of 5 -( 4 - chlorobenzyl ) aminosalicylic acid was obtained as a white solid . : mp 227 - 228 ° c . % c % h % n calculated 60 . 55 4 . 36 5 . 04 found 60 . 43 4 . 21 5 . 02 by following the similar procedure in synthesis example 1 by using 5 - aminosalicylic acid ( 500 mg , 3 . 26 mmole ) and 4 -( trifluoromethyl ) benzyl chloride ( 760 mg , 3 . 92 mmole ), 510 mg ( 50 % yield ) of 5 -( 4 -( trifluoromethyl ) benzyl ) aminosalicylic acid was obtained as a white solid . : mp & gt ; 188 ° c . ( decompose ). elemental analysis for c 14 h 12 f 3 no 3 . % c % h % n calculated 57 . 88 3 . 89 4 . 50 found 57 . 61 3 . 98 4 . 44 by following the similar procedure in synthesis example 1 by using 5 - aminosalicylic acid ( 500 mg , 3 . 26 mmole ) and 4 - fluorobenzyl bromide ( 740 mg , 3 . 92 mmole ), 480 mg ( 44 % yield ) of 5 -( 4 - fluorobenzyl ) aminosalicylic acid was obtained as a white solid . : mp & gt ; 210 ° c . ( decompose ). % c % h % n calculated 64 . 36 4 . 63 5 . 36 found 64 . 10 4 . 42 5 . 07 by following the similar procedure in synthesis example 1 by using 5 - aminosalicylic acid ( 1 . 00 g , 6 . 53 mmole ) and 4 - methoxybenzyl chloride ( 1 . 23 g , 7 . 84 mmole ), 890 mg ( 50 % yield ) of 5 -( 4 - methoxybenzyl ) aminosalicylic acid was obtained as a white solid . : mp 205 - 206 ° c . % c % h % n calculated 65 . 92 5 . 53 5 . 13 found 65 . 83 5 . 45 5 . 07 by following the similar procedure in synthesis example 1 by using 5 - aminosalicylic acid ( 500 mg , 3 . 26 mmole ) and pentafluorobenzyl bromide ( 1 . 02 g , 3 . 92 mmole ), 650 mg ( 60 % yield ) of 5 -( pentafluorobenzyl ) aminosalicylic acid was obtained as a white solid . : mp & gt ; 190 ° c . ( decompose ). elemental analysis for c 14 h 8 f 5 no 3 . % c % h % n calculated 50 . 46 2 . 42 4 . 20 found 50 . 53 2 . 19 4 . 23 to a solution of 5 -( 4 - nitrobenzyl ) aminosalicylic acid ( 1 . 0 g , 3 . 4 mmole ) in ethanol ( 35 ml ) was carefully added conc . h 2 so 4 ( 3 . 5 ml ) at 0 ° c . the reaction mixture was stirred for 6 hr at 80 ° c . and cooled to room temperature . after the solvent was removed in vacuo , the reaction mixture was extracted with ethyl acetate . the organic layer was washed with h 2 o , 10 % nahco 3 solution , 5 % hcl solution and brine . after the organic layer was dried over anhydrous mgso 4 , it was concentrated in vacuo . the residue was purified by column chromatography and recrystalized from ethyl acetate / hexane ( 1 : 2 ) to give 500 mg ( 46 % yield ) of 5 -( 4 - nitrobenzyl ) amino - 2 - hydroxy ethylbenzoate as a yellow solid . : mp 106 . 5 - 107 . 5 ° c . elemental analysis for c 16 h 16 n 2 o 5 . % c % h % n calculated 60 . 75 5 . 10 8 . 86 found 60 . 68 5 . 24 9 . 04 to a solution of 5 -( 4 - nitrobenzyl ) amino - 2 - hydroxy ethylbenzoate ( 500 mg , 1 . 58 mmole ) in dried methanol ( 50 ml ) was carefully added acetic anhydride ( 5 ml ) at 0 ° c . under a nitrogen atmosphere . the reaction mixture was stirred for 2 hr at 10 ° c . after ice chips were slowly added to the reaction mixture , the solvent was removed in vacuo . the reaction mixture was extracted with ethyl acetate and h 2 o , and the organic layer was washed with h 2 o , 10 % nahco 3 ( 30 ml × 3 ) solution , 5 % hcl ( 30 ml × 2 ) solution and brine . the organic solution was dried over anhydrous mgso 4 and evaporated . the residue was purified by column chromatography and recrystalized from ethyl acetate / hexane ( 1 : 3 ) to give 400 mg ( 70 % yield ) of 5 -( 4 - nitrobenzyl )- n - acetylamino - 2 - hydroxy ethylbenzoate as a pale yellow solid . : mp 105 . 5 - 106 . 0 ° c . elemental analysis for c 18 h 18 n 2 o 6 . % c % h % n calculated 60 . 33 5 . 06 7 . 82 found 60 . 54 5 . 35 8 . 12 to a solution of 5 -( 4 - nitrobenzyl ) amino - 2 - hydroxy ethylbenzoate ( 500 mg , 1 . 58 mmole ) in acetic anhydride ( 10 ml ) was carefully added conc . h 2 so 4 ( 0 . 5 ml ) at 0 ° c . under a nitrogen atmosphere . the reaction mixture was stirred for 30 min at 10 ° c . after ice chips were slowly added to the reaction mixture , the solvent was removed in vacuo . the reaction mixture was diluted with water and extracted with ethyl acetate . the organic layer was washed h 2 o , 10 % nahco 3 ( 30ml × 3 ) solution , 5 % hcl ( 30 ml × 2 ) solution and brine and then dried over anhydrous mgso 4 . after evaporation of the solvent , the residue was purified by column chromatography to give 450 mg ( 71 % yield ) of 5 -( 4 - nitrobenzyl )- n - acetylamino - 2 - acetoxy ethylbenzoate as pale yellow oil . elemental analysis for c 20 h 20 n 2 o 7 . % c % h % n calculated 60 . 00 5 . 03 7 . 00 found 59 . 96 4 . 84 7 . 15 by following the similar procedure in synthesis example i by using 5 - aminosalicylic acid ( 500 mg , 3 . 26 mmole ) and 4 - nitrobenzoyl chloride ( 700 mg , 3 . 77 mmole ), 550 mg ( 56 % yield ) of 5 -( 4 - nitrobenzoyl ) aminosalicylic acid was obtained as a pale yellow solid . : mp 270 - 271 ° c . elemental analysis for c 14 h 10 n 2 o 6 . % c % h % n calculated 55 . 63 3 . 33 9 . 27 found 55 . 82 3 . 43 9 . 08 by following the similar procedure in synthesis example 1 by using 5 - aminosalicylic acid ( 500 mg , 3 . 26 mmole ) and 4 - nitrobenzenesulsonyl chloride ( 720 mg , 3 . 26 mmole ), 390 mg ( 35 % yield ) of 5 -( 4 - nitrobenzenesulfonyl ) aminosalicylic acid was obtained as a yellow solid . : mp 239 - 240 ° c . elemental analysis for c 13 h 10 n 2 o 7 s . % c % h % n % s calculated 46 . 15 2 . 98 8 . 28 9 . 48 found 46 . 27 2 . 92 8 . 34 9 . 51 by following the similar procedure in synthesis example 1 by using 5 - aminosalicylic acid ( 500 mg , 3 . 26 mmole ) and 4 - nitrophenethyl bromide ( 900 mg , 3 . 92 mmole ), 890 mg ( 50 % yield ) of 5 -( 4 - nitrophenethyl ) aminosalicylic acid was obtained as a pale yellow solid . : mp 234 - 236 ° c . elemental analysis for c 15 h 14 n 2 o 5 . % c % h % n calculated 59 . 60 4 . 67 9 . 27 found 59 . 77 4 . 79 9 . 24 by following the similar procedure in synthesis example 1 by using 5 - aminosalicylic acid ( 500 mg , 3 . 26 mmole ) and 3 -( 4 - nitrophenyl ) propyl bromide ( 950 mg , 3 . 92 mmole ), 520 mg ( 50 % yield ) of 5 -[ 3 -( 4 - nitrophenyl )- n - propyl ] aminosalicylic acid was obtained as a pale yellow solid . : mp 229 - 231 ° c . elemental analysis for c 16 h 16 n 2 o 5 . % c % h % n calculated 60 . 75 5 . 10 8 . 86 found 60 . 77 5 . 07 8 . 89 primary cortical cell cultures from embryonic mice were prepared and used to examine neuroprotective action of compounds . mouse cortical cell culture system has been extensively used to study mechanisms and pharmacological intervention of neuronal death in neurological diseases . in brief , mouse cerebral cortices were removed from brains of the 15 day - old - fetal mice , in accordance with a protocol approved by our institutional animal care committee . the neocortices were gently triturated and plated on 24 well plates ( 5 hemispheres / plate ) precoated with 100 μg / ml poly - d - lysine and 4 μg / ml laminine . plating media consist of eagles minimal essential media ( mem , earles salts , supplied glutamine - free ) supplemented with 5 % horse serum , 5 % fetal bovine serum , 2 mm glutamine , and 21 mm glucose . cultures were maintained at 37 ° c . in a humidified 5 % co 2 atmosphere . after 7 days in vitro ( div 7 ), cultures were shifted into a growth medium identical to the plating medium but lacking fetal serum . at div 7 - 9 , 10 mm cytosine arabinofuranoside was included to halt overgrowth of glia . mixed cultures of neurons and glia were then fed twice a week . to induce neuronal injury by nmda or zn 2 + , cortical cell cultures were exposed to toxic doses of nmda for 10 min or zn 2 + for 30 min in a hepes - buffered control salt solution ( hcss ): ( in mm ) 120 nacl , 5kcl , 1 . 6 mgcl 2 , 2 . 3 cacl 2 , 15 glucose , 20 hepes and 10 naoh . after exposure , cultures were washed out 3 times and exchanged with mem adjusted to 25 mm glucose and 26 . 2 mm sodium bicarbonate , and placed in the co 2 incubator for the next 20 - 24 hr . to induce free radical neurotoxicity , cortical cell cultures were continuously exposed to fe 2 + or buthionine sulfoximine ( bso ) for 20 - 24 hr , in mem adjusted to 25 mm glucose and 26 . 2 mm sodium bicarbonate . overall cell injury was assessed microscopically under phase - contrast optics or by measuring amount of lactate dehydrogenase ( ldh ) released into the bathing medium 24 hr after neurotoxic insults as previously described ( koh and choi , j neurosci methods 20 : 83 - 90 , 1987 ). the percent neuronal death was normalized to the mean ldh value released 24 hr after continuous exposure 500 μm nmda (= 100 ) or a sham control (= 0 ). to examine anti - oxidant effect , dpph ( 2 , 2 - diphenyl - 1 - picryl - hydrazyl radical ), a stable free radical , was dissolved in ethanol to make a 100 μm solution . a compound was reacted with dpph or ethanol . after incubation for 30 min , relative decrease in dpph absorption at 517 nm was measured by a spectrophotometer . the neuroprotective action of 5 - amino salicylic acid ( as ) was examined in primary cortical cell cultures . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 1 a ), continuously to 50 μm fe 2 + ( 1 b ), or 300 μm zn 2 + for 30 min ( 1 c ), alone or with inclusion of 10 - 300 μm 5 - amino salicylic acid ( as ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the bathing medium , mean ± sem ( n = 9 - 12 ( 1 a ), n = 4 - 8 ( 1 b ) or n = 4 ( 1 c ) culture wells per condition ), scaled to mean ldh efflux value 24 hr after sham wash (= 0 ) and continuous exposure to 500 μm nmda (= 100 ). the result as shown in fig1 indicates significant difference from relevant control at p & lt ; 0 . 05 using anova and student - neuman - keuls &# 39 ; test . inclusion of 10 - 300 μm as did not attenuate neuronal death evolving 24 hr after 10 min - exposure to 300 μm nmda ( fig1 a ). interestingly , addition of 10 - 100 μm as dose - dependently prevented free radical neurotoxicity following 24 hr - exposure to fe 2 + ( fig1 b ). neuronal death 24 hr after 30 min - exposure to 300 μm zn 2 + was not reduced by continuous inclusion of as during and post zn 2 + treatment ( fig1 c ). the neuroprotective action of as against free radical neurotoxicity was attributable to direct anti - oxidant property of the compound as as decreased levels of dpph , a stable free radical ( table 1 ). compared to trolox , a membrane - permeable form of vitamin e , as was a weak anti - oxidant . as or trolox was reacted with 100 um dpph dissolved in ethanol for 30 min . anti - oxidant property was analyzed by measuring changes in the level of dpph at 517 nm , mean ± sem ( n = 3 test tubes per condition ), after subtracting background value resulting from ethanol alone . fig1 indicates significant difference from dpph alone at p & lt ; 0 . 05 , using anova and student - neuman - keuls test . as can protect neurons from free radical injuries without beneficial effects against nmda or zn 2 + neurotoxicity . however , as is weaker than trolox in scavenging free radicals . bas was synthesized and examined against neuronal injuries induced in cortical cell cultures . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 2 a ), continuously to 50 μm fe 2 + ( 2 b ) or 10 mm bso ( 2 c ), or 300 μm zn 2 + for 30 min ( 2 d ), alone or with inclusion of indicated doses of 5 - benzylamino salicylate ( bas ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 7 - 8 ( 2 a ), n = 3 - 6 ( 2 b ), n = 4 ( 2 c ), or n = 4 ( 2 d ) culture wells per condition ). fig2 . indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . concurrent addition of 300 μm 5 - benzylaminosalicylic acid ( bas ) reduced nmda - induced neuronal death approximately by 50 % ( fig2 a ). neuronal death following exposure to 50 μm fe 2 + ( fig2 b ) or 10 mm bso ( fig2 c ) was substantially reduced in the presence of 1 μm bas and near completely blocked by addition of 3 μm bas . administration of 30 - 300 μm bas dose - dependently reduced neuronal death 24 hr following exposure to 300 μm zn 2 + for 30 min ( fig2 d ). like as or trolox , bas acted as a direct anti - oxidant ( table 2 ). the anti - oxidant property of bas was observed at a dose as low as 1 μm . bas was reacted with 100 um dpph dissolved in ethanol for 30 min . anti - oxidant property was analyzed by measuring changes in dpph at 517 nm , mean ± sem ( n = 3 test tubes per condition ), after subtracting background value resulting from ethanol alone . fig1 indicates significant difference from dpph alone ([ bas ]= o ) at p & lt ; 0 . 05 , using anova and student - neuman - keuls test . bas derivatives were synthesized by substituting — h at para position of benzylamino group with — no 2 [ 5 -( 4 - nitrobenzylamino ) salicylic acid , nbas ], — cl [ 5 -( 4 - chlorobenzylamino ) salicylic acid , cbas ], — cf 3 [ 5 -( 4 - trifluoromethylbenzylamino ) salicylic acid , tbas ]. mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 3 a ), continuously to 50 μm fe 2 + ( 3 b ), or 300 μm zn 2 + for 30 min ( 3 c ), alone or with inclusion of indicated doses of 5 -( 4 - nitrobenzyl ) aminosalicylic acid ( nbas ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 7 - 8 ( 3 a ), n = 3 - 6 ( 3 b ), or n = 4 ( 3 c ) culture wells per condition ). the result as shown in fig3 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 4 a ), continuously to 50 μm fe 2 + ( 4 b ), or 300 μm zn 2 + for 30 min ( 4 c ), alone or with inclusion of indicated doses of 5 -( 4 - chlorobenzyl ) aminosalicylic acid ( cbas ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 3 - 4 ( 4 a ), n = 3 - 12 ( 4 b ), or n = 4 ( 4 c ) culture wells per condition ). the result as shown in fig4 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 5 a ), continuously to 50 μm fe 2 + ( 5 b ), or 300 μm zn 2 + for 30 min ( 5 c ), alone or with inclusion of indicated doses of 5 -( 4 - trifluoromethylbenzyl ) aminosalicylic acid ( tbas ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 3 - 4 ( 5 a ), n = 3 - 11 ( 5 b ), or n = 4 ( 5 c ) culture wells per condition ). the result as shown in fig5 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 6 a ) or continuously to 50 μm fe 2 + ( 6 b ) alone or with inclusion of indicated doses of 5 -( 4 - fluorobenzyl ) aminosalicylic acid ( fbas ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 7 - 8 ( 6 a ) or n = 4 - 8 ( 6 b ) culture wells per condition ). the result as shown in fig6 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 7 a ) or continuously to 50 μm fe 2 + ( 7 b ) alone or with inclusion of indicated doses of 5 -( 4 - methoxybenzyl ) aminosalicylic acid ( mbas ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 7 - 8 ( 7 a ) or n = 4 - 8 ( 7 b ) culture wells per condition ). the result as shown in fig7 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . this substitution with electron - withdrawing group did not reduce neuroprotective effects of bas against nmda , zn 2 + , or free radical neurotoxicity ( fig3 - 5 ). these bas derivatives were more potent than bas in preventing free radical neurotoxicity . nbas was reacted with 100 um dpph dissolved in ethanol for 30 min . anti - oxidant property was analyzed by measuring changes in dpph at 517 nm , mean ± sem ( n = 3 test tubes per condition ), after subtracting background value resulting from ethanol alone . table 3 indicates significant difference from dpph alone ([ reactant ]= o ) at p & lt ; 0 . 05 , using anova and student - neuman - keuls test . substituting — no 2 with — f or — och 3 resulted in decreased neuroprotection against nmda toxicity but appeared to increase neuroprotective potential against free radical injury ( fig6 and 7 ). 5 -( pentafluorobenzyl ) amino salicylic acid ( pbas ) was synthesized and tested against neuronal injuries . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 8 a ), continuously to 50 μm fe 2 + ( 8 b ) or 10 mm bso ( 8 c ), or 300 μm zn 2 + for 30 min ( 8 d ), alone or with inclusion of indicated doses of 5 -( pentafluorobenzyl ) amino salicylic acid ( pbas ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 11 - 16 ( 8 a ), n = 3 - 6 ( 8 b ), n = 4 - 11 ( 8 c ), or n = 12 ( 8 d ) culture wells per condition ). the result as shown in fig8 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . concurrent addition of 100 - 1000 μm pbas reduced nmda - induced neuronal death in a dose - dependent manner . treatment with 300 μm pbas reduced nmda neurotoxicity approximately by 65 % ( fig8 a ). increasing doses of pbas up to 1 mm completely blocked neuronal death following exposure to 300 μm nmda . inclusion of 1 μm pbas significantly reduced neuronal death following continuous exposure to 50 μm fe 2 + . fe 2 + - induced neuronal death was near completely blocked in the presence of 3 μm pbas . free radical neurotoxicity resulting from exposure to 10 mm bso was blocked by addition of 1 μm pbas . pbas reduced levels of dpph ( table 4 ), suggesting that pbas blocked free radical neurotoxicity as a direct antioxidant . concurrent addition of 100 - 300 μm pbas attenuated zn 2 + neurotoxicity ( fig8 d ). pbas was reacted with 100 um dpph dissolved in ethanol for 30 min . anti - oxidant property was analyzed by measuring changes in dpph at 517 nm , mean ± sem ( n = 3 test tubes per condition ), after subtracting background value resulting from ethanol alone . table 4indicates significant difference from dpph alone ([ pbas ]= 0 ) at p & lt ; 0 . 05 , using anova and student - neuman - keuls test . several derivatives of pbas such as 5 -( 4 - nitrobenzyl ) amino - 2 - hydroxy ethylbenzoate ( nahe ; r 1 ═ h , r 2 ═ ch 2 ch 3 , r 3 ═ h ), 5 -( 4 - nitrobenzyl )- n - acetylamino - 2 - hydroxy ethylbenzoate ( nnahe ; r 1 ═ coch 3 , r 2 ═ ch 2 ch 3 , r 3 ═ h ), and 5 -( 4 - nitrobenzyl )- n - acetylamino - 2 - acetoxy ethylbenzoate ( nnaae ; r 1 ═ coch 3 , r 2 ═ ch 2 ch 3 , r 3 ═ coch 3 ) were synthesized and their neuroprotection action was examined in cortical cell cultures . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 9 a ) or continuously to 50 μm fe 2 + ( 9 b ), alone or with inclusion of indicated doses of ethyl - 5 -( 4 - nitrobenzyl ) amino - 2 - hydroxy ethylbenzoate ( nahe ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 10 - 12 ( 9 a ) or n = 3 - 4 ( 9 b ) culture wells per condition ). the result as shown in fig9 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 10 a ) or continuously to 50 μm fe 2 + ( 10 b ) alone or with inclusion of indicated doses of 5 -( 4 - nitrobenzyl )- n - acetylamino - 2 - hydroxy ethylbenzoate ( nnahe ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 3 - 4 ( 10 a ) or n = 3 - 4 ( 10 b ) culture wells per condition ). the result as shown in fig1 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 11 a ) or continuously to 50 μm fe 2 + ( 11 b ), alone or with inclusion of indicated doses of 5 -( 4 - nitrobenzyl )- n - acetylamino - 2 - acetoxy ethylbenzoate ( nnaae ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 10 - 12 ( 11 a ), n = 7 - 8 ( 11 b ), or n = 4 ( 11 c ) culture wells per condition ). the result as shown in fig1 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . these derivatives attenuated nmda neurotoxicity at 300 μm ( fig9 - 11 ). inclusion of 10 μm nahe , nnahe , or nnaae blocked fe 2 + - induced free radical neurotoxicity . neuroprotective effects of nbas derivatives ( x ═ co , so 2 , ch 2 ch 2 or ch 2 ch 2 ch 2 ) the group of x ( ch 2 ) of nbas was substituted with co [ 5 -( 4 - nitrobenzoyl ) aminosalicylic acid ; nbaa ], so 2 [ 5 -( 4 - nitrobenzenesulfonyl ) aminosalicylic acid ; nbsaa ], ch 2 ch 2 [ 5 -( 4 - nitrophenethyl ) aminosalicylic acid ; npaa ], or ch 2 ch 2 ch 2 [ 5 -[ 3 -( 4 - nitrophenyl )- n - propyl ] aminosalicylic acid ; nppaa ]. mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 12 a ) or continuously to 50 μm fe 2 + ( 12 b ), alone or with inclusion of indicated doses of 5 -( 4 - nitrobenzonyl ) aminosalicylic acid ( nbaa ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 7 - 8 ( 12 a ) or n = 3 - 4 ( 12 b ) culture wells per condition ). the result as shown in fig1 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 13 a ) or continuously to 50 μm fe 2 + ( 13 b ) alone or with inclusion of indicated doses of 5 -( 4 - nitrobenzenesulfonyl ) aminosalicylic acid ( nbsaa ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 3 - 4 ( 13 a ) or n = 2 - 8 ( 13 b ) culture wells per condition ). the result as shown in fig1 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 14 a ) or continuously to 50 μm fe 2 + ( 14 b ), alone or with inclusion of indicated doses of 5 -[ 2 -( 4 - nitrophenyl )- ethyl ] aminosalicylic acid ( npaa ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 4 ( 14 a ) or n = 4 - 8 ( 14 b ) culture wells per condition ). the result as shown in fig1 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . mouse cortical cell cultures ( div 12 - 14 ) were exposed to 300 μm nmda for 10 min ( 15 a ) or continuously to 50 μm fe 2 + ( 15 b ), alone or with inclusion of indicated doses of 5 -[ 3 -( 4 - nitrophenyl )- n - propyl ] aminosalicylic acid ( nppaa ). neuronal death was analyzed 24 hr later by measuring levels of ldh released into the medium , mean ± sem ( n = 4 ( 15 a ) or n = 3 - 8 ( 15 b ) culture wells per condition ). the result as shown in fig1 indicates significant difference from relevant control , at p & lt ; 0 . 05 using anova and student - neuman - keuls test . nbaa at a dose of 30 μm significantly attenuated nmda neurotoxicity . however , its protective effect against nmda was not further increased up to doses of 300 μm ( fig1 a ). interestingly , inclusion of 30 - 300 μm nbsaa attenuated nmda neurotoxicity in a dose - dependent manner ( fig1 a ). with inclusion of 300 μm nbsaa , 90 - 100 % neuronal death following exposure to 300 μm nmda was markedly reduced . nbaa and nbsaa were still neuroprotective against fe 2 + injury but weaker than nbas in reducing free radical neurotoxicity ( fig1 and 13 ; table 5 ). substitution of ch 2 for x with ch 2 ch 2 or ch 2 ch 2 ch 2 reduced protective potency of nbas against nmda neurotoxicity . administration of 300 μm npaa or nppaa slightly reduced nmda - induced neuronal death in cortical neurons ( fig1 and 15 ). in contrast , npaa or nppaa turned out to be more effective than nbas in blocking free radical neurotoxicity as shown by complete blockade of fe 2 + - induced neuronal death in the presence of 1 μm npaa or nppaa . nbas derivatives ( 100 μm for each ) were reacted with 100 μm dpph dissolved in ethanol for 30 min . anti - oxidant property was analyzed by measuring changes in dpph at 517 nm , mean ± sem ( n = 3 test tubes per condition ), after subtracting background value resulting from ethanol alone . table 5 indicates significant difference from dpph alone at p & lt ; 0 . 05 , using anova and student - neuman - keuls test . the above results show that the compounds of formula ( i ) may be employed efficiently to prevent neurodegenerative diseases in association with excitotoxicity , zn 2 + neurotoxicity and free radical neurotoxicity . administration of compounds within formula ( i ) to humans can be by any technique capable of introducing the compounds into the bloodstream of a human patient or mammals , including oral administration , and by intravenous , intramuscular and subcutaneous injections the mammals include , but not limited to , cats , dogs , poultry , cattle and the like . compounds indicated for prophylactic therapy will preferably be administered in a daily dose generally in a range from about 0 . 1 mg to about 20 mg per kilogram of body weight per day . a more preferred dosage will be a range from about 0 . 2 mg to about 10 mg per kilogram of body weight . most preferred is a dosage in a range from about 0 . 5 to about 5 mg per kilogram of body weight per day . a suitable dose can be administered , in multiple sub - doses per day . these sub - doses may be administered in unit dosage forms . typically , a dose or sub - dose may contain from about 1 mg to about 100 mg of active compound per unit dosage form . a more preferred dosage will contain from about 2 mg to about 50 mg of active compound per unit dosage form . most preferred is a dosage form containing from about 3 mg to about 25 mg of active compound per unit dose . the active compound is usually administered in a pharmaceutically - acceptable formulation . such formulations may comprise the active compound together with one or more pharmaceutically - acceptable carriers or diluents . other therapeutic agents may also be present in the formulation . a pharmaceutically - acceptable carrier or diluent provides an appropriate vehicle for delivery of the active compound without introducing undesirable side effects . delivery of the active compound in such formulations may be performed by various routes including oral , nasal , topical , buccal and sublingual , or by parenteral administration such as subcutaneous , intramuscular , intravenous and intradermal routes . formulations for oral administration may be in the form of capsules containing the active compound dispersed in a binder such as gelatin or hydroxypropylmethyl cellulose , together with one or more of a lubricant , preservative , surface - active or dispersing agent . such capsules or tablets may contain controlled - release formulation as may be provided in a disposition of active compound in hydroxypropylmethyl cellulose . formulations for parenteral administration may be in the form of aqueous or non - aqueous isotonic sterile injection solutions or suspensions . these solutions and suspensions may be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration . although this invention has been described with respect to specific embodiments , the details of these embodiments are not to be construed as limitations . various equivalents , changes and modifications may be made without departing from the spirit and scope of this invention , and it is understood that such equivalent embodiments are part of this invention .
the present invention provides compositions and methods for prevention and prophylaxis of neurological diseases accompanied by neuronal death . the invention includes synthesis of 5 - benzylamino salicylic acid and its derivatives . bas and its derivatives protect cortical neurons from toxic insults by n - methyl - d - aspartate , zn 2 + , and reactive oxygen species . thus , the present invention provides compositions and methods for treating stroke , traumatic brain and spinal cord injury , epilepsy , and neurodegenerative diseases that are accompanied by severe neuronal loss via excitotoxicity , zn 2 + neurotoxicity , and free radical neurotoxicity .