Synthesis and Pharmacological Evaluation of Newer Substituted 2-oxo / thiobarbiturinylbenzoxa / thiazepine Derivatives as Potent Anticonvulsant Agents

A new series of 4-(2'-Oxo/thiobarbiturinyl)-2-(substitutedphenyl)-3-[(substituted aminomethylene)]-2,3-dihydro-1,5-benzothiazepines (5a-5l) and 4-(2'-oxo/thiobarbiturinyl)-2(substituted-phenyl)-3-(substitutedaminomethylene)]-2,3-dihydro-1, 5benzoxazepines (6a-6l) were synthesized. All the newly synthesized compounds were screened in vivo, for their acute toxicity and anticonvulsant activity in MES and PTZ models and were compared with standard drugs phenytoin sodium and sodium valporate. Out of the compounds studied, the most active compound of this series was 5h, showed activity (90%) more potent than the standard drug.


INTRODUCTION
The barbiturates comprise an important and valuable class of central nervous system depressants.
Barbituric acid (2,4,6trioxohexahydrophrimidine)  derivative, phenobarbital and mephobarbital are used for clinical treatment of epilepsy.Further substitution pattern at 5 th position of barbituric acid [3][4][5][6][7][8] by different alkyl, aryl or heteroaryl moieties plays a pivotal role in modulation of anticonvulsant activity.Moreover, compounds containing a fused seven, membered heterocyclic ring i.e. bengzothiazepine/ benzoxazepine nucleus make up a broad class that attracted attention in the past few years owing to its Incorporating these moieties in 5 th position of 2-oxo/thiobarbituric acid nucleus might be thought to yield more potent anticonvulsant compounds as substituted moieties are themselves anticonvulsant and substituting at 5 th position further results in protecting against convulsions.Thus, the substitution by these moieties may be synergistic.The present project is therefore, aimed at synthesizing such compounds.

Anticonvulsant activity (maximum electroshock induced seizures and pentylenetetrazol induced seizure pattern test)
The characteristic feature of the compounds of this series is the incorporation of two heterocyclic moieties, that is 2-oxo/thiobarbituric acid and benzoxazepine/benzothiazepine into a single molecular framework with the aim to develop more potent anticonvulsant agents withminimum or no side effects.5-Acetyl-2-oxo/thiobarbituric acids 1a-1b showed 10-20% anticonvulsant activity at a dose of 50mg/kg i.p. in maximal electroshock and pentylenetetrazole induced seizures, respectively.Screening of step-2 compounds 1-(2'-oxo/ thiobarbiturinyl)-3-arylidenylchalcones 2a-2f revealed that these compoundsshowed somewhat increase in anticonvulsant activity in both the models (ranging from 20 to 50% and 10 to 50% in MES and PTZ models, respectively) in comparison to step-1 compounds when tested at same dose.Compounds 4-(2'-oxo/thiobarbiturinyl)-2s u b s t i t u t e d p h e n y l ) -2 , 3 , -d i h y d r o -1 , 5benzothiazepines 3a-3f showed high percentage protection ranging from 40 to 80% and 30 to 80% in MES and PTZ models, respectively.The most active compound among 3a-3f is compound 3d.This compound was found to be equipotent (80% protection) to phenytoin sodium (standard drug for MES model) and sodium valproate (standard drug for PTZ model) and hence due to its potent nature it was studied in detail at three graded doses (17.5,  25 and 50mg/kg i.p.) for its anticonvulsant activity and was found to possess 20, 40, 80% and 20, 30, 80% protection of seizures in MES and PTZ models, respectively.It was also observed that compound 3c (having 3-methoxyphenyl group) showed least activity 40% while compound 3d (having 2chlorophenyl group) exhibited maximum response 80% in comparison to other substituted compounds.Further compounds having thiobarbituric acid possess more potent activity than the compounds having oxobarbituricacid ring.
Further, the next step compounds(5a-5l) was characterized by presence of different arylaminomethylene substitutions at the third position of benzothiazepine ring.They exhibited potent anticonvulsant activity ranging from 60 to 90% and 50 to 90% inbothmodels, that is MES and PTZ respectively.Out of the twelve compounds 5a-5l the most active compound is 5h.(having 4methoxyphenyl aminomethylen substitution at third position of benzothiazepine ring and 2chlorophenyl substitution at second position of benzodiazepine ring) was found to be most potent compound of this series exhibiting 90% inhibition in both MES and PTZ models.This compound was found to be more potent (90% protection) to phenytoin sodium (standard drug for MES model) and sodium valproate (standard drug for PTZ model).This compound was studied in details at three graded doses (17.5, 25 and 50mg/kg i.p.) for its anticonvulsant activity and was found to possess 20, 50, 90% and 20, 40, 90% protection of seizures in MES and PTZ models, respectively.On the other hand compounds 4a-4f possessed benzoxazepine ring with 2-oxo/thiobarbituric acid, substituted by different moieties at second position exhibited aniconvulsnat activity ranging from 40 to 70% and 30 to 60% in both MES and PTZ models respectively.So the compounds 4a-4f showed a decrease in anticonvulsant activity in comparison to compounds 3a-3f i.e. benzothiazepine compounds.Further the compounds of next step i.e. 6a-6l were characterized by different arylaminomethylene substitutions at the third position of benzoxazepine ring.All the twelve compounds 6a-6l of this step exhibited anticonvulsant activity ranging from 50 to 70% and 40 to 60% protection of seizures in MES and PTZ models, respectively.

ALD 50 Studies
The toxicity study of these compounds indicate their good safety margin.

EXPERIMENTAL Chemistry
Melting points were determined in open capillaries with the help of thermonic melting point apparatus and are uncorrected.1R spectra (KBr) are recorded on Backmann Acculab-10spectrophotometer. 1 H NMR spectra were recorded by Bruker WM 400 FT instrument using CDCl 3 as solvent and tetramethylsilane (TMS) as internal reference standard.All chemical shift (d)are in ppm.The purities of the compounds were checked by thin layer chromatography (TLC) on silicagel-G plates of 0.5mm thickness.The elemental analysis of the compounds were performed on HeracusCarlo Erba 1108 analyser.

Pharmacological evaluation Anticonvulsant activity. Maximum electroshock seizure (MES) test
This test was performed according to the method of Tomen et.al 15 .The group of ten rats was treated with test drugs (50mg/kg i.p.) phenytoin sodium (30mg/kg i.p.).After 1h, they were subjected to the shock of 150mA by convulsiometer through ear electrodes for 0.2s and the presence or absence of extensor response was noted.Animals in which extensor response was abolished were taken as protected rats.
Pentylenetetrazole (PTZ) induced seizures test.This test was performed by following the method of Fischer 16 .The rats were injected with pentylenetetrazol in dose of 70 mg/kg subcutaneously in scruff of neck.After 2-4 min of PTZ injection animals developed sequence of excitement, myoclonic jerks, clonic seizures, one or more maximum tonic seizures.Animals exhibiting these seizures patterns were selected standard drug used in this model was sodium valproate (80 mg/kg i.p) and was injected 60 min prior to PTZ challenge.

Approximate lethal dose (ALD 50 )
Approximate 50% lethal dose (ALD 50 ) of the compounds were determined in albino mice.The mice of either sex 20-25g were used.The test compounds were injected intraperitoneally at different dose levels in groups of 10 animals.After 24h of drug administration, percent mortality in each group was abserved from the data obtained.ALD 50 was calculated by the method of Smith 17 .

CONCLUSION
While considering all the newly synthesized compounds of this series together, we may conclude that: 1.
2-Thiobarbituric acid, containing compounds were found to possess potent anticonvulsant activity in comparison to 2oxobarbituric acid, containing compounds.

2.
Presence of benzothiazepine moiety has shown better anticonvulsant activity than the compounds having benzoxazepine moiety.

3.
Compounds having benzothiazepine moiety with thiobarbituric acid showed better anticonvulsant activity than the compounds having benzoxazepine moiety with oxobarbituric acid.4.
2-Chlorophenyl substitution at second position of benzothiazepines ring showed more potent activity than other substituted benzothiazepines.