Synthesis and Pharmacological Evaluation of New 2-Substituted-5-{ 2-[ ( 2-halobenzyl ) thio ) phenyl }-1 , 3 , 4-oxadiazoles as Anticonvulsant Agents

A new series of 2-substituted-5-{2-[(2-halobenzyl)thio)phenyl}-1,3,4oxadiazoles was designed, synthesized and investigated for anticonvulsant activities. The designed compounds contain the main essential pharmacophore for binding to the benzodiazepine receptors. Conformational analysis and superimposition of energy minima conformers of designed molecules on estazolam, a known benzodiazepine receptor agonist, revealed that the main characteristics of the proposed benzodiazepine pharmacophore were well matched. Electroshock and pentylenetetrazole-induced lethal convulsion tests showed that some of the synthesized compounds had significant anticonvulsant activity. The structureactivity relationship study of these compounds indicated that the introduction of an amino group at position 2 of 1,3,4-oxadiazole ring and a fluoro substituent at the ortho position of the benzylthio moiety had the best anticonvulsant activity. Anticonvulsant effects of active compounds were antagonized by flumazenil, a 186 A. Zarghi et al.: benzodiazepine antagonist, which establishes the involvement of benzodiazepine receptors in these effects.


Introduction
Benzodiazepine (BZD) receptor ligands allosterically modulate the action of GABA on neuronal chloride ion flux, thus eliciting a wide variety of pharmacological actions ranging a continuum from full agonists (anxiolytic, sedative/hypnotic, and anticonvulsant activities) to inverse agonists (proconvulsant and anxiogenic activities), and antagonists, which do not exhibit any pharmacological effects but can antagonize the action of both agonists and inverse agonists [1][2][3].The BZD binding sites in the brain were identified and described by radioligand receptor binding assays and originally it was found that only 1,4-BZD derivatives bind to these receptors.It has since been shown that many groups of compounds bind to the BZD receptor with high affinity, e. g., triazolopyridazines, cyclopyrrolones, quinolines and β-carbolines [4][5][6][7].Several pharmacophore models have been proposed for BZDs, and amongst all models suggested for binding to the BZD receptor at least two features are common: an aromatic ring and a coplanar proton accepting group in suitable distance (5A o ).Also, the presence of a second out-ofplane, aromatic ring could potentiate binding to the receptor [8][9][10][11][12].On this basis, a wide variety of compounds with a chemical structure different from that of benzodiazepines have been synthesized and tested.We recently started a wide research program aimed to design new BZD receptor ligands characterized by a higher degree of flexibility compared with classic BZD ligands.Accordingly, we reported several 1,3,4-oxadiazole derivatives which showed considerable anticonvulsant activity [13][14][15].As part of our ongoing research program to design new anticonvulsant agents, we describe herein the synthesis and biological evaluation of a novel group of 2-substituted-5-[2-(2-halobenzylthio)phenyl]-1,3,4-oxadiazoles (Fig. 1) with a flexible second out-of-plane aromatic ring, benzylthio group which has all the essential pharmacophore groups for binding to the BZD receptors.

Chemistry
The target 1,3,4-oxadiazole derivatives were synthesized according to Scheme 1. Accordingly, reaction of 2-[(2-halobenzyl)thio]benzoic acid methyl ester 1 [15] with hydrazine hydrate in DMF at room temperature afforded the ).The purity of all products was determined by thin layer chromatography using several solvent systems of different polarity.All compounds were pure and stable.The compounds were characterized by 1 H nuclear magnetic resonance, infrared, mass spectrometry and CHN analysis.
Physicochemical data of these compounds are summarized in Table 1.
Conformational analysis of the synthesized compounds and estazolam were preliminarily performed by MMX force field method implemented in Hyperchem 7.0 software.The conformers were optimized further by AM1 calculation using the MOPAC 6.0 program [21].Global energy minima conformers of the designed compounds were superimposed on the corresponding conformer of the estazolam molecule which was considered as a reference BZD agonist.

Pharmacology
The BZD activity of the synthesized compounds was determined through the evaluation of the ability of the compounds to protect mice against convulsion induced by a lethal dose of PTZ and electroshock as two routine models.Diazepam was considered as a reference BZD agonist with anticonvulsant effect in both models.As shown in Table 2, compound 3a with an amino group on position 2 of the oxadiazole ring and a fluoro subtituent at ortho position of the benzylthio moiety has the best anticonvulsant activity in both PTZ and MES models.The activity was antagonized with flumazenil, a benzodiazepine antagonist, which establishes the involvement of benzodiazepine receptors in this effect.Figure 2 shows the superimposition of energy minima conformers of the compound 3a, the most potent synthesized analogues, and estazolam.Obviously, the main BZD pharmacophores, aromatic rings and proton accepting groups (π1 interaction), nitrogen (N-3) of the 1,3,4-oxadiazole and triazolobenzodiazepine rings, are well matched.

Fig. 2.
Replacement of the fluoro substituent with a larger electron-withdrawing group such as Cl (3b) decreases the activity which may be explained by a steric hindrance effect.These results are in good agreement with the classical SAR data of BZDs [22] and our previous studies on 1,3,4-oxadiazoles [14,15].In the series of 2-alkylthio oxadiazoles, compounds 5a and 5b possessing a small alkylthio group at C-2 of the 1,3,4-oxadiazole ring had good anticonvulsant activity in the MES model but showed mild activity against PTZ induced convulsion.Increasing the size of the alkyl group (5c and 5f) significantly decreases the anticonvulsant activity in both PTZ and MES models.Accordingly, compounds 5e and 5f with a bulky benzylthio group did not show any anticonvulsant effects.Similarly, compounds 7a and 7b did not have any considerable anticonvulsant activity in both models.
Therefore, the size and nature of groups at C-2 position of 1,3,4-oxadiazole ring are very important for anticonvulsant activity in both PTZ and MES models.In addition, the size of electron withdrawing substituents at ortho position of the benzylthio moiety is also important for their anticonvulsant effects.

General procedure for preparation of 5-{2-[(2-Halobenzyl)thio]phenyl}-Nphenyl-1,3,4-oxadiazol-2-amines 7
A mixture of 2 (8.5 mmol), phenyl isothiocyanate (8.5 mmol) and dry THF (50 ml) was stirred at room temperature for 10 hours.It was then concentrated and cooled.The obtained solid was filtered and dispersed in ethanol (50 ml) and to this suspension, aqueous sodium hydroxide (5 N, 8 ml) was added with stirring to obtain a clear solution.To this solution, iodine in potassium iodide solution (5%) was added gradually with stirring till the colour of iodine persisted at room temperature.The reaction mixture was refluxed for 2 hour on a steam bath.It was then cooled and poured onto crushed ice.The solid product that separated was filtered, dried and recrystallized from ethanol [20].