Synthesis and Biological Evaluation of Some [1,2,4]Triazolo[4,3-a]quinoxaline Derivatives as Novel Anticonvulsant Agents

2-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylthio)acetic acid hydrazide (10) was used as a precursor for the syntheses of novel quinoxaline derivatives with potential anticonvulsant properties. The newly synthesized compounds have been characterized by IR, 1H NMR, and mass spectral data followed by elemental analysis. The anticonvulsant evaluation was carried out for eleven of the synthesized compounds using metrazol induced convulsions model and phenobarbitone sodium as a standard. Among this set of tested compounds, two of them (14, and 15b) showed the best anticonvulsant activities.

Earlier studies revealed that most of compounds derived from 1,2,4-triazoles have been found to be significant anticonvulsant [19] and tranquillizing agents [20].
Reaction of the potassium salt 7 with substituted aniline in DMF gave the corresponding anilide 8a-d, and its reaction with alkyl chloroacetate in the same solvent gave the corresponding ester 9a-e. The hydrazide derivative 10 was synthesized from the reaction of n-butyl ester with hydrazine hydrate. The hydrazide 10 was used as the key intermediate for the further syntheses. Thus, that hydrazide was allowed to react with ethyl acetoacetate and acetylacetone to give the corresponding methylpyrazolone derivative 11 and dimethylpyrazole derivative 12, respectively. When compound 10 was treated with carbon disulphide and potassium hydroxide, oxadiazole derivative 13 was obtained in an excellent yield. Allowing the same intermediate 10 to react with isocyanate or with isothiocyanates resulted in the formation of semicarbazide 14 and thiosemicarbazides 15, respectively. Acetylation of compound 10 with acetic anhydride and condensation with variety of aromatic aldehydes gave the corresponding N-acetyl derivative 16 and Narylidene derivatives 17a-f, respectively. The structural assignments to the new compounds were based on their elemental analysis and spectral (IR, 1 H NMR, and mass) data.

Biological Activity
3.1. Anticonvulsant Activity. Eleven compounds of the newly synthesized derivatives were selected to be screened for their anticonvulsant activity on different groups of mice. Swiss albino adult male mice, weighing 20-25 g, were used as experimental animals. They were obtained from an animal facility (Animal House, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University). Mice were housed in stainless steel wire-floored cages without any stressful stimuli. Animals were kept under well-ventilated conditions at room temperature (25-30 ∘ C). They were fed on an adequate standard laboratory chow (El-Nasr Co., Abou-Zabal, Egypt) and allowed to acclimatize with free access to food and water for 24-hour period before testing except during the short time they were removed from the cages for testing. Albino mice were randomly arranged in groups, each of six animals. Phenobarbitone sodium was used as a reference drug for comparison. Pentylenetetrazole (PTZ, Sigma Chemicals, USA) was used as convulsant.
The tested compounds were dissolved in DMSO and orally administered in a dose regimen ranging from 200 to 800 mg/kg animal weight, using the dosing volume of 0.2 mL per 20 g. Pentylenetetrazole was dissolved in normal saline in 2% concentration and was given intraperitoneally in a dose of 60 mg/kg body weight (dose that could induce convulsions in at least 80% of the animals without death during the following 24 hours). Phenobarbitone sodium (Alex Co., Egypt) was dissolved in normal saline in 2% concentration and it was intraperitoneally given in doses of 6.25, 12.5, and 25 mg/kg using the same dosing volume. All drugs were freshly prepared to the desired concentration just before use.
Groups of six mice were administered the graded doses of the test compounds and phenobarbitone sodium orally. Control animals received an equal volume of saline (10 mL/kg). After one hour, the animals were subcutaneously injected with the convulsive dose of pentylenetetrazole (60 mg/kg). The criterion of anticonvulsant activity is complete protection against convulsions of any kind. Observations were made at least 60 minutes after the administration of pentylenetetrazole. Doses that gave full protection against the induced convulsions and those which exhibited 50% protection in addition to the relative potencies of the test compounds to phenobarbitone sodium were recorded in Table 1. Observations were made at least 60 min after the administration of metrazol.

General.
Melting points were determined by open capillary and are uncorrected. The IR spectra (in KBr discs) were recorded on potassium bromide discs on a Pye Unicam SP 3300 spectrophotometer. 1 H NMR spectra were recorded either on Gemini 300 MHz (Varian USA) or on Jeol ECA 500 MHz NMR spectrometer (500 MHz) using TMS as an internal standard. All chemical shifts are reported in ppm downfield from TMS. Mass spectra were recorded on a Shimadzu GCMS-QP 1000 EX mass spectrometer operating at 70 eV. Elemental analysis was carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. Progress of the reactions was monitored by TLC sheets precoated with UV fluorescent silica gel Merck 60 F254 plates and was visualized using UV lamp and n-hexane : ethyl acetate 9 : 1 as mobile phase. Starting materials were purchased from Aldrich Chemical Company and used without further purification. Compounds 2-6 were prepared following the procedures reported in the literature [22][23][24]. The used intermediates (chloroacetanilides and alkyl chloroacetates) were prepared as reported [25,26]. [1,2,4]Triazolo[4,3a]quinoxaline-4-thiol (7). Compound 6 (1.5 g, 0.01 mol) was treated with alcoholic solution of potassium hydroxide (0.4 g, 0.01 mol); the reaction mixture was stirred for 1 h; the solid obtained was filtered, washed with absolute ethanol, and airdried to give the potassium salt in quantitative yield. m.p.

Conclusion
In the present investigation, 16 new 4-substituted [1,2,4] triazolo[4,3-a]quinoxaline derivatives were synthesized and characterized by spectral analysis. Few of those derivatives were screened for anticonvulsant activity by PTZ animal model. Compounds 14 and 15b exhibited the highest activity which is comparable to the standard. The activity may be explained by the presence of substituents on position 4 of the condensed heterocyclic system containing quinoxaline, fused to triazole at 1,2 positions in the backbone structure of title compounds. The electronic factors exerted by the substituents and the hydrophobic nature of phenyl nucleus in the title compounds influenced the activity.