Design, synthesis, and pharmacological evaluation of aryl oxadiazole linked 1,2,4-triazine derivatives as anticonvulsant agents

A series of new clubbed aryl oxadiazole-1,2,4-triazine derivatives (6a-l) were designed and synthesized using appropriate chemical routes. The structures were designed to have the required structural elements for any compounds to be potential anticonvulsant. Preliminary screening of anticonvulsant activity was performed using maximal electroshock seizure (MES), subcutaneous pentylenetetrazole-induced seizure (scPTZ) and behavioral activity were assessed by motor impairment test and actophotometer test. The derivatives 6-((5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)amino)-1,2,4-triazine-3(2H)-thione (6f) and 6-((5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)amino)-1,2,4-triazine-3(2H)-thione (6g) revealed significant activity against both MES and scPTZ indicating that the compounds are effective against both generalized tonic-clonic and absence seizure. The lead compound (6g) was further evaluated for quantitative evaluation and emerged as the most effective anticonvulsant with median effective dose of 28.5 mg/kg (MES ED50), 76.6 mg/kg (scPTZ) and toxic dose (TD50) was found to be >500 mg/kg. In the GABA estimation study results showed significantly increased GABA concentration. Graphical abstract Graphical abstract


Introduction
Epilepsy is a complex disorder of CNS characterized by spontaneous and recurrent seizures. An epileptic seizure is a brief episode of altered brain function due to abnormal and excessive electrical discharge from the brain cells [1]. It may involve all parts (generalized seizure) or one part of the brain (focal or partial seizure) [2]. International League against Epilepsy (ILAE) has given the following definition of the patient diagnosed with epilepsy if having (a) two unprovoked or reflex seizures occurring >24 h apart explain that they are at higher risk of further seizure in the future (b) who has a single unprovoked or reflex seizure and the probability of further seizure similar to general recurrence risk (at least 60%) after two unprovoked seizures over the next 10 years means that these also recognizes as seizure even without having actual second seizures (person at risk of having further seizure i.e., CNS infection, stroke, epileptiform activity on EEG and potential epileptogenic abnormality on brain imaging) (c) Diagnosis of an epilepsy syndrome, describe that an epileptic syndrome has been identified by consultation and investigation through clinicians [3,4]. According to WHO, around 50 million peoples worldwide have epilepsy making it the most common neurological disorder and 80% of them are living in low to middle-income countries (https://www.who.int/en/newsroom/fact-sheets/detail/epilepsy) [5,6]. Many newer antiepileptic drugs (AEDs) were made available in the market in the 1990s with favorable tolerability, pharmacokinetics, and potential for drug interactions [7]. Regardless of the newly introduced anti-epileptic drugs (AEDs) such as lamotrigine, felbamate, vigabatrin, tiagabine, zonisamide, and topiramate, still these drugs lack effectiveness in about 20-30% of patients. Drug resistance and undesirable side effects such as neurotoxicity have been associated with nearly all current AEDs. This creates a need for drug companies and scientists around the globe to develop newer AEDs with more efficacies and lesser side effects [8,9]. The 1,3,4-oxadiazole heterocyclic moiety serves as hydrogen bonding domains, having potential sites for the interaction inside the receptors increasing the pharmacological activities and considered as bioisosteres of ester and amide groups [10]. Phenyl oxadiazole derivatives were reported to provide a hydrophobic unit in promising anticonvulsant compounds in tested animal models. On the other hand, the 1,2,4-triazine ring represents a core moiety providing different pharmacological activities, including anticonvulsant (lamotrigine), anticancer (tirapazamine), antiviral (azaribine), and antibacterial (dihydromethyl furalazine) [11]. Lamotrigine (6-(2,3-dichlorophenyl)-1,2,4triazine-3,5-diamine) is an anticonvulsant drug approved for the treatment of partial seizures, secondarily generalized tonic-clonic seizures adjunct therapy for the treatment of generalized seizures associated with Lennox-Gastaut syndrome [12]. The drug acts by the prolongation of voltagesensitive Na + channel's inactivation [13]. It is associated with hypersensitivity reaction due to epoxide generation from the O-dichlorophenyl potential site present in the compound [11].
Dimmock and Pandeya et al. have suggested the pharmacophoric model and indicated that these essential pharmacophoric elements are necessary for good anticonvulsant activity. These are (a) hydrophobic unit i.e., lipophilic aryl ring A (b) hydrogen bonding domain depicted as HBD (c) electron donor D [14][15][16]. Therefore we tried to clubbed aryloxadiazole with 1,2,4-triazine having NH as linker and the proposed skeleton fulfills the pharmacophoric requirements to potentially act as anticonvulsant [17,18]. The rationale behind the design and the pharmacophoric elements in the clubbed aryloxadiazole-1,2,4-triazine derivatives have been represented (Fig. 1). The compounds synthesized were assessed for in vivo anticonvulsant activity using MES, scPTZ test, and motor impairment by using the rotarod test.

Pharmacology
The results of the in vivo preliminary screening are displayed in Table 1. The activities for the compounds to be anticonvulsant were checked by maximal electroshock seizure i.e., MES and chemshock seizure induced by subcutaneously pentylenetetrazole drug i.e., scPTZ test. The results of maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole-induced seizure (scPTZ) model confirm for the compounds to be active against generalized tonic-clonic and absence seizures. The synthesized phenyl oxadiazole clubbed-1,2,4-triazines derivatives were injected into mice intraperitoneally with a dose of 30, 100, and 300 mg/kg of body weight. After 0.5 h and 4.0 h of the test drug administration, the anticonvulsant effects were recorded. The neurological impairment test is performed by the rotarod test. The compound found to be most potent was tested for quantitative determination. In this, protective index (PI) were calculated by dividing the median toxic dose (TD 50 ) with median effective dose (ED 50 ) ( Table 2).

Preliminary studies
In the preliminary study, all the synthesized compounds showed some degree of protection against MES activity indicating the potential of these compounds against generalized tonic-clonic seizure ( Table 1). The compound 6g showed protection against seizure-induced by the MES model at a dose of 30 mg/kg body weight of mice after both 0.5 and 4.0 h of the drug administration. This indicates that compound 6g has a rapid onset and prolonged duration of action at a lower dose. Compounds 6d and 6f showed rapid onset at a lower dose of 30 mg/kg (after 0.5 h) but a long duration of action at a relatively higher dose of 100 mg/kg (after 4.0 h) of the drug administration. Compounds 6c and 6j were more active at 100 mg/kg after both the reported time intervals after both the time interval of 0.5 h and 4.0 h. The compounds 6a, 6b, and 6i were found to be active at higher dose value 100 mg/kg (after 0.5 h) and 300 mg/kg (after 4.0 h). The anticonvulsant activity of compound 6e lasted after 0.5 h at a dose of 100 mg/kg, indicative of shortacting as the compounds do not show any activity after 4.0 h even at the maximum dose of 300 mg/kg. The compounds 6k and 6l were weaker anticonvulsant agents than all other compounds as these compounds were effective at the maximal dose of 300 mg/kg after both time intervals. The compound 6h was found to possess no significant anticonvulsant activity at the maximum dose.
In another chemshock induced seizures test i.e., scPTZ, compounds 6b, 6f, 6g, and 6i showed significant protection. Among these compounds, 6f and 6g raised the seizure threshold against the absence seizure at both the time interval of 0.5 and 4.0 h time intervals at a dose of 100 mg/ kg. Compounds 6b and 6i showed protection after 0.5 h at 100 mg/kg dose and a maximal dose of 300 mg/kg after 4.0 h. The compounds showed rapid onset at 100 mg/kg dose but were longer acting at a higher dose of 300 mg/kg. Compounds 6c, 6d, and 6j raised the seizure threshold at a higher dose of 300 mg/kg after both the time intervals. The compounds 6a and 6e lasted after 0.5 h time interval indicative of short-acting at the maximum dose (300 mg/kg).
The varying substituents on the aryl ring attached at the C2 position of oxadiazole ring in clubbed aryl oxadiazole-1,2,4triazine derivatives were studied for its anticonvulsant potential. The 1,2,4-triazine-2-thione ring was selected rather than 1,2,4-triazine-2-one as according to the literature available [11,19]. The introduction of different electronwithdrawing or electron-donating groups on the aryl ring showed varying degrees of anticonvulsant effect. The most active compounds with -OCH 3 (6f) and -OH (6g) substitution evidenced that electron-donating substituents contribute to anticonvulsant action. Electron-withdrawing groups such as -NO 2 (6i) also lead to an increase in the activity, but the results were more pronounced when the nitro group is at the para position of aryl ring rather than meta or ortho. The other EWG, such as chloro derivatives (6e) led to a decrease in anticonvulsant activity and was also associated with Scheme 1 Synthetic route for the final synthesized compounds 6(a-l) neurotoxicity. This can be in agreement with metabolism to electrophilic arene oxide, which may covalently bind with antioxidant glutathione (GSH) [11,20]. Compounds with naphthyl/furan substitution showed promising activity. This may be reasoned because of an increase in hydrophobicity or electronic parameters and better interaction within the receptors. The SAR of synthesized clubbed aryl-oxadiazole-1,2,4triazine derivatives is represented (Fig. 2).

Quantitative anticonvulsant evaluation in mice
Compound 6g ought to be most potent in preliminary screening with the absence of any neurotoxic (minimal motor impairment) or hepatotoxic effect. This led us to further investigate and quantify its pharmacological properties in quantitative anticonvulsant estimation ( Table 2). The results showed that compound 6g exhibited moderate efficacy with an ED 50 of 28.5 mg/kg against MES screens, which is higher than marketed drugs phenytoin and carbamazepine with ED 50 value of 9.5 mg/kg and 8.8 mg/kg. Conversely, in the scPTZ study the compound 6g showed protection with an ED 50 of 76.6 mg/kg, a lower value than the standard drugs carbamazepine and phenytoin. The median toxic dose (TD 50 ) of the drug 6g in the rotarod test was above 500 mg/kg. Calculation of protective indices (PI) resulted  Each group contains animals used = 10; Drug dissolved in polyethylene glycol (0.1 ml, i.p). a ED 50 median effective dose was found to be effective as an anticonvulsant in 50% or more animals. b TD 50 median toxic dose found to show the absence of behavioral toxicity in 50% or more animals. c PI = Protective index (TD 50 /ED 50 ).
in higher PI values of 17.54 in MES and 6.5 in scPTZ screen, showing that compound 6g is indeed a safer and more effective anticonvulsant agent.

Actophotometer test
The titled compounds were also evaluated for CNS study (locomotor) using an actophotometer (Table 3). Phenytoin did not show a significant behavioral anguish effect after 0.5 (241 ± 10.87) and 1 h (251 ± 13.29) when compared to control (255 ± 11.61). Compound 6d unveiled decreased locomotor activity after post-treatment of 0.5 h interval but did not exhibit remarkable behavioral despair after 1 h of post-treatment. All other compounds were found to display safe and did not show any behavioral anguish effect after both intervals (post-treatment 0.5 and 1 h) when they were compared to 24 h prior to reading.

Hepatotoxicity study
Hepatotoxicity is an adverse drug reaction associated with liver damage or injury and is caused by an increase in levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) than the upper limit of normal. The elevated levels of liver enzymes of AEDs drugs are rare but serious. Abnormalities in the level of the enzymes are signs for early prediction of liver toxicity. Therefore, the most potent compound 6g was selected to study their hepatotoxicity profile. In the study, most potent compound 6g was selected for further analysis of their hepatotoxic profiles. As shown in Table 4 results, the levels of serum glutamicoxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT) remained almost like that with control values indicating a non-toxic effect on the liver (p < 0.01).
Neurochemical study: estimation of the GABA level GABA is an important neurotransmitter and shows CNSinhibitory action. It is strictly related to the manifestations and occurrence of convulsions. To further estimate the possible mechanism of the most potent compound (6g), whole-brain GABA estimation was carried out. The standard drugs clobazam (111.23 ± 4.64) and diazepam (94.72 ± 5.85) significantly increased the GABA concentration as compared to control (47.22 ± 2.52). In a similar manner compound, 6g (80.64 ± 7.74) also increased the GABA level concentrations (Table 5).

Antioxidant activity
The literature study showed evidence of epilepsy in a patient deficient in the antioxidant enzymes such as SOD and catalase [21,22]. In one of the study, the drug The results are represented as mean ± SEM with six animals in each group. phenobarbital was able to reduce oxidative stress against epiletical crisis induced by pilocarpine [23]. The newer epileptic drugs, such as topiramate and zonisamide were very effective in reducing the overproduction of ROS in both human and animal studies [24].

FRAP assay
In FRAP assay, augmented absorbance of the samples with concentration indicates amplified in reducing power. The compounds were assayed for four concentrations (25, 50, 75, 100 µg/ml). The results were presented in IC 50 for each sample (

Computational study
Pharmacophore distance mapping Pharmacophore distance mapping results analysis showed the average distance between (R-D), (R-HBD), and (D-HBD) calculated after optimizing the 3D structures using the help of software ChemDraw Professional PerkinElmer featuring SciFinder/3D viewer version 15.0. According to Unverferth et al. [25] distance between an electron donor (D) and aryl ring (R) and donor (D) and HAD unit is smaller in comparison to the distance between R and HAD, which further depends upon different calculation methods. The distance calculations between the essential structural elements by ab initio MO data obtained by the CHARMm force field are in full agreement. The distance between essential structural elements for standard compounds by molecular dynamic distance calculations are represented (Fig. 3). The distance between essential pharmacophoric elements was also checked for the synthesized compounds (6a-l) to confirm their pharmacophoric models in relation to established AEDs. Our compounds (6a-l) fulfilled the structural distance required for the pharmacophoric features essential for anticonvulsant action. The distance mapping data of compound 6g and standard drugs are represented.

Chemistry
The chemicals used in carrying out the steps in the synthesis were supplied by S.D. Fine and Loba Chemie.   General procedure for 2-(substituted-arylidene)hydrazine-1-carboxoamide (3a-l) In a flat-bottomed flask, a solution of semicarbazide hydrochloride (1.11 g, 0.01 mole) and sodium acetate (1.64 g, 0.02 mole) were dissolved in 15-20 ml of distilled water. Benzaldehyde (1.06 g, 0.01 mole) (1a) was taken in aldehyde-free alcohol and added slowly to the stirred mixture/solution of semicarbazide hydrochloride. The precipitate formed immediately was further stirred for another half an hour with constant stirring. More of the solvent was added if stirring was stopped due to the formation of an immediate precipitate. The precipitate obtained (3a) was filtered and recrystallized from ethanol (95%) in a yield of 90%. A distinct single spot in thin layer chromatography (TLC) was used to initially confirm the reaction completion and purity of the compounds [10]. Similarly, other derivatives 3b-l were synthesized.
General procedure for 2-chloro-N-(5-substituted-aryl-1,3,4oxadiazol-2-yl)acetamide (5a-l) The obtained compounds (4a) (2.57 g; 0.016 moles) were dissolved in DCM (30 ml), and to this solution, powdered anhydrous K 2 CO 3 (1 g) was added. Chloroacetyl chloride (2.38 ml; 0.03 mole) was added dropwise with constant stirring to the reaction mixture maintained at 10°C in an ice-water bath. The reaction mixture was stirred continuously for another 2-4 h. After stirring, it was left open overnight so that excess DCM left over the reaction mixture gets evaporated. After that water was added to the residue and stirred for another 20 min. The crude precipitate obtained was filtered, washed twice or thrice with water, Fig. 3 Three-point pharmacophore model of standard compounds. and then dried (5a) yield of 92%. The obtained product was recrystallized from ethanol. The purity and progress of the reaction were checked throughout by TLC using toluene: ethyl acetate: formic acid (TEF) (5:4:1) as mobile phase [26]. Other derivatives 5b-l were similarly synthesized.
General procedure for 6-((5-substituted-aryl-1,3,4oxadiazol-2-yl)amino)-1,2,4-triazine-3(2H)-thione (6a-l) A mixture of the chloroacetylated derivatives (5a) (2.72 g; 0.01 mole) and thiosemicarbazide (0.91 g; 0.01 mole) in 10% ethanolic sodium hydroxide (20 ml) was refluxed for 8-12 h. After the completion, the reaction mixture was transferred onto the ice and acidified with a few drops of hydrochloric acid. The formed precipitate was shaken with bromine water solution (1.5 g of bromine in 40 ml of water). The solid product obtained (6a) was filtered off and washed with excess water. The compound was treated with 5% sodium thiosulfate (20 mL) and extracted with CHCl 3 : CH 3 OH (9:1) (3 × 30 mL). The organic layer was combined and dried over anhydrous sodium sulfate, concentrated, and recrystallized from ethanol. Similarly, other derivatives 6b-l were synthesized by the above method. program by NINDS, USA. The dose of the test compounds was selected 30, 100, and 300 mg/kg and were given to mice through intraperitoneal route and the response of the animals were recorded after an interval of 0.5 and 4 h [27][28][29]. The compounds found most potent in preliminary screening were further tested for quantitative screening. The results were calculated as ED 50 , TD 50 and further stated in terms of the protective index (PI) [30]. To evaluate the behavioral effects (locomotor activity) the titled compounds 6a-l were also estimated by actophotometer test. The locomotor activity was recorded by actophotometer (IMCORP, Ambala, India) photocell as a digital score. Animals were placed individually in the activity chamber for 3 min as habitation periods before making the actual reading [31]. The liver toxicity results of the potent compound in the series as SGOT and SGPT were evaluated by serum enzyme activity assay [32,33]. For the development of the possible mechanisms of the derivatives. The most potent compounds were screened for estimation of GABA level in extracted tissue of mice brains. The enzymatic spectrophotometric method was very used for the analysis as it is selective and specific. In this method, 2 h after the administration of the drug (30 mg/kg, i.p.) the animals were decapitated. The brains were removed, washed with ice-cold isotonic saline, and then homogenized with 0.1 mmol/L phosphate buffer (pH 7.4). The homogenate (10% w/v) was then centrifuged, and supernatant formed was screened for GABA estimation as previously described [34].

Antioxidant activity
The synthesized compounds were evaluated for in-vitro antioxidant activity by FRAP (Ferric-Reducing antioxidant power) and DPPH (1,1-diphenyl-2-picrylhydrazyl). In the FRAP method, four concentrations (25, 50, 75, 100 µg/ml) of each sample and standard in methanol were prepared and mixed (2.5 ml) with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 1.0% potassium ferricyanide (2.5 ml). The mixture was incubated at 50°C for 20 min. Aliquots of 10% trichloroacetic acid (2.5 ml) were added to the mixture, centrifuged at 5000 rpm for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and a freshly prepared ferric chloride solution (0.5 ml, 0.1%) and allowed to stand for 30 min in dark to complete the reaction. The control solution was prepared as above, taking water in place of samples. The absorbance was measured at λ max 700 nm. The reducing power of each compound was expressed as a percentage of the most active reference compound in the current assay, based on the formula given below and the result obtained was averaged and expressed as mean ± standard deviation. DPPH method principally works on the theory of hydrogen donor is an antioxidant. It measures compounds that are radical scavengers. The antioxidant effect is comparable to the disappearance of DPPH in the test samples. DPPH shows a strong absorption maximum at 517 nm (purple). The color turns from purple to yellow followed by the formation of DPPH upon absorption of hydrogen from an antioxidant. This reaction is stoichiometric to the number of hydrogen atoms absorbed. Therefore, the antioxidant effect can be easily evaluated by following the decrease of UV absorption at 517 nm. Freshly Prepared the DPPH solution 0.1 mM in methanol and was kept in dark for 2 h. Prepared the four different concentrations (5, 10, 15, and 20 µg/mL) of each sample. 2 mL of different concentrations of test samples were taken in a set of test tubes and to this, 2 mL of freshly prepared DPPH solution was added and mixed thoroughly. This final solution is then incubated for 30 min at room temperature, and the absorbance was recorded at 517 nm in UV spectrophotometrically. All tests and analysis were run in triplicates [35].

Conclusion
Various novel 6-((5-substituted-aryl-1,3,4-oxadiazol-2-yl) amino)-1,2,4-triazine-3(2H)-thione derivatives were designed and synthesized. The designed compounds possess structural elements of standard drugs, such as increasing the anticonvulsant potential and decreasing toxicity. The synthesized clubbed aryl oxadiazole-1,2,4triazine derivatives were characterized by mass spectrometry, 13 C NMR, 1 H NMR, IR. After characterization, compounds were assessed for preliminary anticonvulsant activity using MES, scPTZ, and motor impairment test by rotarod test. Compounds were effective and displayed substantial protection against both models i.e., MES and scPTZ. Among them, compounds, 6-((5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)amino)-1,2,4-triazine-3 (2H)-thione (6g) possess to be most potent in preventing seizure spread and elevating seizure threshold. The compound 6g was further subjected to quantitative study, and the results showed a higher protective index as compared to the standard drug. The series of compounds were also revealed no sign of locomotor effect in the actophotometer test. The compounds 6g showed no sign of hepatotoxicity estimated through the liver enzyme estimation. The compound 6g was revealed to increase GABA level in the whole brain GABA estimation test. The synthesized 1,2,4triazine derivatives possess potent anticonvulsant activity with no neurotoxicity as well as hepatotoxicity and may be regarded as strong candidates for future investigation.