Synthesis of N-[ { 5-Aryl-1 , 3 , 4-oxadiazole-2-yl } methyl ]-4-methoxyaniline Derivatives and Their Anticonvulsant Activity

A series of some new 2,5-disubstituted-1,3,4-oxadiazoles 4(a–i) have been conveniently synthesized by intramolecular oxidative cyclization of (E)-2-(arylbenzylidene)-2-[(4-methoxyphenyl)amino]acetohydrazides promoted by iodobenzene diacetate as an oxidant. The structures of the synthesized compounds have been confirmed by H and C NMR, IR, MS, and elemental analysis. All the newly synthesized compounds were screened for their anticonvulsant activity against maximal electroshock (MES) seizure method. Compounds 4g, 4d, and 4awere found to be themost potent of this series.The same compounds showed no neurotoxicity at the maximum dose administered.


Introduction
Epilepsy is one of the most common neurological disorders affecting a large section of people.About 50 million people worldwide have epilepsy, with almost 90% of these people being in developing countries [1].Epilepsy also affects about 4% of individuals over their lifetime.The incidence of epilepsy is highest among children below 7 years of age and in individuals of above 55 years.Epilepsy is the third most wide spread neurological disorder found in the elderly after cerebrovascular disease and dementia.About 20-30% of patients have seizures that are resistant to available medical therapies [2].All currently approved antiepileptic drugs have doserelated toxicity and idiosyncratic side effects [3].Therefore, the search for a newer, more effective, and more selective agent with lesser side effects continues to be an area of investigation of medicinal chemists worldwide.
The anticonvulsant drug design is based on the major characteristics important in newly synthesized compounds which are the inclusion of a hydrophobic site and H-bond donors/acceptors in the compound which is required for the activity in MES.Although several new drugs such as vigabatrin, lamotrigine, gabapentin, tiagabine, felbamate, topiramate, fosphenytoin, and levetiracetam have appeared in the market, the development of novel agents, particularly compounds effective against complex partial seizures, remains a major focus of antiepileptic drug research.

Experimental
All solvents and reagents were purchased from Sigma Aldrich Chemicals Pvt. Ltd.Melting range was determined by Veego Melting Point VMP III apparatus.Elemental analyses were recorded on VarioMICRO superuser V1.3.2Elementar.
The IR spectra were recorded using KBr discs on FT-IR Jasco 4100 infrared spectrophotometer.1H and 13C NMR spectra were recorded on Bruker DRX-500 spectrometer at 400 MHz using DMSO-d 6 as solvent and TMS as an internal standard.The mass spectra of the samples were recorded using the instrument LC-MSD-Trap-XCT.

Synthesis of 2-(4-Methoxyphenylamino)acetohydrazide
(2).To a mixture of ethyl-(4-methoxyphenyl)glycinate 1 (0.01 mmol) and ethanol (25 mL), hydrazine hydrate (0.02 mmol) was added.The reaction mass was heated to reflux for 4 h.The reaction completion was monitored by thin layer chromatography (TLC).The reaction mixture was concentrated to half volume.The solid obtained was filtered and washed with chilled ethanol.The obtained solid was dried to get the pure product.Yield 78%; mp 116-118 ∘ C.

General Procedure for the Synthesis of 2,5-Disubstituted-1,3,4-Oxadiazoles (4a-i). (E)-2-(Arylbenzylidene)-2-[(4methoxyphenyl
)amino]acetohydrazides (3a-i) (0.01 mmol) was dissolved in methanol, and IBD (0.012 mmol) was added to it.The content was stirred for 2 h, and the progress of the reaction was monitored by TLC.Upon completion of the reaction, solvent was removed under reduced pressure, and residue was taken in petroleum ether, and stirred for 30 min.The solid thus obtained was filtered, washed with petroleum ether and dried to afford (4a-i).The crude product was purified by recrystallization method using diethyl ether.Scheme 1: Synthetic route for the target compounds 4(a-i).

Anticonvulsant Evaluation
Animals.Male Wistar rats procured from National Institute of Nutrition, Hyderabad (190-220 g), were used in the present study.The animals were kept in individual cages for one week to acclimatize for the laboratory conditions.They were allowed to free access of water and food.All the experimental procedures were carried out in accordance with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines.The study was reviewed and approved by the Institutional Animal Ethics Committee, G. Pulla Reddy College of Pharmacy, Hyderabad, India.
Maximal Electroshock Seizure (MES) Model.Maximal electroshock seizure model was used in the present study to evaluate the anticonvulsant activity of the compounds on male Wistar rats.Seizures were induced in rats by delivering electroshock of 150 mA for 0.2 s by means of a convulsiometer through a pair of ear clip electrodes.The test compounds (100 mg/kg) were administered by oral route in the form of solution (the compounds were dissolved in 1% sodium carboxymethyl cellulose), 30 minutes before the maximal electroshock seizure test.The animals were observed closely for 2 minutes.The percentage of inhibition of seizure relative to control was recorded and calculated [14].Phenytoin (100 mg/kg) was used as a standard drug.
Neurotoxicity Screening.The minimal motor impairment was measured in mice by the rotarod test.Acute neurological toxicity in mice was evaluated by rotarod test [14].The mice were trained to stay on the accelerating rotarod that rotates at 10 revolutions per minute.The rod diameter was 3.2 cm.Trained animals were administered with the test compounds at dose of 100 mg/kg.Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least one minute in each of the three trails.Phenytoin was used as a standard drug.

Statistical Analysis.
In the present study, data were analyzed by one-way analysis of variance (ANOVA) followed by dunnet test to compare difference between the groups.
The synthesized compounds were characterized by IR, 1 H-NMR, 13 C-NMR, and mass spectral and elemental analyses.The IR spectra of (4a-i) were recorded using KBr pellets in the range of 4000-400 cm −1 .The absorption bands around 3065 cm −1 are assigned to the aromatic C-H stretch.The strong bands around 2924 cm −1 are assigned to the C-H stretch, the appearance of a medium to strong absorption bands above 1610 cm −1 due to a stretching vibration of the azomethine (C=N) bond formation in synthesized compounds.The 1 H NMR spectrum of 4a and 4h showed singlet in the region of , 3.88 and 3.87, respectively, and doublet in the region of , 4.57 and 4.54, respectively.All the compounds showed singlet OCH 3 group in the region of , 3.81-3.88.The proton spectral data of 4a-i shows resonance peak at  6.32-6.36ppm (s, 1H, and NH).The carbon spectral data agree with respect to the number of protons and their chemical shifts with the proposed structures. 13C NMR spectra present the correct number of carbon atoms at the appropriate chemical shift values.The mass spectra of 4a showed molecular ion peak at m/z 317, which is in agreement with the molecular formula C 16 H 14 ClN 3 O 2 .The elemental analyses data showed good agreement between the experimentally determined values and the theoretically calculated values within ±0.4%.The chemical structures and physical data of all the synthesized compounds are tabulated in Table 1.

Anticonvulsant Activity.
In the present study, the anticonvulsant activity of the nine newly synthesized 2,5disubstituted-1,3,4-oxadiazoles 4(a-i) was evaluated by MES induced seizure in rats at the dose of 100 mg/kg, and the results are summarized in Table 2. Compounds 4g, 4d, and 4a demonstrated significant protective effect on MES induced seizure.Similarly, compounds 4e, 4i, and 4h that showed moderate protective effect and a significant difference in protectiveness were observed when compared to standard group.The data in the table represent ratio between the numbers of the animals that exhibited neurotoxicity against the number of tested animals.
Compounds 4f, 4c, and 4b have relatively lower anticonvulsant potencies.All the compounds were examined for their neurotoxicity on mice using rotarod method given in the dose of 100 mg/kg.Except for compounds 4f, 4c, and 4b, none of the compounds showed neurotoxicity.These compounds showed 25% toxicity compared to standard once at 2 h of oral administration (Table 3).The structure activity relationship study of these compounds indicates that the introduction of a benzene ring at position 5 of 1,3,4-oxadiazole ring; trifluoromethyl substituent at the paraposition showed the best anticonvulsant activity in 4g.Compounds 4d and 4a possessing a chloro group had good anticonvulsant activity in the MES model.Both compounds did not exhibit neurotoxicity at the highest administered dose.The fluoro and methoxy groups in 4e resulted in increased anticonvulsant activity.The presence of bromo group in 4i and nitro group in 4h shows moderate anticonvulsant activity.Anticonvulsant activity has increased considerably when methyl group in 4b was replaced with tolyl variation 4c.The presence of electron releasing methoxy group in benzene ring in 4f has more anticonvulsant activity as compared to 4c.

Table 2 :
Effect of the tested compounds in the maximal electroshock seizure test.

Table 3 :
Neurotoxicity screening of the compounds 4(a-i).