Preliminary Anticonvulsant and Toxicity Screening of Substituted Benzylidenehydrazinyl-N-(6-substituted benzo[d]thiazol-2-yl)propanamides

Keeping in view the structural requirements suggested in the pharmacophore model for anticonvulsant activity, a new series of 3-(2-(substitutedbenzylidene)hydrazinyl)-N-(substituted benzo[d]thiazol-2-yl)-propanamides were synthesized with aromatic hydrophobic aryl ring (A), NH–C=O as hydrogen bonding domain (HBD), nitrogen atom as electron donor (D), and phenyl as distal aryl ring (C). Synthesized compounds were characterized by FTIR, 1H NMR, 13C NMR, mass spectroscopy, and elemental analysis. Preliminary in vivo anticonvulsant screening (phase I) was performed by two most adopted seizure models, maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ). Based on anticonvulsant screening results, two compounds, 5h and 5p, were found to be most active; they exhibited activity comparable to standard drugs phenytoin (PHY) and carbamazepine (CBZ). These active compounds were subjected to phase II and phase III screening, where they displayed much higher protective index (PI) in comparison to the standard drugs. In phase IV screening, the bioavailability of active compounds was assessed on oral administration. Further, preliminary safety profiles of 5h and 5p were evaluated by the neurotoxicity testing and liver enzyme estimation.


Introduction
Epilepsy is one of the most prevalent noncommunicable neurological conditions. It is a main cause of disability and mortality [1] and characterized by paroxysmal, excessive, and hyperasynchronous discharges of large numbers of neurons [2]. More than 10 million people in India are afflicted with epilepsy [3]. The prevalence of epilepsy is higher in the rural (1.9%) as compared with the urban population (0.6%) [4,5]. Every year about 2.4 million new cases are added to these figures [6,7]. Several new anticonvulsants like vigabatrin, lamotrigine, gabapentin, topiramate, felbamate, rufinamide, and levetiracetam have been recently introduced in clinical practices. Regardless of the introduction of these new drugs in the past decade, up to one-third of epilepsy patients developed resistance to optimum drug treatment [8]. The therapeutic efficiency of these well-known established drugs in reducing seizure is prevailed over by some detrimental side effects such as headache, nausea, hepatotoxicity, gastrointestinal disturbances, and hirsutism [9,10]. These facts triggered the further scope and search for newer more effective and less toxic anticonvulsants.
Benzothiazole scaffold is amongst the commonly occurring heterocyclic nuclei in many marine as well as natural plant products. It is a promising bicyclic ring system with multiple biological applications [11][12][13][14][15]. In recent years, extensive research has focused on developing novel benzothiazole derivatives to improve anticonvulsant activities.
In view of these facts and as a part of our continuing studies in the area of anticonvulsant agents, it was thought of interest to synthesize some newer derivatives of benzothiazole as anticonvulsant agents. A pharmacophore model along with physicochemical determination provides a useful tool for designing prototypic molecules and explanation of probable interactions. In terms of interaction at binding site, the titled compounds have common structural 2 The Scientific World Journal features such as aromatic hydrophobic aryl ring (A), NH-C=O as hydrogen bonding domain (HBD), nitrogen atom as electron donor (D), and phenyl as distal aryl ring (C) [16]. In the present study, therefore, we hereby describe the synthesis and preliminary anticonvulsant evaluation of some new 3-[2-(substituted benzylidene)hydrazinyl]-N-(substituted benzo[d]thiazol-2yl)-propanamides.

Experimental
2.1. Measurements. The entire chemicals used in the synthesis were procured from E. Merck and S. D. Fine Chemicals. A Thin layer chromatography (TLC) was performed with Silica gel 60 F254 TLC aluminium sheet (Merck) using toluene : ethyl acetate : formic acid (5 : 4 : 1) and benzene : acetone (9 : 1) as eluents. Ashless Whatmann number 1 filter paper was used for vacuum filtration. Melting points were determined by using open capillary tubes in a Hicon melting point apparatus (Hicon, India) and are uncorrected. The purity of the compounds was confirmed through elemental analysis. The elemental analyses (C, H, N, and S) of all compounds were performed on the CHNS Elimentar (Analysen systime, GmbH) Germany Vario EL III and results were within ±0.4% of the theoretical values. Fourier transform infrared (FT-IR) spectra were recorded in KBr pellets on a Shimadzu FT-IR spectrometer. 1 HNMR and 13 CNMR spectra in DMSO-6 /CDCl 3 solutions were, respectively, recorded at 400 and 100 MHz with Bruker 400 Ultrashield TM NMR spectrometer using TMS [(CH 3 ) 4 Si] as internal standard. Splitting patterns are nominated as follows: s, singlet; bs, broad singlet; d, doublet; t, triplet; m, multiplet. The NH protons were D 2 O exchanged for their spectral characterization. The mass spectra were recorded using Waters Micromass ZQ 2000 Spectrophotometer (Jamia Hamdard, New Delhi, India).
Step II: Synthesis of N- (6- Step III: 3-Bromo-N- (6- Step IV: N- Compound 3a (0.1 mol) and hydrazine hydrate (0.3 mol) in ethanol (50 mL) were refluxed for 2 h. The excess of solvent was removed under reduced pressure and recrystallized from chloroform-hexane (3 : 1) to yield crystals of compound 4a. All other compounds of the series (4b-d) were also prepared by the above specified procedure with slight variation in reaction time. Step The solution of compound 4a in glacial acetic acid (5 mL) and ethanol (10 mL) was heated to boiling and refluxed with benzaldehyde (0.12 mol) for 5 h. The refluxed solution was cooled to room temperature and kept overnight. The solid (5a) was collected out, washed with methanol, dried, and recrystallized from methanol to get the pure compound. All other compounds of the series (5b-t) were also prepared by using respective aromatic aldehydes by the above specified procedure with slight variation in reaction time.           conditions and allowed free access to standard pellet diet and water. The pharmacological testing of all the final compounds was performed according to the standard protocol given by epilepsy branch of the National Institute of Neurological Disorders and Stroke (NINDS) following the protocol adopted by the Antiepileptic Drug Development (ADD) program. Phase I pharmacological screening comprised MES, scPTZ, and neurotoxicity. Compounds were administered intraperitoneally as a solution in polyethylene glycol (PEG). The most active compounds were evaluated quantitatively in phase II screening in which the ED 50 and TD 50 of the compounds were determined. These compounds were also tested for their median hypnotic dose (HD 50 ) and median lethal dose (LD 50 ) in phase III screening. To compare the bioavailability of the active compounds, the ED 50 and TD 50 values of the synthesized compounds were also determined after oral administration in phase IV screening.

N-(6-Methoxybenzo[d]thiazol-2-yl)-3-[2-(4-methoxybenzylidene)hydrazinyl]-propanamides (5t
(1) Maximum Electroshock (MES) Test [17]. The compounds were screened for their anticonvulsant activity by electroshock seizure method. Seizures were elicited with a 60 Hz alternating current of 50 mA intensity in mice. The current was applied via ear-clip electrodes for 0.2 s. After i.p. administration of the compounds, the activities were evaluated at two time intervals, 0.5 h and 4 h. Protection against the spread of MES induced seizures was defined as the abolition of the hind limb and tonic maximal extension component of the seizure.
(2) Subcutaneous Pentylenetetrazole (scPTZ) Seizure Threshold Test [18]. The subcutaneous dose of pentylenetetrazole (85 mg/kg) at which 95% of the animals showed convulsive reaction was determined by a dose-percent effect curve. The synthesized compounds were administered at the three graded doses, namely, 30, 100, and 300 mg/Kg, intraperitoneally. At the anticipated time, PTZ was then administered subcutaneously in the posterior midline of mice. Absence of clonic spasm in half or more of the animals in the observed time periods indicates the compounds capacity to terminate the effect of pentylenetetrazole on seizure threshold. [19]. This test was performed using the rotarod method. At 30 min after the administration of the compounds, the animals were tested on a knurled plastic rod of diameter 3.2 cm rotating at 10 rpm for 1 min. Neurotoxicity was indicated by the inability of an animal to maintain equilibrium in each of three trials. [20][21][22][23]. To find out the toxic effects, if any, of the synthesized compounds on liver, the test compounds were administered to mice. After 24 hours, serum samples were taken for estimation of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), albumin, and bilirubin using commercially available kits.

Pharmacology.
A pragmatic approach to synthesize new series of benzothiazole derivatives in satisfactory yields was illustrated in Scheme 1. The result of anticonvulsant activity of the compounds (5a-t) is depicted in Table 2. Phase I preliminary anticonvulsant screening revealed that most of the newly synthesized compounds showed some degree of protection in MES screen, which suggested the good ability of these compounds to stop the seizure spread at a certain dose level. In the MES test, compounds 5h and 5p have shown protection against MES induced seizures at dose of 30 mg/kg after 0.5 h of administration. Fascinatingly, compound 5p exhibited continued protection against seizures at the same dose after 4.0 h also. It signified that compound 5p has rapid onset and long duration of anticonvulsant activity at lower dose and the result is comparable with the standard drug, phenytoin. Compounds that were active at a dose of 100 mg/kg after 0.5 h in MES screen included 5a, 5d, 5f, 5i, 5k, 5n, 5o, 5s, and 5t representing that they have rapid onset and short duration of anticonvulsant activity.
The synthesized compounds challenged the scPTZ test to predict their potential against seizure threshold. Compounds 5a, 5c, 5h, 5i, and 5p were active against seizures after 0.5 h at a dose of 100 mg/kg . Compounds 5d, 5e, 5f, 5j, 5m, 5n, 5r, and 5t were active at 300 mg/kg after the same time period. Only compounds 5a, 5i, 5j, and 5t were active after 4.0 h at a dose of 300 mg/kg indicative of the long duration of action of these compounds at high dose.
In the neurotoxicity screen, most of the compounds did not show any neurotoxicity. Compounds 5i, 5o, and 5r revealed neurotoxicity at a dose of 300 mg/kg after 0.5 h and compounds 5b, 5j, 5n, and 5p exhibited neurotoxicity after 4.0 h.
Scheme 1: Synthetic route to the titled compounds (5a-t).   In phase II anticonvulsant screening, the most active compounds 5h and 5p exhibited, in the MES screen, ED 50 of 27.9 mg/kg and 28.4 mg/kg, respectively, TD 50 of 378.5 mg/kg and 287.1 mg/kg, respectively, and protective index (PI) of 13.5 and 10.1, respectively, which is higher as compared to phenytoin and carbamazepine. In the scPTZ screen, 5h and 5p offered protection with an ED 50 of 188.6 mg/kg and 89.1 mg/kg, respectively, a PI of 2.0 and 3.2, respectively, higher than standard drugs ( Table 3). The protective index showed significant results. Higher PI values in MES and scPTZ screen indicated that compounds 5h and 5p are safer and effective anticonvulsant agents. Since both the compounds have shown potential in both phase I and phase II screening, they were subjected to be further assessed in phase III and phase IV screening.
In phase III screening, the toxicity profile of compounds 5h and 5p was determined and the results are revealed in Table 4. Mice were injected i.p. with the test compounds at different doses in order to determine the HD 50 for the hypnotic activity of the compounds based on loss of the righting reflex. Groups of 10 animals were used for each dose. Solutions were prepared immediately before the test. Logarithmic dose-response curves for test compounds were fitted to calculate the HD 50 using a linear regression analysis. Data are reported as means ± SE. For LD 50 , the selected compounds were administered intraperitoneally to mice at various doses in the multiple of TD 50 and the toxicity persuaded by them was portrayed by diminished motor activity, relaxation of muscles, failure of righting reflex, and decline level of respiration. At higher doses, animals also showed hypnosis, analgesia, and anesthesia. The median hypnotic dose (HD 50 ) of compound 5h was found to be 642 mg/kg, which is nearly twice the TD 50 of the compound. It also showed the 24 h median lethal dose (LD 50 ) of 865 mg/kg.  Compound 5p displayed the HD 50 value 712 mg/kg with LD 50 650 mg/kg. Both compounds showed high safety profile as the HD 50 /ED 50 values of 5h and 5p were found to be 23.01 and 25.07 against MES induced seizures and 3.4 and 8.0 against scPTZ induced seizures. These values are higher than that showed by phenytoin. They displayed a considerable safety profile in scPTZ induced seizure also indicative of the efficiency of both the compounds as broad spectrum anticonvulsants.
In phase IV anticonvulsant screening, the selected compounds 5h and 5p were further evaluated for ED 50 and TD 50 values after oral administration in mice to assess their bioavailability. The result indicated that, on oral administration, the bioavailability of test compounds decreased since the ED 50 values were found to be higher than the ED 50 values in phase II screening on i.p. administration (Table 5).
Phenytoin is a probable cause of acetaminophen hepatotoxicity [24] and anticonvulsants such as carbamazepine [25] and valproic acid [26] were also expected to enhance or show hepatotoxicity as a major side effect. Selected active compounds 5h and 5p were investigated for their hepatotoxic side effects by means of liver function tests (Table 6). Compounds were administered chronically to mice for 2 weeks and the biochemical parameters were estimated. The values of alkaline phosphatase, serum glutamate oxaloacetate