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The Scientific World Journal
Volume 2014, Article ID 194652, 10 pages
http://dx.doi.org/10.1155/2014/194652
Research Article

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

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi 1100062, India

Received 22 July 2014; Accepted 10 November 2014; Published 11 December 2014

Academic Editor: Nimesh Patel

Copyright © 2014 Ruhi Ali and Nadeem Siddiqui. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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.

1. 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 [1115]. 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 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.

2. 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. 1HNMR and 13CNMR spectra in DMSO-d6/CDCl3 solutions were, respectively, recorded at 400 and 100 MHz with Bruker 400 Ultrashield TM NMR spectrometer using TMS [(CH3)4Si] as internal standard. Splitting patterns are nominated as follows: s, singlet; bs, broad singlet; d, doublet; t, triplet; m, multiplet. The NH protons were D2O exchanged for their spectral characterization. The mass spectra were recorded using Waters Micromass ZQ 2000 Spectrophotometer (Jamia Hamdard, New Delhi, India).

2.1.1. Synthesis of 3-[2-(2-Substituted benzylidene)hydrazinyl]-N-(6-substitutedbenzo[d]thiazol-2-yl)propanamides (5at)

Step I: 6-Substituted-1, 3-benzothiazole-2-amines (1ad). A mixture of substituted aniline (0.01 mol) and potassium thiocyanate (0.01 mol) in glacial acetic acid (10%) was cooled and stirred. Bromine (0.01 mol) was added dropwise to this solution at such a rate to keep the temperature below 10°C throughout the addition. For additional 3 h, stirring was continued and the separated hydrochloride salt was filtered, washed with acetic acid, and dried. Reaction mixture was dissolved in hot water and neutralized with aqueous ammonia solution (25%), filtered, washed with water, dried, and recrystallized with benzene to obtain 6-substituted-1, 3-benzothiazole-2-amines (1ad).

Step II: Synthesis of N-(6-Substituted benzo[d]thiazol-2-yl)propanamides (2ad). To the solution of substituted-1, 3-benzothiazole-2-amines (1ad, 0.1 mol) in DMF, propionyl chloride (0.2 mol) was added slowly with continuous stirring. The reaction mixture was stirred for 12 hrs. On cooling, N-(6-substituted benzo[d]thiazol-2-yl)propanamides (2ad) was obtained.

Step III: 3-Bromo-N-(6-substituted benzo[d]thiazol-2-yl)propanamides (3ad). Bromine in glacial acetic acid (10 mL) was added dropwise to the solution of N-(6-substituted benzo[d]thiazol-2-yl)propanamides in DMF at 0°C. Stirring was continued for 24 hrs. Mixture of compounds was obtained which was separated by column chromatography to yield 3-bromo-N-(6-substituted benzo[d]thiazol-2-yl)propanamides (3ad).

Step IV: N-(6-Substituted benzo[d]-thiazol-2yl)-3-hydrazinylpropanamides (4ad). 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 (4bd) were also prepared by the above specified procedure with slight variation in reaction time.

Step V: 3-[2-(2-Substituted benzylidene)hydrazinyl]-N-(6-substituted benzo[d]thiazol-2-yl)propanamides (5at). 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 (5bt) were also prepared by using respective aromatic aldehydes by the above specified procedure with slight variation in reaction time.

3-(2-Benzylidenehydrazinyl)-N-(6-chlorobenzo[d]thiazol-2-yl)propanamides (5a). Yield 65%; IR (KBr) cm−1: 3316 (N–H str.), 1644 (C=O), 1610 (CH=N); 1H NMR (DMSO-) ppm: 2.50 (t, 2H, CH2C=O), 2.98 (t, 2H,  CH2NH), 5.86 (br s, 1H, NH, D2O exchangeable), 7.40–7.80 (m, 5H, Ar–H), 7.50–8.19 (m, 3H, Benzothiazole-H), 8.40 (s, 1H, CH=N), 9.20 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 32.9, 46.2, 118.1, 121.3, 125.9, 128.9, 129.7, 131.1, 132.6, 133.2, 143.9, 151.9, 173.1, 174.5; MS (70 ev), : 358.09 [M+H]+; Anal. Calcd. for C17H15ClN4OS: C, 56.90, H, 4.21, N, 15.61, S, 8.74. Found: C, 56.88, H, 4.19, N, 15.58, S, 8.70.

3-(2-Benzylidenehydrazinyl)-N-(6-flourobenzo[d]thiazol-2-yl)propanamides (5b). Yield 62%; IR (KBr) cm−1: 3320 (N–H str.), 1640 (C=O), 1569 (CH=N); 1H NMR (DMSO-) ppm: 2.45 (t, 2H, CH2), 2.95 (t, 2H,  CH2NH), 6.10 (br s, 1H, NH, D2O exchangeable), 7.42–7.58 (m, 5H, Ar–H), 7.46–8.20 (m, 3H, Benzothiazole-H), 10.02 (s, 1H, CONH, D2O exchangeable), 8.36 (s, 1H, CH=N); 13C NMR (CDCl3) ppm: 32.7, 46.0, 121.8, 123.7, 126.0, 129.1, 129.9, 131.7, 134.8, 147.9, 144.9, 173.1, 174.9, MS (70 ev), : 342.10 [M+H]+; Anal. Calcd. for C17H15FN4OS: C, 59.63, H, 4.45, N, 16.36, S, 9.33. Found: C, 59.65, H, 4.49, N, 16.40, S, 9.29.

3-(2-Benzylidenehydrazinyl)-N-(6-methylbenzo[d]thiazol-2-yl)propanamides (5c). Yield 68%; IR (KBr) cm−1: 3325 (N–H str.), 1680 (C=O), 1580 (CH=N); 1H NMR (DMSO-) ppm: 2.32 (s, 3H, CH3), 2.60 (t, 2H, CH2), 2.93 (t, 2H,  CH2NH), 5.89 (br s, 1H, NH, D2O exchangeable), 7.16–7.58 (m, 5H, Ar–H), 7.56–8.10 (m, 3H, Benzothiazole-H), 8.40 (s, 1H, CH=N), 10.02 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 24.0, 32.9, 46.2, 121.5, 121.7, 124.7, 126.0, 128.9, 129.2, 131.7, 134.8, 143.9, 146.9, 173.7, 174.5; MS (70 ev), : 338.10 [M+H]+; Anal. Calcd. for C18H18N4OS: C, 63.80, H, 5.54, N, 16.68, S, 9.47. Found: C, 63.78, H, 5.56, N, 16.70, S, 9.45.

3-(2-Benzylidenehydrazinyl)-N-(6-methoxybenzo[d]thiazol-2-yl)propanamides (5d). Yield 73%; IR (KBr) cm−1: 3350 (N–H str.), 1665 (C=O), 1601 (CH=N); 1H NMR (DMSO-) ppm: 2.66 (t, 2H, CH2), 2.90 (t, 2H,  CH2NH), 3.70 (s, 3H, OCH3), 5.90 (br s, 1H, NH, D2O exchangeable), 7.26–7.59 (m, 5H, Ar–H), 7.42–8.17 (m, 3H, Benzothiazole-H), 8.20 (s, 1H, CH=N), 10.30 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 32.8, 46.2, 55.9, 105.0, 113.2, 122.5, 125.7, 128.9, 129.2, 131.1, 133.8, 141.9, 143.9, 173.7, 174.5; MS (70 ev), : 354.01 [M+H]+; Anal. Calcd. for C18H18N4O2S: C, 61.10, H, 5.12, N, 15.79, S, 8.98. Found: C, 61.17, H, 5.10, N, 15.76, S, 8.99.

N-(6-Chlorobenzo[d]thiazol-2-yl)-3-[2-(2-hydroxybenzylidene)hydrazinyl]-propanamides (5e). Yield 63%; IR (KBr) cm−1: 3350 (N–H str.), 1665 (C=O), 1609 (CH=N); 1H NMR (DMSO-) ppm: 2.63 (t, 2H, CH2), 2.91 (t, 2H,  CH2NH), 5.40 (s, 1H, OH), 6.12 (br s, 1H, NH, D2O exchangeable), 6.80–7.40 (m, 4H, Ar–H), 7.56–8.17 (m, 3H, Benzothiazole-H), 8.30 (s, 1H, CH=N), 9.20 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 32.9, 46.2, 116.0, 118.2, 121.5, 123.7, 125.9, 129.2, 130.1, 132.8, 143.9, 147.9, 173.9, 174.5; MS (70 ev), : 375.03 [M+H]+; Anal. Calcd. for C17H15ClN4O2S: C, 54.50, H, 4.09, N, 14.96, S, 8.55. Found: C, 54.53, H, 4.00, N, 14.97, S, 8.58.

N-(6-Fluorobenzo[d]thiazol-2-yl)-3-[2-(2-hydroxybenzylidene)hydrazinyl]-propanamides (5f). Yield 69%; IR (KBr) cm−1: 3350 (N–H str.), 1665 (C=O), 1615 (CH=N); 1H NMR (DMSO-) ppm: 2.65 (t, 2H, CH2), 2.90 (t, 2H,  CH2NH), 5.35 (s, 1H, OH), 6.10 (br s, 1H, NH, D2O exchangeable), 6.82–7.45 (m, 4H, Ar–H), 7.26–8.10 (m, 3H, Benzothiazole-H), 8.30 (s, IH, CH=N), 9.15 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 32.9, 46.2, 108.0, 113.2, 116.5, 121.5, 123.7, 126.2, 130.9, 132.9, 143.9, 144.9, 158.1, 173.9, 174.5; MS (70 ev), : 360.09 [M+H]+; Anal. Calcd. for C17H15FN4O2S: C, 56.97, H, 4.29, N, 15.63, S, 8.96. Found: C, 56.99, H, 4.26, N, 15.60, S, 8.90.

3-[2-(2-Hydroxybenzylidene)hydrazinyl]-N-(6-methylbenzo[d]thiazol-2-yl)propanamides (5g). Yield 62%; IR (KBr) cm−1: 3346 (N–H str.), 1669 (C=O), 1585 (CH=N); 1H NMR (DMSO-) ppm: 2.35 (s, 3H, CH3), 2.66 (t, 2H, CH2), 2.93 (t, 2H,  CH2NH), 5.39 (s, 1H, OH), 5.95 (br s, 1H, NH, D2O exchangeable), 6.81–7.40 (m, 4H, Ar–H), 7.26–8.12 (m, 3H, Benzothiazole-H), 8.35 (s, 1H, CH=N), 10.27 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 24.0, 32.7, 46.2, 116.0, 118.9, 121.5, 121.7, 124.4, 126.0, 130.5, 132.0, 134.9, 143.9, 148.1, 173.7, 174.5; MS (70 ev), : 358.12 [M+H]+; Anal. Calcd. for C18H18N4O2S: C, 62.00, H, 5.20, N, 15.90, S, 9.04. Found: C, 62.09, H, 5.24, N, 15.97, S, 9.03.

3-[2-(2-Hydroxybenzylidene)hydrazinyl]-N-(6-methoxybenzo[d]thiazol-2-yl)propanamides (5h). Yield 60%; IR (KBr) cm−1: 3320 (N–H str.), 1640 (C=O), 1600 (CH=N); 1H NMR (DMSO-) ppm: 2.60 (t, 2H, CH2), 2.89 (t, 2H,  CH2NH), 3.50 (s, 3H, OCH3), 5.40 (s, 1H, OH), 5.90 (br s, 1H, NH, D2O exchangeable), 6.81–7.40 (m, 4H, Ar–H), 7.06–8.12 (m, 3H, Benzothiazole-H), 8.36 (s, 1H, CH=N), 9.20 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 32.8, 46.2, 55.8, 105.8, 113.7, 116.0, 121.5, 122.8, 128.9, 130.1, 132.8, 141.8, 143.8, 156.8, 173.7, 174.5; MS (70 ev), : 372.01 [M+H]+; Anal. Calcd. for C18H18N4O3S: C, 59.98, H, 4.84, N, 15.79, S, 8.86. Found: C, 60.01, H, 4.88, N, 15.85, S, 8.84.

N-(6-Chlorobenzo[d]thiazol-2-yl)-3-[2-(4-hydroxybenzylidene)hydrazinyl]-propanamides (5i). Yield 67%; IR (KBr) cm−1: 3355 (N–H str.), 1669 (C=O), 1590 (CH=N); 1H NMR (DMSO-) ppm: 2.63 (t, 2H, CH2), 2.91 (t, 2H,  CH2NH), 5.35 (s, 1H, OH), 6.10 (br s, 1H, NH, D2O exchangeable), 6.82–7.40 (m, 4H, Ar–H), 7.56–8.13 (m, 3H, Benzothiazole-H), 8.40 (s, 1H, CH=N), 10.09 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 32.9, 46.2, 116.0, 121.5, 123.9, 125.8, 126.0, 129.9, 130.6, 143.6, 147.8, 174.0, 175.8; MS (70 ev), : 374.09 [M+H]+; Anal. Calcd. for C17H15ClN4O2S: C, 54.48, H, 4.09, N, 14.96, S, 8.75. Found: C, 54.50, H, 4.10, N, 14.98, S, 8.78.

N-(6-Fluorobenzo[d]thiazol-2-yl)-3-[2-(4-hydroxybenzylidene)hydrazinyl]-propanamides (5j). Yield 66%; IR (KBr) cm−1: 3349 (N–H str.), 1660 (C=O), 1610 (CH=N); 1H NMR (DMSO-) ppm: 2.60 (t, 2H, CH2), 2.95 (t, 2H,  CH2NH), 5.55 (s, 1H, OH), 5.82 (br s, 1H, NH, D2O exchangeable), 6.82–7.40 (m, 4H, Ar–H), 7.26–8.21 (m, 3H, Benzothiazole-H), 9.15 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 32.9, 46.2, 108.1, 113.7, 116.5, 123.7, 126.8, 130.6, 143.3, 144.6, 158.6, 160.6, 173.7, 175.8; MS (70 ev), : 356.9 [M+H]+; Anal. Calcd. for C17H15FN4O2S: C, 56.89, H, 4.28, N, 15.76, S, 8.90. Found: C, 56.91, H, 4.23, N, 15.70, S, 8.93.

3-[2-(4-Hydroxybenzylidene)hydrazinyl]-N-(6-methylbenzo[d]thiazol-2-yl)propanamides (5k). Yield 66%; (KBr) cm−1: 3345 (N–H str.), 1666 (C=O), 1586 (CH=N); 1H NMR (DMSO-) ppm: 2.28 (s, 3H, CH3), 2.59 (t, 2H, CH2), 2.90 (t, 2H,  CH2NH), 5.28 (s, 1H, OH), 6.20 (br s, 1H, NH, D2O exchangeable), 6.81–7.40 (m, 4H, Ar–H), 7.36–8.41 (m, 3H, Benzothiazole-H), 8.52 (s, 1H, CH=N), 10.02 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 23.7, 32.5, 46.9, 116.0, 121.7, 123.8, 126.4, 130.6, 134.3, 143.6, 146.0, 173.7, 175.8; MS (70 ev), : 356.12 [M+H]+; Anal. Calcd. for C18H18N4O2S: C, 61.01, H, 5.13, N, 15.90, S, 9.05. Found: C, 59.88, H, 5.20, N, 15.99, S, 8.99.

3-[2-(4-Hydroxybenzylidene)hydrazinyl]-N-(6-methoxybenzo[d]thiazol-2-yl)propanamides (5l). Yield 76%; IR (KBr) cm−1: 3348 (N–H str.), 1669 (C=O), 1600 (CH=N); 1H NMR (DMSO-) ppm: 2.55 (t, 2H, CH2), 2.98 (t, 2H,  CH2NH), 3.50 (s, 3H, OCH3), 5.32 (s, 1H, OH), 6.18 (br s, 1H, NH, D2O exchangeable), 6.79–7.40 (m, 4H, Ar–H), 7.29–8.45 (m, 3H, Benzothiazole-H), 8.49 (s, 1H, CH=N), 10.24 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 32.9, 46.2, 55.9, 105.0, 113.2, 116.0, 122.5, 125.8, 126.9, 141.8, 143.8, 156.9, 160.1, 173.7, 174.5; MS (70 ev), : 370.11 [M+H]+; Anal. Calcd. for C18H18N4O3S: C, 58.40, H, 4.90, N, 15.12, S, 8.69. Found: C, 58.38, H, 4.88, N, 15.15, S, 8.72.

N-(6-Chlorobenzo[d]thiazol-2-yl)-3-[2-(4-methylbenzylidene)hydrazinyl]-propanamides (5m). Yield 72%; IR (KBr) cm−1: 3352 (N–H str.), 1671 (C=O), 1595 (CH=N); 1H NMR (DMSO-) ppm: 2.30 (s, 3H, CH3), 2.60 (t, 2H, CH2), 2.90 (t, 2H,  CH2NH), 5.80 (br s, 1H, NH, D2O exchangeable), 6.80–7.42 (m, 4H, Ar–H), 7.56–8.75 (m, 3H, Benzothiazole-H), 8.44 (s, 1H, CH=N), 9.15 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 24.3, 32.5, 46.0, 121.3, 123.8, 125.4, 129.1, 129.3, 140.6, 143.0, 147.7, 173.9, 174.0; MS (70 ev), : 389.10 [M+H]+; Anal. Calcd. for C18H17ClN4OS: C, 57.69, H, 4.67, N, 15.07, S, 8.60. Found: C, 57.60, H, 4.60, N, 15.17, S, 8.54.

N-(6-Fluorobenzo[d]thiazol-2-yl)-3-[2-(4-methylbenzylidene)hydrazinyl]-propanamides (5n). Yield 77%; IR (KBr) cm−1: 3354 (N–H str.), 1669 (C=O), 1588 (CH=N); 1H NMR (DMSO-) ppm: 2.35 (s, 3H, CH3), 2.64 (t, 2H, CH2), 2.92 (t, 2H,  CH2NH), 6.20 (br s, 1H, NH, D2O exchangeable), 6.86–7.47 (m, 4H, Ar–H), 7.26–8.47 (m, 3H, Benzothiazole-H), 8.28 (s, 1H, CH=N), 10.27 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 24.3, 32.9, 46.2, 108.3, 113.8, 123.4, 126.1, 129.1, 129.2, 130.6, 143.0, 144.7, 158.6, 173.8, 175.0; MS (70 ev), : 373.01 [M+H]+; Anal. Calcd. for C18H17FN4OS: C, 61.01, H, 4.87, N, 15.65, S, 8.89. Found: C, 59.01, H, 4.90, N, 15.52, S, 8.90.

N-(6-Methylbenzo[d]thiazol-2-yl)3-[2-(4-methylbenzylidene)hydrazinyl] propanamides (5o). Yield 80%; IR (KBr) cm−1: 3355 (N–H str.), 1670 (C=O), 1605 (CH=N); 1H NMR (DMSO-) ppm: 2.30 (s, 6H, 2CH3), 2.60 (t, 2H, CH2), 2.85 (t, 2H,  CH2NH), 5.88 (br s, 1H, NH, D2O exchangeable), 7.10–7.50 (m, 4H, Ar–H), 7.35–8.11 (m, 3H, Benzothiazole-H), 8.55 (s, 1H, CH=N), 10.22 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 23.6, 24.8, 32.1, 46.5, 121.4, 124.1, 126.1, 129.0, 129.2, 130.6, 134.0, 140.7, 146.6, 173.7, 174.9, MS (70 ev), : 356.01 [M+H]+; Anal. Calcd. for C19H20N4O2S: C, 59.76, H, 4.81, N, 15.72, S, 9.00. Found: C, 59.79, H, 4.87, N, 15.68, S, 8.90.

N-(6-Methoxybenzo[d]thiazol-2-yl)-3-[2-(4-methylbenzylidene)hydrazinyl]-propanamides (5p). Yield 79%; IR (KBr) cm−1: 3316 (N–H str.), 1747 (C=O), 1610 (CH=N); 1H NMR (DMSO-) ppm: 2.34 (s, 3H, CH3), 2.62 (t, 2H, CH2), 2.92 (t, 2H,  CH2NH), 3.49 (s, 3H, OCH3), 5.92 (br s, 1H, NH, D2O exchangeable), 6.99–7.44 (m, 4H, Ar–H), 7.23–8.40 (m, 3H, Benzothiazole-H), 8.49 (s, 1H, CH=N), 10.28 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 21.5, 32.8, 46.5, 55.5, 104.9, 114.0, 118.0, 121.1, 123.0, 126.4, 129.9, 130.7, 143.6, 147.8, 163.0, 173.5, 175.8; MS (70 ev), : 385.13 [M+H]+; Anal. Calcd. For C19H20N4O3S: C, 59.28, H, 5.26, N, 14.56, S, 8.49. Found: C, 59.62, H, 5.30, N, 14.66, S, 8.55.

N-(6-Chlorobenzo[d]thiazol-2-yl)-3-[2-(4-methoxybenzylidene)hydrazinyl]-propanamides (5q). Yield 82%, IR (KBr) cm−1: 3350 (N–H str.), 1660 (C=O), 1588 (CH=N); 1H NMR (DMSO-) ppm: 2.66 (t, 2H, CH2), 2.89 (t, 2H,  CH2NH), 3.49 (s, 3H, OCH3), 5.96 (br s, 1H, NH, D2O exchangeable), 7.10–7.44 (m, 4H, Ar–H), 7.15–8.40 (m, 3H, Benzothiazole-H), 8.35 (s, 1H, CH=N), 10.28 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 32.8, 46.5, 55.5, 114.0, 121.1, 123.0, 125.2, 126.4, 129.9, 130.7, 143.6, 147.8, 163.0, 173.5, 175.8; MS (70 ev), : 388.09 [M+H]+; Anal. Calcd. for C18H17FCl4O2S: C, 56.59, H, 4.40, N, 15.11, S, 8.25. Found: C, 56.39, H, 4.35, N, 15.01, S, 8.29.

N-(6-Fluorobenzo[d]thiazol-2-yl)-3-[2-(4-methoxybenzylidene)hydrazinyl]-propanamides (5r). Yield 69%; IR (KBr) cm−1: 3353 (N–H str.), 1670 (C=O), 1599 (CH=N); 1H NMR (DMSO-) ppm: 2.63 (t, 2H, CH2), 2.96 (t, 2H,  CH2NH), 3.58 (s, 3H, OCH3), 6.05 (br s, 1H, NH, D2O exchangeable), 7.10–7.44 (m, 4H, Ar–H), 7.31–8.49 (m, 3H, Benzothiazole-H), 8.99 (s, 1H, CH=N), 10.19 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 32.6, 46.2, 56.5, 108.0, 113.1, 114.0, 123.2, 126.4, 129.9, 130.7, 143.6, 146.8, 158.0, 173.7, 174.8; MS (70 ev), : 372.09 [M+H]+; Anal. Calcd. for C18H17FN4O2S: C, 58.05, H, 4.60, N, 15.04, S, 8.61. Found: C, 58.45, H, 4.68, N, 15.00, S, 8.69.

3-[2-(4-Methoxybenzylidene)hydrazinyl]-N-(6-methylbenzo[d]thiazol-2-yl)propanamides (5s). Yield 77%; IR (KBr) cm−1: 3354 (N–H str.), 1669 (C=O), 1579 (CH=N); 1H NMR (DMSO-) ppm: 2.35 (s, 3H, CH3), 2.58 (t, 2H, CH2), 2.92 (t, 2H,  CH2NH), 3.50 (s, 3H OCH3), 6.15 (br s, 1H, NH, D2O exchangeable), 6.86–7.47 (m, 4H, Ar–H), 7.26–8.47 (m, 3H, Benzothiazole-H), 8.44 (s, 1H, CH=N), 10.27 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 24.0, 32.9, 46.2, 55.5, 114.1, 121.2, 124.4, 126.9, 130.7, 143.9, 146.4, 163.0, 173.7, 174.5; MS (70 ev), : 368.01 [M+H]+; Anal. Calcd. for C19H20N4O2S: C, 61.94, H, 5.45, N, 15.25, S, 8.71. Found: C, 61.99, H, 5.40, N, 15.30, S, 8.70.

N-(6-Methoxybenzo[d]thiazol-2-yl)-3-[2-(4-methoxybenzylidene)hydrazinyl]-propanamides (5t). Yield 69%; IR (KBr) cm−1: 3348 (N–H str.), 1669 (C=O), 1600 (CH=N); 1H NMR (DMSO-) ppm: 2.66 (t, 2H, CH2), 2.93 (t, 2H,  CH2NH), 3.59 (s, 6H, 2OCH3), 5.99 (br s, 1H, NH, D2O exchangeable), 6.79–7.44 (m, 4H, Ar–H), 7.29–8.45 (m, 3H, Benzothiazole-H), 8.36 (s, 1H, CH=N), 10.20 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3) ppm: 32.6, 46.0, 55.9, 105.1, 113.2, 114.4, 122.9, 125.7, 126.9, 130.4, 141.8, 143.0, 156.9, 160.2, 173.7, 174.5; MS (70 ev), : 384.89 [M+H]+; Anal. Calcd. for C19H20N4O3S: C, 59.36, H, 5.24, N, 14.57, S, 8.34. Found: C, 59.39, H, 5.20, N, 14.59, S, 8.37.

2.2. Pharmacology
2.2.1. Anticonvulsant Activity

The anticonvulsant activity was carried out on male albino mice (20–25 g) as experimental animals. The animals were housed under standard 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 ED50 and TD50 of the compounds were determined. These compounds were also tested for their median hypnotic dose (HD50) and median lethal dose (LD50) in phase III screening. To compare the bioavailability of the active compounds, the ED50 and TD50 values of the synthesized compounds were also determined after oral administration in phase IV screening.

(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.

2.2.2. Neurotoxicity Screening [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.

2.2.3. Liver Function Test [2023]

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.

3. Result and Discussion

3.1. Chemistry

A series of new benzothiazole derivatives were synthesized in satisfactory yields (65–80%) as demonstrated in Scheme 1 and their structures were characterized by elemental and spectral analysis. The physicochemical properties of synthesized compounds are presented in Table 1. Substituted anilines are cyclised to 6-substituted benzo[d]thiazole-2-amines (1ad) on treatment with potassium thiocyanates, bromine in glacial acetic acid, which, on treatment with propionyl chloride, afforded N-(6-substituted benzo[d]thiazol-2-yl)propanamides (2ad). This compound on bromination yields 3-bromo-N-(6-substituted benzo[d]thiazol-2-yl)propanamides (3ad). On further treatment with hydrazine, hydrate converted into N-(6-substituted benzo[d]thiazol-2-yl)-3-hydrazinylpropanamide (4ad), which was condensed with different aldehydes to yield the titled compounds 3-[2-(substituted benzylidene)]-N-(6-substituted benzo[d]thiazol-2-yl)propanamides (5at).

Table 1: Physicochemical parameters of the synthesized compounds (5at).
Scheme 1: Synthetic route to the titled compounds (5at).

The synthesized benzothiazole derivatives showed (N–H), (C=O), and (C=N) stretching bands in the region of 3355–3316 cm−1, 1747–1640 cm−1, and 1615–1569 cm−1, respectively, in their IR spectrum, while, in their 1H NMR spectra, these compounds exhibited multiplets for (Ar–H) in the regions of 6.80–7.80 ppm, a singlet in the region of 9.15–10.30 ppm (CONH), and a singlet for (CH=N) in the region of 8.20–8.99 ppm. 13C spectra of prototype compound 5p showed peaks at 21.5, 32.8, 46.5, 55.5, 104.9, 114.0, 118.0, 121.1, 123.0, 126.4, 129.9, 130.7, 143.6, 147.8, 163.0, 173.5, and 175.8 ppm confirming presence of 17 different carbon atoms.

3.2. 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 (5at) 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.

Table 2: Phase I anticonvulsant evaluation of the synthesized compounds (5a–t).

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.

In phase II anticonvulsant screening, the most active compounds 5h and 5p exhibited, in the MES screen, ED50 of 27.9 mg/kg and 28.4 mg/kg, respectively, TD50 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 ED50 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.

Table 3: Phase II quantitative anticonvulsant evaluation of selected active compounds.

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 HD50 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 HD50 using a linear regression analysis. Data are reported as means ± SE. For LD50, the selected compounds were administered intraperitoneally to mice at various doses in the multiple of TD50 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 (HD50) of compound 5h was found to be 642 mg/kg, which is nearly twice the TD50 of the compound. It also showed the 24 h median lethal dose (LD50) of 865 mg/kg. Compound 5p displayed the HD50 value 712 mg/kg with LD50 650 mg/kg. Both compounds showed high safety profile as the HD50/ED50 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.

Table 4: Phase III quantitative toxicity profile of selected compounds.

In phase IV anticonvulsant screening, the selected compounds 5h and 5p were further evaluated for ED50 and TD50 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 ED50 values were found to be higher than the ED50 values in phase II screening on i.p. administration (Table 5).

Table 5: Phase IV quantitative anticonvulsant evaluation of selected active compounds after oral administration.

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 transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), albumin, and bilirubin suggested that none of the compounds had shown any considerable increase or decrease (Figure 1). Further histopathological study of compounds 5h and 5p confirmed that there is no liver toxicity by showing normal hepatic parenchyma with portal triad, central vein, and hepatocytes in comparison to control (Figure 2).

Table 6: Enzyme estimation of the selected compounds.
Figure 1: Hepatic enzymes estimations after treatment with different test compounds. Number of animals tested (). The mean levels were calculated using ANOVA followed by Dunnett’s test.
Figure 2: High power photomicrograph of portal triad area of liver tissue from animals treated with (a) control, (b) compound (5h), and (c) compound (5p) showing a normal histological appearance (HE ×400). PT, portal triad; CV, central vein.

4. Conclusion

In the present study, a series of 3-[2-(2-substituted benzylidene)hydrazinyl]-N-(6-substitutedbenzo[d]thiazol-2-yl)propanamides were synthesized successfully and all compounds were tested for anticonvulsant activity (phases I–IV) using MES and scPTZ screens. Compounds 5h and 5p represent valuable leads in the exploration of agents controlling both treatment of seizures and intoxication during epilepsy. Hence, we may conclude that reported series of substituted benzothiazole derivatives may be promising for the development of potential anticonvulsant agents after further optimization.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

One of the authors (Ruhi Ali) is thankful to the University Grants Commission, Ministry of Human Resource Development, and Government of India for financial support.

References

  1. WHO Epilepsy in the WHO Africa Region: Bridging the Gap: The Global Campaign Against Epilepsy: “Out of the Shadows”, WHO, Geneva, Switzerland, 2004.
  2. C. Wasteralin, G. Siegel, G. Agranoff, R. Albers, and P. Molinoff, Basic Neurochemistry: Molecular, Cellular and Medical Aspects, Raven, New York, NY, USA, 4th edition, 1989.
  3. R. Sridharan and B. N. Murthy, “Prevalence and pattern of epilepsy in India,” Epilepsia, vol. 40, no. 5, pp. 631–636, 1999. View at Publisher · View at Google Scholar · View at Scopus
  4. M. Leonardi and T. B. Ustun, “The global burden of epilepsy,” Epilepsia, vol. 43, no. 6, pp. 21–25, 2002. View at Google Scholar · View at Scopus
  5. K. Pahl and H. M. Boer, Atlas: Epilepsy Care in the World, WHO, Geneva, Switzerland, 2005.
  6. G. Paswerk, “Annual report of the WHO/IBE/ILAE,” Global Campaign Against Epilepsy: Out of the Shadows, International Bureau of Epilepsy, Heemstede, The Netherlands, 2003. View at Google Scholar
  7. A. Sabers and L. Gram, “Newer anticonvulsants: comparative review of drug interactions and adverse effects,” Drugs, vol. 60, no. 1, pp. 23–33, 2000. View at Publisher · View at Google Scholar · View at Scopus
  8. M. Bialer, S. I. Johannessen, H. J. Kupferberg, R. H. Levy, E. Perucca, and T. Tomson, “Progress report on new antiepileptic drugs: a summary of the Seventh Eilat Conference (EILAT VII),” Epilepsy Research, vol. 61, no. 1–3, pp. 1–48, 2004. View at Publisher · View at Google Scholar · View at Scopus
  9. J. F. Wolfe, T. D. Greenwood, and J. M. Mulheron, “Recent trends in the development of new anti-epileptic drugs,” Expert Opinion on Therapeutic Patents, vol. 8, no. 4, pp. 361–381, 1998. View at Publisher · View at Google Scholar · View at Scopus
  10. G. A. Burdock, Encyclopedia of Food and Color Additives, Technology and Engineering, CRC Press, Boca Raton, Fla, USA, 1996.
  11. A. R. Carroll and P. J. Scheuer, “Kuanoniamines A, B, C, and D: pentacyclic alkaloids from a tunicate and its prosobranch mollusk predator Chelynotus semperi,” Journal of Organic Chemistry, vol. 55, no. 14, pp. 4426–4431, 1990. View at Publisher · View at Google Scholar · View at Scopus
  12. G. P. Gunawardana, S. Kohmoto, and N. S. Burres, “New cytotoxic acridine alkaloids from two deep water marine sponges of the family Pachastrellidae,” Tetrahedron Letters, vol. 30, no. 33, pp. 4359–4362, 1989. View at Publisher · View at Google Scholar · View at Scopus
  13. K. P. Bhusari, N. D. Amnerkar, P. B. Khedekar, M. K. Kale, and R. P. Bhole, “Synthesis and in vitro antimicrobial activity of some new 4-amino-N-(1, 3-benzothiazol-2-yl) benzenesulphonamide derivatives,” Asian Journal of Research in Chemistry, vol. 1, pp. 53–58, 2008. View at Google Scholar
  14. G. Turan-Zitouni, Ş. Demirayak, A. Özdemir, Z. A. Kaplancikli, and M. T. Yildiz, “Synthesis of some 2-[(benzazole-2-yl)thioacetylamino]thiazole derivatives and their antimicrobial activity and toxicity,” European Journal of Medicinal Chemistry, vol. 39, no. 3, pp. 267–272, 2004. View at Publisher · View at Google Scholar · View at Scopus
  15. B. Malawska and A. Scatturin, “Application of pharmacophore models for the design and synthesis of new anticonvulsant drugs,” Mini-Reviews in Medicinal Chemistry, vol. 3, no. 4, pp. 341–348, 2003. View at Publisher · View at Google Scholar · View at Scopus
  16. S. N. Pandeya, P. Yogeeswari, and J. P. Stables, “Synthesis and anticonvulsant activity of 4-bromophenyl substituted aryl semicarbazones,” European Journal of Medicinal Chemistry, vol. 35, no. 10, pp. 879–886, 2000. View at Publisher · View at Google Scholar · View at Scopus
  17. R. L. Krall, J. K. Penry, B. G. White, H. J. Kupferberg, and E. A. Swinyard, “Antiepileptic drug development: II. Anticonvulsant drug screening,” Epilepsia, vol. 19, no. 4, pp. 409–428, 1978. View at Publisher · View at Google Scholar · View at Scopus
  18. E. A. Swinyard, J. H. Woodhead, H. S. White, and M. R. Franklin, Antiepileptic Drugs, Raven-Press, New York, NY, USA, 3rd edition, 1989.
  19. N. W. Dunham and T. S. Miya, “A note on a simple apparatus for detecting neurological deficit in rats and mice,” Journal of the American Pharmaceutical Association, vol. 46, no. 3, pp. 208–209, 1957. View at Google Scholar · View at Scopus
  20. S. Reitman and S. Frankel, “A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases,” American Journal of Clinical Pathology, vol. 28, no. 1, pp. 56–63, 1957. View at Google Scholar · View at Scopus
  21. E. J. King and A. R. Armstrong, “A convenient method for determining serum and bile phosphatase activity,” Canadian Medical Association Journal, vol. 31, pp. 376–381, 1934. View at Google Scholar
  22. J. G. Reinhold, in Total Protein Albumin and Globulin. Standard Methods in Clinical Chemistry, M. Reiner, Ed., pp. 88–90, Academic Press, New York, NY, USA, 1953.
  23. H. Varley, in Practical Clinical Biochemistry, pp. 236–238, CBS Publishers and Distributors, New Delhi, India, 1988.
  24. C. C. Brackett and J. D. Bloch, “Phenytoin as a possible cause of acetaminophen hepatotoxicity: case report and review of the literature,” Pharmacotherapy, vol. 20, no. 2, pp. 229–233, 2000. View at Publisher · View at Google Scholar · View at Scopus
  25. M. P. Kalapos, “Carbamazepine-provoked hepatotoxicity and possible aetiopathological role of glutathione in the events—retrospective review of old data and call for new investigation,” Adverse Drug Reactions and Toxicological Reviews, vol. 21, no. 3, pp. 123–141, 2002. View at Publisher · View at Google Scholar · View at Scopus
  26. S. Chitturi and J. George, “Hepatotoxicity of commonly used drugs: nonsteroidal anti-inflammatory drugs, antihypertensives, antidiabetic agents, anticonvulsants, lipid-lowering agents, psychotropic drugs,” Seminars in Liver Disease, vol. 22, no. 2, pp. 169–183, 2002. View at Publisher · View at Google Scholar · View at Scopus