Design, synthesis and evaluation of N-(substituted benzothiazol-2-yl)amides as anticonvulsant and neuroprotective

European Journal of Medicinal Chemistry 58 (2012) 206e213

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Design, synthesis and evaluation of N-(substituted benzothiazol-2-yl)amides as anticonvulsant and neuroprotectiveq Mohd. Zaheen Hassan, Suroor A. Khan, Mohd. Amir* Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi 110062, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 June 2012 Received in revised form 11 September 2012 Accepted 2 October 2012 Available online 10 October 2012

A series of N-(substituted benzothiazol-2-yl)amide derivatives 2aeh and 4aeh were synthesized by the EDC coupling reactions of substituted-benzothiazol-2-amine with 4-oxo-4-phenylbutanoic acid/2benzoyl benzoic acid and evaluated for their anticonvulsant and neuroprotective effect. N-(6methoxybenzothiazol-2-yl)-4-oxo-4-phenylbutanamide (2f) emerged as the most effective anticonvulsant with median doses of 40.96 mg/kg (MES ED50), 85.16 mg/kg (scPTZ ED50) and 347.6 mg/kg (TD50). Furthermore, compound 2f displayed promising neuroprotective effect by lowering the levels of MDA and LDH; therefore, it represents a potential lead in search for safer and effective anticonvulsants having neuroprotective effects. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Benzothiazole Amides Anticonvulsants Neuroprotective agents

1. Introduction Epilepsy is a chronic neurological disorder characterized by an enduring predisposition to generate seizures due to hypersynchronous neuronal activity in the brain. According to epidemiological studies, epilepsy is the third most devastating neurological disorder, affecting more than 50 million people globally with almost 90% of these patients being in developing countries [1]. Antiepileptic drugs (AEDs) comprise a diverse range of molecules acting mostly through: enhancement of g-aminobutyric acid (GABA) mediated inhibitory neurotransmission, modulation of voltage-gated ion channels (Naþ, Caþþ), and reduction of excitatory, particularly glutamate-mediated neurotransmitter [2]. Despite these therapeutic arsenals, there is yet no complete cure for epileptic conditions and current AEDs suppress the seizure symptoms of epilepsy (w70% epileptic patients) but do not affect the natural course of the epileptogenic process [3]. Finally, the efficacy of many AEDs are limited by the dose related toxicities and diverse array of adverse drug reactions from minimal brain impairment, megaloblastic anemia to death from aplastic anemia or hepatic failure [4]. Thus, there is a substantial need for further development

q Part of the work was presented at 8th AFMC International Medicinal Chemistry Symposium "Frontier of Medicinal Science" held in Nov. 2011 at Tokyo, Japan. * Corresponding author. Tel.: þ91 11 2605 9688x5307; fax: þ91 11 26059663. E-mail address: [email protected] (Mohd. Amir). 0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2012.10.002

of safer agents and novel mechanistic approaches in the treatment of epilepsy. Anticonvulsants having neuroprotective actions have received considerable attention in the treatment of epilepsy. Several studies reported that neurodegeneration is the major neurobiological abnormality in epileptic brain [5]. The pathomechanisms of neurodegeneration are complex involving glutamate overload, decreased GABA level due to inhibition of enzyme glutamic acid decarboxylase (GAD) by oxygen free radicals and multiple lipid and protein damages via lipid peroxidation (LPO) [6]. Since epileptic seizure and neurodegeneration share aspects of their underlying pathophysiology therefore, some AEDs are equally effective neuroprotectives [7]. Riluzole (2-amino-6-trifluoromethoxy benzothiazole) is an anticonvulsant drug with phenytoin-like spectrum of activity. It was also found to be effective for treating some forms of neurodegeneration. Although riluzole is known to modulate excitatory neurotransmission, the precise neuroprotective mechanisms remain largely speculative [8]. As riluzole is a simple molecule having benzothiazole moiety in its structure and a wide range of CNS activity, it is a good target for structure modification and structureeactivity relationship (SAR) studies. Many studies have been conducted in an attempt to find derivatives superior to riluzole [9,10]. The synthesis of various riluzole derivatives showed that strong neuroprotective and anti-seizure activity required the presence of six substituents containing either an unchanged or oxidized sulfur atom or a nitrogen atom [9]. On the other hand, a variety of benzothiazole derivatives have been reported to possess

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anticonvulsant activity in a number of animal models. Therefore, these are promising candidates for the design of novel antiepileptic drugs [11,12]. Encouraged by these observations and in continuation of our ongoing research program [13e15], some amide derivatives of benzothiazole were designed and synthesized as anticonvulsant agents. We propose that the GABA like pharmacophore on the benzothiazole nucleus might sufficiently bind with the receptors and the resultant molecules will have a synergistic anticonvulsant effect due to increased lipophilicity. The presence of an amide group as hydrogen bonding domain (HBD) is an optimal anticonvulsant pharmacophoric feature in the title compounds. The additional features include benzothiazolyl hydrophobic-domain (A), S atom as electron donor system (D) with distal phenyl residue (R), which influences the blood brain barrier (BBB) diffusion and pharmacokinetic properties of the anticonvulsants (Fig. 1) [16]. In our present work, we aimed to synthesize a series of N-(substituted benzothiazol-2-yl)amide derivatives in order to investigate their behavior not only as anticonvulsants but as neuroprotective agents too. Since, none of the currently available AEDs is perfect and most of them have chronic and acute adverse effects therefore, the present study also focuses on the toxicity studies of the synthesized compounds.

2. Results and discussion 2.1. Chemistry The synthetic routes of compounds are outlined in Scheme 1. Synthesis of 4-oxo-4-phenylbutanoic acid (1) and 2-benzoyl benzoic acid (3) involves the Lewis acid-catalyzed Friedel Crafts acylation of benzene with succinic anhydride and phthalic anhydride, respectively. Reactions of the above mentioned acids 1 or 3 with 1-(3dimethylaminopropyl)-3-ethyl-carbodiimide (EDC) and hydroxybenzotriazole (HOBt) in presence of DCM resulted in the in situ formation of activated acyl compounds which on further addition of substituted-2-aminobenzothiazole and triethylamine, produced the desired amides 2aeh and 4aeh, respectively in good yields. The structures of the amides 2aeh and 4aeh were confirmed by elemental analyses and spectral data. The IR spectra of compounds 2aeh and 4aeh showed the appearance of C¼O and NeH absorption bands in the range of 1632e1680 and 3265e3345 cm1, respectively. The 1H NMR spectra revealed a broad singlet signal at d 8.77e 10.76 ppm due to CONH proton. The 1H NMR spectra of compounds 2aeh revealed the appearance of a pair of triplets signals at d 2.80e

Fig. 1. Design of the titled compounds as anticonvulsant-neuroprotective agents possessing the essential pharmacophoric elements: aryl unit/Hydrophobic domain (A), electron donor atom (D), and coupled hydrogen donor (HD)ehydrogen acceptor (HA) group.

207

2.98 and 3.44e3.51 ppm due ethylene protons and disappearance of signal of amino protons of substituted-2-aminobenzothiazoles. The 13 C NMR spectra of a prototype compound 2f revealed three upfield peaks at d 28.42, 33.41 and 55.86 due to a pair of ethylene carbons and one methoxy carbon, respectively while carbonyl carbons of amide (CONH) and benzoyl (COPh) groups appeared downfield at d 178.32 and 198.21, respectively. The other peaks of carbon were observed at d 105.53, 113.87, 118.14, 128.08, 128.63, 130.00, 133.27, 136.51, 143.04, 155.74 and 167.03 confirming the presence of eighteen carbons in the compound. 2.2. Pharmacological evaluation The results of the in vivo pharmacological screening are shown in Tables 1e3. The titled compounds were initially evaluated against maximal electroshock seizure (MES) and subcutaneous pentylenetetrazol (scPTZ) (Table 1), the two most widely used seizure models in the search for new AEDs. These tests aim to detect compounds possessing activity against generalized tonicclonic (grand mal) and generalized absence (petit mal) seizures respectively. The most potent compound was also subjected to Phase II quantitative determination of median effective dose (ED50), median toxic dose (TD50) and protective index (PI) (Table 3). In the preliminary screening, all the compounds showed protection against MES test, which indicates the potential of these compounds against generalized tonic-clonic seizure. Compound 2f showed protection against seizures in the MES model at the 30 mg/kg dose after both 0.5 and 4.0 h of the administration. This indicates that compound 2f has a rapid onset and long duration action at a lower dose. Compound 2e was active at 100 mg/kg after 0.5 and 4.0 h in MES tests, which is indicative of its quick onset and long duration of action at relatively higher dose while remaining compounds were active at 300 mg/kg. In the scPTZ screening, only compound 2f raised the seizure threshold at a dose of 100 mg/kg after both 0.5 and 4.0 h time intervals, showing the promising nature of compound against the absence seizure. In the neurotoxicity screen, compounds 2a, 2b, 2eeh, 4a and 4eeg did not exhibit any neurological deficit at the highest dose. Rest of the compounds showed neurotoxicity but at a higher dose. We further explored the CNS depressant effects of all the compounds by Porsolt’s swimming pool test. Their immobility time was compared with respect to the reference drug carbamazepine (Table 1). Compounds 2a, 2e and 2f had no significant CNS depression, whereas rest of the compounds possessed CNS depressant effects as they increased the immobility time significantly compared to control at p < 0.05. Hepatotoxicity is the key limiting factor for the clinical use of AEDs. Therefore, selected compounds 2a, 2e and 2f were further analyzed for their hepatotoxic profiles. Any significant alterations in the level of enzymes and proteins are indicative of liver toxicity. As shown in Table 2, activities of liver enzymes SGOT, SGPT, alkaline phosphatase, albumin and total protein remained almost the same with respect to control values (p > 0.05) except for the compound 2e, which slightly increased the total protein level (p < 0.05). The liver histological examination of compound 2f-treated animals showed no symptoms of necrosis, inflammation or hepatocytes degeneration as compared to control animals. The hepatic parenchyma appeared normal, which indicates the nontoxic effect of compound on liver (Figs. 2 and 3). Compound 2f appeared as the most effective anticonvulsant in phase I screening without any significant toxicities like minimal motor impairment, CNS depression and hepatotoxic effect. This led us to further investigate and quantify its pharmacological properties in phase II anticonvulsant screening (Table 3). In the MES

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O OH

(i) C6H6

AlCl3

O

1 O

S

(iii) R

O

N

OH

(ii)

S

(iii) R

N

3

O N H

2(a-h) O

O

O

N H 4(a-h)

Scheme 1. Reagent and conditions: (i) succinic anhydride, reflux; (ii) phthalic anhydride, reflux, (iii) substituted 2-aminobenzothiazole, EDC, HOBt, DCM, stirring.

(p < 0.01). This GABA-mediatory action might be due the molecular hybridization of lipophilic benzothaizole with GABA like moiety. The neuroprotective effects of the compounds were investigated against the acrylamide (ACR)-induced neurotoxity. ACR is a potent neurotoxin in both humans and experimental animals [18]. The release of tissue malonaldehyde (MDA) and cytosolic enzyme lactate dehydrogenase (LDH) are the peripheral markers of cells intoxication [19]. Neuroprotective agents ameliorate seizure induced neuronal cells intoxications by lowering the LDH and MDA levels which may eventually alters the clinical course of the seizures [20,21]. The data depicted in Fig. 5 demonstrated that, ACR-administration in mice induced degenerative effect on the brain tissue as manifested by the significant increase in the levels of MDA and LDH (224% and 67%, respectively) compared to the control group (p < 0.01). Compounds 2a, 2e and 2f administered 10 min before ACR intoxication revealed the decrease in brain MDA and LDH activity, however significant decrease in the activity was shown by only compound 2f. These results confirmed the powerful neuroprotective actions of compounds and suggested possibly an influence of neuroprotective benzothiazole on intoxicated neuronal cells.

screen, compound 2f exhibited moderate efficacy with an ED50 of 40.96 mg/kg, which is higher than that of phenytoin (9.5 mg/kg) and carbamazepine (8.8 mg/kg) and lower than sodium valproate (272 mg/kg). In the scPTZ screen, it offered protection with an ED50 of 85.16 mg/kg, which is considerably lower than that of phenytoin, carbamazepine and sodium valproate. The median toxic dose (TD50) in rotarod test was also higher (347.6 mg/kg) compared to all the standard drugs. Calculation of protective indices (PI) resulted in higher PI values of 8.4 in MES and 4.0 in scPTZ screen, indicating that compound 2f is indeed a safer and effective anticonvulsant agent. 2.3. GABA assay and neuroprotective studies PTZ produces seizures by inhibiting GABA neurotransmission [17]. The promising anti-PTZ effects of compounds 2a, 2e and 2f prompted us to estimate whole brain GABA levels, to gain insight into the mechanism of anticonvulsant behavior of benzothiazole derivatives. Results of GABA estimation (Fig. 4) showed that oral treatment of compound 2f for 7 days increased the GABA level significantly (by 1.8-fold) compared to the control animals

Table 1 Preliminary anticonvulsant, minimal motor impairment and CNS depression studies of compounds (2aeh and 4aeh). Compound

C log P

i.p. injection in micea MES

2a 2b 2c 2d 2e 2f 2g 2h 4a 4b 4c 4d 4e 4f 4g 4h PHTc CBZc

4.12 3.57 4.29 3.25 3.98 3.44 2.97 4.28 4.60 4.06 4.78 3.74 4.47 3.92 3.45 4.76 e e

Porsolt’s swimming pool test scPTZ

NT

b

Mean average immobility

0.5 h

4h

0.5 h

4h

0.5 h

4h

Control (24 h before)

Post-treatment (60 min after)

300 e e 300 100 30 e 300 e e 300 300 300 e e 300 30 30

300 300 300 e 100 30 100 300 100 100 e e e 100 300 300 30 100

300 e e e 300 100 e e e e e e 300 300 e e e 100

300 e e e 300 100 300 e 300 300 e e e 300 300 e e 300

e e e e e e e e e 300 300 300 e e e 300 100 100

e e 300 300 e e e e e 300 300 300 e e e 300 100 300

120  6.38 127.5  5.22 117  5.13 109  5.33 113.3  5.42 118.5  5.18 115  4.81 125.6  6.20 112  4.87 124  5.03 109.1  5.04 121  5.26 131.5  5.96 104.1  4.66 110  5.16 111.8  5.03 X 112.1  5.85

134.5  5.30NS 160  5.91 162  5.77 166.1  6.27 130  5.46NS 135.6  5.73NS 168.3  6.33 146.6  5.62 157  6.50 147.3  5.57 179.1  6.02 154.3  5.78 180  6.53 177  5.62 172.3  5.78 158.1  6.10 X 179.5  5.53

a Doses of 30, 100 and 300 mg/kg were administered (n ¼ 6). The data indicates the minimum dose whereby bioactivity/neurotoxicity was demonstrated in half or more of the mice while dash indicates the absence of anticonvulsant activity/neurotoxicity at the maximum dose administered (300 mg/kg). b Each value represents the mean average immobility time in seconds  SEM of six mice, significantly different from the control at p < 0.05, and NS denotes not significant at p > 0.05 (Student’s t test). X indicates that the compound was not screened. c PHT, phenytoin; CBZ, carbamazepine.

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Table 2 Effect of selected benzothiazole derivatives on serum enzymatic activity in mice. Compounda

SGOT

SGPT

Alkaline phosphatase

Total albumin

Total protein

2a 2e 2f Controlb

38.20  1.62 35.86  1.40 34.63  1.70 34.08  1.51

28.65  1.60 30.51  1.21 29.36  1.75 27.83  1.37

58.73  1.60 60.41  2.41 57.80  2.09 56.11  2.00

1.63  0.022 1.61  0.024 1.58  0.017 1.56  0.021

5.20  0.26 6.12  0.17* 5.28  0.24 5.21  0.21

a b

Relative to control and data were analyzed by ANOVA followed by Student’s t test for n ¼ 6 at *p < 0.05. Control group were treated with 0.5% methyl cellulose for 15 days.

2.4. Structureeactivity relationship Benzothiazole scaffold was substituted with different functional groups having varied sizes and electronic attributes to assess their stereo-electronic effect on anticonvulsant activity. These substituents were also considered on the basis of lipophilic (p) and electronic (s) attributes as defined by Craig’s plot, like lipophilic electron-withdrawing groups (þs/þp) Cl, F, Br and NO2; hydrophilic electron-donating groups (s/p) OCH3; hydrophilic electron-withdrawing groups (þs/p) COCH3; and lipophilic electron-releasing groups (s/þp) CH3 [22]. The general structureeactivity relationship of these compounds showed that the derivatives having electron-releasing groups were more active than derivatives having electron-withdrawing groups. Among the electron-donating groups, hydrophilic methoxy derivatives were more active than the lipophilic methyl derivatives. On the other hand among the different isoelectronic halogens, the lipophilic chloro substituent at the 6th position of benzothiazole ring was more potent and less neurotoxic than the bromo, fluoro, and nitro derivatives. Also, it is interesting to note that the introduction of hydrophilic electron-withdrawing group such as acetyl group at the benzothiazole ring caused a significant improvement in anticonvulsant activity with abolition of neurotoxicity. 2.5. Molecular docking studies To gain further evidence regarding the GABAergic effect of compound 2f, a molecular docking study was carried out on gaminobutyric acid aminotransferase (GABA-AT) enzyme using Maestro 9.0 program (Schrodinger Inc. USA). GABA-AT is a validated target for AEDs and being a catabolic in nature, its selective inhibition raises GABA concentrations in brain [23]. GABA-AT used in the current study was prepared from the raw PDB structure 1OHV, which has 96% similarity with the human brain enzyme and all active site residues in the vicinity of cofactor have exact counterparts [24]. The best binding modes of vigabatrin, an irreversible GABA-AT inhibitor and compound 2f in the active site of GABA-AT are presented in Figs. 6 and 7. The selectivity of vigabatrin has been shown to result from the carboxylate and amino groups, which are essential for the mechanism-based

inhibition. It binds in a mode very similar to that of GABA, with the g-proton pointing toward the Lys 329 so that it can be successfully abstracted. Furthermore, it forms three hydrogen bonds (one with PLP cofactor and two with Arg 192 protein residue) in the active site. In contrast, compound 2f lacks zwitter ion but hydrogen bonds to the enzyme through its carbonyl oxygen and benzothiazolyl nitrogen atom with the guanidine group of Arg 192 residue i.e. at the same residue where the carboxylate moiety of the natural substrate GABA binds [25]. The amide group of 2f seems to have an important role in strong hydrogen bonding because the lone pair electrons on nitrogen of the amide are delocalized into the carbonyl group. Lipophilicity also appears to play crucial role in 2f inhibitory activity, as benzothiazole group is properly oriented to the more lipophilic area of GABA-AT binding site and formed CHep interaction with Phe 189. All these are corroborated well by the results of the docking calculations. The glide score value of vigabatrin was found to be 3.16, while that of 2f was 4.17, suggesting that molecule 2f interacts better. Therefore, the increased concentration of GABA in brain tissue may be attributed to the inhibition of enzyme GABA-AT. 2.6. Pharmacophore distance mapping A pharmacophore distance mapping provides a useful tool for designing anticonvulsant molecules and explanation of plausible interactions. Therefore, the present work involves the correlation of pharmacophoric pattern of different class of anticonvulsants with the prototype compound 2f. Comparison of pharmacophore distance showed that the compound 2f was in good conformity with the clinically available AEDs (Table 4). 3. Conclusion A series of N-(substitutedbenzothiazol-2-yl)amide derivatives were synthesized and tested for anticonvulsant activity using MES and scPTZ screens. N-(6-methoxybenzothiazol-2-yl)-4-oxo-4phenylbutanamide (2f) represents a valuable lead in the exploration of agents controlling both development of seizure and intoxication during epilepsy. In various toxicity studies, no overt effects were noted that would be considered to confound any findings of

Table 3 Phase II quantitative anticonvulsant evaluation in mice. Compound

ED50a MES

2f Phenytoin Carbamazepine Sod. valproate a b c d

40.96 9.5 8.8 272

PIc

TD50b scPTZ (36.69e45.23)d (8.1e10.4) (5.5e14.1) (247e338)

85.16 (83.32e87) >300 >100 149 (123e177)

347.60 65.5 71.6 426

(343.74e351.46) (52.5e72.9) (45.9e135) (369e450)

MES

scPTZ

8.4 6.9 8.1 1.6

4.0 <0.22 <0.22 2.9

Dose in milligrams per kilogram body mass (n ¼ 10). Minimal toxicity was determined by rotarod test 30 min after the test drug was administered. PI ¼ protective index (TD50/ED50). Data in parentheses are the 95% confidence limits. Probit analyses were done using Statplus 2007 professional 4.0.3 program by Finney method.

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Fig. 2. Control: Section of liver showing normal Portal Triad structures. PV ¼ portal vein, HA ¼ hepatic artery (400).

its efficacy. Compound 2f administration significantly increased the GABA level and also reduced the progression of ACR induced neurotoxicity. Their decreased MDA and LDH enzyme activity suggested that neuroprotective mechanisms may also be involved in observed anticonvulsant activity of the benzothiazole derivatives. However, further molecular and clinical studies are still required to ascertain its effectiveness and mechanism of action during epilepsy. 4. Experimental 4.1. Chemistry The melting points were determined in open capillary tubes in a Hicon melting point apparatus (Hicon, India) and are uncorrected. The elemental analyses (C, H, N) 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 FTIR spectrometer. 1H NMR spectra were recorded on a Brucker Model-300 NMR Spectrometer in CDCl3 and DMSO-d6 using Tetramethysilane (TMS) as the internal reference (chemical shifts in d ppm). 13C NMR spectra were also

Fig. 3. Compound 2f: Section of liver showing normal Portal Triad structures. PV ¼ portal vein, BD ¼ bile duct (400).

Fig. 4. Effects of some selected synthesized derivatives on GABA levels in mice brain tissues (mg/100 mg wet tissue). Each value represents the mean  SEM of six rats, significantly different from the control at *p < 0.05; **p < 0.01 (Student’s t test).

measured on Bruker-300 instrument (75 MHz) with complete proton decoupling. Mass spectra were recorded at Waters Synapt Mass spectrometer. The homogeneity of the compounds was checked by thin layer chromatography (TLC) on silica gel 60 F254coated plates (Merck, Germany) by using ethyl acetate/hexane (1:2) as solvent system. Iodine chamber and UV lamp were used for the visualization of TLC spots. 4.1.1. General procedure for the preparation of N-(substitutedbenzothiazol-2-yl)amide derivatives (2aeh and 4aeh) HOBt (0.01 mol) dissolved in 25 mL of dry DCM was added dropwise to a stirred mixture of EDCl (0.01 mol) and 4-oxo-4phenylbutanoic acid (1) (for the synthesis of compound 2aeh) or 2-benzoyl benzoic acid (3) (for the synthesis of compound 4aeh) (0.01 mol) in 100 mL of dry DCM. The resulting solution was stirred for 15 min and then a mixture of substituted 1,3-benzothiazol2-amine (0.01 mole) and triethylamine (0.01 mole) in dry DCM (25 mL) was added to this. The reaction mixture was further stirred for 5 h at room temperature. Water (15 mL) was added, and the organic layer was separated. The aqueous phase washed three times with 20 mL of DCM. The organic extracts were combined and

Fig. 5. Levels of LPO and LDH enzyme activity in ACR-intoxicated and treated mice brain. Each value represents the mean  SEM of six rats, significantly different from the control at **p < 0.01; NS, p > 0.05 (Student’s t test).

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4.1.3. N-(6-Fluorobenzothiazol-2-yl)-4-oxo-4-phenylbutanamide (2b) Yield 64%; m.p. 112e114  C; IR (KBr) ymax in cm1: 3336 (NeH str.), 1676 (C¼O), 1612 (C¼N). 1H NMR (CDCl3, 300 MHz) d ppm: 2.94 (t, 2H, J ¼ 6.4 Hz, CH2), 3.48 (t, 2H, J ¼ 6.4 Hz, CH2), 7.10e7.14 (m, 2H, 30 and 50 AreH), 7.22e7.25 (d, 1H, J ¼ 8.4 Hz, 5th AreH benzoth.), 7.54e7.59 (t, 1H, J ¼ 7.2, 6.2 Hz, 40 AreH), 7.58e7.61 (d, 1H, J ¼ 8.4 Hz, 4th AreH benzoth.), 7.86e7.88 (d, 2H, J ¼ 7.4 Hz, 20 and 60 AreH), 8.12 (s, 1H, 7th AreH benzoth.), 9.82 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 1, 329 (16), Mþ, 328 (100), 312 (8), 168 (15), 152 (12), 142 (6), 108 (10), 105 (8). Anal. Calcd. for C17H13FN2O2S: C, 62.18; H, 3.99; N, 8.53%. Found: C, 62.22; H, 4.02; N, 8.51%.

Fig. 6. Binding mode of vigabatrin into GABA-AT pocket (PDB code: 1OHV) showing three hydrogen bonds (yellow dotted lines) with PLP (1.46  A) and Arg 192 (1.79  A and 2.00  A).

washed three times with brine, dried over MgSO4, filtered, and evaporated under reduced pressure. The obtained products were purified by crystallization using ethyl acetate/hexane mixture (1:3) to give (6273% yield) of white crystals. 4.1.2. N-(6-Chlorobenzothiazol-2-yl)-4-oxo-4-phenylbutanamide (2a) Yield 68%; m.p. 166e168  C; IR (KBr) ymax in cm1: 3340 (NeH str.), 1672 (C¼O), 1604 (C¼N). 1H NMR (CDCl3, 300 MHz) d ppm: 2.98 (t, 2H, J ¼ 6.3 Hz, CH2), 3.45 (t, 2H, J ¼ 6.3 Hz, CH2), 7.02e7.06 (m, 2H, 30 and 50 AreH), 7.20e7.23 (d, 1H, J ¼ 8.4 Hz, 5th AreH benzoth.), 7.57e7.62 (t, 1H, J ¼ 7.2, 6.3 Hz, 40 AreH), 7.65e7.68 (d, 1H, J ¼ 8.7 Hz, 4th AreH benzoth.), 7.75 (s, 1H, 7th AreH benzoth.), 7.92e8.01 (d, 2H, J ¼ 7.2 Hz, 20 and 60 AreH), 9.66 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 2, 346 (32), Mþ, 344 (100), 328 (10), 183 (22), 168 (10), 158 (8), 124 (16), 105 (7). Anal. Calcd. for C17H13ClN2O2S: C, 59.21; H, 3.80; N, 8.12%. Found: C, 59.24; H, 3.82; N, 8.16%.

4.1.4. N-(6-Bromobenzothiazol-2-yl)-4-oxo-4-phenylbutanamide (2c) Yield 65%; m.p. 124e126  C; IR (KBr) ymax in cm1: 3316 (NeH str.), 1644 (C¼O), 1608 (C¼N). 1H NMR (CDCl3, 300 MHz) d ppm: 2.83 (t, 2H, J ¼ 6.3 Hz, CH2), 3.48 (t, 2H, J ¼ 6.3 Hz, CH2), 7.26e7.52 (m, 6H, 4, 5, 7th, 30 , 40 and 50 AreH and benzoth.), 8.00e8.03 (d, 2H, J ¼ 7.5 Hz, 20 and 60 AreH), 8.82 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 2, 391 (33), Mþ, 389 (100), 373 (12), 229 (20), 213 (8), 203 (10), 169 (7). Anal. Calcd. for C17H13BrN2O2S: C, 52.45; H, 3.37; N, 7.20%. Found: C, 52.47; H, 3.40; N, 7.18%. 4.1.5. N-(6-Nitrobenzothiazol-2-yl)-4-oxo-4-phenylbutanamide (2d) Yield 70%; m.p. 166e168  C; IR (KBr) ymax in cm1: 3320 (NeH str.), 1632 (C¼O), 1602 (C¼N). 1H NMR (DMSO-d6, 300 MHz) d ppm: 2.92 (t, 2H, J ¼ 6.3 Hz, CH2), 3.46 (t, 2H, J ¼ 6.3 Hz, CH2), 7.12e7.16 (d, 1H, J ¼ 8.6 Hz, 5th AreH benzoth.), 7.20e7.48 (m, 3H, 30 , 40 and 50 AreH), 7.60e7.64 (d, 1H, J ¼ 8.1 Hz, 4th AreH benzoth.), 7.82e7.85 (d, 2H, J ¼ 7.6 Hz, 20 and 60 AreH), 7.98 (s, 1H, 7th AreH benzoth.), 9.84 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ, 355 (100), Mþ þ 1, 356 (18), 339 (10) 310 (12), 150 (17), 134 (8), 124 (14), 90 (6), 105 (5). Anal. Calcd. for C17H13N3O4S: C, 57.46; H, 3.69; N, 11.82%. Found: C, 57.50; H, 3.71; N, 11.86%. 4.1.6. N-(6-Methylbenzothiazol-2-yl)-4-oxo-4-phenylbutanamide (2e) Yield 63%; m.p. 178e180  C; IR (KBr) ymax in cm1: 3286 (NeH str.), 1680 (C¼O), 1607 (C¼N). 1H NMR (CDCl3, 300 MHz) d ppm: 2.28 (s, 3H, CH3), 2.80 (t, 2H, J ¼ 6.2 Hz, CH2), 3.44 (t, 2H, J ¼ 6.2 Hz,

Table 4 Calculated distance ranges between the pharmacophoric elements.

-6 4.1

.7

O

A

.

N

A

D

3. 4-

5. 1

HBD 4.0-6.6 A O

Fig. 7. Binding mode of compound (2f) into GABA-AT pocket (PDB code: 1OHV) showing two hydrogen bonds (yellow dotted lines) with Arg 192 (1.88  A and 2.21  A).

OS . . .

AO

O .

N H

.

Compound

A-HBDa

A-Da

D-HBDa

Gabapentin Carbamazepine Phenytoin Remacemide Progabide 2f

4.02 5.11 4.97 5.06 6.65 4.30

4.14 5.22 4.61 6.74 4.49 4.20

4.45 4.63 3.44 4.37 5.12 2.95

a Distance calculated for 3D optimized structures using ACD/Chemsketch/3-D viewer 12.01 version program.

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CH2), 7.07e7.10 (d, 1H, J ¼ 7.8 Hz, 5th AreH benzoth.), 7.38e7.40 (m, 2H, 4 and 7th AreH benzoth.), 7.43e7.48 (t, 2H, J ¼ 7.2, 7.8 Hz, 30 , 50 AreH), 7.54e7.59 (t, 1H, J ¼ 7.5, 7.2 Hz, 40 AreH), 7.97e8.0 (d, 2H, J ¼ 7.5 Hz, 20 , 60 AreH), 8.77 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 1, 325 (19), Mþ, 324 (100), Mþ  1, 323 (10), 308 (12), 163 (16), 147 (7), 104 (12), 105 (6). Anal. Calcd. for C18H16N2O2S: C, 66.64; H, 4.97; N, 8.64%. Found: C, 66.68; H, 5.02; N, 8.60%. 4.1.7. N-(6-Methoxybenzothiazol-2-yl)-4-oxo-4-phenylbutanamide (2f) Yield 72%; m.p. 190e192  C; IR (KBr) ymax in cm1: 3265 (NeH), 1669 (C¼O), 1607 (C¼N); 1H NMR (CDCl3, 300 MHz) d ppm: 2.96 (t, 2H, J ¼ 6.3 Hz, CH2), 3.51(t, 2H, J ¼ 6.3 Hz, CH2), 3.85 (s, 3H, OCH3), 7.02e7.06 (dd, 1H, J ¼ 6.6, 2.4 Hz, 5th AreH benzoth.), 7.27e7.28 (d, 1H, J ¼ 2.7 Hz, 7th AreH benzoth.), 7.47e7.52 (t, 2H, J ¼ 7.5, 7.8 Hz, 30 , 50 AreH), 7.58e7.63 (t, 1H, J ¼ 7.2, 7.5 Hz, 40 AreH), 7.71e7.74 (d, 1H, J ¼ 9.0 Hz, 4th AreH benzoth.), 8.01e8.03 (d, 2H, J ¼ 7.5 Hz, 20 , 60 AreH), 10.39 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3, 75 MHz) d ppm: 28.42 (CH2), 33.41 (CH2), 55.86 (OCH3), 105.53, 113.87, 118.14, 128.08, 128.63, 130.00, 133.27, 136.51, 143.04, 155.74, 167.03, 178.32 (CONH), 198.21 (COPh); MS m/z (%): Mþ þ 2, 342 (5), Mþ þ 1, 341 (18), Mþ, 340 (48), Mþ  1, 339 (100), 324 (8), 268 (6), 180 (6). Anal. Calcd. for C18H16N2O3S: C, 63.51; H, 4.74; N, 8.23%. Found: C, 63.53; H, 4.72; N, 8.28%. 4.1.8. N-(6-Acetylbenzothiazol-2-yl)-4-oxo-4-phenylbutanamide (2g) Yield 73%; m.p. 212e214  C; IR (KBr) ymax in cm1: 3314 (NeH str.), 1660 (C¼O), 1600 (C¼N); 1H NMR (DMSO-d6, 300 MHz) d ppm: 2.50 (s, 3H, COCH3), 2.88 (t, 2H, J ¼ 6.2 Hz, CH2), 3.50(t, 2H, J ¼ 6.2 Hz, CH2), 7.12e7.15 (d, 1H, J ¼ 8.4 Hz, 5th AreH benzoth.), 7.42e7.58 (m, 3H, 30 , 40 and 50 AreH), 7.72e7.75 (d, 1H, J ¼ 8.4 Hz, 4th AreH benzoth.), 7.96e8.0 (d, 2H, J ¼ 7.6 Hz, 20 , 60 AreH), 8.02 (s, 1H, 7th AreH benzoth.), 9.64 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 1, 353 (20), Mþ, 352 (100), 337 (18), 336 (12), 310 (7), 150 (10), 134 (8), 90 (8), 105 (6). Anal. Calcd. for C19H16N2O3S: C, 64.76; H, 4.58; N, 7.95%. Found: C, 64.77; H, 4.59; N, 7.92%. 4.1.9. N-(5-Chloro-6-fluorobenzothiazol-2-yl)-4-oxo-4phenylbutanamide (2h) Yield 67%; m.p. 192e194  C; IR (KBr) ymax in cm1: 3312 (NeH str.), 1646 (C¼O), 1606 (C¼N). 1H NMR (CDCl3, 300 MHz) d ppm: 2.90 (t, 2H, J ¼ 6.1 Hz, CH2), 3.44 (t, 2H, J ¼ 6.1 Hz, CH2), 7.12e7.15 (m, 2H, 30 and 50 AreH), 7.46e7.50 (t, 1H, J ¼ 7.2, 7.2 Hz, 40 AreH), 7.54 (s, 1H, 4th AreH benzo.), 7.76e7.78 (d, 2H, J ¼ 7.5 Hz, 20 and 60 AreH), 8.02 (s, 1H, 7th AreH benzoth.), 9.64 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 2, 364 (37), Mþ þ 1, 363 (22), Mþ, 362 (100), 346 (10), 202 (14), 186 (8), 142 (6), 175 (10), 105 (5). Anal. Calcd. for C17H12ClFN2O2S: C, 56.28; H, 3.33; N, 7.72%. Found: C, 56.30; H, 3.30; N, 7.76%. 4.1.10. 2-Benzoyl-N-(6-chlorobenzothiazol-2-yl)benzamide (4a) Yield 62%; m.p. 220e212  C; IR (KBr) ymax in cm1: 3345 (NeH str.), 1674 (C¼O), 1603 (C¼N). 1H NMR (CDCl3, 300 MHz) d ppm: 7.41e7.43 (d, 1H, J ¼ 7.2 Hz, 5th AreH benzoth.), 7.47e7.76 (m, 10H, 7th benzoth. and AreH), 7.98e8.01 (d, 1H, J ¼ 7.8 Hz, 4th AreH benzoth.), 9.10 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 2, 394 (33), Mþ þ 1, 393 (24), Mþ, 392 (100), 315 (18), 183 (14), 168 (8), 158 (7), 124 (8), 105 (4). Anal. Calcd. for C21H13ClN2O2S: C, 64.20; H, 3.34; N, 7.13 %. Found: C, 64.22; H, 3.38; N, 7.10%. 4.1.11. 2-Benzoyl-N-(6-fluorobenzothiazol-2-yl)benzamide (4b) Yield 70%; m.p. 178e180  C; IR (KBr) ymax in cm1: 3329 (NeH str.), 1671 (C¼O), 1603 (C¼N). 1H NMR (CDCl3, 300 MHz)

d ppm:7.22e7.26 (d, 1H, J ¼ 8.0 Hz, 5th AreH benzoth.), 7.42e7.78 (m, 10H, 4th benzoth. and AreH), 7.94 (s, 1H, 7th AreH benzoth.), 9.42 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 1, 377 (24), Mþ, 376 (100), 299 (17), 168 (18), 152 (10), 108 (8), 142 (7), 105 (7). Anal. Calcd. for C21H13FN2O2S: C, 67.01; H, 3.48; N, 7.44%. Found: C, 67.04; H, 3.50; N, 7.47%. 4.1.12. 2-Benzoyl-N-(6-bromobenzothiazol-2-yl)benzamide (4c) Yield 65%; m.p. 202e204  C; IR (KBr) ymax in cm1: 3316 (NeH str.), 1640 (C¼O), 1608 (C¼N). 1H NMR (CDCl3, 300 MHz) d ppm: 7.12e7.16 (d, 1H, J ¼ 8.1 Hz, 5th AreH benzoth.), 7.36e7.80 (m, 10H, 4th benzoth. and AreH), 7.98 (s, 1H, 7th AreH benzoth.), 9.58 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 2, 439 (30), Mþ, 437 (100), 360 (18), 229 (15), 213 (7), 203 (10), 169 (10), 105 (8). Anal. Calcd. for C21H13BrN2O2S: C, 57.68; H, 3.00; N, 6.41%. Found: C, 57.65; H, 3.06; N, 6.43%. 4.1.13. 2-Benzoyl-N-(6-nitrobenzothiazol-2-yl)benzamide (4d) Yield 65%; m.p. 176e178  C; IR (KBr) ymax in cm1: 3320 (NeH str.), 1656 (C¼O), 1602 (C¼N); 1H NMR (CDCl3, 300 MHz) d ppm: 7.28e7.32 (d, 1H, J ¼ 8.2 Hz, 5th AreH benzoth.), 7.68e7.92 (m, 10H, 4th benzoth. and AreH), 8.10 (s, 1H, 7th AreH benzoth.), 10.02 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 1, 404 (23), Mþ, 403 (100), 358 (12), 281 (17), 150 (20), 134 (16), 124 (10), 90 (8). Anal. Calcd. for C21H13N3O4S: C, 62.52; H, 3.25; N, 10.42%. Found: C, 62.56; H, 3.26; N, 10.46%. 4.1.14. 2-Benzoyl-N-(6-methylbenzothiazol-2-yl)benzamide (4e) Yield 63%; m.p. 216e218  C; IR (KBr) ymax in cm1: 3316 (NeH str.), 1640 (C¼O), 1608 (C¼N). 1H NMR (DMSO-d6, 300 MHz) d ppm: 2.12 (s, 3H, CH3), 6.65e7.89 (m, 12H, AreH and benzoth.), 9.15 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 1, 373 (25), Mþ, 372 (100), Mþ  1, 371 (20), 294 (22), 163 (12), 147 (10), 105 (5), 104 (7). Anal. Calcd. for C22H16N2O2S: C, 70.95; H, 4.33; N, 7.52%. Found: C, 70.98; H, 4.36; N, 7.56%. 4.1.15. 2-Benzoyl-N-(6-methoxybenzothiazol-2-yl)benzamide (4f) Yield 65%; m.p. 172e174  C; IR (KBr) ymax in cm1: 3306 (NeH), 1662 (C¼O), 1600 (C¼N). 1H NMR (CDCl3, 300 MHz) d ppm: 3.83 (s, 3H, OCH3), 7.47e8.14 (m, 12H, AreH and benzoth.), 10.76 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3, 100 MHz) d ppm: 55.86 (OCH3), 105.51, 114.03, 117.66, 127.32, 128.37, 129.38, 129.44, 130.35, 130.81, 132.12, 132.82, 137.49, 142.17, 155.89, 167.11 (CONH), 170.40 (C¼N), 196.78 (COPh); MS m/z (%): Mþ þ 1, 389 (25), Mþ, 388 (100), Mþ  1, 387 (8), 373 (12), 311 (15), 180 (10). Anal. Calcd. for C22H16N2O3S: C, 68.02; H, 4.15; N, 7.21%. Found: C, 68.07; H, 4.18; N, 7.26%. 4.1.16. N-(6-Acetylbenzothiazol-2-yl)-2-benzoylbenzamide (4g) Yield 68%; m.p. 226e228  C; IR (KBr) ymax in cm1: 3312 (NeH str.), 1660 (C¼O), 1605 (C¼N); 1H NMR (DMSO-d6, 300 MHz) d ppm: 3.0 (s, 3H, COCH3), 7.02e8.17 (m, 12H, AreH and benzoth.), 9.83 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 1, 401 (26), Mþ, 400 (100), 386 (22), 358 (10), 281 (18), 150 (17), 134 (7), 124 (8), 105 (6). Anal. Calcd. for C23H16N2O3S: C, 68.98; H, 4.03; N, 7.00%. Found: C, 69.00; H, 4.08; N, 7.02%. 4.1.17. 2-Benzoyl-N-(5-chloro-6-fluorobenzothiazol-2-yl) benzamide (4h) Yield 62%; m.p. 162e164  C; IR (KBr) ymax in cm1: 3316 (NeH), 1670 (C¼O), 1612 (C¼N); 1H NMR (CDCl3, 300 MHz) d ppm: 7.42 (s, 1H, 4th benzoth.), 7.54e7.80 (m, 9H, AreH and benzoth. ), 7.98 (s, 1H, 7th AreH benzoth.), 9.64 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 2, 412 (39), Mþ þ 1, 411 (24), Mþ, 410 (100), 333 (16), 202 (20), 186 (8), 174 (8), 142 (12), 105 (8). Anal. Calcd. for

Mohd.Z. Hassan et al. / European Journal of Medicinal Chemistry 58 (2012) 206e213

C21H12ClFN2O2S: C, 61.39; H, 2.94; N, 6.82%. Found: C, 61.42; H, 2.98; N, 6.80%. 4.2. Pharmacology All the pharmacological experiments were conducted in compliance with ethical principles after Institutional Animal Ethics Committee (IAEC) approval. The investigations were conducted on albino mice of either sex (25e30 g; animals were obtained from central animal house facility, Hamdard University, New Delhi-62. Registration no. 173/CPCSEA and date of registration is 28 January 2000). The animals were housed under standard conditions and allowed free access to standard pellet diet and water (ad libitum). The preliminary anticonvulsant (phase I) screening of the test compounds was assessed by following the standard protocols of Antiepileptic Drug Development (ADD) program by NINDS, US [26] using two widely used models namely, maximal electroshock seizure (MES) [27], subcutaneous pentylenetetrazol (scPTZ) test [28] and minimal neurological toxicity was evaluated by rotarod test [29]. The most potent compound was also subjected to phase II quantitative determination of ED50, TD50, PI and probit calculations were done by means of computer program BioStat 2009 using Finney’s method [30]. Anticonvulsant activity of the title compounds were compared using data of standard AEDs reported in the literature [31,32]. Behavioral depression was measured by evaluating the immobility time of the mice using Porsolt’s swim pool test [33]. Hepatotoxicity studies of the compounds were also carried out using procedures described elsewhere [34e37]. Most active anticonvulsant compounds in phase I screening were also subjected to neurochemical estimation of GABAs levels in the mouse whole brain [38]. Neuroprotective effects of some selected compounds were also evaluated against the ACR induced neurotoxicity [39e41]. Acknowledgment The authors are thankful to the Head of Pharmaceutical Chemistry Department, Hamdard University, New Delhi for providing necessary research facilities. Also we thank Dr. A. K. Tiwary, In charge animal house, Hamdard University for providing experimental animals, and Dr. A. Mukherjee, MD, Department of Pathology, All India Institute of Medical Sciences (AIIMS), New Delhi, for carrying out histopathological studies. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2012.10.002 References [1] The Global Campaign Against Epilepsy, Information Pack for the launch of the Global Campaign’s Second Phase, 12e13 February 2001, World Health Organization, Geneva, 2000. [2] G. Saravanan, V. Alagarsamy, C.R. Prakash, Bioorg. Med. Chem. Lett. 22 (2012) 3072e3078. [3] S. Shorvon, E. Perucca, J. EngelJr (Eds.), The Treatment of Epilepsy, third ed., Wiley-Blackwell, Oxford, 2009, p. 123.

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