Synthesis of benzimidazole-linked-1,3,4-oxadiazole carboxamides as GSK-3β inhibitors with in vivo antidepressant activity

Bioorganic Chemistry 77 (2018) 393–401

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Synthesis of benzimidazole-linked-1,3,4-oxadiazole carboxamides as GSK-3b inhibitors with in vivo antidepressant activity Mushtaq A. Tantray a, Imran Khan a, Hinna Hamid a,⇑, Mohammad Sarwar Alam a, Abhijeet Dhulap b, Abul Kalam c a b c

Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi 110062, India CSIR – Unit for Research and Development of Information Products (URDIP), Pune 411038, India Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi 110062, India

a r t i c l e

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Article history: Received 2 July 2017 Revised 25 January 2018 Accepted 30 January 2018 Available online 1 February 2018 Keywords: Benzimidazole Oxadiazole Anti-depressant Forced swim test Tail suspension test

a b s t r a c t Recent findings of potential implications of glycogen synthase kinase-3b (GSK-3b) dysfunction in psychiatric disorders like depression, have increased focus for development of GSK-3b inhibitors with possible anti-depressant activity. Keeping this in view, we synthesized a series of benzimidazolelinked-1,3,4-oxadiazole carboxamides and evaluated them for in vitro GSK-3b inhibition. Active compounds were investigated for in vivo antidepressant activity in Wistar rats. Docking studies of active compounds have also been performed. Among nineteen compounds synthesized, compounds 7a, 7r, 7j, and 7d exhibited significant potency against GSK-3b in sub-micromolar range with IC50 values of 0.13 lM, 0.14 lM, 0.20 lM, 0.22 lM respectively and significantly reduced immobility time (antidepressant-like activity) in rats compared to control group. Docking study showed key interactions of these compounds with GSK-3b. These compounds may thus serve as valuable candidates for subsequent development of effective drugs against depression and related disorders. Ó 2018 Published by Elsevier Inc.

1. Introduction Glycogen synthase kinase-3 (GSK-3), existing in two major isoforms as GSK-3a and GSK-3b, is a ubiquitously expressed, constitutively active proline-directed serine-threonine kinase found in eukaryotes. Both isoforms are structurally similar, but display non-identical biochemical functions. GSK-3 regulates a broad range of substrates encompassing important metabolic, signaling and structural proteins and transcription factors [1]. These substrates enable GSK-3 as a key convergence point of a multitude of intra-cellular signaling pathways, thereby modulating a broad spectrum of physiological processes such as metabolism, signaling pathways, apoptosis/cell survival, and development [2,3]. Aberrant regulation of GSK-3 has been implicated in the development of severe mental disorders including mood disorders, depression, and bipolar disorder [4,5]. Major depressive disorder is estimated to affect around 16 per cent of the world population at any point in their lives [6]. The potential relationship between impaired inhibitory GSK-3 regulation and depression has been established

⇑ Corresponding author at: Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India. E-mail address: [email protected] (H. Hamid). https://doi.org/10.1016/j.bioorg.2018.01.040 0045-2068/Ó 2018 Published by Elsevier Inc.

by multiple pharmacological and molecular approaches [7–9]. Lithium, a well-known GSK-3b inhibitor, is used as a mood stabilizer in bipolar disorder as well as adjunct therapy for depression and schizophrenia from nearly 50 years exerts its therapeutic action by inhibiting GSK-3b directly as well as indirectly through Akt/b-arrestin/protein phosphatase 2A complex [10–12]. The small molecule GSK-3b inhibitors including, AR-A014418 [13], thiadiazolidinone NP031115 [14], VP2.51 [9], and substrate-competitive peptide inhibitor L803-mts [15], have been shown to exhibit anti-depressive effects in various animal models. Genetic inhibition of GSK-3b has been found to replicate the behavioral effects of these drugs in rodents [16]. Moreover, classical antidepressants, as well as atypical antipsychotics have been found to involve inhibition of GSK-3b, in their mechanism of action [17,18]. Consequently, given the potential role of GSK-3b in depression, there is a great therapeutic potential for the development of GSK-3 inhibitors for the effective treatment of depression and associated mental disorders. In continuation of our efforts to develop new GSK-3b inhibitors with manifold therapeutic applications, particularly depression [3,19–24], we focused in the current study on benzimidazole linked 1,3,4-oxadiazole carboxamides. Since last few years, oxadiazole and benzimidazole derivatives, well known representative

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compounds of pharmaceutical importance, have generated a remarkable interest as potent GSK-3b inhibitors [22,25–33]. There has been an increasing trend in the medicinal chemistry arena of combining scaffolds of different bioactive classes to generate hybrid classes with an aim to modify the efficacy [34]. Suitable structural combinations of benzimidazole and oxadiazole cores with various pharmacophores in a single molecular framework have resulted in improved GSK-3b inhibitory potential and alleviation of associated disorders [26]. We therefore thought it worthwhile to synthesize benzimidazole linked 1,3,4-oxadiazole carboxamides and screen them for GSK-3b inhibition potential. A series of nineteen compounds has been synthesized and evaluated for in vitro GSK-3b inhibition. The compounds exhibiting pronounced GSK-3b inhibitory activity have been further investigated for antidepressant activity by the widely accepted forced swim test (FST) and tail suspension test (TST) models. Docking studies of the active compounds has also been carried out against GSK-3b site to understand the possible modes of molecular interactions. 2. Materials and methods 2.1. General The chemicals and reagents used in this study were obtained from Sigma Aldrich, Merck (India) and Spectrochem. Melting points have been measured using Electro-thermal IA 9100 apparatus (Shimadzu, Japan) and are uncorrected. IR spectra have been recorded on a Bruker spectrophotometer (USA), NMR spectra have been determined on a Bruker (300 and 400 MHz) spectrometer in CDCl3/DMSO-d6 and the chemical shifts expressed as ppm against TMS as internal reference. Mass spectra have been recorded on 70 eV (EI Ms-QP 1000EX, Shimadzu, Japan) or Waters UPLC-TQD (ESI-MS). Elemental analysis was performed using Elemental Vario EL III elemental analyzer and the data is reported in % standard. All the compounds synthesized in this study are novel. 2.2. Chemistry 2.2.1. Procedure for the synthesis of intermediates 2–5 The synthesis of intermediates 2–5 has been done as per the procedure reported in [22]. 2.2.2. Procedure for the synthesis of 2-((5-((2-(trifluoromethyl)-1Hbenzo[d]imidazol-1-yl)methyl)-1,3,4-oxadiazol-2-yl)thio)acetic acid (6) The oxadiazole-2-thiol intermediate 5 was added to a mixture of potassium carbonate in dry acetone and heated to reflux with stirring. After 30 min, bromoacetic acid was added to the reaction mixture and the heating was continued with stirring for 4 h. The reaction mixture was filtered and the filtrate concentrated, cooled and poured on ice-cold water. The solid obtained was filtered and air-dried. 2.2.3. Procedure for the synthesis of benzimidazole substituted oxadiazole carboxamides (7a-7s) Intermediate 5 was dissolved in dry DMF. EDC.HCl and HOBT were added and stirring was continued for 30 min. at room temperature followed by addition of appropriate anilines. The reaction mixture was allowed to stir for 10–12 h with regular monitoring by TLC. After completion of the reaction, the reaction mixture was poured onto crushed ice and the solid obtained was filtered and air dried. The compounds were then crystallized in ethyl acetate to afford purified target compounds (7a-7s). 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-phenylacetamide (7a) Colorless powder; yield: 73%; m. p. 178–180 °C; IR (KBr): m (cm 1) 3374, 3038, 2917, 1674, 1596, 1549, 1473, 1257, 1197,

1145; 1H NMR (400 MHz, DMSO-d6): d 4.26 (s, 2H), 6.04 (s, 2H), 7.05–7.09 (m, 1H), 7.29–7.33 (m, 2H), 7.41–7.45 (m, 1H), 7.49– 7.54 (m, 2H), 7.82–7.88 (m, 2H), 8.31 (s, 1H), 10.37 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 36.81, 111.80, 118.70 (q, J = 270 H z), 119.08, 120.98, 123.61, 124.11, 125.92, 128.80, 135.32, 138.57, 139.31 (q, J = 38.0 Hz), 140.23, 162.44, 164.42, 164.59; LC-MS: 434 (M + 1)+; Anal. Calcd. for C19H14F3N5O2S: C, 52.65; H, 3.26; F, 13.15; N, 16.16; O, 7.38; S, 7.40%; Found: C, 52.59; H, 3.27; N, 16.19; S, 7.41%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(4-ethylphenyl)acetamide (7b) Colorless powder; yield: 78%; m. p. 183–185 °C; IR (KBr): m (cm 1) 3374, 3035, 2925, 1677, 1600, 1545, 1517, 1476, 1247, 1198, 1145; 1H NMR (300 MHz, DMSO-d6): d 1.15 (t, 3H, J = 7.5 H z), 2.51–2.56 (m, 2H, obscured by DMSO peak), 4.24 (s, 2H), 6.03 (s, 2H), 7.14 (d, 2H, J = 8.1 Hz), 7.42–7.55 (m, 4H), 7.81–7.89 (m, 2H), 10.28 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 15.81, 28.22, 37.18, 111.19, 118.87 (q, J = 270 Hz), 119.83, 121.50, 124.38, 124.12, 126.23, 128.10, 135.27, 136.08, 139.31 (q, J = 39.0 Hz), 140.09, 140.79, 161.69, 164.35, 165.72; ESI-MS: 462 (M + 1)+; Anal. Calcd. for C21H18F3N5O2S: C, 54.66; H, 3.93; F, 12.35; N, 15.18; O, 6.93; S, 6.95%; Found: C, 54.70; H, 3.92; N, 18.84; S, 6.92%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(2-methoxyphenyl)acetamide (7c) Colorless powder; yield: 81%; m. p. 129–131 °C; IR (KBr): m (cm 1) 3392, 3002, 2928, 1683, 1600, 1512, 1466, 1256, 1163, 1121; 1H NMR (400 MHz, DMSO-d6): d 3.80 (s, 3H), 4.31 (s, 2H), 6.03 (s, 2H), 6.87–6.89 (m, 1H), 7.03–7.11 (m, 2H), 7.41–7.45 (m, 1H), 7.49–7.53 (m, 1H), 7.82–7.90 (m, 3H), 9.62 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 36.59, 55.67, 111.17, 111.80, 118.71(q, J = 270.0 Hz), 120.23, 120.98, 121.41, 124.11, 124.66, 125.92, 126.81, 135.33, 139.31 (q, J = 39.0 Hz), 140.23, 149.33, 162.47, 164.61, 164.78; ESI-MS: 464 (M + 1)+; Anal. Calcd. for C20H16F3N5O3S: C, 51.83; H, 3.48; F, 12.30; N, 15.11; O, 10.36; S, 6.92%; Found: C, 51.88; H, 3.48; N, 21.35; S, 6.90%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(4-methoxyphenyl)acetamide (7d) Colorless powder; yield: 78%; m. p. 180–182 °C; IR (KBr): m (cm 1) 3374, 3018, 2947, 1642, 1537, 1472, 1244, 1193, 1162; 1 H NMR (400 MHz, DMSO-d6): d 3.64 (s, 3H), 4.15 (s, 2H), 5.96 (s, 2H), 6.81 (d, 2H, J = 8.8 Hz), 7.35–7.38 (m, 3H), 7.42–7.46 (m, 1H), 7.75–7.81 (m, 2H), 10.16 (s, 1H); 13C NMR (100 MHz, DMSOd6): d 36.72, 55.10, 111.77, 113.86, 118.70 (q, J = 270.0 Hz), 120.64, 120.97, 124.08, 125.89, 131.69, 135.31, 139.32 (q, J = 38. 0 Hz), 140.24, 155.43, 162.38, 163.86, 164.63; ESI-MS: 464 (M + 1)+; Anal. Calcd. for C20H16F3N5O3S: C, 51.83; H, 3.48; F, 12.30; N, 15.11; O, 10.36; S, 6.92%; Found: C, 51.81; H, 3.46; N, 15.09; S, 6.92%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(4-chlorophenyl)acetamide (7e) Creamish white powder; yield: 75%; m. p. 154–156 °C; IR (KBr): m (cm 1) 3302, 3035, 2925, 1670, 1607, 1522, 1456, 1244, 1196, 1137; 1H NMR (400 MHz, DMSO-d6): d 4.20 (s, 2H), 5.99 (s, 2H), 7.35 (d, 2H, J = 8.8 Hz), 7.41–7.44 (m, 1H), 7.48–7.53 (m, 3H), 7.78–7.86 (m, 2H), 10.52 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 36.71, 111.78, 118.69 (q, J = 270.0 Hz), 120.64, 120.98, 124.12, 125.92, 127.19, 128.71, 135.30, 137.51, 139.30 (q, J = 38.0 Hz), 140.22, 162.45, 164.53, 164.63; ESI-MS: 468 (M + 1)+; Anal. Calcd. for C19H13ClF3N5O2S: C, 48.78; H, 2.80; Cl, 7.58; F, 12.18; N, 14.97; O, 6.84; S, 6.85%; Found: C, 48.83; H, 2.77; N, 14.94; S, 6.86%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(4-tert-butylphenyl)acetamide (7f) White powder; yield: 71%; m. p. 160–162 °C; IR (KBr): m (cm 1) 3286, 3127, 2964, 1689, 1600, 1528, 1469, 1271, 1185, 1139; 1H NMR (400 MHz, DMSO-d6): d 1.25 (s, 9H), 4.24 (s, 2H), 6.04 (s, 2H), 7.32 (d, 2H, J = 8.4 Hz), 7.43–7.53 (m, 4H), 7.82–7.88 (m,

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2H), 10.30 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 31.12, 33.99, 36.77, 111.79, 118.71 (q, J = 270.0 Hz), 118.89, 120.98, 124.11, 125.31, 125.39, 125.93, 135.33, 136.00, 139.31 (q, J = 39.0 Hz), 140.23, 145.96, 162.44, 164.19, 164.59; ESI-MS: 490 (M + 1)+; Anal. Calcd. for C23H22F3N5O2S: C, 56.43; H, 4.53; F, 11.64; N, 14.31; O, 6.54; S, 6.55%; Found: C, 56.49; H, 4.54; N, 14.27; S, 6.52%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-1-morpholinoethanone (7g) White powder; yield: 70%; m. p. 158–160 °C; IR (KBr): m (cm 1) 3027, 2964, 1678, 1610, 1537, 1439, 1268, 1169, 1110; 1H NMR (400 MHz, DMSO-d6): d 3.42–3.62 (m, 8H), 4.44 (s, 2H), 6.04 (s, 2H), 7.43–7.55 (m, 2H), 7.84–7.89 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 36.49, 45.66, 65.80, 111.82, 118.72 (q, J = 270.0 Hz), 121.00, 124.17, 125.93, 135.33, 139.31 (q, J = 38.0 Hz), 140.24, 162.16, 164.50, 164.70; ESI-MS: 428 (M + 1)+; Anal. Calcd. for C17H16F3N5O3S: C, 47.77; H, 3.77; F, 13.34; N, 16.39; O, 11.23; S, 7.50%; Found: C, 47.70; H, 3.79; N, 16.41; S, 7.51%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-1-(4-methylpiperazin-1-yl)ethanone (7h) White powder; yield: 71%; m. p. 138–140 °C; IR (KBr): m (cm 1) 3027, 2925, 1616, 1532, 1456, 1275, 1192, 1096; 1H NMR (400 MHz, DMSO-d6): d 2.09 (s, 3H), 2.18–2.51 (m, 8H), 4.43 (s, 2H), 6.04 (s, 2H), 7.43–7.55 (m, 2H), 7.84–7.89 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 36.49, 41.99, 45.66, 59.15, 111.81, 118.82 (q, J = 270.0 Hz), 120.98, 124.12, 126.85, 134.99, 140.07 (q, J = 38. 0 Hz), 140.24, 162.79, 164.61, 164.78; ESI-MS: 441 (M + 1)+; Anal. Calcd. for C18H19F3N6O2S: C, 49.09; H, 4.35; F, 12.94; N, 19.08; O, 7.27; S, 7.28%; Found: C, 49.14; H, 4.32; N, 19.04; S, 7.27%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(2-chlorophenyl)acetamide (7i) Creamish white powder; yield: 77%; m. p. 204–206 °C; IR (KBr): m (cm 1) 3275, 3072, 2936, 1661, 1585, 1530, 1469, 1268, 1191, 1120; 1H NMR (400 MHz, DMSO-d6): d 4.14 (s, 2H), 5.87 (s, 2H), 7.05–7.20 (m, 2H), 7.30–7.42 (m, 3H), 7.64–7.73 (m, 3H), 10.21 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 36.23, 111.80, 118.70 (q, J = 270.0 Hz), 120.98, 124.11, 125.68, 125.91, 126.12, 126.53, 127.44, 129.51, 134.35, 135.32, 139.31 (q, J = 38.0 Hz), 140.23, 162.47, 164.46, 165.18; ESI-MS: 468 (M + 1)+; Anal. Calcd. for C19H13ClF3N5O2S: C, 48.78; H, 2.80; Cl, 7.58; F, 12.18; N, 14.97; O, 6.84; S, 6.85%; Found: C, 48.81; H, 2.81; N, 14.99; S, 6.85%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(4-fluorophenyl)acetamide (7j) Creamish white powder; yield: 79%; m. p. 180–182 °C; IR (KBr): m (cm 1) 3288, 3005, 2924, 1670, 1598, 1514, 1469, 1259, 1190, 1117; 1H NMR (400 MHz, DMSO-d6): d 4.63 (s, 2H), 5.27 (s, 2H), 7.20–7.32 (m, 2H), 7.38–7.40 (m, 4H), 7.44–7.53 (m, 1H), 7.79– 7.83 (m, 1H), 10.30 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 33.10, 111.77, 115.89 (d, J = 22.0 Hz), 117.56, 118.90 (q, J = 269.0 Hz), 120.65, 123.54, 125.27, 130.41 (d, J = 9.0 Hz), 130.92, 136.19, 139.78 (q, J = 39.0 Hz), 140.19, 160.71 (d, J = 264.0 Hz), 167.15, 170.68, 171.12; ESI-MS: 452 (M + 1)+; Anal. Calcd. for C19H13F4N5O2S: C, 50.55; H, 2.90; F, 16.84; N, 15.51; O, 7.09; S, 7.10%; Found: C, 50.58; H, 2.91; N, 15.53; S, 7.14%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(2-fluorophenyl)acetamide (7k) Creamish white powder; yield: 78%; m. p. 228–230 °C; IR (KBr): m (cm 1) 3294, 3008, 2920, 1679, 1606, 1544, 1469, 1256, 1194, 1142; 1H NMR (400 MHz, DMSO-d6): d 4.32 (s, 2H), 6.04 (s, 2H), 7.13–7.19 (m, 2H), 7.23–7.28 (m, 1H), 7.41–7.45 (m, 1H), 7.49– 7.53 (m, 1H), 7.82–7.88 (m, 3H), 10.18 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 36.34, 111.79, 115.50 (d, J = 19.0 Hz), 118.70 (q, J = 269.0 Hz), 120.98, 123.69, 124.11, 124.37, 125.53 (d, J = 9.0 Hz), 125.63 (d, J = 14.0 Hz), 125.92, 135.31, 139.31 (q, J = 38.0 Hz), 140.23, 153.32 (d, J = 244 Hz), 162.46, 164.50, 166.11; ESI-MS:

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452 (M + 1)+; Anal. Calcd. for C19H13F4N5O2S: C, 50.55; H, 2.90; F, 16.84; N, 15.51; O, 7.09; S, 7.10%; Found: C, 50.57; H, 2.92; N, 15.49; S, 7.16%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(3,4-dimethylphenyl)acetamide (7l) White powder; yield: 73%; m. p. 148–150 °C; IR (KBr): m (cm 1) 3278, 3129, 2931, 1690, 1611, 1544, 1469, 1270, 1185, 1136; 1H NMR (400 MHz, DMSO-d6): d 2.15 (s, 3H), 2.17 (s, 3H), 4.23 (s, 2H), 6.03 (s, 2H), 7.05 (d, 1H, J = 8.0 Hz), 7.23–7.25 (m, 1H), 7.30 (bs, 1H), 7.41–7.45 (m, 1H), 7.49–7.53 (m, 1H), 7.82–7.88 (m, 3H), 10.21 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 18.74, 19.56, 36.80, 111.80, 116.64, 118.71 (q, J = 270.0 Hz), 120.30, 120.98, 124.12, 125.93, 129.60, 131.40, 135.33, 136.30, 136.40, 139.31 (q, J = 38.0 Hz), 140.23, 162.42, 164.08, 164.62; ESI-MS: 462 (M + 1)+; Anal. Calcd. for C21H18F3N5O2S: C, 54.66; H, 3.93; F, 12.35; N, 15.18; O, 6.93; S, 6.95%; Found: C, 54.62; H, 3.92; N, 15.15; S, 6.96%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(2-bromophenyl)acetamide (7m) White powder; yield: 83%; m. p. 158–160 °C; IR (KBr): m (cm 1) 3280, 3027, 2924, 1660, 1583, 1529, 1467, 1270, 1190, 1120; 1H NMR (400 MHz, DMSO-d6): d 4.23 (s, 2H), 5.98 (s, 2H), 7.07–7.10 (m, 1H), 7.28–7.32 (m, 1H), 7.35–7.39 (m, 1H), 7.43–7.49 (m, 2H), 7.58–7.60 (m, 1H), 7.76–7.82 (m, 2H), 9.83 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 36.15, 111.80, 117.42, 118.70 (q, J = 270. 0 Hz), 120.99, 124.14, 125.94, 126.65, 127.29, 128.04, 132.71, 135.31, 135.64, 139.31 (q, J = 38.0 Hz), 140.23, 162.48, 164.42, 165.09; ESI-MS: 514 (M + 1)+; Anal. Calcd. for C19H13BrF3N5O2S: C, 44.54; H, 2.56; Br, 15.60; F, 11.13; N, 13.67; O, 6.25; S, 6.26%; Found: C, 44.57; H, 2.58; N, 13.69; S, 6.25%. Ethyl 4-(2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl) methyl)-1,3,4-oxadiazol-2-ylthio)acetamido)benzoate (7n) White powder; yield: 75%; m. p. 160–162 °C; IR (KBr): m (cm 1) 3282, 2990, 2916, 1712, 1676, 1593, 1537, 1468, 1261, 1194, 1143, 1094; 1H NMR (400 MHz, DMSO-d6): d 1.28 (t, 3H, J = 7.2 Hz), 4.24 (q, 2H, J = 7.2 Hz), 4.52 (s, 2H), 5.86 (s, 2H), 7.32–7.42 (m, 2H), 7.52 (d, 2H, J = 8.8 Hz), 7.63–7.72 (m, 2H), 7.86 (d, 2H, J = 8.8 Hz), 10.30 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 13.95, 36.77, 60.16, 111.91, 118.33, 118.44 (q, J = 269.0 Hz), 120.93, 123.85, 124.92, 125.68, 129.96, 134.87, 139.37 (q, J = 38.0 Hz), 140.28, 142.35, 161.47, 164.60, 164.90, 165.26; ESI-MS: 506 (M + 1)+; Anal. Calcd. for C22H18F3N5O4S: C, 52.28; H, 3.59; F, 11.28; N, 13.86; O, 12.66; S, 6.34%; Found: C, 52.32; H, 3.60; N, 13.84; S, 6.34%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(4-ethoxyphenyl)acetamide (7o) White powder; yield: 74%; m. p. 184–186 °C; IR (KBr): m (cm 1) 3233, 3035, 2920, 1669, 1598, 1511, 1470, 1244, 1194, 1144, 1120; 1 H NMR (400 MHz, DMSO-d6): d 1.32 (t, 3H, J = 9.2 Hz), 3.92 (q, 2H, J = 9.2 Hz), 4.04 (s, 2H), 5.63 (s, 2H), 7.14–7.19 (m, 2H), 7.28–7.33 (m, 2H), 7.36–7.44 (m, 2H), 7.50–7.53 (m, 1H), 7.83–7.86 (m, 1H), 8.57 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 14.54, 36.71, 63.04, 111.78, 118.71 (q, J = 269.0 Hz), 120.98, 123.53, 125.27, 127.10, 129.26, 131.58, 135.32, 139.32 (q, J = 38.0 Hz), 140.19, 154.70, 162.41, 163.85, 164.62; ESI-MS: 478 (M + 1)+; Anal. Calcd. for C21H18F3N5O3S: C, 52.83; H, 3.80; F, 11.94; N, 14.67; O, 10.05; S, 6.72%; Found: C, 52.79; H, 3.81; N, 14.64; S, 6.71%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(4-bromophenyl)acetamide (7p) White powder; yield: 83%; m. p. 192–194 °C; IR (KBr): m (cm 1) 3295, 3035, 2964, 1649, 1588, 1530, 1461, 1272, 1194, 1126; 1H NMR (400 MHz, DMSO-d6): d 4.28 (s, 2H), 6.05 (s, 2H), 7.42–7.45 (m, 1H), 7.49–7.54 (m, 5H), 7.83–7.89 (m, 2H), 10.53 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 36.78, 111.79, 115.24, 118.71 (q, J = 270.0 Hz), 121.01, 124.10, 125.91, 131.62, 135.31, 137.93, 139.31 (q, J = 38.0 Hz), 140.23, 162.46, 164.54, 164.65; ESI-MS: 512

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(M + 1)+; Anal. Calcd. for C19H13BrF3N5O2S: C, 44.54; H, 2.56; Br, 15.60; F, 11.13; N, 13.67; O, 6.25; S, 6.26%; Found: C, 44.51; H, 2.57; N, 13.66; S, 6.28%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (7q) Grayish black powder; yield: 79%; m. p. 158–160 °C; IR (KBr): m (cm 1) 3305, 3002, 2969, 1683, 1600, 1512, 1466, 1256, 1208, 1163, 1121, 1043; 1H NMR (400 MHz, DMSO-d6): d 3.72 (s, 6H), 4.24 (s, 2H); 6.05 (s, 2H); 6.91 (d, 1H, J = 8.4 Hz), 7.05 (d, 1H, J = 8 .4 Hz), 7.26 (s, 1H), 7.41–7.54 (m, 2H), 7.83–7.89 (m, 2H), 10.23 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 37.27, 55.82, 56.19, 111.63, 112.26, 112.59, 119.22 (q, J = 270.0 Hz), 121.48, 124.60, 126.41, 132.67, 135.82, 139.10, 139.84 (q, J = 38.0 Hz), 140.76, 145.64, 149.08, 162.92, 164.43, 165.12; ESI-MS: 494 (M + 1)+; Anal. Calcd. for C21H18F3N5O4S: C, 51.11; H, 3.68; F, 11.55; N, 14.19; O, 12.97; S, 6.50%; Found: C, 51.18; H, 3.65; N, 14.21; S, 6.49%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(3-(trifluoromethyl)phenyl)acetamide (7r) White powder; yield: 81%; m. p. 168–170 °C; IR (KBr): m (cm 1) 3197, 3019, 2923, 1681, 1611, 1521, 1478, 1263, 1195, 1136; 1H NMR (400 MHz, DMSO-d6): d 4.28 (s, 2H), 6.03 (s, 2H), 7.39–7.43 (m, 2H), 7.47–7.51 (m, 1H), 7.54–7.58 (m, 1H), 7.69 (d, 1H, J = 8. 4 Hz), 7.81–7.86 (m, 2H), 8.02 (s, 1H), 10.71 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 37.18, 112.25, 115.65, 119.19 (q, J = 270. 0 Hz), 120.50, 121.46, 122.68, 123.17, 126.38, 129.81, 130.23, 130.57, 135.81, 139.79, 139.82 (q, J = 38.0 Hz), 140.74, 162.97, 164.97, 165.62; ESI-MS: 502 (M + 1)+; Anal. Calcd. for C20H13F6N5O2S: C, 47.91; H, 2.61; F, 22.73; N, 13.97; O, 6.38; S, 6.40%; Found: C, 47.84; H, 2.59; N, 13.99; S, 6.23%. 2-(5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3, 4-oxadiazol-2-ylthio)-N-(2,4-dichlorophenyl)acetamide (7s) Creamish white powder; yield: 72%; m. p. 164–166 °C; IR (KBr): m (cm 1) 3302, 3035, 2925, 1670, 1607, 1522, 1456, 1244, 1196, 1137; 1H NMR (400 MHz, DMSO-d6): d 5.20 (s, 2H), 5.99 (s, 2H), 7.36–7.44 (m, 2H), 7.52–7.69 (m, 3H), 7.80–7.87 (m, 2H), 10.89 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 36.76, 112.28, 118.77 (q, J = 270.0 Hz), 121.13, 124.10, 125.83, 126.47, 129.05, 130.08, 131.94, 132.86, 135.42, 136.64, 139.31 (q, J = 38.0 Hz), 140.66, 162.50, 164.42, 165.09; ESI-MS: 503 (M + 1)+; Anal. Calcd. for C19H12Cl2F3N5O2S: C, 45.43; H, 2.41; Cl, 14.12; F, 11.35; N, 13.94; O, 6.37; S, 6.38%; Found: C, 45.48; H, 2.42; N, 13.96; S, 6.37%. 2.3. Pharmacology 2.3.1. Chemicals Human recombinant GSK-3b and pre-phosphorylated polypeptide substrate GS-2 were purchased from Merck-Millipore Corporation (India). Kinase-Glo Luminescent Kinase Assay (catalog no. V6713) was brought from Promega Corporation (Madison, WI). Staurosporine was purchased from Sigma-Aldrich Chemicals Pvt. Limited, Bangalore, India. Fluoxetine was procured from the local market. 2.3.2. GSK-3b inhibitory assay The synthesized compounds were screened for their GSK-3b inhibition as per the Kinase-Glo assay method of Baki et al. [35]. In a typical assay, 10 lL of test compound of various concentrations (dissolved in dimethyl sulfoxide [DMSO] and diluted with assay buffer) and 10 lL (20 ng) of enzyme were added to each well followed by 20 lL of assay buffer containing substrate and ATP to obtain a concentration of 25 lM substrate and 1 lM ATP per well. The final DMSO concentration in the reaction mixture was kept less than 1%. After incubation at 30 °C for 30 min, the enzymatic reaction was quenched with 40 lL of Kinase-Glo reagent. Luminescence was recorded after 10 min using Infinite F200ÒPRO multimode reader (Tecan). The activity is proportional to the

difference of the total and consumed ATP. The activity was highest in the absence of inhibitor and was used to calculate the inhibitory activities of test compounds. Staurosporine was used as a standard GSK-3b inhibitor. 2.3.3. Antidepressant activity The antidepressant activity was evaluated by the two widely known behavioral models - Forced swim test and Tail suspension test. 2.3.3.1. Animals. Wistar rats of either sex (150–180 g) were acquired from Central Animal House, Hamdard University (Jamia Hamdard), New Delhi and kept in colony cages at an ambient temperature of 25 ± 2 °C and relative humidity 45–55% with 12 h light/dark cycles after initial acclimatization for about 1 week. They were provided free access to standard rodent pellet diet and water ad libitum. The experimental study was performed in accordance with the Institutional Animal Ethics Committee guidelines. The test compounds were suspended in 0.5% methylcellulose (Methocel) and administered orally 1 h before behavioral testing. 2.3.3.2. Forced swim test. The forced swim test (FST) is the most widely used behavioral test to screen compounds for potential antidepressant activity [36]. The test was conducted as per the procedure described by Vincent Castagne et al. [37]. Rats were shifted from the housing colony room to the testing laboratory and kept undisturbed there for at least 1 h before testing. A full access to standard rodent diet and tap water was provided, except during the test. A controlled temperature of 21 ± 3 °C and standard light/ dark cycle with illumination from 0700 to 1900 h were maintained. Each experimental group consisted of 6 rats. 15-min prior to drug administration, a habituation session was conducted without behavioral recording. The rats were individually forced to swim in a plexiglas cylinder (20 cm in diameter  40 cm in height) filled with water (25 °C) to a depth of 30 cm. After 24 h, each group were administered with the appropriate dose of fluoxetine (30 mg/kg BW) or test compounds (20 mg/kg BW) or their vehicles and followed 1 h later by a 6-min test session. The total immobility time, defined by lack of activity, except for the necessary movements to keep their heads above water, was scored during this session. Data was compared with the data from the control and the standard group. 2.3.3.3. Tail suspension test (TST). The tail suspension test as a model of behavioral testing is related in principle to the forced swimming test, except that the immobility is induced by suspending the rat by its tail [38,39]. Rats were moved to the testing laboratory from the housing colony room and kept undisturbed there for at least 1 h before testing. The dosage, temperature and other environmental conditions were kept from those in the forced swim test. The test was performed using an automated apparatus. Rats were suspended individually upside-down using an adhesive tape to fix its tail to a hook at 60 cm, which was connected to a strain gauge. A square platform was temporarily positioned 20 cm below the apparatus, just below the rat’s forepaws, in order to minimize the weight sustained by the animal’s tail until the start of the recording session. The movements made by the rat were picked up by the strain gauge and transmitted to a central unit, which calculated the total duration of immobility during a 6-min test with the help of a maze software version 3.0. The rat was considered immobile when it failed to show any movements of struggling, attempting to catch the adhesive tape, body torsions, or jerks. Data collected was expressed as a mean of immobility time (in second ± the standard error mean: S.E.M).

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2.4. Molecular docking studies The in silico molecular docking studies were performed along with in vitro studies, using Schrodinger Software (Maestro Version 10). The ligands were virtually prepared by Lig-Prep module (Version 3.3) while as the protein was prepared by Protein Preparation Wizard through Maestro Interface (Version 10). The binding pocket was generated by selecting a grid with respect to reference ligand, 5-[1-(4-methoxyphenyl)-1H-benzimidazol-6-yl]-1,3,4-oxadiazole2(3H)-thione. The docking was carried using Glide Module (Version 6.6) with Extra Precision (XP) mode. The docked molecules were ranked with respect to Glide score. 2.5. Statistical analysis Results are expressed as the mean ± SEM. Different groups were compared using one way analysis of variance (ANOVA) followed by Tukey Kramer test for multiple comparisons. 3. Results and discussion 3.1. Chemistry The compounds were synthesized according to Scheme 1. OPhenylenediamine 1 was treated with trifluoroacetic acid (TFA) for 5–6 h to obtain 2-trifluoromethyl benzimidazole 2. Benzimidazole 2 was then reacted with ethylbromoacetate in presence of potassium carbonate in dry acetone for 4–5 h to obtain ester derivative 3 which was subsequently treated with hydrazine

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hydrate in absolute ethanol to afford the corresponding hydrazide derivative 4. The intermediate 4 was refluxed with carbon disulphide and potassium hydroxide in absolute ethanol for 10 h to form 5-((2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)methyl)1,3,4-oxadiazole-2-thiol 5 which was then treated with bromoacetic acid in the presence of triethylamine in dry THF under refluxing conditions for 5–6 h to obtain 2-(5-((2-(trifluorome thyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3,4-oxadiazol-2-ylthio) acetic acid 6. The intermediate 6 was subsequently treated with various amines in DMF in presence of HOBT and EDC.HCl at room temperature to afford the target compounds, benzimidazole substituted 1,3,4-oxadiazole carboxamides (7a-7s) (Table 1). The structures of all the synthesized compounds were confirmed by the spectral analysis (1H NMR, 13C NMR and mass spectral data). Appearance of signal in the region of d 10.00–10.50 affirmed the presence of amide proton. The presence of two methylene groups was validated by the appearance of singlets in the region of d 4.00–4.50 (s, NACH2) and d 5.75–6.25 (s, SACH2) in the 1H NMR spectra. The corresponding peaks appeared at around d 33.00–36.50 and d 39.00 in the 13C NMR spectra, the latter peak being overlapped by the DMSO (solvent) peak. The trifluoromethyl-group attached to C-2 of benzimidazole ring was identified by the appearance of a quartet in the region of d 118.65–118.75 (J = 268–271 Hz) in the 13C NMR spectra due to CF splitting. Due to adjacent trifluoromethyl group, the C-2 of benzimidazole core appeared as a quartet at d 139–140 (J = 38–40 H z). The presence of oxadiazole core is established by the appearance of two peaks in the region of d 160–165 corresponding to two carbons of oxadiazole. The structures were further verified

Scheme 1. Synthetic approach for Benzimidazole-based 1,3,4-oxadiazole carboxamide conjugates.

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Table 1 Benzimidazole-based 1,3,4-oxadiazole carboxamide conjugates and their GSK-3b inhibitory activity.

N N

CF3 O

S

O

N N

N R2

R1

Compound code

R1 (R2 = H, except for 7g and 7h)

IC50 (lM) ± S.E.

7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 7q 7r 7s –

Phenyl 4-Ethyl Phenyl 2-Methoxy Phenyl 4-Methoxy Phenyl 4-Chloro Phenyl 4-ter-Butyl Phenyl Morpholine N-Methyl Piperazine 2-Chloro Phenyl 4-Fluoro Phenyl 2-Fluoro Phenyl 3,4-Dimethyl Phenyl 2-Bromo Phenyl 4-Ethylbenzoate 4-Ethoxy Phenyl 4-Bromo Phenyl 3,4-Dimethoxy Phenyl 3-Trifluoromethyl Phenyl 2,4-Dichloro Phenyl Staurosporine

0.13 ± 0.01 0.63 ± 0.03 0.29 ± 0.02 0.22 ± 0.01 0.34 ± 0.03 0.85 ± 0.04 17.82 ± 1.23 11.93 ± 0.74 0.38 ± 0. 02 0.20 ± 0.02 1.34 ± 0.11 2.04 ± 0.16 1.67 ± 0.13 0.59 ± 0.04 0.96 ± 0.08 0.51 ± 0.03 0.45 ± 0.02 0.14 ± 0.01 0.28 ± 0.01 0.05 ± 0.01

The compounds with highest GSK-3b inhibitory activities among the synthesized compound series have been highlighted in Bold.

by ESI-MS/LC-MS analysis which showed the presence of [M]+ or [M + 1]+ or [M + 2]+ ion peaks. 3.2. GSK-3b inhibitory activity The synthesized compounds were screened for their GSK-3b inhibition potential in vitro by Kinase-GloTM luminescent method of Baki et al. [35] Staurosporine was used as the standard. Many compounds from the series were found to exhibit potency in the sub-micromolar range (Table 1). Compounds 7a, 7r, 7j, 7d, and 7s were among the most active in inhibiting GSK-3b with IC50

values of 0.13 lM, 0.14 lM, 0.20 lM, 0.22 lM and 0.28 lM respectively. Compounds 7c, 7i, 7q, and 7e also displayed fairly good GSK-3b inhibition profile with corresponding IC50 values below 0.5 lM. A preliminary structure-activity relationship has also been drawn, based on the in vitro GSK-3b inhibition results. Presence of amide-NH was found essential for exhibiting GSK-3b inhibitory activity, as conjugates with secondary amines, 7g (morpholine) and 7h (N-methyl piperazine) led to a considerable loss of activity. Un-substituted phenyl conjugate (7a) demonstrated the best activity against GSK-3b (IC50 = 0.13 lM) among all the synthesized conjugates. The para-halo substituted conjugates exhibited better activity in comparison to ortho-halogenated compounds, except the chloro-substituted compounds. Regarding the effect of the nature of halo-group on activity, p-fluoro substituted conjugate (7j) were found to be more active followed by p-chloro and p-bromo substituted conjugates (7e and 7p), indicating decrease in activity observed with increase in size of halogen on the aryl ring. However, among the ortho-halogenated conjugates, chloro (7i) was more effective than fluoro (7k) or bromo (7m), suggesting the possible interplay of both electronic and steric factors in influencing the activity. In addition, the 2,4-dichloro substituted conjugate (7s) conferred higher activity than presence of chloro at both ortho(7i) or para-position (7e). Among the alkoxy groups introduced, methoxy at para-position (7d) conferred increased activity than at the ortho-position (7c), while among the alkyl substitutions, ethyl group (7b) at para-position imparted higher activity in comparison to bulky tert-butyl group (7f) at the same position, implying decrease in activity with increase in size. 3.3. Antidepressant activity Considering the therapeutic potential of GSK-3b inhibitors as antidepressants, the compounds 7a, 7r, 7j and 7d possessing significant GSK-3b inhibitory activity were investigated further for antidepressant activity by the forced swim test and tail suspension test (TST and FST) models. 3.3.1. Forced swim test (FST) The results depicting the effect of compounds 7a, 7r, 7j and 7d on immobility in the forced swim test is shown in Fig. 1. The immobility time periods in experimental rats were reduced signif-

Fig. 1. Forced swim test. Data is represented as Mean ± SEM of 6 rats and analyzed by one way ANOVA followed by Tukey Kramer multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001 when compared with normal control. Fluoxetine is taken as standard (STD).

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Fig. 2. Tail suspension test. Data is represented as Mean ± SEM of 6 rats and analyzed by one way ANOVA followed by Tukey Kramer multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001 when compared with normal control. Fluoxetine is taken as standard (STD).

icantly (81–100 s) as compared to normal saline treated control group (129 s) (P < 0.05) by all the compounds tested. Compounds 7a, 7r, and 7j lowered the immobility time by 1.4–1.6-fold in comparison to normal saline group, while compound 7d was compara-

Table 2 Glide scores with respect to GSK-3b protein target (PDB ID: 3F88). Compound code 7a 7r 7j 7d Standard

Glide score 8.70 8.04 7.53 7.45 9.01

ble to that of the standard fluoxetine in shortening the mean immobility duration.

3.3.2. Tail suspension test (TST) Fig. 2 describes the effect of the compounds 7a, 7r, 7j, and 7d on immobility in the tail suspension test. The compounds, as in forced swim test, were able to significantly decrease the immobility time periods (142–163 s) in experimental rats in comparison to normal saline treated control group (214 s) (P < 0.05). Compound 7a was particularly most active, reducing the immobility by 1.5-fold relative to the normal saline group. Pre-treatment with 7r and 7j also resulted in a considerable decrease in mean duration of immobility by about 1.35–1.35-fold as compared to normal control group. In

Fig. 3a. Docking pose of ligand 7a with respect to GSK-3b protein (PDB ID: 3F88).

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Fig. 3b. Ligplot of ligand 7a with respect to GSK-3b protein (PDB ID: 3F88).

comparison to fluoxetine, 7a, 7r, and, 7j were more effective while as 7d was slightly less effective. The compounds 7a, 7r, 7j, and 7d thus displayed significant antidepressant activity in both, TST and FST models. Interestingly, the antidepressant activity of the tested compounds was observed in consonance with respect to their GSK-3b inhibitory activity, indicating that these compounds probably exert their antidepressive action through GSK-3b inhibition mechanism. Consequently, these conjugates may hold a significant potential as GSK-3b inhibitors for the treatment of depression and associated disorders. 3.4. Molecular docking studies In order to gain an understanding of the binding interactions with the receptor site, docking studies of the compounds 7a, 7r, 7j and 7d displaying significant GSK-3b inhibition were performed against GSK-3b protein (PDB ID: 3F88). The compounds were docked into the ATP binding pocket of GSK-3b with the ligand 5-[1-(4-methoxyphenyl)-1H-benzimidazol-6-yl]-1,3,4-oxadiazole2(3H)-thione taken as the reference. It forms hydrogen bond with backbone atoms of VAL-135 and fits in the hydrophobic pocket formed by residues like ILE-62, VAL-70, ALA-83 and LEU-188. In order to inhibit GSK-3b phosphorylating the substrate, various inhibitors are designed which bind to GSK-3b in its ATP-binding site so that it is not further available for binding to substrate. It is well documented in literature documents with X-ray crystallographic data, that oxadiazole and benzimidazole derivatives are reported to bind to Val-135 residue in the hinge region of the ATP binding pocket of GSK-3b. The Hydrogen bond formation with Val-135 residue is an important interaction for inactivation of GSK-3b and enhances the activity of the compounds. The designed and synthesized molecules docked against the ATP binding site of GSK-3b shows similar mode of action as shown by the oxadiazole and benzimidozole derivatives. The compounds

showed a good glide score in comparison to reference as shown in Table 2. Compound 7a displayed highest glide score of 8.70 close to the reference with a glide score of 9.01. The NH-amide of 7a formed H-bond with Valine-135 in the hinge-region as shown in Figs. 3a and 3b. The compound also participates in hydrophobic interactions with PHE-67 thereby inhibiting the GSK-3b and contributing to overall activity. These interactions probably account for its in vitro GSK-3b inhibition activity. 4. Conclusions A focused library of nineteen benzimidazole-based 1,3,4oxadiazole carboxamides has been synthesized and assessed for in vitro GSK-3b inhibitory activity. The compounds 7a, 7r, 7j, and 7d exhibited significant GSK-3b inhibition with IC50 values of 0.13 lM, 0.14 lM, 0.20 lM, and 0.22 lM respectively. Given the promising therapeutic potential of GSK-3b inhibitors in the treatment of depression and associated disorders, the compounds 7a, 7r, 7j, and 7d were evaluated for antidepressant activity by FST and TST models. All the test compounds significantly reduced immobility time in experimental rats in comparison to normal control group in both the models. Compound 7a showed maximum activity and was slightly more effective than the standard drug fluoxetine. The docking study of these active compounds showed their key interactions with the GSK-3b protein target. The encouraging results of our work, suggest that the benzimidazole-based 1,3,4-oxadiazole carboxamides as GSK-3b inhibitors may act as a pharmacological tool to explore the pathological implications of GSK-3b and subsequent development of effective drugs against these diseases. Acknowledgements The authors wish to express their thanks to Dr. G.N. Qazi, ViceChancellor, Jamia Hamdard for providing necessary facilities to

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carry out this work. The authors thank DST-SERB, Govt. of India for awarding research project to HH and fellowship to IK. The author MAT is thankful to HNF for providing research assistance. Declaration of interest The authors declare no conflict of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bioorg.2018.01.040. References [1] L. Kockeritz, B. Doble, S. Patel, J.R. Woodgett, Glycogen synthase kinase-3–an overview of an over-achieving protein kinase, Curr. Drug Targets 7 (2006) 1377–1388. [2] W.-J. Song, E.-A.C. Song, M.-S. Jung, S.-H. Choi, H.-H. Baik, B.K. Jin, J.H. Kim, S.-H. Chung, Phosphorylation and inactivation of GSK3b by dual-specificity tyrosine (Y)-phosphorylation-regulated kinase 1A (Dyrk1A), J. Biol. Chem. 290 (2015) 2321–2333. [3] I. Khan, M.A. Tantray, M.S. Alam, H. Hamid, Natural and synthetic bioactive inhibitors of glycogen synthase kinase, Euro. J. Med. Chem. 125 (2017) 464– 477. [4] E.J. Crofton, M.N. Nenov, Y. Zhang, F. Scala, S.A. Page, D.L. McCue, D. Li, J.D. Hommel, F. Laezza, T.A. Green, Glycogen synthase kinase 3 beta alters anxiety-, depression-, and addiction-related behaviors and neuronal activity in the nucleus accumbens shell, Neuropharmacol. 117 (2017) 49–60. [5] M. Pardo, E. Abrial, R.S. Jope, E. Beurel, GSK3b isoform-selective regulation of depression, memory and hippocampal cell proliferation, Genes Brain Behav. 15 (2016) 348–355. [6] P. Zanos, R. Moaddel, P.J. Morris, P. Georgiou, J. Fischell, G.I. Elmer, M. Alkondon, P. Yuan, H.J. Pribut, N.S. Singh, K.S.S. Dossou, Y. Fang, X.-P. Huang, C. L. Mayo, I.W. Wainer, E.X. Albuquerque, S.M. Thompson, C.J. Thomas, C.A. Zarate Jr, T.D. Gould, NMDAR inhibition-independent antidepressant actions of ketamine metabolites, Nature 533 (2016) 481–486. [7] J.F. Costemale-Lacoste, J.P. Guilloux, R. Gaillard, The role of GSK-3 in treatmentresistant depression and links with the pharmacological effects of lithium and ketamine: A review of the literature, Encephale. 42 (2016) 156–164. [8] A. Bureau, J.M. Beaulieu, T. Paccalet, Y.C. Chagnon, M. Maziade, The interaction of GSK3B and FXR1 genotypes may influence the mania and depression dimensions in mood disorders, J Affect. Disord. 213 (2017) 172–177. [9] P. Pérez-Domper, V. Palomo, S. Gradari, C. Gil, M.L. de Ceballos, A. Martínez, J.L. Trejo, The GSK-3-inhibitor VP2.51 produces antidepressant effects associated with adult hippocampal neurogenesis, Neuropharmacol. 116 (2017) 174–187. [10] T.D. Gould, J.A. Quiroz, J. Singh, C.A. Zarate, H.K. Manji, Emerging experimental therapeutics for bipolar disorder: insights from the molecular and cellular actions of current mood stabilizers, Mol. Psychiatry 9 (2004) 734–755. [11] J.M. Beaulieu, S. Marion, R.M. Rodriguiz, I.O. Medvedev, T.D. Sotnikova, V. Ghisi, W.C. Wetsel, R.J. Lefkowitz, R.R. Gainetdinov, M.G. Caron, A beta-arrestin 2 signaling complex mediates lithium action on behavior, Cell. 132 (2008) 125–136. [12] T.D. Gould, C.A. Zarate, H.K. Manji, Glycogen synthase kinase-3: a target for novel bipolar disorder treatments, J. Clin. Psychiatry 65 (2004) 10–21. [13] T.D. Gould, H. Einat, R. Bhat, H.K. Manji, AR-A014418, a selective GSK-3 inhibitor, produces antidepressant-like effects in the forced swim test, Int. J. Neuropsychopharmacol. 7 (2004) 387–390. [14] A.O. Rosa, M.P. Kaster, R.W. Binfare, S. Morales, E. Martin-Aparicio, M.L. Navarro-Rico, A. Martinez, M. Medina, A.G. Garcia, M.G. Lopez, A.L. Rodrigues, Antidepressant-like effect of the novel thiadiazolidinone NP031115 in mice, Prog. Neuropsychopharmacol. Biol. Psychiatry 32 (2008) 1549–1556. [15] O. Kaidanovich-Beilin, A. Milman, A. Weizman, C.G. Pick, H. Eldar-Finkelman, Rapid antidepressive-like activity of specific glycogen synthase kinase-3 inhibitor and its effect on beta-catenin in mouse hippocampus, Biol. Psychiatry 55 (2004) 781–784. [16] J.M. Beaulieu, R.R. Gainetdinov, M.G. Caron, Akt/GSK3 signaling in the action of psychotropic drugs, Annu. Rev. Pharmacol. Toxicol. 49 (2009) 327–347. [17] X. Li, M.A. Frye, R.C. Shelton, Review of pharmacological treatment in mood disorders and future directions for drug development, Neuropsychopharmacol. 37 (2012) 77–101. [18] J.M. Beaulieu, A role for Akt and glycogen synthase kinase-3 as integrators of dopamine and serotonin neurotransmission in mental health, J. Psychiatry Neurosci. 37 (2012) 7–16. [19] I. Khan, M.A. Tantray, H. Hamid, M.S. Alam, A. Kalam, F. Hussain, A. Dhulap, Synthesis of pyrimidin-4-one-1,2,3-triazole conjugates as glycogen synthase kinase-3b inhibitors with anti-depressant activity, Bioorg. Chem. 68 (2016) 41–55.

401

[20] I. Khan, M.A. Tantray, H. Hamid, M.S. Alam, A. Kalam, F. Shaikh, A. Shah, F. Hussain, Synthesis of novel pyrimidin-4-one bearing piperazine ring-based amides as glycogen synthase kinase-3b inhibitors with antidepressant activity, Chem. Biol. Drug Des. 87 (2016) 764–772. [21] M.A. Tantray, I. Khan, H. Hamid, M.S. Alam, S. Umar, Y. Ali, K. Sharma, F. Hussain, Synthesis of novel oxazolo[4,5-b]pyridine-2-one based 1,2,3-triazoles as glycogen synthase kinase-3b inhibitors with anti-inflammatory potential, Chem. Biol. Drug Des. 87 (2016) 918–926. [22] M.A. Tantray, I. Khan, H. Hamid, M.S. Alam, A. Dhulap, A. Kalam, Synthesis of benzimidazole-based 1,3,4-oxadiazole-1,2,3-triazole conjugates as glycogen synthase kinase-3[small beta] inhibitors with antidepressant activity in in vivo models, RSC Adv. 6 (2016) 43345–43355. [23] I. Khan, M.A. Tantray, H. Hamid, M.S. Alam, A. Kalam, A. Dhulap, Synthesis of benzimidazole based thiadiazole and carbohydrazide conjugates as glycogen synthase kinase-3b inhibitors with anti-depressant activity, Bioorg. Med. Chem. Lett. 26 (2016) 4020–4024. [24] M.A. Tantray, I. Khan, H. Hamid, M.S. Alam, A. Dhulap, A. Kalam, Synthesis of Aryl anilinomaleimide based derivatives as glycogen synthase kinase-3[small beta] inhibitors with potential role as antidepressant agents, New J. Chem. 40 (2016) 6109–6119. [25] M. Saitoh, J. Kunitomo, E. Kimura, Y. Hayase, H. Kobayashi, N. Uchiyama, T. Kawamoto, T. Tanaka, C.D. Mol, D.R. Dougan, G.S. Textor, G.P. Snell, F. Itoh, Design, synthesis and structure-activity relationships of 1,3,4-oxadiazole derivatives as novel inhibitors of glycogen synthase kinase-3b, Bioorg. Med. Chem. 17 (2009) 2017–2029. [26] M. Saitoh, J. Kunitomo, E. Kimura, H. Iwashita, Y. Uno, T. Onishi, N. Uchiyama, T. Kawamoto, T. Tanaka, C.D. Mol, D.R. Dougan, G.P. Textor, G.P. Snell, M. Takizawa, F. Itoh, M. Kori, 2-{3-[4-(Alkylsulfinyl)phenyl]-1-benzofuran-5-yl}5-methyl-1,3,4-oxadiazole derivatives as novel inhibitors of glycogen synthase kinase-3b with good brain permeability, J. Med. Chem. 52 (2009) 6270–6286. [27] T. Onishi, H. Iwashita, Y. Uno, J. Kunitomo, M. Saitoh, E. Kimura, H. Fujita, N. Uchiyama, M. Kori, M. Takizawa, A novel glycogen synthase kinase-3 inhibitor 2-methyl-5-(3-{4-[(S)-methylsulfinyl]phenyl}-1-benzofuran-5-yl)-1,3,4oxadiazole decreases tau phosphorylation and ameliorates cognitive deficits in a transgenic model of Alzheimer’s disease, J. Neurochem. 119 (2011) 1330– 1340. [28] M.A. Khanfar, R.A. Hill, A. Kaddoumi, K.A. El Sayed, Discovery of novel GSK3beta inhibitors with potent in vitro and in vivo activities and excellent brain permeability using combined ligand- and structure-based virtual screening, J. Med. Chem. 53 (2010) 8534–8545. [29] F. Lo Monte, T. Kramer, J. Gu, U.R. Anumala, L. Marinelli, V. La Pietra, E. Novellino, B. Franco, D. Demedts, F. Van Leuven, A. Fuertes, J.M. Dominguez, B. Plotkin, H. Eldar-Finkelman, B. Schmidt, Identification of glycogen synthase kinase-3 inhibitors with a selective sting for glycogen synthase kinase-3alpha, J. Med. Chem. 55 (2012) 4407–4424. [30] A.G. Koryakova, Y.A. Ivanenkov, E.A. Ryzhova, E.A. Bulanova, R.N. Karapetian, O.V. Mikitas, E.A. Katrukha, V.I. Kazey, I. Okun, D.V. Kravchenko, Y.V. Lavrovsky, O.M. Korzinov, A.V. Ivachtchenko, Novel aryl and heteroaryl substituted N-[3-(4-phenylpiperazin-1-yl)propyl]-1,2,4-oxadiazole-5carboxamides as selective GSK-3 inhibitors, Bioorg. Med. Chem. Lett. 18 (2008) 3661–3666. [31] D. Shin, S.-C. Lee, Y.-S. Heo, W.-Y. Lee, Y.-S. Cho, Y.E. Kim, Y.-L. Hyun, J.M. Cho, Y.S. Lee, S. Ro, Design and synthesis of 7-hydroxy-1H-benzoimidazole derivatives as novel inhibitors of glycogen synthase kinase-3b, Bioorg. Med. Chem. Lett. 17 (2007) 5686–5689. [32] A.J. Peat, J.A. Boucheron, S.H. Dickerson, D. Garrido, W. Mills, J. Peckham, F. Preugschat, T. Smalley, S.L. Schweiker, J.R. Wilson, T.Y. Wang, H.Q. Zhou, S.A. Thomson, Novel pyrazolopyrimidine derivatives as GSK-3 inhibitors, Bioorg. Med. Chem. Lett. 14 (2004) 2121–2125. [33] S.M. Sondhi, N. Singh, A. Kumar, O. Lozach, L. Meijer, Synthesis, antiinflammatory, analgesic and kinase (CDK-1, CDK-5 and GSK-3) inhibition activity evaluation of benzimidazole/benzoxazole derivatives and some Schiff’s bases, Bioorg. Med. Chem. 14 (2006) 3758–3765. [34] A. Rathore, M.U. Rahman, A.A. Siddiqui, A. Ali, M. Shaharyar, Design and synthesis of benzimidazole analogs endowed with oxadiazole as selective COX-2 inhibitor, Arch. Pharm. (Weinheim) 347 (2014) 923–935. [35] A. Baki, A. Bielik, L. Molnár, G. Szendrei, G.M. Keserü, A high throughput luminescent assay for glycogen synthase kinase-3b inhibitors, Assay Drug Dev. Technol. 5 (2007) 75–83. [36] J.F. Cryan, A. Markou, I. Lucki, Assessing antidepressant activity in rodents: recent developments and future needs, Trends Pharmacol. Sci. 23 (2002) 238– 245. [37] V. Castagne, P. Moser, S. Roux, R.D. Porsolt, Rodent models of depression: forced swim and tail suspension behavioral despair tests in rats and mice, Current protocols in neuroscience/editorial board, Jacqueline N. Crawley ... [et al.] Chapter 8 (2011) Unit 8 10A. [38] R. Chermat, B. Thierry, J.A. Mico, L. Steru, P. Simon, Adaptation of the tail suspension test to the rat, J. Pharmacol. 17 (1986) 348–350. [39] C. Millon, A. Flores-Burgess, M. Narvaez, D.O. Borroto-Escuela, L. Santin, C. Parrado, J.A. Narvaez, K. Fuxe, Z. Diaz-Cabiale, A role for galanin N-terminal fragment (1–15) in anxiety- and depression-related behaviors in rats, Int. J. Neuropsychopharmacol. 18 (2015) 1–13.