ORIGINAL RESEARCH DESIGN, SYNTHESIS AND NEUROPHARMACOLOGICAL EVALUATION OF THIOPHENE INCORPORATED ISOXAZOLE DERIVATIVES AS ANTIDEPRESSANT AND ANTIANXIETY AGENTS Jagdish Kumar1, Mymoona Akhtar2, Chanda Ranjan3, Gita Chawla4,* 1,2,34Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi-110062, India. *Corresponding Author: E-mail: [email protected] ABSTRACT A series of 5-substituted phenyl-3-(thiophen-2-yl)-4, 5-dihydro-1, 2-oxazoles (2a-l) was synthesized by reacting appropriate chalcones of 2-acetyl thiophene with hydroxylamine hydrochloride in the presence of dry pyridine. The newly synthesized compounds were characterized by IR, 1HNMR, 13CNMR and mass spectral data. All the compounds were evaluated for their antidepressant and antianxiety activities in mice by forced swimming test and elevated plus maze method respectively. Test compounds and imipramine were administered intraperitoneally in antidepressant study at dose of 10 mg/kg. Similarly to study antianxiety activity, test compounds at the dose of 10 mg/kg and diazepam at the dose of 2 mg/kg were administered intraperitoneally. However, preliminary antidepressant screening of compounds (2a-l) revealed that none of the compounds showed antidepressant activity except for compound 2k which moderately (P<0.05) reduced the duration of immobility time. This compound was also tested in-vitro for its MAO inhibitory effect. Compound 2f showed highest antianxiety activity compared to diazepam and did not show neurotoxicity in rotarod test. The compounds were also studied for pharmacokinetic parameters and was observed that compound 2f displayed good ADME properties. Keywords: Thiophene, isoxazole, antianxiety and antidepressant. INTRODUCTION the anti-anxiety drugs act primarily by facilitating Depression is a life threatening illness that inhibitory GABAergic transmission [4]. The affects the major part of population across the globe worldwide experience among clinicians and [1]. It is associated with pathological change in mood researchers is that anxiety and depression commonly state. Antidepressants can elevate mood in depressive co-exist in clinical samples and in general population state. Practically, all antidepressants affect [5]. During literature survey it was found that isoxazole monoaminergic transmission in the brain. nucleus is one of the important and widely exploited Antidepressants affect the reuptake/ metabolism of heterocyclic ring for the development of bioactive biogenic amines. Antidepressants are generally molecules. Increasing evidence suggests that classified as tricyclic antidepressants (including NA, isoxazole derivatives possess a broad spectrum of 5HT reuptake inhibitors, selective serotonin reuptake biological activities such as antianxiety, [6-7] inhibitors (SSRIs), noradrenaline reuptake inhibitors antidepressant [8, 9] anti-stress [10], MAO inhibitory [11] (NRIs) and MAO inhibitors [2]. Earlier MAO inhibitors and anticonvulsant [12]. Isocarboxazide is an isoxazole were abandoned due to their side effects such as derivative having antidepressant action available in hepatotoxicity, orthostatic hypotension and the the market which is a non-selective MAO inhibitor. ‘cheese effect’ characterized by hypertensive crisis. Recently antidepressant activity of isoxazole ether This was due to non-selective and irreversible MAO scaffold is also reported [13]. Many isoxazole nucleus inhibition. But identification of two isoforms i.e. containing compounds have also been patented for MAO A and MAO B has renewed the interest in their antidepressant and antianxiety activities [14-18]. biological potential of these compounds. MAO A and Similarly thiophene containing compounds have been MAO B are Flavin adenine dinucleotide (FAD) reported to exhibit antidepressant [19-20] and antianxiety containing enzymes. MAO A preferably metabolizes activities [21]. Duloxetine, Etizolam, Teniloxazine, serotonin (5-HT), adrenaline and noradrenaline Tigabine etc. are the thiophene nucleus containing whereas MAO B metabolizes β-phenyl ethylamine antidepressant and antianxiety drugs being marketed. and benzylamine. MAO A inhibitors are useful in the Thiophene analogues also have been well reported as treatment of depression and anxiety while MAO B MAO inhibitors [22]. could be used to treat Parkinson’s disease and However, during literature survey it was Alzheimer’s disesase. Efforts have been oriented found that thiophene ring has not been linked with towards discovery of reversible and selective isoxazoles so far. This motivated us to synthesize inhibitors of MAO A/ MAO B [3]. Similarly, anxiety is hybrid compounds that comprise both the isoxazole an emotional state associated with uneasiness, and the thiophene ring systems with the hope of discomfort and fear about some future threat. Most of developing potent compounds with antianxiety and International Journal of Pharmaceutical Chemistry and Analysis; April-June 2015;2(2):74-83 74 Kumar et al. Design, Synthesis and Neuropharmacological Evaluation of Thiophene Incorporated Isoxazole… antidepressant effects (Figure 1). We report herein the General procedure for the preparation of synthesis of a series of 5-substituted phenyl-3- 5-substituted phenyl - 3 - (thiophen – 2 - yl) - 4, 5- (thiophen-2-yl)-4,5-dihydro-l, 2-oxazole derivatives dihydro-1, 2-oxazoles (2a-l). To the solution of (2a-l). A computational study for prediction of ADME appropriate chalcone (0.01 mol) in absolute ethanol properties of titled compounds (2a-1) was performed (50 ml) was added dry pyridine (1 ml) and for the prediction of ADME properties. This study hydroxylamine hydrochloride (0.01 mol). The includes lipophilicity data also which is an important contents were refluxed for 8-10 h and left overnight. requirement for neuropharmacological activity. The solvent was evaporated off and the residue was ADME properties of newly synthesized isoxazole poured into ice cold water. The solid mass which was derivatives were calculated using online separated out was filtered, washed with water, dried molinspiration software. These compounds were and crystallized from methanol. tested for their in-vivo antidepressant activity by forced swimming test (FST). Compound 2k showing 5-phenyl-3-(thiophen-2-yl)-4,5-dihydro-1,2-oxazole moderate antidepressant activity was screened for (2a). MAO inhibitory activity. All the twelve compounds FTIR [KBr, cm-1]: 676 (C-S), 1356 (C-O-N stretch), were evaluated for their antianxiety activity by 1659 (C=N stretch). 1H NMR [300 MHz, δ ppm, elevated plus maze method. Neurotoxicity was DMSO-d6]: 3.52 (1H, dd, J = 9.3, 6.8 Hz, CH), 3.74 determined by rotarod toxicity test. Few of the final (1H, dd, J = 11.3, 6.8 Hz, CH), 7.12-7.65 (8H, m, compounds have been reported by Ingle et al. [23] but ArH), 5.92 (1H, dd, J = 9.3, 11.3 Hz, CH), 7.31 and δ have not been tested for their pharmacological 7.73 (dd, 1H, (thiophene C-CH), 7.61 (t, 1H, activities. (thiophene CH-S). 13CNMR [75 δ ppm, MHz, δ ppm, DMSO-d6]: 44.13, 82.09, 125.67, d126.91, 127.41, EXPERIMENTAL d127.57, 130.31, 130.38, 138.17, 140.03, 147.82, MS: + Chemistry: m/z 229 (M ); Anal. Calcd for C13H11NOS: C, 68.09, All the chemicals used were of laboratory H, 4.84, N, 6.11, Found: C, 68.18, H, 4.90, N, 6.16%. grade and procured from E. Merck (Germany) and S.D. Fine Chemicals (India). Melting points were 5-(2-chlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro- determined by the open tube capillary method and are 1,2-oxazole (2b). uncorrected. The Thin layer chromatography (TLC) FTIR [KBr, cm-1]: 676 (C-S), 1357 (C-O-N stretch), plates (silica gel G) were used to confirm the purity of 1662 (C=N stretch). 1H NMR [300 MHz, δ ppm, commercial reagents used, compounds synthesized DMSO-d6]: δ 3.58 (1H, dd, J = 9.4, 7.8 Hz, CH), 3.73 and to monitor the reactions as well. Two different (1H, dd, J = 11.5, 7.8 Hz, CH), 6.00 (1H, dd, J = solvent systems: toluene: ethyl acetate: formic acid 9.4,11.5 Hz, CH), 7.15-7.65 (7H, m, ArH), 7.32 and δ (5:4:1) and benzene: acetone (9:1), were used to run 7.68 (dd, 1H, (thiophene C-CH), 7.62(t, 1H, the TLC and spots were located under iodine (thiophene CH-S); 13CNMR [75 MHz, δ ppm, DMSO- vapors/UV light. IR spectra were obtained on a Perkin- d6]: δ 44.43, 78.63, 126.65, 127.41, 129.21, 129.36, Elmer 1720 FT-IR spectrometer (KBr Pellets). 1H 129.58, 130.35, 130.40, 134.32, 135.36, 138.20, NMR and 13C NMR spectra were recorded in DMSO- 147.82, MS: m/z 263.74 (M+), 265.74 (M+2); Anal. d6/CDCl3 on a Bruker 300 MHz and 75 MHz Calcd for C13H10ClNOS: C, 69.20, H, 3.82, N, 5.31, spectrometer, respectively, using tetramethylsilane Found: C, 69.33, H, 3.96, N, 5.34%. (TMS) as the internal reference (chemical shift was measured in δ ppm). Mass spectra (ESI-Q-TOF) were 5-(3-chlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro- measured on a Waters mass spectrometer with an ESI 1,2-oxazole (2c). (Electron spray ionization) source. FTIR [KBr, cm-1]: 1652 (C=N stretch), 1363 (C-O-N General procedure for the preparation of stretch), 669 (C-S). 1H NMR [300 MHz, δ ppm, 3-(substituted phenyl)-1-(thiophen-2-yl)prop-2-en- DMSO-d6]: 3.57 (1H, dd, J = 9.1, 7.8 Hz, CH), 3.74 1-ones (chalcones) (1a-l) A mixture of 2-acetyl (1H, dd, J = 11.1, 7.8 Hz, CH), 5.99 (1H, dd, J = 9.1, thiophene (1) (0.01 mol) and substituted aromatic 11.1 Hz, CH), δ 6.84-7.64 (7H, m, ArH), 7.35 and δ aldehyde (0.01 mol) in absolute ethanol (30 mL) was 7.71 (dd, 1H, (thiophene C-CH), 7.67(t, 1H, stirred at room temperature in the presence of base (thiophene CH-S). 13CNMR [75 MHz, δ ppm, DMSO- (alc.