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Synthesis, Characterization and Biological Activity of Some New S-Substituted Derivatives of 5-(2-Chlorophenyl)-1,3,4-Oxadiazol-2-Thiol.

Byline: Aziz-ur-Rehman, Shahid Rasool, M. Athar Abbasi, Sabahat Z. Siddiqui, Almas Sattar, Khalid Mohammed Khan, Irshad Ahmad and Saira Afzal

Summary:

Molecules with multi-functional groups are being synthesized to acquire more potent molecules. Two series of S-substituted derivatives of 5-(2-chlorophenyl)-1,3,4-Oxadiazol-2-thiol (4) were synthesized and also evaluated for their biological activities. The 2-chlorobenzoic acid (1) was stepped up to ethyl 2-chlorobenzoate (2), 2-chlorobenzohydrazide (3) and 5-(2-chlorophenyl)-1,3,4- Oxadiazol-2-thiol (4). The molecule 4 was made to react with N-substituted-2-bromoacetamide, 6a-f, to yield N-substituted-2-((5-(2-chlorophenyl)-1,3,4-Oxadiazol-2-yl)sulfanyl)acetamide, 7a-f. Simultaneously, the compound 4 was converted to 2-(5-(2-chlorophenyl)-1,3,4-Oxadiazol-2- ylthio)acetohydrazide (9) which on reaction with different aldehydes, 10a-g, resulted N'-substituted- 2-(5-(2-chlorophenyl)-1,3,4-Oxadiazol-2-ylthio)acetohydrazide, 11a-g.

The proposed structures of the molecules were supported by spectral analysis and the series 7a-f was found to be better antibacterial agents and lipoxygenase enzyme inhibitors as compared to 11a-g.

Keywords: 1,3,4-Oxadiazole, 2-chlorobenzoic acid, Acetamides, Antibacterial activity, Enzyme inhibition activity.

Introduction

The synthesis of molecules bearing multiple functional groups of pharmacological importance is the ongoing research. 1,3,4-Oxadiazole [1-4], acetamides [5-9] and azomethine [10-13] derivatives have been known to possess a broad spectrum of biological activities such as antimicrobial, anticancer, anti-inflammatory, antidepressant, anticonvulsant, antioxidant activities and many others. Our group has synthesized such type of molecules [14-17] bearing multiple functional groups to evaluate their antimicrobial and anti-enzymatic activities. The presented work was an attempt to inaugurate new pharmacologically important compounds with considerable antibacterial and enzyme inhibition activity.

Experimental

General

Alfa Aesar, Merck and Sigma-Aldrich branded synthetic grade chemicals were purchased through local suppliers. Thin layer chromatography (TLC) prepared on silica gel G-25-UV254 coated plates was the tool to check out the purity and reaction completion using CH3COOC2H5 and n- C6H14 solvent system with different ratios. The synthesized molecules were characterized by melting points measured through Gallen-Kamp apparatus with open capillary tube and were uncorrected. IR spectra were recorded through Jasco-320-A spectrophotometer by KBr pellet method. 1H-NMR spectra were recorded on Bruker spectrometer in CHCl3-d1 and DMSO-d6, at 400 and 600 MHz respectively. EIMS spectra were recorded on JMS- HX-110 spectrometer.

Synthesis of Ethyl 2-chlorobenzoate (2)

2-Chlorobenzoic acid (1; 6 g, 0.03 mol) was dissolved in 24 mL absolute ethanol in a 100 mL round bottom (RB) flask, 3.0 mL concentrated H2SO4 was added and refluxed for 3-4 hours. After confirmation by TLC, the contents were taken to a 500 mL separating funnel followed by 120 mL distilled water and concentrated aqueous Na2CO3 solution to set pH of 8-10. The ester, 2, was extracted by 30 mL diethylether and collected after distillating the solvent. Yellow liquid; Yield: 83%; Mol. formula: C9H9ClO2; Mol. Wt.: 184; IR (KBr, vmax/cm- 1): 3105 (Ar C-H), 1738 (C=O), 1593 (Ar C=C), 697 (C-Cl); 1H-NMR (600 MHz, DMSO, d/ppm): 7.93 (dd, J = 8.4, 1.8 Hz, 1H, H-6'), 7.54 (d, J = 7.8 Hz, 1H, H-3'), 7.47 (dt, J = 7.8, 1.2 Hz, 1H, H-5'), 7.39 (dt, J = 8.4, 1.8 Hz, 1H, H-4'), 4.06 (q, J = 7.2 Hz, 2H, -OCH2CH3), 1.04 (t, J = 7.2 Hz, 3H, - OCH2CH3); EIMS (m/z): 186 [M+2]+, 184 [M]+, 139 [C7H4ClO]+, 111 [C6H4Cl]+, 85 [C4H2Cl]+, 51 [C4H3]+.

Synthesis of 2-Chlorobenzohydrazide (3)

Ethyl 2-chlorobenzoate (2; 0.04 mol) was dissolved in 20 mL methanol in a 100 mL RB flask and set to stir with 2.6 mL 80% hydrazine hydrate for 2-3 hours at 28 oC (RT). After supervision by TLC, excess of solvent was distilled off and excess cold distilled water was poured and shook till precipitation. The precipitates were acquired by filtration, washing by n-C6H14 and drying. White amorphous solid; Yield: 81%; M.P.: 118-120 oC; Mol. formula: C7H7ClN2O; Mol. Wt.: 170; IR (KBr, vmax/cm-1): 3327 (N-H), 3122 (Ar C-H), 1655 (C=O), 1613 (Ar C=C), 702 (C-Cl); 1H-NMR (600 MHz, DMSO, d/ppm): 9.36 (s, 1H, CONH), 8.74 (s, 2H, N- H), 7.91 (dd, J = 8.4, 1.2 Hz, 1H, H-6'), 7.52 (d, J = 7.2 Hz, 1H, H-3'), 7.49 (t, J = 7.8 Hz, 1H, H-5'), 7.39 (t, J = 7.8 Hz, 1H, H-4'); EIMS (m/z): 172 [M+2]+, 170 [M]+, 139 [C7H4ClO]+, 111 [C6H4Cl]+, 85 [C4H2Cl]+, 51 [C4H3]+.

Synthesis of 5-(2-Chlorophenyl)-1,3,4-Oxadiazol-2- thiol (4)

2-Chlorobenzohydrazide (3; 0.04 mol) was homogeneously dissolved in 60 mL absolute ethanol in a 250 mL RB flask. The mixture was basified by KOH (0.04 mol) under reflux. Then CS2 (0.08 mol) was added at RT. The reaction contents were set to reflux for other 5-6 hours and monitored through TLC. Excess cold distilled water was added along with shaking to clarify the solution. Then 4-5 mL dilute HCl was added till pH of 2-3 along with shaking and aged for 10-15 minutes. The yielded precipitates were collected through filtration followed by washing and drying. White amorphous solid; Yield: 83%; M.P.: 172-174 oC; Mol. formula: C8H5ClN2OS; Mol. Wt.: 212; IR (KBr, vmax/cm-1): 3122 (Ar C-H), 1670 (C=N), 1593 (Ar C=C), 1254 (C-O-C), 704 (C-Cl), 617 (C-S); 1H-NMR (600 MHz, DMSO, d/ppm):

7.95 (dd, J = 9.0, 1.2 Hz, 1H, H-6'), 7.51 (d, J = 7.8 Hz, 1H, H-3'), 7.49 (dt, J = 7.8, 1.8 Hz, 1H, H-5'), 7.43 (dt, J = 7.8, 1.2 Hz, 1H, H-4'); EIMS (m/z): 214 [M+2]+, 212 [M]+, 153 [C7H4ClNO]+, 139 [C7H4ClO]+, 137 [C7H4ClN]+, 111 [C6H4Cl]+, 85 [C4H2Cl]+, 51 [C4H3]+.

Synthesis of N-Substituted-2-bromoacetamide (6a-f)

The substituted aromatic amines (5a-f; 0.012 mol) were added to 15 mL aqueous Na2CO3 solution (5% w/v) in a 100 mL RB flask and pH was adjusted to 8.0-9.0. To reaction mixture, 2-bromoacetyl bromide (0.012 mol) was added gradually and stirred vigorously for 1 hour till precipitation. The reaction contents were monitored by TLC. The obtained solid precipitates were collected through filtration followed by washing with cold distilled water and drying at RT to yield corresponding electrophiles, N-substituted-2- bromoacetamides, 6a-f.

Synthesis of N-Substituted-2-((5-(2-chlorophenyl)- 1,3,4-Oxadiazol-2-yl)sulfanyl)acetamide (7a-f)

The compound 4 (0.1 g, 0.00047 mol) was completely dissolved in 12 mL N,N- dimethylformamide (DMF) and activated by LiH (0.002 g) by stirring for 30 minutes. The electrophiles, N-substituted-2-bromoacetamide, 6a-f, were added to the flask in equimolar ratios and subjected to stir at RT for 4-5 hours. Progress of reaction was monitored by TLC. Excess of chilled distilled water was added with slight shaking till the appearance of precipitates. Precipitates were then filtered, washed and dried for spectral and antibacterial activity analysis.

2-((5-(2-Chlorophenyl)-1,3,4-Oxadiazol-2- yl)sulfanyl)-N-(2,3-dimethylphenyl)acetamide (7a)

White amorphous solid; Yield: 85%; M.P: 152-154 oC; Mol. formula: C18H16ClN3O2S; Mol. Wt.: 373; IR (KBr, vmax/cm-1): 3347 (N-H), 3095 (Ar C-H), 1683 (C=N), 1642 (C=O), 1590 (Ar C=C), 1270 (C-O-C), 680 (C-Cl), 623 (C-S); 1H-NMR (400 MHz, CDCl3, d/ppm): 8.73 (s, 1H, CONH), 7.92 (d, J = 8.0 Hz, 1H, H-6'), 7.56 (d, J = 8.0 Hz, 1H, H-3'), 7.54 (d, J = 7.6 Hz, 1H, H-6'''), 7.49 (t, J = 7.6 Hz, 1H, H-5'), 7.41 (t, J = 7.6 Hz, 1H, H-4'), 7.07 (t, J = 7.6 Hz, 1H, H-5'''), 6.97 (d, J = 7.6 Hz, 1H, H-4'''), 4.06 (s, 2H, H-2''), 2.32 (s, 3H, CH3-2'''), 2.14 (s, 3H, CH3-3'''); EIMS (m/z): 375 [M+2]+, 373 [M]+, 253 [C10H6ClN2O2S]+, 225 [C9H6ClN2OS]+, 212 [C8H5ClN2OS] , 193 [C10H11NOS] , 179 [C8H4ClN2O]+, 153 [C7H4ClNO]+, 148 [C9H10NO]+, 139 [C7H4ClO]+, 137 [C7H4ClN]+, 121 [C8H11N]+, 111 [C6H4Cl]+, 105 [C8H9]+.

2-((5-(2-Chlorophenyl)-1,3,4-Oxadiazol-2- yl)sulfanyl)-N-(2,4-dimethylphenyl)acetamide (7b)

White amorphous solid; Yield: 82%; M.P: 120-122 C; Mol. formula: C18H16ClN3O2S; Mol. Wt.: 373; IR (KBr, vmax/cm-1): 3326 (N-H), 3077 (Ar C-H), 1663 (C=N), 1643 (C=O), 1592 (Ar C=C), 1222 (C-O-C), 684 (C-Cl), 630 (C-S); 1H-NMR (400 MHz, CDCl3, d/ppm): 8.73 (s, 1H, CONH), 7.90 (d, J = 7.6 Hz, 1H, H-6'), 7.68 (d, J = 7.6 Hz, 1H, H-6'''), 7.55 (d, J = 8.0 Hz, 1H, H-3'), 7.48 (t, J = 7.6 Hz, 1H, H-5'), 7.41 (t, J = 7.6 Hz, 1H, H-4'), 6.99 (d, J = 7.6 Hz, 1H, H-5'''), 6.96 (s, 1H, H-3'''), 4.06 (s, 2H, H- 2''), 2.25 (s, 3H, CH3-2'''), 2.22 (s, 3H, CH3-4'''); EIMS (m/z): 375 [M+2]+, 373 [M]+, 253 [C10H6ClN2O2S]+, 225 [C9H6ClN2OS]+, 212 [C8H5ClN2OS]+, 193 [C10H11NOS]+, 179 [C8H4ClN2O]+, 153 [C7H4ClNO]+, 148 [C9H10NO]+, 139 [C7H4ClO]+, 137 [C7H4ClN]+, 121 [C8H11N]+, 111 [C6H4Cl]+, 105 [C8H9]+.

2-((5-(2-Chlorophenyl)-1,3,4-Oxadiazol-2- yl)sulfanyl)-N-(2,5-dimethylphenyl)acetamide (7c)

White amorphous solid; Yield: 84%; M.P: 139-141 oC; Mol. formula: C18H16ClN3O2S; Mol. Wt.: 373; IR (KBr, vmax/cm-1): 3293 (N-H), 3069 (Ar C-H), 1669 (C=N), 1650 (C=O), 1599 (Ar C=C), 1197 (C-O-C), 680 (C-Cl), 623 (C-S); 1H-NMR (400 MHz, CDCl3, d/ppm): 8.73 (s, 1H, CONH), 7.90 (dd, J = 7.6, 2.4 Hz, 1H, H-6'), 7.53 (d, J = 8.0 Hz, 1H, H- 3'), 7.48 (dt, J = 7.6, 2.4 Hz, 1H, H-5'), 7.41 (t, J = 7.6 Hz, 1H, H-4'), 7.12 (s, 1H, H-6'''), 7.01 (d, J = 7.6 Hz, 1H, H-3'''), 6.87 (d, J = 8.0 Hz, 1H, H-4'''), 4.05 (s, 2H, H-2''), 2.29 (s, 3H, CH3-2'''), 2.21 (s, 3H, CH3- 5'''); EIMS (m/z): 375 [M+2]+, 373 [M]+, 253 [C10H6ClN2O2S]+, 225 [C9H6ClN2OS]+, 212 [C8H5ClN2OS]+, 193 [C10H11NOS]+, 179 [C8H4ClN2O]+, 153 [C7H4ClNO]+, 148 [C9H10NO]+, 139 [C7H4ClO]+, 137 [C7H4ClN]+, 121 [C8H11N]+, 111 [C6H4Cl]+, 105 [C8H9]+.

2-((5-(2-Chlorophenyl)-1,3,4-Oxadiazol-2- yl)sulfanyl)-N-(2,6-dimethylphenyl)acetamide (7d)

White amorphous solid; Yield: 80%; M.P: 149-151 oC; Mol. formula: C18H16ClN3O2S; Mol. Wt.: 373; IR (KBr, vmax/cm-1): 3290 (N-H), 3078 (Ar C-H), 1682 (C=N), 1639 (C=O), 1543 (Ar C=C), 1210 (C-O-C), 685 (C-Cl), 613 (C-S); 1H-NMR (400 MHz, CDCl3, d/ppm): 8.48 (s, 1H, CONH), 7.92 (dd, J = 7.6, 1.6 Hz, 1H, H-6'), 7.56 (dd, J = 8.0, 1.2 Hz, 1H, H-3'), 7.48 (dt, J = 8.0, 1.6 Hz, 1H, H-5'), 7.42 (dt, J = 8.0, 1.2 Hz, 1H, H-4'), 7.06-7.03 (m, 3H, H- 3''' to H-5'''), 4.09 (s, 2H, H-2''), 2.16 (s, 6H, CH3-2''', CH3-6'''); EIMS (m/z): 375 [M+2]+, 373 [M]+, 253 [C10H6ClN2O2S]+, 225 [C9H6ClN2OS]+, 212 [C8H5ClN2OS]+, 193 [C10H11NOS]+, 179 [C8H4ClN2O]+, 153 [C7H4ClNO]+, 148 [C9H10NO]+, 139 [C7H4ClO]+, 137 [C7H4ClN]+, 121 [C8H11N]+, 111 [C6H4Cl]+, 105 [C8H9]+.

2-((5-(2-Chlorophenyl)-1,3,4-Oxadiazol-2- yl)sulfanyl)-N-(3,4-dimethylphenyl)acetamide (7e)

White amorphous solid; Yield: 81%; M.P: 86-88 oC; Mol. formula: C18H16ClN3O2S; Mol. Wt.: 373; IR (KBr, vmax/cm-1): 3311 (N-H), 3110 (Ar C- H), 1655 (C=N), 1641 (C=O), 1585 (Ar C=C), 1200 (C-O-C), 677 (C-Cl), 616 (C-S); 1H-NMR (400 MHz, CDCl 3 , d/ppm): 8.98 (s, 1H, CONH), 7.91 (d, J = 7.6 Hz, 1H, H-6'), 7.55 (d, J = 7.6 Hz, 1H, H-3'), 7.48 (t, J = 7.6 Hz, 1H, H-5'), 7.41 (t, J = 7.6 Hz, 1H, H-4'), 7.29 (s, 1H, H-2'''), 7.26 (d, J = 8.0 Hz, 1H, H-6'''), 7.03 (d, J = 8.0 Hz, 1H, H-5'''), 4.00 (s, 2H, H-2''), 2.27 (s, 3H, CH 3 -3'''), 2.19 (s, 3H, CH 3 -4'''); EIMS (m/z): 375 [M+2] + , 373 [M] + , 253 [C 10 H 6 ClN 2 O 2 S] + , 225 [C 9 H 6 ClN 2 OS] + , 212 [C 8 H 5 ClN 2 OS] + , 193 [C 10 H 11 NOS] + , 179 [C 8 H 4 ClN 2 O] + , 153 [C 7 H 4 ClNO] + , 148 [C 9 H 10 NO] + , 139 [C 7 H 4 ClO] + , 137 [C 7 H 4 ClN] + , 121 [C 8 H 11 N] + , 111 [C 6 H 4 Cl] + , 105 [C 8 H 9 ] + .

2-((5-(2-Chlorophenyl)-1,3,4-Oxadiazol-2- yl)sulfanyl)-N-(3,5-dimethylphenyl)acetamide (7f)

White amorphous solid; Yield: 89%; M.P: 137-139 o C; Mol. formula: C 18 H 16 ClN 3 O 2 S; Mol. Wt.: 373; IR (KBr, v max /cm -1 ): 3377 (N-H), 3060 (Ar C-H), 1680 (C=N), 1655 (C=O), 1589 (Ar C=C), 1295 (C-O-C), 690 (C-Cl), 629 (C-S); 1 H-NMR (400 MHz, CDCl 3 , d/ppm): 9.00 (s, 1H, CONH), 7.92 (dd, J = 7.6, 1.6 Hz, 1H, H-6'), 7.54 (d, J = 8.0 Hz, 1H, H- 3'), 7.49 (dt, J = 7.6, 1.6 Hz, 1H, H-5'), 7.42 (t, J = 8.0 Hz, 1H, H-4'), 7.17 (s, 2H, H-2''', H-6'''), 6.74 (s, 1H, H-4'''), 4.00 (s, 2H, H-2''), 2.26 (s, 6H, CH 3 -3''', CH 3 -5'''); EIMS (m/z): 375 [M+2] + , 373 [M] + , 253 [C 10 H 6 ClN 2 O 2 S] + , 225 [C 9 H 6 ClN 2 OS] + , 212 [C 8 H 5 ClN 2 OS] + , 193 [C 10 H 11 NOS] + , 179 [C 8 H 4 ClN 2 O] + , 153 [C 7 H 4 ClNO] + , 148 [C 9 H 10 NO] + , 139 [C 7 H 4 ClO] + , 137 [C 7 H 4 ClN] + , 121 [C 8 H 11 N] + , 111 [C 6 H 4 Cl] + , 105 [C 8 H 9 ] + .

Synthesis of Ethyl 2-(5-(2-chlorophenyl)-1,3,4- Oxadiazol-2-ylthio)acetate (8)

The compound 4 (0.04 mol) was dissolved in 15 mL DMF in a 100 mL RB flask at RT followed by LiH (0.16 g) and set to stir for 30 minutes. Ethyl 2-bromoacetate (0.04 mol) was added and stirred for 3-4 hours. After supervision by TLC, excess of cold distilled water was added to precipitate the product. The precipitated product was collected through filtration, washed by distilled water and dried. White amorphous solid; Yield: 81%; M.P.: 176-178 o C; Mol. formula: C 12 H 11 ClN 2 O 3 S; Mol. Wt.: 298; IR (KBr, v max /cm -1 ):

3146 (Ar C-H), 1748 (C=O), 1679 (C=N), 1602 (Ar C=C), 1261 (C-O-C), 706 (C-Cl), 607 (C-S); 1 H-NMR (600 MHz, DMSO, d/ppm): 7.94 (dd, J = 7.8, 1.2 Hz, 1H, H-6'), 7.57 (d, J = 8.4 Hz, 1H, H-3'), 7.44 (dt, J = 7.8, 1.8 Hz, 1H, H-5'), 7.39 (dt, J = 7.8, 1.2 Hz, 1H, H-4'), 4.63 (s, 2H, H-2''), 3.96 (q, J = 7.2 Hz, 2H, -OCH 2 CH 3 ), 1.03 (t, J = 7.2 Hz, 3H, -OCH 2 CH 3 ); EIMS (m/z): 300 [M+2] + , 298 [M] + , 253 [C 10 H 6 ClN 2 O 2 S] + , 225 [C 9 H 6 ClN 2 OS] + , 212 [C 8 H 5 ClN 2 OS] + , 179 [C 8 H 4 ClN 2 O] + , 153 [C 7 H 4 ClNO] + , 139 [C 7 H 4 ClO] + , 137 [C 7 H 4 ClN] + , 111 [C 6 H 4 Cl] + , 85 [C 4 H 2 Cl] + , 51 [C 4 H 3 ] + .

Synthesis of 2-(5-(2-Chlorophenyl)-1,3,4-Oxadiazol- 2-ylthio)acetohydrazide (9)

The ester 8 (0.04 mol) was dissolved in 30 mL methanol in a 100 mL RB flask. 2.6 mL 80% hydrazine hydrate was added followed by stirring for 2-3 hours at RT. TLC was processed to monitor reaction completion. The product was acquired by addition of excess cold distilled water and separated by filtration, washed by n-hexane and dried. White amorphous solid; Yield: 81%; M.P.: 180-182 o C;

Mol. formula: C 10 H 9 ClN 4 O 2 S; Mol. Wt.: 284; IR (KBr, v max /cm -1 ): 3373 (N-H), 3092 (Ar C-H), 1665 (C=O), 1691 (C=N), 1617 (Ar C=C), 1235 (C-O-C), 711 (C-Cl), 602 (C-S); 1 H-NMR (600 MHz, DMSO, d/ppm): 9.88 (s, 1H, CONH), 8.76 (s, 2H, N-H), 7.92 (d, J = 8.4 Hz, 1H, H-6'), 7.56 (d, J = 7.8 Hz, 1H, H- 3'), 7.44 (t, J = 8.4 Hz, 1H, H-5'), 7.42 (t, J = 7.8 Hz, 1H, H-4'), 4.68 (s, 2H, H-2''); EIMS (m/z): 286 [M+2] + , 284 [M] + , 253 [C 10 H 6 ClN 2 O 2 S] + , 225 [C 9 H 6 ClN 2 OS] + , 212 [C 8 H 5 ClN 2 OS] + , 179 [C 8 H 4 ClN 2 O] + , 153 [C 7 H 4 ClNO] + , 139 [C 7 H 4 ClO] + , 137 [C 7 H 4 ClN] + , 111 [C 6 H 4 Cl] + , 85 [C 4 H 2 Cl] + , 51 [C 4 H 3 ] + .

Synthesis of N'-Substituted-2-(5-(2-chlorophenyl)- 1,3,4-Oxadiazol-2-ylthio)acetohydrazide (11a-g)

The molecule 9 (0.004 mol) was dissolved in 16 mL methanol in a 50 mL RB flask at RT. The equimolar substituted benzaldehydes (10a-g; 0.004 mol) were added followed by stirring for 2 hours. A few drops of glacial acetic acid were also added as catalyst. The reaction was supervised by TLC. At completion, excess of cold distilled water was added till precipitation. The precipitates were stored after filtration, washed with distilled water and finally dried for analysis.

N'-Benzylidene-2-(5-(2-chlorophenyl)-1,3,4- Oxadiazol-2-ylthio)acetohydrazide (11a)

White amorphous solid; Yield: 78%; M.P.: 156-158 o C; Mol. formula: C 17 H 13 ClN 4 O 2 S; Mol. Wt.: 372; IR (KBr, v max /cm -1 ): 3048 (Ar C-H), 1663 (C=N), 1618 (Ar C=C), 1245 (C-O-C), 696 (C-Cl), 624 (C-S); 1 H-NMR (600 MHz, DMSO, d/ppm): 11.84 (s, 1H, CONH), 8.08 (s, 1H, H-7'''), 7.97 (dd, J = 7.8, 1.2 Hz, 2H, H-2''', H-6'''), 7.91 (dd, J = 8.4, 1.8 Hz, 1H, H-6'), 7.74-7.68 (m, 3H, H-3''' to H-5'''), 7.60 (d, J = 7.8 Hz, 1H, H-3'), 7.44 (dt, J = 7.2, 1.8 Hz, 1H, H-5'), 7.40 (t, J = 7.8 Hz, 1H, H-4'), 4.66 (s, 2H, H-2''); EIMS (m/z): 374 [M+2] + , 372 [M] + , 344 [M- CO] + , 253 [C 10 H 6 ClN 2 O 2 S] + , 225 [C 9 H 6 ClN 2 OS] + , 212 [C 8 H 5 ClN 2 OS] + , 179 [C 8 H 4 ClN 2 O] + , 153 [C 7 H 4 ClNO] + , 147 [C 8 H 7 N 2 O] + , 139 [C 7 H 4 ClO] + , 137 [C 7 H 4 ClN] + , 119 [C 7 H 7 N 2 ] + , 111 [C 6 H 4 Cl] + , 91 [C 7 H 7 ] + , 85 [C 4 H 2 Cl] + , 65 [C 5 H 5 ] + , 51 [C 4 H 3 ] + .

N'-(2-Methylbenzylidene)-2-(5-(2-chlorophenyl)- 1,3,4-Oxadiazol-2-ylthio)acetohydrazide (11b)

White amorphous solid; Yield: 82%; M.P.: 162-164 o C; Mol. formula: C 18 H 15 ClN 4 O 2 S; Mol. Wt.: 386; IR (KBr, v max /cm -1 ): 3067 (Ar C-H), 1652 (C=N), 1618 (Ar C=C), 1191 (C-O-C), 703 (C-Cl), 626 (C-S); 1 H-NMR (600 MHz, DMSO, d/ppm): 11.72 (s, 1H, CONH), 8.34 (s, 1H, H-7'''), 7.94 (dd, J = 8.4, 1.8 Hz, 1H, H-6'), 7.77 (dt, J = 9.0, 1.2 Hz, 1H, H-5'''), 7.74 (d, J = 7.2 Hz, 1H, H-6'''), 7.63 (d, J = 8.4 Hz, 1H, H-3'), 7.43 (dt, J = 7.8, 1.2 Hz, 1H, H- 5'), 7.31 (dt, J = 7.8, 1.2 Hz, 1H, H-4'''), 7.27 (t, J = 7.8 Hz, 1H, H-4'), 7.21 (d, J = 7.2 Hz, 1H, H-3'''), 4.69 (s, 2H, H-2''), 2.43 (s, 3H, CH 3 -2'''); EIMS (m/z): 388 [M+2] + , 386 [M] + , 253 [C 10 H 6 ClN 2 O 2 S] + , 225 [C 9 H 6 ClN 2 OS] + , 212 [C 8 H 5 ClN 2 OS] + , 179 [C 8 H 4 ClN 2 O] + , 161 [C 9 H 9 N 2 O] + , 153 [C 7 H 4 ClNO] + , 139 [C 7 H 4 ClO] + , 137 [C 7 H 4 ClN] + ,

133 [C 8 H 9 N 2 ] + , 111 [C 6 H 4 Cl] + , 105 [C 8 H 9 ] + , 85 [C 4 H 2 Cl] + , 65 [C 5 H 5 ] + , 51 [C 4 H 3 ] + .

N'-(3-Methylbenzylidene)-2-(5-(2-chlorophenyl)- 1,3,4-Oxadiazol-2-ylthio)acetohydrazide (11c)

White amorphous solid; Yield: 80%; M.P.: 158-160 o C; Mol. formula: C 18 H 15 ClN 4 O 2 S; Mol. Wt.: 386; IR (KBr, v max /cm -1 ): 3059 (Ar C-H), 1676 (C=N), 1604 (Ar C=C), 1269 (C-O-C), 699 (C-Cl), 615 (C-S); 1 H-NMR (600 MHz, DMSO, d/ppm): 11.82 (s, 1H, CONH), 8.02 (s, 1H, H-7'''), 7.91 (d, J = 8.4 Hz, 1H, H-6'), 7.60 (d, J = 7.8 Hz, 1H, H-6'''), 7.49 (d, J = 7.8 Hz, 1H, H-3'), 7.34 (t, J = 7.8 Hz, 1H, H-5'), 7.26 (t, J = 8.4 Hz, 1H, H-5'''), 7.17 (d, J = 8.4 Hz, 1H, H-4'''), 7.10 (t, J = 8.4 Hz, 1H, H-4'), 6.74 (s, 1H, H-2'''), 4.64 (s, 2H, H-2''), 2.33 (s, 3H, CH 3 -3'''); EIMS (m/z): 388 [M+2] + , 386 [M] + , 253 [C 10 H 6 ClN 2 O 2 S] + , 225 [C 9 H 6 ClN 2 OS] + , 212 [C 8 H 5 ClN 2 OS] + , 179 [C 8 H 4 ClN 2 O] + , 161 [C 9 H 9 N 2 O] + , 153 [C 7 H 4 ClNO] + , 139 [C 7 H 4 ClO] + , 137 [C 7 H 4 ClN] + ,

133 [C 8 H 9 N 2 ] + , 111 [C 6 H 4 Cl] + , 105 [C 8 H 9 ] + , 85 [C 4 H 2 Cl] + , 65 [C 5 H 5 ] + , 51 [C 4 H 3 ] + .

N'-(4-Methylbenzylidene)-2-(5-(2-chlorophenyl)- 1,3,4-Oxadiazol-2-ylthio)acetohydrazide (11d)

White amorphous solid; Yield: 79%; M.P.: 166-168 o C; Mol. formula: C 18 H 15 ClN 4 O 2 S; Mol. Wt.: 386; IR (KBr, v max /cm -1 ): 3045 (Ar C-H), 1667 (C=N), 1613 (Ar C=C), 1217 (C-O-C), 688 (C-Cl), 621 (C-S); 1 H-NMR (600 MHz, DMSO, d/ppm): 11.76 (s, 1H, CONH), 8.02 (s, 1H, H-7'''), 7.95 (d, J = 8.4 Hz, 1H, H-6'), 7.72 (d, J = 7.8 Hz, 1H, H-3'), 7.67 (t, J = 8.4 Hz, 1H, H-5'), 7.62 (d, J = 8.4 Hz, 2H, H-2''', H-6'''), 7.57 (t, J = 8.4 Hz, 1H, H-4'), 7.22 (d, J = 8.4 Hz, 2H, H-3''', H-5'''), 4.65 (s, 2H, H-2''), 2.35 (s, 3H, CH 3 -4'''); EIMS (m/z): 388 [M+2] + , 386 [M] + , 253 [C 10 H 6 ClN 2 O 2 S] + , 225 [C 9 H 6 ClN 2 OS] + , 212 [C 8 H 5 ClN 2 OS] + , 179 [C 8 H 4 ClN 2 O] + , 161 [C 9 H 9 N 2 O] + , 153 [C 7 H 4 ClNO] + , 139 [C 7 H 4 ClO] + , 137 [C 7 H 4 ClN] + , 133 [C 8 H 9 N 2 ] + , 111 [C 6 H 4 Cl] + , 105 [C 8 H 9 ] + , 85 [C 4 H 2 Cl] + , 65 [C 5 H 5 ] + , 51 [C 4 H 3 ] + .

N'-(2-Hydroxybenzylidene)-2-(5-(2-chlorophenyl)- 1,3,4-Oxadiazol-2-ylthio)acetohydrazide (11e)

Cream white amorphous solid; Yield: 84%; M.P.: 208-210 o C; Mol. formula: C 17 H 13 ClN 4 O 3 S; Mol. Wt.: 388; IR (KBr, v max /cm -1 ): 3065 (Ar C-H), 1677 (C=N), 1616 (Ar C=C), 1249 (C-O-C), 684 (C- Cl), 621 (C-S); 1 H-NMR (600 MHz, DMSO, d/ppm): 11.75 (s, 1H, CONH), 8.34 (s, 1H, H-7'''), 7.97 (d, J = 7.8 Hz, 1H, H-6'), 7.92 (d, J = 7.8 Hz, 1H, H-6'''), 7.71 (d, J = 8.4 Hz, 1H, H-3'), 7.62 (t, J = 8.4 Hz, 1H, H-5'), 7.59 (t, J = 7.8 Hz, 1H, H-4'), 7.54 (dd, J = 7.8, 1.8 Hz, 1H, H-3'''), 7.23 (dt, J = 7.8, 1.8 Hz, 1H, H-4'''), 6.90 (dt, J = 7.2, 1.8 Hz, 1H, H-5'''), 4.67 (s, 2H, H-2''); EIMS (m/z): 390 [M+2] + , 388 [M] + , 253 [C 10 H 6 ClN 2 O 2 S] + , 225 [C 9 H 6 ClN 2 OS] + , 212 [C 8 H 5 ClN 2 OS] + , 179 [C 8 H 4 ClN 2 O] + , 163 [C 8 H 7 N 2 O 2 ] + , 153 [C 7 H 4 ClNO] + , 139 [C 7 H 4 ClO] + , 137 [C 7 H 4 ClN] + , 135 [C 7 H 7 N 2 O] + ,

111 [C 6 H 4 Cl] + , 107 [C 7 H 7 O] + , 85 [C 4 H 2 Cl] + , 65 [C 5 H 5 ] + , 51 [C 4 H 3 ] + .

N'-(3-Hydroxybenzylidene)-2-(5-(2-chlorophenyl)- 1,3,4-Oxadiazol-2-ylthio)acetohydrazide (11f)

Cream white amorphous solid; Yield: 84%; M.P.: 214-216 o C; Mol. formula: C 17 H 13 ClN 4 O 3 S; Mol. Wt.: 388; IR (KBr, v max /cm -1 ): 3036 (Ar C-H), 1683 (C=N), 1614 (Ar C=C), 1216 (C-O-C), 704 (C- Cl), 618 (C-S); 1 H-NMR (600 MHz, DMSO, d/ppm): 11.78 (s, 1H, CONH), 8.47 (s, 1H, HO-3'''), 8.13 (s, 1H, H-7'''), 7.95 (dd, J = 8.4, 1.8 Hz, 1H, H-6'), 7.69 (dd, J = 7.8, 1.2 Hz, 1H, H-6'''), 7.60 (d, J = 7.8 Hz, 1H, H-3'), 7.23 (t, J = 7.8 Hz, 1H, H-5'''), 7.14 (t, J = 8.4 Hz, 1H, H-5'), 7.11 (t, J = 7.8 Hz, 1H, H-4'), 6.85 (dd, J = 9.6, 1.8 Hz, 1H, H-4'''), 6.79 (s, 1H, H-2'''), 4.68 (s, 2H, H-2''); EIMS (m/z): 390 [M+2] + , 388 [M] + , 253 [C 10 H 6 ClN 2 O 2 S] + , 225 [C 9 H 6 ClN 2 OS] + , 212 [C 8 H 5 ClN 2 OS] + , 179 [C 8 H 4 ClN 2 O] + , 163 [C 8 H 7 N 2 O 2 ] + , 153 [C 7 H 4 ClNO] + , 139 [C 7 H 4 ClO] + , 137 [C 7 H 4 ClN] + , 135 [C 7 H 7 N 2 O] + , 111 [C 6 H 4 Cl] + , 107 [C 7 H 7 O] + , 85 [C 4 H 2 Cl] + , 65 [C 5 H 5 ] + , 51 [C 4 H 3 ] + .

N'-(4-Hydroxybenzylidene)-2-(5-(2-chlorophenyl)- 1,3,4-Oxadiazol-2-ylthio)acetohydrazide (11g)

Shiny white crystalline solid; Yield: 78%; M.P.: 230-232 o C; Mol. formula: C 17 H 13 ClN 4 O 3 S; Mol. Wt.: 388; IR (KBr, v max /cm -1 ): 3043 (Ar C-H), 1679 (C=N), 1601 (Ar C=C), 1273 (C-O-C), 705 (C- Cl), 625 (C-S); 1 H-NMR (600 MHz, DMSO, d/ppm): 11.61 (s, 1H, CONH), 9.94 (s, 1H, HO-4'''), 8.12 (s, 1H, H-7'''), 7.96 (dd, J = 8.4, 1.8 Hz, 1H, H-6'), 7.62 (d, J = 7.8 Hz, 1H, H-3'), 7.54 (d, J = 8.4 Hz, 2H, H- 2''', H-6'''), 7.36 (t, J = 8.4 Hz, 1H, H-5'), 7.17 (t, J = 9.0 Hz, 1H, H-4'), 6.78 (d, J = 8.4 Hz, 2H, H-3''', H- 5'''), 4.64 (s, 2H, H-2''); EIMS (m/z): 390 [M+2] + , 388 [M] + , 253 [C 10 H 6 ClN 2 O 2 S] + , 225 [C 9 H 6 ClN 2 OS] + , 212 [C 8 H 5 ClN 2 OS] + , 179 [C 8 H 4 ClN 2 O] + , 163 [C 8 H 7 N 2 O 2 ] + , 153 [C 7 H 4 ClNO] + , 139 [C 7 H 4 ClO] + , 137 [C 7 H 4 ClN] + , 135 [C 7 H 7 N 2 O] + , 111 [C 6 H 4 Cl] + , 107 [C 7 H 7 O] + , 85 [C 4 H 2 Cl] + , 65 [C 5 H 5 ] + , 51 [C 4 H 3 ] + .

Antibacterial Activity Assay

The antibacterial activity was evaluated by using the referenced method but with minor modifications [18-20]. The antibacterial activity was carried out in sterile 96-wells microplates under aseptic circumstances. This technique is based on the principle that as the microbial growth increases in a log phase of growth, the number of microbial cells multiply exponentially which in turn increases absorbance of broth medium. Micro organisms used in this study included; three Gram-negative bacteria i.e. Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi and two Gram-positive bacteria namely Bacillus subtilis and Staphylococcus aureus. The tested strains were nourished on stock agar culture medium. The samples being analyzed were diluted in suitable solvents and 20 g of each sample was pipetted into every well.

Fresh bacterial culture maintained overnight was suitably diluted with fresh nutrient broth and was 180 L quantity of this bacterial culture was poured into every well. The starting absorbance of the culture was strictly maintained at 540 nm between 0.12-0.19. The total volume kept in each well was 200 L. These microplates covered with lids were incubated for 16- 24 hours at 37 C. Before and after incubation, the absorbance was measured at 540 nm using microplate reader, and index of bacterial growth was noted by the difference in absorbance before and after incubation. The formula for calculating the percentage inhibition is:

Equation

where, X = Absorbance in control, containing bacterial culture without test sample; Y = Absorbance of bacterial culture with test sample. Results are mean of three sets of test samples (n=3, SEM). Standard used was Ciprofloxacin. Suitable dilutions ranging from 5-30 g/ well were used to measure the Minimum inhibitory concentration (MIC). EZ-Fitz Perrella Scientific Inc. Amherst USA software was used to calculate the results.

Enzyme Inhibition Activity Assay

The enzyme inhibition activity was assayed against lipoxygenase enzyme because of its role in inflammatory diseases by the reported method with minor modifications [14,15]. 200 A- 10 -6 L volume of mixture was developed by mixing 150 A- 10 -6 L sodium phosphate buffers (0.1 M, pH = 8), 10 A- 10 -6 L sample and 15 A- 10 -6 L enzyme (600 units well -1 ). After thoroughly mixing, mixture was pre-read at 234 nm and then pre-incubated for 0.16 hours at 25 C.

Then 25 A- 10 -6 L substrate was added to initiate the reaction. The absorbance variation was again noted after 0.1 hour at 234 nm by 96-well plate reader, Synergy HT, Biotek, USA. All the reactions were performed in triplicate. Baicalein (5 A- 10 -4 M well -1 ) was a positive control. The inhibition (%age) was calculated by the same formula as discussed for antibacterial activity assay. But here, X = Total enzyme activity without inhibitor and Y = Activity in the presence of test compound. IC 50 values were also estimated by EZFit Enzyme Kinetics software (Perrella Scientific Inc. Amherst, USA).

Statistical Analysis

The results are presented as mean SEM for triplicate calculations after statistical analysis executed by MS Excel 2010.

Results and Discussion

The S-substituted derivatives, 7a-f and 11a- g, of 4 have been synthesized with an aim to evaluate their antibacterial activity against Gram-positive and Gram-negative bacteria and enzyme inhibition activity against lipoxygenase enzyme. The procedures, reaction conditions and spectral characterization of all the synthesized molecules are illustrated in the experimental section. The proposed structures of synthesized molecules have been elucidated by their spectral data analysis and it was found that 7a-f possessed better but 11a-g moderate antibacterial and enzyme inhibition activities.

Chemistry

The significant biological activities of 1,3,4- Oxadiazoles, amides and Schiff bases prompted us to synthesize some new molecules bearing all these functionalities. Using 2-chlorobenzoic acid (1) as precursor, 5-(2-chlorophenyl)-1,3,4-Oxadiazol-2- thiol (4) was prepared after successive step synthesis of ethyl 2-chlorobenzoate (2) and 2- chlorobenzohydrazide (3). The substituted aromatic amines, 5a-f, were treated with 2-bromoacetyl bromide in a weak basic medium to yield corresponding N-substituted-2-bromoacetamides, 6a- f, as electrophiles. The molecules, 6a-f, were further subjected to electrophilic substitution reaction with 4 in an aprotic polar solvent using LiH as an activator.

The acidic proton of thiol group in 4 was replaced by ethyl 2-bromoacetate in LiH/DMF to afford 8 which was stepped to carbohydrazide, 9, by hydrazine in methanol. The compound, 9 was treated with various substituted benzaldehydes (10a-g) in methanol using few drops glacial acetic acid as a catalyst to synthesize the target molecules, 11a-g. The detailed procedures with appropriate conditions are illustrated in the experimental section.

Compound 7a was synthesized as white amorphous solid with melting point of 152-154 o C. The molecular formula C 18 H 16 ClN 3 O 2 S was evaluated by proton integration curve in 1 H-NMR spectrum and [M] + in EIMS at m/z 373. The EIMS presented some other distinct peak at m/z 226 for 2- (2-chlorophenyl)-5-(methylthio)-1,3,4-Oxadiazole. The IR spectrum elaborated definite stretching frequencies at 3347 (N-H), 3095 (Ar C-H), 1683 (C=N), 1642 (C=O), 1590 (Ar C=C), 1270 (C-O-C), 680 (C-Cl) and 623 (C-S) for indicated functionalities of the molecule. Four protons of 2-chlorophenyl ring resonated at d 7.92 (d, J = 8.0 Hz, 1H, H-6'), 7.56 (d, J = 8.0 Hz, 1H, H-3'), 7.49 (t, J = 7.6 Hz, 1H, H-5') and 7.41 (t, J = 7.6 Hz, 1H, H-4') while three protons of 2,3-dimethylphenyl ring at d 7.54 (d, J = 7.6 Hz, 1H, H-6'''), 7.07 (t, J = 7.6 Hz, 1H, H-5''') and 6.97 (d, J = 7.6 Hz, 1H, H-4''').

Three signals of singlet appearing at d 4.06 (s, 2H, H-2''), 2.32 (s, 3H, CH 3 - 2''') and 2.14 (s, 3H, CH 3 -3''') were assigned to two methylene and six dimethyl protons of the molecule. One singlet was also observed at d 8.73 (s, 1H, CONH) for the proton attached to nitrogen of acetamoyl group. The elaborated structural analysis demonstrated the name of 7a as 2-((5-(2- chlorophenyl)-1,3,4-Oxadiazol-2-yl)sulfanyl)-N-(2,3- dimethylphenyl)acetamide. Likewise the compound 11a also executed the same splitting pattern for 2- chlorophenyl group and the other resonating signals were as follows, d 8.08 (s, 1H, H-7'''), 7.97 (dd, J = 7.8, 1.2 Hz, 2H, H-2''', H-6''') and 7.74-7.68 (m, 3H, H-3''' to H-5''') for benzylidene protons, d 11.84 (s, 1H, CONH) for carbamoyl proton and d 4.66 (s, 2H, H-2'') for methylene protons.

The EIMS spectrum showed the characteristics peaks at m/z 147, 119, 91 and 65 for the S-substituted group. The mass fragmentation pattern of 11a has been elaborated in Fig. 1. The functional groups present in the molecule were well supported by IR absorption bands.

Antibacterial Activity (in vitro)

The % age inhibition and MIC values of antibacterial activity have been demonstrated in Table 1 and Table 2 respectively. The both series of compounds have shown moderately better activity against the bacterial strains of Gram-positive and Gram-negative bacteria. Ciprofloxacin was taken as reference standard. The compounds, 7a-e and 11e executed significant activity as predicted by their low MIC values against S. typhi except 7f and 11a-c,f,g, presenting moderately low potential. The most efficient molecule against this strain was 7e with MIC of 9.011.79 g/mL as compared with 8.221.62 g/mL for reference. The molecule, 11d showed no activity at all against this strain.

Against E. coli, 7a-b and 11e were found to possess low MIC values as 10.180.93, 10.091.79 and 11.515.00 g/mL respectively relative to that of reference as 8.872.00 g/mL. and so good inhibitory potential.

Four compounds, 7a-d exhibited better potential against P. aeruginosa and 11d,f-g remained inactive. The most better was 7b executing MIC of 9.531.44 g/mL in comparison of 8.902.50 g/mL. Compounds, 7a-e showed significant inhibition but 7f and 11a-b,d showed no MIC values as compared to reference against B. subtilis. Against S. aureus, 7a- d,f remained better inhibitors but 7e and 11a,e showed moderate activity and the remaining ones were inactive. The MIC results rendered the series 7a-f more efficient against these Gram-positive and Gram-negative bacteria as compared to the series 11a-g.

Table-1: The %age inhibition of antimicrobial activity of the synthesized compounds.

###%age Inhibition

###Compound###Gram-negative bacteria###Gram-positive bacteria

###S. typhi###E. coli###P. aeruginosa###B. subtilis###S. aureus

###7a###84.13 0.29###75.503.70###77.491.43###69.141.41###85.41 0.41

###7b###86.88 0.21###79.304.21###80.892.19###63.143.77###79.74 0.87

###7c###75.212.79###55.251.85###78.360.21###72.821.36###70.87 1.79

###7d###83.250.33###60.402.70###81.720.99###69.503.59###82.50 3.11

###7e###80.083.00###59.053.05###69.180.73###64.362.64###54.234.44

###7f###59.79 2.63###54.404.00###56.622.32###39.412.50###66.941.63

###11a###57.240.36###60.130.13###55.650.05###49.054.59###54.454.23

###11b###54.114.01###63.250.92###52.151.35###48.055.00###45.995.00

###11c###56.151.46###57.921.67###52.350.95###51.412.50###38.195.00

###11d###37.245.00###51.960.21###49.755.00###40.824.45###12.972.53

###11e###67.032.76###65.134.96###52.101.50###57.771.86###51.811.81

###11f###59.320.47###57.540.46###48.350.65###51.411.77###44.953.08

###11g###57.143.70###48.215.00###45.652.05###55.861.32###40.330.66

###Ciprofloxacin###90.42 0.92###90.990.15###91.940.86###91.051.18###91.11 0.26

Table-2: The MIC values of antimicrobial activity of the synthesized compounds.

###MIC ( g/mL )

###Compound###Gram-negative bacteria###Gram-positive bacteria

###S. typhi###E. coli###P. aeruginosa###B. subtilis###S. aureus

###7a###9.72 0.98###10.180.93###10.050.52###9.451.44###9.65 2.29

###7b###9.25 0.38###10.091.79###9.531.44###10.340.06###10.83 0.79

###7c###9.651.09###15.221.36###11.732.81###9.872.39###10.26 0.79

###7d###9.420.49###13.910.36###9.990.05###10.871.61###9.13 1.57

###7e###9.011.79###14.842.14###13.613.12###10.733.17###18.600.72

###7f###16.450.76###17.961.21###15.470.62###-###11.931.07

###11a###12.412.32###16.884.64###15.101.83###-###12.435.00

###11b###17.041.76###15.265.00###17.581.25###-###-

###11c###14.712.04###13.021.34###16.801.25###18.132.04###-

###11d###-###18.572.08###-###-###-

###11e###10.651.31###11.515.00###15.083.50###12.781.65###17.031.76

###11f###12.731.95###15.381.87###-###18.284.37###-

###11g###13.472.37###-###-###17.032.76###-

###Ciprofloxacin###8.221.62###8.872.00###8.902.50###9.021.84###9.321.65

Enzyme Inhibition Activity (in vitro)

All the synthesized molecules have been investigated for lipoxygenase enzyme inhibition activity and their results are presented as %age inhibition and IC50 (concentration for 50% inhibition) values are given in Table-3 with reference to Baicalein, the standard. All the molecules of 7a-f series executed better activity except 7f. The molecules 7a and 7d showed the lowest IC50 values, that is, 28.40.11 and 57.60.51 M relative to reference having IC50 of 22.41.3 M. The molecule 7a, bearing 2,3-dimethylphenyl group, has comparable activity but 7d, bearing 2,6- dimethylphenyl group, has 50% inhibitory action. Among the whole series, 7a-f, the substitution at 3rd, 4th and 5th position of benzene ring attached to nitrogen of acetamoyl resulted in decrement in activity but the remaining substitutions proved to be better inhibitors.

This can be argued here that these substitutions resulted in variation in fitting to active site of enzyme and so different inhibitory potential. The molecules of series, 11a-g, exhibited the least activity and the most probably because of their large size hindering the fitting to active site of enzyme. The only two molecules, 11a and 11f presented the very moderate activity with somewhat high IC50 values.

Table-3: The IC50 values of enzyme inhibition activity of the synthesized compounds.

Compound

###Lipoxygenase enzyme

###Conc. (mM)###Inhibition (%)###IC50 (M)

7a###0.5###79.180.32###28.40.11

7b###0.5###89.290.68###135.31.47

7c###0.5###62.240.33###268.91.28

7d###0.5###94.120.62###57.60.51

7e###0.25###86.670.29###125.41.28

7f###0.5###38.240.18###-

11a###0.5###61.610.12###242.30.13

11b###0.5###90.740.85###-

11c###0.5###45.910.77###greater than 500

11d###0.5###27.710.38###-

11e###0.5###43.600.65###greater than 500

11f###0.5###71.110.76###351.70.45

11g###0.5###20.110.35###-

Baicalein###0.5###93.791.27###22.41.3

Conclusion

Two series bearing acetamoyl and azomethine groups in addition to 1,3,4-Oxadiazole ring have been synthesized and evaluated for antibacterial potential against Gram-positive and Gram-negative bacteria; and lipoxygenase enzyme inhibition activity. The series 7a-f bearing acetamoyl functionality has been found to be more effective than the other series, 11a-g. The molecules, 7a-e and 11e were the inhibitors of all the bacterial strains taken into account and 7a-e were the talented inhibitors of lipoxygenase enzyme. It can be deduced that larger size of the molecules may have some negative effect on the antibacterial and enzyme inhibition potential. These activities of such type of molecules need to be tested for mono-substitutions for series 7 molecules and also di-substituted for series 11 molecules. The molecules, 7a-e and 11e can be further tested for in vivo activity for pharmacological applications.

Acknowledgement

The authors acknowledge the Higher Education Commission (HEC) of Pakistan for the fiscal aid.

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Publication:Journal of the Chemical Society of Pakistan
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Date:Aug 31, 2015
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