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Synthesis and Biological Significances of New Heterocyclic Compounds Containing 5-(4-Chlorobenzyl)-3-(4-Chlorophenyl)-1H-1,2,4-triazol Ring.

Byline: Yusuf Ozdemir, Ergun Gultekin and Olcay Bekircan

Summary: In this study, some novel 5-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol derivatives were synthesized starting from 4-amino-3-(4-chlorobenzyl)-5-(4-chlorophenyl)-4H-1,2,4-triazole (1). The structures of the synthesized compounds were clarified using IR, 1H NMR, 13C NMR, elemental analyses and mass spectral techniques. The synthesized compounds were tested for antimicrobial and anti-lipase inhibitory activities. Among the tested substances, compounds 3, 8c, 8d, 8e and 9d exhibited good activity against Gram-positive bacteria. Furthermore, all the synthesized compounds were investigated with regard to pancreatic lipase inhibition activity, and compounds 5e, 8b, and 9b showed a considerable anti-lipase activity at various concentrations.

Keywords: 1,2,4-Triazole; Schiff base; Mannich base, Antimicrobial activity; Anti-lipase inhibitory activity.

Introduction

Triazole is a quite important heterocyclic five membered ring and possesses aromaticity and rich electrons. Depending on the bonding location of the nitrogen atom, the triazoles exist in two isomeric forms stated 1,2,3-triazole and 1,2,4-triazole [1]. 1,2,4-triazoles known to be pharmacologically more important structures than 1,2,3-triazoles [2]. 1,2,4-Triazole derivatives have attracted a wide attention of many researchers in search for new therapeutic molecules [3]. Presence of 1,2,4-triazole nucleus in numerous categories of therapeutic agents such as antioxidant [4], anti-inflammatory [5], anticancer [6], antimicrobial [7], anti-tubercular [8], antidiabetics [9], antidepressant [10], anticonvulsant [11], analgesic [12], anti-HIV [13], enzyme inhibitory [14-16], antiviral [17], insecticide [18] and fungicide [19] has made it an essential for development of new therapeutic agents.

In addition to these, there are a number of 1,2,4-triazole-containing drugs on the market like letrozole, vorozole and anastrozole (antitumor) [20], ribavirin, viramidine (antiviral) [21], rizatriptan (antimigrane) [22], fluconazole, itraconazole, terconazole (antifungal) [23], loreclezole (sedative and anticonvulsant) [24], hexaconazole, myclobutanil, triadimefon and tebuconazole (fungicide) [25]. Furthermore, 1,2,4-triazoles bearing azomethine (C=N) and hydrazide-hydrazone (-CO-NH-N=CH-) functional groups have potential applications in organic synthesis, medicinal and coordination chemistry [26]. They are also use as catalysts [27], pigments and dyes [28], polymer stabilizers [29], acid-base indicator [30] and corrosion inhibitors [31]. On the other hand, Mannich bases containing N-substitue phenyl piperazine moieties are important structural motif and consider as biologically active compounds [32].

Also, antidepressant drugs are available carrying 1,2,4-triazole and phenyl piperazine ring. For instance, trazodone, etoperidone and nefazodone are used for the treatment of major depressive disorder [33-35]. Drugs such as lidoflazin, prazosin, and urapidil, including the piperazine ring, are also used in cardiovascular treatments [36-37]. In the light of this information, we aimed to synthesize new heterocyclic compounds containing the 1,2,4-triazole ring and to identify the antimicrobial and anti-lipase inhibitory activities of the synthesized compounds.

Experimental

General

Melting points were determined in open capillaries on a Gallenkamp Electrothermal digital melting point apparatus. FT-IR spectra were recorded in a Perkin-Elmer 1600 Frontier FT-IR spectrophotometer using attenuated total reflection (ATR) accessory. 1H NMR and 13C NMR spectra were recorded in DMSO-d6 on a Bruker Avene II 400 MHZ NMR spectrometer (400 MHz for 1H and 100 MHz for 13C). The elemental analysis was performed on a Costech Elemental Combustion System CHNS-O elemental analyzer. All the compounds gave C, H and N analyses within +- 0.4 % of theoretical values. The mass spectra were taken on a Quattro LC-MS (70 eV) instrument. The progress of the reaction was monitored by thin layer chromatography (TLC) on silica gel plates (silica gel 60 F2.54, 0.2 mm thickness). The compound 1 was synthesized by the methods reported earlier [38].

Synthesis of 5-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazole (2)

To the mixture of compound 1 (0.01 mol) in an aqueous solution of hypophosphorous acid solution (50 wt.% in H2O) (30 mL), an aqueous solution of sodium nitrite (0.05 mol in 10 mL of water) was added slowly. Vigorous nitrogen evolution was observed during this addition and the mixture was stirred at room temperature for 1 hour. The precipitate formed was filtered, washed with water and recrystallized from ethanol:water (1:1) to afford the desired compound.

Yield 87%; m.p. 117-118 C; IR (ATR, max, cm-1): 3303 (NH), 1691, 1591 (C=N); 1H-NMR: I' 4.14 (s, 2H, benzyl CH2), Ar-H: [7.33-7.37 (m, 4H), 7.47-7.51 (m, 2H), 7.98 (d, 2H, J = 8.0 Hz)], 13.96 (s, 1H, NH); 13C-NMR: I' 31.67 (benzyl CH2), Ar-C: [127.91 (2CH), 128.99 (2CH), 129.64 (2CH), 130.68, 130.99 (2CH), 131.94, 133.88, 136.23], 156.43 (triazole C5), 160.67 (triazole C3); LC-MS m/z (%): 342.26 ([M+K]+, 4), 304.21 ([M]+, 100), 219.25 (7), 192.14 (18); Anal. Calcd. for C15H11Cl2N3 : C, 59.23; H, 3.65; N, 13.81. Found: C, 59.39; H, 3.31; N, 13.81.

Synthesis of ethyl 2-[5-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]acetate (3)

A solution of compound 2 (0.01 mol) in 100 mL absolute ethanol was refluxed with sodium (0.01 mol) in 100 mL absolute ethanol for 2 h. Then, ethyl bromoacetate (1.2 mL, 0.01 mol) was added and refluxed for an additional 8 h (monitoring by TLC). After evaporation of solvent under reduced pressure, a solid appeared. The crude product was recrystallized from ethanol:water (1:2) to afford the desired compound.

Yield 85%; m.p. 183-184 C; IR (ATR, max, cm-1): 1737 (C=O), 1596 (C=N), 1220 (C-O); 1H-NMR: I' 1.16 (t, 3H, J = 7.42 Hz, CH3), 4.08 (q, 2H, J = 7.42 Hz ,OCH2), 4.23 (s, 2H, benzyl-CH2), 5.27 (s, 2H, N-CH2), Ar-H: [7.34-7.40 (m, 4H), 7.51 (d, 2H, J = 8.4 Hz), 7.95 (d, 2H, J = 8.4 Hz)]; 13C-NMR: I' 14.33 (CH3), 30.57 (benzyl CH2), 50.00 (N-CH2), 61.93 (O-CH2), Ar-C: [127.90 (2CH), 128.86 (2CH), 129.31 (2CH), 129.98, 131.22 (2CH), 131.94, 134.24, 135.51], 156.84 (triazole C3), 159.55 (triazole C5), 167.62 (C=O); LC-MS m/z (%): 412.23 ([M+Na]+, 23), 392.33 ([M+2]+, 78), 390.33 ([M]+, 100), 319.25 (50), 219.26 (30); Anal. Calcd. for C19H17Cl2N3O2: C, 58.47; H, 4.39; N, 10.77. Found: C, 58.45; H, 3.95; N, 11.01.

Synthesis of 2-[5-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]acetohydrazide (4)

A mixture of compound 3 (0.01 mol) and hydrazine hydrate (99%) (0.025 mol) in 50 mL 1-butanol was refluxed for 4 h. The completion of reaction was monitored by TLC. After cooling, the formed precipitate was filtered off and recrystallized from ethanol to give target compound.

Yield 80%; m.p. 231-232C; IR (ATR, max, cm-1): 3342, 3280 (NH + NH2), 1661 (C=O), 1622 (C=N); 1H-NMR: I' 4.22 (s, 2H, benzyl CH2), 4.40 (s, 2H, NH-NH2), 4.92 (s, 2H, N-CH2), Ar-H: [7.37-7.41 (m, 3H), 7.49 (d, 2H, J = 8.4 Hz), 7.91-7.94 (m, 2H)], 9.52 (s, 2H, NH); 13C-NMR: I' 30.82 (benzyl CH2), 49.94 (N-CH2), Ar-C: [127.81 (2CH), 128.82 (2CH), 129.25 (2CH), 130.14, 131.34 (2CH), 131.85, 134.08, 135.90], 156.99 (triazole C5), 159.31 (triazole C3), 165.53 (C=O); LC-MS m/z (%): 416.22 ([M+K]+, 8), 376.24 ([M]+, 45), 302.28 (100), 209.24 (92), 105.19 (43); Anal. Calcd. for C17H15Cl2N5O: C, 54.27; H, 4.02; N, 18.61. Found: C, 54.53; H, 3.87; N, 18.43.

General method for the synthesis of compounds 5a-e

A solution of acetohydrazide 5 (0.01 mol) and the appropriate aldehyde (0.01 mol) in 100 mL ethanol was heated under reflux. After 2h, during the reaction, a precipitate was formed and precipitated product was filtered and crystallized from dimethyl sulfoxide:water (1:2) to yield the target product.

2-[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]-N'-[(4-(fluorobenzylidene)] acetohydrazide (5a): Yield 80%; m.p. 242-243C; IR (ATR, max, cm-1): 3181 (NH), 1671 (C=O), 1621, 1599 (C=N); 1H-NMR: I' 4.23 and 4.26 (s, 2H, benzyl CH2, trans/cis conformers), 5.14 and 5.59 (s, 2H, N-CH2, cis/trans conformers), Ar-H: [7.28 (d, 2H, J = 8.0 Hz), 7.37-7.40 (m, 4H), 7.49 (d, 2H, J= 12.0 Hz), 7.82 (t, 2H, J= 8.0 Hz), 7.96 (d, 2H, J = 8.0 Hz) ], 8.06 and 8.25 (s, 1H, -N=CH, trans/cis conformers), 11.86 and 11.91 (s, 1H, NH, trans/cis conformers); 13C-NMR: I' 30.82 (benzyl CH2), 49.99 and 50.49 (N-CH2 trans/cis conformers),

Ar-C: [116.13 (CH, JC-F = 22.0 Hz), 127.84 (2CH), 128.78 (2CH), 129.24 (2CH), 129.81 (2CH, J = 22.0 Hz), 130.25, 130.96, 131.31 (2CH), 131.87, 134.13 (C, JC-F = 10.0 Hz), 135.79, 163.67 (C, JC-F = 13.0 Hz) ], 143.83 and 147.38 (-N=CH trans/cis conformers), 157.21 (triazole C3), 159.31 (triazole C5), 167.87 (C=O); LC-MS m/z (%): 504.20 ([M+Na]+, 21), 482.18 ([M]+, 17), 381.44 (65), 360.42 (100), 327.26 (89); Anal. Calcd. for C24H18Cl2FN5O: C, 59.76; H, 3.76; N, 14.52. Found: C, 59.52; H, 3.82; N, 14.26.

2-[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]-N'-[(4-(chlorobenzylidene)] acetohydrazide (5b): Yield 84%; m.p. 238-239C; IR (ATR, max, cm-1): 3181-3096 (NH), 1670 (C=O), 1595 (C=N); 1H-NMR: I' 4.21 and 4.25 (s, 2H, benzyl CH2, trans/cis conformers), 5.13 and 5.58 (s, 2H, N-CH2, cis/trans conformers ), Ar-H: [7.36 (m, 4H), 7.50 (m, 4H), 7.77 (d, 2H, J = 8.0 Hz), 7.95 (d, 2H, J = 8.0 Hz)], 8.04 and 8.23 (s, 1H, -N=CH, trans/cis conformers), 11.88 and 11.95 (s, 1H, NH, trans/cis conformers); 13C-NMR: I' 30.78 (benzyl CH2), 49.97 and 50.45 (N-CH2 trans/cis conformers), Ar-C: [127.84 (2CH), 128.78 (2CH), 129.17 (2CH), 129.27 (2CH), 129.34 (2CH), 130.18, 131.31 (2CH), 131.86, 133.26, 134.10, 135.06, 135.76], 143.73 and 147.22 (-N=CH trans/cis conformers), 150.72 (triazole C3), 159.30 (triazole C5), 167.94 (C=O); LC-MS m/z (%): 537.55 ([M+K]+, 3), 381.50 (47), 360.54 (100), 327.32(82). Anal. Calcd. for C24H18Cl3N5O: C, 57.79; H, 3.64; N, 14.04. Found: C, 58.01; H, 3.78; N, 14.17.

2-[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]-N'-[(4-(2,4-difluorobenzylidene)] acetohydrazide (5c): Yield 78%; m.p. 246-248C; IR (ATR, max, cm-1): 3075 (NH), 1678 (C=O), 1611 (C=N); 1H-NMR: I' 4.21 and 4.25 (s, 2H, benzyl CH2, trans/cis conformers), 5.13 and 5.58 (s, 2H, N-CH2, cis/trans conformers), Ar-H: [7.20 (t, 1H, J = 8.0 Hz), 7.34-7.39 (m, 5H) 7.49 (d, 2H, J = 8.0 Hz), 7.95 (d, 2H, J = 8.0 Hz), 8.02-8.08 (m, 1H) ] 8.20 and 8.40 (s, 1H, -N=CH, trans/cis conformers), 11.94 (s, 1H, NH); 13C-NMR: I' 30.77 (benzyl CH2), 49.96 and 50.51 (N-CH2 trans/cis conformers),

Ar-C: [104.93 (CH, J = 25.0 Hz), 113.00 (CH, J = 22.0 Hz), 118.81 (C, JC-F = 10.0 Hz), 127.83 (CH), 128.78 (2CH), 129.25 (2CH), 130.22, 131.30 (2CH), 131.85, 134.07, 135.77, 137.03 (CH), 140.56 (CH), 162.55 (C, J C-F = 12.0 Hz), 162.57 (C, JC-F = 10.0 Hz)], 136,82 and 140.39 (-N=CH trans/cis conformers), 157.22 (triazole C3), 159.32 (triazole C5), 167.68 (C=O). LC-MS m/z (%): 522.22 ([M+Na]+, 6), 502.14 ([M+2]+, 25), 500.20 ([M]+, 38), 113.04 (100); Anal. Calcd. for C24H17Cl2F2N5O: C, 57.61; H, 3.42; N, 14.00. Found: C, 57.73; H, 3.38; N, 13.97.

2-[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]-N'-[(4-(2,4-chlorobenzylidene)] acetohydrazide (5d): Yield 87%; m.p. 253-254C; IR (ATR, max, cm-1): 3075 (NH), 1678 (C=O), 1611 (C=N); 1H-NMR: I' 4.21 and 4.25 (s, 2H, benzyl CH2, trans/cis conformers), 5.14 and 5.60 (s, 2H, N-CH2, cis/trans conformer), Ar-H: [7.36-7.39 (m, 4H), 7.49-7.51 (m, 3H), 7.71 (bs, 2H), 7.95 (d, 2H, J = 8.0 Hz), 8.08 (d, 1H, J = 8.0 Hz)], 8.37 and 8.57 (s, 1H, - N=CH, trans/cis conformers), 12.03 and 12.13 (s, 1H, NH, trans/cis conformers); 13C-NMR: I' 30.75 (benzyl CH2), 50.03 and 50.53 (N-CH2 trans/cis conformers),

Ar-C: [127.83 (2CH), 128.37 (CH), 128.78 (2CH), 128.84 (CH), 129.26 (2CH), 129.83 (CH), 130.21, 130.73, 131.30 (2CH), 131.85, 134.08, 134.23, 135.64, 135.78] 139.95 and 143.35 (-N=CH trans/cis conformers), 157.22 (triazole C3), 159.32(triazole C5), 168.11 (C=O); LC-MS m/z (%): 556.20 ([M+Na]+, 11), 534.24 ([M+1]+, 27), 327.32 (50), 219.26 (100), 114.02 (86); Anal. Calcd. for C24H17Cl4N5O: C, 54.06; H, 3.21; N, 13.13. Found: C, 53.52; H, 2.70; N, 12.93.

2-[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]-N'-[(4-(4-chloro-2-fluorobenzylidene)]acetohydrazide (5e): Yield 88%; m.p. 201-202C; IR (ATR, max, cm-1): 3100-3079 (NH), 1682 (C=O), 1606 (C=N); 1H-NMR: I' 4.21 and 4.25 (s, 2H, benzyl CH2, trans/cis conformers), 5.13 and 5.58 (s, 2H, N-CH2, cis/trans conformers), Ar-H: [7.37-7.39 (m, 5H), 7.50 (d, 2H, J = 8.0 Hz), 7.54-7.57 (m, 1H), 7.95 (d, 2H, J = 8.0 Hz), 8.02 (t, 1H, J = 8.0 Hz)], 8.20 and 8.41 (s, 1H, -N=CH, trans/cis conformers), 11.95 (s, 1H, NH); 13C-NMR: I' 30.74 (benzyl CH2), 49.96 and 50.52 (N-CH2, trans/cis conformers),

Ar-C: [117.16 (CH, J = 25.0 Hz), 121.14 (C, JC-F = 10.0 Hz), 125.79 (CH), 127.84 (2CH), 128.35 (CH), 128.78 (2CH), 129.27 (2CH), 130.08, 130.21, 131.31 (2CH), 131.84, 134.07, 138.80, 160.89 (C, JC-F = 10.0 Hz)], 136.82 and 140.39 (-N=CH, trans/cis conformers), 157.24 (triazole C3), 159.32 (triazole C5), 168.06 (C=O); LC-MS m/z (%); 538.09 ([M+Na]+, 5) , 518.14 ([M+1]+, 8), 113.09 (100), 187.15 (83), 219.18 (30); Anal. Calcd. for C24H17Cl3FN5O: C, 55.78; H, 3.32; N, 13.55. Found: C, 56.01; H, 3.11; N, 13.86.

Synthesis of 4-amino-3-{[5-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]methyl}-1H-1,2,4-triazole-5(4H)-thione (7)

To a solution of KOH (0.015 mol) in absolute ethanol (100 mL), the acid hydrazide 4 (0.01 mol) and CS2 (0.015 mol) were added. The mixture was stirred for 16 h at room temperature. It was then diluted with anhydrous ether (200 mL) and stirred further for 2h. The precipitated potassium dithiocarbazinate 6 was collected by filtration, washed with diethyl ether and dried. The potassium salt 6 was used in the next stage without further purification. Potassium dithiocarbazinate (0.01 mol) and hydrazine hydrate (0.02 mol) in water (100 mL) medium was refluxed for 6 h. The color of the reaction mixture changed to green, hydrogen sulphide was evolved and a homogenous solution resulted. A white solid was precipitated by dilution with cold water (100 mL) and acidified with conc. HCl to pH 1.

The product was filtered, washed with cold water (100 mL) and crystallized from ethanol:water (3:1) to afford the title compound.

Yield 79%; m.p. 209-210 C; IR (ATR, max, cm-1): 3331, 3165 (NH + NH2), 1603, 1578 (C=N), 1293 (C=S); 1H-NMR: I' 4.33 (s, 2H, benzyl CH2), 5.56 (s, 2H, N-CH2), 5.76 (s, 2H, NH2), Ar-H: [7.36 (bs, 4H), 7.48 (d, 2H, J= 8.0 Hz), 7.93 (d, 2H, J= 8.0 Hz)], 13.72 (s, 1H, NH); 13C-NMR: I' 30.93 (benzyl CH2), 43.09 (N-CH2), Ar-C: [127.99 (2CH) , 128.91 (2CH), 129.26 (2CH), 130.00, 131.29 (2CH), 131.98, 134.27, 135.51], 147.83 (triazole second ring, C3), 156.75 (triazole C3), 159.72 (triazole C5), 167.45 (C=S); LC-MS m/z (%): 434.19 ([M+2]+, 27), 432.33 ([M]+, 36), 327.26 (72), 113.14 (100); Anal. Calcd. for C18H15Cl2N7S: C, 50.01; H, 3.50; N, 22.68; S, 7.42. Found: C, 50.46; H, 3.45; N, 22.20; S, 7.57.

General method for the synthesis of compounds 8a-e

A mixture of 4-amino-5-{[3-(4-chlorobenzyl)-5-(4-chlorophenyl)-4H-1,2,4-triazol-4-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione 7 (0.01 mol), substituted aromatic aldehyde (0.01 mol) and 2-3 drops of conc. sulphuric acid in 100 mL ethanol medium was refluxed for 6 h. The product formation was detected by TLC. After evaporating the solvent in reduced pressure, a solid appeared. This was recrystallized from ethanol/water (1:1) to obtain the target compounds.

3-{[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]-methyl}-4-[4-(fluorobenzylidene )amino]-1H-1,2,4-triazole-5(4H)-thione (8a): Yield 78%; m.p. 173-174 C; IR (ATR, max, cm-1): 3104 (NH), 1595 (C=N), 1320 (C=S); 1H-NMR: I' 4.28 (s, 2H, benzyl CH2), 5.75 (s, 2H, N-CH2), Ar-H: [7.24-7.49 (m, 8H), 7.87-7.93 (m, 4H)], 9.89 (s, 1H, - N=CH), 14.08 (s, 1H, NH); 13C-NMR: I' 30.72 (benzyl CH2), 43.42 (N-CH2), Ar-C: [116.81 (d, CH, JC-F = 22.0 Hz), 127.90 (CH), 128.85 (CH), 129.01, 129.28 (CH), 129.82 (CH), 131.00 (CH), 131.71 (d, CH, JC-F = 9.0 Hz), 131.98, 132.47, 135.23, 165.52 (d, C, JC-F = 250.0 Hz)], 146.40 (triazole second ring, C3), 156.64 (triazole C3), 159.62 (triazole C5), 162.52 (C=S), 162.94 (-N=CH); LC-MS m/z (%): 537.49 ([M]+, 4), 381.50 (28), 360.48 (100), 327.36 (56), 233.21 (38), 101.06 (5); Anal. Calcd. for C25H17Cl4N7S: C, 50.95; H, 2.91; N, 16.64; S, 5.44. Found: C, 51.38; H, 2.74; N, 16.68; S, 5.40.

3-{[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]-methyl}-4-[4-(chlorobenzylidene) amino] -1H-1,2,4-triazole-5(4H)-thione (8b): Yield 79%; m.p. 203-204 C; IR (ATR, max, cm-1): 3098 (NH), 1593 (C=N), 1323 (C=S); 1H-NMR: I' 4.28 (s, 2H, benzyl CH2), 5.76 (s, 2H, N-CH2), Ar-H: [7.24-7.37 (m, 4H), 7.48 (d, 2H, J = 12.0 Hz), 7.58 (d, 2H, J = 12.0 Hz), 7.86-7.94 (m, 4H) ], 9.97 (s, 1H, - N=CH), 14.08 (s, 1H, NH); 13C-NMR: I' 30.73 (benzyl CH2), 43.44 (N-CH2), Ar-C: [127.90 (2CH), 128.85 (2CH), 129.55 (2CH), 129.83, 130.48 (2CH), 130.74 (2CH), 130.99 (2CH), 131.29, 131.99, 133.08, 135.22, 137.98 ], 146.46 (triazole second ring, C3), 156.63 (triazole C3), 159.62 (triazole C5), 161.03 (-N=CH), 162.56 (C=S); LC-MS m/z (%): 555.40 ([M+2]+, 10), 553.57 ([M]+, 14), 537.52 (100), 527.46 (60), 511.47 (45); Anal. Calcd. for C25H18Cl3N7S: C, 54.11; H, 3.27; N, 17.67; S, 5.78. Found: C, 54.11; H, 2.85; N, 17.27; S, 5.53.

3-{[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]-methyl}-4-[2,4-(difluorobenzylidene) amino]-1H-1,2,4-triazole-5(4H)-thione (8c): Yield 72%; m.p. 222-223 C; IR (ATR, max, cm-1): 3107 (NH), 1617, 1597(C=N), 1315 (C=S); 1H-NMR: I' 4.29 (s, 2H, benzyl CH2), 5.76 (s, 2H, N-CH2), Ar-H: [7.30-7.51 (m, 8H), 7.90-8.10 (m, 3H)], 10.38 (s, 1H, -N=CH), 14.11 (s, 1H, NH); 13C-NMR: I' 30.72 (benzyl CH2), 43.45 (N-CH2), Ar-C: [106.46 (d, C, JC-F = 26.0 Hz), 113.54 (d, CH, JC-F = 3.0 Hz), 127.87 (2CH), 128.83 (2CH), 129.29 (2CH), 129.83, 130.36 (2CH), 131.21(CH), 131.99, 134.29, 135.20, 131.26 (CH), 162.68 (C, JC-F = 255.0 Hz), 165.59 (C, JC-F = 252.5 Hz)], 146.64 (triazole second ring, C3), 154.36 (-N=CH), 156.65 (triazole C3), 159.60 (triazole C5), 162.43 (C=S); LC-MS m/z (%): 556.09 ([M+1]+, 5), 390.15 (50), 219.12 (100), 130.08 (40); Anal. Calcd. for C25H17Cl2F2N7S: C, 53.96; H, 3.08; N, 17.62; S, 5.76. Found: C, 54.40; H, 3.40; N, 17.63; S, 5.89.

3-{[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]-methyl}-4-[2,4-(dichlorobenzylidene) amino]-1H-1,2,4-triazole-5(4H)-thione (8d): Yield 79%; m.p. 206-207 C; IR (ATR, max, cm ): 3100 (NH), 1584 (C=N), 1339 (C=S); 1H-NMR: I' 4.39 (s, 2H, benzyl CH2), 5.83 (s, 2H, N-CH2), Ar-H: [7.31 (bs, 8H), 7.48 (d, 4H, J= 8.0 Hz), 8.03-8.07 (m, 3H)], 11.08 (s, 1H, -N=CH), 12.93 (s, 1H, NH); 13C-NMR: I' 30.88 (benzyl CH2), 43.29 (N-CH2), Ar-C: [127.54 (2CH), 128.06 (CH), 128.50 (CH), 128.67 (2CH), 128.88 (CH), 129.51, 129.80 (CH), 130.02, 130.41 (2CH), 132.31, 134.41, 134.68, 136.41, 138.26], 146.62 (triazole second ring, C3), 154.38 (-N=CH), 156.07 (triazole C3), 159.75 (triazole C5), 163.44 (C=S); LC-MS m/z (%): 591.58 ([M+2]+, 10), 390.17 (68), 390.22 (100), 219.12 (25), 112.96 (16); Anal. Calcd. for C25H17Cl4N7S: C, 50.95; H, 2.91; N, 16.64; S, 5.44. Found: C, 51.38; H, 2.74; N, 16.68; S, 5.40.

3-{[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]-methyl}-4-[4-(chloro-2-fluorobenzylidene)amino]-1H-1,2,4-triazole-5(4H)-thione (8e): Yield 72%; m.p. 197-198 C; IR (ATR, max, cm-1): 3097 (NH), 1600 (C=N), 1320 (C=S); 1H-NMR: I' 4.29 (s, 2H, benzyl CH2), 5.77 (s, 2H, N-CH2), Ar-H: [7.23-7.66 (m, 8H), 7.90-8.02 (m, 3H)], 10.45 (s, 1H, -N=CH), 14.13 (s, 1H, NH); 13C-NMR: I' 30.71 (benzyl CH2), 43.46 (N-CH2), Ar-C: [117.60 (CH, JC-F = 24.0 Hz), 119.40 (C, JC-F = 10.0 Hz), 126.19 (CH, JC-F = 3.0 Hz), 127.88 (2CH), 127.82 (2CH), 129.29 (2CH), 129.82, 130.95 (2CH), 131.26 (CH), 131.99, 134.29, 135.18, 138.94 (C, JC-F = 11.0 Hz), 161.90 (C, JC-F = 256.0 Hz)], 146.68 (triazole second ring, C3), 153.71 (N=CH), 156.63 (triazole C3), 159.59 (triazole C5), 162.45 (C=S); LC-MS m/z (%): 574.15 ([M+1]+, 8), 219.18 (100), 113.09 (60); Anal. Calcd. for C25H17Cl3FN7S: C, 52.41; H, 2.99; N, 17.11; S, 5.60. Found: C, 52.71; H, 2.60; N, 17.11; S, 6.01.

General method for the synthesis of compounds 9a-e

To the solution of corresponding compound 8a-e (0.01 mol) in dimethyl formamide (20 mL), formaldehyde (37%, 1.55 mL, 0.0155 mol) and 1-phenylpiperazine (0.01 mol) were added and the mixture was stirred at room temperature for 16 h. Water (50 mL) was then added and the mixture was stirred for 10 min. The separated precipitate was filtered, washed with cold water, and crystallized from benzene:petroleum ether (1:4).

3-{[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]methyl}-4-[4-(fluorobenzylidene)amino]-1-[(4-phenylpiperazin-1-yl)-methyl]-1H-1,2,4-triazole-5(4H)-thione (9a): Yield 77%; m.p. 122-123 C; IR (ATR, max, cm-1): 1601 (C=N), 1230 (C=S), 1154 (N-CH2-N); 1H-NMR: I' 2.82 (bs, 4H, N-phenyl piperazine, 2 CH2), 3.10 (bs, 4H, N-phenyl piperazine, 2 CH2), 4.30 (s, 2H, benzyl CH2), 5.11 (s, 2H, N-CH2-N ), 5.80 (s, 2H, N-CH2), Ar-H: [6.77 (t, 1H, J = 8.0 Hz), 6.89 (d, 2H, J = 8.0 Hz), 7.17-7.26 (m, 4H) , 7.30-7.39 (m, 6H), 7.85 (d, 2H, J = 8.0 Hz), 7.91-7.97 (m, 2H)], 9.85 (s, 1H, -N=CH); 13C-NMR: I' 35.54 (benzyl CH2), 48.14 (N-CH2), 53.49 (N-phenyl piperazine C-3, C-5), 55.01 (N-phenyl piperazine C-2, C-6), 73.70 (N-CH2-N),

Ar-C: [120.79 (2CH), 121.57 (d, CH, JC-F = 22.0 Hz), 124.18 (2CH), 132.60 (2CH), 133.62 (2CH), 133.98 (2CH), 134.11 (2CH), 134.55,135.72 (d, H, JC-F = 8.0 Hz), 136.59 (2CH), 135.72 (d, CH, JC-F = 9.0 Hz), 136.72 (2C), 139.02, 139.94, 156.20, 170.04 (d, C, JC-F = 260.0 Hz)], 149.88 (triazole second ring, C3), 161.33 (triazole C3), 164.38 (triazole C5), 168.05 (triazole second ring, C5), 168.62 (-N=CH); LC-MS m/z (%): 750.22 ([M+K]+, 11), 718.58 (59), 718.58 ([M+Na]+, 61), 698.56 (50), 697.68 (100); Anal. Calcd. for C36H32Cl2FN97S: C, 60.67; H, 4.53; N, 17.69; S, 4.50. Found: C, 61.03; H, 4.87; N, 17.58; S, 4.87.

3-{[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]methyl}-4-[4-(chlorobenzylidene )amino]-1-[(4-phenylpiperazin-1-yl)-methyl]-1H-1,2,4-triazole-5(4H)-thione (9b): Yield 72%; m.p. 98-99 C; IR (ATR, max, cm-1): 1597 (C=N), 1232 (C=S), 1158 (N-CH2-N); 1H-NMR: I' 2.82 (bs, 4H, N-phenyl piperazine, 2CH2), 3.09 (bs, 4H, N-phenyl piperazine, 2CH2), 4.30 (s, 2H, benzyl CH2), 5.11 (s, 2H, N-CH2-N ), 5.81 (s, 2H, N-CH2), Ar-H: [6.78 (t, 1H, J = 8.0 Hz), 6.89 (d, 2H, J = 8.0 Hz), 7.18-7.39 (m, 8H), 7.58 (d, 2H, J = 8,0 Hz), 7.86-7.90 (m, 4H)], 9.91 (s, 1H, -N=CH); 13C-NMR: I' 30.78 (benzyl CH2), 43.40 (N-CH2), 48.74 (N-phenyl piperazine C-3, C-5), 50.26 (N-phenyl piperazine C-2, C-6), 68.94 (N-CH2-N),

Ar-C: [116.04 (2CH), 119.43 (2CH), 127.85 (2CH), 128.86 (CH), 129.23 (2CH), 129.36 (2CH), 129.75 (2CH), 129.80, 130.84 (2CH), 130.93 (2CH), 131.16, 131.97, 134.26, 135.19, 138.09, 151.45], 145.17 (triazole second ring, C3), 156.57 (triazole C3), 159.61 (triazole C5), 163.29 (-N=CH),163.42 (triazole second ring, C5); LC-MS m/z 730.13 ([M+1]+, 37), 608.22 (42), 500.38 (100), 360.45 (50); Anal. Calcd. for C36H32Cl3N9S: C, 59.30; H, 4.42; N, 17.29; S, 4.40 Found: C, 58.85; H, 3.93; N, 17.01; S, 4.64.

3-{[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]methyl}-4-[4-(2,4-fluorobenzylidene)amino]-1-[(4-phenylpiperazin-1-yl)-methyl]-1H-1,2,4-triazole-5(4H)-thione (9c): Yield 89%; m.p. 116-118 C; IR (ATR, max, cm-1): 1614, 1599 (C=N), 1234 (C=S), 1165 (N-CH2-N); 1H-NMR: I' 2.82 (bs, 4H, N-phenyl piperazine, 2CH2), 3.10 (bs, 4H, N-phenyl piperazine, 2CH2), 4.31 (s, 2H, benzyl CH2), 5.10 (s, 2H, N-CH2-N ), 5.81 (s, 2H, N-CH2),

Ar-H: [6.75-6.79 (m, 1H), 6.89 (d, 2H, J = 8.0 Hz), 7.17-7.25 (m, 6H), 7.29-7.42 (m, 3H), 7.44-7.51 (m, 1H), 7.89 (d, 2H, J = 8.0 Hz), 8.05-8.11 (m, 1H)], 10.27 (s, 1H, -N=CH); 13C-NMR: I' 30.77 (benzyl CH2), 43.43 (N-CH2), 48.73 (N-phenyl piperazine C-3, C-5), 50.22 (N-phenyl piperazine C-2, C-6), 68.85 (N-CH2-N), Ar-C: [106.50 (d, CH, JC-F = 26.0 Hz), 113.57 (d, CH, JC-F = 22.0 Hz), 116.04 (2CH), 116.95 (d, C, JC-F = 6.0 Hz), 119.42 (CH), 127.82 (CH), 128.77 (CH), 128.85 (2CH), 129.17 (2CH), 129.35 (2CH), 129.81, 130.91 (2CH), 131.96, 134.27, 135.18 (2C), 151.45, 164.73 (d, C, JC-F = 256.0 Hz)], 145.31 (triazole second ring, C3), 155.57 (-N=CH), 156.60 (triazole C3), 159.58 (triazole C5), 163.11 (triazole second ring, C5); LC-MS m/z 733.46 ([M+2]+, 10), 731.07 ([M+1]+,3), 701.51 (100), 680.41 (36), 679.41 (87); Anal. Calcd. for C36H31Cl2F2N9S: C, 59.18; H, 4.28; N, 17.25; S, 4.39; S, 4.40 Found: C, 59.67; H, 4.47; N, 17.63; S, 4.57.

3-{[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]methyl}-4-[2,4-(chlorobenzylidene)amino]-1-[(4-phenylpiperazin-1-yl)-methyl]-1H-1,2,4-triazole-5(4H)-thione (9d): Yield 71%; m.p. 101-102 C; IR (ATR, max, cm-1): 1598, 1584 (C=N), 1237 (C=S), 1165 (N-CH2-N); 1H-NMR: I' 2.81 (bs, 4H, N-phenyl piperazine, 2CH2), 3.10 (bs, 4H, N-phenyl piperazine, 2CH2), 4.30 (s, 2H, benzyl CH2), 5.10 (s, 2H, N-CH2-N ), 5.84 (s, 2H, N-CH2), Ar-H: [6.77 (t, 1H, J = 8.0 Hz), 6.89 (d, 2H, J = 8.0 Hz), 7.17-7.24 (m, 4H), 7.29 (d, 2H, J = 8.0 Hz), 7,37 (bs, 2H), 7.41-7.43 (m, 1H), 7.50-7.52 (m, 1H), 7.90 (d, 2H, J = 8.0 Hz), 8.7-8.10 (m, 1H)], 10.66 (s, 1H, -N=CH); 13C-NMR: I' 30.77 (benzyl CH2), 43.50 (N-CH2), 48.73 (N-phenyl piperazine C-3, C-5), 50.22 (N-phenyl piperazine C-2, C-6), 68.78 (N-CH2-N),

Ar-C: [116.04 (2CH), 119.42 (CH), 127.84 (2CH), 128.71 (CH), 128.78 (CH), 128.82 (2CH), 129.11, 129.28 (2CH), 129.36 (2CH), 129.81, 130.30 (CH), 130.88 (2CH), 131.96, 134.25, 135.96, 136.45, 138.51, 151.45], 145.82 (triazole second ring, C3), 156.59 (triazole C3), 156.66 (-N=CH), 159.54 (triazole C5), 163.03 (triazole second ring, C5); LC-MS m/z 763.34 ([M+1]+, 11), 718.58 (68), 679.61(100); Anal. Calcd. for C36H31Cl4N9S: C, 56.63; H, 4.09; N, 16.51; S, 4.20. Found: C, 56.50; H, 3.73; N, 16.56; S, 3.92

3-{[5-(4-Chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]methyl}-4-[4-chloro-2-fluorobenzylidene)amino]-1-[(4-phenylpiperazin-1-yl)-methyl]-1H-1,2,4-triazole-5(4H)-thione (9e): Yield 92%; m.p. 136-138 C; IR (ATR, max, cm-1): 1600, 1577 (C=N), 1232 (C=S), 1157 (N-CH2-N); 1H-NMR: I' 2.82 (bs, 4H, N-phenyl piperazine, 2 CH2), 3.10 (bs, 4H, N-phenyl piperazine, 2CH2), 4.30 (s, 2H, benzyl CH2), 5.10 (s, 2H, N-CH2-N ), 5.82 (s, 2H, N-CH2), Ar-H: [6.76 (t, 1H, J = 8.0 Hz ), 6.89 (d, 2H, J = 8.0 Hz), 7.19-7.25 (m, 4H), 7.30 (d, 2H, J = 8.0 Hz), 7.36-7.41(m, 3H), 7.64-7.67 (m, 1H), 7.89 (d, 2H, J = 8.0 Hz), 8.00 (t, 1H, J = 8.0 Hz )], 10.34 (s, 1H, -N=CH); 13C-NMR: I' 30.76 (benzyl CH2), 43.44 (N-CH2), 48.73 (N-phenyl piperazine C-3, C-5), 50.22 (N-phenyl piperazine C-2, C-6), 68.83 (N-CH2-N),

Ar-C: [116.04 (2CH), 117.33 (d, CH, JC-F = 25.0 Hz), 119.20 (d, C, JC-F = 10.0 Hz), 119.42 (CH), 126.22 (CH), 127.81 (2CH), 128.84 (2CH), 129.04 (CH), 129.27 (2CH), 129.36 (2CH), 129.80, 130.91 (2CH), 131.96, 134.28, 135.17, 151.45, 160.68], 145.34 (triazole second ring, C3), 154.97 (-N=CH), 156.60 (triazole C3), 159.56 (triazole C5), 163.24 (triazole second ring, C5); LC-MS m/z 770.06 ([M+Na]+,5), 748.15 ([M+1]+,10), 620.53 (43), 619.46 (100); Anal. Calcd. for C36H31Cl3FN9S: C, 57.87; H, 4.18; N, 16.87; S, 4.29. Found: C, 58.07; H, 4.228; N, 16.75; S, 4.44.

Antimicrobial activity assay

The test microorganisms were obtained from the Hifzissihha Institute of Refik Saydam (Ankara, Turkey). The test compounds were weighed and dissolved in DMSO to prepare stock solutions of 20 mg/ml. Agar well diffusion method screening test [39] as adapted earlier [40] was used for all newly synthesized compounds. Each microorganism culture was suspended in Mueller-Hinton (MH) (Difco, Detroit, MI) broth and diluted approximately to 106 colony forming units per milliliter. After that, this colonies (C.albicans and S. cerevisiae) were flood-inoculated onto the surface of MH agar and potato dextrose agar (Difco, Detriot, MI) and dried. Wells of 5 mm-diameter were cut from the agar using a sterile cork-borer, and the substance stock solution (50 ul, 20 mM) was delivered into the wells. The plates were incubated for 18 h at 35AdegC.

Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organism. Ampicillin (10 ug/ml) and fluconazole (5 ug/ml) were used as standard drugs. DMSO was used as solvent control.

Anti-lipase activity assay

The lipase inhibitory effects of the compounds were evaluated against porcine pancreatic lipase (PPL) (Applichem, Germany) (15 ng/ml). Lipase activity assay were done according to literature [41]. The lipase activity was measured using 4-methylumbelliferyl oleate (4-MU oleate) as a substrate. Briefly, compounds were mixed with PPL 1:3 (v/v) and incubated for 30 min. The microtiter plates containing 0.1 mM 4-MU oleate (50 ul), diluted compound-lipase solution (25 uL), distilled H2O (25 ul), and assay buffer (13 mM Tris-HCl, 150 mM NaCl, and 1.3 mM CaCl2, pH 8.0) were incubated at 37AdegC for 20 min. After incubation, in order to stop the reaction, 0.1 M citrate buffer (0.1 ml) was added to the reaction mixture. The amount of 4-methylumbelliferone released by the lipase was measured by using a spectroflourometer (SpectraMax M5, Molecular Devices) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm.

The inhibitory activity of those compounds and orlistat (Xenical, Hoffman La Roche, Segrate, Italy) was measured at various concentrations. Residual activities were calculated by comparing to control without inhibitor. The assays were done in triplicate.

Quantum-chemical calculations

In this work, the electronic properties related to tautomeric forms of compound 2 and isomeric forms of compound 3 were investigated using DFT (density functional theory) B3LYP/DFT 6.31+ G(d) method using Gaussian 0.3 software [42-44]. The reaction pathway was calculated using the compounds 2 and 3, and the reaction coordinate was obtained via minimum energy path (MEP) computations using the DFT/3-21+G** method. Full geometry optimization was carried out employing the Polak-Ribiere (conjugate gradient) algorithm (convergence of 0.00001 kcal mol-1) and an RMS gradient of 0.001 kcalA-1mol-1. The calculations were performed with Hyper Chem 8.0 and Gaussian 03 software.

Results and discussion

Chemistry

The synthetic routes followed for the preparation of compounds 1-9 is outlined in Scheme-1. The starting compound 4-amino-3-(4-chlorobenzyl)-5-(4-chlorophenyl)-4H-1,2,4-triazole (1) has been synthesized and reported in our previous work. [38].

At the first step of the work, synthesis of 5-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazole (2) was achieved by deamination of compound 1 in the presence of H3PO2 (50 wt. % in H2O) and NaNO2 according to literature [14]. This compound has three tautomeric forms. The formation enthalpies of these tautomeric forms were calculated by DFT 6.31+ GD method using Gaussian 0.3 software. The 1H form (I) was determined to be most stable form (Scheme-2). These results are also compatible with the literature [45, 46].

In the second step, the 1,2,4-triazole 2 was transformed into ethyl 2-[5-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]acetate (3) by reacting with BrCH2CO2Et in ethanolic NaOEt medium. The position of substitution on either of the three nitrogen atoms resulting from substitution reaction of compound 2 with ethyl bromoacetate in the presence of NaOEt is not exactly known according to literature data. Because of the possibility of substitution reaction taking place through either of the three nitrogen atoms the formation enthalpies were calculated for each form by DFT 6.31+ GD method using Gaussian 0.3 software. The quantum-chemical calculations indicates that the isomer form (I) is more stable than other isomers (II and III) (Scheme-3) [47].

In the third step, 2-[5-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]acetohydrazide (4) was obtained by reacting compound 3 with hydrazine hydrate in 1-butanol. In the next step, hydrazide-hydrazone derivatives 5a-e were obtained from the reaction of various aldehydes with acetohydrazide 4 in ethanol in the presence of H2SO4 catalyst. In the 1H NMR spectra of these compounds, benzyl CH2, N-CH2, N=CH and NH proton signals and in the 13C NMR spectra benzyl CH2, N-CH2 and N=CH carbon signals were recorded as double singlets. The compounds having -CONHN=CH-functional group may exist as E/Z geometric isomers about the C=N double bonds and cis/trans amide conformers [14, 48-50]. Furthermore, hydrazide-hydrazone derived compounds are dissolved in polar aprotic solvents (DMSO-d6), the geometrical E isomers of these compounds undergo a rapid cis/trans amide equilibrium, in which the trans conformer predominates [14, 48-50].

The E isomers and the cis/trans conformer percentage can be easily determined by 1H NMR integration. The chemical shift values of cis/trans conformers belonging to protons of benzyl CH2, N-CH2, N=CH and NH in the 1H NMR and, to carbons of benzyl CH2, N-CH2 and N=CH in the13C NMR spectra of compounds 5a-e and the percentage ratios of cis/trans conformers are given in Table-1. These data demonstrate the E isomers and trans conformer structures as dominant forms.

Table-1: 1H NMR and 13C NMR chemical shifts and percentage of cis/trans conformers.

###Compound###Conformer###NMR###Benzyl-CH2###N-CH2###N=CH###NH###Percentage of trans/cis

###H###4.23###5.59###8.06###11.86###76.00

###trans

###C###30.82###49.99###143.83###-

###5a

###H###4.26###5.14###8.25###11.91###24.00

###cis

###C###-###50.49###147.38###-

###H###4.21###5.58###8.04###11.88###75.00

###trans

###C###30.78###49.98###143.73###-

###5b

###H###4.25###5.13###8.23###11.95###25.00

###cis

###C###-###50.45###147.22###-

###H###4.21###5.58###8.20###11.94###80.00

###trans

###C###30.77###49.96###136.82###-

###5c

###H###4.25###5.13###8.40###-###20.00

###cis

###C###-###50.51###140.39###-

###H###4.21###5.60###8.37###12.03###77.88

###trans

###C###30.75###50.03###139.95###-

###5d

###H###4.25###5.14###8.57###12.13###22.12

###cis

###C###-###50.53###143.35###-

###H###4.21###5.58###8.20###11.95###77.00

###trans

###C###30.74###49.96###136.82###-

###5e

###H###4.25###5.13###8.41###-###23.00

###cis

###C###-###50.52###140.39###-

After that, acid hydrazide 4 was reacted with CS2 in the ethanolic KOH to give the corresponding potassium dithiocarbazate 6, treatment of dithiocarbazate with hydrazine hydrate in water afforded 4-amino-3-{[5-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]methyl}-1H-1,2,4-triazole-5(4H)-thione (7). After this, the amino-triazole (7) was reacted with appropriate aromatic aldehydes in the presence of a catalytic amount of sulfuric acid in ethanol giving 3-{[5-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]-methyl}-4-[4-(substituebenzylidene)amino]-1H-1,2,4-triazole-5(4H)-thiones (8a-e). Finally, 8a-e compounds reacted with formaldehyde and N-phenylpiperazine in DMF to give Mannich bases (9a-e). All synthesized compounds (1-9) were characterized by IR, 1H NMR, 13C NMR, elemental analyses and mass spectral data, and the spectral data agree with the proposed structures.

Antimicrobial activity

Among the tested substances, 3, 8c, 8d, 8e and 9d compounds exhibited activity against Gram-positive bacteria. These compounds also showed good activity against yeast fungi, especially S. cerevisiae. Compounds 4, 5d and 5e showed considerable activity against S. cerevisiae. Compounds 3 and 8d inhibited S. aureus and B. cereus more than standard antibiotic ampicillin. All the synthesized compounds showed no activity against Gram-negative test microorganisms. The results of antimicrobial activity are summarized in Table-2.

Anti-lipase activity

All synthesized compounds were investigated for their pancreatic lipase inhibition profiles. It was observed that 5e, 8b and 9b among the evaluated compounds considerably inhibited lipase activity at various concentration. Among this compound 8b found to have highest inhibitory activity in terms of IC50 values. This inhibition activity of 8b compared with orlistat that a well-known pancreatic lipase inhibitor used as an anti-obesity drug (Table-3). Although orlistat (Xenical) is known as a unique anti-obesity drug [51] today, it has also some side effects such as fecal incontinence, flatulence, and steatorrhea [52,53]. This situation encourages the researchers to design and discover new organic molecules as potent lipase inhibitor. So, compounds 8b and 9b may further investigated as alternative molecules to orlistat in terms of their low IC50 values.

It was declared in the literature that heterocyclic compounds containing aromatic halogen inhibited lipase activity at different levels [54]. These two compounds contain p-chloro phenyl groups, in addition to other functional heterocyclic moieties.

Conclusions

In this study, nineteen new heterocyclic compounds containing 5-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H-1,2,4-triazole ring were synthesized in good yields by multistep reactions. Newly synthesized compounds were tested against bacterial and fungal strains. Among the tested substances, compounds 3, 8c, 8d, 8e and 9d displayed good activity against Gram-positive bacteria S. aureus, B. cereus. Compounds 4, 5d and 5e showed considerable activity against S. cerevisiae. Furthermore, compounds 3 and 8d were more active than ampicillin (used standard antibiotic) against S. aureus and B. cereus. As a result, tested compounds exhibited in vitro activity against gram-positive bacteria and yeast fungi.

The newly synthesized compounds were also tested for pancreatic lipase inhibitiory activity. Amongst the tested compounds 5e, 8b, and 9b demonstrated a significant anti-lipase activity at different concentrations. Based on the obtained results, compounds 8b (IC50 = 0.11 +- 0.05) and 9b (IC50 = 0.22 +- 0.06) can be speculated as alternative drugs to Orlistat.

Table-2: Screening for antimicrobial activity of the synthesized compounds (Inhibiton zone, mm).

###Bacteria###Fungi

###Compound###E.c.*###Y.p.###P.a.###E.f.###S.a.###B.c.###C.a.###S.c.

###1###-**###-###-###-###-###-###-###-

###2###-###-###-###-###-###-###-###-

###3###-###-###-###-###15###15###17###14

###4###-###-###-###-###-###-###-###20

###5a###-###-###-###-###-###-###-###-

###5b###-###-###-###-###-###-###-###-

###5c###-###-###-###-###-###-###-###-

###5d###-###-###-###-###-###-###-###20

###5e###-###-###-###-###-###-###-###20

###7###-###-###-###-###-###-###-###-

###8a###-###-###-###-###-###-###-###-

###8b###-###-###-###-###-###-###-###-

###8c###-###-###-###-###10###8###8###24

###8d###-###-###-###-###16###14###11###20

###8e###-###-###-###-###12###11###10###20

###9a###-###-###-###-###-###-###-###-

###9b###-###-###-###-###-###9###-###-

###9c###-###-###-###-###-###-###-###-

###9d###-###-###-###-###10###8###8###24

###9e###-###-###-###-###-###-###-###-

###Ampicillin###10###18###10###35###15###10

###Fluconazole###25###>25

###DMSO###-###-###-###-###-###-###-###-

Table-3: Lipase inhibition at final concentration of 100 uM and IC50 of synthesized compounds.

###Compund###Inhibition%###IC50 (uM)

###1###6.1 +- 1.0###-

###2###14.4 +- 6.5###-

###3###81.4 +- 3.3###4.04 +- 0.70

###4###66.7 +- 5.8###-

###5a###97.1 +- 0.7###3.55 +- 0.43

###5b###78.3 +- 5.6###3.71 +- 0.70

###5c###95.2 +- 2.4###1.90 +- 0.23

###5d###90.2 +- 2.3###2.74 +- 0.50

###5e###63.4 +- 3.8###0.85 +- 0.14

###7###9.4 +- 1.4###-

###8a###95.4 +- 0.9###2.68 +- 0.41

###8b###80.9 +- 0.4###0.11 +- 0.05

###8c###86.9 +- 0.8###2.23 +- 0.48

###8d###68.1 +- 0.7###-

###8e###92.2 +- 0.7###2.85 +- 0.13

###9a###64.5 +- 0.5###3.37 +- 0.03

###9b###91.8 +- 0.9###0.22 +- 0.06

###9c###96.3 +- 0.5###3.06 +- 0.20

###9d###92.0 +- 1.2###3.02 +- 0.51

###9e###97.8 +- 0.6###1.73 +- 0.17

Orlistat###99.42 +- 0.24###0.0007+-0.00003

Acknowledgments

This work was produced from PhD thesis of Yusuf Ozdemir. We would like to extend our gratitude to the Karadeniz Technical University Scientific Research Centre for their financial support under Project (BAP, FBA, 2014-89). Furthermore, we would like to thank to Dr. S. Ulker (Recep Tayyip Erdogan Universitesi, Rize) who helped to make biological activity tests.

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Author:Ozdemir, Yusuf; Gultekin, Ergun; Bekircan, Olcay
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Date:Dec 31, 2017
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