3.1. General Experimental Procedures
From common sellable sources, all chemicals and reagents have been procured. By normal steps, all the solvents have been filtered and dried. All reactions have been observed in the silica gel GF254 plate with thin-layer chromatography (TLC). On a Shimadzu GCeMS-QP1000 EX mass spectrometer at 70 eV, mass spectrums were recorded. With an Electrothermal IA 9000 series digital melting point apparatus, melting points were determined. On the PyeUnicamSP 3300 infrared spectrophotometer, IR spectra were recorded on potassium bromide discs. On the VarianMercury 400 MHz spectrophotometer in DMSO-d6, the 1H-NMR and 13C-NMR spectra were measured using TMS as internal. Using a German-made ElementarVario LIII CHNS analyzer, elemental analysis was calculated.
Synthesis of thiazole derivatives 6a-f, and 10a-e
A mixture of maleic anhydride 1 (0.098 g, 1 mmol) and thiosemicarbazide 2 (0.092g, 1 mmol) glacial acetic acid (0.5 mL) in EtOH (15mL) was irradiated under microwave oven at 500 W and 150 °C for 2 min. Then the appropriate hydrazonoyl halides 3a-f or 8a-e and chitosan (0.1 g) were added and the irradiation was continued until all the starting material was consumed (4–8 min. as monitored by TLC). The hot solution was filtered and excess solvent was removed under lower pressures to eliminate chitosan. The solid product was filtered, and crystalized from the appropriate solvent to give pure products 6a-f and 10a-e, respectively. Below are the physical features and spectral data of the products obtained.
1-(4-Methyl-5-(phenyldiazenyl)thiazol-2-yl)-1,2-dihydropyridazine-3,6-dione) (6a)
Red solid; m.p. 213-215 °C (Dioxane); IR (KBr): v 3435 (NH), 3049, 2926 (C-H), 1668, 1654 (2C=O) cm-1; 1H-NMR (DMSO-d6): δ 2.56 (s, 3H, CH3), 6.24 (d, J = 12Hz, 1H, CH=CH), 6.66 (d, J = 12Hz, 1H, CH=CH), 7.01-7.56 (m, 5H, Ar-H), 10.62 (br s, 1H, NH); MS m/z (%) 313 (M+, 12), 250 (7), 233 (18), 149 (23), 133 (30), 128 (41), 113 (60), 98 (39), 73 (100), 65 (41), 55 (91). Anal. Calcd. for C14H11N5O2S (313.33): C, 53.67; H, 3.54; N, 22.35. Found C, 53.55; H, 3.35; N, 22.14%.
1-(4-Methyl-5-(p-tolyldiazenyl)thiazol-2-yl)-1,2-dihydropyridazine-3,6-dione) (6b)
Red solid; m.p. 187-189 °C (EtOH); IR (KBr): v 3429 (NH), 3027, 2921 (C-H), 1690, 1654 (2C=O) cm-1; 1H-NMR (DMSO-d6): δ 2.36 (s, 3H, CH3), 2.56 (s, 3H, CH3), 6.23 (d, J = 12Hz, 1H, CH=CH), 6.52 (d, J = 12Hz, 1H, CH=CH), 7.15-7.31 (m, 4H, Ar-H), 10.66 (br s, 1H, NH); MS m/z (%) 327 (M+, 5), 270 (14), 199 (16), 159 (77), 133 (9), 106 (76), 91 (100), 77 (43), 57 (33). Anal. Calcd. for C15H13N5O2S (327.36): C, 55.04; H, 4.00; N, 21.39. Found C, 55.35; H, 3.70; N, 21.18%.
1-(4-Methyl-5-(m-tolyldiazenyl)thiazol-2-yl)-1,2-dihydropyridazine-3,6-dione) (6c)
Red solid; m.p. 206-208 °C (EtOH); IR (KBr): v 3433 (NH), 3011, 2923 (C-H), 1683, 1669 (2C=O) cm-1; 1H-NMR (DMSO-d6): δ 2.34 (s, 3H, CH3), 2.45 (s, 3H, CH3), 6.23 (d, J = 12Hz, 1H, CH=CH), 6.48 (d, J = 12Hz, 1H, CH=CH), 7.06-7.64 (m, 4H, Ar-H), 10.93 (br s, 1H, NH); MS m/z (%) 327 (M+, 8), 222 (48), 129 (21), 91 (58), 77 (36), 63 (100). Anal.Calcd.for C15H13N5O2S (327.36): C, 55.04; H, 4.00; N, 21.39. Found C, 55.25; H, 3.79; N, 21.17%.
1-(5-((4-Methoxyphenyl)diazenyl)-4-methylthiazol-2-yl)-1,2-dihydropyridazine-3,6-dione (6d). Red solid; m.p. 177-178 °C (EtOH); IR (KBr): v 3423 (NH), 3022, 2924 (C-H), 1676, 1659 (2C=O) cm-1; 1H-NMR (DMSO-d6): δ 2.66 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 6.23 (d, J = 12Hz, 1H, CH=CH), 6.62 (d, J = 12Hz, 1H, CH=CH), 7.03-7.86 (m, 4H, Ar-H), 10.87 (br s, 1H, NH); MS m/z (%) 343 (M+, 3), 313 (6), 199 (5), 129 (11), 108 (15), 97 (27), 73 (40), 57 (100). Anal.Calcd.for C15H13N5O3S (343.07): C, 52.47; H, 3.82; N, 20.40. Found C, 52.48; H, 3.65; N, 20.23%.
1-(5-((4-Chlorophenyl)diazenyl)-4-methylthiazol-2-yl)-1,2-dihydropyridazine-3,6-dione (6e)
Red solid; m.p. 237-239 °C (DMF); IR (KBr): v 3433 (NH), 3042, 2925 (C-H), 1671, 1657 (2C=O) cm-1; 1H-NMR (DMSO-d6): δ 2.56 (s, 3H, CH3), 6.27 (d, J = 12Hz, 1H, CH=CH), 6.64 (d, J = 12Hz, 1H, CH=CH), 7.26-8.12 (m, 4H, Ar-H), 10.84 (br s, 1H, NH); MS m/z (%) 397 (M+, 12), 283 (4), 267 (22), 185 (4), 152 (8), 129 (26), 111 (60), 99 (66), 86 (61), 57 (100). Anal.Calcd.for C14H10ClN5O2S (397.04): C, 48.35; H, 2.90; N, 20.14. Found C, 48.75; H, 2.74; N, 19.98%.
1-(5-((4-Bromophenyl)diazenyl)-4-methylthiazol-2-yl)-1,2-dihydropyridazine-3,6-dione (6f)
Red solid; m.p. 225-227 °C (DMF); IR (KBr): v 3434 (NH), 3032, 2923 (C-H), 1685, 1660 (2C=O) cm-1; 1H-NMR (DMSO-d6): δ 2.43 (s, 3H, CH3), 6.12 (d, J = 12Hz, 1H, CH=CH), 6.46 (d, J = 12Hz, 1H, CH=CH), 7.39-8.20 (m, 4H, Ar-H), 11.23 (br s, 1H, NH); MS m/z (%) 392 (M+, 2), 325 (53), 274 (11), 171 (25), 129 (15), 91 (57), 86 (89), 73 (64), 57 (100). Anal.Calcd. for C14H10BrN5O2S (392.23): C, 42.87; H, 2.57; N, 17.86. Found C, 43.21; H, 2.25; N, 17.55%.
1-(4-Oxo-5-(2-phenylhydrazineylidene)-4,5-dihydrothiazol-2-yl)-1,2-dihydropyridazine-3,6-dione (10a). Yellow solid; m.p. 168-170 °C (EtOH); IR (KBr): v 3429, 3178 (2NH), 3040, 2975 (C-H), 1706, 1680, 1653 (3C=O) cm-1; 1H-NMR (DMSO-d6): δ 6.20 (d, J = 12Hz, 1H, CH=CH), 6.41 (d, J = 12Hz, 1H, CH=CH), 7.04-7.82 (m, 5H, Ar-H), 10.75, 11.00 (2br s, 2H, 2NH); MS m/z (%) 315 (M+, 7), 307 (100), 279 (22), 150 (14), 104 (10), 92 (67), 77 (35), 65 (29). Anal.Calcd. for C13H9N5O3S (315.31): C, 49.52; H, 2.88; N, 22.21. Found C, 49.70; H, 2.57; N, 21.88%.
1-(4-Oxo-5-(2-(p-tolyl)hydrazineylidene)-4,5-dihydrothiazol-2-yl)-1,2-dihydropyridazine-3,6-dione (10b). Yellow solid; m.p. 179-181 °C (EtOH); IR (KBr): v 3431, 3278 (2NH), 3030, 2979 (C-H), 1705, 1679, 1629 (3C=O) cm-1; 1H-NMR (DMSO-d6): δ (s, 3H, CH3), δ 6.23 (d, J = 12Hz, 1H, CH=CH), 6.45 (d, J = 12Hz, 1H, CH=CH), 7.40-8.00 (m, 4H, Ar-H), 10.54, 10.79 (2 br s, 2H, 2NH); MS m/z (%) 329 (M+, 7), 263 (12), 155 (18), 125 (4), 111 (10), 101 (16), 97 (15), 86 (100), 58 (46). Anal. Calcd. for C14H11N5O3S (329.33): C, 51.06; H, 3.37; N, 21.27. Found C, 51.35; H, 3.06; N, 21.03%.
1-(5-(2-(4-Chlorophenyl)hydrazineylidene)-4-oxo-4,5-dihydrothiazol-2-yl)-1,2-dihydropyridazine-3,6-dione (10c). Yellow solid; m.p. 157-159 °C (EtOH); IR (KBr): v 3431, 3219 (2NH), 3039, 2989 (C-H), 1705, 1657, 1629 (3C=O) cm-1; 1H-NMR (DMSO-d6): δ 6.30 (d, J = 12Hz, 1H, CH=CH), 6.54 (d, J = 12Hz, 1H, CH=CH), 6.93-7.58 (m, 4H, Ar-H), 9.96, 12.68 (2 br s, 2H, 2NH); MS m/z (%) 351 (M++2, 2), 349 (M+, 7), 310 (5), 239 (5), 152 (10), 125 (36), 111 (31), 83 (39), 69 (58), 57 (100). Anal.Calcd. for C13H8ClN5O3S (349.75): C, 44.64; H, 2.31; N, 20.02. Found C, 44.93; H, 2.01; N, 19.70%.
1-(5-(2-(2,4-Dichlorophenyl)hydrazineylidene)-4-oxo-4,5-dihydrothiazol-2-yl)-1,2-dihydropyridazine-3,6-dione (10d). Yellow solid; m.p. 168-170 °C (EtOH); IR (KBr): v 3383, 3219 (2NH), 3039, 2983 (C-H), 1696, 1657, 1641 (3C=O) cm-1; 1H-NMR (DMSO-d6): δ 6.30 (d, J = 12Hz, 1H, CH=CH), 6.54 (d, J = 12Hz, 1H, CH=CH), 6.93-7.58 (m, 3H, Ar-H), 9.96, 12.68 (2 br s, 2H, 2NH); MS m/z (%) 382 (M+, 3), 375 (100), 349 (12), 347 (18), 218 (5), 160 (21), 133 (21), 112 (9), 82 (12). Anal.Calcd. for C13H7Cl2N5O3S (382.19): C, 40.64; H, 1.84; N, 18.23. Found C, 40.93; H, 1.55; N, 18.00%.
1-(5-(2-(4-Nitrophenyl)hydrazineylidene)-4-oxo-4,5-dihydrothiazol-2-yl)-1,2-dihydropyridazine-3,6-dione (10e). Yellow solid; m.p. 179-181 °C (EtOH); IR (KBr): v 3426, 3178 (2NH), 3030, 2922 (C-H), 1703, 1649, 1632 (3C=O) cm-1; 1H-NMR (DMSO-d6): δ 2.26 (s, 3H, CH3), 6.27 (d, J = 12Hz, 1H, CH=CH), 6.51 (d, J = 12Hz, 1H, CH=CH), 6.98-7.97 (m, 4H, Ar-H), 10.54, 10.77 (2 br s, 2H, 2NH); MS m/z (%) 360 (M+, 12), 328 (9), 259 (4), 221 (3), 180 (4), 152 (4), 129 (12), 113 (40), 97 (27), 87 (38), 71 (48), 59 (100). Anal. Calcd. for C13H8N6O5S (360.30): C, 43.34; H, 2.24; N, 23.33. Found C, 43.42; H, 2.09; N, 23.15%.
Alternate synthesis of thiazole derivative 6a
i) Synthesis of 3,6-dioxo-3,6-dihydropyridazine-1(2H)-carbothioamide (7).
To a solution of maleic anhydride 1 (0.098 g, 1 mmol), thiosemicarbazide 2 (0.092g, 1 mmol) in ethanol (15 mL), an equivalent amount of glacial acetic acid (0.5 mL) was added. The reaction mixture was heated in a microwave oven at 500 W and 150 °C for 2 min. as monitored by TLC. The reaction mixture was triturated with methanol and the product separated was filtered, washed with methanol, dried and recrystallized from ethanol to give pure carbothioamide derivative 7 as white solid; m.p. 309-311 °C; IR (KBr): v 3387-3314, 3258 (NH2 and NH), 3149, 2963 (C-H), 1687, 1634 (2C=O) cm-1; 1H-NMR (DMSO-d6): δ 6.19 (d, J = 12Hz, 1H, CH=CH), 6.44 (d, J = 12Hz, 1H, CH=CH), 9.30 (br s, 2H, NH2), 10.46 (br s, 1H, NH); MS m/z (%): 171 (M+), 132 (19), 107 (80), 87 (53), 57 (100). Anal. Calcd. for C5H5N3O2S (171.17): C, 35.08; H, 2.94; N, 24.55. Found C, 35.01; H, 2.84; N, 24.49%.
Reaction of 7 with 3a
A mixture of 2-oxo-N-phenylpropane hydrazonoyl chloride 3a (0.196g, mmol) and carbothioamide 7 (0.171 g, l mmol) in EtOH (15 mL) / chitosan (0.1 g) was heated in a microwave oven for 5 min. as monitored by TLC → gave product identical in all respects with compounds 6a.
Alternate synthesis of thiazole derivative 10a
i) 1-(4-Oxo-4,5-dihydrothiazol-2-yl)-1,2-dihydropyridazine-3,6-dione (12).
A solution of maleic anhydride 1 (0.098 g, 1 mmol), thiosemicarbazide 2 (0.092g, 1 mmol), and ethyl 2-bromoacetate 11 (0.0165 g, 1 mmol) in ethanol (15 mL) / chitosan (0.1 g) was heated in microwave oven for 4 min. to give thiazolone 12 as yellow solid; m.p. 157-159 °C (EtOH); IR (KBr): v 3438 (NH), 3168, 2987 (C-H), 1708, 1650, 1648 (3C=O) cm-1; 1H-NMR (DMSO-d6): δ 3.82 (s, 2H, CH2), 6.22 (d, J = 12Hz, 1H, CH=CH), 6.53 (d, J = 12Hz, 1H, CH=CH), 10.25 (s, 1H, NH); MS m/z (%) 211 (M+, 16), 149 (19), 117 (63), 92 (66), 57 (100). Anal.Calcd. for C7H5N3O3S (211.01): C, 39.81; H, 2.39; N, 19.90. Found C, 40.21; H, 2.17; N, 19.64%.
ii) Coupling of 12
Benzenediazonium chloride solution [prepared as usual by diazotizing aniline (1 mmol) in hydrochloric acid (1 ml, 6 M) with sodium nitrite (0.07 g, 1 mmol) in 10 ml water] was added portionwise to a cold solution of 12 (0.211g, 1 mmol) in 10 mL pyridine. The yellow solid which so formed was filtered and then recrystallized from EtOH to give 10a.
Synthesis of bis-thiazole 14 and bis-thiazolone 15
A mixture of maleic anhydride 1 (0.196 g, 2 mmol) and thiosemicarbazide 2 (0.184g, 2 mmol) in ethanol (20 mL) / glacial acetic acid (1mL) was heated in microwave oven for 2 min. Then the appropriate bis-hydrazonoyl halides 13a,b (1mmol for each) and chitosan (0.2 g) were added, the reaction mixture was further heated for 8 min., gave products 14 and 15, respectively.
1,1'-(([1,1'-Biphenyl]-4,4'-diylbis(diazene-2,1-diyl))bis(4-methylthiazole-5,2-diyl))bis(1,2-dihydropyridazine-3,6-dione) (14). Yellow solid; m.p. 168-170 °C (EtOH); IR (KBr): v 3428 (NH), 2922 (C-H), 1699, 1659 (2C=O) cm-1; 1H-NMR (DMSO-d6): δ 2.58 (s, 6H, CH3), 6.27 (d, J = 12Hz, 2H, CH=CH), 6.63 (d, J = 12Hz, 2H, CH=CH), 7.43 (s, 8H, Ar-H), 10.64 (br s, 2H, 2NH); MS m/z (%) 624 (M+, 22), 373 (13), 341 (10), 299 (2), 271 (21), 112 (20), 98 (39), 86 (67), 69 (45), 54 (100). Anal.Calcd. for C28H20N10O4S2 (624.11): C, 53.84; H, 3.23; N, 22.42. Found C, 54.04; H, 3.09; N, 22.27%.
1-(5-(2-(4'-(2-(2-(3,6-Dioxo-3,6-dihydropyridazin-1(2H)-yl)-4-oxothiazol-5(4H)-ylidene)hydrazinyl)-[1,1'-biphenyl]-4-yl)hydrazineylidene)-4-oxo-4,5-dihydrothiazol-2-yl)-1,2-dihydropyridazine-3,6-dione (15). Yellow solid; m.p. 179-181 °C (EtOH); IR (KBr): v 3422, 3032 (2NH), 2978, 2930 (C-H), 1683, 1655, 1636 (3C=O) cm-1; 1H-NMR (DMSO-d6): δ 6.29 (d, J = 12Hz, 2H, CH=CH), 6.53 (d, J = 12Hz, 2H, CH=CH), 7.52 (m, 8H, Ar-H), 10.37, 10.79 (2 br s, 4H, 2NH); MS m/z (%) 628 (M+, 4), 367 (31), 334 (24), 313 (19), 294 (49), 236 (25), 184 (63), 139 (66), 97 (36), 71 (49), 55 (100). Anal.Calcd. for C26H16N10O6S2 (628.60): C, 49.68; H, 2.57; N, 22.28. Found C, 49.59; H, 2.48; N, 22.10%.
3.2 In-vitro XTT assay
XTT assay, a non-radioactive colorimetric assay system, is usually used for measuring cell viability, proliferation and cytotoxicity through the measurement of cellular metabolic activity. This test depends on the reduction of a yellow tetrazolium salt (XTT dye) to an orange formazan dye by metabolically active cells. The minimal inhibitory concentration (MIC) values, which represent the lowest concentrations of samples or standard drugs (Vancomycine for bacteria and Amphotricine B for fungi) that completely inhibit the microbial growth. MICs were determined using the microdilution method. The bacterial inoculum was prepared and the suspensions modified to 106 CFU/mL. The examined samples and the standard drugs were prepared in dimethyl sulfoxide (DMSO), accompanied by two double dilutions in a 96-well plate. Every microplate well included 40 μL of the growth medium, 10 μL of the inoculum and 50 μL of the investigated compounds diluted at final concentrations (1000-0,12 μg/mL), and DMSO was used as a negative monitor. The plates were incubated at 37 °C for 24 hours. Thereafter, 40 μL of tetrazolium salt was applied. The plates were incubated in dark for 1 h at 37 °C, after which colorimetric change in the XTT reduction assay was measured using a μL plate reader at 492 nm. The MIC was detected as the lowest concentration capable of causing the largest color change compared to the negative control [50].
3.3 In-silico studies
The electronic properties of the synthesized derivatives that demonstrated the best biological activities in the in-vitro XTT assay were investigated with density functional theory calculations. The calculations were carried out with the aid of Gaussian 09 [51]. The geometry of the studied molecules was fully optimized using B3LYP/6-311G functional and the obtained molecular orbitals were visualized.
Molecular docking was used to investigate the interaction of the best biologically active molecules with the microbial receptors. We selected the most probable bacterial/fungal proteins that can be affected by the synthesized thiazole ligands based on the results previously reported in the literature. Molecular docking was carried out with the aid of the Molecular Operating Environment (MOE) 2014 software [52]. The geometry-optimized compounds that demonstrated the lowest MIC values in the XTT assay were selected and docked with the corresponding receptors. High-resolution 3D molecular structures of the receptors Secreted Aspartic Proteinase (SAP2; C. albicans; PDB ID: 1EAG), Enoyl-acyl Carrier Protein Reductase (fabI; S. aureus; PDB ID: 3GR6) and Enoyl-acyl Carrier Protein Reductase (FabI; P. aerugiosa; PDB ID: 4NR0) were obtained from the Protein Data Bank (PDB).