Materials and methods
Reagents and chemicals of analytical grade were purchased from commercial sources and used as such without further purification. The melting points were determined on Labtech melting point apparatus and are uncorrected. The progress of reaction was confirmed by TLC performed on silica gel-G plates and the spot was visualized in iodine chamber. Media for antimicrobial activity were obtained from Hi-media Laboratories. Microbial type cell cultures (MTCC) for antimicrobial activity were procured from IMTECH, Chandigarh. Infrared (IR) spectra of the synthesized derivatives were obtained on Bruker 12060280, Software: OPUS 7.2.139.1294 spectrophotometer using KBr disc method covering a range of 4000–400 cm−1. The proton nuclear magnetic resonance (1H NMR) spectra were traced in deuterated dimethyl sulphoxide on Bruker Avance III 600 NMR spectrometer at a frequency of 600 MHz downfield to tetramethylsilane standard. Chemical shifts of 1H NMR were recorded as δ (parts per million). The 13C NMR of the compounds was obtained at a frequency of 150 MHz on Bruker Avance II 150 NMR spectrometer. The LCMS data were recorded on Waters Q-TOF micromass (ESI–MS) while elemental analyses were carried out on a Microprocessor based Thermo Scientific (FLASH 2000) CHNS-O Organic Elemental Analyser.
General procedure for synthesis of ethyl-2-(1H-benzo[d]imidazol-2-ylthio)acetate (2)
A solution containing equimolar (0.03 mol) mixture of 2-mercaptobenzimidazole (1) and potassium hydroxide was heated to 80–90 °C along with stirring in 60 ml ethanol for 15 min. Ethyl chloroacetate (0.03 mol) was then added in one portion that resulted in rise of temperature of 30–40 °C due to exothermic reaction. The reaction mixture was stirred for 24 h at 18–20 °C and poured into 100 g of ice. The mixture was further stirred for 30 min, maintaining the temperature at 0–10 °C. The white product obtained was collected by filtration, washed to render it free of chloride, dried and recrystallized with ethanol to obtain pure product.
General procedure for synthesis of ethyl-2-(1H-benzo[d]imidazol-2-ylthio) acetohydrazide (3)
A mixture of compound 2 (0.01 mol), hydrazine hydrate (0.06 mol) and absolute ethanol was gently refluxed in a round bottom flask on a water bath for an appropriate time. The completion of reaction was checked by TLC. The obtained mixture was concentrated and kept overnight in refrigerator. The creamish white precipitate obtained was separated from the mother liquor, dried and recrystallized from boiling water in order to obtain the pure compound 3.
General procedure for synthesis of Schiff’s bases (4a–4r)
A solution containing equimolar quantities of different aromatic aldehydes (0.01 mol) and compound 3 (0.01 mol) was refluxed for a period of 3–5 h using a few drops of glacial acetic acid as catalyst in ethyl alcohol. The completion of reaction was confirmed by TLC. The excess of solvent was distilled off at low temperature in a rotary evaporator. The resulting solid was washed with dilute ethyl alcohol and recrystallized from rectified spirit.
General procedure for synthesis of benzimidazole-substituted-1,3-thiazolidin-4-ones (5a–5r)
The title compounds benzimidazole-substituted-1,3-thiazolidin-4-ones (5a–5r) were synthesised by refluxing the appropriate Schiff base (4a–4r, 0.015 M) with thioglycolic acid (0.015 M) for 8–10 h in 50 ml dioxane using a pinch of anhydrous zinc chloride as catalyst. The end-point of reaction was ascertained by TLC. The reaction mixture was then cooled to ambient temperature and neutralized with aqueous solution of sodium bicarbonate. The solid obtained was filtered, washed with water and recrystallized from ethanol.
Spectral data of benzimidazole-substituted-1,3-thiazolidin-4-ones (5a–5r)
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(2-methoxyphenyl)-4-oxothiazolidin-3-yl)acetamide (5a)
Yield 90.3%; mp 130–131 °C; Rf 0.46 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 574 OCN deformation, amide present, 1529 ring str. of thiazolidinone, 1595 C=O of thiazolidinone, 3071 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 7.01–7.98 (m, 8H aromatic), 6.99 (s, CH of thiazolidinone), 8.13 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.74 CH2 of thiazolidinone, 40.01 CH2 aliphatic, 55.61 CH of thiazolidinone, 55.77 C of OCH3 (111.63, 111.98, 120.45, 121.03, 122.15, 130.99, 138.60,152.87, 157.19) C aromatic, 158.71 C=O of thiazolidinone, 162.25 C of amide; ESI–MS (m/z) [M + 1] + 415.51; Anal. Calcd. for C19H18N4O3S2: C, 55.05; H, 4.38; N, 13.52; O, 11.58; S, 15.47. Found: C, 55.02; H, 4.42; N, 13.56; O, 11.60; S, 15.50.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(3-methoxyphenyl)-4-oxothiazolidin-3-yl)acetamide (5b)
Yield 60.5%; mp 198–200 °C; Rf 0.34 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 533 OCN deformation, amide present, 1268 C–O–C of asymmetric aralkyl, 1466 ring str. of thiazolidinone, 1593 C=O of thiazolidinone, 2931 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.32 (s, 2H of methylene), 6.94–7.99 (m, 8H aromatic), 6.91 (s, CH of thiazolidinone), 8.00 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.75 CH2 of thiazolidinone, 40.01 CH2 aliphatic, 162.28 C of amide; ESI–MS (m/z) [M + 1]+ 415.52; Anal. Calcd. for C19H18N4O3S2: C, 55.05; H, 4.38; N, 13.52; O, 11.58; S, 15.47. Found: C, 55.06; H, 4.41; N, 13.54; O, 11.59; S, 15.55.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(4-methoxyphenyl)-4-oxothiazolidin-3-yl)acetamide (5c)
Yield 81.2%; mp 205–208 °C; Rf 0.46 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 743 OCN deformation of amide, 1252 C–O–C str. aralkyl asymmetric, 1466 ring str. of thiazolidinone, 1634 C=O of thiazolidinone, 2931 N–H str. of imidazole, 3056 N–H str. secondary amide associated; 1HNMR (DMSO-d6) δ: 3.323 (s, 2H of methylene), 6.80–7.95 (m, 8H aromatic), 6.65 (s, CH of thiazolidinone), 7.98 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.74 CH2 of thiazolidinone, 39.91 CH2 aliphatic, 55.05 CH of thiazolidinone, 55.21 C of OCH3 (114.06, 128.04) C aromatic, 160.04 C=O of thiazolidinone, 162.26 C of amide; ESI–MS (m/z) [M + 1]+ 415.52; Anal. Calcd. for C19H18N4O3S2: C, 55.05; H, 4.38; N, 13.52; O, 11.58; S, 15.47. Found: C, 55.03; H, 4.36; N, 13.52; O, 11.56; S, 15.43.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(2,4-dimethoxyphenyl)-4-oxothiazolidin-3-yl) acetamide (5d)
Yield 94.1%; mp 182–184 °C; Rf 0.31 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 743 OCN deformation of amide, 1034 C–O–C str. symmetric, 1463 ring str. of thiazolidinone, 1636 C=O of thiazolidinone, 2936 N–H str. of imidazole, 3057 N–H str. secondary amide; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 6.96–7.95 (m, 8H aromatic), 6.69 (s, CH of thiazolidinone), 8.03 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.73 CH2 of thiazolidinone, 39.89 CH2 aliphatic, 55.36 CH of thiazolidinone, 55.65 C of OCH3 (97.99, 106.21, 115.32, 126.80, 158.45) C aromatic, 161.85 C=O of thiazolidinone, 162.26 C of amide; ESI–MS (m/z) [M + 1]+ 445.24; Anal. Calcd. for C20H20N4O4S2: C, 55.04; H, 4.53; N, 12.60; O, 14.40; S, 14.43. Found: C, 55.07; H, 4.51; N, 12.57; O, 14.37; S, 14.45.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(4-hydroxyphenyl)-4-oxothiazolidin-3-yl)acetamide (5e)
Yield 73.2%; mp 105–107 °C; Rf 0.48 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 529 OCN deformation, amide present, 1508 ring str. of thiazolidinone, 1658 C=O of thiazolidinone, 2927 O–H associated conjugate chelation intramolecular H-bonded with C=O, 3060 N–H str. of secondary amide (associated), 3224 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 6.91–7.95 (m, 8H aromatic), 6.86 (s, CH of thiazolidinone), 8.55 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.74 CH2 of thiazolidinone, 39.88 CH2 aliphatic, 40.00 CH of thiazolidinone, (115.05, 115.71, 127.52, 130.05) C aromatic, 162.27 C of amide; ESI–MS (m/z) [M + 1]+ 401.34; Anal. Calcd. for C18H16N4O3S2: C, 53.98; H, 4.03; N, 13.99; O, 11.99; S, 16.01. Found: C, 53.96; H, 3.98; N, 13.95; O, 11.96; S, 16.04.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(2-chlorophenyl)-4-oxothiazolidin-3-yl)acetamide (5f)
Yield 62.2%; mp 168–170 °C; Rf 0.42 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 755 C–Cl str. aromatic, 1498 ring str. of thiazolidinone, 1635 C=O of thiazolidinone, 3059 N–H str. of secondary amide (associated), 3206 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 7.00–7.95 (m, 8H aromatic), 8.16 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.74 CH2 of thiazolidinone, 40.02 CH2 aliphatic, 41.14 CH of thiazolidinone, (126.45, 127.03, 127.12, 127.70, 128.16, 129.39, 129.48,130.14, 134.81, 158.21) C aromatic, 162.25 C=O of thiazolidinone, 167.52 C of amide; ESI–MS (m/z) [M + 1]+ 419.04; Anal. Calcd. for C18H15ClN4O2S2: C, 51.61; H, 3.61; N, 13.37; O, 7.64; S, 15.31. Found: C, 51.56; H, 3.59; N, 13.39; O, 7.67; S, 15.34.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(4-chlorophenyl)-4-oxothiazolidin-3-yl)acetamide (5g)
Yield 84.7%; mp 234–236 °C; Rf 0.37 (Toluene: Ethyl acetate:: 3:1); IR (KBr cm−1) νmax: 742 C–Cl str. aromatic, 1490 ring str. of thiazolidinone, 1636 C=O of thiazolidinone, 3059 N–H str. of secondary amide (associated), 3209 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 6.99-7.95 (m, 8H aromatic), 8.03 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.74 CH2 of thiazolidinone, 39.76 CH2 aliphatic, 39.89 CH of thiazolidinone, (99.47, 112.61, 120.66, 128.23, 128.59, 133.46, 133.67,140.91) C aromatic, 162.25 C of amide; ESI–MS (m/z) [M + 1]+ 419.01; Anal. Calcd. for C18H15ClN4O2S2: C, 51.61; H, 3.61; N, 13.37; O, 7.64; S, 15.31. Found: C, 51.54; H, 3.65; N, 13.41; O, 7.65; S, 15.29.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl) acetamide (5h)
Yield 82.6%; mp 218-220 °C; Rf 0.43 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 744 OCN deformation, 1074 C–F str. monoflourinated compound, 1531 ring str. of thiazolidinone, 1632 C=O of thiazolidinone, 3058 N–H str. of secondary amide (associated), 3206 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 6.8–7.95 (m, 8H aromatic), 6.61 (s, CH of thiazolidinone), 8.00 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.74 CH2 of thiazolidinone, 39.88 CH2 aliphatic, 40.00 CH of thiazolidinone, (112.10, 144.26) C aromatic, 162.26 C of amide; ESI–MS (m/z) [M + 1]+ 403.43; Anal. Calcd. for C18H15FN4O2S2: C, 53.72; H, 3.76; N, 13.92; O, 7.95; S, 15.93. Found: C, 53.74; H, 3.76; N, 13.95; O, 7.97; S, 15.91.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(4-bromophenyl)-4-oxothiazolidin-3-yl)acetamide (5i)
Yield 86.9%; mp 140–143 °C; Rf 0.38 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 626 OCN deformation, 744 C–Br str. aromatic, 1469 ring str. of thiazolidinone, 1595 C=O of thiazolidinone, 2815 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.34 (s, 2H of methylene), 7.01–7.95 (m, 8H aromatic), 8.02 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.75 CH2 of thiazolidinone, 39.89 CH2 aliphatic, 40.02 CH of thiazolidinone, (122.32, 128.56, 130.16, 131.50, 131.97, 130.99, 132.88,133.92) C aromatic, 160.68 C=O of thiazolidinone, 162.26 C of amide; ESI–MS (m/z) [M + 1]+ 464.35; Anal. Calcd. for C18H15BrN4O2S2: C, 46.66; H, 3.26; N, 12.09; O, 6.91; S, 13.84. Found: C, 46.64; H, 3.23; N, 12.05; O, 6.95; S, 15.81.
2-(1H-benzo[d]imidazol-2-ylthio)-N-(2-(4-nitrophenyl)-4-oxothiazolidin-3-yl)acetamide (5j)
Yield 88.8%; mp 120–122 °C; Rf 0.46 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 743 OCN deformation, 833 C–N str. aromatic nitro group, 1516 ring str. of thiazolidinone, 1597 C=O of thiazolidinone, 3211 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.35 (s, 2H of methylene), 6.50 (s, CH of thiazolidinone), 6.59–7.95 (m, 8H aromatic), 8.07 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.73 CH2 of thiazolidinone, 39.75 CH2 aliphatic, 39.89 CH of thiazolidinone, (113.43, 113.79, 122.21, 123.80, 127.16, 128.53, 129.40, 150.71) C aromatic, 162.25 C of amide; ESI–MS (m/z) [M + 1]+ 430.43; Anal. Calcd. for C18H15N5O4S2: C, 50.34; H, 3.52; N, 16.31; O, 14.90; S, 14.93. Found: C, 50.29; H, 3.53; N, 16.35; O, 14.95; S, 14.91.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(4-hydroxy-3-methoxyphenyl)-4-oxothiazolidin-3-yl) acetamide (5k)
Yield 67.9%; mp 122–124 °C; Rf 0.76 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 616 OCN deformation, amide present, 1280 C–O–C str. of aralkyl asymmetric, 1465 ring str. of thiazolidinone, 1597 C=O of thiazolidinone, 3206 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.34 (s, 2H of methylene), 6.86–7.98 (m, 8H aromatic), 6.80 (s, CH of thiazolidinone), 8.57 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.74 CH2 of thiazolidinone, 40.01 CH2 aliphatic, 55.48 CH of thiazolidinone, 55.83 C of OCH3 (99.47, 109.43, 115.32, 115.44, 121.37, 122.25, 147.93) C aromatic, 162.26 C of amide; ESI–MS (m/z) [M + 1]+ 431.47; Anal. Calcd. for C19H18N4O4S2: C, 53.01; H, 4.21; N, 13.01; O, 14.87; S, 14.90. Found: C, 52.97; H, 4.23; N, 13.05; O, 14.85; S, 14.94.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(3-ethoxy-4-hydroxyphenyl)-4-oxothiazolidin-3-yl) acetamide (5l)
Yield 89.9%; mp 110–112 °C; Rf 0.32 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 617 OCN deformation, amide present, 1276 C–O–C str. of aralkyl asymmetric, 1469 ring str. of thiazolidinone, 1637 C=O of thiazolidinone, 3063 N–H str. of secondary amide (associated), 3220 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 6.81–7.94 (m, 8H aromatic), 6.66 (s, CH of thiazolidinone), 7.95 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 14.71 C of OCH2
CH3, 35.73 CH2 of thiazolidinone, 39.75 CH2 aliphatic, 39.88 CH of thiazolidinone, 64.03 C of OCH2CH3 (111.20, 115.49, 121.33, 125.86, 147.09, 148.74, 153.45) C aromatic, 162.26 C of amide; ESI–MS (m/z) [M + 1]+ 445.52; Anal. Calcd. for C20H20N4O4S2: C, 54.04; H, 4.53; N, 12.60; O, 14.40; S, 14.43. Found: C, 54.07; H, 4.55; N, 12.65; O, 14.43; S, 14.46.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(4-formylphenyl)-4-oxothiazolidin-3-yl)acetamide (5m)
Yield 69.2%; mp 200–203 °C; Rf 0.31 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 742 OCN deformation, amide present, 952 C-H out of plane bending of aldehyde group, 1468 ring str. of thiazolidinone, 1660 C=O of thiazolidinone, 3052 N–H str. of secondary amide (associated), 3192 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 6.95–7.85 (m, 8H aromatic), 6.91 (s, CH of thiazolidinone), 7.95 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.75 CH2 of thiazolidinone, 40.01 CH2 aliphatic, 39.61 CH of thiazolidinone, 162.27 C of amide; ESI–MS (m/z) [M + 1]+ 413.44; Anal. Calcd. for C19H16N4O3S2: C, 55.32; H, 3.91; N, 13.58; O, 11.64; S, 15.55. Found: C, 55.37; H, 3.95; N, 13.55; O, 11.66; S, 15.58.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(2-hydroxyphenyl)-4-oxothiazolidin-3-yl)acetamide (5n)
Yield 62.4%; mp 196–198 °C; Rf 0.66 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 751 OCN deformation, amide present, 1466 ring str. of thiazolidinone, 1611 C=O of thiazolidinone, 2928 O–H associated with C=O, 3058 N–H str. of secondary amide (associated), 3213 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 6.91–7.70 (m, 8H aromatic), 6.89 (s, CH of thiazolidinone), 7.95 (s, NH of amide), 13C-NMR (DMSO-d6) δ: 35.74 CH2 of thiazolidinone, 39.76 CH2 aliphatic, 40.02 CH of thiazolidinone, (109.44, 116.50, 118.15, 119.56, 122.25, 130.36, 130.80, 133.19, 158.60) C aromatic, 162.26 C=O of thiazolidinone, 162.75 C of amide; ESI–MS (m/z) [M + 1]+ 456.53; Anal. Calcd. for C22H25N5O2S2: C, 58.00; H, 5.53; N, 15.37; O, 7.02; S, 14.08. Found: C, 57.97; H, 5.57; N, 15.39; O, 7.06; S, 14.03.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(4-(dimethylamino)phenyl)-4-oxothiazolidin-3-yl) acetamide (5o)
Yield 64.6%; mp 85–87 °C; Rf 0.60 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 746 OCN deformation of amide, 1362 C–N str. aryl tertiary amine, 1524 ring str. of thiazolidinone, 1600 C=O of thiazolidinone, 3050 N–H str. of secondary amide (associated), 2911 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 6.59 (s, CH of thiazolidinone), 6.65–7.96 (m, 8H aromatic), 8.49 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.73 CH2 of thiazolidinone, 39.64 CH2 aliphatic, 40.13 CH of thiazolidinone, 40.84 CH2 of amide, (109.43, 111.63, 121.52, 124.99, 126.43, 128.22, 129.58, 151.18, 151.91, 153.25) C aromatic, 162.25 C=O of thiazolidinone, 168.41 C of amide; ESI–MS (m/z) [M + 1]+ 401.45; Anal. Calcd. for C18H16N4O3S2: C, 53.98; H, 4.03; N, 13.99; O, 11.99; S, 16.01. Found: C, 53.95; H, 4.07; N, 14.03; O, 11.96; S, 16.03.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(4-(diethylamino)phenyl)-4-oxothiazolidin-3-yl) acetamide (5p)
Yield 91.7%; mp 128–130 °C; Rf 0.38 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 744 OCN deformation of amide, 1357 C–N str. aryl tertiary amine, 1523 ring str. of thiazolidinone, 1633 C=O of thiazolidinone, 2970 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 6.95–7.91 (m, 8H aromatic), 6.58 (s, CH of thiazolidinone), 7.95 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 12.39 C of CH2
CH3, 35.73 CH2 of thiazolidinone, 39.92 CH2 aliphatic, 43.53 CH of thiazolidinone, 39.92 CH2 of amide, 43.93 C of CH2CH3 (99.47, 110.93, 111.36, 120.72, 123.67, 127.73, 128.51, 129.87, 148.46, 153.42) C aromatic, 162.24 C=O of thiazolidinone, 189.39 C of amide; ESI–MS (m/z) [M + 1]+ 428.52; Anal. Calcd. for C20H21N5O2S2: C, 56.18; H, 4.95; N, 16.38; O, 7.48; S, 15.00. Found: C, 56.15; H, 4.97; N, 16.43; O, 7.46; S, 15.03.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(4-oxo-2-styrylthiazolidin-3-yl)acetamide (5q)
Yield 83.2%; mp 210–212 °C; Rf 0.56 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 746 OCN deformation, amide present, 1493 ring str. of thiazolidinone, 1593 C=O of thiazolidinone, 3057 N–H str. of secondary amide (associated), 3206 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 6.58 (d, 2H of CH=CH aliphatic, J = 12 Hz), 6.92 (s, CH of thiazolidinone), 6.99–7.95 (m, 8H aromatic), 8.06 (s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.74 CH2 of thiazolidinone, 39.77 CH2 aliphatic, 39.90 CH of thiazolidinone, (125.72, 126.80, 127.18, 128.56, 128.81) C aromatic, 162.26 C of amide; ESI–MS (m/z) [M + 1]+ 411.47; Anal. Calcd. for C20H18N4O2S2: C, 58.52; H, 4.42; N, 13.65; O, 7.79; S, 15.62. Found: C, 58.55; H, 4.47; N, 13.63; O, 7.76; S, 15.66.
2-(1H-Benzo[d]imidazol-2-ylthio)-N-(2-(4-hydroxynaphthalen-1-yl)-4-oxothiazolidin-3-yl) acetamide (5r)
Yield 74.4%; mp 237–239 °C; Rf 0.82 (Toluene:Ethyl acetate: 3:1); IR (KBr cm−1) νmax: 746 OCN deformation of amide, 1464 ring str. of thiazolidinone, 1599 C=O of thiazolidinone, 3055 N–H str. of secondary amide, 3226 N–H str. of imidazole; 1HNMR (DMSO-d6) δ: 3.33 (s, 2H of methylene), 6.96–7.95 (m, 8H aromatic), 6.91 (s, CH of thiazolidinone), 8.03(s, NH of amide); 13C-NMR (DMSO-d6) δ: 35.74 CH2 of thiazolidinone, 39.92 CH2 aliphatic, 40.83 CH of thiazolidinone, 39.78 CH2 of amide, (109.44, 128.89) C aromatic, 163.61 C=O of thiazolidinone, 168.24 C of amide; ESI–MS (m/z) [M + 1]+ 451.51; Anal. Calcd. for C22H18N4O3S2: C, 58.65; H, 4.03; N, 12.44; O, 10.65; S, 14.23. Found: C, 58.69; H, 4.07; N, 12.42; O, 10.69; S, 14.26.
Antimicrobial activity evaluation
Determination of MIC
The in vitro antimicrobial potential of the synthesized derivatives was assessed using tube dilution method. The micro-organisms used in the study are E. coli (Gram-negative bacterium); B. subtilis MTCC 2063, S. aureus MTCC 2901 (Gram-positive bacterial strain); C. albicans MTCC 227 and A. niger MTCC 8189 (fungal strains) [24]. Serial dilutions of both standard and test compounds were prepared in double strength nutrient broth I.P. (Indian Pharmacopoeia) for bacterial strain and Sabouraud dextrose broth I.P. for fungi [25]. The bacterial cultures were incubated at 37 ± 2 °C for 24 h. The incubation temperature and period for C. albicans was 37 ± 2 °C for 48 h while for A. niger was 25 ± 2 °C for 7 day. The results of antimicrobial activity were compared to the standard antibacterial (norfloxacin) and antifungal (fluconazole) drugs and are expressed in terms of MIC (minimum inhibitory concentration).
Determination of MBC/MFC
The subculturing of 100 µl of culture from each tube that showed no growth in MIC determination onto sterilized petri-plates containing fresh agar medium gave the minimum bactericidal concentration (MBC) and fungicidal concentration (MFC) of the synthesized compounds. After incubation under suitable conditions of temperature and time, the petri-plates were analyzed for microbial growth visually. MBC and MFC denote the minimum quantity of a drug needed to kill nearly 99.9% of the microbes [26].
In vitro cytotoxic evaluation
The cytotoxicity of the synthesized benzimidazole-substituted-1,3-thiazolidin-4-ones was evaluated in vitro on human colorectal carcinoma (HCT116) using Sulforhodamine-B (SRB) assay and the results were compared with that of the standard anticancer drug, 5-fluorouracil. This method is highly cost effective allowing testing of a large number of samples within a short period of time as compared to fluorometric methods [27]. The results of anticancer activity are expressed in terms of µM/ml.
The cells were allowed to attach to the walls of 96-multititre plates for a period of 24 h before treatment with the test compounds. Solutions of the test and standard compounds were prepared in DMSO and made up to appropriate volume with saline. Monolayer cells with different concentrations (5, 12.5, 25 and 50 µg/ml) of the test compounds were then incubated at 37 °C for 48 h in an atmosphere of 5% carbon dioxide. The cells were fixed with trichloroacetic acid for an hour, washed with water and stained with 0.4% w/v solution of pink colored aminoxanthine dye, Sulforhodamine-B, in acetic acid for 30 min. The cultures were washed with 1% acetic acid to remove the excess stain. The attached stain was recovered using Tris-EDTA buffer. The colour intensity was measured using ELISA reader. The experiment was done in triplicate.
Molecular docking studies on CDK-8
All the synthesized derivatives were docked onto the crystal structure of cyclin-dependent kinase 8 (CDK8) using sequential docking procedure on the crystal structure [PDB ID: 5FGK] retrieved from the protein data bank (PDB) [16]. The CDK8 protein structure was optimized using protein preparation wizard by removing the water molecules, hetero-atoms and co-factors. Hydrogen, missing atoms, bonds and charges were computed through Maestro. The synthesized benzimidazole-substituted-1,3-thiazolidin-4-ones were further docked. The structures of synthesized derivatives were built and optimized using LigPrep module implemented in Schrodinger Maestro. Ligand preparation includes generating various tautomers, assigning bond orders, ring conformations and stereochemistry. All the generated conformations were minimized using OPLS2005 force field prior to docking study.
A receptor grid was generated around the active site of CDK8 enzyme by choosing centroid of the enzyme complexed ligand (5XG ligand taken as the reference). The size of grid box was set to 20 Å radius using receptor grid generation implemented in Glide [28]. Docking calculations were accomplished using Glide. All docking calculations were performed using Extra Precision (XP) mode. The Glide docking score determined the best docked structure from the output. The interactions of these docked complexes were further analyzed and imaged using PyMOL [29].