General chemistry
Melting points (uncorrected) were and determined in open capillary on a Gallen Kamp melting point apparatus (Sanyo Gallen Kamp, UK). Precoated silica gel plates (Kieselgel 0.25 mm, 60 F254, Merck, Germany) were used for thin layer chromatography. A developing solvent system of chloroform/methanol (8:2) was used and the spots were detected by ultraviolet light. IR spectra (KBr disc) were recorded using an FT-IR spectrophotometer (Perkin Elmer, USA). 1H-NMR spectra were scanned on an NMR spectrophotometer (Bruker AXS Inc., Switzerland), operating at 500 MHz for 1H- and 125.76 MHz for 13C. Chemical shifts are expressed in δ-values (ppm) relative to TMS as an internal standard, using DMSO-d
6
as a solvent. Elemental analyses were done on a model 2400 CHNSO analyser (Perkin Elmer, USA). All the values were within ±0.4 % of the theoretical values. All reagents used were of AR grads.
Synthesis of thioureidobenzenesulfonamide derivatives (3–17)
General procedure
A mixture of 4-isothiocyanatobenzenesulfonamide 2 (2.14 g, 0.01 mol) and amines (0.012 mol) in dry dimethylformamide (15 ml) containing three drops of triethylamine was refluxed for 24 h, then left to cool. The solid product formed upon pouring onto ice/water was collected by filtration and recrystallized from ethanol–dimethylformamide to give 3–17, respectively.
4-(3-Heptylthioureido)benzenesulfonamide (3)
Yield, 92 %; m.p. 124.7 °C. IR (KBr, cm−1): 3218, 3143 (NH, NH2), 3087 (CH arom.), 2926, 2853 (CH aliph.), 1376, 1150 (SO2), 1254 (C=S). 1H-NMR (DMSO-d2): 0.8 [t, 2H, CH3], 1.2–1.4 [m, 10H, 5CH2], 3.3 [m, 2H, NHCH2], 7.3–7.9 [m, 6H, Ar–H + SO2NH2], 9.3, 10.4 [2 s, 2NH, exchangeable with D2O]. 13C-NMR (DMSO-d6): 14.2, 22.4, 26.2, 28.6, 29.0, 31.5, 43.9, 119.4 (2), 127.4 (2), 134.7, 143.0, 177.4.MS m/z (%): 329 (M+) (14.41), 155 (100). Anal.Calcd. For C14H23N3O2S2 (329): C, 51.03; H, 7.04; N, 12.75. Found: C, 51.29; H, 6.79; N, 12.45.
4-(3-(4-Hydroxyphenyl)thioureido)benzenesulfonamide (4)
Yield, 88 %; m.p.192.9 °C. IR (KBr, cm−1): 3363 (OH), 3280, 3143 (NH, NH2), 3090 (CH arom.), 1393, 1182 (SO2), 1274 (C=S).1H-NMR (DMSO-d2): 6.7–7.9 [m, 10H, Ar–H + SO2NH2], 10.2, 11.4, [2 s, 2H, 2NH, exchangeable with D2O], 13.1 [s, 1H, OH, exchangeable with D2O], 13C-NMR (DMSO-d6): 112.9 (2), 122.8 (2), 126.7 (2), 127.1 (2), 127.9, 139.8, 140.3, 157.6, 180.1. MS m/z (%): 323 (M+) (9.03), 91 (100). Anal.Calcd. For C13H13N3O3S2 (323): C, 48.28; H, 4.05; N, 12.99. Found: C, 48.55; H, 4.31; N, 13.29.
4-(3-(3,5-Dimethoxyphenyl)thioureido)benzenesulfonamide (5)
Yield, 77 %; m.p. 160.3 °C. IR (KBr, cm−1): 3317, 3254, 3173 (NH, NH2), 3100 (CH arom.), 2963, 2938, 2829 (CH aliph.), 1363, 1156 (SO2), 1259 (C=S). 1H-NMR (DMSO-d2): 3.9 [s, 6H, 2OCH3], 6.3–7.8 [m, 8H, Ar–H + SO2NH2], 9.8 [s, 2H, 2NH, exchangeable with D2O].13C-NMR (DMSO-d6): 56.1 (2), 96.8, 102.0 (2), 123.2 (2), 126.6 (2), 141.4 (2), 143.1, 160.6 (2), 179.3. MS m/z (%): 367 (M+) (17.8), 76 (100). Anal.Calcd. For C15H17N3O4S2 (367): C, 49.03; H, 4. 66; N, 11.44. Found: C, 48.74; H, 4.29; N, 11.17.
4-(3-(2-Methyl-6-nitrophenyl)thioureido)benzenesulfonamide (6)
Yield, 81 %; m.p. 226.0 °C. IR (KBr, cm−1): 3353, 3243, 3171 (NH, NH2), 3009 (CH arom.), 1340, 1161 (SO2), 1290 (C=S).1H-NMR (DMSO-d2): 2.2 [s, 3H, CH3], 6.5–7.8 [m, 9H, Ar–H + SO2NH2], 10.3 [s, 2H, 2NH exchangeable with D2O]. 13C-NMR (DMSO-d6): 18.3, 123.3, 123.9 (2), 126.7, 126.8, 127.8 (2), 131.3, 136.5 (2), 139.8, 142.8, 180.1. MS m/z (%): 366 (M+) (15.8), 133 (100). Anal.Calcd. For C14H14N4O4S2 (366): C, 45.89; H, 3.85; N, 15.29. Found: C, 45.57; H, 3.54; N, 15.61.
4-(3-Benzo[d][1,3]dioxol-5-ylthioureido)benzenesulfonamide (7)
Yield, 86 %; m.p. 136.6 °C. IR (KBr, cm−1): 3325, 3241 (NH, NH2), 3100 (CH arom.), 1331, 1156 (SO2), 1241 (C=S). 1 H-NMR (DMSO-d2): 6.0 [s, 2H, CH2], 6.7–7.9 [m, 9H, Ar–H + SO2NH2], 9.5 [s, 2H, 2NH, exchangeable with D2O]. 13C-NMR (DMSO-d6): 101.2, 107.1, 109.1, 117.8, 123.1 (2), 126.5 (2), 133.4, 134.6, 142.4, 143.2, 147.3, 180.6. MS m/z (%): 351 (M+) (34.64), 93 (100). Anal.Calcd. For C14H13N3O4S2 (351): C, 47.85; H, 3.73; N, 11.96. Found: C, 47.49; H, 3.43; N, 11.62.
4-(3-Benzo[d][1,3]dioxol-5-ylmethyl)thioureido)benzenesulfonamide (8)
Yield, 68 %; m.p. 140.8 °C. IR (KBr, cm−1): 3384, 3348, 3206 (NH, NH2), 3003 (CH arom.), 1377, 1185 (SO2), 1294 (C=S).1H-NMR (DMSO-d2): 4.3 [s, 2H, CH2], 6.0 [s, 2H, OCH2O], 6.7–7.7 [m, 9H, Ar–H + SO2NH2], 7.8 [s, 2H, +2NH, exchangeable with D2O]. 13C-NMR (DMSO-d6): 63.8, 101.2, 106.4, 108.2, 120.8, 121.4 (2), 131.2 (2), 133.5, 134.0, 146.4, 147.6, 148.3, 161.1. MS m/z (%): 365 (M+) (18.42), 135 (100). Anal.Calcd. For C15H15N3O4S2 (365): C, 49.30; H, 4.14; N, 11.50. Found: C, 49.05; H, 4.46; N, 11.19.
4-(3-(1-Adamanylamine)thioureidobenzenesulfonamide (9)
Yield, 80 %; m.p. 174.5 °C. IR (KBr, cm−1): 3434, 3354 (NH, NH2), 3100 (CH arom.), 2997, 2906, 2851 (CH aliph.), 1396, 1186 (SO2), 1282 (C=S).1H-NMR (DMSO-d2): 1.6–1.9 [m, 12H, 6CH2], 2.2–2.4 [m, 3H, 3 CH], 6.9–7.9 [m, 6H, Ar–H + SO2NH2], 11.4 [s, 2H, 2NH, exchangeable with D2O].13C-NMR (DMSO-d6): 28.8 (3), 35.3 (3), 40.5 (3), 44.9, 126.4 (2), 129.1 (2), 131.8, 142.7, 179.9. MS m/z (%): 366 (M+) (9.32), 154 (100). Anal.Calcd. For C17H23N3O2S2 (366): C, 55.86; H, 6.34; N, 11.50. Found: C, 55.50; H, 6.68; N, 11.18.
4-(3-(5,6-Dimethylbenzo[d]thiazol-2-yl)thioureido)benzenesulfonamide (10)
Yield, 84 %; m.p. 252.1 °C. IR (KBr, cm−1): 3359, 3257, 3143 (NH, NH2), 3031 (CH arom.), 2954, 2851 (CH aliph.), 1594 (C=N), 1381, 1186 (SO2), 1296 (C=S).1H-NMR (DMSO-d2): 2.2 [s, 6H, 2CH3], 7.2–8.0 [m, 8H, Ar–H + SO2NH2], 10.2, 13.0 [2 s, 2H, 2NH, exchangeable with D2O].13C-NMR (DMSO-d6): 19.9, 20.4, 118.5, 121.5, 123.3 (2), 126.6, 127.1, 127.8 (2), 133.0, 136.2, 139.8, 143.1, 151.9, 180.0 (2).MS m/z (%): 393 (M+) (16.9), 162 (100). Anal.Calcd. For C16H16N4O2S3 (393): C, 48.96; H, 4.11; N, 14.27. Found: C, 48.66; H, 3.85; N, 14.54.
4-(3-(6-Ethoxybenzo[d]thiazol-2-yl)thioureido)benzenesulfonamide (11)
Yield, 78 %;m.p. 153.6 °C. IR (KBr, cm−1): 3410, 3334, 3195 (NH, NH2), 3069 (CH arom.), 2974, 2925, 2843 (CH aliph.), 1595 (C=N), 1393, 1123 (SO2), 1256 (C=S). 1H-NMR (DMSO-d2): 1.3 [t, 3H, CH3], 4.0 [q, 2H, CH2], 6.9–8.0 [m, 9H, Ar–H + SO2NH2], 10.3, 11.2 [2 s, 2H, 2NH, exchangeable with D2O]. 13C-NMR (DMSO-d6): 15.0, 66.8, 106.0, 115.3, 118.2, 120.4 (2), 127.7 (2), 132.7, 138.2, 140.9, 142.1, 157.7, 177.1, 180.1. MS m/z (%): 409 (M+) (1.85), 156 (100). Anal.Calcd. For C16H16N4O3S3 (409): C, 47.04; H, 3.95;N, 13.71. Found: C, 47.34; H, 3.67; N, 13.39.
4-(3-(6-Nitrobenzo[d]thiazol-2-yl))thioureido)benzenesulfonamide (12)
Yield, 65 %; m.p. 205.8 °C. IR (KBr, cm−1): 3384, 3261, 3165 (NH, NH2), 3097 (CH arom.), 1595 (C=N), 1331, 1185 (SO2), 1252 (C=S).1H-NMR (DMSO-d2): 7.1–8.9 [m, 9H, Ar–H + SO2NH2], 10.5, 12.0 [2 s, 2H, 2NH, exchangeable with D2O]. 13C-NMR (DMSO-d6): 119.6 (2), 123.1 (3), 126.6 (3), 139.8 (2), 142.8, 161.2179.9 (2). MS m/z (%): 409 (M+) (13.43), 178 (100). Anal.Calcd. For C14H11N5O4S3 (409): C, 41.07; H, 2.71; N, 17.10. Found: C, 41.31; H, 2.40; N, 17.43.
4-(3-(5-(Bromopyridin-2-yl)thioureido)benzenesulfonamide (13)
Yield, 72 %; m.p. 247.0 °C. IR (KBr, cm−1): 3326, 3175 (NH, NH2), 3088 (CH arom.), 1572 (C=N), 1356, 1192 (SO2), 1211 (C=S). 1H-NMR (DMSO-d2): 6.8–8.3 [m, 8H, Ar–H + SO2NH2], 12.4 [s, 2H, 2NH, exchangeable with D2O]. 13C-NMR (DMSO-d6): 105.5, 124.8 (2), 128.9 (2), 134.6, 140.8, 158.5, 159.3 (2), 178.6.MS m/z (%): 388 (M+) (11.81), 157 (100). Anal.Calcd. For C11H10BrN5O2S2 (388): C, 34.03; H, 2.60; N, 18.04. Found: C, 34.28; H, 2.27; N, 18.37.
4-(3-Pyrazin-2-ylthioureido)benzenesulfonamide (14)
Yield, 80 %; m.p. 185.3 °C. IR (KBr, cm−1): 3378, 3240, 3155 (NH, NH2), 3100 (CH arom.), 1601 (C=N), 1346, 1199 (SO2), 1270 (C=S).1H-NMR (DMSO-d2): 7.2–8.7 [m, 9H, Ar–H + SO2NH2], 11.3, 13.0 [2 s, 2H, 2NH, exchangeable with D2O]. 13C-NMR (DMSO-d6): 123.1 (2), 126.7 (2), 137.1, 138.4, 138.5, 139.9, 140.3, 149.7, 179.0. MS m/z (%): 309 (M+) (12.83), 79 (100). Anal.Calcd. For C11H11N5O2S2 (309): C, 42.71; H, 3.58; N, 22.64. Found: C, 42.38; H, 3.84; N, 22.29.
4-(3-(5,6,7,8-Tetrahydronaphthalen-1-yl)thioureido)benzenesulfonamide (15)
Yield, 76 %; m.p. 171.8 °C. IR (KBr, cm−1): 3413, 3354, 3152 (NH, NH2), 3083 (CH arom.), 2982, 2935, 2831 (CH aliph.), 1351, 1159 (SO2), 1264 (C=S).1H-NMR (DMSO-d2): 1.8–2.8 [m, 8H, 4CH2, cyclo], 7.0–8.0 [m, 9H, Ar–H + SO2NH2], 9.0[s, 2H, 2NH, exchangeable with D2O]. 13C-NMR (DMSO-d6): 22.7 (2), 24.8, 29.6, 117.4, 120.8 (2), 124.0, 125.7, 127.4 (2), 134.5, 137.1, 137.4, 137.6, 146.4, 181.5.MS m/z (%): 361 (M+) (26.34), 177 (100). Anal.Calcd. For C17H19N3O2S2 (361): C, 56.48; H, 5.30; N, 11.62. Found: C, 56.12; H, 5.03; N, 11.36.
4-(3-Quinolin-3-ylthioureido)benzenesulfonamide (16)
Yield, 66 %; m.p. 214.6 °C. IR (KBr, cm−1): 3373, 3246, 3164 (NH, NH2), 3077 (CH arom.), 1595 (C=N), 1365, 1150 (SO2), 1293 (C=S). 1H-NMR (DMSO-d2): 6.8–8.5 [m, 12H, Ar–H + SO2NH2], 10.8 [s, 2H, 2NH, exchangeable with D2O].13C-NMR (DMSO-d6): 127.3, 127.7, 128.6, 129.1 (2), 130.0 (2), 132.0, 134.3 (2), 137.9 (2), 142.8, 178.6. MS m/z (%): 358 (M+) (17.53), 156 (100). Anal.Calcd. For C16H14N4O2S2 (358): C, 53.61; H, 3.94; N, 15.63. Found: C, 53.36; H, 3.62; N, 15.36.
4-(3-(2-Methylquinolin-4-yl)thioureido)benzenesulfonamide (17)
Yield, 71 %; m.p. 192.3 °C. IR (KBr, cm−1): 3363, 3218, 3154 (NH, NH2), 3034 (CH arom.), 2943, 2836 (CH aliph.), 1590 (C=N), 1324, 1154 (SO2), 1241 (C=S). 1H-NMR (DMSO-d2): 2.6 [s, 3H, CH3], 6.6–8.8 [m, 11H, Ar–H + SO2NH2], 10.1, 13.8[2 s, 2H, 2NH, exchangeable with D2O]. 13C-NMR (DMSO-d6): 19.9, 102.0, 108.3, 121.1 (2), 122.9, 124.0, 126.1, 127.8, 128.0 (2), 137.3, 139.5, 143.1, 151.7, 158.0, 179.3. MS m/z (%): 372 (M+) (21.22), 141 (100). Anal.Calcd. For C17H16N4O2S2 (372): C, 54.82; H, 4.33; N, 15.04. Found: C, 54.51; H, 4.09; N, 15.31.
In vitro anticancer evaluation
Cell culture
Human cancer cell lines HeLa (cervical), A549 (lungs) and Lovo (colorectal) were grown in DMEM + GlutaMax (Invitrogen), and MDA MB321 (breast) were grown in DMEM-F12 + GlutaMax) medium (invitrogen), supplemented with 10 % heat-inactivated bovine serum (Gibco) and 1× penicillin–streptomycin (Gibco) at 37 °C in a humified chamber with 5 % CO2 supply.
Cytotoxicity assay
The in vitro anticancer screening was done at pharmacognosy Department, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. Cells were seeded (105 cells/well) in 96-well flat-bottom plates (Becton–Dickinson Labware) a day before treatment and grown overnight. Compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma) and finally prepared as 1.0 mg ml−1 stocks, respectively in the culture media. The final concentration of DMSO never exceeded 0.1 % in the treatment doses. Four different doses of compounds (50, 25, 12.5 and 6.25 µg ml−1) were further prepared by diluting the stocks in culture media, and cells were treated (in triplicate/dose). 2′7′ dichlorofluorescein (DCF) was included as standard reference drug (positive control) and untreated culture was considered as negative control. The treated cultures were further incubated for 48 h. At 48 h post-treatment, cell viability test was performed using TACS MTT Cell Proliferation and Viability Assay Kit (TACS) as per manufacturer’s instructions. The optical density (OD) was recorded at 570 nm in a microplate reader (BioTek, ELx800) and cell survival fraction was determined. The cell survival fraction was calculated as [(A − B)/A], where A and B are the OD of untreated and of treated cells, respectively [42]. The IC50 values of the tested compound were estimated using the best fit regression curve method in Excel.
Microscopy
A direct visual investigation was made under an inverted microscope (Optica, 40× and 100×) to observe any morphological changes in the cells cultured with different treatment doses at 24 and 48 h.
Molecular docking
All the molecular modeling studies were carried out on an Intel Pentium 1.6 GHz processor, 512 MB memory with Windows XP operating system using Molecular Operating Environment (MOE, 10.2008) software. All the minimizations were performed with MOE until a RMSD gradient of 0.05 kcal mol−1 Å with MMFF94X force field and the partial charges were automatically calculated. The protein data bank file (PDB: 3WI6) was selected for this purpose. The file contains MK-2 enzyme co-crystalized with a ligand obtained from protein data bank. The enzyme was prepared for docking studies where: (1) Ligand molecule was removed from the enzyme active site. (2) Hydrogen atoms were added to the structure with their standard geometry. (3) MOE Alpha Site Finder was used for the active sites search in the enzyme structure and dummy atoms were created from the obtained alpha spheres. (4). The obtained model was then used in predicting the ligand enzymes interactions at the active site.