Experimental
All chemical compounds were purchased from Merck and Sigma Aldrich. Melting points were measured with a Thermoscientific 9300 melting point apparatus and were uncorrected. Infrared (IR) Spectra were recorded using Shimadzu FTIR 8400S spectrophotometer. 1H-NMR and 13C-NMR experiments were carried out using BRUKER NMR spectrometer, and chemical shifts (δH) are reported relative to TMS as the internal standard. Mass spectra were recorded using Agilent LC/MSD and Xevo G2-XS QTof spectrometer. All reagents and solvents were dried and purified by the standard techniques. Chemical shift (δ) values of rotameric hydrogens whenever identified are presented within parentheses by assigning an asterisk (*) along with that of other forms [28]. Compounds 1a-n and 2a-b are already recorded in the literature [4, 13–21]. Compounds 1i and its ester derivative, 3e and 3j have CAS Registry Numbers but no reference, analytical, or spectral data.
Synthesis
Methyl 4-[(thiophene-2-carbonyl)amino]benzoate
White cream solid; yield: 56%; m.p.185-187oC; FTIR υmax (cm−1): 3360 (N-H), 1666 (C=O), 1593 (N-H bending); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 1.32 (t, 3H, CH2-CH3), 4.30 (q, 2H, CH2-CH3), 7.23-8.08 (m, 7H, Ar-H), 10.52 (s, 1H, CONH); Anal. calcd. for C14H13NO3S.1/2C2H5OH: C: 60.38, H: 5.41, N: 4.69, S: 10.75. Found: C: 60.51, H: 5.19, N: 4.87, S: 10.51.
N -[4-(hydrazinecarbonyl)phenyl]thiophene-2-carboxamide (1i)
White cream solid; yield: 76%; m.p. 230-231oC; FTIR υmax (cm−1): 3306 (N-H), 1633 (C=O), 1600 (N-H bending); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 4.46 (s, 2H, CONHNH2); 7.23-8.06 (m, 7H, Ar-H); 9.69 (s, 1H, CONH); 10.41 (s, 1H, CONH); Anal. calcd. for C12H11N3O2S.1/5H2O: C: 54.41, H: 4.34, N: 15.86, S: 12.10. Found: C: 54.85, H: 4.45, N: 15.71, S: 12.34.
General procedure for the synthesis of target compounds
The mixture of aldehydes 2a-b (1 mmol) and 1a-n derivatives (1 mmol) was refluxed in ethanol (20 ml) in the presence of glacial acetic acid as the catalyst. After completion of the reaction (checked by TLC, t:250C, acetone/petroleum ether 50:50 (v/v)), ethanol was evaporated, and the solid compounds 3a-n and 4a-n were dried and recrystallized with ethanol.
Synthetic route and chemical structures of compounds 3a-n and 4a-n are presented in Scheme 1.
Scheme 1. Synthetic route to target compounds
4-((2-(2-(6-methoxynaphthalen-2-yl)propanoyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3a)
White cream solid; yield: 81%; m.p.155-156 oC; FTIR υmax (cm−1): 3185 (N-H), 1660 (C=O), 1600 (C=N), 1373&1174 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 1.46 (t, 3H, J=7.2 Hz, CH-CH3); 2.43 (2.41*, s, 3H, Ar-CH3); 3.85- 4.74 (m, 4H, OCH3 & CH-CH3); 7.28-7.95 (m, 14H, Ar-H); 8.16 (7.85*, s, 1H, CH=N); 11.63 (11.34*, s, 1H, CONH); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 18.9 (CH-CH3), 21.6 (Ar- CH3), 44.4 (CH-CH3), 55.6 (O-CH3), 106.1, 119.0, 119.1, 122.9, 123.3, 125.9, 126.1, 126.7, 127.1, 127.2, 127.3, 128.7, 128.8, 128.9, 129.4, 129.5, 130.7, 131.6, 131.7, 131.9, 133.5, 133.7, 133.9, 137.0, 137.5, 141.5, 145.5&146.3 (CH=N), 150.0&150.2 (C-OSO2), 157.4&157.5 (C-OCH3), 170.4&175.5 (C=O); Anal. calcd. for C28H26N2O5S.1/3H2O: C: 66.03, H: 5.85, N: 5.33, S: 6.52. Found: C: 66.12, H: 5.28, N: 5.51, S: 6.30; LCMS (ES-API) m/z: 504 [M + H]+, 527 [M+H+Na]+.
4-((2-(2',4'-difluoro-4-hydroxy-[1,1'-biphenyl]-3-carbonyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3b)
White solid; yield: 79%; m.p. 218-219oC; FTIR υmax (cm−1): 3185 (N-H), 1637 (C=O), 1595 (C=N), 1373&1166 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.42 (s, 3H, Ar-CH3); 7.07-8.01 (m, 14H, Ar-H); 8.42 (s, 1H, CH=N); 11.92 (s, 2H, Ar- OH&CO-NH); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (Ar-CH3), 104.9, 112.3, 116.8, 118.0, 123.1, 125.5, 128.7, 129.2, 129.4, 130.7, 131.6, 132.1, 133.7, 146.4, 147.7 (CH=N), 150.5 (C-OSO2), 158.9, 157.7&161.0 (C-F, J=246 Hz), 160.2&163.5 (C-F, J=252 Hz), 164.8 (CONH); Anal. calcd. for C27H20F2N2O5S.1/4 H2O: C: 61.53, H: 3.92, N: 5.32, S: 6.08. Found: C: 61.88, H: 4.06, N: 5.39, S: 6.17; LCMS (ES-API) m/z: 523 [M + H]+, 546 [M +H+Na]+.
4-((2-(2-(2-fluoro-[1,1'-biphenyl]-4-yl)propanoyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3c)
White solid; yield: 87%; m.p. 146-147oC; FTIR υmax (cm−1): 3228 (N-H), 1651 (C=O), 1599 (C=N), 1345&1170 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 1.06 (CH3CH2OH, ethanol); 1.43 (1.40*, d, 3H, J=6.9 Hz, CH-CH3); 2.42 (s, 3H, Ar-CH3); 3.46 (CH3CH2OH, ethanol); 4.36 (CH3CH2OH, ethanol); 4.74 (3.77*, q, 1H, J=6.9 Hz, CH-CH3); 7.07-7.76 (m, 16H, Ar-H); 8.19 (7.92*, s, 1H, CH=N); 11.65 (11.44*, s, 1H, CO-NH); 13C-NMR (150 MHz), (DMSO-d6/TMS) δ ppm: 18.6 (CH3CH2OH), 18.7&19.0 (CH-CH3), 21.6 (Ar-CH3), 43.9 (CH-CH3), 56.5 (CH3CH2OH), 115.3, 115.4, 115.5, 115.7, 122.9,123.0, 126.8, 126.9, 127.1, 127.2, 128.2, 128.2, 128.7, 128.7, 129.0, 129.0, 129.0, 129.1, 130.7, 131.08, 131.1, 131.1, 131.1, 131.6, 131.7, 133.8, 135.3, 142.0, 143.7, 143.8, 144.2, 144.2, 145.9, 146.3&146.4 (CH=N), 150.1&150.3 (C-OSO2), 158.5&160.1 (C-F, J=245 Hz), 169.7&174.9 (CONH); Anal. calcd. for C29H25FN2O4S.1/3 C2H5OH: C: 66.98, H: 5.12, N: 5.27, S: 6.03. Found: C: 66.06, H: 4.86, N: 5.46, S: 6.18; LCMS (ES-API) m/z: 539 [M +Na]+.
4-((2-(2-(1-methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl)acetyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3d)
White solid; yield: 80%; m.p. 183-184oC; FTIR υmax (cm−1): 3352 (O-H), 3230 (N-H), 1651 (C=O), 1599 (C=N), 1373&1170 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.39 (s, 3H, ArCH3); 2.42 (s, 3H, ArCH3); 3.75-4.17 (m, 5H, N-CH3 ve CH2CO); 6.11 (d, 1H, J=3.9 Hz, CH2, pyrrole); 6.57 (d, 1H, J= 3.9 Hz, pyrrole); 7.07-7.76 (m, 12H, Ar-H); 8.19 (8.00*, s, 1H, -CH=N); 11.64 (s, 1H, - CONH); 13C-NMR (150 MHz), (DMSO-d6/TMS) δ ppm: 21.5 (Ar-CH3), 21.6 (Ar-CH3), 31.1 (N-CH3), 33.0&33.4 (CH2CO), 109.7, 110.0, 122.1, 122.2, 123.0, 128.7, 128.8, 129.0, 129.1, 129.4, 129.4, 130.7, 131.6, 131.7, 133.7, 133.8, 137.3, 137.4, 137.5, 137.7, 141.9, 141.9, 142.3, 145.7, 146.4&146.4 (CH=N), 150.2&150.3 (C-OSO2), 165.2&170.8 (CONH), 184.7&184.8 (C=O); Anal. calcd. for C29H27N3O5S.1/2 H2O: C: 64.67, H: 5.24, N: 7.80, S: 5.95. Found: C: 64.50, H: 5.33, N: 7.91, S: 6.22; LCMS (ES-API) m/z: 553 [M +H+Na]+.
4-((2-(2-(4-isobutylphenyl)propanoyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3e)
White solid; yield: 82%; m.p. 99-101oC; FTIR υmax (cm−1): 3230 (N-H), 1651 (C=O), 1599 (C=N), 1346,&1170 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 0.81 (m, 6H, -CH(CH3)2); 1.36 (t, 3H, J=6.9 Hz, -CHCH3); 1.72-1.83 (m, 1H, -CH(CH3)2); 2.36-2.42 (m, 5H, ArCH3&CH2CH(CH3)2); 4.60 (3.64*, q, 1H, J=6.9 Hz, CHCH3); 7.04-7.76 (m, 12H, Ar-H); 8.16 (7.87*, s, 1H, -CH=N); 11.56 (11.04*, s, 1H, -CONH); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 18.8&18.9 (CHCH3), 21.6 (Ar-CH3), 22.6 (CH(CH3)2), 30.0 (CH(CH3)2), 44.0 (CH2CH(CH3)2), 44.6 (CHCH3), 122.9, 127.4, 127.7, 128.6, 128.7, 128.9, 129.3, 129.4, 130.7, 131.6, 131.7, 133.9, 139.1, 139.5, 139.6, 140.0, 141.5, 145.5, 146.3&146.4 (CH=N), 150.0&150.2 (C-OSO2), 170.5& 175.6 (C=O); Anal. calcd. for C27H30N2O4S: C: 67.76, H: 6.32, N: 5.85, S: 6.70. Found: C: 68.15, H: 5.96, N: 5.99, S: 6.73; LCMS (ES-API) m/z: 501 [M + Na]+.
4-((2-(2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)acetyl) hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3f)
Dark yellow solid; yield: 77%; m.p. 107-108oC; FTIR υmax (cm−1): 3223 (N-H), 1651 (C=O), 1599 (C=N), 1346&1170 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 0.65 (0.57*, t, 3H, J=7.2 Hz, -CH2-CH3); 1.06 (t, ethanol); 1.24 (t, 3H, -CH2-CH3); 2.03-2.15 (m, 2H, CH2-CH3); 2.42 (s, 3H, Ar-CH3); 2.58-2.68 (m, 2H, -CH2-CH3); 2.78-2.98 (m, 4H, - CH2CONH & -CH2); 3.45 (m, ethanol); 3.69-4.00 (m, 2H, -CH2); 4.36 (t, ethanol); 6.86-7.75 (m, 11H, Ar-H); 8.19 (7.94*, s, 1H, N=CH); 10.51 (10.49*, s, 1H, indole N-H); 11.37 (11.24*, s, 1H, CO-NH); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 8.3 (pyran CH2CH3), 14.9 (indole CH2CH3), 21.6 (ArCH3), 24.2 (pyran -CH2CH2O-), 22.3 (indole CH2CH3), 31.2 (pyran CH2CH3), 6.2&76.5 (- CH2OCH-), 107.5, 107.7, 115.8, 119.1, 12.9, 126.5, 127.0, 128.6, 128.7, 128.9, 130.7, 131.7, 134.0, 134.9, 136.7, 137.0, 146.4 (CH=N), 150.0& 150.2 (C-OSO2), 166.1,&171.9 (C=O); Anal. calcd. for C31H33N3O5S.3/2 C2H5OH: C: 65.78, H: 6.32, N: 7.12, S: 5.43. Found: C: 65.16, H: 5.67, N: 7.14, S: 5.54; LCMS (ES-API) m/z: 582 [M + Na]+.
4-((2-(6-(2-(4-(tosyloxy)benylidene)hydrazine-1-carbonyl)picolinoyl) hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3g)
White solid; yield: 82%; m.p. 227-228oC; FTIR υmax (cm−1): 3230, 3171 (N-H), 1668, 1651, 1599 (C=O & C=N), 1344,1170 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.44 (s, 6H, CH3), 7.16-7.84 (m, 16H, Ar- H), 8.26-8.38 (m, 3H, pyridine-H), 8.74 (s, 2H, CH=N), 12.37 (s, 2H, CONH-N=); 13C-NMR (150 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (2Ar-CH3), 123.1, 126.1, 128.7, 129.3, 130.7, 131.7, 133.7, 140.5, 146.4, 148.6 (pyridin 2C-N), 149.0 (2CH=N), 150.6 (2C-OSO2), 160.0 (2C=O); Anal. calcd. for C35H29N5O8S2.4H2O: C: 53.63, H: 4.76, N: 8.93, S: 8.18. Found: C: 53.99, H: 4.37, N: 9.21, S: 8.54; LCMS (ES-API) m/z: 734 [M + Na]+.
4-((2-(2-(2-((2,6-dichlorophenyl)amino)phenyl)acetyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3h)
White solid; yield: 88%; m.p. 228-229; FTIR υmax (cm−1): 3317 (N-H), 1658 (C=O), 1614 (C=N), 1357&1178 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.43 (s, 3H, CH3), 4.12 (3.70*, s, 2H, CH2CO), 6.27-8.04 (m, 16H, Ar-H& CH=N), 8.20 (s, 1H, NH), 11.89 (11.68*, s, 1H, CONH-N=); 13C-NMR (150 MHz), (DMSO-d6/TMS) δ ppm: 21.6&21.7 (Ar-CH3), 35.8&36.0 (CH2CO), 146.4&146.5 (CH=N), 150.2&150.4 (C-OSO2), 168.3 (C=O); Anal. calcd. for C28H23Cl2N3O4S2.1/2H2O: C: 58.24, H: 4.19, N: 7.28, S: 5.55. Found: C: 58.84, H: 4.29, N: 7.42, S: 5.80; LCMS (Q-TOF) m/z: 568 [M + H]+.
4-((2-(4-(thiophene-2-carboxamido)benzoyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3i)
White cream solid; yield: 76%; m.p. 281-283oC; FTIR υmax (cm−1): 3354, 3292 (N-H), 1703, 1641 (C=O), 1599 (C=N), 1340&1172 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.43 (s, 3H, Ar-CH3), 7.25-8.09 (m, 15H, Ar-H), 8.43 (s, 1H, CH=N), 10.49 (s,1H, thiophene-CONH), 11.87 (s, 1H, CONH); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (Ar-CH3), 119.9, 123.0, 128.4, 128.6, 128.7, 129.0, 130.0, 130.7, 131.6, 132.9, 134.0, 140.0, 142.4, 146.3, 146.4 (C-OSO2),150.3 (CH=N), 160.6 (C=O), 163.0 (C=O); Anal. calcd. for C26H21N3O5S2: C: 60.10, H: 4.07, N: 8.09, S: 12.34. Found: C: 59.68, H: 4.16, N: 8.06, S: 12.91; LCMS (Q-TOF) m/z: 520 [M + H]+.
4-((2-(4-benzamidobenzoyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3j)
White solid; yield: 70%; m.p. 286oC; FTIR υmax (cm−1): 3338, 3230 (N-H), 1668, 1651, 1599 (C=O & C=N), 1373&1172 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.43 (s, 3H, Ar-CH3), 7.53-8.00 (m, 17H, Ar-H), 8.43 (s, 1H, CH=N), 10.53 (s, 1H, amide-CONH), 11.87 (s, 1H, CONH); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (Ar-CH3), 119.9, 123.0, 128.2, 128.7, 128.9, 130.7, 131.6, 132.3, 134.0, 135.0, 146.4 (CH=N), 150.2 (C-OSO2), 163.0 (C=O), 165.3 (C=O); Anal. calcd. for C28H23N3O5S.1/2H2O: C: 64.35, H: 4.63, N: 8.04, S: 6.14. Found: C: 64.79, H: 4.13, N: 8.20, S: 6.11; LCMS (ES-API) m/z: 537 [M +H+Na]+.
4-((2-((5-(propylthio)-2,3-dihydro-1H-benzimidazol-2-yl)carbamoyl) hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3k)
White solid; yield: 79%; m.p. 228-230oC; FTIR υmax (cm−1): 3365, 3236 (N-H), 1699, 1626 (C=O & C=N), 1367&1174 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 0.95 (t, 3H, J=7.2 Hz, propyl CH3); 1.48-1.60 (m, 2H, propyl CH2); 2.43 (s, 3H, Ar-CH3); 2.86 (t, 2H, J=7.2 Hz, propyl CH2); 7.02-7.97 (m, 12H, Ar- H&CH=N); 10.53 (s, 1H, NHCONHN=); 11.28 (s, 1H, benzimidazole NH); 11.94 (s, 1H, NHCONHN=); 13C-NMR (150 MHz), (DMSO-d6/TMS) δ ppm: 13.5 (propyl CH3), 21.6 (Ar- CH3), 22.5 (propyl CH2), 37.2 (propyl CH2), 122.7, 128.7, 129.2, 130.7, 131.7, 133.9, 141.5, 146.3 (CH=N), 150.0 (C-OSO2), 159.1 (C=N, benzimidazole); Anal. calcd. for C25H25N5O4S2: 57.34, H: 4.81, N: 13.37, S: 12.25. Found: C: 57.84, H: 5.03, N: 13.22, S: 12.69; LCMS (ES-API) m/z: 525 [M + 2H]+.
4-((2-((4-bromophenyl)sulfonyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3l)
Dark yellow solid; yield: 73%; m.p. 127-129oC; FTIR υmax (cm−1): 3234 (N-H), 1599 (C=N), 1346&1177 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.42 (s, 3H, CH3), 7.47-7.85 (m, 12H, Ar-H), 7.90 (s, 1H, CH=N), 11.72 (s, 1H, CONH-N=); 13C-NMR (150 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (Ar-CH3), 128.0, 123.1, 123.5, 128.2, 128.7, 128.9, 129.6, 130.8, 131.6, 131.9, 132.8, 133.1, 138.5, 146.4, 146.6 (CH=N), 150.4 (C-OSO2); Anal. calcd. for C20H17BrN2O5S2.1/2H2O: C: 46.34, H: 3.50, N: 5.40, S: 12.37. Found: C: 46.75, H: 3.95, N: 5.33, S: 12.77; LCMS (ES-API) m/z : 546 [M +K]+.
4-((2-((4-chlorophenyl)sulfonyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3m)
Dark yellow solid; yield: 73%; m.p. 127-129oC; FTIR υmax (cm−1): 3223 (N-H), 1599 (C=N), 1346&1161 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.41 (s, 3H, CH3), 7.47-7.91 (m, 12H, Ar-H), 8.66 (s, 1H, CH=N), 11.74 (s, 1H, CONH-N=); 13C-NMR (150 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (Ar-CH3), 123.0, 123.1, 127.9, 128.1, 128.7, 129.6, 129.9, 130.5, 130.7, 131.6, 133.1, 133.2, 138.1, 138.3, 138.5, 146.4, 146.5, 146.6 (CH=N), 150.4 (C-OSO2); Anal. calcd. for C20H17ClN2O5S2. 1/2C2H5OH: C: 51.69, H: 4.13, N: 5.74, S: 13.14. Found: C: 51.02, H: 4.54, N: 5.79, S: 13.29; LCMS (ES-API) m/z : 464 [M +2H]+.
4-((2-((4-nitrophenyl)sulfonyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (3n)
White cream solid; yield: 86%; m.p. 153-154oC; FTIR υmax (cm−1): 3185 (N-H), 1599 (C=N), 1348&1174 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.42 (s, 3H, CH3), 7.58-8.45 (m, 13H, Ar-H&CH=N), 11.98 (s, 1H, CONH-N=);13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (Ar-CH3), 123.0, 125.1, 123.6, 129.0, 129.2, 130.7, 131.6, 132.9, 144.6, 146.4, 147.2 (CH=N), 150.4 (C-NO2), 150.5 (C-OSO2); Anal. calcd. for C20H17N3O7S2.H2O: C: 48.67, H: 3.88, N: 8.51, S: 12.99. Found: C: 48.82, H: 3.81, N: 8.06, S: 13.08; LCMS (ES-API) m/z: 476 [M +H]+, 497 [M +Na]+.
2-methoxy-4-((2-(2-(6-methoxynapthalen-2-yl)propanoyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (4a)
Off-white solid; yield: 90%; m.p. 164-165oC; FTIR υmax (cm−1): 3176 (N-H), 1660 (C=O), 1604 (C=N), 1373&1169 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 1.42-1.49 (m, 3H, CH-CH3); 2.43 (s, 3H, Ar-CH3); 3.51 (s, 3H, ArOCH3); 3.57-4.76 (m, 4H, OCH3 & CH-CH3); 7.25-7.79 (m, 13H, Ar-H); 8.15 (7.81*, s, 1H, CH=N); 11.65 (11.39*, s, 1H, CONH); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 18.9&19.2 (CH-CH3), 21.6 (Ar-CH3), 44.3 (CH-CH3), 55.6 (O-CH3), 56.0&56.1 (ArOCH3), 106.1, 110.6, 119.0, 119.1, 120.0, 120.6, 124.2, 125.9, 126.7, 127.3, 128.7, 128.8, 128.9, 129.3, 129.5, 130.2, 130.4, 132.4, 132.4, 133.5, 133.7, 134.9, 136.3, 137.0, 137.8, 138.7, 139.0, 141.5, 145.9 (C-OSO2), 146.0&146.1 (CH=N), 151.9 (C-OCH3), 157.4&157.5 (C-OCH3), 170.4&175.5 (C=O); Anal. calcd. for C29H28N2O6S.1/2H2O: C: 64.31, H: 5.40, N: 5.17, S: 5.92. Found: C: 64.33, H: 5.62, N: 4.96, S: 6.10; LCMS (ES-API) m/z: 555 [M + Na]+.
2-methoxy-4-((2-(2',4'-difluoro-4-hydroxy-[1,1'-biphenyl]-3-carbonyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (4b)
White solid; yield: 72%; m.p. 221-222oC; FTIR υmax (cm−1): 3244 (N-H), 1643 (C=O), 1606 (C=N), 1354&1169 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.43 (s, 3H, Ar-CH3); 3.56 (s, 3H, ArOCH3); 7.29-8.00 (m, 13H, Ar-H); 8.41 (s, 1H, CH=N); 11.94 (s, 2H, CONH&Ar-OH; 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (Ar-CH3), 56.1 (O-CH3), 110.8, 117.0, 118.0, 121.0, 124.4, 125.5, 128.7, 129.5, 130.3, 132.4, 134.7, 139.3, 146.2 (C-OSO2), 148.0 (CH=N), 157.6&161.0 (C-F, J=253 Hz), 158.8 (C-OH), 160.2&163.5 (C-F, J=243 Hz). 164.7 (C=O) Anal. calcd. for C28H22F2N2O6S: C: 60.86, H: 4.01, N: 5.07, S: 5.80. Found: C: 60.59, H: 3.85, N: 5.09, S: 5.61; LCMS (ES-API) m/z: 574.9 [M +Na]+.
2-methoxy-4-((2-(2-(2-fluoro-[1,1'-biphenyl]-4-yl)propanoyl)hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (4c)
White solid; yield: 86%; m.p. 150-151oC; FTIR υmax (cm−1): 3232 (N-H), 1651 (C=O), 1599 (C=N), 1373&1170 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 1.45 (1.40*, d, 3H, J=6.9 Hz, CH-CH3); 2.42 (2.38*, s, 3H, Ar-CH3); 3.57 (3.52*, s, 3H, ArOCH3); 4.71 (3.77*, q, 1H, J=6.9Hz, CH-CH3); 7.37-7.71 (m, 15H, Ar-H); 8.17 (7.86*, s, 1H, CH=N); 11.68 (11.48*, s, 1H, CO-NH); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 18.6&19.0 (CH-CH3), 21.5&21.6 (Ar-CH3), 43.9 (CH-CH3), 56.0 (Ar-OCH3), 110.5, 110.7, 115.5, 115.8, 120.2, 120.6, 124.2, 128.2, 128.7, 129.0, 129.0, 129.1, 130.2, 131.1, 132.4, 132.4, 134.9, 135.3, 138.8, 139.0, 142.0, 146.0 (ArOCH3), 146.1 (CH=N), 152.0 (C-OSO2), 157.6&160.9 (C-F, J=252 Hz), 169.7&174.9 (CONH); Anal. calcd.for C30H27FN2O5S: C: 65.92, H: 4.98, N: 5.12, S: 5.87. Found: C: 65.23, H: 4.68, N: 5.07, S: 5.56; LCMS (ES-API) m/z: 571 [M+H+Na]+.
2-methoxy-4-((2-(2-(1-methyl-5-(4-methylbenzoyl)-1 H -pyrrol-2-yl)acetyl) hydrazinylidene)methyl)phenyl 4-methylbenzenesulfonate (4d)
White solid; yield: 83%; m.p. 215-216oC; FTIR υmax (cm−1): 3190 (N-H), 1662 (C=O), 1620 (C=N ), 1365&1170 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.38 (s, 3H, ArCH3); 2.42 (s, 3H, ArCH3); 3.55 (s, 3H, ArOCH3); 3.86 (s, 3H, N-CH3), 4.18 (s, 2H, CH2CO); 6.12 (d, 1H, J= 3.9 Hz, CH2, pyrrole); 6.58 (d, 1H, J=3.9 Hz, C=O, pyrrole); 7.16-7.72 (m, 11H, Ar-H); 8.17 (7.97*, s, 1H, -CH=N); 11.66 (s, 1H, -CONH); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 21.5 (Ar-CH3), 21.6 (Ar-CH3), 31.2 (N-CH3), 33.4 (CH2CO), 56.1 (Ar-OCH3), 110.0, 111.2, 120.0, 122.2, 124.2, 124.3, 128.7, 129.1, 129.4, 130.3, 130.6, 132.4, 132.4, 134.8, 137.3, 137.5, 137.7, 138.9, 139.1, 141.9, 142.5 (C-OCH3), 146.1 (CH=N), 152.0 (C-OSO2), 170.9 (CONH), 184.7 (C=O); Anal. calcd. for C30H29N3O6S: C: 64.39, H: 5.22, N: 7.51, S: 5.73. Found: C: 64.34, H: 5.21, N: 7.52, S: 5.55; LCMS (ES-API) m/z: 582 [M +Na]+.
4-((2-(2-(4-isobutylphenyl)propanoyl)hydrazinylidene)methyl)-2-methoxyphenyl 4- methylbenzenesulfonate (4e)
White solid; yield: 76%; m.p. 125-127oC; FTIR υmax (cm−1): 3230 (N-H ), 1668 (C=O), 1597 (C=N), 1375&1172 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 0.80-0.86 (m, 6H, -CH(CH3)2); 1.36 (q, 3H, J=7.2 Hz, -CHCH3); 1.72-1.84 (m, 1H, -CH(CH3)2); 2.36-2.43 (m, 5H, ArCH3&CH2CH(CH3)2); 3.57 (3.52*, s, 3H, Ar-OCH3); 4.57 (3.65*, q, 1H, J=6.9Hz, CHCH3); 7.04-7.72 (m, 11H, Ar-H); 8.14 (7.82*, s, 1H, -CH=N); 11.58 (11.34*, s, 1H, -CONH); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 18.8&19.2 (CHCH3), 21.6 (Ar-CH3), 22.6 (CH(CH3)2), 30.0&30.0 (CH(CH3)2), 44.0 (CH2CH(CH3)2), 44.6 (CHCH3), 56.0 ( Ar-OCH3), 110.6, 120.6,. 124.2, 127.4, 127.6, 128.7, 129.3, 130.2, 132.4, 132.5, 135.0, 138.7, 139.0, 139.1, 139.6, 139.8, 140.0, 141.4, 145.8 (C-OCH3), 146.0&146.1 (CH=N), 151.9 (C-OSO2), 170.4& 175.6 (C=O); Anal. calcd. for C28H32N2O5S.1/3 H2O: C: 65.35, H: 6.40, N: 5.44, S: 6.03. Found: C: 65.31, H: 6.06, N: 5.45, S: 6.11; LCMS (ES-API) m/z :509 [M+H]+, 531 [M + Na]+.
4-((2-(2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)acetyl) hydrazinylidene)methyl)-2-methoxyphenyl 4-methylbenzenesulfonate (4f)
Off-white solid; yield: 78%; m.p. 175-176oC; FTIR υmax (cm−1): 3342 (O-H), 3223 (N-H), 1645 (C=O); 1599 (C=N); 1369&1175 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 0.66 (0.57*, t, 3H, J=7.2 Hz, -CH2-CH3); 1.25 (t, 3H, J=7.2 Hz, -CH2-CH3); 2.02-2.15 (m, 2H, CH2-CH3); 2.42 (s, 3H, Ar-CH3); 2.63-2.69 (m, 2H, -CH2-CH3); 2.79-3.01 (m, 4H, -CH2CONH & -CH2); 3.48-4.03 (m, 5H, -CH2 & -OCH3); 6.87-7.71 (m, 10H, Ar-H); 8.17 (7.93*, s, 1H, N=CH); 10.51 (s, 1H, indole N-H); 11.43 (11.25*, s, 1H, CO-NH); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 8.3 (pyran CH2CH3), 14.9 (indole CH2CH3), 21.6 (ArCH3), 24.2 (pyran -CH2CH2O-), 22.3 (indole CH2CH3), 31.2 (pyran CH2CH3), 43.6 (CH2CO), 55.9&56.0 (ArOCH3), 60.5&60.7 (pyran -CH2CH2O-), 76.2&76.5 (-CH2OCH-), 107.5, 107.7, 115.8, 119.1, 12.9, 126.5, 127.0, 128.6, 128.7, 128.9, 130.7, 131.7, 134.0, 134.9, 136.7, 137.0, 146.4 (CH=N), 150.0& 150.2 (C-OSO2), 166.1&171.9 (C=O); Anal. calcd. for C32H35N3O6S.1/2H2O: C: 64.20, H: 5.91, N: 7.05, S: 5.39. Found: C: 64.76, H: 6.06, N: 7.02, S: 5.36; LCMS (ES-API) m/z: 582 [M + Na]+.
4-((2-(6-(2-(3-methoxy-4-(tosyloxy)benzylidene)hydrazine-1-carbonyl)picolinoyl) hydrazinylidene)methyl)-2-methoxyphenyl 4-methylbenzenesulfonate (4g)
White solid; yield: 88%; m.p. 263-264oC; FTIR υmax (cm−1): 3574 (O-H), 3379, 3244 (N-H), 1668, 1583 (C=O & C=N), 1356&1165 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.44 (s, 6H, CH3), 3.61 (s, 6H, ArOCH3), 7.23-7.76 (m, 14H, Ar-H), 8.27-8.39 (m, 3H, pyridine-H), 8.75 (s, 2H, CH=N), 12.39 (s, 2H, CONH-N=); 13C-NMR (150 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (2Ar-CH3), 56.1 (2ArOCH3), 110.9, 121.0, 124.4, 126.1, 128.7, 130.3, 132.5, 134.8, 139.4, 140.5, 146.2 (pyridine 2C-N), 148.6 (2C-OCH3), 149.3 (2CH=N), 152.1 (2C-OSO2), 160.0 (2C=O); Anal. calcd. for C37H33N5O10S2.4H2O: C: 52.66, H: 4.90, N:8.30, S: 7.60. Found: C: 52.98, H: 5.04, N: 8.54, S: 7.91; LCMS (ES-API) m/z: 794 [M + Na]+.
4-((2-(2-(2-((2,6-dichlorophenyl)amino)phenyl)acetyl)hydrazinylidene)methyl)-2-methoxyphenyl 4-methylbenzenesulfonate (4h)
White solid; yield: 83%; m.p. 224-225oC; FTIR υmax (cm−1): 3329 (N-H), 1660 (C=O), 1600 (C=N), 1369&1182 (S=O.); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.43 (s, 3H, CH3), 3.54 (s, 3H, OCH3), 4.13 (3.71*, s, 2H, CH2CO), 6.27-8.19 (m, 16H, Ar-H& NH& CH=N), 11.89 (11.74*, s, 1H, CO-NH); 13C-NMR (150 MHz), (DMSO-d6/TMS) δ ppm: 21.6&21.6 (Ar-CH3), 35.8&36.0 (CH2CO), 56.0 (Ar- OCH3), 111.1, 111.3, 116.1, 120.0, 120.7, 121.0, 121.3, 124.3, 124.4, 125.1, 125.7, 126.0, 127.8, 127.9, 128.6, 129.7, 130.0, 130.9, 131.6, 132.5, 134.7, 137.4, 138.9, 139.2, 143.1, 143.3, 143.5&146.1 (CH=N), 146.8, 152.0 (C-OSO2), 168.3, 173.6 (C=O). Anal. calcd. for C29H25Cl2N3O5S: C: 58.20, H: 4.21, N: 7.02, S: 5.36. Found: C: 58.20, H: 4.59, N: 6.96, S: 5.39.
4-((2-(4-(thiophene-2-carboxamido)benzoyl)hydrazinylidene)methyl)-2-methoxyphenyl 4-methylbenzenesulfonate (4i)
White solid; yield: 88%; m.p. 242-243oC; FTIR υmax (cm−1): 3315, 3234 (N-H), 1703, 1645 (C=O), 1608 (C=N), 1377&1174 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.43 (s, 3H, Ar-CH3), 3.57 (s, 3H, Ar-OCH3), 7.26-8.09 (m, 14H, Ar-H), 8.41 (s, 1H, CH=N), 10.49 (s, 1H, thiophene-CONH), 11.89 (s, 1H, CONH); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (Ar-CH3), 56.0 (ArOCH3),110.6, 120.7, 124.3, 128.6, 129.0, 130.3, 132.9, 135.1, 139.0, 140.0, 142.5, 146.1, 146.6 (CH=N), 152.0 (C-OSO2), 160.6, 163.0 (C=O); Anal. calcd. for C27H23N3O6S2: C: 59.00, H: 4.22, N: 7.65, S: 11.67. Found: C: 58.80, H: 4.26, N: 7.68, S: 11.94; LCMS (Q-TOF) m/z: 550 [M + H]+.
4-((2-(4-benzamidobenzoyl)hydrazinylidene)methyl)-2-methoxyphenyl 4-methylbenzenesulfonate (4j)
White solid; yield: 79%; m.p. 276-278oC; FTIR υmax (cm−1): 3336, 3273 (N-H), 1647 (C=O), 1585 (C=N), 1352&1178 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.43 (s, 3H, CH3), 3.57 (s, 3H, Ar-OCH3), 7.18-8.00 (m, 16H, Ar-H), 8.42 (s, 1H, CH=N), 10.53&11.88 (s, 2H, CONH); 13C-NMR (150 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (Ar-CH3), 56.1 (Ar-OCH3), 110.7, 120.0, 120.7, 124.3, 128.2, 128.4, 128.7, 128.9, 130.3, 132.3, 132.4, 135.1, 135.1, 139.1, 142.9, 146.1 (CH=N), 146.7 (C-OCH3), 152.0 (C-OSO2), 163.1 (C=O), 166.3 (C=O); Anal. calcd. for C29H25N3O6S. H2O: C: 62.02, H: 4.85, N: 7.48, S: 5.71. Found: C: 62.70, H: 4.67, N: 7.77, S: 5.97; LCMS (ES-API) m/z: 567 [M +H+Na]+.
4-((2-((5-(propylthio)-2,3-dihydro-1 H -benzo[d]imidazol-2-yl)carbamoyl) hydrazinylidene)methyl)-2-methoxyphenyl 4-methylbenzenesulfonate (4k)
White solid; yield: 72%; m.p. 182-184oC; FTIR υmax (cm−1): 3398, 3252 (N-H); 1703 (C=O), 1627 (C=N); 1354&1172 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 0.95 (t, 3H, J= 7.2Hz, CH3), 1.48-1.61 (m, 2H, CH2), 2.43 (s, 3H, Ar-CH3), 2.86 (t, 2H, J=7.2 Hz, CH2), 3.60 (s, 3H, OCH3), 7.09-7.75 (m, 10H, Ar-H); 7.96 (s, 1H, CH=N), 10.52 (s, 1H, NHCONHN=), 11.29 (s, 1H, benzimidazole NH), 11.96 (s, 1H, NHCONHN=); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 13.5 (propyl -CH2-CH2-CH3), 21.5 (Ar-CH3), 22.5 (propyl -CH2-CH2-CH3), 37.1 (propyl -CH2-CH2-CH3), 56.2 (OCH3),120.6, 123.9, 124.4, 128.7, 130.2, 132.4, 134.8, 138.7, 141.9, 146.0 (CH=N), 151.9 (C-OSO2); Anal. calcd. for C26H27N5O5S2: C: 56.40, H: 4.92, N: 12.65, S: 11.58. Found: C: 56.47, H: 5.01, N: 12.44, S: 11.76; LCMS (Q-TOF) m/z: 554 [M + H]+.
4-((2-((4-bromophenyl)sulfonyl)hydrazinylidene)methyl)-2-methoxyphenyl 4-methylbenzenesulfonate (4l)
Yellow solid; yield: 82%; m.p. 147-148oC; FTIR υmax (cm−1): 3234 (N-H), 1599 (C=N), 1346&1151 (SO2); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.42 (s, 3H, CH3), 3.52 (s, 3H, Ar-OCH3), 7.09-7.87 (m, 13H, Ar-H&CH=N), 11.73 (s, 1H, CONH-N=); 13C-NMR (150 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (Ar-CH3), 56.1 (Ar-OCH3), 111.5, 119.7, 127.6, 128.2, 128.7, 129.7, 130.3, 130.4, 132.4, 132.8, 134.1, 138.5, 139.1, 146.1 (CH=N), 146.8 (C-OCH3), 151.9 (C-OSO2); Anal. calcd. for C21H19BrN2O6S2: C: 46.76, H: 3.55, N: 5.19, S: 11.89. Found: C: 46.81, H: 3.45, N: 4.65, S: 11.31; LCMS (Q-TOF) m/z: 540 [M + 2H]+.
4-((2-((4-chlorophenyl)sulfonyl)hydrazinylidene)methyl)-2-methoxyphenyl 4-methyl benzene sulfonate (4m)
Dark yellow solid; yield: 87%; m.p. 136-137oC; FTIR υmax (cm−1): 3252 (N-H); 1583 (C=N); 1357&1172 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.42 (s, 3H, CH3), 3.51 (s, 3H, Ar-OCH3), 7.10-7.89 (m, 12H, Ar-H&CH=N), 11.74 (s, 1H, CONH-N=); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (Ar-CH3); 56.0 (OCH3), 111.5, 119.6, 124.3, 128.1, 128.7, 129.6, 129.9, 130.3, 132.3, 134.1, 138.5, 139.0, 146.5,146.8 (CH=N), 151.9 (C-OSO2); Anal. calcd. for C21H19ClN2O6S2: C: 50.96, H: 3.84, N: 5.74, S: 11.91. Found: C: 49.82, H: 3.87, N: 5.66, S: 12.96. LCMS (ES-API) m/z: 495 [M +H]+.
4-((2-((4-nitrophenyl)sulfonyl)hydrazinylidene)methyl)-2-methoxyphenyl 4-methylbenzenesulfonate (4n)
Yellow solid; yield: 90%; m.p. 104-105oC; FTIR υmax (cm−1): 3230 (N-H), 1668 (C=N), 1346&1170 (S=O); 1H-NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.42 (s, 3H, CH3), 3.53 (s, 3H, Ar-OCH3), 7.09-8.46 (m, 12H, Ar-H&CH=N), 11.99 (s, 1H, CONH-N=); 13C-NMR (75 MHz), (DMSO-d6/TMS) δ ppm: 21.6 (Ar-CH3), 56.1 (Ar-OCH3), 111.6, 119.8, 124.3, 125.1, 128.6, 129.3, 130.3, 132.4, 133.9, 139.2, 144.5, 146.1 (C-OCH3), 147.4 (CH=N), 150.4 (C-NO2), 152.0 (C-OSO2); Anal. calcd. for C21H19N3O8S2.2H2O: C: 46.57, H: 3.43, N: 7.76, S: 11.84. Found: C: 46.90, H: 3.44, N: 7.56, S: 11.86; LCMS (ES-API) m/z: 528 [M +Na]+.
Biological assay
Cell culture studies
Human lung adenocarcinoma cell line (A549), human breast cancer cell line (MCF-7), human colorectal adenocarcinoma cell line (HT-29), human prostate cell line (PC-3), human liver cancer cell line (Hep3B), human cervical cancer cell (HeLa) and nontumorigenic mouse embryonic fibroblast cell line (NIH 3T3) were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were cultured in Dulbecco’s modified eagle medium (DMEM) (Gibco, Rockville, MD, USA) containing 10% fetal bovine serum (FBS) (Gibco, Rockville, MD, USA) and maintained in a 37°C, 5% CO2 incubator. Cell passage was conducted at 80-90% confluence.
Cell viability assay
Cell viability was determined by the MTT assay as described previously [29].
Apoptosis studies
Annexin V binding, caspase-3 and caspase-9 activation measurement and detection of the loss of mitochondrial membrane potential procedures have been previously described in detail [30].