H.M. Refaat / European Journal of Medicinal Chemistry 45 (2010) 2949e2956
2955
exchangeable); Anal. Calcd. for C19H18N4O3S (382.42): C, 59.66, H,
4.74, N, 14.65. Found: C, 59.79, H, 4.76, N, 14.70.
(aliph. CH), 2230 (C^N); 1H NMR (DMSO-d6):
d
2.06 (brs, 2H, CH2),
4.14 (t, 2H, J ¼ 7.1 Hz, N-CH2), 4.39 (s, 2H, S-CH2), 6.2e6.9 (m, 5H,
ArH), 7.20 (d,1H, J ¼ 8.7 Hz, benzimidazole-C7-H), 7.87 (d, 1H,
J ¼ 8.7 Hz, benzimidazole-C6-H), 7.94 (s, 1H, benzimidazole-C4-H),
12.80 (brs,1H, NH, D2O exchangeable); Anal. Calcd. for C19H15Cl N4S
(366.85): C, 62.20, H, 4.12, N, 15.27. Found: C, 62.48, H, 4.10, N, 15.31.
5.1.6.2. 2-[(3-Phenyl-5-oxothiazolidin-2-ylidene)cyanomethyl] benz-
imidazole-5-carboxylic acid (6b). Yield: 72%; mp: 296e298 ꢂC; IR
(cmꢀ1): 2900, 2870 (CH aliph.), 2230 (C^N),1720,1660 (2C]O); 1H
NMR (DMSO-d6): d 4.44 (s, 2H, thiazolidinone-C4-H), 7.44e7.64 (m,
5H, ArH), 7.84 (d, 1H, J ¼ 8.4 Hz, benzimidazole-C7-H), 7.93 (dis-
torted d, 1H, benzimidazole-C6-H), 8.30 (s, 1H, benzimidazole-C4-
H), MS: m/z 376 (Mþ, 0.7%); Anal. Calcd. for C19H12N4O3S (376.38):
C, 60.62, H, 3.21, N, 14.88. Found: C, 60.41, H, 3.09, N, 14.92.
5.1.6.9. 2-[(3-Phenyl[1,3]thiazin-2-ylidene)cyanomethyl]benzimid-
azole-5-carboxylic acid (9b). Yield: 83%; mp: 210e212 ꢂC; IR
(cmꢀ1): 2919, 2853 (aliph. CH), 2228 (C^N), 1711 (C]O); 1H NMR
(DMSO-d6):
d
2.24 (brs, 2H, CH2), 3.64 (t, 2H, J ¼ 6.9 Hz, N-CH2),
4.44 (brs, 2H, S-CH2), 7.07e7.59 (m, 5H, ArH), 7.84e7.97 (m, 2H,
benzimidazole-C7,6-H), 8.29 (s, 1H, benzimidazole-C4-H), 13.02 (brs,
2H, NH and COOH, D2O exchangeable); MS: m/z 367 (Mþ, 0.41%);
Anal. Calcd. for C20H16N4O2S (376.42): C, 63.81, H, 4.28, N, 14.88.
Found: C, 63.62, H, 4.31, N, 14.83.
5.1.6.3. 5-Chloro-2-[(3-phenyl-4-(4-chlorophenyl)thiazol-2-ylidene)
cyanomethyl]benzimidazole (7a). Yield: 82%; mp: 198e200 ꢂC; IR
(cmꢀ1): 2225 (C^N), 1H NMR (DMSO-d6):
d 6.35 (s, 1H, thiazole-C5-
H), 7.02e7.11 (m, 4H, ArH), 7.28e7.35 (m, 5H, ArH), 7.59e7.64 (m,
2H, benzimidazole-C6,7-H), 8.01 (s, 1H, benzimidazole-C4-H); Anal.
Calcd. for C24H14Cl2N4S (461.35): C, 62.47, H, 3.05, N,12.14. Found: C,
62.29, H, 3.08, N, 12.17.
5.1.7. Cytotoxic activity studies
Anticancer activity studies were done at Cairo University,
National Cancer Institute, Cancer Biology Department, Pharma-
cology Unit.
Compounds 3a, 4a, 5b, 6a, 7c, 8a and 9b were tested at concen-
trations between 1 and 10 mg/ml using SulfoRhodamine-B (SRB)
5.1.6.4. 2-[(4-(4-Bromophenyl)-3-cyclohexylthiazol-2-ylidene) cyano-
methyl]benzimidazole-5-carboxylic acid (7b). Yield: 87%; mp:
233e235 ꢂC; IR (cmꢀ1): 2220 (C^N), 1695 (C]O); 1H NMR (DMSO-
d6):
d
1.4e2.04 (m, 11H, cyclohexyl-H), 6.44 (s, 1H, thiazole-C5-H),
assay for cytotoxic activity against the following tumor cell lines:
7.60e8.18 (m, 6H, benzimidazole-C7,6-H and 4 ArH), 8.32 (s, 1H,
benzimidazole-C4-H), 12.96e13.68 (brs, 2H, NH and COOH, D2O
exchangeable); Anal. Calcd. for C25H21Br N4O2S (521.42): C, 57.58, H,
4.06, N, 10.74. Found: C, 57.74, H, 4.03, N, 10.71.
1. Liver carcinoma cell line (HEPG2)
2. Breast carcinoma cell line (MCF7)
3. Colon carcinoma cell line (HCT 116)
5.1.8. Measurement of potential cytotoxicity by SRB assay
Potential cytotoxicity of the compounds was tested using the
method of Skehan et al. [42], as follows:
5.1.6.5. 2-[3-(4-Bromophenyl)-4-(2-methoxyphenylthiazol-2-ylidene)
cyanomethyl]benzimidazole-5-carboxylic acid (7c). Yield: 91%; mp:
198e200 ꢂC; IR (cmꢀ1): 3228 (C^N), 1673 (C]O); 1H NMR (DMSO-
Cells were plated in 96 multiwell plate (104 cells/well) for 24 h
before treatment with the compound(s) to allow attachment to the
wall of the plate. Different concentrations of the compounds (0, 1,
d6): d3.71 (s, 3H,OCH3), 6.40 (s, 1H, thiazole-C5-H), 6.84e6.89 (m,
2H, 2-methoxyphenyl-C4,5-H), 7.09, 7.24 (2d, each 1H, J ¼ 8.4 Hz, 2-
methoxyphenyl-C3,6-H), 7.45 (d, 2H, J ¼ 8.7 Hz, 4-bromophenyl-
C2,6-H), 7.52 (d, 2H, J ¼ 8.7 Hz, 4-bromopenyl-C3,5-H), 7.61 (d, 1H,
J ¼ 8.4, benzimidazole-C7-H), 7.79 (distorted d, 1H, benzimidazole-
C6-H), 7.95 (s, 1H, benzimidazole-C4-H), 12.01e13.22 (brs, 2H, NH
and COOH, D2O exchangeable); MS: m/z 545 (Mþ, 7.5%); Anal.
Calcd. for C26H17Br N4O3S (545.40): C, 57.25, H, 3.14, N,10.27. Found:
C, 57.41, H, 3.15, N, 10.32.
2.5, 5 and 10 mg/ml) were added to the cell monolayer triplicate
wells were prepared for each individual dose. Monolayer cells were
incubated with the compound(s) for 48 h at 37 ꢂC in atmosphere of
5% CO2. After 48 h, cells were fixed, washed and stained with Sul-
foRhodamine-B stain. Excess stain was washed with acetic acid and
attached stain was recovered with Tris EDTA buffer. Color intensity
was measured in an ELISA reader.
The relation between surviving fraction and drug concentration
is plotted to get the survival curve of each tumor cell line after the
specified compound.
5.1.6.6. 5-Chloro-2-[(3-phenyl-4-oxothiazolidin-2-ylidene)cyano-
methyl] benzimidazole (8a). Yield: 88%; mp: 246e248 ꢂC; IR
(cmꢀ1): 2230 (C^N), 1707 (C]O); 1H NMR (DMSO-d6):
d 4.19 (s,
2H,thiazolidinone-C5-H), 6.91e7.12 (m, 2H, benzimidazole-C7,6-H),
7.35e7.45 (m, 5H, ArH), 8.06 (s, 1H, benzimidazole-C4-H), 10.96 (s,
1H, NH, D2O exchangeable); Anal. Calcd. for C18H11Cl N4OS
(366.81): C, 58.93, H, 3.02, N, 15.27. Found: C, 58.74, H, 3.03, N,
15.24.
5.1.9. Statistical analysis
Differences between different treatment groups were analyzed
using ANOVA followed by Dunnett t-test. P values of less than 0.05
were considered to represent a significant difference.
References
5.1.6.7. 2-[(3-Cyclohexyl-4-oxothiazolidin-2-ylidene)cyanomethyl]
benzimidazole-5-carboxylic acid (8b). Yield: 93%; mp < 300 ꢂC; IR
(cmꢀ1): 2925 (aliph. CH), 2223 (C^N), 1717, 1670 (2C]O); 1H NMR
[1] M. Prudhomme, Recent Pat. Anti-Canc. Drug Discovery 1 (2006) 55e68.
[2] D. Kumar, M.R. Jacob, M.B. Reynolds, S.M. Kerwin, Bioorg. Med. Chem. 10
(2002) 3997e4004.
(DMSO-d6):
d 1.11e1.40 (m, 6H, cyclohexyl-C3,4,5-H), 1.80e1.90 (m,
[3] M. Hranjec, M. Kralj, I. Piantanida, M. Sedic, L. Suman, K. Pavelic, G. Karminski-
Zamola, J. Med. Chem. 50 (2007) 5696e5711.
[4] A.K. Piskin, Z. Ates-Alagoz, F.B. Atac, Y. Musdal, E. Buyukbingol, Turk. J. Bio-
chem. 34 (2009) 39e43.
[5] J.S. Kim, B. Gatto, C. Yu, A. Liu, L.F. Liu, E. LaVioe, J. Med. Chem. 39 (1996)
992e998.
[6] S. Neidle, J. Mann, E.L. Rayner, A. Baron, Y. Opoku-Boahen, I.J. Simpson, N.
J. Smith, K.R. Fox, J.A. Hartley, L.R. Kelland, Chem. Commun. (1999) 929e930.
[7] C. Le Sann, A. Baron, J. Mann, H. van den Berg, M. Gunaratnam, S. Neidle, Org.
Biomol. Chem. 4 (2006) 1305e1312.
4H, cyclohexyl-C2,6-H), 4.18 (m, 1H, J ¼ 7.2 Hz, cyclohexyl-C1-H),
4.91 (d, 2H, J ¼ 15.6 Hz, thiazolidinone-C5-H), 7.62 (d, 1H, J ¼ 6.9 Hz,
benzimidazole-C7-H), 7.83 (d, 1H, J ¼ 6.9 Hz, benzimidazole-C6-H),
8.18 (s, 1H, benzimidazole-C4-H), 12.80 (brs, 1H, NH, D2O
exchangeable), 13.89 (brs, 1H, COOH, D2O exchangeable); Anal.
Calcd. for C19H18N4O3S (382.42): C, 59.66, H, 4.74, N, 14.65. Found:
C, 59.51, H, 4.76, N, 14.68.
[8] A.S. Alpan, S. Zencir, I. Zupkó, G. Coban, B. Réthy, H.S. Gunes, Z. Topcu, J.
Enzym. Inhib. Med. Ch. 24 (2009) 844e849.
[9] S. Huang, I. Hsei, C. Chen, Bioorg. Med. Chem. 14 (2006) 6106e6119.
5.1.6.8. 5-Chloro-2-[(3-phenyl[1,3]thiazin-2-ylidene)cyanomethyl]
benzimidazole (9a). Yield: 78%; mp: 196e198 ꢂC; IR (cmꢀ1): 2900