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S.A. Galal et al. / European Journal of Medicinal Chemistry 46 (2011) 327e340
4.1.9.2. Quinoxalino[2,3-d]benzo[b]imidazolium chloride mono-
hydrate (16). Rf ¼ 0.18 (petroleum ether/ethyl acetate, 1: 3), yield:
72%, m.p. > 300 ꢀC.
(m, 2H), 7.60(m, 2H); 7.65(m, 2H); 7.83(m, 2H); 12.33(br., NH,
D2O exchangeable). 13C NMR (500 MHz, DMSO-d6): 35.9, 115.3,
123.7, 128.2, 129.6, 131.3, 138.2, 141.4, 142.1, 143.4, 148.7, 152.1. IR
(cmꢁ1): 3426 (NH); 3043(CH, aromatic), 2839 (CH, aliphatic), 1625
(C]N), 1571(C]C). MS: [m/z(rel. abundance)]: 326 (Mþ, 19%), 132
(m*, 100). Anal. Calcd for C16H11ClN4S (FW: 326.04): C, 58.80; H,
3.39; Cl, 10.85; N, 17.14; S, 9.81. Found: C, 58.89; H, 3.42; Cl, 10.87; N,
17.15; S, 9.90
4.1.10. Preparation of compounds 17e21
A mixture of 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carbonitrile derivatives [27], 1H-benzimidazole-2-thiol [28] or 2-
thiomethyl-1H-benzimidazole [29] (4 mmol), K2CO3 (4.2 mmol)
and compound 3 (4.2 mmol) in 10 mL of DMF was stirred for
10e12 h. The inorganic salt was filtered off and the solution is
evaporated under the reduced pressure. The solid residue was
washed with water and crystallized from acetone.
4.2. Chemopreventive activity
4.2.1. Cells
EBV genome-carrying lymphoblastoid cells (Raji cells derived
Burkitt’s lymphoma) were cultured in 10% fetal bovine serum (FBS)
in RPMI-1640 under the conditions described previously [30].
Spontaneous activation of EBV-EA in our subline of Raji cells was
less than 0.1%.
4.1.10.1. 2-(3-Chloroquinoxalin-2-ylthio)-6-oxo-4-phenyl-1,6-dihy-
dropyrimidine-5-carbonitrile (17). Rf ¼ 0.42 (petroleum ether/ethyl
acetate, 1: 3), yield: 63.4%, m.p. 269e271 ꢀC. 1H NMR (500 MHz,
DMSO-d6): 7.49(m, 3H), 7.51(m, 2H), 7.82 (m, 2H), 8.12(m, 2H),11.86
(s, 1H, NH, D2O exchangeable). 13C NMR (500 MHz, DMSO-d6): 95.3,
115.7, 127.9, 128.5, 129.2, 131.5, 134.7, 135.5,136.9,141.3, 143.5, 145.8,
153.1, 161.4, 164.2, 170.2 IR (cmꢁ1): 3344 (NH), 3053(CH aromatic),
2222 (CN), 1603 (C]N), 1576 (C]N, C]C pyrimidine). MS: [m/z(rel.
abundance)]: 390 (Mþ ꢁ 1, 24%), 196(m*, 100). Anal. Calcd for
C19H10ClN5OS (FW: 391.03): C, 58.24; H, 2.57; Cl, 9.05; N, 17.87; S,
8.18. Found: C, 58.31; H, 2.61; Cl, 9.16; N, 17.82; S, 8.21.
4.2.2. Animals
Specific pathogen-free (SPF) female ICR and female SENCAR
mice (6 weeks old, respectively) were obtained from Japan SLC, Inc.
(Hamamatsu, Japan) and maintained under SPF conditions in
Animal Center of Kyoto Prefectural University of Medicine. The
mice were housed five per polycarbonate cage in a temperature
controlled room at 24 ꢃ 2 ꢀC and given food, Oriental MF (Oriental
Yeast Co., Tokyo, Japan), and water or aqueous sample solution
adlibitum during the experiments. All animal experiments were
conducted according to the Guidelines for Animal Experimentation
at Kanazawa University of Medicine.
4.1.10.2. 2-(3-Chloroquinoxalin-2-ylthio)-4-(4-hydroxyphenyl)-6-
oxo-1,6-dihydropyrimidine-5-carbonitrile (18). Rf ¼ 0.51 (petroleum
ether/ethyl acetate, 1:3), yield: 52%, m.p. > 300 ꢀC. 1H NMR
(500 MHz, DMSO-d6): 6.88(m, 2H), 7.32(m, 2H), 7.68 (m, 2H), 7.97
(m, 2H), 10.54(s, 1H, OH, D2O exchangeable), 11.33 (br., 1H, NH, D2O
exchangeable). 13C NMR (500 MHz, DMSO-d6): 92.3, 115.4, 127.1,
128.3,129.1,129.6,130.4,140.2,142.5,146.7,145.8,153.1,158.4,163.9,
165.3, 170.3. IR (cmꢁ1): 3452 (OH), 32836 (NH), 3037(CH aromatic),
2210 (CN),1661.7 (C]O),1610 (C]N),1579 (C]N, C]C pyrimidine).
MS: [m/z(rel. abundance)]: 407(Mþ, 8%), 245(m*, 100). Anal. Calcd
for C19H10ClN5O2S (FW: 407.02): C, 55.96; H, 2.47; Cl, 8.69; N,17.17; S,
7.86. Found: C, 55.82; H, 2.39; Cl, 8.72; N, 17.21; S, 7.87.
4.2.3. Inhibition of EBV-EA activation assay
Inhibition of EBV-EA activation was assayed using Raji cells (Virus
nonproducer type), an EBV genome-carrying human lymphoblastoid
cell, which were cultivated in 10% fetal bovine serum. (FBS) RPMI-
1640 medium. The indicator cells (Raji,1
37 ꢀC for 48 h in 1 mL of medium containing n-butyric acid (4 mM as
trigger), TPA (32 pM ¼ 20 ng in 2 L of DMSO as inducer), and various
amounts of the test compounds dissolved in 5 L of DMSO (ca. 0.7%
m
106/mL) were incubated at
m
m
4.1.10.3. 2-(3-Chloroquinoxalin-2-ylthio)-4-(4-fluorophenyl)-6-oxo-
1,6-dihydropyrimidine-5-carbonitrile (19). Rf ¼ 0.48 (petroleum
ether/ethyl acetate, 1: 3), yield: 49.3%, m.p. 277e279 ꢀC. 1H NMR
(500 MHz, DMSO-d6): 6.99(m, 2H,); 7.46 (m, 2H); 7.78 (m, 2H); 8.09
(m, 2H); 11.33 (br., 1H, NH, D2O exchangeable). 13C NMR (500 MHz,
DMSO-d6): 93.3, 114.2, 115.9, 127.1, 128.4, 129.0, 129.7, 130.6, 132.4,
140.2, 143.5, 146.7, 145.8, 151.6, 158.4, 161.8, 163.3, 165.7, 170.2. IR
(cmꢁ1): 32836 (NH pyrimidine); 2208. (CN); 1661 (C]O); 1610.
(C]N); 1549 (C]N, C]C). MS: [m/z(rel. abundance)]: 409(Mþ,
11.6%), 247(m*, 100). Anal. Calcd for C19H9ClFN5OS (FW: 409.02): C,
55.68; H, 2.21; Cl, 8.65; F, 4.64; N, 17.09; S, 7.82. Found: C, 55.75; H,
2.31; Cl, 8.55; N, 17.17; S, 7.89.
DMSO). Smears were made from the cell suspension. The EBV-EA
inducing cells were stained with high titer EBV-EA positive serum
from NPC patients and detected by an indirect immunofluorescence
technique. In each assay, at least 500 cells were counted, and the
number of stained cells (positive cells) was recorded. Triplicate
assays were performed for each data point. as a relative ratio to the
positive control experiment (100%), which was carried out with n-
butyric acid (4 mM) plus TPA (32 pM). In the experiments, the EBV-
EA induction was normally around 35%, and this value was taken as
the positive control (100%). n-Butyric acid (4 mM) alone induced 0.1%
EA-positive cells. The viability of treated Raji cells was assayed by the
trypan blue staining method. The cell viability of the TPA positive
control was greater than 80%. Therefore, only the compounds that
induced less than 80% (% of control) of the EBV-activated cells (those
with a cell viability of more than 60%) were considered able to inhibit
the activation caused by promoter substances. Student’s t-test was
used for all statistical analyses [30,31].
4.1.10.4. 2-(1H-Benzo[d]imidazol-2-ylthio)-3-chloroquinoxaline
(20). Rf ¼ 0.28 (petroleum ether/ethyl acetate, 1: 3), yield: 52%,
m.p. > 300 ꢀC. 1H NMR (500 MHz, DMSO-d6): 7.19(m, 2H), 7.55(m,
2H); 7.61(m, 2H); 7.81(m, 2H); 12.91(br., NH, D2O exchangeable).
13C NMR (500 MHz, DMSO-d6): 115.3, 123.7, 128.5, 129.7, 130.1,
130.8, 133.0, 138.2, 140.4, 142.1, 143.4, 147.7, 152.1. IR (cmꢁ1): 3421
(NH); 3041(CH, aromatic), 1622 (C]N), 1565(C]C). MS: [m/z(rel.
abundance)]: 312 (Mþ, 22%), 150(m*, 100). Anal. Calcd for
C15H9ClN4S (FW: 312.02): C, 57.60; H, 2.90; Cl, 11.33; N, 17.91; S,
10.25. Found: C, 57.53; H, 2.85; Cl, 11.28; N, 17.86; S, 10.29.
4.2.4. Two-stage mouse skin carcinogenesis model induced by
DMBA/TPA
Animals (6 weeks old SPF female ICR mice for 1, 6 weeks old SPF
female SENCAR mice for 6 and 7) were divided into five experimental
groups of 15 mice each. The back of each mouse was shaved with
surgical clippers, and the mice were treated topically with DMBA
(100 mg, 390 nmol) in acetone (0.1 mL) as an initiation treatment. For
groupIa (positive control group of the ICRmice) and groupIb (positive
control group of the SENCAR mice), one week after the initiation,
4.1.10.5. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-3-chloroquinoxa-
line (21). Rf ¼ 0.61 (petroleum ether/ethyl acetate, 1: 3), yield: 57%,
m.p. > 300 ꢀC. 1H NMR (500 MHz, DMSO-d6): 4.72 (s, 2H, CH2), 7.23