Synthesis of novel 4-(1H-benzimidazol-2-yl)benzene-1,3-diols and their cytotoxic activity against human cancer cell lines

Article abstract of DOI:10.1007/s12272-011-1008-0

One-pot synthesis of new biologically active 4-(1H-benzimidazol-2-yl) benzene-l,3-diols has been developed. The compounds were prepared by the reaction of aryl-modified sulfinylbis[(2,4-dihydroxyphenyl)methanethione]s with benzene-l,2-diamines. Their stru

Full text of DOI:10.1007/s12272-011-1008-0

Arch Pharm Res Vol 34, No 10, 1639-1647, 2011
DOI 10.1007/s12272-011-1008-0
Synthesis of Novel 4-(1H-benzimidazol-2-yl)benzene-1,3-diols and
their Cytotoxic Activity against Human Cancer Cell Lines
Monika M. Karpin´ska1, Joanna Matysiak2, and Andrzej Niewiadomy1,2
2
1Institute of Industrial Organic Chemistry, Annopol 6, 03-236 Warsaw, Poland and Department of Chemistry, Univer-
sity of Life Sciences, Akademicka 15, 20-950 Lublin, Poland
(Received April 20, 2011/Revised June 16, 2011/Accepted June 27, 2011)
One-pot synthesis of new biologically active 4-(1H-benzimidazol-2-yl)benzene-l,3-diols has been
developed. The compounds were prepared by the reaction of aryl-modified sulfinylbis[(2,4-
dihydroxyphenyl)methanethione]s with benzene-l,2-diamines. Their structures were identified
l
using elemental, IR, H-NMR, and mass spectra analyses. The developed method offers short
reaction times, relatively large-scale synthesis, easy and quick isolation of the products, and
good yields. The cytotoxicity in vitro against the 4 human cancer cell lines: SW707 (rectal),
HCV29T (bladder), A549 (lung), and T47D (breast) was determined. The antiproliferative
properties of some compounds studies were stronger than those of cisplatin, which was used
as a comparator drug.
Key words: 4-(l
H
-Benzimidazol-2-yl)benzene-l,3-diols, Synthesis, Antiproliferative activity,
Cytotoxic activity
the PARP-1 enzyme, showing activity at a concentra-
tion of 1 nM. In addition, it was orally bioavailable and
INTRODUCTION
Compounds exhibiting the functionality of benzim- possessed good in vivo efficacy in a murine melanoma
idazole have been extensively employed in the area of model (Penning et al., 2010). Other benzimidazole-4-
pharmaceuticals (Singh et al., 2010). They can be found carboxamide analogues as PARP-1 inhibitors with high
in many commercial drugs such as omeprazole (Prilosec), cellular activity were also presented (Tong et al., 2009).
pantoprazole (Protonix), Vermox, fenbendazole, Atacand
Benzimidazoles are described as inhibitors of the
and mibefradil (Posicor) (Fig. 1), as well as in numerous kinesin spindle protein (KSP) (Lahue et al., 2009),
experimental drug candidates in a wide range of thera- hypoxia-inducible factor (HIF-1) (Won et al., 2009),
peutic areas (Biron, 2006; Pescovitz, 2008). The anti- the enzyme 17
cancer properties of the benzimidazole based com- and others (Bhattacharya et al., 2010), which are im-
pounds are also very valuable (Thomas et al portant targets for the development of new anticancer
, 2007).
α-hydroxylase/17,20 (Bruno et al., 2008),
.
A decade ago, a class of benzimidazole-4-carboxamides drugs. The derivatives of bis-benzimidazoles (Hoechst-
was identified as poly(ADP-ribose) polymerase (PARP) 33342, Hoechst-33258) are inhibitors of DNA topoi-
inhibitors and used for the design of potential anti- somerase I (Kazimierczuk and Shugar, 1989; Kraut et
cancer drugs (White et al., 2000); Among them, the clin- al., 1991).
ical candidates, A-620223 and ABT-888 were developed
(Penning et al., 2009). Next, ( )-2-(2-fluoro-4-(pyrrolidin- screening of compounds with a benzimidazole core. For
2-yl)phenyl)-l -benzimidazole-4-carboxamide (A-966492) example, Vitale and co-workers described 2-naphthyl
There are numerous studies on preliminary cytotoxic
S
H
was identified as a compound with high potency against and 2-biphenyl derivatives acting as cytotoxic agents
against human cell lines derived from hematological
Correspondence to: Joanna Matysiak, Department of Chemi-
stry, University of Life Sciences, Akademicka 15, 20-950 Lublin,
Poland
Tel: 48-81-445-6816, Fax: 48-81-533-3549
E-mail: joanna.matysiak@up.lublin.pl
and solid tumors (Vitale et al
., 2008, 2009). 2-Hetero-
cyclic benzimidazoles exhibit cytotoxic effects against
a panel of human cancer cell lines (Refaat, 2010). Other
derivatives are described as precursors of antileuke-
1639

1640
M. M. Karpin´ ska et al.
Fig. 1. Structures of 1H-benzimidazole-based commercial drugs
mic agents (Ranganatha et al., 2009). Benzimidazole- analyses were performed on a Perkin-Elmer 2400
4,7-diones show cytotoxicity against colon, breast and instrument; the determined values (C, H, and N) were
lung cancer cell lines (Gellis et al., 2008). The broad within ± 0.4% of the theoretical values. IR spectra
profile of therapeutic applications of compounds with were measured with a Perkin-Elmer FT-IR 1725X spec-
a benzimidazole core and their valuable biological pro- trophotometer (in KBr) in the range of 600-4000 cm−1.
perties has prompted extensive studies of their syn- 1H-NMR spectra were recorded in DMSO-d6 using a
thesis (Perry and Wilson, 1993; Brain and Brunton, Bruker DRX 500 instrument. Chemical shifts (
2002; Yang et al , 2005; Zhang et al., 2009; Bahrami et were described in relation to tetramethylsilane (TMS).
al , 2010; Peng et al., 2010). MS (EI, 70 eV) were recorded with an AMD-604 in-
We previously reported the synthesis and antipro- strument.
liferative activity of 1,3,4-thiadiazoles and 4 -3,1-ben- The purity of the compounds was examined by a
zothiazines with a 2,4-dihydroxyphenyl residue against liquid chromatograph (Knauer) with a dual pump, a
human cancer cell lines (Matysiak, 2006; Matysiak 20 L simple injection valve, and a UV-visible detector
and Opolski, 2006). Taking into account their valuable at 280 nm. A Hypersil Gold C18 (1.9 m, 100 2.1
δ, ppm)
.
.
H
µ
µ
×
biological properties and the frequent incidence of a mm) column was used as the stationary phase. The
benzimidazole core in the anticancer compounds, we mobile phase included different contents of MeOH and
developed an efficient synthesis of novel differently acetate buffer (pH 4, 20 nM) as the aqueous phase.
substituted 4-(lH-benzimidazol-2-yl)benzene-1,3-diols. The flow rate was 0.5 mL/min at room temperature.
We first obtained a series of compounds modified at The retention time of an unretained solute (t0) was
positions 5, 6 or 7 of the benzimidazole core at the determined by the injection of a small amount of acet-
constant C-2 substitution. Next, we tried to find the one dissolved in water. Log k values for 70% of metha-
best modification of a resorcinol moiety to produce a nol (v/v) in the mobile phase are presented.
more active structure.
4-(7-Methyl-1H-benzimidazol-2-yl)benzene-1,3-diol
(1)
MATERIALS AND METHODS
Chemistry
A mixture of 3-methylbenzene-1,2-diamine (0.002 mol)
and STB (0.002 mol) in MeOH (10 mL) was heated to
Melting points (m.p.) were determined using a BÜCHI ref1ux for 3 h, and was left at room temperature (24
B-540 (Flawil) melting point apparatus. Elemental h). The formed solid was filtered via a Büchner funnel

Synthesis of (lH-benzimidazol-2-yl)benzenediols
1641
and recrystallized from MeOH (6 mL).
Yield 87%; m.p. 158-160oC; IR (KBr)
(OH), 1624 (C=N), 1596 (C=C), 1517 (C=C), 1470, 1379,
Anal. calcd. for C15H14N2O2 (254.28): C, 70.85; H, 5.55;
ν
[cm−1]: 3175 N, 11.02. Found: C, 71.02; H, 5.52; N, 10.97%.
1331, 1302, 1248, 1214 (C-OH), 1178, 1131, 1080, 1025, 4-(5,6-Dichloro-1
H-benzimidazol-2-yl)benzene-1,3-
l
1002, 978, 950, 846, 823, 734; H-NMR (DMSO-d6):
δ
diol (4)
13.21 (s, 1H, HN), 13.02 (s, 1 H, HO-3), 10.52 (s, 1H, A mixture of 4,5-dichlorobenzene-1,2-diamine (0.0015
HO-1), 8.24 (m, 1H, H-5), 7.33 (m, 2H), 7.26 (m, 1H), mol) and STB (0.0015 mol) in MeOH (8 mL) was heat-
6.49 (m, 1H, H-2), 6.31 (m, 1H, H-6), 2.39 (s, 3H, CH3); ed to reflux for 3 h and was left at room temperature
EI-MS (m/z, %): 240 (M+, 100), 211 (6), 183 (22), 169 (24 h). The formed solid was filtered via a Büchner
(3), 154 (3), 106 (9), 91 (8), 77 (5), 51 (2); log k =
Anal. calcd. for C14H12N2O2 (240.26): C, 69.99; H, 5.03; tration of the filtrate. The compound was recrystallized
−0.331. funnel and combined with that removed after concen-
N, 11
.
66
.
Found: C, 69.87; H, 4.92; N, 11.73%.
from MeOH (4 mL).
Yield 80%; m.p. 279-281oC; IR (KBr)
ν
[cm−1]: 3313
4-(5,6-Dimethyl-1
diol (2)
H-benzimidazol-2-yl)benzene-1,3-
(OH), 3256 (OH), 2861 (CH), 1622 (C=N), 1504 (C=C),
1498, 1450, 1389, 1331, 1251 (C-OH), 1198, 1136, 1084,
A mixture of 4,5-dimethylbenzene-1,2-diamine (0.0018 1069, 1027, 984, 950, 917, 892, 846, 812, 779, 764; 1H-
mol) and STB (0.0018 mol) in MeOH (9 mL) was heat- NMR (DMSO-d6):
ed to reflux for 2.5 h, and was filtered via a Büchner 8.8 Hz, 1H, H-5), 7.96 (s, 2H, H-4',7'), 6.71 (d,
funnel. The filtrate was concentrated. The formed Hz, 1H, H-2), 6.55 (dd, = 8.8, 2.0 Hz, 1H, H-6); EI-
solid was recrystallized from MeOH (6 mL).
MS (m/z, %): 295 (M+, 63), 294 (100), 265 (4), 239 (11),
Yield 81%; m.p. 212-215oC; IR (KBr) [cm−1]: 3445 237 (12), 231 (3), 202 (4); log k = 0.470. Anal. calcd. for
δ
10.72 (s, 1H, HO-1), 8.07 (d,
J =
J
= 2.0
J
ν
(OH), 3251 (OH), 3148 (OH), 2920 (CH), 2856 (CH), C13H8Cl2N2O2 (295.12): C, 52.91; H, 2.73; N, 9.49.
1621 (C=N), 1598 (C=C), 1502 (C=C), 1474, 1453, 1383, Found: C, 53.01; H, 2.75; N, 9.44%.
1325, 1297, 1263, 1240, 1208 (C-OH), 1178, 1139, 1082,
1030, 1004, 980, 953, 851, 817, 739; 1H-NMR (DMSO- Methyl 2-(2,4-dihydroxyphenyl)-1
H-benzimidazole-
d6):
1H, HO-1), 8.06 (d,
H-4',7'), 6.77 (d, = 2.2 Hz, 1H, H-2), 6.55 (dd,
δ
14.00 (s, 1H, HN), 11.87 (s, 1H, HO-3), 10.69 (s, 5-carboxylate (5)
= 8.8 Hz, 1H, H-5), 7.55 (s, 2H, A mixture of methyl 3,4-diaminobenzoate (0.0015 mol)
= 8.8, and STB (0.0015 mol) in MeOH (7.5 mL) was heated
J
J
J
2.2 Hz, 1H, H-6), 2.37 (s, 6H, CH3); EI-MS (m/z, %): to reflux for 2.5 h and was left at room temperature
254 (M+, 100), 239 (16), 225 (7), 197 (4), 169 (3), 127 (4), (24 h). The formed solid was filtered via a Büchner
123 (5), 91 (7), 77 (2), 65 (3), 38 (24), 36 (68); log k =
−
funnel and combined with that removed after concen-
0.144. Anal. calcd. for C15H14N2O2 (254.28): C, 70.85; tration of the filtrate. The compound was recrystallized
H, 5.55; N, 11.02. Found: C, 71.02; H, 5.58; N, 10.98%. from MeOH (4 mL).
Yield 88%; m.p. 233-236oC; IR (KBr)
ν
[cm−1]: 3187
4-(6,7-Dimethyl-1H-benzimidazol-2-yl)benzene-1,3- (OH), 3038 (CH), 1686 (C=O), 1619 (C=N), 1578 (C=C),
diol (3)
1505 (C=C), 1485, 1461, 1440, 1420, 1400, 1359, 1334,
A mixture of 3,4-dimethylbenzene-1,2-diamine (0.0018 1296, 1244, 1235 (C-OH), 1180, 1140, 1098, 979, 950,
1
mol) and STB (0.0018 mol) in MeOH (9 mL) was heat- 900, 862, 840, 809, 766; H-NMR (DMSO-d6):
ed to ref1ux for 2.5 h. The hot mixture was filtered via (s, 1H, HN), 11.51 (s, 1H, HO-2), 10.82 (s, 1H, HO-4),
a Büchner funnel and the filtrate was concentrated. 8.35 (s, 1H, H-4), 8.16 (d, = 8.8 Hz, 1H, H-6), 8.03 (dd,
= 8.5, 1.4 Hz, 1H, H-6'), 7.86 (d, = 8.5 Hz, 1H, H-
7'), 6.76 (d, = 2.1 Hz, 1H, H-3), 6.56 (dd, = 8.8, 2.2
δ 14.03
J
The formed solid was recrystallized from MeOH (6
mL).
J
J
J
J
Yield 78%; m.p. 236-238oC; IR (KBr)
ν
[cm−1]: 3395 Hz, 1H, H-5), 3.90 (s, 3H, CH3); EI-MS (m/z, %): 284
(OH), 3212 (OH), 2985 (CH), 2860 (CH), 1617 (C=N), (M+, 100), 270 (12), 253 (63), 225 (24), 198 (3), 142 (4),
1578 (C=C), 1507 (C=C), 1491, 1458, 1398, 1326, 1240 126 (12), 112 (5), 98 (3), 90 (2), 63 (5); log k = 0.137.
(C-OH), 1197, 1132, 1088, 1072, 1021, 983, 952, 915, Anal. calcd. for Cl5H12N2O4 (284.27): C, 63.38; H, 4.25;
−
1
897, 840, 809, 772, 758; H-NMR (DMSO-d6):
(s, 1H, HN), 11.70 (s, 1H, HO-3), 10.62 (s, 1H, HO-1),
8.12 (d, = 8.6 Hz, 1H, H-5), 7.52 (d, = 8.2 Hz, 1H, Ethyl 2-(2,4-dihydroxyphenyl)-1
H-4'), 7.28 (d, = 8.2 Hz, 1H, H-5'), 6.75 (s, 1H, H-2), 5-carboxylate (6)
6.55 (dd, = 8.7, 2.3 Hz, 1H, H-6), 2.54 (s, 3H, CH3), A mixture of ethyl 3,4-diaminobenzoate (0.0014 mol)
2.38 (s, 3H, CH3); EI-MS (m/z, %): 254 (M+, 100), 239 and STB (0.0014 mol) in MeOH (7 mL) was heated to
(22), 225 (5), 187 (8), 113 (5), 36 (26); log k = 0.002. reflux for 3.5 h. The hot mixture was filtered via a
δ 13.80 N, 9.85. Found: C, 63.60; H, 4.27; N, 9.80%.
J
J
H-benzimidazole-
J
J
−

1642
M. M. Karpin´ ska et al.
Büchner funnel and the filtrate was concentrated. The d6):
δ 13.89 (s, 1H, HN), 10.68 (s, 1H, HO-3), 7.95 (s,
formed solid was recrystallized from MeOH (4 mL).
1H, H-5), 7.54 (s, 2H, H-4',7'), 6.83 (s, 1H, H-2), 2.53
= 7.5 Hz, CH2CH3), 2.37 (s, 6H, CH3), 1.20 (t,
Yield 89%; m.p. 231-233oC; IR (KBr) [cm−1]: 3192 (q, 2H,
ν
J
(OH), 3051 (CH), 1690 (C=O), 1616 (C=N), 1579 (C=C), 3H,
J
= 7.5 Hz, CH2CH3); EI-MS (m/z, %): 282 (M+,
1508 (C=C), 1487, 1462, 1438, 1423, 1399, 1362, 1329, 49), 267 (M+-CH3, 100), 239 (5), 225 (2), 210 (2), 197
1289, 1250 (C-OH), 1184, 1148, 1087, 980, 947, 896, (4), 182 (2), 160 (4), 141 (3), 126 (4), 91 (4), 77 (3), 38
l
860, 838, 811, 762, 744; H-NMR (DMSO-d6):
δ 14.20 (4), 36 (11); log k = 0.040. Anal. calcd. for C17H18N2O2
(s, 1H, HN), 11.20 (s, 1H, HO-2), 10.68 (s, 1H, HO-4), (282.34): C, 72.32; H, 6.43; N, 9.92. Found: C, 72.48;
8.35 (d, = 1.0 Hz, 1H, H-4'), 8.12 (d, = 8.8 Hz, 1H, H, 6.46; N, 9.83%.
H-6), 8.04 (dd, = 8.5, 1.4 Hz, 1H, H-6'), 7.86 (d,
8.5 Hz, 1H, H-7'), 6.73 (d, = 2.2 Hz, 1H, H-3), 6.58 4-Chloro-6-(5,6-dimethyl-1
(dd, = 8.8, 2.2 Hz, 1H, H-5), 4.37 (q, 2H, = 7.1 Hz, zene-1,3-diol (9)
CH2CH3), 1.37 (t, 3H, = 7.1 Hz, CH2CH3); EI-MS A mixture of 4,5-dimethylbenzene-1,2-diamine (0.0018
m/z, %): 298 (M+, 100), 270 (35), 253 (33), 225 (13), mol) and SClTB (0.0018 mol) in MeOH (9 mL) was
213 (6), 169 (2), 126 (3), 90 (2), 36 (16); log k = 0.082. heated to reflux for 2.5 h. The hot mixture was filtered
Anal. calcd. for C16H14N2O4 (298.29): C, 64.42; H, 4.73; via a Büchner funnel and the filtrate was concentrat-
J
J
J
J =
J
H-benzimidazol-2-yl)ben-
J
J
J
(
−
N, 9.39. Found: C, 64.58; H, 4.70; N, 9.36%.
ed. The formed solid was recrystallized from MeOH (6
mL).
4-(5,6-Dimethyl-1
benzene-1,3-diol (7)
A mixture of 4,5-dimethylbenzene-1,2-diamine (0.0018 1473, 1408, 1320, 1283, 1246 (C-OH), 1171, 1083, 1019,
H-benzimidazol-2-yl)-2-methyl-
Yield 89%; m.p. 241-243oC; IR (KBr) [cm−1]: 3156
ν
(OH), 2927 (CH), 1622 (C=N), 1577 (C=C), 1512 (C=C),
1
mol) and S3MTB (0.0018 mol) in MeOH (9 mL) was 1006, 949, 897, 849, 784, 726; H-NMR (DMSO-d6):
δ
heated to reflux for 2.5 h. The hot mixture was filtered 13.98 (s, 1H, HN), 11.51 (s, 1H, HO-1), 11.40 (s, 1H,
via a Büchner funnel. The obtained solid was combin- HO-3), 8.23 (s, 1H, H-5), 7.55 (s, 2H, H-4',7'), 7.02 (s,
ed with that removed after concentration of the fil- 1H, H-2), 2.55 (s, 6H, CH3); EI-MS (m/z, %): 288 (M+,
trate. The compound was recrystallized from MeOH- 100), 258 (12), 243 (5), 231 (3), 225 (6), 207 (3), 197
H2O (3:1, 8 mL).
(10), 192 (15), 187 (8), 169 (6), 140 (7), 130 (5), 118 (5),
Yield 83%; m.p. 287-288oC; IR (KBr)
ν
[cm−1]: 3143 105 (4), 91 (4), 80 (9), 77 (9), 51 (7), 45 (28), 39 (7); log
(OH), 1608 (C=N), 1568 (C=C), 1503 (C=C), 1471, 1382, k = 0.179. Anal. calcd. for Cl5H13ClN2O2 (288.73): C,
1311, 1269, 1205 (C-OH), 1027, 1039, 1002, 937, 914, 62.40; H, 4.54; N, 9.70. Found: C, 62.52; H, 4.56; N,
1
859, 809, 757, 736, 703; H-NMR (DMSO-d6):
δ 13.95 9.65%.
(s, 1H, HN), 11.11 (s, 1H, HO-3), 10.44 (s, 1H, HO-1),
7.82 (d, = 8.8 Hz, 1H, H-5), 7.52 (s, 2H, H-4',7'), 6.67 4-(5,6-Dimethyl-1
(d, = 8.7 Hz, 1H, H-6), 2.37 (s, 6H, CH3); EI-MS (m/ 2,3-triol (10)
, %): 268 (M+, 100), 254 (10), 239 (27), 223 (10), 197 A mixture of 4,5-dimethylbenzene-1,2-diamine (0.0018
J
H-benzimidazol-2-yl)benzene-1,
J
z
(6), 169 (2), 134 (4), 103 (3), 91 (6), 65 (3), 36 (26); log mol) and S3TTB (0.0018 mol) in MeOH (9 mL) was
k = 0.220. Anal. calcd. for C16H16N2O2 (268.31): C, heated to reflux for 2.5 h. The hot mixture was filtered
71.62; H, 6.01; N, 10.44. Found: C, 71.80; H, 5.88; N, via a Büchner funnel and the filtrate was concentrat-
10.40%.
ed. The formed solid was recrystallized from MeOH (6
mL).
4-(5,6-Dimethyl-1
zene-1,3-diol (8)
H ν
-benzimidazol-2-yl)-6-ethylben- Yield 64%; m.p. 223-225oC; IR (KBr) [cm−1]: 3216
(OH), 1623 (C=N), 1577 (C=C), 1511 (C=C), 1451, 1260
1
A mixture of 4,5-dimethylbenzene-1,2-diamine (0.0018 (C-OH), 1178, 1082, 954, 878, 844, 785, 759, 717; H-
mol) and SETB (0.0018 mol) in MeOH (9 mL) was NMR (DMSO-d6): 14.09 (s, 1H, HN), 10.39 (s, 1H,
heated to reflux for 2.5 h. The hot mixture was filtered HO-3), 9.35 (s, 1H, HO-2), 7.62 (d, = 8.8 Hz, 1H, H-5),
via a Büchner funnel and the filtrate was concentrat- 7.55 (s, 2H, H-4',7'), 6.66 (d, = 8.8 Hz, 1H, H-6), 2.37
ed. The formed solid was recrystallized from MeOH (6 (s, 6H, CH3); EI-MS (m/z, %): 270 (M+, 100), 255 (6),
δ
J
J
mL).
241 (8), 236 (4), 197 (3), 171 (6), 159 (3), 135 (4), 121
Yield 84%; m.p. 212-214oC; IR (KBr)
ν
[cm−1]: 3248 (4), 91 (5), 77 (7), 44 (4); log k =
−0.781. Anal. calcd. for
(OH), 2967 (CH), 1621 (C=N), 1567 (C=C), 1521 (C=C), C15H14N2O3 (270.28): C, 66.66; H, 5.22; N, 10.36.
1469, 1404, 1313, 1272, 1239 (C-OH), 1165, 1074, Found: C, 66.49; H, 5.25; N, 10.32%.
1023, 1002, 950, 893, 853, 788, 731; 1H-NMR (DMSO-

Synthesis of (lH-benzimidazol-2-yl)benzenediols
1643
2-(5,6-Dimethyl-1
3,5-triol (11)
H-benzimidazol-2-yl)benzene-1,
wells filled with the medium, without the cells. The
cellular material fixed with TCA was stained with
A mixture of 4,5-dimethylbenzene-l,2-diamine (0.0018 0.4% sulforhodamine B (SRB, Sigma-Aldrich Chemie
mol) and S6TTB (0.0018 mol) in MeOH (10 mL) was GmbH) dissolved in 1% acetic acid (POCh) for 30 min.
heated to reflux for 3 h. The hot mixture was filtered The unbound dye was removed by rinsing (4 times)
via a Büchner funnel and the filtrate was concentrat- with 1% acetic acid, and the protein-bound dye was ex-
ed. The formed solid was recrystallized from MeOH- tracted with 10 mM unbuffered Tris base (tris(hydroxy-
H2O (3:1, 8 mL).
methyl)aminomethane, POCh) for determination of
Yield 62%; m.p. 259-261oC; IR (KBr)
ν
[cm−1]: 3260 optical density (at 540 nm) in a computer-interfaced,
(OH), 1627 (C=N), 1578 (C=C), 1515 (C=C), 1453, 1268 96-well microtiter plate reader Uniskan II (Labsys-
(C-OH), 1190, 1076, 998, 870, 846, 782, 743; 1H-NMR tems). The compounds were tested in triplicate for
(DMSO-d6):
δ 11.67 (s, 2H, HO-1,3), 10.09 (s, 1H, HO- each experiment, and the experiments were repeated
5), 7.21 (s, 2H, H-4',7'), 5.86 (s, 2H, H-4,6), 2.40 (s, 6H, at least 3 times.
CH3); EI-MS (m/z, %): 270 (M+, 56), 248 (5), 240 (4),
The investigations were carried out in the Depart-
229 (5), 285 (6), 200 (13), 186 (49), 171 (12), 159 (45), ment of Experimental Oncology, Institute of Immuno-
145 (43), 126 (46), 110 (23), 98 (12), 91 (19), 83 (25), 77 logy and Experimental Therapy, Polish Academy of
(15), 69 (25), 55 (38), 43 (30), 39 (100), 36 (6); log k = Sciences, Wroclaw, Poland.
´
−
0.838. Anal. calcd. for C15H14N2O3 (270.28): C, 66.66;
H, 5.22; N, 10.36. Found: C, 66.74; H, 5.19; N, 10.40%.
RESULTS AND DISCUSSION
4-(lH-Benzimidazol-2-yl)benzene-1,3-diols were pro-
Antiproliferative assay in vitro
The following established in vitro human cell lines duced by the reaction of benzene-1,2-diamines with a
were used in this study: T47D (breast cancer), SW707 series of aryl-modified thioacylating reagents, i.e. sul-
(rectal adenocarcinoma), A549 (non-small cell lung finylbis[(2,4-dihydroxyphenyl)methanethione]s: STB,
carcinoma) from the American Type Culture Collec- S3MTB, SETB, SClTB, S3TTB, and S6TTB (Scheme 1).
tion and HCV29T (bladder cancer) from the Fibiger Their specifically substituted aromatic rings can pro-
Institute, Copenhagen, Denmark. Twenty-four h before duce electrophilic activation of a thiocarbonyl group.
the addition of the tested agent, the cells were plated Favored thermodynamically released in situ carboca-
in 96-well plates (Sarstedt Inc) at a density of 104 tions transformed the substrates into monothioamides.
cells/well. All cell lines were maintained in the opti- In general, the reaction is carried out according to the
MEM medium supplement with 2 mM glutamine electrophilic substitution (Scheme 2). The type and
(Gibco), streptomycin (50 µg/mL), penicillin (50 U/mL) manner of the benzene ring substitutions influence
(Polfa, Tarchomin), and 5% fetal calf serum (Gibco). the equilibrium states of the thion-thiole rearrange-
The cells were incubated at 37oC in a humid atmos- ment and the ability to easily release outgoing groups.
phere saturated with 5% CO2. The solutions of com- The properties of the equilibrium thiol-imine groups of
pounds (1 mg/mL) were prepared ex tempore by dissolv- the intermediate product were taken into account dur-
ing the substance in 100
addition of 900 L of tissue culture medium. Afterwards, a typical acid-base reactivity. The interaction of the
the compounds were diluted in the culture medium to ring substituents of benzene-1,2-diamines is also de-
final concentrations ranging from 0.1 to 100 g/mL. cisive in the synthesis of 1 -benzimidazoles. It deter-
µL of DMSO followed by ing development of the synthesis. They allow to reverse
µ
µ
H
The solvent (DMSO) used at the highest concentration mines the basicity of amine groups and the structures
in the test did not reveal any cytotoxic activity. Cis- of the products. Electrophilic reagents were obtained
platin was used as a test reference agent. The cytotox- according to the method previously described, i.e. by
icity assay was performed after 72 h exposure of the treatment of the corresponding dithioic acids with
cultured cells at concentrations of tested agents rang- SOCl2 (Matysiak and Niewiadomy, 2006).
ing from 0.1 to 100
measure inhibition of cell proliferation in vitro (Skehan phase (RP-18) HPLC chromatography with methanol-
et al , 1990). The cells attached to the plastic were water as a mobile phase. There were characteristic
fixed with cold 50% TCA (trichloroacetic acid, Sigma- O-H and N-H vibration frequencies between 3450
Aldrich Chemie GmbH) added on the top of the culture and 3150 cm−1 in the IR spectra of the compounds. The
µg/mL. The SRB test was used to
Purity of compounds was monitored by reversed-
.
ν
ν
medium in each well. The plates were incubated at
ν C-H and δ C-H bands of ring residues are observed
4oC for 1 h and then washed 5 times with tap water. in the wave region between 3060-3038 cm−1 and 850-
The background optical density was measured in the 700 cm−1, respectively. C=N asymmetric stretching fre-

1644
M. M. Karpin´ ska et al.
Scheme 1. Synthetic pathway for compounds 1-11
corresponding to H-3 and H-6 protons, respectively (
8
and
9).
4-(1H-Benzimidazol-2-yl)benzene-1,3-diols presented
in Scheme 1 were evaluated for their antiproliferative
activity against human bladder cancer HCV29T cells.
The cytotoxic activity in vitro was expressed as IC50
[µg/mL], which is the concentration of the compound
that inhibited the proliferation rate of tumor cells by
50% as compared to the untreated control cells. Cis-
platin was used as a reference drug. Previously de-
scribed unsubstituted 4-(1H-benzimidazol-2-yl)benzene-
1,3-diol (bib-1,3-diol) was obtained and used for com-
parison (Tavman and Birteksoz, 2009). The results of
the screening are summarized in Table I. The activity
of compounds varied and evidently depended on the
type of substitution on both rings. From the group of
Scheme 2. Reaction mechanism of the formation of 4-(lH-
benzimidazol-2-yl)benzene-l,3-diols
1
quencies appear at 1627-1608 cm−1. In the H-NMR compounds not containing a modified resorcinol moi-
spectra, the -NH- proton is detected as a broad singlet ety (
1
-
6
) it is evident that the highest effect against
in the range >13 ppm, similar to other analogues HCV29T cells was exhibited by compound
2, with two
(Refaat, 2010). The resonance bands of protons of Me substituents in positions 5 and 6 (Table I). Strong
hydroxyl groups usually appear in the range of ~11.5 antiproliferative activity was also exhibited by com-
and 10.6 ppm (Tavman and Birteksoz, 2009). They are pounds
sometimes invisible in the background of the base line The structure of compound
). In the H-NMR spectra of 5,6- modified in the resorcinol moiety in positions 2, 4 or 6
-benzimidazoles (compounds and 7-11), by the hydrophobic and hydrophilic substituents of
3
and
7
(IC50 < 5
µ
g/mL).
2
as the most active, was
1
(compounds
dimethyl-1
4 and 8
H
2
there is the characteristic singlet in the range of ~7.55 the contrary electronic effect (Scheme 1). In this way
ppm corresponding to two protons of H-4 and H-7. In compounds 7-11 were obtained. Table I reveals that
the case of 5,6-dichloro-1
tons are recorded at 8 ppm (
of the 5-substituted resorcinol moiety appear as char-
acteristic two singlets in the range of ~7.9 and 6.6 ppm, against HCV29T were also tested against A549 (non-
H
-benzimidazole, these pro- none of new compounds showed better activity than
). The resonance signals compound
The selected compounds with the highest activity
4
2.

Synthesis of (lH-benzimidazol-2-yl)benzenediols
1645
Table I. Antiproliferative activity of 4-(l
H-benzimidazol-2-
beneficial properties were shown for the compounds
with two Me substituents in positions 5 and 6 or 6 and
7. The SAR analysis shows that the additional sub-
stituent in the resorcinol moiety of 5,6-dimethylbenzi-
midazoles decreases antiproliferative activity. This
was especially evident in the case of the derivatives
yl)benzene-l,3-diols against human cancer cell line HCV29T
expressed as IC50
No.
[µ
g/mL]a
IC50 [µg/mL]
01
02
03
04
05
06
07
08
09
22.13 ± 3.28
0.53 ± 0.17
4.43 ± 0.67
6.54 ± 2.23
14.34 ± 2.16
14.33 ± 2.15
2.26 ± 1.29
5.92 ± 1.97
4.92 ± 0.67
23.13 ± 7.62
33.20 ± 2.22
28.50 ± 0.04
2.66 ± 1.15
with the third hydroxyl group (10, 11). This was a
general trend observed across different groups of
compounds with the 2,4-dihydroxyphenyl substituent.
At the same time, this group of derivatives does not
confirm the earlier finding that the presence of a Cl-
atom or Me (Et, i-Pr) substituents in position 5 of the
resorcinol moiety of resorcinol azoles improves their
anticancer properties (Brough et al., 2005; Kreusch et
al., 2005).
10
11
bib-1,3-diolb
cisplatin
In conclusion, a synthetic method for 1H-benzimida-
zoles which offers short reaction times, relatively large-
scale synthesis, easy and quick isolation of the pro-
ducts, and good yields has been developed. It provides
aIC50
[µg/mL] indicates the compound concentration that
inhibits the proliferation rate of tumor cells by 50% as
compared to the untreated control cells. The values are the opportunity to obtain a wide range of the compounds
the means ± S.D. of 9 independent experiments.
with (modified) resorcinol substituents, and in this
b4-(1
H-Benzimidazol-2-yl)benzene-1,3-diol previously describ-
way it makes it possible to add new analogues to the
library of 1 -benzimidazoles. The presented results of
ed (Tavman and Birteksoz, 2009).
H
biological studies indicate a high level of antiprolifera-
tive activity against human cancer cell lines for sev-
Table II. Antiproliferative activity of compounds against
the human cancer lines: A549, T47D and SW707 express-
ed as IC50
eral of these compounds (IC50 = 2-3
µM). Therefore
further studies including design, synthesis, and an-
alysis of subsequent derivatives as well as extension
of anticancer activity will be conducted.
IC50 [µg/mL]
No.
A 549
T47D
SW 707
2
3
7
8
9
3.51 ± 037
7.64 ± 1.04
4.76 ± 0.30
4.97 ± 0.18
0.65 ± 0.17
4.37 ± 1.84
3.51 ± 0.11
3.41 ± 0.34
5.94 ± 2.56
2.03 ± 1.61
1.72 ± 0.67
8.47 ± 3.57
3.39 ± 0.08
4.92 ± 0.08
8.98 ± 0.70
3.13 ± 1.49
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