Synthesis and in vitro cytotoxic evaluation of some thiazolylbenzimidazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.11.014

A novel kind of thiazolylbenzimidazole derivatives were designed and synthesized and evaluated for their antitumor activity against SMMC-7721 and A549 cell lines. Most compounds showed good antitumor activities, and compound 11b displayed remarkable in vi

Full text of DOI:10.1016/j.ejmech.2010.11.014

European Journal of Medicinal Chemistry 46 (2011) 417e422
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and in vitro cytotoxic evaluation of some thiazolylbenzimidazole
derivatives
,
,
b
,
,
d
,
c
Yu Luo a 1, Feng Xiao a 1, Shijing Qian c, Wei Lu a , Bo Yang
*
a Department of Chemistry, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China
b State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, 555 Zu Chongzhi Road, Shanghai 201203, China
c College of Pharmaceutical Science, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, China
d Institute of Drug Discovery and Development, East China Normal University, Shanghai 200062, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel kind of thiazolylbenzimidazole derivatives were designed and synthesized and evaluated for
their antitumor activity against SMMC-7721 and A549 cell lines. Most compounds showed good anti-
tumor activities, and compound 11b displayed remarkable in vitro anticancer activity comparable to
taxol. The preliminary structureeactivity relationship of these benzimidazole derivatives was discussed
based on the experimental data obtained.
Received 22 June 2010
Received in revised form
6 November 2010
Accepted 9 November 2010
Available online 27 November 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Thiazolyl benzimidazole
Anticancer
Synthesis
SMMC-7721
A549
1. Introduction
R'
R'
N
N
S
N
N
N
S
Benzimidazole derivatives are recognized as a class of important
heterocycles for their pharmacological properties in the past years
[1]. Existing studies have proved their wide range of biological
activity as antiviral [2e7], antibacterial [8,9], antifungal [10] and
antitumor [11e13] agents. On the other hand, many substituted
thiazole derivatives were also proven to exhibit anticancer and
antimicrobial activities [14,15]. In addition, other studies on thia-
zole-containing antitumor agents indicate that substituents on the
thiazole ring play an important role for their biological activity [16].
Despite many 2-(thiazol-4-yl)-1H-benzoimidazole compounds
(compound 1, Fig. 1) have been synthesized and studied for their
biological activity, such as antifungal, antibacterial and anthelmintic
activities [17e20] 2-(thiazole-2-yl)-1H-benzimidazol derivatives
(compound 2, Fig. 1) have been seldom reported in the literature.
Recently, Garuti and co-workers reported thiazolylbenzimidazole-
4,7-diones showed good antiproliferative activity against some
tumor cell lines [21]. However, it is well known that quinones could
N
R''
R
R
1
2
Fig. 1. The structures of two kinds of 2-thiazole-1H-benzimidazol.
precede reductive alkylation process [22], making quinone deriva-
tives potentially toxic both to tumor cell lines and to normal
counterparts.
In the light of the above reports and our continued interest in
studying benzimidazole derivatives, we decided to synthesize
a series of thiazolylbenzimidazole compounds and assess their
antitumor activity. To increase the antitumor activity, efforts were
exerted on the structure modification of the thiazole ring. Herein, we
report the synthesis and antitumor activity of novel thiazolylbenzi-
midazole compounds related to the general structure 2. And the
preliminary structureeactivity relationship was discussed (Fig. 1).
2. Chemistry
* Corresponding author. Department of Chemistry, East China Normal University,
3663 North Zhongshan Road, Shanghai 200062, China. Tel./fax: þ86 21 62602475.
E-mail address: wlu@chem.ecnu.edu.cn (W. Lu).
The synthesis of this kind of thiazolylbenzimidazoles started
from 2-chloro-1H-benzoimidazole (3) (Scheme 1), which was
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.014

418
Y. Luo et al. / European Journal of Medicinal Chemistry 46 (2011) 417e422
N
N
S
c
N
N
N
N
b
N
a
Cl
CN
Cl
NH2
N
H
R
R
R
5a-d
3
6a-d
a: R = CH3;
4a-d
b: R = C2H5
c: R = CH(CH3)2; d: R = Bn
Scheme 1. Conditions and reagents: (a) organic iodides and bromides, NaH, DMF, 47e97%; (b) DMF, NaCN, 100 ꢀC, 62e97%; (c) (NH4)2S, DMF, TEA, 50 ꢀC, 47e90%.
prepared according the published method [23]. Reaction of 3 with
organic iodide (methyl iodide) or bromides (ethyl bromide, 2-bro-
mopropane and benzyl bromide) gave the corresponding N-alkyl
benzimidazoles 4aed, which were subsequently treated with NaCN
in DMF to afford the desired 2-cyano-benzimidazole intermediates
5aed. Using (NH4)2S as sulfuration reagent [24], these cyano
compounds were smoothly converted into thioformamides 6aed.
One subseries of the thiazolylbenzimidazoles was first synthe-
sized from the key intermediate compounds 6aed [25]. Thus,
benzyl group resulted in a dramatically increased activity (7d,
IC50 ¼ 2.78), suggesting that the lipophilic character of substituents
in N-1 position was an important determinant for the cytotoxicity.
However, compound 7c with an isopropyl group in N-1 position
displayed much reduced antitumor potency (IC50 ¼ 98.35).
To identify the effects of the thiazole ring on the biological
profiles, this subseries of compounds 7e9 has different patterns of
substitution on the thiazole’s 4,5 positions. Compound 8a and 8b
displayed moderate activity, while 9 showed slightly less potent
cytotoxicity. However, both 8 and 9 were generally less potent
antitumoractivitythan compounds 7 (withthe exception of 7c). This
result suggested that both the electronic character and the position
of substituents played important roles for the antitumor activity.
Although most of the above compounds exhibited antitumor
activity against SMMC-7721 cell, none of them were active against
A549 cell line. Nevertheless, 7d, for its good antitumor activity, was
selected as the benchmark compound for subsequent optimization.
Dramatic change in antitumor activity was observed, when the
ester group of 7d was transformed into its derivatives. The acid 10
reaction of compounds 6aed with three kinds of
a-haloketones
yielded the target compounds 7aed, 8aec and 9, respectively
(Scheme 2). This cyclization with ethyl 2-chloroacetoacetate pro-
ceeded smoothly and the yields were from moderate to good.
However, reaction with ethyl bromopyruvate only resulted in poor
to moderate yields. Reaction of 6aed with ethyl 4-chlor-
oacetoacetate was also attempted, while only one thiazolylbenzi-
midazole compound 9 could be synthesized in a moderate yield.
Another subseries of the title compounds was prepared from 7d
(Scheme 3). Thus, 7d was hydrolyzed to give the acid 10, which was
condensed with different primary or secondary amines to afford
the amides 11aeg in 42e94% yields.
displayed reduced activity (IC50 ¼ 15.30
m
M) against SMMC-7721
cell, whereas exhibited low antitumor activity (IC50 ¼ 68.05
mM)
against A549 cell line. Condensation of acid 10 with different
primary or secondary amines afforded the amides 11aeg (Scheme
3). As shown in Table 1, the amides from primary amines (11aed)
were more toxic than those from secondary amines (11eeg).
Compounds (11aec) were not only active for SMMC-7721 cell, but
also exhibited good cytotoxicity against A549 cell, suggesting the
hydrophilic feature of the thiazole ring was closely related with the
antitumor activity.
3. Results and discussion
The cytotoxicity of the synthesized benzimidazoles, together
with the reference drug taxol was evaluated in SMMC-7721 and
A549 cell lines. The results are summarized in Table 1. The cyto-
toxicity of each compound was expressed as the concentration of
compound required to kill 50% of the tumor cells.
Compound 11b, with a flexible and basic alkyl chain, exhibited
the potent antitumor activity against both SMMC-7721 cell and
A549 cell. Replacement of the flexible basic side chain with phenyl
group resulted in a slight loss of cytotoxicity (IC50 ¼ 3.70 and 3.96,
respectively). However, replacement of 2-diethylamino-ethyl side
chain with hydrophilic cyclohexyl ring (11d) resulted in a 12-fold
As shown in Table 1, most of these compounds exhibited good to
moderate antitumor potency (IC50 from 1.14 to 33.31) against
SMMC-7721 cell. The different kinds of substituents in N-1 position
were first investigated. The N-CH3 compound 7a and N-C2H5
compound 7b exhibited moderate activity (IC50 ¼ 13.88 and 9.85,
respectively), while replacement of the methyl or ethyl group with
O
a
N
N
S
N
b
N
N
R
6a-d
S
N
N
S
N
OEt
CH
OEt
NH
2
3
R
R
O
8a: R = CH
3
7a: R = CH
3
8b: R = Bn
7b: R = C H
2
5
c
8c: R = C H
2
5
7c: R = CH(CH )
3 2
7d: R = Bn
N
S
O
N
N
OEt
9
Scheme 2. Conditions and reagents: (a) ethyl 2-chloroacetoacetate, ethanol, reflux, 32e70%; (b) ethyl bromopyruvate, ethanol, reflux, 25e42%; (c) ethyl 4-chloroacetoacetate,
ethanol, reflux, 32%.

Y. Luo et al. / European Journal of Medicinal Chemistry 46 (2011) 417e422
419
O
O
O
R
N
N
S
N
N
N
S
N
OEt
CH3
N
N
S
N
a
OH
CH3
N
H
b
CH3
Bn
Bn
Bn
7d
10
11a: R = isopropyl
11b: R = -CH2CH2N(C2H5)2
11c: R = phenyl
11d: R = cyclohexyl
b
O
N
N
S
N
N
A
CH3
Bn
11e: A = CH2
11f: A = O
11g: A = NCH3
Scheme 3. Conditions and reagents: (a) KOH, methanol, H2O, reflux, 72%; (b) primary and secondary amines, CDI (N,N-carbonyldiimidazole), THF, 42e94%.
(ppm). Data are reported as follows: chemical shift, multiplicity (br
s ¼ broad singlet, d ¼ doublet, dd ¼ doublet of doublet, dt ¼ doublet
of triplet, m ¼ multiple, s ¼ singlet and t ¼ triplet), coupling
constants (Hz), integration.
Table 1
The Inhibition Concentration 50% of Compounds 7e11.
Compounds
SMMC-7721
IC50 M]
A549
[
m
95% confidence
IC50
[
m
M]
95% confidence
interval
interval
5.2. General procedures for the synthesis of compounds 4aed
7a
7b
7c
7d
8a
8b
8c
9
13.88
9.85
> 20
2.78
16.48
13.00
> 20
> 20
15.30
6.90
1.14
3.70
12.42
>20
>20
6.94e27.74
2.27e43.37
26.80e361.1
0.79e4.45
4.35e61.78
3.44e48.84
9.78e221.9
5.21e86.28
4.47e52.29
6.07e7.74
>20
>20
>20
>20
>20
>20
>20
>20
>20
6.64
2.43
3.96
5.23
e
e
e
e
e
2-Chloro-1H-benzoimidazole (3.04 g, 20 mmol) was dissolved
in dry DMF (15 mL) at 0 ꢀC, to the solution was added NaH (0.91 g,
22.7 mmol), and the mixture was stirred for 1 h at 0 ꢀC, then halide
(21.6 mmol) was added. The mixture was stirred overnight at room
temperature and was poured into water (50 mL) and stirred for 1 h,
filtrated, washed with water and dried to afford 4aed.
e
e
10
26.07e177.4
3.92e11.28
0.65e9.35
2.45e7.75
2.90e9.39
19.42e127.8
19.56e501.6
15.64e49.98
0.19e2.13
11a
11b
11c
11d
11e
11f
11g
Taxol
0.67e1.95
1.46e9.36
5.2.1. 1-Methyl-2-chloro-1H-benzoimidazole (4a)
4a(1.75 g, 53%), mp 113e114 ꢀC. 1H NMR (CDCl3):
d 3.79 (s, 3H),
3.63e42.44
9.88e54.76
13.99e146.7
13.25e30.23
0.43e4.63
7.28e7.30 (m, 3H), 7.69 (d, J ¼ 7.6 Hz, 1H). EI-MS: m/z 166 [M]þ.
>20
>20
>20
20.0
1.42
5.2.2. 1-Ethyl-2-chloro-1H-benzoimidazole (4b)
0.63
4b (2.64 g, 73%), mp 52e53 ꢀC. 1H NMR (CDCl3):
d 1.75 (t,
J ¼ 7.8 Hz, 3H), 4.96 (q, J ¼ 7.8 Hz, 2H), 7.27e7.31 (m, 3H), 7.55 (d,
decrease of activity against SMMC-7721 and a 2-fold decrease of
activity against A549 cell. This result suggested again the hydro-
philic feature of the amide groups played a pivotal role for the
antitumor potency.
J ¼ 7.6 Hz, 1H). EI-MS: m/z 180 [M]þ.
5.2.3. 1-Isopropyl-2-chloro-1H-benzoimidazole (4c)
4c (1.84 g, 47%), mp 53e54 ꢀC. 1H NMR (CDCl3):
d 1.65 (d,
J ¼ 7.1 Hz, 6H), 4.90e4.93 (m, 1H), 7.24e7.26 (m, 2H), 7.49 (dd,
J ¼ 6.0 Hz, J ¼ 2.0 Hz, 1H), 7.69 (dd, J ¼ 6.0 Hz, J ¼ 2.0 Hz, 1H). EI-MS:
m/z 194 [M]þ.
4. Conclusions
A series of thiazolylbenzimidazole derivatives was synthesized
and assessed for antitumor activity in vitro against SMMC-7721 and
A549 cells. Compounds 11aec displayed the optimal profiles, with
5.2.4. 1-Benzyl-2-chloro-1H-benzoimidazole (4d)
4d (4.72 g, 97%), mp 107e109 ꢀC. 1H NMR (CDCl3):
d 5.39 (s, 2 H),
IC50 in mM range. The most promising compound 11b with potent
7.18 (d, J ¼ 6.5 Hz, 2H), 7.23e7.34 (m, 6H), 7.72 (d, J ¼ 7.0 Hz, 1H).
EI-MS: m/z 242 [M]þ.
antitumor activity was an attractive candidate for further assess-
ment in vivo as antitumor agent. These results also indicate that the
thiazolylbenzimidazole compounds with no quinone moiety could
also serve as potent antitumor agents, which might exhibit fewer
side-effects than the quinone counterparts.
5.3. General procedures for the synthesis of compounds 5aed
4 (8.7 mmol) was dissolved in dry DMF (15 mL), to the solution
was added NaCN (9.2 mol), and the mixture was stirred for 2 h at
100 ꢀC. The mixture was poured into water (50 mL), filtrated,
washed with water and dried to afford 5.
5. Experimental
5.1. Chemistry
5.3.1. 1-Methyl-2-cyano-1H-benzoimidazole (5a)
1H NMR spectra were recorded on a Bruker DRX-500 (500 MHz).
Mass spectra (MS) were determined on a Finnigan MAT-95 mass
spectrometer. Chemical shifts (d) are reported in parts per million
5a (1.16 g, 75%), mp 179e180 ꢀC. 1H NMR (CDCl3):
d 4.01 (s, 3H),
7.41e7.45(m, 2H), 7.48e7.50(m, 1H), 7.86 (d, J ¼ 8.0 Hz, 1H). EI-MS:
m/z 157 [M]þ.

420
Y. Luo et al. / European Journal of Medicinal Chemistry 46 (2011) 417e422
5.3.2. 1-Ethyl-2-cyano-1H-benzoimidazole (5b)
5.5.2. 4-Methyl-2-(1-ethyl-1H-benzoimidazol-2-yl)-thiazole-5-
5b (0.92 g, 62%), mp 86e88 ꢀC. 1H NMR (CDCl3):
d
1.56 (t,
carboxylic acid ethyl ester (7b)
J ¼ 7.1 Hz, 3H), 4.45 (q, J ¼ 7.1 Hz, 3H), 7.40e7.49 (m, 3H), 7.87 (d,
7b (0.10 g, 32%), mp 111e114 ꢀC. 1H NMR (CDCl3):
d
1.35 (t,
J ¼ 8.3 Hz, 1H). EI-MS: m/z 171 [M]þ.
J ¼ 7.1 Hz, 3H),
d
: 1.53 (t, J ¼ 7.1 Hz, 3H), 2.84 (s, 3H), 4.40 (q,
J ¼ 7.1 Hz, 2H), 4.92 (q, J ¼ 7.1 Hz, 2H), 7.34e7.42 (m, 2H), 7.48 (d,
5.3.3. 1-Isopropyl-2-cyano-1H-benzoimidazole (5c)
J ¼ 7.9 Hz, 1H), 7.86 (d, J ¼ 8.1 Hz, 1H). 13C NMR (CDCl3):
d 162.30,
5c (1.35 g, 84%), mp 67e68 ꢀC. 1H NMR (CDCl3):
d
1.76 (d,
161.86, 160.88, 145.01, 144.12, 135.22, 132.40, 124.10, 129.95, 121.23,
111.87, 61.53, 45.83, 22.66, 17.67, 14.42. EI-MS: m/z 315 [M]þ. HRMS
(EI): Cal. for: 315.1041, found: 315.1039.
J ¼ 7.0 Hz, 6H), 4.98e5.02 (m, 1H), 7.38e7.46 (m, 2H), 7.57 (d,
J ¼ 9.0 Hz, 1H), 7.86e7.88 (m, 1H). EI-MS: m/z 185 [M]þ.
5.3.4. 1-Benzyl-2-cyano-1H-benzoimidazole (5d)
5.5.3. 4-Methyl-2-(1-isopropyl-1H-benzoimidazol-2-yl)-thiazole-
5d (2.02 g, 97%), mp 139e141 ꢀC. 1H NMR (CDCl3, 500 MHz):
5-carboxylic acid ethyl ester (7c)
d
5.55 (s, 2H), 7.23e7.26 (m, 2H), 7.33e7.44 (m, 6H), 7.87 (d,
7c (0.14 g, 42%), mp 149e152 ꢀC. 1H NMR (CDCl3):
d
1.38 (t,
: 1.71 (d, J ¼ 7.0 Hz, 6H), 2.80 (s, 3H), 4.36 (q,
J ¼ 7.2 Hz, 2H), 6.40e7.45 (m, 1H), 7.28e7.32 (m, 2H), 7.66e7.68 (m,
1H), 7.82e7.84 (m, 1H). 13C NMR (CDCl3):
162.29, 161.85, 160.88,
J ¼ 7.5 Hz, 1H). EI-MS: m/z 233 [M]þ.
J ¼ 7.2 Hz, 3H),
d
d
5.4. General procedures for the synthesis of compounds 6aed
144.53, 143.97, 135.06, 132.49, 124.01, 123.17, 121.03, 113.31, 61.59,
48.92, 21.51, 17.87, 14.45. EI-MS: m/z 329 [M]þ. HRMS (EI): Cal. for:
329.1198, found: 329.1196.
To a solution of 5 (7.6 mmol) in dry DMF (15 mL) was added TEA
(2.2 mL) and 20% (NH4)2S (2.2 mL). The mixture was stirred for 2 h
at 50 ꢀC, and then poured into water (60 mL). The precipitation was
filtrated, washed with water and dried to afford 6aed.
5.5.4. 4-Methyl-2-(1-benzyl-1H-benzoimidazol-2-yl)-thiazole-5-
carboxylic acid ethyl ester (7d)
7d (0.26 g, 70%), mp 179e181 ꢀC. 1H NMR (CDCl3, 500 MHz)
d:
5.4.1. 1-Methyl-1H-benzoimidazole-2-carbothioic acid amide (6a)
1.38 (t, J ¼ 7.2 Hz, 3H),
2H), 7.20e7.38 (m, 8H), 7.83e7.86 (m, 1H); 13C NMR (CDCl3):
d 163.13, 158.12, 157.54, 148.50, 144.46, 143.05, 140.57, 136.83,
129.90, 128.20, 127.25, 124.82, 123.70, 120.64, 111.06, 61.60, 39.64,
32.35, 14.43. EI-MS: 377 [M]þ, 348, 332, 300, 286, 272, 258, 234,
219, 207, 188, 175, 169, 152, 144, 129, 116, 102, 91, 85, 77, 71, 65, 51,
45. HRMS: Cal. for: 377.1198, found: 377.1199.
d
: 2.76 (s, 3H), 4.36 (q, J ¼ 7.2 Hz, 2H), 6.16 (s,
6a (1.14 g, 82%), mp 156e158 ꢀC. 1H NMR (CDCl3):
d 4.34 (s, 3H),
7.34e7.44 (m, 3H), 7.74 (br, 1H), 7.77 (d, J ¼ 8.0 Hz, 1H); 9.15 (br, 1H).
EI-MS: m/z 191 [M]þ.
5.4.2. 1-Ethyl-1H-benzoimidazole-2-carbothioic acid amide (6b)
6b (1.13 g, 73%), mp 156e157 ꢀC. 1H NMR (CDCl3):
d 1.53 (t,
J ¼ 7.1 Hz, 3H), 5.05 (q, J ¼ 7.1 Hz, 3H), 7.35e7.47 (m, 3H), 7.51 (br,
1H), 7.78 (d, J ¼ 8.0 Hz, 1H); 9.03 (br, 1H). EI-MS: m/z 205 [M]þ.
5.6. General procedures for the synthesis of compounds 8aec
5.4.3. 1-Isopropyl-1H-benzoimidazole-2-carbothioic acid amide
6 (1 mmol) and ethyl bromopyruvate (1.2 mmol) were added
into EtOH (10 mL). The reaction mixture was heated at reflux for
8 h, and then poured into water (30 mL), extracted 3 times with
CHCl3. The combined organic layer was washed with saturated
brine, dried over anhydrous Na2SO4, and evaporated under reduced
pressure. The residue was subjected to silica column chromatog-
raphy (EtOAc/hexane) to give 8 as white solids.
(6c)
6c (0.79 g, 47%), mp 163e164 ꢀC. 1H NMR (CDCl3):
d 1.65 (d,
J ¼ 7.0 Hz, 6H), 6.50e6.53 (m, 1H), 7.29e7.33 (m, 2H), 7.64e7.66 (m,
1H), 7.73e7.75 (m, 1H), 8.01 (br, 1H), 9.35 (br, 1H). EI-MS: m/z 219
[M]þ.
5.4.4. Benzyl-1H-benzoimidazole-2-carbothioic acid amide (6d)
6d (1.83 g, 90%), mp 162e164 ꢀC. 1H NMR (CDCl3):
d
6.36 (s, 2H),
5.6.1. 2-(1-Methyl-1H-benzoimidazol-2-yl)-thiazole-4-carboxylic
7.14 (d, J ¼ 7.1 Hz, 2H), 7.24e7.36 (m, 6H), 7.61 (br, 1H), 8.01 (d,
acid ethyl ester (8a)
J ¼ 6.1 Hz, 1H), 9.08 (br, 1H). EI-MS: m/z 267 [M]þ.
8a (0.12 g, 42%), mp 197e201(dec)ꢀC. 1H NMR (CDCl3):
d 1.36 (t,
J ¼ 7.0 Hz, 3H), 4.32 (s, 3H), 4.39 (q, J ¼ 7.0 Hz, 2H), 7.34 (t, J ¼ 8.0 Hz,
1H), 7.41 (t, J ¼ 8.0 Hz, 1H), 7.71 (d, J ¼ 8.0 Hz,1H), 7.76 (d, J ¼ 8.0 Hz,
5.5. General procedures for the synthesis of compounds 7aed
1H), 8.73 (s, 1H). 13C NMR (CDCl3):
d 160.32, 151.88, 149.37, 139,97,
133.79, 132.59, 132.32, 127.90, 127.77, 117.13, 111.81, 62.35, 34.07,
29.87, 14.50. ESI-MS: 288 [Mþ1]þ. HRMS: Cal. for: 287.0728, found:
287.0723.
6 (1 mmol) and ethyl 2-chloroacetoacetate (1.2 mmol) were
added into EtOH (10 mL). The reaction mixture was heated at reflux
for 8 h, and then poured into water (30 mL), extracted with CHCl3.
The combined organic layer was washed with saturated brine,
dried over anhydrous Na2SO4, and evaporated under reduced
pressure. The residue was subjected to silica column chromatog-
raphy (EtOAc/hexane) to give 7aed as white solids.
5.6.2. 2-(1-Benzyl-1H-benzoimidazol-2-yl)-thiazole-4-carboxylic
acid ethyl ester (8b)
8b (0.13 g, 35%), mp 163e166 ꢀC. 1H NMR (CDCl3, 500 MHz)
d: 1.43
(t, J ¼ 7.2Hz, 3H), 4.44(q, J¼ 7.2 Hz, 2H), 6.17 (s, 2H), 7.23e7.27 (m, 3H),
7.32e7.36 (m, 4H), 7.46e7.48 (m, 1H), 7.86e7.88 (m, 1H), 8.26 (s, 1H);
5.5.1. 4-Methyl-2-(1-methyl-1H-benzoimidazol-2-yl)-thiazole-5-
13C NMR (CDCl3):
d 160.96, 158.41, 157.65, 148.58, 143.43, 140.58,
carboxylic acid ethyl ester (7a)
136.13, 130.20, 128.88, 127.69, 125.51, 124.59, 119.87, 111.38, 61.75,
49.04,14.49.EI-MS:363[M]þ, 334, 318, 258, 205,161,154,144,129,116,
102, 90, 8377, 71, 65, 51, 45. HRMS: Cal. for: 363.1041, found: 363.1036.
7a (0.16 g, 57%), mp 105e107 ꢀC. 1H NMR (CDCl3):
d 1.33 (t,
J ¼ 7.1 Hz, 3H), 2.81 (s, 3H), 4.27 (s, 3H), 4.33 (q, J ¼ 7.1 Hz, 2H), 7.32
(t, J ¼ 8.0 Hz, 1H), 7.41 (t, J ¼ 8.0 Hz, 1H), 7.69 (d, J ¼ 8.0 Hz, 1H), 7.74
(d, J ¼ 8.0 Hz, 1H). 13C NMR (CDCl3):
d
162.25, 161.38, 160.98, 145.08,
5.6.3. 2-(1-Ethyl-1H-benzoimidazol-2-yl)-thiazole-4-carboxylic
142.87, 137.24, 133.92, 124.58, 123.51, 120.59, 110.22, 61.60, 32.35,
17.79, 14.42. EI-MS: m/z 301 [M]þ. HRMS (EI): Cal. for: 301.0885,
found: 301.0879.
acid ethyl ester (8c)
8c (80 mg, 25%), mp 181e184 ꢀC. 1H NMR (CDCl3):
d
1.36 (t,
J ¼ 7.0 Hz, 3H),
d: 1.55 (t, J ¼ 7.1 Hz, 3H), 4.38 (q, J ¼ 7.1 Hz, 2H), 4.45

Y. Luo et al. / European Journal of Medicinal Chemistry 46 (2011) 417e422
421
(q, J ¼ 7.0 Hz, 2H), 7.36 (t, J ¼ 8.0 Hz, 1H), 7.43 (t, J ¼ 8.0 Hz, 1H), 7.72
(d, J ¼ 8.0 Hz, 1H), 7.78 (d, J ¼ 8.0 Hz, 1H), 8.75 (s, 1H). 13C NMR
157.47, 154.58, 143.78, 142.18, 137.07, 136.28, 128.65, 127.45, 127.07,
124.45, 123.32, 119.73, 111.54, 51.17, 47.59, 46.64, 17.03, 16.87, 11.94.
EI-MS: m/z 447 [M]þ. HRMS (EI): Cal. for: 447.2093, found:
447.2096.
(CDCl3):
d 160.49, 152.80, 149.32, 140,24, 134.06, 132.53, 132.25,
127.96, 127.71, 116.93, 111.82, 65.27, 61.18, 40.29, 37.57, 15.87. EI-
MS: m/z 287 [M]þ. HRMS (EI): Cal. for: 287.0728, found: 287.0827.
5.9.3. 2-(1-Benzyl-1H-benzoimidazol-2-yl)-4-methyl-thiazole-5-
5.7. [2-(1-Methyl-1H-benzoimidazol-2-yl)-thiazol-4-yl]-acetic acid
ethyl ester (9)
carboxylic acid phenylamide (11c)
11c (80 mg, 66%), mp 212e214 ꢀC. 1H NMR (DMSO-d6):
d 2.74 (s,
3 H), 6.19 (s, 2 H), 7.16e7.20 (m, 6 H), 7.29e7.37 (m, 5H), 7.52 (d,
J ¼ 7.0 Hz, 1H), 7.65 (d, J ¼ 6.8 Hz, 1H), 7.70 (d, J ¼ 7.0 Hz, 1H), 7.89 (s,
A mixture of 6a (1 mmol) and ethyl 4-chloroacetoacetate
(1.2 mmol) in EtOH (10 mL) was heated at reflux for 8 h. Then the
mixture was poured into water (30 mL), extracted with CHCl3. The
combined organic layer was washed with saturated brine, dried
over anhydrous Na2SO4, and evaporated to dryness under reduced
pressure. The residue was subjected to silica column chromatog-
raphy (EtOAc/hexane) to give 9 as a white solid (0.10 g, 3%), mp
1H). 13C NMR (DMSO-d6):
d 160.55, 157.42, 152.48, 139.66, 138.16,
136.92, 130.74, 128.71, 128.63, 127.54, 127.03, 126.97, 124.87, 123.61,
121.74, 119.95, 118.33, 118.14, 47.76, 17.64. EI-MS: m/z 424 [M]þ.
HRMS (EI): Cal. for: 424.1358, found: 424.1363.
5.9.4. 2-(1-Benzyl-1H-benzoimidazol-2-yl)-4-methyl-thiazole-5-
95e97 ꢀC. 1H NMR (CDCl3):
d
1.30 (t, J ¼ 7.2 Hz, 3H), 3.93 (s, 2H),
carboxylic acid cyclohexylamide (11d)
4.24 (q, J ¼ 7.2 Hz, 2H), 4.30 (s, 3H), 7.30e7.38 (m, 3H), 7.43 (d,
11d (0.12 g, 94%), mp 228e230 ꢀC. 1H NMR (CDCl3):
d 1.07e1.09
J ¼ 7.9 Hz, 1H), 7.82 (d, J ¼ 7.8 Hz, 1H), 8.73 (s, 1H). 13C NMR (CDCl3):
(m, 4H), 1.66e1.72 (m, 6H), 2.70 (s, 3H), 3.45e3.48 (m, 1H), 5.65 (s,
1H), 6.13 (s, 2H), 7.19e7.24 (m, 3H), 7.29e7.35 (m, 5H), 7.80e7.82
d
159.21, 150.06, 144.71, 142.80, 135.99, 124.14, 123.73, 123.25,
120.32, 118.94, 110.19, 66.49, 61.02, 37.94, 15.19. EI-MS: m/z 301
(m, 1H). 13C NMR (CDCl3):
d 160.79, 157.99, 156.86, 144.56, 143.09,
[M]þ. HRMS (EI): Cal. for: 301.0885, found: 301.0883.
136.84, 136.72, 128.98, 127.98, 127.61, 127.37, 124.79, 123.75, 120.66,
111.11, 48.70, 26.46, 24.67, 16.57. EI-MS: m/z 430 [M]þ. HRMS (EI):
Cal. for: 430.1827, found: 430.1826.
5.8. 2-(1-Benzyl-1H-benzoimidazol-2-yl)-4-methyl-thiazole-5-
carboxylic acid (10)
5.9.5. [2-(1-Benzyl-1H-benzoimidazol-2-yl)-4-methyl-thiazol-5-
KOH (1.6 g) was dissolved in H2O/MeOH (40 mL/8 mL), then 7d
(3.0 g, 8.6 mmol) was added. The reaction mixture was heated at
reflux for 2.5 h, cooled and adjusted to pH ¼ 3e4 with 2 N HCl. The
resultant precipitation was filtrated, washed with water and dried
to give 10 as a yellow solid (2.0 g, 72%), mp 225e228 ꢀC (dec). 1H
yl]-piperidin-1-yl-methanone (11e)
11e (90 mg, 76%), mp 180e181 ꢀC. 1H NMR (CDCl3):
d 1.68e1.70
(m, 6H), 2.48 (s, 3H), 3.57e3.59 (m, 4H), 6.14 (s, 2H), 7.21e7.31 (m,
7H), 7.35 (d, J ¼ 8.1 Hz, 1H), 7.84 (d, J ¼ 8.1 Hz, 1H). 13C NMR (CDCl3):
d
162.20, 158.47, 153.27, 144.81, 143.08, 136.90, 136.78, 128.91,
NMR (DMSO-d6):
d
2.62 (s, 3H), 6.17 (s, 2H), 7.20e7.29 (m, 7H), 7.58
127.86, 127.28, 127.04, 124.57, 123.56, 120.60, 111.01, 48.70, 26.46,
24.67, 16.57. EI-MS: m/z 416 [M]þ. HRMS (EI): Cal. for: 416.1671,
found: 416.1673.
(d, J ¼ 7.0 Hz, 1H), 7.72 (d, J ¼ 6.8 Hz, 1H). 13C NMR (DMSO-d6):
d
164.74, 155.86, 152.84, 144.84, 142.33, 137.35, 136.16, 128.56,
127.41, 127.07, 124.03, 123.80, 122.92, 119.53, 111.33, 66.76, 47.54,
16.58. EI-MS: m/z 349 [M]þ. HRMS (EI): Cal. for: 349.0883, found:
349.0885.
5.9.6. [2-(1-Benzyl-1H-benzoimidazol-2-yl)-4-methyl-thiazol-5-
yl]-morpholin-4-yl-methanone (11f)
11f (0.10 g, 83%), mp 126e130 ꢀC. 1H NMR (CDCl3):
d
2.50 (s, 3H),
3.67e3.73 (m, 8H), 6.13 (s, 2H), 7.20e7.24 (m, 1H), 7.29e7.37 (m,
5H), 7.82e7.84 (m, 1H). 13C NMR (DMSO-d6):
162.31, 158.79,
5.9. General procedures for the synthesis of compounds 11aeg
d
A mixture of 10 (100 mg, 0.29 mmol) and CDI (100 mg,
0.62 mmol) in dry THF (10 mL) was stirred at room temperature for
0.5 h. Then amine (0.58 mmol) was added and the resultant solu-
tion was stirred at room temperature overnight. After the reaction
was completed, the mixture was poured into water. The precipi-
tation was filtrated, washed with water, dried and recrystallized
from ethanol to give 11aeg as white solids.
153.75, 144.61, 143.00, 136.80, 136.74, 128.86, 127.85, 127.21, 126.31,
124.62, 123.58, 120.56, 110.99, 55.14, 48.65, 46.09, 17.14, 16.64. EI-
MS: m/z 418 [M]þ. HRMS (EI): Cal. for: 418.1463, found: 418.1464.
5.9.7. [2-(1-Benzyl-1H-benzoimidazol-2-yl)-4-methyl-thiazol-5-
yl]-(4-methyl-piperazin-1-yl)-methanone (11g)
11g (70 mg, 57%), mp 188e191 ꢀC. 1H NMR (CDCl3):
d 2.33 (s,
3H), 2.42e2.46 (m, 4H), 2.49 (s, 3H), 3.67e3.69 (m, 4H), 6.13 (s, 2H),
7.20e7.33 (m, 7H), 7.35 (d, J ¼ 7.4 Hz, 1H), 7.84 (d, J ¼ 7.4 Hz, 1H).
ESI-MS: m/z 432 [M þ 1]þ. HRMS (EI): Cal. for: 432.1867, found:
432.1866.
5.9.1. 2-(1-Benzyl-1H-benzoimidazol-2-yl)-4-methyl-thiazole-5-
carboxylic acid isopropylamide (11a)
11a (50 mg, 42%), mp 238e241 ꢀC. 1H NMR (CDCl3):
d 1.26 (d,
J ¼ 6.6 Hz, 6H), 2.71 (s, 3H), 4.21e4.25 (m, 1H), 5.59e5.61 (m, 1H),
6.14 (s, 2H), 7.12e7.24 (m, 2H), 7.25e7.28 (m, 3H), 7.30e7.32 (m,
2H), 7.36e7.38 (m, 1H), 7.81e7.84 (m, 1H). 13C NMR (DMSO-d6):
5.10. Antitumor activity assay
d
159.72, 157.31, 154.40, 143.84, 142.21, 137.08, 136.28, 128.73,
Compounds 7e11 were studied for their in vitro anticancer
activities against HCT-116 cells by MTT-based assay. Cells were
maintained as a suspension in RPMI-1640 medium supplemented
with 10% fetal calf serum at 37 ꢀC in a humidified atmosphere
containing 5% CO2. Then the cells were planted onto standard 96-
well plates at the concentration 104 cells per well and allowed to
proliferate for 24 h under the above conditions. The compounds
128.66, 127.49, 127.06, 124.36, 123.28, 119.73, 111.52, 47.57, 41.44,
22.17, 16.83. ESI-MS: m/z 391 [M þ 1]þ. HRMS (EI): Cal. for:
390.1514, found: 390.1512.
5.9.2. 2-(1-Benzyl-1H-benzoimidazol-2-yl)-4-methyl-thiazole-5-
carboxylic acid (2-diethylamino-ethyl)-amide (11b)
11b (0.10 g, 78%), mp 136e138 ꢀC. 1H NMR (CDCl3):
d
1.05 (t,
were added in six threefold dilutions (10
taxol co-assayed as a positive control. After 72 h exposure period,
then 15 L of 5 mg/mL MTT were added to each well and the plates
were incubated for 4 h at 37 ꢀC. The medium was then aspirated
mg/mL to 0.04 mg/mL) with
J ¼ 7.1 Hz, 6H), 2.59 (q, J ¼ 7.1 Hz, 4H), 2.65 (t, J ¼ 5.4 Hz, 2H), 2.73 (s,
3H), 3.47e3.49 (m, 2H), 6.14 (s, 2H), 6.84 (s, 1H), 7.19e7.31 (m, 7H),
m
7.33e7.35 (m,1H), 7.82e7.84 (m,1H). 13C NMR (DMSO-d6):
d 160.45,

422
Y. Luo et al. / European Journal of Medicinal Chemistry 46 (2011) 417e422
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and the formazan product was solubilized by 100 mL DMSO. The
absorbance was measured at 570 nm using an ELISA Reader. Each
assay was carried out at least three times, and the results of the
experiment were summarized in the Table 1.
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We gratefully acknowledge the financial support for Shanghai
Municipal Natural Science Foundation (10ZR1409600). And we also
thank Lab of Organic Functional Molecules, the Sino-French Insti-
tute of ECNU for supports.
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