Discovery and molecular modeling of novel 1-indolyl acetate - 5-Nitroimidazole targeting tubulin polymerization as antiproliferative agents

Article abstract of DOI:10.1016/j.ejmech.2014.07.082

A series of 18 novel 1-indolyl acetate-5-nitroimidazole 3a-3r were designed, synthesized, and evaluated for their in vitro biological activities as potential tubulin polymerization inhibitors. Among these compounds, 3p displayed strong antitumor activity with IC₅₀ of 2.00, 1.05, 0.87 μM against A549, Hela and U251 respectively, and also showed the most potent PLK1 inhibitory activity with IC₅₀ of 2.4 μM. Molecular docking studies within the colchicine binding site of tubulin were in good agreement with the tubulin polymerization inhibitory data and confirmed the importance of the configuration of the synthesized 1-indolyl acetate-5-nitroimidazolefor potential tubulin polymerization inhibitors.

Full text of DOI:10.1016/j.ejmech.2014.07.082

European Journal of Medicinal Chemistry 85 (2014) 341e351
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery and molecular modeling of novel 1-indolyl acetate e
5-Nitroimidazole targeting tubulin polymerization as antiproliferative
agents
Yong-Tao Duan, Ya-Li Sang, Jigar A. Makawana, Shashikant B. Teraiya, Yong-Fang Yao,
Dan-Jie Tang, Xiang-Xiang Tao, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 18 novel 1-indolyl acetate-5-nitroimidazole 3ae3r were designed, synthesized, and evaluated
for their in vitro biological activities as potential tubulin polymerization inhibitors. Among these com-
Received 14 February 2014
Received in revised form
23 July 2014
Accepted 23 July 2014
Available online 30 July 2014
pounds, 3p displayed strong antitumor activity with IC50 of 2.00, 1.05, 0.87
U251 respectively, and also showed the most potent PLK1 inhibitory activity with IC50 of 2.4
m
M against A549, Hela and
M. Mo-
m
lecular docking studies within the colchicine binding site of tubulin were in good agreement with the
tubulin polymerization inhibitory data and confirmed the importance of the configuration of the syn-
thesized 1-indolyl acetate-5-nitroimidazolefor potential tubulin polymerization inhibitors.
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Indolyl acetate
Nitroimidazole
Tubulin
Docking
1. Introduction
of CA-4 and its analogues have a lower activity than that of the
corresponding cis isomers [11]. And more, a limitation to the clin-
Antimitotic agents, one of the major classes of cytotoxic drugs
for cancer treatment, have received increasing attention recently, in
part, due to the clinical success of taxol [1e4]. Microtubule (MT)
polymerization dynamics can greatly affect a cell's ability to change
and maintain its shape and undergo critical processes such as cell
signaling and mitosis. The major protein component found in mi-
crotubules is tubulin. Interference with microtubule assembly,
either by inhibition of tubulin polymerization or by blocking
microtubule disassembly, leads to an increase in the number of cells
in metaphase arrest. Because of their crucial role in the formation of
the mitotic spindle during cell division, MTs are a highly attractive
target for the development of new effective anticancer agents
[5e9].
ical development of CA-4 is its poor water solubility. Hence, some
bioavailable cis-restricted analogues and more soluble structural
analogues have been developed, such as compounds 1 [12] and 2
[13] which inhibit MT assembly by preventing tubulin
polymerization.
It is well-known that CA-4 analogues have three important
pharmacophore components, a linking cis double bond bridge and
two hydrophobic rings(rings A and B, Fig. 1), and the appropriate
dihedral angle and cis-configuration of the two rings are important.
However, the cis double bond in CA-4 and its cis analogues easily
undergoes isomerization during storage and administration, so a
large number of modifications of CA-4 focused on the linking
bridge. Imidazole,
a five-membered hetero aromatic, as the
Over the years, a large number of natural and synthetic MT in-
hibitor shave been identified. Combretastatin A-4 (CA-4, Fig. 1) [10]
is the most active member of the combretastatins A family, isolated
from bark of the South African tree Combretum caffrum. A reported
critical structural requirement for the activity of these compounds
is the cis configuration of the double bond. In fact, the trans isomer
bioavailable cis-restricted analogue and bioisosteric replacement of
the olefinic double bond, has been developed [13e17]. And more,
some excellent work from Keira's group showed no necessity of the
three methoxyl groups for biological activity [18]. Besides, some
SAR analysis showed modification of the trimethoxybenzene with
groups of higher lipophilicity could lead to decreased cytotoxicity
and increased antitubulin activity. Some reports implied that indole
core was a very important building block for many biological active
agents including tubulin inhibitors [19e21], and the carbonyl group
near the linking bridge can improve its solubility and may be
* Corresponding author.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
http://dx.doi.org/10.1016/j.ejmech.2014.07.082
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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Y.-T. Duan et al. / European Journal of Medicinal Chemistry 85 (2014) 341e351
Fig. 1. Structure of CA-4 and common structural characteristic of CA-4.
favorable for activity because of its polar interactions with amino
acids at the tubulin colchicine site [12,22,23].
Based on the data obtained, we surveyed a variety of sub-
stituents at different positions on the phenyl ring, and found that
various substituents of such as halogen, methyl and methoxyl
groups led to distinct anticancer activities of these target com-
pounds. Compounds 3qe3r whoseIC50 value were 7.02, 10.12,12.79
Therefore, here we describe the synthesis of some novel 1-
indolyl
acetate-2-styryl-5-nitroimidazolederivatives
(3a-3r).
Several were found to be highly potent inhibitors of tubulin poly-
merization. Docking simulation was also performed using X-ray
crystallographic structure of the tubulin to explore the binding
modes of the synthetic compounds at the active site of the enzyme.
and 8.43, 13.56, 16.89 mM against A549, Hela and U251 respectively,
with a bare phenyl and naphthyl ring, presented 10-fold decrease
compared to positive control CA-4. The introduction of electron-
withdrawing groups produced compounds 3ae3i, which showed
more potent activity than compounds 3qe3r. However, a different
behavior was observed that compounds with a NO2 moiety had a
nearly 3-fold increase in inhibition of cancer growth than com-
pounds 3ae3f whose substituted group was Cl and Br. More
interesting, based on the data we find that these compounds with
different substituted groups, the potency order was
eNO2 > halogen substitutions > eCH3/eOCH3 > Ph/H. Meanwhile,
a comparison of the substitution on the benzene ring was
demonstrated as follows: when the compounds were halogen-
2. Results and discussion
2.1. Chemistry
A
series of 1-indolyl acetate-2-styryl-5-nitroimidazole de-
rivatives were synthesized by the routes outlined in Scheme 1.
Compounds 1ae1r were prepared by the reaction of metronidazole
with different substituted benzaldehyde in DMSO by adding rapidly
a stirred solution of sodium methoxide in methanol at room tem-
perature. Synthesized compounds 1ae1r were treated with4-
toluene sulfonyl chloride using triethylamine as catalyst in CH2Cl2
which led to the formation of compounds 2ae2r. The refined
compounds were finally obtained by subsequent purification with
chromatography. Then, indole-3-carboxylic acid, compounds
2ae2r, and K2CO3 were taken in DMF and refluxed to provide the
desired compounds 3ae3r.
substituted
derivatives,
the
activity
gradient
was
ortho > para > meta. Notably, compound 3p, with two chlorine
groups on the phenyl ring, showed the most potent activity than all
other compounds, nearly the same potency with positive control.
2.3. Cytotoxicity
All compounds were evaluated for their toxicity against human
macrophage with the median cytotoxic concentration (CC50) data
of tested compounds by the MTT assay. As shown in Table 1, most of
the analogues possessed low cytotoxicity.
2.2. Biological activity
2.2.1. Antiproliferative activity
The cytotoxicity of compounds 3ae3r wasevaluated against
three human tumor cell lines by CKK assay: human pulmonary
adenocarcinoma cells (A549), human Glioma Cells (U251) and hu-
man cervical cancer cell line (Hela). The cytotoxicity levels of the
target compounds are reported in Table 1.
2.4. Tubulin polymerization inhibition
After these results, we checked the abilities of the synthesized
compounds in inhibiting the polymerization of tubulin. As shown

Y.-T. Duan et al. / European Journal of Medicinal Chemistry 85 (2014) 341e351
343
Scheme 1. General method of the preparation of compounds 3ae3r. Reagents and conditions: (I) Sodium methoxide, different aldehydes, DMSO, methanol, room temperature, 4 h;
(II) 4-toluene sulfonyl chloride; CH2Cl2, TEA, room temperature, 5 h. (III) Indole-3-carboxylic acid, DMF, K2CO3, reflux, overnight.
in Table 2, IC50 against tubulin polymerization corresponded with
IC50 value against cancer cells in general. Compound 6p showed
strong inhibitory effect and its 50% tubulin polymerization inhibi-
The end group of LYS352 was formed one charge interaction with
one nitrogen atom. Meanwhile, the end amino of LYS254 formed
one
p-cation interaction with Ph ring. The docking calculation of
tion concentration of 2.4
m
M. This result suggested that the potent
all the compounds was also depicted in Table 2. The CDocker
Energy (energy of the ligandereceptor complexes) agreed with
the telomerase inhibitory trend for all the synthesized
compounds.
inhibitory effects of the synthetic compounds on the cell prolifer-
ation assay were causally related to their kinase inhibitory
activities.
2.5.2. 3D-QSAR model
2.5. Computational
In order to acquire a systematic SAR profile on18 compounds as
antitumor agents and to explore the more powerful tubulin in-
hibitors, 3D-QSAR model was built to choose activity conformation
of the designed molecular and reasonably evaluated the designed
molecules by using the corresponding pIC50 values which were
2.5.1. Molecular docking
To help understand the SARs observed at the tubulin and guide
further SAR studies, we proceeded to examine the interaction of
compound 3p with tubulin (PDB code:1SA0). The molecular
docking was performed by simulation of synthetic compounds
into the colchicine binding site in tubulin. All docking runs were
applied the Lamarckian genetic algorithm of Auto-Dock 3.5. The
binding model of compound 3pand tubulin is depicted in Fig. 2. In
the binding model, compound 3p is nicely bound to the colchicine
site of tubulin via three hydrogen bonds (angle O$$$HeN ¼ 122.9ꢀ,
converted from the obtained IC50 (mM) values of tubulin inhibition
and performed by built-in QSAR software of DS 3.5 (Discovery
Studio 3.5, Accelrys, Co. Ltd). The way of this transformation was
derived from an online calculator developed from an Indian dicinal
chemistry lab (http://www.sanjeevslab.org/tools-IC50.html). The
training and test set was divided by the random diverse molecules
method of DS 3.5, in which the test set accounted for 83% while the
training set was set to 17%. The training set composed4 agents and
14 agents were consisted of the relative test set. The success of this
model depended on docking study and the reliability of previous
study about activities data.
distance
¼
2.23 Å with amino hydrogen SER178, angle
O$$$HeO ¼ 118.1ꢀ,distance ¼ 2.35 Å with oxygen group of ALA317,
and angle O$$$HeO ¼ 117.2ꢀ,distance ¼ 2.42 Å with oxygen group
of LYS352), one charge interaction and one
p-cation interaction.
In default situation, the alignment conformation of each mole-
cule that possessed the lowest CDOCKER_INTERACTION_ENENGY
among the ten docked poses. The 3D-QSAR model generated from
DS 3.5, defined the critical regions (steric or electrostatic) affecting
the binding affinity. It was a PLS model that set up 180 independent
variables (conventional R2 ¼ 0.87). The graphical relationship of
observed and predicted values had been illustrated inFig. 3a, in
Table 1
In vitro anticancer activities (IC50
cell lines and cytotoxicity assay of the compounds on macrophages cells (CC50
,
m
M) of compounds3ae3ragainst human tumor
M).
,
m
Compounds
CC50
IC50
A549
Hela
U251
3a
3b
3c
3d
3e
3f
3g
3h
3i
132
156
104
145
164
98
109
123
200
202
154
123
77
4.24
5.02
4.51
5.11
6.03
5.55
2.12
2.86
2.01
4.02
4.32
3.88
3.11
4.09
3.97
2.00
7.02
8.43
2.79
3.10
4.21
3.77
4.12
4.81
4.80
1.24
2.01
1.79
4.43
3.77
2.28
3.23
4.01
3.88
1.05
10.12
13.56
1.44
2.98
4.08
3.23
3.09
3.87
3.89
1.11
2.81
2.03
2.79
3.02
3.11
4.09
2.76
3.07
0.87
12.79
16.89
0.54
Table 2
Tubulin inhibitory activity and docking calculation of compounds 3ae3r.
Compounds IC50
(
m
M)
D
Gb
Compounds IC50
(
m
M)
DGb
(ꢁkcal/mol)
(ꢁkcal/mol)
Tubulin
inhibition energy
CDocker
Tubulin
inhibition energy
CDocker
3j
3k
3l
3a
3b
3c
3d
3e
3f
3g
3h
3i
71.2
103.0
98.8
112.2
162.0
123.3
6.3
19.5
6.9
67.2
36.88
33.44
34.70
31.32
26.99
28.64
58.43
50.15
56.20
40.08
3k
3l
3m
3n
3o
75.3
45.2
21.4
67.7
50.3
2.4
35.45
45.55
48.33
38.56
41.78
62.11
26.1
3m
3n
3o
3p
3q
3r
89
106
287
158
105
e
3p
3q
186.5
204.1
1.8
3r
24.2
e
CA-4a
CA-4a
3j
a
a
Used as a positive control.
Used as a positive control.

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Y.-T. Duan et al. / European Journal of Medicinal Chemistry 85 (2014) 341e351
Fig. 2. Molecular docking modeling of compound 3p with tubulin: (A) 2D model of the interaction between compound 3p and the tubulin binding site. (B) 3D model of the
interaction between compound 3p and the tubulin binding site.
which the plot of the observed IC50 versus the predicted values
showed that this model could be used in prediction of activity.
A contour plot of the electrostatic field region favorable (in blue)
or unfavorable (red) for anticancer activity based on telomerase
target was shown in Fig. 3b while the energy grids corresponding to
the favorable (in green) or unfavorable (yellow) steric effects for the
telomerase affinity were shown in Fig. 3c. It was widely acceptable
that a better inhibitor based on the 3D-QSAR model should have
strong Van der Waals attraction in the green areas and a polar
group in the blue electrostatic potential areas (which were domi-
nant close to the skeleton). As shown in these two pictures, this
promising model would provide a guideline to design and optimize
more effective telomerase inhibitors based on the 1-indolyl
acetate-2-styryl-5-nitroimidazole derivatives and pave the way for
us to further study in future.
3. Conclusion
In this investigation a series of novel CA-4 analogues have been
synthesized that exhibited significant anticancer activity, and also
performed SARs study. Compound 3pshowed the most potent
inhibitory activity that inhibited the growth of cancer cells with
IC50 of 2.00, 1.05, 0.87
mM against A549, Hela and U251 respectively,
and inhibited the activity of tubulin with IC50 ¼ 2.4
m
M. Docking
simulation was performed to position compound 3p into the
tubulin active site to determine the probable binding conformation

Y.-T. Duan et al. / European Journal of Medicinal Chemistry 85 (2014) 341e351
345
apparatus (Taike Corp, Beijing, China). All the 1H NMR spectra were
recorded on a Bruker DPX 300 model Spectrometer in DMSO-d6
and chemical shifts were reported in ppm (d). ESI-MS spectra were
recorded on a Mariner System 5304 Mass spectrometer.
4.2. General method of synthesis of compounds 3ae3r
Reaction of metronidazole (12 mmol) with different substituted
benzaldehyde (16 mmol) in 6 mL DMSO by adding rapidly a stirred
solution of sodium methoxide (12.8 mmol) in methanol at room
temperature resulted in the formation of target compounds
1ae1r.To a stirred solution of compounds 1ae1r (7 mmol) in
CH2Cl2 (15 mL), 4-toluene sulfonyl chloride (7 mmol) and trie-
thylamine (10 mL) at room temperature were added, stirring for
5 h. The precipitate was purified by column chromatography using
EtOAc/hexane (1:6) as eluent to give the puried compounds 2ae2r.
Equimolar portions of the synthesized compounds 2ae2r
(5 mmol), Indole-3-carboxylic acid (5 mmol) and K2CO3 (5 mmol)
were dissolved in approximately 15 mL of DMF. The reaction so-
lution was allowed to stir overnight at room temperature, and the
precipitate was purified by column chromatography using EtOAc/
hexane (1:9) as eluent to give the puried compounds 3ae3r.
4.2.1. (Z)-2-(2-(2-chlorostyryl)-5-nitro-1H-imidazol-1-yl) ethanol
(1a)
Yield 65.6%. m.p. 79e81 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.21 (d, J ¼ 4.5 Hz, 1H, CH), 7.86e7.74 (m, 3H, ArH), 7.39e7.21 (m,
3H, ArH, CH),5.04e4.97 (m, 1H, OH), 4.24e4.21 (m, 2H, CH2),
3.70e3.61 (m, 2H, CH2); 13C NMR
d: 48.03, 60.70, 116.54, 123.85,
125.32, 128.73, 129.70, 131.90, 135.07, 139.21, 150.67, 159.21, 161.64;
ESI-MS: 293.72 [MþH]þ. Anal. Calcd for C13H12ClN3O3: C, H, N.
4.2.2. (Z)-2-(2-(3-chlorostyryl)-5-nitro-1H-imidazol-1-yl) ethanol
(1b)
Yield 62.8%. m.p. 80e82 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d: 8.22
(s, 1H, CH), 8.08 (s, 1H, ArH), 7.76e7.71 (m, 2H, ArH), 7.57e7.36 (m,
3H, ArH, CH), 4.99 (t, J ¼ 4.0 Hz,1H, OH), 4.65 (t, J ¼ 5.7 Hz, 2H, CH2),
3.73e3.68 (m, 2H, CH2); 13C NMR
d: 48.03, 60.36, 116.32, 123.22,
125.09, 128.45, 129.04, 131.65, 135.43, 139.18,150.80, 159.90, 160.21;
ESI-MS: 293.81 [MþH]þ. Anal. Calcd for C13H12ClN3O3: C, H, N.
4.2.3. (Z)-2-(2-(4-chlorostyryl)-5-nitro-1H-imidazol-1-yl) ethanol
(1c)
Yield 74.6.0%; m.p. 71e72 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Fig. 3. (A) Using linear fitting curve to compare the predicted pIC50 value with that of
experiment. (B) Isosurface of the 3D-QSAR model coefficients on electrostatic potential
grids. The blue triangle mesh represents positive electrostatic potential and the red
area represents negative electrostatic potential. (C) Isosurface of the 3D-QSAR model
coefficients on Van der Waals grids. The green triangle mesh representation indicates
positive coefficients; the yellow triangle mesh indicates negative coefficients. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
8.22 (d, J ¼ 6.8 Hz, 1H, CH), 7.97e7.78 (m, 3H, ArH), 7.54e7.39 (m,
3H, ArH, CH), 5.01 (t, J ¼ 4.5 Hz,1H, OH), 4.70 (t, J ¼ 6.0 Hz, 2H, CH2),
0.72e3.65 (m, 2H, CH2); 13C NMR
d: 48.05, 60.59, 116.21, 123.08,
125.00,128.11,131.22,135.15,139.80,150.73,159.20; ESI-MS: 293.56
[MþH]þ. Anal. Calcd for C13H12ClN3O3: C, H, N.
4.2.4. (Z)-2-(2-(2-bromostyryl)-5-nitro-1H-imidazol-1-yl) ethanol
and the result indicated that compound 3p was a potent inhibitor of
tubulin. Finally, QSAR models were built with previous activity data
and binding conformations to begin our work in this paper as well
as to provide a reliable tool for reasonable design of telomerase
inhibitors in the future.
(1d)
Yield 68.6%. m.p. 79e82 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.22 (d, J ¼ 4.7 Hz, 1H, CH), 7.86e7.75 (m, 3H, ArH), 7.39e7.25 (m,
3H, ArH, CH),5.04e4.97 (m, 1H, OH), 4.64e4.49 (m, 2H, CH2),
3.73e3.68 (m, 2H, CH2); 13C NMR
d: 48.03, 60.46, 117.27, 128.00,
129.22, 130.44, 131.41, 132.87, 134.09, 135.56, 136.53, 139.53, 150.67;
4. Experiments
ESI-MS: 338.01 [MþH]þ. Anal. Calcd for C13H12BrN3O3: C, H, N.
4.1. Chemistry
4.2.5. (Z)-2-(2-(3-bromostyryl)-5-nitro-1H-imidazol-1-yl) ethanol
(1e)
All chemicals and reagents used in the current study were of
analytical grade. The reactions were monitored by thin layer
chromatography (TLC) on Merck precoated silica GF254 plates.
Melting points (uncorrected) were determined on a XT4 MP
Yield 64.8%. m.p. 81e83 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.22 (s, 1H, CH), 8.08 (s, 1H, ArH), 7.76e7.71 (m, 2H, ArH),
7.57e7.36 (m, 3H, ArH, CH), 4.99 (t, J ¼ 4.0 Hz, 1H, OH), 4.65 (t,
J ¼ 5.7 Hz, 2H, CH2), 3.73e3.68 (m, 2H, CH2); 13C NMR
d: 48.03,

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Y.-T. Duan et al. / European Journal of Medicinal Chemistry 85 (2014) 341e351
60.55, 116.20, 128.05, 129.31, 130.40, 131.89, 132.31, 134.90, 135.08,
CH2), 2.61 (s, 3H, CH3); 13C NMR
d: 21.65, 48.03, 60.49, 116.15,
136.02, 139.78, 150.39; ESI-MS: 338.01 [MþH]þ. Anal. Calcd for
123.21, 128.90, 129.00, 135.13, 139.09, 150.44, 159.52, 161.63; ESI-
C
13H12BrN3O3: C, H, N.
MS: 273.32 [MþH]þ. Anal. Calcd for C14H15N3O3: C, H, N.
4.2.6. (Z)-2-(2-(4-bromostyryl)-5-nitro-1H-imidazol-1-yl) ethanol
(1f)
4.2.13. (Z)-2-(2-(2-methoxystyryl)-5-nitro-1H-imidazol-1-yl)
ethanol (1m)
Yield 77.0%; m.p. 76e79 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 68.1%; m.p. 80e82 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.22 (d, J ¼ 6.8 Hz, 1H, CH), 7.97e7.78 (m, 3H, ArH), 7.54e7.39 (m,
8.15 (s, 1H, CH), 7.81e7.43 (m, 4H, ArH), 6.93 (d, J ¼ 6.4 Hz, 2H, CH),
3H, ArH, CH), 5.01 (t, J ¼ 4.5 Hz, 1H, OH), 4.70 (t, J ¼ 6.0 Hz, 2H, CH2),
5.01 (s, 1H, OH), 4.53 (t, J ¼ 5.6 Hz, 2H, CH2), 3.79 (m, 2H, CH2), 3.18
0.72e3.65 (m, 2H, CH2); 13C NMR
d: 48.03, 60.46, 117.55, 128.74,
(s, 3H, CH3); 13C NMR
d: 40.19, 48.04, 60.72, 116.43, 123.87, 125.14,
129.20, 132.89, 134.00, 135.64, 136.51, 139.90, 150.10; ESI-MS:
128.32, 129.10, 131.29, 135.42, 139.50, 150.46, 159.03, 161.21; ESI-
338.11 [MþH]þ. Anal. Calcd for C13H12BrN3O3: C, H, N.
MS: 290.11 [MþH]þ. Anal. Calcd for C14H15N3O4: C, H, N.
4.2.7. Z)-2-(5-nitro-2-(2-nitrostyryl)-1H-imidazol-1-yl) ethanol
4.2.14. (Z)-2-(2-(3-methoxystyryl)-5-nitro-1H-imidazol-1-yl)
(1g)
ethanol (1n)
Yield 72.1%. m.p. 77e80 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 73.2%; m.p. 79e82 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.25 (d, J ¼ 6.7 Hz, 1H, CH), 8.03e7.94 (m, 3H, ArH, CH), 7.83e7.58
8.22 (s, 1H, CH), 7.76e7.65 (m, 1H, CH), 7.60e7.44 (m, 4H, ArH), 6.83
(d, J ¼ 6.0 Hz, 1H, CH), 5.02 (s, 1H, OH), 4.62e4.50 (m, 2H, CH2), 3.71
(m, 3H, ArH, CH), 4.99 (t, J ¼ 6.3 Hz, 1H, OH), 4.54 (t, J ¼ 5.4 Hz, 2H,
CH2), 3.74e3.52 (m, 2H, CH2); 13C NMR
d
: 48.09, 60.12, 115.27,
(t, J ¼ 6.2 Hz, 2H, CH2), 3.14 (s, 3H, CH3); 13C NMR
d: 40.34, 48.03,
126.18, 128.02, 130.44, 132.19, 132.93, 134.17, 135.30, 137.53, 140.29,
60.70, 115.40, 123.21, 126.04, 128.10, 129.22, 131.90, 135.29, 139.42,
153.60; ESI-MS: 305.08 [MþH]þ. Anal. Calcd for C13H12N4O5: C, H,
150.61, 159.50, 160.09; ESI-MS: 290.11 [MþH]þ. Anal. Calcd for
N.
C14H15N3O4: C, H, N.
4.2.8. (Z)-2-(5-nitro-2-(3-nitrostyryl)-1H-imidazol-1-yl) ethanol
4.2.15. (Z)-2-(2-(4-methoxystyryl)-5-nitro-1H-imidazol-1-yl)
ethanol (1o)
(1h)
Yield 74.1%. m.p. 81e82 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 65.3%; m.p. 82e85 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.31e7.73 (m, 3H, ArH, CH), 7.70e7.54 (m, 2H, ArH), 7.35e7.16 (m,
2H, CH), 4.85 (t, J ¼ 7.3 Hz, 1H, OH), 4.53 (t, J ¼ 6.4 Hz, 2H, CH2), 3.65
7.94 (s, 1H, CH),7.81e7.62 (m, 4H, ArH), 7.44 (d, J ¼ 7.9 Hz, 2H, CH),
4.86 (s, 1H, OH), 4.35 (t, J ¼ 6.4 Hz, 2H, CH2), 3.63 (t, J ¼ 7.2 Hz, 2H,
(m, 2H, CH2); 13C NMR
d
: 48.09, 60.12, 115.09, 125.79, 128.71, 131.04,
CH2), 3.22 (s, 3H, CH3); 13C NMR
d: 40.08, 48.08, 60.70, 116.32,
132.90, 133.13, 134.41, 135.00, 137.42, 140.10,153.21; ESI-MS: 305.08
125.90, 128.07, 129.18, 135.32, 139.43, 150.55, 159.60, 161.48; ESI-
[MþH]þ. Anal. Calcd for C13H12N4O5: C, H, N.
MS: 290.11 [MþH]þ. Anal. Calcd for C14H15N3O4: C, H, N.
4.2.9. (Z)-2-(5-nitro-2-(4-nitrostyryl)-1H-imidazol-1-yl) ethanol
4.2.16. (E)-2-(2-(2,4-dichlorostyryl)-5-nitro-1H-imidazol-1-yl)
(1i)
ethanol (1p)
Yield 73.1%. m.p.79 ~ 81 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 65.6%. m.p. 74e76 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d: 8.20
8.29e8.20 (m, 4H, ArH, CH), 8.05e7.87 (m, 2H, ArH), 7.61 (d,
J ¼ 13.8 Hz, 1H, CH), 5.01 (t, J ¼ 5.6 Hz, 1H, OH), 4.8 (t, J ¼ 6.0 Hz, 2H,
(d, J ¼ 4.4 Hz, 1H, CH), 7.85e7.74 (m, 2H, ArH), 7.34e7.20 (m, 3H,
ArH, CH), 5.02e4.97 (m, 1H, OH), 4.22e4.20 (m, 2H, CH2), 3.71e3.64
CH2), 3.75e3.70 (m, 2H, CH2); 13C NMR
d
: 48.09, 60.12, 115.42,
(m, 2H, CH2); 13C NMR
d: 48.03, 60.70, 117.02, 124.18, 125.75, 128.30,
127.10,128.99,130.12,132.70,136.30,137.52,142.10,150.20; ESI-MS:
129.19, 134.11, 135.20, 139.69, 150.46, 159.55, 161.34; ESI-MS: 328.12
305.08 [MþH]þ. Anal. Calcd for C13H12N4O4: C, H, N.
[MþH]þ. Anal. Calcd for C13H11Cl2N3O3: C, H, N.
4.2.10. (Z)-2-(2-(2-methylstyryl)-5-nitro-1H-imidazol-1-yl)
4.2.17. (Z)-2-(2-(2-(naphthalen-2-yl)vinyl)-5-nitro-1H-imidazol-
ethanol (1j)
1-yl) ethanol (1q)
Yield 78.2%; m.p. 80e81 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 71.4%. m.p. 86e89 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.17 (s, 1H, CH), 7.81e7.42 (m, 4H, ArH), 6.90 (d, J ¼ 6.4 Hz, 2H, CH),
8.05e7.92 (m, 3H, ArH, CH), 7.89e7.64 (m, 2H, ArH), 7.61 (t,
J ¼ 6.5 Hz, 2H, ArH), 7.43 (t, J ¼ 7.3 Hz, 1H, ArH), 7.09e6.87 (m, 2H,
CH), 5.02 (t, J ¼ 7.6 Hz, 1H, OH), 4.41 (t, J ¼ 6.0 Hz, 2H, CH2),
5.03 (s, 1H, OH), 4.51 (t, J ¼ 5.6 Hz, 2H, CH2), 3.73 (m, 2H, CH2), 2.68
(s, 3H, CH3); 13C NMR
d: 21.70, 48.03, 60.70, 116.04, 123.29, 125.02,
128.43,129.71,131.00,135.20,139.12,150.58,159.10,161.32; ESI-MS:
3.70e3.58 (m, 2H, CH2); 13C NMR
d: 48.03, 60.70, 115.19, 126.30,
273.27 [MþH]þ. Anal. Calcd for C14H15N3O3: C, H, N.
127.12, 130.19, 131.56, 134.29, 136.98, 141.45, 153.16, 159.76, 163.28;
ESI-MS: 310.11 [MþH]þ. Anal. Calcd for C17H15N3O3: C, H, N.
4.2.11. (Z)-2-(2-(3-methylstyryl)-5-nitro-1H-imidazol-1-yl)
ethanol (1k)
4.2.18. (Z)-2-(5-nitro-2-styryl-1H-imidazol-1-yl) ethanol (1r)
Yield 72.3%; m.p. 79e80 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 83.1%; m.p. 75e78 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.22 (s, 1H, CH), 7.76e7.65 (m, 1H, CH), 7.62e7.41 (m, 4H, ArH), 6.82
(d, J ¼ 6.1 Hz, 1H, CH), 5.12 (s, 1H, OH), 4.62e4.51 (m, 2H, CH2), 3.71
8.22 (s, 1H, CH), 7.79e7.61 (m, 2H, ArH), 7.52 (d, J ¼ 7.6 Hz, 1H, CH),
7.51e7.40 (m, 3H, ArH), 7.30e7.19 (m, 1H, CH), 4.96 (s, 1H, OH), 4.61
(t, J ¼ 6.3 Hz, 2H, CH2), 2.64 (s, 3H, CH3); 13C NMR
d: 21.32, 48.09,
(t, J ¼ 5.1 Hz, 2H, CH2), 3.70 (m, 2H, CH2); 13C NMR
d: 48.03, 60.49,
60.70, 116.20, 123.40, 125.14, 128.78, 129.24, 131.90, 135.13, 139.64,
116.42, 123.98, 128.09, 129.12, 135.54, 139.72, 150.87, 159.46, 161.59;
150.08, 159.13, 161.51; ESI-MS: 273.24 [MþH]þ. Anal. Calcd for
ESI-MS: 260.10 [MþH]þ. Anal. Calcd for C13H13N3O3: C, H, N.
C
14H15N3O3: C, H, N.
4.2.19. (Z)-2-(2-(2-chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl-4-
4.2.12. (Z)-2-(2-(4-methylstyryl)-5-nitro-1H-imidazol-1-yl)
ethanol (1l)
methylbenzenesulfonate (2a)
Yield 77.2%. m.p. 251e253 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 75.1%; m.p. 84e86 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.15 (d, J ¼ 15.0 Hz,1H, CH), 7.92 (s,1H, ArH), 7.71e7.63 (m,1H, ArH),
7.62e7.51 (m, 3H, ArH), 7.32 (t, J ¼ 7.2 Hz, 1H, ArH), 7.21e7.14 (m,
3H, ArH, CH), 6.92 (d, J ¼ 7.7 Hz, 1H, CH), 4.70 (t, J ¼ 9.1 Hz, 2H, CH2),
7.92 (s, 1H, CH),7.77e7.52 (m, 4H, ArH), 7.41 (d, J ¼ 7.8 Hz, 2H, CH),
4.86 (s, 1H, OH), 4.35 (t, J ¼ 6.3 Hz, 2H, CH2), 3.60 (t, J ¼ 7.1 Hz, 2H,

Y.-T. Duan et al. / European Journal of Medicinal Chemistry 85 (2014) 341e351
347
4.43 (t, J ¼ 9.8 Hz, 2H, CH2), 2.35 (s, 3H, CH3); 13C NMR
d
: 21.67,
ArH, CH), 7.14 (d, J ¼ 9.0 Hz, 2H, ArH, CH), 4.73 (t, J ¼ 7.8 Hz, 2H,
44.48, 62.14, 114.31, 116.80, 120.15, 122.37, 125.30, 126.10, 129.21,
132.90, 134.12, 135.20, 135.54, 136.39, 137.13, 139.68, 151.20; ESI-
MS: 447.81 [MþH]þ. Anal. Calcd for C20H18ClN3O5S: C, H, N.
CH2), 4.42 (t, J ¼ 9.3 Hz, 2H, CH2), 2.32 (s, 3H, CH3); 13C NMR
d:
21.09, 40.05, 62.29, 114.53, 116.17, 120.29, 122.19, 125.44, 126.57,
129.18, 132.39, 134.63, 135.25, 135.89, 136.30, 137.35, 139.88, 151.71;
ESI-MS: 459.09 [MþH]þ. Anal. Calcd for C20H18N4O7S: C, H, N.
4.2.20. (Z)-2-(2-(3-chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl-4-
methylbenzenesulfonate (2b)
4.2.26. (Z)-2-(5-nitro-2-(3-nitrostyryl)-1H-imidazol-1-yl)ethyl-4-
Yield 65.3%. m.p. 265e267 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
methylbenzenesulfonate (2h)
8.11 (s, 1H, CH), 8.03 (s, 1H, ArH), 7.76e7.65 (m, 2H, ArH), 7.62e7.55
(m, 2H, ArH), 7.51e7.44 (m, 3H, ArH) 7.32 (t, J ¼ 7.3 Hz, 1H, CH),
7.20e7.14 (m, 1H, CH), 4.71 (t, J ¼ 8.4 Hz, 2H, CH2), 4.41 (t, J ¼ 9.2 Hz,
Yield 66.5%. m.p. 261e263 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.53 (s, 1H, CH), 8.38 (d, J ¼ 15.0 Hz, 2H, ArH), 8.21e8.09 (m, 4H,
ArH) 7.92e7.81 (m, 4H, ArH, CH), 4.79 (t, J ¼ 9.0 Hz, 2H, CH2), 4.46 (t,
2H, CH2), 2.34 (s, 3H, CH3); 13C NMR
d: 21.14, 44.31, 62.47, 114.20,
J ¼ 8.4 Hz, 2H, CH2), 2.37 (s, 3H, CH3); 13C NMR
d: 21.30, 40.06, 62.27,
116.07, 120.15, 122.90, 125.16, 126.00, 129.29, 132.64, 134.23, 135.57,
135.79, 136.61, 137.08, 139.22, 151.52; ESI-MS: 447.89 [MþH]þ. Anal.
Calcd for C20H18ClN3O5S: C, H, N.
114.56, 116.48, 120.99, 122.07, 125.21, 126.48, 129.59, 132.73, 134.03,
135.07, 135.25, 136.20, 137.53, 139.90, 151.38; ESI-MS: 459.01
[MþH]þ. Anal. Calcd for C20H18N4O7S: C, H, N.
4.2.21. (Z)-2-(2-(4-chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl-4-
4.2.27. (Z)-2-(5-nitro-2-(4-nitrostyryl)-1H-imidazol-1-yl)ethyl-4-
methylbenzenesulfonate (2c)
methylbenzenesulfonate (2i)
Yield 62.5%. m.p. 252e254 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 67.5%. m.p. 255e257 ꢀC; 1H NMR (DMSO-d6,300 MHz)
d:
7.86 (s, 1H, CH), 7.74 (d, J ¼ 6.3 Hz, 1H, ArH), 7.52e7.48 (m, 4H, ArH),
7.31 (t, J ¼ 12.8 Hz, 2H, ArH), 7.17 (d, J ¼ 7.6 Hz, 2H, ArH, CH), 6.83 (d,
J ¼ 12.4 Hz, 1H, CH), 4.75 (t, J ¼ 9.7 Hz, 2H, CH2), 4.41 (t, J ¼ 9.2 Hz,
8.17 (s, 1H, CH), 8.03 (d, J ¼ 6.3 Hz, 2H, ArH), 7.82 (d, J ¼ 9.0 Hz, 1H,
ArH), 7.57e7.47 (m, 4H, ArH), 7.31 (d, J ¼ 6.0 Hz, 2H, ArH, CH), 7.11
(d, J ¼ 6.0 Hz, 1H, CH), 4.94 (t, J ¼ 2.7 Hz, 2H, CH2), 4.43 (t, J ¼ 3.0 Hz,
2H, CH2), 2.34 (s, 3H, CH3); 13C NMR
d
: 21.39, 44.46, 62.70, 114.29,
2H, CH2), 2.24 (s, 3H, CH3); 13C NMR
d: 21.20, 40.03, 62.39, 114.30,
116.00, 120.24, 122.29, 125.13, 126.67, 129.89, 132.47, 134.30, 135.80,
116.45, 120.29, 122.80, 125.64, 129.20, 132.14, 134.55, 135.78, 136.27,
137.27, 139.39, 151.76; ESI-MS: 447.81 [MþH]þ. Anal. Calcd for
137.30, 139.21, 151.20; ESI-MS: 459.08 [MþH]þ. Anal. Calcd for
C
20H18ClN3O5S: C, H, N.
C20H18N4O7S: C, H, N.
4.2.22. (Z)-2-(2-(2-bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl-
4-methylbenzenesulfonate (2d)
4.2.28. (Z)-2-(2-(2-methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl-
4-methylbenzenesulfonate (2j)
Yield 67.4%. m.p. 253e256 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 64.4%. m.p. 221e222 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.17 (d, J ¼ 15.0 Hz, 1H, CH), 7.96 (s, 1H, ArH), 7.72e7.65 (m, 1H,
ArH), 7.60e7.54 (m, 3H, ArH), 7.32 (t, J ¼ 7.5 Hz, 1H, ArH), 7.21e7.17
(m, 3H, ArH, CH), 6.93 (d, J ¼ 7.9 Hz, 1H, CH), 4.71 (t, J ¼ 9.3 Hz, 2H,
7.91 (s, 1H, CH), 7.62 (d, J ¼ 9.2 Hz, 2H, ArH), 7.46e7.41 (m, 2H, ArH),
7.31 (d, J ¼ 8.3 Hz, 2H, ArH), 7.27e7.24 (m, 1H, ArH), 7.04 (d,
J ¼ 7.2 Hz,1H, CH), 6.91 (t, J ¼ 7.6 Hz, 2H, ArH, CH), 4.52 (t, J ¼ 5.1 Hz,
CH2), 4.43 (t, J ¼ 9.9 Hz, 2H, CH2), 2.35 (s, 3H, CH3); 13C NMR
d:
2H, CH2), 4.13 (m, 2H, CH2), 2.39 (s, 6H, CH3); 13C NMR
d: 19.32,
21.50, 44.40, 62.23, 114.17, 116.35, 120.67, 122.08, 125.28, 126.57,
129.69, 132.21, 134.54, 135.59, 135.20, 136.12, 137.35, 139.20, 151.38;
ESI-MS: 494.01 [MþH]þ. Anal. Calcd for C20H18BrN3O5S: C, H, N.
21.29, 40.05, 62.34, 115.06, 116.29, 120.89, 122.71, 125.75, 126.35,
129.49, 132.15, 134.38, 135.10, 135.50, 136.29, 137.33, 139.40, 151.12;
ESI-MS: 427.42 [MþH]þ. Anal. Calcd for C21H21N3O5S: C, H, N.
4.2.23. (Z)-2-(2-(3-bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl-
4.2.29. (Z)-2-(2-(3-methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl-
4-methylbenzenesulfonate (2e)
4-methylbenzenesulfonate(2k)
Yield 66.3%. m.p. 254e257 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 74.4%. m.p. 254e255 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.12 (s, 1H, CH), 8.01 (s, 1H, ArH), 7.76e7.67 (m, 2H, ArH), 7.61e7.56
(m, 2H, ArH), 7.55e7.34 (m, 3H, ArH) 7.32 (t, J ¼ 7.5 Hz, 1H, CH),
7.21e7.17 (m, 1H, CH), 4.75 (t, J ¼ 8.3 Hz, 2H, CH2), 4.47 (t, J ¼ 9.1 Hz,
8.11 (s, 1H, CH), 7.72e7.61 (m, 3H, ArH), 7.52e7.41 (m, 4H, ArH), 7.22
(t, J ¼ 7.1 Hz, 2H, ArH, CH), 6.82 (d, J ¼ 6.1 Hz, 1H, CH), 4.62e4.51 (m,
2H, CH2), 3.71 (t, J ¼ 6.3 Hz, 2H, CH2), 2.36 (s, 3H, CH3), 2.14 (s, 3H,
2H, CH2), 2.39 (s, 3H, CH3); 13C NMR
d
: 21.39, 44.55, 62.59, 114.49,
CH3); 13C NMR
d: 19.25, 21.43, 40.05, 62.34, 115.78, 116.60, 120.39,
116.04, 120.29, 122.61, 125.43, 126.09, 129.21, 132.34, 134.78, 135.90,
135.10, 136.24, 137.65, 139.27, 151.19; ESI-MS: 494.03 [MþH]þ. Anal.
Calcd for C20H18BrN3O5S: C, H, N.
122.35, 125.20, 126.31, 129.48, 132.67, 134.15, 135.90, 136.08, 136.15,
137.53, 139.40, 151.31; ESI-MS: 427.47 [MþH]þ. Anal. Calcd for
C21H21N3O5S: C, H, N.
4.2.24. (Z)-2-(2-(4-bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl-
4.2.30. (Z)-2-(2-(4-methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl-
4-methylbenzenesulfonate (2l)
4-methylbenzenesulfonate (2f)
Yield 67.5%. m.p. 261e264 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 75.1%. m.p. 246e247 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
7.96 (s, 1H, CH), 7.72 (d, J ¼ 6.6 Hz, 1H, ArH), 7.58e7.48 (m, 4H, ArH),
7.42 (t, J ¼ 13.8 Hz, 2H, ArH), 7.19 (d, J ¼ 7.8 Hz, 2H, ArH, CH), 6.99 (d,
J ¼ 12.6 Hz, 1H, CH), 4.72 (t, J ¼ 9.9 Hz, 2H, CH2), 4.43 (t, J ¼ 9.3 Hz,
7.83 (s, 1H, CH), 7.80e7.67 (m, 4H, ArH), 7.64e7.51 (m, 4H, ArH, CH),
7.45 (d, J ¼ 7.7 Hz, 2H, ArH, CH), 4.32 (t, J ¼ 6.1 Hz, 2H, CH2), 3.73 (t,
J ¼ 7.2 Hz, 2H, CH2), 2.54 (s, 3H, CH3), 2.22 (s, 3H, CH3); 13C NMR
d:
2H, CH2), 2.36 (s, 3H, CH3); 13C NMR
d
: 21.17, 44.31, 62.90, 114.34,
19.25, 21.30, 40.20, 62.17, 115.55, 116.20, 120.38, 125.21, 126.60,
129.27,132.35,135.49,136.31,136.50,137.31,139.44,151.60; ESI-MS:
427.42 [MþH]þ. Anal. Calcd for C21H21N3O5S: C, H, N.
116.20, 120.31, 122.67, 125.43, 126.39, 129.22, 132.21, 134.15, 135.20,
137.48, 139.63, 151.30; ESI-MS: 494.05 [MþH]þ. Anal. Calcd for
C
20H18BrN3O5S: C, H, N.
4.2.31. (Z)-2-(2-(2-methoxystyryl)-5-nitro-1H-imidazol-1-yl)ethyl-
4.2.25. (Z)-2-(5-nitro-2-(2-nitrostyryl)-1H-imidazol-1-yl)ethyl-4-
methylbenzenesulfonate (2g)
4-methylbenzenesulfonate (2m)
Yield 79.4%. m.p. 258e261 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 64.1%. m.p. 261e263 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
7.94 (s, 1H, CH), 7.54 (d, J ¼ 9.0 Hz, 2H, ArH), 7.46e7.42 (m, 2H, ArH),
7.33 (d, J ¼ 8.1 Hz, 2H, ArH), 7.25e7.21 (m, 1H, ArH), 7.08 (d,
J ¼ 7.5 Hz,1H, CH), 6.94 (t, J ¼ 7.6 Hz, 2H, ArH, CH), 4.57 (t, J ¼ 5.6 Hz,
8.28 (d, J ¼ 6.3 Hz, 1H, CH), 8.03 (d, J ¼ 8.4 Hz, 1H, ArH), 7.94 (d,
J ¼ 6.9 Hz, 2H, ArH), 7.73 (d, J ¼ 7.8 Hz, 2H, ArH), 7.59e7.52 (m, 3H,

348
Y.-T. Duan et al. / European Journal of Medicinal Chemistry 85 (2014) 341e351
2H, CH2), 4.17 (m, 2H, CH2), 3.76 (s, 3H, CH3), 2.39 (s, 3H, CH3); 13
NMR : 21.43, 38.56, 42.28, 62.09, 115.22, 116.31, 120.40, 122.35,
C
(t, J ¼ 4.4 Hz, 2H); 13C NMR
d: 44.32, 61.18, 106.65, 112.31, 114.81,
d
120.45, 121.78, 122.05, 125.47, 126.21, 129.59, 132.88, 134.32, 135.20,
135.54, 136.70, 137.37, 139.28, 151.30, 164.56; ESI-MS: 437.09
[MþH]þ. Anal. Calcd for C22H17ClN4O4: C, H, N.
125.68, 126.09, 129.43, 132.29, 134.11, 135.38, 136.55, 136.68, 137.20,
139.19, 151.65; ESI-MS: 444.12 [MþH]þ. Anal. Calcd for
C
21H21N3O6S: C, H, N.
4.2.38. (E)-2-(2-(3-chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
4.2.32. (Z)-2-(2-(3-methoxystyryl)-5-nitro-1H-imidazol-1-yl)
ethyl-4-methylbenzenesulfonate (2n)
1H-indole-3-carboxylate (3b)
Yield 66.5%. m.p. 188e190 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 72.4%. m.p. 253e255 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
11.68 (s, 1H), 8.26 (t, J ¼ 7.9 Hz 2H), 8.06 (d, J ¼ 8.4 Hz, 2H), 7.76 (t,
8.12 (s,1H, CH), 7.76e7.65 (m, 3H, ArH), 7.58e7.44 (m, 4H, ArH), 7.12
(t, J ¼ 7.5 Hz, 2H, ArH, CH), 6.83 (d, J ¼ 6.0 Hz,1H, CH), 4.62e4.54 (m,
2H, CH2), 3.81 (t, J ¼ 6.2 Hz, 2H, CH2), 3.56 (s, 3H, CH3), 2.14 (s, 3H,
J ¼ 8.4 Hz, 4H), 7.29 (t, J ¼ 8.5 Hz, 1H), 7.19e7.00 (m, 3H), 5.03 (t,
J ¼ 4.3 Hz, 2H), 4.65 (t, J ¼ 4.4 Hz, 2H); 13C NMR
d: 44.19, 61.15,
106.20, 112.36, 114.89, 120.08, 121.14, 122.21, 125.47, 126.10, 129.31,
132.90, 134.12, 135.20, 135.99, 136.23, 137.05, 139.26, 151.18, 164.74;
ESI-MS: 437.09 [MþH]þ. Anal. Calcd for C22H17ClN4O4: C, H, N.
CH3); 13C NMR
d: 21.15, 38.45, 42.09, 62.35, 115.19, 116.57, 120.90,
122.61, 125.08, 126.37, 129.15, 132.42, 134.58, 135.60, 136.20, 136.31,
137.34, 139.50, 151.12; ESI-MS: 444.12 [MþH]þ. Anal. Calcd for
C
21H21N3O6S: C, H, N.
4.2.39. (E)-2-(2-(4-chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
1H-indole-3-carboxylate (3c)
4.2.33. (Z)-2-(2-(4-methoxystyryl)-5-nitro-1H-imidazol-1-yl)
ethyl-4-methylbenzenesulfonate (2o)
Yield 67.5%. m.p. 188e189 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
11.77 (s, 1H), 8.22 (s, 1H), 7.89 (t, J ¼ 10.1 Hz, 2H), 7.77 (d, J ¼ 2.7 Hz,
Yield 65.4%. m.p. 256e259 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
1H), 7.69e7.59 (m, 3H), 7.44e7.31 (m, 3H), 7.14e7.02 (m, 2H), 4.99
7.99 (s, 1H, CH), 7.85e7.67 (m, 4H, ArH), 7.61e7.52 (m, 4H, ArH, CH),
7.46 (d, J ¼ 7.9 Hz, 2H, ArH, CH), 4.36 (t, J ¼ 6.4 Hz, 2H, CH2), 3.73 (t,
(t, J ¼ 4.4 Hz, 2H), 4.62 (t, J ¼ 4.3 Hz, 2H); 13C NMR
d: 44.18, 61.15,
106.37, 112.10, 114.02, 120.10, 122.57, 125.31, 126.40, 129.57, 132.69,
134.21, 135.09, 136.12, 137.38, 139.29, 151.90, 164.18; ESI-MS: 437.09
[MþH]þ. Anal. Calcd for C22H17ClN4O4: C, H, N.
J ¼ 7.5 Hz, 2H, CH2), 3.54 (s, 3H, CH3), 2.22 (s, 3H, CH3); 13C NMR
d:
21.43, 38.60, 42.07, 62.20, 115.29, 116.38, 120.40, 125.60, 126.14,
129.39, 132.55, 134.49, 135.30, 136.22, 137.30, 139.44, 151.61; ESI-
MS: 444.12 [MþH]þ. Anal. Calcd for C21H21N3O6S: C, H, N.
4.2.40. (Z)-2-(2-(2-bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
1H-indole-3-carboxylate (3d)
4.2.34. (Z)-2-(2-(2,4-dichlorostyryl)-5-nitro-1H-imidazol-1-yl)
Yield 68.5%. m.p. 188e190 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
ethyl-4-methylbenzenesulfonate (2p)
11.79 (s, 1H), 8.24 (d, J ¼ 3.3 Hz, 1H), 7.87 (d, J ¼ 7.6 Hz, 2H), 7.77 (d,
J ¼ 2.5 Hz, 2H), 7.70e7.59 (m, 3H), 7.43 (d, J ¼ 7.9 Hz, 1H), 7.28 (t,
J ¼ 8.4, 1H), 7.22e7.15 (m, 2H), 5.04 (t, J ¼ 4.4 Hz, 2H), 4.66 (t,
Yield 72.1%. m.p. 261e263 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.12 (s, 1H, CH), 8.05 (s, 1H, ArH), 7.63e7.51 (m, 3H, ArH), 7.51e7.41
(m, 3H, ArH) 7.21 (t, J ¼ 7.2 Hz, 1H, CH), 7.24e7.13 (m, 1H, CH), 4.70
(t, J ¼ 8.1 Hz, 2H, CH2), 4.45 (t, J ¼ 9.3 Hz, 2H, CH2), 2.32 (s, 3H, CH3);
J ¼ 4.4 Hz, 2H); 13C NMR
d: 44.56, 62.37, 106.12, 112.54, 114.61,
120.78, 121.07, 122.31, 125.00, 126.59, 129.10, 132.21, 134.49, 135.26,
135.31, 136.08, 137.15, 139.26, 151.17, 164.35; ESI-MS: 481.04
[MþH]þ. Anal. Calcd for C22H17BrN4O4: C, H, N.
13C NMR
d: 21.40, 44.29, 62.80, 114.18, 116.09, 120.15, 122.76, 125.49,
126.60, 129.31, 132.14, 134.79, 135.36, 136.08, 137.55, 139.04, 143.18,
151.57; ESI-MS: 482.34 [MþH]þ. Anal. Calcd for C20H17Cl2N3O5S: C,
H, N.
4.2.41. (Z)-2-(2-(3-bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
1H-indole-3-carboxylate (3e)
4.2.35. (Z)-2-(2-(2-(naphthalen-2-yl)vinyl)-5-nitro-1H-imidazol-
Yield 67.5%. m.p. 188e190 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
1-yl)ethyl-4-methylbenzenes-ulfonate (2q)
11.80 (s, 1H), 8.22 (d, J ¼ 6.3, 1H), 7.95 (s, 1H), 7.87 (d, J ¼ 7.6 Hz, 1H),
7.77 (d, J ¼ 2.1, 1H), 7.70e7.62 (m, 1H), 7.58e7.46 (m, 3H), 7.35
(d,J ¼ 7.9 Hz, 1H),7.25 (t, J ¼ 8.2, 1H), 7.12e6.99 (m, 2H), 5.02 (t,
Yield 63.4%. m.p. 255e258 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.15e7.98 (m, 2H, ArH, CH), 7.89e7.67 (m, 4H, ArH), 7.67 (t,
J ¼ 6.9 Hz, 2H, ArH), 7.45 (t, J ¼ 7.5 Hz, 2H, ArH), 7.19e6.82 (m, 4H,
ArH, CH), 4.45 (t, J ¼ 6.0 Hz, 2H, CH2), 3.72e3.57 (m, 2H, CH2), 2.27
J ¼ 4.4 Hz, 2H), 4.62 (t, J ¼ 4.3 Hz, 2H); 13C NMR
d: 44.88, 62.01,
106.11, 112.75, 114.38, 120.85, 121.73, 122.87, 125.95, 126.29, 129.70,
132.86, 134.33, 135.48, 135.90, 136.75, 137.01, 139.19, 151.01, 164.28;
ESI-MS: 481.04 [MþH]þ. Anal. Calcd for C22H17BrN4O4: C, H, N.
(s, 3H, CH3); 13C NMR
d: 21.38, 44.44, 62.07, 114.31, 116.56, 120.39,
122.30, 125.21, 126.50, 127.58, 129.49, 132.25, 134.29, 135.71, 136.68,
137.98,139.30,143.29,145.60,151.55; ESI-MS: 464.12 [MþH]þ. Anal.
Calcd for C24H21N3O5S: C, H, N.
4.2.42. (Z)-2-(2-(4-bromostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
1H-indole-3-carboxylate (3f)
4.2.36. (Z)-2-(5-nitro-2-styryl-1H-imidazol-1-yl)ethyl-4-
Yield 62.5%. m.p. 189e191 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
methylbenzenesulfonate (2r)
11.80 (s, 1H), 8.22 (d, J ¼ 6.3, 1H), 7.95 (s, 1H), 7.87 (d, J ¼ 7.6 Hz, 1H),
7.77 (d, J ¼ 2.1, 3H), 7.70e7.62 (m, 1H), 7.58e7.46 (m, 3H), 7.35 (d,
J ¼ 7.9 Hz, 1H),7.25 (t, J ¼ 8.2, 1H), 7.12e6.99 (m, 2H), 5.02 (t,
Yield 67.3%. m.p. 254e257 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.12 (s, 1H, CH), 7.83e7.72 (m, 5H, ArH), 7.52 (d, J ¼ 6.0 Hz, 1H, ArH),
7.31 (t, J ¼ 9.0 Hz, 3H, ArH, CH), 7.11 (d, J ¼ 4.8 Hz, 2H, ArH, CH), 4.88
(t, J ¼ 6.0 Hz, 2H, CH2), 4.41 (t, J ¼ 6.9 Hz, 2H, CH2), 2.27 (s, 3H, CH3);
J ¼ 4.4 Hz, 2H), 4.62 (t, J ¼ 4.3 Hz, 2H); 13C NMR
d: 44.67, 62.29,
106.38, 112.54, 114.90, 120.75, 121.00, 122.25, 125.05, 129.17, 132.83,
134.18, 135.09, 135.47, 137.31, 139.50, 151.29, 164.55; ESI-MS: 481.04
[MþH]þ. Anal. Calcd for C22H17BrN4O4: C, H, N.
13C NMR
d: 21.20, 44.38, 62.45,114.09,116.26,120.39,122.18,125.20,
126.31, 129.40, 132.35, 134.66, 135.90, 136.23, 137.15, 139.37, 143.08,
151.40; ESI-MS: 414.10 [MþH]þ. Anal. Calcd for C20H19N3O5S: C, H,
N.
4.2.43. (Z)-2-(5-nitro-2-(2-nitrostyryl)-1H-imidazol-1-yl)ethyl 1H-
indole-3-carboxylate (3g)
4.2.37. (E)-2-(2-(2-chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl
Yield 66.5%. m.p. 189e190 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
1H-indole-3-carboxylate (3a)
11.82 (s, 1H), 8.21 (s, 1H), 8.02 (s, 1H), 7.93 (t, J ¼ 6.6 Hz, 1H), 7.83 (d,
J ¼ 2.5 Hz, 1H), 7.75e7.68 (m, 3H), 7.58 (d, J ¼ 4.7 Hz, 2H), 7.35 (t,
J ¼ 6.2 Hz, 1H), 7.15e6.99 (m, 2H), 5.01 (t, J ¼ 4.4 Hz, 2H), 4.67 (t,
Yield 64.5%. m.p. 189e191 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
11.71 (s, 1H), 8.23 (s, 1H), 7.95e7.69 (m, 4H), 7.58 (t, J ¼ 7.9 Hz, 2H),
7.33 (t, J ¼ 7.5 Hz, 2H), 7.14e7.02 (m, 3H), 5.02 (t, J ¼ 4.3 Hz, 2H), 4.65
J ¼ 4.3 Hz, 2H); 13C NMR
d: 44.58, 62.29, 106.10, 112.28, 114.86,

Y.-T. Duan et al. / European Journal of Medicinal Chemistry 85 (2014) 341e351
349
120.57, 121.90, 122.05, 125.32, 126.49, 129.61, 132.54, 134.20, 135.17,
135.90, 136.38, 137.40, 139.10, 151.31, 164.88; ESI-MS: 448.12
[MþH]þ. Anal. Calcd for C22H17N5O6: C, H, N.
135.43, 136.12, 139.16, 151.09, 157.61, 164.19; ESI-MS: 433.14
[MþH]þ. Anal. Calcd for C23H20N4O5: C, H, N.
4.2.50. (Z)-2-(2-(3-methoxystyryl)-5-nitro-1H-imidazol-1-yl)ethyl
1H-indole-3-carboxylate (3n)
4.2.44. (Z)-2-(5-nitro-2-(3-nitrostyryl)-1H-imidazol-1-yl)ethyl 1H-
Yield 67.5%. m.p. 184e185 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
indole-3-carboxylate (3h)
Yield 65.5%. m.p. 189e191 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
11.81 (s, 1H), 8.23 (s, 1H), 7.92 (d, J ¼ 6.6 Hz, 2H), 7.82 (t, J ¼ 4.5 Hz,
1H), 7.74 (d, J ¼ 4.9 Hz, 1H), 7.49e7.38 (m, 1H), 7.26 (t, J ¼ 7.5 Hz,
3H), 7.16e7.04 (m, 2H), 6.94e6.86 (m, 1H), 5.02 (t, J ¼ 4.4 Hz, 2H),
11.81 (s, 1H), 8.21 (s, 1H), 8.05e7.99 (m, 1H), 7.89 (d, J ¼ 7.6 Hz, 1H),
7.38e7.29 (m, 3H), 7.12 (t, J ¼ 14.7 Hz, 1H), 7.07e6.99 (m, 2H), 6.82
4.62 (t, J ¼ 4.3 Hz, 2H), 3.77 (s, 3H); 13C NMR
d: 44.82, 56.03, 62.05,
(t, J ¼ 7.5 Hz, 1H), 4.95 (t, J ¼ 4.3 Hz, 2H), 4.62 (t, J ¼ 4.4 Hz, 2H); 13
C
106.09, 112.74, 113.32, 120.82, 120.94, 121.74, 122.85, 125.98, 127.75,
131.46, 132.82, 133.40, 135.58, 136.74, 139.10, 151.39, 157.64, 164.27;
ESI-MS: 433.14 [MþH]þ. Anal. Calcd for C23H20N4O5: C, H, N.
NMR d: 44.68, 62.70, 106.29, 112.19, 114.09, 120.28, 121.61, 122.89,
125.34, 126.16, 129.80, 132.29, 134.00, 135.07, 135.67, 136.20, 137.49,
139.30, 151.11, 164.37; ESI-MS: 448.12 [MþH]þ. Anal. Calcd for
C
22H17N5O6: C, H, N.
4.2.51. (Z)-2-(2-(4-methoxystyryl)-5-nitro-1H-imidazol-1-yl)ethyl
1H-indole-3-carboxylate (3o)
4.2.45. (Z)-2-(5-nitro-2-(4-nitrostyryl)-1H-imidazol-1-yl)ethyl 1H-
Yield 63.5%. m.p. 182e185 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
indole-3-carboxylate (3i)
Yield 62.5%. m.p. 187e189 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
11.80 (s, 1H), 8.22 (s, 1H), 7.87 (d, J ¼ 7.6 Hz, 1H), 7.77 (d, J ¼ 2.5 Hz,
1H), 7.70 (d, J ¼ 14.9 Hz, 1H), 7.46e7.35 (m, 2H), 7.23 (t, J ¼ 7.5 Hz,
3H), 7.14e7.01 (m, 2H), 6.93e6.89 (m, 1H), 5.00 (t, J ¼ 4.2 Hz, 2H),
11.80 (s, 1H), 8.23 (s, 1H), 7.98 (t, J ¼ 6.6 Hz, 2H), 7.85 (d, J ¼ 3.5 Hz,
1H), 7.77e7.69 (m, 2H), 7.58 (t, J ¼ 4.4 Hz, 3H), 7.39 (t, J ¼ 6.6 Hz,
1H), 7.18e6.99 (m, 2H), 5.03 (t, J ¼ 4.4 Hz, 2H), 4.68 (t, J ¼ 4.3 Hz,
4.62 (t, J ¼ 4.3 Hz, 2H), 3.74 (s, 3H); 13C NMR
d: 44.78, 56.18, 62.30,
2H); 13C NMR
d: 44.45, 62.49, 106.03, 112.67, 114.28, 120.34, 121.10,
112.42, 120.80, 121.09, 123.74, 124.32, 125.65, 127.31, 131.02, 132.19,
133.47, 135.22, 136.30, 139.89, 151.68, 157.14, 164.00; ESI-MS: 433.14
[MþH]þ. Anal. Calcd for C23H20N4O5: C, H, N.
122.76, 125.55, 126.20, 129.39, 132.12, 134.30, 135.21, 135.89, 136.11,
137.30, 139.14, 151.56, 164.39; ESI-MS: 448.12 [MþH]þ. Anal. Calcd
for C22H17N5O6: C, H, N.
4.2.52. (Z)-2-(2-(2,4-dichlorostyryl)-5-nitro-1H-imidazol-1-yl)
ethyl 1H-indole-3-carboxylate (3p)
4.2.46. (Z)-2-(2-(2-methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl
Yield 63.5%. m.p. 192e195 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
1H-indole-3-carboxylate (3j)
11.78 (s, 1H), 8.23 (d, J ¼ 6.7 Hz, 2H), 8.09 (d, J ¼ 8.3 Hz, 2H), 8.01 (d,
Yield 66.5%. m.p. 189e191 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
J ¼ 9.5 Hz, 2H), 7.71 (d, J ¼ 2.9 Hz, 1H), 7.58e7.49 (m, 3H), 7.44 (s,
11.81 (s, 1H), 8.23 (s, 1H), 7.94e7.79 (m, 3H), 7.60 (d, J ¼ 7.6 Hz, 1H),
1H), 4.98 (t, J ¼ 4.1 Hz, 2H), 4.63 (t, J ¼ 4.3 Hz, 2H); 13C NMR
d: 44.23,
7.30e7.10 (m, 3H), 7.03 (t, J ¼ 7.3 Hz, 4H), 4.98 (t, J ¼ 4.4 Hz, 2H),
61.20, 106.36, 112.78, 114.90, 120.21, 121.45, 122.67, 125.79, 126.09,
129.31, 132.33, 134.27, 135.00, 135.67, 136.69, 137.32, 139.11, 151.4,
164.56; ESI-MS: 471.05 [MþH]þ. Anal. Calcd for C22H16Cl2N4O4: C,
H, N.
4.62 (t, J ¼ 4.3 Hz, 2H), 2.26 (s, 3H); 13C NMR
d: 19.68, 40.59, 62.01,
106.11, 112.75, 114.38, 120.85, 121.73, 122.87, 126.29, 126.56, 129.70,
131.01, 132.86, 134.33, 135.48, 135.90, 136.75, 137.01, 151.01, 164.28;
ESI-MS: 417.15 [MþH]þ. Anal. Calcd for C23H20ClN4O4: C, H, N.
4.2.53. (Z)-2-(2-(2-(naphthalen-1-yl)vinyl)-5-nitro-1H-imidazol-
4.2.47. (Z)-2-(2-(3-methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl
1-yl)ethyl 1H-indole-3-carboxylate (3q)
Yield 67.5%. m.p. 190e192 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
1H-indole-3-carboxylate (3k)
Yield 68.5%. m.p. 189e191 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
11.80 (s, 1H), 8.23 (s, 1H), 8.11 (d, J ¼ 4.3 Hz, 3H), 8.05 (t, J ¼ 4.5 Hz,
11.75 (s, 1H), 8.23 (s, 1H), 7.91 (t, J ¼ 4.4 Hz, 2H), 7.57 (d, J ¼ 7.2 Hz,
3H), 7.75 (d, J ¼ 4.9 Hz, 2H), 7.59e7.47 (m, 4H), 7.47 (m, 2H), 4.99 (t,
J ¼ 4.4 Hz, 2H), 4.65 (t, J ¼ 4.3 Hz, 2H); 13C NMR
d: 44.25, 62.21,
2H), 7.35e7.21 (m, 3H), 7.15e7.03 (m, 4H), 4.98 (t, J ¼ 4.3 Hz, 2H),
4.63 (t, J ¼ 4.4 Hz, 2H), 2.26 (s, 3H); 13C NMR
d: 19.18, 40.17, 62.43,
106.34, 112.34, 114.89, 120.20, 121.21, 122.00, 125.32, 126.98, 129.32,
130.78, 132.17, 134.56, 135.59, 135.67, 136.13, 137.04, 139.20, 145.72,
151.31, 164.89; ESI-MS: 453.15 [MþH]þ. Anal. Calcd for C26H20N4O4:
C, H, N.
106.51, 112.21, 114.34, 120.10, 121.11, 122.00, 125.25, 126.47, 129.78,
132.09, 134.23, 135.17, 135.00, 136.27, 137.67, 139.61, 151.49, 164.07;
ESI-MS: 417.15 [MþH]þ. Anal. Calcd for C23H20ClN4O4: C, H, N.
4.2.48. (Z)-2-(2-(4-methylstyryl)-5-nitro-1H-imidazol-1-yl)ethyl
4.2.54. (Z)-2-(5-nitro-2-styryl-1H-imidazol-1-yl)ethyl 1H-indole-
1H-indole-3-carboxylate (3l)
3-carboxylate (3r)
Yield 65.5%. m.p. 189e190 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
Yield 67.5%. m.p. 188e190 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
11.79 (s, 1H), 8.21 (s, 1H), 7.91e7.66 (m, 3H), 7.51 (d, J ¼ 7.9 Hz, 2H),
11.80 (s, 1H), 8.21 (s, 1H), 8.13 (d, J ¼ 5.3 Hz, 2H), 8.08 (t, J ¼ 7.5 Hz,
7.35 (t, J ¼ 7.9 Hz, 2H), 7.14e7.02 (m, 4H), 4.98 (t, J ¼ 4.4 Hz, 2H), 4.61
2H), 7.76e7.67 (m, 3H), 7.55 (d, J ¼ 4.6 Hz, 2H), 7.47 (m, 3H), 4.99 (t,
(t, J ¼ 4.3 Hz, 2H), 2.29 (s, 3H); 13C NMR
d: 19.57, 40.35, 62.21,
J ¼ 4.4 Hz, 2H), 4.65 (t, J ¼ 4.3 Hz, 2H); 13C NMR
d: 42.20, 60.18,
106.45, 112.00, 114.19, 120.23, 121.41, 122.28, 125.67, 126.78, 129.80,
132.24, 134.36, 135.70, 136.51, 139.01, 151.12, 164.52; ESI-MS: 417.15
[MþH]þ. Anal. Calcd for C23H20ClN4O4: C, H, N.
112.30, 115.15, 119.34, 121.09, 122.21, 125.63, 126.18, 129.55, 132.79,
134.32, 135.19, 136.24, 137.57, 139.31, 151.66, 164.10; ESI-MS: 403.13
[MþH]þ. Anal. Calcd for C22H18N4O4: C, H, N.
4.2.49. (Z)-2-(2-(2-methoxystyryl)-5-nitro-1H-imidazol-1-yl)ethyl
5. Bioassay conditions
1H-indole-3-carboxylate (3m)
Yield 64.5%. m.p. 183e185 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d:
5.1. Effects on tubulin polymerization
11.80 (s, 1H), 8.25 (s, 1H), 7.89 (t, J ¼ 7.6 Hz, 2H), 7.76 (d, J ¼ 5.5 Hz,
1H), 7.48e7.36 (m, 2H), 7.27 (d, J ¼ 4.5 Hz, 2H), 7.16e7.07 (m, 3H),
6.97e6.89 (m, 1H), 5.02 (t, J ¼ 4.4 Hz, 2H), 4.65 (t, J ¼ 4.3 Hz, 2H),
Bovine brain tubulin was purified as described previously
[24,25]. To evaluate the effect of the compounds on tubulin as-
sembly in vitro, varying concentrations were preincubated with
3.76 (s, 3H); 13C NMR
d: 44.32, 56.03, 62.05, 106.57, 112.41, 114.90,
120.45, 121.51, 123.89, 124.70, 125.98, 127.05, 129.75, 132.58, 133.19,
10 m
M tubulin in glutamate buffer at 30 ꢀC and then cooled to 0 ꢀC.

350
Y.-T. Duan et al. / European Journal of Medicinal Chemistry 85 (2014) 341e351
After addition of GTP, the mixtures were transferred to 0 ꢀC cuvettes
in a recording spectrophotometer and warmed up to 30 ꢀC and the
assembly of tubulin was observed turbidimetrically. The IC50was
defined as the compound concentration that inhibited the extent of
assembly by 50% after 20 min incubation.
times and subject to leave-one-out validation test, respectively.
The models were also validated by test sets, in which the com-
pounds are not included in the training sets. Usually, one can
believe that the modeling is reliable, when the R2 for test sets is
larger than 0.6.
5.2. Cell proliferation assay
Acknowledgments
CCK-8 is much more convenient and helpful than MTT for
analyzing cell proliferation, because it can be reduced to soluble
formazan by dehydrogenase in mitochondria and has little toxicity
to cells. Cell proliferation was determined using CCK-8 dye (Beyo-
timeInst Biotech, China) according to manufacture's instructions.
Briefly, 1e5 ꢂ 103 cells per well were seeded in a 96-well plate,
grown at 37 ꢀC for 12 h. Subsequently, cells were treated with the
target compounds at increasing concentrations in the presence of
This work was supported by the project (KYLX_0035) from
Jiangsu Province Innovation for Ph.D candidate & Major Projects on
Control and Rectification of Water Body Pollution (no.2011ZX07204-
001-004), and Supported by PCSIR (IRT1020).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.082.
10% FBS for 24 or 48 h. After 10 mL CCK-8 dye was added to each
well, cells were incubated at 37 ꢀC for 1e2 h and plates were read in
a Victor-V multilabel counter (PerkineElmer) using the default
europium detection protocol. Percent inhibition or IC50 values of
compounds were calculated by comparison with DMSO-treated
control wells.
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