Synthesis, biological evaluation, and molecular docking studies of novel 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives as potential tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.05.021

A series of 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives were designed, synthesized and evaluated as potential tubulin polymerization inhibitors and for the cytotoxicity against anthropic cancer cell lines. Among the novel compounds, compound 11f was demonstrated the most potent tubulin polymerization inhibitory activity (IC₅₀ = 1.5 μM) and antiproliferative activity against A549, HepG2 and MCF-7 (GI₅₀ = 2.4, 3.8 and 5.1 μM, respectively), which was compared with the positive control colchicine and CA-4. We also evaluated that compound 11f could effectively induce apoptosis of A549 associated with G2/M phase cell cycle arrest. Docking simulation and 3D-QSAR model in these studies provided more information that could be applied to design new molecules with more potent tubulin inhibitory activity.

Full text of DOI:10.1016/j.ejmech.2015.05.021

Accepted Manuscript
Synthesis, Biological Evaluation, and Molecular Docking Studies of Novel 1-
benzene acyl-2-(1-methylindol-3-yl)-benzimidazole Derivatives as Potential Tubulin
Polymerization Inhibitors
Yan-Ting Wang, Ya-Juan Qin, Na Yang, Ya-Liang Zhang, Chang-Hong Liu, Hai-Liang
Zhu
PII:
S0223-5234(15)30048-9
10.1016/j.ejmech.2015.05.021
EJMECH 7900
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 March 2015
Revised Date: 11 May 2015
Accepted Date: 13 May 2015
Please cite this article as: Y.-T. Wang, Y.-J. Qin, N. Yang, Y.-L. Zhang, C.-H. Liu, H.-L. Zhu, Synthesis,
Biological Evaluation, and Molecular Docking Studies of Novel 1-benzene acyl-2-(1-methylindol-3-yl)-
benzimidazole Derivatives as Potential Tubulin Polymerization Inhibitors, European Journal of Medicinal
Chemistry (2015), doi: 10.1016/j.ejmech.2015.05.021.
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ACCEPTED MANUSCRIPT
Synthesis, Biological Evaluation, and Molecular Docking Studies of
Novel 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole
Derivatives as Potential Tubulin Polymerization Inhibitors
Yan-Ting Wang, Ya-Juan Qin, Na-Yang, Ya-Liang Zhang, Chang-Hong Liu*,
Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing210093, P. R. China
*Corresponding author. Tel. & fax: +86-25-89682572; e-mail: zhuhl@nju.edu.cn
Crystal structure diagram of compound 10c
1

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3D diagram of the interaction between Compound 11f and the colchicine binding
site
Compounds of novel 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole
derivatives containing different substituent groups were designed, synthesized and
evaluated for the inhibitory activity against tubulin polymerization and cancer cell
inhibitory activity. Docking simulation and the QSAR study were conducted.
2

ACCEPTED MANUSCRIPT
Abstract: A series of 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole
derivatives were designed, synthesized and evaluated as potential tubulin
polymerization inhibitors and for the cytotoxicity against anthropic cancer cell lines.
Among the novel compounds, compound 11f was demonstrated the most potent
tubulin polymerization inhibitory activity (IC50 = 1.5 ꢀM) and antiproliferative
activity against A549, HepG2 and MCF-7 (GI = 2.4, 3.8 and 5.1 ꢀM, respectively),
5
0
which was compared with the positive control colchicine and CA-4. We also
evaluated that compound 11f could effectively induce apoptosis of A549 associated
with G2/M phase cell cycle arrest. Docking simulation and 3D-QSAR model in these
studies provided more information that could be applied to design new molecules with
more potent tubulin inhibitory activity.
Keywords: tubulin polymerization inhibitors; N-methylindole; benzimidazole;
molecular docking; 3D-QSAR.
3

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With the changes in the living environment, cancer has become one of the major
causes of death worldwide. [1] The common treatments were surgery, radiotherapy,
chemotherapy and gene et al., and the main method was chemotherapy. However the
majority of chemotherapy agents were restrict for their high toxicity and more adverse
reaction. [2] Therefore, seeking high-efficiency, low toxicity anticancer agents have
become one of the research subjects in the current pharmacy field.
Tubulin is an essential eukaryotic protein that plays critical roles in cell division
and microtubule-targeted drugs are now indispensable for the therapy of various
cancer types worldwide. [3, 4, 5, 6] The formation of microtubules is a dynamic
process that involves the polymerization and depolymerization of R and â tubulin
heterodimers. [7] Compounds that interfere with the microtubulin equilibria in cells
are useful in the treatment of human cancer. [8, 9] They interfere with this dynamic
equilibrium by binding to tubulins and induce cell cycle arrest, resulting in cell death.
[
10, 11]
Antimitotic agents largely fall into three major classes. The taxanes such as
paclitaxel and docetaxel stabilize microtubules by preventing the depolymerization of
tubulin. [10, 12] The Vinca alkaloids (e.g., vincristine, vinblastine, and vinorelbine)
and colchicine inhibit the polymerization of tubulin. [10, 12] Disruption of tubulin
dynamics leads to cell cycle arrest in the G2/M phase and induction of apoptosis. [13,
1
4]
There are a great quantity of evidences, derived from molecular modeling studies
and experimental data, demonstrating how these inhibitors bind to the active site of
tubulin. [15-18] Colchicine binding is thought to be a universal property of higher
eukaryotic tubulin. [19] Compounds that bind to the colchicine site have been the
subject of intense investigation by researcher seeking to identify new agents capable
of addressing the limitations of existing tubulin targeting drugs. [20, 21]
Combretastatin A-4 (drawed in Figure 1), a natural product isolated from the South
African bush willow tree Combretum caffrum, binds to the colchicine binding site and
inhibits the polymerization of microtubules. Recently, some excellent tubulin
inhibitors have been reported. Nocodazole [22], D-24851, D-64131, D-68144 [23],
4

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ATIs [24] and BPROL075 [25] (Figure 1) exhibit potent tubulin polymerization
inhibitory activity by binding to the colchicine binding site. According to their
chemical structure characteristics, it could be found that indole structure and amide
structure play important role in tubulin inhibition. What’s more, it has been reported
that the indole nucleus is the core structure of a great number of tubulin
polymerization inhibitors. [26, 27] Moreover, insertion of N-methylindole would
yield potent cytotoxicity and tubulin polymerization inhibition. [4, 28] Nocodazole
mentioned above owned another important group, benzimidazole. Some studies
showed that agents containing benzimidazole possessed good antitumor activity. [29]
They could affect the dynamic loop of the tubulin polymerization and
depolymerization, [30] then cell apoptosis was resulted in. [29] What's more, Docking
simulations were performed using the X-ray crystallographic structure of the tubulin
(PDB code: 1SA0) in complex with an inhibitor to explore the binding modes of these
compounds at the active site.
We are presently utilizing insights gleaned from these researches and molecular
modeling studies to design and synthesis new tubulin inhibitors: compounds 10a -
1
2h. Our group previously did some researches about antitubulin agents, [31, 32, 33]
and the structures of novel 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole
derivatives were similar with the agents. So the work in this paper is a continuation of
them. The docking study based on tubulin crystal structure (PDB code: 1SA0)
indicated that compounds 11f, 10f , 11e, 12f, 10e and 12e exhibit more affinity for
tubulin than colchicine, which rationally proved the reason why these compounds also
possess effective inhibitory activity profile against tubulin.
In this paper, some work has been done: discuss the synthetic method of this
series of compounds, depict the results of reactivity studies; evaluate their anticancer
and antitubulin activities; research the influence on the cell division cycle and cell
apoptosis and analysis the structure-activity relationship. Additionally, molecular
docking and 3D-QSAR model provided more information that could be applied to
design new molecules with more potent tubulin inhibitory activity. [34]
(
Figure 1)
5

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2
2
. Results and Discussion
.1. Chemistry.
The synthetic route for compounds 10a - 12h is outlined in Scheme 1. These
compounds were synthesized from amine 10 - 12 and acid a - h. And compounds 10 -
2 were synthesized from aldehyde 7 - 9 and o-phenylenediamine. Compounds 7 - 9
1
were prepared according to the procedure reported by Mark A. Seefeld et al. and
Debajyoti Saha et al. with some modifications. [35, 36]
Firstly, a solution of substituted indole 1 - 3 in DMF was added dropwise to a
stirring suspension of phosphorus oxychloride in DMF. And then we obtained the
corresponding compounds 4 - 6, respectively. Secondly, compounds 7 - 9 were
synthesized from the corresponding compounds 4 - 6. A solution of compounds 4 - 6
in THF were added dropwise to a suspension of NaH in THF, and were also treated
with iodomethane. Then compounds 7 - 9 were obtained, respectively. Thirdly,
compounds 7 - 9 reacted with o-phenylenediamine together with Sodium pyrosulfite
in DMF, respectively. The heterogeneous mixture was stirred at 110ꢀ for 4h, then
compounds 10 - 12 were obtained. Fourthly, The acids a - h and SOCl were mixed
2
and stirred at 80ꢀ for 4 h to give reactive acyl chloride, respectively. Finally, the
corresponding acyl chloride of acids (a - h) were added dropwise to the amine
compounds (10 - 12) in ethyl acetate containing triethylamine at 0ꢀ, respectively.
The mixture was stirred overnight. The corresponding crude products were
crystallized with ethanol to give target compounds 10a - 12h (Table 1). In addition,
compounds 10a - 12h could be synthesized from compounds (10 - 12) and acids (a - h)
in the presence of EDC·HCl and HOBt in CH
2
Cl . Compounds 10a - 12h were fully
2
1
characterized by H NMR, ESI-MS and elemental analysis.
(Scheme 1)
(Table 1)
2.2. Crystal structure determination
6

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Among them, a crystal structure of compound 10c was determined by X-ray
diffraction analysis. The crystal data presented in Table S1 and Figure 2 gave
perspective views of 10c with the atomic labeling system.
(Table S1)
(Figure 2)
2.3. Biological Evaluation
2.3.1. In Vitro Antiproliferative Activities (Cell viability was assessed by MTT
assay)
To test the anticancer activities of the synthesized compounds, firstly we
evaluated antiproliferative activities of compounds 10a - 12h against A549
carcinomic human alveolar basal epithelial cell), HepG2 (human liver hepatocellular
(
carcinoma) and MCF-7 (breast cancer) cancer cells. The results were summarized in
Table 2. From the results of the MTT assay, it was found that some synthesized novel
compounds showed remarkable antiproliferative effects mainly for A549 cells. The
new compounds 11f, 10f and 11e showd more active than CA-4 (GI = 2.8 ꢀM for
5
0
A549, GI = 7.4 ꢀM for HepG2, and GI = 9.4 ꢀM for MCF-7, respectively) in
5
0
50
antiproliferativivities.
Structure-activity relationships in these novel derivatives demonstrated as
follows:
Analysis of substituent groups in the A-ring: in the case of the same substituent
groups in the B-ring, compounds with different substituent groups in the A-ring were
compared. The results showed that the antiproliferative activity of these compounds
with different substituents in the A-ring increased in the following order: OCH > H >
3
Br. It demonstrated that electron-donating groups in the A-ring were necessary for
antiproliferative activity.
Analysis of substituent groups in the B-ring: in the case of the same substituent
groups in the A-ring, compounds with different substituent groups in the B-ring were
compared. The antiproliferative activity of these compounds with different
7

ACCEPTED MANUSCRIPT
substituents in the B-ring increased in the following order: 10c > 10d > 10b, 11c >
11d > 11b, 12c > 12d > 12b, 10h > 10g, 11h > 11g, 12h > 12g. These proved that
meta-position is better than para-position, and para-position is better than
ortho-position. More interestingly, electron-donating groups in the B-ring were better
than electron-withdrawing groups, which could be seen from the comparison: 10c >
1
0a > 10h, 11c > 11a > 11h, 12c > 12a > 12h. What’s more, the more
electron-donating substituted groups in the B-ring, the better antiproliferative activity
it was, and this could be demonstrated from the comparison: 10f > 10e > 10c, 11f >
11e > 11c, 12f > 12e > 12c.
Strikingly, the most potent small molecule screened via these antiproliferation
assays was compound 11f which suppresses the A549, HepG2 and MCF-7 cells with
the GI = 2.4 ꢀM, GI = 3.8 ꢀM and GI = 5.1 ꢀM respectively.
5
0
50
50
(Table 2)
2.3.2. Inhibition of tubulin polymerization and the binding of Colchicine to
tubulin
In the second set of experiments, we assessed the ability of novel 1-benzene
acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives 10a - 12h to inhibit tubulin
polymerization
in
vitro
(Table
3).
The
new
1-benzene
tubulin
acyl-2-(1-methylindol-3-yl)-benzimidazole
derivatives
inhibited
polymerization with IC values ranging from 1.5 to 45.6 ꢀM, as compared with 1.8
5
0
ꢀM for CA-4. As tubulin polymerization inhibitors, compounds 11f, 10f and 11e were
stronger than CA-4 (IC =1.8 ꢀM); compounds 11f, 10f , 11e, 12f, 10e and 12e were
5
0
stronger than Colchicine (IC =2.62 ꢀM). The experimental results showed that the
5
0
most potent novel compound as inhibitor was 11f, which had an IC of 1.5 ꢀM.
5
0
The new 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives were
3
also examined for potential inhibition of the binding of [ H]Colchicine to tubulin
(Table 3). All except 10g and 12g were strong inhibitors of the binding reaction (51 -
9
6% inhibition ratio). What’s more, 11f, 10f and 11e were stronger than CA-4 (93%
8

ACCEPTED MANUSCRIPT
inhibition ratio). In this assay, it was observed that compound 11f was the strongest
inhibitor.
(Table 3)
2.3.3. Cell Cycle Analysis
We hypothesized
that
the
novel
1-benzene
acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives have the mechanism of action
that they arrest the process of mitosis. To test this hypothesis, we first performed the
cell cycle analysis after the treatment of the novel compounds on A549 cells. Cell
cycle distribution was determined by propidium iodide (PI) staining. Compound 11f
was treated on A549 cells for 24 h. The results indicated that these novel compounds
arrested cells in the G2/M phase (Figure. 3). And there was a concomitant decrease of
cells in the other phases of the cell cycle (G1 and S).
In the vehicle treated group, about 13.26% of A549 cells were distributed in the
G2/M phase. Compound 11f increased the proportion of cells in G2/M phase in a
concentration-dependent manner (Figure. 3). About 27.32% of cells were found in
G2/M phase when treated with 1 ꢀM compound 11f for 24 h; about 33.21% of cells
for 2 ꢀM; and about 39.46% of cells for 5 ꢀM.
(Figure 3)
2.3.4. Analysis of apoptosis by fluorescence-activated cell sorting
To characterize the mode of cell death induced by 11f, a biparametric
cytofluorimetric analysis was performed using propidium iodide (PI), which stains
DNA and enters only dead cells, and fluorescent immunolabeling of the protein
annexin-V, which binds to phosphatidyl serine (PS) in a highly selective manner. [37]
Dual staining for annexin-V and with PI permits discrimination between live
cells (annexin-V−/PI−), early apoptotic cells (annexin-V+/PI−), late apoptotic cells
(annexin-V+/PI+), and necrotic cells (annexin-V−/PI+). Here A549 cells were used to
test cell apoptosis induced by the treatment of compound 11f. As depicted in Figure 4,
9

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every compound concentration induced an accumulation of annexin-V positive cells
in comparison with the control. About 19.57% of cells were found to be apoptotic
(annexin-V positive) when treated with 1 ꢀM compound 11f for 24 h. And with the
increasing of the compound concentration, 22.13% of cells were found to be apoptotic
for 2 ꢀM; 23.23% of cells were found to be apoptotic for 5 ꢀM.
(Figure 4)
2.4. Docking simulations
To gain better understanding on the potency of the synthesized compounds and
guide further SAR studies, we proceeded to examine the interaction of the novel
-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives with tubulin crystal
1
structure (PDB code: 1SA0).
Data was provided for the molecular docking simulations for the synthesized
compounds 10a - 12h in Table 4. The predicted binding interaction energy was used
as the criterion for ranking. The molecular docking was performed by inserting the
compound into the colchicine binding site of tubulin. All docking runs were applied
by Discovery Studio 3.1. As depicted in Table 4, compound 11f showed the biggest
interaction energy, which meant that compound 11f exhibited the most potent affinity
for tubulin in prediction. The binding models of compound 11f and tubulin were
depicted in Figure 5. The amino acid residues which had interaction with tubulin
were labeled.
The obtained results were presented in the two groups of pictures. Figure 5
showed the binding mode of compound 11f interacting with 1SA0 protein and the
docking results revealed that four amino acids Ser178, Cys241, Leu248 and Lys352
located in the binding pocket of protein played vital roles in the conformation with
compound 11f, which were stabilized by two Pi-cation bonds, and two hydrogen
bonds that shown in 2D diagram. One Pi-cation bond with 6.9 Å was formed between
amino acid Lys352 and five-membered ring of indole in portion A. The other
Pi-cation bond with 2.7 Å was formed between amino acid Leu248 and benzene ring
1
0

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C of compound 11f. One hydrogen bond connected amino acid Ser178 to methoxy
oxygen atom of portion A with 2.4 Å; the second hydrogen bond with 2 Å was
formed between Cys241 and oxygen atom of portion C.
This molecular docking result in a molecular level foundation, along with the
biological assay data, could suggest that compound 11f was the most potential
inhibitors of tubulin.
(Table 4)
(
Figure 5)
2.5. QSAR model
To evaluate the synthesized compounds as tubulin inhibitors systematically and
explore more potent inhibitors, twenty-four compounds with definite IC50 values
against tubulin were selected as the model dataset by using the Create 3D QSAR
protocol of Discovery Studio 3.1. 3D-QSAR model was built by using the
corresponding pIC values which were converted from the obtained IC (ꢀM) values
5
0
50
of tubulin inhibition and performed by built-in QSAR software of DS 3.1 (Discovery
Studio 3.1, Accelrys, Co. Ltd). The way of this transformation was derived from an
online calculator developed from an Indian medicinal chemistry lab
(http://www.sanjeevslab.org/tools-IC50.html). [38]
The training and test set were chosen by the Diverse Molecules method in
Discovery Studio. Considering a good alignment is very important for the analysis of
molecular fields, we applied CDOCKER protocol to explore each molecule with
lowest
energy
before
alignment
conformation.
1-benzene
acyl-2-(1-methylindol-3-yl)-benzimidazole was selected as substructure to build
alignment conformation before building the QSAR model.
Among all the 24 compounds, 83.3% (that is 20) were utilized as a training set
for QSAR modeling and the remaining 16.7% (that is 4) were chosen as an external
test subset for validating the reliability of the QSAR model by the Diverse Molecules
protocol in Discovery Studio 3.1. The selected test compounds were: 10a, 11b, 11f
11

ACCEPTED MANUSCRIPT
and 12h, which had been presented in Table 5. The main purpose to create 3D-QSAR
model was to choose activity conformation of the designed molecular and reasonably
evaluated the designed molecules. The success of this model depended on docking
study and the reliability of previous study about activities data. [39]
2
It was a PLS model set up 400 independent variables (conventional R = 0.980).
The observed and predicted values, corresponding residual values for the training set
and test set molecules in 3D-QSAR model, were presented in Table 5. The well
agreement between predicted pIC value and experimental pIC value for both test
5
0
50
sets and training sets were shown in Figure 6.
Also the molecules aligned with the iso-surfaces of the 3D-QSAR model
Organic & Biomolecular Chemistry Page 10 of 349 coefficients on Van der Waals
grids (Figure 7A) and electrostatic potential grids (Figure 7B) were listed.
Electrostatic map indicated red contours around regions where high electron density
(negative charge) is expected to increase activity, and blue contours represented areas
where low electron density (partial positive charge) is expected to increase activity.
Similarly, steric map indicates areas where steric bulk is predicted to increase (green)
or decrease (yellow) activity. 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 dominant close
to the skeleton). As shown in the two pictures, the potent compound 11f not only
could circumvent the red subregion or the unfavorable yellow steric subregion but can
also get more close to the favorable blue and green spaces. Thus, this promising
model would provide a guideline to design and optimize more effective tubulin
inhibitors based on the 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole
analogues skeleton and pave the way for us to further study in future.
(Table 5)
(
Figure 6)
Figure 7)
(
3. Conclusion
1
2

ACCEPTED MANUSCRIPT
We
have
synthesized
a
comprehensive
series
of
1-benzene
acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives that showed potent potent
antiproliferative activity and tubulin polymerization inhibition activity. Mechanism of
action studies confirmed that 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole
analogues maintained their ability to inhibit tubulin polymerization at colchicine
binding site, arrest cells in G2/M phase and induce cell apoptosis. And the docking
study based on tubulin crystal structure (PDB code: 1SA0) also indicated that
compound 11f exhibited the most potent affinity for tubulin. These results strongly
suggested that novel 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole analogues
can be further developed as a promising antitumor agent for the more efficacious
treatment of advanced cancers.
4. Experimental Section
4.1. Materials and measurements
All chemicals and reagents used in current study were analytical grade. Thin
1
layer chromatography (TLC), proton nuclear magnetic resonance ( H NMR) and
elemental microanalyses (CHN) were usually used. Analytical thin-layer
chromatography (TLC) was performed on the glass-backed silica gel sheets (silica gel
6
0 Å GF254). All compounds were detected using UV light (254 nm or 365 nm).
Separation of the compounds by column chromatography was carried out with silica
gel 60 (200 – 300 mesh ASTM, E. Merck). The quantity of silica gel used was 50-100
times the weight charged on the column. Melting points were determined on a XT4
1
MP apparatus (Taike Corp., Beijing, China). H NMR spectra were measured on a
Bruker AV-300 or AV-500 spectrometer at 25 ꢀ and referenced to Me Si. Chemical
4
shifts are reported in ppm (δ) using the residual solvent line as internal standard.
Splitting patterns are designed as s, singlet; d, doublet; t, triplet; m, multiplet. ESI-MS
spectra were recorded on a Mariner System 5304 Mass spectrometer. Elemental
analyses were performed on a CHN-O-Rapid instrument and were within ± 0.4% of
the theoretical values.
1
3

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4.2. General procedure for preparation of 1H-indole-3-carboxaldehyde (4 - 6)
A solution of substituted indole 1 - 3 (82.6 mmol) in DMF (20 mL) was added
dropwise to a stirring suspension of phosphorus oxychloride (7.0 mL, 75.0 mmol) in
DMF (35 mL). After stirring at room temperature for 2 h, the reaction mixture was
poured onto ice. The mixture was made basic with a solution of NaOH (330 mmol,
1
3.2 g) in H O (44 mL), then was extracted with Et O (2 × 50 mL). The combined
2 2
organic layers were washed with brine, dried (MgSO ), and concentrated under
4
vacuum. Flash chromatography on silica gel (10% ethyl acetate/hexanes) gave the
title compounds 4 - 6 as an off-white solid, respectively.
4.3.
General
procedure
for
preparation
of
1
-Methyl-1H-indole-3-carboxaldehyde (7 - 9)
Compounds 7 - 9 were synthesized from the corresponding compounds 4 - 6. A
solution of compounds 4 - 6 (60 mmol) in THF (30 mL) were added dropwise to a
suspension of NaH (3.60 g, 60% dispersion in mineral oil, 150 mmol) in THF (30
mL) at 0ꢀ. After stirring for 15 min, the heterogeneous mixture was treated with
iodomethane (5.04 mL, 79.2 mmol) at room temperature for 1h. Then the reaction
mixture was cooled to 0ꢀ, quenched with saturated NH Cl (60 mL), and extracted
4
with ether (3 × 50 mL). The organic layers were combined, washed with brine, dried
over
-Methyl-1H-indole-3-carboxaldehyde (7 - 9), a light brown solid. The crude 7 - 9
were used in the next step without any further purification.
anhydrous
Na SO4
and
concentrated
in
vacuo
to
give
2
1
4.4. General procedure for preparation of 2-(1-methylindol-3-yl)-benzimidazole
(10 - 12)
A solution of o-phenylenediamine (5.41 g, 50 mmol) in DMF (20 mL) was
added dropwise to a suspension of 7 - 9 (50 mmol) and Sodium pyrosulfite (19.09 g,
00 mmol) in DMF (50 mL). The heterogeneous mixture was stirred at 110 ꢀ for 4h,
then cooled and pured onto a lot of ice. A large amount of solid would emerge, and
1
1
4

ACCEPTED MANUSCRIPT
was filtered to give 2-(1-methylindol-3-yl)-benzimidazole (10 - 12), a yellow powder.
4
.5.
General
synthesis
method
of
1-benzene
acyl-2-(1-methylindol-3-yl)-benzimidazole (10a - 12h)
The first method: the starting materials (acids a - h) for the synthesis of amides
should be activated. The compounds a - h (1.0 mM) and SOCl (6 - 10 mL) were
2
mixed and stirred at reflux 80 ꢀfor 4 h, then cooled and evaporated to give reactive
acyl chloride, respectively. A solution of acyl chloride (1.0 mM) in ethyl acetate (5 - 6
mL) was added dropwise to the corresponding suspension of amine compounds (10 -
1
2) (0.5 mM) and triethylamine (0.5 mL) in 20 mL ethyl acetate at 0ꢀ.After stirring
overnight, the reaction mixture was then poured in excess of diluted NaOH and
extracted with EtOAc. The extraction liquid was purified by a flash chromatography
with
EtOAc/petroleum
ether
(3:1,
v/v)
to
give
1-benzene
acyl-2-(1-methylindol-3-yl)-benzimidazole (10a - 12h), a yellow or white powder,
respectively with yields of 40% - 85%.
The other method: 1.2 mM EDC·HCl (0.23g) and 1.2 mM HOBt (0.162g) were
added to a stirred solution of 2-(1-methylindol-3-yl)-benzimidazole (10 - 12, 1.0 mM)
and the corresponding acids (a - h, 1.0 mM) in CH
2
Cl , then the reaction mixture was
2
stirred at room temperature for 24 h. After that, the solvent was evaporated under
reduced pressure to afford a residue, which was extracted with EtOAc (3 × 20mL).
The combined organic phases were dried with anhydrous Na
2
SO . Removal of all the
4
solvent under reduced pressure distillation to get the residue, which was purified with
recrystallization to give target products 10a - 12h with yields of 62% - 70%.
4
.5.1. 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole (10a)
1
White powder, yield: 77%. M. p: 127 – 129 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.72 (s, 3H); 7.23 – 7.27 (m, 4H); 7.33 – 7.37 (m, 1H); 7.37 – 7.47 (m, 3H); 7.61 –
7
7
1
.65 (m, 1H); 7.66 (s, 1H); 7.72 (s, 1H); 7.74 (s, 1H); 7.81 (d, J = 8, 1H); 8.16 (d, J =
1
3
.2, 1H). C NMR (DMSO-d , 100 MHz)
ꢀ
: 170.06, 149.94, 143.66, 136.90, 134.68,
6
34.53, 133.37, 132.98, 130.70, 129.25, 126.56, 124.38, 123.94, 122.83, 121.26,
1
5

ACCEPTED MANUSCRIPT
1
21.23, 119.56, 112.73, 110.74, 104.67, 56.50, 33.15, 19.04. ESI-MS: 351.40
+
(
C H N O, [M+H] ). Anal. Calcd for C H N O: C, 78.60; H, 4.87; N, 11.97; O,
2
3
17
3
23 17
3
4
4
.56; Found: C, 78.61; H, 4.88; N, 11.96; O, 4.55.
.5.2. 1-(2-methoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10b)
1
Yellow powder, yield: 84%. M. p: 137 – 138 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.84 (s, 6H); 6.88 – 7.00 (m, 2H); 7.13 – 7.16 (m, 4H); 7.46 – 7.52 (m, 3H); 7.63 –
+
7
.65 (m, 2H); 7.88 – 7.91 (m, 2H). ESI-MS: 381.43 (C H N O , [M+H] ). Anal.
2
4
19
3
2
Calcd for C H N O : C, 75.57; H, 5.02; N, 11.02; O, 8.39; Found: C, 75.56; H, 5.02;
2
4
19
3
2
N, 11.04; O, 8.38.
4
.5.3. 1-(3-methoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10c)
1
Yellow powder, yield: 87%. M. p: 146 – 147 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.65 (s, 3H); 3.72 (s, 3H); 7.16 – 7.18 (m, 2H); 7.21 (s, 1H); 7.26 (s, 4H); 7.34 –
7
.36 (m, 2H); 7.45 – 7.47 (m, 1H); 7.64 (s, 1H); 7.80 – 7.82 (m, 1H); 8.15 – 8.17 (m,
1
3
1
H). C NMR (DMSO-d , 100 MHz)
ꢀ: 169.81, 159.58, 149.92, 143.67, 136.87,
6
1
1
3
1
4
34.80, 134.53, 132.99, 130.36, 126.54, 124.44, 124.00, 123.07, 122.82, 121.24,
21.21, 120.96, 119.56, 114.84, 112.88, 110.75, 104.71, 55.78, 33.12. ESI-MS:
+
81.43 (C H N O , [M+H] ). Anal. Calcd for C H N O : C, 75.55; H, 5.03; N,
2
4
19
3
2
24 19
3
2
1.03; O, 8.39; Found: C, 75.57; H, 5.02; N, 11.02; O, 8.39.
.5.4. 1-(4-methoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10d)
1
Yellow powder, yield: 77%. M. p: 155 – 156 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.69 (s, 3H); 3.76 (s, 3H); 7.21 – 7.23 (m, 1H); 7.28 – 7.29 (m, 2H); 7.22 – 7.23 (m,
1
1
H); 7.25 – 7.27 (m, 1H); 7.32 – 7.35 (m, 2H); 7.37 – 7.38 (m, 1H); 7.48 – 7.50 (m,
+
H); 7.62 (s, 1H); 7.80 (m, 2H); 8.25 (s, 1H). ESI-MS: 381.43 (C H N O , [M+H] ).
2
4
19
3
2
Anal. Calcd for C H N O : C, 75.57; H, 5.02; N, 11.02; O, 8.39; Found: C, 75.55; H,
2
4
19
3
2
5
.01; N, 11.05; O, 8.39.
4
.5.5. 1-(3, 4-dimethoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10e)
1
Yellow powder, yield: 87%. M. p: 205 – 206 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.69 (s, 3H); 3.76 (s, 3H); 3.82 (s, 3H); 7.01 – 7.03 (m, 1H); 7.18 – 7.19 (m, 1H);
.20 – 7.23 (m, 2H); 7.25 – 7.27 (m, 1H); 7.32 – 7.35 (m, 2H); 7.37 – 7.38 (m, 1H);
7
1
6

ACCEPTED MANUSCRIPT
1
3
7
.48 – 7.50 (m, 1H); 7.62 (s, 1H); 7.80 (d, J = 7.6, 1H); 8.25 (d, J = 7.6, 1H). C
NMR (DMSO-d , 100 MHz) : 169.28, 154.70, 149.84, 149.13, 143.59, 136.95,
34.90, 132.44, 126.56, 126.21, 125.01, 124.00, 123.70, 122.86, 121.49, 121.24,
ꢀ
6
1
1
19.43, 113.11, 112.39, 111.61, 110.81, 104.67, 56.39, 55.97, 33.22. ESI-MS: 411.45
+
(
C H N O , [M+H] ). Anal. Calcd for C H N O : C, 72.96; H, 5.13; N, 10.24; O,
2
5
21
3
3
25 21
3
3
1
4
1.67; Found: C, 72.98; H, 5.14; N, 10.21; O, 11.67.
.5.6. 1-(3, 4, 5-trimethoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole
(10f)
1
Yellow powder, yield: 72%. M. p: 183 – 184 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.58 (s, 6H); 3.63 (s, 3H); 3.69 (s, 3H); 6.95 (s, 2H); 7.25 – 7.30 (m, 2H); 7.32 –
7
7
.34 (m, 1H); 7.37 – 7.43 (m, 1H); 7.45 (s, 1H); 7.50 – 7.52 (m, 1H); 7.57 (s, 1H);
1
3
.82 (d, J = 8, 1H); 8.09 (d, J = 7.6, 1H). C NMR (DMSO-d , 100 MHz)
ꢀ: 169.33,
6
1
1
1
53.12, 152.82, 149.86, 143.71, 142.45, 136.76, 134.63, 133.01, 128.45, 126.49,
24.50, 124.10, 122.79, 121.11, 121.01, 119.59, 113.18, 110.62, 108.38, 106.96,
+
04.94, 60.66, 56.33, 33.01. ESI-MS: 441.48 (C H N O , [M+H] ). Anal. Calcd for
2
6
23
3
4
C H N O : C, 70.72; H, 5.26; N, 9.52; O, 14.50; Found: C, 70.73; H, 5.25; N, 9.52;
26
23
3
4
O, 14.50.
4
.5.7. 1-(2-bromo-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10g)
1
White powder, yield: 82%. M. p: 160 ꢀ, H NMR (DMSO-d , 400 MHz) δ:
6
3
7
1
6
9
4
.71 (s, 3H); 7.20 – 7.21 (m, 1H); 7.22 – 7.25 (m, 2H); 7.26 – 7.27 (m, 1H); 7.39 –
.42 (m, 3H); 7.60 (m, 1H); 7.67 (s, 1H); 7.70 (m, 1H); 7.81 – 7.83 (m, 2H); 8.01 (s,
+
H). ESI-MS: 430.30 (C H BrN O, [M+H] ). Anal. Calcd for C H BrN O: C,
23
16
3
23 16
3
4.19; H, 3.74; Br, 18.57; N, 9.78; O, 3.73; Found: C, 64.20; H, 3.73; Br, 18.57; N,
.77; O, 3.74.
.5.8. 1-(3-bromo-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10h)
1
White powder, yield: 79%. M. p: 153 – 154 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.71 (s, 3H); 7.19 – 7.21 (m, 1H); 7.22 – 7.23 (m, 1H); 7.24 – 7.25 (m, 1H); 7.26 –
7
.32 (m, 1H); 7.39 – 7.45 (m, 3H); 7.60 (d, J = 8, 1H); 7.67 (s, 1H); 7.70 (d, J = 0.8,
H); 7.81 – 7.83 (m, 2H); 8.03 (d, J = 7.6, 1H). ESI-MS: 430.30 (C H BrN O,
1
2
3
16
3
1
7

ACCEPTED MANUSCRIPT
+
[
M+H] ). Anal. Calcd for C H BrN O: C, 64.19; H, 3.74; Br, 18.57; N, 9.78; O,
23 16 3
3
4
.73; Found: C, 64.20; H, 3.75; Br, 18.57; N, 9.77; O, 3.72.
.5.9. 1-benzene acyl-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11a)
1
White powder, yield: 86%. M. p: 161 – 162 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.70 (s, 3H); 3.71 (s, 3H); 6.87 – 6.89 (m, 1H); 7.15 – 7.17 (m, 1H); 7.20 – 7.24 (m,
1
H); 7.32 – 7.36 (m, 2H); 7.43 – 7.47 (m, 2H); 7.58 – 7.59 (m, 1H); 7.63 – 7.67 (m,
H); 7.77 (s, 1H); 7.78 – 7.79 (m, 1H); 7.82 (s, 1H). ESI-MS: 381.43 (C H N O ,
2
2
4
19
3
2
+
[
M+H] ). Anal. Calcd for C H N O : C, 75.55; H, 5.01; N, 11.03; O, 8.41; Found: C,
24 19 3 2
7
4
5.57; H, 5.02; N, 11.02; O, 8.39.
.5.10.
1-(2-methoxy-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11b)
1
Yellow powder, yield: 76%. M. p: 158 – 159 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.79 (s, 6H); 3.68 – 3.70 (m, 6H); 3.72 – 3.73 (m, 3H); 6.78 – 6.79 (m, 1H); 7.23 –
7
.25 (m, 3H); 7.34 – 7.36 (m, 4H); 7.61 – 7.63 (m, 3H); 7.81 (s, 1H). ESI-MS: 411.45
+
(
C H N O , [M+H] ). Anal. Calcd for C H N O : C, 72.97; H, 5.15; N, 10.21; O,
2
5
21
3
3
25 21
3
3
1
4
1.67; Found: C, 72.98; H, 5.14; N, 10.21; O, 11.67.
.5.11. 1-(3-methoxy-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11c)
1
Yellow powder, yield: 74%. M. p: 153 – 154 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.77 (s, 6H); 3.68 – 3.73 (m, 9H); 6.86 – 6.89 (m, 1H); 7.23 – 7.24 (m, 3H); 7.31 –
7
.36 (m, 5H); 7.61 – 7.63 (m, 2H); 7.80 – 7.82 (m, 1H). ESI-MS: 411.45 (C H N O ,
25 21 3 3
+
[
M+H] ). Anal. Calcd for C H N O : C, 72.97; H, 5.15; N, 10.21; O, 11.67; Found:
25 21 3 3
C, 72.98; H, 5.14; N, 10.21; O, 11.67.
.5.12.
4
1-(4-methoxy-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11d)
1
Yellow powder, yield: 87%. M. p: 195 – 196 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.76 (s, 3H); 3.78 (s, 3H); 3.82 (s, 3H); 7.01 – 7.03 (m, 1H); 7.16 – 7.18 (m, 1H);
7
7
.20 – 7.22 (m, 2H); 7.29 – 7.31 (m, 2H); 7.32 – 7.35 (m, 1H); 7.37 – 7.38 (m, 1H);
.48 – 7.50 (m, 1H); 7.62 (s, 2H); 8.25 (s, 1H). ESI-MS: 411.45 (C H N O ,
25
21
3
3
+
[
M+H] ). Anal. Calcd for C H N O : C, 72.96; H, 5.13; N, 10.24; O, 11.67; Found:
25 21 3 3
1
8

ACCEPTED MANUSCRIPT
C, 72.97; H, 5.13; N, 10.23; O, 11.67.
.5.13. 1-(3, 4-dimethoxy-benzene
4
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11e)
1
Yellow powder, yield: 83%. M. p: 126 – 127 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.71 – 3.73 (m, 9H); 3.83 (s, 3H); 6.89 (s, 1H); 7.02 – 7.04 (m, 1H); 7.14 – 7.16 (m,
1
2
H); 7.19 – 7.23 (m, 1H); 7.30 – 7.34 (m, 1H); 7.37 – 7.41 (m, 3H); 7.61 – 7.65 (m,
1
3
H); 7.79 (d, J = 8, 1H). C NMR (DMSO-d , 100 MHz)
ꢀ: 169.21, 155.16, 154.77,
6
1
1
5
7
4
50.17, 149.19, 143.57, 134.87, 132.66, 132.15, 126.99, 126.32, 124.97, 123.96,
23.60, 119.39, 113.11, 112.80, 112.32, 111.69, 111.63, 104.42, 102.94, 56.41, 56.01,
+
5.68, 33.38. ESI-MS: 441.48 (C H N O , [M+H] ). Anal. Calcd for C H N O : C,
2
6
23
3
4
26 23
3
4
0.72; H, 5.24; N, 9.54; O, 14.50; Found: C, 70.73; H, 5.25; N, 9.52; O, 14.50.
.5.14. 1-(3, 4, 5-trimethoxy-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11f)
1
Yellow powder, yield: 72%. M. p: 131 – 132 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.83 (s, 12H); 6.87 – 6.89 (m, 1H); 6.90 – 6.92 (m, 1H); 7.07 – 7.12 (m, 4H); 7.40
–
7.43 (m, 3H); 7.96 – 7.97 (m, 2H); 8.05 (s, 2H). ESI-MS: 471.18 (C H N O ,
27 25 3 5
+
[
M+H] ). Anal. Calcd for C H N O : C, 68.78; H, 5.34; N, 8.91; O, 16.97; Found: C,
27 25 3 5
6
4
8.77; H, 5.33; N, 8.93; O, 16.97.
.5.15. 1-(2-bromo-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11g)
1
Yellow powder, yield: 82%. M. p: 185 – 187 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.63 (s, 3H); 3.78 (s, 3H); 3.87 (s, 1H); 6.83 – 6.86 (m, 1H); 7.25 – 7.26 (m, 1H);
7
.29 (s, 1H); 7.30 – 7.32 (m, 1H); 7.34 – 7.39 (m, 2H); 7.42 (s, 1H); 7.44 (s, 1H); 7.57
1
3
(
s, 2H); 7.81 (d, J = 8, 1H). C NMR (DMSO-d , 100 MHz)
ꢀ
: 168.18, 155.06,
6
1
1
5
49.99, 143.82, 136.06, 134.06, 133.70, 133.12, 133.03, 131.88, 131.80, 127.68,
27.45, 125.26, 124.76, 119.90, 119.80, 113.95, 112.61, 111.29, 104.00, 102.65,
+
5.87, 33.17. ESI-MS: 460.32 (C H BrN O , [M+H] ). Anal. Calcd for
2
4
18
3
2
C H BrN O : C, 62.60; H, 3.93; Br, 17.37; N, 9.13; O, 6.97; Found: C, 62.62; H,
24
18
3
2
3
.94; Br, 17.36; N, 9.13; O, 6.95.
1
9

ACCEPTED MANUSCRIPT
4
.5.16. 1-(3-bromo-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11h)
1
Yellow powder, yield: 76%. M. p: 142 – 143 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.87 (s, 6H); 6.92 – 6.95 (m, 1H); 7.13 – 7.15 (m, 2H); 7.45 – 7.50 (m, 2H); 7.53 –
7
.56 (m, 2H); 7.83 – 7.84 (m, 1H); 7.95 – 7.96 (m, 1H); 8.03 (s, 1H); 8.07 (s, 2H).
+
ESI-MS: 460.32 (C H BrN O , [M+H] ). Anal. Calcd for C H BrN O : C, 62.60;
24
18
3
2
24 18
3
2
H, 3.93; Br, 17.37; N, 9.13; O, 6.97; Found: C, 62.62; H, 3.94; Br, 17.36; N, 9.13; O,
6
.95.
4
.5.17. 1-benzene acyl-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12a)
1
Yellow powder, yield: 81%. M. p: 127 – 129 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.83 (s, 3H); 6.94 – 6.96 (m, 1H); 7.15 – 7.16 (m, 2H); 7.35 – 7.37 (m, 2H); 7.45 –
7
1
.46 (m, 1H); 7.53 – 7.56 (m, 2H); 7.83 – 7.84 (m, 1H); 7.95 – 7.96 (m, 1H); 8.03 (s,
+
H); 8.07 (s, 2H). ESI-MS: 430.30 (C H BrN O, [M+H] ). Anal. Calcd for
23
16
3
C H BrN O: C, 64.20; H, 3.75; Br, 18.57; N, 9.77; O, 3.72; Found: C, 64.21; H,
23
16
3
3
.76; Br, 18.56; N, 9.77; O, 3.71.
4
.5.18.
1-(2-methoxy-benzene
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12b)
1
Yellow powder, yield: 91%. M. p: 134 – 136 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.86 (s, 6H); 6.91 – 6.95 (m, 2H); 7.13 – 7.15 (m, 2H); 7.45 – 7.47 (m, 1H); 7.52 –
7
.54 (m, 2H); 7.83 – 7.84 (m, 2H); 8.03 (s, 1H); 8.05 (s, 2H). ESI-MS: 460.32
+
(
C H BrN O , [M+H] ). Anal. Calcd for C H BrN O : C, 62.62; H, 3.94; Br,
2
4
18
3
2
24 18
3
2
1
7.36; N, 9.13; O, 6.95; Found: C, 62.61; H, 3.95; Br, 17.37; N, 9.14; O, 6.95.
.5.19. 1-(3-methoxy-benzene
4
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12c)
1
Yellow powder, yield: 69%. M. p: 136 – 137 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.84 (s, 3H); 3.86 (s, 3H); 6.92 – 6.95 (m, 2H); 7.15 – 7.17 (m, 1H); 7.48 – 7.50 (m,
2
H); 7.53 – 7.56 (m, 2H); 7.83 – 7.84 (m, 1H); 7.95 – 7.96 (m, 1H); 8.03 (s, 2H); 8.08
+
(
s, 1H). ESI-MS: 460.32 (C H BrN O , [M+H] ). Anal. Calcd for C H BrN O : C,
2
4
18
3
2
24 18
3
2
6
2.62; H, 3.94; Br, 17.36; N, 9.13; O, 6.95; Found: C, 62.63; H, 3.95; Br, 17.37; N,
9
.11; O, 6.94.
2
0

ACCEPTED MANUSCRIPT
4
.5.20.
1-(4-methoxy-benzene
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12d)
1
Yellow powder, yield: 85%. M. p: 151 – 152 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.83 (s, 3H); 3.86 (s, 3H); 7.12 – 7.14 (m, 2H); 7.45 – 7.48 (m, 2H); 7.50 – 7.52 (m,
2
H); 7.73 – 7.74 (m, 1H); 7.95 – 7.96 (m, 2H); 7.98 – 8.01 (m, 1H); 8.07 (s, 2H).
+
ESI-MS: 460.32 (C H BrN O , [M+H] ). Anal. Calcd for C H BrN O : C, 62.62;
2
4
18
3
2
24 18
3
2
H, 3.94; Br, 17.36; N, 9.13; O, 6.95; Found: C, 62.64; H, 3.96; Br, 17.36; N, 9.11; O,
6
.93.
4
.5.21.
1-(3,
4,
-dimethoxy-benzene
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12e)
1
Yellow powder, yield: 79%. M. p: 121 – 123 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.71 (s, 3H); 3.76 (s, 3H); 3.83 (s, 3H); 7.01 – 7.03 (m, 1H); 7.16 – 7.18 (m, 1H);
7
7
.22 – 7.26 (m, 1H); 7.31 – 7.35 (m, 2H); 7.39 – 7.45 (m, 2H); 7.49 – 7.51 (m, 1H);
.66 (s, 1H); 7.83 – 7.86 (m, 1H); 8.5 (s, 1H). ESI-MS: 490.35 (C H BrN O ,
2
5
20
3
3
+
[
M+H] ). Anal. Calcd for C H BrN O : C, 61.24; H, 4.11; Br, 16.30; N, 8.57; O,
25 20 3 3
9
4
.79; Found: C, 61.23; H, 4.12; Br, 16.30; N, 8.57; O, 9.79.
.5.22. 1-(3, 4,
5-trimethoxy-benzene
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12f)
Yellow powder, yield: 76%. M. p: 142 – 143 ꢀ, 1H NMR (DMSO-d6, 400
MHz) δ: 3.62 (s, 6H); 3.64 (s, 3H); 3.70 (s, 3H); 6.89 (s, 2H); 7.30 – 7.41 (m, 3H);
13
7
.45 – 7.52 (m, 2H); 7.64 (s, 1H); 7.83 – 7.85 (s, 1H); 8.27 (s, 1H). C NMR
(
DMSO-d , 100 MHz) : 169.23, 152.90, 149.18, 143.58, 142.55, 135.54, 134.61,
ꢀ
6
1
1
34.21, 128.42, 128.21, 125.28, 124.57, 124.29, 123.25, 119.72, 113.94, 113.28,
12.88, 108.45, 104.57, 60.69, 56.37, 33.25. ESI-MS: 520.37 (C H BrN O ,
2
6
22
3
4
[
M+H]+). Anal. Calcd for C H BrN O : C, 60.01; H, 4.26; Br, 15.36; N, 8.07; O,
26 22 3 4
1
4
2.30; Found: C, 62.63; H, 3.96; Br, 17.36; N, 9.11; O, 6.94.
.5.23. 1-(2-bromo-benzene acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole
(12g)
1
White powder, yield: 86%. M. p: 155 – 157 ꢀ, H NMR (DMSO-d , 400 MHz)
6
2
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δ: 3.65 (s, 3H); 7.25 – 7.28 (m, 3H); 7.38 – 7.40 (m, 2H); 7.44 – 7.48 (m, 1H); 7.65 (s,
1
2
5
8
4
H); 7.76 – 7.79 (m, 1H); 7.82 – 7.83 (m, 1H); 7.97 – 7.99 (m, 1H); 7.99 – 8.01 (m,
+
H). ESI-MS: 509.19 (C H Br N O, [M+H] ). Anal. Calcd for C H Br N O: C,
23
15
2
3
23 15
2
3
4.25; H, 2.97; Br, 31.38; N, 8.25; O, 3.14; Found: C, 54.23; H, 2.98; Br, 31.38; N,
.26; O, 3.14.
.5.24. 1-(3-bromo-benzene acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole
(12h)
1
White powder, yield: 76%. M. p: 163 – 164 ꢀ, H NMR (DMSO-d , 400 MHz)
6
δ: 3.64 (s, 3H); 7.15 – 7.23 (m, 3H); 7.37 – 7.39 (m, 1H); 7.42 – 7.52 (m, 2H); 7.67 (s,
1
1
H); 7.78 – 7.81 (m, 1H); 7.84 – 7.86 (m, 1H); 7.98 – 7.99 (m, 1H); 8.03 – 8.07 (m,
+
H); 8.18 – 8.21 (m, 1H). ESI-MS: 509.19 (C H Br N O, [M+H] ). Anal. Calcd for
2
3
15
2
3
C H Br N O: C, 54.25; H, 2.97; Br, 31.38; N, 8.25; O, 3.14; Found: C, 54.23; H,
23
15
2
3
2
.98; Br, 31.38; N, 8.26; O, 3.14.
4
.6. Crystal structure determination
Crystal structure determination of compound 10c was carried out on a Nonius
CAD4 diffractometer equipped with graphite-mono chromated MoKa (k = 0.71073 Å)
2
radiation. The structures were solved by direct methods and refined on F by
fullmatrix least-squares methods using SHELX-97. [40] All non-hydrogen atoms of
compound 10c were refined with anisotropic thermal parameters. All hydrogen atoms
were placed in geometrically idealized positions and constrained to ride on their
parent atoms.
4
.7. Cell proliferation assay (Cell viability was assessed by MTT assay)
We evaluated the antiproliferative activities of compounds 10a - 12h against
A549 (human lung adenocarcinoma cells), HepG2 (human liver hepatocellular
carcinoma) and MCF-7 (human breast carcinoma cells) with analysis. Cell
proliferation was determined using MTT dye (Beyotime Inst Biotech, China)
3
according to the instructions of manufacture. Briefly, (1 – 5) × 10 cells per well were
2
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seeded in a 96-well plate, grown at 37 ꢀ for 12 h. Subsequently, cells were treated
with compounds (Table 2) at increasing concentrations in the presence of 10% FBS
for 24 h. After 10 ꢀL MTT dye was added to each well, cells were incubated at 37 ꢀ
for 3 – 4 h. Then all the solution in the wells was poured out and 150 ꢀL DMSO was
added to every well. Plates were read in a Victor-V multilabel counter (Perkin-Elmer)
using the default europium detection protocol. Percent inhibition or GI values of
5
0
compounds were calculated by comparison with DMSO-treated control wells. The
results are shown in Table 2.
4
.8. Effects on Tubulin Polymerization and on Colchicine Binding to Tubulin.
To evaluate the effect of the compounds on tubulin polymerization in vitro,
different concentrations of 26 compounds (containing Colchicine and CA-4) were
pre-incubated with 10 ꢀM bovine brain tubulin in glutamate buffer at 30 ꢀ and then
cooled to 0 ꢀ. After addition of 0.4 mM GTP, the mixtures were transferred to 0 ꢀ
cuvettes in a recording spectrophotometer and warmed to 30 ꢀ . Tubulin
polymerization was followed turbidimetrically at 350 nm. The IC was defined as the
5
0
compound concentration that inhibited the extent of polymerization by 50% after 20
min incubation.
The capacity of the test compounds to inhibit Colchicine binding to tubulin was
measured as described above (tubulin polymerization effect) except that the reaction
3
mixtures contained 1 ꢀM tubulin, 5 ꢀM [ H]Colchicine, and 5 ꢀM test compound.
4
4
.9. Flow cytometry
.9.1. Cell Cycle Analysis
The synchronization was performed by incubating A549 cells in DMEM
supplemented with 0.5% FBS for 12h. Then cells were incubated in fesh DMEM
supplemented with compound 11f for 24h. Except control culture, three different
concentrations: 1 ꢀM, 2 ꢀM and 5 ꢀM of compound 11f, were chosen to examine the
dose effect. The control cells were teated with DMSO. After incubation, cells were
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harvested, washed with cold PBS and fixed with 70% ethanol at 4 ꢀ overnight. The
fixed cells were washed with PBS, stained with 50 ꢀg/ml of propidium iodide (PI)
containing 100 ꢀg/ml of Rnase A and 1% Triton X-100 in the dark for 45 min, and
then subjected to flow cytometric analysis.
4
.9.2. Analysis of apoptosis
5
Approximately 10 cells/well were plated in a 12 well plate and allowed to
adhere. After 12 h, the medium was replaced with fresh culture medium containing
compound 11f at final concentrations of 0, 1 ꢀM, 2 ꢀM and 5 ꢀM. Then cells were
harvested after 24 h.
They were trypsinized, washed in PBS and centrifuged at 2000rpm for 5 min.
The pellet was then resuspended in 500 ꢀL of staining solution (containing 5 ꢀL
Annexin V-PE and 5 ꢀL PI in Binding Buffer), mixed gently and incubated for 15 min
at room temperature (15 – 25 ꢀ) in dark. The samples were then read in a
FACScalibur flow cytometer (USA) at 488 nm excitation. Analyses were performed
by the software supplied in the instrument.
4
.10. Docking simulations
The three-dimensional X-ray structure of tubulin (PDB code: 1SA0) was chosen
as the template for the modeling study of compound 11f. The pdb file about the
crystal structure of the tubulin domain bound to colchicine (CN2700) (1SA0.pdb) was
obtained from the RCSB protein data bank (http://www.pdb.org). The molecular
docking procedure was performed by using CDOCKER protocol for receptor-ligand
interactions section of Discovery Studio 3.1 (Accelrys Software Inc, Organic &
Biomolecular Chemistry Page 20 of 3419 San Diego, CA). [41] All bound water and
ligands were eliminated from the protein and the polar hydrogen was added. The
whole tubulin complex was defined as a receptor and the site sphere was selected
based on the ligand binding location of colchicine (CN2700), then the CN2700
molecule was removed and compound 11f was placed during the molecular docking
2
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procedure. Types of interactions of the docked protein with ligand were analyzed after
the end of molecular docking.
4
.11. 3D-QSAR
Ligand-based 3D-QSAR approach was performed by QSAR software of DS 3.1
(Discovery Studio 3.1, Accelrys, Co. Ltd). The training sets were composed of
inhibitors with the corresponding pIC50 values which were converted from the
obtained IC (ꢀM), and test sets comprised compounds of data sets as list in Table 5.
5
0
All the definition of the descriptors can be seen in the “Help” of DS 3.1 software and
they were calculated by QSAR protocol of DS 3.1. The alignment conformation of
each molecule was the one with lowest interaction energy in the docked results of
CDOCKER. The predictive ability of 3D-QSAR modeling can be evaluated based on
the cross-validated correlation coefficient, which qualifies the predictive ability of the
models. Scrambled test (Y scrambling) was performed to investigate the risk of
chance correlations. The inhibitory potencies of compounds were randomly reordered
for 30 times and subject to leave-one-out validation test, respectively. The models
were also validated by test sets, in which the compounds were not included in the
2
training sets. Usually, one can believe that the modeling is reliable, when the R for
test sets is larger than 0.6, respectively.
Acknowledgments
The work was financed by a grant from Major Projects on Control and
Recti□cation of Water Body Pollution (no. 2011ZX07204-001-004), and Supported by
“PCSIRT” (IRT1020).
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