Accepted Manuscript Synthesis, Biological Evaluation, and Molecular Docking Studies of Novel 1benzene 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
DOI:
10.1016/j.ejmech.2015.05.021
Reference:
EJMECH 7900
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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Synthesis, Biological Evaluation, and Molecular Docking Studies of Novel 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole
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Derivatives as Potential Tubulin Polymerization Inhibitors
Yan-Ting Wang, Ya-Juan Qin, Na-Yang, Ya-Liang Zhang, Chang-Hong Liu*, Hai-Liang Zhu*
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State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
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Nanjing210093, P. R. China
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*Corresponding author. Tel. & fax: +86-25-89682572; e-mail:
[email protected] Crystal structure diagram of compound 10c
1
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site
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3D diagram of the interaction between Compound 11f and the colchicine binding
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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
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inhibitory activity. Docking simulation and the QSAR study were conducted.
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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.
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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
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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
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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;
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molecular docking; 3D-QSAR.
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ACCEPTED MANUSCRIPT 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
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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
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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
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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
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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
14]
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dynamics leads to cell cycle arrest in the G2/M phase and induction of apoptosis. [13,
There are a great quantity of evidences, derived from molecular modeling studies
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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
ACCEPTED MANUSCRIPT 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
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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
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showed that agents containing benzimidazole possessed good antitumor activity. [29] They could affect the dynamic loop of the tubulin polymerization and
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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
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modeling studies to design and synthesis new tubulin inhibitors: compounds 10a 12h. 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
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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
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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
ACCEPTED MANUSCRIPT 2. Results and Discussion 2.1. Chemistry. The synthetic route for compounds 10a - 12h is outlined in Scheme 1. These
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compounds were synthesized from amine 10 - 12 and acid a - h. And compounds 10 12 were synthesized from aldehyde 7 - 9 and o-phenylenediamine. Compounds 7 - 9 were prepared according to the procedure reported by Mark A. Seefeld et al. and Debajyoti Saha et al. with some modifications. [35, 36]
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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
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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
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in DMF, respectively. The heterogeneous mixture was stirred at 110
for 4h, then
compounds 10 - 12 were obtained. Fourthly, The acids a - h and SOCl2 were mixed and stirred at 80
for 4 h to give reactive acyl chloride, respectively. Finally, the
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corresponding acyl chloride of acids (a - h) were added dropwise to the amine compounds (10 - 12) in ethyl acetate containing triethylamine at 0
, respectively.
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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 CH2Cl2. Compounds 10a - 12h were fully characterized by 1H NMR, ESI-MS and elemental analysis. (Scheme 1) (Table 1) 2.2. Crystal structure determination
6
ACCEPTED MANUSCRIPT 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)
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(Figure 2) 2.3. Biological Evaluation
2.3.1. In Vitro Antiproliferative Activities (Cell viability was assessed by MTT
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assay)
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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
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compounds showed remarkable antiproliferative effects mainly for A549 cells. The new compounds 11f, 10f and 11e showd more active than CA-4 (GI50 = 2.8 µM for A549, GI50 = 7.4 µM for HepG2, and GI50 = 9.4 µM for MCF-7, respectively) in antiproliferativivities.
follows:
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Structure-activity relationships in these novel derivatives demonstrated as
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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: OCH3 > H > 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
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than electron-withdrawing groups, which could be seen from the comparison: 10c > 10a > 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 >
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11e > 11c, 12f > 12e > 12c.
Strikingly, the most potent small molecule screened via these antiproliferation
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assays was compound 11f which suppresses the A549, HepG2 and MCF-7 cells with the GI50 = 2.4 µM, GI50 = 3.8 µM and GI50 = 5.1 µM respectively. (Table 2)
2.3.2. Inhibition of tubulin polymerization and the binding of Colchicine to
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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 in
vitro
(Table
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polymerization
acyl-2-(1-methylindol-3-yl)-benzimidazole
3).
The
derivatives
new inhibited
1-benzene tubulin
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polymerization with IC50 values ranging from 1.5 to 45.6 µM, as compared with 1.8 µM for CA-4. As tubulin polymerization inhibitors, compounds 11f, 10f and 11e were stronger than CA-4 (IC50=1.8 µM); compounds 11f, 10f , 11e, 12f, 10e and 12e were stronger than Colchicine (IC50=2.62 µM). The experimental results showed that the most potent novel compound as inhibitor was 11f, which had an IC50 of 1.5 µM. The new 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives were also examined for potential inhibition of the binding of [3H]Colchicine to tubulin (Table 3). All except 10g and 12g were strong inhibitors of the binding reaction (51 96% 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)
We
hypothesized
that
the
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2.3.3. Cell Cycle Analysis 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
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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
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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
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concentration-dependent manner (Figure. 3). About 27.32% of cells were found in G2/M phase when treated with 1 µM compound 11f for 24 h; about 33.21% of cells for 2 µM; and about 39.46% of cells for 5 µM.
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(Figure 3)
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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
ACCEPTED MANUSCRIPT 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 µM compound 11f for 24 h. And with the increasing of the compound concentration, 22.13% of cells were found to be apoptotic
(Figure 4) 2.4. Docking simulations
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for 2 µM; 23.23% of cells were found to be apoptotic for 5 µM.
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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
structure (PDB code: 1SA0).
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1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives with tubulin crystal
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
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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
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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
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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 10
ACCEPTED MANUSCRIPT 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
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biological assay data, could suggest that compound 11f was the most potential inhibitors of tubulin. (Table 4)
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(Figure 5) 2.5. QSAR model
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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 pIC50 values which were converted from the obtained IC50 (µM) values
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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
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(http://www.sanjeevslab.org/tools-IC50.html). [38] The training and test set were chosen by the Diverse Molecules method in
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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]
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It was a PLS model set up 400 independent variables (conventional R2 = 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 pIC50 value and experimental pIC50 value for both test
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sets and training sets were shown in Figure 6.
Also the molecules aligned with the iso-surfaces of the 3D-QSAR model
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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.
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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
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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
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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 12
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
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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
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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
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treatment of advanced cancers. 4. Experimental Section
4.1. Materials and measurements
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All chemicals and reagents used in current study were analytical grade. Thin layer chromatography (TLC), proton nuclear magnetic resonance (1H NMR) and elemental
microanalyses
(CHN)
were
usually
used.
Analytical
thin-layer
chromatography (TLC) was performed on the glass-backed silica gel sheets (silica gel
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60 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
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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 MP apparatus (Taike Corp., Beijing, China). 1H NMR spectra were measured on a Bruker AV-300 or AV-500 spectrometer at 25
and referenced to Me4Si. Chemical
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. 13
ACCEPTED MANUSCRIPT 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
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poured onto ice. The mixture was made basic with a solution of NaOH (330 mmol, 13.2 g) in H2O (44 mL), then was extracted with Et2O (2 × 50 mL). The combined organic layers were washed with brine, dried (MgSO4), and concentrated under
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vacuum. Flash chromatography on silica gel (10% ethyl acetate/hexanes) gave the title compounds 4 - 6 as an off-white solid, respectively. General
procedure
for
preparation
of
Compounds 7 - 9 were synthesized from the corresponding compounds
4 - 6. A
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4.3.
1-Methyl-1H-indole-3-carboxaldehyde (7 - 9)
solution of compounds 4 - 6 (60 mmol) in THF (30 mL) were added dropwise to a
mL) at 0
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suspension of NaH (3.60 g, 60% dispersion in mineral oil, 150 mmol) in THF (30 . 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 0 , quenched with saturated NH4Cl (60 mL), and extracted
over
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with ether (3 × 50 mL). The organic layers were combined, washed with brine, dried anhydrous
Na2SO4
and
concentrated
in
vacuo
to
give
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1-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. 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, 100 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 14
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)
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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 SOCl2 (6 - 10 mL) were 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
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mL) was added dropwise to the corresponding suspension of amine compounds (10 12) (0.5 mM) and triethylamine (0.5 mL) in 20 mL ethyl acetate at 0
.After stirring
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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%.
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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 CH2Cl2, then the reaction mixture was
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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).
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The combined organic phases were dried with anhydrous Na2SO4. Removal of all the 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) White powder, yield: 77%. M. p: 127 – 129
, 1H NMR (DMSO-d6, 400 MHz)
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 – 7.65 (m, 1H); 7.66 (s, 1H); 7.72 (s, 1H); 7.74 (s, 1H); 7.81 (d, J = 8, 1H); 8.16 (d, J = 7.2, 1H). 13C NMR (DMSO-d6, 100 MHz)
: 170.06, 149.94, 143.66, 136.90, 134.68,
134.53, 133.37, 132.98, 130.70, 129.25, 126.56, 124.38, 123.94, 122.83, 121.26, 15
ACCEPTED MANUSCRIPT 121.23, 119.56, 112.73, 110.74, 104.67, 56.50, 33.15, 19.04. ESI-MS: 351.40 (C23H17N3O, [M+H]+). Anal. Calcd for C23H17N3O: C, 78.60; H, 4.87; N, 11.97; O, 4.56; Found: C, 78.61; H, 4.88; N, 11.96; O, 4.55. 4.5.2. 1-(2-methoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10b) , 1H NMR (DMSO-d6, 400 MHz)
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Yellow powder, yield: 84%. M. p: 137 – 138
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 (C24H19N3O2, [M+H]+). Anal. Calcd for C24H19N3O2: C, 75.57; H, 5.02; N, 11.02; O, 8.39; Found: C, 75.56; H, 5.02;
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N, 11.04; O, 8.38.
4.5.3. 1-(3-methoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10c) , 1H NMR (DMSO-d6, 400 MHz)
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Yellow powder, yield: 87%. M. p: 146 – 147
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, 1H).
13
C NMR (DMSO-d6, 100 MHz)
: 169.81, 159.58, 149.92, 143.67, 136.87,
134.80, 134.53, 132.99, 130.36, 126.54, 124.44, 124.00, 123.07, 122.82, 121.24,
TE D
121.21, 120.96, 119.56, 114.84, 112.88, 110.75, 104.71, 55.78, 33.12. ESI-MS: 381.43 (C24H19N3O2, [M+H]+). Anal. Calcd for C24H19N3O2: C, 75.55; H, 5.03; N, 11.03; O, 8.39; Found: C, 75.57; H, 5.02; N, 11.02; O, 8.39.
EP
4.5.4. 1-(4-methoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10d) Yellow powder, yield: 77%. M. p: 155 – 156
, 1H NMR (DMSO-d6, 400 MHz)
AC C
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, 1H); 7.25 – 7.27 (m, 1H); 7.32 – 7.35 (m, 2H); 7.37 – 7.38 (m, 1H); 7.48 – 7.50 (m, 1H); 7.62 (s, 1H); 7.80 (m, 2H); 8.25 (s, 1H). ESI-MS: 381.43 (C24H19N3O2, [M+H]+). Anal. Calcd for C24H19N3O2: C, 75.57; H, 5.02; N, 11.02; O, 8.39; Found: C, 75.55; H, 5.01; N, 11.05; O, 8.39. 4.5.5. 1-(3, 4-dimethoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10e) Yellow powder, yield: 87%. M. p: 205 – 206
, 1H NMR (DMSO-d6, 400 MHz)
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); 7.20 – 7.23 (m, 2H); 7.25 – 7.27 (m, 1H); 7.32 – 7.35 (m, 2H); 7.37 – 7.38 (m, 1H); 16
ACCEPTED MANUSCRIPT 7.48 – 7.50 (m, 1H); 7.62 (s, 1H); 7.80 (d, J = 7.6, 1H); 8.25 (d, J = 7.6, 1H). NMR (DMSO-d6, 100 MHz)
13
C
: 169.28, 154.70, 149.84, 149.13, 143.59, 136.95,
134.90, 132.44, 126.56, 126.21, 125.01, 124.00, 123.70, 122.86, 121.49, 121.24, 119.43, 113.11, 112.39, 111.61, 110.81, 104.67, 56.39, 55.97, 33.22. ESI-MS: 411.45
11.67; Found: C, 72.98; H, 5.14; N, 10.21; O, 11.67.
RI PT
(C25H21N3O3, [M+H]+). Anal. Calcd for C25H21N3O3: C, 72.96; H, 5.13; N, 10.24; O,
4.5.6. 1-(3, 4, 5-trimethoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole
Yellow powder, yield: 72%. M. p: 183 – 184
, 1H NMR (DMSO-d6, 400 MHz)
SC
(10f)
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 –
M AN U
7.34 (m, 1H); 7.37 – 7.43 (m, 1H); 7.45 (s, 1H); 7.50 – 7.52 (m, 1H); 7.57 (s, 1H); 7.82 (d, J = 8, 1H); 8.09 (d, J = 7.6, 1H). 13C NMR (DMSO-d6, 100 MHz)
: 169.33,
153.12, 152.82, 149.86, 143.71, 142.45, 136.76, 134.63, 133.01, 128.45, 126.49, 124.50, 124.10, 122.79, 121.11, 121.01, 119.59, 113.18, 110.62, 108.38, 106.96,
TE D
104.94, 60.66, 56.33, 33.01. ESI-MS: 441.48 (C26H23N3O4, [M+H]+). Anal. Calcd for C26H23N3O4: C, 70.72; H, 5.26; N, 9.52; O, 14.50; Found: C, 70.73; H, 5.25; N, 9.52; O, 14.50.
4.5.7. 1-(2-bromo-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10g)
EP
White powder, yield: 82%. M. p: 160
, 1H NMR (DMSO-d6, 400 MHz) δ:
3.71 (s, 3H); 7.20 – 7.21 (m, 1H); 7.22 – 7.25 (m, 2H); 7.26 – 7.27 (m, 1H); 7.39 –
AC C
7.42 (m, 3H); 7.60 (m, 1H); 7.67 (s, 1H); 7.70 (m, 1H); 7.81 – 7.83 (m, 2H); 8.01 (s, 1H). ESI-MS: 430.30 (C23H16BrN3O, [M+H]+). Anal. Calcd for C23H16BrN3O: C, 64.19; H, 3.74; Br, 18.57; N, 9.78; O, 3.73; Found: C, 64.20; H, 3.73; Br, 18.57; N, 9.77; O, 3.74.
4.5.8. 1-(3-bromo-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10h) White powder, yield: 79%. M. p: 153 – 154
, 1H NMR (DMSO-d6, 400 MHz)
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, 1H); 7.81 – 7.83 (m, 2H); 8.03 (d, J = 7.6, 1H). ESI-MS: 430.30 (C23H16BrN3O, 17
ACCEPTED MANUSCRIPT [M+H]+). Anal. Calcd for C23H16BrN3O: C, 64.19; H, 3.74; Br, 18.57; N, 9.78; O, 3.73; Found: C, 64.20; H, 3.75; Br, 18.57; N, 9.77; O, 3.72. 4.5.9. 1-benzene acyl-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11a) White powder, yield: 86%. M. p: 161 – 162
, 1H NMR (DMSO-d6, 400 MHz)
RI PT
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, 1H); 7.32 – 7.36 (m, 2H); 7.43 – 7.47 (m, 2H); 7.58 – 7.59 (m, 1H); 7.63 – 7.67 (m, 2H); 7.77 (s, 1H); 7.78 – 7.79 (m, 1H); 7.82 (s, 1H). ESI-MS: 381.43 (C24H19N3O2, [M+H]+). Anal. Calcd for C24H19N3O2: C, 75.55; H, 5.01; N, 11.03; O, 8.41; Found: C,
SC
75.57; H, 5.02; N, 11.02; O, 8.39. 4.5.10.
1-(2-methoxy-benzene
M AN U
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11b) Yellow powder, yield: 76%. M. p: 158 – 159
, 1H NMR (DMSO-d6, 400 MHz)
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 (C25H21N3O3, [M+H]+). Anal. Calcd for C25H21N3O3: C, 72.97; H, 5.15; N, 10.21; O,
TE D
11.67; Found: C, 72.98; H, 5.14; N, 10.21; O, 11.67. 4.5.11. 1-(3-methoxy-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11c) , 1H NMR (DMSO-d6, 400 MHz)
EP
Yellow powder, yield: 74%. M. p: 153 – 154
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 (C25H21N3O3,
AC C
[M+H]+). Anal. Calcd for C25H21N3O3: C, 72.97; H, 5.15; N, 10.21; O, 11.67; Found: C, 72.98; H, 5.14; N, 10.21; O, 11.67. 4.5.12.
1-(4-methoxy-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11d) Yellow powder, yield: 87%. M. p: 195 – 196
, 1H NMR (DMSO-d6, 400 MHz)
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); 7.20 – 7.22 (m, 2H); 7.29 – 7.31 (m, 2H); 7.32 – 7.35 (m, 1H); 7.37 – 7.38 (m, 1H); 7.48 – 7.50 (m, 1H); 7.62 (s, 2H); 8.25 (s, 1H). ESI-MS: 411.45 (C25H21N3O3, [M+H]+). Anal. Calcd for C25H21N3O3: C, 72.96; H, 5.13; N, 10.24; O, 11.67; Found: 18
ACCEPTED MANUSCRIPT C, 72.97; H, 5.13; N, 10.23; O, 11.67. 4.5.13. 1-(3, 4-dimethoxy-benzene acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11e) Yellow powder, yield: 83%. M. p: 126 – 127
, 1H NMR (DMSO-d6, 400 MHz)
RI PT
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, 1H); 7.19 – 7.23 (m, 1H); 7.30 – 7.34 (m, 1H); 7.37 – 7.41 (m, 3H); 7.61 – 7.65 (m, 2H); 7.79 (d, J = 8, 1H). 13C NMR (DMSO-d6, 100 MHz)
: 169.21, 155.16, 154.77,
150.17, 149.19, 143.57, 134.87, 132.66, 132.15, 126.99, 126.32, 124.97, 123.96,
SC
123.60, 119.39, 113.11, 112.80, 112.32, 111.69, 111.63, 104.42, 102.94, 56.41, 56.01, 55.68, 33.38. ESI-MS: 441.48 (C26H23N3O4, [M+H]+). Anal. Calcd for C26H23N3O4: C,
4.5.14.
M AN U
70.72; H, 5.24; N, 9.54; O, 14.50; Found: C, 70.73; H, 5.25; N, 9.52; O, 14.50. 1-(3,
4,
5-trimethoxy-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11f) Yellow powder, yield: 72%. M. p: 131 – 132
, 1H NMR (DMSO-d6, 400 MHz)
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
TE D
– 7.43 (m, 3H); 7.96 – 7.97 (m, 2H); 8.05 (s, 2H). ESI-MS: 471.18 (C27H25N3O5, [M+H]+). Anal. Calcd for C27H25N3O5: C, 68.78; H, 5.34; N, 8.91; O, 16.97; Found: C, 68.77; H, 5.33; N, 8.93; O, 16.97.
EP
4.5.15. 1-(2-bromo-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11g)
AC C
Yellow powder, yield: 82%. M. p: 185 – 187
, 1H NMR (DMSO-d6, 400 MHz)
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 (s, 2H); 7.81 (d, J = 8, 1H).
13
C NMR (DMSO-d6, 100 MHz)
: 168.18, 155.06,
149.99, 143.82, 136.06, 134.06, 133.70, 133.12, 133.03, 131.88, 131.80, 127.68, 127.45, 125.26, 124.76, 119.90, 119.80, 113.95, 112.61, 111.29, 104.00, 102.65, 55.87, 33.17. ESI-MS: 460.32 (C24H18BrN3O2, [M+H]+). Anal. Calcd for C24H18BrN3O2: C, 62.60; 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. 19
ACCEPTED MANUSCRIPT 4.5.16. 1-(3-bromo-benzene acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11h) Yellow powder, yield: 76%. M. p: 142 – 143
, 1H NMR (DMSO-d6, 400 MHz)
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 –
RI PT
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 (C24H18BrN3O2, [M+H]+). Anal. Calcd for C24H18BrN3O2: C, 62.60; 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.
Yellow powder, yield: 81%. M. p: 127 – 129
SC
4.5.17. 1-benzene acyl-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12a)
, 1H NMR (DMSO-d6, 400 MHz)
M AN U
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 – 7.46 (m, 1H); 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: 430.30 (C23H16BrN3O, [M+H]+). Anal. Calcd for C23H16BrN3O: C, 64.20; H, 3.75; Br, 18.57; N, 9.77; O, 3.72; Found: C, 64.21; H, 3.76; Br, 18.56; N, 9.77; O, 3.71.
TE D
4.5.18.
1-(2-methoxy-benzene
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12b) Yellow powder, yield: 91%. M. p: 134 – 136
, 1H NMR (DMSO-d6, 400 MHz)
EP
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 (C24H18BrN3O2, [M+H]+). Anal. Calcd for C24H18BrN3O2: C, 62.62; H, 3.94; Br,
AC C
17.36; N, 9.13; O, 6.95; Found: C, 62.61; H, 3.95; Br, 17.37; N, 9.14; O, 6.95. 4.5.19.
1-(3-methoxy-benzene
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12c) Yellow powder, yield: 69%. M. p: 136 – 137
, 1H NMR (DMSO-d6, 400 MHz)
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, 2H); 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 (C24H18BrN3O2, [M+H]+). Anal. Calcd for C24H18BrN3O2: C, 62.62; 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. 20
ACCEPTED MANUSCRIPT 4.5.20.
1-(4-methoxy-benzene
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12d) , 1H NMR (DMSO-d6, 400 MHz)
Yellow powder, yield: 85%. M. p: 151 – 152
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,
RI PT
2H); 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 (C24H18BrN3O2, [M+H]+). Anal. Calcd for C24H18BrN3O2: C, 62.62; 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. 1-(3,
4,
-dimethoxy-benzene
SC
4.5.21.
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12e)
, 1H NMR (DMSO-d6, 400 MHz)
M AN U
Yellow powder, yield: 79%. M. p: 121 – 123
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); 7.22 – 7.26 (m, 1H); 7.31 – 7.35 (m, 2H); 7.39 – 7.45 (m, 2H); 7.49 – 7.51 (m, 1H); 7.66 (s, 1H); 7.83 – 7.86 (m, 1H); 8.5 (s, 1H). ESI-MS: 490.35 (C25H20BrN3O3, [M+H]+). Anal. Calcd for C25H20BrN3O3: C, 61.24; H, 4.11; Br, 16.30; N, 8.57; O,
4.5.22.
TE D
9.79; Found: C, 61.23; H, 4.12; Br, 16.30; N, 8.57; O, 9.79. 1-(3,
4,
5-trimethoxy-benzene
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12f) , 1H NMR (DMSO-d6, 400
EP
Yellow powder, yield: 76%. M. p: 142 – 143
MHz) δ: 3.62 (s, 6H); 3.64 (s, 3H); 3.70 (s, 3H); 6.89 (s, 2H); 7.30 – 7.41 (m, 3H);
AC C
7.45 – 7.52 (m, 2H); 7.64 (s, 1H); 7.83 – 7.85 (s, 1H); 8.27 (s, 1H). (DMSO-d6, 100 MHz)
13
C NMR
: 169.23, 152.90, 149.18, 143.58, 142.55, 135.54, 134.61,
134.21, 128.42, 128.21, 125.28, 124.57, 124.29, 123.25, 119.72, 113.94, 113.28, 112.88, 108.45, 104.57, 60.69, 56.37, 33.25. ESI-MS: 520.37 (C26H22BrN3O4, [M+H]+). Anal. Calcd for C26H22BrN3O4: C, 60.01; H, 4.26; Br, 15.36; N, 8.07; O, 12.30; Found: C, 62.63; H, 3.96; Br, 17.36; N, 9.11; O, 6.94. 4.5.23. 1-(2-bromo-benzene acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12g) White powder, yield: 86%. M. p: 155 – 157 21
, 1H NMR (DMSO-d6, 400 MHz)
ACCEPTED MANUSCRIPT 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, 1H); 7.76 – 7.79 (m, 1H); 7.82 – 7.83 (m, 1H); 7.97 – 7.99 (m, 1H); 7.99 – 8.01 (m, 2H). ESI-MS: 509.19 (C23H15Br2N3O, [M+H]+). Anal. Calcd for C23H15Br2N3O: C, 54.25; H, 2.97; Br, 31.38; N, 8.25; O, 3.14; Found: C, 54.23; H, 2.98; Br, 31.38; N,
RI PT
8.26; O, 3.14. 4.5.24. 1-(3-bromo-benzene acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12h) White powder, yield: 76%. M. p: 163 – 164
, 1H NMR (DMSO-d6, 400 MHz)
SC
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, 1H); 7.78 – 7.81 (m, 1H); 7.84 – 7.86 (m, 1H); 7.98 – 7.99 (m, 1H); 8.03 – 8.07 (m,
M AN U
1H); 8.18 – 8.21 (m, 1H). ESI-MS: 509.19 (C23H15Br2N3O, [M+H]+). Anal. Calcd for C23H15Br2N3O: C, 54.25; H, 2.97; Br, 31.38; N, 8.25; O, 3.14; Found: C, 54.23; H, 2.98; Br, 31.38; N, 8.26; O, 3.14.
4.6. Crystal structure determination
TE D
Crystal structure determination of compound 10c was carried out on a Nonius CAD4 diffractometer equipped with graphite-mono chromated MoKa (k = 0.71073 Å) radiation. The structures were solved by direct methods and refined on F2 by
EP
fullmatrix least-squares methods using SHELX-97. [40] All non-hydrogen atoms of compound 10c were refined with anisotropic thermal parameters. All hydrogen atoms
AC C
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) according to the instructions of manufacture. Briefly, (1 – 5) × 103 cells per well were 22
ACCEPTED MANUSCRIPT 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 µL 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 µL DMSO was
RI PT
added to every well. Plates were read in a Victor-V multilabel counter (Perkin-Elmer) using the default europium detection protocol. Percent inhibition or GI50 values of compounds were calculated by comparison with DMSO-treated control wells. The
SC
results are shown in Table 2.
4.8. Effects on Tubulin Polymerization and on Colchicine Binding to Tubulin.
M AN U
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 µM bovine brain tubulin in glutamate buffer at 30 cooled to 0
and then
. After addition of 0.4 mM GTP, the mixtures were transferred to 0
cuvettes in a recording spectrophotometer and warmed to 30
. Tubulin
TE D
polymerization was followed turbidimetrically at 350 nm. The IC50 was defined as the compound concentration that inhibited the extent of polymerization by 50% after 20 min incubation.
EP
The capacity of the test compounds to inhibit Colchicine binding to tubulin was measured as described above (tubulin polymerization effect) except that the reaction
AC C
mixtures contained 1 µM tubulin, 5 µM [3H]Colchicine, and 5 µM test compound. 4.9. Flow cytometry
4.9.1. 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 µM, 2 µM and 5 µM of compound 11f, were chosen to examine the dose effect. The control cells were teated with DMSO. After incubation, cells were 23
ACCEPTED MANUSCRIPT harvested, washed with cold PBS and fixed with 70% ethanol at 4
overnight. The
fixed cells were washed with PBS, stained with 50 µg/ml of propidium iodide (PI) containing 100 µg/ml of Rnase A and 1% Triton X-100 in the dark for 45 min, and then subjected to flow cytometric analysis.
RI PT
4.9.2. Analysis of apoptosis
Approximately 105 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
SC
compound 11f at final concentrations of 0, 1 µM, 2 µM and 5 µM. Then cells were harvested after 24 h.
M AN U
They were trypsinized, washed in PBS and centrifuged at 2000rpm for 5 min. The pellet was then resuspended in 500 µL of staining solution (containing 5 µL Annexin V-PE and 5 µL 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
TE D
by the software supplied in the instrument. 4.10. Docking simulations
EP
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
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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 24
ACCEPTED MANUSCRIPT procedure. Types of interactions of the docked protein with ligand were analyzed after the end of molecular docking. 4.11. 3D-QSAR
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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 IC50 (µM), and test sets comprised compounds of data sets as list in Table 5.
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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
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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
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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 training sets. Usually, one can believe that the modeling is reliable, when the R2 for
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test sets is larger than 0.6, respectively.
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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).
25
ACCEPTED MANUSCRIPT Notes and references
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ACCEPTED MANUSCRIPT 26. G. G. Dark, S. A. Hill, V. E. Prise, G. M. Tozer, G. R. Pettit and D. J. Chaplin, Cancer. Rres. 57 (1997) 1829-1834. 27. C. Nien, Y. Chen, C. Kuo, H. Hsieh, C. Chang, J. Wu, S. Wu, J. Liou and J. Chang, J. Med. Chem. 53 (2010) 2309-2313.
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28. R. Romagnoli, P. G. Baraldi, T. Sarkar, M. D. Carrion, C. L. Cara, O. Cruz-Lopez, D. Preti, M. A. Tabrizi, M. Tolomeo and S. Grimaudo, J. Med. Chem. 51 (2008) 1464-1468.
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35. M. A. Seefeld, W. H. Miller, K. A. Newlander, W. J. Burgess, W. E. DeWolf, P. A. Elkins, M. S. Head, D. R. Jakas, C. A. Janson and P. M. Keller, J. Med. Chem. 46 (2003) 1627-1635.
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36. D. Saha, R. Ghosh and A. Sarkar, Tetrahedron 69 (2013) 3951-3960. 37. J. Piette, C. Volanti, A. Vantieghem, J. Matroule, Y. Habraken and P. Agostinis, Biochem. Pharmacol. 66 (2003) 1651-1659.
38. C. Selvaraj, S. K. Tripathi, K. K. Reddy and S. K. Singh, Current Trends in Biotechnology and Pharmacy 5 (2011) 1104-1109. 39. I. Vermes, C. Haanen, H. Steffens-Nakken and C. Reutellingsperger, J. Immunol. Methods 184 (1995) 39-51. 40. G. M. Sheldrick, Germany (1997). 41. G. Wu, D. H. Robertson, C. L. Brooks and M. Vieth, J. Comput. Chem. 24 (2003) 28
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1549-1562.
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ACCEPTED MANUSCRIPT Figure Captions
Table 1. Structures of compounds 10a - 12h Table 2. Inhibition of growth of A549, HepG2 and MCF-7 cells by compounds 10a -
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12h, combretastatin A-4 and colchicine Table 3. Inhibition of Tubulin Polymerization, and Colchicine Binding by compounds 10a - 12h, Combretastatin A-4 and Colchicine
Table 4. Molecular docking simulations data for the synthesized compounds 10a -
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12h and Colchicine
Table 5. Experimental, predicted inhibitory activity of compounds 10a - 12h by
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3D-QSAR models based upon active conformation achieved by molecular docking Figure 1. Potential Inhibitors and new Inhibitor of Tubulin Polymerization. Figure 2. Crystal structure diagram of compound 10c. H atoms are shown as small spheres of arbitrary radii.
Figure 3. Effects of compound 11f on cell cycle progression of A549 cells were
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determined by flow cytometry analysis. A549 cells were treated with different concentrations of compound 11f for 24 h. The percentage of cells in each cycle phase was indicated.
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Figure 4. Representative Scatter Plot of A549 cells treated with 11f (0, 1, 2 and 5 µM) for 24 h and analyzed by flow cytometry after double staining of the cells with
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Annexin-V-FITC and PI.
Figure 5. The binding mode between the active conformation of compound 11f and tubulin. (5A). 2D diagram of the interaction between compound 11f and the colchicine binding site. The H-bond (blue arrows) is displayed as dotted arrows, and the π-cation interaction is shown as orange lines. (5B). 3D diagram of the interaction between compound 11f and the colchicine binding site. For clarity, only interacting residues are displayed. The H-bond (green arrows) is displayed as dotted arrows, and the π-cation interaction is shown as orange lines. (5C). The receptor surface model with compound 11f. Figure 6. Using linear fitting curve to compare the predicted pIC50 value (tubulin 30
ACCEPTED MANUSCRIPT inhibitory activities) with that of experimental pIC50. Figure 7. (7A). 3D-QSAR model coefficients on electrostatic potential grids. Blue represents positive coefficients; red represents negative coefficients. (7B). 3D-QSAR model coefficients on Van der Waals grids. Green represents positive coefficients;
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Scheme 1. General synthesis of compounds (10a – 12h).
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yellow represents negative coefficients.
31
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R1
R2
R3
R4
R5
10a
H
H
H
H
H
10b
H
H
H
H
10c
H
H
H
H
10d
H
H
10e
H
H
10f
H
H
10g
H
10h
H -OCH3
11b
-OCH3
H
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-OCH3
-OCH3
H
-OCH3
-OCH3
H
-OCH3
-OCH3
-OCH3
Br
H
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
H
-OCH3
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11a
-OCH3
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Compounds
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Table 1. Structures of compounds 10a - 12h
-OCH3
H
-OCH3
H
-OCH3
H
11f
-OCH3
H
11g
-OCH3
Br
H
H
H
11h
-OCH3
H
Br
H
H
12a
-Br
H
H
H
H
12b
-Br
H
H
H
12c
-Br
H
H
H
12d
-Br
H
12e
-Br
H
12f
-Br
H
12g
-Br
Br
H
H
H
12h
-Br
H
Br
H
H
11c 11d
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11e
-OCH3
32
-OCH3 H
-OCH3
H
-OCH3
-OCH3
H
-OCH3
-OCH3
-OCH3 H
-OCH3
-OCH3
H
-OCH3
-OCH3
H
-OCH3
-OCH3
-OCH3
ACCEPTED MANUSCRIPT Table 2. Inhibition of growth of A549, HepG2 and MCF-7 cells by compounds 10a 12h, Combretastatin A-4 and Colchicine GI50 ± SD(µM) Compounds HepG2a
MCF-7a
10a
19.6 ± 2.1
42.3 ± 0.17
43.7 ± 0.32
10b
12.3 ± 0.32
31.8 ± 0.45
10c
10.2 ± 0.76
13.6 ± 0.32
10d
10.4 ± 0.17
15.2 ± 0.44
10e
3.1 ± 0.35
8.4 ± 0.27
10f
2.6 ± 0.79
4.3 ± 2.8
5.4 ± 0.45
10g
43.2 ± 2.8
68.2 ± 0.41
70.1 ± 0.76
10h
34.2 ± 0.43
61.4 ± 0.38
63.4 ± 0.27
11a
16.7 ± 0.56
37.9 ± 0.29
38.4 ± 0.31
11b
11.9 ± 0.71
23.6 ± 0.36
23.9 ± 0.42
11c
4.6 ± 0.92
11.7 ± 0.67
14.6 ± 0.52
11d
4.9 ± 1.6
12.8 ± 0.6
14.8 ± 0.55
11e
2.7 ± 2.6
5.1 ± 0.32
6.3 ± 0.23
11f
2.4 ± 0.42
3.8 ± 0.5
5.1 ± 0.42
29.5 ± 0.54
54.2 ± 0.27
55.8 ± 0.15
24.8 ± 0.67
51.3 ± 0.22
52.7 ± 2.4
23.1 ± 0.65
48.1 ± 0.34
50.3 ± 0.51
12.8 ± 0.32
34.6 ± 0.23
36.2 ± 0.18
11h 12a
15.7 ± 0.87 16.3 ± 2.5
10.2 ± 2.8
12c
10.8 ± 2.7
17.8 ± 1.8
18.2 ± 2.6
12d
11.3 ± 3.8
18.9 ± 0.28
19.2 ± 0.08
12e
3.4 ± 4.1
9.7 ± 0.71
11.3 ± 0.13
12f
3.0 ± 4.2
8.2 ± 2.2
9.7 ± 0.37
12g
46.7 ± 3.7
72.1 ± 0.37
73.6 ± 0.44
12h
38.5 ± 5.2
67.3 ± 0.77
68.2 ± 0.51
Colchicineb
4.4 ± 3.8
10.5 ± 0.57
13.5 ± 0.66
CA-4b
2.8 ± 0.04
7.4 ± 0.04
9.4 ± 0.04
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32.1 ± 0.34
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11g
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A549a
A549 (human lung adenocarcinoma cells), HepG2 (human liver hepatocellular
carcinoma) and MCF-7 (human breast carcinoma cells). Cancer cells were purchased from NanJing KeyGen Biotech Co., Ltd., which subcultured by State Key Laboratory of Pharmaceutical Biotechnology. 33
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Used as positive controls.
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b
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Inhibition ratio of Colchifine Bindingb (% ± SD)
10a
23.8 ± 0.33
61 ± 1.3
10b
15.9 ± 1.21
10c
4.4 ± 0.36
10d
4.6 ± 0.34
10e
2.1 ± 0.22
10f
1.6 ± 0.32
10g
42.9 ± 0.55
10h
35.2 ± 0.36
19.6 ± 0.34
64 ± 3.3
8.4 ± 2.32
73 ± 0.6
3.3 ± 2.73
84 ± 1.4
3.5 ± 3.35
82 ± 2.9
1.7 ± 4.51
94 ± 0.9
1.5 ± 0.56
96 ± 0.2
11b 11c 11d 11e
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11f
80 ± 1.3 87 ± 4.1
95 ± 0.6 50 ± 0.6 52 ± 0.3
55 ± 0.6
29.2 ± 0.44
57 ± 1.6
25.3 ± 0.52
60 ± 0.1
17.3 ± 2.56
68 ± 0.7
12c
5.7 ± 4.67
78 ± 0.9
12d
6.3 ± 4.23
75 ± 3.1
12e
2.4 ± 5.76
85 ± 3.6
12f
2.0 ± 6.45
91 ± 4.8
12g
45.6 ± 6.32
48 ± 0.4
39.7 ± 7.83
51 ± 4.2
Colchicine
2.62 ± 4.82
—
CA-4c
1.8 ± 0.20
93% ± 0.4
11h 12a
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12h
c
81 ± 2.4
32.3 ± 0.97
11g
c
Inhibition of tubulin polymerization.
b
70 ± 0.7
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a
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Compounds
Tubulin Assemblya IC50 ± SD (µM)
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10a - 12h, Combretastatin A-4 and Colchicine
Inhibition of Colchifine Binding.
Used as a positive controls.
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-54.3 -56.7 -59.6 -58.8 -63.2 -65.9 -49.5 -51.8 -54.7 -57.1 -61.6 -60.8 -65.2 -67.6 -52.6 -52.8 -53.1 -55.2 -57.6 -57.3 -62.7 -63.8 -48.7 -50.2 -62.2
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-16.3 -18.2 -19.6 -17.4 -19.2 -21.9 -20.5 -18.8 -16.7 -19.7 -21.6 -20.8 -17.7 -16.6 -16.6 -18.2 -20.1 -17.7 -17.6 -19.4 -20.5 -17.8 -19.5 -20.5 -20.8
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Residual error Actual pIC50
Predicted pIC50
10a
4.623
4.557
10b
4.799
4.815
10c
5.357
5.355
10d
5.337
5.373
10e
5.678
5.645
10f
5.796
5.86
-0.060
10g
4.368
4.403
-0.035
10h
4.453
11a
4.708
11b
5.076
11c
5.481
11d
5.456
11e
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0.066 -0.016 0.002
-0.036 0.033
-0.093
4.560
0.148
4.935
0.141
5.304
0.177
5.369
0.095
5.770
5.779
-0.009
5.824
5.799
0.025
4.491
4.455
0.0361
4.535
4.572
-0.037
4.597
4.525
0.072
4.762
4.857
-0.095
12c
5.244
5.249
-0.005
12d
5.201
5.238
-0.037
12e
5.620
5.673
-0.053
12f
5.699
5.716
-0.017
12g
4.341
4.411
-0.070
12h
4.401
4.457
-0.056
11g 11h 12a
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12b
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4.546
Underlined compounds were selected as the test sets, while the rest ones were in the training sets.
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Figure 1. Potential Inhibitors and new Inhibitor of Tubulin Polymerization.
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spheres of arbitrary radii.
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Figure 2. Crystal structure diagram of compound 10c. H atoms are shown as small
39
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Figure 3. Effect of compound 11f on cell cycle progression of A549 cells was determined by flow cytometry analysis. A549 cells were treated with different concentrations of compound 11f for 24 h. The percentage of cells in each cycle phase
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Figure 4. Representative Scatter Plot of A549 cells treated with 11f (0, 1, 2 and 5 µM) for 24 h and analyzed by flow cytometry after double staining of the cells with
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Figure 5. The binding mode between the active conformation of compound 11f and tubulin. (5A). 2D diagram of the interaction between compound 11f and the colchicine binding site. The H-bond (blue arrows) is displayed as dotted arrows, and the π-cation interaction is shown as orange lines. (5B). 3D diagram of the interaction between compound 11f and the colchicine binding site. For clarity, only interacting residues are displayed. The H-bond (green arrows) is displayed as dotted arrows, and 42
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Figure 6. Using linear fitting curve to compare the predicted pIC50 value (tubulin
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Figure 7. (7A). 3D-QSAR model coefficients on electrostatic potential grids. Blue represents positive coefficients; red represents negative coefficients. (7B). 3D-QSAR model coefficients on Van der Waals grids. Green represents positive coefficients; yellow represents negative coefficients.
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Scheme 1. General synthesis of compounds (10a – 12h). Reagents and conditions: (a) , 4h; (b) SOCl2, 80
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Sodium metabisulfite, DMF, 110
, 4h; (c) triethylamine,
CH2Cl2, room temperature, overnight; (d) EDC·HCl, HOBT, CH2Cl2, room
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temperature, 24h.
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> 24 novel 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives have been synthesized. > Their biological activities were evaluated as potential tubulin assembling inhibitors. > Compound 11f showed the most potent inhibitory activity against cancer cell and tubulin. > Crystal structure of compound 10c was determined.
ACCEPTED MANUSCRIPT Supporting information: complete spectras for novel compounds (including 1H NMR, 13C NMR, HRMS and ESI-MS spectras) and tabulated X-ray crystal structure data for compound 10c.
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complete spectras 4.5.1. 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole (10a)
White powder, yield: 77%. M. p: 127 – 129 ℃, 1H NMR (DMSO-d6, 400 MHz) 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 –
SC
7.65 (m, 1H); 7.66 (s, 1H); 7.72 (s, 1H); 7.74 (s, 1H); 7.81 (d, J = 8, 1H); 8.16 (d, J = 7.2, 1H). 13C NMR (DMSO-d6, 100 MHz) δ: 170.06, 149.94, 143.66, 136.90, 134.68,
M AN U
134.53, 133.37, 132.98, 130.70, 129.25, 126.56, 124.38, 123.94, 122.83, 121.26, 121.23, 119.56, 112.73, 110.74, 104.67, 56.50, 33.15, 19.04. HRMS: C23H17N3O for +, calculated 351.4026, found 351.4028. ESI-MS: 351.40 (C23H17N3O, [M+H]+). Anal. Calcd for C23H17N3O: C, 78.60; H, 4.87; N, 11.97; O, 4.56; Found: C, 78.61; H, 4.88; N, 11.96; O, 4.55.
TE D
4.5.2. 1-(2-methoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10b) Yellow powder, yield: 84%. M. p: 137 – 138 ℃, 1H NMR (DMSO-d6, 400 MHz) 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). HRMS: C23H17N3O for +, calculated 381.4331,
EP
found 381.4342. ESI-MS: 381.43 (C24H19N3O2, [M+H]+). Anal. Calcd for C24H19N3O2: C, 75.57; H, 5.02; N, 11.02; O, 8.39; Found: C, 75.56; H, 5.02; N, 11.04;
AC C
O, 8.38.
4.5.3. 1-(3-methoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10c) Yellow powder, yield: 87%. M. p: 146 – 147 ℃, 1H NMR (DMSO-d6, 400 MHz)
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, 1H).
13
C NMR (DMSO-d6, 100 MHz) δ: 169.81, 159.58, 149.92, 143.67, 136.87,
134.80, 134.53, 132.99, 130.36, 126.54, 124.44, 124.00, 123.07, 122.82, 121.24, 121.21, 120.96, 119.56, 114.84, 112.88, 110.75, 104.71, 55.78, 33.12. HRMS: C23H17N3O for +, calculated 381.4323, found 381.4307. ESI-MS: 381.43 (C24H19N3O2,
ACCEPTED MANUSCRIPT [M+H]+). Anal. Calcd for C24H19N3O2: C, 75.55; H, 5.03; N, 11.03; O, 8.39; Found: C, 75.57; H, 5.02; N, 11.02; O, 8.39. 4.5.4. 1-(4-methoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10d) Yellow powder, yield: 77%. M. p: 155 – 156 ℃, 1H NMR (DMSO-d6, 400 MHz)
RI PT
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, 1H); 7.25 – 7.27 (m, 1H); 7.32 – 7.35 (m, 2H); 7.37 – 7.38 (m, 1H); 7.48 – 7.50 (m, 1H); 7.62 (s, 1H); 7.80 (m, 2H); 8.25 (s, 1H). HRMS: C23H17N3O for +, calculated
381.4325, found 381.4307. ESI-MS: 381.43 (C24H19N3O2, [M+H]+). Anal. Calcd for
SC
C24H19N3O2: C, 75.57; H, 5.02; N, 11.02; O, 8.39; Found: C, 75.55; H, 5.01; N, 11.05; O, 8.39.
M AN U
4.4.5. 1-(3, 4-dimethoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10e) Yellow powder, yield: 87%. M. p: 205 – 206 ℃, 1H NMR (DMSO-d6, 400 MHz) 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); 7.20 – 7.23 (m, 2H); 7.25 – 7.27 (m, 1H); 7.32 – 7.35 (m, 2H); 7.37 – 7.38 (m, 1H); 7.48 – 7.50 (m, 1H); 7.62 (s, 1H); 7.80 (d, J = 7.6, 1H); 8.25 (d, J = 7.6, 1H).
13
C
TE D
NMR (DMSO-d6, 100 MHz) δ: 169.28, 154.70, 149.84, 149.13, 143.59, 136.95, 134.90, 132.44, 126.56, 126.21, 125.01, 124.00, 123.70, 122.86, 121.49, 121.24, 119.43, 113.11, 112.39, 111.61, 110.81, 104.67, 56.39, 55.97, 33.22. HRMS: C23H17N3O for +, calculated 411.4526, found 411.4523. ESI-MS: 411.45 (C25H21N3O3,
EP
[M+H]+). Anal. Calcd for C25H21N3O3: C, 72.96; H, 5.13; N, 10.24; O, 11.67; Found:
AC C
C, 72.98; H, 5.14; N, 10.21; O, 11.67. 4.4.6. 1-(3, 4, 5-trimethoxy-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10f)
Yellow powder, yield: 72%. M. p: 183 – 184 ℃, 1H NMR (DMSO-d6, 400 MHz)
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 – 7.34 (m, 1H); 7.37 – 7.43 (m, 1H); 7.45 (s, 1H); 7.50 – 7.52 (m, 1H); 7.57 (s, 1H); 7.82 (d, J = 8, 1H); 8.09 (d, J = 7.6, 1H). 13C NMR (DMSO-d6, 100 MHz) δ: 169.33, 153.12, 152.82, 149.86, 143.71, 142.45, 136.76, 134.63, 133.01, 128.45, 126.49, 124.50, 124.10, 122.79, 121.11, 121.01, 119.59, 113.18, 110.62, 108.38, 106.96, 104.94, 60.66, 56.33, 33.01. HRMS: C23H17N3O for +, calculated 441.4809, found
ACCEPTED MANUSCRIPT 441.4813. ESI-MS: 441.48 (C26H23N3O4, [M+H]+). Anal. Calcd for C26H23N3O4: C, 70.72; H, 5.26; N, 9.52; O, 14.50; Found: C, 70.73; H, 5.25; N, 9.52; O, 14.50. 4.4.7. 1-(2-bromo-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10g) White powder, yield: 82%. M. p: 160 ℃, 1H NMR (DMSO-d6, 400 MHz) δ:
RI PT
3.71 (s, 3H); 7.20 – 7.21 (m, 1H); 7.22 – 7.25 (m, 2H); 7.26 – 7.27 (m, 1H); 7.39 – 7.42 (m, 3H); 7.60 (m, 1H); 7.67 (s, 1H); 7.70 (m, 1H); 7.81 – 7.83 (m, 2H); 8.01 (s, 1H). HRMS: C23H17N3O for +, calculated 430.3048, found 430.3045. ESI-MS: 430.30 (C23H16BrN3O, [M+H]+). Anal. Calcd for C23H16BrN3O: C, 64.19; H, 3.74; Br, 18.57;
SC
N, 9.78; O, 3.73; Found: C, 64.20; H, 3.73; Br, 18.57; N, 9.77; O, 3.74.
4.4.8. 1-(3-bromo-benzene acyl)-2-(1-methylindol-3-yl)-benzimidazole (10h)
M AN U
White powder, yield: 79%. M. p: 153 – 154 ℃, 1H NMR (DMSO-d6, 400 MHz) 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, 1H); 7.81 – 7.83 (m, 2H); 8.03 (d, J = 7.6, 1H). HRMS: C23H17N3O for +, calculated 430.3072, found 430.3068. ESI-MS: 430.30 (C23H16BrN3O, [M+H]+). Anal. Calcd for
TE D
C23H16BrN3O: C, 64.19; H, 3.74; Br, 18.57; N, 9.78; O, 3.73; Found: C, 64.20; H, 3.75; Br, 18.57; N, 9.77; O, 3.72.
4.4.9. 1-benzene acyl-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11a)
EP
White powder, yield: 86%. M. p: 161 – 162 ℃, 1H NMR (DMSO-d6, 400 MHz) 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, 1H); 7.32 – 7.36 (m, 2H); 7.43 – 7.47 (m, 2H); 7.58 – 7.59 (m, 1H); 7.63 – 7.67 (m,
AC C
2H); 7.77 (s, 1H); 7.78 – 7.79 (m, 1H); 7.82 (s, 1H). HRMS: C23H17N3O for +, calculated 381.4332, found 381.4345. ESI-MS: 381.43 (C24H19N3O2, [M+H]+). Anal. Calcd for C24H19N3O2: C, 75.55; H, 5.01; N, 11.03; O, 8.41; Found: C, 75.57; H, 5.02; N, 11.02; O, 8.39. 4.4.10.
1-(2-methoxy-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11b) Yellow powder, yield: 76%. M. p: 158 – 159 ℃, 1H NMR (DMSO-d6, 400 MHz) 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). HRMS:
ACCEPTED MANUSCRIPT C23H17N3O for +, calculated 411.4539, found 411.4532. ESI-MS: 411.45 (C25H21N3O3, [M+H]+). Anal. Calcd for C25H21N3O3: C, 72.97; H, 5.15; N, 10.21; O, 11.67; Found: C, 72.98; H, 5.14; N, 10.21; O, 11.67. 4.4.11. 1-(3-methoxy-benzene
RI PT
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11c) Yellow powder, yield: 74%. M. p: 153 – 154 ℃, 1H NMR (DMSO-d6, 400 MHz) 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). HRMS: C23H17N3O for +,
SC
calculated 411.4572, found 411.4552. ESI-MS: 411.45 (C25H21N3O3, [M+H]+). Anal. Calcd for C25H21N3O3: C, 72.97; H, 5.15; N, 10.21; O, 11.67; Found: C, 72.98; H,
M AN U
5.14; N, 10.21; O, 11.67. 4.4.12.
1-(4-methoxy-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11d) Yellow powder, yield: 87%. M. p: 195 – 196 ℃, 1H NMR (DMSO-d6, 400 MHz) 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);
TE D
7.20 – 7.22 (m, 2H); 7.29 – 7.31 (m, 2H); 7.32 – 7.35 (m, 1H); 7.37 – 7.38 (m, 1H); 7.48 – 7.50 (m, 1H); 7.62 (s, 2H); 8.25 (s, 1H). HRMS: C23H17N3O for +, calculated 411.4527, found 411.4525. ESI-MS: 411.45 (C25H21N3O3, [M+H]+). Anal. Calcd for
EP
C25H21N3O3: C, 72.96; H, 5.13; N, 10.24; O, 11.67; Found: C, 72.97; H, 5.13; N, 10.23; O, 11.67.
AC C
4.4.13. 1-(3, 4-dimethoxy-benzene acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11e) Yellow powder, yield: 83%. M. p: 126 – 127 ℃, 1H NMR (DMSO-d6, 400 MHz)
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, 1H); 7.19 – 7.23 (m, 1H); 7.30 – 7.34 (m, 1H); 7.37 – 7.41 (m, 3H); 7.61 – 7.65 (m, 2H); 7.79 (d, J = 8, 1H). 13C NMR (DMSO-d6, 100 MHz) δ: 169.21, 155.16, 154.77, 150.17, 149.19, 143.57, 134.87, 132.66, 132.15, 126.99, 126.32, 124.97, 123.96, 123.60, 119.39, 113.11, 112.80, 112.32, 111.69, 111.63, 104.42, 102.94, 56.41, 56.01, 55.68, 33.38. HRMS: C23H17N3O for +, calculated 441.4806, found 441.4814. ESI-MS: 441.48 (C26H23N3O4, [M+H]+). Anal. Calcd for C26H23N3O4: C, 70.72; H,
ACCEPTED MANUSCRIPT 5.24; N, 9.54; O, 14.50; Found: C, 70.73; H, 5.25; N, 9.52; O, 14.50. 4.4.14.
1-(3,
4,
5-trimethoxy-benzene
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11f) Yellow powder, yield: 72%. M. p: 131 – 132 ℃, 1H NMR (DMSO-d6, 400 MHz)
RI PT
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). HRMS: C23H17N3O for +, calculated 471.1842, found 471.1836. ESI-MS: 471.18 (C27H25N3O5, [M+H]+). Anal. Calcd for C27H25N3O5: C, 68.78; H, 5.34; N, 8.91; O, 16.97; Found: C, 68.77; H, 5.33;
SC
N, 8.93; O, 16.97. 4.4.15. 1-(2-bromo-benzene
M AN U
acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11g)
Yellow powder, yield: 82%. M. p: 185 – 187 ℃, 1H NMR (DMSO-d6, 400 MHz) 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 (s, 2H); 7.81 (d, J = 8, 1H).
13
C NMR (DMSO-d6, 100 MHz) δ: 168.18, 155.06,
TE D
149.99, 143.82, 136.06, 134.06, 133.70, 133.12, 133.03, 131.88, 131.80, 127.68, 127.45, 125.26, 124.76, 119.90, 119.80, 113.95, 112.61, 111.29, 104.00, 102.65, 55.87, 33.17. HRMS: C23H17N3O for +, calculated 460.3211, found 460.3208.
EP
ESI-MS: 460.32 (C24H18BrN3O2, [M+H]+). Anal. Calcd for C24H18BrN3O2: C, 62.60; 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,
AC C
6.95.
4.4.16. 1-(3-bromo-benzene acyl)-2-(5-methoxy-1-methylindol-3-yl)-benzimidazole (11h) Yellow powder, yield: 76%. M. p: 142 – 143 ℃, 1H NMR (DMSO-d6, 400 MHz)
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). HRMS: C23H17N3O for +, calculated 460.3216, found 460.3212. ESI-MS: 460.32 (C24H18BrN3O2, [M+H]+). Anal. Calcd for C24H18BrN3O2: C, 62.60; 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. 4.4.17. 1-benzene acyl-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12a)
ACCEPTED MANUSCRIPT Yellow powder, yield: 81%. M. p: 127 – 129 ℃, 1H NMR (DMSO-d6, 400 MHz) 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 – 7.46 (m, 1H); 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). HRMS: C23H17N3O for +, calculated 430.3036, found 430.3051.
RI PT
ESI-MS: 430.30 (C23H16BrN3O, [M+H]+). Anal. Calcd for C23H16BrN3O: C, 64.20; H, 3.75; Br, 18.57; N, 9.77; O, 3.72; Found: C, 64.21; H, 3.76; Br, 18.56; N, 9.77; O, 3.71. 4.4.18.
1-(2-methoxy-benzene
SC
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12b)
Yellow powder, yield: 91%. M. p: 134 – 136 ℃, 1H NMR (DMSO-d6, 400 MHz)
M AN U
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). HRMS: C23H17N3O for +, calculated 460.3242, found 460.3237. ESI-MS: 460.32 (C24H18BrN3O2, [M+H]+). Anal. Calcd for C24H18BrN3O2: C, 62.62; H, 3.94; Br, 17.36; N, 9.13; O, 6.95; Found: C, 62.61; H, 3.95; Br, 17.37; N, 9.14; O, 6.95.
1-(3-methoxy-benzene
TE D
4.4.19.
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12c) Yellow powder, yield: 69%. M. p: 136 – 137 ℃, 1H NMR (DMSO-d6, 400 MHz)
EP
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, 2H); 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). HRMS: C23H17N3O for +, calculated 460.3219, found 460.3235. ESI-MS:
AC C
460.32 (C24H18BrN3O2, [M+H]+). Anal. Calcd for C24H18BrN3O2: C, 62.62; 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. 4.4.20.
1-(4-methoxy-benzene
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12d) Yellow powder, yield: 85%. M. p: 151 – 152 ℃, 1H NMR (DMSO-d6, 400 MHz) 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, 2H); 7.73 – 7.74 (m, 1H); 7.95 – 7.96 (m, 2H); 7.98 – 8.01 (m, 1H); 8.07 (s, 2H). HRMS: C23H17N3O for +, calculated 460.3243, found 460.3252. ESI-MS: 460.32 (C24H18BrN3O2, [M+H]+). Anal. Calcd for C24H18BrN3O2: C, 62.62; H, 3.94; Br,
ACCEPTED MANUSCRIPT 17.36; N, 9.13; O, 6.95; Found: C, 62.64; H, 3.96; Br, 17.36; N, 9.11; O, 6.93. 4.4.21.
1-(3,
4,
-dimethoxy-benzene
acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12e) Yellow powder, yield: 79%. M. p: 121 – 123 ℃, 1H NMR (DMSO-d6, 400 MHz)
RI PT
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); 7.22 – 7.26 (m, 1H); 7.31 – 7.35 (m, 2H); 7.39 – 7.45 (m, 2H); 7.49 – 7.51 (m, 1H); 7.66 (s, 1H); 7.83 – 7.86 (m, 1H); 8.5 (s, 1H). HRMS: C23H17N3O for +, calculated 490.3527, found 490.3523. ESI-MS: 490.35 (C25H20BrN3O3, [M+H]+). Anal. Calcd
4.12; Br, 16.30; N, 8.57; O, 9.79. 1-(3,
4,
M AN U
4.4.22.
SC
for C25H20BrN3O3: C, 61.24; H, 4.11; Br, 16.30; N, 8.57; O, 9.79; Found: C, 61.23; H,
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); 7.45 – 7.52 (m, 2H); 7.64 (s, 1H); 7.83 – 7.85 (s, 1H); 8.27 (s, 1H).
13
C NMR
TE D
(DMSO-d6, 100 MHz) δ: 169.23, 152.90, 149.18, 143.58, 142.55, 135.54, 134.61, 134.21, 128.42, 128.21, 125.28, 124.57, 124.29, 123.25, 119.72, 113.94, 113.28, 112.88, 108.45, 104.57, 60.69, 56.37, 33.25. HRMS: C23H17N3O for +, calculated
EP
520.3734, found 520.3737. ESI-MS: 520.37 (C26H22BrN3O4, [M+H]+). Anal. Calcd for C26H22BrN3O4: C, 60.01; H, 4.26; Br, 15.36; N, 8.07; O, 12.30; Found: C, 62.63;
AC C
H, 3.96; Br, 17.36; N, 9.11; O, 6.94. 4.4.23. 1-(2-bromo-benzene acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole (12g)
White powder, yield: 86%. M. p: 155 – 157 ℃, 1H NMR (DMSO-d6, 400 MHz)
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, 1H); 7.76 – 7.79 (m, 1H); 7.82 – 7.83 (m, 1H); 7.97 – 7.99 (m, 1H); 7.99 – 8.01 (m, 2H). HRMS: C23H17N3O for +, calculated 509.1921, found 509.1925. ESI-MS: 509.19 (C23H15Br2N3O, [M+H]+). Anal. Calcd for C23H15Br2N3O: C, 54.25; H, 2.97; Br, 31.38; N, 8.25; O, 3.14; Found: C, 54.23; H, 2.98; Br, 31.38; N, 8.26; O, 3.14. 4.4.24. 1-(3-bromo-benzene acyl)-2-(5-bromo-1-methylindol-3-yl)-benzimidazole
ACCEPTED MANUSCRIPT (12h) White powder, yield: 76%. M. p: 163 – 164 ℃, 1H NMR (DMSO-d6, 400 MHz) 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, 1H); 7.78 – 7.81 (m, 1H); 7.84 – 7.86 (m, 1H); 7.98 – 7.99 (m, 1H); 8.03 – 8.07 (m,
RI PT
1H); 8.18 – 8.21 (m, 1H). HRMS: C23H17N3O for +, calculated 509.1926, found 509.1921. ESI-MS: 509.19 (C23H15Br2N3O, [M+H]+). Anal. Calcd for C23H15Br2N3O: C, 54.25; H, 2.97; Br, 31.38; N, 8.25; O, 3.14; Found: C, 54.23; H, 2.98; Br, 31.38; N,
AC C
EP
TE D
M AN U
SC
8.26; O, 3.14.
ACCEPTED MANUSCRIPT X-ray table Table S1. Crystallographic and Experimental Data for compound 10c 10c
Formula
C24H19N3O2
Formula weight
381.43
RI PT
Compound
Crystal system
Monoclinic
Space group
P21/n
8.2150(11)
SC
a (Å)
c (Å) α(o) β(o) γ(o)
TE D
V(Å3)
10.1925(17)
11.9396(16)
M AN U
b (Å)
85.409(5) 76.051(4) 80.847(5) 957.0(2) 19
Dc(g•cm-3)
1.419
µ(mm-1)
0.128
F(000)
418
θrang(o)
2.58- 25.11
Reflns collected
8693
Reflns unique
3364
Goodness-of-fit on F2
1.027
R1, wR2[I>2σ(I)]
0.0538, 0.1310
R1, wR2[all data]
0.0913, 0.1495
Max, min∆ρ(e Å-3)
0.352, -0.189
AC C
EP
Z