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Design, synthesis and biological evaluation of novel 2-phenyl-4,5,6,7-tetrahydro-1H-indole derivatives as potential anticancer agents and tubulin polymerization inhibitors
⁎Corresponding Author. wanggch123@163.com (Guangcheng Wang), pengzhiyun1986@163.com (Zhiyun Peng)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
A new series of 2-phenyl-4,5,6,7-tetrahydro-1H-indole derivatives as tubulin polymerization inhibitors were synthesized and evaluated for the anti-proliferative activities. All newly prepared compounds were tested for their antiproliferative activity in vitro on the human breast cancer cell line (MCF-7) and human lung adenocarcinoma cell line (A549). Among them, compound 7b with a 4-methoxyl substituent at the phenylhydrazone moiety exhibited the most potent anticancer activity against MCF-7 and A549 with IC50 values of 1.77 ± 0.37 and 3.75 ± 0.11 μM, respectively. Interestingly, 7b displayed significant selectivity in inhibiting cancer cells over LO2 (normal human liver cells). Further mechanism studies revealed that 7b significantly arrested cell cycle at G2/M phase and induced apoptosis in a dose-dependent manner. Additionally, 7b effectively inhibited tubulin polymerization with an inhibitory manner similar to that of colchicine. Furthermore, molecular docking study suggested that 7b had high binding affinities for the colchicine binding pocket of tubulin. Hence, this study demonstrates for the first time that tetrahydroindole can be used as a functional group for the design and development of new tubulin polymerization inhibitors.
Keywords
Tetrahydro-1H-indole
Hydrazone
Tubulin polymerization inhibitors
Anticancer activity
1 Introduction
Microtubules are key components of the cytoskeleton in eukaryotic cells, which mainly consist of α- and β-tubulin heterodimers (Downing & Nogales, 1998a, 1998b). They play important roles in a series of essential cellular processes including intracellular transportation, regulation of motility, cell signaling, formation and maintenance of cell shape, and secretion (Honore et al., 2005). It was found that the formation of microtubules is a dynamic process related to the polymerization and depolymerization of α- and β-tubulin heterodimers (Amos, 2004). The interruption of the dynamic equilibrium of α- and β-tubulin heterodimers hinders cell division at mitosis and thus resulting in cell cycle arrest at metaphase, which leads to the tumor cell death by apoptosis (Jordan et al., 1996). Hence, microtubules have become one of the most successful therapeutic targets for the treatment of human cancer (Dumontet & Jordan, 2010; Jordan & Wilson, 2004; Stanton et al., 2011).
Pyrroles are an important class of nitrogen-containing heterocyclic compounds, which are present in numerous naturally occurring and synthetic compounds, such as Vitamin B12, Chlorophyll, Tolmetin, Zomepirac, Pyrvinium, and Prodigiosin (Ahmad et al., 2018; Singh et al., 2021). Previous studies revealed that pyrrole derivatives exhibit a wide range of pharmacological activities including anticancer, antimalarial, anti-HIV, anti-inflammatory, anti-depressant, and anti-ulcer activities (Ahmad et al., 2018). Specifically, pyrrole fused form indole is a very common skeleton in natural products and synthetic compounds, which have attracted great attention in medicinal chemistry over the past decade (Singh & Singh, 2018; Sravanthi & Manju, 2016). It is interesting to point that indole has been proven to be an important pharmacophore for the design and development of anticancer agents and tubulin polymerization inhibitors (Patil et al., 2016; Sang et al., 2017; Sunil & Kamath, 2016; Wan et al., 2019). Over the last few years, numbers of indole-containing tubulin polymerization inhibitors have been used in clinical (vinblastine and vincristine) or in clinical trials (indibulin, MKC-118, and LP-261) (Wang et al., 2014).
In recent years, tetrahydroindole derivatives have aroused increased interest due to their enhanced biological activity. For instance, Vojacek et al. found that 2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxamides are potent and selective SIRT2 inhibitors, and could serve as an exquisite starting point for hit-to-lead profiling (Fig. 1, I) (Vojacek et al., 2019). Andreev et al. discovered that the 2-phenyl-4,5,6,7-tetrahydro-1H-indole could be used as a novel anti-hepatitis C virus targeting scaffold (Fig. 1, II) (Andreev et al., 2015). Sun et al. reported that a series of new 3-substituted indolin-2-one derivatives containing a tetrahydroindole moiety (Fig. 1, III) can be used as specific inhibitors of receptor tyrosine kinases (Sun et al., 2000). Fatahala et al reported the synthesis of a series of tetrahydroindole derivatives and some of them shown promising antioxidant activity besides their anticancer activity (Fig. 1, IV) (Fatahala et al., 2015). Recently, Gülçin et al. reported the synthesis of sulfur-containing tetrahydroindole derivatives (Fig. 1, V and VI) which showed excellent inhibitory potential against the human erythrocyte carbonic anhydrase I, and II isoenzymes, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glycosidase enzymes (Gulcin et al., 2020).
Some tetrahydroindole derivatives with diverse pharmacological activities.
However, to the best of our knowledge, there is no report about tetrahydroindole derivatives as tubulin polymerization inhibitors. Hence, prompted by these observations and in continuation to our interest in design and synthesis of novel tubulin polymerization inhibitors (Wang et al., 2020a, Wang et al., 2020c, Wang et al., 2020d; Wang et al., 2018a; Wang et al., 2018b), herein we report for the first time the synthesis of a novel series of 2-phenyl-4,5,6,7-tetrahydro-1H-indole derivatives as potential anticancer agents and tubulin polymerization inhibitors. The mechanism of antiproliferative effects of this class of compounds was studied by using in vitro tubulin polymerization, cell cycle arrest, and cell apoptosis assay. Furthermore, molecular modeling study was also performed to elucidate the binding mode of the inhibitor with tubulin.
2 Results and discussion
2.1 Synthesis
A novel series of 2-phenyl-4,5,6,7-tetrahydro-1H-indole derivatives 7a-7o were synthesized according to the pathways described in Schemes 1. The commercially available 1-pyrrolidinocyclohexene 1 was reacted with 2-bromo-1-(4-methoxyphenyl)ethan-1-one 2 (commercially from Bide Pharm) in DMF and then hydrolyzed at room temperature to give the corresponding 3 (Naik et al., 2014). The subsequent cyclization of 3 with NH4Ac under reflux in EtOH as a solvent to generate 2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indole 4. Then, according to Duff reaction, intermediate 4 reacted with hexamethylenetetramine under reflux in acetic acid to afford the key intermediate 5. Finally, condensation of intermediate 5 with appropriate benzoyl hydrazines 6a-6o in the presence of acetic acid in ethanol to afford the title compounds (7a-7o) in high yields (69%-87%).
(a) DMF, room temperature, 4 h, then H2O, room temperature, 12 h; (b) NH4Ac, EtOH, reflux, 1 h; (c) hexamethylenetetramine, AcOH, reflux, 3 h; (d) AcOH (c), EtOH, reflux, 5 h.
The chemical structures of these title compounds were characterized by spectroscopic methods (1H NMR and 13C NMR), as well as HRMS spectrometry (see Supporting Information). Such as the 1H NMR spectrum of 7b shown two singlets at δ 3.80 and 3.81 ppm due to two methoxy groups on the benzene ring. The methylene protons (–CH2–) of tetrahydroindole moiety were appeared at δ 1.72–1.75 (m, 4H), 2.56 (t, 2H, J = 6.8 Hz) and 2.73 ppm (t, 2H, J = 6.8 Hz). Four doublet peaks at δ 7.00, 7.02, 7.34 and 7.84 ppm with coupling constant of 8.4 Hz were attributed to two 4-methoxybenzyl parts. The singlet peak of CH proton of hydrazone moiety was observed at 8.44 ppm, and the signals of NH were shown as two single peaks at δ 10.88 and 11.14 ppm. In 13C NMR spectrum of 7b the signals at δ 22.84, 23.25, 23.83 and 24.33 ppm were assigned to the methylene carbon in tetrahydroindole moiety. The signals at δ 55.76 and 55.88 ppm were attributed to the carbon in two methoxyl groups. The multiple signals observed at δ 113.51–133.64 ppm were assigned to aromatic ring. The signals observed at δ 146.21 ppm was assigned to CH carbon of hydrazone moiety. The signals at δ 158.89–162.11 ppm attributed to carbonyl group or the aromatic carbon connected with the methoxy. The high-resolution mass spectrum of 7b shown a molecular ion peak at m/z 404.1847 as [M−H]-. Therefore, the spectral data (1H NMR, 13C NMR, and HRMS) were in full agreement with the expected structure of the compound 7b.
2.2 Biological evaluation
2.2.1 In vitro anticancer activity and cytotoxicity
The growth inhibitory effects of the newly synthesized 2-phenyl-4,5,6,7-tetrahydro-1H-indole derivatives 7a-7o were evaluated for their in vitro cytotoxicity profiles against human breast cancer cell line (MCF-7) and human lung adenocarcinoma cell line (A549) using the CCK-8 assay with cisplatin, 5-fluorouracil (5-Fu), tamoxifen, and combretastatin A-4 (CA-4) as reference standards. The IC50 values (50% inhibitory concentrations) of all the tested compounds for both cancer cell lines are summarized in Table 1. In comparison to the standard drug cisplatin, 5-Fu, tamoxifen, and CA-4, all tested compounds showed potent antiproliferative activity on the MCF-7 and A549 cancer cell lines with IC50 ranging from 1.77 ± 0.37–4.73 ± 0.61 μM and 3.74 ± 0.21–7.47 ± 0.57 μM, respectively. Among the series, compound 7b with a 4-methoxyl substituent at the phenylhydrazone moiety exhibited the most potent anticancer activity against MCF-7 and A549 with IC50 values of 1.77 ± 0.37 and 3.75 ± 0.11 μM, respectively. These results indicated that 4-methoxy group plays an important role in the improvement of antiproliferative activity for this class of compounds, which was consistent with the previously reported study (Li et al., 2017; Xia et al., 2020). The replacement of 4-methoxy group with other substituents (F, Cl, Br, Me, tBu, etc) resulted in a slight decrease in antiproliferative activity. It is particularly noteworthy that although 3,4,5-trimethoxy fragment has been proved to be the optimal group for tubulin inhibitors (Li et al., 2018). Many tubulin inhibitors contain this fragment in their structure. However, the replacement of 4-methoxy group (7b) with 3,4, 5-trimethoxy (7 k) resulted in a slight decrease in antitumor activity on MCF-7 cells, but a significant decrease in antitumor activity on A549 cells.
Compound
R
IC50 (μM)a
MCF-7
A549
7a
4-F
3.63 ± 0.39
4.12 ± 0.29
7b
4-MeO
1.77 ± 0.37
3.75 ± 0.11
7c
4-tBu
4.73 ± 0.61
6.68 ± 0.27
7d
H
2.21 ± 0.54
3.08 ± 0.66
7e
4-Br
3.50 ± 0.50
7.17 ± 0.33
7f
4-Cl
3.30 ± 0.38
4.38 ± 0.12
7 g
4-Me
2.97 ± 0.78
4.04 ± 0.13
7 h
3-Me
3.54 ± 0.56
3.93 ± 0.16
7i
3-Cl
4.04 ± 0.44
7.18 ± 0.90
7j
3-NO2
3.03 ± 0.32
4.68 ± 0.06
7 k
3,4,5-MeO3
2.07 ± 0.17
7.47 ± 0.57
7 l
2-NH2
2.33 ± 0.40
7.40 ± 0.45
7 m
4-NH2
1.90 ± 0.31
3.74 ± 0.21
7n
4-OH
2.21 ± 0.17
7.42 ± 0.11
7o
3-Br
4.02 ± 0.18
5.09 ± 0.40
Cisplatin
–
11.15 ± 0.75
4.92 ± 0.56
5-Fu
–
11.61 ± 0.60
2.75 ± 0.31
Tamoxifen
–
14.28 ± 0.40
20.20 ± 0.65
CA-4
–
5.55 ± 0.11
0.029 ± 0.004
In order to evaluate the safety of these new synthetic compounds, we selected the most active compound 7b, and tested its toxicity to human normal liver cell line (LO2). The result was shown that 7b displayed moderate cytotoxicity against LO2 with an IC50 value of 44.71 ± 2.71 μM. Due to compound 7b exhibited strong antiproliferative activity against human cancer cell lines with IC50 values of 1.77 ± 0.37 (MCF-7) and 3.75 ± 0.11 μM (A549), respectively. These results indicated that compound 7b showed higher selectivity to tumor cells than normal cells. Hence, it can be concluded that these compounds have good safety for potential application in the treatment of human cancer.
2.2.2 Cell cycle analysis
Encouraged by the preliminary in vitro antiproliferative screening results, the anticancer mechanism of this series of compounds was further explored, the most potent compound 7b was selected to investigate its effect on the cell cycle progression of MCF-7 cancer cells using flow cytometry analysis. In this study, MCF-7 cancer cells were treated with DMSO (control group) or increased concentrations of 7b (0.625, 1.25, and 2.5 μM) for 24 h. As depicted in Fig. 2, the percentage of cells at the G2/M phase in presence of the compound was 30.67%, 42.24%, and 71.31%, respectively, while 25.98% of the G2/M phase was detected for the control group. These findings demonstrated that compound 7b concentration-dependently caused a significant G2/M arrest, which was a representative characteristic for tubulin polymerization inhibitors (Wang et al., 2021; Wang et al., 2020b).
Effect of compound 7b on cell cycle in MCF-7 cells. Flow cytometry of MCF-7 cells treated with 7b for 24 h. (A) Control; (B) 7b, 0.625 μM; (C) 7b, 1.25 μM; (D) 7b, 2.5 μM.
2.2.3 Cell apoptosis study
Due to breast cancer is the most common invasive cancer in women, and many tubulin polymerization inhibitors are commonly used in clinical for the treatment of breast cancer, we selected MCF-7 cells to evaluate the antitumor activity of these compounds. It has been demonstrated that tubulin polymerization inhibitors are able to induce cellular apoptosis (Mustafa et al., 2019; Wang et al., 2020c), we also examined whether compound 7b can induce cell apoptosis. In this work, cell apoptosis analysis of MCF-7 cells incubated with the increasing concentrations of 7b (0.5, 2.0, and 5.0 μM) was performed using an Annexin V-FITC/PI assay. As shown in Fig. 3, after treatment with 7b at the concentrations of 0.5, 2.0, and 5.0 μM for 24 h, the total numbers of early (Q3) and late apoptotic (Q2) cells were 28.4%, 31.4%, and 53.3%, respectively, whereas that of the control group was only 9.21%. Hence, the results revealed that compound 7b exhibited antiproliferative activity through dose-dependently inducing cellular apoptosis.
Effect of compound 7b on cell apoptosis in MCF-7 cells. Flow cytometric analysis of apoptotic cells after treatment of MCF-7 cells with 7b for 24 h. (A) Control; (B) 7b, 0. 5 μM; (C) 7b, 2.0 μM; (D) 7b, 5.0 μM. The diverse cell stages were given as live (Q4), early apoptotic (Q3), late apoptotic (Q2), and necrotic cells (Q1).
2.2.4 In vitro inhibition of the tubulin polymerization
To obtain an insight into the molecular mechanism of action of these compounds and to investigate whether their antiproliferative activities are related to an interaction with tubulin, the most potent compound 7b was tested for its in vitro ability to inhibit tubulin polymerization using the typical tubulin inhibitor colchicine as the positive control (Wang et al., 2020e). As shown in Fig. 4, after tubulin was incubated with 7b at various concentrations (3.0, 6.0, 12.5, 25, 50, and 100 μM), the absorbance values decreased compared with the control. Besides, compound 7b shown a similar inhibitory manner with the positive control colchicine. The results revealed that the effect on the tubulin polymerization positively correlated well with antiproliferative activity, indicating that these 2-phenyl-4,5,6,7-tetrahydro-1H-indole derivatives were potent tubulin polymerization inhibitors.
Effect of compound 7b on the in vitro tubulin polymerization. Purified tubulin protein and GTP in a reaction buffer incubated at 37 °C in the presence of 7b (3.0, 6.0, 12.5, 25, 50, and 100 μM), colchicine (12.5 μM) or vehicle (DMSO). Tubulin polymerization reaction was monitored at OD 340 nm every minute at 37 °C over a 20 min period.
2.2.5 Molecular modeling study
To investigate the possible binding mode of these newly synthesized 2-phenyl-4,5,6,7-tetrahydro-1H-indole derivatives with tubulin, molecular docking study of compound 7b was performed at the colchicine binding site of the tubulin crystal structure (PDB ID: 1SA0) using Autodock vina 1.1.2 (Trott & Olson, 2010). The binding pose of 7b and tubulin was shown in Fig. 5, and the estimated binding energy was −9.6 kcal·mol−1. As shown in Fig. 5, compound 7b adopted a “Y-shaped” conformation in the pocket of the tubulin. Compound 7b located at the hydrophobic pocket, surrounded by the residues A/Ala-180, A/Val-181, B/Leu-248, B/Ala-250, B/Leu-252, B/Leu-255, B/Ala-316, B/Ala-317, and B/Val-318, forming a strong hydrophobic binding. Detailed analysis showed that the phenyl group of 7b formed cation-π interactions with the residue Lys-352. It was shown that the residue A/Thr-179 (bond length: 2.2 Å) formed a hydrogen bond with 7b, which was the main interaction between 7b and tubulin. All these interactions helped 7b to anchor in the colchicine binding site of the tubulin.
Compound 7b was docked to the colchicine binding site of the tubulin (α: green; β: cyan). (A) The overall structure of tubulin with 7b. (B) Binding pose of 7b at colchicine binding site. (C) Superimposed pose of 7b (pink) and colchicine (yellow-orange) in the binding site. (D) Binding pose of 7b in the surface of colchicine binding pocket.
3 Conclusions
In this investigation, we report a novel series of 2-phenyl-4,5,6,7-tetrahydro-1H-indole derivatives as potential anticancer agents and tubulin polymerization inhibitors. All newly prepared compounds were tested for their antiproliferative activity in vitro on the human breast cancer cell line (MCF-7) and human lung adenocarcinoma cell line (A549). The SAR analysis of these compounds revealed that compound 7b with a 4-methoxyl substituent at the phenylhydrazone moiety exhibited the most potent anticancer activity against MCF-7 and A549 with IC50 values of 1.77 ± 0.37 and 3.75 ± 0.11 μM, respectively. Further mechanism studies revealed that 7b significantly arrested cell cycle at G2/M phase, induced apoptosis with a dose-dependent manner, and inhibited tubulin polymerization. Molecular docking study suggested that 7b binds well to the colchicine site of tubulin. These preliminary results demonstrate that compound 7b is a new tubulin polymerization inhibitor and is worthy of further investigation aiming to the development of new potential anticancer agents.
4 Experimental section.
4.1 Chemistry.
The starting materials (compound 1, 2 and 6a-6o), solvents or reagents were purchased from commercial suppliers. Nuclear magnetic resonance spectra (NMR) were recorded on a JNM spectrometer (400 MHz) with TMS as an external reference and reported in parts per million. High-resolution mass spectra (HRMS) were recorded on Bruker MicroQTOF Ⅱ using ESI method.
4.1.1 2-(2-(4-Methoxyphenyl)-2-oxoethyl)cyclohexan-1-one (3)
To a solution of compound 1 (1.0 mmol) in DMF (10 mL) were added and compound 2 (1.0 mmol) and the reacting mixture was stirred at room temperature for 4 h. Then, 100 mL of water was added to the reaction solution and stirred at room temperature for 12 h. The white solid precipitate was collected by filtration and purified by chromatography on silica gel with EtOAc/petroleum ether to give compound 3 as white solid.
4.1.2 2-(4-Methoxyphenyl)-4,5,6,7-tetrahydro-1H-indole (4)
A mixture of 3 (1 mmol) and NH4Ac (5 mmol) in ethanol (10 mL) was stirred at reflux for 1 h. After the completion of the reaction (monitored by TLC), the mixture was poured into water and extracted 3 times for ethyl acetate. The combined organic layers were dried over Na2SO4 and concentrated under vacuum. The residue was purified by chromatography to give compound 4 as brown solid.
4.1.3 2-(4-Methoxyphenyl)-4,5,6,7-tetrahydro-1H-indole-3-carbaldehyde (5)
A mixture of compound 4 (227 mg, 1.0 mmol) and hexamethylenetetramine (281 mg, 2 mmol) in acetic acid (10 mL) was stirred at reflux for 3 h. After the completion of the reaction, the mixture was poured into water (50 mL) and the precipitate was collected by filtration and purified by chromatography to give the key intermediate 5 as yellow solid (88%). 1H NMR (DMSO‑d6, 400 MHz) δ: 1.67–1.74 (m, 4H), 2.50–2.54 (m, 2H), 2.65 (t, 2H, J = 5.6 Hz), 3.80 (s, 3H), 7.03 (d, 2H, J = 8.8 Hz), 7.45 (d, 2H, J = 8.8 Hz), 9.69 (s, 1H), 11.38 (s, 1H).
4.1.4 General procedure for the synthesis of 7
A mixture of 5 (1.0 mmol) and different benzoyl hydrazines 6 (1.0 mmol) was refluxed in ethanol (10 mL) for 6 h in the presence of one drop of glacial acetic acid. After the completion of the reaction, the solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (petroleum ether/EtOAc) to give the title compounds 7a-7o. The spectroscopic and analytical data of these compounds are as follows:
4.1.4.1 (E)-4-Fluoro-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)benzohydrazide (7a)
Yellow solid; yield = 72%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.61–1.77 (m, 4H), 2.49–2.54 (m, 2H), 2.67–2.72 (m, 2H), 3.75 (s, 3H), 6.98 (d, 2H, J = 8.8 Hz), 7.26–7.32 (m, 4H), 7.88–7.91 (m, 2H), 8.40 (s, 1H), 10.89 (s, 1H), 11.24 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 22.81, 23.21, 23.79, 24.31, 55.75, 113.33, 114.64, 115.65, 115.86, 116.64, 125.37, 128.83, 129.68, 129.77, 130.49, 130.57, 131.09, 133.96, 146.88, 158.92, 161.63; HRMS (ESI) calcd for [M−H]- C23H21FN3O2-: 390.1623 found 390.1653.
4.1.4.2 (E)-4-methoxy-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)benzohydrazide (7b)
Brown solid; yield = 84%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.72–1.75 (m, 4H), 2.56 (t, 2H, J = 6.8 Hz), 2.73 (s, 2H, J = 6.8 Hz), 3.80 (s, 3H), 3.81 (s, 3H), 7.00 (d, 2H, J = 8.4 Hz), 7.02 (d, 2H, J = 8.4 Hz), 7.34 (d, 2H, J = 8.8 Hz), 7.84 (d, 2H, J = 8.4 Hz), 8.44 (s, 1H), 10.88 (s, 1H), 11.14 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 22.84, 23.25, 23.83, 24.33, 55.76, 55.88, 113.51, 114.04, 114.64, 116.62, 124.80, 125.50, 126.77, 128.74, 129.74, 133.64, 146.21, 158.89, 162.11; HRMS (ESI) calcd for [M−H]− C24H26N3O3-: 404.1980 found 404.1847.
4.1.4.3 (E)-4-(tert-butyl)-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)benzohydrazide (7c)
Light yellow solid; yield = 75%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.26 (s, 9H), 1.69 (s, 4H), 2.51 (s, 2H), 2.69 (s, 2H), 3.75 (s, 3H), 6.97 (d, 2H, J = 8.0 Hz), 7.29 (d, 2H, J = 8.0 Hz), 7.44 (d, 2H, J = 8.0 Hz), 7.74 (d, 2H, J = 8.0 Hz), 8.40 (s, 1H), 10.87 (s, 1H), 11.17 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 22.83, 23.21, 23.80, 24.32, 31.46, 35.14, 55.72, 113.39, 114.61, 125.39, 125.57, 127.75, 128.77, 129.73, 130.56, 131.87, 133.79, 146.57, 154.47, 158.87, 162.69; HRMS (ESI) calcd for [M−H]- C27H30N3O2-: 428.2344 found 428.2383.
4.1.4.4 (E)-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)benzohydrazide (7d)
Yellow solid; yield = 69%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.70 (s, 4H), 2.52 (s, 2H), 2.70 (s, 2H), 3.75 (s, 3H), 6.98 (d, 2H, J = 8.4 Hz), 7.30 (d, 2H, J = 8.4 Hz), 7.42–7.50 (m, 3H), 7.81–7.83 (m, 2H), 8.41 (s, 1H), 10.88 (s, 1H), 11.24 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 22.82, 23.22, 23.80, 24.32, 55.74, 113.38, 114.63, 116.65, 125.40, 127.89, 128.81, 129.75, 131.69, 133.88, 134.64, 146.80, 158.90, 162.69; HRMS (ESI) calcd for [M−H]- C23H22N3O2–: 372.1718 found 372.1751.
4.1.4.5 (E)-4-bromo-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)benzohydrazide (7e)
Yellow solid; yield = 73%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.70 (s, 4H), 2.52 (s, 2H), 2.70 (s, 2H), 3.76 (s, 3H), 6.98 (d, 2H, J = 8.8 Hz), 7.31 (d, 2H, J = 8.8 Hz), 7.64 (d, 2H, J = 8.4 Hz), 7.77 (d, 2H, J = 8.4 Hz), 8.41 (s, 1H), 10.82 (s, 1H), 11.23 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 22.79, 23.20, 23.77, 24.30, 55.75, 113.30, 114.65, 116.64, 125.32, 125.40, 128.86, 129.16, 129.78, 130.03, 130.57, 131.83, 133.68, 134.08, 147.11, 158.94, 161.69; HRMS (ESI) calcd for [M−H]- C23H21BrN3O2-: 450.0823 found 450.0867.
4.1.4.6 (E)-4-chloro-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)benzohydrazide (7f)
Light yellow solid; yield = 82%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.68–1.70 (m, 4H), 2.50–2.51 (m, 2H), 2.65–2.71 (m, 2H), 3.75 (s, 3H), 6.98 (d, 2H, J = 8.8 Hz), 7.29 (d, 2H, J = 8.8 Hz), 7.51 (d, 2H, J = 8.4 Hz), 7.84 (d, 2H, J = 8.4 Hz), 8.40 (s, 1H), 10.91 (s, 1H), 11.30 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 22.79, 23.20, 23.78, 24.30, 55.75, 113.31, 114.65, 116.65, 125.34, 128.86, 128.90, 129.78, 129.84, 133.33, 134.07, 136.49, 147.10, 158.94, 161.58; HRMS (ESI) calcd for [M−H]- C23H21ClN3O2-: 406.1328 found 406.1367.
4.1.4.7 (E)-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)-4-methylbenzohydrazide (7 g)
Light yellow solid; yield = 87%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.68–1.70 (m, 4H), 2.32 (s, 3H), 2.49–2.54 (m, 2H), 2.67–2.72 (m, 2H), 3.75 (s, 3H), 6.98 (d, 2H, J = 8.8 Hz), 7.23 (d, 2H, J = 8.0 Hz), 7.30 (d, 2H, J = 8.8 Hz), 7.72 (d, 2H, J = 8.4 Hz), 8.41 (s, 1H), 10.88 (s, 1H), 11.18 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 21.51, 22.82, 23.22, 23.80, 24.32, 25.95, 55.74, 113.43, 114.63, 115.02, 116.63, 125.42, 127.90, 128.77, 129.34, 129.74, 131.73, 133.77, 134.31, 141.61, 143.66, 146.52, 158.88, 162.51; HRMS (ESI) calcd for [M−H]- C24H24N3O2-: 386.1874 found 386.1915.
4.1.4.8 (E)-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)-3-methylbenzohydrazide (7 h)
Light yellow solid; yield = 79%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.72–1.75 (m, 4H), 2.37 (s, 3H), 2.53–2.58 (m, 2H), 2.71–2.75 (m, 2H), 3.79 (s, 3H), 7.02 (d, 2H, J = 8.8 Hz), 7.34–7.37 (m, 4H), 7.64–7.67 (m, 2H), 8.44 (s, 1H), 10.93 (s, 1H), 11.25 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 21.49, 22.82, 23.22, 23.80, 24.32, 55.74, 113.40, 114.64, 116.63, 125.09, 125.41, 128.34, 128.72, 128.78, 129.75, 132.27, 133.84, 134.64, 138.08, 146.62, 158.89, 162.77; HRMS (ESI) calcd for [M−H]- C24H24N3O2-: 386.1874 found 386.1919.
4.1.4.9 (E)-3-chloro-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)benzohydrazide (7i)
Yellow solid; yield = 82%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.72–1.75 (m, 4H), 2.54–2.58 (m, 2H), 2.71–2.76 (m, 2H), 3.80 (s, 3H), 7.03 (d, 2H, J = 8.8 Hz), 7.34 (d, 2H, J = 8.8 Hz), 7.53 (t, 1H, J = 8.0 Hz), 7.61–7.63 (m, 1H), 7.82 (d, 1H, J = 8.0 Hz), 7.91 (s, 1H), 8.44 (s, 1H), 10.97 (s, 1H), 11.37 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 22.80, 23.20, 23.78, 24.30, 55.76, 113.27, 114.66, 116.66, 125.32, 126.76, 127.56, 128.87, 129.80, 130.87, 131.54, 133.64, 134.16, 136.63, 147.24, 158.96, 161.16; HRMS (ESI) calcd for [M−H]- C23H21ClN3O2-: 406.1328 found 406.1376.
4.1.4.10 (E)-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)-3-nitrobenzohydrazide (7j)
Orange solid; yield = 76%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.74 (s, 4H), 2.56 (s, 2H), 2.75 (s, 2H), 3.80 (s, 3H), 7.04 (d, 2H, J = 8.8 Hz), 7.35 (d, 2H, J = 8.8 Hz), 7.80 (t, 1H, J = 8.0 Hz), 8.31 (d, 1H, J = 8.0 Hz), 8.39 (d, 1H, J = 8.0 Hz), 8.47 (s, 1H), 8.70 (s, 1H), 11.00 (s, 1H), 11.60 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 22.80, 23.19, 23.77, 24.30, 55.77, 113.23, 114.69, 116.68, 122.55, 125.28, 126.35, 128.93, 129.72, 129.86, 130.65, 134.56, 136.04, 147.67, 148.24, 159.02, 160.44; HRMS (ESI) calcd for [M−H]- C23H21N4O4-: 417.1568 found 417.1611.
4.1.4.11 (E)-3,4,5-trimethoxy-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)benzohydrazide (7 k)
Brown solid; yield = 78%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.72–1.75 (m, 4H), 2.56 (s, 2H), 2.73 (s, 2H), 3.70 (s, 3H), 3.79 (s, 3H), 3.83 (s, 6H), 7.02 (d, 2H, J = 8.8 Hz), 7.17 (s, 2H), 7.34 (d, 2H, J = 8.8 Hz), 8.42 (s, 1H), 10.95 (s, 1H), 11.17 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 22.81, 23.21, 23.81, 24.30, 55.76, 56.58, 60.63, 105.46, 113.39, 114.69, 116.65, 125.40, 128.81, 129.82, 133.90, 140.44, 146.83, 153.10, 158.96, 162.27; HRMS (ESI) calcd for [M−H]- C26H28N3O5-: 462.2034 found 462.2075.
4.1.4.12 (E)-2-amino-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)benzohydrazide (7 l)
Yellow solid; yield = 81%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.69 (s, 4H), 2.51 (s, 2H), 2.69 (s, 2H), 3.75 (s, 3H), 6.28 (s, 2H), 6.47 (t, 1H, J = 8.0 Hz), 6.65 (d, 1H, J = 8.0 Hz), 6.97 (d, 2H, J = 8.8 Hz), 7.10 (t, 1H, J = 8.0 Hz), 7.29 (d, 2H, J = 8.8 Hz), 7.43 (d, 1H, J = 8.0 Hz), 8.37 (s, 1H), 10.36 (s, 1H), 11.04 (s, 1H); 13C NMR (DMSO‑d6, 150 MHz) δ: 22.78, 23.18, 23.76, 24.30, 55.68, 113.46, 114.55, 114.94, 116.53, 116.62, 125.47, 128.51, 128.65, 129.63, 131.49, 132.10, 132.51, 133.49, 145.91, 150.20, 158.77; HRMS (ESI) calcd for [M−H]- C23H23N4O2-: 387.1826 found 387.1887.
4.1.4.13 (E)-4-amino-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)benzohydrazide (7 m)
Yellow solid; yield = 74%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.67–1.70 (m, 4H), 2.51 (s, 2H), 2.67 (s, 2H), 3.75 (s, 3H), 5.64 (s, 2H), 6.49 (d, 2H, J = 8.4 Hz), 6.97 (d, 2H, J = 8.4 Hz), 7.29 (d, 2H, J = 8.4 Hz), 7.55 (d, 2H, J = 8.4 Hz), 8.37 (s, 1H), 10.83 (s, 1H), 10.86 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 22.83, 23.23, 23.83, 24.31, 55.72, 113.08, 114.60, 116.56, 120.87, 125.54, 128.66, 129.46, 129.65, 133.24, 145.22, 152.25, 158.77, 162.73; HRMS (ESI) calcd for [M−H]- C23H23N4O2-: 387.1826 found 387.1879.
4.1.4.14 (E)-4-hydroxy-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)benzohydrazide (7n)
Brown solid; yield = 80%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.68 (s, 4H), 2.51 (s, 2H), 2.68 (s, 2H), 3.75 (s, 3H), 6.76 (d, 2H, J = 8.0 Hz), 6.97 (d, 2H, J = 8.0 Hz), 7.29 (d, 2H, J = 8.0 Hz), 7.69 (d, 2H, J = 8.0 Hz), 8.38 (s, 1H), 9.98 (s, 1H), 10.86 (s, 1H), 11.04 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 22.84, 23.24, 23.83, 24.33, 55.74, 113.53, 114.61, 115.33, 116.59, 125.18, 125.51, 128.70, 129.70, 129.82, 133.50, 145.91, 158.84, 160.65, 162.34; HRMS (ESI) calcd for [M−H]- C23H22N3O3–: 388.1667 found 388.1702.
4.1.4.15 (E)-3-bromo-N'-((2-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-indol-3-yl)methylene)benzohydrazide (7o)
Yellow solid; yield = 83%; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.65–1.65 (m, 4H), 2.50 (s, 2H), 2.68 (s, 2H), 3.74 (s, 3H), 6.97 (d, 2H, J = 8.8 Hz), 7.29 (d, 2H, J = 8.8 Hz), 7.41 (t, 1H, J = 8.0 Hz), 7.68–7.71 (m, 1H), 7.80 (d, 1H, J = 8.0 Hz), 7.98–7.98 (m, 1H), 8.38 (s, 1H), 10.90 (s, 1H), 11.32 (s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 22.78, 23.18, 23.77, 24.28, 55.75, 113.25, 114.67, 116.67, 122.14, 125.28, 127.13, 128.90, 129.79, 130.38, 131.15, 134.20, 134.46, 136.77, 147.31, 158.96, 161.15; HRMS (ESI) calcd for [M−H]- C23H21BrN3O2-: 450.0823 found 450.0877.
4.2 Cell proliferation and cytotoxicity assays
Cell proliferation and cytotoxicity of these newly synthesized compounds were evaluated following our previous reports using CCK-8 assay (Wang et al., 2021). Briefly, cells (MCF-7, A549 and LO2) were seeded in 96-well plates at 1 × 104 cells/well and cultured in RPMI-1640 with 10% fetal bovine serum for 24 h. Then, the cells were treated with different concentrations (0.3125, 0.625, 1.25, 2.5, 5.0, 10 and 20 μM) of tested compounds or standard drugs for 48 h. After incubation, cell viability was determined by the CCK-8 method after incubation. The cell proliferation and cytotoxicity were expressed as the IC50 values.
4.3 Cell cycle assay
Human breast cancer cells (MCF-7) were treated with DMSO or different concentrations of compound 7b (0.625, 1.25 and 2.5 μM) for 24 h. After incubation, the cells were collected, washed with PBS, and fixed by ice-cold ethanol (75%) at 4 °C overnight. Subsequently, cells were then washed, treated using RNase (50 μg/mL) for 30 min at 37 °C, and stained with propidium iodide. The DNA content of the cells was analyzed using a flow cytometry.
4.4 Cell apoptosis assay
After treatment with different concentrations of 7b (0.5, 2.0 and 5.0 μM) and vehicle for 24 h, MCF-7 cells were harvested and incubated with 5 μL Annexin-V/FITC in binding buffer (containing 140 mM NaCl, 10 mM HEPES, 2.5 mM CaCl2, pH 7.4) and 10 μL PI staining solution at room temperature for 15 min. Thereafter, the stained cells were analyzed by flow cytometry.
4.5 Tubulin polymerization assay
Tubulin protein was purified from pig brain, and experimental details for tubulin polymerization assay were reported in our previous work (Wang et al., 2018b). The purified tubulin protein was incubated with different concentrations of 7b, colchicine) or DMSO in PEM buffer (100 mM PIPES, 1 mM MgCl2, and 1 mM EGTA) containing 1 mM GTP and 5 % glycerol. The absorbance value at 340 nm was monitored by a SPECTRA MAX 190 (Molecular Device) spectrophotometer. The plateau absorbance values were used for calculations.
4.6 Molecular modeling study
According to our previous work, molecular modeling study was performed by Autodock vina 1.1.2. The x-ray crystal structure of tubulin (PDB ID: 1SA0) was downloaded from the Protein Data Bank (PDB). In brief, the search grid of the tubulin was identified as center_x: 118.921, center_y: 89.718, and center_z: 5.932 with dimensions size_x: 15, size_y: 15, and size_z: 15. The value of exhaustiveness was set to 20. The top-score docking poses were selected and visually analyzed using PyMoL 1.7.6 software (www.pymol.org).
4.7 Statistical analysis
All experiments were carried out at least in three independent trials. The experimental data were expressed as the mean ± standard deviation.
Acknowledgements
This work was supported by One Thousand Talents Program of Guizhou Province (the fifth group, [2019]4), Academic New Seedling Project of Guizhou Medical University (19NSP030).
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Appendix A
Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2021.103504.
Appendix A
Supplementary material
The following are the Supplementary data to this article:Supplementary Data 1
Supplementary Data 1
