Synthesis, biological evaluation and molecular docking studies of novel 1-(4,5-dihydro-1H-pyrazol-1-yl)ethanone-containing 1-methylindol derivatives as potential tubulin assembling inhibitors

Abstract

A series of novel compounds (6a–6v) containing 1-methylindol and 1-(4,5-dihydro-1H-pyrazol-1-yl)ethanone skeletons were designed, synthesized and biologically evaluated as potential tubulin polymerization inhibitors and anticancer agents. Among them, compound 6q showed the most potent tubulin polymerization inhibitory activity (IC₅₀ = 1.98 μM) and in vitro growth inhibitory activity against A549, MCF-7 and HepG2 cell lines, with IC₅₀ values of 0.15 μM, 0.17 μM, and 0.25 μM respectively, comparable to the positive control. Furthermore, compound 6q was a potent inducer of apoptosis in A549 cells and it had typical cellular effects for microtubule interacting agents, causing arrest of the cell cycle in the G2/M phase. Confocal microscopy assay and molecular docking results further demonstrated that 6q could bind tightly to the colchicine site of tubulin and act as an anti-tubulin agent. These studies, along with 3D-QSAR modeling provided an important basis for further optimization of compound 6q as a potential anticancer agent.

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1. Introduction

Microtubules (MTs) are a broadly exploited target for the development of new effective anticancer agents.^1–3 MTs are highly dynamic polymers mainly composed of α,β-tubulin heterodimers that turn over continuously. Their polymerization–depolymerization dynamics play a crucial role in various fundamental cellular processes including mitotic spindle formation, organization of intracellular structure, and intracellular transport, as well as cell signaling and secretion.⁴ Hence, interfering with MTs function can block cell division and cause cell damage, which is an established strategy to inhibit the proliferation of cancer cells.^2,3,5

There are many agents that bind to tubulin and interfere with microtubule assembly. Colchicine (compound 1, Fig. 1), one of the earliest tubulin-targeting drugs, can effectively interfere with microtubule polymerization and its binding site on tubulin has been characterized already.^6,7 However, due to its highly adverse effects, the clinical development of colchicine for cancer therapy was not successful.⁸ Another well-known potent anti-tubulin agent is CA-4 (compound 2, Fig. 1), which was isolated from the bark of the South African tree Combretum caffrum. It can potently bind to the colchicine binding site, leading to severe cytotoxicity against multiple human cancer cell lines.⁹ But unfortunately, it easily undergoes cis–trans isomerization under natural light, causing a sharp reduction in the anti-tubulin activity, which limits its further applications.¹⁰ Despite a long history of characterization, colchicine binding site inhibitors have not yet reached the commercial phase as anticancer drugs to date. Thus, further studies are required.

Fig. 1 Chemical structures of inhibitors and potential inhibitors of tubulin polymerization.

In the last two decades, an ever increasing number of synthetic indoles have been reported, which are able to inhibit tubulin polymerization, such as compound 3 (ATIs),¹¹ 4 (BPROL075)¹² and 5 (ref. 13) (Fig. 1). It could be found that the indole structure is a crucial structural motif for tubulin inhibition. The indole nucleus also has been reported to be one of the core nucleuses for potent inhibitors of tubulin polymerization.^14,15 Furthermore, the addition of methyl to the N atom of indole would be better for the inhibition of tubulin polymerization and cytotoxicity.^16,17

Also, pyrazolines have gained prominence due to its broad spectrum of biological activities including anticancer, antimicrobial and anti-inflammatory,^18,19 as well as producing useful intermediates in several organic preparations. It was also reported some pyrazoline derivatives can inhibit tubulin polymerization by binding to the colchicine-site, such as compound 6.²⁰ Besides, the presence of acetyl on the N atom of the five-membered ring enhances the inhibition of cell proliferation and the binding of the colchicine-site. For example, compound 7 (A-204197)²¹ and compound 8 (ref. 22) (Fig. 1) showed potent anti-proliferative activities against multiple cancer cell lines and tightly bound to the colchicine-site of tubulin.

Therefore, due to these previous researches and molecule modeling studies, we attempt to integrate 1-methylindol with 1-(4,5-dihydro-1H-pyrazol-1-yl)ethanone to screen novel compounds (6a–6v) as potential anti-tubulin agents (Fig. 1). These coupling structures might possess enhanced anticancer activities. A series of novel 1-methylindol derivatives targeting tubulin were recently reported as potent anticancer agents in our group.²³ To extend our research and gain further insight in the ensuing structure–activity relationships (SARs), here we search more novel agents. Our strategy for further development of active anti-tubulin agents was to perform modifications on the two aromatic rings (A, B ring). Once the new combined scaffold was identified as the minimum structural requirement for activity in this new series of compounds, then some substituents were introduced onto the aromatic rings, and the modifications have been performed to screen a series of active anticancer agents.

So far, few reports have been dedicated to design and synthesize any anticancer compound containing 1-methylindol and 1-(4,5-dihydro-1H-pyrazol-1-yl)ethanone. Herein, we discussed the synthetic method and structure–activity relationship (SAR) of this series of compounds, evaluated their anticancer and anti-tubulin activities, and learned the influence on cell cycle and cell apoptosis. In addition, confocal microscopy assay provided a more directly view of the effect on microtubule system. Docking simulations are performed using the X-ray crystallographic structure of the tubulin in complex with an inhibitor to explore the binding modes of these compounds at the active site.

2. Results and discussion

2.1. Chemistry

The synthetic route of compounds 6a–6v (Table 1) was outlined in Scheme 1. First of all, the substituted 1H-indole (1a–1c) dissolved in DMF was added to a mixture of POCl₃ and DMF at 0 °C to generate compounds 2a–2c after two-hour stirring. Then the substituted indole-3-carbaldehyde (2a–2c) was dissolved in THF and reacted with NaH and CH₃I to introduce the methyl group to the N atom of indole and obtain compounds 3a–3c. Subsequently, the obtained compounds (3a–3c) and the corresponding acetophenone were dissolved in ethanol in the presence of KOH to give products 4a–4v. After that, the obtained chalcone (4a–4v) was stirred with hydrazine hydrate in ethanol and refluxed for 2 h, and then crude compounds 5a–5v were gained. Finally, to a stirred solution of acetic acid, EDC·HCl and HOBt in acetonitrile or dichloromethane, obtained compounds (5a–5v) were added with the protection of N₂ to get the target compounds 6a–6v. All synthesized compounds 6a–6v are reported for the first time and determined by ¹H NMR, melting test, ESI-MS and elemental analysis, which were consistent with the assigned structures.

Table 1 Structures of compounds 6a–6v

Compounds	R1	R2	R3	R4	R5
6a	H	H	H	H	H
6b	H	H	H	OCH3	H
6c	H	H	OCH3	OCH3	H
6d	H	H	OCH3	OCH3	OCH3
6e	H	H	H	Cl	H
6f	H	H	H	Br	H
6g	H	H	H	CF3	H
6h	OCH3	H	H	H	H
6i	OCH3	H	H	OCH3	H
6j	OCH3	H	OCH3	OCH3	H
6k	OCH3	H	H	F	H
6l	OCH3	H	H	Cl	H
6m	OCH3	H	H	Br	H
6n	Br	H	H	H	H
6o	Br	OCH3	H	H	H
6p	Br	H	OCH3	H	H
6q	Br	H	H	OCH3	H
6r	Br	H	OCH3	OCH3	H
6s	Br	H	OCH3	OCH3	OCH3
6t	Br	H	H	F	H
6u	Br	H	H	Cl	H
6v	Br	H	H	Br	H

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Scheme 1 General synthesis of compounds 6a–6v. Reagents and conditions: (i) DMF, POCl₃, 0 °C, 2 h; (ii) CH₃I, NaH, THF, 0 °C – r.t., 24 h; (iii) substituted acetophenone, KOH, EtOH, r.t., 24 h; (iv) N₂H₄·H₂O, EtOH, reflux, 2 h; (v) AcOH, EDC·HCl, HOBt, CH₃CN/CH₂Cl₂, r.t., 24 h.

2.2. Crystal structure of compound 6o

The crystal structure of compound 6o was determined by X-ray diffraction analysis. The crystal data presented in Table 2 and Fig. 2 gave perspective views of compound 6o with the atomic labeling system.

Table 2 Crystallographic and experimental data for compound 6o

Compound	6o
Formula	C21H20BrN3O2
Formula weight	426.31
Crystal system	Monoclinic
Space group	P21/c
a (Å)	16.5763(14)
b (Å)	8.0259(7)
c (Å)	15.6946(13)
α (°)	90
β (°)	109.896(2)
γ (°)	90
V (Å^3)	1963.4(3)
Z	4
Dc (g cm^−3)	1.442
F (000)	872
θ range (°)	2.62–25.06
Reflections collected/unique	16 844/3457
Data/restraints/parameters	3457/0/247
Absorption coefficient (mm^−1)	2.114
Goodness-of-fit on F^2	1.050
R1, wR2 [I > 2σ(I)]	0.0494, 0.1100
R1, wR2 [all data]	0.0821, 0.1265
Max, min Δρ (e Å^−3)	0.578, −0.653

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Fig. 2 Crystal structure diagram of compound 6o. H atoms are shown as small spheres of arbitrary radii.

2.3. Biological activity

2.3.1. In vitro anti-proliferative activity. We prepared three different human cancer cell lines, including A549 (human nonsmall cell lung carcinoma), MCF-7 (human breast adenocarcinoma) and HepG2 (human liver hepatocellular carcinoma), and one normal cell line (293T) to evaluate the anti-proliferative activities of all the synthesized compounds (6a–6v). The results were summarized in Table 3. Among all the 22 tested compounds, 6b, 6i, 6q, 6t, 6u and 6v possessed the highest overall potency with IC₅₀ values ranging from 0.15 to 0.34 μM against all the three typical cancer cell lines. Compound 6q was found to be the most active among all compounds with the level of IC₅₀ values of 0.15 μM, 0.17 μM, and 0.25 μM against A549, MCF-7 and HepG2 cell lines respectively, superior to the positive compound colchicine. Besides, the results of 293T cell line indicated that these compounds possessed high safety.

Table 3 In vitro inhibitory effects of compounds 6a–6v against human tumor cell lines, normal cell line and tubulin polymerization^a

Compounds	IC50 ± SD (μM)
	A549^b	MCF-7^b	HepG2^b	293T^c	Tubulin^d
a All experiments were independently performed at least three times.b Inhibition of the growth of tumor cell lines.c Inhibition of the growth of normal cell line.d Inhibition of tubulin polymerization.e Used as positive control.
6a	5.18 ± 1.03	6.35 ± 0.33	9.22 ± 1.98	105.41 ± 4.04	32.53 ± 1.97
6b	0.19 ± 0.02	0.22 ± 0.01	0.26 ± 0.08	129.37 ± 3.93	4.33 ± 0.24
6c	4.52 ± 0.12	5.27 ± 0.68	5.46 ± 0.29	234.10 ± 4.50	23.19 ± 1.46
6d	2.36 ± 0.23	3.03 ± 0.23	3.12 ± 0.20	269.48 ± 3.84	11.45 ± 0.53
6e	1.68 ± 0.39	1.43 ± 0.05	2.24 ± 0.08	48.68 ± 6.05	22.02 ± 1.19
6f	0.87 ± 0.11	1.46 ± 0.36	1.71 ± 0.11	>300	14.32 ± 0.41
6g	1.24 ± 0.18	2.40 ± 0.31	1.92 ± 0.56	139.34 ± 0.82	26.14 ± 0.47
6h	11.75 ± 1.29	13.21 ± 0.13	14.09 ± 0.15	298.46 ± 3.19	34.96 ± 1.34
6i	0.22 ± 0.02	0.31 ± 0.11	0.34 ± 0.09	243.59 ± 6.11	5.75 ± 0.17
6j	8.31 ± 0.41	10.09 ± 0.52	9.76 ± 1.78	187.09 ± 1.41	19.62 ± 0.55
6k	4.92 ± 0.73	5.78 ± 0.07	6.63 ± 0.17	168.46 ± 5.85	13.05 ± 0.20
6l	6.43 ± 0.14	8.12 ± 0.36	7.93 ± 0.16	>300	19.76 ± 1.95
6m	1.42 ± 0.29	1.60 ± 0.27	1.88 ± 0.24	134.51 ± 1.89	10.39 ± 0.18
6n	1.08 ± 0.27	1.51 ± 0.47	1.20 ± 0 03	32.79 ± 0.68	20.54 ± 0.40
6o	0.71 ± 0.34	0.85 ± 0.13	0.92 ± 0.13	>300	15.92 ± 0.81
6p	1.24 ± 0.26	0.97 ± 0.25	1.14 ± 0.21	205.86 ± 2.67	29.64 ± 2.68
6q	0.15 ± 0.03	0.17 ± 0.05	0.25 ± 0.05	245.92 ± 3.44	1.98 ± 0.25
6r	0.51 ± 0.01	0.55 ± 0.02	0.54 ± 0.09	89.18 ± 2.52	10.03 ± 0.50
6s	0.31 ± 0.04	0.38 ± 0.09	0.42 ± 0.06	>300	7.81 ± 0.39
6t	0.21 ± 0.10	0.19 ± 0.04	0.29 ± 0.01	100.23 ± 4.67	4.22 ± 0.13
6u	0.24 ± 0.07	0.27 ± 0.12	0.34 ± 0.04	79.68 ± 2.88	5.85 ± 0.37
6v	0.18 ± 0.01	0.20 ± 0.04	0.26 ± 0.05	46.46 ± 1.34	3.16 ± 0.42
Colchicine^e	0.21 ± 0.06	0.22 ± 0.12	0.32 ± 0.09	172 ± 5.52	2.35 ± 0.34

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Based on the data of Table 3, we found that the substituent group on the A-ring was of great importance to the activity of the tested compounds. Obviously, the anti-proliferative activities of compounds with different substituents in the A-ring increased in the following order: Br > H > OCH₃, which could be observed from the comparisons: 6n > 6a > 6h, 6q > 6i > 6b and 6v > 6f > 6m.

The inhibitory activity of compounds with –OCH₃ on different positions of the B-ring increased in the following order: 4-OCH₃ (6q) > 3,4,5-(OCH₃)₃ (6s) > 3,4-(OCH₃)₂ (6r) > 2-OCH₃ (6o) > 3-OCH₃ (6p). Thus, we could draw a conclusion that compounds with p-substituted group showed the most potent activity, following by those of o-substituted, while m-substituted compounds had the lowest activity. Among all p-substituted (B-ring) compounds, 6n, 6q, 6t, 6u and 6v (IC₅₀ = 1.08 μM, 0.15 μM, 0.21 μM, 0.24 μM, 0.18 μM) exhibited significant anti-proliferative activities in the order of –OCH₃> –Br > –F > –Cl > –H in A549 cell line, while 6a, 6b, 6e and 6f, 6h, 6i, 6k, 6l and 6m showed the similar order.

2.3.2. Inhibition of tubulin polymerization. To investigate whether the anti-proliferative activities of compounds 6a–6v were resulted from their interaction with tubulin, we evaluated the ability of these agents to inhibit tubulin polymerization in vitro (Table 3). These agents inhibited tubulin assembling with IC₅₀ values ranging from 1.98 to 34.96 μM. Among them, compound 6q showed the most potent activity of anti-tubulin (IC₅₀ = 1.98 μM), better than the positive control colchicine (2.35 μM). We also found that the IC₅₀ values of tubulin polymerization of these compounds displayed a similar tendency with their relevant IC₅₀ values of the anti-proliferative assay. Fig. 3 presented the relationship between the anti-proliferative activity against A549 cell line and the tubulin polymerization inhibitory activity of the top 10 compounds (6b, 6f, 6i, 6o, 6q, 6r, 6s, 6t, 6u and 6v), which indicated that there was a moderate correlation between them with an R² value of 0.90. Therefore, we could conclude that most of the synthesized compounds could inhibit the tubulin assembly and the anti-proliferative effect was produced partly by their interaction with tubulin.

Fig. 3 Correlation between the anti-proliferative activity against A549 cell line and the tubulin polymerization inhibitory activity of the top 10 compounds.

2.3.3. Compound 6q induced cell cycle arrest and cell apoptosis. To explore whether compound 6q could arrest the process of mitosis, A549 cells were treated with 6q of increasing concentrations (0.02 μM, 0.05 μM, 0.1 μM, 0.15 μM, 0.2 μM) for 24 h and then incubated with propidium iodide (PI) to analyze their genomic DNA content in flow cytometry assays (Fig. 4). We found that, compound 6q induced a gradual accumulation of cells in G2/M phase of the cell cycle in a dose-dependent manner with a rise in G2/M cells occurring at a concentration as low as 0.02 μM. When the concentration increased to 0.20 μM, up to 90% of the cells were arrested in the G2/M phase. In contrast, there was a continuing decrease in the proportion of cells in other phase (G1 and S) of the cell cycle.

Fig. 4 Effect of compound 6q on cell cycle progression of A549 cells was determined by flow cytometry analysis (G1 phase, blue; S phase, yellow and G2/M phase, green).

To explore the correlation between cell cycle arrest and cell death induced by compound 6q, a biparametric cytofluorimetric analysis using PI and annexin-V (stain DNA and phosphatidylserine residues, respectively) was performed. Dual staining of annexin-V and PI permits discrimination between early apoptotic cells (annexin-V⁺/PI⁻), late apoptotic cells (annexin-V⁺/PI⁺), live cells (annexin-V⁻/PI⁻) and necrotic cells (annexin-V⁻/PI⁺). A549 cells were treated with 0, 0.05, 0.1 and 0.15 μM of 6q for 24 h. As shown in Fig. 5, the percentage of both early and late apoptotic cells increased as the concentration of 6q became higher, rising from 2.34% and 1.76% at 0 μM initially to 9.0% and 16.2% at 0.15 μM eventually. Thus, we could draw a conclusion that compound 6q could induce cell apoptosis in a dose-dependent manner.

Fig. 5 Representative scatter plot of A549 cells treated with 6q at 0, 0.05, 0.1 and 0.15 μM and analyzed by flow cytometry after double staining of the cells with annexin-V-FITC and PI.

2.3.4. Compound 6q inhibited tubulin polymerization and disrupted the microtubule system. Our results suggested that 6q can target microtubules in A549 cells. To test this directly, we treated cells with 0.2 μM of 6q for 24 h and analyzed microtubules by immunocytochemistry staining. As shown in Fig. 6, the microtubule network of control cells displayed normal arrangement and organization, which was slim and fibrous. After treatment with 0.2 μM of 6q, microtubules became short and wrapped around the nucleus. We also noted a significant reduction in the density of cellular microtubules and the radial organization of microtubules was perturbed, which could also be found in colchicine (0.5 μM)-treated cells which were used as positive control in this assay. In contrast, 0.5 μM of paclitaxel dramatically enhanced microtubule polymerization, with an increase in the density of cellular microtubules and formation of long thick microtubule bundles. These results further confirmed that the observed effects were due to interaction with tubulin, resulting in a prolonged mitotic arrest that ultimately leads to cell death.

Fig. 6 Effects of paclitaxel (0.5 μM), colchicine (0.5 μM) and 6q (0.2 μM) on cellular architecture and microtubule network of A549 cells. Microtubules tagged with Cy3 (red) and nuclei tagged with DAPI (blue) were observed with a confocal microscope.

2.4. Computational

2.4.1. Molecular docking. To gain a better understanding on the binding mode of the synthesized compounds, we proceeded to examine the interaction of compounds 6a–6v with tubulin crystal structure (PDB code: 1SA0) using the DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd). The molecular docking was performed by inserting the compounds into the colchicine binding site of tubulin. Among them, compound 6q showed the lowest interaction energy, at −54.27 kcal mol⁻¹. The binding models of compound 6q and tubulin were depicted in Fig. 7. The enzyme surface model (Fig. 7A) revealed that the compound 6q was well embedded in the active pocket via hydrophobic interaction. The amino acid residues interacting with tubulin were labeled. The oxygen atom of acetyl contributes to the hydrogen bonding interaction with the hydrogen atom on the main chain of Ala B: 250 (angle N–H⋯O = 127.1°, distance = 2.47 Å). The 2D diagram of the interactions between the colchicine binding site and colchicine or compound 6q were shown in Fig. 7C and D, respectively. Significant similarities could be found in their tubulin-binding models. For example, residues Ala B: 250, Met B: 259, Cys B: 241, Leu B: 255, Leu B: 248 and Lys B: 352 were predicted to make contacts with colchicine or compound 6q. Besides, Fig. 7B presented a superimposition of these two compounds. This molecular docking results along with the biological assay data suggested that compound 6q was a potential inhibitor of tubulin.

Fig. 7 The binding mode between the active conformation of compound 6q and the target protein tubulin (PDB code: 1SA0) provided by the CDOCKER protocol (Discovery Studio 3.5, Accelrys, Co. Ltd). (A) The receptor surface model with compound 6q; (B) 3D diagram of the interactions between colchicine (burlywood) & 6q (cyan) and the colchicine binding site. Only interacting residues are displayed. The H-bond (green arrow) is displayed as dotted arrows; (C) 2D diagram of the interactions between colchicine and the colchicine binding site; (D) 2D diagram of the interactions between compound 6q and the colchicine binding site. The H-bond (green arrow) is displayed as dotted arrows.

2.4.2. 3D-QSAR model. To obtain a systematic SAR profile on the synthesized compounds as antitumor agents and explore more potent and selective tubulin inhibitors, 3D-QSAR model was built by using the corresponding pIC₅₀ values which were converted from the obtained IC₅₀ (μM) values of tubulin inhibition (the way of this transformation was derived from an online calculator developed by an Indian Medicinal Chemistry Lab (http://www.sanjeevslab.org/tools-IC50.html)) and performed by built-in QSAR software of DS 3.5. The training and test set were divided by the random diverse molecules method, where the training set accounted for 80% of all the molecules while the test was set to 20%, with 18 agents and 4 agents respectively. The observed and predicted values, corresponding residual values for the training set and test set molecules in 3D-QSAR model, were presented in Table 4. The graphical relationship of observed and predicted values was showed in Fig. 8, in which the plot of the observed IC₅₀ versus the predicted values showed that this model could be used in prediction of activity for 1-methylindol- and pyrazoline-containing derivatives. Also the molecules aligned with the iso-surfaces of the 3D-QSAR model coefficients on electrostatic potential grids (Fig. 9A) and van der Waals grids (Fig. 9B) were listed. Electrostatic map indicated red contours around regions where high electron density (negative charge) was expected to increase activity, while blue contours represent areas where low electron density (partial positive charge) was expected to increase activity. This model is consistent with the docking results. For example, the red region near the B ring corresponds to the Lys B: 352 residue. Compounds 6b, 6i and 6q could fit the red subregion, and this is part of the reason why these small molecules show potent tubulin inhibitory activity. Similarly, steric map indicated areas where steric bulk was predicted to increase (green) or decrease (yellow) activity. It was widely acceptable that a better inhibitor based on the 3D-QSAR model should have strong van der Waals attraction in the green areas and a polar group in the blue electrostatic potential areas (which were dominant close to the skeleton). This promising model would provide a guideline to design and optimize more effective tubulin inhibitors and pave the way for us in the further study.

Table 4 The experimental and predicted inhibitory activity of compounds (6a–6v) by 3D-QSAR models based upon active conformation achieved by molecular docking

Compounds^a	Tubulin assembly		Residual error
	Actual pIC50	Predicted pIC50
a The italicised for the test set, and the rest for training.
6a	4.488	4.480	0.008
6b	5.364	5.380	−0.016
6c	4.635	4.640	−0.005
6d	4.941	4.930	0.011
6e	4.657	4.793	−0.136
6f	4.844	4.885	−0.041
6g	4.583	4.556	0.027
6h	4.456	4.797	−0.341
6i	5.240	5.219	0.021
6j	4.707	4.713	−0.006
6k	4.884	4.857	0.027
6l	4.704	4.719	−0.015
6m	4.983	4.989	−0.006
6n	4.687	4.718	−0.031
6o	4.772	4.682	0.090
6p	4.528	4.525	0.003
6q	5.703	5.688	0.015
6r	4.999	4.988	0.011
6s	5.107	5.098	0.009
6t	5.375	5.385	−0.010
6u	5.233	5.238	−0.005
6v	5.500	5.504	−0.004

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Fig. 8 Comparing the predicted pIC₅₀ value of tubulin inhibitory activities with that of the experiment by linear fitting curve.

Fig. 9 (A) Isosurface of the 3D-QSAR model coefficients on electrostatic potential grids. Blue represents positive coefficients; red represents negative coefficients. (B) Isosurface of the 3D-QSAR model coefficients on van der Waals grids. Green represents positive coefficients; yellow represents negative coefficients.

3. Conclusions

In this study, a series of novel compounds (6a–6v) containing 1-methylindol and 1-(4,5-dihydro-1H-pyrazol-1-yl)ethanone skeleton were designed, synthesized and evaluated as potential inhibitors of tubulin polymerization. Most of them showed potent anti-proliferative activity, and compound 6q displayed the most potent activity against tubulin assembling, A549, MCF-7 and HepG2 cell lines with IC₅₀ values of 1.98 μM, 0.15 μM, 0.17 μM, and 0.25 μM respectively, which was comparable to the positive compound colchicine. Furthermore, the mechanism of action studies proved that compound 6q could cause accumulation of cells in the G2/M phase of the cell cycle and potently induce apoptosis in A549 cells, as well as severe disruption of the microtubule system. Besides, molecular docking and 3D-QSAR were performed to learn the inhibitor–tubulin interactions. According to the analysis of the binding model of compound 6q with tubulin, several interactions were observed with the protein residues in the colchicine binding site, which may contribute to its anti-tubulin polymerization and anti-proliferative activities. The research of these well designed compounds might be greatly helpful for the development of tubulin polymerization inhibitors with stronger activities in the future.

4. Experiments

4.1. Chemistry general

All reagents and solvents were of analysis or synthesis grade. Melting points were determined on a XT4MP apparatus (Taike Corp, Beijing, China). ¹H NMR spectra were recorded on a Bruker DPX300 model spectrometer using tetramethylsilane (TMS) as internal standard and DMSO or CDCl₃ as solvents (chemical shifts in ppm (d)). TLC was performed on the glass backed silica gel sheets (Silica Gel 60 GF254) and visualized in UV light (254 nm and 365 nm). 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.

4.1.1. General procedure for the synthesis of 1H-indole-3-carbaldehyde (2a). To a stirred and completely mixed solution of POCl₃ (20 mL) in DMF (40 mL), 1a (35 mmol) in DMF (40 mL) was added at 0 °C. After a further 2 h reaction, the mixture was poured into 300 mL ice water and mixed completely. Then NaOH pellets were added and adjusted the pH to 9.0. The reaction mixture was filtered, and washed with ethyl acetate. Then the filtrate was extracted by ethyl acetate. The organic layer was dried over Na₂SO₄ and concentrated to give a brown solid, yield 98.1%. The crude 2a was used in the next step without any further purification. Following this way, we got crude compounds 2a–2c with yields of 95.3–98.1%.

4.1.2. General procedure for the synthesis of 1-methyl-1H-indole-3-carbaldehyde (3a). To a stirred solution of 2a (25 mmol) in THF (15 mL) at 0 °C was added NaH (62.5 mmol) in THF (15 mL) slowly and stirred for 15 min. Then CH₃I (33 mmol) was added and the reaction mixture was moved to room temperature to react for further 24 h. The reaction was stopped after the consumption of the starting material monitored by TLC. Then the solution was removed and the mixture was extracted by ethyl acetate. The organic layer was washed with brine, dried over Na₂SO₄ and concentrated to give crude residue, 3a, yield 97.5%. Following this way, we got crude compounds 3a–3c with yields of 93.4–97.5%.

4.1.3. General procedure for the synthesis of (E)-3-(1-methyl-1H-indol-3-yl)-1-phenylprop-2-en-1-one (4a). To a stirred solution of 3a (2 mmol) and acetophenone (2 mmol) in ethanol (20 mL), KOH (6 mmol, 336 mg) was added at room temperature. After the reaction was completed (24 h), the reaction mixture was filtered and washed with water and cool ethanol. The crude product was purified by recrystallization from ethanol and dichloromethane to give 4a, a yellow crystal, yield 93.1%. Following this way, we got pure compounds 4a–4v with yields of 38.5–95.7%.

4.1.4. General procedure for the synthesis of 1-methyl-3-(3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-1H-indole (5a). A mixture of 4a (1 mmol) and hydrazine hydrate (2 mmol) was dissolved in ethanol (5 mL) and refluxed for 2 h. The reaction mixture was cooled and kept at −20 °C overnight. Then the solid obtained was filtered off and washed with petroleum ether and taken for the next step immediately. Following this way, we got crude compounds 5a–5v.

4.1.5. General procedure for the synthesis of 1-(5-(1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethanone (6a). To a stirred solution of acetic acid (2 mmol), EDC·HCl (3 mmol) and HOBt (1.2 mmol) in acetonitrile or dichloromethane (5 mL), 5a (1 mmol) was added at room temperature with the protection of N₂. After 24 h, the reaction mixture was filtered and washed with cool ethanol and water. The crude product was purified by recrystallization from ethanol and dichloromethane to give 6a, a white powder, yield 30.2%. Following this way, we got pure compounds 6a–6v.

White powder. Yield: 30.2%. Mp 171–172 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.93–7.76 (m, 2H), 7.57–7.43 (m, 3H), 7.40 (d, J = 8.2 Hz, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.25 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 6.97 (t, J = 7.3 Hz, 1H), 5.81 (dd, J = 11.8, 4.4 Hz, 1H), 3.84 (dd, J = 18.0, 11.9 Hz, 1H), 3.72 (s, 3H), 3.25 (dd, J = 18.0, 4.4 Hz, 1H), 2.26 (s, 3H). MS (ESI) m/z: 318.15 (C₂₀H₁₉N₃O, [M + H]⁺). Anal. calcd for C₂₀H₁₉N₃O: C, 75.69; H, 6.03; N, 13.24. Found: C, 75.81; H, 6.04; N, 13.22.

4.1.6. 1-(3-(4-Methoxyphenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6b). Brown crystal. Yield: 34.5%. Mp 156–158 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.77 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 8.2 Hz, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.23 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 7.03 (d, J = 8.8 Hz, 2H), 6.96 (t, J = 7.4 Hz, 1H), 5.77 (dd, J = 11.8, 4.2 Hz, 1H), 3.87–3.75 (m, 4H), 3.72 (s, 3H), 3.22 (dd, J = 17.9, 4.3 Hz, 1H), 2.23 (s, 3H). MS (ESI) m/z: 348.16 (C₂₁H₂₁N₃O₂, [M + H]⁺). Anal. calcd for C₂₁H₂₁N₃O₂: C, 72.60; H, 6.09; N, 12.10. Found: C, 72.54; H, 6.09; N, 12.11.

4.1.7. 1-(3-(3,4-Dimethoxyphenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6c). Brown crystal. Yield: 14.3%. Mp 169–171 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.39 (d, J = 8.4 Hz, 2H), 7.36–7.29 (m, 2H), 7.23 (s, 1H), 7.13 (t, J = 7.6 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 5.78 (dd, J = 11.7, 4.2 Hz, 1H), 3.88–3.75 (m, 7H), 3.72 (s, 3H), 3.25 (dd, J = 17.8, 4.3 Hz, 1H), 2.25 (s, 3H). MS (ESI) m/z: 378.17 (C₂₂H₂₃N₃O₃, [M + H]⁺). Anal. calcd for C₂₂H₂₃N₃O₃: C, 70.01; H, 6.14; N, 11.13. Found: C, 69.90; H, 6.15; N, 11.15.

4.1.8. 1-(5-(1-Methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6d). White powder. Yield: 43.1%. Mp 224–226 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.38 (t, J = 8.7 Hz, 2H), 7.22 (s, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.09 (s, 2H), 6.99 (t, J = 7.4 Hz, 1H), 5.81 (dd, J = 11.7, 4.2 Hz, 1H), 3.87–3.76 (m, 7H), 3.72 (d, J = 5.5 Hz, 6H), 3.32 (dd, J = 17.9, 4.4 Hz, 1H), 2.27 (s, 3H). MS (ESI) m/z: 408.18 (C₂₃H₂₅N₃O₄, [M + H]⁺). Anal. calcd for C₂₃H₂₅N₃O₄: C, 67.80; H, 6.18; N, 10.31. Found: C, 67.95; H, 6.17; N, 10.33.

4.1.9. 1-(3-(4-Chlorophenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6e). White powder. Yield: 11.1%. Mp 182–183 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.84 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.40 (d, J = 8.2 Hz, 1H), 7.32 (d, J = 7.9 Hz, 1H), 7.25 (s, 1H), 7.13 (t, J = 7.6 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 5.81 (dd, J = 11.8, 4.4 Hz, 1H), 3.83 (dd, J = 18.0, 11.9 Hz, 1H), 3.72 (s, 3H), 3.25 (dd, J = 18.0, 4.5 Hz, 1H), 2.25 (s, 3H). MS (ESI) m/z: 352.11 (C₂₀H₁₈ClN₃O, [M + H]⁺). Anal. calcd for C₂₀H₁₈ClN₃O: C, 68.28; H, 5.16; N, 11.94. Found: C, 68.21; H, 5.15; N, 11.96.

4.1.10. 1-(3-(4-Bromophenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6f). White powder. Yield: 17.7%. Mp 183–185 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.77 (d, J = 8.6 Hz, 2H), 7.68 (d, J = 8.6 Hz, 2H), 7.39 (d, J = 8.2 Hz, 1H), 7.32 (d, J = 7.9 Hz, 1H), 7.25 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 5.81 (dd, J = 11.9, 4.4 Hz, 1H), 3.83 (dd, J = 18.0, 11.9 Hz, 1H), 3.72 (s, 3H), 3.25 (dd, J = 18.0, 4.5 Hz, 1H), 2.25 (s, 3H). MS (ESI) m/z: 396.06 (C₂₀H₁₈BrN₃O, [M + H]⁺). Anal. calcd for C₂₀H₁₈BrN₃O: C, 60.62; H, 4.58; N, 10.60. Found: C, 60.67; H, 4.59; N, 10.58.

4.1.11. 1-(5-(1-Methyl-1H-indol-3-yl)-3-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6g). White crystal. Yield: 61.6%. Mp 218–220 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.04 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.2 Hz, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.28 (s, 1H), 7.14 (t, J = 7.5 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 5.85 (dd, J = 11.9, 4.5 Hz, 1H), 3.89 (dd, J = 18.1, 12.0 Hz, 1H), 3.73 (s, 3H), 3.33–3.22 (m, 1H), 2.28 (s, 3H). MS (ESI) m/z: 386.14 (C₂₁H₁₈F₃N₃O, [M + H]⁺). Anal. calcd for C₂₁H₁₈F₃N₃O: C, 65.45; H, 4.71; N, 10.90. Found: C, 65.31; H, 4.70; N, 10.87.

4.1.12. 1-(5-(5-Methoxy-1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6h). White crystal. Yield: 45.0%. Mp 175–176 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.93–7.79 (m, 2H), 7.56–7.42 (m, 3H), 7.29 (d, J = 8.8 Hz, 1H), 7.20 (s, 1H), 6.80 (s, 1H), 6.77 (d, J = 8.8 Hz, 1H), 5.78 (dd, J = 11.7, 3.8 Hz, 1H), 3.82 (dd, J = 17.9, 11.7 Hz, 1H), 3.68 (s, 3H), 3.54 (s, 3H), 3.25 (dd, J = 17.9, 3.9 Hz, 1H), 2.26 (s, 3H). MS (ESI) m/z: 348.16 (C₂₁H₂₁N₃O₂, [M + H]⁺). Anal. calcd for C₂₁H₂₁N₃O₂: C, 72.60; H, 6.09; N, 12.10. Found: C, 72.71; H, 6.10; N, 12.12.

4.1.13. 1-(5-(5-Methoxy-1-methyl-1H-indol-3-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6i). Light yellow powder. Yield: 46.7%. Mp 158–160 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.78 (d, J = 8.8 Hz, 2H), 7.28 (d, J = 8.8 Hz, 1H), 7.18 (s, 1H), 7.03 (d, J = 8.9 Hz, 2H), 6.81 (s, 1H), 6.77 (d, J = 8.8 Hz, 1H), 5.75 (dd, J = 11.6, 3.8 Hz, 1H), 3.84–3.69 (m, 4H), 3.68 (s, 3H), 3.56 (s, 3H), 3.21 (dd, J = 17.8, 3.9 Hz, 1H), 2.23 (s, 3H). MS (ESI) m/z: 378.17 (C₂₂H₂₃N₃O₃, [M + H]⁺). Anal. calcd for C₂₂H₂₃N₃O₃: C, 70.01; H, 6.14; N, 11.13. Found: C, 69.91; H, 6.16; N, 11.13.

4.1.14. 1-(3-(3,4-Dimethoxyphenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6j). Grey solid. Yield: 10.2%. Mp 186–187 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.42 (s, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 7.17 (s, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.83 (s, 1H), 6.78 (d, J = 8.8 Hz, 1H), 5.76 (dd, J = 11.5, 3.4 Hz, 1H), 3.86–3.72 (m, 7H), 3.68 (s, 3H), 3.58 (s, 3H), 3.24 (dd, J = 17.7, 3.6 Hz, 1H), 2.25 (s, 3H). MS (ESI) m/z: 408.18 (C₂₃H₂₅N₃O₄, [M + H]⁺). Anal. calcd for C₂₃H₂₅N₃O₄: C, 67.80; H, 6.18; N, 10.31. Found: C, 67.93; H, 6.17; N, 10.32.

4.1.15. 1-(3-(4-Fluorophenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6k). Gray powder. Yield: 29.8%. Mp 178–180 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.90 (dd, J = 8.7, 5.5 Hz, 2H), 7.31 (dd, J = 18.0, 8.9 Hz, 3H), 7.19 (s, 1H), 6.85–6.73 (m, 2H), 5.78 (dd, J = 11.7, 3.8 Hz, 1H), 3.82 (dd, J = 17.9, 11.8 Hz, 1H), 3.68 (s, 3H), 3.57 (s, 3H), 3.25 (dd, J = 17.9, 3.9 Hz, 1H), 2.25 (s, 3H). MS (ESI) m/z: 366.15 (C₂₁H₂₀FN₃O₂, [M + H]⁺). Anal. calcd for C₂₁H₂₀FN₃O₂: C, 69.03; H, 5.52; N, 11.50. Found: C, 69.31; H, 5.51; N, 11.48.

4.1.16. 1-(3-(4-Chlorophenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6l). White crystal. Yield: 36.7%. Mp 169–170 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.86 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.29 (d, J = 8.6 Hz, 1H), 7.20 (s, 1H), 6.78 (d, J = 8.7 Hz, 2H), 5.79 (dd, J = 11.7, 3.9 Hz, 1H), 3.81 (dd, J = 18.0, 11.8 Hz, 1H), 3.68 (s, 3H), 3.57 (s, 3H), 3.24 (dd, J = 17.9, 4.0 Hz, 1H), 2.25 (s, 3H). MS (ESI) m/z: 382.12 (C₂₁H₂₀ClN₃O₂, [M + H]⁺). Anal. calcd for C₂₁H₂₀ClN₃O₂: C, 66.05; H, 5.28; N, 11.00. Found: C, 65.98; H, 5.27; N, 10.98.

4.1.17. 1-(3-(4-Bromophenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6m). White crystal. Yield: 38.6%. Mp 172–173 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.78 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.5 Hz, 1H), 7.19 (s, 1H), 6.78 (d, J = 8.6 Hz, 2H), 5.79 (dd, J = 11.7, 3.9 Hz, 1H), 3.81 (dd, J = 18.0, 11.9 Hz, 1H), 3.68 (s, 3H), 3.58 (s, 3H), 3.24 (dd, J = 18.0, 4.0 Hz, 1H), 2.25 (s, 3H). MS (ESI) m/z: 426.07 (C₂₁H₂₀BrN₃O₂, [M + H]⁺). Anal. calcd for C₂₁H₂₀BrN₃O₂: C, 59.17; H, 4.73; N, 9.86. Found: C, 59.33; H, 4.71; N, 9.85.

4.1.18. 1-(5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6n). White powder. Yield: 59.0%. Mp 173–174 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.94–7.74 (m, 2H), 7.61 (s, 1H), 7.57–7.41 (m, 3H), 7.39 (d, J = 8.7 Hz, 1H), 7.29 (s, 1H), 7.25 (d, J = 8.7 Hz, 1H), 5.81 (dd, J = 11.7, 4.2 Hz, 1H), 3.84 (dd, J = 18.0, 11.8 Hz, 1H), 3.72 (s, 3H), 3.25 (dd, J = 18.0, 4.3 Hz, 1H), 2.27 (s, 3H). MS (ESI) m/z: 396.06 (C₂₀H₁₈BrN₃O, [M + H]⁺). Anal. calcd for C₂₀H₁₈BrN₃O: C, 60.62; H, 4.58; N, 10.60. Found: C, 60.79; H, 4.57; N, 10.63.

4.1.19. 1-(5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(2-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6o). Light yellow crystal. Yield: 58.5%. Mp 180–181 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.85 (d, J = 6.1 Hz, 1H), 7.63 (s, 1H), 7.46 (t, J = 7.0 Hz, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.30 (s, 1H), 7.25 (d, J = 6.9 Hz, 1H), 7.14 (d, J = 8.3 Hz, 1H), 7.06 (t, J = 7.9 Hz, 1H), 5.74 (dd, J = 11.8, 4.2 Hz, 1H), 3.88 (dd, J = 18.5, 11.8 Hz, 1H), 3.81 (s, 3H), 3.73 (s, 3H), 3.28 (dd, J = 18.5, 4.2 Hz, 1H), 2.23 (s, 3H). MS (ESI) m/z: 426.07 (C₂₁H₂₀BrN₃O₂, [M + H]⁺). Anal. calcd for C₂₁H₂₀BrN₃O₂: C, 59.17; H, 4.73; N, 9.86. Found: C, 59.31; H, 4.74; N, 9.84.

4.1.20. 1-(5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6p). White crystal. Yield: 59.9%. Mp 184–185 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.60 (s, 1H), 7.44–7.36 (m, 3H), 7.34 (s, 1H), 7.29 (s, 1H), 7.26 (d, J = 6.9 Hz, 1H), 7.11–7.02 (m, 1H), 5.80 (dd, J = 11.7, 4.3 Hz, 1H), 3.89–3.78 (m, 4H), 3.72 (s, 3H), 3.26 (dd, J = 18.0, 4.3 Hz, 1H), 2.27 (s, 3H). MS (ESI) m/z: 426.07 (C₂₁H₂₀BrN₃O₂, [M + H]⁺). Anal. calcd for C₂₁H₂₀BrN₃O₂: C, 59.17; H, 4.73; N, 9.86. Found: C, 59.24; H, 4.72; N, 9.87.

4.1.21. 1-(5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6q). White crystal. Yield: 75.8%. Mp 208–209 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.77 (d, J = 8.8 Hz, 2H), 7.59 (s, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.28 (s, 1H), 7.25 (d, J = 7.0 Hz, 1H), 7.03 (d, J = 8.8 Hz, 2H), 5.77 (dd, J = 11.6, 4.1 Hz, 1H), 3.91–3.75 (m, 4H), 3.72 (s, 3H), 3.21 (dd, J = 17.9, 4.2 Hz, 1H), 2.25 (s, 3H). MS (ESI) m/z: 426.07 (C₂₁H₂₀BrN₃O₂, [M + H]⁺). Anal. calcd for C₂₁H₂₀BrN₃O₂: C, 59.17; H, 4.73; N, 9.86. Found: C, 59.19; H, 4.72; N, 9.85.

4.1.22. 1-(5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6r). White powder. Yield: 64.8%. Mp 229–231 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.61 (s, 1H), 7.43–7.36 (m, 2H), 7.33 (d, J = 8.3 Hz, 1H), 7.26 (d, J = 9.1 Hz, 2H), 7.03 (d, J = 8.4 Hz, 1H), 5.78 (dd, J = 11.6, 4.0 Hz, 1H), 3.85–3.75 (m, 7H), 3.72 (s, 3H), 3.24 (dd, J = 17.9, 4.1 Hz, 1H), 2.27 (s, 3H). MS (ESI) m/z: 456.08 (C₂₂H₂₂BrN₃O₃, [M + H]⁺). Anal. calcd for C₂₂H₂₂BrN₃O₃: C, 57.90; H, 4.86; N, 9.21. Found: C, 57.98; H, 4.87; N, 9.22.

4.1.23. 1-(5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6s). Pink powder. Yield: 40.6%. Mp 228–229 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.65 (s, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.26 (t, J = 5.1 Hz, 2H), 7.09 (s, 2H), 5.82 (dd, J = 11.6, 4.1 Hz, 1H), 3.87–3.76 (m, 7H), 3.72 (s, 6H), 3.30–3.28 (m, 1H), 2.29 (s, 3H). MS (ESI) m/z: 486.10 (C₂₃H₂₄BrN₃O₄, [M + H]⁺). Anal. calcd for C₂₃H₂₄BrN₃O₄: C, 56.80; H, 4.97; N, 8.64. Found: C, 56.81; H, 4.97; N, 8.62.

4.1.24. 1-(5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6t). White solid. Yield: 38.6%. Mp 199–200 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.89 (dd, J = 8.7, 5.5 Hz, 2H), 7.60 (s, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.33 (t, J = 8.9 Hz, 2H), 7.30 (s, 1H), 7.25 (d, J = 6.9 Hz, 1H), 5.81 (dd, J = 11.7, 4.2 Hz, 1H), 3.83 (dd, J = 18.0, 11.7 Hz, 1H), 3.72 (s, 3H), 3.26 (dd, J = 18.0, 4.3 Hz, 1H), 2.26 (s, 3H). MS (ESI) m/z: 414.05 (C₂₀H₁₇BrFN₃O, [M + H]⁺). Anal. calcd for C₂₀H₁₇BrFN₃O: C, 57.99; H, 4.14; N, 10.14. Found: C, 57.88; H, 4.14; N, 10.16.

4.1.25. 1-(5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6u). White powder. Yield: 48.5%. Mp 200–201 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.84 (d, J = 8.5 Hz, 2H), 7.60 (s, 1H), 7.54 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.7 Hz, 1H), 7.29 (s, 1H), 7.25 (d, J = 8.7 Hz, 1H), 5.81 (dd, J = 11.7, 4.1 Hz, 1H), 3.82 (dd, J = 18.0, 11.8 Hz, 1H), 3.71 (s, 3H), 3.25 (dd, J = 18.0, 4.3 Hz, 1H), 2.27 (s, 3H). MS (ESI) m/z: 430.02 (C₂₀H₁₇BrClN₃O, [M + H]⁺). Anal. calcd for C₂₀H₁₇BrClN₃O: C, 55.77; H, 3.98; N, 9.76. Found: C, 55.91; H, 3.97; N, 9.78.

4.1.26. 1-(5-(5-Bromo-1-methyl-1H-indol-3-yl)-3-(4-bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6v). White powder. Yield: 37.0%. Mp 206–207 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.77 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 8.6 Hz, 2H), 7.59 (s, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.30 (s, 1H), 7.25 (d, J = 7.0 Hz, 1H), 5.81 (dd, J = 11.7, 4.3 Hz, 1H), 3.82 (dd, J = 18.0, 11.8 Hz, 1H), 3.72 (s, 3H), 3.25 (dd, J = 18.0, 4.4 Hz, 1H), 2.27 (s, 3H). MS (ESI) m/z: 473.97 (C₂₀H₁₇Br₂N₃O, [M + H]⁺). Anal. calcd for C₂₀H₁₇Br₂N₃O: C, 50.55; H, 3.61; N, 8.84. Found: C, 50.42; H, 3.61; N, 8.86.

4.2. Crystal structure determination

Crystal structure determination of compound 6o was carried out on a Nonius CAD4 diffractometer equipped with graphite-mono chromated Mo-Kα (k = 0.71073 Å) radiation. The structure was solved by direct methods and refined on F² by full-matrix least-squares methods using SHELX-97.²⁴ All non-hydrogen atoms of compound 6o were refined anisotropically. All hydrogen atoms were placed in geometrically idealized positions and constrained to ride on their respective parent atoms.

4.3. Biological assays

4.3.1. Anti-proliferation activity. Human nonsmall cell lung carcinoma (A549), human breast adenocarcinoma (MCF-7), human liver hepatocellular carcinoma (HepG2) and human renal epithelial cells (293T) were cultured in DMEM supplemented with 10% fetal bovine serum, 115 units per mL of penicillin G, and 115 μg mL⁻¹ of streptomycin, in 5% CO₂ cell incubator at 37 °C. Then 100 μL cell suspensions (1000 to 5000 cells) were added into each well of 96-well plates depending on growth rate and allowed to adhere in the incubator. After that, tested samples at pre-set concentrations were added to 96 wells with colchicine as positive reference. After 48 h incubation, 10 μL of PBS containing 5 mg mL⁻¹ of MTT was added to each well. The plates were incubated for further 4 h and then were centrifuged at 1500 rpm for 10 min to remove supernatant. After 150 mL of DMSO was added to each well, the plates were shaken vigorously for 10 min to ensure complete solubilization. The absorbance was measured and recorded on an ELISA reader (ELx800, BioTek, USA) at a test wavelength of 490 nm. In all the experiments three replicate wells were used for each drug concentration. Each assay was carried out at least three times.

4.3.2. Tubulin polymerization assay. Tubulin was purified from bovine brain as described previously.^25,26 To characterize the effect of the compounds on tubulin assembly in vitro, different concentrations of compounds were incubated with 10 μM bovine brain tubulin in glutamate buffer at 30 °C and then cooled to 0 °C. After 0.4 mM GTP added, the mixtures were transferred to cuvettes at 0 °C in a recording spectrophotometer and warmed up to 30 °C. Finally, the assembly of tubulin was observed turbidimetrically at 350 nm. The IC₅₀ was defined as the compound concentration inhibited the extent of assembly by 50% after 20 min incubation.

4.3.3. Cell cycle analysis. A549 cells were plated in a 6-well plate (5.0 × 10⁵ cells per well) and incubated at 37 °C for 24 h. Then the cells were treated with 6q at 0, 0.02, 0.05, 0.1, 0.15 and 0.2 μM for 24 h. Treated cells were washed with PBS and fixed with 70% ice-cold ethanol overnight. Then, fixed cells were resuspended in PBS containing 0.1 mg mL⁻¹ RNase A and 5 μg mL⁻¹ propidium iodide (PI) and stained at 37 °C for 30 min. Cell cycle distribution was analyzed by BD Accuri C6 Flow Cytometer (BD, USA). The percentage of cells in the G1, S and G2/M phases of the cell cycle were determined using the Flowjo 7.6.1 software after cell debris exclusion.

4.3.4. Annexin-V assay. A549 cells were plated into a 12-well plate (2.0 × 10⁵ cells per well) and were allowed to adhere for 12 h. After that, the cells were treated with 6q at final concentrations of 0, 0.05, 0.1 and 0.15 μM for 24 h. Then cells were collected and washed twice with precooled PBS and strained with 10 μL of annexin V-FITC and 5 μL of PI (5 μg mL⁻¹) in 1× binding buffer for 30 min in the dark. Apoptotic cells were monitored on BD Accuri C6 Flow Cytometer (BD, USA). Statistics were analyzed by Flowjo 7.6.1 software.

4.3.5. Confocal microscopy assay. A549 cells were seeded on glass coverslips and incubated with 0.5 μM paclitaxel, 0.5 μM colchicine and 0.2 μM 6q, for 24 h respectively. Cells were then rinsed with PBS for three times, fixed with 4% paraformaldehyde for 20 min and permeabilized with cold formaldehyde for 10 min. After that, fixed cells were washed with PBS and blocked with 5% albumin from bovine serum (BSA) for at least 1 h. After washed with PBS once, the cells were incubated with anti-tubulin antibody (1 : 500) in 3% BSA overnight at 4 °C, followed by Cy3-labeled goat anti-mouse IgG (H + L) (1 : 500) in 3% BSA and incubated for 1 h. Subsequently, the cells were incubated with DAPI (5 ng mL⁻¹). Images were captured with Olympus confocal microscope and data was analyzed using FV-10-ASW.

4.4. Docking simulations

Molecular docking of compound 6q into the three dimensional X-ray structure of tubulin (PDB code: 1SA0) was carried out using the DS 3.5 (Discovery Studio 3.5) as implemented through the graphical user interface DS-CDOCKER protocol. 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 replaced by 6q during the molecular docking procedure. Types of interactions of the docked protein with ligand were analyzed after the end of molecular docking.

4.5. 3D-QSAR

Ligand-based 3D-QSAR approach was performed by QSAR software of DS 3.5. The training sets consisted of inhibitors with the corresponding pIC₅₀ values which were converted from the obtained IC₅₀ (μM), and test sets comprised compounds of data sets. All the definition of the descriptors could be seen in the “Help” of DS 3.5 software and they were calculated by QSAR protocol of DS 3.5. The alignment conformation of each molecule was the one with lowest interaction energy in the docked results of CDOCKER. The predictive ability of 3D-QSAR modeling can be evaluated based on the cross-validated correlation coefficient, which qualifies the predictive ability of the models. Scrambled test (Y scrambling) was performed to investigate the risk of chance correlations. The inhibitory potencies of compounds were randomly reordered for 30 times and subject to leave-one-out validation test respectively. The models were also validated by test sets, in which the compounds were not included in the training sets.

Acknowledgements

The work was financed by the Projects (No. CXY1409 & CG1305) from the Science & Technology Bureau of Lianyungang City of Jiangsu Province.

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Footnotes

† CCDC 1444877. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra28141e

‡ Both authors contributed equally to this work.

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