Design, synthesis, and biological evaluation of diarylpyrazole derivatives as antitumor agents targeting microtubules

2.2.1 In vitro anti-proliferative activity

All of the target compounds, 9a-9t, along with the positive control, CA-4 (2), were evaluated for their anti-proliferative activity against two human cancer cell lines: human cervical cancer cells HeLa, gastric adenocarcinoma cells SGC-7901. Some target compounds displayed anti-proliferative activity with IC₅₀ values in the micromolar range. This indicates that the pyrazole moiety is suitable for mimicking the cis-olefin moiety in CA-4, as analogues with the pyrazole moiety maintain potent anti-proliferative activities against multiple cancer cell lines.

As illustrated in Table 1, some target compounds showed moderate anti-proliferative activities with potencies in the micromolar range. In general, the strongest anti-proliferative activity against the two cell lines with IC₅₀ value of 1.9–3.2 μM was observed when the B ring was substituted with indole ring (9s), while a sharp decrease in anti-proliferative activity was observed when the B ring was substituted with other aryl/heteroaryl groups, such as thiophene (9p), pyridine (9r) and naphthalene (9t). Comparison of the IC₅₀ values of the corresponding compounds 9a-9o with benzene or substituted benzene revealed that compounds 9a, 9h, 9i, and 9l significantly inhibited the growth of the HeLa and SGC-7901 cell lines at 10 μM. The target compounds contain halide and nitro substituents that have been widely used by medicinal chemists in microtubule destabilizers research, so we synthesize these compounds to evaluate the potential anti-proliferative activities. Unexpectedly, we found that these compounds (9m, 9n, and 9o) showed weak anti-proliferative activities.

2.2.2 Effect on tubulin polymerization

To explore the anti-proliferative activity of these compounds in relation to microtubulin, the most active compound 9s, was evaluated for its effect on microtubulin polymerization and compared with the positive control CA-4 and the negative control paclitaxel. As shown in Fig. 3, both CA-4 and compound 9s inhibited microtubulin polymerization. In contrast, paclitaxel increased the rate of microtubulin polymerization. These results indicate that compound 9s has an interfering effect on microtubulin polymerization.

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Fig. 3

Effects of compound 9s on tubulin polymerization. Tubulin had been pre-incubated for 1 min with 9s at 15 µM, 5 µM of CA-4, Paclitaxel at 5 µM or vehicle DMSO at room temperature before GTP was added to start the tubulin polymerization reactions. The reaction was monitored at 37 °C.

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2.2.3 Analysis of immunofluorescence staining

To clarify the mechanism of action of compound 9s, immunofluorescence staining was performed to further evaluate the effect of compound 9s on microtubule protein assembly. HeLa cells were treated with CA-4 and compound 9s at their respective 2-fold IC₅₀ concentrations for 24 h. As shown in Fig. 4, the cells treated with compound 9s showed changes in the shape of the nucleus, and the microtubule network became disorganized and shrunken or even appeared multinucleated compared with the control group. These results demonstrate that the mechanism of action of compound 9s is similar to that of CA-4 in that both inhibit microtubule assembly and disrupt the cytoskeleton.

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Fig. 4

Effects of compound 9s and CA-4, at their respective 2-fold IC₅₀ concentrations, on the cellular microtubule network and microtubule reassemble by immunofluorescence. HeLa cells were treated with compound 9s or CA-4 for 24 h, and then direct microscopy detection of the fixed and stained cell was performed. Microtubules and unassembled microtubule proteins were stained with anti-a-tubulin-FITC specific antibodies (green). Nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI, blue).

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2.2.4 Cell cycle analysis

As we all know, tubulin inhibitors arrest cell cycle distribution in G2/M phase (Wu et al., 2017). To explore the possible antitumor mechanisms of diarylpyrazole derivatives, the most promising compound 9s, was selected and its effect on HeLa cell cycle progression was analyzed by flow cytometry. In the present work, HeLa cells were treated with 9s at concentrations of 2-fold IC₅₀, 5-fold IC₅₀, and 10-fold IC₅₀ and control for 24 h, respectively. As shown in Fig. 5, the percentage of cells in the G2/M phase of the cell cycle increased from 18.17% to 50.63% under the treatment with the indicated concentration of 9s, compared to 5.48% in the control. The results show that the compound 9s caused a significant G2/M phase block in a concentration-dependent manner, which is typical for inhibitors of microtubule protein polymerization.

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Fig. 5

Effects of compound 9s on cell cycle. HeLa cells lines treated with compound 9s for 24 h. (A) Control; (B) 9s, 2-fold IC₅₀; (C) 9s, 5-fold IC₅₀; (D) 9s, 10-fold IC₅₀.

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2.2.5 Cell apoptosis assay

The apoptosis-inducing potential of 9s, the most cytotoxic compound, on HeLa cells was evaluated using Annexin V-FITC/PI. Examination of flow cytometry data allowed to distinguish between apoptotic (early and late) and necrotic cell populations. Compound 9s induced apoptosis in HeLa cells already at its IC₅₀ concentration, as shown in Fig. 6. The total number of early (Q3) and late (Q2) cells was 15.13%, 22.73% and 32.7% after 48 h of compound 9s action at 1-fold IC₅₀, 2-fold IC_50, and 3-fold IC₅₀ concentrations, respectively, compared to 7.1% in the control group. Thus, the results illustrate that compound 9s has anti-proliferative activity by dose-dependently inducing apoptosis.

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Fig. 6

Effect of compound 9s on cell apoptosis in HeLa cells. Flow cytometric analysis of apoptotic cells after treatment of HeLa cells with 9s or control for 48 h. (A) Control; (B) 9s, 1-fold IC₅₀; (C) 9s, 2-fold IC₅₀; (D) 9s, 3-fold IC₅₀. Different cell stages were given as live (Q4), early apoptotic (Q3), late apoptotic (Q2), and necrotic cells (Q1).

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4.1.1 Materials and methods

All reagents and solvents we use are purchased from commercial sources. We monitored the reaction using silica gel thin layer color (TLC) at UV wavelengths (365 nm and 254 nm). At room temperature, ¹H (500 MHz) and ¹³C NMR (125 MHz) were measured on Agilent ProPulse 500 MHz (Agilent Inc. USA) using CDCL₃ as the solvent. Agilent Accurate-Mass Q-TOF 6530 instrument (Agilent Inc. USA) in ESI mode was used to record high resolution mass spectrometry (HRMS). The microwave were carried out in a single mode cavity microwave synthesiser (CEM MATTHEWS, NC, USA). (Wang et al., 2021).

4.1.2 General synthetic procedures for 4-bromo-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole (11)

To a round-bottom flask equipped with a magnetic stir bar, 4-bromo-1H-pyrazole (10) (0.20 mmol), 3,4,5-trimethoxybenzeneboronic acid (0.20 mmol), Cu(OAc)₂ (0.40 mmol), pyridine (0.40 mmol), and dried DCM (10.0 mL) were added. The flask was evacuated and back-filled with nitrogen. The mixture was stirred at room temperature for 4 h. After completion of the reaction, the reaction mixture was extracted with DCM (25 mL × 3). The organic layer was washed with brine and dried over Na₂SO₄. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to give 11.

4.1.3 General synthetic procedures for diarylpyrazole derivatives (9)

A mixture of 11 (0.10 mmol), Pd(PPh₃)₄ (0.01 mmol), and K₂CO₃ (0.12 mmol), and substituted phenylboronic acid (0.11 mmol) in 1,4-dioxane/H₂O (5 mL, 3:1) was degassed and purged with N₂ for about three times. The reaction mixture was stirred at irradiated in a microwave reactor for 25 min at 126 °C (indicated by TLC) under N₂ atmosphere. The reaction mixture was poured into H₂O (50 mL) and extracted with ethyl acetate (EtOAc) (80 mL × 3). The combined organics were washed with brine (10 mL × 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure to give a residue, then the residue was purified by column chromatography (petroleum ether/EtOAc = 3:1) on silica gel to afford pure product 9.

4.2.1 Cell culture

Human gastric adenocarcinoma SGC-7901 cells and human cervical cancer carcinoma HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U/mL streptomycins and 100 U/mL penicillin at 37 °C in a humid environment containing 5% CO₂. All cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) (Wang et al., 2021).

4.2.2 In vitro anti-proliferative activity

The in vitro anti-proliferative activity of CA-4 and all target compounds were determined by MTT assay (meilunbio®, China). Briefly, cells were grown into 96-well plates at a density of 5000 cells/well, depending on the growth rate of the cell line. 24 h later, cells in 96-well plates were subjected to control treatment and compound testing. After 72 h incubation at 37 °C in 5% CO₂, 20 μL containing MTT solution (5 mg/mL) was added to the 96-well plates. After 4 h of incubation, the culture solution containing MTT was removed and 100 mL of dimethyl sulfoxide (DMSO) was added to each well. The plates were gently stirred and incubated for 30 min at 37 °C in an incubator protected from light until the purple formazan crystals were dissolved and the OD₄₉₀ values were measured using a microplate reader. Data were calculated and plotted as percent survival compared to the control (using GraphPad Prism version 8.3.0). In the MTT assay, the half-inhibitory concentration (IC₅₀) was defined as the concentration of the drug that brought the absorbance to 50% of the untreated wells (Wang et al., 2016; Wang et al., 2019).

4.2.3 Effect on tubulin polymerization

The tubulin polymerization inhibition kit (Cytoskeleton-Cat.#BK011P) was used to detect the influencing factors of compound 9s on tubulin polymerization. We re-suspended tubulin in a cold G-PEM buffer (80 mM PIPES, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM GTP, 20% (v/v) glycerol) and added it to the well of a 96-well plate containing the specified concentration of 9s or blank. The controls group were the cells treated with vehicle DMSO, the positive controls were the cells treated with CA-4, and the negative controls were the cells treated with paclitaxel. We used microplate reader (Tecan, Austria) to monitor tubulin polymerization for 61 min at 37 °C at 1 min intervals (emission wavelength: 450 nm, excitation wavelength: 360 nm) and calculated using absorbance values. Data processing was performed using GraphPad Prism 8.3.0 version (Wang et al., 2019).

4.2.4 Analysis of immunofluorescence staining

HeLa cells were inoculated in 6-well plate at a cell density of 1 × 10⁴ ∼ 4 × 10⁴ and grown for 24 h. The cells were treated for 24 h with compound 9s and CA-4 at their respective 2-fold IC₅₀ concentrations. The blank controls were untreated cells and the positive controls were cells treated with CA-4. Then each well was washed with PBS, which were fixed with cooled 4% formaldehyde solution for 20 min. Formaldehyde solution was discarded and 0.5% Triton-X/min was added to each well. 0.5% Triton-X was discarded and the wells were washed three times with PBST for 5 min. Added 2 mL of glycine to each well and blocked with 3% bovine serum albumin (BSA) at room temperature for 1 h. Antibody to α-microtubulin (Aclonal, China) was diluted with 3% BSA (1:100) and incubated for 3 h. The cells were washed three times with PBST solution for 10 min each to remove unbound primary antibodies. Then the FITC-coupled anti-mouse secondary antibody (1:100) and 4′,6-diamidino-2-phenylindole DAPI (1:100) were then diluted with 3% BSA and incubated for 1 h. Washed three times again with PBST for 5 min. Finally, the slices were sealed with Prolongold and photographed under a fluorescence confocal microscopy (Nikon, Japan) for a period of time (Wang et al., 2019; Liu et al., 2021).

4.2.5 Cell cycle analysis

3 × 10⁵ Human cervical cancer cell line (HeLa) was treated with compound 9s at concentrations of 2-fold IC₅₀, 5-fold IC₅₀ and 10-fold IC₅₀ and the control for 24 h. Then cells were collected by centrifugation, washed with PBS, and fixed in 70% ethanol on ice. The cells were treated with Ribonuclease A (RNase) (50 mg/mL) for 30 min at 37 °C and incubated with pyridinium iodide (PI) solution (Solarbio, China) for 15 min at 4 °C. Then flow cytometry (Beckman Coulter, USA) assay was performed. We repeated these experiments at least three times (Alvarez et al., 2013).

4.2.6 Cell apoptosis assay

2 × 10⁵ Human cervical cancer cell line (HeLa) was treated with compound 9s at concentrations of 1-fold IC₅₀, 2-fold IC₅₀ and 3-fold IC₅₀ for 48 h. Cells were collected using EDTA-free trypsin, centrifuged at 300r for 5 min, and the supernatant was discarded and centrifuged at 300r for 5 min at 4 °C using PBS. 100 μL of 1 × Binding Buffer was added to each sample to mix the cells. Then the cells were added with 5 μL Annexin V-FITC and 10 μL PI Staining Solution and mixed gently. After the reaction at room temperature for 15 min, 400 μL of 1 × Binding Buffer was added, mixed and placed on ice. The samples were detected by flow cytometry (Beckman Coulter, USA) within 1 h. Apoptosis was analyzed by FlowJo_v 10.8.1 software (Huo et al., 2021; Liu et al., 2021).