Design, synthesis, and biological evaluation of 4-aryl-9H-carbazoles as tubulin polymerization inhibitors with potent anticancer activities

2.2.1 In vitro antiproliferative activity

The antiproliferative activities of the newly synthesized compounds 13a-u were evaluated against three cancer cell lines (HeLa, HepG2, and SGC-7901) using a conventional MTT assay with CA-4 as the positive control. Most of the compounds exhibited moderate antiproliferative activities against the three tested cell lines with IC₅₀ values in the micromolar range. These results indicated that the introduction of the carbazole moiety as a suitable mimic for the ternary heterocyclic ring present in NSC676693 maintained the effective antiproliferative activity.

We first introduced different rigid aromatic groups such as phenyl (13a), naphthyl (13q), thienyl (13r), pyridyl (13s and 13t), and indolyl (13u) into the A-ring to explore the effect of different skeletons on antiproliferative activities against three different cell lines. As shown in Table 1, benzene derivative (13a), naphthalene derivative (13q), thiophene derivative (13r), pyridine derivatives (13s and 13t), and indole derivative (13u) showed lower potencies as compared to diminished activities, which indicated the importance of the A-ring scaffold. Among them, benzene derivative (13a) exhibited the highest cytotoxicity with an average IC₅₀ value of 11.6 μM against the three cancer cells. Subsequently, we concentrated on systematic modification of phenyl to explore the variations in position and type of substituents. Replacement of hydrogen atom in 13a with methyl (13b, 13c, and 13d), methoxyl (13f, 13g, and 13h), ethyoxyl (13l), or hydroxyl (13p) resulted in compounds with similar or stronger potencies than 13a. Compound 13h exhibited the highest antiproliferative activity with mean IC₅₀ value ranging from 3.4 μM to 5.5 μM, at least 3-fold more potent than 13a, indicating that 4-position methoxyl in phenyl A-ring is optimal for activity. Next, the introduction of electron-withdrawing groups, such as fluorine (13m), chlorine (13n), or nitro (13o) in the 4-position methoxy portion of compound 13h reduced the inhibitory activities to varying degrees. In addition, the triple substituted compound showed increased activity, which could be confirmed by compound 13k. Among the target compounds, compound 13k (A-ring = 3,4,5-trimethoxyphenyl) showed the best antiproliferative activity against HeLa cells with an IC₅₀ value of 1.2 ± 0.09 μM.

2.2.2 Effect on tubulin polymerization

To investigate the relationship between the antiproliferative activities of these compounds and tubulin, we evaluated the effect of the most active compound 13k on tubulin polymerization compared to the positive control CA-4 and the negative control paclitaxel. As shown in Fig. 3, both CA-4 and 13k exhibited significant inhibition of tubulin polymerization. In comparison, paclitaxel enhanced the rate of tubulin polymerization. These results demonstrated that 13k interferes with tubulin polymerization.

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Fig. 3

Effects of compound 13k on tubulin polymerization.

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2.2.3 Analysis of immunofluorescence staining

To examine whether compound 13k can interfere with microtubule dynamics, an immunofluorescence assay was performed in HeLa cells treated with 13k. As presented in Fig. 4, the control cells exhibited normal organization and arrangement with hairy, elongated, and fibrous microtubules (green) wrapped around the nucleus (blue). However, after treatment with CA-4 (30 nM) or 13k (1 μM, 2 μM, and 3 μM) for 24 h, the microtubules became short and disintegrated, indicating that the microtubule network was disrupted. These results implied that 13k could destabilize microtubules by inducing the depolymerization and collapse of the microtubule network, similar to CA-4.

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Fig. 4

Effects of compound 13k (1 μM, 2 μM, and 3 μM) and CA-4 (30 nM), on the cellular microtubule network and microtubule reassemble by immunofluorescence.

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2.2.4 Cell cycle analysis

Tubulin polymerization inhibitors are known to induce cell cycle arrest in the G2/M phase (Shi et al., 2022). To explore the effect of compound 13k on the cell cycle, flow cytometric analysis was performed on HeLa cells treated with 13k. As demonstrated in Fig. 5, the percentage of cells stalled in G2/M phase increased from 10.03% to 61.06% after treatment with different concentrations of 13k (1 μM, 2 μM, and 3 μM) for 24 h compared to the control (8.59%). Thus, 13k could concentration-dependently induce G2/M phase arrest in HeLa cells.

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Fig. 5

Effects of CA-4 and compound 13k on cell cycle. HeLa cell lines were treated with CA-4 (30 nM) and compound 13k (1 μM, 2 μM, and 3 μM) for 24 h.

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2.2.5 Induction of cell apoptosis

It has been shown that tubulin polymerization inhibitors can induce apoptosis (Wang et al., 2019; Yang et al., 2022). Therefore, we investigated the effect of compound 13k on apoptosis in HeLa cells. As illustrated in Fig. 6, when HeLa cells were treated with different concentrations (1 μM, 2 μM, and 3 μM) of 13k for 48 h, the percentage of total apoptotic cells increased significantly and concentration-dependently, from 5.20% (control) to 13.20% (1 μM), 22.62% (2 μM), and 27.50% (3 μM), respectively.

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Fig. 6

Analyses of apoptosis induction in Hela cells. Cells were harvested and stained with Annexin-V/PI for analysis after treatment with different concentrations of compound 13k (1, 2, and 3 μM) and control for 48 h. The diverse cell stages were given as live (Q4), early apoptotic (Q3), late apoptotic (Q2), and necrotic cells (Q1).

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2.2.6 Molecular docking analysis

To understand the possible binding modes of the target compounds, a molecular docking analysis of compound 13k was conducted by using the CDOCKER program of Discovery Studio 3.0 software (PDB: 1SA0). The docking study indicated that 13k occupied the colchicine binding sites of α- and β-tubulin and was mainly buried in the β-subunit (Fig. 7). For 13k, a hydrogen bond was formed between the oxygen atom of methoxy and the residue of ALAβ250. In addition, the nitrogen atom of carbazole formed another hydrogen bond with the residue of VALβ238. These results point to a possible binding of 13k to the colchicine binding site on tubulin.

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Fig. 7

(A) Predicted mode for compound 13k (yellow) binding in the colchicine binding site of tubulin (PDB: 1SA0); (B and C) Docking conformation of compound 13k in the colchicine binding site of tubulin.

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2.2.7 Physicochemical properties

To explore the drug-like properties of the target compounds, some physicochemical properties of 9 and 13k were predicted using the ChemBioDraw Ultra 14.0 software or free online website (https://www.swissadme.ch/index.php) for their adaptability with Lipinski's rule of five. As shown in Table 2, 9 and 13k conform well to the Lipinski's rule of five. Additionally, 13k exists three hydrogen bond acceptors and one hydrogen bond donor, which helps to reduce the binding energy between the compound and the action site.

4.1.1 Materials and methods

All of the reagents and solvents were purchased from chemical companies. ¹H NMR and ¹³C NMR spectra were tested in CDCl₃ with TMS as the internal reference on a Bruker AVANCE (¹H at 500 MHz, ¹³C at 126 MHz). Mass spectra (MS) were measured on an Agilent 1100-sl mass spectrometer with an electrospray ionization source from Agilent Co. Ltd.. TLC analysis was used for determining the extent of reactions under UV light (wavelength: 365 nm and 254 nm).

4.1.2 General synthetic procedure for 2-bromo-2′-nitro-1,1′-biphenyl (16)

To a stirring solution of 1-bromo-2-nitrobenzene (14, 4.95 mmol) and (2-bromophenyl)boronic acid (15, 5.45 mmol) in THF (15 ml) were added Pd(PPh₃)₄ (0.25 mmol) followed by K₂CO₃ (0.05 mmol in 10 ml water) at room temperature. The reaction mixture was degassed and purged with N₂ about three times and was stirred at irradiated in a microwave reactor for 20 min at 116 °C after which it was cooled and extracted with dichloromethane. The combined organic layer was dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude material was purified by column chromatography over silica gel using n-hexane/ethyl acetate (9:1) as a solvent to give 2-bromo-2′-nitro-1,1′-biphenyl (16).

4.1.3 General synthetic procedure for4-bromo-9H-carbazole (17)

2-bromo-2′-nitro-1,1′-biphenyl (16, 1.01 mmol) and triphenylphosphine (2.6 mmol) were dissolved in 1,2-dichlorobenzene (10 ml), and the resulting solution was stirred at 170 °C for 12 h, after which it was cooled and extracted with dichloromethane/H₂O. The combined organic layer was dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude material was purified by column chromatography over silica gel using n-hexane/ethyl acetate (8:1) to yield 4-bromo-9H-carbazole (17).

4.1.4 General synthetic procedure for 4-aryl-9H-carbazoles (13a-u)

A mixture of 4-bromo-9H-carbazole (17, 0.10 mmol), Pd(PPh₃)₄ (0.005 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. After stirring at irradiated in a microwave reactor for 25 min at 130 °C (indicated by TLC) under N₂ atmosphere, H₂O (50 ml) was added to the reaction mixture and extracted with ethyl acetate. The combined organics were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to give a residue, which was purified by column 400 chromatography using a mixture of n-hexane/ethyl acetate (3:1) as an eluent to provide the target compounds 4-aryl-9H-carbazoles (13a-v).

4.2.1 Cell culture

HeLa, HepG2, and SGC-7901 cells were cultured in RPMI-1640 medium containing 10% FBS, 100 U/mL streptomycin, and 100 U/mL penicillin at 37 °C in a humidified atmosphere containing 5% CO₂. All cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

4.2.2 In vitro antiproliferative activity

The in vitro antiproliferative activities of CA-4 and all of the target compounds were determined by MTT assay (Tan et al., 2023). Briefly, cells were seeded into 96-well plates at a density of 2 × 10⁴/well, depending on the growth rate of the cell line. 24 h later, triplicate wells were treated with media and the compounds being tested. After 72 h of incubation at 37℃ in 5% CO₂, the drug-containing medium was removed and replaced with 100 μL of fresh medium containing 5 mg/mL MTT solution. After 4 h of incubation, the medium with MTT was removed, and 100 μL of dimethyl sulfoxide was added to each well. The plates were gently agitated until the purple formazan crystals were dissolved, and the OD₄₉₀ values were determined using a microplate reader. The data were calculated and plotted as the percent viability compared to the control. The IC₅₀ was defined as the drug concentration that resulted in an absorbance of 50% of that of the untreated wells in the MTT assay.

4.2.3 Effect on tubulin polymerization

The effects of compound 13k and CA-4 on tubulin polymerization were determined using a fluorescence-based tubulin polymerisation assay kit (Cytoskeleton-Cat.#BK011P) according to the manufacturer's protocol (Chen et al., 2020). Tubulin was resuspended in ice-cold G-PEM buffer and added to wells of a 96-well plate containing the designated concentrations of the drug or vehicle. The samples were mixed well, and tubulin assembly was monitored at 1 min intervals for 90 min at 37℃ using a plate reader. The IC₅₀ values were calculated after 20 min using SPSS software.

4.2.4 Analysis of immunofluorescence staining

Immunostaining was performed to detect the microtubule-associated tubulin protein after exposure to CA-4 and the investigated compound 13 k (Deng et al., 2022). HeLa cells were seeded in a 24-well plate at 1 × 10⁴ cells per well and grown for 24 h. The cells were treated with the vehicle, CA-4, or 13 k for 24 h. The control and treated cells were fixed with 4% formaldehyde in PBS for 30 min at −20℃, washed twice with PBS and permeabilized with 0.1% (v/v) Triton X-100 in PBS for 5 min. The cells were then blocked with 5% bovine serum albumin in PBS for 10 min. The primary α-tubulin antibody was diluted (1/100) with 2% bovine serum albumin in PBS, and the plates were incubated overnight at 4℃. The cells were washed with PBS to remove unbound primary antibody, and the cells were then incubated with FITC-conjugated antimouse secondary antibody diluted (1/1000) with 2% BSA in PBS for 3 h at 37℃. The cells were washed with PBS to remove unbound secondary antibody, and the nuclei were stained with DAPI. Then, immunofluorescence was detected using a fluorescence microscope.

4.2.5 Cell cycle analysis

Cell cycle analysis studies were performed according to procedures described previously (Deng et al., 2022). Briefly, HeLa cells (8 × 10⁴ cells) were incubated with various concentrations of CA-4, 13 k or 0.05% DMSO for the indicated time. The cells were collected by centrifugation, washed with PBS and fixed in ice-cold 75% ethanol. The fixed cells were harvested by centrifugation and resuspended in 530 μL buffer containing 10 μL RNase (50 × ) and 25 μL PI (20 × ). After incubation at 37℃ in the dark for 30 min, the samples were then analyzed by FACScan flow cytometry (BectoneDickinson, Franklin Lakes, NJ, USA).

4.2.6 Induction of cell apoptosis

To investigate whether the target compound can induce apoptosis, an Annexin Van -FITC/PI experiment was carried out (Shi et al., 2022). HeLa cells were grown in 6-well plates (3 × 10⁵ cells/well) and incubated with various concentrations of 13 k, CA-4, or vehicle control for 48 h. Subsequently, cells were harvested by centrifugation, washed with PBS, and resuspended in binding buffer. Then, 10 μL of PI Staining Solution and 5 μL of Annexin V-FITC were added to the cell suspension for 15 min at room temperature in the dark. Finally, the samples were detected by a CytoFLEX flow cytometer and the percentage of apoptotic cells was calculated using Flowjo 10.8 software.