Design, synthesis and biological evaluation of isoxazole-naphthalene derivatives as anti-tubulin agents

2.1 Chemistry

All starting materials and reagents were purchased from commercial suppliers. TLC was performed on 0.20 mm Silica Gel 60 F₂₅₄ plates (Qingdao Ocean Chemical Factory, Shandong, China). Nuclear magnetic resonance spectra (NMR) were recorded on a Bruker spectrometer (400 MHz) with TMS as an external reference and reported in parts per million.

2.2 MTT assay

Human breast cancer cells (MCF-7) were seeded in 96-well plates and incubated for 24 h. Then treated with DMSO, cisplatin, or this series of compounds at the different concentrations (0.3125, 0.625, 1.25, 2.5, 5.0, 10.0 and 20.0 μM) for 48 h. MTT (tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide) was added to each well, and incubated with cells at 37 °C for 4 h. Then, the formazan crystals were dissolved in DMSO, and the absorbency at 570 nm were measured by the Spectramax M5 Microtiter Plate Luminometer (Molecular Devices, USA). The IC₅₀ value were presented as mean ± standard deviation.

2.3 Tubulin polymerization assay

Tubulin protein was purified from fresh pig brain three cycles of temperature-dependent assembly/disassembly according to Shelanski et al in 100 mM PIPES (pH 6.5), 1 mM MgSO4, 2 mM EGTA, 1 mM GTP and 1 mM 2-mercaptoethanol (Shelanski et al., 1973). In the first cycle of polymerization, glycerol and phenylmethylsulfonyl fluoride were added to 4 M and 0.2 mM, respectively. Homogeneous tubulin was prepared from microtubule protein by phosphocellulose (P11) chromatography.

After mixed tubulin protein with different concentrations of compound in PEM buffer (100 mM PIPES, 1 mM MgCl2, and 1 mM EGTA) containing 1 mM GTP and 5% glycerol at 37 °C, the fluorescence intensity was monitored at 340 nm using a SPECTRA MAX 190 (Molecular Device) spectrophotometer. The plateau absorbance values were used for calculations.

2.4 Cell cycle analysis

Human breast MCF-7 cancer cells were seeded into the 6-well plate and treated with compound 5j (2.0 μM) or DMSO for 24 h. After incubation, the cells were washed twice on ice with PBS and fixed with 70% ethanol at 4 °C overnight. Finally, the cells were treated with RNAse and stained with propidium iodide (PI). The samples were analyzed by flow cytometry (TASC240, USA).

2.5 Cell apoptosis assay

Human breast MCF-7 cancer cells were seeded into the 6-well plate and treated with compound 5j (2.0 μM) or DMSO. Cells were collected, washed, and stained with AnnexinV-FITC and PI. Then the sample was analyzed via flow cytometry (TASC240, USA).

2.6 Molecular docking study

In this study, the tubulin structure (PDB ID: 1SA0) was downloaded from Protein Data Bank (www.rcsb.org). All bound waters and ligands were removed from the protein. The docking procedure was performed by using Autodock vina 1.1.2. 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 best-scoring pose as judged by the Vina docking score was chosen and visually analyzed using PyMoL 1.7.6 software (www.pymol.org).

3.1 Chemistry

The synthetic pathway of the title compounds 5a-5u was shown in Scheme 1. Condensation of 1-methoxynaphthalene 1 with various substituted phenylacetic acids 2 in the presence of trifluoroacetic acid (TFA) and trifluoroacetic anhydride (TFAA) to afford the corresponding deoxybenzoins 3 (Liu and Xu, 2018). Condensation of 3 with N,N-dimethylformamide dimethylacetal (DMF-DMA) to afford the enamines 4 in high yields, which reacted with hydroxylamine hydrochloride to provide the title compounds 5a-5u.

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Scheme 1

Reagents and conditions: Reagents and conditions: (a) TFAA, TFA, room temperature, 12 h; (b) DMF-DMA, 80 °C, 24 h; (c) HONH₂·HCl, Na₂CO₃, AcOH, MeOH/H₂O, 70 °C, 24 h.

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All the synthesized compounds 5a-5u were characterized by ¹H NMR, ¹³C NMR and HRMS spectroscopy (Supplemental Material, Figs. S1–S63). The ¹H NMR of compound 5a showed a singlet at δ 8.63 ppm representing the CH proton of isoxazole. The aromatic protons of 3,4,5-trimethoxyphenyl have appeared as a singlet for two protons at δ 6.39 ppm. The remaining six aromatic signals were assigned to the naphthalene nucleus in the compound 5a. Characteristic methoxy protons appeared as three singlets at δ 3.47, 3.77 and 4.05 ppm. In ¹³C NMR spectrum of compound 5a the methoxy carbons appeared at δ 55.9, 55.9, and 61.0 ppm. All the aromatic carbons resonate around δ 103.3–164.7 ppm. The HRMS of compound 5a showed a molecular ion peak at m/z 392.14603 as [M + H]⁺ which also supports the proposed structure of the compound. A similar pattern was observed in ¹H NMR, ¹³C NMR and HRMS of all the title compounds.

3.2 In vitro antiproliferative activity against breast cancer cell line (MCF-7)

The synthesized compounds 5a-5u were tested for their antiproliferative activity against the human MCF-7 breast cancer cell line by using the standard MTT assay. Cisplatin was used as positive control drug. The IC₅₀ values (concentration required to cause 50% inhibition of the viability of cancer cells) of test compounds 5a-5u and the positive control cisplatin are shown in Table 1. According to the experimental data, the majority of the tested compounds (except 5e, 5l, 5n and 5s) showed low antiproliferative activity with IC₅₀ values less than 10.0 μM. Compound 5b and 5j displayed the most potent antiproliferative activity with IC₅₀ values of 1.27 ± 0.13 μM and 1.23 ± 0.16 μM, respectively.

According to the anti-proliferative activity evaluations of these compounds mentioned above, the preliminary structure-activity relationships (SAR) of this class of compounds can be summarized. The results suggested that electron-donating group (Me, OMe, and OEt) located at the 4-position of the phenyl ring resulted in the best activity (5a-5c, 5g, 5i, 5j, 5m, 5p and 5u). Shifting methoxyl or methyl group to the 2- or 3- position decreased the activity (5f, 5h and 5k). Furthermore, introduction of electron withdrawing group (F, Cl, Br and NO₂) into the phenyl ring results in a significant decrease the inhibitory activity (5d, 5e, 5l, 5n, 5o, 5q, and 5r-5t). In particular, the substituent group located at the 2-position of the phenyl ring resulted in a decrease of inhibitory activity (5e, 5k, 5l and 5q). Among this series of synthesized compounds, 5j (IC₅₀ = 1.23 ± 0.16 μM) with 4-ethoxy substitution at phenyl ring was found to be the most active compound. Additionally, 5b (IC₅₀ = 1.27 ± 0.13 μM) containing 4-methoxyl substitution at the phenyl ring was found to be the second most active compound.

3.3 Effect on tubulin polymerization

To investigate whether the antiproliferative activities of this class of compounds derived from interaction with tubulin, the most potent active compound 5j was selected and evaluated for its inhibition of tubulin polymerization. For comparison, colchicine was used as a positive control. The results of this assay were shown in Fig. 3. After incubated tubulin protein with various concentrations of compound 5j (0.8–25 μM), the increased tendency of the fluorescence intensity was obviously slowed down as compared with the control, which showed a trend similar to the positive colchicine. In addition, compound 5j showed better inhibition of tubulin polymerization (IC₅₀ = 3.4 μM) than colchicine (IC₅₀ = 7.5 μM). These data confirmed that tubulin might be the molecular target of this series of compounds.

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Fig. 3

Effect of compound 5j and colchicine on tubulin polymerization.

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3.4 Cell cycle arrest

Based on the literature report, tubulin inhibitors could inhibit the polymerization of tubulin and thus cause cell cycle arrest in the G2/M phase (Jordan, 2002). To verify whether these newly synthesized compounds could prevent mitosis, the effects of compound 5j on cell cycle progression were examined by flow cytometry. MCF-7 cells were treated with DMSO or 5j (2.0 μM) for 24 h, and the proportion of tested cells at different cell cycle phases was analyzed by flow cytometry. As illustrated in Fig. 4, there was an accumulation of 83.30% of cells in the G2/M phase of the cell cycle observed in 5j treatment group, in contrast to that of untreated control (9.54%). Therefore, these observations indicated that compound 5j could block the cell in the G2/M phase and affect the normal mitosis of the cell.

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Fig. 4

Compound 5j induced the cell cycle arrest and cell apoptosis in MCF-7 cells. (A, B) Cell cycle analysis of MCF-7 cells with or without treatment of 5j for 24 h; (C, D) Cell apoptosis induction of MCF-7 cells with or without treatment of 5j for 24 h.

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3.5 Cell apoptosis

To verify whether compound 5j could induce apoptosis of cancer cells, the effect of compound 5j on cell apoptosis in MCF-7 was evaluated by using Annexin V-FITC/PI assay. As shown in Fig. 4, the cells were incubated with compound 5j at 2.0 μM for 24 h, the percentages of early and late apoptosis cells were 15.3% and 6.44%, respectively, being elevated from the control group. These results confirmed that compound 5j could induce apoptosis of MCF-7 cells, similar to other tubulin polymerization inhibitors.

3.6 Molecular docking

To elucidate the binding mode and type of interactions with tubulin (PDB code 1SA0), molecular docking simulations of the most active compound 5j in the colchicine site of tubulin were performed by using Autodock vina 1.1.2 (Trott and Olson, 2010). Colchicine was first docked into the colchicine binding site of tubulin. As shown in Fig. 5A, the docked conformation of colchicine is similar to the crystal conformation, indicating that this protocol of molecular docking is capable of reproducing the crystallographic pose. Then, we investigate the binding mode of compound 5j with tubulin. The resulting binding mode is shown in Fig. 5B–D, Compound 5j adopted an “L-shaped” conformation in the pocket of the tubulin. The estimated binding energy of compound 5j with the enzyme was −8.7 kcal·mol⁻¹. The naphthyl group of 5j located at the hydrophobic pocket, surrounded by the residues Cys-241, Leu-248, Ala-250, Leu-255, Met-259, Val-315, Ala-316, Val-318, and Ala-354, forming a strong hydrophobic binding. Detailed analysis showed that the phenyl group of 5j formed cation-π interactions with the residues Lys-254 and Lys-352. Altogether, compound 5j exhibited efficient binding to the colchicine site of tubulin, which provided a rational explanation of its inhibitory effect against tubulin.

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Fig. 5

(A) Redocked conformation superimposed on crystal conformation, the colchicine was shown in violet (docking conformation) or pink sticks (crystal conformation), respectively; (B) docked conformation of compound 5j (rose red) superimposed on crystal conformation of the colchicine (pink); (c) Chemical Structure of compound 5j; (d) Docking model of the most potent compound 5j (magenta colour stick) in the colchicine binding site of tubulin.

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