Synthesis, biological evaluation and molecular docking investigation of new sulphonamide derivatives bearing naphthalene moiety as potent tubulin polymerisation inhibitors

Abstract A new series of sulphonamide derivatives bearing naphthalene moiety were synthesised and evaluated for their antiproliferative and tubulin polymerisation inhibitory activities. These new compounds were evaluated for their in vitro antiproliferative activity against MCF-7 and A549 by using CCK-8 method. Among all the tested compounds, compound 5c with naphthalen-1-yl moiety exhibited the most potent antiproliferative activity against MCF-7 and A549 cell line, with IC50 values of 0.51 ± 0.03 µM and 0.33 ± 0.01 µM, respectively. The results of tubulin polymerisation assay shown that 5c exhibited a significant ability to inhibit tubulin polymerisation with IC50 value of 2.8 μM. Consistent with its antitubulin activity, 5c can significantly arrest the cell cycle at G2/M phase and induce apoptosis in MCF-7 cancer cells. Molecular docking study indicated that compound 5c inhibited tubulin polymerisation through interacting at the colchicine-binding site of tubulin. Furthermore, 5c exhibited low cytotoxic activity on human normal cell line.


Introduction
Microtubules are crucial elements of the cytoskeleton in eukaryocyte, which are polymerised by aand b-tubulin heterodimers in a head-to-tail manner to form hollow cylindrical filaments 1 . The microtubule system of eukaryotic cells plays important roles in numerous essential cellular functions, such as cell growth, division, motility, maintenance of cell shape, and intracellular vesicle transport 2,3 . There is an increasing evidence showing that the disruption of microtubule will result in the cycle arrest in G2/M phase and lead to the apoptosis of cell 4 . Therefore, microtubule has become an attractive molecular target in anti-tumour drug discovery [5][6][7] . Up to the present, a larger number of anti-tubulin agents have been developed and some of them (e.g., paclitaxel, docetaxel, vinblastine, and vincristine) have been approved by FDA for clinical treatment of cancer 8 . However, these natural products and their derivatives are facing severe disadvantages, such as toxicity, drug resistance, and difficult synthesis 9 . Therefore, the search of novel molecules with potent tubulin polymerisation inhibitory activity is still in progress.
On the other hand, the trimethoxyphenyl has been proved to be an important pharmacophoric group of tubulin inhibitors by binding at the colchicine binding site of tubulin 16 . Based on this, the trimethoxyphenyl moiety has been chosen as a core for the design and development of novel tubulin polymerisation inhibitors 16 . In the last few decades, a large number of tubulin polymerisation inhibitors containing trimethoxyphenyl moiety have been reported in the literature, and some of them have entered clinical trials, such as BNC-105p, CA-4P, CKD-516, and AVE8062 ( Figure 1) [17][18][19][20] .
Hence, in continuation of our interest on the design and development of novel tubulin polymerisation inhibitors 21-24 , a new series of sulphonamide derivatives (5a-5e and 8a-8i) were designed based on the molecular hybridisation approach (Figure 2) 25,26 . All the newly synthesised target compounds were evaluated for their antiproliferative activity to explore the preliminary structure-activity relationships (SAR). Tubulin polymerisation inhibition assay, cell cycle analysis, and cell apoptosis assay were performed to illuminate the pharmacologic mechanism. Additionally, molecular modelling was carried out to elucidate its possible binding mode in tubulin.

Chemistry
The sulphonamide derivatives (5a-5e and 8a-8i) were synthesised according to the synthetic route illustrated in Scheme 1. Firstly, 3,4,5-trimethoxyaniline 1 was condensed with 4-methoxybenzoyl chloride 2 in the presence of Et 3 N as base at room temperature to afford intermediate 3 in high yields, followed by the carbonyl reduction reaction with LiAlH 4 to give the key intermediate 4 [27] . Then, condensation of compound 4 with appropriate commercially available aryl sulphonyl chloride in the presence of Et 3 N and DMAP in THF to generate the title compounds (5a-5e) in high yields. On the other hand, treatment of 3,4,5-trimethoxyaniline 1 with naphthalene-1-sulphonyl chloride 6 in the presence of Et 3 N in CH 2 Cl 2 to afforded key intermediate 7, which reacted with commercially available benzyl halide in the presence of KI and K 2 CO 3 to provide the title compounds (8a-8i). All target derivatives were fully characterised by 1 H NMR, 13 C NMR, HRMS, and elemental analysis (see Supporting Information).
To study the structure-activity relationships (SAR) of this class of compounds, the aryl substituents on sulphonamides were discussed firstly. Based on the antiproliferative activity of compounds 5a-5e, it can be seen that the aryl substituents on sulphonamides affected on antiproliferative activity of this class of compounds. Among these molecules, 5 b with a phenyl ring displayed low antiproliferative activity with IC 50 value of > 30.0 mM. Introduction of electron-donating (5a and 5e) groups into the phenyl ring, results in significantly increased the antiproliferative activity. The replacement of phenyl ring with naphthalen-1-yl or naphthalen-2yl led to compounds 5c and 5d, resulting in significantly increased the antiproliferative activity. In particular, compound 5c with naphthalen-1-yl moiety was found to be the most active compound in this series. These inhibitory results indicate that naphthalen-1-yl group seems to be the optimal substituent on the position.
In order to verify the safety profile of this class of compounds, the most potent compound 5c was selected to test its cytotoxicity against human normal liver cell line (LO2). The result was shown that compound 5c exhibited moderate cytotoxic activity against human normal liver cell (LO2) with IC 50 value of 12.73 ± 3.26 mM.
While, 5c displayed potent anticancer activity against MCF-7 and A549 cell lines with IC 50 value of 0.51 ± 0.03 and 0.33 ± 0.01 mM, respectively. Hence, we could conclude that these compounds have good safety for potential application in the treatment of tumour cells.

Inhibition of tubulin polymerisation
To examine whether tubulin is the target of this class of compounds, the in vitro tubulin polymerisation inhibitory activity of 5c was evaluated using tubulin polymerisation assay 28 . Meanwhile, tubulin polymerisation inhibitor colchicine was used as positive control. As shown in Figure 5, compared with the control, the absorbance values at 340 nm of tubulin gradually decreased after incubation with different concentrations of 5c or colchicine. The results shown that 5c exhibited a significant ability to inhibit tubulin polymerisation in a concentration-dependent manner with IC 50 values of 2.8 mM, as compared to colchicine (IC 50 ¼ 9.3 mM). Besides, 5c and colchicine have similar effects on inhibit tubulin polymerisation, indicating that 5c was a microtubulin-destabilizing agent.

Cell cycle arrest
Due to microtubules play an important role in eukaryotic cell division, tubulin polymerisation inhibitors can disrupt regulated cell cycle distribution and block the cell cycle in G2/M phase 21,29 . Therefore, the effect of compound 5c on the cell cycle of MCF-7 cancer cells was evaluated by using flow cytometry. As shown in Figure 6, after treated with different concentrations of compound 5c (0.125 mM, 0.25 mM or 0.5 mM), the G2/M population in MCF-7 cancer cells increased from 21.19% (control) to 30.77% (0.125 mM), 57.50% (0.25 mM), and 82.22% (0.5 mM), respectively. The results indicate that compound 5c can arrest cell cycle at G2/M phase in a dose-dependent manner.

Cell apoptosis
Since many literatures have reported that tubulin polymerisation inhibitors are able to induce cellular apoptosis 22,30 , the Annexin V-FITC/PI assay was carried out to examine the influence of compound 5c on cell apoptosis in MCF-7 cancer cells. As shown in

Molecular docking
To elucidate the binding mode of this class of compounds, molecular docking simulations of compound 5c with tubulin were performed. Colchicine was first docked into the colchicine binding site of tubulin. The result was shown that co-crystallized conformation of colchicine was reproduced approximately (RMSD: 1.10 Å), indicating that this protocol of molecular docking is credible. Then, the theoretical binding mode between 5c and tubulin was investigated, and the estimated binding energy was À9.6 kcalÁmol À1 . As shown in Figure 8, Compound 5c adopted an "Y-shaped" conformation in the colchicine pocket of tubulin.    naphthyl groups of 5c formed cation-p interactions with the residues Lys-254 and Lys-352, respectively. All these interactions helped 5c to anchor in the colchicine binding site of tubulin.

Molecular dynamics (MD) simulations
To explore the potential binding mode between 5c and tubulin, molecular docking and molecular dynamics simulations were performed using the AutoDock vina 1.1.2 and Amber12 software package. The preferential binding mechanism of tubulin with 5c was determined by 30-ns molecular dynamics simulations based on the docking results. To explore the dynamic stability of the models and to ensure the rationality of the sampling strategy, the root-mean-square deviation (RMSD) value of the protein backbone based on the starting structure along the simulation time was calculated and plotted in Figure 9(A). The result was shown that the protein structure of the system was stabilised during the simulation. The theoretical binding mode between 5c and tubulin was shown in Figure 9(B). Compound 5c adopted a compact conformation in the pocket of tubulin. The naphthyl group of 5c located at the hydrophobic pocket, surrounded by the residues A/Ala-180, A/Val-181, B/Val-238, B/Leu-248, B/Ala-250, B/Leu-255, B/Ala-316, B/Ala-317, B/Val-318 and B/Ala-354, forming a strong hydrophobic binding. Detailed analysis showed that the phenyl group of 5c formed a cation-p interaction with the residue Lys-254. It was shown that the residue Lys-254 (bond length: 2.7 Å) formed a hydrogen bond with 5c, which was the main interaction between 5c and tubulin. All in all, the above molecular dynamics simulation gave us rational explanation of the interaction between 5c and tubulin, which provided valuable information for further development of tubulin polymerisation inhibitors.

Conclusion
In summary, a new series of sulphonamide derivatives bearing naphthalene moiety have been synthesised and and characterised by 1 H NMR, 13 C NMR, HRMS, and elemental analysis. All of the title compounds were screened for antiproliferative activity against human breast cancer cells (MCF-7) and human non-small cell lung carcinoma cells (A549) by using CCK-8 method. Among all synthesised compounds, compound 5c with naphthalen-1-yl moiety exhibited the most potent antiproliferative activity against MCF-7 and A549 cell line, with IC 50 values of 0.51 ± 0.03 mM and 0.33 ± 0.01 mM, respectively. SAR studies suggested that the naphthalen-1-yl and 4-methoxybenzyl at the sulphonamide played an important role for the potent antiproliferative activity. Tubulin polymerisation assay revealed that compound was a microtubulindestabilizing agent with IC 50 value of 2.8 mM. Further mechanism evaluation demonstrated that 5c can significantly arrest the cell cycle at G2/M phase and induce apoptosis in MCF-7 cancer cells. Additionally, molecular modelling results showed that 5c binds well to the colchicine-binding site of tubulin. Hence, these results suggest that 5c could be used as a promising lead compound for further investigation in anticancer drug development.

4-Methoxy-N-(3,4,5-trimethoxyphenyl)benzamide (3)
To a solution of 3,4,5-trimethoxyaniline 1 (10 mmol) and Et 3 N (20 mmol) in THF (50 ml) were added 4-methoxybenzoyl chloride 2 (10 mmol) and the reacting mixture was stirred at room temperature for 4 h. Then, the solvent was removed under reduced pressure, and water was added to the reaction mixture and extracted 3 times with ethyl acetate. The combined organic layers were dried, filtered, and concentrated. The residue was purified by chromatography on silica gel with EtOAc/petroleum ether ¼ 1:1 to give compound 3 as white solid (yield 88%).

3,4,5-Trimethoxy-N-(4-methoxybenzyl)aniline (4)
A mixture of LiAlH 4 (2 mmol) in anhydrous THF (10 ml) was stirred in an ice bath for 10 min. Then, a solution of compound 3 (1 mmol) in 10 ml of THF was added dropwise to the mixture. After completion of dropwise addition, the mixture was stirred at 0 C for 30 min then refluxed at 70-80 C for 2 h. After completion of the reaction, the solvent was evaporated and the organic product was extracted with ethyl acetate. The combined organic layers were dried over sodium sulphate and evaporated to afford black oil, which was used directly for the next step without further purification.

General procedure for the synthesis of 5a-5e
To a solution of compound 4 (2 mmol), DMAP (0.2 mmol), and Et 3 N (2 mmol) in THF (50 ml) was added commercially available aryl sulphonyl chloride (2 mmol) and the reacting mixture was stirred at room temperature for 12 h. The solvent was evaporated and water was added to the reaction mixture. The organic material was extracted with ethyl acetate, and the organic layer was dried over Na 2 SO 4 and concentrated under vacuum. The residue was purified by chromatography to give the title product 5a-5e.

General procedure for the synthesis of 8a-8i
A mixture of 7 (1.0 mmol), K 2 CO 3 (2.0 mmol), KI (1.0 mmol), and commercially available benzyl halide (1.0 mmol) in acetone (10 ml) was stirred at reflux for 5 h. After completion of the reaction, the mixture was concentrated under reduced pressure and the residue was purified by chromatography to give the title compounds 8a-8i.

Disclosure statement
No potential conflict of interest was reported by the author(s).