Discovery of novel arylamide derivatives containing piperazine moiety as inhibitors of tubulin polymerisation with potent liver cancer inhibitory activity

Abstract

In this work, a series of novel arylamide derivatives containing piperazine moiety were designed and synthesised as tubulin polymerisation inhibitors. Among 25 target compounds, compound 16f (MY-1121) exhibited low nanomolar IC₅₀ values ranging from 0.089 to 0.238 μM against nine human cancer cells. Its inhibitory effects on liver cancer cells were particularly evident with IC₅₀ values of 89.42 and 91.62 nM for SMMC-7721 and HuH-7 cells, respectively. Further mechanism studies demonstrated that compound 16f (MY-1121) could bind to the colchicine binding site of β-tubulin and directly act on β-tubulin, thus inhibiting tubulin polymerisation. Additionally, compound 16f (MY-1121) could inhibit colony forming ability, cause morphological changes, block cell cycle arrest at the G2 phase, induce cell apoptosis, and regulate the expression of cell cycle and cell apoptosis related proteins in liver cancer cells. Overall, the promising bioactivities of compound 16f (MY-1121) make the novel arylamide derivatives have the value for further development as tubulin polymerisation inhibitors with potent anticancer activities.

Keywords: Tubulin; antiproliferative activities; arylamide; piperazine.

Figure 1.

Structures of typical CBSIs under clinical trials and reported CBSIs.

Figure 2.

Structures of piperazine-based tubulin inhibitors.

Figure 3.

Design strategy of arylamide derivatives as inhibitors of tubulin polymerisation.

Scheme 1.

Synthesis of target compounds 16a–16y.

Figure 4.

Effects of compound MY-1121 on tubulin polymerisation under cell free condition. The vertical coordinate indicates the degree of tubulin polymerisation. The experiments were repeated thrice.

Figure 5.

Compound MY-1121 binds to β-tubulin directly on colchicine binding site. (A, B) EBI competition assay, the affinity of the compound with colchicine binding site was negatively correlated with the level of the tubulin adduct band; (C, D) alkaline protease assay. Cells were treated with alkaline proteinase and different concentrations of compound MY-1121. The band signal of β-tubulin was negatively correlated with the ability of alkaline protease to hydrolyse β-tubulin. The binding of the compound to β-tubulin was able to inhibit the hydrolysis of β-tubulin by alkaline protease.

Figure 6.

Effects of compound MY-1121 on microtubule network in liver cancer cells SMMC-7721 and HuH-7. Cells were treated for 48 h. β-tubulin was stained green and cell nuclei were stained blue.

Figure 7.

Molecular docking results of compound MY-1121 with tubulin (PDB: 1AS0). The hydrogen bond, ionic interactions, and hydrophobic interactions are depicted as yellow, magentas, and green dashed lines, respectively.

Figure 8.

Effects of compound MY-1121 on cell cycle distribution. Liver cancer cells SMMC-7721 (A, B) and HuH-7 (C, D) were treated for 48 h. The cells were stained with propidium iodide and analysed via flow cytometry to measure the cell cycle profile.

Figure 9.

Effects of compound MY-1121 on colony formatting ability. Liver cancer cells SMMC-7721 (A) and HuH-7 (B) were treated for seven days.

Figure 10.

The effects of compound MY-1121 on cell cycle related proteins in SMMC-7721 cell were conducted via Western blotting assay. Cells were treated with indicated concentrations of compound MY-1121 for 48 h.

Figure 11.

Effects of compound MY-1121 on morphology changes of liver cancer cells. Liver cancer cells were treated for 48 h. (A) Cell morphology in the bright field; (B) number of live cells (green) to dead cells (red); and (C) cell nuclei changes.

Figure 12.

Cell apoptosis induced by compound MY-1121. Liver cancer cell SMMC-7721 (A, B) and HuH-7 (C, D) were treated indicated concentrations of compound MY-1121 for 48 h.

Figure 13.

The effects of compound MY-1121 on apoptosis-related proteins in SMMC-7721 cell were conducted via Western blotting assay. Cells were treated indicated concentrations of compound MY-1121 for 48 h.