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

Figures & data

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.

Table 1. Antiproliferative activities of compounds 16a–16k and colchicine.

Comp.		IC50 (μM)^a
		MGC-803	HCT-116	SMMC-7721
16a		0.25 ± 0.010	0.32 ± 0.032	0.18 ± 0.022
16b		0.26 ± 0.023	0.15 ± 0.014	0.21 ± 0.018
16c		0.78 ± 0.033	0.88 ± 0.078	0.65 ± 0.018
16d		0.88 ± 0.046	0.91 ± 0.065	0.8 ± 0.021
16e		0.094 ± 0.006	0.12 ± 0.027	0.092 ± 0.008
16f (MY-1121)		0.092 ± 0.005	0.098 ± 0.034	0.088 ± 0.006
16g		0.089 ± 0.004	0.104 ± 0.013	0.095 ± 0.004
16h		0.095 ± 0.002	0.098 ± 0.004	0.097 ± 0.005
16i		0.17 ± 0.022	0.16 ± 0.026	0.10 ± 0.012
16g		0.11 ± 0.007	0.098 ± 0.008	0.091 ± 0.004
16k		0.15 ± 0.023	0.16 ± 0.012	0.13 ± 0.014
Colchicine		0.066 ± 0.003	0.077 ± 0.002	0.087 ± 0.002

^a Cells were treated with different concentrations of compounds for 48 h to obtain the IC₅₀ values. IC₅₀ values were indicated as the mean ± SD of three independent experiments.

Table 2. Antiproliferative activities of compounds 16l–16s and colchicine.

Comp.		IC50 (μM)^a
		MGC-803	HCT-116	SMMC-7721
16l		1.21 ± 0.14	0.88 ± 0.21	1.58 ± 0.28
16m		1.44 ± 0.38	1.57 ± 0.41	1.79 ± 0.32
16n		1.48 ± 0.011	1.66 ± 0.18	1.77 ± 0.15
16o		2.17 ± 0.25	2.41 ± 0. 18	2.21 ± 0.19
16p		5.36 ± 0.42	3.53 ± 0.27	3.77 ± 0.31
16q		6.21 ± 0.38	5.91 ± 0.52	5.68 ± 0.48
16r		6.29 ± 0.38	6.31 ± 0.17	6.69 ± 0.11
16s		2.86 ± 0.014	2.28 ± 0.12	2.87 ± 0.066
Colchicine		0.066 ± 0.003	0.077 ± 0.002	0.087 ± 0.002

^a Cells were treated with different concentrations of compounds for 48 h to obtain the IC₅₀ values.

^b IC₅₀ values are indicated as the mean ± SD of three independent experiments.

Table 3. Antiproliferative activities of compounds 16t–16y and colchicine.

Comp.		IC50 (μM)^a
		MGC-803	HCT-116	SMMC-7721
16t		5.87 ± 0.39b	5.75 ± 0.42	6.82 ± 0.44
16u		7.55 ± 0.36	5.45 ± 0.42	7.12 ± 0.67
16v		6.37 ± 0.54	5.29 ± 0.38	8.16 ± 0.48
16w		5.48 ± 0.28	5.18 ± 0.41	6.11 ± 0.46
16x		5.13 ± 0.27	6.07 ± 0.16	5.12 ± 0.28
16y		5.18 ± 0.14	6.22 ± 0.28	5.01 ± 0.25
Colchicine		0.066 ± 0.003	0.077 ± 0.002	0.087 ± 0.002

^a Cells were treated with different concentrations of compounds for 48 h to obtain the IC₅₀ values. IC₅₀ values are indicated as the mean ± SD of three independent experiments.

Table 4. Antiproliferative activities of compound MY-1121 against six additional cancer cell lines.

Cell types	Cell lines	IC50 (nM)^a
Liver cancer	MHCC-97H	131.9 ± 10.31
	HuH-7	91.6 ± 5.92
	HepG2	100.8 ± 6.09
Gastric cancer	BGC-823	146.1 ± 23.38
	NCI-N87	110.1 ± 4.30
Oesophageal cancer	KYSE450	238.4 ± 14.75
Normal liver cells	HL-7702	3276.2 ± 531.23

^a Cells were treated with different concentrations of compounds for 48 h to obtain the IC₅₀ values. IC₅₀ values are indicated as the mean ± SD of three independent experiments.

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 bind 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 concentration of compound MY-1121. The band signal of β-tubulin was negatively correlated with the ability of Alkaline Protease to hydrolyze 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.