Synthesis and anticancer properties of celastrol derivatives involved in the inhibition of VEGF

Figures & data

Scheme 1. Synthesis route of Celastrol derivatives 1–14

Scheme 2. Diagram of Structure–activity relationship

Table 1. The solubility of celastrol and compound 2.

Compound	Water solubility (µg/ml)
Celastrol	6.2
2	11.8

Table 2. Antiproliferative efficacy of celastrol derivatives 1–14 in six human cancer cell lines.

Compds.	IC50 values (µM)
	BEL-7402	AGS	HCT-116	HepG-2	MGC-803	A549
1	0.68 ± 0.09	0.47 ± 0.14	0.47 ± 0.02	0.62 ± 0.07	0.37 ± 0.05	3.19 ± 0.66
2	0.58 ± 0.07	0.32 ± 0.06	0.32 ± 0.04	0.75 ± 0.10	0.29 ± 0.04	4.30 ± 0.20
3	0.63 ± 0.11	0.35 ± 0.08	0.39 ± 0.09	0.63 ± 0.13	0.27 ± 0.06	3.06 ± 0.39
4	0.59 ± 0.11	0.43 ± 0.09	0.48 ± 0.07	10.10 ± 0.43	0.32 ± 0.03	>100
5	1.22 ± 0.49	0.36 ± 0.09	0.53 ± 0.06	10.73 ± 0.51	0.39 ± 0.03	>100
6	0.74 ± 0.11	0.41 ± 0.04	0.93 ± 0.12	7.21 ± 1.06	0.40 ± 0.04	>100
7	1.16 ± 0.23	0.42 ± 0.04	0.58 ± 0.07	3.30 ± 0.59	0.38 ± 0.03	4.13 ± 0.56
8	0.72 ± 0.10	0.36 ± 0.08	0.52 ± 0.09	1.32 ± 0.18	0.40 ± 0.05	4.95 ± 0.38
9	0.59 ± 0.13	0.43 ± 0.08	0.47 ± 0.11	2.55 ± 0.45	0.41 ± 0.05	3.19 ± 0.12
10	0.81 ± 0.07	0.39 ± 0.05	0.49 ± 0.08	0.94 ± 0.22	0.40 ± 0.01	3.22 ± 0.32
11	1.33 ± 0.13	1.36 ± 0.29	1.34 ± 0.29	3.04 ± 0.77	0.46 ± 0.08	5.39 ± 0.68
12	5.42 ± 0.68	2.10 ± 0.73	2.75 ± 0.23	>100	1.09 ± 0.24	>100
13	0.77 ± 0.14	0.32 ± 0.05	0.57 ± 0.03	1.76 ± 0.32	0.34 ± 0.06	7.83 ± 1.30
14	0.83 ± 0.05	0.37 ± 0.06	0.64 ± 0.04	1.27 ± 0.16	0.39 ± 0.09	3.65 ± 0.14
celastrol	0.79 ± 0.08	0.39 ± 0.12	0.43 ± 0.14	1.23 ± 0.13	0.35 ± 0.09	5.34 ± 0.39

Table 3. In vitro antiproliferative activities of celastrol derivatives 1–14 against normal cell line (LO2) and selectivity index.

Compounds	IC50 for LO2 (µM)^a	Selectivity index^b
		BEL-7402	AGS	HCT-116	HepG-2	MGC-803	A549
1	0.46 ± 0.05	0.68	0.98	0.98	0.74	1.24	0.14
2	0.39 ± 0.03	0.67	1.22	1.22	0.52	1.34	0.09
3	0.43 ± 0.04	0.68	1.23	1.10	0.68	1.59	0.14
4	1.28 ± 0.30	2.17	2.98	2.67	0.13	4.00	<0.01
5	0.48 ± 0.07	0.39	1.33	0.91	0.04	1.23	<0.00
6	0.71 ± 0.09	0.96	1.73	0.76	0.10	1.78	<0.01
7	0.73 ± 0.17	0.63	1.74	1.26	0.22	1.92	0.18
8	0.61 ± 0.09	0.85	1.69	1.17	0.46	1.53	0.12
9	0.67 ± 0.07	1.14	1.56	1.43	0.26	1.63	0.21
10	0.64 ± 0.09	0.79	1.64	1.31	0.68	1.60	0.20
11	1.63 ± 0.24	1.23	1.20	1.22	0.54	3.54	0.30
12	4.56 ± 0.79	0.84	2.17	1.65	<0.05	4.18	<0.05
13	0.38 ± 0.04	0.49	1.19	0.67	0.22	1.12	0.05
14	0.71 ± 0.11	0.86	1.92	1.11	0.56	1.82	0.19
celastrol	0.47 ± 0.19	0.59	1.21	1.09	0.38	1.34	0.09

^aIC₅₀: concentration that inhibits 50%.

^bSI: selective index (IC₅₀ against normal cells/IC₅₀ against tumour cells).

Figure 1. Cell cycle arrestation in MGC-803 cells after treatment for 18 h with 0.1% DMSO (vehicle), 2 (0.1 µM, 0.5 µM, 1 µM), and Celastrol(0.1 µM, 0.5 µM, 1 µM), respectively.

Figure 2. Apoptosis induction in MGC-803 cells after treatment for 18 h with 0.1% DMSO (vehicle), 2(0.1 µM, 0.5 µM, 1 µM), Celastrol (0.1 µM, 0.5 µM, 1 µM), respectively.

Figure 3. Compound 2 and celastrol inhibited the expression quantity of VEGF in MGC-803 cells. (A) The effects of compound 2, and Celastrol (0.1 µM, 0.5 µM, 1 µM) on the secretion of VEGF in Hypoxia-induced MGC-803. (B) The effects of compound 2, and Celastrol (0.1 µM, 0.5 µM, 1 µM) on the secretion of VEGF in MGC-803. Data are presented as ng.ml⁻¹ and were analysed using one-way ANOVA with Graphpad Prism 7. **p < 0.01, ***p < 0.001 when compared to control group (1%DMSO) (n ≥ 3).

Figure 4. The effects of compound 2 (0.1 µM, 0.5 µM, 1 µM) and celastrol (0.1 µM, 0.5 µM, 1 µM) on the secretion of of MMP-9 in MGC-803. Data are presented as ng ml⁻¹ and were analysed using one-way ANOVA with Graphpad Prism 7. **p < 0.01, ***p < 0.001 when compared to control group (0.1%DMSO) (n ≥ 3).

Figure 5. The effects of compound 2 (1 mg/kg, 2.5 mg/kg) and 5-FU (10 mg/kg) on the tumour volume of mice. *p < 0.05, and **p < 0.01 compared with Control group.

Figure 6. The effects of compound 2 (1 mg/kg, 2.5 mg/kg) and 5-FU (10 mg/kg) on the tumour weight and body weight of mice. (A) Compound 2 and 5-FU inhibited the growth of tumours in mice. ***p < 0.001, and ****p < 0.0001 compared with the control group, ^##p < 0.01 when comparing the 5-FU group and high dose group of compound 2; (B) Body weight changes of mice during administration.

Figure 7. The effects of compound 2 (1 mg/kg, 2.5 mg/kg) and 5-FU (10 mg/kg) on the TNF- α content of tumour mice. *p < 0.05, and **p < 0.01 compared with the control group.

Figure 8. Histological examination of tumour in compound 2 (1 mg/kg, 2.5 mg/kg) and 5-FU (10 mg/kg) treated mice by H&E staining. Original magnification, 400×.

Figure 9. The effects of compound 2 (1 mg/kg, 2.5 mg/kg) and 5-FU (10 mg/kg) on the VEGF expression in tumour mice. (A) Immunohistochemical and dyeing pictures in each group; (B) Quantitative analysis of VEGF expression in each group. Original magnification, 400×.

Figure 10. The effects of compound 2 (1 mg/kg, 2.5 mg/kg) and 5-FU (10 mg/kg) on the VEGF expression in Western blotting analysis. Data are represented as the mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01when compared to the control.

Figure 11. The death rate of zebrafish embryos treated with celastrol (A) and compound 2 (B).

Figure 12. Hatching rate of zebrafish embryos treated with celastrol (A) and compound 2 (B).

Figure 13. Death rate of adult zebrafish treated with celastrol (abbreviated as C) and compound 2 (abbreviated as T) analysed at 24 and 72 hpf.