Design, synthesis and biological evaluation of novel diosgenin–benzoic acid mustard hybrids with potential anti-proliferative activities in human hepatoma HepG2 cells

Figures & data

Figure 1. Structure and numbering scheme of diosgenin (DSG, 1).

Figure 2. Chemical structures of several examples of natural product and benzoic acid mustard hybrids.

Table 1. Primer sequences for qRT-PCR.

Gene	Sequence	Length (bp)
CDK2	Forward: 5′-TGCCTGATTACAAGCCAAGTTTCCC-3′	98
	Reverse: 5′-TTGCGATAACAAGCTCCGTCCATC-3′
CDK4	Forward: 5′-TTGCCAGCCGAAACGATCAAGG-3′	123
	Reverse: 5′-TCCACCACTTGTCACCAGAATGTTC-3′
CDK6	Forward: 5′-GTGACCAGCAGCGGACAAATAAAAC-3′	86
	Reverse: 5′-ACGACCACTGAGGTTAGAGCCATC-3′
Cyclin D1	Forward: 5′-GCCCTCGGTGTCCTACTTCAAATG-3′	111
	Reverse: 5′-TCCTCCTCGCACTTCTGTTCCTC-3′
Cyclin E1	Forward: 5′-ACACCAGCCACCTCCAGACAC-3′	174
	Reverse: 5′-CGCAACCACCTGCTCCACTTG-3′
Bax	Forward: 5′-AGCTTCTTGGTGGACGCAT-3′	101
	Reverse: 5′-CAGAGGCGGGGTTTCATC-3′
Bcl-2	Forward: 5′-GAGAAATCAAACAGAGGCCG-3′	106
	Reverse: 5′-CTGAGTACCTGAACCGGCA-3′
Caspase 9	Forward: 5′-CATGCTCAGGATGTAAGCCA-3′	93
	Reverse: 5′-AGGTTCTCAGACCGGAAACA-3′
Caspase 3	Forward: 5′-TCGCTTCCATGTATGATCTTTG-3′	110
	Reverse: 5′- CTGCCTCTTCCCCCATTCT-3′

Scheme 1. Synthesis of diosgenin–benzoic acid mustard trihybrids 8−10. Reagents and conditions: (a) ethylene oxide, H₂O, CH₃COOH, rt, 24 h; (b) POCl₃, 50 °C, 0.5 h; (c) 10% HCl, 12 h; (d) Ac₂O, dry pyridine, dry CH₂Cl₂, rt, 6 h; (e) NaBH₃CN, AcOH, CH₂Cl₂, rt, 8 h; (f) KOH, CH₃OH, rt, 6 h; (g) (h) Benzoic acid mustard, EDCI, DMAP, CH₂Cl₂, rt, 24 h.

Scheme 2. Synthesis of diosgenin–benzoic acid mustard hybrids 14a−14f and 15a−15f. Reagents and conditions: (a) Jones reagent, THF/acetone (1/1), rt, 3 h; (b) N-Boc-protected amines, TBTU, DIPEA, CH₂Cl₂, rt, 8 h; (c) CF₃COOH, CH₂Cl₂, rt, 4 h; (d) Benzoic acid mustard, DMAP, EDCI, CH₂Cl₂, rt, 24 h; (e) NaOH, CH₃OH/THF (1/1.5), rt, 6 h.

Table 2. Cytotoxic activities of target hybrids 8– 10, 14a–14f and 15a–15f in different cell lines.

Compound	IC50^a (μM)				SI^b
	HepG2	MCF-7	HeLa	GES-1
8	>50	>50	>50	>100	N.D.
9	–	>50	>50	>100	N.D.
10	>50	28.06 ± 1.52	>50	>100	N.D.
14a	10.19 ± 1.01	48.33 ± 2.21	17.24 ± 2.81	>100	9.81
14b	25.29 ± 1.40	30.30 ± 2.08	>100	46.97 ± 2.34	1.86
14c	12.22 ± 1.09	24.59 ± 1.11	>100	80.79 ± 3.11	6.61
14d	17.47 ± 0.98	>100	>100	46.99 ± 3.24	2.69
14e	19.27 ± 1.29	42.69 ± 2.25	>50	58.76 ± 1.34	3.05
14f	2.26 ± 0.87	11.07 ± 1.01	41.63 ± 3.12	>100	>44.25
15a	13.25 ± 1.01	–	>50	83.17 ± 3.12	6.28
15b	14.00 ± 1.15	37.20 ± 2.23	49.81 ± 2.13	>100	>7.14
15c	14.57 ± 1.16	>100	>50	>100	>6.86
15d	>50	>100	>50	>100	N.D.
15e	13.99 ± 1.12	>50	28.46 ± 2.13	–	N.D.
15f	18.93 ± 0.93	>100	>50	>100	>5.28
4	–	25.58 ± 1.31	>100	>100	N.D.
Diosgenin	33.87 ± 1.37	23.91 ± 1.34	55.05 ± 2.11	>100	>2.95
Mitomycin C	32.63 ± 1.22	16.71 ± 1.02	22.03 ± 1.12	23.43 ± 1.11	0.72

^a IC₅₀: concentrations inhibit 50% of cell growth measured by the MTT assay. The values are expressed as average ± SD of three independent experiments.

“–”: not active.

N.D.: Not determined.

^b SI = IC₅₀ for GES-1 cell line/IC₅₀ for HepG2 cell line.

Figure 3. Analysis of the effects of 14f on the cell cycle in HepG2 cells. HepG2 cells were treated with indicated concentrations (5, 10, and 20 μM) of 14f for 24 h, harvested, stained with PI, and assessed using a flow cytometer.

Figure 4. Effect of 14f on the levels of cell cycle-related genes and proteins in HepG2 cells. GAPDH served as the loading control. (A) Relative mRNA level. (B) The expression of proteins was detected using Western blot. Values have been represented as mean ± SD (n = 3). *p<0.05; **p<0.01; ***p<0.001 versus control group.

Figure 5. Fluorescence microscopy images of HepG2 cells stained using Hoechst 33342/PI. HepG2 cells were treated with different concentrations (5, 10, and 20 μM) of 14f for 48 h.

Figure 6. Flow cytometry analysis of 14f-induced apoptosis in HepG2, as assessed using Annexin V-FITC/PI assay. The percent of apoptotic cells is indicated on the right. Values have been represented as mean ± SD (n = 3). **p<0.01; ***p<0.001 versus control group.

Figure 7. 14f induced the collapse of mitochondrial membrane potential (ΔΨm) in HepG2 cells. After treatment with 14f for 48 h, the cells stained with JC-1 were determined by means of flow cytometric analysis. Values have been represented as mean ± SD (n = 3). **p<0.01; ***p<0.001 versus control group.

Figure 8. 14f activated the mitochondrial pathway, to regulate the apoptosis of HepG2 cells. (A) Relative mRNA level. (B) The activities of caspase 9 and caspase 3 were detected using ELISA. (C) The relative protein expression levels were detected using western blot analyses. (D) Quantitative analysis of the protein expression data mentioned in (C). Values have been represented as mean ± SD (n = 3). **p<0.01; ***p<0.001 versus control group.

Figure 9. (A) Binding mode of ABT-199 to Bcl-2 protein; (B) Binding mode of 14f to Bcl-2 protein.