Synthesis and evaluation of anticancer activity of quillaic acid derivatives: A cell cycle arrest and apoptosis inducer through NF- κ B and MAPK pathways

Abstract

A series of quillaic acid derivatives with different substituents on the 28-carboxyl group were designed and synthesized. Five human cancer cell lines (HCT116, BEL7402, HepG2, SW620, and MCF-7) were evaluated for their antitumor activity in vitro. Some of the tested derivatives showed improved antiproliferative activity compared to the lead compound, quillaic acid. Among them, compound E (IC₅₀ = 2.46 ± 0.44 μM) showed the strongest antiproliferative activity against HCT116 cells; compared with quillaic acid (IC₅₀ > 10 μM), its efficacy against HCT116 cancer cells was approximately 4-fold higher than that of quillaic acid. Compound E also induces cell cycle arrest and apoptosis by modulating NF-κB and MAPK pathways. Therefore, the development of compound E is certainly valuable for anti-tumor applications.

Keywords: Western blot; antitumor; apoptosis; cell-cycle arrest; quillaic acid.

FIGURE 1

(A) Structures of quillaic acid. (B) Structures of natural-product derivatives containing phenyl-1,2,3-triazole; (C) Structures of compounds containing phenyl-1,3,4-oxadiazole; (D) Structures of compounds containing 1,2,4-triazol-3-one.

FIGURE 2

Designing quillaic-acid derivatives containing a nitrogen-containing heterocycle to achieve potential anti-proliferative activity.

SCHEME 1

Reagents and conditions: (A) (i) HCl, NaNO₂, H₂O, 0–5°C, 30 min; (ii) NaN₃, H₂O, 0–5°C, 2–4 h; (B) propynol, Na ascorbate, CuSO₄•5H₂O, H₂O, t-BuOH/H₂O, 25°C, overnight; (C) thionyl chloride, CH₂Cl₂, r. t., overnight; (D) NH₂-NH₂•H₂O, EtOH, reflux, overnight; (E) chloroacetic acid, POCl₃, reflux, 4 h; (F) (I) methyl hydrazinocarboxylate, triethyl orthoformate, EtOH, reflux, 48 h; (ii) NaOCH₃, EtOH, reflux, 48 h; (G) 1,2-dibromoethane, DMF, K₂CO₃, 60°C, 4–6 h; (H) NH₂-OH•HCl, K₂CO₃, CH₃OH:H₂O = 1:1, r. t., overnight; (I) NaClO, CH₂Cl₂, H₂O, 0°C, 8 h.

SCHEME 2

Reagents and conditions: (A) DMF, K₂CO₃, 60°C, 4–6 h; (B) 1,2-dibromoethane, DMF, K₂CO₃, KI, 60°C, 4–6 h; (C) 5-phenyl-1H-tetrazole, DMF, K₂CO₃, KI, 60°C, 8 h.

FIGURE 3

Colony formation of HCT116 cells inhibited by compound E. (A) HCT116 cells were incubated with varying concentrations of E (0, 2.5, 5, 10 μM) and stained with crystal violet. (B) The micrographic differences between the colonies. Images were taken of stained single colonies observed under a microscope.

FIGURE 4

Compound E induced cell cycle arrest at G1 phase in HCT116.

FIGURE 5

Cell morphological alterations and nuclear changes associated with HCT116 cells after incubation with varying concentrations E (0, 2.5, 5, 10 μM) for 24 h were assessed by staining with H&E and visualized by microscopy.

FIGURE 6

Compound E induced cell apoptosis in a concentration-dependent manner.

FIGURE 7

Effect of compound E on the levels of Bax, and Bcl-2 in HCT116 cells. Cells were pretreated with the various concentrations of compound E (2.5, 5 and 10 μM) for 24 h. The expression of Bax, and Bcl-2 were analyzed by Western blot. ***p < 0.001, **p < 0.01, *p < 0.05 vs. control group. The error bars represent the mean ± SD for three independent experiments.

FIGURE 8

Effect of compound E on NF-κB signaling pathway in HCT116 cells. Cells were pretreated with the various concentrations of compound E (2.5, 5 and 10 μM) for 24 h. The expression of phospho-NF-κB p65, NF-κB p65, phospho-IκB, IκB were analyzed by Western blot. ***p < 0.001, **p < 0.01, *p < 0.05 vs. control group. The error bars represent the mean ± SD for three independent experiments.

FIGURE 9

Effect of compound E on NF-κB signaling pathway in HCT116 cells. Cells were pretreated with the various concentrations of compound E (2.5, 5 and 10 μM) for 24 h. The expression of phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38 were analyzed by Western blot. ***p < 0.001, **p < 0.01, *p < 0.05 vs. control group. The error bars represent the mean ± SD for three independent experiments.