Synthesis of MeON-Glycoside Derivatives of Oleanolic Acid by Neoglycosylation and Evaluation of Their Cytotoxicity Against Selected Cancer Cell Lines

Abstract

A series of C-3 and C-28 MeON-neoglycosides of oleanolic acid were designed and synthesized by neoglycosylation as potential antiproliferative agents. Their cytotoxicity was evaluated in vitro against five human cancer cell lines: human non-small cell lung cancer cell line (A549), human melanoma cell line (A375), human colon cancer cell line (HCT116), human liver carcinoma cell line (HepG2), human breast adenocarcinoma cell line (MCF-7) by the Cell Counting Kit-8 (CCK-8) assay. Most of C-3 and C-28 MeON-neoglycosides of oleanolic acid exhibited notably inhibitory effects against the tested cancer cells and more sensitive to HepG2 cells than 5-Fluorouracil (5-FU). Structure-activities relationship (SAR) analysis revealed that sugar types and the d/l configuration of sugars would significantly affect their antiproliferative activities of neoglycosides. Among them, compound 8a (28-N-methoxyaminooleanane-β-d-glucoside) exhibited the most potent antiproliferative activities against HepG2 cells with IC₅₀ values of 2.1 µM. Further pharmacological experiments revealed that compound 8a could cause morphological changes and cell cycle arrest at G0/G1 phase and induce apoptosis in HepG2 cells. These results suggested that neoglycosylation could provide a rapid strategy for the discovery of potential antiproliferative agents and their possible pharmacological mechanisms need more further research.

Keywords: anticancer; antiproliferation; apoptosis; neoglycosides; oleanolic acid.

Scheme 1

Synthesis of oleanolic acid C-3-neoaglycone 3 and neoglycosides 4a–4r. Reagents and conditions: (a) ClCH₂COCl, DIPEA, DMAP, THF, rt, 2 h; (b) NaI, EtOH, 60 °C, MeONH₂ in THF, every 2 h; (c) reducing sugars, MeOH: CHCl₃ (6:1), 40 °C, 48 h.

Scheme 2

Synthesis of oleanolic acid C-28-neoaglycone 7 and neoglycosides 8a–8r. Reagents and conditions: (a) MeON(H)Me, NMM, EDAC, 0 °C, 2 h; (b) LAH, THF; CH₃ONH₂·HCl, Pyridine, MeOH: CH₂Cl₂ (4:1), reflux, 55 °C, 8 h; (c) NaCNBH₃, AcOH, rt, 10 h; (d) reducing sugars, MeOH: CHCl₃ (4:1), AcOH, 40 °C, 48 h.

Scheme 3

Biotransformation of oleanolic acid, erythrodiol by Bacillus subtilis ATCC 6633.

Figure 1

HepG2 cells morphological changes (a) and Hoechst 33342 staining (b) after treated with compound 8a (1, 5, 10 µM) for 24 h. Scale bar, 50 μm.

Figure 2

Compound 8a induces G0/G1 arrest in HepG2 cells. (a). Flow cytometric plots show the cell cycle distribution when the cells were treated with 8a (1, 5, 10 µM) for 24 h; (b). Bar graphs show the cell cycle distribution when the cells were treated with compound 8a; ** p < 0.01, *** p < 0.001 vs. the control group. All data are expressed as mean ± SD of the three independent experiments.

Figure 3

Compound 8a induces apoptosis in HepG2 cells. (a). Flow cytometric plots show cells in the different stages when the cells were treated with compound 8a (1, 5, 10 µM) for 24 h; (b). Bar graphs show the percentage of apoptosis cell populations when the cells were treated with 8a; *** p < 0.001 vs. the control group. All data are expressed as mean ± SD of the three independent experiments.