Synthesis and anti-cancer activities of glycosides and glycoconjugates of diterpenoid isosteviol

Abstract

A series of glycosides and glycoconjugates of diterpenoid isosteviol (16-oxo-ent-beyeran-19-oic acid) with various monosaccharide residues were synthesized and their cytotoxicity against some human cancer and normal cell lines was assayed. Most of the synthesized compounds demonstrated moderate to significant cytotoxicity against human cancer cell lines M-HeLa and MCF-7. Three lead compounds exhibited selective cytotoxic activities against M-HeLa (IC₅₀ = 10.0-15.1 μM) that were three times better than the cytotoxicity of the anti-cancer drug Tamoxifen (IC₅₀ = 28.0 μM). Moreover, the lead compounds were not cytotoxic with respect to the normal human cell line Chang liver (IC₅₀ > 100 μM), whereas Tamoxifen inhibited the viability of normal human Chang liver cells with an IC₅₀ value of 46.0 μM. It was determined that the cytotoxicity of the lead compounds was due to induction of apoptosis proceeding along the mitochondrial pathway. The cytotoxic activity of the synthesized compounds substantially depended on the nature of the monosaccharide residue and its position, that is, whether the monosaccharide residue was attached directly to the isosteviol skeleton or was moved away from it by means of a polymethylene linker.

Scheme 1. Synthesis of isosteviol glycosides. Reagents and conditions: (i) K₂CO₃, TBAB, CH₂Cl₂/H₂O = 10 : 1, 20–48 h, 50 °C; (ii) MeONa, MeOH, 2 h, 20 °C; (iii) Zn powdered, AcOH, Ac₂O, 20 °C, 24 h; (iv) NH₂NH₂·H₂O, MeOH, 20 °C.

Scheme 2. Synthesis of isosteviol glycoconjugates with monosaccharides moved from the diterpenoid skeleton. Reagents and conditions: (i) K₂CO₃, TBAB, CH₂Cl₂/H₂O, 20–48 h, 50 °C; (ii) Zn powdered, AcOH, Ac₂O, 20 °C, 24 h.

Fig. 1. Apoptotic effects of compounds 9, 20 and 22 on M-HeLa cells. M-HeLa cells were treated with the compounds at indicated concentrations for 24 h. Apoptotic effects were measured by flow cytometry using annexin V-FITC staining protocol. The values are presented as the mean ± SD (n = 3).

Fig. 2. Representative histograms for the numbers of cells (% of total) in the early and late stages of apoptosis for the control and treatment groups. The values are presented as the mean ± SD (n = 3); (*) P < 0.01 compared to control.

Fig. 3. Flow cytometry analysis of M-HeLa cells treated with compounds 9, 20 and 22, along with the quantification of % of cells with red aggregates. The values are presented as mean ± SD.