Synthesis and Biological Evaluation of 4β-N-Acetylamino Substituted Podophyllotoxin Derivatives as Novel Anticancer Agents

Abstract

A series of novel podophyllotoxin derivatives obtained by 4β-N-acetylamino substitution at C-4 position was designed, synthesized, and evaluated for in vitro cytotoxicity against four human cancer cell lines (EC-9706, HeLA, T-24 and H460) and a normal human epidermal cell line (HaCaT). The cytotoxicity test indicated that most of the derivatives displayed potent anticancer activities. In particular, compound 12h showed high activity with IC₅₀ values ranging from 1.2 to 22.8 μM, with much better cytotoxic activity than the control drug etoposide (IC₅₀: 8.4 to 78.2 μM). Compound 12j exhibited a promising cytotoxicity and selectivity profile against T24 and HaCaT cell lines with IC₅₀ values of 2.7 and 49.1 μM, respectively. Compound 12g displayed potent cytotoxicity against HeLA and T24 cells with low activity against HaCaT cells. According to the results of fluorescence-activated cell sorting (FACS) analysis, 12g induced cell cycle arrest in the G2/M phase accompanied by apoptosis in T24 and HeLA cells. Furthermore, the docking studies showed possible interactions between human DNA topoisomerase IIα and 12g. These results suggest that 12g merits further optimization and development as a new podophyllotoxin-derived lead compound.

Keywords: C-4 substitutions; anticancer agent; biological evaluation; podophyllotoxin derivatives; structure–activity relationships (SAR).

Figure 1

Structures of PPT, DPPT, and related compounds.

Figure 2

Rational design of the target compounds.

Graphical Abstract

The diagrammatic sketch of our study on podophyllotoxin derivatives.

Scheme 1

Synthesis of 4β-N-acetylamino substituted PTT and DPPT derivatives 12a-t.

Figure 3

Fluorescent images of Hoechst 33258 staining showing compound 12g induced cell death with 0.1% DMSO vehicle as the control. Cells were examined under a fluorescence microscope. The arrows indicated the formation of apoptotic bodies with condensed chromatin or fragmented chromatin. (A) Treatment of T24 cells with 12g for 24 h. (B) Treatment of HeLA cells with 12g for 24 h. The apoptosis rates of T24 and HeLA cells with treatment of 12g were quantify and analyzed by One-way ANOVA (n = 3 in each treatment). ^*P< 0.05, ^***P < 0.001, and ^****P < 0.0001 compared with the vehicle (0.1% DMSO). The statistical data are presented as mean ± S.D. Scale bar = 50 μm.

Figure 4

Compound 12g affected the cancer cell cycle distribution at three different gradient concentrations with 0.1% DMSO vehicle as the control. (A) T24 cells treated with 12g for 24 h. (B) HeLA cells treated with 12g for 24 h.

Figure 5

Human DNA topoisomerase IIα active site in complex with its ligand AMP-PNP or synthesized compound 12g. (A) X-ray crystal structure of human DNA topoisomerase IIα active site in complex with AMP-PNP. (B) The 3D chemical structure of compound 12g. (C) The surface structure of human DNA topoisomerase IIα binding pocket in complex with 12g.

Figure 6

Docking poses for compound 12g in the active site of human DNA topoisomerase IIα. (A) X-ray crystal structure of human DNA topoisomerase IIα active site in complex with 12g (green). (B) Inhibitor 12g was bound to the hydrophobic groove of DNA topoisomerase IIα. (C,D) View of compound 12g (green) docked in the binding residues of human topoisomerase IIα from different perspectives.