Synthesis and Anticancer Activity Evaluation of Novel Phenanthridine Derivatives

Abstract

Based on the structure of sanguinarine, fourteen phenanthridine derivatives were designed and synthesized in the current study. The cytotoxic activities of synthesized compounds were evaluated against five human cancer cell lines (MCF-7, PC3, Hela, A549, and HepG2 cell lines) via MTT assay. Among all the compounds tested, molecule 8a exhibited significant cytotoxic activity against MCF-7 cells with a IC₅₀ value of 0.28 μM. A following up enzymatic assay indicated that compound 8a could inhibit the activity of DNA topoisomerase I/II. Further mechanistic studies performed in the MCF-7 cell line revealed that compound 8a could arrest cell cycle in S phase and induce cell apoptosis via downregulation of Bcl-2 and upregulation of Bax. Collectively, a potent DNA topoisomerase inhibitor (8a) was discovered, which exhibited potential as a candidate chemotherapeutic agent for the management of tumors in the present study.

Keywords: anticancer; apoptosis; cell cycle arrest; phenanthridine; topoisomerase.

Figure 1

The structures of SA and phenanthridine.

Scheme 1

Synthesis of SA derivatives 8a-n: (i) IPA, Boc₂O, ice-bath; (ii) ACN, NBS; (iii) DCM, TFA; (iv) [Pd], K₂CO₃, DME, 80°C; (v) HCOOH, THF, 60°C; (vi) POCl₃, NEt₃, THF, 0°C; (vii) BPO, AcONa, reflux.

Figure 2

Effects of phenanthridine derivatives and positive control on human Top I (A)/IIa (B). Native superhelix pBR322 was incubated at 37°C for 30 min with 2 units of human Top I/IIα in the absence (lane 2) or presence of compound at concentration 100 μM. One hundred micromolar OPT, VP 16, and SA were used as positive controls, respectively. Negatively supercoiled pBR322 (SC) and relaxed DNA (RLX) were shown. DNA samples were run on agarose gel followed by Genecolour I TM staining.

Figure 3

Cell cycle analysis using PI staining of compound 8a on MCF-7 cells. Cells were treated with compound 8a at 0.15 (B), 0.3 (C), and 0.6 (D) μM for 24 h, compared with the control (A). Cell cycle were detected by flow cytometry.

Figure 4

Pro-apoptotic effect of compound 8a on MCF-7 cells. (A) Apoptotic assay by Hoechst 33258. MCF-7 cells were treated with compound 8a at 0.15, 0.3, and 0.6 μM for 24 h, and then cells were stained with Hoechst 33258 and visualized under a fluorescent microscope. (B) Apoptotic assay by flow cytometry. MCF-7 cells were treated with compound 8a at 0.15, 0.3, and 0.6 μM for 24 h. Then cells were stained with Annexin V-FITC/PI and were detected by flow cytometry analysis.

Figure 5

Effects of 8a on the expressions of Bcl-2 (A), Bax (B) and the expression ratio (C) in MCF-7 cells. The cells were treated with different concentrations (0.15, 0.3, and 0.6 μM) for 24 h; β-actin served as an internal control. All date were represented as mean ± SD (n = 3). **p < 0.01, compared with control group.