Synthesis and Antiproliferative Effect of Halogenated Coumarin Derivatives

Abstract

A series of 6- and 6,8-halocoumarin derivatives have been investigated as potential antiproliferative compounds against a panel of tumor and normal cell lines. Cytotoxic effects were determined by the MTT method. To investigate the potential molecular mechanism involved in the cytotoxic effect, apoptosis assay, cell cycle analysis, reactive oxygen species (ROS), and reduced glutathione analysis were performed. Among the screened compounds, coumarins 6,8-dibromo-2-oxo-2H-chromene-3-carbonitrile 2h and 6,8-diiodo-2-oxo-2H-chromene-3-carbonitrile 2k exhibited the most antiproliferative effect in thyroid cancer-derived cells TPC-1. The apoptosis assay showed that both 2h and 2k induced apoptosis in TPC-1 thyroid cancer cells. According to these experiments, both coumarins induced a slight increase in TPC-1 cells in the G2/M phase and a decrease in the S phase. A significant increase in ROS levels was observed in TPC-1 treated with diiodocoumarin 2k, while the dibromocoumarin 2h induced a decrease in ROS in a dose and time-dependent manner.

Keywords: ROS; TPC-1 cells; antiproliferative activity; apoptosis; coumarins.

Scheme 1

Reagents (i) diethyl malonate, piperidine, AcOH, EtOH, 50 °C, 16 h, yield 73–90%; (ii) malonitrile, aq sodium carbonate, EtOH, r.t., 24, then 37% aq HCl, 80 °C, 6 h, yield 68–87%; (iii) ethyl 2-(methylsulfonyl) acetate piperidine, AcOH, EtOH, 50 °C, 16 h, yield 75%; (iv) LiOH, H₂O/MeOH (1:5 v/v), yield 81%.

Figure 1

Detection of apoptosis by flow cytometry. (A): Flow cytometry histograms of Annexin V-FITC/PI staining in TPC-1cells treated with 20 μM solutions of 2k and 2h. (B): Flow cytometry histograms of Annexin V-FITC/PI staining in control cells treated with 20 μM solutions of 2k and 2h. The results shown represent the mean ± SD of three independent experiments. Significant differences between untreated vs. each treated cells are indicated by * p < 0.05.

Figure 2

Flow cytometric analysis of coumarins 2k, 2h effects on TPC-1 and Nthy-ori-3-5 cell cycle. (A): representative profiles of the cell cycle and percentage of cells in various phases of the TPC-1 cell cycle. (B): representative profiles of the cell cycle and percentage of cells in various phases of the control cell cycle. Data represent the mean ± SD of three experiments. Statistical analysis was performed by Student t-test. ** p < 0.01.

Figure 3

Effects of coumarins 2k and 2h on intracellular DCF (ROS). A and B: Graphs illustrate DCF fluorescence (ROS) levels (%) in thyroid cancer cell line TPC-1 (A) and thyroid normal cells Nthy-ori-1-3 (B) compared with each group of control (untreated) cells. Data represent the mean ± SD of three independent experiments. Statistical analysis was performed by Student t-test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Effects of coumarins 2k and 2h on intracellular levels of GSH/GSSG ratio. (A,B): Graphs illustrate GSH/GSSG levels (%) in thyroid cancer cell line TPC-1 (A) and thyroid normal cells Nthy-ori-1-3 (B) compared with each group of control (untreated) cells. Peak areas of intracellular aminothiols were normalized using protein contents (ng of aminothiols per μg of proteins) and expressed as the percentage of the ratio of reduced and oxidized forms of the untreated cells (control, 100%). Data are expressed as mean ± SD. All experiments were performed three times independently, each time in triplicate to confirm the results. Statistical analysis was performed by Student t-test. ** = p < 0.01 and *** = p < 0.001, respectively, for 2k and 2h vs. control. # = p < 0.05 and ### = p < 0.001 2k vs. 2h.