Phenanthrotriazine Derivatives Containing Arylidine Hydrazone Moieties as Novel Potential c-Met Inhibitors with Anticancer Effect

Abstract

Cancer is the second cause of death in the world and the discovery of novel anticancer agents is of vital importance to provide better therapeutic options for cancer patients. In this study, a new series of 12 arylidene hydrazone phenanthrotriazine derivatives were designed, synthesized, and tested in-vitro for antiproliferative activity against three cancer cell lines including colorectal cancer (HT-29), breast cancer (MCF-7) and leukemia (MOLT-4) cells and also against Vero normal cells. The effect of derivatives on cell cycle and apoptosis induction were studied by flow cytometric propidium iodide/RNase assay and Hoechst 33258 staining, respectively, while docking analysis was used to investigate the interactions of synthesized derivatives with the c-Met receptor kinase domain. Some compounds showed considerable antiproliferative activity against tested cancer cells. The most potent derivative was 9k bearing pyrrole moiety with IC₅₀ values of 14.3, 4.7 and 1.7 µM against HT-29, MCF-7 and MOLT-4 cells, respectively, while it showed negligible activity against Vero normal cells (IC₅₀: 95.4 µM). Derivatives bearing 2-nitrophenyl (9g), 4-cyanophenyl (9j), pyrrole (9k), and thiophene (9l) moieties induced G0/G1 cell cycle arrest and also apoptosis at higher doses in MCF-7 cells. Docking study showed that the phenanthrotriazine backbone form H-bond interactions with Asn1209, while phenyl moieties of the pendants generate different hydrophobic interactions with the Asp1164 and Asp1231 residues of c-Met. In conclusion, phenanthrene 1,2,4-triazines, especially the ones with less influence on normal cells, may constitute promising compounds for the discovery of antiproliferative agents with potential c-Met inhibitory capacity.

Keywords: Antiproliferative; Breast cancer; Cell cycle block; Receptor tyrosine kinase inhibitor; Schiff base; Targeted therapy.

Figure 1

Design strategy of target compounds with arylidine hydrazone phenanthrenotriazine structure as anti-cancer agents

Figure 2

Representative cell cycle histograms of MCF-7 cells treated with synthesized derivatives. Analysis of the percentage of cells in different phases of cell cycle was performed using propidium iodide (PI)-RNase flow cytometric assay. MCF-7 cells were seeded in 6-well microplates and treated with synthesized derivatives for 48 h. The cells were then collected and fixed in 70% ethanol overnight at -20 °C. In the end, the cells were stained with DNA staining solution (PI 20 µg/mL and RNase 200 µg/mL) for 30 min and 20,000 events were analyzed by a FACS Calibur flow cytometer (BD Biosciences). Representative histograms of cells treated with compounds 9g, 9j, 9k and 9l are shown

Figure 3

Detection of apoptosis in MCF-7 cells by Hoechst staining. Hoechst 33258 was used as a DNA stain to detect apoptosis in MCF-7 cells treated with synthesized compounds. The cells were seeded in a 6-well plates and treated with different concentrations of test compounds 9g, 9j, 9k and 9l for 72 h. After removing the whole medium, 4% cold freshly prepared paraformaldehyde (PFA) was added for 20 min at room temperature, the cells were then washed twice with PBS, and incubated with Hoechst 33258 2.5 µg/mL for 30 min at room temperature in the dark. Finally, the cells were washed with PBS again and the plates were imaged with a fluorescence microscope

Figure 4

Molecular docking study of the interactions of synthesized derivatives with c-Met receptor active site. Interactions and binding modes of compounds 9k and 9l with the c-Met receptor active site (PDB code: 3ZZE) were studied by molecular docking. 3D Stereo view of the overlaid model (A), 2D docking models and possible interactions of 9k (B) 9l (C) are shown

Scheme 1

Synthesis procedure of phenanthrotriazine derivatives