Synthesis and Biological Activity Screening of Newly Synthesized Trimethoxyphenyl-Based Analogues as Potential Anticancer Agents

Abstract

A group of novel trimethoxyphenyl (TMP)-based analogues were synthesized by varying the azalactone ring of 2-(3,4-dimethoxyphenyl)-4-(3,4,5-trimethoxybenzylidene)oxazolone 1 and characterized using NMR spectral data as well as elemental microanalyses. All synthesized compounds were screened for their cytotoxic activity utilizing the hepatocellular carcinoma (HepG2) cell line. Compounds 9, 10 and 11 exhibited good cytotoxic potency with IC₅₀ values ranging from 1.38 to 3.21 μM compared to podophyllotoxin (podo) as a reference compound. In addition, compounds 9, 10 and 11 exhibited potent inhibition of β-tubulin polymerization. DNA flow cytometry analysis of compound 9 shows cell cycle disturbance at the G2/M phase and a significant increase in Annexin-V-positive cells compared with the untreated control. Compound 9 was further studied regarding its apoptotic potential in HepG2 cells; it decreased the level of MMP and Bcl-2 as well as boosted the level of p53 and Bax compared with the control HepG2 cells.

Keywords: apoptosis; cell cycle analysis; cytotoxicity; diamide; imidazolone; oxazolone; triazinone; tubulin.

Figure 1

Chemical structure of col I, podo II, Sinensetin III, Imidazole IV, and 1,2,4-triazine V as anti-cancer agents and some reported tubulin polymerization inhibitors, VI and VII.

Scheme 1

Synthesis of the target compounds 2–11. Reagents and conditions: (i) Et₃N, respective alcohol; (ii) respective amino acid, Et₃N, EtOH; (iii) respective aryl amine, NaOAc, AcOH; (iv) phenyl hydrazine, EtOH; (v) isonicotinic acid hydrazide, EtOH; (vi) respective aryl amine, NaOAc, AcOH; (vii) thiosemicarbazide, NaOAc, AcOH; (viii) phenyl hydrazine, EtOH; (ix) isonicotinic acid hydrazide, NaOAc, AcOH; (x) 2-hydrazinyl-4-phenylthiazole, AcOH, DMF.

Figure 2

Compounds 9, 10, and 11 inhibited β-tubulin in HepG2 cells. (A) HepG2 cells were treated with 1.38, 2.52, 3.21, and 2.08 μM of compounds 9, 10, 11, and podo, respectively, and tubulin percentage was determined by ELISA assay. (B) β-Tubulin polymerization inhibition percentage induced by compounds 9, 10, and 11 compared to podo at their IC₅₀ concentration (μM).

Figure 3

Compound 9 induces G2/M phase disturbance in HepG2 cells. (A) HepG2 cells were treated with 1.38 μM of compound 9 for 48 h. Cell apoptosis was quantized by PI and FACS analysis using image-based cytometry. (B) The percentage of cells in different phases was quantized.

Figure 4

Compound 9 induces apoptosis in HepG2 cells. (A) HepG2 cells were treated with 1.38 μM of compound 9 for 48 h. Cell apoptosis was quantized by Annexin V-FITC/PI dual staining assay using image-based cytometry. (B) The quantification of HepG2 cell apoptosis.

Figure 5

Compound 9 promotes MMP dissipation in HepG2 cells. (A) HepG2 cells were treated with 1.38 μM of compound 9 for 48 h. MMP was quantized using image-based flow cytometry. (B) The quantification of MMP in HepG2 cells after treatment with compound 9 at its IC₅₀ concentration (μM).

Figure 6

(A) The quantification of p53. (B) The quantification of Bax. (C) The quantification of Bcl-2. HepG2 cells were treated with 1.38 μM of compound 9 for 48 h and apoptotic markers were determined by ELISA assay.

Figure 7

The binding poses (2D and 3D) of N-phenyl triazinone 9 in tubulin active site (PDB ID: 5LYJ).