Design and Synthesis of Potent in Vitro and in Vivo Anticancer Agents Based on 1-(3',4',5'-Trimethoxyphenyl)-2-Aryl-1H-Imidazole

Abstract

A novel series of tubulin polymerization inhibitors, based on the 1-(3',4',5'-trimethoxyphenyl)-2-aryl-1H-imidazole scaffold and designed as cis-restricted combretastatin A-4 analogues, was synthesized with the goal of evaluating the effects of various patterns of substitution on the phenyl at the 2-position of the imidazole ring on biological activity. A chloro and ethoxy group at the meta- and para-positions, respectively, produced the most active compound in the series (4o), with IC50 values of 0.4-3.8 nM against a panel of seven cancer cell lines. Except in HL-60 cells, 4o had greater antiproliferative than CA-4, indicating that the 3'-chloro-4'-ethoxyphenyl moiety was a good surrogate for the CA-4 B-ring. Experiments carried out in a mouse syngenic model demonstrated high antitumor activity of 4o, which significantly reduced the tumor mass at a dose thirty times lower than that required for CA-4P, which was used as a reference compound. Altogether, our findings suggest that 4o is a promising anticancer drug candidate that warrants further preclinical evaluation.

Figure 1. Lead structures of tubulin polymerization inhibitors.

Figure 2. General synthetic procedure followed for the preparation of compounds 4a–q.

Reagents. (a) 1-bromo-3,4,5-trimethoxybenzene, Cs₂CO₃, CuI, DMF, 120 °C, 40 h; (b) NBS, CH₃CN, rx; (c) PdCl₂(DPPF), ArB(OH)₂, CsF, 1,4-dioxane, 65 °C.

Figure 3. Proposed binding for compound 4k (in grey) in the colchicine site.

Co-crystallized DAMA-colchicine is shown in green. The hydrophobic subpocket is highlighted with a red surface.

Figure 4

Percentage of cells in each phase of the cell cycle in HeLa (Panel A) and Jurkat cells (Panel B) treated with compound 4o at the indicated concentrations for 24 h. Cells were fixed and labeled with PI and analyzed by flow cytometry as described in the Experimental Section. Data are represented as mean ± SEM of three independent experiments. Representative histograms of mitotic cells with phosphorylated histone-H3 (Panel C) and quantitative analysis (Panel D) after treatment with 4o at the indicated concentrations in HeLa cells. Data are represented as mean ± SEM of two independent experiments.

Figure 5

Effect of 4o on cell cycle and spindle assembly checkpoint proteins (Panel A), and expression of p-H2A.X^Ser139 (Panel B). HeLa cells were treated for 24 or 48 h with the indicated concentrations of 4o. The cells were harvested and lysed for detection of the expression of the indicated protein by western blot analysis. To confirm equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.

Figure 6

Flow cytometric analysis of apoptotic cells after treatment of HeLa (Panels A,B) and Jurkat (Panels C,D) cells with 4o at the indicated concentrations after incubation for 24 (Panels A,C) or 48 h (Panels B,D). The cells were harvested and labeled with annexin-V-FITC and PI and analyzed by flow cytometry.

Figure 7

Assessment of mitochondrial membrane potential (Δψ_mt) and production of ROS after treatment of HeLa (Panels A,B) or Jurkat (Panels C,D) cells with compound 4o. Cells were treated with the indicated concentration of compound for 24 or 48 h and then stained with the fluorescent probe JC-1 for analysis of mitochondrial potential or H₂-DCFDA for the evaluation of ROS levels. Cells were then analyzed by flow cytometry as described in the experimental section.

Figure 8. Western blot analysis of caspase-3, PARP, Bcl-2 and Mcl-1 after treatment of HeLa cells with 4o at the indicated concentrations and for the indicated times.

To confirm equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.

Figure 9. Inhibition of mouse allograft growth in vivo by compound 4o.

Male C57BL/6 mice were injected subcutaneously at their dorsal region with 10⁷ BL6-B16 murine melanoma cells. Tumor-bearing mice were administered the vehicle, as control, or 1, 3 or 7.5 mg/kg of 4o or CA-4P as reference compound at the dose of 30 mg/kg. Injections were given intraperitoneally at the days indicated by the arrows. Data are presented as mean ± SEM of tumor volume at each time point for 5 animals per group. *p < 0.05, **p < 0.01 ***p < 0.001 vs. control.