Design, synthesis and biological evaluation of imidazopyridine-propenone conjugates as potent tubulin inhibitors

Abstract

A library of imidazopyridine-propenone conjugates (8a-8u) were synthesized and evaluated for their antitumor activity against four human cancer cell lines, namely, prostate (DU-145), lung (A549), cervical (Hela) and breast (MCF-7) cancer cell lines. These conjugates showed good to moderate activity against the tested cell lines. Among them, two conjugates (8m and 8q) showed significant antiproliferative activity against the human lung cancer cell line (A549) with IC₅₀ values of 0.86 μM and 0.93 μM, respectively. Flow cytometry analysis revealed that these compounds arrested the cell cycle at the G₂/M phase in the human lung cancer cell line (A549), inhibiting tubulin polymerization leading to apoptosis. Further, Hoechst staining, decrease in mitochondrial membrane potential and Annexin V-FITC assay suggested that the cell death was due to apoptosis induction. Overall, the present investigation demonstrated that the synthesized imidazopyridine-propenone conjugates are promising tubulin inhibitors and apoptotic inducers.

Fig. 1. Chemical structures of microtubule targeting agents: colchicine (I), nocodazole (II), imidazopyridine guanylhydrazones (III), imidazopyridine–benzimidazoles (IV), aryl propenones (V) and imidazopyridine–propenone conjugates 8(a–u).

Scheme 1. Synthesis of imidazopyridine–propenones.

Fig. 2. Structure–activity relationship of imidazopyridine–propenone conjugates.

Fig. 3. Flow cytometric analysis of the A549 lung cancer cell line after treatment with compounds 8m and 8q at 0.5 and 1 μM concentrations for 48 h. A: Control cells (A549); B: nocodazole (1 μM); C: 8m (0.5 μM); D: 8m (1 μM); E: 8q (0.5 μM) and F: 8q (1 μM).

Fig. 4. Effect of conjugates on tubulin polymerization: tubulin polymerization was monitored by means of the increasing fluorescence at 360 nm (excitation) and 420 nm (emission) for 1 h at 37 °C.

Fig. 5. Immunohistochemistry analysis of the microtubule network in A549 cells treated with conjugates 8m and 8q at 0.5 μM concentration for 48 h followed by staining with an antitubulin antibody and an FITC conjugated secondary antibody. A: Control (A549); B: nocodazole (0.5 μM); C: 8m (0.5 μM) and D: 8q (0.5 μM).

Fig. 6. Hoechst staining of the A549 lung cancer cell line. A: Control cells (A549); B: nocodazole (0.5 μM); C: 8m (0.5 μM) and D: 8q (0.5 μM). The arrows indicate apoptotic cells.

Fig. 7. Compounds 8m and 8q trigger mitochondrial injury. The drop in membrane potential (ΔΨm) was assessed by JC-1 staining of A549 cells treated with the test compounds, and the samples were then subjected to flow cytometry analysis using a FAC scan (Becton Dickinson) in the FL1 and FL2 channels to determine the mitochondrial potential. A: Control cells (A549); B: nocodazole (1 μM); C: 8m (0.5 μM); D: 8m (1 μM); E: 8q (0.5 μM) and F: 8q (1 μM).

Fig. 8. Annexin V-FITC staining assay. Quadrants: Upper left (necrotic cells), lower left (live cells), lower right (early apoptotic cells) and upper right (late apoptotic cells). A: Control cells (A549); B: nocodazole (1 μM); C: 8m (0.5 μM); D: 8m (1 μM); E: 8q (0.5 μM) and F: 8q (1 μM).

Fig. 9. Binding model of ligands at the interface of the α,β-tubulin heterodimer. Ligands are shown in ball and stick models (yellow colour). Hydrogen bonding interactions are shown as green coloured dashed lines and the residues involved are represented by thin tube models. 9A) Represents the binding mode of 8m; 9B) represents the binding mode of 8q.