A pyridone-fused imidazo[1,2-a]pyridine hybrid binds to tubulin at the vinblastine site, depolymerizes microtubules and inhibits Cancer cell proliferation.

Tubulin remains a key target in anticancer therapy, motivating the development of structurally novel small-molecule inhibitors. The vinblastine-binding site on tubulin is a well-established and druggable pocket that accommodates structurally diverse ligands, including small-molecule inhibitors. Imidazo[1,2-a]pyridine represents an important scaffold in medicinal chemistry, particularly in anticancer research. CH activation on this scaffold provides an efficient approach to access structurally diverse and bioactive fused heterocycles. Herein, we report the regioselective Ru (II)-catalyzed [4 + 2] C-H/alkyne annulation of imidazo [1,2-a]pyridine-3-methoxyamides, enabling efficient construction of pyridone-fused imidazo[1,2-a]pyridine frameworks which are structurally reminiscent of carbazole-based anticancer motifs. A library of thirty novel compounds of previously inaccessible species was synthesized and evaluated for anticancer activity. Several derivatives exhibited significant antiproliferative effects, with C-18 and C-28 identified as the most active against HeLa and MDA-MB-231 cell lines. Mechanistic studies demonstrated that C-18 inhibits tubulin polymerization in vitro, disrupts microtubule assembly in cells, and induces mitotic arrest. Molecular docking studies indicated that C-18 binds at the vinblastine site on tubulin, consistent with its mechanism of action. Furthermore, ADMET analysis suggested that most of the compounds possess favorable drug-like properties with acceptable pharmacokinetic profiles. SIGNIFICANCE STATEMENT: We report an efficient, oxidizing directing group-assisted CH activation strategy that overcomes severe synthetic bottlenecks to access novel pyridone-fused imidazo[1,2-a]pyridines. With the help of cell-free and phenotypic assays, we validate C-18 as a rare small molecule that targets the tubulin vinblastine site via competitive displacement. Validated structure and mode of formation by single-crystal X-ray data and DFT studies, C-18 drives G2/M mitotic arrest and successfully suppresses aggressive MDA-MB-231 breast cancer cell migration, offering a distinct chemical blueprint for microtubule-targeted oncology drug discovery.