A Pyrroloquinoline-Based Colchicine-Binding Site Inhibitor Drives Mitotic Arrest and Caspase-Dependent Apoptosis

Abstract Targeting the colchicine-binding site on tubulin represents a promising strategy for developing anticancer agents capable of overcoming drug resistance and alleviating limitations of current microtubule-targeting therapies. Building on the pharmacological potential of quinoline scaffolds, we designed pyrroloquinoline-based compounds aimed at targeting the colchicine-binding site on tubulin. A hypothesis-driven design strategy, guided by structure- and ligand-based molecular modeling, was employed to prioritize the designed molecules for strong predicted binding and pharmacophoric complementarity within the colchicine-binding site. A concise, efficient synthetic strategy was employed to access the target molecules, which were subsequently evaluated for antiproliferative activity across various cancer cell lines. The hit compounds reported in this paper, 4a and 5b, exhibited potent cytotoxicity, competitively inhibited the formation of the EBI−β-tubulin adduct, and induced pronounced disruption of the intracellular microtubule network. Compound 4a, in particular, consistently displayed significant biological activity, as mechanistic studies revealed that it triggers sustained G2/M phase arrest, suppresses proliferative signaling, and activates DNA damage–associated stress responses, followed by caspase-3 activation and PARP cleavage, indicative of caspase-dependent apoptosis. These results reinforce the utility of the quinoline scaffold for the development of colchicine-site microtubule inhibitors and highlight their potential in advancing new chemotherapeutic candidates.