A series of novel pyridyl acridone derivatives comprised of a pseudo-five-cyclic system to extend the π-conjugated acridone chromophore, were designed and synthesized as potent DNA binding antitumor compounds. Most synthesized compounds displayed good activity against human leukemia K562 cells in MTT tests, with compound 6d exhibiting the highest activity with IC50 value at 0.46 μM. Moreover, 6d showed potent activities against solid tumor cell lines (0.16-3.79 μM). Several experimental studies demonstrated that the antitumor mode of action of compound 6d involves DNA intercalation, topoisomerase I inhibition, and apoptosis induction through the mitochondrial pathway. In summary, compound 6d represents a novel and promising lead structure for the development of new potent anticancer DNA-binding agents.