Synthesis and In Vitro Bioactivity of Symmetrical Enediynes Based on Maleimide and Propargylamides

Abstract Natural enediynes, characterized by the (Z)‐1,5‐diyn‐3‐ene core, are among the most potent antitumor agents, functioning through DNA‐damaging diradicals generated via Bergman or Myers–Saito cyclization. Despite their promising therapeutic potential, their clinical application is limited due to their scarcity. To address this limitation, we developed a new class of symmetrical maleimide‐based acyclic enediynes featuring propargylamide moieties, which exhibit excellent thermal reactivity through a maleimide‐assisted rearrangement and cycloaromatization mechanism. These compounds demonstrated high efficiency in generating biradicals at physiological temperatures, as confirmed by electron paramagnetic resonance (EPR) analysis. Additionally, in vitro assays revealed their potent DNA cleavage ability and significant cytotoxicity against tumor cells, with half‐maximal cytotoxic concentrations (CC 50 ) as low as 1.98 µM. Confocal laser scanning microscopy indicated substantial cellular uptake and DNA damage in HeLa cells. Our findings underscore the potential of these novel enediynes in targeted cancer therapy, positioning them as candidates for both traditional chemotherapy and antibody‐drug conjugate (ADC) payloads.