Abstract

A series of new dihydropyrano[2,3- b ]indoles were designed, synthesized, and meticulously evaluated for their antiproliferative activities against multiple human tumor cell lines, including A549, MCF-7, DU145, HeLa, and HepG2 cells. Among these derivatives, compound 4k exhibited the most potent antitumor activity against non-small cell lung cancer (NSCLC) A549 cells. Further mechanistic investigations showed that 4k arrested the A549 cell cycle at the S phase but triggered only weak apoptosis. Instead, 4k eliminated cells through non-apoptotic mechanisms involving the simultaneous inhibition of autophagy and induction of ferroptosis in NSCLC by elevating reactive oxygen species (ROS) levels, reducing mitochondrial membrane potential (MMP), and downregulating glutathione peroxidase 4 (GPX4) expression. Moreover, 4k also showed potent in vivo antitumor activity in a fluorescent zebrafish xenograft model. Additionally, the potential target proteins were predicted and preliminarily validated using network pharmacology and molecular docking. Collectively, our findings identified 4k as a dual modulator that blocks autophagy and sequentially triggers ferroptosis, offering a promising therapeutic strategy to overcome apoptosis resistance.