Oxopyrrolidine–Acylhydrazone Scaffolds as Dual Inducers of Autophagy and Ferroptosis in the Breast Cancer via JNK/Beclin‐1/GPX4 Regulation

Autophagy and ferroptosis are evolutionarily conserved stress‐response pathways that critically regulate tumor progression and therapeutic responsiveness. In breast cancer, the autophagy initiator Beclin‐1 acts as a haploinsufficient tumor suppressor, whereas susceptibility to ferroptosis is largely governed by the GPX4–SLC7A11–TFRC redox axis. Therapeutic strategies capable of coordinately modulating these pathways remain largely unexplored. This study aimed to identify novel redox‐active anticancer agents by designing and synthesizing a focused library of oxopyrrolidine‐tethered N ‐acylhydrazone derivatives PCH (01–17). The lead compound, PCH‐03, was investigated to elucidate its ability to induce ROS‐dependent JNK3 signaling and coordinate autophagy–ferroptosis crosstalk in breast cancer cells through integrated computational and experimental analyses. Computational investigations, including molecular docking, molecular dynamics simulations, and frontier molecular orbital (FMO) and molecular electrostatic potential (MESP) analyses, were performed to evaluate the structural compatibility of PCH‐03 with the conserved JNK kinase domain and its electronic properties. Biological activity was assessed in four human breast cancer cell lines (MCF‐7, SK‐BR‐3, BT‐474, and MDA‐MB‐231) and normal breast epithelial MCF‐10A cells using MTT assays, mitochondrial membrane potential analysis, intracellular ROS measurement, immunocytochemical analysis of LC3 and GPX4, MDC staining, flow cytometry, and intracellular Fe 2+ quantification. Western blotting, together with chloroquine‐mediated autophagic flux analysis and p62/SQSTM1 evaluation, was used to characterize autophagy, while ferroptosis involvement was validated using ferrostatin‐1 (Fer‐1). Pharmacological inhibition with SP600125 was employed to investigate the contribution of JNK3 signaling. PCH‐03 inhibited the viability of breast cancer cells with IC 50 values of 35.24 μM in MCF‐7, 35.46 μM in SK‐BR‐3, 50.81 μM in BT‐474, and 35.71 μM in MDA‐MB‐231 cells, whereas a higher IC 50 value of 141.85 μM was observed in normal breast epithelial MCF‐10A cells, indicating preferential cytotoxicity toward breast cancer cells. PCH‐03 induced intracellular ROS accumulation, mitochondrial membrane depolarization, and oxidative stress, accompanied by activation of JNK phosphorylation. The compound increased Atg7, Beclin‐1 and phosphorylated Beclin‐1 (p‐Beclin‐1), and LC3‐II expression, and chloroquine‐mediated flux assays together with p62/SQSTM1 analysis confirmed activation of functional autophagic flux. Concurrently, PCH‐03 suppressed GPX4 and SLC7A11, increased TFRC expression, and promoted intracellular Fe 2+ accumulation, consistent with ferroptosis induction. SP600125 significantly attenuated autophagy‐associated signaling and partially restored GPX4 expression, supporting the involvement of JNK3 signaling in mediating autophagy–ferroptosis crosstalk, whereas ferrostatin‐1 partially rescued cell viability, reduced iron accumulation, and attenuated JNK3 phosphorylation, indicating that ferroptosis functionally contributes to PCH‐03‐induced cell death alongside JNK3 pathway activation. PCH‐03 is a novel oxopyrrolidine‐ N ‐acylhydrazone derivative that exerts antibreast cancer activity by disrupting redox homeostasis and promoting coordinated autophagy and ferroptosis with the contribution of ROS‐dependent JNK signaling. Although additional genetic validation is warranted to define isoform‐specific JNK regulation, these findings identify PCH‐03 as a promising lead scaffold for developing anticancer therapeutics that coordinately modulate autophagy and ferroptosis to potentially overcome therapeutic resistance in breast cancer.