6-(2-Hydroxybenzoyl)pyrazolo[1,5-a]pyrimidine-3‑carbonitriles as novel reactive oxygen species-generating anticancer agents inducing ER stress and DNA damage in EGFR-mutant non-small cell lung cancer.

A series of twenty-one 6-(2-hydroxybenzoyl)pyrazolo[1,5-a]pyrimidine-3‑carbonitrile derivatives (HBPs), incorporating a pyrazolo[1,5-a]pyrimidine scaffold with a Michael acceptor moiety, were designed and synthesized as novel reactive oxygen species (ROS)-generating anticancer agents. Following structural characterization via NMR and mass spectrometry, anti-proliferative screening in EGFR-mutant (L858R/T790M) H1975 non-small cell lung cancer cells identified HBP-10 as the hit compound. Molecular docking predicted favorable binding of HBP-10 to the active site of glutathione S-transferase P (GST-P). In order to evaluate the stability of the HBP-10-GST-P complex, molecular dynamics simulations were carried out. Mechanistic studies demonstrated that HBP-10 induces intracellular ROS accumulation, triggering endoplasmic reticulum stress, DNA damage, and caspase-dependent apoptosis. Notably, scavenging ROS with N-acetylcysteine markedly attenuated these cytotoxic effects, confirming ROS generation as the upstream mediator of cell death. In conclusion, these findings establish HBP-10 as a highly promising anticancer hit compound for the development of next-generation therapeutic agents, targeting ROS-mediated cellular stress in EGFR-mutant NSCLC.