Triazole-substituted pyrazole–pyrimidine hybrids as anticancer agents: synthesis, cytotoxicity, apoptosis mechanisms, and JAB1-targeted structure-based design

Abstract

In this study, twelve new triazole substituted pyrazole-pyrimidine hybrid compounds were synthesized through click reaction and evaluated for their cytotoxic activity against pancreatic, breast, and gastric cancer cell lines. Among the tested compounds, 4e demonstrated the most potent cytotoxic activity, with IC₅₀ values below 10 μM across all evaluated cancer cell lines: Breast cancer cell lines MCF-7 (5.6 ± 1.01 μM), MDA-MB-231 (8.18 ± 1.26 μM), and gastric cancer cell line HGC-27 (5.68 ± 0.45 μM). Furthermore, RT-qPCR analysis revealed that 4e significantly modulated the expression of apoptosis-related genes, notably inducing a marked downregulation of BIRC3, implicating the activation of the mitochondria-mediated intrinsic apoptotic pathway. Flow cytometry confirmed apoptosis induction. Furthermore, computational metabolomic pathway analysis indicated that 4e altered glucose metabolism, notably affecting genes and metabolites associated with glycolysis and fatty acid biosynthesis. These results highlight compound 4e as a promising anticancer candidate with dual action on apoptotic signaling and metabolic pathways. Given the potent biological activity of 4e , further optimization was pursued through a structure-based drug design strategy targeting the oncogenic regulator JAB1, 4e was designed as a scaffold for targeting JAB1. A virtual library of analogues was generated, and all derivatives were docked against the JAB1 crystal structure (PDB ID: 5JOG). Several compounds showed higher docking scores than the co-crystallized ligand (CSN5i-3), suggesting enhanced binding affinity. In parallel, binary QSAR models were developed using the MetaCore/MetaDrug platform to predict anticancer activity. Based on the combined docking and QSAR analyses, several promising analogues were identified and proposed for synthesis and subsequent biological evaluation in future studies.