Concise synthesis and biological activity evaluation of novel pyrazinyl–aryl urea derivatives against several cancer cell lines, which can especially induce T24 apoptotic and necroptotic cell deathElectronic supplementary information (ESI) available. See DOI: 10.1039/d1md00306b

Based on the structural modification of regorafenib, 28 pyrazinyl–aryl urea derivatives were synthesized and their in vitroantiproliferative activities were evaluated. Six compounds (5-16, 5-17, 5-18, 5-19, 5-22, and 5-23) exhibited favorable inhibitory activity against the human bladder cancer T24 cell line, and 5-23demonstrated the strongest inhibitory activity (IC50= 4.58 ± 0.24 μM) with high selectivity. Compound 5-23induced apoptosis in the low concentration range (≤7.5 μM) combined with shorter incubation time (≤10 h) viathe activation of caspases, while high concentrations and prolonged incubation times led to necroptotic cell death by activating the RIPK1/RIPK3/MLKL signaling pathway. Induced apoptosis and necroptosis were closely associated with intracellular reactive oxygen species generation and decreased mitochondrial membrane potential. Compared with regorafenib, 5-23displayed improved pharmacokinetic profiles in an in vivorat model. Molecular docking and structure–activity relationship analyses were in agreement with the biological data. Compound 5-23may be a potent anti-bladder cancer agent and this small molecule can be considered as a promising structure for further optimization.