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 death

Based on the structural modification of regorafenib, 28 pyrazinyl-aryl urea derivatives were synthesized and their in vitro antiproliferative 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 5-23 demonstrated the strongest inhibitory activity (IC₅₀ = 4.58 ± 0.24 μM) with high selectivity. Compound 5-23 induced Apoptosis in the low concentration range (≤7.5 μM) combined with shorter incubation time (≤10 h) via the 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, 5-23 displayed improved pharmacokinetic profiles in an in vivo rat model. Molecular docking and structure-activity relationship analyses were in agreement with the biological data. Compound 5-23 may be a potent anti-bladder Cancer agent and this small molecule can be considered as a promising structure for further optimization.