Bisarylureas Based on 1H-Pyrazolo34-dpyrimidine Scaffold as Novel Pan-RAF Inhibitors with Potent Anti-Proliferative Activities: Structure-Based Design Synthesis Biological Evaluation and Molecular Modelling Studies

摘要

RAF (Ras activating factor) kinases are important and attractive targets for cancer therapy. With the aim of discovering RAF inhibitors that bind to DFG-out inactive conformation created by the movement of Asp-Phe-Gly (DFG), we conducted structure-based drug design using the X-ray cocrystal structures of BRAF (v-raf murine sarcoma viral oncogene homolog B1), starting from bisarylurea derivative based on 1H-pyrazolo[3,4-d]pyrimidine scaffold >1a. Most of the synthesized compounds showed good to excellent inhibitory activities against BRAF^V600E kinase, possessed moderate to potent anti-proliferative activities against four tumor cell lines (A375, HT-29, PC-3 and A549) and good selectivity towards cancer cells rather normal cells (Madin-Darby canine kidney, MDCK). The most promising compound, >1v, exhibited potent inhibitory activity against not only BRAF^V600E (half maximal inhibitory concentration, IC50 = 23.6 nM) but also wild-type BRAF (IC50 = 51.5 nM) and C-RAF (IC50 = 8.5 nM), and effective cellular anti-proliferative activities against A375, HT-29, PC-3 and A549 cell lines as well as a very good selectivity profile. Moreover, compound >1v mainly arrested the A375 cell line in the G0/G1 stage, and showed significant suppression of MEK (mitogen-activated protein kinase kinase) phosphorylation in A375 and HT-29 cell lines. Taken together, the optimal compound >1v showed excellent in vitro potency as a pan-RAF inhibitor. In addition, the promise of compound >1v was further confirmed by molecular dynamics simulation and binding free energy calculations.