Development of Highly Potent and Selective Covalent FGFR4 Inhibitors Based on S_NAr Electrophiles

Fibroblast growth factor receptor 4 (FGFR4) is thought to be a driver in several cancer types, most notably in hepatocellular carcinoma. One way to achieve high potency and isoform selectivity for FGFR4 is covalently targeting a rare cysteine (C552) in the hinge region of its kinase domain that is not present in other FGFR family members (FGFR1–3). Typically, this cysteine is addressed via classical acrylamide electrophiles. We demonstrate that noncanonical covalent “warheads” based on nucleophilic aromatic substitution (SNAr) chemistry can be employed in a rational manner to generate highly potent and (isoform-)selective FGFR4 inhibitors with a low intrinsic reactivity. Key compounds showed low to subnanomolar potency, efficient covalent inactivation kinetics, and excellent selectivity against the other FGFRs, the kinases with an equivalent cysteine, and a representative subset of the kinome. Moreover, these compounds achieved nanomolar potencies in cellular assays and demonstrated good microsomal stability, highlighting the potential of SNAr-based approaches in covalent inhibitor design.成纤维细胞生长因子受体4（Fibroblast growth factor receptor 4, FGFR4）被认为是多种癌症的驱动基因，在肝细胞癌中尤为关键。实现FGFR4高效力与亚型选择性的一种可行路径，是共价靶向其激酶结构域铰链区中一类罕见的半胱氨酸残基（C552）——该位点在其余FGFR家族成员（FGFR1~3）中均未出现。常规而言，这类半胱氨酸残基可通过经典丙烯酰胺亲电试剂进行靶向修饰。本研究证实，基于亲核芳香取代（nucleophilic aromatic substitution, SNAr）化学的非经典共价弹头，可通过理性设计策略，制备得到本征反应活性较低、兼具高效力与（亚型）选择性的FGFR4抑制剂。核心化合物展现出低至亚纳摩尔级的活性效力、高效的共价失活动力学，且对其他FGFR亚型、携带等效半胱氨酸残基的激酶，以及具有代表性的激酶组子集均表现出优异的选择性。此外，这类化合物在细胞实验中可达到纳摩尔级的活性效力，同时具备良好的微粒体稳定性，凸显了基于SNAr的策略在共价抑制剂研发设计中的应用潜力。