Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1–10 μM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton’s tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.

用小分子对疾病相关蛋白的不可逆抑制是获得增强和持续的药理效力的有力方法。在这里，我们介绍α-氯氟乙酰胺（CFA）作为靶向共价抑制剂（TCI）的新型战斗部。尽管固有​​反应性弱，但添加CFA的喹唑啉对表皮生长因子受体（EGFR）的Cys797具有高反应性。在细胞中，CFA-喹唑啉在较宽的浓度范围（0.1–10μM）中显示出比相应的Michael受体更高的EGFR靶标特异性。CFA衍生物的半胱氨酸加成物易于水解，可逆地产生完整的硫醇，但在EGFR的溶剂隔离的ATP结合口袋中稳定。这种与环境有关的水解作用可能会降低基于CFA的药物对脱靶蛋白的修饰作用。在小鼠异种移植模型中，口服CFA喹唑啉NS-062可显着抑制肿瘤的生长。此外，添加CFA的吡唑并嘧啶不可逆地抑制布鲁顿酪氨酸激酶，并具有更高的靶标特异性。这些结果证明了CFA作为TCI新型战斗部的用途。