Macrocyclization of Broad-Spectrum Kinase Inhibitor Bosutinib Leads to Potent and Selective Quinoline-Based HIPK4 Inhibitor AZ137

Abstract

Homeodomain-interacting protein kinase 4 (HIPK4) remains an understudied member of the dark kinome. While genetic knockout studies suggest its involvement in spermiogenesis and cutaneous squamous cell carcinoma, whether these cellular functions can be recapitulated by pharmacological inhibition remains to be determined. These investigations are currently hampered by a lack of high-quality chemical tools. To address this, we employed a rational design strategy utilizing macrocyclization of a bosutinib-based scaffold. Systematic optimization led to the discovery of AZ137 (28e), a potent and selective HIPK4 inhibitor (IC50: 11 nM; cellular EC50: 76 nM). AZ137 exhibits exceptional selectivity across three comprehensive orthogonal panels, high solubility, and no detectable cytotoxicity. Its cellular activity was confirmed in cell-based assays of HIPK4-dependent F-actin remodeling. Together with a negative control compound, this probe set provides a foundational framework for validating HIPK4 as a therapeutic target and a high-quality resource to elucidate its roles in normal physiology and disease.