Discovery of 2, 4-disubstituted pyrimidine derivatives as selective HIPK2 inhibitors for potential treatment of chronic kidney disease.
Chronic kidney disease (CKD) remains a major global health burden, and renal fibrosis is a key pathological driver of CKD progression. Homeodomain-interacting protein kinase 2 (HIPK2) has emerged as a potential therapeutic target because of its involvement in TGF-β/Smad3-mediated fibrotic signaling and NF-κB-driven inflammatory responses. In this study, a series of 2, 4-disubstituted pyrimidine derivatives were designed and synthesized as HIPK2 inhibitors. Among them, A6 (AF-1) and B11 (AF-2) exhibited potent HIPK2 inhibitory activity, with IC50 values of 0.36 and 0.56 μM, respectively, together with improved subtype selectivity over HIPK1 and HIPK3. Cellular thermal shift assays showed that AF-1 and AF-2 increased the thermal stability of endogenous HIPK2 in both NRK-49F and HK-2 cells, supporting intracellular target engagement. In TGF-β1-stimulated NRK-49F cells and HK-2 cells, both compounds reduced the expression of fibrosis-related markers, including fibronectin, collagen I, and α-SMA. AF-1 and AF-2 also inhibited the proliferation and migration of NRK-49F cells and attenuated TNF-α-induced NF-κB activation in HK-2 cells. In a 0.2% adenine-diet-induced mouse model of CKD, AF-1 and AF-2 alleviated renal tubular injury and collagen deposition, demonstrating significant renoprotective and antifibrotic effects in vivo. Collectively, AF-1 and AF-2 represent antifibrotic candidates with a new chemical scaffold, submicromolar HIPK2 inhibitory activity, and improved subtype selectivity within the HIPK family. Although further optimization is required to improve their pharmacokinetic and drug-like properties, these findings provide a new structural basis for the development of HIPK2 inhibitors with enhanced HIPK family selectivity for the treatment of CKD-associated renal fibrosis.