Design, synthesis, and in vitro anti-renal fibrotic effects of imidazopyridazine-based homeodomain-interacting protein kinase 2 inhibitors.

Renal fibrosis, a progressive pathology in chronic kidney disease (CKD), is primarily driven by HIPK2 (homeodomain-interacting protein kinase 2)-mediated activation of the TGF-β/Smad3 and NF-κB signaling pathways, leading to excessive extracellular matrix deposition and inflammation. Current therapeutic strategies targeting HIPK2 show limited efficacy, highlighting the need for more effective inhibitors. We developed compound c4 through rational optimization of the lead compound CHR-6494. This derivative exhibits potent dual activities, with IC50 values of 0.68 ± 0.18 μM for HIPK2 inhibition and 0.15 ± 0.02 μM for anti-proliferative effects in NRK-49F cells. Molecular dynamics simulations confirmed the stable binding of the c4-HIPK2 complex. Functional assays in TGF-β-stimulated NRK-49F cells and TNF-α-stimulated HK-2 cells demonstrated that c4, even at low concentrations, significantly downregulated fibrosis markers (Collagen I, Fibronectin, α-SMA) and inflammatory mediators (p-P65, IL-6), while suppressing fibrotic responses (cell proliferation, migration). These findings establish c4 as a promising HIPK2 kinase inhibitor for developing effective anti-fibrotic therapies targeting HIPK2 in CKD.