Chronic kidney disease (CKD) affects 10–13 % of the global population and is characterized by persistent inflammation and renal fibrosis [1]. Its causes include hypertension [2], chronic glomerulonephritis [3] and polycystic kidney disease [4]. The nature of the disease in CKD is renal fibrosis with associated inflammation, and therapies for fibrosis are not yet available, with treatment strategies focusing on slowing disease progression and alleviating symptoms [1]. Finerenone, a selective mineralocorticoid receptor antagonist, is commonly prescribed for CKD associated with type II diabetes mellitus [5]. However, its clinical use is limited by adverse effects, including hyperkalemia. Dapagliflozin, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, has emerged as an effective and safer option for CKD management. In addition to improving glycaemic control, it may also slow the progression of CKD through non-glycaemic mechanisms such as anti-inflammatory and anti-fibrotic [6,7]. But its efficacy is still limited; therefore, there remains an urgent need for the development and optimization of safe and effective therapies for CKD.

Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear dual-specific serine/threonine and tyrosine kinase with important functions in the regulation of fibrosis and inflammation [8]. Studies have demonstrated that HIPK2 mediates fibrosis and inflammation-related biological effects by regulating key signaling pathways such as NF-κB, TGF-β/Smad3, p53 and Wnt/β-catenin [9]. Elevated HIPK2 expression has been observed in various renal fibrosis models and CKD patient samples [8], being suggested as a potential therapeutic target for CKD.

BT173 (1) (Fig. 1) is an inhibitor of the protein-protein interaction of HIPK2 with Smad3 and exhibits moderate inhibitory activity against HIPK2 [10]. As an analogue of BT173(1), RLA-23174 (structure not disclosed) is in Phase I clinical trials for the treatment of kidney fibrosis-related diseases [11]. Phosphate Niclosamide (2) inhibits HIPK2 expression by disrupting the binding of Smad3 to the HIPK2 gene promoter sequence. In recent years, HIPK2 kinase inhibitors have been extensively studied [9]. Several multi-target inhibitors, such as STO-609 [12] (3), TAE-226 [13] (4) and CTx-0294857 [14] (5), have been reported to exhibit moderate inhibitory activity against HIPK2. MU135 [15] (6) and CX-4945 [16] (7) have been crystallized in complex with HIPK2. MU135 exhibits potent inhibition of HIPK2, whereas for CX-4945, despite evidence of structural binding, quantitative data on its inhibitory activity against HIPK2 remain unavailable. TBID (8) [17], a phthalimide derivative, displays moderate inhibitory activity against HIPK2 and has been used as a tool molecule for the study of HIPK2 inhibition. CHR-6494 [18] (9) and 15q [19] (10) are potent HIPK2 kinase inhibitors developed and characterized within our group. CHR-6494, initially identified through virtual screening, functions as a dual-action inhibitor by not only suppressing HIPK2 kinase activity but also promoting its proteasomal degradation. In vitro assays demonstrated that CHR-6494 exerts anti-inflammatory and anti-fibrotic effects through HIPK2 inhibition. Compound 15q, derived from the structural scaffold of TAE-226 by cyclizing the 2-aminobenzamide moiety into a benzimidazole core, selectively targets HIPK2 and displays robust inhibitory activity both in vitro and in vivo.

In this study, to further enhance the inhibitory activity of compounds against HIPK2, we performed structural modifications based on 15q as follows (Fig. 2): (1) to retain the core structures of pyrimidine and piperazine rings, replacing isopentyl group with smaller substituents (R1); (2) to modify the pyrimidine N-2 position (R2) with different groups; and (3) to link the pyrimidine N-4 position to the 3-pyridine ring directly (n = 0). All final compounds are screened with a kinase assay and a cell viability assay of NRK-49F. The screening result suggests 7a (XRF-1021) as a potential candidate for further research. Molecular docking, molecular dynamics simulations, drug affinity responsive target stability (DARTS), cell-based mechanisms of action and in vivo druggability studies were conducted. XRF-1021 significantly reduced the expression levels of pro-fibrotic factors (e.g., FN and Collagen I) in NRK-49F cells, exhibited significant anti-fibrotic effects in the unilateral ureteral obstruction (UUO) model and adenine-induced kidney injury model. The LD50 of XRF-1021 was 1943 mg/kg in the adenine model of renal injury, and the NOAEL was 269.0 mg/kg in the UUO model. The results obtained suggest that XRF-1021 has the potential to be developed as an anti-CKD drug against HIPK2.