Structure-guided discovery of 1H-pyrazolo[3,4-c]pyridin-3-amines as a novel class of hematopoietic progenitor kinase 1 (HPK1) inhibitors.

Hematopoietic progenitor kinase 1 (HPK1) is an intracellular negative regulator of immune responses, particularly in T cell receptor (TCR) signaling. Compelling genetic evidence indicates that HPK1 impairs multiple stages of antitumor immunity largely via its kinase activity, thereby positioning it as a promising target for cancer immunotherapy. Starting from two 3-aminopyrazole hits identified through in-house screening, we initiated a structure-guided medicinal chemistry campaign. Through multi-stage optimization to enhance potency by engaging the Asp101 residue and to improve kinase selectivity via molecular hybridization, we developed a novel series of 1H-pyrazolo[3,4-c]pyridin-3-amine derivatives, from which D5 emerged as a key representative. Compound D5 exhibited potent HPK1 inhibitory activity (IC50 = 26.3 nM), and inhibited SLP76 phosphorylation and promoted IL-2 secretion in cell-based assays. Importantly, D5 showed favorable selectivity across diversity and immune-focused kinase panels, representing a marked improvement over its earlier analogue C4. Furthermore, D5 significantly suppressed tumor growth in the CT26 syngeneic mouse model without causing observable body weight loss. Overall, this work provides both a promising lead and a novel chemical scaffold, offering valuable insights and a concrete starting point for HPK1-targeted drug discovery.