Discovery of a Novel Pyrido[2,1- h ]pteridin-6-one Derivative as a Potent and Orally Bioavailable HPK1 Inhibitor

Abstract Hematopoietic progenitor kinase 1 (HPK1), a key negative regulator of T-cell activation, represents a highly promising yet challenging target for cancer immunotherapy. To date, no HPK1 inhibitor has received marketing approval, underscoring the need for innovative scaffolds. Here, we report the structure-based drug discovery of a potent HPK1 inhibitor featuring a unique pyrido[2,1-h]pteridin-6-one core. Through a series of chemical modifications, including scaffold hopping and ring-system optimization, starting from a known diaminopyrimidine carboxamide structure, we successfully constructed this key tricyclic core. The lead compound 29 exhibited remarkable potency against HPK1, along with outstanding selectivity. It effectively suppressed HPK1-mediated SLP-76 phosphorylation, thereby enhancing T-cell activation in both Jurkat cells and human PBMCs. Moreover, 29 displayed excellent oral bioavailability and markedly enhanced antitumor efficacy in combination with anti-PD-1 in a CT26.WT syngeneic mouse model. Collectively, these findings identify a novel, potent, and selective HPK1 inhibitor, offering a promising candidate for advancing cancer immunotherapy.