Structure-Guided Optimization of 2-Aminoquinazoline Hematopoietic Progenitor Kinase 1 Inhibitors for Improved Oral Bioavailability and Synergistic Antitumor Immunity.

Hematopoietic progenitor kinase 1 (HPK1), a pivotal T-cell immunity suppressor, offers transformative potential to overcome immune checkpoint resistance, yet existing inhibitors fail to balance potency, selectivity, and pharmacokinetics. We developed a spatially resolved strategy within a unified chemical framework of our 2-aminoquinazoline core, integrating (1) high-affinity engagement of the HPK1 hinge-region subpocket (Leu23/Phe93/Gly95) through bidentate hydrogen bonding and hydrophobic packing with (2) strategic occupation of a solvent-exposed allosteric site to sterically block CYP3A4/2C9/2D6-mediated oxidative metabolism. Optimized compound 39 demonstrated subnanomolar binding affinity (IC50 = 0.70 nM) with moderate selectivity, combined with high metabolic stability in human liver microsomes (CLint < 1 mL/min/kg) and favorable oral bioavailability (>100%) in mice. In CT26 models, compound 39 synergized with anti-PD-1 (60% tumor growth inhibition) by expanding IFN-γ+CD8+ tumor-infiltrating lymphocytes (7-fold) and enhancing splenic IFN-γ production (3-fold). This work validates 2-aminoquinazolines as a novel HPK1 chemotype addressing metabolic instability─a key hurdle in kinase drug discovery.