Design, Synthesis, and Biological Evaluation of a Series of Spiro Analogues as Novel HPK1 Inhibitors.

Hematopoietic progenitor kinase 1 (HPK1) negatively affects T cell activation and proliferation and is a promising target for immunotherapy. Although HPK1 inhibitors have shown promising efficacy in preclinical models, none have been approved for clinical use. One significant challenge in developing an HPK1 inhibitor is the difficulty in designing a potent inhibitor with good kinase selectivity and pharmacokinetic properties. Here, we report a series of spiro HPK1 inhibitors with good potency and selectivity. Specifically, compound 16 exhibited potent HPK1 inhibition (IC₅₀ = 2.67 nM), adequate selectivity toward the MAP4K family (>100-fold), and good selectivity against selected kinases (>300-fold). Compound 16 demonstrated moderate in vivo clearance and reasonable oral exposure in mice and rats. Notably, compound 16 possessed good antitumor efficacy in the CT26 murine colon cancer and a synergistic effect when combined with anti-PD-1. These exciting preclinical results support the continued development of this class of HPK1 inhibitors.