Discovery of 1,2,4-benzotriazine derivatives as new hematopoietic progenitor kinase 1 (HPK1) inhibitors.

Hematopoietic progenitor kinase 1 (HPK1), which negatively regulates immune signaling, has emerged as an attractive small-molecule drug target for tumor immunotherapy. Herein, we report the discovery of the 1,2,4-benzotriazine derivatives as new potent HPK1 inhibitors. Notably, compound A29 exhibited improved HPK1 inhibitory activity relative to compound 1 in the ADP-Glo kinase assay (IC50 = 2.70 and 13.6 nM, respectively). The pronounced inhibitory activity of A29 against downstream p-SLP76 in Jurkat T cells (IC50 = 8.1 nM) as well as the ability to induce the production of interleukin 2 (IL-2) in human peripheral blood mononuclear cells (PBMCs) confirmed its cellular target engagement and immune stimulatory effect. Consistently, this lead compound significantly enhanced T-cell killing ability against murine colon cancer cells CT26 or MC38 in a co-culture system. Furthermore, A29 was efficacious in a CT26 xenograft mouse model alone, and significantly enhanced the antitumor efficacy of an anti-PD-1 antibody. This work provides a promising lead for the development of effective HPK1 inhibitors for tumor immunotherapy.