Hematopoietic progenitor kinase 1 (HPK1, also known as MAP4K1), a member of the Ste20-related serine/threonine kinase family of MAP4Ks, is predominantly expressed in hematopoietic organs and cells, including T cells, B cells and dendritic cells [1]. Previous studies have verified HPK1 as an intracellular negative regulator of T cell receptor (TCR) and B cell receptor (BCR) signal transduction via phosphorylation of SH2 domain-containing leukocyte protein of 76 kD (SLP76) and B-cell linker (BLNK), respectively [[2], [3], [4]]. HPK1-ablated T cells demonstrated increased proliferation, sustained TCR-induced calcium flux and elevated cytokine (e.g., IL-2 and TNF-α) production underscoring the essential role of HPK1 in regulating T cell activation and immune function [2,5]. More recently, HPK1 has been shown to be positively correlated with tumor-infiltrating T cell exhaustion through HPK1-NFκB-Blimp1 axis, which would dampen the antitumor immune response [6]. Furthermore, results from the kinase-dead knock-in transgenic mice model confirmed that the kinase activity of HPK1 is crucial for the negative modulation of TCR signaling and secretion of effector cytokines. Loss of HPK1 kinase function was able to suppress tumor growth and boost anti-tumor immune responses in vivo [7]. Indeed, the negative regulation exerted by HPK1 is involved in nearly every step of the cancer-immunity cycle [8]. Additionally, survival analysis revealed that higher HPK1 expression is strongly correlated with worse survival outcomes across cancer types including lower-grade glioma, renal clear cell carcinoma, pancreatic adenocarcinoma, breast invasive carcinoma and non-small cell lung cancer [6,9]. Therefore, HPK1, a druggable kinase, is deemed to be an attractive candidate target for cancer immunotherapies.

Currently, distinct strategies have been exploited to inhibit or eliminate HPK1 function, including small-molecule inhibitors, proteolytic targeting chimera (PROTAC) molecules and chimeric antigen receptor T (CAR-T) cells with edited endogenous HPK1 [6,10]. Discovery and development of HKP1 small-molecule inhibitors are of particular interest both in academia and industry, with numerous diverse structures being reported, which have been well-reviewed [[11], [12], [13], [14], [15], [16]]. Notably, 10 HPK1 small-molecule inhibitors have entered Phase I/II clinical trials (Table S1, Supplementary Data), among which CFI-402411 [17], NDI-101150 [18], BGB-15025 [19] and PRJ1-3024 [20] have shown encouraging clinical benefits. However, no HPK1 inhibitor has been approved yet and the clinical trials of PF-07265028 (Pfizer) and RGT-264 (Regor Pharmaceuticals Inc.) have been terminated. The progress of HPK1 inhibitors/PROTACs development is primarily hindered by poor selectivity, impotent immuno-stimulation and unsatisfactory efficacy. Given highly conserved kinase ATP-binding pockets among HPK1 and other Ste20 family members, it is challenging to develop a highly selective small molecule. Besides, several kinases in this family, such as HPK1 and GLK (MAP4K3) play opposite biological functional roles in modulating T-cell activation and immune responses [21]. In fact, most HPK1 inhibitors in the literatures with in vivo efficacy reported had undisclosed or low to moderate selectivity over GLK (Fig. 1 and Table S2, Supplementary Data) [6,[22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35]]. Hence, developing a highly selective HPK1 inhibitor, especially over GLK, is urgently needed.

In this study, we report the discovery and optimization of a series of tetrahydroisoquinoline derivatives as selective and potent HPK1 inhibitors. The optimal compound displays excellent selectivity over GLK, favorable pharmacokinetic properties and anti-tumor efficacy in preclinical models.