Discovery of Pyrazolopyridine Derivatives as HPK1 Inhibitors.

pubs.acs.org/acsmedchemlett Letter Discovery of Pyrazolopyridine Derivatives as HPK1 Inhibitors Qinda Ye,* Kai Liu,* Hai-Fen Ye, Jun Pan, Alexander Sokolsky, Anlai Wang, Ke Zhang, Joshua R. Hummel, Ling Kong, Elham Behshad, Xin He, Patricia Conlen, Kristine Stump, Min Ye, Sharon Diamond, Maryanne Covington, Swamy Yeleswaram, Onur Atasoylu, Oleg Vechorkin,* and Wenqing Yao Cite This: ACS Med. Chem. Lett. 2023, 14, 5−10 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: In spite of the great success of immune checkpoint inhibitors in immune-oncology therapy, an urgent need still exists to identify alternative approaches to broaden the scope of therapeutic coverage. Hematopoietic progenitor kinase 1 (HPK1), also known as MAP4K1, functions as a negative regulator of activation signals generated by the T cell antigen receptor. Herein we report the discovery of novel pyrazolopyridine derivatives as selective inhibitors of HPK1. The structure−activity relationship campaign led to the discovery of compound 16, which has shown promising enzymatic and cellular potency with encouraging kinome selectivity. The outstanding pharmacokinetic profiles of 16 in rats and monkeys supported further evaluations of its efficacy and safety in preclinical models. KEYWORDS: HPK1, MAP4K1, tyrosine kinase, cancer immunotherapy I treat HPK1.kd mice.10 These results, coupled with emerging studies of small-molecule HPK1 inhibitors,12−18 suggest that HPK1 could be an excellent drug target for enhancing antitumor immunity. Herein we describe the design and development of novel pyrazolopyridine derivatives as ATPcompetitive inhibitors of HPK1. The program commenced with a high-throughput screen (HTS) of Incyte’s internal compound collection against the kinase domain of HPK1. Binding affinity Ki was determined by incubating the tested compound with HPK1 kinase domain followed by measuring the homogeneous time-resolved fluorescence (HTRF) of the resulting solution. Compound A (Figure 1) with HPK1 Ki = 91 nM was identified from an earlier fibroblast growth factor receptor (FGFR) inhibitor mmuno-oncology (IO) therapy has transformed the cancer treatment landscape and fueled hope for long-term survivorship and even cure in some cancers.1 However, the response rates to IO therapy vary widely across tumor types. Thus, there is a need to develop alternative approaches to broaden the scope of therapeutic coverage.2 One such approach is targeting the enzymes that can modulate the immune response. This approach, when used in combination with immune checkpoint inhibitors, could potentially result in a synergistic effect and thus improve the response rate and reduce the resistance developed by tumor cells. Hematopoietic progenitor kinase 1 (HPK1), also known as MAP4K1, functions as a negative regulator of activation signals generated by the T cell antigen receptor (TCR).3−9 HPK1 acts through phosphorylation of SLP76 and has an immunosuppressive function across a variety of cell types, including CD4+ and CD8+ T cells as well as dendritic cells. Loss of HPK1 kinase function in HPK1 kinase-dead (HPK1.kd) knockin mice enhanced T cell receptor signaling and cytokine secretion in a T-cell-intrinsic manner.10,11 A synergistic effect in controlling tumor growth was also observed when anti-PD-L1 was used to © 2022 American Chemical Society Received: May 19, 2022 Accepted: December 7, 2022 Published: December 12, 2022 5 https://doi.org/10.1021/acsmedchemlett.2c00238 ACS Med. Chem. Lett. 2023, 14, 5−10 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Letter Figure 1. Discovery of the pyrazolopyridine scaffold. program.19 Initial efforts focused on improving the HPK1 potency while attenuating the activity against other kinases. However, structure−activity relationship (SAR) efforts to replace the 2,6-difluoro-3,5-dimethoxyphenyl group, which is known to be specifically important for FGFR binding, led to significant loss of binding affinity against HPK1. Further replacing the pyrazolopyrimidine scaffold with other bicyclic structures such as pyrazolopyridine did not result in any improvement (e.g., compound 1 in Figure 1, Ki = 1151 nM). These unsuccessful attempts prompted us to redesign the topography of the molecules. Our design was guided by compound B, a compound derived from another series identified by another HTS hit from Incyte’s internal compound library. Its structure suggested that migration of the top phenyl ring to the 5-position might potentially be tolerated. This modification resulted in the discovery of compound 2 with moderate binding affinity (Ki = 223 nM). In order to reduce the molecular planarity of 2, twisting the conformation of the biaryl system by incorporation of a fluoro group at the 2′-postion to obtain 3 (Table 1) provided a remarkable 8-fold improvement in binding affinity (Ki = 28 nM). To our delight, micromolar cellular potency of 3 (IC50 = 2744 nM) was observed in Jurkat cells as determined by inhibition of phospho-SLP76. Encouraged by this success, we next examined a small library of 2′,6′-disubstituted compounds, of which fluoromethoxy analogue 4 rapidly emerged as a promising lead with single-digit binding affinity (Ki = 3.0 nM) and improved cellular potency (IC50 = 395 nM). With the promising improvement of HPK1 potency, our attention turned to exploring additional bicyclic scaffolds adopting a similar conformation, as exemplified by 5 and 6. Both of these compounds were potent in the binding assay, with Ki values of 2.5 and <1.0 nM, respectively. Further profiling in the cellular assay showed that 1H-pyrazolo[3,4c]pyridine 6 was almost 2-fold more potent (IC50 = 144 nM) compared to 4 and 5. A similar trend was also observed with 1methylpyrazol-4-yl analogues, as compound 7 (IC50 = 148 nM) was significantly more potent than 8 (IC50 = 640 nM). Having become a milestone of the program, 7 served as a prototype for the following round of the SAR campaign at the R2 position in light of its relatively lower molecular weight and lower human intrinsic clearance (IntCl) (Table 1). While many additional molecules with a variety of R2 groups were prepared, finding the right balance between HPK1 cellular potency and physicochemical properties remained a challenge. We hypothesized that the frequently observed high clearance was related to the high lipophilicity of the molecule. Thus, our exploration then shifted to the introduction of polar substituents on the phenyl ring. Consequently, several lead compounds were discovered, of which benzylic amine 9 (Table 2) was the most promising, with significantly improved human intrinsic clearance (<0.5 L h−1 kg−1). Although initial kinase profiling of 9 revealed submicromolar potency against several other kinases, its good HPK1 cellular potency (IC50 = 219 nM) warranted further investigation of the benzylic amine moiety. The goal was to maintain the desired HPK1 potency and at the same (...truncated)