Potent and Selective Biaryl Amide Inhibitors of Hematopoietic Progenitor Kinase 1 (HPK1).

pubs.acs.org/acsmedchemlett Note Potent and Selective Biaryl Amide Inhibitors of Hematopoietic Progenitor Kinase 1 (HPK1) Alexander Sokolsky,* Oleg Vechorkin, Joshua R. Hummel, Evan D. Styduhar, Anlai Wang, Minh H. Nguyen, Hai Fen Ye, Kai Liu, Ke Zhang, Jun Pan, Qinda Ye, Onur Atasoylu, Elham Behshad, Xin He, Patricia Conlen, Kristine Stump, Min Ye, Sharon Diamond, Maryanne Covington, Swamy Yeleswaram, and Wenqing Yao Cite This: ACS Med. Chem. Lett. 2023, 14, 116−122 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: Herein we report the discovery of a novel biaryl amide series as selective inhibitors of hematopoietic protein kinase 1 (HPK1). Structure−activity relationship development, aided by molecular modeling, identified indazole 5b as a core for further exploration because of its outstanding enzymatic and cellular potency coupled with encouraging kinome selectivity. Late-stage manipulation of the right-hand aryl and amine moieties surmounted issues of selectivity over TRKA, MAP4K2, and STK4 as well as generating compounds with balanced in vitro ADME profiles and promising pharmacokinetics. KEYWORDS: HPK1, tyrosine kinase inhibition, selectivity profile, MAP4K2, STK4 I mmune checkpoint inhibition has emerged as a highly effective strategy in cancer treatment, with durable responses observed across a variety of tumor types.1 Nevertheless, a significant proportion of patients do not respond effectively to immune checkpoint blockade or develop resistance after an initial response. Thus, additional targets that can modulate the immune response synergistically with checkpoint inhibitors are of great interest.2 One such target is hematopoietic progenitor kinase 1 (HPK1/MAP4K1),3−5 a serine/threonine kinase of the Ste20 family that is involved in the downregulation of T-cell and B-cell antigen receptor signaling.6 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.7,8 HPK1 knockout mice showed increased T-cell activation, improved antitumor immunity, and increased propensity for autoimmune disease, all suggesting a critical role of HPK1 in immune modulation.6 Recent studies with single-point kinase active-site mutants have shown that the promising results with knockout mice can be recapitulated by inhibiting kinase activity,9−11 suggesting that a small-molecule ATP-competitive approach to HPK1 inhibition could lead to effective immune activation. Intrigued by the possibilities of HPK1 blockade, we and others12−17 initiated a program to develop small-molecule ATP-competitive inhibitors of HPK1. The preceding paper described the identification of a series of 1H-pyrazolo[3,4-c]pyridines, culminating in the discovery of compound 1 (Figure 1). In an effort to identify additional leads from a structurally distinct scaffold, we reanalyzed the screen of our in-house compound collection in light of the © 2022 American Chemical Society Figure 1. Scaffold hop from 1H-pyrazolo[3,4-c]pyridine to a picolinamide-based core. structure−activity relationship (SAR) uncovered in the identification of 1. In particular, compound (±)-2 caught our attention, as it had satisfactory preliminary binding potency in a homogeneous time-resolved fluorescence (HTRF)-based binding assay but no clear traditional hinge binder. When tested in an in-house kinase panel, (±)-2 had promising selectivity. Furthermore, when tested in an HTRF-based cellular assay measuring the phosphorylation of SLP76 in Jurkat cells, (±)-2 displayed micromolar potency (p-SLP76 Received: May 20, 2022 Accepted: December 7, 2022 Published: December 13, 2022 116 https://doi.org/10.1021/acsmedchemlett.2c00241 ACS Med. Chem. Lett. 2023, 14, 116−122 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Note Figure 2. (A) Docking model of compound 2 in complex with HPK1. The R isomer is shown. Color coding: green, P-loop; yellow, hinge; red, catalytic loop. (B) Overlay of compounds 1 (magenta) and (R)-2 (cyan). IC50 = 4.6 μM). Based on these promising results, (±)-2 was selected for follow-up studies. To initiate our efforts, we modeled 2 in the active site of the HPK1 protein. Our modeling was based on the hypothesis that the carbonyl of the amide binds to the hinge, as exemplified in a recent crystal structure of an IRAK1 inhibitor (PDB entry 6BFN18). Docking of (R)-2 in HPK1 (PDB entry 6CQF19) using a similar binding mode is shown in Figure 2a. The R isomer is shown based on observed data (vide infra), but initial modeling did not suggest a clear preference for one isomer. In this model, 2 displays a monodentate interaction with the hinge region of HPK1, in which the amide carbonyl forms a hydrogen bond with the backbone N-H of Cys-94. The ofluorophenyl group extends into a lipophilic pocket under the P-loop. Finally, the primary amine forms a hydrogen bond with Asp-101. An overlay of (R)-2 and our lead 1 (Figure 2b) suggests that the fluorophenyl and fluoromethylphenyl rings overlap significantly, whereas the interaction with Asp-101 is unique, representing a promising area for further exploration. The previously disclosed SAR in the P-loop region of 1 revealed that enzyme potency was extremely sensitive to the dihedral angle between rings A and B (see Figure 1 for labeling). While the exact substitution pattern for maximum potency was empirical, substantial improvements could be realized. Specifically, 2′,6′-disubstitution of ring B frequently led to improved potency and general kinase selectivity. Furthermore, a recently disclosed HPK1 crystal structure (PDB entry 7L2512) suggested that the pyridine nitrogen in the pyrazolopyridine core in 1 formed a water-mediated hydrogen bond with Asp-155 of HPK1. By applying these findings to our new core, we discovered that pyrimidine 3 possessing a fluoromethoxyphenyl substituent as ring A and pyrimidine as ring B (Figure 1, right) exhibited the greatest potency gain in this series. Compound 3 showed greatly improved (>20-fold) enzyme potency in the primary binding assay relative to our initial racemic hit (±)-2 and displayed ∼1 μM potency in the cellular assay. We next examined the interaction with Asp-101 in an attempt to further improve the cellular and enzymatic potency. A preliminary SAR study evaluated a focused set of replacements for the aminopiperidine as well as a set of small substituents around ring C. Gratifyingly, compound 4 adorned with 3-aminopyrrolidine and fluoro substitution maintained enzyme potency while greatly improving selectivity in our in-house panel, albeit with significant activity remaining for TRKA (Table 1, entry 1). Next, a number of ring C alternatives were evaluated (Table 1). The key finding of SAR Table 1. SAR Study of Replacements for Ring C a Measured at [ATP] = 1 mM. studies in this binding pocket was the identification of 1Hindazole 5a (entry 2), which improv (...truncated)