Currently, surgery, radiotherapy, and chemotherapy are still the three major traditional methods for treating cancer worldwide[1]. However, as treatment durations extend, malignant tumors often develop drug resistance and are prone to recurrence, leading to significant threats to patients' survival and formidable challenges to clinical treatments.

Immunotherapies, for instance, immune checkpoint blockade (ICB) and adoptive T-cell therapy (ACT) have provided new treatment options for cancer patients who have limited effectiveness with traditional treatments[2], [3]. In the past decade, immune checkpoint inhibitors (ICIs), especially PD-1/PD-L1 inhibitors, have demonstrated significant clinical efficacy in the treatment of various malignant tumors[4]. Nonetheless, a high proportion of cancer patients exhibited resistance to single-agent ICIs due to immune resistance mechanisms, tumor heterogeneity, and the complexity of the tumor microenvironment[5]. Hence, there is a pressing imperative to explore alternative strategies for broadening the scope of ICI treatment. Hematopoietic progenitor kinase 1 (HPK1), a serine/threonine protein kinase, [6], [7]also known as mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), was highly expressed in many immune cell types, including T cells, B cells, and dendritic cells [8], [9]. HPK1 plays a pivotal role as a negative regulatory factor in the signaling pathway of T-cell receptors (TCR)[10]. Upon receiving antigenic stimuli, TCR prompts the translocation of HPK1 from the cytoplasm to the proximity of the cell membrane. This relocation triggers the phosphorylation of the Ser376 residue on the SLP76 protein, resulting in the destabilization of the TCR complex and subsequent downregulation of TCR signaling[11], [12]. Furthermore, HPK1 kinase-dead (HPK1-kd) and HPK1 knockout (HPK1-ko) mouse models have revealed enhanced T-cell functionality and improved immune response to PD-1/PD-L1 therapy compared to HPK1 wild-type (wt) mice[13]. Notably, mice with HPK1-kd or HPK1-ko did not manifest fatal inflammation in contrast to the mice with functional deficiencies in CTLA-4[14]. Moreover, due to the limited expression of HPK1 in hematopoietic cells, the potential for systemic side effects resulting from HPK1 inhibition is significantly reduced. These studies demonstrated that HPK1 inhibitors represent a promising approach to enhancing the immune response of T cells against tumors[13], [15], [16].

In recent years, collaborative endeavors from both academia and industry and the research on HPK1 inhibitors have experienced substantial growth[13], [17]. Nonetheless, the development of FDA-approved HPK1 inhibitors remained unrealized. Currently, nine HPK1 inhibitors have entered clinical research (Table 1), however, Pfizer's PF-07265028 (1) was the only one for which the molecular structure has been disclosed (Fig. 1).

To obtain the detailed structure–activity relationships of HPK1 inhibitors, we summarized the structural types of HPK1 inhibitors reported in the literature. The mode of HPK1 inhibitors was identified and expressed as shown in Fig. 1[17]. The core group of the compounds occupies the hinge region, with two distinct substituents extending into the hydrophilic solvent region and the hydrophobic P-loop region, respectively. For instance, compound 2 with a 2,4-diaminopyrimidine scaffold, was reported by Merck Company, as a representative HPK1 inhibitor. In this compound, the amide group at the 5-position of the pyrimidine scaffold acts as a crucial active group, establishing hydrogen bonds with the hinge region. Simultaneously, the aniline fragment at the 4-position occupies the P-loop region, while the tetrahydroisoquinoline segment is oriented towards the solvent region. While compound 2 exhibited a high affinity for HPK1 (IC50 = 0.061 nM), its advancement was hindered by suboptimal pharmacokinetic properties[18]. Compound 3, disclosed by Bristol-Myers Squibb Company, demonstrated remarkable selectivity (54-fold) for the HPK1 homolog kinase GLK. The isobenzofuranone moiety resides within the P-loop and DFG regions, while the hydroxyl group at the 4-position forms a hydrogen bond with Asp101 in the solvent region. Simultaneously, compound 3 effectively inhibited the phosphorylation of the SLP76 protein (IC50 = 290 nM). Moreover, compound 3 demonstrated moderate metabolic stability in mouse and human liver microsomes, along with a considerable free fraction in human serum protein. In the MC38 homologous tumor model, the combination of compound 3 and PD-1 monoclonal antibody achieved a 100 % cure rate[19].

Building upon compound 3, Yang et al. employed a cyclization strategy to restrict the compound's conformation, resulting in compound 4[20]. Compound 4 exhibited more than 100-fold selectivity towards GLK while maintaining inhibitory activity against HPK1 (IC50 = 0.8 nM) and demonstrating excellent metabolic stability in human liver microsomes. Furthermore, in the CT26 mouse tumor model, the combination of compound 4 and PD-1 inhibitor significantly enhanced the anti-tumor effect without evident toxic or side effects.

Zhang et al. modified the structure of compound 3 to yield compound 5 with further improved PK properties[21]. Specific strategies included replacing the isobenzofuranone fragment with the more stable isoindoline fragment, introducing a fluorine atom at the para position of the benzene ring of phenylglycinol, and deuterating the methylene group adjacent to the hydroxyl group to shield metabolic sites. Compound 5 exhibited enhanced inhibitory activity against HPK1 and improved selectivity towards GLK. Additionally, compound 5 reversed the inhibitory effects of immunosuppressive factors such as prostaglandin E2 and adenosine on IFN-γ secretion.

Compound 6 (A-745), developed by AbbVie Company, was a pyrazine-2-formamide compound as an HPK1 inhibitor. The substituent at the 6-position on the pyrazine scaffold extends into the back pocket through the “gatekeeper” amino acid Met91, forming hydrogen bonds with Ala154 and Lys46[22]. Compound 7, with an indazole core, was another HPK1 inhibitor reported by Merck & Co Company[23]. The bridge ring group of compound 7 is connected to Asp101 through a salt bridge, which was the reason for the remarkable improvement of activity and selectivity. Moreover, the nitrogen atom at the 5-position forms a hydrogen bond with the Asp155 residue of the DFG motif through water network mediation. Compound 8 was a selective HPK1 inhibitor reported by Pfizer[24]. Its pyrrolopyrimidine core occupies the hinge region, and the incorporation of a fluorine atom into the piperidine ring enhanced steric hindrance, thereby achieving selectivity towards GLK. Although these reports about HPK1 inhibitors have increased significantly in recent years, no drugs were available on the market. It is noteworthy that Pfizer concluded the research pipeline of PF-07265028 in the third quarter of 2023, and specific reasons for this termination have not been disclosed[25].

In summary, there exists a considerable research gap in the field of HPK1 inhibitors, presenting substantial untapped potential for further investigation. In this paper, we describe our research process in the development of HPK1 inhibitors featuring a 2,4-diaminopyrimidine scaffold. Simultaneously, we conducted a comprehensive exploration of the structure–activity relationships and carried out biological evaluations, with the aspiration that our efforts will contribute to the progress of HPK1 inhibitors.