Hematopoietic progenitor kinase 1 (HPK1, also known as MAP4K1) is a serine/threonine kinase belonging to the Ste20-related MAP4K family and plays a pivotal role in negatively regulating immune cell signaling pathways [[1], [2], [3], [4], [5]]. Predominantly expressed in hematopoietic cells, HPK1 acts as a key suppressor of T-cell receptor (TCR) and B-cell receptor (BCR) signaling by phosphorylating adaptor proteins such as SLP-76 and BLNK, thereby inhibiting downstream NF-κB and AP-1 activation [[6], [7], [8]]. Preclinical studies have demonstrated that HPK1 knockout or kinase inactivation enhances T-cell proliferation, cytokine production, and antitumor immunity, suggesting its potential as an immunotherapeutic target [9]. Given its immunosuppressive function in the tumor microenvironment, HPK1 inhibition has emerged as a promising strategy to augment immune checkpoint blockade and adoptive cell therapies in oncology [10]. A key challenge in developing effective small-molecule HPK1 inhibitors is achieving selectivity while preserving the activity of other kinases critical for intracellular homeostasis and general signal transduction [11,12]. For instance, within the MAP4K family, HPK1 and GLK are both activated by TCR signaling and bind SLP-76, yet they perform distinct functions in the regulation of T cell activation and immune responses [13,14].

Recent advances in HPK1 inhibitor development have demonstrated promising therapeutic potential [[15], [16], [17], [18], [19], [20]] (Fig. 1a). The first HPK1 inhibitor to enter clinical studies was developed by Treadwell Therapeutics, and its molecular structure has not been publicly disclosed. Compound DD203-1 was disclosed by University Health Network in a patent (WO2016205942A1). Pfizer successfully developed PF-07265028, a highly selective HPK1 inhibitor with favorable pharmacological properties [21]. This compound exhibits potent HPK1 inhibition both in vitro and in vivo, along with suitable pharmacokinetic profiles, and has advanced into Phase 1 clinical trials. Another notable inhibitor, GNE-6893, enhances antitumor immunity by augmenting TCR signaling, thereby improving T-cell-mediated immune responses [22]. Additionally, AZ3246, a pyrazine carboxamide-based HPK1 inhibitor, effectively stimulates IL-2 secretion (EC50 = 90 nM) with minimal off-target kinase inhibition. It demonstrates oral bioavailability and significant antitumor activity in the EMT6 syngeneic mouse model [23]. Further structural optimization has led to the discovery of ZA-19 a pyridine-2-carboxamide derivative exhibiting excellent drug metabolism and pharmacokinetic (DMPK) properties [24]. When combined with anti-PD-1 therapy, this inhibitor shows robust in vivo efficacy in two murine colorectal cancer models (CT26 and MC38). Moreover, it exhibits high Kinome selectivity, with minimal inhibition of hERG and 44 other safety-related off-targets, suggesting a wide therapeutic window. Beyond inhibitors, significant research on HPK1 has also focused on the discovery of degraders. Our prior work identified DD205-291 as a novel HPK1 degrader with potent activity, strong immunomodulatory effects, and excellent in vivo antitumor efficacy, demonstrating synergy with anti-PD1 therapy [25] (Fig. 1b). This compound is currently in Phase 1 clinical trials. Wu et al. [26,27] discovered two potent HPK1 degraders, C3 and E3, both of which exhibit favorable drug-like properties. These findings highlight HPK1 as a tractable target for immunotherapy.

Herein, we employed an integrated AIDD/CADD approach to design and optimize novel pyrrolopyrazine derivatives as HPK1 inhibitors. Using structure-based drug design guided by the HPK1 co-crystal structure, we systematically investigated the SAR to simultaneously enhance both potency and selectivity. Our lead optimization strategy prioritized three key parameters: (1) improved safety profiles, (2) optimized ADME properties, and (3) enhanced pharmacokinetic characteristics. Through comprehensive in vitro and in vivo evaluations, we identified compound 37 as the preclinical candidate, which demonstrates both intrinsic antitumor activity and synergistic effects when combined with PD-1/PD-L1 checkpoint inhibitors, establishing it as a promising candidate for cancer immunotherapy applications.