Kinases are enzymes that transfer terminal phosphate group of the adenosine triphosphate (ATP) to specific substrate to regulate cellular signaling, thus playing a significant role in oncogenesis [1,2]. The ATP binding site of kinase located between the N-lobe and C-lobe connected through hinge region [3]. The hinge region serves as a critical structural element for the proper positioning and stable binding of ATP within the kinase active pocket through forming hydrogen-bond interactions with the adenine ring of ATP [4,5]. Kinase has become the prime target to design inhibitors for cancer treatment. The majority of FDA-approved kinase inhibitors form interactions with hinge region, underscoring the significance of hinge region in drug design [3,[5], [6], [7]]. Proviral insertion site in Moloney murine leukemia virus (PIM) kinases are serine/threonine kinases that are frequently overexpressed in acute myeloid leukemia (AML). PIM kinases contribute to the disease progression by regulating cell survival, proliferation, apoptosis and cycle, making them attractive therapeutic target for AML [[8], [9], [10], [11]]. The PIM kinase family comprises three members: PIM-1, PIM-2, and PIM-3, which display functional redundancy in oncogenesis [[12], [13], [14]]. The compensatory mechanisms among PIM isoforms suggest that pan-PIM inhibitor rather than isoform-selective inhibitor may be more clinically beneficial [15,16]. Structurally, all three PIM kinases are constitutively active and adopt a classic bilobal protein kinase fold, with ATP binding site located in the deep cavity enclosed by the two lobes and hinge region [9,17]. The development of PIM inhibitors has been guided by structural insights from crystallography studies on ATP binding site, revealing key interactions with catalytic residues (Lys67), salt-bridge forming site (Asp128/Glu171) in ribose patch, hydrophobic pockets and hinge region (Fig. 1A) [18,19].

In recent years, several PIM kinase inhibitors have advanced into clinical research, though none have yet received approval for cancer treatment [[20], [21], [22], [23]]. For instance, SGI-1776, an imidazo[1,2-b]pyridazine derivative, was the first PIM inhibitor assessed clinically for replased/refractory leukemias [24,25]. However, its phase Ⅰ clinical research was terminated due to the cardiotoxicity caused by hERG inhibition (ClinicalTrials.gov ID: NCT01239108). Similarly, the thiazolidine-2,4-dione derivative AZD1208, demonstrated antitumor potential in preclinical models [26], but failed to achieve desired efficacy and exhibited P450 inhibition in clinical trials (ClinicalTrials.gov ID: NCT01588548). In comparison, the N-pyridinyl amide has emerged as a particularly promising scaffold. Representative inhibitors derived from this scaffold have progressed to clinical trials and demonstrating anti-tumor efficacy in patients with hematological malignancies [23,27,28]. For example, PIM447 exhibited promising efficacy in phase Ⅰ/Ⅱ studies for replased/refractory multiple myeloma (ClinicalTrials.gov ID: NCT01456689). INCB053914 has completed phase Ⅰ study in diffuse large B-cell lymphoma (ClinicalTrials.gov ID: NCT03688152). Another clinical candidate GDC-0339 deriving from N-pyridinyl amide, is also underclinical evaluation for multiple myeloma, underscoring the translational potential of this chemotype [14]. The N-pyridinyl amide PIM inhibitors derived from lead compound LGB321, which was obtained by hit optimization study and revealed (i) the difluorophenyl group in the hinge region is beneficial for the hydrophobic interaction; (ii) the amino group on the solvent fragment and pyridine nitrogen make key hydrogen bonding interactions with Asp128/Glu171 and Lys67 (Fig. 1B) [29]. Further optimization on LGB321 improves its metabolic stability thus leading to the discovery of PIM447 [22]. Subsequent optimization based on PIM447 emphasized the importance of basic amine in retaining potency [30] and tetra-substituted cyclohexyl diol in improving permeability [31]. In the aforementioned study, it has been observed that modification (especially the nitrogen positional isomerization) of fragment towards hinge region affected inhibitory potency. Notwithstanding these observations, a systematic investigation into SAR specifically on the hinge-binding motif remains largely unexplored.

Our previous work developed structurally novel PIM inhibitors based on N-pyridinyl amide, revealing positional isomerization of N toward to Lys67 would decrease activity, whereas increasing freedom of solvent fragment by introducing a rotatable ether group is beneficial for potency. The SAR studies focused on solvent fragment of compounds ultimately identifying FD1024 as a potent pan-PIM kinase inhibitor [32,33]. Based on the scaffold of FD1024, whose thiazole ring showed structural proximity to the PIM kinase hinge region, we proposed to explore additional interactions in this domain. In this work, we used molecular structure hopping construction, replacing thiazole of FD1024 with a six-membered ring, and presented a comprehensive SAR study on heterocycle targeting interactions with hinge region. The design rationale from FD1024 to lead compound FD2024 is illustrated in Fig. 2A. Our investigation underscored the importance of nitrogen positional isomerization within the six-membered ring. SAR study revealed that the presence of nitrogen at 2-position facilitated intramolecular hydrogen bond and achieved the highest potency, with a 139- to 5936-fold increase compared to its isomers. We further demonstrated that 6-amino group could act as a hydrogen bond donor. This amino successfully formed a key additional hydrogen bond with Glu121 residue compared with FD1024. And this interaction significantly enhanced pan-PIM inhibitory activity. We tuned this hydrogen bond strength and found that substituent electronegativity on the ring had negligible impac on binding affinity. Notably, the second nitrogen atom adjacent to the 6-amino in pyrazine ring, demonstrated unique ability to strengthen this hydrogen bond interaction by its electron-withdrawing nature. This work culminated in the discovery of FD2024 (compound 27) with potent PIM inhibition (IC50 = 0.17, 1.86, 0.38 nM for PIM-1, PIM-2, PIM-3 respectively) and anti-AML efficacy both in vitro (IC50 = 0.05, 0.19, 0.02, 0.25, 0.21 μM for MV-4-11, EOL-1, MOLM-13, MOLM-16, HL-60 cells respectively) and in vivo (MV-4-11 tumor regression and prolongation of mice survival to 44 days), validating the hinge region as a highly promising frontier for developing next-generation PIM kinase inhibitors with optimized potency.