Pyrazine-Based Syk Kinase Inhibitors
A series of aminopyrazines as inhibitors of Syk kinase activity and showing inhibition of LAD2 cells degranulation is described. Optimization of the carboxamide motif with aminomethylpiperidines provided high potency inhibiting Syk but low cellular activity. Amides of cis and trans adamantanol showed good inhibitory activity against Syk as well as remarkable activity in the LAD2 cells degranulation assay.
Introduction
Spleen tyrosine kinase (Syk) is a cytosolic non-receptor protein tyrosine kinase that plays an essential role in immunoreceptor signaling, mainly in B cell receptor, Fc receptors, integrin, and lectin receptors. Syk is highly expressed in hematopoietic cells, including B cells, mast cells, macrophages, and neutrophils. Engagement of these receptors with their ligands activates Syk, which orchestrates several cellular responses such as proliferation, differentiation, cell adhesion, cytokine production, and mast cell degranulation.
Therefore, Syk is considered a promising therapeutic target for the treatment of immune-mediated disorders such as allergy, autoimmune diseases, and hematological malignancies. A number of ATP-competitive Syk inhibitors have been described, and it has been reported that pharmacological inhibition of Syk kinase activity modulates mast cell degranulation, leukocyte immune function, and suppresses inflammation in vivo. Interestingly, since Syk is positioned upstream in the cell signaling pathway of several immunoreceptors, therapies with Syk inhibitors may be more advantageous than drugs that inhibit a single downstream event.
To date, several compounds have entered into clinical development. R-788 is currently in phase II for rheumatoid arthritis and other autoimmune diseases and lymphoma. Few molecules are in early phases of clinical studies, including R-343 for asthma and PRT062607 for autoimmune diseases.
Hit Identification and Initial SAR
Random screening of our internal compound library identified compound 1 as an ATP-competitive Syk inhibitor. After docking studies, we envisaged the 2-aminopyrazine moiety as a key H-bonding motif in the hinge region of the kinase. Initial structure–activity relationship (SAR) optimization on the pyrazine scaffold focused on replacing the aniline group with a substituted carboxamide to allow planarity of the molecule and facilitate SAR expansion to engage key interactions in the ATP-binding pocket.
Synthetic Chemistry
The analogs were prepared following a general synthetic route. Functional group transformations from commercially available aminopyrazine provided key intermediates that, upon activation with HATU, formed amide intermediates. The final step of a Suzuki reaction allowed access to the desired compounds. This straightforward synthetic pathway enabled rapid exploration of SAR in both R1 and R2 positions.
The methylsulfonamidophenyl group, which we speculated was pointing toward the solvent-exposed area, was left for later optimization. Exploration of amide substituents in the R1 position included aliphatic amino alcohols and aminopiperidines.
SAR and Biological Evaluation
A series of piperidines bearing diverse linkers were synthesized to capture a potential salt bridge interaction with Asp512. Aminomethylpiperidine derivatives provided the best Syk inhibitory activity, likely due to their optimal orientation for interaction with Asp512. Aliphatic amines in R1, such as trans-aminocyclohexanol, showed some potency. The introduction of adamantane motifs, similar to those used in JAK inhibitors, yielded promising results. Adamantane and cis or trans adamantanol derivatives showed good potency. Tertiary amides were not tolerated, possibly due to incorrect orientation of the hydroxypiperidine. Thus, the primary amide was identified as a critical binding element.
Further SAR exploration focused on varying the substituent at position 5 while maintaining the best substitutions at R1. The most potent Syk inhibitors were evaluated in a degranulation assay in the human LAD2 mast cell line.
An increase in enzymatic potency was observed with m-trifluoromethoxy analogs as well as indazolyl compounds. There were opportunities to explore the solvent-exposed region using C4 substitution to engage Lys458. Biaryl-substituted compounds were very potent Syk inhibitors, although they failed to show acceptable potency in the LAD2 functional assay, likely due to low permeability. The presence of the ionizable piperidine motif may also have contributed to poor LAD2 activity.
Adamantanol Derivatives and Cell Activity
Focus then shifted to non-basic substituents at the amide motif. Since one adamantane derivative was cytotoxic in CHO cells, non-cytotoxic cis and trans adamantanol analogs were pursued. Among them, the 3-(4-pyrazolyl)phenyl substituents at R2 gave the best Syk inhibitory activity, ranging from 30–80 nM. Functional activity in LAD2 was most potent in compounds with activities around 100 nM. Diverse substitutions at the pyrazole ring were tolerated, including aminoethyl, hydroxyethyl, and carboxamide groups. However, comparison of cell versus enzyme IC50 values indicated that ionizable groups negatively impacted cellular potency.
Similar SAR trends were observed with trans-adamantanol derivatives, which showed Syk inhibitory activities as low as 20 nM and maximum LAD2 potency of 120 nM.
X-ray Crystallography
X-ray structures of selected compounds bound to Syk confirmed the proposed binding modes. Key H-bond interactions were observed between the aminopyrazole motif and the hinge region residues Glu449 and Ala451. In one compound, a salt bridge with Asp512 and an H-bond with Asn499 were seen. In another, the hydroxyl group of the adamantanol motif formed an H-bond with Asp512. In both cases, the R2 group projected toward the solvent-exposed region, and one compound captured an additional H-bond interaction with Lys458 through the pyrazole motif.
Physicochemical Properties and Selectivity
Compound 13f was not cytotoxic in CHO cells and showed good permeability in CaCo-2 assays. It had moderate aqueous solubility at pH 7.4 and a measured logD7.4 of 1.5. In a selectivity panel of 42 kinases, 13f showed less than 50% inhibition at 1 µM for 20 kinases. The most potently inhibited kinases included Aurora A, FGFR3, Fms, TrkA, and Yes. Lyn kinase was inhibited with an IC50 of 325 nM, suggesting a possible contribution of off-target effects to the observed cellular activity.
Conclusion
A hit compound was successfully optimized to yield potent Syk inhibitors with strong cellular activity. Incorporation of cis and trans adamantanol motifs into the R2 position was critical for achieving good cellular potency. Introduction of 3-(4-pyrazolyl)phenyl groups into R1 further enhanced potency. Compounds such as 13d, 13f, 13i, 14b, and 14d exhibited excellent enzyme and cellular activity. Among them, compound 13f demonstrated a favorable selectivity profile,Cevidoplenib good permeability, and no cytotoxicity.