Virtual Screening, Synthesis and In Vitro Characterization of Histamine H4 Receptor Ligands Based on Pyrimidine Scaffolds.

Histamine is a biologically active monoamine acting through four G protein-coupled receptors (H1R-H4R), which represent attractive therapeutic targets for a range of diseases. Nowadays, H4R is recognized as a key player in inflammation and cancer. Here, we describe the design, syntheses and characterization of new H4R ligands containing pyrido[2,3-d]pyrimidine or pyrimidine scaffold. Candidate structures were scored in silico by docking to the structure of the human inactive H4R. Favorable structures were synthesized and, after confirmation of identity, their affinities were verified in in vitro screenings. Tested compounds did not exhibit any relevant cytotoxic (PrestoBlue) or antiproliferative (BrdU incorporation) effects at concentrations used in the functional assays. In a radioligand displacement assay (H4R affinity) the pyrimidine series showed lower binding, whereas several pyrido[2,3-d]pyrimidine derivatives and one pyrimidine analog produced >60% inhibition at 1 µM. Two similar compounds with the highest and moderate affinities were selected for further studies. In the Gi-cAMP pathway, both compounds behaved as moderate H4R antagonists, but in contrast, their profiles diverged in the β-arrestin pathway. These findings suggest ligand-dependent biased differences in signaling across G-protein and β-arrestin pathways at H4R. Overall, YAN-153 emerges as a promising lead structure for further optimization and for more detailed in vitro (e.g., metabolic stability, selectivity) and in vivo studies.