P2Y₁₄Receptor Antagonists: Piperidine Bioisosteres and Mutagenesis-Supported Molecular Modeling

The human P2Y₁₄receptor (hP2Y₁₄R) has emerged as a promising target for inflammation and pain treatment, but its zwitterionic antagonists have low bioavailability. We extended the naphthalene-based antagonist series’ structure–activity relationship (SAR) by replacing an outward-facing piperidine moiety with small heteroaromatics. Notably, C-linked 1,2,3-triazol-4-yl (10, MRS4916) and pyrazol-3-yl (11, MRS4917) substitutions yielded antagonists with IC₅₀values of 3.69 and 2.88 nM, respectively. In contrast, incorporation of a second triazole in the phenyl-triazolyl series (16) significantly reduced affinity. Charged phosphate groups were strategically placed at two positions of potent triazole derivative 7 to explore the ligand’s binding site vicinity and detect potential proximity to cationic side chains but neither increased affinity. Site-directed mutagenesis was used to probe the antagonist binding site vicinity. However, residues that were previously predicted to participate in the binding of antagonist 1 were found to be nonessential. Molecular dynamics based on SAR and mutagenesis identified a critical interaction between the ligand’s carboxylate and R253, defining a binding pose where the aromatic core inserts into a hydrophobic cleft between TM6 and TM7. This interaction supports a minimally orthosteric antagonist mechanism. Compound 11 demonstrated oral efficacy in reversing mechanoallodynia in mice. Additionally, a selective P2Y₁₄R agonist, 2-thiouridine-5′-O-(α,β-methylene)diphosphate (MRS2905), caused acute hypothermia in mice, likely via mast cell activation, while antagonists 1 and 11 had no such effect. Our study refines the P2Y₁₄R antagonist binding model and introduces new drug-like scaffolds with improved solubility and CNS penetration. This work provides a platform for future SAR optimization and virtual screening campaigns targeting P2Y₁₄R.