P2Y₁₄ Receptor Antagonists: Piperidine Bioisosteres and Mutagenesis-Supported Molecular Modeling

The human P2Y₁₄ receptor (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, <i>C</i>-linked 1,2,3-triazol-4-yl (<b>10</b>, MRS4916) and pyrazol-3-yl (<b>11</b>, MRS4917) substitutions yielded antagonists with IC₅₀ values of 3.69 and 2.88 nM, respectively. In contrast, incorporation of a second triazole in the phenyl-triazolyl series (<b>16</b>) significantly reduced affinity. Charged phosphate groups were strategically placed at two positions of potent triazole derivative <b>7</b> 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 <b>1</b> 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 <b>11</b> demonstrated oral efficacy in reversing mechanoallodynia in mice. Additionally, a selective P2Y₁₄R agonist, 2-thiouridine-5'-<i>O</i>-(α,β-methylene)-diphosphate (MRS2905), caused acute hypothermia in mice, likely via mast cell activation, while antagonists <b>1</b> and <b>11</b> 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.