The P2Y₁₄ nucleotide receptor, a subtype of P2Y receptors, is implicated in many human inflammatory diseases. Based on the identification of favorable residues of two screening hits in the almost symmetrical P2Y₁₄ binding domain, we describe the structural optimization of previously identified virtual screening hits 6 and 7 that result in the development of P2Y₁₄R antagonists with a novel 2-phenyl-benzoxazole acetamide chemical scaffold. Notably, compound 52 showed potent P2Y₁₄R antagonistic activity (IC₅₀ = 2 nM), and a stronger inhibitory effect on MSU-induced inflammatory in vitro, better than a previously described P2Y₁₄R antagonist PPTN. In vivo evaluation demonstrated that compound 52 also had satisfactory inhibitory activity on the inflammatory response of gout flares in mice. Moreover, P2Y₁₄R antagonist 52 decreased paw swelling and inflammatory cell infiltration through cAMP/NLRP3/GSDMD signaling pathways in MSU-induced acute gouty arthritis mice. The discussions on the binding mechanism that employ MM/GBSA free energy calculations/decompositions also provide some useful clues for further structural designing of compound 52. Taken together, 2-phenyl-benzoxazole acetamide derivative 52 with potent P2Y₁₄R antagonistic activity and in vivo potency could be a promising strategy for gout therapy and deserves further optimization.

P2Y ₁₄核苷酸受体是 P2Y 受体的一种亚型，与许多人类炎症性疾病有关。基于对几乎对称的 P2Y ₁₄结合域中两个筛选命中的有利残基的鉴定，我们描述了先前确定的虚拟筛选命中6和7的结构优化，导致开发具有新型 2-苯基的 P2Y ₁₄ R 拮抗剂-苯并恶唑乙酰胺化学支架。值得注意的是，化合物52显示出有效的 P2Y ₁₄ R 拮抗活性 (IC _{50 = 2 nM)，并且}在体外 对 MSU 诱导的炎症具有​​更强的抑制作用，优于先前描述的 P2Y ₁₄ R 拮抗剂 PPTN。体内评估表明，化合物52对小鼠痛风发作的炎症反应也具有令人满意的抑制活性。此外，P2Y ₁₄ R 拮抗剂52通过 cAMP/NLRP3/GSDMD 信号通路减少 MSU 诱导的急性痛风性关节炎小鼠的爪肿胀和炎症细胞浸润。对采用 MM/GBSA 自由能计算/分解的结合机制的讨论也为化合物52的进一步结构设计提供了一些有用的线索。总之，2-苯基-苯并恶唑乙酰胺衍生物52与强效 P2Y ₁₄R 拮抗活性和体内效力可能是痛风治疗的一种有前途的策略，值得进一步优化。