PURPOSE: The P2Y(14) receptor (P2Y(14)R) is closely associated with several inflammatory diseases in humans. Although several P2Y(14)R antagonists have been reported to date, few have been successfully developed as therapeutic drugs, and none have entered clinical trials. We aimed to obtain P2Y(14)R antagonists with high antagonistic activity and druggability for further investigation into anti-inflammatory drugs. METHODS: Three series of novel P2Y(14)R antagonists were screened. The druggability of the most promising compounds was evaluated through assays for the inhibition of cytochrome P450 and hERG (human Ether-à-go-go-Related Gene) channels, as well as pharmacokinetic experiments. The in vivo efficacy of the lead compound was assessed in a Lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. RESULTS: We designed a series of N-acyl tryptophan derivatives as novel and potent P2Y(14)R antagonists based on the hit compound 7. Among them, compound II-3 with an IC(50) value of 1.2 nM, was a better antagonist than PPTN with an IC(50) value of 2.0 nM. Through structural modification, the zwitterionic character was eliminated, resulting in significantly improved solubility and oral bioavailability compared to PPTN. We have confirmed that P2Y(14)R is highly expressed in macrophages of ALI lung tissue. II-3, as a P2Y(14)R antagonist, can alleviate the pathological progression of ALI by inhibiting the activation of the NLRP3 inflammasome pathway and reducing the release of inflammatory factors, thus providing direct evidence for P2Y(14)R as a therapeutic target. CONCLUSION: Compound II-3 with potent P2Y(14)R antagonistic activity, may be a promising candidate for further investigation as an anti-inflammatory drug.