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4H-chromones (inset) are competitive FPR1 antagonists and inhibit formyl peptide binding and cell activation. SAR analysis of active compounds facilitated development of a model for antagonist docking in FPR1.Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) expressed on a variety of cell types. Because FPRs play an important role in the regulation of inflammatory reactions implicated in disease pathogenesis, FPR antagonists may represent novel therapeutics for modulating innate immunity. Previously, 4H-chromones were reported to be potent and competitive FPR1 antagonists. In the present studies, 96 additional chromone analogs, including related synthetic and natural isoflavones were evaluated for FPR1 antagonist activity. We identified a number of novel competitive FPR1 antagonists that inhibited fMLF-induced intracellular Ca2+ mobilization in FPR1-HL60 cells and effectively competed with WKYMVm-FITC for binding to FPR1 in FPR1-HL60 and FPR1-RBL cells. Compound 10 (6-hexyl-2-methyl-3-(1-methyl-1H-benzimidazol-2-yl)-4-oxo-4H-chromen-7-yl acetate) was found to be the most potent FPR1-specific antagonist, with binding affinity Ki ˜ 100 nM. These chromones inhibited Ca2+ flux and chemotaxis in human neutrophils with nanomolar-micromolar IC50 values. In addition, the most potent novel FPR1 antagonists inhibited fMLF-induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in FPR1-RBL cells. These antagonists were specific for FPR1 and did not inhibit WKYMVM/WKYMVm-induced intracellular Ca2+ mobilization in FPR2-HL60 cells, FPR3-HL60 cells, RBL cells transfected with murine Fpr1, or interleukin 8-induced Ca2+ flux in human neutrophils and RBL cells transfected with CXC chemokine receptor 1 (CXCR1). Moreover, pharmacophore modeling showed that the active chromones had a significantly higher degree of similarity with the pharmacophore template as compared to inactive analogs. Thus, the chromone/isoflavone scaffold represents a relevant backbone for development of novel FPR1 antagonists.