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Growing evidence supporting the anti inflammatory and tissue
Growing evidence supporting the anti-inflammatory and tissue-protective effects of FPR antagonists led to the screening of commercial libraries for novel small-molecule FPR antagonists. As result of these screening efforts and/or structure–activity relationship (SAR)-directed design and synthesis, a number of synthetic non-peptide FPR1/FPR2 antagonists with a wide range of chemical diversity have been identified ([26], [27], [28], [29], [30], [31], [32], [33]). Structures of the most potent small-molecule FPR1 antagonists are shown in Fig. 1. Among these competitive FPR1 antagonists are some compounds with a 4H-chromen-4-one scaffold (Fig. 1, compounds 1–4) [26], [27]. However, activities of these chromones in primary cells, SAR analysis of related chromones and isoflavones, as well as molecular modeling have not been described.
In the present study, we evaluated 96 4H-chromen-4-ones, including synthetic and naturally occurring isoflavones, for their ability to antagonize FPR-dependent signaling in neutrophils and FPR-transfected Phenytoin sodium and identified novel and potent FPR1-specific antagonists. These antagonists were specific for FPR1 and did not inhibit FPR2-, FPR3-, or CXCR1-dependent responses. SAR analysis of these compounds revealed the importance of a small hydrophobic group at position 2 and the type of substituent at position 7 of the 4H-chromen-4-one scaffold. In addition, molecular modeling showed a high degree of similarity for low-energy conformations of these antagonists to the pharmacophore model for FPR1 antagonists. Overall, the isoflavone scaffold represents an appropriate backbone to develop novel FPR1 antagonists.
Materials and methods
Results
Discussion
4H-Chromones are an important class of synthetic and natural compounds that exhibit a broad range of biological activities. Their closely related derivatives are isoflavones, a large family of secondary plant metabolites with several variants of a heterocyclic ring substitution pattern. These compounds, along with their synthetic analogues, possess a wide variety of biological activities, including antiinflammatory, antiproliferative, antiosteoporotic, antihyperglycemic, antifungal, antiviral, antiparasitic, antioxidant, and cardiovascular effects [50], [51], [52], [53], [54], [55]. Although reactive oxidant scavenging activity of flavonoids/isoflavones may contribute to modulation of phagocyte functional activity [56], [57], these compounds also interact with other cellular targets, including receptors, enzymes, and other macromolecules (for review [58], [59], [60]). Such properties include actions upon peroxisome proliferator-activated receptor isoforms, estrogen receptors, tyrosine kinases, DNA topoisomerase II, and NF-κB activation [61], [62], [63]. There are a few reports regarding regulation of GPCRs by isoflavones, although most describe effects on estrogen receptor 1 (GPER1/GPR30) [64], [65].
Previously, several 4H-chromen-4-one derivatives were identified as small-molecule competitive FPR1 antagonists [26], [27]. In the current work, we report further characterization and development of related analogs with improved potency and FPR1 antagonist activity in functional tests using transfected cells and primary neutrophils. Screening of a library of 96 4H-chromen-4-ones, which are structural derivatives of four known chromone-derived competitive FPR1 antagonists 1–4, resulted in the discovery of novel potent FPR1 antagonists. We also showed that these compounds can compete with FITC-labeled WKYMVm for binding with FPR1 in both FPR1-HL-60 and FPR1-RBL transfected cells. Compound 10 was the most potent FPR1 antagonist and exhibits the highest binding affinity among all known chromone analogs, including previously reported compounds 1–4.
A review of the current literature regarding small-molecule FPR1 antagonists [66] showed that pyrazole-4-carboxamides are even more active at inhibition of fMLF-induced Ca2+ flux in human neutrophils, with the most potent being compound 6 (see the structure in Fig. 1), but competition binding at FPR1 for these compounds was not evaluated [29]. In addition, all independent conformations generated for molecule 6 in our modeling experiments did not exhibit good correspondence with the molecular fields of the other three antagonists with different chemical scaffolds (1, 5, and 7) that were used for building the FPR1 pharmacophore template. Thus, inhibition of fMLF-induced Ca2+ flux in human neutrophils by pyrazole-4-carboxamides may involve FPR1-independent mechanisms.