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Formyl peptide receptor 2 (FPR2) agonists can boost the resolution of inflammation and can offer alternative approaches for the treatment of pathologies with underlying chronic neuroinflammation, including neurodegenerative disorders. Starting from the FPR2 agonist 2 previously identified in our laboratory and through fine-tuning of FPR2 potency and metabolic stability, we have identified a new series of ureidopropanamide derivatives endowed with a balanced combination of such properties. Computational studies provided insights into the key interactions of the new compounds for FPR2 activation. In mouse microglial N9 cells and in rat primary microglial cells stimulated with lipopolysaccharide, selected compounds inhibited the production of pro-inflammatory cytokines, counterbalanced the changes in mitochondrial function, and inhibited caspase-3 activity. Among the new agonists, ( S )- 11l stands out also for the ability to permeate the blood-brain barrier and to accumulate in the mouse brain in vivo , thus representing a valuable pharmacological tool for studies in vivo .

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint inflammation, cartilage damage and bone destruction. Although the pharmacological treatment of RA has evolved over the last few years, the new drugs have serious side effects and are very expensive. Thus, the research has been directed in recent years towards new possible targets. Among these targets, N-formyl peptide receptors (FPRs) are of particular interest. Recently, the mixed FPR1/FPR2 agonist Cpd43 , the FPR2 agonist AT-01-KG , and the pyridine derivative AMC3 have been shown to be effective in RA animal models. As an extension of this research, we report here a new series of pyridinone derivatives containing the (substituted)phenyl acetamide chain, which was found to be essential for activity, but with different substitutions at position 5 of the scaffold. The biological results were also supported by molecular modeling studies and additional pharmacological tests on AMC3 have been performed in a rat model of RA, by repeating the treatments of the animals with 10 mg/kg/day of compound by 1 week.

N-formyl peptide receptors (FPR1, FPR2, and FPR3) play key roles in the regulation of inflammatory processes, and recently, it was demonstrated that FPR1 and FPR2 have a dual role in the progression/suppression of some cancers. Therefore, FPRs represent an important therapeutic target for the treatment of both cancer and inflammatory diseases. Previously, we identified selective or mixed FPR agonists with pyridazinone or pyridinone scaffolds showing a common 4-(bromophenyl)acetamide fragment, which was essential for activity. We report here new pyrazole and pyrazolone derivatives as restricted analogues of the above 6-membered compounds, all exhibiting the same 4-bromophenylacetamide side chain. Most new products had low or absent FPR agonist activity, suggesting that the pyrazole nucleus was not appropriate for FPR agonists. This hypothesis was confirmed by molecular modeling studies, which highlighted that the five-membered scaffold was responsible for a worse arrangement of the molecules in the receptor binding site., (© 2021 The Authors. Chemical Biology & Drug Design published by John Wiley & Sons Ltd.)

The resolution of inflammation is an active response involving the interaction of pro-resolving mediators with specific receptors, such as N-formyl peptide receptor 2 (FPR2). FPRs represent potentially important therapeutic targets for the treatment of some pathologies, including asthma and rheumatoid arthritis. Previously, we identified selective or mixed FPR agonists with a pyridazin-3(2H)-one scaffold, all containing a 4-bromophenylacetamide fragment at N-2. The most effective compounds in this series were EC3, a potent mixed FPR1/FPR2/FPR3 agonist, and EC10, which had a preference for FPR1. We report here a new series of pyridinone and pyrimidindione derivatives containing the 4-(bromophenyl)acetamide substituent that was essential for activity in the pyridazinone series. All new compounds were evaluated for FPR agonist activity in HL60 cells transfected with FPR1 or FPR2 and in human neutrophils. While most of the pyridinone derivatives had reasonable FPR agonist activity in the submicromolar/micromolar range, the pyrimidindione derivatives were less active. Compound 2a (N-(4-bromophenyl)-2-[3-cyano-5-(3-methoxyphenyl)-6-methyl-2-oxopyridin-1(2H)-yl]acetamide) was the most active pyridinone derivative and had a 10-fold preference for FPR2 (EC 50  = 120 nM) versus FPR1 (EC 50  = 1.6 μM). To assess their therapeutic activity, compounds 2a, EC3, and EC10 were evaluated in vivo using a rat model of rheumatoid arthritis. All three compounds increased the pain threshold and reduced pain hypersensitivity in the treated rats versus control rats, although 2a and EC10 were much more effective than EC3. Thus, these FPR agonists represent potential leads to develop for the treatment of inflammatory diseases such as rheumatoid arthritis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Formyl peptide receptor 2 (FPR2) is a G protein coupled receptor belonging to the N-formyl peptide receptor (FPR) family that plays critical roles in peripheral and brain inflammatory responses. FPR2 has been proposed as a target for the development of drugs that could facilitate the resolution of chronic inflammatory reactions by enhancing endogenous anti-inflammation systems. Starting from lead compounds previously identified in our laboratories, we designed a new series of ureidopropanamide derivatives with the goal of converting functional activity from agonism into antagonism and to develop new FPR2 antagonists. Although none of the compounds behaved as antagonists, some of the compounds were able to induce receptor desensitization and, thus, functionally behaved as antagonists. Evaluation of these compounds in an in vitro model of neuroinflammation showed that they decreased the production of reactive oxygen species in mouse microglial N9 cells after stimulation with lipopolysaccharide. These FPR2 ligands may protect cells from damage due to inflammation-associated oxidative stress., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Formyl peptide receptors (FPRs) are expressed on a variety of leukocytes and play important roles in inflammation. Thus, FPR antagonists may represent novel therapeutics for modulating innate immunity and treating inflammatory diseases. Previously, 1H-pyrrol-2(5H)-ones were reported to be potent and competitive FPR1 antagonists. In the present studies, 42 additional 1H-pyrrol-2(5H)-one analogs were evaluated for FPR1 antagonist activity. We identified a number of novel competitive FPR1 antagonists that inhibited N-formylmethionyl-leucyl-phenylalanine (fMLF)-induced intracellular Ca 2+ mobilization in FPR1-transfected HL60 cells and effectively competed with WKYMVm-FITC for binding to FPR1 in FPR1-transfected RBL cells. The most active pyrroles inhibited human neutrophil Ca 2+ flux, chemotaxis, and adhesion to human epithelial cells, with the most potent being compounds 14 (4-benzoyl-1-hexyl-3-hydroxy-5-(4-hydroxy-3-methoxyphenyl)-2,5-dihydro-1H-pyrrol-2-one) and 17 (4-benzoyl-5-(2,5-dimethoxyphenyl)-3-hydroxy-1-(2-methoxyethyl)-2,5-dihydro-1H-pyrrol-2-one). In addition, these FPR1 antagonists inhibited fMLF-induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in FPR1-RBL cells, differentiated HL-60 cells, and human neutrophils. Most of the antagonists were specific for FPR1 and did not inhibit WKYMVM/WKYMVm-induced intracellular Ca 2+ mobilization in FPR2-HL60 cells, FPR3-HL60 cells, or interleukin 8-induced Ca 2+ flux in human neutrophils. Moreover, molecular modeling showed that the active pyrroles had a significantly higher degree of similarity with the FPR1 antagonist pharmacophore template as compared to inactive analogs. Thus, the 4-aroyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-one scaffold represents an important backbone for the development of novel FPR1 antagonists and could provide important clues for understanding the molecular structural requirements of FPR1 antagonists., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Preclinical Research Formyl peptide receptors (FPRs) are G-protein-coupled receptors that play an important role in the regulation of inflammatory process and cellular dysfunction. In humans, three different isoforms are expressed (FPR1, FPR2, and FPR3). FPR2 appears to be directly involved in the resolution of inflammation, an active process carried out by specific pro-resolving mediators that modulate specific receptors. Previously, we identified 2-arylacetamido pyridazin-3(2H)-ones as FPR1- or FPR2-selective agonists, as well as a large number of mixed-agonists for the three isoforms. Here, we report a new series of 2-arylacetamido pyridazinones substituted at position 5 and their development as FPR agonists. We also synthesized a new series of 2-oxothiazolones bearing a 4-bromophenylacetamido fragment, which was fundamental for activity in the pyridazinone series. The compounds of most interest were 4a, a potent, mixed FPR agonist recognized by all three isotypes (FPR1 EC 50  = 19 nM, FPR2 EC 50  = 43 nM, FPR3 EC 50  = 40 nM), and 4b, which had potent activity and a preference for FPR2 (EC 50  = 13 nM). These novel compounds may represent valuable tools for studying FPR activation and signaling. Drug Dev Res 78 : 49-62, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Formyl peptide receptor 1 (FPR1) regulates a wide variety of neutrophil functional responses and plays an important role in inflammation and the pathogenesis of various diseases. To date, a variety of natural and synthetic molecules have been identified as FPR1 ligands. Here, we review current knowledge on natural products and natural product-inspired small molecules reported to antagonize and/or inhibit the FPR1-mediated responses. Based on this literature, additional screening of selected commercially available natural compounds for their ability to inhibit fMLF-induced Ca(2+) mobilization in human neutrophils and FPR1 transfected HL-60 cells, and pharmacophore modeling, natural products with potential as FPR1 antagonists are considered and discussed in this review. The identification and characterization of natural products that antagonize FPR1 activity may have potential for the development of novel therapeutics to limit or alter the outcome of inflammatory processes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

N-Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) that play critical roles in inflammatory reactions, and FPR-specific interactions can possibly be used to facilitate the resolution of pathological inflammatory reactions. We here report the synthesis and biological evaluation of six pairs of chiral ureidopropanamido derivatives as potent and selective formyl peptide receptor-2 (FPR2) agonists that were designed starting from our lead agonist (S)-3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(5-methoxy-2-pyridinyl)cyclohexyl]methyl]propanamide ((S)-9a). The new compounds were obtained in overall yields considerably higher than (S)-9a. Several of the new compounds showed agonist properties comparable to that of (S)-9a along with higher selectivity over FPR1. Molecular modeling was used to define chiral recognition by FPR2. In vitro metabolic stability of selected compounds was also assessed to obtain preliminary insight on drug-like properties of this class of compounds., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The synthesis of three racemates and the corresponding non-chiral analogues of a C5-methyl pyridazine series is described here, as well as the isolation of pure enantiomers and their absolute configuration assignment. In order to obtain optically active compounds, direct chromatographic methods of separation by HPLC-UV were investigated using four chiral stationary phases (CSPs: Lux Amylose-2, Lux Cellulose-1, Lux Cellulose-2 and Lux Cellulose-3). The best resolution was achieved using amylose tris(5-chloro-2-methylphenylcarbamate) (Lux Amylose-2), and single enantiomers were isolated on a semipreparative scale with high enantiomeric excess, suitable for biological assays. The absolute configuration of optically active compounds was unequivocally established by X-ray crystallographic analysis and comparative chiral HPLC-UV profile. All compounds of the series were tested for formyl peptide receptor (FPR) agonist activity, and four were found to be active, with EC50 values in the micromolar range., (© 2013 Wiley Periodicals, Inc.)

Formyl peptide receptors (FPRs) play an essential role in the regulation of endogenous inflammation and immunity. In the present studies, a large series of pyridazin-3(2H)-one derivatives bearing an arylacetamide chain at position 2 was synthesized and tested for FPR agonist activity. The pyridazin-3(2H)-one ring was confirmed to be an appropriate scaffold to support FPR agonist activity, and its modification at the 4 and 6 positions led to the identification of additional active agonists, which induced intracellular Ca(2+) flux in HL-60 cells transfected with either FPR1, FPR2, or FPR3. Seven formyl peptide receptor 1 (FPR1)-specific and several mixed FPR1/FPR2 dual agonists were identified with low micromolar EC50 values. Furthermore, these agonists also activated human neutrophils, inducing intracellular Ca(2+) flux and chemotaxis. Finally, molecular docking studies indicated that the most potent pyridazin-3(2H)-ones overlapped in their best docking poses with fMLF and WKYMVM peptides in the FPR1 and FPR2 ligand binding sites, respectively. Thus, pyridazinone-based compounds represent potential lead compounds for further development of selective and/or potent FPR agonists., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

N-formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) that play critical roles in inflammatory reactions, and FPR-specific interactions can possibly be used to facilitate the resolution of pathological inflammatory reactions. Recent studies indicated that FPRs have stereo-selective preference for chiral ligands. Here, we investigated the structure-activity relationship of 24 chiral ureidopropanamides, including previously reported compounds PD168368/PD176252 and their close analogs, and used molecular modeling to define chiral recognition by FPR2. Unlike previously reported 6-methyl-2,4-disubstituted pyridazin-3(2H)-ones, whose R-forms preferentially activated FPR1/FPR2, we found that four S-enantiomers in the seven ureidopropanamide pairs tested preferentially activated intracellular Ca(2+) flux in FPR2-transfected cells, while the R-counterpart was more active in two enantiomer pairs. Thus, active enantiomers of FPR2 agonists can be in either R- or S-configurations, depending on the molecular scaffold and specific substituents at the chiral center. Using molecular modeling approaches, including field point methodology, homology modeling, and docking studies, we propose a model that can explain stereoselective activity of chiral FPR2 agonists. Importantly, our docking studies of FPR2 chiral agonists correlated well with the FPR2 pharmacophore model derived previously. We conclude that the ability of FPR2 to discriminate between the enantiomers is the consequence of the arrangement of the three asymmetric hydrophobic subpockets at the main orthosteric FPR2 binding site with specific orientation of charged regions in the subpockets., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A series of chiral pyridazin-3(2H)-ones was synthesized, separated as pure enantiomers, and evaluated for N-formyl peptide receptor (FPR) agonist activity. Characterization of the purified enantiomers using combined chiral HPLC and chiroptical studies (circular dichroism, allowed unambiguous assignment of the absolute configuration for each pair of enantiomers). Evaluation of the ability of racemic mixtures and purified enantiomers to stimulate intracellular Ca(2+) flux in FPR-transfected HL-60 cells and human neutrophils and to induce β-arrestin recruitment in FPR-transfected CHO-K1 cells showed that many enantiomers were potent agonists, inducing responses in the sub-micromolar to nanomolar range. Furthermore, FPRs exhibited enantiomer selectivity, generally preferring the R-(-)-forms over the S-(+)-enantiomers. Finally, we found that elongation of the carbon chain in the chiral center of the active compounds generally increased biological activity. Thus, these studies provide important new information regarding molecular features involved in FPR ligand preference and report the identification of a novel series of FPR agonists., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human N-formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor (GPCR) involved in host defense and sensing cellular damage. Since structure-based ligand design for many GPCRs, including FPR1, is restricted by the lack of experimental three dimensional structures, homology modeling has been widely used to study GPCR-ligand binding. Indeed, receptor-ligand binding mode predictions can be derived from homology modeling with supporting ligand information. In the present work, we report comparative docking studies of 2-(benzimidazol-2-ylthio)-N-phenylacetamide derived FPR1 agonists, identified here and previously, with several known FPR1 peptide agonists in a FPR1 homology model that is based on the crystal structure of bovine rhodopsin. We found that the binding pocket of the most active molecules shares some common features with high affinity FPR1 peptide agonists, suggesting that they may bind to similar binding sites. Classification tree analysis led to the derivation of a good recognition model based on four amino acid descriptors for distinguishing FPR1 ligands from inactive analogs. Hence, the corresponding residues (Thr199, Arg201, Gly202, and Ala261) can be considered as markers of important areas in the ligand binding site. Concurrently, we identified several unique binding features of benzimidazole derivatives and showed that alkoxy-substituents of the benzimidazole ring are located within a FPR1 hole bounded by Thr199, Thr265, Ile268, and Leu271 or in a groove in the vicinity of Leu198, Arg201, Gly202, and Arg205. The understanding of these molecular features will likely prove beneficial in future design of novel FPR1 agonists based on the benzimidazole scaffold.

N-Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) involved in host defense and sensing cellular dysfunction. Thus, FPRs represent important therapeutic targets. In the present studies, we screened 32 ligands (agonists and antagonists) of unrelated GPCRs for their ability to induce intracellular Ca²+ mobilization in human neutrophils and HL-60 cells transfected with human FPR1, FPR2, or FPR3. Screening of these compounds demonstrated that antagonists of gastrin-releasing peptide/neuromedin B receptors (BB₁/BB₂) PD168368 [(S)-a-methyl-a-[[[(4-nitrophenyl)amino]carbonyl]amino]-N-[[1-(2-pyridinyl) cyclohexyl]methyl]-1H-indole-3-propanamide] and PD176252 [(S)-N-[[1-(5-methoxy-2-pyridinyl)cyclohexyl]methyl]-a-methyl-a-[[-(4-nitrophenyl)amino]carbonyl]amino-1H-indole-3-propanamide] were potent mixed FPR1/FPR2 agonists, with nanomolar EC₅₀ values. Cholecystokinin-1 receptor agonist A-71623 [Boc-Trp-Lys(ε-N-2-methylphenylaminocarbonyl)-Asp-(N-methyl)-Phe-NH₂] was also a mixed FPR1/FPR2 agonist, but with a micromolar EC₅₀. Screening of 56 Trp- and Phe-based PD176252/PD168368 analogs and 41 related nonpeptide/nonpeptoid analogs revealed 22 additional FPR agonists. Most were potent mixed FPR1/FPR2/FPR3 agonists with nanomolar EC₅₀ values for FPR2, making them among the most potent nonpeptide FPR2 agonists reported to date. In addition, these agonists were also potent chemoattractants for murine and human neutrophils and activated reactive oxygen species production in human neutrophils. Molecular modeling of the selected agonists using field point methods allowed us to modify our previously reported pharmacophore model for the FPR2 ligand binding site. This model suggests the existence of three hydrophobic/aromatic subpockets and several binding poses of FPR2 agonists in the transmembrane region of this receptor. These studies demonstrate that FPR agonists could include ligands of unrelated GPCR and that analysis of such compounds can enhance our understanding of pharmacological effects of these ligands.

N-formyl peptide receptors (FPRs) are important in host defense. Because of the potential for FPRs as therapeutic targets, recent efforts have focused on identification of non-peptide agonists for two FPR subtypes, FPR1 and FPR2. Given that a number of specific small-molecule agonists have recently been identified, we hypothesized that computational structure-activity relationship (SAR) analysis of these molecules could provide new information regarding molecular features required for activity. We used a training set of 71 compounds, including 10 FPR1-specific agonists, 36 FPR2-specific agonists, and 25 non-active analogs. A sequence of (1) one-way analysis of variance selection, (2) cluster analysis, (3) linear discriminant analysis, and (4) classification tree analysis led to the derivation of SAR rules with high (95.8%) accuracy for correct classification of compounds. These SAR rules revealed key features distinguishing FPR1 versus FPR2 agonists. To verify predictive ability, we evaluated a test set of 17 additional FPR agonists, and found that the majority of these agonists (>94%) were classified correctly as agonists. This study represents the first successful application of classification tree methodology based on atom pairs to SAR analysis of FPR agonists. Importantly, these SAR rules represent a relatively simple classification approach for virtual screening of FPR1/FPR2 agonists., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

N-formyl peptide receptor (FPR1) and N-formyl peptide receptor-like 1 (FPRL1, now known as FPR2) are G protein-coupled receptors involved in host defense and sensing cellular dysfunction. Because of the potential for FPR1/FPR2 as a therapeutic target, our recent high-throughput screening efforts have focused on the identification of unique nonpeptide agonists of FPR1/FPR2. In the present studies, we screened a chemolibrary of drug-like molecules for their ability to induce intracellular calcium mobilization in RBL-2H3 cells transfected with human FPR1 or FPR2. Screening of these compounds resulted in the identification of novel and potent agonists that activated both FPR1 and FPR2, as well as compounds that were specific for either FPR1 or FPR2 with EC(50) values in the low micromolar range. Specificity of the compounds was supported by analysis of calcium mobilization in HL-60 cells transfected with human FPR1 and FPR2. In addition, all but one agonist activated intracellular calcium flux and chemotaxis in human neutrophils, irrespective of agonist specificity for FPR1 or FPR2. Molecular modeling of the group of FPR1 and FPR2 agonists using field point methodology allowed us to create pharmacophore models for ligand binding sites and formulate requirements for these specific N-formyl peptide receptor agonists. These studies further demonstrate that agonists of FPR1/FPR2 include compounds with wide chemical diversity and that analysis of such compounds can enhance our understanding of their ligand/receptor interaction.

Following a ligand-based drug design approach, a potent mixed formyl peptide receptor 1 (FPR1) and formyl peptide receptor-like 1 (FPRL1) agonist (14a) and a potent and specific FPRL1 agonist (14x) were identified. These compounds belong to a large series of pyridazin-3(2H)-one derivatives substituted with a methyl group at position 6 and a methoxy benzyl at position 4. At position 2, an acetamide side chain is essential for activity. Likewise, the presence of lipophilic and/or electronegative substituents in the position para to the aryl group at the end of the chain plays a critical role for activity. Affinity for FPR1 receptors was evaluated by measuring intracellular calcium flux in HL-60 cells transfected with FPR1, FPRL1, and FPRL2. Agonists were able to activate intracellular calcium mobilization and chemotaxis in human neutrophils. The most potent chemotactic agent (EC(50) = 0.6 microM) was the mixed FPR/FPRL1 agonist 14h.

Development of immunomodulatory agents that enhance innate immune responses represents a promising strategy for combating infectious diseases. In the present studies, we screened a series of 71 arylcarboxylic acid hydrazide derivatives for their ability to induce macrophage tumor necrosis factor alpha (TNF-alpha) production and identified six such compounds, including one compound previously shown to be a formyl peptide receptor (FPR/FPRL1) agonist. The two most potent compounds [compound 1, nicotinic acid [5-(3-bromophenyl)-2-furyl]methylene-hydrazide; compound 2, 4-fluoro-benzoic acid [5-(3-trifluoromethyl-phenyl)-2-furyl]-methylene-hydrazide] were selected for further analysis. These compounds induced de novo production of TNF-alpha in a dose- and time-dependent manner in human and murine monocyte/macrophage cell lines and in primary macrophages. These compounds also induced mobilization of intracellular Ca(2+), production of reactive oxygen species, and chemotaxis in human and murine phagocytes. Induction of macrophage TNF-alpha production was pertussis toxin-sensitive, and analysis of the cellular target of these compounds showed that they were FPRL1-specific agonists and that this response was blocked by FPR/FPRL1 and FPRL1-specific antagonists. In addition, pharmacophore modeling showed a high degree of similarity for low-energy conformations of these two compounds to the current pharmacophore model for FPR ligands ( Mol Pharmacol 68: 1301-1310, 2005 ). Overall, these compounds represent novel FPRL1 agonists that induce TNF-alpha, a response distinct from those induced by other known FPR and FPRL1 agonists.

We screened a chemolibrary of drug-like molecules for their ability to activate reactive oxygen species (ROS) production in murine phagocytes, and we identified 26 novel compounds with potent neutrophil activating properties. We used substructure screening, fragment-focusing, and structure-activity relationship analyses to further probe the parent library and defined at least two groups of activators of ROS production in murine neutrophils: t-butyl benzene and thiophene-2-amide-3-carboxylic ester derivatives. Further studies of the active compounds revealed 11 compounds that activated ROS production in human neutrophils, and six of these compounds also activated intercellular Ca(2+) mobilization and chemotaxis in human neutrophils. Of the latter compounds, compound 14 (1,3-benzodioxolane-5-carboxylic acid 4'-benzyloxy-3'-methoxybenzylidene-hydrazide) activated neutrophils at nanomolar concentrations, and Ca(2+) mobilization was inhibited by pertussis toxin and N-t-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc-2), an antagonist of formyl peptide receptors (FPR/FPRL1). Likewise, activation by compound 14 was desensitized after N-formyl-Met-Leu-Phe pretreatment. Similar biological activities were found for compound 104 (1,3-benzodioxolane-5-carboxylic acid 3'-bromo-5'-ethoxy-4'-hydroxybenzylidene-hydrazide), an analog of compound 14. Furthermore, conformational analysis of the activators of chemotaxis and Ca(2+) mobilization showed a high degree of similarity in distances between pharmacophore points of compounds 14 and 104 with a model of FPR published by Edwards et al. (Mol Pharmacol 68:1301-1310, 2005), indicating that conformational features of the agonists identified here are structurally compatible with steric constraints of the ligand-binding pocket of the receptor. Based on these results, we conclude that compounds 14 and 104 represent novel small-molecule agonists of FPR. These studies enhance our understanding of FPR ligand/receptor interactions and structure/activity relationships of phagocyte agonists.