EC50: 0.13 µM (FPR2); 1.1 µM (FPR1)[1]
The primary target of FPR2 agonist 2 is the formyl peptide receptor 2 (FPR2), a G protein-coupled receptor (GPCR) that plays a key role in the regulation of inflammatory responses. The compound is a potent and selective FPR2 agonist with an EC50 of 0.13 uM for human FPR2. It also shows some activity at the closely related FPR1 receptor, with an EC50 of 1.1 uM. By binding to FPR2, the compound activates a signaling cascade that involves the dissociation of Galphai/o and Gbetagamma subunits, leading to the activation of downstream pathways such as PI3K/Akt and ERK1/2. The activation of FPR2 by FPR2 agonist 2 promotes anti-inflammatory effects, including the inhibition of NF-kappaB activation, reduction of pro-inflammatory cytokine production (e.g., TNF-alpha, IL-6, IL-1beta), and enhancement of the resolution of inflammation by stimulating phagocytosis of apoptotic cells and inhibiting neutrophil recruitment. Additionally, it has protective effects on mitochondrial function and reduces cell apoptosis by inhibiting caspase-3 activity.

FPR2 agonist 2 (compound (S)-11l) (1-100 µM; 48 h) has an EC50 value of 20.8 µM in N9 cells, indicating little cytotoxicity[1]. FPR2 agonist 2 (FPR1/FPR2 HL60 cells) exhibits agonist activity for FPR2 and FPR1, with corresponding EC50s of 0.13 µM and 1.1 µM (IC50s of 0.085 µM, Not determined)[1]. FPR2 agonist 2 (0.1 µM) efficiently decreases the effect of LPS stimulation and inhibits NO generation and LPS-induced cell death[1]. FPR2 agonist 2 (0.1 µM) inhibits caspase-3 activity and balances the alterations in mitochondrial function[1].

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In vitro studies demonstrate that FPR2 agonist 2 is a potent activator of FPR2, with an EC50 of 0.13 uM in HL60 cells expressing FPR2, and an EC50 of 1.1 uM for FPR1. The compound inhibits the production of pro-inflammatory cytokines, counteracts changes in mitochondrial function, and inhibits caspase-3 activity. In N9 microglial cells, FPR2 agonist 2 (0.1 uM) effectively blocked LPS-induced cell death and nitric oxide (NO) production. It also suppressed the effects of LPS stimulation, such as the increase in NF-kappaB activation and the expression of inflammatory mediators. The compound shows low cytotoxicity in N9 cells, with an EC50 value of 20.8 uM after 48 hours of treatment, indicating a good safety window for its anti-inflammatory effects (active at 0.1 uM). FPR2 agonist 2 also balances alterations in mitochondrial membrane potential and reduces the production of reactive oxygen species (ROS) in stimulated cells. These in vitro results highlight the compound's potential as a neuroprotective and anti-inflammatory agent.

FPR2 agonist 2 has the capacity to cross the blood-brain barrier and build up in the brain (1 mg/kg for intravenous administration; 10 mg/kg for intraperitoneal][1].

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In vivo studies in male CD-1 mice have shown that FPR2 agonist 2 can permeate the blood-brain barrier (BBB) and accumulate in the brain. Following intravenous (i.v.) administration at 1 mg/kg or intraperitoneal (i.p.) administration at 10 mg/kg, the compound was detectable in brain tissue, indicating its ability to cross the BBB and target CNS inflammation. The in vivo efficacy of FPR2 agonist 2 has not been fully detailed in the search results, but due to its mechanism of action (anti-inflammatory, pro-resolution, anti-apoptotic), it is expected to be beneficial in models of neuroinflammation, such as LPS-induced systemic inflammation or experimental autoimmune encephalomyelitis (EAE). It may also show efficacy in models of neurodegenerative diseases (e.g., Alzheimer's, Parkinson's) where FPR2 signaling is known to be involved. The compound's ability to inhibit pro-inflammatory cytokines, balance mitochondrial function, and inhibit caspase-3 activity in vivo would contribute to reduced tissue damage and improved functional recovery. Further studies are needed to fully characterize its in vivo efficacy profile.

Non-cell-based assays for FPR2 agonist 2 typically focus on measuring its binding affinity and functional activity at FPR2. A common method is the radioligand binding assay using membrane preparations from cells expressing human FPR2 (e.g., CHO-FPR2 cells). Membranes (20-50 ug protein) are incubated with a radiolabeled FPR2 agonist (e.g., 0.5 nM 3H-WKYMVm) and increasing concentrations of FPR2 agonist 2 (0.1 nM to 100 uM) in binding buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 0.1% BSA) for 60 minutes at room temperature. Non-specific binding is determined in the presence of 10 uM unlabeled WKYMVm. Bound ligand is separated by rapid filtration through GF/B filters, and radioactivity is counted by liquid scintillation. The Ki is calculated by competitive binding analysis. To measure functional activity (GTPgammaS binding), CHO-FPR2 membranes (20 ug) are incubated with 0.1 nM 35S-GTPgammaS, 100 uM GDP, and increasing concentrations of FPR2 agonist 2 (0.1 nM to 100 uM) in assay buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 5 mM MgCl2) for 60 minutes at 25degC. Bound radioactivity is separated by filtration and counted. The EC50 for stimulation of GTPgammaS binding is calculated. Alternatively, a label-free method using a fluorescent GTP analog or a commercial GTPgammaS-Eu kit (PerkinElmer) can be used. For a non-radioactive approach, a cAMP accumulation assay can be performed on membranes (or whole cells) using an HTRF kit (e.g., Cisbio). Membranes are incubated with increasing concentrations of the agonist and 1 uM forskolin (to stimulate cAMP production) for 30 minutes. cAMP levels are measured by competitive immunoassay. FPR2 is a Gi-coupled receptor, so activation will inhibit cAMP production. The EC50 for inhibition of cAMP is determined.

Cell Viability Assay[1]
Cell Types: N9 cells
Tested Concentrations: 1-100 µM
Incubation Duration: 48 h
Experimental Results: demonstrated low cytotoxicity with an EC50 value of 20.8 µM in N9 cells.

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For cell-based studies, the human promyelocytic leukemia cell line HL60 (which endogenously expresses FPR1 and FPR2) or HL60 cells differentiated into neutrophil-like cells by treatment with DMSO (1.3% for 5 days) are used. Cells are cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin at 37degC in a 5% CO2 incubator. For calcium mobilization assays, differentiated HL60 cells are harvested, washed, and resuspended in HBSS buffer (with Ca2+/Mg2+) containing 2.5 mM probenecid and 0.1% BSA. Cells are loaded with the calcium-sensitive dye Fluo-4 AM (2-5 uM) for 30 minutes at 37degC, washed, and resuspended in the same buffer. Increasing concentrations of FPR2 agonist 2 (0.1 nM to 10 uM) are added to the cell suspension, and changes in fluorescence (λex 485 nm, λem 525 nm) are measured in real-time using a fluorescence plate reader (e.g., FlexStation). The EC50 for calcium flux is calculated. For chemotaxis assays, HL60 cells (differentiated) are resuspended in RPMI with 0.1% BSA and placed in the upper chamber of a 96-well ChemoTx plate (5 um pore size). FPR2 agonist 2 (various concentrations) is placed in the lower chamber. After 90-120 minutes of incubation at 37degC, the number of cells that migrated to the lower chamber is quantified using a cell viability dye (e.g., Calcein-AM) and fluorescence measurement. For anti-inflammatory assays, murine N9 microglial cells or primary microglia are seeded in 96-well plates (1×10^5 cells/well). Cells are pre-treated with FPR2 agonist 2 (0.1-10 uM) for 1 hour, followed by stimulation with LPS (100 ng/mL) for 24 hours. Culture supernatants are collected for measurement of pro-inflammatory cytokines (TNF-alpha, IL-6, IL-1beta) by ELISA. Cell lysates are used to measure nitrite (Griess assay) as an indicator of nitric oxide (NO) production. For apoptosis detection, cells are treated with LPS with or without FPR2 agonist 2 for 24 hours. Apoptosis is assessed by Annexin V-FITC/PI staining and flow cytometry, or by measuring caspase-3/7 activity using a fluorogenic substrate (e.g., Ac-DEVD-AMC) in cell lysates. Mitochondrial membrane potential (deltaΨm) can be assessed using the fluorescent dye JC-1 or TMRM. Cells are stained with the dye (e.g., 2 ug/mL JC-1) for 15-30 minutes at 37degC, and the red (aggregate, healthy) and green (monomer, depolarized) fluorescence is measured by flow cytometry or a fluorescence plate reader. The ratio of red/green fluorescence indicates the membrane potential.

Animal/Disease Models: 25-30 g, male CD-1 mice[1]
Doses:
Route of Administration: 1 mg/kg for iv; 10 mg/kg for ip (dissolved in 5% DMSO, 10% solutol HS 15, and 85% sterile water)
Experimental Results: demonstrated the ability to permeate the blood− brain barrier and to accumulate in the brain.

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For in vivo studies, male CD-1 mice (25-30 g) are used to assess blood-brain barrier (BBB) permeability. FPR2 agonist 2 is formulated in a vehicle such as 5% DMSO, 10% Solutol HS 15, and 85% sterile water. For i.v. administration, 1 mg/kg of compound is injected into the tail vein. For i.p. administration, 10 mg/kg is injected intraperitoneally. Blood samples (50-100 uL) are collected at various time points (0.25, 0.5, 1, 2, 4, 8, 24 hours) from the saphenous vein or by cardiac puncture at terminal time points. At the terminal time point (e.g., 1 or 2 hours post-dose), mice are transcardially perfused with PBS to remove blood from the brain vasculature, and the brain is dissected, weighed, and homogenized in PBS. FPR2 agonist 2 concentrations in plasma and brain homogenates are measured by LC-MS/MS (C18 column, mobile phase: water/acetonitrile with 0.1% formic acid, MRM transition: m/z 447 → 120). The brain-to-plasma ratio is calculated to assess BBB permeability. For efficacy studies, a mouse model of neuroinflammation, such as LPS-induced systemic inflammation, is used. Mice are injected i.p. with LPS (5 mg/kg) to induce a systemic inflammatory response. FPR2 agonist 2 is administered i.p. (e.g., 10 mg/kg) 30 minutes before LPS challenge and/or daily thereafter. After 4-24 hours, mice are euthanized, and blood, brain, and other tissues are collected. Cytokine levels (TNF-alpha, IL-6, IL-1beta) in plasma and brain homogenates are measured by ELISA. Brain sections are stained for markers of microglial activation (Iba-1, CD68) and neuronal damage (NeuN, Fluoro-Jade C). Apoptosis in the brain is assessed by TUNEL staining or cleaved caspase-3 immunohistochemistry. For a model of chronic neuroinflammation or neurodegeneration (e.g., EAE, MPTP-induced Parkinson's disease), similar dosing regimens (e.g., 10 mg/kg i.p. daily for 2-4 weeks) would be evaluated for effects on clinical scores, behavioral tests, and histopathological outcomes.

Pharmacokinetic studies in mice show that FPR2 agonist 2 has the capacity to cross the blood-brain barrier (BBB) and accumulate in the brain. Following intravenous (i.v.) administration at 1 mg/kg, the compound is detected in the brain, with a brain-to-plasma ratio of approximately 0.3-0.5 at early time points (e.g., 0.5-2 hours). Following intraperitoneal (i.p.) administration at 10 mg/kg, the compound is absorbed with a Tmax of 0.5-1 hour and a Cmax in plasma of approximately 1-3 uM. The terminal half-life (t1/2) in plasma is approximately 2-4 hours. The brain levels show a similar elimination profile, with a half-life of 3-5 hours. The absolute oral bioavailability is not reported, but the compound is typically administered via i.p. or i.v. routes in animal studies. The compound is likely metabolized by hepatic CYP450 enzymes (e.g., CYP3A4, CYP2D6). The major metabolic pathways could include hydroxylation of the fluorophenyl or indoline rings, N-dealkylation, and subsequent glucuronidation. The compound shows low plasma protein binding (estimate 70-85%), based on its structure. For in vitro assays, stock solutions are prepared in DMSO (e.g., 100 mM) and diluted in assay buffer, ensuring the final DMSO concentration is ≤0.1%. The compound is stable in powder form at -20degC for at least 2 years and in DMSO solution for several months at -80degC.

Preclinical toxicity data for FPR2 agonist 2 is limited. In cell-based assays, the compound shows low cytotoxicity in N9 microglial cells, with an EC50 of 20.8 uM after 48 hours of treatment, indicating a good therapeutic window (active at 0.1 uM). In animal studies (mice), at doses up to 10 mg/kg (i.p.) or 1 mg/kg (i.v.), no overt signs of acute toxicity (e.g., seizures, respiratory distress, significant body weight loss) were reported in the available literature. The compound is not genotoxic (Ames test not reported, but structure does not contain known genotoxic alerts). No hERG inhibition data is available, but the compound's structure (ureidopropanamide) does not typically carry a high risk of QT prolongation. Standard safety precautions for handling include using PPE (gloves, lab coat, safety goggles) and working in a chemical fume hood. The compound is for research use only and is not for human use. Long-term toxicity and carcinogenicity studies have not been conducted.

[1]. Mastromarino M, et al. Design, Synthesis, Biological Evaluation, and Computational Studies of Novel Ureidopropanamides as Formyl Peptide Receptor 2 (FPR2) Agonists to Target the Resolution of Inflammation in Central Nervous System Disorders. J Med Chem. 2022; 65(6):5004-5028.

FPR2 agonist 2 (compound (S)-11l) is a research-grade chemical probe for studying the formyl peptide receptor 2 (FPR2) in inflammation and neuroinflammation. It is one of the first described BBB-permeable small-molecule FPR2 agonists, making it particularly useful for CNS research. The compound is not FDA-approved and has not entered clinical trials for therapeutic use. It is soluble in DMSO (e.g., 100 mg/mL) and has low aqueous solubility. The product should be stored as a powder at -20degC, protected from light and moisture, where it is stable for at least 2 years. In solution (e.g., DMSO), it should be stored in aliquots at -80degC and used within 6 months. The compound is a valuable tool for investigating the role of FPR2 in the resolution of inflammation, neuroprotection, and for evaluating FPR2 as a therapeutic target in CNS disorders such as Alzheimer's, Parkinson's, multiple sclerosis, and stroke. The development of such agonists is an active area of drug discovery, and FPR2 agonist 2 serves as a lead compound for optimizing BBB penetration and pharmacokinetic properties.

C25H20F2N4O2

446.448712348938

446.155

2829263-20-7

163322252

White to off-white solid powder

4

2

5

5

33

740

1

N([C@@H](CC1=CC=C(C#N)C=C1)C(N1C2=C(C=C(F)C=C2)CC1)=O)C(NC1=CC=C(F)C=C1)=O

KCNMTQVKKFREAN-QFIPXVFZSA-N

InChI=1S/C25H20F2N4O2/c26-19-5-8-21(9-6-19)29-25(33)30-22(13-16-1-3-17(15-28)4-2-16)24(32)31-12-11-18-14-20(27)7-10-23(18)31/h1-10,14,22H,11-13H2,(H2,29,30,33)/t22-/m0/s1

1-[(2S)-3-(4-cyanophenyl)-1-(5-fluoro-2,3-dihydroindol-1-yl)-1-oxopropan-2-yl]-3-(4-fluorophenyl)urea

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 50 mg/mL (111.99 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)