CCR2
(s)-PF-4136309 is an inactive isomer that does not bind effectively to the chemokine receptor CCR2 (C-C chemokine receptor type 2). CCR2 is a G-protein-coupled receptor (GPCR) expressed on monocytes and macrophages, and it is a key mediator of inflammation. Its endogenous ligand is MCP-1 (CCL2). The active (R)-enantiomer acts as a potent, reversible, and competitive antagonist of CCR2, blocking the recruitment of inflammatory cells. Because the (S)-enantiomer cannot form the necessary stereospecific interactions with the receptor's orthosteric binding pocket, it is considered an inactive control compound and does not inhibit MCP-1-induced chemotaxis, calcium flux, or ERK phosphorylation.

In vitro activity: PF-4136309 exhibits potency in human chemotaxis activity (IC50 = 3.9 nM) and in the whole blood assay (IC50 = 19 nM). In mouse and rat chemotaxis assays, its IC50 values are 16 and 2.8 nM, respectively. With IC50 values of 3.3 and 0.5 nM, respectively, PF-4136309 exhibits potent inhibition of CCR2 mediated signaling events, including intracellular calcium mobilization and ERK (extracellular signal-regulated kinase) phosphorylation. With an IC50 of 20 μM, PF-4136309 suppresses the hERG potassium current in the hERG patch clamp assay. With IC50 values greater than 30 μM against the five main CYP isozymes CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, PF-4136309 is not a cytochrome P450 (CYP) inhibitor. Moreover, at concentrations up to 30 μM, PF-4136309 is not a CYP inducer[1].

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The active (R)-enantiomer (PF-4136309) demonstrates potent in vitro activity, with IC50 values of 5.2 nM, 17 nM, and 13 nM for human, mouse, and rat CCR2, respectively, in a membrane-based binding assay. It also potently inhibits MCP-1-induced chemotaxis in human monocytes (IC50 = 3.9 nM) and in whole blood (IC50 = 19 nM). In functional cell-based assays, it blocks CCR2-mediated intracellular calcium mobilization (IC50 = 3.3 nM) and ERK phosphorylation (IC50 = 0.5 nM). The (S)-enantiomer is reported to be inactive (IC50 > 10 microM) and is used as a negative control to validate that observed biological effects are due to specific target engagement rather than non-specific compound interactions.

PF-4136309 (2 mg/kg) shows a moderate half-life (2.5 and 2.4 hours) following intravenous administration in both species. When taken orally, PF-4136309 (10 mg/kg) is quickly absorbed, with rats and dogs experiencing peak concentration times (Tmax) of 1.2 and 0.25 hours, respectively. When administering IV and po doses to both species, a comparable half-life is seen. In both species, PF-4136309 exhibits a high degree of oral bioavailability, measuring 78%[1].

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The active (R)-enantiomer has been evaluated in multiple in vivo models of inflammatory disease. In a mouse model of delayed-type hypersensitivity (DTH), oral administration of PF-4136309 potently inhibited ear swelling (ED50 = 4.5 mg/kg). In a rat model of collagen-induced arthritis (CIA), it significantly reduced paw swelling and disease scores. It has also shown efficacy in models of multiple sclerosis (EAE) and allograft rejection. The compound is known to be orally bioavailable and has entered human clinical trials for the treatment of pancreatic tumors and various inflammatory conditions. The (S)-enantiomer is inactive in these models and serves as an important negative control.

The binding affinity of the active enantiomer for CCR2 is determined using a standard radioligand binding assay. Membranes prepared from Chinese hamster ovary (CHO) cells expressing human, mouse, or rat CCR2 are incubated with [125I]-MCP-1 or a radiolabeled small molecule antagonist in the presence of varying concentrations of the test compound. After a 1-2 hour incubation at room temperature, the reaction is terminated by rapid filtration onto glass fiber filters. The filters are washed and the retained radioactivity is counted. The IC50 and Ki values are calculated by nonlinear regression using a competitive binding model. The assay is performed in the presence of a final DMSO concentration of 1% to maintain compound solubility. For the (S)-enantiomer, saturation binding experiments confirm negligible displacement of the radioligand even at the highest concentrations tested (up to 30 microM).

Functional in vitro cellular assays are performed to assess CCR2 antagonism. The most common is a chemotaxis assay using human peripheral blood monocytes (PBMCs) or THP-1 cells. Cells are loaded with a fluorescent dye and placed in the upper chamber of a transwell plate (5 microm pore size). The lower chamber contains buffer with 10 nM MCP-1 (CCL2) and varying concentrations of the test compound. After incubation for 1-2 hours at 37degC, the number of cells that migrated to the lower chamber is quantified using a fluorescence plate reader. IC50 values are calculated from the dose-response curves. An alternative functional assay measures MCP-1-induced intracellular calcium flux. Cells are loaded with a calcium-sensitive dye (e.g., Fluo-4 AM), and the fluorescence signal is monitored in real-time on a fluorometric imaging plate reader (FLIPR) following the addition of agonist in the presence or absence of the test compound.

In vivo efficacy is typically tested using the active (R)-enantiomer in a mouse model of thioglycollate-induced peritonitis. Female BALB/c mice are injected intraperitoneally (i.p.) with 1 mL of 4% sterile thioglycollate solution. Four hours later, the mice are orally gavaged with the test compound or vehicle control. After 20 hours, the mice are euthanized, and the peritoneal cavity is lavaged with 5 mL of PBS. The collected lavage fluid is analyzed by flow cytometry to quantify the number of infiltrated monocytes/macrophages (CD11b+). The ED50 for inhibition of monocyte recruitment is calculated. For a tumor model, human pancreatic cancer cells (e.g., BxPC-3, MIAPaCa-2) are implanted orthotopically or subcutaneously in immunodeficient mice, and the compound is administered orally daily. Tumor volume, metastasis, and survival are monitored to assess efficacy.

In vitro ADME (absorption, distribution, metabolism, and excretion) analysis showed that compound 17 (INCB8761/PF-4136309) had moderate permeability on Caco-2 monolayers, with a permeability coefficient of 3.1 × 10⁻⁶ cm/s. In protein binding assays, the free fraction of compound 17 in human serum was 23%. After incubation with human liver microsomes, compound 17 exhibited moderate intrinsic clearance with a half-life (t₁/₂) of 89 min. No glutathione adduct was detected when compound 17 was incubated with human S9 protein in the presence or absence of NADPH and the cofactor glutathione. Compound 17 is not a cytochrome P450 (CYP) inhibitor, and its IC50 values against the five major CYP isoenzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 are all >30 μM. Compound 17 is not a CYP inducer at concentrations up to 30 μM. [1] The pharmacokinetics of compound 17 (INCB8761/PF-4136309) were evaluated in rats and dogs (Table 4). After intravenous administration of compound 17, total systemic clearance was moderate in rats and low in dogs. The apparent steady-state volume of distribution (Vss) followed the same trend as clearance, with higher Vss in rats and lower Vss in dogs. Thus, compound 17 showed moderate half-lives in both animals after intravenous administration (2.5 h and 2.4 h, respectively). After oral administration, compound 17 was rapidly absorbed, with a time to peak concentration (Tmax) of 1.2 h in rats and 0.25 h in dogs. The half-lives of intravenous and oral administration were similar in both animals. Compound 17 was well absorbed, with an oral bioavailability of 78% in both animals. [1]

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The active (R)-enantiomer exhibits excellent pharmacokinetic properties: it is highly soluble (>10 mg/mL) in aqueous buffers, has a high fraction of unbound drug in human plasma (23% free fraction), and is metabolically stable. In mice, the compound has a plasma half-life (t1/2) of 2.6 hours, a low clearance (Cl) of 23 mL/min/kg, and high oral bioavailability (F%) of 46%. It is not a potent inhibitor (IC50 > 30 microM) or inducer of major CYP450 isoforms, reducing the risk of drug-drug interactions. The inactive (s)-PF-4136309 isomer is expected to have very similar absorption and distribution properties but lacks the specific target engagement that drives the pharmacological activity of its (R)-antipode.

The toxicity profile is derived from the active enantiomer. PF-4136309 has a clean preclinical safety profile with no significant off-target activities. It exhibits a high selectivity index (>100-fold) over other chemokine receptors (CCR1, CCR3, CCR5, CXCR4). In a hERG (human Ether-à-go-go-Related Gene) patch-clamp assay, the IC50 is 20 microM, indicating a low risk for QT prolongation at therapeutic doses. The compound is not genotoxic in the Ames test and has no significant cytotoxicity in normal human cell lines up to 30 microM. The inactive (S)-enantiomer, being chemically similar, is expected to have a benign toxicity profile, lacking the mechanism-based toxicities of the antagonist. As a control compound, it is handled under standard laboratory safety conditions.

[1]. Xue CB, et al. Discovery of INCB8761/PF-4136309, a Potent, Selective, and Orally Bioavailable CCR2 Antagonist. ACS Med. Chem. Lett., 2011, 2 (12), pp 913-918.

new class of (S)-3-aminopyrrolidine CCR2 antagonists has been discovered. Structure-activity relationship studies were conducted on this series of compounds, ultimately identifying compound 17 (INCB8761/PF-4136309), which exhibits potent CCR2 antagonistic activity, high selectivity, weak hERG activity, and excellent in vitro and in vivo ADMET properties. INCB8761/PF-4136309 has entered the human clinical trial stage. [1]

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(s)-PF-4136309 is a research tool used to dissect the role of CCR2 signaling. The active (R)-enantiomer, PF-4136309 (also known as INCB8761), is a first-in-class, potent, and selective small-molecule antagonist of CCR2. It has been developed for the treatment of inflammatory diseases, neuropathic pain, and various cancers (particularly pancreatic cancer and metastatic breast cancer). PF-4136309 has been evaluated in Phase I and Phase II clinical trials. Although its development as an anti-cancer monotherapy has been limited, it remains a valuable research tool and may be used in combination regimens. The enantiomer is critical for confirming that observed biological activities are due to specific CCR2 antagonism rather than nonspecific compound-related effects. This product is supplied as a research-grade compound, not for clinical use.

C29H31F3N6O3

568.59

568.24

C, 61.26; H, 5.50; F, 10.02; N, 14.78; O, 8.44

1372407-07-2

PF-4136309;1341224-83-6;(Rac)-PF-4136309;857679-55-1

11192346

Typically exists as solid at room temperature

1.4±0.1 g/cm3

712.2±60.0 °C at 760 mmHg

384.5±32.9 °C

0.0±2.4 mmHg at 25°C

1.626

2.1

3

10

7

41

894

1

FC(C1=CC=CC(=C1)C(NCC(N1CCC(C1)NC1CCC(C2C=CC(C3N=CC=CN=3)=CN=2)(CC1)O)=O)=O)(F)F

ZNSVOHSYDRPBGI-CBQRAPNFSA-N

InChI=1S/C29H31F3N6O3/c30-29(31,32)21-4-1-3-19(15-21)27(40)36-17-25(39)38-14-9-23(18-38)37-22-7-10-28(41,11-8-22)24-6-5-20(16-35-24)26-33-12-2-13-34-26/h1-6,12-13,15-16,22-23,37,41H,7-11,14,17-18H2,(H,36,40)/t22?,23-,28?/m0/s1

N-[2-[(3S)-3-[[4-hydroxy-4-(5-pyrimidin-2-ylpyridin-2-yl)cyclohexyl]amino]pyrrolidin-1-yl]-2-oxoethyl]-3-(trifluoromethyl)benzamide

1341224-83-6; PF-4136309; INCB8761; 1372407-07-2; INCB8761(PF-4136309); 857679-55-1; INCB-8761; PF4136309;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)