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Purity: =98.21%
INCB3344 (INCB-3344 ) is a novel, potent, selective, orally bioavailable small molecule antagonist of the CCR2 receptor with anti-inflammatory activity. It inhibits CCR with IC50 values of 3.8 nM (hCCR2) and 7.8 nM (mCCR2) in antagonism of chemotaxis activity and 5.1 nM (hCCR2) and 9.5 nM (mCCR2) in binding antagonistic activity. Pharmacological studies of CCR2 antagonists and their potential therapeutic utility have been hindered by the inherent lack of rodent cross-reactivity in small molecule antagonists found to date. With nanomolar potency (IC(50) = 10 nM), INCB3344 inhibits CCL2 binding to mouse monocytes in vitro. It also exhibits dose-dependent inhibition of CCL2-mediated functional responses, including ERK phosphorylation and chemotaxis, with a similar potency. INCB3344 is at least 100-fold more selective for CCR2 than other G protein-coupled receptors against which other CC chemokine receptors are present. Because of its high oral bioavailability and systemic exposure in rodents, INCB3344 is suitable for in vivo pharmacological research. INCB3344 treatment results in a dose-dependent inhibition of macrophage influx in a mouse model of delayed-type hypersensitivity. The histopathological examination of tissues from the delayed-type hypersensitivity model indicates that CCR2 inhibition significantly reduces tissue inflammation, pointing to a potential orchestration role for macrophages in immune-based inflammatory responses. The investigation of INCB3344 in inflammatory disease models was prompted by these findings. In mice with experimental autoimmune encephalomyelitis, a model of multiple sclerosis, and in rats with inflammatory arthritis, therapeutic dosage of INCB3344 dramatically ameliorates disease. In conclusion, these findings lend support to the idea of treating chronic inflammatory diseases by targeting this receptor.
| Targets |
hCCR2 ( IC50 = 5.1 nM ); mCCR2 ( IC50 = 9.5 nM )
In vitro activity: INCB3344 exhibits strong antagonistic effects on both rat and cynomolgus CCR2; the latter two exhibit IC50 values of 2.7 and 6.2 nM in chemotaxis activity antagonistic activity and 7.3 and 16 nM in binding antagonistic activity, respectively. IC50 values of more than 1 μM are demonstrated by INCB3344, a selective antagonist of hCCR2, against a panel of more than 50 ion channels, transporters, chemokine receptors, and other specific GPCRs. It is also a selective mCCR2 antagonist, showing IC50 values of >1 μM and >3 μM against murine CCR1 and murine CCR5, respectively, the two most homologous chemokine receptors to mCCR2[1]. In this assay, the binding IC50 of INCB3344 is found to be 10±5 nM, and at a concentration of 90 nM, inhibition of >90% binding is seen[2]. |
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| ln Vitro |
In vitro activity: INCB3344 exhibits strong antagonistic effects on both rat and cynomolgus CCR2; the latter two exhibit IC50 values of 2.7 and 6.2 nM in chemotaxis activity antagonistic activity and 7.3 and 16 nM in binding antagonistic activity, respectively. IC50 values of more than 1 μM are demonstrated by INCB3344, a selective antagonist of hCCR2, against a panel of more than 50 ion channels, transporters, chemokine receptors, and other specific GPCRs. It is also a selective mCCR2 antagonist, showing IC50 values of >1 μM and >3 μM against murine CCR1 and murine CCR5, respectively, the two most homologous chemokine receptors to mCCR2[1]. In this assay, the binding IC50 of INCB3344 is found to be 10±5 nM, and at a concentration of 90 nM, inhibition of >90% binding is seen[2].
INCB3344 exhibited an IC₅₀ of 5.1 nM in a human MCP-1 binding assay using peripheral blood monocytes and an IC₅₀ of 3.8 nM in a human MCP-1 stimulated chemotaxis assay. [1] Against murine CCR2, INCB3344 exhibited an IC₅₀ of 9.5 nM in a binding assay and 7.8 nM in a chemotaxis assay using a murine monocytic cell line. [1] INCB3344 showed >1000-fold selectivity over a panel of >50 ion channels, transporters, chemokine receptors and other selected GPCRs. Specifically, it showed IC₅₀ values of >1 μM against murine CCR1 and >3 μM against murine CCR5. [1] In a dofetilide binding assay for hERG channel activity, INCB3344 showed an IC₅₀ of 13 μM. [1] The free fraction of INCB3344 in protein binding assays was 24% in human serum and 15% in mouse serum. [1] |
| ln Vivo |
INCB3344 has a brief half-life of one hour when given intravenously to CD-1 mice due to its high clearance and moderate volume of distribution. Good oral exposure is nevertheless obtained despite its high clearance, with an AUC at 2664 nM h at a dose of 10 mg/kg. There is a 47% oral bioavailability. In comparison, oral administration of the same dose to Balb/c mice results in slightly better oral exposure (AUC=3888 nM h). This PK property, couple with its potent anti-mCCR2 activity and good selectivity, makes this compound suitable for model studies in rodents[1]. INCB3344 inhibits the alterations in CCR2 vascular expression brought on by deoxycorticosterone acetate and/or salt. In an independent set of tests, CCR2 expression is significantly lower in the vehicle-treated group (P<0.05, n=6) than in the aortas of mice that receive INCB3344 from days 7 to 21 of the Deoxycorticosterone acetate/salt treatment period, despite being elevated (approximately 1.5-fold higher) when compared to sham animals. Similarly, mice given INCB3344 have reduced levels of increased expression of its receptor ligand CCL2 (P<0.05, n=6) compared to mice given Deoxycorticosterone acetate/salt. In contrast, mice treated with Deoxycorticosterone acetate and salt and given either vehicle or INCB3344 exhibit comparable increases in CCL7, CCL8, and CCL12 levels[3].
INCB3344 has been used as a tool compound for target validation and was effective in lowering macrophage content in target tissues and suppressing disease development in a mouse model of multiple sclerosis, a rat model of inflammatory arthritis, and a mouse model of obesity. [1] |
| Enzyme Assay |
Human MCP-1 Binding Assay: Human peripheral blood mononuclear cells (200,000 to 500,000) were incubated with a low concentration of ¹²⁵I-labeled MCP-1 in a binding buffer containing HEPES and BSA, with or without unlabeled competitor MCP-1 or various concentrations of the test compounds. The reaction was carried out for 30 minutes at room temperature and terminated by rapid filtration through glass fiber filters presoaked in polyethyleneimine or PBS. The filters were rinsed with buffer containing NaCl, dried, and the bound radioactivity was quantified using a gamma counter. [1]
Murine MCP-1 Binding Assay: A similar binding assay was performed using a murine monocytic cell line that expresses CCR2. [1] |
| Cell Assay |
WEHI-274.1, in a 96-well modified Boyden chamber, cells (5×105) in RPMI 1640 (VWR) are loaded into the wells above an 8-μm polycarbonate filter, either with or without different concentrations of INCB3344 in RPMI 1640. A matching 96-well plate containing 30 nM mCCL2—with or without INCB3344—or media is positioned underneath the filter. The sealed chambers are incubated for 45 min at 37°C, 5% CO2. Wright-Giemsa staining is applied to the filters, and microscopy is used to count the number of cells that migrate toward mCCL2 in the bottom chamber. The ability of INCB3344 to antagonize CCR2-mediated chemotaxis is reported as the inhibitor concentration required for IC50 values of specific migration to mCCL2. The total migration less the background migration is known as specific migration. Utilizing mouse MIP-1α as a ligand, an analogous assay is employed to evaluate the effect of INCB3344 on CCR1-mediated chemotaxis of WEHI-274.1 cells. Furthermore, C5a, FMLP, and RANTES are examined in a comparable manner for WEHI-274.1 cell migration when INCB3344 is present. Using mouse MIP-1β as the ligand, murine T cells are employed as the cell system in investigations on the effects of INCB3344 on CCR5-mediated chemotaxis[2].
Human MCP-1 Chemotaxis Assay: The capacity of compounds to antagonize CCR2 function was determined using human peripheral blood mononuclear cells in a modified Boyden chamber. Cells in serum-free medium, with or without inhibitors, were placed in the top chamber. MCP-1 was added to the bottom chamber. The chamber was incubated at 37°C with 5% CO₂ for 30 minutes. After incubation, cells on the top of the filter were washed away, and the filter was air-dried and stained. The number of cells that migrated to the bottom side of the filter was counted microscopically. Antagonist potency was determined by comparing migration in wells containing the antagonist to control wells containing only MCP-1. [1] Murine MCP-1 Chemotaxis Assay: A similar chemotaxis assay was performed using a murine monocytic cell line. [1] |
| Animal Protocol |
Mice: The CCR2 antagonist, INCB3344 (30 mg/kg per day; Haoyuan Chemexpress Co Ltd), or the vehicle (10% DMSO/0.9% carboxymethylcellulose) are given intraperitoneally every day to mice treated with deoxycorticosterone acetate and salt in a subset of experiments. The injections are given 10 days after the induction of hypertension and continue until the end of the 21-day treatment period. During days 10 to 21, sham-treated mice that receive a vehicle comprise the normotensive control group in these experiments.
Rats: The rats used are adult male Sprague-Dawleys weighing 200–250 g. 1 μg of CCL2 and/or 1 mM of INCB3344 is administered intrathecally between L5 and L6 vertebrae. One test is conducted on the animals at 30, 60, 90, 120, and 240 minutes after the drug is administered. For each time point, the percentage of the maximal potential effect (MPE) is determined. Pharmacokinetic Study in Mice: The pharmacokinetics of INCB3344 was assessed in CD-1 and Balb/c mice. For intravenous administration in CD-1 mice, a dose of 5 mg/kg was used. For oral administration, doses of 10 mg/kg were given to both CD-1 and Balb/c mice. The specific formulation for administration is not detailed in the provided text. [1] |
| ADME/Pharmacokinetics |
In CD-1 mice, after intravenous injection of INCB3344 (5 mg/kg), its clearance (CL) was 5.1 L/h/kg, its steady-state volume of distribution (Vdₛₛ) was 2.8 L/kg, and its half-life (T₁/₂) was 1.0 h. [1]
In CD-1 mice, after oral administration of INCB3344 (10 mg/kg), its maximum concentration (Cₘₐₓ) was 1886 nM, its area under the curve (AUC) was 2664 nM·h, and its oral bioavailability (F) was 47%. [1] In Balb/c mice, after oral administration of INCB3344 (10 mg/kg), its Cₘₐₓ was 961 nM, and its AUC was 3888 nM·h. [1] |
| Toxicity/Toxicokinetics |
In the multifenestrone competitive binding assay, INCB3344 showed an IC₅₀ inhibition of the hERG channel of 13 μM. [1]
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| References |
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| Additional Infomation |
INCB3344 was discovered through rational design based on the common pharmacophore of CCR2 antagonists. [1]
Due to its potent anti-mouse CCR2 activity, good selectivity, and oral bioavailability, INCB3344 is considered a tool compound suitable for target validation in rodent models. [1] |
| Molecular Formula |
C29H34N3O6F3
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| Molecular Weight |
577.59196
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| Exact Mass |
577.24
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| Elemental Analysis |
C, 60.30; H, 5.93; F, 9.87; N, 7.28; O, 16.62
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| CAS # |
1262238-11-8
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| Related CAS # |
INCB3344 R-isomer; cis-INCB3344; 1285539-85-6; 709018-37-1 (isomers)
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| PubChem CID |
10008367
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| Appearance |
White to yellow solid powder
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
41
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| Complexity |
915
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CCO[C@H]1CN(C[C@@H]1NC(=O)CNC(=O)C2=CC(=CC=C2)C(F)(F)F)C3CCC(CC3)(C4=CC5=C(C=C4)OCO5)O
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| InChi Key |
MZEOSVPWMSEFPW-XYCDVDSTSA-N
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| InChi Code |
InChI=1S/C29H34F3N3O6/c1-2-39-25-16-35(21-8-10-28(38,11-9-21)19-6-7-23-24(13-19)41-17-40-23)15-22(25)34-26(36)14-33-27(37)18-4-3-5-20(12-18)29(30,31)32/h3-7,12-13,21-22,25,38H,2,8-11,14-17H2,1H3,(H,33,37)(H,34,36)/t21?,22-,25-,28?/m0/s1
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| Chemical Name |
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 6 mg/mL (10.39 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
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. Solubility in Formulation 2: 6 mg/mL (10.39 mM) in 5% DMSO + 95% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. View More
Solubility in Formulation 3: ≥ 2.75 mg/mL (4.76 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 5%DMSO + Corn oil: 5.0mg/ml (8.66mM) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7313 mL | 8.6567 mL | 17.3133 mL | |
| 5 mM | 0.3463 mL | 1.7313 mL | 3.4627 mL | |
| 10 mM | 0.1731 mL | 0.8657 mL | 1.7313 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Effects of INCB3344 and INCB3090 on binding and functional responses of WEHI-274 cells to CCL2.J Immunol.2005 Oct 15;175(8):5370-8. |
Lack of inhibition of INCB3344 on MIP-1β binding or signaling.J Immunol.2005 Oct 15;175(8):5370-8. |
INCB3344 is efficacious in rat AIA. Treatment of rats with INCB3344 100 mg/kg BID s.c. from day 9 postimmunization resulted in significant inhibition of clinical and histological disease.J Immunol.2005 Oct 15;175(8):5370-8. |
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