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.

Physicochemical Properties

Molecular Formula	C29H34N3O6F3
Molecular Weight	577.59196
Exact Mass	577.24
Elemental Analysis	C, 60.30; H, 5.93; F, 9.87; N, 7.28; O, 16.62
CAS #	1262238-11-8
Related CAS #	INCB3344 R-isomer; cis-INCB3344; 1285539-85-6; 709018-37-1 (isomers)
PubChem CID	10008367
Appearance	White to yellow solid powder
LogP	3.3
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	10
Rotatable Bond Count	8
Heavy Atom Count	41
Complexity	915
Defined Atom Stereocenter Count	2
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
InChi Key	MZEOSVPWMSEFPW-XYCDVDSTSA-N
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
Chemical Name	N-[2-[[(3S,4S)-1-[4-(1,3-benzodioxol-5-yl)-4-hydroxycyclohexyl]-4-ethoxypyrrolidin-3-yl]amino]-2-oxoethyl]-3-(trifluoromethyl)benzamide
Synonyms	INCB-3344; INCB3344; INCB 3344
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	hCCR2 ( IC50 = 5.1 nM ); mCCR2 ( IC50 = 9.5 nM )
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].
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].
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].
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.
References	[1]. Discovery of INCB3344, a potent, selective and orally bioavailable antagonist of human and murine CCR2. Bioorg Med Chem Lett. 2010 Dec 15;20(24):7473-8. [2]. Discovery and pharmacological characterization of a novel rodent-active CCR2 antagonist, INCB3344. J Immunol. 2005 Oct 15;175(8):5370-8. [3]. Reversal of vascular macrophage accumulation and hypertension by a CCR2 antagonist in deoxycorticosterone/salt-treated mice. Hypertension. 2012 Nov;60(5):1207-12. [4]. Spinal CCL2 pronociceptive action is no longer effective in CCR2 receptor antagonist-treated rats. J Neurochem. 2008 Jul;106(2):757-69. [5]. Enrichment of Ly6Chi monocytes by multiple GM-CSF injections with HBV vaccine contributes to viral clearance in a HBV mouse model. Hum Vaccin Immunother. 2017 Dec 2;13(12):2872-2882. [6]. CCR2 upregulation in DRG neurons plays a crucial role in gastric hyperalgesia associated with diabetic gastropathy. Mol Pain. 2018 Jan-Dec;14:1744806917751322. [7]. Mcp1 Promotes Macrophage-Dependent Cyst Expansion in Autosomal Dominant Polycystic Kidney Disease. J Am Soc Nephrol. 2018 Oct;29(10):2471-2481.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 200 mg/mL Water: < 1mg/mL Ethanol: N/A

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. 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. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 4: 5%DMSO + Corn oil: 5.0mg/ml (8.66mM)  (Please use freshly prepared in vivo formulations for optimal results.)

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.