NBI-74330 855527-92-3_CXCR_GPCR & G Protein_Signaling Pathways_Products

NBI-74330 (NBI74330) is a novel, potent and selective antagonist for CXCR3 (CXC chemokine receptor 3) with potential anticancer, immunomodulatory and antiinflammatory activities. It shows strong inhibition of (125I)CXCL10 and (125I)CXCL11 specific binding with Ki of 1.5 and 3.2 nM, respectively, and inhibits CXCR3 with IC50 values ranging from 7 to 18 nM. In mice lacking the LDL receptor, NBI-74330 reduces the development of atherosclerotic plaque. Glioma growth is stimulated by the chemokine receptor CXCR3. Since CXCR3 antagonism directly inhibits gliomas, treating human GBM may benefit from targeting this receptor as a therapeutic target.

Physicochemical Properties

Molecular Formula	C32H27F4N5O3
Molecular Weight	605.5821
Exact Mass	605.205
Elemental Analysis	C, 63.47; H, 4.49; F, 12.55; N, 11.56; O, 7.93
CAS #	855527-92-3
Related CAS #	473722-68-8 (racemate);855527-92-3 (R-isomer)
PubChem CID	45784923
Appearance	Light yellow to yellow solid powder
LogP	6.064
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	10
Rotatable Bond Count	9
Heavy Atom Count	44
Complexity	1020
Defined Atom Stereocenter Count	1
SMILES	[C@H](C1=NC2N=CC=CC=2C(=O)N1C1C=CC(OCC)=CC=1)(C)N(C(=O)CC1C=CC(F)=C(C(F)(F)F)C=1)CC1C=NC=CC=1
InChi Key	XMRGQUDUVGRCBS-UHFFFAOYSA-N
InChi Code	InChI=1S/C32H27F4N5O3/c1-3-44-24-11-9-23(10-12-24)41-30(39-29-25(31(41)43)7-5-15-38-29)20(2)40(19-22-6-4-14-37-18-22)28(42)17-21-8-13-27(33)26(16-21)32(34,35)36/h4-16,18,20H,3,17,19H2,1-2H3
Chemical Name	N-[1-[3-(4-ethoxyphenyl)-4-oxopyrido[2,3-d]pyrimidin-2-yl]ethyl]-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-(pyridin-3-ylmethyl)acetamide
Synonyms	NBI74330; NBI 74330; NBI-74330.
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

[¹²⁵I]CXCL10-CXCR3 ( Ki = 1.5 nM ); [¹²⁵I]CXCL11-CXCR3 ( Ki = 3.2 nM )

ln Vitro

NBI74330 (also known as NBI 74330; NBI-74330) exhibits a strong suppression of [¹²⁵I]CXCL11-specific binding to membranes derived from CXCR3-expressing transfected CHO cells (CXCR3-CHO) at a Ki=3.6 nM. Compared to CXCL9 (Ki=45.2 nM) and CXCL10 (Ki=12.5 nM), NBI-74330 is 12- and 3.5-fold more potent, respectively, at inhibiting [¹²⁵I]CXCL11 binding to CXCR3-CHO cell membranes. NBI-74330 inhibits calcium mobilization in response to CXCL11 and CXCL10 with an IC50 value of 7 nM for both ligands used at their EC80 concentrations (1 nM for CXCL11 and 30 nM for CXCL10). Specifically, CXCR3-mediated calcium mobilization is inhibited by NBI-74330. Additionally, NBI-74330 inhibits [³⁵S]GTPγS binding in the membranes of cells that naturally express CXCR3 (H9 cells; IC50 value: 5.5 nM) in a dose-dependent manner. BI-74330 inhibits CXCL11-induced chemotaxis in these cells with an IC50 of 3.9 nM[1]. NBI-74330 (30-300 nm, 1-10 μM) causes parallel, concentration-dependent rightward shifts in the CXCL11 E/[A] curve, but the maximal response of the E/[A] curve is unaffected[2].

ln Vivo

NBI74330 (NBI 74330; NBI-74330) (100 mg/kg) causes the production of an N-oxide metabolite that is also a CXCR3 antagonist, in mice[2]. The serum concentrations of mice treated with 100 mg/kg NBI-74330 (in 1% Na Doc in 0.5% 400Cp Methylcellulose) are about 1 μM. To fully block the CXCR3 receptor in vivo, this concentration is adequate[3].

Enzyme Assay

In order to use cell membrane fractions in competitive radioligand binding reactions, they are resuspended in 50 mM HEPES, 10 mM MgCl₂, 100 mM NaCl, and 1 mM CaCl₂ at pH 7.2. The assay buffer (50 mM HEPES, 10 mM MgCl₂, 100 mM NaCl, 1 mM CaCl₂, 0.1% BSA, pH 7.2) is gradually added to low-binding 96-well plates containing 5-μL membrane protein (5 μg). The reactions are carried out in duplicate and comprise 25 μL unlabeled chemokine at indicated concentrations and 25 μL radiolabeled chemokine ligand (approx 70 nM; [¹²⁵I]CXCL11 and [¹²⁵I]CXCL10 with specific activities of 1500 and 2200 Ci/mmol, respectively). The reaction is shaken and incubated for 45 minutes at room temperature in order to reach equilibrium. The extraction of membranes through filtration through a UniFilter GF/C filter plate using a UniFilter-96 vacuum manifold (pretreated filters with 1% polyethylenimine) yields the amount of bound radioligand. The membranes are then washed twice with 400-μL wash buffer (10 mM HEPES, 5 mM MgCl₂, 1 mM CaCl₂, and 500 mM NaCl, pH 7.3), and radioactivity is measured using liquid scintillation with a TopCount NXT. The measurement of the dissociation half-life of [¹²⁵I]CXCL11 is conducted using CXCR3-CHO membranes, which are first allowed to equilibrate with radiolabel (approximately 70 nM) for 30 minutes. After that, excess cold CXCL11 (31 nM final) is added, either in the presence or absence of varying concentrations of NBI-74330. Membrane-bound [¹²⁵I]CXCL11 is measured in duplicate on the same plate at each time point, along with total binding ([¹²⁵I]CXCL11 without inhibitors) and nonspecific binding ([¹²⁵I]CXCL11 plus excess cold CXCL11 added at the same time).

Animal Protocol

Two weeks prior to collar insertion, female LDLr^−/− mice, aged 10 weeks (n=8–12 per group), are given a Western-xstyle diet containing 15% cocoa butter and 0.25% cholesterol. For the duration of the experiment, 20 mice receive daily subcutaneous injections of 100 mg/kg NBI-74330. The mice are put to sleep after eight weeks of being fed a diet typical of the West and having their organs removed for histology, fluorescence-activated cell sorter (FACS) analysis, and RNA isolation. Blood is drawn from nonfasted animals by tail bleeding, and enzymatic colorimetric techniques are used to measure the levels of serum cholesterol and triglycerides.

References

[1]. Pharmacological characterization of CXC chemokine receptor 3 ligands and a small molecule antagonist. J Pharmacol Exp Ther. 2005 Jun;313(3):1263-71.

[2]. Analysis of the pharmacokinetic/pharmacodynamic relationship of a small molecule CXCR3 antagonist, NBI-74330, using a murine CXCR3 internalization assay. Br J Pharmacol. 2007 Dec;152(8):1260-71.

[3]. CXCR3 antagonist NBI-74330 attenuates atherosclerotic plaque formation in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol. 2008 Feb;28(2):251-7.

Additional Infomation

See also: Nbi-74330 (annotation moved to).

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (~165.1 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 2.5 mg/mL (4.13 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

Solubility in Formulation 2: 2.5 mg/mL (4.13 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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 3: ≥ 2.5 mg/mL (4.13 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.6513 mL	8.2565 mL	16.5131 mL
	5 mM	0.3303 mL	1.6513 mL	3.3026 mL
	10 mM	0.1651 mL	0.8257 mL	1.6513 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.

PeptideDB

NBI-74330 855527-92-3

CAS No.: 855527-92-3

Physicochemical Properties

Biological Activity

Solubility Data