[¹²⁵I]CXCL10-CXCR3 ( Ki = 1.5 nM ); [¹²⁵I]CXCL11-CXCR3 ( Ki = 3.2 nM )
Target: NBI-74330 specifically targets CXCR3, a seven-transmembrane GPCR that is predominantly expressed on Th1 cells and involved in inflammatory diseases. Its three endogenous ligands—CXCL9, CXCL10, and CXCL11—are produced at inflammation sites and elicit migration of pathological Th1 cells. NBI-74330 inhibits [125I]CXCL10 and [125I]CXCL11 specific binding with Ki values of 1.5 nM and 3.2 nM respectively. It is highly selective for CXCR3, showing no significant inhibition of other chemokine receptors and no inhibition of a panel of nonchemokine GPCRs.

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].

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In Vitro Activity: NBI-74330 potently inhibits [125I]CXCL11 binding to membranes from CXCR3-CHO cells with Ki=3.6 nM, 12-fold more potent than CXCL9 (Ki=45.2 nM). It inhibits ligand-induced [35S]GTPγS binding, calcium mobilization, and chemotaxis with IC50 values of 7-18 nM. For calcium flux, IC50 against both CXCL11 and CXCL10 is 7 nM. In human glioma lines (A172, U118, T98G, U87, U138) and murine GL261 cells, NBI-74330 reduces CXCR3-mediated proliferation and migration. It also inhibits CXCL11-induced chemotaxis with IC50 of 3.9 nM in H9 cells.

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].

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In Vivo Activity: In LDL receptor-deficient mice, NBI-74330 reduces atherosclerotic plaque formation. At 100 mg/kg (in 1% sodium deoxycholate/0.5% methylcellulose), it produces an N-oxide metabolite that also acts as a CXCR3 antagonist in mice. Serum concentrations reach ~1 μM, sufficient to fully block CXCR3 in vivo. In glioma models, CXCR3 antagonism may offer therapeutic benefit since CXCR3 signaling stimulates glioma growth. The compound has been evaluated in inflammatory disease models to reduce pathological Th1 cell migration.

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).

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In Vitro Enzyme/Receptor Binding Protocol: For competition binding, membranes from CXCR3-CHO cells are incubated with [125I]CXCL11 and varying concentrations of NBI-74330. Bound radioactivity is measured after filtration and washing to determine Ki. For [35S]GTPγS binding, H9 T lymphoma cell membranes (naturally expressing CXCR3) are incubated with [35S]GTPγS, GDP, and compound in presence of CXCL11 or CXCL10. Bound [35S]GTPγS is quantified by scintillation counting. All assays include appropriate positive and negative controls.

In Vitro Cell-Based Assay Protocol: CXCR3-CHO or H9 cells are seeded in culture media and stimulated with CXCL11 or CXCL10 at EC80. For calcium mobilization, cells are loaded with fluorescent dye (e.g., Fluo-4) and fluorescence is measured after addition of compound at 0.1-1000 nM. For chemotaxis, cells are placed in upper transwell chambers with CXCL11 in lower chamber; migrated cells are counted after treatment. For glioma studies, human lines are treated with NBI-74330 and assessed for proliferation (BrdU) and migration (wound healing or Boyden chamber).

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.

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In Vivo Animal Assay Protocol: LDL receptor-deficient mice receive NBI-74330 orally or via gavage. For PK, mice get 100 mg/kg in vehicle and serum is collected at timed intervals. Atherosclerosis is assessed by aortic plaque area after treatment. In glioma xenografts, tumor-bearing mice are treated with compound and tumor volume measured caliper-wise. Dosing schedules vary (daily or intermittent). Endpoints include tumor growth inhibition, survival, and histopathological analysis of plaques or tumors.

Pharmacokinetics: After oral administration, NBI-74330 reaches serum concentrations of ~1 μM at 100 mg/kg in mice. It is metabolized to an N-oxide metabolite that retains CXCR3 antagonist activity. The compound shows 4-fold higher affinity for human and rhesus CXCR3 compared to rat or mouse orthologs. Detailed parameters (t1/2, bioavailability, Vd, Cl) are not fully published. As a research compound, comprehensive PK profiling would be required for therapeutic development.

Toxicity: No detailed toxicity data are publicly available for NBI-74330. In preclinical studies at doses up to 100 mg/kg in mice, no significant adverse effects were reported. It is not intended for human use, and standard toxicological assessments (acute, chronic, genotoxicity, carcinogenicity) would be needed before clinical development. The compound is for research use only and has not been approved by any regulatory agency.

[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.

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

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Additional Information: NBI-74330 is also known as NBI74330 and VUF10472, with molecular formula C32H27F4N5O3 and MW 605.58. Glioma growth is stimulated by CXCR3, and antagonism may benefit glioblastoma treatment. There is a striking difference in potencies among CXCR3 ligands (CXCL11 >> CXCL10 > CXCL9), but weaker affinities are compensated by elevated expression. The compound is a valuable tool for studying Th1-mediated inflammatory diseases and cancer. It is not approved for therapy.

C32H27F4N5O3

605.5821

605.205

C, 63.47; H, 4.49; F, 12.55; N, 11.56; O, 7.93

855527-92-3

473722-68-8 (racemate);855527-92-3 (R-isomer)

45784923

Light yellow to yellow solid powder

6.064

0

10

9

44

1020

1

[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

XMRGQUDUVGRCBS-UHFFFAOYSA-N

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

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

NBI74330; NBI 74330; NBI-74330.

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: ~100 mg/mL (~165.1 mM)

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.

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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.

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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.

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