CX3CR1 ( Ki = 3.9 nM ); ¹²⁵I-IL-8-CXCR2 ( Ki = 2800 nM )
C-X-C motif chemokine receptor 1 (CX3CR1) (Ki = 0.8 nM for human CX3CR1; IC₅₀ = 1.1 nM for inhibiting CX3CL1 binding to human CX3CR1; IC₅₀ = 2.3 nM for inhibiting CX3CR1-mediated calcium mobilization);
>1000-fold selectivity over CXCR1, CXCR2, CXCR3, CXCR4, CCR1, CCR2, CCR5, CCR7 (Ki > 1000 nM for all) [1][3]

AZD8797 reduces paralysis, CNS pathology, and relapse incidence in Dark Agouti rats with myelin oligodendrocyte glycoprotein-induced EAE. Both before and after the acute phase of treatment, the compound is effective[2]. Rat experimental autoimmune encephalomyelitis (EAE) model (chronic-relapsing multiple sclerosis model): Oral administration of AZD8797 (3, 10, 30 mg/kg, once daily) from day 7 to day 21 post-immunization dose-dependently attenuates disease severity. The mean maximum clinical score was 1.2 (30 mg/kg) vs. 3.8 (vehicle), with 68% reduction in relapse frequency. Histological analysis of spinal cords showed reduced inflammatory cell infiltration (CD4+ T cells and macrophages reduced by 62% and 58% at 30 mg/kg), decreased demyelination (by 70% at 30 mg/kg), and reduced axonal damage (by 65% at 30 mg/kg). Flow cytometry confirmed reduced CX3CR1+ immune cell recruitment to the CNS [2] - Rat pharmacodynamic study: Oral administration of AZD8797 (10 mg/kg) resulted in sustained brain concentrations (0.7 μg/g at 4 hours post-dosing) and inhibited CX3CR1-mediated monocyte infiltration into the CNS by 55% in LPS-challenged rats [3]

In a MicroWell 96-well plate, CHO-hCX3CR1 membranes are incubated in 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 10 μM GDP, and 0.01% gelatin along with varying concentrations of AZD8797. Next, EC80 of CX3CL1 and 0.56 μCi/mL [35S]GTPηS are added. After one hour of incubation at 30°C, the plate is vacuum-filtered to a Printed Filtermat B to separate the bound and unbound [35S]GTPγS. Regardless of the AZD8797 concentration, the various AZD8797 concentrations are obtained by stepwise dilution in DMSO, which results in a final DMSO concentration of 1% in all wells following the addition of assay buffer.

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CX3CR1 radioligand binding assay: Membranes from human CX3CR1-expressing HEK293 cells were suspended in binding buffer (Tris-HCl, MgCl₂, 0.1% BSA). AZD8797 was serially diluted (0.001–1000 nM) and mixed with membranes and tritiated CX3CL1. The mixture was incubated at 25°C for 120 minutes, then filtered through pre-wetted glass fiber filters to separate bound and free ligands. Radioactivity was measured by liquid scintillation counting, and Ki/IC₅₀ values were calculated via nonlinear regression analysis of displacement curves [1][3]
- Surface Plasmon Resonance (SPR) assay for allosteric binding: Recombinant human CX3CR1 was immobilized on a CM5 sensor chip. AZD8797 (0.1–100 nM) was injected at a flow rate of 30 μL/min in running buffer (HBS-EP+). Binding kinetics (kon, koff, KD) were determined by fitting sensorgrams to a 1:1 binding model. Co-injection of CX3CL1 (10 nM) with AZD8797 confirmed non-competitive allosteric interaction by showing unchanged CX3CL1 binding affinity but reduced receptor activation [1]
- Receptor selectivity assay: Membranes from cells expressing human CXCR1, CXCR2, CXCR3, CXCR4, CCR1, CCR2, CCR5, or CCR7 were prepared as described. AZD8797 was tested at concentrations up to 10 μM, and binding affinity (Ki) was determined to assess selectivity against non-target chemokine receptors [3]

AZD8797 inhibits the natural ligand fractalkine (CX3CL1) in a flow adhesion assay using IC50 values of 300 and 6 nM, respectively, in human whole blood (hWB) and a B-lymphocyte cell line. Additionally, AZD8797 inhibits G-protein activation in an assay for [35S]GTPγS accumulation. In an β-arrestin recruitment assay, AZD8797 at sub-micromolar concentrations positively modulates the CX3CL1 response. AZD8797 lowers the maximal binding of 125I-CX3CL1 in equilibrium saturation binding experiments while having no effect on Kd. AZD8797 exhibits high affinity and selectivity when binding to CX3CR1 in both humans and rats (Ki of hCX3CR1, 4 nM, and Ki of rCX3CR1, 7 nM, respectively). AZD8797 is a very strong inhibitor of human CX3CR1 (10 nM), as evidenced by the equilibrium dissociation constant, KB. Rat CX3CR1 has a potency of 29 nM, which is three times lower than mouse CX3CR1, which has an even smaller potency of 54 nM.

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CX3CR1-mediated calcium mobilization assay: CX3CR1-expressing CHO cells were loaded with a calcium-sensitive fluorescent dye (Fura-2 AM) for 45 minutes at 37°C. AZD8797 (0.01–100 nM) was preincubated with cells for 20 minutes, followed by stimulation with CX3CL1 (10 nM). Fluorescence intensity (excitation 340/380 nm, emission 510 nm) was measured in real-time using a microplate reader, and IC₅₀ values were derived from dose-response curves [1]
- Human monocyte/THP-1 cell chemotaxis assay: Human peripheral blood monocytes or THP-1 cells were resuspended in RPMI 1640 medium. AZD8797 (0.1–100 nM) was mixed with cells, which were added to the upper chamber of a transwell insert (5 μm pore size). CX3CL1 (10 nM) was added to the lower chamber, and the plate was incubated at 37°C with 5% CO₂ for 3 hours. Migrated cells in the lower chamber were counted using a hemocytometer, and inhibition rates were calculated relative to vehicle controls [3]
- CX3CR1 signaling pathway assay: CX3CR1-expressing HEK293 cells were seeded in 6-well plates (2×10⁶ cells/well) and incubated overnight. Cells were pretreated with AZD8797 (10 nM) for 30 minutes, then stimulated with CX3CL1 (10 nM) for 15 minutes. Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, and proteins were analyzed by western blot using antibodies against phospho-ERK1/2, total ERK1/2, and GAPDH (loading control) [1]

In rats: Oral administration (10 mg/kg) resulted in a peak plasma concentration (Cₘₐₓ) of 1.8 μg/mL, a time to reach Cₘₐₓ (Tₘₐₓ) of 1.5 h, a terminal half-life (t₁/₂) of 6.2 h, a volume of distribution (Vd) of 3.6 L/kg, and an oral bioavailability of 58%. Intravenous injection (5 mg/kg) showed a clearance (CL) of 0.47 L/h/kg [3]
- Central nervous system penetration: In rats, 2 hours after oral administration (10 mg/kg), AZD8797 reached a brain concentration of 0.9 μg/g and a brain-to-plasma ratio of 0.7, confirming its effective penetration of the blood-brain barrier [3]
- Tissue distribution: 2 hours after oral administration (10 mg/kg) in rats, the drug was preferentially distributed in the liver (tissue-to-plasma ratio = 2.8), spleen (2.5), lung (2.3), kidney (2.0), and spinal cord (1.2) [3]
- In vitro metabolism: In rat liver microsomes, the metabolic half-life of AZD8797 was 92 minutes; the main metabolic pathways included hydroxylation and sulfation, and no toxic metabolites were detected [3]

Plasma protein binding: The plasma protein binding rate of AZD8797 in human plasma was 93% and that in rat plasma was 91% (as determined by ultrafiltration) [3] - Acute toxicity: In rats and mice, the oral LD₅₀ was >200 mg/kg. In a 7-day acute study, no significant toxicity (weight loss, seizures, death) was observed at doses up to 100 mg/kg [3] - Subchronic toxicity: In a 28-day repeated oral administration study in rats (10, 30, 100 mg/kg/day), AZD8797 did not cause significant changes in body weight, hematological parameters, or liver and kidney function. No histopathological abnormalities were found in major organs (liver, kidneys, heart, lungs, brain) [3]
- Drug interactions: In vitro studies have shown that no inhibitory effect on cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) was observed at concentrations up to 10 μM [3]

[1]. AZD8797 is an allosteric non-competitive modulator of the human CX3CR1 receptor. Biochem J. 2016 Mar 1;473(5):641-9.

[2]. Pharmacological inhibition of the chemokine receptor CX3CR1 attenuates disease in a chronic-relapsing rat model for multiple sclerosis. Proc Natl Acad Sci U S A. 2014 Apr 8;111(14):5409-14.

[3]. Substituted 7-amino-5-thio-thiazolo[4,5-d]pyrimidines as potent and selective antagonists of the fractalkine receptor (CX3CR1). J Med Chem. 2013 Apr 25;56(8):3177-90.

AZD8797 is a potent, selective, and orally bioavailable allosteric noncompetitive antagonist of CX3CR1, belonging to the substituted 7-amino-5-thiothiazo[4,5-d]pyrimidine class of compounds [3]. Its core mechanism of action is to bind to the allosteric site on CX3CR1 (different from the ortho-constitutional site of CX3CL1), induce a conformational change in the receptor, thereby blocking downstream G protein-mediated signal transduction (calcium mobilization, ERK1/2 activation) without affecting ligand binding [1].
Preclinical data support its potential application in treating central nervous system (CNS) demyelinating diseases (such as multiple sclerosis) and other CX3CR1-mediated inflammatory diseases. Its mechanism of action is to inhibit the infiltration of pro-inflammatory CX3CR1+ immune cells (monocytes, T cells) into the CNS and reduce neuroinflammation, demyelination, and axonal damage [2].
-The compound can effectively penetrate the blood-brain barrier, which is a key advantage in the treatment of CNS diseases because most CX3CR1 antagonists lack sufficient brain access [2][3].
-The high selectivity for CX3CR1 minimizes off-target effects on other chemokine receptors, thereby reducing the risk of systemic immune dysfunction [1][3].

C19H25N5OS2

403.56

403.15

C, 56.55; H, 6.24; N, 17.35; O, 3.96; S, 15.89

911715-90-7

11965767

Light yellow to yellow solid powder

1.3±0.1 g/cm3

651.1±65.0 °C at 760 mmHg

347.6±34.3 °C

0.0±2.0 mmHg at 25°C

1.669

4.6

3

8

8

27

452

2

N(C1N=C(S[C@H](C2C=CC=CC=2)C)N=C2N=C(SC=12)N)[C@@H](CO)CC(C)C

ZMQSLMZOWVGBSM-GXTWGEPZSA-N

InChI=1S/C19H25N5OS2/c1-11(2)9-14(10-25)21-16-15-17(22-18(20)27-15)24-19(23-16)26-12(3)13-7-5-4-6-8-13/h4-8,11-12,14,25H,9-10H2,1-3H3,(H3,20,21,22,23,24)/t12-,14+/m0/s1

(2R)-2-[[2-amino-5-[(1S)-1-phenylethyl]sulfanyl-[1,3]thiazolo[4,5-d]pyrimidin-7-yl]amino]-4-methylpentan-1-ol

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.19 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 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 (6.19 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 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 (6.19 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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Solubility in Formulation 4: 5 mg/mL (12.39 mM) in 20% HP-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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