AZD8797 (AZD-8797) is a novel, selective, orally bioavailable and allosteric non-competitive modulator of the human CX3CR1 receptor with potential anti-Inflammatory and immunomodulatory activity. It irritates CX3CR1 and CXCR2, with corresponding Ki values of 3.9 and 2800 nM. AZD8797 inhibited 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. In an assay for [(35)S]GTPηS (guanosine 5'-[γ-thio]triphosphate) accumulation, AZD8797 also inhibited G-protein activation. By contrast, AZD8797 agonism was found to be weakly Gαi-dependent through dynamic mass redistribution (DMR) experiments. Furthermore, in an β-arrestin recruitment assay, AZD8797 positively modulated the CX3CL1 response at sub-micromolar concentrations. AZD8797 decreased the maximum binding of (125)I-CX3CL1 in equilibrium saturation binding experiments while having no effect on Kd. Kinetic studies that determined the kon and koff of AZD8797 showed that this was a genuine non-competitive mechanism rather than an artifact of irreversible or insurmountable binding. Ultimately, we demonstrate that GTPγS and AZD8797 both raise the rate at which CX3CL1 separates from CX3CR1 in a comparable way, suggesting a link between AZD8797 and the G-protein bound to CX3CR1. All together, these findings demonstrate that AZD8797 functions as a non-competitive allosteric modulator of CX3CL1, binding CX3CR1 and causing biased effects on G-protein signaling and β-arrestin acquisition.

Physicochemical Properties

Molecular Formula	C19H25N5OS2
Molecular Weight	403.56
Exact Mass	403.15
Elemental Analysis	C, 56.55; H, 6.24; N, 17.35; O, 3.96; S, 15.89
CAS #	911715-90-7
Related CAS #	911715-90-7
PubChem CID	11965767
Appearance	Light yellow to yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	651.1±65.0 °C at 760 mmHg
Flash Point	347.6±34.3 °C
Vapour Pressure	0.0±2.0 mmHg at 25°C
Index of Refraction	1.669
LogP	4.6
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	8
Heavy Atom Count	27
Complexity	452
Defined Atom Stereocenter Count	2
SMILES	N(C1N=C(S[C@H](C2C=CC=CC=2)C)N=C2N=C(SC=12)N)[C@@H](CO)CC(C)C
InChi Key	ZMQSLMZOWVGBSM-GXTWGEPZSA-N
InChi Code	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
Chemical Name	(2R)-2-[[2-amino-5-[(1S)-1-phenylethyl]sulfanyl-[1,3]thiazolo[4,5-d]pyrimidin-7-yl]amino]-4-methylpentan-1-ol
Synonyms	AZD-8797; AZD8797; AZD 8797
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	CX3CR1 ( Ki = 3.9 nM ); 125I-IL-8-CXCR2 ( Ki = 2800 nM )
ln Vitro	In vitro activity: 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[1]. 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). As evidenced by the equilibrium dissociation constant, Kb, AZD8797 is a highly effective inhibitor of human CX3CR1 (10 nM). At rat CX3CR1 (29 nM), the potency is three times lower, and at mouse CX3CR1 (54 nM), it is even lower[2].
ln Vivo	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].
Enzyme Assay	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.
Cell Assay	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.
Animal Protocol	Rats: AZD8797 is given subcutaneously via osmotic minipumps and is formulated with 30- 35% (wt/wt) hydroxy-propyl-beta-cyklodextrin. The operator is blind to the treatment. Two analyses of each rat's plasma concentration of AZD8797 are conducted[2].
References	[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.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 150 mg/mL Water: <1mg/mL Ethanol: <1mg/mL

Solubility (In Vivo)

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.4779 mL	12.3897 mL	24.7795 mL
	5 mM	0.4956 mL	2.4779 mL	4.9559 mL
	10 mM	0.2478 mL	1.2390 mL	2.4779 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.