AZD-5069 (AZD5069) is a novel and potent CXCR2 chemokine receptor antagonist with potential anticancer and antiinflammatory activities. The proliferation and progression of tumor cells are facilitated by the upregulation of the CXC chemokine receptor CXCR2 in a range of distinct tumor cell types. Reduced tumorigenesis and metastasis were the results of CXCR2 inhibition. At a pIC50 of 9.1, AZD-5069 prevents radiolabeled CXCL8 from binding to human CXCR2. In phase Ib/II studies, AZD5069, an antagonist of CXCR2, is presently being studied in combination with tremelimumab for patients with metastatic squamous cell carcinoma of the head and neck and advanced solid tumors. AZD-5069 has the potential to treat individuals suffering from COPD and other inflammatory diseases.

Physicochemical Properties

Molecular Formula	C18H22F2N4O5S2
Molecular Weight	476.51
Exact Mass	476.099
Elemental Analysis	C, 45.37; H, 4.65; F, 7.97; N, 11.76; O, 16.79; S, 13.46
CAS #	878385-84-3
Related CAS #	878385-84-3
PubChem CID	56645576
Appearance	White to off-white solid powder
Density	1.6±0.1 g/cm3
Boiling Point	680.5±65.0 °C at 760 mmHg
Flash Point	365.4±34.3 °C
Vapour Pressure	0.0±2.2 mmHg at 25°C
Index of Refraction	1.651
LogP	2.43
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	12
Rotatable Bond Count	10
Heavy Atom Count	31
Complexity	670
Defined Atom Stereocenter Count	2
SMILES	N(C1C=C(O[C@H](C)[C@@H](O)CO)N=C(SCC2C=CC=C(F)C=2F)N=1)S(N1CCC1)(=O)=O
InChi Key	QZECRCLSIGFCIO-RISCZKNCSA-N
InChi Code	InChI=1S/C18H22F2N4O5S2/c1-11(14(26)9-25)29-16-8-15(23-31(27,28)24-6-3-7-24)21-18(22-16)30-10-12-4-2-5-13(19)17(12)20/h2,4-5,8,11,14,25-26H,3,6-7,9-10H2,1H3,(H,21,22,23)/t11-,14+/m1/s1
Chemical Name	N-[2-[(2,3-difluorophenyl)methylsulfanyl]-6-[(2R,3S)-3,4-dihydroxybutan-2-yl]oxypyrimidin-4-yl]azetidine-1-sulfonamide
Synonyms	AZD 5069; AZD-5069; AZD5069
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-IL-8-CXCR2
ln Vitro	In vitro activity: AZD-5069 is a novel and potent antagonist of CXCR2 chemokine receptor with potential anticancer and antiinflammatory activities. The proliferation and progression of tumor cells are facilitated by the upregulation of the CXC chemokine receptor CXCR2 in a range of distinct tumor cell types. Reduced tumorigenesis and metastasis were the results of CXCR2 inhibition. At a pIC50 of 9.1, AZD-5069 prevents radiolabeled CXCL8 from binding to human CXCR2. In phase Ib/II studies, AZD5069, an antagonist of CXCR2, is presently being studied in combination with tremelimumab for patients with metastatic squamous cell carcinoma of the head and neck and advanced solid tumors. AZD-5069 has the potential to treat individuals suffering from COPD and other inflammatory diseases.
ln Vivo	Rat Airway LPS Challenge Model: Oral dosing of AZD5069, AZD8309, or AZ10397767 or dexamethasone (5.8 μmol/kg) was administered to treatment groups of eight rats one hour prior to LPS challenge. 0.9% saline was given to the rats in group 1. Groups of rats were given 0.1 mg/ml LPS in either a saline (0.9%) or saline vehicle control. After being housed in perspex boxes for thirty minutes, the rats were exposed to an aerosol produced by two jet nebulizers running at a 12 l/min airflow rate. Four hours after the LPS challenge, the airway was lavaged with three aliquots of sterile PBS at room temperature, and the trachea was cannulated. For cell counting, an aliquot of lavage fluid was taken out. Using a Hema-Tek-2000 automatic slide stainer (Fisher Scientific Ltd, UK, Loughborough, UK), slides were stained with Wright-Giemsa stain, and 200 cells were usually counted under a microscope. The cells were divided into mononuclear, neutrophil, and eosinophil subtypes. Monocytes, macrophages, and lymphocytes were examples of mononuclear cells. To calculate the quantity of neutrophils, the cell count was expressed as a percentage of the total count. Each treatment group's average neutrophil count was calculated, and the result was given as the mean ± S.E.M. GraphPad InStat was used to compare the results between treatment groups using nonparametric statistics, Mann-Whitney, or Kruskal-Wallis methodology. Rats were put to sleep using isofluorane, and then the animals' abdominal vena cava was used to draw blood samples (0.5 and 2 ml). Subsequently, the animals were put to death intraperitoneally with 1.0 ml of pentobarbitone sodium. An Advia Haematology System (Siemens, London, UK) was used to determine the differential cell numbers in one set of blood samples. The remaining blood sample (2 ml) was centrifuged for 10 minutes at 4°C at 2800g. Following its removal, the plasma was kept in storage at -20°C until the compound concentration could be ascertained.
Enzyme Assay	AZD-5069 functions as a CXCR2 antagonist by preventing radiolabelled [125I]-IL-8 from binding to human CXCR2 receptors. It also prevents GROα-induced Ca2+ flux in human neutrophils that have been loaded with fluo-3 dye.
Cell Assay	In a humidified incubator at 37°C and 5% CO2, human embryonic kidney 293 (HEK293) cells expressing recombinant human CXCR2 or CXCR1 were grown to about 80% confluence in Dulbecco's modified Eagle’s medium–Glutamax medium (Life Technologies Ltd, Paisley, UK) containing 10% (v/v) fetal calf serum and 0.5 mg/ml geneticin. Accutase (Sigma-Aldrich Company Ltd., Dorset, UK) was used to extract cells from the flask after it had been at 37°C for three to five minutes.
Animal Protocol	p.o. Rat Airway LPS Challenge Model.
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.
Additional Infomation	CXC Chemokine Receptor 2 Antagonist AZD5069 is an orally bioavailable, selective and reversible antagonist of CXC chemokine receptor 2 (CXCR2), with potential anti-inflammatory and antineoplastic activities. Upon administration, CXC chemokine receptor 2 antagonist AZD5069 directly binds to CXCR2 and inhibits its activation. This inhibits CXCR2-mediated signaling and may inhibit tumor cell proliferation in CXCR2-overexpressing tumor cells. In addition, AZD5069 reduces both neutrophil recruitment and migration from the systemic circulation into sites of inflammation, including the lung mucosa; it may also prevent neutrophil migration from the bone marrow. This results in the reduction of inflammation, mucus production, and neutrophil proteinase-mediated tissue destruction in the lung. CXCR2, a G protein-coupled receptor protein also known as IL-8 receptor B (IL-8RB), is upregulated in a variety of tumor cell types and plays a key role in tumor cell proliferation and progression; it is known to be elevated in several inflammatory diseases, such as chronic obstructive pulmonary disease (COPD), asthma and fibrotic pulmonary disorders.

Solubility Data

Solubility (In Vitro)

DMSO: 90~95 mg/mL (188.9~199.4 mM) Water: N/A Ethanol: ~19 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: 2.62 mg/mL (5.50 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.25 mg/mL (4.72 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 22.5 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 3: ≥ 2.25 mg/mL (4.72 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 22.5 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 4: ≥ 2.25 mg/mL (4.72 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 22.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly. Solubility in Formulation 5: 5%DMSO + 40%PEG300 + 65%ddH2O: 8.0mg/ml (16.79mM) Solubility in Formulation 6: 12.5 mg/mL (26.23 mM) in 50% PEG300 50% 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	2.0986 mL	10.4930 mL	20.9859 mL
	5 mM	0.4197 mL	2.0986 mL	4.1972 mL
	10 mM	0.2099 mL	1.0493 mL	2.0986 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.