AMG-487 (AMG487) is a novel, potent, selective and oral antagonist of chemokine receptor 3 CXCR3 with potential immunomodulatory and antiinflammatory activities. It has IC50s of 8.0 and 8.2 nM for I-IP-10 and I-ITAC, respectively, which inhibits the binding of CXCL10 and CXCL11 to CXCR3. I-IP-10 and I-ITAC, two chemokines, are prevented from binding to CXCR3 by AMG 487. AMG 487 has been shown in the cellular assays to inhibit CXCR3-mediated cell migration, with IC50 values for I-IP-10, I-ITAC, and MIG of 8 nM, 15 nM, and 36 nM, respectively.

Physicochemical Properties

Molecular Formula	C32H28F3N5O4
Molecular Weight	603.591037750244
Exact Mass	603.209
Elemental Analysis	C, 63.68; H, 4.68; F, 9.44; N, 11.60; O, 10.60
CAS #	473719-41-4
Related CAS #	AMG 487 (S-enantiomer); 473720-30-8; (±)-AMG 487; 947536-03-0
PubChem CID	24957182
Appearance	White to yellow solid powder
LogP	5.805
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	10
Rotatable Bond Count	10
Heavy Atom Count	44
Complexity	997
Defined Atom Stereocenter Count	1
SMILES	O=C(N([C@@H](C1=NC2=NC=CC=C2C(N1C3=CC=C(OCC)C=C3)=O)C)CC4=CC=CN=C4)CC5=CC=C(OC(F)(F)F)C=C5
InChi Key	WQTKNBPCJKRYPA-OAQYLSRUSA-N
InChi Code	InChI=1S/C32H28F3N5O4/c1-3-43-25-14-10-24(11-15-25)40-30(38-29-27(31(40)42)7-5-17-37-29)21(2)39(20-23-6-4-16-36-19-23)28(41)18-22-8-12-26(13-9-22)44-32(33,34)35/h4-17,19,21H,3,18,20H2,1-2H3/t21-/m1/s1
Chemical Name	N-[(1R)-1-[3-(4-ethoxyphenyl)-4-oxopyrido[2,3-d]pyrimidin-2-yl]ethyl]-N-(pyridin-3-ylmethyl)-2-[4-(trifluoromethoxy)phenyl]acetamide
Synonyms	AMG487; AMG-487; AMG 487
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-IP10-CXCR3 ( IC50 = 8 nM ); 125I-ITAC-CXCR3 ( IC50 = 8.2 nM )
ln Vitro	AMG-487 (AMG487) suppresses the three CXCR3 chemokines' ability to migrate cells through CXCR3 (IP-10 IC50=8 nM, ITAC IC50=15 nM, and MIG IC50=36 nM). AMG 487 also suppresses the release of calcium in response to ITAC (IC50=5 nM)[1]. Reduced lung metastases and significantly smaller lungs compared to vehicle-treated lungs are observed with AMG487 (1 μM) treatment[2]. AMG487 stops C26 tumor cells from proliferating or surviving[3].
ln Vivo	AMG-487 (AMG487) (0.03-10 mg/kg, s.c.) significantly reduces cellular infiltration into the lungs in a dose dependent manner[1]. Compared to mice given a vehicle, AMG487 (5 mg/kg, s.c., twice daily) causes fewer metastases to form[2]. In both models, mice treated with AMG487 (5 mg/kg, s.c.) show fewer pulmonary nodules than the control group. AMG487 reduces the tumour volume[3].
Enzyme Assay	In order to examine the harmful impact of AMG487 on bone marrow cells and how it affects BMDC development, the bone marrow cells are exposed to AMG487 at concentrations of 3 μM, 15 μM, 30 μM, and 60 μM starting on day 0 and getting appropriate doses of AMG487 every three days. After two rounds of washing, LPS is added to the cells to promote maturation. We only add different concentrations of AMG487 (10 μM, 30 μM, and 60 μM) along with the LPS on day 6 in order to study its impact on BMDC activation. In every instance, the vehicle is added in equal parts as a control.
Cell Assay	At a density of 104 cells cm2, colon cancer cells are seeded and then incubated in serum-enriched medium or base medium (containing 0.1% bovine serum albumin, BSA) supplemented or not with different concentrations of rCXCL9, rCXCL10, and rCXCL11 for the indicated times. After that, they are either trypsin-detached, collected, and counted, or they are re-fed with fresh medium for three days, harvested, and counted. On day seven, photos are taken and the morphology of the CRC cells are examined using an inverted optical microscope at ×20 magnification.
Animal Protocol	Injection of 3×105 viable tumor cells s.c. near the right abdominal mammary gland of syngeneic female mice is used to assess local tumor growth and spontaneous metastasis. Tumor diameters are measured twice a week using a caliper, and mice are put to death individually if their s.c. tumor measures 18 mm in diameter or earlier if they appear moribund. Under a dissecting microscope, surface tumor colonies are measured blindly while the lungs are removed and weighed. To assess experimental metastasis, 9×104 viable tumor cells are injected intraperitoneally (i.v.) into the lateral tail vein of syngeneic female mice. On day 21 after transplantation, or earlier if the mice appeared moribund, all of the mice are put to death. Under a dissecting microscope, surface tumor colonies are measured blindly while the lungs are removed and weighed. A 50% hydroxypropyl-β-cyclodextrin solution is made; this solution acts as the vehicle at 20%. Following the addition of AMG487 to the 50% solution, the mixture is incubated for two hours in a sonicating water bath with periodic vortexing. To achieve the proper final concentration of AMG487 in 20% hydroxypropyl-β-cyclodextrin, distilled water is added.
References	[1]. Discovery and optimization of a series of quinazolinone-derived antagonists of CXCR3. Bioorg Med Chem Lett. 2007 Jun 15;17(12):3339-43. [2]. Antagonism of CXCR3 inhibits lung metastasis in a murine model of metastatic breast cancer. Cancer Res. 2006 Aug 1;66(15):7701-7. [3]. Organ-specific inhibition of metastatic colon carcinoma by CXCR3 antagonism. Br J Cancer. 2009 Jun 2;100(11):1755-64.

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (~165.7 mM) Ethanol: ~100 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.14 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 (4.14 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 (4.14 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%DMSO + 40%PEG300 + 5%Tween 80 + 50%ddH2O: 5.0mg/ml (8.28mM) Solubility in Formulation 5: 5 mg/mL (8.28 mM) in 20% HP-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), Suspened 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	1.6568 mL	8.2838 mL	16.5675 mL
	5 mM	0.3314 mL	1.6568 mL	3.3135 mL
	10 mM	0.1657 mL	0.8284 mL	1.6568 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.