PF-06260933 is a highly selective small-molecule inhibitor of MAP4K4 (Mitogen-activated protein kinase kinase kinase 4) with IC50 values of 3.7 and 160 nM for cell-freel assay and cell assay, respectively. Additionally, with IC50 values of 8 and 13 nM, it inhibits MINK and TNIK. Fasting hyperglycemia in mice has been shown to be reduced by PF-06260933. Recent research on the serine/threonine protein kinase MAP4K4, which is found in adipose tissue, the pancreas, muscle, and macrophages, suggests that it may be a promising target for anti-diabetic medications. The evaluation of MAP4K4 as a potential new anti-diabetic target is being done with PF-6260933.

Physicochemical Properties

Molecular Formula	C16H13CLN4
Molecular Weight	296.08
Exact Mass	296.08
Elemental Analysis	C, 64.76; H, 4.42; Cl, 11.95; N, 18.88
CAS #	1811510-56-1
Related CAS #	1883548-86-4 (2HCl);1811510-56-1;2118243-34-6 (HCl);
PubChem CID	118701008
Appearance	White to off-white solid powder
LogP	3
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	2
Heavy Atom Count	21
Complexity	332
Defined Atom Stereocenter Count	0
InChi Key	KHPCIHZXOGHCLY-UHFFFAOYSA-N
InChi Code	InChI=1S/C16H13ClN4/c17-13-4-1-10(2-5-13)14-7-12(9-21-16(14)19)11-3-6-15(18)20-8-11/h1-9H,(H2,18,20)(H2,19,21)
Chemical Name	5-(6-aminopyridin-3-yl)-3-(4-chlorophenyl)pyridin-2-amine
Synonyms	PF-06260933; PF 06260933; PF06260933
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	MAP4K4 (IC50 = 3.7 nM)
ln Vitro	PF-6260933 demonstrates good physicochemical characteristics and a moderate HLM clearance[1].
ln Vivo	PF-6260933 exhibits a plasma exposure after oral dosing (10 mg/kg) that provided free drug concentrations above the cell IC50 value for about 4-6 h after administration in a mouse model. For use as a tool in an in vivo diabetes model, PF-6260933's PK properties in mice are suitable[1].
Enzyme Assay	Aortas were lysed in 1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 50 mM EDTA, and 1 × HALT protease and phosphatase inhibitors (Thermo Scientific). Immunoprecipitates were then made using Bethyl MAP4K4 antibodies (503 A; 1 μg) or regular rabbit IgG (Cell Signaling 2729; 1 μg). The immunoprecipitates were combined with 1 μg of myelin basic protein (MBP) and 10 μCi of [γ-32P]ATP, and then incubated for 30 min at 30 °C in kinase buffer (20 mM HEPES, 10 nM MgCl2, 1 mM dithiothreitol (DTT), and protease and phosphatase inhibitor cocktail. Samples were electrophoretically separated on a 12% SDS-polyacrylamide gel and then autoradiographically viewed.
Cell Assay	In EGM2 media, HUVECs are kept alive at 5% CO2 and 37°C. To ascertain whether pharmacological inhibition of MAP4K4 alters MAPK signaling in response to TNF-α, HUVECs or peritoneal macrophages are treated in vitro with vehicle or PF-06260933.
Animal Protocol	Compound PF-06260933 (10 mg/kg, dissolved in dH2O) is orally administered to 8 to 10-week-old male Apoe-/- mice twice daily for 6 weeks. Male Ldlr-/- mice are subjected to a high-fat diet (HFD) for ten weeks prior to medication administration. Male Ldlr-/- mice aged 8 to 10 weeks receive the same dosage of compound PF-06260933 for 10 weeks. In all studies, the vehicle control is the oral administration of water. CO2 inhalation and bilateral pneumothorax are used to put mice to sleep.
References	[1]. Discovery of an in Vivo Tool to Establish Proof-of-Concept for MAP4K4-Based Antidiabetic Treatment. ACS Med Chem Lett. 2015 Oct 6;6(11):1128-33. [2]. Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis. Nat Commun. 2015 Dec 21;6:8995.

Solubility Data

Solubility (In Vitro)

DMSO: ~30 mg/mL Water: N/A Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.42 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 (8.42 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.3775 mL	16.8873 mL	33.7747 mL
	5 mM	0.6755 mL	3.3775 mL	6.7549 mL
	10 mM	0.3377 mL	1.6887 mL	3.3775 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.