WZ4003 is a novel, potent and highly specific inhibitor of NUAK kinases. It exhibits little inhibition against 139 other kinases but inhibits NUAK1 and NUAK2 with IC50s of 20 nM and 100 nM in cell-base assays.

Physicochemical Properties

Molecular Formula	C25H29CLN6O3
Molecular Weight	496.9892
Exact Mass	496.199
Elemental Analysis	C, 60.42; H, 5.88; Cl, 7.13; N, 16.91; O, 9.66
CAS #	1214265-58-3
Related CAS #	1214265-58-3
PubChem CID	72200024
Appearance	White to beige solid powder
LogP	4.1
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	8
Heavy Atom Count	35
Complexity	667
Defined Atom Stereocenter Count	0
InChi Key	SDGJBAUIGHSMRI-UHFFFAOYSA-N
InChi Code	InChI=1S/C25H29ClN6O3/c1-4-23(33)28-17-6-5-7-19(14-17)35-24-20(26)16-27-25(30-24)29-21-9-8-18(15-22(21)34-3)32-12-10-31(2)11-13-32/h5-9,14-16H,4,10-13H2,1-3H3,(H,28,33)(H,27,29,30)
Chemical Name	N-[3-[5-chloro-2-[2-methoxy-4-(4-methylpiperazin-1-yl)anilino]pyrimidin-4-yl]oxyphenyl]propanamide
Synonyms	WZ 4003; WZ4003; WZ-4003
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	NUAK1 (IC50 = 20 nM); NUAK2 (IC50 = 100 nM)
ln Vitro	WZ4003 (3–10 μM) significantly reduces MYPT1 phosphorylation caused by NUAK1 in HEK-293 cells that are overexpressing wild-type NUAK1. Furthermore, WZ4003 (10 μM) inhibits MYPT1 Ser445 phosphorylation as well as cell migration, invasion, and proliferation to a similar extent as NUAK1 knockout in MEFs or knockdown in U2OS cells.[1] While having a significantly lower cellular IC50 against T790M-containing Ba/F3 cells, WZ4003 also shows a high, specific affinity to the L858R/T790M mutant EGFR. [2]
ln Vivo	NA
Enzyme Assay	The reactions take place in a 50 μL reaction volume for 30 min at 30°C, and they are stopped by sprinkling 40 μL of the reaction mix onto P81 paper and then immediately submerging it in 50 mM orthophosphoric acid. Three 50 mM orthophosphoric acid washings are performed on the samples, followed by one acetone rinse and air drying. Cerenkov counting is used to measure the [γ-32P]ATP incorporation into Sakamototide caused by kinases. One unit of activity is the amount of time it took to catalyze the incorporation of 1 nmol of [32P]phosphate into the substrate over the course of one hour.
Cell Assay	In 96-well plates, colorimetric cell proliferation assays are conducted. U2OS cells and MEFs are initially seeded with 2000 and 3000 cells, respectively, per well. Over the course of five days, the proliferation assays are performed with or without 10 M HTH-01-015 or WZ4003.
Animal Protocol	NA;
References	[1]. Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases. Biochem J. 2014 Jan 1;457(1):215-25. [2]. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature. 2009 Dec 24;462(7276):1070-4.

Solubility Data

Solubility (In Vitro)

DMSO: ~7 mg/mL (~14.1 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.03 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 2: ≥ 2.5 mg/mL (5.03 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	2.0121 mL	10.0606 mL	20.1211 mL
	5 mM	0.4024 mL	2.0121 mL	4.0242 mL
	10 mM	0.2012 mL	1.0061 mL	2.0121 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.