BIX02189 is a novel, potent and selective inhibitor of MEK5 with important biological activity. With an IC50 of 1.5 nM, it inhibits MEK5. It does not inhibit closely related kinases like MEK1, MEK2, ERK2, or JNK2, but it does inhibit ERK5 with an IC50 of 59 nM in cell-free assays. Purified MEK5 enzyme's catalytic activity was said to be inhibited by BIX02189. As a downstream substrate of the MEK5/ERK5 signaling cascade, MEF2C's transcriptional activation was inhibited by BIX02189 in a cellular trans-reporter assay system. To better understand how the MEK5/ERK5 pathway functions in various biological systems, BIX02189 may provide novel pharmacological tools.

Physicochemical Properties

Molecular Formula	C27H28N4O2
Molecular Weight	440.54
Exact Mass	440.221
Elemental Analysis	C, 73.61; H, 6.41; N, 12.72; O, 7.26
CAS #	1094614-85-3
Related CAS #	BIX02189;1265916-41-3
PubChem CID	135659062
Appearance	Solid powder
Density	1.2±0.1 g/cm3
Boiling Point	653.4±55.0 °C at 760 mmHg
Flash Point	349.0±31.5 °C
Vapour Pressure	0.0±2.0 mmHg at 25°C
Index of Refraction	1.659
LogP	2.05
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	6
Heavy Atom Count	33
Complexity	688
Defined Atom Stereocenter Count	0
SMILES	O([H])C1=C(/C(/C2C([H])=C([H])C([H])=C([H])C=2[H])=N/C2=C([H])C([H])=C([H])C(=C2[H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])C2C([H])=C([H])C(C(N(C([H])([H])[H])C([H])([H])[H])=O)=C([H])C=2N1[H]
InChi Key	ZGXOBLVQIVXKEB-UHFFFAOYSA-N
InChi Code	InChI=1S/C27H28N4O2/c1-30(2)17-18-9-8-12-21(15-18)28-25(19-10-6-5-7-11-19)24-22-14-13-20(27(33)31(3)4)16-23(22)29-26(24)32/h5-16,29,32H,17H2,1-4H3InChI=1S/C27H28N4O2/c1-30(2)17-18-9-8-12-21(15-18)28-25(19-10-6-5-7-11-19)24-22-14-13-20(27(33)31(3)4)16-23(22)29-26(24)32/h5-16,29,32H,17H2,1-4H3
Chemical Name	3-[N-[3-[(dimethylamino)methyl]phenyl]-C-phenylcarbonimidoyl]-2-hydroxy-N,N-dimethyl-1H-indole-6-carboxamide
Synonyms	BIX 02189; BIX-02189; BIX02189
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	MEK5 (IC50 = 1.5 nM); ERK5 (IC50 = 59 nM)
ln Vitro	BIX02189 blocks MEK5 and ERK5 catalytic activity with IC50 values of 1.5 nM and 59 nM, respectively. With IC50 values of 4.3 nM and 810 nM, respectively, they are more potent than the effect brought on by BIX02188. BIX02189 exhibits inhibitory activity against CSF1R (FMS) with an IC50 of 46 nM but exhibits no activity against related kinases MEK1, MEK2, ERK1, p38α, JNK2, EGFR, and STK16 with IC50 values of >3.7 μM. Pretreatment with BIX02189 inhibits sorbitol-induced ERK5 phosphorylation in HeLa cells in a dose-dependent manner but has no inhibitory effect on ERK1/2, p38, or JNK1/2 MAPK phosphorylation. In HeLa or HEK293 cells, BIX02189 treatment alone for 24 hours had no cytotoxic effects. In HeLa and HEK293 cells, BIX02189 inhibits MEK5/ERK5/MEF2C-driven luciferase expression with IC50 values of 0.53 μM and 0.26 μM, respectively. This has a greater impact than what BIX02188 has to offer. [1] In human umbilical vein endothelial cells (HUVECs) exposed to 15d-PGJ2, BIX02189 reverses the protective effect brought on by laminar flow (L-flow) by suppressing C-terminus of Hsc70-interacting protein (CHIP)-mediated p53 ubiquitination. [2] In neonatal rat cardiomyocytes (NRCMs) stimulated by isoproterenol, BIX02189 (10 uM) inhibits ERK5 phosphorylation and decreases myocyte enhancer factor 2 (MEF2) transcriptional activity. By accelerating sorbitol-induced apoptosis in NRCMs, BIX02189 supports the protective function of ERK5 in cardiomyocytes. [3]
ln Vivo	Mice are treated with either 10 mg/kg of BIX02189 (in 25% DMSO) or vehicle control (same volume of 25% DMSO) by intraperitoneal injection. The nuclear localization of Nrf2 is inhibited in aortic endothelial cells from mice treated with BIX02189.
Enzyme Assay	Utilizing the PKLight ATP Detection Reagent, the MEK5 protein isolated from the baculovirus expression system is used to assess kinase activity. The assay is carried out in the presence of varying concentrations of BIX02189 using 15 nM GST-MEK5 and 0.75 μM ATP in an assay buffer made up of 25 mM Hepes, pH 7.5, 10 mM MgCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 μM Na3VO4, 0.5 mM DTT, and 1% DMSO. The kinase reaction mixture is incubated for 90 minutes at room temperature before 10 μL of ATP detection reagent is added. For the purpose of determining the IC50 value, the relative light unit (RLU) signal is measured and converted to percent of control (POC) values.
Cell Assay	Before being stimulated with sorbitol at a final concentration of 0.4 M for 20 minutes at 37 °C, the cells are serum-starved for 20 hours. 1.5 hours go by between the addition of BIX02189 and the sorbitol. At 4 °C for 5 to 10 minutes, the cells are collected and lysed in 50 μL of RIPA buffer containing Halt protease and phosphate inhibitors. The lysates are centrifuged at 14,000 rpm for 10 minutes, and then 50 μL of the lysate is mixed with 50 μ 2× sample buffer and boiled at 95 °C for 4 minutes. A sample of 20 microliters is run on 10% Tris-glycine SDS-PAGE gels before being transferred to nitrocellulose. With the appropriate antibodies, western blotting is carried out.
Animal Protocol	Dissolved in 25% DMSO; 10 mg/kg; i.p. C57BL/6-specific pathogen-free mice
References	[1]. Biochem Biophys Res Commun . 2008 Dec 5;377(1):120-5 [2]. Anat Cell Biol . 2011 Dec;44(4):265-73. [3]. Circ Res . 2010 Mar 19;106(5):961-70.
Additional Infomation	3-[[3-[(dimethylamino)methyl]anilino]-phenylmethylidene]-N,N-dimethyl-2-oxo-1H-indole-6-carboxamide is an indolecarboxamide.

Solubility Data

Solubility (In Vitro)	DMSO: ~60 mg/mL (~136.2 mM) Water: <1 mg/mL Ethanol: ~80 mg/mL (~181.6 mM)
Solubility (In Vivo)	2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 10mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2699 mL	11.3497 mL	22.6994 mL
	5 mM	0.4540 mL	2.2699 mL	4.5399 mL
	10 mM	0.2270 mL	1.1350 mL	2.2699 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.