XMD17-109 (XMD-17-109), a pyrimido-diazepinone analog, is a novel, potent, specific, cell-permeable and ATP-competitive inhibitor of ERK-5 with important biological/anticancer activity. In HEK293 cells, it has an EC50 of 4.2 μM and inhibits ERK-5. In cellular assays, XMD17-109 is able to prevent ERK5 from becoming autophosphorylated. The pharmacokinetic characteristics of XMD17-109 when administered to mice orally and intravenously are as follows: the oral bioavailability is 90%, the oral clearance is 15745 h*ng/mL, the plasma clearance is 8.64 mL/min/Kg, and the T1/2 is 8.2 h.

Physicochemical Properties

Molecular Formula	C36H46N8O3
Molecular Weight	638.81
Exact Mass	638.369
Elemental Analysis	C, 67.69; H, 7.26; N, 17.54; O, 7.51
CAS #	1435488-37-1
Related CAS #	1435488-37-1
PubChem CID	71604307
Appearance	White to beige solid powder
Density	1.3±0.1 g/cm3
Boiling Point	831.6±75.0 °C at 760 mmHg
Flash Point	456.8±37.1 °C
Vapour Pressure	0.0±3.0 mmHg at 25°C
Index of Refraction	1.640
LogP	2.39
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	7
Heavy Atom Count	47
Complexity	1060
Defined Atom Stereocenter Count	0
SMILES	O=C1C2=C(C=CC=C2)N(C3CCCC3)C4=NC(NC5=C(OCC)C=C(C(N6CCC(N7CCN(C)CC7)CC6)=O)C=C5)=NC=C4N1C
InChi Key	XVBGRTMNFNMINE-UHFFFAOYSA-N
InChi Code	InChI=1S/C36H46N8O3/c1-4-47-32-23-25(34(45)43-17-15-26(16-18-43)42-21-19-40(2)20-22-42)13-14-29(32)38-36-37-24-31-33(39-36)44(27-9-5-6-10-27)30-12-8-7-11-28(30)35(46)41(31)3/h7-8,11-14,23-24,26-27H,4-6,9-10,15-22H2,1-3H3,(H,37,38,39)
Chemical Name	11-cyclopentyl-2-[2-ethoxy-4-[4-(4-methylpiperazin-1-yl)piperidine-1-carbonyl]anilino]-5-methylpyrimido[4,5-b][1,4]benzodiazepin-6-one
Synonyms	XMD-17109; XMD 17-109; XMD17109; XMD 17109; XMD17-109; XMD-17-109
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	ERK5 (IC50 = 162 nM); LRRK2[G2019S] (IC50 = 339 nM)
ln Vitro	XMD17-109 (Compound 26) blocks pidermal growth factor-induced ERK5 autophosphorylation in cells with an EC50 of 0.09 ± 0.03 μM and inhibits ERK5 biochemically with an IC50 of 0.162 0.006 M. Additionally, XMD17-109 blocks LRRK2[G2019S] with an IC50 value of 339 nM[1]. By significantly dose-dependently reducing the mobility shifted phosphorylated ERK5 bands from sorbitol stimulated cells, XMD17-109 demonstrates low nanomolar cellular activity. At 30 M and an EC50 of 4.2 μM, XMD17-109 completely blocks ERK5-mediated AP1 transcriptional activity[2].
ln Vivo	NA
Enzyme Assay	In kinase buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 1 mM 2-mercaptoethanol) with 200 ng of pure active ERK5 and the specified amount of inhibitor, kinase activity was assessed in an assay volume of 40 μL. 10 mM magnesium acetate, 50 μM [γ-32P]-ATP (500 cpm/pmol), and 250 μM PIMtide (ARKKRRHPSGPPTA) were added as substrates to begin the reaction. Assays were conducted for 20 min at 30 °C, followed by the application of the reaction mixture onto p81 paper and the measurement of the incorporated radioactivity as previously described.
Cell Assay	HeLa cells are maintained in 10% FBS, 2 mM l-glutamine, 50 U/mL penicillin G, and 50 μg/mL streptomycin-supplemented DMEM. Before use HeLa cells are serum starved for 16 h in DMEM supplemented with 2 mM l-glutamine, 50 U/mL penicillin G, and 50 μg/mL streptomycin. After that, HeLa cells are exposed to ERK5-IN-1 at the indicated concentrations for 1 hour before being stimulated with 0.5mol/L sorbitol for 30 minutes. Triton lysis buffer, which contains 20 μg of protein per well and contains 50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and 1 mM sodium pyrophosphate, as well as 0.27 mol/L sucrose and 1 M microcystin-LR, is used to lyse cells. Standard procedures are used to run samples on 8% polyacrylamide gels. Proteins are transferred onto nitrocellulose membranes, and specific proteins are found using immunoblotting.
Animal Protocol	NA NA
References	[1]. Structural determinants for ERK5 (MAPK7) and leucine rich repeat kinase 2 activities of benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones. Eur J Med Chem. 2013;70:758-67. [2]. X-ray Crystal Structure of ERK5 (MAPK7) in Complex with a Specific Inhibitor. Journal of Medicinal Chemistry (2013), 56(11), 4413-4421. [3]. Extracellular signal-regulated kinase 5 promotes acute cellular and systemic inflammation. Sci Signal. 2015 Aug 25;8(391):ra86.

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (~156.5 mM) Water: <1 mg/mL Ethanol: ~100 mg/mL (~156.5 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.91 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 (3.91 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 3: ≥ 2.5 mg/mL (3.91 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	1.5654 mL	7.8271 mL	15.6541 mL
	5 mM	0.3131 mL	1.5654 mL	3.1308 mL
	10 mM	0.1565 mL	0.7827 mL	1.5654 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.