Chk2 Inhibitor II (also known as BML-277) is an ATP-competitive inhibitor of Chk2 (checkpoint kinase 2) with an IC50 of 15 nM. The selectivity towards Chk2 is 1000 times higher than that of Chk1 and Cdk1/B kinases. Human CD4+ and CD8+ T-cells are protected from ionizing radiation-induced apoptosis by BML-277 in a dose-dependent manner. With an observed EC50 of 3−7.6 μM, it can effectively rescue both T-cell populations from radiation-induced apoptosis in a dose-dependent manner. The biochemical measurement of chk2 inhibition is consistent with the concentration of BML-277 needed for radioprotection.

Physicochemical Properties

Molecular Formula	C20H14CLN3O2
Molecular Weight	363.80
Exact Mass	363.077
Elemental Analysis	C, 66.03; H, 3.88; Cl, 9.74; N, 11.55; O, 8.80
CAS #	516480-79-8
Related CAS #	516480-79-8; 516480-80-1;
PubChem CID	9969021
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Boiling Point	637.1±63.0 °C at 760 mmHg
Flash Point	339.1±33.7 °C
Vapour Pressure	0.0±1.9 mmHg at 25°C
Index of Refraction	1.703
LogP	4.61
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	4
Heavy Atom Count	26
Complexity	489
Defined Atom Stereocenter Count	0
SMILES	ClC(C=C1)=CC=C1OC(C=C2)=CC=C2C3=NC4=CC(C(N)=O)=CC=C4N3
InChi Key	UXGJAOIJSROTTN-UHFFFAOYSA-N
InChi Code	InChI=1S/C20H14ClN3O2/c21-14-4-8-16(9-5-14)26-15-6-1-12(2-7-15)20-23-17-10-3-13(19(22)25)11-18(17)24-20/h1-11H,(H2,22,25)(H,23,24)
Chemical Name	2-[4-(4-chlorophenoxy)phenyl]-3H-benzimidazole-5-carboxamide
Synonyms	BML-277; BML277; Chk2 Inhibitor II; BML 277
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	Chk2 (IC50 = 15 nM)
ln Vitro	The CHK2 Inhibitor II was initially found to be a potent, selective small molecule exhibiting radioprotection towards human T cells. It demonstrates 1,000-fold greater selectivity for the CHK2 serine/threonine kinase than for the Cdk1/B and CK1 kinases. While CHK1 phosphorylation is not specifically inhibited by varying doses of CHK2 inhibitor II, CHK2 phosphorylation at Thr68 is at different times specifically inhibited. When CHK2 inhibitor II and ERK inhibitor are used together, treatment causes a significant increase in apoptosis when compared to when either drug is used alone[2].
ln Vivo	No significant weight loss or gross abnormalities were observed in SUDHL6 DLBCL xenograft mice treated intraperitoneally with either vehicle, ERK inhibitor (5 mg kg−1), CHK2 inhibitor II (1 mg kg−1), or both for 20 days. While both 1 mg/kg CHK2 inhibitor II and 5 mg/kg ERK inhibitor modestly inhibit tumor growth, combined treatment with both inhibitors produces a statistically significant suppression of tumor growth[2].
Enzyme Assay	In order to ascertain the activity of chk2 inhibitors, the following conditions must be met: 5 nM recombinant human Chk2, 50 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 25 μM synthetic peptide substrate (biotin-SGLYRSPSMPENLNRPR), 1 μM ATP, 50 μCi/mL [γ-33P] ATP, and a combination of protease inhibitors. The peptide substrate is bound to streptavidin-conjugated agarose beads by the reaction mixtures after three hours of incubation at 37°C. A 0.1% Tween-20 solution in phosphate-buffered saline (pH 7.4) is used to repeatedly wash the agarose beads. The amount of radioactive phosphate bound to the substrate peptide at various concentrations of BML-277 (6.25, 12.5, 25, 50, 100, and 200 nM) is measured by scintillation counting, which is used to determine the enzyme activity. Kinetic experiments involve varying the concentration of ATP while maintaining a constant ratio between unlabeled and [γ-33P] labeled ATP. Samples are kept on ice for additional processing and reactions are halted at various times by adding 50 mM cold ATP[1].
Cell Assay	Purified T-cells are incubated at 100,000 cells per well in BML-277 (102.5 nM, 1 μM, 100.5 μM, 10 μM, and 101.5 μM) or vehicle (DMSO) at different concentrations in 96-well stripwells for one hour in order to assess the radioprotective effect of Chk2 inhibitors. The cells are then placed back into the incubator for an additional 24 hours after being exposed to a dose of 0 or 10 Gy gamma irradiation from a 137Cs source at a dose rate of 3.65 Gy/min. Propidium iodide and Annexin V-FITC are used to stain cells in accordance with the manufacturer's instructions. Using a FACSCalibur FACS instrument, apoptotic and surviving cells are quantified. The number of survivors from treatment groups less the number of cells surviving in the irradiated control group divided by the number of surviving cells in the untreated control groups is the percentage of recovery used to report data[1].
Animal Protocol	Formulated in alcohol and diluted in PBS; 1 mg/kg; i.p. mice bearing SUDHL6 DLBCL xenografts
References	[1]. J Med Chem . 2005 Mar 24;48(6):1873-85. [1]. Nat Commun . 2011 Jul 19:2:402.

Solubility Data

Solubility (In Vitro)

DMSO: ~72 mg/mL (~197.9 mM) Water: < 1mg/mL Ethanol: ~21 mg/mL (~57.7 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1.67 mg/mL (4.59 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 16.7 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: ≥ 1.67 mg/mL (4.59 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 16.7 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: ≥ 1.67 mg/mL (4.59 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 16.7 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	2.7488 mL	13.7438 mL	27.4876 mL
	5 mM	0.5498 mL	2.7488 mL	5.4975 mL
	10 mM	0.2749 mL	1.3744 mL	2.7488 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.