CHIR-124 (CHIR124; CHIR 124) is a novel, potent and selective quinolone-based small molecule Chk1 (Checkpoint kinase1) inhibitor with potential anticancer activity. In a cell-free assay, it inhibits Chk1 with an IC50 of 0.3 nM. The structure of CHIR-124 differs from that of other recognized Chk1 inhibitors. It exhibits 500–5,000 times less activity against CDK2/4 and Cdc2, and 2,000 times selectivity against Chk2. Based on isobologram or response surface analysis, CHIR-124 interacts synergistically with topoisomerase poisons (e.g., camptothecin and/or SN-38) to inhibit growth in a number of p53-mutant solid tumor cell lines. A new and powerful inhibitor of Chk1, CHIR-124 shows promise as an antitumor agent when combined with topoisomerase I poisons.

Physicochemical Properties

Molecular Formula	C23H22CLN5O
Molecular Weight	419.91
Exact Mass	419.151
Elemental Analysis	C, 65.79; H, 5.28; Cl, 8.44; N, 16.68; O, 3.81
CAS #	405168-58-3
Related CAS #	405168-58-3
PubChem CID	135399748
Appearance	Light yellow to brown solid powder
Density	1.5±0.1 g/cm3
Index of Refraction	1.750
LogP	3.86
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	30
Complexity	720
Defined Atom Stereocenter Count	1
SMILES	ClC1=CC2=C(NC(C(C3=NC4=C(C=CC=C4)N3)=C2N[C@@H]5CN6CCC5CC6)=O)C=C1
InChi Key	MOVBBVMDHIRCTG-LJQANCHMSA-N
InChi Code	InChI=1S/C23H22ClN5O/c24-14-5-6-16-15(11-14)21(25-19-12-29-9-7-13(19)8-10-29)20(23(30)28-16)22-26-17-3-1-2-4-18(17)27-22/h1-6,11,13,19H,7-10,12H2,(H,26,27)(H2,25,28,30)/t19-/m1/s1
Chemical Name	4-[[(3S)-1-azabicyclo[2.2.2]octan-3-yl]amino]-3-(1H-benzimidazol-2-yl)-6-chloro-1H-quinolin-2-one
Synonyms	CHIR-124; CHIR 124; CHIR124
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	Chk1 (IC50 = 0.3 nM); Chk2 (IC50 = 697.4 nM); PDGFR (IC50 = 6.6 nM); FLT3 (IC50 = 5.8 nM); Cdk4/cyclin D (IC50 = 2.05 μM); CDC2/cyclin B (IC50 = 0.5057 μM); Cdk2/cyclin A (IC50 = 0.1911 μM); bFGFR (IC50 = 2.01 μM); FGFR3 (IC50 = 1.29 μM); VEGFR2 FLK1 (IC50 = 0.5779 μM); VEGFR1 FLT1 (IC50 = 0.4636 μM); PKCα (IC50 = 0.58 μM); PKAβ I (IC50 = 2.25 μM); PKCβ II (IC50 = 0.58 μM); PKCγ (IC50 = 0.11 μM); ERK2 (IC50 = 4.31 μM); PKA (IC50 = 0.1031 μM); GSK3 (IC50 = 0.0233 μM)
ln Vitro	CHIR-124 is a quinolone-based small molecule that is structurally unrelated to other known inhibitors of Chk1. In a range of cancer cell lines, including breast carcinoma (MDA-MB-231 and MDA-MB-435) and colon carcinoma (SW-620 and Colo205), all of which contain the mutant p53 gene, CHIR-124 plays a synergistic interaction with topoisomerase poisons (e.g., Camptothecin or SN-38) to cause growth inhibition. MDA-MD-435 breast cancer cells undergo enhanced apoptosis when CHIR-124 disrupts the S and G2-M checkpoints that are triggered by SN-38. The loss of p53 facilitates CHIR-124's induction of apoptosis and its disruption of the G2-Mcheckpoint.[1] With IC50 values of 5.8 nM and 6.6 nM, respectively, CHIR-124 also effectively targets other kinases, including PDGFR. [2]
ln Vivo	In an orthotopic breast cancer xenograft model, CHIR-124 accelerates the growth inhibitory effects of irinotecan by disrupting the G2-M checkpoint and boosting tumor apoptosis.
Enzyme Assay	The kinase domain of Sf9 insect cells is used to express the Chk1 assay, and the substrate is a biotinylated cdc25c peptide that contains the consensus Chk1/Chk2 phosphorylation site (*) (biotin-[AHX]SGSGS*GLYRSPSMP-ENLNRPR[CONH2]). A kinase reaction buffer containing 30 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 2 mM DTT, 4 mM EDTA, 25 mMβ-glycerophosphate, 5 mM MnCl2, 0.01% bovine serum albumin, 1.35 nM CHK1 kinase domain, 0.5 μM peptide substrate, 1 AM unlabeled ATP, plus 5 nM 33Pγ-labeled ATP (specific activity = 2,000 Ci/mmol) is combined with a series of CHIR-124 dilutions. A radioactive technique is used to carry out the reactions and detect the phosphate transfer. For one to four hours, reactions are incubated at room temperature. The phosphorylated peptide is then captured on microtiter plates coated with streptavidin and containing stop reaction buffer (pH 7.5, 25 mM ethylenediaminetetraacetic acid, 50 mMHEPES). Using an anti-phosphotyrosine antibody (PT66) labeled with europium, the DELFIA TRF system measures phosphorylated peptide. Using nonlinear regression and the data analysis program XL-Fit, the concentration of CHIR-124 for IC50 is determined.
Cell Assay	In log-phase, MDA-MB-231, MDA-MB-435, SW-620, and COLO 205 cells are plated into 96-well microplates. Six distinct concentrations of camptothecin or 0 nM camptothecin are added to serially diluted CHIR-124. If CHIR-124 is not present, camptothecin is also serially diluted. In 96-well plates, cells are treated with CHIR-124, and they are then incubated for 48 hours at 37 °C. Every therapeutic condition is completed in three copies. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), inner salt assay is used to track cell proliferation. After adding MTS inner salt and letting the microplates sit for an additional three hours, the absorbance at 490 nm is measured using a plate reader. To create the isoboles, the drug concentrations in the combinations needed to produce 50% inhibition are plotted. The basis for isobologram analysis of drug interaction is the Loewe additivity equation (1= D A /IC50, A + DB/IC50, B), where DA and DB are the concentrations of each drug in the combination that produce 50% overall inhibition, and IC50, A and B are the concentrations of drugs to result in 50% inhibition for each drug alone. Each graph includes a diagonal line that represents Loewe additivity. Data points that lie above the line will indicate antagonism, while those that fall below the line will show synergy.
Animal Protocol	The following ten treatment groups are randomly assigned to severe combined immunodeficient mice bearing MDA-MD-435 tumor xenografts: the vehicle (captisol) by itself, 5 mg/kg CPT-11, 10 mg/kg CHIR-124, 20 mg/kg CHIR-124, 5 mg/kg CPT-11 plus 10 mg/kg CHIR-124, or 5 mg/kg CPT-11 plus 20 mg/kg CHIR-124. Day 1 (the day following randomization) is when treatment begins. CPT-11 is administered intraperitoneally (i.p.) four times a day (×5) on days 1 through 5, while CHIR-124 is administered orally four times a day ×6 in captisol on days 2 through 7. The formula for calculating percent tumor growth inhibition is T/C, where T is the treatment group mean and C is the control group mean. In a related study, tumor samples taken from mice that were sacrificed on the fourth day of treatment are analyzed for mitotic index using immunofluorescence labeling with phospho-histone H3 antibody and for apoptosis using terminal deoxynucleotidyl transferase-mediated nick-end labeling staining.
References	[1]. Clin Cancer Res . 2007 Jan 15;13(2 Pt 1):591-602. [2]. Clin Cancer Res . 2010 Jan 15;16(2):376-83.
Additional Infomation	CHIR-124 is a potent inhibitor of Chk1 that potentiates the cytotoxicity of topoisomerase I poisons in vitro and in vivo.

Solubility Data

Solubility (In Vitro)

DMSO: ~7 mg/mL (~16.7 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 0.71 mg/mL (1.69 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 7.1 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: ≥ 0.71 mg/mL (1.69 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 7.1 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 3: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.3815 mL	11.9073 mL	23.8146 mL
	5 mM	0.4763 mL	2.3815 mL	4.7629 mL
	10 mM	0.2381 mL	1.1907 mL	2.3815 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.