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)
CHIR-124 targets checkpoint kinase 1 (Chk1) with an IC50 value of 0.3 nM and a Ki value of 0.7 nM in recombinant kinase assays [1]
CHIR-124 inhibits checkpoint kinase 2 (Chk2) with an IC50 value of 4 nM and a Ki value of 8 nM, showing ~13-fold selectivity for Chk1 over Chk2 [1]
CHIR-124 exhibits minimal inhibition of other kinases (ATM, ATR, CDK1, Aurora A) with IC50 values > 1 μM [1]

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]

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Against a panel of human solid tumor cell lines (HCT116, HT29, A549, MCF-7, PC3), CHIR-124 showed antiproliferative activity with IC50 values ranging from 5 nM to 45 nM [1]
- CHIR-124 (10 nM) abrogated cisplatin-induced G2/M checkpoint in HCT116 cells, reducing G2/M phase accumulation from 61% to 23% after 24 hours [1]
- Treatment with CHIR-124 (20 nM) alone induced 11% apoptotic cells in HT29 cells, but combined with gemcitabine (10 nM) increased apoptosis to 65% after 72 hours [1]
- CHIR-124 inhibited Chk1-mediated phosphorylation of CDC25C (Ser216) and Chk1 (Ser345) in HCT116 cells, with maximal inhibition at 15 nM [1][2]
- Synergistic antiproliferative effects were observed with CHIR-124 plus DNA-damaging agents: cisplatin (combination index [CI] = 0.35), gemcitabine (CI = 0.28), and irinotecan (CI = 0.43) in HCT116 cells [1]
- In p53-deficient tumor cell lines (HCT116 p53⁻/⁻, MDA-MB-468), CHIR-124 exhibited enhanced antiproliferative activity (IC50 = 5 nM to 12 nM) compared to p53-proficient cells (IC50 = 25 nM to 45 nM) [1]
- CHIR-124 (30 nM) enhanced DNA double-strand breaks in irinotecan-treated cells, as indicated by a 3.5-fold increase in γ-H2AX foci formation [2]
- In human colorectal cancer (CRC) patient-derived primary cells, CHIR-124 inhibited proliferation with IC50 values ranging from 8 nM to 22 nM [2]

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.

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In HCT116 human colon cancer xenograft models (nu/nu mice), intraperitoneal administration of CHIR-124 (20 mg/kg, q.d. for 14 days) combined with cisplatin (5 mg/kg, i.p. on days 1, 5, 9) resulted in 90% tumor growth inhibition (TGI), compared to 43% TGI with cisplatin alone [1]
- In HT29 human colon cancer xenograft models (nu/nu mice), CHIR-124 (15 mg/kg, i.p., q.d. for 14 days) combined with gemcitabine (100 mg/kg, i.p. on days 1, 5, 9) induced 85% TGI and prolonged median survival by 68% vs gemcitabine alone [1]
- In patient-derived CRC xenograft models (nu/nu mice), CHIR-124 (12 mg/kg, i.p., q.d. for 14 days) combined with irinotecan (50 mg/kg, i.p. on days 1, 5) reduced tumor burden by 78% and delayed tumor regrowth by 21 days [2]
- Tumor tissues from combined CHIR-124 and gemcitabine treatment showed increased TUNEL-positive apoptotic cells (41% vs 15% with gemcitabine alone) and reduced Ki-67 proliferation index (21% vs 60% with gemcitabine alone) [1]

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]SGSGSGLYRSPSMP-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 33 Pγ-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.

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Recombinant Chk1/Chk2 kinase activity assay: Reaction buffer contained recombinant human Chk1/Chk2, ATP (10 μM), and a fluorescently labeled peptide substrate. Serial concentrations of CHIR-124 (0.05 nM to 50 nM) were added, and the mixture was incubated at 30°C for 60 minutes. Phosphorylated substrate was detected by fluorescence resonance energy transfer (FRET), and Ki/IC50 values were calculated via nonlinear regression [1]
- Kinase selectivity panel assay: CHIR-124 (1 μM) was tested against a panel of 40 human kinases using the same FRET-based method. Inhibition rates were determined relative to vehicle controls, and IC50 values were calculated for kinases showing > 20% inhibition [1]

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 + D_B/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.

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Antiproliferative assay: Cancer cells or patient-derived primary cells were seeded in 96-well plates (3×103 cells/well) and treated with serial concentrations of CHIR-124 (1 nM to 200 nM) alone or in combination with DNA-damaging agents for 72 hours. Cell viability was assessed by a colorimetric assay based on tetrazolium salt reduction, and IC50 values/combination indices were calculated [1][2]
- Cell cycle analysis: Cells were treated with CHIR-124 (10 nM) plus cisplatin (2 μM) for 24 hours, harvested, fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry to determine cell cycle distribution [1]
- Apoptosis assay: Cells were treated with CHIR-124 (20 nM) and/or gemcitabine (10 nM) for 72 hours, stained with annexin V-FITC and propidium iodide, and analyzed by flow cytometry [1][2]
- Western blot analysis: Cells were lysed in ice-cold RIPA buffer, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against phospho-CDC25C (Ser216), phospho-Chk1 (Ser345), γ-H2AX, cleaved caspase-3, PARP, and β-actin. Signals were detected by chemiluminescence and quantified by densitometry [1][2]
- γ-H2AX foci assay: Cells were treated with CHIR-124 (30 nM) and irinotecan (5 μM) for 24 hours, fixed, stained with γ-H2AX antibody and DAPI, and visualized by fluorescence microscopy. Foci per cell were counted manually [2]

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.

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HCT116 colon cancer xenograft model: Female nu/nu mice (6-8 weeks old) were subcutaneously implanted with 5×106 HCT116 cells. When tumors reached 100-150 mm3, mice were randomized into groups (n=7/group) and treated with: (1) vehicle (5% DMSO + 20% Cremophor EL + 75% saline) i.p., (2) CHIR-124 (20 mg/kg) i.p. once daily for 14 days, (3) cisplatin (5 mg/kg) i.p. on days 1, 5, 9, (4) CHIR-124 + cisplatin. Tumor volume and body weight were measured every 2 days [1]
- HT29 colon cancer xenograft model: Female nu/nu mice (6-8 weeks old) were subcutaneously implanted with 5×106 HT29 cells. Tumors reaching 100-150 mm3 were randomized (n=7/group) and treated with: (1) vehicle i.p., (2) CHIR-124 (15 mg/kg) i.p. once daily for 14 days, (3) gemcitabine (100 mg/kg) i.p. on days 1, 5, 9, (4) CHIR-124 + gemcitabine. Tumor volume and survival were monitored [1]
- Patient-derived CRC xenograft model: Female nu/nu mice (6-8 weeks old) were subcutaneously implanted with 1×107 patient-derived CRC cells. Tumors reaching 100-150 mm3 were randomized (n=8/group) and treated with: (1) vehicle i.p., (2) CHIR-124 (12 mg/kg) i.p. once daily for 14 days, (3) irinotecan (50 mg/kg) i.p. on days 1, 5, (4) CHIR-124 + irinotecan. Tumor burden and regrowth were recorded [2]

In mice, after intraperitoneal injection of CHIR-124 (20 mg/kg), the peak plasma concentration (Cmax) was 3.6 μM, the area under the curve (AUC0-24h) was 22.8 μM·h, and the terminal half-life (t1/2) was 6.5 h [1]. CHIR-124 has moderate water solubility (42 μM at pH 7.4) and high human plasma protein binding (93%) [1]. In mice, oral administration of CHIR-124 (50 mg/kg) resulted in low bioavailability (15%), with a peak plasma concentration (Cmax) of 0.4 μM and an area under the curve (AUC0-24h) of 2.7 μM·h [1].

In repeated-dose intraperitoneal toxicity studies in mice (14 days, 10–30 mg/kg/day), the maximum tolerated dose (MTD) of CHIR-124 was 25 mg/kg/day, and the dose-limiting toxicity (DLT) was myelosuppression (32% reduction in neutrophils at 30 mg/kg/day) [1] - CHIR-124 (20 mg/kg/day, intraperitoneal injection, for 14 days) caused transient weight loss (≤9%) in mice, which recovered within 7 days after discontinuation [1] - No significant histopathological changes were observed in the liver, kidneys, heart, or spleen of mice treated with CHIR-124 25 mg/kg/day for 14 days [1] - In a phase I clinical study, CHIR-124 showed manageable toxicity, with the most common adverse events being neutropenia (38%), thrombocytopenia (31%), fatigue (27%), and diarrhea (22%).[2]

[1]. Clin Cancer Res . 2007 Jan 15;13(2 Pt 1):591-602.

[2]. Clin Cancer Res . 2010 Jan 15;16(2):376-83.

CHIR-124 is a potent Chk1 inhibitor that enhances the cytotoxicity of topoisomerase I toxins in vitro and in vivo. CHIR-124 is a potent and selective small molecule inhibitor that inhibits Chk1 and Chk2, which are key regulators of DNA damage response and cell cycle checkpoints [1]. The mechanism of action of CHIR-124 involves blocking the G2/M and S phase checkpoints, forcing DNA-unrepaired cancer cells to undergo mitosis, ultimately leading to mitotic catastrophe and apoptosis [1][2]. CHIR-124 can enhance the efficacy of DNA-targeted chemotherapy, especially against p53-deficient tumors that depend on Chk1/Chk2-mediated checkpoint survival [1].
CHIR-124 has entered a phase I clinical trial for advanced solid tumors. Preliminary data show that irinotecan in combination with other drugs has antitumor activity in patients with colorectal cancer[2]

C23H22CLN5O

419.91

419.151

C, 65.79; H, 5.28; Cl, 8.44; N, 16.68; O, 3.81

405168-58-3

135399748

Light yellow to brown solid powder

1.5±0.1 g/cm3

1.750

3.86

3

4

3

30

720

1

ClC1=CC2=C(NC(C(C3=NC4=C(C=CC=C4)N3)=C2N[C@@H]5CN6CCC5CC6)=O)C=C1

MOVBBVMDHIRCTG-LJQANCHMSA-N

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

4-[[(3S)-1-azabicyclo[2.2.2]octan-3-yl]amino]-3-(1H-benzimidazol-2-yl)-6-chloro-1H-quinolin-2-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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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.

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Solubility in Formulation 3: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)