Chk1 (IC50 = 7.7 nM); PIM1 (IC50 = 42 nM); CK2 (IC50 = 215 nM)

CCT244747 poorly inhibits CHK2 (IC50 >10 μM) and CDK1 (IC50 >10 μM). CCT244747 exhibits strong inhibitory effects (>80%) on CHK1, RSK1, RSK2, AMPK, BRSK1, IRAK1, and TrkA. hNav1.5, hKv4.3/hKChIP2, hCav1.2, hKv1.5, hKCNQ1/hminK, and hHCN4 are among the other ion channels that are <25% inhibited by CCT244747 (10 μM)[1]. At an IC50 of 600 nM, CCT244747 inhibits FLT3. In human colon cancer cell lines, CCT244747 (0.5 μM) overcomes genotoxic-induced S and G2 cell cycle arrest. In HT29, SW620, MiaPaCa-2, and Calu6 cell lines, CCT244747 inhibits cellular CHK1 function with IC50s ranging from 29 nM to 170 nM for cellular G2 checkpoint abrogation (mitosis induction assay); the GI50s are between 0.33 and 3μM. In HT29 and SW620 colon cancer cell lines, CCT244747 (0.3 μM) inhibits SN38 and gemcitabine-induced CHK1 activity, which is correlated with the abrogation of cell cycle arrest, induction of DNA damage, and apoptosis[2]. Radiation sensitivity is increased in bladder and head and neck cancer cell lines (T24, RT112, and Cal27) by CCT244747 (0.5-2.0 μM)[3].

In human tumor xenografts, CCT244747 (100 mg/kg po, qd7d) dramatically lowers tumor burden. In HT29 colon tumor xenografts, CCT244747 (100–300 mg/kg, po) inhibits gemcitabine-induced pS296 CHK1 for a maximum of 24 hours[1]. In HT29 colon tumor xenografts and Calu6 human lung cancer xenografts, CCT244747 (75 mg/kg, p.o.) combined with gemcitabine exhibits strong antitumor effects. In SW620 human colon tumor xenografts, CCT244747 (150 mg/kg p.o.) demonstrates antitumor activities with irinotecan as well[2]. In Cal27 xenografts, CCT244747 (100 mg/kg, p.o.) demonstrates radiosensitization activity[3].

A microfluidic assay that tracks the separation of a phosphorylated product from its substrate is used to measure CHK1 kinase activity. CR-8 (500 nM)-containing separation buffer is used to run the assay on an EZ Reader II. Duplicate 8 pt dilution curves are created using an ECHO^® 550 acoustic dispenser and are then directly transferred into 384 polypropylene test plates. Each compound is used at a stock concentration of 50 μM in 100% DMSO. A final assay concentration of 2.5% DMSO and compound concentrations in the range of 0.5-1000 nM are obtained by dispensing a total of 250 nL of DMSO per well. This assay plate is loaded with 6 PL CHK1 (2 nM final concentration, prepared in-house), 2 PL peptide 10 (5-FAM-KKKVSRSGLYRSPSMPENLNRPR-COOH, 1.5 PM final concentration), and 2 PL ATP (90 PM final concentration), all diluted in kinase buffer (HEPES 50 mM, NaN3 0.02%, BSA 0.01%, sodium orthovanadate 0.1 mM, DTT 1 mM, MgCl² 2 mM, Tween20 0.1%). Before being incubated for one hour at room temperature, the plate is sealed and centrifuged for one minute at 1000 rpm. Separation buffer (90 PL) is added to halt the reaction. Using a 12-sipper chip and instrument settings of -1.5 psi and 1750 ΔV, the plate is read on an EZ Reader II. The percentage inhibition is computed in relation to total wells (which contain all reagents and 2.5% DMSO) and blank wells (which contain no enzyme). The percentage conversion of product from substrate is computed automatically. Using a nonlinear regression fit of the log (inhibitor) vs. response with variable slope equation, IC50 values are computed in GraphPad Prism5[1].

Using a 96-hour sulforhodamine B assay (SRB), compound cytotoxicity and the capacity of CHK1 inhibitors to increase SN38 (the active metabolite of the topoisomerase I inhibitor irinotecan) and gemcitabine (an antimetabolite) cytotoxicity are evaluated. Before treatment, HT29 or SW620 cells are seeded in 96-well plates at 1.6 to 3.2 × 10³ cells per well in a volume of 160 μL medium, and they are given 36 hours to attach. In order to perform cytotoxicity assays, 10 mM stock in DMSO of CHK1 inhibitors is serially diluted in medium from a starting concentration of 250 PM. 40 PL is then added in quadruplicate to the appropriate wells to provide a final concentration range of 50-0.1 PM (10 concentrations). After serial dilution in medium from a starting concentration of 2 PM, genotoxic agents (SN38; 10 mM stock in DMSO) are added to each well in quadruplicate, resulting in final concentrations ranging from 200-0.39 nM (10 concentrations). After 96 hours (four doublings) of incubation at 37°C in a humidified 5% CO2 environment, the cells are fixed and stained with SRB. Results are expressed as the concentration of test compound needed to inhibit cell growth by 50% relative to untreated controls (SRB IC50), with appropriate controls included. In potentiation assays, a fixed SRB IC50 concentration of either SN38 or gemcitabine was added in quadruplicate to each well in a volume of 20 μL of medium (10× final concentration), followed by one minute of mixing. In order to obtain a final concentration range of 5-0.039 PM (8 concentrations), CHK1 inhibitor (10 mM stock) is serially diluted from a starting concentration of 50 PM in medium and 20 PL is added per well in quadruplicate. The cells are mixed for one minute and then incubated for ninety-six hours (four doublings) at 37°C in a humidified environment before being fixed and stained with SRB. The results are expressed as the concentration of CHK1 inhibitor needed to inhibit cell growth by 50% (potentiation IC50), with untreated and genotoxic alone treated controls included. The ratio of the SRB IC50 versus potentiation IC50 (PI = SRB IC50 / Potentiation IC50) is used to calculate the potentiation index (PI), which is used to assess the ability of the CHK1 inhibitor to increase SN38 or gemcitabine cytotoxicity[1].

Six-week-old female BALB/c mice are housed in a controlled environment with unlimited access to food and sterile water. The animals' weight during the experiment was 20 (±2) g. The compounds are dissolved in 10% DMSO and 5% Tween20 in 85% saline to create dosing solutions. Each of the compounds is given both intravenously and orally. Before administering a single intravenous bolus injection into a lateral tail vein, the animals are warmed. Oral gavage is used for oral administration. Plasma is extracted from blood at predetermined intervals (1 and 6 hours post-dosage) using cardiac punctures performed under anesthesia, transferred to microcentrifuge tubes, and centrifuged for 2 minutes at 4500 × g. Olomoucine is used as an internal standard for multiple reaction monitoring of selected transitions in high performance liquid chromatography tandem mass spectrometry on a triple quadrupole apparatus for quantitative analysis. The measured matrix's concentrations are measured between 2 and 1000 nM using a standard curve as a guide. At the levels of 25, 250, and 750 nM, quality controls are present. Samples are diluted in the relevant matrix as needed[1].

[1]. Discovery of 3-alkoxyamino-5-(pyridin-2-ylamino)pyrazine-2-carbonitriles as selective, orally bioavailable CHK1 inhibitors. J Med Chem. 2012 Nov 26;55(22):10229-40.

[2]. CCT244747 is a novel potent and selective CHK1 inhibitor with oral efficacy alone and in combination with genotoxic anticancer drugs. Clin Cancer Res. 2012 Oct 15;18(20):5650-61.

[3]. An orally bioavailable Chk1 inhibitor, CCT244747, sensitizes bladder and head and neck cancer cell lines to radiation. Radiother Oncol. 2017 Mar;122(3):470-475.

C20H24N8O2

408.46

408.202

C, 58.81; H, 5.92; N, 27.43; O, 7.83

1404095-34-6

1404095-34-6

54758482

Yellow solid powder

2.297

1

9

8

30

584

1

C1N=C(NC2N=C(O[C@@H](CN(C)C)C)C(C#N)=NC=2)C=C(OC)C=1C1=CN(C)N=C1

IENLGMOXAQMNEH-CYBMUJFWSA-N

InChI=1S/C20H24N8O2/c1-13(11-27(2)3)30-20-16(7-21)22-10-19(26-20)25-18-6-17(29-5)15(9-23-18)14-8-24-28(4)12-14/h6,8-10,12-13H,11H2,1-5H3,(H,23,25,26)/t13-/m1/s1

3-[(2R)-1-(dimethylamino)propan-2-yl]oxy-5-[[4-methoxy-5-(1-methylpyrazol-4-yl)pyridin-2-yl]amino]pyrazine-2-carbonitrile

CCT244747; CCT-244747; CCT 244747

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)

DMSO: ~50 mg/mL (~122.4 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.12 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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.

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