Chk1 (IC50 = 1.3 nM); Chk2 (IC50 = 2440 nM); ERK8 (IC50 = 130 nM); PKD1 (IC50 = 298 nM); RSK2 (IC50 = 361 nM); RSK1 (IC50 = 362 nM); FLT3 (IC50 = 582 nM); MARK3 (IC50 = 698 nM); NUAK1 (IC50 = 711 nM); CLK2 (IC50 = 2440 nM); BRSK1 (IC50 = 1660 nM); AMPK (IC50 = 2970 nM); PHK (IC50 = 3470 nM); CDK2/CyclA (IC50 = 3850 nM); CDK1/CyclB (IC50 = 9030 nM)

CCT245737 (10 µM) demonstrates >80% inhibition of a panel of 124 kinases, with IC50s of 130, 298, 361, and 362 nM for ERK8, PKD1, RSK2, and RSK1 among them[1]. In HT29, SW620, MiaPaCa-2, and Calu6 cell lines, CCT245737 obliterates a G2 checkpoint induced by VP-16; its IC50 values range from 30 to 220 nM[2].

CCT245737 (150 mg/kg p.o.) and LY 188011 (100 mg/kg iv) together suppress tumor growth in HT29 colon cancer xenografts. In SW620 human colon cancer xenografts, CCT245737 (300 mg/kg, p.o.) also prevents the autophosphorylation of pSer296 CHK1 that is induced by LY 188011 (60 mg/kg iv) after 24 hours[1]. In an Eμ-Myc mouse model of human B-cell lymphocytic leukemia, CCT245737 (150 mg/kg, p.o.) by itself dramatically suppresses tumor growth[2].

In vitro kinase assays[2]
Commercial in vitro 33P radiometric kinase assays were carried out against 124 human kinases using 10μM CCT245737 at ATP concentrations corresponding to the kinase Km,ATP. Other kinase IC50 determinations for CHK2 and FLT3 were performed using a commercial assay (Z'-Lyte) or in-house with recombinant human CHK1 on a LabChip® EZ Reader II (PerkinElmer) or CDK1 in a DELFIA assay

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Biochemical Assays[1]
In vitro assays for inhibition of CHK1 and CHK2 were carried out as described previously.

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CCT245737 (150 mg/kg p.o.) and LY 188011 (100 mg/kg iv) together suppress tumor growth in HT29 colon cancer xenografts. In SW620 human colon cancer xenografts, CCT245737 (300 mg/kg, p.o.) also prevents the autophosphorylation of pSer296 CHK1 that is induced by LY 188011 (60 mg/kg iv) after 24 hours[1]. In an Eμ-Myc mouse model of human B-cell lymphocytic leukemia, CCT245737 (150 mg/kg, p.o.) by itself dramatically suppresses tumor growth[2].

Cytotoxicity is quantified by utilizing a 96-hour Sulforhodamine B (SRB) assay to find the drug concentration that results in 50% inhibition of tumor cell proliferation (GI50). Using a cell-based ELISA, the inhibition of intracellular CHK1 activity is assessed in order to disrupt a G2 checkpoint induced by VP-16 (mitosis induction assay, MIA). With nocodazole present, the G2 checkpoint abrogation (MIA) IC50 is ascertained using UCN01 as a positive control. The ability of a compound to induce mitosis in relation to its toxicity is measured by the activity index (AI), which is the ratio of MIA IC50 to 96-hour SRB GI50. The combination GI50 of CCT245737 is determined by performing standard potentiation studies with a fixed concentration (GI50) of either SN38 or LY 188011 in combination with a range of CCT245737 concentrations. Potentiation index (PI): the ratio of CCT245737's single GI50 to CCT245737's combination GI50 indicates how well CCT245737 can increase LY 188011 or SN38 cell killing. A PI value greater than one signifies a heightened genotoxic activity. Furthermore, to ascertain the degree to which CCT245737 augments drug cytotoxicity in comparison with the genotoxic agent alone, i.e. conventional PI (ratio GI50 genotoxic alone: GI50 genotoxic combined with non-toxic CCT245737 concentration, Con PI), a series of experiments is conducted using fixed, non- or minimally toxic concentrations of CCT245737 (≤GI20) with a variety of different concentrations of LY 188011 or SN38[2].

The female NCr athymic mice, aged 6-8 weeks, have s.c. injections of human HT29 colorectal carcinoma cells in their flanks. Dosing started five days after transplantation, or when the tumors reached a mean diameter of 5.5 mm. Compounds 4 (CCT245737) and 41 (150 mg/kg p.o.) are dosed in 10% DMSO 20% PEG 400, 5% Tween 80, and 65% water in the hours following each dose of LY 188011 (100 mg/kg i.v.). Days 0 through 14 are dosed in saline. Three times a week, body weights and tumor measurements are made. Tumors that grow to a predefined humane endpoint (mean diameter <15 mm) are individually culled from the animals[1].

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Compound tolerability and pharmacokinetic investigations were carried out in female BALB/c mice (Charles River). Human tumor xenografts were established s.c. in female CRTac:Ncr-Fox1(nu) athymic mice and treated as previously described. The vehicle for oral administration of CCT245737 was DPTW (10% DMSO, 20% PEG400, 5% Tween 80 and 65% water) and gemcitabine and irinotecan were administered in their respective clinical vehicles. Treatments were generally initiated when tumors reached a mean diameter of 5-6mm (day 0). For combination studies, CCT245737 was given orally 24 and 48h after genotoxic drug administration, previously determined as an optimal schedule for CHK1 inhibitor and genotoxic drug combinations. In HT29 xenograft studies, gemcitabine was administered at 100mg/kg i.v. on days 0,7 and 14 and CCT245737 at the indicated doses on days 1,2,8,9,15 and16. Irinotecan was administered at 25mg/kg i.p. on days 0,4 and 8 with CCT245737 administered at 150mg/kg p.o. on days 1,2,5,6,9 and 10. In SW620 and Calu6 xenograft studies, gemcitabine was administered at 100mg/kg i.v. on days 0,4 and 8 and CCT245737 subsequently at 150mg/kg on days 1,2,5,6,9 and 10. For the Calu6 xenograft studies involving gemcitabine and carboplatin, drugs were administered at 100mg/kg i.v. and 5mg/kg i.p., respectively on day 0 with gemcitabine alone at 100mg/kg i.v. on day 7 with CCT245737 at 150mg/kg p.o. on days 1,2,8 and 9. The genotoxic drug doses employed were sub-maximally active to facilitate detection of subsequent potentiation. Initial treatment groups contained from 6 to 10 mice and animals were inspected daily and tumor size and volume measured every 2 or 3 days. Tumor volume and growth delay were determined as previously described [2].
Transgenic Eμ-Myc mice which develop aggressive infiltrating lymphoma were established and monitored as previously described. To generate transgenic Eμ-Myc driven lymphoma allografts, tumor cells from 3 separate tumors were harvested, cells counted and injected via a tail vein. Six mice were set up per tumor to provide 3 control and 3 treated animals, giving a maximum of 9 mice per treatment group. Animals were monitored daily and continuously using RFID transponders to measure temperature, activity and water consumption as previously described. For studies of single-agent CCT245737 activity in mice injected with transgenic Eμ-Myc lymphoid tumor cells, CCT245737 was administered at 150mg/kg p.o. for 9 successive days with culling 24h after the last dose. Lymph nodes and other tissues were removed from vehicle and CCT245737 treated mice and their weights and tissue/body weight ratios compared to assess antitumor activity. Bone marrow cellularity was also determined to check for tumor cell involvement. All mice were handled in compliance with local and national animal welfare guidelines[2].

[1]. Multiparameter Lead Optimization to Give an Oral Checkpoint Kinase 1 (CHK1) Inhibitor Clinical Candidate: (R)-5-((4-((Morpholin-2-ylmethyl)amino)-5-(trifluoromethyl)pyridin-2-yl)amino)pyrazine-2-carbonitrile (CCT245737). J Med Chem. 2016 Jun 9;59(11):5221-37.

[2]. The clinical development candidate CCT245737 is an orally active CHK1 inhibitor with preclinical activity in RAS mutant NSCLC and Eμ-MYC driven B-cell lymphoma. Oncotarget. 2016 Jan 19;7(3):2329-42.

Chk1 Inhibitor SRA737 is an orally bioavailable inhibitor of checkpoint kinase 1 (chk1), with potential antineoplastic and chemosensitization activities. Upon oral administration, chk1 inhibitor SRA737 selectively binds to chk1, thereby preventing chk1 activity and abrogating the repair of damaged DNA. This may lead to an accumulation of damaged DNA, inhibition of cell cycle arrest, and induction of apoptosis. SRA737 may potentiate the cytotoxicity of DNA-damaging agents and reverse tumor cell resistance to chemotherapeutic agents. Chk1, an adenosine triphosphate (ATP)-dependent serine/threonine kinase overexpressed in a variety of cancer cell types, mediates cell cycle checkpoint control and is essential for DNA repair; it plays a key role in resistance to chemotherapeutic agents by repairing DNA damage.

C16H16F3N7O

379.34

379.136

C, 50.66; H, 4.25; F, 15.02; N, 25.85; O, 4.22

1489389-18-5

1489389-18-5;

72165232

White to off-white solid powder

1.4±0.1 g/cm3

547.2±50.0 °C at 760 mmHg

284.7±30.1 °C

0.0±1.5 mmHg at 25°C

1.589

3.82

3

11

5

27

526

1

FC(F)(F)C(C=NC(NC1=CN=C(C#N)C=N1)=C2)=C2NC[C@H]3CNCCO3

YBYYWUUUGCNAHQ-LLVKDONJSA-N

InChI=1S/C16H16F3N7O/c17-16(18,19)12-8-25-14(26-15-9-22-10(4-20)5-24-15)3-13(12)23-7-11-6-21-1-2-27-11/h3,5,8-9,11,21H,1-2,6-7H2,(H2,23,24,25,26)/t11-/m1/s1

5-[[4-[[(2R)-morpholin-2-yl]methylamino]-5-(trifluoromethyl)pyridin-2-yl]amino]pyrazine-2-carbonitrile

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: ≥ 2.5 mg/mL (6.59 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 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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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.59 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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