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)
Checkpoint Kinase 1 (CHK1) (Ki = 0.3 nM; IC50 = 0.9 nM for recombinant CHK1 kinase activity; >1000-fold selectivity over CHK2, CDK2, ATR, and 200+ other kinases) [1]

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].

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CCT245737 (0.01-10 nM) dose-dependently inhibited recombinant human CHK1 kinase activity, with 95% inhibition at 5 nM; it blocked CHK1-mediated phosphorylation of CDC25C (Ser216) and histone H3 (Ser10) in A549 cells by 80% and 75% respectively at 10 nM [1]
- CCT245737 suppressed proliferation of RAS mutant NSCLC cell lines (A549, H460, Calu-6) with GI50 values of 0.8 μM, 1.2 μM, and 0.6 μM respectively after 72 hours; it also inhibited Eμ-MYC-driven B-cell lymphoma cells (WEHI-231, BCL1) with GI50 = 0.5 μM and 0.7 μM [2]
- CCT245737 (1 μM) enhanced DNA damage-induced cell death: combined with cisplatin (10 μM) in A549 cells, apoptotic rate increased from 22% (cisplatin alone) to 58% (combination); Western blot showed increased γ-H2AX and cleaved caspase-3 expression [2]
- CCT245737 (0.5 μM) induced G2/M cell cycle arrest in H460 cells (48% increase in G2/M phase cells) by abrogating CHK1-mediated cell cycle checkpoint activation [1]
- CCT245737 showed low cytotoxicity to normal human fibroblasts (CCD-18Co) and bronchial epithelial cells (BEAS-2B): GI50 > 50 μM, resulting in therapeutic indices >50 for RAS mutant NSCLC cells [2]
- CCT245737 (1 μM) reduced colony formation of A549 cells by 82% and WEHI-231 lymphoma cells by 78% compared to control [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].

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Nude mice (BALB/c-nu) bearing A549 (RAS mutant NSCLC) xenografts were administered CCT245737 (25, 50 mg/kg, oral gavage, once daily for 21 days). The 50 mg/kg group showed 72% tumor growth inhibition and 30% extension of median survival (from 32 days to 42 days) [2]
- In Eμ-MYC-driven B-cell lymphoma xenograft mice, CCT245737 (50 mg/kg, po, qd×14) induced 68% tumor volume reduction and increased apoptotic index (TUNEL-positive cells) by 4.2-fold in tumor tissues [2]
- Combination of CCT245737 (25 mg/kg, po) and cisplatin (5 mg/kg, ip, q7d×3) in H460 xenograft mice resulted in 85% tumor growth inhibition, with 2/8 mice achieving complete tumor regression (CR) [2]
- CCT245737 (50 mg/kg, po) treatment in A549 xenograft mice reduced p-CHK1 (Ser345) and p-CDC25C (Ser216) expression in tumor tissues by 70% and 65% respectively, as detected by immunohistochemistry [1]

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].

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Recombinant human CHK1 was incubated with ATP (5 μM) and synthetic CDC25C-derived peptide (substrate) in reaction buffer (pH 7.4). Serial concentrations of CCT245737 (0.001-50 nM) were added, and the mixture was incubated at 30°C for 60 minutes. Phosphorylated peptide was detected using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay kit, and IC50/Ki values were calculated by nonlinear regression [1]
- Surface Plasmon Resonance (SPR) assay: Recombinant CHK1 was immobilized on a sensor chip. CCT245737 (0.1-20 nM) was injected over the chip at 25°C, and binding affinity (Ki) was determined by analyzing sensorgrams of resonance signal changes [1]
- Kinase selectivity panel assay: CCT245737 (1 μM) was tested against a panel of 200+ kinases including CHK2, CDK2/cyclin A, ATR, ATM, and PKA. Kinase activity was measured using kinase-specific substrates and detection systems, and selectivity was determined as the ratio of IC50 for off-target kinases to IC50 for CHK1 [1]

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].

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Anti-proliferation assay: A549, H460, Calu-6 (NSCLC), and WEHI-231, BCL1 (lymphoma) cells were cultured in RPMI 1640 or DMEM medium supplemented with fetal bovine serum. Cells were treated with CCT245737 (0.01-50 μM) for 72 hours, and cell viability was assessed by MTT assay; GI50 values were derived from dose-response curves [2]
- Western blot assay: A549 and H460 cells were treated with CCT245737 (0.1-1 μM) for 24 hours, or combined with cisplatin (10 μM) for 48 hours. Total protein was extracted, and blots were probed with antibodies against p-CHK1 (Ser345), p-CDC25C (Ser216), γ-H2AX, cleaved caspase-3, and GAPDH (loading control) [1][2]
- Cell cycle and apoptosis analysis: H460 cells treated with CCT245737 (0.5 μM) for 24 hours were stained with propidium iodide (cell cycle) or Annexin V-FITC/PI (apoptosis) and analyzed by flow cytometry [1]
- Colony formation assay: A549 and WEHI-231 cells were seeded in 6-well plates at low density, treated with CCT245737 (0.1-1 μM) for 14 days, fixed with methanol, stained with crystal violet, and visible colonies were counted [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].

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NSCLC xenograft model: 6-8 weeks old BALB/c-nu nude mice were subcutaneously injected with A549 or H460 cells (5×10⁶ cells/mouse) to establish xenograft tumors. When tumors reached 100-150 mm³, mice were randomly divided into control (0.5% carboxymethylcellulose sodium) and CCT245737 groups (25, 50 mg/kg). The drug was suspended in 0.5% carboxymethylcellulose sodium and administered via oral gavage once daily for 21 days. Tumor volume was measured every 3 days; mice were monitored for survival, and tumor tissues were collected for immunohistochemical and Western blot analysis [2]
- Eμ-MYC lymphoma model: Transgenic Eμ-MYC mice (6-8 weeks old) with spontaneous B-cell lymphoma were treated with CCT245737 (50 mg/kg, po, qd×14). Tumor growth was monitored by palpation; mice were euthanized at endpoint, and lymphoma tissues were collected for apoptotic index detection [2]
- Pharmacokinetic study in Sprague-Dawley rats: Rats received CCT245737 (10 mg/kg iv; 30 mg/kg po) dissolved in 10% DMSO/90% polyethylene glycol 400. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-dosing; plasma drug concentrations were measured by LC-MS/MS [1]
- Combination therapy protocol: H460 xenograft mice were treated with CCT245737 (25 mg/kg, po, qd×21) plus cisplatin (5 mg/kg, ip, every 7 days for 3 doses). Control groups received vehicle, single-agent CCT245737, or cisplatin alone. Tumor growth and CR rate were evaluated [2]

The oral bioavailability of CCT245737 in rats was 68% (30 mg/kg dose)[1] - The terminal elimination half-life (t1/2) of the drug in rat plasma was 6.2 hours (intravenous injection) and 8.5 hours (oral administration); the peak plasma concentration (Cmax) was 450 ng/mL (intravenous injection, 10 mg/kg) and 180 ng/mL (oral administration, 30 mg/kg)[1] - CCT245737 was widely distributed in tissues, and the highest concentrations were found in the liver (820 ng/g), kidney (650 ng/g), and tumor tissue (320 ng/g) 2 hours after oral administration in mice[1] - Human liver microsomal metabolism studies showed that the drug was mainly metabolized by CYP3A4 and CYP2D6. Mediated oxidative metabolism; approximately 70% of the dose is excreted in feces within 72 hours, and approximately 20% is excreted in urine (as metabolites) [1]
- CCT245737 has a plasma protein binding rate of 96% in human plasma and 94% in rat plasma [1]

CCT245737 (≤5 μM) showed low cytotoxicity to normal human fibroblasts (CCD-18Co) and BEAS-2B cells, with cell viability >85% after 72 hours [2]
- Acute toxicity in mice: oral LD50 >300 mg/kg; no treatment-related deaths were observed at doses ≤200 mg/kg [1]
- In a subchronic toxicity study in rats (28 days), administration of CCT245737 (10, 30, 100 mg/kg/day, orally) resulted in mild thrombocytopenia (15% reduction at 100 mg/kg) and transient elevation of serum AST (12% above normal), but no significant hepatotoxicity or nephrotoxicity was observed [1]
- In nude mice, CCT245737 (50 mg/kg/day, orally, for 21 days) did not cause significant weight loss or organ pathological damage [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.

SRA737, a Chk1 inhibitor, is an orally bioavailable checkpoint kinase 1 (Chk1) inhibitor with potential antitumor and chemosensitizing activities. After oral administration, SRA737 selectively binds to Chk1, thereby inhibiting Chk1 activity and blocking DNA damage repair. This may lead to DNA damage accumulation, cell cycle arrest inhibition, and apoptosis induction. SRA737 may enhance the cytotoxicity of DNA-damaging agents and reverse tumor cell resistance to chemotherapeutic drugs. Chk1 is an ATP-dependent serine/threonine kinase overexpressed in various cancer cells, mediating cell cycle checkpoint control and playing a crucial role in DNA repair. It exerts a key influence on chemosensitivity by repairing DNA damage.

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CCT245737 is an orally active, highly selective CHK1 inhibitor and a candidate drug for clinical development, targeting the ATP-binding pocket of CHK1[1][2]
- Its antitumor mechanism includes inhibiting CHK1-mediated G2/M cell cycle checkpoints, enhancing DNA damage-induced apoptosis, and making cancer cells more sensitive to chemotherapeutic drugs (cisplatin, gemcitabine)[2]
- The drug has shown strong preclinical activity against RAS-mutant non-small cell lung cancer (NSCLC) and Eμ-MYC-driven B-cell lymphoma, supporting its clinical development for these indications[2]
- CCT245737 has good physicochemical properties, including good water solubility (65 μg/mL) and stability in simulated gastric and intestinal fluids.[1]
- The drug has entered an early clinical trial (NCT02555642) for the treatment of advanced solid tumors and hematologic malignancies[2]

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.)