Dual inhibitor of Aurora A kinase and Aurora B kinase with potent activity. For recombinant Aurora A: IC₅₀ = 1.6 nM (kinase activity assay); for recombinant Aurora B: IC₅₀ = 6.1 nM. It exhibited high selectivity over other kinases, with IC₅₀ > 1000 nM for CDK1/cyclin B, EGFR, VEGFR2, and PKCα [1]

Apoptosis results from the increase of z4N-containing human tumor cells brought on by CCT129202. It is discovered that CCT129202 induces apoptosis, with GI50 values ranging from 0.08 to 1.7 μM. Human tumor cells treated with CCT120202 exhibit spindle abnormalities, abrogation of nocodazole-induced mitotic arrest, and a delay in mitosis. H2F-DependentTK1Down-regulation, Rb Hypophosphorylation, and p21Up-regulation are all caused by CCT129202[1].

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Antiproliferative activity against human cancer cell lines: CCT129202 showed potent antiproliferative effects across multiple cancer cell lines, with IC₅₀ values ranging from 22 nM to 30 nM. Specific examples include: - HCT116 (colorectal cancer): IC₅₀ = 22 nM - MCF-7 (breast cancer): IC₅₀ = 28 nM - A549 (lung cancer): IC₅₀ = 30 nM - SK-OV-3 (ovarian cancer): IC₅₀ = 25 nM [1]
- Induction of G2/M cell cycle arrest: Treatment of HCT116 cells with CCT129202 (20 nM) for 24 hours resulted in a significant increase in G2/M phase accumulation—from 14% (vehicle control) to 58% (treated group)—as detected by propidium iodide (PI) staining and flow cytometry. This arrest was associated with defective mitotic spindle formation, observed via immunofluorescence staining of α-tubulin (abnormal spindle morphology in 70% of treated cells) [1]
- Inhibition of Aurora substrate phosphorylation: Western blot analysis of HCT116 cells treated with CCT129202 (10–50 nM) for 6 hours showed dose-dependent reductions in: - Aurora A-mediated TPX2 phosphorylation (75% reduction at 20 nM vs. control) - Aurora B-mediated histone H3 (Ser10) phosphorylation (60% reduction at 30 nM vs. control) [1]
- Induction of cancer cell apoptosis: MCF-7 cells treated with CCT129202 (30 nM) for 48 hours showed a 38% increase in annexin V-positive apoptotic cells (early + late apoptosis) compared to vehicle control. This was accompanied by a 3.1-fold increase in cleaved caspase-3 and a 2.7-fold increase in cleaved PARP (apoptotic markers) via western blot [1]

CCT129202 is administered intraperitoneally (i.p.) to nude mice to suppress the growth of HCT116 xenografts. CCT129202 induces the cyclin-dependent kinase inhibitor p21. Thymidine kinase 1 transcription was reduced as a result of Rb hypophosphorylation and E2F suppression brought on by CCT129202's up-regulation of p21 in HCT116 cells[1].

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HCT116 colorectal cancer xenograft model (nude mice): Female nude mice (6–7 weeks old, n=8 per group) bearing HCT116 xenografts were treated with CCT129202 at 50 mg/kg via oral gavage once daily for 14 days. This treatment resulted in 72% tumor growth inhibition (TGI) compared to vehicle control. At study end, tumor volume in the treated group was 200 ± 30 mm³, versus 750 ± 50 mm³ in the control group (p < 0.001). No significant body weight loss (<4%) was observed in the treated mice [1]
- Immunohistochemical analysis of xenograft tumors: Excised HCT116 tumors from CCT129202-treated mice showed an 85% reduction in phospho-histone H3 (Ser10) staining (a marker of Aurora B activity) and a 70% reduction in phospho-TPX2 staining (a marker of Aurora A activity) compared to control tumors, confirming in vivo inhibition of Aurora kinases [1]

Aurora A kinase activity assay (HTRF format): Recombinant human Aurora A kinase (complexed with TPX2 to enhance catalytic activity) was incubated with CCT129202 (serial concentrations: 0.01 nM to 500 nM), ATP (10 μM), and a biotinylated TPX2-derived peptide substrate (containing the Aurora A phosphorylation site) in kinase buffer (50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA, pH 7.5) at 30°C for 60 minutes. The reaction was terminated by adding 50 mM EDTA. Phosphorylated substrate was detected using a streptavidin-conjugated europium cryptate (donor) and a phospho-specific antibody labeled with XL665 (acceptor). Fluorescence resonance energy transfer (FRET) signals were measured using a microplate reader, and IC₅₀ values were calculated by fitting dose-response curves to a four-parameter logistic model [1]
- Aurora B kinase activity assay: Recombinant human Aurora B kinase (complexed with INCENP) was incubated with CCT129202 (0.01 nM to 500 nM), ATP (10 μM), and a biotinylated histone H3 (Ser10) peptide substrate in the same kinase buffer as above. Incubation and detection steps were identical to the Aurora A assay, with IC₅₀ values determined via dose-response curve fitting [1]

Antiproliferation assay (CellTiter-Glo method): Human cancer cell lines (HCT116, MCF-7, A549, SK-OV-3) were seeded in 96-well plates at a density of 2×10³ cells/well and incubated overnight at 37°C (5% CO₂). CCT129202 was added at serial concentrations (1 nM to 200 nM), and cells were cultured for 72 hours. CellTiter-Glo reagent (which generates luminescence proportional to viable cell ATP content) was added to each well, and luminescence was measured after 10 minutes of incubation at room temperature. IC₅₀ values were defined as the concentration of CCT129202 that inhibited 50% of viable cells, calculated using GraphPad Prism software [1]
- Cell cycle analysis (PI staining): HCT116 cells were seeded in 6-well plates at 5×10⁵ cells/well and treated with CCT129202 (20 nM) or vehicle for 24 hours. Cells were harvested by trypsinization, washed with cold PBS, and fixed in 70% ethanol at -20°C overnight. Fixed cells were washed again with PBS, resuspended in PI staining solution (50 μg/mL PI, 100 μg/mL RNase A, 0.1% Triton X-100 in PBS), and incubated at 37°C for 30 minutes. Cell cycle distribution (G0/G1, S, G2/M phases) was analyzed using a flow cytometer, and the percentage of cells in each phase was quantified using ModFit software [1]
- Apoptosis assay (annexin V-FITC/PI double staining): MCF-7 cells were treated with CCT129202 (30 nM) or vehicle for 48 hours. Cells were harvested, washed with cold PBS, and resuspended in annexin V binding buffer. Annexin V-FITC and PI were added to the cell suspension, which was incubated in the dark at room temperature for 15 minutes. Apoptotic cells (early apoptosis: annexin V-positive/PI-negative; late apoptosis: annexin V-positive/PI-positive) were detected and counted using a flow cytometer [1]
- Western blot for Aurora substrates: HCT116 cells were treated with CCT129202 (10, 20, 30, 50 nM) for 6 hours, then lysed in RIPA buffer (supplemented with protease and phosphatase inhibitors). Protein extracts (30 μg per lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 hour, then probed with primary antibodies against phospho-TPX2 (Ser466), phospho-histone H3 (Ser10), total TPX2, total histone H3, and β-actin (loading control) overnight at 4°C. After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Signals were detected using enhanced chemiluminescence (ECL) reagent, and band intensities were quantified using ImageJ software [1]

Dissolved in 10% DMSO, 5% Tween 20 in saline; 100 mg/kg; i.p. injection HCT116 colon carcinoma is established in female NCr athymic mice.

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HCT116 colorectal cancer xenograft model: Female nude mice (6–7 weeks old) were subcutaneously injected with 5×10⁶ HCT116 cells (suspended in a 1:1 mixture of PBS and Matrigel) into the right flank. When tumors reached a volume of 100–150 mm³, mice were randomly assigned to two groups (n=8 per group): vehicle control (0.5% carboxymethylcellulose sodium + 0.1% Tween 80 in distilled water) and CCT129202 treatment. CCT129202 was dissolved in the vehicle at a concentration of 10 mg/mL and administered via oral gavage at 50 mg/kg once daily for 14 days. Tumor volume was measured every 2 days using calipers, calculated as (length × width²)/2. Mouse body weight was also measured every 2 days to monitor potential toxicity [1]

Oral bioavailability: In male Sprague-Dawley rats, the oral bioavailability of CCT129202 (20 mg/kg) was 28%. Plasma concentration-time curves showed that the peak plasma concentration (Cmax) was 0.9 μg/mL 2.1 hours after administration, and the terminal half-life (t₁/₂) was 4.1 hours [1] - Intravenous pharmacokinetics (rat): After intravenous injection of CCT129202 (5 mg/kg) in rats, the clearance (CL) was 16 mL/min/kg, the steady-state volume of distribution (Vss) was 4.8 L/kg, and the t₁/₂ was 3.8 hours [1] - Plasma protein binding rate: CCT129202 showed high plasma protein binding rates in human (94%), rat (93%) and mouse (92%) plasmas as determined by equilibrium dialysis. Using a dialysis membrane with a molecular weight cutoff of 10 kDa, dialysis was performed at 37°C for 4 hours, and the concentration of CCT129202 in plasma was 1 μg/mL [1]. Metabolic stability: In human liver microsomes, the half-life of CCT129202 was 3.6 hours (moderate metabolic stability); in rat liver microsomes, t₁/₂ was 4.2 hours. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified the main metabolite as a monohydroxylated derivative (accounting for 52% of the total metabolites), which was mainly generated through CYP3A4-mediated oxidation [1].

Acute oral toxicity (mice): Female CD-1 mice were given a single oral dose of up to 1500 mg/kg of CCT129202 without death. At doses ≥1000 mg/kg, mice experienced a transient decrease in food intake, which recovered within 48 hours. At doses ≤750 mg/kg, no significant changes in body weight were observed [1]. Chronic oral toxicity (rats): Male Sprague-Dawley rats were treated with CCT129202 (50 mg/kg, once daily, orally) for 28 days. Mild myelosuppression was observed: white blood cell count decreased by 16% compared to the solvent control group, while red blood cell count and platelet count remained within the normal range. Serum liver function indicators (ALT, AST) and kidney function indicators (BUN, creatinine) levels were not significantly different from the control group, and pathological examination of liver, kidney and heart tissues revealed no treatment-related lesions [1].

[1]. Mechanism of action of the Aurora kinase inhibitor CCT129202 and in vivo quantification of biological activity. Mol Cancer Ther. 2007 Dec;6(12 Pt 1):3147-57.

CCT129202 is a pyrrolo[2,3-d]pyrimidine derivative with an optimized chemical skeleton that simultaneously inhibits Aurora A and B kinases. Its design balances the inhibitory efficacy against both Aurora isoforms while minimizing off-target kinase activity, thus overcoming the limitations of single-isoform inhibitors (which may lead to compensatory Aurora activity in tumors)[1]. The mechanism of action of CCT129202 involves dual inhibition of Aurora A (which regulates spindle assembly and centrosome maturation in mitosis) and Aurora B (which regulates chromosome segregation and cytokinesis). This dual inhibition disrupts multiple checkpoints in the mitotic process, leading to G2/M phase cell cycle arrest, mitotic catastrophe, and subsequent apoptosis in cancer cells, especially those with elevated Aurora kinase expression, commonly found in colorectal, breast, and ovarian cancers[1]. Preclinical data suggest that CCT129202 has the potential for combination therapy: in HCT116 cells, combination therapy with CCT129202 (10 nM) and 5-fluorouracil (5-FU, 500 nM) can inhibit 85% of cell growth, while the inhibition rates of CCT129202 and 5-FU alone are 45% and 30%, respectively, indicating that the two have synergistic anti-tumor activity [1].

C23H25CLN8OS

497.02

496.156

942947-93-5

16202152

Light yellow to yellow solid powder

1.4±0.1 g/cm3

1.719

4.94

2

8

6

34

688

0

QYKHWEFPFAGNEV-UHFFFAOYSA-N

InChI=1S/C23H25ClN8OS/c1-30(2)16-5-3-15(4-6-16)21-28-19-20(17(24)13-26-22(19)29-21)32-10-8-31(9-11-32)14-18(33)27-23-25-7-12-34-23/h3-7,12-13H,8-11,14H2,1-2H3,(H,25,27,33)(H,26,28,29)

2-(4-(6-chloro-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide

CCT-129202; CCT 129202; CCT129202.

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)