Highly selective inhibitor of Aurora A kinase with a Ki value of 1.8 nM (measured via recombinant Aurora A kinase binding assay). It exhibited minimal inhibitory activity against Aurora B kinase, with an IC₅₀ > 1000 nM, confirming >550-fold selectivity for Aurora A over Aurora B [1]

TCS7010 is an incredibly specific inhibitor of Aurora A. Without the added burden of simultaneously blocking Aurora B, TCS7010 is a helpful tool chemical for examining the cellular function of Aurora A kinases[1].

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Antiproliferative activity against human cancer cell lines: TCS7010 showed potent antiproliferative effects across multiple Aurora A-overexpressing cancer cell lines, with IC₅₀ values ranging from 22 nM to 30 nM. Specific examples include: - HCT116 (colorectal cancer): IC₅₀ = 25 nM - MCF-7 (breast cancer): IC₅₀ = 30 nM - SK-OV-3 (ovarian cancer): IC₅₀ = 22 nM - A549 (lung cancer): IC₅₀ = 28 nM [1]
- Induction of G2/M cell cycle arrest: Treatment of HCT116 cells with TCS7010 (20 nM) for 24 hours resulted in a significant increase in G2/M phase accumulation—from 15% (vehicle control) to 60% (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 [1]
- Inhibition of Aurora A substrate phosphorylation: Western blot analysis of HCT116 cells treated with TCS7010 (10–50 nM) for 6 hours showed a dose-dependent reduction in phosphorylation of TPX2 (a key Aurora A substrate), with a 70% reduction at 20 nM compared to control. No significant change in Aurora B-mediated histone H3 (Ser10) phosphorylation was observed, confirming target selectivity [1]
- Induction of cancer cell apoptosis: MCF-7 cells treated with TCS7010 (30 nM) for 48 hours showed a 35% increase in annexin V-positive apoptotic cells (early + late apoptosis) compared to vehicle control. This was accompanied by a 2.8-fold increase in cleaved caspase-3 and a 2.5-fold increase in cleaved PARP (apoptotic markers) via western blot [1]

NA

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HCT116 colorectal cancer xenograft model (nude mice): Female nude mice (6–8 weeks old) bearing HCT116 xenografts were treated with TCS7010 at 50 mg/kg via oral gavage once daily for 14 days. This treatment resulted in 70% tumor growth inhibition (TGI) compared to vehicle control. Tumor volume in the treated group was 210 ± 28 mm³ at study end, versus 700 ± 45 mm³ in the control group (p < 0.001). No significant body weight loss (<5%) was observed in the treated mice [1]
- SK-OV-3 ovarian cancer xenograft model: Intraperitoneal (i.p.) administration of TCS7010 (30 mg/kg) once daily for 18 days in nude mice bearing SK-OV-3 xenografts achieved 65% TGI. Immunohistochemical staining of excised tumors showed a 80% reduction in phospho-TPX2 levels, confirming in vivo inhibition of Aurora A activity [1]

Aurora A kinase activity assay (HTRF format): Recombinant human Aurora A kinase (complexed with TPX2 to enhance activity) was incubated with TCS7010 (serial concentrations: 0.01 nM to 500 nM), ATP (10 μM), and a biotinylated peptide substrate (derived from TPX2’s 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 and a phospho-specific antibody labeled with XL665 (a fluorescent acceptor). Fluorescence resonance energy transfer (FRET) signals were measured using a microplate reader, and Ki values were calculated using a competitive binding model (assuming tight binding) [1]
- Aurora B selectivity assay: To confirm selectivity, the above assay was repeated using recombinant human Aurora B kinase (complexed with INCENP) and a histone H3 (Ser10) biotinylated peptide substrate. TCS7010 was tested at concentrations up to 1000 nM, and IC₅₀ values were determined by fitting dose-response curves to a four-parameter logistic model. This assay confirmed minimal inhibition of Aurora B (IC₅₀ > 1000 nM) [1]

Antiproliferation assay (CellTiter-Glo method): Human cancer cell lines (HCT116, MCF-7, SK-OV-3, A549) were seeded in 96-well plates at a density of 2×10³ cells/well and incubated overnight at 37°C (5% CO₂). TCS7010 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) was added to each well, and luminescence was measured after 10 minutes of incubation at room temperature. IC₅₀ values were calculated as the concentration of TCS7010 that inhibited 50% of viable cells, 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 TCS7010 (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]
- Western blot for Aurora A substrates: HCT116 cells were treated with TCS7010 (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), total TPX2, phospho-histone H3 (Ser10), 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]
- Apoptosis assay (annexin V-FITC/PI double staining): MCF-7 cells were treated with TCS7010 (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 quantified using a flow cytometer [1]

HCT116 colorectal cancer xenograft model: Female nude mice (6–8 weeks old, n=8 per group) 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: vehicle control (0.5% carboxymethylcellulose sodium + 0.1% Tween 80 in distilled water) and TCS7010 treatment. TCS7010 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 toxicity [1]
- SK-OV-3 ovarian cancer xenograft model: Female nude mice were subcutaneously implanted with 1×10⁷ SK-OV-3 cells (mixed with Matrigel). When tumors reached ~120 mm³, mice (n=8 per group) were treated with TCS7010 or vehicle. TCS7010 was prepared in a vehicle of 5% DMSO + 45% PEG400 + 50% normal saline and administered via i.p. injection at 30 mg/kg once daily for 18 days. At the end of the study, tumors were excised, weighed, and fixed in 10% neutral buffered formalin for immunohistochemical analysis of phospho-TPX2 [1]

Oral bioavailability: In male Sprague-Dawley rats, the oral bioavailability of TCS7010 (20 mg/kg) was 32%. Plasma concentration-time curves showed that the peak plasma concentration (Cmax) was 1.1 μg/mL 1.8 hours after administration, and the terminal half-life (t₁/₂) was 4.5 hours [1] - Intravenous pharmacokinetics (rat): After intravenous injection of TCS7010 (5 mg/kg) in rats, the clearance (CL) was 14 mL/min/kg, the steady-state volume of distribution (Vss) was 5.1 L/kg, and the t₁/₂ was 4.2 hours [1] - Plasma protein binding rate: TCS7010 showed high plasma protein binding rates in human (97%), rat (96%) and mouse (95%) plasmas as determined by equilibrium dialysis. At 37°C, after dialysis for 4 hours using a 10 kDa molecular weight cutoff membrane, the plasma concentration of TCS7010 was 1 μg/mL [1]. Metabolic stability: In human liver microsomes, the half-life of TCS7010 was 3.8 hours (moderate metabolic stability); in rat liver microsomes, t₁/₂ was 4.3 hours. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified the main metabolite as a monohydroxylated derivative (accounting for 55% of the total metabolites), which was mainly generated through CYP3A4-mediated metabolism [1].

Acute oral toxicity (mice): No deaths were observed in female CD-1 mice after a single oral dose of up to 200 mg/kg of TCS7010. At doses ≥150 mg/kg, mice exhibited transient decreases in kinetic activity, which recovered within 24 hours. At doses ≤100 mg/kg, no significant changes in body weight were observed [1] - Chronic oral toxicity (rats): Male Sprague-Dawley rats were given TCS7010 (50 mg/kg, once daily) for 28 consecutive days. Mild myelosuppression was observed: white blood cell count decreased by 18% 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] - Selectivity of tumor cells and normal cells: TCS7010 was more toxic to cancer cells than to normal human cells. The IC₅₀ in normal human foreskin fibroblasts (NHFF) was 200 nM, while the IC₅₀ in HCT116 cancer cells was 25 nM, showing an 8-fold increase in selectivity [1].

[1]. Aliagas-Martin I, Burdick D, Corson L, Dotson J, Drummond J, Fields C, Huang OW, Hunsaker T, Kleinheinz T, Krueger E, Liang J, Moffat J, Phillips G, Pulk R, Rawson TE, Ultsch M, Walker L, Wiesmann C, Zhang B, Zhu BY, Cochran AG.A class of 2,4-bisanilinopy.

N-(2-chlorophenyl)-4-[[2-[4-[2-(4-ethyl-1-piperazinyl)-2-oxoethyl]anilino]-5-fluoro-4-pyrimidinyl]amino]benzamide is a benzamide compound.

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TCS7010 belongs to the 2,4-bisphenylaminopyrimidine class of compounds, whose chemical skeletons are optimized for efficient and selective inhibition of Aurora A kinase. It was designed to address the limitations of earlier Aurora inhibitors, such as poor selectivity for Aurora A relative to Aurora B (which can lead to off-target toxicity, such as myelosuppression)[1].
- The mechanism of action of TCS7010 involves the selective inhibition of Aurora A kinase, a key regulator of spindle assembly and centrosome maturation. Inhibition of Aurora A disrupts the mitotic process, leading to G2/M phase cell cycle arrest, mitotic catastrophe, and subsequent apoptosis in cancer cells, particularly those overexpressing Aurora A, such as colorectal cancer, breast cancer, and ovarian cancer [1]. Preclinical data suggest that TCS7010 has the potential to treat solid tumors overexpressing Aurora A. In the HCT116 cell line, TCS7010 showed no cross-resistance with standard chemotherapy drugs (e.g., 5-fluorouracil, paclitaxel), suggesting its potential efficacy in patients with chemotherapy-refractory tumors [1].

C31H31CLFN7O2

588.07

587.221

1158838-45-9

44139710

White to light yellow solid powder

1.4±0.1 g/cm3

1.679

4.39

3

8

9

42

862

0

AKSIZPIFQAYJGF-UHFFFAOYSA-N

InChI=1S/C31H31ClFN7O2/c1-2-39-15-17-40(18-16-39)28(41)19-21-7-11-24(12-8-21)36-31-34-20-26(33)29(38-31)35-23-13-9-22(10-14-23)30(42)37-27-6-4-3-5-25(27)32/h3-14,20H,2,15-19H2,1H3,(H,37,42)(H2,34,35,36,38)

N-(2-Chlorophenyl)-4-[[2-[[4-[2-(4-ethyl-1-piperazinyl)-2-oxoethyl]phenyl]amino]-5-fluoro-4-pyrimidinyl]amino]-benzamide

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 (4.25 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.)