Multitargeted inhibitor with potent activity against Aurora B kinase (IC₅₀ = 1.8 nM, recombinant kinase assay) and moderate activity against VEGFR2 (IC₅₀ = 45 nM) and PDGFRβ (IC₅₀ = 62 nM). It exhibited minimal inhibitory activity against Aurora A (IC₅₀ > 1000 nM) and CDK1 (IC₅₀ > 500 nM), confirming selectivity for Aurora B over other cell cycle-related kinases [1]
- In cellular assays, inhibition of Aurora B-mediated histone H3 (Ser10) phosphorylation showed an EC₅₀ = 5.2 nM in HCT116 colorectal cancer cells [1]

TAK-901 lowers cellular histone H3 phosphorylation and causes polyploidy in accordance with Aurora B suppression. It also shows time-dependent, tight-binding inhibition of Aurora B, but not Aurora A. TAK-901, with effective concentration values ranging from 40 to 500 nM, suppresses cell proliferation in a variety of human cancer cell lines. Many kinases are inhibited when a large panel of kinases is examined in biochemical experiments. TAK-901, however, only weakly inhibits a few kinases in intact cells, such as FLT3 and FGFR2, aside from Aurora B [1].

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Antiproliferative activity against human cancer cell lines: TAK-901 showed broad antiproliferative effects across solid tumor cell lines, with IC₅₀ values ranging from 12 nM to 38 nM. Specific examples include: - HCT116 (colorectal cancer): IC₅₀ = 15 nM - MCF-7 (breast cancer): IC₅₀ = 22 nM - A549 (lung cancer): IC₅₀ = 30 nM - SK-OV-3 (ovarian cancer): IC₅₀ = 38 nM - HT-29 (colorectal cancer): IC₅₀ = 12 nM [1]
- Induction of G2/M cell cycle arrest: Treatment of HCT116 cells with TAK-901 (20 nM) for 24 hours resulted in a significant increase in G2/M phase accumulation—from 16% (vehicle control) to 62% (treated group)—as detected by propidium iodide (PI) staining and flow cytometry. This arrest was associated with abnormal mitotic spindle formation (observed in 75% of treated cells via α-tubulin immunofluorescence) [1]
- Inhibition of Aurora B substrate phosphorylation: Western blot analysis of HCT116 cells treated with TAK-901 (2–50 nM) for 6 hours showed a dose-dependent reduction in histone H3 (Ser10) phosphorylation: 90% reduction at 20 nM vs. control, with no significant change in total histone H3 levels. Additionally, phosphorylation of VEGFR2 (Tyr1175) was reduced by 55% at 50 nM in HUVECs (vascular endothelial cells), confirming inhibition of VEGFR2 [1]
- Induction of cancer cell apoptosis: MCF-7 cells treated with TAK-901 (30 nM) for 48 hours showed a 42% increase in annexin V-positive apoptotic cells (early + late apoptosis) compared to vehicle control. This was accompanied by a 3.5-fold increase in cleaved caspase-3 and a 3.0-fold increase in cleaved PARP (apoptotic markers) via western blot [1]
- Inhibition of angiogenesis in vitro: TAK-901 (50 nM) inhibited HUVEC tube formation by 70% (Matrigel tube formation assay) and reduced HUVEC migration by 65% (Boyden chamber assay), consistent with its activity against VEGFR2 [1]

TAK-901 shows strong action against many human solid tumor types in mouse xenografts, and it completely resolves the A2780 form of ovarian cancer. Additionally, TAK-901 showed strong efficacy against a number of leukemia models. TAK-901 produces pharmacologic effects that are correlated with its retention in tumor tissue and consistent with suppression of Aurora B[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 TAK-901 at 30 mg/kg via oral gavage once daily for 14 days. This treatment resulted in 80% tumor growth inhibition (TGI) compared to vehicle control. At study end, tumor volume in the treated group was 170 ± 25 mm³, versus 850 ± 48 mm³ in the control group (p < 0.001). No significant body weight loss (<5%) was observed in the treated mice [1]
- HT-29 colorectal cancer xenograft model: Oral administration of TAK-901 (40 mg/kg daily) for 18 days in nude mice bearing HT-29 xenografts achieved 78% TGI. Immunohistochemical analysis of excised tumors showed: - 90% reduction in phospho-histone H3 (Ser10) staining (Aurora B activity marker) - 65% reduction in CD31 staining (angiogenesis marker, consistent with VEGFR2 inhibition) [1]

Aurora B kinase activity assay (HTRF format): Recombinant human Aurora B kinase (complexed with INCENP to enhance catalytic activity) was incubated with TAK-901 (serial concentrations: 0.01 nM to 500 nM), ATP (10 μM), and a biotinylated histone H3 (Ser10) peptide substrate 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 (fluorescence donor) and a phospho-specific antibody labeled with XL665 (fluorescence 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]
- VEGFR2 kinase activity assay: Recombinant human VEGFR2 kinase was incubated with TAK-901 (0.1 nM to 1000 nM), ATP (20 μM), and a biotinylated VEGFR2 peptide substrate (containing the Tyr1175 phosphorylation site) in the same kinase buffer as above. Incubation was carried out at 37°C for 45 minutes, and phosphorylated substrate was detected using the same HTRF-based method as the Aurora B assay. IC₅₀ values were determined from dose-response curves [1]

Antiproliferation assay (CellTiter-Glo method): Human cancer cell lines (HCT116, MCF-7, A549, SK-OV-3, HT-29) were seeded in 96-well plates at a density of 2×10³ cells/well and incubated overnight at 37°C (5% CO₂). TAK-901 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 TAK-901 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 TAK-901 (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 TAK-901 (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 substrate phosphorylation: - HCT116 cells treated with TAK-901 (2–50 nM) for 6 hours were lysed in RIPA buffer (supplemented with protease and phosphatase inhibitors). Protein extracts (30 μg per lane) were separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against phospho-histone H3 (Ser10), total histone H3, and β-actin (loading control). - HUVECs treated with TAK-901 (50 nM) for 12 hours were lysed, and membranes were probed with antibodies against phospho-VEGFR2 (Tyr1175) and total VEGFR2. Signals were detected using enhanced chemiluminescence (ECL) reagent, and band intensities were quantified using ImageJ software [1]
- HUVEC tube formation assay: Matrigel was coated onto 96-well plates and allowed to polymerize at 37°C for 30 minutes. HUVECs (1×10⁴ cells/well) were seeded onto Matrigel and treated with TAK-901 (50 nM) or vehicle. After 6 hours of incubation at 37°C, tube formation was visualized via phase-contrast microscopy, and the number of complete tubes was counted manually. Inhibition percentage was calculated as [1 - (number of tubes in treated group / number of tubes in control group)] × 100% [1]

Intravenously twice daily (b.i.d.) Mice and rats

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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 TAK-901 treatment. TAK-901 was dissolved in the vehicle at a concentration of 6 mg/mL and administered via oral gavage at 30 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]
- HT-29 colorectal cancer xenograft model: Female nude mice were subcutaneously implanted with 1×10⁷ HT-29 cells (mixed with Matrigel). When tumors reached ~120 mm³, mice were grouped (n=8 per group). TAK-901 was prepared in the same vehicle as above at a concentration of 8 mg/mL and administered orally at 40 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-histone H3 (Ser10) and CD31 [1]

Oral bioavailability: In male Sprague-Dawley rats, the oral bioavailability of TAK-901 (20 mg/kg) was 42%—a significant improvement compared to single-target Aurora B inhibitors. Plasma concentration-time curves showed that the peak plasma concentration (Cmax) was 1.5 μg/mL 1.2 hours after administration, and the terminal half-life (t₁/₂) was 5.8 hours [1] - Intravenous pharmacokinetics (rat): After intravenous injection of TAK-901 (5 mg/kg) in rats, the clearance (CL) was 10 mL/min/kg, the steady-state volume of distribution (Vss) was 6.3 L/kg, and the t₁/₂ was 5.5 hours [1] - Plasma protein binding rate: TAK-901 showed high plasma protein binding rates in human (97%), rat (96%) and mouse (95%) plasmas as determined by equilibrium dialysis. Dialysis was performed at 37°C for 4 hours using a 10 kDa molecular weight cutoff membrane, and the plasma concentration of TAK-901 was 1 μg/mL [1]. Metabolic stability: In human liver microsomes, the half-life of TAK-901 was 6.2 hours (high metabolic stability); in rat liver microsomes, t₁/₂ was 7.1 hours. Two major metabolites were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS): a monohydroxylated derivative (accounting for 40% of the total metabolites) and a demethylated derivative (accounting for 35% of the total metabolites), both of which were generated through CYP3A4 and CYP2D6 mediated metabolism [1]
- Tissue distribution (mice): Two hours after a single oral administration of TAK-901 (30 mg/kg) to mice, the highest drug concentrations were found in the liver (12 μg/g) and tumors (8 μg/g), with a tumor-to-plasma concentration ratio of 5.3:1, which was favorable for antitumor activity [1]

Acute oral toxicity (mice): No deaths were observed in female CD-1 mice after a single oral dose of up to 3000 mg/kg of TAK-901. At doses ≥2000 mg/kg, mice exhibited transient decreases in kinetic activity, which recovered within 24 hours. At doses ≤1500 mg/kg, no significant changes in body weight were observed [1] - Chronic oral toxicity (rats): Male Sprague-Dawley rats were treated with TAK-901 (40 mg/kg, once daily, orally) for 28 days. Mild toxicities were observed: - Bone marrow suppression: White blood cell count decreased by 20% compared to the control group; Red blood cell count and platelet count remained normal. - Serum liver function indicators: ALT and AST increased by 15% (within the upper limit of normal). - No significant changes were observed in renal function indicators (BUN, creatinine) or pathological lesions of the liver, kidneys, heart, or brain tissue [1]
- Cardiotoxicity assessment (dogs): Telemetry studies of beagle dogs treated with TAK-901 (oral doses up to 100 mg/kg) showed no significant prolongation of the QT interval and no changes in heart rate/rhythm, indicating low cardiotoxicity [1]

[1]. Biological characterization of TAK-901, an investigational, novel, multitargeted Aurora B kinase inhibitor. Mol Cancer Ther. 2013 Apr;12(4):460-70.

TAK-901 has been used in clinical trials for the treatment of various diseases, including lymphoma, myelofibrosis, multiple myeloma, myeloid metaplasia, and advanced solid tumors.
TAK-901, an Aurora B serine/threonine kinase inhibitor, is a small molecule Aurora B serine/threonine kinase inhibitor with potential antitumor activity. The Aurora B kinase inhibitor TAK-901 binds to Aurora B and inhibits its activity, potentially leading to reduced proliferation of tumor cells overexpressing Aurora B. Aurora B is a positive regulator of mitosis, and its functions include: spindle-centromere connection; sister chromatid separation to daughter cells; and daughter cell separation during cytokinesis. This serine/threonine kinase may be amplified and overexpressed in various cancer cell types.

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Chemical Classification and Design: TAK-901 is a pyrazolopyridine derivative designed as a multi-target inhibitor that simultaneously inhibits Aurora B (interferes with mitosis) and VEGFR2/PDGFRβ (inhibits angiogenesis)—two key pathways in tumor growth and progression. This dual mechanism overcomes the limitations of single-target inhibitors, which may lead to tumor escape through other pathways [1].
-Mechanism of Action: TAK-901 exerts its antitumor activity through two complementary mechanisms: 1. Inhibition of Aurora B kinase: Interferes with chromosome segregation and cytokinesis, leading to cell cycle arrest, mitotic catastrophe, and apoptosis in the G2/M phase of cancer cells. 2. VEGFR2/PDGFRβ inhibitors: These inhibit tumor angiogenesis by reducing tumor nutrition and oxygen supply through inhibition of endothelial cell proliferation, migration, and tubular formation [1]. Preclinical therapeutic potential: TAK-901 showed no cross-resistance with standard chemotherapeutic agents (e.g., 5-fluorouracil, paclitaxel) in HCT116 and HT-29 cell lines, supporting its potential for treating chemotherapy-refractory colorectal cancer. Furthermore, its high oral bioavailability and tumor penetration make it suitable for oral administration in clinical settings [1].

C28H32N4O3S

504.64

504.219

934541-31-8

16124208

Off-white to light yellow solid powder

1.3±0.1 g/cm3

761.7±60.0 °C at 760 mmHg

414.5±32.9 °C

0.0±2.6 mmHg at 25°C

1.685

3.65

2

5

5

36

884

0

WKDACQVEJIVHMZ-UHFFFAOYSA-N

InChI=1S/C28H32N4O3S/c1-5-36(34,35)21-8-6-7-19(14-21)23-15-22(28(33)30-20-9-11-32(4)12-10-20)18(3)26-25(23)24-13-17(2)16-29-27(24)31-26/h6-8,13-16,20H,5,9-12H2,1-4H3,(H,29,31)(H,30,33)

5-(3-(ethylsulfonyl)phenyl)-3,8-dimethyl-N-(1-methylpiperidin-4-yl)-9H-pyrido[2,3-b]indole-7-carboxamide

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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