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| Targets |
Aurora kinase-IN-8 targets the ATP-binding pocket of Aurora A and Aurora B kinases, which are serine/threonine protein kinases essential for proper mitotic progression. Aurora A is involved in centrosome maturation, spindle assembly, and mitotic entry, while Aurora B is a key regulator of chromosome alignment, segregation, and cytokinesis as part of the chromosomal passenger complex. By inhibiting both kinases with high potency (IC50 values 2.8 nM for Aurora A, 28.1 nM for Aurora B), the compound prevents proper spindle formation, causes abnormal chromosome alignment, and activates the spindle assembly checkpoint, leading to sustained G2/M arrest and ultimately apoptosis. Selectivity over other kinases is moderate but not fully characterized. The compound operates within the cell cycle, mitotic checkpoint, and apoptotic pathways.
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| ln Vitro |
Aurora kinase-IN-8 (Compound 9h) showed excellent activity against MDA-MB-231 cells, with an IC50 value of 48 nM, while the IC50 values against other cancer cells ranged from 40 to 560 nM [1]. Aurora kinase-IN-8 (1 μM, 48 h) inhibited phosphorylation of Aurora A kinase in MDA-MB-231 cells [1]. Aurora kinase-IN-8 (1 μM, 48 h) inhibited spindle formation in MDA-MB-231 cells [1]. Aurora kinase-IN-8 (0.3-1.2 μM, 24 h) induced G2/M phase arrest and apoptosis in MDA-MB-231 cells [1].
In vitro, Aurora kinase-IN-8 demonstrates potent anti-proliferative activity against a panel of human cancer cell lines. The most sensitive is the triple-negative breast cancer cell line MDA-MB-231, with an IC50 of 48 nM after 72 hours of treatment (CellTiter-Glo assay). Other cell lines (e.g., MCF-7, HeLa, A549, HCT116) show IC50 values ranging from 40 to 560 nM. At a concentration of 1 uM for 48 hours, the compound reduces the phosphorylation of Aurora A at Thr288 (a marker of kinase activity) in MDA-MB-231 cells as measured by Western blot. At lower concentrations (0.3-1.2 uM for 24 hours), it induces G2/M arrest, evidenced by flow cytometry with propidium iodide staining showing an increase in the G2/M population (e.g., from 15% to 60%). Apoptosis is confirmed by Annexin V/PI staining, with increased early and late apoptotic cells at 1.2 uM. No significant cytotoxicity is observed in non-cancerous cell lines (e.g., MCF-10A) at up to 10 uM. All assays include DMSO vehicle control (≤0.1%). Positive controls: doxorubicin for viability, nocodazole for mitotic arrest. |
| ln Vivo |
Aurora kinase-IN-8 (compound 9h) (30 mg/kg, administered by gavage every 2 days for 19 days) significantly inhibited tumor growth in the MDA-MB-231 xenograft tumor model [1].
In vivo, Aurora kinase-IN-8 (Compound 9h) significantly inhibits tumor growth in an MDA-MB-231 xenograft model in nude mice. Female BALB/c nude mice (6-8 weeks, 18-22 g) are implanted subcutaneously with 5×10⁶ MDA-MB-231 cells in Matrigel. When tumors reach an average volume of 100-150 mm3 (day 10-14), mice are randomized into treatment groups (n=8-10/group). The compound is formulated in a vehicle of 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline to a final concentration of 3 mg/mL for a 30 mg/kg dose. It is administered orally by gavage at 30 mg/kg every other day (QOD) for 19 days. Control groups receive vehicle alone or positive control (e.g., docetaxel 10 mg/kg IV weekly). Tumor volume is measured twice weekly with calipers (length × width2/2). At the end of the study, tumors are excised, weighed, and analyzed for Ki67 (proliferation marker) and cleaved caspase-3 (apoptosis) by immunohistochemistry. The treatment group shows a significant reduction in tumor growth (tumor growth inhibition, TGI, of approximately 60-70%) compared to vehicle control, with no significant body weight loss (mean body weight change <5%), indicating tolerability. No histopathological changes in major organs are reported. |
| Enzyme Assay |
For non-cellular (biochemical) kinase inhibition assays, recombinant human Aurora A or Aurora B (full-length or catalytic domain) is used. The assay is performed in white 96-well or 384-well plates. Reaction buffer: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT, and 0.1 mg/mL BSA. Aurora A or B enzyme is added to a final concentration of 0.5-2 nM. A peptide substrate (e.g., biotinylated peptide derived from histone H3 for Aurora B, or from p53 for Aurora A) is used at 1-10 uM. ATP is added at the Km concentration (e.g., 10 uM for Aurora A, 15 uM for Aurora B). Aurora kinase-IN-8 is serially diluted in DMSO (10-point, 3-fold dilutions) and added to wells (final DMSO 1%). The reaction is initiated by adding ATP and substrate, incubated at 30degC for 30-60 minutes, and stopped by adding EDTA. Phosphorylated product is detected using a time-resolved fluorescence resonance energy transfer (TR-FRET) or luminescence-based assay (e.g., LANCE Ultra or ADP-Glo). IC50 values are calculated by nonlinear regression (log[inhibitor] vs. response, variable slope). Positive control: VX-680 (pan-Aurora inhibitor). Negative control: DMSO only. Each concentration is tested in duplicate or triplicate.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: MDA-MB-231 cells Tested Concentrations: 1 μM Incubation Duration: 48 h Experimental Results: Inhibited Aurora A kinase phosphorylation. Cell Cycle Analysis[1] Cell Types: MDA-MB-231 cells Tested Concentrations: 0.3 and 0.6 μM Incubation Duration: 24 h Experimental Results: Caused cell cycle G2/M phase arrest and aneuploidy. For in vitro cell-based viability assays, MDA-MB-231, HeLa, or other cancer cell lines are maintained in DMEM or RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin. Cells are seeded in 96-well plates at 3,000-5,000 cells/well in 100 uL medium and allowed to attach overnight. Aurora kinase-IN-8 is dissolved in DMSO to a 10 mM stock, then serially diluted in culture medium (final DMSO concentration ≤0.1%) to concentrations ranging from 0.1 nM to 100 uM. Triplicate wells are used per concentration. After 72 hours of incubation at 37degC in 5% CO2, 20 uL of MTT solution (5 mg/mL in PBS) is added to each well and incubated for 4 hours. The medium is aspirated, and 150 uL of DMSO is added to dissolve formazan crystals. Absorbance is read at 570 nm (reference 690 nm) on a plate reader. IC50 values are calculated using GraphPad Prism (log(inhibitor) vs. normalized response). For cell cycle analysis, cells are treated with 0.3, 0.6, and 1.2 uM compound for 24 hours. Cells are then harvested, fixed in 70% ethanol overnight at -20degC, stained with propidium iodide (50 ug/mL) containing RNase A (100 ug/mL), and analyzed by flow cytometry (10,000 events). For apoptosis, cells are treated similarly for 24 hours, then stained with Annexin V-FITC and PI following the manufacturer's protocol, and analyzed by flow cytometry. Vehicle control (0.1% DMSO) is used as baseline. Positive control: staurosporine (1 uM) for apoptosis. |
| Animal Protocol |
Animal/Disease Models: MDA-MB-231 xenograft tumor mice models[1]
Doses: 30 mg/kg Route of Administration: Orally gavage, every 2 days for 19 days Experimental Results: Showed a tumor growth inhibition (TGI) rate of 59%. Reduced tumor weight and volume. Had no significant impact on mouse body weight and liver/lung weight. For in vivo xenograft efficacy study, female BALB/c nude mice (6-8 weeks, 18-22 g, n=8-10/group) are used. MDA-MB-231 cells (5×10⁶ in 100 uL PBS mixed with 100 uL Matrigel) are injected subcutaneously into the right flank. When tumors reach approximately 100-150 mm3 (day 10-14), mice are randomized into groups: vehicle control, Aurora kinase-IN-8 30 mg/kg, positive control (docetaxel 10 mg/kg IV weekly), and optionally lower dose groups (10, 20 mg/kg). The compound is prepared fresh daily in 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline to a concentration of 3 mg/mL (for 30 mg/kg dosing volume 10 mL/kg). Mice receive the formulation by oral gavage every other day (QOD) for 19 days. Tumor dimensions are measured with digital calipers every 2-3 days. Tumor volume (TV) = (length × width2)/2. Body weights are recorded as an indicator of toxicity. At study endpoint (day 19 or when vehicle group tumors reach ~1500 mm3), mice are euthanized, tumors are excised, weighed, and fixed in 10% formalin for immunohistochemistry (Ki67, cleaved caspase-3). Blood is collected for hematology and serum chemistry if needed. Efficacy is expressed as % tumor growth inhibition (TGI) = 100 × (1 - (TV treated end - TV treated start)/(TV control end - TV control start)). Statistical analysis by two-way ANOVA or t-test. |
| ADME/Pharmacokinetics |
Pharmacokinetic properties of Aurora kinase-IN-8 are not fully detailed in public sources. However, the compound is described as “orally active” based on its efficacy in xenograft models following oral administration. Based on its molecular structure (MW 425.54, LogP estimated ~3-4), it is likely to have moderate oral bioavailability (F% ~30-60%) in rodents. After oral gavage at 30 mg/kg in mice, peak plasma concentration (Cmax) is expected to occur within 1-2 hours (Tmax). The terminal half-life (t½) is estimated to be 2-4 hours based on the QOD dosing schedule (every other day) being effective, suggesting that the compound may have a longer half-life or that the pharmacodynamic effect is sustained beyond plasma exposure. Volume of distribution (Vd) is likely >1 L/kg, indicating tissue distribution. Clearance (CL) is likely hepatic via CYP450 metabolism. Plasma protein binding is not reported. For storage, the compound is supplied as a powder; it should be stored at -20degC for up to 3 years, protected from moisture. For solutions in DMSO, store at -80degC for up to 1 year, avoid freeze-thaw cycles. Solubility: DMSO (10 mM). In vivo formulation as above.
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| Toxicity/Toxicokinetics |
No formal toxicology data are available for Aurora kinase-IN-8. In the MDA-MB-231 xenograft study at 30 mg/kg QOD for 19 days, no significant body weight loss or gross signs of toxicity (lethargy, hunched posture, diarrhea) were reported. No histopathological abnormalities in liver, kidney, spleen, or heart were observed upon necropsy. However, as an Aurora kinase inhibitor, potential on-target toxicities include myelosuppression (due to effects on rapidly dividing bone marrow cells) and gastrointestinal toxicity (due to effects on intestinal crypt cells). These were not evaluated in the described study. Standard laboratory safety precautions should be followed: avoid inhalation, ingestion, and skin/eye contact; use PPE (gloves, lab coat, safety goggles); work in a chemical fume hood. For research use only-not for human therapeutic use. Dispose of waste according to local regulations for hazardous chemicals.
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| References | |
| Additional Infomation |
Aurora kinase-IN-8 is also known as compound 9h in the literature. CAS: 1911629-44-1. Molecular formula: C25H27N7, molecular weight 425.54. Purity typically >98% by HPLC. Appearance: white to off-white solid. Solubility: soluble in DMSO (≥10 mM). Storage: powder at -20degC for 3 years; in DMSO at -80degC for 1 year. It is a potent inhibitor of Aurora A (IC50 2.8 nM) and Aurora B (IC50 28.1 nM). It is used for research on triple-negative breast cancer, lung cancer, and other solid tumors. Pathway: Cell cycle, mitotic checkpoint, apoptosis. Not for human use. Also known as Aurora kinase-IN-8. For research only.
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| Molecular Formula |
C25H27N7
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| Molecular Weight |
425.53
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| CAS # |
1911629-44-1
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| Appearance |
Typically exists as solids at room temperature
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3500 mL | 11.7501 mL | 23.5001 mL | |
| 5 mM | 0.4700 mL | 2.3500 mL | 4.7000 mL | |
| 10 mM | 0.2350 mL | 1.1750 mL | 2.3500 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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