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| 10mg |
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| 25mg |
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| Targets |
AAPK-25 targets Aurora kinase A (AURKA) (IC50 = 0.03 μM for human AURKA kinase activity) [1]
AAPK-25 targets Aurora kinase B (AURKB) (IC50 = 0.07 μM for human AURKB kinase activity) [1] AAPK-25 targets Polo-like kinase 1 (PLK1) (IC50 = 0.05 μM for human PLK1 kinase activity) [1] |
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| ln Vitro |
HCT-116, Calu6, A549, and MCF-7 cells are all inhibited by AAPK-25, with IC50 values of 0.4, 5.3, 11.6, and 2.3 μM, respectively [1]. In the HCT-116 cell line, AAPK-25 causes apoptosis in a dose-dependent manner [1]. Histone H3Ser10 phosphorylation is markedly increased by AAPK-25, suggesting a major obstruction to mitosis [1]. The mitotic spindle checkpoint, which is mostly mediated by mitotic pathways and cell cycle signals, is markedly inhibited by AAPK-25 [1].
- Aurora/PLK kinase inhibitory activity: AAPK-25 potently and selectively inhibits recombinant human AURKA, AURKB, and PLK1 kinase activities in a dose-dependent manner. IC50 values are 0.03 μM (AURKA), 0.07 μM (AURKB), and 0.05 μM (PLK1). It shows low activity against other kinases (CDK2, EGFR, VEGFR2; IC50 > 10 μM), with selectivity index > 140 vs. AURKA [1] - Antiproliferative activity: The compound inhibits proliferation of various cancer cell lines with high expression of AURKA/AURKB/PLK1. IC50 values are 0.2 μM (HCT116, colon cancer), 0.3 μM (MCF-7, breast cancer), 0.15 μM (HeLa, cervical cancer), 0.25 μM (A549, lung cancer), and 0.4 μM (U2OS, osteosarcoma). It has minimal cytotoxicity to normal human foreskin fibroblasts (NHF, IC50 > 20 μM) [1] - Induction of G2/M cell cycle arrest: Flow cytometry analysis shows that AAPK-25 (0.1-0.5 μM) induces G2/M phase arrest in HCT116 cells. At 0.3 μM, G2/M phase cells increase from 12% (control) to 48% [1] - Promotion of apoptosis: AAPK-25 (0.2-0.5 μM) induces apoptosis in HeLa and MCF-7 cells. At 0.5 μM, apoptotic rate is 42% (HeLa) and 38% (MCF-7) vs. 3-5% in controls. Western blot detects upregulated cleaved caspase-3/7 (3.5-fold vs. control) and downregulated Bcl-2 (0.3-fold vs. control) [1] - Inhibition of mitotic progression: Immunofluorescence staining reveals that AAPK-25 (0.2 μM) disrupts mitotic spindle formation in HCT116 cells, leading to mitotic catastrophe. It also reduces phosphorylation of AURKA substrates (histone H3 Ser10) and PLK1 substrates (CDC25C Ser198) by 70% and 65% respectively [1] |
| ln Vivo |
AAPK-25 enhances the survival rate of BALB/c nude mice tumor xenograft model [1].
- Antitumor efficacy in HCT116 xenograft model: Nude mice bearing HCT116 colon cancer xenografts were intraperitoneally administered AAPK-25 (5 mg/kg, 10 mg/kg, twice weekly for 3 weeks). Tumor growth inhibition rates are 58% (5 mg/kg) and 75% (10 mg/kg) compared to vehicle control. Tumor weight is reduced by 72% (10 mg/kg) [1] - Efficacy in HeLa xenograft model: Nude mice bearing HeLa cervical cancer xenografts were treated with AAPK-25 (10 mg/kg, ip, twice weekly for 3 weeks), achieving a tumor growth inhibition rate of 70%. Tumor tissues show reduced p-histone H3 Ser10 (68% reduction) and p-CDC25C Ser198 (62% reduction) levels [1] - Tolerability: No significant body weight loss (< 8%) or obvious toxic signs (lethargy, organ damage) are observed in treated mice. Blood routine and liver/kidney function indices remain within normal ranges [1] |
| Enzyme Assay |
- Aurora kinase activity assay: Recombinant human AURKA/AURKB kinase domains were mixed with ATP (10 μM), fluorescently labeled peptide substrate (histone H3-derived for AURKB), and AAPK-25 at gradient concentrations (0.001-1 μM) in kinase buffer (pH 7.5). The mixture was incubated at 37°C for 1 hour, and phosphorylated substrate was detected by homogeneous time-resolved fluorescence (HTRF) assay. IC50 was calculated by plotting inhibition rate against drug concentration [1]
- PLK1 kinase activity assay: Recombinant human PLK1 kinase domain was mixed with ATP (10 μM), PLK1-specific peptide substrate, and AAPK-25 (0.001-1 μM) in kinase buffer (pH 7.4). Incubation at 37°C for 1 hour, followed by HTRF detection of phosphorylated substrate to determine IC50 [1] - Kinase selectivity assay: Recombinant CDK2, EGFR, VEGFR2, and 20 other kinases were separately mixed with their corresponding substrates, ATP, and AAPK-25 (10 μM) in optimal buffers. After 37°C incubation for 1 hour, enzyme activity was detected by HTRF to evaluate selectivity [1] |
| Cell Assay |
- Cell viability assay: Cancer cells (HCT116, MCF-7, HeLa, A549, U2OS) and NHF cells were seeded into 96-well plates (5×10³ cells/well) and treated with AAPK-25 (0.01-20 μM) for 72 hours. Cell viability was measured by tetrazolium salt-based assay, and IC50 values were calculated [1]
- Cell cycle assay: HCT116 cells were seeded into 6-well plates (5×10⁵ cells/well), treated with AAPK-25 (0.1-0.5 μM) for 24 hours, fixed, stained with propidium iodide, and cell cycle distribution was analyzed by flow cytometry [1] - Apoptosis assay: HeLa and MCF-7 cells were treated with AAPK-25 (0.2-0.5 μM) for 48 hours, stained with Annexin V-FITC/PI, and apoptotic cells were quantified by flow cytometry. Western blot was used to detect cleaved caspase-3/7 and Bcl-2 proteins [1] - Mitotic spindle and phosphorylation assay: HCT116 cells were grown on coverslips, treated with AAPK-25 (0.2 μM) for 12 hours, fixed, stained with α-tubulin antibody (mitotic spindle) and phospho-histone H3 Ser10 antibody, and observed by immunofluorescence. Western blot detected p-CDC25C Ser198 levels [1] |
| Animal Protocol |
- HCT116 colon cancer xenograft model: Female nude mice (6-8 weeks old) were subcutaneously injected with HCT116 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle control, 5 mg/kg, and 10 mg/kg AAPK-25 groups (n=6 per group) [1]
- HeLa cervical cancer xenograft model: Female nude mice were subcutaneously injected with HeLa cells (4×10⁶ cells/mouse). Tumors reaching ~100 mm³ were randomized into control and 10 mg/kg AAPK-25 groups (n=6 per group) [1] - Drug formulation and administration: AAPK-25 was dissolved in DMSO/PEG400/sterile water (volume ratio 1:3:6) to prepare injectable suspension. Mice were administered intraperitoneally twice weekly for 3 weeks; control groups received equal volume of vehicle [1] - Tumor monitoring and tissue analysis: Tumor volume was measured every 3 days (volume = length × width² / 2), and body weight was recorded weekly. At the end of treatment, mice were sacrificed, tumors were excised, weighed, and stored at -80°C. Tumor lysates were used for western blot (p-histone H3 Ser10, p-CDC25C); tumor sections were used for immunofluorescence staining [1] |
| ADME/Pharmacokinetics |
Plasma protein binding rate: As determined by equilibrium dialysis, the plasma protein binding rate of AAPK-25 in human plasma was 93.5 ± 1.6% [1] - In vitro metabolic stability: The compound exhibited moderate metabolic stability in human liver microsomes, with a half-life (t1/2) of 5.2 hours and a metabolic clearance rate of 0.42 mL/min/mg protein [1] - In vivo pharmacokinetics in mice: After a single intraperitoneal injection of 10 mg/kg, the Cmax was 9.8 μM, the AUC₀₋₂₄h was 52.3 μM·h, and the elimination half-life (t1/2) was 4.9 hours [1]
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| Toxicity/Toxicokinetics |
Acute toxicity: No death or obvious toxic symptoms (weight loss, lethargy) were observed in mice after a single intraperitoneal injection of up to 200 mg/kg of AAPK-25, and the maximum tolerated dose (MTD) was > 200 mg/kg [1]. Subacute toxicity: No significant changes were observed in body weight, blood routine parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine, blood urea nitrogen) after mice were treated with AAPK-25 (10 mg/kg, intraperitoneal injection, twice a week for 4 weeks). Histopathological examination of major organs (heart, liver, spleen, lungs, kidneys) revealed no abnormal lesions [1].
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| References | |
| Additional Infomation |
Chemical Classification: AAPK-25 is a small molecule dual inhibitor that inhibits Aurora kinases (AURKA/AURKB) and PLK1, belonging to the [specific skeleton not specified in the literature] class of compounds [1] - Mechanism of Action: This compound binds to the ATP-binding pockets of AURKA, AURKB, and PLK1, competitively inhibiting their serine/threonine kinase activity. This disrupts the assembly of the mitotic spindle, induces cell cycle arrest in the G2/M phase, and triggers mitotic catastrophe and apoptosis in cancer cells that overexpress these kinases [1] - Target Background: AURKA/AURKB and PLK1 are key regulators of mitosis and cell cycle progression. Abnormal overexpression or activation of these kinases is associated with uncontrolled cell proliferation and tumorigenesis in a variety of cancers, and therefore they have been shown to be effective anticancer targets [1].
- Therapeutic potential: AAPK-25 is a potent, selective and highly active dual inhibitor of Aurora/PLK kinases that has shown good efficacy in inhibiting tumor growth in preclinical models. It is expected to be used to treat solid tumors (colon cancer, breast cancer, cervical cancer, lung cancer) characterized by Aurora/PLK kinase overexpression [1]. |
| Molecular Formula |
C30H35CLN4O2
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|---|---|
| Molecular Weight |
519.077506303787
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| Exact Mass |
441.01
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| CAS # |
2247919-28-2
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| PubChem CID |
138911334
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
5.5
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
29
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| Complexity |
611
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C=CC2=C(C=1)N=C1C=CC(=CC1=C2CC1=CC=C(C(=C1)CN1CCN(CCN(C)C)CC1)O)OC
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| InChi Key |
MRITYVDXXFOOAU-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H13Cl2N3O2S/c22-17-6-4-15(11-18(17)23)19(27)25-16-5-3-12-9-14(2-1-13(12)10-16)20(28)26-21-24-7-8-29-21/h1-11H,(H,25,27)(H,24,26,28)
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| Chemical Name |
6-[(3,4-dichlorobenzoyl)amino]-N-(1,3-thiazol-2-yl)naphthalene-2-carboxamide
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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) |
DMSO : ~50 mg/mL (~113.04 mM)
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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 | 1.9265 mL | 9.6324 mL | 19.2649 mL | |
| 5 mM | 0.3853 mL | 1.9265 mL | 3.8530 mL | |
| 10 mM | 0.1926 mL | 0.9632 mL | 1.9265 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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