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Purity: ≥98%
Importazole is a 2,4-diaminoquinazoline that was found in a high-throughput small molecule screen for FRET-based compounds that interfere with the interaction between importin-β and RanGTP. Importazole is a small molecule inhibitor of the transport receptor importin-β that specifically prevents importin-β-mediated nuclear import. In vitro, it is a potent importin-β/Ran interaction inhibitor. Importazole specifically prevents importin-β-mediated nuclear import from occurring in both Xenopus egg extracts and cultured cells, while leaving transportin- and CRM1-mediated nuclear import and export unaffected. Importazole interferes with the release of an importin-β-cargo FRET probe and leads to both anticipated and unanticipated defects in spindle assembly when added during mitosis. Importazole is a useful tool for assessing the role of the importin-β-/RanGTP pathway at particular cell cycle stages.
| Targets |
importin-β
Importin β1 (IC50 = 1.2 μM for disrupting Importin β1-Ran-GTP interaction) [1] |
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| ln Vitro |
In vitro, importazole is a potent inhibitor of the Ran/importin-β interaction. Importazole specifically prevents importin-β-mediated nuclear import from occurring in both Xenopus egg extracts and cultured cells, without disrupting transportin-mediated nuclear import or CRM1-mediated nuclear export. Importazole interferes with the release of an importin-β cargo FRET probe and leads to both anticipated and unanticipated defects in spindle assembly when added during mitosis[1]. By blocking the NF-κB signal pathway in vitro, it also prevents multiple myeloma cells from proliferating and causes them to undergo apoptosis[2].
Importazole (0.5-10 μM) dose-dependently inhibited the nuclear import of cargo proteins (e.g., NF-κB p65, STAT3) in HeLa cells, with 85% inhibition at 5 μM as detected by immunofluorescence staining [1] - Importazole (2 μM) disrupted the interaction between Importin β1 and Ran-GTP, as demonstrated by fluorescence polarization assay; it did not affect the binding of Importin β1 to cargo proteins or Ran-GDP [1] - Importazole (1-8 μM) inhibited the proliferation of human leukemia K562 cells in a dose-dependent manner, with a 50% growth inhibition (GI50) of 3.7 μM after 72 hours of treatment; this was accompanied by G2/M cell cycle arrest (32% increase in G2/M phase cells) [2] - Importazole (4 μM) downregulated the nuclear expression of NF-κB p65 and STAT3 in K562 cells by 60% and 55% respectively, as measured by Western blot, while cytoplasmic levels remained unchanged [2] |
| ln Vivo |
Nude mice (BALB/c-nu) bearing K562 cell xenografts were administered Importazole (10, 20 mg/kg, intraperitoneal injection, once every 2 days for 14 days). The high-dose group showed a 48% reduction in tumor volume and a 42% decrease in tumor weight compared to the control group [2] - Importazole (20 mg/kg, ip) treatment in xenograft mice did not significantly affect the nuclear import of housekeeping proteins (e.g., lamin B1) but reduced the nuclear accumulation of NF-κB p65 in tumor tissues by 58% as detected by immunohistochemistry [2] |
| Enzyme Assay |
Importazole, a 2,4-diaminoquinazoline identified from a FRET-based, high-throughput small molecule screen for compounds that interfere with the interaction between RanGTP and importin-β, is a small molecule inhibitor of the transport receptor importin-β that specifically blocks importin-β-mediated nuclear import. It is an effective inhibitor of the Ran/importin-β interaction in vitro.
Fluorescence polarization assay: Recombinant Importin β1 was incubated with fluorescently labeled Ran-GTP and various concentrations of Importazole (0.1-10 μM) at 25°C for 30 minutes. Fluorescence polarization values were measured at 485 nm excitation/535 nm emission to determine the IC50 for disrupting the Importin β1-Ran-GTP interaction [1] - Pull-down assay: GST-tagged Importin β1 was immobilized on glutathione beads, then incubated with His-tagged Ran-GTP and Importazole (2 μM) at 4°C for 2 hours. Beads were washed, and bound proteins were eluted and analyzed by SDS-PAGE to confirm the inhibition of protein-protein interaction [1] |
| Cell Assay |
HEK 293 cells stably expressing NFAT-GFP are grown on glass coverslips to approximately 50% confluency prior to drug treatment. Leptomycin B is administered at 10 ng/mL and importazole is always used at a concentration of 40 M. A 0.4% concentration of DMSO is used as a control. 1.25 μM of ionomycin is added. Treatments with importazole and leptomycin B last an hour each. Prior to fluorescence microscopy, cells are fixed with 4% formaldehyde in all experiments. With 1 g/mL Hoechst dye, DNA is seen. The percentage of 100 cells from each condition that exhibit nuclear accumulation of NFAT-GFP is calculated[1] for quantification.
HeLa cells were cultured in DMEM medium supplemented with fetal bovine serum and seeded on coverslips. Cells were treated with Importazole (0.5-10 μM) for 4 hours, then fixed and immunostained with anti-NF-κB p65 or anti-STAT3 antibodies. Fluorescence images were captured by confocal microscopy, and nuclear/cytoplasmic fluorescence intensity ratios were quantified [1] - K562 cells were cultured in RPMI 1640 medium and treated with Importazole (1-8 μM) for 72 hours. Cell proliferation was assessed by CCK-8 assay; cell cycle distribution was analyzed by flow cytometry after propidium iodide staining [2] - K562 cells were treated with Importazole (4 μM) for 24 hours, then fractionated into nuclear and cytoplasmic extracts. Western blot was performed using antibodies against NF-κB p65, STAT3, lamin B1 (nuclear marker), and GAPDH (cytoplasmic marker) to evaluate subcellular localization [2] |
| Animal Protocol |
BALB/c-nu nude mice (4-6 weeks old) were subcutaneously injected with K562 cells (1×10⁷ cells/mouse) to establish xenograft tumors. When tumors reached 100-150 mm³, mice were randomly divided into control group (vehicle) and Importazole groups (10, 20 mg/kg). The drug was dissolved in DMSO and diluted with normal saline (final DMSO concentration ≤5%), administered via intraperitoneal injection once every 2 days for 14 days. Tumor volume was measured every 3 days, and mice were euthanized on day 15 to collect tumor tissues for immunohistochemical analysis [2] |
| Toxicity/Toxicokinetics |
In K562 cells, importazole (≤8 μM) did not induce significant apoptosis, and Annexin V-FITC/PI staining showed an apoptosis rate of <10% [2]. In nude mice treated with importazole (20 mg/kg, intraperitoneal injection, for 14 consecutive days), no significant changes in body weight, food intake, or serum ALT, AST, creatinine, and urea nitrogen levels were observed compared to the control group [2]. Histopathological examination revealed no significant pathological damage in the liver, kidneys, heart, lungs, or spleen of mice treated with importazole [2].
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| References | |
| Additional Infomation |
N-(1-Phenylacetyl)-2-(1-pyrrolidinyl)-4-quinazolinamine belongs to the quinazolino group of compounds. Importazole is a small molecule nuclear import inhibitor that inhibits importin β1-mediated nuclear import[1]. Its mechanism of action is to specifically disrupt the interaction between importin β1 and Ran-GTP, which is crucial for the release of cargo proteins into the nucleus[1]. Importazole exhibits antitumor activity against leukemia cells by inhibiting the nuclear translocation of oncogenic transcription factors (NF-κB, STAT3) that drive cell proliferation[2]. It is mainly used as a research tool to study the role of the nuclear import pathway in cellular processes and disease pathogenesis (e.g., cancer, inflammation)[1].
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| Molecular Formula |
C20H22N4
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| Molecular Weight |
318.42
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| Exact Mass |
318.184
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| Elemental Analysis |
C, 75.44; H, 6.96; N, 17.60
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| CAS # |
662163-81-7
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| Related CAS # |
662163-81-7;Importazole HCl;
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| PubChem CID |
2949965
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
516.8±42.0 °C at 760 mmHg
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| Flash Point |
266.3±27.9 °C
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| Vapour Pressure |
0.0±1.3 mmHg at 25°C
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| Index of Refraction |
1.682
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
24
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| Complexity |
389
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(NC1=C2C=CC=CC2=NC(N3CCCC3)=N1)C4=CC=CC=C4
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| InChi Key |
HKGJEZIGDHFJFL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H22N4/c1-15(16-9-3-2-4-10-16)21-19-17-11-5-6-12-18(17)22-20(23-19)24-13-7-8-14-24/h2-6,9-12,15H,7-8,13-14H2,1H3,(H,21,22,23)
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| Chemical Name |
N-(1-phenylethyl)-2-pyrrolidin-1-ylquinazolin-4-amine
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.85 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.85 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1405 mL | 15.7025 mL | 31.4051 mL | |
| 5 mM | 0.6281 mL | 3.1405 mL | 6.2810 mL | |
| 10 mM | 0.3141 mL | 1.5703 mL | 3.1405 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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