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.

Physicochemical Properties

Molecular Formula	C20H22N4
Molecular Weight	318.42
Exact Mass	318.184
Elemental Analysis	C, 75.44; H, 6.96; N, 17.60
CAS #	662163-81-7
Related CAS #	662163-81-7;Importazole HCl;
PubChem CID	2949965
Appearance	White to off-white solid powder
Density	1.2±0.1 g/cm3
Boiling Point	516.8±42.0 °C at 760 mmHg
Flash Point	266.3±27.9 °C
Vapour Pressure	0.0±1.3 mmHg at 25°C
Index of Refraction	1.682
LogP	3.3
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	4
Heavy Atom Count	24
Complexity	389
Defined Atom Stereocenter Count	0
SMILES	CC(NC1=C2C=CC=CC2=NC(N3CCCC3)=N1)C4=CC=CC=C4
InChi Key	HKGJEZIGDHFJFL-UHFFFAOYSA-N
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)
Chemical Name	N-(1-phenylethyl)-2-pyrrolidin-1-ylquinazolin-4-amine
Synonyms	Importazole
HS Tariff Code	2934.99.9001
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)

Biological Activity

Targets	importin-β
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].
ln Vivo
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.
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.
Animal Protocol
References	[1]. ACS Chem Biol . 2011 Jul 15;6(7):700-8. [2]. Zhonghua Xue Ye Xue Za Zhi . 2013 Apr;34(4):323-6.
Additional Infomation	N-(1-phenylethyl)-2-(1-pyrrolidinyl)-4-quinazolinamine is a member of quinazolines.

Solubility Data

Solubility (In Vitro)

DMSO: ~63 mg/mL (~197.9 mM) Water: <1 mg/mL Ethanol: ~31 mg/mL (~97.4 mM)

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.