Apoptozole (formerly known as Apoptosis Activator VII) is a potent and selective inhibitor of heat shock protein 70(HSP70) and Hsc70. It can cause apoptosis and inhibits the ATPase domain of Hsp70 and Hsc70, with Kd values of 0.14 μM and 0.21 μM, respectively.causes caspase-dependent apoptosis by preventing HSP70 from interacting with APAF-1; this interaction is unaffected by HSP70's interactions with ASK1, JNK, BAX, and AIF. Nevertheless, in aqueous environments, apoptozole may clump together and interact non-specifically with HSP70 proteins, possibly producing inconsistent results and false positives.
Physicochemical Properties
| Molecular Formula | C33H25F6N3O3 | |
| Molecular Weight | 625.56 | |
| Exact Mass | 625.18 | |
| Elemental Analysis | C, 63.36; H, 4.03; F, 18.22; N, 6.72; O, 7.67 | |
| CAS # | 1054543-47-3 | |
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| PubChem CID | 24894064 | |
| Appearance | White to off-white solid powder | |
| LogP | 8.786 | |
| Hydrogen Bond Donor Count | 1 | |
| Hydrogen Bond Acceptor Count | 10 | |
| Rotatable Bond Count | 8 | |
| Heavy Atom Count | 45 | |
| Complexity | 936 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | FC(C1C([H])=C(C(F)(F)F)C([H])=C(C=1[H])C1=NC(C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])=C(C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])N1C([H])([H])C1C([H])=C([H])C(C(N([H])[H])=O)=C([H])C=1[H])(F)F |
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| InChi Key | ZIMMTPFXOMAJTQ-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C33H25F6N3O3/c1-44-26-11-7-20(8-12-26)28-29(21-9-13-27(45-2)14-10-21)42(18-19-3-5-22(6-4-19)30(40)43)31(41-28)23-15-24(32(34,35)36)17-25(16-23)33(37,38)39/h3-17H,18H2,1-2H3,(H2,40,43) | |
| Chemical Name | 4-[[2-[3,5-bis(trifluoromethyl)phenyl]-4,5-bis(4-methoxyphenyl)imidazol-1-yl]methyl]benzamide | |
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| 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 |
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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) |
Biological Activity
| Targets | HSP70 ( Kd = 0.14 μM ); HSC70 ( Kd = 0.21 μM ) | |
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| Enzyme Assay | Ammonium heptamolybdate tetrahydrate (5.7% w/v in 6 M HCl), polyvinyl alcohol (2.3% w/v), and malachite green (0.081% w/v) stock solutions are made and kept at 4°C. To make the malachite green reagent, three solutions are combined with water in a 2:1:1:1:2 ratio. A master mixture of the ATPase domain of Hsc70 is prepared in assay buffer (100 mM Tris-HCl, 20 mM KCl, and 6 mM MgCl2, pH 7.4) at a final concentration of 1 mM in order to measure the ATPase activity of Hsc70. In a 96-well plate, aliquots (10 mL) of this mixture are added to each well. Each compound, including apoptozole, is added to this solution in assay buffer, and the plate is then allowed to sit at room temperature for 30 minutes. One milliliter of 4 mM ATP is added to the mixture to initiate the reaction. 200 mM ATP and 1 mM protein in 20 mL of assay buffer are the final concentrations. Each well receives 80 mL of the malachite green reagent after three hours of 37°C incubation. After thoroughly combining the samples, they are incubated for 15 minutes at 37°C. Afterwards, 10 milliliters of 34% sodium citrate are added to halt the nonenzymatic hydrolysis of ATP. The SpectraMax 340 PC 384 is used to measure the absorbance at 620 nm[1]. | |
| Cell Assay | In triplicate, 5 × 105 cells are plated in 96-well plates with 0.1 mL of culture media containing 10% FBS. Cells are treated with different concentrations of Apoptozole (0-15 μM) in culture media containing 3% FBS (final volume: 0.2 mL per well) for 24, 48, and 72 hours prior to being treated with MTT. This is done after the 24-hour mark. A UV microplate reader is used to measure absorbance at 570 nm[3]. | |
| Animal Protocol |
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| References |
[1]. An apoptosis-inducing small molecule that binds to heat shock protein 70. Angew Chem Int Ed Engl. 2008;47(39):7466-9. [2]. Probing the effect of an inhibitor of an ATPase domain of Hsc70 on clathrin-mediated endocytosis. Mol Biosyst. 2015 Oct;11(10):2763-9. [3]. A small molecule inhibitor of ATPase activity of HSP70 induces apoptosis and has antitumor activities. Chem Biol. 2015 Mar 19;22(3):391-403. |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.00 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 (4.00 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 20% SBE-β-CD physiological saline solution and mix evenly. 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (4.00 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 | 1.5986 mL | 7.9928 mL | 15.9857 mL | |
| 5 mM | 0.3197 mL | 1.5986 mL | 3.1971 mL | |
| 10 mM | 0.1599 mL | 0.7993 mL | 1.5986 mL |