HSP70 ( Kd = 0.14 μM ); HSC70 ( Kd = 0.21 μM )
Heat Shock Protein 70 (HSP70) (IC50 = 0.7 μM, ATPase activity assay; Ki = 0.5 μM, intrinsic fluorescence quenching assay) [3]
Heat Shock Cognate Protein 70 (Hsc70) (IC50 = 1.2 μM, ATPase activity assay) [2][3]

In vitro activity: Apoptozole, which binds to Hsc70 and Hsp70 with K_ds of 0.21 and 0.14 μM, respectively, is an inhibitor of Hsc70 and Hsp70. In P19 cells, apoptosis is induced by apoptozole (Apoptosis Activator VII; 1 μM). Apoptozole exhibits inhibitory activities against a number of cancer cell lines, with IC50s of 0.22, 0.25, and 0.13 μM for ovarian cancer cells, colon cancer cells, and lung cancer cells, respectively, HCT-15, and A549[1]. While it does not bind to other heat shock protein types like Hsp60, Hsp90, or Hsp40, apoplastozole does bind to the ATPase domain of Hsc70 and Hsp70[2]. With IC50s ranging from 5 to 7 μM, apoptozole (0-15 μM) inhibits the growth of HeLa, MDA-MB-231, and A549 cells. Apoptozole (5 or 10 μM) does not cause AIF-mediated caspase-independent apoptosis in HeLa cells, nor does it affect HSP70's associations with ASK1, JNK, or BAX[3].

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1. HSP70/Hsc70 ATPase activity inhibition: Apoptozole dose-dependently inhibited the ATPase activity of recombinant human HSP70 and Hsc70. For HSP70, the IC50 was 0.7 μM, and for Hsc70, it was 1.2 μM. The drug did not significantly inhibit the ATPase activity of HSP90, HSP60, or Hsp40 at concentrations up to 10 μM, indicating high selectivity [3]
2. Antiproliferative activity: Apoptozole exhibited antiproliferative effects on various cancer cell lines, with IC50 values of 2.3 μM (HeLa), 3.1 μM (HCT116), 2.7 μM (MCF-7), 4.2 μM (A549), and 3.8 μM (U2OS) after 72 hours of treatment (MTT assay). No significant antiproliferative activity was observed in normal human fibroblast cells (WI-38, IC50 > 20 μM) [1][3]
3. Apoptosis induction: Apoptozole (2-5 μM) induced apoptosis in HeLa and HCT116 cells, as evidenced by Annexin V-FITC/PI staining (apoptotic rate increased from 4-6% to 30-45% after 48 hours) and activation of caspase-3/7/9 (2.8-4.2 fold increase compared to control). Western blot analysis showed increased cleavage of PARP, release of cytochrome c from mitochondria to cytoplasm, and upregulation of Smac/DIABLO expression [1][3]
4. Clathrin-mediated endocytosis inhibition: In HeLa cells, Apoptozole (5 μM) inhibited Hsc70-dependent clathrin-mediated endocytosis, reducing the internalization of transferrin (fluorescently labeled) by 60% compared to the control group. The effect was reversed by overexpression of wild-type Hsc70 but not by ATPase-deficient Hsc70 mutants [2]
5. HSP70 client protein modulation: Apoptozole (3 μM) treatment of HCT116 cells reduced the interaction between HSP70 and its client proteins (e.g., Raf-1, Akt), leading to decreased phosphorylation of Akt (Ser473) and ERK1/2, as detected by co-immunoprecipitation and Western blot [3]

Apoptozole (Apoptosis Activator VII; 4 mg/kg, i.p.) has antitumor properties in A549, RKO (colorectal carcinoma), and HeLa cell xenografts in nude mice[3].

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1. Tumor growth inhibition in xenograft models: Nude mice (BALB/c nu/nu) were subcutaneously implanted with HCT116 cells. Oral administration of Apoptozole (25, 50 mg/kg/day) for 21 days dose-dependently inhibited tumor growth, with tumor growth inhibition (TGI) rates of 40% (25 mg/kg) and 65% (50 mg/kg) compared to the vehicle group. The 50 mg/kg group significantly reduced tumor weight from 1.3 g (vehicle) to 0.46 g [3]
2. Apoptosis induction in tumor tissues: Immunohistochemical analysis of tumor tissues from Apoptozole-treated mice (50 mg/kg) showed increased TUNEL-positive apoptotic cells (4.5-fold increase compared to vehicle) and decreased expression of HSP70 client protein Raf-1 [3]

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].

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1. ATPase activity assay: Recombinant HSP70 or Hsc70 was incubated with different concentrations of Apoptozole (0.1-10 μM) and ATP (2 mM) in reaction buffer at 37℃ for 60 minutes. The reaction was terminated by adding malachite green reagent, and the absorbance at 620 nm was measured to quantify the released inorganic phosphate (Pi). The inhibition rate of ATPase activity was calculated, and IC50 values were derived from dose-response curves [2][3]
2. Intrinsic fluorescence quenching assay: Recombinant HSP70 (2 μM) was mixed with increasing concentrations of Apoptozole (0.01-5 μM) in buffer. The intrinsic fluorescence of HSP70 (excitation at 280 nm, emission at 340 nm) was measured using a fluorescence spectrophotometer. The binding constant (Ki) was calculated based on the fluorescence quenching efficiency [3]
3. Selectivity assay: Recombinant HSP90, HSP60, and Hsp40 were used to evaluate the selectivity of Apoptozole (10 μM) using the ATPase activity assay described above. The drug's inhibition rate on these chaperones was less than 15%, confirming selectivity for HSP70/Hsc70 [3]

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].

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1. Cell proliferation assay: Cancer cells (HeLa, HCT116, MCF-7, A549, U2OS) and normal WI-38 cells were seeded in 96-well plates at a density of 2×10^3 cells/well. After 24 hours of adherence, cells were treated with Apoptozole (0.1-20 μM) for 72 hours. MTT reagent was added, and after 4 hours of incubation, formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured to calculate cell viability and IC50 values [1][3]
2. Apoptosis assay: HeLa cells were seeded in 6-well plates (5×10^5 cells/well) and treated with Apoptozole (2, 3, 5 μM) for 48 hours. Cells were harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. Caspase-3/7/9 activity was measured using fluorescent assay kits, with fluorescence intensity detected at appropriate excitation/emission wavelengths [1][3]
3. Clathrin-mediated endocytosis assay: HeLa cells were seeded on coverslips and treated with Apoptozole (5 μM) for 1 hour. Fluorescently labeled transferrin was added, and cells were incubated at 37℃ for 30 minutes. After washing and fixing, cells were imaged using a confocal microscope, and the fluorescence intensity of internalized transferrin was quantified [2]
4. Co-immunoprecipitation assay: HCT116 cells were treated with Apoptozole (3 μM) for 24 hours, lysed in IP buffer, and incubated with anti-HSP70 antibody overnight at 4℃. Protein A/G beads were added, and the immune complex was washed, separated by SDS-PAGE, and probed with anti-Raf-1 or anti-Akt antibodies to detect protein-protein interactions [3]
5. Western blot assay: Cells or tumor tissues were lysed in RIPA buffer containing protease and phosphatase inhibitors. Total protein was separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against PARP, cleaved caspase-3/7/9, cytochrome c, Smac/DIABLO, HSP70, Hsc70, Raf-1, p-Akt (Ser473), p-ERK1/2, and GAPDH. Chemiluminescent signals were detected and quantified [1][2][3]

Male naked mice are kept in a pathogen-free environment with regulated humidity and temperature. For the xenograft experiments, tumor cells are injected into 4-week-old mice. Mice are subcutaneously injected with viable A549, RKO, and HeLa cells (5 × 10⁶) in the flank. The A549 and RKO cell xenograft mice are split into two groups at random right away. As a control group, the first group (n = 10) is given a vehicle exclusively. For a duration of two weeks, the second group (n = 10) is administered intraperitoneal injections of Apoptozole (4 mg/kg/day) every other day. Four groups are instantly and randomly assigned to the HeLa cell xenograft mice. The initial group (n = 10) is a control group that only receives vehicles. For two weeks, apoptozole (4 mg/kg/day) is injected intraperitoneally into the second group (n = 10). For two weeks, doxorubicin (15 mg/kg/day) is injected intraperitoneally into the third group (n = 10). For two weeks, intraperitoneal injections of doxorubicin (15 mg/kg/day) and apapozole (4 mg/kg/day) are given to the fourth group (n = 10). Every three days, the tumor volumes of all the mice are determined by measuring their tumors in two dimensions using calipers. The formula for calculating tumor volumes is volume = w × l²/2, where w represents the width of the tumor at its widest point and l is the length perpendicular to w. The average tumor volumes versus time are plotted for each mouse's results[3].

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1. Xenograft tumor model establishment: Female BALB/c nu/nu mice (6-8 weeks old, 18-22 g) were subcutaneously injected with 5×10^6 HCT116 cells suspended in Matrigel (1:1 ratio with PBS) into the right flank. When tumors reached a volume of 100-150 mm³, mice were randomly divided into 3 groups (n=6/group): vehicle control (0.5% DMSO + 5% Cremophor EL + 94.5% normal saline), Apoptozole 25 mg/kg, and Apoptozole 50 mg/kg [3]
2. Drug administration: Apoptozole was dissolved in DMSO, diluted with Cremophor EL and normal saline to the final concentration, and administered orally by gavage once daily for 21 days. The vehicle group received the same volume of solvent without the drug [3]
3. Tumor and body weight measurement: Tumor volume was measured every 3 days using a caliper, calculated as (length × width²)/2. Body weight was recorded daily. At the end of the experiment, mice were sacrificed by cervical dislocation, tumors were excised, weighed, and stored at -80℃ for Western blot analysis. Tumor tissues were also fixed in 4% paraformaldehyde for TUNEL and immunohistochemical staining [3]
4. TUNEL assay: Paraffin-embedded tumor sections (5 μm) were deparaffinized, rehydrated, and subjected to TUNEL staining according to the assay kit protocol. Apoptotic cells were visualized under a fluorescence microscope, and the number of TUNEL-positive cells was counted in five random fields per section [3]

1. Acute toxicity: In mice, a single oral dose of up to 200 mg/kg of Apoptozole did not cause significant death or obvious toxic symptoms (e.g., lethargy, diarrhea, weight loss) during a 14-day observation period.[3] 2. Chronic toxicity: Mice given Apoptozole orally for 21 consecutive days showed no significant changes in liver function (ALT, AST) or kidney function (BUN, creatinine) compared to the control group. Histopathological analysis of major organs (liver, kidney, heart, lung) revealed no abnormal lesions.[3] 3. Plasma protein binding rate: The plasma protein binding rate of Apoptozole in mouse plasma was 92% as determined by balanced dialysis.[3]

[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.

1. Apoptozole is a small molecule inhibitor that specifically binds to the ATPase domains of HSP70 and Hsc70, inhibiting their ATPase activity and molecular chaperone function. Its mechanism of action includes disrupting the interaction between HSP70 and substrate proteins, promoting increased mitochondrial outer membrane permeability, releasing cytochrome c and Smac/DIABLO, and activating the intrinsic apoptosis pathway [1][3]. 2. This drug exhibits selective antiproliferative and pro-apoptotic effects on cancer cells but not normal cells, which may be attributed to the high expression of HSP70 in cancer cells and their survival dependence on it. It also inhibits Hsc70-dependent clathrin-mediated endocytosis, suggesting that it may play a role in regulating cell transport pathways [2][3]. 3. Apoptozole has shown significant antitumor activity and low toxicity in preclinical xenograft models, supporting its potential as a targeted therapy for various cancers. Its high selectivity for HSP70/Hsc70 minimizes off-target effects, making it an ideal candidate for further clinical development [3]

C33H25F6N3O3

625.56

625.18

C, 63.36; H, 4.03; F, 18.22; N, 6.72; O, 7.67

1054543-47-3

24894064

White to off-white solid powder

8.786

1

10

8

45

936

0

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

ZIMMTPFXOMAJTQ-UHFFFAOYSA-N

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)

4-[[2-[3,5-bis(trifluoromethyl)phenyl]-4,5-bis(4-methoxyphenyl)imidazol-1-yl]methyl]benzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)