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| Targets |
mTOR
- mTOR inhibitor-1 targets the mammalian target of rapamycin (mTOR) kinase (IC50 for mTOR kinase activity inhibition: 8.7 μM; IC50 for inhibiting A549 lung carcinoma cell proliferation: 12.3 μM) [1] |
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| ln Vitro |
- Anti-proliferative activity on A549 cells: mTOR inhibitor-1 (concentrations: 5–20 μM) inhibited A549 cell proliferation in a dose-dependent manner. MTT assay showed the IC50 was 12.3 μM after 72 hours of treatment. Clone formation assay revealed that 10 μM and 15 μM mTOR inhibitor-1 reduced the number of cell clones by ~50% and ~75%, respectively, compared to the vehicle control [1]
- Autophagy induction in A549 cells: mTOR inhibitor-1 (10–15 μM) induced autophagy in A549 cells. Western blot analysis showed increased LC3-II/LC3-I protein ratio (15 μM treatment for 24 hours increased the ratio by ~3.2-fold) and decreased p62 protein expression (downregulated by ~60% at 15 μM). Immunofluorescence staining confirmed the accumulation of autophagosomes (marked by LC3 puncta) in cells treated with 15 μM mTOR inhibitor-1 [1] - mTOR pathway inhibition: mTOR inhibitor-1 (5–15 μM) dose-dependently suppressed the activation of the mTOR signaling pathway in A549 cells. Western blot detected decreased phosphorylation of mTOR (p-mTOR), p70 ribosomal S6 kinase (p-p70S6K), and eukaryotic translation initiation factor 4E-binding protein 1 (p-4E-BP1). At 15 μM, p-mTOR, p-p70S6K, and p-4E-BP1 protein levels were downregulated by ~70%, ~65%, and ~62%, respectively, compared to the control [1] |
| ln Vivo |
- Antitumor activity in nude mouse xenograft model: BALB/c nude mice (4–6 weeks old) were subcutaneously inoculated with A549 cells (1×10⁷ cells/mouse) to establish xenograft tumors. When tumors reached ~100 mm³, mice were randomly divided into 3 groups (n=6/group): vehicle control (DMSO + saline), low-dose mTOR inhibitor-1 (10 mg/kg), and high-dose mTOR inhibitor-1 (20 mg/kg). The drug was administered via intraperitoneal injection once every 2 days for 21 days. At the end of treatment, high-dose mTOR inhibitor-1 reduced tumor volume by ~68% and tumor weight by ~65% compared to the control. Western blot of tumor tissues showed decreased p-mTOR, p-p70S6K, p-4E-BP1, and p62, as well as increased LC3-II/LC3-I ratio, confirming mTOR pathway inhibition and autophagy induction in vivo [1]
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| Enzyme Assay |
- mTOR kinase activity assay: Recombinant human mTOR kinase was mixed with reaction buffer containing ATP (10 μM), fluorescently labeled substrate peptide (specific for mTOR), and mTOR inhibitor-1 (concentrations: 1–30 μM). The mixture was incubated at 37°C for 60 minutes. The reaction was terminated by adding stop buffer, and the fluorescence intensity of phosphorylated substrate was measured using a microplate reader. The inhibition rate of mTOR kinase activity was calculated by comparing with the vehicle control, and the IC50 value (8.7 μM) was determined via nonlinear regression analysis of the concentration-inhibition curve [1]
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| Cell Assay |
- A549 cell proliferation assay (MTT method): A549 cells were seeded in 96-well plates (5×10³ cells/well) and cultured overnight. mTOR inhibitor-1 (5, 10, 15, 20 μM) was added, and cells were incubated for 24, 48, 72 hours. MTT reagent was added and incubated for 4 hours, then the supernatant was removed, and dimethyl sulfoxide was added to dissolve formazan crystals. Absorbance at 490 nm was measured to calculate cell viability and IC50 [1]
- A549 cell clone formation assay: A549 cells were seeded in 6-well plates (200 cells/well) and cultured for 24 hours. mTOR inhibitor-1 (5, 10, 15 μM) was added and incubated for 14 days. Colonies were fixed with paraformaldehyde, stained with crystal violet, and counted. The clone formation rate was calculated by comparing with the control group [1] - A549 cell autophagy and mTOR pathway detection (western blot): A549 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with mTOR inhibitor-1 (5, 10, 15 μM) for 24 hours. Cells were lysed to extract total protein, which was separated by SDS-PAGE, transferred to a membrane, and incubated with primary antibodies (against mTOR, p-mTOR, p70S6K, p-p70S6K, 4E-BP1, p-4E-BP1, LC3, p62) and secondary antibodies. Chemiluminescence detection was performed, and band intensity was quantified using image analysis software [1] - A549 cell autophagy detection (immunofluorescence): A549 cells were seeded on coverslips, treated with 15 μM mTOR inhibitor-1 for 24 hours, fixed with paraformaldehyde, permeabilized with Triton X-100, and incubated with anti-LC3 primary antibody and fluorescent secondary antibody. Nuclei were stained with DAPI, and LC3 puncta (autophagosomes) were observed under a fluorescence microscope [1] |
| Animal Protocol |
- Nude mouse A549 xenograft model: BALB/c nude mice (4–6 weeks old, male) were maintained under specific pathogen-free conditions. A549 cells (1×10⁷ cells in 0.2 mL PBS + Matrigel) were subcutaneously injected into the right flank of each mouse. When tumors grew to ~100 mm³, mice were divided into 3 groups: vehicle group (0.1% DMSO + 0.9% saline), 10 mg/kg mTOR inhibitor-1 group, 20 mg/kg mTOR inhibitor-1 group (n=6/group). The drug was prepared by dissolving mTOR inhibitor-1 in DMSO first, then diluting with saline to the final concentration (DMSO content ≤0.1%). Administration was via intraperitoneal injection, once every 2 days for 21 days. Tumor volume (measured with calipers, volume = length × width² / 2) and mouse body weight were recorded every 3 days. After treatment, mice were euthanized, tumors were excised and weighed, and tumor tissues were stored at -80°C for western blot analysis [1]
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| Toxicity/Toxicokinetics |
In vitro cytotoxicity: mTOR inhibitor-1 (at concentrations up to 15 μM) showed no significant cytotoxicity to normal human lung fibroblasts (MRC-5 cells); MTT assay showed that cell viability was >80% after 72 hours of treatment [1]
- In vivo toxicity: In a 21-day nude mouse experiment, mTOR inhibitor-1 (10, 20 mg/kg) did not cause significant changes in mouse weight (weight change <5% compared to the control group), and no pathological abnormalities were observed in major organs (heart, liver, spleen, lung, kidney) by hematoxylin-eosin staining [1] |
| References | |
| Additional Infomation |
- mTOR inhibitor-1 is a small molecule identified by computer virtual screening (using molecular docking analysis of mTOR kinase crystal structure)[1]
- Its anti-tumor mechanism in A549 cells involves a dual action: inhibiting the mTOR signaling pathway to suppress cell proliferation and inducing autophagy to promote tumor cell death[1] - mTOR inhibitor-1 shows potential as a candidate drug for the treatment of lung cancer, especially for non-small cell lung cancer (NSCLC) represented by A549 cells[1] |
| Molecular Formula |
C16H15BRN2O3
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|---|---|
| Molecular Weight |
363.205903291702
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| Exact Mass |
363.21
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| Elemental Analysis |
C, 52.91; H, 4.16; Br, 22.00; N, 7.71; O, 13.21
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| CAS # |
468747-17-3
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| Related CAS # |
468747-17-3
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| PubChem CID |
135418721
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| Appearance |
Light yellow to yellow solid
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| Melting Point |
262 °C
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| LogP |
3.4
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| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
22
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| Complexity |
430
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| Defined Atom Stereocenter Count |
0
|
| SMILES |
BrC1C=C(C(N/N=C(/C)\C2C=CC(=CC=2O)O)=O)C=CC=1C
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| InChi Key |
NKMSVTGHOVMMHV-VCHYOVAHSA-N
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| InChi Code |
InChI=1S/C16H15BrN2O3/c1-9-3-4-11(7-14(9)17)16(22)19-18-10(2)13-6-5-12(20)8-15(13)21/h3-8,20-21H,1-2H3,(H,19,22)/b18-10+
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| Chemical Name |
3-bromo-N-[(E)-1-(2,4-dihydroxyphenyl)ethylideneamino]-4-methylbenzamide
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| Synonyms |
mTOR inhibitor-1; E74766; BCP32690
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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 |
| 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: 73~83.3 mg/mL (201~229.4 mM)
Ethanol: ~2 mg/mL (~5.5 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.73 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 20.8 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: ≥ 2.08 mg/mL (5.73 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (5.73 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7532 mL | 13.7661 mL | 27.5323 mL | |
| 5 mM | 0.5506 mL | 2.7532 mL | 5.5065 mL | |
| 10 mM | 0.2753 mL | 1.3766 mL | 2.7532 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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