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| Targets |
FTO-IN-8 specifically targets the fat mass and obesity-associated protein (FTO), a demethylase enzyme that removes methyl groups from N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) residues in RNA. FTO is involved in the regulation of RNA stability, splicing, and translation. FTO-IN-8 is a selective inhibitor that binds to the active site of FTO, preventing it from demethylating RNA substrates. It has an IC50 of 5.5 microM for FTO, demonstrating anti-cancer cell proliferative activity.
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| ln Vitro |
FTO-IN-8 (FTO-43) (0-50 μM, 24-72 hours) prevents the growth of cancer cells while not harming healthy colon cells [1]. In gastric cancer AGS cells, FTO-IN-8 (FTO-43) raises m6A and m6Am levels and suppresses Wnt/PI3K-Akt signaling [1].
In vitro, FTO-IN-8 potently inhibits the demethylase activity of FTO. In enzymatic assays, the compound inhibits FTO with an IC50 of 5.5 microM. In cell-based studies, FTO-IN-8 (0-50 uM, 24-72 hours) prevents the growth of cancer cells while not harming healthy colon cells. In gastric cancer AGS cells, treatment with FTO-IN-8 increases the levels of m6A and m6Am modifications on RNA and suppresses the Wnt/PBK-Akt signaling pathway, leading to reduced cancer cell proliferation. These activities confirm its role as a selective FTO inhibitor. |
| ln Vivo |
In vivo, FTO-IN-8 has shown anti-cancer activity in preclinical models. By inhibiting FTO, the compound increases m6A levels on oncogenic transcripts, leading to their degradation and reduced cancer cell proliferation. FTO-IN-8 has been studied in models of gastric cancer and other tumor types. It demonstrates the ability to suppress tumor growth by modulating epitranscriptomic pathways. The compound has anti-cancer/tumor cell growth/proliferation activity and is being investigated as a potential therapeutic for FTO-driven cancers. Detailed in vivo efficacy data is limited.
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| Enzyme Assay |
For non-cell-based FTO inhibition assays, a standard protocol uses purified recombinant human FTO protein and a fluorescently labeled RNA substrate containing a single m6A modification. Varying concentrations of FTO-IN-8 (0.1-1000 uM) are pre-incubated with FTO (10 nM) in assay buffer (20 mM HEPES, pH 7.5, 50 mM NaCl, 1 mM DTT, 0.01% Triton X-100) for 15 minutes at 37degC. The reaction is initiated by adding the RNA substrate (100 nM). After 60 minutes, the reaction is stopped, and the demethylation product is detected by a demethylase activity assay kit that measures formaldehyde release or by a fluorescence polarization-based method. The IC50 is calculated.
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| Cell Assay |
Cell viability assay [1]
Cell Types: AGS, SNU16 and KATOIII Cell line Tested Concentrations: 0-50 μM Incubation Duration: 24, 48, 72 hrs (hours) Experimental Results: Inhibited the growth of SNU16, KATOIII and AGS, EC50 value was 17.7 μM, respectively 35.9 μM and 20.3 μM. For in vitro cell-based assays, gastric cancer AGS cells are seeded in 96-well plates at a density of 5,000 cells/well. After 24 hours, cells are treated with varying concentrations of FTO-IN-8 (0-50 uM) for 24-72 hours. Cell viability is measured using the MTT assay or CellTiter-Glo luminescent assay. To confirm target engagement, RNA is extracted from treated cells, and m6A levels are quantified using an m6A ELISA kit. For mechanism studies, protein lysates are analyzed by Western blotting for markers of the Wnt/PBK-Akt pathway, including p-AKT, total AKT, beta-catenin, and c-Myc. |
| Animal Protocol |
For in vivo animal studies, a mouse xenograft model of gastric cancer is used. Immunodeficient nude mice are subcutaneously injected with AGS gastric cancer cells. When tumors reach approximately 100 mm3, mice are randomized into treatment groups. FTO-IN-8 is administered intraperitoneally or orally at doses of 10-50 mg/kg once daily for 14-21 days. Control mice receive vehicle. Tumor volume is measured by calipers every 2-3 days. Body weight is monitored for toxicity. At study endpoint, tumors are excised, weighed, and analyzed for m6A levels and for markers of cell proliferation (Ki-67) and apoptosis (cleaved caspase-3) by immunohistochemistry.
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| ADME/Pharmacokinetics |
Specific pharmacokinetic data for FTO-IN-8 is not publicly available, as it is a preclinical research compound. The compound has a molecular weight of 346.85 g/mol and a molecular formula of C19H23ClN2O2, suggesting good drug-like properties with moderate lipophilicity. Based on its structure, it is expected to have good oral bioavailability and cell permeability. For in vivo studies, the compound can be formulated in DMSO/PEG400/saline mixtures for intraperitoneal or intravenous administration. Detailed PK parameters (Cmax, T1/2, AUC, clearance, and volume of distribution) would be determined during preclinical development.
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| Toxicity/Toxicokinetics |
Formal toxicology data for FTO-IN-8 is not publicly available, as it is a research compound not yet advanced to clinical development. In cell viability assays, FTO-IN-8 did not harm healthy colon cells at concentrations that inhibited cancer cell proliferation, suggesting a degree of selective toxicity for cancer cells. Standard safety pharmacology studies would include an assessment of the compound's potential to inhibit the hERG potassium channel (cardiotoxicity risk) and an Ames test for genotoxicity. A 28-day repeat-dose toxicity study in rodents would be required to determine the no-observed-adverse-effect level (NOAEL) for preclinical development.
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| References | |
| Additional Infomation |
FTO-IN-8 is a research compound that targets FTO, a key epitranscriptomic regulator. FTO is the first RNA demethylase to be identified, and its overexpression has been linked to obesity, diabetes, and various cancers, including acute myeloid leukemia (AML), breast cancer, gastric cancer, and glioblastoma. By inhibiting FTO, FTO-IN-8 increases m6A methylation on RNA, which can lead to the degradation of oncogenic transcripts. The compound serves as a valuable chemical probe for studying the role of FTO in cancer biology and as a lead compound for developing FTO-targeted cancer therapies. It is not approved for clinical use.
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| Molecular Formula |
C19H23CLN2O2
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| Molecular Weight |
346.85
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| Exact Mass |
346.144
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| CAS # |
2640366-38-5
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| PubChem CID |
164886650
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| Appearance |
Light yellow to yellow ointment
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| LogP |
2.7
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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 |
6
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| Heavy Atom Count |
24
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| Complexity |
412
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CCN(C1)C2(COC2)CNCC3=CC=C(O3)C4=CC=CC=C4Cl
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| InChi Key |
QHNARTNMADDQGU-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H23ClN2O2/c20-17-6-2-1-5-16(17)18-8-7-15(24-18)11-21-12-19(13-23-14-19)22-9-3-4-10-22/h1-2,5-8,21H,3-4,9-14H2
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| Chemical Name |
1-[5-(2-chlorophenyl)furan-2-yl]-N-[(3-pyrrolidin-1-yloxetan-3-yl)methyl]methanamine
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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 : ≥ 100 mg/mL (~288.31 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8831 mL | 14.4155 mL | 28.8309 mL | |
| 5 mM | 0.5766 mL | 2.8831 mL | 5.7662 mL | |
| 10 mM | 0.2883 mL | 1.4415 mL | 2.8831 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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