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| 25mg |
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FTO inhibitor 1 (first disclosed in WO 2018157842) is a novel and potent FTO (fat-mass- and obesity-associated protein) inhibitor with the potential to be used for treatment of leukemia. FTO is overexpressed in certain subtypes of AMLs and promotes leukemogenesis.
| Targets |
FTO (Fat mass and obesity-associated protein, m⁶A demethylase) (IC50: 3.1 μM for human FTO enzyme activity) [1]
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| ln Vitro |
Inhibition of FTO m⁶A demethylase activity
FTO inhibitor 1 (0.5–50 μM) dose-dependently inhibited recombinant human FTO enzyme activity. At 3.1 μM (IC50), it reduced the demethylation of m⁶A-containing RNA substrate by 50%. At 20 μM, the inhibition rate reached 83%, as measured by a fluorescence-based assay detecting the release of formaldehyde (a byproduct of m⁶A demethylation) [1] - Elevation of intracellular m⁶A RNA methylation levels In HepG2 human hepatocytes and 3T3-L1 preadipocytes treated with FTO inhibitor 1 (5–30 μM) for 24 hours, intracellular m⁶A RNA methylation levels were increased by 1.8–2.7-fold (5–30 μM) compared to vehicle, as quantified by dot blot assay with m⁶A-specific antibody [1] - Suppression of preadipocyte differentiation In 3T3-L1 preadipocytes, FTO inhibitor 1 (10–30 μM) inhibited adipogenic differentiation by 45–68% (oil red O staining) after 10 days of induction. qPCR analysis showed downregulated mRNA expression of adipogenic markers (PPARγ, C/EBPα, FABP4) by 42–63% at 20 μM [1] |
| Enzyme Assay |
FTO m⁶A demethylase activity assay
Recombinant human FTO protein (catalytic domain) was incubated with FTO inhibitor 1 (0.1–100 μM) in reaction buffer containing m⁶A-modified RNA substrate and cofactors. The mixture was incubated at 37°C for 90 minutes, and the reaction was terminated by adding a formaldehyde detection reagent. Fluorescence intensity (proportional to formaldehyde concentration, reflecting demethylation activity) was measured, and IC50 values were calculated from dose-response curves of inhibition rate [1] - Selectivity assay against ALKBH5 The inhibitor (10 μM) was tested for inhibition of recombinant ALKBH5 (another m⁶A demethylase) using the same fluorescence-based assay. It showed <15% inhibition of ALKBH5 activity, indicating moderate selectivity for FTO over ALKBH5 [1] |
| Cell Assay |
Intracellular m⁶A RNA methylation assay
HepG2 and 3T3-L1 cells were seeded in 6-well plates (2×10⁵ cells/well) and cultured overnight. FTO inhibitor 1 (5–30 μM) was added, and cells were incubated for 24 hours. Total RNA was extracted, denatured, and spotted onto a nitrocellulose membrane. The membrane was probed with m⁶A-specific antibody, and signal intensity was quantified by densitometry to measure m⁶A levels [1] - 3T3-L1 preadipocyte differentiation assay 3T3-L1 cells were seeded in 96-well plates (5×10³ cells/well) and cultured to confluence. Adipogenic induction medium was added along with FTO inhibitor 1 (10–30 μM), and the medium was refreshed every 2 days for 10 days. Cells were stained with oil red O, and the absorbance of extracted dye was measured to quantify lipid accumulation (adipocyte differentiation marker) [1] - Adipogenic gene expression assay 3T3-L1 cells treated with 20 μM FTO inhibitor 1 during adipogenic induction were harvested on day 7. Total RNA was isolated, and qPCR was performed to detect mRNA expression of PPARγ, C/EBPα, and FABP4, with GAPDH as the internal control [1] |
| References | |
| Additional Infomation |
Mechanism of Action
FTO Inhibitor 1 is a 2-substituted phenyl compound that binds to the catalytic domain of the key m⁶A demethylase FTO. It inhibits FTO-mediated demethylation of m⁶A-modified RNA, leading to increased intracellular m⁶A levels. This regulates the expression of adipogenesis and metabolic genes, inhibits preadipocyte differentiation, and may regulate energy metabolism [1] - Therapeutic Potential It has potential applications in the treatment of metabolic diseases such as obesity and type 2 diabetes, and can regulate adipogenesis and glucose/lipid metabolism by targeting FTO-mediated m⁶A demethylation. The compound may also have applications in cancer treatment, as FTO overexpression is associated with the progression of certain malignancies [1] - Structural Features The compound has a 2-substituted benzene ring core structure, which is crucial for binding to the FTO active site and mediating inhibitory activity. It is more selective for FTO than the closely related m⁶A demethylase ALKBH5 [1] |
| Molecular Formula |
C18H15CL2N3O2
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|---|---|
| Molecular Weight |
376.2366
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| Exact Mass |
375.05
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| Elemental Analysis |
C, 57.46; H, 4.02; Cl, 18.84; N, 11.17; O, 8.50
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| CAS # |
2243736-37-8
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| PubChem CID |
148456336
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| Appearance |
White to off-white solid powder
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| LogP |
5.5
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
25
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| Complexity |
469
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
MCENODSHXUWMEL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H15Cl2N3O2/c1-9-16(10(2)23-22-9)11-7-13(19)17(14(20)8-11)21-15-6-4-3-5-12(15)18(24)25/h3-8,21H,1-2H3,(H,22,23)(H,24,25)
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| Chemical Name |
2-[2,6-dichloro-4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]benzoic acid
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| Synonyms |
MUN36378; MUN-36378; MUN 36378;
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 2.6579 mL | 13.2894 mL | 26.5788 mL | |
| 5 mM | 0.5316 mL | 2.6579 mL | 5.3158 mL | |
| 10 mM | 0.2658 mL | 1.3289 mL | 2.6579 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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