FTO (fat mass and obesity-associated protein, an RNA m6A demethylase) – IC50 = 1.3 ± 0.2 μM (PAGE-based assay); IC50 = 43.7 ± 4.2 nM (cell-free dot blot assay); exhibits high selectivity over ALKBH5 and ALKBH3 (no inhibition at 50 μM) [1]

FTO-IN-15 (compound 8a) (0-50 μM) is a competitive inhibitor of the 2-OG binding pocket of FTO, as evidenced by the dose-dependent reversal of its inhibitory activity in cell-free enzyme assays with increasing 2-OG concentration[1]. FTO-IN-15 exhibits potent FTO inhibitory activity with an IC50 of 43.7 nM in cell-free dot blot assays and 1.3 μM in PAGE-based demethylation assays[1].

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Compound 8a inhibited FTO demethylase activity with an IC50 of 1.3 ± 0.2 μM in a PAGE-based ssDNA demethylation assay, and an IC50 of 43.7 ± 4.2 nM in a cell-free dot blot assay using an m6A-modified ssRNA substrate. [1]
Compound 8a showed high selectivity for FTO over the related ALKBH family members ALKBH5 and ALKBH3, demonstrating no inhibition at 50 μM in a PAGE-based demethylation assay. [1]
In differential scanning fluorimetry (DSF) experiments, compound 8a concentration-dependently stabilized recombinant FTO ΔN31 folding, yielding a ΔTm of 1.9°C at an 80-fold molar excess. [1]
Compound 8a exhibited synergistic inhibitory activity compared to its individual fragments: the MA analog showed modest activity (IC50 = 22.2 ± 5.8 μM) while fumaric acid was inactive (IC50 > 50 μM). [1]
The prodrug ester 8a-1 (mixed ethyl/methyl ester of 8a) exhibited potent antiproliferative activity across AML cell lines with IC50 values ranging from 2.3 ± 0.6 μM to 5.5 ± 0.7 μM in CCK-8 viability assays. In MOLM13 cells, 8a-1 (8 μM) induced significant growth suppression after 72 h treatment. [1]
In NB4 cells, 8a-1 treatment induced dose-dependent suppression of colony formation and promoted apoptosis. [1]
8a-1 treatment increased global m6A levels in both NB4 and MOLM13 cells. Western blot analysis showed upregulation of tumor suppressors RARA and ASB2, and downregulation of oncoproteins CEBPA and c-Myc. qPCR analysis in NB4 cells confirmed increased ASB2 expression accompanied by decreased c-Myc and CEBPA transcripts. The downregulation of c-Myc protein was observed across seven AML cell lines (NB4, MOLM13, HEL, MV4-11, OCI-AML3, NOMO-1, KG-1). 8a-1 treatment did not alter protein levels of FTO itself, ALKBH5, or METTL3. [1]
In a 2-OG competition PAGE-based assay, the inhibition by 8a could be dose-dependently reversed by 2-OG, confirming engagement with the 2-OG binding pocket. [1]

In an NB4 xenograft model in female BALB/c nude mice, daily intraperitoneal administration of 8a-1 (30 or 60 mg/kg for 9 days) significantly suppressed tumor growth in a dose-dependent manner compared to vehicle controls. Final tumor weights were significantly reduced by 8a-1 treatment. Molecular analysis of excised tumors revealed upregulation of tumor suppressors (RARA, ASB2) and downregulation of oncoproteins (CEBPA, c-Myc), as well as a marked increase in global RNA m6A modifications by dot blot assay. No body weight loss or organ toxicity (heart, liver, spleen, lung, kidney) was observed. [1]

PAGE-based demethylation assay: A synthetic 39-nucleotide single-stranded DNA substrate containing a central DpnII recognition site and an m6A modification was used. Reactions (50 mM Tris-HCl, pH 7.5) contained 1 μM ssDNA substrate, 1 μM FTO ΔN31, 300 μM 2-OG, 280 μM (NH4)2Fe(SO4)2, 2 mM L-ascorbic acid, and 20 μM test compound. After 2 h at room temperature, reactions were heat-inactivated. The substrate was then annealed to its complementary DNA strand to enable DpnII cleavage. Digestion products were separated on 15% native polyacrylamide gels, visualized with nucleic acid stain, and quantified using image analysis software. [1]
Cell-free dot blot assay: Reaction mixtures (50 mM HEPES, pH 7.0) contained 200 nM FTO, 283 μM (NH4)2Fe(SO4)2, 75 μM 2-OG, 2 mM L-ascorbic acid, 50 μg/mL BSA, and different concentrations of inhibitor. After 10 min preincubation at room temperature, the m6A-modified ssRNA substrate (5'-AUUGUCA(m6A)CAGCAGC-3') was introduced. Reactions proceeded for 3 h at 37°C before termination with 0.5 M EDTA. RNA was ethanol-precipitated overnight at -80°C. Aliquots were immobilized on membranes via a Bio-Dot apparatus, followed by UV cross-linking. Membranes were probed with anti-m6A antibody, then with HRP-conjugated secondary antibody. m6A signals were developed using ECL substrate and captured on a chemiluminescence imaging system. [1]
Differential scanning fluorimetry (DSF): Assay mixtures (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) contained 2 μM recombinant FTO ΔN31, test compounds, and SYPRO Orange dye. Samples underwent thermal denaturation via a 0.5°C/min ramp from 25 to 95°C. Fluorescence emission at 610 nm (excitation: 492 nm) was continuously recorded. Denaturation curves were processed to derive melting temperatures (Tm). [1]
Crystallization and structure determination of FTO/8j complex: Crystals of FTO ΔN31 with compound 8j were obtained. The structure was determined at 2.3 Å resolution (PDB: 9VG4). Key interactions included hydrogen bonds between the A-ring carboxylic acid and Ser229, a chlorine substituent with Thr92, the amide NH with His232, and the fumaramide carbonyl oxygen with His231. [1]
Molecular docking: MOE docking simulations indicated that 8a fully occupies both the substrate- and 2-OG-binding pockets, with hydrogen bonds involving Ser229, Arg96, Asp233, Arg316, and Ser318, as well as CH-π interactions with Thr92 and Leu109. [1]

Cellular thermal shift assay (CETSA): NB4 AML cells were lysed in prechilled RIPA buffer with protease inhibitor cocktail. After centrifugation, the soluble fraction was incubated with 50 μM 8a or vehicle control (DMSO) for 1 h at 25°C. The treated lysates were then aliquoted and subjected to thermal challenge with heating at incremental temperatures (55-80°C). Following centrifugation, remaining soluble proteins were analyzed by immunoblotting. 8a induced thermal stabilization of endogenous FTO but not ALKBH5. [1]
Cell viability assay (CCK-8): AML cell lines (NB4, MOLM13, HEL, MV4-11, OCI-AML3, NOMO-1, KG-1) were treated with various concentrations of prodrug 8a-1 for 72 h. IC50 values ranged from 2.3 ± 0.6 μM to 5.5 ± 0.7 μM. [1]
Colony formation assay: NB4 cells were treated with 8a-1 at indicated concentrations. Dose-dependent suppression of colony formation was observed. [1]
Apoptosis assay: NB4 cells treated with 8a-1 showed promoted apoptosis (method details not specified). [1]
Western blot analysis: Cells or tumor tissues were lysed and probed with antibodies against RARA, ASB2, CEBPA, c-Myc, FTO, ALKBH5, METTL3, and loading controls. 8a-1 treatment upregulated RARA and ASB2, downregulated CEBPA and c-Myc, and did not affect FTO, ALKBH5, or METTL3 levels. [1]
qPCR analysis: NB4 cells treated with 8a-1 showed increased ASB2 mRNA and decreased c-Myc and CEBPA transcripts. [1]
RNA m6A dot blot: Total RNA was extracted from cells or tumors, immobilized on membranes, and probed with anti-m6A antibody. 8a-1 treatment increased global m6A levels. [1]

NB4 xenograft model: Female BALB/c nude mice (6-8 weeks old, 18-22 g) were housed under standardized conditions (12 h light/dark cycle, 22 ± 2°C, 50 ± 10% humidity). Subcutaneous AML xenografts were established by inoculating 5 × 10^6 NB4 cells suspended in 100 μL of 50% Matrigel/RPMI-1640 medium into the right flank. Tumor dimensions were measured daily using digital calipers, with volume calculated as 0.5 × (length × width^2). When tumors reached 100-150 mm^3, mice were randomly assigned to three cohorts (n = 6/group) receiving daily intraperitoneal injection of 8a-1 (30 or 60 mg/kg) or vehicle control (5% DMSO, 5% Tween-80, 40% PEG400, 50% saline) for 9 days. Terminal procedures included CO2 euthanasia followed by tumor excision for gravimetric analysis and molecular profiling. [1]

The parent dicarboxylic acid 8a showed poor cellular permeability (antiproliferative activity <10% inhibition at 10 μM in AML cells), which was overcome by prodrug esterification. [1]

In the NB4 xenograft model, daily intraperitoneal administration of 8a-1 at 30 or 60 mg/kg for 9 days caused no body weight loss or organ toxicity (heart, liver, spleen, lung, kidney weights showed no treatment-related abnormalities). [1]

[1]. Structure-Based Design of a Highly Potent Dual-Competitive FTO Inhibitor for Targeted m6A Demethylase Inhibition in AML. J Med Chem. 2025 Nov 13;68(21):22779-22798.

Compound 8a is a substrate/2-OG dual-competitive FTO inhibitor developed through fragment linking of meclofenamic acid (MA) and 2-OG mimetics. The fumaric acid derivative 8a was highly potent (IC50 = 1.3 ± 0.2 μM), while its saturated analog succinic acid-based 8b lost all inhibitory activity, underscoring the essential role of the trans-double bond for effective 2-OG mimicry. The ethylenediamine linker was optimal; extending to propyl- or butylenediamine resulted in complete loss of activity. The 3,5-dichloro motif in 8a and the chloro/cyclopropyl substitution pattern in 8k (IC50 = 0.6 ± 0.1 μM) were identified as optimal for FTO inhibition. The prodrug ester 8a-1 (mixed ethyl/methyl ester) was synthesized to overcome the poor cellular permeability of the dicarboxylate parent compound. 8a-1 demonstrated potent antileukemic activity by effectively modulating the m6A epitranscriptomic machinery, representing a promising lead for clinical translation in AML. [1]

C20H17CL2N3O6

466.27

Typically exists as solids at room temperature

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)

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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