FB23-2 is a potent and selective inhibitor of the mRNA N6-methyladenosine (m6A) demethylase FTO with an IC50 of 2.6 μM.

FTO:2.6 μM (IC50)

FB23-2 directly bind to FTO and selectively inhibit FTO's m6A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells in vitro[1].

FB23-2 significantly inhibits the progression of human AML cell lines and primary cells in xeno-transplanted mice[1].

Quantitation of FB23 and FB23-2 in AML cells: NB4 and MONOMAC6 cells were treated with 10 μM FB23 or FB23-2 for 24 hr, respectively. Viable cells were distinguished with 0.1% trypan blue, counted and then harvested with PBS by several washings. Cells were diluted into 100 μl with 50% (v/v) water/methanol and followed by several shock freeze-thaw cycles. The supernatants were collected for analysis. The Ultimate 3000 system coupled with a TSQ Quantiva mass spectrometer was applied to determine the cellular concentration of compound FB23 and FB23-2. Analytes were separated on a XSELECT? HSS T3 column (100 mm × 3.0 mm, 2.5 μm). The mobile phases used for elution were (A) 0.1% (v/v) formic acid/water and (B) 0.1% (v/v) formic acid/acetonitrile. The mass spectrometer was operated in the negative MRM mode. Parent-to-product transitions were m/z 375.1→339.1, 375.1→298.1 for FB23, and m/z 390.3→318.0, 390.3→289.9 for FB23-2, respectively[1].

The NSGS mice were bred and subjected to the xeno-transplantation model. For the AML mouse model, 0.2 × 10^6 MONOMAC6 cells were directly transplanted into NSGS mice via tail vein. After 10 days, FB23-2 (2 mg/kg/day) and DMSO vehicle were intraperitoneally injected into the mice for a continuous 10 days. The mice were euthanized by CO2 inhalation if they exhibited classical AML symptoms including hunched posture, paralysis, and reduced body weight. Meanwhile, the PB, spleen, and liver samples were collected for further analysis[1].