Kinase experiment:

The activity of the CDKs and FLT3 are assayed in reaction buffer (20 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO) at room temperature at a final ATP concentration of 10 mM. Then FLT3, dissolved in 100% DMSO at the indicated doses, are delivered into the kinase reaction mixture by acoustic technology and incubated for 20 min at room temperature. After 10 μM [γ-33P] ATP (specific activity 10 Ci/μL) is added to initiate the reaction, the reactions are carried out at 25℃ for 120 min. The kinase activities are detected by the filterbinding method. IC50 values and curve fits are obtained by Prism[1].

Cell experiment:

The human AML cell line MV4-11 is cultured in IMDM media with 10% FBS and supplemented with 2% l-glutamine and 1% penicillin/streptomycin. The MV4-11 cell line is maintained in culture media at 37℃ with 5% CO2. The effects of FN-1501 on MV4-11 proliferation are performed. Cells are cultured in 96-well culture plates (10 000 cells/well). FN-1501 at various concentrations is added to the plates. Cell proliferation is determined after treatment with FN-1501 for 72 h. Cell viability is measured using the CellTiter-Glo assay, and luminescence is measured in a multilabel reader. Data are normalized to control groups (DMSO) and represented as the means of three independent measurements with standard errors of<20%. IC50 values are calculated using Prism 5.0[1].

Animal experiment:

Mice[1]Six-week-old female nu/nu mice are housed in a specific pathogen-free facility. Prior to implantation, cells are harvested during exponential growth. Five million MV4-11 cells in PBS are formulated as a 1:1 mixture with a Matrigel and injected into the subcutaneous space on the right flank of each nu/nu mouse. Daily intravenous injections are initiated when MV4-11 tumors have reached sizes of 100-200 mm3. The animals are then randomized into treatment groups of 8 mice each for the efficacy studies and dosed with FN-1501 (0, 15, 30, or 40 (mg/kg)/d) or cytarabine (50 (mg/kg)/d). The compounds (FN-1501, etc.) are dissolved in a solution of PEG400 (25%), ethanol (3.7%), glucose (5%), and acetic acid/sodium acetate buffer (pH 4.5, 7.5%). Tumor growth is measured every 3 days using Vernier calipers for the duration of the treatment. The volume is calculated as follows: tumor volume = a × b2/2, where a is the long diameter, and b is the short diameter. The percentage of tumor-growth inhibition (GI) is calculated as follows: GI = 100% × {1 - [(tumor volumefinal - tumor volumeinitial for the compound-treated group)/(tumor volumefinal - tumor volumeinitial for the vehicle-treated group)]}. The percent tumor regression (PTR) is calculated as follows: PTR = 100% × (tumor volumeinitial - tumor volumefinal)/(tumor volumeinitial)[1].

FN-1501 is a potent inhibitor of FLT3 and CDK, with IC50s of 2.47, 0.85, 1.96, and 0.28 nM for CDK2/cyclin A, CDK4/cyclin D1, CDK6/cyclin D1 and FLT3, respectively; FN-1501 has anticancer activity.

FN-1501 is a potent inhibitor of FLT3 and CDK, with IC50s of 2.47 ± 0.21, 0.85 ± 0.28, 1.96 ± 0.08 and 0.28 ± 0.01 nM for CDK2/cyclin A, CDK4/cyclin D1, CDK6/cyclin D1 and FLT3, respectively. FN-1501 shows potent inhibitory activity against several tumor cells, such as MGC803, RS4;11, MCF-7, HCT-116, and NCI-H82, with GI50s of 0.37 ± 0.04, 0.05 ± 0.01, 2.84 ± 0.25, 0.09 ± 0.04, 0.11 ± 0.02 nM, respectively[1].

FN-1501 exhibits potent antitumor activity, and shows little cytotoxicity on normal lymphocyte cells, with LD50 of 185.67 mg/kg in ICR mice. FN-1501 (15. 30, or 40 mg/kg/d, i.v.) dose-dependently and significantly suppresses the growth of tumor in MV4-11-cell-inoculated-xenograft mice[1].

[1]. Wang Y, et al. Discovery of 4-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (FN-1501), an FLT3- and CDK-Kinase Inhibitor with Potentially High Efficiency against Acute Myelocytic Leukemia. J Med Chem. 2018 Feb 22;61(4):1499-1518.