Molecular Weight (MW)

455.5

Formula

C23H22FN3O4S

CAS No.

1251156-08-7

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 23 mg/mL (50.5 mM)

Water: <1 mg/mL

Ethanol: <1 mg/mL

Solubility (In vivo)

30% PEG400+0.5% Tween80+5% propylene glycol: 30 mg/mL

Synonyms

Synonym: XL388; XL 388; XL-388

Chemical Name: (7-(6-aminopyridin-3-yl)-2,3-dihydrobenzo[f][1,4]oxazepin-4(5H)-yl)(3-fluoro-2-methyl-4-(methylsulfonyl)phenyl)methanone

InChi Key: LNFBAYSBVQBKFR-UHFFFAOYSA-N

InChi Code: InChI=1S/C23H22FN3O4S/c1-14-18(5-7-20(22(14)24)32(2,29)30)23(28)27-9-10-31-19-6-3-15(11-17(19)13-27)16-4-8-21(25)26-12-16/h3-8,11-12H,9-10,13H2,1-2H3,(H2,25,26)

SMILES Code: O=C(N1CCOC2=CC=C(C3=CC=C(N)N=C3)C=C2C1)C4=CC=C(S(=O)(C)=O)C(F)=C4C

In Vitro

Kinase Assay: The measurement of mTOR enzyme activity is performed in an ELISA format following the phosphorylation of 4E-BP1 protein. All experiments are performed in the 384-well format. Generally, 0.5 μL of DMSO containing varying concentrations of the test compound is mixed with 15 μL of the enzyme solution. Kinase reactions are initiated with the addition of 15 μL of a solution containing the substrate. The assay conditions are as follows: 0.2 nM mTOR, 10 μM ATP, and 50 nM NHis-tagged 4E-BP1 in 20 mM Hepes, pH 7.2, 1 mM DTT, 50 mM NaCl, 10 mM MnCl2, 0.02 mg/mL BSA, 0.01% CHAPS, 50 mM β-glycerophophate. Following an incubation of 120 min at ambient temperature, 20 μL of the reaction mixture is transferred to a Ni-chelate-coated 384-well plate. The binding step of the 4E-BP1 protein proceeded for 60 min, followed by washing four times each with 50 μL of Tris-buffered saline solution (TBS). Anti-phospho-4E-BP1 rabbit immunoglobulin G (IgG; 20 μL, 1:5000) in 5% BSA-TBST (0.2% Tween-20 in TBS) is added, and the reaction mixuture is further incubated for 60 min. Incubation with a secondary horseradish peroxidase (HRP)-tagged anti-IgG is similarly performed after the primary antibody is washed off (four washes of 50 μL). Following the final wash step with TBST, 20 μL of SuperSignal ELISA Femto is added and the luminescence measured using an EnVision plate reader. Data are reported as the mean (n≥2)

Cell Assay: In MCF-7 cells, XL388 blocks mTORC1 phosphorylation of p70S6K (T389)with an IC50 value of 94 nM and blocks mTORC2 phosphorylationof AKT (S473) with an IC50 value of 350 nM. In vitro, XL388 inhibits the viability of solid and hematopoietic tumor cell lines. The proliferation IC50 is 1.37 μM in MCF-7 cell line. XL388 also synergizes with chemotherapeutics in cell-based assays to block cell viability.

In Vivo

When dosed orally once daily in mice, XL388 shows robust anti-tumor activity in multiple xenograft models including> 100% tumor growth inhibition in the MCF-7 xenograft model. XL388 displays good pharmacokinetics and oral exposure in multiple species with moderate bioavailability. The mean plasma protein binding of XL388 in human, monkey, dog, rat, and mouse plasma is evaluated at 5 μM and is determined to be 86%, 90%, 89%, 85%, and 84%, respectively. Oral administration of XL388 to athymic nude mice implanted with human tumor xenografts afforded significant and dose-dependent antitumor activity. Strong inhibition of both mTORC1 and mTORC2 is achieved 4–8 h following administration orally at 100 mg/kg. Modest inhibition (39–45%) of phosphorylation of the PI3K target AKT (T308) is also observed 4–8 h post dose.

Animal model

MCF-7 xenograft tumors

Formulation & Dosage

Formulated in solution/fine suspension in water (with a 1:1 molar ratio of 1 N HCl); 50, 100 mg/kg; p.o.

References

J Med Chem. 2013 Mar 28;56(6):2218-34.