Molecular Weight (MW)

460.45

Formula

C26H19F3N4O

CAS No.

742112-33-0

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 11 mg/mL (23.9 mM)

Water: <1 mg/mL

Ethanol: <1 mg/mL

Solubility (In vivo)

0.5% methylcellulose+0.2% Tween 80: 30mg/mL

Other info

Synonym: AR12; AR 12; AR-12; OSU-03012; OSU03012; OSU 03012.

Chemical Name: 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl} acetamide

InChi Key: YULUCECVQOCQFQ-UHFFFAOYSA-N

InChi Code: InChI=1S/C26H19F3N4O/c27-26(28,29)24-14-23(33(32-24)20-10-8-19(9-11-20)31-25(34)15-30)18-7-12-22-17(13-18)6-5-16-3-1-2-4-21(16)22/h1-14H,15,30H2,(H,31,34)

SMILES Code: O=C(NC1=CC=C(N2N=C(C(F)(F)F)C=C2C3=CC=C4C5=CC=CC=C5C=CC4=C3)C=C1)CN

In Vitro

In vitro activity: OSU-03012 induces apoptotic death in PC-3 cells with IC50 of 5 μM and reduces the activity of immunoprecipitated p70S6K. OSU-03012 completely suppress cell growth in a diverse range of tumor cell lines at concentrations of 3–5 μm, as compared with the concentration of at least 50 μm required for celecoxib. OSU-03012 promotes cell killing to a greater extent in glioma cells than in nontransformed astrocytes. OSU-03012 causes a dose-dependent induction of cell death that is not altered by p53 mutation, expression of ERBB1 VIII, or loss of phosphatase and tensin function due to a homolog deletion on chromosome 10. OSU-03012 and ionizing radiation cause an additive, caspase-independent elevation in cell killing. OSU-03012 lethality as a single agent or when combined with signaling modulators is not modified in cells lacking expression of BIM or of BAX/BAK. OSU-03012 promotes the release of cathepsin B from the lysosomal compartment and that of AIF from mitochondria. The lethality of OSU-03012 is attenuated in protein kinase R-like endoplasmic reticulum kinase-/- cells, which correlated with the reduced cleavage of BID and suppression of cathepsin B and AIF release into the cytosol. OSU-03012 inhibits thyroid cancer cell (NPA, WRO, and ARO cells) proliferation, migration and induces apoptosis, which results in an increase of cells in the S phase without an increase of cells in G2. OSU-03012 is an ATP-competitive inhibitor of PAK activity and suppresses the phosphorylation of AKT in thyroid cancer cells. OSU-03012 inhibits cell growth of hepatocellular carcinoma cell lines including Huh7, Hep3B and HepG2 cells with IC50 values below 1 μM. OSU-03012 does not suppress PDK1 or AKT activity or induce cellular apoptosis but induces autophagy in Huh7 cells. Moreover, accumulation of reactive oxygen species (ROS) is detected after OSU-03012 treatment. A recent study shows that OSU-03012 could enhance the susceptibility of (Bcr)-Abl mutant cell lines to imatinib-induced apoptosis.

Kinase Assay: This in vitro assay is performed using a PDK-1 kinase assay kit. This cell-free assay is based on the ability of recombinant PDK-1, in the presence of DMSO vehicle or OSU-03012, to activate its downstream serum- and glucocorticoid-regulated kinase which, in turn, phosphorylates the Akt/serum- and glucocorticoid-regulated kinase-specific peptide substrate RPRAATF with [γ-32P]ATP. The 32P-phosphorylated peptide substrate is then separated from the residual [γ-32P]-ATP by using P81 phosphocellulose paper and quantitated in a scintillation counter after three washes with 0.75% phosphoric acid.

Cell Assay: The effect of OSU-03012 on PC-3 cell viability is assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay in six replicates. Cells are grown in 10% FBS- supplemented RPMI 1640 in 96-well, flat-bottomed plates for 24 hours. They are exposed to various concentrations of OSU-03012 (0-10 μM) dissolved in DMSO (final concentration ≤0.1%) in 1% serum-containing RPMI 1640 for different time intervals (~72 hours). Controls receive DMSO vehicle at a concentration equal to that in OSU-03012-treated cells. The medium is removed and replaced by 200 μL of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide in 10% FBS-containing RPMI 1640. The cells are incubated in the CO2 incubator at 37 °C for 2 hours. Supernatants are removed from the wells, and the reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide dye is solubilized in 200 μL DMSO per well. Absorbance at 570 nm is determined by using a plate reader.

In Vivo

OSU-03012 suppresses tumor growth by 57.59% and increases cleaved LC3 in Huh7 tumor xenografts at 200 mg/kg. OSU-03012 remarkably decreases expression of EGFR protein in the tumors by 48% compared with vehicle controls and also prevents YB-1 from binding to the EGFR promoter in MDA-MB-435/LCC6 xenografts. OSU-03012 is well tolerated and inhibits the growth of HMS-97 schwannoma xenografts by 55% after oral administration

Animal model

Huh7 tumor xenografts in male BALB/c nude mice

Formulation & Dosage

Dissolved in 0.5% methylcellulose, 0.1% Tween 80; 100-200 mg/kg; i.p.

References

Cancer Res. 2004 Jun 15;64(12):4309-18; Cancer Res. 2004 Jun 15;64(12):4309-18; Mol Pharmacol. 2007 Nov;72(5):1124-31.