Molecular Weight (MW)

452.55

Formula

C24H32N6O3

CAS No.

1207360-89-1

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 91 mg/mL (201.1 mM)

Water: <1 mg/mL

Ethanol: 6 mg/mL (13.3 mM)

Other info

Chemical Name: (S)-1-ethyl-3-(4-(4-(3-methylmorpholino)-7-(oxetan-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)urea
InChi Key: RGJOJUGRHPQXGF-INIZCTEOSA-N
InChi Code: InChI=1S/C24H32N6O3/c1-3-25-24(31)26-18-6-4-17(5-7-18)22-27-21-12-29(19-14-33-15-19)9-8-20(21)23(28-22)30-10-11-32-13-16(30)2/h4-7,16,19H,3,8-15H2,1-2H3,(H2,25,26,31)/t16-/m0/s1
SMILES Code: O=C(NC1=CC=C(C2=NC(N3[C@@H](C)COCC3)=C4C(CN(C5COC5)CC4)=N2)C=C1)NCC

Synonyms

RG-7603; RG7603; RG 7603; GDC0349; GDC 0349; GDC-0349

In Vitro

Kinase Assay: The kinase activity of mTOR enzyme is assessed by incubating purified recombinant enzyme (mTOR(1360-2549)+GBL, prepared in-house) in a reaction mixture containing ATP, MnCl2, and a fluorescently labeled mTOR substrate, e.g., GFP-4E-BP1. The reaction is stopped by an addition of a Terbium-labeled phospho-specific antibody, e.g., Tb-labeled anti-p4E-BP1 T37/T46, EDTA, and TR-FRET buffer solution. Product formation is detected by way of time-resolved fluorescence resonance energy transfer (TR-FRET), which occurs when the phosphorylated substrate and labeled antibody are in close proximity due to phosphospecific binding. Enzymatic activity is measured as an increase in TR-FRET signal using a Perkin Elmer Envision plate reader. The assay is performed in a 384-well Proxiplate Plus using the following protocol: Compound activity is tested in 10 point dose curves starting at the highest final concentration of 10 uM. They are serially diluted in 100% DMSO prior to further dilution with assay buffer. The reaction mixture (8μL) containing 0.25 nM mTOR+GBL enzyme, 400 nM GFP-4E-BP1, 8 uM ATP, 50 mM Hepes pH 7.5, 0.01% Tween 20, 10 mM MnCl2, 1 mM EGTA, 1 mM DTT, 1% DMSO (±compound) is incubated at room temperature for 30 minutes. 8 μL of solution containing 2 nM Tb-anti-p4E-BP1 antibody & 10 mM EDTA diluted TR-FRET buffer is then added and incubated for 30 minutes to stop the reaction. The plate is scanned with the Envision plate reader. Ki values are calculated in Assay Explorer using the Morrison ATP-competitive tight binding equation for Kiapparent determination.

Cell Assay: GDC-0349 has remarkable selectivity over 266 kinases, including all isoforms of PI3K. GDC-0349 inhibits downstream markers of mTOR, including phospho-4EBP1 and phospho-Akt(S473) in an in vivo PK/PD study in mouse, consistent with an inhibition of both mTORC1 and mTORC2 complexes.

In Vivo

GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models. When dosed orally once daily in athymic mice in a MCF7-neo/Her2 tumor xenograft model (PI3K mutation), GDC-0349 inhibits tumor growth in a dose-dependent manner. It is also efficacious in other xenograft models, including PC3 (PTEN null) and 786-O (VHL mutant).

Animal model

MCF7-neo/Her2 tumor xenograft

Formulation & Dosage

Formulated in 0.5% methylcellulose/0.2% Tween 80; 10, 20, 30, 40,50, 60, 70, 80 mg/kg; p.o.

References

J Med Chem. 2013 Apr 11;56(7):3090-101.