Molecular Weight (MW)

570.65

Formula

C29H34N10O3

CAS No.

1173204-81-3

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 0.4 mg/mL (0.7 mM)

Water:<1 mg/mL

Ethanol: <1 mg/mL

Solubility (In vivo)

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

Chemical Name/Synonym

1-[4-(3-ethyl-7-morpholin-4-yltriazolo[4,5-d]pyrimidin-5-yl)phenyl]-3-[4-(4-methylpiperazine-1-carbonyl)phenyl]urea; PKI402; PKI 402; PK-I402

In Vitro

Kinase Assay: Enzyme assays are done in fluorescent polarization (FP) format, adapted from the Echelon K-1100 PI3K FP assay kit protocol. Assay reaction buffer is 20 mM Hepes, pH 7.5, 2 mM MgCl2, 0.05% CHAPS, and 0.01% βME. Assay STOP/detection buffer is 100 mM Hepes, pH 7.5, 4 mM EDTA, 0.05% CHAPS. FP assays are run in Nunc 384-well black polypropylene fluor plates. The FP reaction is run in 20 μL of reaction buffer containing 20 μM PIP2, 25 μM ATP, and>4% DMSO. The reaction is run for 30 minutes at room temp. The reaction is stopped with 20 μL of STOP/detection buffer containing 10 nM probe and 40 nM GST-GRP. Assay plates are incubated for 2 hours, and fluorescence polarization is measured in a Perkin-Elmer Envision plate reader with TAMRA-FP filters.

Cell Assay: Cells: MDA-MB-361, MDA-MB-468, T47D, MCF7, BT474, HT29, HCT116, DLD1, U87MG, H157, NCI-H460, A549, NCI-H1975, NCI-H1650, NCIH2170, KB, 786-0, A498, MIA-PaCa-2, and PC3; Cell growth inhibition is determined using the CellTiter 96 Aqueous nonradioactive cell proliferation assay. This homogeneous colorimetric method determined the number of viable cells in proliferation assays. The assay is carried out in 96-well format, with cell number numbers per well being adjusted on the basis of growth characteristics of the various cell lines used. Assay end point data are quantitated after 72 hours of PKI-402 exposure using a Victor2 V (Wallac) model 1420 multilabel HTS counter.

Equivalent to the IC50 for wild-type PI3Kα, PKI-402 inhibits E545K and H1047R PI3Kα mutants with IC50 of 3 nM. In a panel of 236 human protein kinases, PKI-402 only displays inhibitory activity against C-Raf and B-Raf with IC50 of 7 μM, and displays little activity against all other kinases with IC50 of> 10 μM. PKI-402 inhibits the growth of human tumor cell lines with IC50 of 6-349 nM. Consistently, PKI-402 inhibits phosphorylation of PI3K and mTOR effector proteins, particularly phosphorylated Akt (p-Akt) at T308 and S473 with IC50 of<10 nM and <30 nM, respectively. PKI-402 inhibits both p70S6K and 4EBP1 phosphorylation with IC50 of <10 nM. PKI-402 inhibits Akt phosphorylation of PRAS40 at T246 with IC50 of <30 nM, and inhibits Akt phosphorylation of ENOS at S1177 and GSK3α/GSK3β at S9/S21 with IC50 of <10 nM. In MDAMB-361, a breast tumor line with mutant PI3K-α (E545K) and elevated levels of Her2 receptor, PKI-402 treatment induces cleaved poly(ADP-ribose) polymerase (PARP), a marker for apoptosis. Less than 10% of MDAMB-361 cells exposed to PKI-402 at 0.3 μM (or higher) for 24 hours remain viable.

In Vivo

Single dose of PKI-402 (100 mg/kg) suppresses Akt phosphorylation (at T308) and induces cleaved PARP in MDA-MB-361 tumors. In normal tissue (heart and lung), PKI-402 (100 mg/kg) has minimal effect on p-Akt, with no detectable cleaved PARP. Consistently, PKI-402 at 100 mg/kg (daily for 5 days, one round) reduces initial tumor volume of 260 mm3 to 129 mm3 and prevents tumor regrowth for 70 days in MDA-MB-361. PKI-402 significantly inhibits the growth of A549 tumors in nude mice at 25 mg/kg and 50 mg/kg. PKI-402 at 100 mg/kg (daily for 5 days, one round) causes significant (P < 0.01) reduction in tumor growth of U87MG.

Animal model

Female nude mice injected subcutaneously with MDA-361, U87MG, or A549 cells

Formulation & Dosage

Dissolved in 0.5% methylcellulose and 0.4% polysorbate 80 (Tween-80); 100 mg/kg; i.v. administration

References

[1] Mallon R, et al. Mol Cancer Ther, 2010, 9(4), 976-984.

In vitro profile of PKI-402. MDA-MB-361 [Her2+/PIK3CA (E545K)] cells were exposed to PKI-402 (0.003–0.3 μmol/L) for 4 h. Mol Cancer Ther, 2010, 9(4), 976-984.

PKI-402 suppression of p-Akt-T308, induction of cleaved PARP, and activation of caspase-3/7 alone, and with 0.1 μmol/L PD0325901 (MEK) in HCT116 (K-Ras and

PIK3CA) at 24 h.

In vivo efficacy against MDA-MB-361 xenografts and in vivo biomarkers in tumor and heart tissue.