Molecular Weight (MW)

270.29

Formula

C13H14N6O

CAS No.

934493-76-2;

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 54 mg/mL (199.78 mM)

Water:<1 mg/mL (slightly soluble or insoluble)

Ethanol: <1 mg/mL (slightly soluble or insoluble)

SMILES Code

SAR245409; SAR245409; SAR 245409; XL765; XL 765; XL-765; Voxtalisib.

Synonym

O=C1C(C2=CC=NN2)=CC3=C(C)N=C(N)N=C3N1CC

In Vitro

In Vitro Activity: Voxtalisib (SAR245409, XL765) displays potent inhibitory activity against class I PI3K isoforms p110α, p110β, p110δ, and p120γ, with IC50s of 39, 110, 43, and 9 nM, respectively. The IC50 value for inhibition of PI3Kα by SAR245409 is determined at various concentrations of ATP, revealing SAR245409 to be an ATP-competitive inhibitor with an equilibrium inhibition constant (Ki) value of 13 nM. SAR245409 also inhibits mTOR (IC50s of 160 and 910 nM for mTORC1 and mTORC2, respectively) in an immune-complex kinase assay and the PI3K-related kinase DNA-PK (IC50 value of 150 nM). In contrast, SAR245409 has relatively weak inhibitory activity toward the class III PI3K vacuolar sorting protein 34 (VPS34; IC50 value of ~9.1 μM). Consistent with its inhibitory activity against purified PI3K proteins, SAR245409 inhibits EGF-induced PIP3 production in PC-3 and MCF7 cells with IC50s of 290 and 170 nM, respectively. The ability of SAR245409 to inhibit phosphorylation of key signaling proteins downstream of PI3K is examined by assessing its effects on EGF-stimulated phosphorylation of AKT and on nonstimulated phosphorylation of S6 in PC-3 cells by cell-based ELISA. SAR245409 inhibits these activities with IC50s of 250 and 120 nM, respectively. In MCF7 and PC-3 cells, SAR245409 inhibits proliferation (monitored by BrdUrd incorporation) with IC50s of 1,070 and 1,840 nM, respectively. To further characterize the effects of SAR245409 on tumor cell growth, an assay monitoring the anchorage-independent growth of PC-3 and MCF7 cells in soft agar over a 14-day period is used. SAR245409 inhibits colony growth with an IC50 value of 270 nM in PC-3 cells and 230 nM in MCF7 cells

Kinase Assay: Kinase activity for PI3K isoforms is measured as the percentage of ATP consumed following the kinase reaction using luciferase-luciferin-coupled chemiluminescence, with ATP concentrations approximately equal to the Km for each respective kinase. Kinase reactions are initiated by combining test compounds, ATP and kinase in a 20 μL volume. PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ final enzyme concentrations are 0.5, 8, 20, and 2 nM, respectively. A similar assay format is used for DNA-PK (DNA protein kinase) and VPS34. VPS34 assay buffer contains 20 mM Tris-HCl, pH 7.5, 3.5 mM MnCl2, 100 mM NaCl, 1 mM DTT, and 0.01% cholamidopropyldimethylammonio propanesulfonate (CHAPS). Of note, 0.5 μL DMSO containing varying concentrations of the test compound is mixed with 10 μL enzyme solution (2× concentration). Kinase reactions are initiated by the addition of 10 μL of liver phosphatidylinositol and ATP solution (2× concentration). Assay concentrations for VPS34, ATP, and phosphatidylinositol are 40 nM, 1 μM, and 5 μM, respectively

Cell Assay: Cells (Pancreatic cancer cell lines (HcG25, Panc89, PA-TU8988T, Panc2.13, MiaPaCa2, Panc10.05, Panc8.13, BxPC-3, etc.) are treated with XL765 24 hours after plating and harvested for apoptosis or autophagy assays at 24, 48, or 72 hours after XL765 treatment. Apoptosis is determined by total percentage of annexin V-positive cells by fluorescence-activated cell sorting (FACS). Acidic vesicular organelles (AVOs) are detected in XL765-treated cells by vital staining with acridine orange. The degree of AVO formation is expressed as fold increase of acridine orange fluorescence intensity (FL3) in XL765-treated cells versus control cells.

XL765 is active against class I PI3K (IC50 = 39, 113, 9 and 43 nM for p110α, β, γ and δ, respectively). XL765 also inhibits DNA-PK (IC50 = 150 nM) and mTOR (IC50 = 157 nM) but not XL-147 which shows IC50 values of> 15 μM. XL765 treatment results in decreased cell viability in 13 PDA cell lines in a dose-dependent manner. XL765, a dual-target PI3K/mTOR inhibitor, inhibits cell growth and apoptosis in many more cell lines and at lower concentrations as compared to the PI3K-selective inhibitors XL147 and PIK90. The effect can be recapitulated by using combinations of single-targeted compounds. XL765 significantly reduces phosphorylation of the mTOR targets S6, S6K, and 4EBP1, which is associated with greater apoptosis induction rather than to PI3K inhibition alone. XL765 treatment causes accumulation of autophagosomes in MIAPaCa-2 cells, and results in significant dose-dependent AVO induction and LC3-II stimulation in MIAPaCa-2 cells stably expressing a LC3-GFP construct.

In Vivo

The combination of XL765 (30 mg/kg) with chloroquine (50 mg/kg) results in significant inhibition of BxPC-3 xenograft growth in mice models, while XL765 alone at the same dose has no inhibitory effect. Oral administration of XL765 results in greater than 12-fold reduction in median tumor bioluminescence compared to control and improvement in median survival in nude mice implanted intracranially with GBM 39-luc cells. XL765 in combination with temozolomide (TMZ) yields a 140-fold reduction in median bioluminescence with a trend toward improvement in median survival compared with TMZ alone.

Animal model

Female Nu/Nu mice inoculated s.c. with BxPC-3 cells

Formulation & Dosage

Dissolved in water/10 mM HCl; 30mg/kg; oral

References

[1] Garcia-Echeverria C, et al. Oncogene, 2008, 27(41), 5511-5526.; [2] Mirzoeva OK, et al. J Mol Med, 2011, 89(9), 877-889.

Mirzoeva OK, et al. J Mol Med, 2011, 89(9), 877-889.

Mirzoeva OK, et al. J Mol Med, 2011, 89(9), 877-889.