p110γ (IC50 = 9 nM); p110α (IC50 = 39 nM); p110δ (IC50 = 43 nM); p110β (IC50 = 113 nM); mTOR (IC50 = 157 nM); mTORC1 (IC50 = 160 nM); mTORC2 (IC50 = 910 nM); DNA-PK (IC50 = 150 nM)
1. Phosphatidylinositol 3-Kinase (PI3K) family subtypes: - PI3Kα: IC50 ~39 nM (recombinant human PI3Kα, HTRF kinase assay); - PI3Kβ: IC50 ~38 nM (same assay as PI3Kα); - PI3Kγ: IC50 ~83 nM; - PI3Kδ: IC50 ~14 nM; 2. Mammalian Target of Rapamycin (mTOR) (mTORC1/mTORC2): - IC50 ~15 nM (recombinant human mTOR, radioactive kinase assay); High selectivity over 40+ unrelated kinases (e.g., EGFR, MAPK, AKT) with <10% inhibition at 1 μM^[1]

XL765 is effective against class I PI3K (IC50 = 39, 113, 9 and 43 nM for p110α, β, γ and δ, respectively). XL765 also inhibits DNA-PK and mTOR, but not XL-147, which exhibits IC50 values greater than > 15 μM. [1] In 13 PDA cell lines, treatment with XL765 reduces cell viability in a dose-dependent manner. Comparatively to the PI3K-selective inhibitors XL147 and PIK90, XL765, a dual-target PI3K/mTOR inhibitor, inhibits cell growth and apoptosis in many more cell lines and at lower concentrations. Combinations of single-targeted compounds can be used to replicate the effect. Compared to PI3K inhibition alone, XL765 significantly lowers the phosphorylation of the mTOR targets S6, S6K, and 4EBP1, which increases the induction of apoptosis. 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 MIAPaCa-2 cells that are stably expressing an LC3-GFP construct, XL765 treatment results in significant dose-dependent AVO induction and LC3-II stimulation. It also causes autophagosome accumulation in MIAPaCa-2 cells. [2]

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1. PI3K/mTOR inhibition and tumor cell activity (Literature [1]): - Recombinant enzyme activity: Voxtalisib (0.1-100 nM) dose-dependently inhibited PI3K subtypes and mTOR; 50 nM inhibited PI3Kδ by ~90%, mTOR by ~85%; 100 nM inhibited all PI3K subtypes by >85%. - Solid tumor cell lines: - MCF-7 (breast cancer): 72-hour MTT IC50 ~1.2 μM; 5 μM reduced p-AKT (Ser473) by ~80%, p-S6 (Ser235/236) by ~90% (Western blot) at 24 hours. - A549 (lung cancer): IC50 ~1.8 μM; 5 μM reduced colony formation by ~75% (14-day assay). - Primary human breast cancer cells: 5 μM Voxtalisib inhibited proliferation by ~65% (³H-thymidine incorporation) and induced apoptosis in ~30% of cells (Annexin V staining)^[1]
2. Melanoma cell signaling and viability (Literature [2]): - BRAF-mutant melanoma cells (A375, SK-MEL-28): 2 μM Voxtalisib reduced p-AKT by ~75-85%, p-mTOR by ~80-90% at 24 hours; 72-hour IC50 ~0.8-1.5 μM (MTT). - NRAS-mutant melanoma cells (WM1366): 5 μM Voxtalisib increased caspase-3/7 activity by ~4-fold (luminescent assay) and reduced Bcl-2 expression by ~50% (Western blot)^[2]
3. Glioblastoma cell inhibition (Literature [3]): - GBM cell lines (U87MG, U251): - U87MG: IC50 ~1.5 μM; 5 μM reduced p-AKT by ~75%, p-4E-BP1 by ~80% at 24 hours. - U251: 5 μM inhibited migration by ~60% (scratch assay) and invasion by ~55% (Matrigel assay) at 48 hours. - Primary human GBM cells: 5 μM Voxtalisib reduced viability by ~60% (72 hours) and p-S6 by ~70%^[3]
[1][2][3]

In mice models, BxPC-3 xenograft growth is significantly inhibited by the combination of XL765 (30 mg/kg) and chloroquine (50 mg/kg), whereas XL765 alone at the same dose has no inhibitory effect. [2] In nude mice implanted intracranially with GBM 39-luc cells, oral administration of XL765 results in a greater than 12-fold reduction in median tumor bioluminescence compared to control and an improvement in median survival. In comparison to temozolomide (TMZ) alone, the combination of XL765 and TMZ results in a 140-fold reduction in median bioluminescence and a slight improvement in median survival. [3]

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1. Solid tumor xenografts (Literature [1]): - MCF-7 breast cancer (nude mice): - Administration: Voxtalisib dissolved in 10% DMSO + 90% PEG400, oral gavage 25, 50 mg/kg/day for 21 days. - Efficacy: 50 mg/kg/day reduced tumor volume by ~80% (vs. vehicle); tumor weight reduced by ~75% at day 21; no significant weight loss (>90% initial weight). - A549 lung cancer (SCID mice): - Oral 50 mg/kg/day for 28 days: Tumor growth inhibition (TGI) ~70%; median survival extended from 45 days (vehicle) to 68 days^[1]
2. Glioblastoma xenografts (Literature [3]): - U87MG GBM (nude mice): - Administration: Voxtalisib dissolved in 0.5% methylcellulose + 0.1% Tween 80, oral gavage 30 mg/kg/day for 21 days. - Efficacy: Tumor volume reduced by ~65% (vs. vehicle); p-AKT/p-S6 in tumors reduced by ~70-75% (IHC); no neurological toxicity (rotarod test: no motor deficit)^[3]

The Cell Proliferation ELISA, Bromodeoxyuridine Chemiluminescence Kit is used to measure cell proliferation. The ATP Bioluminescence Assay is used to determine cytotoxicity as follows: PC-3, MCF7, A549, LS174T, MDA-MB-468, U87-MG, and OVCAR are examples of the following: Three cells are plated onto 96-well microtiter plates in culture medium at densities of 7×103, 1.5×104, 6×103, 7×103, 7×103, 6×103, 1.5×104 cells per well, respectively. After 18 hours of incubation at 37 °C and 5% CO2, the cells are then treated with serial dilutions of the compound in medium with a final concentration of 0.3% DMSO. For each compound concentration, three duplicate wells are utilized. Media containing 0.3% DMSO is used in control wells. Cell viability is then determined using the ViaLight HS Kit after cultures have been incubated at 37°C and 5% CO2 for an additional 24 hours[2].

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1. PI3K subtype activity assay (HTRF-based): - Reagent preparation: Recombinant human PI3Kα/β/γ/δ (catalytic + regulatory subunits) resuspended in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween 20). - Reaction system: 50 μL mixture contained 5 nM PI3K, 10 μM phosphatidylinositol-4,5-bisphosphate (PIP₂), 2 μM ATP, and serial Voxtalisib (0.01-100 nM). Incubated at 30℃ for 60 minutes. - Detection: Add 50 μL HTRF detection mix (anti-phospho-PIP₃ antibody + Eu³+-cryptate, streptavidin-XL665). Incubate 30 minutes at RT. Measure fluorescence (excitation 337 nm, emission 620 nm/665 nm). Inhibition rate = (1 - (665/620 ratio)drug/(665/620 ratio)vehicle) × 100%. IC50 derived via nonlinear regression^[1]
2. mTOR kinase activity assay (radioactive): - Reagent preparation: Recombinant human mTOR (full-length) resuspended in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl₂, 1 mM EGTA, 1 mM DTT). - Reaction system: 25 μL mixture contained 10 nM mTOR, 1 μg 4E-BP1 (substrate), 1 μCi [γ-³²P]-ATP, and serial Voxtalisib (0.05-500 nM). Incubated at 37℃ for 45 minutes. - Detection: Reaction terminated by 5×SDS loading buffer. Proteins separated by SDS-PAGE, transferred to PVDF membrane. Membrane exposed to autoradiography film; radioactivity quantified via phosphorimager. IC50 calculated via dose-response curve^[1]
[1]

Cells are treated with XL765 24 hours after plating and harvested for apoptosis or autophagy assays at 24, 48, or 72 hours after XL765 treatment. Fluorescence-activated cell sorting (FACS) measures the overall percentage of annexin V-positive cells to determine the level of apoptosis. Acridine orange vital staining is used to identify acidic vesicular organelles (AVOs) in cells that have been exposed to XL765. The amount of AVO formation is measured by the ratio of the increase in acridine orange fluorescence intensity (FL3) in XL765-treated cells to control cells.

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1. Tumor cell proliferation and signaling assay (Literature [1]): - Cell culture: MCF-7/A549 cells maintained in RPMI 1640/DMEM + 10% FBS, seeded in 96-well plates (5×10³ cells/well) overnight. - Treatment: Incubated with Voxtalisib (0.1-10 μM) for 72 hours (viability) or 24 hours (signaling). - Detection: - Viability: MTT (5 mg/mL) added for 4 hours, DMSO dissolved formazan, absorbance 570 nm measured. - Signaling: Cells lysed, Western blot for p-AKT, p-S6, and GAPDH (loading control); band intensity quantified via ImageJ^[1]
2. Melanoma cell apoptosis assay (Literature [2]): - Cell culture: A375/SK-MEL-28/WM1366 cells seeded in 24-well plates (1×10⁵ cells/well) overnight. - Treatment: Incubated with Voxtalisib (0.5-5 μM) for 48 hours. - Detection: Caspase-3/7 activity measured via luminometer (caspase substrate with DEVD peptide); apoptosis confirmed by Annexin V-FITC/PI staining (flow cytometry)^[2]
3. GBM cell migration/invasion assay (Literature [3]): - Migration (scratch assay): U251 cells seeded to confluency, scratch made with pipette tip. Incubated with Voxtalisib (1-5 μM) for 48 hours; scratch closure measured via microscope. - Invasion (Matrigel assay): U251 cells (5×10⁴ cells/well) seeded in Matrigel-coated transwell inserts, incubated with Voxtalisib (1-5 μM) for 24 hours. Invaded cells stained with crystal violet, counted under microscope^[3]
[1][2][3]

Mice: Mice that are athymic and nude are used for in vivo efficacy tests. At 37°C in a humidified 5% CO2 environment, tumor cells are cultured in DMEM enriched with 10% FBS (20% for PC-3 and OVCAR-3 cells), Penicillin-Streptomycin, and non-essential amino acids. In 0.1 mL of ice-cold water, 1 to 5×106 cells are harvested by brief trypsinization on day 0. Hanks Female athymic nude mice have Balanced Salt Solutions implanted subcutaneously (OVCAR-3) or intradermally (MCF7 and U-87 MG) into the hind flank. When tumor cells are implanted in the MCF7 model, an estrogen pellet (IRA) is inserted subcutaneously at the nape of the neck. Male nude mice between the ages of 5 and 8 weeks old have their hind-flanks subcutaneously implanted with a total of 3×106 PC-3 cells. Up until staging and dose initiation, tumor growth is measured every week with calipers. Body and tumor weights are measured throughout the dosing period. Voxtalisib (XL-765) is formulated in sterile water/10 mM HCl or water and given orally via gavage at the prescribed doses and schedules with a dose volume of 10 mL/kg.

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1. MCF-7/A549 xenograft protocols (Literature [1]): - Animals: Female nude/SCID mice (6-8 weeks old), 5 mice/group; acclimated 7 days (12h light/dark, ad libitum food/water). - Tumor induction: 5×10⁶ MCF-7/A549 cells injected subcutaneously (right flank). - Drug preparation: Voxtalisib dissolved in 10% DMSO + 90% PEG400 (sonicated 5 minutes for dissolution). - Administration: Oral gavage (10 μL/g body weight) 25/50 mg/kg/day, starting when tumors reached ~100 mm³ (volume = length×width²/2). - Assessment: Tumor volume measured twice weekly; body weight weekly; mice euthanized at day 21/28, tumor lysed for Western blot/IHC^[1]
2. U87MG GBM xenograft protocol (Literature [3]): - Animals: Female nude mice (6-8 weeks old), 6 mice/group. - Tumor induction: 1×10⁷ U87MG cells injected subcutaneously. - Drug preparation: Voxtalisib dissolved in 0.5% methylcellulose + 0.1% Tween 80 (stirred 2 hours at RT). - Administration: Oral gavage 30 mg/kg/day for 21 days (tumor ~150 mm³ at start). - Assessment: Tumor volume measured 3 times weekly; neurological function tested via rotarod (no deficit observed); tumor IHC for p-AKT/p-S6 at euthanasia^[3]

1. Oral bioavailability: - Rats: Comparison of a single oral dose of 50 mg/kg with an intravenous dose of 10 mg/kg. Oral AUC₀-∞ is approximately 3,200 ng·h/mL, while intravenous AUC₀-∞ is approximately 4,000 ng·h/mL; bioavailability is approximately 80%. - Mice: Comparison of a single oral dose of 50 mg/kg with an intravenous dose of 10 mg/kg. Bioavailability is approximately 75%. 2. Half-life (t₁/₂): - Rats: Approximately 5.2 hours after oral administration and approximately 4.8 hours after intravenous administration. - Mice: Approximately 4.5 hours after oral administration and approximately 4.1 hours after intravenous administration. 3. Distribution: - Volume of distribution (Vd) in rats: Approximately 2.5 L/kg after intravenous administration, indicating good tissue penetration. - MCF-7 xenograft tumor/plasma ratio: approximately 4.0 (50 mg/kg/day orally, day 7). 4. Excretion: - Rats: approximately 60% of the oral dose was excreted in feces within 72 hours (35% of which was the unchanged drug); approximately 20% was excreted in urine (10% of which was the unchanged drug) ^[1]>

1. In vitro toxicity: - All tested cell lines (MCF-7, A549, melanoma, GBM): No nonspecific cytotoxicity was observed at Voxtalisib concentrations up to 10 μM (LDH release <10%); no morphological changes were observed. [1] [2] [3] 2. In vivo toxicity (Reference [1]): - Rats: Oral doses up to 100 mg/kg/day for 28 days: No deaths; body weight maintained above 90% of initial values; serum ALT/AST (liver) and creatinine/BUN (kidney) were within the normal range. - Mice: Oral doses of 50 mg/kg/day for 21 days: No hematological abnormalities (white blood cells, red blood cells, platelets); no histopathological damage to the liver/kidneys. 3. Plasma protein binding rate: - Human plasma: ~97% (ultrafiltration); Rat plasma: ~96%; Mouse plasma: ~95% [1]

[1]. Oncogene . 2008 Sep 18;27(41):5511-26.

[2]. J Mol Med (Berl) . 2011 Sep;89(9):877-89.

[3]. Neuro Oncol . 2011 Apr;13(4):384-92.

2-Amino-8-ethyl-4-methyl-6-(1H-pyrazol-5-yl)-7-pyrido[2,3-d]pyrimidinone is a pyrazolopyridine compound. Voxtalisib has been used in clinical trials for the treatment of various cancers, including melanoma, lymphoma, glioblastoma, and breast cancer. Voxtalisib is a small molecule drug with high oral bioavailability that targets phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) kinase in the PI3K/mTOR signaling pathway, exhibiting potential antitumor activity. Voxtalisib inhibits PI3K and mTOR kinases, leading to apoptosis and growth inhibition in susceptible tumor cells. Activation of the PI3K/mTOR pathway promotes cell growth, survival, and resistance to chemotherapy and radiotherapy; mTOR is a serine/threonine kinase downstream of PI3K and may also be activated in a PI3K-independent manner under conditions of nutrient and energy deprivation. Therefore, this drug may be more effective than drugs that inhibit PI3K kinase or mTOR kinase alone.

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1. Mechanism of action: Voxtalisib is a dual PI3K/mTOR inhibitor that binds to the ATP-binding pockets of PI3K (all class I subtypes) and mTOR (mTORC1/mTORC2). It blocks the PI3K-AKT-mTOR signaling pathway, inhibits tumor cell proliferation, migration and invasion, and induces apoptosis—effective against PI3K/mTOR-activated tumors (e.g., breast cancer, glioblastoma, melanoma) ^[1]
[2][3]
2. Preclinical significance: - Literature [1]: Confirmed that Voxtalisib is an orally effective dual inhibitor with broad efficacy against a variety of solid tumors. [1]
- Literature [2]: Confirmed that Voxtalisib is effective against BRAF/NRAS mutant melanoma, providing a new treatment option for subtypes with limited targeted therapy options. [2] - Reference [3]: Voxtalisib was identified as a candidate drug for glioblastoma (a highly aggressive brain tumor with a poor prognosis). [3]

C13H14N6O

270.29

270.122

C, 57.77; H, 5.22; N, 31.09; O, 5.92

934493-76-2

1349796-36-6;934493-76-2;

16123056

White solid powder

1.4±0.1 g/cm3

1.674

5.95

2

5

2

20

425

0

O=C1N(CC)C2N=C(N)N=C(C=2C=C1C1C=CNN=1)C

RGHYDLZMTYDBDT-UHFFFAOYSA-N

InChI=1S/C13H14N6O/c1-3-19-11-8(7(2)16-13(14)17-11)6-9(12(19)20)10-4-5-15-18-10/h4-6H,3H2,1-2H3,(H,15,18)(H2,14,16,17)

2-amino-8-ethyl-4-methyl-6-(1H-pyrazol-5-yl)pyrido[2,3-d]pyrimidin-7-one

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~54 mg/mL (199.78 mM)
Water: <1 mg/mL (slightly soluble or insoluble)
Ethanol: <1 mg/mL (slightly soluble or insoluble)

Solubility in Formulation 1: ≥ 1 mg/mL (3.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1 mg/mL (3.70 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1 mg/mL (3.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)