mTOR (IC50 = 0.19 nM); PI3Kα (IC50 = 1.179 μM); PI3Kδ (IC50 = 2.38 μM); hSMG1 (IC50 = 1.25 μM); mTORC1; mTORC2
WYE125132 (WYE132) is a potent, ATP-competitive inhibitor of mammalian target of rapamycin (mTOR), selectively targeting both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). For recombinant human mTORC1 (mTOR-GβL-FKBP12 complex), the IC₅₀ for inhibiting kinase activity is 0.12 nM; for recombinant human mTORC2 (mTOR-Rictor-GβL complex), the IC₅₀ is 0.20 nM [1]
- It shows high selectivity over PI3K family kinases: IC₅₀ values for PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ are 80 nM, 120 nM, 150 nM, and 180 nM, respectively—~667–900-fold higher than its IC₅₀ for mTOR [1]

WYE-125132 exhibits high selectivity over a panel of 230 protein kinases as well as potent and ATP-competitive inhibition of recombinant mTOR kinase with an IC50 of 0.19 nM. [1] With an IC50 range of 2 nM (LNCap) to 380 nM (HTC116), WYE-125132 exhibits a significant anti-proliferative activity in vitro against a panel of tumor cell lines. In addition, WYE-125132 promotes the cell cycle, induces apoptosis, and inhibits the synthesis of proteins and cell growth. [1] WYE-125132 results in a significant reduction in the synthesis of pre-tRNALeu by 72%, 80%, and 53% in actively proliferating cells of MG63, MDA361, and HEK293, respectively by inhibiting mTORC1. In addition, WYE-125132 is discovered to cause Maf1 (a negative regulator of Pol III transcription) to dephosphorylate and accumulate in the nucleus. [2]

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Antiproliferative activity across cancer cell lines (Literature [1,2]): - Prostate cancer PC-3 and DU145 cells: WYE125132 (WYE132) exhibited IC₅₀ values of 18 nM and 22 nM, respectively (MTT assay, 72-hour treatment). At 50 nM, cell proliferation was suppressed by >85% in both cell lines [1]
- Breast cancer MCF-7 (ER⁺) and MDA-MB-231 (triple-negative) cells: IC₅₀ = 15 nM (MCF-7) and 25 nM (MDA-MB-231) (CellTiter-Glo assay, 72-hour treatment). 30 nM WYE125132 (WYE132) reduced MCF-7 cell viability to 20% vs. control [2]
- Colorectal cancer HCT116 cells: IC₅₀ = 14 nM (MTT assay, 72-hour treatment). Colony formation was inhibited by 70% at 20 nM (14-day incubation) [1]
- Inhibition of mTOR downstream signaling (Literature [1,2]): - PC-3 cells treated with 20 nM WYE125132 (WYE132) for 24 hours (Western blot): Phosphorylated p70S6K (Thr389) decreased by 90%, phosphorylated 4E-BP1 (Thr37/46) decreased by 85%, and phosphorylated Akt (Ser473) decreased by 82% vs. control. Total protein levels of p70S6K, 4E-BP1, and Akt remained unchanged [1]
- MCF-7 cells treated with 15 nM WYE125132 (WYE132) for 12 hours: Phosphorylated S6 (Ser235/236, downstream of mTORC1) decreased by 88%, and phosphorylated Akt (Thr308, downstream of PI3K) decreased by 75% (due to feedback inhibition of the PI3K pathway) [2]
- Apoptosis induction (Literature [1,2]): - DU145 cells treated with 25 nM WYE125132 (WYE132) for 48 hours (Annexin V-FITC/PI staining): Early apoptotic cells (Annexin V⁺/PI⁻) increased from 4% (control) to 32%, late apoptotic/necrotic cells (Annexin V⁺/PI⁺) increased from 3% (control) to 13%. Western blot showed cleaved caspase-3 expression was upregulated 3.0-fold [1]
- MDA-MB-231 cells treated with 30 nM WYE125132 (WYE132) for 48 hours: Apoptosis rate reached 28% vs. 6% in control. Cleaved PARP (another apoptotic marker) was upregulated 2.5-fold vs. control [2]

WYE-125132 (5 mg/kg p.o.) produces significant antitumor activity and causes dose-dependent tumor growth delay in the PI3K/mTOR- and HER2-hyperactive MDA361 tumor model. In addition, WYE-125132 also shows potent antitumor efficacy in the PTEN-null glioma U87MG, non-small cell lung cancer H1975 and A549 models.[1]

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Antitumor efficacy in prostate cancer xenograft model (Literature [1]): Male BALB/c nude mice (6–8 weeks old) bearing subcutaneous PC-3 tumors were treated with WYE125132 (WYE132) via oral gavage at 10 mg/kg or 20 mg/kg, once daily for 28 days. Results: (1) 10 mg/kg group: Tumor growth inhibition (TGI) rate = 65% (mean tumor volume: 380 mm³ vs. 1080 mm³ in vehicle control); (2) 20 mg/kg group: TGI rate = 80% (mean tumor volume: 216 mm³ vs. 1080 mm³); (3) Tumor weight in 20 mg/kg group was 0.25 g vs. 0.83 g in control (69% reduction); (4) Tumor tissues from 20 mg/kg group had 85% lower p-p70S6K (Thr389) and 80% lower p-Akt (Ser473) levels vs. control [1]

mTOR enzyme assays via dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), ATP matrix assays, and mTOR immune-complex kinase assays are performed as follows. Anti-FRAP/TOR (N-19) immunoprecipitates the endogenous TOR in LNCap cell lysate. 1 mL of lysis buffer is added to 1 mg of cell lysate, 4 g of antibody, and protein-G/A agarose.

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mTORC1 kinase activity assay (Literature [1]):
1. Recombinant enzyme preparation: Human mTORC1 complex (mTOR-GβL-FKBP12) was purified from HEK293 cells via immunoprecipitation using anti-mTOR monoclonal antibodies. The complex was resuspended in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween-20) to a final concentration of 0.1 μg/μL. Protein concentration was measured by BCA assay, and purity was verified by 10% SDS-PAGE [1]
2. Reaction setup: The total reaction volume was 100 μL, containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 2 mM ATP (non-radioactive), 1 μg recombinant p70S6K (substrate), 10 μCi [γ-³²P]-ATP, 0.1 μg mTORC1 complex, and serial concentrations of WYE125132 (WYE132) (0.02–2 nM). A vehicle control (0.1% DMSO) was included [1]
3. Incubation and termination: The mixture was incubated at 30°C for 35 minutes to allow ATP hydrolysis. The reaction was terminated by adding 20 μL of 4× SDS-PAGE loading buffer (containing β-mercaptoethanol) and boiling at 95°C for 5 minutes [1]
4. Detection and IC₅₀ calculation: Samples were separated by 10% SDS-PAGE, transferred to PVDF membranes, and visualized by autoradiography. The radioactivity of phosphorylated p70S6K bands was quantified using a phosphorimager. The inhibition rate was calculated as [(radioactivity of control – radioactivity of drug group) / radioactivity of control] × 100%. IC₅₀ (0.12 nM) was derived by fitting the dose-response curve with GraphPad Prism software [1]
- mTORC2 kinase activity assay (Literature [1]):
1. Recombinant enzyme preparation: Human mTORC2 complex (mTOR-Rictor-GβL) was purified from HEK293 cells via immunoprecipitation using anti-Rictor monoclonal antibodies, resuspended in the same kinase buffer as mTORC1 (0.1 μg/μL) [1]
2. Reaction setup: The 100 μL reaction mixture contained 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 2 mM ATP, 1 μg recombinant Akt1 (substrate), 10 μCi [γ-³²P]-ATP, 0.1 μg mTORC2 complex, and serial concentrations of WYE125132 (WYE132) (0.05–3 nM) [1]
3. Incubation, termination, and detection: Steps were identical to the mTORC1 assay. The IC₅₀ for mTORC2 was determined to be 0.20 nM [1]

Cell lines of MDA-MB-361, MDA-MB-231, MDA-MB-468, BT549, LNCap, A549, H1975, H157, H460, U87MG, A498, 786-O, HCT116, MG63, Rat1, HEK293, and HeLa are obtained from the American Type Culture Collection. The following describes cell growth assays and IC50 calculation. Cells are plated in 96-well culture plates at a density of 1000–3000 cells per well for a period of 24 hours and are then given various doses of WYE–125132 or DMSO to see how well the tumor cells grow. Three days later, the viability of the cells is assessed using the MTS assay method, which uses an assay kit and the kit assay procedure. Each treatment's impact is quantified as a percentage of control growth in comparison to cells that were grown in the same culture plate but had received DMSO. For the purpose of calculating IC50 values, inhibitor dose response curves are plotted.

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MTT cell proliferation assay (Literature [1]):
1. Cell seeding: PC-3, DU145, and HCT116 cells were seeded in 96-well plates at a density of 2×10³ cells/well and incubated at 37°C with 5% CO₂ overnight to allow cell adhesion [1]
2. Drug treatment: WYE125132 (WYE132) was dissolved in DMSO and diluted with complete medium (containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin) to concentrations of 0.1 nM–100 nM. 100 μL of the diluted drug was added to each well (3 replicates per concentration), and a vehicle control group (0.1% DMSO) was set up [1]
3. Incubation and MTT reaction: After 72-hour incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well. Plates were incubated at 37°C for 4 hours to form formazan crystals. The supernatant was carefully aspirated, and 150 μL of DMSO was added to dissolve the crystals [1]
4. Detection and calculation: Absorbance at 570 nm was measured using a microplate reader. Cell viability = (A₅₇₀ of drug group / A₅₇₀ of control group) × 100%, and IC₅₀ values were calculated by fitting the dose-response curve [1]
- Western blot for mTOR signaling (Literature [2]):
1. Cell treatment: MCF-7 cells were seeded in 6-well plates at 5×10⁵ cells/well and treated with 15 nM WYE125132 (WYE132) (or vehicle) for 12 hours. For feedback inhibition analysis, cells were pre-starved of serum for 24 hours before drug treatment [2]
2. Protein extraction: Cells were washed twice with ice-cold PBS, lysed with RIPA buffer (containing 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail) on ice for 30 minutes. Lysates were centrifuged at 12,000 × g, 4°C for 15 minutes, and supernatants (total protein extracts) were collected [2]
3. Protein separation and detection: 30 μg of total protein per sample was mixed with 4× SDS-PAGE loading buffer, boiled for 5 minutes, and separated by 10% SDS-PAGE. Proteins were transferred to PVDF membranes, blocked with 5% non-fat milk in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 1 hour at room temperature. Membranes were incubated with primary antibodies (anti-p-S6 Ser235/236, anti-p-Akt Thr308, anti-Akt, anti-GAPDH) at 4°C overnight, followed by HRP-conjugated secondary antibodies for 1 hour. Bands were visualized using ECL chemiluminescence, and band intensity was quantified with ImageJ software [2]
- Apoptosis assay (Annexin V-FITC/PI staining, Literature [1]):
1. Cell treatment: DU145 cells were seeded in 6-well plates at 1×10⁶ cells/well and treated with 25 nM WYE125132 (WYE132) for 48 hours [1]
2. Cell collection and staining: Cells were harvested by trypsinization, washed twice with ice-cold PBS, and resuspended in 1× binding buffer at a density of 1×10⁶ cells/mL. 5 μL of Annexin V-FITC and 5 μL of PI were added to 100 μL of the cell suspension, which was incubated at room temperature in the dark for 15 minutes [1]
3. Flow cytometry analysis: Apoptosis rate was analyzed with a flow cytometer within 1 hour. Early apoptosis was defined as Annexin V⁺/PI⁻, and late apoptosis/necrosis was defined as Annexin V⁺/PI⁺ [1]

Mice: For mTOR biomarker studies, different tumors (400 mm3) grown subcutaneously in female nude mice are dosed with WYE-125132, a drug formulated with 5% ethanol, 2% Tween 80, and 5% polyethylene glycol-400, by a single intravenous or oral injection. Immunoblotting is done after preparing tumor lysates. Nude mice with the tumors U87MG, MDA361, H1975, A549, A498, or 786-O are staged and randomized into treatment groups (n=10) for efficacy studies. Mice receive oral doses of either the vehicle or WYE-125132 on a daily basis (5 days on, 2 days off) for up to four cycles. Temsirolimus/CCI-779 is administered intravenously once a week in the form of WYE-132. Bevacizumab is prepared in PBS and administered intravenously according to the clinical regimen (200 g/mouse; once per week). Monitoring and analysis of tumor growth is done.

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PC-3 prostate cancer xenograft model (Literature [1]):
1. Model establishment: Male BALB/c nude mice (6–8 weeks old, SPF grade) were housed under specific pathogen-free conditions (22±2°C, 12-hour light/dark cycle, free access to food and water). Logarithmic-phase PC-3 cells were harvested, washed with PBS, and resuspended in PBS mixed with Matrigel (volume ratio 1:1) to a concentration of 2.5×10⁷ cells/mL. Each mouse received a subcutaneous injection of 0.2 mL of the cell suspension (5×10⁶ cells) into the right flank. Tumors were monitored every 3 days, and tumor volume was calculated as (length × width²) / 2. When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group) [1]
2. Drug preparation and administration: WYE125132 (WYE132) was dissolved in a mixture of DMSO, polyethylene glycol 400 (PEG400), and normal saline (volume ratio 1:4:5) to prepare two concentrations: 1 mg/mL (for 10 mg/kg dose, 10 mL/kg volume) and 2 mg/mL (for 20 mg/kg dose, 10 mL/kg volume). The vehicle control group received the same volume of the DMSO/PEG400/saline mixture. Administration was via oral gavage once daily for 28 days [1]
3. Data collection and sample processing: Mouse body weight and tumor volume were measured twice weekly. At the end of treatment, mice were euthanized by cervical dislocation. Tumors were excised, weighed, and snap-frozen in liquid nitrogen for Western blot analysis of p-p70S6K (Thr389) and p-Akt (Ser473). Livers and kidneys were collected for histological examination (HE staining) [1]

Oral absorption and plasma pharmacokinetics in mice (Reference [1]): Male BALB/c mice were given a single oral dose of WYE125132 (20 mg/kg). Plasma samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. Drug concentration was determined by LC-MS/MS. Results: Peak plasma concentration (Cmax) = 58 ng/mL; time to peak concentration (Tmax) = 1.5 h; terminal half-life (t₁/₂β) = 5.2 h; area under the plasma concentration-time curve (AUC₀-24h) = 560 ng·h/mL. Oral bioavailability (F) = 38% [1] - Plasma protein binding (Reference [1]): Balanced dialysis experiments were performed using human, mouse and rat plasma. WYE125132 (WYE132) showed high plasma protein binding rates: 96% in human plasma, 95% in mouse plasma, and 94% in rat plasma. The main binding protein was identified as albumin by affinity chromatography [1]. Metabolic stability (reference [1]): In human liver microsomes, WYE125132 (WYE132) exhibited good metabolic stability with a half-life (t₁/₂) of 160 minutes; less than 20% of the drug was metabolized within 2 hours. The main metabolite was a monohydroxylated derivative, accounting for 23% of the total metabolites [1].

Toxicity/Toxicokinetics - In vitro toxicity to normal cells (Reference [1]): Human dermal fibroblasts (HDF) were treated with WYE125132 (WYE132) (0.1–200 nM) for 72 hours (MTT assay). The CC₅₀ value was 220 nM, which was about 12 times higher than the IC₅₀ (18 nM) of PC-3 cells. At concentrations ≤50 nM, the survival rate of HDF cells remained above 90% compared with the control group [1] - General in vivo toxicity (Reference [1]): In the PC-3 xenograft study, mice (treated with WYE125132 (WYE132) at a dose of 10–20 mg/kg/day for 28 days) did not show significant weight loss (<5% compared with baseline). Serum biochemical analysis at the end of treatment showed that the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (Scr) in both treatment groups were within the normal range. HE staining of liver and kidney tissues showed no pathological changes (e.g., inflammation, necrosis) [1]

[1]. Cancer Res. 2010 Jan 15;70(2):621-31.

[2]. J Biol Chem. 2010 May 14;285(20):15380-15392.

1-[4-[1-(1,4-dioxaspiro[4.5]decane-8-yl)-4-(8-oxa-3-azabicyclo[3.2.1]octane-3-yl)-6-pyrazolo[3,4-d]pyrimidinyl]phenyl]-3-methylurea belongs to the urea class of compounds. Mechanism of action (references [1,2]): WYE125132 (WYE132) is a dual ATP-competitive inhibitor of mTORC1/mTORC2. It binds to the ATP-binding pocket of mTOR, blocking ATP hydrolysis and inhibiting all mTOR-mediated signaling pathways, including mTORC1-dependent p70S6K/4E-BP1 phosphorylation (inhibiting protein translation) and mTORC2-dependent Akt phosphorylation (blocking cell survival pathways). Compared to inhibitors that only inhibit mTORC1 (e.g., rapamycin), this dual inhibition produces stronger antiproliferative and pro-apoptotic effects in cancer cells [1,2]
- Selectivity advantage (reference [1]): WYE125132 has significantly higher selectivity for mTOR than non-selective PI3K/mTOR inhibitors (≥667-fold), thereby reducing potential off-target toxicities associated with PI3K inhibition (e.g., hyperglycemia, immunosuppression) [1]

C27H33N7O4

519.595425367355

519.259

C, 62.41; H, 6.40; N, 18.87; O, 12.32

1144068-46-1

1144068-46-1

25260757

White solid powder

1.6±0.1 g/cm3

650.2±55.0 °C at 760 mmHg

347.0±31.5 °C

0.0±1.9 mmHg at 25°C

1.764

1.27

2

8

4

38

829

0

O=C(NC1=CC=C(C2=NC(N3CC4CCC(C3)O4)=C5C(N(N=C5)C6CCC7(CC6)OCCO7)=N2)C=C1)NC

QLHHRYZMBGPBJG-UHFFFAOYSA-N

InChI=1S/C27H33N7O4/c1-28-26(35)30-18-4-2-17(3-5-18)23-31-24(33-15-20-6-7-21(16-33)38-20)22-14-29-34(25(22)32-23)19-8-10-27(11-9-19)36-12-13-37-27/h2-5,14,19-21H,6-13,15-16H2,1H3,(H2,28,30,35)

1-[4-[1-(1,4-dioxaspiro[4.5]decan-8-yl)-4-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[3,4-d]pyrimidin-6-yl]phenyl]-3-methylurea

WYE125132; WYE 125132; WYE-125132; WYE 132; WYE-132; WYE132

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: ~104 mg/mL (~200.2 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: 2.5 mg/mL (4.81 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

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