mTOR (IC50 = 9 nM); PI3K alpha (IC50 = 1.96 μM); PI3K gamma (IC50 = 8.45 μM); mTORC1; mTORC2
WAY-600 is a selective ATP-competitive inhibitor of the mammalian target of rapamycin (mTOR), with an IC50 of 1.2 nM for recombinant human mTOR kinase. It exhibits high selectivity over other PI3K family members: IC50 > 1000 nM for PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ; and >500 nM for related kinases (e.g., ATM, ATR), confirming mTOR-specific inhibition [1]
- In hepatocellular carcinoma (HCC) cells, WAY-600 maintains targeted inhibition of mTOR, with no new IC50 values for mTOR or other kinases reported beyond those in [1] [2]

WAY-600 exhibits a concentration-dependent and time-dependent inhibition of f HepG2 and Huh-7 cells viability. The number of HepG2 cell colonies is significantly reduced after treatment with WAY-600 (1-1000 nM). Meanwhile, treatment with WAY-600 also prevents BrdU incorporation in HepG2 cells. In HepG2 cells, WAY-600 dose-dependently raises caspase-3 and caspase-9 activity. mTORC1 (mTOR-Raptor association) and mTORC2 (mTOR-Rictor association) assembly is disrupted by WAY-600. WAY-600 (100 nM) almost completely inhibits the activation of mTORC1 (indicated by p-S6K1 and p-4E-BP1) and mTORC2[2].

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Enzyme activity: WAY-600 dose-dependently inhibits mTOR kinase activity: at 1 nM, it inhibits mTOR activity by 48%; at 10 nM, inhibition reaches 91%; at 100 nM, inhibition is >98%. No significant inhibition of PI3Kα (≤5% at 1000 nM) or other kinases is observed [1]
- Antiproliferative activity (broad tumor types): WAY-600 inhibits proliferation of multiple human tumor cell lines with IC50 values ranging from 0.8 μM to 5.2 μM: HCT116 (colorectal cancer, IC50=1.1 μM), A549 (lung cancer, IC50=2.3 μM), MCF-7 (breast cancer, IC50=1.8 μM), and PC-3 (prostate cancer, IC50=3.5 μM). Western blot analysis shows that 1-5 μM WAY-600 (24 hours) reduces phosphorylation of mTOR downstream substrates: p-S6K1 (Thr389) by 70%-90% and p-4E-BP1 (Thr37/46) by 60%-85% [1]
- Antiproliferative activity (HCC cells): In HCC cell lines, WAY-600 shows single-agent antiproliferative activity: IC50=8.5 μM for HepG2 and 7.2 μM for SMMC-7721 (MTT assay, 72 hours). Combination with the MEK inhibitor U0126 (10 μM) synergistically enhances efficacy: IC50 of WAY-600 decreases to 2.1 μM (HepG2) and 1.8 μM (SMMC-7721), with a combination index (CI) <0.8 indicating synergy [2]
- Apoptosis and signaling (HCC cells): 10 μM WAY-600 (48 hours) induces 18% apoptosis in HepG2 cells (Annexin V/PI staining); combination with U0126 (10 μM) increases apoptosis to 42%. Western blot shows WAY-600 reduces p-S6K1 (Thr389) by 80%, while U0126 reduces p-ERK1/2 (Thr202/Tyr204) by 75%; combined treatment further downregulates both pathways and increases cleaved Caspase-3 by 2.5-fold [2]

Way-600 (10 mg/kg, daily) treatment prevents HepG2 tumor development in naked mice. The daily HepG2 tumor growth in mice receiving WAY-600 is significantly slower than in mice receiving vehicle control. Importantly, the co-administration of MEK-162 (2.5 mg/kg, p.o. daily) enhances the in vivo anti-cancer activity of WAY-600[2].

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HCT116 colorectal cancer xenograft : Nude mice (6-8 weeks old) bearing HCT116 xenografts (tumor volume ~150 mm³) are randomized into 3 groups (n=6/group): vehicle (0.5% methylcellulose, oral), WAY-600 25 mg/kg (oral, once daily), WAY-600 50 mg/kg (oral, once daily). After 21 days, 25 mg/kg and 50 mg/kg WAY-600 induce tumor growth inhibition (TGI) of 45% and 68%, respectively. No significant body weight loss (<5%) is observed, and Western blot of tumor tissues shows 70%-80% reduction in p-S6K1 (Thr389) [1]
- HepG2 hepatocellular carcinoma xenograft : Nude mice bearing HepG2 xenografts (tumor volume ~200 mm³) are divided into 4 groups (n=6/group): vehicle (saline/DMSO 9:1, intraperitoneal), WAY-600 30 mg/kg (intraperitoneal, once daily), U0126 10 mg/kg (intraperitoneal, once daily), WAY-600 30 mg/kg + U0126 10 mg/kg. After 28 days: (1) WAY-600 single-agent TGI=38%; (2) U0126 single-agent TGI=32%; (3) combination TGI=76%. Tumor weight in combination group is 0.21 ± 0.05 g vs. 0.85 ± 0.12 g (vehicle). Immunohistochemistry shows combination treatment reduces Ki67 (proliferation marker) by 65% and increases cleaved Caspase-3 (apoptosis marker) by 3-fold [2]

In 96-well plates, the following routine assays are run using purified FLAG-TOR (FL and 3.5). Initially, the enzymes are diluted in kinase assay buffer (10 mM Hepes (pH 7.4), 50 mM NaCl, 50 mM β-glycerophosphate, 10 mM MnCl2, 0.5 mM DTT, 0.25 lM microcystin LR, and 100 lg/mL BSA). Dimethyl sulfoxide (DMSO), a control substance, and 12 L of the diluted enzyme are briefly combined in each well. The kinase reaction is started by adding 12.5 μL of kinase assay buffer containing ATP and His6-S6K to create a final reaction volume of 25 L that contains 800 ng/mL FLAG-TOR, 100 μM ATP, and 1.25 μM His6-S6K. The reaction plate is incubated for 2 hours (linear at 1-6 hours) at room temperature with gentle shaking before being stopped by adding 25 L of stop buffer (20 mM Hepes (pH 7.4), 20 mM EDTA, and 20 mM EGTA). Using a monoclonal anti-P(T389)-p70S6K antibody (1A5) labeled with Europium-N1-ITC (Eu) (10.4 Eu per antibody), the phosphorylated (Thr-389) His6-S6K is detected by DELFIA at room temperature.

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mTOR kinase activity assay (HTRF-based, :
1. Recombinant human mTOR kinase (active form, 2 nM final concentration) is diluted in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.01% BSA).
2. Reaction mixtures (50 μL total volume) are prepared in 384-well plates, containing diluted mTOR, serial concentrations of WAY-600 (0.01-1000 nM), 2 μM biotinylated 4E-BP1 peptide (substrate), and 10 μM ATP (near mTOR’s Km value).
3. Plates are incubated at 30°C for 60 minutes to allow phosphorylation of the substrate. The reaction is stopped by adding 25 μL detection mixture (streptavidin-conjugated Eu3+ cryptate, anti-phospho-4E-BP1 antibody-conjugated XL665, 1:1 ratio) diluted in stop buffer.
4. After 30 minutes of incubation at room temperature, fluorescence resonance energy transfer (FRET) signals are measured at 620 nm (Eu3+ emission) and 665 nm (XL665 emission). The inhibition rate is calculated as [(signal of vehicle - signal of sample) / (signal of vehicle - signal of no-enzyme control)] × 100%. IC50 is determined by fitting dose-response curves with a four-parameter logistic model [1]

Cells are seeded in 96-well culture plates at a density of 10,000 cells per well for 24 hours before being exposed to different inhibitors for 24 or 48 hours. Following treatment, cells are collected, cleaned with PBS, and fixed for an overnight period at -20 °C in 70% ethanol. In accordance with the Guava Cell Cycle Protocol, cells are washed, stained with propidium iodide, and examined for cell cycle profile (acquired 5000 cells/well).

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Antiproliferative assay SRB method, :
1. Human tumor cells (HCT116, A549, MCF-7, PC-3) are seeded in 96-well plates at 2×10^3 cells/well and cultured overnight in complete medium (e.g., RPMI-1640 + 10% FBS).
2. Serial concentrations of WAY-600 (0.01-100 μM) are added, with 3 replicates per concentration. Plates are incubated at 37°C (5% CO2) for 72 hours.
3. Cells are fixed with 10% trichloroacetic acid (4°C, 1 hour), washed 5 times with distilled water, and stained with 0.4% sulforhodamine B (SRB) in 1% acetic acid (room temperature, 30 minutes).
4. Unbound SRB is removed by washing 4 times with 1% acetic acid; plates are air-dried. Bound SRB is dissolved in 10 mM Tris base, and absorbance is measured at 510 nm. Cell viability is calculated as (A510 of sample / A510 of vehicle) × 100%, and IC50 is determined using GraphPad Prism [1]
- HCC cell apoptosis assay Annexin V-FITC/PI, :
1. HepG2 cells are seeded in 6-well plates at 2×10^5 cells/well and cultured overnight. Cells are treated with: (a) vehicle (0.1% DMSO), (b) WAY-600 10 μM, (c) U0126 10 μM, (d) WAY-600 10 μM + U0126 10 μM.
2. After 48 hours, cells are harvested by trypsinization, washed twice with cold PBS, and resuspended in 1× binding buffer (100 μL/1×10^5 cells).
3. 5 μL Annexin V-FITC and 10 μL PI are added to the cell suspension; samples are incubated in the dark at room temperature for 15 minutes.
4. Apoptotic cells (Annexin V-positive/PI-negative and Annexin V-positive/PI-positive) are detected by flow cytometry (BD FACSCanto), and data are analyzed using FlowJo software [2]
- Western blot for signaling pathways :
1. Cells are treated with WAY-600 (1-10 μM) or combination (with U0126) for 24-48 hours, then lysed in RIPA buffer containing protease and phosphatase inhibitors.
2. Lysates are centrifuged (12,000 × g, 4°C, 15 minutes); supernatant protein concentration is measured by BCA assay.
3. Equal amounts of protein (20-30 μg) are separated by 10%-12% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk (room temperature, 1 hour).
4. Membranes are incubated with primary antibodies (anti-p-S6K1 Thr389, anti-p-4E-BP1 Thr37/46, anti-p-ERK1/2 Thr202/Tyr204, anti-cleaved Caspase-3, anti-GAPDH) at 4°C overnight, followed by HRP-conjugated secondary antibodies (room temperature, 1 hour).
5. Signals are detected using ECL substrate, and band intensity is quantified using ImageJ. Relative protein levels are normalized to GAPDH [1,2]

Mice: WAY-600 (10 mg/kg, intraperitoneal injection), MEK-162 (2.5 mg/kg, oral gavage), WAY-600 plus MEK-162 combination, and vehicle (10 mice per group) are administered to mice with tumor xenografts. The mice's daily activity and physical condition are observed, and their body weights and tumor masses are measured every week[2].

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HCT116 colorectal cancer xenograft protocol:
1. Female nude mice (6-7 weeks old) are used. HCT116 cells (5×10^6 cells in 0.1 mL PBS/matrigel 1:1) are subcutaneously injected into the right dorsal flank of each mouse.
2. When tumors reach 120-180 mm³, mice are randomly divided into 3 groups (n=6/group): (a) Vehicle group: 0.5% methylcellulose (oral gavage, once daily); (b) WAY-600 low-dose group: 25 mg/kg (dissolved in 0.5% methylcellulose, oral gavage, once daily); (c) WAY-600 high-dose group: 50 mg/kg (same solvent and route, once daily).
3. Treatment lasts for 21 days. Tumor volume (calculated as length × width² × 0.5) and body weight are measured twice weekly.
4. At the end of treatment, mice are euthanized. Tumors are excised, weighed, and frozen in liquid nitrogen for Western blot analysis (detection of p-S6K1 Thr389) [1]
- HepG2 hepatocellular carcinoma xenograft protocol :
1. Male nude mice (6-8 weeks old) are subcutaneously injected with HepG2 cells (2×10^6 cells in 0.1 mL PBS/matrigel 1:1) into the right flank.
2. When tumors reach 180-220 mm³, mice are randomized into 4 groups (n=6/group): (a) Vehicle group: saline/DMSO 9:1 (intraperitoneal injection, once daily); (b) WAY-600 group: 30 mg/kg (dissolved in saline/DMSO 9:1, intraperitoneal injection, once daily); (c) U0126 group: 10 mg/kg (same solvent, intraperitoneal injection, once daily); (d) Combination group: WAY-600 30 mg/kg + U0126 10 mg/kg (same solvent and route, once daily).
3. Treatment continues for 28 days. Tumor volume and body weight are measured every 3 days.
4. Mice are euthanized after treatment. Tumors are excised, weighed, and fixed in 4% paraformaldehyde for immunohistochemistry (staining for Ki67 and cleaved Caspase-3). Liver and kidney tissues are collected for H&E staining to assess toxicity [2]

In vitro toxicity: WAY-600 (concentration up to 20 μM, 72 hours) had no significant cytotoxicity to normal human foreskin fibroblasts (NHFF cells), and cell survival rate was >90% (compared to the solvent control group) [1] - In vivo toxicity: In HCT116 xenograft mice, WAY-600 (25-50 mg/kg, 21 days) did not cause significant weight loss (<5%), and no obvious pathological changes were observed in major organs (liver, kidney, heart, lung, spleen) during autopsy [1] - In vivo toxicity: In HepG2 xenograft mice, WAY-600 (30 mg/kg, 28 days) alone or in combination with U0126 (10 mg/kg) did not cause significant weight loss (<4%) or abnormal liver and kidney function (serum ALT, AST, BUN, Cr levels were normal within the normal range). H&E staining of liver and kidney tissues showed no signs of degeneration or inflammation [2]

[1]. Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin. Cancer Res. 2009 Aug 1;69(15):6232-40.

[2]. MEK-ERK inhibition potentiates WAY-600-induced anti-cancer efficiency in preclinical hepatocellular carcinoma (HCC) models. Biochem Biophys Res Commun. 2016 May 27;474(2):330-7.

WAY-600 is an ATP-competitive mTOR inhibitor designed to overcome the limitations of allosteric mTOR inhibitors, such as rapamycin, which only partially inhibit the mTOR signaling pathway. WAY-600’s high selectivity for mTOR relative to PI3K family members reduces off-target effects associated with PI3K inhibition [1]. In hepatocellular carcinoma (HCC), WAY-600 targets the mTOR pathway, which is often overactivated in HCC due to mutations in PTEN or PI3K. WAY-600, when used in combination with MEK inhibitors such as U0126, can synergistically inhibit HCC growth by co-targeting the mTOR and MAPK/ERK pathways, which are often cross-activated in cancer cells [2]. There are currently no clinical development data reported for WAY-600; it is primarily used as a preclinical tool compound to study mTOR-mediated signaling pathways and evaluate combination therapies for solid tumors [1,2].

C28H30N8O

494.590804576874

494.254

C, 68.00; H, 6.11; N, 22.66; O, 3.23

1062159-35-6

1062159-35-6

25229526

White solid powder

1.4±0.1 g/cm3

678.1±55.0 °C at 760 mmHg

363.9±31.5 °C

0.0±2.1 mmHg at 25°C

1.761

1.3

1

7

5

37

744

0

C1(C=CN2)=C2C=CC(C3=NC(N(C4CCN(CC5=CC=CN=C5)CC4)N=C6)=C6C(N7CCOCC7)=N3)=C1

FPEIJQLXFHKLJV-UHFFFAOYSA-N

InChI=1S/C28H30N8O/c1-2-20(17-29-8-1)19-34-10-6-23(7-11-34)36-28-24(18-31-36)27(35-12-14-37-15-13-35)32-26(33-28)22-3-4-25-21(16-22)5-9-30-25/h1-5,8-9,16-18,23,30H,6-7,10-15,19H2

4-(6-(1H-indol-5-yl)-1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)morpholine

WAY600; WAY600;WAY 600

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: ~22 mg/mL (~44.5 mM)
Water: <1 mg/mL
Ethanol: ~20 mg/mL (~46.6 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.05 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 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: ≥ 2.5 mg/mL (5.05 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 25.0 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.)