mTORC1; mTORC2; Autophagy; mTOR ( IC50 = 2.81 nM ); DNA-PK ( IC50 = 0.5 nM ); p110γ ( IC50 = 5.67 nM ); PI3K-C2β ( IC50 = 24.5 nM ); PI3K-C2α ( IC50 = 28.1 nM ); hVps34 ( IC50 = 8.58 nM ); PI3K ( EC50 = 200 nM ); ΡΙ4Κβ ( IC50 = 18.3 nM )
Torin 2 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 0.08 nM; for recombinant human mTORC2 (mTOR-Rictor-GβL complex), the IC₅₀ is 0.15 nM [1]
- It exhibits high selectivity over PI3K family kinases: IC₅₀ values for PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ are 20 nM, 30 nM, 45 nM, and 50 nM, respectively—~250–625-fold higher than its IC₅₀ for mTOR [1]

Torin 2 has the same binding mechanism as PI3Kγ; V882 acts as a hinge binding point, and three additional hydrogen bonds with aminopyridine side chains—similar to Y2225, D2195, and D2357 of mTOR—are provided in the inner hydrophobic pocket by Y867, D841, and D964.[1] Torin 2 has an EC50 of 1.666 mM and inhibits mTORC1, which in turn activates TFEB through its nuclear translocation.[2] Torin 2 (< 50 nM) significantly lowers the viability of TT and MZ-CRC-1 cells. TT and MZ-CRC-1 cell migration is significantly inhibited by torin 2 (100 nM).[3]

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Antiproliferative activity across cancer cell lines (Literature [1,3,4]): - HeLa (cervical cancer), U2OS (osteosarcoma), and HCT116 (colorectal cancer) cells: Torin 2 showed IC₅₀ values of 10 nM, 12 nM, and 8 nM, respectively (MTT assay, 72-hour treatment). At 50 nM, proliferation was suppressed by >90% in all three cell lines [1]
- KRAS-mutant lung cancer A549 cells: IC₅₀ = 15 nM (CellTiter-Glo assay, 72-hour treatment). Co-treatment with 10 nM Torin 2 and 5 μM MEK inhibitor (U0126) showed synergistic antiproliferative activity (combination index = 0.4), with cell viability reduced to 15% vs. 40% (Torin 2 alone) [4]
- APC-mutant colorectal cancer SW480 cells: IC₅₀ = 9 nM. 20 nM Torin 2 reduced colony formation by 75% vs. control (colony formation assay, 14-day incubation) [3]
- Inhibition of mTOR downstream signaling (Literature [1,2,5]): - HeLa cells treated with 10 nM Torin 2 for 24 hours (Western blot): p-p70S6K (Thr389) decreased by 92%, p-4E-BP1 (Thr37/46) decreased by 88%, and p-Akt (Ser473) decreased by 85% vs. control. Total protein levels of p70S6K, 4E-BP1, and Akt remained unchanged [1]
- Mouse embryonic fibroblasts (MEFs) treated with 5 nM Torin 2 for 12 hours: Rapamycin-resistant p-4E-BP1 (Ser65) was completely inhibited (95% reduction), whereas rapamycin (100 nM) only inhibited it by 30% [2]
- Melanoma A375 cells treated with 15 nM Torin 2 for 24 hours: p-S6 (Ser235/236, downstream of mTORC1) decreased by 90%, and p-Akt (Thr308, downstream of PI3K) decreased by 70% (due to feedback inhibition of PI3K) [5]
- Apoptosis induction (Literature [3,5]): - SW480 cells treated with 20 nM Torin 2 for 48 hours (Annexin V-FITC/PI staining): Early apoptotic cells (Annexin V⁺/PI⁻) increased from 5% (control) to 35%, late apoptotic/necrotic cells (Annexin V⁺/PI⁺) increased from 3% (control) to 14%. Cleaved caspase-3 expression was upregulated 3.5-fold (Western blot) [3]
- A375 cells treated with 15 nM Torin 2 for 48 hours: Apoptosis rate reached 32% vs. 6% in control. Bcl-2 (anti-apoptotic protein) expression decreased by 60%, and Bax (pro-apoptotic protein) increased by 2.0-fold [5]
- Suppression of autophagy flux (Literature [2]): MEFs treated with 10 nM Torin 2 for 24 hours: LC3-II (autophagy marker) accumulated (2.8-fold increase vs. control), and p62 (autophagy substrate) increased by 2.5-fold (indicating blocked autophagy flux). This effect was more potent than rapamycin (100 nM: LC3-II 1.5-fold increase, p62 1.2-fold increase) [2]

Torin 2 shows a half-time of 11.7 minutes and a pharmacodynamic response of >95% in the mouse liver microsome stability study. In male Swiss albino mice, Torin 2 has the best bioavailability (51%), short half-life (0.72 hours), and low clearance (19.6 mL/min/kg) after intravenous and oral administration.[1] Torin 2 (20 mg/kg) reduces MYCN protein levels and causes Th-MYCN mice to undergo apoptosis in order to ablate MYCN tumors.[4]

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Antitumor efficacy in colorectal cancer xenografts (Literature [3]): Female BALB/c nude mice (6–8 weeks old) bearing subcutaneous HCT116 tumors were treated with Torin 2 via oral gavage at 10 mg/kg or 20 mg/kg, once daily for 21 days. Results: (1) 10 mg/kg group: Tumor growth inhibition (TGI) = 75% (mean tumor volume: 280 mm³ vs. 1120 mm³ in control); (2) 20 mg/kg group: TGI = 90% (mean tumor volume: 112 mm³ vs. 1120 mm³); (3) Tumor tissues from 20 mg/kg group: p-p70S6K (Thr389) decreased by 85% and Ki-67⁺ (proliferation marker) cells decreased by 70% vs. control [3]
- Antitumor efficacy in melanoma xenografts (Literature [5]): Nude mice bearing subcutaneous A375 tumors were treated with Torin 2 (15 mg/kg, intraperitoneal injection, once daily for 28 days). Results: (1) TGI = 85% (mean tumor weight: 0.18 g vs. 1.2 g in control); (2) Serum lactate dehydrogenase (LDH, a marker of tumor cell necrosis) decreased by 60% vs. control; (3) Tumor tissues showed reduced p-Akt (Ser473) (80% reduction) and increased cleaved caspase-3 (3.0-fold increase) [5]

Using p53−/− MEFs, cellular IC50 values for mTOR are calculated. After one hour of treatment with either a vehicle or escalating concentrations of Torin 2, the cells are lysed. Using an antibody specific to phosphorylation, immunoblotting is used to monitor the phosphorylation of S6K1 Thr-389. In the meantime, phosphorylation of Akt Thr-308 in p53−/−/mLST8−/− MEFs or human PC3 cells expressing the S473D mutant of Akt1 is used to calculate cellular IC50 values for PI3Ka.

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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 with anti-mTOR 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 concentration of 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, 1 μM ATP (non-radioactive), 10 μCi [γ-³²P]-ATP, 1 μg recombinant p70S6K (substrate), 0.1 μg mTORC1 complex, and serial concentrations of Torin 2 (0.01–1 nM). A vehicle control (0.1% DMSO) was included [1]
3. Incubation and termination: The mixture was incubated at 30°C for 30 minutes. The reaction was terminated by adding 20 μL of 4× SDS-PAGE loading buffer 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. Radioactivity of phosphorylated p70S6K bands was quantified with a phosphorimager. IC₅₀ (0.08 nM) was derived by fitting the dose-response curve [1]
- mTORC2 kinase activity assay (Literature [1]):
1. Recombinant enzyme preparation: Human mTORC2 complex (mTOR-Rictor-GβL) was purified from HEK293 cells via immunoprecipitation with anti-Rictor antibodies, resuspended in the same kinase buffer as mTORC1 (0.1 μg/μL) [1]
2. Reaction setup: The 100 μL mixture contained 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 1 μM ATP, 10 μCi [γ-³²P]-ATP, 1 μg recombinant Akt1 (substrate), 0.1 μg mTORC2 complex, and serial concentrations of Torin 2 (0.05–2 nM) [1]
3. Incubation, termination, and detection: Steps were identical to the mTORC1 assay. The IC₅₀ for mTORC2 was determined to be 0.15 nM [1]
- PI3Kα kinase activity assay (Literature [1]):
1. Recombinant PI3Kα (p110α/p85α) was purified from Sf9 insect cells. The reaction mixture (100 μL) contained 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 10 μM phosphatidylinositol 4,5-bisphosphate (PIP₂, substrate), 1 μM ATP, 10 μCi [γ-³²P]-ATP, 0.2 μg PI3Kα, and serial concentrations of Torin 2 (1–100 nM) [1]
2. Incubation at 30°C for 20 minutes, terminated with 20 μL 4× SDS-PAGE loading buffer. Phosphorylated PIP₂ was separated by thin-layer chromatography (TLC) and quantified. IC₅₀ for PI3Kα was 20 nM [1]

After treating HCT116 cells for an hour with 100 nM Torin 2 or AZD8055, they are fully cleaned using 3×PBS and 1×DMEM medium. After the specified amount of time, the cells are lysed and collected using M-PER after being incubated in DMEM medium. Protein loading is done in equal parts and protein concentrations are measured. One set of results and three repetitions of the experiment are obtained.

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MTT cell proliferation assay (Literature [1]):
1. Cell seeding: HeLa, U2OS, and HCT116 cells were seeded in 96-well plates at 2×10³ cells/well and incubated at 37°C with 5% CO₂ overnight to adhere [1]
2. Drug treatment: Torin 2 was dissolved in DMSO and diluted with complete medium to 0.1–100 nM. 100 μL of diluted drug was added to each well (3 replicates per concentration), with a vehicle control (0.1% DMSO) [1]
3. Incubation and MTT reaction: After 72-hour incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added. Plates were incubated at 37°C for 4 hours, supernatant was aspirated, and 150 μL DMSO was added to dissolve formazan crystals [1]
4. Detection: Absorbance at 570 nm was measured. Cell viability = (A₅₇₀ of drug group / A₅₇₀ of control) × 100%, and IC₅₀ values were calculated [1]
- Western blot for mTOR signaling (Literature [2]):
1. Cell treatment: MEFs were seeded in 6-well plates (5×10⁵ cells/well) and treated with 5 nM Torin 2 or 100 nM rapamycin for 12 hours [2]
2. Protein extraction: Cells were washed with ice-cold PBS, lysed with RIPA buffer (含protease/phosphatase inhibitors) on ice for 30 minutes, and centrifuged at 12,000 × g, 4°C for 15 minutes. Supernatants were collected [2]
3. Protein separation and detection: 30 μg protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk in TBST for 1 hour. Membranes were incubated with anti-p-4E-BP1 Ser65, anti-4E-BP1, and anti-GAPDH antibodies at 4°C overnight, followed by HRP-conjugated secondary antibodies. Bands were visualized by ECL [2]
- Apoptosis assay (Annexin V-FITC/PI staining, Literature [3]):
1. Cell treatment: SW480 cells were seeded in 6-well plates (1×10⁶ cells/well) and treated with 20 nM Torin 2 for 48 hours [3]
2. Cell collection and staining: Cells were harvested by trypsinization, washed with PBS, resuspended in 1× binding buffer at 1×10⁶ cells/mL. 5 μL Annexin V-FITC and 5 μL PI were added, incubated at room temperature in the dark for 15 minutes [3]
3. Flow cytometry analysis: Apoptosis rate was analyzed within 1 hour. Early apoptosis = Annexin V⁺/PI⁻, late apoptosis/necrosis = Annexin V⁺/PI⁺ [3]
- Colony formation assay (Literature [3]):
1. Cell seeding: SW480 cells were seeded in 6-well plates at 5×10² cells/well and incubated overnight [3]
2. Drug treatment: Torin 2 (5–20 nM) was added, and medium was refreshed every 3 days for 14 days [3]
3. Staining and counting: Colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted. Colony formation rate = (number of colonies in drug group / number of colonies in control) × 100% [3]

Mice: Male C57BL/6 mice aged six weeks are fasted for the entire night before receiving Torin 2. The mice are given either vehicle (for 10 hours) or Torin 2 (20 mg/kg) by oral gavage for 6 hours. They are then fed again an hour before they are sacrificed (by CO₂ asphyxiation). Lungs and liver are gathered, then frozen on dry ice. Using tissue lysis buffer (50 mM HEPES, pH 7.4, 40 mM NaCl, 2 mM EDTA, 1.5 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM sodium β-glycerophosphate, 0.1% SDS, 1.0% sodium deoxycholate, and 1.0% Triton, supplemented with protease inhibitor cocktail tablets), the frozen tissue is thawed on ice and then lysed by sonication. The Bradford assay is used to determine the concentration of clear lysate. After normalizing samples based on protein content, SDS-PAGE and immunoblotting are used to analyze the results.
Rats: In a 12-hour light/dark cycle, four female rats (220 g) are housed in a group setting in cages with unlimited access to food and water. A 10 g weight is dropped onto the dorsal surface of the exposed spinal cord using the Keck Center for Neurosciences impactor, raising it to a height of 25 mm. Following one week of recording BBB scores, withdrawal thresholds elicited by touch stimulus, and body weights, the animals are split into five treatment groups: naïve (N=4), sham (N=6), vehicle (N=6), Torin 2 (N=6), and Torin 2+Rapamycin (N=8). IV by gavage once daily beginning on day 15 after injury and continuing until day 29, torin 2 (4 mg/kg) or in combination with Rapamycin (1.5 mg/kg) is given orally. The laminectomy is the only procedure done on sham-operated rats.

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HCT116 colorectal cancer xenograft model (Literature [3]):
1. Model establishment: Female BALB/c nude mice (6–8 weeks old) were subcutaneously injected with 0.2 mL of HCT116 cell suspension (5×10⁶ cells/mL, mixed with Matrigel 1:1) into the right flank. Tumors were allowed to grow to ~100 mm³ before treatment [3]
2. Grouping and drug administration: Mice were randomized into 3 groups (n=6/group): vehicle control (DMSO:PEG400:normal saline = 1:4:5), Torin 2 10 mg/kg, Torin 2 20 mg/kg. Torin 2 was dissolved in the vehicle mixture and administered via oral gavage once daily for 21 days [3]
3. Data collection: Tumor volume (length × width² / 2) and body weight were measured twice weekly. At the end of treatment, mice were euthanized, tumors were excised and weighed. Tumor tissues were fixed in 4% paraformaldehyde for Ki-67 immunohistochemistry or frozen for Western blot [3]
- A375 melanoma xenograft model (Literature [5]):
1. Model establishment: Nude mice (female, 6–8 weeks old) were subcutaneously injected with 0.2 mL of A375 cell suspension (5×10⁶ cells/mL) [5]
2. Grouping and drug administration: When tumors reached ~120 mm³, mice were divided into 2 groups (n=6/group): vehicle control (0.5% methylcellulose), Torin 2 15 mg/kg. Torin 2 was suspended in 0.5% methylcellulose and administered via intraperitoneal injection once daily for 28 days [5]
3. Data collection: Tumor volume and body weight were measured twice weekly. Serum LDH levels were detected by biochemical assay. Tumor tissues were collected for Western blot (p-Akt, cleaved caspase-3) [5]

Oral bioavailability in mice (Reference [1]): BALB/c mice were given a single oral dose of Torin 2 (10 mg/kg). Plasma samples were collected 0.25–24 hours after administration, and drug concentrations were determined by LC-MS/MS. Results: Cmax = 75 ng/mL, Tmax = 1.0 h, t₁/₂β = 4.2 h, AUC₀-24h = 680 ng·h/mL, oral bioavailability (F) = 45% [1] - Plasma protein binding rate (Reference [1]): Balanced dialysis experiments showed that Torin 2 had a high binding rate: 97% in human plasma, 96% in mouse plasma, and 95% in rat plasma. It mainly binds to albumin[1]
- Metabolic stability (Reference[1]): In human liver microsomes, the half-life (t₁/₂) of Torin 2 is 190 minutes, and the metabolic rate is <15% within 2 hours. The main metabolite is a dihydroxylated derivative (accounting for 20% of the total metabolites)[1]
- Tissue distribution in mice (Reference[5]): After a single intraperitoneal injection of 15 mg/kg Torin 2, the tissue concentrations 2 hours after administration were: liver = 350 ng/g, kidney = 300 ng/g, tumor = 220 ng/g, brain = 15 ng/g (poor blood-brain barrier penetration). The ratio of tumor to plasma concentration was 2.8:1[5]

In vitro toxicity to normal cells (Reference [1]): Human dermal fibroblasts (HDF) and normal mammary epithelial cells (HMEC) were treated with Torin 2 (0.1–200 nM) for 72 hours. The CC₅₀ values were 220 nM (HDF) and 250 nM (HMEC), respectively, which were about 25 times higher than the IC₅₀ values of cancer cells (e.g., HCT116, 8 nM). Cell viability remained above 90% at ≤50 nM [1] - General in vivo toxicity (Reference [3,5]): - In the HCT116 xenograft study, mice (20 mg/kg Torin 2, administered by gavage for 21 days) did not show significant weight loss (<5% compared to baseline). Serum ALT, AST, BUN, and Scr were all within the normal range [3]
- No obvious pathological changes were observed in the liver, kidney, or spleen of mice in the A375 xenograft study (intraperitoneal injection of 15 mg/kg Torin 2 for 28 days). Hematological parameters (white blood cell count, platelet count) were normal [5]

[1]. J Med Chem . 2011 Mar 10;54(5):1473-80.

[2]. EMBO J . 2012 Mar 7;31(5):1095-108.

[3]. Clin Cancer Res . 2012 Jul 1;18(13):3532-40.

[4]. Cancer Cell . 2012 Jul 10;22(1):117-30.

[5]. Cancer Res . 2013 Apr 15;73(8):2574-86.

Torin 2 belongs to the pyridoquinoline class of compounds with the structure benzo[h][1,6]naphthid-2-one, with 3-(trifluoromethyl)phenyl and 6-aminopyridin-3-yl substituents at positions 1 and 9, respectively. It is a potent mTOR inhibitor with anticancer properties. It can function as both an mTOR inhibitor and an antitumor drug. It is an organofluorine compound, pyridoquinoline, aminopyridine, and primary amino compound.

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Mechanism of action advantage (references [1,2]): Torin 2 is a dual mTORC1/mTORC2 inhibitor with higher potency and selectivity than its analogue Torin 1. It binds to the ATP-binding pocket of mTOR, completely blocking mTOR-mediated signal transduction, including rapamycin-resistant mTORC1 function (e.g., complete inhibition of 4E-BP1 phosphorylation) and mTORC2-dependent Akt activation. This results in stronger inhibition of protein translation and cell proliferation, and induces apoptosis [1,2].
-Application as a research tool (References [2,4]): Torin 2 is widely used as a research tool for studying mTOR signaling, especially for studying the activity of mTORC1 and the biological function of mTORC2 insensitive to rapamycin. It can also be used as a positive control in studies of mTOR-targeted cancer therapy [2,4]
-Synergistic effects with other inhibitors (Reference [4]): In KRAS-mutant cancers (e.g., A549 lung cancer), Torin 2 synergistically inhibits tumor growth with MEK inhibitors. This is because mTOR inhibition reverses the MEK inhibitor-induced feedback activation of the PI3K/mTOR pathway, thereby enhancing antiproliferative efficacy [4]

C24H15F3N4O

432.3973

432.119

C, 66.67; H, 3.50; F, 13.18; N, 12.96; O, 3.70

1223001-51-1

51358113

White to light yellow solid powder

1.4±0.1 g/cm3

623.7±55.0 °C at 760 mmHg

331.0±31.5 °C

0.0±1.8 mmHg at 25°C

1.676

4.2

1

7

2

32

729

0

FC(C1C([H])=C([H])C([H])=C(C=1[H])N1C(C([H])=C([H])C2=C([H])N=C3C([H])=C([H])C(C4=C([H])N=C(C([H])=C4[H])N([H])[H])=C([H])C3=C12)=O)(F)F

GUXXEUUYCAYESJ-UHFFFAOYSA-N

InChI=1S/C24H15F3N4O/c25-24(26,27)17-2-1-3-18(11-17)31-22(32)9-6-16-13-29-20-7-4-14(10-19(20)23(16)31)15-5-8-21(28)30-12-15/h1-13H,(H2,28,30)

9-(6-aminopyridin-3-yl)-1-[3-(trifluoromethyl)phenyl]benzo[h][1,6]naphthyridin-2-one

Torin-2; Torin 2; Torin2

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: 15.6~41 mg/mL (36.1~94.8 mM)

Solubility in Formulation 1: ≥ 1.56 mg/mL (3.61 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 15.6 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: 5%DMSO+ 40%PEG300+ 5%Tween80+ 50%ddH2O: 1.5 mg/mL (3.47mM)

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Solubility in Formulation 3: ≥ 2 mg/mL (4.63 mM) (saturation unknown) in 10% 1-Methyl-2-pyrrolidinone 90% PEG300 (add these co-solvents sequentially from left to right, and one by one), clear solution.

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