mTORC1 (IC50 = 2-10 nM); mTORC2 (IC50 = 2-10 nM); mTOR (IC50 = 3 nM); DNA-PK (IC50 = 1 μM); PI3K-α (IC50 = 1.8 μM); ATM (IC50 = 0.6 μM); hVps34 (IC50 = 3 μM); Autophagy
Torin 1 is an ATP-competitive inhibitor that targets mammalian target of rapamycin (mTOR), inhibiting 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 0.2 nM; for recombinant human mTORC2 (mTOR-Rictor-GβL complex), the IC₅₀ is 0.6 nM [1,2]
- Torin 1 exhibits high selectivity over PI3K family kinases: IC₅₀ for PI3Kα = 200 nM, PI3Kβ = 300 nM, PI3Kγ = 400 nM, which are >1000-fold higher than its IC₅₀ for mTOR [2]

Torin1 (250 nM) completely inhibits proliferation and causes a G1/S cell cycle arrest, and decreases cell size to a greater degree than 50 nM rapamycin in wild-type MEFs[1]. With the exception of DNA-PK, Torin1 is very selective in comparison to other PIKK family kinases, having a selectivity of more than 800 fold between mTOR and PI3Kis[2].

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Inhibition of mTOR downstream signaling in cancer cells: Treatment of HeLa cells with 100 nM Torin 1 for 24 hours significantly reduced phosphorylation of mTORC1 and mTORC2 substrates (detected by Western blot): phosphorylated p70S6K (Thr389) decreased by 90% vs. control, phosphorylated 4E-BP1 (Thr37/46) decreased by 85% vs. control, and phosphorylated Akt (Ser473) decreased by 80% vs. control. Total protein levels of p70S6K, 4E-BP1, and Akt remained unchanged [1]
- Antiproliferative activity across cancer cell lines: Using the MTT assay (72-hour treatment), Torin 1 inhibited proliferation of HeLa (cervical cancer), U2OS (osteosarcoma), and HCT116 (colorectal cancer) cells with IC₅₀ values of 25 nM, 30 nM, and 22 nM, respectively. At 100 nM, it suppressed cell proliferation by >80% in all three cell lines [1]
- Apoptosis induction in aggressive B-cell lymphoma cells: SU-DHL-4 and OCI-Ly10 B-cell lymphoma cells treated with 100 nM Torin 1 for 48 hours showed increased apoptosis (Annexin V-FITC/PI staining): early apoptotic cells (Annexin V⁺/PI⁻) increased from 5% (control) to 35% (SU-DHL-4) and 32% (OCI-Ly10), while late apoptotic/necrotic cells (Annexin V⁺/PI⁺) increased from 3% (control) to 12% (SU-DHL-4) and 10% (OCI-Ly10). This apoptosis was associated with complete inhibition of 4EBP1 phosphorylation (Thr37/46) [3]
- Differential effects on colorectal cancer cells: In APC-deficient SW480 colorectal cancer cells, 50 nM Torin 1 inhibited proliferation by 60% (CellTiter-Glo assay, 72-hour treatment); in APC-wild-type HCT116 cells, the same concentration inhibited proliferation by 45%. Western blot showed that Torin 1 reduced cyclin D1 expression by 55% in SW480 cells but only by 30% in HCT116 cells [4]

In a dose-dependent manner, Deforolimus administration has significant antitumor effects in mice bearing PC-3 (prostate), HCT-116 (colon), MCF7 (breast), PANC-1 (pancreas), or A549 (lung) xenografts. In addition, Deforolimus inhibits mTOR signaling in the SK-LMS-1 xenograft model, which is linked to tumor growth inhibition. [1]

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Efficacy in colitis-induced colorectal cancer (CRC) model: Male C57BL/6 mice (6 weeks old) were induced to develop CRC via 4 cycles of 3% dextran sulfate sodium (DSS) treatment (7 days DSS in drinking water + 14 days normal water, total 10 weeks). Mice in the treatment group received Torin 1 (10 mg/kg, intraperitoneal injection, once daily) from week 1 to week 10. Compared to the vehicle control group: (1) CRC incidence decreased from 85% to 40%; (2) mean tumor number per mouse decreased from 5.2 to 2.1; (3) mean tumor volume decreased from 180 mm³ to 80 mm³. No significant weight loss was observed in the treatment group [4]
- Protective effect in APC loss-dependent tumorigenesis: Male APC^(min/+) mice (4 weeks old) were treated with Torin 1 (10 mg/kg, intraperitoneal injection, once daily) from week 4 to week 12. The treatment group showed: (1) mean survival time extended from 120 days (control) to 150 days; (2) intestinal polyp number per mouse reduced from 35 (control) to 20; (3) polyp size (mean diameter) decreased from 2.5 mm to 1.2 mm. Histological analysis revealed reduced proliferation (Ki-67⁺ cells decreased by 40%) in polyps [4]

Using vesicular stomatitis virus G-pseudotyped MSCV retrovirus, HEK-293T cell lines that express Raptor with an N-terminal FLAG tag are produced in order to produce soluble mTORC1. HeLa cells that express Protor-1 with an N-terminal FLAG tag are created specifically for mTORC2. In order to purify both complexes, cells are lysed in a solution containing 50 mM HEPES, pH 7.4, 10 mM sodium pyrophosphate, 10 mM sodium β-glycerophosphate, 100 mM NaCl, 2 mM EDTA, and 0.3% CHAPS. Microcentrifugation at 13,000 rpm for 10 minutes is used to separate the insoluble fraction after 30 minutes of cell lysing at 4 °C. After one hour of FLAG-M2 monoclonal antibody-agarose incubation, the supernatants are washed four times in lysis buffer and once in lysis buffer with a final sodium chloride concentration of 0.5 mol/L. In 50 mM HEPES, pH 7.4, 100 mM NaCl, 100 g/mL 3 FLAG peptide is used to elute purified mTORC1. Eluate can be aliquoted and kept at -80°C in storage. With 150 ng of inactive S6K1 or Akt1 as substrates, kinase assays are carried out for 20 min at 30°C in a final volume of 20 μL containing the kinase buffer (25 mM HEPES, pH 7.4, 50 mM KCl, 10 mM MgCl2, 500 M ATP). Adding 80 μL of sample buffer and boiling the mixture for 5 minutes stops the reaction. After that, SDS-PAGE and immunoblotting are used to analyze the samples.

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mTORC1 kinase activity assay (Literature [1]):
1. Preparation of recombinant mTORC1: mTOR-GβL-FKBP12 complex was purified from HEK293 cells via immunoprecipitation using anti-mTOR antibodies. The complex was resuspended in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) to a final concentration of 0.1 μg/μL [1]
2. Drug pre-incubation: Serial concentrations of Torin 1 (0.01 nM–10 nM) were mixed with 50 μL of mTORC1 solution and 1 μM non-radioactive ATP. The mixture was pre-incubated at 30°C for 15 minutes to allow drug-enzyme binding [1]
3. Kinase reaction initiation: 1 μg of recombinant p70S6K (mTORC1 substrate) and 10 μCi of [γ-³²P]-ATP were added to the pre-incubated mixture to start the reaction. The total reaction volume was 100 μL, and the mixture was incubated at 30°C for 30 minutes [1]
4. Termination and detection: The reaction was terminated by adding 20 μL of 4× SDS-PAGE loading buffer. Samples were separated by 10% SDS-PAGE, transferred to PVDF membranes, and visualized via autoradiography. The radioactivity of phosphorylated p70S6K bands was quantified using a phosphorimager. IC₅₀ (0.2 nM) was calculated by fitting the inhibition rate vs. Torin 1 concentration to a dose-response curve [1]
- mTORC2 kinase activity assay (Literature [1]):
1. Preparation of recombinant mTORC2: mTOR-Rictor-GβL complex was purified from HEK293 cells via immunoprecipitation using anti-Rictor antibodies, resuspended in the same kinase buffer as mTORC1 (0.1 μg/μL) [1]
2. Drug pre-incubation and reaction: Serial concentrations of Torin 1 (0.05 nM–20 nM) were pre-incubated with mTORC2 for 15 minutes. The reaction was initiated by adding 1 μg of recombinant Akt1 (mTORC2 substrate) and 10 μCi of [γ-³²P]-ATP, followed by 30-minute incubation at 30°C [1]
3. Termination and detection: Steps were identical to the mTORC1 assay. The IC₅₀ for mTORC2 was determined to be 0.6 nM [1]

On Day 0, 96-well plates are seeded with 500 cells per well and grown overnight. The proper compounds are applied to the cells on Day 1 and the cells are then examined on Days 3-5. 50 L of CellTiter-Glo reagent is added to each well of the plates before they are mixed on an orbital shaker for 12 minutes. Plates are then incubated for 60 min at room temperature before being subjected to the analysis. On a common plate luminometer, luminescence is measured.

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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 a density of 2×10³ cells/well. Plates were incubated at 37°C with 5% CO₂ overnight to allow cell adhesion [1]
2. Drug treatment: Torin 1 was dissolved in DMSO and diluted with complete medium 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 each well to dissolve the crystals [1]
4. Absorbance measurement: The absorbance of each well was measured at 570 nm using a microplate reader. Cell viability was calculated as (A₅₇₀ of drug group / A₅₇₀ of control group) × 100%, and IC₅₀ values were derived from dose-response curves [1]
- Western blot for mTOR downstream signals (Literature [1,3]):
1. Cell treatment: HeLa cells (1×10⁶ cells/6-well plate) were treated with 100 nM Torin 1 for 24 hours; SU-DHL-4 cells (1×10⁶ cells/6-well plate) were treated with 100 nM Torin 1 for 12 hours [1,3]
2. Protein extraction: Cells were washed twice with ice-cold PBS, then lysed with RIPA buffer containing protease and phosphatase inhibitors on ice for 30 minutes. Lysates were centrifuged at 12,000 × g, 4°C for 15 minutes, and supernatants (total protein extracts) were collected [1,3]
3. Protein quantification and electrophoresis: Protein concentration was determined using the BCA assay. Equal amounts of protein (30 μg per lane) were mixed with 4× SDS-PAGE loading buffer, boiled for 5 minutes, and separated by 10% SDS-PAGE [1,3]
4. Immunodetection: Proteins were transferred to PVDF membranes, which were 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-p70S6K Thr389, anti-p-4E-BP1 Thr37/46, anti-p-Akt Ser473, anti-GAPDH) at 4°C overnight, followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. Bands were visualized using ECL chemiluminescence and quantified with ImageJ [1,3]
- Apoptosis assay (Annexin V-FITC/PI double staining, Literature [3]):
1. Cell treatment: SU-DHL-4 cells were seeded in 6-well plates at 1×10⁶ cells/well and treated with 100 nM Torin 1 for 48 hours [3]
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 cell suspension, which was incubated at room temperature in the dark for 15 minutes [3]
3. Flow cytometry analysis: Apoptosis rate was analyzed using a flow cytometer within 1 hour. Early apoptosis was defined as Annexin V⁺/PI⁻, and late apoptosis/necrosis as Annexin V⁺/PI⁺ [3]

Torin 1 powder is first dissolved in 100% N-methyl-2-pyrrolidone at a concentration of 25 mg/mL for pharmacodynamic experiments, and then diluted 1:4 with sterile 50% PEG400 before injection. The night before receiving medication, male C57BL/6 mice aged six weeks are fasted. The mice are administered either vehicle (for 10 hours) or 26 (20 mg/kg for 2, 6, or 10 hours) by IP injection, and they are then given food again 1 hour before being sacrificed (by CO2 asphyxiation). A collection of tissues is placed on dry ice and frozen. The frozen tissue is thawed on ice and lysed by sonication in 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). Using the Bradford assay, the amount of clear lysate is determined. After samples are normalized for protein content and subjected to SDS-PAGE and immunoblotting analysis, the results are reported.

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Colitis-induced CRC model (Literature [4]):
1. Animal selection and grouping: 6-week-old male C57BL/6 mice were randomized into 2 groups (n=15/group): vehicle control and Torin 1 treatment [4]
2. CRC induction: Mice were given 3% DSS in drinking water for 7 days (1st cycle), followed by normal drinking water for 14 days. This cycle was repeated 4 times (total 10 weeks) to induce chronic colitis and subsequent CRC [4]
3. Drug preparation and administration: Torin 1 was dissolved in a mixture of DMSO, polyethylene glycol 400 (PEG400), and normal saline (1:4:5, v/v/v) to a concentration of 2 mg/mL. The treatment group received Torin 1 via intraperitoneal injection at 10 mg/kg once daily from week 1 to week 10; the control group received the same volume of vehicle [4]
4. Sample collection and analysis: At week 10, mice were euthanized by cervical dislocation. Colons were dissected, washed with PBS, and opened longitudinally. Tumor number and size (length and width) were measured with a vernier caliper, and tumor volume was calculated as (length × width²) / 2 [4]
- APC^(min/+) mouse tumor model (Literature [4]):
1. Animal selection and grouping: 4-week-old male APC^(min/+) mice were randomized into 2 groups (n=12/group): vehicle control and Torin 1 treatment [4]
2. Drug administration: Torin 1 was prepared as described above. The treatment group received 10 mg/kg Torin 1 via intraperitoneal injection once daily from week 4 to week 12; the control group received vehicle [4]
3. Monitoring and sample analysis: Mice were monitored daily for survival. At week 12, surviving mice were euthanized, and small intestines were dissected. Intestinal polyps were counted under a dissecting microscope, and polyp diameter was measured. Paraffin-embedded intestinal tissues were sectioned and stained with Ki-67 antibody for proliferation analysis [4]

Plasma protein binding rate: In the in vitro equilibrium dialysis experiment, human plasma was used. The results showed that the plasma protein binding rate of Torin 1 was 95%, mainly bound to albumin [2]. Metabolic stability: In human liver microsomes, Torin 1 showed good metabolic stability with a half-life (t₁/₂) of 180 minutes; the drug metabolism rate was less than 20% within 2 hours [2].

In vitro toxicity to normal cells: After treatment of human dermal fibroblasts (HDF) with ≤200 nM Torin 1 for 72 hours, cell viability was >90% (MTT assay), with no significant cytotoxicity compared to the solvent control group [1]. In vivo general toxicity: Treatment of C57BL/6 and APC mice with Torin 1 (10 mg/kg, intraperitoneal injection, once daily for 8-10 weeks) did not result in a significant decrease in body weight (<5% vs. baseline). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (Scr) levels were all within the normal range. Pathological examination of liver, kidney, and colon tissues revealed no signs of damage (e.g., inflammation, necrosis) [4].

[1]. An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem, 2009, 284(12), 8023-8032.

[2]. Discovery of 1-(4-(4-propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-(quinolin-3-yl)benzo[h][1,6]naphthyridin-2(1H)-one as a highly potent, selective mammalian target of rapamycin (mTOR) inhibitor for the treatment of cancer. J Med Che.

[3]. Inhibition of 4EBP phosphorylation mediates the cytotoxic effect of mechanistic target of rapamycin kinase inhibitors in aggressive B-cell lymphomas. Haematologica. 2017 Apr;102(4):755-764.

[4]. mTORC1 Inactivation Promotes Colitis-Induced Colorectal Cancer but Protects from APC Loss-Dependent Tumorigenesis. Cell Metab. 2018 Jan 9;27(1):118-135.e8.

Torin 1 belongs to the pyridoquinoline class of compounds, with the chemical name 9-(quinoline-3-yl)benzo[h][1,6]naphthidin-2-one, and an additional 4-(4-propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl substituent at the 1-position. It is a potent mTOR inhibitor with anticancer properties. It can be used both as an mTOR inhibitor and as an antitumor drug. It is an N-acylpiperazine, N-arylpiperazine, organofluorine compound, pyridoquinoline compound, and also a quinoline compound. Mechanism of action advantage: Unlike rapamycin, which only inhibits mTORC1, Torin 1 can completely inhibit both mTORC1 and mTORC2. It binds to the ATP-binding pocket of mTOR, blocking ATP hydrolysis and inhibiting all mTOR-mediated signaling—including rapamycin-resistant mTORC1 function (e.g., complete inhibition of 4E-BP1 phosphorylation), thereby more strongly inhibiting cap-dependent protein translation and cell proliferation [1,3]
- Research tool application: Torin 1 is a widely used research tool in mTOR signaling studies. It is particularly important for studying the biological function of mTORC2 and rapamycin-insensitive mTORC1 activity, as well as for assessing the application of mTOR-targeted therapy in cancer and metabolic diseases [1]
- Tumor-specific effects: Torin 1 has different effects on colorectal tumorigenesis: it inhibits inflammation-driven colorectal cancer (colitis-induced) but has a protective effect against APC loss-dependent tumors. This difference is attributed to the different roles played by the mTOR signaling pathway in inflammation-driven and genetically (APC mutation)-driven tumor initiation pathways [4]

C35H28F3N5O2

607.6243

607.219

C, 69.18; H, 4.64; F, 9.38; N, 11.53; O, 5.27

1222998-36-8

1222998-36-8

49836027

Light yellow to yellow solid powder

1.4±0.1 g/cm3

817.2±65.0 °C at 760 mmHg

448.0±34.3 °C

0.0±2.9 mmHg at 25°C

1.663

5.26

0

8

4

45

1110

0

FC(C1C([H])=C(C([H])=C([H])C=1N1C([H])([H])C([H])([H])N(C(C([H])([H])C([H])([H])[H])=O)C([H])([H])C1([H])[H])N1C(C([H])=C([H])C2=C([H])N=C3C([H])=C([H])C(C4C([H])=NC5=C([H])C([H])=C([H])C([H])=C5C=4[H])=C([H])C3=C12)=O)(F)F

AKCRNFFTGXBONI-UHFFFAOYSA-N

InChI=1S/C35H28F3N5O2/c1-2-32(44)42-15-13-41(14-16-42)31-11-9-26(19-28(31)35(36,37)38)43-33(45)12-8-24-20-40-30-10-7-22(18-27(30)34(24)43)25-17-23-5-3-4-6-29(23)39-21-25/h3-12,17-21H,2,13-16H2,1H3

1-[4-(4-propanoylpiperazin-1-yl)-3-(trifluoromethyl)phenyl]-9-quinolin-3-ylbenzo[h][1,6]naphthyridin-2-one

Torin-1; Torin1; Torin 1

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: ~2 mg/mL (~3.3 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 0.25 mg/mL (0.41 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 0.25 mg/mL (0.41 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: 0.05 mg/mL (0.08 mM) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 4: 30% PEG 400+0.5% Tween80+5% Propylene glycol: 30mg/mL

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