Torin 2 is a novel, potent and selective ATP-competitive inhibitor of mTOR (mammalian target of rapamycin) with potential antitumor activity. It exhibits ~800-fold higher selectivity for mTOR over PI3K and has favorable pharmacokinetics. In p53−/− MEFs cell line, it inhibits mTOR with an IC50 of 0.25 nM.

Physicochemical Properties

Molecular Formula	C24H15F3N4O
Molecular Weight	432.3973
Exact Mass	432.119
Elemental Analysis	C, 66.67; H, 3.50; F, 13.18; N, 12.96; O, 3.70
CAS #	1223001-51-1
PubChem CID	51358113
Appearance	White to light yellow solid powder
Density	1.4±0.1 g/cm3
Boiling Point	623.7±55.0 °C at 760 mmHg
Flash Point	331.0±31.5 °C
Vapour Pressure	0.0±1.8 mmHg at 25°C
Index of Refraction	1.676
LogP	4.2
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	2
Heavy Atom Count	32
Complexity	729
Defined Atom Stereocenter Count	0
SMILES	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
InChi Key	GUXXEUUYCAYESJ-UHFFFAOYSA-N
InChi Code	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)
Chemical Name	9-(6-aminopyridin-3-yl)-1-[3-(trifluoromethyl)phenyl]benzo[h][1,6]naphthyridin-2-one
Synonyms	Torin-2; Torin 2; Torin2
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	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 )
ln Vitro	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]
ln Vivo	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]
Enzyme Assay	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.
Cell Assay	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.
Animal Protocol	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.
References	[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.
Additional Infomation	Torin 2 is a member of the class of pyridoquinolines that is benzo[h][1,6]naphthyridin-2-one carrying additional 3-(trifluoromethyl)phenyl and 6-aminopyridin-3-yl substituents at positions 1 and 9 respectively. It is a potent inhibitor of mTOR and exhibits anti-cancer properties. It has a role as a mTOR inhibitor and an antineoplastic agent. It is an organofluorine compound, a pyridoquinoline, an aminopyridine and a primary amino compound.

Solubility Data

Solubility (In Vitro)

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

Solubility (In Vivo)

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. Solubility in Formulation 2: 5%DMSO+ 40%PEG300+ 5%Tween80+ 50%ddH2O: 1.5 mg/mL (3.47mM) 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.3127 mL	11.5634 mL	23.1267 mL
	5 mM	0.4625 mL	2.3127 mL	4.6253 mL
	10 mM	0.2313 mL	1.1563 mL	2.3127 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.