mTOR inhibitor-8 is an mTOR inhibitor and autophagy inducer. mTOR inhibitor-8 inhibits mTOR activity through FKBP12 and induces autophagy in A549 human lung cancer cells.

Physicochemical Properties

Molecular Formula	C24H19CLN4OS
Molecular Weight	446.95
Exact Mass	446.096
CAS #	2489196-70-3
PubChem CID	154573769
Appearance	White to yellow solid powder
LogP	5.6
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	5
Heavy Atom Count	31
Complexity	615
Defined Atom Stereocenter Count	0
SMILES	C1C=C(OC)C=CC=1C1N(C2=NC(=CS2)C2=CC=NC=C2)N=C(C2C=CC(Cl)=CC=2)C1
InChi Key	YYORGGAMBAZQIJ-UHFFFAOYSA-N
InChi Code	InChI=1S/C24H19ClN4OS/c1-30-20-8-4-18(5-9-20)23-14-21(16-2-6-19(25)7-3-16)28-29(23)24-27-22(15-31-24)17-10-12-26-13-11-17/h2-13,15,23H,14H2,1H3
Chemical Name	2-[5-(4-chlorophenyl)-3-(4-methoxyphenyl)-3,4-dihydropyrazol-2-yl]-4-pyridin-4-yl-1,3-thiazole
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	mTOR Autophagy
ln Vitro	Compound 5e, mTOR inhibitor-8 (0.1-10 μM; 24 and 48 hours) inhibits A549 cell growth in a dose-dependent manner[1]. Autophagy is induced by mTOR inhibitor-8 in a mTOR-dependent way. Autophagy is induced in a time-dependent manner by mTOR inhibitor-8 (10 μM; 3-24 hours). There is an increase in LC3B-II levels[1]. Two crucial mTOR substrates, ribosomal protein S6 kinase (RPS6KB1) and eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1), are phosphorylated less when treated with mTOR inhibitor-8 (10 μM; 3–24 hours)[1].
ln Vivo	In vivo tumor development is significantly inhibited by mTOR inhibitor-8 (25 and 50 μM; 6 days) without negatively impacting normal chick chorioallantoic membrane angiogenesis.
Cell Assay	Cell Viability Assay[1] Cell Types: A549 cells Tested Concentrations: 0.1, 1, 5, 10 μM Incubation Duration: 24 and 48 hrs (hours) Experimental Results: Suppressed the growth of A549 cells with an IC50 of 2.6±0.11 μM. Western Blot Analysis[1] Cell Types: A549 cells Tested Concentrations: 10 μM Incubation Duration: 3, 6, 12 and 24 hrs (hours) Experimental Results: The levels of phosphorylation of RPS6KB1 and EIF4EBP1 were Dramatically diminished after treatment.
Animal Protocol	Animal/Disease Models: Fertile chicken eggs (7-9 days old)[1] Doses: 25 and 50 μM Route of Administration: 6 days Experimental Results: Significant xenograft tumor remission was observed in eggs compared with the DMSO-treated eggs.
References	[1]. Discovery of new fluorescent thiazole-pyrazoline derivatives as autophagy inducers by inhibiting mTOR activity in A549 human lung cancer cells. Cell Death Dis. 2020 Jul 20;11(7):551.

Solubility Data

Solubility (In Vitro)

DMSO : 25 mg/mL (55.93 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 2.08 mg/mL (4.65 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 20.8 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: ≥ 2.08 mg/mL (4.65 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2374 mL	11.1869 mL	22.3739 mL
	5 mM	0.4475 mL	2.2374 mL	4.4748 mL
	10 mM	0.2237 mL	1.1187 mL	2.2374 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.