Autophinib is a novel and potent autophagy inhibitor, which inhibits starvation-induced or rapamyc induced autophagy. It acts by targeting the lipid kinase VPS34 with IC50 values of 90, 40 and 19 nM, respectively. Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small-molecule autophagy modulators such as autophinib are in high demand. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy-induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34.

Physicochemical Properties

Molecular Formula	C14H11CLN6O3
Molecular Weight	346.73
Exact Mass	346.06
Elemental Analysis	C, 48.50; H, 3.20; Cl, 10.22; N, 24.24; O, 13.84
CAS #	1644443-47-9
Related CAS #	1644443-47-9
PubChem CID	129626605
Appearance	Light yellow to yellow solid powder
LogP	3.8
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	4
Heavy Atom Count	24
Complexity	431
Defined Atom Stereocenter Count	0
InChi Key	CEUMAXLRGBKFQP-UHFFFAOYSA-N
InChi Code	InChI=1S/C14H11ClN6O3/c1-8-6-13(20-19-8)17-12-7-11(15)16-14(18-12)24-10-4-2-9(3-5-10)21(22)23/h2-7H,1H3,(H2,16,17,18,19,20)
Chemical Name	6-Chloro-N-(5-methyl-1H-pyrazol-3-yl)-2-(4-nitrophenoxy)pyrimidin-4-amine
Synonyms	Autophinib
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

ln Vitro	In starved MCF7-LC3 cells, autophinib (0.01-1 μM) suppresses LC3 lipidation to generate LC3-II in a dose-dependent manner. Autophinib dose-dependently suppressed autophagy-induced p62 degradation in MCF7-LC3 cells, which is consistent with its inhibition of autophagic flux [1]. By triggering apoptosis (EC50 234 nM) in MCF7 cells, autophinib increases cell death (EC50 264 nM) in starved cells relative to fed cells [1].
ln Vivo
Animal Protocol
References	[1]. Phenotypic Identification of a Novel Autophagy Inhibitor Chemotype Targeting Lipid Kinase VPS34. Angew Chem Int Ed Engl. 2017 Jul 3;56(28):8153-8157.

Solubility Data

Solubility (In Vitro)

DMSO:≥ 30mg/mL Water:N/A Ethanol:N/A

Solubility (In Vivo)

Solubility in Formulation 1: 0.5 mg/mL (1.44 mM) in 10% DMSO + 90% (20% SBE-β-CD in 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 5.0 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. Solubility in Formulation 2: ≥ 0.5 mg/mL (1.44 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 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.8841 mL	14.4204 mL	28.8409 mL
	5 mM	0.5768 mL	2.8841 mL	5.7682 mL
	10 mM	0.2884 mL	1.4420 mL	2.8841 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.