EX229 (EX-229; AMPK Activator 991), a benzimidazole analog, is a novel, potent and allosteric AMPK (AMP-activated protein kinase) activator with Kd values of 0.06 μM, 0.06 μM and 0.51 μM for α1β1γ1, α2β1γ1 and α1β2γ1 in biolayer interferometry, respectively. AMPK plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. EX229 activates AMPK in incubated rat epitrochlearis skeletal muscle and incubation of rat skeletal muscle with ex229 increases glucose uptake. The stimulation of skeletal muscle glucose uptake by ex229 is PI3K/PKB-independent, while AMPK-dependent. ex229 increases fatty acid oxidation and glucose uptake in L6 myotubes.

Physicochemical Properties

Molecular Formula	C24H18CLN3O3
Molecular Weight	431.8710
Exact Mass	431.103
Elemental Analysis	C, 66.75; H, 4.20; Cl, 8.21; N, 9.73; O, 11.11
CAS #	1219739-36-2
Related CAS #	1219739-36-2
PubChem CID	45256693
Appearance	Off-white to pink solid powder
LogP	5.6
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	4
Heavy Atom Count	31
Complexity	666
Defined Atom Stereocenter Count	0
InChi Key	FHWSAZXFPUMKFL-UHFFFAOYSA-N
InChi Code	InChI=1S/C24H18ClN3O3/c1-13-3-5-16(10-17(13)23(29)30)31-24-26-20-11-18(19(25)12-21(20)27-24)14-4-6-22-15(9-14)7-8-28(22)2/h3-12H,1-2H3,(H,26,27)(H,29,30)
Chemical Name	5-{[6-chloro-5-(1-methylindol-5-yl)-1H-benzimidazol-2-yl]oxy}-2-methyl-benzoic acid
Synonyms	EX229; EX 229; EX-229; AMPK Activator 991
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	AMPK α1β1γ1 (Kd = 0.06 μM); AMPK α2β1γ1 (Kd = 0.06 μM); AMPK α1β2γ1 (Kd = 0.51 μM)
ln Vitro	EX229 activates AMPK in incubated rat epitrochlearis skeletal muscle and incubation of rat skeletal muscle with ex229 increases glucose uptake. Ex229 stimulates skeletal muscle glucose uptake, but this stimulation is AMPK-dependent and PI3K/PKB-independent. In L6 myotubes, ex229 boosts fatty acid oxidation and glucose uptake[1].
Cell Assay	Vehicle or 50 μM 991 were applied to cells transfected with Myc-Rabep1 WT or Ser407Ala for 60 min.
References	[1]. Biochem J. 2014 Jun 15;460(3):363-75. [2]. Cell Rep. 2019 Dec 3;29(10):3331-3348.e7.

Solubility Data

Solubility (In Vitro)

DMSO: >10 mg/mL Water: <1mg/mL Ethanol: <1mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1.3 mg/mL (3.01 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 13.0 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: ≥ 1.3 mg/mL (3.01 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 13.0 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 3: ≥ 1.3 mg/mL (3.01 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 13.0 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	2.3155 mL	11.5776 mL	23.1551 mL
	5 mM	0.4631 mL	2.3155 mL	4.6310 mL
	10 mM	0.2316 mL	1.1578 mL	2.3155 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.