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| Targets |
AMPK activator 4 targets AMP-activated protein kinase (AMPK). AMPK is a heterotrimeric serine/threonine protein kinase composed of a catalytic α subunit and regulatory β and γ subunits. Activation of AMPK leads to phosphorylation of downstream targets, including acetyl-CoA carboxylase (ACC), which regulates fatty acid synthesis and oxidation. The compound selectively activates AMPK in muscle tissue without inhibiting mitochondrial complex I, distinguishing it from other AMPK activators that may have off-target effects on mitochondrial function. The compound has a molecular formula of C24H21ClN2O3 and a molecular weight of 420.89 g/mol.
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| ln Vitro |
AMPK activator 4 (compound B10) (0–20 μM; 12–24 hours) causes AMPK and its downstream protein ACC to become phosphorylated[1]. LKB1 is necessary for AMPK phosphorylation mediated by AMPK activator 4[1]. When HepG2 cells are treated with AMPK activator 4 at 10–20 μM for 72 hours, their viability is slightly decreased[1].
In vitro studies have demonstrated that AMPK activator 4 induces dose-dependent phosphorylation of ACC in C2C12 myotube cells, which is consistent with AMPK phosphorylation. At concentrations of 10-20 µM for 72 hours, the compound slightly decreases the viability of HepG2 cells. It also increases AMPK and ACC phosphorylation in HepG2 and Huh1-7 cells. These findings confirm that the compound activates AMPK signaling in multiple cell types. The compound’s ability to activate AMPK without inhibiting mitochondrial complex I suggests a favorable selectivity profile. |
| ln Vivo |
AMPK activator 4 (100 mg/kg; administered intragastrically; daily for 9 weeks) exhibits anti-hyperglycemic properties[1].
AMPK activator 4 has demonstrated significant in vivo efficacy in animal models. In normal mice, the compound dose-dependently improves glucose tolerance. In db/db diabetic mice, oral administration of AMPK activator 4 at 100 mg/kg daily for 9 weeks significantly lowers fasting blood glucose levels (observed at 2-6 weeks) and significantly improves insulin resistance (observed for 9 weeks). Importantly, the compound does not significantly affect body weight or serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicating a favorable safety profile with no evidence of hepatotoxicity. These results demonstrate the compound’s anti-hyperglycemic effects. |
| Enzyme Assay |
The AMPK activating activity of the compound can be assessed using in vitro kinase assays. In a typical assay, recombinant AMPK is incubated with a peptide substrate, ATP, and varying concentrations of the compound. The phosphorylation of the substrate is measured, and the EC50 for AMPK activation is calculated from dose-response curves. Alternatively, cellular AMPK activation can be assessed by measuring the phosphorylation of AMPK and its downstream target ACC using Western blotting with phospho-specific antibodies. The compound’s selectivity for AMPK over other kinases can be assessed using kinase profiling services.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: C2C12 myotubes, HepG2 and HuH-7 cells Tested Concentrations: 0-20 μM Incubation Duration: 12-24 hrs (hours) Experimental Results: Induced dose-dependently phosphorylation of ACC in C2C12 myotube cells, and the phosphorylation of ACC was highly consistent with the phosphorylation of AMPK. Increased the phosphorylation of AMPK and ACC in human hepatocarcinoma HepG2 and HuH-7 cells. The cellular activity of AMPK activator 4 is assessed using C2C12 myotube cells, HepG2 cells, and Huh1-7 cells. Cells are cultured in appropriate media and treated with escalating concentrations of the compound (0-20 µM) for 12-24 hours. AMPK activation is assessed by Western blotting using antibodies specific for phospho-AMPK (Thr172) and phospho-ACC (Ser79). Cell viability is assessed using MTT assays. The compound induces dose-dependent phosphorylation of ACC in C2C12 myotube cells, which is consistent with AMPK phosphorylation. At 10-20 µM for 72 hours, the compound slightly decreases the viability of HepG2 cells. |
| Animal Protocol |
Animal/Disease Models: db/db mice (5-week old)[1]
Doses: 100 mg/kg Route of Administration: intragastric (po)administration; daily for 9 weeks Experimental Results: Dramatically lowered the fasting blood glucose in db/db mice (2-6 weeks). Dramatically improved insulin resistance in db /db mice (for 9 weeks). The body weight and the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (two markers of liver injury) were not Dramatically affected. AMPK activator 4 has been evaluated in db/db diabetic mice. In these studies, the compound was administered by intragastric administration at a dose of 100 mg/kg daily for 9 weeks. Fasting blood glucose levels were measured weekly, and insulin resistance was assessed using appropriate methods. The compound significantly lowered fasting blood glucose levels (2-6 weeks) and significantly improved insulin resistance (for 9 weeks) without significantly affecting body weight or serum ALT and AST levels. These results demonstrate the compound’s anti-hyperglycemic effects and favorable safety profile. |
| ADME/Pharmacokinetics |
The pharmacokinetic properties of AMPK activator 4 have been characterized to some extent in preclinical studies. The compound is orally bioavailable and can be administered by intragastric administration. It has a molecular weight of 420.89 g/mol and is soluble in DMSO at 50 mg/mL (118.8 mM). The compound’s half-life, maximum concentration (Cmax), area under the curve (AUC), and other pharmacokinetic parameters have not been detailed in the available literature. Further studies would be needed to fully characterize the absorption, distribution, metabolism, and excretion (ADME) properties of AMPK activator 4.
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| Toxicity/Toxicokinetics |
In the db/db diabetic mouse model, AMPK activator 4 did not significantly affect body weight or serum levels of ALT and AST at a dose of 100 mg/kg daily for 9 weeks, indicating a favorable safety profile with no evidence of hepatotoxicity. No other detailed toxicity data has been published in the available literature. As a research compound, AMPK activator 4 has not been subjected to comprehensive toxicology studies. Further studies would be needed to assess the compound’s safety profile for potential therapeutic development.
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| References | |
| Additional Infomation |
AMPK activator 4 (CAS#: 2493239-46-4) is a potent and selective activator of AMPK that does not inhibit mitochondrial complex I. The compound selectively activates AMPK in muscle tissue and demonstrates anti-hyperglycemic effects. In db/db diabetic mice, oral administration at 100 mg/kg daily for 9 weeks significantly lowers fasting blood glucose and improves insulin resistance without affecting body weight or liver enzyme levels. The compound has a molecular formula of C24H21ClN2O3 and a molecular weight of 420.89 g/mol. It is a valuable research tool for studying AMPK signaling and metabolic diseases. The compound was discovered as an analogue of AdipoRon.
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| Molecular Formula |
C24H21CLN2O3
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| Molecular Weight |
420.888145208359
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| Exact Mass |
420.124
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| CAS # |
2493239-46-4
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| PubChem CID |
154573764
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| Appearance |
White to off-white solid powder
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| LogP |
5.2
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
30
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| Complexity |
593
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C=CC(=CC=1)OC(C(NC1C=CC(=CC=1)OCC1C=CC(C#N)=CC=1)=O)(C)C
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| InChi Key |
OSSMPMZJYDZMFX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H21ClN2O3/c1-24(2,30-22-11-7-19(25)8-12-22)23(28)27-20-9-13-21(14-10-20)29-16-18-5-3-17(15-26)4-6-18/h3-14H,16H2,1-2H3,(H,27,28)
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| Chemical Name |
2-(4-chlorophenoxy)-N-[4-[(4-cyanophenyl)methoxy]phenyl]-2-methylpropanamide
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO : 250 mg/mL (593.98 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.94 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 20.8 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 | 2.3759 mL | 11.8796 mL | 23.7592 mL | |
| 5 mM | 0.4752 mL | 2.3759 mL | 4.7518 mL | |
| 10 mM | 0.2376 mL | 1.1880 mL | 2.3759 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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