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| Targets |
The target is AMPK (AMP-activated protein kinase), a key energy sensor and regulator of cellular metabolism. AMPK is a heterotrimeric enzyme complex. AMPK-IN-3 potently inhibits the kinase activity of both the AMPK alpha1 and alpha2 subunits. It shows selectivity over the kinase KDR (VEGFR2), indicating a focused mechanism on energy metabolism pathways.
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| ln Vitro |
AMPK(α2), FLT1, JAK1 JH2-pseudokinase, and AMPK(α1) are all inhibited by AMPK-IN-3 (100 nM) by 64%, 43%, 41%, and 29%, respectively[1]. K562 cells with AMPK-IN-3 (0.195313, 0.78125, 3.125, 12.5, 50 µM; 2 h) exhibit a reduction in p-ACC levels [1]. Without compromising the viability of the cells, AMPK-IN-3 (1-100 µM; 24, 48, and 72 hours) can efficiently decrease cellular AMPK activity [1].
AMPK-IN-3 inhibits AMPK enzymatic activity at low nanomolar concentrations in biochemical assays. In cellular assays, AMPK-IN-3 inhibits AMPK but does not affect the viability of K562 leukemia cells nor cause significant cytotoxicity. This suggests that its inhibitory effect on AMPK is not broadly toxic to all cell types, making it a useful tool for studying AMPK-specific functions in metabolism and cancer. |
| ln Vivo |
Specific in vivo data for AMPK-IN-3 are not published. As a tool compound targeting a key metabolic regulator, its in vivo use would be carefully considered due to the central role of AMPK in whole-body metabolism. It could be used in murine studies of metabolic diseases or cancer to understand the contribution of AMPK signaling.
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| Enzyme Assay |
A typical AMPK biochemical assay measures the phosphorylation of a peptide substrate. Purified recombinant human AMPK alpha1/beta1/gamma1 or alpha2/beta1/gamma1 complex is pre-incubated with varying concentrations of AMPK-IN-3. The reaction is initiated by adding ATP and a fluorescein-labeled SAMS peptide substrate (HMR SAMS). After incubation, the reaction is stopped, and the ratio of phosphorylated to non-phosphorylated peptide is quantified using a Mobility Shift Assay or a time-resolved fluorescence resonance energy transfer (TR-FRET) system.
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| Cell Assay |
Cell Viability Assay [1]
Cell Types: K562 Cell Tested Concentrations: 0.195313, 0.78125, 3.125, 12.5, 50 µM Incubation Duration: 2 hrs (hours) Experimental Results: diminished cellular levels of p-ACC(Ser79) in K562 cells. Cell viability assay [1] Cell Types: K562 Cell Tested Concentrations: 1-100 µM Incubation Duration: 24, 48, 72 hrs (hours) Experimental Results: Culturing under hypoxic conditions for 72 hrs (hours) had no measurable effect on the cell viability of K562 cells. A cellular assay would be performed in K562 cells or other cell lines where AMPK is active. Cells are treated with varying concentrations of AMPK-IN-3 for 4-6 hours. The compound's ability to inhibit AMPK is assessed by Western blot analysis of its downstream target, Acetyl-CoA Carboxylase (ACC). A reduction in p-ACC (Ser79) levels compared to a vehicle control indicates effective AMPK inhibition. Cell viability is measured after 24-72 hours of treatment using a standard MTT or CellTiter-Glo assay. |
| Animal Protocol |
No detailed in vivo protocols are available. A typical study would involve administering AMPK-IN-3 to mice via intraperitoneal injection to explore its acute effects on metabolism. For example, after a fasting period, mice would be injected with the compound, and then tissues such as liver and skeletal muscle would be harvested to measure p-ACC levels as a marker of AMPK activity.
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| ADME/Pharmacokinetics |
Specific pharmacokinetic parameters for AMPK-IN-3 are not provided in the available literature. As a research chemical intended for studying the role of AMPK, its characterization would typically involve assessing its solubility, stability, and initial in vivo exposure to determine appropriate dosing for future animal studies.
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| Toxicity/Toxicokinetics |
Specific toxicological data for AMPK-IN-3 are not detailed. However, it is noted that the compound does not affect cell viability or cause significant cytotoxicity in K562 cancer cells, suggesting that at certain concentrations, its effects are specific to AMPK signaling rather than inducing general toxicity. This selectivity is a key feature for a target validation tool.
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| References | |
| Additional Infomation |
AMPK is a central regulator of cellular energy balance and a major target for metabolic diseases like diabetes and for cancer research. AMPK-IN-3 is a potent and selective research tool, particularly valuable because it inhibits both alpha1 and alpha2 isoforms with high potency. As of the latest updates, it is an experimental research chemical for pre-clinical studies and has not yet been approved for clinical use.
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| Molecular Formula |
C25H33N5O3
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| Molecular Weight |
451.561225652695
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| Exact Mass |
451.258
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| CAS # |
2417674-27-0
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| PubChem CID |
162664520
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| Appearance |
Yellow to brown solid powder
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| LogP |
1.6
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
33
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| Complexity |
750
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N1C2=C(C=C(CCC(N)=O)C=C2)/C(=C/C2=C(C)C(C(NCCN(CC)CC)=O)=C(C)N2)/C1=O
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| InChi Key |
IQUHNNUWFFXNMM-RGEXLXHISA-N
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| InChi Code |
InChI=1S/C25H33N5O3/c1-5-30(6-2)12-11-27-25(33)23-15(3)21(28-16(23)4)14-19-18-13-17(8-10-22(26)31)7-9-20(18)29-24(19)32/h7,9,13-14,28H,5-6,8,10-12H2,1-4H3,(H2,26,31)(H,27,33)(H,29,32)/b19-14-
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| Chemical Name |
5-[(Z)-[5-(3-amino-3-oxopropyl)-2-oxo-1H-indol-3-ylidene]methyl]-N-[2-(diethylamino)ethyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide
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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 : ~115 mg/mL (~254.67 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2145 mL | 11.0727 mL | 22.1455 mL | |
| 5 mM | 0.4429 mL | 2.2145 mL | 4.4291 mL | |
| 10 mM | 0.2215 mL | 1.1073 mL | 2.2145 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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