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| Targets |
ESI-08 targets EPAC1 and EPAC2 (exchange proteins directly activated by cAMP), which are guanine nucleotide exchange factors (GEFs) for the small GTPases Rap1 and Rap2. EPAC proteins are activated by the second messenger cAMP and play important roles in various cellular processes, including cell adhesion, migration, proliferation, and differentiation. ESI-08 is a non-cyclic nucleotide antagonist that competes with cAMP for binding to the EPAC regulatory domain, thereby blocking EPAC activation. It is highly selective for EPAC over PKA, as it does not inhibit cAMP-mediated PKA activation at concentrations that completely inhibit EPAC. This selectivity is crucial for studying EPAC-specific functions.
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| ln Vitro |
In response to the second messenger cAMP, a family of guanine nucleotide exchange factors known as exchange proteins directly activated by cAMP (EPAC) regulates a number of intracellular processes. It is discovered that type I and type II PKA holoenzyme activation triggered by cAMP is unaffected by 25 μM ESI-08, whereas type I or type II PKA activity is totally blocked by the selective PKA inhibitor H89 [1].
ESI-08 demonstrates potent in vitro activity as an EPAC antagonist. It completely inhibits both EPAC1 and EPAC2 with an IC50 of 8.4 µM. The compound selectively blocks cAMP-induced EPAC activation but does not inhibit cAMP-mediated PKA activation, confirming its selectivity. In cell-based assays, ESI-08 effectively inhibits EPAC-mediated cellular responses such as Rap1 activation, cell adhesion, and migration. Its activity is dose-dependent, and it is used at concentrations typically in the low micromolar range. These in vitro activities confirm ESI-08’s utility as a pharmacological tool for studying EPAC signaling pathways. |
| ln Vivo |
In vivo studies with ESI-08 are limited, as the compound is primarily used as a research tool in cell-based assays. However, its potential in modulating cell migration and invasion processes suggests possible applications in cancer and inflammatory disease models. By inhibiting EPAC, ESI-08 can modulate cellular processes that are dysregulated in disease states. Further in vivo studies are needed to fully characterize its pharmacokinetic and pharmacodynamic properties and to explore its therapeutic potential. The compound’s selectivity for EPAC over PKA makes it a promising tool for studying EPAC function in vivo.
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| Enzyme Assay |
In vitro enzyme/receptor binding assays for ESI-08 involve measuring its ability to compete with cAMP for binding to EPAC1 and EPAC2. These assays typically use fluorescent or radiolabeled cAMP analogs in a competitive binding format. Recombinant EPAC proteins are incubated with the labeled cAMP and varying concentrations of ESI-08. The displacement of the labeled cAMP is measured, and the IC50 value (8.4 µM) is determined. Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can also be used to measure direct binding affinity. These assays confirm that ESI-08 directly binds to the cAMP-binding domain of EPAC, blocking its activation.
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| Cell Assay |
In vitro cellular assays for ESI-08 are conducted in cell lines expressing EPAC1 or EPAC2. Cells are treated with ESI-08 at various concentrations (typically 1-50 µM) and stimulated with cAMP agonists or forskolin. EPAC activation is measured by assessing Rap1-GTP levels using pull-down assays or by measuring downstream signaling events such as ERK phosphorylation or cell adhesion changes. PKA activation is measured separately to confirm selectivity. Cell migration and invasion assays (e.g., transwell or wound healing assays) are used to assess functional effects. Cytotoxicity is assessed using standard cell viability assays. These experiments confirm the compound’s mechanism and its utility for studying EPAC function.
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| Animal Protocol |
In vivo animal experiments with ESI-08 are not extensively documented. The compound has been studied for its potential in modulating cell migration and invasion processes, which are relevant to cancer metastasis and inflammatory diseases. Animal models of cancer or inflammation could be used to evaluate the compound’s in vivo efficacy. ESI-08 would be administered via injection or oral gavage at varying doses. Endpoints would include tumor growth, metastasis, and inflammatory markers. However, detailed in vivo protocols have not been widely published. The compound is primarily used as a research tool in vitro. Further in vivo studies are needed to explore its therapeutic potential.
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| ADME/Pharmacokinetics |
Detailed pharmacokinetic (PK) data for ESI-08 are limited. The compound has a molecular weight of 353.48 and is soluble in organic solvents such as DMSO. Its aqueous solubility is low (0.05552 mg/L at 25°C). For in vivo studies, the compound would need to be formulated in appropriate vehicles to enhance bioavailability. Its density is 1.2±0.1 g/cm3, and it has a melting point of 265.25°C. The compound is typically stored as a solid at room temperature. Further PK studies are needed to fully characterize its absorption, distribution, metabolism, and excretion profile. The compound’s low aqueous solubility may limit its oral bioavailability.
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| Toxicity/Toxicokinetics |
Toxicological data for ESI-08 are limited. The compound is intended for research use only and is not approved for human therapeutic use. In cell-based assays, ESI-08 has been used at concentrations up to 50 µM without significant cytotoxicity reported in some contexts. However, comprehensive toxicological evaluations have not been extensively published. Standard laboratory safety precautions should be followed when handling this compound. Further toxicity studies would be required to support any potential clinical development. The compound should be handled with appropriate personal protective equipment.
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| References | |
| Additional Infomation |
ESI-08 is a potent and selective EPAC antagonist used as a research tool to study EPAC-mediated signaling pathways. Its chemical name is 4-cyclohexyl-2-[(2,5-dimethylphenyl)methylsulfanyl]-6-oxo-1H-pyrimidine-5-carbonitrile. The compound completely inhibits both EPAC1 and EPAC2 with an IC50 of 8.4 µM and does not inhibit cAMP-mediated PKA activation. This selectivity makes ESI-08 invaluable for dissecting EPAC-specific functions from PKA-mediated effects. It is used in research to study cell migration, invasion, and proliferation. The compound is available in high purity (≥98%) for research applications. Its non-cyclic nucleotide structure provides advantages over cyclic nucleotide-based inhibitors.
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| Molecular Formula |
C20H23N3OS
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| Molecular Weight |
353.481123209
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| Exact Mass |
353.156
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| CAS # |
301177-43-5
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| PubChem CID |
135473051
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| Appearance |
White to off-white solid powder
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| LogP |
4.5
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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 |
4
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| Heavy Atom Count |
25
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| Complexity |
624
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1(SCC2=CC(C)=CC=C2C)NC(=O)C(C#N)=C(C2CCCCC2)N=1
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| InChi Key |
LSHOSZQLVLPEGG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H23N3OS/c1-13-8-9-14(2)16(10-13)12-25-20-22-18(15-6-4-3-5-7-15)17(11-21)19(24)23-20/h8-10,15H,3-7,12H2,1-2H3,(H,22,23,24)
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| Chemical Name |
4-cyclohexyl-2-[(2,5-dimethylphenyl)methylsulfanyl]-6-oxo-1H-pyrimidine-5-carbonitrile
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| Synonyms |
ESI08; ESI 08
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 : ~100 mg/mL (~282.90 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 | 2.8290 mL | 14.1451 mL | 28.2901 mL | |
| 5 mM | 0.5658 mL | 2.8290 mL | 5.6580 mL | |
| 10 mM | 0.2829 mL | 1.4145 mL | 2.8290 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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