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Purity: ≥98%
Dabigatran ethyl ester is the ethyl ester form of dabigatran which is an antithrombotic drug used to treat blood clot. Dabigatran (also known as BIBR 953 and BIBR 953ZW) is a potent, nonpeptide, reversible, selective and direct thrombin inhibitor with an IC50 of 9.3 nM in a cell-free assay. Dabigatran is designed to be converted into an orally active prodrug BIBR 1048 due to its highly polar, zwitterionic nature and poor oral absorption. Dabigatran inhibits thrombin in a competitive fashion. This inhibition is rapid and reversible. Dabigatran inhibits both clot-bound and free thrombin. Dabigatran is demonstrated to have an anticoagulant efficacy both in vitro and ex vivo.
| Targets |
Thrombin; ribosyldihydronicotinamide dehydrogenase (NQO2)
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|---|---|
| ln Vitro |
1. NQO2 Inhibition Potency: Dabigatran ethyl ester (2) is a potent inhibitor of ribosyldihydronicotinamide dehydrogenase (NQO2). It exhibits significantly higher inhibitory activity than the parent compound dabigatran (1), with a calculated inhibition constant (K_i) of 0.9 µM and a half-maximal inhibitory concentration (IC_50) of 0.8 µM in the NADH-dependent mitomycin C metabolism functional assay using recombinant NQO2. This represents an approximately 75-fold increase in potency compared to dabigatran (K_i ~70 µM, IC_50 ~60 µM).[1]
2. Thrombin Inhibition Potency: Compound 2 also demonstrates increased inhibitory potency against its primary target, thrombin, compared to dabigatran (1). The IC_50 value for thrombin inhibition by Compound 2 was determined to be 1.2 nM, which is approximately 11.5-fold lower (more potent) than dabigatran's IC_50 of 13.8 nM.[1] 3. Binding Affinity to NQO2: Competition experiments against Capture Compound 5 (CC 5, derived from dabigatran) for binding to recombinant NQO2 showed that Compound 2 displaces CC 5 with much higher affinity than dabigatran itself. The concentration causing half-maximal competition of NQO2 binding was 1.5 µM for Compound 2, compared to 11 µM for dabigatran.[1] 4. Binding Mode Analysis (Molecular Modeling): Molecular docking predicted that the ethyl ester group of Compound 2 forms favorable hydrophobic van der Waals interactions with residues Ile128 and Met154 in the NQO2 binding pocket, explaining its higher affinity compared to the carboxylic acid of dabigatran (1). This increased lipophilic interaction surface also correlated well with the observed affinity ratio between Compounds 1 and 2. Ligand efficiency (LE) and fit quality (FQ) calculations for thrombin and NQO2 binding were similar for both compounds (FQ ~0.6 for thrombin, ~0.4 for NQO2)[1]. |
| Enzyme Assay |
1. NQO2 Functional Inhibition Assay: The functional inhibition of NQO2 by Dabigatran ethyl ester/Compound 2 was determined using the NADH-dependent metabolism of mitomycin C as the enzymatic reaction. Recombinant human NQO2 (0.5 µM) was incubated with mitomycin C (50 µM) and varying concentrations of Compound 2 in 100 mM potassium phosphate buffer (pH 5.8) at room temperature for 5 minutes. The reaction was initiated by adding increasing concentrations of NADH (the co-substrate). The enzymatic activity (consumption of NADH) was monitored photometrically by measuring the decrease in absorbance at 340 nm over 30 minutes at room temperature. Michaelis-Menten kinetics analysis (using Sigma Plot 10) of the data obtained at different inhibitor concentrations was used to determine the K_i and IC_50 values for Compound 2's competitive inhibition of NQO2.[1]
2. Thrombin Functional Inhibition Assay: The inhibitory potency of Compound 2 against human α-thrombin was assessed using a fluorogenic substrate assay. Boc-Val-Pro-Arg-AMC substrate (15 µM) in assay buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) was supplemented with varying concentrations of Compound 2 (serial dilutions from 2.5 mM to 75 nM in DMSO). Thrombin was added to a final concentration of 5.5 ng/mL and the reaction incubated for 2 hours at room temperature. The enzymatic cleavage of the substrate, releasing the fluorescent AMC moiety, was measured on a plate reader (excitation 355 nm, emission 460 nm). Dose-response curves were generated to calculate the IC_50 value [1]. |
| Cell Assay |
1. Specific Binding to Endogenous NQO2: Western Blot analysis confirmed that Compound 2/Dabigatran ethyl ester effectively competes with Capture Compound 5 (CC 5) for binding to endogenous NQO2 protein within whole cell lysates (HepG2 cells) and lysates derived from human liver microsomes. This demonstrates the specific interaction occurs in complex cellular environments, not just with purified protein.[1]
2. Cross-Competition for NQO2 Binding Pocket: Capture experiments using recombinant NQO2 and visualized by silver-stained SDS-PAGE demonstrated that Compound 2 competes effectively for the same binding site on NQO2 as dabigatran (1) and the known NQO2 inhibitor imatinib (4). Pre-incubation with Compound 2 significantly reduced the capture of NQO2 by CC 5 [1]. |
| References |
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| Additional Infomation |
Recent studies have revealed that compounds believed to be highly selective frequently address multiple target proteins. We investigated the protein interaction profile of the widely prescribed thrombin inhibitor dabigatran (1), resulting in the identification and subsequent characterization of an additional target enzyme. Our findings are based on an unbiased functional proteomics approach called capture compound mass spectrometry (CCMS) and were confirmed by independent biological assays. 1 was shown to specifically bind ribosyldihydronicotinamide dehydrogenase (NQO2), a detoxification oxidoreductase. Molecular dockings predicted and biological experiments confirmed that Dabigatran ethyl ester (2) inhibits NQO2 even more effectively than the parent 1 itself. Our data show that 1 and 2 are inhibitors of NQO2, thereby revealing a possible new aspect in the mode of action of 1. We present a workflow employing chemical proteomics, molecular modeling, and functional assays by which a compound’s protein-interaction profile can be determined and used to tune the binding affinity.[1]
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| Molecular Formula |
C27H29N7O3
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|---|---|
| Molecular Weight |
499.56426
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| Exact Mass |
499.233
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| Elemental Analysis |
C, 64.91; H, 5.85; N, 19.63; O, 9.61
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| CAS # |
429658-95-7
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| Related CAS # |
Dabigatran;211914-51-1;Dabigatran-d4 hydrochloride;Dabigatran etexilate;211915-06-9;Dabigatran etexilate mesylate;872728-81-9;Dabigatran ethyl ester hydrochloride;211914-50-0;Dabigatran-d4;1618637-32-3
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| PubChem CID |
446804
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
759.5±70.0 °C at 760 mmHg
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| Flash Point |
413.1±35.7 °C
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| Vapour Pressure |
0.0±2.6 mmHg at 25°C
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| Index of Refraction |
1.658
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| LogP |
1.78
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
37
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| Complexity |
788
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CCOC(=O)CCN(C1=CC=CC=N1)C(=O)C2=CC3=C(C=C2)N(C(=N3)CNC4=CC=C(C=C4)C(=N)N)C
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| InChi Key |
BGLLICFSSKPUMR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H29N7O3/c1-3-37-25(35)13-15-34(23-6-4-5-14-30-23)27(36)19-9-12-22-21(16-19)32-24(33(22)2)17-31-20-10-7-18(8-11-20)26(28)29/h4-12,14,16,31H,3,13,15,17H2,1-2H3,(H3,28,29)
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| Chemical Name |
ethyl 3-[[2-[(4-carbamimidoylanilino)methyl]-1-methylbenzimidazole-5-carbonyl]-pyridin-2-ylamino]propanoate
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| Synonyms |
Dabigatran ethyl ester
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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) |
H2O : < 0.1 mg/mL
DMSO :< 1 mg/mL |
|---|---|
| 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 | 2.0018 mL | 10.0088 mL | 20.0176 mL | |
| 5 mM | 0.4004 mL | 2.0018 mL | 4.0035 mL | |
| 10 mM | 0.2002 mL | 1.0009 mL | 2.0018 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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