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Purity: ≥98%
EMD638683 is a potent inhibitor of SGK1 with an IC50 of 3 μM. EMD638683 treatment significantly augmented the radiation-induced decrease of forward scatter, increase of phosphatidylserine exposure, decrease of mitochondrial potential, increase of caspase 3 activity, increase of DNA fragmentation and increase of late apoptosis. EMD638683 promotes radiation-induced suicidal death of colon tumor cells in vitro and decreases the number of colonic tumors following chemical carcinogenesis in vivo.
| Targets |
Serum- and glucocorticoid-inducible kinase 1 (SGK1) [1]
Other kinases inhibited at higher concentrations: MSK1 (IC50 ratio 37 relative to SGK1? actual IC50 not given), SGK2, SGK3, PRK2, PKA [1] |
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| ln Vitro |
EMD638683 is a conjugate of SGK1. Half-maximum effect (IC50) of EMD638683's inhibition of NDRG1 (N-Myc downstream regulatory gene 1) needs the addition of 3.35 μM EMD638683 to the cell culture medium. Moreover, three cAMP topologies in control and EMD638683 (50 μM) are inhibited by EMD63868: SGK isoforms SGK2 and SGK3, mitogen- and casein-activated protein kinase 1 (MSK1), and protein kinase C-related protein kinase 2 (PKR2) [1]. irradiation markedly raised the proportion of CaCo-2 cells treated with late cells. ) treated CaCo-2 cells. The percentage of CaCo-2 cells tended to rise with treatment with EMD638683 alone. A small number received EMD638683 treatment following radiation. In light of this, EMD638683 therapy greatly increased UV radiation following irradiation [2].
In HeLa cells, EMD638683 inhibited SGK1-dependent phosphorylation of NDRG1 with an IC50 of 3.35 ± 0.32 µM (cell culture medium concentration) [1]. In CaCo-2 colon carcinoma cells, EMD638683 (50 µM for 24 h) alone did not significantly modify forward scatter (cell volume) but significantly augmented radiation-induced (3 Gray) cell shrinkage (forward scatter decrease) [2]. EMD638683 alone tended to increase the percentage of CaCo-2 cells with depolarized mitochondria; radiation alone also tended to increase it; the combination significantly increased depolarization [2]. EMD638683 alone tended to enhance caspase-3 activity; radiation alone also tended to enhance it; the combination significantly increased caspase-3 activity [2]. EMD638683 alone tended to enhance annexin V binding; radiation significantly increased annexin V binding both in absence and presence of EMD638683, with significantly higher binding in the presence of EMD638683 [2]. EMD638683 alone tended to enhance late apoptosis; radiation significantly increased late apoptosis; the combination further increased the percentage of apoptotic cells [2]. |
| ln Vivo |
In comparison to the night-treated group, the EMD638683-treated group's night weight was much lower and its night length was significantly longer. This result suggests that, following chemical action, EMD638683 has an impact on tumor growth. Additionally, the group receiving EMD treatment saw a considerable decrease in stomach weight. was given EMD638683 treatment, which considerably lowered the quantity of tumors that grew as a result of food processing [2]. In lung cancer patients, monocrotaline (MCT)-guided vascular transplantation is stopped from progressing by EMD638683 (20 mg/kg, gavage). According to the hemodynamic profile, the EMD638683 therapy was found to lower the right ventricular hypertrophy index (RVHI) (0.27 vs. 0.41; P<0.05; n = 6) and right ventricular systolic pressure (15.8 vs. 28.2 mmHg; P<0.05; n = 6) as compared to the control dose [3].
In fructose/saline-treated hyperinsulinemic mice, EMD638683 (4460 ppm in chow, ~600 mg/kg/day) for 4 days significantly decreased systolic blood pressure from 111±4 mmHg to 87±3 mmHg; replacement with placebo food caused blood pressure to rise back to 106±11 mmHg [1]. EMD638683 treatment did not modify blood pressure in tap water-drinking wild-type mice (normoinsulinemic) nor in SGK1 knockout mice [1]. Chronic (4 weeks) co-administration of EMD638683 with fructose/high salt prevented the rise in blood pressure caused by fructose/high salt [1]. In a chemical carcinogenesis model (1,2-dimethylhydrazine + dextran sulfate sodium), EMD638683 treatment (4460 ppm in chow, ~600 mg/kg/day) significantly reduced the number of colonic tumors compared to placebo [2]. EMD638683 treatment significantly increased urinary flow rate and significantly decreased body weight in fructose/saline-treated mice [1]. |
| Enzyme Assay |
EMD638683 was screened against a panel of 69 protein kinases. Kinase activities were measured robotically at room temperature (21°C) in 96-well format using MgATP as cofactor. Reactions were stopped by addition of orthophosphoric acid and spotted onto P81 filter plates. For each kinase, activity in the presence of 1 µM EMD638683 was expressed as percentage of activity without inhibitor. Kinases showing <50% residual activity (e.g., MSK1, SGK2, SGK3, PRK2) were further evaluated. IC50 values were determined using ten different concentrations of EMD638683 for each kinase, with each data point in triplicate. The relative inhibitory effect (selectivity ratio) was calculated by dividing the IC50 for each kinase by the IC50 for SGK1 [1].
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| Cell Assay |
For NDRG1 phosphorylation assay: HeLa cells were plated at 10-20×10³ cells/cm² in DMEM with 10% FCS. After 24 h, cells were treated with various concentrations of EMD638683 (diluted in DMSO, final DMSO 1%) for 24 h. Cells were lysed in lysis buffer containing Triton X-100, protease inhibitors, and phosphatase inhibitors. Lysates were sonicated, denatured, separated by SDS-PAGE (4-12% Bis-Tris gels for P-NDRG1, 7% gels for NDRG1), transferred to nitrocellulose membranes, and immunoblotted with NDRG1 or phospho-NDRG1 antisera. Bands were detected by peroxidase-conjugated secondary antibody and chemiluminescence. P-NDRG1 levels were normalized to total NDRG1. The IC50 for EMD638683 was determined from the dose-response curve (3.35±0.32 µM) [1].
For CaCo-2 cell apoptosis assays: Cells were grown in DMEM with 10% FBS. 10⁵ cells/well in 6-well plates were treated with 50 µM EMD638683 or solvent control (0.2 µl DMSO) for 24 h, then exposed to 3 Gray radiation (3.18 min) and further incubated for 72 h. Cells were detached with trypsin-EDTA, washed, and analyzed by flow cytometry. Cell volume was estimated from forward scatter. Mitochondrial potential was measured using JC-9 dye (10 mg/ml, 10 min at 37°C in dark); depolarized cells showed fluorescence shift. Active caspase-3 was detected using a fluorescent active caspase-3 staining kit (1 h incubation). Apoptosis was determined by annexin V FITC and propidium iodide staining after fixation in 70% ethanol and incubation in hypotonic buffer [2]. |
| Animal Protocol |
For hypertension studies: Mice were fed control diet (0.2% Na⁺, 0.7% K⁺). EMD638683 was mixed into chow at 4460 ppm, resulting in a dose of approximately 600 mg/kg/day. Placebo food had identical composition without the inhibitor. Hyperinsulinemia was induced by replacing drinking water with 10% fructose for 3 weeks, then adding isotonic saline for another 14 days. Systolic blood pressure was measured by tail-cuff method after appropriate training, prewarming to 29°C, in a quiet semi-dark environment, between 10-12 am. Five measurements per session were taken; session accepted if deviation <5 mmHg. For metabolic cage studies, mice were habituated for 3 days, then 12-hour urine collections (7 pm to 7 am) were performed. Urinary Na⁺ and K⁺ were measured by flame photometry [1].
For carcinogenesis studies: Wild-type mice (8 weeks old) received intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine in 0.9% saline, followed by three cycles of 30 g/L dextran sulfate sodium in drinking water for 7 days alternating with 14 days of normal water. EMD638683 was administered as 4460 ppm in chow (~600 mg/kg/day) starting before carcinogen treatment. At 20 weeks of age, mice were sacrificed, colons were examined, measured, and tumors counted [2]. |
| Toxicity/Toxicokinetics |
In fructose/saline-treated mice, EMD638683 treatment for 4 days did not significantly modify fluid and food intake; urinary Na⁺ and K⁺ excretion were not significantly altered; body weight significantly decreased [1].
EMD638683 did not affect blood pressure in normoinsulinemic wild-type mice or SGK1 knockout mice, indicating low off-target cardiovascular effects [1]. In carcinogenicity studies, mortality: 3 out of 11 mice died in placebo group; 1 out of 12 died in EMD638683-treated group. Body weight at the end of the study was similar between groups (placebo: 23.73±0.53 g; EMD638683: 23.99±0.40 g) [2]. |
| References |
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| Additional Infomation |
EMD638683 is a water-soluble SGK1 inhibitor. It reverses hypertension caused by hyperinsulinism/salt excess but does not affect blood pressure in normoinsulinemic mice or mice without salt excess. It may serve as a template for drugs counteracting hypertension in individuals with type II diabetes and metabolic syndrome [1].
EMD638683 promotes radiation-induced apoptosis of colon carcinoma cells and decreases colonic tumor growth following chemical carcinogenesis in vivo, suggesting SGK1 as an attractive pharmacological target for cancer treatment [2]. |
| Molecular Formula |
C18H18F2N2O4
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|---|---|
| Molecular Weight |
364.3488
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| Exact Mass |
364.123
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| CAS # |
1181770-72-8
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| Related CAS # |
EMD638683 R-Form;1184940-47-3;EMD638683 S-Form;1184940-46-2
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| PubChem CID |
44182398
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| Appearance |
Off-white to light brown solid powder
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| LogP |
3.267
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
26
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| Complexity |
498
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
SSNAPUUWBPZGOY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H18F2N2O4/c1-3-13-9(2)15(23)5-4-14(13)17(25)21-22-18(26)16(24)10-6-11(19)8-12(20)7-10/h4-8,16,23-24H,3H2,1-2H3,(H,21,25)(H,22,26)
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| Chemical Name |
N'-[2-(3,5-difluorophenyl)-2-hydroxyacetyl]-2-ethyl-4-hydroxy-3-methylbenzohydrazide
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| Synonyms |
EMD638683 EMD 638683 EMD-638683.
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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 : ≥ 50 mg/mL (~137.23 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.86 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 25.0 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: ≥ 2.5 mg/mL (6.86 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (6.86 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7446 mL | 13.7231 mL | 27.4461 mL | |
| 5 mM | 0.5489 mL | 2.7446 mL | 5.4892 mL | |
| 10 mM | 0.2745 mL | 1.3723 mL | 2.7446 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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