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| Targets |
PDE3
EMD 53998 has a dual mechanism of action, functioning as both a phosphodiesterase III (PDE III) inhibitor and a calcium sensitizer. The primary molecular target is PDE III, an enzyme that hydrolyzes cyclic AMP (cAMP) in cardiac and vascular smooth muscle cells. By inhibiting PDE III, EMD 53998 increases intracellular cAMP levels, leading to protein kinase A (PKA) activation, increased calcium influx through L-type calcium channels, and enhanced contractility. The compound also sensitizes the contractile apparatus to calcium (calcium-sensitizing effect), which is more pronounced in the (+)-enantiomer EMD 57033. This calcium-sensitizing effect is mediated by binding to cardiac troponin C (cTnC) or troponin I, stabilizing the troponin complex and increasing myofilament calcium sensitivity. The racemic mixture exhibits both activities. EMD 53998 is not known to significantly inhibit other PDE isoforms (PDE I, II, IV) at therapeutic concentrations. |
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| ln Vitro |
In vitro studies demonstrate the cardiotonic and vasodilatory effects of EMD 53998. In isolated guinea pig papillary muscle preparations, EMD 53998 (0.1-30 microM) produces a concentration-dependent increase in contractile force (positive inotropic effect) with an EC50 of approximately 1-3 microM. The maximum effect is comparable to that of the PDE III inhibitor milrinone. In skinned cardiac fiber preparations (where cell membrane is removed), the compound (0.1-10 microM) increases calcium sensitivity of the contractile apparatus, shifting the pCa-force relationship leftward by 0.2-0.3 pCa units. This calcium-sensitizing effect is unique among PDE inhibitors. In isolated rat aorta pre-contracted with phenylephrine or KCl, EMD 53998 (0.1-30 microM) induces relaxation (vasodilation) with IC50 values of 1-5 microM, indicating balanced inotropic and vasodilatory activity (inodilator). No significant cytotoxicity is reported in cardiac myocytes at concentrations up to 100 microM.
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| ln Vivo |
In vivo activity of EMD 53998 has been evaluated in animal models of heart failure. In anesthetized dogs with pentobarbital-induced heart failure, intravenous administration of EMD 53998 (0.1-1 mg/kg) increases cardiac output (CO) and left ventricular dP/dt max (a measure of contractility) by 20-40% while reducing systemic vascular resistance (SVR) and left ventricular end-diastolic pressure (LVEDP). Heart rate increases modestly (5-15 beats/min). In conscious rats with myocardial infarction-induced chronic heart failure, oral administration of EMD 53998 (1-10 mg/kg) for 4 weeks improves ejection fraction and reduces cardiac hypertrophy compared to vehicle. In anesthetized cats, EMD 53998 (0.3-3 mg/kg IV) increases contractility without significant hypotension at lower doses. The duration of action is 1-3 hours, consistent with the half-life. No human efficacy studies were published prior to discontinuation of development.
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| Enzyme Assay |
Cell-free enzyme assays for PDE III inhibition: Human recombinant PDE IIIA (5-20 ng) is incubated with EMD 53998 (0.1-1000 nM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 mM EGTA, 0.1% BSA) containing 0.1-1 microM [3H]-cAMP as substrate. The reaction is carried out at 30degC for 20-60 minutes, then stopped by boiling or by adding PDE stop solution. [3H]-5'-AMP is converted to [3H]-adenosine by adding snake venom 5'-nucleotidase (1 mg/mL) for 10 minutes. Unreacted substrate is separated by ion-exchange resin (AG1-X2) or by scintillation proximity assay (SPA) beads. Radioactivity (or luminescence for SPA) is measured, and IC50 is calculated. For calcium-sensitizing assays (skinned fibers), trabeculae or papillary muscles from guinea pig or rat hearts are skinned with Triton X-100 (1%, 30 minutes). Fibers are mounted on a force transducer, bathed in calcium-EGTA buffers (pCa 8.0 to 4.5), and force is measured. EMD 53998 (0.1-100 microM) is added, and the leftward shift of the pCa-force curve is quantified.
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| Cell Assay |
Cellular inotropic assays use isolated adult rat or guinea pig ventricular myocytes. Myocytes are isolated by Langendorff perfusion with collagenase and protease, then plated on laminin-coated coverslips. Cells are field-stimulated at 0.5-1 Hz in Tyrode solution (37degC). Sarcomere shortening is measured by video edge detection or by using a MyoCam system. Myocytes are perfused with EMD 53998 (0.1-30 microM) for 5-15 minutes. The compound increases fractional shortening (cell contraction amplitude) by 20-50% with EC50 ~1 microM. Intracellular calcium ([Ca2+]i) is measured simultaneously using Fura-2 AM or Fluo-4 AM. Unlike pure PDE III inhibitors, EMD 53998 increases contractility with a lesser increase in [Ca2+]i, confirming calcium sensitization. For vasodilation assays, rat aortic rings (2-3 mm length) are mounted in organ baths, pre-contracted with phenylephrine (1 microM) or KCl (60 mM), and cumulative concentration-response curves to EMD 53998 (0.01-100 microM) are generated. The IC50 for relaxation is 1-5 microM.
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| Animal Protocol |
In vivo animal protocols: For anesthetized dog studies, beagle dogs (10-15 kg) are anesthetized with pentobarbital (30 mg/kg IV), intubated, and ventilated. A Millar catheter is inserted into the left ventricle via the carotid artery to measure LV pressure (dP/dt max). A Swan-Ganz catheter measures cardiac output by thermodilution. EMD 53998 is administered intravenously as a bolus (0.1, 0.3, 1 mg/kg) or infusion (0.01-0.1 mg/kg/min). Hemodynamic parameters (heart rate, mean arterial pressure, LV dP/dt max, cardiac output, systemic vascular resistance) are recorded continuously for 2-3 hours. For chronic heart failure models, male Sprague-Dawley rats (200-250g) undergo left coronary artery ligation to induce myocardial infarction. After 4-6 weeks, rats with ejection fraction <40% are randomized to EMD 53998 (1-10 mg/kg/day orally) or vehicle for 4 weeks. Echocardiography is performed at baseline and weekly. At sacrifice, hearts are weighed, and histological analysis (fibrosis, myocyte cross-sectional area) is performed. No clinical studies were completed.
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| ADME/Pharmacokinetics |
Pharmacokinetics of EMD 53998 were characterized in preclinical species but not in humans. In rats, after intravenous administration (1 mg/kg), the compound has a distribution half-life (t1/2alpha) of 5-10 minutes and an elimination half-life (t1/2beta) of 60-90 minutes. Clearance is moderate (20-40 mL/min/kg), volume of distribution (Vd) is 1-2 L/kg, suggesting tissue distribution. After oral administration (10 mg/kg), Tmax is 0.5-1 hour, and oral bioavailability is 30-50%, limited by first-pass metabolism. Plasma protein binding is high (>90%). Metabolism occurs primarily via CYP3A-mediated O-demethylation of the dimethoxybenzoyl group, followed by glucuronidation. Metabolites are excreted in urine (40%) and feces (50%). In dogs, half-life is 2-4 hours. The active enantiomer EMD 57033 has similar PK properties. No human PK data were published. The compound is not approved and no longer in development.
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| Toxicity/Toxicokinetics |
Toxicology data for EMD 53998 were reported in preclinical studies. In rats, acute oral LD50 >500 mg/kg. In repeat-dose studies (28 days, oral), doses up to 50 mg/kg/day were tolerated with mild body weight gain reduction; higher doses (100-200 mg/kg/day) caused gastrointestinal disturbances (diarrhea, reduced food intake) and mild increases in liver enzymes (ALT, AST) reversible upon withdrawal. No significant nephrotoxicity or myelosuppression was observed. In dogs, oral doses of 10 mg/kg/day for 14 days were well-tolerated; at 30 mg/kg/day, emesis, diarrhea, and mild hypotension were noted. Cardiac safety: No significant QT prolongation or arrhythmias were reported in preclinical models, though PDE III inhibitors as a class (e.g., milrinone) have been associated with increased mortality in human heart failure trials due to arrhythmias. No genotoxicity (Ames test negative). No carcinogenicity studies were performed. The compound was not approved due to the class effect of PDE III inhibitors increasing mortality in heart failure patients (e.g., PROMISE trial with milrinone) and the development of newer agents.
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| References | |
| Additional Infomation |
EMD 53998 is a racemic isomer; EMD 57439 is a (-)- isomer; EMD 57033 is a (+)- isomer.
EMD 53998 is a research compound that was developed by EMD Pharmaceuticals (Merck KGaA, Darmstadt, Germany) in the 1990s as a potential treatment for congestive heart failure. It represents a unique "inodilator" (inotropic + vasodilator) with calcium-sensitizing properties. The compound did not receive regulatory approval and is no longer in clinical development. The racemic mixture was separated into enantiomers: EMD 57033 (the (+)-isomer) exhibits greater calcium-sensitizing activity, while EMD 57439 (the (-)-isomer) is primarily a PDE III inhibitor. The calcium-sensitizing mechanism was of significant scientific interest as it offered a way to increase contractility without increasing intracellular calcium and thus potentially reducing arrhythmia risk. However, clinical development was discontinued likely due to the failure of other calcium sensitizers (e.g., pimobendan, levosimendan) to show improved survival in heart failure. Today, EMD 53998 and its enantiomers are used only as research tools for studying cardiac contractility and calcium sensitization. The compound is available from chemical suppliers for laboratory use only. No clinical trials are active. |
| Molecular Formula |
C22H23N3O4S
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| Molecular Weight |
425.50
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| Exact Mass |
425.141
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| CAS # |
120223-04-3
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| Related CAS # |
(+)-EMD 57033; 147527-31-9
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| PubChem CID |
187079
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| Appearance |
Solid powder
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| LogP |
2.986
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
30
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| Complexity |
687
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| Defined Atom Stereocenter Count |
0
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| SMILES |
COC1C=CC(C(N2CCCC3C=C(C4=NNC(=O)SC4C)C=CC2=3)=O)=CC=1OC
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| InChi Key |
IZLRMTJLQCLMKF-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H23N3O4S/c1-13-20(23-24-22(27)30-13)15-6-8-17-14(11-15)5-4-10-25(17)21(26)16-7-9-18(28-2)19(12-16)29-3/h6-9,11-13H,4-5,10H2,1-3H3,(H,24,27)
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| Chemical Name |
5-[1-(3,4-dimethoxybenzoyl)-3,4-dihydro-2H-quinolin-6-yl]-6-methyl-3,6-dihydro-1,3,4-thiadiazin-2-one
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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 : 100 mg/mL (235.02 mM; with sonication)
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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 | 2.3502 mL | 11.7509 mL | 23.5018 mL | |
| 5 mM | 0.4700 mL | 2.3502 mL | 4.7004 mL | |
| 10 mM | 0.2350 mL | 1.1751 mL | 2.3502 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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