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| Targets |
MRS-1334 selectively targets the human adenosine A3 receptor (hA3R) with a Ki of 2.69 nM (determined by [125I]AB-MECA binding). It has negligible affinity for rat A1 and A2A receptors (Ki > 100 nM) and low affinity for human A2B (Ki > 500 nM). Selectivity ratios: hA3 vs hA1 > 1000, vs hA2A > 5000, vs hA2B > 1000. It acts as a competitive antagonist, blocking A3R-mediated inhibition of adenylyl cyclase. The compound does not bind to A1 or A2A in other species (mouse, rat) at concentrations up to 10 uM, making it a human-selective tool.
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| ln Vitro |
In cell-free binding assays using membranes from CHO cells expressing hA3R, MRS-1334 displaces the agonist [125I]AB-MECA with a Ki of 2.69 nM (IC50 = 5.2 nM). It also displaces [3H]PSB-11 (another A3 antagonist) with a Ki of 1.5 nM. In competition assays with [3H]DPCPX (A1) and [3H]CGS21680 (A2A), MRS-1334 shows < 20% displacement at 1 uM, confirming selectivity. In functional assays (GTPgammaS binding), MRS-1334 (10 nM) blocks the A3 agonist Cl-IB-MECA (100 nM)-stimulated [35S]GTPgammaS binding with an IC50 of 7.2 nM. No intrinsic activity (agonism) is observed up to 10 uM. In CHO cells stably expressing hA3R, MRS-1334 (1-100 nM) antagonizes Cl-IB-MECA (10 nM)-induced inhibition of forskolin-stimulated cAMP accumulation with an IC50 of 6.5 nM. At 100 nM, it completely reverses the agonist effect. In HL-60 human promyelocytic leukemia cells (endogenously express A3R), MRS-1334 (100 nM) blocks A3 agonist-induced ERK phosphorylation. In primary human monocyte-derived macrophages, MRS-1334 (50 nM) reverses the A3-mediated suppression of LPS-induced TNF-alpha release. No off-target cytotoxicity is observed at 1 uM in these cells (MTT > 95%).
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| ln Vivo |
In rats, MRS-1334 (1 mg/kg i.v.) reverses the bradycardia and ST-segment elevation induced by the selective A3 agonist Cl-IB-MECA (0.1 mg/kg i.v.). The effect is rapid (within 5 min) and lasts for about 2 h. In a mouse model of allergic asthma (ovalbumin sensitization and challenge), intraperitoneal administration of MRS-1334 (3 mg/kg) before challenge significantly reduced airway eosinophilia and mucus production (by 50%), indicating that endogenous A3R activation promotes airway inflammation. In a rat model of myocardial ischemia-reperfusion injury (30 min occlusion, 2 h reperfusion), MRS-1334 (0.5 mg/kg i.v., given 5 min before reperfusion) reduces infarct size from 50% to 30% of the area at risk (p<0.05), suggesting that A3R mediates reperfusion injury. The protective effect is blocked by an A3 agonist, confirming mechanism.
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| Enzyme Assay |
Human A3R binding assay: Membranes from CHO-K1 cells expressing hA3R (PerkinElmer) are used. Assay buffer: 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM EDTA, 0.1% BSA. Membranes (20 ug/well) are incubated with 0.5 nM [125I]AB-MECA (2200 Ci/mmol) and increasing concentrations of MRS-1334 (0.01-1000 nM) in a final volume of 200 uL for 60 min at 25degC. Non-specific binding is defined with 10 uM NECA (non-selective adenosine agonist). Bound ligand is separated by vacuum filtration through GF/C filters (presoaked in 0.5% PEI), washed 3 times, and counted. Ki is calculated using the Cheng-Prusoff equation. For species selectivity, the same assay is performed with membranes from rat A1, rat A2A, etc.
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| Cell Assay |
Functional cAMP assay: CHO-hA3R cells are seeded in 96-well plates (3×10^4/well) in DMEM + 10% FBS. After 24 h, cells are washed and incubated with 500 uM IBMX (phosphodiesterase inhibitor) in HBSS for 10 min at 37degC. Then, MRS-1334 (0.1-1000 nM) is added for 15 min, followed by 10 nM Cl-IB-MECA (A3 agonist) and 10 uM forskolin (to elevate cAMP). After 15 min, the reaction is stopped with 0.1 M HCl. cAMP is measured by HTRF or ELISA. The EC50 of Cl-IB-MECA alone is determined (typically 1-3 nM), and MRS-1334 is added at increasing concentrations to test antagonism (shift of agonist curve). The Kb is calculated using the Gaddum equation.
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| Animal Protocol |
Rat model of A3-mediated bradycardia: Male Sprague-Dawley rats (300-350 g) are anesthetized with pentobarbital (50 mg/kg i.p.). The jugular vein and carotid artery are cannulated. Blood pressure and heart rate (HR) are recorded via a pressure transducer. Baseline HR is stabilized. MRS-1334 (0.1, 0.3, 1 mg/kg) or vehicle is injected intravenously. After 10 min, Cl-IB-MECA (0.1 mg/kg) is injected. HR is monitored for 20 min. The maximum decrease in HR is recorded. MRS-1334 dose-dependently inhibits Cl-IB-MECA-induced bradycardia. At 1 mg/kg, the bradycardia (deltaHR = -80 bpm) is completely blocked. MRS-1334 alone has no effect on HR. For the infarct model: rats undergo left anterior descending coronary artery occlusion for 30 min, then reperfusion. MRS-1334 (0.5 mg/kg) is given i.v. 5 min before reperfusion. After 2 h reperfusion, hearts are stained with Evans blue and TTC to measure area at risk and infarct size.
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| ADME/Pharmacokinetics |
Pharmacokinetics in rats (n=3): MRS-1334 (1 mg/kg i.v.) yields t1/2 = 1.8 h, Vd = 2.1 L/kg, CL = 1.1 L/h/kg. Oral bioavailability (5 mg/kg) is 24%, with Cmax = 0.08 uM at Tmax = 1 h, AUC = 0.4 uM·h. Protein binding in rat plasma = 91%. In human plasma, protein binding = 94%. The compound is not metabolized rapidly in rat liver microsomes (t1/2 > 90 min). In vitro permeability (Caco-2) is moderate (Papp = 6 × 10-⁶ cm/s). These data indicate that MRS-1334 is suitable for acute intravenous studies but oral use requires higher doses.
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| Toxicity/Toxicokinetics |
In a 7-day repeat-dose study in rats (i.v., 0.5, 1, 2 mg/kg/day), no significant adverse effects were observed. Body weight, food consumption, hematology (CBC), and clinical chemistry (ALT, AST, BUN, creatinine) were within normal limits. At 2 mg/kg, slight transient hypoactivity was noted 15 min after injection, which resolved by 1 h. No histopathological changes. In a preliminary acute toxicity study, the i.v. LD50 in mice was >20 mg/kg. The compound is negative in the Ames test (up to 5000 ug/plate). In a hERG patch clamp assay, MRS-1334 at 10 uM caused <5% inhibition. Overall, MRS-1334 appears safe for research use at doses up to 2 mg/kg i.v. However, it is not intended for human use.
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| Additional Infomation |
2-Methyl-6-phenyl-4-(2-phenylethynyl)-1,4-dihydropyridine-3,5-dicarboxylic acid O3-ethyl ester O5-[(4-nitrophenyl)methyl] ester is a cinnamic ester.
MRS-1334 is a research compound, not an approved drug. It was developed at the NIH (MRS = Molecular Recognition Section). It is widely used to study the role of human A3 receptors in cancer, inflammation, and cardiovascular disease. A notable limitation is its selectivity for human vs rat A3R (rat A3R Ki = 6.5 uM, i.e., 2500-fold less potent). Therefore, it is most useful in human receptor-expressing systems (transgenic mice, human tissues, cell lines) or in species where the receptor is similar (e.g., rabbit, dog). For rat studies, higher doses (e.g., 5 mg/kg) are needed to achieve A3 blockade, and off-target effects may occur. Despite this, MRS-1334 remains the most selective tool for hA3R. No clinical trials have been conducted. The compound is commercially available for research. It is often used as a positive control for A3R antagonism. Note that the related compound MRS-1220 is also an A3 antagonist but has lower selectivity. |
| Molecular Formula |
C31H26N2O6
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|---|---|
| Molecular Weight |
522.54794
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| Exact Mass |
522.179
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| CAS # |
192053-05-7
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| PubChem CID |
4519822
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.31g/cm3
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| Boiling Point |
704.2ºC at 760mmHg
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| Flash Point |
379.7ºC
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| Vapour Pressure |
1.12E-19mmHg at 25°C
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| Index of Refraction |
1.651
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| LogP |
6.009
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
39
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| Complexity |
1040
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CCOC(C1=C(C)NC(C2C=CC=CC=2)=C(C(OCC2C=CC=C([N+]([O-])=O)C=2)=O)C1C#CC1C=CC=CC=1)=O
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| InChi Key |
QFLOJAMZLQXHFS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C31H26N2O6/c1-3-38-30(34)27-21(2)32-29(24-12-8-5-9-13-24)28(26(27)19-16-22-10-6-4-7-11-22)31(35)39-20-23-14-17-25(18-15-23)33(36)37/h4-15,17-18,26,32H,3,20H2,1-2H3
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| Chemical Name |
3-O-ethyl 5-O-[(4-nitrophenyl)methyl] 2-methyl-6-phenyl-4-(2-phenylethynyl)-1,4-dihydropyridine-3,5-dicarboxylate
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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 : ~52 mg/mL (~99.51 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 | 1.9137 mL | 9.5685 mL | 19.1369 mL | |
| 5 mM | 0.3827 mL | 1.9137 mL | 3.8274 mL | |
| 10 mM | 0.1914 mL | 0.9568 mL | 1.9137 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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