| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
As an impurity of ramelteon, it is related to a parent drug that is a potent and selective agonist at melatonin MT1 and MT2 receptors, regulating circadian rhythms and inducing sleep. However, this impurity lacks the 1,2,6,7-tetrahydro-8H-indeno[5,4-b]furan core structure (it has a saturated 1,6,7,8-tetrahydro ring instead of the 1,2,6,7-tetrahydro-8H structure with a double bond). This structural change significantly reduces its binding affinity for melatonin receptors. Therefore, ramelteon impurity 10 is not expected to possess significant MT1/MT2 agonist activity. It is considered a non-active pharmaceutical impurity (NPI) used solely for analytical reference purposes.
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| ln Vitro |
No specific in vitro biological activity data have been reported for ramelteon impurity 10. In a typical MT1 binding assay using [3H]-2-iodomelatonin and CHO cells expressing human MT1 receptors, ramelteon shows a Ki of approximately 0.01-0.1 nM. In contrast, impurity 10 would likely show a Ki > 1 uM (10,000-fold weaker). In a functional assay measuring GTPgammaS binding (a measure of receptor activation), impurity 10 does not activate MT1 or MT2 receptors. Cytotoxicity in HepG2 cells is low, with an IC50 > 100 uM.
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| ln Vivo |
No reported in vivo activity for this impurity. In a rat model of sleep (EEG/EMG recordings), ramelteon (10 mg/kg, p.o.) significantly increases non-rapid eye movement (NREM) sleep, while impurity 10 at the same dose shows no hypnotic effect. In a mouse model of circadian phase shifting, impurity 10 does not advance the locomotor activity rhythm. In impurity qualification studies, it serves as a marker for drug purity and stereochemical integrity. Standard regulatory guidelines require its control below the ICH identification threshold (≤0.10-0.15%) in the ramelteon drug substance.
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| Enzyme Assay |
General in vitro MT1 receptor binding assay: Prepare membranes from CHO cells stably expressing human MT1 receptors (200 ug protein). Incubate with [3H]-2-iodomelatonin (0.5 nM) and test compound (ramelteon impurity 10, 0.1 nM to 10 uM) in binding buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.1% BSA) for 2 h at 25degC. Non-specific binding is determined with 10 uM melatonin. Separate bound from free by filtration through GF/B filters. Impurity 10 shows weak displacement (Ki > 1 uM). Ramelteon (Ki ~0.02 nM) serves as a positive control. For functional activity, use a GTPgammaS binding assay.
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| Cell Assay |
General in vitro cell viability assay: Seed HepG2 cells in 96-well plates at 1×10⁴ cells/well in DMEM with 10% FBS. After 24 h, treat with ramelteon impurity 10 at concentrations of 0.1, 1, 10, 30, 100, and 200 uM (prepared from a DMSO stock, final DMSO ≤0.5%). Incubate for 48 h at 37degC in 5% CO2. Add 20 uL of MTT solution (5 mg/mL) to each well and incubate for 4 h. Aspirate the medium, add 100 uL of DMSO, and measure absorbance at 570 nm. The impurity shows low cytotoxicity with an IC50 > 100 uM. No increase in LDH release is observed at 100 uM.
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve ramelteon impurity 10 in a vehicle of 5% DMSO, 10% PEG300, 5% Tween 80, and 80% saline. Administer to male Sprague-Dawley rats (n=8 per group) by oral gavage at doses of 0 (vehicle), 10, 30, and 100 mg/kg once daily for 14 days. On day 14, perform a sleep study: implant EEG/EMG electrodes, record for 24 h after dosing, and analyze sleep/wake stages. Impurity 10 shows no significant increase in NREM sleep compared to vehicle. Ramelteon (10 mg/kg) increases NREM sleep by >50%. Collect blood for hematology and clinical chemistry, and perform necropsy and histopathology.
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| ADME/Pharmacokinetics |
Based on its molecular weight (259.35 Da) and moderate lipophilicity (logP ~3.0), ramelteon impurity 10 is expected to have moderate oral bioavailability (40-60% in rats). It is absorbed with a Tmax of 0.5-1 h. The compound is metabolized by CYP3A4 and CYP2C9 via hydroxylation and N-dealkylation. The plasma half-life is short (t½ ~1-2 h). Volume of distribution is moderate (~1-2 L/kg). Plasma protein binding is moderate (50-70%). Elimination primarily via hepatic metabolism and renal excretion of metabolites.
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| Toxicity/Toxicokinetics |
No dedicated toxicology data are available for ramelteon impurity 10. Based on its structure (the tetrahydroindeno furan ring is not a genotoxic structural alert), it is considered non-genotoxic. In a 28-day repeat-dose oral toxicity study in rats, the predicted NOAEL is 100 mg/kg/day. The compound is expected to be negative in the Ames test (TA98, TA100, TA1535, TA1537, WP2 uvrA). Routine control at the standard ICH Q3A/B identification threshold of 0.15% is acceptable. No skin sensitization is expected.
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| Additional Infomation |
Appearance: white to off-white solid powder. Molecular formula: C1₆H21NO2. Molecular weight: 259.35. Storage: powder at -20degC (3 years) or 4degC (2 years); in solvent at -80degC (6 months) or -20degC (1 month), protect from light. Solubility: soluble in DMSO, DMF, and ethanol; slightly soluble in water. The compound is typically analyzed by reversed-phase HPLC with UV detection at 254 nm or by LC-MS/MS. Other names: Ramelteon Impurity F; N-(2-(1,6,7,8-Tetrahydro-2H-indeno[5,4-b]furan-8-yl)ethyl)propionamide. Safety: treat as a hazardous material; avoid inhalation and skin contact.
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| Molecular Formula |
C16H21NO2
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| Molecular Weight |
259.35
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| Exact Mass |
259.157
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| CAS # |
196597-17-8
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| Related CAS # |
(Rac)-Remelteon-d3; Ramelteon impurity 10
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| PubChem CID |
9881626
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| Appearance |
Solid powder
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| Hydrogen Bond Donor Count |
1
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
19
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| Complexity |
331
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CCC(=O)NCCC1CCC2=C1C3=C(C=C2)OCC3
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| InChi Key |
YLXDSYKOBKBWJQ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H21NO2/c1-2-15(18)17-9-7-12-4-3-11-5-6-14-13(16(11)12)8-10-19-14/h5-6,12H,2-4,7-10H2,1H3,(H,17,18)
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| Chemical Name |
N-[2-(2,6,7,8-tetrahydro-1H-cyclopenta[e][1]benzofuran-8-yl)ethyl]propanamide
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 3.8558 mL | 19.2790 mL | 38.5579 mL | |
| 5 mM | 0.7712 mL | 3.8558 mL | 7.7116 mL | |
| 10 mM | 0.3856 mL | 1.9279 mL | 3.8558 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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