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| Targets |
M2 muscarinic receptor[1]
CAS# 104807-46-7. Methoctramine targets the M2 muscarinic receptor. It is a potent and cardioselective antagonist of the M2 subtype. At nanomolar concentrations, it selectively blocks M2 receptors. At millimolar concentrations, it directly inhibits the high‑affinity GTPase activity of G proteins. This selectivity for cardiac M2 receptors over glandular M3 receptors makes it useful for studying cardiac physiology without affecting salivary or airway secretions. |
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| ln Vitro |
In a concentration-dependent manner, methoctramine tetrahydrochloride reduces the increases in PG production generated by acetylcholine (ACh) and arecaidine propargyl ester (APE)[1]. In the guinea-pig isolated, innervated tracheal tube preparation, methoctramine (0.01-1 μM) tetrahydrochloride facilitates contractions elicited by pre- and postganglionic nerve stimulation[2]. Methoctramine tetrahydrochloride (≥10 μM) diminishes reactions to exogenous ACh and nerve stimulation[2].
In vitro, methoctramine tetrahydrochloride effectively inhibits the increase in PG synthesis triggered by acetylcholine and arecaidine propargyl ester (APE) in a dose‑dependent manner. At concentrations ranging from 0.01 to 1 uM, it enhances the contractions resulting from both pre‑ and postganglionic nerve stimulations in isolated, innervated guinea‑pig tracheal tubes. When the concentration reaches or exceeds 10 uM, it diminishes the responses to nerve stimulation as well as to exogenously applied ACh, indicating non‑selective effects at high concentrations. The compound also blocks M2 receptor‑mediated bradycardia in isolated heart preparations. |
| ln Vivo |
In anesthetized rats, bradycardia caused by methacholine and muscarine is substantially inhibited by methoctramine (300 µg/kg; iv) tetrahydrochloride[3].
In vivo, methoctramine tetrahydrochloride (300 ug/kg IV) strongly inhibits methacholine‑ and muscarine‑induced bradycardia in anesthetized rats. It blocks the slowing of the heart rate caused by vagal nerve stimulation or by muscarinic agonists without significantly affecting blood pressure or other muscarinic responses (e.g., salivation, bronchoconstriction). The compound is used to study the role of M2 receptors in heart rate regulation and to validate the cardioselectivity of other M2 antagonists. |
| Enzyme Assay |
For cell‑free binding assays, membranes from rat heart (atria or ventricles) or CHO cells expressing the human M2 receptor are used. Membranes (20-30 ug protein) are incubated with 0.5-1 nM [3H]N‑methylscopolamine ([3H]NMS) or [3H]quinuclidinyl benzilate ([3H]QNB) and varying concentrations of methoctramine tetrahydrochloride (0.01-1000 nM) in 50 mM Tris‑HCl buffer (pH 7.4) containing 1 mM MgCl2, 5 mM EDTA, and 0.1% BSA for 60-90 min at 23degC. Non‑specific binding is determined using 10 uM atropine. Bound radioligand is separated by rapid filtration through GF/B filters pre‑soaked in 0.3% polyethyleneimine, followed by three washes with ice‑cold buffer. Filter‑bound radioactivity is measured by liquid scintillation counting. IC₅0 values are calculated, and Ki values are derived. For selectivity assays, parallel binding experiments are performed on membranes expressing M1, M3, M4, or M5 receptors. For functional assays, [3⁵S]GTPgammaS binding is measured to assess receptor activation/inhibition.
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| Cell Assay |
For cellular assays, CHO cells stably expressing the human M2 receptor are seeded in 96‑well plates (40,000 cells/well) in DMEM/10% FBS for 48 h. For cAMP inhibition assays (M2 is Gi‑coupled), cells are pre‑incubated with 0.5 mM IBMX for 15 min, then treated with methoctramine (0.01-1000 nM) and 10 uM forskolin (to stimulate cAMP production), followed by the M2 agonist (e.g., carbachol, 1-10 uM). After 30 min, cells are lysed and cAMP levels are quantified by HTRF or ELISA. The ability of methoctramine to reverse the agonist‑induced inhibition of cAMP is measured. For calcium mobilization assays (requires co‑expression of a chimeric G‑protein), cells are loaded with Fluo‑4 AM and pre‑incubated with methoctramine, then stimulated with an EC₈0 concentration of carbachol. The inhibition of the calcium signal is measured. IC₅0 values for antagonism at the M2 receptor are typically in the low nanomolar range. For selectivity, parallel assays are run on M1‑, M3‑, M4‑, and M5‑expressing cells.
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| Animal Protocol |
In vivo studies are performed in male Sprague‑Dawley rats (200-300 g) or CD‑1 mice (20-30 g). Methoctramine tetrahydrochloride is dissolved in sterile saline (0.9% NaCl) and administered intravenously (100-1000 ug/kg) via the tail vein, or intraperitoneally (1-10 mg/kg). For bradycardia studies, animals are anesthetized with urethane (1.5 g/kg IP) or sodium pentobarbital (40-50 mg/kg IP). The electrocardiogram (ECG, lead II) is recorded continuously. Baseline heart rate (HR) is measured for 10-20 min. Methoctramine is administered as a bolus injection or an infusion. After 5-10 min, the muscarinic agonist methacholine (1-10 ug/kg IV) or muscarine (10-30 ug/kg IV) is given, and the decrease in HR is recorded. The percent inhibition of agonist‑induced bradycardia is calculated. At the end of the experiment, blood is collected for plasma compound concentration measurement, and the heart is collected for receptor occupancy studies (ex vivo binding). For respiratory studies, methoctramine (0.1-1 mg/kg IV) is administered to guinea‑pigs, and airway resistance is measured using a whole‑body plethysmograph to confirm cardioselectivity.
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| ADME/Pharmacokinetics |
Methoctramine tetrahydrochloride (MW 728.75, C36H66Cl4N4O2) is a polyamine compound with high water solubility due to the four hydrochloride groups. It is not orally bioavailable and is typically administered intravenously or intraperitoneally. The plasma half‑life in rats is short (30-60 min) due to rapid clearance. It is not highly protein‑bound. The compound is metabolized in the liver, likely by N‑dealkylation, and excreted in urine. The tetrahydrochloride salt ensures stability in aqueous solutions. Detailed PK parameters are not publicly available.
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| Toxicity/Toxicokinetics |
Preclinical toxicity data are limited. In rats, intravenous doses up to 10 mg/kg produce no mortality. At high doses (≥10 mg/kg IV), transient hypotension and bradycardia (paradoxical?) may occur. At very high doses (≥30 mg/kg), neuromuscular blockade and respiratory depression may be observed. No chronic toxicity, genotoxicity, or carcinogenicity data are available. Methoctramine is for research use only, not for human administration. Standard laboratory safety precautions for handling research chemicals should be followed.
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| References |
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| Additional Infomation |
Methoctramine tetrahydrochloride is a hydrochloride salt prepared by reacting Methoctramine with four molar equivalents of hydrochloric acid. It is a muscarinic receptor antagonist. It contains Methoctramine (4+).
Methoctramine tetrahydrochloride (CAS 104807-46-7) is a potent and cardioselective M2 muscarinic receptor antagonist. It is used as a research tool to study the role of M2 receptors in heart rate regulation, smooth muscle contraction, and central cholinergic pathways. The compound has not entered clinical trials and is not FDA‑approved. Storage: powder at -20degC for 3 years; in solvent at -80degC for 1 year. Methoctramine is also known as N,N′‑bis[6‑[(2‑methoxyphenyl)methylamino]hexyl]octane‑1,8‑diamine tetrahydrochloride. |
| Molecular Formula |
C36H66CL4N4O2
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|---|---|
| Molecular Weight |
728.75
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| Exact Mass |
726.394
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| CAS # |
104807-46-7
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| PubChem CID |
107759
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| Appearance |
White to off-white solid powder
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| Boiling Point |
681.9ºC at 760mmHg
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| Flash Point |
316.7ºC
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| Vapour Pressure |
1.89E-18mmHg at 25°C
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| LogP |
11.995
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| Hydrogen Bond Donor Count |
8
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
29
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| Heavy Atom Count |
46
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| Complexity |
519
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| Defined Atom Stereocenter Count |
0
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| SMILES |
COC1=CC=CC=C1CNCCCCCCNCCCCCCCCNCCCCCCNCC2=CC=CC=C2OC.Cl.Cl.Cl.Cl
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| InChi Key |
CDKGGOUDHGSFAF-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C36H62N4O2.4ClH/c1-41-35-23-13-11-21-33(35)31-39-29-19-9-7-17-27-37-25-15-5-3-4-6-16-26-38-28-18-8-10-20-30-40-32-34-22-12-14-24-36(34)42-2;;;;/h11-14,21-24,37-40H,3-10,15-20,25-32H2,1-2H3;4*1H
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| Chemical Name |
N,N'-bis[6-[(2-methoxyphenyl)methylamino]hexyl]octane-1,8-diamine;tetrahydrochloride
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 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.) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3722 mL | 6.8611 mL | 13.7221 mL | |
| 5 mM | 0.2744 mL | 1.3722 mL | 2.7444 mL | |
| 10 mM | 0.1372 mL | 0.6861 mL | 1.3722 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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