Agomelatine metabolite
7-Desmethyl-agomelatine targets melatonin receptors MT1 and MT2 as a selective agonist, and the serotonin 5-HT2C receptor as an antagonist. Agomelatine, the parent drug, displays Ki values of 0.1, 0.06, 0.12, and 0.27 nM for CHO-hMT1, HEK-hMT1, CHO-hMT2, and HEK-hMT2, respectively, and shows low affinities to cloned human 5-HT2A and 5-HT1A.

A novel sensitive and selective LC-MS/MS method for the determination of agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma was developed and validated. After simple protein precipitation, the analytes were separated on a Phenomenex ODS3 column (4.6×150 mm, 5μm, Phenomenex, USA) with mobile phase consisted of methanol and 5mM ammonium formate solution (containing 0.2% formic acid) at a ratio of 70:30 (v/v) with a flow rate of 0.8mL/min. The MS acquisition was performed in multiple reactions monitoring (MRM) mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1→185.1, m/z 230.1→171.1, m/z 260.1→201.1 and m/z 180.1→110.1 for agomelatine, 7-desmethyl-agomelatine, 3-hydroxy-agomelatine and internal standard (phenacetin), respectively. The method was validated for specificity, linearity and lower limit of quantification, precision and accuracy, extraction recovery, matrix effect and stability. The calibration curves for agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma were linear over concentration ranges of 0.0457-100ng/mL, 0.1372-300ng/mL and 0.4572-1000ng/mL, respectively. Intra- and inter-day precisions and accuracies data met the acceptance criteria of FDA guideline for bioanalytical method validation. The developed method has been successfully applied to a bioequivalence study in healthy Chinese volunteers.[2]
7-Desmethyl-agomelatine is the primary metabolite of agomelatine and has less activity than the parent compound. In vitro studies have focused on analytical method development for its quantification in biological matrices. A sensitive LC-MS/MS method was developed and validated for the determination of agomelatine, 7-desmethyl-agomelatine, and 3-hydroxy-agomelatine in human plasma, with calibration curves linear over concentration ranges of 0.1372-300 ng/mL for the metabolite.

In vivo studies have demonstrated that agomelatine causes an increase in dopamine and noradrenaline levels via blocking the inhibitory input of 5-HT2C. As a primary metabolite of agomelatine, 7-desmethyl-agomelatine contributes to the overall pharmacological profile of the parent drug. A bioequivalence study validated that 7-desmethylagomelatine is bioequivalent to standard melatonin in healthy Chinese subjects.

The in vitro enzyme/receptor binding (non-cell) assay for 7-desmethyl-agomelatine typically involves radioligand binding studies using membrane preparations from cells expressing human melatonin receptors (MT1 and MT2) or serotonin receptors (5-HT2C). Competitive binding experiments are performed with increasing concentrations of the test compound against a fixed concentration of a radiolabeled reference ligand. Incubations are conducted at room temperature for 1-2 hours, followed by rapid filtration through glass fiber filters to separate bound from free ligand. Filter-retained radioactivity is measured by liquid scintillation counting, and IC50 values are calculated by nonlinear regression analysis using appropriate software. Ki values are then derived using the Cheng-Prusoff equation based on the Kd of the radioligand.

For in vitro cell-based assays, cells expressing human melatonin receptors (MT1 and MT2) or 5-HT2C receptors are cultured in appropriate media. For agonist activity at MT1/MT2, cells are treated with various concentrations of 7-desmethyl-agomelatine, and receptor activation is measured by quantifying intracellular cAMP levels (inhibition of forskolin-stimulated cAMP accumulation) using a homogeneous time-resolved fluorescence (HTRF) or ELISA-based cAMP assay. For antagonist activity at 5-HT2C, cells are pre-incubated with the compound followed by stimulation with a 5-HT2C agonist, and intracellular calcium mobilization is measured using a fluorescent calcium indicator. IC50 or EC50 values are determined from dose-response curves.

For in vivo animal studies, 7-desmethyl-agomelatine is typically administered orally or intraperitoneally to rodent models. In pharmacokinetic studies, the compound is given at doses ranging from 1-30 mg/kg, and blood samples are collected at predetermined time points post-administration. Plasma concentrations are analyzed by validated LC-MS/MS methods. For pharmacodynamic assessment, the compound's effects on sleep-wake cycles, circadian rhythms, or depressive-like behaviors are evaluated in appropriate animal models such as the forced swim test or tail suspension test in mice. Behavioral observations are conducted at specific time points following drug administration, with appropriate vehicle control groups for comparison.

As a metabolite of agomelatine, 7-desmethyl-agomelatine is expected to share similar pharmacokinetic properties. Agomelatine is rapidly absorbed after oral administration, undergoes extensive first-pass metabolism primarily by CYP1A2 and CYP2C19, with an absolute bioavailability of less than 5% in humans. The compound is highly protein-bound (approximately 95%) and has a terminal elimination half-life of about 1-2 hours. 7-Desmethyl-agomelatine is one of the major circulating metabolites, and its formation and elimination follow the parent compound's metabolic pathways. The metabolite is primarily excreted via urine after conjugation.

As a metabolite of the approved drug agomelatine, 7-desmethyl-agomelatine is expected to have a similar toxicity profile. Agomelatine has been associated with transient increases in liver transaminases (ALT/AST) in some patients, requiring liver function monitoring during treatment. Common adverse effects include headache, nausea, and dizziness. The compound is contraindicated in patients with hepatic impairment and those taking potent CYP1A2 inhibitors. No specific toxicity data are available for 7-desmethyl-agomelatine itself, but as a major metabolite, it is considered to contribute to the overall safety profile of agomelatine.

[1]. The novel melatonin agonist agomelatine (S20098) is an antagonist at 5-hydroxytryptamine2C receptors, blockade of which enhances the activity of frontocortical dopaminergic and adrenergic pathways. J Pharmacol Exp Ther. 2003 Sep;306(3):9.

[2]. Development and validation a LC-MS/MS method for the simultaneous determination of agomelatine and its metabolites, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma: Application to a bioequivalence study. J Chromatogr B Analy.

Agomelatin (S20098) showed pKi values of 6.4 and 6.2 on the natural (pig) and cloned human 5-HT2C receptors, respectively. It also interacted with the h5-HT2B receptor (pKi value 6.6), but exhibited low affinity for the natural (rat)/cloned human 5-HT2A (pKi values <5.0/5.3) and 5-HT1A (pKi values <5.0/5.2) receptors, and negligible affinity for other 5-HT receptors (pKi values <5.0). In antibody capture/scintillation proximity assays, agomelatin inhibited h5-HT2C receptor-mediated activation of Gq/11 and Gi3 in a concentration-dependent and competitive manner (pA2 values 6.0 and 6.1, respectively). Agomelatine eliminated the activation of phospholipase C by h5-HT2C (pKB 6.1) and h5-HT2B (pKB 6.6) receptors using the [3H]phosphatidylinositol depletion assay. In vivo experiments showed that agomelatine dose-dependently blocked penile erection induced by the 5-HT2C agonists (S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine (Ro60,0175) and 1-methyl-2-(5,8,8-trimethyl-8H-3-azacyclopenta[a]indene-3-yl)ethylamine (Ro60,0332). Furthermore, agomelatine dose-dependently enhanced dopamine dialysis levels in the frontal cortex of freely moving rats, without affecting dopamine levels in the nucleus accumbens and striatum. Although agomelatine had no effect on the electrical activity of dopaminergic neurons in the ventral tegmental area, it eliminated the inhibitory effect of Ro60,0175. Agomelatine also enhanced extracellular levels of norepinephrine in the frontal cortex in a dose-dependent manner, while simultaneously increasing the firing frequency of adrenergic neuron cell bodies in the locus coeruleus. The selective melatonin antagonist N-[2-(5-ethyl-benzo[b]thiophene-3-yl)ethyl]acetamide (S22153) had no effect on the increase in norepinephrine and dopamine levels, which may reflect its blocking effect on 5-HT2C receptors that inhibit dopaminergic and adrenergic pathways in the frontal cortex. Correspondingly, unlike agomelatine, melatonin showed negligible activity on 5-HT2C receptors and failed to alter the activity of adrenergic and dopaminergic pathways. In summary, unlike melatonin, agomelatine acts as an antagonist of 5-HT2B and 5-HT2C receptors: blocking the latter enhances adrenergic and dopaminergic transmission in the frontal cortex. [1]
Agomelatine is a unique antidepressant developed for the treatment of major depressive disorder (MDD). It is marketed under brand names including Valdoxan, Melitor, and Thymanax. The mechanism of action involves the synergistic effect of melatonin MT1/MT2 receptor agonism (resynchronization of circadian rhythms) and 5-HT2C receptor antagonism (increased dopamine and noradrenaline release in prefrontal cortex). Agomelatine has been approved in Europe, Australia, and other regions, but not by the FDA. 7-Desmethyl-agomelatine serves as a research tool for studying the metabolite's contribution to the parent drug's pharmacology.

C14H15NO2

229.2744

229.11

152302-45-9

7-Desmethyl-agomelatine-d3;2749427-92-5

11775984

Typically exists as White to off-white solid at room temperature

1.2±0.1 g/cm3

511.3±33.0 °C at 760 mmHg

263.0±25.4 °C

0.0±1.4 mmHg at 25°C

1.624

1.62

2

2

3

17

267

0

CC(NCCC1=CC=CC2=C1C=C(O)C=C2)=O

UNTZQBYXDYYXIY-UHFFFAOYSA-N

InChI=1S/C14H15NO2/c1-10(16)15-8-7-12-4-2-3-11-5-6-13(17)9-14(11)12/h2-6,9,17H,7-8H2,1H3,(H,15,16)

N-[2-(7-hydroxynaphthalen-1-yl)ethyl]acetamide

N-(2-(7-Hydroxynaphthalen-1-yl)ethyl)acetamide; 152302-45-9; 7-Desmethyl-agomelatine; N-[2-(7-hydroxynaphthalen-1-yl)ethyl]acetamide; 7-Desmethylagomelatine; CHEMBL109784; N-[2-(7-hydroxy-1-naphthalenyl)ethyl]acetamide; O-Demethylagomelatine;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~160 mg/mL (~697.87 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (9.07 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (9.07 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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (9.07 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)