5-HT2C Receptor (IC50 = 3.2 μM); 5-HT2C Receptor (Ki = 1.8 μM)

The affinities of Agomelatine and S 21517 for the 5-HT2C receptor are moderately strong, with Ki values of 0.21 μM and 0.13 μM, respectively. The affinity of the metabolite 3-hydroxyagomelatine (S 21540) is 1.8 μM, which is ten times lower. Five-HT2C receptor antagonists, namely agomelatine, S 21517, and 3-hydroxyagomelatine (10-7-10-4 M), are arranged in the following order of efficacy: S 21517 > agomelatine > 3-hydroxyagomelatine[1].

The mCPP- and Ro 60-0175-induced penile erection in Wistar rats was not affected by increasing the doses of 3-hydroxyagomelatine (S 21540) from 1.25 to 40 mg/kg, i.p. [1].

In vitro binding[1]
Agomelatine and its three metabolites S 21540 (3-Hydroxy agomelatine ), S 21517 and S 22380 (10−9 to 10−4 M) were assessed on pig choroid plexus membranes incubated with 3H-mesulergine. Pig choroid plexi were homogenized with a Polytron homogenizer in 10 vol of 0.32 M sucrose in 50 mM TRIS HCl (pH 7.7 at 25°C) and were centrifuged at 900 g for 10 min. Supernatant was centrifuged at 70,000 g for 15 min and the pellet re-suspended in 10 vol buffer. The suspension was vortexed and incubated at 37°C for 15 min and centrifuged again at 70,000 g for 15 min. The final pellets were re-suspended in 50 mM TRIS HCl (pH 7.7 at 25°C) containing 4 mM CaCl2, 0.1% ascorbic acid and 10 μM pargyline.
3H-Mesulergine (TRK 845, 85 Ci/mmol) was used to measure 5-HT2C receptors following the method of Pazos et al. (1984). 3H-Mesulergine (0.5 nM) was incubated with choroid plexi membranes in 500 μl of buffer in the absence or presence of agomelatine or the metabolites, or 1.0 μM serotonin (5-HT), which was used to define the non-specific binding. The incubation was carried out for 30 min at 37°C before the reaction was stopped by filtration through Whatman GF/C glass fiber filters. Filters were washed quickly with three 5 ml portions of ice-cold 50 mM TRIS buffer and were counted in 5 ml Ready Safe by scintillation counting at an efficiency of 45%. The ten concentrations of agomelatine and its metabolites were tested in duplicate assays. Inhibition curves were analysed with the software EBDA and results were expressed as IC50 and Ki values.

Penile erections were measured in male Wistar rats (WI, Charles River, weighing 200–250 g) using experimental conditions described previously (Protais et al. 1983, 1995). Briefly, rats housed in an air-conditioned room under controlled 12-h light-dark cycle (light on at 8 a.m.) were injected IP with 5-HT antagonists or melatonin derivatives. Thirty minutes later, they received an SC injection of mCPP (0.75 mg/kg) or Ro 60-0175 (2.5 mg/kg). Dosages of mCPP and Ro 60-0175 were chosen from dose-response curves performed in previous experiments (Protais et al. 1995; Millan et al. 1997). For the observations, rats were introduced into rectangular cages (L=25 cm; W=18 cm; H=30 cm) with vertical walls of wire netting. Immediately after the injection of the 5-HT2C agonists, penile erections (rats in an upright position presenting an emerging and engorged penis) were counted by direct observation during 60 min. All the experiments were carried out between 9 a.m. and 7 p.m., without taking care of phases of circadian rhythms of rats. Melatonin, agomelatine and its metabolites were suspended in a 1% hydroxyethylcellulose solution. Other drugs were dissolved in saline or in distillated water. All the solutions were prepared extemporaneously. All doses, expressed as the free base of respective salts, were injected in a volume of 5 ml/kg. An analysis of variance (ANOVA), followed by Student's t-test, was applied to evaluate the significance of means.[1]

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Agomelatine, an antidepressant with melatonin agonist and 5-HT(2C) antagonist properties, as well as two of its main metabolites, S 21517 (N-[2-(7-hydroxy-1-naphtyl)ethyl]acetamide) and S21540 (N-[2-(3-hydroxy-7-methoxynaphtalen-1-yl)ethyl]acetamide), have been assessed in vitro on pig choroid plexus preparations to determine their affinities for 5-HT(2C) receptors and their effects on inositol phosphate production. These compounds were also tested for their ability to inhibit the penile erections induced by the 5-HT(2C) receptor agonists, m-(chlorophenyl)piperazine (mCPP, 0.75 mg/kg, SC) and Ro 60-0175 (2.5 mg/kg, SC) in Wistar rats. These in vivo effects were compared to those of melatonin and the 5-HT antagonists pizotifen and SB 206,553. Agomelatine and S 21517 had moderate affinity for 5-HT(2C) receptors and behaved in vitro as weak antagonists at this receptor subtype. S 21540 had a 10-fold lower affinity. Pizotifen and SB 206,553 antagonized mCPP- and Ro 60-0175-induced penile erections, suggesting that penile erections induced by mCPP or Ro 60-0175 resulted from the stimulation of 5-HT(2C) receptors. Whereas increasing doses (from 1.25 to 40 mg/kg, IP) of melatonin were unable to modify the penile erections induced by mCPP and Ro 60-0175, agomelatine (from 1.25 to 40 mg/kg, IP) dose-dependently decreased mCPP- as well Ro 60-0175-induced penile erections. Furthermore, increasing doses (from 1.25 to 40 mg/kg, IP) of S 21517 and S 21540, the two main metabolites of agomelatine, did not affect the penile erections induced by mCPP and Ro 60-0175. Considering the similar activity of melatonin and agomelatine at melatonin receptors, these data suggested that the reported effects were not due to the stimulation of melatonin receptors and that, contrary to melatonin, agomelatine exerted 5-HT(2C) receptor antagonist properties in addition to its agonist activity at melatonin receptors. Finally, neither S 21517 nor S 21540 seemed to participate to the observed inhibition of penile erections by agomelatine.[1]

[1]. Agomelatine(S 20098) antagonizes the penile erections induced by the stimulation of 5-HT2C receptors in Wistar rats. Psychopharmacology (Berl). 2003 Oct;170(1):17-22.

In the present study, neither 3-hydroxylated (S 21540 (3-Hydroxy agomelatine )) and demethylated (S 21517) metabolites of agomelatine, were able to modify the penile erections induced by mCPP or Ro 60-0175. Both metabolites of agomelatine have at least 100 times less affinity than the native compound to melatonin receptors, and indeed, unlike agomelatine, they cannot re-entrain free running rhythms of Long Evans rats in constant dim light (Redman and Francis 1998). The present in vitro binding results also show that the metabolites' affinities for 5-HT2C receptors were equal or lower than that of agomelatine. It could be suggested that the doses of S 21517 and S 21540 used in the present study (up to 40 mg/kg) were too low to efficiently inhibit mCPP or Ro 60-0175-induced penile erections. However, it seems unlikely that the metabolism of the highest tested dose of agomelatine (40 mg/kg), which completely antagonizes the penile erections induced by 5-HT2C receptor agonists, could lead to plasma concentrations of S 21517 or S 21540 which are higher than those obtained following the administration of 40 mg/kg S 21517 or S 21540. Indeed, metabolism studies at both pharmacological and toxicological doses in rat have shown only a weak plasma amount of non-conjugated S 21517 (<5% of native compound).[1]

C15H17NO3

259.300

259.12

166526-99-4

3-Hydroxy agomelatine-d3;1079774-23-4

10400344

Off-white to light brown solid powder

1.2±0.1 g/cm3

545.8±40.0 °C at 760 mmHg

283.9±27.3 °C

0.0±1.5 mmHg at 25°C

1.606

1.53

2

3

4

19

308

0

VUBBOOVHTBZRTJ-UHFFFAOYSA-N

InChI=1S/C15H17NO3/c1-10(17)16-6-5-12-8-13(18)7-11-3-4-14(19-2)9-15(11)12/h3-4,7-9,18H,5-6H2,1-2H3,(H,16,17)

N-[2-(3-hydroxy-7-methoxynaphthalen-1-yl)ethyl]acetamide

3-Hydroxy Agomelatine; 166526-99-4; 3-Hydroxyagomelatine; N-[2-(3-hydroxy-7-methoxynaphthalen-1-yl)ethyl]acetamide; N-[2-(7-methoxy-3-hydroxynaphth-1-yl)ethyl]-acetamide; n-(2-(3-hydroxy-7-methoxynaphthalen-1-yl)ethyl)acetamide; S 21540; N-[2-(3-Hydroxy-7-methoxy-1-naphthalenyl)ethyl]acetamide; Agomelatine 3-Hydroxy Impurity; SCHEMBL6247493;

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 : ~250 mg/mL (~964.13 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.02 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 (8.02 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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 (Please use freshly prepared in vivo formulations for optimal results.)