Novel Conformationally Constrained Analogues of Agomelatine as New Melatoninergic Ligands

Novel conformationally restricted analogues of agomelatine were synthesized and pharmacologically evaluated at MT1 and MT2 melatoninergic receptors. Replacement of the N-acetyl side chain of agomelatine by oxathiadiazole-2-oxide (compound 3), oxadiazole-5(4H)-one (compound 4), tetrazole (compound 5), oxazolidinone (compound 7a), pyrrolidinone (compound 7b), imidazolidinedione (compound 12), thiazole (compounds 13 and 14) and isoxazole moieties (compound 15) led to a decrease of the melatoninergic binding affinities, particularly at MT1. Compounds 7a and 7b exhibiting nanomolar affinity towards the MT2 receptors subtypes have shown the most interesting pharmacological results of this series with the appearance of a weak MT2-selectivity.


Introduction
Melatonin or N-acetyl-5-methoxytryptamine (Figure 1), is a neurohormone that is synthesized and secreted from the pineal gland during the period of darkness following a circadian rhythm [1]. Since the demonstration of its role in many physiological processes such as the regulation of immune functions [2], retinal physiology [3], circadian and seasonal rhythms [4] research efforts to identify new melatoninergic OPEN ACCESS ligands grow up continuously. However, much more efforts must be done to clarify the various functions exerted by melatonin and its mechanisms of action.
This neurohormone exerts its multiple pharmacological actions through two G-protein-coupled receptors MT 1 and MT 2 which were cloned in the mid-1990s [2][3][4][5]. A third melatonin binding site, MT 3 , having lower affinity than MT 1 and MT 2 has been characterized as the hamster homologue of quinone reductase 2 (QR2 EC 1.6.99.2) [6]. Exploring the exact physiological role of each of these binding sites requires selective MT 1 , MT 2 and MT 3 ligands. Melatoninergic MT 1 receptors are expressed in several areas of the brain in particular in the suprachiasmatic nuclei (SCN) and the pars tuberalis. The MT 2 receptors are localized in the SCN and retina. The low affinity binding site, MT 3 is closely related to the detoxifying enzyme quinone reductase 2 and its exact biological relevance in melatonin's effects is still uncertain [7]. Nonetheless, MT 3 has shown to be involved in acute inflammatory responses in the rat [8] and in the regulation of intraocular pressure in the rabbit [9]. In fact and in order to clarify MT 1 , MT 2 and MT 3 biological functions, over the last years several ligands were synthesized [10][11][12][13][14][15][16][17][18][19]. Only ramelteon (Rozerem ® ) [20] and agomelatine (Valdoxan ® ) [21], two MT 1 and MT 2 receptor agonists, are respectively marketed for the treatment of insomnia and major depressive disorders. Agomelatine is the first antidepressant which does not block the reuptake of monoamines. Therefore it might represent the prototype of a new class of antidepressant drugs. The development of new derivatives is of importance in order to increase efficacy and reduce side effects. Agomelatine, which is also a 5-HT 2C selective antagonist was revealed to be also potent in resynchronization of circadian rhythms [22,23].
The importance of melatonin as a promising therapeutic target has led to the investigation of the pharmacophoric requirements for its receptors' binding and activation in order to develop selective ligands. Early SAR studies showed that both methoxy group and the N-acetylamino side chain of melatonin are crucial for high receptor affinity and that the relative spatial distance between these groups is also an important factor [24]. In addition, 3D-QSAR analysis of melatoninergic ligands revealed that MT 1 and MT 2 binding affinity could be enhanced by replacement and/or conformationally restriction of the amide substituent [25]. This approach could help probing the existing pharmacophore for potent MT 1 and MT 2 selective ligands, and open new therapeutic perspectives by targeting a specific receptor.
In our continuing efforts to develop new melatonin ligands using the agomelatine as a lead, we previousely reported the design and synthesis of melatoninergic MT 1 [26,27], MT 2 [28][29][30][31] and MT 3 -selective ligands [32][33][34]. We also were the pioneers in preparing non-selective MT 1 and MT 2 ligands with 5-HT 2C activity [17]. In this paper we describe the synthesis and pharmacological evaluation of a novel small series of naphtalenic constrained compounds issued from the incorporation of the amide side chain into heterocycles.
Finally, synthesis of compounds 12-15 was carried out as illustrated in Scheme 3. 2-(7-methoxynaphth-1-yl)acetaldehyde (11) was obtained from (7-methoxynaphth-1-yl)acetic acid [21] via esterification followed by reduction and Dess Martin oxidation [42]. Compound 11 was then converted to the desired imidazolidine-dione 12 by treatment with potassium cyanide and ammonium carbonate. Compounds 13-15 were prepared under the same conditions by condensation of 11 with the appropriate heterocyclic amine, followed by reduction of the imine generated in situ by use of sodium cyanoborohydride in the presence of zinc iodide.  I]Iodomelatonin binding assay conditions were essentially as previously described [43]. Briefly, binding was initiated by addition of membrane preparations from transfected CHO cells stably expressing the human melatonin MT 1 or MT 2 diluted in binding buffer (50 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCl 2 ) to 2-[ 125 I]iodomelatonin (20 pM for MT 1 and MT 2 receptors expressed in CHO cells) and the tested drug. Non-specific binding was defined in the presence of 1 µM melatonin. After 120 min incubation at 37 °C, reaction was stopped by rapid filtration through GF/B filters presoaked in 0.5% (v/v) polyethylenimine. Filters were washed three times with 1 mL of ice-cold 50 mM Tris-HCl buffer, pH 7.4. Data from the dose-response curves (seven concentrations in duplicate) were analysed using the program PRISM (Graph Pad Software Inc., San Diego, CA, USA) to yield IC 50 (inhibitory concentration 50). Results are expressed as pK i (pK i = −Log10 (K i )) with K i = IC 50 /1 + ([L]/KD), where [L] is the concentration of radioligand used in the assay and KD, the dissociation constant of the radioligand characterizing the membrane preparation.

Discussion
Conformationally restricted ligands for melatonin receptors were synthesized and their binding affinities at human MT 1 and MT 2 receptors were determined. The data summarized in Table 1 emphasized the lack of good affinities for the MT 1 and MT 2 receptors of the prepared compounds. In fact, in comparison with agomelatine the lock of the ethylamido side chain conformation by its incorporation in rigid structures led to the decrease of the binding affinities at both receptors. This decrease of the melatoninergic binding affinities is more noticeable for the MT 1 than the MT 2 leading to the appearance of a weak MT 2 -selectivity. Only compounds 7a and 7b showed an interesting pharmacological profile by conserving a good binding affinity (10 −8 M) at MT 2 receptors subtypes.

General
All common reagents and solvents were obtained from commercial sources (Sigma-Aldrich Alfa Aesar or Acros Organics) and used without further purification. Compounds were purified on a glass column using Merck Silica Gel 60 (230-400 mesh). Their purity and mass spectra were determined on a Surveyor MSQ Thermoelectron spectrometer (+cAPCI corona sid = 30.00, det = 1400.00 Full ms [100.00-1000.00]). Melting points were determined with a büchi 510 capillary apparatus and are uncorrected. 1 H-NMR spectra were recorded on a Bruker AC300P spectrometer using (Me) 4 Si as internal standard and with DMSO-d 6 or CDCl 3 as solvents; The chemical shifts are reported in ppm (parts per million) δ and constant (J) values are given in Hertz (Hz). Signal multiplicities are represented by: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), dt (doublet of triplet), q (quartet) and m (multiplet). Infrared spectra were obtained on a Perkin-Elmer FT-IR S1000 in KBr pellets. Elemental analyses for final compounds were performed by CNRS Laboratory (Vernaison, France). (1). A mixture of 2-(7-methoxynaphth-1-yl)acetonitrile (5 g, 25.3 mmol) and hydroxylamine hydrochloride (3.52 g, 50.6 mmol) in DMSO (20 mL) was treated with 25% NaOMe solution in methanol (11.5 mL, 50.6 mmol) and heated at 80 °C for 5 h. After cooling, the solvent was evaporated under reduced pressure. The crude was taken with water, the white solid obtained was washed with water and recrystallized from toluene to afford 3.   (5). A mixture of 2-(7-methoxynapht-1-yl)acetonitrile (2 g, 1.01 mmol), sodium azide (2.62 g, 40.4 mmol) and tributyltin chloride (10.9 mL, 40.4 mmol) in dry DMF (20 mL) was heated to reflux and monitored by TLC until the reaction was complete (~7 h). After being cooled to room temperature, 1 M HCl (50 mL) was added to precipitate the crude product. The white solid product was collected, washed with water and ether, and dried with phosphorous pentoxide under vacuum and recrystallized from cyclohexane affording 1.21 g (50% yield) of tetrazole 5 as a beige solid, mp: 177-179 °C (decomposition). 1

General Protocol for the Preparation of Compounds 9a and 9b
A solution of agomelatine (4 g, 16.4 mmol) in DMSO (100 mL) was refluxed for 15 h. The reaction mixture was poured into ice and extracted twice with ether, organic phase was washed with water and brine and then concentrated under reduced pression. The crude was purified by column chromatography (SiO 2 ) using ether as eluant.  (10). Thionyl chloride (27 mL, 370 mmol) was added dropwise to a solution of (7-methoxynaphth-1-yl) acetic acid (20 g, 92.5 mmol) in methanol (350 mL) at 0 °C. After stirring for 5 h, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in ethyl acetate and washed with 10% aqueous potassium carbonate solution and water. The organic layer was dried over MgSO 4 , filtered and concentrated under reduced pressure affording the intermediate ester. The residue was dissolved in ether (100 mL) and added dropwise to a suspension of lithium aluminium hydride (12.4 g, 327 mmol) in ether (200 mL) at 0 °C. The stirring was maintained at room temperature for 4 h. Water (50 mL) and 20% NaOH aqueous solution (12 mL) were added and the mixture was stirred and filtered. After evaporation under reduced pressure, the residue was recrystallized from cyclohexane to furnish 13.7 g (73% yield) of alcohol 10 as a white solid, mp: 79-81 °C. 1  2-(7-Methoxynaphth-1-yl)acetaldehyde (11). To a stirred solution of alcohol 10 (6 g, 29.6 mmol) in anhydrous CH 2 Cl 2 (300 mL) under argon atmosphere was added Dess-Martin periodinane (25 g, 59.2 mmol) and stirred at room temperature for 5 h. The reaction mixture was quenched by adding saturated Na 2 S 2 O 4 (60 mL) and saturated NaHCO 3 (8 mL). The heterogeneous mixture was extracted with CH 2 Cl 2 and the organic layer was washed with saturated NaHCO 3 and water. The combined organic layers were dried over MgSO 4 and the solvent removed under reduced pressure. The residue was purified by column chromatography (SiO 2 , CH 2 Cl 2 ) to give 5.2 g (87% yield) of 11 as a yellow oil.  (12). Ammonium carbonate (1.67 g, 17.4 mmol) and potassium cyanide (341 mg, 5.24 mmol) were added to a stirred solution of aldehyde 14 (700 mg, 3.49 mmol) in 20 mL (16 mL/4 mL) of ethanol/water, the mixture was refluxed overnight. After cooling, the reaction mixture was poured into cold water. The resulting precipitate was filtrated, washed with water and recrystallized from isopropyl ether affording 400 mg (42% yield) of 15 as a white solid, mp: 236-238 °C (decomp.). 1

General Protocol for the Preparation of Compounds 13-15
To a mixture of compound 11 (0.5 g, 2.49 mmol) and the appropriate heterocyclic amine (10 mmol) in methanol (20 mL) and 0.2 mL of DMF, were added a solution of sodium cyanoborohydride (172 mg, 2.73 mmol) and zinc iodide (437 mg, 1.36 mmol) in methanol (5 mL). The reaction was stirred at room temperature for 5 h and concentrated under reduced pressure to dryness, hydrolyzed and extracted with ethyl acetate. The organic layer was dried (MgSO 4 ), filtered, and concentrated under reduced pressure. (13). The crude was purified by column chromatography (SiO 2 ) using cyclohexane/ethyl acetate; 7/3 as eluant, treatment with gaseous HCl in ether provided 280 mg (35% yield) of hydrochloride salt 13 as a white solid, mp: 156-158 °C.  (15). Recrystallized from cyclohexane to afford 333 mg (42% yield) of 15 as a white solid, mp 144-146 °C (decomp.).

Conclusions
In the search for pharmacological tools for the elucidation of MT 1 and MT 2 biological functions, we synthesized and pharmacologically evaluated a new series of constrained naphthalenic compounds. Indeed, replacement of the N-acetyl side chain of agomelatine by oxathiadiazole-2-oxide, oxadiazole-5(4H)-one, oxazolidinone, 2-oxopyrrolidine, imidazolidin-2,4-dione, thiazole, 5-methyl-1,3-thiazole, or 5-methyl-1,2-isoxazole hydrochloride resulted in a decrease of the melatoninergic binding affinities particularly towards the MT 1 receptors leading to the appearance of a weak MT 2 -selectivity. Compound 7b conserves good affinity for both melatonin receptors subtypes and exhibited a selectivity of about 33-fold for the MT 2 receptor subtype.