No abstract

The synthesis of several novel indole melatonin analogues substituted at the 2-position with acylaminomethyl (8-11), acylaminoethyl (5a-k), or acylaminopropyl (13) side chains is reported. On the basis of a novel in vitro functional assay (specific binding of [35S]GTPgammaS), which can discriminate agonist from partial agonist, antagonist, and inverse agonist ligands, 5a,g, h,j and 13 were shown to be partial agonists, 5d,e and 8-11 competitive antagonists, and 5b,c,k putative inverse agonists. Binding and functional assays were performed on cloned human MT1 receptor. Structure-activity relationship considerations indicate that N-[1-aryl-2-(4-methoxy-1H-indol-2-yl)(C1-C2)alkyl]alkanamides represent a lead structure for this type of ligands.

The cyclic recurrence of cluster periods and the regular timing of headache occurrence in cluster headache (CH) induced us to study the circadian secretion of melatonin and cortisol in 12 patients with episodic CH, during a cluster period, and compare them with 7 age- and sex-matched healthy controls. Blood was sampled every 2 h for 24 h. All subjects were confined to a dark room from 22.00 to 08.00. Plasma melatonin levels were significantly reduced in CH patients (repeated measures ANOVA p < 0.03; mesor p < 0.02), and the cortisol mesor was significantly increased (p < 0.03). Amplitudes and acrophases did not differ between the groups. Individual cosinor analysis showed that 4/12 (33.3%) CH patients had no significant melatonin rhythm, and that 5/11 (45.5%) had no cortisol rhythm. Group analysis of cosinor revealed significantly rhythmicity of melatonin and cortisol secretion in both groups. In controls, the timing of melatonin and cortisol acrophase significantly correlated with each other, indicating that the biorhythm controllers for the secretion of these hormones were synchronized. Such correlation was not found in the CH patients; mesor, amplitude and acrophase of melatonin and cortisol did not correlate with duration of illness, duration of headache in course, or time since last headache attack.

1 Mouse ®broblasts (NIH3T3) transfected with the full-length coding region of the Mel 1a melatonin receptor stably expressed the receptor, coupled to a pertussis toxin-sensitive G-protein(s) and exhibiting high a nity and adequate pharmacological pro®le. 2 The receptor protein had the tendency of a strong coupling to the G-protein and therefore lowa nity state was induced by uncoupling the receptor from its G-protein in presence of high concentrations of NaCl (500 ± 700 mM) and/or GTPgS (100 mM). Thereafter, the a nity of a series of melatonin analogues was determined to both, high-and low-a nity receptor states, thus providing a basis for the prediction of their e cacy, according to the ternary complex model. 3 The cells were subsequently used to study the agonist-induced G-protein activation, determined by calculating the rate of GDP-GTP exchange measured in presence of 35 S-labelled GTPgS. The natural ligand melatonin induced a signi®cant increase in the GDP-GTP exchange rate, the presence of GDP and NaCl being necessary to observe this e ect. 4 The full agonists 2-phenylmelatonin, 2-bromomelatonin and 6-chloromelatonin equally induced an increase of the GDP-GTP exchange. 5-Hydroxy-N-acetyltryptamine activated the GTP-GDP exchange to a much lesser extent (53%) than melatonin, thus behaving as a partial agonist. As predicted by the model, the melatonin antagonist (N-[(2-phenyl-1H-indol-3-yl)ethyl]cyclobutanecarboxamide) was without e ect on basal G protein activation. Coincubation of this compound with melatonin induced a dosedependent rightward shift in the melatonin concentration-e ect curve, thus exhibiting the behaviour of a competitive and surmountable antagonist. 5 Using the equation proposed by Venter (1997) we were able to determine that there were no`spare' receptors in the system. Therefore, the approach proposed in the present work can be successfully used for the determination of`drug action' at the level of the human Mel 1a melatonin receptor and evaluation of the e cacy of new selective melatonin analogues.

A novel series of melatonin receptor ligands, characterized by a N-(substituted-anilinoethyl)amido scaffold, along with preliminary structure-activity relationships (SARs), is presented. MT1 and MT2 receptor binding affinity and intrinsic activity have been modulated by the introduction of different substituents on the aniline nitrogen, on the benzene ring, and on the amide side chain. Modulation of intrinsic activity and MT2 selectivity of the newly synthesized compounds has been achieved by applying SAR models previously developed, providing compounds with different binding and intrinsic activity profiles. Compound 3d, with a bulky ss-naphthyl group, behaves as an MT2-selective antagonist with sub-nM affinity. Size reduction of the substituent enhances intrinsic activity, as in the nonselective N-methyl-anilino agonist 3i. The phenyl derivative 3g is an MT2-selective partial agonist, with MT2 binding affinity higher than melatonin, showing promising sleep-promoting and antianxiety properties in animal models.

1 NIH3T3 ®broblast cells transfected with the full-length coding region of the MT 2 human melatonin receptor stably expressed the receptor that is coupled to a pertussis toxin-sensitive G protein and exhibits high anity for melatonin (K I =261 pM). 2 The order of apparent anity for selected compounds was: 5 The other two mt 1 antagonists used here, ethyl]cyclobutanecarboxamide, behaved as partial agonists at the MT 2 subtype, with relative intrinsic activities of 0.37 and 0.39, respectively. 6 These ®ndings show, for the ®rst time, important dierences in the intrinsic activity of analogues between the human mt 1 and MT 2 melatonin receptor subtypes.

The design, synthesis, and biological profile of several indole melatonin analogues with a conformationally restricted C3 amidoethane side chain are presented. Examination of the accessible conformations of the melatonin side chain led us to explore some of its fully or partially restricted analogues, 2-12, the binding affinity values of which were utilized to gain further insight on the melatonin binding site. Two pharmacophoric models have been devised for melatonin and the active compounds by conformational analysis and superimposition performed using the DISCO program. In these models, the melatonin side chain can adopt a gauche/anti conformation out of the indole plane. Another contribution of this study regards the observation of a possible binding point interaction around the C2 position of the indole, as suggested by the remarkably increased binding affinity observed in the C2-substituted analogues 6 and 9 and especially in the more rigid analogue 5. The biological activity and the efficacy of the new compounds were tested by measuring the inhibition of the forskolin-stimulated cAMP accumulation and the GTP gamma S index. Both analyses demonstrated that all of the compounds were full agonists with the exception of 4 and 9, which showed a slight reduction in efficacy and would seem to be partial agonists.

The CoMFA methodology was applied to melatonin receptor ligands in order to establish quantitative structure-affinity relationships. One hundred thirty-three compounds were considered: they were either collected from literature or newly synthesized in order to gain information about the less explored positions. To this end, various melatonin derivatives were prepared and their affinity for quail optic tecta melatonin receptor was tested. Compounds were aligned on the putative active conformation of melatonin proposed by our previously reported pharmacophore search, and their relative affinities were calculated from the displacement of 2-[125I]-iodomelatonin on different tissues expressing aMT receptors. Compounds were grouped into three sets according to their topology. Subset A: melatonin-like compounds; subset B: N-acyl-2-amino-8-methoxytetralins and related compounds; subset C:N-acyl-phenylalkylamines and related compounds. CoMFA models were derived for each set, using the steric, electrostatic, and lipophilic fields as structural descriptors; the PLS analyses were characterized by good statistical parameters, taking into account the heterogeneity of the binding data, obtained with different experimental protocols. From the CoMFA model for the melatonin-like compounds, besides the well-known positive effect of 2-substitution, a low steric tolerance for substituents in 1, 6, and 7, and a negative effect of electron-rich 4-substituents were observed; the information provided by the newly synthesized compounds was essential for these results. Moreover, a comprehensive model for the 133 compounds, accounting for a common alignment and a common mode of interaction at the melatonin receptor, was derived (Q2 = 0.769, R2 = 0.905). This model validates our previously reported pharmacophore search and offers a clear depiction of the structure-affinity relationships for the melatonin receptor ligands.