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.

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.