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Activation of melatonin receptors and inhibition of fatty acid amide hydrolase (FAAH) have both shown potential benefits for the treatment of glaucoma. To exploit the combination of these biological activities in single therapeutic agents, we designed dual-acting compounds sharing the pharmacophore elements required for the two targets, in search for balanced potencies as MT 1 /MT 2 agonists and FAAH inhibitors. In particular, the N-anilinoethylamide scaffold, previously developed for melatonergic ligands, was decorated at meta position with a polymethylene linker bound to an O-arylcarbamate group, substituted according to known structure-activity relationships for FAAH inhibition. For the most active series, the N-anilinoethylamide portion was also replaced with the indole scaffold of melatonin. O-Biphenyl-3-ylcarbamate derivatives were characterized by remarkable and balanced activity at both targets, in the nanomolar range for compound 29. Topical administration reduced elevated intraocular pressure in rabbits, with a longer action and improved efficacy compared to the reference compounds melatonin and URB597.

A new family of melatonin receptor ligands, characterized by a tetrahydroquinoline (THQ) scaffold carrying an amide chain in position 3, was devised as conformationally constrained analogs of flexible N-anilinoethylamides previously developed. Molecular superposition models allowed to identify the patterns of substitution conferring high receptor binding affinity and to support the THQ ring as a suitable scaffold for the preparation of melatonin ligands. The biological activity of 3-acylamino-THQs was compared with that of the corresponding tetralin derivatives. The THQ ring proved to be a versatile scaffold for easy feasible MT 1 and MT 2 ligands, which resulted as more polar bioisosteres of their tetralin analogs. Potent partial agonists, with subnanomolar binding affinity for the MT 2 receptor, were obtained, and a new series of THQ derivatives is presented. The putative binding mode of potent THQs and tetralines was discussed on the basis of their conformational equilibria as inferred from molecular dynamics simulations and experimental NMR data.

Molecular superposition models guided the design of novel melatonin receptor ligands characterized by a 2-acylaminomethyltetrahydroquinoline scaffold. Starting from the structure of N-anilinoethylamide ligands, the flexible chain was conformationally constrained to reproduce the bioactive conformation of melatonin. Structure-activity relationships were investigated, focusing on the substituent at the nitrogen atom, the position of the methoxy group, and the replacement of the amide side chain by urea and thiourea groups. The compounds were tested for binding affinity and intrinsic activity at human MT1 and MT2 receptors. Structural optimization resulted in N-[(1-benzyl-1,2,3,4-tetrahydro-5-methoxyquinolin-2-yl)methyl]propionamide (UCM1014), with picomolar MT2 binding affinity (K(i) = 0.001 nM), more than 10000-fold selectivity over the MT1 receptor, and a full agonist profile (GTPγS test), being the most potent MT2-selective full agonist reported to date. Molecular dynamics simulations provided a rationale for high binding affinity, stereoselectivity, and agonist behavior of these novel melatonin receptor ligands based on superposition models and conformational preference.

New derivatives of 4-phenyl-2-propionamidotetralin (4-P-PDOT) were prepared and tested on cloned MT1 and MT2 receptors, with the purpose of merging previously reported pharmacophores for nonselective agonists and for MT2-selective antagonists. A 8-methoxy group increases binding affinity of both (±)-cis- and (±)-trans-4-P-PDOT, and it can be bioisosterically replaced by a bromine. Conformational analysis of 8-methoxy-4-P-PDOT by molecular dynamics, supported by NMR data, revealed an energetically favored conformation for the (2S,4S)-cis isomer and a less favorable conformation for the (2R,4S)-trans one, fulfilling the requirements of a pharmacophore model for nonselective melatonin receptor agonists. A new superposition model, including features characteristic of MT2-selective antagonists, suggests that MT1/MT2 agonists and MT2 antagonists can share the same arrangement for their pharmacophoric elements. The model correctly predicted the eutomers of (±)-cis- and (±)-trans-4-P-PDOT. The model was validated by preparing three dihydronaphthalene derivatives, either able or not able to reproduce the putative active conformation of 4-P-PDOT.

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.

A novel series of melatonin receptor ligands was discovered by opening the cyclic scaffolds of known classes of high affinity melatonin receptor antagonists, while retaining the pharmacophore elements postulated by previously described 3D-QSAR and receptor models. Compounds belonging to the classes of 2,3- and [3,3-diphenylprop(en)yl]alkanamides and of o- or [(m-benzyl)phenyl]ethyl-alkanamides were synthesized and tested on MT(1) and MT(2) receptors. The class of 3,3-diphenyl-propenyl-alkanamides was the most interesting one, with compounds having MT(2) receptor affinity similar to that of MLT, remarkable MT(2) selectivity, and partial agonist or antagonist behavior. In particular, the (E)-m-methoxy cyclobutanecarboxamido derivative 18f and the di-(m-methoxy) acetamido one, 18g, have sub-nM affinity for the MT(2) subtype, with more than 100-fold selectivity over MT(1), 18f being an antagonist and 18g a partial agonist on GTPgammaS test. Docking of 18g into a previously developed MT(2) receptor model showed a binding scheme consistent with that of other antagonists. The MT(2) expected binding affinities of the new compounds were calculated by a previously developed 3D-QSAR CoMFA model, giving satisfactory predictions.

The three-dimensional quantitative structure-activity relationship comparative molecular field analysis (3D-QSAR CoMFA) approach was applied to some classes of melatonin (MLT) membrane receptor ligands, with the principal aim of exploring the correlation between their steric features and MT(2)-selective antagonism. Binding data obtained from cloned MT(1) and MT(2) receptor subtypes were used to develop 3D-QSAR models for agonists and for antagonists at the two receptor subtypes, looking for the structural requirements for receptor subtype selectivity. In particular, we superposed the compounds showing antagonist activity, or very low intrinsic activity at the GTPgammaS test, following the hypothesis that the occupation of an additional pocket positioned out of the plane of MLT is one of the major determinants for MT(2) selectivity; the statistical models obtained confirmed this hypothesis. Structure-intrinsic activity relationship studies, applied to a set of compounds homogeneously tested, allowed the identification of the structural features whose modulation shifts the behavior from that of the agonist to that of the antagonist. The pocket out of the plane of MLT was identified as one of the key features for obtaining selective MT(2) antagonists. The reliability of our statistical models was further confirmed by the correct prediction of the pharmacological behavior of some N-substituted melatonin derivatives, which were prepared and tested on cloned receptor subtypes.

Several indole analogues of melatonin (MLT) were obtained by moving the MLT side chain from C(3) to C(2) of the indole ring. Binding and in vitro functional assays were performed on cloned human MT1 and MT2 receptors, stably transfected in NIH3T3 cells. Quantitative structure-activity relationship studies showed that 4-methoxy-2-(N-acylaminomethyl)indoles, with a benzyl group in position 1, were selective MT2 antagonists and, in particular, N-[(1-p-chlorobenzyl-4-methoxy-1H-indol-2-yl)methyl]propanamide (12) behaved as a pure antagonist at MT1 and MT2 receptors, with a 148-fold selectivity for MT2. We present a topographical model that suggests a lipophilic group, located out of the plane of the indole ring of MLT, as the key feature of the MT2 selective antagonists.

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.

A series of 2-substituted 5-methoxy-N-acyltryptamines was synthesized and their affinity for the melatonin receptor, isolated from whole quail brains, was tested in a succession of in vitro ligand-receptor binding experiments, using 2-[125I]iodomelatonin as a labeled ligand. Optimization of the C2 substituent and the N-acyl group resulted in compounds having picomolar affinity for the receptor (vs nanomolar affinity for melatonin). In two tests for evaluation of the biological activity (effects on the spontaneous firing activity of single neurons in the rabbit parietal cortex in situ, and the Syrian hamster gonadal regression model in vivo) most of the analogs behaved as agonists. Isopropyl substitution at C2 alone, or concomitantly with cyclopropyl substitution at the N-acyl position, resulted in much lower affinity and weaker biological effect, or lack of activity in the latter case. Of interest are the compounds 4d (R = phenyl, R1 = CH3) and 4g (R = phenyl, R1 = cyclopropyl), which expressed high affinity for the receptor and apparent antagonistic activity under the conditions of the experimental models employed, though the analog 4g (R = phenyl, (R1 = cyclopropyl) seemingly was a weak antagonist and in situ expressed mixed activity in the higher concentration range. Cyclopropyl substitution at the N-acyl position inevitably resulted in lower affinity for the receptor and weaker biological activity. These data demonstrate that the N-acetyl group is important for both affinity and agonist biological activity. The substituents at C2 are crucial for the affinity of the compound for the receptor and can be utilized to create putative high-affinity agonists or antagonists.