Design and synthesis of benzofuranic derivatives as new ligands at the melatonin-binding site MT3

Benzofuranic analogues of MCA-NAT (5-methoxycarbonylamino-N-acetyltryptamine) have been synthesized and evaluated as melatonin receptor ligands. Introduction of a methoxycarbonylamino substituent in the C-5 position of the benzofurane nucleus obtains MT₃ selective ligands. This selectivity can be modulated with suitable variations of the C-5 position and the acyl group on the C-3 side chain.     

Synthesis and biological activity of new melatonin dimeric derivatives

A new series of melatonin (MLT) dimers were obtained by linking together two melatonin units with a linear alkyl chain through the MLT acetamido group or through a C-2 carboxyalkyl function. The binding properties of these ligands were evaluated in in vivo experiments on cloned human MT(1) and MT(2) receptors expressed in NIH3T3 rat fibroblast cells. The class of 2-carboxyalkyl dimers was the most interesting one with compounds having good MT(1)/MT(2) nanomolar affinity. The data obtained suggest that the spacer length is crucial for optimal interaction at both receptor subtypes as well as to determine functional activity of the resulting dimers.     

Design and synthesis of 1-(2-alkanamidoethyl)-6-methoxy-7-azaindole derivatives as potent melatonin agonists

A series of 7-azaindolic ligands bearing a methoxy group and a N-acetyl chain as melatoninergic pharmacophores were synthesized and their binding affinities towards MT₁ and MT₂ receptors were evaluated. Compounds 7a-c and 12 (cyclohexyl ring connected at C-2 and C-3 position) appears as important melatonin MT₂ and MT₁ receptors agonists. On the other hand, the presence of basic groups (amines) at position C-3 was detrimental to the melatoninergic affinities.     

Indole melatonin agonists and antagonists derived by shifting the melatonin side chain from the C-3 to the N-1 or to the C-2 indole position

This article reviews our efforts in the development of indole melatonin (MLT) agonist and antagonist compounds. Evidence is presented which indicates that high-affinity melatonergic agonists were obtained by shifting the MLT amido side chain from the C-3 to the N-1 indole position. Conversely, by moving the side chain from the C-3 to the C-2 indole position it is possible to produce MLT antagonist compounds.     

Synthesis and conformational study of 3,4-carbocyclic bridged indole melatonin and serotonin analogues

Tetrahydrobenz[cd]indole, has been usually assumed to be a rigid scaffold of arylethylamines of pharmaceutical interest, such as melatonin and serotonin. A series of molecules containing this scaffold has been synthesized and their conformation in solution has been determined by 1H NMR. The values of the coupling constants show that the carbocycle fused with the indole ring is a mixture of the two conformers with substituent in equatorial or axial orientation. The molar fraction of the conformers appears to be sensibly affected by the bulkiness of the C-2 indole substituent. A pseudo-axial orientation of the C-3 alkylamido side chain is important for melatonin ligands to access the binding site and exhibit potent in vitro affinity, as illustrated for melatonin ligand 1 (pK(i)=9.32).     

Application of the bridgehead fragments for the design of conformationally restricted melatonin analogues

Conformationally constrained analogues of the hormone melatonin with a side chain incorporated into the bicyclic bridgehead core were synthesized based on the homology modeling and molecular docking studies performed for the MT₂ melatonin receptor. The methoxy-indole derivative fused with exo-N-acetamino-substituted bicyclo[2.2.2]octane was found to possess nanomolar MT₂ receptor affinity.     

O-linked melatonin dimers as bivalent ligands targeting dimeric melatonin receptors

A series of dimeric melatonin analogues 3a-e obtained by connecting two melatonin molecules through the methoxy oxygen atoms with spacers spanning 16–24 atoms and the agomelatine dimer 7 were synthesized and characterized in 2-[¹²⁵-I]-iodomelatonin binding assays, bioluminescence resonance energy transfer (BRET) experiments, and in functional cAMP and β-arrestin recruitment assays at MT₁ and MT₂ receptors. The binding affinity of 3a-e generally increased with increasing linker length. Bivalent ligands 3a-e increased BRET signals of MT₁ dimers up to 3-fold compared to the monomeric control ligand indicating the simultaneous binding of the two pharmacophores to dimeric receptors. Bivalent ligands 3c and 7 exhibited important changes in functional properties on the G_i/cAMP pathway but not on the β-arrestin pathway compared to their monomeric counterparts. Interestingly, 3c (20 atoms spacer) shows inverse agonistic properties at MT₂ on the G_i/cAMP pathway. In conclusion, these findings indicate that O-linked melatonin dimers are promising tools to develop signaling pathway-based bivalent melatonin receptor ligands.     

Synthesis of 3-phenylnaphthalenic derivatives as new selective MT(2) melatoninergic ligands

A series of naphthalenic analogues of melatonin were prepared and evaluated as melatonin receptor MT(2) selective ligands. Activity and MT(2) selectivity can be modulated with suitable variations of the C-3 phenyl and the acyl group on the C-1 side chain. Surprisingly, in contrast with what had been previously described in other series (2-benzylindoles, 2-benzylbenzofurans and 3-phenyltetralins), the presence of a C-3 phenyl with a functional group on the meta position seems to be primordial for MT(2) affinity and selectivity. Indeed, N-[2-(3-(3-hydroxymethylphenyl)-7-methoxynaphth-1-yl)ethyl]acetamide (21) is one of the best MT(2) selective ligands described until now and behaves as an antagonist.     

Structure-affinity relationships of indole-based melatonin analogs

This paper reviews our progress made in characterizing structure-affinity relationships of indole-based melatonin analogs. Evidence is presented suggesting a preferred folded conformation for the amido side chain, almost orthogonal to the plane of indole. A 3D-QSAR comparative molecular field analysis (CoMFA) model, accounting for the observed differences in binding affinity within different classes of melatonergic ligands, and capable of quantitatively predicting the binding affinity of new compounds, is also reported.     

Modification of the Phe3 aromatic moiety in delta receptor-selective dermorphin/deltorphin-related tetrapeptides. Effects on opioid receptor binding.

The previously described cyclic delta opioid receptor-selective tetrapeptide H-Tyr-D-Cys-Phe-D-Pen-OH (JOM-13) was modified at residue 3 by incorporation of both natural and unnatural amino acids with varying steric, electronic, and lipophilic properties. Effects on mu and delta opioid receptor binding affinities were evaluated by testing the compounds for displacement of radiolabeled receptor-selective ligands in a guinea pig brain receptor binding assay. Results obtained with the bulky aromatic 1-Nal3 and 2-Nal3 substitutions suggest that the shape of the receptor subsite with which the side chain of the internal aromatic residue interacts differs for delta and mu receptors. This subsite of either receptor can accommodate the transverse steric bulk of the 1-Nal3 side chain but only the delta receptor can readily accept the more elongated 2-Nal3 side chain. Several analogs with pi-excessive heteroaromatic side chains in residue 3 were examined. In general, these analogs display diminished binding to mu and delta receptors, consistent with previous findings for analogs with residue 3 substitutions of modified electronic character. Several analogs with alkyl side chains in residue 3 were also examined. While delta receptor binding affinity is severely diminished with Val3, Ile3, and Leu3 substitutions, Cha3 substitution is very well tolerated, indicating that, contrary to the widely held belief, an aromatic side chain in this portion of the ligand is not required for delta receptor binding. Where possible, comparison of results in this delta-selective tetrapeptide series with those reported for analogous modification in the cyclic delta-selective pentapeptide [D-Pen2, D-Pen5]enkephalin (DPDPE) and linear pentapeptide enkephalins reveals similar trends.     

Structure-activity relationships in a series of melatonin analogues with the low-density lipoprotein oxidation model

Despite an increasing number of publications concerning the antioxidant activity of melatonin, little is known about the structural features responsible for this kind of activity. To understand the role played by the different elements of melatonin structure in its antioxidant activity, we have designed and tested several compounds related to this molecule in the low-density lipoprotein peroxidation model. We present here the results of this study in terms of structure-activity relationships focusing on the influence of the acetamidoethyl side chain, the methoxy group, and the indole heterocycle. In this model, we found that changing the acyl residue generally resulted in more active products. We obtained particularly good results with the nonanoyl derivative which showed a level of activity comparable to that of phenols despite lacking a phenolic function. The presence of a methoxy group in position 5 generally had a beneficial influence on the activity, but when located in position 6, the effects were various. The substitution of a hydroxy for the methoxy group led to phenolic compounds endowed with very high antioxidant activity. Replacing the amide with a ketone function did not affect the activity while replacement with an amine group in some cases resulted in prooxidant compounds. Finally, we compared the efficacy of different aromatic rings. The indole heterocycle proved to be better than benzofurane and naphthalene rings.     

Therapeutic potential of melatonin ligands

Melatonin is a neurohormone that is believed to be involved in a wide range of physiological functions. In humans, appropriate clinical trials confirm the efficacy of melatonin or melatoninergic agonists for the MT1 and MT2 receptor subtypes in circadian rhythm sleep disorders only. Nevertheless, preclinical animal model studies relevant to human pathologies involving validated reference compounds lead to other therapeutic possibilities. Among these is a recently developed treatment concept for depression, which has been validated by the clinical efficacy of agomelatine, an agent having both MT1 and MT2 agonist and 5-HT2C antagonist activity. A third melatonin binding site has been purified and characterized as the enzyme quinone reductase 2 (QR2). The physiological role of this enzyme is not yet known. Recent results obtained by different groups suggest: (1) that inhibition of QR2 may lead to "protective" effects and (2) that over-expression of this enzyme may have deleterious effects. The inhibitory effect of melatonin on QR2 observed in vitro may explain the protective effects reported for melatonin in different animal models, such as cardiac or renal ischemia-effects that have been attributed to the controversial antioxidant properties of the hormone. The development of specific ligands for each of these melatonin binding sites is necessary to link physiological and/or therapeutic effects.     

Knock-down of RGS4 and beta tubulin in CHO cells expressing the human MT1 melatonin receptor prevents melatonin-induced receptor desensitization

Previously, it has been shown that chronic melatonin exposure in MT1-CHO cells results in receptor desensitization while at the same time producing drastic morphological changes. The addition of a depolymerizing agent during the melatonin pretreatment period prevents MT1 receptor desensitization and the changes in cellular morphology. The lack of morphological change in the presence of a depolymerizing agent is easily explained by the inability of the microtubules to polymerize, however, the prevention of receptor desensitization is a little more complex and may involve G-protein activation. The goal of this study was to determine whether melatonin-induced MT1 receptor desensitization is regulated by proteins known to regulate G-protein activation states, beta-tubulin and RGS4,using anti sense knockdown approaches. The expression of RGS4 mRNA in CHO cells was confirmed using RT PCR and successful knockdown of each was confirmed by western blot analysis or quantitative PCR. Pretreatment of MT1-CHO cells, transfected with the nonsense probes and exposed to melatonin, resulted in a desensitization of the receptor, an increase in forskolin-induced cAMP accumulation, an increase in 2-[125I]-iodomelatonin binding and no change in the affinity of melatonin for the MT1 receptor. However, knockdown of either beta-tubulin or RGS4 in MT1-CHO cells followed by pretreatment with melatonin attenuated the desensitization of melatonin receptors, decreased total 2-[125I]-iodomelatonin binding, and did not affect neither the forskolin response nor the affinity of melatonin for the MT1 receptor. Perhaps RGS4 and beta-tubulin modulate Galpha-GDP and Galpha-GTP states thus modulating MT1 melatonin receptor function.     

Synthesis, pharmacological characterization and QSAR studies on 2-substituted indole melatonin receptor ligands

A number of 6-methoxy-1-(2-propionylaminoethyl)indoles, carrying properly selected substituents at the C-2 indole position, were prepared and tested as melatonin receptor ligands. Affinities and intrinsic activities for the human cloned mt1 and MT2 receptors were examined and compared with those of some 2-substituted melatonin derivatives recently described by us. A quantitative structure activity relationship (QSAR) study of the sixteen 2-substituted indole compounds, 5a-k, 1, 8-11, using partial least squares (PLS) and multiple regression analysis (MRA) revealed the existence of an optimal range of lipophilicity for the C2 indole substituent. There are also indications that planar, electron-withdrawing substituents contribute to the affinity by establishing additional interactions with the binding pocket. No mt1/MT2 subtype selectivity was observed, with the relevant exception of the 2-phenethyl derivative 5e, which exhibited the highest selectivity for the h-MT2 receptor among all the compounds tested (MT2/mt1 ratio of ca. 50). Conformational analysis and superposition of 5e to other reported selective MT2 ligands revealed structural and conformational similarities that might account for the MT2/mt1 selectivity of 5e.     

Binding affinity and biological activity of oxygen and sulfur isosteres at melatonin receptors as a function of their hydrogen bonding capability

Analogues of melatonin (1) and of N-acetyl 5-ethoxytryptamine (3) in which the oxygen atoms are replaced by sulfur have been prepared and tested against human and amphibian melatonin receptors. All sulfur analogues show a decreased binding affinity at human MT1 and MT2 receptors and a reduced potency as melatonin agonists on the Xenopus melanophore assay. The 5-methoxy oxygen of melatonin is significantly more important for receptor binding than the amide oxygen. N-Acetyl 5-ethoxytryptamine shows a decrease in both binding affinity and potency in comparison with melatonin. In this series, replacing either the ethoxy or amide oxygen by sulfur has a similar but smaller effect on both binding affinity and potency. Using K(B)(H) values from Abraham's equations we have assessed the possibility of estimating EC50 values for sulfur isosteres from the EC50 values of their oxygen analogues.     

Synthesis of β-Substituted Naphth-1-yl Ethylamido Derivatives as New Melatoninergic agonists

Naphthalene melatoninergic ligands with alkyl groups (Me, Et, Pr, Bz) in the β position of the ethylamido chain were synthesised. The affinity of the compounds for chicken brain melatonin receptors was evaluated using 2-[¹²⁵I]-iodomelatonin as the radioligand. An increase in the affinity was observed with the β-methyl derivatives and the greatest increase was seen with the (−) enantiomers. The introduction of a 2- or 7-MeO group on the naphthalene ring and the lengthening (Et, Pr) of the alkylamido chain gave potent compounds such as (−)1h (K_i=24 pM). The functional activity of these compounds was evaluated by the aggregation of melanophores in Xenopus laevis tadpoles. The potency to produce lightening of the skin of Xenopus laevis was related to the affinities values of the molecules at melatonin chicken brain receptors. The most potent ligands were found to be full agonists and compound 1h was 25 fold more potent than melatonin in this bioassay.     

Effects of water salinity on melatonin levels in plasma and peripheral tissues and on melatonin binding sites in European sea bass (Dicentrarchus labrax)

Sea bass is an euryhaline fish that lives in a wide range of salinities and migrates seasonally from lagoons to the open sea. However, to date, the influence of water salinity on sea bass melatonin levels has not been reported. Here, we evaluated the differences in plasma and tissue melatonin contents and melatonin binding sites in sea bass under four different salinity levels: seawater (36‰), isotonic water (15‰), brackish water (4‰) and freshwater (0‰). The melatonin content was evaluated in plasma, whole brain, gills, intestine and kidney, while melatonin binding sites were analyzed in different brain regions and in the neural retina. Plasma melatonin levels at mid-dark varied, the lowest value occurring in seawater (102 pg/mL), and the highest in freshwater (151 pg/mL). In gills and intestine, however, the highest melatonin values were found in the seawater group (209 and 627 pg/g tissue, respectively). Melatonin binding sites in the brain also varied with salinity, with the highest density observed at the lower salinities in the optic tectum, cerebellum and hypothalamus (30.3, 13.0, and 8.0 fmol/mg protein, respectively). Melatonin binding sites in the retina showed a similar pattern, with the highest values being observed in freshwater. Taken together, these results reveal that salinity influences melatonin production and modifies the density of binding sites, which suggests that this hormone could play a role in timing seasonal events in sea bass, including those linked to fish migration between waters of different salinities for reproduction and spawning.     

Therapeutic Potential of Melatonin Ligands

Melatonin is a neurohormone that is believed to be involved in a wide range of physiological functions. In humans, appropriate clinical trials confirm the efficacy of melatonin or melatoninergic agonists for the MT₁ and MT₂ receptor subtypes in circadian rhythm sleep disorders only. Nevertheless, preclinical animal model studies relevant to human pathologies involving validated reference compounds lead to other therapeutic possibilities. Among these is a recently developed treatment concept for depression, which has been validated by the clinical efficacy of agomelatine, an agent having both MT₁ and MT₂ agonist and 5‐HT_2C antagonist activity. A third melatonin binding site has been purified and characterized as the enzyme quinone reductase 2 (QR2). The physiological role of this enzyme is not yet known. Recent results obtained by different groups suggest: (1) that inhibition of QR2 may lead to “protective” effects and (2) that over‐expression of this enzyme may have deleterious effects. The inhibitory effect of melatonin on QR2 observed in vitro may explain the protective effects reported for melatonin in different animal models, such as cardiac or renal ischemia—effects that have been attributed to the controversial antioxidant properties of the hormone. The development of specific ligands for each of these melatonin binding sites is necessary to link physiological and/or therapeutic effects.