Synthesis of substituted N-[3-(3-methoxyphenyl)propyl] amides as highly potent MT2-selective melatonin ligands

A series of substituted N-[3-(3-methoxyphenyl)propyl] amides were synthesized and their binding affinities towards human melatonin MT₁ and MT₂ receptors were evaluated. It was discovered that a benzyloxyl substituent incorporated at C6 position of the 3-methoxyphenyl ring dramatically enhanced the MT₂ binding affinity and at the same time decreased MT₁ binding affinity.     

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.     

Design,synthesis and pharmacological evaluation of novel naphthalenic derivatives as selective MT1 melatoninergic ligands

Novel heterodimer analogues of melatonin were synthesized, when agomelatine (1) and various aryl units are linked via a linear alkyl chain through the methoxy group. The compounds were tested for their actions at melatonin receptors. Several of these ligands are MT₁-selective with nanomolar or subnanomolar affinity. In addition, while most of the derivatives behave as partial agonists on one or both receptor subtypes, N-[2-(7-{4-[6-(1-methoxycarbonylethyl)naphthalen-2-yloxy]butoxy}naphthalen-1-yl)ethyl]acetamide (36), a subnanomolar MT₁ ligand with an 11-fold preference over MT₂ receptors, is a full antagonist on both receptors. Our results also confirm that the selectivity seen for the MT₁ receptor arises predominantly from steric factors and is not a consequence of the bridging of melatonin receptor dimers.     

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.     

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.     

Synthesis of new N-(arylcyclopropyl)acetamides and N-(arylvinyl)acetamides as conformationally-restricted ligands for melatonin receptors

N-(Arylcyclopropyl)acetamides and N-(arylvinyl)acetamides or methyl ureas have been prepared as constrained analogues of melatonin. The affinity of these new compounds for chicken brain melatonin receptors and recombinant human MT₁ and MT₂ receptors was evaluated using 2-[¹²⁵I]-iodomelatonin as radioligand. Strict ethylenic or cyclopropyl analogues of the commercialized agonist agomelatine (Valdoxan®) were equipotent to agomelatine in binding bioassays. However, the ethylenic analogue was more effective than the cyclopropyl one in the melanophore aggregation bioassay, but was still less potent than the disubstituted 2,7-dimethoxy-naphtalenic compounds.     

Design and synthesis of 3-phenyltetrahydronaphthalenic derivatives as new selective MT2 melatoninergic ligands. Part II

Following our studies of the melatoninergic receptors, we have developed new tetrahydronaphthalenic derivatives of melatonin that have been tested as selective melatonin receptors ligands. Regarding the role of the phenyl substituent to obtain selective ligands, modulation of selectivity and activity have been achieved by modifications of the acyl group and substitutions on the phenyl ring. Ten of the seventeen evaluated derivatives have MT₂ receptor affinity similar to that of melatonin. Moreover, we have achieved remarkable MT₂ selectivity over MT₁ (selectivity >100) and have been able to further extend the RSA of the tetrahydrophthalenic series. However, the compounds presented here display partial agonist or antagonist behavior instead of full agonist.     

Melatonin Signaling Controls the Daily Rhythm in Blood Glucose Levels Independent of Peripheral Clocks

Melatonin is rhythmically secreted by both the pineal gland and retina in a circadian fashion, with its peak synthesis occurring during the night. Once synthesized, melatonin exerts its effects by binding to two specific G-protein coupled receptors–melatonin receptor type 1(MT₁) and melatonin receptor type 2(MT₂). Recent studies suggest the involvement of MT₁ and MT₂ in the regulation of glucose homeostasis; however the ability of melatonin signaling to impart timing cues on glucose metabolism remains poorly understood. Here we report that the removal of MT₁ or MT₂ in mice abolishes the daily rhythm in blood glucose levels. Interestingly, removal of melatonin receptors produced small effects on the rhythmic expression patterns of clock genes within skeletal muscle, liver, and adipose tissue. Taken together, our data suggest that the loss of the daily rhythm in blood glucose observed in MT₁^-/- and MT₂^-/- mice does not occur as a consequence of ‘disrupted’ clocks within insulin sensitive tissues. Finally our results highlight a diurnal contribution of melatonin receptor signaling in the daily regulation of blood glucose levels.     

Convergence of Melatonin and Serotonin (5-HT) Signaling at MT2/5-HT2C Receptor Heteromers

Inasmuch as the neurohormone melatonin is synthetically derived from serotonin (5-HT), a close interrelationship between both has long been suspected. The present study reveals a hitherto unrecognized cross-talk mediated via physical association of melatonin MT₂ and 5-HT_2C receptors into functional heteromers. This is of particular interest in light of the “synergistic” melatonin agonist/5-HT_2C antagonist profile of the novel antidepressant agomelatine. A suite of co-immunoprecipitation, bioluminescence resonance energy transfer, and pharmacological techniques was exploited to demonstrate formation of functional MT₂ and 5-HT_2C receptor heteromers both in transfected cells and in human cortex and hippocampus. MT₂/5-HT_2C heteromers amplified the 5-HT-mediated G_q/phospholipase C response and triggered melatonin-induced unidirectional transactivation of the 5-HT_2C protomer of MT₂/5-HT_2C heteromers. Pharmacological studies revealed distinct functional properties for agomelatine, which shows “biased signaling.” These observations demonstrate the existence of functionally unique MT₂/5-HT_2C heteromers and suggest that the antidepressant agomelatine has a distinctive profile at these sites potentially involved in its therapeutic effects on major depression and generalized anxiety disorder. Finally, MT₂/5-HT_2C heteromers provide a new strategy for the discovery of novel agents for the treatment of psychiatric disorders.     

Identification of Pathway-Biased and Deleterious Melatonin Receptor Mutants in Autism Spectrum Disorders and in the General Population

Melatonin is a powerful antioxidant and a synchronizer of many physiological processes. Alteration of the melatonin pathway has been reported in circadian disorders, diabetes and autism spectrum disorders (ASD). However, very little is known about the genetic variability of melatonin receptors in humans. Here, we sequenced the melatonin receptor MTNR1A and MTNR1B, genes coding for MT₁ and MT₂ receptors, respectively, in a large panel of 941 individuals including 295 patients with ASD, 362 controls and 284 individuals from different ethnic backgrounds. We also sequenced GPR50, coding for the orphan melatonin-related receptor GPR50 in patients and controls. We identified six non-synonymous mutations for MTNR1A and ten for MTNR1B. The majority of these variations altered receptor function. Particularly interesting mutants are MT₁-I49N, which is devoid of any melatonin binding and cell surface expression, and MT₁-G166E and MT₁-I212T, which showed severely impaired cell surface expression. Of note, several mutants possessed pathway-selective signaling properties, some preferentially inhibiting the adenylyl cyclase pathway, others preferentially activating the MAPK pathway. The prevalence of these deleterious mutations in cases and controls indicates that they do not represent major risk factor for ASD (MTNR1A case 3.6% vs controls 4.4%; MTNR1B case 4.7% vs 3% controls). Concerning GPR50, we detected a significant association between ASD and two variations, Δ502–505 and T532A, in affected males, but it did not hold up after Bonferonni correction for multiple testing. Our results represent the first functional ascertainment of melatonin receptors in humans and constitute a basis for future structure-function studies and for interpreting genetic data on the melatonin pathway in patients.     

MT2-like melatonin receptor modulates amplitude receptor potential in visual cells of crayfish during a 24-hour cycle

Retinular photoreceptors are structures involved in the expression and synchronization of the circadian rhythm of sensitivity to light in crayfish. To determine whether melatonin possesses a differential effect upon the receptor potential (RP) amplitude of retinular photoreceptors circadian time (CT)-dependent, we conducted experiments by means of applying melatonin every 2 h during a 24-hour cycle. Melatonin with 100 nM increased RP amplitude during subjective day to a greater degree than during subjective night. To determine whether MT₂ melatonin receptors regulate the melatonin-produced effect, we carried out two experiments, circadian times (CTs) 6 and 18, which included the following: (1) application of the MT₂ receptor selective agonist 8-M-PDOT and antagonist DH97, and (2) the specific binding of [¹²⁵I]-melatonin in eyestalk membranes. The amount of 10 nM of 8-M-PDOT increased RP amplitude in a similar manner to melatonin, and 1 nM DH97 abolished the increase produced by melatonin and 8-M-PDOT. Binding of [¹²⁵I]-melatonin was saturable and specific. Scatchard analysis revealed an affinity constant (K_d) of 1.1 nM and a total number of binding sites (B_max) of 6 fmol/mg protein at CT 6, and a K_d of 1.46 nM and B_max of 7 fmol/mg protein at CT 18. Our results indicate that melatonin increased RP amplitude of crayfish retinular photoreceptors through MT₂-like melatonin receptors. These data support the idea that melatonin is a signal of darkness for the circadian system in crayfish retinular cells.     

Development of substituted N-[3-(3-methoxylphenyl)propyl] amides as MT2-selective melatonin agonists: Improving metabolic stability

A series of novel and selective N-[3-(6-benzyloxy-3-methoxyphenyl)propyl] amides has recently been shown to possess sub-nanomolar range binding affinity to the type 2 melatonin receptor (MT₂). Pharmacokinetics studies suggested that these compounds were subject to vigorous CYP450-mediated metabolism, resulting in a series of metabolites with significantly decreased or diminished binding affinities toward MT₂ receptor. The ether bonds were found to be the major positions susceptible to metabolism. In this study, the benzyl ether bond was either removed or replaced with a carbon–carbon bond in an attempt to improve metabolic stability and enhance their resistance towards phase I oxidation. The synthesis, receptor binding affinity, intrinsic potency and metabolic stability of modified structures are reported in this article. By removal or replacement of metabolic labile ether linkerage with carbon linkers, a novel compound was identified with good potency and MT₂ selectivity, and with increased metabolic stability.     

Synthesis of new melatoninergic hexahydroindenopyridines

Hexahydroindenopyridine (HHIP) is an interesting heterocyclic framework that contains an indene core similar to ramelteon. This type of tricyclic piperidines aroused our interest as potential melatoninergic ligands. Melatonin receptor ligands have applications in insomnia and depression. We report herein an efficient two-step method to prepare new HHIP by the reaction of an enamine with 3-bromopropylamine hydrobromide. Some synthesized compounds showed moderate affinity for melatonin receptors in the nanomolar or low micromolar range. Furthermore, the methylenedioxy HHIPs 2d (N-phenylacetamide) and 2f (N,N-diethylacetamide), exhibited high selectivity at MT₁ or MT₂ receptors, respectively, when compared with melatonin. It seems that the methylenedioxy group on the indene ring system and the N-acetamide substituent are important structural features to bind selectively MT₁ or MT₂ subtypes.     

Role of the MT1 and MT2 melatonin receptors in mediating depressive‐ and anxiety‐like behaviors in C3H/HeN mice

Melatonin is a neurohormone primarily synthesized by the pineal gland following a circadian rhythm with a high level during the night and a low level during the day. Alterations in the synthesis and secretion of melatonin have been reported in various mood disorders, including major depressive disorder. However, the role of endogenous melatonin in the pathophysiology of depressive disorder is unclear. Melatonin primarily acts through two G protein‐coupled receptors, termed MT₁ and MT₂. The present study investigated the effect of genetic deletion of the MT₁ and/or MT₂ receptors on tests associated with depression‐ and anxiety‐like behaviors in C3H/HeN mice. Deletion of the MT₁ and/or MT₂ receptors caused a deficit in hedonic and social interaction behavior, and increased anxiety‐like behavior. It is likely that dysregulations of the MT₁ and/or MT₂ melatonin receptors could be involved in the pathophysiology of depression and anxiety.     

Design,synthesis, and pharmacological effects of structurally simple ligands for MT1 and MT2 melatonin receptors

A series of phenoxyalkyl and phenylthioalkyl amides were prepared as melatoninergic ligands. Modulation of affinity of the newly synthesized compound by applying SARs around the terminal amide moiety, the alkyl chain, and the methoxy group on the aromatic ring provides compounds with nanomolar affinity for both melatonin receptor subtypes. Affinity towards MT₁ and MT₂ receptors were modulated also exploiting chirality. The investigation of intrinsic activity revealed that all the tested compounds behave as full or partial agonists.     

New melatonin (MT1/MT2) ligands: Design and synthesis of (8,9-dihydro-7H-furo[3,2-f]chromen-1-yl) derivatives

Herein we describe the synthesis of novel tricyclic analogues issued from the rigidification of the methoxy group of the benzofuranic analogue of melatonin as MT₁ and MT₂ ligands. Most of the synthesized compounds displayed high binding affinities at MT₁ and MT₂ receptors subtypes. Compound 6b (MT₁, K_i = 0.07 nM; MT₂, K_i = 0.08 nM) exhibited with the vinyl 6c and allyl 6d the most interesting derivatives of this series. Functional activity of these compounds showed full agonist activity with EC₅₀ in the nanomolar range. Compounds 6a (EC₅₀ = 0.8 nM and E_max = 98%) and 6b (EC₅₀ = 0.2 nM and E_max = 121%) exhibited good pharmacological profiles.     

Distribution of MT1 Melatonin Receptor Promoter-Driven RFP Expression in the Brains of BAC C3H/HeN Transgenic Mice

The pineal hormone melatonin activates two G-protein coupled receptors (MT₁ and MT₂) to regulate in part biological functions. The MT₁ and MT₂ melatonin receptors are heterogeneously distributed in the mammalian brain including humans. In the mouse, only a few reports have assessed the expression of the MT₁ melatonin receptor expression using 2-iodomelatonin binding, in situ hybridization and/or polymerase chain reaction (PCR). Here, we described a transgenic mouse in which red fluorescence protein (RFP) is expressed under the control of the endogenous MT₁ promoter, by inserting RFP cDNA at the start codon of MTNR1a gene within a bacterial artificial chromosome (BAC) and expressing this construct as a transgene. The expression of RFP in the brain of this mouse was examined either directly under a fluorescent microscope or immunohistochemically using an antibody against RFP (RFP-MT₁). RFP-MT₁ expression was observed in many brain regions including the subcommissural organ, parts of the ependyma lining the lateral and third ventricles, the aqueduct, the hippocampus, the cerebellum, the pars tuberalis, the habenula and the habenula commissure. This RFP-MT₁ transgenic model provides a unique tool for studying the distribution of the MT₁ receptor in the brain of mice, its cell-specific expression and its function in vivo.     

The Presence and Localization of Melatonin Receptors in the Rat Aorta

Melatonin is involved in blood pressure modulation in rats and humans. Some of the effects of melatonin are presumably mediated via two G-protein-coupled receptors (MT₁ and MT₂), but the distribution of MT₁ and MT₂ in the cardiovascular system remains to be explored comprehensively. We investigated the expression of both the receptors in the rat aorta on mRNA level by RT-PCR and real time RT-PCR as well as on protein level via western blotting and immunofluorescence microscopy. We verified MT₁ mRNA expression in the rat aorta and demonstrated the absence of MT₂ mRNA in this vessel type. MT₁ receptors were confirmed also at the protein level, and surprisingly they were preferentially localized to the tunica adventitia. Since no daily changes in MT₁ mRNA expression were detected, we suppose that the circadian changes in circulating melatonin concentrations are sufficient to mediate circadian effects of melatonin in the aorta. The localization of MT₁ in the tunica adventitia suggests an influence of melatonin on vasa vasorum function and signal transduction in the aorta wall.     

Polybenzyls from Gastrodia elata,their agonistic effects on melatonin receptors and structure-activity relationships

Gastrodia elata is a famous traditional Chinese herb with medicinal and edible application. In this study, nine polybenzyls (1?9), including six new ones (2?5, 7 and 9), were isolated from the EtOAc extract of G. elata. Five compounds 1, 3, 4, 6 and 8 were found to activate melatonin receptors. Especially, compound 1 showed agonistic effects on MT₁ and MT₂ receptors with EC₅₀ values of 237 and 244 μM. For better understanding their structure-activity relationships (SARs), ten polybenzyl analogs were further synthesized and assayed for their activities on melatonin receptors. Preliminary SARs study suggested that two para-hydroxy groups were the key pharmacophore for maintaining activity. Molecular docking simulations verified that compound 1 could strongly interact with MT₂ receptor by bonding to Phe 118, Gly 121, His 208, Try 294 and Ala 297 residues.     

Human Gastroenteropancreatic Expression of Melatonin and Its Receptors MT1 and MT2

Background and Aim

The largest source of melatonin, according to animal studies, is the gastrointestinal (GI) tract but this is not yet thoroughly characterized in humans. This study aims to map the expression of melatonin and its two receptors in human GI tract and pancreas using microarray analysis and immunohistochemistry.

Method

Gene expression data from normal intestine and pancreas and inflamed colon tissue due to ulcerative colitis were analyzed for expression of enzymes relevant for serotonin and melatonin production and their receptors. Sections from paraffin-embedded normal tissue from 42 individuals, representing the different parts of the GI tract (n=39) and pancreas (n=3) were studied with immunohistochemistry using antibodies with specificity for melatonin, MT₁ and MT₂ receptors and serotonin.

Results

Enzymes needed for production of melatonin are expressed in both GI tract and pancreas tissue. Strong melatonin immunoreactivity (IR) was seen in enterochromaffin (EC) cells partially co-localized with serotonin IR. Melatonin IR was also seen in pancreas islets. MT₁ and MT₂ IR were both found in the intestinal epithelium, in the submucosal and myenteric plexus, and in vessels in the GI tract as well as in pancreatic islets. MT₁ and MT₂ IR was strongest in the epithelium of the large intestine. In the other cell types, both MT₂ gene expression and IR were generally elevated compared to MT₁. Strong MT₂, IR was noted in EC cells but not MT₁ IR. Changes in gene expression that may result in reduced levels of melatonin were seen in relation to inflammation.

Conclusion

Widespread gastroenteropancreatic expression of melatonin and its receptors in the GI tract and pancreas is in agreement with the multiple roles ascribed to melatonin, which include regulation of gastrointestinal motility, epithelial permeability as well as enteropancreatic cross-talk with plausible impact on metabolic control.