New MT2 melatonin receptor-selective ligands: Agonists and partial agonists

Article abstract of DOI:10.3390/ijms18071347

The search for melatonin receptor agonists and antagonists specific towards one of the receptor subtypes will extend our understanding of the role of this system in relaying circadian information to the body. A series of compounds derived from a hit compound discovered in a screening process led to powerful agonists specific for one of the isoform of the melatonin receptor namely, MT2. The compounds are based on a poorly explored skeleton in the molecular pharmacology of melatonin. By changing the steric hindrance of one substituent (i.e., from a hydrogen atom to a tributylstannyl group), we identified a possible partial agonist that could lead to antagonist analogues. The functionalities of these compounds were measured with a series of assays, including the binding of GTPγS, the inhibition of the cyclic AMP production, the β-arrestin recruitment, and the cell shape changes as determined by cellular dielectric spectroscopy (CellKey). The variations between the compounds are discussed.

Full text of DOI:10.3390/ijms18071347

International Journal of
Molecular Sciences
Article
New MT Melatonin Receptor-Selective Ligands:
2
Agonists and Partial Agonists
Jean A. Boutin 1,2,* , Anne Bonnaud , Chantal Brasseur , Olivier Bruno , Nolwenn Lepretre ,
1
1
1
3
3
1
4
1,2
Peter Oosting , Sophie Coumailleau , Philippe Delagrange , Olivier Nosjean
and
Céline Legros 1
1
Pôle d’Expertise Biotechnologie, Chimie, Biologie, Institut de Recherches SERVIER, 78290 Croissy-sur-Seine,
France; anne.bonnaud@servier.com (A.B.); chantal.brasseur@servier.com (C.B.);
olivier.bruno@servier.com (O.B.); sophie.coumailleau@servier.com (S.C.); olivier.nosjean@servier.com (O.N.);
celine.legros@servier.com (C.L.)
2
3
4
Pôle d’Expertise Recherches & BioPharmacie, Institut de Recherches Internationales SERVIER,
9
2150 Suresnes, France
DIVERCHIM S.A., 95700 Roissy-en-France, France; nolwenn.lepretre@diverchim.com (N.L.);
peter.oosting@diverchim.com (P.O.)
Pôle d’Innovations Thérapeutiques en Neurosciences, Institut de Recherches, SERVIER,
7
8290 Croissy-sur-Seine, France; philippe.delagrange@servier.com
*
Correspondence: jean.boutin@servier.com; Tel.: +33-155-724-400; Fax: +33-155-722-810
Received: 26 March 2017; Accepted: 20 June 2017; Published: 23 June 2017
Abstract: The search for melatonin receptor agonists and antagonists specific towards one of the
receptor subtypes will extend our understanding of the role of this system in relaying circadian
information to the body. A series of compounds derived from a hit compound discovered in
a screening process led to powerful agonists specific for one of the isoform of the melatonin receptor
namely, MT . The compounds are based on a poorly explored skeleton in the molecular pharmacology
2
of melatonin. By changing the steric hindrance of one substituent (i.e., from a hydrogen atom
to a tributylstannyl group), we identified a possible partial agonist that could lead to antagonist
analogues. The functionalities of these compounds were measured with a series of assays, including
the binding of GTP
γ
S, the inhibition of the cyclic AMP production, the
β
-arrestin recruitment, and
®
the cell shape changes as determined by cellular dielectric spectroscopy (CellKey ). The variations
between the compounds are discussed.
Keywords: molecular pharmacology; melatonin receptors; agonist; partial agonist; functional assays
1
. Introduction
The neurohormone melatonin was discovered as N-acetyl-5-methoxytryptamine more than fifty
years ago by Lerner and coworkers [
performed by the pineal gland [ ]. Other regions of the central nervous system and other tissues
and cells such as the retina [ ], skin, bone marrow, lymphocytes [ ], and gastrointestinal tract [
are capable of melatonin synthesis. The main acitivities of melatonin are mediated by two receptors
named MT and MT ) and a cytosolic protein (quinone reductase 2).
1]. Its biosynthetic route follows a circadian rhythm, and is mainly
2
3
4
5]
(
1
2
Melatonin plays a major role in almost all physiological processes, including modulation of
hormonal secretions [ ], regulation of cardiovascular functions [ ], pain perception [ ], depression [ ],
and immune system and core body temperature control [10 11]. Melatonin is a key participant in
6
7
8
9
,
several pathological processes (see Boutin for recent review [12]) such as sleep disturbances and
insomnia [13], cancer and inflammation [14], neurodegenerative diseases, diabetes, depression and
anxiety [15–17]. Some cases of specificities were reported in which one particular isoform was
Int. J. Mol. Sci. 2017, 18, 1347; doi:10.3390/ijms18071347
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2017, 18, 1347
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implicated in a given pathology. In particular, several reports point to the possible key role of the
receptor isotype MT in sleep [18] and in neuropathic pain [19], after initially referring to its implication
2
in depression in mice [20]. Despite these reported numerous actions, ramelteon, agomelatine, and
tasimelteon constitute the only melatonin analogues commercialized up to now [21
–23] together with
circadin, a melatonin galenic alternative form [24].
Although the endogenous role(s) and mechanism(s) of action of melatonin have not yet been fully
elucidated, its therapeutic potential appears to be mediated via the activation of its two receptors (MT1
and MT ) that belong to the G protein-coupled receptors superfamily (GPCRs), although the inhibition
2
of the enzyme quinone reductase 2 (previously known as MT ) might also have some role in the
3
numerous therapeutic effects of melatonin [12]. Numerous studies of the two recombinant receptors
from several species (human, sheep, rat or mouse) have revealed only minor differences between
species. Particularly intriguing is that only a few agonist ligands with selective specificity towards the
receptor subtypes have been found, and even fewer ligands with antagonistic functionality. Therefore,
a better understanding of the melatoninergic system could lead to the discovery of new compounds,
particularly potent ligands that are selective for the receptor subtypes [25
affinity non-selective ligands are described in the literature, among which ligands with clear receptor
subtype selectivity, especially for MT , remain elusive, despite a series of dimeric compounds claimed
–27]. A large number of high
1
to be specific of this isotype (see Zlotos et al. [28]). To the contrary, MT specific ligands have been
2
discovered and described more frequently.
In the present work, we sought to prepare a series of putative specific ligands for the MT2
melatonin receptor, based on a hit compound (DIV0879) from our high throughput screening (HTS)
campaigns [29]. Herein, we describe the analogues of DIV0880, a compound that we have used
as a selective ligand in previous studies [30,31]. We attempted to characterize this compound and
its derivatives as possible selective ligands for MT by observing their effects on the main protein
2
Gi-mediated signaling pathways of melatonin receptors. We show that whatever the size of the
R-substituent of the biphenyl moiety (see Figure 1), the compounds remain, at best, only partial
agonists specific for the MT receptor while the original compounds (DIV0879 and DIV0880) were
2
full agonists [30,31], a feature confirmed in the present work. However, as the size/hindrance of the
substituents decreases, the agonistic partiality also decreases, rendering the compounds less and less
effective as agonists.
Figure 1. Structures of the compounds.
2
. Results
For the syntheses of these new compounds based on the biphenylmethane moiety, we questioned
if the size of the substituent would lead to enhanced antagonistic activity specific toward the MT2
melatonin receptor. In the early phase of our designs, we did not anticipate that the compounds could
be used in vivo at a later stage. The basic structures of the compounds were based on an initial result

Int. J. Mol. Sci. 2017, 18, 1347
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(
(
from a random HTS). The model compound lacked the moiety frequently found in melatonin ligands
i.e., CH CH NHCOR), which was shown for agonists at this receptor by Mor et al. [32]. Another
2
2
interesting feature of this molecule was its specificity toward the MT receptor, with no measurable
2
affinity for the MT receptor. Although this feature was not unusual in this domain, it is worth
1
pointing out that in this small series of compounds, none of the analogues regained affinity for the
other subtype.
We decided to introduce more and more structural hindrance into the region of the molecule
bearing a bromide (see Figure 1 for structures). We simply added larger and larger substituents at
this position: from H, F, Br, I, Phenyl, to tri-n-butylstannyl [Sn(But) ]. We prepared these analogues
3
without regard for the physical properties of the various substituents.
We first determined the binding affinities of those compounds that bound to the MT receptor,
2
as evaluated by a binding assay using 2-[1 I]-iodo-melatonin as a ligand (Table 1). All the compounds,
25
interestingly, presented binding affinities in the nanomolar range, with the exception of the compound
with the largest substituent, the Sn(But) derivative, which displayed a binding affinity for MT that
3
2
was lower by a factor of a log value. As pointed out previously, all compounds remained MT -specific,
2
as binding at MT was recorded in the low micromolar range, presenting, therefore, a difference of 3
1
log values between the two types of receptors.
Table 1. Affinity of the compounds at human melatonin MT and MT receptors.
1
2
Compounds
Substituents
K MT (nM)
K MT (nM)
i
2
i
1
Melatonin
DIV4288
DIV6518
DIV0879
DIV0880
DIV6519
DIV6520
-
H
F
Br
I
Ph
0.22 ± 0.01
1300 ± 97
630 ± 55
0.33 ± 0.01
1.4 ± 0.1
1.4 ± 0.3
1.4 ± 0.1
1.0 ± 0.1
0.49 ± 0.01
36 ± 3
1779 ± 118
1202 ± 143
44 ± 7
Sn(But)3
5000 ± 160
Binding parameters of the compounds were obtained with 2-[125I]-melatonin as the radioligand. The data were
1 2
obtained using membranes from CHO-K1 cells expressing either human melatonin receptors (hMT or hMT )
receptors. Results are given as the mean ± SEM for at least three experiments. SEM is standard error of the mean.
A sketch of the signaling pathways visited in the present work is provided in Figure 2. Because
affinity does not presume the functional nature of the compounds (whether agonist or antagonist),
we turned to several functional assays: GTPγS is a test that is dependent on the activation of the Gi
pathway [33]. As can be seen from Table 2, the compounds activated this particular pathway up to
the iodine-bearing compound, DIV0880, but larger derivatives did not, as the presence of even larger
substituents (phenyl or Sn(But) impaired the respective compound ability to activate this particular
3
Gi-dependent pathway. Furthermore, while they all acted as agonists (i.e., being able to activate the
pathway by themselves, without melatonin), they were only partial, poorly effective agonists, as their
potencies were in the 30% range of the melatonin potency with respect to the same receptor, within the
same pathway.

Int. J. Mol. Sci. 2017, 18, 1347
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Figure 2. Schematic representation of the melatonin receptor pathways worked out in this paper.
Red: the binding of the radio ligand 2-[125I]-melatonin onto the receptor gives an affinity of the
receptor for the compound; Green: The binding of the nonhydrolysable, radioactive GTP analogue
3
5
[
S]GTPγS is a measure of the capacity of the ligand molecule to recruit the induced the Gi protein,
that leads to the Gi-mediated inhibition of the adenylate cyclase; Orange: The measure of the cyclic
AMP (cAMP) production by adenylate cyclase is a finer image of the capacity of the ligand at the
receptor to recruit Gi and to inhibit this production; Blue: Ligands that are agonist tends to provoke
the recruitment of
β
-arrestin, as a first step towards the desensitization of the receptor; Purple
:
®
Cellular dielectric spectroscopy (Cellkey ) is a technology capable of detecting whole-cell response to
a treatment—such as the binding of an agonist at a receptor by measuring modifications in electric
impedance in a dose-dependent manner.
3
5
Table 2. Melatoninergic agonist potencies and efficacies on [ S]-GTPγS binding.
MT1
MT2
Compounds
Substituents
pEC50
Emax
pEC50
Emax
Melatonin
DIV4288
DIV6518
DIV0879
DIV0880
DIV6519
DIV6520
-
H
F
Br
I
Ph
8.77 ± 0.66
100
ND
ND
ND
ND
ND
ND
9.20 ± 0.04
8.69 ± 0.37
8.93 ± 0.66
8.86 ± 0.87
9.48 ± 0.16
<5
100
34 ± 11
30 ± 5
39 ± 9
37 ± 5
ND
<5
<5
<5
<5
<5
<5
Sn(But)3
<5
ND
Functional binding parameters of the compounds were obtained with [35S]-GTP
γ
S as the radioligand. The data
were obtained using membranes from CHO-K1 cells expressing either hMT1 or hMT2 receptors. Results are given
as the mean SEM. for at least three experiments. ND: not defined. SEM is standard error of the mean. EC50 is
±
defined as the half maximal effective concentration. It is sometimes expressed as its Log, pEC50. The Emax is the
maximum possible effect for the agonist (melatonin).
35
The [ S]-GTPγS binding assay measures directly the activation of Gi. This activation can—but
does not always—lead to the inhibition of the cAMP production, once the adenylate cyclase is induced
by forskolin. To better evaluate this parameter, with the goal to characterize the compounds as agonists
at the MT receptor, we chose to measure the variations of the cAMP production as it can be induced
2
by the various agonists (Table 3). The first four compounds activated the Gi-dependent pathway
35
with potencies similar to those obtained while measuring the [ S]-GTP
γS binding pathway. EC50
is defined as the half maximal effective concentration. It is sometimes expressed as its Log, pEC .
5
0

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For the last two larger compounds, however, if activation of the pathway occurred, they were with
pEC50 values that remained poorer by 2 log units than the first compounds. The Emax is the maximum
possible effect for the agonist. Interestingly, the Emax values of the compounds were all close to 100%,
the score of the melatonin itself. This discrepancy is due to the nature of the assay. Indeed, the GTPγS
binding assay is a static one, limited roughly by the number of binding sites, and the cAMP assay
is an enzymatic one, the only limit of which is the time during which the enzyme produces cAMP.
In other words, the result at a given time, for the cAMP assay, is similar from an agonist to another,
while the pEC50 will be dependent on the potency of the molecule. It is therefore slightly wrong to
use cAMP as a potency assay. It should be limited to an “affinity measurement” one. This outcome
strongly suggests that the compounds were full agonists of this pathway. Interestingly, when one
compares the values obtained with this cAMP production assay with the previous test, one finds the
higher sensitivity of the latter to be key. Indeed, the two larger compounds seemed to be completely
unable to activate the Gi pathway, while the cAMP production, a more dynamic assay, reveals they can.
Table 3. Potency and effect of the compounds on the cAMP production (inhibition).
cAMP MT1
cAMP MT2
Compounds
Substituents
pEC50
Emax (%)
pEC50
Emax (%)
Melatonin
DIV4288
DIV6518
DIV0879
DIV0880
DIV6519
DIV6520
-
H
F
Br
I
Ph
9.46 ± 0.06
100
ND
ND
ND
ND
ND
ND
9.6 ± 0.12
8.74 ± 0.19
8.55 ± 0.19
8.93 ± 0.19
8.28 ± 0.22
7.24 ± 0.56
7.31 ± 0.10
100
<5
<5
<5
<5
<5
<5
88 ± 6
92 ± 14
100 ± 6
90 ± 10
143 ± 25
94 ± 3
Sn(But)3
The data were obtained using CHO-K1 cells expressing either hMT
1 2
or hMT receptors. Results are given as the
mean SEM for at least three experiments. SEM is standard error of the mean. EC50 is defined as the half maximal
±
effective concentration. It is sometimes expressed as its Log, pEC50. The Emax is the maximum possible effect for
the agonist (melatonin).
To summarize, the compounds of identical chemical structure but differing in the size of a single
substituent (from the hindrance point of view) could activate, as agonists, the Gi pathway. Despite
their differences in potencies, they remained strong agonists toward only the MT receptor. As is
2
often the case with the melatonin receptors, the complexity of the pathways makes it difficult to find
pure agonists or antagonists, even if they are not specific of one of the subtypes. In the present work,
we identified strong binders, specific for MT but, for some of them, they were only partially effective,
2
depending on the signaling pathway measured.
The investigation of those compounds continued with determination of the β-arrestin recruitment.
This test evaluates the agonist capacity of the compound. Indeed, viewed in a simple way,
the compounds that are agonists lead to the desensitization of the pathway. For desensitization
to occur, the receptor must be internalized. The first step of the internalization is the recruitment of the
β-arrestin (see Claing et al. [34], and Reiter and Lefkovitz [35] , for further details). This desensitization
process is used by the vast majority of 7TMRs (see reviews and references thereof: Pitcher et al. [36],
and Lefkowitz and Shenoy [37]).
The data were obtained using CHO-K1 cells expressing either hMT or hMT receptors. Results
1
2
are given as the mean ± S.E.M. for at least three experiments.
We developed a complementary assay to measure the recruitment of
β-arrestin as a function of
the concentration of the compound. Because the test is built with brand new cell lines using enzyme
®
fragment complementation assay Technology (PathHunter , DiscovereX) [38], the pharmacology of
these cell lines should be validated and the findings with reference compounds double checked with
standard methodology. This process has been done and confirms the similarity of the cell lines, as well
as the robustness of the assay (Dupré and Boutin, in preparation). Only agonists can elicit some

Int. J. Mol. Sci. 2017, 18, 1347
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recruitment in this model, although some agonists do not, while antagonists never do. As indicated in
Table 4, despite the low affinity of the compounds for MT (in the micromolar range), they elicited this
1
recruitment. This is a phenomenon often seen with complementary assays: the affinity of the two parts
of the enzyme for each other tends to drive, at least in part, the agonist-induced pathway. The efficacy
of these compounds, compared to melatonin, was partial, in the 60% range. Furthermore, these pEC50
values are of the same range as the affinity of the compounds for the MT receptor. In other words,
1
the test is clearly very sensitive and can be used to evaluate poor affinity phenomena. To the contrary,
for the MT receptor, the same compounds were able to recruit β-arrestin in the same manner as
2
for melatonin itself (namely, 100%), and even in some cases, beyond that (140%), despite a slightly
diminished pEC50 in this test. The same ability applies for the two most hindered compounds that
were ineffective, partial agonists in this test.
Table 4. Potency and effect of the compounds on the β-arrestin recruitment.
β-Arrestin MT1
β-Arrestin MT2
Compounds
Substituents
pEC50
Emax (%)
pEC50
Emax (%)
Melatonin
DIV4288
DIV6518
DIV0879
DIV0880
DIV6519
DIV6520
-
H
F
Br
I
Ph
9.49 ± 0.49
5.65 ± 0.20
5.66 ± 0.12
5.61 ± 0.48
5.75 ± 0.25
<5
100
65 ± 1
47 ± 0
65 ± 15
41 ± 2
ND
9.57 ± 0.06
7.65 ± 0.39
7.00 ± 0.53
7.18 ± 0.59
6.70 ± 0.57
8.21 ± 0.76
5.81 ± 0.49
100
112 ± 16
98 ± 20
141 ± 41
101 ± 10
16 ± 5
Sn(But)3
<5
ND
25 ± 6
The data were obtained using cells expressing either hMT
1
or hMT
2
receptors. Results are given as the mean ± SEM
for at least three experiments. SEM is standard error of the mean. EC50 is defined as the half maximal effective
concentration. It is sometimes expressed as its Log, pEC50. The Emax is the maximum possible effect for the
agonist (melatonin).
To further characterize these compounds, we tested them using the cellular assay, Cellkey®,
described elsewhere [39] and applied to systems other than the melatoninergic receptors (e.g.,
δ opioid
receptors [40], and lysophosphatidic acid receptor 1 [41]). This methodology is a label-free cellular
technique that measures the changes in the conductance (dielectric impedance) of cells that occur
when the cellular receptor binds a ligand, resulting in a change in the shape of the cell. Depending on
the signaling pathway, it has been shown that the variation of this impedance curve as a function of
time (and of concentration) is somewhat specific of one of the canonic GPCR signaling pathways (see
Figure 3).
As the conformation of the cell is altered, its ability to conduct a current is also altered. The method
has been previously assessed and applied to the melatoninergic system in our laboratory [42]. In this
study, we have used cells that were transfected with the MT or MT receptors and compared the
1
2
results to naïve CHO cells. Figure 4 compare the changes in electric impedance of each category of
cells as a function of the concentration of the compounds. In control non-transfected cells, none of
the compounds elicited a response, indicating that in the absence of melatonin receptors, there was
a baseline level of specificity for endogenous receptors spontaneously expressed in the cell line
(
Figure 4A). For cells containing MT (Figure 4B), the compounds were not potent enough toward this
1
receptor to elicit any signal, while melatonin itself did. It is interesting to notice that there are three
kinds of compounds in this series, excepting melatonin. Indeed, melatonin clearly shows a capacity
to recruit the
Figure 4B,C). The first compound is DIV0879 (orange trace, Figure 4C) that is very similar to melatonin
in this test, as it recruits -arrestin with identical potency and efficiency, when compared to the natural
agonist. The next group comprises DIV4288 (blue trace, Figure 4C), DIV6518 (purple trace, Figure 4C)
and DIV880 (green trace, Figure 4C). They all are less potent in recruiting -arrestin, but remained
capable of doing so with almost 100% potency, close to the natural agonist. The last group is formed of
β-arrestin on both receptors, as expected, and as a natural agonist should do (red trace,
β
β

Int. J. Mol. Sci. 2017, 18, 1347
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the two last compounds, DIV6519 and DIV6520 (pink and grey traces, Figure 4C), the most hindered
ones, and they showed poor capacity to act as agonists at the MT receptor.
2
An overall comparison can be made at this stage between the various signaling pathways.
The features of the
with the data obtained first with the GTP
DIV6519 and DIV6520 are similar and they do not result in the binding of GTP
β
-arrestin recruitment results are also interesting when they are observed together
S assay. Indeed, the behavior of the two last compounds,
S, as if they were acting
γ
γ
like potent antagonists, according to the fact that their affinities for MT are 0.5 and 36 nM, respectively.
2
When comparisons were made with the cAMP production assay, though, the situation was less clear.
Indeed, both compounds, DIV6519 and DIV6520, could induce a production of cAMP, albeit with
a poorer efficiency, compared to the other compounds of the series and melatonin itself, reinforcing the
idea that they are poorly potent agonists to a point that they can behave as antagonists. The final point
®
would be to comment on the Cell Key assay. Due to the relative newness, it is hard to know exactly
what to expect. It is certain that melatonin, on both isoforms of the receptor, behaves roughly the
same way, even though on MT receptor, a peak of respond appears at low concentrations, a feature
1
that melatonin on MT does not induce. For the other compounds, the overall picture is the same:
2
concentration-dependent enhanced response of impedance dielectric signal shape (red traces, bottom
of Figure 3), with enhanced amplitude at high concentrations. The last two compounds (DIV6519 and
DIV6520), behave differently, with a poor increase in the signal, if any.
Figure 3. Screen capture of the CellKey® experiments. Within a square, the changes in impedance
translate as a function of time (20 min total) and as a function of concentration of ligands (within
−
12
−5
a column, from the top line (1
inflection point could be used to construct graphs, leading to half maximal inhibitory concentration
IC ) calculations. Note the absence of response in naïve cells ( ), the response of melatonin (MLT)
) and the various curves for the MT -specific ligands ( ). These captures
×
10
M) to the bottom line (1
×
10 M) are monitored, and the
(
A
5
0
only in MT -expressing cells (
B
C
1
2
are examples taken from a series of 2–5 experiments, depending on the compounds.

Int. J. Mol. Sci. 2017, 18, 1347
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Figure 4. CellKey measurements melatonin receptors MT and MT response elicited by DIV0879
1
2
analogues. CHO cells transfected with MT (
A
) and MT (
B) as well as naïve CHO cells (C) were
1
2
treated with melatonin (MLT) and the analogues as indicated in the color key in the figure over the
−
12
−5
concentration range of 10
to 10 M. At each concentration, the change in electrical impedance was
measured as described in the Experimental section. The data were corrected for baseline response and
are normalized with respect to the response to melatonin at the same concentration. The data represent
the mean ± SEM of five trials. SEM is standard error of the mean.
3
. Discussion
The search for ligands for the melatonin receptors has been a quest for many laboratories since
the pioneering work on melatonin analogues by Taborsky and McIsaac [43], and Kobayashi et al. [44].
The compounds discovered over recent decades fall into four categories: (1) the bicyclic compounds,
loose analogues of the indole cycle of melatonin; (2) the tricyclic compounds that are simply one
more cycle fused to the bicyclic ones; (3) the unusual case of the unique and selective MT antagonist
1
verapamil-derived D600 [45] that is everything but specific (it affects many other targets including the
voltage-dependent calcium channels); and (4) the present compound(s) in which the two cycles (of the
first category) have been separated by a methylene bridge (notably, the series described by Hu et al. [46]
encompasses the same global scheme). Most of these compounds bear the acetylamidoethyl side chain.
A complete discussion of the melatonin receptor ligands can be found in the International
Union of Basic and Clinical Pharmacology (IUPHAR) compendium [47], as well as in the review of
Zlotos et al. [28] where DIV0880 is mentioned as a selective MT radioligand. The present series of
2
compounds exemplifies further the series of diphenylmethylene-based compounds [46], described by
Hu et al. in a study mainly dealing with phenoxyphenyl compounds [46]. These compounds differ
from the current agonists in that they do not bear the canonic melatonin chain (i.e., acetylamidoethyl,
simply referred to as “the amide moiety” [25]) that most of the agonists of melatonin receptors possess
(see review by Spadoni et al. [25]).
Antagonists at melatonin receptor(s) have been difficult to obtain. Most of the compounds
described thus far are partial agonists that behave completely differently—from a functional point
of view—whether they are characterized by one or another of the numerous functional pathways
downstream of the seven trans-membrane domain G-coupled receptors (Legros, C.; Boutin, J.A.,
in preparation).
At that stage, the notions of agonists, antagonists and partial agonists should be briefly discussed.
Agonists, often natural compounds, are capable of eliciting a full activation of the receptor pathway in
a functional assay. This is followed very rapidly by a desensitization process in which the receptor

Int. J. Mol. Sci. 2017, 18, 1347
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is internalized in the cell, in order to make it unavailable to further activation. This process is
often targeted by the recruitment of -arrestin. Antagonists are compounds that impair the agonist
β
to bind (and to act) without having its own capacity to activate the receptor. Partial agonists are
defined by their partial capacity to activate the receptor. The IUPHAR recommendations state that
“
. . . a partial agonist that when occupying all the receptors produces a maximal response that is half
that of a full agonist (under the same experimental conditions), has an efficacy of unity. Efficacy is
both agonist- and tissue-dependent” [48], and hence, less than 50%. To these notions, one can add the
notion of ineffective partial agonists [49] that translate simply by a very poor activation of the receptor,
being then different from an antagonist, since those ones are not able to activate the receptor while
partial agonists do, albeit ineffectively. Unfortunately, none of those authors launched a mathematical
limit between ineffective and effective partial agonists; neither did the IUPHAR ad hoc committees
(M. Spedding, personal communication). We tried to respect these general ideas based on common
sense in the present paper.
In multi-domain receptors that mediate multiple functions, the notion of biased agonism has
been introduced. Biased agonists are ligands that selectively affect only one signaling pathway within
a multifunctional receptor. We have described new and original compounds exhibiting partial agonism
favoring the MT receptor functionality. While the concept of biased agonism is not new, as far as we
2
know, it has never been applied to melatonin receptors.
We wanted to explore the behavior of simple analogues of the powerful original compound
discovered from an HTS campaign, with a nanomolar affinity at MT2. Despite our goal to
generate antagonists simply by enhancing the steric hindrance of a substituent at a given position,
the compounds we prepared were partial to very partial agonists (from effective to ineffective agonists),
which were still able to generate internalization of the receptor, a feature that antagonists do not possess.
Most of the ligands described in the literature over the last three decades have been fused bicyclic
compounds with various heteroatoms in the cycle(s), bearing the so-called “amide” moiety [25],
and having a methoxy substituent on the aromatic rings. All of these specific features of melatonin
ligands—particularly agonists—have been described with a large number of variations within these
features and in their proximity (see Zlotos et al. [28] for a review of the literature).
The present series of compounds is slightly different in structure. First, rather than bearing
the characteristic ethylacetamide moiety, these new derivatives have a shorter and more hindered
CH COOtBu moiety. It seems unlikely that this substituent is a bioisostere of the CH CH NHCOR
2
2
2
chain. The second structural difference is that the new compounds do not have a fused cyclic structure,
but instead, a methylene chain separates the rings.
We were surprised to see large substituents such as Sn(But)3 and phenyl directing the
corresponding compound toward the behavior of ineffective partial agonists (e.g., less than 20%
of melatonin at MT receptor), remaining slightly agonistic for MT . Although there are several
2
2
models of the receptors that have been published, it has not been easy, so far, to elaborate on this
structure–function relationship.
We hope to further explore this series of compounds by using various approaches that can analyze
the melatonin receptor structural biology, e.g., the purification of a functional receptor [50
,51] (albeit
MT only, at the moment) and the models that have been described recently for the receptors [52
,53]).
1
Hopefully, the combination of new chemical series with strong affinity and clear functionality toward
the melatonin receptor(s) will help assess new tools to improve our understanding of the role of
melatonin within and outside its receptor routes.
4
. Materials and Methods
4
.1. Synthesis of the Compounds
The synthesis of compounds DIV0879 (the bromide derivative) and DIV0880 (the iodinated
derivative) have been previously described [30]. All the compounds were purified and analyzed by

Int. J. Mol. Sci. 2017, 18, 1347
10 of 17
mass spectrometry and nuclear magnetic resonance NMR. The crude graphs corresponding to the six
compounds are added as supplementary files (Figures S1–S11).
4
.1.1. DIV6518
Compound DIV6518 was prepared in three steps according to the scheme (Figure 5).
Figure 5. Schematic representation of the synthesis of DIV6518.
Preparation of Compound 2
Compound
1
(51.4 g) was mixed with a solution of acetic acid (150 mL) and concentrated hydrochloric
◦
acid (50 mL). Formalin (50 mL) was added and the mixture was heated at 90 C for 2 h. The solution
was allowed to cool to room temperature and poured into water (1.2 L). The aqueous layer was
extracted three times with dichloromethane. The combined organic layers were washed successively with
a saturated sodium bicarbonate solution (three times) and brine, dried over magnesium sulfate, filtered
and concentrated to give a yellow solid. The crude solid was triturated in isopropyl ether to give the
expected product: a light yellow solid (33.3 g, yield 61%).
Preparation of Compound 4
Compound
2
(2.64 g) was dissolved in formic acid (26 mL) and compound
3
(Alfa Aesar, Haysham,
◦
UK, A17936): 4-Fluoro-1,2-dimethoxybenzene, 2 g) was added. The mixture was heated at 90 C
overnight. A thin layer chromatography TLC analysis (dichloromethane/ethyl acetate 8/2) indicated
that compound 2 had been totally consumed. The mixture was allowed to cool to room temperature
and poured into water. The aqueous phase was extracted three times with ethyl acetate. The combined
organic phases were washed with water until the pH of the aqueous washings was neutral. The organic
phase was finally washed with brine, dried over magnesium sulfate, filtered and concentrated to
give a crude product, (4.57 g). The crude solid was triturated in isopropyl ether to give the expected
product: brown solid (2.7 g, yield 58%).

Int. J. Mol. Sci. 2017, 18, 1347
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Preparation of the Final Compound DIV6518
Compound
4
(2 g) and compound
5
(Diverchim, Roissy, France (DIV0439) 2-tert-Butyl-1,
3
-diisopropylisourea, 1.65 g) were dissolved in dichloromethane (25.5 mL). The mixture was stirred
at room temperature overnight. An aliquot was analyzed by NMR showing the presence of residual
starting material. An additional batch of compound (1.65 g) was added and stirring was continued
5
overnight. An aliquot was analyzed by NMR indicating complete conversion of starting material.
Water (25 mL) was added and the biphasic mixture was stirred for 45 min. The solid was filtered and the
filter cake was rinsed with dichloromethane. The filtrate was collected and the phases were separated
and the aqueous phase was further extracted with dichloromethane. The combined organic phases
were washed with brine, dried over magnesium sulfate, filtered and concentrated to give a semi solid
compound (2.46 g). The resulting crude solid was purified twice by column chromatography (Silica
gel, heptane/ethyl acetate 3/1 and dichloromethane/diisopropyl ether 98/2) to give the expected
product: white solid (270 mg, yield 11%).
4
.1.2. DIV4288
Compound DIV4288 was prepared in two steps from intermediate
scheme (Figure 6).
2
according to the synthesis
Figure 6. Schematic representation of the synthesis of DIV4288.
Preparation of Compound 7
Compound
2
(12.1 g) was dissolved in formic acid (121 mL) and compound
6
(8.9 mL) was
◦
added. The mixture was heated at 90 C for 4 h. An aliquot was analyzed by NMR, showing complete
consumption of the starting materials. The mixture was allowed to cool to room temperature and
poured into water. The aqueous phase was extracted four times with ethyl acetate. The combined
organic phases were washed with brine, dried over magnesium sulfate, filtered and concentrated to
give a brown oil (29 g, 178-080-B). The crude oil was purified by column chromatography (Silica gel,
dichloromethane/ethyl acetate 9/1) to give the expected product: brown solid (2 g, yield 10%).
Preparation of Compound DIV4288
Compound
7 (2 g) and compound 5 (Diverchim, Roissy, France (DIV0439) 2-tert-Butyl-1,
3-diisopropylisourea, 3.71 g) were dissolved in dichloromethane (30 mL). The mixture was stirred at

Int. J. Mol. Sci. 2017, 18, 1347
12 of 17
room temperature for 5 h. A TLC analysis (dichloromethane/methanol 95/5) showed the presence of
residual starting material. An additional batch of compound (1.86 g) was added and stirring was
5
continued overnight. An aliquot was analyzed by HPLC indicating virtually complete conversion
of starting material. The suspension was filtered and the filtrate was concentrated. The residue was
triturated in diethyl ether (1 V) and the filtrate from this operation was concentrated to give a brown
oil (3.9 g, 178-092-Ef). The resulting crude solid was combined with a previously obtained batch and
purified by column chromatography (Silica gel, dichloromethane) to give a brown semi solid that was
triturated in pentane to result in the expected product: white solid (2.5 g, yield 42%).
4
.1.3. DIV6519
Compound DIV6519 was prepared in three steps from intermediate
scheme (Figure 7).
2
according to the synthesis
Figure 7. Schematic representation of the synthesis of DIV6519.
Preparation of Compound 9
Compound
2
(5 g) was dissolved in formic acid (25 mL) and compound
8
(Acros, Geel, Belgium
◦
(
107,540,250, 6 g) was added. The mixture was heated at 100 C for 3 h and for 7 h overnight. The
mixture was poured into water and extracted three times with ethyl acetate. The combined organic
phases were washed with brine, dried over magnesium sulfate, filtered and concentrated to give
a brown semi solid. The crude product was triturated in a mixture of diisopropyl ether/ethyl acetate
7
/3 to give a brown solid. The solid was crystalized from toluene to give the expected product: brown
solid (6 g, yield 59%). A second crop of product was obtained by trituration of the different mother
liquors in a mixture of heptane/ethyl acetate 1/1 to give a brown solid (1.05 g, yield 10%).

Int. J. Mol. Sci. 2017, 18, 1347
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Preparation of DIV0879
Compound
9
(1.04 g) and compound
5
(1.58 g) were dissolved in dichloromethane (30 mL).
The mixture was stirred at room temperature overnight. A TLC analysis (dichloromethane/methanol
/2) showed the presence of residual starting material. An additional batch of compound
0.79 g) was added and stirring was continued overnight. The reaction mixture was concentrated to
8
5
(
dryness to give a crude product. The product was purified by column chromatography (Silica gel,
dichloromethane/ethyl acetate 85/15) to give a product that was not pure (320-006-C1). The product
was triturated in a mixture of pentane/ether. Concentration of the filtrate led to a product that was
further purified by column chromatography (Silica gel, gradient heptane/ethyl acetate) to give the
expected product, DIV0879 as an off-white solid (749 mg, yield 63%).
Preparation of Compound DIV6519
DIV0879 (199 mg, 0.41 mmol), sodium carbonate (123 mg, 1.16 mmol) and phenyl boronic acid
(86 mg, 0.71 mmol) were dissolved in the ethanol/water mixture (5 mL). The mixture was placed
under nitrogen atmosphere and palladium tetrakis triphenylphosphine (25 mg, 0.02 mmol) was
◦
added. The reaction was heated at 85 C overnight. A TLC analysis (dichloromethane/methanol 99/1)
indicated that all the starting materials had been consumed. The mixture was concentrated to dryness
to give a crude product (600 mg, 210-164-B). The product was purified by column chromatography
(
(
Silica gel, dichloromethane/methanol 99/1) resulting in a colorless oil that solidified upon standing
200 mg, 210-164-C1). The product was triturated in pentane (15 mL) to give the expected compound:
white solid (90 mg, yield 46%).
4
.1.4. DIV6520
Compound DIV6520 was prepared in one step from intermediate 10 according to the synthesis
scheme (Figure 8).
Figure 8. Schematic representation of the synthesis of DIV6520.
Preparation of Compound DIV6520
DIV0879 (200 mg, 0.41 mmol) and hexabutyl tin (603 mg, 1.04 mmol) were dissolved in the
ethanol/water mixture (5 mL). The mixture was placed under nitrogen atmosphere and palladium
◦
tetrakis triphenylphosphine (25 mg, 0.02 mmol) was added. The reaction was heated at 100 C
overnight. A TLC analysis (toluene/ethyl acetate 9/1) indicated that virtually all the starting materials
had been consumed. The mixture was allowed to cool to room temperature and was poured into
a solution of potassium fluoride (20 mL). The suspension was filtered over Celite and the filtrate
was extracted with ethyl acetate. The organic phase was dried over magnesium sulfate, filtered and
concentrated to give a brown paste (0.73 g). The product was purified by column chromatography
(Silica gel, gradient toluene/ethyl acetate) resulting in colorless oil that was further purified by
preparative HPLC to give the expected compound: light yellow oil (60 mg, yield 22%).

Int. J. Mol. Sci. 2017, 18, 1347
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4
.2. Biological Experiments
4
.2.1. Cell Line and Membrane Preparation
We described these methodologies previously and used them as we described [30].
4
.2.2. 2-[125I]-Iodomelatonin Binding Assay
We used these assays exactly as we described previously [54].
4
.2.3. Homogeneous Time Resolved Fluorescence HTRF cAMP Assay
For the cellular cAMP production measurement, we used the assay we validated and described
previously [30].
35
4
.2.4. [ S]-GTPγS Binding Assays
3
5
The [ S]-GTP
γS binding assay we used has been thoroughly described previously by us on other
systems [33] or on melatonin receptors [30].
4
.2.5. β-Arrestin Recruitment Assay
The -Arrestin recruitment was monitored using PathHunter CHO-K -MT1 R β-arrestin cell
β
1
2
®
line from DiscoveRx (Birmingham, UK), essentially as described by Sakurai et al. [55] and recently
partly assessed by Gdahou et al. [56]. Cells were cultured at 37 C, 5% CO in F12-GlutaMAX medium
◦
2
(
Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat inactivated fetal bovine
serum. Engineered MT -2R and -arrestin expression was maintained using antibiotic selection
g/mL, Geneticin 800 g/mL). For assays, cells were washed in Dulbecco’s
β
1
(
Hygromycin B 300
µ
µ
Phosphate-Buffered Saline (DPBS), resuspended using Cell Dissociation Solution (Sigma-Aldrich,
St. Louis, MO, USA) and seeded into CELLSTAR 384-well microplates (Greiner Bio-one) at 6500 cells
◦
per well in 20
µ
L PathHunter Cell Plating Reagent, then incubated at 37 C, 5% CO for 24 h.
2
After compound incubation (in 1.5% final concentration dimethyl sulfoxide (DMSO) for a final volume
◦
of 25
µ
L) for 90 min at 37 C, 5% CO , PathHunter detection reagent was added and the enzyme
2
fragment complementation reaction was carried out for 120 min with shaking at room temperature.
Chemiluminescent signal was measured on a MicroBeta TriLux (PerkinElmer, Villebon-sur-Yvette,
France). This approach has been further validated and characterized and will be published in
a forthcoming paper [57].
4
.2.6. Cellular Dielectric Spectroscopy
The impedance measurement assay uses CHO-K1 cells expressing human MT and MT receptors.
1
2
We previously described this approach in our work on sheep melatonin receptor cloning [58].
This test was also previously assessed, and used in our group with the human receptors [42].
It was used here with minor modifications: the cells were plated (40,000 cells per well) onto MDS
Analytical 96-well assay plates with embedded electrodes (MDS Analytical, Concord, ON, Canada).
◦
The plates were incubated at 37 C, CO 6% for 48 h. Prior to the measurement, cells were washed
2
three times with Hanks balanced salt solution, 0.1% bovine serum albumin (BSA), 20 mm HEPES
[
2
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)], pH 7.4. The cells were left to equilibrate at
8 C for 30 min. The impedance measurement was performed on a CellKey instrument (MDS
◦
Analytical, Concord, ON, Canada). The signal was recorded for 5 min before the addition of the
compounds, and 15 min thereafter. The cells in each well were stimulated once with a single
concentration of compounds. The resulting data are expressed as the maximal signal corrected for the
baseline and represented as a percentage of the full agonist effect (melatonin that serves as a positive
control in these experiments). Naïve CHO-K1 cells were used as controls in these experiments as they
do not show any signal after melatonin treatment.

Int. J. Mol. Sci. 2017, 18, 1347
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Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/7/1347/s1.
Author Contributions: Céline Legros, Olivier Nosjean and Jean A. Boutin conceived and designed the
experiments; Anne Bonnaud, Chantal Brasseur, Olivier Bruno and Sophie Coumailleau performed the
experiments; Nolwenn Lepretre and Peter Oosting synthesized the compounds; Jean A. Boutin, Céline Legros and
Philippe Delagrange wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Lerner, A.B.; Case, J.D.; Takahashi, Y.; Lee, T.J.; Mori, W. Isolation of melatonin, the pineal gland factor that
lightens melanocytes. J. Am. Chem. Soc. 1958, 80, 2587–2588. [CrossRef]
2
3
.
.
Vanecek, J. Cellular mechanisms of melatonin action. Physiol. Rev. 1998, 78, 687–721. [PubMed]
Tosini, G.; Menaker, M. The clock in the mouse retina: Melatonin synthesis and photoreceptor degeneration.
Brain Res. 1998, 789, 221–228. [CrossRef]
4
.
Carrillo-Vico, A.; Calvo, J.R.; Abreu, P.; Lardone, P.J.; Garcia-Maurino, S.; Reiter, R.J.; Guerrero, J.M. Evidence
of melatonin synthesis by human lymphocytes and its physiological significance: Possible role as intracrine,
autocrine, and/or paracrine substance. FASEB J. 2004, 18, 537–539. [CrossRef] [PubMed]
5
.
.
Bubenik, G.A. Gastrointestinal melatonin: Localization, function, and clinical relevance. Dig. Dis. Sci. 2002
7, 2336–2348. [CrossRef] [PubMed]
Falcon, J.; Besseau, L.; Fazzari, D.; Attia, J.; Gaildrat, P.; Beauchaud, M.; Boeuf, G. Melatonin modulates
secretion of growth hormone and prolactin by trout pituitary glands and cells in culture. Endocrinology 2003
44, 4648–4658. [CrossRef] [PubMed]
,
4
6
,
1
7.
8.
9.
Favero, G.; Franceschetti, L.; Buffoli, B.; Moghadasian, M.H.; Reiter, R.J.; Rodella, L.F.; Rezzani, R. Melatonin:
Protection against age-related cardiac pathology. Ageing Res. Rev. 2016, 35, 336–349. [CrossRef] [PubMed]
Srinivasan, V.; Lauterbach, E.C.; Ho, K.Y.; Acuna-Castroviejo, D.; Zakaria, R.; Brzezinski, A. Melatonin in
antinociception: Its therapeutic applications. Curr. Neuropharmacol. 2012, 10, 167–178. [CrossRef] [PubMed]
De Berardis, D.; Di, I.G.; Acciavatti, T.; Conti, C.; Serroni, N.; Olivieri, L.; Cavuto, M.; Martinotti, G.; Janiri, L.;
Moschetta, F.S.; et al. The emerging role of melatonin agonists in the treatment of major depression: Focus
on agomelatine. CNS Neurol. Disord. Drug Targets 2011, 10, 119–132. [CrossRef] [PubMed]
1
1
1
1
1
1
1
0. Calvo, J.R.; Gonzalez-Yanes, C.; Maldonado, M.D. The role of melatonin in the cells of the innate immunity:
A review. J. Pineal Res. 2013, 55, 103–120. [CrossRef] [PubMed]
1. Cagnacci, A.; Krauchi, K.; Wirz-Justice, A.; Volpe, A. Homeostatic versus circadian effects of melatonin on
core body temperature in humans. J. Biol. Rhythm. 1997, 12, 509–517. [CrossRef] [PubMed]
2. Boutin, J.A. Quinone reductase 2 as a promising target of melatonin therapeutic actions. Expert Opin. Ther.
Targets 2016, 20, 303–317. [CrossRef] [PubMed]
3. Fisher, S.P.; Davidson, K.; Kulla, A.; Sugden, D. Acute sleep-promoting action of the melatonin agonist,
ramelteon, in the rat. J. Pineal Res. 2008, 45, 125–132. [CrossRef] [PubMed]
4. Su, S.C.; Hsieh, M.J.; Yang, W.E.; Chung, W.H.; Reiter, R.J.; Yang, S.F. Cancer metastasis: Mechanisms of
inhibition by melatonin. J. Pineal Res. 2017, 62. [CrossRef] [PubMed]
5. Wang, J.Z.; Wang, Z.F. Role of melatonin in Alzheimer-like neurodegeneration. Acta Pharmacol. Sin. 2006, 27,
4
1–49. [CrossRef] [PubMed]
6. Lyssenko, V.; Nagorny, C.L.; Erdos, M.R.; Wierup, N.; Jonsson, A.; Spegel, P.; Bugliani, M.; Saxena, R.; Fex, M.;
Pulizzi, N.; et al. Common variant in MTNR1B associated with increased risk of type 2 diabetes and impaired
early insulin secretion. Nat. Genet. 2009, 41, 82–88. [CrossRef] [PubMed]
1
7. Lanfumey, L.; Mongeau, R.; Hamon, M. Biological rhythms and melatonin in mood disorders and their
treatments. Pharmacol. Ther. 2013, 138, 176–184. [CrossRef] [PubMed]
1
8. Comai, S.; Gobbi, G. Unveiling the role of melatonin MT2 receptors in sleep, anxiety and other
neuropsychiatric diseases: A novel target in psychopharmacology. J. Psychiatry Neurosci. 2014, 39, 6–21.
[CrossRef] [PubMed]
1
9. Posa, L.; de Gregorio, D.; Gobbi, G.; Comai, S. Targeting melatonin MT receptors: A novel pharmacological
2
avenue for inflammatory and neuropathic pain. Curr. Med. Chem. 2017. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2017, 18, 1347
16 of 17
2
2
2
0. Sumaya, I.C.; Masana, M.I.; Dubocovich, M.L. The antidepressant-like effect of the melatonin receptor ligand
luzindole in mice during forced swimming requires expression of MT but not MT melatonin receptors.
2
1
J. Pineal Res. 2005, 39, 170–177. [CrossRef] [PubMed]
1. Hirai, K.; Kato, K.; Nishikawa, H.; Yukuhiro, N.; Nishiyama, K.; Miyamoto, M. Preclinical pharmacological
profiles and clinical outcome of the novel melatonin-receptor agonist ramelteon (Rozerem 8 mg). Nihon
Yakurigaku Zasshi 2010, 136, 51–60. [CrossRef] [PubMed]
2. De Bodinat, C.; Guardiola-Lemaitre, B.; Mocaer, E.; Renard, P.; Munoz, C.; Millan, M.J. Agomelatine, the first
melatonergic antidepressant: Discovery, characterization and development. Nat. Rev. Drug Discov. 2010, 9,
6
28–642. [CrossRef] [PubMed]
2
2
3. Dhillon, S.; Clarke, M. Tasimelteon: First global approval. Drugs 2014, 74, 505–511. [CrossRef] [PubMed]
4. Lemoine, P.; Zisapel, N. Prolonged-release formulation of melatonin (Circadin) for the treatment of insomnia.
Expert Opin. Pharmacother. 2012, 13, 895–905. [CrossRef] [PubMed]
2
2
2
2
2
3
5. Spadoni, G.; Bedini, A.; Piersanti, G.; Mor, M.; Rivara, S.; Tarzia, G. Strategies leading to MT selective
melatonin receptor antagonists. Adv. Exp. Med. Biol. 2003, 527, 577–585. [PubMed]
6. Mor, M.; Rivara, S.; Pala, D.; Bedini, A.; Spadoni, G.; Tarzia, G. Recent advances in the development of
2
melatonin MT and MT receptor agonists. Expert Opin. Ther. Pat. 2010, 20, 1059–1077. [CrossRef] [PubMed]
1
2
7. Zlotos, D.P. Recent progress in the development of agonists and antagonists for melatonin receptors.
Curr. Med. Chem. 2012, 19, 3532–3549. [CrossRef] [PubMed]
8. Zlotos, D.P.; Jockers, R.; Cecon, E.; Rivara, S.; Witt-Enderby, P.A. MT and MT melatonin receptors: Ligands,
1
2
models, oligomers, and therapeutic potential. J. Med. Chem. 2014, 57, 3161–3185. [CrossRef] [PubMed]
9. Yan, J.H.; Su, H.R.; Boutin, J.A.; Renard, M.P.; Wang, M.W. High-throughput screening assay for new ligands
at human melatonin receptors. Acta Pharmacol. Sin. 2008, 29, 1515–1521. [CrossRef] [PubMed]
0. Legros, C.; Matthey, U.; Grelak, T.; Pedragona-Moreau, S.; Hassler, W.; Yous, S.; Thomas, E.; Suzenet, F.;
Folleas, B.; Lefoulon, F.; et al. New radioligands for describing the molecular pharmacology of MT and
1
MT melatonin receptors. Int. J. Mol. Sci. 2013, 14, 8948–8962. [CrossRef] [PubMed]
2
3
3
3
1. Legros, C.; Brasseur, C.; Delagrange, P.; Ducrot, P.; Nosjean, O.; Boutin, J.A. Alternative radioligands for
investigating the molecular pharmacology of melatonin receptors. J. Pharmacol. Exp. Ther. 2016, 356, 681–692.
[CrossRef] [PubMed]
2. Mor, M.; Rivara, S.; Silva, C.; Bordi, F.; Plazzi, P.V.; Spadoni, G.; Diamantini, G.; Balsamini, C.; Tarzia, G.;
Fraschini, F.; et al. Melatonin receptor ligands: Synthesis of new melatonin derivatives and comprehensive
comparative molecular field analysis (CoMFA) study. J. Med. Chem. 1998, 41, 3831–3844. [CrossRef] [PubMed]
3. Audinot, V.; Fabry, N.; Nicolas, J.P.; Beauverger, P.; Newman-Tancredi, A.; Millan, M.J.; Try, A.; Bornancin, F.;
Canet, E.; Boutin, J.A. Ligand modulation of [35S]GTP
γ
S binding at human
α
2A,
α
and
α
adrenoceptors.
2C
2B
Cell Signal. 2002, 14, 829–837. [CrossRef]
3
3
3
3
3
3
4. Claing, A.
013, 118, 149–174. [PubMed]
5. Reiter, E.; Lefkowitz, R.J. GRKs and
Endocrinol. Metab. 2006, 17, 159–165. [CrossRef] [PubMed]
6. Pitcher, J.A.; Freedman, N.J.; Lefkowitz, R.J. G protein-coupled receptor kinases. Annu. Rev. Biochem. 1998
β-Arrestins: Modulators of small GTPase activation and function. Prog. Mol. Biol. Transl. Sci.
2
β-arrestins: Roles in receptor silencing, trafficking and signaling. Trends
,
6
7, 653–692. [CrossRef] [PubMed]
7. Lefkowitz, R.J.; Shenoy, S.K. Transduction of receptor signals by
CrossRef] [PubMed]
8. Van der Lee, M.M.; Bras, M.; van Koppen, C.J.; Zaman, G.J.
β-arrestins. Science 2005, 308, 512–517.
[
β-arrestin recruitment assay for the identification of
agonists of the sphingosine 1-phosphate receptor EDG1. J. Biomol. Screen. 2008, 13, 986–998. [CrossRef] [PubMed]
9. Peters, M.F.; Knappenberger, K.S.; Wilkins, D.; Sygowski, L.A.; Lazor, L.A.; Liu, J.; Scott, C.W. Evaluation of
cellular dielectric spectroscopy, a whole-cell, label-free technology for drug discovery on Gi-coupled GPCRs.
J. Biomol. Screen. 2007, 12, 312–319. [CrossRef] [PubMed]
4
0. Fujii, H.; Hayashida, K.; Saitoh, A.; Yokoyama, A.; Hirayama, S.; Iwai, T.; Nakata, E.; Nemoto, T.; Sudo, Y.;
Uezono, Y.; et al. Novel
δ opioid receptor agonists with oxazatricyclodecane structure. ACS Med. Chem. Lett.
2
014, 5, 368–372. [CrossRef] [PubMed]
4
1. Kajitani, N.; Miyano, K.; Okada-Tsuchioka, M.; Abe, H.; Itagaki, K.; Hisaoka-Nakashima, K.; Morioka, N.;
Uezono, Y.; Takebayashi, M. Identification of lysophosphatidic acid receptor 1 in astroglial cells as a target

Int. J. Mol. Sci. 2017, 18, 1347
17 of 17
for glial cell line-derived neurotrophic factor expression induced by antidepressants. J. Biol. Chem. 2016, 291,
7364–27370. [CrossRef] [PubMed]
2. Devavry, S.; Legros, C.; Brasseur, C.; Delagrange, P.; Spadoni, G.; Cohen, W.; Malpaux, B.; Boutin, J.A.;
2
4
Nosjean, O. Description of the constitutive activity of cloned human melatonin receptors hMT and hMT2
1
and discovery of inverse agonists. J. Pineal Res. 2012, 53, 29–37. [CrossRef] [PubMed]
4
3. Taborsky, R.G.; Mcisaac, W.M. The synthesis and preliminary pharmacology of some 9H-pyrido(3,4-b)indoles
1
2
(β
-carbolines) and tryptamines related to serotonin and melatonin . J. Med. Chem. 1964, 7, 135–141.
CrossRef] [PubMed]
4. Kobayashi, T.; Spande, T.F.; Aoyagi, H.; Witkop, B. Tricyclic analogs of melatonin. J. Med. Chem. 1969, 12,
36–638. [CrossRef] [PubMed]
5. Legros, C.; Devavry, S.; Caignard, S.; Tessier, C.; Delagrange, P.; Ouvry, C.; Boutin, J.A.; Nosjean, O. Melatonin
MT and MT receptors display different molecular pharmacologies only in the G-protein coupled state.
[
4
4
6
1
2
Br. J. Pharmacol. 2014, 171, 186–201. [CrossRef] [PubMed]
4
4
4
6. Hu, Y.; Zhu, J.; Chan, K.H.; Wong, Y.H. Development of substituted N-3-(3-methoxylphenyl)propyl amides
as MT -selective melatonin agonists: Improving metabolic stability. Bioorg. Med. Chem. 2013, 21, 547–552.
2
[CrossRef] [PubMed]
7. Alexander, S.P.; Benson, H.E.; Faccenda, E.; Pawson, A.J.; Sharman, J.L.; McGrath, J.C.; Catterall, W.A.;
Spedding, M.; Peters, J.A.; Harmar, A.J.; et al. The concise guide to pharmacology 2013/14: Overview. Br. J.
Pharmacol. 2013, 170, 1449–1458. [CrossRef] [PubMed]
8. Neubig, R.R.; Spedding, M.; Kenakin, T.; Christopoulos, A. International union of pharmacology committee
on Receptor nomenclature and drug classification. XXXVIII. Update on terms and symbols in quantitative
pharmacology. Pharmacol. Rev. 2003, 55, 597–606. [CrossRef] [PubMed]
4
5
9. Riese, D.J. Ligand-based receptor tyrosine kinase partial agonists: New paradigm for cancer drug discovery?
Expert Opin. Drug Discov. 2011, 6, 185–193. [CrossRef] [PubMed]
0. Logez, C.; Berger, S.; Legros, C.; Baneres, J.L.; Cohen, W.; Delagrange, P.; Nosjean, O.; Boutin, J.A.; Ferry, G.;
Simonin, F.; et al. Recombinant human melatonin receptor MT1 isolated in mixed detergents shows
pharmacology similar to that in mammalian cell membranes. PLoS ONE 2014, 9, e100616. [CrossRef] [PubMed]
5
1. Logez, C.; Damian, M.; Legros, C.; Dupre, C.; Guery, M.; Mary, S.; Wagner, R.; M'Kadmi, C.; Nosjean, O.;
Fould, B.; et al. Detergent-free isolation of functional G protein-coupled receptors into nanometric lipid
particles. Biochemistry 2016, 55, 38–48. [CrossRef] [PubMed]
5
5
5
2. Chan, K.H.; Wong, Y.H. A molecular and chemical perspective in defining melatonin receptor subtype
selectivity. Int. J. Mol. Sci. 2013, 14, 18385–18406. [CrossRef] [PubMed]
3. Pala, D.; Lodola, A.; Bedini, A.; Spadoni, G.; Rivara, S. Homology models of melatonin receptors: Challenges
and recent advances. Int. J. Mol. Sci. 2013, 14, 8093–8121. [CrossRef] [PubMed]
4. Audinot, V.; Mailliet, F.; Lahaye-Brasseur, C.; Bonnaud, A.; Le, G.A.; Amosse, C.; Dromaint, S.; Rodriguez, M.;
Nagel, N.; Galizzi, J.P.; et al. New selective ligands of human cloned melatonin MT and MT receptors.
1
2
Naunyn Schmiedebergs Arch. Pharmacol. 2003, 367, 553–561. [CrossRef] [PubMed]
5. Sakurai, T.; Koike, T.; Nakayama, M. Pharmacological characterization of a highly selective and potent
5
5
partial agonist of the MT melatonin receptor. Pharmacology 2014, 93, 244–252. [CrossRef] [PubMed]
2
6. Gbahou, F.; Cecon, E.; Viault, G.; Gerbier, R.; Jean-Alphonse, F.; Karamitri, A.; Guillaumet, G.; Delagrange, P.;
Friedlander, R.M.; Vilardaga, J.P.; et al. Design and validation of the first cell-impermeant melatonin receptor
agonist. Br. J. Pharmacol. 2017, in press. [CrossRef] [PubMed]
5
5
7. Dupré, C.; Bruno, O.; Bonnaud, A.; Giganti, A.; Nosjean, O.; Legros, C.; Boutin, J.A. Assessments of
cellular melatonin receptor signaling pathways:
spectroscopy. Unpublished work. 2017.
β-arrestin recruitment, internalization and cellular dielectric
8. Coge, F.; Guenin, S.P.; Fery, I.; Migaud, M.; Devavry, S.; Slugocki, C.; Legros, C.; Ouvry, C.; Cohen, W.;
Renault, N.; et al. The end of a myth: Cloning and characterization of the ovine melatonin MT receptor.
2
Br. J. Pharmacol. 2009, 158, 1248–1262. [CrossRef] [PubMed]
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