N-(substituted-anilinoethyl)amides: Design, synthesis, and pharmacological characterization of a new class of melatonin receptor ligands

Article abstract of DOI:10.1021/jm700957j

A novel series of melatonin receptor ligands, characterized by a N-(substituted-anilinoethyl)amido scaffold, along with preliminary structure-activity relationships (SARs), is presented. MT₁ and MT₂ receptor binding affinity and intr

Full text of DOI:10.1021/jm700957j

6618
J. Med. Chem. 2007, 50, 6618–6626
N-(Substituted-anilinoethyl)amides: Design, Synthesis, and Pharmacological Characterization of
a New Class of Melatonin Receptor Ligands
Silvia Rivara,† Alessio Lodola,† Marco Mor,† Annalida Bedini,‡ Gilberto Spadoni,*,‡ Valeria Lucini,§ Marilou Pannacci,§
Franco Fraschini,§ Francesco Scaglione,§ Rafael Ochoa Sanchez,|, Gabriella Gobbi,|, and Giorgio Tarzia‡
Dipartimento Farmaceutico, UniVersità degli Studi di Parma, V.le G. P. Usberti 27/A, Campus UniVersitario, 43100 Parma, Italy, Istituto di
Chimica Farmaceutica e Tossicologica, UniVersitá degli Studi di Urbino “Carlo Bo”, Piazza Rinascimento 6, 61029 Urbino, Italy,
Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, UniVersità degli Studi di Milano, Via VanVitelli 32, 20129 Milano, Italy,
Department of Psychiatry, McGill UniVersity, Montréal, QC, Canada H1N 3V2, and Department of Psychiatry, Centre de Recherche Fernand
Seguin, UniVersité de Montréal, Montreal, QC, Canada H3A 1A1
ReceiVed August 2, 2007
A novel series of melatonin receptor ligands, characterized by a N-(substituted-anilinoethyl)amido scaffold,
along with preliminary structure–activity relationships (SARs), is presented. MT1 and MT2 receptor binding
affinity and intrinsic activity have been modulated by the introduction of different substituents on the aniline
nitrogen, on the benzene ring, and on the amide side chain. Modulation of intrinsic activity and MT2 selectivity
of the newly synthesized compounds has been achieved by applying SAR models previously developed,
providing compounds with different binding and intrinsic activity profiles. Compound 3d, with a bulky
ß-naphthyl group, behaves as an MT2-selective antagonist with sub-nM affinity. Size reduction of the
substituent enhances intrinsic activity, as in the nonselective N-methyl-anilino agonist 3i. The phenyl derivative
3g is an MT2-selective partial agonist, with MT2 binding affinity higher than melatonin, showing promising
sleep-promoting and antianxiety properties in animal models.
Introduction
human enzyme quinone reductase 2.15 Moreover, calmodulin
has also been identified as an intracellular binding site for
MLT.16 MT1 and MT2 receptors are widely distributed in
different areas of the central nervous system, as well as in
peripheral organs.17 The limited availability of subtype-selective
ligands has hampered an exhaustive elucidation of their patho/
physiological role. Indeed, the MT1 receptor produces vaso-
constriction, inhibits prolactin secretion from the hypophyseal
pars tuberalis, and reduces neuronal firing of the hypothalamic
suprachiasmatic nucleus, suggesting a role in the sleep-promot-
ing effect of MLT; the MT2 receptor induces vasodilation and
produces a phase shift in circadian rhythms, probably sustaining
the resynchronizing activity of MLT.18 Irreversible melatonin
receptor ligands, such as N-[2-(2-bromoacetyl-7-methoxynaph-
thyl)ethyl]propionamide (BMNEP) that permanently activates
the MT2 receptor, may help the elucidation of MLT receptor
functions.19
Melatonin (N-acetyl-5-methoxytryptamine, MLTa, Figure 1)
is a tryptophan-derived hormone characterized by a circadian
rhythm of secretion, with peak levels occurring during the period
of darkness. It is mainly synthesized by the pineal gland,
although other cells and organs, such as retina, gut, testes, bone
marrow cells, and human lymphocytes, produce minor amounts
of MLT, which may be of some local importance. Besides the
well-known chronobiotic and sleep-inducing properties,1 many
other physiological and behavioral effects have been ascribed
to MLT, such as the modulation of cardiovascular2 and immune
systems3 and the influence on metabolism, bone formation,4 and
hormone secretion.5 For this reason, the potential benefits of
MLT administration have been evaluated in a variety of
pathological conditions, such as sleep disturbances,6 cancer,7
neurodegenerative diseases,8,9 headache disorders,10 and
stroke.11
During the past decades, many series of melatonin receptor
ligands have been reported in the literature and in patents.
Compounds with agonist activity have been mainly evaluated
for their sleep inducing properties or for circadian phase shift
action. Ramelteon is at present the only melatonin receptor
agonist marketed for the treatment of insomnia20 and other
compounds,21 such as LY-15673522 or VEC-162,23 have been
tested in clinical trials for their hypnotic properties. Agomelatine,
an MT1/MT2 agonist and 5HT2c antagonist is under evaluation
for the treatment of major depressive disorders.24 Examples of
subtype-selective agonists are still limited.25 Evaluation of
melatonin receptor antagonists has been limited to preclinical
studies, for example, S22153 in circadian rhythm entrainment
experiments,26 luzindole for its antidepressant-like properties,27
or ML-23 in the treatment and management of Parkinson’s
disease.28 While only few examples of MT1-selective antagonists
are reported, several series of MT2-selective antagonists have
been discovered.25,29 The main classes of MT2-selective an-
tagonists are represented in Figure 1; they comprise benzofuran30
MLT exerts its effects by multiple mechanisms. It acts as an
antioxidant and radical scavenger and it regulates the activity
of a series of enzymes affecting the redox state of the cell.12 In
mammals, MLT activates two G-protein-coupled receptors,
named MT1 and MT2,13,14 at nanomolar concentrations, and it
binds with lower affinity to the so-called MT3 binding site,
recently characterized as a melatonin-sensitive form of the
* To whom correspondence should be addressed. Gilberto Spadoni,
Istituto di Chimica Farmaceutica, Università degli Studi di Urbino “Carlo
Bo”, Piazza Rinascimento 6, I-61029 Urbino, Italy. Tel.: ++39 0722
303337. Fax: ++39 0722 303313. E-mail: gilberto.spadoni@uniurb.it.
†
Università degli Studi di Parma.
Universitá degli Studi di Urbino “Carlo Bo”.
Università degli Studi di Milano.
McGill University.
‡
§
|
Université de Montréal.
aAbbreviations: MLT, melatonin; MT1, melatonin receptor subtype 1;
MT2, melatonin receptor subtype 2; MT3, third binding site of melatonin;
4P-PDOT, 4-phenyl-propionamidotetralin; [35S]GTPγS, [35S]guanosine-5′-
O-(3-thio-triphosphate); IAr, relative intrinsic activity.
10.1021/jm700957j CCC: $37.00
2007 American Chemical Society
Published on Web 12/06/2007

N-(Substituted-anilinoethyl)amides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6619
Figure 1. Melatonin and representative MT2 selective antagonists.
Scheme 1a
Figure 2. N-(3,3-Diphenylpropyl)acetamide and N-(3,3-diphenylpro-
penyl)acetamide derivatives; see ref 38.
and tetrahydronaphthalenic31 derivatives, 6,7-dihydro-5H-ben-
zo[c]azepino[2,1-a]indoles,32 2-N-acylaminoalkylindoles,33 and
tetrahydroisoquinoline derivatives.34
Our research in the field of melatonin receptor ligands is
dedicated to the study of their structure–activity relationships
and to the synthesis of new agonists and antagonists endowed
with selectivity for each of the two receptor subtypes. Molecular
modeling on representative ligands has led us to hypothesize
that a bulky substituent in an area corresponding to positions
1–2 of the indole nucleus of MLT, and “out-of-plane” from the
indole ring, relates to selectivity for the MT2 receptor and
reduces intrinsic activity at melatonin receptors.33 The impor-
tance of this structural element has been confirmed by 3D-
QSAR35 and by docking within three-dimensional models of
the MT2 and MT1 receptors; the putative receptor binding sites
have a lipophilic pocket available, where the lipophilic “out-
of-plane” substituents of MT2 antagonists can be accom-
modated.36 This information has been applied to the design of
a novel series of tricyclic dihydrodibenzocycloheptene antago-
nists, characterized by a skewed scaffold, which fulfills the
requirements for MT2-selective antagonism (compound a in
Figure 1).37 An open chain analog approach has been subse-
quently applied to the rigid structures of compound a and of
the tetralin antagonist 4-phenyl-propionamidotetralin (4P-PDOT,
Figure 1), maintaining the pharmacophoric elements for MT2
binding. This structural simplification has led to the new classes
of N-(3,3-diphenylpropyl)alkanamides and N-(3,3-diphenylpro-
penyl)alkanamides (Figure 2), some of which have MT2 binding
affinity higher than MLT and a remarkable selectivity for the
MT2 receptor.38 The two aromatic rings of these classes are
linked to an alkyl or alkenyl portion, resulting in different
enantiomers or E/Z diastereoisomers, respectively. The N-
(substituted-anilinoethyl)amides obtained by replacing the ben-
zhydryl carbon with a nitrogen atom fulfill the structural
requirements for MLT receptor binding, while avoiding isomer-
ism and likely leading to a desirable improvement in water
solubility. We report here the synthesis, receptor binding
a Reagents and conditions: (a) Cu(OAc)2, CH2Cl2, pyridine, room
temperature; (b) Ac2O, AcOH, room temperature; (c) 3-bromoanisole, CuI,
K2CO3, 220 °C; (d) KOH, EtOH, reflux; (e) CuI, K2CO3, L-proline, DMSO,
90 °C.
characterization, and preliminary structure–activity relationships
for a series of these compounds.
Starting from this scaffold, the acylating group has been
slightly modulated, a methoxy or a bromine substituent has been
introduced on the phenyl ring to mimic the 5-methoxy group
of MLT, the effect of lengthening of the ethyl chain was
examined, and the anilinoethyl moiety has been replaced by a
phenoxyethyl group.
Moreover, different substituents have been added at the
aniline nitrogen to try to reproduce the cited SARs for the out-
of-plane group of MT2-selective antagonists. The synthesized
compounds have been tested for their binding affinity for MT1
and MT2 receptors and for their intrinsic activity. A representa-
tive compound has been preliminarily evaluated in animal
models to investigate potential sleep-inducing properties.
Chemistry
The (anilinoalkyl)amido derivatives (3a-m) are prepared by
N-cyanoalkylation of anilines (1a-j) with bromoacetonitrile
(chloroacetonitrile for 2j) or 3-bromoproprionitrile (for 2k) in
the presence of sodium hydride (for 2a-d and 2f-h) or in the
absence of a base (for 2e, 2i-k), followed by reduction of
the intermediate nitriles (2a-k) and N-acylation of the crude
diamines with anhydrides or acid chlorides (Scheme 2). The
cyano group of nitriles 2a-k is easily reduced using standard
procedures. Briefly, Raney nickel hydrogenation of nitriles
2a-d, 2f-i, or 2k and concomitant N-acylation of the corre-
sponding crude anilinoalkylamine with acetic or n-butyric

6620 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
RiVara et al.
Scheme 2a
Scheme 3a
a Reagents and conditions: (a) ClCH2CN, K2CO3, CH3CN, reflux; (b)
H2, Ni/Raney, Ac2O, THF, 60 °C.
solubility sufficient to perform the pharmacological tests on the
free bases, and the possibility to prepare water-soluble salts, if
necessary. The synthetic procedure allows insertion on the
aniline nitrogen of groups of different size and shape and, in
general, modification of the scaffold according to classical
medicinal chemistry procedures.
The preliminary binding data for the N-phenyl-N-(3-meth-
oxyphenyl) derivative 3g, which showed partial agonist activity
with good MT2 selectivity, supported the bioisosteric role of
nitrogen, leading us to further explore the SARs of this new
class of compounds. Groups with different size and shape were
thus inserted on the aniline nitrogen, and other more classical
changes were also performed, such as the substitution of the
methoxy with a bromine atom and the use of different acylating
groups. Methylation of the amide nitrogen and lengthening of
the ethyl spacer were also evaluated. The MT1 and MT2 binding
affinity of the newly synthesized compounds and their effect
on GTPγS binding (intrinsic activity) are reported in Table 1.
Compound 3f, having two unsubstituted phenyl rings, displays
moderate receptor affinity, with some selectivity for the MT2
receptor; it behaves as an MT2 partial agonist, while it is an
antagonist at the MT1 receptor. This profile is similar to those
observed for N-(3,3-diphenylpropyl)acetamide and N-(3,3-
diphenylpropenyl)acetamide derivatives,38 being closer to that
of the unsaturated compounds and with higher intrinsic activity
at the MT2 receptor. The introduction of a methoxy group in
the meta-position, superposable to the 5-methoxy group of MLT
(Figure 3, left panel), induces a remarkable increase in binding
affinity at both receptor subtypes, while maintaining MT2
selectivity. Compound 3g has one of the highest binding
affinities ever reported for MT2 receptor ligands (pKi ) 10.18).
The meta-methoxy group influences intrinsic activity at the MT1
receptor only, with a modest or null effect at the MT2; compound
3g thus behaves as a partial agonist at both receptor subtypes.
It must be noted that the previously obtained 3D-QSAR models
evidence a role for the methoxy group in both MT2 intrinsic
activity and selectivity, which is not observed in the present
series (see 3f and 3g). Comparison with the corresponding
propyl and propenyl derivatives (Figure 2) evidences highest
resemblance with the binding profile of the (E)-unsaturated
derivative, with the (Z)-isomer and the saturated analog showing
lower affinity for both subtypes. Replacement of the methoxy
group with bromine is tolerated, affording 3 h with binding
affinities and intrinsic activities only slightly lower than those
of the methoxy analog 3g. This behavior is consistent with what
was observed for other series of melatonin receptor ligands.43
Shift of the methoxy group to the para-position (3c) greatly
reduces binding affinity at both receptor subtypes; the pKi values
obtained for 3c are even lower than those of the unsubstituted
3f; intrinsic activity at the MT2 receptor is also abolished. The
negative role of the para-methoxy group, seen in 3c, is
confirmed in the di-methoxyphenyl derivative 3b. This behavior
resembles what was observed for MLT derivatives, where
moving the methoxy group from position 5 to position 6 led to
micromolar binding affinities and to partial agonist behavior.43
Reduction of binding affinity and intrinsic activity because of
a Reagents and conditions: (a) BrCH2CN (ClCH2CN) or BrCH2CH2CN,
DMF, NaH, 100 °C (NaH is not necessary for e, i-k); (b) H2, Ni/Raney,
(R2CO)2O, THF, or H2, Ni/Raney, NH3/EtOH, then Ac2O/TEA for 3j or
c-butanoyl chloride/TEA for 3m; (c) LiAlH4, THF for 3e; (d) Ac2O, TEA,
THF; (e) MeI, NaH, DMF.
anhydride provides the desired melatonin ligands 3a-d, 3f-i,
and 3k,l. The cyclobutanecarboxamido derivative 3m and the
monosubstituted aniline derivative 3j are prepared by hydro-
genation of the nitriles 2g and 2j over Raney nickel in the
presence of 1 M NH3-EtOH, followed by N-acylation with
c-butanoyl chloride or acetic anhydride, respectively, in the
presence of triethylamine (TEA). To prepare the N-benzyl
derivative 3e, the corresponding nitrile 2e is reduced with lithium
aluminum hydride, and the resulting crude amine was N-acylated
with acetic anhydride. Compound 4 is prepared by N-alkylation
of 3g with methyl iodide in the presence of NaH as a base.
The key starting anilines are commercially available (1f-j)
or have been synthesized (1a-e, Scheme 1) using known
procedures. Briefly, 3-methoxy-N-(3-methoxyphenyl)benze-
namine 1a is synthesized by reacting 3-bromoanisole with
3-methoxyacetanilide and subsequent alkaline hydrolysis of
the intermediate N,N-bis(3-methoxyphenyl)acetamide.39 The
N-substituted-anilines 1b, c, and e are prepared in good yield
by CuI-catalyzed coupling reaction of the suitable amine with
bromobenzene or 3-bromoanisole, in the presence of L-
proline.40 Alternatively, the new compound (3-methoxy)-N-
(naphthalen-2-yl)aniline 1d is obtained by coupling m-ani-
sidine and 2-naphthaleneboronic acid in the presence of
cupric acetate and pyridine, according to a previously reported
procedure,41 Scheme 1.
(3-Methoxyphenoxy)acetonitrile42 is obtained by alkylation
of 3-hydroxyanisole with chloroacetonitrile and then converted
to the target compound 5 by hydrogenation over Raney nickel
of the cyano group and concomitant N-acetylation with acetic
anhydride, as depicted in Scheme 3.
Results and Discussion
N-(Substituted-anilinoethyl)amides have some advantages
relative to the previously reported N-(3,3-diphenylpropyl)al-
kanamides, that is, a shorter and higher yielding reaction
pathway, the absence of optical or geometrical isomers, a water

N-(Substituted-anilinoethyl)amides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6621
Table 1. Experimental Binding Affinity and Intrinsic Activity of Newly Synthesized Compounds for Human MT1 and MT2 Melatonin Receptors Stably
Expressed in NIH3T3 Cells
MT1
MT2
cmpd
R
R1
n
R2
pKia
IAr ( SEMb
pKia
IAr ( SEMb
MLT
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
4
5
9.85 ( 0.09
7.72 ( 0.13
7.00 ( 0.01
6.11 ( 0.12
6.88 ( 0.07
7.30 ( 0.10
6.90 ( 0.04
8.38 ( 0.01
7.77 ( 0.19
9.09 ( 0.10
8.28 ( 0.01
9.08 ( 0.04
8.38 ( 0.01
6.49 ( 0.50
5.89 ( 0.07
8.14 ( 0.03
1.0 ( 0.01
0.59 ( 0.05
0.06 ( 0.03
0.02 ( 0.01
0.17 ( 0.03
0.82 ( 0.06
0.10 ( 0.03
0.79 ( 0.03
0.43 ( 0.04
0.95 ( 0.08
0.84 ( 0.01
0.87 ( 0.05
1.01 ( 0.04
0.22 ( 0.01
-0.01 ( 0.03
0.83 ( 0.02
9.62 ( 0.10
10.56 ( 0.10
9.06 ( 0.10
7.56 ( 0.10
9.95 ( 0.64
9.12 ( 0.05
8.41 ( 0.13
10.18 ( 0.32
9.70 ( 0.43
9.19 ( 0.01
8.13 ( 0.18
8.70 ( 0.26
9.98 ( 0.26
8.43 ( 0.40
7.28 ( 0.06
7.90 ( 0.05
1.01 ( 0.01
0.15 ( 0.01
0.03 ( 0.01
0.06 ( 0.03
-0.20 ( 0.03
0.31 ( 0.02
0.57 ( 0.04
0.61 ( 0.04
0.37 ( 0.01
1.06 ( 0.05
0.95 ( 0.03
1.07 ( 0.06
0.74 ( 0.02
0.29 ( 0.01
-0.01 ( 0.03
0.97 ( 0.05
3-OMe
3-OMe
4-OMe
3-OMe
3-OMe
H
3-OMe-Ph
4-OMe-Ph
Ph
ꢀ-naphthyl
-CH2Ph
Ph
Ph
Ph
Me
H
Me
Ph
1
1
1
1
1
1
1
1
1
1
2
1
1
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
n-Pr
c-Bu
3-OMe
3-Br
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
Ph
a pKi values were calculated from IC50 values obtained from competition curves by the method of Cheng and Prusoff,61 and are the mean of at least three
determinations performed in duplicate. b The relative intrinsic activity values were obtained by dividing the maximum analogue-induced G-protein activation
by that of MLT.
Figure 3. Left panel: superposition of MLT (purple carbons) and 3d (orange carbons) within CoMFA regions related to MT2 intrinsic activity.35
Occupation of green regions is related to an increase of intrinsic activity, and occupation of yellow regions is related to a decrease of intrinsic
activity. Middle panel: MT2-selective antagonists 3d (orange carbons) and UCM454 (Figure 1, white carbons) surrounded by the CoMFA regions,
whose occupancy is related to MT1 (cyan) or MT2 (red) selectivity, in accordance with the 3D-QSAR models described in ref 35. Right panel:
energy-minimized conformation of the last step of 1 ns MD simulation for the complex between MT2 receptor model and 3d.
the shifting of the methoxy group has also been observed in
other series of MLT receptor ligands.33,34 Insertion of an
additional meta-methoxy group on the second phenyl ring of
3g (i.e., compound 3a) leads to the reduction of binding at MT1
and to an increase in MT2 binding affinity, thus enhancing MT2
selectivity. Compound 3a is characterized by the highest binding
affinity for the MT2 receptor of the whole series, about ten times
greater than that of MLT, and by about seven hundred times
selectivity over the MT1 receptor. The second methoxy group
decreases intrinsic activity, more at the MT2 receptor than at
the MT1. The different behavior of 3g and 3a is consistent with
the putative role of the out-of-plane group for MT2-selective
antagonism. As hypothesized on the basis of 3D models of
melatonin receptor subtypes, the corresponding pocket within
the MT1 receptor is smaller, leading to a reduction of binding
affinity for compounds with bulky substituents.36 This is also
consistent with the data observed for 3e and 3d, having a benzyl
and a ꢀ-naphthyl group, respectively; the increase of substituent
bulk parallels the decrease of MT1 binding affinity and of MT2
intrinsic activity. In Figure 3 (left panel), compound 3d is
surrounded by the CoMFA coefficients correlated with intrinsic
activity at the MT2 receptor, previously obtained from a set of
ligands evaluated by the GTPγS binding assay.35 The N-phenyl
substituent of 3g is halfway between the green region, corre-
sponding to position 2 of MLT, which correlates with an
increase in intrinsic activity, while the yellow one denotes a
decrease in intrinsic activity; this alignment is consistent with
the observed MT2 partial agonist behavior. Conversely, com-
pound 3d, which possesses a naphthyl substituent deeply
inserted into the yellow region, behaves as an antagonist.
Moreover, the naphthyl substituent occupies the red region,
which is related to MT2 selectivity, as depicted in Figure 3

6622 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
RiVara et al.
(middle panel). Indeed, the ꢀ-naphthyl derivative 3d has a
remarkable binding affinity for the MT2 receptor, with more
than 1000× selectivity over the MT1 receptor.
liminary results indicate that this compound, when administered
(40 mg/kg s.c.) to freely moving rats, promotes sleep, as
demonstrated by the decrease in the latency and the increase in
the duration of slow wave sleep, and by the increase of
paradoxical sleep latency, without significantly altering its
duration and quantity.50 Moreover, 3g displayed activity in the
Elevated Plus Maze Test in Sprague–Dawley rats,51 an experi-
mental animal model of anxiety.52 Additionally, pharmacological
characterization of this compound, which may become a
candidate hypnotic drug, is now ongoing.53
In conclusion, substitution of benzhydryl carbon atom of the
previously reported N-(3,3-diphenylpropyl)alkanamido series of
melatonin receptor ligands with a nitrogen atom results in a
novel series of N-(substituted-anilinoalkyl)amido ligands, whose
binding affinity and intrinsic activity can be modulated by
application of SAR derived from earlier 3D-QSAR and molec-
ular modeling studies. A methoxy group in the meta-position,
corresponding to the position of the methoxy group in MLT,
greatly improves receptor binding affinity. Intrinsic activity and
MT1/MT2 subtype selectivity can be modulated by the introduc-
tion of substituents with different sizes on the aniline nitrogen.
This chemical modification has afforded compounds 3a and 3d,
carrying bulky 3-methoxyphenyl and ꢀ-naphthyl substituents,
which are highly selective antagonists, with MT2 binding
affinities greater than MLT. Insertion of a phenyl ring on the
nitrogen atom leads to the MT2-selective partial agonist 3g,
while the smaller methyl substituent gives compound 3i,
behaving as a nonselective full agonist. The partial agonist 3g
shows sleep-inducing and antianxiety properties in animal
models, which will be reported elsewhere.
When 3d is docked within the MT2 receptor model, by means
of an automated docking procedure implemented in the program
Glide,44 the solution characterized by the best E-model score
gives a complex that is stable during 1 ns MD simulation (Figure
3, right panel). Compound 3d occupies the same region as other
previously docked MT2 antagonists and undertakes similar
interactions.36 In particular, the ꢀ-naphthyl substituent is deeply
inserted into the lipophilic pocket, close to Trp264 of the CWXP
motif in TM6, known to be involved in the process of receptor
activation. No significant correlation is found between calculated
E-model values and receptor affinity for this series of com-
pounds. Even if this model provides only partial explanation
for the observed SARs, its qualitative application has proved
useful for the design and refinement of potent and selective MT2
antagonists.
The effect of acyl chain modulation has been evaluated on
the basis of compounds 3l and 3m. The propyl substituent is
tolerated at both receptor subtypes and it increases MT1 intrinsic
activity; in fact, compound 3l behaves as an MT1 full agonist.
The cyclobutyl group has a negative effect on both binding
affinity and intrinsic activity, as already observed for MLT
derivatives45,46 and consistent with the results of a 3D-QSAR
analysis performed on an extended set of melatonin receptor
ligands.35 Methylation of the amide nitrogen in compound 4
produces a huge drop in binding affinity at both receptors and
abolishes intrinsic activity. This behavior underlines that the
amide NH group is important for receptor binding and activation.
On the other hand, this cannot be considered a general rule for
MT2-selective agonists, as a series of indanyl-piperazine deriva-
tives, in which the amide nitrogen is part of the piperazine ring,
are reported as nanomolar MT2-receptor agonists.47
Experimental Section
General Methods. Melting points were determined on a Büchi
1
B-540 capillary melting point apparatus and are uncorrected. H
Chain elongation is tolerated at the MT1 receptor, while it
produces a limited reduction in MT2 binding affinity, leading
to a modest MT1 selectivity for 3k. As its shorter homologue,
3i, compound 3k behaves as a melatonin receptor agonist.
NMR spectra were recorded on a Bruker AVANCE 200 spectrom-
eter, using CDCl3 as solvent unless stated otherwise. Chemical shifts
(δ scale) are reported in parts per million (ppm) relative to the
central peak of the solvent. Coupling constants (J values) are given
in hertz (Hz). EI-MS spectra (70 eV) were taken on a Fisons Trio
1000 instrument. Only molecular ions (M+) and base peaks are
given. Infrared spectra were obtained on a Nicolet Avatar 360 FT-
IR spectrometer; absorbances are reported in ν (cm-1). Elemental
analyses for C, H, and N were performed on a Carlo Erba analyzer,
and the results are within 0.4% of the calculated values. Column
chromatography purifications were performed under “flash” condi-
tions using Merck 230–400 mesh silica gel. Analytical thin-layer
chromatography (TLC) was carried out on Merck silica gel 60 F254
plates. Diphenylamine (1f), 3-methoxydiphenylamine (1g), 3-bro-
modiphenylamine (1h), 3-methoxy-N-methylaniline (1i), m-anisi-
dine (1j), and all the others chemicals were purchased from
commercial suppliers and used directly without any further
purification.
Replacement of one of the phenyl rings with the smaller
methyl group (compound 3i) mainly influences receptor intrinsic
activity, while retaining good binding affinity. Once again, this
effect is probably related to the size of the substituent on the
nitrogen atom. The smaller group is preferred at the MT1
receptor, giving a moderate increase of binding affinity; MT2
binding affinity decreases about ten times, while intrinsic activity
increases. Compound 3i is, therefore, a potent nonselective
melatonin receptor agonist. Removal of the methyl group has a
negative effect on binding affinity, as the monosubstituted
aniline derivative 3j has lower pKi at both receptor subtypes.
Substitution of the NH group of 3j with an oxygen atom
provides the N-(2-phenoxyethyl)acetamide derivative 5, with
similar binding affinities and intrinsic activities to 3j. A series
of propyl-alkanamido melatonin receptor ligands, differing from
our N-methyl-anilino derivative 3i for a methylene group
replacing the NCH3 portion, is described in literature.48 Their
profile, with nanomolar binding affinities for the 3-methoxy
derivatives and agonist activity on the melanophore pigment
aggregation assay49 is similar to that of the compounds of this
study.
The two radioligands 2-[125I]iodomelatonin (specific activity,
2000 Ci/mmol) and [35S]GTPγS ([35S]guanosine-5′-O-(3-thio-
triphosphate); specific activity, 1000 Ci/mmol) were purchased from
Amersham Pharmacia Biotech (Italy).
3-Methoxy-N-(3-methoxyphenyl)benzenamine (1a). Com-
pound 1a was prepared as previously described.39
3-Methoxy-N-(4-methoxyphenyl)benzenamine (1b). Com-
pound 1b was prepared as previously described.40 1H NMR
(CDCl3): δ 3.77 (s, 3H), 3.81 (s, 3H), 6.39–6.52 (m, 3H), 6.85–6.91
(m, 2H), 7.08–7.17 (m, 3H).
The respective roles of MT1 and MT2 receptors in the CNS
as well as in other organs have not been clarified. Considering
that ramelteon is a nonselective MT1/MT2 agonist, we have
thought it could be useful to see if the moderately MT2 selective
partial agonist 3g would have sleep promoting activity. Pre-
4-Methoxy-N-phenylbenzenamine (1c). Compound 1c was
prepared as previously described.40
3-Methoxy-N-(naphthalen-2-yl)benzenamine (1d). Cupric ac-
etate (2.54 g, 14 mmol) and dry pyridine (2 mL) were added to a
vigorously stirred solution of m-anisidine (0.86 g, 7 mmol) and

N-(Substituted-anilinoethyl)amides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6623
2-naphthaleneboronic acid (2.41 g, 14 mmol) in dry CH2Cl2 (22
mL), under a nitrogen atmosphere. After stirring at room temper-
ature for 72 h, the reaction mixture was filtered on Celite, and the
filtrate was evaporated to give a residue that was partitioned between
water and EtOAc. The combined organic phases were washed with
brine, dried (Na2SO4), and evaporated under reduced pressure to
give a crude residue that was purified by flash chromatography
(silica gel; cyclohexane/EtOAc 8:2 as eluent). Amorphous solid,
51% yield. MS (EI): m/z 249 (M+, 100). 1H NMR (CDCl3): δ 3.81
(s, 3H), 5.92 (br s, 1H), 6.53–6.58 (m, 1H), 6.74–6.79 (m, 2H),
7.20–7.52 (m, 8H). IR (cm-1, Nujol): 3380, 1594.
crude residue that was purified by flash chromatography (silica gel;
cyclohexane/EtOAc 8:2 as eluent).
[Benzyl-(3-methoxyphenyl)amino]acetonitrile (2e). Oil, 59%
yield. MS (EI): m/z 252 (M+), 91 (100). 1H NMR (CDCl3): δ 3.81
(s, 3H), 4.10 (s, 2H), 4.53 (s, 2H), 6.50–6.61 (m, 3H), 7.20–7.41
(m, 6H). IR (cm-1, neat): 2230.
[(3-Methoxyphenyl)methylamino]acetonitrile (2i). White solid,
88% yield, mp 65–6 °C (petroleum ether; lit.55 mp 67–68 °C). MS
1
(EI): m/z 176 (M+, 100). H NMR (CDCl3): δ 3.00 (s, 3H), 3.82
(s, 3H), 4.15 (s, 2H), 6.40–6.53 (m, 3H), 7.24 (t, 1H).
(3-Methoxyphenylamino)acetonitrile (2j). Oil (lit.56 137 °C/
1
0.4 mmHg), 54% yield. MS (EI): m/z 162 (M+, 100). H NMR
N-(3-Methoxyphenyl)benzenemethanamine (1e). Compound
1e was prepared as previously described.40 1H NMR (CDCl3): δ
3.72 (s, 3H), 4.01 (br s, 1H), 4.29 (s, 2H), 6.15–6.30 (m, 3H),
7.02–7.20 (m, 6H).
(CDCl3): δ 3.80 (s, 3H), 4.05 (s, 2H), 6.25–6.48 (m, 3H), 7.18 (t,
1H). IR (cm-1, neat): 3381, 2235.
3-[(3-Methoxyphenyl)methylamino]propionitrile (2k). Oil
(lit.57 175 °C/5 mmHg), 48% yield. MS (EI): m/z 190 (M+), 150
(100). 1H NMR (CDCl3): δ 2.57 (t, 2H), 3.02 (s, 3H), 3.70 (t, 2H),
3.80 (s, 3H), 6.24–6.3 (m, 3H), 7.18 (t, 1H). IR (cm-1, neat): 2241.
General Procedure for Hydrogenation of Nitriles and
Concomitant N-Acylation. A solution of the suitable nitrile 2a-d,
2f-i, 2k, or (3-methoxyphenoxy)acetonitrile42 (1 mmol), in THF
(10 mL) and acetic anhydride (16 mmol, butyric anhydride for 3l),
was hydrogenated over Raney nickel at 4 atm of H2 for 5 h at 60
°C (1 atm of H2 for 3 h at room temperature for 3h). The catalyst
was filtered on Celite, the filtrate was concentrated in vacuo, and
the residue was partitioned between EtOAc and 2 N NaOH. The
organic layer was washed with brine, dried (Na2SO4), and evapo-
rated under reduced pressure to give a crude residue that was
purified by flash chromatography (silica gel; EtOAc as eluent, unless
otherwise specified).
N-[2-(Bis-3-methoxyphenylamino)ethyl]acetamide (3a). White
solid, 53% yield; mp 84–5 °C (diethyl ether/petroleum ether). MS
(EI): m/z 314 (M+), 242 (100). 1H NMR (CDCl3): δ 1.94 (s, 3H),
3.50 (q, 2H, J ) 6.3), 3.77 (s, 6H), 3.87 (t, 2H, J ) 6.3), 5.63 (br
s, 1H), 6.51–6.68 (m, 6H), 7.23 (t, 2H). IR (cm-1, Nujol): 3304,
1646. Anal. (C18H22N2O3) C, H, N.
N-{2-[(4-Methoxyphenyl)-3-methoxyphenylamino]ethyl}acet-
amide (3b). White solid, 84% yield; mp 115–6 °C (CH2Cl2/
petroleum ether). MS (EI): m/z 314 (M+), 242 (100). 1H NMR
(CDCl3): δ 1.94 (s, 3H), 3.48 (q, 2H, J ) 6.2), 3.74 (s, 3H), 3.76
(t, 2H, J ) 6.2), 3.82 (s, 3H), 5.67 (br s, 1H), 6.33–6.41 (m, 3H),
6.88–6.93 (m, 2H), 7.05–7.14 (m, 3H). IR (cm-1, Nujol): 3227,
1640. Anal. (C18H22N2O3) C, H, N.
General Procedure for the Synthesis of Nitriles 2a-d and
2f-h. A solution of the suitable N,N-diarylamine 1a-d or 1f-h
(2 mmol) in dry DMF (5 mL) was added dropwise to a stirred
ice-cooled suspension of sodium hydride (0.15 g of an 80%
dispersion in mineral oil, 5 mmol) in dry DMF (5 mL) under a N2
atmosphere. The mixture was stirred at 0 °C for 30 min, then
bromoacetonitrile (0.65 mL, 9.3 mmol) was added dropwise, and
the resulting mixture was heated at 100 °C for 24 h. After pouring
the reaction mixture into ice/water, the aqueous solution was
extracted with EtOAc, and the combined extracts were washed with
brine, dried (Na2SO4), and concentrated under reduced pressure to
give a crude residue of the desired product.
[(Bis-3-methoxyphenyl)amino]acetonitrile (2a). Purification by
flash chromatography (silica gel; cyclohexane/EtOAc 7:3 as eluent).
Oil, 37% yield. MS (EI): m/z 268 (M+, 100). 1H NMR (CDCl3): δ
3.78 (s, 6H), 4.51 (s, 2H), 6.58–6.69 (m, 6H), 7.26 (t, 2H). IR
(cm-1, neat): 2236.
[(3-Methoxyphenyl)-4-methoxyphenylamino]acetonitrile (2b).
Purification by flash chromatography (silica gel, cyclohexane/EtOAc
9:1 as eluent). Oil, 58% yield. MS (EI): m/z 268 (M+), 131 (100).
1H NMR (CDCl3): δ 3.75 (s, 3H), 3.84 (s, 3H), 4.46 (s, 2H),
6.31–6.52 (m, 3H), 6.92–6.97 (m, 2H), 7.15–7.23 (m, 3H).
[(4-Methoxyphenyl)phenylamino]acetonitrile (2c). Purification
by flash chromatography (silica gel; cyclohexane/EtOAc 9:1 as
eluent) and crystallization. White solid, 65% yield; mp 103–4 °C
(diethyl ether/petroleum ether; lit.54 mp 94 °C). MS (EI): m/z 238
(M+), 154 (100).
[(3-Methoxyphenyl)-2-naphthylamino]acetonitrile (2d). Pu-
rification by flash chromatography (silica gel, cyclohexane/EtOAc
8:2 as eluent). Solid, 11% yield, mp 74 °C (subl.). MS (EI): m/z
N-{2-[(4-Methoxyphenyl)phenylamino]ethyl}acetamide (3c).
White solid, 30% yield; mp 85–6 °C (diethyl ether/petroleum ether).
1
MS (EI): m/z 284 (M+), 212 (100). H NMR (CDCl3): δ 1.94 (s,
1
288 (M+, 100). H NMR (CDCl3): δ 3.77 (s, 3H), 4.64 (s, 2H),
3H), 3.49 (q, 2H, J ) 6.2), 3.81 (t, 2H, J ) 6.2), 3.82 (s, 3H), 5.70
(br s, 1H), 6.75–6.93 (m, 5H), 7.07–7.20 (m, 4H). IR (cm-1, Nujol):
3221, 1636. Anal. (C17H20N2O2) C, H, N.
6.62–6.72 (m, 3H), 7.18–7.81 (m, 8H).
N,N-Diphenylaminoacetonitrile (2f). Purification by flash chro-
matography (silica gel; cyclohexane/EtOAc 9:1 as eluent) and
crystallization. White solid, 36% yield; mp 44–5 °C (diethyl ether/
petroleum ether; lit.54 oil). MS (EI): m/z 208 (M+), 167 (100).
[(3-Methoxyphenyl)phenylamino]acetonitrile (2g). Purification
by flash chromatography (silica gel; cyclohexane/EtOAc 9:1 as
eluent) and crystallization. Pink solid, 59% yield; mp 53–4 °C
N-{2-[(3-Methoxyphenyl)-2-naphthylamino]ethyl}acetamide
(3d). Mp 92–3 °C, 48% yield. MS (EI): m/z 334 (M+), 262 (100).
1H NMR (CDCl3): δ 1.92 (s, 3H), 3.54 (q, 2H, J ) 6.2), 3.76 (s,
3H), 3.99 (t, 2H, J ) 6.2), 5.89 (br t, 1H), 6.52–6.69 (m, 3H),
7.15–7.48 (m, 5H), 7.69–7.78 (m, 3H). IR (cm-1, film): 3298, 1649.
Anal. (C21H22N2O2) C, H, N.
1
(petroleum ether). MS (EI): m/z 238 (M+), 154 (100). H NMR
N-[2-(Diphenylamino)ethyl]acetamide (3f). White solid, 78%
(CDCl3): δ 3.78 (s, 3H), 4.52 (s, 2H), 6.55 (t, 1H), 6.59–6.65 (m,
yield; mp 102–103 °C (diethyl ether/petroleum ether). MS (EI):
2H), 7.08–7.41 (m, 6H). IR (cm-1, neat): 2235.
1
m/z 254 (M+), 182 (100). H NMR (CDCl3): δ 1.93 (s, 3H), 3.50
[(3-Bromophenyl)phenylamino]acetonitrile (2h). Purification
by flash chromatography (silica gel; cyclohexane/EtOAc 9:1 as
eluent). Oil, 67% yield. MS (EI): m/z 286–288 (M+), 167 (100).
1H NMR (CDCl3): δ 4.50 (s, 2H), 6.82–6.92 (m, 1H), 7.08–7.46
(m, 8H). IR (cm-1, neat): 2235.
(q, 2H, J ) 6.2), 3.90 (t, 2H, J ) 6.2), 5.77 (br s, 1H), 6.95–7.07
(m, 6H), 7.25–7.33 (m, 4H). IR (cm-1, Nujol): 3304, 1643. Anal.
(C16H18N2O) C, H, N.
N-{2-[(3-Methoxyphenyl)phenylamino]ethyl}acetamide (3g).
White solid, 85% yield; mp 73–74 °C (isopropyl ether). MS (EI):
1
m/z 248 (M+), 212 (100). H NMR (CDCl3): δ 1.93 (s, 3H), 3.50
General Procedure for the Synthesis of Nitriles 2e and
2i-k. Bromoacetonitrile (0.21 mL, 3 mmol; chloroacetonitrile for
2j or 3-bromopropionitrile for 2k) was added dropwise to a solution
of the suitable aniline 1e or 1i,j (1.5 mmol) in dry DMF (3 mL),
and the resulting mixture was heated at 100 °C for 3 h (8 h for 2e
and 2j). The mixture was poured into water and extracted with
ethyl acetate. The combined organic phases were washed with brine,
dried (Na2SO4), and concentrated under reduced pressure to give a
(q, 2H, J ) 6.2), 3.76 (s, 3H), 3.89 (t, 2H, J ) 6.2), 5.77 (br s,
1H), 6.50–6.63 (m, 3H), 6.99–7.35 (m, 6H). IR (cm-1, Nujol): 3298,
1650. Anal. (C17H20N2O2) C, H, N.
N-{2-[(3-Bromophenyl)phenylamino]ethyl}acetamide (3h). Pu-
rification by flash chromatography (silica gel; CH2Cl2/acetone 95:5
as eluent), mp 73–74 °C, 43% yield. MS (EI): m/z 332–334 (M+),
77 (100). 1H NMR (CDCl3): δ 1.94 (s, 3H), 3.48 (q, 2H, J ) 6.2),

6624 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
RiVara et al.
3.87 (t, 2H, J ) 6.2), 5.65 (br s, 1H), 6.81–6.89 (m, 1H), 6.97–7.01
(m, 1H), 7.05–7.14 (m, 5H), 7.32–7.41 (m, 2H). IR (cm-1, Nujol):
3292, 1638. Anal. (C16H17BrN2O) C, H, N.
used without any further purification. Acetic anhydride (0.08 mL,
0.85 mmol) was added to a solution of the amine in THF (3 mL)
and TEA (0.12 mL, 0.87 mmol), and the mixture was stirred at
room temperature for 16 h. The solvent was evaporated in vacuo,
and the residue was dissolved in EtOAc. The solution was washed
with 2 N NaOH and then with brine and dried (Na2SO4). After
distillation of the solvent, a crude residue was obtained that was
purified by flash chromatography (silica gel; EtOAc as eluent). Oil,
N-{2-[(3-Methoxyphenyl)methylamino]ethyl}acetamide (3i).
White solid, 49% yield, mp 70–71 °C (diethyl ether/petroleum
1
ether). MS (EI): m/z 222 (M+), 150 (100). H NMR (CDCl3): δ
1.97 (s, 3H), 3.01 (s, 3H), 3.45 (m, 4H), 3.81 (s, 3H), 5.73 (br s,
1H), 6.30–6.41 (m, 3H), 7.11–7.27 (m, 1H). IR (cm-1, Nujol): 3243,
1654. Anal. (C12H18N2O2) C, H, N.
1
20% yield. MS (EI): m/z 298 (M+), 91 (100). H NMR (CDCl3):
δ 1.84 (s, 3H), 3.52 (m, 4H), 3.78 (s, 3H), 4.57 (s, 2H), 5.63 (br t,
1H), 6.29–6.42 (m, 3H), 7.11–7.38 (m, 6H). IR (cm-1, neat): 3282,
1655. Anal. (C18H22N2O2) C, H, N.
N-{3-[(3-Methoxyphenyl)methylamino]propyl}acetamide (3k).
Oil, 43% yield. MS (EI): m/z 236 (M+), 150 (100). 1H NMR
(CDCl3): δ 1.80 (m, 2H), 1.95 (s, 3H), 2.91 (s, 3H), 3.24–3.40 (m,
4H), 3.80 (s, 3H), 5.62 (br s, 1H), 6.24–6.37 (m, 3H), 7.15 (t, 1H).
IR (cm-1, neat): 3285, 1655. Anal. (C13H20N2O2) C, H, N.
N-{2-[(3-Methoxyphenyl)phenylamino]ethyl}butanamide (3l).
Purification by flash chromatography (silica gel; EtOAc/cyclohexane
1:1 as eluent). White solid, 80% yield, mp 50–2 °C (petroleum
N-Methyl-N-{2-[(3-methoxyphenyl)-phenylamino]ethyl}acet-
amide (4). A solution of 3g (0.284 g, 1 mmol) in dry DMF (2.5
mL) was added dropwise to an ice-cooled stirred suspension of
sodium hydride (0.075 g of an 80% dispersion in mineral oil, 2.5
mmol) in dry DMF (2.5 mL) under a N2 atmosphere. The mixture
was stirred at 0 °C for 40 min, then iodomethane (0.075 mL, 1.2
mmol) was added, and the resulting mixture was stirred at room
temperature for 16 h. The reaction mixture was poured into ice/
water and then extracted with EtOAc. The combined organic phases
were washed with brine, dried (Na2SO4), and concentrated under
reduced pressure to give a crude residue that was purified by flash
chromatography (silica gel; cyclohexane/EtOAc 9:1 as eluent); oil,
1
ether). MS (EI): m/z 312 (M+), 212 (100). H NMR (CDCl3): δ
0.92 (t, 3H, J ) 7.3), 1.52–1.71 (m, 2H), 2.10 (t, 2H, J ) 7.2),
3.52 (q, 2H, J ) 6.2), 3.76 (s, 3H), 3.89 (t, 2H, J ) 6.2), 5.71 (br
s, 1H), 6.55–6.61 (m, 3H), 7.02–7.31 (m, 6H). IR (cm-1, Nujol):
3277, 1638. Anal. (C19H24N2O2) C, H, N.
N-[2-(3-Methoxyphenoxy)ethyl]acetamide (5). Oil, 51% yield.
1
MS (EI): m/z 209 (M+), 86 (100). H NMR (CDCl3): δ 2.01 (s,
1
3H), 3.65 (q, 2H, J ) 5.4), 3.79 (s, 3H), 4.02 (t, 2H, J ) 5.4), 6.08
(br s, 1H), 6.45–6.56 (m, 3H), 7.20 (t, 1H). IR (cm-1, neat): 3291,
1653. Anal. (C11H15NO3) C, H, N.
72% yield. MS (EI): m/z 298 (M+), 212 (100). H NMR (CDCl3,
registered at 55 °C): δ 2.04 (s, 3H), 2.96 (s, 3H), 3.61 (m, 2H),
3.77 (s, 3H), 3.93 (m, 2H), 6.49–6.64 (m, 3H), 6.99–7.33 (m, 6H).
IR (cm-1, neat): 3449, 1646. Anal. (C18H22N2O2) C, H, N.
Pharmacology. Binding affinities of compounds 3a-m, 4, and
5 were determined using 2-[125I]iodomelatonin as the labeled ligand
in competition experiments on cloned human MT1 and MT2
receptors expressed in NIH3T3 rat fibroblast cells. The character-
ization of NIH3T3-MT1 and -MT2 cells was already described in
detail.58,59 Membranes were incubated for 90 min at 37 °C in
binding buffer (Tris/HCl 50 mM, pH 7.4). The final membrane
concentration was 5–10 µg of protein per tube. The membrane
protein level was determined in accordance with a previously
reported method.60 2-[125I]Iodomelatonin (100 pM) and different
concentrations of the new compounds were incubated with the
receptor preparation for 90 min at 37 °C. Nonspecific binding was
assessed with 10 µM MLT; IC50 values were determined by
nonlinear fitting strategies with the program PRISM (GraphPad
SoftWare Inc., San Diego, CA). The pKi values were calculated
from the IC50 values in accordance with the Cheng-Prusoff
equation.61 The pKi values are the mean of at least three independent
determinations performed in duplicate.
N-[2-(3-Methoxyphenylamino)ethyl]acetamide (3j). A solution
of the nitrile 2j (0.19 g, 1.1 mmol) in THF (7 mL) and 1 M NH3
in EtOH (1.5 mL) was hydrogenated over Raney nickel at 4 atm
of H2 for 6 h at 60 °C. The catalyst was filtered on Celite, the
filtrate was concentrated in vacuo, and the residue was partitioned
between EtOAc and water. The organic phase was washed with
brine, dried (Na2SO4), and evaporated under reduced pressure to
give a crude oily amine, which was used without any further
purification.
TEA (0.14 mL, 1 mmol) and acetic anhydride (0.095 mL, 1
mmol) were added to a cold solution of the above crude amine in
THF (4 mL) and the resulting mixture was stirred at room
temperature for 1 h. The solvent was evaporated under reduced
pressure, and the residue was taken up in EtOAc and washed with
a saturated aqueous solution of NaHCO3, followed by brine. After
drying over Na2SO4, the solvent was removed by distillation in
vacuo to give a crude product that was purified by flash chroma-
tography (silica gel; EtOAc as eluent). Oil, 51% yield. MS (EI):
1
m/z 208 (M+), 136 (100). H NMR (CDCl3): δ 2.00 (s, 3H), 3.27
(t, 2H, J ) 6.2), 3.50 (m, 2H), 3.78 (s, 3H), 5.84 (br s, 1H),
6.17–6.32 (m, 3H), 7.09 (t, 1H). IR (cm-1, neat): 3402, 3314, 1655.
Anal. (C11H16N2O2) C, H, N.
To define the functional activity of the new compounds at MT1
and MT2 receptor subtypes, [35S]GTPγS binding assays in NIH3T3
cells expressing human-cloned MT1 or MT2 receptors were
performed. The amount of bound [35S]GTPγS is proportional to
the level of the analogue-induced G-protein activation and is related
to the intrinsic activity of the compound under study. The detailed
description and validation of this method were reported elsewhere.58,62
Membranes (15–25 µg of protein, final incubation volume 100 µL)
were incubated at 30 °C for 30 min in the presence and in the
absence of MLT analogues in an assay buffer consisting of
[35S]GTPγS (0.3–0.5 nM), GDP (50 µM), NaCl (100 mM), and
MgCl2 (3 mM). Nonspecific binding was defined using [35S]GTPγS
(10 µM). In cell lines expressing human MT1 or MT2 receptors,
MLT produced a concentration-dependent stimulation of basal
[35S]GTPγS binding with a maximal stimulation, above basal levels,
of 370% and 250% in MT1 and MT2 receptors, respectively. Basal
stimulation is the amount of [35S]GTPγS specifically bound in the
absence of compounds, and it was taken as 100%. The maximal
G-protein activation was measured in each experiment by using
MLT (100 nM). Compounds were added at three different
concentrations (one concentration was equivalent to 100 nM MLT,
a second one was 10 times smaller, and a third one was 10 times
larger), and the percent stimulation above basal was determined.
The equivalent concentration was estimated on the basis of the ratio
of the affinity of the test compound over that of MLT. It was
N-{2-[(3-Methoxyphenyl)-phenylamino]ethyl}cyclobutancar-
boxamide (3m). This compound was prepared according to the
above-described procedure for 3j, starting from the nitrile 2g and
using cyclobutanecarbonyl chloride (1.0 mmol) instead of acetic
anhydride to acylate the intermediate crude amine. Purification by
flash chromatography (silica gel; cyclohexane/EtOAc 9:1 as eluent)
and crystallization. White solid, 56% overall yield, mp 64–5 °C
(diethyl ether/petroleum ether). MS (EI): m/z 324 (M+), 55 (100).
1H NMR (CDCl3): δ 1.82–2.26 (m, 6H), 2.84–2.97 (m, 1H), 3.52
(q, 2H, J ) 6.2), 3.76 (s, 3H), 3.90 (t, 2H, J ) 6.2), 5.63 (br s,
1H), 6.50–6.67 (m, 3H), 7.03–7.35 (m, 6H). IR (cm-1, Nujol): 3292,
1642. Anal. (C20H24N2O2) C, H, N.
N-{2-[(3-Methoxyphenyl)-benzylamino]ethyl}acetamide (3e).
A solution of the nitrile 2e (0.15 g, 0.6 mmol) in dry THF (3 mL)
was added dropwise to an ice-cooled suspension of LiAlH4 (0.04
g, 1 mmol) in dry THF (3 mL), and the resulting mixture was stirred
at room temperature for 5 h. The reaction mixture was cooled to 0
°C and water was added dropwise to destroy the excess of hydride.
The resulting mixture was filtered on Celite, and the filtrate was
concentrated in vacuo and partitioned between EtOAc and water.
The combined organic phases were washed with brine, dried
(Na2SO4), and evaporated to afford the crude amine which was

N-(Substituted-anilinoethyl)amides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6625
P.; Seely, D.; Guyatt, G. Melatonin in the treatment of cancer: A
systematic review of randomized controlled trials and meta-analysis.
J. Pineal Res. 2005, 39, 360–366.
assumed that at the equivalent concentration the test compound
occupies the same number of receptors as 100 nM MLT. All of
the measurements were performed in triplicate. The relative intrinsic
activity (IAr) values were obtained by dividing the maximum ligand-
induced stimulation of [35S]GTPγS binding by that of MLT, as
measured in the same experiment. By convention, the natural ligand
MLT has an efficacy (Emax) of 100%. Full agonists stimulate
[35S]GTPγS binding with a maximum efficacy close to that of MLT
itself. If Emax is between 30% and 70% that of MLT (0.3 < IAr <
0.7), the compound is considered a partial agonist, whereas if Emax
is lower than 30% (IAr < 0.3), the compound is considered an
antagonist.30
(8) Srinivasan, V.; Pandi-Perumal, S.; Cardinali, D.; Poeggeler, B.;
Hardeland, R. Melatonin in Alzheimer’s disease and other neurode-
generative disorders. BehaV. Brain Funct. 2006, 2, 15.
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Docking and Molecular Dynamics (MD) Simulations. Dock-
ing studies were performed with Glide 3.5,44 employing our
previously developed MT2 receptor model.36 Ligand geometry
was optimized using the MMFF94s force field,63 as implemented
in Macromodel.64 Docking experiments were performed starting
from minimum energy conformations of the ligands placed in
arbitrary position within a region centered on amino acids
His208, Trp264, and Tyr183, using enclosing and bounding
boxes of 46 and 14 Å on each side, respectively. Van der Waals
radii of the protein atoms were not scaled, while Van der Waals
radii of the ligand atoms with partial atomic charges lower than
|0.15| were scaled by 0.8. Standard precision mode was applied,
with the amide bond of the ligand maintained fixed in anti
disposition. Poses with a Coulomb-Van der Waals score greater
than 0 kcal/mol were rejected, while the other docking solutions
were ranked according to their Emodel value.
The best scoring solution of 3d was merged into the MT2 receptor
model, and the complex was submitted to geometry optimization
with MMFF94s force field, to an energy gradient of 0.01 kJ/
(mol·Å), and with fixed protein backbone. A MD simulation was
performed on the complex, with MMFF94 force field,65 1 fs time
step, for 1 ns after 100 ps of equilibration, with a fixed protein
backbone. The last step of MD was energy minimized, and it is
depicted in Figure 3 (right panel).
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Acknowledgment. This work was supported by the Italian
Mi.U.R. (Ministero dell’Università e della Ricerca). The CIM
(Centro Interdipartimentale Misure) and S.I.T.I. (Settore In-
novazione Tecnologie Informatiche) of the University of Parma
are gratefully acknowledged for providing the Sybyl software
license.
Supporting Information Available: Elemental analysis for
compounds 3a-m, 4, and 5. This material is available free of charge
via the Internet at http://pubs.acs.org.
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