Design and synthesis of N-(3,3-diphenylpropenyl)alkanamides as a novel class of high-affinity MT2-selective melatonin receptor ligands

Article abstract of DOI:10.1021/jm060850a

A novel series of melatonin receptor ligands was discovered by opening the cyclic scaffolds of known classes of high affinity melatonin receptor antagonists, while retaining the pharmacophore elements postulated by previously described 3D-QSAR and recepto

Full text of DOI:10.1021/jm060850a

J. Med. Chem. 2006, 49, 7393-7403
7393
Design and Synthesis of N-(3,3-Diphenylpropenyl)alkanamides as a Novel Class of High-Affinity
MT2-Selective Melatonin Receptor Ligands
Annalida Bedini,† Gilberto Spadoni,*,† Giuseppe Gatti,† Simone Lucarini,† Giorgio Tarzia,† Silvia Rivara,‡ Simone Lorenzi,‡
Alessio Lodola,‡ Marco Mor,‡ Valeria Lucini,§ Marilou Pannacci,§ and Francesco Scaglione§
Istituto di Chimica Farmaceutica e Tossicologica, UniVersita` degli Studi di Urbino “Carlo Bo”, Piazza Rinascimento 6, 61029 Urbino, Italy,
Dipartimento Farmaceutico, UniVersita` degli Studi di Parma, V.le G. P. Usberti 27/A Campus UniVersitario, 43100 Parma, Italy, and
Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, UniVersita` degli Studi di Milano, Via VanVitelli 32, 20129 Milano, Italy
ReceiVed July 20, 2006
A novel series of melatonin receptor ligands was discovered by opening the cyclic scaffolds of known
classes of high affinity melatonin receptor antagonists, while retaining the pharmacophore elements postulated
by previously described 3D-QSAR and receptor models. Compounds belonging to the classes of 2,3- and
[3,3-diphenylprop(en)yl]alkanamides and of o- or [(m-benzyl)phenyl]ethyl-alkanamides were synthesized
and tested on MT1 and MT2 receptors. The class of 3,3-diphenyl-propenyl-alkanamides was the most
interesting one, with compounds having MT2 receptor affinity similar to that of MLT, remarkable MT2
selectivity, and partial agonist or antagonist behavior. In particular, the (E)-m-methoxy cyclobutanecar-
boxamido derivative 18f and the di-(m-methoxy) acetamido one, 18g, have sub-nM affinity for the MT2
subtype, with more than 100-fold selectivity over MT1, 18f being an antagonist and 18g a partial agonist on
GTPγS test. Docking of 18g into a previously developed MT2 receptor model showed a binding scheme
consistent with that of other antagonists. The MT2 expected binding affinities of the new compounds were
calculated by a previously developed 3D-QSAR CoMFA model, giving satisfactory predictions.
Introduction
both centrally (suprachiasmatic nucleus, cortex, pars tuberalis,
etc.) and peripherally (kidney, adipocytes, retina, blood vessels,
etc.),18 the physiological roles of these receptors are not as yet
well defined. There is evidence that MT1 receptors might be
implicated in the sleep promoting effects of MLT19,20 and in
mediating vasoconstriction,21 whereas MT2 receptors appear to
play a major role in the resynchronizing activity of MLT19,22
and in mediating vasodilation. Moreover, an antidepressive
effect has been reported for the antagonist luzindole in a mice
model, being ascribed to its selective action at the MT2
receptor.23 However, an accurate characterization of MLT
receptors-mediated functions in native tissues can only be made
by using subtype-selective ligands. Therefore, a substantial share
of current drug discovery efforts in the melatonin area is being
directed toward the development of subtype-selective MLT
receptor agonists and antagonists, which will be valuable tools
in understanding the role of this enigmatic hormone in health
and disease.
Melatonin (N-acetyl-5-methoxytryptamine, MLT, 1) is a
neurohormone primarily secreted by the pineal gland at night
in all species.1 The circadian pattern of MLT secretion, coupled
with the localization of specific MLT binding sites in the brain
region associated with the “biological clock”, suggests that MLT
may play an important role in modulation of the sleep-wake
cycle and of circadian rhythms in humans.2 Some MLT receptor
agonists, with improved properties in comparison to MLT, are
now in clinical trials for treatment of insomnia or circadian
rhythm sleep disorders,3-5 and ramelteon (TAK-375) has been
recently approved for insomnia.6 Other effects of MLT described
in the literature include its anti-inflammatory,7 pain modulatory,8
retinal,9vascular,10 antitumor,11 and antioxidant12 properties. A
remarkable efficacy of MLT in animal models of focal cerebral
ischemia had also been reported, suggesting the hormone as a
candidate neuroprotective drug for human stroke.13 However,
the functions of MLT in mammals are still a matter of
investigation, and more rigorous clinical studies are needed to
demonstrate the potential benefits of MLT assumption and to
rule out the possibility of toxic effects.
Most physiological MLT effects result from the activation
of high affinity G protein-coupled receptors. MT1 and MT2
receptors14-16 have been found in mammals, including humans,
and subsequently cloned. A third subtype (Mel1c), first cloned
from Xenopous laeVis, has been found only in nonmammalian
species.14 In addition to these high-affinity MLT receptors (Ki
= 0.1 nM), another low-affinity MLT binding site, termed MT3
(Ki = 60 nM), has recently been characterized as a melatonin-
sensitive form of the human enzyme quinone reductase 2.17
Whereas it is known that MT1 and MT2 receptors are expressed
In the course of our studies, various modifications of the MLT
structure had been examined to determine which structural
features are required for receptor affinity, intrinsic activity, and/
or subtype selectivity at MLT receptors.24,25 However, marked
subtype selectivity is still a challenge, and only recently this
field has registered some important advances, leading to the
identification of a small number of selective compounds.26 While
27
28
only a few examples of MT1 and MT3 ligands have been
reported, the majority of subtype-selective compounds behave
as MT2 receptor antagonists. These compounds belong to
different structural classes (Figure 1) and display various degrees
of binding affinity and selectivity. A consistent structural motif
found in most of these MLT ligands is the presence of a
lipophilic substituent, which can be located out-of-the-plane of
their core nucleus (i.e., the indole ring in luzindole or the tetralin
scaffold in 4P-PDOT) in a position corresponding to positions
1 and 2 of the indole in MLT, and we had hypothesized that
this arrangement confers selectivity for the MT2 receptor and
* To whom correspondence should be addressed. Tel.: ++39 0722
303323. Fax: ++39 0722 303313. E-mail: gilberto@uniurb.it.
† Universita` degli Studi di Urbino “Carlo Bo”.
‡ Universita` degli Studi di Parma.
§ Universita` degli Studi di Milano.
10.1021/jm060850a CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/15/2006

7394 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Bedini et al.
Figure 1. Chemical structures of MLT and representative MT2-selective antagonists.
Figure 2. Application of the open-chain analog approach leading to the newly synthesized compounds.
leads to a reduction of intrinsic activity.29,30 This hypothesis
had been statistically validated by a three-dimensional quantita-
tive structure-activity relationship (3D-QSAR) analysis on a
series of structurally different MT1 and MT2 receptor ligands,31
revealing a correlation between the occupation of the out-of-
plane region and both MT2 selectivity and antagonist behavior.
Moreover, three-dimensional homology models of the MT1 and
MT2 receptors allowed the rationalization, at the receptor level,
of the structure-activity relationships (SARs) found for several
ligands, providing a common interaction pattern and a possible
explanation for their MT2 selectivity and low intrinsic activity.32
This information had been applied to the identification of a novel
class of MT2 antagonists, derived from 10,11-dihydrodibenzo-
cycloheptene,33 whose MT2 selectivity was attributed to the
skewed shape of the tricyclic scaffold, fulfilling the out-of-plane
requirement previously defined. The dihydrodibenzocyclohep-
tene derivative 7 (Figure 1) is endowed with one of the highest
binding affinities reported to date, comparable to that of the
tetralin derivative 4P-PDOT (3).34
As a further step in the process of exploiting SAR information
accumulated on MT2-selective antagonism, we applied an “open-
chain analog” approach35 to this tricyclic scaffold and to the
traditional tetralin scaffold of 4P-PDOT, looking for more
flexible structures retaining the requirements described above.
In the present paper, we report the design, synthesis, receptor
binding characterization, and SAR analysis of novel classes of
MLT receptor ligands of the N-[diphenylprop(en)yl]alkanamido
type and benzyl-phenylethyl-alkanamides, whose structures are
shown in Figure 2. These compounds were designed by
structural simplification of the potent and selective cyclic
antagonists 3 and 7, and their structures appeared promising
for MT2 antagonism when superposed to the pharmacophore
and 3D-QSAR-based models or docked into the MT2 receptor
binding site model.

MT2-SelectiVe Melatonin Receptor Ligands
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7395
Scheme 1a
trile43 with iodobenzene or of trans-cinnamonitrile with 3-io-
doanisole, respectively (Scheme 3). The relative stereochemistry
of the double bond was assigned through 1H NMR NOE
experiments. Whereas selective irradiation of the vinyl proton
of compound 17 enhances H-2′ and H-6′ signals, the same
irradiation in the case of compound 16 enhances ortho proton
signals of the unsubstituted phenyl ring, showing unambiguously
the (E)- and (Z)-configuration, respectively (see Experimental
Section). Hydrogenation of the unsaturated nitriles 16 and 17
in the presence of Raney nickel and of the suitable anhydride
gave the target N-(3,3-diphenylpropyl)alkanamides 19b-d
(Scheme 3).
Results and Discussion
a Reagents and conditions: (a) H2, Raney-Ni, 4 atm, Ac2O or (EtCO)2O,
The tricyclic antagonist 7 and the tetralin derivative 3 were
the starting structures used to generate novel ligands, according
to the “open-chain analog” approach represented in Figure 2.
Deletion of the lower methylene group in the tricycle of 7 gave
the open structure 9, while removal of the upper one afforded
that of 13, which can also be derived from the cyclic structure
of 3 by deleting the lower methylene. On the other hand,
structure 19 results from the deletion of the upper methylene
from 3. In our study, we also considered compound 11, which
could be superposed to the pharmacophore elements for MT2
antagonism despite its different topology. Finally, the effect of
a conformational constraint by a double bond (structures 10 and
18) and of some groups typical of MLT receptor ligands was
investigated for the most promising structures. We thus syn-
thesized (2,3-diphenylpropyl)alkanamido (9a-c) and (3,3-
diphenylpropyl)alkanamido derivatives (19a-d), the corre-
sponding unsaturated derivatives 10a,b and 18a-g, and the two
phenylethyl alkanamido derivatives carrying a benzyl substituent
in different positions (11 and 13).
The spatial requirements for selective MT2 receptor binding,
as defined by our previous molecular modeling studies,31,32 were
checked for the new MLT receptor ligands here described. All
the selected compounds were endowed with the three pharma-
cophore features previously identified, two aromatic rings and
an amide function, connected through an acyclic scaffold that
fulfilled the antagonist pharmacophore model and could be
docked into the three-dimensional MT2 receptor model in at
least one accessible conformation.
MT1 and MT2 binding affinity for compounds 9-11, 13, 18,
and 19 and their effect on GTPγS binding (intrinsic activity)
are reported in Table 1. Chiral compounds 9 and 19b-d were
tested as racemic mixtures. The group of [2,3-diphenylprop-
(en)yl]alkanamides (9a-c and 10a,b) showed an antagonist/
weak inverse agonist behavior, but they failed to demonstrate
high affinity for MLT receptors (pKi 5.44-6.83). The presence
of the methoxy substituent did not improve the binding affinity
in the saturated series, and its effect was therefore not further
evaluated in the unsaturated one. In contrast, the isomeric [3,3-
diphenylprop(en)yl]alkanamides (18 and 19), resembling the
carbon framework of 4P-PDOT (3), had good to excellent
affinity for the MT2 receptor, with lower affinity for the MT1
receptor. Within this series, the role of a methoxy group and
changes in the N-acyl side chain were considered to investigate
structure-affinity and structure-intrinsic activity relationships for
MT1 and MT2 receptors. The effect of different geometries of
the acylamino side chain was also evaluated by testing the (E)-
and (Z)-propenyl derivatives (18c-f) and the dihydro analogues
(19a-d). The binding affinities of compounds with these
scaffolds showed that derivatives with a methoxy group (18c-f
vs 18a,b and 19b-d vs 19a) placed in a position that can be
THF, 60-80 °C; (b) n-BuLi, benzyltriphenylphosphonium chloride, Et2O.
Scheme 2a
a Reagents and conditions: (a) AlCl3, benzyl chloride, DCE; (b) H2,
Raney-Ni, 4 atm, Ac2O, THF, 60 °C.
Chemistry
N-(3,3-Diphenylpropyl)acetamide (19a)36 was prepared by
N-acetylation of the commercially available 3,3-diphenylpro-
panamine with Ac2O/NaOAc in AcOH. Synthetic routes to the
other target compounds are described in Schemes 1-3. Briefly,
melatonin receptor ligands 9a-c and 11 were prepared by
hydrogenation over Raney nickel of suitable nitriles (com-
mercially available R-cinnamonitrile or its 3-methoxy analogue37
for 9a-c and 3-benzoyl-phenylacetonitrile38 for 11) and con-
comitant N-acylation with acetic or propionic anhydride (Scheme
1). N-(2-Oxo-2-phenylethyl)acetamide39 was subjected to Wittig
reaction conditions40 using benzyltriphenylphosphonium chloride
in the presence of n-BuLi to give an E/Z mixture of N-(2,3-
diphenylpropenyl)acetamide (10a,b) from which each diaste-
reoisomer was separated by flash chromatography (Scheme 1).
Friedel-Crafts benzylation (benzyl chloride/AlCl3) of 3-meth-
oxyphenylacetonitrile gave a complex mixture from which
2-benzyl-5-methoxy-phenylacetonitrile (12) could be separated
in low yield (6%) by flash chromatography. The structure of
the intermediate 12 was confirmed by two NOE experiments
(see Experimental Section). The nitrile 12 was then converted
to the target compound 13 by hydrogenation over Raney nickel
and concomitant N-acetylation with acetic anhydride (Scheme
2).
The N-(3,3-diphenylpropenyl)alkanamides 18a-g were ob-
tained by reduction of the suitable nitriles 14-17 with LiAlH4/
AlCl3, followed by N-acylation of the crude intermediate amine
with acetic anhydride or cyclobutanecarbonyl chloride; (E)- and
1
(Z)-configurations were determined by H NMR NOE experi-
ments. Partial isomerization of the double bond was observed
during the reduction step; the best diastereoselective reduction
(retention of configuration >90%, determined by 1H NMR) was
achieved by using an 1:2 LiAlH4/AlCl3 molar ratio. 3,3-
Diphenylacrylonitrile (14)41 and 3,3-bis(3-methoxyphenyl)-
acrylonitrile (15)42 were prepared by submitting the appropriate
benzophenone to Horner-Emmons reaction with diethyl (cya-
nomethyl)phosphonate, as previously described. The key (Z)-
16 and (E)-17 nitriles were synthesized by diastereoselective
Pd-catalyzed Heck reaction of (E)-3-(3-methoxyphenyl)acriloni-

7396 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Bedini et al.
Scheme 3a
a Reagents and conditions: (a) Pd(OAc)2, AcO-K+, (n-Bu)4N+Br-, DMF, 80 °C; (b) AlCl3, LiAlH4, dry Et2O, room temperature, 1 h; (c) Ac2O or
cyclobutanecarbonyl chloride, TEA, THF, room temperature; (d) H2, Raney-Ni, 4 atm, (R1CO)2O, THF, 60 °C. bNitriles 1441 and 1542 were prepared as
c
previously described. 19a36 was prepared by N-acetylation of the commercially available 3,3-diphenylpropanamine.
Table 1. Experimental Binding Affinity and Intrinsic Activity (IAr) of Newly Synthesized Compounds for Human MT1 and MT2 Melatonin Receptors
Stably Expressed in NIH3T3 Cells and Calculated MT2 pKi Values
human MT1
IAr ( SEMb
human MT2
cmpd
R
R1
R2
pKia
pKia
IAr ( SEMb
pKi calcdc
MLT
9a
9b
9.49
6.15
6.16
6.28
5.92
5.44
6.41
7.16
6.29
6.04
6.64
8.57
6.59
7.03
7.23
5.38
5.66
6.44
6.26
1.00 ( 0.01
-0.30 ( 0.04
-0.23 ( 0.08
-0.13 ( 0.03
0.11 ( 0.02
-0.23 ( 0.01
0.24 ( 0.01
0.24 ( 0.04
-0.03 ( 0.03
-0.18 ( 0.06
0.45 ( 0.05
0.54 ( 0.06
-0.04 ( 0.01
0.29 ( 0.04
0.20 ( 0.02
-0.19 ( 0.02
0.24 ( 0.05
0.31 ( 0.05
0.21 ( 0.05
9.59
6.38
6.69
6.72
6.83
6.28
6.18
8.22
7.94
7.88
8.60
9.66
8.81
9.28
9.56
6.85
8.27
8.30
8.58
1.01 ( 0.02
-0.43 ( 0.2
-0.18 ( 0.04
-0.25 ( 0.2
0.18 ( 0.01
-0.15 ( 0.03
0.48 ( 0.04
0.06 ( 0.02
0.19 ( 0.14
-0.20 ( 0.15
0.30 ( 0.18
0.38 ( 0.06
-0.02 ( 0.01
0.13 ( 0.01
0.34 ( 0.02
-0.23 ( 0.13
0.04 ( 0.12
0.28 ( 0.02
0.27 ( 0.01
H
OMe
OMe
Me
Me
Et
7.49
7.49
7.90
6.81
7.39
8.12
8.68
7.81
8.06
7.94
8.45
7.76
8.68
8.60
7.59
8.70
8.91
8.69
9c
10a-(Z)
10b-(E)
11
13
18a
H
H
Me
c-Bu
Me
H
H
H
H
H
H
OMe
H
H
18b
18c-(Z)
18d-(E)
18e-(Z)
18f-(E)
18g
19a
19b
19c
19d
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
Me
c-Bu
c-Bu
Me
Me
Me
Et
n-Pr
H
H
a pKi values were calculated from IC50 values, obtained from competition curves by the method of Cheng and Prusoff56 and are the mean of at least three
determinations performed in duplicate. SEM of pKi values were lower than 0.06. b The relative intrinsic activity values were obtained by dividing the
maximum analogue-induced G-protein activation by that of MLT. c MT2 pKi values, calculated with the CoMFA model described in the text (training set
of 34 compounds), for the new compounds as an external test set.
easily superposed to position 5 of the indole ring of MLT,
usually have the highest binding affinity. The methoxy group
led to a higher increase in MT2 than in MT1 binding affinity
(18c vs 18a and 19b vs 19a), causing a MT2/MT1 selectivity
ratio of more than two log units for compounds 18f, 18g, 19b,
and 19d. The presence of a 2,3 double bond in compounds 18
increased the binding affinity at both receptor subtypes com-
pared to that of the corresponding saturated compounds 19.
Analysis of the two diastereoisomers (Z)-18c and (E)-18d (or
(Z)-18e and (E)-18f) revealed that the (E)-isomer has higher
binding affinity at both receptor subtypes, suggesting that the
relative spatial orientation of the phenyl group and the acylamino
side chain affects optimal receptor binding, even if this
difference is less evident, at the MT2 receptor, for the cyclobu-
tanamides 18e and 18f. Structural variations at the N-acylamino
group were found to be less important than in other series of

MT2-SelectiVe Melatonin Receptor Ligands
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7397
affinity and MT2 selectivity, the meta-benzyl derivative 11
showed little affinity for both MT1 and MT2 receptor subtypes.
Even if the reduced binding affinity of 11 could be, at least in
part, explained by the lack of the methoxy substituent, these
two compounds were considered less promising than 18 and
19.
The binding mode for the potent and selective representative
of the (3,3-diphenylpropenyl)alkanamido series, 18g, was in-
ferred by an automated docking procedure, implemented in the
program Glide,50 within our previously developed MT2 receptor
model,32 and by evaluating the stability of receptor-ligand
complexes by molecular dynamics (MD) simulations. The
docking solution giving the best Emodel score was employed
to build the complex, which proved to be stable during 1 ns of
MD simulation (Figure 4, left). Compound 18g occupied the
same region of space as previously docked antagonists and
undertook similar interactions with the putative MT2 binding
cavity. The amide oxygen is engaged in a hydrogen bond with
Tyr183, belonging to the extracellular loop 2, and one phenyl
ring is involved in a T-shaped interaction with His208 in TM5,
while the other one is inserted into the lipophilic pocket close
to Trp264 of the CWXP motif in TM6. One of the two
symmetrical methoxy groups reinforced the T-shape interaction,
occupying a region of space as the 5-methoxy group of MLT,
while the second one could be accommodated within the
lipophilic pocket also receiving the out-of-plane substituents of
MT2-selective antagonists.32 In fact, this second methoxyl, even
if not giving any affinity improvement (see 18g vs 18d in Table
1), is tolerated similarly to what was observed for p-methyl and
p-methoxyluzindole,34 and for some benzofuran51 (e.g., 6) and
2-acylaminomethylindole derivatives30 (e.g., 4), compared to
their unsubstituted analogues. Contrary to expectations, the
methoxy substituent was preferentially accommodated within
the lipophilic pocket when the monomethoxy derivatives were
docked into the model. Actually, the precise role of the
5-methoxy group of MLT-like ligands is still partially unex-
plained by this model.
Figure 3. Schild plot for 18f in the [35S]GTPγS assay.
MLT analogues.44 Homologation of the methyl group of the
amido side chain to ethyl (19c) or propyl (19d) did not
considerably modify the binding affinity and efficacy for each
of the two receptor subtypes. Replacement of the methyl group
of the acetamido side chain by cyclobutyl in (Z)-18e or (E)-18f
retains a high MT2 binding affinity but causes a loss of intrinsic
activity, a finding that parallels previous results about the role
of a cycloalkyl substituent on the acylamino side chain.45,46
N-[3,3-Bis(3-methoxyphenyl)propenyl]acetamide (18g) retains
the same MT2 binding affinity of the monomethoxy derivative
(E)-18d, but it shows lower MT1 binding affinity (ca. 22-fold),
leading to an enhancement of the MT2/MT1 selectivity ratio from
10 to 214. All the studied compounds behaved as antagonists
or partial agonists. The presence of the methoxy group of the
double bond and the nature of the N-acyl substituent allowed
the modulation of MT2 selectivity and intrinsic activity.
Compound (E)-18f (MT2/MT1 ratio 178) is about 12-fold more
selective for MT2 than the lead 18d; it presents an affinity for
MT2 similar to that of melatonin itself and acts as an MT2
antagonist.
The ability of compound 18f to antagonize MLT-stimulated
[35S]GTPγS binding was further investigated by carrying out a
series of MLT concentration-response curves in the presence
of four different concentrations of compound 18f (1, 10, 100,
and 1000 nM). Compound 18f causes a dose-dependent parallel
rightward shift of the concentration-response curve for MLT
stimulation of [35S]GTPγS binding. As can be seen from the
Schild plot in Figure 3, the slope was not significantly different
from unity (0.90 ( 0.09, R2 ) 0.98), being consistent with a
competitive antagonism, with an X intercept corresponding to
a pA2 value of 9.41 and a pKB value, obtained by forcing the
intercept to 1, of 9.28, identical to the pKi value from binding
experiments.
As stated in the introduction, the design of the putative MT2-
selective antagonists of this study had been driven by pharma-
cophore and 3D-QSAR models, which showed a satisfactory
predictive ability across structurally different classes of ligands.25,33
Therefore, within this work, the binding affinity expected for
the newly synthesized compounds was calculated applying a
previously defined 3D-QSAR model31 based on thirty-four in-
house and literature antagonists. The former CoMFA model,
however, was revised to be fully consistent with our MT2
receptor model,32 where the out-of-plane group and the acy-
lamino chain had a different mutual orientation than in the
original alignment (see Experimental Section). The statistical
parameters of the new PLS analysis were very similar to those
of the previous model: Q2 ) 0.62, SDEP ) 0.52, R2 ) 0.87,
and s ) 0.33 for 34 compounds and 5 latent variables (previous
model: Q2 ) 0.59, SDEP ) 0.53, R2 ) 0.87, and s ) 0.34, 5
LVs), and the coefficient contour plots, applied to the new
alignment, carried the same information (Figure 4, right). The
predicted MT2 pKi values for the new compounds 9-11, 13,
18, and 19 are reported in Table 1. From a structural point of
view, the CoMFA training set greatly differs from our test set,
being mainly composed by indole-based antagonists. Despite
this structural difference, binding affinities were generally
adequately predicted, with the greatest over- or under-estimation
being around 10 times the experimental values. An important
exception was represented by compound 11, whose binding
affinity was greatly overestimated. This 2-phenylethyl-propi-
A series of propyl-alkanamido derivatives with only one
phenyl substituent linked to the propyl side chain had previously
been described.47,48 Unlike [3,3-diphenylprop(en)yl]alkanamides
18 and 19, which are MT2-selective, they showed no selectivity,
having about the same binding affinity at MT1 and MT2
receptors.47 Moreover, N-[3-(3-methoxyphenyl)propyl]acetamide
had been described as an agonist in Xenopus melanophore
assay,49 while the presence of the second phenyl ring in 18 and
19 greatly affected intrinsic activity.
As for the 2-phenylethyl-alkanamides 11 and 13, whereas
the ortho-benzyl derivative 13 retained significant binding

7398 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Bedini et al.
Figure 4. Left: energy-minimized conformation of the last step of 1 ns MD simulation for the complex between MT2 receptor model and 18g
(green carbons); the reference compound 4 (orange carbons), docked as described in ref 32, is represented for comparison. Right: superposition of
3 (orange carbons), 18c (purple carbons), and 18d (white carbons) in the new CoMFA alignment; regions of positive (>0.0045) and negative
(<-0.0045) standard deviation × coefficients for the model with five latent variables (see text) are illustrated in green and yellow, respectively.
In conclusion, the constrained cycloalkyl scaffolds of the
dihydrodibenzocycloheptene compound 7 and of the 4-phe-
nyltetralin 3 were successfully replaced by a conformationally
flexible di-phenyl-prop(en)yl side chain. This structural modi-
fication led to the discovery of a series of N-[3,3-diphenylprop-
(en)yl]alkanamides (18 and 19), having a structural motif
fulfilling the key features outlined by our previous 3D-QSAR
models (i.e., two aromatic rings and an amide side chain in a
suitable arrangement), as novel MT2-selective ligands. Modula-
tion of the selectivity and of the intrinsic activity can be obtained
by proper modification on the N-acyl, phenyl substituents, and
stereochemistry of the side chain. The most interesting results
are obtained with the (E)-propenyl derivatives 18d and 18f and
for the symmetrical propenyl derivative 18g. They are very good
MT2 ligands, with affinities as strong as that of MLT itself,
and the last two compounds show MT2/MT1 selectivity ratios
higher than one hundred. Compounds 18d and 18g behave as
MT1 and MT2 partial agonists, whereas the N-cyclobutyl
analogue 18f is an MT2 antagonist. This class represents a
successful example of molecular simplification based on a
pharmacophore hypothesis.
Figure 5. Experimental pKi values at the MT2 receptor vs those
predicted by the CoMFA model described in the text (training set of
34 compounds).
Experimental Section
onamido derivative has a unique structure within the newly
synthesized compounds; its high conformational freedom may
account for the poor binding affinity, despite its good superposi-
tion to the 3D-QSAR model and high predicted activity.
Excluding the outlier 11, the correlation between experimental
and predicted pKi values for the external test set gave R2 )
0.54 (Figure 5), acceptable if compared to the predictive ability
of the CoMFA model, estimated by leave-one-out cross-
validation (Q2 ) 0.62). The limited accuracy of quantitative
predictions can be attributed to some SAR information for the
present series that was not fully addressed by the old training
set. If a comprehensive model with all the 52 compounds
included is built, a significant improvement in Q2 was achieved
(Q2 ) 0.70, SDEP ) 0.53, R2 ) 0.90, and s ) 0.33 with 5
LVs), even if the shape of regions with negative and positive
coefficients was highly conserved (data not shown). More im-
portantly, some relevant SAR elements for the new compounds
were properly explained by the CoMFA model. Thus, among
the 3,3-diphenyl-propenyl-alkanamido derivatives 18, the (E)-
isomers were predicted more potent than the (Z)-isomers, and
the presence of the methoxy substituent led to a higher predicted
binding affinity in both the 18 and 19 series (Table 1).
General Methods. Melting points were determined on a Buchi
B-540 capillary melting point apparatus and are uncorrected. H
1
NMR spectra were recorded on a Bruker AVANCE 200 spectrom-
eter, using CDCl3 as solvent unless otherwise noted. 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). NOE spectra were measured using the Bruker pulse
program Selnogp, based on double pulsed field gradient spin echo
nuclear Overhauser experiment (DPFGSE),52 pre-irradiation time:
5 s. 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 FTIR
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. All chemicals were purchased from commercial suppliers
and used directly without any further purification. 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).

MT2-SelectiVe Melatonin Receptor Ligands
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7399
1
N-(2,3-Diphenylpropyl)acetamide (9a): A solution of R-phe-
nylcinnamonitrile (0.41 g, 2 mmol) and acetic anhydride (3 mL,
31 mmol) in THF (10 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 2 N NaOH. The organic layer was
washed twice with 2 N NaOH and once with brine, dried (Na2-
SO4), and evaporated under reduced pressure to give a residue that
was purified by flash chromatography (silica gel; EtOAc/cyclo-
hexane 1:1 as eluent) and crystallization (Et2O/petroleum ether);
(100). H NMR (CDCl3): δ 1.09 (t, 3H, J ) 7.5), 2.10 (q, 2H, J
) 7.5), 2.77 (t, 2H, J ) 6.7), 3.48 (q, 2H, J ) 6.7), 3.95 (s, 2H),
5.4 (br s, 1H), 7.02-7.32 (m, 9H). IR (cm-1, Nujol): 3302, 1639.
Anal. (C18H21NO) C, H, N.
(2-Benzyl-5-methoxyphenyl)acetonitrile (12): Benzyl chloride
(2 mL, 17.3 mmol) was added to an ice-cooled stirred solution of
3-methoxyphenylacetonitrile (1.47 g, 10 mmol) in dry DCE (20
mL). Aluminum trichloride (2.12 g, 16 mmol) was added portion-
wise (30 min), and the resulting mixture was stirred for 30 min at
0 °C and then at room temperature for 5 h. The reaction mixture
was poured into an ice-cooled 2 N HCl aqueous solution, filtered
on Celite, and the filtrate was extracted three times with EtOAc.
The combined organic phases were washed with brine, dried (Na2-
SO4), and evaporated under reduced pressure, to give a residue that
was purified by flash chromatography (silica gel; EtOAc/cyclo-
hexane 1:9 as eluent) to give a 6% yield of an oil. MS (EI): m/z
1
59% yield; mp ) 68 °C. MS (EI): m/z 253 (M+), 194 (100). H
NMR (CDCl3): δ 1.83 (s, 3H), 2.92 (m, 2H), 3.09 (m, 1H), 3.33
(m, 1H), 3.77 (m, 1H), 5.19 (br s, 1H), 7.03-7.35 (m, 10H). IR
(cm-1, Nujol): 3272, 1635. Anal. (C17H19NO) C, H, N.
N-[2-(3-Methoxyphenyl)-3-phenylpropyl]acetamide (9b): Com-
pound 9b was obtained following the procedure above-described
by using 2-(3-methoxyphenyl)-3-phenyl-acrylonitrile37 instead of
R-phenylcinnamonitrile. Purification by flash chromatography (silica
gel; EtOAc as eluent) gave a 27% yield of an oil. MS (EI): m/z
283 (M+), 224 (100). 1H NMR (CDCl3): δ 1.83 (s, 3H), 2.91 (m,
2H), 3.07 (m, 1H), 3.30 (m, 1H), 3.74 (m, 1H), 3.77 (s, 3H), 5.21
237, (M+), 165 (100). H NMR (CDCl3): δ 3.53 (s, 2H), 3.83 (s,
1
3H), 3.98 (s, 2H), 6.86 (dd, 1H, H-4′, J ) 2.7 and J ) 8.3), 6.99
(d, 1H, H-2′, J ) 2.7), 7.17 (d, 1H, H-5′, J ) 8.3), 7.09 (m, 2H,
phenylortho), 7.20-7.35 (m, 3H, phenyl). Selective irradiation of
signal due to CH2CN at δ ) 3.53 resulted in enhancement of signal
due to CH2Ph (1%) and of signal due to H-2′ (1.4%). Moreover,
irradiation of CH2Ph (at δ ) 3.98) resulted in enhancement of H-5′
(1.2%), CH2CN (1%), and Hortho of the unsubstituted phenyl ring
(2.4%). IR (cm-1, neat): 2241.
(br s, 1H), 6.67-6.79 (m, 3H), 7.04-7.22 (m, 6H). IR (cm-1
neat): 3292, 1651. Anal. (C18H21NO2) C, H, N.
,
N-[2-(3-Methoxyphenyl)-3-phenylpropyl]propanamide (9c):
Compound 9c was obtained following the above procedure
described for 9b, by using propionic anhydride instead of acetic
anhydride. Purification by flash chromatography (silica gel; EtOAc/
cyclohexane 1:1 as eluent) gave a 44% yield of an oil. MS (EI):
N-[2-(2-Benzyl-5-methoxyphenyl)ethyl]acetamide (13): A so-
lution of 12 (1 mmol) and acetic anhydride (1.5 mL, 15.5 mmol)
in THF (6 mL) was hydrogenated over Raney nickel at 4 atm of
H2 for 6 h at 60 °C. Standard workup gave a residue that was
purified by flash chromatography (silica gel; EtOAc as eluent) and
crystallization from diethyl ether to give a 44% yield; mp 84-85
°C. MS (EI): m/z 283 (M+), 209 (100). 1H NMR (CDCl3): δ 1.88
(s, 3H), 2.76 (m, 2H), 3.36 (m, 2H), 3.80 (s, 3H), 3.98 (s, 2H),
5.38 (br s, 1H), 6.76-7.31 (m, 8H). IR (cm-1, Nujol): 3306, 1647.
Anal. (C18H21NO2) C, H, N.
(Z)-3-(3-Methoxyphenyl)-3-phenylacrylonitrile (16): (E)-3-
Methoxyphenyl-acrylonitrile43 (1.37 g, 8.6 mmol) was added to a
mixture of iodobenzene (1.85 mL, 16.5 mmol), potassium acetate
(2.11 g, 21.5 mmol), tetrabutylammonium bromide (3.03 g, 9.4
mmol), and palladium acetate (0.095 g, 0.42 mmol) in dry DMF
(30 mL) under N2. After stirring at 80 °C for 3 days, the reaction
mixture was cooled to room temperature, poured into water, and
extracted three times with diethyl ether. The combined organic
phases were washed with brine, dried (Na2SO4), and evaporated
under reduced pressure to give a residue that was purified by flash
chromatography (silica gel; cyclohexane/EtOAc, 95:5 as eluent)
and crystallization from diethyl ether/petroleum ether to give a 79%
yield; mp 79 °C. MS (EI): m/z 235 (M+, 100). 1H NMR (acetone-
d6): δ 3.83 (s, 3H), 6.13 (s, 1H), 6.96 (ddd, 1H, H-6′, J ) 1.0, J
) 1.7, and J ) 7.6), 6.98 (dd, 1H, H-2′, J ) 1.0 and J ) 2.4), 7.09
(ddd, 1H, H-4′, J ) 1.0, J ) 2.4, and J ) 8.3), 7.35-7.55 (m, 6H,
H-5′ and unsubstituted phenyl). Selective irradiation of the vinylic
proton (δ ) 6.13) resulted in 3.0% enhancement of the signal at δ
) 7.4 due to the ortho protons of the unsubstituted phenyl ring. IR
(cm-1, Nujol): 2211, 1569.
1
m/z 297 (M+), 57 (100). H NMR (CDCl3): δ 1.03 (t, 3H, J )
7.3), 2.04 (q, 2H, J ) 7.3), 2.92 (m, 2H), 3.13 (m, 1H), 3.32 (m,
1H), 3.81 (m, 1H), 3.90 (s, 3H), 5.20 (br s, 1H), 6.73 (m, 3H),
7.05-7.22 (m, 6H). IR (cm-1, neat): 3293, 1644. Anal. (C19H23-
NO2) C, H, N.
N-(2,3-Diphenyl-2-propenyl)acetamides (10a and 10b): n-BuLi
(10 M in hexane, 0.157 mL, 1.57 mmol) was added dropwise to a
stirred ice-cooled suspension of benzyltriphenylphosphonium chlo-
ride (0.61 g, 1.57 mmol) in diethyl ether (4 mL) under N2. The
mixture was stirred at 0 °C for 30 min, and a solution of N-(2-
oxo-2-phenyl-ethyl)acetamide39 (0.27 g, 1.52 mmol) in Et2O (2.5
mL) and CH2Cl2 (2.5 mL) was then added dropwise. The resulting
mixture was stirred at room temperature for 90 min and filtered,
and the filter cake was washed with diethyl ether. The combined
filtrates were evaporated to give a crude residue from which the
two (E)- and (Z)-diastereoisomers were separated by flash chro-
matography (silica gel; cyclohexane/EtOAc, 1:1 as eluent).
(Z)-N-(2,3-Diphenyl-2-propenyl)acetamide (10a): Compound
10a was obtained in a 31% yield; mp 113 °C (Et2O/hexane). MS
(EI): m/z 251 (M+, 100). 1H NMR (CDCl3): δ 1.96 (s, 3H), 4.30
(d, 2H), 5.54 (br s, 1H), 6.60 (s, 1H), 6.95-7.4 (m, 10H). Selective
irradiation of the CH2 allylic protons (δ ) 4.30) resulted in 3.2%
enhancement of the signal at δ ) 6.60 due to the CHd vinylic
proton. IR (cm -1, Nujol): 3289, 1649, 1544. Anal. (C17H17NO)
C, H, N.
(E)-N-(2,3-Diphenyl-2-propenyl)acetamide (10b): Compound
10b was obtained in a 13% yield; mp 180 °C (trituration from Et2O).
1
MS (EI): m/z 251 (M+, 100). H NMR (CDCl3): δ 1.89 (s, 3H),
(E)-3-(3-Methoxyphenyl)-3-phenylacrylonitrile (17): trans-
Cinnamonitrile (0.28 mL, 2.24 mmol) was added to a mixture of
3-iodoanisole (0.56 mL, 4.25 mmol), potassium acetate (0.55 g,
5.6 mmol), tetrabutylammonium bromide (0.79 g, 2.45 mmol), and
palladium acetate (0.025 g, 0.11 mmol) in dry DMF (8 mL) under
N2. After stirring at 80 °C for 24 h, the reaction mixture was cooled
to room temperature, poured into water, and extracted three times
with diethyl ether. The organic combined phases were washed with
brine, dried (Na2SO4), and evaporated under reduced pressure to
give a residue that was purified by flash chromatography (silica
gel; cyclohexane/EtOAc, 95:5 as eluent) and crystallization from
diethyl ether/petroleum ether to give a 62% yield; mp 60-61 °C.
4.57 (d, 2H), 5.37 (br s, 1H), 7.01 (s, 1H), 7.3-7.55 (m, 10H).
Selective irradiation of the CH2 allylic protons (δ ) 4.57) did not
enhance the signal at δ ) 7.01 due to the CHd vinylic proton. IR
(cm-1, Nujol): 3291, 1632, 1536. Anal. (C17H17NO) C, H, N.
N-{2-[(3-Benzyl)phenyl]ethyl}propanamide (11): A solution
of (3-benzoylphenyl)acetonitrile38 (0.15 g, 0.68 mmol) and propionic
anhydride (1 mL, 7.8 mmol) in THF (7.5 mL) was hydrogenated
over Raney nickel at 4 atm of H2 for 5 h at 80 °C. 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; cyclohexane/EtOAc,
1:1 as eluent) and crystallization (diethyl ether/petroleum ether) to
give a 38% yield; mp 47-48 °C. MS (EI): m/z 267 (M+), 194
MS (EI): m/z 235 (M+, 100). H NMR (acetone-d6): δ 3.80 (s,
3H), 6.14 (s, 1H), 6.89 (ddd, 1H, H-6′, J ) 1.0, J ) 1.7, and J )
7.6), 6.97 (dd, 1H, H-2′, J ) 1.5 and J ) 2.4), 7.06 (ddd, 1H,
H-4′, J ) 1.5, J ) 2.7, and J ) 8.3), 7.34 (dd, 1H, H-5′, J ) 7.8
1

7400 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Bedini et al.
and J ) 8.1), 7.40-7.55 (m, 5H, unsubstituted phenyl). Selective
irradiation of the vinylic proton (δ ) 6.14) resulted in 3.3%
enhancement of signals at δ ) 6.89 and δ ) 6.97 due to H-6′ and
H-2′, respectively. IR (cm-1, Nujol): 2212, 1583.
H-6′ protons. IR (cm-1, Nujol): 3269, 1644, 1557. Anal. (C18H19-
NO2) C, H, N.
(Z)-N-[3-(3-Methoxyphenyl)-3-phenyl-2-propenyl]cyclobutan-
carboxamide (18e): Compound 18e was obtained by reduction of
the nitrile (Z)-16, followed by N-acylation of the corresponding
crude amine with cyclobutanecarbonyl chloride, according to the
above-described general procedure. Purification by flash chroma-
tography (silica gel; EtOAc/cyclohexane 3:7 as eluent) and crystal-
lization from diethyl ether/ petroleum ether gave a 61% yield; mp
121-122 °C. MS (EI): m/z 321 (M+), 222 (100). 1H NMR
(CDCl3): δ 1.81-2.37 (m, 6H), 2.98 (m, 1H), 3.80 (s, 3H), 3.95
(dd, 2H, J ) 5.9 and J ) 6.7), 5.38 (br t, 1H), 6.07 (t, 1H, J )
7.0), 6.74 (m, 2H, H-2′ and H-6′), 6.89 (ddd, 1H, H-4′, J ) 0.8, J
) 2.4, and J ) 8.3), 7.14-7.43 (m, 6H, H-5′ and unsubstituted
phenyl). IR (cm-1, Nujol): 3315, 1635, 1532. Anal. (C21H23NO2)
C, H, N.
(E)-N-[3-(3-Methoxyphenyl)-3-phenyl-2-propenyl]cyclobutan-
carboxamide (18f): Compound 18f was obtained by reduction of
the nitrile (E)-17, followed by N-acylation of the corresponding
crude amine with cyclobutanecarbonyl chloride, according to the
above-described general procedure. Purification by flash chroma-
tography (silica gel; EtOAc/cyclohexane 1:1 as eluent) and crystal-
lization from diethyl ether/petroleum ether gave a 62% yield; mp
89-90 °C MS (EI): m/z 321 (M+), 222 (100). 1H NMR (CDCl3):
δ 1.82-2.32 (m, 6H), 2.98 (m, 1H), 3.76 (s, 3H), 3.95 (dd, 2H, J
) 5.9 and J ) 6.7), 5.36 (br t, 1H), 6.07 (t, 1H, J ) 7.0), 6.74-
6.85 (m, 3H, H-2′, H-4′, and H-6′), 7.14-7.45 (m, 6H, H-5′ and
unsubstituted phenyl). IR (cm-1, Nujol): 3308, 1636, 1540. Anal.
(C21H23NO2) C, H, N.
General Procedure for the Synthesis of N-(3,3-Diphenyl-2-
propenyl)alkanamides 18a-g: A suspension of aluminum trichlo-
ride (0.293 g, 2.2 mmol) in dry Et2O (2.8 mL) was added to a
stirred ice-cooled suspension of LiAlH4 (0.041 g, 1.1 mmol) in dry
Et2O (1.3 mL) under a nitrogen atmosphere. The mixture was stirred
for 10 min, a solution of the suitable nitrile 14-17 (1.0 mmol) in
dry Et2O (0.9 mL) was then added dropwise, and the resulting
mixture was stirred at room temperature for 1 h. Water was carefully
added at 0 °C, and the resulting mixture was filtered through a
Celite pad. The filtrate was concentrated in vacuo and partitioned
between water and EtOAc. The combined organic phases were
washed with brine, dried (Na2SO4), and evaporated to yield the
crude (3,3-diphenyl)propenylamines, which were used without any
further purification for the preparation of 10a-g. TEA (0.15 mL,
1.07 mmol) and acetic anhydride (0.1 mL, 1.07 mmol for 18a,
18c,d, and 18g), or cyclobutanecarbonyl chloride (0.114 mL, 1
mmol for 18b, 18e,f) were added to a stirred solution of the
corresponding crude amine in dry THF (12 mL), and the mixture
was stirred at room temperature for 6 h (1 h for 18b, 18e,f). The
solvent was evaporated in vacuo, and the residue was dissolved in
EtOAc. The solution was washed with 2 N NaOH and brine and
then dried (Na2SO4). After distillation of the solvent, a crude residue
was obtained, which was purified by crystallization or flash
chromatography.
N-(3,3-Diphenyl-2-propenyl)acetamide (18a): Compound 18a
was obtained with purification by crystallization from EtOAc/
petroleum ether, giving a 39% yield; mp 139 °C. MS (EI): m/z
251 (M+), 192 (100). 1H NMR (CDCl3): δ 1.98 (s, 3H), 3.95 (dd,
2H, J ) 6.0 and J ) 6.8), 5.48 (br s, 1H), 6.08 (t, 1H, J ) 6.8),
7.15-7.43 (m, 10H). IR (cm -1, Nujol): 3308, 1643, 1541. Anal.
(C17H17NO) C, H, N.
N-[3,3-Bis-(3-methoxyphenyl)-2-propenyl]acetamide (18g):
Compound 18g was obtained by reduction of the nitrile 15,42
followed by N-acylation of the corresponding crude amine with
acetic anhydride, according to the general procedure. Purification
by flash chromatography (silica gel; EtOAc/cyclohexane 7:3 as
eluent) gave a 70% yield of an oil. MS (EI): m/z 311 (M+), 252
1
(100). H NMR (CDCl3): δ 1.99 (s, 3H), 3.77 (s, 3H), 3.80 (s,
N-(3,3-Diphenyl-2-propenyl)cyclobutanecarboxamide (18b):
Compound 18b was obtained with purification by crystallization
from EtOAc, giving a 53% yield; mp 131 °C. MS (EI): m/z 291
3H), 3.95 (m, 2H), 5.54 (br t, 1H), 6.08 (t, 1H, J ) 6.9), 6.70-
6.91 (m, 6H), 7.17 (dd, 1H, J ) 7.8 and J ) 1.0), 7.30 (t, 1H, J )
7.8). IR (cm-1, neat): 3281, 1651, 1577. Anal. (C19H21NO3) C, H,
N.
1
(M+), 192 (100). H NMR (CDCl3): δ 1.83-2.37 (m, 6H), 2.95
(m, 1H), 3.96 (dd, 2H, J ) 5.9 and J ) 6.8), 5.36 (br t, 1H), 6.07
(t, 1H, J ) 7.0), 7.16-7.38 (m, 10H). IR (cm -1, Nujol): 3325,
1638, 1531. Anal. (C20H21NO) C, H, N.
N-(3,3-Diphenyl-propyl)acetamide (19a): Compound 19a was
prepared by N-acetylation of the commercially available 3,3-
diphenylpropanamine with Ac2O/NaOAc in AcOH, as previously
described;36 mp 103 °C (EtOAc/petroleum ether; lit.36 mp 101-
102 °C, from benzene-petroleum ether). 1H NMR (CDCl3): δ 1.87
(s, 3H), 2.28 (m, 2H), 3.23 (m, 2H), 3.95 (t, 1H, J ) 7.8), 5.48 (br
t, 1H), 7.15-7.34 (m, 10H).
General Procedure for the Synthesis of (()-N-[3-(3-methoxy-
phenyl)-3-phenylpropyl]alkanamides 19b-d: A solution of the
nitrile (Z)-16 or (E)-17 (0.3 g, 1.27 mmol) in THF (7 mL) and
acetic, propionic, or butyric anhydride (21 mmol) 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 2 N NaOH. The
organic layer was washed twice with 2 N NaOH and once with
brine, dried (Na2SO4), and evaporated under reduced pressure to
give a crude residue, which was purified by flash chromatography
(silica gel; EtOAc/cyclohexane 1:1 as eluent).
(Z)-N-[3-(3-Methoxyphenyl)-3-phenyl-2-propenyl]aceta-
mide (18c): Compound 18c was obtained by reduction of the nitrile
(Z)-16, followed by N-acylation of the corresponding crude amine
with acetic anhydride, according to the above-described general
procedure. Purification by flash chromatography (silica gel; EtOAc/
cyclohexane 1:1 as eluent) gave 57% yield of an oil. MS (EI): m/z
1
281 (M+), 222 (100). H NMR (acetone-d6): δ 1.88 (s, 3H), 3.80
(s, 3H), 3.84 (dd, 2H, J ) 5.6 and J ) 7.1), 6.10 (t, 1H, J ) 7.0),
6.75 (m, 2H, H-2′ and H-6′), 6.93 (ddd, 1H, H-4′, J ) 1.5, J )
2.2, and J ) 8.0), 7.15-7.43 (m, 7H, NH, H-5′and unsubstituted
phenyl). Selective irradiation of vinylic proton (δ ) 6.10) resulted
in 2.0% enhancement of resonance due to ortho protons of the
unsubstituted phenyl ring. IR (cm-1, neat): 3281, 1651, 1550. Anal.
(C18H19NO2) C, H, N.
(E)-N-[3-(3-Methoxyphenyl)-3-phenyl-2-propenyl]aceta-
mide (18d): Compound 18d was obtained by reduction of the nitrile
(E)-17, followed by N-acylation of the corresponding crude amine
with acetic anhydride, according to the above-described procedure.
Purification by flash chromatography (silica gel; EtOAc/cyclohex-
ane 1:1 as eluent) and crystallization from diethyl ether gave a 60%
(()-N-[3-(3-Methoxyphenyl)-3-phenylpropyl]acetamide (19b):
Compound 19b was obtained as an oil, 35% yield. MS (EI): m/z
1
283, (M+), 73 (100). H NMR (CDCl3): δ 1.89 (s, 3H), 2.27 (m,
2H), 3.24 (m, 2H), 3.78 (s, 3H), 3.92 (t, 1H, J ) 7.8), 5.36 (br t,
1H), 6.71-6.86 (m, 3H), 7.18-7.32 (m, 6H). IR (cm-1, neat):
3296, 1642. Anal. (C18H21NO2) C, H, N.
1
yield; mp 128 °C. MS (EI): m/z 281 (M+), 222 (100). H NMR
(acetone-d6): δ 1.85 (s, 3H), 3.73 (s, 3H), 3.83 (dd, 2H, J ) 5.0
and J ) 7.0), 6.12 (t, 1H, J ) 7.0), 6.76 (dd, 1H, H-2′, J ) 2.5 and
J ) 2.6), 6.78 (ddd, 1H, H-6′, J ) 1.0, J ) 1.6, and J ) 7.0), 6.84
(ddd, 1H, H-4′, J ) 2.0, J ) 2.5, and J ) 8.0), 7.21 (t, 1H, H-5′,
J ) 8.0), 7.15-7.48 (m, 6H, NH and unsubstituted phenyl).
Selective irradiation of vinylic proton (δ ) 6.12) resulted in 4.2%
enhancement of signals at δ ) 6.76 and δ ) 6.78 due to H-2′ and
(()-N-[3-(3-Methoxyphenyl)-3-phenylpropyl]propanamide
(19c): Compound 19c was obtained as an oil, 75% yield. MS (EI):
1
m/z 297 (M+), 87 (100). H NMR (CDCl3): δ 1.09 (t, 3H, J )
7.6), 2.10 (q, 2H, J ) 7.6), 2.27 (m, 2H), 3.25 (m, 2H), 3.77 (s,
3H), 3.92 (t, 1H, J ) 7.8), 5.38 (br t, 1H), 6.69-6.90 (m, 3H),
7.14-7.35 (m, 6H). IR (cm-1, neat): 3293, 1644. Anal. (C19H23-
NO2) C, H, N.

MT2-SelectiVe Melatonin Receptor Ligands
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7401
previously developed MT2 receptor model.32 Ligand geometries
were optimized using the MMFF94s force field,58 as imple-
mented in Macromodel.59 Docking experiments were performed
starting from the minimum energy conformations of the ligands
placed in arbitrary positions, within a region centered on amino
acids His208, Trp264, and Tyr183, using enclosing and bound-
ing boxes of 46 and 14 Å on each side, respectively. Van der
Waals radii of protein and ligand atoms were not scaled. The
amide bond of the ligand was maintained fixed in anti
disposition. Docking solutions were ranked according to their
Emodel value.
(()-N-[3-(3-Methoxyphenyl)-3-phenylpropyl)butanamide (19d):
Compound 19d was obtained as an oil, 70% yield. MS (EI): m/z
1
311 (M+), 101 (100). H NMR (CDCl3): δ 0.92 (t, 3H, J ) 7.5),
1.62 (m, 2H), 2.05 (dd, 2H, J ) 7.0 and J ) 7.8), 2.29 (m, 2H),
3.25 (m, 2H), 3.77 (s, 3H), 3.92 (t, 1H, J ) 7.8), 5.38 (br s, 1H),
6.73 (dd, 1H, J ) 2.4 and J ) 8.0), 6.79 (t, 1H, J ) 2.1), 6.85 (d,
1H, J ) 7.8), 7.14-7.35 (m, 6H). IR (cm-1, neat): 3294, 1643.
Anal. (C20H25NO2) C, H, N.
Pharmacology
Binding affinities of compounds 9-11, 13, 18, and 19 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 char-
acterization of NIH3T3-MT1 and MT2 cells was already
described in detail.53,54 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.55 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.56 The pKi values are the mean of
at least three independent determinations performed in duplicate.
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 recep-
tors 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.53,57 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, respectively. Data from
[35S]GTPγS binding experiments are given as percentage of
basal binding, where the basal binding is the amount of [35S]-
GTPγS specifically bound in the absence of compounds and
was taken as 100%. The maximal G-protein activation was
measured in each experiment by using MLT (100 nM). The
relative intrinsic activity (IAr) values of the new compounds
were obtained by dividing the maximum ligand-induced stimu-
lation of [35S]GTPγS binding by that of MLT, as measured in
the same experiment. All of the measurements were performed
in triplicate. Compounds were added at a concentration equiva-
lent to 100 nM MLT; the equivalent concentration was estimated
on the basis of the ratio of the affinity of the test compound
over that of MLT. It was assumed that at this concentration the
test compound occupies the same number of receptors as 100
nM MLT. Concentrations 10 times smaller and 10 times higher
were also tested to verify that a plateau in [35S]GTPγS binding
had been reached.
The best scoring solution of 18g 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), with fixed protein backbone. A MD
simulation was performed on the complex, with MMFF9460
force field, 1 fs time step, for 1 ns after 100 ps of equilibration,
with fixed protein backbone.
3D-QSAR
3D-QSAR analysis was performed with the CoMFA61 module
of Sybyl.62 A former 3D-QSAR model built from a set of 34
MT2 antagonists (model 7 in Table 4 of ref 31) was rebuilt,
changing ligand conformations and orientations, to get an
alignment consistent with docking into the previously described
receptor model.32 Thus, the side chain of acylaminoethyl-indole
antagonists (e.g., luzindole), adopted a τ1 (C3a-C3-Câ-CR)
≈ 270° (instead of the previous ≈90°), with unmodified τ2
(C3-Câ-CR-N) ≈ 180° and τ3 (Câ-CR-N-C) ≈ 180°. The
new CoMFA models were calculated within the same grid
region and applying the parameters previously described. Leave-
one-out cross-validation was applied to PLS analysis63 to select
the model with the number of latent variables giving the first
maximum of Q2 value, calculated with the SAMPLS algo-
rithm.64 Standard deviation of error in prediction (SDEP)65 was
calculated as SDEP ) [∑(y - yPRED)2/N]1/2
.
The newly synthesized compounds were aligned on the
reference 4P-PDOT (3) by means of a rigid fit procedure,
superposing the four atoms of the amide group and the two
centroids of the phenyl rings. The minimum energy conforma-
tion giving the best fit in terms of root-mean-square distances
(rmsd) was selected for binding affinity prediction. Although
for chiral compounds only affinity data for racemic mixtures
were available, the R-isomers of compounds 9 and the S-isomers
of 19b-d were chosen, as they gave better superpositions.
Geometry optimization was achieved applying the Tripos force
field66 with the Powell method67 to an energy gradient of 0.01
kcal/mol‚Å, without the electrostatic contribution.
Acknowledgment. This work was supported by the Italian
M.I.U.R. (Ministero dell’Istruzione, dell’Universita` e della
Ricerca). The C.I.M. (Centro Interdipartimentale Misure) and
C.C.E. (Centro di Calcolo Elettronico) of the University of
Parma are gratefully acknowledged for providing the Sybyl
software license.
Supporting Information Available: Elemental analyses for
compounds 9a-c, 10a,b, 11, 13, 18a-g, and 19b-d. This material
is available free of charge via the Internet at http://pubs.acs.org.
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