Toward the definition of stereochemical requirements for MT 2-selective antagonists and partial agonists by studying 4-phenyl-2-propionamidotetralin derivatives

Article abstract of DOI:10.1021/jm200790v

New derivatives of 4-phenyl-2-propionamidotetralin (4-P-PDOT) were prepared and tested on cloned MT₁ and MT₂ receptors, with the purpose of merging previously reported pharmacophores for nonselective agonists and for MT₂-selective antagonists. A 8-methoxy group increases binding affinity of both (±)-cis- and (±)-trans-4-P-PDOT, and it can be bioisosterically replaced by a bromine. Conformational analysis of 8-methoxy-4-P-PDOT by molecular dynamics, supported by NMR data, revealed an energetically favored conformation for the (2S,4S)-cis isomer and a less favorable conformation for the (2R,4S)-trans one, fulfilling the requirements of a pharmacophore model for nonselective melatonin receptor agonists. A new superposition model, including features characteristic of MT₂- selective antagonists, suggests that MT₁/MT₂ agonists and MT₂ antagonists can share the same arrangement for their pharmacophoric elements. The model correctly predicted the eutomers of (±)-cis- and (±)-trans-4-P-PDOT. The model was validated by preparing three dihydronaphthalene derivatives, either able or not able to reproduce the putative active conformation of 4-P-PDOT. (Figure presented)

Full text of DOI:10.1021/jm200790v

Article
pubs.acs.org/jmc
Toward the Definition of Stereochemical Requirements for
MT2-Selective Antagonists and Partial Agonists by Studying
4-Phenyl-2-propionamidotetralin Derivatives
Annalida Bedini,† Simone Lucarini,† Gilberto Spadoni,† Giorgio Tarzia,† Francesco Scaglione,‡
Silvana Dugnani,‡ Marilou Pannacci,‡ Valeria Lucini,‡ Caterina Carmi,§ Daniele Pala,§ Silvia Rivara,*,§
and Marco Mor§
†Dipartimento di Scienze Biomolecolari, Universita
̀
degli Studi di Urbino “Carlo Bo”, Piazza Rinascimento 6, I-61029 Urbino, Italy
‡Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Universita
Milano, Italy
̀
degli Studi di Milano, Via Vanvitelli 32, I-20129
§Dipartimento Farmaceutico, Universita
̀
degli Studi di Parma, Viale G. P. Usberti 27/A Campus Universitario, I-43124 Parma, Italy
S
* Supporting Information
ABSTRACT: New derivatives of 4-phenyl-2-propionamidotetralin (4-P-PDOT) were prepared and
tested on cloned MT1 and MT2 receptors, with the purpose of merging previously reported
pharmacophores for nonselective agonists and for MT2-selective antagonists. A 8-methoxy group
increases binding affinity of both (±)-cis- and (±)-trans-4-P-PDOT, and it can be bioisosterically
replaced by a bromine. Conformational analysis of 8-methoxy-4-P-PDOT by molecular dynamics,
supported by NMR data, revealed an energetically favored conformation for the (2S,4S)-cis isomer and
a less favorable conformation for the (2R,4S)-trans one, fulfilling the requirements of a pharmacophore
model for nonselective melatonin receptor agonists. A new superposition model, including features
characteristic of MT2-selective antagonists, suggests that MT1/MT2 agonists and MT2 antagonists can
share the same arrangement for their pharmacophoric elements. The model correctly predicted the eutomers of (±)-cis- and
(±)-trans-4-P-PDOT. The model was validated by preparing three dihydronaphthalene derivatives, either able or not able to
reproduce the putative active conformation of 4-P-PDOT.
and other agonists are currently being tested in clinical trials.16
INTRODUCTION
■
Besides nonselective ligands, different series of MT1- or MT2-
selective agents have also been described.17−19 Investigations
on the role of melatonin receptors depend on the availability of
potent and selective ligands. The MT2-selective antagonist/
partial agonist 4-phenyl-2-propionamidotetralin20 (4-P-PDOT,
Figure 1), together with luzindole,20 played a key role as a
pharmacological tool for the evaluation of specific effects of
melatonin and melatonin receptor ligands. In particular, 4-P-
PDOT has been widely employed to discriminate the role of
MT1 and MT2 receptors in melatonin-mediated effects. 4-P-
PDOT revealed 100-fold selectivity for the MT2 receptor and
antagonist behavior on electrically evoked dopamine release in
rabbit retina.20 However, it showed different degrees of intrinsic
activity in different experiments; e.g., it behaved as an MT1
antagonist/MT2 partial agonist in tests based on GTPγS
binding or cAMP formation.21−23 Because of its two
asymmetric carbons, there are two couples of 4-P-PDOT
enantiomers, corresponding to the cis and trans isomers. In
many cases, the use of 4-P-PDOT had been reported without
clear indications about composition of the mixture employed.
Melatonin (Figure 1) is a tryptophan-derived hormone secreted
by the pineal gland following a circadian rhythm, with peak
levels occurring during the period of darkness. Besides its well-
known chronobiotic and sleep-inducing properties,1 many
other effects have been ascribed to melatonin, such as the
modulation of cardiovascular2 and immune systems,3 bone
formation,4 and hormone secretion.5 For this reason melatonin
has been proposed as a therapeutic agent in a variety of
pathological conditions, such as sleep disturbances,6 cancer,7
neurodegenerative diseases,8 and stroke.9 Melatonin binds to
and activates two G-protein-coupled receptors, MT1 and MT2,
mainly expressed in the central nervous system but also present
in peripheral organs.10,11 Moreover, an additional melatonin
binding site, named MT3, has been reported as the hamster
homologue of the human enzyme quinone reductase 2, which is
thought to be involved in melatonin-mediated antioxidant
activity.12
The past decade has brought significant advancements in the
design and development of MT1 and MT2 receptor ligands, as
well as in their therapeutic application. Melatonin, in a
prolonged-release formulation,13 and ramelteon14 (Figure 1)
are prescribed for the treatment of insomnia; agomelatine15
(Figure 1), an MT1/MT2 agonist and 5HT2C antagonist, has
been approved for the treatment of major depressive disorders,
Received: June 17, 2011
Published: November 2, 2011
© 2011 American Chemical Society
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to rotate, and the active orientation of this pharmacophore
element was therefore ill-defined. Furthermore, no evidence has
been presented so far that the common pharmacophore
elements have the same mutual arrangement for both the
“agonist” and the “MT2-selective antagonist” binding modes.
The purpose of the present work is to develop a unique
superposition model for both classes of ligands, based on
stereoselectivity data for 4-P-PDOT derivatives. Such a model
could be useful not only to drive the development of new
classes of ligands with tailored subtype selectivity and/or
intrinsic activity but also to select the bioactive conformation of
MT2-selective ligands that can be used to refine MT2 receptor
models built by homology modeling. We therefore prepared 8-
methoxy derivatives of both cis- and trans-4-P-PDOT and the
corresponding bromine bioisoster for the more potent
racemate. Once assessed that the role of these groups was
the same as observed in known classes of agonists, we explored
the accessible conformational space of the two diastereomers by
stochastic dynamics simulations and NMR. A superposition of
energetically accessible conformations, consistent with both
cited pharmacophore models, also was consistent with the
affinity ratios for pure (2S,4S)-, (2R,4S)-, (2R,4R)-, and
(2S,4R)-4-P-PDOT, which had been recently separated and
characterized.30 The superposition model was further validated
by preparing dihydronaphthalene analogues that either could or
could not be closely superposed to the putative active
conformation of 4-P-PDOT.
Figure 1. Melatonin and selected melatonin receptor ligands.
In spite of the importance of 4-P-PDOT, structure−activity
relationships (SARs) for 4-phenyl-2-acylaminotetralin deriva-
tives have been poorly explored. Only two analogues of 4-P-
PDOT, characterized by minor modifications at the acylating
group, have been described in the literature, showing that both
the N-acetyl and N-chloroacetyl derivatives have binding
affinities similar to that of 4-P-PDOT.20 Furthermore it was
reported that (±)-cis-4-P-PDOT displays higher MT2 binding
affinity than the (±)-trans-isomer and that both racemic
mixtures showed selectivity for the MT2 receptor.24 No
information about the role of a key element for the
pharmacophore of melatonin receptor ligands, i.e., the 5-
methoxy group of melatonin, had been reported so far.
Moreover, while the structure of 4-P-PDOT can be easily
superposed to the putative active conformations of other MT2-
selective antagonists,25 the relevance of amide group orientation,
with respect to the lipophilic portions, has not been assessed
so far.
The aim of this work is to focus on the active conformation
and configuration of 4-P-PDOT derivatives, to test the
hypothesis that it is possible to merge a pharmacophore
model for melatonin receptor agonists26 and one for MT2-
selective antagonists.25,27 These models share an aromatic
nucleus and an amide fragment, whereas a lipophilic group with
an out-of plane arrangement is typical for MT2-selective
antagonists. While the MT2-antagonists pharmacophore
model led to the design of new MT2-selective ligands,28,29 it
contains no information about stereoselectivity or about the
conformation of the alkylamide side chain. On the other hand,
the pharmacophore model for nonselective melatonin receptor
agonists26 accounted for stereoselectivity and side chain
conformation. Starting from the observed stereoselectivity for
the two enantiomers of a conformationally constrained tricyclic
melatonin analogue, it had been observed that a putative active
conformation of melatonin, having the acetylaminoethyl chain
below the plane of indole ring (as represented in Figure 1),
could be efficiently superposed to the eutomers of stereo-
selective agonists. In spite of the conformational constraint in
the tricyclic structure, however, the acetylamino group was free
RESULTS AND DISCUSSION
■
Synthesis of 4-P-PDOT Derivatives. Compounds 3a,b
and 4b,c were prepared in a reaction sequence already employed
by our group31 for the synthesis of cis-4-P-PDOT (Scheme 1),
involving condensation of the suitable tetralone 2a−c with
propionamide in the presence of PTSA and azeotropic removal of
water, followed by ionic diastereoselective reduction (Et3SiH/
TFA) of the cyclic enamides 3a−c. The key tetralones 2a−c were
obtained by reaction of styrene with the appropriate phenylacetyl
chloride 1a−c in the presence of aluminum chloride.
Target compound 6 was synthesized by a four-step procedure
starting from the tetralone 2a.31 As shown in Scheme 1, the first
step involved the reaction of 2a with trimethylsilyl cyanide and
ZnI2 followed by treatment with phosphorus oxychloride in
pyridine to give the intermediate nitrile 5 which was converted
into the final product 6 by reduction with LiAlH4 and acylation
of the intermediate amine with propionic anhydride.
Starting from the tetralone 2b the (±)-trans-propionamidote-
tralin derivative 10 was prepared in a reaction sequence already
applied for similar compounds,32 involving a diastereoselective
(85:15 cis/trans) ketone reduction using NaBH4, followed by
tosylation of the resulting 2-tetralol 7 with p-toluensulfonyl
chloride in pyridine. The cis-tosylate 8 was then converted to
the corresponding (±)-trans-propionamido derivative 10 by
reaction with sodium azide in aqueous DMF, followed by
catalytic (10% Pd/C) hydrogenation and acylation of the
intermediate amine with propionic anhydride (Scheme 2).
Pharmacological Characterization of 8-Substituted 4-
P-PDOT Derivatives. Binding affinity and intrinsic activity at
human MT1 and MT2 receptors for newly synthesized compounds
and for recently separated four 4-P-PDOT single stereoisomers are
reported in Tables 1 and 2. To improve data homogeneity for
SAR comparisons, the racemic mixtures (±)-cis- and (±)-trans-4-
P-PDOT were tested during the experimental runs carried out for
new compounds. The introduction of a methoxy group in position
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a
Scheme 1
a
Reagents and conditions: (a) AlCl3, CH2Cl2, 0 °C, 15 min; (b) propionamide, PTSA, toluene, reflux, 4 h; (c) TES, TFA, −10 °C, 10 min; (d)
(CH3)3SiCN, ZnI2, toluene, room temp, 18 h; (e) POCl3, Py, reflux, 1 h; (f) LiAlH4, Et2O, room temp, 15 min; (g) propionic anhydride, TEA, THF,
room temp, 16 h.
a
Scheme 2
a
Reagents and conditions: (a) NaBH4, MeOH, reflux, 16 h; (b) TsCl, Py, 5 °C, 3 days; (c) NaN3, DMF/H2O, 50 °C, 5 h; (d) H2 (4 atm), 10% Pd/
C, i-PrOH/CH2Cl2, room temp, 16 h; (e) propionic anhydride, TEA, THF, room temp, 4 h.
8 caused a moderate increase of MT1 and MT2 binding affinity for
both the cis and trans racemic mixtures of 4-P-PDOT derivatives
(compounds 4b and 10), consistent with what had been observed
for a series of 2-propionamidotetralins, including the agonist 8-
M-PDOT (Figure 1).33 The racemic mixtures 4b and 10 showed
low to medium intrinsic activity at the GTPγS test, with no
increase of intrinsic activity compared to the respective 4-P-PDOT
racemates. The affinity ratio between cis and trans racemates was
fully conserved, as well as selectivity for receptor subtypes. Thus,
the positive effect of the 8-methoxy group on binding affinity
resembles what had been observed in other series of melatonin
receptor ligands.25,28,29,34−38 Replacement of the 8-methoxy group
of the better racemate, 4b, by a bromine atom gave 4c, showing
MT1 and MT2 affinities comparable to, or slightly better than,
those of the methoxy derivative 4b and a moderate increase of
intrinsic activity. This is consistent with what had been previously
observed for other series of melatonin ligands, e.g., in the series of
melatonin derivatives39 and of N-anilinoethylamides.29
NMR Analysis and SD Simulations on cis- (4b) and
trans-8-Methoxy-4-P-PDOT (10). The observation that 4-
P-PDOT derivatives show SAR patterns (constant effect of the
8-methoxy group, bioisosterism with bromine) matching those
of typical melatonin receptor agonists prompted us to apply a
chiral pharmacophore model, previously developed for stereo-
selective, MT1/MT2 nonselective agonists,26 to the search for
active conformations of 4-P-PDOT stereoisomers. In previous
work, it had been observed that conformational equilibrium of
the tetralin nucleus can qualitatively explain the different
affinity for cis and trans isomers, but no experimental data on
single stereoisomers were available, and, regarding conforma-
tional aspects, the role of amide orientation was not
addressed.24
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Table 1. Binding Affinity and Intrinsic Activity of Newly Synthesized Compounds 4b,c, 10, and of 4-P-PDOT Stereoisomers for
Human MT1 and MT2 Melatonin Receptors
MT1
MT2
a
b
a
b
compd
melatonin
R
stereochemistry
pKi ± SEM
IAr ± SEM
pKi ± SEM
IAr ± SEM
9.58 ± 0.15
7.83 ± 0.07
7.04 ± 0.06
7.99 ± 0.16
7.12 ± 0.22
6.65 ± 0.07
7.02 ± 0.11
6.59 ± 0.06
6.89 ± 0.09
5.67 ± 0.19
1.00 ± 0.05
0.20 ± 0.02
0.02 ± 0.01
0.49 ± 0.08
0.23 ± 0.06
0.41 ± 0.07
0.15 ± 0.08
0.06 ± 0.02
0.33 ± 0.04
9.47 ± 0.12
1.00 ± 0.03
0.44 ± 0.07
0.21 ± 0.03
0.65 ± 0.09
0.46 ± 0.02
0.40 ± 0.01
0.45 ± 0.01
0.11 ± 0.02
0.48 ± 0.05
4b
OCH3
OCH3
Br
(±)-cis
(±)-trans
(±)-cis
(±)-cis
(±)-trans
(+)-cis
10.22 ± 0.38
9.71 ± 0.42
10.85 ± 0.20
9.16 ± 0.06
8.06 ± 0.08
9.26 ± 0.14
7.01 ± 0.05
8.02 ± 0.15
6.10 ± 0.15
10
4c
c
(±)-cis-4-P-PDOT
(±)-trans-4-P-PDOT
(2S,4S)-4-P-PDOT
H
c
H
d
H
d
(2R,4R)-4-P-PDOT
H
(−)-cis
(+)-trans
d
(2R,4S)-4-P-PDOT
H
d
(2S,4R)-4-P-PDOT
H
(−)-trans
−0.23 ± 0.03
−0.14 ± 0.04
pKi values were calculated from IC50 values, obtained from competition curves by the method of Cheng and Prusoff,45 and are the mean of at least
three determinations performed in duplicate. The relative intrinsic activity values were obtained by dividing the maximum analogue-induced G-
a
b
c
d
protein activation by that of melatonin. See ref 24. See ref 30.
Table 2. Binding Affinity and Intrinsic Activity of Newly Synthesized Compounds 3a,b and 6 for Human MT1 and MT2
Melatonin Receptors
MT1
MT2
a
b
a
b
compd
R
pKi ± SEM
IAr ± SEM
pKi ± SEM
IAr ± SEM
melatonin
(±)-3a
(±)-3b
(±)-6
9.58 ± 0.15
7.30 ± 0.12
6.74 ± 0.32
7.69 ± 0.09
1.00 ± 0.05
0.14 ± 0.03
0.32 ± 0.06
0.01 ± 0.06
9.47 ± 0.12
7.43 ± 0.31
7.11 ± 0.28
8.46 ± 0.12
1.00 ± 0.03
0.59 ± 0.11
0.49 ± 0.05
0.02 ± 0.05
H
OCH3
H
pKi values were calculated from IC50 values, obtained from competition curves by the method of Cheng and Prusoff,45 and are the mean of at least
three determinations performed in duplicate. The relative intrinsic activity values were obtained by dividing the maximum analogue-induced G-
a
b
protein activation by that of melatonin.
To explore the accessible conformations of 4-P-PDOT
derivatives, detailed conformational analysis for compounds 4b
and 10 was performed, complementing molecular dynamics
simulations with NMR experiments. The cis racemic mixture 4b
NMR spectra of the trans isomer 10 are consistent with the
presence of two conformations in rapid exchange, similar to what
had been observed for trans-4-P-PDOT.24 In fact, H−1H vicinal
1
coupling constants (3J, Table 3) for the cyclohexene ring showed
intermediate values consistent with an equilibrium between two half-
chair conformations, one with equatorial amide side chain and
pseudoaxial phenyl ring and the other with axial amide and
pseudoequatorial phenyl ring. Cross-peaks in the NOESY spectrum
(Supporting Information, Figure S2) also supported the presence of
a conformational equilibrium. In fact, cross-peaks consistent with
both conformations were detected with similar intensities (equatorial
amide side chain: between H1a and H3a, between H2 and Ho; axial
amide side chain: between H1b and H3b, between H4 and NH).
Intense NOEs were also observed between O-methyl protons at
position 8 and H7; between aromatic Ho and H3a, H4 and H5;
and between NH and H1a, H2, H3a, and the propionamidic
α-CH2.
1
showed H−1H vicinal coupling constants (3J, Table 3) for the
cyclohexenyl ring consistent with a half-chair conformation, with
equatorial amide chain and pseudo-equatorial phenyl ring. 2D
NOESY data (Supporting Information, Figure S1) also
supported the presence of a single conformation for the tetralin
scaffold. In particular, by reference to the structure of 4b
reported in Figure 2, strong NOE cross-peaks were observed
between protons H1b and H3b and between H2 and H4, all in
pseudoaxial or axial arrangement, while no cross-peak was
detected between the equatorial H1a and H3a. Additional
NOEs were detected: between the O-methyl protons at position
8 and H7; between Ho and H3b, H4 and H5; between NH and
H1b, H3b, and the α-CH2 of the propionamide side chain.
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Monitoring the dihedral angle τ1 (C1−C2−C3−C4, Figure 2),
we observed that the half-chair conformation of 4b, having both
substituents in equatorial arrangement (Figure 3, cis-I and cis-
II), was conserved during the simulation (τ1 = 60° for (2S,4S)-
4b, Figure 4a) and only a small number of snapshots (<2%)
presented a conformation with diaxial arrangement (τ1 = 300°
in Figure 4a, cis-III in Figure 3). (2R,4S)-10 underwent
repeated conversions between the two cyclohexene conforma-
tions characterized by τ1 = 60° (Figure 4b, trans-I in Figure 3)
and τ1 = 300° (Figure 4b, trans-II and trans-III in Figure 3).
The ratio between these two cyclohexene conformations was
approximately 30/70, with the more abundant having the 2-
propionamido substituent in axial arrangement and the 4-
phenyl ring in pseudo-equatorial one. By collection of 50 000
conformations (snapshots) during SD simulation (one every 20 ns)
and by represention of cyclohexene arrangements by τ1 and
amide rotamers around the C2−N bond by τ2 = H2−C2−N−
H (Figure 2), the vast majority of snapshots could be grouped
in the conformation families reported in Table 4. Considering
this set as representative of the dynamic conformational
equilibrium (for both 4b and 10, more than 200 conversions
among different conformation families were observed), we
estimated J values for vicinal protons as ensemble averages of
those calculated from dihedral angles measured for each
snapshot. The calculated values are reported in Table 3 and
here compared to those experimentally observed. While for 4b
there is good agreement between experimental and theoretical
data, some J values calculated for 10 were slightly different from
experimental ones. In particular, the experimental values of
JH3a‑H2 and JH3b‑H4, which strongly depend on the relative
abundance of the two cyclohexene conformations, were 7.8 and
7.2 Hz, respectively, compared to those calculated as averages
of SD conformations, being 5.8 and 8.4, respectively. As stated
above, SD simulation showed a 30/70 ratio between
confromations with the amide chain in equatorial and axial
arrangements. In fact, the best agreement between experimental
and calculated J values for 10 corresponds to a ratio of
45/55, giving Jcalc,H3a‑H2 = 7.1 and Jcalc,H3b‑H4 = 6.9. Although
significant, this discrepancy corresponds to limited differences
in free energies and does not affect the following superposition
hypothesis. The 8-methoxy group mostly assumed one
preferred orientation, with the methyl group pointing toward
H7 (Supporting Information, Figure S3). This is consistent
with the presence of strong NOE signals between methyl
Table 3. Geminal and Vicinal Coupling Constants Values
Observed by NMR Spectroscopy (600 MHz) and Calculated
for Compounds 4b and 10
10, coupling constant (Hz)
4b, coupling
constant (Hz)
calcd
type of
coupling
a
b
b
c
c
protons
obsd calcd
obsd
av
ax
eq
H1a-H1b
H3a-H3b
H1b-H2
H1a-H2
H3b-H2
H3a-H2
H3b-H4
H3a-H4
NH-H2
geminal (2J)
geminal (2J)
vicinal (3J)
vicinal (3J)
vicinal (3J)
vicinal (3J)
vicinal (3J)
vicinal (3J)
vicinal (3J)
16.8
12.0
17.4
12.6
5.4
6.0
3.0
7.8
7.2
6.0
7.2
10.8
5.4
10.7
4.5
4.2
5.0
3.0
5.8
8.4
5.2
3.8
2.9
5.3
10.3
3.2
12.0
2.4
11.0
3.1
2.9
3.8
11.2
2.0
12.0
4.8
11.1
4.8
11.0
5.2
5.2
7.2
a
b
Refer to Figure 2 for proton numbering scheme. Average values for
50 000 conformations collected from SD (see Experimental Section).
c
Calculated from conformations of 10 having the amide side chain in
axial or in equatorial arrangement.
Stochastic dynamics (SD) trajectories of two single
enantiomers of 4b and 10 [(2S,4S)-4b and (2R,4S)-10 were
employed as reference structures and are represented in Figures
2 and 3] were registered for a simulation time of 1 μs.
Figure 2. Chemical structures and atom labeling for 4b and 10. The
(2S,4S) and the (2R,4S) enantiomers are reported as representatives of
the cis-(4b) and trans-(10) racemic mixtures, respectively. Protons
below the plane of the tetralin ring are labeled with “a” and those
above the plane with “b”.
Figure 3. Representation of minimum-energy conformations of (2S,4S)-4b (cis) and (2R,4S)-10 (trans), explored during SD simulations.
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Figure 4. Graphical representation of τ1 (C1−C2−C3−C4) dihedral angle values for cis-(2S,4S)-4b (a) and trans-(2R,4S)-10 (b).
Table 4. Families of Conformations and Their % Abundance
in a Set of 50 000 Snapshots Collected during SD
Simulations for Compounds 4b and 10
range of dihedral angle (deg)
a
b
b
compd
conformation
τ1
τ2
%
(2S,4S)-4b
cis-I
30−90
30−90
270−330
120−240
330−360 and 0−60
120−240
96.29
1.04
1.59
cis-II
cis-III
Figure 5. Superposition of (a) melatonin (green carbons), (S)-8-M-
PDOT (yellow carbons), and cis-(2S,4S)-4b (orange carbons); (b) cis-
(2S,4S)-4b (orange carbons) and UCM454 (white carbons); (c) cis-
(2S,4S)-4b (orange carbons) and trans-(2R,4S)-10 (cyan carbons, in
conformation trans-III).
(2R,4S)-10
trans-I
30−90
270−330
270−330
120−240
120−240
285−360 and 0−15
70.60
26.72
0.45
trans-II
trans-III
a
Corresponding to the minimum-energy conformations represented in
fragment, the methoxy group and the phenyl ring was observed
(Figure 5b). These superpositions imply that pharmacophore
models for agonists and for MT2-selective antagonists share the
same arrangement of the common elements. This hypothesis
obviously needs to be supported by further experimental data.
The enantiomer of 4b that best fits the requirements of the
chiral pharmacophore model for agonists is the (2S,4S) one
(Figure 5a). This is consistent with the fact that the agonist 8-
M-PDOT showed high stereoselectivity, with the (S)-
enantiomer being the eutomer.41 The trans-isomer 10 able to
give a strict superposition of its 4-phenyl ring with that of the
(2S,4S)-enantiomer of 4b is the (2R,4S)-enantiomer, having
opposite configuration of the amide chain (Figure 5c). The
same orientation of the amide fragments of the two
stereoisomers was achieved by selecting for 10 a H2−C2−
N−H dihedral that, although consistent with NMR data, was
unfavored at SD simulation. In particular, the conformation of
10 reported in Figure 5c has an equatorial arrangement of the
amide chain and the NH bond pointing toward H2. This was a
minimum-energy conformation, belonging to a family of similar
snapshots, accounting for about 0.5% of the simulation
ensemble (Table 4, trans-III in Figure 3). This unfavorable
energetic state could explain, at least in part, the lower potency
observed for the trans racemate 10.
b
Figure 3. τ1 = C1−C2−C3−C4. τ2 = H2−C2−N−H. See Figure 2
for atom numbering.
protons and H7. The amide NH group was oriented anti to H2
during most of the simulation time (>99%) for both 4b and 10,
with a small number of snapshots presenting an opposite
orientation (Supporting Information, Figure S4). On the other
hand, both the coupling constant H2−NH (7.2 Hz) and the
absence of strong NOE signals between NH and tetralin
protons are more consistent with free rotation around the C2−
N bond.
Therefore, combined information from NMR data and SD
simulations pointed to (i) a preferred diequatorial arrangement
of substituents in 2 and 4 for the cis isomer 4b; (ii) an
equilibrium, with no overwhelming abundance, between the
two half-chair conformations for the trans isomer 10; (iii) a
prevalent anti orientation of the H2−C2−N−H dihedral, with
possible rotation, for both isomers.
Superposition of 8-Methoxy-4-P-PDOT Stereoisomers
on Melatonin Active Conformation. The most abundant
conformer of 4b, resulting from SD simulation, could be
superposed on the active conformation of melatonin which had
been inferred from a pharmacophore model based on
stereoselective MT1/MT2 agonists, including (S)-8-M-PDOT
(Figure 5a).26 The 4-phenyl ring of 4b represents an element,
not present in the agonist pharmacophore, related to MT2-
selectivity and reduced intrinsic activity.40 When the same
conformer of 4b was superimposed to the MT2-selective
antagonist UCM454, a strict superposition of the amide
Model Validation: 4-P-PDOT Stereoisomers and
Dihydronaphthalene Derivatives. The present availability
of all four single 4-P-PDOT stereoisomers30 allowed us to test
them in the same experimental conditions employed for
racemates 4b, 4c, and 10. Their binding affinity and intrinsic
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activity for MT1 and MT2 receptors are reported in Table 1. As
predicted by the superpositions discussed above, the eutomers
for the cis and trans racemic mixtures were the (2S,4S) and the
(2R,4S) ones, respectively. The present data suggest that, also
for MT2-selective antagonists or partial agonists, the NH bond
of the amide fragment should be arranged in an orientation
perpendicular to the aromatic portion of the tetralin nucleus,
pointing toward the same direction identified for MT1/MT2
agonists (Figure 5b). This hypothesis was further validated by
testing compounds 3a and 3b, where the introduction of a
double bond between positions 1 and 2 of the tetralin nucleus
caused partial conjugation with the amide NH, preventing its
perpendicular orientation. As expected, 3a showed a significant
loss of MT2 receptor affinity (Table 2) compared to 4a, 10, and
4-P-PDOT racemates, in spite of the coplanar arrangement of
the amide chain, that could resemble the equatorial one of 4-P-
PDOT. Moreover, the introduction of the 8-methoxy group in
3b did not improve MT2 binding affinity, suggesting that these
compounds interact with their binding site with poses differing
from those required by the pharmacophore model discussed
above. To further validate the hypothesis that this behavior is
due to the nonperpendicular orientation of the amide fragment,
a new compound was devised conserving the dihydronaph-
thalene nucleus but introducing a flexible methylene spacer,
thus allowing close superposition of the aromatic and polar
pharmacophore elements on those of cis-4-P-PDOT (Figure 6).
agonists, prompted the superposition of accessible conforma-
tions of 8-methoxy-4-P-PDOT on the recently proposed active
conformation of melatonin. The best matches of the common
binding elements were observed for the most populated
conformation of the (2S,4S)-cis-tetralin derivative and for an
energetically unfavored conformation of the (2R,4S)-trans one.
Binding studies on each single 4-P-PDOT stereoisomer and on
appropriately designed, conformationally constrained dihydro-
naphthalene derivatives validated the proposed pharmacophore
model.
EXPERIMENTAL SECTION
Chemistry. Melting points were determined on a Buchi B-540
capillary melting point apparatus and are uncorrected. H NMR (200
■
1
or 600 MHz) and 13C NMR (50 MHz) spectra were recorded on a
Bruker AVANCE 200 or on a Varian INOVA 600 MHz spectrometer,
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) 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. ESI-MS spectra
were taken on a Waters Micromass Zq instrument; only molecular
ions (M + 1) are given. Infrared spectra were obtained on a Nicolet
Avatar 360 FT-IR spectrometer; absorbances are reported in cm−1
.
Elemental analyses for C, H, and N were performed on a Carlo Erba
analyzer. Purity of tested compounds was greater than 95%. Column
chromatography purifications were performed under “flash” conditions
using Merck 230−400 mesh silica gel. Analytical thin-layer
chromatography (TLC) was carried out on Merck silica gel 60 F254
plates. The two radioligands 2-[125I]iodomelatonin (specific activity,
2000 Ci/mmol) and [35S]GTPγS ([35S]guanosine 5′-O-(3-thiotriphos-
phate); specific activity, 1000 Ci/mmol) were purchased from Perkin-
Elmer (Italy).
General Procedure for the Synthesis of Tetralones 2a−c. Freshly
distilled styrene (0.69 mL, 6 mmol) in dry dichloromethane (70 mL)
was added dropwise under nitrogen to an ice-cooled and stirred
suspension of anhydrous aluminum chloride (1.6 g, 12 mmol) and the
suitable phenylacetyl chloride (6 mmol) in dry dichloromethane (80 mL)
(for the synthesis of tetralone 2b we needed to change the adding
order of the reagents: anhydrous aluminum trichloride was added to a
cooled solution of styrene and 2-methoxyphenylacetyl chloride42).
The mixture was stirred at 0 °C for a further 15 min, quenched by
water, and extracted with CH2Cl2. The organic phases were combined,
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure to afford the crude tetralone which was purified by
flash chromatography (silica gel, cyclohexane−EtOAc, 9:1).
Figure 6. Superposition of cis-(2S,4S)-4b (orange carbons) and 6
(purple carbons).
Again, the predicted recovery of MT2 binding affinity was
observed for 6, which behaved as a moderately selective MT2
antagonist. Although the preferred way to confirm bioactive
conformations is to prepare active compounds with constrained
conformations, in this case we had indirect evidence on the role
of NH orientation: compounds 3a and 3b, where rotation of
the amide group is constrained in an orientation different from
the putative active one, were inactive, while the flexible
analogue 6, topologically different from starting compounds
and yet easily superimposable to the putative active
conformation of 4b, significantly recovered receptor affinity.
(±)-4-Phenyl-3,4-dihydronaphtalen-2(1H)-one (2a). Chemi-
cal physical data were identical with those previously reported.31
(±)-8-Methoxy-4-phenyl-3,4-dihydronaphtalen-2(1H)-one
1
(2b). Oil; 35% yield. ESI MS (m/z): 253 (M + 1). H NMR (200
MHz, CDCl3): δ 2.94 (d, 2H, J = 6.2), 3.53 (d, 1H, J = 21.8), 3.65 (d,
1H, J = 21.8), 3.87 (s, 3H), 4.47 (dd, 1H, J = 6.0), 6.66 (d,1H, J = 7.7),
6.81 (d, 1H, J = 8.1), 7.08−7.35 (m, 6H). 13C NMR (50 MHz,
CDCl3): δ 38.2, 45.4, 46.5, 55.4, 108.4, 120.6, 122.2, 126.9, 127.4,
127.8, 128.8, 139.9, 142.2, 156.8, 209.5.
(±)-8-Bromo-4-phenyl-3,4-dihydronaphtalen-2(1H)-one
1
(2c). Oil; 34% yield. MS (EI): m/z 300−302 (M+), 178 (100). H
CONCLUSIONS
■
NMR (200 MHz, CDCl3): δ 2.92 (dd, 1H, J = 6.2 and J = 15.7), 3.01
(dd, 1H, J = 6.2 and = 15.7), 3.66 (d, 1H, J = 21.4), 3.79 (d, 1H, J =
21.4), 4.50 (dd, 1H, J = 6.2), 6.81−7.12 (m, 5H), 7.30−7.34 (m, 2H),
7.54 (dd, 1H, J = 1.4 and J = 7.6). 13C NMR (50 MHz, CDCl3): δ
44.7, 45.6, 46.1, 124.6, 127.2, 127.7, 128.1, 128.2, 129.0, 131.3, 133.2,
141.2, 141.4, 207.7.
General Procedure for the Synthesis of Enamides 3a−c. A
solution of the suitable tetralone 2a−c (6.8 mmol), propionamide (1.2
g, 16.4 mmol), and p-toluenesulfonic acid (0.13 g, 0.7 mmol) in
toluene (30 mL) was refluxed for 4 h under nitrogen atmosphere in a
50 mL round-bottom flask equipped with a Dean−Stark apparatus.
After the mixture was cooled to room temperature, the propionamide
The present work reports some evidence supporting the
hypothesis that melatonin receptor agonists and MT2-selective
antagonists or partial agonists share the same arrangement of
their common pharmacophore elements. In particular, a 8-
methoxy group on the scaffold of 4-P-PDOT conferred a
significant increase of binding affinity, similar to what had been
reported for the nonselective agonist 8-M-PDOT, conserved
the cis > trans affinity rank, and could be bioisosterically
replaced by a bromine atom. The effect of this group,
suggesting the same binding role as in melatonin receptor
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was precipitated and filtered off. The organic phase was washed with a
saturated solution of NaHCO3 (3 × 50 mL) and water (3 × 50 mL),
dried over Na2SO4, and concentrated under reduced pressure to give a
crude residue that was purified by flash chromatography (silica gel,
cyclohexane−EtOAc, 7:3) and crystallization.
(±)-N-(4-Phenyl-3,4-dihydronaphtalen-2-yl)propionamide
(3a). Chemical physical data were identical with those previously
reported.31
(±)-N-(8-Methoxy-4-phenyl-3,4-dihydronaphtalen-2-yl)-
propionamide (3b). White solid, mp 195−196 °C (acetone/Et2O);
52% yield. ESI MS (m/z): 308 (M + 1). 1H NMR (200 MHz, CDCl3):
δ 1.18 (t, 3H, J = 7.4), 2.29 (q, 2H, J = 7.4), 2.74 (dd, 1H, J = 8.6 and
J = 16.4), 2.84 (dd, 1H, J = 7.2 and J = 16.4), 3.86 (s, 3H), 4.19 (dd,
1H, J = 8.6 and J = 7.2), 6.44 (d, 1H, J = 7.6), 6.54 (br s, 1H), 6.74 (d,
1H, J = 8.2), 7.00 (dd, 1H, J = 7.2 and J = 8.5), 7.20−7.35 (m, 6H).
13C NMR (50 MHz, CDCl3): δ 9.5, 30.8, 35.3, 44.4, 55.5, 109.0, 120.0,
145.4, 173.1. IR (cm−1, Nujol): 3302, 1639. Anal. (C19H20BrNO) C,
H, N.
4-Phenyl-3,4-dihydronaphtalene-2-carbonitrile (5). A solu-
tion of 2a (0.75 g, 3.38 mmol), trimethylsilyl cyanide (0.58 mL, 4.35
mmol), and ZnI2 (0.027 g, 0.081 mmol) in toluene (2.5 mL) was
stirred at room temperature for 18 h. Pyridine (5 mL) and afterward
POCl3 (1 mL, 11 mmol) were added to the above solution, and the
resulting mixture was refluxed for 1 h. After cooling, the reaction
mixture was poured into 5% HCl/ice and the aqueous phase was
extracted with EtOAc. The combined organic extracts 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, 38% yield. ESI MS
1
(m/z): 232 (M + 1). H NMR (200 MHz, CDCl3): δ 2.81 (ddd, 1H,
J = 1.5, 9.5 and J = 16.8), 2.90 (ddd, 1H, J = 1.5, 7.5 and J = 16.8), 4.25
(dd, 1H, J = 7.5 and J = 9.5), 6.80−7.65 (m, 10H).
N-[(4-Phenyl-3,4-dihydronaphthalen-2-yl)methyl]propion-
amide (6). A 1 M solution of LiAlH4 in diethyl ether (1.3 mL,
1.3 mmol) was added dropwise to a solution of 5 (0.3 g, 1.3 mmol) in
dry diethyl ether (4 mL). The reaction mixture was stirred at room
temperature for 15 min and then quenched by water and a 15% NaOH
aqueous solution. The mixture was extracted with EtOAc, and the
combined organic phases were washed with brine, dried (Na2SO4),
and concentrated under reduced pressure. The obtained crude residue
was dissolved in THF (14 mL), and to the mixture were added TEA
(0.23 mL, 1.64 mmol) and propionic anhydride (0.21 mL, 1.64 mmol).
The resulting solution was stirred at room temperature for 16 h. After
distillation of the solvent under reduced pressure, the residue was
partitioned between EtOAc and 2 N NaOH. The organic layer was
washed with brine, dried (Na2SO4), and concentrated under reduced
pressure to give a crude residue which was purified by flash chroma-
tography (silica gel, CH2Cl2−acetone, 95:5) and crystallization.
White solid, mp 118−120 °C (EtOAc−petroleum ether); 46%
123.6, 126.4, 126.6, 128.3, 128.5, 130.0, 133.0, 136.5, 143.6, 154.7,
172.1. IR (cm−1, Nujol): 3344, 1662. Anal. (C20H21NO2) C, H, N.
(±)-N-(8-Bromo-4-phenyl-3,4-dihydronaphtalen-2-yl)-
propionamide (3c). White solid, mp 133−134 °C (acetone/
petroleum ether); 55% yield. MS (EI): m/z 355−357 (M+), 57
1
(100). H NMR (200 MHz, acetone-d6): δ 1.18 (t, 3H, J = 7.5), 2.31
(q, 2H, J = 7.5), 2.76 (dd, 1H, J = 8.3 and J = 16.4), 2.87 (dd, 1H, J =
8.3 and J = 16.4), 4.19 (dd, 1H, J = 8.3), 6.46 (br s, 1H), 6.75−6.90
(m, 3H), 7.16−7.47 (m, 6H). 13C NMR (50 MHz, CDCl3): δ 9.3,
30.6, 35.1, 44.8, 109.7, 121.9, 126.6, 126.7, 126.9, 128.2, 128.6, 131.2,
134.1, 135.9, 137.7, 142.8, 172.3.
General Procedure for the Synthesis of Tetralines 4a−c. Triethyl-
silane (0.4 mL, 2.5 mmol) was added dropwise to a cooled (−10 °C)
solution of the suitable enamide 3a−c (0.25 mmol) in CF3COOH
(3.25 mL), and the resulting mixture was stirred for a further 10 min at
−10 °C. A saturated aqueous solution of NaHCO3 was carefully added
until neutral pH, and the resulting mixture was extracted with CH2Cl2
(3 × 50 mL). The combined organic extracts were washed with brine
(3 × 50 mL), dried over Na2SO4, and concentrated under reduced
pressure to give a crude residue that was purified by flash
cromatography (silica gel, cyclohexane−ethyl acetate, 7:3).
1
yield. MS (EI): m/z 291 (M+), 218 (100). H NMR (200 MHz,
CDCl3): δ 1.10 (t, 3H, J = 7.6), 2.14 (q, 2H, J = 7.6), 2.51 (dd, 1H,
J = 8.0 and J = 16.5), 2.67 (dd, 1H, J = 7.0 and J = 16.5), 3.90 (dd,
1H, J = 4.5, and J = 15.0), 4.03 (dd, 1H, J = 6.5, and J = 15.0), 4.16
(dd, 1H, J = 7.0, and J = 8.0), 5.25 (br t, 1H), 6.41 (s, 1H), 6.89
(d, 1H, J = 6.8), 7.12−7.33 (m, 8H). 13C NMR (50 MHz, CDCl3):
δ 9.8, 29.7, 34.0, 43.9, 44.3, 124.1, 126.2, 126.5, 127.0, 127.4, 127.9,
128.2, 128.4, 134.0, 135.6, 137.0, 144.0, 173.7. IR (cm−1, Nujol):
3307, 1643. Anal. (C20H21NO) C, H, N.
(±)-cis-N-(4-Phenyl-1,2,3,4-tetrahydronaphtalen-2-yl)-
propionamide (4a, 4-P-PDOT). Chemical physical data were
identical with those previously reported.31
(±)-cis-N-(8-Methoxy-4-phenyl-1,2,3,4-tetrahydronaphta-
len-2-yl)propionamide (4b). Mixture of cis and trans diastereomers
(cis/trans ratio 80/20 by HPLC analysis) was obtained from which the
pure (±)-cis-4b can be obtained by a single recrystallization (acetone/
n-hexane). White solid, mp 220−222 °C; 60% yield. ESI MS (m/z):
(±)-cis-8-Methoxy-4-phenyl-1,2,3,4-tetrahydronaphtalen-2-
ol (7). NaBH4 (0.105 g, 2.8 mmol) was added portionwise to an ice-
cooled stirred solution of 2b (0.2 g, 0.8 mmol) in dry MeOH (6 mL),
and the resulting mixture was heated at reflux for 16 h. After the
mixture was cooled to room temperature, water was added and MeOH
was removed by distillation under reduced pressure. The residue was
extracted with CH2Cl2 (3 × 50 mL). The combined organic phases
were washed with brine, dried (Na2SO4), and concentrated to afford
the crude alcohols (cis/trans, 85/15, by 1H NMR analysis) which were
purified by flash chromatography (silica gel, cyclohexane−EtOAc, 7:3).
1
310 (M + 1). H NMR (600 MHz, CDCl3): δ 1.10 (t, 3H, J = 7.2,
CH2CH3), 1.71 (ddd, 1H, J = 12.0, H3b), 2.31 (m, 2H, CH2CH3),
2.34 (ddd, 1H, J = 2.4, 4.8, and 12.0, H3a), 2.38 (dd, 1H, J = 10.8 and
16.8, H1b), 3.29 (dd, 1H, J = 5.4 and 16.8, H1a), 3.79 (s, 3H, OCH3),
4.19 (dd, 1H, J = 4.8 and 12.0, H4), 4.29 (m, 1H, H2), 5.38 (br d, 1H,
J = 7.2, NH), 6.50 (d, 1H J = 7.8, H5), 6.64 (d, 1H, J = 8.7, H7), 6.97
(dd, 1H, J = 7.8 and 8.4, H6), 7.10 (d, 2H, J = 7.8, Ho), 7.18 (dd, 1H,
J = 7.2, Hp), 7.25 (dd, 2H, J = 7.2, Hm). 13C NMR (50 MHz, CDCl3):
δ 9.9, 29.9, 30.7, 39.9, 45.5, 46.2, 55.3, 107.2, 121.6, 124.1, 126.4,
128.4, 128.5, 128.6, 140.1, 146.0, 156.9, 173.1. IR (cm−1, Nujol): 3336,
1646. Anal. (C20H23NO2) C, H, N.
1
Oil, 59% yield. ESI MS (m/z): 255 (M + 1). H NMR (±)-cis-isomer
(200 MHz, CDCl3): δ 1.64 (brs, 1H), 1.91 (ddd, 1H, J = 12.0), 2.37
(ddd, 1H, J = 3.0, 5.5 and J = 12.0), 2.55 (dd, 1H, J = 10.0 and J =
16.5), 3.37 (dd, 1H, J = 5.5 and J = 16.5), 3.86 (s, 3H), 4.10−4.21 (m,
2H), 6.37 (d, 1H, J = 7.8), 6.69 (d, 1H, J = 7.8), 7.01 (t, 1H, J = 7.8),
7.15−7.38 (m, 5H). 13C NMR (50 MHz, CDCl3): δ 33.4, 42.6, 46.5,
55.4, 67.9, 107.3, 121.3, 126.3, 126.4, 128.5, 128.7, 140.2, 146.0, 157.0,
157.9.
(±)-cis-N-(8-Bromo-4-phenyl-1,2,3,4-tetrahydronaphtalen-
2-yl)propionamide (4c). Mixture of cis and trans diastereomers (cis/
trans ratio 90/10 by HPLC analysis) was obtained from which the
pure (±)-cis-4c can be obtained by a single recrystallization (acetone/
n-hexane). White solid, mp 196−197 °C; 54% yield. ESI MS (m/z):
(±)-cis-8-Methoxy-4-phenyl-1,2,3,4-tetrahydronaphtalen-2-
yl-4-methylbenzensulfonate (8). A solution of p-toluensulfonyl
chloride (0.15 g, 0.78 mmol) in dry pyridine (1.15 mL) was added to a
solution of (±)-cis-7 (0.100 g, 0.39 mmol) in dry pyridine (1.15 mL),
and the resulting mixture was stirred at 5 °C for 3 days. The mixture
was poured into ice−water. The resulting precipitate was filtered,
washed with water, and dried to afford the desired cis-tosylate which
was used for the next step without any further purification. Amorphous
1
358−360 (M + 1). H NMR (200 MHz, CDCl3): δ 1.16 (t, 3H, J =
7.6), 1.76 (ddd, 1H, J = 12.0), 2.21 (q, 2H, J = 7.6), 2.35 (ddd, 1H, J =
3.0, J = 5.0, and J = 12.0), 2.59 (dd, 1H, J = 11.3 and J = 16.0), 3.39
(dd, 1H, J = 5.0 and J = 16), 4.25 (dd, 1H, J = 5.0 and J = 12), 4.41
(dddd, 1H, J = 3.0, 5.0, 8.5 and J = 12), 5.49 (br d, 1H, J = 8.4), 6.75
(d, 1H, J = 8.0), 6.92 (dd, 1H, J = 8.0), 7.10−7.15 (m, 2H), 7.21−7.44
(m, 4H). 13C NMR (50 MHz, CDCl3): δ 9.8, 29.8, 37.6, 39.8, 45.6,
46.6, 125.3, 126.6, 127.3, 128.6, 128.7, 128.8, 130.4, 134.6, 141.6,
1
solid; 82% yield. ESI MS (m/z): 426 (M + NH4). H NMR (±)-cis-
isomer (200 MHz, CDCl3): δ 2.09 (ddd, 1H, J = 12.0), 2.31−2.40 (m,
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1H), 2.46 (s, 3H), 2.79 (dd, 1H, J = 10.5, J = 16.5), 3.28 (dd, 1H, J =
16.5 and J = 6.0), 3.81 (s, 3H), 4.09 (dd, 1H, J = 5.5 and J = 12.0),
4.85 (dddd, 1H, J = 6.0, 10.5 and J = 12.0), 6.32 (d, 1H, J = 7.9), 6.66
(d, 1H, J = 7.9), 7.00 (t, 1H, J = 7.9), 7.09−7.31 (m, 5H), 7.35 (d, 2H,
J = 8.3), 7.83 (d, 2H, J = 8.3).
expressing human MT1 or MT2 receptors, melatonin 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 melatonin (100 nM). Compounds were
added at three different concentrations (one concentration was
equivalent to 100 nM melatonin, a second one 10 times smaller, and a
third one 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 to that of melatonin. It
was assumed that at the equivalent concentration the test compound
occupies the same number of receptors as 100 nM melatonin. 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 melatonin as
measured in the same experiment. By convention, the natural ligand
melatonin has an efficacy (Emax) of 100%. Full agonists stimulate
[35S]GTPγS binding with a maximum efficacy, close to that of
melatonin itself. If Emax is between 30% and 70% that of melatonin
(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.37
(±)-trans-N-(8-Methoxy-4-phenyl-1,2,3,4-tetrahydronaphta-
len-2-yl)propionamide (10). A solution of sodium azide (0.106 g,
1.7 mmol) in water (1 mL) was added dropwise to a stirred solution of
8 (0.13 g, 0.32 mmol) in DMF (1.1 mL), and the resulting mixture
was stirred for 5 h at 45−50 °C. The reaction mixture was poured into
ice−water and extracted with CH2Cl2 (3 × 50 mL). The combined
organic extracts were washed with brine, dried (Na2SO4), and
concentrated to afford the crude azido intermediate 9 which was
sufficiently pure to be used without any further purification. EI MS
1
(m/z): 279 (M+), 236 (M+, 100). H NMR (±)-trans-isomer (200
MHz, CDCl3): δ 2.17 (dd, 1H, J = 6.0 and J = 14.0), 2.38−2.47 (m,
1H), 2.73−2.86 (m, 1H), 3.18 (dd, 1H, J = 5.0 and J = 18.0), 3.88 (s,
3H), 3.88−4.04 (m, 1H), 4.35 (t, 1H, J = 6.0), 6.54 (d, 1H, J = 7.8),
6.74 (d, 1H, J = 7.8), 6.97−7.31 (m, 6H).
A solution of the above crude azide 9 in i-PrOH (7.8 mL) and
CH2Cl2 (0.4 mL) was hydrogenated in the presence of 10% Pd/C at 4 atm
of H2 for 16 h at room temperature. The catalyst was removed by
filtration on Celite. The filtrate was concentrated to afford a crude oily
amine, which was used without any further purification. TEA (0.06
mL, 0.43 mmol) and propionic anhydride (0.05 mL, 0.4 mmol) were
added to a cold solution of the above crude amine in THF (4.5 mL),
and the resulting mixture was stirred at room temperature for 4 h. The
solvent was removed by distillation under reduced pressure, and the
resulting crude amide was purified by flash chromatography (silica gel,
CH2Cl2−acetone, 95:5). Amorphous solid; 25% overall yield. ESI MS
(m/z): 310 (M + 1). 1H NMR (600 MHz, CDCl3): δ 1.09 (t, 3H, J =
7.8, CH2CH3), 1.99 (ddd, 1H, J = 3.0, 7.2 and 12.6, H3a), 2.12 (q, 2H,
J = 7.6, CH2CH3), 2.17 (ddd, 1H, J = 6.0, 7.8 and 12.6, H3b), 2.56
(dd, 1H, J = 6.0 and J = 17.4, H1b), 3.16 (dd, 1H, J = 5.4 and J = 17.4,
H1a), 3.80 (s, 3H, OCH3), 4.17 (dd, 1H, J = 6.6, H4), 4.32 (m, 1H,
H2), 5.40 (br d, 1H, J = 7.2, NH), 6.50 (d, 1H, J = 7.8, H5), 6.67 (d,
1H, J = 7.8, H7), 7.02 (d, 2H, J = 7.2, Ho), 7.04 (t, 1H, J = 7.8, H6),
7.15 (dd, 1H, J = 7.2, Hp), 7.22 (dd, 2H, J = 7.8, Hm). 13C NMR (50
MHz, CDCl3): δ 9.8, 29.80, 29.83, 36.9, 42.2, 43.0, 55.3, 107.3, 122.2,
123.7, 126.5, 126.6, 128.4, 128.7, 139.0, 146.0, 157.4, 173.2. Anal.
(C20H23NO2) C, H, N.
NMR Spectroscopy. NMR spectra of compounds 4b and 10 were
recorded on a Varian INOVA 600 MHz spectrometer. The sample
concentration used in NMR studies was about 5 mM dissolved in
deuterated chloroform. 1D and 2D NMR spectra were measured at
298 K. 1H chemical shifts were given on the δ scale and were calibrated
to the residual solvent signal of CDCl3 at δ 7.22. NOESY spectra are
reported in Supporting Information (Figures S1 and S2).
Molecular Modeling. Molecular modeling studies were per-
formed with Macromodel 9.7.47 Starting structures of 4b and 10
stereoisomers were optimized with the MM2 force field,48 using a
convergence criterion of 0.2 kJ mol−1 Å−1, applying the GB/SA49
CHCl3 solvation treatment. Stochastic dynamics (SD) simulations
were performed employing the MM2 force field with implicit CHCl3,
applying a time step of 1 fs and a temperature of 298 K for 1 μs after
1000 ps of equilibration; 50 000 snapshots were collected for data
analysis. SD had already proved to be able to reproduce the
conformational equilibrium of small molecules in CHCl3, applying
an implicit treatment of solvent molecules.50−56 Preliminary SD
simulations performed on both enantiomers of 4b and 10 gave
comparable results for each couple of enantiomers in terms of relative
abundances of different conformations. Therefore, full-length SD
simulations were conducted on two reference enantiomers only, i.e.,
(2S,4S)-4b and (2R,4S)-10. For the trans derivative (2R,4S)-10,
different starting conformations were tested, with axial amide side
chain and pseudoequatorial phenyl substituent or with equatorial
amide side chain and pseudoaxial phenyl substituent. The starting
point did not significantly affect the results.
Proton−proton coupling constants of 4b and 10 were calculated
with the software Maestro 9.0,57 applying the implemented version of
the Karplus equation58 to the dihedrals of single conformations
collected during SD simulations. The expected values of coupling
constants reported in Table 3 are average values for 50 000
conformations, unless otherwise indicated.
Superposition of compounds was performed by means of a rigid fit
procedure, superimposing the four atoms of the amide function, the
centroid of the phenyl rings, and the oxygen atom of the methoxy
group, when present.
Pharmacology. Binding affinities 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 characterization of NIH3T3-MT1 and -MT2 cells
had been already described in detail.21,43 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.44 2-[125I]Iodomelatonin (100 pM) and
different concentrations of melatonin (10−10−10−6 M) or of the new
compounds were incubated with the receptor preparation for 90 min
at 37 °C. Nonspecific binding was assessed with 10 μM melatonin;
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.45 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 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.43,46 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 melatonin
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
ASSOCIATED CONTENT
■
S
* Supporting Information
NOESY spectra for compounds 4b and 10, results of stochastic
dynamics simulations on 4b and 10, and elemental analytical
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
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dx.doi.org/10.1021/jm200790v | J. Med. Chem. 2011, 54, 8362−8372

Journal of Medicinal Chemistry
Article
Enzyme Catalysis and Regulation. J. Biol. Chem. 2000, 275, 31311−
31317.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +39 0521 905062. Fax: +39 0521 905006. E-mail:
silvia.rivara@unipr.it.
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ACKNOWLEDGMENTS
■
We are grateful to the Centro Interdipartimentale Misure
“Giuseppe Casnati” of the University of Parma, Italy, for
providing NMR instrumentation.
ABBREVIATIONS USED
■
8-M-PDOT, 8-methoxy-2-propionamidotetralin; 4-P-PDOT, 4-
phenyl-2-propionamidotetralin; cAMP, cyclic adenosine mono-
phosphate; DMF, dimethylformamide; GTPγS, guanosine 5′-O-
(3-thiotriphosphate); IAr, relative intrinsic activity; MT1,
melatonin receptor subtype 1; MT2, melatonin receptor
subtype 2; NOE, nuclear Overhauser effect; NOESY, nuclear
Overhauser effect spectroscopy; PTSA, p-toluenesulfonic acid;
SAR, structure−activity relationship; SD, stochastic dynamics;
TEA, triethylamine; THF, tetrahydrofuran; TFA, trifluoroacetic
acid
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NOTE ADDED AFTER ASAP PUBLICATION
■
After this paper was published online November 18, 2011,
several minor corrections were made to the text. The corrected
version was published November 28, 2011.
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