Bivalent ligand approach on N-{2-[(3-methoxyphenyl)methylamino]ethyl} acetamide: Synthesis, binding affinity and intrinsic activity for MT1 and MT2 melatonin receptors

Article abstract of DOI:10.1016/j.bmc.2011.06.063

We report the synthesis, binding properties and intrinsic activity at MT₁ and MT₂ melatonin receptors of new dimeric melatonin receptor ligands in which two units of the monomeric agonist N-{2-[(3-methoxyphenyl)methylamino]ethyl}acet

Full text of DOI:10.1016/j.bmc.2011.06.063

Bioorganic & Medicinal Chemistry 19 (2011) 4910–4916
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bivalent ligand approach on N-{2-[(3-methoxyphenyl)methylamino]ethyl}-
acetamide: Synthesis, binding affinity and intrinsic activity for
MT1 and MT2 melatonin receptors
Gilberto Spadoni a, , Annalida Bedini a, Pierfrancesco Orlando a, Simone Lucarini a, Giorgio Tarzia a,
⇑
Marco Mor b, Silvia Rivara b, Valeria Lucini c, Marilou Pannacci c, Francesco Scaglione c
a Dipartimento di Scienze Biomolecolari, Università degli Studi di Urbino ‘Carlo Bo’, Piazza Rinascimento 6, 61029 Urbino, Italy
b Dipartimento Farmaceutico, Università degli Studi di Parma, V.le G.P. Usberti 27A, Campus Universitario, 43124 Parma, Italy
c Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Università degli Studi di Milano, Via Vanvitelli 32, 20129 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis, binding properties and intrinsic activity at MT1 and MT2 melatonin receptors of
new dimeric melatonin receptor ligands in which two units of the monomeric agonist N-{2-[(3-methoxy-
phenyl)methylamino]ethyl}acetamide (1) are linked together through different anchor points. Dimeriza-
tion of compound 1 through the methoxy substituent leads to a substantial improvement in selectivity
for the MT1 receptor, and to a partial agonist behavior. Compound 3a, with a trimethylene linker, was
the most selective for the MT1 subtype (112-fold selectivity) and compound 3d, characterized by a
hexamethylene spacer, had the highest MT1 binding affinity (pKiMT1 = 8.47) and 54-fold MT1-selectivity.
Dimerization through the aniline nitrogen of 1 abolished MT1 selectivity, leading to compounds with
either a full agonist or an antagonist behavior depending on the nature of the linker.
Received 29 April 2011
Revised 21 June 2011
Accepted 22 June 2011
Available online 28 June 2011
Keywords:
Melatonin
Melatoninergic dimers
MT1 and MT2 receptors
Bivalent ligand
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
superfamily and exhibit subnanomolar affinity for MLT.10 Elucida-
tion of the distinct functions of MT1 and MT2 receptors in many
Melatonin (N-acetyl-5-methoxytryptamine, MLT, Fig. 1) is a
neurohormone primarily secreted by the pineal gland at night in
all species.1 The ability of MLT to synchronize the ‘circadian biolog-
ical clock’, by its direct action on the suprachiasmatic nucleus has
led to the investigation of MLT and its analogs as a remedy for
treating disordered circadian rhythms that occur in jet lag, shift
work, certain types of insomnia, and some neuropsychiatric dis-
eases. Some MLT receptor agonists are currently under clinical
evaluation or have been very recently approved. The MT1/MT2 mel-
atonin receptor agonist ramelteon (RozeremÒ) was approved and
launched in 2005 in the U.S. for the treatment of primary insom-
nia,2 and other compounds,3 such as Neu-P11,4 TIK-3015 or tasi-
melteon6 are undergoing evaluation in clinical trials for their
hypnotic properties. Moreover, the naphthalenic MLT bioisostere
agomelatine is a novel antidepressant with an innovative pharma-
cological profile (MT1/MT2 agonist and 5HT2c antagonist) which
was recently approved by the EU-EMA for the treatment of major
depressive disorders and is available in several European coun-
tries.7–9 In mammals, two melatonin receptors, MT1 and MT2, have
been identified. They belong to the G-protein-coupled receptor
target tissues is still under investigation and requires a continual
development of specific and selective affinity ligands. Whereas
some selective MT2 receptor ligands have been recently de-
scribed,11,12 the limited availability of MT1 subtype-selective li-
gands has hampered an exhaustive elucidation of the MT1
receptor patho/physiological role. Although, a few monomeric li-
gands displaying moderate MT1-selectivity were reported,13 the
most applied approach for the design of MT1 selective receptor li-
gands relies in the preparation of symmetric dimers, by coupling
two moieties deriving from known MLT receptor ligands.
Accumulating evidence indicates that most GPCRs (classically
considered to function as monomers) exist as functional dimers
or higher oligomeric units.14 Oligomerization may occur in na-
tive tissues and may have important consequences on receptor
function. As evidenced for several other GPCRs the formation
of MT1 and MT2 homodimers and MT1/MT2 heterodimers has
been shown in heterologous expression systems at physiological
expression levels.15,16 Although MT1/MT2 heterodimers remain to
be identified in native tissues, their formation has to be taken
into account by virtue of the documented co-expression of MT1
and MT2 receptors in many melatonin-sensitive tissues, such as
the hypothalamic suprachiasmatic nucleus, retina, arteries, and
adipose tissue.17,18
⇑
Corresponding author. Tel.: +39 0722 303337; fax: +39 0722 303313.
E-mail address: gilberto.spadoni@uniurb.it (G. Spadoni).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.063

G. Spadoni et al. / Bioorg. Med. Chem. 19 (2011) 4910–4916
4911
H
H
N
NHCOCH3
N
C
CO2 (CH2)n O2
H3CO
OCH3
H3CO
N
NHCOCH3
H
NHCOCH3
MLT
Indole dimers
NHCOCH3
NHCOCH3
NHCOCH3
O
O
(CH2)n
Agomelatine dimers
CH3
H3C
N
N
S 24268
NHCOCH3
NHCOCH3
(CH2)n
N
N
O
O
O
NHCOCH3
NHCOCH3
O
Azaindole dimers
(CH2)4
CO2CH3
Naphthalene heterodimers
Figure 1. Melatonin and dimeric melatonin receptor ligands.
Homodimerization and heterodimerization processes could
generate receptors with novel characteristics, and pharmacological
properties different from any of the monomers, providing new
opportunities for rational drug design and discovery.19 Different
strategies have been developed to specifically target GPCR dimers.
Bivalent ligands, which are composed of two functional pharmaco-
‘bivalent ligand’ approach to this new scaffold by synthesizing
the new dimeric melatonin receptor ligands highlighted in Figure 2.
We linked two units of the monomeric agonist (Fig. 2, R1 = OMe,
R2 = Me)25 through different anchor points, in order to evaluate
the effects of the length and position of the spacers on their ability
to bind to and activate MT1 and MT2 receptors.
phores linked by
approaches.
a spacer, are among the most promising
2. Chemistry
It was postulated that these ligands would have distinct proper-
ties, such as increased selectivity, potency and different efficacy,
when compared to the activity of each monomer; therefore, there
seems to be a great potential in developing new leads compounds
by linking two single chemical entities to generate bivalent ligands.
To date, only few reports on dimeric molecules designed to target
MLT-receptors appeared in the literature. Dimers of melatonin20
and agomelatine21 were prepared and tested for their activity on
MT1 and MT2 melatonin receptors, showing interesting pharmaco-
logical profiles. Melatonin dimers linked through position 2 of the
indole ring showed greater MT1 and MT2 binding affinity than di-
mers linked through the acylamino side chain. The most potent
derivatives displayed nanomolar affinity for MT1 and MT2 recep-
tors, while the intrinsic activity strongly varied with the linker
length. Agomelatine dimers are characterized by a polymethylene
chain connecting the oxygen atoms in position 7 on the naphtha-
lenic nucleus. These derivatives showed a moderate to good MT1-
selectivity, with an antagonist behavior reported for the compound
with a trimethylene linker. Other azaindoles22 or naphthalene23 di-
meric melatonin receptor ligands were described, but they showed
lower affinity and weaker selectivity when compared to the ago-
melatine dimers (Fig. 1). Following the same ‘bivalent ligands’ ap-
proach, a series of novel asymmetric heterodimers was recently
reported to be selective partial agonists (Ki MT2/MT1 = 70–90) with
subnanomolar affinity (Fig. 1).24
The synthesis of the new compounds is described in Schemes 1
and 2. Key intermediate in the synthesis of the target compounds
3a–f and 4 is the phenol derivative 2, obtained from the previously
described methoxy analog 125 by cleavage of the methyl ether
using boron tribromide. Homodimers 3a–f were prepared by reac-
tion of the phenol derivative 2 with 0.5 equiv of the appropriate
dibromoalkane in the presence of K2CO3 in acetonitrile. Small
amounts (ca. 10–12%) of
a by-product (N-[2-(3-allyoxyphe-
nyl)methylamino)ethyl]acetamide) resulting from HBr elimination
of the monoalkylated starting material 2, was also observed during
the homodimerization. The monovalent ligand 4 was obtained by
O-alkylation of the phenol 2 with 6-bromo-1-phenoxyhexane26
in the presence of sodium hydride as a base.
The dimeric ligands linked through the aniline nitrogen (5a–b
and 6) were synthesized by N-alkylation, or N-acylation, of N-[2-
(3-methoxyphenylamino)ethyl]acetamide25 with 0.5 equiv of the
opportune dibromoalkane or adipoyl chloride, respectively
(Scheme 2).
3. Biological results and discussion
The chemical structures, binding affinities at MT1 and MT2
receptors and the intrinsic activity of the new compounds 3a–f, 4,
5a–b and 6 are reported in the Table 1. Length, position and chem-
ical properties of the spacer connecting each pharmacophore signif-
icantly affect affinity and activity at melatonin receptors. We
decided to connect the two anilinoethylamides by inserting the
Recently, we reported a new class of high affinity melatonin
receptor ligands, structurally characterized by a N-(substituted-
anilinoethyl)amide scaffold (A, Fig. 2).25 We decided to apply the

4912
G. Spadoni et al. / Bioorg. Med. Chem. 19 (2011) 4910–4916
R1
O
(CH2)n
O
OCH3
OCH3
NHCOCH3
NHCOCH3
N
N
X
N
N
H3C
H3C
N
X
R2
(CH2)n
(A)
NHCOCH3
NHCOCH3
X=CH2, CO
NHCOCH3
(B)
(C)
Figure 2. Approaches to new dimeric melatonin receptor ligands.
OCH3
OH
O
(CH2)n
O
NHCOCH3
NHCOCH3
b
a
N
N
N
N
4
H3C
H3C
CH3
CH3
1
2
NHCOCH3
NHCOCH3
5 6 8 10
c
n
3
3a-f
3a 3b 3c 3d 3e 3f
O(CH2)6O
NHCOCH3
N
CH3
4
Scheme 1. Reagents and conditions: (a) BBr3, CH2Cl2, rt; (b) Br(CH2)nBr, K2CO3, CH3CN, reflux; (c) NaH, PhO(CH2)6Br, DMF.
more complex manner. All these dimers exhibited lower affinity
OCH3
OCH3
than the monomer 1, but, interestingly, they showed pronounced
MT1 subtype selectivity. The best MT1 affinity values were obtained
for the dimers with 6 and 10 methylene units (compounds 3d and
3f, pKi = 8.47 and 8.40, respectively). The MT1 affinity of compound
3d was slightly lower than that of the monomer 1 (four-fold), but it
displayed an 54-fold selectivity for the MT1 receptor, while mono-
mer 1 was a non-selective ligand. The SARs observed for this series
of compounds resembled those for agomelatine analogs21, even if
agomelatine derivatives were reported to be generally more potent
and more MT1-selective than our compounds. In particular, also in
the agomelatine series the most selective compounds had a tri-
methylene linker (KiMT2/KiMT1 = 224), while longer chains gave sig-
nificant increases in MT1 affinity, with lower selectivity: the
hexamethylene derivative of agomelatine, corresponding to 3d,
has pKiMT1 = 10.1 and KiMT2/KiMT1 = 30. For the sake of comparison,
it should be noted that binding data may also depend on the cell
line employed. For the agomelatine series, tests were performed
on HEK 293 cells, and in those conditions melatonin was more
MT1-selective than in our test (KiMT2/KiMT1 = 4). In order to evaluate
the influence of the second amido pharmacophore unit to the bind-
ing, the monovalent ligand 4, characterized by a phenoxyhexane
fragment, was synthesized. Compound 4, having a hexamethylene
spacer as the most interesting dimer 3d, showed the same MT2
affinity and a significantly lower MT1 affinity, even if its pKi (7.84)
was among the highest values obtained on MT1 receptors for com-
pounds 3a–f. While the irregular trend of pKi values on MT1 recep-
tors does not allow a conclusive assessment of the role of a second
MLT-like fragment, the relatively high MT1 affinity of compound 4
suggests that a bivalent ligand is not necessary to generate MT1
selectivity, as also shown in a recent series of agomelatine ana-
logs.24 Taken together, melatonin receptor affinity values of dimers
3a–f and of the monovalent ligand 4 indicate that the two pharma-
OCH3
NHCOCH3
NHCOCH3
N
a
N
N
H
(CH2)n
b
NHCOCH3
5a: n=6
5b: n=10
OCH3
NHCOCH3
O
OCH3
N
O
(CH2)4
N
6
NHCOCH3
Scheme 2. Reagents and conditions: (a) Br(CH2)nBr, K2CO3, DMF, 100 °C; (b)
adipoyl chloride, TEA, THF, rt.
linker between the methoxy groups (compounds 3a–f) or between
the aniline nitrogens (5a–b and 6) because large substituents in
these positions have been shown to be tolerated.22 Several spacers
have been generally used for dimer formation, including polymeth-
ylene, polyalkyloxy ether, polyesters, etc. We have initially selected
a polymethylene spacer because of the ease of coupling of the phar-
macophores to the spacer and the possibility to modulate the linker
length by small increases. With the aim to find out the optimal
length, we evaluated compounds 3a–f bearing a spacer of 3, 4, 5,
6, 8 and 10 methylene units, respectively. Analysis of MT1 and
MT2 binding data for compounds 3a–f showed a gradual increase
in MT2 affinity with chain length, while MT1 affinity varied in a

G. Spadoni et al. / Bioorg. Med. Chem. 19 (2011) 4910–4916
4913
Table 1
Binding affinity (pKi)a and intrinsic activity (IAr)b of new compounds for human MT1 and MT2 receptors stably expressed in NIH3T3 cells
OCH3
O
(CH2)n
O
OCH3
OCH3
NHCOMe
N
NHCOCH3
O
O(CH2)6O
OCH3
NHCOCH3
N
N
N
N
H3C
(CH2)n
H3C
O
(CH2)4
N
N
NHCOCH3
NHCOCH3
NHCOCH3
CH3
n=6, 10
n=3, 4, 5, 6, 8, 10
NHCOCH3
3a-f
4
5a-b
6
Compound
n
Human MT1
Human MT2
KiMT2/KiMT1
pKi
IAr SEM
pKi
IAr SEM
MLT
1c
9.58 0.18
1.00 0.01
0.95 0.08
0.26 0.03
0.21 0.03
0.46 0.07
0.44 0.01
0.47 0.02
0.42 0.02
0.49 0.02
ꢀ0.04 0.02
0.12 0.05
0.94 0.08
9.47 0.13
9.19 0.01
5.68 0.10
6.00 0.12
6.09 0.04
6.74 0.09
7.03 0.12
7.39 0.32
6.80 0.07
7.44 0.33
6.51 0.02
6.61 0.22
1.00 0.02
1.06 0.05
0.33 0.04
0.18 0.04
0.64 0.04
0.33 0.06
0.38 0.03
0.57 0.02
0.54 0.04
ꢀ0.09 0.01
0.37 0.12
0.91 0.05
1.3
0.8
102
23
14
54
4.7
10
11
0.05
0.8
0.85
9.09 0.10
7.69 0.14
7.37 0.13
7.24 0.09
8.47 0.05
7.70 0.31
8.40 0.18
7.84 0.07
6.13 0.19
6.42 0.04
6.54 0.35
3a
3b
3c
3d
3e
3f
3
4
5
6
8
10
4
5a
5b
6
6
10
a
pKi values were calculated from IC50 values, obtained from competition curves by the method of Cheng and Prusoff32 and are the mean of at least three determinations
performed in duplicate.
b
The relative intrinsic activity values were obtained by dividing the maximum analogue-induced G-protein activation by that of MLT.
Ref. 25.
c
cophores did not bind at two neighboring MLT binding sites be-
cause none of the dimers showed higher receptor affinity than that
of the monomer 1. The actual presence of receptor dimers has not
been assessed in the transfected NIH3T3 cells employed for our
in vitro tests. However, in these cells the MT1 or MT2 receptor
was expressed at high density levels (>100 fmol/mg protein27,28),
consistent with the formation of dimers, which had been observed
in HEK293 cells at 20–100 fmol/mg protein.15 Therefore, on the ba-
sis of the above data, it can be supposed that both the linker and one
pharmacophore are bound to the receptor in a region of steric tol-
erance, that resulted more suitable in the MT1 binding site. The
compounds show moderate MT2 binding affinity, slightly better
for longer derivatives. This is different from what observed for ago-
melatine dimers, whose derivatives with longer linkers retained
subnanomolar MT2 affinity.21
Compound 6, despite its low binding affinity, behaves as a full ago-
nist at both receptor subtypes.
4. Conclusions
We have synthesized the new MLT receptor ligands 3a–f, 5a–b
and 6, that were designed according to the ‘bivalent ligand’
approach by linking two moieties of the MT1/MT2 melatonin ago-
nist N-{2-[(3-methoxyphenyl)methylamino]ethyl}acetamide (1),
through their methoxy substituent or their aniline nitrogen, by
polymethylene chains of variable length, with the aim to increase
affinity and MT1/MT2 subtype selectivity. The dimers did not show
MLT receptor affinities higher than that of the monomer 1, and the
similar binding profile shown by the asymmetric analog 4 suggests
that they do not interact with two independent recognition sites;
therefore, the bivalent ligand approach failed, in this case, to
achieve more potent compounds. On the contrary, it is clear that
dimerization of compound 1 through the methoxy substituent
leads to an improvement in selectivity for MT1 receptors. Consider-
ing that the development of MT1 melatonin selective ligands can be
still considered a difficult task to achieve, the most selective C3-
dimer 3a (112-fold MT1-selectivity) and dimer 3d with a C6 spacer
(pKiMT1 = 8.47 and 54-fold MT1 preference over MT2 receptors)
could be considered interesting lead molecules in the development
of selective MT1 ligands, the most selective C3-dimer 3a and dimer
3d with a C6 spacer (pKiMT1 = 8.47 and 54-fold MT1 preference over
MT2 receptors) could be considered interesting lead molecules in
the development of selective MT1 ligands.
The functional activity of the new compounds has been evalu-
ated on both receptors (GTPcS assay) in comparison with MLT.
None of the cited compounds (3a–f, 4) showed full agonist activity,
but they behave as partial agonists on both MT1 and MT2 receptors.
We also evaluated the possibility to link the two monomers 1
through their aniline nitrogens rather than their methoxy substitu-
ent, as we previously showed that N-substitution with a large
hydrophobic substituent is favorable for binding to MT2 receptor.25
Compounds 5a and 5b (n = 6 and 10 respectively) displayed affinity
values at MT2 receptor in the same range as the corresponding di-
mers (3d, 3f) connected through the methoxy substituent, but they
exhibited considerably lower MT1 binding affinity. This drop of
MT1 affinity is consistent with the reduced steric tolerance in the
corresponding region of previously developed pharmacophore
and 3D-QSAR models.29 In fact, the shorter derivative, 5a, is a mod-
erately MT2-selective full antagonist, even if the longer derivative,
5b, also loses affinity for the MT2 receptor. The steric arrangement
of the linker chain with respect to the aniline scaffold also proved
important for interaction with the melatonin receptors. Indeed, a
linker connected through two planar amide groups led to a lack
of subtype selectivity and to an increase in intrinsic activity.
5. Experimental section
5.1. Chemistry
Melting points were determined on a Büchi SMP-510 capillary
melting point apparatus and are uncorrected. 1H NMR spectra were

4914
G. Spadoni et al. / Bioorg. Med. Chem. 19 (2011) 4910–4916
recorded on a Bruker AC 200 spectrometer; chemical shifts (d
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). ESI-MS spectra were taken on a Waters Micromass ZQ instru-
ment; only molecular ions (M+1) are given. EI-MS spectra (70 eV)
were taken on a Fisons Trio 1000 spectrometer; only molecular
ions (M+) and base peaks are given. Infrared spectra were obtained
on a Nicolet Avatar 360 FT-IR spectrometer; absorbances are re-
4H), 6.16–6.30 (m, 6H), 7.01 (t, 2H J = 8.0 Hz), 7.95 (br t, 2H). IR
(cmꢀ1, Nujol): 3319,1650, 1616. Anal. Calcd for C27H40N4O4: C,
66.92; H, 8.32; N, 11.56. Found: C, 66.67; H, 8.45; N, 11.84.
5.1.2.4.
N-(2-{[3-(6-{3-[(2-Acetylaminoethyl)methylamino]
phenoxy}hexyloxy)phenyl]methylamino}ethyl)acetamide (3d)
White solid, 48% yield; mp 118–120 °C (EtOAc). ESI-MS (m/z):
499 (M+1). 1H NMR (DMSO-d6): d 1.46 (m, 4H), 1.71 (m, 4H),
1.76 (s, 6H), 2.85 (s, 6H), 3.14 (m, 4H), 3.33 (m, 4H), 3.92 (t, 4H
J = 6.5 Hz), 6.16–6.32 (m, 6H), 7.02 (t, 2H J = 8.0 Hz), 7.93 (br t,
ported in
m
(cmꢀ1). Analyses indicated by the symbols of the ele-
ments (C, H, N) are within 0.4% of the theoretical 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.
2H). IR (cmꢀ1
,
Nujol): 3322, 1650, 1615. Anal. Calcd for
C28H42N4O4: C, 67.44; H, 8.49; N, 11.24. Found: C, 67.52; H, 8.57;
N, 11.08.
5.1.2.5.
N-(2-{[3-(8-{3-[(2-Acetylaminoethyl)methylamino]
5.1.1. N-{2-[(3-Hydroxyphenyl)methylamino]ethyl}acetamide
phenoxy}octyloxy)phenyl]methylamino}ethyl)acetamide (3e)
White solid, 60% yield; mp 108–110 °C (EtOAc). ESI-MS (m/z):
527 (M+1). 1H NMR (DMSO-d6): d 1.35 (m, 8H), 1.67 (m, 4H),
1.76 (s, 6H), 2.85 (s, 6H), 3.14 (m, 4H), 3.30 (m, 4H), 3.89 (t, 4H
J = 6.5 Hz), 6.15–6.30 (m, 6H), 7.01 (t, 2H J = 8.0 Hz), 7.94 (br t,
(2)
A solution of BBr3 (12.6 mmol) in dry CH2Cl2 (60 mL) was added
dropwise to
a solution of N-{2-[(3-methoxyphenyl)methyl-
amino]ethyl}acetamide25 (1.4 g, 6.3 mmol) in dry CH2Cl2 (47 mL)
at 0 °C and the resulting mixture was stirred at room temperature
for 18 h. The solvent was evaporated, the residue was neutralized
with an aqueous saturated solution of NaHCO3 and extracted with
EtOAc. The organic phases were combined, washed once with
brine, dried (Na2SO4) and concentrated under reduced pressure
to give a crude residue, which was purified by flash chromatogra-
phy (CH2Cl2–MeOH, 98:2 as eluent) and crystallization. Pink solid,
76% yield; mp 74–76 °C (Et2O–light pet.). MS (EI): m/z 208 (M+),
136 (100). 1H NMR (CDCl3) is in line with previous report.30
2H). IR (cmꢀ1
,
Nujol): 3321, 1650, 1615. Anal. Calcd for
C30H46N4O4: C, 68.41; H, 8.80; N, 10.64. Found: C, 68.21; H, 8.59;
N, 10.48.
5.1.2.6.
N-(2-{[3-(10-{3-[(2-Acetylaminoethyl)methylamino]
phenoxy}decyloxy)phenyl]methylamino}ethyl)acetamide (3f)
White solid, 63% yield; mp 105–107 °C (EtOAc). ESI-MS (m/z):
555 (M+1). 1H NMR (DMSO-d6): d 1.28 (m, 12H), 1.66 (m, 4H),
1.76 (s, 6H), 2.84 (s, 6H), 3.14 (m, 4H), 3.28 (m, 4H), 3.88 (t, 4H
J = 6.5 Hz), 6.15–6.30 (m, 6H), 7.01 (t, 2H J = 8.0 Hz), 7.94 (br t,
5.1.2. General procedure for the synthesis of dimeric derivatives
3a–f
2H). IR (cmꢀ1
C
,
Nujol): 3320, 1649, 1615. Anal. Calcd for
32H50N4O4: C, 69.28; H, 9.08; N, 10.10. Found: C, 69.20; H, 9.01;
N, 10.26.
K2CO3 (0.300 g, 2.17 mmol) was added to a solution of 2
(0.150 g, 0.72 mmol) in acetonitrile (2.5 mL). The resulting mixture
was refluxed for 30 min and then the required dibromo-derivative
(0.36 mmol) was added dropwise. The reaction mixture was
refluxed 16 h, quenched with water and extracted with EtOAc.
The organic phases were combined, washed once with brine, dried
(Na2SO4) and concentrated under reduced pressure to yield a crude
residue which was purified by flash chromatography
(EtOAc–MeOH, 95:5 as eluent) and crystallization.
5.1.3. N-(2-{Methyl[3-(6-phenoxyhexyloxy)phenyl]amino}
ethyl)acetamide (4)
Sodium hydride (80% in mineral oil, 0.02 g, 0.66 mmol) and 6-
bromo-1-phenoxyhexane26 (0.265 g, 1.03 mmol) were added to a
solution of 2 (0.13 g, 0.62 mmol) in dry DMF (2.6 mL) at ꢀ10 °C un-
der nitrogen atmosphere. The reaction mixture was stirred at room
temperature for 18 h, poured onto ice cooled water and extracted
three times with EtOAc. The organic phases were combined, dried
(Na2SO4), and evaporated to give a crude residue which was puri-
fied by flash chromatography (EtOAc as eluent) and crystallization.
White solid, 79% yield; mp 67–68 °C (Et2O–light pet.). MS (EI): m/z
384 (M+), 94 (100). 1H NMR (CDCl3): d 1.59 (m, 4H), 1.83 (m, 4H),
1.94 (s, 3H), 2.94 (s, 3H), 3.46 (m, 4H), 3.98 (m, 4H), 5.60 (br s, 1H),
6.29–6.40 (m, 3H), 6.88–6.97 (m, 3H), 7.14 (t, 1H J = 8.0 Hz), 7.25–
7.32 (m, 2H). IR (cmꢀ1, Nujol): 3266, 1641, 1611. Anal. Calcd for
5.1.2.1.
N-(2-{[3-(3-{3-[(2-Acetylaminoethyl)methylamino]
phenoxy}propoxy)phenyl]methylamino}ethyl)acetamide (3a)
White solid, 61% yield; mp 120–121 °C (EtOAc–light pet.). ESI-MS
(m/z): 457 (M+1). 1H NMR (DMSO-d6): d 1.76 (s, 6H), 2.11 (m,
2H), 2.85 (s, 6H), 3.14 (m, 4H), 3.31 (m, 4H), 4.07 (t, 4H
J = 6.0 Hz), 6.19–6.32 (m, 6H), 7.03 (t, 2H, J = 8.0 Hz), 7.93 (br t,
2H). IR (cmꢀ1
, Nujol): 3314, 1635, 1614. Anal. Calcd for
C25H36N4O4: C, 67.77; H, 7.95; N, 12.27. Found: C, 67.61; H, 7.80;
N, 12.31.
C23H32N2O3: C, 71.84; H, 8.39; N, 7.29. Found: C, 72.05; H, 8.53;
N, 7.51.
5.1.2.2.
N-(2-{[3-(4-{3-[(2-Acetylaminoethyl)methylamino]
(3b)
phenoxy}butoxy)phenyl]methylamino}ethyl)acetamide
5.1.4. General procedure for the synthesis of dimeric ligands
5a,b
White solid, 29% yield; mp 137–138 °C (EtOAc). ESI-MS (m/z):
471 (M+1). 1H NMR (DMSO-d6): d 1.76 (s, 6H), 1.83 (m, 4H), 2.86
(s, 6H), 3.15 (m, 4H), 3.31 (m, 4H), 3.98 (m, 4H), 6.18–6.32 (m,
6H), 7.03 (t, 2H J = 8.0 Hz), 7.93 (br t, 2H). IR (cmꢀ1, Nujol): 3321,
1649, 1614. Anal. Calcd for C26H38N4O4: C, 66.36; H, 8.14; N,
11.91. Found: C, 66.29; H, 7.95; N, 12.05.
K2CO3 (0.36 g, 2.61 mmol) followed by the opportune dibromo-
derivative (0.87 mmol) were added to a solution of N-[2-(3-
methoxyphenylamino)ethyl]acetamide25 (0.358 g, 1.74 mmol) in
DMF (2 mL). The resulting mixture was heated at 100 °C for 24 h,
quenched with water and extracted with EtOAc. The combined or-
ganic phases were washed once with brine, dried (Na2SO4) and
concentrated under reduced pressure to yield a crude residue
which was purified by flash chromatography (EtOAc as eluent)
and crystallization. About 65% of non-reacted starting aniline was
recovered after chromatography.
5.1.2.3.
N-(2-{[3-(5-{3-[(2-Acetylaminoethyl)methylamino]
phenoxy}pentyloxy)phenyl]methylamino}ethyl)acetamide (3c)
White solid, 49% yield; mp 109–111 °C (EtOAc). ESI-MS (m/z):
485 (M+1). 1H NMR (DMSO-d6): d 1.55 (m, 2H), 1.76 (s, 6H),
1.80 (m, 4H), 2.85 (s, 6H), 3.14 (m, 4H), 3.29 (m, 4H), 3.93 (m,

G. Spadoni et al. / Bioorg. Med. Chem. 19 (2011) 4910–4916
4915
5.1.4.1.
N-{2-[{6-[(2-Acetylaminoethyl)-(3-methoxyphenyl)
(50
was defined using [35S]GTP
human MT1 or MT2 receptors, melatonin produced a concentra-
tion dependent stimulation of basal [35S]GTP
S binding with a
l
M), NaCl (100 mM), and MgCl2 (3 mM). Nonspecific binding
amino]hexyl}-(3-methoxyphenyl)amino]ethyl}acetamide (5a)
White solid, 15% yield; mp 123–124 °C (EtOAc–Et2O). ESI-MS (m/
z): 499 (M+1). 1H NMR (CDCl3): d 1.33 (m, 4H), 1.58 (m, 4H),
1.95 (s, 6H), 3.25 (m, 4H), 3.42 (m, 8H), 3.79 (s, 6H), 5.74 (br s,
2H), 6.25–6.36 (m, 6H), 7.13 (t, 2H J = 8.5 Hz). IR (cmꢀ1, Nujol):
3256, 1638, 1609. Anal. Calcd for C28H42N4O4: C, 67.44; H, 8.49;
N, 11.24. Found: C, 67.73; H, 8.72; N, 11.60.
c
S (10 M). In cell lines expressing
l
c
maximal stimulation, above basal levels, of 370% and 250% in
MT1 and MT2 receptors, respectively. Basal stimulation is the
amount of [35S]GTP
cS specifically bound in the absence of com-
pounds and it was taken as 100%. The maximal G-protein activa-
tion was measured in each experiment by using melatonin
(100 nM). Compounds were added at three different concentra-
tions (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 over that of melatonin. It
was assumed that at the equivalent concentration the test com-
pound occupies the same number of receptors as 100 nM mela-
tonin. All of the measurements were performed in triplicate. The
relative intrinsic activity (IAr) values were obtained by dividing
5.1.4.2.
N-{2-[{10-[(2-Acetylaminoethyl)-(3-methoxyphenyl)
amino]decyl}-(3-methoxyphenyl)amino]ethyl}acetamide (5b)
White solid, 13% yield; mp 101–102 °C (EtOAc–Et2O). ESI-MS (m/
z): 555 (M+1). 1H NMR (CDCl3): d 1.28 (m, 12H), 1.55 (m, 4H),
1.95 (s, 6H), 3.25 (m, 4H), 3.42 (m, 8H), 3.79 (s, 6H), 5.64 (br s,
2H), 6.24–6.37 (m, 6H), 7.13 (t, 2H J = 8.5 Hz). IR (cmꢀ1, Nujol):
3256, 1638, 1608. Anal. Calcd for C32H50N4O4: C, 69.28; H, 9.08;
N, 10.10. Found: C, 68.98; H, 8.95; N, 10.01.
5.1.5. Hexanedioic acid bis[(2-acetylaminoethyl)-(3-methoxy-
phenyl)amide] (6)
the maximum ligand-induced stimulation of [35S]GTP
cS binding
To a solution of N-[2-(3-methoxyphenylamino)ethyl]acetam-
ide25 (0.14 g, 0.67 mmol) and TEA (0.12 mL, 0.86 mmol) in dry
THF (4 mL) was added adipoyl chloride (0.05 mL, 0.34 mmol).
The resulting mixture was stirred at room temperature for 2 h,
quenched with water and extracted three times with hot EtOAc.
The organic phases were combined, dried (Na2SO4) and concen-
trated under reduced pressure to yield a crude solid which was
triturated with EtOAc and filtered. White solid, 45% yield; mp
219–221 °C. ESI-MS (m/z): 527 (M+1). 1H NMR (DMSO-d6): d 1.35
(m, 4H), 1.75 (s, 6H), 1.90 (m, 4H), 3.17 (m, 4H), 3.62 (m, 4H),
3.80 (s, 6H), 6.84–7.00 (m, 6H), 7.36 (t, 2H J = 8.0 Hz), 7.92 (br t,
by that of melatonin as measured in the same experiment.
The two radioligands 2-[125I]iodomelatonin (specific activity,
2000 Ci/mmol) and [35S]GTP S ([35S]guanosine-50-O-(3-thio-tri-
c
phosphate); specific activity, 1000 Ci/mmol) were purchased from
Amersham Pharmacia Biotech (Italy).
Acknowledgments
This work was supported by grants from the Italian Ministero
dell’Università e della Ricerca and University of Urbino ‘Carlo Bo’.
7.92). IR (cmꢀ1
28H38N4O6: C, 63.86; H, 7.27; N, 10.64. Found: C, 63.56; H, 7.35;
N, 10.79.
, Nujol): 3307, 1647, 1604. Anal. Calcd for
References and notes
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