Preparation and pharmacological evaluation of a novel series of 2-(phenylthio)benzo[b]thiophenes as selective MT2 receptor ligands

Article abstract of DOI:10.1016/j.ejmech.2011.02.044

A series of N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl) ethyl)acylamides was synthesized and evaluated for binding affinity and intrinsic activity at melatonin receptors. The affinity of each compound for the melatonin receptors was determined by binding studies on cloned human MT ₁ and MT₂ receptors expressed in CHO cells. Agonist and antagonist potency was measured on the [³⁵S]GTPγS binding assay for the most interesting compounds. The new derivatives 8-14 showed modest to high selectivity (between 4 and 220) for MT₂ receptors. The most selective compound, N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl) ethyl)but-3-enamide (14), an MT₂ ligand with affinity for the MT ₂ receptor similar to that of melatonin and a 220-fold preference over MT₁ receptors, acts as a partial agonist. In addition, N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl)ethyl)propionamide (9), a nanomolar MT₂ ligand with a good selectivity ratio (MT ₁/MT₂ = 51) shows antagonist activity on both melatonin receptors.

Full text of DOI:10.1016/j.ejmech.2011.02.044

European Journal of Medicinal Chemistry 46 (2011) 1835e1840
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Preparation and pharmacological evaluation of a novel series of 2-(phenylthio)
benzo[b]thiophenes as selective MT2 receptor ligands
,
b
,
a,
Christophe Mésangeau a , Mikaël Fraise b, Philippe Delagrange c, Daniel Henri Caignard c,
*
Jean Albert Boutin d, Pascal Berthelot a b, Saïd Yous a
,
,b
a Université Lille Nord de France, F-59000 Lille, France
b UDSL, EA GRIIOT, UFR Pharmacie, F-59000 Lille, France
c Département des Sciences Expérimentales, Institut de Recherches Servier, 11 rue des Moulineaux, 92150 Suresnes, France
d Division de Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl)ethyl)acylamides was synthe-
sized and evaluated for binding affinity and intrinsic activity at melatonin receptors. The affinity of each
compound for the melatonin receptors was determined by binding studies on cloned human MT1 and
Received 28 September 2010
Received in revised form
16 February 2011
Accepted 17 February 2011
Available online 23 February 2011
MT2 receptors expressed in CHO cells. Agonist and antagonist potency was measured on the [35S]GTP
gS
binding assay for the most interesting compounds. The new derivatives 8-14 showed modest to high
selectivity (between 4 and 220) for MT2 receptors. The most selective compound, N-(2-(5-fluoro-2-(4-
fluorophenylthio)benzo[b]thiophen-3-yl)ethyl)but-3-enamide (14), an MT2 ligand with affinity for the
MT2 receptor similar to that of melatonin and a 220-fold preference over MT1 receptors, acts as a partial
agonist. In addition, N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl)ethyl)propionamide
(9), a nanomolar MT2 ligand with a good selectivity ratio (MT1/MT2 ¼ 51) shows antagonist activity on
both melatonin receptors.
Keywords:
Melatonin
MT2
Antagonist
Selective ligands
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
compounds are non-selective melatonin ligands and are the
product of the early efforts to develop stable derivatives of
Melatonin (MLT, Chart 1) is a hormone released by the pineal
gland during the night [1]. Its major role lies with the regulation
of the circadian rhythms but many studies also pointed out the
involvement of this hormone in several physio-pathological
processes [2]. Melatonin exerts most of its effect through the
activation of two high-affinity G-protein coupled receptors (MT1
and MT2) cloned in the mid-1990s [3e5]. Another MLT binding
site with a lower affinity profile (MT3) has been later character-
ized as a melatonin-sensitive form of the enzyme quinone re-
ductase 2 [6]. Melatonin is readily available in the U.S. and in
other countries as an over-the-counter dietary supplement and is
used primarily as a sleep-aid. Ramelteon (RozeremÒ), an MT1 and
MT2 receptor agonist, is marketed for the treatment of insomnia
associated with delayed sleep onset [7]. Another melatonin
receptors agonist, Agomelatine (ValdoxanÒ), which is also
a 5-HT2C antagonist, has recently been granted approval for the
treatment of moderate to severe depression [8,9]. These two
melatonin. Since their first synthesis, numerous studies have
addressed and overcame their lack of selectivity, and MT1 [10e13]
MT2 [14e25] and MT3 [26,27] selective ligands have been
described. These ligands will aid in the further elucidation of
melatonin receptors mechanisms and open new therapeutic
perspectives by targeting a specific receptor.
Herein, we report how the unexpected result of a reaction led us
to prepare and evaluate a small series of novel 2-(phenylthio)
benzothiophenes as selective MT2 ligands.
2. Results and discussion
2.1. Chemistry
During the course of our work on the preparation of novel ben-
zothiophene structures as inhibitors of serotonin N-acetyltransfer-
ase [28,29], a key enzyme in the biosynthesis of melatonin, we
prepared in two steps several 5-sustituted-3-methylbenzo[b]thio-
phenes from the corresponding para-substituted thiophenols
(Scheme 1). In a typical manner, a nucleophilic addition reaction
between the thiophenol and chloroacetone gave 1-(4-subsituted-
* Corresponding author. Present address: Department of Medicinal Chemistry,
425 Faser Hall, The University of Mississippi, University, MS 38677, USA. Tel.: þ1
662 915 1663; fax: þ1 662 915 5638.
E-mail address: cmmesang@olemiss.edu (C. Mésangeau).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.044

1836
C. Mésangeau et al. / European Journal of Medicinal Chemistry 46 (2011) 1835e1840
protons, carbons and fluorines of this compound and allowed us to
discard any other potential structure.
NHCOCH3
NHCOCH3
The similitude of this intermediate with the core template of the
selective MT2 ligand luzindole [14,33] prompted us to use this
interesting new intermediate for chemical modifications in an
attempt to prepare a new series of MT2 selective ligands (8e14,
Chart 1). This series would also be closely related to N-(2-(5-fluo-
robenzo[b]thiophen-3-yl)ethyl)acetamide (S27239), a melatonin
analogue previously synthesized in our laboratory [unpublished
data]. Previous work in the MT2 selective ligand field showed that
acetyl was not the optimum group on the C-3 side chain and that
variations of the acyl group can modulate affinity, selectivity and
agonist or antagonist potency of a melatoninergic ligand [19,34].
Therefore, we synthesized a series of N-((fluorophenylthio)benzo
[b]thiophen-3-yl)ethylacylamide with different acylating groups
on the side chain amide in order to systematically investigate their
biological properties.
O
N
H
N
H
Melatonin
Luzindole
NHCOR
S
NHCOCH3
F
F
S
With this in mind, we first decided to optimized the formation of
4, and we successfully increased the yield of the reaction from 20% to
40% by decreasing the temperature and the reaction time. Bromi-
nation of 4 by photochemical reaction with N-bromosuccinimide in
the presence of benzoyl peroxyde then provided 3-(bromomethyl)-
5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophene (5), which was
converted to the corresponding nitrile 6 bynucleophilic substitution
with sodium cyanide (Scheme 2) [32]. Finally, 6 was reduced with
boranes and the N-acylated target compounds 8e14 were prepared
by reaction of the amine 7 with the appropriate acyl chloride in the
presence of potassium carbonate or with vinylacetic acid in the
presence of EDC [32].
S
F
8-14
S27239
Chart 1. Chemical structures of melatonin, luzindole, S27239 and general structure of
compounds 8e14.
phenylsulfanyl)-propan-2-one (2aef) in nearly quantitative yields
and the benzothiophene cycles werethenprepared in good yields by
cyclization of the intermediate in neat polyphosphoric acid. While
this reaction worked in the case of 5-hydrogen (3a), -methyl (3b)
[30], -methoxy (3c), -bromo (3d) [31] and echloro (3e) [31,32]
derivates, treatment of 1-(4-fluoro-phenylsulfanyl)-propan-2-one
(2f) in similar conditions did not lead to the corresponding 5-fluoro-
3-methylbenzo[b]thiophene but gave the unexpected 5-fluoro-2-
(4-fluoro-phenylsulfanyl)-3-methyl-benzo-[b]-thiophene (4). The
structure of this unexpected product was assigned on the basis of 1H,
19F, 13C NMR, 2-D NMRexperiments, IR and MS. Indeed, inspection of
the 1H NMR spectrum of 4 showed the presence of 7 aromatic
protons with coupling to a fluorine and the absence of the H-2
proton seen in the spectrum of 5-fluoro-3-methylbenzo[b]thio-
phene [unpublished data]. These results suggested the presence of
a 4-fluorophenyl group at the C-2 position. Based on the mass
spectroscopy showing a parent peak at m/z 292, we assigned the
structure of 4 to this new compound.19F NMR, HMBC (heteronuclear
multiple bond correlation) and HSQC (heteronuclear single
quantum correlation) analyses made possible the assignment of all
2.2. Pharmacology
The compounds synthesized in this manuscript were subjected
to binding studies against human MT1 and MT2 receptors
transfected in Chinese Hamster Ovarian (CHO) cells, using 2-[125I]-
iodomelatonin as radioligand [11]. The [35S]guanosine-50-O-(3-
thiotriphosphate) ([35S]GTP
gS) binding assay using CHO cell lines
stably expressing the human MT1 or MT2 receptors was used to
determine their activity and efficacy. In this test, which measures
the interaction between the melatonin receptors and the G proteins,
an agonist stimulates [35S]GTP
g
S binding and the stimulation is
proportional to the efficacy and intrinsic activity of the molecule.
Full agonists stimulate [35S]GTP
S binding with a maximum efficacy
g
(Emax), close to that of melatonin itself (Emax ¼ 100%). A compound is
considered a partial agonist if its Emax is between 30% and 70%. If
Emax is below 30%, the compound is considered an antagonist and
the antagonist potency is expressed as KB.
The chemical structures, binding affinities and MT1/MT2 selec-
tivity ratios of the new compounds are presented in Table 1. The
results of the evaluation of the intrinsic activity of the best ligands
are shown in Table 2.
R
R
O
S
a
R
b
SH
S
As entries S27239 and 8 in Table 1 clearly illustrate, the intro-
duction of a 4-fluorophenylthio group at the C-2 position of S27239
leads to a decrease in the affinity for MT1 and an increase to sub-
nanomolar level of the affinity for MT2, resulting in a 13-fold
increase in selectivity for MT2. The compound behaves as a partial
agonist on both melatonin receptors (Table 2). The homologation of
the methyl group of 8 for ethyl (compound 9) causes a decrease in
affinity for both receptors. The selectivity of the compound
(S ¼ 51.4) remains comparable to that of 8. This small modification
in the structure causes the derivative 9 to be an antagonist. The
introduction of a terminal cyclobutyl gives the worst ligand in this
series: 10 exhibits very weak MT1 and MT2 binding affinities
(915 nM and 194 nM, respectively). The introduction of a haloge-
R = F
/
1a-f
2a-f
3a-e
R = H (1a); CH3 (1b); OCH3 (1c)
1d 1e 1f)
b
R = F
Br ( ); Cl ( ); F (
F
S
S
4
F
Scheme 1. Synthesis of 3aee and unexpected formation of 4. Reagents and conditions:
(a) ClCH2COCH3, NaOH, water; (b) PPA, 160 ꢀC.
nated alkyl side chain shows the effect of a bulky group.

C. Mésangeau et al. / European Journal of Medicinal Chemistry 46 (2011) 1835e1840
1837
Br
CN
S
b
a
F
F
F
S
S
S
S
S
4
5
6
F
F
F
c
NHCOR
S
NH2
8
R = CH3
9
R = CH2CH3
R = c-C4H7
d
F
F
10
S
11 R = CH2Br
12
S
S
R = (CH2)3Cl
13 R = CH=CH2
14 R = CH2CH=CH2
8-14
7
F
F
Scheme 2. Synthesis of compounds 8e14. Reagents and conditions: (a) NBS, BP, CCl4, reflux; (b) NaCN, DMSO, r.t.; (c) BH3-THF then HCl, reflux; (d) RCOCl, K2CO3, H2O, CH2Cl2 for
8e13 or CH2 ¼ CHCH2COOH, EDC, CH2Cl2 for 14.
Substituting an acetyl group for the bromomethyl induces
a reduced affinity for MT2 (5.2 nM) and an important loss of
selectivity. This compound (11) appears to be an antagonist on MT1
and a partial agonist on MT2 receptors. Furthermore, the chlor-
opropyl group causes a sharp decrease in MT1 (50-fold) and MT2
(28-fold) affinities, leading to a selective ligand (S ¼ 115) but with
a weak affinity for the MT2 receptor subtype (24.8 nM). Replace-
ment of the acetyl group by a vinyl gives derivative 13 which
exhibits another weak binding profile. On the other hand, substi-
tution with an allyl group (compound 14) causes a slight decrease
in MT2 binding affinity, to a value lower than that of melatonin
(0.24 nM), and leads to an increased selectivity ratio (S ¼ 220). As
shown in Table 2, 14 behaves as a partial agonist on both melatonin
receptors.
3. Conclusion
These results confirm the presence of a hydrophobic pocket at
the MT2 receptor [19,24,34]. Indeed, introduction of a phenylthio
group at the C-2 position of S27239, a benzothiophenic ligand with
mixed affinity for both MT1 and MT2 receptors, allows access to
a MT2 selective ligand. The nature of the acyl substituent on the C-3
side chain also influences greatly affinity and activity in this series.
The most interesting compounds 9 and 14 show MT1/MT2 selec-
tivity ratios of 51 and 220 respectively. They also present good
affinities for the MT2 receptor (4.4 nM for 9 and 0.24 nM for 14) but
differ in their intrinsic activities (antagonist and partial agonist,
respectively).
4. Experimental
Table 1
4.1. Analysis and instruments
MT1 and MT2 binding affinities of 2-(phenylthio)benzo[b]thiophene compounds.
Melting points were determined on a Büchi SMP-535 apparatus
and are uncorrected. Flash column chromatography was performed
using silica gel 60 (70-230 Mesh, ASTM, Merck). Infrared spectra
were recorded using a Vector 22 Bruker spectrophotometer. 1H, 13C,
and 19F NMR spectra were recorded on a Bruker AC300 spectrom-
NHCOR
F
S
S
eter. Chemical shifts are reported in
d units (parts per million)
relative to TMS. Electronic impact mass spectra were measured on
a Ribermag 10-106 Finnigan spectrometer. Elemental analyses were
performed by the "Service Central de Microanalyses", CNRS, Ver-
naison, France.
F
Compound
R
Ki ꢂ SEM (nM) Ki ꢂ SEM (nM)
S
MT1
MT2
(MT1/MT2)
4.2. Syntheses
Melatonine
S27239
8
9
10
11
12
13
14
e
0.22 ꢂ 0.01
24.6 ꢂ 0.7
57 ꢂ 3.6
0.35 ꢂ 0.01
4.87 ꢂ 0.81
0.87 ꢂ 0.25
4.4 ꢂ 0.75
194 ꢂ 24.3
5.21 ꢂ 1.91
24.8 ꢂ 2.45
10.7 ꢂ 0.5
0.24 ꢂ 0.06
0.8
5
65.5
51.4
4.7
4.2
115.3
22
e
4.2.1. 1-(4-fluoro-phenylsulfanyl)propan-2-one (2f)
CH3
Chloroacetone (8.7 mL, 109.2 mmol) was slowly added to a stir-
red solution of 4-fluorothiophenol (1) (10 g, 78 mmol) and sodium
hydroxyde (3.75 g, 93.6 mmol) in water (40 mL) at 0 ꢀC. The reac-
tion mixture was stirred for 1 h before being extracted twice with
ether. The combined organic layers were then washed with brine,
dried over magnesium sulfate and evaporated under reduced
pressure. The crude oil was purified by column chromatography
(SiO2) using cyclohexane as the eluent to give 10.35 g (72%) of 2f as
CH2CH3
c-C4H7
CH2Br
(CH2)3Cl
CH]CH2
CH2eCH ¼ CH2
226 ꢂ 29.5
915 ꢂ 86.8
22.1 ꢂ 7.7
2860 ꢂ 595
236 ꢂ 4.5
52.8 ꢂ 5.7
220
Concentration-response curves were analyzed by non-linear regression. Binding
affinities (nM) are expressed as mean Ki ꢂ SEM of a least three independent
experiments. The selectivity ratio between MT1 and MT2 receptors is calculated for
each compound.
colorless oil. IR (
(s, 3H), 3.98 (s, 2H), 7.17 (m, 2H), 7.38 (m, 2H).
n d 2.20
cmꢁ1) 1704 (CO). 1H NMR (DMSO-d6):

1838
C. Mésangeau et al. / European Journal of Medicinal Chemistry 46 (2011) 1835e1840
Table 2
Functional assays.
Compd.
MT1
MT2
EC50(nM)
Emax(%)
KB(nM)
Imax (%)
EC50(nM)
Emax(%)
KB(nM)
Imax(%)
S27239
8
9
11
14
23.8 ꢂ 5.3
202 ꢂ 44
87 ꢂ 3.2
33 ꢂ 2.5
nd
nd
45 ꢂ 7.0
62.8 ꢂ 14
nd
nd
nd
0.68 ꢂ 0.17
0.64 ꢂ 0.1
61 ꢂ 6.7
42 ꢂ 3.1
nd
>10
nd
m
M
nd
nd
>10
>10
m
m
M
M
42 ꢂ 1.9
115 ꢂ 5.5
>10
m
M
0.67 ꢂ 0.15
92 ꢂ 4.5
nd
nd
nd
7.27 ꢂ 3.56
47 ꢂ 10
nd
nd
nd
188 ꢂ 76.4
nd
2.15 ꢂ 0.72
52.4 ꢂ 6.3
nd
Concentration-response curves were analyzed by non-linear regression. Agonist potency was expressed as EC50 ꢂ S.E.M. (nM) while the maximal efficacy, Emax ꢂ S.E.M. was
expressed as a percentage of that observed with melatonin 1
M (¼ 100%). Antagonist potency to inhibit the effect of melatonin (30 or 3 nM respectively for MT1 and MT2
receptors) was expressed as KB ꢂ S.E.M. Data are mean of at least 3 independent experiments. nd : not determined.
m
4.2.2. 5-fluoro-2-(4-fluorophenylthio)-3-methylbenzo[b]thiophene
(4)
(dd, 1H, J ¼ 8.8, 4.7 Hz). Anal. Calcd. for C16H9F2NS2: C, 60.55; H, 2.86;
N, 4.41. Found: C, 60.62; H, 2.85; N, 4.41.
Compound 2f (3 g, 13.6 mmol) was added to polyphosphoric
acid (30 g), and the reaction mixture was stirred vigorously at 80 ꢀC
for 1 h. The mixture was then carefully poured onto ice and
extracted with ethyl acetate (3 ꢃ 40 mL). The combined organic
layers were washed with water, dried over magnesium sulfate, and
evaporated to dryness. The residue was purified by column chro-
matography (SiO2) using heptane as the eluent to give 0.8 g (40%) of
4.2.5. 2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl)
ethanamine hydrochloride (7)
To a solution of 6 (1.2 g, 3.78 mmol) in anhydrous tetrahydro-
furan was added a solution of boraneetetrahydrofuran complex in
THF (1M,11.35 mL,11.35 mmol). The reaction mixture was heated to
reflux under argon. After 3 h, a 6N solution of HCl (2.8 mL, 17 mmol)
was added dropwise and the reaction was refluxed for another
hour. The solvent was then removed under reduced pressure. The
residue was taken up in ethylic ether, and the precipitate was
collected by filtration. Recrystallization from a mixture of ethyl
acetate and methanol (4:1 ratio) gave 0.88 g (65%) of 7 as a white
n
4 as a white solid. Mp ¼ 40 ꢀC. IR ( cmꢁ1) 3100e2850 (CH). 1H NMR
(CDCl3):
d
2.47 (s, 3H, CH3), 6.99 (dd, 2H, Jortho ¼ JHeF ¼ 9.1 Hz, H-30,
H-50), 7.14 (td, 1H, J6e7 ¼ J6eF ¼ 8.8 Hz, J6e4 ¼ 2.6 Hz, H-6), 7.26 (dd,
2H, Jortho ¼ 9.1 Hz, JHeF ¼ 5.2 Hz, H-20, H-60), 7.37 (dd, 1H,
J4e6 ¼ 2.6 Hz, J4eF ¼ 9.5 Hz, H-4), 7.68 (dd, 1H, J7e6 ¼ 8.8 Hz,
J7eF
¼
4.9 Hz, H-7). 13C NMR(CDCl3):
d
12.7 (CH3), 108.1
solid. Mp > 200 ꢀC. IR (
d6):
1H, J ¼ 8.9, 2.5 Hz), 7.42 (dd, 2H, J ¼ 8.9, 5.1 Hz), 7.95 (dd, 1H,
J ¼ 10.2, 2.5 Hz), 8.01 (dd, 1H, J ¼ 8.9, 5.1 Hz), 8.26 (br s, 3H). Anal.
Calcd. for C16H13F2NS2. HCl: C, 53.70; H, 3.94; N, 3.91. Found:
C, 53.65; H, 3.86; N, 3.96.
n
cmꢁ1) 3200e2500 (NH). 1H NMR (DMSO-
2.90 (m, 2H), 3.31 (m, 2H), 7.25 (dd, 2H, J ¼ 8.9 Hz), 7.35 (td,
(2J ¼ 22.9 Hz, C-4), 114.0 (2J ¼ 25.1 Hz, C-6), 116.2 (2J ¼ 21.8 Hz, C-30,
C-50), 123.3 (3J ¼ 8.7 Hz, C-7), 130.6 (3J ¼ 7.6 Hz, C-20, C-60), 131.4 (C-
10, C-1 or C-2), 136.2 (C-8), 137.4 (C-2 or C-1), 140.9 (C-3) 160.8
(1J ¼ 243.0 Hz, C-5), 161.9 (1J ¼ 246.3 Hz, C-40). F NMR(CDCl3):
d
d
ꢁ122.2 (td, JFe(Ce4) ¼ JFe(Ce6) ¼ 9.3 Hz, JFe(Ce7) ¼ 5.3 Hz, F), 119.3
(tt, JFe(Ce3’) ¼ JFe(Ce5’) ¼ 8.6 Hz, JFe(Ce2’) ¼ JFe(Ce6’) ¼ 5.3 Hz, F). EI-MS
m/z 292. Anal. Calcd. for C15H10F2S2: C, 61.62; H, 3.45; F, 13.00.
Found: C, 61.73; H, 3.52; F, 12.95.
4.2.6. General Procedure for the Preparation of compounds 8e13
Potassium carbonate (0.27 g, 1.96 mmol) and methylene chlo-
ride (20 mL) were added to a solution of 7 (0.2 g, 0.56 mmol) in
water (10 mL). The reaction mixture was cooled to 0 ꢀC, and
a solution of the appropriate acyl chloride (0.78 mmol) in methy-
lene chloride (2 mL) was added dropwise. The reaction was then
allowed to warm to room temperature and was stirred for another
2 h. The layers were separated and the aqueous layer was extracted
with methylene chloride (2 ꢃ 20 mL). The combined organic layers
were washed with water, dried over magnesium sulfate, and
evaporated in vacuo.
4.2.3. 3-(bromomethyl)-5-fluoro-2-(4-fluorophenylthio)benzo[b]
thiophene (5)
To a solution of 4 (3 g, 10.26 mmol) in anhydrous carbon tetra-
chloride (150 mL) was added N-bromosuccinimide (1.92 g,
10.77 mmol) and benzoyl peroxide (0.19 g, 0.8 mmol). The reaction
mixture was heated to gentle reflux for 4 h while irradiating with
a 250 W lamp. The reaction was allowed to cool to room temper-
ature, and filtered to remove the succinimide. The filtrate was
evaporated and the solid residue was recrystallized from acetoni-
trile to give 2.3 g (60%) of 5 as a white solid. Mp ¼ 113 ꢀC. IR (
n
cmꢁ1
)
4.2.6.1. N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-
yl)ethyl)acetamide (8). Recrystallization from toluene, yield 60%,
3100e2850 (CH). 1H NMR (CDCl3):
d 4.84 (s, 2H), 7.03 (dd, 2H,
J ¼ 8.9 Hz), 7.16 (td, 1H, J ¼ 8.9, 2.6 Hz), 7.41 (dd, 2H, J ¼ 9.1, 5.2 Hz),
7.52 (dd, 1H, J ¼ 9.4, 2.6 Hz), 7.66 (dd, 1H, J ¼ 8.9, 4.7 Hz). Anal.
Calcd. for C15H9BrF2S2: C, 48.53; H, 2.44. Found: C, 48.62; H, 2.50.
white solid. Mp ¼ 118e119 ꢀC. IR (
n
cmꢁ1) 3306 (NH), 1643 (CO). 1H
1.75 (s, 3H), 3.07 (t, 2H, J ¼ 6.3 Hz), 3.21 (m, 2H),
NMR (DMSO-d6):
d
7.31 (m, 5H), 7.77 (dd,1H, J ¼ 9.8, 2.6 Hz), 7.98 (dd,1H, J ¼ 8.8, 4.9 Hz),
8.05 (br s, 1H). Anal. Calcd. for C18H15F2NOS2: C, 59.48; H, 4.16; N,
3.85. Found: C, 59.50; H, 4.12; N, 3.86.
4.2.4. 2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl)
acetonitrile (6)
To a stirred suspension of sodium cyanide (0.58 g, 11.85 mmol) in
dimethylsulfoxide (30mL)was added compound 5 (2.2 g, 5.92mmol).
The reaction mixture was stirred at room temperature for 1 h, poured
into water (100 mL) and extracted with ethyl acetate (3 ꢃ 40 mL). The
combined organic layers were washed with brine, dried over
magnesium sulfate, and removed in vacuo. The residue was recrys-
tallized from a mixture of acetonitrile and toluene (9:1 ratio) to give
4.2.6.2. N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-
yl)ethyl)propionamide (9). Recrystallization fromcyclohexane, yield
92%, white solid. Mp ¼ 141e144 ꢀC. IR (
n
cmꢁ1) 3318 (NH),1641 (CO).
1H NMR (CDCl3):
d
1.13 (t, 3H, J ¼ 7.5 Hz), 2.17 (q, 2H, J ¼ 7.5 Hz), 3.19
(t, 2H, J ¼ 6.7 Hz), 3.50 (m, 2H), 5.65 (br s, 1H), 7.00 (m, 2H), 7.11 (td,
1H, J ¼ 8.7, 2.6 Hz), 7.28e7.30 (m, 2H), 7.52 (dd, 1H, J ¼ 9.4, 2.3 Hz),
7.64 (dd, 1H, J ¼ 8.7, 4.6 Hz). 13C NMR (CDCl3):
d 9.6, 27.5, 29.6, 39.2,
1.3 g (70%) of 6 as awhite solid. Mp ¼ 128 ꢀC. IR (
n
cmꢁ1) 2244 (CN).1H
108.1 (d, 2J ¼ 23.1 Hz), 114.1 (d, 2J ¼ 24.9 Hz), 116.4 (d, 2J ¼ 22.0 Hz),
123.4 (d, 3J ¼ 9.0 Hz), 130.9, 131.2 (d, 3J ¼ 8.1 Hz), 133.4, 136.4, 137.9,
140.3 (d, 3J ¼ 9.0 Hz),160.9 (d, 1J ¼ 241.8 Hz),162,1 (d, 1J ¼ 246.3 Hz),
NMR(CDCl3):
d
4.05(s,2H), 7.05(dd, 2H, J¼ 8.8Hz),7.21(td,1H,J¼8.8,
2.3 Hz), 7.35 (dd, 2H, J ¼ 8.8, 4.7 Hz), 7.51 (dd,1H, J ¼ 8.8, 2.3 Hz), 7.72

C. Mésangeau et al. / European Journal of Medicinal Chemistry 46 (2011) 1835e1840
1839
173.9. Anal. Calcd. for C19H17F2NOS2: C, 60.46; H, 4.54; N, 3.71.
Found: C, 60.50; H, 4.52; N, 3.66.
(t, 2H, J ¼ 6.5 Hz), 3.27 (m, 2H), 5.05 (m, 2H), 5.85 (m, 1H), 7.31 (m,
5H), 7.77 (dd, 1H, J ¼ 10.0, 2.7 Hz), 7.98 (dd, 1H, J ¼ 8.7, 5.2 Hz), 8.06
(br s, 1H). Anal. Calcd. for C20H17F2NOS2: C, 61.68; H, 4.40; N, 3.60.
Found: C, 61.88; H, 4.43; N, 3.53
4.2.6.3. N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl)
ethyl)cyclobutanecarboxamide (10). Recrystallization from toluene,
yield 71%, white solid. Mp ¼ 157e160 ꢀC. IR (
n
cmꢁ1) 3304 (NH),
4.3. Melatonin pharmacology
1637 (CO). 1H NMR (CDCl3):
d 1.89 (m, 2H), 2.11 (m, 2H), 2.25 (m,
2H), 2.91 (qu, 1H, J ¼ 8.5 Hz), 3.18 (t, 2H, J ¼ 6.9 Hz), 3.48 (m, 2H),
5.55 (br s, 1H), 7.00 (dd, 2H, J ¼ 8.7 Hz), 7.13 (td, 1H, J ¼ 8.7, 2.6 Hz),
7.29 (m, 2H), 7.53 (dd, 1H, J ¼ 9.2, 2.6 Hz), 7.66 (dd, 1H, J ¼ 8.7,
4.3.1. Reagents and chemicals
2-[125I]-Iodomelatonin (2200 Ci/mmol) was purchased from
NEN (Boston, MA). Other drugs and chemicals were purchased from
SigmaeAldrich (Saint Quentin, France).
4.6 Hz). 13C NMR (CDCl3):
d 18.0, 25.2, 27.4, 39.1, 39.8, 108.2 (d,
2J ¼ 23.0 Hz), 114.1 (d, 2J ¼ 24.9 Hz), 116.3 (d, 2J ¼ 22.1 Hz), 123.3 (d,
3J ¼ 9.0 Hz), 130.9, 131.1 (d, 3J ¼ 8.1 Hz), 133.2, 136.3, 138.0, 140.2 (d,
3J ¼ 9.0 Hz), 160.8 (d, 1J ¼ 241.7 Hz), 162,0 (d, 1J ¼ 246.3 Hz), 175.1.
Anal. Calcd. for C21H19F2NOS2: C, 62.51; H, 4.75; N, 3.47. Found: C,
62.60; H, 4.72; N, 3.56.
4.3.2. Cell Culture
CHO cell lines stably expressing the human melatonin MT1 or
MT2 receptors were grown in DMEM medium supplemented with
10% fetal calf serum, 2 mM glutamine, 100 IU/mL penicillin and
100 m
g/mL streptomycin. Grown at confluence at 37 ꢀC (95% O2/5%
4.2.6.4. N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl)
ethyl)bromoacetamide (11). Recrystallization from a mixture of
cyclohexane and toluene (1:2 ratio), yield 71%, white solid.
CO2), they were harvested in PBS containing EDTA 2 mM and
centrifuged at 1000 ꢃ g for 5 min (4 ꢀC). The resulting pellet was
suspended in TRIS 5 mM (pH 7.5), containing EDTA 2 mM and
homogenized using a Kinematica polytron. The homogenate was
then centrifuged (95000 g, 30 min, 4 ꢀC) and the resulting pellet
suspended in 75 mM TRIS (pH 7.5),12.5 mM MgCl2 and 2 mM EDTA.
Aliquots of membrane preparations were stored at ꢁ80 ꢀC until use.
Mp ¼ 133 ꢀC. IR (
n
cmꢁ1) 3293 (NH), 1655 (CO). 1H NMR (DMSO-d6):
d
3.09 (t, 2H, J ¼ 7.5 Hz), 3.28 (m, 2H), 3.82 (s, 2H), 7.22 (dd, 2H,
J ¼ 8.6 Hz), 7.32 (td, 1H, J ¼ 8.7, 2.4 Hz), 7.39 (m, 2H), 7.77 (dd, 1H,
J ¼ 9.6, 2.4 Hz), 7.97 (dd, 1H, J ¼ 8.7, 4.8 Hz), 8.26 (br s, 1H). 13C NMR
(CDCl3):
d
27.0, 29.0, 39.7,107.9 (d, 2J ¼ 23.1 Hz),114.1 (d, 2J ¼ 24.9 Hz),
116.4(d, 2J ¼22.0Hz),123.4(d, 3J¼ 9.0Hz),130.0,131.3(d, 3J¼ 8.1 Hz),
133.9,136.3,137.0,140.0 (d, 3J ¼ 9.0 Hz),160.9 (d,1J ¼ 241.8 Hz),162,1
(d,1J ¼ 246.5 Hz),165.5. Anal. Calcd. for C18H14BrF2NOS2: C, 48.88; H,
3.19; N, 3.17. Found: C, 48.84; H, 3.21; N, 3.18.
4.3.3. Binding assays
2-[125I]-iodomelatonin binding assay conditions were essen-
tially as previously described [11]. Briefly, binding was initiated by
addition of membrane preparations from stable transfected CHO
cells diluted in binding buffer (50 mM TriseHCl buffer, pH 7.4
containing 5 mM MgCl2) to 2-[125I]-iodomelatonin (20 pM for MT1
and MT2 receptors) and the tested drug. Nonspecific binding was
4.2.6.5. 4-chloro-N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thio-
phen-3-yl)ethyl)butanamide (12). Recrystallization from toluene-
cyclohexane, yield 40%, white solid. Mp ¼ 74e75 ꢀC. IR (
n
cmꢁ1
)
defined in the presence of 1 mM melatonin. After 120 min incubation
3307 (NH), 1639 (CO). 1H NMR (DMSO-d6):
d
1.89 (m, 2H), 2.16 (t,
at 37 ꢀC, reaction was stopped by rapid filtration through GF/B filters
presoaked in 0.5% (v/v) polyethylenimine. Filters were washed three
times with 1 mL of ice-cold 50 mM TriseHCl buffer, pH 7.4.
Data from the dose-response curves (7 concentrations in
duplicate) were analysed using the program PRISM (Graph Pad
Software Inc., San Diego, CA) to yield IC50 (inhibitory concentration
50). Results are expressed as Ki ¼ IC50/1 þ ([L]/KD), where [L] is the
concentration of radioligand used in the assay and KD, the disso-
ciation constant of the radioligand characterising the membrane
preparation.
2H, J ¼ 7.2 Hz), 3.07 (t, 2H, J ¼ 6.8 Hz), 3.24 (m, 2H), 3.60 (t, 2H,
J ¼ 6.3 Hz), 7.31 (m, 5H), 7.77 (dd, 1H, J ¼ 10.0, 1.8 Hz), 7.98 (dd, 1H,
J ¼ 9.0, 4.8 Hz), 8.10 (br s, 1H). Anal. Calcd. for C20H18ClF2NOS2: C,
56.51; H, 4.27; N, 3.29. Found: C, 56.18; H, 4.48; N, 3.30.
4.2.6.6. N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-yl)
ethyl)acrylamide (13). Purification by column chromatography
(SiO2) using methylene chloride/ethyl acetate (9:1) as the eluent,
yield 45%, white solid. Mp ¼ 100e102 ꢀC. IR (
n
cmꢁ1) 3295 (NH),
3.12 (t, 2H, J ¼ 7.1 Hz), 3.32 (m, 2H),
1654 (CO). 1H NMR (DMSO-d6):
d
[ gSbinding assaywas performed according topublished
35S]-GTP
methodology [11]. Briefly, membranes from transfected CHO cells
expressing MT1 or MT2 receptor subtype and compounds were
5.58 (dd, 1H, J ¼ 9.1, 3.5 Hz), 6.11 (m, 2H), 7.31 (m, 5H), 7.78 (dd, 1H,
J ¼ 10.1, 2.5 Hz), 7.98 (dd, 1H, J ¼ 8.6, 5.1 Hz), 8.33 (br s, 1H). Anal.
Calcd. for C19H15F2NOS2: C, 59.48; H, 4.16; N, 3.85. Found: C, 59.38;
H, 4.00; N, 3.92.
diluted in binding buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 3
GDP, 3 mM MgCl2, and 20 g/mL saponin). Incubationwas started by
the addition of 0.2 nM [35S]-GTP
S to membranes (20 g/mL) and
mM
m
g
m
4.2.7. N-(2-(5-fluoro-2-(4-fluorophenylthio)benzo[b]thiophen-3-
yl)ethyl)but-3-enamide (14)
drugs, and further followed for 1 h at room temperature. For
experiments with antagonists, membranes were pre-incubated
with both the melatonin (30 nM or 3 nM for hMT1 and hMT2
receptors, respectively) and the antagonist for 30 min prior the
Compound 7 (0.2 g, 0.56 mmol) was dissolved in water (50 mL).
Sodium hydroxide (0.05 g, 1.1 mmol) was added and the reaction
mixture was stirred for 1 h at room temperature. The mixture was
then extracted with methylene chloride and dried over MgSO4.
Vinylacetic acid (0.07 mL, 0.84 mmol) and N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide (0.13 g, 0.84 mmol) were dissolved in
methylene chloride (10 mL) at 0 ꢀC. After 30 min, the solution of
amine 7 was added dropwise. The reaction mixture was stirred at
0 ꢀC for 30 min and at room temperature for 3 h. It was then washed
with a 1M HCl solution and water. The organic layer was dried over
magnesium sulfate, and removed under reduced pressure. The solid
residue was recrystallized from toluene-cyclohexane to give 0.09 g
addition of [35S]-GTP
GTP S (10
B filters followed by three successive washes with ice-cold buffer.
Usual levels of [35S]-GTP
S binding (expressed in dpm) were for
CHO-MT2 membranes : 2000 for basal activity, 8000 in the pres-
ence of melatonin 1 M and 180 in the presence of GTP S 10
gS. Nonspecific binding was defined using cold
g
mM). Reaction was stopped by rapid filtration through GF/
g
m
g
mM
which defined the non specific binding. Data from the dose-
response curves (7 concentrations in duplicate) were analysed by
using the program PRISM (Graph Pad Software Inc., San Diego, CA)
to yield EC50 (Effective concentration 50%) and Emax (maximal
(40%) of 14 as a white solid. Mp ¼ 119e121 ꢀC. IR (
n
cmꢁ1) 3308
effect) for agonists. Antagonist potencies are expressed as KB ¼ IC50
/
(NH), 1643 (CO). 1H NMR (DMSO-d6):
d
2.83 (d, 2H, J ¼ 7.0 Hz), 3.10
1 þ ([Ago]/EC50 ago), where IC50 is the inhibitory concentration of

1840
C. Mésangeau et al. / European Journal of Medicinal Chemistry 46 (2011) 1835e1840
antagonist that gives 50% inhibition of [35S]-GTP
gS binding in the
presence of a fixed concentration of melatonin ([Ago]) and EC50 ago
is the EC50 of the molecule when tested alone.
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