Synthesis and structure - Activity relationships of novel naphthalenic and bioisosteric related amidic derivatives as melatonin receptor ligands

Article abstract of DOI:10.1016/S0968-0896(98)00147-3

A previous paper reported the synthesis of melatonin receptor ligands. In order to complete the structure-activity relationships and to obtain antagonists to the melatonin receptor, a new series of naphthalenic analogues of melatonin have been synthesized

Full text of DOI:10.1016/S0968-0896(98)00147-3

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1875±1887
Synthesis and Structure±Activity Relationships of Novel
Naphthalenic and Bioisosteric Related Amidic Derivatives as
Melatonin Receptor Ligands
Veronique Leclerc,a Eric Fourmaintraux,a Patrick Depreux,a
Daniel Lesieur,a Peter Morgan,b H. Edward Howell,b Pierre Renard,c
Daniel-Henri Caignard,c,* Bruno Pfei€er,c Philippe Delagrange,c
Beatrice Guardiola-Lemaõtrec and Jean Andrieuxd
aInstitut de Chimie Pharmaceutique. Lille, France
bRowett Research Institute. Aberdeen, Scotland UK
c
Â
Institut de Recherches Internationales Servier, 1 rue Carle Hebert, 92415 Courbevoiez Cedex, France
d
Â
Faculte de Pharmacie, Paris Sud. Chatenay-Malabry, France
Received 10 October 1997; accepted 2 June 1998
AbstractÐA previous paper reported the synthesis of melatonin receptor ligands. In order to complete the structure±
activity relationships and to obtain antagonists to the melatonin receptor, a new series of naphthalenic analogues of
melatonin have been synthesized. Modi®cations include deletion of the 7-methoxy group, replacement of the ethylene
moiety, replacement of the amidic function by bioisosteres, and replacement of the naphthalenic nucleus by other
bicyclic rings. Almost all the structural modi®cations lead to decreased anity for the melatonin receptor. However,
the N-n propyl urea derivative (27) is a very potent ligand at this receptor (pKi=14.3). Most interestingly deletion of
the methoxy group resulted in the ®rst antagonist in this series. This molecule, compound 12, or N-[2-(1-naphthyl)-
ethyl]cyclobutyl carboxamide has been selected for preclinical development. # 1998 Published by Elsevier Science Ltd.
All rights reserved.
Introduction
lenic analogue of melatonin has been selected for
clinical development.
In a previous paper1 we reported that although the exact
functions of melatonin in man were not fully elucidated,
recent work had suggested its potential usefulness in a
number of therapeutic areas. However the very short
half-life and lack of selectivity of this natural molecule
justi®ed the development of novel analogues that would
overcome these limitations.
To continue this work, we have further developed new
compounds in order to gain improved structure±activity
relationships for melatonin analogues and better charac-
terize the melatonin receptor. We additionally hoped to
obtain potent antagonist derivatives as the previous
work only obtained agonist derivatives. This second aim
is fully justi®ed as compound 12, in this paper has been
the subject of extensive studies that con®rmed its
antagonist activity. This compound restored the neuro-
nal response of surprachiasmatic neurones to a light
¯ash in presence of melatonin leading to the possibility
that such compounds may be of use in the treatment of
seasonal a€ective disorders.2 Moreover, this compound
was able to prevent weight gain in an animal model of
For these reasons, we initiated and have reported
work on the synthesis of bicyclic analogues of melato-
nin. These studies described very potent agonists for
the melatonin receptor and among these, the naphtha-
*Corresponding author. Tel.: 33 1 46 41 60 00; Fax: 33 1 46 41
60 11.
0968-0896/98/$Ðsee front matter # 1998 Published by Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00147-3

1876
V. Leclerc et al./Bioorg. Med. Chem. 6 (1998) 1875±1887
seasonal obesity indicating that a melatonin antagonist
could be useful in the treatment of obesity.3
Table 1. Structure of starting primary amines
We have synthesized 50 new derivatives, among which
are thirty six naphthalenic bioisosteres. Following our
previous report we studied direct naphthalenic analo-
gues of melatonin by deleting the 7-methoxy group to
check its importance for biological activity as previously
hypothesized.4 We have also extended our study related
to the importance of the amidic function by replacing it
with various bioisosteric systems: ureas, thioureas, car-
bamates, esters, ethers and retroamides. It has been
reported that this part of the molecule has a primary
importance for binding anity.4 Thirdly, in studies to
investigate the role of the side chain, we replaced the
ethylene moiety by lower and higher homologues and
also added a rami®cation. Finally, 13 indolic, benzo-
furanic and benzothiophenic derivatives with the most
representative structural modi®cations introduced in the
naphthalenic family were studied to verify the equiva-
lence of these variations in these series. The organic
synthesis, melatonin receptor binding data and agonist
activity properties for these bicyclic compounds are
described in this paper.
Ar
R
H
n
0
3a
3b
H
1
2
1
1
1
1
1
1
1
1
H
3c
3d
3e
3f
H
COOCH3
H
H
H
H
H
H
Chemistry
Key intermediates in the synthesis of the target com-
pounds described here are the corresponding primary
amines, alcohols or carboxylic acids (Tables 1 and 2).
Some of the primary amines (Table 1) such as 3b, 3f, 3g,
3h, 3i, 3j, and 3k are commercially available or pre-
viously described.1,5±8 Primary amine (3a) was prepared
(Scheme 1) through Ho€man's degradation9 of the pre-
viously described8 7-methoxy-1-naphthylacetamide. To
form10 (Scheme 2) the homologous derivative (3c), the
acid precursor (2a) was esteri®ed with methanol and
thionyl chloride. The ester (4a) was reduced with
LiAlH4 to the alcohol (5a), that reacted with methane
sulfonyl chloride (94% yield) to give the mesylate (6a).
Nucleophilic displacement11 of (6a) with potassium
cyanide in DMSO provided (95% yield) the nitrile (7a),
which was catalytically reduced to the amine (3c).
Compound 3d was obtained using the same procedure
as for the attainment of its 7-methoxy analogue (3c).1
Amino-ester (3e) was synthesized (Scheme 3) from
commercially available 1-naphthylacetonitrile via
methyl-2-cyano(1-naphthyl) acetate (7c) according to a
previously described procedure.8 Alkaline hydrolysis of
the nitrile (7a) gave (Scheme 4) the propanoic acid deri-
vative (2b), which was transformed by the same route to
the butyryl homologue (2c). Etheri®cation of the alco-
hols (5a and 5b) with various dialkylsulfates under
phase transfer catalysis conditions12 with benzyl-
triethylammonium chloride (BTEAC) as catalyst gave
(Scheme 5) compounds (39±41). The ester (42) was
3g
3h
3i
3j
3k
obtained by heating the alcohol (5a) with acetic anhy-
dride. Treatment (Scheme 6) of the acids (2b and 2c)
with thionyl chloride in chloroform yielded in situ the
corresponding acyl chlorides, which were then reacted
without further puri®cation with ammonia or primary
amines to give the carboxamides (43±46). Mono-
substituted ureas (24 and 30) could be synthezised by
reaction of amines (3b and 3d) with a large excess of
potassium cyanate in acidic aqueous medium, or more
eciently, with a slight excess of potassium cyanate

V. Leclerc et al./Bioorg. Med. Chem. 6 (1998) 1875±1887
1877
alone in water. Dissymmetric disubstituted analogues
(25±28; 31±38; 52; 54±56; 58) were obtained (Scheme 7)
by reaction with various alkylisocyanates of the corre-
sponding primary amines in ether or pyridine. Sym-
metric disubstituted ureas (29 and 53) were obtained
(Scheme 7) by coupling two equivalents of the suitable
amines with carbonyldiimidazole as reactant. Carbamates
(22 and 23) were synthezised from 3b in THF by action
of an appropriate alkylchloroformate in the presence of
triethylamine. The N-acylated derivatives (10±12; 14±21;
Table 2. Physical properties of key intermediates
Compd
R1
R2
n
X
Mp (ꢀC)
Recrys solvent
Formula
2b
2c
3a
3c
3d
3e
4a
4b
5b
5b
6a
6b
7a
7b
7c
OCH3
OCH3
OCH3
OCH3
H
H
1
2
0
2
1
1
0
1
1
2
1
2
1
2
1
COOH
COOH
NH2,HCl
NH2,HCl
NH2,HCl
NH2,HCl
COOCH2
COOCH2
OH
154±155
104±106
>260
toluene
toluene-cyclohexane
ethanol
C14H14O3
C15H16O3
H
H
C12H14ClNO
C14H18ClNO
C12H14ClN
C14H16ClNO2
C14H14O3
H
197±200
230±233
217±219
51±52
cyclohexane-ethanol
ethanol
acetonitrile
H
COOCH3
H
OCH3
OCH3
OCH4
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
hexane
Ða
H
Oil
80±82
C15H16O3
C13H14O2
C14H16O2
H
cyclohexane
cyclohexane
cyclohexane
toluene-cyclohexane
hexane
H
OH
38±39
62±63
H
OSO2CH3
OSO2CH2
CN
C14H16O4S
C15H18O4S
C14H13NO
C15H15NO
C14H11NO2
H
52±54
62±64
H
H
CN
CN
52±54
89±90
ethanol-water
cyclohexane
COOCH3
aPuri®ed by column chromatography: SiO2, acetone/toulene/cyclohexane (4/2/2).
Scheme 1. Reagents: (a) SOCl2, NH3; (b) Br2, NaOH.
Scheme 2. Reagents: (a) SOCl2, CH3OH; (b) LiALH4, ether; (c) CH3SO2Cl, (C2H5)3N, CH2Cl2; (d) KCN, DMSO; (e) H2, Ni, EtOH,
NH3.

1878
V. Leclerc et al./Bioorg. Med. Chem. 6 (1998) 1875±1887
47±51; 57; 59±60) were prepared (Scheme 7) from the
appropriate primary amine (3) by treatment with
the suitable acid chloride in the presence of K2CO3 as
base and a biphasic medium according to a variant1
of the Schotten±Baumann reaction. The tri¯uoro-
acetamido compound 13 was obtained by treatment of
the corresponding amine (3d) with tri¯uoroacetic anhy-
dride13 in pyridine.
Results and Discussion
For all compounds, the chemical structures, melatonin
receptor binding characteristics and biological activities
(expressed as a cAMP index determined by the degree of
melatonin mediated inhibition of forskolin-stimulated
cAMP production) under single dose conditions14 are
shown in Table 4.
Scheme 3. Reagents: (a) (CH3O)2CO, Na; (b) NaBH4, CoCl2.
Scheme 4. Reagents: (a) 1. NaOH, MeOH; 2. HCl; (b) SOC12, CH3OH; (c) LIAIH4, ether; (d) CH3SO2Cl, (C2H5)3N, CH2Cl2; (e)
KCN, DMSO.
Scheme 6. Reagents: (a) SOCl2, CHCl3; (b) NH3 or RNH3,
ether.
Scheme 5. Reagents: (a) BTEAC, 50% NaOH, toluene,
(CH3O)2SO2 or (C2H5O)SO2.

V. Leclerc et al./Bioorg. Med. Chem. 6 (1998) 1875±1887
1879
Scheme 7. Reagents: (a) R0COCl, K2CO3, H2O, CHCl3; (b) R0NCO or R0NCS, ether or pyridine; (c) carbonyldiimidazole, CHCl3.
Binding data were analyzed by non-linear regression to
curves for one or two-state receptor models14,17 to
determine best least-squares ®t based on an F-ratio test
for relative reduction in the Sum of Squares for each
®t.15 All binding experiments were carried out under
Zone A conditions16 whereby free ligand concentration
approximates total concentration in the reaction. Fitted
mono or biphasic binding anity parameter estimates
for compounds are given as the negative log (pKi) of
molar inhibition constants, Ki, calculated by application
of Cheng±Pruso€ correction factors to observed IC50
values. As for some G-protein coupled receptors, the
2-state estimates may provide an indication of the ability
of di€erent agonists to descriminate di€erent con-
formational states of the recepto.18 The binding method
however does not exclude the possibility of binding to
distinct sites on a multivalent receptor as has been
described for other G-protein coupled receptors19 or to
distinct receptor subtypes.
(F/M/D) activity index. For each experiment, the cAMP
responses are normalized against the response induced
by forskolin alone which is taken as the 100% e€ect.
The indexed activity of melatonin (M) is de®ned from
the normalized response ratio [F±F/M]/[F±F/M]=1 and
the indexed activity of the various treatments are calcu-
lated as the ratio [F±treatment]/[F±F/M] where treat-
ments are: basal response melatonin alone, drug alone,
drug/forskolin or drug/forskolin/melatonin at ®nal con-
centrations indicated above. The index values reported
are averages taken from three separate experiments and
error estimates are derived from standard statistical
formulae for error propogation through sums and quo-
tients.24 As these experiments are performed at only a
single dose, these indices provide only semi-quantitative
information. In some cases, rather than mimicking the
inhibition activity of melatonin, drug induced potentia-
tion of the forskolin response is seen resulting in a
negative activity value (e.g. [F±F/D]=1.0±1.5) in the
numerator of the ratio giving rise to a negative activity
index value. A compound that has no agonist activity or
that blocks the e€ect of melatonin will have indices
approaching 0, while one that fully mimics the activity
of melatonin will have an agonist activity index
approaching 1. There are also cases where a compound
has a negative `agonist' index and a corresponding
positive `antagonist' index showing that the potentiating
e€ect of the compound is blocked by melatonin. At
present, we have no experimental information which
allows us to explain the potentiation of forskolin sti-
mulated cAMP by these compounds but we suggest
these observations may possibly re¯ect inverse agonism.
The criteria used to used to categorize the biological
properties of the compounds are as follows:
The binding data are complemented by in vitro screen-
ing experiments using ovine pars tuberalis primary cul-
tures to characterize the activity of the reported
compounds on forskolin stimulated cAMP production.
The bioassay is described elsewhere in detail,14 but
brie¯y, forskolin (1 mM) stimulates a robust increase in
cAMP production (typically 20- to 50-fold) in these cells
which is inhibited (typically 80%) by 1 nM melatonin.
The e€ect of 10 mM drug (D) treatment alone (a high
dose chosen to maximize the likelihood of observing a
signi®cant response in the screening assay), with for-
skolin (F/D), and with the forskolin/melatonin (F/M/D)
is compared to the e€ect of forskolin/melatonin (F/M)
in order to derive an `agonist' (F/D) and an `antagonist'
AGONIST INDEX
(Drug Alon cf. Melatonin)
ANTAGONIST INDEX
(Drug/Melatonin cf. Melatonin)
Activity*
>0.8
0.4<<0.8
0.4<<0.8
<0.4
>0.8
>0.8
<0.8
Full agonist
Weak agonist
} Partial agonist/antag.
0.2<<0.8
<0.2
>0.8
<0.4
<0.4
Antagonist
No activity
*Activity on the melatonin receptor coupled to cAMP transduction pathway.

1880
V. Leclerc et al./Bioorg. Med. Chem. 6 (1998) 1875±1887
Table 3. Physical properties of melatonin analogues
Compd
R1
R2
n
X
R
Mp (ꢀC)
Recryst solvent
Yield (%)
Formula
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
1
1
1
1
1
1
1
1
0
2
2
2
1
1
1
1
1
1
1
1
1
1
0
0
2
1
1
1
1
2
2
3
2
2
2
3
3
1
-
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
CH3
C3H5
C4H7
85±87
117±118
88±89
79±80
79±80
51±53
140±142
88±90
165±166
66±68
75±76
toluene-cyclohexane
toluene-cyclohexane
toluene-cyclohexane
cyclohexane
cyclohexane
cyclohexane
toluene-cyclohexane
toluene-cyclohexane
toluene
cyclohexane
cyclohexane
cyclohexane
toluene-cyclohexane
cyclohexane
ethylacetate
toluene-cyclohexane
ethanol-water
toluene-cyclohexane
toluene-cyclohexane
toluene
90
88
89
85
70
78
80
79
94
56
56
92
35
90
65
80
69
93
80
56
44
72
77
99
70
80
81
80
80
53
50
38
96
43
78
45
38
75
78
86
87
76
85
59
59
87
30
88
65
78
75
C15H13NO
C17H15NO
C17H19NO
C14H12F3NO
C20H19NO
C22H23NO
CF3
H
CH2C6H5
(CH2)3C6H5
C3H5
C4H7
CH3
CH3
nC4H9
C3H5
OCH3
O-nC3H7
NH2
H
COOCH3
COOCH3
C18H19NO3
C19H21NO3
C14H15NO2
C16H19NO2
C19H25NO2
C18H21NO2
C15H17NO3
C17H21NO3
C14H16N2O2
C15H18N2O2
C16H16N2O2
C17H22N2O2
C18H24N2O2
C27H28N2O3
C13H14N2O
C14H16N2O
C16H20N2O2
C17H22N2O2
C18H24N2O2
C15H18N2OS
C16H20N2OS
C17H22N2O2S
C18H24N2OS
C14H16O2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
121±123
70±71
87±88
187±188
139±141
134±135
104±105
106±107
187±189
141±143
104±106
161±162
154±156
120±121
105±107
114±115
95±97
NHCH3
NHC2H5
NHnC3H7
NHnC4H9
NHCH2CH2Napha
NH2
toluene
H
NHCH3
NHnC3H7
NHnC4H9
NHnC3H7
NHCH3
NHC2H5
NHnC3H7
NHnC4H9
CH3
C2H5
CH3
COCH3
H
CH3
toluene-cyclohexane
95 ethanol
95 ethanol
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
toluene
toluene-cyclohexane
toluene-cyclohexane
toluene-n.hexane
toluene-cyclohexane
Ðb
S
S
S
65±68
Oil
Oil
Oil
Ðc
C15H18O2
C15H18O2
C15H16O3
Ðb
hexane
95 ethanol
51±52
125±126
98±100
96±98
109±112
156±157
91±92
99±100
102±103
99±100
79±80
125±127
163±165
153±155
126±127
64±67
C14H15NO2
C15H17NO2
C16H19NO2
C18H23NO2
C22H23NO2
C15H20N2O
C14H16N2O
C15H18N2O
C16H20N2O
C15H21N3O2
C13H16N2O3
C13H16N2O3
C14H18N2O3
C15H20N2O3
C15H17NO2
C15H20N2SO2
C14H15NOS
C15H17NOS
cyclohexane
cyclohexane
water-ethanol
cyclohexane
toluene-cyclohexane
toluene
CH3
nC3H7
(CH2)3C6H5
nC4H9
C3H5
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NH
NH
NH
NH
NH
NH
O
O
O
O
S
-
-
-
-
-
-
-
-
-
-
-
-
C4H7
C5H9
toluene
toluene
toluene
toluene
toluene
toluene
NHnC3H7
NHCH2CH2Indd
NHCH3
NHC2H5
NHnC3H7
C4H7
cyclohexane-toluene
ethanol-water
cyclohexane
cyclohexane
n.hexane-toluene
NHC3H7
C3H5
C4H7
103±105
98±99
80±82
S
S
aNaph: 7-methoxy-1-naphthyl.
bPuri®ed by column chromatography: SiO2, cyclohexane-ethyl acetate (4/1).
cPuri®ed by column chromatography: SiO2, dichloromethane.
dInd: 5-methoxy-3-indolyl.

V. Leclerc et al./Bioorg. Med. Chem. 6 (1998) 1875±1887
1881
Table 4. Binding and biological data for new melatonin analogues
Compd R1
R2
n
X
R
1-State (site)
Model
2- State (site)
Model
Agonist
Index
(‹ sd)
Antagonist
Index
(‹ sd)
pKi‹
est. sd
pKi (Hi) ‹ pKi (Lo) ‹
est. sd
est. sd
1
OCH3
H
H
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
CH3
CH3
9.57‹0.13
8.40‹0.49
7.38‹0.19
7.40‹1.77
7.18‹0.14
5.67‹1.09
5.84‹0.26
6.04‹0.11
5.16‹0.13
6.34‹0.28
8.34‹0.16
6.71‹0.10
7.62‹0.15
7.72‹0.21
1.00
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
H
1
1
1
1
1
1
1
1
0
2
2
2
1
1
1
1
1
1
1
1
1
1
0
0
2
1
1
1
1
2
2
3
2
2
2
3
3
1
-
0.75‹0.31
0.33‹0.13
0.10‹0.03
0.38‹0.15
0.13‹0.02
0.12‹0.09
0.85‹0.36
0.28‹0.11
0.01‹0.00
0.37‹0.14
0.63‹0.13
0.85‹0.36
H
H
C3H5
C4H7
CF3
H
H
H
H
H
H
CH2-C6H5
(CH2)3C6H5
C3H5
H
H
COOCH3
COOCH3
H
0.10‹0.03 0.16‹0.05
H
C4H7
CH3
N.D.
N.D.
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.26‹0.15
1.28‹0.42
6.10‹2.29
10.00‹3.39
1.16‹0.33
1.01‹0.28
1.15‹1.41
1.27‹0.34
1.33‹0.26
0.95‹0.22
1.19‹0.06
0.61‹0.13
0.47‹0.17
0.55‹0.13
0.12‹0.02
1.43‹0.23
0.47‹0.16
0.98‹0.29
0.99‹0.23
0.96‹0.22
N.D.
1.00‹0.75
1.07‹0.41
5.39‹1.83
4.75‹1.74
1.00‹0.33
1.00‹0.30
1.19‹0.42
1.18‹0.38
1.29‹0.36
0.99‹0.30
0.75‹0.22
1.00‹0.24
0.91‹0.33
0.92‹0.25
0.52‹0.10
0.83‹0.16
0.82‹0.29
0.98‹0.34
1.01‹0.34
0.96‹0.50
N.D.
CH3
nC4H9
C3H5
OCH3
O-nC3H7
NH2
12.99‹0.68 7.29‹0.16
14.31‹0.09 7.62‹0.33
8.45‹0.31
8.66‹0.46
8.17‹0.14
NHCH3
NHC2H5
NHnC3H7
NHnC4H9
NHCH2CH2MN
NH2
5.61‹0.10
5.92‹0.55
6.62‹0.12
6.97‹0.13
7.28‹0.16
5.70‹0.15
6.79‹0.05
8.05‹0.05
H
NHCH3
NHnC3H7
NHnC4H9
NHnC3H7
NHCH3
NHC2H5
NHnC3H7
NHnC4H9
CH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
S
12.43‹0.40 7.37‹0.06
12.48‹0.63 7.38‹0.50
S
S
5.44‹1.08
5.94‹0.13
6.09‹0.09
6.70‹0.19
6.16‹0.11
6.36‹0.13
8.15‹0.14
8.29‹0.02
7.37‹0.04
1.63‹0.19
0.40‹0.09
0.93‹0.45
0.52‹0.19
0.89‹0.33
1.14‹0.47
0.19‹0.08
0.03‹0.01
0.45‹0.19
0.70‹0.23
1.04‹0.38
1.05‹0.41
0.09‹0.03
0.09‹0.05
1.02‹0.34
N.D.
C2H5
COCH3
COCH3
H
CH3
CH
0.05‹0.01
nC3H7
0.02‹0.01
0.95‹0.34
N.D.
CH2CH2CH2C6H5 7.16‹0.86
H
H
NH
NH
NH
NH
NH
nC4H9
C3H5
C4H7
5.60‹0.60
6.81‹0.07
5.65‹0.34
5.57‹0.67
-
0.40‹0.16
N.D.
N.D.
0.50‹0.20
N.D.
N.D.
H
-
H
OCH3
-
C5H9
NHnC3H7
-
11.50‹0.64 7.92‹0.14
1.03‹0.48
1.08‹0.52
(continued)

1882
V. Leclerc et al./Bioorg. Med. Chem. 6 (1998) 1875±1887
Table 4Ðcontd
Compd R1
R2
n
X
R
1-State (site)
Model
2- State (site)
Model
Agonist
Index
(‹ sd)
Antagonist
Index
(‹ sd)
pKi‹
est. sd
pKi (Hi) ‹ pKi (Lo) ‹
est. sd
est. sd
53
54
55
56
57
58
59
60
OCH3
OCH3
OCH3
OCH3
H
-
-
-
-
-
-
-
-
NH NHCH2CH2MI
6.40‹0.55
8.22‹0.03
8.37‹0.16
7.33‹0.28
5.80‹0.07
0.89‹0.25
1.01‹0.19
0.95‹0.24
1.32‹0.32
2.16‹0.79
0.53‹0.28
1.13‹0.58
0.48‹0.16
0.98‹0.23
0.98‹0.22
0.99‹0.20
0.98‹0.23
0.62‹0.19
0.77‹0.37
0.65‹0.33
1.00‹0.33
O
O
O
O
S
NHCH3
NHC2H5
NHnC3H7
C4H7
OCH3
H
NHC3H7
C3H5
C4H7
12.44‹0.68 7.43‹0.22
S
7.53‹0.08
6.27‹0.24
H
S
MN: 7-Methoxy-1-naphthyl.
MI: 5-Methoxy-3-indolyl.
Binding data
receptor as in the amidic series.1 In the urea family we
observed a decrease in the anity following deletion of
the 7-methoxy group (30, 31 v/v 24, 25) and by increas-
ing (34 v/v 27) or decreasing (32 v/v 27) in the length of
the side chain between the naphthalenic system and the
urea function similar to the amidic series. However, that
is not the case when comparing 33 to 28 but for both
compounds, anities are very low and this result cannot
be considered as signi®cant.
The structure±anity relationships for the naphthalenic
analogues of melatonin from this study con®rms our
previous ®nding that the 7-methoxy group is essential
for high anity binding to the melatonin receptor. For
9
example, 10 has a Ki50 of 4.0Â10 as compared to
11
8.9Â10
for its methoxylated counterpart.1 Some
modi®cations to the acetyl side chain have been shown
to improve the anity of 7-methoxy analogues for the
melatonin receptor.1 However similar modi®cations to
the non-methoxylated analogues, such as replacement of
the methyl group of the amidic function by a cyclopro-
pyl group (11) or a tri¯uoromethyl group (13), failed to
improve the anity. Instead, a slight decrease in anity
was observed. It is therefore not surprising that the
introduction of a bulky arylalkylgroup, which is detri-
mental for binding of 7-methoxylated analogues1 to
melatonin receptors, also had a negative impact on the
anity of the nonmethoxylated analogues (14 and 15).
Increasing 19 or decreasing 18 the length of the side
chain between the naphthalenic system and the amidic
function results in a loss of binding anity. This is also
the result of the introduction of a rami®cation of the
side chain: 16 and 17 are only poor ligands, which is
consistent with the results of our previous study where
the introduction of a methyl group on the side chain
reduced the anity.
In the indolic, benzofuranic and benzothiophenic famil-
ies, deletion of the methoxy group as well as replace-
ment of the amidic function by urea also decreased
anity (48±60) for the melatonin receptor.
Some of the compounds studied gave biphasic binding
curves. These include carbamate, ureas or thioureas
substituted by ethyl (36) or bulky n-propyl groups (23,
27, 37, 52, and 58). The lack of correlation between
biphasic binding curves and nonsterically hindered
derivatives contrast with our previous study.1 In addi-
tion, most of the bulky substituents introduced in the
present study onto amidic, urea, and thiourea functions
in the naphthalenic, indolic, benzofuranic or benzothio-
phene series failed to show biphasic binding curves
under the conditions employed here. Thus at present
there is no structural clue for the basis of biphasic
binding curves.
Substitution of the amidic function by a urethane (22), a
urea (25), a thiourea (35), an alkoxy group (39), a car-
boxy (42) or a retroamide function (44) dramatically
reduces the anity for the melatonin receptor when
compared to the amidic analogue.1
Biological data
From the two last columns of Table 4 it ®rst appears
that the 7-methoxy group is of primary importance to
biological activity as seen previously.20 Deletion of this
group permitted us to obtain our ®rst antagonist: com-
pound 12. Moreover most of compounds lacking this
group are poor agonists with only derivative 10 having
Introduction of an n-propyl group (27) as a urea sub-
stituent increases the anity binding to the melatonin

V. Leclerc et al./Bioorg. Med. Chem. 6 (1998) 1875±1887
1883
any intrinsic activity. However, its anity for the mela-
tonin receptors is approximately 4500 times lower than
that of the corresponding methoxylated analogue.1 This
result con®rms the importance of the 7-methoxy group
for biological activity as previously hypothesized.4 Oth-
ers are either weak agonists (30, 31, and 49) or partial
agonists (11 and 13). The properties of compounds (14,
15, 57 and 59) in this group are less clear. While com-
pound 15 has no measurable activity, others potentiate
the e€ect of forskolin raising the possibility that they
may be acting as inverse agonists in this system.
naphthalenic ring by indole, benzothiophene or benzo-
furan ring has little e€ect on the previously described
structure±activity relationships.
Among the potential antagonists we note that we have:
.
the 7-demethoxy compound 12 with a bulky
cyclobutyl group as substituent of the carbonyl of
the amidic function (The corresponding cyclopro-
pyl derivative is a weak antagonist and the corre-
sponding methyl derivative is a weak agonist);
a compound having a rami®cation on the side
chain 16 and a cyclopropyl substituent on the
carbonyl of the amidic function (The analogue
(11) without a rami®cation is an agonist);
the three amidic compounds (19, 20, and 21) we
made with an extended side chain, a propyl link
replacing the ethyl one (Analogues with an ethyl
side chain are agonists);
.
.
Deletion of the 7-methoxy group from the full agonists
(24 and 25) gives the weak agonists (30 and 31) while
introduction of a carboxymethyl group lowers the
binding anity for melatonin receptors (16 and 17).
Replacement of the amidic group by an urethane (22), a
urea (25), a thiourea (35), or a retroamide (44) does not
modify the biological activity. The carboxy derivative
(42) is only a partial agonist while the alkoxy compound
39 is an antagonist.
.
.
the two ether compounds (39 and 40);
an ester compound (41) with an extended propyl
side chain (The analogue 42 with the ethyl side
chain is a partial agonist);
.
the two retroamidic derivatives (45 and 46) having
an extended propyl side chain. The analogue of
the derivative 45 having an ethyl side chain (44) is
a full agonist;
In the urea family we note that the n-propyl derivative
(32) is a weak antagonist but its higher homologue, the
n-butyl derivative (33) may be an inverse agonist. All the
urea derivatives studied are full agonists except com-
pounds 32 and 33, which have a shortened methylene
side chain and the n-propyl benzothiophene compound
58, which surprisingly has no agonist properties even
though the naphthalenic, indolic and benzofuranic ana-
logues are full agonists.
Lastly, it is important to compare the binding values
obtained with compounds 46 and 27. In 46 the NH
group of the original amidic function (which is also one
of the two NH groups of the urea function of com-
pound 27 is replaced by a CH2 group. Compound 46 is
a retroamidic derivative with a propylene side chain and
the binding inhibition pro®les are di€erent.
Changing the length of the side chain strongly decreases
the biological activity of the corresponding compounds.
For instance amides (19, 20, and 21) with a propylene
side chain are not full agonists in contrast to the ethyl-
ene homologues. These compounds also potentiate for-
skolin induced cAMP production. The same result is
seen when retroamidic compound 44 is compared to its
lengthened side chain analogue 45 and when the side
chain is shortened in urea compound 27 to give deriva-
tive 32.
Derivative 27 gives a biphasic binding curve with a very
high anity (pKi(Hi)=14.3) component and is an ago-
nist whereas compound 46 gives a much lower anity
monophasic response (pKi=7.37) and is an antagonist.
This provides evidence for the importance of the NH
group of the amidic function, which could provide a
hydrogen bond, for full functional activation of the
receptor response. It appears that the presence of this
group, separated from the aromatic ring by an ethylene
side chain is necessary to obtain ligands with full agonist
activity and with good anity whereas compounds,
which do not possess this NH group (39±42), are poor
ligands.
We note that the retroamide with lengthened side chain
46 is an antagonist but that its anity for the receptor is
quite low. A similar result was obtained after shortening
the side chain; 32 is a weak antagonist and no activity
was seen for 18 and 33.
If we take into account the above mentioned Table 4 it
appears that reasoning strictly according to agonist and
antagonist indexes we have obtained several antagonist
derivatives, especially compounds 12, 16, 19, 20, 21, 39,
40, 41, 45, and 46. Weak antagonists are compounds 11,
13, 14, 32, 49, 57, 58 and 59. Lastly, replacement of the
These results con®rm the importance of the secondary
amide site as well as the methoxy group ®xed on the
heterocyclic system for binding of the molecule to the
melatonin receptor as was previously reported.1 It also
appears that the respective position of these two structural
features is similarly important since any modi®cation

1884
V. Leclerc et al./Bioorg. Med. Chem. 6 (1998) 1875±1887
of the length of the ethylene moiety of the side chain
dramatically reduces binding anity and modi®es the
biological activity of the compounds. For full agonists,
a 7-methoxy group as well as an ethyl side chain
between the NH group of the amidic function and an
aromatic ring are necessary. Deletion of this 7-methoxy
group, an increase in the global steric size of the mole-
cule, modi®cations concerning the amidic function itself
and its position relative to the aromatic ring can result
in full or partial agonist. For these antagonists, we have
yet to compare the binding data with Schild analyses of
the full functional responses curves. However, com-
pound 12 appears to be the best antagonist from this
series and it has been selected for preclinical development.
organic layer was evaporated yielding a residue, that
was crystallized from n-hexane a€ording 4.98 g (94%) of
4a: mp 51±52 ꢀC, H NMR (80 MHz, CDCl3) 7.90±7.60
1
(m, 2H), 7.45±7.10 (m, 4H), 4.00 (s, 2H), 3.90 (s, 3H),
3.65 (s, 3H). Anal. (C14H14O3) C, H, O.
General procedure for the synthesis of the (7-methoxy-1-
naphthyl)alkanols derivatives (5a±5b). The method
adopted for the synthesis of 2-(7-methoxy-1-naphthyl)
ethanol (5a) is described. To a stirred suspension of
3.35 g (0.088 mol) of LiAlH4 in 50 mL of dry ether was
added a solution of 5 g (0.022 mol) of (4a) in 50 mL of
dry ether and the reaction was stirred at room tempera-
ture for 30 min. Addition of water, ®ltration, ether
extraction of the ®ltrate and evaporation of the organic
layer gave a solid residue, that was crystallized from
cyclohexane a€ording 3.65 g (82%) of 5a: mp 80±82 ꢀC,
1H NMR (80 MHz, CDCl3) 7.80±7.60 (m, 2H) 7.40±7.00
(m, 4H), 4.00 (t, 2H), 3.90 (1, 3H), 3.30 (t, 2H), 1.60 (s,
1H). Anal. (C13H14O2), C, H, O.
Experimental
Chemistry
Melting points were determined on a BUCHI 510 cap-
illary apparatus and are uncorrected. IR spectra were
1
recorded on a Perkin±Elmer 297 spectrophotometer. H
General procedure for the synthesis of the (7-methoxy-1-
naphthyl)alkanol mesylates derivatives (6a±6b). The
method adopted for the synthesis of 2-(7-methoxy-1-
naphthyl)ethanol mesylate (6a) is described. A solution
of 4 g (0.02 mol) of (5a) in 100 mL of dichloromethane
and 3.3 mL (0.024 mol) of triethylamine at 10 ꢀC was
treated with 2.75 g (0.024 mol) of mesyl chloride and, the
mixture was stirred at 0 ꢀC for 30 min. The reaction
mixture was washed with H2O, 1 N HCl and the organic
layer dried. Concentration a€orded the crude mesylate
which was crystallised from cyclohexane to give 5.27 g
NMR spectra were recorded on a WP 80-54 or a AC
300 Brucker spectrometer. Chemical shifts are reported
in d units (parts per million) relative to (CH3)4Si. Ele-
mental analyses for new substances were performed by
CNRS Laboratories (Vernaison, France). Obtained
results were within‹0.4% of the theoretical values.
(7-Methoxy-1-naphthyl) methylamine hydrochloride (3a).
To a 10% NaOH solution (30 mL), 0.65 mL of bromine
and 2.15 g (0.01 mol) of 7-methoxy-1-naphthylacetamide
were slowly added, under cooling and stirring. The
mixture was stirred for 30 min at room temperature and
then for a further 16 h at 80 ꢀC. After cooling, the mix-
ture was ether extracted and 1 N HCl was added until
pH 2±3. Aqueous phases were neutralized with NaOH
and extracted with ether. Evaporation of the organic
layer yielded an oil that, by treatment with HCl gas in
anhydrous ether, a€orded a solid, that was recrys-
tallized from ethanol to give 1.61 g (72%) of (3a): mp >
260 ꢀC, 1H NMR (80 MHz, DMSO d6) 8.75 (s, 3H),
8.00±7.00, (m, 6H), 4.50 (s, 2H), 4.00 (s, 3H). Anal.
(C12H14Cl NO) C, H, N.
ꢀ
1
(94%) of 6a: mp 62±63 C, H NMR (80 MHz, CDCl3)
7.80±7.60 (m, 2H), 7.40±7.10 (m, 4H), 4.55 (t, 2H), 3.95
(s, 3H), 3.50 (t, 2H), 2.85 (s, 3H). Anal. (C14H16O4) C,
H, O.
General procedure for the synthesis of the (7-methoxy-1-
naphthyl)alkyl nitriles derivatives (7a±7b). The method
adopted for the synthesis of 3-(7-methoxy-1-naphthyl)
propionitrile (7a) is described. 4.2 g (0.015 mole) of the
mesylate (6a) was dissolved in 20 mL of DMSO and
treated under re¯ux for 2 h with 2.90 g (0.045 mol) of
KCN. The reaction mixture was cooled and poured into
ice/H2O precipitating the crude nitrile which was crys-
tallised from n-hexane to give 3 g (95%) of 7a: mp 62±
64 ꢀC, 1H NMR (80 MHz, CDCl3) 7.90±7.70 (m, 2H),
7.40±7.20 (m, 4H), 4.00 (s, 3H), 3.40 (t, 2H), 2.75 (t,
2H). Anal. (C14H13NO) C, H, O.
General procedure for the synthesis of the methyl (7-
methoxy-1-naphthyl)alkylcarboxylates derivatives (4a±
4b). The method adopted for the synthesis of methyl
(7-methoxy-1-naphthyl)acetate (4a) is described.
A
solution of 5 g (0.023 mol) of the acid (2a) in 150 mL of
methanol was cooled to 10 ꢀC. Under stirring, 6.75 mL
(0.092 mol) of SOCl2 were added dropwise and the
mixture was stirred for 30 min at room temperature.
After evaporation, the oil was dissolved in ethylacetate
and the solution washed with 10% potassium carbonate
aqueous solution and then with water. After drying, the
General procedure for the synthesis of the (7-methoxy-1-
naphthyl)alkylamine derivatives (3c and 3d). The method
adopted for the synthesis of 3-(7-methoxy-1-naphthyl)-
propylamine hydrochloride (3c) is described. An over
NH3 saturated solution of 1.50 g (0.006 mol) of (7a) in
150 mL of ethanol was hydrogenated over Raney nickel

V. Leclerc et al./Bioorg. Med. Chem. 6 (1998) 1875±1887
1885
under pressure (50 bars) at 60 ꢀC for 12 h. After ®ltra-
tion and evaporation, the oil was dissolved in dry ether
and treated with gazeous HCl to give after ®ltration and
crystallisation from cyclohexane/ethanol (1:1) 1.37 g
(91%) of 3c: mp 197±200 ꢀC, 1H NMR (300 MHz,
DMSO-d6) 8.10 (s, 3H), 7.85 (d, 1H), 7.75 (d, 1H), 7.40±
7.15 (m, 4H), 3.95 (s, 3H), 3.10 (t, 2H), 2.90 (t, 2H), 2.00
(m, 2H). Anal (C14H18ClNO) C, H, N, O.
ethyl methyl ether (39) is described. 5 mL (0.05 mol) of
dimethylsulfate and 0.04 g (0.00014 mol) of benzyl-
triethylammonium chloride were added to a mixture of
0.6 g (0.003 mol) of the alcohol (5a) in 1.2 mL of 50%
aqueous sodium hydroxyde solution and 5 ml of
toluene. The reaction mixture was vigorously stirred for
24 h. Toluene was separated and the aqueous layer
extracted with toluene. The organic layer was separated,
washed with water, dried and evaporated under reduced
pressure to a€ord crude (39), which was puri®ed by
column chromatography (SiO2, cyclohexane/ethylace-
tate, 4/1) to give 0.34 g (53%) of pure 39: 1H NMR
(300 MHz, CDCl3) 7.73 (d, 1H), 7.65 (d, 1H), 7.44±7.13
(m, 4H), 3.93 (s, 3H), 3.73 (t, 2H), 3.40 (s, 3H), 3.30 (t,
2H). Anal. (C14H16O2) C, H, N.
Methyl-2-cyano(1-naphthyl) acetate (7c). To
a hot
(110 ꢀC) solution of 16.7 g (0.1 mol) of 1-naphthylaceto-
nitrile in 120 mL of dimethylcarbonate, 2.3 g (0.1 atg) of
sodium was added slowly during 30 min. The mixture
was maintained at this temperature for 1 h and then
evaporated. After extraction with ethylacetate, the
organic layer was dried and evaporated. The solid resi-
due was recrystallised from cyclohexane to give 19.8 g
(7-Methoxy-1-naphthyl) ethyl acetate (42). A solution of
2 g (0.01 mol) of the alcohol (5a) in 20 g (0.2 mol) of
acetic anhydride was re¯uxed for 2 h. After cooling and
evaporation under reduced pressure, the oily residue
was puri®ed by column chromatography (SiO2, cyclo-
hexane/ethylacetate, 4/1) to give an oil, which then
a€orded a solid that was crystallized from hexane to
(88%) of 7c: mp 89±90 ꢀC, H NMR (80 MHz, DMSO-
1
d6) 8.10±7.40,(m, 7H), 5.40 (s, 1H), 3.75 (s, 3H). Anal.
(C14H11NO2) C, H, N.
Methyl-3-amino-2-(1-naphthyl)propionate (3e) hydro-
chloride. To a solution of 4.5 g (0.02 mol) of the nitrile
(7c) in 250 mL of methanol, 9.5 g (0.04 mol) of CoCl2
was added, followed by slow addition of 5.29 g
(0.14 mol) of NaBH4 at room temperature. The mixture
was stirred at this temperature for 2 h and then made
acidic with 3 N HCl. Solvent was evaporated and the
solution extracted with ethylacetate. The aqueous solu-
tion was made basic with aqueous ammonia and
extracted several times with ethylacetate. The organic
layers were evaporated and the obtained residue dis-
solved in ethanol. Gazeous HCl was bubbled into the
solution to a€ord a solid, that was recrystallized from
acetonitrile to give 2.38 g (42%) of 3e: mp 217±219 ꢀC,
1H NMR (80 MHz, DMSO-d6) 8.40 (s, 3H), 8.25±7.40,
(m, 7H), 5.10 (dd, 1H), 3.60 (s, 3H), 3.60±3.00 (m, 4H).
Anal. (C14H16ClNO2, H2O) C, H, N.
give 0.235 g (96%) of pure 42: mp 51±52 ꢀC; H NMR
1
(300 MHz, CDCl3) 7.77 (d, 1H), 7.67 (d, 1H), 7.47±7.13
(m, 4H), 4.41 (t, 2H), 3.98 (s, 3H), 3.35 (t, 2H), 2.07 (s,
3H). Anal. (C15H16O3) C, H, N.
General procedure for the synthesis of the (7-methoxy-1-
naphthyl)alkyl carboxamides derivatives (43±46). The
method adopted for the synthesis of 2-(7-methoxy-1-
naphthyl)ethyl carboxamide (43) is described. To a
solution of 1.1 g (0.0048 mol) of the acid (2b) in 20 mL
of chloroform, 5.3 mL (0.072 mol) of SOCl2 were
added dropwise under stirring and the mixture was then
re¯uxed for 2 h. After cooling and evaporation, the
residue was taken o€ with anhydrous ether and ®ltrated.
To the cooled ®ltrate, 8.8 mL (0.096 mol) of 20% aqu-
eous solution of NH3 was added in one portion. Stirring
was continued for 1 h. After ®ltration, the crude amide
was crystallized from 95 ꢀC ethanol a€ording 0.47 g
(43%) of 43: mp 125±126 ꢀC, 1H NMR (80 MHz,
CDCl3) 7.80±7.60 (m, 2H), 7.40±7.00 (m, 4H), 5.40 (s,
2H), 3.95 (s, 3H), 3.40 (t, 2H), 2.60 (t, 2H). Anal.
(C14H15NO2) C, H, N.
General procedure for the synthesis of the (7-methoxy-1-
naphthyl)alkanoic acids derivatives (2b and 2c). The
method adopted for the synthesis of 3-(7-methoxy-1-
naphthyl)propionic acid (2b) is described. One gramme
(0.0047 mol) of 7a was dissolved in 10 mL of methanol
and 10 ml (0.06 mol) of a 6 N aqueous solution of
sodium hydroxyde was then added. The mixture was
re¯uxed for 15 h. After cooling and acidi®cation with
6 N HCl, the precipitate was separated and then crys-
tallized from toluene a€ording 0.97 g (90%) of 2b: mp
154±155 ꢀC, 1H NMR (80 MHz, DMSO-d6) 12.20 (s,
1H), 7.95±7.60 (m, 2H), 7.10±7.40 (m, 4H), 3.90 (s, 3H),
3.30 (t, 2H), 2.65 (t, 2H). Anal. (C14H14O3) C, H, O.
General procedure for the synthesis of the N-mono-
substituted ureas derivatives (24 and 30). The method
adopted for the synthesis of N-[2-(7-methoxy-1-naph-
thyl)ethyl] urea (24) is described. 0.55 g (0.0067 mol) of
potassium cyanate was added to a solution of 1.5 g
(0.006 mol) of 3b hydrochloride in 8 mL of water. The
reaction mixture was then stirred at room temperature
for 30 min. The solid formed was separated and then
crystallized from ethylacetate, a€ording 0.79 g (65%) of
24: mp 187±188 ꢀC; 1H NMR (300 MHz, DMSO-d6)
General procedure for the synthesis of the (7-methoxy-1-
naphthyl)alkyl ethers derivatives (39±41). The method
adopted for the synthesis of 2-(7-methoxy-1-naphthyl)-

1886
V. Leclerc et al./Bioorg. Med. Chem. 6 (1998) 1875±1887
7.82 (d, 1H), 7.69 (m, 2H), 7.29 (m, 2H), 7.15 (dd, 1H),
6.14 (m, 1H), 5.52 (s, 2H), 3.94 (s, 3H), 3.28 (m, 2H),
3.10 (t, 2H). Anal. (C14H16N2O2) C, H, N.
dropwise at this temperature. The reaction mixture was
then stirred at room temperature for 1 h. After acid-
i®cation with 2 N HCl and stirring for 15 min, the aqu-
eous layer was extracted three times with chloroform.
The organic layers were separated, washed with water,
dried and evaporated under reduced pressure. The resi-
due was crystallized from toluene a€ording 0.21 g (94%)
of 18: mp 165±166 ꢀC; 1H NMR (80 MHz, CDCl3) 7.80±
7.00 (m, 6H), 5.70 (s, 1H), 4.80 (d, 2H), 3.90 (s, 3H),
2.00 (s, 3H). Anal. (C14H15NO2) C, H, N.
General procedure for the synthesis of the dissymetric
N,N0-disubstituted ureas derivatives (25±28; 31±38; 52;
54±56; 58). The method adopted for the synthesis of N-
[2-(7-methoxy-1-naphthyl)ethyl]-N0-methyl urea (25) is
described. 0.23 mL (0.0055 mol) of methyl isocyanate
was added to a solution of 1 g (0.005 mol) of 3b in 8 mL
of ether. The reaction mixture was then stirred at room
temperature for 30 min. The solid formed was separated
and then crystallized from toluene/cyclohexane (9/1)
N-[2-(1-naphthyl)ethyl] tri¯uoroacetamide (13). A suspen-
sion of 2.10 g (0.01 mol) of 3d hydrochloride in 6 mL of
pyridine was cooled to 5 ꢀC. 2.1 g (0.01 mol) of tri¯uoro-
acetic anhydride was then added dropwise at this tempera-
ture. After stirring for 30 min at room temperature, the
solution was poured into ice water. The resulting precipitate
was ®ltered, washed with water and crystallised from
cyclohexane a€ording 0.23 g (85%) of 13: mp 79±80 ꢀC 1H
NMR (80 MHz, CDCl3) 8.15±7.30 (m, 7H), 6.50 (s, 1H),
3.70 (m, 2H), 3.32 (t, 2H). Anal. (C14H12F3NO), C, H, N.
a€ording 1.03 g (80%) of 25: mp 139±141 ꢀC; H NMR
1
(80 MHz, DMSO-d6) 7.85±7.05 (m, 6H), 4.55 (t, 1H),
3.95 (s, 3H), 3.55 (m, 2H), 3.20 (t, 2H), 2.65 (d, 3H).
Anal. (C15H18N2O2) C, H, N.
General procedure for the synthesis of the symetric N,N0-
disubstituted ureas derivatives (29 and 53). The method
adopted for the synthesis of bis N,N0-[2-(7-methoxy-1-
naphthyl)ethyl urea (29) is described. 0.8 g (0.005 mol)
of N,N0-carbonyl diimidazole was added to a solution of
2 g (0.01 mol) of 3b in 100 mL of chloroform. The reac-
tion mixture was then re¯uxed for 4 h. The organic layer
was separated, washed with 2 N HCl, then with water,
dried and evaporated under reduced pressure. The resi-
due was crystallized from toluene, a€ording 1.20 g
(56%) of 29: mp 187±189 ꢀC; 1H NMR (80 MHz,
CDCl3) (7.80±7.10 (m, 12H), 7.95 (s, 6H), 3.50 (m, 4H),
3.20 (m, 4H). Anal. (C27H28N2O3) C, H, N.
Pharmacology. Reagents and chemicals. 2-[125I]-Iodome-
latonin was purchased from a commercial source (Dupont-
NEN) at a speci®c activity of approximately 2000 Ci/
mmol. [125I]AMP was also prepared as described.21
Stock chemicals and media were purchased from Sigma
Chemical Co. and BRL Life Technologies. Novel com-
pounds were synthesized as described in the present paper.
Cell and membrane preparations. Fresh ovine pars
tuberalis tissue was obtained at a local abattoir from
animals of mixed sex, breed, and age. Preparation of
crude membranes22 and primary culture of pars tuber-
alis cells21 have been extensively described. For binding
studies, washed membranes were resuspended in ice-
cold assay bu€er (0.025 M Tris±HCl, 1 mM EGTA, pH
7.5) at a ®nal concentration of 0.04 pars tuberalis equiv/
50 mL (ca. 6±12 mg of protein by the method of Brad-
ford23). For cAMP inhibition studies, cells were har-
vested after a 24 h culture in Dulbecco's modi®ed
Eagle's medium (25 mM HEPES, 4500 g of glucose/L,
penicillin (100 units/mL), streptomycin (100 mg/mL),
fungizone (0.25 mg/mL)) at 37 ꢀC, 95%/5% air/CO2.
Single-cell suspensions were washed and resuspended at
a concentration of 1.0Â106 cells/mL of medium for
experimental use.
General procedure for the synthesis of the alkyl N-2-(7-
methoxy-1-naphthyl)ethyl carbamates derivatives (22±
23). The method adopted for the synthesis of methyl N-
2-(7-methoxy-1-naphthyl)ethyl carbamate (22) is descri-
bed. Methyl chloroformate (0.0055 mol) was added
dropwise to a cooled solution of 1.25 g (0.005 mol) of 3b
hydrochloride in 10 mL of THF and 1.1 mL (0.015 mol)
of triethylamine. The reaction mixture was then stirred
at room temperature for 1 h and ®ltered. The ®ltrate was
evaporated under reduced pressure and the residue
crystallized from toluene/cyclohexane (9/1) a€ording
0.45 g (35%) of 22: mp 70±71 ꢀC; 1H NMR (80 MHz,
DMSO-d6) 7.90±7.05 (m, 6H), 4.75 (m, 1H), 3.80 (s,
3H), 3.70 (s, 3H), 3.55 (m, 2H), 3.30 (t, 2H). Anal.
(C15H17NO3) C, H, N.
General procedure for the synthesis of the N-acylated
derivatives (10±12; 14±21; 47±51; 57; 59±60). The
method adopted for the synthesis of N-(7-methoxy-1-
naphthyl)methyl acetamide (18) is described. Potassium
carbonate (0.003 mol) was added to a solution of 0.24 g
(0.001 mol) of 3a hydrochloride in 15 mL of water and
50 mL of chloroform. The mixture was cooled to 0 ꢀC.
0.12 mL (0.0016 mol) of acetyl chloride was added
Binding studies. For competitive binding experiments,
50 mL membrane suspension was added to wells of spe-
cially designed 96-well plates (Milipore Multiscreen
Assay System, Millipore Inc, Bedford, MA, USA) con-
taining 150 mL bu€er with approximately 50 pM 2-[125I]-
iodomelatonin and varying concentrations of competing
ligand (0, 10 14±10 4 M ®nal concentration). Reactions

V. Leclerc et al./Bioorg. Med. Chem. 6 (1998) 1875±1887
1887
(2 h, 37 ꢀC) were stopped by rapid ®ltration (ca. 15 sec/
plate) on a vacuum manifold and the wells were washed
once with 200 mL cold bu€er. Plates were then dried and
the ®lter disks, with trapped membrane bound radio-
ligand, were punched from the bottom of each well
directly into tubes which were then counted on a Packard
Instrument Co Model 5010 autogamma counter at 82%
eciency. Count data were analyzed by non-linear
regressions as described earlier. Individual drugs were
assayed at least three times and the values reported are
the means and estimated standard deviations of logarithms
of the calculated inhibition constants.
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200 mL cell suspensions (200 000 cells) were added to
microtubes containing 50 mL of medium alone or with
appropriately concentrated drug solution. Reactions
(15 min, 37 ꢀC) were stopped in a boiling water bath
(2 min), and the tubes were sonicated and then frozen
( 20 ꢀC) until cAMP was measured by radioimunno-
assay using a highly speci®c antibody (anti-cAMP
serum NO. 338; Drs D. C. Klein and A. K. Ho,
National Institutes of Health, Bethesda, MA) and [125I]
cAMP. Sodium acetate (0.05 M, pH 6.0) was used as the
diluent bu€er. Samples and standards (100 mL) were
acetylated by addition of 10 mL of triethylamine/acetic
anhydride (2/1) prior to the addition of iodinated tracer
(100 mL, 15 000 cpm) and antibody (100 mL). Antibody-
binding reactions were carried out overnight at 4 ꢀC and
separation of the bound fraction was accomplished by
precipitation of carrier protein (100 mL of 10% (w/v
bu€er) bovine serum albumin) with 2 mL of ice-cold
ethanol. Data were analysed on-line (Packard Instru-
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coecients of variation of this assay are less than 15%,
and the sensitivity (lowest amount statistically distin-
guishable from zero dose) is 4 fmol/tube. The biological
activity of each compound was assessed relative to the
e€ect of melatonin inhibition (activity index = 1.0) of
forskolin-stimulated cAMP production.
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