New trisubstituted 1,2,4-triazole derivatives as potent ghrelin receptor antagonists. 3. Synthesis and pharmacological in vitro and in vivo evaluations

Article abstract of DOI:10.1021/jm701292s

Ghrelin receptor ligands based on trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the replacement of the α- aminoisobutyryl moiety by aromatic or heteroaromatic groups. Compounds 5 and 34 acted as potent in vivo antagonists of hexarelin-stimulated food intake. These two compounds did not stimulate growth hormone secretion in rodents and did not antagonize growth hormone secretion induced by hexarelin.

Full text of DOI:10.1021/jm701292s

J. Med. Chem. 2008, 51, 689–693
689
New Trisubstituted 1,2,4-Triazole Derivatives as Potent Ghrelin Receptor Antagonists.
3. Synthesis and Pharmacological in Vitro and in Vivo Evaluations
Aline Moulin,† Luc Demange,† Joanne Ryan,† Delphine Mousseaux,† Pierre Sanchez,† Gilbert Bergé,† Didier Gagne,†
Daniel Perrissoud,§ Vittorio Locatelli,# Antonio Torsello,# Jean-Claude Galleyrand,† Jean-Alain Fehrentz,*,†,‡ and
Jean Martinez†,‡
Institut des Biomolécules Max Mousseron, UMR 5247, CNRS, UniVersités Montpellier 1, Montpellier 2, BP 14491, 15 AVenue Charles
Flahault, 34093 Montpellier Cedex 5, France, Zentaris GmbH, Weismuellerstrasse 50, 60314 Frankfurt am Main, Germany, and Department of
Experimental Medicine, School of Medicine, UniVersity of Milano-Bicocca, Via Cadore 48, 20052 Monza (MI), Italy
ReceiVed October 15, 2007
Ghrelin receptor ligands based on trisubstituted 1,2,4-triazole structure were synthesized and evaluated
for their in vitro binding and biological activity. In this study, we explored the replacement of the
R-aminoisobutyryl moiety by aromatic or heteroaromatic groups. Compounds 5 and 34 acted as potent
in vivo antagonists of hexarelin-stimulated food intake. These two compounds did not stimulate growth
hormone secretion in rodents and did not antagonize growth hormone secretion induced by hexarelin.
Introduction
Ghrelin1 and its receptor (GHS-R1aa )2 are nowadays inten-
sively studied. Ghrelin exhibits a wide range of biological
activities.3,4 It not only increases growth hormone (GH) plasma
levels, it also stimulates food intake. Ghrelin affects body weight
and adiposity. Chronic administration of ghrelin in freely fed
mice and rats results in increased body weight and decreased
fat utilization.5 Antagonizing the ghrelin effect with a peptide
Figure 1. General formula for the trisubstituted 1,2,4-triazoles.
antagonist results in reduction of food intake and body weight
gain in diet-induced obese mice.6 For these reasons, small non-
peptide GHS-R1a antagonists became of great interest as
potential drugs for the treatment of obesity.7–9
We recently described new series of ghrelin receptor–li-
gands based on trisubstituted 1,2,4-triazoles of general
formula shown in Figure 1. In an early study,10 we reported
the synthesis and biological evaluation of potent GHS-R1a
ligands; the structure–activity relationship study on positions
R1 and R2 led to the conclusion that a benzyl group in position
4 of the triazole ring or a two-carbon atom chain bearing a
phenyl or an indole group in position 5 of the triazole ring,
keeping an R-aminoisobutyryl (Aib) moiety as the R3 group
(see Figure 1), yields the most potent compounds. Further-
more, the introduction of a 4-methoxybenzyl or a 2,4-
dimethoxybenzyl group in position 4 of the triazole ring
yielded GHS-R1a antagonists.10 Thus, compounds JMV2806
Figure 2. Previously described 1,2,4-triazole based GHS-R1a ligands.
and JMV2844 (Figure 2) (respectively, compounds 18a and
19c in ref 10) were found to be potent GHS-R1a ligands
with agonist and antagonist activities, respectively.
we have shown that it was possible to maintain or even enhance
More recently,11 we investigated the significance of the Aib
GHS-R1a affinity by introducing a large variety of substituents
moiety by replacing it with natural or unnatural R-amino acids
in place of the Aib moiety in this triazole series. We were also
and piperidine- and piperazine-carboxyl groups. Contrary to
able to modulate the agonist or antagonist character of the
previous unsuccessful attempts with compound JMV1843,12
a
molecule by modifying this R3 group. We described subnano-
molar GHS-R1a agonists such as compound JMV2951 and also
potent antagonists such as compound JMV3008 (Figure 2)
(respectively 40 and 20 in ref 11).
In our ongoing efforts to target new potent selective and
orally active GHS-R1a ligands, and particularly antagonists,
we now describe the replacement of the Aib moiety by
aromatic or heteroaromatic groups such as benzyl, phenyl-
carboxyl, pyridinyl-carboxyl, pyridinyl-acetyl, or pyrazine-
carboxyl groups.
potent GHS-R1a agonist active in man by oral administration,13,14
* To whom correspondence should be addressed. Phone: 33 4 67 54 86
51. Fax: 33 4 67 54 86 54. E-mail: jean-alain.fehrentz@univ-montp1.fr.
†
Universités Montpellier 1.
Zentaris GmbH.
University of Milano-Bicocca.
Equal contributors.
§
#
‡
a Abbreviations: GHS-R1a, growth hormone secretagogue receptor type
1a; GH, growth hormone; Aib, R-aminoisobutyric acid; CHO, Chinese
hamster ovary.
10.1021/jm701292s CCC: $40.75
2008 American Chemical Society
Published on Web 01/15/2008

690 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Brief Articles
Table 1. Binding Affinity Constants and Biological Activities of Trisubstituted 1,2,4-Triazoles
% of maximum [Ca2+
]
i
compd
R1
R2
R3
IC50 (nM)a
response at 10 µMb
EC50 (nM)c Kb (nM)d
1
2
3
4
5
6
7
8
2,4-dimethoxybenzyl 1H-indole-3-yl-ethyl aminoisobutyryl
108 ( 17
11 ( 4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
6
4
0
3
3
3
6
5
14 ( 2
5 ( 1
4-methoxybenzyl
2,4-dimethoxybenzyl phenethyl
phenethyl
aminoisobutyryl
aminoisobutyryl
60 ( 10
1.8 ( 0.4
1.1 ( 0.5
33 ( 14
67 ( 15
49 ( 9
1.9 ( 0.6
0.7 ( 0.2
105 ( 19
160 ( 20
58 ( 1
>1000
220 ( 70
34 ( 6
120 ( 30
51 ( 20
96 ( 33
7.9 ( 0.0
3 ( 1
17 ( 7
19 ( 11
25 ( 3
65 ( 9
513 ( 45
129 ( 27
102 ( 20
12 ( 3
ND
2,4-dimethoxybenzyl 1H-indole-3-yl-ethyl (pyridin-2-yl)carboxyl
2,4-dimethoxybenzyl phenethyl
2,4-dimethoxyphenyl phenethyl
(pyridin-2-yl)carboxyl
(pyridin-2-yl)carboxyl
(pyridin-2-yl)carboxyl
4-ethylbenzyl
4-ethylphenyl
4-methoxybenzyl
4-methoxybenzyl
phenyl
phenethyl
1H-indole-3-yl-ethyl (pyridin-2-yl)carboxyl
1H-indole-3-yl-ethyl (pyridin-2-yl)carboxyl
phenethyl
phenethyl
9
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
(pyridin-2-yl)carboxyl
(pyridin-2-yl)carboxyl
phenyl
1H-indole-3-yl-ethyl (pyridin-2-yl)carboxyl
ND
93 ( 23
ND
2,4-dimethoxybenzyl phenethyl
(pyridin-4-yl)carboxyl
(pyridin-4-yl)carboxyl
(pyridin-4-yl)carboxyl
4-ethylbenzyl
phenethyl
phenethyl
4-methoxybenzyl
ND
2,4-dimethoxybenzyl 1H-indole-3-yl-ethyl (pyridin-2-yl)acetyl
14 ( 3
35 ( 2
124 ( 44
34 ( 11
12 ( 4
6 ( 3
41 ( 7
ND
ND
2,4-dimethoxyphenyl phenethyl
(pyridin-2-yl)acetyl
(pyridin-2-yl)acetyl
4-ethylbenzyl
4-ethylphenyl
4-methoxybenzyl
4-methoxybenzyl
benzyl
phenethyl
1H-indole-3-yl-ethyl (pyridin-2-yl)acetyl
1H-indole-3-yl-ethyl (pyridin-2-yl)acetyl
phenethyl
(pyridin-2-yl)acetyl
1H-indole-3-yl-ethyl (pyridin-2-yl)acetyl
61 ( 34
700 ( 250
480 ( 120
29 ( 0
phenyl
phenethyl
phenethyl
phenethyl
(pyridin-2-yl)acetyl
(pyridin-3-yl)acetyl
(pyridin-3-yl)acetyl
4-ethylbenzyl
4-methoxybenzyl
4-methoxybenzyl
4-methoxybenzyl
4-methoxybenzyl
4-methoxybenzyl
93 ( 2
10 ( 3
ND
1H-indole-3-yl-ethyl (pyridin-4-yl)acetyl
76 ( 1
phenethyl
phenethyl
phenethyl
(pyridin-4-yl)acetyl
(pyridin-3-yl)propionyl
phenylcarboxyl
phenylcarboxyl
benzyl
270 ( 50
177 ( 78
80 ( 20
11.8 ( 4.0
>1000
ND
5
23 ( 0.3
16 ( 4
ND
2,4-dimethoxybenzyl phenethyl
2,4-dimethoxybenzyl phenethyl
31 ( 7
20 ( 2
2,4-dimethoxybenzyl 1H-indole-3-yl-ethyl 2-aminophenylcarboxyl
2,4-dimethoxybenzyl 1H-indole-3-yl-ethyl (cis)2-aminocylcohexylcarboxyl 140 ( 10
2,4-dimethoxybenzyl 1H-indole-3-yl-ethyl pyrazine-2-carboxyl
112 ( 17
7
4
0
0
0
0
1
ND
140 ( 50
97 ( 24
24 ( 5
530 ( 110
76 ( 11
ND
18.5 ( 5.0
5 ( 1
14 ( 8
89 ( 26
300 ( 70
2,4-dimethoxybenzyl phenethyl
pyrazine-2-carboxyl
pyrazine-2-carboxyl
pyrazine-2-carboxyl
pyrazine-2-carboxyl
4-methoxybenzyl
4-ethylbenzyl
phenethyl
phenethyl
2,4-dimethoxyphenyl phenethyl
a Specific binding was determined by incubation of membranes from GHS-R1a transfected LLC PK-1 cells with 125I-His9-ghrelin in the presence of
increasing concentrations of compounds. b The activation percentage for each compound (10-5 M) was assessed relatively to the maximal response, obtained
with ghrelin at 10-7 M (100%). c The signaling through the GHS-R1a, as determined by the accumulation of intracellular calcium and thus fluorescence
output, was measured in the presence of increasing concentrations of agonists. d The ability of the antagonists to inhibit ghrelin signaling through the
GHS-R1a (measurement of flurorescence output) was assessed using Schild plots, with increasing concentrations of ghrelin alone or in the presence of
antagonist compound at 10-8, 10-7, or 10-6 M.
Chemistry
percent of the maximal response induced by 10-7 M ghrelin
(Table 1). Compounds 1-3, containing the Aib moiety, are
given as reference compounds.10 The best compounds were
tested in vivo for their ability to stimulate food intake or to
inhibit hexarelin-stimulated food intake.
To study the effect of Aib replacement, 1,2,4-triazoles were
prepared as previously described10,15 from Boc-D-Trp-OH and
various substitutes of Aib were introduced after deprotection
of the primary amine.
We previously described11 that the best GHS-R1a ligands of
our series had an isonipecotyl moiety in position R3, i.e., a six-
atom ring with a nitrogen atom in a position para to the carbonyl
group. We so evaluated the influence of the introduction of
heteroaryl group containing a nitrogen atom in the same position
on the binding affinity and we first introduced a pyridyl moiety.
(Pyridin-2-yl)carboxyl group replaced favorably the Aib residue,
yielding compounds with high affinity for the ghrelin receptor.
When comparing 1, 2, and 3 possessing an Aib with 4, 10, and
5 having a (pyridin-2-yl)carboxyl group with R1 and R2
unchanged, the affinity constants were found to be 60-, 56-,
and 13-fold better for the last compounds with nanomolar
affinity (4, IC50 ) 1.8 ( 0.4 nM; 5, IC50 ) 1.1 ( 0.5 nM; 10,
IC50 ) 0.7 ( 0.2 nM) (Table 1). As previously described,
4-methoxybenzyl or 2,4-dimethoxybenzyl groups in position R1
gave the best ligands. When 2,4-dimethoxyphenyl (6), 4-eth-
ylbenzyl (7), and 4-ethylphenyl (8) groups were placed in the
R1 position, the affinities decreased from 33 to 67 nM.
All compounds described in this paper were obtained by
coupling a carboxylic function on the primary amine, except
compound 31 which was obtained by reaction of benzyl chloride
with the corresponding amine in dichloromethane in the presence
of sodium bicarbonate as a base. Keeping the starting material
Boc-D-Trp-OH and in a few steps, we could prepare interesting
molecules representing three points of diversity, thus allowing
a structure–activity relationship study.
Results and Discussion
The synthesized compounds were tested for their ability to
displace 125I-His9-ghrelin from the cloned hGHS-1a receptor
transiently expressed in LLC PK-1 cells. Binding affinities of
human ghrelin and MK-0677 on these cells were in accordance
with values reported in the literature. The biological in vitro
activity of the compounds (10-5 M) was then evaluated on
intracellular calcium mobilization [Ca2+]i using CHO cells
transiently expressing GHS-R1a. Results are expressed as a

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 691
Table 2. Cumulative Food Intake in Rats after Subcutaneous Administration of 160 µg/kg Compound and/or 80 µg/kg Hexarelin
cumulative food intakea at 2 h
for 160 µg of compound
(cumulative food intake at 2 h
for 80 µg of hexarelin)
cumulative food
intakea at 2 h for 160
µg compound + 80
µg of hexarelin
cumulative food intakea
at 6 h for 160 µg compound
(cumulative food intake at 6 h
for 80 µg of hexarelin)
cumulative food
intakea at 6 h for 160
µg compound + 80
µg of hexarelin
% variation versus
hexarelin at 6 hb
compd
saline
1
2
4
5
0.18 ( 0.11
0.49 ( 0.18
0.02 ( 0.01 (0.83 ( 0.30)
0.01 ( 0.0 (0.63 ( 0.23)
0.30 ( 0.11 (0.57 ( 0.17)
0.01 ( 0.0 (0.43 ( 0.16)
0.14 ( 0.14 (1.03 ( 0.21)
0.29 ( 0.17 (0.58 ( 0.13)
0.01 ( 0.0 (0.41 ( 0.12)
0.02 ( 0.01 (0.43 ( 0.16)
0.01 ( 0.0 (1.06 ( 0.15)
0.01 ( 0.0 (0.26 ( 0.11)
0.01 ( 0.0 (0.46 ( 0.15)
0.01 ( 0.0 (0.54 ( 0.17)
0.01 ( 0.0 (0.28 ( 0.08)
0.52 ( 0.28 (0.69 ( 0.27)
0.01 ( 0.0 (0.29 ( 0.11)
0.41 ( 0.33 (0.58 ( 0.13)
0.06 ( 0.03
0.53 ( 0.21
0.72 ( 0.16
0.01 ( 0.0
0.06 ( 0.04 (0.91 ( 0.38)
0.20 ( 0.19 (0.70 ( 0.21)
0.32 ( 0.13 (0.98 ( 0.19)
0.11 ( 0.10 (0.69 ( 0.20)
0.35 ( 0.15 (1.45 ( 0.19)
0.43 ( 0.25 (0.88 ( 0.09)
0.02 ( 0.01 (0.73 ( 0.21)
0.02 ( 0.01 (0.69 ( 0.20)
0.62 ( 0.30 (1.39 ( 0.29)
0.01 ( 0.0 (0.73 ( 0.11)
0.28 ( 0.17 (0.72 ( 0.17)
0.02 ( 0.0 (0.59 ( 0.17)
0.08 ( 0.06 (0.88 ( 0.26)
0.53 ( 0.28 (0.73 ( 0.27)
0.14 ( 0.08 (0.60 ( 0.15)
0.91 ( 0.17 (0.88 ( 0.09)
0.33 ( 0.21
0.63 ( 0.20
1.03 ( 0.12
0.02 ( 0.01
0.73 ( 0.24
0.75 ( 0.20
0.87 ( 0.14
0.64 ( 0.23
0.91 ( 0.32
0.87 ( 0.38
0.46 ( 0.25
0.56 ( 0.33
0.48 ( 0.14
0.20 ( 0.09
1.04 ( 0.15
0.81 ( 0.20
-64
-10
+5
-97
-50
-15
+19
-7
-34
+19
-36
-5
-45
-73
+73
-8
9
0.47 ( 0.20
0.37 ( 0.11
0.56 ( 0.10
0.37 ( 0.18
0.89 ( 0.32
0.43 ( 0.23
0.13 ( 0.13
0.55 ( 0.34
0.01 ( 0.01
0.11 ( 0.07
0.42 ( 0.15
0.72 ( 0.18
10
13
16
18
21
25
27
33
34
35
36
a Expressed as g of food intake per 100 g of body weight. b 100 × (cumulative food intake at 6 h for compound and hexarelin minus cumulative food
intake at 6 h for hexarelin)/(cumulative food intake at 6 h for hexarelin).
Substitution with a phenyl group in R1 position (11 and 12)
resulted in a significant decrease of affinity. When the pyridin-
2-yl moiety was replaced with the pyridin-4-yl group, the affinity
constant decreased from 1.1 ( 0.5 nM (5) to 58 ( 1 nM (13)
and from 0.7 ( 0.2 nM (10) to 220 ( 70 nM (15). Compound
14 with a 4-ethylbenzyl group in the R1 position did not present
any affinity toward the GHS-1a receptor (compared to 7). These
results pointed out the significance of the nitrogen atom position
in the R3 group for affinity to the GHS-R1a.
When tested for their ability to induce intracellular calcium
mobilization, most of the compounds were not able to activate
the receptor. This ability to activate the GHS-1a receptor and
promote a biological response was determined for all compounds
by assessing the increase of intracellular calcium levels. The
activation percentage for each compound (10-5 M) was assessed
relative to the maximal response obtained with ghrelin (10-7
M, 100%). EC50 for agonist compounds with high affinity and
Kb for antagonist compounds with high affinity were determined
as described,10,11 and these values are reported in Table 1.
We then introduced a methylene group between the pyridyl
and the carbonyl moieties (16-27). Clearly, the introduction
of more flexibility in this part of the molecule produced less
affine compounds (compare 4 and 16 or 5 and 17, for example).
However, 20 and 21 are exceptions because they displayed a
nanomolar range affinity. (Pyridin-3-yl)- and (pyridin-4-yl)acetyl
containing compounds (24-27) were found to be less affine
toward the GHS-1a receptor, even with the optimized R1 and
R2 substituents. As previously observed, replacement of the
4-methoxybenzyl group in the R1 position by phenyl (23) or
4-ethylbenzyl groups (24) yielded compounds with decreased
affinity. Addition of an extra methylene between the heteroaryl
and the carbonyl group led to a weak ligand (28). Suppression
of the heteroatom in R3 produced less affine compounds
(compare 29 and 10 or 30 and 5) showing the significance of
the nitrogen atom for interaction with the GHS-R1a. When the
carbonyl group in R3 was reduced to a methylene group, the
resulting 31 presented an IC50 superior to 1000 nM, demonstrat-
ing the significance of the carbonyl group for binding to the
receptor. Introduction of an amine function as a substituent in
position 2 of the aryl ring in R3 moiety did not improve the
binding (32) while dearomatization did not yield to a significant
loss of affinity (33). The replacement of the carbonyl group by
a sulfonyl moiety in R3 position led to moderate or bad ligands
(data not shown in Table 1). Introduction of a second nitrogen
atom in the aromatic ring of R3 was well tolerated for the
binding to the GHS-1a receptor as assessed by compounds
containing a pyrazine ring, the best compound exhibiting an
IC50 of 5 ( 1 nM (35 to compare with 2 and 10). As previously
observed, replacement of the 4-methoxybenzyl group by an
4-ethylbenzyl in the R1 position decreased the affinity constant
(37) while suppression of the methylene moiety in the R1 benzyl
group led to 38 with a lower IC50.
Most of the compounds described in this paper exhibited an
antagonist activity toward the GHS-R1a except 30 and 31.
Compound 31 exhibited a very low binding affinity. We only
determined the EC50 for 30 (Table 1), which was found to be a
weak partial agonist with a maximal response of about 30% of
the maximal response induced by 10-7 M ghrelin. Most of
the compounds that were synthesized were not able to promote
[Ca2+]i accumulation, although they were able to bind to the
GHS-1a receptor with moderate to high affinities. They were
able to antagonize the [Ca2+]i accumulation induced by 10-7
M ghrelin. Compounds 4 (Kb ) 19 ( 11 nM), 5 (Kb ) 25 ( 3
nM), 9 (Kb ) 102 ( 20 nM), 10 (Kb ) 12 ( 3 nM), 16 (Kb )
14 ( 3 nM), 20 (Kb ) 12 ( 4 nM), 21 (Kb ) 6 ( 3 nM), 29
(Kb ) 23 ( 0.3 nM), and 35 (Kb ) 24 ( 5 nM) were among
the most potent ghrelin receptor antagonists. They were able to
antagonize dose-dependently ghrelin-induced [Ca2+]i accumula-
tion in CHO cells transiently transfected with the GHS-R1a.
Interestingly, in the presence of various concentration of these
compounds, the dose-response curves of ghrelin on [Ca2+
]
i
accumulation were shifted rightward in a parallel manner,
indicating a competitive antagonism.
Selected compounds were then tested in vivo in rats for their
activity on food intake (subcutaneous injection) (Table 2).
Compounds were first evaluated alone for their effect on food
intake and then for their ability to inhibit hexarelin-stimulated
food intake (80 µg of hexarelin). A significant increase in
cumulative food intake was found for 34 and 36 when
administered alone at 2 and 6 h (Table 2). These compounds
behaved as ghrelin antagonists in vitro with Kb values of 97
and 530 nM, respectively. All other compounds did not
significantly increase food intake when administered alone. Eight
of them were able to inhibit food intake even at 6 h (1, 5, 13,

692 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Brief Articles
Table 3. Inhibitory Activity by Graded Doses of Compounds 5 and 34
on Feeding Stimulated by 80 µg/kg Hexarelina
Table 4. GH Secretion in Infant Rat (sc Injection)a
compd [rGH], ng/mL
saline 5.55 ( 1.62
compd
food intake at 2 h
food intake at 6 h
hexarelin, 80 µg
5, 20 µg/kg
1.00 ( 0.26
0.56 ( 0.11
0.02 ( 0.01
0.21 ( 0.11
1.23 ( 0.37
1.01 ( 0.26
0.56 ( 0.11
0.02 ( 0.01
0.21 ( 0.11
1.23 ( 0.37
hexarelin
2
2 + hexarelin
saline
187.58 ( 22.68
5.28 ( 0.60
220.52 ( 15.52
5.24 ( 0.73
5, 80 µg/kg
5, 160 µg/kg
5, 320 µg/kg
hexarelin
5
5 + hexarelin
saline
hexarelin
34
34 + hexarelin
170.10 ( 13.23
13.06 ( 2.17
138.39 ( 14.58
10.72 ( 2.02
253.82 ( 12.27
15.32 ( 1.35
173.61 ( 18.44
hexarelin, 80 µg
34, 20 µg/kg
34, 80 µg/kg
34, 160 µg/kg
34, 320 µg/kg
1.25 ( 0.26
0.66 ( 0.23
0.23 ( 0.13
0.29 ( 0.09
0.20 ( 0.09
1.26 ( 0.26
0.66 ( 0.23
0.23 ( 0.13
0.31 ( 0.10
0.21 ( 0.09
a Cumulative food intake at 2 and 6 h expressed in g of food per 100 g
of body weight.
a Hexarelin was tested at a dose of 80 µg/kg, and compounds were tested
at a dose of 160 µg/kg.
GH secretion is not yet activated at the hypothalamic level, and
therefore, a constant GH blood level is measured in these young
animals.
As expected, when administered alone, these GHS-R1a
antagonists did not elicit significant GH release. Unexpectedly,
these GHS-R1a antagonists were unable to significantly inhibit
GH secretion stimulated by hexarelin.
When regarding both GH secretion and food intake responses,
5 and 34, both GHS-R1a antagonists of this new series, inhibited
food intake induced by hexarelin but not hexarelin-stimulated
GH secretion. These results support our previous observations10,11
showing the possible modulation of the ghrelin effect on appetite
without altering GH secretion. To assess the medical relevance
of this observation, these compounds will be tested in various
obesity animal models.
16, 21, 27, 33, and 35). When considering the coadministration
with hexarelin, only 5 and 34 were able to inhibit hexarelin-
induced food intake by -97% and -73%, respectively, at 6 h.
Some other compounds were found to be less efficient at
inhibiting hexarelin-stimulated food intake (1, -64%; 9, -50%;
18, -34%; 25, -36%; 33, -45%). However, unexpectedly, 35,
without effect when administered alone, was found to be able
to potentiate food intake induced by hexarelin (+73%). Com-
pounds 13 and 21, without effect alone, also seemed to stimulate
food intake induced by hexarelin but in a weaker way (+19%).
Although, as it was reported previously,10,11 some of the
discrepancies between in vitro and in vivo results on food intake
were difficult to understand and explain (i.e., 35), it clearly
appeared in this study that in vitro potent GHS-R1a antagonists
such as 5 and 34 were also efficient in antagonizing hexarelin-
stimulated food intake.
Conclusion
A dose-response study for the two most active compounds
(5 and 34) was conducted with rats at various doses (20, 80,
160, and 320 µg/kg) along with a 80 µg/kg hexarelin-stimulated
food intake. Results are in Table 3. Compound 34 displayed a
well correlated progressive dose-response curve inhibition of
hexarelin stimulated cumulative food intake. The lowest 20 µg/
kg dose was able to inhibit 47% of hexarelin-stimulated feeding
behavior at 6 h, while higher doses inhibited 75-83% of
hexarelin-stimulated food intake. Compound 5 was found to
be efficient in suppressing feeding at very low doses: -44%
inhibition of food intake at 20 µg/kg and -98% inhibition at
80 µg/kg. Surprisingly, its efficiency decreased at a dose of 160
µg/kg and no effect was obtained at the highest dose (320 µg/
kg).
Information on the toxicity of the compounds could be
obtained by the observation of adult rats that received subcu-
taneous doses of the compounds (20–320 µg/kg) for assessing
the effect on food intake. We did not obtain mortality with any
of the tested compounds, and the observation of the animals
during the 6 h of the food intake experiments did not reveal
altered locomotion behavior.
Replacement of the Aib moiety, often present in GHS-R1a
ligands, by a heteroaryl-carbonyl or a heteroarylalkyl-carbonyl
group led to potent antagonists of the GHS-1a receptor. These
compounds were easily synthesized from commercially available
materials and contain only one asymmetric center. The most
potent compounds in this series were tested in vivo for their
activity on food intake in the rat. Compounds 5 and 34, both in
vitro antagonists of the GHS-R1a, were found to be the most
potent compounds in inhibiting hexarelin-induced food intake
in the rat. Furthermore, they did not show any activity on GH
secretion in the rat and did not inhibit hexarelin-stimulated GH
secretion. It has been previously demonstrated that the ghrelin
receptor couples to Gq/11 and to G12/13 G proteins,16 and
a recent study describes compounds able to activate the ghrelin
receptor through one or the other signaling pathway.17 Our
results suggest that some of the compounds described in this
study selectively target the mechanism of action of the ghrelin
receptor that correlated with food intake and not with GH
secretion. Further investigations are ongoing to explain these
observations.
The binding affinity of a model compound, 5, was tested at
10 µM versus a panel of 92 GPCRs including cannabinoids,
melanocortins, and NPY receptors (MDS Pharma Services’
GPCR screen). Only GHS-R1a, CCKI, motilin, tachykinin 2,
and vasopressin 1a receptors showed significant binding with
this compound. However, the binding affinity to the ghrelin
receptor GHS-R1a was 1000 times higher than the affinity to
the other receptors.
Three selected antagonists (2, 5, and 34) were tested for their
ability to stimulate/inhibit GH secretion in infant rats after sc
administration, in the presence or not of hexarelin (Table 4).
Indeed, it is known that in the baby rats the pulsatility of the
Experimental Section
Chemistry. Compounds were synthesized as previously de-
scribed.10,11 All final compounds were purified by reversed-phase
HPLC. The purity assessed by analytical reversed-phase C18 HPLC
was found to be greater than 95% for target compounds and greater
than 98% for key target compounds (4, 5, 10, 13, 16, 20, 21, 34,
35, and 36), and the structures were confirmed by MS (electrospray),
1H NMR, and 13C NMR for the most interesting compounds.
(R)-N-(1-(4-(2,4-Dimethoxybenzyl)-5-phenethyl-4H-1,2,4-tri-
azol-3-yl)-2-(1H-indol-3-yl)ethyl)picolinamide (5). 1H NMR (300
MHz, DMSO-d6, 300 K): δ 2.83 (m, 2H, CH2-CH2-phenyl), 2.90
(m, 2H, CH2-CH2-benzyl), 3.48 (m, 2H, CH2 ꢀTrp), 3.57 (s, 3H,

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 693
(2) Howard, A. D.; Feighner, S. D.; Cully, D. F.; Arena, J. P.; Liberator,
P. A.; Rosenblum, C. I.; Hamelin, M. J.; Hreniuk, D. L.; Palyha, O. C.;
Anderson, J.; Paress, P. S.; Diaz, C.; Chou, M.; Liu, K.; Mckee, K. K.;
Pong, S. S.; Chaung, L. Y.; Elbrecht, A.; Heavens, R.; Rigby, M.;
Sirinathsinghji, D. J. S.; Dean, D. C.; Melillo, D. G.; Patchett, A. A.;
Nargund, R.; Griffin, P. R.; DeMartino, J. A.; Gupta, S. K.; Schaeffer,
J. M.; Smith, R. G.; Van Der Ploeg, L. H. T. A receptor in pituitary
and hypothalamus that functions in growth hormone release. Science
1996, 273, 974–977.
OCH3), 3.61 (s, 3H, OCH3), 4.97 (d, 1H, J ) 17 Hz, CH2 o,p-
dimethoxybenzyl), 5.09 (d, 1H, J ) 17 Hz, CH2-o,p-dimethoxy-
benzyl), 5.56 (m, 1H, CH RTrp), 6.18 (dd, 1H, Jo ) 8 Hz and Jm
) 2 Hz, H5 o,p-dimethoxybenzyl), 6.41 (d, 1H, Jm ) 2 Hz, H3
o,p-dimethoxybenzyl), 6.55 (d, 1H, Jo ) 8 Hz, H6 o,p-dimethoxy-
benzyl), 6.87 (t, 1H, Jo ) 8 Hz, H5 Trp), 7.01 (t, 1H, Jo ) 8 Hz,
H6 Trp), 7.08 (m, 3H, H2 Trp, H2 and H6 phenyl), 7.14 (d, 1H, Jo
) 7 Hz, H4 Trp), 7.19–7.31 (m, 4H, H7 Trp, H3, H4 and H5 phenyl),
7.56 (t, 1H, J ) 8 Hz, NH amide), 7.91 (m, 2H, H4 and H5
o-pyridyl), 8.57 (d, 1H, JRꢀ ) 5 Hz, H6 o-pyridyl), 9.16 (d, 1H, Jo
) 8 Hz, H3 o-pyridyl), 10.80 (s, 1H, NH indole Trp). 13C NMR
(75 MHz, DMSO-d6, 300 K): δ 26.2 (CH2-CH2-phenyl), 28.8
(C ꢀTrp), 32.1 (CH2-CH2-phenyl), 43.0 (CH2 o,p-dimethoxy-
benzyl), 45.5 (C RTrp), 55.6 (OCH3), 55.8 (OCH3), 98.9 (C3 o,p-
dimethoxybenzyl), 105.0 (C5 o,p-dimethoxybenzyl), 109.5 (C3 Trp),
111.8 (C7 Trp), 114.4 (C1 o,p-dimethoxybenzyl), 118.4 (C4 Trp),
118.8 (C5 Trp), 121.4 (C6 Trp), 122.4 (C3 o-pyridyl), 124.4 (C2
Trp), 126.7 (C6 o,p-dimethoxybenzyl), 127.2 (C5 o-pyridyl), 127.5
(C9 Trp), 128.6–128.8 (C2, C3, C4, C5, and C6 phenyl), 136.4 (C8
Trp), 138.1 (C4 o-pyridyl), 140.2 (C1 phenyl), 148.8 (C6 o-pyridyl),
149.3 (C2 o-pyridyl), 155.2 (Cq triazole), 155.4 (Cq triazole), 157.9
(C2 o,p-dimethoxybenzyl), 161.0 (C4 o,p-dimethoxybenzyl), 163.9
(CO amide).
(3) Kojima, M.; Jangawa, K. Ghrelin: structure and function. Physiol. ReV.
2005, 85, 495–522.
(4) Van der Lely, A. J.; Tschop, M.; Heiman, M. L.; Ghigo, E. Biological,
physiological, pathophysiological, and pharmacological aspects of
ghrelin. Endocr. ReV. 2004, 25 (3), 426–457.
(5) Nakazato, M.; Murakami, N.; Date, Y.; Kojima, M.; Matsuo, H.;
Kangawa, K.; Matsukura, S. A role for ghrelin in the central regulation
of feeding. Nature 2001, 409, 194–198.
(6) Asakawa, A.; Inui, A.; Kaga, T.; Katsuura, G.; Fujimiya, M.; Fujin,
M. A.; Kasuga, M. Antagonism of ghrelin receptor reduces food intake
and body weight gain in mice. Gut 2003, 52, 947–952.
(7) Xin, Z.; Serby, M. D.; Zhao, H.; Kosogof, C.; Szczepankiewicz, B. G.;
Liu, M.; Liu, B.; Hutchins, C. W.; Sarris, K. A.; Hoff, E. D.; Falls,
H. D.; Lin, C. W.; Ogiela, C. A.; Collins, C. A.; Brune, M. E.; Bush,
E. N.; Droz, B. A.; Fey, T. A.; Knourek-Segel, V. E.; Shapiro, R.;
Jacobson, P. B.; Beno, D. W. A.; Turner, T. M.; Sham, H. L.; Liu, G.
Discovery and pharmacological evaluation of growth hormone secre-
tagogue receptor antagonists. J. Med. Chem. 2006, 49, 4459–4469.
(8) Serby, M. D.; Zhao, H.; Szczepankiewicz, B. G.; Kosogof, C.; Xin,
Z.; Liu, B.; Liu, M.; Nelson, L. T. J.; Kaszubska, W.; Falls, H. D.;
Schaefer, V.; Bush, E. N.; Shapiro, R.; Droz, B. A.; Knourek-Segel,
V. E.; Fey, T. A.; Brune, M. E.; Beno, D. W. A.; Turner, T. M.;
Collins, C. A.; Jacobson, P. B.; Sham, H. L.; Liu, G. 2,4-Diaminopy-
rimidine derivatives as potent growth hormone secretagogue receptor
antagonists. J. Med. Chem. 2006, 49, 2568–2578.
(9) Rudolph, J.; Esler, W. P.; O’Connor, S.; Coish, P. D. G.; Wickens,
P. L.; Brands, M.; Bierer, D. E.; Bloomquist, B. T.; Bondar, G.; Chen,
L.; Chuang, C.-Y.; Claus, T. H.; Fathi, Z.; Fu, W.; Khire, U. R.; Kristie,
J. A.; Liu, X.-G.; Lowe, D. B.; McClure, A. C.; Michels, M.; Ortiz,
P. D.; Ramsden, R. W.; Schoenleber, T. E.; Shelekhin, A.; Vaka-
lopoulos, W.; Tang, L.; Wang, A. A.; Yi, L.; Gardell, S. J.; Livingston,
J. N.; Sweet, L. J.; Bullock, W. H. Quinazolinone derivatives as orally
available ghrelin receptor antagonists for the treatment of diabetes and
obesity. J. Med. Chem. 2007, 50, 5202–5216.
(10) Demange, L.; Boeglin, D.; Moulin, A.; Mousseaux, D.; Ryan, J.; Bergé,
G.; Gagne, D.; Heitz, A.; Perrissoud, D.; Locatelli, V.; Torsello, A.;
Galleyrand, J.-C.; Fehrentz, J.-A.; Martinez, J. Synthesis and phar-
macological in vitro and in vivo evaluations of novel triazole
derivatives as ligands of the ghrelin receptor. 1. J. Med. Chem. 2007,
50, 1939–1957.
(11) Moulin, A.; Demange, L.; Bergé, G.; Gagne, D.; Ryan, J.; Mousseaux,
D.; Heitz, A.; Perrissoud, D.; Locatelli, V.; Torsello, A.; Galleyrand,
J.-C.; Fehrentz, J.-A.; Martinez, J. Towards potent GHS-R1a ligands
based on trisubstituted 1,2,4-triazole structure. synthesis and pharma-
cological in vitro and in vivo evaluations. 2. J. Med. Chem. 2007, 50,
5790–5806.
(R)-N-(1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(2,4-dimethoxybenzyl)-
4H-1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)pyrazine-2-carbox-
1
amide (34). H NMR (300 MHz, DMSO-d6, 300 K): δ 3.01 (m,
2H, CH2-CH2-indole), 3.10 (m, 2H, CH2-CH2-indole), 3.51
(m, 2H, CH2 ꢀTrp), 3.55 (s, 3H, OCH3), 3.57 (s, 3H, OCH3), 5.15
(d, 2H, J ) 7 Hz, CH2 o,p-dimethoxybenzyl), 5.63 (m, 1H, CH
RTrp), 6.08 (dd, 1H, Jo ) 8 Hz and Jm ) 2 Hz, H5 o,p-
dimethoxybenzyl), 6.35 (d, 1H, Jm ) 2 Hz, H3 o,p-dimethoxyben-
zyl), 6.53 (d, 1H, Jo ) 8 Hz, H6 o,p-dimethoxybenzyl), 6.89 (m,
2H, H5 indole and H5 Trp), 6.99 (m, 2H, H6 indole and H6 Trp),
7.08 (m, 2H, H2 indole and H2 Trp), 7.29 (m, 3H, H4 Trp, H4 and
H7 indole), 7.41 (d, 1H, Jo ) 8 Hz, H7 Trp), 8.61 (t, 1H, J ) 2 Hz,
H3 o-pyrazinyl), 8.79 (d, 1H, J ) 2 Hz, H5 o-pyrazinyl), 8.94 (d,
1H, J ) 1 Hz, H6 o-pyrazinyl), 9.43 (d, 1H, J ) 8 Hz, NH amide),
10.84 (s, 2H, NH indole and NH indole Trp). 13C NMR (75 MHz,
DMSO-d6, 300 K):
δ
22.0 (CH2-CH2-indole), 25.3
(CH2-CH2-indole), 27.9 (C ꢀTrp), 44.1 (CH2 o,p-dimethoxyben-
zyl), 45.5 (C RTrp), 55.5 (OCH3), 55.8 (OCH3), 98.8 (C3 o,p-
dimethoxybenzyl), 104.9 (C5 o,p-dimethoxybenzyl), 109.3 (C3 Trp),
111.8 (C7 indole and C7 Trp), 112.1 (C3 indole), 113.4 (C1 o,p-
dimethoxybenzyl), 118.4 (C4 indole), 118.5 (C4 Trp), 118.8 (C5
indole and C5 Trp), 121.5 (C6 indole and C6 Trp), 127.0 (C9 indole),
127.4 (C9 Trp), 136.4 (C8 Trp), 136.6 (C8 indole), 141.2 (C6
o-pyrazinyl), 144.1 (C2 o-pyrazinyl), 144.3 (C3 o-pyrazinyl), 146.8
(C5 o-pyrazinyl), 155.4 (Cq triazole), 156.0 (Cq triazole), 157.8
(C2 o,p-dimethoxybenzyl), 161.0 (C4 o,p-dimethoxybenzyl), 163.2
(CO amide).
(12) Guerlavais, V.; Boeglin, D.; Mousseaux, D.; Oiry, C.; Heitz, A.;
Deghenghi, R.; Locatelli, V.; Torsello, A.; Ghe, C.; Catapano, F.;
Muccioli, G.; Galleyrand, J. C.; Fehrentz, J. A.; Martinez, J. New active
series of growth hormone secretagogues. J. Med. Chem. 2003, 46,
1191–1203.
(13) Broglio, F.; Boutignon, F.; Benso, A.; Gottero, C.; Prodam, F.; Arvat,
E.; Ghe, C.; Catapano, F.; Torsello, A.; Locatelli, V.; Muccioli, G.;
Boeglin, D.; Guerlavais, V.; Fehrentz, J. A.; Martinez, J.; Ghigo, E.;
Deghenghi, R. EP1572: a novel peptido-mimetic GH secretagogue
with potent and selective GH-releasing activity in man. J. Endocrinol.
InVest. 2002, 25 (8), RC26–RC28.
Expression of the receptor, in vitro determination of the binding
affinities and intracellular calcium mobilization assay, in vivo
experiments in the rat for GH secretion, and food intake experiments
were previously described.10,11
Acknowledgment. The authors thank Aeterna-Zentaris and
the CNRS for financial support of this work and for providing
a research grant for the Ph.D. thesis project of A.M. (Grant BDI
752776/01).
(14) Piccoli, F.; Degen, L.; MacLean, C.; Peter, S.; Baselgia, L.; Larsen,
F.; Beglinger, C.; Drewe, J. Pharmacokinetics and pharmacodynamic
effects of an oral ghrelin agonist in healthy subjects. J. Clin.
Endocrinol. Metab. 2007, DOI 10.1210/jc.2006-2160.
(15) Boeglin, D.; Cantel, S.; Heitz, A.; Martinez, J.; Fehrentz, J.-A. Solution
and solid-supported synthesis of 3,4,5-trisubstituted 1,2,4-triazole-based
peptidomimetics. Org. Lett. 2003, 5, 4465–4468.
Supporting Information Available: Biological data; LC-MS
chromatograms of 4-38; table of physicochemical properties of
1-38; general preparation of thioamides and triazoles; 1H and 13
C
(16) Holst, B.; Holliday, N. D.; Bach, A.; Elling, C. E.; Cox, H. M.;
Schwartz, T. W. Common structural basis for constitutive activity of
the ghrelin receptor family. J. Biol. Chem. 2004, 53806–53817.
(17) Holst, B.; Mokrosinski, J.; Lang, M.; Brandt, E.; Nygaard, R.; Frimurer,
T. M.; Beck-Sickinger, A.; Schwartz, T. W. Identification of an efficacy
switch region in the ghrelin receptor responsible for interchange
between agonism and inverse agonism. J. Biol. Chem. 2007, 282,
15799–15811.
NMR data of compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
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JM701292S