New trisubstituted 1,2,4-triazoles as ghrelin receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2014.11.031

Ghrelin receptor ligands based on a trisubstituted 1,2,4-triazole scaffold were recently synthesized and evaluated for their in vitro affinity for the GHS-R1a receptor and their biological activity. In this study, replacement of the α-aminoisobutyryl (Aib

Full text of DOI:10.1016/j.bmcl.2014.11.031

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New trisubstituted 1,2,4-triazoles as ghrelin receptor antagonists
Anne-Laure Blayo a, Mathieu Maingot a, Babette Aicher b, Céline M’Kadmi a, Peter Schmidt b,
Gilbert Müller b, Michael Teifel b, Eckhard Günther b, Didier Gagne a, Séverine Denoyelle a, Jean Martinez a,
Jean-Alain Fehrentz a,
⇑
a Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS—Université Montpellier I et Université Montpellier II, BP 14491, Faculté de Pharmacie, bât. E, 3ème étage,
15 avenue Charles Flahault, 34093 Montpellier Cedex 5, France
b Æterna Zentaris GmbH, Weismuellerstrasse 50, 60314 Frankfurt am Main, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Ghrelin receptor ligands based on a trisubstituted 1,2,4-triazole scaffold were recently synthesized and
evaluated for their in vitro affinity for the GHS-R1a receptor and their biological activity. In this study,
replacement of the a-aminoisobutyryl (Aib) moiety (a common feature present in numerous growth hor-
mone secretagogues described in the literature) by aromatic and heteroaromatic groups was explored.
We found potent antagonists incorporating the picolinic moiety in place of the Aib moiety. In an attempt
to increase affinity and activity of our lead compound 2, we explored the modulation of the pyridine ring.
Herein we report the design and the structure–activity relationships study of these new ghrelin receptor
ligands.
Received 19 September 2014
Revised 7 November 2014
Accepted 8 November 2014
Available online xxxx
Keywords:
1,2,4-Triazole scaffold
Structural modulation
GHS-R1a, ghrelin receptor
Antagonists
Ó 2014 Elsevier Ltd. All rights reserved.
SAR study
Ghrelin, the natural ligand of the Growth Hormone Secreta-
gogue Receptor type 1a (GHS-R1a),1 was recently discovered and
characterized.2 Its main biological activities were found to be the
stimulation of both growth hormone release and food intake.
Ghrelin also regulates the metabolic balance by decreasing fat
use, affecting body weight and adiposity. Thus, ghrelin functions
as an orexigenic hormone3 and it can be of interest to find agonists
and antagonists of its receptor.
Before the discovery of ghrelin, research focused on the finding
of agonists able to elicit the stimulation of GH secretion. Among
many compounds, we can cite the peptidic precursor component
GHRP-6 described by Bowers,4 the non peptidic Merck compound,
MK-0677, which allowed the characterization of the GHS-R1a in its
S35 labeled version,5 and the CP-424,391 (or Capromorelin),
developed by Pfizer.6 Our team also described the JMV 1843, a
pseudo-peptide which was able to stimulate GH secretion by oral
administration in man.7 This compound will be soon commercial-
ized for the diagnostic of GH deficiency in adults. When multiple
ghrelin biological activities were exhibited, it appears that antago-
nist (and/or inverse agonist) compounds of the GHS-R1a receptor
could be useful to treat obesity, for example. Ipsen developed ghre-
lin analogs and identified a competitive GHS-R1a antagonist, BIM
28163,8 while Tranzyme Pharma has extensively claimed conform-
ationally defined macrocyclic compounds incorporating peptides
as modulators of the GHS-R1a, including antagonists.9 Numerous
nonpeptide small molecules such as benzodiazepine,10 piperazine
bisamide,11 azaquinazolinone12 and indolinone13 derivatives have
been described as potent antagonists of the ghrelin receptor.14
In our effort to find new ghrelin receptor ligands, we recently
described a family of peptidomimetics based on a decorated
1,2,4-triazole scaffold that led to potent agonists and antagonists
of this receptor.15–17 These compounds were synthesized from
(D)tryptophan residue as starting material and present in their final
structure only one asymmetric carbon atom, whose configuration
was controlled during the synthetic process. Replacement of the
a-aminoisobutyryl (Aib) moiety, a common feature often present
in GHS-R1a ligands,5–7 by groups such as glycyl, propyl, isonipeco-
tyl or piperazyl-2-carbonyl groups was first explored.16 One of our
front runners, compound JMV 2959 (compound 1, Fig. 1), a trisub-
stituted 1,2,4-triazole comprised of a glycyl group in place of the
Aib moiety, was found to be a potent GHS-R1a antagonist. There-
fore 1 has been extensively studied in various in vivo animal
models18 and exhibits a large range of interesting properties such
as suppression of food intake induced by ghrelin or by fasting19
and suppression of fat mass accumulation.20 It also presents
in vivo effects on addictive behavior, providing evidence that the
central ghrelin signaling system is required in the reward induced
⇑
Corresponding author. Tel.: +33 4 11 75 96 07; fax: +33 4 11 75 96 41.
E-mail address: jean-alain.fehrentz@univ-montp1.fr (J.-A. Fehrentz).
http://dx.doi.org/10.1016/j.bmcl.2014.11.031
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Blayo, A.-L.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.11.031

2
A.-L. Blayo et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
H
N
H
N
HN
HN
O
O
O
(i)
(ii)
O
H
N
OH
BocNH
BocNH
N
N
HN
N
HN
O
O
H2N
N
3
N
N
Boc-(D)Trp-OH
N
O
O
1
2
H
N
H
N
IC50: 32 nM; Kb: 19 nM
MW: 508; CLogD: 2.34 (pH=7)
IC50: 0.7 nM; Kb: 12 nM
MW: 556; CLogD: 5.45 (pH=7)
O
H
(iii)
Figure 1. Structures of compounds 1 (JMV 2959) and 2 (JMV 3018).
N
N
BocNH
BocNH
N
S
N
4
by addictive drugs such as alcohol,21 amphetamine22 and
cocaine.23,24
5
H
N
O
When replacing the glycine residue on the N-terminal part of
compound 1 by a picolinic moiety, we obtained a very potent
antagonist (compound 2, Fig. 1).17 As shown in Table 1, the phar-
macokinetic studies of the two compounds 1 and 2 exhibited a
very different pattern. Indeed, compound 2 showed a much better
pharmacokinetic profile than compound 1, with a high maximal
plasma concentration (Cmax) reached 5 h after administration and
a high AUC value reflecting a long drug exposure to the body with
a slow clearance.
(iv)
(v)
N
H2N
HCl
final compounds
7-24
N
N
6
Scheme 1. Reagents and conditions: (i) BOP, DIEA, 4-methoxybenzylamine, DCM,
rt, (ii) Lawesson’s reagent, DME, 80 °C, (iii) 3-phenyl-propanehydrazide, PhCOÀ2 Ag+,
AcOH, DCM, rt, (iv) 4N HCl/dioxane, rt, (v) substituted pyridyl acid, BOP, DIEA, DCM,
rt.
These results prompted us to conserve the picolinic moiety on
the N-terminal amino function of our compounds and introduce
various substituents on the pyridine ring in an attempt to modu-
late the physicochemical properties of the ligands. In this Letter,
we report on the consequences of these structural modifications
on both the affinity and the biological activity of the new ligands
toward the ghrelin receptor.
compounds to bind to the receptor. So at first, the effect of the posi-
tion of the nitrogen atom into that pyridyl ring was investigated.
When the picolinamide (compound 2) was replaced by an isonico-
tinamide (compound 7), a loss of binding affinity was observed
(IC50 = 220 nM). A similar decrease was found with a nicotinamide
moiety (compound 8) even when substituted with 5-bromo,
2-bromo, 6-chloro or 2-amino substituent (compounds 9–12,
respectively). The position of the nitrogen atom in the pyridyl
moiety has therefore proved to be determinant for the binding to
GHS-R1a. Indeed, it has to be in ortho position to the carbonyl to
allow the best binding to the receptor. Thus, all the following com-
pounds were designed with a picolinamide moiety decorated with
various substituents. Incorporation of an electron withdrawing
group (fluorine atom) in position 3 or 6 of the pyridyl ring (com-
pounds 13 and 14, respectively) or even of 2 fluorine atoms in posi-
tion 3 and 5 (compound 15) was well tolerated as it led to very
potent compounds with nanomolar affinities (IC50 64 nM). Various
substituents such as methyl-, methoxy- or amino-group were also
well tolerated in position 6 as shown with compounds 16–18,
respectively (IC50 66 nM). Unfortunately, the presence of a hydro-
xyl group in position 3 (compound 19) led to a significant loss of
binding affinity (IC50 = 240 nM) while the introduction of a N-oxide
into the pyridyl ring (compound 20) was better tolerated
(IC50 = 42 nM), which remains interesting as it lowers the ClogD
value. At last, in order to explore the size of the binding site of
the N-terminal substituent, commercially available picolinic acids
substituted by sterically hindered groups were used to design
new ligands. Pyrollidin-1-yl group in position 5 of the pyridyl ring
(compound 21) as well as phenylmethanone in position 3 (com-
pound 22) led to a significant loss of affinity for GHS-R1a
(IC50 = 65 nM and IC50 = 240 nM, respectively) compared to parent
compound 2. This indicates that steric hindrance on this side of the
molecule might be a limitation for binding to the receptor as it
decreases the binding affinity of the ligands. The same results were
observed with isoquinoline-1 or isoquinoline-3. Indeed, com-
pounds 23 and 24, respectively, exhibited less potent binding
affinities than unsubstituted picolinamide 2.
Compounds were synthesized as previously described.25 Briefly,
triazole derivatives were obtained in five steps starting from Boc-
(D)-Trp-OH as shown in Scheme 1. After coupling with 4-methoxy-
benzylamine, the amide 3 was transformed into the thioamide 4
using the Lawesson’s reagent. Cyclization into the triazole
heterocycle (compound 5) was performed in the presence of
3-phenylpropanehydrazide and silver benzoate.26 After removal
of the Boc protecting group using 4N HCl in dioxane, the structural
diversity was introduced on compound 6 by coupling various
substituted picolinic, isonicotinic and nicotinic acids. All final com-
pounds were purified by reversed-phase preparative HPLC. The
purity assessed by analytical reversed phase C18 HPLC was found
to be greater than 98% for most of the target compounds and greater
than 95% for the rest, and the structures of the compounds were
confirmed by MS (electrospray), 1H NMR, and 13C NMR.
The synthesized compounds 7–24 along with intermediate salt
6 were first tested for their ability to displace 125I-[His9]-ghrelin
from the cloned hGHS-R1a receptor transiently expressed in LLC
PK-1 cells as previously described15 and compared to the parent
compound 2 (Table 2). Overall, the binding affinities were in the
nanomolar range for the most potent ligands (compounds 13–18)
with IC50 values between 0.7 and 6 nM while compound 6, with
a free amine, lost its binding affinity (IC50 >1000 nM). This indi-
cates that substitution of the N-terminal amino function by an
acylating group such as the pyridyl ring remains essential for the
Table 1
Pharmacokinetic profiles of compounds 1 and 2
Compounds
AUCa
Cmax (ng/mL)b
Tmax (h)c
1
2
15
7250
5
1064
1.2
5.3
a
b
c
The biological in vitro activity of the compounds was then eval-
uated. First, the compounds were tested for their ability to induce
intracellular calcium [Ca2+]i mobilization in GHS-R1a expressing
Area under the curve (integral of the plasma concentration–time curve).
Peak concentration (max plasma concentration after drug administration).
Peak time (time to reach Cmax).
Please cite this article in press as: Blayo, A.-L.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.11.031

A.-L. Blayo et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 2
SAR study on various substituted pyridine rings
H
N
O
R
N
N
H
N
N
Compounds
R
IC50 (nM)
ClogD
(pH = 7)d
Competitive bindinga
CRE/Luc reporter gene assayb
Calcium releasec
O
N
2
6
0.7 0.2
>1000
6.0 3.1
nd
4.5
nd
5.45
3.45
H (HCl salt)
O
7
8
220 60
122 20
828
411
nd
5.06
5.06
N
O
337
N
O
Br
9
84 12
250 52
130 21
380 63
222
193
771
427
88
5.83
6.04
5.89
5.47
N
O
10
11
12
510
1010
671
N
Br
O
Cl
N
O
N
N
NH2
O
13
14
15
3
2
4
0.6
0.2
0.8
8.8 1.1
2.1 0.7
2.8 0.2
7.0
2.1
4.6
5.59
5.99
5.74
F
O
F
F
N
N
O
F
O
H3C
N
16
17
18
3.4 1.2
7.5 0.4
9.0 2.7
21 6.6
5.4
7.8
25
5.58
5.89
5.22
O
N
O
2
6
0.5
1.6
H3C
H2N
O
N
O
N
19
20
240 38
42 12
108
79
165
112
5.44
4.24
OH
O
O
N
(continued on next page)
Please cite this article in press as: Blayo, A.-L.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.11.031

4
A.-L. Blayo et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 2 (continued)
Compounds
R
IC50 (nM)
ClogD
(pH = 7)d
Competitive bindinga
65 21
CRE/Luc reporter gene assayb
Calcium releasec
2115
O
N
21
22
1210
5.96
6.91
N
O
N
N
240 33
150 21
100 12
313
73
230
82
O
O
23
6.44
6.44
O
N
24
126
404
a
IC50 values are the mean of at least two independent experiments, each of them were conducted in triplicate.
IC50 values were determined by one experiment for the less efficient compounds and are the mean of at least two independent experiments for compounds in the
b
nanomolar range. Mean data points of each experiment are based on quadruplicate measurements. (nd for not determined).
c
IC50 values were determined by one experiment and the mean data points are based on quadruplicate measurements. (nd for not determined).
ClogD values were calculated using the Marvin software provided by http://www.chemaxon.com.
d
cells.15 We considered as potential antagonist any compound
unable to elicit at 10À5 M an increase superior to 10% of the intra-
cellular calcium level compared to the maximal response (fixed at
100% when elicited by 10À7 M ghrelin). In that case, the IC50 value
was determined using antagonist inhibition curves in the presence
of 10À7 M ghrelin (submaximal concentration) and calculated as
the molar concentration of antagonist that reduced the maximal
response of ghrelin by 50%. Then, the compounds were tested in
a cyclic AMP response element CRE-luciferase reporter gene
assay.27 This assay was conducted on mouse LTK-cells stably
expressing the human GHS-R1a and uses a luciferase reporter gene
under the control of CRE elements and the CMV minimal promoter.
Receptor-mediated reporter gene expression was calculated in %
inhibition according to cells treated with saturating concentrations
of ghrelin (0% inhibition), and nontreated cells as positive control
(100% inhibition). Results are reported in Table 2 and show a good
correlation between the two assays (calcium and CRE). All the new
ligands were found to act as ghrelin receptor antagonists except
compound 6 which was not tested as it showed no binding affinity
for the receptor. As expected, compounds with poor binding affin-
ities revealed low efficacy with IC50 values sometimes in the micro-
molar range. However, compounds designed with the picolinamide
moiety decorated with 3-fluoro, 6-fluoro, 3,5-difluoro, 6-methyl or
6-methoxy substituent (compounds 13–17, respectively) revealed
great efficacy with IC50 values in the nanomolar range (IC50
<10 nM). Even the presence of an amino group at position 6 (com-
pound 18) was well tolerated with only a slight decrease of
potency (IC50 = 25 nM). These results correlate with the fact that
compounds 13–18 have high affinity for the GHS-R1a receptor.
Replacement of the glycyl moiety present on compound 1 by a
pyridyl-carbonyl group led to antagonists of the GHS-R1a receptor.
These compounds were easily synthesized from commercially
available materials and contain only one asymmetric center. The
most potent compounds in this series were obtained with a picoli-
namide moiety indicating the nitrogen atom of the pyridyl group
must be in ortho position to the carbonyl to obtain good affinity
and efficacy. Various substituents on the pyridyl ring were well
tolerated, but compounds 13, 14, and 15 containing fluorine atoms
and compounds 16 and 17 containing respectively a methyl or a
methoxy group, showed the best results. As compounds 1 and 2,
our trisubstituted 1,2,4-triazole front runner ligands, are not able
to inhibit GH secretion induced by an agonist (hexarelin, ghre-
lin),16,17 we have good hopes that this new series of antagonists
will present the same properties. This may be particularly interest-
ing for the development of potential anti-obesity (or anti-addic-
tion) treatment. These compounds are currently under further
in vivo investigations.
Acknowledgments
The authors thank Æterna-Zentaris (Germany) and the CNRS
(France) for financial support of this work, and for providing a
research grant for the Ph.D. thesis project of A.-L. Blayo. (Grant
BDI 021121).
Supplementary data
Supplementary data (complete set of data related to 1H and 13
NMR, LC/MS and analytical HPLC studies, and copies of 1H and 13
C
C
NMR of compounds 2, 7–25) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2014.11.031. These data include MOL files and InChiKeys
of the most important compounds described in this article.
References and notes
1. Howard, A. D.; Feighner, S. D.; Cully, D. F.; Arena, J. P.; Liberator, P. A.;
Rosenblum, C. I.; Hamelin, M.; Hreniuk, D. L.; Palyha, O. C., et al. Science 1996,
273, 974.
2. Kojima, M.; Kangawa, K. Physiol. Rev. 2005, 85, 495.
3. Cummings, D. E.; Purnell, J. Q.; Frayo, R. S.; Schmidova, K.; Wisse, B. E.; Weigle,
D. S. Diabetes 2001, 50, 1714.
4. Bowers, C. Y.; Momany, F. A.; Reynolds, G. A.; Hong, A. Endocrinology 1984, 114,
1537.
5. Patchett, A. A.; Nargund, R. P.; Tata, J. R.; Chen, M. H.; Barakat, K. J.; Johnston, D.
B.; Cheng, K.; Chan, W. W.; Butler, B.; Hickey, G. Proc. Natl. Acad. Sci. U.S.A. 1995,
92, 7001.
Please cite this article in press as: Blayo, A.-L.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.11.031

A.-L. Blayo et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
6. Pan, L. C.; Carpino, P. A.; Lefker, B. A.; Ragan, J. A.; Toler, S. M.; Pettersen, J. C.;
Nettleton, D. O.; Ng, O.; Pirie, C. M.; Chidsey-Frink, K.; Lu, B.; Nickerson, D. F.;
Tess, D. A.; Mullins, M. A.; MacLean, D. B.; Da Silva-Jardine, P. A.; Thompson, D.
D. Endocrine 2001, 14, 437.
7. 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. J. Med. Chem. 2003, 46, 1191.
18. Moulin, A.; Brunel, L.; Boeglin, D.; Demange, L.; Ryan, J.; M’Kadmi, C.;
Denoyelle, S.; Martinez, J.; Fehrentz, J.-A. Amino Acids 2013, 44, 301.
19. Salome, N.; Haage, D.; Perrissoud, D.; Moulin, A.; Demange, L.; Egecioglu, E.;
Fehrentz, J.-A.; Martinez, J.; Dickson, S. L. Eur. J. Pharmacol. 2009, 612, 167.
20. Salome, N.; Hansson, C.; Taube, M.; Gustafsson-Ericson, L.; Egecioglu, E.;
Karlsson-Lindahl, L.; Fehrentz, J. A.; Martinez, J.; Perrissoud, D.; Dickson, S. L. J.
Neuroendocrinol. 2009, 21, 777.
8. Zizzari, P.; Halem, H.; Taylor, J.; Dong, J. Z.; Datta, R.; Culler, M. D.; Epelbaum, J.;
Bluet-Pajot, M. T. Endocrinology 2005, 146, 3836.
21. Jerlhag, E.; Egecioglu, E.; Landgren, S.; Salome, N.; Heilig, M.; Moechars, D.;
Datta, R.; Perrissoud, D.; Dickson, S. L.; Engel, J. A. Proc. Natl. Acad. Sci. U.S.A.
2009, 106, 11318.
22. Jerlhag, E.; Egecioglu, E.; Dickson, S. L.; Engel, J. A. Psychopharmacology 2010,
211, 415.
23. Clifford, P. S.; Rodriguez, J.; Schul, D.; Hughes, S.; Kniffin, T.; Hart, N.; Eitan, S.;
Brunel, L.; Fehrentz, J.-A.; Martinez, J.; Wellman, P. J. Addict. Biol. 2012, 17, 956.
24. Wellman, P. J.; Clifford, P. S.; Rodriguez, J.; Hughes, S.; Eitan, S.; Brunel, L.;
Fehrentz, J.-A.; Martinez, J. Regul. Pept. 2011, 172, 77.
9. Hoveyda, H. R.; Marsault, E.; Gagnon, R.; Mathieu, A. P.; Vezina, M.; Landry, A.;
Wang, Z. G.; Benakli, K.; Beaubien, S.; Saint-Louis, C.; Brassard, M.; Pinault, J. F.;
Ouellet, L.; Bhat, S.; Ramaseshan, M.; Peng, X. W.; Foucher, L.; Beauchemin, S.;
Bherer, P.; Veber, D. F.; Peterson, M. L.; Fraser, G. L. J. Med. Chem. 2011, 54, 8305.
10. Mihalic, J. T.; Kim, Y. J.; Lizarzaburu, M.; Chen, X. Q.; Deignan, J.; Wanska, M.;
Yu, M.; Fu, J.; Chen, X.; Zhang, A.; Connors, R.; Liang, L.; Lindstrom, M.; Ma, J.;
Tang, L.; Dai, K.; Li, L. Bioorg. Med. Chem. Lett. 2012, 22, 2046.
11. Yu, M.; Lizarzaburu, M.; Beckmann, H.; Connors, R.; Dai, K.; Haller, K.; Li, C.;
Liang, L.; Lindstrom, M.; Ma, J.; Motani, A.; Wanska, M.; Zhang, A.; Li, L.;
Medina, J. C. Bioorg. Med. Chem. Lett. 2010, 20, 1758.
12. Hanrahan, P.; Bell, J.; Bottomley, G.; Bradley, S.; Clarke, P.; Curtis, E.; Davis, S.;
Dawson, G.; Horswill, J.; Keily, J.; Moore, G.; Rasamison, C.; Bloxham, J. Bioorg.
Med. Chem. Lett. 2012, 22, 2271.
25. Boeglin, D.; Cantel, S.; Heitz, A.; Martinez, J.; Fehrentz, J.-A. Org. Lett. 2003, 5,
4465.
26. Bibian, M.; Blayo, A. L.; Moulin, A.; Martinez, J.; Fehrentz, J. A. Tetrahedron Lett.
2010, 51, 2660.
27. In the CRE/Luc reporter gene assay, mouse LTK-cells were stably transfected
with a plasmid containing the CMV minimal promotor linked to three cAMP
response elements (CRE) followed by a luciferase reporter gene. Based on this
parental cell line, single cell clones stably overexpressing the human GHS-R1A
13. Puleo, L.; Marini, P.; Avallone, R.; Zanchet, M.; Bandiera, S.; Baroni, M.; Croci, T.
Bioorg. Med. Chem. 2012, 20, 5623.
14. Cameron, K. O.; Bhattacharya, S. K.; Loomis, A. K. J. Med. Chem. 2014. http://
dx.doi.org/10.1021/jm5003183.
15. Demange, L.; Boeglin, D.; Moulin, A.; Mousseaux, D.; Ryan, J.; Berge, G.; Gagne,
D.; Heitz, A.; Perrissoud, D.; Locatelli, V.; Torsello, A.; Galleyrand, J. C.; Fehrentz,
J. A.; Martinez, J. J. Med. Chem. 1939, 2007, 50.
were established and characterized. The cells were incubated for 6 h with 1 lM
rolipram in the presence of different concentrations of the tested compounds
and saturating concentration of ghrelin. Subsequently, cells were lysed and
ATP bioluminescence was measured in the luminescence mode on FlexStation3
(Molecular Devices). All data were assessed in quadruplicate measurements
16. Moulin, A.; Demange, L.; Berge, G.; Gagne, D.; Ryan, J.; Mousseaux, D.; Heitz, A.;
Perrissoud, D.; Locatelli, V.; Torsello, A.; Galleyrand, J. C.; Fehrentz, J. A.;
Martinez, J. J. Med. Chem. 2007, 50, 5790.
17. Moulin, A.; Demange, L.; Ryan, J.; Mousseaux, D.; Sanchez, P.; Berge, G.; Gagne,
D.; Perrissoud, D.; Locatelli, V.; Torsello, A.; Galleyrand, J. C.; Fehrentz, J. A.;
Martinez, J. J. Med. Chem. 2008, 51, 689.
and calculated in % inhibition according to cells treated with saturating
concentrations of ghrelin (Polypeptide #sc1357) as negative (0% inhibition),
and nontreated cells as positive control (100%). IC50 values were determined by
using the GraphPad Prism analysis program (GraphPad Software).
Please cite this article in press as: Blayo, A.-L.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.11.031