New ligands of the ghrelin receptor based on the 1,2,4-triazole scaffold by introduction of a second chiral center

Article abstract of DOI:10.1016/j.bmcl.2016.04.003

Introducing a second chiral center on our previously described 1,2,4-triazole, allowed us to increase diversity and elongate the 'C-terminal part' of the molecule. Therefore, we were able to explore mimics of the substance P analogs described as inverse agonists. Some compounds presented affinities in the nanomolar range and potent biological activities, while one exhibited a partial inverse agonist behavior similar to a Substance P analog.

Full text of DOI:10.1016/j.bmcl.2016.04.003

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New ligands of the ghrelin receptor based on the 1,2,4-triazole
scaffold by introduction of a second chiral center
a
a
a
a
a
Mathieu Maingot , Anne-Laure Blayo , Séverine Denoyelle , Céline M’Kadmi , Marjorie Damian ,
a
a
a
b
b
b
Sophie Mary , Didier Gagne , Pierre Sanchez , Babette Aicher , Peter Schmidt , Gilbert Müller ,
Michael Teifel , Eckhard Günther , Jacky Marie , Jean-Louis Banères , Jean Martinez ,
Jean-Alain Fehrentz
b
b
a
a
a
a,
⇑
a
ème
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS, ENSCM, Université de Montpellier, BP 14491, Faculté de Pharmacie, bât. E, 3
Flahault, 34093 Montpellier Cedex 5, France
Æterna Zentaris GmbH, Weismuellerstrasse 50, 60314 Frankfurt am Main, Germany
étage, 15 avenue Charles
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Introducing a second chiral center on our previously described 1,2,4-triazole, allowed us to increase
diversity and elongate the ‘C-terminal part’ of the molecule. Therefore, we were able to explore mimics
of the substance P analogs described as inverse agonists. Some compounds presented affinities in the
nanomolar range and potent biological activities, while one exhibited a partial inverse agonist behavior
similar to a Substance P analog.
Received 16 February 2016
Revised 1 April 2016
Accepted 3 April 2016
Available online xxxx
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
1
,2,4-Triazole scaffold
Ghrelin receptor (GHS-R1a)
Structural modulation
Chirality
Inverse agonist
Antagonist
1
Ghrelin, an orexigenic hormone essentially synthesized in the
stomach, is the endogeneous ligand of the growth hormone secre-
which could lead to potential anti-obesity agents. We decided to
explore non-peptide ligands of the GHS-R1a through heterocycles
bearing the pharmacophore groups included in JMV 1843. We dis-
covered that the 1,2,4-triazole scaffold presented an interesting
approach to obtain ligands with high affinity toward the GHS-
2
tagogue receptor (GHS-R1a). Ghrelin is a peptide composed of 28
amino-acids, octanoylated on the seryl residue in position 3. This
lipidation is essential for both binding to the receptor and biolog-
ical activity. Among its various biological functions, ghrelin stim-
ulates the secretion of GH (Growth Hormone), and food intake,
3
15,16
R1a (Fig. 1).
3
4
The synthesis of the 1,2,4-triazole scaffold was efficiently per-
formed starting from commercially available compounds. An easy
access to tri-substituted 1,2,4-triazoles with four points of diver-
5
6
controls energy homeostasis and gastrointestinal motility. It
has effects on cellular proliferation,7 cardiovascular,8 pancreatic,
9
17
pulmonary and immune functions, memory and sleep. More
sity was developed and optimized. The first step of the synthesis
recently, it was established that ghrelin plays a role in addiction
included a N-protected a-amino acid whose configuration was
1
0
processes. In our effort to find efficient ghrelin receptor ligands,
conserved during the entire synthetic route. Several derivatives
of these new active non-peptide compounds were synthesized.
An intensive SAR study involving the four putative points of diver-
sity was carried out. The following conclusions were made: (i) the
11
we have developed a pseudo-peptide (JMV 1843), which is a
potent in vitro and in vivo agonist of the GHS-R1a. This compound
contains a gem-diamino moiety (Fig. 1) and is orally active in
1
2
13
man as exemplified by an initial validation study. Named maci-
morelin, it is evaluated in a test that detects adult growth hormone
deficiency. A pivotal confirmatory study is currently recruiting par-
preferred amino-acid starting material was (D)tryptophan; (ii) on
position 4 of the triazole, a 4-methoxy- or 2,4-dimethoxy-benzyl
group preferentially led to receptor antagonists; (iii) a 2 carbon
chain bearing a phenyl or an indole group was preferred in position
1
4
ticipants. We then focused on the search of GHS-R1a antagonists,
3
; (iv) numerous acyl groups including a-aminoisobutyryl (Aib),
pyridin-2-ylcarboxyl, and glycyl groups, could be introduced in
R , modulating both the binding affinity and the biological activity.
⇑
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).
3
http://dx.doi.org/10.1016/j.bmcl.2016.04.003
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0
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2
M. Maingot et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
NH
2
O
O
O
HN
H
N
H
N
H
N
HN
(R)
(R)
N
H
N
H
NH2
O
R1
H
N
H
N
H
O
O
O
O
N
H
H N
N
NH
NH
2
O
O
N
H
n
In
2
H N
2
H N
N
N
JMV 1843
NH
1
,2,4-triazole scaffold
(
n = 1-3)
KwFwLL-NH
2
O
2
-hydroxylacetyl
Acetyl-Leu-Leu
For myl
O
2
-hydroxyl-2-methylpropionyl
isonipecotyl
Acetyl
R3
R4
Aib
morpholine-2-carboxyl
Lys
mLe u-OMe
mLe u-Leu-OMe
mLe u-Leu-NH
2
HN
NH
HN
R4
N
HN
(R)
(R)
R3
N
4
3
R2
*
N
N
N
5
H
1
2
24-34
N
N
NH
HN
1
-34
2
Figure 3. Similarity between KwFwLL-NH and triazoles 24–34.
Figure 1. From pseudo-peptide to peptido-mimetic.
It is also interesting to notice that a simple atom change could shift
an agonist compound into an antagonist (Fig. 2). Indeed, when the
piperidine moiety of compound JMV 2951 [EC50 (Ca ) = 1.6 nM]
1843 compound: the presence of two (D)tryptophan residues
(Fig. 3). When comparing our triazole scaffold with this peptide,
we can hypothesize that the benzyl group in position 4 of our
scaffold can play the role of the phenylalanine residue. In that
case, the new chiral center incorporating an amino function in
position 3 of our triazole scaffold could allow the elongation with
the Leu-Leu dipeptide sequence.
A new series of triazole derivatives was therefore designed and
tested in order to find new efficient ligands, potentially inverse
agonists, of the GHS-R1a.
2+
was replaced by a tetrahydro-2H-pyran group (compound JMV
3
168), the agonist character of the ligand was lost to the benefit
2+
of the antagonist character [IC50 (Ca ) = 60 nM].
As compound JMV 1843 includes in its C-terminal part a gem-
diamino function, we then decided to introduce a chiral center in
position 3 of our 1,2,4 triazole scaffold allowing the presence of
an amine function at this position. This modification should lead
to closer structures of compound JMV 1843 than the previous tri-
substituted triazoles and could allow an additional point of diver-
sity (Fig. 1). Moreover, a characteristic of this receptor is its high
constitutive (ligands independent) activity, which reach about
In a first set of experiments (Table 1), the new chiral center was
introduced by the reaction of the thioamide Boc-(
(pOMe)Benzyl with Cbz-( ) or ( )phenylalanine hydrazide or Cbz-
) or ( )tryptophan hydrazide in the presence of silver benzoate
D)Trp[S]-NH
L
D
(L
D
5
0% of the maximal activity induced by ghrelin.18 Although ghrelin
receptor antagonists are able to reduce meal-associated food
to form the triazole scaffold bearing two chiral centers (Scheme 1).
After removal of the Boc protecting group, diverse interesting R3
acids were introduced to the amine function by conventional cou-
pling. The Cbz protection was then removed by hydrogenolysis and
the amino function was acylated or not with a formyl or acetyl
group to lead to the final compounds. All final compounds were
1
9
intake, inverse agonists of the ghrelin receptor, by blocking the
constitutive receptor activity, are expected to lower the set-point
for hunger between meals.20 The first GHS-R1a inverse agonist
1
was reported by Holst et al. as a Substance P analog: [(
Phe ,(
D
)Arg ,(
D)
5
7,9
11
18
22
D
)Trp ,Leu ]-substance P. Later, extensive SAR studies
purified by reversed-phase preparative HPLC. These compounds
identified new inverse agonist peptides with better specificity
were tested for their affinity toward the GHS-R1a, their ability to
induce intracellular calcium mobilization and for confirmation
toward GHS-R1a than the Substance P analog.21 In these papers,
15
a core pentapeptide wFwLL-NH
active sequence maintaining the inverse agonist activity. When a
lysine residue was introduced at the N-terminal of this pentapep-
tide, a potent inverse agonist of the receptor was obtained.
striking structural similarity could be found between the
2
was described as the minimal
of their agonist/antagonist character in a cyclic AMP response ele-
2
3
ment CRE-luciferase reporter gene assay.
2
Compounds 1 to 8 with the indole group as R clearly showed
that the R configuration of the new chiral center leads to ligands
with a higher affinity than compounds with the S configuration
of this carbon. The most potent compounds were obtained with
Aib or picolinic acid at the N-terminus (compounds 5 and 8 with
2
1a
A
2
hexapeptide KwFwLL-NH described by Holst et al. and our JMV
i
K values of 12 and 9 nM, respectively). As previously described,
2
4
the picolinic acid was well tolerated at this position. For com-
2
pounds 9 to 18, all containing the phenyl group as R , the best
affinities were also obtained when the second chiral center was
of R configuration. For this reason, compounds 19–23 were only
synthesized in the R series. Acetylation of the amino function
was preferred and only the N-terminal group was modulated. We
introduced at this position acyl groups that gave good results in
other series: 3-fluoro(pyridin-2-yl)carboxylic acid, 4,6-difluoro
O
O
HN
HN
N
N
HN
HN
N
N
N
N
O
O
HN
O
JMV 2951
JMV 3168
(
pyridin-2-yl)carboxylic acid, 3,4-dihydro-2H-pyran-6-carboxylic
IC50 (affinity): 0.6 nM
IC50 (affinity): 5 nM
2
+
2+
acid, 2-hydroxylacetic acid, and (S) morpholine-2-carboxylic
EC50 (Ca ): 1.6 nM
IC50 (Ca ): 60 nM
1
5
acid. These modifications led to compounds with a high affinity
Figure 2. Shift from agonist (JMV2951) to antagonist (JMV3168).
toward the receptor, particularly compounds 22 and 23, exhibiting
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M. Maingot et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 1
Binding affinity constants and biological activities of 1,2,4-triazole antagonists
O
HN
R4
HN
N
R2
*
3
R -NH
N
N
R2
R3
R4
C
⁄
configuration
K
(nM)a
IC50 [Ca2+ (nM)b
]
i
IC50 CRE/Luc (nM)c
Compounds
i
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
Indole
Indole
Indole
Indole
Indole
Indole
Indole
Indole
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Aib
Aib
Aib
Aib
H
H
S
>1000
131 ± 16
46 ± 6
>1000
12 ± 3
154 ± 20
62 ± 9
9 ± 2
>1000
154 ± 18
59 ± 7
>1000
46 ± 5
62 ± 8
18 ± 8
17 ± 4
10 ± 5
8 ± 3
>1000
90
33
>1000
19
1200
190
3
>1000
135
43
>1000
90
150
4
70
2
3
2
4
4
2
3
>1000
130
60
>1000
22
410
100
2
>1000
115
113
>1000
70
320
7
360
4
8
2
R
R
S
R
S
S
R
S
R
R
S
R
S
Formyl
Acetyl
Acetyl
Formyl
Acetyl
Acetyl
H
Aib
(Pyridin-2-yl)carboxyl
(Pyridin-2-yl)carboxyl
(Pyridin-2-yl)carboxyl
Aib
Aib
Aib
0
1
2
3
4
5
6
7
8
9
0
1
2
3
H
Formyl
Acetyl
Acetyl
H
Aib
Aib
(Pyridin-2-yl)carboxyl
(Pyridin-2-yl)carboxyl
(Pyridin-2-yl)carboxyl
(Pyridin-2-yl)carboxyl
(Pyridin-2-yl)carboxyl
3-Fluoro(pyridin-2-yl)carboxyl
4,6-Difluoro(pyridin-2-yl)carboxyl
3,4-Dihydro-2H-pyran-6-carboxyl
2-Hydroxylacetyl
(S) morpholine-2-carboxyl
H
R
S
Acetyl
Acetyl
Formyl
Acetyl
Acetyl
Acetyl
Acetyl
Acetyl
R
R
R
R
R
R
R
4 ± 1
11 ± 4
9 ± 3
0.8 ± 0.2
2 ± 0.4
4
3
8
9
a
Specific binding was determined by competition binding on membranes prepared from GHS-R1a transfected LLC PK-1 cells incubated with 125I-His -ghrelin (0.3 nM) in
9
i
the presence of increasing concentrations of compounds: K values were obtained from binding curves using GraphPad Prism Software.
b
2+
The ability of the antagonists to inhibit ghrelin signaling [Ca
i
] through the GHS-R1a (measurement of fluorescence output), was assessed using Schild Plots, with
À8
À7
À6
increasing concentrations of ghrelin, alone or in the presence of a fixed antagonist concentration (10 M, 10 M or 10 M). Experiments were performed in quadruplicate.
c
IC50 values were determined by competition using saturated dose of ghrelin and increasing amounts of tested compounds. Experiments were performed in quadruplicate.
R2
In
In
O
H
N
and potent biological activities were still of R configuration for the
new chiral center. The most potent ligands with IC50s in the
nanomolar range were compounds 8, 15 and 17–23. Compounds
Cbz-HN
NH
2
H
N
OH
(i)
ii)
O
Boc-HN
Boc-HN
(
(iii)
O
S
1
7–23 were latter evaluated in the IP3 turnover assay (data not
À5
O
O
shown) and they were all found partial agonists at 10 M, except
compound 22 (containing hydroxyl-acetyl moiety at the N-termi-
nus) which acted as a neutral antagonist, being unable to increase
or decrease the constitutive IP3 activity of the ghrelin receptor.
In a second set of syntheses, the amino function of the second
chiral center was elongated to mimic the Substance P analog and
to potentially obtain inverse agonists. A first series of compounds,
incorporating at the C-terminal part the acetyl-Leu-Leu sequence,
was designed. This led to an inversed dipeptide sequence at the
C-terminus compared to the peptidic sequence of substance P ana-
log. For the synthesis, a first approach consisting in the condensa-
In
In
Cbz
Cbz
HN
(iv)
(v)
HN
(vi)
N
R2
N
R2
3
Boc-HN
R -HN
N
N
N
N
O
O
In
In
(
vii)
R
4
NH
2
HN
N
R2
N
N
R2
3
3
R -HN
R -HN
tion of N-acetyl-Leu-Leu-(D)tryptophan hydrazide with the usual
N
N
N
thioamide Boc-( )Trp[S]-NH(pOMe)Benzyl led to a poor yield of
D
1
,2,9,10,14,15
3-8,11-13,16-23
the desired triazole after a long time reaction (more than two
months). As the 1,2,4-triazole scaffold includes a symmetry axis,
R2 = phenyl, indol
3
R
= Aib, (pyridin-2-yl)carboxyl, 3-fluoro(pyridin-2-yl)carboxyl, 4,6-difluoro(pyridin-2-yl)carboxyl,
3
,4-dihydro-2H-pyran-6-carboxyl, 2-hydroxylacetyl, (s)morpholine-2-carboxyl
we proposed a different synthetic pathway (Scheme 2). Boc-(D)-
R4 = CHO, CH
CO
3
Trp-OH was coupled with the 4-methoxybenzylamine and then
thionated as previously described. The obtained thioamide was
then elongated step by step by conventional peptide synthesis to
yield the corresponding tripeptide containing a thioamide bond:
Scheme 1. Reagents and conditions: (i) 4-methoxybenzyl amine, BOP, DIEA, DCM,
rt; (ii) Lawesson’s reagent, DME, 85 °C; (iii) AgBz, AcOH, DCM, rt; (iv) 4 N
HCl/dioxanne, rt; (v) appropriate acid group, BOP, DIEA, DCM, rt; (vi) H
2
, Pd/c,
EtOH, rt; (vii) appropriate acylating agent, DCM, rt.
Ac-Leu-Leu-(
reacted with the Cbz-(
D
)Trp[S]-NH(pOMe)Benzyl. This thioamide was
)Trp-NH-NH to yield the desired triazole
D
2
K
i
values of 0.8 and 2 nM, respectively. All these compounds
within two days. After hydrogenolysis of the Z protecting group,
six different acylating moieties (R in Table 2) were then intro-
duced at the N-terminal part (compounds 24–29). Keeping the
3
behaved as receptor antagonists, when tested in two functional
assays. The best compounds of the series, presenting good affinities
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M. Maingot et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
In
In
O
high-affinity red fluorescent ghrelin ligand that was previously
described. As we were looking for compounds acting as inverse
(
i)
(iii)
(iv)
26
H
N
(
ii)
Boc-NH
COOH
Boc-NH
agonists, the constitutive activity was evaluated with the IP3 turn-
over assay as it could not be detected using measurement of intra-
S
1
8
cellular calcium. As reference, the hexapeptide KwFwLL-NH
described by Holst et al. was tested and included in Table 2. Con-
cerning affinity, some compounds exhibited K in the nanomolar
2
,
In
In
O
H
N
i
Cbz-NH
CO-NH-NH
(v)
2
Ac-Leu-Leu-NH
range (compounds 24–26, 30 and 31). In the first series of ligands
with the Ac-Leu-Leu moiety, compounds 25, 26, 27 and 28 contain-
S
ing 2-hydroxyl-2-methylpropionyl, isonipecotyl,
a-aminoisobu-
O
O
tyryl and (S)morpholin-2-carboxyl groups respectively behaved
as agonists in the IP3 assay, while compound 24 (containing the
hydroxyacetyl moiety) presented a neutral antagonist character.
Introduction of a lysine residue at the N-terminal part (compound
29) induced a loss of binding affinity. However, this compound
behaved as a partial inverse agonist, being able to partially inhibit
the constitutive activity of the receptor (Emax = À37%). This con-
firmed the observation of Holst et al. on the significance of this
In
In
Leu-Leu-Ac
In
Leu-Leu-Ac
In
HN
HN
(
vi)
N
N
Cbz-NH
R3-HN
(
vii)
N
N
N
N
2
4-29
2
1b
Scheme 2. Reagents and conditions: (i) 4-methoxybenzyl amine, BOP, DIEA, DCM,
rt; (ii) Lawesson’s reagent, DME, 85 °C; (iii) deprotection steps: 4 N HCl/dioxane,
coupling steps: Boc-Leu-OH, BOP, DIEA, DCM; (iv) Ac
residue for inverse agonist activity.
With this interesting
result, we decided to keep the lysine residue at the N-terminal
part, and we then modulated the C-terminal part (compounds
2
O, DCM, rt; (v) AgBz, AcOH,
DCM, rt; (vi) H , Pd/c, EtOH, rt; (vii) appropriate acid group, BOP, DIEA, DCM, rt.
2
3
0–34). We first suppressed the two leucine residues and just
formylated or acylated the amino-function. Both compounds (30,
31) revealed good binding affinity in the nanomolar range but
exhibited a partial agonist character in the IP3 assay, showing
that the presence of the lysine residue at the N-terminus is not
sufficient to get inverse agonists. To recover the peptide
backbone similarity, we introduced 2-isobutylmalonic methyl
ester residue (as a racemic mixture) to get compound 32, which
lysine residue at the N-terminus, another series of compounds was
designed to evaluate the influence of the C-terminal part. We first
suppressed the two leucine residues and just formylated (com-
pound 30) or acetylated (compound 31) the amino function. Then,
to recover the peptide backbone similarity at the C-terminal part,
we introduced a 2-isobutylmalonic methyl ester residue (as a race-
mic mixture) to get compound 32. After saponification of the ester,
elongation of the C-terminal part with a leucine methyl ester or
leucine amide led to compounds 33 and 34. All final compounds
were purified by reversed-phase preparative HPLC and fully
i
showed a slight decrease in affinity (K : 17 nM) and a partial
agonist activity. Elongation of the C-terminal part with a leucine
methyl ester or leucine amide (compounds 33, 34) gave a
negative shift of one log in the affinity constants versus
compound 32. Both compounds were partial agonists. As a
2
5
characterized.
Pharmacological characterization of compounds 24–34 is
reported in Table 2. To determine the binding affinities of the com-
pounds, we used a fluorescence-based assay, called Tag-lite bind-
ing assay. This assay is based on a fluorescence resonance energy
transfer (FRET) process between a terbium cryptate covalently
attached to a SNAP-tag fused GHS-R1a (SNAP-GHS-R1a) and a
comparison, the
reported by Holst et al.
this compound exhibited a K value of 255 nM and 55% inhibition
i
of the basal response.
We have shown in this study that the introduction of a second
chiral center in position 3 of our 1,2,4-triazole scaffold could lead
i 2
K value of the hexapeptide KwFwLL-NH ,
2
1a
was inserted in Table 2. In our hands,
Table 2
Evaluation of the biological activity of the ligands after structural modulations on the second chiral center
O
H
N
R4
HN
NH
N
3
R -NH
N
N
Compounds
R3
R4
K
i
(nM)a
E
max (%)b
EC50 (nM)c
Behavior
2
2
2
2
2
2
3
3
3
3
3
4
5
6
7
8
9
0
1
2
3
4
2-Hydroxylacetyl
2-Hydroxyl-2-methylpropionyl
Isonipecotyl
Aib
Acetyl-Leu-Leu–
Acetyl-Leu-Leu
Acetyl-Leu-Leu
Acetyl-Leu-Leu
Acetyl-Leu-Leu
Acetyl-Leu-Leu
Formyl
Acetyl
mLeu-OMe
mLeu-Leu-OMe
3 ± 0.2
3 ± 0.5
2 ± 0.3
30 ± 8
50 ± 7
110 ± 12
8 ± 2
2.5 ± 0.8
17 ± 6
220 ± 32
210 ± 28
255 ± 31
0
75
108
80
75
—
Antagonist
Agonist
Agonist
Agonist
Agonist
Inverse agonist
Agonist
Agonist
Agonist
Agonist
74
4.4
0.8
60
70
10
4
20
82
170
100
(S)morpholine-2-carboxyl
Lys
Lys
Lys
Lys
Lys
Lys
À37
89
89
81
82
60
mLeu-Leu-NH
2
Agonist
Inverse agonist
KwFwLL-NH
2
À55
a
K
i
values of compounds were determined from binding fluorescent assays as described in Ref. 26.
b
The activity was determined by HTRF inositol phosphate assay: Emax values are expressed for agonists and antagonists as % of maximal stimulation promoted by ghrelin
over basal, and for inverse agonists as % of maximal inhibition of basal (basal represents 50–60% of the maximal ghrelin stimulation).
c
EC50 values were determined from analysis of dose response curves using GraphPad Prism Software.
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M. Maingot et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
to potent GHS-R1a ligands presenting good to high affinities
toward the receptor. Some compounds (8, 15, 17–23) exhibited a
nanomolar value of IC50 in two functional tests (calcium and CRE
reporter assays). Furthermore, the presence of an amino function
at the new chiral center allowed us to elongate the C-terminal part
of the molecule and to introduce the Leu-Leu dipeptide sequence
that is included in potent described inverse agonists. Unfortu-
nately, most of the compounds incorporating this dipeptide
behaved as agonists except compound 24 which is a neutral antag-
onist and compound 29 which exhibits a partial inverse agonist
16. (a) 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; (b) 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,
6
89.
7. (a) Boeglin, D.; Cantel, S.; Heitz, A.; Martinez, J.; Fehrentz, J.-A. Org. Lett. 2003, 5,
465; (b) Bibian, M.; Blayo, A. L.; Moulin, A.; Martinez, J.; Fehrentz, J. A.
1
4
Tetrahedron Lett. 2010, 51, 2660.
18. Holst, B.; Cygankiewicz, A.; Jensen, T. H.; Ankersen, M.; Schwartz, T. W. Mol.
Endocrinol. 2003, 17, 2201.
1
9. Holubova, M.; Nagelova, V.; Lacinova, Z.; Haluzik, M.; Sykora, D.; Moulin, A.;
Blayo, A. L.; Fehrentz, J. A.; Martinez, J.; Stofkova, A.; Jurcovicova, J.; Zelezna, B.;
Maletinska, L. Mol. Cell. Endocrinol. 2014, 393, 120.
behavior similar to the hexapeptide KwFwLL-NH
the literature. Some discrepancies between the functional assays
calcium mobilization, CRE-luciferase and IP3 turnover) were
2
described in
2
2
0. Holst, B.; Schwartz, T. W. Trends Pharmacol. Sci. 2004, 25, 113.
1. (a) Holst, B.; Lang, M.; Brandt, E.; Bach, A.; Howard, A.; Frimurer, T. M.; Beck-
Sickinger, A.; Schwartz, T. W. Mol. Pharmacol. 2006, 70, 936; (b) Holst, B.;
Mokrosinski, J.; Lang, M.; Brandt, E.; Nygaard, R.; Frimurer, T. M.; Beck-
Sickinger, A. G.; Schwartz, T. W. J. Biol. Chem. 2007, 282, 15799.
2. 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
(
observed, indicating the complex signaling of this receptor which
is likely responsible for the diversity in biological effects elicited
by ghrelin. We are currently investigating to try to understand
how signaling efficacy and selectivity are regulated.27 In conclu-
sion, we demonstrated that the trisubstituted 1,2,4-triazole scaf-
fold bearing a second chiral center can be an alternative to
introduce more diversity and to obtain high affinity ligands.
2
the rest, and the structures of the compounds were confirmed by MS
13
(
electrospray), 1
H
and/or
C NMR.1 As an example: compound 8. White
powder, 51 mg (0.067 mmol, 32%). H NMR (300 MHz, DMSO-d
ppm) 1.62 (s, 3H, COCH ), 3.21–3.43 (m, 3H, CH Trp (2H), CH
3.47–3.62 (m, 4H, CH Trp (1H), OCH
.12–5.22 (m, 2H, CH (1H), CH Trp), 5.36–5.45 (m, 1H, CH Trp), 6.50 (d,
6
, 303 K): d
(
3
2
2
Trp (1H)),
2
3 2
), 4.95 (d, J = 16.8 Hz, 1H, CH U (1H)),
5
2
U
J = 8.7 Hz, 2H, CH aryl), 6.62 (d, J = 8.7 Hz, 2H, CH aryl), 6.76–6.90 (m, 2H, CH
aryl), 6.91–7.07 (m, 4H, CH aryl), 7.16 (d, J = 7.9 Hz, 1H, CH aryl), 7.22 (d,
J = 7.9 Hz, 1H, CH aryl), 7.24–7.31 (m, 2H, CH aryl), 7.51–7.58 (m, 1H, CH
pyridin-2-yl), 7.82 (d, J = 7.7 Hz, 1H, CH pyridin-2-yl), 7.91 (dt, J = 7.7,
J = 1.6 Hz, 1H, CH pyridin-2-yl), 8.53-8.61 (m, 2H, NH acetyl, CH pyridin-2-
Acknowledgments
The authors thank Æterna-Zentaris (Germany), the CNRS
(
France) and the University of Montpellier (France) for financial
13
yl), 8.95 (d, J = 8.7 Hz, 1H, NHCO), 10.72–10.79 (m, 2H, NH Trp). C NMR
support of this work, and for providing a research grant for the
Ph.D. thesis projects of M. Maingot and A.-L. Blayo.
(75 MHz, DMSO-d
6
, 303 K): d (ppm) 22.1, 28.6, 28.7, 44.2, 44.8, 45.2, 54.9,
1
1
1
09.3, 109.7, 111.2, 111.3, 113.6 (2C), 118.0 (2C), 118.2, 118.3, 120.7, 120.9,
21.9, 123.9, 124.0, 126.6, 126.7 (2C), 126.9, 127.0, 127.1, 135.9, 136.0, 137.6,
48.2, 148.8, 154.9, 155.8, 158.3, 163.2, 168.8. Purity >98% (reverse phase
+
+
References and notes
HPLC). LC–MS (ES): m/z 533.2 [M+H-4-methoxybenzyl] , 653.4 [M+H] .
3. 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
2
1
.
.
Kojima, M.; Hosoda, H.; Date, Y.; Nakazato, M.; Matsuo, H.; Kangawa, K. Nature
999, 402, 656.
Howard, A. D.; Feighner, S. D.; Cully, D. F.; Arena, J. P.; Liberator, P. A.;
1
2
Rosenblum, C. I.; Hamelin, M.; Hreniuk, D. L.; Palyha, O. C., et al. Science 1996,
were established and characterized. The cells were incubated for 6 h with 1 lM
2
73, 974.
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
and calculated in % inhibition according to cells treated with saturating
concentrations of ghrelin (Polypeptide #sc1357) as negative (0% inhibition),
and non-treated cells as positive control (100%). IC50 values were determined
by using the GraphPad Prism analysis program (GraphPad Software).
3.
4.
5.
Takaya, K.; Ariyasu, H.; Kanamoto, N.; Iwakura, H.; Yoshimoto, A.; Harada, M.;
Mori, K.; Komatsu, Y.; Usui, T.; Shimatsu, A.; Ogawa, Y.; Hosoda, K.; Akamizu,
T.; Kojima, M.; Kangawa, K.; Nakao, K. J. Clin. Endocrinol. Metab. 2000, 85, 4908.
Akamizu, T.; Takaya, K.; Irako, T.; Hosoda, H.; Teramukai, S.; Matsuyama, A.;
Tada, H.; Miura, K.; Shimizu, A.; Fukushima, M.; Yokode, M.; Tanaka, K.;
Kangawa, K. Eur. J. Endocrinol. 2004, 150, 447.
(a) Guan, X. M.; Yu, H.; Palyha, O. C.; McKee, K. K.; Feighner, S. D.;
Sirinathsinghji, D. J.; Smith, R. G.; Van der Ploeg, L. H.; Howard, A. D. Mol.
Brain Res. 1997, 48, 23; (b) Willesen, M. G.; Kristensen, P.; Romer, J.
Neuroendocrinology 1999, 70, 306.
24. Blayo, A.-L.; Maingot, M.; Aicher, B.; M’Kadmi, C.; Schmidt, P.; Müller, G.; Teifel,
M.; Günther, E.; Gagne, D.; Denoyelle, S.; Martinez, J.; Fehrentz, J.-A. Bioorg.
Med. Chem. Lett. 2015, 25, 20.
6
7
8
9
.
.
.
.
Masuda, Y.; Tanaka, T.; Inomata, N.; Ohnuma, N.; Tanaka, S.; Itoh, Z.; Hosoda,
H.; Kojima, M.; Kangawa, K. Biochem. Biophys. Res. Commun. 2000, 276, 905.
Cassoni, P.; Papotti, M.; Catapano, F.; Ghe, C.; Deghenghi, R.; Ghigo, E.; Muccioli,
G. J. Endocrinol. 2000, 165, 139.
Isgaard, J.; Johansson, I.; Tivesten, A. In Endocrine Updates; Ghigo, E., Ed.;
Kluver: Norwel, 2004; Vol. 23, p 113.
(a) Davenport, A. P.; Bonner, T. I.; Foord, S. M.; Harmar, A. J.; Neubig, R. R.; Pin, J.
P.; Spedding, M.; Kojima, M.; Kangawa, K. Pharmacol. Rev. 2005, 57, 541; (b)
Leite-Moreira, A. F.; Soares, J. B. Drug Discovery Today 2007, 12, 276; (c) Van der
Lely, A. J.; Tschop, M.; Heiman, M. L.; Ghigo, E. Endocr. Rev. 2004, 25, 426.
25. 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
1
13
(electrospray), H and/or C NMR. As an example: compound 29. White
1
powder, 38 mg (0.067 mmol, 27%). H NMR (400 MHz, DMSO-d
(ppm) 0.68 (d, J = 6.4 Hz, 3H, CH Leu), 0.70 (d, J = 6.4 Hz, 3H, CH Leu), 0.76 (d,
J = 6.4 Hz, 3H, CH Leu), 0.83 (d, J = 6.4 Hz, 3H, CH Leu), 0.97 (m, 2H, CH Lys),
1.22–1.33 (m, 3H, CH Lys, CH(CH ), 1.36–1.40 (m, 4H, 2 CH Leu), 1.52 (m,
1H, CH(CH ), 1.81 (s, 3H, COCH ), 2.51 (m, 2H, CH Lys), 3.09 (dd, J = 6.0 Hz,
J = 14.0 Hz, 1H, CH Trp (1H)), 3.18–3.29 (m, 2H, CH
3.38 (dd, J = 6.8 Hz, J = 14.0 Hz, 1H, CH Trp (1H)), 3.56 (m, 1H, CH
3.71 (m, 4H, OCH , CH
4.92 (d, J = 16.4 Hz, 1H, CH
6
, 303 K): d
3
3
3
3
2
2
3
)
2
2
3
)
2
3
2
2
2
Trp (1H), CH
2
Trp (1H),
1
0. (a) Jerlhag, E.; Egecioglu, E.; Dickson, S. L.; Andersson, M.; Svensson, L.; Engel, J.
A. Addict. Biol. 2006, 11, 45; (b) 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.
2
2
Lys (1H)),
3
2
Lys (1H)), 4.15–4.27 (m, 3H, CH Lys, CH Leu, CH Leu),
(1H)), 5.06 (d, J = 16.4 Hz, 1H, CH (1H)), 5.17
2
U
2
U
(dd, J = 8.8 Hz, J = 14.8 Hz, 1H, CH Trp), 5.30 (dd, J = 8.0 Hz, J = 15.6 Hz, 1H, CH
Trp), 6.77 (d, J = 8.8 Hz, 2H, CH aryl), 6.81–6.91 (m, 4H, CH aryl), 6.98–7.06 (m,
5H, CH aryl), 7.26–7.32 (m, 3H, CH aryl), 7.56 (d, J = 8.4 Hz, 1H, NH-CO), 7.71 (s,
1
1. 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.
+
+
3H, NH
3 3
), 7.94 (s, 3H, NH ), 7.98 (d, J = 8.4 Hz, 1H, NH-CO), 8.63 (d, J = 8.8 Hz,
1
2. 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. J. Endocrinol. Invest. 2002,
1H, NH-CO), 9.17 (d, J = 8.8 Hz, 1H, NH-CO), 10.76 (d, J = 8.4 Hz, 2H, NH Trp).
13
6
C NMR (100 MHz, DMSO-d , 303 K): d (ppm) 20.44, 21.31, 21.59, 22.37,
22.85, 22.89, 23.87, 24.06, 26.44, 28.78, 29.58, 30.07, 38.36, 40.21, 40.65, 43.95,
44.51, 45.00, 50.71, 50.95, 51.64, 54.99, 109.11, 109.67, 111.20, 111.24, 113.50,
114.00, 117.85, 118.00, 118.22, 118.26, 120.73, 120.93, 124.01, 124.20, 126.71,
126.96, 127.39, 127.49, 128.26, 135.88, 135.90, 154.63, 155.12, 158.73, 167.92,
169.28, 171.65, 171.72. Purity >96% (reverse phase HPLC). LC–MS (ES): m/z
25, 6.
1
1
3. Garcia, J. M.; Swerdloff, R.; Wang, C.; Kyle, M.; Kipnes, M.; Biller, B. M.; Cook, D.;
Yuen, K. C.; Bonert, V.; Dobs, A.; Molitch, M. E.; Merriam, G. R. J. Clin. Endocrinol.
Metab. 2013, 98, 2422.
4. Validation of macimorelin as a test for adult growth hormone deficiency (Phase
III). This study is currently recruiting participants. For more details, see the
451.89 [M+2H]2 , 782.51 [M+H-4-methoxybenzyl] , 902.53 [M+H] .
26. Leyris, J. P.; Roux, T.; Trinquet, E.; Verdie, P.; Fehrentz, J. A.; Oueslati, N.;
Douzon, S.; Bourrier, E.; Lamarque, L.; Gagne, D.; Galleyrand, J. C.; M’kadmi, C.;
Martinez, J.; Mary, S.; Baneres, J. L.; Marie, J. Anal. Biochem. 2011, 408, 253.
27. M’Kadmi, C.; Leyris, J. P.; Onfroy, L.; Gales, C.; Sauliere, A.; Gagne, D.; Damian,
M.; Mary, S.; Maingot, M.; Denoyelle, S.; Verdie, P.; Fehrentz, J. A.; Martinez, J.;
Baneres, J. L.; Marie, J. J. Biol. Chem. 2015, 290, 27021.
+
+
+
following
link:
https://www.clinicaltrials.gov/ct2/show/NCT02558829?
term=macrilen&rank=1.
1
5. 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. 2007, 1939, 50.
Please cite this article in press as: Maingot, M.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.04.003