Synthesis and pharmacological evaluation of indolinone derivatives as novel ghrelin receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2012.07.018

The ghrelin receptor is a G-protein-coupled receptor (GPCR) widely expressed in the brain, stomach and the intestine. It was firstly identified during studies aimed to find synthetic modulators of growth hormone (GH) secretion. GHSR and its endogenous ligand ghrelin were found to be involved in hunger response. Through food intake regulation, they could affect body weight and adiposity. Thus GHSR antagonists rapidly became an attractive target to treat obesity and feeding disorders. In this study we describe the biological properties of new indolinone derivatives identified as a new, chiral class of ghrelin antagonists. Their synthesis as well as the structure-activity relationship will be discussed herein. The in vitro identified compound 14f was a potent GHSR1a antagonist (IC₅₀ = 7 nM). When tested in vivo, on gastric emptying model, 14f showed an inhibitory intrinsic effect when given alone and it dose dependently inhibited ghrelin stimulation. Compound 14f also reduced food intake stimulated both by fasting condition (high level of endogenous ghrelin) and by icv ghrelin. Moreover this compound improved glucose tolerance in ipGTT test.

Full text of DOI:10.1016/j.bmc.2012.07.018

Bioorganic & Medicinal Chemistry 20 (2012) 5623–5636
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological evaluation of indolinone derivatives
as novel ghrelin receptor antagonists
⇑
Letizia Puleo , Pietro Marini, Roberta Avallone, Marco Zanchet, Silvio Bandiera,
Marco Baroni, Tiziano Croci
Sanofi-Midy Research Centre, Exploratory Unit, Sanofi Research, Via G. Sbodio 2, 20134 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The ghrelin receptor is a G-protein-coupled receptor (GPCR) widely expressed in the brain, stomach and
the intestine. It was firstly identified during studies aimed to find synthetic modulators of growth hor-
mone (GH) secretion. GHSR and its endogenous ligand ghrelin were found to be involved in hunger
response. Through food intake regulation, they could affect body weight and adiposity. Thus GHSR
antagonists rapidly became an attractive target to treat obesity and feeding disorders. In this study we
describe the biological properties of new indolinone derivatives identified as a new, chiral class of ghrelin
antagonists. Their synthesis as well as the structure–activity relationship will be discussed herein. The in
vitro identified compound 14f was a potent GHSR1a antagonist (IC50 = 7 nM). When tested in vivo, on
gastric emptying model, 14f showed an inhibitory intrinsic effect when given alone and it dose depen-
dently inhibited ghrelin stimulation. Compound 14f also reduced food intake stimulated both by fasting
condition (high level of endogenous ghrelin) and by icv ghrelin. Moreover this compound improved
glucose tolerance in ipGTT test.
Received 9 March 2012
Revised 1 June 2012
Accepted 11 July 2012
Available online 24 July 2012
Keywords:
Ghrelin
GHSR1a antagonists
Indolinone derivatives
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
was the result of an extensive research on synthetic modulators of
GH secretion seen as a new alternative treatment to growth hor-
Ghrelin and its receptor (GHSR) have been shown to affect sev-
eral important physiological activities such as food intake regula-
tion, energy balance, gastric motility and secretion, as well as
glucose metabolism, fat accumulation and cell proliferation.1,2
Ghrelin is a 28-amino acid hormone predominantly produced
by P/D1 cells of the stomach and small intestine, but is also present
in minor quantity in the kidneys, pancreas and hypothalamus.3 It
was discovered in 1999 and recognized as the endogenous bioac-
tive ligand for the Growth Hormone Secretagogue receptor
(GHSR).4 Ghrelin presents an unusual n-octanoyl acetylation at ser-
ine 3 essential for its biological activity. It has been postulated that
this acetylation is also critical for the transport of the ghrelin mol-
ecule across the blood–brain barrier into the brain where the
receptor is highly expressed.5
The ghrelin receptor, GHSR, is a member of the G-protein-cou-
pled receptor (GPCR) superfamily.6 Two different splice forms of
the human GHSR are known; however, only GHSR1a is activated
by ghrelin and its mimetics, whereas the role of GHSR1b, a trun-
cated form of GHSR1a, is unknown.7 GHSR1a is widely expressed
in the brain and peripheral tissues, especially in the stomach. It
was identified and cloned by Howard et al. in 1996.8 Its discovery
mone replacement therapy.9–11 Interestingly, GHSR1a showed a
constitutive activity, independent from the presence of its ligand,
being able to change into an active conformation in the absence
of the agonist.12,13
GHSR1a, with its ligand, ghrelin, is predominantly involved in
the hunger perception prior to mealtimes. Ghrelin circulating
levels decrease with feeding and increase by fasting achieving con-
centrations sufficient to stimulate hunger and food intake. Admin-
istration of exogenous ghrelin potently stimulates food intake; this
effect was more efficient than that of any other molecule, with the
exception of neuropeptide Y.14 Chronic administration of ghrelin
in freely feeding mice and rats, increased body weight and
adiposity.15
Due to its involvement in feeding behavior, in energy homeo-
stasis and body weight regulation, ghrelin has become an attrac-
tive target to treat obesity and feeding disorders. Moreover,
ghrelin seems to play a direct role on glucose homeostasis through
regulation of insulin secretion.16–18
Those findings support the potential therapeutic role of ghrelin
antagonists in diabetes.
However, while GHSR agonists have been extensively studied,
due to their role in GH release, relatively fewer antagonists are de-
scribed in literature.19–23 Some of these compounds are shown in
Figure 1.
⇑
Corresponding author.
E-mail addresses: letizia.puleo@sanofi.com, letizia.puleo@virgilio.it (L. Puleo).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.018

5624
L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
Cl
N
H
N
O
N
N
N
NH
N
O
N
NH
N
NH2
N
O
N
F
O
O
H2N
O
A-778193
JMV3002
Figure 1. Structure of known GHSR1a antagonists.
YIL-781
R5
As part of our efforts to develop ghrelin receptor antagonists we
identified, through HTS screen of the Sanofi-Aventis compound li-
brary, an indolinone scaffold as exemplified by the general formula
1 (Fig. 2).
This class of compounds has been previously identified as Vaso-
pressin (V1) receptor antagonists.24 We designed new molecules
devoid of vasopressin activity, with lower molecular weight and
improved in vitro ADME profile. We first investigated the influence
of N-sulphonylation of the indolinone nitrogen atom on GHSR1a
antagonism. This group was known to be essential for V1 antago-
nism. The urea linker was modified with the aim to ameliorate bio-
availability. This synthetic strategy also led to the identification of
novel, enantiopure compounds.
Cl
(+)
O
N
R4
R3
N
N
N
O
H
N
H
N
R2
R1
Cl
Cl
O
O
N
N
H
O
S
O
Ar
1
2
Figure 2. Hit identification. R1, R2, R3, R4 are independently hydrogen, chlorine or
methyl groups, R5 is a C 1–3 alkyl group or, at least, a phenyl. Ar represents a
benzene substituted with small groups as chlorine or methoxy groups.
The hit compound 2 (Fig. 2), with an IC50 of 47 nM in functional
CreLuc assay, represented an excellent starting point for new leads
identification.25
Interestingly, during our SAR studies, we could observe that
slight modifications in scaffold decoration, could lead to agonist
activity. Indeed a series of oxindole derivatives, here represented
by SM-130686 (Fig. 3), have already been published by Sumitomo,
and reported to be GHSR agonists.26
CF
3HO
Cl
O
NC
N
O
Our compounds, however, basically differ from Sumitomo’s
class of molecules, especially for the presence of a substituted
amide at the indolinone C-3 position and for this carbon
configuration.
N
HCl
Figure 3. Structure of SM-130686.
Looking for new molecules we focused our attention on com-
pounds possessing a full GHSR1a antagonist activity only. Modifi-
cations both at the basic moiety and at the aromatic portions
were largely evaluated and will be discussed hereafter.
(ꢀ)-2-phenylglycinol, thus obtaining the 5c, 5d pair. The two
compounds were easily separated by flash chromatography. X-
ray diffraction of compound 5d found the absolute configuration
at the C-3 position to be (R) (Fig. 4).
2. Chemistry
Subsequent two steps amine dealkylation provided the (ꢀ)-3-
aminoindolinone 6b.
The synthetic pathway leading to the Hit compound 2 is re-
ported on Scheme 1.
The two epimers were well distinguishable each other for their
Rf on TLC plate. Also NMR spectra showed several relevant differ-
ences between the two diastereoisomers, with a general pattern
characterizing each enantiomer. This behavior resulted similar
for all couples of diastereoisomeric derivatives synthesized. It
was also possible to assess the diastereoisomeric ratio, simply by
comparing NMR signal integrals in the mixture of the two com-
pounds. In the same way we controlled the enantioenrichment of
separated pairs.
For the 5c, 5d pair we found a diastereoisomeric ratio of 40:60,
respectively, providing the (R)-(ꢀ) amine in major quantity after
amine deprotection. By performing the same reaction with (S)-
(+)-2-phenylglycinol we obtained the 5a, 5b pair with a 60:40 dia-
stereoisomeric ratio. As both 5b and 5d provide the same (R)-(ꢀ)
amine, is easy to identify the C-3 configuration to be (R) for 5b
and (S) for 5a (Scheme 1). Being mainly interested on the (S)-(+)
amine, the use of (S)-(+)-2-phenylglycinol resulted preferable.
Key intermediate (S)6a was then opportunely transformed
through classical methods into compound 2.
Starting from commercially available 1,2-dichloro-4-fluoro-5-
nitrobenzene and methyl-4-chlorophenylacetate, an aromatic SN
reaction provided the intermediate 3, which was transformed in
the halogen derivative 4 by the use of phenyltrimethylammonium
tribromide. Other reagents more commonly used for benzylic halo-
genation, such as NBS, failed in giving the desired product. In some
cases the halogenations proceeded also to the aromatic ring posi-
tions. Moreover, it was observed that halogen derivative easily
undergoes degradation to form several byproducts. Due to its
instability it was therefore rapidly used for the next reaction step
without further purification.
To obtain enantiopure compounds, the synthetic strategy used
herein mainly relied on functionalization of racemic indolinone
intermediate 4 to form an epimeric pair, through reaction with
optically active 2-phenylglycinol. As we aimed to identify which
of the two optically active reagents was preferable for our
synthetic purposes, we initially performed the reaction with (R)-

L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
Cl
5625
Cl
H
N
Cl
Cl
Cl
(S,S)5a, (R,S)5b
60:40
O
OH
N
H
d
Cl
Cl
F
R
Cl
Cl
COOMe
a,b
O
Cl
N
H
NO2
e
3: R=H
c
4:
R=Br
H
N
(S,R)5c, (R,R)5d
Cl
Cl
40:60
O
OH
N
H
Cl
Cl
(+)
O
(+)
N
H
N
N
NH2
O
Cl
Cl
Cl
Cl
f,g
h,i
O
5a or 5c
N
N
H
H
(S)6a
2
Cl
(-)
NH2
O
Cl
Cl
f,g
5b or 5d
N
H
(R)6b
Scheme 1. Reagents and conditions: (a) NaH, DMF, 3 h, ꢀ10 °C/rt, (b) Fe, AcOH, MeOH, 4 h, reflux, (c) PhMe3NBr3, CH2Cl2, rt, 12 h, (d) (S)-(+)-2-phenylglycinol, CH2Cl2, rt, 18 h,
(e) (R)-(ꢀ)-2-phenylglycinol, CH2Cl2, rt, 18 h, (f) Pb(OAc)4, CH2Cl2 MeOH, rt, 3 h, (g) MeOH/HCl 3 N, MeOH, rt, 18 h, (h) chloroacetylchloride, pyridine, toluene, 80 °C, 2 h,
(i) N-methylpiperazine, K2CO3, NaI, DMF, 100 °C, 3 h.
that is, properly substituted 1-fluoro-2-nitro-benzenes and pheny-
lacetates for the synthesis of the scaffold, and the appropriate basic
moiety to obtain in particular compounds 2i–o, as outlined in the
following Tables. All enantiopure compounds were prepared by
following the previously described strategy. In this way we ob-
tained epimeric pairs with a diastereoisomeric ratio varying from
60:40 to 70:30 according to the substrate (NMR calculation). The
enantioenrichment in the final products thus achieved was deter-
mined through optical rotation measurements. Unless otherwise
noted, following synthetic schemes and SAR tables refers to the
(+)-enantiomer. In some other cases the diastereoisomeric mixture
was not separated thus isolating a (+/-) mixture at the end of the
synthesis, which ratio was not further defined.
This synthetic methodology resulted preferable as this two step
indolinone ring formation allow to control from the beginning the
desired end position of aromatic substitution. However, an impor-
tant drawback is the difficult accessibility of suitable substituted
1-fluoro-2-nitro-benzenes, considering both commercial availabil-
ity and synthesis. In this situation a different pathway, was applied.
Compounds 10a–c were prepared as reported in Scheme 2.
p-Chloromandelic acid was protected at the free oxydrile and
transformed into the corresponding acyl chloride. Reaction of 7
Figure 4. X-ray structure of 5d.
All compounds 2a–o listed below, were synthesized according
to this synthetic pathway starting from the following precursors,

5626
L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
with the required commercially available aniline gave compounds
8a–c which cyclized under acidic conditions. The symmetrical
3,4,5-trichloroaniline provided, using these conditions, a single pro-
duct, whereas starting either from 4-chloro-3-methylaniline or
from 3-chloro-4-methylaniline a mixture of the two regioisomers
is obtained, generally enriched in one of the two regioisomers.
Synthesis, accomplished as previously depicted, furnished com-
pounds 10a–c.
a
b
N
Et
N Et
O
N
Et
NC
NC
Cl
15
16
Cl
Cl
(+)
Cl
Cl
(+)
NH2
O
Cl
N
Cl
N
H
N
Through substituted isatines commercially available, the syn-
thesis of compounds 14a–l, was easier. Actually, this useful start-
13H
HOOC
c
N
Et
O
O
N
H
ing material can be transformed in
a few steps in the
Cl
d
halogenated key intermediate. Scheme 3 describes the synthesis
of intermediate 12 as referring methodology.
17
18
Scheme 4. Reagents and conditions: (a) (OEt)2P(O)CH2CN, K2CO3, THF, 16 h, 70 °C,
(b) Mg, MeOH, 4 h, rt, (c) HClc, 20 h, 100 °C, (d) 13, PCl5, pyridine, CH2Cl2, rt, 3 h.
The obtained compound was easily functionalized as reported
above and used to prepare compounds 14e–l after reaction with
the appropriate base as indicated in the following Tables. Com-
pounds 14a–d were also obtained through this methodology, start-
ing from suitably substituted isatines and Grignard reagents.
The synthesis of piperidine analogue 18 required the prepara-
tion of aminoacid 17. A Wittig reaction on the N-ethyl-piperidone
provided the acrylonitril derivative. Hydrogenation of 15 per-
formed with classical methodologies failed in giving the desired
product. Compound 16 could be obtained through reduction with
magnesium.27 Further acidic hydrolysis of cyano group led to the
carboxylic acid, which was coupled with the previously described
amine 13 (Scheme 4).
3. Results and discussion
3.1. In vitro SAR studies
Compounds potency and efficacy on GHSR1a were determined
by Luciferase reporter assay system employing a hGHSR-1a CHO-
Creluc cell line. Binding was performed on membrane preparations
from the same cell line in presence of [125I]-ghrelin.
In our preliminary investigation we identified the hit 2; the (+)-
enantiomer showed an IC50 of 47 nM, whereas, the corresponding
(ꢀ)-enantiomer was completely inactive (IC50 >10,000 nM). This
The use of commercially available 1-methyl-3-piperidone led to
compound 19 following the same synthetic procedure.
R3
R2
R1
Cl
R3
O
Cl
R2
R1
Cl
O
NH2
O
a, b
Cl
OH
O
N
H
c,d
OH
OH
O
8a: R1=R2=R3=Cl
8b: R1=CH3, R2=Cl, R3=H
8c: R1=H, R2=CH3, R3=Cl
7
Cl
Cl
R3
N
H
N
R3
N
R2
R1
R2
R1
e
O
O
O
N
N
H
H
(+/-)10a: R1=R2=R3=Cl
(+)10b: R1=CH3, R2=Cl,
(+/-)10c: R1=H, R2=CH3, R3=Cl
9a: R1=R2=R3=Cl
9b: R1=CH3, R2=Cl, R3=H
9c: R1=H, R2=CH3, R3=Cl
Scheme 2. Reagents and conditions: (a) CH3COCl, CH2Cl2, 50 °C, 3 h, (b) SOCl2, CH2Cl2, 65 °C, 2 h, (c) K2CO3, CH2Cl2, rt, (d) MeOH, toluene, 80 °C, 2 h, (e) H2SO4, rt, 4 h.
Cl
Cl
Cl
Cl
Cl
Cl
(+)
Cl
Cl
O
Cl
MgBr
R
NH2
O
O
O
14e-
a
N
N
H
Cl
Cl
N
H
Cl
H
11: R=OH
12: R=Cl
b
13
Scheme 3. Reagents and conditions: (a) THF, rt, 4 h, (b) SOCl2, pyridine, CH2Cl2, rt, 1 h.

L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
5627
Table 1
Table 1 (continued)
SAR investigation of aromatic substitutions
No.
R1 R2
R3
R4 R5
Synthetic
method
(Scheme n°)
GHSR1a
(+)
antagonism
CreLuca (IC50
nM)
R4
R5
N
H
N
,
N
R3
R2
O
O
Cl
Cl
N
H
R1
10cb
H
H
H
CH3 Cl
2
1160 260
No.
R1 R2
R3
R4 R5
Synthetic
method
(Scheme n°)
GHSR1a
antagonism
CreLuca (IC50
nM)
,
1(Pathway
d)
2db,c
Cl
CH3
H
213 18
Cl
Cl
Cl
Cl
Cl
Cl
Cl
1(Pathway
d)
2
H
H
Cl
H
H
H
H
H
H
H
Cl
Cl
H
Cl
H
H
H
H
Cl
H
Cl
Cl
H
H
H
47
7
Cl
Cl
1(Pathway
d)
2eb
2fb
H
H
Cl
Cl
CH3
CH3
H
H
1180 157
>10,000
14a
14b
10ab
14c
14d
14e
2a
3
3
2
3
3
3
140 15
1390 180
273 35
111 12
72 14
1(Pathway
d)
Cl
Cl
Cl
Cl
Cl
Cl
H
1(Pathway
d)
2gb
H
H
Cl
Cl
CH3
CH3
H
H
220 31
>10,000
Cl
Cl
Cl
Cl
Cl
1(Pathway
d)
2hb
a
b
c
Reported in vitro values are an average of at least two replicates.
(+/-) Mixture.
Compound is reported for a better comparison with analogs 2e–2h.
Cl
big difference in activity was observed for several pairs of enanti-
omers and appears to be a general trend for this class of molecules.
Considering substitutions on the aromatic ring a strictly related
family of analogs was prepared by varying both the aromatic moi-
eties number and position of chlorine substituents. In addition very
few other substituents were used to modulate the molecule elec-
tronegativity, that is, alkyl vs methoxy groups. These compounds
were then tested for GHSR1a antagonism (Table 1).
H
Cl
H
24
7
At first glance it is clear that of the limited numbers of substit-
uents examined, chlorine is preferred. In particular substitution at
the position 6 of the indolinone core (R2 substituent) is important
for potency. Actually its removal causes a major activity loss, as can
be observed for compound 14b and 10c. When referring to the aro-
matic in position 3 of the indolinone, Table 1 clearly shows that an
ortho-substitution causes a dramatic potency loss as shown for
both compounds 2f and 2h, whereas a para-substitution is prefer-
able in comparison to a meta-substitution. The double 3, 4 substi-
tution is well tolerated and becomes interesting when coupled
with the 3, 4 disubstitution on the other indolinone ring, as was
observed for compound 14e.
All compounds reported in Table 1, showing nanomolar activity,
were further tested for in vitro metabolic stability, using hepatic
microsomal fractions from mouse, rat and human. These studies
demonstrated that the N-methyl piperazinyl moiety is the major
site of methabolization (data not shown). The replacement with
a series of cyclic or acyclic bases was then explored with the aim
to reduce the high rate of metabolization.
OMe
Cl
1(Pathway
d)
Cl
700 120
251 37
525 140
10b
2b
CH3 Cl
2
OMe
1(Pathway
d)
Cl
Cl
CH3
1(Pathway
d)
2c
H
Cl
H
1470 220
Some representative compounds are reported in Table 2.
The data shown in Table 2, demonstrates that the piperazine
moiety is optimal for GHSR1a activity, as small structural modifica-

5628
L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
Table 2
SAR investigation at the basic moiety
Cl
R4
(+)
R1
R3
H
N
R2
Cl
O
O
N
H
No.
R1
R2
H
R3
Cl
R4
Synthetic method (Scheme n°)
GHSR1a antagonism CreLuca (IC50, nM)
N
N
14f
Cl
Cl
3
7
5
N
N
N
14g
14h
H
H
Cl
Cl
3
3
38
9
4
N
Cl
Cl
64
N
14i
H
Cl
3
239 21
N
N
N
N
N
14j
H
H
Cl
Cl
Cl
Cl
3
3
25
7
14k
110 27
47 11
19
H
H
Cl
Cl
Cl
Cl
4
3
N
O
N
N
N
14l
2780 280
N
N
18
H
Cl
H
Cl
H
4
1
0.6
2ib,c
CH3
1(Pathway d)
129 21
>10,000
>10,000
H
N
N
2jb
CH3
CH3
H
H
H
H
1(Pathway d)
1(Pathway d)
N
N
2kb
N
2lb
CH3
H
H
1(Pathway d)
>10,000
N
N
N
N
N
N
N
2mb,d
2nb
Cl
Cl
Cl
H
H
H
H
H
H
1(Pathway d)
1(Pathway d)
1(Pathway d)
110 12
>10,000
2ob
2540 150
a
b
c
Reported in vitro values are an average of at least two replicates.
(+/-) Mixture.
Compound is reported for a better comparison with analogs 2j–2l.
d
Compound is reported for a better comparison with analogs 2n–2o.

L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
5629
Table 3
Comparison of GHSR1a CreLuc antagonism and human binding of some selected compounds
Cl
R4
(+)
R1
R3
H
N
R2
Cl
O
O
N
H
No.
R1
R2
Cl
R3
H
R4
GHSR1a antagonism CreLuca (IC50, nM)
GHSR1a human bindinga (Ki, nM)
N
N
N
N
N
2
H
H
H
Cl
Cl
Cl
Cl
H
H
47
7
249 26
N
N
N
N
N
14d
2db
2gb
14f
14g
18
H
Cl
H
72 16
213 18
220 15
44
8
CH3
CH3
H
392 28
>1000
H
Cl
Cl
Cl
H
7
38
1
2
8
2
N
N
N
N
H
5
121 10
N
H
0.8
3
1
2ib
CH3
Cl
129 21
110 12
286 25
202 21
N
N
2mb
H
a
Reported in vitro values are an average of at least two replicates.
(+/-) Mixture.
b
tions causes a substantial loss in activity. In particular ring rigidity
is required. The corresponding open chain analogue 2n is for in-
stance completely inactive. Alkyl substitution on the external
nitrogen of the piperazine ring is essential for activity; as shown
in compound 2j, an unsubstituted piperazine results in a loss of
activity. Only small alkyl substituents are well tolerated, bulky
groups such as benzyl or pyridinyl groups (compounds 2k and
2l) cause a significant loss of activity. Replacement of the pipera-
zine with 4-methyl piperidine 2o or modification of the basicity
of the external piperazine nitrogen by introduction of a carbonyl
alpha to the nitrogen (compound 14l) resulted in a dramatic de-
crease in potency.
On the other hand, compounds like 14j, and above all 14f and
18 stand out against other values for their activity. Those results
were consistent in all the series checked.
Few representative molecules were compared in the two assays,
GHSR1a CreLuc and binding. The results are showed in Table 3.
Although, some discrepancies observed were difficult to under-
stand and explain (i.e., 2g vs 2d, or 14d results), data obtained from
the two assays where generally in line and it clearly appeared that
compounds 14f and 18, with a Ki of 8 and 3 nM, respectively, were
in vitro potent GHSR-1a antagonists.
The 4-piperidine analogues, exemplified by compound 18,
exhibited excellent potency but they showed an high Pgp affinity
Table 4
In vitro ADME profiling of 14f
Cl
Cl
(+)
N
N
H
Cl
N
O
O
N
H
Cl
No.
Caco-2/TC-7 permeability
% Recovery
Ptotal (10ꢀ8cm secꢀ1
51.8 38.45 7.5
Liver microsomes (5
Mouse Rat
44 45
l
M) total metabolisation (%)
Hepatocytes (5 lM) intrinsinc clearance
Clint (mL hꢀ1 (106hep)ꢀ1
)
)
Human
41
14f
8
0.097 0.027

5630
L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
100
90
80
70
60
50
40
30
20
10
0
vehicle 14f ip 2 ml/kg plus ghrelin vehicle 15 µl/rat icv
vehicle 14f 2ml/kg ip plus ghrelin 10µg/rat icv
Vehicle 2 ml/kg i.p. n=11
14f 3 mg/kg i.p. n=9
14f 10 mg/kg i.p. n=6
6
5
4
3
2
1
0
14f 10mg/kg ip plus ghrelin10 µg/rat icv
***
###
***
***
°
**
***
°°
**
**
*
**
**
Rat Ghrelin
(20 µg/kg iv)
(-)
(+)
###P<0.0001 VS Vehicle (-) Ghrelin, one way Anova Newman-Keuls test
*,**P<0.001,0.0005, VS Vehicle (-) Ghrelin Vehicle, one way Anova Newman-Keuls test
°,°°P<0.005 and 0.0001 VS Vehicle (+) Ghrelin, one way Anova Newman-Keuls test
1 h
2 h
3 h
4 h
Time (h), after ICV treatment
**,***P<0.01 and 0.0001 VS vehicle, two-way Anovarep Newman-Keuls test
Figure 5. Effect of compounds 14f on gastric emptying in CD rats in absence and in
presence of ghrelin (20 lg/kg iv).
Figure 7. Effect of compound 14f on rat cumulative food intake stimulated by ICV
ghrelin. The compound was dosed 30 min before icv treatment.
(efflux ratio = 58); this suggests that 18 could display in vivo a low
bioavailability and poor brain penetration. Consequently, piperi-
dine series was abandoned despite of the large numbers of com-
pounds synthesized. Piperazines showed a lower efflux ratio and
they were chosen as the preferred basic moiety. Indeed 14f dis-
played a 10 time lower Pgp value then compound 18 (efflux
ratio = 6.6).
Compound 14f was also investigated for in vitro ADME proper-
ties (Table 4). The trans-epithelial intestinal transport (Ptotal) on
Caco-2/TC-7 cell monolayers of 14f was very high. These results
suggests that 14f will show a good bioavailability when oral
administered. 14f also showed an intermediary metabolism, fol-
As part of our SAR we also explored some modifications at the
alkyl linker from the indolinone core to the basic unit. Several com-
pounds from both the piperidine and the piperazine series were
prepared to examine the importance of the alkyl chain lenght.
However shortening or lengthening of the alkyl chain resulted in
a loss of activity. All the reported molecules were devoid of agonist
or inverse agonist properties.
On the basis of all those considerations, compound 14f was se-
lected for further investigation because its selectivity at high con-
centration over a panel of 33 different receptors (Cerep 33). The
lowing a 24 h incubation at 5 lM on isolated hepatic microsomal
fractions prepared from mouse, rat and humans (see Table 4). In
all species the metabolism was similar (range 41–45%). In human
hepatocytes the 14f intrinsic metabolic clearance (Clint) was also
intermediary, 0.097 0.027 mL hꢀ1 (106 hep)ꢀ1
.
Because the good in vitro profile, 14f was selected for a further
in vivo characterization. The interesting obtained results should be
considered as a first activity evaluation of the new indolinone
derivative, further studies were commenced to well characterize
pharmacokinetic properties for a better contextualization of those
results.
compound had no significant binding at 10 lM, including adeno-
sine, adrenergic, cannabinoids, dopamine, histamine, muscarinics,
nicotinic, opioids, serotonin, and vasopressin receptors.
3.2. In vivo activity
Ghrelin is a potent stimulator of gastric emptying.28–30 In 24 h
fasted CD male rats the lead GHS-R1a antagonist 14f (3 and
10 mg/kg, ip) dose dependently inhibited ghrelin effect on gastric
emptying by 55% and 62%, respectively. This antagonist also slo-
wed gastric emptying in absence of ghrelin treatment (68% at
3 mg/kg and 55% at 10 mg/kg); this intrinsic, not dose dependently
effect, suggests a physiological role of endogenous ghrelin in regu-
lating gastric functions (Fig. 5).31
2.5
Vehicle 10ml/kg, n=9
2.0
14f 10mg/kg ip n=10
**
1.5
There is strong evidence to suggest that ghrelin plays a critical
role in modulating body weight through the control of food intake
and/or regulation of fuel substrate efficiency.32 We tested the effect
of compound 14f in two different models of food intake: the fasted
refed mouse model and the icv ghrelin rat model. In the former
high plasmatic levels of ghrelin were physiologically induced
whereas in the latter ghrelin was injected directly in the central
nervous system. As shown in Figure 6, 14f dosed ip at 10 mg/kg
caused a significant reduction in food intake in fasted C57/BL6N
mice out to 6 h.
**
1.0
*
*
0.5
0.0
1 h
2 h
4 h
6 h
Time (h)
*,**P<0.05 and 0.005 VS vehicle, two-way Anovarep Newman-Keuls test
The compound at 10 mg/kg inhibited the food intake stimulated
by icv (10
lg/rat) ghrelin injection (Fig. 7) in fed male rats. The
Figure 6. Effect of compound 14f on food intake in the mouse fasted-refed model
(C57/BL6N mice).
ghrelin effect was assessed for 4 h, the compound inhibited the

L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
5631
A
vehicle 10 ml/kg n=6
300
270
240
210
180
150
120
90
glimepiride 2 mg/kg po n=7
14f 30 mg/kg po n=7
*
*
*
*
60
*
*
30
-30'
0'
20'
40
Time (h)
60'
80'
100'
120'
* P< 0.05 vs Vehicle two-way Anovarep Newman-Keuls test
B
20000
vehicle 2 ml/kg n=6
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
glimepiride 2 mg/kg po n=7
14f 30 mg/kg po n=7
*
*
* P< 0.05 vs Vehicle one-way Anova
Newman-Keuls test
Figure 8. Effect of compound 2 on glucose tolerance in GTT test in C57/BL6N mice.
ghrelin stimulation by 58%, 60%, 61%, 53%, respectively, at 1, 2, 3,
4 h.
The metabolic effect of compound 14f was assessed on glucose
homeostasis in vivo,28 using an ip Glucose Tolerance Test (ipGTT)
in fasted C57/BL6N mice.
showed a significant improvement in glucose tolerance. Encour-
aged by the initial promising results, 14f has gone forward for fur-
ther more detailed pharmacological profiling and a large lead
optimization programme has been commenced to build on these
results.33,34
Compound 14f, dosed 30 mg/kg po was shown to significantly
improve glucose tolerance (Fig. 8A), reducing the glucose excursion
by 32% when compared to vehicle. This was similar in magnitude
to the effect of the insulin secretagogue glimepiride, (Fig. 8B).
5. Experimental
5.1. General chemistry
4. Conclusion
Chemicals were purchased from commercial sources and used
as supplied, unless otherwise indicated. All reactions were moni-
tored by TLC on VWR glass plates precoated with silica gel 60
F254; spots were visualized through UV light at 220 nm or by
treatment with 1% aq KMnO4. Products were purified by flash chro-
matography on BiotageÒ SNAP Cartridge KP-Sil or on Supelco Ver-
Following the HTS identification of new indolinone derivatives
with GHSR-1a receptor antagonist properties, we began a compre-
hensive synthetic program to improve hits profile and establish a
robust structure–activity relationship. Compound 14f was identi-
fied in vitro as a selective and potent lead (7 nM). Thus it was se-
lected for in vivo further characterization. 14f inhibited the effect
of GHSR1a agonist ghrelin on rats gastric emptying and food intake
attesting its specificity. It also inhibited food intake in the mouse
fasted-refed model in which high plasmatic level of endogenous
ghrelin are induced. When tested under ipGTT conditions 14f
saPak™ Cartridges, Spherical Silica (20–45
lm). Melting points
have been measured with Buchi B-540 instrument. ½a D
ꢁ
measure-
ments were determined trough a Perkin Elmer polarimeter PE341.
1H and 13C NMR spectroscopy was performed on 400 MHz Bru-
ker spectrometers. Chemical shifts (d) are expressed in ppm rela-
tive to TMS frequency.

5632
L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
Liquid chromatography mass spectrometry (LC–MS) was per-
1,2-dichloro-4-[2-(methy-l-4-chlorophenyl acetate)]-5-nitroben-
zene (3.18 g, 36%) as a colourless oil.
formed on a Thermo Electron Surveyor system equipped by a diode
array detector and LCQ DecaXP Max ion trap mass spectrometer
with electrospray ionization. The mass detection was used in par-
allel with UV/vis detector and the eluent coming from HPLC was
splitted with an intent to optimize both chromatography and ion-
ization at the MS interface.
Two micro liters of a 0.1 mg/ml of considered sample was dis-
solved in acetonitrile/water (9/1) and analyzed according with
the following LC–MS methods.
A solution of 1,2-dichloro-4-[2-(methy-l-4-chlorophenyl ace-
tate)]-5-nitrobenzene (4,6 g, 11.8 mmol), acetic acid (15 ml), pow-
dered iron (1.7 g, 30.4 mmol) in MeOH (60 ml) was mechanically
stirred under nitrogen atmosphere and heated at reflux for 1 h
30. Ice and the mixture was then treated with ice, basified with
10% NaHCO3aq and extracted with ethyl acetate. The organic layer
was dried over Na2SO4, filtered and concentrated in vacuo. Diiso-
propylether was then added, and the mixture was further stirred
for 10 min. The precipitate was collected by filtration and washed
with cold diisopropylether, and the wet solid was dried in vacuo to
give 3 (2.65 g, 100%) as a white solid.
Method A: The HPLC column was an Xterra C-18 (2.1 ꢂ 50 mm,
3.5 lM) The eluents were Milli Q water containing trifluoracetic
acid 0.01% (solvent A) and acetonitrile as organic modifier (solvent
B).The total run time was 20 min at a flow rate of 0.5 mL/min. with
a gradient elution started from 98% of A to 95% of B over 10 min
with a final hold at 95% of B of 5 min and additional 5 min of post
run. Spectra were scanned from 100 to 1500 amu using a variable
ion time according to the number of ions in the source and ioniza-
tion obtained in electrospray in positive mode (ESI+).
Pf: 214–215 °C; 1H NMR (DMSO-d6, 313 K) d 4.88 (bs,1H), 7.10
(s,1H), 7.20 (m, 2H), 7.30 (s,1H), 7.42 (m, 2H), 10.76 (s, H), MS
(ESIꢀ): m/z = 310 [Mꢀ].
5.2.2. 3-Bromo-5,6-dichloro-1,3-dihydro-3-(4-chlorophenyl)-
indole-2-one (4)
Method B: The HPLC run was preformed with a Phenomenex
Compound 3 (2.75 g, 8.8 mmol) was dissolved in CH2Cl2
Gemini C-18 (2.1 ꢂ 100 mm, 5.0
l
M) column. The eluents were
(100 ml) under nitrogen atmosphere. The solution was cooled with
ice bath and a solution of PhMe3NBr3 (3.93 g, 10.5 mmol) in CH2Cl2
(100 ml) was added. The reaction was stirred 3 h at room temper-
ature. The mixture was then washed with HCl 1 M and water. The
organic layer was dried over Na2SO4, filtered and concentrated in
vacuo to give 4 (3.4 g, 100%) as a yellow oil, which was used di-
rectly for the next step reaction without further purification.
1H NMR (DMSO-d6, 313 K) d 7.20 (s, 1H), 7.50 (m, 2H), 7.61 (m,
2H), 7.81 (s, 1H), 11.29 (bs, 1H), MS (ESI+): compound did not ion-
ize under these conditions.
Milli Q water containing 0.005 M ammonium acetate pH 6.5 (sol-
vent A) and acetonitrile as organic modifier (solvent B). The total
run time was 27 min at a flow rate of 0.3 mL/min, with a gradient
elution started from 95% of A to 90% of B over 17 min with a final
hold at 95% of B (5 min), and additional 5 min of post run. Spectra
were scanned from 100 to 1500 amu using a variable ion time
according to the number of ions in the source and ionization ob-
tained in electrospray in negative mode (ESIꢀ).
The retention time (RT) is reported in min at the apex of the
peak as detected at 220 nm by the UV detector.
X-Ray diffraction(XRD) measurements havebeen conductedon a
single crystal sample. Crystals of 5a were grown in a methanol/acet-
one mixture. The molecule crystallized in the tetragonal P41/P43
space group, the asymmetric unit in the crystal was found to be
made up of two molecules of the compound. The structure did not
contain any additional solvate molecule (organic or water). Crystal
data, data collection and refinement parameters for C22H17C13N2O2
are below summarized: MW = 447.73 g/mol, unit cell dimensions;
5.2.3. 5,6-Dichloro-3-[(1S)-2-hydroxy-1-phenylethyl]amino1,3-
dihydro-3-(4-chlorophenyl)-indole-2-one (5a,5b)
3-Bromo-5,6-dichloro-1,3-dihydro-3-(4-chlorophenyl)-indole-
2-one (3.4 g, 8.7 mmol) and (S)-(+)-2-phenylglycinol (2.9 g,
21.1 mmol) were dissolved in dichloromethane (50 ml) under
nitrogen atmosphere. The reaction was stirred 2 h at room temper-
ature. The precipitate was filtered off and the mother liquor was
concentrated in vacuo. The residue was purified by column chro-
matography on silica gel (hexane/EtOAc = 7/3) to give 5b
(Rf = 0.35, 1.7 g, 44%) and 5a (Rf = 0.20, 2.2 g, 56%).
a = 21,636(3) Å, b = 21,636(3) Å, c = 9.0850(18) Å,
90,00°,
= 90,00°, V = 4252.8(12) Å3, Z = 8, calcd = 1.399 mg/m3,
k = 0.71073 Å, T = 133(2) K,
= 0.452 mmꢀ1, F(000) = 1840, hrange
a = 90,00°, b =
c
q
l
=
Compound 5a: pf: 107–109 °C, 1H NMR (DMSO-d6, 313 K) d
3.30–3.47 (m, 3H), 3.56 (m, 1H), 4.85 (t, 1H, J = 5.7 Hz,), 6.99 (s,
1H), 7.08–7.20 (m, 5H), 7.22 (s, 1H), 7.41 (m, 4H), 10.41 (bs, 1H),
LC–MS (ESI+) m/z 447 [M+H]+, RT(min) = 7.2, ½aꢁD: +78° (c 0.1% in
MeOH).
1.33°–25.50°, limiting indices = ꢀ19<h<26, ꢀ26<k<24, ꢀ11<l<9, re-
flections collected = 24177, independent reflections = 7807 [R(int) =
0.1060], refinement method = full-matrix least-squares on F2, data/
restraints/parameters = 7807/1/524, goodness-of-fit on F2 = 0.991,
final R indices [I < 2
(all data) R1 = 0.1270, wR2 = 0.1651, peak/hole = 0.299/ꢀ0.254
eÅꢀ3
r
(I)]; R1 = 0.0558, wR2 = 0.1242, final R indices
Compound 5b: 1H NMR (DMSO-d6, 313 K) d 3.29–3.49 (m, 3H),
3.52–3.63 (m, 1H), 4.94 (m, 1H), 6.48 (s, 1H), 6.88 (s, 1H), 6.99–7.09
(m, 5H), 7.36–7.53 (m, 4H), 10.87 (bs, 1H), MS (ESI+) m/z 447
[M+H]+.
.
5.2. Synthetic procedures and compound characterization
5.2.4. 5,6-Dichloro-3-[(1R)-2-hydroxy-1-phenylethyl]amino1,3-
dihydro-3-(4-chlorophenyl)-indole-2-one (5c,5d)
The title compounds were prepared from 4 by the method de-
scribed in the preparation of 5a,5b pairs.
5.2.1. 5,6-Dichloro-1,3-dihydro-3-(4-chlorophenyl)-indole-2-
one (3)
Sodium hydrate 60% (2.85 g, 71.2 mmol) at ꢀ10 °C and under
nitrogen atmosphere was suspended in N,N-dimethylformamide
(DMF) (45 ml) before being added to a solution of 1,2-dichloro-4-
fluoro-5-nitrobenzene (5 g, 23.8 mmol) and methyl-4-chloro-
phenyl acetate (4.4 g, 23.8 mmol). in DMF (70 ml) The mixture
was stirred at ꢀ5 °C for 2 h and then allowed to warm up to room
temperature. After quenching with ice, an aqueous solution of 10%
NH4Cl was added and the mixture was partitioned between EtOAc
and water. The organic layer was dried over Na2SO4, filtered and
concentrated in vacuo. The residue was purified using column
chromatography on silica gel (hexane/EtOAc = 3/1) to give
Compound 5c: 1H NMR (DMSO-d6, 313 K) d 10.4 (bs, 1H), 7.41
(m, 4H), 7.22 (s, 1H), 7.14 (m, 5H), 6.99 (s, 1H), 4.85 (m, 1H),
3.56 (m, 1H), 3.34 (d, J = 4 Hz, 1H), 3.4 (m, 2H), MS (ESI+) m/z
447 [M+H]+.
Compound 5d: 1H NMR (DMSO-d6, 313 K) d 10.9 (bs, 1H), 7.44
(m, 4H), 7.0 (m, 5H), 6.87 (s, 1H), 6.48 (s, 1H), 4.94 (bs, 1H), 3.57
(bs, 1H), 3.44 (m, 1H), 3.4 (m. 1H), 3.3 (m, 1H), MS (ESI+) m/z
447 [M+H]+. Single crystal X-ray diffraction allowed to assign to
this compound the absolute (R,R) configuration.
The structure was of good quality, the agreement factor value
was R1 = 0.056.

L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
5633
5.2.5. (S)-(+)-3-Amino-5,6-dichloro-1,3-dihydro-3-(4-
chlorophenyl)-indole-2-one ((S)6a)
5.2.9. 4-Chloro-N-(3,4,5-trichlorophenyl)mandelamide (8a)
In a three-necked flask, provided with mechanical stirrer and
under nitrogen atmosphere, 3,4,5-trichloroaniline (4.04 g,
20.6 mmol) were dissolved in toluene (50 ml) and cooled at 0 °C.
K2CO3 (9.6 g, 69.5 mmol) and a mixture of 7 (6.8 g, 27.5 mmol) in
toluene (10 ml) were added and the mixture was stirred for 1 h
at room temperature. MeOH (4.15 ml) was added, and the mixture
was further stirred 2 h at 80 °C. A solution of HCl 1 N was then
added. After extraction with EtOAc, the organic phase was dried
over Na2SO4, filtered and concentrated in vacuo to obtain 8a
(5.7 g, 95%) as a yellow solid, which was used directly for the next
step reaction without further purification.
Compond 5a (1.8 g, 4 mmol) and lead tetraacetate (1.9 g,
4.3 mmol) were dissolved in CH2Cl2 (28 ml) and MeOH (12 ml).
The reaction was stirred 3 h at room temperature before being con-
centrated in vacuo. The residue was then partitioned between
EtOAc and aqueous NaHCO3. The organic phase was dried over
Na2SO4, filtered and evaporated and the residue was dissolved in
MeOH (3.7 ml) and HCl 3 N (36 ml). The mixture was stirred at
room temperature overnight, then concentrated in vacuo. After
partitioning between EtOAc and water, the organic phase was
washed with HCl 1 N. The collected aqueous phase was basified
with ammonium hydroxide and extracted with CH2Cl2. The organic
phase is dried over Na2SO4, filtered and dried in vacuo to obtain 6
(0.54 g, 41%) as a white solid, which was used directly for the next
step reaction without further purification.
1H NMR (DMSO-d6, 313 K) d 5.16 (d, J = 4.3 Hz, 1H), 6.68 (d,
J = 4.3 Hz, 1H), 7.43 (m, 2H), 7.52 (m, 2H), 7.07 (s, 2H), 10.37 (bs,
1H), MS (ESIꢀ): m/z 362 [Mꢀ].
1H NMR (DMSO-d6, 313 K) d 2.78 (s, 2H), 7.10 (s, 1H), 7.35 (s,
1H), 7.38 (m, 4H), 10.67 (bs, 1H), MS (ESI+) m/z 327 [M+H]+, ½aꢁD:
+32.9° (c 0.5% in MeOH).
5.2.10. 4,5,6-Trichloro-1,3-dihydro-3-(4-chlorophenyl)-indol-2-
one (9a)
At 0 °C, 8a (5.7 g, 15.6 mmol) was gradually added to H2SO4 96%
(22 ml) and oleum (5 ml). The reaction was stirred 4 h at room
temperature then iced water was added. The solution was strongly
basified and extracted with CH2Cl2 .The organic phase was dried
over Na2SO4, filtered and concentrated in vacuo. The residue was
crystallized from diethyl ether to obtain 9a (4.2 g, 77%) as a yellow
powder.
5.2.6. (R)-(ꢀ)-3-Amino-5,6-dichloro-1,3-dihydro-3-(4-
chlorophenyl)-indole-2-one ((R)6b)
The title compound was prepared from 5b by the method de-
scribed in the preparation of 6a. The spectral properties of 6b were
identical with those of 6a. ½aꢁD: ꢀ21.9° (c 0.3% in MeOH).
1H NMR (DMSO-d6, 313 K) d 4.95 (s, 1H), 7.12–7.20 (m, 3H),
7.40 (m, 2H), 10.92 (bs, 1H), LC–MS (ESIꢀ): m/z = 344 [Mꢀ],
RT(min) = 13.05.
5.2.7. (+)-N-[5,6-Dichloro-3-(4-chlorophenyl)-2-oxo-2,3-
dihydro-1H-indol-3-yl]-2-(4-méthyl-piperazin-1-yl)-acetamide
(2)
5.2.11. 5-Chloro-6-methyl-1,3-dihydro-3-(4-chlorophenyl)-
indol-2-one (9b)
Compound (S)6a (1.3 g, 4 mmol) and pyridine (0.32 ml, 4 mmol)
were dissolved in toluene (47 ml) under nitrogen atmosphere.
After 5 min stirring, chloroacetylchloride (0.31 ml, 4.56 mmol)
was slowly added, and the reaction was stirred 2 h at 80 °C. The
mixture was quenched with water and extracted with ethyl ace-
tate; the organic layer was dried on Na2SO4, filtered and evapo-
rated under reduced pressure. The residue was purified by
column chromatography (cyclohexane/EtOAc = 8/2) to obtain 2-
chloro-N-[5,6-dichloro-3-(4-chloro-phenyl)-2-oxo-2,3-dihydro-
1H-indol-3-yl]-acetamide (0.4 g, 25%).
1H NMR (DMSO-d6, 313 K): d 2.32 (s, 3H), 4.80 (s, 1H), 6.89 (s,
1H), 7.06(bs, 1H), 7.18 (m, 2H), 7.41 (m, 2H), 10.59 (s, 1H), MS
(ESIꢀ): m/z 290 [Mꢀ].
5.2.12. 4-Chloro-5-methyl-1,3-dihydro-3-(4-chlorophenyl)-
indol-2-one (9c)
1H NMR (DMSO-d6, 313 K) d 2.25 (s, 3H), 4.80 (s, 1H), 6.83 (d,
J = 7.8 Hz, 1H) 7.12 (m, 2H), 7.26 (d, J = 7.8 Hz, 1H), 7.39 (m, 2H),
10.56 (bs, 1H), LC–MS (ESIꢀ): m/z = 290 [Mꢀ], RT(min) = 11.
2-Chloro-N-[5,6-dichloro-3-(4-chloro-phenyl)-2-oxo-2,3-dihy-
dro-1H-indol-3-yl]-acetamide (0.4 g, 1 mmol), N-methylpiperazine
(0.11 ml, 1 mmol), K2CO3 (0.14 g, 1 mmol) and NaI (70 mg,
0.47 mmol) were dissolved DMF (8 ml). The reaction was stirred
3 h at 100 °C, quenched with water and extracted with ethyl ace-
tate. The organic phase was dried over Na2SO4, filtered and concen-
trated in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/MeOH = 8/2) to obtain 2
(40 mg, 80%) as a white solid.
5.2.13. 3-Hydroxy-4,6-dichloro-1,3-dihydro-3-(3,4-
dichlorophenyl)-indol-2-one (11)
A
solution of 4,6-dichloroisatine (7.2 g, 33 mmol) in THF
(120 ml) was dropwise added under nitrogen atmosphere to 3,4-
dichlorophenylmagnesium bromide (200 ml, 0.5 M in THF). The
mixture was stirred 4 h at 70 °C, then NH4Claq was added. After
extraction in EtOAc, the organic phase was washed three times
with NaOH 1 N, then dried over Na2SO4, filtered and concentrated
in vacuo. The residue was purified by column chromatography on
silica gel (cyclohexane/EtOAc = 8/2) to give 11 (6.73 g, 56%) as a
white solid.
Pf: 207.1–207.6 °C, 1H NMR (DMSO-d6, 313 K) d 2.36 (m, 3H),
2.58 (m, 8H), 2.96–3.16 (m, 2H), 7.08 (s, 1H), 7.31 (m, 2H), 7.48
(m, 2H), 7.52 (s, 1H), 8.70 (sb, 1H), 10.70 (bs, 1H), ½aꢁD: +141° (c
0.25% in MeOH), LC–MS (ESI+): m/z = 467 [M+H]+, RT(min) = 8.3.
1H NMR (DMSO-d6, 313 K) d 6.95 (d, J = 1.8 Hz, 1H), 7.03 (s, 1H),
7.10 (dd, J = 8.3 and 2.1 Hz, 1H), 7.14 (d, J = 1.8 Hz, 1H), 7.55–7.60
(m, 2H), 10.86 (bs, 1H), MS (ESI+): m/z 345 [M+H]+–H2O.
5.2.8. 4-Chloro-O-acyl-mandelic-chloride (7)
In a two-necked flask provided with magnetic stirring, 4-chloro-
DL mandelic acid (10 g, 53.6 mmol) and acetyl chloride (4.2 ml,
59 mmol) were dissolved in CH2Cl2 (80 ml). The reaction was stir-
red 3 h at 50 °C. Thionyl chloride (7.8 ml, 107.5 mmol) was drop-
wise added. After 2 h of stirring at reflux the solvent was
evaporated under vacuum to obtain 7 (10.7 g, 100%) as an oil,
which was used directly for the next step reaction without further
purification.
5.2.14. 3,4,6-Trichloro-1,3-dihydro-3-(3,4-dichlorophenyl)-
indol-2-one (12)
Compound 11 (1.2 g, 3.3 mmol) was dissolved in CH2Cl2 (8 ml)
under nitrogen atmosphere. The mixture was cooled at 0 °C and
pyridine (0.47 ml, 5.8 mmol) was added. After 5 min stirring, a
solution of thionyl chloride (0.34 ml, 4.7 mmol) in CH2Cl2 (4 ml)
was slowly added. The reaction was further stirred 1 h at rt. A sat-
urated solution of ammonium chloride was then added. The organ-
ic phase was dried over Na2SO4, filtered and concentrated in vacuo
1H NMR (CDCl3, 300 K) d 2.25 (s, 3H), 6.08 (s, 1H), 7.43–7.48 (m,
4H), MS (ESIꢀ): compound did not ionized under these conditions.

5634
L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
to obtain 12 (1.2 g, 94%) as a yellow oil, which was used directly for
the next step reaction without further purification.
9.99–10.65 (bs, NH+, 1H), 10.86 (bs, 1H) LC–MS (ESI+): m/z = 514
[M+H]+, RT(min) = 8.1.
1H NMR (DMSO-d6, 313 K): 7.08 (d, J = 1.7 Hz, 1H), 7.31–7.35
(m, 2H), 7.63 (d, J = 2.3 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 11.47 (bs,
1H), MS (ESI+): compound did not ionize under these conditions.
5.2.19. N-[6-Chloro-5-methyl-3-(3,4-dichlorophenyl)-2-oxo-
2,3-dihydro-1H-indol-3-yl]-2-(4-methyl-piperazin-1-yl)-
acetamide (2g)
White solid, Pf: 229.4–232.6 °C, 1H NMR (DMSO-d6, 313 K) d
2.16 (s, 3H), 2.30 (s, 3H), 2.31–2.39 (m, 4H), 2.41–2.55 (m,
4H+dmso), 2.91 (d, J = 15 Hz,1H), 3.03 (d, J = 15 Hz,1H) 6.91 (s,
1H), 7.09 (dd, J1 = 8.5 Hz, J2 = 2.3 Hz, 1H), 7.26 (s, 1H), 7.54 (d,
J = 2.3 Hz, 1H), 7.64 (d, J = 8.5 Hz, 1H), 8.68 (s, 1H), 10.52 (bs, 1H),
LC–MS (ESI+): m/z = 481 [M+H]+, RT(min) = 8.7.
5.2.15. (1-Ethyl-piperidin-4-ylidene)-acetonitrile (15)
Diethyl(cyanomethyl)phosphonate (8.89 ml, 54.9 mmol) and
K2CO3 (9.36 g, 67.7 mmol) were dissolved under nitrogen atmo-
sphere in THF (12 ml). The reaction was stirred 15 minute at room
temperature and 20 min at reflux, then was cooled to room tem-
perature. 1-Ethyl-4-piperidone (6.5 ml, 55.9 mmol) was added
and the mixture was stirred 16 h at reflux. The mixture was parti-
tioned between EtOAc and water. The organic phase was dried over
Na2SO4, filtered and concentrated in vacuo to obtain 15 (6.8 g, 82%)
as a colorless oil, which was used directly for the next step reaction
without further purification.
5.2.20. N-[6-Chloro-5-methyl-3-(4-chlorophenyl)-2-oxo-2,3-
dihydro-1H-indol-3-yl]-2-piperazinyl-acetamide (2j)
White solid, Pf: 264–265 °C, 1H NMR (DMSO-d6, 313 K) d 2.30 (s,
3H), 3.00–4.10 (m, 8H+H2O), 6.93 (s, 1H), 7.24 (s, 1H), 7.33 (m, 2H),
7.47 (m, 2H), 9.43 (bs, 1H), 9.53 (bs, 1H), 10.59 (s, 1H), LC–MS
(ESI+): m/z = 433 [M+H]+, RT(min) = 8.9.
1H NMR (DMSO-d6, 313 K) d 1.01 (t, J = 7.2 Hz, 3H), 2.31–2.50
(m, 10H + dmso), 5.45 (m, 1H), MS (ESI+) m/z 151 [M+H]+.
5.2.16. (1-Ethyl-piperidin-4-yl)-acetonitrile (16)
5.2.21. N-[6-Chloro-5-methyl-3-(4-chlorophenyl)-2-oxo-2,3-
dihydro-1H-indol-3-yl]-2-(4-benzyl-piperazin-1-yl-acetamide
(2k)
Compound 15 (1 g, 6.7 mmol) was dissolved in MeOH (70 ml)
and cooled at 0 °C. Magnesium (7.2 g, 296 mmol) was then added
and the mixture was stirred for 4 h before being filtrate. The filtrate
phase was concentrated in vacuo and partitioned between CH2Cl2
and brine. The organic layer was dried over Na2SO4, filtered and
concentrated in vacuo to obtain 16 (0.45 g, 44%) as a colorless oil,
which was used directly for the next step reaction without further
purification.
White solid, Pf: 218.7–220.2 °C, 1H NMR (DMSO-d6, 313 K) d
2.28 (s, 3H), 2.36–2.45 (m, 4H), 2.45–2.55 (m, 4H+dmso), 2.93 (d,
J = 15 Hz, 1H), 3.02 (d, J = 15 Hz, 1H), 3.47 (m, 2H), 6.88 (s, 1H),
7.20–7.36 (m, 8H), 7.46 (m, 2H), 8.54 (s, 1H), 10.43 (bs, 1H), LC–
MS (ESI+): m/z = 523 [M+H]+, RT(min) = 9.2.
1H NMR (DMSO-d6, 313 K) d 0.98 (t, J = 7.3 Hz, 3H), 2.26 (m, 2H),
1.57 (m, 1H), 1.68 (m, 2H), 1.84 (m, 2H), 2.30 (q, J = 7.3 Hz, 2H),
2.46 (d, J = 6.4 Hz, 2H), 2.85 (m, 2H), MS (ESI+) m/z 153 [M+H]+.
5.2.22. N-[6-Chloro-5-methyl-3-(4-chlorophenyl)-2-oxo-2,3-
dihydro-1H-indol-3-yl]-2-(4-(4-pyridyl)-piperazin-1-yl-
acetamide (2l)
White solid, Pf: 314–316 °C, 1H NMR (DMSO-d6, 313 K) d 2.30 (s,
3H), 2.55–2.66 (m, 4H), 3.02 (d, J = 15 Hz, 1H), 3.12 (d, J = 15 Hz,
1H), 3.32–3.40 (m, 4H), 6.82 (m, 2H), 6.89 (s, 1H), 7.23 (s, 1H),
7.30 (m, 2H) 7.46 (m, 2H) 8.16 (m, 2H), 8.70 (s, 1H), 10.44 (s,
1H), LC–MS (ESI+): m/z = 510 [M+H]+, RT(min) = 12.4.
5.2.17. (1-Ethyl-piperidin-4-yl) acetic acid (17)
Compound 16 (3.65 g, 24.3 mmol) was dissolved in HClconc
(47 ml) and stirred at reflux for 20 h. The mixture was partitioned
between water CH2Cl2. The pH was adjusted to 6 with an aqueous
solution of NH4Cl. The aqueous phase was washed with CH2Cl2 and
concentrated in vacuo. The residue was dissolved in ethanol and
filtrated. The filtrate, concentrated in vacuo, gave 17 (3.6 g, 87%)
as a yellow solid.
5.2.23. N-[5,6-Dichloro-3-(4-chlorophenyl)-2-oxo-2,3-dihydro-
1H-indol-3-yl]-2-N-methyl-(2-N-dimethyl)-ethyl-acetamide
(2n)
1H NMR (DMSO-d6, 313 K) d 1.06 (t, J = 7.1 Hz, 3H), 1.29 (m, 2H),
1.71 (m, 2H), 2.10–2.25 (m, 3H), 2.55 (m, 2H+DMSO), 3.00 (m, 2H),
MS (ESI+) m/z 172 [M+H]+.
White solid, Pf: 204.7–205.4 °C, 1H NMR (DMSO-d6, 313 K) d
2.01 (s, 6H), 2.26–2.34 (m, 5H), 2.46–2.53 (m, 2H + DMSO), 2.99
(d, J = 16 Hz, 1H), 3.06 (d, J = 16 Hz, 1H), 7.07 (s, 1H), 7.33 (m,
2H), 7.45–7.51 (m, 3H), 9.10 (s, 1H), 10.72 (bs, 1H), LC–MS
(ESI+): m/z = 469 [M+H]+, RT(min) = 8.3.
5.2.18. (+)N-[4,6-Dichloro-3-(3,4-dichlorophenyl)-2-oxo-2,3-
dihydro-1H-indol-3-yl]-2-(1-ethyl-piperidin-4-yl)-acetamide
(18)
5.2.24. N-[4-Chloro-5-methyl-3-(4-chlorophenyl)-2-oxo-2,3-
dihydro-1H-indol-3-yl]-2-(1-methyl-piperazin-4-yl)-acetamide
(10c)
In a two-necked flask under nitrogen atmosphere, PCl5 (0.2 g,
9.6 mmol) was suspended at 0 °C in CH2Cl2 (9 ml), then compound
17 (0.16 g, 9.6 mmol) was added. The suspension was stirred
10 min at 0 °C and 2 h at room temperature (Solution 1). Under
nitrogen atmosphere, at 0 °C, compound 13 (0.25 g, 0.7 mmol)
and pyridine (0.6 ml, 7.4 mmol) were dissolved in CH2Cl2 (9 ml)
and stirred 10 min (Solution 2). Solution 1 was then added to Solu-
tion 2 at 0 °C and the mixture was stirred 10 min at 0 °C and 3 h at
room temperature. The mixture was partitioned between is aque-
ous NaHCO3 and CH2Cl2, and the organic phase was dried over
Na2SO4, filtered and concentrated in vacuo. The residue was puri-
fied by column chromatography on silica gel (EtOAc/MeOH = 8/2
to obtain 18 (0.11 g, 28%) as a beige powder.
White solid, Pf: 282.2–284.6 °C, 1H NMR (DMSO-d6, 313 K) d
2.15 (s, 3H), 2.28 (s, 3H), 3.33 (m, 4H), 2.45–2.59 (m, 4H+DMSO),
2.98 (d, J = 15 Hz, 1H), 3.06 (d, J = 15 Hz, 1H), 6.80 (d, J = 7.8 Hz,
1H), 7.22 (m, 2H), 7.29 (d, J = 7.8 Hz, 1H), 7.49 (m, 2H), 8.53 (s,
1H), 10.48 (s, 1H), LC–MS (ESI+): m/z = 447 [M+H]+, RT(min) = 7.8.
5.2.25. (+)N-[6-Chloro-3-(4-chlorophenyl)-2-oxo-2,3-dihydro-
1H-indol-3-yl]-2-(1-methyl-piperazin-4-yl)-acetamide (14a)
White solid, Pf: 159–160 °C, 1H NMR (DMSO-d6, 313 K) d 2.16 (s,
3H), 2.33 (m, 4H), 2.47 (m, 4H), 2.92 (d, J = 15 Hz, 1H), 3.01(d,
J = 15 Hz, 1H), 6.89 (d, J = 2 Hz,1H), 7.08 (dd, J1 = 8 Hz, J2 = 2 Hz,
1H), 7.23–7.31 (m, 3H), 7.47 (m, 2H), 8.60 (s, 1H), 10.56 (bs, 1H),
LC–MS (ESI+): m/z = 433 [M+H]+, RT(min) = 5.7, ½aꢁD: +134° (c
0.25% in MeOH).
Pf = 211–213 °C, 1H NMR (DMSO-d6, 313 K) d 1.17 (m, 3H),
1.36–1.56 (m, 2H), 1.61–1.94 (m, 3H), 2.22 (m, 2H), 2.70–3.03
(m, 4H), 3.11–3.48 (m, 2H + DHO), 6.93 (s, 1H), 7.04 (m, 1H), 7.19
(s, 1H), 7.59 (bs, 1H), 7.65 (d, J = 8.6 Hz, 1H), 9.25 (bs, 1H),

L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
5635
5.2.26. (+)N-[4,6-Dichloro-3-(3,4-dichlorophenyl)-2-oxo-2,3-
dihydro-1H-indol-3-yl]-2-(1-methyl-piperazin-4-yl)-acetamide
(14e)
gene to human ghrelin (Tocris). Cell lines were therefore subcloned
by limited dilution, and the subclone CHO-CREluc-hGHSR1a was
selected for functional assays.
White solid, Pf: 165–166 °C, 1H NMR (DMSO-d6, 313 K) d 2.13 (s,
3H), 2.30 (m, 4H), 2.40–2.56 (m, 4H+DMSO), 3.04 (m, 2H), 6.92 (s,
1H), 7.05 (d, J = 8.6 Hz, 1H), 7.20 (s, 1H), 7.54 (s, 1H), 7.66 (d,
J = 8.6 Hz, 1H), 8.86 (s, 1H) 10.86 (s, 1H), LC–MS (ESI+): m/z = 501
[M+H]+, RT(min) = 5.2, ½aꢁD: +222° (c 0.23% in MeOH).
5.3.2. Cell stimulation and luciferase assay
Cells were plated onto white 96-well microplates in complete
medium (mimimal essential medium supplemented with 10% v/v
dialysed foetal bovine serum, 0.5 mM sodium pyruvate 0,01 mg/
ml gentamicin, 0,3 mg/ml geneticin, 0,04 mg/ml L-Proline, all prod-
5.2.27. (+)N-[4,6-Dichloro-3-(3,4-dichlorophenyl)-2-oxo-2,3-
dihydro-1H-indol-3-yl]-2-(4-N-dimethylamino-piperidin-1-yl)-
acetamide (14i)
ucts from Gibco) and incubated for 24 h at 37 °C/5%CO2. The next
day, cells were stimulated for 4 h in the presence or absence of var-
ious concentrations of compounds in complete medium supple-
White solid, Pf: 128–130 °C, 1H NMR (DMSO-d6, 313 K) d 1.37
(m, 2H), 1.70 (m, 2H), 2.00 (m, 1H), 2.05–2.13 (m, 2H), 2.15 (s,
6H) 2.81 (m, 1H), 2.91 (m, 1H), 3.02 (m, 2H), 6.92 (d, 1H), 7.06
(dd, J1 = 8.5 Hz, J2 = 2.3 Hz, 1H) 7.20 (d, J = 1.7 Hz, 1H), 7.55 (d,
J = 2.3 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 8.81 (s. 1H), 10.88 (bs, 1H),
LC–MS (ESI+): m/z = 529 [M+H]+, RT(min) = 5.4, ½aꢁD: +193° (c
0.12% in MeOH).
mented with 0.1 lM forskolin (Sigma) and 3 nM human ghrelin
(Tocris). Luciferase activities were determined using the Luclite™
assay system (Perkin Elmer) and quantification of light emission
was obtained by photon counting in a TopCount™ Microplate Scin-
tillation and Luminescence Counter (Perkin Elmer).
5.3.3. Radioligand binding assay
Radioligand binding assay was performed on crude cell mem-
brane extracts obtained from CHO–CREluc-hGHSR1a as previously
described.36 Cells grown to confluence were collected and spun at
200g for 10 min at 4 °C. Crude membranes were prepared by
homogenization of cells in TM buffer (10 mM Tris–HCl pH 7.4,
10 mM MgCl2), centrifugation at 1000g for 5 min, and further cen-
trifugation of the supernatants at 40,000g for 40 min at 4 °C. Pellets
were resuspended in TM buffer supplemented with a protease
inhibition cocktail (4-(2-aminoethyl) benzenesulfonyl fluoride
5.2.28. (+)N-[4,6-Dichloro-3-(3,4-dichlorophenyl)-2-oxo-2,3-
dihydro-1H-indol-3-yl]-2-(R)-(3-N-dimethylamino-pirrolidin-
1-yl)-acetamide (14j)
White solid, Pf: 108–113 °C, 1H NMR (DMSO-d6, 313 K) d 1.52–
1.64 (m, 1H), 1.76–1.87 (m, 1H), 2.08 (s, 6H), 2.41 (m, 1H), 2.53–
2.76 (m, 4H), 3.15 (m, 2H), 6.92 (d, J = 1.7 Hz, 1H), 7.06 (dd,
J1 = 8.5 Hz, J2 = 2.2 Hz,1H), 7.21 (d, J = 1.7 Hz, 1H), 7.57 (d,
J = 2.2 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 8.89 (s, 1H), 10.84 (bs, 1H),
LC–MS (ESI+): m/z = 515 [M+H]+, RT(min) = 4.8, ½aꢁD: +230° (c
0.13% in MeOH).
(AEBSF) 2
lM f.c., E-64 14 nM f.c., bestatin 130 nM f.c., leupeptin
1 nM f.c., aprotinin 0.3 nM f.c., and sodium EDTA 1
lM f.c., Sigma),
and stored at ꢀ80 °C until use. To determine the effect of the se-
5.2.29. (+)N-[4,6-Dichloro-3-(3,4-dichlorophenyl)-2-oxo-2,3-
dihydro-1H-indol-3-yl]-2-(S)-(3-N-dimethylamino-pirrolidin-1-
yl)-acetamide (14k)
lected compounds on displacement of [125I] human ghrelin, mem-
branes (2 lg of protein) were incubated at room temperature for
White solid, Pf: 113–115 °C, 1H NMR (DMSO-d6, 313 K) d 1.53–
1.63 (m, 1H), 1.77–1.88 (m, 1H), 2.07 (s, 6H), 2.42 (m, 1H), 2.53–
2.59 (m, 1H+DMSO), 2.62–2.76 (m, 3H), 3.11 (d, J = 1.5 Hz, 1H),
3.18 (d, J = 1.5 Hz, 1H), 6.92 (d, J = 1.7 Hz, 1H), 7.06 (dd,
J1 = 8.5 Hz, J2 = 2.3 Hz, 1H), 7.20 (d, J = 1.7 Hz, 1H), 7.55 (d,
J = 2.3 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 8.89 (s, 1H), 10.85 (bs, 1H),
LC–MS (ESI+): m/z = 515 [M+H]+, RT(min) = 4.8, ½aꢁD: +205° (c
0.11% in MeOH)
1 h in TM buffer supplemented with 0.25% BSA w/v and 10% v/v
dialysed foetal bovine serum added of 0.25 nM [125I] human ghre-
lin (Perkin Elmer), in the presence or absence of increasing concen-
trations of selected compounds. A rapid filtration technique using
Whatman GF/B filters (pretreated with 0.5% (w/v) polyethyleneim-
ine) and a 96-well filtration apparatus (Unifilter-96 Harvester, Per-
kin Elmer) was used to harvest and rinse labeled membranes with
cold TM buffer. Following a 900/60 °C incubation, filter-bound
radioactivity was counted upon addition of 50 ll/well Microscint™
20 liquid scintillation cocktail (Perkin Elmer) using a TopCount™
Microplate Scintillation and Luminescence Counter (Perkin Elmer).
5.2.30. (+)N-[4,6-Dichloro-3-(3,4-dichlorophenyl)-2-oxo-2,3-
dihydro-1H-indol-3-yl]-2-(4-ethyl-piperazin-3-one)-acetamide
(14l)
Nonspecific binding was determined in the presence of 1
man ghrelin (Tocris).
lM hu-
White solid, Pf: 126–130 °C, 1H NMR (DMSO-d6, 313 K) d 1.01 (t,
J = 7.2 Hz, 3H), 1.18 (m, 2H), 2.74 (m, 2H), 3.08 (m, 2H), 3.19 (m,
2H), 3.29 (m, 2H + DMSO), 6.92 (d, J = 1.7 Hz, 1H), 7.05 (dd,
J1 = 8.5 Hz, J2 = 2.3 Hz, 1H), 7.22 (d, J = 1.7 Hz, 1H), 7.58 (d,
J = 2.3 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 9.16 (s, 1H), 10.86 (s, 1H),
LC–MS (ESI+): m/z = 531 [M+H]+, RT(min) = 6.3, ½aꢁD: +191° (c
0.11% in MeOH)
5.4. In vivo assays
5.4.1. Gastric emptying
To assess gastric emptying, a bolus (1 ml/100 g body weight) of
a standard meal (BaSO4, Prontobario Esofago, Bracco, Italia; 75%
W/V with 0.5% Na–carboxymethylcellulose (CMC) was given by ga-
vage and the rats were euthanized by cervical dislocation immedi-
ately (t = 0) or 60 min after the meal.21 The pylorus and the
esophageal junction were clamped before excision of the stomach.
The gastric content was assessed by weighing the stomachs before
and after removing their contents and then cutting, rinsing and
blotting them on dry tissues. At each time, the stomach content
remaining after drug treatment was compared to that of control
drug-free rats, euthanized at t = 0. The percentage inhibition of gas-
tric emptying was calculated for each rat: % gastric empty-
ing = (content of treated rats/mean content of control rats)ꢀ100.
5.3. In vitro assays
5.3.1. Stable CHO double transformants
Stable CHO double transformants were obtained according to
the methods previously described.35 Briefly, human GHSR1a
(NM_198407.1) was cloned into a CHO-derived cell line stably
transfected with a CRE-Luc reporter gene cassette (six consensus
cAMP responsive elements (CRE) linked upstream to the firefly
luciferase sequence). Cells were screened for hGHSR1a membrane
expression and dose-dependent responsiveness of the reporter

5636
L. Puleo et al. / Bioorg. Med. Chem. 20 (2012) 5623–5636
7. Smith, R. G.; Jiang, H.; Sun, Y. Trends Endocrinol. Metab. 2005, 16, 436.
5.4.2. Mouse fasted refed model
8. 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.; Anderson, J.; Paress,
P. S.; Diaz, C.; Chou, M.; Liu, K. K.; McKee, K. K.; Pong, S. S.; Chaung, L. Y.;
Elbrecht, A.; Dashkevicz, M.; Heavens, R.; Rigby, M.; Sirinathsinghji, D. J.; 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. Science 1996, 273,
974.
Male C57BL/6 mice were fasted overnight and dosed the follow-
ing day, 1 h before refeeding. The cumulative food intake was re-
corded for a period of 4 h after food return.
5.4.3. Food intake stimulated by central administration of
ghrelin
9. Bowers, C. Y.; Chang, J.; Momany, F.; Folkers, K. Mol. Endocrinol. Proc.; Elsevier:
Amsterdam, 1977; pp 287–292.
For icv injection of solutions, adult male CD rats, weighing 200–
300 g were implanted with stainless steel cannulae in the lateral
ventricle. Animals were anesthetized (sodium pentobarbital,
60 mg/kg body weight, ip injection) and then placed in a stereo-
taxic frame. A stainless steel guide cannula (550 lm outer diameter,
10 mm length) was implanted stereotaxically at the following
coordinates: 0.8 mm posterior to the bregma, 1.4 mm lateral to
midline, and 2.0 mm below the surface of the left cortex such that
a tip of the cannula was 1.0 mm above the left cerebral ventricle.
Two stainless steel anchoring screws were fixed to the skull, and
the cannula was secured in place by acrylic dental cement. The ani-
mals were then returned to their cages and allowed to recover for
at least 7 days. During the recovery period, the animals were han-
dled daily.
10. Rosen, T.; Johannsson, G.; Johansson, J. O.; Bengtsson, B. A. Horm. Res. 1995, 43,
93.
11. Nargund, R. P.; Patchett, A. A.; Bach, M. A.; Murphy, M. G.; Smith, R. G. J. Med.
Chem. 1998, 41, 3103.
12. Barazzoni, R.; Bosutti, A.; Stebel, M.; Cattin, M. R.; Roder, E.; Visintin, L.; Cattin,
L.; Biolo, G.; Zanetti, M.; Guarnieri, G. Am. J. Physiol. Endocrinol. Metab. 2005,
288, E228.
13. Holst, B.; Cygankiewicz, A.; Jensen, T. H.; Ankersen, M.; Schwartz, T. W. Mol.
Endocrinol. 2003, 17, 2201.
14. Wren, A. M.; Seal, L. J.; Cohen, M. A.; Brynes, A. E.; Frost, G. S.; Murphy, K. G.;
Dhillo, W. S.; Ghatei, M. A.; Bloom, S. R. J. Clin. Endocrinol. Metab. 2001, 86, 5992.
15. Nakazato, M.; Murakami, N.; Date, Y.; Kojima, M.; Matsuo, H.; Kangawa, K.;
Matsukura, S. Nature 2001, 409, 194.
16. Wierup, N.; Yang, S.; McEvilly, R. J.; Mulder, H.; Sundler, F. J. Histochem.
Cytochem. 2004, 52, 301.
17. Dezaki, K.; Hosoda, H.; Kakei, M.; Hashiguchi, S.; Watanabe, M.; Kangawa, K.;
Yada, T. Diabetes 2004, 53, 3142.
18. Egido, E. M.; Rodriguez-Gallardo, J.; Silvestre, R. A.; Marco, J. Eur. J. Endocrinol.
2002, 146, 241.
19. 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.; Turner, T. M.; Sham, H. L.; Liu, G. J.
Med. Chem. 2006, 49, 4459.
20. 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.
21. Rudolph, J.; Esler, W. P.; O’connor, S.; Coish, P. D.; 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, A. A.; Ramsden, P. D.; Schoenleber, R. W.; Shelekhin, T. E.;
Vakalopoulos, A.; Tang, W.; Wang, L.; Yi, L.; Gardell, S. J.; Livingston, J. N.;
Sweet, L. J.; Bullock, W. H. J. Med. Chem. 2007, 50, 5202.
22. Pasternak, A.; Goble, S. D.; deJesus, R. K.; Hreniuk, D. L.; Chung, C. C.; Tota, M.
R.; Mazur, P.; Feighner, S. D.; Howard, A. D.; Mills, S. G.; Yang, L. Bioorg. Med.
Chem. Lett. 2009, 19, 6237.
23. 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.
24. Schoentjes, B.; Serradeil-Le Gal, C.; Wagnon, J. (Sanofi-Synthelabo, Fr).
Preparation of 1,3-dihydro-2H-indol-2-ones with selective binding affinity
for the V1b arginine-vasopressin receptor for pharmaceutical use, [WO
2001055134], 2011.
25. Baroni, M.; Puleo, L. (Sanofi-Aventis, Fr). 3-Disubstituted indol-2-one
derivatives as ghrelin receptor antagonists, [WO/2009/056707], 2009.
26. Tokunaga, T.; Hume, W. E.; Umezome, T.; Okazaki, K.; Ueki, Y.; Kumagai, K.;
Hourai, S.; Nagamine, J.; Seki, H.; Taiji, M.; Noguchi, H.; Nagata, R. J. Med. Chem.
2001, 44, 4641.
For icv injection of ghrelin, or vehicle (sterile 0.9% saline), a
stainless steel injector was introduced through the cannula at a
depth of 1.0 mm beyond the end of the guide. The total volume
of injected solution of ghrelin (10
lg/rat), or saline into the lateral
ventricle was 15 l. Rat ghrelin was purchased from Tocris.
l
Thirty minutes before icv injection of ghrelin or vehicle, the
compound 14f was dosed at 10 mg/kg ip and animals were put into
the cages. The cumulative food intake was measured for 4 h after
icv injection of ghrelin or vehicle. The number of rats was 6–7 in
each group.
5.4.4. Glucose tolerance test
Male C57/Bl6N were fasted overnight (16–18 h) and then given
ghrelin antagonist at 30 mg/kg or vehicle by oral gavage. Thirty
minutes after dosing, the fasting blood glucose level was measured
from tail-tip blood using a Glucometer (Roche), and after 30 min
the animals were given 2 g/kg of glucose by intraperitoneal injec-
tion (IPGTT). Blood glucose was measured again after 20, 40, 60,
80, 100, 120 min. The area under the glucose curve (AUC) from 0
to 80 min, was calculated using the trapezoidal method, and the ef-
fect of the compound on the AUC was expressed as a percentage of
the AUC for the vehicle-treated group.
Acknowledgment
27. Contreras, J.; Rival, Y. M.; Chayed, S.; Bourguignon, J. J.; Wermuth, C. J. J. Med.
Chem. 1999, 42, 730.
28. Ohno, T.; Mochiki, E.; Kuwano, H. Int. J. Pept. 2010, 2010.
29. Ogiso, K.; Asakawa, A.; Amitani, H.; Inui, A. J. Gastroenterol. Hepatol. 2011,
26(Suppl 3), 67.
We thank Valentina Ghidelli, Elena Sabbadin, Donatella Picci,
Giordano Lesma, Alessandra Silvani, Antonella Larovere, Raffaella
Panigada, Massimo Meleri, Elisabetta Ottolina, Giovanni Boccardi,
Andrea Bertario, Fausto Gesualdi, Silvia Milanesi, Philippe Ochsen-
bein, Marc Bianciotto, Laurence Fajas and Sabina Improta for their
contribution to this work.
30. Levin, F.; Edholm, T.; Ehrstrom, M.; Wallin, B.; Schmidt, P. T.; Kirchgessner, A.
M.; Hilsted, L. M.; Hellstrom, P. M.; Naslund, E. Regul. Pept. 2005, 131, 59.
31. De Smet, B.; Mitselos, A.; Depoortere, I. Pharma. Ther. 2009, 123, 207–223.
32. Esler, W. P.; Rudolph, J.; Claus, T. H.; Tang, W.; Barucci, N.; Brown, S. E.; Bullock,
W.; Daly, M.; Decarr, L.; Li, Y.; Milardo, L.; Molstad, D.; Zhu, J.; Gardell, S. J.;
Livingston, J. N.; Sweet, L. J. Endocrinology 2007, 148, 5175–5185.
33. Baroni, M.; Puleo, L. (Sanofi-Aventis, Fr). Substituted 3-Benzofuranyl-indol-2-
one-3-acetamidopiperazine Derivatives, Preparation thereof, and therapeutic
use thereof, [WO/2010/092288], 2010.
References and notes
1. Kojima, M.; Kangawa, K. Physiol. Rev. 2005, 85, 495.
2. van der Lely, A. J.; Tschop, M.; Heiman, M. L.; Ghigo, E. Endocrinol. Rev. 2004, 25,
426.
34. Baroni, M.; Puleo, L. (Sanofi-Aventis, Fr). 3-Benzofuranyl-indol-2-one
derivatives substituted at the 3 position, preparation thereof, and therapeutic
use thereof, [WO/2010/092289], 2010.
3. Korbonits, M.; Grossman, A. B. Eur. J. Endocrinol. 2004, 151, S67.
4. Kojima, M.; Hosoda, H.; Date, Y.; Nakazato, M.; Matsuo, H.; Kangawa, K. Nature
1999, 402, 656.
35. Portier, M.; Rinaldi-Carmona, M.; Pecceu, F.; Combes, T.; Poinot-Chazel, C.;
Calandra, B.; Barth, F.; Le, F. G.; Casellas, P. J. Pharmacol. Exp. Ther. 1999, 288,
582.
5. Banks, W. A.; Tschop, M.; Robinson, S. M.; Heiman, M. L. J. Pharmacol. Exp. Ther.
2002, 302, 822.
6. Thomsen, W. J.; Behan, D. P. Comprehensive Medicinal Chemistry II; Elsevier Ltd:
Oxford, UK, 2006; pp 771–826.
36. Bouaboula, M.; Perrachon, S.; Milligan, L.; Canat, X.; Rinaldi-Carmona, M.;
Portier, M.; Barth, F.; Calandra, B.; Pecceu, F.; Lupker, J.; Maffrand, J. P.; Le, F. G.;
Casellas, P. Biol. Chem. 1997, 272, 22330.