Novel isoxazole carboxamides as growth hormone secretagogue receptor (GHS-R) antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.06.060

Novel isoxazole carboxamides have been identified as growth hormone secretagogue receptor (GHS-R) antagonists. Substituent modification off the 5-position of the isoxazole ring led to analogues with potent binding affinity and functional antagonism of GHS-R. A potent analogue (32) with high aqueous solubility and good GPCR selectivity was also identified as a potential pharmacological tool for in vivo studies.

Full text of DOI:10.1016/j.bmcl.2004.06.060

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5223–5226
Novel isoxazole carboxamides as growth hormone secretagogue
receptor (GHS-R) antagonists
Bo Liu, Gang Liu,* Zhili Xin, Micheal D. Serby, Hongyu Zhao, Verlyn G. Schaefer,
H. Douglas Falls, Wiweka Kaszubska, Christine A. Collins and Hing L. Sham
Metabolic Disease Research, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, IL 60064-6098, USA
Received 10 May 2004; revised 17 June 2004; accepted 18 June 2004
Available online 24 August 2004
Abstract—Novel isoxazole carboxamides have been identified as growth hormone secretagogue receptor (GHS-R) antagonists. Sub-
stituent modification off the 5-position of the isoxazole ring led to analogues with potent binding affinity and functional antagonism
of GHS-R. A potent analogue (32) with high aqueous solubility and good GPCR selectivity was also identified as a potential phar-
macological tool for in vivo studies.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Ghrelin, a 28 amino acid peptide with an unique n-octa-
noyl modification on Ser 3, was recently identified as an
endogenous ligand for growth hormone secretagogue
weight over a five-day period.9 A recent study using a
peptidic GHS-R antagonist, [D-Lys-3]-GHRP-6, showed
a reduction of food intake and body weight gain in diet
induced obese mice.10 Based on the above observations
in rodents and humans, administration of a selective
small molecule GHS-R antagonist can potentially lead
to reduced food intake, decreased adiposity, and body
weight reduction in humans.
receptor (GHS-R),
a G-protein coupled receptor
(GPCR).1 Ghrelin is produced mainly in the upper intes-
tinal tract as well as in the hypothalamus.2 Besides being
a potent growth hormone secretagogue and regulator of
other endocrine functions, ghrelin is also implicated in
the short- and long-term regulation of energy balance.3
Its administration in near physiological doses in
rodents4 and humans5 stimulates food intake. Chronic
administration of ghrelin in freely feeding mice and rats
results in increased body weight and decreased fat
utilization.6
Only one small molecule GHS-R antagonist has been
reported in the literature. Compound 1 is a 3-amino-
2,3,4,5-tetrahydro-benzo[b]azepin-2-one derivative used
in the preparation of a nonpeptidyl GHS.11 We recently
identified a novel isoxazole carboxamide derivative (2)
from high throughput screening. It was confirmed as a
pure, competitive GHS-R antagonist with an IC50 of
180nM in a calcium flux cellular assay. Extensive medi-
cinal chemistry efforts were initiated to explore the struc-
ture–activity relationship (SAR) of this series of
compounds. In this paper we present the synthesis and
biological profiles of analogues through substituent
modification off the 5-position of the isoxazole core,
which led to compounds with potent functional antago-
nism of GHS-R and good GPCR selectivity (Fig. 1).
Consistent with ghrelinÕs involvement in long-term regu-
lation of body weight, accumulating data suggest that
ghrelin antagonism may lead to desired effects in an
anti-obesity therapeutic regimen. It has been shown that
single intracerebroventricular (icv) or intraperitoneal
(ip) administration of anti-ghrelin IgG suppresses acute
feeding in lean rats.7,8 In addition, chronic twice-daily
icv administration of anti-ghrelin IgG reduces body
Keywords: Growth hormone secretagogue receptor; Ghrelin; Anta-
gonist.
The isoxazole carboxamide derivatives were prepared in
a straightforward fashion as outlined in Scheme 1. Var-
ious b-ketoesters 3 were prepared through reacting the
imidazolide of the corresponding carboxylic acids with
magnesium monoethylmalonate followed by spontane-
ous decarboxylation.12 The [2+3] cyclization between
*
Corresponding author. Tel.: +1-847-935-1224; fax: +1-847-938-
1674; e-mail: gang.liu@abbott.com
Present address: Serono International S.A., Corporate Global Pro-
duct Development, chemin des Mines 15bis, 1211 Geneva 20,
Switzerland.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.060

5224
B. Liu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5223–5226
logues of GHS-Rs were also cloned and stable cell
lines were generated for use in species selectivity assays
on selected compounds.
N
HN
O
N
O
The original screening lead 2 showed submicromolar
activity in both binding and FLIPR assays. Initial exten-
sion of the 5-methyl group of the isoxazole ring revealed
interesting SAR and prompted us to further explore this
observation. Elongation of the methyl group of 2 to
ethyl (9), propyl (10), and isopropyl (11) groups seemed
to have little impact on the binding affinity of the result-
ant analogues, but such changes appeared to cause a
decrease in FLIPR activity (Table 1). Compound 12
seemed to suggest that more hydrophilic groups, such
as a hydroxyl group, would be tolerated off the extended
alkyl groups. Additionally, methoxyethyl group-derived
13 appeared to be equipotent to 2 in terms of both bind-
ing affinity and FLIPR activity. Therefore, we decided
to explore analogues featuring a variety of hydrogen
bond acceptor-containing cyclic structures linked
through an ethylene group to the isoxazole core.
Cl
O
N
N
H
O
Cl
O
1
2
Figure 1. GHS-R antagonists from both literature and internal
screening.
O
R1
O
Cl
N
O
O
a
b
CO2H
R1
R1
O
O
Cl
3
4
c
O
O
R1
O
R1
O
Cl
N
Cl
N
When 1,3-dioxane ethyl (14) was introduced into the
5-position of isoxazole ring, an over 20-fold increase in
binding potency was observed; a similar degree of
FLIPR activity improvement was also noted (Table 2).
Corresponding 2-tetrahydropyran and 4-tetrahydropy-
ran analogues (15–16) were only slightly better than 2
in binding affinity and were comparable in FLIPR activ-
ity. Five-membered dioxalane-derived analogue 17 was
3–4-fold weaker than the six-membered 14, while tetra-
hydrofuran analogue 18 was almost equipotent to 14.
The largest potency improvement came from introduc-
tion of piperidinonylethyl to give antagonist 19 with
low nanomolar IC50 values in both binding and FLIPR
assays. In comparison, five-membered pyrrolidinone-
d
HN
OH
Cl
Cl
R2
6
5
Scheme 1. Reagents and conditions: (a) CDI, THF, MgCl2,
EtO2CCH2COOK; (b) 2,6-dichloro-N-hydroxybenzenecarboximidoyl
chloride, NaOMe, MeOH, rt; (c) NaOH, MeOH, H2O; (d) R2NH2,
TBTU, HOBT, Et3N, DMF, rt.
ketoesters 3 and 2,6-dichloro-N-hydroxyarylcarboximi-
doyl chloride was accomplished with sodium methoxide
in methanol to yield the isoxazole carboxylates 4. Base
hydrolysis of the esters 4 afforded the key isoxazole acids
5, which were then coupled with amines to furnish is-
oxazole carboxamide derivatives 6.13
Table 1. SAR of 5-alkyl modification in isoxazole carboxamides
R1
The amine 8 was prepared from trans-1,4-cyclohexyldi-
amine through monoprotection of the amine group to
yield compound 7 (Scheme 2). Subsequent reductive
amination and deprotection generated the amine 8 for
coupling with the isoxazole acid core.
N
HN
O
N
O
Cl
Cl
The primary binding assay for the compounds described
in this study was a measurement of their ability to dis-
place 125I-radiolabeled ghrelin from its receptor using
Chinese hamster ovarian (CHO)-K cell membranes per-
manently expressing human GHS-R. Ghrelin was found
to have a Kd of 0.4nM in this assay. A fluorescent cal-
cium indicator assay (FLIPR) measuring the com-
poundsÕ ability to inhibit a ghrelin-induced increase in
intracellular [Ca2+] in CHO-K cells was utilized to deter-
mine functional antagonism.14 Mouse and rat ortho-
Compounds
R1
Binding IC50
(nM)a
FLIPR IC50
(nM)a
2
CH3
C2H5
130
147
131
150
132
110
180
319
492
861
308
115
9
10
11
12
13
n-C3H7
i-C3H7
n-C3H6OH
C2H4OCH3
a Values are the means of at least two experiments against human
GHS-R.
a
b,c
H2N
NH2
H2N
NHBoc
N
NH2
7
8
Scheme 2. Reagents and conditions: (a) Boc2O (0.5equiv), CHCl3, rt; (b) CH3CHO, Na(OAc)3BH, AcOH, CH2ClCH2Cl, rt; (c) 4N HCl in
1,4-dioxane, rt.

B. Liu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5223–5226
5225
Table 2. SAR of 5-alkyl modification in isoxazole carboxamides
acyclic acetamide (23) analogues. In contrast, pyrrolidi-
none linked via a methylene group to the isoxazole core
(24) was almost 50-fold weaker than the corresponding
ethylene analogue 20.
R1
N
HN
O
N
O
Cl
A variety of methoxy-substituted phenyl groups were
also examined as alternatives to H-bond acceptor-con-
taining heterocycles. 3-Benzo[1,3]dioxolyl replacement
yielded a slightly more potent analogue (25, Table 2)
than 2, while 2-benzo[1,3]dioxolyl substituent yielded a
slightly less potent analogue 26. In comparison, 2-meth-
oxyphenylethyl substituent resulted in analogue 27 with
a 4-fold loss of FLIPR activity.
Cl
Compounds R1
Binding
IC50 (nM)a
FLIPR
IC50 (nM)a
O
O
14
6
19
15b
39
102
O
For in vivo studies we would like to inject GHS-R
antagonists directly into the brain of rodents, which re-
quires small dosing volumes and highly aqueous soluble
agents. In the course of SAR exploration of phenylene-
diamine amide off the isoxazole core of 2, it was found
that the phenylenediamine motif could be replaced with
trans-cyclohexyldiamine with slight improvement of
activity, as shown with compound 28 in Table 3. Ana-
logue 28 was also found to possess much-improved
aqueous solubility as determined by turbidity assay
(>1.0mM @ pH7.4). Therefore, trans-cyclohexyldi-
amine was incorporated in selected isoxazole core modi-
fications to explore SAR of more soluble analogues. In
the context of trans-cyclohexyldiamine amides, 1,3-diox-
ane ethyl derivative (29) was found to be a fairly potent
binder with strong ghrelin-like agonistic response.15
Moderately potent pure antagonists were generated with
tetrahydrofuranyl (30) and piperidinonyl (31)-based
core modifications. Benzo[1,3]dioxyl-based core modifi-
cation yielded potent, pure ghrelin antagonist 32 when
O
16
17
29
25
146
56
O
O
18b
19
57
9
57
8
O
N
O
N
20
21
18
39
18
15
O
N
O
N
N
22
23
21
34
10
19
Table 3. SAR of the isoxazole trans-cyclohexyl diamine amides
O
R1
N
O
N
HN
O
N
O
Cl
N
Cl
24
25
122
47
98
93
O
Compounds
R1
Binding
IC50 (nM)a
FLIPR
IC50 (nM)a
O
O
O
28
29
CH3
53
57
3b
O
O
87
26
27
162
389
564
714
O
30c
31
194
139
103
190
O
O
O
N
a Values are means of at least two experiments against human GHS-R.
b 1:1 mixture of two enantiomers.
O
32
32
54
O
derived analogue 20 was about 2-fold less potent than the
six-membered 19 in both binding and FLIPR. Further
modification of the piperidinone only maintained the
low double-digit nanomolar potency with 5,5-dimethyl
pyrrolidinone (21), 3-N-methylimidazolinone (22), and
a Values are means of at least two experiments.
b Exhibited a strong ghrelin-like agonistic response in the FLIPR
assay.
c 1:1 mixture of two enantiomers.

5226
B. Liu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5223–5226
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combined with trans-cyclohexyldiamine amide. It had
aqueous solubility of greater than 1mg/mL, making it
suitable for dosing icv through catheter injection.
Compound 32 was found to have FLIPR IC50 values of
12 and 7nM, respectively, against rat and mouse GHS-
Rs. Compound 32 was also tested against a panel of
seven GPCRs, including adrenergic, histaminergic, mu-
scarinic, and dopaminergic receptors. The FLIPR Ki
values for 32 exceeded 4000nM in these assays, suggest-
ing good GPCR selectivity for this potent GHS-R
antagonist.
8. Nakazato, M.; Murakami, N.; Date, Y.; Kojima, M.;
Matsuo, H.; Kangawa, K.; Matsukura, S. Nature 2001,
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In summary, a novel series of isoxazole carboxamides
have been identified as potent GHS-R antagonists. Sub-
stituent modification off the 5-position of the isoxazole
led to analogues with potent binding affinity and func-
tional antagonism of GHS-R. The high aqueous solubil-
ity of 32 and its good selectivity profile suggest that this
compound could be a useful pharmacological tool for
investigating the effects of GHS-R antagonism in vivo.
12. Brooks, D. W.; Lu, L. D.-L.; Masamune, S. Angew.
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13. All target compounds were determined to be >95% pure
1
by both H NMR and LC/MS spectroscopy; the spectral
data were consistent with the reported structure.
14. FLIPR assay: CHO-K cells expressing human GHS-R
(Euroscreen) were cultured in Ultra-CHO medium from
BioWhittaker supplemented with 1% dialyzed FCS, 1%
penicillin/streptomycin/fungizone, and 400lg/mL G418
(all from Life Technologies) at 37ꢁC in a humidified cell
incubator containing 5% CO2. Cells were plated in black
96-well plates with clear bottom (Costar) and cultured to
confluency overnight. Prior to assay, cells were incubated
in 100lL of DulbeccoÕs phosphate-buffered saline (DPBS)
containing 1000mg/L D-glucose, 36mg/L sodium pyru-
vate, without phenol red (Life Technologies) with 1.14mM
Fluo-4 AM (Molecular Probes) and 0.25M probenecid
(Sigma) for 1–3h in the darkat room temperature. The
dye solution was aspirated and the cells were washed twice
with DPBS using the EL-450X cell washer (BioTech).
After the last wash, 100lL of DPBS was added to each
well. Cell plates were then transferred to the FLIPR unit
(Molecular Probes). Compound additions were 50lL in
duplicate or triplicate of 4· final concentration in DPBS
containing 0.1% BSA and 4% DMSO. Fluorescence
emissions from 96 wells were measured simultaneously at
excitation and emission wavelength of 488 and 520nm,
respectively, for 3min in 1-s intervals for the first minute
and 5-s intervals thereafter. During this time agonist
responses, if any, were recorded in the absence of ghrelin.
Next, 50lL in duplicate or triplicate of 4· final concen-
tration of ghrelin in DPBS containing 0.1% BSA and 4%
DMSO were delivered within 1s by an integrated 96-well
pipettor. Fluorescence emissions were measured for
another 3min as above. During this time the antagonist
effects of compounds on ghrelin-stimulated calcium flux
were recorded. Sigmoidal curves were fitted and IC50 and
EC50 values were determined by GraphPad Prism soft-
ware. Ghrelin shows an EC50 of 0.2nM in this assay.
15. Compound 29 was found to give a strong ghrelin-like
agonistic response in the FLIPR assay as described in Ref.
14 with a calculated EC50 value of 9nM.
Acknowledgement
The authors thankMs. April Miao of Millennium Phar-
maceuticals, Inc. for generating the GPCR selectivity
data.
References and notes
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