Synthesis and structure-activity relationships of isoxazole carboxamides as growth hormone secretagogue receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.11.075

A series of isoxazole carboxamide derivatives has been developed as potent ghrelin receptor antagonists. The synthesis and structure-activity relationship (SAR) are described.

Full text of DOI:10.1016/j.bmcl.2004.11.075

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1201–1204
Synthesis and structure–activity relationships of isoxazole
carboxamides as growth hormone secretagogue receptor antagonists
Zhili Xin,* Hongyu Zhao, Michael D. Serby, Bo Liu, Verlyn G. Schaefer, Douglas H. Falls,
Wiweka Kaszubska, Christine A. Colins, Hing L. Sham and Gang Liu
Metabolic Disease Research, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, IL 60064-6098, USA
Received 19 October 2004; revised 19 November 2004; accepted 30 November 2004
Available online 25 December 2004
Abstract—A series of isoxazole carboxamide derivatives has been developed as potent ghrelin receptor antagonists. The synthesis
and structure–activity relationship (SAR) are described.
Ó 2004 Elsevier Ltd. All rights reserved.
Ghrelin, an octanoylated 28 amino acid peptide, is an
endogenous ligand for growth hormone secretagogue
receptor (GHS-R), a member of G-protein coupled
receptors.1 Ghrelin is secreted primarily in the stomach
and the upper intestinal tract. It stimulates growth hor-
mone secretion and increases food intake in humans.2
Ghrelin plasma level increases sharply on fasting in
rodents3 as well as prior to each meal in humans and
decreases after feeding.4 A chronic intracerebroventri-
cular (icv) infusion of ghrelin significantly increases food
intake and body weight in rats.5 It has also been shown
that subcutaneous administration of ghrelin for 12 days
in mice reduces fat utilization.3 Since endogenous ghre-
lin appears to play an important role in the long-term
regulation of energy balance, blocking the actions of
ghrelin is expected to reduce food intake, adiposity,
and body weight.
decreased food intake and body weight gain in ob/ob ob-
ese mice.8
These studies have provided some evidence that selective
ghrelin receptor antagonists may possibly be useful in
the prevention of weight gain and treatment of obesity,
which is becoming increasingly prevalent worldwide.
One example of nonpeptidyl ghrelin receptor antago-
nist, a 3-amino-1,3,4,5-tetrahydro-benzo[b]azepin-2-one
derivative, was reported in the literature a few years
ago.9 High-throughput screening for small molecule
ghrelin receptor antagonists in our labs identified com-
pound 1 (Fig. 1) with an IC50 of 130 nM in a binding
assay and 180 nM in a cell-based functional assay
(FLIPR). Preliminary structure–activity relationship
(SAR) revealed that the replacement of the isoxazole
core was not successful, while extension at the 5-position
of the isoxazole ring led to analogs with >10-fold
improvement of potency.10 The modification of amide
linkage was also not tolerated. Accordingly, medicinal
chemistry efforts were focused on the variation of substi-
tution on both N,N-diethylaniline and dichlorophenyl
rings.
It has been demonstrated that acute or chronic icv
administration of anti-ghrelin IgG suppresses feeding
in lean rats.5,6 In addition, transgenic rats expressing
an antisense ghrelin receptor mRNA have been reported
to exhibit ꢀ10% reduction in body weight and up to
80% reduction in adipose tissue than control rats.7 Re-
cently, Asakawa et al. found that peripherally adminis-
tered peptidic ghrelin antagonist, [D-Lys-3]-GHRP-6,
Synthetic routes to these compounds were relatively
straightforward (Scheme 1). Substituted isoxazole acids
9 (commercially available, or prepared by [2 + 3] cycliza-
tion between b-keto esters 8 and N-hydroxyarylcarbox-
imidoyl chloride followed by hydrolysis) were coupled
with anilines by activation of TBTU or HATU to
furnish isoxazole carboxamide derivatives (1–7).
Keyword: Growth hormone secretagogue receptor antagonist.
*
Corresponding author. Tel.: +1 847 935 7307; fax: +1 847 938
1674; e-mail: zhili.xin@abbott.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.075

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Z. Xin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1201–1204
Table 1. SAR of the 2,6-dichlorophenyl group
5
HN
N
O
N
HN
N
O
Cl
O
N
O
Cl
R2
R1
1
R3
IC50 = 130 nM (Binding)
IC50 = 180 nM (FLIPR)
R4
Figure 1. Ghrelin antagonist from high-throughput screening.
Compd
R1
R2
R3
R4
Binding
IC50 (lM)a
FLIPR
IC50 (lM)a
1
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
Cl
0.13
8.2
0.18
7.6
2a
2b
2c
2d
2e
2f
2g
2h
H
Cl
Alternatively, ortho-substituted acid 12, prepared by
acylation of 2-amino benzoate (10) and deprotection
of allyl eater 11, could be coupled to various amines
to provide analogs 4j–n in a parallel fashion.
0.69
0.34
0.55
1.3
0.25
0.28
0.62
6.3
Br
NO2
C6H6
OMe
H
4.0
6.8
NDb
6.0
The ghrelin antagonists were assessed in a primary bind-
ing assay, monitoring for the displacement of radio-
labeled ghrelin from its receptor. Functional activity
was determined in a fluorescent calcium indicator assay,
measuring compoundsÕ ability to inhibit ghrelin-induced
increase of intracellular [Ca2+] in CHO–K cells. The
binding and FLIPR data for 2,6-dichlorophenyl
modifications are shown in Table 1. Compared to its
18.5
7.1
H
a Values are means of at least two experiments against human GHS-R.
b For the compounds with binding IC50s > 10 lM, FLIPR data were
not determined.
2,6-dichloro counterpart
1
(IC50 = 0.2 lM), the
deficient group, would be beneficial to enhance receptor
affinity and functional activity, whereas substitution on
meta or para position has little impact on the potency
of resultant analogs. Since compound 1 remained the
most potent one among these analogs, 2,6-dichlorophe-
nyl moiety was kept for the subsequent SAR studies.
unsubstituted phenyl analog (2a) showed diminished
potency (IC50 = 8 lM). However, the monochloro-
substituted derivative 2b retained similar FLIPR antago-
nist activity to the parent 1. Compounds 2a–h seemed to
suggest that ortho-substitution, especially with electron-
R2
R
R
OH
O
HN
O
O
N
O
N
b, c
O
O
O
a
d
R
OH
R
OEt
8
R1
R1
9
1-7
O
O
Cl
O
N
e
f
HN
O
O
N
Cl
Cl
O
Cl
Cl
10
11
OH
NR'R"
O
O
HN
HN
O
N
O
N
d
O
Cl
O
Cl
Cl
Cl
12
4j-n
Scheme 1. Reagents and conditions: (a) CDI, MeCN, then potassium ethyl malonate, Et3N, MgCl2, 38–64%; (b) N-hydroxyarylcarboximidoyl
chloride, NaOMe, MeOH, rt, 59–73%; (c) NaOH, MeOH, H2O, 100%; (d) amines, TBTU or HATU, DIPEA, DMF, rt, 78–92%; (e) 2-aminobenzoic
acid allyl ester, NaHCO3, THF, rt, 49%; (f) Pd(PPh3)4, morpholine, dichloromethane, rt, 100%.

Z. Xin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1201–1204
Table 2. SAR of substituted phenylenediamine Table 3. Replacement of N,N-diethylaniline
1203
R1
R1
R2
HN
R2
HN
N
O
N
O
N
O
Cl
O
Cl
Cl
Cl
Compd R1
R2
Binding IC50 (lM) FLIPR IC50 (lM)
Compd R1
R2
Binding FLIPR
IC50
IC50
(lM)
1
H
H
0.13
9.0
0.18
4.5
(lM)
3a
3b
3c
3d
3e
3f
3g
CN
CF3
Me
Et
H
H
4.1
>10
1
H
H
H
0.13
>10
5.5
0.18
ND
N
H
0.037
0.37
0.60
0.039
0.041
2.9
H
4a
4b
OMe
H
H
NH
N
Cl
Me
>10
>0.1
ND
>10
4.7
H
4c
4d
4e
H
H
H
0.41
0.51
N
N
N
The effect of substitution on N,N-diethylaniline ring was
also explored (Table 2). Compounds 3a and 3b, with an
ortho-electron-withdrawing group (CN or CF3) next to
the amide linker, displayed weaker affinity for ghrelin
receptor. In contrast, Compounds (3c–e) showed little
or no loss in binding affinity. In particular, compound
3c bearing an ortho-Me group displayed improved po-
tency (IC50 = 0.04 lM, ꢀ4-fold) in both binding and
functional activity. The substitution on meta-position
(3f–g) seemed to be less favorable.
>10
ND
9.2
3.1
OH
N
O
4f
H
H
>10
ND
ND
4g
>10
>10
Despite the efforts to increase the potency as ghrelin
antagonist, the oral bioavailability of these isoxazole
carboxamides remains low (4% for compound 1 and
1% for compound 3c). A number of factors potentially
contribute to the poor bioavailability. Rat liver micro-
somal incubation results of compound 1 indicated that
this compound is rapidly metabolized in 30 min as
shown by the rate of 800 pmol/mg protein/minute. The
major metabolite seemed to be the de-ethylation of the
N,N-diethylaniline. Therefore, the replacement of the
N,N-diethylaniline group was evaluated.
4h
4i
H
ND
1.7
N
H
0.98
N
4j
H
H
1.9
2.2
2.5
2.3
N
N
O
O
It was found that N,N-diethylaniline portion of mole-
cule is very sensitive to modifications (4a–h in Table
3). Compound 4a, the metabolite of compound 1, had
no binding affinity for receptor. Only slightly modified
N,N-dialkylaniline groups are tolerated (4b–e). The
attempt to replace one of the ethyl groups by acyl group
led to inactive compound 4f. The replacement of nitro-
gen with carbon (4g) or insertion of an extra carbon
between the aniline (4h) was also synthesized but led
to completely inactive analogs.
4k
4l
H
H
0.86
1.2
0.51
N
O
O
HN
4m
10.5
4.9
HN
We then turned our attention to broadly screen amide
derivatives from available amine library without attach-
ment of N,N-diethylamino moiety. It was found that the
ortho-substituted analog 4i displayed modest affinity.
Further investigation identified compound 4j with
ortho-amide substituent. The improved affinity of 4j
prompted the preparation of numerous analogs with
other amide substituents. Compound 4l, for example,
showed submicromolar efficacy in both binding and
O
4n
H
0.35
O
O
FLIPR data. While benzylamide analogs displayed im-
proved binding affinity, the functional activity did not
demonstrate the same preference.

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Z. Xin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1201–1204
Table 4. The combination of two sites modification
However, the combination of substitutions from both
sides of the amide linker did not seem to offer any
advantage over single substituted analogs. The major
challenge in this series of compounds remains to im-
prove PK profile while maintaining the potency of
receptor antagonists.
O
O
R1
R2
HN
R3
O
N
O
Cl
Acknowledgements
Cl
The authors thankDavid Beno and Kennan Marsh for
obtaining the rat pharmacokinetic data, and the Abbott
Analytical Department for assistance in acquiring H
Compd
R1
R2
R3
Binding
IC50 (lM)
FLIPR
IC50 (lM)
1
NMR and mass spectra for the compounds discussed
in this report.
5
6
H
Me
H
H
NEt2
NEt2
0.006
0.009
0.014
0.010
7
H
H
7.5
7.4
N
References and notes
O
1. Kojima, M.; Hosoda, H.; Date, Y.; Nakazato, M.;
Matsuo, H.; Kangawa, K. Nature 1999, 402, 656; For
reviews on ghrelin, see: (a) Kojima, M.; Kangawa, K.
Curr. Opin. Pharmacol. 2002, 2, 665; (b) Horvath, T. L.;
Castaneda, T.; Tang-Christensen, M.; Pagotto, U.;
Tschop, M. H. Curr. Pharm. Des. 2003, 9, 1383.
2. Wren, A. M.; Seal, L. J.; Cohen, M. A.; Brynes, A. E.;
Frost, G. S.; Murphy, K. G.; Dhillo, W. S.; Ghatei, M. A.;
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3. Tschop, M.; Smiley, D. L.; Heiman, M. L. Nature 2000,
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4. Cummings, D. E.; Purnell, J. Q.; Frayo, R. S.; Schmidova,
K.; Wisse, B. E.; Weigle, D. S. Diabetes 2001, 50, 1714.
5. Nakazato, M.; Murakami, N.; Date, Y.; Kojima, M.;
Matsuo, H.; Kangawa, K.; Matsukura, S. Nature 2001,
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6. Murakami, N.; Hayashida, T.; Kuroiwa, T.; Nakahara,
K.; Ida, T.; Mondal, M. S.; Nakazato, M.; Kojima, M.;
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Ohata, H.; Tamura, H.; Kamegai, J.; Sugihara, H.;
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To summarize the SAR from different sites, it was obvi-
ous that successful modification comes from the ortho-
substitution on the aniline ring. We have previously
demonstrated that extension of isoxazole side chain at
5-position led to >10-fold boost in potency (compound
5). The combination of these two optimized modifica-
tions was tried (Table 4). To our disappointment, com-
pound 6 only maintained equipotent nanomolar IC50
value of 5. Compound 7 was designed to add 1,3-diox-
anepropyl moiety on the top of analog 4k (ꢀ2 lM) to
achieve improved potency over 4k. However, it dis-
played decreased binding affinity and functional activity
(ꢀ8 lM). These results implicated that both ortho-sub-
stituents adjacent to the amide linker may compete for
the same binding site. When the size of the substituent
on isoxazole ring increased from methyl to 1,3-dioxane-
propyl, small groups like Me on aniline ring are just
barely tolerated, however, more bulky substituents like
amide group in compound 7 will become detrimental
to providing favorable binding affinity.
8. Asakawa, A.; Inui, A.; Kaga, T.; Katsuura, G.; Fujimiya,
M.; Fujino, M. A.; Kasuga, M. Gut 2003, 52, 947.
9. Cheng, K.; Chan, W. W. S.; Butler, B.; Wei, L.; Smith, R.
G. Horm. Res. 1993, 40, 109.
10. Liu, B.; Liu, G.; Xin, Z.; Serby, M.; Zhao, H.; Schaefer,
V.; Falls, D.; Kaszubska, W.; Collins, C.; Sham, H.
Bioorg. Med. Chem. Lett. 2004, 14, 5223.
In conclusion, the SAR of isoxazole carboxamides as
ghrelin receptor antagonists was explored. The presence
of ortho-substituent on aniline ring seemed to be benefi-
cial to improve binding affinity and functional activity.