Synthesis and biological evaluation of an orally active ghrelin agonist that stimulates food consumption and adiposity in rats

Article abstract of DOI:10.1016/j.bmcl.2004.09.027

LY444711 is an orally active ghrelin agonist that binds with good affinity and is a potent activator of the growth hormone secretagogue receptor 1a (GHS-R1a) receptor. In rat models of feeding behavior and pharmacology, LY444711 creates a positive energy

Full text of DOI:10.1016/j.bmcl.2004.09.027

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5873–5876
Synthesis and biological evaluation of an orally active ghrelin
agonist that stimulates food consumption and adiposity in rats
Charles W. Lugar, Michael P. Clay, Terry D. Lindstrom, Andrea L. Woodson,
David Smiley, Mark L. Heiman and Jeffrey A. Dodge*
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 5 August 2004; revised 9 September 2004; accepted 10 September 2004
Available online 1 October 2004
Abstract—2-(2-Amino-2-methyl-propionylamino)-5-phenyl-pentanoic acid {1-[1-(4-methoxy-phenyl)-1-methyl-2-oxo-2-pyrrolidin-
1-yl-ethyl]-1H-imidazol-4-yl}-amide (LY444711, 6) is an orally active ghrelin agonist that binds with high affinity to and is a potent
activator of the growth hormone secretagogue receptor 1a (GHS-R1a) receptor. In rat models of feeding behavior and pharmacol-
ogy, 6 creates a positive energy balance and induces adiposity by stimulating food consumption and sparing fat utilization. As an
orally active ghrelin agonist, 6 represents a new pharmacological tool to investigate the orexigenic role of ghrelin in regulating
energy homeostasis.
Ó 2004 Elsevier Ltd. All rights reserved.
Ghrelin (1) is a 28 amino acid peptide produced pre-
dominantly by the stomach and intestines that has re-
O
GSSFLSPEHQRVQQRKESKKPPAKLQPR
Ghrelin
1a Ghrelin amide (C-terminal CONH2)
cently been identified as the endogenous ligand for the
growth hormone secretagogue receptor (GHS-R1a).1 It
is uniquely characterized by an n-octanoyl moiety on
the Ser3 residue that is derived from a post-translation
modification and is essential for biological activity.
Ghrelin has been shown to induce adiposity in rodents
by increasing food intake and decreasing the utilization
of fat tissue.2 In humans, ghrelin stimulates appetite and
induces food intake.3 Other physiological effects of this
hormone include the stimulation of GH secretion from
the pituitary in rats and humans.4,5 Recent studies sug-
gests that the adipogenic properties of ghrelin may be
independent from its ability to stimulate GH secretion
from the pituitary.2 Ghrelin likely induces its effects on
feeding and energy balance by binding the GHS-R1a
on the NPY and Agouti-related protein (AGRP) neu-
rons of the arcuate nucleus of the hypothalamus6 and in-
duces the synthesis7 and release of these anabolic
neuropeptides. While extensive efforts have been direc-
ted toward optimizing molecules that stimulate pituitary
1
2
3
GHRP-6
His-D-2-CH3-Trp-Ala-Trp-D-Phe-Lys-NH2
ipamorelin Aib-His-D3-NaI-D-Phe-Lys-NH2
O
O
O
N
N
S
O
N
N
H2N
N
O
N
O
O
O
N
H2N
O
4 ibutamoren
5 capromorelin
O
O
N
N
N
N
H
HN
O
O
NH2
Keywords: Ghrelin; Growth hormone secretagogues; Energy expend-
iture; Food consumption.
6
LY444711
*
Figure 1. Structures of ghrelin (human) analogs and growth hormone
secretagogues.
Corresponding author. Tel.: +1 3172768252; fax: +1 3172772035;
e-mail: jdodge@lilly.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.027

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C. W. Lugar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5873–5876
GH release, such as growth hormone releasing peptide
GHRP-6 (2), ipamorelin (3), ibutamoren (4), capromo-
relin (5), and others,8 the effects of GHSs on appetite
regulation and energy homeostasis have remained con-
siderably less characterized. In fact, despite the fact that
orally active small molecule GHS ligands are well docu-
mented, only peptide ligands with poor oral activity,
such as ghrelin, 2, and 3, have been prospectively char-
acterized for their adipogenic effects to date.2,9 Herein,
we describe the synthesis and the pharmacology of 6, a
ghrelin agonist that represents an orally active pharma-
cological tool to investigate the orexigenic and anabolic
role of ghrelin in regulating energy homeostasis (Fig. 1).
of 9 with the lithium anion of (4R,5S)-(+)-4-methyl-5-
phenyl-2-oxazolidinone. The resulting 1:1 mixture of
diastereomers, 10 and 11, were readily separated by sil-
ica gel chromatography. The absolute stereochemistry
of the chiral quaternary center was assigned as S for
10 and R for 11 based on the X-ray crystal structure
obtained for 10. Conversion to 12 was accomplished
by removal of the oxazolidinone and formation of
the corresponding pyrrolidine amide. Reduction of 12
with hydrogen in the presence of palladium yielded a
relatively unstable 4-aminoimidazole, which was not
isolated but coupled directly with (R)-2-(2-tert-
butoxycarbonylamino-2-methyl-propionylamino)-5-
phenyl-pentanoic acid11 to yield 13. Deprotection of
13 using HCl yielded 6.
A convergent synthesis of 6 is outlined in Scheme 1. Bro-
mination of ethyl 4-methoxyphenyl acetic acid (7) with
N-bromosuccinamide followed by regioselective N-alkyl-
ation10 of 4-nitroimidazole yielded 8. Treatment of 8
with sodium bis(trimethylsilyl)amide followed by methyl
iodide gave 9, which was subsequently saponified with
lithium hydroxide to give the corresponding carboxylic
acid. This racemic acid was resolved by formation of
diastereomers 10 and 11 by reaction of the acid chloride
Compound 6 was evaluated for its ability to bind to the
human ghrelin receptor (GHS-R1a) and to act as a func-
tional agonist. In a competitive binding assay,1 6 dis-
placed I125-ghrelin with an affinity constant (Ki) of
10.6 3.2nM (n = 23). In AV-12 cells transfected with
the GHS-R1a receptor,12 6 is a potent agonist with an
EC50 of 1.2 0.2nM (n = 5) in stimulating calcium
O2N
O2N
N
N
OEt
N
N
C
a,b
OEt
OEt
O
O
O
O
O
O
7
9
8
d,e
O2N
O2N
N
N
Ph
N
N
N
Xc =
O
+
Xc
Xc
O
O
O
O
O
11
10
f, g
N
O2N
O
N
O
N
h,i
N
N
N
N
HN
O
O
O
O
NHBoc
12
13
O
N
O
N
N
j
N
H
O
HN
O
NH2
6
Scheme 1. Reagents and conditions: (a) N-bromosuccinamide, AIBN; (b) 4-nitroimidazole, K2CO3; 61% two steps; (c) sodium bis(trimethylsil-
yl)amide, methyl iodide, 92%; (d) LiOH; (e) oxalyl chloride, (4R,5S)-(+)-4-methyl-5-phenyl-2-oxazolidinone (Xc), n-BuLi gave 10 (41%), and 11
(41%); (f) LiOH; (g) oxalyl chloride, pyrrolidine; (h) H2, 5% Pd/C; (i) R-2-(2-tert-butoxycarbonylamino-2-methyl-propionylamino)-5-phenyl-
pentanoic acid, DCC, HOBt; (j) step 1 HCl; step 2 NaHCO3.

C. W. Lugar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5873–5876
5875
Table 1. Daily fuel balance (kcal/kg/d) for male Long–Evans rats fed a
caloric dense dieta
mobilization as determined by fluorescence light induced
polarization.
Caloric source Dose, mg/kg Caloric intake Caloric utilization
In order to assess the viability of 6 as an orally active
chemical tool for studying ghrelin pharmacology, we ini-
tially evaluated the stability of 6 versus that of ghrelin
amide 1a in rat plasma at 37°C. We used 1a since C-ter-
minal modifications of peptides are generally known to
decrease their susceptibility to enzymatic degradation.
We found that 1a undergoes rapid decomposition when
incubated in rat plasma. After a 1h incubation period,
only 61% of the parent peptide remained and after 6h
18% remained. In contrast, 6 remained unchanged after
6h when exposed to rat plasma under conditions identi-
cal to that of ghrelin amide. The oral bioavailability of 6
was subsequently evaluated in rats and dogs. Oral
administration of a 30mg/kg dose of 6 to male F344 rats
(n = 3 rats/time point) resulted in a maximal concentra-
tion (Cmax) of 211 76ng/mL in 2h. The area under the
curve AUC (0-inf.) was 633ngh/mL with a half-life of
1h. The bioavailability of 6 was determined to be 10%
in rats and 37% in beagle dogs.
Protein
Fat
Vehicle
3
42.7 5.2
51.1 1.5
52.2 1.9
55.7 2.2
33.0 1.0
32.5 0.2
32.9 0.7
32.9 0.7
10
30
Vehicle
3
91.3 11.1
109.3 3.2
111.6 4.1
119.2 4.8
61.7 13.6
32.3 3.6
27.9 2.4*
27.9 6.3*
10
30
Carbohydrate Vehicle
3
94.3 12.8
112.8 3.4
115.2 4.2
123.0 5.0
81.7 11.8
109.0 3.9
115.1 5.5*
124.4 6.2*
10
30
* p < 0.05.
a Animals were treated (n = 4/group) with LY444711 or vehicle by oral
gavage.
energy requirements results in the sparing of glycogen
synthesis and fat stores, data which is consistent with
the increase in body fat observed after two weeks of
dosing (vide infra). Food consumption tended to increase
in proportion to the amount of compound administered,
with the food intake at 30mg/kg exceeding that of vehi-
cle-treated controls by 130% (p = 0.06).
The pharmacological profile of 6 as a ghrelin agonist
was assessed by measuring carbohydrate and fat utiliza-
tion in rodents by indirect calorimetry using respiratory
quotient (RQ) over a 24h period (Fig. 2).13–15 A single
oral administration of 6 induced an increase of the RQ
in a dose-dependent manner. This increase is especially
evident during the dark photoperiod when the animals
are consuming the majority of their daily fuel intake.
As shown in Table 1, significant decreases in lipid oxida-
tion at the 10mg/kg (27.9 2.44kcal/kg/d, p = 0.04) and
30mg/kg (27.9 6.32kcal/kg/d, p = 0.04) groups are
observed when compared to vehicle-treated animals
(61.66 13.62kcal/kg/d). These doses significantly in-
creased carbohydrate utilization (115 5.49kcal/kg/d,
p = 0.02 and 124 6.18kcal/kg/d, p = 0.008, respec-
Chronic treatment of LY444711 (po, 10mg/kg/d) for
2 weeks in Long–Evans rats (n = 5/group) significantly
increased body weight gain by day 2 of treatment
(p = 0.037) and by day 14, animals receiving 6 had
gained 23% more body weight than vehicle-treated con-
trol animals (p = 0.049) (Fig. 3). Cumulative food con-
sumption of treated animals was significantly greater
than that of vehicle-treated control rats beginning on
day 4 (p = 0.018) (Fig. 4).
tively)
compared
to
vehicle-treated
controls
Analysis of body composition before and following 2
weeks of treatment with 6 was performed by dual-energy
X-ray absorptiometry (DEXA) to determine the relative
(81.7 11.8kcal/kg/d). The observed increase in the
use of carbohydrates of the food consumed to meet
1.0
0.9
80
Vehicle Control
*
*
10mg/kg 444711
*
*
60
40
20
0
R
0.9
Q
V
C
*
*
0.8
0.8
0.7
0.7
O
2/
V
*
*
O
*
Vehicle Control
3 mg/kg
10 mg/kg
30 mg/kg
Dark Photo
0
5
1
1
2
2
-20
0
2
4
6
8
10
12
14
16
Time Relative to Treatment
Days of Treatment
Figure 2. Respiratory quotient (RQ) over a 24h period after oral
administration of LY444711. RQ (y-axis) is defined as the volume of
carbon monoxide produced (VCO )/volume of oxygen consumed (V O ).
Figure 3. Oral administration of LY444711 increases body weight in
rats.
2
2

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C. W. Lugar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5873–5876
350
300
250
200
150
100
50
Acknowledgements
*
*
Vehicle Control
10mg/kg 444711
We wish to thank the Lead Optimization Biology group
for conducting binding and functional assays.
*
*
*
*
*
*
References and notes
*
*
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12
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Figure 4. Oral administration of LY444711 increases cumulative food
intake in male rats.
5. Takaya, K.; Ariyasu, H.; Kanamoto, N.; Iwakura, H.;
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contributions of changes in fat and lean mass to the
observed increase in body weight gain.16 Administration
of 6 resulted in an increase of fat mass (74.7g 6.6),
which was significantly greater than the gain of fat mass
observed in vehicle-treated control animals (47.2g 7.6,
p = 0.026). No change in lean mass (22.7g 5.7 and
26.7g 4.9, respectively, p = 0.61) or bone area was
noted (19.0cm2 2.5 and 14.7cm2 2.0, respectively,
p = 0.2). Serum IGF-1 levels did not differ between
groups at the end of the experimental period (p = 0.59)
indicating that the observed changes in body composi-
tion and energy balance induced with 6 may be inde-
pendent of its ability to stimulate pituitary GH release.
However, 6 elevated plasma GH and IGF-1 in beagle
dogs after a single oral dose of 1mg/kg.17
LY444711 (6) is an orally active ghrelin agonist. This
compound binds with high affinity to the GHS-R1a
receptor and stimulates calcium mobilization in AV-12
cells transfected with the human ghrelin receptor. This
compound is significantly more stable in rat plasma than
ghrelin amide 1a and has good plasma exposure after
oral administration to rats. Like ghrelin, 6 creates a pos-
itive energy balance when administered acutely to
rodents. When administered chronically, 6 induces adi-
posity by stimulating food consumption and sparing
fat utilization. As an orally active ghrelin agonist with
a prolonged half-life relative to ghrelin, 6 represents a
new pharmacological tool to investigate the orexigenic
role of ghrelin in regulating energy homeostasis.
15. Flatt, J. P. J. Nutr. Biochem. 1991, 2, 193–202.
16. Rose, B. S.; Flatt, W. P.; Martin, R. J.; Lewis, R. D. J.
Nutr. 1998, 128, 246–250.
17. Seyler, D. E.; Dodge, J. A.; Osborne, J. J.; Cox, K. L.;
Viswanath, D.; Wilmot, A. F.; Keaton, M. J.; Heiman, M.
L.; Bryant, H. U.; Cutler, G. B. Drug Dev. Res. 2000, 49,
260–265.