Pyrazolinone-piperidine dipeptide growth hormone secretagogues (GHSs): Discovery of capromorelin

Article abstract of DOI:10.1016/S0968-0896(02)00433-9

Novel pyrazolinone-piperidine dipeptide derivatives were synthesized and evaluated as growth hormone secretagogues (GHSs). Two analogues, capromorelin (5, CP-424391-18, hGHS-R1a K_i=7 nM, rat pituicyte EC₅₀=3 nM) and the des-methyl an

Full text of DOI:10.1016/S0968-0896(02)00433-9

Bioorganic & Medicinal Chemistry 11 (2003) 581–590
Pyrazolinone-piperidine Dipeptide Growth Hormone
Secretagogues (GHSs):
Discovery of Capromorelin
Philip A. Carpino,* Bruce A. Lefker, Steven M. Toler, Lydia C. Pan, John R. Hadcock,
Ewell R. Cook, Joseph N. DiBrino, Anthony M. Campeta, Shari L. DeNinno,
Kristin L. Chidsey-Frink, William A. Hada, John Inthavongsay, F. Michael Mangano,
Michelle A. Mullins, David F. Nickerson, Oicheng Ng, Christine M. Pirie,
John A. Ragan, Colin R. Rose, David A. Tess, Ann S. Wright, Li Yu,
Michael P. Zawistoski, Paul A. DaSilva-Jardine, Theresa C. Wilson
and David D. Thompson
Pfizer Global Research and Development, Groton Labs, MS8220-3004, Eastern Point Rd, Groton, CT 06340, USA
Received 24 May 2002; accepted 12 August 2002
Abstract—Novel pyrazolinone-piperidine dipeptide derivatives were synthesized and evaluated as growth hormone secretagogues
(GHSs). Two analogues, capromorelin (5, CP-424391-18, hGHS-R1a Ki=7 nM, rat pituicyte EC50=3 nM) and the des-methyl
analogue 5c (hGHS-R1a Ki=17 nM, rat pituicyte EC50=3 nM), increased plasma GH levels in an anesthesized rat model, with
ED50 values less than 0.05 mg/kg iv. Capromorelin showed enhanced intestinal absorption in rodent models and exhibited superior
pharmacokinetic properties, including high bioavailabilities in two animal species [F(rat)=65%, F(dog)=44%]. This short-dura-
tion GHS was orally active in canine models and was selected as a development candidate for the treatment of musculoskeletal
frailty in elderly adults.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
synthesis by the liver. Recently, the endogenous ligand
for the GHS-R1a has been identified as a pleiotrophic
hormone synthesized by the stomach called ghrelin.4
Growth hormone secretagogues (GHSs) are a new class
of orally active drugs which increase plasma levels of
growth hormone (GH) and insulin-like growth factor-1
(IGF-1), two hormones that play important roles in the
regulation of body composition and metabolism.1 These
drugs stimulate GH secretion from the pituitary gland
via a heptahelical G-protein coupled receptor called the
type 1a growth hormone secretagogue receptor (GHS-
R1a).2 They also promote release of a GH stimulatory
hormone called GH-releasing hormone (GHRH) from
the hypothalamus, possibly by antagonizing the actions
of a hormone that inhibits GH release called somato-
trophin release inhibitory factor (SRIF, somatostatin).3
Elevation of plasma GH levels leads to increased IGF-1
The first GHSs were discovered in the early 1980s by
Bowers and co-workers and were called growth hor-
mone-releasing peptides (GHRPs).5,6 Many structurally
distinct GHSs have since been reported in the literature,
including tripeptidyl compounds such as NN703 (1),7
dipeptidyl derivatives such as the spiroindane MK-0677
(2)8 and L-163540 (3),9 and several non-peptidyl small
molecules (see Fig. 1).10,11 Prominent features of dipep-
tidyl GHSs include groups such as a-amino-isobutyric
(Aib) acid and various (d)-amino acids (i.e., Aib-(d)-O-
Bn-Ser, Aib-(d)-Trp) that minimize protease degrada-
tion in the intestinal tract following oral administration.
Only a few of the peptidyl and peptidomimetic com-
pounds have exhibited sufficient bioavailability in ani-
mal models to advance into human clinical studies. The
most extensively studied GHS, MK-0677, has been
*Corresponding author. Tel.: +1-860-441-5694; fax: +1-860-715-
8067; e-mail: philip_a_carpino@groton.pfizer.com
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00433-9

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P. A. Carpino et al. / Bioorg. Med. Chem. 11 (2003) 581–590
shown to restore GH and IGF-1 levels in elderly sub-
jects to ‘youthful’ levels,12 increase fat-free mass and
markers of bone formation in healthy obese men,13,14
and stimulate growth velocity in growth hormone-defi-
cient children.15
(C-terminal) region was derived from compounds 2 and
3 using a pharmacokinetic-oriented screening strategy
that focused first on the identification of compounds
with good absorption properties in rodent models and
second on the optimization of the in vitro and in vivo
GH secretagogue activities. Several PP analogues with
an Aib-(d)-O-Bn-Ser dipeptide subunit as in 2, rather
than a Aib-(d)-Trp dipeptide moiety as in 3, exhibited
A new series of dipeptidyl GHSs with pyrazolinone-
piperidine (PP) heterocycles (4) in the carboxy-terminal
Figure 1. Peptidyl and peptidomimetic GHSs and pyrazolinone-piperidine (PP) heterocycles.
Scheme 1. Syntheses of GHSs 5, 5a and 5b. Reagents and conditions: (a) Boc2O, DMAP, CH2Cl2, 0 ꢀC; (b) NaH, PhCH2Br, DMF; (c) R1NHNH2,
EtOH, reflux; (d) toluene, reflux; (e) TFA, CH2Cl2, 0 ꢀC or concd HCl, EtOH; (f) HOAt, EDC, NEt3, CH2Cl2, 0 ꢀC; (g) TFA, CH2Cl2, 0 ꢀC; (h)
concd HCl, EtOH; (i) (l)-tartaric acid, MeOH; (j) EtOAc, reflux.

P. A. Carpino et al. / Bioorg. Med. Chem. 11 (2003) 581–590
583
high intestinal absorption in rats and showed good
biological activities in dog efficacy models. We have
previously disclosed the preliminary pharmacological
characterization of a short-duration GHS from this
series, capromorelin (5, CP-424391–18).16 Herein, we
describe the discovery, biological activities, and
syntheses of PP dipeptides and report the physicochem-
ical and pharmacokinetic properties of one GHS,
capromorelin, that led to its selection as a development
candidate.
GHS analogue 5c was prepared by an alternate route
that involved coupling dipeptide 11 with piperidine
derivative 14, obtained from the N-Boc derivative 8 by
treatment with trifluoroacetic acid (see Scheme 2).
Introduction of the PP heterocycle using standard
methodology provided 12d. Subsequent acid-catalyzed
cleavage of the N-Boc protecting group yielded dipep-
tide 16. The (l)-tartrate salt 5c was prepared as described
previously for 5.
Results and Discussion
Chemistry
Several different dipeptidyl GHSs such as the clinical
candidate MK-0677 (2) and the pre-clinical compound
L-163540 (3) have been disclosed over the past decade.
These compounds have generally exhibited good oral
activities in canine models, but have required high
pharmacological doses or shown unsatisfactory absorp-
tion properties in rat models used either for chronic oral
efficacy studies or for prediction of absorption proper-
ties in humans.1 The poor rat absorption was often
ascribed to low aqueous solubility and low intestinal
permeation, due in part to several unfavorable struc-
tural features in the molecules, including multiple amide
bonds and high number of H-bond donor and acceptor
groups in the polar dipeptide backbone. For some
GHSs, high intestinal metabolism also contributed to
the poor absorption.
The synthesis of the pyrazolinone-piperidine dipeptide
capromorelin 5 is shown in Scheme 1. The N-tert-
butoxycarbonyl (N-Boc) piperidine derivative 7, pre-
pared from methyl 4-oxo-3-piperidinecarboxylate 6 and
di-tert-butyldicarbonate (Boc2O), was alkylated with
benzyl bromide using sodium hydride as a base. The
keto ester product 8 was then treated with methylhy-
drazine to provide the PP heterocycle 9a. Acid-catalyzed
cleavage of the N-Boc protecting group in 9a yielded the
amine 10a, which was coupled with Boc-Aib-(d)-O-Bn-
Ser-OH (11)17 using 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (EDC) and 1-hydroxy-
7-azabenzotriazole (HOAt)18 to give two diastereomers,
(3aR)-12a and (3aS)-12b. Analysis of the reaction mix-
ture by thin layer chromatography (TLC) revealed rapid
formation of the more polar isomer (3aS)-12b, followed
by slow appearance of (3aR)-12a. HOAt was added to
the coupling reaction to minimize racemization of the
dipeptide moiety. Removal of the N-Boc group in
(3aR)-12a with aqueous hydrochloric acid gave (3aR)-
13a. The (l)-tartrate salt of (3aR)-13a was prepared and
crystallized from hot ethyl acetate to provide capro-
morelin. The absolute configuration of capromorelin
was established by coupling the (3aR)-isomer of 10a
(confirmed by X-ray crystallographic analysis) with the
(d)-isomer of dipeptide 11 under non-racemizing condi-
tions, the results of which will be described separately in
due course. Compound 5a was obtained in two steps
from (3aS)-12b by the procedure used to prepare 5 from
(3aR)-12a. GHS 5b was synthesized by first coupling
dipeptide 11 with 10b and then removing the N-Boc
protecting group.
The dipeptidyl backbones in GHSs 2 and 3 could not be
easily modified without resulting in loss of biological
activity. The amino group in the C-terminal region was
therefore used to improve the physicochemical proper-
ties of the compounds and enhance oral absorption.
New compounds were initially screened for intestinal
absorption in rodent models and then evaluated as
GHSs if exposure was found to be acceptable (a phar-
macokinetic-oriented strategy). Intestinal absorption
was assessed by measuring portal vein drug concentra-
tions after oral dosing using protocols described in the
literature for the identification of bioavailable peptider-
gic molecules.19 Dipeptidyl analogues with an Aib-(d)-
O-Bn-Ser moiety generally showed better intestinal
absorption than analogues with an Aib-(d)-Trp group,
possibly because they contained one less hydrogen bond
and lacked a metabolically labile indole ring.
Because pyrazolinone-piperidine 10c (HCl salt) was
chemically unstable in CH2Cl2 or DMF solutions in the
presence of bases such as triethylamine, the des-methyl
An unprecedented pyrazolinone-piperidine (PP, 3a-benzyl-
2,3a,4,5,6,7-hexahydro- pyrazolo[4,3-c]pyridin -3-one)
ꢀ
ꢀ
.
Scheme 2. Synthesis of GHS 5c. Reagents and conditions: (a) TFA, CH2Cl2, 0 C; (b) 11, EDC, HOAt, CH2Cl2, 0 C; (c) NH2NH2 H2SO4, NaOAc,
EtOH, 78 ꢀC; (d) concd. HCl, EtOH; (e) saturated aqueous NaHCO3; (f) (l)-tartaric acid, MeOH; (g) EtOAc, 80 ꢀC.

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P. A. Carpino et al. / Bioorg. Med. Chem. 11 (2003) 581–590
heterocycle 4 was identified as a suitable replacement for
the C-terminal amino group in prototypical dipeptidyl
GHSs. The PP heterocycle 4 was conceived as an enzy-
matically stable surrogate for the ethyl 3-benzylnipeco-
tate moiety found in the short-duration GHS 3, a
compound that was designed to rapidly undergo
esterase hydrolysis in the plasma to give a less active
metabolite.9 The PP group with its critical 3a-benzyl
substituent proved sufficiently lipophilic to counter-
balance the hydrophilic nature of the core dipeptidyl
backbone. In the Aib-(d)-O-Bn-Ser series, analogues
such as 5 and 5b containing substituted PP heterocycles
(4, R=Me, Et) possessed especially good physicochem-
ical properties and exhibited high intestinal absorption
in multiple animal models. An Aib-(d)-O-Bn-Ser analo-
gue 5c with an unsubstituted PP group (4, R=H)
showed lower, but still satisfactory, absorption in the rat
models, most likely due to the extra H-bond capacity. A
representative substituted PP dipeptide derivative,
capromorelin (5), was not only highly soluble in deio-
nized water (>100 mgA/mL) and in a pH 7 buffer
solution (4.9 mgA/mL) but also moderately lipophilic,
with a calculated partition coefficient (clog P) in the
optimal range for transcellular diffusion (clog P=2.9).
When administered orally to rats (1 mg/kg of free base
equivalents or 1 mgA/kg), the calculated fraction of
drug absorbed was approximately 1.0 (assuming most of
the plasma clearance was due to hepatic elimination).20
The unsubstituted PP dipeptide derivative 5c (R=H,
Fig. 1) exhibited similar physicochemical properties, but
a lower calculated fraction absorbed (Fa=0.6).
found to be 17-fold less potent than ghrelin, the endo-
genous ligand for the GHS-R1a (see Table 1). The
binding activity of capromorelin was shown to be
stereospecific, with the 3aR-isomer of the pyrazolinone-
piperidine heterocyclic ring preferred over the 3aS-iso-
mer. Removal of the N-methyl group on the PP group
led to a two-fold loss in activity. The N-ethyl PP deri-
vative 5b was equipotent to ghrelin in the assay.
Functional activities of the pyrazolinone-piperidine
dipeptide GHSs were evaluated in a GH release assay in
rat pituitary cell cultures. Despite exhibiting a 30-fold
range of binding affinities, capromorelin and analogues
5b and 5c stimulated GH release with similar potencies
(see Table 1). The 3aS-diastereomer of capromorelin
(5a) showed significantly weaker activity, with an EC50
value greater than 1 mM. Interestingly, both the 3aR-
and 3aS-diastereomers of compound 3 (structural pre-
cursors of PP dipeptides) potently stimulated GH
secretion (EC50s<25 nM), though the 3aS-diastereomer
was slightly preferred.9 With the exception of 5a, the
binding affinities of the GHSs did not predict activity in
the cellular assay, possibly because of the potentially con-
founding effects of protein binding in the whole cell assay.
The in vivo GH activities of the PP dipeptide analogues
were measured in an anesthesized rat model following iv
administration. Capromorelin and compounds 5b and
5c stimulated GH secretion after a single 1 mg/kg dose,
though the mean GH peak heights for capromorelin
and 5c were significantly higher than the mean GH peak
height for 5b (data not shown). Capromorelin and 5c
showed similar dose–response relationships in the
model, with ED50 values less than 0.05 mg/kg iv. The
weaker in vivo activity of the N-ethyl PP derivative 5b
was attributed to its increased lipophilicity, which could
have reduced the amount of unbound drug in the
plasma compartment capable of interacting with the
GHS-R1a.
The biological activities and pharmacokinetic properties
of several pyrazolinone-piperidine dipeptide derivatives
with good absorption properties (capromorelin, 5a–c)
were characterized. Binding affinities were measured via
competitive binding assays in HEK293 cells over-
expressing the cloned human GHS-R1a (hGHS-R1a)
using [125I]-ghrelin as a radioligand. Capromorelin was
Table 1. Binding affinities and GHS activities of PP dipeptide GHSs
Compound
C-3a stereochemistry
R1
Ki (nM)a
EC50 (nM)b
ED50 (mg/kg iv)c
Ghrelin
5 (Capromorelin)
5a
5b
5c
0.42
7
1430
0.66
17
R
S
R
R
Me
Me
Et
3
>1000
4
3
0.05
NDd
ND
H
0.04
aKis for displacement of [125I]-ghrelin in HEK293/hGHS-R1a cell membranes, calculated from average of duplicate experiments.
bGH release in rat pituitary cell cultures, mean EC50 values calculated from triplicate determinations.
cGH release in anesthesized rat model.
dNot determined.

P. A. Carpino et al. / Bioorg. Med. Chem. 11 (2003) 581–590
585
Pharmacokinetic properties of two PP dipeptide GHSs,
Conclusions
capromorelin and 5c, were measured in female Sprague–
Dawley rats. For capromorelin, plasma clearance (CL),
volume of distribution (Vd) and plasma elimination
half-life (t1/2) following a 1 mgA/kg iv dose were 34Æ5
mL/min/kg, 1.7 L/kg and 0.79Æ0.21 h respectively.
Capromorelin was rapidly absorbed after a 1 mgA/kg
oral (po) dose, reaching maximum systemic concentra-
tions (Cmax) of 329Æ158 ng/mL in 0.25 h. The unbound
plasma free fraction was high (Fu=27%), possibly
explaining the robust GHS activity in the in vivo anes-
thesized rat model. Oral bioavailability was 65%, far
greater than the reported bioavailabilities of other pep-
tidyl or peptidomimetic GHSs in the literature. Com-
pound 5c was detected as a circulating metabolite,
though exposure generally represented less than 10% of
parent drug. Pharmacokinetic values for 5c were similar
to those for capromorelin. Following a 1 mgA/kg iv dose,
systemic clearance was high and volume of distribution
was moderate, resulting in a short half-life (CL=56.7
mL/min/kg; Vd=2.6 L/kg; t1/2=0.83 h). Oral bioavail-
ability was low, most likely due to the combination of
incomplete intestinal absorption and high CL (F=12%).
A novel series of pyrazolinone-piperidine (PP) dipeptide
GHSs has been discovered using a pharmacokinetic-
oriented screening strategy that focused on identifying
compounds with good absorption properties. Several
PP analogues with Aib-(d)-O-Bn-Ser subunits exhibited
high intestinal absorption in rat models and possessed
favorable physicochemical properties, including high
aqueous solubility and moderate lipophilicity. One
short-acting compound, capromorelin (5, CP-424391-
18), stimulated GH secretion in several animal species
following intravenous and oral administration and was
selected as a clinical candidate for the treatment of
musculoskeletal frailty in elderly subjects, the results of
which will be reported elsewhere.
Experimental
General chemistry methods
Proton magnetic spectra were recorded on Bruker AC-
250, Bruker AC-300, Varian XL-300 or Varian Unity
400 spectrometers. Chemical shifts are expressed in
parts per million (d) downfield from trimethylsilane; the
deuterium lock signal of the deuterated solvent was used
as an internal standard. Low resolution mass spectra
(LRMS) were obtained on a Hewlett-Packard 5989A
spectrometer using ammonia as the source of chemical
ionization. All reagents, solvents and drying agents were
obtained from commercial sources and were used with-
out further purification unless otherwise specified. All
moisture- or air-sensitive reactions were performed in
flame-dried glassware equipped with septum inlets
under a positive pressure of nitrogen. Analytical thin-
layer chromatography was carried out using silica plates
(E. Merck Kieselgel 60 F254). Preparative chromato-
graphic separations were performed using flash chro-
matography with Amicon silica gel (30 mM, 60 A pore
size). Melting points were recorded on a Buchi 510
apparatus and are uncorrected. Combustion analyses
were obtained from Micromass.
As the only PP GHS with superior bioavailability and
good absorption properties in a rat model, capromor-
elin was selected for pharmacokinetic evaluation in the
dog. After a 1 mgA/kg iv dose, the plasma clearance of
the compound was 19Æ5 mL/min/kg, the volume of
distribution was 2.0Æ0.4 L/kg and the elimination half-
life was 1.3 h. Mean values for Cmax and Tmax following
a 1 mgA/kg po dose were 180Æ66 ng/mL and 1 h. The
plasma free fraction of drug was 51% and the bioavail-
ability was 44%. Compound 5c was also identified as a
circulating plasma metabolite.
Pharmacokinetic characterization of capromorelin in
rats and dogs revealed short plasma elimination half-
lives. In dogs, the short half-life was attributed to a
moderate volume of distribution and a moderate-to-
high clearance due in part to de-methylation of the PP
ring and oxidation of both the benzyl group in the (d)-
O-Bn-Ser moiety and a methyl group in the Aib moiety
(data not shown). The compound did not undergo pro-
teolytic degradation in plasma. The des-methyl meta-
bolite 5c exhibited a similarly short half-life, but
because of its lower rat bioavailability, it did not offer
any advantages over capromorelin. A short half-life was
considered pharmacologically desirable for stimulating
GH secretion (rather than increasing IGF-1 levels) and
preventing attenuation of the post-dose GH response
during repeat administration.9
4-Oxo-piperidine-1,3-dicarboxylic acid 1-tert-butyl ester
3-methyl ester (7). To a mixture of 4-oxo-piperidine-3-
carboxylic acid methyl ester (6, 7.0 g, 36.2 mmol) and
4,4-dimethylaminopyridine (8.82 g, 72.3 mmol) in
CH2Cl2 (200 mL) at 0 ꢀC was added a solution of di-
tert-butyldicarbonate (7.88 g, 36.2 mmol) in CH2Cl2
(150 mL). The reaction mixture was allowed to warm to
room temperature and stir for 19 h. The solvents were
removed in vacuo and the residue was diluted with
CHCl3. The organic solution was washed with 10% aqu-
eous HCl and saturated aqueous NaHCO3, dried over
MgSO4, and concentrated in vacuo to yield the desired
The oral activity of capromorelin was examined in a
dog model that was found to be predictive of GHS
activity in humans.8 Following a single 1 mg/kg po
dose, capromorelin rapidly increased plasma GH levels,
with a maximum peak height of 73 ng/mL. Significant
GHS activity was observed at doses as low as 0.05 mg/
kg. When administered at 1 mg/kg po for 5 days,
capromorelin stimulated GH secretion on the first and
last days of the study; however, the post-dose GH
response on day 5 was somewhat attenuated.16
1
product as an amorphous solid (9.18 g, 99%). H NMR
(CDCl3) d 4.0 (s, 2H), 3.75 (s, 3H), 3.74 (s, 1H), 3.55 (t,
2H), 2.32 (t, 2H), 1.45 (s, 9H). LRMS m/z 258 (M++1).
3-Benzyl-4-oxo-piperidine-1,3-dicarboxylic acid 1-tert-
butyl ester 3-methyl ester (8). Sodium hydride (745 mg,
7.4 mmol, 60% oil dispersion) was added to a solution

586
P. A. Carpino et al. / Bioorg. Med. Chem. 11 (2003) 581–590
1
of 7 (5.0 g, 19.4 mmol) in DMF (10 mL). The reaction
mixture was stirred at room temperature for 0.25 h, then
benzyl bromide (3.32 g, 19.4 mmol) in DMF (15 mL) was
added. The reaction mixture was stirred for 42 h at room
temperature and diluted with ethyl acetate. The organic
solution was washed with H2O and saturated aqueous
NaCl, dried over MgSO4, and concentrated in vacuo
to give the desired product (6.0 g, 89%) as a yellow
(7.8 g, 80%). H NMR (CDCl3) d 7.24–7.13 (m, 5H),
3.50 (m, 1H), 3.40 (d, 1H, J=12.45 Hz), 3.33 (d, 1H,
J=13.29 Hz), 3.16 (d, 1H, J=13.29), 3.08 (s, 3H), 3.05
(m, 1H), 2.60 (m, 3H). LRMS m/z 244 (M++1).
3a-Benzyl-2-ethyl-2,3a,4,5,6,7-hexahydro-pyrazolo[4,3-
c]pyridin-3-one (10b). The title compound 10b was pre-
pared from 9b by the procedure described for 10a. H
NMR (CDCl3) d 7.22–7.07 (m, 10H), 3.58 (m, 1H), 3.46
(m, 1H), 3.35 (m, 1H), 3.28 (m, 2H), 3.10 (d, 1H, J=13.7
Hz), 2.55 (m, 4H), 1.73 (bs, 1H), 0.82 (t, 3H). LRMS m/z
258 (M++1).
1
1
oil. H NMR (CDCl3) d 7.26–7.12 (m, 5H), 4.57 (bs,
1H), 4.17 (bs, 1H), 3.6 (s, 3H), 3.23 (d, 1H, J=14.1
Hz), 3.14 (t, 1H), 3.01 (d, 2H, J=13.29 Hz), 2.7 (m,
1H), 2.42 (d, 1H, J=14 Hz), 1.43 (s, 9H). LRMS m/z
348 (M++1).
3a-Benzyl-2,3a,4,5,6,7-hexahydro-pyrazolo[4,3-c]pyridin-
3-one hydrochloride (10c). A solution of 9c (1.0 g, 3
mmol) in a 2:1 ethanol/concentrated HCl solution (15
mL) was stirred at room temperature for 2 h. The reac-
tion mixture was diluted with ethanol (30 mL) and
concentrated under vacuum to give the desired product
3a-Benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyra-
zolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester (9a).
A mixture of 8 (4.0 g, 11.5 mmol) and methylhydrazine
(530 mg, 11.5 mmol) in ethanol (100 mL) was heated
under reflux for 8 h. The reaction was cooled to room
temperature and the ethanol was removed in vacuo. The
residue was dissolved in toluene (100 mL) and heated
under reflux for 17 h. After cooling to room tempera-
ture, the reaction mixture was concentrated under
vacuum and the residue was purified on silica gel using a
solvent gradient (15:85 v/v ethyl acetate/hexanes to
75:25 v/v ethyl acetate/hexanes) to give the desired pro-
duct (2.6 g, 66%) as a colorless oil. 1H NMR (CDCl3) d
7.25–7.02 (m, 5H), 4.65 (bs, 1H), 4.55 (bs, 1H), 3.2 (d,
1H, J=12.87 Hz), 3.05 (s, 3H), 3.0 (d, 2H, J=13.27
Hz), 2.7 (bs, 3H), 2.52 (d, 1H, J=9.97), 1.51 (s, 9H).
LRMS m/z 344 (M++1).
1
as a colorless solid (0.6 g, 100%). H NMR (MeOH-d4)
d 7.3–7.0 (m, 5H), 4.7 (d, 1H), 4.5 (d, 1H), 3.8–3.6 (m),
3.1–2.6 (m). LRMS m/z 230 (M++1).
{1-[2-(3a-(R)-Benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahy-
dro-pyrazolo[4,3-c]pyridin-5-yl)-1-(R)-benzyloxymethyl-
2-oxo-ethylcarbamoyl]-1-methyl-ethyl}-carbamic acid
tert-butyl ester [(3aR)-12a] and {1-[2-(3a-(S)-benzyl-2-
methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyri-
din-5-yl)-1-(R)-benzyloxymethyl-2-oxo-ethylcarbamoyl]-
1-methyl-ethyl}-carbamic acid tert-butyl ester [(3aS)-
12b]. To a solution of the PP derivative 10a (7.8 g, 32
mmol) in CH2Cl2 (120 mL) at 0 ꢀC was sequentially
added Boc-Aib-(d)-O-Bn-Ser-OH (11, 18.2 g, 48 mmol),
1-hydroxy-7-azabenzotriazole (6.4 g, 48 mmol), 1-[3-
3a-Benzyl-2-ethyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyra-
zolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester (9b).
The title compound 9b was prepared from 8 by the
procedure described for 9a except ethylhydrazine was
(dimethylamino)propyl]-3-ethylcarbodiimide
hydro-
chloride (9.2 g, 48 mmol), and triethylamine (6.7 mL, 48
mmol). The reaction mixture was allowed to warm to
room temperature and stir for 17 h. Ethyl acetate (100
mL) was added and the organic solution was washed
with 10% HCl, saturated aqueous NaHCO3, saturated
aqueous NaCl, dried over Na2SO4, and concentrated
under vacuum. The crude residue was purified by silica
gel chromatography using a solvent gradient (1:1 v/v
ethyl acetate/hexanes to 100% ethyl acetate) to give a
less polar diastereomer, (3aR)-12a (9 g, 46%), and a
more polar diastereomer, (3aS)-12b (9.5 g, 49%). Com-
1
used. H NMR (CDCl3) d 7.24–7.02 (m, 5H), 4.6 (bs,
1H), 4.4 (bs, 1H), 3.55 (m, 1H), 3.35 (m, 1H), 3.16 (d,
1H, J=13.29 Hz), 3.0 (d, 1H, J=13.29 Hz), 2.7 (bs,
3H), 2.53 (d, 1H, J=9.55 Hz), 1.5 (s, 9H), 0.8 (t, 3H).
LRMS m/z 358 (M++1).
3a-Benzyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]-
pyridine-5-carboxylic acid tert-butyl ester (9c). The title
compound 9c was prepared from 8 by the procedure
1
described for 9a except hydrazine hydrate was used. H
1
NMR (CDCl3) d 8.08 (s, 1H), 7.2–7.07 (m, 5H), 4.66 (s,
1H), 4.46 (s, 1H), 3.2 (d, 1H, J=12.46 Hz), 3.03 (d, 1H,
J=13.75 Hz), 2.7 (bs, 3H), 2.56 (d, 1H, J=9.14 Hz), 1.5
(s, 9H). LRMS m/z 330 (M++1).
pound (3aR)-12a: H NMR (CDCl3) d 7.30–7.14 (m,
10H), 7.05 (d, 1H, J=7.47 Hz), 5.22 (q, 1H), 5.05 (d,
1H, J=12.04 Hz), 4.85 (d, 2H, J=3.74 Hz), 3.68 (m,
1H), 3.62 (t, 1H), 3.13 (d, 1H, J=13.28 Hz), 3.05 (s,
3H), 2.90 (m, 2H), 2.53 (d, 1H, J=10.80 Hz), 2.47 (d,
1H, J=13.29 Hz), 1.47 (s, 3H), 1.44 (s, 3H), 1.35 (s,
9H). LRMS m/z 606 (M++1). Compound (3aS)-12b:
1H NMR (CDCl3) d 7.30–7.06 (m, 10H), 6.75 (d, 1H,
J=6.65 Hz), 5.3 (m, 1H), 5.05 (d, 1H, J=12.46 Hz),
4.61 (dd, 2H, J=11.21, 29 Hz), 3.8 (m, 1H), 3.65 (t,
1H), 3.1 (d, 1H, J=13.7 Hz), 2.99 (s, 3H), 2.90 (d, 1H,
J=13.29 Hz), 1.54 (s, 3H), 1.45 (s, 3H), 1.4 (s, 9H).
LRMS m/z 606 (M++1).
3a-Benzyl-2-methyl-2,3a,4,5,6,7-hexahydro-pyrazolo[4,3-
c]pyridin-3-one (10a). To a solution of 9a (13.8, 40
ꢀ
mmol) in CH2Cl2 (140 mL) at 0 C was added cold tri-
fluoroacetic acid (70 mL). The reaction mixture was
stirred for 2.5 h and then concentrated under vacuum.
Ethyl acetate was added and the solution was con-
centrated to dryness. The residue was then diluted with
CHCl3 and the organic solution was washed with satu-
rated aqueous NaHCO3, dried over MgSO4, and con-
centrated. The crude residue was chromatographed on
silica gel using a gradient of 1:99 v/v MeOH/CH2Cl2 to
1:10 v/v MeOH/CH2Cl2 to give the desired product 10a
{1-[2-(3a-(R)-Benzyl-2-ethyl-3-oxo-2,3,3a,4,6,7-hexahy-
dro-pyrazolo[4,3-c]pyridin-5-yl)-1-(R)-benzyloxymethyl-
2-oxo-ethylcarbamoyl]-1-methyl-ethyl}-carbamic
acid

P. A. Carpino et al. / Bioorg. Med. Chem. 11 (2003) 581–590
587
tert-butyl ester [(3aR)-12c]. The title compound was
prepared by coupling 10b with 11 using the procedure
described for (3aR)-12a. The desired compound was
obtained as the less polar compound of the diaster-
eomeric mixture after silica gel chromatography using a
solvent gradient of 1:1 v/v ethyl acetate/hexanes to
100% ethyl acetate. 1H NMR (CDCl3) d 7.5–7.1
(m, 10H), 5.25 (t, 1H), 5.05 (d, 1H, J=13.7 Hz),
4.48 (d, 2H), 3.7 (m, 1H), 3.6 (m, 1H), 3.35 (m,
1H), 3.13 (d, 1H, J=13.7 Hz), 2.9 (m, 2H), 2.58
(m, 1H), 2.47 (d, 1H, J=13 Hz), 1.54 (s), 1.47 (s, 3H),
1.43 (s, 3H), 1.35 (s, 9H), 0.8 (t, 3H). LRMS m/z 621
(M++1).
6.75 (d, 1H, J=6.65 Hz), 5.3 (bs, 1H), 5.06 (1H, dd,
J=12.88, 23.89 Hz), 4.7–4.45 (m, 3H), 3.85–3.7 (m,
2H), 3.05 (q, 1H), 2.99 (s, 3H), 2.89 (t, 1H), 2.6 (m, 1H),
2.47 (d, 1H, J=13.28 Hz), 2.4 (d, 1H, J=11.2 Hz), 1.55
(s, 6H). LRMS m/z 506 (M++1).
2-Amino-N-[2-(3a-(S)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-
hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-1-(R)-benzyloxyme-
thyl-2-oxo-ethyl]-isobutyramide (L)-tartrate (5a). (l)-(+)-
Tartaric acid (122 mg, 0.81 mmol) in MeOH (5 mL) was
added to a solution of (3aS)-13b (410 mg, 0.81 mmol) in
MeOH (5 mL). The reaction mixture was stirred at
room temperature for 17 h and concentrated under
vacuum to give the desired product (530 mg, 100%). 1H
NMR (MeOH-d4) d 7.37–7.0 (m, 9H), 6.8 (d, 1H,
J=6.65 Hz), 5.30 (t, 1H), 4.60 (m, 2H), 4.37 (s, 2H),
3.88 (t, 1H), 3.80 (t, 1H), 3.72 (m, 1H), 3.42 (d, 1H,
J=13.7 Hz), 3.2–3.07 (m, 2H), 2.96 (s, 3H), 2.75 (m,
1H), 2.6 (d, 1H, J=12.87 Hz), 2.5 (d, 1H, J=13.29 Hz),
1.55 (s, 6H). LRMS m/z 506 (M++1).
2-Amino-N-[2-(3a-(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-
hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-1-(R)-benzyloxyme-
thyl-2-oxo-ethyl]-isobutyramide [(3aR)-13a]. To a solution
of (3aR)-12a (10 g, 17 mmol) in CH2Cl2 (69 mL) at 0 ꢀC
was slowly added trifluoroacetic acid (35 mL). The
reaction mixture was allowed to warm to room tem-
perature. After 3 h, the organic solution was con-
centrated under vacuum. The residue was diluted with
CH2Cl2 and washed with saturated aqueous NaHCO3,
dried over MgSO4 and concentrated in vacuo to afford
(3aR)-13a (7.4 g, 86%). 1H NMR (MeOH-d4) d 7.35–7.1
(m, 10H), 5.2 (t, 1H), 4.55 (s, 2H), 3.75 (d, 2H), 3.05
(m), 2.61 (d, 1H), 2.55 (d, 1H), 1.45 (s, 3H), 1.42 (s, 3H).
LRMS m/z 506 (M++1).
2-Amino-N-[2-(3a-(R)-benzyl-2-ethyl-3-oxo-2,3,3a,4,6,7-
hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-1-(R)-benzyloxy-
methyl-2-oxo-ethyl]-isobutyramide hydrochloride (5b). A
solution of dipeptide (3aR)-12c (300 mg, 0.48 mmol) in
a 2:1 ethanol/concentrated HCl solution (30 mL) was
stirred at room temperature for 5 h. The reaction mix-
ture was diluted with ethanol (30 mL) and concentrated
under vacuum. Additional ethanol was added to the
residue and the solution was concentrated in vacuo.
This process was repeated until the product was
2-Amino-N-[2-(3a-(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-
hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-1-(R)-benzyloxyme-
thyl-2-oxo-ethyl]-isobutyramide (L)-tartrate (5). To a solu-
tion of (3aR)-13a (385 g, 0.761 mol) in methanol (4 L) at
room temperature was added (l)-(+)-tartaric acid (114
g, 0.761 mol). The reaction mixture was stirred for 17 h,
filtered to remove any insoluble residues, and con-
centrated under vacuum. Ethyl acetate (12 L) was added
and the solution was concentrated in vacuo. The residue
was re-dissolved in ethyl acetate (12 L) and heated
under reflux for 16 h. After slowly cooling to room
temperature, a crystalline white solid slowly precipitated
out of solution and was collected by filtration to give the
desired product (482 g, 96.8%), mp 174–176 ꢀC. 1H
NMR (MeOH-d4) d 7.3–7.1 (m, 10H), 5.2 (t, 1H), 4.55
(s, 2H), 4.4 (s), 3.78 (m, 2H), 3.1 (d, 1H, J=13.3 Hz),
3.01 (s, 3H), 2.97 (d, 1H), 2.65 (d, 1H, J=12.4 Hz),
2.55 (d, 1H, J=11 Hz), 1.59 (s, 3H), 1.58 (s, 3H).
LRMS m/z 506 (M++1). Anal. calcd for C32H41N5O10:
C, 58.62; H, 6.30; N, 10.68. Found: C, 58.65; H, 6.14; N,
10.78.
1
obtained as a solid residue (250 mg, 100%). H NMR
(MeOH-d4) d 7.3–7.1 (m, 10H), 5.18 (t, 1H), 4.58 (s,
2H), 3.77 (m, 2H), 3.56 (m, 2H), 3.36 (m, 2H), 3.13 (d,
1H, J=13 Hz), 3.0 (m, 2H), 2.6 (t, 2H), 1.6 (s, 3H), 1.59
(s, 3H), 0.8 (t, 3H). LRMS m/z 521 (M++1).
3-Benzyl-4-oxo-piperidine-3-carboxylic acid methyl ester
(14). To a solution of 8 (20.0 g, 5.8 mmol) in CH2Cl2
(200 mL) at 0 ꢀC was added cold trifluoroacetic acid
(100 mL). The reaction mixture was stirred for 2 h at
0 ꢀC and for 3 h at room temperature and was then
concentrated under vacuum. Ethyl acetate was added
and the solution was concentrated again in vacuo. The
residue was then diluted with CHCl3 and the organic
solution was washed with saturated aqueous NaHCO3,
dried over MgSO4, and concentrated under vacuum to
give the desired product as a white solid (13.8 g, 97%).
1H NMR (CDCl3) d 7.3–7.25 (m, 4H), 7.05 (m, 1H),
3.69 (s, 3H), 3.45 (m, 1H), 3.36 (d, 1H, J=14.12 Hz),
3.2 (m, 1H), 2.95 (m, 1H), 2.85 (d, 1H, J=14.12 Hz),
2.68 (d, 1H, J=13.71 Hz), 2.5 (m, 2H). LRMS m/z 248
(M++1).
2-Amino-N-[2-(3a-(S)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-
hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-1-(R)-benzyloxyme-
thyl-2-oxo-ethyl]-isobutyramide [(3aS)-13b]. A solution of
dipeptide (3aS)-12b (0.5 g, 0.83 mmol) in a 3:1 ethanol/
concentrated HCl solution (26 mL) was stirred at room
temperature for 5 h. The reaction mixture was diluted
with ethanol (50 mL) and concentrated under vacuum.
CHCl3 (10 mL) was added and the organic solution was
washed with saturated aqueous NaHCO3, saturated
aqueous NaCl, dried over MgSO4, and concentrated in
vacuo to give the desired product as a colorless solid
3-(R)-Benzyl-1-[3-benzyloxy-2-(R)-(2-tert-butoxycarbo-
nylamino-2-methyl-propionylamino)-propionyl]-4-oxo-pi-
peridine-3-carboxylic acid methyl ester (15). To
a
solution of 14 (13.8 g, 56 mmol) in CH2Cl2 (200 mL) at
0 ꢀC was added Boc-Aib-(d)-O-Bn-Ser-OH (11, 21 g,
56 mmol), 1-hydroxy-7-azabenzotriazole (11.4 g, 84
mmol) and 1-[3-(dimethylamino)propyl]-3-ethylcarbo-
diimide hydrochloride (11 g, 56 mmol). The reaction
mixture was allowed to warm to room temperature and
1
(0.41 g, 98%). H NMR (CDCl3) d 7.36–7.1 (m, 9H),

588
P. A. Carpino et al. / Bioorg. Med. Chem. 11 (2003) 581–590
stir for 72 h. The organic solution was washed with
saturated aqueous NaHCO3 and saturated aqueous
NaCl, dried over MgSO4, and concentrated under
vacuum. The crude residue was chromatographed on
silica gel using a solvent gradient (2:1 v/v ethyl acetate/
hexanes to 100% EtOAc) to give the desired product as
the less polar isomer of the diastereomeric mixture (2.6
Hz), 3.0 (m, 2H), 2.63 (d, 1H, J=12.87 Hz), 2.56 (d, 1H,
J=10.8 Hz), 1.59 (s, 3H), 1.58 (s, 3H). LRMS m/z 492
(M++1).
Biological methods
Binding assay. Membranes were prepared from
HEK293 cells (ATCC) stably transfected with the
human GHS-R1a receptor cDNA in the plasmid
pcDNA3.1neo (Invitrogen).21 Competition radioligand
binding assays were performed in 96-well format with
GF/C filters pre-soaked in 0.3% polyethyleneimine.
Assays were performed at room temperature for 1 h in
duplicate using 50 pM [125I]-ghrelin (Perkin–Elmer
NEN-Life Sciences) and 1 mg membrane per well in 50
mM HEPES, pH 7.4, 10 mM MgCl2, 0.2% bovine
serum albumin and the following protease inhibitors:
100 mg/mL bacitracin, 100 mg/mL benzamidine, 5 mg/
mL aprotinin, 5 mg/mL leupeptin. The membranes were
harvested and washed three times with ice-cold wash
buffer containing 50 mM HEPES, pH 7.4 and 10 mM
MgCl2. IC50 and Ki values were determined using Prism
by GraphpadTM. The Kd of [125I]-ghrelin at membranes
expressing human GHS receptors was calculated to be
0.2 nM. No detectable binding was observed in un-
transfected HEK293 cell membranes (data not shown).
1
g, 28%). H NMR (MeOH-d4) d 7.35–7.15 (m, 10H),
5.40–4.70 (m, 3H), 4.42 (s, 2H), 3.70–3.40 (m, 5H), 3.2
(d, 1H, J=14 Hz), 2.88 (m, 1H), 2.55 (m, 2H), 1.45 (s,
3H), 1.42 (s, 1H), 1.38 (s, 9H). LRMS m/z 611
(M++1).
{1-[2-(3a-(R)-Benzyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyra-
zolo[4,3-c]pyridin-5-yl)-1-(R)-benzyloxymethyl-2-oxo-
ethylcarbamoyl]-1-methyl-ethyl}-carbamic acid tert-butyl
ester (12d). A solution of 15 (820 mg, 1.32 mmol),
hydrazine sulfate (343 mg, 2.6 mmol) and sodium ace-
tate (431 mg, 5.26 mmol) in EtOH (25 mL) was heated
at 78 ꢀC for 17 h. The reaction mixture was cooled to
room temperature and concentrated under vacuum. The
crude residue was diluted with EtOAc and washed with
saturated aqueous NaHCO3 and saturated aqueous
NaCl, dried over MgSO4, and concentrated under
vacuum. The crude residue was chromatographed on
silica gel using a solvent gradient (3:1 v/v EtOAc/hexane
to 100% EtOAc) to give the desired product as a white
1
solid (550 mg, 14%). H NMR (CDCl3) d 9.0 (bs, 1H),
Functional activity in vitro (GH release in rat pituitary
cell cultures). Primary pituitary cell cultures were estab-
lished by enzymatic dissociation of anterior pituitary
glands from 6-week-old male Wistar rats.16 Cells were
suspended in Dulbecco’s modified Eagle’s medium
(DMEM, 4.5 g/L glucose supplemented with 1 mM
sodium pyruvate, 1% MEM non-essential amino acids,
10% heat-inactivated horse serum, 2.5% fetal bovine
serum plus antibiotics), plated at 1Â105 cells per well in
24-well tissue culture plates (Costar, Corning, NY) and
incubated in a humidified 5% CO2/95% air incubator at
37 ꢀC. Hormone release was performed 3–4 days after
plating. Cell cultures were rinsed twice and then equili-
brated at 37 ꢀC in release medium (DMEM with 25 mM
HEPES buffer, pH 7.4 and 5 mg/mL bovine serum
albumin) for 30 min. This medium was aspirated and
replaced with pre-warmed release medium containing
test agents. After a 15 min incubation period at 37 ꢀC,
the medium was removed and assayed for GH. Results
are expressed as meanÆSEM of quadruplicate wells.
Groups were compared by unpaired Student’s 2-tailed t-
test unless otherwise indicated. The data are reported as
the concentration necessary to produce a half-maximum
response (EC50). MK-0677 was used as a reference
standard in control plates.
7.30–7.10 (m, 10H), 5.3 (q, 1H), 4.96 (d, 1H, J=12 Hz),
4.50 (s, 2H), 3.75–2.80 (m, 2H), 3.12 (d, 1H, J=13 Hz),
2.92 (m, 2H), 2.5 (m, 2H), 1.46 (s, 3H), 1.42 (s, 3H), 1.37
(s, 9H). LRMS m/z 592 (M++1).
2-Amino-N-[2-(3a-(R)-benzyl-3-oxo-2,3,3a,4,6,7-hexahy-
dro-pyrazolo[4,3-c]pyridin-5-yl)-1-(R)-benzyloxymethyl-
2-oxo-ethyl]-isobutyramide hydrochloride (16). The title
compound was obtained from 12d using the method
1
described for the preparation of 5b. H NMR (MeOH-
d4) d 7.36–7.17 (m, 10H), 5.26 (t, 1H), 4.56 (s, 2H),
3.80 (d, 2H, J=4.27 Hz), 3.1 (d, 1H, J=13.67 Hz), 3.0
(m, 2H), 2.63 (d, 1H, J=12.6 Hz), 2.56 (d, 1H,
J=10.26 Hz), 1.61 (s, 3H), 1.60 (s, 3H). LRMS m/z
492 (M++1).
2-Amino-N-[2-(3a-(R)-benzyl-3-oxo-2,3,3a,4,6,7-hexahy-
dro-pyrazolo[4,3-c]pyridin-5-yl)-1-(R)-benzyloxymethyl-
2-oxo-ethyl]-isobutyramide (L)-tartrate (5c). Compound
16 (343 mg, 0.65 mmol) was added to CHCl3 (10 mL) in
a separatory funnel and the organic solution was
washed with saturated aqueous NaHCO3, saturated
aqueous NaCl, dried over MgSO4, and concentrated in
vacuo. The residue was dissolved in MeOH (5 mL) and
a solution of (l)-tartaric acid (99 mg, 0.65 mmol) in
MeOH (3 mL) was added. The reaction mixture was
stirred at room temperature for 17 h and concentrated
under vacuum. The residue was dissolved in EtOAc (5
mL) and heated at 80 ꢀC for 1 h and 62 ꢀC for 17 h, after
which time a white, finely crystalline solid precipitated
out of solution. After cooling to room temperature, the
solid was collected by filtration to give the desired pro-
duct (320 mg, 77%), mp 211–213 ꢀC. 1H NMR (MeOH-
d4) d 7.35–7.16 (m, 10H), 5.19 (t, 1H), 4.55 (s, 2H), 4.37
(s, 2H), 3.77 (d, 2H, J=5.39 Hz), 3.10 (d, 1H, J=13.7
Functional activity in vivo (GH release in anesthesized
rat and canine models). Wistar rats (Charles River
Laboratories, Wilmington, MA) were anesthesized with
pentobarbital. Compounds were administered intrave-
nously and the serum GH levels were measured 10 min
post-dose. The ratio of the serum GH concentration at
10 min to the serum GH level prior to injection was
calculated and used to determine ED50 values. Beagle
dogs, male and female, 2–5 years old, were purchased
from Marshall Farms (North Rose, NY). Dogs were

P. A. Carpino et al. / Bioorg. Med. Chem. 11 (2003) 581–590
589
fasted overnight, dosed in the early morning by oral
gavage, then fed approximately 3 h after dosing. Access
to water was provided at all times. Canine blood sam-
ples were withdrawn by direct venipuncture of the
jugular vein into heparinized vacutainers. Serum and
plasma samples were stored frozen pending hormone
assays. Rat and canine GHs were measured by species-
specific double antibody radioimmunoassays using
reagents and protocols obtained from the NIDDK
National Hormone and Pituitary Program (Dr. A. F.
Parlow, Harbor-UCLA Medical Center Torrance, CA).
All animal studies were conducted under approved
protocols in accordance with NIH Guidelines for the
Care and Use of Laboratory.
gradient elution program. The mobile phases consisted
of A=10:90:0.01 (v/v/v) acetonitrile/water/formic acid
and B=90:10:0.01 (v/v/v) acetonitrile/water/formic
acid. A switching valve was used to divert the HPLC
flow after the column to waste from 0 to 4 min and 8 to
13 min. Samples were detected via a Hewlett-Packard
5989B Mass Spectrometer (equipped with a hexapole
ion guide driver). Ions were generated with a Hewlett-
Packard 59987A Electrospray source. Capromorelin
and internal standard (MK-0677) were monitored at
m/z ratios of 506.05 and 529.00, respectively. Capro-
morelin and internal standard had retention times of
approximately 5.5 min. The dynamic range was 5–1000
ng/mL with a run time of 13 min. Pharmacokinetic
parameters were determined using PK_PARAM version
1.040.
Pharmacokinetics of capromorelin in rats. Eight adult
female Sprague–Dawley rats were prepared for use by
surgical implantation of a cannula in the femoral vein
while under methoxyflurane anesthesia the day before
study initiation. Study rats were fasted overnight prior
to dosing, allowed free access to water and housed in
standard polycarbonate rodent cages. The rodents were
allowed access to food 4 h post-dose. The dose (intra-
venous and oral) was prepared as a 0.5 mg/mL base
equivalent (0.5 mgA/kg) solution of capromorelin in
deionized water. Four Sprague–Dawley rats (weighing
0.280–0.305 kg) received an iv dose of 1 mgA/kg of
capromorelin. The dose was administered via the
femoral vein catheter followed by a 1 mL infusion of
normal saline to rinse the catheter. Blood samples (0.5
mL) were taken from the femoral vein catheter pre-
dose, 0.083, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6 and 8 h post-
dose and transferred into heparinized microtainers. The
blood volume withdrawn was replaced with normal sal-
ine. Plasma was harvested from the centrifuged micro-
tainers and stored frozen at À70 ꢀC in 500 mL
polypropylene microcentrifuge tubes. Four Sprague–
Dawley rats (weighing 0.275–0.290 kg) received an oral
dose of 1 mgA/kg capromorelin. The dose was admi-
nistered via an 18-gauge, 3 in., curved gavage needle.
Blood samples (0.5 mL) were taken from the femoral
vein catheter pre-dose, 0.083, 0.17, 0.25, 0.5, 0.75, 1, 2,
4, 6 and 8 h post-dose and transferred into hepar-
inized microtainers. The blood volume withdrawn
was replaced with normal saline. Plasma was
harvested from the centrifuged microtainers and
stored frozen at À70 ꢀC in 500 mL polypropylene
microcentrifuge tubes.
Pharmacokinetics of compound 5c in rat. The protocol
described for capromorelin was followed in preparing
and dosing animals.
Sample extraction of compound 5c and analysis. A 100
mL aliquot of plasma was added to a 5 mL glass conical
tube. The sample was vortexed with 100 mL of deionized
water (containing 250 ng/mL MK-0677 as the internal
standard), 25 mL of 1.0 M sodium hydroxide and 1.5 mL
of ethyl acetate followed by centrifugation. The organic
layer was transferred to a 15 mL conical tube and dried
under a stream of nitrogen at 60 ꢀC. The samples were
reconstituted in 100 L of water, vortexed, centrifuged
and transferred into injection vials. The samples (40 mL)
were injected onto a Keystone Inertsil ODS-2 2.0Â50
mm 5 mm C18 analytical HPLC column. The com-
pounds of interest were eluted from the column using a
gradient elution program. The mobile phases consisted
of A=10:90:0.01 (v/v/v) acetonitrile/water/formic acid
and B=90:10:0.01 (v/v/v) acetonitrile/water/formic
acid. Samples were detected via a Hewlett-Packard
5989B Mass Spectrometer. Ions were generated with a
Hewlett-Packard 59987A Electrospray source. Com-
pound 5c and internal standard (MK-0677) were mon-
itored at m/z ratios of 492.05 and 529.00, respectively.
Compound 5c and internal standard had retention
times of approximately 5.5 min. The dynamic range was
5–1000 ng/mL with a run time of 13 min.
Pharmacokinetics of capromorelin in dogs. Four adult
beagle dogs (2 males and 2 females weighing 8.9–12.9
kg) were fasted overnight prior to dosing but were
allowed free access to water. The dogs were allowed
access to food 4 h post-dose. The iv dose was prepared
as a 10 mgA/mL solution of capromorelin in deionized
water and filter sterilized. The oral dose was prepared as
a 2 mgA/mL solution of capromorelin in deionized
water. Four beagle dogs (weighing 8.9–12.9 kg) received
an oral dose of 1 mgA/kg of capromorelin. The dose
was administered via an oral gavage tube. Blood sam-
ples (2 mL) were taken from the jugular vein pre-dose,
0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6 and 8 h post-dose. Blood
samples were collected directly into heparinized vacu-
tainers. Plasma was harvested from the centrifuged
vacutainers and stored frozen at À70 ꢀC in 500 mL
polypropylene microcentrifuge tubes. Following a 2 day
Sample extraction of capromorelin and analysis. A 100
mL aliquot of plasma (obtained as described above) was
added to a 5 mL glass conical tube. The sample was
vortexed with 100 mL of deionized water (containing
250 ng/mL MK-0677 as the internal standard), 25 mL of
1.0 M sodium hydroxide and 1.5 mL of ethyl acetate
followed by centrifugation. The organic layer was
transferred to a 15 mL conical tube and dried under a
stream of nitrogen at 60 ꢀC. The samples were recon-
stituted in 100 mL of water, vortexed, centrifuged and
transferred into injection vials. The samples (40 mL)
were injected onto a Keystone Inertsil ODS-2 2.0Â50
mm 5 mm C18 analytical HPLC column. The com-
pounds of interest were eluted from the column using a

590
P. A. Carpino et al. / Bioorg. Med. Chem. 11 (2003) 581–590
wash-out period, the same four beagle dogs received an
iv dose of 1 mgA/kg of capromorelin. The dose was
administered via the cephalic vein. Blood samples (2
mL) were taken from the jugular vein pre-dose, 0.083,
0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6 and 8 h post-dose. Blood
samples were collected directly into heparinized vacu-
tainers. Plasma was harvested from the centrifuged
vacutainers and stored frozen at À70 ꢀC in 500 mL
polypropylene microcentrifuge tubes.
8. Patchett, A. A.; Nargund, R. P.; Tata, J. R.; Chen, M.-H.;
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2439.
10. Schoen, W. R.; Pisano, J. M.; Prendergast, K.; Wyvratt,
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Solubility measurements. Capromorelin (an amount in
excess of its solubility) was added to 2 mL of deionized
water. The sample was gently agitated for 24 h at room
temperature (25 ꢀC). After this time the samples were
filtered, and the filtrate concentration of capromorelin
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lowed to measure the solubility of capromorelin in 2 mL
of a 0.02 M sodium phosphate/0.02 sodium acetate
combination buffer at pH 7.
References and Notes
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