Spiro(indoline-3,4′-piperidine) growth hormone secretagogues as ghrelin mimetics

Article abstract of DOI:10.1016/S0960-894X(01)00324-9

A series of small molecules derived from MK-0677, a potent synthetic GHS, mimicking the N-terminal Gly-Ser-O-(n-octanoyl)-L-Ser-Phe segment of ghrelin was synthesized and tested in a binding and in a functional assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Replacement of Phe in this tetrapeptide with a spiro(indoline-3,4′-piperidine) group, Gly-Ser with 2-aminoisobutyric acid, and O-(n-octanoyl)-L-Ser with O-benzyl-D-Ser provided synthetic GHS agonists with similar functional potency as ghrelin.

Full text of DOI:10.1016/S0960-894X(01)00324-9

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1955–1957
Spiro(indoline-3,40-piperidine) Growth Hormone Secretagogues
as Ghrelin Mimetics
Brenda L. Palucki,a,* Scott D. Feighner,b Sheng-Shung Pong,b Karen Kulju McKee,b
Donna L. Hreniuk,b Carina Tan,b Andrew D. Howard,b Lex H. Y. Van der Ploeg,b
Arthur A. Patchetta and Ravi P. Nargunda
aDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Obesity Research, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 23 April 2001; accepted 9 May 2001
Abstract—Aseries of small molecules derived from MK-0677, a potent synthetic GHS, mimicking the N-terminal Gly-Ser- O-(n-
octanoyl)-l-Ser-Phe segment of ghrelin was synthesized and tested in a binding and in a functional assay measuring intracellular
calcium elevation in HEK-293 cells expressing hGHSR1a. Replacement of Phe in this tetrapeptide with a spiro(indoline-3,40-
piperidine) group, Gly-Ser with 2-aminoisobutyric acid, and O-(n-octanoyl)-l-Ser with O-benzyl-d-Ser provided synthetic GHS
agonists with similar functional potency as ghrelin. # 2001 Elsevier Science Ltd. All rights reserved.
Over the past decade, extensive research has been
focused on identifying synthetic, nonpeptidyl growth
hormone secretagogues (GHSs) as an alternative treat-
ment to growth hormone replacement therapy.1 For
example, research at Merck & Co. has led to the dis-
covery of MK-0677, one of the most potent peptidomi-
metic GHSs.2 Acomparison of the structural
architecture of synthetic MK-0677 with natural endo-
genous ligand(s) was not previously possible because
natural endogenous ligand(s) were not yet identified. A
systematic comparison of the structural architecture of
natural and synthetic GHSs has the potential to be uti-
lized in the identification of new, perhaps more potent
peptidomimetic GHSs.
for its interaction with GHSR1a. Evaluation of peptidyl
ghrelin analogues revealed that the N-terminal tetra-
peptide, Gly-Ser-O-(n-octanoyl)-l-Ser-Phe, is the smal-
lest segment of ghrelin (28-amino acid peptide) which
has agonist activity at hGHSR1a.4 Based upon these
studies, we wanted to examine if its structural features
can be used with advantage in synthetic peptidomimetic
GHS compounds (i.e., MK-0677).
We speculated that our peptidomimetic GHS com-
pounds may bind to the cloned hGHS1a receptor
mimicking the Gly-Ser-O-(n-octanoyl)-l-Ser-Phe N-
terminal sequence of ghrelin [ghrelin(1-4)]. In particular,
we envisioned that the spiro(indoline-3,40-piperidine)
group binds to the receptor in the same region as Phe in
the truncated N-terminal tetrapeptide of ghrelin, and
that the dipeptide part-structure of MK-0677 binds to
the receptor in the same region as Gly-Ser-O-(n-octa-
noyl)-l-Ser. Herein, we report the synthesis and evalua-
tion of novel MK-0677 analogues containing N-
terminal analogues of ghrelin, which show similar
functional potency as ghrelin.
Ghrelin was recently isolated and identified as an endo-
genous ligand of the GHS receptor(s).3 This discovery
has sparked interest in the structural features responsible
The spiro(indoline-3,40-piperidine) compounds were
prepared in a linear fashion as illustrated in Scheme 1.
*Corresponding author. Tel.: +1-732-594-6188; fax: +1-732-594-
3007; e-mail: brenda_palucki@merck.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00324-9

1956
B. L. Palucki et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1955–1957
butyric acid (AIB), and final removal of the Boc pro-
tecting group.
The spiro(indoline-3,40-piperidine) analogues 8–12
shown in Figure 1 were prepared by modifications to
the synthetic route described in Scheme 1. For instance,
N-Boc-d-serine-O-benzyl was utilized in the first amino
acid coupling reaction. This intermediate provided ana-
logues 8–12 via the acylation, coupling, and deprotection
methods shown in Scheme 1.
The spiro(indoline-3,40-piperidine) compounds (6–12)
were evaluated for their binding affinities to the cloned
hGHSR1a via
a competitive binding assay with
[35S]MK-0677 as the radiolabeled ligand. In addition,
these compounds were also examined for functional
potency via their ability to stimulate inositol tripho-
sphate-coupled mobilization of intracellular calcium in
HEK-293 cells expressing hGHSR1a. The data is
presented in Table 1.
Scheme 1. Reagents and conditions: (a) N-Boc-l-serine-O-benzyl,
EDC, HOBt, DIEA, CH2Cl2; (b) H2, Pd/C, EtOH; (c) capryloyl chlo-
ride, pyridine, CH2Cl2; (d) 1:1 TFA–CH2Cl2; (e) N-Boc-l-serine-O-
benzyl, EDC, HOBt, DIEA, CH2Cl2; (f) N-Boc-glycine, EDC, HOBt,
DIEA, CH2Cl2; (g) N-Boc-aminoisobutyric acid, EDC, HOBt, DIEA,
CH2Cl2.
As shown in Table 1, replacement of Phe with N-
methanesulfonyl spiro(indoline-3,40-piperidine) (6) led
to a 14- and 6-fold decrease in functional potency in
comparison to ghrelin and ghrelin(1-4), respectively.
MK-0677, however, contains an O-benzyl-d-serine
amino acid, and this d-amino acid contributes impor-
tantly to its binding and functional potency.2 Likewise,
replacement of O-(n-octanoyl)-l-Ser with O-(n-octa-
noyl)-d-Ser provided a GHS agonist (9) with similar
functional potency as ghrelin and ghrelin(1-4), but with
a 7-fold improvement in binding potency in comparison
with ghrelin(1-4). Therefore, it appears the spiro(indo-
line-3,40-piperidine) group can serve as a replacement
for Phe in ghrelin(1-4).
Spiro(indoline-3,40-piperidine) 15 was coupled to N-
Boc-l-serine-O-benzyl using 1-hydroxybenzotriazole
hydrate (HOBt) and 1-[3-(dimethylamino)propyl]-3-
.
ethylcarbodiimide HCl (EDC). Removal of the benzyl
protecting group via hydrogenation provided alcohol 2,
which was acylated with capryloyl chloride to yield ester
3. Ester 3 was treated with trifluoroacetic acid (TFA) in
methylene chloride to give the free amine which was
then coupled with N-Boc-l-serine-O-benzyl to provide
compound 4. Another Boc deprotection reaction fol-
lowed by a third amino acid coupling reaction with N-
Boc-glycine afforded compound 5. Final benzyl removal
via hydrogenation and Boc removal with TFAprovided
spiro(indoline-3,40-piperidine) 6 as a TFAsalt. Analo-
gue 7 was synthesized from ester 3 via removal of the
Boc protecting group, coupling with N-Boc-aminoiso-
We also examined the binding and functional properties
of additional ghrelin(1-4)/MK-0677-hybrid compounds
with the cloned hGHS1a receptor. Thus, replacement of
O-(n-octanoyl)-l-Ser in ghrelin(1-4) with the O-benzyl-
d-serine amino acid in MK-0677 (12) led to a notable
improvement in both binding and functional potency.
The benzyl ether analogue (12) is 4- and 9-fold more
potent than ghrelin and ghrelin(1-4), respectively, with
moderate binding affinity. Interestingly, removal of the
Table 1. Binding and functional data of spiro(indoline-3,40-piper-
idine) analogues of the ghrelin tetrapeptide segment
Compound
Binding assaya
IC50 (nM)
Functional assayb
EC50 (nM)
Human ghrelin
Ghrelin(1-4)c
MK-0677
6
7
8
9
10
11
12
0.25c
889c
0.63
2670
590.5
828.5
128
32c
72c
1.43
448.5
378.1
432.2
68.2
19.6
69.2
7.95
8.12
209
36.2
[
a 35S]MK-0677 binding assay.
bAequorin bioluminescence assay.
cSee ref 4.
Figure 1. Spiro(indoline-3,40-piperidine) analogues.

B. L. Palucki et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1955–1957
1957
glycine amino acid led to a compound (11) with similar
functional potency to ghrelin and ghrelin(1-4), but with
a 4-fold improvement in binding affinity compared to
ghrelin(1-4).
than the truncated N-terminal ghrelin tetrapeptide,
ghrelin(1-4), which is the smallest segment of ghrelin
displaying agonist activity.
Encouraged by the results of the dipeptidyl spiro(indo-
line-3,40-piperidine) analogue (11), we evaluated whe-
ther aminoisobutyric acid (AIB) can be a replacement
for Gly-Ser. Indeed, analogue 10 shows similar func-
tional potency as ghrelin, but with a 32-fold decrease in
binding affinity. In comparison with ghrelin(1-4), how-
ever, the AIB replacement (10) improves binding
potency about 100-fold. More importantly, this AIB
analogue (10) binds to the cloned hGHSR1a better than
the corresponding Gly-Ser analogue (9). As discussed
earlier, the O-(n-octanoyl)-l-Ser analogue (7) is less
potent than the d-analogue (10). These studies empha-
size the importance of stereochemistry in truncated ver-
sions of ghrelin as was the case with MK-0677 and its
analogues.
References and Notes
1. (a) Nargund, R. P.; Patchett, A. A.; Bach, M. A.; Murphy,
M. G.; Smith, R. G. J. Med. Chem. 1998, 41, 3103. (b) Rosen,
T.; Johannsson, G.; Johansson, J.-O.; Bengtsson, B.-A. Horm.
Res. 1995, 43, 93. (c) Strobl, J. S.; Thomas, M. J. Pharmacol.
Rev. 1993, 46, 1.
2. Patchett, A. A.; Nargund, R. P.; Tata, J. R.; Chen, M.-H.;
Barakat, K. J.; Johnston, D. B. R.; Cheng, K.; Chan, W. W.-
S.; Butler, B.; Hickey, G.; Jacks, T.; Schleim, K.; Pong, S.-S.;
Chaung, L.-Y. P.; Chen, H. Y.; Frazier, E.; Leung, K. H.;
Chiu, S.-H. L.; Smith, R. G. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 7001.
3. (a) Kojima, M.; Hosoda, H.; Date, Y.; Nakazato, M.;
Matsuo, H.; Kangawa, K. Nature 1999, 402, 656. (b) Hosoda,
H.; Kojima, M.; Matsuo, H.; Kangawa, K. Biochem. Biophys.
Res. Commun. 2000, 279, 909.
4. Bednarek, M. A.; Feighner, S. D.; Pong, S.-S.; McKee,
K. K.; Hreniuk, D. L.; Silva, M. V.; Warren, V. A.; Howard,
A. D.; Van der Ploeg, L. H. Y.; Heck, J. V. J. Med. Chem.
2000, 43, 4370.
5. Intermediate 1 was prepared via sulfonylation and then
demethylation of N0-methyl spiro(indoline-3,40-piperidine).
For the synthesis of N0-methyl spiro(indoline-3,40-piperidine),
see: Ong, H. H.; Profitt, J. A.; Fortunato, J.; Glamkowski,
E. J.; Ellis, D. B.; Geyer, H. M., III; Wilker, J. C.; Burghard,
H. J. Med. Chem. 1983, 26, 981.
In summary, we report the synthesis and evaluation of a
series of MK-0677/ghrelin(1-4)-hybrid compounds as
ghrelin mimetics. Replacement of Phe in the tetrapep-
tide with a spiro(indoline-3,40-piperidine) group, Gly-
Ser with 2-aminoisobutyric acid, and O-(n-octanoyl)-l-
Ser with O-benzyl-d-Ser provided synthetic GHS ago-
nists with similar functional potency as ghrelin. The
binding affinities on the cloned hGHSR1a for the d-
serine hybrid compounds (8–12) are equal to or greater