Design of a new peptidomimetic agonist for the melanocortin receptors based on the solution structure of the peptide ligand, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH2

Article abstract of DOI:10.1016/S0960-894X(03)00412-8

The solution structure of a potent melanocortin receptor agonist, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH₂ (1) was calculated using distance restraints determined from ¹H NMR spectroscopy. Eight of the lowest energy conformations f

Full text of DOI:10.1016/S0960-894X(03)00412-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2337–2340
Design of a New Peptidomimetic Agonist for the Melanocortin
Receptors Based on the Solution Structure of the Peptide Ligand,
Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH2
Christopher Fotsch,* Duncan M. Smith, Jeffrey A. Adams, Janet Cheetham,
Michael Croghan, Elizabeth M. Doherty, Clarence Hale, Mark A. Jarosinski,
Michael G. Kelly,y Mark H. Norman, Nuria A. Tamayo, Ning Xi
and James W. Baumgartner
Departments of Small Molecule Drug Discovery and Metabolic Disorders, Amgen Inc., One Amgen Center Drive,
Thousand Oaks, CA 91320, USA
Received 12 February 2003; revised 16 April 2003; accepted 22 April 2003
Abstract—The solution structure of a potent melanocortin receptor agonist, Ac-Nle-cyclo[Asp-Pro-dPhe-Arg-Trp-Lys]-NH2 (1)
was calculated using distance restraints determined from HNMR spectroscopy. Eight of the lowest energy conformations from
1
this study were used to identify non-peptide cores that mimic the spatial arrangement of the critical tripeptide region, dPhe-Arg-
Trp, found in 1. From these studies, compound 2a, containing the cis-cyclohexyl core, was identified as a functional agonist of the
melanocortin-4 receptor (MC4R) with an IC50 and EC50 below 10 nM. Compound 2a also showed 36- and 7-fold selectivity over
MC3R and MC1R, respectively, in the binding assays. Subtle changes in cyclohexane stereochemistry and removal of functional
groups led to analogues with lower affinity for the MC receptors.
# 2003 Elsevier Science Ltd. All rights reserved.
The melanocortin receptors (MCRs)1,2 are a class of
G-protein coupled receptors that are involved in
inflammation,3 skin pigmentation,4 steroidogenesis,5 fat
metabolism,6 sexual function,7,8 feeding behavior,9,10
and exocrine gland secretion.11 Their function is regu-
lated by peptide agonists (e.g., a-, b-, g- melanocyte sti-
mulating hormones (MSH), and adrenocorticotropic
hormone (ACTH)) and antagonists (e.g., agouti and
agouti-related protein). The MCRs are attractive targets
for drug discovery,12,13 but the use of their endogenous
ligands as therapeutic agents is not practical. In general,
peptides are metabolically unstable, poorly absorbed,
and are costly to prepare. In this report we describe our
initial efforts to design and synthesize non-peptide ago-
nists for the melanocortins. Our approach was to deter-
mine the structure of a peptide ligand, and use that
structure to design non-peptide agonists for the recep-
tor.14 Since our group was interested in developing new
therapies for obesity, we focused on agonists for MC4R
because of its role in feeding behavior.9,10
Endogenous agonists for the MCRs all contain the tetra-
peptide sequence, His-Phe-Arg-Trp, which has been
labeled the ‘message sequence’, that is, the minimal
peptide sequence necessary for activating the receptor.15
Further studies with MCR agonists have shown that
histidine can be replaced with alanine without much loss
in activity,16 and phenylalanine when replaced with its
d-stereoisomer has better activity at the MCRs.17
Therefore, we focused on mimicking the tripeptide,
dPhe-Arg-Trp. This tripeptide is only weakly active at
the MCRs,18 but the cyclic peptide, Ac-Nle-cyclo[Asp-
Pro-dPhe-Arg-Trp-Lys]-NH2 (1), reported by Bednarek
and co-workers,19 has low nM affinity at MC3, 4, and
5R. Apparently, in the cyclic peptide (1), dPhe-Arg-Trp
is positioned in a way that better complements the
MCRs. To understand the bioactive conformation of
the dPhe-Arg-Trp, we set out to determine the solution
structure of peptide 1. After we determined the structure
of 1, we would prepare compounds that could position
groups in close proximity to side-chains found on the
dPhe-Arg-Trp.
*Corresponding author. Tel.: +1-805-447-7746; fax: +1-805-480-
1337; e-mail: cfotsch@amgen.com
yCurrent address: Renovis, 270 Littlefield Avenue, South San Fran-
sico, CA 94080, USA.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00412-8

2338
C. Fotsch et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2337–2340
20
We used 600MHz 1HNMR spectroscopy
to deter-
mine the solution structure of peptide 1. The structure
was calculated using eighty-two distance restraints
between hydrogen atoms at least four bonds apart. One
dihedral angle (Lys w1 fixed at 180Æ30o), and one
hydrogen bond (Lys aNHto Asp C ¼O, obtained from
NHtemperature coefficients) were also included in the
calculations. Through this analysis, an ensemble of 35
low energy structures were calculated, and eight of the
lowest energy structures were used for modeling (see
Fig. 1). Peptide 1 did not exist in any classical turn
structure, but we saw a significant number of short-
range nuclear Overhauser effects that suggested con-
formational preferences for the peptide backbone.
These findings are in contrast to the NMR structure of a
closely related analogue Ac-Nle-cyclo[Asp-His-dPhe-
Arg-Trp-Lys]-NH2, MT-II, reported by Bednarek and
co-workers.16 They concluded that the peptide back-
bone did not have any preferred conformation. The
structure of peptide 1 was determined at 5 ꢀC in aqueous
buffer rather than in DMSO-d6 or CD3OHat 25 ꢀC
used for the NMR of MT-II, which could account for
the differing observations.
Figure 2.
butyl guanidine on the 1-position mimicking arginine,
and d-phenylalanine at the 1-position. Since the back-
bone of peptide 1 has no charge, acyl groups were added
to d-phenylalanine and to the b-nitrogen on the trypta-
mine to keep the compound neutral at those positions.
Compound 2a can adopt one of two chair conforma-
tions that have either H(a) or H(b) in the equatorial
position. Isomer 2b (trans) exists as one chair con-
formation with H(a) and H(b) both in the axial orien-
tation. All three conformations of 2 overlapped with the
tripeptide region of 1 equally well.
With the structure of 1 in hand, various ring systems
(carbocyclic and heterocyclic) appended with groups that
mimicked the side-chains on dPhe-Arg-Trp were over-
lapped with our NMR structure. Those compounds that
showed the best overlap were synthesized. Two examples,
cyclohexanes 2a and 2b (see Fig. 2), contain a tryptamine
group at the 4-position intended to mimic tryptophan, a
Compound 2 was synthesized (see Scheme 1) by first
appending tryptamine onto 1,4-cyclohexanedione ethyl-
ene ketal (3) through a reductive amination. Inter-
mediate 4 was then acylated and deprotected to give
ketone 5. A second reductive amination was performed
Scheme 1. Synthesis of Compound 2a: (a) tryptamine, NaHB(OAc)3,
DCE; (b) (i) Ac2O, Et3N; (ii) aq AcOH, (65% from 3);
(c) H2N(CH2)4NHC(=NCbz)NHCbz, NaHB(OAc)3, DCE (89%);
(d) FmocDPheCl, BSA, CH2Cl2 (66%); (e) (i) TAEA, CH2Cl2; (ii)
Ac2O, Et3N, CH2Cl2 (46%, 2 steps); (iii) H2, Pd-C, CH3OH; AcOH
(50%).
Figure 1. Ensemble of eight lowest energy conformations of (1) (in
purple); compound 2a is overlaid (carbon atoms are in green, hydro-
gen atoms are removed for clarity).

C. Fotsch et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2337–2340
2339
Compounds were tested at the mouse MC1R, human
MC3R, and human MC4R. Table 1 summarizes the
results of a ligand (NDP-MSH) displacement assay.24
For comparison the activity of peptide 1 is included in
Table 1. The cis-isomer 2a had sub-micromolar IC50s at
all of the MCRs, and was the most potent at MC4R
with an IC50=7.7 nM. Compound 2a was also 36- and
7-fold selective over MC3R and MC1R, respectively.
The IC50s for the trans isomer 2b were >10,000 nM for
MC1R and MC3R and ca. 3000 nM at MC4R. The
functional activities of 2a and 2b, measured by cAMP
production of HEK 293 cells transfected with recombi-
nant human MC4R,25 were 4 and 680 nM, respectively.
The large difference in activity between the cis and
trans isomers of 2 cannot be explained by their over-
lap with the structure of 1. There seems to be enough
flexibility in the side chains of dPhe-Arg-Trp that
either isomer of 2 could be positioned to overlap with
the functionality found on the key tripeptide. Com-
pound 2a has one group appended in the axial orien-
tation, so the indole group and the phenylalanine/
guanidine groups are closer together than in the trans
isomer 2b. Apparently, the closer arrangement of these
groups on 2a better approximates the active conformation
of 1.
Scheme 2. Analogues of Compound 2a.
Table 1. Biological activity of analogues of compound 2
Compd mMC1R,
hMC3R,
hMC4R,
IC50, nMa
hMC4R,
EC50, nMb
IC50, nMa IC50, nMa
1
nd
23 (Æ6)c
3.0 (Æ2)
7.7 (Æ2)f
0.17 (Æ0.1)c
2a
2b
8
9
10
11
51 (Æ20)d 280 (Æ80)e
4 (Æ3) (93%)g
>10,000
>10,000
9000
>10,000
nd
>10,000
>10,000
>10,000
>10,000
nd
3500 (Æ500) 680 (Æ90) (90%)g
>10,000
>10,000
nd
nd
nd
nd
9250 (Æ750)
500 (Æ90)
a125I-NDP-a-MSHbinding to the melanocortin receptors, SEM of two
IC50s determined over six dilutions except where noted.
bIntracellular levels of cAMP in cells expressing melanocortin recep-
tors, SEM of two EC50s determined over six dilutions.
cData from ref 19.
dn=4.
en=3.
fn=8.
Analogues of 2a were also prepared to determine the
significance of each functional group (compounds 8–11,
see Scheme 2). Compounds 8–10 were weakly active
(IC50=9000 to >10,000 nM) at each of the receptor
sub-types. The replacement of either the d-phenyl-
alanine group with acetyl (8) or the replacement of the
tryptamine group with a methyl (9) was detrimental to
activity. Bednarek and co-workers reported16 similar
results in their work with the potent melanocortin pep-
tide agonist MTII (Ac-Nle-cyclo[Asp-His-dPhe-Arg-
Trp-Lys]-NH2). In their studies they also showed that
removing the amino acid side chains on d-phenyl-
alanine or tryptophan, by substituting with alanine, was
deleterious to activity at the MCRs. In compound 10,
the amino group replacement for the guanidine also
adversely affected activity even though both groups are
positively charged at physiological pH. Compound 2a
may be more potent than 10 since the guanidine group
is able to form additional hydrogen bonding inter-
actions with the receptor. Finally, the lPhe analogue,
compound 11, was 65-fold less potent than 2a, which is
similar to the trend observed for peptide ligands.17
gPercentage of maximal response with respect to a-MSH.
with the bis-benzylcarbamate (Cbz) protected (4-amino-
butyl)guanidine21 to provide a mixture of cis and trans
isomers 6a and 6b that were separable by flash chroma-
tography. Isomers 6a and 6b were stereochemically
assigned by examining the coupling constants of the
1
H(a)-signal. The aliphatic region in the HNMR con-
tained many overlapping signals, so to simplify the
spectrum, H(b) (d 3.6 ppm, CD3OD) was irradiated in a
TOCSEY experiment to enhance the peaks on the
cyclohexane ring. The H(a) peak (d=2.9 ppm, CD3OD)
on the cis-isomer 6a was a multiplet, but after irradiating
the H(b) peak, a Jꢁ4 Hz was observed for H(a), which
is an indication of either an equatorial/equatorial or
equatorial/axial coupling. Doing the same for the trans-
isomer 6b, a Jꢁ10 Hz was measured for the H(a) peak
(d=2.4 ppm, CD3OD) which correlates to an axial/axial
coupling. Each isomer was then independently carried
through the remainder of the synthesis (the cis-isomer is
shown for the remaining part of the synthesis in Scheme
1). The secondary amine on 6 proved to be a difficult
coupling partner for Fmoc-dPhe-OH. After extensive
experimentation with standard coupling reagents, we
found that by pre-treating 6 with N,O-bis(trimethyl-
silyl)acetamide (BSA)22 and then adding the acid chlo-
ride of Fmoc-dPhe-OH, we were able to obtain 7 in
adequate yields (66% for 7a). The Fmoc protecting
group was removed with tris-(2-aminoethyl)amine
(TAEA) using the protocol described by Carpino and
co-workers,23 the a-amino group was acetylated, and
the Cbz protecting groups were then removed by
hydrogenolysis to provide the desired product 2. Ana-
logous synthetic methods were used to prepare com-
pounds 8–11.
By using a set of low energy structures calculated from
NMR data collected for the potent MCR peptide ligand
(1), we were able to design molecules that overlapped
with the tripeptide region of dPhe-Arg-Trp. Among
those compounds were those that contained the cyclo-
hexane core. When substituted with d-phenylalanine,
guanidine, and indole we were able to identify com-
pound 2a, which had an IC50 <10 nM at MC4R, and
showed selectivity versus MC3R and MC1R. Com-
pound 2a was also a low nM functional agonist of
MC4R. The overall strategy for identifying a peptido-
mimetic agonist from the structure of a peptide ligand
was successful. Compound 2a still has liabilities that
may prevent it from being well absorbed in vivo (e.g.,

2340
C. Fotsch et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2337–2340
presence of polar guanidine group), but it could be used as
a lead to discover compounds that are more ‘drug-like’.
19. Peptide 1 is linked through the side-chains of Asp and Lys
to form a 23-membered lactam. Bednarek, M. A.; MacNeil,
T.; Kalyani, R. N.; Tang, R.; Van der Ploeg, L. H. T.; Wein-
berg, D. H. Biochem. Biophys. Res. Commun. 1999, 261, 209.
20. The sample was dissolved in 90% H2O/10% D2O, and the
pH was adjusted to 5.0 with dilute HCl or NaOH. The spectra
were acquired at 5 ꢀC, and the resonances were assigned using
TOCSEY and ROESY spectra. Intranuclear distances were
obtained from NOESY data, and structures were calculated
using XPLOR.
21. The bis-Cbz protected (4-aminobutyl)guanidine was pre-
pared using a method analogous to one reported in the litera-
ture. See: Preville, P.; He, J. X.; Tarazi, M.; Siddiqui, M. A.;
Cody, W. L.; Doherty, A. M. Bioorg. Med. Chem. Lett. 1997,
7, 1563.
References and Notes
1. Haskell-Luevano, C.; Hadley, M. E. Drug News Perspect.
1999, 12, 197.
2. The Melanocortin Receptors. Cone, R. D.; Ed.; Humana:
Totowa, N. J., 2000.
3. Huang, Q.-H.; Hruby, V. J.; Tatro, J. B. Am. J. Physiol.
1998, 275, R524.
4. Roubos, E. W. Comp. Biochem. Physiol., Part A: Mol.
Integr. Physiol. 1997, 118A, 533.
5. Grahame-Smith, D. G.; Butcher, R. W.; Ney, R. L.;
Sutherland, E. W. J. Biol. Chem. 1967, 242, 5535.
6. Ng, T. B. Comp. Biochem. Physiol., B: Comp. Biochem.
1990, 97B, 441.
7. Bertolini, A.; Gessa, G. L.; Vergoni, W.; Ferrari, W. Life
Sci. 1968, 7, 1203.
22. Carpino, L. A.; Ionescu, D.; El-Faham, A.; Henklein, P.;
Wenschuh, H.; Bienert, M.; Beyermann, M. Tetrahedron Lett.
1998, 39, 241. Wenschuh, H.; Beyermann, M.; Winter, R.;
Bienert, M.; Ionescu, D.; Carpino, L. A. Tetrahedron Lett.
1996, 37, 5483.
23. Carpino, L. A.; Sadat-Aalaee, D.; Beyermann, M. J. Org.
Chem. 1990, 55, 1673.
8. Bertolini, A.; Vergoni, W.; Gessa, G. L.; Ferrari, W. Nature
1969, 221, 667.
24. Ligand Binding Assay. Binding assays were performed in
96-well U-bottom plates. Membranes (200 mg tissue) were
incubated at 30 ꢀC for 2 h in assay buffer with various com-
pounds in the presence of 0.2 nM 125I-NDP-a-MSH(Amer-
shamPharmacia Biotech, Piscataway, NJ) in 100 mL total
volume. Non-specific binding was assessed in the presence of 1
mM cold NDP–MSH. The reaction was terminated by rapid
filtration through Unfilter-96 GF/C glass fiber filter plates
(FilterMate 196 Harvester, Packard Instrument Company,
Meriden, CT) followed by three washes with 300 mL of ice-
cold water. Bound radioactivity was determined using a Top-
Count microplate scintillation and luminescence counter
(Packard Instrument Company, Meriden, CT). Nonlinear
regression analyses of test compound concentration curves
were performed using GraphPad Prism (GraphPad Software,
Inc., San Diego, CA).
25. Functional (cAMP) Assay: Human embryonic kidney cells
(HEK 293) expressing the melanocortin 4 receptor were see-
ded into 96-well fibronectin coated plates to a density of
75,000 cells/well. Plates were used for assay ca. 48 h after
seeding. Assay was initiated by removing media from cells and
replacing with 90 mL of assay buffer (DMEM+100 mM
3-Isobutyl-1-Methylxanthine, IBMX), followed by addition of
10 mL of the test peptide ranging in concentration from 0.1
nM to 10 mM. Cells were then incubated for 30 min at 37 ꢀC.
Removal of assay media and addition of 100 mL Tropix Lysis
Buffer terminated the reaction. Production of cAMP was
measured using Tropix cAMP-Screen 96-well assay (Applied
Biosystems, Foster City, CA). The kit used was a chemilumi-
nescent ELISA system designed for quantitation of cAMP from
mammalian cell extracts. Chemiluminescent activity was mea-
sured using a Labsystems Luminoscan (Labsystems, Inc.,
Franklin, MA). Nonlinear regression analyses of test compound
concentration curves were performed using GraphPad Prism.
9. Fan, W.; Boston, B. A.; Kesterson, R. A.; Hruby, V. J.;
Cone, R. D. Nature 1997, 385, 165.
10. Huszar, D.; Lynch, C. A.; Fairchild-Huntress, V.; Dun-
more, J. H.; Fang, Q.; Berkemeier, L. R.; Gu, W.; Kesterson,
R. A.; Boston, B. A.; Cone, R. D.; Smith, F. J.; Campfield,
L. A.; Burn, P.; Lee, F. Cell 1997, 88, 131.
11. Chen, W.; Kelly, M. A.; Opitz-Araya, X.; Thomas, R. E.;
Low, M. J.; Cone, R. D. Cell 1997, 91, 789.
12. Wikberg, J. E. S. Eur. J. Pharmacol. 1999, 375, 295.
13. Adan, R. A.; Gispen, W. H. Eur. J. Pharmacol. 2000, 405, 13.
14. A group at Merck recently reported another example of
deriving a non-peptide lead from the structure of an MCR pep-
tide agonist. See: Sebhat, I. K.; Martin, W. J.; Ye, Z.; Barakat,
K.; Mosley, R. T.; Johnston, D. B. R.; Bakshi, R.; Palucki, B.;
Weinberg, D. H.; MacNeil, T.; Kalyani, R. N.; Tang, R.;
Stearns, R. A.; Miller, R. R.; Tamvakopoulos, C.; Strack, A. M.;
McGowan, E.; Cashen, D. E.; Drisko, J. E.; Hom, G. J.;
Howard, A. D.; MacIntyre, D. E.; van der Ploeg, L. H. T.;
Patchett, A. A.; Nargund, R. P. J. Med. Chem. 2002, 45, 4589.
15. Hruby, V. J.; Wilkes, B. C.; Hadley, M. E.; Al-Obeidi, F.;
Sawyer, T. K.; Staples, D. J.; de Vaux, A. E.; Dym, O.; Cas-
trucci, A. M.; Hintz, M. F.; Riehm, J. P.; Rao, R. J. Med.
Chem. 1987, 30, 2126.
16. Bednarek, M. A.; Silva, M. V.; Arison, B.; MacNeil, T.;
Kalyani, R. N.; Huang, R.-R. C.; Weinberg, D. H. Peptides
(New York) 1999, 20, 401.
17. For a comparison of Phe and dPhe analogues of peptide
ligands for the MCRs see: Haskell-Luevano, C.; Nikiforovich,
G.; Sharma, S. D.; Yang, Y.-K.; Dickinson, C.; Hruby, V. J.;
Gantz, I. J. Med. Chem. 1997, 40, 2740.
18. Haskell-Luevano, C.; Hendrata, S.; North, C.; Sawyer,
T. K.; Hadley, M. E.; Hruby, V. J.; Dickinson, C.; Gantz, I.
J. Med. Chem. 1997, 40, 2133.