Design and microwave-assisted synthesis of novel macrocyclic peptides active at melanocortin receptors: Discovery of potent and selective hMC5R receptor antagonists

Article abstract of DOI:10.1021/jm701181n

Differentiation of the physiological role of the melanocortin receptor 5 MC5R from that of other melanocortin receptors will require development of high affinity and selective antagonists. To date, a few synthetic antagonist ligands active at hMC5 receptor are available, but most do not have appreciable selectivity. With the aim to gain more potent and selective antagonists for the MC5R ligands, we have designed, synthesized, and pharmacologically characterized a series of alkylthioaryl-bridged macrocyclic peptide analogues derived from MT-II and SHU9119. These 20-membered macrocycles were synthesized by a tandem combination using solid phase peptide synthesis and microwave-assisted reactions. Biological assays for binding affinities and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues, compounds, 9, 4, and 7, are selective antagonists at the hMC5 receptor. In particular, compound 9 (PG-20N) is a selective and competitive hMC5R antagonist, with IC₅₀ of 130 ± 11 nM, and a pA₂ value of 8.3, and represents an important tool for further biological investigations of the hMC5R. Compounds 4 and 7 (PG14N, PG17N) show potent and selective allosteric inhibition at hMC5R with IC₅₀ values of 38 ± 3 nM and 58 ± 6 nM, respectively. Compound 9 will be used to further investigate and more clearly understand the physiological roles played by the MC5 receptor in humans and other animals.

Full text of DOI:10.1021/jm701181n

J. Med. Chem. 2008, 51, 2701–2707
2701
Design and Microwave-Assisted Synthesis of Novel Macrocyclic Peptides Active at Melanocortin
Receptors: Discovery of Potent and Selective hMC5R Receptor Antagonists
Paolo Grieco,†,‡,§ Minying Cai,†,‡ Lu Liu,‡ Alexander Mayorov,‡ Kevin Chandler,‡ Dev Trivedi,‡ Guangxin Lin,‡
Pietro Campiglia,| Ettore Novellino,§ and Victor J. Hruby*,‡
Department of Chemistry, UniVersity of Arizona, Tucson, Arizona 85721, Department of Pharmaceutical Chemistry and Toxicology, UniVersity
of Napoli “Federico II”, Naples, Italy, and Department of Pharmaceutical Science, UniVersity of Salerno, Fisciano, Salerno, Italy
ReceiVed September 19, 2007
Differentiation of the physiological role of the melanocortin receptor 5 MC5R from that of other melanocortin
receptors will require development of high affinity and selective antagonists. To date, a few synthetic
antagonist ligands active at hMC5 receptor are available, but most do not have appreciable selectivity. With
the aim to gain more potent and selective antagonists for the MC5R ligands, we have designed, synthesized,
and pharmacologically characterized a series of alkylthioaryl-bridged macrocyclic peptide analogues derived
from MT-II and SHU9119. These 20-membered macrocycles were synthesized by a tandem combination
using solid phase peptide synthesis and microwave-assisted reactions. Biological assays for binding affinities
and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues,
compounds, 9, 4, and 7, are selective antagonists at the hMC5 receptor. In particular, compound 9
(PG-20N) is a selective and competitive hMC5R antagonist, with IC50 of 130 ( 11 nM, and a pA2 value of
8.3, and represents an important tool for further biological investigations of the hMC5R. Compounds 4 and
7 (PG14N, PG17N) show potent and selective allosteric inhibition at hMC5R with IC50 values of 38 ( 3
nM and 58 ( 6 nM, respectively. Compound 9 will be used to further investigate and more clearly understand
the physiological roles played by the MC5 receptor in humans and other animals.
Introduction
similar to MC1R and MC4R in its ability to respond to all
melanocortins, except γ-MSH. The MC5R mRNA is expressed
at high levels in the exocrine glands, such as lacrimal and
Harderian glands.25 It is also expressed in skin tissues,
particularly in sebaceous glands, and in skeletal muscles. The
functions of MC5R are still not very well understood; they are
speculated to include neuro/myotropic, gastric, and anti-inflam-
matory effects, and regulation of aldosterone secretion.23,24,26,27
In the present study we synthesized a series of novel 20-
membered macrocycles formally derived from MTII and
SHU9119, containing an alkylthioaryl bridge within the main
ring by a tandem combination using solid phase peptide
synthesis and microwave-assisted reactions. The 19 systemati-
cally modified analogues were tested in binding assays and in
functional assays for cAMPa accumulation at human melano-
cortin receptors 1, 3, 4, and 5. The resulting compounds 4, 7,
and 9 (PG14N, PG17N, and PG20N) were found to be selective
and potent antagonists at the hMC5R.
The melanocortins are a group of structurally related peptides
derived from proopiomelanocortin (POMC) that derive their
name from their melanotropic and corticotropic activities and
are comprised of adrenocorticotropic hormone (ACTH), R-mel-
anocyte stimulating hormone (R-MSH), ꢀ-MSH, and γ-MSH.1,2
Post-translational modification of POMC by proteolytic cleavage
generates many bioactive peptides, including the melanocortins,
lipotropic hormone (ꢀ-LPH), and ꢀ-endorphin.3 Melanocortins
are best known for their ability to stimulate eumelanin synthesis
in mammalian melanocytes and steroidogenesis in adrenocortical
cells.4,5 These two bioactivities are by no means the exclusive
effects of these hormones. Other effects include regulation of
food intake and energy metabolism, and neuronal regeneration.6–9
Melanocortins act as endogenous antipyretic agents, and have
systemic as well as peripheral anti-inflammatory effects.10,11
Melanocortins have sebotrophic effects,12 induce lipolysis,4
regulate exocrine glands, cardiac and testicular functions,13,14
natriuresis, and many others.15,16
The effects of melanocortins are mediated by activation of a
family of melanocortin receptors (MCRs). The cloning of the
MCR genes has led to tremendous progress in understanding
the biological effects of melanocortins. So far, five MCR
(MC1R, MC2R, MC3R, MC4R, and MC5R) genes have been
cloned and the receptors pharmacologically characterized.16–23
The MC5R gene was the last of the melanocortin receptor gene
family to be cloned.23,24 This receptor has the most sequence
homology to MC4R and the least homology to MC2R. It is
Chemistry. Peptide Synthesis. Preparation of the macrocy-
clic peptides (Table 1) began with a series of HBTU/HOBt
a Abbreviations: Abbreviations used for amino acids and designation of
peptides follow the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature in J. Biol. Chem. 1972, 247, 977–983. The following
additional abbreviations are used: Boc, tert-butyloxycarbonyl; cAMP,
adenosine 3′,5′-cyclic monophosphate; DMF, N,N-dimethylformamide;
FAB-MS, fast-atom bombardment mass spectrometry; Fmoc, 9-fluorenyl-
methoxycarbonyl; HOBt, N-hydroxybenzotriazole; HBTU, 2-(1H-ben-
zotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HEPES,
N-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid); Pbf, 2,2,4,6,7-
pentamethyldihydrobenzo-furan-5-sulfonyl; RP-HPLC, reversed-phase high
performance liquid chromatography; TFA, trifluoroacetic acid; Tris, 2-amino-
2-(hydroxymethyl)-1,3-propanediol; Trt, triphenylmethyl (trityl); Cit, cit-
rulline; Nal(2), 2-naphtylalanine; Aic, 2-aminoindane-2-carboxylic acid;
Hyp, hydroxyl-L-proline; Cpa, cyclopentyl-L-alanine; Paf, p-amino-Phe;
MALDI, matrix-assisted laser desorption/ionization; Fmoc, fluorenyl-
methoxycarbonyl; amino acid symbols denote L-configuration unless
indicated otherwise.
* To whom correspondence should be addressed: Tel.: 520-621-6332.
Fax: 520- 621-8407. E-mail: hruby@u.arizona.edu.
†
These authors equally contributed to this paper.
University of Arizona.
University of Napoli “Federico II”.
University of Salerno.
‡
§
|
10.1021/jm701181n CCC: $40.75
2008 American Chemical Society
Published on Web 04/15/2008

2702 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Grieco et al.
Scheme 1. General Synthetic Method for Macrocyclic
Melanocortins
couplings performed using a ACT mod 348 Ω synthesizer and
following a conventional Fmoc approach28 to obtain the
appropriate open chain intermediates. Then, the peptides were
capped with fluoronitrobenzoic acid (as indicated in Scheme 1
for compound 1) as previously reported.29 Before the cyclization
step, the S-Trt group of the Cys residue were removed with
dilute trifluoroacetic acid (5% in CH2Cl2) without cleavage of
the peptide from the resin. At this stage, all peptides were
transferred in a Milestone Ethos CombiChem microwave

Peptides ActiVe at Melanocortin Receptors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2703
cyclization reaction. 2-Fluoro-5-nitro benzoic acid is a useful
electrophile for nucleophilic aromatic substitution reactions
(SNAr) and has been used in several solid phase syntheses for
various purposes.34 The macrocyclization step, carried out by
our methodology, was performed in only 10 min compared to
the conventional thermal heating, performed with potassium
carbonate in DMF for 20 h and using identical stoichiometry.
The macrocyclizations were performed between positions 5 and
10 of the peptide as in MTII,35 which was chosen as a reference
compound. Thus, conserving the sequence His-Phe-Arg-Trp is
important, which is responsible for biological activities. All
synthesized compounds were evaluated for their binding affini-
ties to the human melanocortin receptors 1, 3, 4, and 5 in
competitive binding assays using the radiolabeled ligand [125I]-
NDP-R-MSH and for their agonist potency in cAMP assays
employing the HEK293 cells expressing those receptors. All
the data is compiled in Table 1.
The primary intent of the current study was to examine
alkylthioaryl-type macrocyclization as an approach toward
development of MT-II analogues with enhanced receptor
selectivity. MCMM-low mode/OPLS-2005 simulations revealed
that the 3D structure of analogue 1 significantly deviates from
that of the superagonist MT-II (Figure 2), displaying a type I
ꢀ-turn spanning the Arg8-Trp9 residues compared to type II
ꢀ-turn between the His6-D-Phe7 residues in MT-II.36 Such a
significant change in the peptide backbone fold may stem from
the apparent face-to-face aromatic stacking interactions between
the electron-rich indole ring of Trp9 and the electron-deficient
aromatic linker (Figure 2). This also may result in steric
crowding of the Trp9, which can be expected to strongly
influence the biological activity of the peptides derived from
this template. Interestingly, despite these structural deviations
from the parent peptide MT-II, analogue 1 exhibited potent full
agonist activities at all four melanocortin receptor subtypes. On
the other hand, replacement of His6 with conformationally
constrained hydrophobic amino acids Cha and Pro produced
analogues 4 and 7, respectively, 3D structures of which showed
minimal differences with the structure of analogue 1 (Figure
3). However, biological evaluation of these peptides revealed
substantially diminished binding affinities and agonist potencies
at the human melanocortin receptors 1, 3, and 4, but good
binding affinity to the hMC5R (IC50 ) 38 and 58 nM,
respectively) was retained, thus making these two compounds
potent and quite selective hMC5R antagonists. pA2 value assay
studies showed that both analogues 4 and7 are allosteric
inhibitors of hMC5R. These findings suggest an essential role
for His6 for potent full agonist activity of the peptides derived
from this template, which sets it apart from other previously
reported cyclic R- and γ-MSH templates that have been
demonstrated to have no such structural requirement for His6.37
The ꢀ-turn position shift observed in our molecular modeling
experiments may prove to be highly beneficial as another
approach for development of highly selective melanocortin
receptor agonists and antagonists. Subsequently, we synthesized
a series of compounds (2-19, Table 1) where we performed
modifications at positions 6-9 by replacing with several
different amino acids to obtain a rational SAR study.
Figure 1. Design of macrocyclic compounds using L-Cys.
synthesizer (Milestone, Bergamo, Italy) to perform the macro-
cyclization reactions. The nucleophilic aromatic substitution was
performed by irradiation with 450 W, 50 °C, in DMF for 10
min. These optimized conditions were suitable to provide more
than 70% conversion of all linear peptides in cyclic peptide
without decomposition of the products. The one-pot cyclization
was easily monitored by analytical HPLC. Products isolated after
cleavage from the resin with TFA were reasonably pure, as
assessed by LC/MS, and characterized by analytical RP-HPLC.
Results and Discussion
The natural melanotropin hormones have relatively lower nM
affinities for the hMC3–5R and are not particularly selective.16
Elucidation of the physiological role of the melanocortin receptor
5 will require development of high affinity and selective hMC5R
antagonists. To date only a few synthetic ligands that are
antagonist for hMC5 receptor are available and even those are
not very selective.30 We chose a cyclic lactam heptapeptide
SHU9119, Ac-Nle4-cyclo(Asp5-His6-DNal(2′)7-Arg8-Trp9-Lys10)-
NH2,30 a highly potent hMC3/4R antagonist and an agonist at
melanocortin receptors 1 and 5, as the starting template for the
development of more selective hMC5R antagonists described
in this report. Thus, with the aim to discover new potent and
selective ligands at hMC5R, we designed and synthesized a
series of 20-membered macrocyclic peptides in which the
structures conserved the melanocortin core sequence His-Phe/
Nal(2′)-Arg-Trp (Table 1). Macrocyclic peptides containing a
cystine bridge have been the subject of considerable study.31
In fact, various kinds of macrocycles often have improved
pharmacokinetic and conformational properties relative to their
cognate peptides.32
A tandem combination was employed using solid phase
peptide synthesis and microwave-assisted reaction to perform
macrocyclization reactions to prepare peptidomimetics of the
type shown in Figure 1. The application of microwave technol-
ogy to speed up the synthesis of biologically significant
molecules on solid support is of great value for library
generation33 and it has recently been recognized as a useful tool
for drug-discovery program. Recently, we have demonstrated
that microwave irradiation combined with the solid-phase
peptide synthesis represents a powerful technique for accelerat-
ing thermal organic reactions to perform macrocyclization
reactions.29
The compounds 2-7 differ from 1 only at position 6, where
His has been replaced with Aic, Cpa, Cha, DPro, Hyp, and Pro
residues. Compound 2, having an Aic residue in position 6, was
found to be a weak agonist at the hMC1 receptor and inactive
at hMC3 and hMC4 receptors but an antagonist at the hMC5R
(IC50 ) 565 nM). Compound 3, with a Cpa residue in position
6, and compound 6, with a Hyp residue in the same position,
In this study we prepared a number of melanotropin analogues
in which 2-fluoro-5-nitrobenzoic acid was used for the macro-

2704 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Grieco et al.
Figure 2. Stereoview of the superimposed NMR structure of MTII (red) with MCMM/LMCS-OPLS 2005-derived structure of 1 (blue).
Figure 3. Stereoview of the superimposed MCMM/LMCS-OPLS 2005-derived structure of peptide 4 (orange), 7 (green) and 1 (blue). The aromatic
linker is highly stacked with the indole ring of Trp9. This caused the peptide to adopt a new conformation favorable to hMC5R binding.
were found to be inactive at all melanocortin receptors tested.
On the other hand, compound 5, with a DPro residue, resulted
in a weak antagonist at the MC1 receptor. Surprisingly, in this
series of macrocyclic derivatives, the compounds 4 and 7 had
potent antagonist activity at the hMC5 receptor. In fact,
compound 4 showed low affinity and potency as an agonist at
the MC1 and MC3 receptors (IC50 ) >2000 and >1000,
respectively) but high potency as antagonist at the MC5R (IC50
) 38 nM). Interestingly, compound 7 was found to be a potent
and selective antagonist at the hMC5R (IC50 ) 58 nM) but a
weak agonist (IC50 ) 380 nM) at the hMC3R, and inactive at
the other two melanocortin receptors. The additional compounds
8-11 were modified in position 7 where the DPhe residue has
been replaced with Nal(2′), DNal(2′), Cit, and Paf. Compound
8 was a poor agonist at the hMC1R and inactive at the hMC4R,
but it was found to be a weak antagonist at the hMC3 and the
hMC5 receptors (IC50 ) 190 and 760 nM, respectively). Figure
3 shows a stereoview of the superimposed global minimum of
compounds 4, 7, and peptide 1. The compounds 4 and 7 are
highly selective hMC5R antagonists (Table 1). The aromatic
linker is stacked with the indole ring of Trp9 or Xaa9. This
caused the Trp9 to take a new conformation that is not in the ꢀ
turn region of MTII, which has been proposed36 as critical for
binding to the hMC1R, hMC3R and hMC4. Thus, our new
ligands are found to have selective binding to the hMC5R. In
earlier observations,38 we obtained hMC3R and hMC4R an-
tagonists and hMC5R agonists when His6 was replaced with
Pro6 or with many similar conformationally restricted amino
acids. This is in contrast with the present study where 20-
membered macrocycle peptides 4, 7, and 9 produced hMC5R
selective antagonists, which are weak hMC3R agonists and
totally inactive at the hMC4R.
Compound 9 in which DNal(2′) residue was replaced with
its isomer LNal(2′) resulted in a weak agonist at the hMC3R,
no activity at the hMC4R, and a very weak antagonist activity
at the hMC1R. However, 9 is a potent, selective, and competitive
antagonist at the hMC5R (IC50 ) 130 nM) with a pA2 value of
8.3. Compounds 10 and 11, where the DPhe residue was
replaced by Paf and Cit, respectively, were completely inactive
at all melanocortin receptors tested. These results are in line
with previous results showing the importance of an aromatic
residue in position 7. Compounds 12-14 in which Arg residue
was replaced by other basic amino acids such as Lys, DArg,
and Orn were completely inactive. These results represent an

Peptides ActiVe at Melanocortin Receptors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2705
Table 2. Physiochemical Properties of the Melanocortin
Peptidomimetics
additional demonstration that modification of the Arg residue
in position 8 can be highly detrimental for biological activity.
Finally, we explored position 9, replacing the Trp residue with
DNal(2′), Nal(2′), DPhe, Phe, and DTrp residues. Compound
15 (with DNal(2′)9) showed a weak agonist activity at the
hMC3R but was inactive at all other receptors. Compound 16
with Nal(2′)9 was a good agonist at the hMC1R and a weak
agonist at hMC3R (IC50 ) 180 and 610 nM, respectively) but
was inactive at the hMC4R and a weak antagonist at the hMC5R
(IC50 ) 340 nM). Compounds 17 and 18 containing Phe and
DPhe residues, respectively, in position 9 were inactive at all
melanocortin receptors. Finally, compound 19 containing a DTrp
in position 9 was a weak agonist at the hMC1R and totally
inactive at all other melanocortin receptors.
In conclusion, we have designed, synthesized, and biologically
evaluated new 20-membered macrocyclic peptidomimetics with
interesting bioactivities. The binding affinity and adenylate
cyclase activity assays of these novel macrocycles at human
melanocortin receptors showed that three of the new R-MSH
analogues act as selective antagonists toward the human
melanocortin-5 receptor. In particular, we have identified
compounds 4, 7, and 9, which represent important new tools to
investigate the physiological role played by hMC5 receptor in
vivo. The identification of these new ligands was enabled by
the versatile and powerful implementation of solid-phase peptide
strategy and microwave-assisted synthesis. Further applications
of this methodology will be investigated to perform the synthesis
of selective ligands at all melanocortin receptors.
code
solv. Aa solv. Ba
solv. Ca
M.W.
MS
HPLCb
1
2
3
4
5
6
7
8
0.78
0.82
0.78
0.85
0.82
0.85
0.86
0.80
0.88
0.77
0.75
0.70
0.71
0.70
0.84
0.86
0.83
0.87
0.80
0.04
0.04
0.04
0.08
0.04
0.04
0.05
0.04
0.10
0.05
0.06
0.05
0.04
0.06
0.05
0.06
0.07
0.07
0.04
0.38
0.41
0.38
0.43
0.41
0.47
0.46
0.39
0.55
0.37
0.35
0.36
0.36
0.36
0.45
0.46
0.44
0.47
0.39
893.72
915.66
938.26
909.81
853.70
869.70
853.70
943.80
943.80
899.71
894.71
865.71
893.72
851.68
904.76
904.76
854.68
854.68
893.72
894.11
916.15
938.84
910.23
854.24
870.25
854.24
944.31
944.33
900.25
895.23
866.25
984.24
852.18
905.27
907.28
856.17
855.19
894.23
4.15
4.81
5.11
4.32
4.63
4.54
4.71
4.75
4.91
4.87
4.14
4.10
4.18
4.11
4.89
4.91
4.66
4.87
4.20
9
10
11
12
13
14
15
16
17
18
19
a Solvent systems: (A) 1-butanol/HOAc/pyridine/H2O (5:5:1:4); (B)
EtOAc/pyridine/AcOH/H2O (5:5:1:3); (C) 1-butanol/AcOH/H2O (4:1:1).
b Analytical HPLC performed on a C18 column (Vydac 218TP104) using
a gradient of acetonitrile in 0.1% aqueous TFA at 1 mL/min. The following
gradient was used: 10–90% acetonitrile in 40 min.
washed three times with DMF and the next coupling step was then
initiated in a stepwise manner. All reactions were done under a
nitrogen atmosphere. Then the linear peptide sequence was capped
with fluorobenzoic acid (as indicated in Scheme 1 for compound
1). Before the cyclization step, the S-Trt group of Cys residue was
removed with dilute trifluoroacetic acid (5% in CH2Cl2), without
cleavage of the peptidomimetic from the resin. At this stage all
peptides were transferred in a Milestone Ethos CombiChem
microwave synthesizer to perform the macrocyclization reactions.
The nucleophilic aromatic substitution was performed by irradiation
with 450 W, 50 °C, in DMF for 10 min. These optimized conditions
were suitable to provide more than 70% conversion of all linear
peptides in cyclic peptide without decomposition of the products.
The one-pot cyclization was easily monitored by analytical HPLC.
In particular, all reactions were carried out in vessels of 4 mL
volume, using DMF as solvent. In all irradiation experiments,
rotation of the rotor, irradiation time, temperature, and power were
monitored with the “easyWAVE” software package. Temperature
was monitored with the aid of an optical fiber inserted into one of
the reaction containers. Once 50 °C was reached, the reaction
mixture was held at this temperature for 10 min and then cooled
rapidly to room temperature. The reaction vessels were opened and
the contents were poured into a separating funnel. The compounds
were washed and subjected to the final cleavage from the resin.
The resin was removed by filtration and the crude peptide recovered
by precipitation using cold anhydrous ethyl ether to give a white
powder, which was purified by HPLC on a C18-bonded silica
column (Vydac 218TPP1010, 1.0 × 25 cm), eluting with a linear
gradient of acetonitrile in aqueous 0.1% TFA. The products were
obtained by lyophilization of the appropriate fractions after removal
of the acetonitrile by rotary evaporation. Analysis by analytical
HPLC and TLC (three solvents) showed the peptides to be pure
(>98%; Table 2). The structures were further confirmed by high
resolution mass spectroscopy (Table 2) and amino acid analysis.
Biological Activity Assays. Receptor Binding Assay. Competi-
tion binding experiments were carried out using whole HEK293
cells stably expressing human MC1, MC3, MC4, and MC5
receptors. HEK293 cells transfected with hMCRs39 were seeded
on 96-well plates 48 h before assay (50000 cells/well). For the assay,
the cell culture medium was aspirated and the cells were washed
once with a freshly prepared MEM buffer containing 100%
minimum essential medium with Earle’s salt (MEM, GIBCO) and
25 mM sodium bicarbonate. Next, the cells were incubated for 40
min at 37 °C with different concentrations of unlabeled peptide
and labeled [125I]-[Nle4,D-Phe7]-R-MSH (Perkin-Elmer Life Sci-
Experimental Section
Materials. NR-Fmoc-protected amino acids and resin were
purchased from Advanced ChemTech (Louisville, KY). HBTU and
HOBt were purchased from Quantum Biotechnologies (Montreal,
Quebec, Canada). For the NRFmoc-protected amino acids, the
following side chain protecting groups were used: Cys(Trt), Arg(Nγ-
Pbf), His(Nim-Trt), and Trp(Nin-Boc). All protected amino acid
derivatives were analyzed for purity by thin-layer chromatography
before use. Peptide synthesis solvents, reagents, as well as CH3CN
for HPLC were reagent grade and were acquired from commercial
sources and used without further purification unless otherwise noted.
TLC was done on Analtech, Inc. (Newark, DE) silica gel 60 F254
plates using the following solvent systems: (A) 1-butanol/acetic
acid/pyridine/water (5:5:1:4); (B) ethyl acetate/pyridine/acetic acid/
water (5:5:1:3); (C) upper phase of 1-butanol/acetic acid/water (4:
1:1). The peptides were detected on the TLC plates using iodine
vapor. Amino acid analyses were carried out using a Pico-Tag work
station (Waters-Millipore, Waltham, MA). Peptide structures were
confirmed by MALDI-TOF MS analyses. The purity of the finished
peptides was checked by analytical RP-HPLC using a Shimadzu
model CL-10AD VP system with a built-in diode array detector.
In all cases, the purity of the finished peptides was greater than
95% as determined by these methods. The analytical data for the
peptide is given in Table 2.
General Method for Peptide Synthesis and Purification. The
protected peptide resins used to make the cyclic melanotropins were
prepared using 0.5 g of Rink amide resin (0.7 mmol of NH2/g of
resin) by first coupling NR-Fmoc-Cys(Trt)-OH to the resin previ-
ously deprotected by a 25% piperidine solution in DMF for 30 min.
In this strategy automated solid phase synthesis was performed on
the Advanced ChemTech ACT 348 Ω instrument. The following
protected amino acids were then added stepwise NR-Fmoc-Trp(Nin-
Boc)-OH, NR-Fmoc-Arg(Nγ-Pbf)-OH, NR-Fmoc-DNal(2′)-OH, Fmoc-
DPhe-OH, NR-Fmoc-His(Nim-Trt)-OH or NR-Fmoc-Xaa-OH. Each
coupling reaction was accomplished using a 3-fold excess of amino
acid with HBTU (HOBt) in the presence of diisopropylethyl amine
(DIEA). The NR-Fmoc protecting groups were removed by treating
the protected peptide resin with 25% piperidine solution in DMF
twice (1 × 5 min and 1 × 25 min). The peptide resin was then

2706 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Grieco et al.
ence, 20000 cpm/well, 33.06 pM) diluted in 125 µL of freshly
prepared binding buffer containing 100% MEM, 25 mM HEPES
(pH 7.4), 0.2% bovine serum albumin, 1 mM 1,10-phenanthrolone,
0.5 mg/L leupeptin, 200 mg/L bacitracin. The assay medium was
subsequently removed, the cells were washed once with basic
medium, and then lysed by the addition of 100 µL of 0.1 M NaOH
and 100 µL of 1% Triton X-100. The lysed cells were transferred
to 12 × 75 mm borosilicate glass tubes, and the radioactivity was
measured by a Wallac 1470 WIZARD Gamma Counter.
automatically within Maestro graphical user interface. A total of
20000 search steps were performed and the conformations with
energy difference of 50 kJ/mol from the global minimum were
saved. The superimpositions of peptide structures were performed
using the R-carbons of the core sequence -His-D-Phe-Arg-Trp- with
Xa6-Xb7-Xc8-Xd9-.
Acknowledgment. This research was supported primarily
by the U.S. Public Health Service, National Institute of Health,
DK17420 and DA06284. We thank Erin Palmer for the pA2
values.
Adenylate Cyclase Assay. HEK 293 cells transfected with
human melanocortin receptors39 were grown to confluence in MEM
medium (GIBCO) containing 10% fetal bovine serum, 100 units/
mL penicillin and streptomycin, and 1 mM sodium pyruvate. The
cells were seeded on 96-well plates 48 h before assay (50000 cells/
well). For the assay, the cell culture medium was removed and the
cells were rinsed with 100 µL of MEM buffer (GIBCO). An aliquot
(100 µL) of the Earle’s balanced salt solution with 0.5 mM
isobutylmethylxanthine (IBMX) was placed in each well along for
1 min at 37 °C. Next, aliquots (25 µL) of melanotropin peptides of
varying concentration were added, and the cells were incubated
for 3 min at 37 °C. The reaction was stopped by aspirating the
assay buffer and adding 60 µL ice-cold Tris/EDTA buffer to each
well, then placing the plates in a boiling water bath for 7 min. The
cell lysates were then centrifuged for 10 min at 2300 g. A 50 µL
aliquot of the supernatant was transferred to another 96-well plate
and placed with 50 µL [3H] cAMP and 100 µL protein kinase A
(PKA) buffer in an ice bath for 2–3 h. The PKA buffer consisted
of Tris/EDTA buffer with 60 µg/mL PKA and 0.1% bovine serum
albumin by weight. The incubation mixture was filtered through
1.0 µm glass fiber filters in MultiScreen-FB 96-well plates
(Millipore, Billerica, MA). The total [3H] cAMP was measured by
a Wallac MicroBeta TriLux 1450 LSC and Luminescence Counter
(PerkinElmer Life Science, Boston, MA). The cAMP accumulation
data for each peptide analogue was determined with the help of a
cAMP standard curve generated by the same method as described
above. IC50 and EC50 values represent the mean of two experiments
performed in triplicate. IC50 and EC50 estimates and their associated
standard errors were determined by fitting the data using a nonlinear
least-squares analysis, with the help of GraphPad Prism 4 (GraphPad
Software, San Diego, CA). The maximal cAMP produced at 10
µM concentration of each ligand was compared to the amount of
cAMP produced at 10 µM concentration of the standard agonist
MT-II, and is expressed in percent (as % max effect) in Table 1.
The antagonist properties of the lead compounds were evaluated
by their ability to competitively displace the MT-II agonist in a
dose-dependent manner at four different doses of the antagonist
for 10-10 to 10-6 M. The pA2 values were obtained using the Schild
analysis method.40
Supporting Information Available: 1H NMR spectra of peptide
analogues in DMSO-d6 and the list of chemical shifts and coupling
constants. This material is available free of charge via the Internet
at http://pubs.acs.org.
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