Design, synthesis, and biological evaluation of a new class of small molecule peptide mimetics targeting the melanocortin receptors

Article abstract of DOI:10.1016/j.bmcl.2006.07.015

A new bicyclic template has been developed for the synthesis of peptide mimetics. Straightforward synthetic steps, starting from amino acids, allow the facile construction of a wide range of analogs. This system was designed to target the melanocortin receptors (MCRs), with functional group selection based on a known pharmacophore and guidance from molecular modeling to rationally identify positional and stereochemical isomers likely to be active. The functions of hMCRs are critical to myriad biological activities, including pigmentation, steroidogenesis, energy homeostasis, erectile activity, and inflammation. These G-protein-coupled receptors (GPCRs) are targets for drug discovery in a number of areas, including cancer, pain, and obesity therapeutics. All compounds from this series tested to date are antagonists which bind with high affinity. Importantly, many are highly selective for a particular MCR subtype, including some of the first completely hMC5R-selective antagonists reported.

Full text of DOI:10.1016/j.bmcl.2006.07.015

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5462–5467
Design, synthesis, and biological evaluation of a new class of small
molecule peptide mimetics targeting the melanocortin receptors
James P. Cain, Alexander V. Mayorov, Minying Cai, Hui Wang, Bahar Tan,
Kevin Chandler, YeonSun Lee, Ravil R. Petrov, Dev Trivedi and Victor J. Hruby*
Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA
Received 2 May 2006; revised 23 June 2006; accepted 5 July 2006
Available online 22 August 2006
Abstract—A new bicyclic template has been developed for the synthesis of peptide mimetics. Straightforward synthetic steps, start-
ing from amino acids, allow the facile construction of a wide range of analogs. This system was designed to target the melanocortin
receptors (MCRs), with functional group selection based on a known pharmacophore and guidance from molecular modeling to
rationally identify positional and stereochemical isomers likely to be active. The functions of hMCRs are critical to myriad biolog-
ical activities, including pigmentation, steroidogenesis, energy homeostasis, erectile activity, and inflammation. These G-protein-
coupled receptors (GPCRs) are targets for drug discovery in a number of areas, including cancer, pain, and obesity therapeutics.
All compounds from this series tested to date are antagonists which bind with high affinity. Importantly, many are highly selective
for a particular MCR subtype, including some of the first completely hMC5R-selective antagonists reported.
Ó 2006 Elsevier Ltd. All rights reserved.
The human melanocortin receptors (hMCRs) comprise
a family of five Type I, or rhodopsin-like, G-protein-
coupled receptors (GPCRs) to which a wide array of
biological functions has been ascribed.1 Some examples
include nociception, inflammation, energy balance, and
sexual function. From the early understanding of the
role MCRs play in pigmentation to recent revelations
concerning their relevance to pain, new studies have
continually uncovered crucial but previously unknown
actions of this receptor system. Beyond advancing our
knowledge of basic biology, the understanding and
modulation of MCR function also has clinical relevance,
with potential therapeutic value for addressing obesity,2
cachexia,3 pain,4 inflammatory diseases,5 and sexual
dysfunction,6 as well as the diagnosis and treatment of
certain cancers.7
contains the His-Phe-Arg-Trp tetrad, the minimum se-
quence necessary for activation of all melanocortin
receptors.9 Both endogenous antagonists contain an
Arg-Phe-Phe sequence. Extensive melanotropin peptide
structure–activity relationship (SAR) studies by our
group and others have identified modifications which
enhance potency, stability, or selectivity.10 The value
of the ligands generated—particularly the standard ago-
nists NDP-a-MSH and MT-II, and the antagonist,
SHU9119, is hard to overestimate.
Nevertheless, with respect to certain applications in biol-
ogy and medicine, the possession of small molecules
with activity at the MCRs and properties complementa-
ry to those of peptides would be advantageous. Consid-
erable effort in both academic and industrial
laboratories has been directed toward this end.11 Suc-
cessful examples have come from screening libraries,12
‘privileged structure’ design strategies,13 and ligand-
based rational design using computational chemistry.14
Much research to date has relied on natural and synthet-
ic peptide ligands for these receptors. The MCRs are
unique in that both endogenous agonists (a-, b-,
c-MSH, ACTH) and antagonists (agouti, AGRP) for
the system have been discovered.8 Each of the agonists
We decided to employ this latter modeling approach to
guide our design of small molecule peptide mimetics.
Careful consideration of potential molecular scaffolds
for b-turn mimetics led us to the bicyclic structure in
Figure 1. From a synthetic point of view, compounds
of this type are easily constructed from amino acids,
Keywords: Melanocortins; Peptide mimetics; GPCRs.
*
Corresponding author. Tel.: +1 520 621 6332; fax: +1 520 621
8407; e-mail: hruby@u.arizona.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.015

J. P. Cain et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5462–5467
5463
R2
R3
of multiple analogs, some with better overlap than oth-
ers, would allow us to simultaneously probe the struc-
tural requirements of MCRs and to test the validity of
our model.
N
R1
N
Figure 1. General structure of peptide mimetics.
The synthesis of our first set of analogs began with the
construction of the core template from L-proline and
D- or L-phenylalanine (Scheme 1, L-phenylalanine
shown). The dipeptide of these amino acids was easily
obtained by BOP-mediated coupling. Deprotection of
the Boc-protected amine using TFA was followed by
cyclization in triethylamine and methanol to yield the
diketopiperazine. Reduction with LAH provided the
diamine core structure.
allowing us to take advantage both of the naturally
available chiral pool and our group’s extensive reper-
toire of unnatural amino acid syntheses.15
Interestingly, none of our initial target molecules had
been previously reported. Despite the prevalence of
piperazine-based structures in medicinal chemistry,16,17
the pyrrolopiperazine moiety remains surprisingly
underutilized.18 On the other hand, diketopiperazines,
including the cyclodipeptides that are key precursors
to our final structures, are found in a wide variety of
natural products and synthetic ligands.19–21 As a conse-
quence, a significant body of methodology for the syn-
thesis of these molecules has been developed.22
Functionalization of this core structure could be achieved
with a variety of acylation chemistries (Scheme 2). Reac-
tion with phenylacetyl chloride provided HMC001
(3S,6S) and HMC002 (3S,6R). A convenient means of
introducing a spacer was the reaction with succinic anhy-
dride, followed by amide bond coupling, to generate
HMC013 (3S, 6S) and HMC014 (3S,6R). This coupling
reaction itself, using BOP, was convenient and effective
for functionalizing the core secondary amine, and was
used with indolepropionic acid to access HMC009 (3S,
6S) and HMC010 (3S,6R). The same coupling chemistry
was used to produce HMC021 and HMC025. All com-
pounds were purified using RP-HPLC.
The functional groups appended to our template were
chosen based on SAR both for peptide ligands and pre-
vious MCR-targeted small molecules. As noted above,
peptides active at all five MCRs contain the His-Phe-
Arg-Trp sequence, while the minimum chemical features
seemingly common to all active small molecules are the
presence of two hydrophobic aromatic groups and a ba-
sic nitrogen. In the design of our first set of compounds,
we intended to explore the effect of variations in the type
and orientation of the hydrophobic groups and of the
presence or absence of arginine.
Biological assays of these compounds were performed
using human melanocortin receptors (hMCRs)
All four stereoisomeric variants of a number of such
compounds were modeled and compared to the solution
conformations23 of MT-II, a superpotent, nonselective
agonist for the MCRs.24 One relatively simple example
is shown in Figure 2. Spectroscopy and molecular
modeling have suggested that the receptors recognize a
b-turn structure in MT-II, probably centered at the his-
tidine and phenylalanine residues.23 The new structures
with the best overlap against MT-II were predicted to
have the best biological activity. We also hoped to iden-
tify structurally distinct features of molecules within this
subset which demonstrated greater receptor subtype
selectivity. Thus, the synthesis and biological evaluation
Scheme 1. Reagents: (a) BOP, HOBt, DIEA, DMF; (b) TFA; (c) TEA,
MeOH; (d) LiAlH4, THF.
Figure 2. Superimposition of computational model of HMC001 (orange) and NMR-derived structure of MT-II (cyan).23

5464
J. P. Cain et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5462–5467
O
O
O
O
a
NH
*
N
*
HMC001(6S)
HMC002(6R)
N
N
N
N
N
N
N
N
N
N
H
N
b,c
NH
*
N
*
O
NH
HMC013(6S)
HMC014(6R)
NH
*
d
N
*
NH
HMC009(6S)
HMC010(6R)
NH
H
N
e,f,g
h,i,j
NH
NH
N
O
HMC021
NH
O
H
N
N
O
HMC025
NH
HN NH2
Scheme 2. Reagents: (a) Phenylacetyl chloride, DIEA, DMAP, DCM;
(b) succinic anhydride, DMAP; (c) tryptamine, BOP, HOBt, DIEA,
DMF; (d) indolepropioinic acid, BOP, HOBt, DIEA, DMF; (e) Boc-6-
aminohexanoic acid, BOP, HOBt, DIEA, DMF; (f) TFA; (g)
indolepropionic acid, BOP, HOBt, DIEA, DMF; (h) Boc-L-arginine,
BOP, HOBt, DIEA, DMF; (i) TFA; (j) indolepropionic acid, BOP,
HOBt, DIEA, DMF.
expressed in whole HEK293 cells.25 Binding was deter-
mined by measuring competition with 125I-labeled
NDP-a-MSH.26 Binding efficiency refers to the degree
of displacement of the radiolabeled ligand by the com-
pound being assayed, relative to the maximum displace-
ment caused by excess amounts of MT-II. Functional
activity (activation of adenylate cyclase) was determined
by measuring the cAMP produced by the cells after its
incubation with ligand.26
The assay results for our first set of compounds (Table
1) are very encouraging. Each of the analogs tested thus
far binds to one or more of the receptor subtypes, and
most with high selectivity. HMC001, for instance, binds
strongly to MC1, with greater than 150-fold selectivity
over MC3, and no measurable binding at other sub-
types. HMC002, with the opposite stereochemistry at
C6, also binds strongly to MC1. The effect of switching
the chirality of C6 in the analogs containing an indole-
propionyl group is dramatic. HMC009 binds only to
MC5, while HMC010 binds to MC1 and MC3. For both
HMC009 and HMC010, only 50% displacement of radi-
oligand is observed at each relevant receptor subtype.
Each of the remaining members of this test set contains
an indole ring separated from the bicyclic core by a rel-
atively long linker group. The biological results for all of
these compounds are remarkably similar, exhibiting
complete MC5 selectivity, with subnanomolar IC50
values and 50–65% binding efficiency. The presence of

J. P. Cain et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5462–5467
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The discovery of five new MC5-selective antagonists
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HMC025) may prove useful for the study of this rela-
tively unexplored receptor subtype. The most widely ex-
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naling in mice.29 Chemical biology using a small mole-
cule may offer advantages over the use of MC5R
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