Design, synthesis, and evaluation of proline based melanocortin receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2005.03.120

A series of proline based melanocortin ligands has been developed on the basis of initial piperazine leads by using a more conformationally rigid scaffold. A number of these novel ligands showed significant binding affinity for MC3 and MC4 receptors.

Full text of DOI:10.1016/j.bmcl.2005.03.120

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2819–2823
Design, synthesis, and evaluation of proline based
melanocortin receptor ligands
Xinrong Tian,* Timothy Field, Adam W. Mazur, Frank H. Ebetino, John A. Wos,
Doreen Crossdoersen, Beth B. Pinney and Russell J. Sheldon
Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason Montgomery Rd., Mason, OH 45040, USA
Received 18 January 2005; revised 21 March 2005; accepted 25 March 2005
Available online 4 May 2005
Abstract—A series of proline based melanocortin ligands has been developed on the basis of initial piperazine leads by using a more
conformationally rigid scaffold. A number of these novel ligands showed significant binding affinity for MC3 and MC4 receptors.
Ó 2005 Elsevier Ltd. All rights reserved.
The melanocortin receptors (MCRs) are a family of five
7-transmembrane G-protein coupled receptors (MC1R–
MC5R). These receptors are activated by the peptide
ligands: a, b, c-melanocyte stimulating hormones (MSH),
and adrenocorticotropin (ACTH), which are derived
from a common precursor protein, proopiomelanocor-
tin (POMC) by post-translational cleavage.1 The MCRs
have been found to mediate a variety of physiological re-
sponses that include skin pigmentation, inflammation,
steroidogenesis, feeding behavior, sexual function, and
exocrine gland secretion. Recently, many research
groups have invested intensive effort toward the design
of selective, non-peptidic small molecule ligands as po-
tential therapeutic agents for melanocortin-mediated
diseases.2 While the majority of this activity has been
focused on developing MC4R agonists as a treatment
for obesity or sexual dysfunction, small-molecule MC4R
antagonists have also been pursued as possible thera-
peutics to treat disease associated involuntary weight
loss.2d,3
that were common to all of these leads included two aro-
matic side chains attached at the 2,4-positions of the
piperazine ring and an acylated Arg residue at the top
side chain as exemplified by compounds 1 and 2. Despite
being devoid of functional activity, these leads provided
a viable starting point for small molecule MCR ligand
design so as to further improve affinity and, more
importantly, to achieve melanocortin functional activity
(Fig. 1).
Among a variety of potential ways envisioned to modify
structures of the initial piperazine lead molecules, we
chose first to explore the use of a more rigid five-mem-
bered proline ring to replace the 2-carboxylate piper-
azine motif as the constraining element for two aromatic
moieties, while keeping a flexible Arg side chain. The
goal was to gain insight into the effect of reducing con-
formational mobility of two phenyl side chains on
The initial leads for our drug discovery effort emerged
from screening libraries that were designed to capture
key recognition elements from the ÔmessageÕ sequence,
His-Phe-Arg-Trp, present in endogenous melanocortin
agonists.4 A particularly successful library was built on
a piperazine-2-carboxamide scaffold and ultimately led
to the identification of numerous leads with sub-micro-
molar binding to the MC4 receptor. Chemotype features
*
Corresponding author. Tel.: +1 513 622 1444; fax: +1 513 622
1195; e-mail: tian.x@pg.com
Figure 1. Piperazine leads.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.120

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X. Tian et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2819–2823
Scheme 1. Synthesis of trans proline analogs. Reagents and conditions: (a) LiOH, CH3OH/H2O, 18 h, 92%; (b) R1NH2, EDCI, HOBt, NMM, DMF,
4 h, 54–79%; (c) TFA/CH2Cl2, 3 h, ꢀ100%; (d) Pbf-(Fmoc)-Arg-OH, EDCI, HOBt, NMM, DMF, 4 h, ꢀ90%; (e) piperidine, DMF, 0.5 h, ꢀ91%; (f)
R2CO2H, EDCI, NMM, HOBt, DMF, 3 h, 61–81%; (g) EDCI, HOBt, NMM, DMF, 5 h, 54–62%; (h) H2, Pd/BaSO4, HOAc, CH3OH, 10 h, 30–50%.
biological activity. Herein, we would like to report on
synthesis, binding affinity, and functional activity of a
class of proline based MCR ligands.
using a cell based assay that is specific for each subtype
of MCR (MC1R, MC3R, MC4R). Each subtype of
receptor was stably transfected into HEK293 cells. The
MCR expressing cells were stably transfected with a re-
porter system consisting of a cyclic-AMP responsive ele-
ment (CRE) coupled to a luciferase reporter gene.
Responses were compared to the effect of NDP-MSH
(MT-I) and expressed as a % of maximum activity of
MT-1 (Emax). MT-1 is considered to be a full agonist
at each of the three MCR subtypes.
The synthesis of trans L-proline analogs was carried out
using approaches shown in Scheme 1 and all reactions
starting from 4 were performed at room temperature.
The (2S,4R) 4-benzyl proline ester 4 was prepared from
3 by a three-step sequence as reported in the literature.5
Hydrolysis of the ester followed by the amide formation
afforded the proline core structure 6 possessing two aro-
matic side chains. Cleavage of the Boc group gave rise to
7, which was then transformed into the final compounds
for assay by means of two approaches. Coupling 7 with
the pbf-(Fmoc)-Arg-OH followed by treatment of the
product with piperidine afforded 8. Acylation of the
amino group of Arg moiety and cleavage of protection
groups led to the desired analogs. Alternatively, amine
7 was coupled with an acylated Arg 10, which was pre-
pared from 9, followed by hydrogenation to deliver final
products in a more convergent fashion.
Binding affinity at the MC3 and MC4 receptors for trans
proline ligands are listed in Table 1. The Ki values at the
MC1R for all the compounds are >5000 nM. For com-
parison, a potent and selective 4,4-disubstituted piperi-
dine containing MC4 agonist (MK-1) reported by
Sebhat and co-workers2j was also tested in our assays
and the binding data at the MC3 and MC4 receptors
are included in Table 1 (Ki for MC1R, 591 nM). The
first analog synthesized in this series was compound
11, which was found to possess Ki values of 139 nM at
MC4R and 250 nM at MC3R. Interestingly, it was also
one of a few analogs that exhibited weak partial MC1
agonism (EC50, 1684 nM; Emax, 51%), although being
inactive in MC3 and MC4 functional assays. In fact,
none of analogs in this series showed functional activity
for MC3 and MC4 receptors. Replacement of 2-naphth-
ylacetic capping group of the Arg residue with 1-naphth-
ylacetyl resulted in a ꢀ5-fold increase in affinity at
MC4R (12, 29 nM), while maintaining MC3 and MC1
affinity. Although 12 was inactive in functional assays
across the three receptors, it represents a significant
improvement over the piperazine 2-carboxamide lead
compounds in terms of binding affinity. Based on the
MC4 binding affinity value and lack of functional
MC4 activity for 12, it was evaluated as an antagonist
of the MC4 receptor. Toward this end, MC4-mediated
luciferase responses to the MT-1 were determined in
the absence and presence of 12 at five different concen-
trations.8 Compound 12 caused a rightward-shift of
the MT-1 concentration-effect curve with reduction of
the Emax (Fig. 2), suggesting that it is an insurmountable
(2S,4S)-cis L-proline analogs were synthesized from 10
and 28 in a similar convergent manner as described in
Scheme 1. cis Proline derivative 28 was, in turn, pre-
pared from 3 in four steps6 (Scheme 2).
All analogs were screened in binding and functional
assays against the human MC1R, MC3R, and MC4R.
Binding affinity (calculated as IC50 and Ki values) was
determined by measuring the displacement of a constant
concentration of europium labeled NDP-a-MSH7 with
competing unlabeled ligands. The agonist activity of
MCR ligands was evaluated at three human hMCR
Scheme 2. Synthesis of cis proline analogs.

X. Tian et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2819–2823
Table 1. Binding affinity of trans proline analogs
2821
O
N
R2
O
O
HN
HN
N
H
H
N
N
H2N
N
O
N
N
O
NH
NHR1
Bn
Cl
MK-1
Compound
R1
R2
MC4 Ki (nM)a
MC3 Ki (nM)a
MK-1
9
1
736 49
250 38
11
12
138 47
29
47
8
9
258 11
13
871 242
14
15
243 91
1110 110
2422 503
1057 296
16
17
667 143
226 85
1486 93
527 136
18
19
1845 85
1360 435
4861 616
8017 2242
20
21
22
2780 547
255 62
333 81
10,733 752
900 109
270 55
23
24
310 76
109 17
692 171
66 13
25
26
27
31,392 6392
4756 266
7780 894
11,250
0
10,788 5133
7061 358
a The Ki values represent the mean of the at least three experiments SEM.

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X. Tian et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2819–2823
MC4R and MC3R in each case. The trend was observed
as well when 3,4-dichlorobenzyl (21), 3,4-(methylenedi-
oxy)phenyl methyl (22), and 2-chlorobenzyl (23) were
employed. Nevertheless, when 4-chlorobenzyl was used
in place of the naphthyl methyl of 11, the resulting ana-
log (24) retained the MC4 affinity but showed 4-fold bet-
ter affinity at MC3R with a Ki value of 66 nM, the best
MC3 affinity achieved in this series.
Use of an amino acid as the capping group of the Arg
residue was briefly explored to probe if introducing chi-
rality in conjunction with the hydrogen bonding capac-
ity would promote affinity and functional activity. Three
amino acids, Phe (25), His (26), and Tic (27), were exam-
ined along this line and all of them significantly de-
creased affinity at the MC3 and MC4 receptors as
compared to the bicyclic naphthylacetyl group.
To determine the stereochemistry requirement at the C-4
position of the proline ring, (2S,4S)-cis analog 29, a ste-
reoisomer of 11, was synthesized and screened. How-
ever, it did not show significant affinity at MC1R,
MC3R, and MC4R. Similarly, inverting the chirality
of the Arg residue of 11 to R configuration (30) led to
a complete loss of binding as well (Fig. 3).
Figure 2. MT-1 dose response curves compound 12: luciferase
response in MC4cre Cells.
antagonist at the MC4 receptor. Introduction of a meth-
oxyl group to the para position of C-2 amide phenyl ring
of 12 afforded an analog, 13 that maintained MC4 bind-
ing but was 3-fold less potent at MC3R relative to 12.
Fostch et al. recently reported a conceptually similar ap-
proach of the design of cis-cyclohexane diamine based
MC4 agonists from a potent D-Phe-Arg-Trp containing
MCR peptide ligand.9 Their strategy was to use a set of
low energy structures derived from NMR data for the
peptide ligand to identify ring systems that could posi-
tion groups in close proximity to the side chains of the
D-Phe-Arg-Trp tripeptide. Lack of the functional activ-
ity with our proline analogs is likely because spatial ori-
entation of four groups intended to mimic the side
chains of the tetrapeptide could not result in bioactive
conformations required for activating MCRs under cur-
rent topographical pattern and conformational restric-
tion mode. Our subsequent efforts toward investigating
a variety of approaches of introducing conformational
restriction between two adjacent amino acid residues
of the tetrapeptide His-D-Phe-Arg-Trp have identified
an optimum constraining mode for designing potent
MCR peptidomimetic agonists, which will be reported
in due course.
Substitution of the naphthyl group present in 12 for a
hydrogen-bonding capable indole ring led to a ꢀ8-fold
loss in MC4 affinity and a ꢀ4-fold loss in MC3 affinity
(14). Additional indole analogs were then prepared to
examine whether the affinity could be regained by vary-
ing the linkage length for the C-2 phenyl ring. Interest-
ingly, shortening (15) or lengthening (16) the space
between the phenyl ring and the nitrogen atom of C-2
amide moiety by one-carbon resulted in 4-fold and 3-
fold loss, respectively, in binding at MC4R and a slight
drop in MC3R. However, lengthening the space by two-
carbon (17) had no effect on MC4 affinity but led to a
slight increase at MC3 affinity.
A fused bicyclic naphthyl moiety present in capping
group of the Arg residue in 11 appears to be important
for significant binding. Replacement of the naphthyl
methyl group with benzyl (18), 2-phenyl ethyl (19),
and 2-biphenyl (20) significantly reduced binding at
Figure 3. Stereochemical effect on affinity.

X. Tian et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2819–2823
2823
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In summary, a series of proline-based MCR ligands dis-
playing significant binding affinity at the MC3 and MC4
receptors has been prepared and evaluated. The discov-
ery of these compounds originated with lead compounds
that emerged from screening libraries built on an analo-
gous piperazine-2-carboxamide scaffold. Key features of
these compounds include two conformationally re-
stricted aromatic side chains and a N-capped Arg moi-
ety. Although significant MCR agonism was not
achieved with this type of structures, some of these ana-
logs displayed high affinity at MC4R (12, 29 nM) and
MC3R (24, 66 nM). Further, this work has identified a
useful scaffold for further MCR ligand designs.
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