Discovery of tyrosine-based potent and selective melanocortin-1 receptor small-molecule agonists with anti-inflammatory properties

Article abstract of DOI:10.1021/jm025600i

The melanocortin-1 receptor (MC-1R) is a G-protein-coupled receptor involved in inflammation and skin pigmentation. Compound 2 is the first highly potent and selective MC-1R small-molecule agonist reported. Compound 2 showed efficacy in an acute model of inflammation, which has demonstrated the role of MC-1R in modulation of inflammation.

Full text of DOI:10.1021/jm025600i

J . Med. Chem. 2003, 46, 1123-1126
1123
cyclic peptide, has full agonist activity at MC-1R and
MC-5R and a weak partial agonist/antagonist activity
at the MC-3R and MC-4R receptors.13 HP-228 is a
peptide analogue with affinity for all four receptors but
a preference for MC-1R.14 HP-228 was in clinical trials
for the treatment of pain and inflammation associated
with surgery.15 There is a clear opportunity for the dis-
covery of receptor-selective melanocortin receptor agents.
Discover y of Tyr osin e-Ba sed P oten t a n d
Selective Mela n ocor tin -1 Recep tor
Sm a ll-Molecu le Agon ists w ith
An ti-in fla m m a tor y P r op er ties
Timothy F. Herpin, Guixue Yu,* Kenneth E. Carlson,
George C. Morton, Ximao Wu, Liya Kang,
Huji Tuerdi, Ashish Khanna, J ohn S. Tokarski,
R. Michael Lawrence, and J ohn E. Macor
Several small-molecule ligands for the melanocortin
receptors have been disclosed.15 Most of the activity in
small-molecule drug discovery has been directed at MC-
4R agonists as a treatment for obesity or sexual dysfunc-
tion. Scientists at Merck have disclosed a series of po-
tent and selective MC-4R agonists exemplified by struc-
ture 1.16 Trega and Melacure also have patented small-
molecule ligands for melanocortins.17 Recently, selective
small-molecule MC-1R agonists with micromolar affin-
ity have been identified from â-turn libraries.18
Bristol-Myers Squibb Pharmaceutical Research Institute,
P.O. Box 5400, Princeton, New J ersey 08543
Received October 23, 2002
Abstr a ct: The melanocortin-1 receptor (MC-1R) is a G-
protein-coupled receptor involved in inflammation and skin
pigmentation. Compound 2 is the first highly potent and
selective MC-1R small-molecule agonist reported. Compound
2 showed efficacy in an acute model of inflammation, which
has demonstrated the role of MC-1R in modulation of inflam-
mation.
Although MC-4R has received most of the medicinal
chemistry attention to date, we were interested in MC-
1R as a target for the development of anti-inflammatory
agents. MC-1R is expressed on the surfaces of mono-
cytes, and activation by R-MSH has been shown to
increase expression of the anti-inflammatory cytokine
IL-10 and stabilization of I-κB. These effects, in turn,
negatively modulate the activation of NF-κB, resulting
in the suppression of cytokine production and i-NOS
activity.19,20 In addition, R-MSH has been demonstrated
to be a potent anti-inflammatory agent in vivo in both
acute and chronic inflammatory disease models (e.g.,
inflammatory bowel disease, rheumatoid arthritis, and
renal ischemia/reperfusion injury).21 Administration of
R-MSH in these disease models resulted in substantial
tissue protection from inflammation-mediated damage,
a significant decrease in leukocyte infiltration, and a
dramatic reduction (near baseline level) of elevated
levels of cytokines (e.g., TNF-R), chemokines (e.g., MCP-
1, IL-8), and inflammatory mediators (e.g., i-NOS and
ICAM-1). Although there are strong indications that
these effects were mediated by MC-1R, the lack of potent
and selective MC-1R agonists has prevented an unam-
biguous identification of MC-1R as the melanocortin
receptor mediating this anti-inflammatory response.
Recently, highly selective and potent peptide MC-1R
agonists have been reported,22 but to the best of our
knowledge, their in vivo evaluation has not been pub-
lished.
In tr odu ction . There are five receptor subtypes known
in the melanocortin family. Activation of these G-
protein-coupled receptors (GPCR) stimulates the pro-
duction of cAMP in various tissues.1 The melanocortin-1
receptor (MC-1R) is expressed in melanocytes, mono-
cytes, mast cells, and neutrophils and is involved in
blocking inflammation and augmenting pigmentation.2
The melanocortin 2 receptor (MC-2R) is expressed in
adipocytes and adrenal cells and is involved in steroido-
genesis.3 MC-3R is present in the brain, hypothalamus,
heart, gut, and placenta and has been associated with
energy homeostasis and inflammation.4 MC-4R is ex-
pressed in the brain and has been associated with
feeding behavior, energy homeostasis, and erectile func-
tion.5 MC-5R is found in a wide range of tissues and is
involved in exocrine gland function.6 With the plethora
of melanocortin receptors, there is a significant need for
receptor-selective agents to further define and delineate
the roles of the individual receptors.
The endogenous agonists for these receptors are all
derived from the pro-opiomelanocortin (POMC) gene
transcript by post-translational cleavage.7 Differential
processing of the gene products generates R-melanocyte
stimulating hormone (R-MSH), â-MSH, γ-MSH, δ-MSH,
and adrenocorticotropic hormone (ACTH). All of these
melanocortin peptide agonists contain the His-Phe-Arg-
Trp sequence that is believed to be essential for stimu-
lation of four out of five melanocortin receptors.8 There
also exists two endogenous antagonists for the melano-
cortin receptors, agouti and the agouti-related protein
(AGRP).9 Several synthetic peptides or peptide ana-
logues have been used to characterize the function of
these receptors. NDP-RMSH (also called melanotan-I or
MT-I) is a high-affinity, nonselective agonist of MC-1R,
MC-3R, MC-4R, and MC-5R.10 Melanotan-II (MT-II),11
a cyclic peptide currently in phase II clinical trial for
the treatment of erectile dysfunction, is an agonist with
a similar nonselective profile.12 SHU-9119, another
Starting from the known16 MC-4R agonist 1 (Figure
1) and through extensive use of parallel synthesis
techniques, we have identified compound 2 as a low-
nanomolar, selective MC-1R full agonist. Compound 2
was evaluated in a mouse model of lipopolysaccharide-
induced (LPS-induced) TNF-R production, where it dose-
dependently inhibited cytokine production.
Ch em istr y. The chemistry to prepare compound 2
and its analogues is depicted in Scheme 1. Typical
EDC-HOAt coupling was used to assemble the three
fragments: the piperidine moiety (4), O-methyltyrosine
(3), and the histidine derivative (6). For the production
of libraries, SCX cartridge purification was used to
* To whom correspondence should be addressed. Phone: 609-818-
6528. Fax: 609-818-3450. E-mail: guixue.yu@bms.com.
10.1021/jm025600i CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/20/2003

1124 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Letters
Ta ble 1. In Vitro Activity of O-Methyltyrosine MC-1R
Agonists (n ) 4 Unless Otherwise Stated)
F igu r e 1. Known MC-4R agonist and novel selective MC-1R
agonist (IA ) intrinsic activity).
Sch em e 1. Synthesis of Compound 2
a
(a) (i) EDC, HOAt, DCM-DMF (1:1), overnight, SCX extrac-
tion, (ii) TFA/DCM, 1 h, SCX extraction; (b) (i) EDC, HOAt, DCM-
DMF (1:1), overnight, SCX extraction, (ii) TFA/DCM, 1 h.
remove most of the byproducts, followed by reverse-
phase HPLC purification.
Biology. Functional activity at human MC-1R was
measured by a SPA-based cAMP assay in HBL mela-
noma cells. HBL cells express only MC-1R among the
melanocortin receptors.23 Functional activity at MC-3R,
MC-4R, and MC-5R was tested in the SPA-based cAMP
assay using CHO cells overexpressing the individual
melanocortin receptors. Functional activity at MC-2R
was measured in a mouse Y1-adenocarcinoma cell line,
which expresses MC-2R endogenously. Results were
normalized to the level of c-AMP obtained after stimula-
tion with either 100 nM NDP-RMSH (for MC-1,2,3, and
4R) or 100 nM ACTH (for MC-2R). Binding affinities
were measured with a SPA-based radioligand binding
assay in intact cells using [125I]-NDP-R-MSH.
In Vitr o SAR. An initial library designed around the
known MC-4R agonist 1 was executed. This effort
identified the combination of the piperidine moiety 4
linked with tyrosine and histidine (compound 10, Table
1) as a potent and full agonist at MC-1R. A subsequent
small library of close analogues was then used to probe
the structure-activity relationship of the basic moiety
in this series. Table 1 summarizes the functional and
binding activities of selected compounds from this effort.
The R-amino group of the histidine in 10 could be
removed (compound 8) or replaced by a hydroxy group
(compound 9) without a loss in potency or intrinsic
activity. However, when the amino group was present,
the S configuration (10) was much more potent than
the corresponding R isomer (compound 11). N-methy-
lation of the imidazole ring of histidine provided two
isomers with very different activity. Isomer 2 retained
most of the activity of the parent compound, whereas
isomer 12 was only a partial agonist and 50-fold less
active than 10. Finally, acylation of the R-amino group
of the histidine of compound 2 resulted in an improve-
a
b
EC50 ( SEM (nM). IA ) intrinsic activity ( SEM (%). c IC50
d
( SEM (nM). n ) 2.
ment in functional activity by about 2 orders of mag-
nitude (compounds 6 and 7).
Table 2 summarizes the selectivity data for some of
these compounds. All compounds had no agonist activity
at MC-2R when tested at concentrations up to 40 µM.
Compounds 6-10 were found to activate the MC-3R and
MC-4R receptors, albeit with EC50 values that were 2
orders of magnitude less potent than for MC-1R. They
also had mixed activities at MC-5R with 6 and 7 being
full agonists and 8-10 being only partial agonists.
Compound 2 was found to be the most selective among
the MC-1R agonists. It did not activate MC-3R and was
a very weak partial agonist at MC-4R and MC-5R with
potencies of 3 and 4 µM. Compound 2 was selected for
in vivo evaluation based on its selectivity profile and
on results from a preliminary pharmacokinetic screen
in mice (data not shown).
P K P r ofile of Com p ou n d 2. Before the compound
was subjected to in vivo efficacy studies, the pharma-

Letters
Ta ble 2. In Vitro Selectivity for All Four Melanocortin Receptors (n ) 2 Unless Otherwise Stated)a
MC-1R MC-3R MC-4R
EC50 (nM)b EC50 (nM)b EC50 (nM)b
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1125
MC-5R
EC50 (nM)b
compd
IA (%)c
IA (%)c
IA (%)c
IA (%)c
2
6
7
8
9
28 ( 12d
0.19 ( 0.02
0.35 ( 0.07
2.5 ( 1.3d
3.8 ( 1.3d
1.5 ( 0.7d
92 ( 3d
98 ( 2
NA
2.1 ( 0.2e
100 ( 6
2600 ( 200
2.9 ( 0.04
23 ( 12
39 ( 3
95 ( 0.01
96 ( 0.8
92 ( 3
95 ( 0.4
103 ( 3
4400 ( 1300
2100 ( 400
3900 ( 900
9400 ( 200
2700 ( 100
10000 ( 500
15 ( 3
250 ( 170
2200 ( 1000
7000 ( 1700
3900 ( 3000
1700 ( 1000
65 ( 12
90 ( 15
32 ( 0.2
20 ( 2
96 ( 4
90 ( 0.01
85 ( 14
97 ( 1d
90 (1.3d
93 ( 4d
840 ( 150
77 ( 25
85 ( 14
98 ( 0.01
10
630 ( 340
40 ( 4
a
b
All of these compounds had no activity at MC-2R when tested at concentrations up to 40 µM. EC50 ( SEM (nM). c IA ) intrinsic
activity ( SEM (%). n ) 4. e Tested up to 40 µM.
d
Ta ble 3. Pharmacokinetic (PK) Profile of Compound 2 Dosed
this class of compounds and will be reported elsewhere.
Evaluation of compound 2 in other animal models of
acute and chronic inflammation is currently underway.
Intravenously in Balb/c Mice (n ) 3)a
dose,
µmol/kg
6.8
Cmax
nM
2200
,
AUC,b T1/2
,
MRT,
h
1.2
Cl,
mL min-1 kg-1L kg-1
110 8.2
Vss,
nM h
1000
h
1.5
Ack n ow led gm en t. The authors thank Dr. J anet
Caceres-Cortes for the complete NMR analysis of com-
pound 8, Dr. Graham Poindexter and Dr. Ildiko Antal
for valuable suggestions, Ms. Celia Darienzo for LC-
MS analysis, and Ms. Yifan Yang for conducting TNF-R
enzyme linked immunosorbent assays.
a
Coefficient of variation of the concentration determinations
b
is less than 26%. Area under the curve, 0-6 h.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedure for the preparation of 10, high-temperature 1H NMR
for compound 8, and 1H NMR, HPLC, and HRMS data for
compounds 2, 6-10, and assay protocols. This material is
available free of charge via the Internet at http://pubs.acs.org.
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