A potent and selective nonpeptide antagonist of the melanocortin-4 receptor induces food intake in satiated mice

Article abstract of DOI:10.1016/j.bmcl.2005.03.053

Optimization on a series of piperazinebenzylamines resulted in analogues with low nanomolar binding at the human MC4 receptor but weak affinity (K _i > 500 nM) at the MC3 receptor. Compound 14c was identified to be a potent MC4R antagonist (K_i = 3.2 nM) with a selectivity of 240-fold over MC3R. It proved to be an insurmountable antagonist in a cAMP assay. Compound 14c potently stimulated food intake in satiated mice when given by intracerebroventricular administration.

Full text of DOI:10.1016/j.bmcl.2005.03.053

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2541–2546
A potent and selective nonpeptide antagonist of the
melanocortin-4 receptor induces food intake in satiated mice
Joseph Pontillo,a Joseph A. Tran,a Stacy Markison,c Margaret Joppa,c Beth A. Fleck,b
Dragan Marinkovic,a Melissa Arellano,a Fabio C. Tucci,a Marion Lanier,a Jodie Nelson,a
John Saunders,a Sam R. J. Hoare,b Alan C. Fosterc and Chen Chena,*
aDepartment of Medicinal Chemistry, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
bDepartment of Pharmacology, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
cDepartment of Neuroscience, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
Received 20 January 2005; revised 10 March 2005; accepted 15 March 2005
Available online 9 April 2005
Abstract—Optimization on a series of piperazinebenzylamines resulted in analogues with low nanomolar binding at the human
MC4 receptor but weak affinity (Ki > 500 nM) at the MC3 receptor. Compound 14c was identified to be a potent MC4R antagonist
(Ki = 3.2 nM) with a selectivity of 240-fold over MC3R. It proved to be an insurmountable antagonist in a cAMP assay. Compound
14c potently stimulated food intake in satiated mice when given by intracerebroventricular administration.
Ó 2005 Elsevier Ltd. All rights reserved.
Among the five melanocortin receptors (MC1-5R),
MC3R and MC4R are centrally expressed and are be-
lieved to play important roles in mediating feeding
behavior, metabolism, and energy homeostasis.1 It has
been well documented that nonselective peptide MC4
antagonists such as SHU9119 and AgRP have a pro-
found effect in inducing feeding behavior in animals.2
Selective peptide MC4R antagonists such as MBP10
and HS131 have been discovered and data from these
peptides suggest the feeding effect is mediated by the
MC4 receptor.3 In addition, recent studies have showed
that MC4R also has a role in anxiety and pain.4,5 Thus,
potent and selective MC4R antagonists may be useful in
human diseases such as cachexia, anxiety, and neuro-
pathic pain.
mal models.4a In addition, a recent publication has
shown that an MC4R antagonist 2 (Ki = 160 nM) can
protect tumor-induced weight loss in mice following
peripheral administration.7
We have reported substituted phenylpiperazines as
MC4R-selective ligands which, interestingly, contain
both agonists and antagonists having only subtly differ-
ent chemical structures.8 For example, molecule 3 is
a
potent MC4R-selective agonist (EC50 = 4.7 nM,
IA = 100%),8b whereas compound 4 (Ki = 15 nM) is a
potent antagonist. The latter compound did, however,
stimulate cAMP release in cells expressing the human
MC4 receptor with 12% maximal level of a-MSH at
high concentrations.8c,9 Recently we demonstrated that
a
2,4-dichlorophenylalanine analogue of 5a (Ki =
Several small molecule MC4R antagonists have been re-
ported. MCL0129 (1b), which is structurally related to
an earlier reported series as exemplified by 1a (Fig.
1),6 inhibited the NDP-MSH binding to MC4R with a
Ki value of 7.9 nM. This compound exhibits anxio-
lytic-like and antidepressant-like activity in several ani-
21 nM) possesses high affinity (5b, Ki = 1.8 nM) with
inability to stimulate cAMP release (IA < 3%).10 Here
we report compounds, derived from this structure,
as potent antagonists of the human melanocortin-4
receptor (hMC4R) with high selectivity over the melano-
cortin-3 receptor.11 In addition, we demonstrate that
one of these compounds, 14c, is an insurmountable
antagonist, and is able to stimulate food intake in sati-
ated mice.
Keywords: Melanocortin-4; Antagonist; Piperazinebenzylamine; Food
intake.
*
The synthesis of targeted compounds started from pip-
erazinebenzaldehyde 6, which was obtained from the
Corresponding author. Tel.: +1 858 617 7600; fax: +1 858 617
7967; e-mail: cchen@neurocrine.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.053

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J. Pontillo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2541–2546
F
F
Br
O
N
N
N
NH
N
N
N
N
N
N
F
O
O
1a
1b (MCL0129)
2 (ML253764)
X
Cl
Cl
Cl
N
N
N
N
N
N
HN
S
NH
HN
S
NH
HN
NH
O
O
O
O
NH2
O
O
NH
HN
N
H
5a: X = H
5b: X = Cl
4
3
Figure 1. Example small molecule MC4R ligands.
condensation of 2-fluorobenzaldehyde and N-Cbz-
piperazine in DMSO at 120 °C (Scheme 1). Compound
6 was subjected to reductive amination with 2-(2-thien-
yl)ethylamine in the presence of sodium triacetoxy-
borohydride in dichloromethane, followed by
protection of the resulting secondary amine with a
Boc-group in 82% yield. The protected diamine interme-
diate was selectively deprotected with palladium-cata-
lyzed hydrogenation in methanol to give the amine 7
in 35% yield. Coupling reaction of 7 with N-Cbz-(R)-
arylalanines (9) under the standard peptide coupling
conditions (EDC/HOBt/CH2Cl2), followed by Boc-
deprotection with HCl/dioxane afforded the desired
products 8a–g in 60–80% yields (Table 1). Compound
7 was also coupled with N-Fmoc-(R)-(2,4-Cl)Phe-OH,
followed by removal of the Fmoc-group with diethyl-
Table 1. SAR of arylalanines at hMC4Ra
Compound
Ar
Ki (nM)
23 3
8a
8b
8c
8d
8e
8f
2,4-ClPh
1-Naphthyl
4-HOPh
420 280
5700 490
3600 1100
>10,000
4-Imidazolyl
4-Thiazolyl
3-Indolyl
2300 210
1100 190
8g
3-Benzothienyl
a Binding affinity at the human melanocortin-4 receptor stably trans-
fected in HEK 293 cells, using [125I]-NDP-MSH as the radiolabeled
ligand.
amine to give 10 in 87% yield, which served as the key
intermediate to be derivatized in several ways as follows.
Ar
b
a
c, d
N
N
N
F
Boc
NH
N
N
N
S
O
Cbz
HN
S
NHCbz
O
O
6
7
8a-g
e
Cl
Cl
Cl
Cl
Cl
Cl
f, d
g, d
N
N
N
N
R
Boc
N
N
HN
S
N
H
N
NH2
HN
NH
O
O
O
S
O
O
S
S
11a-f
12
10
Scheme 1. Reagents and conditions: (a) N-Cbz-piperazine/K2CO3/DMSO/120 °C, 19 h, 53%. (b) (i) 2-(2-Thienyl)ethylamine/NaBH(OAc)3/CH2Cl2/
rt, 16 h; (ii) (Boc)2O/CH2Cl2/rt, 6 h, 82%; (iii) Pd/C–H2 (40 psi)/NH3/MeOH/rt, 23 h, 35%. (c) N-Cbz-(R)-(Ar)Ala-OH (9)/EDC/HOBt/CH2Cl2/rt,
18 h, 60–80%. (d) HCl/dioxane/rt, 2 h; (e) (i) N-Fmoc-(R)-(2,4-Cl)Phe-OH/EDC/HOBt/CH2Cl2/rt, 18 h; (ii) Et2NH/CH2Cl2/rt, 3 h, 87%. (f)
Aldehyde/NaBH(OAc)3/CH2Cl2/rt. (g) MsCl/pyridine/rt.

J. Pontillo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2541–2546
2543
Table 2. SAR on the N-alkylation of the 2,4-dichlorophenylalaninea
Table 3. Binding affinity (Ki, nM) of compounds 13–15 at the human
MC4 receptor and selectivity over the human MC3 receptora
Compound
R
Ki (nM)b
Cl
Cl
11a
11b
11c
11d
11e
11f
12
H
i-Bu
94 18
23
18
16
31
18
14
6
3
4
9
5
6
N
c-PrCH2
2-ThiazoleCH2
Bn
N
HN
S
NH
O
O
R'
CH2CH2NH2
SO2Me
a Binding affinity at the human melanocortin-4 receptor stably trans-
fected in HEK 293 cells, using [125I]-NDP-MSH as the radiolabeled
ligand.
5b, 13-15
Compound
R0
MC4Rb
MC3Rb
b Data are average of three or more independent measurements.
5b
CH2CH2NH2
1.8 0.2
2.4 0.4
3.1 1.1
2.0 0.8
2.8 0.2
5.0 0.9
4.6 1.8
3.2 1.4
4.5 1.0
1.7 1.0
2.1 0.4
1.4 0.1
2.4 0.5
5.0 0.3
2.9 0.3
1.3 0.3
2.0 0.2
1.5 0.3
640 190
570 210
1600 500
950 500
830 150
810 210
800 10
13a
13b
13c
13d
14a
14b
14c
14d
15a
15b
15c
15d
15e
15f
15g
15h
15i
Me
Ph
CH2CH2CH2NH2
CH2CH2COOMe
OMe
Compound 11a (R = H) was obtained by deprotection
of 10 with HCl/dioxane. Reductive alkylation of 10 with
various aldehydes afforded, after HCl treatment, the
diamines 11b–f (Table 2). Compound 10 was also
coupled with methanesulfonyl chloride under basic
conditions to give the sulfonamide 12 after Boc-
deprotection. Alternatively, coupling reactions of 10
with various carboxylic acids, including N-Boc-amino
acids, gave the final compounds 13a–d in 34–65% yields
after Boc-deprotection (Scheme 2). Reaction of 10 with
alkyl chloroformates under basic conditions, followed
by HCl-deprotection provided carbamates 14a–d, whilst
reaction with isocyanates afforded the ureas 15a–e. The
ureas 15f–i were obtained from the reaction of 10 with
phosgene in toluene at 0 °C, followed by alkylamines
at room temperature (Table 3). All final compounds
were purified using a HPLC-MS system as previous
described.10
OEt
OCH2CH2F
O(i-Pr)
NHEt
790 120
1300 450
790 120
860 150
450 80
NH(c-Pr)
NH(i-Pr)
NH(t-Bu)
1000 190
2300 720
390 150
580 100
560 41
NH(2-FBn)
NHCH2CH2OH
NH(cyclopentyl)
NMe2
1-Pyrrolidinyl
670 41
a Binding affinity at the human melanocortin-4 or melanocortin-3
receptor stably transfected in HEK 293 cells, using [125I]-NDP-MSH
as the radiolabeled ligand.
b Data are average of three or more independent measurements.
measured for their ability to inhibit a-MSH-stimulated
cAMP production in the same cell lines.
The competition binding experiments were performed
using HEK293 cells stably transfected with the human
melanocortin receptors as previously described, using
[
tional antagonist activity of selected compounds was
125I]-NDP-MSH as the radiolabeled ligand.12 The func-
Previously we have showed that replacing the 4-chloro-
phenylalanine of 5a (Ki = 21 nM, IA = 14%) with the
Cl
Cl
Cl
Cl
N
N
a, b
Boc
N
N
N
S
NH2
HN
S
NH
R1
O
O
O
10
13a-d
d, b
c, b
Cl
Cl
Cl
Cl
N
N
N
N
HN
S
NH
HN
S
NH
R4
R2
O
O
O
N
O
O
R3
15a-i
14a-d
Scheme 2. Reagents and conditions: (a) R1COOH/EDC/HOBt/CH2Cl2/rt, 16 h, 34–65%. (b) HCl/dioxane/rt, 2 h. (c) R2OCOCl/Et3N/CH2Cl2/0 °C to
rt. (d) R3NCO/CH2Cl2/rt; or (i) COCl2/Et3N/toluene/0 °C to rt; (ii) R3R4NH/THF, rt.

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J. Pontillo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2541–2546
Table 4. Binding affinity (Ki, nM) of selected compounds at the human
melanocortin receptor subtypesa
2,4-dichloro variant not only increases its binding affin-
ity over 10-fold, but also diminishes the intrinsic activity
in stimulating cAMP release of this compound (5b,
Ki = 1.8 nM, IA < 3%).10 A brief survey on the phenyl-
alanine moiety of 8 revealed that the highly lipophilic
2,4-dichlorophenyl compound (8a, Ki = 23 nM) was
more potent in binding than other analogues including
the 1-naphthyl variant (8b, Ki = 420 nM). The relatively
polar 4-hydroxyphenyl derivative (8c, Ki = 5.7 lM) was
only weakly active. All heteroaromatic analogues 8d–g
exhibited poor binding affinity (Ki > 1 lM, Table 1).
Compound MC1Rb
MC3R
MC4R
MC5R
13a
14c
14d
15a
15f
15h
15i
(31%)
570 210 2.4 0.4 730 90
790 120 3.2 1.4 560 110
1300 450 4.5 1.0 680c
790 120 1.7 1.0 300c
1000 340
(9%)
(18%)
(8%)
6800 2200
6900 1100
390 150 2.9 0.3 350 42
560 190 2.0 0.2 350 38
670 41
1.5 0.3 160 16
a Human melanocortin receptors stably expressed in HEK 293 cells.
b Data in parenthesis are percentage of inhibition at 10 lM concen-
tration, and average of 2–3 measurements.
We also demonstrated in this series that the amide func-
tionality of the 2,4-chlorophenylalaninyl nitrogen has a
profound effect on both binding affinity and, more sig-
nificantly, functional activity. Thus, the primary amine
11a displayed a Ki value of 94 nM, which was over 50-
fold reduction in affinity caused by the removal of the
b-alanine of 5b. Introduction of a 2-aminoethyl group
on 11a restored affinity by about 4-fold (11f,
Ki = 18 nM). Incorporation of thiazolemethyl (11d,
Ki = 16 nM) and a small lipophilic isobutyl group
(11b, Ki = 23 nM), or sulfonylation (12, Ki = 14 nM) of
11a resulted in compounds with affinity similar to 11f.
These results indicate that the 2-aminoethyl group of
b-alanine 5b has minimal effect on binding, and that
the amide functionality (–CONH–) plays an important
role either by direct interaction with MC4R, or by forc-
ing a more favored binding conformation of the 2,4-
dichlorophenylalanine moiety.
c Single measurement.
selected compounds from this series dose-dependently
inhibited a-MSH-stimulated cAMP production as
exemplified by a representative 14c (Fig. 2). Interest-
ingly, unlike 5b which is a competitive antagonist in
the cAMP assay with a pA2 of 7.9,10 compound 14c
was demonstrated by a Schild analysis to be an insur-
mountable antagonist on the inhibition of a-MSH-stimu-
lated cAMP release (Fig. 3). Thus, the small structural
Amides (13a–d), carbamates (14a–d), and ureas (15a–i)
derived from 10 displayed potent binding affinity at
MC4R (Table 3). Since all amides (13a–d) had Ki values
of 2.0–3.1 nM, irrespective of the acyl substituent, it is
obvious that the only important element of the amide
group is the –CONH– moiety. Interestingly, this region
tolerated groups with different properties, such as phe-
nyl (13b) and amino alkyl (5b, 13c). Additionally, since
the carbamates 14 and ureas 15 were also potent
binders, this locus offers an opportunity for optimiza-
tion of this series of compounds towards desirable phys-
icochemical properties as potential CNS agents.
Figure 2. Dose-dependent inhibition of a-MSH-stimulated cAMP
accumulation by 14c in HEK 293 cells expressing the human
melanocortin-4 receptor. Results are average of two independent
experiments.
Since the MC3 receptor is also centrally localized and
may also play a role in control of food intake and
metabolism,1 we tested compounds 13–15 in the
MC3R binding assay, and found that these compounds
showed weak binding affinity (Ki P 530 nM). For exam-
ple, while compound 14c displayed a Ki value of 3.2 nM
at hMC4R, it possessed low binding affinity at hMC3R
(Ki = 790 nM). Thus, 14c exhibited a 240-fold selectivity
of MC4R over the MC3 receptor subtype.
Selected compounds were also tested on other melano-
cortin receptor subtypes (MC1R and MC5R) and
MC4R selectivity was demonstrated (Table 4). In addi-
tion, the compounds derived from 2,4-dichlorophenyl-
alanine showed no significant stimulation of cAMP
accumulation at 10 lM concentration (<3% of a-MSH
maximal levels, data not shown) in cells expressing
MC4R. These data demonstrate that these compounds
do not behave as functional MC4R agonists. Moreover,
Figure 3. a-MSH-stimulated cAMP accumulation in the presence of
different concentrations of the MC4R antagonist 14c. Results are
average of two independent experiments.

J. Pontillo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2541–2546
2545
difference in the amide group between 5b and 14c causes
profound pharmacological changes in these two
compounds.
References and notes
1. (a) Gantz, I.; Fong, T. M. Am. J. Physiol. Endocrinol.
Metab. 2003, 284, E468; (b) Goodfellow, V.; Saunders, J.
Curr. Top. Med. Chem. 2003, 3, 855.
In addition to high affinity on human MC4R, com-
pound 14c bound to the mouse MC4R with a Ki value
of 3.7 nM, demonstrating no selectivity between these
two species. Thus, we were able to utilize compounds
from this series to examine the ability of a small mole-
cule MC4R antagonist to stimulate food intake in mice.
Unfortunately, many of the potent MC4R antagonists
listed in Table 3 suffered from either poor absorption
associated with high hydrophilicity (5b, logD = 1.1,
F = 1%), or metabolic instability in liver microsomes
(14c, Clint = 1750 mL/min kg in mouse liver micro-
somes), thus precluding them from peripheral adminis-
tration. Therefore, we measured the efficacy by directly
delivering the compound into the mouse brain via intra-
cerebroventricular (icv) administration. Female CD-1
mice were divided into four groups (n = 10/group) and
injected, with distilled water, 0.3, 3, or 10 nmol of 14c,
into the lateral/third cerebral ventricles using a free-
hand injection method.13 Bonferroni-adjusted t-test post
hoc comparisons indicated that compound 14c signifi-
cantly increased food consumption at the highest dose
given at all the time points measured (all Ps < 0.01)
(Fig. 4).
2. Adage, T.; Scheurink, J. W.; de Boer, S. F.; de Vries, K.;
Konsman, J. P.; Kuipers, F.; Adan, R. A. H.; Baskin, D.
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3. (a) Foster, A. C.; Joppa, M.; Markinson, S.; Gogas, K. R.;
Fleck, B. A.; Murphy, B. J.; Wolff, M.; Cismowski, M. J.;
Ling, N.; Goodfellow, V. S.; Chen, C.; Saunders, J.;
Conlon, P. J. Ann. N.Y. Acad. Sci. 2003, 994, 103; (b)
Schioth, H. B.; Kask, A.; Mutulis, F.; Muceniece, R.;
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Baumgartner, J. W.; Zambrano, M.; Cheetham, J.;
Tamayo, N. A. J. Med. Chem. 2003, 46, 9.
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Forsyth, N. E.; Drabic, S. V.; Horlick, R. A.; Lamppu, D.;
Yowe, D. L.; Balani, S.; Li, P.; Zeng, H.; Joseph, I. B. J.
K.; Rodriguez, L. E.; Claiborne, C. F. J. Med. Chem.
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In conclusion, we have synthesized a series of (R)-2,4-
dichlorophenylalanine derivatives as potent antagonists
of MC4R. These compounds are highly selective over
MC3R, as well as other human melanocortin receptor
subtypes. One compound from the series, 14c, had Ki
values of 3.2 and 790 nM, respectively, at the melano-
cortin-4 and -3 receptors. This compound was also dem-
onstrated to be an insurmountable antagonist in the
inhibition of a-MSH-stimulated cAMP release by a
Schild analysis, possibly due to slow dissociation rate.14
Compound 14c potently stimulated food intake in sati-
ated mice when given intracerebroventricularly.
vehicle
0.8
0.3 nmol
*
3 nmol
10 nmol
*
*
0.6
0.4
0.2
0.0
*
1
2
4
6
11. Several phenylpiperazines bearing an amide side-chain
have been reported to be potent and selective antagonists
of the melanocortin-4 receptor, see Ref. 9.
12. Nickolls, S. A.; Cismowski, M. I.; Wang, X.; Wolff, M.;
Conlon, P. J.; Maki, R. A. J. Pharmacol. Exp. Ther. 2003,
304, 1217.
Time (h)
Figure 4. Effect of 14c on cumulative food intake in satiated mice.
Mice (n = 10/group) were given vehicle (distilled water) or the MC4R
antagonist, 14c, via icv injection and food intake was measured over
the following 6 h. Cumulative food intake was significantly increased
by the 10 nmol dose of 14c relative to vehicle at all time points
(*P < 0.01).
13. Briefly, following a short anesthetization with isoflurane
gas, a 30-gauge needle modified to a length of 4 mm was

2546
J. Pontillo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2541–2546
placed at the apex of a triangle between the eyes and back
as an interaction (F(9,159) = 2.04, P < 0.041). Pelley-
mounter, M. A.; Joppa, M.; Carmouche, M.; Cullen, M.
J.; Brown, B.; Murphy, B.; Grigoriadis, D. E.; Ling, N.;
Foster, A. C. J. Pharmacol. Exp. Ther. 2000, 293,
799.
of head and 5 lL of vehicle or compound was injected
over 30 s. Fifteen minutes after the injection, food was put
in the cage and intake was measured 1, 2, 4, and 6 h later.
A
two-way repeated measures analysis of variance
revealed significant main effects of dose (F(3,36) = 13.64,
P < 0.001) and time (F(3,108) = 79.31, P < 0.001) as well
14. Vauquelin, G.; Van Liefde, I.; Vanderheyden, P. Trends
Pharmacol. Sci. 2002, 23, 514.