Potent and orally active non-peptide antagonists of the human melanocortin-4 receptor based on a series of trans-2-disubstituted cyclohexylpiperazines

Article abstract of DOI:10.1016/j.bmcl.2005.06.071

The melanocortin-4 receptor (MC4R) plays an important role in the regulation of energy homeostasis. Recent studies have shown that blockade of the MC4R reverses tumor-induced weight loss in mice. Herein, we describe the synthesis and identification of potent and selective non-peptide antagonists of the human MC4R from a series of 2-ethoxycarbonylcyclohexyl-piperazines. Compound 12i was found to possess low nanomolar affinity for the MC4R, and exhibit oral bioavailability in rats. More importantly, when administered orally to mice (10 mg/kg), it led to statistically significant increases in food intake over a 24-h period.

Full text of DOI:10.1016/j.bmcl.2005.06.071

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4389–4395
Potent and orally active non-peptide antagonists of the human
melanocortin-4 receptor based on a series
of trans-2-disubstituted cyclohexylpiperazines
Fabio C. Tucci,a,* Nicole S. White,a Stacy Markison,d Margaret Joppa,d
Joe A. Tran,a Beth A. Fleck,b Ajay Madan,c Brian P. Dyck,a Jessica Parker,a
Joseph Pontillo,a L. Melissa Arellano,a Dragan Marinkovic,a Wanlong Jiang,a
Caroline W. Chen,a Kathleen R. Gogas,d Val S. Goodfellow,a
John Saunders,a Alan C. Fosterd 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 Preclinical Development, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
dDepartment of Neuroscience, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
Received 26 April 2005; revised 8 June 2005; accepted 9 June 2005
Abstract—The melanocortin-4 receptor (MC4R) plays an important role in the regulation of energy homeostasis. Recent studies have
shown that blockade of the MC4R reverses tumor-induced weight loss in mice. Herein, we describe the synthesis and identification of
potent and selective non-peptide antagonists of the human MC4R from a series of 2-ethoxycarbonylcyclohexyl-piperazines. Com-
pound 12i was found to possess low nanomolar affinity for the MC4R, and exhibit oral bioavailability in rats. More importantly,
when administered orally to mice (10 mg/kg), it led to statistically significant increases in food intake over a 24-h period.
Ó 2005 Elsevier Ltd. All rights reserved.
Cachexia is a syndrome of weight loss associated with
debilitating diseases such as cancer, renal failure, heart
failure, and chronic infections.1 The severity of cachexia
in these illnesses is considered to be a primary determi-
nant of the deteriorating quality of life and eventual
mortality, and is characterized by a significant decrease
in lean body mass.2 Current available treatments for
cachexia rely heavily on the use of high doses
(>800 mg) of megestrol acetate, which leads primarily
to an increase in fat mass and water content and not
the desired increase in lean body mass.3 The high doses
used also may lead to undesired side-effects due to the
shut down of the hypothalamic–pituitary axis (HPA).4
Consequently, there is a need for the development of
an effective therapy for the management of cachexia.
The melanotropins are small peptide-hormones, which
are involved in regulating skin pigmentation, the im-
mune system, steroid production, central sexual behav-
ior, feeding behavior, and exocrine gland secretion.5
They consist of melanocyte-stimulating hormones
(MSH) and the adrenocorticotropic hormone (ACTH),
which are derived from a large precursor peptide, pro-
opiomelanocortin (POMC).6 MSH appears in three dif-
ferent forms: a-MSH, b-MSH, and c-MSH. In addition
to these agonists, two endogenous antagonists, agouti-
protein and agouti-related protein (AgRP) have been
identified. All melanocortin peptides possess a His-
Phe-Arg-Trp (HFRW) motif, which is crucial for activa-
tion of the melanocortin receptors.
The melanocortin peptides exert their biological effects
by activating five distinct melanocortin receptors
(MC1-5 R), which belong to the class A G-protein-
coupled receptor superfamily.7
Keywords: Melanocortin-4 receptor; Antagonists; GPCR; Food intake;
SAR.
*
Corresponding authors. Tel.: +1 858 617 7677 (F.C.T.), +1 858 617
7634 (C.C.); fax: +1 858 617 7967; e-mail addresses:
ftucci@neurocrine.com; cchen@neurocrine.com
Compelling evidence suggests that the centrally ex-
pressed melanocortin-4 receptor (MC4R) plays an
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.071

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F. C. Tucci et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4389–4395
important role in the regulation of energy homeostasis
associated with food intake and metabolism.8 In animal
models of feeding, it has been demonstrated that peptide
antagonists of the MC4R such as AgRP and SHU9119
are effective in stimulating food intake in mice.9 Studies
have shown that icv administration of peptide MC4R
antagonists leads to an increase of food intake in tu-
mor-bearing mice, and reverses the weight loss induced
by tumor growth.10,11 A recent study has also shown
that ML00253764 (1), a small molecule antagonist of
the MC4R, effectively reduces tumor-induced weight
loss in a mouse model by a peripheral route of adminis-
tration.12 Thus, a potent, selective, and orally bioavail-
able MC4R antagonist should have considerable
potential as a novel therapy for cachexia.13
(EC50 = 3.8 nM with stimulation of cAMP levels com-
parable to that of a-MSH).18 Structure–activity relation-
ship studies in these series reveal that the basic nitrogen
of the benzylamine portion plays an important role in
the interactions of these compounds with the receptor,
perhaps through charge–charge attraction with an acidic
residue, possibly Asp-122.19 While the presence of the
benzylamine leads to highly potent antagonists and ago-
nists such as 5a and 5b, respectively, the resulting com-
pounds possess multiple highly basic centers, and as a
consequence are poorly absorbed when given orally.18
In search of potent and selective MC4R antagonists
with the potential for oral administration, we screened
several different substituted piperazines as replacements
for the highly basic benzylamines, such as in 5a and 5b.
During this effort, we discovered a novel series of cyclo-
hexylpiperazines that once combined with various N-ac-
ylated D-2,4-dichlorophenylalanines generated several
potent MC4R antagonists.20 In this letter, we describe
the synthesis and structure–activity relationships of this
series of compounds, and also the pharmacokinetic pro-
file of a selected compound as well as its oral efficacy in a
murine feeding model.
In addition to compound 1, only a small number of
small molecule MC4R antagonists have been recently
described in the literature. A series of alkylpiperazines
exemplified by 2 (IC50 = 52 nM) were reported to pre-
vent AgRP binding to the MC4R. These compounds
also exhibit moderate affinity (2, IC50 = 220 nM) against
NDP–MSH binding.14 MCL0129 (3), having a structure
similar to 2, is the only potent non-peptide antagonist
reported, which possesses low nanomolar affinity on
the inhibition of NDP–MSH binding (Ki = 7.9 nM).
Interestingly, 3 exhibits anxiolytic-like and antidepres-
sant-like activities in several animal models.15 Finally,
a structure–activity relationship study on a series of imi-
dazoles related to 1, such as 4 (Ki = 180 nM) has also
been reported (Fig. 1).16
In our studies, the key intermediate 11 was synthesized
according to the sequence depicted in Scheme 1. Com-
mercially available b-keto-ester 6 was subjected to
reductive amination with N-Boc-piperazine, in the pres-
ence of sodium (triacetoxy)borohydride in dichloro-
methane containing 1 equiv of acetic acid, to give
compound 7. The predominantly cis-isomer 7 was con-
verted to the trans-form 8 under basic conditions (NaO-
Et/EtOH).21 The relative stereochemistry was confirmed
by analyzing the coupling constant of the 1,2-protons
(J = 4.1 Hz for cis-form, 11.6 Hz for trans-form) of the
proton NMR spectrum.21 Additionally, ROESY NMR
experiments were performed on both compounds and
the spatial relationship of the 1,2-protons was estab-
lished leading to the conclusive structural assignment
of 7 and 8. Finally, treatment of cis-isomer 7 with
LiAlH4 in THF resulted in the crystalline cis-alcohol
In our efforts to identify potent and selective antagonists
of the MC4R, we have discovered a series of piperazi-
nebenzylamines that possesses subtype selectivity and
high binding affinity at the human receptor. Both ago-
nists and antagonists have been identified from this ser-
ies of compounds. For example, 5a (Ki = 21 nM) is a
potent MC4R antagonist (IC50 = 90 nM, on the inhibi-
tion of a-MSH-stimulated cAMP production),17 and
5b (Ki = 6.4 nM) is
a
potent MC4R agonist
F
F
Br
O
N
N
N
NH
N
N
N
N
N
F
O
O
N
3 (MCL0129)
2
1 (ML00253764)
S
HN
Cl
Cl
O
NH
N
NH
N
Br
O
N
N
O
N
N
N
H
NH
2
N
H
O
O
HN
5b
5a
4
Figure 1. Small molecule ligands for the melanocortin-4 receptor.

F. C. Tucci et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4389–4395
4391
N
N
Boc
HO
9
c
Cl
Cl
e
a
b
d
N
N
N
O
N
N
N
EtO
O
Boc
EtO
O
NHBoc
EtO
O
Boc
EtO
EtO
O
O
O
8
7
6
10
Cl
Cl
Cl
Cl
Cl
Cl
f
N
N
N
N
N
N
NH
EtO
O
NH
EtO
O
NH
2
O
O
O
1
O
R
O
NH
2
11
12
13
Scheme 1. Reagents: (a) N-Boc-piperazine/Na(OAc)3BH/CH2Cl2/HOAc; (b) NaOEt/EtOH; (c) LiAlH4, THF; (d) i—TFA/CH2Cl2, ii—N-Boc-D-
(2,4-Cl)Phe-OH/HBTU/DIEA/DMF; (e) TFA/CH2Cl2; (f) R1COOH/EDC/Et3N/CH2Cl2; for R1 containing BocNH, TFA/CH2Cl2.
9, which was subjected to X-ray diffraction studies, thus
confirming the cis-relative stereochemistry.22 Com-
pound 8 was then deprotected with TFA and the result-
ing free amine was coupled with N-Boc-D-2,4-
dichlorophenylalanine in the presence of HBTU to give
10. Deprotection of 10 with TFA afforded the free amine
11, which was coupled with various carboxylic acids to
give the designed compounds 12. When N-Boc-protect-
ed amino acids, such as N-Boc-glycine, were used in
the final amide-forming step, the resulting compounds
12 were further deprotected with TFA to afford the
desired products (Scheme 1).
To explore the role of the ethyl ester in compound 12a,
an alternative synthetic route was devised as shown in
Scheme 2. Thus, compound 8 was saponified under
alkaline conditions to give the corresponding carboxylic
acid, which was then deprotected in the presence of HCl
in CH2Cl2 and diethyl ether to give hydrochloride salt
14. N-acetyl-2,4-dichlorophenylalanine was preactivated
by HBTU in DMF, and then treated with 14, to afford
acid 15. This, in turn, was subjected to coupling reac-
tions with various alcohols or amines to give the desired
compounds 16 and 17, respectively.
We first examined amides 12 in a competition-binding as-
say with radiolabeled [125I]-NDP–MSH, using HEK293
cells stably transfected with the human MC4R as previ-
ously described.23 Acetylation of amine 11 (Ki = 120 nM)
resulted in an analog (12a, Ki = 11 nM) with an increase
of almost 10-fold in binding affinity. These results indicate
that either the NH or the carbonyl moiety of this amide
For comparison purposes, the cis-cyclohexylpiperazine
derivative 7 was also converted to the final compound
13 (cis-12i) by TFA deprotection in CH2Cl2, followed
by coupling with the dipeptide N-(Boc-b-Ala)-D-(2,4-
Cl2)Phe-OH in the presence of HBTU. Final TFA
deprotection afforded 13.
c
a,b
N
N
N
O
NH
.HCl
O
EtO
HO
O
O
HO
O
8
14
Cl
Cl
Cl
Cl
N
N
d
N
N
R2
O
NH
NH
O
O
O
O
15
16,17
Scheme 2. Reagents: (a) KOH/EtOH/H2O; (b) HCl/Et2O/CH2Cl2; (c) N-Ac-(2,4-Cl)Phe-OH/HBTU/DIEA/DMF; (d) alcohol or amine/EDC/Et3N/
DMF.

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F. C. Tucci et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4389–4395
Table 1. Binding affinity of amides at the human MC4 receptora
Table 2. Binding affinity of ethyl ester replacements at the human
MC4 receptora
Cl
Cl
Cl
Cl
Cl
Cl
O
N
N
N
N
2
N
R
O
NH
N
O
O
NH
O
O
NH
O
O
O
1
O
1
O
R
R
13
12a-l
15, 16a-c,17a-m
R2
Ki (nM)b
Compound
R1
Ki (nM)b
Compound
11
—
120 11
15
–OH
580 260
12a
12b
–CH3
11 0.3
H3C
O
16a
12a
91
6
11 0.3
46c
O
O
18
2
16b
16c
12c
10.0
19
3
2
N
48
9
O
H3C
HO
17a
17b
17c
17d
1600 30
1150 440
850 210
790 220
12d
N
H
N
H
H2N
12e
9.8 4.9
N
H
Me2N
H2N
12f
11
10
17
5
3
4
N
H
12g
17e
17f
700 59
H2N
N
H
12h
640 29
640 11
H2N
H2N
N
H
12i
6.3 0.6
53 21
O
17g
17h
17i
N
H
O
13 (cis-12i)
420
6
N
H
12j
12
2
N
H
S
92 24
N
H
12k
12l
2.8 0.6
4.7 1.0
N
H
17j
160 19
N
N
17k
160
4
HN
a Receptor stably expressed in HEK293 cells.
17l
480 160
N
O
b Ki values (n = 2 or 3) SEM.
17m
1000 100
N
H
play a major role in binding to the receptor. Small alkyl-
(12b), pyridinyl-(12c), and hydroxymethyl-(12d) amides
had comparable Ki values to 12a. Introduction of a basic
amino group led to a slight increase in binding affinity
a Receptor stably expressed in HEK293 cells.
b Ki values (n = 2 or 3) SEM.
c Single determination.

F. C. Tucci et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4389–4395
4393
Table 3. Binding affinity (Ki, nM) of selected compounds at the melanocortin receptor subtypesa
Compound
MC1R
MC3R
MC4R
MC5R
IC50 (nM)b,c
12a
12i
3300 390
2800 1250
620 40
1200 18
460 72
950 30
11.0 0.3
6.3 0.6
2.8 0.6
4.7 1.0
840 120
310 74
190 50
290 60
41d
12
13d
10d
4
12k
12l
600 54
987.0 0.1
a Melanocortin receptors stably expressed in HEK293 cells. The data are mean SEM (n = 2 or 3).
b Inhibition of a-MSH-stimulated cAMP production.
c No significant stimulation of cAMP production at 10 lM concentration (<5% to the level of a-MSH).
d Single determination.
over 12a. Thus, 12i, 12k, and 12l had Ki values of 6.3, 2.8,
and 4.7 nM, respectively (Table 1). While the additional
basic nitrogen played a small beneficial role in binding
to the receptor, the relative configuration of the 1,2-disub-
stituted cyclohexane ring was much more important.
Thus, the cis-isomer 13 (Ki = 53 nM) was almost 10-fold
less potent than its corresponding trans-analog 12i in
MC4R binding.
ing tertiary ones, in general possessed higher Ki values
than the ester derivatives (Table 2).
All compounds were tested for their ability to stimulate
cAMP production in HEK293 cells expressing the hu-
man MC4 receptor.23 No significant stimulation was ob-
served for any compound at 10 lM concentration (less
than 10% of the maximum a-MSH stimulation, data
not shown), which indicates that these compounds are
not functional agonists at the human MC4R. Selected
compounds were then tested for their ability to inhibit
a-MSH-stimulated cAMP accumulation, to assess func-
tional antagonism. Thus, 12a, 12i, 12k, and 12l were
found to dose-dependently inhibit a-MSH-stimulated
cAMP production with IC50 values of 41, 12, 13, and
10 nM, respectively (Table 3). 12i shifted a-MSH-stimu-
lated cAMP dose-curve rightward and had a Kb value of
47 nM in a Schild plot. These data demonstrated that 12i
was a competitive functional antagonist at the MC4R.
A small lipophilic group at the 2-carboxylate was re-
quired for high binding affinity to the receptor. Thus,
the free carboxylic acid 15 (Ki = 580 nM) lost more than
50-fold affinity when compared to its ethyl ester 12a.
The smaller methyl ester 16a also showed a reduction
in affinity when compared to 12a. However, the slightly
larger isopropyl and isobutyl esters 16b and 16c dis-
played lower potency than 12a, suggesting that the ethyl
group was optimal at this position. Amides (17), includ-
Table 4. Pharmacokinetic properties of 12i in ratsa
The selectivity profile of 12i at the other melanocortin
receptors was also determined. Thus, it had Ki values
of 2800, 460, and 310 nM, respectively, at the MC1R,
MC3R, and MC5R. These data demonstrated that 12i
was very selective at the MC4R over these melanocortin
subtypes, with an approximated ratio of 70 at the
MC3R and 50 at the MC5R. Compounds 12a, 12k,
and 12l also had similar selectivity. These results are
summarized in Table 3.
iv (5 mg/kg)
CL (mL/min kg)
t1/2 (h)
Vd (L/kg)
57 13
5.8 0.6
28.7 7.4
Cplasma (1 h) (ng/mL)
Cbrain (1 h) (ng/g)
B/P ratio (1 h)
88
5
158 73
1.8 0.7
po (10 mg/kg)
Tmax (h)
Cmax (ng/mL)
2.8 1.6
117 40
AUC (ng/mL h)
Oral bioavailability (%)
365.5 90.3
12.2 3.6
Compound 12i was chosen for in vivo evaluation due
to its favorable in vitro profile, as well as its desirable
calculated physicochemical properties.24 The calculated
a Average of three animals.
*
*
vehicle
compound 12i
2.0
1.5
1.0
0.5
0.0
4
3
2
1
1
2
4
6
24
time (h)
time (h)
Figure 2. Effect of orally administered compound 12i (10 mg/kg, PO, dosed in distilled water at pH 5) on night phase feeding in mice. Cumulative
food intake was significantly increased at the 6- and 24-h measurement intervals (*p < 0.05). Values are means SEM.

4394
F. C. Tucci et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4389–4395
3. Argiles, J. M.; Meijsing, S. H.; Pallares-Trujillo, J.;
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logD value (2.2) agreed well with the experimental result
(measured logD value of 1.8). After intravenous admin-
istration in Sprague–Dawley rats (5 mg/kg), 12i had
high clearance (CL = 57 ng/mL kg), and a high volume
of distribution (Vd = 28.7 L/kg). This resulted in a
half-life of 5.8 h in this species. The brain concentration
at the 1 h time point was 158 ng/g, which was 1.8-fold of
the plasma concentration at the same time point. After
oral administration (10 mg/kg), the maximal concentra-
tion (Cmax = 117 ng/mL) of 12i was reached at the 2.8 h
time point (Tmax). The area under curve (AUC) was
365.5 ng/mL h, and this resulted in a calculated oral
bioavailability of 12.2% (Table 4).
´
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Next, we examined the effects of a single administration
on normal food intake in mice over a 24 h period. Male
C57BL/6 mice (7–8 weeks of age; n = 10 per group) were
orally administered vehicle or 10 mg/kg of compound
12i at the onset of the dark phase of the light/dark cycle,
a time when rodents are active and normally eat. Food
intake was then measured 1, 2, 4, 6, and 24 h after lights
were out. Compound 12i significantly increased cumula-
tive food intake at the 6- and 24-h measurement inter-
vals (p < 0.05; Fig. 2). This finding demonstrates that a
selective MC4R antagonist with oral bioavailability
has the ability to increase feeding significantly over a
24-h period and suggests that MC4R antagonists may
be useful to treat diseases with an anorexia component
such as cachexia. In addition, it has been described
recently that 12i reverses tumor-induced weight loss
upon peripheral administration in mice.25
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In conclusion, a series of cyclohexylpiperazines (12, 16,
and 17) was synthesized. Compounds from this series
were identified to be potent and selective antagonists
of the human MC4R. Functionally, 12i was found to
be a competitive MC4R antagonist versus a-MSH-stim-
ulated cAMP production. In rats, 12i displayed reason-
able plasma exposure from oral administration and
penetrated into the brain. We have shown that com-
pound 12i, when orally administered, increases food
intake in mice over a 24-h period.
17. Pontillo, J.; Tran, J. A.; Fleck, B. A.; Marinkovic, D.;
Arellano, M.; Tucci, F. C.; Lanier, M.; Nelson, J.; Parker,
J.; Saunders, J.; Murphy, B.; Foster, A. C.; Chen, C.
Bioorg. Med. Chem. Lett. 2004, 14, 5605.
Acknowledgments
18. Pontillo, J.; Tran, J. A.; Arellano, M.; Fleck, B. A.;
Huntley, R.; Marinkovic, D.; Lanier, M.; Nelson, J.;
Parker, J.; Saunders, J.; Tucci, F. C.; Jiang, W.; Chen, C.
W.; White, N. S.; Foster, A. C.; Chen, C. Bioorg. Med.
Chem. Lett. 2004, 14, 4417.
We thank Dr. Michael Mesleh (Neurocrine Biosciences)
for running the ROESY experiments and Dr. John
C. Huffman (Indiana University—Molecular Structure
Center) for the X-ray data.
19. Chen, C.; Pontillo, J.; Fleck, B. A.; Gao, Y.; Wen, J.;
Tran, J. A.; Tucci, F. C.; Foster, A. C.; Saunders, J.
J. Med. Chem. 2004, 49, 6821.
20. For the rationale behind the design of these MC4R
antagonists, including the role of the D-2,4-dichlorophe-
nylalanine, please see Ref. 19.
Supplementary data
Supplementary data associated with this article can be
found in the online version at doi:10.1016/
j.bmcl.2005.06.071.
21. Cimarelli, C.; Palmieri, G. J. Org. Chem. 1996, 61,
5557.
22. See supporting information for X-Ray data on compound
9. Crystallographic data (excluding structure factors) for
the structures in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplemen-
tary publication numbers CCDC 270115. Copies of the
data can be obtained, free of charge, on application to
References and notes
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F. C. Tucci et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4389–4395
4395
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK
[fax: +44 (0)1223 336033 or e-mail: deposit@ccdc.cam.
ac.uk].
24. Calculated logP = 3.7; caculated logD = 2.2; calculated
pKaÕs = 8.9 and 6.6. Calculations were performed with
ACD/Labs 8.00. Advanced Chemistry Development Inc.
25. Markison, S.; Foster, A. C.; Chen, C.; Brookhart, G. B.;
Hesse, A.; Hoare, S. R. J.; Fleck, B. A.; Brown, B. T.;
Marks, D. L. Endocrinology 2005, 146, 2766.
23. Nickolls, S. A.; Cismowski, M. I.; Wang, X.; Wolff, M.;
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