4-{(2R-[3-Aminopropionylamido]-3-(2,4-dichlorophenyl)propionyl} -1-{2-[(2-thienyl)ethylaminomethyl]phenyl}piperazine as a potent and selective melanocortin-4 receptor antagonist - Design, synthesis, and characterization

Article abstract of DOI:10.1021/jm049278i

Recent studies have demonstrated that melanocortin-4 receptor (MC4R) antagonists can prevent weight loss in tumor-bearing mice, which indicates clinical usage for the treatment of cachexia. In our efforts to develop potent and selective antagonists of the human MC4R, we designed piperazinebenzylamines bearing a 2,4-dichlorophenylalanine, by utilizing information derived from structure-activity relationships of MC4R agonists and mutagenesis results of the MC4R and peptide ligands. On the basis of known MC4R agonists such 6, we successfully synthesized potent MC4R antagonists exemplified by 10, which possesses a K_i value of 1.8 nM in binding affinity. 10 does not stimulate cAMP release in HEK 293 cells expressing the human MC4 receptor at 10 μM concentration. It was demonstrated by Schild analysis that 10 was a competitive functional antagonist with a pA₂ value of 7.9 in the inhibition of α-MSH-stimulated cAMP accumulation. 10 also penetrated into the brain when dosed intravenously in rats.

Full text of DOI:10.1021/jm049278i

J. Med. Chem. 2004, 47, 6821-6830
6821
4-{(2R)-[3-Aminopropionylamido]-3-(2,4-dichlorophenyl)propionyl}-1-
{2-[(2-thienyl)ethylaminomethyl]phenyl}piperazine as a Potent and Selective
Melanocortin-4 Receptor AntagonistsDesign, Synthesis, and Characterization
Chen Chen,* Joseph Pontillo, Beth A. Fleck, Yinghong Gao, Jenny Wen, Joe A. Tran, Fabio C. Tucci,
Dragan Marinkovic, Alan C. Foster, and John Saunders
Neurocrine Biosciences, Inc. 12790 El Camino Real, San Diego, California 92130
Received September 2, 2004
Recent studies have demonstrated that melanocortin-4 receptor (MC4R) antagonists can prevent
weight loss in tumor-bearing mice, which indicates clinical usage for the treatment of cachexia.
In our efforts to develop potent and selective antagonists of the human MC4R, we designed
piperazinebenzylamines bearing a 2,4-dichlorophenylalanine, by utilizing information derived
from structure-activity relationships of MC4R agonists and mutagenesis results of the MC4R
and peptide ligands. On the basis of known MC4R agonists such 6, we successfully synthesized
potent MC4R antagonists exemplified by 10, which possesses a Ki value of 1.8 nM in binding
affinity. 10 does not stimulate cAMP release in HEK 293 cells expressing the human MC4
receptor at 10 µM concentration. It was demonstrated by Schild analysis that 10 was a
competitive functional antagonist with a pA2 value of 7.9 in the inhibition of R-MSH-stimulated
cAMP accumulation. 10 also penetrated into the brain when dosed intravenously in rats.
Introduction
pigmentation and the immune system. The MC2R
(ACTH receptor) controls steroid production. The MC3R
might be involved in regulation of central sexual be-
havior, the MC4R controls feeding behavior, and the
MC5R has a role for regulating exocrine gland secre-
tion.11 The melanotropin peptides are the natural
ligands for the melanocortin receptors and consist of the
melanocyte-stimulating hormones R-, â-, and γ-MSH
and the adrenocorticotropin ACTH. In addition, two
endogenous antagonists, agouti-protein and agouti-
related protein (AgRP), have been identified.12 All the
melanotropin peptides possess a His-Phe-Arg-Trp
(HFRW) motif, which has been proven to be crucial for
activation of the melanocortin receptors.13 Agouti-
protein and AgRP, on the other hand, have an Arg-Phe-
Phe motif located in a loop known to interact with the
melanocortin receptors14 and believed to be important
for functional antagonism.15
While current research of the melanocortin system
has focused on MC4R agonists for the possible treat-
ment of obesity,16 recent studies have showed that
peptide MC3/MC4 receptor antagonists effectively pre-
vent tumor-bearing mice from reduction of food intake
and, more importantly, weight loss.17 Studies have also
shown that MC4R knock-out mice resist tumor-induced
loss of lean body mass.17a These positive results in
animal models indicate a potential clinical treatment
of cachexia by using a melanocortin antagonist, which
is significant since this wasting syndrome occurs in a
large population of cancer patients.18 Very recently a
small molecule MC4R antagonist ML253764 (2) has
been shown, upon peripheral administration in tumor-
bearing mice, to protect against weight loss.19 However,
while this molecule displays moderate potency at the
human MC4R (IC50 ) 106 nM in inhibition of cAMP
production), its activity at the MC3R is not clear.
G-protein-coupled receptors (GPCRs) are a family of
structurally related membrane-bound proteins that play
a central role in the recognition and signal transduction
of hormones and neurotransmitters. GPCRs share
general structural motifs, including seven transmem-
brane helices connected by intra- and extracellular
loops, an extracellular amino terminus, and a cytoplas-
mic carboxyl terminus.1 GPCRs are activated by light,
small molecules such as histamine, peptides, and pro-
teins.2 Although many of them are considered to be drug
targets, GPCRs activated by peptides and proteins,3 due
to a lack of structural information, have been particu-
larly challenging to medicinal chemists to design non-
peptide small-molecule agonists or antagonists of these
receptors.3b The recent resolution of the crystal struc-
ture of bovine rhodopsin shines some light on GPCR
research, since it provides the first three-dimensional
structure of any GPCR,4 thereby making it possible to
analyze experimental data of other GPCRs from a more
informed and structural perspective.1b,5 Because there
are many conserved residues in the transmembrane
domains across the class A GPCRs,6 the crystal struc-
ture of rhodopsin should offer a good template for
computational modeling of many of these receptors.7 In
addition, site-directed mutagenesis has made it possible
to characterize the ligand interaction sites of GPCRs.8
A binding pocket in the transmembrane domain of
several class A GPCRs, including the adrenergic and
neurokinin receptors,9 has been identified via mutation
studies.
Five subtypes of melanocortin receptors, MC1-5R,
that belong to the class A GPCR superfamily have been
identified and cloned.10 The MC1R regulates skin
* Corresponding author. Telephone: 858-617-7600. Fax: 858-617-
7967. E-mail: cchen@neurocrine.com.
10.1021/jm049278i CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/09/2004

6822 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Chen et al.
Figure 1. Small molecule antagonists of the melanocortin-4 receptor.
Figure 2. Chemical structure of MTII, SHU9119, and small molecule MC4R ligands 3 and 4.
Small molecule antagonists of the melanocortin-4
receptor have been discovered recently (Figure 1).
Compound 1a, first reported to be an inhibitor of AgRP
binding to the MC4R (IC50 ) 52 nM), also exhibits
moderate inhibition (IC50 ) 220 nM) of NDP-MSH
binding and reduces R-MSH-stimulated cAMP produc-
tion at high concentration.20 MCL0129 (1b), which has
structural similarity to 1a, binds to the MC4R with high
affinity (Ki ) 7.9 nM). Interestingly, 1b has shown
anxiolytic-like and antidepressant-like activities in
several animal models.21 The benzamidine 2a
(ML253764) is reported to have a Ki value of 160 nM in
binding affinity at the MC4R.19 A series of imidazoles
such as 2b (Ki ) 180 nM) as antagonists of the MC4R
has also been reported.22
On the other hand, several agonists from different
chemical classes have been discovered since compound
4 (EC50 ) 1.2 nM)23 was disclosed as the first potent
and selective small molecule MC4R agonist in 2002.24
4 is reported to be developed from the initial leads
3a,b,23 which are in turn designed by mimicking the
His-D-Phe-Arg-Trp motif of MT-II by replacing the Arg-
Trp residues with a privileged structure for GPCRs
(Figure 2).25 Interestingly, the triazole moiety of 4 can
be replaced by a N-methyltetrazole (5b or 5c, Figure 3)
but not tetrazole itself (5a). Thus, 5b has an EC50 of
0.6 nM, while 5a is a poor MC4R agonist (EC50 ) 771
nM).
Recently, a series of substituted phenylpiperazines
such as 6 (6a: EC50 ) 110 nM), which are characterized
as MC4R-selective agonists, has been reported from our
laboratory (Figure 3).24d We then optimized this series
into highly potent derivatives such as 8b (EC50 ) 4.7
nM).26 Because of the potential clinical importance for
the possible treatment of cancer cachexia, and anxiety/
depression, we are interested in the development of
potent and selective small molecule MC4R antagonists.
Herein we report our design, synthesis, and character-
ization of 4-{(2R)-[3-aminopropionylamido]-3-(2,4-dichlo-
rophenyl)propionyl}-1-{2-[(2-thienyl)ethylaminomethyl]-
phenyl}piperazine 10 as such a molecule (see Scheme
1). Our design strategies are based upon information
derived from structure-activity relationships of known
small molecule and peptide MC4R agonists, in combina-

A Potent and Selective MC4R Antagonist
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6823
Figure 3. Chemical structure of some MC4R ligands with a Tic group.
Scheme 1a
Table 1. Summary of Binding and Function of 4 and 6-10 at
the Human MC4 Receptora
EC50
compd
Ki (nM)b
(cAMP, nM)c
IA (%)c,d
3a
3b
4
5a
5b
5c
6a
7
8a
8b
9a
9b
9c
10e
(108)
n/a
n/a
n/a
(530)
(118)
100 (97)
(79)
(103)
(87)
105
131
105
100
102
14
9.8 ( 2.1 (1.2)
(238)
(0.3)
(1.2)
270 ( 65
380 ( 10
10.8 ( 2.3
6.4 ( 1.4
44.0 ( 1.1
20.5 ( 0.6
69.5 ( 0.8
1.8 ( 0.2
1.45 ( 0.6 (2.1)
(771)
(0.6)
(9.6)
110 ( 61
890 ( 160
290 ( 48
4.77 ( 0.7f
580 ( 81
16
3
a The human melanocortin-4 receptor stably expressed in HEK
293 cells. b Values in parentheses are IC50 (nM) cited from ref 23.
c Values in parentheses are cited from ref 23. d Intrinsic activity
(IA): percentage of the maximal cAMP levels by R-MSH stimula-
tion. e pA2 ) 7.9 in inhibition of R-MSH-stimulated cAMP produc-
tion. f An EC50 of 3.8 nM was obtained on CHO cells.
a Reagents and conditions: (a) (i) 2-(2-thienyl)ethylamine/NaB-
H(OAC)3/DCM; (ii) (Boc)2O, 82%; (b) Pd/C-H2/NH3/MeOH, 35%.
(c) N-Fmoc-(R)-(4-Cl)Phe-OH or N-Fmoc-(R)-(2,4-Cl)Phe-OH/EDC/
HOBt/DCM; (d) (i) Et2NH/DCM, 87%; (ii) R′CH2COOH/EDC/
HOBt/DCM; (iii) HCl/dioxane, 34-65%.
production in the same cell lines as previously de-
scribed,27 and these results are depicted in Table 1.
Compound 4 had a Ki value of 9.8 nM in our competi-
tion binding assay (an IC50 of 1.2 nM was reported)23
and an EC50 value of 1.45 nM, which matches well with
the reported value of 2.1 nM. The discrepancy in Ki
value could be caused by different assay conditions. The
parent primary benzylamine 7 had moderate affinity
and agonist potency. Incorporation of a 2-(2-thienyl)-
ethyl side chain on 7 resulted in 8a with much better
binding affinity (35-fold increase) but only 3-fold in-
crease in agonist potency. Replacement of the R-1,2,3,4-
tetrahydroisoquinolin-3-ylcarbonyl (Tic) moiety of 8a
with a glycine gave compound 9a with 4-fold reduction
in affinity (Ki ) 44 nM) but little change in agonist
potency and efficacy. Its close analogue 9b from â-ala-
nine possessed slightly increased affinity (Ki ) 20.5 nM),
and the methoxyacetamide 9c (Ki ) 69.5 nM) displayed
about 2-fold reduction. However, unlike 8a and 9a,
compounds 9b and 9c exhibited very low efficacy in
stimulation of cAMP release at 10 µM concentration
(14-16% of R-MSH maximal level). Using a 2,4-dichlo-
rophenyl group to replace the 4-chlorophenyl moiety of
9b not only increased binding affinity by over 10-fold
but also completely diminished agonist efficacy (Table
1). Thus, 10 showed no significant stimulation of cAMP
release at 10 µM concentration (3% of R-MSH maximal
level). 10 was also demonstrated by a Schild analysis
to function as a competitive antagonist with a pA2 value
tion with site-directed mutagenesis of the MC4R and
melanotropin peptides.
Chemistry
The syntheses of 9 and 10 started from the protected
piperazinebenzaldehyde 11, which was subjected to a
reductive amination with 2-(2-thienyl)ethylamine and
sodium triacetoxyborohydride, followed by Boc protec-
tion of the resulting secondary amine to give the
intermediate 12 in 82% yield. Removal of the Cbz
protecting group of 12 by palladium-catalyzed hydro-
genolysis provided a free amine, which was coupled
under standard peptide coupling conditions (EDC/
HOBt) with N-FMOC-D-(4-Cl)Phe-OH or N-FMOC-D-
(2,4-Cl)Phe-OH to afford the amides 13a or 13b, re-
spectively. After FMOC-deprotection of 13 with diethyl-
amine, the resulting free amines were coupled with
N-Boc-glycine, N-Boc-â-alanine, or methoxyacetic acid
to give the final compounds 9a-c and 10 after TFA-
deprotection.
Results
In Vitro Binding and Agonist and Antagonist
Activities at the Human MC4 Receptor. The syn-
thesized compounds were tested for their ability to
compete with radiolabeled NDP-MSH in HEK 293 cells
stably expressing the human MC4R. These compounds
were also measured for their ability to stimulate cAMP

6824 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Chen et al.
Figure 4. Dose-response curves and Schild analysis of compound 10 in cAMP assay.
Table 2. Binding Affinity (Ki, NM) of 8-10 at the Human
Table 3. Pharmacokinetic Profile of 10 in Ratsa
Intravenous (10 mg/kg)
Melanocortin Receptor Subtypesa
compd
hMC1
hMC3
hMC4
hMC5
CL (mL/min kg)
24
Vd (L/kg)
t1/2 (h)
9.5
7
9a
9b
10
(27%)b
(50%)b
1300
(34%)b
1900
1400
640
380
44
2300
990
1000
280
4.6
21
Intravenous (2 mg/kg)
4200
1.8
Cbrain at 1.5 h (ng/g)
Cplasma at 1.5 h (ng/mL)
b/p ratio at 1.5 h
75
71
1.0
a Data are the average of two or more independent experiments.
b Percentage inhibition at 10 µM concentration.
Oral (10 mg/kg)
Tmax (h)
0.5
18
51
1
of 7.9 in inhibition of R-MSH-stimulated cAMP ac-
cumulation (Figure 4).
Cmax (ng/mL)
AUC (ng/mL h)
F (%)
Selectivity at the Melanocortin Subtype Recep-
tors. Compounds 8-10 were profiled for selectivity in
binding affinity at the human melanocortin receptor
subtypes. These competition binding assays were per-
formed on the HEK 293 cells stably transfected with
the human MC1, MC3, and MC5 receptors, using [125I]-
NDP-MSH as the radiolabeled ligand. The tested com-
pounds displayed good selectivity of MC4R over other
receptor subtypes. For example, compound 10 exhibited
Ki values of 4200, 640, 1.8, and 280 nM, respectively,
at the human MC1R, MC3R, MC4R, and MC5R (>130-
fold selectivity). The results are summarized in Table
2.
Pharmacokinetic Property of Compound 10. The
pharmacokinetic profile of 10 was assessed in rats (N
) 3). This compound exhibited limited plasma exposure
when given orally (1% at 10 mg/kg dose). This was most
likely caused by poor intestinal absorption,28 which
could be associated with the high hydrophilicity of the
molecule (measured log D value of 1.1 at pH 7.4). After
an intravenous administration at 10 mg/kg, the clear-
ance of 10 was 24 mL/min kg, the volume distribution
was 9.5 L/kg, and the half-life was 4.6 h in this species.
Moreover, 10 penetrated to the brain. Thus, when given
intravenously at 2 mg/kg dose, the brain and plasma
concentrations of 10 were respectively 75 ng/g and 71
ng/mL at the 1.5 h time point (Table 3).
a Data are the average of three animals.
one of the most widely used nonselective melanocortin
agonists, and it has high binding affinity (Ki ) 1.32 nM)
and agonist potency (EC50 ) 0.27 nM) at the human
MC4R. SHU9119 is a very close analogue of the cyclic
peptide superagonist MT-II (Figure 2). However,
SHU9119 is a potent antagonist (IC50 ) 1.8 nM, pA2 )
9.3) at the human MC4R.30 Chemically, the only differ-
ence between these cyclic peptides is the D-(2′)Nal
residue of SHU9119 and the D-Phe of MT-II.
An alanine-scan of NDP-MSH has revealed that
D-Phe7 is the most important residue of this peptide in
both binding and function, followed by Arg8. Thus,
D-alanine replacement of D-Phe7 results in an over 1100-
fold reduction in binding (Ki ) 1.6 µM) and function
(EC50 ) 320 nM). Norleucine replacement of Arg8 gives
the peptide with a 200-fold decrease in binding affinity
(Ki ) 270 nM) and about 55-fold loss in agonist potency
(EC50 ) 15 nM). Alanine replacement of the Trp9 residue
also results in a 100-fold decrease in binding (Ki ) 130
nM), but much less reduction in function (EC50 ) 3.45
nM), and this mutant can be rescued by a phenylala-
nine.31
Mutagenesis of the Melanocortin-4 Receptor
and NDP-MSH Peptide. Site-directed mutagenesis
studies of the mouse and human melanocortin receptors
have established several key residues that are involved
in the binding and function of R-MSH and other peptide
ligands such as NDP-MSH and MT-II.31,32 In parallel
to the poor interaction of the (D-Phe)7(D-Ala) NDP-MSH
mutant with the wild-type human MC4R, NDP-MSH
is unable to bind the His264Ala mutant of the human
MC4R and exhibited low agonist potency (almost 500-
fold increase in EC50 value).32 The binding affinity of
Discussion
Structural Information of Melanotropin Pep-
tides. All the melanotropin peptides, R-, â-, and γ-MSH
and ACTH, possess a HFRW motif, which has been
proven to be crucial for the activation of the melano-
cortin receptors.13,29 One common feature for potent
synthetic melanocortin agonists is the replacement of
the L-Phe7 of R-MSH with its D-isomer. NDP-MSH is

A Potent and Selective MC4R Antagonist
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6825
NDP-MSH to the Phe261Ala human MC4R mutant also
decreases over 5-fold. In the mouse MC4R, the binding
affinity of NDP-MSH to the Phe253Ser mutant is
reduced over 10-fold (Phe253 of the mouse MC4R is
equivalent to Phe261 of the human MC4R). More
interestingly, NDP-MSH can only partially activate this
mutant (i.e. acts as a partial agonist).32 Because of the
good match between the magnitudes of reduction in
binding and function for the (D-Phe)7(D-Ala) NDP-MSH
and the His264Ala human MC4R mutants, it has been
suggested that the phenyl group of the NDP-MSH Phe7
residue mainly interacts with the His264 of the human
MC4R. Since Phe261 is located very close to His264 (one
loop below in TM-6), it also has a direct or indirect effect
on the NDP-MSH binding and function.32
One interesting mutant of the mouse MC4 receptor
is the Phe254Ser, at which the MC3/MC4 receptor
antagonist SHU9119 acts like a partial agonist with an
EC50 value of 0.83 nM. The Phe254 residue (equal to
the Phe262 of the human MC4R) is located very closely
to the Phe253 of the mouse MC4R. Data from mutagen-
esis suggest that a π-rich lipophilic pocket formed by
Phe261, Phe262, His264, and other lipophilic amino
acids in TM-5 and TM-6 of the human MC4R may host
the Phe7 residue of the melanocortin peptides during
their interactions.31,32
displays full efficacy and moderate agonist potency (IA
) 97%, EC50 ) 500 nM, IC50 value is not reported).
These results are parallel to that observed in peptide
ligands. For example, the only difference between the
agonist MT-II and the antagonist SHU9119 is a D-Phe
residue in MT-II and a D-2′-Nal moiety in SHU9119.30
Increased potency by introducing a 4-chlorine on the
Phe7 residue of R-MSH analogues has also been ob-
served.33 Apparently the His residue of 3b is optimized
to the R-Tic moiety of 4 for better binding and functional
activity (Figure 2). These data strongly suggest that the
D-4-chlorophenylanaline of 4 is an equivalent of the Phe7
residue of the melanotropin peptides. Computational
superposition of MT-II and 4 indicates the 4-chlorophe-
nyl group of 4 overlays with D-Phe of MT-II, and the
Tic group of 4 sits between His and Trp residues of MT-
II.23 The role of the triazole moiety of 4, however, is not
clear based on this model. In a similar study, superposi-
tion of the solution structure of a constrained melano-
cortin peptide analogue and 4 results in a different
orientation,34 in which, the Tic group takes both Arg
and Trp functionalities. Since the triazole of 4 does not
mimic any of the HFRW residues of the melanotropin
peptide, the authors speculated that this moiety binds
in a lipophilic pocket in order to explain the large
difference in binding affinity between 5a and 5b.30
One striking data set is from the small molecule
MC4R agonists of the tetrazoles 5a-c (Figure 3). While
the N-methylated tetrazole 5b is a potent agonist (EC50
) 0.6 nM, IA ) 103%) with high binding affinity (IC50
) 0.3 nM), the parent tetrazole 5a exhibited poor
binding (IC50 ) 238 nM) and poor agonist potency (EC50
) 771 nM, IA ) 79%). In addition, the regioisomeric
tetrazole 5c (IC50 ) 1.2 nM, EC50 ) 9.6 nM, IA ) 87%)
is also a potent MC4R agonist. The poor interaction of
5a with the MC4R is most likely associated with the
acidic nature of the tetrazole. We believe that these
results imply that the triazole of 4 or the N-methyltet-
razole of 5b or 5c may interact with acidic residues of
the MC4 receptor by partially mimicking, through
hydrogen-bonding, the Arg8 function of the melanotropin
peptides. Interestingly, the basicity of the guanidine
moiety of the Arg8 residue of melanotropin peptides
seems to be nonessential for interactions with the
receptor; thus, an acylguanidine analogue retains the
agonist activity of the corresponding guanidine com-
pound.35
Three-Dimensional Model of the Human MC4
Receptor. The homology model of the human MC4R
based on the crystal rhodopsin template has been built
by several different groups.36 From these three-dimen-
sional structure models, several key residues, which line
a putative binding pocket, are identified between TM-
3, TM-6, and TM-7 of the transmembrane domains.
These amino acids include Phe261, Phe262, and His264
of the TM-6 and Asp122 and Asp126 of the TM-3 (Figure
5).
Three acidic residues, Glu100, Asp122, and Asp126,
toward the extracellular surface of TM-2 and TM-3 of
the human MC4R are believed to form a negatively
charged cage and interact cooperatively with the posi-
tively charged guanidinyl moiety of the NDP-MSH Arg8
residue.32 The affinity of NDP-MSH binding to the
Asp122Ala mutant decreased, albeit only 6-fold, from
the wild-type receptor (Ki ) 1.54 nM). However, NDP-
MSH is unable to bind to either Asp126Ala or Asp126Asn
mutant. In addition, the Asp126 mutants respond
weakly to NDP-MSH in cAMP stimulation, although the
Asp126Asn mutant can be activated by NDP-MSH at a
much higher concentration (EC50 ) 342 vs 0.27 nM at
the wild-type). In comparison, Asp122Ala mutation had
small effect on NDP-MSH-induced activation (EC50
)
1.67 nM). These results suggest that the basic residue
of NDP-MSH interacts with both Asp122 and Asp126,
but mainly the latter; or the loss of the Asp122 function
can be compensated by the Asp126 residue, but not vice
versa. The involvement of the Arg8 residue of NDP-MSH
in this interaction is evidenced by the peptide mutant
Nle8[NDP-MSH], which binds to the human MC4R with
a much lower affinity (Ki ) 264 nM). A double-mutation
study has further confirmed the interaction between the
Asp122 of the human MC4R and the Arg8 of NDP-MSH.
Thus, the binding affinity of Nle8[NDP-MSH] at the
Asp122Ala mutant is exactly the same as that at the
wild-type receptor (Ki ) 273 nM, vs 264 nM).
Key Interactions of Compound 4 with the MC4
Receptor. The first reported potent and selective small
molecule MC4R agonist 4 was developed from the initial
leads 3a,b (Figure 2),23 which were in turn designed by
replacing the Arg-Trp residues of the His-Phe-Arg-Trp
motif with a privileged structure for GPCRs.24 3a, which
has a D-2′-naphthylalanine moiety, possesses an IC50
value of 108 nM, but little agonist efficacy at the human
MC4R. However, 3b, a D-phenylanaline derivative,
Bondensgaard and co-workers suggest that the TM-6
lipophilic cavity hosts the privileged structure part of
the MC4R agonist 4 on the basis of their computational
model.35b Alternatively, from the experimental results
discussed above, we believe that the 4-chlorophenyl
group of 4 might interact with these π-rich residues of

6826 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Chen et al.
enhance its possible interaction with an acidic residue
of the receptor from hydrogen-bonding to a charge-
charge attraction. Not surprisingly, 7 has a Ki value of
380 nM,38 which is very comparable to the triazole 6
(Ki ) 270 nM). From the receptor model, several
lipophilic residues, such as Thr101 and Ile104 of TM-2,
and Ile125, Ile129, Leu133, and Leu134 of TM-3 could
be found near the Asp122 and Asp126 residues. Thus,
in addition to hydrogen bonding with Asp122, the
triazole of 6a may also contribute binding energy
through lipophilic interaction with these residues. In
addition, an empty cavity between TM-3 and TM-4 could
be identified from the receptor model (Figure 5). This
cavity was lined by several lipophilic residues such as
Leu133 and Leu134 of TM-3 and Phe184 of TM-4. On
the basis of this observation, we then introduced a
lipophilic side chain on the benzylamine 7. One of these
analogues, the 2-(2-thienyl)ethylamine 8a, exhibits a Ki
value of 10.8 nM, a 35-fold increase over 7. Interestingly,
the agonist potency of 8a only increases by 3-fold (EC50
) 290 nM).
After successfully improving the binding affinity of
compounds 8, we then focused on the R-Tic group, since
this moiety could be important for receptor activation.
A possible interaction of the basic nitrogen of the Tic
group with the acidic residue of Asp126 on TM-3 was
predicted from a preliminary receptor modeling study
(Figure 5). On the basis of mutagenesis studies, Asp126
is very important residue for receptor function. To test
this hypothesis of the Tic interaction, a glycine analogue
9a, in which the possible lipophilic interaction of 8a is
eliminated, was synthesized. 9a has a Ki value of 44
nM, which is only about a 4-fold decrease from 8a. Like
8a, compound 9a also possesses full agonist efficacy
(EC50 ) 580 nM, intrinsic activity ) 102%). Very
interestingly, by changing the amino functionality of the
glycine moiety of 9a, the agonist efficacy of its analogues
is greatly reduced. Thus, the â-alanine analogue 9b
displays slightly increased affinity (Ki ) 20.5 nM) but
loses most efficacy (cAMP stimulation 14% of R-MSH).
Similarly, replacing the glycine of 9a with a methoxy-
acetyl group (9c) results in a slight reduction in affinity
(Ki ) 69.5 nM), but much lower efficacy (IA ) 16%).
While the Asp122 and Asp126 residues of the human
MC4R may function cooperatively, mutagenesis studies
suggest that Asp126 is a more important residue than
Asp122 in receptor activation by agonists. This could
be used to explain why the benzylamine 8a (Ki ) 10.8
nM, EC50 ) 290 nM) is a weak agonist and the diamine
8b (Ki ) 6.4 nM, EC50 ) 4.7 nM) is a potent one, while
both possesses similar binding affinities. The basic
nitrogen of benzylamine in 8a probably mainly contacts
Asp122; however, the second nitrogen of 8b might
interact directly with the Asp126 residue.
Next, we turned our attention to the 4-chlorophenyl
group of 9b. We believe this aromatic ring is an
equivalent of the Phe7 residue of the melanotropin
peptides and thus interacts with His264/Phe261 and the
adjacent aromatic/lipophilic residues of the human
MC4R. The involvement of the Phe261 residue in
binding of 4 is evidenced by an almost 50-fold decrease
in affinity of 4 binding to the Phe261Ala mutant (Ki )
479 nM).27 Since the agonist activity of MT-II and
compound 3b can be “switched off” simply by replacing
Figure 5. Illustration of small molecule ligand 6b binding
in the putative binding pocket of human MC4 receptor.
TM-6, which is considered to be the primary helix, in
addition to TM-3, for GPCRs’ activation.37 The aromatic
group of the phenylalanine of 3a and 3b is responsible
for switching between agonist and antagonist activity.
The lipophilic cyclohexyl group of 4, which is part of the
privileged structure, could reside in an area close to TM-
7, which has several hydrophobic residues, such as
Phe284, Leu288, Ile289, Ile291, and Met292, facing the
putative binding pocket. Most importantly, the triazole
of 4 now sits very closely to the Asp122 residue of TM-
3. Figure 5 illustrates the orientation and location of
6b in the putative binding pocket of the human MC4R.
With one loop separation, the Asp122 residue directly
points to the binding pocket, while Asp126 faces TM-5.
Asp126 may interact with the basic nitrogen of the Tic
group of 4, which functions as both Arg and His residues
of the HFRW motif. The triazole of 4 mainly interacts
with the Asp122 residue through hydrogen-bonding.
Small Molecule MC4R Agonist 4 at the Mutant
Receptor. Mutagenesis results from our lab have
demonstrated that several key residues such as Asp122
and Phe261 of the human MC4R impact on the binding
and function of the small molecule MC4R agonist 4.27
Mutation of Asp126 to a nonacidic residue abolishes the
radiolabeled NDP-MSH binding to the mutant recep-
tor31,32 and greatly diminishes the cAMP stimulation by
melanotropin peptides as well as 4. Asp122Ala and
Asp122Asn mutants, however, are still fully functioning
receptors. While the binding affinity of NDP-MSH at
the Asp122Ala mutant only decreases 6-fold, 4 has a Ki
value of 646 nM at this mutant, a 65-fold reduction from
the wild-type. The EC50 value of 4 is also increased over
30-fold (EC50 ) 49 nM, IA ) 78%). These results indicate
a major involvement of the Asp122 residue in the
interaction with small molecule 4. In addition, the
binding affinities of 4 at the Phe261Ala and Phe284Ala
mutants are reduced by 48- and 28-fold, respectively.
Rational Design of Piperazinebenzylamines as
MC4R Antagonists. Results from mutagenesis, SAR
of peptide and small molecule MC4R agonists, and
receptor modeling point to a possible interaction be-
tween the Asp122 of the human MC4R and the triazole
motif of 4 or 6a. This hypothesis prompted us to
synthesize the benzylamine 7 in which the nonbasic
triazole of 6a was replaced with a basic amino group to

A Potent and Selective MC4R Antagonist
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6827
g, 80.54 mmol), and potassium carbonate (16.75 g. 121.16
mmol) in DMF (81 mL) was heated at 150 °C for 8 h with
constant stirring. The reaction mixture was cooled to room
temperature, diluted with ethyl acetate (200 mL), and washed
with water (3 × 150 mL) and saturated NaCl solution (150
mL). The organic layer was dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The yellow oil solidified
under vacuum overnight, giving a bright yellow solid. The solid
was washed with hexanes (3 × 100 mL) to removed impurities,
collected, and dried under high vacuum. 2-[4-(tert-Butoxycar-
bonyl)-piperazin-1-yl]benzaldehyde was obtained as a bright
yellow solid (17.5 g, 75% yield).
2-[4-(tert-Butoxycarbonyl)piperazin-1-yl]benzaldehyde (304.3
mg, 1 mmol) was dissolved in a mixture of methylene chloride/
trifluoroacetic acid (2 mL/2 mL) and was stirred vigorously
for 30 min at room temperature. Solvents were evaporated,
and the residue was dissolved in methylene chloride (5 mL).
Diisopropylethylamine (3 mL) was added and evaporation
under vacuum gave the free amine intermediate, which (190
mg, 1 mmol), dissolved in DMF (2 mL), was added to a mixture
of Boc-D-Tic-D-(4-Cl)Phe-OH 24g (459 mg, 1 mmol) and HBTU
(457 mg, 1.2 mmol), previously stirred in DMF (4 mL) for 30
min at 40 °C. The mixture was stirred at 40 °C for 6 additional
hours. Water (5 mL) was added, and the product was extracted
with diethyl ether and purified on silica (hexanes/ethyl acetate
1:1) to yield 4-{(2R)-[(3R)-1,2,3,4-tetrahydroisoquinolinylcar-
boxamido]-3-(4-chlorophenyl)propionyl}-1-[2-formylphenyl]pi-
perazine (415 mg, 66%).
the phenyl ring with a more lipophilic 2′-naphthyl
group, we hypothesized that a similar change could
switches off any agonist efficacy of compounds 9b.
Indeed, replacing the 4-chlorophenyl ring of 9b with a
more lipophilic 2,4-dichlorophenyl group (10) not only
abolishes its intrinsic activity but also increases the
binding affinity over 10-fold. Thus, compound 10 has a
Ki value of 1.8 nM in binding affinity, and it does not
stimulate significant cAMP release mediated by the
human MC4 receptor at 10 µM concentration (∼3% of
the R-MSH level). We also proved that 10 is a competi-
tive antagonist in inhibiting R-MSH-stimulated cAMP
production by the Schild plot analysis (pA2 ) 7.9, Figure
4). Like 4, compound 10 is also very selective over the
other melanocortin subtypes. Thus, 10 has Ki values of
4200, 640, and 280 nM on the human MC1, MC3, and
MC5 receptors, respectively (Table 2). 10 also inhibits
the radiolabeled AgRP(87-132) binding to the human
MC4R with high potency (Ki ) 1.5 nM).
The pharmacokinetic profile of 10 was assessed in
rats. After intravenous administration (10 mg/kg), it
shows a good half-life of 4.6 h in this species. Most
importantly, 10 penetrated to the brain with a brain/
plasma (b/p) ratio of about 1 at 1.5 h time point. In
human liver microsomes in vitro, 10 exhibited a low
intrinsic clearance (CLint ) 23 mL/min kg).39
A solution of the above compound (30 mg, 0.048 mmol) and
ammonia/methanol (7 M, 0.023 mL, 0.16 mmol) in methanol
(0.5 mL) was stirred for 24 h. Sodium borohydride (3 mg, 0.08
mmol) was added, and the mixture was stirred overnight. The
mixture was then diluted with ethyl acetate (5 mL), washed
with saturated aqueous sodium bicarbonate (2 mL), dried over
sodium sulfate, and concentrated. To the residue were added
dichloromethane (0.5 mL) and trifluoroacetic acid (0.5 mL),
and stirring was continued for 15 min. The solution was
concentrated and then purified by preparative HPLC to give
the title compound 7 (TFA salt) as a pale yellow oil (12 mg,
33%). An analytical sample of the free base of 7 was prepared
by dissolving in dichloromethane (1 mL), washing with satu-
rated aqueous sodium bicarbonate (1 mL), drying over mag-
nesium sulfate, and evaporating to give a white solid: purity
Conclusion
We have successfully designed and synthesized a
potent and selective antagonist, 10, of the human MC4
receptor, based on information derived from the receptor
structure, mutagenesis, and the structure-activity
relationships of peptide ligands and small molecule
agonists. The recognition of an acidic residue of the
receptor that may be involved in the interaction with
small molecule agonists prompted us to introduce a
basic moiety into the initial molecule 6 to maximize its
binding energy. We were also able to identify a site that
may involve receptor activation from the results of
peptide agonists and antagonists and applied this
information to modify the piperazinebenzylamine into
a potent MC4R antagonist. Thus, compound 10 pos-
sesses a Ki value of 1.8 nM in binding affinity and a
pA2 value of 7.9 in competitive inhibition of R-MSH-
stimulated cAMP production. This potent MC4R an-
tagonist 10 is very selective over the other melanocortin
receptor subtypes. It possesses a good half-life in rats,
and most importantly, it is also able to penetrate into
the brain. The effects of 10 and its analogues on the
mutant receptors of Asp122 and other residues lining
the binding pocket will be reported elsewhere.
1
99% (HPLC); H NMR 7.96 (d, J ) 8 Hz, 1H), 7.23-7.34 (m,
4H), 7.09-7.21 (m, 6H), 7.02-7.10 (m, 1H), 6.94-7.02 (m, 1H),
5.14-5.24 (m, 1H), 3.80-4.00 (m, 4H), 3.20-3.65 (m, 4H), 3.18
(d, J ) 5 Hz, 1H), 3.13 (d, J ) 5 Hz, 1H), 2.92-3.08 (m, 3H),
2.54-2.92 (m, 3H), 2.06-2.35 (m, 1H), 1.23-1.29 (m, 1H),
0.82-0.95 (m, 2H); HRMS m/z calcd for C30H35N5ClO2 (MH)+
532.2474, found 532.2459.
4-{(2R)-[(3R)-1,2,3,4-Tetrahydroisoquinolinylcarbox-
amido]-3-(4-chlorophenyl)propionyl}-1-{2-[2-(2-thienyl)-
ethylaminomethyl]phenyl}piperazine (8a). To a solution
of 4-{(2R)-[(3R)-1,2,3,4-tetrahydroisoquinolinylcarboxamido]-
3-(4-chlorophenyl)propionyl}-1-[2-formylphenyl]piperazine (25
mg, 0.040 mmol) and 2-(2-thienyl)ethylamine (10 mg, 0.080
mmol) in dichloromethane (0.5 mL) was added sodium triace-
toxyborohydride (12 mg, 0.080 mmol), and the mixture was
stirred overnight. The mixture was then quenched with
saturated aqueous sodium bicarbonate (0.5 mL) and separated,
and the aqueous layer was extracted with ethyl acetate (1 mL).
The combined organics were dried over sodium sulfate and
concentrated. To the residue were added dichloromethane (0.5
mL) and trifluoroacetic acid (0.3 mL), and stirring was
continued for 30 min. The solution was concentrated then
purified by preparative HPLC to give the title product 8a (TFA
salt) as a pale yellow oil (21 mg, 60%). An analytical sample
of the free base of 8a was prepared by dissolving in dichlo-
romethane (1 mL), washing with saturated aqueous sodium
bicarbonate (1 mL), drying (magnesium sulfate), and evaporat-
Experimental Section
Chemistry. General Methods. NMR spectra were re-
corded on a Varian 300 MHz spectrometer with TMS as an
internal standard and CDCl3 as the solvent. Chemical shifts
are reported in parts per million (δ), and signals are expressed
as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
or br (broad). High-resolution mass spectra were measured at
the Scripps Center for Mass Spectrometry using MALDI-
FTMS. Purity measurements were performed on an HP
Agilent 1100 HPLC-MS (detection at 254 nm).
4-{(2R)-[(3R)-1,2,3,4-Tetrahydroisoquinolinylcarbox-
amido]-3-(4-chlorophenyl)propionyl}-1-[2-(aminometh-
yl)phenyl]piperazine (7). A mixture of 2-fluorobenzaldehyde
(8.54 mL, 80.54 mmol), 1-(tert-butoxycarbonyl)-piperazine (15
1
ing to give a white solid: purity 95% (HPLC); H NMR 7.95
(d, J ) 12 Hz, 1H), 7.68-7.72 (m, 1H), 7.51-7.56 (m, 1H),
7.25-7.30 (m, 2H), 7.04-7.23 (m, 7H), 6.89-6.95 (m, 2H),
6.78-6.81 (m, 1H), 5.13-5.22 (m, 1H), 4.16-4.27 (m, 2H), 3.98

6828 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Chen et al.
(d, J ) 6 Hz, 1H); 3.82 (s, 2H), 3.62-3.76 (m, 1H), 3.54 (d, J
) 6 Hz, 1H), 3.58 (d, J ) 6 Hz, 1H), 3.24-3.52 (m, 2H), 3.18
(d, J ) 5 Hz, 1H), 3.15 (d, J ) 5 Hz, 1H), 2.95-3.15 (m, 2H),
2.95 (d, J ) 6 Hz, 1H), 2.88 (t, J ) 6 Hz, 2H), 2.53-2.82 (m,
2H), 2.15-2.25 (m, 1H), 1.59-1.76 (m, 1H), 1.23-1.49 (m, 2H),
0.83-0.98 (m, 1H); HRMS m/z calcd for C36H41N5ClO2S (MH)+
642.2664, found 642.2651.
4-{(2R)-[2-Aminoacetamido]-3-(4-chlorophenyl)propio-
nyl}-1-{2-[(2-thienyl)ethylaminomethyl]phenyl}-
piperazine (9a). To a solution of N-Cbz-piperazine (14.2 g,
64.5 mmol) and 2-fluorobenzaldehyde (8.10 g, 65.3 mmol) in
dry, degassed DMSO (60 mL) in a pressure tube was added
potassium carbonate (12.2 g, 88.3 mmol). The mixture was
heated with stirring at 120 °C for 19 h. The mixture was
cooled, diluted with ethyl acetate (200 mL), and washed with
saturated aqueous ammonium chloride (100 mL). The aqueous
layer was extracted with ethyl acetate (100 mL), and the
combined organics were dried over sodium sulfate, concen-
trated in vacuo, and purified by flash column chromatography
(10-20% ethyl acetate/dichloromethane) to give 2-(4-Cbz-
piperazine)benzaldehyde 11 as a viscous yellow oil (11.0 g,
53%): MS m/z 325.0 (MH)+.
Sodium triacetoxyborohydride (4.50 g, 21.2 mmol) was
added in portions to a solution of 2-(4-Cbz-piperazine)benzal-
dehyde (11, 4.93 g, 15.2 mmol) and 2-(2-thienyl)ethylamine
(2.04 g, 16.0 mmol) in dry dichloromethane (60 mL) over 5 min.
The mixture was stirred for 16 h and then was quenched with
aqueous saturated sodium bicarbonate (30 mL). The mixture
was separated, and the aqueous layer was extracted with
dichloromethane (2 × 30 mL). The combined organics were
washed with brine (60 mL), dried over magnesium sulfate, and
concentrated to give the crude amine (6.79 g). The amine was
immediately dissolved in dichloromethane (30 mL), and di-
tert-butyl dicarbonate (3.49 g, 16.0 mmol) was added. The
solution was stirred for 6 h and then diluted with dichlo-
romethane (100 mL), washed with saturated sodium bicarbon-
ate (50 mL) and brine (50 mL), dried over magnesium sulfate,
and concentrated. The crude was purified by flash column
chromatography (25% ethyl acetate/hexane) to give N-Boc-N-
[2-(2-thienyl)ethyl]-2-(4-Cbz-piperazine)benzylamine 12 as a
viscous, pale yellow oil (6.60 g, 82% over two steps): MS m/z
536.1 (MH)+.
A mixture of N-Boc-N-[2-(2-thienyl)ethyl]-2-(4-Cbz-piper-
azine)benzylamine(12, 6.30 g, 11.8 mmol) and 10% Pd/C (650
mg) in ammonia/methanol (7 M, 80 mL) was hydrogenated in
a Parr apparatus at 40 psi for 1 h. A second batch of catalyst
(650 mg) was added, and the mixture was hydrogenated for 4
h. A third batch of catalyst (650 mg) was added, and the
mixture was hydrogenated for 18 h and then filtered through
Celite, concentrated in vacuo, and purified by flash column
chromatography. The remaining starting material was eluted
first (50% ethyl acetate/hexane) followed by N-Boc-N-[2-(2-
thienyl)ethyl]-2-piperazinebenzylamine as a viscous, pale yel-
low oil (10% methanol/dichloromethane) (1.65 g, 35%): MS m/z
402.0 (MH)+.
To a mixture of 4-[(2R)-amino-3-(4-chlorophenyl)propionyl]-
1-{2-[N-Boc-(2-thienyl)ethylaminomethyl]phenyl}piperazine (30
mg, 0.052 mmol) and N-Boc-glycine (10 mg, 0.057 mmol) in
dichloromethane (1 mL) was added HOBt (9.0 mg, 0.057
mmol), and the mixture was stirred for 10 min. EDC (11 mg,
0.057 mmol) was added, and stirring was continued overnight.
The mixture was washed with saturated aqueous sodium
bicarbonate (1 mL) and separated, and the aqueous layer was
extracted with ethyl acetate (1 mL). The combined organics
were dried over sodium sulfate and concentrated. To the
residue was added 4 M HCl/dioxane (1 mL), and the mixture
was stirred for 2 h and then concentrated and purified by
preparative HPLC to give the title product 9a (TFA salt) as a
pale yellow oil (26 mg, 65%). An analytical sample of the free
base of 9a was prepared by dissolving in dichloromethane (1
mL), washing with saturated aqueous sodium bicarbonate (1
mL), drying over magnesium sulfate, and evaporating to give
1
a white solid: purity 100% (HPLC); H NMR 7.88 (d, J ) 9
Hz, 1H), 7.06-7.33 (m, 9H), 6.88-6.93 (m, 2H), 6.79-6.81 (m,
1H), 5.15-5.23 (m, 1H), 3.82 (bs, 2H), 3.61-3.72 (m, 1H),
3.19-3.45 (m, 3H), 3.36 (s, 2H), 2.94-3.09 (m, 4H), 2.88 (t, J
) 7 Hz, 2H), 2.69-2.77 (m, 1H), 2.50-2.64 (m, 2H), 1.90-
2.20 (m, 2H), 1.22-1.29 (m, 1H); HRMS m/z calcd for C28H35N5-
ClO2S (MH)+ 540.2194, found 540.2188.
4-{(2R)-[3-Aminopropionylamido]-3-(4-chlorophenyl)-
propionyl}-1-{2-[(2-thienyl)ethylaminomethyl]phenyl}-
piperazine (9b). This compound was synthesized using a
procedure similar to that described for 9a with N-Boc-â-
1
alanine: white solid (100% purity, HPLC); H NMR 7.71 (bs,
1H), 7.02-7.36 (m, 7H), 6.86-6.96 (m, 2H), 6.78-6.82 (m, 1H),
5.15-5.21 (m, 1H), 3.80 (bs, 1H), 3.51-3.55 (m, 2H), 3.48 (s,
1H), 2.11-3.47 (m, 18H), 1.17-1.29 (m, 1H), 0.83-0.94 (m,
1H); HRMS m/z calcd for C29H37N5ClO2S (MH)+ 554.2351,
found 554.2350.
4-{(2R)-[2-Methoxyacetamido]-3-(4-chlorophenyl)pro-
pionyl}-1-{2-[(2-thienyl)ethylaminomethyl]phenyl}-
piperazine (9c). This compound was synthesized using a
procedure similar to that described for 9a with 2-methoxy-
1
acetic acid: colorless oil (95% purity, HPLC); H NMR 7.06-
7.34 (m, 7H), 6.87-6.93 (m, 2H), 6.79-6.81 (m, 1H), 5.17-
5.25 (m, 1H), 3.90 (d, J ) 4.2 Hz, 2H), 3.80 (s, 2H), 3.65-3.94
(m, 3H), 3.42 (s, 3H), 3.15-3.35 (m, 3H), 2.91-3.08 (m, 4H),
2.87 (t, J ) 6.0 Hz, 2H); 2.68-2.80 (m, 1H), 2.49-2.65 (m, 2H),
2.02-2.18 (m, 1H), 1.25 (s, 1H); HRMS m/z calcd for C29H35N4-
ClO3S (MH)+ 555.2191, found 555.2199.
4-{(2R)-[3-Aminopropionylamido]-3-(2,4-dichlorophen-
yl)propionyl}-1-{2-[(2-thienyl)ethylaminomethyl]phenyl}-
piperazine (10). To a mixture of N-Boc-N-[2-(2-thienyl)ethyl]-
2-piperazinebenzylamine (880 mg, 2.19 mmol) and N-Fmoc-
D-(2,4-dichlorophenyl)alanine (1.02 g, 2.41 mmol) in dichloro-
methane (30 mL) was added HOBt (325 mg, 2.41 mmol), and
the mixture was stirred for 20 min. EDC (460 mg, 2.41 mmol)
was added, and stirring was continued for 18 more hours. The
mixture was then washed with saturated sodium bicarbonate
(2 × 15 mL) and brine (15 mL), dried over magnesium sulfate,
and concentrated in vacuo. The crude 13b was filtered through
silica gel (10% ethyl acetate/dichloromethane), concentrated,
and dissolved in a 1:1 mixture of diethylamine/dichlo-
romethane (20 mL). After stirring for 3 h, the solution was
concentrated and the product isolated by flash column chro-
matography (9:1 ethyl acetate/dichloromethane to 94:5:1 dichlo-
romethane/methanol/triethylamine) to give 4-[(2R)-amino-
3-(2,4-dichlorophenyl)propionyl]-1-{2-[N-Boc-(2-thienyl)ethyl-
aminomethyl]phenyl}piperazine as a white foam (1.11 g, 87%
over two steps): MS m/z 583.2 (MH)+.
To a mixture of 4-[(2R)-amino-3-(2,4-dichlorophenyl)propio-
nyl]-1-{2-[N-Boc-(2-thienyl)ethylaminomethyl]phenyl}piper-
azine (26 mg, 0.036 mmol) and N-Boc-â-alanine (7.0 mg, 0.036
mmol) in dichloromethane (1 mL) was added HOBt (5.0 mg,
0.036 mmol), and the mixture was stirred for 10 min. EDC
(7.0 mg, 0.036 mmol) was added, and stirring was continued
overnight. The mixture was washed with saturated aqueous
sodium bicarbonate (1 mL) and separated, and the aqueous
layer was extracted with ethyl acetate (1 mL). The combined
To a mixture of N-Boc-N-[2-(2-thienyl)ethyl]-2-piperazineben-
zylamine (880 mg, 2.19 mmol) and N-Fmoc-D-(4-chlorophenyl)-
alanine (1.02 g, 2.41 mmol) in dichloromethane (30 mL) was
added HOBt (325 mg, 2.41 mmol), and the mixture was stirred
for 20 min. EDC (460 mg, 2.41 mmol) was added, and stirring
was continued for 18 more hours. The mixture was then
washed with saturated sodium bicarbonate (2 × 15 mL) and
brine (15 mL), dried over magnesium sulfate, and concentrated
in vacuo. The crude 13a was filtered through silica gel (10%
ethyl acetate/dichloromethane), concentrated, and dissolved in
a 1:1 mixture of diethylamine/dichloromethane (20 mL). After
stirring for 3 h, the solution was concentrated and the product
isolated by flash column chromatography (9:1 ethyl acetate/
dichloromethane to 94:5:1 dichloromethane/methanol/triethyl-
amine) to give 4-[(2R)-amino-3-(4-chlorophenyl)propionyl]-1-
{2-[N-Boc-(2-thienyl)ethylaminomethyl]phenyl}piperazine as
a white foam (1.11 g, 87% over two steps): MS m/z 583.2
(MH)+.

A Potent and Selective MC4R Antagonist
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6829
(4) Palczewski, K.; Kumasaka, T.; Hori, T.; Nehnke, C. A.; Mo-
toshima, H.; Fox, B. A.; Le Trong, I.; Teller, D. C.; Okada, T.;
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of rhodopsin: A G protein-coupled receptor. Science 2000, 289,
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receptors: Crystals clarify. Trends Pharmacol. Sci. 2002, 23,
140-146.
organics were dried over sodium sulfate and concentrated. To
the residue was added 4 M HCl/dioxane (1 mL), and the
mixture was stirred for 2 h and then concentrated and purified
by preparative HPLC to give the title product 10 (TFA salt)
as a pale yellow oil (10 mg, 34%). An analytical sample of the
free base of 10 was prepared by dissolving in dichloromethane
(1 mL), washing with saturated aqueous sodium bicarbonate
(1 mL), drying over magnesium sulfate, and evaporating to
give the title compound as a white solid: purity 100% (HPLC);
1H NMR 7.63 (d, J ) 7 Hz, 1H), 7.41 (s, 1H) 7.07-7.26 (m,
6H), 6.85-6.95 (m, 2H), 6.80 (d, J ) 3 Hz, 1H), 5.30-5.39 (m,
2H), 3.80 (bs, 2H), 3.17-3.79 (m, 4H), 2.90-3.17 (m, 5H),
2.83-2.90 (m, 2H), 2.65-2.81 (m, 3H), 2.42-2.65 (m, 2H),
2.15-2.42 (m, 3H), 1.21-1.32 (m, 1H); HRMS m/z calcd for
C29H36N5Cl2O2S (MH)+ 588.1961, found 588.1980.
Receptor Binding. Receptor binding was performed on
HEK293 cells stably expressing the human melanocortin
receptors, using [125I]NDP-MSH as the radiolabeled ligand.
The cAMP stimulation and inhibition assays were performed
in the same cell lines using R-MSH (as the standard in agonist
assay). The assay conditions were similar to those previously
reported.27
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Dixon, R. A. Structure and function of
G protein-coupled
receptors. Annu. Rev. Biochem. 1994, 63, 101-132. (b) Kris-
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The Three-Dimensional Structure of the MC4 Recep-
tor and Small Molecule Docking. The homology 3D struc-
ture of the human MC4 receptor has been built by several
groups.36 The receptor model used in this study was provided
by Yang and co-workers.36a The small molecule with a low-
energy conformation, selected from random conformation
search results, was initially placed in the transmembrane
domain. The MMFF94 force field with implicit solvation model
implemented in MOE was used. The receptor was rigid and
the ligand was flexible. A Tabu search with default settings
in MOE-dock was performed aiming to optimize both spatial
contacts and electostatic interactions. It used a stochastic
searching algorithm and wrote the resulting docking confor-
mations and their energies to a molecular database file. The
resulting docking conformations were ranked by both total
energy and ligand energy. The top ranked solutions were
visually evaluated, and the one that best explains the mu-
tagenesis study results is illustrated in Figure 5.
Pharmacokinetic Characterization. Compound was dosed
as a hydrochloride salt in aqueous solution. The pharmacoki-
netic profile of 10 was determined in male mice (N ) 3/time
points at a dose of 10 mg/kg). The dosing solution was prepared
in purified water and filtered through a 0.2 µm Nylon filter
before administration (2 mL/kg) via the tail vain (iv) or oral
gavage (po). The bioanalytical method applied for the mea-
surement of test articles in plasma along with added internal
standard consisted of precipitation with 200 µL of acetonitrile
from 50 µL of plasma, centrifugation, and recovery the
supernatant, which was evaporated in a vacuum and then
reconstituted in acetonitrile/water solution before introduction
into an LC-MS/MS system for analysis. The lower limit of
quantification (LLOQ) for the analytical methods was 5 ng/
mL of test article in plasma. All pharmacokinetic parameters
were calculated from a noncompartmental model using Win-
Nonlin program version 3.2.
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