Design, synthesis, in vitro, and in vivo characterization of phenylpiperazines and pyridinylpiperazines as potent and selective antagonists of the melanocortin-4 receptor

Article abstract of DOI:10.1021/jm701137s

Benzylamine and pyridinemethylamine derivatives were synthesized and characterized as potent and selective antagonists of the melanocortin-4 receptor (MC4R). These compounds were also profiled in rodents for their pharmacokinetic properties. Two compounds with diversified profiles in chemical structure, pharmacological activities, and pharmacokinetics, 10 and 12b, showed efficacy in an established murine cachexia model. For example, 12b had a K_i value of 3.4 nM at MC4R, was more than 200-fold selective over MC3R, and had a good pharmacokinetic profile in mice, including high brain penetration. Moreover, 12b was able to stimulate food intake in the tumor-bearing mice and reverse their lean body mass loss. Our results provided further evidence that a potent and selective MC4R antagonist with appropriate pharmacokinetic properties might potentially be useful for the treatment of cancer cachexia.

Full text of DOI:10.1021/jm701137s

6356
J. Med. Chem. 2007, 50, 6356–6366
Design, Synthesis, In Vitro, and In Vivo Characterization of Phenylpiperazines and
Pyridinylpiperazines as Potent and Selective Antagonists of the Melanocortin-4 Receptor
Joe A. Tran, Wanlong Jiang, Fabio C. Tucci,† Beth A. Fleck, Jenny Wen, Yang Sai, Ajay Madan, Ta Kung Chen,
Stacy Markison, Alan C. Foster, Sam R. Hoare, Daniel Marks,# John Harman, Caroline W. Chen, Melissa Arellano,
Dragan Marinkovic, Haig Bozigian, John Saunders,‡ and Chen Chen*
Department of Medicinal Chemistry, Department of Pharmacology, Department of Neuroscience and Department of Preclinical DeVelopment,
Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, California 92130
ReceiVed September 12, 2007
Benzylamine and pyridinemethylamine derivatives were synthesized and characterized as potent and selective
antagonists of the melanocortin-4 receptor (MC4R). These compounds were also profiled in rodents for
their pharmacokinetic properties. Two compounds with diversified profiles in chemical structure,
pharmacological activities, and pharmacokinetics, 10 and 12b, showed efficacy in an established murine
cachexia model. For example, 12b had a Ki value of 3.4 nM at MC4R, was more than 200-fold selective
over MC3R, and had a good pharmacokinetic profile in mice, including high brain penetration. Moreover,
12b was able to stimulate food intake in the tumor-bearing mice and reverse their lean body mass loss. Our
results provided further evidence that a potent and selective MC4R antagonist with appropriate pharmaco-
kinetic properties might potentially be useful for the treatment of cancer cachexia.
Introduction
feeding behavior and energy homeostasis in animals and humans.9
MC4 receptors are widely expressed in the brain, and MC4R
mutations that impair receptor function have been associated with
binge eating and obesity in humans.10,11 In addition, many studies
have demonstrated that MC4R agonists suppress food intake
and reduce body weight in animals.12
Cachexia is a condition characterized by weight loss, wasting
of muscle, loss of appetite, and general debility that accompanies
many chronic diseases, affects various patient populations,
including those with cancer, and is estimated to be responsible
for over 20% of all cancer-related deaths.1 Cancer cachexia
significantly impairs quality of life and response to antineoplastic
therapies and increases morbidity and mortality of cancer
patients. Most current therapeutic strategies to counteract cancer
cachexia have proven to be only partially effective.2 In the past
decade, research into the processes leading to cachexia have
provided ideas for more effective therapeutic intervention that
have been attempted pharmacologically with encouraging results
in animal models and preliminary clinical trials. While recent
studies have linked cancer cachexia to endocannabinoid,3
ghrelin,4 and other biological systems associated with appetite
stimulation and food intake, one key center that is likely
involved in the propagation of symptoms of cachexia is the
melanocortin system in the hypothalamus and brainstem.5
The melanocortin system consists of five cell surface receptors
that belong to the class A G-protein-coupled receptor super-
family.6 These receptors are activated by the family of melanocyte-
stimulating hormone peptide agonists produced from the post-
translational processing of pro-opiomelanocortin (POMCa), including
R-, ꢀ-, and γ-MSH and adrenocorticotropin hormone (ACTH).7
In addition, these receptors are also regulated by the endogenous
antagonists agouti-protein and agouti-related protein (AgRP).8 The
melanocortin-4 receptor (MC4R)a plays a very important role in
In contrast, recent studies have shown that MC4R antagonists
promote food intake and increase weight gain in animals.13
Moreover, evidence suggests that cachexia brought about by a
variety of illnesses can be attenuated or reversed by blocking
MC4R activation within the central nervous system.14 For
example, Wisse and co-workers demonstrate that the peptide
MC4R antagonist Ac-Nle(4)-c[Asp(5)-2′-Nal(7)-Lys(10)]-R-
MSH(4–10)-NH2 (SHU9119)15 reverses body weight loss in
mice after intracerebroventricular administration.16 This effect,
however, is diminished in MC4R knock out mice,17 demonstrat-
ing MC4R involvement. Central infusion of AgRP(83–132) also
prevents cachexia-related symptoms induced by radiation and
colon-26 tumors in mice.18
While potent peptide antagonists for the MC4 receptor have
been known for years, small nonpeptide molecules that antago-
nize the receptor were only recently discovered.19 One advantage
of nonpeptide molecules is the possibility of delineating
pharmacological activity in animals without intracerebroventrical
injection if the molecules achieve sufficient brain penetration
after peripheral administration. For example, a 2-phenylimida-
zoline 1 (ML00253764, Figure 1) is a functional MC4R
antagonist (IC50 ) 103 nM) with poor selectivity versus MC3R,
a receptor subtype that is also believed to have a role in feeding
regulation20 and has demonstrated efficacy in a cachexia model
via subcutaneous administration.21,22 Additionally, we have
shown that a ꢀ-alanine-(2,4-Cl)phenylalanine dipeptide deriva-
tive 2 is a potent functional MC4R antagonist with negligible
affinity at MC3R.23 Intraperitoneal administration of 2 ef-
fectively stimulates daytime (satiated) food intake and decreases
basal metabolic rate in normal animals. Furthermore, this
compound attenuates cachexia and preserves lean body mass
in a murine cancer model.24 These data provide evidence for
* To whom correspondence should be addressed. Phone: 858-617-7634.
Fax: 858-617-7602. E-mail: cchen@neurocrine.com.
†
Current address: Department of Medicinal Chemistry, Tanabe Research
Laboratories, U.S.A., Inc., 4540 Towne Centre Ct., San Diego, CA 92121.
#
Department of Pediatrics, Oregon Health and Science University, 3181
Southwest Sam Jackson Park Road, Portland, OR 97239.
‡
Current address: Vertex Pharmaceuticals Inc., 11010 Torreyana Road,
San Diego, CA 92121.
a
Abbreviations: MC4R, type 4 melanocortin receptor; MSH, melanocyte-
stimulating hormone; AgRP, agouti-related protein; cAMP, cyclic adenosine
monophosphate; i.p., intraperitoneal; b/p, brain/plasma.
10.1021/jm701137s CCC: $37.00
2007 American Chemical Society
Published on Web 11/10/2007

Antagonists of the Melanocortin-4 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6357
Figure 1. Chemical structures of some small molecule MC4R antagonists.
the potential utility of small molecule MC4R antagonists,
particularly in the treatment of cancer cachexia.25
10 after Boc-deprotection.33 Compound 13 was synthesized from
6b by a sequence for 9, followed by a coupling reaction with
3-(dimethylamino)propionic acid.
Several MC4R antagonists from different chemical classes
have been reported. For example, the structure–activity relation-
ship of bispiperazine derivatives exemplified by 3 has been
extensively studied.26,27 A series of acylguanidines exemplified
by 4 are reported as highly potent MC4R antagonists with brain
penetration in rodents.28 Very recently, a series of benzimida-
zoles are described as potent and selective MC4R antagonists.29
We have previously reported the discovery of a series of
piperazinebenzylamines such as 5 (Ki ) 75 nM) as MC4R
antagonists.30 By using 5 as a lead, we started a research effort
to improve the MC4R antagonist properties associated with this
compound in several categories including potency, metabolism,
and pharmacokinetics. Here we report the design, synthesis, and
in vitro and in vivo characterization of several compounds based
on this initial lead. Among them, N-(1S-[2-{4-[(2R)-methyl-3-
(4-chlorophenyl)propionyl]-1-piperazinyl}-5-chlorophenyl]-3-
methylbutyl)-3-(dimethylamino)propionamide (12b) was iden-
tified as potent and selective at the MC4 receptor. It also
exhibited suitable pharmacokinetic properties for further evalu-
ation and demonstrated anticachectic activity in a murine
cachexia model.
The optically pure 2R-methyl-3-(4-chlorophenyl)propionic
acid 16a and 2R-methyl-3-(2,4-dichlorophenyl)propionic acid
16b were synthesized according to Scheme 2. Stereoselective
alkylation of (S)-3-propionyl-4-benzyloxazolidine 14 with 4-chlo-
robenzyl or 2,4-dichlorobenzyl bromide, followed by hydrolyzis
of the diastereomer 15 promoted by hydrogen peroxide under
basic conditions afforded the desired acid 16a or 16b in good
yield.34
The synthesis of the pyridine derivatives 20–23 is outlined
in Scheme 3. 2-Bromo-3-pyridylcarboxaldehyde 17a was ob-
tained by a formylation of 2-bromopyridine, which was achieved
by quenching the carbanion, formed by LDA at -78 °C in THF,
with DMF in 19% yield.35 2-Chloro-6-methyl-3-pyridylcarbox-
aldehyde 17b was obtained by the reduction of 2-chloro-3-
cyano-6-methylpyridine with Dibal-H in toluene at -10 to 0
°C in 53% yield. These aldehydes were condensed with (S)-
tert-butanesulfinamide to the corresponding pyridylmethylidene
sulfinylamides S-18a-b. Reactions of S-18 with isobutyllithium
afforded the adducts R-19a,b using conditions similar to those
for 6a-d. Unexpectedly, the stereoselectivity of this reaction
was reversed from the benzene analogs 6, and the major
stereoisomer had an R-configuration at the newly formed chiral
center. The stereochemistry of R-19b was confirmed by X-ray
crystal structure determination (Figure 2). The reason for this
reversed stereoselectivity was possibly caused by the participa-
tion of the pyridine nitrogen in the chelating process.31 The
corresponding S-19a was then synthesized from (R)-tert-
butanesulfinamide using the same procedure. After a TFA
treatment of R-19a, the resultant amine intermediate was coupled
with 2,4-dichlorophenylpropionic acid to give R-20a after an
HCl/MeOH treatment. Compound R-20b was obtained from
R-19a and 2-methyl-3-(2,4-dichlorophenyl)propionic acid. Simi-
larly, a coupling reaction of the free amine derived from R-19b
with (2R)-methyl-3-(2,4-dichlorophenyl)propionic acid provided
the amide R-21. Compound R-20b was further derivatized to
R-22 by coupling with glycine, while R-21 was converted to
the N,N-dimethylpropionamide R-23. The S-isomers, S-20a and
S-22, were also synthesized from S-19a for comparison.
Chemistry
The target benzylamine compounds were synthesized from
the protected piperazinebenzylamines 6,31 as shown in Scheme
1. A coupling reaction of the free amine derived from 6a with
3-(2,4-dichlorophenyl)propionic acid afforded the amides 5 after
deprotection with HCl in methanol, which was coupled with
N-Boc-glycine to give 11 after a TFA treatment.32 Similarly,
6a was also coupled with 2-methyl-3-(2-methoxy-4-chlorophe-
nyl)propionic acid to provide 7,30 and compound 8 was obtained
from 6b and 2R-methyl-3-(4-chlorophenyl)propionic acid. Reac-
tion of 8 with 3-(N,N-dimethylamino)propionic acid under
coupling conditions gave the amide 12a. Compound 12b was
prepared using the same procedure from 6c. Compound 9 was
synthesized from 6d by the following sequence. A coupling
reaction of the free amine of 6d with N-Boc-D-(2,4-Cl)Phe-OH
provided an amide that was selectively Boc-deprotected with
TFA and reacted with succinaldehydic acid under reductive
conditions, followed by a cyclization promoted by EDC under
coupling conditions to provide the desired product after depro-
tection with HCl in methanol.33 Reductive alkylation of 9 with
(2-oxoethyl)carbamic acid tert-butyl ester afforded the diamine
Structure–Activity Relationship
The synthesized compounds were tested in a binding assay
using membranes from HEK293 cells stably expressing the

6358 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25
Tran et al.
Scheme 1a
a Reagents and conditions: (a) TFA/CH2Cl2, r.t., 1 h; (b) 2,4-ClC6H3CH2CH2COOH/EDC/HOBt/NaHCO3/DMF/CH2Cl2, r.t., 16 h; (c) HCl/MeOH, r.t.,
1 h; (d) 2-MeO,4-ClC6H3CH2CHMeCOOH/EDC/HOBt/NaHCO3/DMF/CH2Cl2, r.t., 16 h; (e) R-(4-ClC6H4)CH2CHMeCOOH/EDC/ HOBt/NaHCO3/DMF/
CH2Cl2, r.t., 16 h; (f) (i) N-Boc-D-(2,4-Cl)Phe-OH/EDC/HOBt/NaHCO3/DMF/CH2Cl2, r.t., 16 h; (ii) TFA/CH2Cl2, r.t., 1 h; (iii) OCHCH2CH2COOH/
NaBH(OAc)3/HOAc/ClCH2CH2Cl, r.t., 24 h; (iv) EDC/HOBt/NaHCO3/DMF/CH2Cl2, r.t., 16 h; (g) N-Boc-GlyOH/EDC/HOBt/NaHCO3/DMF/CH2Cl2, r.t.,
16 h; (h) Me2NCH2CH2COOH/EDC/HOBt/NaHCO3/DMF, r.t., 16 h; (i) N-Boc-NHCH2CHO/NaBH(OAc)3/CH2Cl2/r.t. 14 h.
Scheme 2a
For the pyridine compounds 20a, the R-configured compound
R-20a (Ki ) 260 nM) was more potent than its S-isomer (S-
20a, Ki ) 890 nM), the opposite of what was seen in the
benzylamine series.30 Incorporating a 2-methyl moiety at the
3-(2,4-dichlorophenyl)propionyl group of R-20a resulted in a
more potent molecule (R-20b, Ki ) 36 nM). Adding a glycine
to R-20b improved its potency by 6-fold (R-22, Ki ) 6 nM),
parallel to the result of benzylamines 5 and 11. Incorporating a
a Reagents and conditions: (a) NaHMDS/THF, -70°C, 1 h, then 2-Y-
methyl group at the 6-position of the pyridine R-20b had little
4-ClC6H4CH2Br, -78 °C to r.t., 6 h; (b) LiOH/H2O2/THF/H2O, 0 °C to
effect on its binding affinity (R-21, Ki ) 44 nM). An almost
20-fold increase in binding affinity from R-21 was observed
for the N,N-dimethylaminopropionamide derivative (R-23, Ki
) 2.5 nM). The potencies of the pyridine derivatives R-20–23
were quite similar to those of the phenyl analogs (5 and 7–13).
In the solid state, the piperazine plane was orthogonal to the
pyridine ring in 2-piperazinepyridine R-19b reflected by its
X-ray crystal structure (Figure 2). This conformational feature
is also observed for benzene analogs such as Boc-6a,31 although
the overlay of these two X-ray structures indicates that the
sulfinylamides point in different directions (Figure 3).
r.t., 1.5 h.
human melanocortin-4 receptor as previously described,36 and
the results are depicted in Table 1. Compounds 7, 8, 10–13 and
R-23 were also tested in a whole cell cAMP assay to study their
functional antagonist activity. Selectivity profiles of these
compounds at the other melanocortin receptor subtypes were
determined in binding assays, and the results are summarized
in Table 2.
While the benzylamine 5 displayed a Ki of 75 nM, its glycine
derivative 11 (Ki ) 19 nM) exhibited increased potency. The
2-methyl-3-(2-methoxy-4-chlorophenyl)propionyl analog 7 (Ki
) 14 nM) was over 5-fold better than 5 in binding affinity.
Incorporating a N,N-dimethylpropionyl group at the benzyl
nitrogen of 8 (Ki ) 25 nM) improved the potency by about
7-fold (12a, Ki ) 3.7 nM). Replacing the methyl group of 12a
with a chlorine gave an analog with the similar potency (12b,
Ki ) 3.4 nM). The pyrrolidinone 9 possessed a Ki value of 9.7
nM, which was increased by 10-fold when an aminoethyl group
was attached (10, Ki ) 0.9 nM). In comparison, the N,N-
dimethylaminopropionamide 13 (Ki ) 0.6 nM) also exhibited
subnanomolar binding affinity.
Most of these compounds (7, 8, 10–13 and R-23) were very
selective at MC4R over the other melanocortin receptor subtypes
(Table 2). For example, the pyridine compound R-23 displayed
at least 500-fold selectivity over the other receptor subtypes.
The only exception was 7, which only exhibited 6-fold selectiv-
ity at the MC5 receptor compared to MC4R. All of the
compounds tested in the functional agonist assay showed no
significant stimulation of cAMP accumulation via MC4R at up
to 10 µM (data not shown). In the functional antagonist assay,
all compounds displayed dose-dependent inhibition of R-MSH-
stimulated cAMP accumulation with various potencies (Table
2). The functional IC50 values were typically 20- to 30-fold

Antagonists of the Melanocortin-4 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6359
Scheme 3a
a Reagents and conditions: (a) DMF/DIEA/4-Boc-piperazine, 100°C, 8 h; (b) S-Me3CSONH2/Ti(OEt)4/THF, r.t., 8 h; (c) (i) Me3Al/THF, -40 °C, 20 min;
(ii) i-BuLi/THF, -78 °C, 5–8 h; (d) (i) TFA/CH2Cl2, r.t., 1 h; (ii) (2,4-Cl2C6H3)CH2CH(R′)COOH/EDC/HOBt/CH2Cl2, r.t., 8 h; (e) N-Boc-Gly-OH/EDC/
HOBt/NaHCO3/DMF/CH2Cl2, r.t., 8 h; (ii) TFA/CH2Cl2, r.t., 1 h; (f) Me2NCH2CH2COOH/EDC/HOBt/NaHCO3/DMF/CH2Cl2, r.t., 8 h.
Table 2. Binding Affinity (Ki, nM) at the Melanocortin Receptor
Subtypes,a Functional Activity at MC4R and Measured LogD Values of
Some MC4R Antagonists
binding affinity (Ki, nM)
MC4R function
c
cmpd
MC1b
MC3
MC4
MC5
IC50 (nM)
LogDd
7
8
10
11
12a
12b
13
(23%)
3100
320
1300
750
1200
2800
710
14
25
86
1000
850
500
710
600
520
1200
1700
570
21
1300
160
930
19
4.5
3.0
1.8
4.5
2.4
3.9
3.9
3.3
(38%)
(18%)
(34%)
(20%)
8400
0.9
19
3.7
3.4
0.6
2.5
820
3300
R-23
6400
49
Figure 2. X-ray crystal structure of R-19b.
a Data are the average of two or more independent measurements. b The
percentage inhibition at 10 µM concentration is indicated in parenthesis.
c Dose-dependent inhibition of R-MSH-stimulated cAMP production via
MC4R. d The logD value was measured using a shake-flake method.
Table 1. Binding Affinity of 5, 7–13, and 20–23 at hMC4Ra
compound
Ki (nM)
compound
Ki (nM)
5
7
8
9
10
11
12a
12b
75
14
25
9.7
0.9
19
3.7
3.4
13
0.6
260
890
36
44
6.0
39
R-20a
S-20a
R-20b
R-21
R-22
S-22
R-23
2.5
a Data are the average of two or more independent measurements.
higher than the binding affinity Ki values, possibly due to the
different assay conditions. However, compounds 7, 11, and 12b
exhibited a much larger separation between their Ki and IC50
values. It is worth noting that these compounds were highly
lipophilic, with measured logD values of >3.9 (Table 2).
Figure 3. Overlay of pyridylpiperazine R-19 and phenylpiperazine 6a.
Pharmacokinetics
Compounds 7, 8, 10, 11, 12b, and R-22 were studied in mice
for their pharmacokinetic properties (Table 3). After an intra-
venous (i.v.) injection at a 5 mg/kg dose, the benzylamine 7
displayed a high plasma clearance (CL ) 62.3 mL/min.kg).
Despite its high volume of distribution (Vd ) 10.3 L/kg), 7 had
a short half-life (t1/2 ) 1.9 h), indicative of fast elimination in
this species. A concentration of 735 ng/g was detected in the
brain at the 1 h time point post-dosing, indicating high brain
penetration. A brain/plasma (b/p) ratio of 2.3 was calculated at
this time point. The absorption of this compound was very fast
after a 10 mg/kg oral gavage (p.o.), and a maximal concentration
(Cmax ) 99 ng/mL) appeared at 0.25 h (Tmax), resulting in an
area under curve (AUC) of 249 ng/mL ·h and a low oral
bioavailability (F% ) 8.6). In comparison, the benzylamine 8
had a moderate plasma clearance (CL ) 33.3 mL/min ·kg) and
a high Vd value of 10.2 L/kg, resulting in a t1/2 of 3.5 h. The
lipophilicity of 8 (LogD ) 3.0) was significantly lower than
that of 7 (LogD ) 4.5), which might contribute to its lower
plasma clearance. The brain penetration of 8 was high (b/p )
2.9 and 1.4 at 1 h and 4 h, respectively) and its oral
bioavailability increased to 34% compared to 7. The less

6360 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25
Tran et al.
Table 3. Pharmacokinetic Profiles of MC4R Antagonists in CD-1 Micea
cmpd CL (mL/min ·kg) Vd (L/kg) t1/2 (h) Cb (ng/g) at 1, 4 h b/p ratio at 1, 4 h Tmax (h) Cmax (ng/mL) oral AUC (ng/mL.h) F (%)
7
8
10
11
12b
R-22
62.3
33.3
15
26.9
36
10.3
10.2
13
8.8
11
1.9
3.5
10
3.8
3.5
5.2
735, 122
940, 330
63, 53
43, 33
800, 177
35, 14
2.3, 1.9
2.9, 1.4
0.14, 0.23
0.08, 0.17
2.4, 2.2
0.25
0.5
2.0
2.0
0.5
0.5
99
267
552
115
132
288
249
1762
1950
687
1160
551
8.6
34.4
19.5
11.2
23.0
12.6
37.4
16.8
0.07, 0.15
a Three animals were dosed intravenously at 5 mg/kg and orally at 10 mg/kg; brain concentrations were taken from the i.v. dosing.
Table 4. Pharmacokinetic Profiles of 12a,b, 13, and R-23 in Sprague–Dawley Ratsa
cmpd CL (mL/min ·kg) Vd (L/kg) t1/2 (h) Tmax (h) Cmax (ng/mL) oral AUC (ng/mL ·h) F(%) Cb (ng/g) at 1, 4 h b/p ratio at 1, 4 h
12a
12b
13
15.0
13.0
38.7
64.9
4.1
2.5
8.8
3.1
2.2
2.6
3.3
5.3
6.0
4.0
2.0
196
163
40
2358
1867
393
19
18
8.7
19.6
12, 36
3, 27
1.5, 15
15, 29
0.2, 0.3
0.1, 0.3
0.2, 0.3
0.2, 0.2
R-23b
18.6
60
517
a Three animals were dosed intravenously at 5 mg/kg and orally at 10 mg/kg; brain concentrations were taken from p.o. dosing. b i.v. dose at 2.5 mg/kg.
Table 5. Pharmacokinetic Properties of 12b in Dogs and Monkeysa
lipophilic diamine 10 (logD ) 1.8) had a low plasma clearance
despite its high tissue distribution (Vd ) 13 L/kg), resulting in
a very long t1/2 of 10 h in mice. However, this compound had
low brain penetration, with a b/p ratio of 0.14–0.23. In an in
vitro Caco-2 assay, 10 exhibited a Papp of 20 nm/s from apical
to basolateral direction, which was not significantly different
from that of compound 8 (Papp ) 18 nm/s). Its permeation from
the basolateral to apical direction was about 3.4-fold higher than
the apical to basolateral direction (ba/ab ratio ) 3.4), suggesting
a possible efflux mechanism involving P-glycoprotein.37 How-
ever, this value was also similar to that of 8 (ba/ab ratio ) 4.4),
indicating that the P-glycoprotein transporter might not be the
only determinant for the difference between these two com-
pounds in brain penetration. The plasma exposure of 10 after
oral dosing was high (AUC ) 1950 ng/mL.h), and the absolute
bioavailability was about 20%.
The aminoacetamide 11 had a moderate plasma clearance,
but its brain penetration was low, similar to 10. This phenom-
enon might be associated with the high polar surface area of
these two compounds (PSA ) 79 and 82 Å2 for 11 and 10,
respectively). The N,N-dimethylpropionamide 12b displayed a
moderate clearance and a t1/2 of 3.5 h. Its brain penetration was
much better (b/p ratio ) 2.2–2.4, PSA ) 56 Å2) than the
diamine 10 and the aminoacetamide 11. The oral absorption of
12b was fast (Tmax ) 0.5 h) and the oral bioavailability was
23% in this species. The pyridine R-22 was also profiled in this
study, and its PK parameters were not significantly different
from that of its close phenyl analog 11, except for a higher Vd
species
dog
5
29.4
11.8
4.7
monkey
i.v. dose (mg/kg)
CL (ml/min ·kg)
Vd (L/kg)
5
24.3
8.5
t1/2 (h)
4.1
AUC (ng/ml ·h)
p.o. dose (mg/kg)
Cmax (ng/mL)
Tmax (h)
AUC (ng/mL ·h)
F (%)
2509
10
142
1.0
1475
29.4
3700
10
108
2.7
1070
13.1
a Data are the average of three animals.
(logD ) 3.3) was only slightly lower than its close phenyl
analog 12b (logD ) 3.9). The plasma clearance of R-23 was
high (CL ) 64.9 mL/min.kg), which resulted in a moderate t1/2
of 3.3 h. Its plasma exposure and brain concentration after oral
dosing was low. Overall, this pyridine compound did not show
a better PK profile than its phenyl analogs such as 12b.
The pharmacokinetic properties of 12b were also profiled in
monkeys and dogs (Table 5). The volume of distribution of 12b
was high in both dogs (Vd ) 11.8 L/kg) and monkeys (Vd )
8.5 L/kg), which matched that in mice (Vd ) 11 L/Kg), but not
in rats (Vd ) 2.5 L/kg). The low Vd value in rats may be
associated with a species-specific phenomenon (e.g., high plasma
protein binding). The half-life (4.7 and 4.1 h, respectively, for
dogs and monkeys) was moderate and the oral bioavailability
was good in dogs, but lower in monkeys.
value, which resulted in a longer t1/2
.
In Vivo Efficacy in Mouse Cachexia Model
Because the N,N-dimethylpropionamide 12b exhibited a
desirable PK profile in mice (high brain penetration, good oral
bioavailability, and moderate t1/2), this compound and its close
analogs 12a, 13, and R-23 were also profiled in rats for their
pharmacokinetic properties (Table 4). Compounds 12a and 12b
displayed very similar profiles in rats. The volume of distribution
of 12b (Vd ) 2.5 L/kg) was much lower than that in mice, which
resulted in a shorter t1/2 (2.2 h). Absorption was slow and the
Cmax appeared at 6 h after oral dosing. The whole brain
concentration of 12b in rats was much lower than that in mice,
and the b/p ratio was observed to be 0.1 and 0.3, respectively,
at the 1 and 4 h time points after an oral administration.
Compound 13, which had a similar lipophilic profile to 12b,
displayed a large Vd in this species. However, its t1/2 was not
much longer than that of 12b due to the high plasma clearance.
The pyridine compound R-23 had a very large Vd of 18.6 L/kg,
which might be associated with its moderate basicity of the
2-aminopyridine structure (calculated pKa ) 9.3 for pipera-
zinepyridine), although its measured lipophilicity at pH 7.4
Previously we have shown that when administered twice daily
(3 mg/kg s.c.) for 4 days to C57BL6, mice bearing subcutaneous
Lewis lung carcinoma tumors, 10, stimulated food intake by
82% relative to vehicle-treated controls. The lean body mass
of the tumor-bearing mice treated with 10 (-0.5%) significantly
increased (9%) over that of tumor-vehicle treated animals
(-9.5%), demonstrating a positive effect in this cachexia
model.38
For the current in vivo study, C57BL/6J male mice were
inoculated with Lewis lung carcinoma (LLC) tumor cells.
Beginning 11 days after LLC inoculation, animals were treated
over 4 days with 12b twice daily (3 and 9 mg/kg, i.p.). LLC
tumor bearing mice treated with a high dose of 12b showed a
significant increase in food intake relative to vehicle-treated
tumor bearing controls (Figure 4A). Body weight was also
significantly increased in mice treated with 12b in both dose
groups. Analysis of body composition with dual-energy X-ray
absorbatometry (DEXA) demonstrated that the greater increase

Antagonists of the Melanocortin-4 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6361
Figure 4. The effects of compound 12b (i.p., 3 and 9 mg/kg twice per day) on food intake (4A, left) and lean body mass (4B, right) in tumor-
bearing mice.
MC4R over the ghrelin receptor (Ki of 0.7 nM and 720 nM,
respectively) and 12b had a Ki value of 460 on the ghrelin
receptor. The extent to which the ghrelin component of these
compounds contributes to the efficacy in the mouse cachexia
model is unclear, but unlikely to be significant, especially for
compound 12b due to its low intrinsic activity.
Metabolic Profile of 12b
Compound 12b was a fairly lipophilic molecule with a logD
value of 3.9, measured by a shake-flask method. It did not show
significant inhibitory activity at the major liver enzymes
CYP1A2, CYP3A4, CYP2D6, CYP2C9, and CYP2C19 (IC50
Figure 5. Time–concentration curve of 12b after an i.p. administration
> 10 µM). In liver microsomes of various species, 12b showed
moderate metabolic stability and the scaled systemic clearances
were 57, 35, 38.1, 17.1, and 18.5 mL/min ·kg, respectively, in
mouse, rat, monkey, dog, and human. After incubation with
human liver microsomes, two major metabolites were observed.
The metabolic profiles were similar in liver microsomes of CD-1
mice, Sprague–Dawley rats, rhesus monkeys, and beagle dogs.
One of the major metabolites was identified as the N-demethy-
lation product. The other was characterized as the hydroxylation
of the piperazine ring based on MS-MS analyses.
(10 mg/kg) to CD-1 mice (n ) 3; Cmax ) 1521 ng/mL; Tmax ) 0.3 h;
AUC ) 3073 ng/mL ·h).
of body weight in 12b-treated mice was due to sparing of lean
body mass (Figure 4B). Tumor bearing animals treated with
vehicle demonstrated an ∼2% increase in lean body mass
(LBM) over the course of the 14 day experiment, whereas
tumor-bearing animals treated with 12b increased their LBM
by ∼7% for the 3 mg/kg group and ∼15% for the 9 mg/kg
group, demonstrating a dose–response effect.
Conclusion
Pharmacokinetic and Pharmacodynamic Relationship
In conclusion, a series of R-isobutylbenzylamine derivatives
7–13 were synthesized and studied as potent and selective
antagonists of the melanocortin-4 receptor. Pyridine alternatives
20–23 were also studied to compare to their benzene analogs.
Although potent and selective MC4R antagonists such as R-23
were identified, and these pyridine derivatives did not exhibit
an advantage in pharmacokinetic properties over their phenyl
analogs. Compounds 10 and 12b were tested in a murine
cachexia model and efficacy was demonstrated. Our results
provide more evidence that a potent and selective MC4R
antagonist has a potential utility in the treatment of cancer
cachexia.
The binding affinity (Ki) of 12b at the mouse melanocortin-4
receptor was determined to be 18 nM, which was much lower
than that of 10 (Ki ) 0.71 nM at mouse MC4R). It also bound
to the mouse melanocortin-3 receptor with a moderate affinity
(Ki ) 340 nM). We profiled the pharmacokinetics of 12b in
CD-1 mice via an intraperitoneal (i.p.) administration of a 10
mg/kg dose. Compound 12b reached a maximal plasma
concentration (Cmax) of 1521 ng/mL (∼2.7 µM, Figure 5). At
8 h post-dosing, the plasma concentration was about 100 ng/
mL (∼0.18 µM). Therefore, although the free fraction of the
compound in the brain was unknown, the total brain concentra-
tion of 12b at any time point during the in vivo efficacy study
should have been 5-fold above its Ki value (18 nM) for a twice
a day administration at a 3 mg/kg dose, considering its high
brain/plasma ratio of ∼2 and assuming linear pharmacokinetics.
Because ghrelin agonists have demonstrated efficacy in
cachexia models,39 we also measured these MC4 antagonists
against the ghrelin receptor in vitro. Compound 10 was a
moderately potent full agonist of the ghrelin receptor, with an
EC50 value of 79 nM (107% intrinsic activity). In comparison,
12b was a much weaker partial agonist with an EC50 of 720
nM and IA of 47% at the ghrelin receptor.
Experimental Section
Chemistry. General Methods. NMR spectra were recorded on
a Varian 300 MHz spectrometer with TMS as an internal standard.
13C NMR spectra were recorded at 75 MHz. 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 mea-
surements were performed on an HP Agilent 1100 HPLC-MS
(detection at 220 and 254 nm).
Both 10 and 12b exhibited low binding affinity at the ghrelin
receptor. Compound 10 had 100-fold selectivity for the mouse
(1S)-[2-{4-[3-(2,4-Dichlorophenyl)propionyl]-1-piperazinyl}-5-
(trifluoromethyl)phenyl]-3-methylbutylamine Trifluoroacetate (5).

6362 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25
Tran et al.
Trifluoroacetic acid (0.2 mL) was added to a solution of 4-{2-[(1S)-
((S)-tert-butanesulfinylamino)-3-methylbutyl]- 4-(trifluorometh-
yl)phenyl}-1-piperazinecarboxylic acid tert-butyl ester (6a, 50 mg,
0.096 mmol) in dichloromethane (0.8 mL), and the mixture was
stirred at r.t. for 50 min. The reaction mixture was basified with
saturated aqueous NaHCO3 solution (5 mL) and extracted with
EtOAc (30 mL). The organic layer was dried over Na2SO4 and
concentrated in vacuo to provide 4-{2-[(1S)-((S)-tert-butanesulfi-
nylamino)-3-methylbutyl]-4-trifluoromethylphenyl}-1-piperazine as
white foam, which was dissolved in DMF/dichloromethane (1:3, 1
mL). NaHCO3 (16.2 mg, 0.192 mmol), 3-(2,4-dichlorophenyl)pro-
pionic acid (25.3 mg, 0.12 mmol), HOBt (15.5 mg, 0.12 mmol),
and EDC (22.0 mg, 0.12 mmol) were sequentially added to this
solution. The reaction mixture was stirred at r.t. overnight, diluted
with EtOAc (20 mL), washed with 5% aqueous HCl (5 mL),
saturated aqueous NaHCO3 (5 mL), and brine (5 mL), and dried
over Na2SO4. The solution was concentrated in vacuo to provide a
crude product that was dissolved in MeOH (2 mL) and treated with
HCl (58 µL 4 N HCl in dioxane). The mixture was stirred at r.t.
for 1 h and concentrated in vacuo. The residue was purified by
flash column chromatography (5∼15% MeOH in dichloromethane)
to provide the titled compound 5 as a yellowish foam (55.2 mg,
93%). HPLC purity: 100% (220 and 254 nm); 1H NMR (CD3OD,
TFA salt): 0.96 (d, J ) 6.6 Hz, 3H), 1.04 (d, J ) 6.6 Hz, 3H),
1.38–1.52 (m, 1H), 1.68–1.80 (m, 1H), 1.80–1.92 (m, 1H), 2.77 (t,
J ) 7.5 Hz, 2H), 2.82–3.12 (m, 5H), 3.21–3.27 (m, 2H), 3.50–3.80
(m, 3H), 5.00–5.08 (m, 1H), 7.22–7.38 (m, 3H), 7.46 (d, J ) 1.8
Hz, 1H), 7.73 (dd, J ) 1.8, 8.3 Hz, 1H), 7.83 (d, J ) 1.8 Hz, 1H).
MS: 516 (MH+).
1H NMR (DMSO-d6, free base): 0.84 (d, J ) 6.0 Hz, 3H), 0.89 (d,
J ) 6.0 Hz, 3H), 1.04 (d, J ) 6.6 Hz, 3H), 1.43 (m, 1H), 1.58 (m,
1H), 2.26 (s, 3H), 2.45 (m, 1H), 2.62 (dd, J ) 5.7, 12.6 Hz, 1H),
2.80 (m, 3H), 2.90–3.50 (m, 8H), 4.56 (t, J ) 6.9 Hz, 1H), 6.40
(brs, 1H), 6.94 (brs, 1H), 7.07 (d, J ) 8.1 Hz, 1H), 7.24 (d, J )
8.1 Hz, 2H), 7.30 (s, 1H), 7.36 (d, J ) 8.1 Hz, 2H). MS: 442
(MH+). Anal. (C26H36ClN3O·1.3MeSO3H·2.5H2O) C, H, N, S.
(1S)-(2-{4-[(2R)-(2-Oxo-1-pyrrolidinyl)-3-(2,4-dichlorophenyl)
propionyl]-1-piperazinyl}-3-fluorophenyl)-3-methylbuty-
lamine (9). TFA (4.5 mL) was added to a solution of 4-{2-[(1S)-
((S)-tert-butanesulfinylamino)-3-methylbutyl]- 6-fluorophenyl}-1-
piperazinecarboxylic acid tert-butyl ester (6d, 1.02 g, 2.17 mmol)
in dichloromethane (18 mL), and the mixture was stirred at r.t. for
45 min. The reaction mixture was basified with saturated aqueous
NaHCO3 solution (100 mL) and extracted with EtOAc (2 × 100
mL). The organic layer was dried over Na2SO4 and concentrated
in vacuo to provide 4-{6-fluoro-2-[(1S)-((S)-tert-butylsulfinylamino)-
3-methylbutyl]phenyl}-1-piperazine as white foam, which was
dissolved in DMF/CH2Cl2 (1:3, 12 mL). NaHCO3 (0.365 g, 4.34
mmol), N-Boc-D-(2,4-Cl)Phe-OH (0.871 g, 2.61 mmol), HOBt
(0.352 g, 2.61 mmol), and EDC (0.50 g, 2.61 mmol) were
sequentially added to this solution. The reaction mixture was stirred
at r.t. overnight. The mixture was diluted with EtOAc (60 mL),
washed with 5% aqueous HCl (15 mL), saturated aqueous NaHCO3
(15 mL) and brine (15 mL), and was dried over Na2SO4. The
solution was concentrated in vacuo, and the residue was purified
by flash column chromatography (30∼60% EtOAc in hexanes) to
provide N-((1S)-[2-{4-[(2R)-(tert-butoxycarbonylamino)-3-(2,4-
dichlorophenyl)propionyl]-1-piperazinyl}-3-fluorophenyl]-3-meth-
ylbutyl)-(S)-tert-butanesulfinamide as a white solid (1.295 g, 87%).
N-(1S-[2-{4-[3-(4-Chlorophenyl)propionyl]-1-piperazinyl}-5-
(trifluoromethyl)phenyl]-3-methylbutyl)-2-aminoacetamide (11).
In a 4 dram vial, the crude (1S)-(2-{4-[3-(2,4-dichlorophenyl)pro-
pionyl]-1-piperazinyl}-5-(trifluoromethyl)phenyl)-3-methylbuty-
lamine (5, 0.70 g), N-Boc-glycine (0.19 g, 1.35 mmol), NaHCO3
(208 mg, 2.48 mmol), and HOBt (0.183 g, 1.35 mmol) were
combined and dissolved in dichloromethane (3 mL). The mixture
was capped and stirred at r.t. for 15 min. EDC (0.258 g, 1.35 mmol)
was added, and the mixture was stirred for 1 h. The mixture was
diluted with dichloromethane (2 mL) and washed with saturated
aqueous NaHCO3 (2 × 1 mL) and brine (1 mL). The organic layer
was dried over anhydrous Na2SO4, filtered, and concentrated by a
stream of nitrogen. The residue was dissolved in (1:1) TFA/CH2Cl2,
and the solution was stirred at r.t. for 30 min. The mixture was
concentrated in vacuo, and the residue was purified by column
chromatography on silica using 1:1 hexane/ethyl acetate as the
eluent to afford the titled compound as a light yellow solid (55
mg, 70% yield). HPLC purity: 98% (220 nm) and 97% (254 nm).
1H NMR (CD3OD, TFA salt): 0.97 (d, J ) 6.2 Hz, 3H), 0.97 (d,
J ) 6.2 Hz, 3H), 1.22–1.35 (m, 1H), 1.37–1.50 (m, 1H), 1.52–1.68
(m, 1H), 2.56–2.70 (m, 2H), 2.77 (t, J ) 7.5 Hz, 2H), 3.07 (t, J )
7.5 Hz, 2H), 3.15–3.27 (m, 2H), 3.50–3.90 (m, 6H), 5.65–5.72 (m,
1H), 7.24–7.38 (m, 3H), 7.46 (d, J ) 2.2 Hz, 1H), 7.53 (dd, J )
1.8, 8.3 Hz, 1H), 7.60 (d, J ) 1.8 Hz, 1H). MS: 573 (MH+). HRMS
(MH+) calcd for C27H34Cl2F3N4O2, 573.2011; found, 573.2009.
(1S)-[2-{4-[2-Methyl-3-(2-methoxy-4-chlorophenyl)propionyl]-
1-piperazinyl}-5-(trifluoromethyl)phenyl]-3-methylbutylamine
Mesylate (7). This compound was prepared using a procedure
similar to that for 5 from 6a and 2-methyl-3-(2-methoxyl-4-
chlorophenyl)propionic acid. White powder; HPLC purity: 100%
(220 and 254 nm). 1H NMR (DMSO-d6): 0.85 (d, J ) 6.6 Hz,
3H), 0.92 (d, J ) 6.3 Hz, 3H), 0.99 and 1.00 (d, J ) 6.6 Hz, 3H),
1.38 (m, 1H), 1.53 (m, 1H), 1.76 (m, 1H), 2.29 (s, 3H, MsOH),
2.30–3.20 (m, 3H), 3.30–3.70 (m, 8H), 3.80 and 3.83 (s, 3H), 4.79
(m, 1H), 6.92–7.12 (m, 3H), 7.35 (t, J ) 8.7 Hz, 1H), 7.72 (d, J )
8.1 Hz, 1H), 7.93 (s, 1H), 8.20 (brs, 3H). MS: 526 (MH+). Anal.
(C27H35ClF3N3O2 ·MeSO3H·H2O) C, H, N.
TFA (1 mL) was added to a solution of the above compound
(338 mg, 0.494 mmol) in dichloromethane (4 mL), and the mixture
was stirred at r.t. for 1 h. The reaction mixture was basified with
saturated aqueous NaHCO3 solution (30 mL) and extracted with
EtOAc (2 × 30 mL). The organic layer was dried over Na2SO4
and concentrated in vacuo to provide N-((1S)-{2-[4-[(2R)-amino-
3-(2,4-dichlorophenyl)propionyl]-1-piperazinyl}-3-fluorophenyl]-
3-methylbutyl)-(S)-tert-butanesulfinamide as a white foam, which
was dissolved in 1,2-dichloroethane (5 mL). This solution was
treated with acetic acid (118 µL, 1.98 mmol) and succinic
semialdehyde (374 µL 15 wt% solution in water, 0.592 mmol) and
stirred for 30 min NaBH(OAc)3 (220 mg, 0.987 mmol) was added,
and the mixture was stirred at r.t. for 24 h. LC-MS showed the
reductive amination was completed and the ratio of lactam product
(M.W. ) 653) to carboxylic acid product (M.W. ) 671) was 1:1.
The reaction was quenched with brine (10 mL), and the products
were extracted with EtOAc (40 mL), dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was dissolved in DMF/
CH2Cl2 (1:3, 10 mL), and NaHCO3 (0.083 g, 0.988 mmol), HOBt
(0.080 g, 0.592 mmol), and EDC (0.113 g, 0.592 mmol) were
sequentially added. The reaction mixture was stirred at r.t. overnight,
diluted with EtOAc (60 mL), washed with 5% aqueous HCl (10
mL), saturated aqueous NaHCO3 solution (10 mL), and brine (10
mL), and dried over Na2SO4. After filtration, the solution was
concentrated in vacuo, and the residue was purified by flash column
chromatography (40∼70% EtOAc in hexanes) to provide N-((1S)-
{2-[4-[(2R)-(2-oxo-1-pyrrolidinyl)-3-(2,4-dichlorophenyl)propionyl]-
1-piperazinyl}-3-fluorophenyl]-3-methylbutyl)-(S)-tert-butanesulfi-
namide as a white solid (0.239 g, 74%).
To a solution of the above compound (239 mg, 0.366 mmol) in
MeOH (4 mL) was added 2 equiv HCl (183 µL, 4 N HCl in
dioxane), and the mixture was stirred at r.t. for 30 min. The mixture
was concentrated in vacuo to give the crude product. A small sample
was purified using HPLC-MS. Light yellow foam; HPLC purity:
1
100% (220 nm). H NMR (CD3OD, free base): 0.91 (d, J ) 6.2
Hz, 3H), 0.95 (d, J ) 6.2 Hz, 3H), 1.22–1.36 (m, 1H), 1.45–1.63
(m, 2H), 2.00–2.15 (m, 2H), 2.22–2.37 (m, 2H), 2.72–2.99 (m, 5H),
3.12–3.30 (m, 5H), 3.40–3.53 (m, 1H), 3.60–3.77 (m, 1H),
3.90–4.00 (m, 1H), 4.42–4.63 (m, 2H), 5.41–5.55 (m, 1H),
6.90–7.00 (m, 1H), 7.18–7.24 (m, 2H), 7.24–7.35 (m, 2H), 7.45
(1S)-(2-{4-[(2R)-Methyl-3-(4-chlorophenyl)propionyl]-1-pip-
erazinyl}-5-methylphenyl)-3-methylbutylamine Mesylate (8).
This compound was prepared using a procedure similar to that for
5 from 6b and 2R-methyl-3-(4-chlorophenyl)propionic acid (16a).
White powder; HPLC purity: 97.9% (220 nm) and 97.7% (254 nm).

Antagonists of the Melanocortin-4 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6363
(dd, J ) 1.8, 10.1 Hz, 1H). MS: 549 (MH+). HRMS (MH+) calcd
for C28H36Cl2FN4O2, 549.2244; found, 549.2266.
evaporate residual HCl, and the remaining solvent was removed in
vacuo. The residue was dissolved in dichloromethane (250 mL)
and washed with saturated NaHCO3 (3 × 250 mL) and brine (250
mL). The organic layer was separated, dried over anhydrous
MgSO4, filtered, and concentrated in vacuo to give N-((1S)-(2-{4-
[(2R)-methyl-3-(4-chlorophenyl)propionyl]-1-piperazinyl}-5-chlo-
rophenyl)-3-methylbutylamine as an off-white foam in quantitative
yield. This compound (11.3 g, 24.32 mmol) was dissolved in
dichloromethane (122 mL) along with 3-dimethylaminopropionic
acid hydrochloride (3.74 g, 24.32 mmol) and triethylamine (3.42
mL, 24.32 mmol). The reaction mixture was stirred at r.t. for 5
min and then HOBt (3.28 g, 24.3 mmol) was added. After another
5 min, EDC (4.66 g, 24.32 mmol) was added to the reaction
mixture, and stirring was continued at r.t. for an additional 8 h.
The reaction mixture was washed with saturated NaHCO3 (3 ×
250 mL) and brine (250 mL). The organic layer was dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The residue
was purified by column chromatography on silica using 5%
methanol/dichloromethane as the eluent (Rf ) 0.3) to give the
desired product as a free base (10.35 g, 18.42 mmol, 76% yield).
HPLC purity: 98.4% (220 nm) and 97.6% (254 nm). 1H NMR
(CDCl3): 0.93 (d, J ) 6.3 Hz, 3H), 0.94 (d, J ) 6.3 Hz, 3H), 1.18
(d, J ) 5.7 Hz, 3H), 1.22–1.60 (m, 3H), 2.20–2.70 (m, 10H) 2.35
(s, 6H), 2.70–3.62 (m, 5H), 5.44 (m, 1H), 6.89 (d, J ) 8.7 Hz,
1H), 7.08 (d, J ) 2.4 Hz, 1H), 7.16 (m, 3H), 7.27 (d, J ) 8.4 Hz,
2H), 8.94 (d, J ) 8.4 Hz, 1H). MS: 561 (MH+).
N-(1-Aminoethyl)-N-[(1S)-(2-{4-[(2R)-(2-oxo-1-pyrrolidinyl)-
3-(2,4-dichlorophenyl)propionyl]-1-piperazinyl}-3-fluorophenyl)-
3-methylbutyl]amine Mesylate (10). A solution of (1S)-(2-{4-
[(2R)-(2-oxo-1-pyrrolidinyl)-3-(2,4-dichlorophenyl)propionyl]-1-
piperazinyl}-3-fluorophenyl)-3-methylbutylamine (9, 34.0 mg, 0.0615
mmol) in CH2Cl2 (1 mL) was treated with glacial acetic acid (7.4
µL) and tert-butyl-N-(2-oxoethyl)carbamate (14.7 mg, 0.0923
mmol).The mixture was stirred at r.t. for 30 min, and then
NaBH(OAc)3 (26.1 mg, 0.123 mmol) was added. The resulting
suspension was stirred at room temperature for 14 h. The reaction
mixture was diluted with CH2Cl2 (10 mL), transferred to a
separatory funnel, and washed with saturated NaHCO3 aqueous
solution (2 × 5 mL). The organic layers were dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel, eluting with
ethyl acetate. The product was obtained as white foam, which was
treated with 2 mL of TFA/CH2Cl2 (1:1) for 1 h. The excess of
TFA and solvent were removed in vacuo, and the crude product
was purified by flash column chromatography (5∼10% MeOH
/CH2Cl2) to provide the titled product as a white solid (20 mg, 54%).
The free base was converted to the mesylate salt by treatment with
1 equiv of methanesulfonic acid as a white solid; HPLC purity:
1
99% (220 nm) and 97.8% (254 nm). H NMR (DMSO-d6): 0.86
(d, J ) 6.6 Hz, 3H), 0.92 (d, J ) 6.6 Hz, 3H), 1.95 (m, 2H), 2.13
(m, 2H), 2.34 (s, 3H, MeSO3H), 2.60–3.45 (m, 16H), 3.60 (m, 2H),
4.38 (m, 1H), 4.43 (m, 1H), 5.25 (m, 1H), 5.31 (t, J ) 7.8 Hz,
1H), 7.30 (brs, 3H), 7.39 (m, 5H), 7.57 (m, 1H). MS: 592 (MH+).
Anal. (C30H40Cl2FN5O2 ·MeSO3H·3H2O) C, H, N.
N-((1S)-[2-{4-[(2R)-Methyl-3-(4-chlorophenyl)propionyl]-1-pip-
erazinyl}-5-chlorophenyl]-3-methylbutyl)-3-(dimethylamino)propi-
onamide (10.02 g, 17.84 mmol) was dissolved in dichloromethane
(90 mL). With constant stirring, HCl (2 M in ether, 13.38 mL, 26.76
mmol) was added in one portion. The reaction mixture was stirred
at r.t. for 5 min and then nitrogen gas was bubbled through the
reaction mixture for 10 min to evaporate excess HCl. The remaining
solvent was removed in vacuo. Ether (200 mL) was then added to
the solid residue, and the mixture was evaporated to dryness
(repeated three times). The residual solid powder was then filtered
off and washed with additional ether (3 × 200 mL). The solid was
dried in a vacuum oven at 40 °C for 4 h and then at r.t. for 2 days.
The product was recovered as a hydrochloride salt in 98% yield
(10.49 g, 17.55 mmol) as an off-whiter powder. [R]25D ) -22.65
N-(1S-[2-{4-[(2R)-Methyl-3-(4-chlorophenyl)propionyl]-1-piper-
azinyl}-5-chlorophenyl]-3-methylbutyl)-3-(dimethylamino)propi-
onamide Hydrochloride (12b). 4-{2-[(1S)-((S)-tert-Butanesulfinyl-
amino)-3-methylbutyl]-4-chlorophenyl}-1-piperazinecarboxylic acid
tert-butyl ester (6c, 21.39 g, 44.1 mmol) was dissolved in CH2Cl2
(440 mL) with magnetic stirring. Trifluoroacetic acid (88 mL) was
added slowly, and the resulting solution was stirred at r.t. for 1 h.
The reaction mixture was slowly poured into 0.5 M K2CO3 (500
mL). After all the bubbling had ceased, the mixture was placed in
a separatory funnel and the organic layer was separated. The
aqueous layer was extracted with CH2Cl2 (100 mL), and the
combined organics were washed with water and brine, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo to give 4-{2-
[(1S)-((S)-tert-butylsulfinylamino)-3-methylbutyl]-4-chlorophenyl}-
1-piperazine as a pale yellow foam (18 g), which was used in the
next step without further purification.
(2R)-methyl-3-(4-chlorophenyl)propionic acid (16a, 10.1 g, 50.9
mmol), diisopropylethylamine (17.6 mL, 101 mmol), and HOBt
(10.27 g, 76.1 mmol) were sequentially added to a stirring solution
of the above compound (16.98 g, 44.1 mmol) in CH2Cl2 (220 mL)
under N2. The resulting mixture was stirred at r.t. for 0.5 h. Then
EDC (14.58 g, 76.1 mmol) was added portionwise, and the resulting
solution was stirred at r.t. overnight. The reaction mixture was
placed in a separatory funnel and washed with 0.1 N HCl (200
mL), water, saturated aqueous NaHCO3, and brine. The organics
were dried over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel,
eluting with a 3:1 v/v mixture of hexanes and ethyl acetate, which
was gradually increased to a 1:2 ratio, to give N-((1S)-{2-[4-[(2R)-
methyl-3-(2,4-dichlorophenyl)propionyl]-1-piperazinyl}-5-chlo-
rophenyl]-3-methylbutyl)-(S)-tert-butanesulfinamide (13.80 g, 55%
over 2 steps). Rf ) 0.36 (1:1 v/v hexanes/EtOAc). 1H NMR
(CDCl3): 0.87 (d, J ) 6.6 Hz, 3H), 0.92 (d, J ) 6.6 Hz, 3H), 1.19
(d, J ) 5.7 Hz, 3H), 1.20 (s, 9H), 1.54–1.39 (m, 2H), 1.75–1.70
(m, 1H), 2.71–2.63 (m, 2H), 2.89–2.82 (m, 1H), 3.01–2.95 (m, 2H),
4.00–3.00 (br, 6H), 4.85 (br, 1H), 6.95 (br, 1H), 7.38–7.13 (m,
7H). MS: 566 (MH+).
1
(1.04, MeOH). H NMR (CDCl3): 0.86 (d, J ) 5.1 Hz, 6H), 1.02
(d, J ) 6.6 Hz, 3H), 1.22 (m, 1H), 1.48 (m, 2H), 2.28 (m, 1H),
2.60 (m, 2H), 2.70 (s, 3H), 2.71 (s, 3H), 2.79 (m, 2H), 3.02 (m,
2H), 3.20 (m, 2H), 3.40 (m, 2H), 4.15 (brs, 4H), 5.33 (m, 1H),
6.97 (d, J ) 8.1 Hz, 1H), 7.22 (m, 3H), 7.34 (m, 3H), 8.68 (d, J )
8.1Hz,1H),10.2(brs,1H).MS:561.3(MH+).Anal.(C30H42Cl2N4O2·HCl·2/
3H2O) C, H, N.
N-(1S-[2-{4-[(2R)-Methyl-3-(4-chlorophenyl)propionyl]-1-pip-
erazinyl}-5-methylphenyl]-3-methylbutyl)-3-(dimethylamino)pro-
panamide Mesylate (12a). This compound was synthesized using
a procedure similar to that for 12b from 6b. HPLC purity: 100%
(220 nm) and 95.1% (254 nm). 1H NMR (DMSO-d6): 0.86 (d, J )
6.6 Hz, 6H), 1.04 (d, J ) 6.6 Hz, 3H), 1.22 (m, 1H), 1.46 (m, 2H),
2.22 (s, 3H), 2.30 (s, 3H, MeSO3H), 2.40 (m, 1H), 2.56 (m, 4H),
2.60 (s, 6H), 2.80 (m, 1H), 2.80–3.36 (m, 10H), 5.37 (m, 1H), 6.85
(brs, 1H), 6.98 (dd, J ) 1.8, 8.4 Hz, 1H), 7.08 (d, J ) 1.8 Hz, 1H),
7.24 (d, J ) 8.1 Hz, 2H), 7.35 (s, J ) 8.4 Hz, 2H), 8.44 (d, J )
8.1 Hz, 1H). MS: 541 (MH+). Anal. (C31H45ClN4O2 ·MeSO3H·1/
2H2O) C, H, N, S.
N-[(1S)-1-[2-[4-[(2R)-3-(2,4-Dichlorophenyl)-2-(2-oxo-1-pyr-
rolidinyl)propionyl]-1-piperazinyl]-5-methylphenyl]-3-methyl-
butyl]-3-(dimethylamino)propanamide Mesylate (13). This com-
pound was synthesized from 6b using a procedure similar to that
for 9, followed by a procedure similar to that for 12b. White solid;
HPLC purity: 98.7% (220 nm) and 96.6% (254 nm). 1H NMR
(DMSO-d6): 0.88 (d, J ) 6.6 Hz, 6H), 1.04 (d, J ) 6.6 Hz, 3H),
1.27 (m, 1H), 1.46 (m, 2H), 1.91 (m, 1H), 2.09 (m, 1H), 2.23 (s,
3H), 2.28 (s, 3H, MeSO3H), 2.35–2.60 (m, 9H), 2.63 (s, 6H), 2.80
(m, 1H), 2.88–3.50 (m, 6H), 5.37 (m, 1H), 6.86 (brs, 1H), 6.98
(dd, J ) 1.8, 8.4 Hz, 1H), 7.08 (d, J ) 1.8 Hz, 1H), 7.24 (d, J )
The above compound (13.78 g, 24.3 mmol) was dissolved in
MeOH (243 mL), and HCl (2 M in ether, 15.81 mL, 31.61 mmol)
was added. The reaction mixture was stirred at r.t. for 45 min.
Nitrogen gas was then bubbled through the reaction mixture to

6364 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25
Tran et al.
8.1 Hz, 2H), 7.35 (d, J ) 8.4 Hz, 2H), 8.44 (d, J ) 8.1 Hz, 1H).
MS: 644 (MH+). Anal. (C34H47Cl2N5O3 ·MeSO3H) C, H, N, S.
as the eluent (Rf ) 0.3). The product 2-(4-Boc-1-piperazinyl)-3-
formylpyridine was obtained as a yellow solid (9.8 g, 33.5 mmol,
67% yield).
4S-Benzyl-3-[3-(4-chlorophenyl)-2R-methylpropionyl]oxazo-
lidin-2-one (15a). (S)-4-benzyl-3-propionyloxazolidin-2-one (14,
46.7g, 200 mmol) was dissolved in THF (870 mL) under an inert
atmosphere (N2). This was then cooled to -70 °C (dry ice/acetone)
and treated with sodium hexamethyldisilazide (110 mL of a 2.0 M
solution in THF, 220 mmol) in a dropwise fashion (addition lasted
for ∼45 min). The resulting mixture was stirred at -70 °C for 1 h.
A solution of 4-chlorobenzyl bromide (53.4 g, 260 mmol) in THF
(160 mL) was then added dropwise over 30 min. The resulting
mixture was stirred at -70 °C for 6 h and then allowed to warm to
r.t. overnight. The reaction was carefully quenched with water (100
mL), and the solvent was removed in vacuo. The resulting slurry
was suspended in water (200 mL) and filtered. The solid was rinsed
with EtOAc and air-dried to give the desired product (36.67 g, 102.6
mmol, 51%). A second crop of product was obtained from the
filtrates after the organic layer was separated, washed with brine,
dried over MgSO4, and concentrated in vacuo. The resulting brown
solid was suspended in MeOH and filtered to give 17.22 g (48.2
mmol, 24%) of the desired product. Total yield, 53.89 g (75%).
Only one diastereomer was observed by 1H NMR, and stereochem-
istry was confirmed by single crystal X-ray analysis. 1H NMR
(CDCl3): 1.18 (d, J ) 6.3 Hz, 3H), 2.66–2.56 (m, 2H), 3.16–3.07
(m, 2H), 4.22–4.02 (m, 3H), 4.71–4.63 (m, 1H), 7.08–7.05 (m, 2H),
7.32–7.21 (m, 7H).
2R-Methyl-3-(4-chlorophenyl)propionic Acid (16a). 4S-Ben-
zyl-3-[3-(4-chlorophenyl)-2R -methylpropionyl]oxazolidin-2-one
(15a, 53.89 g, 150.7 mmol) was dissolved in a 4:1 v/v THF/H2O
mixture (750 mL) and cooled to 0 °C (ice/water bath). Hydrogen
peroxide 50% (60 mL) was added slowly, followed by a solution
of lithium hydroxide monohydrate (11.08 g, 263.7 mmol) in H2O
(380 mL). The resulting mixture was stirred at 0 °C for 1.5 h.
Na2SO3 ·7H2O (155.70 g, 618.0 mmol) dissolved in H2O (250 mL)
was added at 0 °C, and the resulting mixture was allowed to slowly
reach r.t. The volatiles were removed in vacuo, and the aqueous
residue was extracted with CH2Cl2 (2 × 300 mL). The aqueous
layer was separated, made acidic with 2.0 N HCl, and then extracted
with EtOAc (2 × 250 mL). The organics were washed with brine,
dried over MgSO4, and concentrated in vacuo to give the titled
compound as an oil that solidified upon standing (29.80 g, 150.1
mmol, 99%). 1H NMR (CDCl3): 1.18 (d, J ) 6.3 Hz, 3H),
2.80–2.62 (m, 2H), 3.02 (dd, J ) 6.0, 12.6 Hz, 1H), 7.12 (d, J )
8.7 Hz, 2H), 7.26 (d, J ) 8.7 Hz, 2H). [R]25D ) -27.930 (c 8.685
mg/cc, MeOH).40
2-(4-Boc-1-piperazinyl)-3-formylpyridine (3 g, 10.3 mmol) was
dissolved in THF (51 mL) along with (S)-tert-butanesulfinamide
(1.4 g, 11.3 mmol) and titanium(IV) ethoxide (8.6 mL, 41.2 mmol).
The reaction mixture was allowed to stir at r.t. for 8 h and then
brine (20 mL) was added. The reaction mixture was filtered, and
the solid was washed with ethyl acetate (3 × 75 mL). The organic
layer was collected, dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo to give the titled compound as a yellow solid
in quantitative yield without further purification (4.1 g, 10.3 mmol).
The above intermediate (4.1 g, 10.3 mmol) in THF (30 mL) was
cooled to –40 °C, and Me3Al (15.45 mL, 30.9 mmol) was added.
The reaction mixture was allowed to stir at -40 °C under nitrogen
atmosphere for 20 min and was then cooled to -78 °C. To the
reaction mixture, isobutyl lithium (12.9 mL, 20.6 mmol, 1.6 M in
heptane) was added slowly. After the addition was complete, the
reaction was warmed to r.t. and carefully quenched with water.
The mixture was then concentrated in vacuo and diluted with
dichloromethane (150 mL). The organic layer was then washed
with saturated NaHCO3 solution (2 × 100 mL) and brine (100 mL),
dried over anhydrous MgSO4, filtered, and concentrated in vacuo.
The residue was purified by column chromatography on silica using
75% ethyl acetate/hexanes as the eluent (Rf ) 0.3). The titled
product was obtained as a yellow solid (2.8 g, 6.15 mmol, 60%).
1H NMR (CDCl3): 0.89 (d, J ) 6.3 Hz, 3H), 0.94 (d, J ) 6.0 Hz,
3H), 1.21 (s, 9H), 1.47 (s, 9H), 1.24–1.30 (m, 2H), 1.5–1.58 (m,
1H), 2.80–3.40 (m, 4H), 3.40–3.70 (m, 4H), 4.69 (dd, J ) 6.9 Hz,
1H), 7.06 (dd, J ) 4.8 Hz, 1H), 7.61 (dd, J ) 1.8 Hz, 1H), 8.29
(dd, J ) 1.8 Hz, 1H).
4-{3-[(1R)-((S)-tert-Butanesulfinylamino)-3-methylbutyl]-6-
methyl-2-pyridinyl}-1-piperazinecarboxylic Acid tert-Butyl Es-
ter (R-19b). This compound was synthesized from 2-bromo-6-
methylpyridine and S-tert-butanesulfinamide using a procedure
1
similar to that for 19a. H NMR (CDCl3): 0.87 (d. J ) 6.0 Hz,
3H), 0.94 (d. J ) 6.0 Hz, 3H), 1.12 (s. 9H), 1.46 (s. 9H), 1.48–1.58
(m, 2H), 1.70–1.78 (m, 1H), 2.43 (s, 3H), 2.8–3.05 (m, 4H),
3.40–3.78 (m, 4H), 4.64 (dd, J ) 6.9 Hz, 1H), 6.89 (d, J ) 7.8 Hz,
1H), 7.46 (d, J ) 7.8 Hz, 1H). A small sample was crystallized in
ether/hexanes to give single crystals for X-ray determination.
1-{3-[(1R)-Amino-3-methylbutyl]-2-pyridinyl}-4-[3-(2,4-dichlo-
rophenyl)-propionyl]piperazine Trifluoroacetate (R-20a). 4-{3-
[(1R)-((S)-tert-Butanesulfinylamino)-3-methylbutyl]-2-pyridinyl}-
1-piperazinecarboxylic acid tert-butyl ester (R-19a, 452.6 mg, 1
mmol) was allowed to stir at r.t. for 1.5 h in a 20% TFA/CH2Cl2
mixture (20 mL). The reaction was quenched with saturated
NaHCO3 solution (5 mL). The organic layer was washed with
saturated NaHCO3 solution (2 × 10 mL) and brine(10 mL), dried
over anhydrous MgSO4, filtered, and concentrated in vacuo. The
deprotected intermediate was recovered in quantitative yield. A
small portion of this piperazine intermediate (35.2 mg, 0.1 mmol)
was dissolved in dichloromethane (0.5 mL) along with HOBt (13.5
mg, 0.1 mmol), NaHCO3 (8.4 mg, 0.1 mmol), and 3-(2,4-
dichlorophenyl)propanic acid (21.9 mg, 0.1 mmol). The reaction
mixture was allowed to stir at r.t. for 10 min and then EDC (19.2
mg, 0.1 mmol) was added. The reaction was then stirred for an
additional 8 h followed by quenching with saturated NaHCO3
solution. The organic layer was separated, washed with brine (2
mL), dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The resulting residue was dissolved in MeOH (2 mL), and
0.2 M HCl/ether (1 mL) was added. The reaction was stirred at r.t.
for 1 h and then solvent was removed under a stream of nitrogen.
The crude product was purified by prep HPLC to yield the desired
product as the TFA salt (15 mg, 0.026 mmol, 26% yield). Light
yellow foam; HPLC purity: 98% (220 nm) and 95% (254 nm). 1H
NMR (CD3OD): 0.95 (d, J ) 6.6 Hz, 3H), 1.03 (d, J ) 6.6 Hz,
3H), 1.37–1.53 (m, 1H), 1.69–1.82 (m, 1H), 1.82–1.93 (m, 1H),
2.76 (t, J ) 7.7 Hz, 2H), 2.85–2.96 (m, 4H), 3.00–3.13 (m, 4H),
3.55–3.64 (m, 1H), 3.64–3.84 (m, 3H), 4.83 (t, J ) 7.5 Hz, 1H),
7.24–7.38 (m, 3H), 7.44 (d, J ) 4.1 Hz, 1H), 7.90 (dd, J ) 1.8,
4-{3-[(1R)-((S)-tert-Butanesulfinylamino)-3-methylbutyl]-2-py-
ridinyl}-1-piperazinecarboxylic Acid tert-Butyl Ester (R-19a).
Lithium diisopropylamide (131 mL, 262 mmol, 2 M in THF) was
added to a stirring solution of 2-bromopyridine (25 mL, 262 mmol)
in THF (208 mL) at –78 °C under nitrogen. The reaction mixture
was stirred at -78 °C for 2 h and then a solution of DMF (20.3
mL, 262 mmol) in THF (104 mL) was added. After the addition,
the reaction mixture was allowed to warm to r.t. and was neutralized
by adding to a saturated solution of ammonium chloride. The crude
product was extracted with ethyl acetate (3 × 200 mL), the organic
layers were combined, dried over anhydrous Na2SO4, and filtered,
and solvent was removed in vacuo. The residue was purified by
column chromatography on silica using 15% ethyl acetate/hexanes
as the eluent (Rf ) 0.3). The product 2-bromo-3-formylpyridine
17a was obtained as yellow oil (9.4 g, 50.5 mmol, 19% yield).
2-Bromo-3-formylpyridine (17a, 9.4 g, 50.5 mmol) was dissolved
in DMF (100 mL) along with diisopropylethylamine (8.8 mL, 50.5
mmol) and 1-Boc-piperazine (9.4 g, 50.5 mmol) in a reaction flask.
The reaction mixture was heated at 100 °C for 8 h, then cooled to
r.t., and quenched with saturated NaHCO3 (150 mL). The product
was extracted with ethyl acetate (3 × 100 mL), the organic layers
were combined, dried over anhydrous Na2SO4, and filtered, and
the solvent was removed in vacuo. The residue was purified by
column chromatography on silica using 25% ethyl acetate/hexanes

Antagonists of the Melanocortin-4 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6365
7.7 Hz, 1H), 8.39 (dd, J ) 1.8, 4.8 Hz, 1H). MS: 449 (MH+).
Hz, 1H), 7.19 (d, J ) 8.5 Hz, 1H), 7.33 (m, 2H), 8.60 (brs, 1H).
MS: 576 (MH+). Anal. (C30H43Cl2N3O2 ·H2O) C, H, N.
HRMS (MH+) calcd for C23H31Cl2N4O, 449.1875; found, 449.1892.
1-{3-[(1S)-Amino-3-methylbutyl]-2-pyridinyl}-4-[3-(2,4-dichlo-
rophenyl)-propionyl]piperazine Trifluoroacetate (S-20a). This
compound was synthesized using the same method for R-20a from
S-tert-butanesulfinamide. Light yellow foam, HPLC purity: 92%
(220 nm) and 96% (254 nm). 1H NMR (CD3OD): 0.95 (d, J ) 6.6
Hz, 3H), 1.03 (d, J ) 6.6 Hz, 3H), 1.37–1.53 (m, 1H), 1.69–1.82
(m, 1H), 1.82–1.93 (m, 1H), 2.76 (t, J ) 7.7 Hz, 2H), 2.85–2.96
(m, 4H), 3.00–3.13 (m, 4H), 3.55–3.64 (m, 1H), 3.64–3.84 (m, 3H),
4.83 (t, J ) 7.5 Hz, 1H), 4.83 (t, J ) 7.5 Hz, 1H), 7.24–7.38 (m,
3H), 7.44 (d, J ) 4.1 Hz, 1H), 7.90 (dd, J ) 1.8, 7.7 Hz, 1H), 8.39
(dd, J ) 1.8, 4.8 Hz, 1H). MS: 449 (MH+).
1-{3-[(1R)-1-Amino-3-methylbutyl]-2-pyridinyl}-4-[2-methyl-
3-(2,4-dichlorophenyl)propionyl]piperazine Trifluoroacetate (R-
20b). This compound was synthesized from R-19a and 2-methyl-
3-(2,4-dichlorophenyl)propionic acid using a procedure similar to
that for R-20a. Light yellow foam; HPLC purity: 97% (220 and
254 nm). 1H NMR (CD3OD): 0.94 (d, J ) 6.6 Hz, 3H), 1.01 (d, J
) 6.6 Hz, 3H), 1.19 and 1.20 (d, J ) 6.6 Hz, 3H), 1.34–1.50 (m,
1H), 1.67–1.91 (m, 2H), 2.40–2.54 (m, 1H), 2.60–2.76 (m, 1H),
2.80–3.08 (m, 5H), 3.32–3.46 (m, 2H), 3.52–3.88 (m, 4H), 4.81 (t,
J ) 7.5 Hz, 1H), 7.24–7.30 (m, 3H), 7.42–7.47 (m, 1H), 7.83–7.89
(m, 1H), 8.39 (dd, J ) 1.8, 4.8 Hz, 1H). MS: 463 (MH+).
Supporting Information Available: Synthetic procedure for the
preparation of compound 6c, analytic data of key compounds, and
description of pharmacokinetic studies and protocol for the murine
cachexia model. This material is available free of charge via the
Internet at http:/pubs.acs.org.
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1-{3-[(1R)-1-Amino-3-methylbutyl]-6-methyl-2-pyridinyl}-4-
[2R-methyl-3-(2,4-dichlorophenyl)propionyl]piperazine Trifluo-
roacetate (R-21). This compound was synthesized from R-19b and
2R-methyl-3-(2,4-dichlorophenyl)propionic acid using a procedure
similar to that for R-20a. Light yellow foam; HPLC purity: 100%
1
(220 and 254 nm). H NMR (CD3OD): 0.92 (d, J ) 6.6 Hz, 3H),
1.00 (d, J ) 6.6 Hz, 3H), 1.21 (d, J ) 6.6 Hz, 3H), 1.32–1.47 (m,
1H), 1.65–1.88 (m, 2H), 2.39–2.50 (m, 1H), 2.47 (s, 3H), 2.78–3.04
(m, 6H), 3.32–3.45 (m, 2H), 3.54–3.80 (m, 4H), 4.75 (t, J ) 7.5
Hz, 1H), 7.12 (d, J ) 7.9 Hz, 1H), 7.24–7.28 (m, 2H), 7.43 (d, J
) 1.3 Hz, 1H), 7.71 (d, J ) 7.9 Hz, 1H). MS: 477 (MH+). HRMS
(MH+) calcd for C25H34Cl2N4O, 477.2188; found, 477.2165.
N-((1R)-[2-{4-[2R-Methyl-3-(2,4-dichlorophenyl)propionyl]-
1-piperazinyl}-3-pyridinyl]-3-methylbutyl)-2-aminoacetamide Me-
sylate Trifluoroacetate (R-22). This compound was synthesized
from R-20b using a procedure similar to that for 11. White solid;
HPLC purity: 99% (220 nm) and 98% (254 nm). 1H NMR
(CD3OD): 0.94 (d, J ) 6.1 Hz, 3H), 0.96 (d, J ) 6.1 Hz, 3H), 1.18
and 1.20 (d, J ) 6.6 Hz, 3H), 1.34–1.50 (m, 1H), 1.50–1.66 (m,
2H), 2.42–2.54 (m, 1H), 2.60–2.70 (m, 1H), 2.74–3.14 (m, 5H),
3.20–3.42 (m, 2H), 3.52–3.86 (m, 7H), 5.36–5.45 (m, 1H),
7.12–7.19 (m, 1H), 7.23–7.26 (m, 2H), 7.42 and 7.46 (d, J ) 1.8
Hz, 1H), 7.72–7.78 (m, 1H), 8.17–8.22 (m, 1H). MS: 520 (MH+).
(15) Li, S. J.; Varga, K.; Archer, P.; Hruby, V. J.; Sharma, S. D.
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Reversal of cancer anorexia by blockade of central melanocortin
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Differential role of melanocortin receptor subtypes in cachexia.
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infusion of the melanocortin receptor antagonist agouti-related peptide
(AgRP(83–132)) prevents cachexia-related symptoms induced by
radiation and colon-26 tumors in mice. Peptides 2007, 28, 636–642.
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therapeutics in the treatment of cachexia. Curr. Top. Med. Chem.
2007, 7, 1131–1136.
N-((1S)-[2-{4-[2R-Methyl-3-(2,4-dichlorophenyl)propionyl]-1-
piperazinyl}-3-pyridinyl]-3-methylbutyl)-2-aminoacetamide Trif-
luoroacetate (S-22). This compound was synthesized from S-20
using a procedure similar to that for 11. Light yellow foam; HPLC
purity: 100% (220 and 254 nm). 1H NMR (CD3OD): 0.94 (d, J )
6.1 Hz, 3H), 0.96 (d, J ) 6.1 Hz, 3H), 1.18 and 1.20 (d, J ) 6.6
Hz, 3H), 1.34–1.50 (m, 1H), 1.50–1.66 (m, 2H), 2.42–2.54 (m, 1H),
2.60–2.70 (m, 1H), 2.74–3.14 (m, 5H), 3.20–3.42 (m, 2H),
3.52–3.86 (m, 7H), 5.36–5.45 (m, 1H), 7.12–7.19 (m, 1H),
7.23–7.26 (m, 2H), 7.42 and 7.46 (d, J ) 1.8 Hz, 1H), 7.72–7.78
(m, 1H), 8.17–8.22 (m, 1H). MS: 520 (MH+). HRMS (MH+) calcd
for C26H36Cl2N5O2, 520.2246; found, 520.2236.
N-((1R)-[2-{4-[2R-Methyl-3-(2,4-dichlorophenyl)propionyl]-
1-piperazinyl}-6-methyl-3-pyridinyl]-3-methylbutyl)-3-(dimethy-
lamino)propionamide (R-23). This compound was synthesized
from R-21 using a procedure similar to that for 12b. White solid;
HPLC purity: 96.1% (220 nm) and 96.5 (254 nm). 1H NMR
(CDCl3): 0.91 (d, J ) 6.5 Hz, 3H), 0.93 (d, J ) 6.5 Hz, 3H), 1.17
(d, J ) 7.0 Hz, 3H), 1.40 (m, 1H), 1.47 (m, 2H), 2.37 (s, 6H), 2.42
(s, 3H), 2.43 (m, 2H), 2.52 (m, 1H), 2.67 (m, 2H), 2.83 (m, 2H),
3.03 (dd, J ) 8.0, 13.0 Hz, 1H), 3.18 (dd, J ) 7.0, 14.5 Hz, 1H),
3.22 (m, 1H), 3.35 (m, 1H), 3.42 (m, 1H), 3.58 (m, 2H), 3.88 (m,
1H), 5.28 (m, 1H), 6.83 (d, J ) 7.5 Hz, 1H), 7.17 (dd, J ) 2.0, 8.5
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