Novel piperazines: Potent melanocortin-4 receptor antagonists with anxiolytic-like activity

Article abstract of DOI:10.1016/j.bmc.2007.01.019

In the present study, we found that a novel piperazine compound, 11a, showed a moderate affinity (IC₅₀ = 333 nM) for the MC4 receptor. We developed the new type of piperazine compounds and found that mono-piperazine 11b exhibited a high-affinity (IC₅₀ = 40.3 nM) for the MC4 receptor. We also found that a series of biphenyl analogues exhibited a high-affinity for the receptor, and in particular, compound 11j exhibited the highest affinity for the MC4 receptor with an IC₅₀ value of 14.5 nM. Furthermore, some of these compounds, when administered orally, significantly reversed the stress-induced anxiety-like behavior in rats. In this paper, we report the synthesis, structure-activity relationships, and oral activity of the novel mono-piperazines as MC4 receptor antagonists.

Full text of DOI:10.1016/j.bmc.2007.01.019

Bioorganic & Medicinal Chemistry 15 (2007) 2375–2385
Novel piperazines: Potent melanocortin-4 receptor
antagonists with anxiolytic-like activity
Dai Nozawa,* Taketoshi Okubo, Takaaki Ishii, Kazuaki Takamori,
Shigeyuki Chaki, Shigeru Okuyama and Atsuro Nakazato
Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd,
1-403 Yoshino-cho, Kita-ku, Saitama, Saitama 331-9530, Japan
Received 22 December 2006; revised 12 January 2007; accepted 13 January 2007
Available online 18 January 2007
Abstract—In the present study, we found that a novel piperazine compound, 11a, showed a moderate affinity (IC50 = 333 nM) for
the MC4 receptor. We developed the new type of piperazine compounds and found that mono-piperazine 11b exhibited a high-
affinity (IC50 = 40.3 nM) for the MC4 receptor. We also found that a series of biphenyl analogues exhibited a high-affinity for
the receptor, and in particular, compound 11j exhibited the highest affinity for the MC4 receptor with an IC50 value of 14.5 nM.
Furthermore, some of these compounds, when administered orally, significantly reversed the stress-induced anxiety-like behavior
in rats. In this paper, we report the synthesis, structure-activity relationships, and oral activity of the novel mono-piperazines as
MC4 receptor antagonists.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
the adrenocorticotropic hormone (ACTH), all of which
are derived from proopiomelanocortin (POMC).4 Two
endogenous antagonistic proteins, namely, agouti and
agouti-related protein (AGRP), have also been identi-
fied.5,6 All of the melanocortin peptides possess a core
His-Phe-Arg-Trp (HFRW) terapeptide sequence, which
has been shown to be essential for activation of the mel-
anocortin receptors.7,8
Since five subtypes of melanocortin receptors (MC1–
MC5 receptors) belonging to a family of seven-trans-
membrane G-protein-coupled receptors were first cloned
and characterized, numerous studies have been conduct-
ed to investigate the distribution and functions of
these receptors.1–3 The MC1 receptor is prominently
expressed in the skin and melanoma cells, and plays a
major role in regulating skin pigmentation. The MC2
receptor is prominently expressed in the adrenal cortex
and is involved in steroidogenesis. The MC3 receptor
is widely expressed in the central nervous system as well
as placenta and plays a role in fat metabolism and
energy homeostasis together with the MC4 receptor,
which is primarily in the brain. The MC5 receptor is
expressed in various peripheral tissues and plays a role
in exocrine gland function.1–3
Among the MC1–MC5 receptors, the MC4 receptor
plays an important role in the regulation of feeding
behavior and energy homeostasis.9–15 It has also been
reported to be involved in the regulation of sexual func-
tions,16,17 and to protect against tumor-induced decrease
of body weight.18,19 In addition, this receptor has been a
focus of interest in terms of its relationship to stress and
regulation of emotional behavior such as depression and
anxiety.20–26 These findings untill date indicate that the
MC4 receptor could be a promising target for the devel-
opment of drugs for the above-mentioned conditions,
and numerous ligands of the MC4 receptor, both peptidic
and nonpeptidic, have been developed. Although several
peptidic MC4 receptor agonists and antagonists designed
based on the core His-Phe-Arg-Trp terapeptide sequence
have been reported,27 nonpeptidic ligands of the MC4
receptor have been sought since peptidic ligands tend to
have properties unsuitable for oral administration, such
as poor absorption from the gastrointestinal tract.
The melanocortin peptides, the natural ligands for the
melanocortin receptors, consist of the melanocyte-stim-
ulating hormones (a-MSH, b-MSH, and c-MSH) and
Keywords: Melanocortin-4 receptor antagonist; Piperazine; Anxiety;
Depression.
*
Corresponding author. Tel.: +81 48 663 1111; fax: +81 48 652
7254; e-mail: dai.nozawa@po.rd.taisho.co.jp
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.019

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D. Nozawa et al. / Bioorg. Med. Chem. 15 (2007) 2375–2385
Numerous studies focused on the development of non-
peptidic agonists28 and antagonists29–36 for the MC4
receptor have been reported (Fig. 1). Compound 1,
having a 1,2-bispiperazinylethane core, inhibited the
binding of AGRP (IC50 = 52 nM), and also Nle4-D-
Phe7-a-MSH (NDP-MSH) (IC50 = 217 nM), to the
MC4 receptor.29 Compound 2, having a succinamide
core, exhibited a high-affinity for the MC4 receptor
(IC50 = 1.4 nM) although it showed no effect of activat-
ing the receptor in a functional assay.30 Compound 3
(ML00253764) exhibited a moderate affinity (Ki =
160 nM) and antagonist activity (IC50 = 103 nM) for
the receptor; subcutaneous administration of this com-
pound in mice protected the animals against tumor-in-
duced weight loss,18 suggesting its potential clinical
usefulness for the treatment of cachexia.18,19 Compound
4, having an imidazole ring instead of the amidine moi-
ety of compound 3, antagonized the MC4 receptor with
a functional IC50 value of 51 nM.32 Compound 5, de-
signed by using information derived from the SAR of
the MC4 agonists, and mutagenesis results of the MC4
receptor structure and peptidic ligands, exhibited a high
binding affinity (Ki = 1.8 nM) for the MC4 receptor.33
Compound 6 also exhibited a high-affinity for the
MC4 receptor (Ki = 3.2 nM), with a 240-fold selectivity
for this receptor over the MC3 receptor; intracerebroven-
tricular administration of this compound potently stimu-
lated food intake in satiated mice.31 Compound 7, having
a guanidine part, exhibited a high-affinity for the MC4
receptor (Ki = 0.58 nM).34 Furthermore, 3-arylpropio-
nylpiperazine, compound 8, exhibited a good binding
affinity (Ki = 13 nM) for the MC4 receptor and also good
oral bioavailability (F = 26.1%).35
We previously demonstrated that the bis-piperazine
compound, MCL0129 (Fig. 1), exhibited a high-affinity
(IC50 = 8.13 nM) for the MC4 receptor, and acts as an
antagonist at the receptor.23 Studies have also shown
that this compound exhibits antidepressant and anxio-
lytic-like activities in various rodent models.23 These
findings suggest that blockade of the MC4 receptor
may be a useful approach for treating subjects with
depressive disorders and anxiety. This hypothesis is sup-
ported by the results of studies showing that other MC4
receptor antagonists also exhibit antidepressant and
anxiolytic effects in various rodent models of depression
and anxiety.24,25 In this paper, we report a new class of
MC4 receptor antagonists, namely, the mono-piperazine
compounds, with reduced basicity relative to the bis-pip-
erazines. The synthesis, structure–activity relationships,
and anxiolytic-like activities of the novel mono-pipera-
zine compounds are described in this study.
Cl
O
F
O
N
O
N
O
N
N
2. Chemistry
N
O
N
N
N
N
S
Compounds 11a–j were prepared via two synthetic
routes (Methods A and B) from 4-fluorophenylacetic
acid 12, which was the common starting material for
both (Schemes 1 and 2). Compounds 11a and 11b were
synthesized from compound 15 (Scheme 1). Reaction of
12 with LDA and N-Boc-piperizin-4-one 9 yielded 13.
Dehydration of 13 with H2SO4 in CHCl3, followed by
protection with a Boc group, yielded 14. After hydroge-
nation of 14 with Pd(OH)2 as the catalyst, condensation
with N-(Ar1–(CH2)4)-piperazine 10 in the presence of
HOBt and EDC yielded 16. After removal of the Boc
group of 16 under acidic conditions, treatment with
alkyl iodide in the presence of K2CO3 yielded 17. Com-
pounds 11a and 11b were obtained by reduction of 17
with LiAlH4 (Method A).
OO
1
2
Cl
Cl
O
Br
O
O
Br
O
N
NH2
N
H
N
NH
N
N
F
N
S
N
H
3
4
5
Cl
Cl
Cl
O
O
NH
F
O
O
N
H
N
H
N
N
O
Cl
Compounds 11c–j were synthesized from compound 15
by another synthetic pathway (Scheme 2). Compound
20 was obtained by coupling 15 with N-CBZ piperazine
18 in the presence of HOBt and EDC. After removal of
the Boc group of 20 under acidic conditions, reaction
with iso-propyl iodide in the presence of K2CO3 yielded
21. After removal of the CBZ group of 21 with Pd(OH)2
as the catalyst, condensation with Ar1–(CH2)2– CO2H
19 in the presence of HOBt and EDC yielded 22. Com-
pounds 11c–j were obtained by reduction of 22 with
LiAlH4 (Method B).
N
H
N
N
N
O
O
O
N
H
NH2
6
CF3
7
8
F
MeO
N
N
N
N
The syntheses of 27 and 30 are shown in Scheme 3.
Condensation of 13 with N-(1-Nap-(CH2)4)-piperazine
23 in the presence of HOBt and EDC yielded 25. After
MCL0129
Figure 1. Small molecule antagonists of the MC4 receptor.

D. Nozawa et al. / Bioorg. Med. Chem. 15 (2007) 2375–2385
2377
F
F
F
F
CO2H
a
c
CO2H
OH
b
CO2H
CO2H
12
N
N
N
Boc
Boc
Boc
15
13
14
d
F
F
F
O
O
N
f
N
e
N
N
Ar1
N
(CH2)4
Ar1
N
(CH2)4
Ar1
(CH2)4
N
N
N
R1
R1
Boc
16
11
17
Scheme 1. Synthesis of mono-piperazine compound 11 (Method A). Reagents and conditions: (a) N-Boc-piperizin-4-one (9), LDA, THF, HMPA,
ice-cooling to rt; (b) H2SO4, CHCl3, reflux and then Boc2O, 4 M aq NaOH, 1,4-dioxane, rt; (c) H2, Pd(OH)2, MeOH, rt; (d) N-(Ar1–(CH2)4)-
piperazine-2HCl (10), EDC–HCl, HOBt–H2O, Et3N, DMF; (e) 4 M HCl/1,4-dioxane, MeOH, rt and then R1-I, K2CO3, DMF, rt; (f) LiAlH4, THF,
50 ꢁC.
F
F
F
O
O
g
h
CO2H
N
N
N
N
CBZ
CBZ
N
N
N
Boc
Boc
15
20
21
i
F
F
O
j
N
N
N
Ar1
(CH2)3
N
(CH2)2
Ar1
N
O
N
11
22
Scheme 2. Synthesis of mono-piperazine compounds 11 (Method B). Reagents and conditions: (g) N-CBZ-piperazine (18), EDC–HCl, HOBt–H2O,
Et3N, DMF; (h) 4 M HCl/1,4-dioxane, MeOH, rt; and then iso-propyl iodide, K2CO3, DMF, rt; (i) H2, Pd(OH)2, MeOH, rt, and then Ar1–(CH2)2–
CO2H (19), EDC–HCl, HOBt–H2O, Et3N, DMF; (j) LiAlH4, THF, 50 ꢁC.
removal of the Boc group of 25 under acidic conditions,
treatment with formaldehyde and NaBH(OAc)3 in the
presence of AcOH yielded 26. Compound 27 was
obtained by reduction of 26 with LiAlH4. Compound
30 was also prepared from 14 by the same synthetic
method as that for 27 (Method C).
rat cortical membrane (for NET), [3H]prazopsin binding
to the rat cortical membrane (for a1), [3H]raclopride
binding to the rat striatal membrane (for D2), [3H]pyril-
amine binding to the rat whole brain membrane (for
H1), [3H]DAMGO binding to the rat brain membrane
(for l), and [3H]DPDPE binding to the rat brain mem-
brane (for d) (Table 2).
3. Results and discussion
Since the bis-piperazine compound, MCL0042, reported
previously by us,24 had very strong basicity, we attempt-
ed to explore MC4 antagonists with varied physico-
chemical properties (Fig. 2). During our investigation
of other structural types of compounds with less basic
amines, we found that mono-piperazine compound,
11a, in which one of the piperazine nitrogen atoms of
the bis-piperazine compound MCL0042 is replaced with
a carbon, showed no significant decrease of binding
affinity (IC50 = 333 nM) as compared to MCL0042
(IC50 = 118 nM). These results suggest that at least
The affinities of all the mono-piperazines for the MC4
receptor were evaluated by assessing their binding affin-
ity to the membranes of COS-1 cells expressing the
human MC4 receptor, calculated from the inhibition
curve of [125I]Nle4-D-Phe7-a-MSH binding,23 and the
IC50 values are shown in Table 1. In addition, the affin-
ities of compounds ( )-11b–d, 11g, 11i, and 11j were
tested as follows: [3H]paroxetine binding to the rat cor-
tical membrane (for SET), [3H]nisoxetine binding to the

2378
D. Nozawa et al. / Bioorg. Med. Chem. 15 (2007) 2375–2385
F
F
F
O
O
k
l
N
N (CH2)4
CO2H
OH
N
N (CH2)4
OH
OH
N
N
N
Boc
Boc
26
13
25
m
F
F
O
F
k
CO2H
N
N (CH2)4
N
N
OH
(CH2)4
N
N
N
Boc
Boc
14
28
27
l
F
F
O
N
N
m
N
N
(CH2)4
(CH2)4
N
N
30
29
Scheme 3. Synthesis of mono-piperazine compounds 27 and 30 (Method C). Reagents and conditions: (k) N-(Nap-(CH2)4)-piperazine-2HCl (23),
EDC–HCl, HOBt–H2O, Et3N, DMF; (l) 4 M HCl/1,4-dioxane, MeOH, rt and then HCHO, AcOH, NaBH(OAc)3, CHCl3, rt; (m) LiAlH4, THF,
50 ꢁC.
Table 1. Binding affinity for the MC4 receptor of compounds 11
F
R1
N
N
N (CH2)n
Ar1
R1
Compounda
Method
Ar1
n
IC50 (nM)
( )-11a
( )-11b
( )-11c
( )-11d
( )-11e
( )-11f
( )-11g
( )-11h
( )-11i
( )-11j
A
A
B
B
B
B
B
B
B
B
1-Nap
2-MeO-1-Nap
2-Ph–Ph
Me
4
4
3
3
3
3
3
3
3
3
333
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
40.3
19.7
19.8
52.7
52.2
18.3
98.6
23.4
14.5
2-Ph-3-F-Ph
2-Ph-4-F-Ph
2-Ph-5-F-Ph
2-Ph-6-F-Ph
2-(2-F-Ph)-Ph
2-(3-F-Ph)-Ph
2-(4-F-Ph)-Ph
a All compounds are of 3 hydrochloride form.
Table 2. In vitro receptor profiles of compounds 11
Compound
IC50 (nM)
H1
MC4
SET
NET
a1
D2
Opiate l
Opiate d
( )-11b
( )-11c
( )-11d
( )-11g
( )-11i
( )-11j
40.3
19.7
19.8
18.3
23.4
14.5
877
953
2830
1180
3940
491
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
423
482
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>1000
>1000
>1000
>1000
>1000
3130
723
>1000
805
820
2140
3940
543
729

D. Nozawa et al. / Bioorg. Med. Chem. 15 (2007) 2375–2385
2379
on the terminal benzene ring (B ring) in the biphenyl
compounds (Fig. 2). Introduction of a fluorine atom at
4-position of the terminal benzene ring resulted in a
slightly higher affinity (11j; IC50 = 14.5 nM) as com-
pared with that of the nonsubstituted compound 11c,
while 11i with a substitution at 3-position exhibited a
slightly lower affinity (IC50 = 23.4 nM). In contrast,
compound 11h with a substitution at 2-position of the
terminal benzene ring showed a much lower affinity
(IC50 = 98.6 nM) as compared with that of the nonsub-
stituted compound 11c, indicating that a substituent at
2-position might have a negligible effect on the confor-
mation for binding to the MC4 receptor.
F
F
N
N
N
N
N
N
N
11a
MCL0042
A ring
F
5
6
4
3
2
F
N
N
N
O
N
N
3
N
4
Finally, we focused our attention on compounds having
different frameworks related structurally to the original
mono-piperazine 11a. We prepared compounds 27 and
30 as shown in Scheme 1, and evaluated the binding
affinity of the compounds for the MC4 receptor. These
compounds did not show any significant decrease of
the affinity (27, IC50 = 487 nM and 30, 563 nM) for
the receptor as compared to that of 11a
(IC50 = 333 nM), supporting our hypothesis that the
presence of one nitrogen atom in the piperazine may
have little impact on the binding affinity for the MC4
receptor. These results indicate that these structural
changes also do not cause any important change in the
conformation of the molecule.
B ring
11c
11b
Figure 2. Lead discovery and optimization.
one nitrogen atom in the piperazine ring does not play
an important role in binding to the MC4 receptor. Thus,
we began to explore the mono-piperazine analogues
with reduced basicity relative to the bis-piperazines to
identify compounds with higher affinity for the MC4
receptor.
Initially, the binding affinity for the MC4 receptor was
optimized by replacement of a methyl group at R1
(Table 1). We prepared compound 11b with an iso-pro-
pyl group at R1, designed based on the results of our
previous investigations,23 and evaluated the affinity of
the compound for the receptor. As expected, compound
11b showed a much higher affinity (IC50 = 40.3 nM) for
the MC4 receptor as compared to compound 11a
(IC50 = 333 nM).
3.1. Selectivity for other stress- and anxiety/depression-
related receptors and transporters
To investigate the in vitro receptor-binding profiles of
the mono-piperazine compounds described above, sev-
eral compounds with a high affinity for the MC4 recep-
tor were evaluated for their affinities for the serotonin
transporter (SET), norepinephrine transporter (NET),
a1-adrenoceptor (a1), dopamine D2 receptor (D2), his-
tamine H1 receptor (H1), and opiate l and d receptors
(Table 2). The results indicate that none of the com-
pounds showed a significant affinity for the receptors
or transporters examined, revealing the high selectivity
of these mono-piperazine compounds for the MC4
receptor.
As the next structural conversion to increase the affinity,
we considered that the naphthyl group could be replaced
with a biphenyl (2-Ph–Ph) group. We prepared 11c with
the biphenyl (2-Ph–Ph) group and evaluated the affinity
of this compound for the MC4 receptor. Fortunately,
11c showed a higher affinity (IC50 = 19.7 nM) for the
MC4 receptor than compound 11b (IC50 = 40.3 nM),
therefore, we started to explore biphenyl analogues
further.
3.2. Effect on stress-induced anxiety-like behavior in rats
We investigated the effects of substituents on the core
benzene ring (A ring) in the biphenyl compounds
(Fig. 2). Compounds 11d and 11g substituted with a
fluorine atom at 3- or 6-position of the core benzene
ring exhibited almost the same affinity (IC50 = 19.8
and 18.3 nM, respectively) for the MC4 receptor as
the nonsubstituted compound 11c (IC50 = 19.7 nM),
while 11e and 11f substituted with a fluorine atom at
4- or 5-position of the core benzene ring showed a
decreased affinity (IC50 = 52.7 and 52.2 nM, respective-
ly) as compared to 11c. These findings suggest that the
sterically acceptable space at 4- or 5-position on the
core benzene ring for binding to the MC4 receptor
was limited.
We examined the ability of selected compounds from
this series to reverse anxiety-like behavior in the elevated
plus-maze task in rats. Exposure of rats to swim stress
for 2 min markedly reduced the time spent on the open
arms of the maze in the elevated plus-maze task, sugges-
tive of anxiety-like behavior (Fig. 3). The stress-induced
anxiety-like behavior in the elevated plus-maze task has
been used as a useful model to evaluate the anxiolytic
and antistress activity of such compounds.23 Com-
pounds 11c and 11d, when administered orally at
1 mg/kg, significantly reversed the stress-induced reduc-
tion in the time spent in the open arms of the maze
(Fig. 3), while bis-piperazine compound, MCL0129,
reversed at 10 mg/kg.23 These results indicate that these
mono-piperazine compounds exhibited anxiolytic effects
in the rats.
To optimize the binding affinity of this series of com-
pounds, we next investigated the effects of substitution

2380
D. Nozawa et al. / Bioorg. Med. Chem. 15 (2007) 2375–2385
substituting one of the piperazine nitrogen atoms of
##
50
45
40
35
30
25
20
15
10
5
MCL0042 with a carbon, showed a moderate affinity
for the MC4 receptor. Then, structural conversion led
to the development of compound 11b, which showed
optimal binding affinity (IC50 = 40.3 nM) for the MC4
receptor. We also found another class of compounds,
the biphenyl analogues, some of which showed a high-
affinity for the MC4 receptor. Among these compounds,
11j exhibited the highest affinity for the MC4 receptor
(IC50 = 14.5 nM), with an over 20-fold higher affinity
for this receptor than compound 11a. In addition, com-
pounds 11c and 11d, having a high affinity for the MC4
receptor, significantly reversed anxiety-like behavior
caused by swim stress when administered po, indicating
that these mono-piperazine compounds exhibited anxio-
lytic effects in the rats. These various mono-piperazine
compounds that have been demonstrated to show a high
affinity for the MC4 receptor can be useful tools for
investigating the involvement of the MC4 receptor in
the development of stress-related disorders, such as anx-
iety, and for elucidating the actions mediated by this
receptor.
#
#
**
0
vehicle vehicle
1
3
10
11c (mg/kg, p.o.)
Swim stress
5. Experimental
##
55
50
45
40
35
30
25
20
15
10
5
Melting points were determined on a Yanaco MP-500D
melting point apparatus and are uncorrected. Proton
nuclear magnetic resonance (1H NMR) spectra were ob-
tained using Varian Gemini 2000 (200 MHz) or Varian
Unity Inova 300 (300 MHz). Chemical shifts are report-
ed in parts per million relative to tetramethylsilane as an
internal standard. Mass spectra (MS) were obtained on
Micromass Platform LC (IonSpray). Elemental analyses
were performed by a Perkin–Elmer 2400 or a Yanaco
MT-6. Silica gel C-200 (100–200 mesh, Wako Pure
Chemical) and Chromatorex NH (100–200 mesh, Fuji
Silysia Chemical Ltd.)) were used for column chroma-
tography, using the solvent systems (volume ratios) indi-
cated below.
#
#
**
5.1. General methods for the synthesis of 11a and 11b
(Method A)
5.1.1. ( )-[1-(tert-Butoxycarbonyl)-4-hydroxypiperidin-4-
yl](4-fluorophenyl)acetic acid (( )-13). A solution of
(4-fluorophenyl)acetic acid 12 (25.2 g, 163 mmol) in
THF (100 mL) was added dropwise to a solution of
LDA prepared by treatment of iPr2NH (48.3 mL,
343 mmol) in THF (200 mL) with 2.50 M solution of
n-BuLi in hexane (137 mL, 343 mmol) with ice-cooling.
To the mixture was added HMPA (28.4 mL) and the
mixture was stirred at room temperature for 30 min.
The mixture was cooled in ice-bath, and a solution of
0
vehicle vehicle
1
3
10
11d (mg/kg, p.o.)
Swim stress
Figure 3. Effects of 11c and 11d on swim stress-induced reduction of
time spent in open arms in the elevated plus-maze task in the rats. Data
represent means SE (n = 10 or 15). **P < 0.01 versus nonstress group
(Dunnett’s test). #P < 0.05, ##P < 0.01 versus swim stress group
(Dunnett’s test).
tert-butyl 4-oxopiperidine-1-carboxylate
9
(32.5 g,
163 mmol) in THF (100 mL) was added dropwise to
the cooled mixture. After stirring at room temperature
for 3 h, the mixture was acidified by treatment with 5%
KHSO4 aq and extracted with AcOEt. The extract was
washed with brine, dried over Na2SO4, filtered, and con-
centrated in vacuo. The residue was stirred in Et2O and
the resulting precipitate was collected by filtration to
obtain ( )-[1-(tert-butoxycarbonyl)-4-hydroxypiperi-
4. Conclusion
We have reported on the synthesis, SAR, and biological
activity of a new series of MC4 receptor antagonists,
namely, the mono-piperazines, with reduced basicity rel-
ative to the bis-piperazines. Compound 11a, obtained by

D. Nozawa et al. / Bioorg. Med. Chem. 15 (2007) 2375–2385
2381
din-4-yl](4-fluorophenyl)acetic acid ( )-13 (30.0 g, 50%):
1H NMR (300 MHz, CDCl3) d 1.18–1.30 (2H, m), 1.42
(9H, s), 1.59–1.81 (2H, m), 2.91–3.22 (2H, m), 3.27–
3.43 (1H, m), 3.70–3.99 (3H, m), 6.98–7.07 (2H, m),
7.29–7.42 (2H, m); MS (ESI, Neg) m/z 352 (MꢀH)ꢀ.
1.50–1.70 (6H, m), 1.80–2.02 (1H, m), 2.19–2.41 (6H,
m), 2.48–2.82 (2H, m), 3.02–3.09 (2H, m), 3.35–3.72
(5H, m), 3.92 (3H, s), 3.94–4.15 (2H, m), 6.96–7.04
(2H, m), 7.42 (5H, t, J = 7.5 Hz), 7.70–7.90 (2H, m),
7.92 (1H, d, J = 9.5 Hz); MS (ESI, Pos) m/z 618 (M+H)+.
5.1.2. ( )-[1-(tert-Butoxycarbonyl)-1,2,3,6-tetrahydropy-
ridin-4-yl](4-fluorophenyl)acetic acid (( )-14). To
5.1.5. ( )-1-[(4-Fluorophenyl) (1-isopropylpiperidin-4-
yl)acetyl]-4-[4-(2-methoxy-1-naphthyl)butyl]piperazine
(( )-17). To a solution of ( )-tert-butyl 4-(1-(4-fluor-
ophenyl)-2-{4-[4-(2-methoxy-1-naphthyl)butyl]pipera-
zin-1-yl}-2-oxoethyl)piperidine-1-carboxylate ( )-16 (2.1 g,
3.4 mmol) in MeOH (10 mL) was added 4 M HCl in 1,4-
dioxane (10 mL), and the mixture was stirred at room
temperature for 2 h. The mixture was concentrated in
vacuo and the resulting residue was crystallized from
Et2O to obtain ( )-1-[(4-fluorophenyl) (piperidin-4-yl)-
a
solution of ( )-[1-(tert-butoxycarbonyl)-4-hydroxypi-
peridin-4-yl](4-fluorophenyl)acetic acid ( )-13 (20.0 g,
56.7 mmol) in CHCl3 (40 mL) was added dropwise con-
centrated H2SO4 (40 mL). The mixture was stirred at re-
flux for 3 h and cooled in ice-bath. To the cooled
mixture were added 4 M NaOH aq (250 mL), 1,4-diox-
ane (200 mL), and (Boc)2O (14.8 g, 68.0 mmol). The
mixture was stirred at room temperature for 30 min.
The mixture was acidified by treatment with 5% KHSO4
aq and extracted with CHCl3. The extract was washed
with brine, dried over Na2SO4, filtered, concentrated
in vacuo, and chromatographed on Silica gel C-200
(CHCl3/MeOH, 10:1) to obtain ( )-[1-(tert-butoxycar-
bonyl)-1,2,3,6-tetrahydropyridin-4-yl](4-fluorophenyl)-
acetyl]-4-[4-(2-methoxy-1-naphthyl)butyl]piperazine
2
hydrochloride (1.7 g, 85%). 1H NMR (DMSO-d6) d
1.02–1.61 (4H, m), 1.65–1.92 (3H, m), 2.02–2.31 (2H,
m), 2.60–3.82 (16H, m), 3.92 (3H, m), 4.10–4.52 (2H,
m), 7.11–7.24 (2H, m), 7.30–7.53 (5H, m), 7.79–7.98
(3H, m); MS (ESI, Pos) m/z 518 (M + H)+. A mixture
of ( )-1-[(4-fluorophenyl) (piperidin-4-yl)acetyl]-4-[4-(2-
methoxy-1-naphthyl)butyl]piperazine 2 hydrochloride
(0.20 g, 0.34 mmol), 2-iodopropane (41 lL, 0.41 mmol),
K2CO3 (93 mg, 0.68 mmol) in DMF (2 mL) was stirred
at room temperature for 15 h. The mixture was parti-
tioned between EtOAc and satd aq NaHCO3, and sepa-
rated organic phase was washed with brine. The organic
phase was dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was chromatographed on
Chromatorex NH (hexane/EtOAc, 4:1) to obtain ( )-1-
[(4-fluorophenyl) (1-isopropylpiperidin-4-yl)acetyl]-4-[4-
(2- methoxy-1-naphthyl)butyl]piperazine ( )-17 (0.15 g,
1
acetic acid ( )-14 (18.0 g, 95%): H NMR (300 MHz,
CDCl3) d 1.43 (9H, s), 2.01–2.10 (2H, m), 3.22–3.40
(2H, m), 3.81–4.02 (2H, m), 4.27–4.32 (1H, m), 5.51–
5.62 (1H, m), 6.99–7.08 (2H, m), 7.21–7.29 (2H, m);
MS (ESI, Pos) m/z 358 (M+Na)+.
5.1.3. ( )-[1-(tert-Butoxycarbonyl)piperidin-4-yl](4-fluor-
ophenyl)acetic acid (( )-15). A suspension of ( )-[1-
(tert-butoxycarbonyl)-1,2,3,6-tetrahydropyridin-4-yl](4-
fluorophenyl)acetic acid ( )-14 (5.00 g, 14.9 mmol) and
Pd(OH)2/C (20 wt% Pd on carbon, wet) (0.50 g) in
MeOH (50 mL) was stirred under hydrogen atmosphere
for 2 days. The mixture was filtered through Celite and
the filtrate was concentrated in vacuo to obtain crude
[1-(tert-butoxycarbonyl)piperidin-4-yl](4-fluorophenyl)-
1
78%) as an oily product: H NMR (300 MHz, CDCl3)
d 1.00 (6H, d, J = 6.5 Hz), 1.10–1.30 (2H, m), 1.50–1.70
(5H, m), 1.84–2.42 (8H, m), 2.59–3.10 (6H, m),
3.37–3.70 (6H, m), 3.91 (3H, s), 6.95–7.01 (2H, m),
7.19–7.36 (4H, m), 7.42 (1H, t, J = 7.5 Hz), 7.68–7.79
(2H, m), 7.91 (1H, d, J = 9.4 Hz); MS (ESI, Pos) m/z
560 (M+H)+.
1
acetic acid ( )-15 (3.60 g, 72%): H NMR (300 MHz,
CDCl3) d 0.77–1.09 (1H, m), 1.17–1.29 (2H, m), 1.42
(9H, s), 1.79–1.90 (1H, m), 1.98–2.20 (1H, m), 2.47–
2,.82 (2H, m), 3.18–3.25 (1H, m), 3.28–3.55 (1H, m),
3.87–4.20 (1H, m), 6.96–7.08 (2H, m), 7.18–7.36 (2H,
m); MS (ESI, Neg) m/z 336 (MꢀH)ꢀ.
5.1.6. ( )-1-[2-(4-Fluorophenyl)-2-(1-isopropylpiperidin-
4-yl)ethyl]-4-[4-(2-methoxy-1-naphthyl)butyl]piperazine 3
hydrochloride (( )-11b). A mixture of ( )-1-[(4-fluor-
ophenyl) (1-isopropylpiperidin-4-yl)acetyl]-4-[4-(2-meth-
oxy-1-naphthyl)butyl]piperazine ( )-17 (0.14 g, 0.25
mmol) and LiAlH4 (10 mg, 0.25 mmol) in THF (5 mL)
was stirred at 50 ꢁC for 15 min. To the mixture was add-
ed dropwise 25% aq NH3 (1.0 mL) at room temperature.
The resulting solid was excluded by the filtration
through Celite. The filtrate was concentrated in vacuo
and chromatographed on Chromatorex NH (hexane/
EtOAc, 4:1) to obtain ( )-1-[2-(4-fluorophenyl)-2-(1-iso-
propylpiperidin-4-yl)ethyl]-4-[4-(2-methoxy-1-naphthyl)-
5.1.4. ( )-tert-Butyl 4-(1-(4-fluorophenyl)-2-{4-[4-(2-
methoxy-1-naphthyl)butyl]piperazin-1-yl}-2-oxoethyl)pi-
peridine- 1-carboxylate (( )-16). To a mixture of crude
( )-[1-(tert-butoxycarbonyl)piperidin-4-yl](4-fluorophenyl)-
acetic acid ( )-15 (2.2 g, 6.5 mmol), 1-[4-(2-methoxy-
1-naphthyl)butyl]piperazine 2 hydrochloride 10 (2.0 g,
5.4 mmol), HOBtÆH2O (1.5 g, 9.8 mmol), and Et3N
(3.5 mL, 25 mmol) in DMF (20 mL) was added ED-
CÆHCl (1.9 g, 9.8 mmol) and the mixture was stirred at
room temperature for 15 h. The mixture was partitioned
between EtOAc and satd aq NaHCO3, and separated
organic phase was washed with brine. The organic phase
was dried over Na2SO4, filtered, and concentrated in
vacuo. The residue was chromatographed on Chromato-
rex NH (hexane/EtOAc, 3:1) to obtain ( )-tert-butyl
4-(1-(4-fluorophenyl)-2-{4-[4-(2-methoxy-1-naphthyl)bu-
tyl]-piperazin-1-yl}-2-oxoethyl)piperidine-1-carboxyl-
1
butyl]piperazine (0.13 g, 95%) as an oily product: H
NMR (300 MHz, CDCl3) d 0.99 (6H, d, J = 6.5 Hz),
1.03–1.18 (1H, m), 1.30–1.52 (2H, m), 1.55–1.68 (4H,
m), 1.73–1.85 (1H, m), 1.90–2.05 (1H, m), 2.20–2.50
(10H, m), 2.50–2.92 (8H, m), 3.01–3.12 (2H, m), 3.91
(3H, s), 6.88–7.09 (4H, m), 7.22–7.32 (2H, m), 7.42
(1H, t, J = 7.5 Hz), 7.69–7.81 (2H, m), 7.97 (1H, d,
J = 8.5 Hz); MS (ESI, Pos) m/z 546 (M+H)+. The above
1
ate ( )-16 (2.4 g, 73%) as an oily product: H NMR
(300 MHz, CDCl3) d 0.86–1.22 (2H, m), 1.42 (9H, s),

2382
D. Nozawa et al. / Bioorg. Med. Chem. 15 (2007) 2375–2385
free base (0.11 g, 0.20 mmol) was dissolved in MeOH
(4 mL), treated with 4 M HCl in AcOEt (1 mL) at room
temperature, and the mixture was concentrated in vacuo.
The residue was crystallized from AcOEt, and the pre-
cipitate was collected by filtration to obtain ( )-1-[2-(4-
fluorophenyl)-2-(1-isopropylpiperidin-4-yl)ethyl]-4-[4-
(2-methoxy-1-naphthyl)butyl]piperazine 3 hydrochlo-
ride ( )-11b (0.12 g, 91%) as a crystal: mp 233–235 ꢁC
phase was washed with brine. The organic phase was
dried over Na2SO4, filtered, and concentrated in vacuo
to give crude ( )-benzyl 4-[(4-fluorophenyl) (piperidin-
4-yl)acetyl]piperazine-1-carboxylate (9.30 g, quantitative
1
yield). H NMR (300 MHz, CDCl3) d 0.83–1.19 (3H,
m), 1.43 (9H, s), 1.81–1.92 (1H, m), 2.19–2.37 (1H, m),
2.49–2.92 (3H, m), 3.18–3.61 (8H, m), 3.70–3.93 (1H,
m), 3.93–4.17 (1H, m), 5.10 (2H, m), 6.97–7.03 (2H,
m), 7.18–7.40 (7H, m). This product was used in the next
step without further purification. A mixture of crude
( )-benzyl 4-[(4-fluorophenyl) (piperidin-4-yl)acetyl]pi-
perazine-1-carboxylate (9.30 g, 19.6 mmol), 2-iodopro-
pane (2.95 mL, 29.5 mmol), and K2CO3 (8.14 g,
59.0 mmol) in DMF (100 mL) was stirred at room tem-
perature for 15 h. The mixture was partitioned between
EtOAc and saturated water, and separated organic
phase was washed with brine. The organic phase was
dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was chromatographed on Chromatorex NH
(hexane/EtOAc, 1:1) to obtain ( )-benzyl 4-[(4-fluor-
ophenyl) (1-isopropylpiperidin-4-yl)acetyl]piperazine-1-
1
(EtOAc); H NMR (200 MHz, DMSO-d6) d 1.20 (6H,
d, J = 6.6 Hz), 1.20–1.70 (5H, m), 1.70–2.10 (4H, m),
2.60–4.00 (20H, m), 3.92 (3H, s), 7.19–7.52 (7H, m),
7.79–7.88 (2H, m), 7.96 (1H, d, J = 8.6 Hz); MS (ESI,
Pos) m/z 546 (M + H)+; Anal.(C35H48FN3OÆ3HClÆ2.5-
H2O) C, H, N.
Compound ( )-11a was prepared by the same synthetic
method as that for ( )-11b.
5.1.7. ( )-1-[2-(4-Fluorophenyl)-2-(1-methylpiperidin-4-
yl)ethyl]-4-[4-(1-naphthyl)butyl]piperazine 3 hydrochlo-
ride (( )-11a). Compound ( )-11a was obtained as a
1
1
crystal. Mp 190–193 ꢁC (AcOEt); H NMR (300 MHz,
carboxylate ( )-21 (6.20 g, 66%) as an oily product: H
DMSO-d6) d 1.22–1.55 (2H, m), 1.62–1.93 (7H, m),
2.64 (3H, s), 2.70–3.75 (19H, m), 7.21 (2H, t, J = 8.9
Hz), 7.33–7.58 (6H, m), 7.78 (1H, d, J = 7.8 Hz), 7.90–
7.94 (1H, m), 8.07 (1H, d, J = 7.8 Hz); MS (ESI, Pos)
m/z 519 (M + H)+; Anal. (C32H42FN3Æ3HClÆ1.2H2O)
C, H, N.
NMR (300 MHz, CDCl3) d 1.01 (6H, d, J = 6.6 Hz),
1.05–1.19 (2H, m), 1.83–2.22 (3H, m), 2.59–2.98 (5H,
m), 3.18–3.61 (8H, m), 3.63–3.83 (1H, m), 5.10 (2H,
m), 6.96–7.03 (2H, m), 7.18–7.39 (7H, m); MS (ESI,
Pos) m/z 482 (M+H)+.
5.2.3. ( )-1-[3-(40-Fluorobiphenyl-2-yl)propanoyl]-4-[(4-
fluorophenyl) (1-isopropylpiperidin-4-yl)acetyl]piperazine
(( )-22). A suspension of ( )-benzyl 4-[(4-fluorophenyl)
(1-isopropylpiperidin-4-yl)acetyl]piperazine-1-carboxyl-
ate ( )-21 (6.1 g, 13 mmol) and Pd(OH)2/C (20 wt% Pd
on carbon, wet) (0.61 g) in MeOH (60 mL) was stirred
under a hydrogen atmosphere for 15 h. The mixture
was filtered through Celite and the filtrate was concen-
trated in vacuo to obtain crude ( )-1-[(4-fluorophe-
nyl) (1-isopropylpiperidin-4-yl)acetyl]piperazine (4.8 g,
quantitative yield). This product was used in the next
step without further purification. To a mixture of crude
( )-1-[(4-fluorophenyl) (1-isopropylpiperidin-4-yl)acetyl]-
piperazine (0.30 g, 0.86 mmol), 3-(40-fluorobiphenyl-2-
yl)propanoic acid 19 (0.23 g, 0.95 mmol), HOBtÆH2O
(0.20 g, 1.3 mmol), and Et3N (0.14 mL, 1.0 mmol) in
DMF (5 mL) was added EDCÆHCl (0.20 g, 1.0 mmol)
and the mixture was stirred at room temperature for
3 h. The mixture was partitioned between EtOAc and
satd aq NaHCO3, and separated organic phase was
washed with brine. The organic phase was dried over
Na2SO4, filtered, and concentrated in vacuo. The
residue was chromatographed on Chromatorex NH
(hexane/EtOAc, 1:2) to obtain ( )-1-[3-(40-fluorobiphe-
nyl-2-yl)propanoyl]-4-[(4-fluorophenyl) (1-isopropylpi-
peridin-4-yl)acetyl]piperazine ( )-22 (0.30 g, 61%) as
5.2. General methods for the synthesis of 11c–j (Method B)
5.2.1. ( )-Benzyl 4-[[1-(tert-butoxycarbonyl)piperidin-4-
yl](4- fluorophenyl)acetyl]piperazine-1-carboxylate (( )-20).
To a mixture of ( )-[1-(tert-butoxycarbonyl)piperidin-4-
yl](4-fluorophenyl)acetic acid ( )-15 (9.2 g, 27 mmol),
benzyl piperazine-1-carboxylate 18 (6.6 g, 30 mmol),
HOBtÆH2O (6.3 g, 41 mmol), and Et3N (4.5 mL,
33 mmol) in DMF (20 mL) was added EDCÆHCl
(6.3 g, 33 mmol) and the mixture was stirred at room
temperature for 3 h. The mixture was partitioned
between EtOAc and satd aq NaHCO3, and separated
organic phase was washed with brine. The organic phase
was dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was chromatographed on
Chromatorex NH (hexane/EtOAc, 4:1) to obtain ( )-
benzyl 4-[[1-(tert-butoxycarbonyl)piperidin-4-yl](4-fluor-
ophenyl)acetyl]piperazine-1-carboxylate ( )-20 (11 g,
1
75%) as an oily product: H NMR (300 MHz, CDCl3)
d 0.83–1.19 (3H, m), 1.43 (9H, s), 1.81–1.92 (1H, m),
2.19–2.37 (1H, m), 2.49–2.92 (3H, m), 3.18–3.61 (8H,
m), 3.70–3.93 (1H, m), 3.93–4.17 (1H, m), 5.10 (2H,
m), 6.97–7.03 (2H, m), 7.18–7.40 (7H, m); MS (ESI,
Pos) m/z 562 (M+Na)+.
1
5.2.2. ( )-Benzyl 4-[(4-fluorophenyl) (1-isopropylpiperi-
din-4-yl)acetyl]piperazine-1- carboxylate (( )-21).
Compound ( )-Benzyl 4-[[1-(tert-butoxycarbonyl)pip-
eridin-4-yl](4-fluorophenyl)acetyl]piperazine-1-carboxyl-
ate ( )-20 (10.6 g, 19.6 mmol) was added with 4 M HCl
in 1,4-dioxane (50 mL), and the mixture was stirred at
room temperature for 1 h. The mixture was concentrat-
ed in vacuo and the resulting residue was partitioned be-
tween EtOAc and NaOH aq, and separated organic
an oily product: H NMR (300 MHz, CDCl3) d 1.02
(6H, d, J = 6.5 Hz), 1.04–1.22 (2H, m), 1.84–2.51 (9H,
m), 2.59–3.01 (8H, m), 3.03–3.76 (3H, m), 6.95–7.42
(12H, m); MS (ESI, Pos) m/z 574 (M+H)+.
5.2.4. ( )-1-[3-(40-Fluorobiphenyl-2-yl)propyl]-4-[2-(4-flu-
orophenyl)-2-(1-isopropylpiperidin-4-yl)ethyl]piperazine 3
hydrochloride (( )-11j). A mixture of ( )-1-[3-(40-fluoro-
biphenyl-2-yl)propanoyl]-4-[(4-fluorophenyl) (1-isopro-

D. Nozawa et al. / Bioorg. Med. Chem. 15 (2007) 2375–2385
2383
pylpiperidin-4-yl)acetyl]piperazine
( )-22
(0.29 g,
5.2.8. ( )-1-[3-(4-Fluorobiphenyl-2-yl)propyl]-4-[2-(4-flu-
orophenyl)-2-(1-isopropylpiperidin-4-yl)ethyl]piperazine 3
hydrochloride (( )-11f). Compound ( )-11f was obtained
as a crystal. Mp 229–231 ꢁC (AcOEt–MeOH); 1H NMR
(300 MHz, DMSO-d6) d 1.19 (6H, d, J = 6.5 Hz), 1.23–
1.60 (3H, m), 1.79–2.03 (4H, m), 2.52–2.61 (1H, m),
2.70–3.90 (18H, m), 4.20–4.45 (1H, m), 7.03–7.45 (12H,
m); MS (ESI, Pos) m/z 546 (M+H)+; Anal.
(C35H45F2N3Æ3HClÆ0.5H2O) C, H, N.
0.52 mmol) and LiAlH4 (41 mg, 1.0 mmol) in THF
(5 mL) was stirred at 50 ꢁC for 30 min. To the mixture
was added dropwise 25% aq NH3 (1.0 mL) at room
temperature. The resulting solid was excluded by the
filtration through Celite. The filtrate was concentrated
in vacuo and chromatographed on Chromatorex NH
(hexane/EtOAc, 1:1) to obtain ( )-1-[3-(40-fluorobiphe-
nyl-2-yl)propyl]-4-[2-(4-fluorophenyl)-2-(1-isopropylpi-
peridin-4-yl)ethyl]piperazine (0.25 g, 88%) as an oily
1
product: H NMR (300 MHz, CDCl3) d 1.00 (6H, d,
5.2.9. ( )-1-[3-(3-Fluorobiphenyl-2-yl)propyl]-4-[2-(4-flu-
orophenyl)-2-(1-isopropylpiperidin-4-yl)ethyl]piperazine 3
hydrochloride (( )-11g). Compound ( )-11g was ob-
tained as a crystal. Mp 226–228 ꢁC (AcOEt–MeOH);
1H NMR (300 MHz, DMSO-d6) d 1.19 (6H, d,
J = 6.5 Hz), 1.22–1.60 (3H, m), 1.74–2.03 (4H, m),
2.50–2.59 (1H, m), 2.70–3.70 (18H, m), 4.18–4.37 (1H,
m), 7.03 (2H, d, J = 7.5 Hz), 7.13–7.47 (10H, m); MS
(ESI, Pos) m/z 546 (M+H)+; Anal. (C35H45F2N3Æ3H-
ClÆ0.5H2O) C, H, N.
J = 6.5 Hz), 1.03–1.16 (1H, m), 1.31–1.82 (6H, m),
1.91–2.41 (9H, m), 2.50–2.91 (10H, m), 6.89–6.99
(2H, t, J = 7.5 Hz), 7.00–7.27 (10H, m); MS (ESI,
Pos) m/z 546 (M+H)+. The above free base (0.22 g,
0.40 mmol) was dissolved in EtOH (4 mL), treated
with 4 M HCl in AcOEt (1 mL) at room temperature,
and the mixture was concentrated in vacuo. The resi-
due was crystallized from a mixture of AcOEt and
MeOH, and the precipitate was collected by filtration
to obtain ( )-1-[3-(40-fluorobiphenyl-2-yl)propyl]-4-[2-
(4-fluorophenyl)-2-(1-isopropylpiperidin-4-yl)ethyl]pi-
perazine 3 hydrochloride ( )-11j (0.23 g, 81%) as a
crystal: mp 221–223 ꢁC (AcOEt–MeOH); 1H NMR
(300 MHz, DMSO-d6) d 1.19 (6H, d, J = 6.5 Hz),
1.23–1.60 (3H, m), 1.80–2.02 (4H, m), 2.50–2.59 (1H,
m), 2.70–4.10 (19H, m), 7.15–7.60 (12H, m); MS
(ESI, Pos) m/z 546 (M+H)+; Anal. (C35H45F2N3Æ3H-
ClÆ0.5H2O) C, H, N.
5.2.10. ( )-1-[3-(20-Fluorobiphenyl-2-yl)propyl]-4-[2-(4-
fluorophenyl)-2-(1-isopropylpiperidin-4-yl)ethyl]pipera-
zine 3 hydrochloride (( )-11h). Compound ( )-11h was
obtained as a crystal. Mp 221–223 ꢁC (AcOEt–MeOH);
1H NMR (300 MHz, DMSO-d6) d 1.19 (6H, d,
J = 6.5 Hz), 1.22–1.59 (3H, m), 1.78–2.03 (4H, m), 2.40–
2.49 (1H, m), 2.70–4.00 (19H, m), 7.14–7.49 (12H, m);
MS (ESI, Pos) m/z 546 (M+H)+; Anal. (C35H45F2N3Æ3H-
ClÆ0.7H2O) C, H, N.
Compound ( )-11c–i were prepared by the same syn-
thetic method as that for ( )-11j.
5.2.11. ( )-1-[3-(30-Fluorobiphenyl-2-yl)propyl]-4-[2-(4-flu-
orophenyl)-2-(1-isopropylpiperidin-4-yl)ethyl]piperazine
hydrochloride (( )-11i). Compound ( )-11i was obtained
as a crystal. Mp 218–220 ꢁC (AcOEt–MeOH); H NMR
(300 MHz, DMSO-d6) d 1.20 (6H, d, J = 6.5 Hz), 1.22–
1.61 (3H, m), 1.79–2.03 (4H, m), 2.52–2.61 (1H, m),
2.75–3.70 (19H, m), 7.10–7.52 (12H, m); MS (ESI, Pos)
m/z 546 (M+H)+; Anal. (C35H45F2N3Æ3HClÆ0.5H2O) C,
H, N.
3
5.2.5. ( )-1-(3-Biphenyl-2-ylpropyl)-4-[2-(4-fluorophenyl)-2-
(1-isopropylpiperidin-4-yl)ethyl]piperazine 3 hydrochloride
(( )-11c). Compound ( )-11c was obtained as a crystal.
1
1
Mp 211–213 ꢁC (AcOEt–MeOH); H NMR (300 MHz,
DMSO-d6)
d 1.18 (6H, d, J = 6.5 Hz), 1.22–1.60
(3H, m), 1.78–2.03 (4H, m), 2.52–2.61 (1H, m),
2.77–4.03 (19H, m), 7.17–7.50 (13H, m); MS (ESI, Pos)
m/z 528 (M+H)+; Anal. (C35H46FN3Æ3HClÆ0.5H2O)
C, H, N.
5.3. General methods for the synthesis of ( )-27 and ( )-
30 (Method C)
5.2.6. ( )-1-[3-(6-Fluorobiphenyl-2-yl)propyl]-4-[2-(4-flu-
orophenyl)-2-(1-isopropylpiperidin-4-yl)ethyl]piperazine 3
hydrochloride (( )-11d). Compound ( )-11d was ob-
tained as a crystal. Mp 227–229 ꢁC (AcOEt–MeOH);
1H NMR (300 MHz, DMSO-d6) d 1.19 (6H, d,
J = 6.5 Hz), 1.23–1.61 (3H, m), 1.77–2.03 (4H, m),
2.41–2.61 (1H, m), 2.70–4.93 (19H, m), 7.17–7.50
(12H, m); MS (ESI, Pos) m/z 546 (M+H)+; Anal.
(C35H45F2N3Æ3HCl) C, H, N.
5.3.1. 4-(1-(4-Fluorophenyl)-2-{4-[4-(1-naphthyl)butyl]-
piperazin-1-yl}ethyl)- 1-methylpiperidin-4-ol 3 hydrochlo-
ride (( )-27). Compound ( )-27 was prepared from ( )-
13 by the same synthetic method as that for ( )-11b.
1
Compound ( )-27 was obtained as an amorphous. H
NMR (300 MHz, DMSO-d6) d 1.21–1.34 (1H, m),
1.59–1.84 (6H, m), 2.68 (3H, d, J = 4.7 Hz), 2.90–3.85
(21H, m), 7.14–7.25 (2H, m), 7.37–7.57 (6H, m), 7.78
(1H, d, J = 7.6 Hz), 7.91–7.94 (1H, m), 8.07 (1H, d,
J = 7.6 Hz); MS (ESI, Pos) m/z 504 (M+H)+; Anal.
(C32H42FN3OÆ3HClÆ3.5H2O) C, H, N.
5.2.7. ( )-1-[3-(5-Fluorobiphenyl-2-yl)propyl]-4-[2-(4-flu-
orophenyl)-2-(1-isopropylpiperidin-4-yl)ethyl]piperazine 3
hydrochloride (( )-11e). Compound ( )-11e was ob-
tained as a crystal. Mp 219–221 ꢁC (AcOEt–MeOH);
1H NMR (300 MHz, DMSO-d6) d 1.19 (6H, d,
J = 6.5 Hz), 1.23–1.60 (3H, m), 1.78–2.03 (4H, m),
2.51–2.59 (1H, m), 2.70–3.65 (18H, m), 4.00–4.18 (1H,
m), 6.99 (2H, dd, J = 1.5, 6.0 Hz), 7.12–7.49 (10H, m);
MS (ESI, Pos) m/z 546 (M+H)+; Anal. (C35H45F2N3Æ
3HClÆ0.5H2O) C, H, N.
5.3.2. 1-[2-(4-Fluorophenyl)-2-(1-methyl-1,2,3,6-tetrahy-
dropyridin-4-yl)ethyl]-4-[4-(1-naphthyl)butyl]piperazine 3
hydrochloride (( )-30). Compound ( )-30 was prepared
from ( )-14 by the same synthetic method as that for
( )-11b. Compound ( )-30 was obtained as an amor-
phous. 1H NMR (300 MHz, DMSO-d6) d 1.63–1.91
(4H, m), 2.68–2.86 (3H, m), 2.94–3.90 (19H, m),

2384
D. Nozawa et al. / Bioorg. Med. Chem. 15 (2007) 2375–2385
3.91–4.18 (2H, m), 7.16–7.25 (2H, m), 7.33–7.59 (6H,
m), 7.79 (1H, d, J = 7.8 Hz), 7.91–7.94 (1H, m), 8.08
(1H, d, J = 7.8 Hz); HRMS; 486.3287 (M+1).
For behavioral studies, these compounds were dissolved
in 0.3% Tween 80/saline solution.
5.5.2. Stress-induced anxiogenic-like behavior in rats. The
swim stress consists of placing rats in a 40-cm tall, 20 cm
wide cylindrical plastic container containing 25 cm of
water maintained at 25 1 ꢁC. Duration of the swim
stress was 2 min, and the elevated plus-maze test was
performed 5 min after the swim stress. The elevated
plus-maze test was based on that validated for the rat
by Guimaraes et al.37 The apparatus consisted of a
plus-maze elevated 50 cm high from the floor. The maze
has four arms such that two opposite open arms,
50 · 10 cm, were crossed at right angles by the same
dimensions and enclosed by 40 cm-high walls with an
open roof. In addition, a 1-cm high edge made of Plex-
iglas surrounded the open arms to avoid falls. Luminos-
ity measured at the center of the maze was 40 lux.
During the observation, the experimenter always sat in
the same place, next to the apparatus. Each rat was
placed in the center of the plus-maze facing one enclosed
arm. The amount of time spent in open arms of the maze
was recorded. Rats were naive to the apparatus. Testing
compounds were administered po 30 min prior to swim
stress.
5.4. Binding test
5.4.1. Materials. [125I][Nle4,D-Phe7]a-melanocyte stimu-
lating hormone ([Nle4,D-Phe7]a-MSH) (specific radioac-
tivity: 81.4 TBq/mmol) was purchased from Amersham
International (Buckinghamshire, England). COS-1 cells
were purchased from American Type Culture Collection
(Rocksville, MD, USA). [Nle4,D-Phe7]a-MSH was
purchased from Peninsula Laboratories (Belmont, CA,
USA). All other chemicals used in this study were
obtained commercially, and all were of the highest purity
available.
5.4.2. [125I][Nle4,D-Phe7]a-MSH binding to the recombi-
nant MC4 receptor. COS-1 cells expressing the MC4
receptor, prepared according to the method reported
previously,23 were washed with phosphate-buffered
saline, scraped, and pelleted by centrifugation. Cell
pellets were homogenized with 50 mM Tris–HCl buff-
er (pH 7.4) containing 2 mM EDTA, 10 mM CaCl2,
and 100 lM phenylmethylsulfonylfluoride, and centri-
fuged at 48,000g for 20 min at 4 ꢁC. The pellet was
washed twice with the buffer, and the final pellet
was suspended in an assay buffer (50 mM Tris–HCl
buffer (pH 7.4) containing 2 mM EDTA, 10 mM
CaCl2, 100 lM phenylmethylsulfonylfluoride, and
0.1% bovine serum albumin (BSA)) and served as
crude membrane preparation for binding studies.
Binding assays of [125I][Nle4,D-Phe7]a-MSH were per-
formed according to Chaki et al.23 Membranes were
5.5.3. Statistical analysis. Data from in vivo experiment
were analyzed by one-way ANOVA and significant dif-
ferences between groups were determined, using Dun-
nett’s test.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2007.01.019.
incubated with
[
125I][Nle4,D-Phe7]a-MSH (0.2 nM)
for 120 min at 25 ꢁC, and the reaction was terminat-
ed by rapid filtration over a GF/C filter presoaked
with 0.5% BSA, after which the filters were washed
three times with the buffer. Radioactivity was quanti-
fied in a c-counter. Nonspecific binding was deter-
mined in the presence of 1 lM [Nle4,D-Phe7]a-MSH.
Specific binding was determined by subtracting non-
specific from total binding. In the competition assay,
concentration of the test compound that caused 50%
inhibition of the specific binding (IC50 value) was
determined from each concentration–response curve.
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