Novel N-biphenyl-2-ylmethyl 2-methoxyphenylpiperazinylalkanamides as 5-HT7R antagonists for the treatment of depression

Article abstract of DOI:10.1016/j.bmc.2014.07.026

5-HT₇ receptor (5-HT₇R) is a promising target for the treatment of depression and neuropathic pain. 5-HT₇R antagonists exhibited antidepressant effects, while the agonists produced strong anti-hyperalgesic effects. In our efforts to discover selective 5-HT ₇R antagonists or agonists, N-biphenylylmethyl 2- methoxyphenylpiperazinylalkanamides 1 were designed, synthesized, and biologically evaluated against 5-HT₇R. Among the synthesized compounds, N-2′-chlorobiphenylylmethyl 2- methoxyphenylpiperazinylpentanamide 1-8 showed the best binding affinity with a K_i value of 8.69 nM and it was verified as a novel antagonist according to functional assays. The compound 1-8 was very selective over 5-HT_1DR, 5-HT_2AR, 5-HT₃R, 5-HT_5AR and 5-HT₆R and moderately selective over 5-HT_1AR, 5-HT_1BR and 5-HT_2CR. The novel 5-HT₇R antagonist 1-8 exhibited an antidepressant effect at a dose of 25 mg/kg in the forced swimming test in mice and showed a U-shaped dose-response curve which typically appears in 5-HT₇R antagonists such as SB-269970 and lurasidone.

Full text of DOI:10.1016/j.bmc.2014.07.026

Bioorganic & Medicinal Chemistry 22 (2014) 4587–4596
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel N-biphenyl-2-ylmethyl
2-methoxyphenylpiperazinylalkanamides
as 5-HT7R antagonists for the treatment of depression
Youngjae Kim a,b, Jinsung Tae b, Kangho Lee c,d, Hyewhon Rhim c,d, Il Han Choo e, Heeyeong Cho f,
Woo-Kyu Park f, Gyochang Keum a,g, , Hyunah Choo a,g,
⇑
,
⇑
,
a Center for Neuro-Medicine, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, Republic of Korea
b Department of Chemistry, Yonsei University, Seodaemun-gu, Seoul 120-749, Republic of Korea
c Center for Neuroscience, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, Republic of Korea
d Department of Neuroscience, University of Science and Technology, Yuseong-gu, Daejeon 305-350, Republic of Korea
e School of Medicine, Chosun University, Dong-gu, Kwangju 501-759, Republic of Korea
f Pharmacology Research Center, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejeon 305-343, Republic of Korea
g Department of Biological Chemistry, University of Science and Technology, Yuseong-gu, Daejeon 305-350, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
5-HT7 receptor (5-HT7R) is a promising target for the treatment of depression and neuropathic pain. 5-HT7R
antagonists exhibited antidepressant effects, while the agonists produced strong anti-hyperalgesic effects.
In our efforts to discover selective 5-HT7R antagonists or agonists, N-biphenylylmethyl 2-meth-
oxyphenylpiperazinylalkanamides 1 were designed, synthesized, and biologically evaluated against
5-HT7R. Among the synthesized compounds, N-20-chlorobiphenylylmethyl 2-methoxyphenylpiperazinyl-
pentanamide 1–8 showed the best binding affinity with a Ki value of 8.69 nM and it was verified as a novel
antagonist according to functional assays. The compound 1–8 was very selective over 5-HT1DR, 5-HT2AR,
5-HT3R, 5-HT5AR and 5-HT6R and moderately selective over 5-HT1AR, 5-HT1BR and 5-HT2CR. The novel
5-HT7R antagonist 1–8 exhibited an antidepressant effect at a dose of 25 mg/kg in the forced swimming test
in mice and showed a U-shaped dose–response curve which typically appears in 5-HT7R antagonists such as
SB-269970 and lurasidone.
Received 2 July 2014
Revised 16 July 2014
Accepted 17 July 2014
Available online 25 July 2014
Keywords:
Serotonin receptor
5-HT7R
Antagonist
Depression
Antidepressant
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, attention has been given to 5-HT7Rs because of their
potential roles in depression, sleep disorders, stress, anxiety, learn-
Serotonin (5-hydroxytryptamine, 5-HT), first isolated in 1948, is
one of neurotransmitters and exerts signal transduction by activat-
ing its receptors to effect on the central and peripheral nervous
system.1 Based on the structure, pharmacology and amino acid
sequence, serotonin receptors (5-HTR) have been divided into
seven families 5-HT1R–5-HT7R with total fourteen distinct
subtypes. Except 5-HT3R, a member of the superfamily of ligand-
gated ion channels, most of serotonin receptors belong to G protein
coupled receptors (GPCRs). 5-HT7R is the most recently cloned
subtype of the serotonin receptor family and has four isoforms of
5-HT7aR, 5-HT7bR, 5-HT7cR, and 5-HT7dR, which are all coupled to
ing and memory deficiencies, and neuropathic pain. In particular,
there have been observations such as down regulation of 5-HT7Rs
after chronic antidepressant administration and antidepressant-
like behavioral effects in 5-HT7R-knock-out mice, which suggested
the use of 5-HT7R antagonists as possible antidepressants.5–7 On
the other hand, it has been reported that 5-HT7R activation by sys-
temically administered agonists produced strong anti-hyperalgesic
effects in animal models.8 Recent studies have also reported that
treatment of 5-HT7R agonists 8-OH-DPAT and LP-211 has made
significant neurite outgrowth and suggested that selective activa-
tion of 5-HT7R may represent a novel therapeutic strategy in the
treatment of Fragile X syndrome and autistic spectrum disorder.9
Until now, only a few 5-HT7R modulators have been reported.10
5-HT7R antagonists such as DR-4004 and SB-269970 are being
developed for the treatment of depression and sleep disorders
(Fig. 1).11 On the other hand, LP-211 and LP-44 are known as
5-HT7R agonists, of which an amide and an arylpiperazine group
G
a
protein.2–4
s
⇑
Corresponding authors.
E-mail addresses: gkeum@kist.re.kr (G. Keum), hchoo@kist.re.kr (H. Choo).
These two corresponding authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.07.026
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

4588
Y. Kim et al. / Bioorg. Med. Chem. 22 (2014) 4587–4596
NC
H
N
N
N
O
N
O
N
H
DR-4004
LP-211
LP-44
H
N
N
SCH3
N
S
O
N
HO
N
O
O
SB-269970
Figure 1. 5-HT7R antagonists and agonists.
are reminiscent of a 5-HT7R antagonist, DR-4004.12 In recent pub-
lications,13 we have demonstrated the pharmacological efficacy of
aryl biphenylylmethylpiperazines as 5-HT7R antagonists with their
biphenyl and o-methoxyphenylpiperazine moieties being the key
elements for the 5-HT7R activity. Structure–activity relationship
study in conjunction with molecular docking study proposed that
there should be a specific hydrophobic binding site for aromatic
o-methoxy group within the arylpiperazine. Therefore, we
designed new biphenyl 5-HT7R modulators 1 with the o-methoxy-
phenylpiperazine moiety combined with the characteristic amide
linkages of DR-4004, LP-211 and LP-44 (Fig. 2). A series of N-biphenyl-
2-ylmethyl 2-methoxyphenylpiperazinylalkanamides (1) were
synthesized and tested for binding affinity as well as antagonistic
effects against 5HT7R. The antidepressant effects of the title com-
pounds were then further tested in the forced swimming test.
hexafluorophosphate (HATU) in acetonitrile at room temperature
to provide the desired compounds, N-biphenyl-2-ylmethyl
2-methoxyphenylpiperazinylalkanamides 1 (Scheme 2). For the
purpose of optimizing the distances between the amide and
the piperazine functionalities of 1, four different alkyl spacers
(n = 2–5) were employed. Also, various aryl substituents (R) were
imposed on the biphenyl moiety to find the substituent with the
best binding affinity to the 5-HT7R.
2.2. Pharmacology
The binding affinities of the synthesized compounds 1–1 to
1–19 to 5-HT7R were determined by [3H]D-lysergic acid diethyl-
amide ([3H]LSD) radioligand binding assays in transfected
CHO-K1 cells.15 SB-269970 was used as a reference compound. To
find the proper carbon chain length, unsubstituted N-biphenylylm-
ethyl 2-methoxyphenylpiperazinylalkanamides 1–1 to 1–4 (n =
2–5) were evaluated against 5-HT7R and the binding affinity values
(Kis) were between 37.9 nM and 186.5 nM (Table 1). The longer
carbon chain gave the better binding affinity to 5-HT7R until
n = 4. The compound 1–3 with n = 4 showed the best binding affin-
ity with a Ki value of 37.9 nM and its homologue 1–4 (n = 5) also
showed good binding affinity with a Ki value of 43.7 nM.
The most potent 5-HT7R binder 1–3 was then further elaborated
by introduction of various substituents (R) on the biphenyl moiety
(Table 2). The binding affinities of compounds 1–5, 1–6 and 1–7
with fluorine substituents are almost the same regardless of sub-
stitution positions (ortho, meta and para-positions) with Ki values
of 36.5 nM, 34.7 nM and 34.7 nM, respectively. In the case of chlo-
rine substituent, binding affinities were dependent on chlorine
positions; the 20-Cl-biphenyl analogue 1–8 was the strongest bin-
der to 5-HT7R with a Ki value of 8.69 nM, while the 30-Cl-biphenyl
congener 1–9 was the weakest binder among all the synthesized
compounds. The compounds 1–10 to 1–14 with monomethoxy or
dimethoxy group had similar binding affinities to the unsubstitu-
ent biphenyl compound 1–3. The meta-methoxy compound 1–12
showed good binding affinity with a Ki value of 22.0 nM, and the
o-methyl, m-methyl and p-methyl compounds (1–15, 1–16 and
1–17) also showed good binding affinities with Ki values of
26.1 nM, 47.6 nM and 66.1 nM, respectively.
2. Results and discussion
2.1. Chemistry
Synthesis of the titled compounds, N-biphenyl-2-ylmethyl
2-methoxyphenylpiperazinylalkanamides 1 (Fig. 1) was carried
out via amide coupling with biphenyl-2-ylmethylamines 5 and
2-methoxyphenylpiperazinylalkanoic acids 9 (Schemes 1 and 2).
The biphenyl-2-ylmethylamines 5 were synthesized in 2 steps by
Suzuki coupling reaction followed by cyanide reduction
(Scheme 1). Thus, 2-iodobenzonitrile 2 and arylboronic acids 3
underwent Suzuki coupling to afford biphenyl-2-carbonitriles 4
which were reduced to the primary amines 5 using lithium alumi-
num hydride and aluminum chloride in THF (Scheme 1).14 The
2-methoxyphenylpiperazinylalkanoic acids 9 were synthesized
from 2-methoxyphenylpiperazine
6 via alkylation with ethyl
bromoalkanoates 7 followed by hydrolysis. 2-Methoxyphenylpip-
erazinylalkanoic acids 9, thus obtained, were coupled with
biphenyl-2-ylmethylamines
zotriazol-1-yl)-1,1,3,3-tetramethyluronium
5
by treating with 2-(1H-ben-
hexafluorophosphate
(HBTU) or 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
MeO
Variation of the alkyl spacer of 1–8 (n = 4) resulted in 2- (1–19,
n = 5) to 5-fold (1–18, n = 3) decrease in binding affinity to 5-HT7R
(Table 3), which confirmed that the compound 1–8 has the opti-
mum length of the alkyl spacer.
For the compound 1–8 and 1–19, which had the best binding
affinities in this series, the selectivity profile over other subtype
serotonin receptors was examined and the results are summarized
in Table 4. The compound 1–8 showed significant selectivities over
R
O
N
N
N
H
n
1
Figure 2. Designed compound 1.

Y. Kim et al. / Bioorg. Med. Chem. 22 (2014) 4587–4596
4589
R
R
I
R
a
b
CN
+
CN
NH2
B(OH)2
2
3
4
5
Scheme 1. Synthesis of biphenylylmethylamines 5. Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, DMF, reflux, overnight, 15–99%; (b) LAH, AlCl3, THF, 0 °C ? rt, 60–75%.
MeO
N
MeO
N
MeO
N
O
a
b
O
O
Br
n
+
EtO
N
N
HN
EtO
HO
n
n
6
7
8
9
c
MeO
N
R
O
N
N
H
n
1
Scheme 2. Synthesis of the targeted compound 1. Reagents and conditions: (a) EtOH, reflux, NaHCO3, 59–74%; (b) 5% NaOH, dioxane, rt, 18–73%; (c) 5, HBTU or HATU, Et3N,
MeCN, rt, 70–86%.
Table 1
Table 2
Binding affinities (Kis) to 5-HT7 receptor depending on the carbon chain (n)
Binding affinities (Ki’s) to 5-HT7 receptor depending on various substituents on the
biphenyl group
R
O
N
O
N
n
OMe
N
H
N
H
N
OMe
N
Entry
Compd
n
Kia in nM
1
2
3
4
1–1
1–2
1–3
1–4
2
3
4
5
186.5 29.8
64.5 10.3
37.9 5.4
43.7 5.2
Entry
Compd
R
Kia in nM
1
2
3
4
5
6
7
8
9
10
11
12
13
1–5
1–6
1–7
1–8
20-F
36.5 5.9
34.7 4.9
34.7 5.0
8.69 0.77
405.3 55.2
25.9 3.5
31.4 3.2
22.0 2.2
43.7 6.4
32.4 5.4
26.1 2.7
47.6 7.9
66.1 9.8
30-F
a
40-F
Values are the mean SEM of triplicate binding experiments.
20-Cl
30-Cl
40-Cl
1–9
1–10
1–11
1–12
1–13
1–14
1–15
1–16
1–17
5-HT1DR, 5-HT2AR, 5-HT3R, 5-HT5AR and 5-HT6R, but it was only
moderately selective over 5-HT1AR, 5-HT1BR and 5-HT2CR with
selectivity indexes (Ki for other subtype 5-HTR/Ki for 5-HT7R) of
2.3, 15 and 3.0, respectively. On the other hand, the compound
1–19 showed more potent binding affinities to 5-HT1AR (Ki = 11.0
nM) and 5-HT1BR (Ki = 7.0 nM) than to 5-HT7R (Ki = 15.9 nM), and
it showed only moderate selectivity over 5-HT1DR, 5-HT2AR, and
5-HT2CR. But still, the compound 1–19 was selective over 5-HT3R,
5-HT5AR and 5-HT6R. Taken together, the compound 1–8 was the
most potent 5-HT7R binder and it showed broad selectivity profile
over other subtype serotonin receptors.
In functional assays to classify the 5-HT7R binders as antago-
nists or agonists, the compound 1–8 was evaluated for adenylate
cyclase activity by using HEK293 cells transiently transfected with
human 5-HT7R (Fig. 3).16 In the antagonist test, 5-HT (serotonin)-
induced increase in cAMP level by the HEK293 cells was monitored
upon addition of 1–8 and the positive control, SB-269970, and both
20-OMe
30-OMe
40-OMe
20,60-diOMe
20-Me
30-Me
40-Me
a
Values are the mean SEM of triplicate binding experiments.
compounds showed potent inhibition of cAMP production (76.2%
and 102% inhibitions at 10 M, respectively) (Fig. 3a). In the ago-
nist test, in which a nonselective 5-HT7R agonist 5-CT was used
as positive control, the compound 1–8 showed only 22.4% cAMP
level increase, compared with cAMP level increase by 5-CT at
l
12.5
lM (Fig. 3b). According to these functional assays, the
compound 1–8 could be defined as a 5-HT7R antagonist.

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Y. Kim et al. / Bioorg. Med. Chem. 22 (2014) 4587–4596
Table 3
3. Conclusion
Binding affinities (Kis) to 5-HT7 receptor depending on the carbon chain (n) with
20-chlorobiphenyl group
Various N-biphenylylmethyl 2-methoxyphenylpiperazinylal-
kanamides 1 were designed, synthesized, and biologically evalu-
ated against 5-HT7R. Among the synthesized compounds, the
compound 1–8 showed the best binding affinity with a Ki value
of 8.69 nM and it was verified as a novel antagonist according to
functional assays. In the selectivity profile, the compound 1–8
was very selective over 5-HT1DR, 5-HT2AR, 5-HT3R, 5-HT5AR and
5-HT6R and moderately selective over 5-HT1AR, 5-HT1BR and
5-HT2CR. The novel 5-HT7R antagonist 1–8 exhibited an antide-
pressant effect at a dose of 25 mg/kg in the forced swimming test
in mice and a U-shaped dose–response curve which typically
appears in 5-HT7R antagonists such as SB-269970 and lurasidone.
In this study, we discovered a novel 5-HT7R antagonist, N-20-chlo-
Cl
O
N
N
n
OMe
N
H
Entry
Compd
n
Kia in nM
1
2
3
4
1–18
1–8
1–19
SB-269970
3
4
5
46.6 7.7
8.69 0.77
15.9 1.7
0.34 0.4
a
Values are the mean SEM of triplicate binding experiments.
robiphenylylmethyl
2-methoxyphenylpiperazinylpentanamide
1–8 and further pharmacokinetic and pharmacodynamic profile
studies are in progress and will be reported in due course.
The compound 1–8 and fluoxetine were evaluated in the forced
swimming test in mice which is one of the most used tools for
screening potential antidepressants (Fig. 4).17 Fluoxetine, a selec-
tive serotonin reuptake inhibitor, was intraperitoneally (ip) admin-
istrated and the total duration of immobility was reduced to 87.8%
at a dose of 10 mg/kg. The compound 1–8 was also ip injected at
three doses of 10, 25 and 50 mg/kg and showed antidepressant
effect only at a dose of 25 mg/kg (immobility reduction to 84.9%).
According to the in vivo animal study, the 5-HT7R antagonist, com-
pound 1–8, showed antidepressant effect only at a certain dose,
where the dose–response curves were U-shaped. In the literature,
5-HT7R antagonists, SB-269970 and lurasidone also showed a
U-shaped dose–response curve both in forced swimming test
and tail suspension test.5c,18 The U-shaped or bell-shaped
dose–response relationship of SB-269970 occurred in other
in vivo animal studies such as reversal of the hypothermic effect
by a nonselective 5-HT receptor agonist 5-CT and a 5-HT1A/7 recep-
tor agonist 8-OH-DPAT.19 However, it is difficult to explain the
cause of the U-shaped or bell-shaped dose–response dependency
of the 5-HT7R antagonist SB-269970.
4. Experimental section
4.1. Chemicals and instrumentation
General: All reactions were carried out under dry nitrogen or
argon unless otherwise indicated. Commercially available reagents
were used without further purification. Solvents and gases were
dried according to standard procedures. Organic solvents were
evaporated with reduced pressure using a rotary evaporator.
Analytical thin layer chromatography (TLC) was performed using
glass plates precoated with silica gel (0.25 mm). TLC plates were
visualized by exposure to UV light (UV), and then were visualized
with a KMnO4 stain followed by brief heating on hot plate. Flash
column chromatography was performed using silica gel 60
(230–400 mesh, Merck) with the indicated solvents. 1H and 13C
spectra were recorded on Bruker 300, Bruker 400 or Varian 300
NMR spectrometers. 1H NMR spectra are represented as follows:
Table 4
Selectivities of the compounds 1–8 and 1–19 over other serotonin receptors
Compd
Kia in nM
5-HT7
5-HT1A
5-HT1B
5-HT1D
5-HT2A
5-HT2C
5-HT3
5-HT5A
5-HT6
1–8
1–19
8.69
15.9
20.0
11.0
131.0
7.0
418.0
122.0
478.0
77.0
26.0
70.0
>10,000
>10,000
1178.0
517.0
1517.0
1321.0
a
Values are the mean value of triplicate binding experiments.
(b) Agonist test
(a) Antagonist test
120.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
100.0
80.0
60.0
40.0
20.0
0.0
compound 1-8 SB-269970
compound 1-8
5-CT
Figure 3. Functional assays against 5-HT7R: (a) %inhibitions of cAMP production by compounds 1–8 and SB-269970 and (b) relative increase of cAMP level by compounds
1–8 compared with 5-CT.

Y. Kim et al. / Bioorg. Med. Chem. 22 (2014) 4587–4596
4591
Figure 4. Forced swimming test for fluoxetine (10 mg/kg) and the compounds 1–8 (10, 25 and 50 mg/kg). Each drug was injected intraperitoneally (ip) 30 min before the
testing, and the total duration of immobility was recorded (%-control) during the last 5 min of the 6-min testing period. Values are means SEM (n = 8). #P <0.05, when
compared to the vehicle control group.
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet,
q = quartet, m = multiplet, dd = doublet of doublet, td = triplet of
doublet, br s = broad singlet, br t = broad triplet), integration, and
coupling constant (J) in Hertz (Hz). 1H NMR chemical shifts are
reported relative to CDCl3 (7.26 ppm). 13C NMR was recorded rela-
tive to the central line of CDCl3 (77.0 ppm). LC/MS analyses were
performed on either Micromass QUATTRO micro™ or Agilent
6410 Triple Quad system. Purity was checked on a Waters HPLC
e2695 instrument equipped with a UV/Visible detector 2489 and
4.3. General procedure of various biphenyl-2-ylmethanamines
(5)
A stirring suspension of lithium aluminum hydride (1.1 equiv)
in anhydrous THF was treated dropwise under N2 with a solution
of aluminum chloride (0.9 equiv) in THF. The reaction mixture
was maintained at room temperature during the addition by
means of a water bath. After addition was complete, the reaction
mixture was stirred vigorously for 20 min and then cooled to 0°C
before adding dropwise a solution of biphenyl-2-carbonitrile 4
(1.0 equiv) in THF. After complete addition, stirring was continued
for 2 h and the reaction was quenched by slowly adding methanol
followed by water. The mixture was concentrated under reduced
pressure, and the residue was extracted with 1 N HCl. The aqueous
phase were combined, washed with EtOAc and neutralized by
addition of 10 N NaOH. The mixture was extracted with CH2Cl2,
the organic phase was combined, washed with water, dried over
Na2SO4 and evaporated under reduced pressure. The residue was
filtered through a pad of silica gel, and the filtrate was evaporated
and dried to give the product 5 in 60–75% yields.
a SunFire™ C18, 4.6 ꢀ 150 mm, 5
lm, reversed-phase column.
Standard conditions were as follows: eluent system A (MeCN),
system B (water/0.1 M ammonium acetate), a flow rate of 1 mL/
min and a gradient of (30–100%) A over 20 min were used with
detection at 254 and 280 nm.
4.2. General procedure for preparation of biphenyl-2-
carbonitriles (4)
To a solution of 2-iodobenzonitrile 2 (1.0 equiv) in DMF were
added boronic acid 3 (1.2 equiv), tetrakis-(triphenylphosphine)-
palladium (0.05 equiv) and Na2CO3 (1.5 equiv). The resulting
mixture was stirred and refluxed for 6 h. After termination of the
reaction, the solution was cooled to room temperature, partitioned
between sat. NaHCO3 soln and EtOAc, and then extracted with
EtOAc. The organic phase was washed with brine and dried over
MgSO4 and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (n-hexane/diethyl ether = 5:1) to
afford the desired product 4 as colorless solid in 15–99% yields.13c
4.3.1. Biphenyl-2-ylmethanamine (5–1)
The reaction of lithium aluminum hydride (2.15 ml, 2.15 mmol)
in anhydrous THF, aluminum chloride (234 mg, 1.76 mmol), and
biphenyl-2-carbonitrile (350 mg, 1.95 mmol) in THF (5 ml) gave
the title compound 5–1 (256 mg, 1.40 mmol, 72% yield). 1H NMR
(400 MHz, CDCl3) d 7.46 (d, J = 7.5 Hz, 1H), 7.41–7.40 (m, 2H),
7.37–7.32 (m, 3H), 7.30 (td, J = 7.5, 1.3 Hz, 1H), 7.24 (dd, J = 8.1,
2.1 Hz, 1H), 3.81 (s, 2H), 1.34 (s, 2H).
4.2.1. Biphenyl-2-carbonitrile (4–1)
The reaction of 2-iodobenzonitrile (500 mg, 2.18 mmol), phen-
ylboronic acid (320 mg, 2.62 mmol), tetrakis-(triphenylphos-
phine)-palladium (25 mg, 0.022 mmol), and Na2CO3 (346 mg,
3.27 mmol) in DMF (20 ml) gave the title compound 4–1
(372 mg, 2.08 mmol, 95% yield). 1H NMR (400 MHz, CDCl3) d 7.77
(dd, J = 7.7, 1.2 Hz, 1H), 7.64 (td, J = 7.7, 1.2 Hz, 1H), 7.58–7.42
(m, 7H).
4.3.2. (20-Chlorobiphenyl-2-yl)methanamine (5–2)
The reaction of lithium aluminum hydride (0.93 ml, 0.93 mmol)
in anhydrous THF, aluminum chloride (101 mg, 0.76 mmol), and
20-chlorobiphenyl-2-carbonitrile (180 mg, 0.84 mmol) in THF
(1 ml) gave the title compound 5–2 (120 mg, 0.55 mmol, 66%
yield). 1H NMR (300 MHz, CDCl3) d 7.53–7.26 (m, 7H), 7.17 (d,
J = 7.5 Hz, 1H), 3.66 (q, J = 14.4 Hz, 2H), 1.28 (br s, 2H).
4.2.2. 20-Chlorobiphenyl-2-carbonitrile (4–2)
4.4. General procedure of various ethyl 2-
methoxyphenylpiperazinylalkanoates (8)
The reaction of 2-iodobenzonitrile (500 mg, 2.18 mmol), 2-chlo-
rophenylboronic acid (410 mg, 2.62 mmol), tetrakis-(triphenyl-
phosphine)-palladium (25 mg, 0.022 mmol), and Na2CO3 (346 mg,
3.27 mmol) in DMF (20 ml) gave the title compound 4–2
(290 mg, 1.36 mmol, 62% yield). 1H NMR (400 MHz, CDCl3) d 7.77
(dd, J = 7.8, 1.1 Hz, 1H), 7.66 (td, J = 7.7, 1.3 Hz, 1H), 7.54–7.45
(m, 3H), 7.40–7.35 (m, 3H).
Ethyl bromoalkanoate (1 equiv) was added to the solution of
1-(2-methoxyphenyl)piperazine (1 equiv) in ethanol at room tem-
perature and the resulting solution was refluxed under nitrogen
atmosphere for 6 h. After the completion of reaction, the mixture
was cooled to room temperature. Sat. NaHCO3 was added to the

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Y. Kim et al. / Bioorg. Med. Chem. 22 (2014) 4587–4596
reaction mixture and the resulting solution was extracted with
CH2Cl2. The combined organic solution was dried over MgSO4
and concentrated. The residue was purified by silica gel column
chromatography (n-hexane/EtOAc = 1:1) to give the product 8 in
63–82% yields.
4.5.2. 4-(4-(2-Methoxyphenyl)piperazin-1-yl)butanoic acid
(9–2)
The reaction of ethyl 4-(4-(2-methoxyphenyl)piperazin-1-
yl)butanoate (500 mg, 1.63 mmol), and 5% NaOH (8.5 ml) in diox-
ane (8.5 ml) gave the title compound 9–2 (417 mg, 1.50 mmol,
92% yield). 1H NMR (400 MHz, CDCl3) d 7.07–6.87 (m, 4H), 3.87
(s, 3H), 3.23 (br s, 4H), 3.02 (br s, 4H), 2.81 (t, J = 5.7 Hz, 2H),
2.61 (t, J = 5.9 Hz, 2H), 1.91 (m, 2H).
4.4.1. Ethyl 3-(4-(2-methoxyphenyl)piperazin-1-yl)propanoate
(8–1)
The reaction of ethyl 3-bromopropionate (1.88 g, 10.4 mmol),
and 1-(2-methoxyphenyl)piperazine (2 g, 10.4 mmol) in ethanol
(100 mL) gave the title compound 8–1 (2.09 g, 7.15 mmol, 69%
yield). 1H NMR (400 MHz, CDCl3) d 7.26–6.85 (m, 4H), 4.15 (q,
J = 7.1 Hz, 2H), 3.86 (s, 3H), 3.09 (br s, 4H), 2.77 (t, J = 7.3 Hz, 2H),
2.68 (br s, 4H), 2.55 (t, J = 7.1 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H).
4.5.3. 5-(4-(2-Methoxyphenyl)piperazin-1-yl)pentanoic acid
(9–3)
The reaction of ethyl 5-(4-(2-methoxyphenyl)piperazin-1-
yl)pentanoate (3.6 g, 11.2 mmol), and 5% NaOH (20 ml) in dioxane
(20 ml) gave the title compound 9–3 (2.4 g, 8.21 mmol, 73% yield).
1H NMR (400 MHz, CDCl3) d 7.02–6.85 (m, 4H), 3.86 (s, 3H), 3.22
(br s, 4H), 3.01 (br s, 4H), 2.67 (t, J = 7.4 Hz, 2H), 2.30 (t,
J = 5.7 Hz, 2H), 1.72–1.64 (m, 4H).
4.4.2. Ethyl 4-(4-(2-methoxyphenyl)piperazin-1-yl)butanoate
(8–2)
The reaction of ethyl 4-bromobutanoate (507 mg, 2.60 mmol),
and 1-(2-methoxyphenyl)piperazine (500 mg, 2.60 mmol) in etha-
nol (40 mL) gave the title compound 8–2 (500 mg, 1.63 mmol, 63%
yield). 1H NMR (400 MHz, CDCl3) d 7.01–6.84 (m, 4H), 4.15 (q,
J = 7.1 Hz, 2H), 3.86 (s, 3H), 3.09 (br s, 4H), 2.65 (br s, 4H), 2.43
(t, J = 7.0 Hz, 2H), 2.36 (t, J = 7.4 Hz, 2H), 1.87 (quintet, J = 7.3 Hz,
2H), 1.26 (t, J = 7.1 Hz, 3H).
4.5.4. 6-(4-(2-Methoxyphenyl)piperazin-1-yl)hexanoic acid
(9–4)
The reaction of ethyl 6-(4-(2-methoxyphenyl)piperazin-1-
yl)hexanoate (3.4 g, 10.2 mmol), and 5% NaOH (50 ml) in dioxane
(50 ml) gave the title compound 9–4 (2.6 g, 8.49 mmol, 83% yield).
1H NMR (300 MHz, CDCl3) d 7.09–6.87 (m, 4H), 3.87 (s, 3H), 3.40
(br s, 4H), 3.28 (br s, 4H), 2.95 (t, J = 8.0 Hz, 2H), 2.32 (t,
J = 7.1 Hz, 2H), 1.88–1.81 (m, 2H), 1.72–1.63 (m, 2H), 1.48–1.40
(m, 2H).
4.4.3. Ethyl 5-(4-(2-methoxyphenyl)piperazin-1-yl)pentanoate
(8–3)
The reaction of ethyl 5-bromopentanoate (2.94 g, 14.0 mmol),
and 1-(2-methoxyphenyl)piperazine (3 g, 15.6 mmol) in ethanol
(100 mL) gave the title compound 8–3 (3.69 g, 11.5 mmol, 82%
yield). 1H NMR (300 MHz, CDCl3) d 7.03–6.86 (m, 4H), 4.15 (q,
J = 7.1 Hz, 2H), 3.87 (s, 3H), 3.12 (br s, 4H), 2.66 (br s, 4H), 2.44
(t, J = 7.4 Hz, 2H), 2.36 (t, J = 7.1 Hz, 2H), 1.75–1.58 (m, 4H), 1.27
(t, J = 7.1 Hz, 3H).
4.6. General procedure of various N-biphenylylmethyl
2-methoxyphenylpiperazinylalkanamides (1)
HBTU or HATU (1.3 equiv) and Et3N (4 equiv) were added to a
solution of (4-(2-methoxyphenyl)piperazin-1-yl)alkanoic acid 9
(1.1 equiv) in acetonitrile at room temperature. The resulting mix-
ture was stirred at room temperature for 30 min. A solution of
biphenyl-2-ylmethanamine (1 equiv) in acetonitrile was added to
the reaction mixture. The resulting mixture was stirred for addi-
tional 6 h. After reaction was completed, MeOH was added to the
reaction mixture and the resulting solution was evaporated. The
residue was purified with by silica gel column chromatography
(CH2Cl2/mixture (CH2Cl2/MeOH/H2O/NH3 = 80:20:1:1) = 8/1) to
give the desired product 1 in 23–98 % yields.
4.4.4. Ethyl 6-(4-(2-methoxyphenyl)piperazin-1-yl)hexanoate
(8–4)
The reaction of ethyl 6-bromohexanoate (3.57 g, 16.0 mmol),
and 1-(2-methoxyphenyl)piperazine (4 g, 20.8 mmol) in ethanol
(150 mL) gave the title compound 8–4 (3.52 g, 10.5 mmol, 66%
yield). 1H NMR (400 MHz, CDCl3) d 7.00–6.84 (m, 4H), 4.13
(q, J = 7.2 Hz, 2H), 3.87 (s, 3H), 3.10 (br s, 4H), 2.65 (br s, 4H),
2.40 (t, J = 7.6 Hz, 2H), 2.31 (t, J = 7.5 Hz, 2H), 1.66 (quintet,
J = 7.7 Hz, 2H), 1.56–1.54 (m, 2H), 1.38–1.36 (m, 2H), 1.26
(t, J = 7.1 Hz, 3H).
4.6.1. N-(Biphenyl-2-ylmethyl)-3-(4-(2-methoxyphenyl)
piperazin-1-yl)propanamide (1–1)
The reaction of 3-(4-(2-methoxyphenyl)piperazin-1-yl)propa-
noic acid (80 mg, 0.30 mmol), biphenyl-2-ylmethanamine (50 mg,
4.5. General procedure of various 2-
methoxyphenylpiperazinylalkanoic acids (9)
0.27 mmol), HBTU (133 mg, 0.35 mmol) and Et3N (151 ll,
1.08 mmol) in acetonitrile (3 ml) gave the titled compound 1–1
(60 mg, 0.14 mmol, 52% yield). 1H NMR (400 MHz, CDCl3) d 8.57
(br s, 1H), 7.46–7.25 (m, 9H), 7.01 (td, J = 7.8, 1.5 Hz, 1H), 6.92
(td, J = 7.6 Hz, 1.5 Hz, 1H), 6.85 (dd, J = 7.8, 1.3 Hz, 1H), 6.82 (dd,
J = 7.8, 1.5 Hz, 1H), 4.40 (d, J = 5.2 Hz, 2H), 3.84 (s, 3H), 2.91 (br s,
4H), 2.66–2.63 (m, 6H), 2.40 (t, J = 6.3 Hz, 2H), 1.69 (qd, J = 10.8,
3.6 Hz, 2H); 13C NMR (100 MHz, CDCl3) d 172.1, 152.3, 141.7,
140.9, 140.7, 135.8, 130.3, 129.1, 128.8 (2C), 128.4, 127.7, 127.4,
127.3, 123.2, 121.0, 118.2, 111.2, 55.4, 53.9, 52.6, 50.5, 41.1, 31.9;
LC/MS (ESI+): m/z: calcd for C27H31N3O2: 429.56, [M+H]+; found:
430.55.
5% NaOH was added to the solution of ethyl 2-methoxyphenyl-
piperazinyl alkanoates (1 equiv) in dioxane at room temperature.
The resulting solution was stirred at room temperature for 2 h.
After removing solvent under reduced pressure, the residue was
dissolved in minimum amount of water and acidified with 1 N
HCl solution until pH 6 or 5. The precipitate was filtered and dried
to give the product 9 in 73–95% yields.
4.5.1. 3-(4-(2-Methoxyphenyl)piperazin-1-yl)propanoic acid
(9–1)
The reaction of ethyl 3-(4-(2-methoxyphenyl)piperazin-1-
yl)propanoate (2 g, 6.84 mmol), and 5% NaOH (10 ml) in dioxane
(10 ml) gave the title compound 9–1 (1.72 g, 6.51 mmol, 95%
yield). 1H NMR (400 MHz, CDCl3) d 7.07–7.03 (m, 1H), 6.97–6.88
(m, 3H), 3.88 (s, 3H), 3.20 (br s, 4H), 2.93 (br s, 4H), 2.86 (t,
J = 6.3 Hz, 2H), 2.58 (t, J = 6.1 Hz, 2H).
4.6.2. N-(Biphenyl-2-ylmethyl)-4-(4-(2-methoxyphenyl)
piperazin-1-yl)butanamide (1–2)
The reaction of 4-(4-(2-methoxyphenyl)piperazin-1-yl)buta-
noic acid (83 mg, 0.30 mmol), biphenyl-2-ylmethanamine (50 mg,
0.27 mmol), HBTU (133 mg, 0.35 mmol) and Et3N (151
1.08 mmol) in acetonitrile (3 ml) gave the titled compound 1–2
ll,

Y. Kim et al. / Bioorg. Med. Chem. 22 (2014) 4587–4596
4593
(48 mg, 0.11 mmol, 40% yield). 1H NMR (400 MHz, CDCl3) d 7.41–
7.28 (m, 8H), 7.22 (dd, J = 7.3, 1.5 Hz, 1H), 7.05 (m, 1H), 6.91 (t,
J = 7.5 Hz, 1H), 6.88–6.86 (m, 2H), 6.65 (br s, 1H), 4.37 (d,
J = 5.6 Hz, 2H), 3.85 (s, 3H), 3.13 (br s, 8H), 2.98 (t, J = 5.6 Hz, 2H),
2.48 (t, J = 5.7 Hz, 2H), 1.89 (m, 2H); 13C NMR (100 MHz, CDCl3) d
174.2, 152.2, 141.7, 140.7, 139.3, 134.7, 130.2, 129.1, 128.6,
128.4, 127.9, 127.6, 124.1, 121., 118.6, 111.5, 58.1, 55.5, 52.9,
48.6, 41.9, 34.5, 20.1; LC/MS (ESI+): m/z: calcd for C28H33N3O2:
443.59, [M+H]+; found: 444.55.
1–6 (27 mg, 0.057 mmol, 23% yield). 1H NMR (300 MHz, CDCl3) d
7.44–7.29 (m, 4H), 7.21 (dd, J = 7.2 Hz, 1.5 Hz, 1H), 7.08–6.85 (m,
7H), 6.29 (br s, 1H), 4.37 (d, J = 5.6 Hz, 2H), 3.86 (s, 3H), 3.18 (br
s, 4H), 3.02 (br s, 4H), 2.83 (br s, 2H), 2.30–2.26 (m, 2H), 1.74–
1.62 (m, 4H); 13C NMR (75 MHz, CDCl3) d 173.5, 162.5 (d,
J = 245 Hz), 152.3, 142.9, 142.8, 140.2, 139.7, 135.1, 130.0 (d,
J = 4 Hz), 128.4 (d, J = 10 Hz), 127.5, 124.9 (d, J = 3 Hz), 123.9,
121.1, 118.6, 116.0 (d, J = 21 Hz), 114.2 (d, J = 21 Hz), 111.5, 57.1,
55.5, 53.1, 48.8, 41.6, 34.8, 24.3, 21.9; LC/MS (ESI+): m/z: calcd
for C29H34FN3O2: 475.61, [M+H]+; found: 476.55.
4.6.3. N-(Biphenyl-2-ylmethyl)-5-(4-(2-methoxyphenyl)
piperazin-1-yl)pentanamide (1–3)
4.6.7. N-((40-Fluorobiphenyl-2-yl)methyl)-5-(4-(2-
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (88 mg, 0.30 mmol), biphenyl-2-ylmethanamine (50 mg,
0.27 mmol), HBTU (133 mg, 0.35 mmol) and Et3N (151 ll,
methoxyphenyl)piperazin-1-yl)pentanamide (1–7)
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (88 mg, 0.30 mmol), HBTU (122 mg, 0.32 mmol), TEA
1.08 mmol) in acetonitrile (3 ml) gave the titled compound 1–3
(90 mg, 0.20 mmol, 73% yield). 1H NMR (400 MHz, CDCl3) d 7.43–
7.23 (m, 9H), 7.03 (td, J = 7.0, 2.0 Hz, 1H), 6.94–6.86 (m, 3H), 5.87
(br s, 1H), 4.41 (d, J = 5.6 Hz, 2H), 3.86 (s, 3H), 3.12 (br s, 4H),
2.81 (br s, 4H), 2.62–2.59 (m, 2H), 2.21–2.18 (m, 2H), 1.63 (m,
4H); 13C NMR (100 MHz, CDCl3) d 173.0, 152.3, 141.6, 140.7,
130.3, 129.0, 128.6, 128.5, 127.9, 127.5, 127.4, 123.5, 121.0,
118.4, 111.3, 100.0, 57.6, 55.4, 53.3, 49.7, 41.6, 35.5, 28.3, 22.6;
LC/MS (ESI+): m/z: calcd for C29H35N3O2: 457.62, [M+H]+; found:
458.55.
(140 l
l, 1.00 mmol), and (40-fluorobiphenyl-2-yl)methanamine
(50 mg, 0.25 mmol) in acetonitrile (3 ml) gave the titled compound
1–7 (74 mg, 0.16 mmol, 62% yield). 1H NMR (300 MHz, CDCl3) d
7.43–7.19 (m, 6H), 7.12–7.04 (m, 3H), 6.96–6.85 (m, 3H), 6.30 (br
s, 1H), 4.36 (d, J = 5.6 Hz, 2H), 3.87 (s, 3H), 3.21 (br s, 4H), 3.08
(br s, 4H), 2.91 (br s, 2H), 2.30–2.26 (m, 2H), 1.77–1.67 (m, 4H);
13C NMR (75 MHz, CDCl3) d 173.6, 159.9 (d, J = 247 Hz), 152.3,
140.5, 139.4, 136.6 (d, J = 3.6 Hz), 135.2, 130.7 (d, J = 8.3 Hz),
130.3, 128.4, 128.1, 127.5, 124.1, 121.6, 121.1, 118.6, 115.3 (d,
J = 21 Hz), 111.6, 56.9, 55.5, 53.0, 48.5, 41.7, 34.5, 23.9, 21.5; LC/
MS (ESI+): m/z: calcd for C29H34FN3O2: 475.61, [M+H]+; found:
476.55.
4.6.4. N-(Biphenyl-2-ylmethyl)-6-(4-(2-methoxyphenyl)
piperazin-1-yl)hexanamide (1–4)
The reaction of 6-(4-(2-methoxyphenyl)piperazin-1-yl)hexa-
noic acid (100 mg, 0.33 mmol), biphenyl-2-ylmethanamine
4.6.8. N-((20-Chlorobiphenyl-2-yl)methyl)-5-(4-(2-
methoxyphenyl)piperazin-1-yl)pentanamide (1–8)
(50 mg, 0.27 mmol), HBTU (133 mg, 0.35 mmol) and Et3N (151
l
l,
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (73.1 mg, 0.25 mmol), HBTU (113.7 mg, 0.30 mmol),
1.08 mmol) in acetonitrile (3 ml) gave the titled compound 1–4
(45 mg, 0.10 mmol, 35% yield). 1H NMR (300 MHz, CDCl3) d 7.47–
7.26 (m, 9H), 7.06–6.99 (m, 1H), 6.95–6.87 (m, 3H), 5.65 (br t,
J = 5.1 Hz, 1H), 4.43 (d, J = 5.4 Hz, 2H), 3.88 (s, 3H), 3.13 (br s,
4H), 2.74 (br s, 4H), 2.50 (t, J = 7.8 Hz, 2H), 2.13 (t, J = 7.5 Hz 2H),
1.52–1.66 (m, 4H), 1.38–1.33 (m, 2H); 13C NMR (75 MHz, CDCl3)
d 172.7, 152.2, 141.6, 141.0, 140.7, 135.5, 130.3, 129.0, 128.7,
128.5, 127.8, 127.5, 127.4, 123.1, 121.0, 118.3, 111.1, 58.5, 55.4,
53.5, 50.3, 41.5, 36.4, 27.0, 26.2, 25.3; LC/MS (ESI+): m/z: calcd for
C30H37N3O2: 471.64, [M+H]+; found: 472.60.
TEA (129 l
l, 0.92 mmol), and (20-chlorobiphenyl-2-yl)methan-
amine (50 mg, 0.23 mmol) in acetonitrile (3 ml) gave the titled
compound 1–8 (87 mg, 0.18 mmol, 77% yield). 1H NMR
(300 MHz, CDCl3) d 7.48–7.39 (m, 3H), 7.37–7.29 (m, 3H), 7.26–
7.20 (m, 1H), 7.16 (d, J = 7.1 Hz, 1H), 7.07 (td, J = 7.9 Hz, 1.9 Hz,
1H), 6.97–6.88 (m, 3H), 6.21 (br t, J = 5.6 Hz, 1H), 4.29–4.16 (m,
2H), 3.87 (s, 3H), 3.23 (br s, 4H), 3.13 (br s, 4H), 2.93 (br s, 2H),
2.26 (t, J = 6.0 Hz, 2H), 1.76–1.65 (m, 4H); 13C NMR (75 MHz,
CDCl3) d 173.5, 152.3, 139.4, 139.2, 138.7, 135.6, 133.1, 131.1,
130.0, 129.2, 128.5, 128.3, 127.5, 127.0, 124.1, 121.1, 118.7,
111.6, 56.9, 55.5, 53.0, 48.4, 41.8, 34.5, 23.9, 21.5; LC/MS (ESI+):
m/z: calcd for C29H34ClN3O2: 492.06, [M+H]+; found: 492.55.
4.6.5. N-((20-Fluorobiphenyl-2-yl)methyl)-5-(4-(2-
methoxyphenyl)piperazin-1-yl)pentanamide (1–5)
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (146.2 mg, 0.50 mmol), HATU (248 mg, 0.65 mmol), TEA
4.6.9. N-((30-Chlorobiphenyl-2-yl)methyl)-5-(4-(2-
methoxyphenyl)piperazin-1-yl)pentanamide (1–9)
(283
l
l, 2.00 mmol), and (20-fluorobiphenyl-2-yl)methanamine
(100 mg, 0.50 mmol) in acetonitrile (5 ml) gave the titled com-
pound 1–5 (170 mg, 0.36 mmol, 72% yield). 1H NMR (400 MHz,
CDCl3) d 7.46 (dd, J = 7.6 Hz, 1.5 Hz, 1H), 7.42–7.33 (m, 3H), 7.26–
7.12 (m, 4H), 7.06–7.02 (m, 1H), 6.95–6.87 (m, 3H), 6.00 (br s,
1H), 4.33 (br s, 2H), 3.86 (s, 3H), 3.15 (br s, 4H), 2.88 (br s, 4H),
2.69 (br s, 2H), 2.24 (t, J = 6.6 Hz, 2H), 1.67–1.65 (m, 4H); 13C
NMR (100 MHz, CDCl3) d 172.5, 159.5 (d, J = 243 Hz), 152.3,
141.4, 136.9, 135.2, 131.6 (d, J = 4 Hz), 130.5, 129.6 (d, J = 8 Hz),
128.8, 128.6, 128.0 (d, J = 16 Hz), 127.5, 124.4 (d, J = 4 Hz), 122.9,
121.1, 118.2, 115.6 (d, J = 22 Hz), 111.2, 58.2, 55.4, 53.5, 50.6,
41.4, 36.4, 26.4, 23.7; LC/MS (ESI+): m/z: calcd for C29H34FN3O2:
475.61, [M+H]+; found: 476.55.
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (148 mg, 0.50 mmol), HBTU (228 mg, 0.60 mmol), TEA
(259 l
l, 1.84 mmol), and (30-chlorobiphenyl-2-yl)methanamine
(100 mg, 0.46 mmol) in acetonitrile (5 ml) gave the titled com-
pound 1–9 (127 mg, 0.26 mmol, 56% yield). 1H NMR (300 MHz,
CDCl3) d 7.45–7.30 (m, 6H), 7.25–7.18 (m, 2H), 7.05–6.87 (m,
4H), 5.80 (br s, 1H), 4.42 (d, J = 5.7 Hz, 2H), 3.88 (s, 3H), 3.09 (br
s, 4H), 2.69 (br s, 4H), 2.48 (t, J = 7.2 Hz, 2H), 2.21 (t, J = 7.5 Hz,
2H), 1.70–1.58 (m, 4H); 13C NMR (75 MHz, CDCl3) d 172.6, 152.3,
142.5, 141.3, 140.2, 135.6, 134.3, 130.4, 129.6, 129.1, 128.7,
128.3, 127.5, 127.5, 127.2, 122.9, 121.0, 118.2, 111.2, 58.1, 55.4,
53.5, 50.6, 41.3, 36.4, 26.3, 23.6; LC/MS (ESI+): m/z: calcd for
C29H34ClN3O2: 492.06, [M+H]+; found: 492.55.
4.6.6. N-((30-Fluorobiphenyl-2-yl)methyl)-5-(4-(2-
methoxyphenyl)piperazin-1-yl)pentanamide (1–6)
4.6.10. N-((40-Chlorobiphenyl-2-yl)methyl)-5-(4-(2-
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (88 mg, 0.30 mmol), HBTU (122 mg, 0.32 mmol), TEA
(140
(50 mg, 0.25 mmol) in acetonitrile (3 ml) gave the titled compound
methoxyphenyl)piperazin-1-yl)pentanamide (1–10)
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (148 mg, 0.50 mmol), HBTU (228 mg, 0.60 mmol), TEA
l
l, 1.00 mmol), and (30-fluorobiphenyl-2-yl)methanamine
(259 l
l, 1.84 mmol), and (40-chlorobiphenyl-2-yl)methanamine

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Y. Kim et al. / Bioorg. Med. Chem. 22 (2014) 4587–4596
(100 mg, 0.46 mmol) in acetonitrile (5 ml) gave the titled com-
pound 1–10 (170 mg, 0.35 mmol, 75% yield). 1H NMR (400 MHz,
CDCl3) d 7.42–7.30 (m, 5H), 7.26–7.19 (m, 3H), 7.03–6.98 (m,
1H), 6.91–6.85 (m, 3H), 5.85 (br s, 1H), 4.40 (d, J = 5.6 Hz, 2H),
3.86 (s, 3H), 3.07 (br s, 4H), 2.66 (br s, 4H), 2.45 (t, J = 7.2 Hz,
2H), 2.19 (t, J = 7.3 Hz, 2H), 1.64–1.55 (m, 4H); 13C NMR (75 MHz,
CDCl3) d 172.7, 152.2, 141.1, 140.3, 139.0, 135.5, 133.4, 130.4,
130.2, 128. 7, 128.6, 128.2, 127.6, 123.1, 121.0, 118.2, 111.1, 58.0,
55.4, 53.4, 50.4, 41.3, 36.2, 26.1, 23.5; LC/MS (ESI+): m/z: calcd for
amine (50 mg, 0.21 mmol) in acetonitrile (3 ml) gave the titled
compound 1–14 (92 mg, 0.18 mmol, 85% yield). 1H NMR
(400 MHz, CDCl3) d 7.40–7.30 (m, 4H), 7.16–7.13 (m, 1H), 7.08–
7.03 (m, 1H), 6.95–6.87 (m, 3H), 6.68 (s, 1H), 6.66 (s, 1H), 6.02
(br s, 1H), 4.16 (d, J = 5.1 Hz, 2H), 3.86 (s, 3H), 3.71 (s, 6H), 3.24
(br s, 4H), 3.07 (br s, 4H), 2.87 (br s, 2H), 2.17 (br t, J = 6.3 Hz,
2H), 1.74–1.64 (m, 4H); 13C NMR (100 MHz, CDCl3) d 172.9,
157.5, 152.3, 136.6, 134.3, 131.4, 129.5, 129.0, 127.8, 127.6,
124.1, 121.1, 118.7, 111.5, 104.4, 57.1, 56.0, 55.5, 53.1, 48.7, 42.9,
34.7, 21.5; LC/MS (ESI+): m/z: calcd for C31H39N3O4: 517.67,
[M+H]+; found: 518.65.
C
29H34ClN3O2: 492.06, [M+H]+; found: 492.55.
4.6.11. N-((20-Methoxybiphenyl-2-yl)methyl)-5-(4-(2-
methoxyphenyl)piperazin-1-yl)pentanamide (1–11)
4.6.15. 5-(4-(2-Methoxyphenyl)piperazin-1-yl)-N-((20-
methylbiphenyl-2-yl)methyl)pentanamide (1–15)
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (76 mg, 0.26 mmol), HBTU (118 mg, 0.31 mmol), TEA
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (54 mg, 0.18 mmol), HBTU (74 mg, 0.20 mmol), TEA
(135 l
l, 0.96 mmol), and (20-methoxybiphenyl-2-yl)methanamine
(50 mg, 0.23 mmol) in acetonitrile (3 ml) gave the titled compound
1–11 (110 mg, 0.23 mmol, 98% yield). 1H NMR (400 MHz, CDCl3) d
7.42–7.31 (m, 4H), 7.21–7.18 (m, 1H), 7.14 (dd, J = 7.3 Hz, 1.8 Hz,
1H), 7.04–6.97 (m, 3H), 6.95–6.91 (m, 2H), 6.86 (d, J = 8.1 Hz,
1H), 5.85 (br s, 1H), 4.39–4.15 (m, 2H), 3.86 (s, 3H), 3.78 (s, 3H),
3.11 (br s, 4H), 2.73 (br s, 4H), 2.51 (t, J = 7.1 Hz, 2H), 2.15 (t,
J = 7.1 Hz, 2H), 1.67–1.53 (m, 4H); 13C NMR (100 MHz, CDCl3) d
174.0, 152.3, 141.6, 138.2, 136.8, 131.1, 130.5, 129.7, 129.2,
128.6, 127.9, 127.5, 121.0, 121.0, 120.6, 118.3, 117.5, 111.2,
111.0, 58.0, 55.6, 55.4, 53.4, 50.2, 45.6, 41.9, 36.1, 24.8; LC/MS
(ESI+): m/z: calcd for C30H37N3O3: 487.64, [M+H]+; found: 488.60.
(85 l
l, 0.60 mmol), and (20-methybiphenyl-2-yl)methanamine
(30 mg, 0.15 mmol) in acetonitrile (2 ml) gave the titled compound
1–15 (45 mg, 0.095 mmol, 64% yield). 1H NMR (300 MHz, CDCl3) d
7.45–7.41 (m, 1H), 7.39–7.21 (m, 5H), 7.17–7.11 (m, 2H), 7.04–6.98
(m, 1H), 6.96–6.93 (m, 2H), 6.87 (d, J = 7.5 Hz, 1H), 5.64 (br t,
J = 5.3 Hz, 1H), 4.36–4.06 (m, 2H), 3.87 (s, 3H), 3.09 (br s, 4H),
2.63 (br s, 4H), 2.41 (t, J = 7.0 Hz, 2H), 2.15 (t, J = 7.1 Hz, 2H), 2.08
(s, 3H), 1.69–1.48 (m, 4H); 13C NMR (75 MHz, CDCl3) d 172.4,
152.3, 141.3, 141.0, 140.2, 136.0, 135.8, 130.2, 129.8, 129.2,
128.4, 127.7, 127.3, 125.8, 122.9, 121.0, 118.2, 111.2, 58.5, 55.4,
53.5, 50.6, 41.4, 36.4, 26.4, 23.6, 20.1; LC/MS (ESI+): m/z: calcd for
C
30H37N3O2: 471.64, [M+H]+; found: 472.55.
4.6.12. N-((30-Methoxybiphenyl-2-yl)methyl)-5-(4-(2-
methoxyphenyl)piperazin-1-yl)pentanamide (1–12)
4.6.16. 5-(4-(2-Methoxyphenyl)piperazin-1-yl)-N-((30-
methylbiphenyl-2-yl)methyl)pentanamide (1–16)
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (145 mg, 0.52 mmol), HBTU (232 mg, 0.61 mmol), TEA
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (82 mg, 0.28 mmol), HBTU (124 mg, 0.33 mmol), TEA
(264 l
l, 1.88 mmol), and (30-methoxybiphenyl-2-yl)methanamine
(100 mg, 0.47 mmol) in acetonitrile (5 ml) gave the titled com-
pound 1–12 (90 mg. 0.18 mmol, 39%yield). 1H NMR (400 MHz,
CDCl3) d 7.43 (dd, J = 7.6 Hz, 1.5 Hz, 1H), 7.39–7.31 (m, 3H), 7.27–
7.25 (m, 1H), 7.07 (td, J = 7.6 Hz, 1.8 Hz, 1H), 6.97–6.85 (m, 6H),
6.06 (br s, 1H), 4.43 (d, J = 5.8 Hz, 2H), 3.88 (s, 3H), 3.83 (s, 3H),
3.22 (br s, 4H), 3.03 (br s, 4H), 2.84 (br s, 2H), 2.27 (t, J = 6.3 Hz,
2H), 1.75–1.67 (m, 4H); 13C NMR (100 MHz, CDCl3) d 173.4,
159.5, 152.3, 141.4, 135.1, 130.1, 129.5, 128.5, 128.0, 127.5,
124.0, 121.4, 121.1, 118.6, 114.7, 112.8, 111.4, 57.1, 55.5, 55.3,
53.1, 48.8, 41.8, 34.7, 24.3, 21.7; LC/MS (ESI+): m/z: calcd for
(140 l
l, 1.00 mmol), and (30-methybiphenyl-2-yl)methanamine
(50 mg, 0.25 mmol) in acetonitrile (3 ml) gave the titled compound
1–16 (106 mg, 0.22 mmol, 90% yield). 1H NMR (300 MHz, CDCl3) d
7.42–7.22 (m, 5H), 7.17 (d, J = 7.1 Hz, 1H), 7.11–7.00 (m, 3H), 6.95–
6.86 (m, 3H), 6.24 (br t, J = 4.9 Hz, 1H), 4.40 (d, J = 5.3 Hz, 2H), 3.86
(s, 3H), 3.11 (br s, 4H), 2.79 (br s, 4H), 2.60 (br s, 2H), 2.34 (s, 3H),
2.21 (br s, 2H), 1.65–1.62 (m, 4H); 13C NMR (75 MHz, CDCl3) d
173.1, 152.3, 141.7, 140.6, 140.5, 138.0, 135.4, 130.2, 129.8,
128.3, 128.1, 127.7, 127.3, 126.1, 123.4, 121.1, 118.4, 111.4, 57.6,
55.4, 53.2, 49.7, 41.5, 35.6, 25.3, 22.9, 21.5; LC/MS (ESI+): m/z: calcd
for C30H37N3O2: 471.64, [M+H]+; found: 472.55.
C
30H37N3O3: 487.64, [M+H]+; found: 488.60.
4.6.13. N-((40-Methoxybiphenyl-2-yl)methyl)-5-(4-(2-
methoxyphenyl)piperazin-1-yl)pentanamide (1–13)
4.6.17. 5-(4-(2-Methoxyphenyl)piperazin-1-yl)-N-((40-
methylbiphenyl-2-yl)methyl)pentanamide (1–17)
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (76 mg, 0.26 mmol), HBTU (118 mg, 0.31 mmol), TEA
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (89 mg, 0.30 mmol), HBTU (128 mg, 0.34 mmol), TEA
(135
l
l, 0.96 mmol), and (40-methoxybiphenyl-2-yl)methanamine
(142 l
l, 1.00 mmol), and (40-methybiphenyl-2-yl)methanamine
(50 mg, 0.23 mmol) in acetonitrile (3 ml) gave the titled compound
1–13 (103 mg, 0.21 mmol, 92% yield). 1H NMR (300 MHz, CDCl3) d
7.48–7.29 (m, 5H), 7.23–7.20 (m, 1H), 7.13–6.98 (m, 4H), 6.94–6.86
(m, 2H), 6.02 (br s, 1H), 4.39 (d, J = 5.6 Hz, 2H), 3.86 (s, 3H), 3.81 (s,
3H), 3.14 (br s, 4H), 2.87 (br s, 4H), 2.27 (br s, 2H), 2.24 (br s, 2H),
1.66–1.65 (m, 4H); 13C NMR (75 MHz, CDCl3) d 174.1, 158.6, 152.3,
141.1, 139.2, 135.2, 132.9, 130.3, 130.1, 128.0, 127.6, 127.4, 124.2,
121.1, 118.6, 113.9, 111.7, 56.7, 55.5, 55.3, 53.5, 48.1, 41.9, 34.4,
23.5, 21.4; LC/MS (ESI+): m/z: calcd for C30H37N3O3: 487.64,
[M+H]+; found: 488.60.
(50 mg, 0.25 mmol) in acetonitrile (3 ml) gave the titled compound
1–17 (55 mg, 0.12 mmol, 47%yield). 1H NMR (300 MHz, CDCl3) d
7.41–7.26 (m, 3H), 7.22–7.15 (m, 5H), 7.06 (td, J = 7.5 Hz, 1.2 Hz,
1H), 6.94–6.82 (m, 3H), 6.49 (br t, J = 5.7 Hz, 1H), 4.36 (d,
J = 5.7 Hz, 2H), 3.86 (s, 3H), 3.16 (br s, 4H), 3.05 (br s, 4H), 2.87
(t, J = 6.6 Hz, 2H), 2.38 (s, 3H), 2.26 (t, J = 6.0 Hz, 2H), 1.68–1.65
(m, 4H); 13C NMR (75 MHz, CDCl3) d 173.7, 152.2, 141.4, 139.5,
137.6, 137.0, 135.2, 130.2, 129.1, 128.9, 128.0, 127.7, 127.4,
124.0, 121.3, 118.5, 111.4, 56.9, 55.5, 52.9, 48.5, 41.7, 34.7, 24.0,
21.8, 21.2; LC/MS (ESI+): m/z: calcd for C30H37N3O2: 471.64,
[M+H]+; found: 472.55.
4.6.14. N-((20,60-Dimethoxybiphenyl-2-yl)methyl)-5-(4-(2-
methoxyphenyl)piperazin-1-yl)pentanamide (1–14)
4.6.18. N-((20-Chlorobiphenyl-2-yl)methyl)-4-(4-(2-
The reaction of 5-(4-(2-methoxyphenyl)piperazin-1-yl)penta-
noic acid (66 mg, 0.23 mmol), HBTU (102 mg, 0.27 mmol), TEA
methoxyphenyl)piperazin-1-yl)butanamide (1–18)
The reaction of 4-(4-(2-methoxyphenyl)piperazin-1-yl)buta-
noic acid (70.3 mg, 0.25 mmol), HBTU (113.7 mg, 0.30 mmol),
(116 l
l, 0.82 mmol), and (20,60-dimethoxybiphenyl-2-yl)methan-

Y. Kim et al. / Bioorg. Med. Chem. 22 (2014) 4587–4596
4595
TEA (129
l
l, 0.92 mmol), and (20-chlorobiphenyl-2-yl)methan-
which blocks phosphodiesterase enzyme degraded cAMP. Cells
were stimulated by 5-HT for 30 min and with or without pretreat-
ment of compounds for 10 min. After 30 min, cAMP labeled with the
dye d2 and anti-cAMP antibodies labeled with cryptate were added
into the cell plates. The plates were incubated at room temperature
for 1 h. The fluorescence intensity of accumulated cAMP level was
measured using Flexstation3 microplate reader (Molecular Devices,
Downingtown, PA).
amine (50 mg, 0.23 mmol) in acetonitrile (3 ml) gave the titled
compound 1–18 (75 mg, 0.16 mmol, 70% yield). 1H NMR
(300 MHz, CDCl3) d 7.46–7.26 (m, 5H), 7.24–7.20 (m, 1H), 7.12
(d, J = 7.2 Hz, 1H), 7.07–7.02 (m, 2H), 6.94–6.86 (m, 3H), 4.28–
4.14 (m, 2H), 3.84 (s, 3H), 3.20 (br s, 8H), 3.05 (br t, J = 5.6 Hz,
2H), 2.50 (br s, 2H), 1.85 (br s, 2H); 13C NMR (75 MHz, CDCl3) d
174.1, 152.3, 139.2, 139.2, 138.8, 135.3, 133.8, 131.3, 130.0,
129.4, 129.1, 128.5, 128.4, 127.5, 126.9, 124.2, 121.1, 118.6,
111.7, 58.1, 55.5, 52.8, 48.3, 41.9, 34.3, 19.8; LC/MS (ESI+): m/z:
calcd for C28H32ClN3O2: 478.03, [M+H]+; found: 478.50.
4.9. Forced swimming test in mice
ICR (CD-1) male mice (21–25 g) were obtained from Orient (Ori-
ent Corporation, Seoul, Korea). They were housed in a humidity
(60 5%) and temperature (21 2 °C)-controlled animal facility on
a 12:12 h light/dark cycle and were given a solid diet and tap water
ad libitum. All experimental procedures were performed during the
light cycle, and all experimental protocols were consistent with the
guidelines issued by Institutional Animal Care and Use Committee of
Korea Research Institute of Chemical Technology.
The forced swimming test was performed according to the
methods described by Porsolt et al.17c Each mouse was placed in
a 25-cm glass cylinder (10 cm diameter) containing 15 cm of water
maintained at 23 1 °C, and was forced to swim for 10 min.
Twenty-four hours later, the mouse was replaced into the cylinder
and the total duration of immobility was recorded during the last
5 min of the 6-min testing period. Mice are judged immobile when
they float in an upright position and make only small movements
to keep their head above water. Drugs were dissolved in 20%-
DMSO in 10% cremophor solution, and administered intraperitone-
ally 30 min before the testing.
4.6.19. N-((20-Chlorobiphenyl-2-yl)methyl)-6-(4-(2-
methoxyphenyl)piperazin-1-yl)hexanamide (1–19)
The reaction of 6-(4-(2-methoxyphenyl)piperazin-1-yl)hexa-
noic acid (78 mg, 0.25 mmol), HBTU (113.7 mg, 0.30 mmol), TEA
(129 l
l, 0.92 mmol), and (20-chlorobiphenyl-2-yl)methanamine
(50 mg, 0.23 mmol) in acetonitrile (3 ml) gave the titled compound
1–19 (69 mg, 0.16 mmol, 59% yield). 1H NMR (300 MHz, CDCl3) d
7.49–7.30 (m, 6H), 7.23–7.20 (m, 1H), 7.16 (d, J = 6.8 Hz, 1H),
7.05 (td, J = 7.5 Hz, 1.5 Hz, 1H), 6.94–6.84 (m, 3H), 6.03 (br t,
J = 4.9 Hz, 1H), 4.22 (d, J = 5.3 Hz, 2H), 3.86 (s, 3H), 3.22 (br s,
4H), 3.13 (br s, 4H), 2.90 (br t, J = 7.5 Hz, 2H), 2.17 (t, J = 7.1 Hz,
2H), 1.77–1.67 (m, 2H), 1.65–1.51 (m, 2H), 1.41–1.32 (m, 2H);
13C NMR (75 MHz, CDCl3) d 173.2, 152.2, 139.6, 139.2, 138.7,
135.7, 133.08, 131.1, 130.0, 129.5, 129.2, 128.5, 128.3, 127.5,
127.0, 124.0, 121.1, 118.6, 111.5, 57.6, 55.5, 53.2, 48.5, 41.7, 35.6,
25.7, 24.2; LC/MS (ESI+): m/z: calcd for C30H36ClN3O2: 506.09,
[M+H]+; found: 506.55.
4.7. Serotonin receptor binding affinity assay15c
Acknowledgements
Eleven dilutions (5 X assay concentration) of the test and refer-
ence compounds were prepared in Standard Binding Buffer
(50 mM Tris–HCl, 10 mM MgCl2, 1 mM EDTA, pH7.4) by serial dilu-
tion: 0.05 nM, 0.5 nM, 1.5 nM, 5 nM, 15 nM, 50 nM, 150 nM,
500 nM, 1.5 lM, 5 lM, 50 lM. Radioligand, which was properly
chosen for each serotonin subtype receptor, was diluted to five
Binding affinity data were generously provided by the US
National Institute of Mental Health (NIMH) Psychoactive Drug
Screening Program (Contract: HHSN-271-2008-00025-C). This
research was supported by the Basic Science Research Program
(NRF-2013-R1A1A2A10009907) funded by the National Research
Foundation of Korea (NRF). Additional funding was provided by
the Korea Institute of Science and Technology (KIST) Institutional
Program (2E24510).
times the assay concentration in Standard Binding Buffer. Aliquots
(50
wells of a 96-well plate containing 100
Buffer. Then, triplicate 50- l aliquots of the test and reference
compound dilutions were added. Finally, crude membrane 50-
fractions of cells expressing recombinant target are dispensed into
each well. The 250- l reactions are incubated at room temperature
ll) of the appropriate radioligand were dispensed into the
l
l of Standard Binding
l
l
l
References and notes
l
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ethyleneimine using a 96-well Brandel harvester. Four rapid
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