Synthesis and T-type calcium channel blocking activity of novel diphenylpiperazine compounds, and evaluation of in vivo analgesic activity

Article abstract of DOI:10.1016/j.bmc.2010.06.082

Novel diphenylpiperazine derivatives were synthesized and evaluated for their inhibitory activity against T-type calcium channel by whole-cell patch clamp recordings on HEK293 cells. Among the test compounds, 2 and 3d were effective in decreasing the response to formalin in both the first and second phases and demonstrated antiallodynic effects in a rat model of neuropathic pain.

Full text of DOI:10.1016/j.bmc.2010.06.082

Bioorganic & Medicinal Chemistry 18 (2010) 5938–5944
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and T-type calcium channel blocking activity of novel
diphenylpiperazine compounds, and evaluation of in vivo analgesic activity
Yoo Lim Kam a, Hee-Kyung Rhee b, Hyewhon Rhim c, Seung Keun Back d, Heung Sik Na d,
,
*
Hea-Young Park Choo a,
*
a School of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
b Department of Biological Science, Sookmyung Women’s University, Seoul 140-742, Republic of Korea
c Life Sciences Division, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea
d Department of Physiology, Korea University College of Medicine, Seoul 136-705, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel diphenylpiperazine derivatives were synthesized and evaluated for their inhibitory activity against
T-type calcium channel by whole-cell patch clamp recordings on HEK293 cells. Among the test com-
pounds, 2 and 3d were effective in decreasing the response to formalin in both the first and second
phases and demonstrated antiallodynic effects in a rat model of neuropathic pain.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 3 May 2010
Revised 23 June 2010
Accepted 24 June 2010
Available online 30 June 2010
Keywords:
T-type calcium channel blocker
Diphenylpiperazine
Formalin test
Neuropathic pain
1. Introduction
Antipsychotic drugs such as flunarizine and pimozide are used
clinically for the treatment of various psychiatric disorders
Low-voltage-activated (LVA or T-type) calcium channels are ex-
pressed in peripheral and central neurons of the pain pathway.
Three isoforms of T-type channel including Cav3.1 (a1G), Cav3.2
(Fig. 1). Flunarizine is a neuroleptic of the diphenylpiperazine class.
It has been described as one of the most potent neuronal T-type
calcium channel blockers.11 Flunarizine is known to block prefer-
(
a
1H), and Cav3.3 (
a
1I) have been identified and cloned. Recent evi-
entially Cav3.1 and Cav3.3 channels (Kd = 0.53 and 0.84
lM, respec-
À=À
dence using the
a
1G-deficient mice (
a
) suggested that Cav3.1
tively), compared with CaV3.2 channels (Kd = 3.6
l
M).12 Pimozide,
1G
played a pivot role in the mediation of physiological and patholog-
an antipsychotic, is a diphenylbutylpiperidine derivative that
blocks dopaminergic D2 receptors as other antipsychotics and neu-
roleptics. Pimozide markedly blocks the recombinant CaV3 chan-
nels (ꢀ40 nM) in the same concentration range as it binds to D2
receptors (Kd ꢀ29 nM).12,13 Also, a mixed N-type and T-type cal-
cium antagonist NP078585 (Fig. 1) was founded efficacious in ani-
mal models of chronic pain as well as attenuated ethanol
intoxication.14
In attempts to have T-type channel blocking activity, we de-
signed novel compounds possessing diphenylpiperazine moiety
with a flexible alkyl chain as a linker attached with hydrophobic
groups by mimicking flunarizine, pimozide, and mibefradil
(Fig. 2). In this study, we report the synthesis of novel series of
diphenylpiperazine derivatives and evaluation of their T-type cal-
cium channel blocking activities in whole-cell patch clamp record-
ings. According to their T-type calcium channel blocking activities,
two compounds (2 and 3d) were chosen for further in vivo studies
of inflammatory pain and also evaluated their analgesic efficacies
in a rat model of neuropathic pain.
ical pain.1–5
Mibefradil (PosicorÒ, Roche, Fig. 1), withdrawn from the mar-
ket due to several drug interactions, is often described as a selec-
tive T-type calcium channel blocker (IC50 values: ꢀ1
lM for
Cav3.1, Cav3.2, and Cav3.3; ꢀ10–20 M for L-type) and used
l
widely as a research tool to examine the functional role of T-type
calcium channels. It was reported that mibefradil showed an
antinociceptive property in both the first and second phases of
the formalin response.6–9 In the formalin test, biphasic behavioral
responses are induced by formalin when injected subcutane-
ously. An acute nociceptive injury is thought to be involved in
the first phase, while the second phase represents a model of
central sensitization.10
* Corresponding authors.
E-mail addresses: hsna@korea.ac.kr (Heung Sik Na), hypark@ewha.ac.kr
(Hea-Young Park Choo).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.082

Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 5938–5944
5939
O
F
N
O
N
N
O
H
N
F
N
F
mibefradil
flunarizine
F
F
F
N
O
N
O
O
N
NH
N
O
F
pimozide
NP078585
Figure 1. Structures of mibefradil, flunarizine, pimozide, and NP078585.
R = 3f =
3g =
R = 2 = H
3a =
NO2
O
H
N
R
N
3b =
3c =
O
N
3h =
Cl
3i
=
3d
=
N
Cl
3e =
3j =
Cl
Figure 2. Structures of novel diphenylpiperazine derivatives.
Scheme 1. The coupling reaction of 1-((4-chlorobenzhydryl)piper-
azine with 4-(Boc-amino)butyric acid and PyBOP afforded com-
pound 1. N-Boc deprotection of compound 1 using HCl–dioxane
gave compound 2, which was treated with aldehydes and sodium
triacetoxyborohydride to provide compounds 3a–j via reductive
amination. Each individual compound synthesized was character-
2. Results and discussion
2.1. Chemistry
The synthetic procedures are consisted of two main reactions:
coupling reaction and reductive amination as illustrated in
O
H
N
N
Boc
NH
O
i
N
H
N
N
+
HO
Boc
1
Cl
Cl
O
O
H
N
NH2
N
N
R
iii
N
N
ii
3a j
-
2
Cl
Cl
Scheme 1. Reagents and conditions: (i) PyBOP, DIEA, DMF, rt, 16 h; (ii) 4 M HCl–dioxane, CHCl3, 3 h; (iii) aldehydes, NaBH(OAc)3, TEA, DCE.

5940
Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 5938–5944
ized by 1H NMR, 13C NMR and high resolution MS. The structures of
different linkers and amines in both ends are illustrated in
Table 1.
Table 1
Pharmacological data of diphenylpiperazine derivatives
O
H
N
N
N
R
2.2. Effects on T-type and N-type calcium channels
The biological activity of the synthesized compounds was eval-
uated against HEK293 cells which stably express Cav3.1 (a1G
)
T-type calcium channels. The IC50 value of Ca2+ current was mea-
sured. As a result, five novel compounds (3a, 3b, 3d, 3f, and 3h)
Cl
inhibited T-type calcium current with IC50 values 61
the % inhibition of Ca2+ current on HEK293 cells which stably ex-
press Cav3.1 ( 1B) N-type calcium channels was measured at
10 M concentration of the compounds, and three compounds
lM. Also
Compds
R
HEK293 cell (T-
HEK293 cell (N-type:
a
1B) % inhibitionb
a
type:
M)
a
1G) IC50
a
(
l
(10 lM)
l
2
H
Ethyl
Isobutyl
3.71 0.67
0.42 0.04
0.36 0.01
1.54 0.16
0.19 0.01
1.67 0.13
0.60 0.08
16.10 0.41
0.28 0.03
2.32 0.04
10.03 3.58
20.03 5.68
13.30 4.50
16.80 5.30
84.86 2.37
69.84 1.85
88.75 2.03
49.60 9.17
80.24 2.68
10.79 1.38
(3d, 3f, and 3h) showed over 80% inhibition
3a
3b
3c
3d
3e
3f
3g
3h
3i
Compounds 3a and 3b containing ethyl and isobutyl group,
respectively, showed good selectivity on T-type over N-type cal-
cium channel antagonism. On the other hand, 3d, 3f, and 3h, with
cyclohexyl, 4-nitrobenzyl or 4-isopropyl amines, respectively,
inhibited not only T-type channel but also N-type calcium channel
2-Ethylbutyl
Cyclohexylmethyl
Benzyl
4-Nitrobenzyl
4-Methoxybenzyl
4-Isopropylbenzyl
4-
Dimethylaminobenzyl
2,6-Dichlorobenzyl
Mibefradil
ranged from 80% to 89% at 10 lM. In this respect, T-type calcium
channel antagonism is favored over N-type calcium channel inhibi-
tion when alkyl groups rather than benzyl groups are attached as R
group. The compound 3d with the most favorable T-type calcium
3j
1.02 0.16
1.34 0.49
78.41 4.04
67.63 1.21
channel blocking activity (IC50 = 0.19 0.01 lM) was further se-
a
IC50 value was determined from the dose–response curve.
% Inhibition value ( SE) was obtained by repeated procedures (n P 3) under
lected to verify in vivo analgesic effect. In addition, compound 2
with unsubstituted R group was also selected for in vivo study to
compare analgesic effects between substituted and unsubstituted
b
patch–clamp assay.
A
100
80
60
40
20
0
500
Comp 2 (60 g, n=6)
vehicle (n=12)
Comp 2 (60 g, n=6)
vehicle (n=12)
400
300
200
*
100
*
*
*
*
*
*
*
*
0
0
5
0-10 min
10 15 20 25 30 35 40 45 50 55 60
10-60 min
Post-injection time (min)
B
100
80
60
40
20
0
500
400
300
200
100
0
Comp 3d (6 g, n=6)
vehicle (n=12)
Comp 3d (60 g, n=6)
vehicle (n=12)
*
**
**
*
*
**
*
*
**
5
*
0
5
0-10 min
10-60 min
10 15 20 25 30 35 40 45 50
5
60
Post-injection time (min)
Figure 3. The effects of subcutaneous injection (60
biting in each 5-min interval following subcutaneous injection of formalin (5%, 50
and late (10–60 min) phases after formalin injection. Data are expressed as means SEM. Significance of differences was analyzed by Student‘s unpaired t-test. P <0.05.
lg) of compounds 2 (A) and 3d (B) on formalin-induced pain response. Values represent the time (s) spent paw licking or
ll). In the bar graph, values represent the response duration during the early (0–10 min)
*

Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 5938–5944
5941
R groups in rodent models of inflammatory and neuropathic pain.
The observed pharmacological data are represented in Table 1.
old and latencies in response to mechanical, cold and warm
water stimuli, respectively [P <0.05 vs presurgical value (À1),
one-way repeated measured ANOVA followed by Bonferroni’s t-
test]. We regarded these increased sensitivities as the signs of
mechanical, cold and warm allodynia, and these neuropathic ani-
mals were subjected to the treatment of compound 2 or 3d. As
shown in Figure 4, Compound 2 (60 mg/kg, ip) and gabapentin
(60 mg/kg, ip) significantly relieved the mechanical, cold and
2.3. Effects on formalin-induced pain
The rat formalin test, an inflammatory pain model, was exam-
ined for compounds 2 and 3d (Fig. 3). The rats treated with the
mixture of formalin and vehicle showed significant increase of
the time spent in licking, shaking or biting the affected paw during
both the early and late phases. However, compounds 2 and 3d
*
warm allodynia unlike vehicle group ( P <0.05 vs baseline, one-
way repeated measured ANOVA followed by Bonferroni’s t-test).
On the other hand, compound 3d (60 mg/kg, ip) significantly re-
lieved the cold and warm, but not mechanical, allodynia (Fig. 5,
(60 lg, sc) injected with formalin clearly reduced the response
duration in both phases. The response duration of pain behaviors
in the vehicle, 2 and 3d in the early phase was 47.05 8.53,
2.41 1.77, and 1.80 1.14 s, respectively. In the late phase, the
time spent in licking, shaking or biting the paw in each group
was 334.21 51.10, 62.57 20.07, and 24.32 14.08 s, respec-
tively. This result indicates that compounds 2 and 3d are effective
in attenuating both nociceptive and inflammatory pain when trea-
ted locally.
*
P <0.05 vs baseline, one-way repeated measured ANOVA fol-
lowed by Bonferroni’s t-test). Even though both 3d and 2 showed
analgesic effects, the behavioral results are not correlated with
the biological activities of the compounds to T-(a1G) type calcium
channels. Compound 3d, shown more robust inhibition against T-
type calcium channels, less effectively relieved the neuropathic
behaviors, especially mechanical allodynia than compound 2.
However, it is not unusual to observe this kind of results, be-
cause in vivo experiments the compounds were given ip and
the results could be affected by pharmacokinetic factors of com-
pounds, such as absorption, distribution, metabolism, and excre-
tion. Additionally, compound 2 may possess other antiallodynic
activities except the inhibitory effect against T-type or N-type
calcium channels.
2.4. Effects on neuropathic pain
Before nerve injury, animals rarely exhibited tail withdrawal
responses to von Frey filaments, cold (4 °C) and warm (40 °C)
water stimuli (Figs. 4 and 5). However, two weeks after nerve in-
jury, rats exhibited significant decrease of tail withdrawal thresh-
Anti-mechanical allodynic effect
Anti-cold allodynic effect
A
B
*
*
15
#
#
*
#
*
15
12
9
*
Comp 2 (n=5)
GBP (n=7)
12
9
*
Vehicle (n=7)
6
#
6
#
Comp 2 (n=4)
GBP (n=7)
#
3
3
Vehicle (n=7)
0
0
-1
1
7
14 1h 3h 5h 24h
(BL)
-1
1
7
14 1h 3h 5h 24h
(BL)
Experimental time (day or hour)
Experimental time (day or hour)
C
Anti-warm allodynic effect
* * *
#
#
#
15
12
9
6
Comp 2 (n=5)
GBP (n=7)
Vehicle (n=7)
3
0
-1
1
7
14 1h 3h 5h 24h
(BL)
Experimental time (day or hour)
Figure 4. The effect of compound 2 (compd 2) on mechanical (A), cold (B), and warm (C) allodynia induced by rat tail nerve injury. ‘À1’ represents the 1 day prior to nerve
injury. On 14 days after nerve injury, rats were given baseline (BL) test and then were subjected to the injection of compd 2 (60 mg/kg, ip), gabapentin (GBP, 60 mg/kg, ip) or
vehicle (methyl pyrrolidone/Tween 80/saline = 1:1:8). Behavioral tests were re-performed 1, 3, 5, and 24 h after the injection. P <0.05 (compd 2); #P <0.05 (GBP) versus BL
*
(one-way repeated measured ANOVA followed by Bonferroni’s t-test).

5942
Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 5938–5944
A
B
Anti-mechanical allodynic effect
Anti-cold allodynic effect
Comp 3d (n=8)
GBP (n=7)
Vehicle (n=7)
*
#
15
12
9
15
12
9
#
#
#
#
#
6
6
Comp 3d (n=8)
GBP (n=7)
Vehicle (n=7)
3
3
0
0
-1
1
7
14 1h 3h 5h 24h
(BL)
-1
1
7
14 1h 3h 5h 24h
(BL)
Experimental time (day or hour)
Experimental time (day or hour)
Anti-warm allodynic effect
C
#
*
#
*
15
12
9
#
6
Comp 3d (n=7)
GBP (n=7)
Vehicle (n=7)
3
0
-1
1
7
14 1h 3h 5h 24h
(BL)
Experimental time (day or hour)
Figure 5. The effect of compound 3d (compd 3d) on mechanical (A), cold (B), and warm (C) allodynia induced by rat tail nerve injury. ‘À1’ represents the 1 day prior to nerve
injury. On 14 days after nerve injury, rats were given baseline (BL) test and then were subjected to the injection of compd 3d (60 mg/kg, ip), gabapentin (GBP, 60 mg/kg, ip) or
vehicle (methyl pyrrolidone/Tween 80/saline = 1:1:8). Behavioral tests were re-performed 1, 3, 5, and 24 h after the injection. *P <0.05 (compd 3d); #P <0.05 (GBP) versus BL
(one-way repeated measured ANOVA followed by Bonferroni’s t-test).
from the Korea Basic Science Institute (Daegu) on a Jeol JMS 700
high resolution mass spectrometer. Reagents were of commercial
grade and were purchased from Sigma–Aldrich Co., Merck, Duck-
san Pure Chemical Co.
3. Conclusion
A series of novel diphenylpiperazine compounds were designed
and synthesized by a simple synthetic method and evaluated their
T-type calcium channel antagonism using path-clamp assay. Most
of the test compounds inhibited T-type calcium current with IC50
4.2. Synthesis of tert-butyl 4-(4-((4-chlorophenyl)(phenyl)
methyl)piperazin-1-yl)-4-oxobutylcarbamate (1)
values 61 lM and compound 3d showed the most favorable T-
type calcium channel antagonism. In addition, compound 2 and
3d produced statistically significant pain-relieving effects in both
the first and second phases induced by formalin. Furthermore,
compounds 2 and 3d demonstrated analgesic effects in rodent
model of neuropathic pain induced by peripheral nerve injury.
In 3 mL of DMF, 4-(Boc-amino)butyric acid (2.46 mmol), 1-((4-
chlorophenyl)(phenyl)methyl)piperazine (2.71 mmol) and PyBOP
(2.71 mmol) were dissolved. Then, 4.92 mmol of DIEA was added
and stirred at room temperature for 16 h. Twenty milliliters of
10% HCl was put into the reaction solution, and extracted with
30 mL of EtOAc. The organic layer was washed with 20 mL of
10% HCl, 20 mL of a saturated NaHCO3 solution twice and with
20 mL of a saturated NaCl solution twice. The organic layer was
collected, dried over anhydrous MgSO4, and filtered. The organic
solvent in the filtrate was removed under reduced pressure. The
residue was recrystallized from EtOAc to afford desired com-
pound. White solid, yield: 80%; 1H NMR (acetone-d6, 400 MHz)
4. Experimental
4.1. Materials and methods
Melting points were measured on an electrothermal digital
melting point (Buchi, Germany) without calibration. 1H NMR and
13C NMR spectra were recorded on Varian NMR AS and Varian
Unity Inova 400 NMR spectrometers. Chemical shifts were re-
ported in parts per million (d) units relative to the solvent peak.
The 1H NMR data were reported as peak multiplicities: s for sin-
glet; d for doublet; t for triplet; and m for multiplet. Coupling con-
stants were recorded in hertz. Mass spectral data were obtained
d
7.46–7.53 (m, 4H), 7.29–7.35 (m, 4H), 7.19–7.22 (m, 1H),
5.96 (br s, 1H), 4.39 (s, 1H), 3.53–3.56 (m, 4H), 3.07 (q,
J = 6.4 Hz, 2H), 2.32–2.40 (m, 6H), 1.70–1.77 (m, 2H), 1.38 (s,
9H); HR-FABMS calcd for C26H35O3N3Cl (M+H)+: 472.2367, found:
472.2365.

Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 5938–5944
5943
4.3. Synthesis of 4-amino-1-(4-((4-chlorophenyl)(phenyl)
methyl)piperazin-1-yl)butan-1-one (2)
(d, J = 6.4 Hz, 2H), 2.42 (t, J = 7.0 Hz, 2H), 2.35–2.39 (m, 4H),
1.78–1.85 (m, 2H), 1.62–1.74 (m, 4H), 1.12–1.28 (m, 4H), 0.84–
0.96 (m, 3H); 13C NMR (CDCl3, 100 MHz) d 171.59, 141.59,
140.79, 133.15, 129.32, 129.07, 129.01, 127.98, 127.71, 75.25,
54.27, 51.83, 51.48, 48.60, 35.02, 31.91, 31.14, 30.91, 26.17,
25.53; HR-FABMS calcd for C28H37ON3Cl (M+H)+: 468.2782, found:
468.2778.
CHCl3 (5 mL) and 4 M HCl–dioxane (5 mL) were added to com-
pound 1 (0.76 mmol) and was allowed to stand for 3 h. The solvent
and excess acid were removed under reduced pressure. White so-
lid, yield: 99%; mp 115–118 °C; 1H NMR (D2O, 400 MHz) d 7.63–
7.66 (m, 4H), 7.51–7.57 (m, 5H), 5.41 (s, 1H), 3.82–3.87 (m, 4H),
3.28–3.33 (m, 4H), 3.04 (t, J = 7.6 Hz, 2H), 2.60 (t, J = 7.2 Hz, 2H),
1.92–1.99 (m, 2H); 13C NMR (D2O, 100 MHz) d 172.96, 135.35,
133.63, 132.50, 130.10, 129.98, 129.75, 128.14; HR-FABMS calcd
for C21H27ON3Cl (M+H)+: 372.1843, found: 372.1842.
4.4.5. 4-(Benzylamino)-1-(4-((4-chlorophenyl)(phenyl)
methyl)piperazin-1-yl)-butan-1-one (3e)
Yellow oil; yield: 84%; 1H NMR (CDCl3, 400 MHz) d 7.21–7.42
(m, 14H), 4.21 (s, 1H), 3.9 (s, 2H), 3.56–3.62 (m, 2H), 3.42–3.46
(m, 2H), 2.80 (t, J = 6.4 Hz, 2H), 2.41 (t, J = 6.8 Hz, 2H), 2.32–2.38
(m, 4H), 1.88–1.94 (m, 2H); 13C NMR (CDCl3, 100 MHz) d 171.13,
141.57, 140.81, 139.52, 130.60, 129.13, 128.78, 128.72, 128.59,
128.41, 128.28, 128.04, 127.78, 127.38, 127.10, 75.17, 51.98,
51.51, 48.49, 45.63, 41.66, 30.61, 26.55; HR-FABMS calcd for
4.4. General procedure for diphenylpiperazine derivatives (3a–
j)
In 1,2-dichloroethane (DCE) 9 mL, 4-amino-1-(4-((4-chloro-
phenyl)(phenyl)methyl)piperazin-1-yl)butan-1-one (2, 0.5 mmol)
and aldehydes (0.5 mmol) were dissolved. Then, 0.7 mmol of so-
dium triacetoxyborohydride (NaBH(OAc)3) and 0.5 mmol of trieth-
ylamine were added and reacted at room temperature overnight
under nitrogen. The reaction mixture was quenched by saturated
NaHCO3 solution and extracted with 30 mL of EtOAc. The organic
layer was collected, dried over anhydrous MgSO4, and filtered.
The organic solvent in the filtrate was removed under reduced
pressure.
C
28H33ON3Cl (M+H)+: 462.2312, found: 462.2316.
4.4.6. 4-(4-Nitrobenzylamino)-1-(4-((4-chlorophenyl)(phenyl)
methyl)piperazin-1-yl)butan-1-one (3f)
Yellow oil; yield: 76%; 1H NMR (CDCl3, 400 MHz) d 8.24 (d,
J = 8.8 Hz, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H),
7.49 (d, J = 8.4 Hz, 1H), 7.21–7.37 (m, 9H), 4.21 (s, 1H), 3.88 (s,
2H), 3.60–3.65 (m, 2H), 3.44–3.48 (m, 2H), 2.66 (t, J = 6.8 Hz, 2H)
2.32–2.38 (m, 6H), 1.81–1.88 (m, 2H); 13C NMR (CDCl3, 100 MHz)
d 171.00, 149.02, 141.73, 141.60, 140.85, 132.85, 129.19, 128.86,
128.82, 127.84, 127.49, 124.34, 123.91, 75.26, 52.06, 51.62, 48.75,
45.72, 41.78, 30.63, 26.41; HR-FABMS calcd for C28H32O3N4Cl
(M+H)+: 507.2163, found: 507.2162.
4.4.1. 1-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-4-
(ethylamino)butan-1-one (3a)
Brown solid, yield: 79%; mp 141–143 °C; 1H NMR (CDCl3,
400 MHz) d 7.34–7.38 (m, 4H), 7.21–7.30 (m, 5H), 4.21 (s, 1H),
3.58–3.63 (m, 2H), 3.44–3.48 (m, 2H), 2.64–2.67 (m, 2H), 2.44–
2.56 (m, 2H), 2.31–2.38 (m, 6H), 1.74–1.82 (m, 2H), 1.05 (t,
J = 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz) d 173.19, 141.56,
140.72, 131.47, 129.14, 128.82, 128.76, 127.79, 127.44, 75.10,
51.89, 51.47, 45.42, 41.85, 29.22, 26.29, 14.21; HR-FABMS calcd
for C23H31ON3Cl (M+H)+: 400.2156, found: 400.2156.
4.4.7. 4-(4-Methoxybenzylamino)-1-(4-((4-
chlorophenyl)(phenyl)methyl)piperazin-1-yl)butan-1-one (3g)
Pale yellow gel; yield: 19%; 1H NMR (CDCl3, 400 MHz) d 7.19–
7.38 (m, 11H), 6.84–6.91 (m, 2H), 4.21 (s, 1H), 3.90 (s, 2H), 3.81
(s, 3H), 3.55–3.60 (m, 2H), 3.43–3.49 (m, 2H), 2.80–2.88 (m, 2H),
2.30–2.47 (m, 6H), 1.92–2.02 (m, 2H); 13C NMR (CDCl3, 100 MHz)
d 171.37, 160.85, 141.66, 140.90, 132.85, 312.39, 132.06, 129.68,
129.40, 129.21, 128.87, 128.82, 127.86, 127.47, 114.40, 113.86,
75.27, 55.65, 52.07, 51.62, 48.68, 45.74, 41.76, 30.96, 25.54; HR-
FABMS calcd for C29H35O2N3Cl (M+H)+: 492.2418, found: 492.2420.
4.4.2. 1-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-4-
(isobutylamino)butan-1-one (3b)
Pale yellow solid; yield: 43%; mp 66–68 °C; 1H NMR (CDCl3,
400 MHz) d 7.34–7.38 (m, 4H), 7.21–7.31 (m, 5H), 4.21 (s, 1H),
3.58–3.64 (m, 2H), 3.44–3.48 (m, 2H), 2.64 (t, J = 6.8 Hz, 2H),
2.32–2.42 (m, 6H), 1.71–1.84 (m, 4H), 1.26 (t, J = 7.2 Hz, 1H), 0.90
(d, J = 6.8 Hz, 6H); 13C NMR (CDCl3, 100 MHz) d 171.25, 141.63,
140.86, 132.91, 129.23, 128.91, 128.85, 127.88, 127.52, 75.29,
57.08, 52.04, 51.61, 48.98, 45.78, 41.85, 31.02, 27.65, 20.68; HR-
FABMS calcd for C25H35ON3Cl (M+H)+: 428.2469, found: 428.2469.
4.4.8. 4-(4-Isopropylbenzylamino)-1-(4-((4-chlorophenyl)
(phenyl)methyl)piperazin-1-yl)butan-1-one (3h)
Pale yellow solid; yield: 81%; mp 65–66 °C; 1H NMR (CDCl3,
400 MHz) d 7.61 (d, J = 8.0 Hz, 1H), 7.16–7.40 (m, 11H), 7.18 (d,
J = 8.0 Hz, 1H) 4.21 (d, J = 6.4 Hz, 1H), 3.74 (s, 2H), 3.58–3.64 (m,
2H), 3.44–3.48 (m, 2H), 2.86–2.96 (m, 1H), 2.68 (t, J = 6.8 Hz, 2H),
2.32–2.41 (m, 6H), 1.98–2.04 (m, 2H), 1.23–1.30 (m, 6H); 13C
NMR (CDCl3, 100 MHz) d 171.40, 147.72, 141.69, 140.91, 132.91,
130.11, 129.24, 128.91, 128.84, 128.27, 127.89, 127.51, 127.25,
126.81, 126.55, 126.25, 75.33, 53.60, 52.12, 51.67, 48.80, 45.77,
41.79, 33.88, 31.00, 25.53, 24.16; HR-FABMS calcd for C31H39ON3Cl
(M+H)+: 504.2782, found: 504.2776.
4.4.3. 4-(2-Ethylbutylamino)-1-(4-((4-
chlorophenyl)(phenyl)methyl)piperazin-1-yl)butan-1-one (3c)
Green solid; yield: 25%; mp 91–93 °C; 1H NMR (CDCl3,
400 MHz) d 7.34–7.4 (m, 4H), 7.21–7.34 (m, 5H), 4.21 (s, 1H),
3.39–3.66 (m, 6H), 2.31–2.38 (m, 6H), 1.88–2.0 (m, 2H), 1.80–
1.82 (m, 1H), 1.60–1.69 (m, 4H), 1.41–1.47 (m, 2H), 0.78–0.96
(m, 6H); 13C NMR (CDCl3, 100 MHz) d 171.29, 141.54, 140.80,
132.77, 129.13, 128.78, 128.72, 128.51, 127.77, 127.39, 75.18,
52.43, 51.97 51.53, 49.44, 45.68, 41.70, 40.43, 30.93, 25.06, 23.89,
10.86; HR-FABMS calcd for C27H39ON3Cl (M+H)+: 456.2782, found:
456.2778.
4.4.9. 4-(4-(Dimethylamino)benzylamino)-1-(4-((4-chloro-
phenyl)(phenyl)methyl)piperazin-1-yl)butan-1-one (3i)
Light brown solid; yield: 33%; mp 85–87 °C; 1H NMR (acetone-
d6, 400 MHz) d 7.45–7.72 (m, 5H), 7.16–7.35 (m, 5H), 6.70–6.82 (m,
3H), 4.31 (s, 1H), 3.50–3.58 (m, 6H), 3.01 (s, 6H), 2.88–2.9 (m, 2H),
2.38 (t, J = 7.4 Hz, 2H), 2.31–2.36 (m, 4H), 1.86–1.93 (m, 2H); 13C
NMR (CDCl3, 100 MHz) d 171.59, 150.54, 141.82, 141.03, 132.96,
129.65, 129.52, 129.32, 129.17, 128.95, 128.81, 127.97, 127.56,
124.51, 112.80, 111.80, 75.43, 52.23, 51.80, 45.89, 41.83, 40.84,
40.27, 30.88, 27.00; HR-FABMS calcd for C30H38ON4Cl (M+H)+:
505.2734, found: 505.2738.
4.4.4. 1-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-4-
(cyclohexylmethylamino)butan-1-one (3d)
White solid; yield: 20%; mp 136–139 °C; 1H NMR (CDCl3,
400 MHz) d 7.34–7.38 (m, 4H), 7.21–7.31 (m, 5H), 4.21 (s, 1H),
3.58–3.62 (m, 2H), 3.44–3.48 (m, 2H), 2.77 (t, J = 6.8 Hz, 2H), 2.52

5944
Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 5938–5944
4.4.10. 4-(2,6-Dichlorobenzylamino)-1-(4-((4-chlorophenyl)
(phenyl)methyl)piperazin-1-yl)butan-1-one (3j)
filaments (3.92, 5.88, 9.80, 19.60, 39.20, 58.80, 78.40, and
147.00 mN, Stoeling, Wood Dale, IL, USA; equivalent to 0.4, 0.6,
1.0, 2.0, 4.0, 6.0, 8.0, and 15.0 g). The 50% withdrawal threshold
was determined using the up-down method.19 In brief, testing
was initiated with a filament whose bending force was 19.60 mN
in the middle of the series. When a withdrawal response was ob-
tained, next weaker filament was used, whereas next stronger fila-
ment was administered when no response was obtained.
Interpolation of the 50% threshold was carried out using the Dixon
method.20 A brisk tail withdrawal to von Frey filament application
was regarded as a positive response. Testing for cold or warm allo-
dynia was performed by measuring tail withdrawal latency to cold
(4 °C) or warm (40 °C) water stimulation, respectively.18 After
immersing the tail into cold or warm water bath, the tail was con-
tinuously observed to find out whether it moved abruptly, and the
latency of tail movement was measured within a cut-off time of
15 s. An abrupt tail movement within the cut-off time was consid-
ered as a positive withdrawal response, whereas a lack of tail move-
ment until the cut-off time or slow tail movement within the cut-off
time was considered to be not a positive response. The testing was
repeated five times with 5 min intervals, and the average latency of
tail response was calculated. At 2 weeks after TNI, rats were given a
baseline test and thereafter test compounds (60 mg/kg), gabapentin
(60 mg/kg), which is widely used for the treatment of neuropathic
pain or vehicle (Methyl pyrrolidone:Tween80:Saline = 1:1:8) was
administered intraperitoneally. Behavioral tests for mechanical,
cold and warm allodynia were conducted at 1, 3, and 5 h after the
injection. Behavioral testing was carried out by an investigator
who was unaware of the treatment status of the rats.
Pale yellow gel; yield: 25%; 1H NMR (CDCl3, 400 MHz) d 7.19–
7.38 (m, 12H), 4.21 (s, 1H), 3.74–3.78 (m, 2H), 3.58–3.64 (m, 2H),
3.44–3.49 (m, 2H), 2.44 (t, J = 7.6 Hz, 2H), 2.32–2.39 (m, 4H),
2.04–2.12 (m, 2H), 1.79–1.84 (m, 2H); 13C NMR (CDCl3, 100 MHz)
d 170.86, 141.69, 140.90, 136.48, 136.16, 135.93, 132.97, 129.92,
129.31, 128.97, 128.92, 128.68, 128.62, 127.93, 127.58, 75.19,
51.94, 51.55, 45.78, 31.03, 29.85; HR-FABMS calcd for C28H31ON3Cl3
(M+H)+: 530.1533, found: 530.1530.
4.5. Effect on T-type calcium channel
For the recordings of a1G T-type Ca2+ currents and N-type Ca2+
channels, the standard whole-cell patch–clamp method was uti-
lized.15 Briefly, borosilicate glass electrodes with a resistance of
3–4 MX were pulled and filled with the internal solution contain-
ing (in mM): 130 KCl, 11 EGTA, 5 Mg-ATP, and 10 Hepes (pH 7.4).
The external solution contained (in mM): 140 NaCl, 2 CaCl2, 10
Hepes, and 10 glucose (pH 7.4). a1G T-type Ca2+ currents were
evoked every 15 s by a 50 ms depolarizing voltage step from
À100 mV to À30 mV. The molar concentrations of test compounds
required to produce 50% inhibition of peak currents (IC50) were
determined from fitting raw data into dose–response curves. The
current recordings were obtained using an EPC-9 amplifier and
Pulse/Pulsefit software program (HEKA, Germany).
4.6. Nociceptive and inflammatory pain
4.6.1. Behavioral testing for formalin test
4.8. Statistical analysis
To investigate the effect of compounds 2 and 3d on both noci-
ceptive and inflammatory pain, the formalin test was carried out.
Each rat was placed in the observation chamber (20 Â 20
 20 cm) equipped with a mirror placed behind the chamber to al-
low an unobstructed view of the paw for a 30 min habituation per-
iod. Thereafter, animals were assigned to the two groups; the first
group was subjected to the subcutaneous injection of the mixture
All data are presented as mean SEM. Student’s unpaired t-test,
repeated one-way ANOVA (Bonferroni t-test) and paired t-test
were used wherever appropriate. A difference of P <0.05 was con-
sidered to be statistically significant.
Acknowledgments
of formalin (5%, 50
Saline = 1:1:8, 10
paw using a 30-gage syringe, the second group was treated with
the mixture of formalin (5%, 50 l) plus test compounds (60 g/
10 l) by the same way described above. After the injection, ani-
mals were returned to the chamber immediately and the time spent
in licking, shaking or biting the injected paw was recorded for every
5 min over a 60-min period. The early phase was defined as the first
10 min after formalin injection, and the late phase was the follow-
ing 50 min.10,16 The observations in this test were carried out by
three investigators who were unaware of the treatment status.
l
l) and vehicle (Methyl pyrrolidone: Tween80:-
l
l) under the plantar surface of the right hind
This study was supported in part by the Korea Research Foun-
dation (Grant KRF-2004-E00406) and Grant (M103KV010015-
08K2201-01510) from Brain Research Center of the 21st Century
Frontier Research Program funded by the Ministry of Education,
Science and Technology, the Republic of Korea.
l
l
l
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4.7.2. Behavioral testing for neuropathic pain
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