Lead discovery and optimization of T-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmc.2006.11.004

A series of compounds were designed as T-type calcium channel blocker containing 6 or 5 pharmacophore features from structure-based virtual screening. To optimize the suggested structure, over 130 derivatives were synthesized and their inhibitory activities on T-type calcium channel were assayed using in vitro screening system with α1_G and α1_H clones. For the compounds with higher activities in FDSS assay system, the efficacy was measured by patch-clamp method. Among the library with 5 features, alkaneamide derivatives (7b, 9j, 11b, 11g, 11h) with 4-arylsubstituted piperazine showed better IC₅₀ values than Mibefradil.

Full text of DOI:10.1016/j.bmc.2006.11.004

Bioorganic & Medicinal Chemistry 15 (2007) 1409–1419
Lead discovery and optimization of T-type calcium channel blockers
Jung Hwan Park,a,b Jin Kyu Choi,a Eunjung Lee,a Jae Kyun Lee,a Hyewhon Rhim,a
Seon Hee Seo,a Yoonjee Kim,a Munikumar Reddy Doddareddy,a Ae Nim Pae,a
Jahyo Kangb and Eun Joo Roha,*
aLife Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
bDepartment of Chemistry, Sogang University, Shinsu-dong 1, Mapo-gu, Seoul 121-742, Republic of Korea
Received 29 September 2006; revised 1 November 2006; accepted 3 November 2006
Available online 10 November 2006
Abstract—A series of compounds were designed as T-type calcium channel blocker containing 6 or 5 pharmacophore features from
structure-based virtual screening. To optimize the suggested structure, over 130 derivatives were synthesized and their inhibitory
activities on T-type calcium channel were assayed using in vitro screening system with a1G and a1H clones. For the compounds with
higher activities in FDSS assay system, the efficacy was measured by patch–clamp method. Among the library with 5 features,
alkaneamide derivatives (7b, 9j, 11b, 11g, 11h) with 4-arylsubstituted piperazine showed better IC50 values than Mibefradil.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
treatment of hypertension, and angina pectoris.12–14
Unfortunately, the drug had to be withdrawn by the
manufacturer due to its interaction with the cytochrome
P-450 3A4 enzyme, an effect unrelated to T-type Ca2+
channel blockade. T-type Ca2+ channels participate in
cardiac pacemaking,15 regulation of vascular tone and
hormone secretion.16,17 Involvement of T-type Ca2+
channels in cell growth and proliferation has been pro-
posed.18 The distribution and properties of T-type
Ca2+ channels have been reported to be altered in path-
ophysiological conditions such as ventricular hypertro-
phy19,20 and cardiomyopathy.21 Thus, T-type Ca2+
channels are now considered to be novel therapeutic tar-
gets for the treatment of various cardiovascular disorders
such as heart failure, arrhythmia, and hypertension and
neuronal disorders such as epilepsy and pain. Even with
these properties as promising drug target, the speed of
development is surprisingly slow compared to other
areas. It is because of non-availability of X-ray structure
and existence of several subtypes of the channel. To ob-
tain a lead compound as a T-type calcium channel block-
er, the strategy we used involved the pharmacophore
model generated from structure-based virtual screening
and hypothetical mapping with the designed structure.
Voltage-gated calcium channels are transmembrane pro-
teins involved in the regulation of calcium influx in a
number of cell types.1,2 A number of different types of
calcium channels have been identified in native tissues
and divided into various categories based on functional
and pharmacological criteria.3,4 High-voltage-activated
(HVA) channels which can be subdivided further into
L-, N-, P/Q-, and R-types5,6 require strong depolariza-
tion for activation, whereas low-voltage-activated or
T-type (transient) channels first activate at relatively
more negative voltage range and rapidly inactivate.7–9
The main structural component of the voltage-gated cal-
cium channel is the a1 subunit, which forms the pore and
the channel gate. So far, 10 a1 subunits have been identi-
fied by molecular cloning. a1A–a1E and a1S encode HVA
channels Ca2+, whereas a1G–a1I encode T-type channels.
Pharmacological agents that have selective activity on a1
subtypes have been key in studying calcium channel
function in physiological systems, but a selective antago-
nist of T-type calcium channel is not yet available.
Mibefradil, which blocks T-type Ca2+ channels at con-
centration lower than that needed to block L-type Ca2+
channels,10,11 appeared to be a promising drug for the
The first set of compounds with 6 pharmacophore
features (5) was prepared and their activities were
assayed with calcium imaging screening system.
Disappointingly, they showed weaker activity than
*
Corresponding author. Tel.: +82 2 958 6779; fax: +82 2 958 5189;
e-mail: r8636@kist.re.kr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.004

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J. H. Park et al. / Bioorg. Med. Chem. 15 (2007) 1409–1419
mibefradil. Then we need to understand the relative
importance of the pharmacophores by comparing the
activities of the compounds with 5 features without
one hydrogen bond acceptor or the linker carbon length
is varied. The 2nd set of libraries (7, 13, 15) in which the
number and position of the proposed features were var-
ied have been designed and synthesized. The synthetic
procedure of the compounds with 5 features without
one carbonyl group was simplified and the library was
easily prepared. The FDSS results of this library showed
relatively better activity than that of the compounds
with initial 6 features. The 3rd set of libraries (9, 11,
17, 19, 21) in which the number of rotatable carbon
and halogen of aniline moiety are varied were prepared.
The in vitro screening assays to figure out the selectivity
and hERG channel blocking activity are still going on.
A series of compounds with 6 features were prepared by
routine procedures shown in Scheme 1. To obtain the
derivatives easily, the common intermediates from ani-
line moiety were used as starting material and the piper-
azine derivatives were introduced at final step. All the
steps showed acceptable yields (40–80%) and purity.
The 2nd set of library was designed to fit the essential
5 features (Fig. 2) and synthesized easily from piperazine
derivatives and bromovaleryl chloride (Scheme 2) or
aniline derivatives and piperazine units(Scheme 3). To
evaluate the importance of distance of two features
and number of halogens in aniline moiety, the 3rd set
was prepared as same as Scheme 2.
2.2. Biological data and structure–activity relationships
As shown in Table 1, the in vitro activities of initial 5
series were assayed with T-type channels stably ex-
pressed in Xenopus oocytes (a1H). After setting up the
HTS system utilizing calcium imaging plate reader sys-
tem (FDSS), the primary assay was performed with
HEK293 cells (a1G). For the compounds with high inhi-
bition value in FDSS assay, the efficacy was measured as
IC50 value by patch–clamp method. The in vitro activi-
ties of 1st set of compounds (5) were weaker than expect-
ed. Also the synthetic procedure was not enough
2. Results and discussion
2.1. Chemistry
The synthesis of suggested compound which generated
from statistically best 6-pharmacophore features hypoth-
esis was not successful. Then it was modified to fit the
6-features (Fig. 1) and increase conformational rigidity.
HBA
PI
8.36-10.36
3.00-5.00
11.83-13.83
3.74-5.74
7.62-9.62
HP
4.40-6.40
4.33-6.33
HP
3.56-5.56
5.00-7.00
HP
HBA
˚
Figure 1. (L) 6-feature hypothesis for T-type calcium channel blockers with distance constraints in armstrong units (A) (HBA, hydrogen bond
acceptor; HP, hydrophobic; PI, positive ionizable). (R) Mapping of 5a with 6-feature hypothesis.
X
X
O
Boc
Boc
iii
N
HN
HN
X
N
iv
i
Br
N
HN
N
NH
NH
N
O
4a-d
O
v
1
3a-d
H
X
NH2
N
O
N
ii
Br
O
N
HN
X
2a-d
N
N
R
O
5a-o
Scheme 1. Reagents and conditions: (i) (Boc)2O, TEA, CH2Cl2, 0 ꢁC to rt; (ii) bromoacetyl chloride, NEt3, 0 ꢁC to rt; (iii) NEt3, CH2Cl2, rt; (iv)
TFA, CH2Cl2, rt; (v) 1-substituted piperazine derivatives, NEt3, 0 ꢁC to rt.

J. H. Park et al. / Bioorg. Med. Chem. 15 (2007) 1409–1419
1411
˚
Figure 2. (L) 5-feature hypothesis for T-type calcium channel blockers with distance constraints in Armstrong units (A) (HBA, hydrogen bond
acceptor; HP, hydrophobic; PI, positive ionizable). (R) Mapping of 11g with 5-feature hypothesis.
i
ii
O
O
X
NH2
N
Br
X
X
N
H
7a-b
N
n
H
N
6 (X = 2-Cl, n=3)
8 (X = 4-Cl, n=2)
10 (X = 2,4-diCl, n=2)
16 (X = 2,4-diF, n=1)
18 (X = 2,4-diF, n=2)
20 (X = 3,4,5-triCl, n=2)
R
9a-l
11a-j
17a-l
19a-l
21a-l
Scheme 2. Reagents and conditions: (i) n-bromoalkyl chloride, benzene, rt; (ii) 1-substituted piperazine derivatives, NEt3, CH2Cl2, reflux.
O
O
N
Br
i
N
N
ii
N
NH
N
N
N
R
X
12 (X = 2-F)
14 (X = H)
X = H, 2-F
X
13a-b
15a-b
X
Scheme 3. Reagents and conditions (i) 4-bromovaleryl chloride, benzene, rt: (ii) 1-substituted piperazine derivatives, NEt3, CH2Cl2, reflux.
efficient to obtain a library easily. If one of the hydrogen
bond acceptors were omitted to have 5 features, the
synthetic procedure will be simplified. Also, we need to
figure out the relative importance of two hydrogen bond
acceptors, the distance of features, and number of halo-
gens, the suggested structure was modified and they fit-
ted the 5-feature model previously suggested by Dr.
Pae’s modeling group in the same institute.22 From the
activity data in Table 2, the compounds with 5 features
showed higher activity than those with 6 features and
some of them has better IC50 value than mibefradil.
Among the two carbonyl groups in the 6 features, that
of the amide contributed more in terms of activity than
that attached to piperazine ring. Examination of the
substituents of R group of compounds 7, 9, 11 (Table
1) reveals a moderate structure–activity relationship:
the halogen substituents on the phenyl or benzyl ring in-
crease the efficacy of these compounds. The halogen
substitution at the same position (para) of phenyl and
benzyl ring is preferred, because the hydrophobic fea-
ture is well matched with para position of aromatic ring.
And the optimal length of linear carbons including car-
bonyl is 4 or 5 carbons. If it is shorter than 3, the activ-
ities were slightly decreased as shown in Table 3. The
in vitro activities of compound 19 series were better than
those of 17 series and the halogen (Cl) substituent
increases the activities. Also the general tendency was
found that the derivatives with one or two chlorines in
aniline moiety had higher activities than three-chlorine
derivatives as in Table 4.
In conclusion, the new scaffolds starting from the hit
compound by virtual screening were designed to fit the
5- or 6-pharmacophore hypothesis and evaluated as T-
type calcium channel blocker. Even with the preliminary
biological data, this approach has proved its usefulness in
designing new ligands especially when the crystal struc-
ture of the target protein is unknown. The derivatization

1412
J. H. Park et al. / Bioorg. Med. Chem. 15 (2007) 1409–1419
Table 1. In vitro calcium channel blocking effects of derivatives with 6 features
X
O
N
N
HN
N
N
R
O
5
Compound
X
R
Xenopus oocyte
(T-type a1H) %
HEK 293 cell
(T-type a1G) %
inhibitiona (10 lM)
inhibitiona (100 lM)
5a
5b
5c
5d
5e
5f
3-Chloro
3-Chloro
3-Chloro
Phenyl
Methyl
Ethyl
75.35
No blockb
71.61
52.98
3-Ttrifluoromethyl
3-Trifluoromethyl
3-Trifluoromethyl
3-Methyl
Phenyl
Methyl
Ethyl
No block
23.60
5g
5h
5i
Ethyl
Phenyl
2-Fluorophenyl
À1.36
25.17
22.13
26.11
7.38
2-Chloro
2-Chloro
5j
2-Chloro
2-Chloro
3-Methoxyphenyl
2-Cyanophenyl
4-Chlorobenzyl
3-Chlorobenzyl
4-Fluorophenyl
Benzyl
5k
5l
2-Chloro
2-Chloro
26.24
40.94
34.89
15.89
78.17
5m
5n
5o
2-Chloro
2-Chloro
Mibefradil
86.0
a % Inhibition value was obtained by FDSS assay.
b No block means the inhibition was less than 1%.
with 9j, 11g, and 11h, and IC50 assay for the compounds
with higher value in FDSS assay are in progress to in-
crease the number of hit compounds and diversity.
Hex = 1:3) to give product 6 (0.82 g, 59.4%) as a white
color solid: H NMR (400 MHz, CDCl3) d = 1.86–2.04
(4H, m, 2CH2), 2.49 (2H, t, J = 16.64 Hz, CH2), 3.45
(2H, t, J = 6.25 Hz, CH2), 7.63 (1H, br s, NH), 6.98–
8.35 (4H, m, Ph); 13C NMR (100 MHz, CDCl3)
d = 23.95, 31.95, 33.05, 36.69, 121.73, 122.68, 124.69,
127.75, 129.00, 134.48, 170.51.
1
3. Experimental
3.1. Chemistry
3.1.3. 4-Bromo-N-(4-chlorophenyl)butyramide (8). 4-
Bromobutyryl chloride (5.3 mL, 0.043 mol) was added
3.1.1. Materials and methods. All commercially available
chemicals were of reagent grade and used as purchased
unless stated otherwise. All reactions were performed
under an inert atmosphere of dry argon or nitrogen
using distilled dry solvents. Reactions were monitored
by TLC analysis using Merck silica gel 60 F-254 thin
layer plates. Flash column chromatography was carried
out on Merck silica gel 60 (230–400 mesh) by prepara-
tive LC system. 1H and 13C NMR spectra were recorded
on a spectrometer operating at Bruker 400 and
100 MHz, respectively. High-resolution mass spectra
were recorded on a 4.7 T Ion Spec ESI-FTMS or a
Micromass LCT ESI-TOF mass spectrometer.
dropwise to
a solution of 4-chloroaniline (5.0 g,
0.039 mol) in benzene (100 mL) at 0 ꢁC. Then the mix-
ture was stirred at room temperature for 12 h. The
resulting mixture was quenched by addition of water
and extracted with dichloromethane. The combined
organic layer was dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. The residue
was purified by recrystallization (EtOAc and hexane)
to give product 8 (9.84 g, 90.8%) as a gray color solid:
1H NMR (400 MHz, CDCl3) d = 2.27 (2H, m, CH2),
2.56 (2H, t, J = 7.02 Hz, CH2), 3.52 (2H, t,
J = 6.18 Hz, CH2), 7.41 (1H, br s, NH), 7.26–7.47 (4H,
m, Ph); 13C NMR (100 MHz, CDCl3) d = 27.82, 33.31,
35.26, 121.11, 129.02, 129.40, 136.17, 169.90.
3.1.2. 5-Bromo-N-(2-chlorophenyl)pentanamide (6). 4-
Bromovaleryl chloride (0.7 mL, 5.23 mmol) was added
dropwise to a solution of 2-chloroaniline (0.5 mL,
4.75 mmol) in benzene (10 mL) at 0 ꢁC. Then the mixture
was stirred at room temperature for 12 h. The resulting
mixture was quenched by addition of water and extract-
ed with dichloromethane. The combined organic layer
was dried over anhydrous MgSO4, filtered, and concen-
trated under reduced pressure. The residue was purified
by flash column chromatography (SiO2, EtOAc/n-
3.1.4. 4-Bromo-N-(2,4-dichlorophenyl)butyramide (10).
4-Bromobutyryl chloride (1.05 mL, 8.55 mmol) was
added dropwise to a solution of 2,4-dichloroaniline
(1.26 g, 7.78 mmol) in benzene (20 mL) at 0 ꢁC. Then
the mixture was stirred at room temperature for 12 h.
The resulting mixture was quenched by addition of
water and extracted with dichloromethane. The com-
bined organic layer was dried over anhydrous MgSO4,

J. H. Park et al. / Bioorg. Med. Chem. 15 (2007) 1409–1419
Table 2. In vitro calcium channel blocking effects of derivatives with 5 features
1413
O
X
O
N
N
N
N
N
N
n
R
13, 15
H
N
7, 9, 11
R
X
Compound
X
n
R
FDSS HEK 293 cell (T-type a1G) %
inhibition (10 lM)
Patch–clamp HEK 293 cell
(T-type a1G)
% inhibition (10 lM)
IC50 (lM)
7a
7b
2-Cl
2-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2,4-Cl
2-F
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3-Chlorophenyl
3,4-Dichlorobenzyl
Phenyl
53.99
55.93
30.38
36.06
32.50
37.34
48.27
51.81
40.61
30.80
38.33
33.65
39.84
33.36
38.53
38.15
42.70
44.22
44.87
50.84
61.26
47.61
49.80
49.61
48.82
48.29
50.38
56.23
74.29
93.17 1.48
91.00 1.86
75.1 2.3
79.2 1.5
76.1 0.9
60.5 2.8
94.6 0.9
84.3 2.3
84.1 2.0
78.0 0.4
92.4 0.9
92.6 3.2
72.8 1.0
81.9 0.8
76.2 1.2
95.4 1.4
79.1 1.1
80.3 0.8
79.3 1.0
87.3 1.1
91.1 2.7
94.4 1.1
88.1 2.3
89.6 0.3
87.13 1.27
89.70 1.53
79.70 1.23
73.80 2.30
95.9 1.7
1.91 0.21
1.01 0.10
4.47 0.36
3.63 0.21
3.49 0.27
6.01 0.89
2.64 0.59
2.61 0.14
2.78 0.18
3.52 0.31
1.80 0.09
0.37 0.04
3.27 0.36
2.73 0.13
4.21 0.28
1.01 0.10
3.34 0.10
3.81 0.24
3.14 0.40
1.48 0.20
0.28 0.06
0.75 0.04
1.67 0.12
2.40 0.06
2.44 0.24
1.81 0.11
3.4 0.18
9a
9b
2-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
4-Chlorophenyl
2-Methoxyphenyl
3-Methoxyphenyl
Benzyl
9c
9d
9e
9f
9g
9h
9i
3-Chlorobenzyl
4-Chlorobenzyl
9j
9k
2-Chloro-6-fluorobenzyl
Piperonyl
Phenyl
9l
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
13a
13b
15a
15b
3-Chlorophenyl
2-Methoxyphenyl
3-Methoxyphenyl
Benzyl
3-Chlorobenzyl
4-Chlorobenzyl
3,4-Dichlorobenzyl
Piperonyl
2-Chloro-6-fluorobenzyl
3-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
3,4-Dichlorobenzyl
Mibefradil
2-F
H
H
2.91 0.25
1.34 0.49
Table 3. In vitro calcium channel blocking effects of derivatives of 5 features with different chain lengths
F
O
F
R
O
N
N
N
N
N
H
F
N
H
17
19
F
R
Compound
R
FDSSa
Compound
FDSSa
17a
17b
17c
17d
17e
17f
17g
17h
17i
Phenyl
33.18
30.29
29.68
28.36
26.40
37.14
29.50
21.09
34.74
33.16
29.71
34.60
75.19
19a
19b
19c
19d
19e
19f
19g
19h
19i
36.80
32.21
38.14
42.00
51.04
33.00
40.57
23.22
30.24
48.04
38.93
59.92
2-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
3-Chlorophenyl
2-Methoxyphenyl
3-Methoxyphenyl
Benzyl
3-Chlorobenzyl
4-Chlorobenzyl
17j
19j
17k
17l
2-Chloro-6-fluorobenzyl
3,4-Dichlorobenzyl
Mibefradil
19k
19l
a % inhibition value was obtained with T-type a1G (HEK 293 cell)and 10 lM of compounds.

1414
J. H. Park et al. / Bioorg. Med. Chem. 15 (2007) 1409–1419
Table 4. In vitro calcium channel blocking effects of derivatives of 5 features with different numbers of halogen
H
H
N
Cl
Cl
N
N
N
Cl
H
O
N
O
N
N
Cl
R
R
N
9
21
Cl
O
N
Cl
R
11
R
Compound
FDSSa
Compound
FDSSa
Compound
FDSSa
Phenyl
9a
9b
9c
9d
30.38
36.06
32.50
37.34
11a
11k
11l
38.53
27.35
29.06
21a
21b
21c
21d
21e
21f
21g
21h
21i
38.31
20.08
20.68
13.62
16.78
33.74
21.65
49.41
36.98
27.48
35.15
2-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
3-Chlorophenyl
2-Methoxyphenyl
3-Methoxyphenyl
Benzyl
11b
11c
11d
11e
11f
11g
11j
38.15
42.70
44.22
44.87
50.84
61.26
49.61
9f
9g
9h
9i
51.81
40.61
30.80
38.33
33.65
39.84
3-Chlorobenzyl
4-Chlorobenzyl
2-Chloro-6-fluorobenzyl
9j
9k
21j
21k
a % Inhibition value was obtained with T-type a1G (HEK 293 cell) and 10 lM of compounds.
filtered, and concentrated under reduced pressure. The
residue was purified by recrystallization (EtOAc and hex-
ane) to give product 10 (2.17 g, 89.7%) as a pink color sol-
id: 1H NMR (400 MHz, CDCl3) d = 2.29 (2H, m, CH2),
2.65 (2H, t, J = 7.02 Hz, CH2), 3.54 (2H, t, J = 6.23 Hz,
CH2), 7.24 (1H, J = 2.36 Hz, CH in Ph), 7.39 (1H,
J = 2.36 Hz, CH in Ph), 7.60 (1H, br s, NH), 8.34 (1H,
J = 8.89 Hz, CH in Ph); 13C NMR (100 MHz, CDCl3)
d = 27.76, 33.01, 35.53, 122.28, 123.13, 127.91, 128.73,
129.18, 133.14, 169.80.
was purified by recrystallization (EtOAc and hexane)
to give product 14 (2.64 g, 67.6%) as a pink color solid:
1H NMR (400 MHz, CDCl3) d = 1.83 (2H, m, CH2),
1.93 (2H, m, CH2), 2.41 (2H, t, J = 7.30 Hz, CH2), 3.17
(4H, m, 2CH2), 3.45 (2H, t, J = 6.55 Hz, CH2), 3.63
(2H, t, J = 4.92 Hz, CH2), 3.78 (2H, t, J = 4.98 Hz,
CH2), 6.89–7.31 (5H, m, Ph); 13C NMR (100 MHz,
CDCl3) d = 23.68, 32.12, 32.18, 33.35, 41.46, 45.45,
49.40, 49.76, 116.64, 120.60, 129.21, 150.82, 170.87.
3.1.7. 3-Bromo-N-(2,4-difluorophenyl)propionamide (16).
3-Bromopropionyl chloride (2.7 mL, 0.027 mol) was
added dropwise to a solution of 2,4-difluoroaniline
(2.5 mL, 0.025 mol) in benzene (50 mL) at 0 ꢁC. Then
the mixture was stirred at room temperature for 12 h.
The resulting mixture was quenched by addition of
water and extracted with dichloromethane. The com-
bined organic layer was dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The
residue was purified by flash column chromatography
(SiO2, EtOAc/n-Hex = 1:8) to give product 16 (4.9 g,
75.6%) as a white color solid: 1H NMR (400 MHz,
CDCl3) d = 2.99 (2H, t, J = 6.48 Hz, CH2), 3.71 (2H, t,
J = 6.50 Hz, CH2), 7.33 (1H, br s, NH), 6.86–8.26 (3H,
m, Ph); 13C NMR (100 MHz, CDCl3) d = 26.53, 40.51,
103.36, 103.60, 103.86, 111.18, 111.40, 122.99, 167.86.
3.1.5. 5-Bromo-1-[4-(2-fluorophenyl)piperazin-1-yl]pen-
tan-1-one (12). 5-Bromovaleryl chloride (1.10 mL,
0.010 mol) was added dropwise to a solution of 2-fluor-
ophenylpiperazine (1.5 mL, 0.009 mol) in benzene
(30 mL) at 0 ꢁC. Then the mixture was stirred at room
temperature for 12 h. The resulting mixture was
quenched by addition of water and extracted with dichlo-
romethane. The combined organic layer was dried over
anhydrous MgSO4, filtered, and concentrated under re-
duced pressure. The residue was purified by flash column
chromatography (SiO2, EtOAc/n-Hex = 1:6) to give
1
product 12 (2.02 g, 62.0%) as a white color solid: H
NMR (400 MHz, CDCl3) d = 1.83 (2H, m, CH2), 1.95
(2H, m, CH2), 2.41 (2H, t, J = 7.24 Hz, CH2), 3.07
(4H, m, 2CH2), 3.45 (2H, t, J = 6.47 Hz, CH2), 3.64
(2H, s, CH2), 3.80 (2H, s, CH2), 6.91–7.09 (4H, m, Ph);
13C NMR (100 MHz, CDCl3) d = 23.76, 32.22, 33.50,
41.69, 45.76, 50.69, 116.27, 119.24, 123.19, 124.55,
139.50, 170.92.
3.1.8. 4-Bromo-N-(2,4-difluorophenyl)butyramide (18).
4-Bromobutyryl chloride (8.4 mL, 0.069 mol) was added
dropwise to a solution of 2,4-difluoroaniline (7.0 mL,
0.069 mol) in benzene (100 mL) at 0 ꢁC. Then the mix-
ture was stirred at room temperature for 12 h. The
resulting mixture was quenched by addition of water
and extracted with dichloromethane. The combined
organic layer was dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. The residue
was purified by flash column chromatography (SiO2,
EtOAc/n-Hex = 1:8) to give product 18 (18.0 g, 94.1%)
as a white color solid: 1H NMR (400 MHz, CDCl3)
d = 2.29 (2H, m, CH2), 2.61 (2H, t, J = 7.03 Hz, CH2),
3.1.6. 5-Bromo-1-(4-phenylpiperazin-1-yl)pentan-1-one
(14). 5-Bromovaleryl chloride (1.65 mL, 0.013 mol) was
added dropwise to a solution of 1-phenylpiperazine
(1.5 mL, 0.012 mol) in benzene (30 mL) at 0 ꢁC. Then
the mixture was stirred at room temperature for 12 h.
The resulting mixture was quenched by addition of water
and extracted with dichloromethane. The combined
organic layer was dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. The residue

J. H. Park et al. / Bioorg. Med. Chem. 15 (2007) 1409–1419
1415
3.54 (2H, t, J = 6.22 Hz, CH2), 7.30 (1H, br s, NH),
6.84–8.23 (3H, m, Ph); 13C NMR (100 MHz, CDCl3)
d = 27.79, 33.08, 35.20, 103.58, 111.13, 111.35, 122.43,
122.92, 169.80.
(t, J = 7.4 Hz, 2H), 2.64 (d, J = 2.7 Hz, 8H), 3.11
(t, J = 4.4 Hz, 4H), 3.22 (s, 2H), 3.59 (t, J = 4.4 Hz, 2H),
3.74 (br s, 2H), 6.90–7.07 (m, 5H), 7.29 (m, 1H), 7.38
(dd, J = 1.4, 8.0 Hz, 1H), 8.46 (dd, J = 1.4, 8.3 Hz, 1H),
9.87 (s, 1H); 13C NMR (100 MHz, CDCl3) d = 23.1,
26.4, 33.0, 41.6, 45.6, 50.41, 50.43, 53.1, 53.2, 53.4, 58.1,
61.9, 115.9, 116.1, 118.8, 120.8, 122.3, 122.4, 122.6,
124.3, 124.4, 124.6, 127.8, 129.0, 134.2, 140.0, 140.1,
154.4, 156.8, 167.8, 171.4; MS (EI) (m/z) 352 (M+, 100),
281 (23), 268 (27), 105 (21), 83 (18), 71 (26), 57 (34); MS
(FAB) (m/z) 516 ([M+H]+, 45), 647 (5), 328 (23), 176
(100), 154 (41); HRMS (FAB) calcd for C27H36O2N5ClF:
[M+H]+ = 516.2463, found = 516.2538.
3.1.9. 4-Bromo-N-(3,4,5-trichlorophenyl)butyramide (20).
4-Bromobutyryl chloride (1.4 mL, 0.011 mol) was added
dropwise to a solution of 3,4,5-trichloroaniline (2.0 g,
0.010 mol) in benzene (50 mL) at 0 ꢁC. Then the mixture
was stirred at room temperature for 12 h. The resulting
mixture was quenched by addition of water and extract-
ed with dichloromethane. The combined organic layer
was dried over anhydrous MgSO4, filtered, and concen-
trated under reduced pressure. The residue was purified
by flash column chromatography (SiO2, EtOAc/n-
Hex = 1:8) to give product 20 (2.36 g, 67.1%) as a white
3.1.11. General procedure of compounds 7a and 7b. Piper-
azine derivative (0.95 mmol) was added to a solution of
5-bromo-N-(2-chlorophenyl)pentanamide (6) (250 mg,
0.86 mmol) and Et3N (0.18 mL, 1.29 mmol) in CH2Cl2
(5.0 mL) at room temperature. Then the mixture was
stirred at reflux for overnight. The reaction mixture
was cooled to room temperature and quenched by addi-
tion of water (10 mL). The mixture was extracted with
dichloromethane (3· 10 mL), dried over anhydrous
MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by flash column chroma-
tography (SiO2, CH2Cl2/MeOH = 20:1) to give product
7a and 7b.
1
color solid: H NMR (400 MHz, CDCl3) d = 2.29 (2H,
m, CH2), 2.58 (2H, t, J = 6.95 Hz, CH2), 3.53 (2H, t,
J = 6.12 Hz, CH2), 7.28 (1H, br s, NH), 7.64 (2H, s,
Ph); 13C NMR (100 MHz, CDCl3) d = 27.64, 33.08,
35.25, 119.75, 126.58, 134.34, 136.76, 170.14.
3.1.10. General procedure of compounds 5a–o. To a
solution of 1-substituted piperazine (0.2879 mmol) in
CH2Cl2 (0.5 mL) was added triethylamine (0.05 mL,
0.3840 mmol) at room temperature. After 20 min, 4
(0.1920 mmol) in CH2Cl2 (1.5 mL) was added to the reac-
tion mixture. The reaction mixture was stirred at room
temperature for 24 h, and then the reaction mixture was
quenched by addition of saturated NH4Cl aqueous solu-
tion. The combined organic layer was washed with brine,
dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure. The residue was purified by flash
column chromatography (SiO2, CH2Cl2/MeOH = 9:1) to
give product 5a–o as a white solid.
Compound 7a: 54%; mp 102.9 ꢁC; 1H NMR (400 MHz,
CDCl3) d = 1.63 (2H, m, CH2), 1.81 (2H, m, CH2), 2.43–
2.50 (4H, m, 2CH2), 2.60 (4H, s, 2CH2), 3.20 (4H, t,
J = 4.78 Hz, 2CH2), 7.66 (1H, br s, NH), 6.77–8.38
(8H, m, Ph); 13C NMR (100 MHz, CDCl3) d = 23.40,
26.26, 37.67, 48.61, 53.04, 58.06, 113.83, 115.73,
119.24, 121.67, 124.60, 127.77, 128.99, 130.00, 134.57,
134.94, 152.32, 170.64; IR (KBr) 3282 (NH), 2939,
2822, 1660 (C@O), 1594, 1488 (amide), 1440, 1241,
767 cmÀ1; HRMS (ES): m/z calcd for C21H25Cl2N3NaO:
[M+Na]+ = 428.1273, found = 428.1268.
Compound 5g: 1H NMR (400 MHz, CDCl3) d = 1.10 (t,
J = 7.1 Hz, 3H), 1.56–1.69 (m, 4H), 2.00–2.61 (m, 21H),
3.17 (s, 2H), 3.56 (br s, 2H), 3.71 (br s, 2H), 6.95 (d,
J = 7.4 Hz, 1H), 7.23 (t, J = 7.7 Hz, 1H), 7.37 (m, 2H),
8.91 (br s, 1H); 13C NMR (100 MHz, CDCl3) d = 11.8,
21.5, 23.2, 26.4, 33.0, 41.6, 45.5, 52.3, 52.6, 53.0, 53.2,
53.6, 58.1, 62.0, 116.6, 120.1, 125.2, 128.9, 137.2, 139.1,
167.5, 171.4; MS (FAB) (m/z) 430 ([M+H]+, 100), 428
(30), 154 (95), 136 (61); HRMS (FAB) calcd for
C24H40O2N5: [M+H]+ = 430.3104, found = 430.3177.
1
Compound 7b: 58%; mp 87.4 ꢁC; H NMR (400 MHz,
CDCl3) d = 1.59 (2H, m, CH2), 1.77 (2H, m, CH2),
2.38–2.47 (12H, m, 6CH2), 3.44 (2H, s, CH2(benzyl)),
7.65 (1H, br s, NH), 7.02–8.36 (7H, m, Ph); 13C NMR
(100 MHz, CDCl3) d = 23.45, 26.27, 37.69, 52.96,
53.14, 58.04, 61.73, 121.70, 124.57, 127.75, 128.31,
128.97, 130.15, 130.79, 132.29, 134.59, 138.75, 171.73;
IR (KBr) 3271 (NH), 2944, 2810, 1656 (C@O), 1584,
1532 (amide), 1471, 1441, 1128, 756 cmÀ1; HRMS
(ES): m/z calcd for C22H26Cl3N3NaO: [M+Na]+ =
476.1039, found = 476.1032.
1
Compound 5h: H NMR (400 MHz, CDCl3) d = 1.58–
1.72 (m, 4H), 2.41 (m, 4H), 2.64 (m, 4H), 3.20 (d,
J = 5.1 Hz, 4H), 3.22 (s, 2H), 3.59 (t, J = 4.6 Hz, 2H),
3.74 (br s, 2H), 6.85 (t, J = 7.3 Hz, 1H), 6.93 (d,
J = 7.9 Hz, 2H), 7.09 (td, J = 1.5, 7.8 Hz, 1H), 7.28 (m,
3H), 7.38 (dd, J = 1.4, 9.4 Hz, 1H), 8.46 (dd, J = 1.5,
8.2 Hz, 1H), 9.87 (s, 1H); 13C NMR (100 MHz, CDCl3)
d = 23.1, 26.5, 30.0, 41.7, 45.7, 49.1, 53.1, 53.2, 53.5,
58.1, 62.0, 116.0, 119.7, 120.9, 122.6, 124.7, 127.9,
129.1, 134.3, 151.2, 167.9, 171.4; MS (FAB) (m/z) 498
([M+H]+, 99), 496 (34), 154 (100), 136 (68); HRMS
(FAB) calcd for C27H37O2N5Cl: [M+H]+ = 498.2558,
found = 498.2635.
3.1.12. General procedure of compounds 9a–l. Piperazine
derivative (1.00 mmol) was added to a solution of
4-bromo-N-(4-chlorophenyl)butyramide (8) (250 mg,
0.90 mmol) and Et3N (0.19 mL, 1.36 mmol) in CH2Cl2
(5.0 mL) at room temperature. Then the mixture was
stirred at reflux for overnight. The reaction mixture
was cooled to room temperature and quenched by
addition of water (10 mL). The mixture was extracted
with dichloromethane (3· 10 mL), dried over anhydrous
MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified by flash column
1
Compound 5i: H NMR (400 MHz, CDCl3) d = 1.56–
1.74 (m, 4H), 2.38 (t, J = 7.2 Hz, 2H), 2.44

1416
J. H. Park et al. / Bioorg. Med. Chem. 15 (2007) 1409–1419
chromatography (SiO2, CH2Cl2/MeOH = 20:1) to give
product 9a–l.
m/z calcd for C21H25Cl2N3NaO: [M+Na]+ = 428.1273,
found = 428.1278.
1
Compound 9f: 80%; 1H NMR (400 MHz, CDCl3)
d = 1.94 (2H, m, CH2), 2.48 (2H, t, J = 5.15 Hz, CH2),
2.54 (2H, t, J = 7.87 Hz, CH2), 2.69 (4H, s, 2CH2), 3.12
(4H, s, 2CH2), 3.86 (3H, s, OCH3), 6.86–7.49 (8H, m,
Ph), 8.80 (1H, br s, NH); 13C NMR (100 MHz, CDCl3)
d = 22.08, 35.77, 50.60, 53.24, 55.40, 56.95, 111.23,
111.31, 111.36, 118.18, 118.45, 121.00, 121.05, 128.83,
128.96, 152.28, 171.47; IR (KBr) 3257 (NH), 2939,
2815, 1665 (C@O), 1595, 1500 (amide), 1239, 1027,
748 cmÀ1; HRMS (ES): m/z calcd for C21H26ClN3NaO2:
[M+Na]+ = 410.1611, found = 410.1611.
Compound 11f: 40%; mp 82.2 ꢁC; H NMR (400 MHz,
CDCl3) d = 1.94 (2H, m, CH2), 2.42–2.49 (12H, m,
6CH2), 3.44 (2H, s, CH2(benzyl)), 8.02 (1H, br s, NH),
7.16–8.31 (7H, m, Ph); 13C NMR (100 MHz, CDCl3)
d = 22.35, 35.70, 52.95, 53.02, 57.04, 63.37, 122.75,
127.17, 127.22, 127.86, 128.73, 129.03, 129.45, 133.54,
134.15, 140.31, 171.36; IR (KBr) 3300 (NH), 2939,
2820, 1656 (C@O), 1578, 1519 (amide), 777 cmÀ1
;
HRMS (ES): m/z calcd for C21H24Cl3N3NaO:
[M+Na]+ = 462.0883, found = 462.0891.
3.1.14. General procedure of compounds 13a and b. Piper-
azine derivative (0.801 mmol) was added to a solu-
tion of 5-bromo-1-[4-(2-fluorophenyl)piperazin-1-yl]-
pentan-1-one (12) (250 mg, 0.728 mmol) and Et3N
(0.15 mL, 1.09 mmol) in CH2Cl2 (5.0 mL) at room
temperature. Then the mixture was stirred at reflux
for overnight. The reaction mixture was cooled to
room temperature and quenched by addition of water
(10 mL). The mixture was extracted with dichloro-
methane (3· 10 mL), dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure.
The residue was purified by flash column chromatog-
raphy (SiO2, CH2Cl2/MeOH = 20:1) to give product
13a and 13b.
3.1.13. General procedure of compounds 11a–j. Pipera-
zine derivative (0.88 mmol) was added to a solution
of 4-bromo-N-(2,4-dichlorophenyl)butyramide (10)
(250 mg, 0.80 mmol) and Et3N (0.22 mL, 1.61 mmol)
in CH2Cl2 (5.0 mL) at room temperature. Then the mix-
ture was stirred at reflux for overnight. The reaction
mixture was cooled to room temperature and quenched
by addition of water (10 mL). The mixture was extracted
with dichloromethane (3· 10 mL), dried over anhydrous
MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by flash column chroma-
tography (SiO2, CH2Cl2/MeOH = 20:1) to give product
11a–j.
1
Compound 11c: 44%; mp 114.4 ꢁC; 1H NMR (400 MHz,
CDCl3) d = 1.96 (2H, m, CH2), 2.50–2.53 (4H, m, 2CH2),
2.66 (4H, s, 2CH2), 3.07 (4H, s, 2CH2), 3.86 (3H, s,
OCH3), 8.05 (1H, br s, NH), 6.85–8.34 (7H, m, Ph);
13C NMR (100 MHz, CDCl3) d = 22.35, 35.71, 50.59,
53.32, 55.35, 57.11, 111.20, 118.17, 120.99, 122.70,
122.96, 123.38, 127.87, 128.73, 129.04, 133.55, 141.25,
152.28, 171.40; IR (KBr) 3246 (NH), 2959, 2935, 2811,
Compound 13a: 48%; mp 85.0 ꢁC; H NMR (400 MHz,
CDCl3) d = 1.56 (2H, m, CH2), 1.68 (2H, m, CH2), 1.80
(4H, s, 2CH2), 2.38 (4H, t, J = 7.40 Hz, 2CH2), 2.48
(4H, s, 2CH2), 3.05 (4H, m, 2CH2), 3.47 (2H, s,
CH2(benzyl)), 3.64 (2H, t, J = 4.84 Hz, CH2), 3.79
(2H, t, J = 4.91 Hz, CH2), 6.90–7.33 (8H, m, Ph); 13C
NMR (100 MHz, CDCl3) d = 23.27, 26.54, 33.10,
41.61, 45.74, 50.72, 53.07, 58.11, 62.39, 116.17, 116.38,
119.19, 123.12, 124.54, 127.20, 129.06, 129.45, 134.14,
140.39, 171.45; IR (KBr) 2940, 2815, 1641 (C@O),
1505, 1436, 1234, 1207, 758 cmÀ1; HRMS (ES): m/z
calcd for C26H34ClFN4NaO: [M+Na]+ = 495.2303,
found = 495.2304.
1660 (C@O), 1580, 1521 (amide), 1243, 742 cmÀ1
;
HRMS (ES): m/z calcd for C21H25Cl2N3NaO2:
[M+Na]+ = 444.1221, found = 444.1227.
Compound 11d: 51%; mp 63.8 ꢁC; 1H NMR (400 MHz,
CDCl3) d = 1.97 (2H, m, CH2), 2.49 (2H, t,
J = 4.83 Hz, CH2), 2.51 (2H, t, J = 5.14 Hz, CH2),
2.60 (4H, t, J = 4.96 Hz, 2CH2), 3.18 (4H, t,
J = 4.97 Hz, 2CH2), 3.79 (3H, s, OCH3), 7.94 (1H, br
s, NH), 6.41–8.35 (7H, m, Ph); 13C NMR (100 MHz,
CDCl3) d = 22.31, 35.58, 49.03, 53.07, 55.19, 57.02,
102.51, 104.46, 108.86, 122.57, 127.90, 128.74, 129.78,
133.48, 152.64, 160.58, 171.29; IR (KBr) 3307 (NH),
2954, 2826, 2807, 1665 (C@O), 1586, 1511 (amide),
1210, 1181, 971, 825 cmÀ1; HRMS (ES): m/z calcd
for C21H25Cl2N3NaO2: [M+Na]+ = 444.1221, found =
444.1225.
1
Compound 13b: 43%; mp 86.1 ꢁC; H NMR (400 MHz,
CDCl3) d = 1.55 (2H, m, CH2), 1.67 (2H, m, CH2), 1.75
(4H, s, 2CH2), 2.38 (4H, t, J = 7.46 Hz, 2CH2), 2.47 (4H,
s, 2CH2), 3.04 (2H, t, J = 5.13 Hz, CH2), 3.07 (2H, t,
J = 5.05 Hz, CH2), 3.50 (2H, s, CH2(benzyl)), 3.63 (2H,
t, J = 4.90 Hz, CH2), 3.79 (2H, t, J = 4.99 Hz, CH2),
6.92–7.29 (8H, m, Ph); 13C NMR (100 MHz, CDCl3)
d = 23.26, 26.51, 33.08, 41.62, 45.77, 50.74, 53.05,
58.12, 62.24, 116.17, 116.38, 119.22, 123.12, 124.58,
128.35, 130.43, 132.74, 136.70, 171.44; IR (KBr) 2942,
2818, 1638 (C@O), 1505, 1438, 1237, 1208, 754 cmÀ1
;
HRMS (ES): m/z calcd for C26H34ClFN4NaO:
1
Compound 11e: 43%; mp 81.0 ꢁC; H NMR (400 MHz,
[M+Na]+ = 495.2303, found = 495.2301.
CDCl3) d = 1.91 (2H, m, CH2), 2.41–2.49 (12H, m,
6CH2), 3.48 (2H, s, CH2(benzyl)), 8.06 (1H, br s, NH),
7.22–8.30 (8H, m, Ph); 13C NMR (100 MHz, CDCl3)
d = 22.36, 35.71, 52.96, 53.06, 57.05, 63.05, 122.82,
127.05, 127.84, 128.20, 128.73, 129.05, 129.20, 133.56,
137.98, 171.40; IR (KBr) 3313 (NH), 2816, 1660
(C@O), 1578, 1509 (amide), 743 cmÀ1; HRMS (ES):
3.1.15. General procedure of compounds 15a and b.
Piperazine derivative (0.85 mmol) was added to a
solution of 5-bromo-1-(4-phenylpiperazin-1-yl)pentan-
1-one (14) (250 mg, 0.77 mmol) and Et3N (0.16 mL,
1.15 mmol) in CH2Cl2 (5.0 mL) at room temperature.
Then the mixture was stirred at reflux for overnight.

J. H. Park et al. / Bioorg. Med. Chem. 15 (2007) 1409–1419
1417
The reaction mixture was cooled to room temperature
and quenched by addition of water (10 mL). The mix-
ture was extracted with dichloromethane (3· 10 mL),
dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure. The residue was purified by
flash column chromatography (SiO2, CH2Cl2/
MeOH = 20:1) to give product 15a and 15b.
Compound 17j: 49%; mp 111.2 ꢁC; 1H NMR (400 MHz,
CDCl3) d = 2.55–2.72 (12H, m, 6CH2), 3.51 (2H, s,
CH2(benzyl)), 6.82–8.38 (7H, m, Ph), 11.27 (1H, br s,
NH); 13C NMR (100 MHz, CDCl3) d = 32.54, 52.36,
52.59, 53.54, 62.07, 103.29, 110.94, 122.44, 123.63,
128.40, 130.36, 132.82, 136.57, 170.80; IR (KBr) 3118
(NH), 3049, 2939, 2819, 1680 (C@O), 1613, 1555, 1506
(amide), 1491, 1432, 1234, 1132, 1010, 961, 807 cmÀ1
;
Compound 15a: 38%; 1H NMR (400 MHz, CDCl3)
d = 1.54 (2H, m, CH2), 1.67 (2H, m, CH2), 2.35–2.46
(8H, m, 4CH2), 3.16 (4H, m, 2CH2), 3.45 (2H, s,
CH2(benzyl)), 3.62 (2H, t, J = 4.90 Hz, CH2), 3.77
(2H, t, J = 5.00 Hz, CH2), 6.89–7.30 (9H, m, Ph); 13C
NMR (100 MHz, CDCl3) d = 23.28, 26.58, 33.11,
41.48, 45.55, 49.65, 53.10, 58.15, 62.26, 116.02, 116.64,
120.56, 128.34, 129.25, 130.42, 132.71, 136.73, 150.97,
171.42; HRMS (ES): m/z calcd for C26H35ClN4NaO:
[M+Na]+ = 477.2397, found = 477.2391.
HRMS (ES): m/z calcd for C20H22ClF2N3NaO:
[M+Na]+ = 416.1317, found = 416.1313.
Compound 17l: 69%; mp 104.3 ꢁC; 1H NMR (400 MHz,
CDCl3) d = 2.56–2.72 (12H, m, 6CH2), 3.50 (2H, s,
CH2(benzyl)), 6.83–8.39 (6H, m, Ph), 11.24 (1H, br s,
NH); 13C NMR (100 MHz, CDCl3) d = 32.53, 52.31,
52.58, 53.51, 61.55, 103.27, 110.96, 122.40, 123.60,
128.23, 130.19, 130.71, 130.97, 132.34, 138.56, 170.74;
IR (KBr) 3182 (NH), 2940, 2823, 1678 (C@O),
1612, 1568, 1504 (amide), 1468, 1435, 1254, 1136,
960, 840, 809 cmÀ1; HRMS (ES): m/z calcd for
C20H21Cl2F2N3NaO: [M+Na]+ = 450.0927, found =
450.0925.
3.1.16. General procedure of compounds 17a–l. Pipera-
zine derivative (1.04 mmol) was added to a solution
of 3-bromo-N-(2,4-difluorophenyl)propionamide (16)
(250 mg, 0.95 mmol) and Et3N (0.20 mL, 1.42 mmol)
in CH2Cl2 (5.0 mL) at room temperature. Then the mix-
ture was stirred at reflux for overnight. The reaction
mixture was cooled to room temperature and quenched
by addition of water (10 mL). The mixture was extract-
ed with dichloromethane (3· 10 mL), dried over anhy-
drous MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash col-
umn chromatography (SiO2, only EtOAc) to give prod-
uct 17a–l.
3.1.17. General procedure of compounds 19a–l. Pipera-
zine derivative (1.80 mmol) was added to a solution
of 4-bromo-N-(2,4-difluorophenyl)butyramide (18)
(500 mg, 1.80 mmol) and Et3N (0.30 mL, 2.16 mmol)
in CH2Cl2 (10.0 mL) at room temperature. Then the
mixture was stirred at reflux for overnight. The
reaction mixture was cooled to room temperature and
quenched by addition of water (20 mL). The mixture
was extracted with dichloromethane (3· 15 mL), dried
over anhydrous MgSO4, filtered, and concentrated un-
der reduced pressure. The residue was purified by flash
column chromatography (SiO2, only EtOAc) to give
product 19a-l.
Compound 17a: 96%; mp 91.7 ꢁC; 1H NMR (400 MHz,
CDCl3) d = 2.61 (2H, t, J = 5.74 Hz, CH2), 2.77 (6H, s,
3CH2), 3.30 (4H, s, 2CH2), 6.78–8.39 (8H, m, Ph), 11.22
(1H, br s, NH); 13C NMR (100 MHz, CDCl3) d = 32.57,
48.91, 52.44, 53.65, 103.30, 110.99, 116.21, 120.01,
122.32, 123.48, 129.18, 170.64; IR (KBr) 3113
(NH), 2827, 1674 (C@O), 1599, 1555, 1503 (amide),
1248, 1096, 959, 753 cmÀ1; HRMS (ES): m/z calcd
for C19H21F2N3NaO: [M+Na]+ = 368.1550, found =
368.1548.
Compound 19b: 46%; mp 105.2 ꢁC; 1H NMR
(400 MHz, CDCl3) d = 1.96 (2H, m, CH2), 2.52 (4H,
m, 2CH2), 2.60 (4H, s, 2CH2), 3.12 (4H, s, 2CH2),
6.82–8.26 (7H, m, Ph), 8.29 (1H, br s, NH); 13C NMR
(100 MHz, CDCl3) d = 22.10, 35.38, 50.35, 53.08,
56.67, 103.50, 111.19, 116.09, 118.87, 122.51, 122.81,
123.21, 124.45, 139.97, 171.42; IR (KBr) 3274 (NH),
2756, 1654 (C@O), 1613, 1535, 1502 (amide), 1429,
Compound 17b: 98%; mp 102.3 ꢁC; 1H NMR (400 MHz,
CDCl3) d = 2.61 (2H, t, J = 5.76 Hz, CH2), 2.79 (6H, s,
3CH2), 3.21 (4H, s, 2CH2), 6.83–8.42 (7H, m, Ph),
11.25 (1H, br s, NH); 13C NMR (100 MHz, CDCl3)
d = 32.53, 50.15, 52.49, 53.66, 103.28, 111.01, 116.16,
122.37, 122.75, 123.52, 139.75, 170.69; IR (KBr) 3124
(NH), 2929, 2830, 1687 (C@O), 1610, 1561, 1500
(amide), 1244, 1137, 923, 758 cmÀ1; HRMS (ES): m/z
calcd for C19H20F3N3NaO: [M+Na]+ = 386.1456,
found = 386.1450.
1354, 1251, 1207, 1139, 1099, 959, 844, 747 cmÀ1
;
HRMS (ES): m/z calcd for C20H22F3N3NaO:
[M+Na]+ = 400.1613, found = 400.1617.
1
Compound 19c: 43%; mp 86.7 ꢁC; H NMR (400 MHz,
CDCl3) d = 1.96 (2H, m, CH2), 2.52 (4H, m, 2CH2), 2.62
(4H, t, J = 4.95 Hz, 2CH2), 3.12 (4H, t, J = 4.95 Hz,
2CH2), 8.18 (1H, br s, NH), 6.82–8.25 (7H, m, Ph);
13C NMR (100 MHz, CDCl3) d = 22.14, 35.35, 50.07,
53.03, 56.58, 103.49, 111.20, 115.51, 117.80, 123.07,
147.86, 171.34; IR (KBr) 3288 (NH), 2945, 2820,
1667 (C@O), 1531, 1509 (amide), 1267, 1239, 1229,
Compound 17i: 89%; 1H NMR (400 MHz, CDCl3)
d = 2.54–2.78 (12H, m, 6CH2), 3.52 (2H, s, CH2(ben-
zyl)), 6.83–8.41 (7H, m, Ph), 11.28 (1H, br s, NH); 13C
NMR (100 MHz, CDCl3) d = 32.56, 52.36, 52.65,
53.56, 62.24, 103.28, 110.94, 122.46, 123.50, 127.21,
128.99, 129.51, 134.21, 140.32, 170.81; HRMS (ES): m/
z calcd for C20H22ClF2N3NaO: [M+Na]+ = 416.1317,
found = 416.1321.
1153, 825, 816 cmÀ1
;
HRMS (ES): m/z calcd
for C20H22F3N3NaO: [M+Na]+ = 400.1613, found =
400.1618.
Compound 19d: 33%; mp 123.1 ꢁC; 1H NMR
(400 MHz, CDCl3) d = 1.97 (2H, m, CH2), 2.51 (2H, t,

1418
J. H. Park et al. / Bioorg. Med. Chem. 15 (2007) 1409–1419
J = 6.93 Hz, CH2), 2.55 (2H, t, J = 6.66 Hz, CH2), 2.67
(4H, s, 2CH2), 3.08 (4H, s, 2CH2), 6.85–8.28 (7H, m,
Ph), 8.34 (1H, br s, NH); 13C NMR (100 MHz,
CDCl3) d = 22.12, 35.43, 51.03, 53.16, 56.65, 103.49,
111.19, 120.27, 123.20, 123.72, 127.56, 128.75, 130.62,
149.10, 171.43; IR (KBr) 3276 (NH), 2961, 2820,
1665 (C@O), 1614, 1504 (amide), 1480, 1261, 1142,
854, 756, 690 cmÀ1; HRMS (ES): m/z calcd for
C20H22ClF2N3NaO: [M+Na]+ = 416.1317, found =
416.1313.
(1H, br s, NH); 13C NMR (100 MHz, CDCl3)
d = 22.12, 35.57, 52.29, 52.50, 52.95, 56.64, 103.53,
111.11, 113.92, 123.76, 125.40, 129.09, 136.60, 171.56;
IR (KBr) 3303 (NH), 2943, 2821, 1680 (C@O),
1661, 1610, 1556, 1538, 1500 (amide), 1453, 1430,
1141, 1007, 854 cmÀ1; HRMS (ES): m/z calcd for
C21H23ClF3N3NaO: [M+Na]+ = 4448.1379, found =
448.1373.
Compound 19l: 50%; 1H NMR (400 MHz, CDCl3)
d = 1.91 (2H, m, CH2), 2.44 (12H, m, 6CH2),
3.42 (2H, s, CH2(benzyl)), 6.83–8.24 (6H, m, Ph),
8.36 (1H, br s, NH); 13C NMR (100 MHz,
CDCl3) d = 22.13, 35.45, 52.88, 52.92, 56.59,
61.70, 103.50, 111.15, 123.35, 128.24, 130.15, 130.72,
132.29, 138.65, 171.46; HRMS (ES): m/z calcd for
C21H23Cl2F2N3NaO: [M+Na]+ = 464.1084, found =
464.1087.
1
Compound 19e: 30%; mp 90.3 ꢁC; H NMR (400 MHz,
CDCl3) d = 1.98 (2H, m, CH2), 2.52 (4H, t, J = 6.66 Hz,
2CH2), 2.64 (4H, s, 2CH2), 3.22 (4H, s, 2CH2), 8.13 (1H,
br s, NH), 6.76–8.24 (7H, m, Ph); 13C NMR (100 MHz,
CDCl3) d = 21.97, 35.18, 48.43, 52.77, 56.58, 103.27,
111.21, 113.90, 115.80, 119.45, 123.15, 130.03, 134.94,
152.12, 171.22; IR (KBr) 3245 (NH), 2962, 2816, 1666
(C@O), 1593, 1533 (amide), 1485, 1258, 1242, 1143,
1099, 943, 849, 680 cmÀ1; HRMS (ES): m/z calcd
for C20H22ClF2N3NaO: [M+Na]+ = 416.1317, found =
416.1315.
3.1.18. General procedure of compounds 21a–l. Pipera-
zine derivative (0.80 mmol) was added to a solution of
4-bromo-N-(3,4,5-trichlorophenyl)butyramide
(20)
(250 mg, 0.72 mmol) and Et3N (0.15 mL, 1.09 mmol)
in CH2Cl2 (5.0 mL) at room temperature. Then the mix-
ture was stirred at reflux for overnight. The reaction
mixture was cooled to room temperature and quenched
by addition of water (10 mL). The mixture was extracted
with dichloromethane (3· 10 mL), dried over anhydrous
MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by flash column chroma-
tography (SiO2, CH2Cl2/MeOH = 20:1) to give product
21a–l.
1
Compound 19f: 53%; mp 83.9 ꢁC; H NMR (400 MHz,
CDCl3) d = 1.96 (2H, m, CH2), 2.52 (4H, m, 2CH2), 2.67
(4H, s, 2CH2), 3.09 (4H, s, 2CH2), 3.87 (3H, s, OCH3),
6.82–8.23 (7H, m, Ph), 8.43 (1H, br s, NH); 13C NMR
(100 MHz, CDCl3) d = 22.13, 35.48, 50.49, 53.23,
55.32, 56.70, 103.49, 111.18, 118.11, 120.95, 122.97,
123.26, 141.15, 152.22, 171.52; IR (KBr) 3289 (NH),
2941, 2818, 1660 (C@O), 1595, 1531, 1505 (amide),
1430, 1243, 1130, 960, 743 cmÀ1; HRMS (ES): m/z calcd
for C21H25F2N3NaO2: [M+Na]+ = 412.1812, found =
412.1817.
Compound 21f: 47%; mp 178.0 ꢁC; 1H NMR (400 MHz,
CDCl3) d = 1.94 (2H, m, CH2), 2.50 (2H, t, J = 6.61 Hz,
CH2), 2.56 (2H, t, J = 6.22 Hz, CH2), 2.72 (4H, s,
2CH2), 3.15 (4H, s, 2CH2), 3.87 (3H, s, OCH3), 6.87–
7.68 (6H, m, Ph), 9.34 (1H, br s, NH); 13C NMR
(100 MHz, CDCl3) d = 21.69, 36.25, 50.54, 53.31,
55.41, 57.17, 111.24, 118.27, 119.74, 121.09, 123.37,
125.81, 134.20, 137.64, 140.80, 152.27, 171.69; IR
(KBr) 3247 (NH), 2941, 2820, 1673 (C@O), 1588,
Compound 19g: 53%; mp 55.3 ꢁC; 1H NMR (400 MHz,
CDCl3) d = 1.96 (2H, m, CH2), 2.51 (4H, m, 2CH2), 2.61
(4H, t, J = 4.78 Hz, 2CH2), 3.20 (4H, t, J = 4.78 Hz,
2CH2), 3.80 (3H, s, OCH3), 6.41–8.26 (8H, m, Ph and
NH); 13C NMR (100 MHz, CDCl3) d = 22.11, 35.34,
48.94, 52.98, 55.16, 56.62, 102.50, 103.52, 104.44,
108.83, 111.28, 123.17, 129.77, 152.58, 160.55, 171.38;
IR (KBr) 3294 (NH), 2944, 2822, 1670 (C@O),
1611, 1580, 1524, 1501 (amide), 1465, 1258, 1203,
1139, 1010, 844, 759 cmÀ1; HRMS (ES): m/z calcd
for C21H25F2N3NaO2: [M+Na]+ = 412.1812, found =
412.1818.
1522, 1498 (amide), 1443, 1240, 1186, 1150, 754 cmÀ1
;
HRMS (ES): m/z calcd for C21H24Cl3N3NaO2:
[M+Na]+ = 478.0832, found = 478.0836.
Compound 21i: 54%; mp 115.8 ꢁC; 1H NMR
(400 MHz, CDCl3) d = 1.90 (2H, m, CH2), 2.49 (12H,
m, 6CH2), 3.51 (2H, s, CH2(benzyl)), 7.00–7.73 (6H,
m, Ph), 9.43 (1H, br s, NH); 13C NMR (100 MHz,
CDCl3) d = 21.65, 36.38, 52.83, 53.05, 57.28, 62.35,
119.84, 127.13, 127.04, 128.98, 129.57, 134.15, 134.27,
137.65, 140.10, 171.67; IR (KBr) 3307 (NH),
2951, 2809, 1663 (C@O), 1577, 1507 (amide), 1439,
1383, 1148, 857, 778 cmÀ1; HRMS (ES): m/z calcd
for C21H23Cl4N3NaO: [M+Na]+ = 496.0493, found =
496.0486.
1
Compound 19j: 59%; mp 79.0 ꢁC; H NMR (400 MHz,
CDCl3) d = 1.90 (2H, m, CH2), 2.45 (12H, m, 6CH2),
3.44 (2H, s, CH2(benzyl)), 6.85–8.19 (7H, m, Ph), 8.44
(1H, br s, NH); 13C NMR (100 MHz, CDCl3)
d = 22.09, 35.40, 52.80, 52.91, 56.60, 62.14, 103.46,
111.10, 123.38, 128.30, 130.32, 132.67, 136.57, 171.48;
IR (KBr) 3328 (NH), 2953, 2821, 1792, 1671 (C@O),
1610, 1535, 1520, 1500 (amide), 1431, 1259, 1137,
1097, 1009, 849 cmÀ1
;
HRMS (ES): m/z calcd
for C21H24ClF2N3NaO: [M+Na]+ = 430.1474, found =
430.1480.
3.2. Biological assay
Compound 19k: 65%; mp 69.3 ꢁC; 1H NMR (400 MHz,
CDCl3) d = 1.89 (2H, m, CH2), 2.46 (12H, m, 6CH2),
3.70 (2H, s, CH2(benzyl)), 6.82–8.19 (6H, m, Ph), 8.49
3.2.1. FDSS 6000 assay (calcium imaging plate reader).
HEK293 cells which stably express both a1G and Kir2.1
subunits23 were grown in Dulbecco’s modified Eagle’s

J. H. Park et al. / Bioorg. Med. Chem. 15 (2007) 1409–1419
1419
medium supplemented with 10% (v/v) fetal bovine
Supplementary data
serum, penicillin (100 U/ml), streptomycin (100 lg/ml),
geneticin (500 lg/ml), and puromycin (1 lg/ml) at
37 ꢁC in a humid atmosphere of 5% CO2 and 95%
air. Cells were seeded into 96-well black wall clear-
bottomrf plates at a density of 40,000 cells/well and
were used on the next day for high-throughput screen-
ing (HTS) FDSS 6000 assay.24 For FDSS6000 assay,
cells were incubated for 60 min at room temperature
with 5 lM fluo3/AM and 0.001% Pluronic F-127 in
a HEPES-buffered solution composed of (in mM):
115 NaCl, 5.4 KCl, 0.8 MgCl2, 1.8 CaCl2, 20 HEPES,
and 13.8 glucose (pH 7.4). During fluorescence-based
FDSS6000 assay, a1G T-type Ca2+ channels were acti-
vated using a high concentration of KCl (70 mM) in
10 mM CaCl2 containing HEPES-buffered solution,
and the increase in [Ca2+]i by KCl-induced depolariza-
tion was detected. All data were collected and
analyzed using FDSS6000 and related software
(Hamamatsu, Japan).
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2006.11.004.
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Acknowledgments
We thank to Prof. Sang-gi Lee and Dr. Bang Sook
Lee in Ewha Women’s University and Min Jung
Park for their contribution in this work. We also
acknowledge Prof. Jung-Ha Lee’s group in Sogang
University for biological assay of compounds (5a–f).
This study was supported by Vision 21 Program
(2E 18970) and National Research Laboratory Pro-
gram (2E 19310) from Korea Institute of Science
and Technology.