Synthesis and biological evaluation of 1-(2-hydroxy-3-phenyloxypropyl) piperazine derivatives as T-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmcl.2012.12.072

To obtain selective and potent inhibitor for T-type calcium channel by ligand based drug design, 2-hydroxy-3-phenoxypropyl piperazine derivatives were synthesized and evaluated for in vitro activities. Compound 6m and 6q showed high selectivity over hERG

Full text of DOI:10.1016/j.bmcl.2012.12.072

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 1-(2-hydroxy-3-phenyloxy-
propyl)piperazine derivatives as T-type calcium channel blockers
Jung-eun Park a,b, Wan Keun Ji a,c, Jae Wan Jang d, Ae Nim Pae d, Keehyun Choi a, Ki Hang Choi c,
Jahyo Kang b, Eun Joo Roh a,
⇑
a Chemical Kinomics Research Center, Korea Institute of Science and Technology, Wolsong-gil 5, Seongbuk-gu, Seoul, Republic of Korea
b Department of Chemistry, Sogang University, Baekbeomno 35, Mapo-gu, Seoul, Republic of Korea
c Department of Chemistry, Korea University, Anam-dong, Seongbuk-gu, Seoul, Republic of Korea
d Center for Neuro-Medicine, Korea Institute of Science and Technology, Wolsong-gil 5, Seongbuk-gu, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
To obtain selective and potent inhibitor for T-type calcium channel by ligand based drug design, 2-
hydroxy-3-phenoxypropyl piperazine derivatives were synthesized and evaluated for in vitro activities.
Compound 6m and 6q showed high selectivity over hERG channel (IC50 ratio of hERG/a1G 6m = 8.5,
6q = 18.38) and they were subjected to measure pharmacokinetics profiles. Among them compound
6m showed an excellent pharmacokinetic profile in rats.
Received 24 September 2012
Revised 10 December 2012
Accepted 21 December 2012
Available online xxxx
Ó 2013 Elsevier Ltd. All rights reserved.
Voltage-dependent calcium channels are the main pathway for
calcium ion influx to the cellular membrane. The mechanism used
is the difference in membrane potential, and this plays an
important physiological role in the excitability cells.1 Based on
electrophysiological and pharmacological characteristics, voltage-
dependent calcium channels are classified as either high-voltage-
activated (L, N, P, Q/R type) channel or low-voltage-activated
mented in the CATALYST software was employed for hypothesis
generation and the derived query was used for screening of com-
mercial library such as ion channel and Maybridge 2001. The vir-
tual screening identified hit compound (1) which overlaid well
on our five feature phamacophore including three hydrophobic
groups, one hydrogen bond acceptor and one positive ionizable
feature (Fig. 1). Two hydrophobic groups matched with halogen
substituents on benzyl moiety and another hydrophobic group
mapped well onto phenyl ring of phenoxy moiety. Positive ioniz-
able group mapped onto piperazine moiety and hydrogen bond
acceptor matched well with hydroxyl group. From the above fea-
ture, we designed and synthesized a series of 1-(2-hydroxy-3-
phenoxypropyl)piperazine derivatives (4a–q, 5a–r, 6a–r).
Synthesis of 1-(2-hydroxy-3-phenoxypropyl)piperazine deriva-
tives is elucidated in Scheme 1. The phenols (2) were reacted with
chiral epichlorohydrin to give oxirane (3a–f),7 which was subjected
to SN2 displacement with derivatives of piperazine to give the tar-
get compounds 4a–q, 5a–r and 6a–r (Scheme 1).8
As shown in Table 1, the first in vitro activities of the synthe-
sized compounds 4a–q, 5a–r and 6a–r were assayed using the
FDSS6000 HTS (high-throughput screening) system.9 Our initial
synthetic plan was to fix the chloride on phenyl group (X = Cl) sim-
ilar to compound 1, and to change various piperazines. Also we’d
like to figure out the relation between biological activities and
compound chirality (4a–q). Most compounds in series of 4a–q
exhibited greater than 40% inhibitory activity against the a1G cal-
(T-type) channels. Calcium channels consist of four subunits: a1
2d, b, and . The main structural component of voltage-dependent
calcium channels is the 1 subunit, which can be divided by molec-
,
a
c
a
ular cloning into 10 subtypes; Cav 1.1–1.4 (L-type), Cav 2.1–2.3 (N,
P, Q/R-type), Cav 3.1–3.3 (T-type).2
T-type calcium channels are expressed throughout the body,
including nervous tissue, heart, kidney, smooth muscle, and many
endocrine organs. Therefore, selective T-type calcium channel
blockers may have high potentials for the treatment of some types
of cancer, hypertension, cardiac arrhythmia, and CNS-related dis-
orders3 such as epilepsy, neurophatic pain3 and insomnia.4
Mibefradil, the first marketed selective T-type calcium channel
blocker, was withdrawn from the market due to its drug–drug
interaction.5 Unfortunately, there are no potent and selective T-
type calcium channel blockers which are available for clinical use
until now.
To discover a new structural motif of T-type calcium channel
blockers, a three-dimensional common feature pharmacophore
model was derived from eight structurally diverse T-type calcium
channel blockers including mibefradil.6 HipHop algorithm imple-
cium channel at 10 lM concentration. However, %inhibition values
of compounds 4a–q did not reach the hit compound (1, %inhibition
54.41) and mibefradil (%inhibition 81.00). Thus, the substituent on
the phenyl group was changed to fluoride 5a–r or to trifluromethyl
group 6a–r. The %inhibition values increased with both fluoride
⇑
Corresponding author.
E-mail address: r8636@kist.re.kr (E.J. Roh).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.072
Please cite this article in press as: Park, J.-e.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.072

2
J. Park et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
F
N
OH
O
N
Cl
Cl
1
% Inhibition : 54.41
(in HEK 293 cell)
1G - IC50 (μM) : 1.42 0.09
a
R
N
OH
O
N
∗
X
4a-q, 5a-r, 6a-r
Figure 1. Designed structures of T-type calcium channel blocker and the structural mapping with 2 of 1. Red sphere: positive ionizable, yellow sphere: hydrogen bond
acceptor; Blue sphere: hydrophobic group.
R
N
OH
O
OH
O
O
N
*
ii
*
i
X
X
X
2
3a-f
4a-q, 5a-r, 6a-r
Scheme 1. Reagents and conditions: (i) (R/S)-epichlorohydrin, K2CO3, 2-butanone, reflux; (ii) piperazine derivatives, MeOH, rt.
Table 1
In vitro activities for T-type calcium channel blockers of 2-hydroxy-3-phenoxypropyl piperazine derivatives using FDSS6000 HTS system
R
N
O∗H
O
N
X
4a-q, 5a-r, 6a-r
Entry
Compound (R/S)
X
R
%inhibitiona (10
lM)
R-configuration
S-configuration
1
2
3
4
5
6
7
8
4a/4j
4b/4k
4c/4l
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
F
F
F
F
2-Fluorophenyl
4-Fluorophenyl
2-Methoxyphenyl
3-Methoxyphenyl
3-Chlorobenzyl
4-Chlorobenzyl
3,4-Dichlorobenzyl
2-Chloro-6-fluorobenzyl
2-Fluorobenzyl
2-Fluorophenyl
4-Fluorophenyl
2-Methoxyphenyl
3-Methoxyphenyl
3-Chlorobenzyl
46.93
39.69
47.38
44.60
45.95
42.12
48.61
40.86
35.76
51.15
47.48
50.67
61.30
61.10
50.14
69.45
56.45
23.77
50.21
53.74
56.24
43.94
43.13
35.90
28.32
48.81
45.99
52.18
56.46
41.17
54.41
41.46
42.00
49.99
41.02
44.46
65.79
65.76
64.18
62.07
20.34
48.88
59.22
50.84
68.55
43.42
4d/4m
4e/4n
4f/4o
4g/4p
4h/1b
4i/4q
5a/5j
5b/5k
5c/5l
5d/5m
5e/5n
5f/5o
5g/5p
5h/5q
5i/5r
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
4-Chlorobenzyl
3,4-Dichlorobenzyl
2-Chloro-6-fluorobenzyl
2-Fluorobenzyl
2-Fluorophenyl
4-Fluorophenyl
2-Methoxyphenyl
3-Methoxyphenyl
3-Chlorobenzyl
6a/6j
6b/6k
6c/6l
6d/6m
6e/6n
CF3
CF3
CF3
CF3
CF3
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J. Park et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
Table 1 (continued)
Entry
Compound (R/S)
X
R
%inhibitiona (10
lM)
R-configuration
S-configuration
24
25
26
27
6f/6o
6g/6p
6h/6q
6i/6r
CF3
CF3
CF3
CF3
4-Chlorobenzyl
61.54
38.81
44.57
45.46
81.00
56.57
45.62
55.09
48.58
3,4-Dichlorobenzyl
2-Chloro-6-fluorobenzyl
2-Fluorobenzyl
Mibefradil
a
%inhibition value was obtained at 10
Hit compound from pharmacophore modeling.
l
M.
b
Table 2
Inhibitory activities of selected compounds against T-type calcium channel and hERG
meaning of the IC50 of hERG/a1G shows selective inhibition of the
T-type calcium channel. Most compounds showed good activities
against T-type calcium channel (Table 2). When R group was 4-
Compound
a
1G-IC50
(
lM)
hERG-IC50
(lM)
hERG-IC50/a1G-IC50 (lM)
chlorobenzyl, it had good potency in IC50 values of a1G, but didn’t
4o
5d
5e
5g
5h
5n
5o
5p
5q
6c
6g
6k
6m
6o
6q
1.08 0.02
12
0.32 0.09
—
NA
0.87 0.13
—
—
1.35 0.05
0.29 0.02
—
3.95 0.15
9.2 0.5
—
4.08 1.28
—
13.97 8.3
2.07 5.04
1.40 0.29
0.30
showed selectivity between a1G and hERG channel (4o, 5o).
Whereas some compounds (5g, 5p, 6m, 6q) showed strong
potency than hit compound (1) and mibefradil. 3,4-Dichlorobenzyl
substituent on R group showed that the compounds including
(R)-configuration (5g, 6g) showed better potency and selectivity
than (S)-configuration. Biological activities of compound 6m
(R = 3-methoxyphenyl) and 6q (2-chloro-6-fluorobenzyl) were af-
fected by X group on phenyl, not R group on piperazine. Moreover
2.1 0.78
0.1 0.05
12.33 1.2
2.58 1.23
1.56 0.89
0.23 0.01
11.38 3.03
1.79 1.1
1.27 0.37
15.5 6.3
0.48 0.27
4.23 0.42
0.76 0.62
1.42 0.09
0.83 0.19
8.70
0.87
1.26
2.21
7.24
compound 6m (IC50 of
and 6q (IC50 of 1G = 0.76 0.62, IC50 of hERG = 13.97 8.3) IC50 ra-
tio of hERG/a1G channel than mibefradil (IC50 of 1G = 0.48 0.27,
a1G = 0.83 0.19, IC50 of hERG = 4.08 1.28)
a
8.50
a
18.38
1.46
1.69
IC50 of hERG = 1.40 0.29) and they were chosen to perform fur-
ther biological assay.
1
Mibefradil
In order to determine the potential of compounds 6m and 6q as
a lead compound for therapeutic targets, these were subjected to
measure pharmacokinetic profiles. The pharmacokinetic data for
6m and 6q after oral administration in rats are presented Figure 2
and Table 3. Both compounds 6m and 6q were observed to be rap-
idly absorbed in plasma (Tmax). Concentration–time profiles
showed that compound 6m lasted longer than 6q in plasma and
brain (Fig. 2). Moreover, compound 6m possessed good oral bio-
availability (F, 67.25%) and showed a good AUCbrain/AUCplasma ratio
of 346.5% (Table 3).
5a–r and trifluoromethyl group 6a–r, indicating that the presence
of fluoride on the phenyl ring is important for the inhibition. In
addition, there was no significant effect between the stereochem-
istry of the compounds and respect to %inhibitory activities.
Compounds that showed above 55% inhibitory activity against
T-type calcium channels were evaluated with the whole-cell
patch-clamp method to measure the IC50 values for a1G in HEK
293 cell and IC50 values for hERG channel.10 With this data ob-
tained we could choose with the selective lead compound over
In summary, the series of 2-hydroxy-3-phenoxyproyl deriva-
tives 4a–q, 5a–r and 6a–r was designed, synthesized and evaluated
as T-type calcium channel blockers. Using the FDSS assay system,
we rapidly screened the title compounds and selected several hav-
hERG/a1G channel. Blocking of the hERG channel can lead to a heart
rhythm disorder, such as long QT syndrome which is characterized
by prolonged action potential of ventricular muscle.11 Thus, the
Figure 2. Mean plasma (ꢀ) or brain (°) concentration–time profiles after oral administration of 6m and 6q at a dose of 10 mg/kg to mice (n = 3 per time point). Bars represent
standard deviation.
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4
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Yang, Z.-Q.; Schlegel, K.-A. S.; Shu, Y.; Mattern, C.; Cube, R.; Rittle, K. E.;
McGaughey, G. B.; Hartman, G. D.; Tang, C.; Ballard, J.; Kuo, Y.; Prueksaritanont,
T.; Nuss, C. E.; Doran, S. M.; Fox, S. V.; Garson, S. L.; Li, Y.; Kraus, R. L.; Uebele, V.
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Brain pharmacokinetic parameters after oral administration of 6m (10 mg/kg) and 6q
(10 mg/kg) to ICR mice (n = 3 per time point)
6m
Plasma
6q
Brain
Plasma
Brain
AUC0–1
AUClast
Terminal half-life (min)
Cmax g/mL)
(
l
g min/ml or
l
g min/g)
198.1
188.5
102.4
1.566
15
686.6
497.4
474.3
4.662
15
16.55
15.61
56.54
0.1974
15
—
(l
g min/ml or
l
g min/g)
119.1
185.9
0.8702
30
(
l
Tmax (min)
AUCbrain/AUCplasma (%)
F (%)
346.5
67.25
763.0
9.935
Values are presented as mean. Cmax, peak plasma and brain concentration; Tmax
,
time to reach Cmax; AUC (area under the plasma concentration versus time cur-
ve) = dose/clearance; F, bioavailability; AUCbrain/AUCplasma, equal to B/P ratio.
8. All data of compounds such as experimental procedure, and spectroscopy data
are mentioned on Supplementary data.
9. Experimental procedure for the FDSS6000 assay: HEK 293cells which express
both stable
medium supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U/
mL), streptomycin (100 g/mL), geneticin (500 g/mL), and puromycin (1 g/
mL) at 37 °C in a humid atmosphere of 5% CO2 and 95% air. Cells were seeded
into 96-well black wall clear bottom plates at a density of 40,000 cells/well and
were used on the next day for high-throughput screening (HTS) FDSS 6000
assay. For FDSS6000 assay, cells were incubated for 60 min at room
a1G and Kir2.1 subunits were grown in Dulbecco’s modified Eagle’s
ing high potency. Especially compound 6m and 6q showed high
selectivity over hERG channel and against T-type calcium channel
(IC50 ratio of hERG/a1G 6m = 8.5, 6f = 18.38). Compound 6m
showed excellent pharmacokinetic profile in rats. Our results show
promise because we gained proof of concept using a ligand-based
drug design with a new target. Further assays with neuropathic
pain models and structural optimization will be followed with this
scaffold.
l
l
l
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, 13.8 glucose (pH 7.4). During fluorescence-based FDSS6000 assay,
a1G T-type Ca2+ channels were activated using a high concentration of KCl
(70 mM) in 10 mM CaCl2 contained a HEPES-buffered solution, and the increase
in [Ca2+]i by KCl-induced depolarization was detected. Throughout the entire
procedure, cells were washed in a BIO-TEK 96-well washer. All data were
collected and analyzed using FDSS6000 and related software (Hamamatsu,
Japan).
Acknowledgment
We thank Medifron DBT for pain animal test. This work was
supported by the research fund of Korea Institute of Science and
Technology (2E 22760).
10. Experimental procedure for the patch-clamp test (electro-physiological
recording): For the recording of a1G T-type Ca2+ currents, the standard
whole-cell patch-clamp method was utilized. Briefly, borosilicate glass
electrodes with
a resistance of 3–4 MX were pulled and filled with the
internal solution containing (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 ꢁ100 to ꢁ30 mV. The molar
concentration 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). For more details, see: (a)
Rhim, H.; Lee, Y. S.; Park, S. J.; Chung, B. Y.; Lee, J. Y. Bioorg. Med. Chem. Lett.
2005, 15, 283; (b) Choi, K. H.; Song, C.; Shin, D.; Park, S. Biochim. Biophys. Acta,
Biomembr. 2011, 1808, 1560.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.12.
072.
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Please cite this article in press as: Park, J.-e.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.072