Facile synthesis and biological evaluation of 3,3-diphenylpropanoyl piperazines as T-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmcl.2010.11.033

We have developed a facile synthesis of 3,3-diphenylpropanamides using Meldrum's acid derivatives as amide coupling components. The in vitro biological evaluation of the title compounds led to the identification of compound 1h, which has good inhibitory a

Full text of DOI:10.1016/j.bmcl.2010.11.033

Bioorganic & Medicinal Chemistry Letters 21 (2011) 215–219
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Facile synthesis and biological evaluation of 3,3-diphenylpropanoyl
piperazines as T-type calcium channel blockers
Yeon-hee Choi a,b, Du Jong Baek b, Seon Hee Seo a, Jae Kyun Lee a, Ae Nim Pae a,c, Yong Seo Cho a,c,
,
⇑
Sun-Joon Min a,c,
⇑
a Life/Health Division, Korea Institute of Science and Technology, PO Box 131, Cheongyang, Seoul 130-650, Republic of Korea
b Department of Chemistry, College of Natural Sciences, Sangmyung University, Seoul 110-743, Republic of Korea
c School of Science, University of Science and Technology (UST), Daejeon 305-333, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed a facile synthesis of 3,3-diphenylpropanamides using Meldrum’s acid derivatives as
amide coupling components. The in vitro biological evaluation of the title compounds led to the identi-
Received 8 September 2010
Revised 21 October 2010
Accepted 4 November 2010
Available online 12 November 2010
fication of compound 1h, which has good inhibitory activity against T-type calcium channel (IC50
0.83 M).
=
l
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
T-Type calcium channel blockers
Meldrum’s acid
3,3-Diphenylpropanoyl piperazine
HTS assays
Voltage gated calcium channels regulate the calcium influx into
cells in response to membrane depolarization.1 On the basis of
encoding of the pore-forming a-subunit, they are divided into three
main families: CaV 1.x (L-type), CaV 2.x (N-, P/Q-, R-type), and CaV
3.x (T-type). Among them, T-type calcium channels, also known
as low voltage activated calcium channels, play a crucial role in
the excitability of both central and peripheral neurons.1 In particu-
lar, they are mainly expressed in the CNS regions, such as the dorsal
root ganglion (DRG), the dorsal horn, and the thalamus, and take
responsibility for modulating neuronal excitability. Three different
pimozide, also showed T-type calcium channel blocking activity
with high binding affinity (Kd = ꢀ40 nM).5 While these compounds
are not selective calcium channel blockers, mibefradil (PosicorÒ,
Roche), approved by the FDA for the treatment of hypertension,
is described as the first selective T-type calcium channel blocker.6
Although it was withdrawn from the market due to unfavorable
drug–drug interaction,7 it is still used widely as a biological probe
to investigate the function of T-type calcium channels in neuronal
tissues. Recently, it was reported that the intraperitoneal adminis-
tration of mibefradil yields efficacy in animal models of neuro-
pathic pain.8
genes coding for T-type calcium channels, CaV 3.1 (
a1G), CaV 3.2
(a
1H) and CaV 3.3 ( 1I) have been identified and the physiological
a
and pathological role of each subtype in nervous system has not
been clearly understood due to lack of selective blocker for them.2
Recent genetic studies indicated that they involve the expression
of spontaneous absence epilepsy, the enhancement of sleep, and
the attenuation of neuropathic pain.3 Therefore, selective T-type
calcium channel blockers may have high potentials for treatment
of CNS related disorders such as epilepsy, insomnia, and pain.
Several T-type calcium channel blockers have been discovered
as described in Figure 1. Flunarizine, a neuroleptic, exhibited
potent inhibitory activities against a1G and a1I subtypes (Kd =
F
F
O
HN
N
N
N
N
F
F
Flunarizine
Pimozide
O
MeO
O
0.53 and 0.84 l
M, respectively).4 Another antipsychotic agent,
N
N
NH
F
⇑
Mibefradil
Figure 1. Structures of flunarizine, pimozide, and mibefradil.
Corresponding authors. Tel.: +82 2 958 5156 (Y.S.C.); tel.: +82 2 958 5173; fax:
+82 2 958 5189 (S.-J.M.).
E-mail addresses: ys4049@kist.re.kr (Y.S. Cho), sjmin@kist.re.kr (S.-J. Min).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.033

216
Y. Choi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 215–219
R2
R2
Inspired by such compounds, together with our compounds re-
O
O
ported in the recent literatures,9–11 we have designed new molec-
ular scaffolds 1 and 2 having a 3,3-diphenylpropanamide and an
oxazole or benzoxazole to search for new T-type calcium channel
blockers (Fig. 2). Based upon analysis of the common features of
those compounds, we hypothesized that the hydrophobic diphe-
nyl-propanoyl group and the piperazine ring probably contribute
to T-type calcium channel blocking activity. It is expected that
the incorporation of various substituents into the diphenyl moiety
of this target molecule might affect its affinity for T-type calcium
channel. In addition, we could examine the effect of the function-
alized heteroaromatic rings such as oxazole or benzoxazole on
the T-type calcium channel blockade.
N
N
N
N
N
N
R3
O
O
Me
R1
R1
Figure 2. Structures of our target molecules 1 and 2.
1
2
To achieve the synthesis of a library of target molecules, we
need more convenient methods for substituted benzhydryl forma-
tion and amide coupling reactions.12 To this end, we conceived a
synthetic plan using Meldrum’s acid as the key functional material
(Scheme 1).13 Indeed, alkylidene Meldrum’s acids 3 are strong elec-
trophiles, which can readily react with various phenyl cuprates to
afford substituted benzhydryl Meldrum’s acids 4. Nucleophilic
cleavage of this intermediate by piperazine would give the desired
3,3-diphenylpropanamide 5. It should be noted that there would
be no harmful by-products generated in this amide formation pro-
cess. Accordingly, the use of Meldrum’s acid would provide the fi-
nal amide compounds in a practical and expedient manner. In this
Letter, we report a facile synthesis of the 3,3-diphenylpropanoyl
piperazine derivatives 1 and 2 using Meldrum’s acid as the
coupling component and in vitro biological activities of these com-
pounds against a1G subtype of T-type calcium channels.
Scheme 1. Synthetic plan toward 3,3-diphenylpropanoylamides.
Table 1
Synthesis of the 3,3-diarylpropanoylpiperazine derivatives 1 and 2 from alkylidene Meldrum’s acid
R2
O
N
N
N
R2
MgBr
CO2Me
R2
R2
NBoc
O
R1
O
O
O
O
O
O
NBoc
HN
1) TFA
CuI, THF
O
N
O
1
R1
CH3CN, 90 °C
0 °C to rt, 2 h
2) iPr2NEt
O
O
R2
Cl
O
N
N
3
N
N
R1
R1
Me
or
CO2Me
O
O
4
5
Cl
N
Me
R1
2
Entry
Code
R1
R2
H
4-F
3-F
H
4-F
3-F
H
4-F
3-F
H
4-F
3-F
H
4-F
3-F
Yielda (%)
4
5
1
2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
F
F
F
Cl
Cl
Cl
Br
Br
77
64
59
42
91
51
68
59
49
50
43
27
39
71
75
35
63
69
54
64
54
97
76
77
64
54
34
47
53
54
74
74
64
70
89
74
84
71
62
69
86
78
81
65
85
91
71
73
84
87
68
90
61
65
72
77
65
91
55
32
Br
CF3
CF3
CF3
MeO
MeO
MeO
a
Isolated yields.

Y. Choi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 215–219
217
Table 2
The synthesis of substituted diphenylmethyl Meldrum’s acid is
described in Table 1. Following the procedure reported by Fillion,14
we carried out a Knoevenagel condensation of Meldrum’s acid with
benzaldehydes under pyrrolidinium acetate catalysis to obtain a
series of alkylidene Meldrum’s acids 3. The conjugate addition of
substituted phenyl Grignard reagents to 3 in the presence of cop-
per(I) iodide successfully afforded substituted benzhydryl Mel-
drum’s acids 4 in good to excellent yield.15 In this case, a
stoichiometric amount of the arylcuprate reagents is not necessary
because the alkylidene Meldrum’s acids are electrophilic enough to
accept the phenylmagnesium bromide with a catalytic amount of a
copper species.
Next, we explored the coupling reactions of compounds 4 with
N-Boc-piperazine (Table 1). Several examples describe the forma-
tion of amides from the reaction between Meldrum’s acids and
nitrogen-containing nucleophiles.16,17 Indeed, it is crucial to con-
trol the amount of amine to avoid formation of the piperazine salt
due to the high acidity of the Meldrum’s acid derivatives. There-
fore, we attempted several reaction conditions, including thermal
aminolysis using excess piperazine (either Boc-protected or unpro-
tected), TFA promoted reaction, and microwave irradiation. Fortu-
nately, we found that treatment of the Meldrum’s acid derivatives
4 with one equivalent of N-Boc-piperazine in acetonitrile at 90 °C
afforded the corresponding amides 5 in 34–97% yields, generating
CO2 and acetone as by-products.18 It is noted that the reaction con-
ditions are so mild and practical that we could easily isolate the
products by simple purification procedures such as solvent evapo-
ration and column chromatography. Having established the opti-
mized conditions for amide formation, we then removed the Boc
protecting group. Subsequent alkylation of the corresponding sec-
ondary amine with chloromethyl-oxazole19a or chloromethyl
benzoxazole19b produced the final target molecules 1 and 2 in good
yield.20
In vitro T-type calcium channel blocking activity of 1, 2, and 6–8
Entry
Compd
% Inhibitiona
Entry
Compd
% Inhibitiona
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1a
1b
1c
1d
1e
1f
1g
1h
1i
27.69
33.53
44.54
35.01
45.06
24.53
42.83
42.12
36.60
60.40
51.77
57.65
22.82
24.54
25.47
42.67
30.95
44.93
25.94
15.50
32.24
13.61
11.15
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
2i
2j
18.02
22.39
9.84
2k
2l
2m
2n
2o
6c
6f
6i
6l
6o
7b
7e
7h
7k
7n
8c
8f
8i
8l
26.42
63.53
23.26
52.02
13.66
19.20
21.32
19.24
4.90
3.84
3.62
5.42
3.62
1j
1k
1l
1m
1n
1o
2a
2b
2c
2d
2e
2f
7.68
36.87
48.63
44.64
53.41
19.69
78.92
2g
2h
8o
Mibefradil
a
% Inhibition value was obtained at 10 lM.
of the synthesized compounds are summarized in Table 2. In gen-
eral, the % inhibition values of oxazole esters 1 are relatively higher
than those of benzoxazole 2. However, it seems that the electronic
character and position of the R1 substituents in the diphenyl units
have a crucial effect on structure–activity relationship. When
R1 = F, the potencies of compound 1a–c are almost similar to those
of 2a–c (entries 1–3 and 16–18). The oxazole esters 1 are more po-
tent than the corresponding benzoxazoles 2 when the R1 substitu-
ents are other electron-withdrawing groups such as Cl, Br, and CF3.
In the case of electron-donating methoxy group (R1 = OMe), how-
ever, the benzoxazoles 2m–o exhibit significantly higher %-inhibi-
tion compared with the oxazole esters 1m–o (entries 13–15 and
28–30).
On the other hand, the functional group of oxazole component
also plays an important role for the inhibition. In fact, when the
substituent on the oxazole moiety was changed into the alcohol
or the acid (compounds 6 or 7), lower inhibitory activity in FDSS
assay was observed. This result indicated that the binding site of
oxazole substituents in 6 and 7 may occupy a hydrophobic region
of T-type calcium channel pores. In addition, morpholine amides 8
exhibited better channel blocking activities in comparison to 6 and
7 even if they were similar to those of the esters 1.
With a series of the compounds 1 in hand, we also prepared
additional analogues replacing the ester functional group of 1 with
alcohol, acid, and amide groups (Scheme 2). Reduction of 1 with
LiBH4 generated the corresponding alcohols 6 in low to moderate
yields. Saponification of 1 under basic condition gave high yields
of the acids 7, which was subsequently coupled with morpholine
to afford the amide derivatives 8. In the case of R1 = CF3, R2 = 3-F,
the amide 8l was alternatively obtained from the direct coupling
reaction of the ester 1l with morpholine in the presence of TBD
(1,5,7-triazabicyclo[4.4.0]dec-5-ene) at 75 °C.
Using the FDSS6000 HTS (high-throughput screening) system,21
we evaluated the calcium channel inhibitory activity of the 3,3-
diphenylpropamide derivatives 1, 2, 6, 7, and 8 against the T-type
calcium channel a1G subtype in HEK293 cells. The in vitro efficacies
Scheme 2. Transformation of 1 to alcohols 6, acids 7, and amides 8.

218
Y. Choi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 215–219
10. Oh, Y.; Kim, Y.; Seo, S. H.; Lee, J. K.; Rhim, H.; Pae, A. N.; Jeong, K. S.; Choo, H.;
Table 3
Cho, Y. S. Bull. Korean Chem. Soc. 2008, 29, 1881.
11. Gu, S. J.; Lee, J. K.; Pae, A. N.; Chung, H. J.; Rhim, H.; Han, S. Y.; Min, S. J.; Cho, Y.
S. Bioorg. Med. Chem. Lett. 2010, 20, 2705.
12. Montalbetti, C. A. G. N.; Falque, V. Tetrahedron 2005, 61, 10827.
13. For recent reviews of the use of Meldum’s acids, see: (a) Dumas, A. M.; Fillion,
E. Acc. Chem. Res. 2010, 43, 440; (b) Lipson, V. V.; Gorobets, N. Y. Mol. Divers.
2009, 13, 399; (c) Ivanov, A. S. Chem. Soc. Rev. 2008, 37, 789; Chem, B.-C.
Heterocycles 1991, 32, 529.
Inhibitory activities of selected compounds against T-type calcium and hERG
channels
a
Entry
Compd
IC50
(l
M)
hERGa
(lM)
b
1
2
3
4
5
6
7
8
1e
1g
1h
1j
1k
2a
2m
Mibefradil
8.80 2.20
21.15 2.31
0.86 0.23
17.05 0.93
1.73 0.24
1.29 0.12
2.48 0.13
1.34 0.49
—
b
—
2.23 0.90
b
—
14. Dumas, A. M.; Seed, A.; Zorzitto, A. K.; Fillion, E. Tetrahedron Lett. 2007, 48,
7072.
3.83 1.13
2.27 0.63
1.16 0.21
1.40 0.29
15. Huang, X.; Chan, C.-C.; Wu, Q.-L. Tetrahedron Lett. 1982, 23, 75.
16. Sørensen, U. S.; Falch, E.; Krogsgaard-Larsen, P. J. Org. Chem. 2000, 65, 1003.
17. (a) Hansen, K. B.; Hsiao, Y.; Xu, F.; Rivera, N.; Clausen, A.; Kubryk, M.; Krska, S.;
Rosner, T.; Simmons, B.; Balsells, J.; Ikemoto, N.; Sun, Y.; Spindler, F.; Malan, C.;
Grabowski, E. J. J.; Armstrong, J. D., III J. Am. Chem. Soc. 2009, 131, 8798; (b)
Knöpfel, T. F.; Zarotti, P.; Ichikawa, T.; Carreira, E. M. J. Am. Chem. Soc. 2005, 127,
9682.
a
IC50 value ( SD) was obtained from a dose–response curve.
Not available.
b
18. Boisbrun, M.; Vassileva, E.; Raoul, M.; Laronze, J.-Y.; Sapi, J. Monatsh. Chem.
2003, 134, 1641.
19. (a) Hermitage, S. A.; Cardwell, K. S.; Chapman, T.; Cooke, J. W. B.; Newton, R.
Org. Process Res. Dev. 2001, 5, 37; (b) Saari, W. S.; Wai, J. S.; Fisher, T. E.; Thomas,
C. M.; Hoffman, J. M.; Rooney, C. S.; Smith, A. M.; Jones, J. H.; Bamberger, D. L.;
Goldman, M. E.; O’Brien, J. A.; Nunberg, J. H.; Quintero, J. C.; Schleif, W. A.;
Emini, E. A.; Anderson, P. S. J. Med. Chem. 1992, 35, 3792.
Next, we selected several compounds with over 40% inhibition
of Ca2+ current in FDSS assay and examined the IC50 values of them
using the whole-cell patch-clamp method.22 While 2m exhibited
the highest % of inhibition values in the preliminary FDSS assay,
20. Experimental procedure for compound 4h, 5h, and 1h. Compound 4h: To a
solution of the alkylidene Meldrum’s acid 3h (600 mg, 1.93 mmol) and copper
iodide (37 mg, 0.193 mmol) in THF (19 ml) cooled to 0 °C was added 4-
fluorophenylmagnesium bromide (2.0 M, 1.93 ml, 3.86 mmol). The reaction
mixture was stirred for 2 h and then quenched with aq NH4Cl (20 mL). The
solution was neutralized with 1 N HCl (up to pH 7), extracted with CH2Cl2
(3 Â 25 mL), washed with brine (30 mL) and dried over MgSO4. The solvent
was removed under reduced pressure to give a crude oil, which was purified by
column chromatography on silica gel (hexane/EtOAc = 4:1) to give
diphenylmethyl Meldrum’s acid 4h (462 mg, 59%) as a yellowish oil. 1H NMR
(CDCl3, 300 MHz) d 7.46–7.41 (m, 2H), 7.27 (m, 2H), 7.17 (d, J = 8.5 Hz, 2H),
7.04–6.97 (m, 2H), 5.34 (d, J = 2.2 Hz, 1H), 4.24 (d, J = 2.6 Hz, 1H), 1.78 (s, 3H),
1.57 (s, 3H). 13C NMR (CDCl3, 75 MHz) d 164.4, 164.3, 162.0 (1J = 245.3 Hz),
139.0, 135.2 (4J = 3.3 Hz), 131.6, 131.0 (3J = 8.1 Hz), 130.8, 121.3, 115.5
(2J = 21.2 Hz), 105.3, 51.1, 47.6, 28.3, 27.5. Compound 5h: A solution of 4h
(438 mg, 1.08 mmol) and tert-butyl piperazine-1-carboxylate (200 mg,
1.08 mmol) in CH3CN (11 mL) was stirred at 75 °C for 15 h. The solvent was
removed under reduced pressure. The remaining crude oil was purified by
column chromatography (hexane/EtOAc = 1:1) on silica gel to afford the 3,3-
diphenylpropanoylamide 5h (404 mg, 76%) as a clear pinkish oil. 1H NMR
(CDCl3, 300 MHz) d 7.42–7.35 (m, 2H), 7.21–6.91 (m, 6H), 4.63 (t, J = 7.4 Hz,
1H), 3.51–3.25 (m, 8H), 2.99 (d, J = 7.4 Hz, 2H), 1.45 (s, 9H). 13C NMR (CDCl3,
75 MHz) d 169.3, 161.5 (1J = 244.0 Hz), 154.5, 142.7, 139.2 (4J = 3.2 Hz), 131.7,
129.5, 129.2 (3J = 8.0 Hz), 120.5, 115.5 (2J = 21.1 Hz), 80.4, 45.8, 45.5, 41.6, 38.8,
28.4. HRMS (m/z): [M+H+] calcd for C24H29BrFN2O3 491.1346, Found 491.1349.
Compound 1h: To a solution of 5h (306 mg, 0.623 mmol) in CH2Cl2 (6 mL) at
23 °C was added TFA (1.53 mL, 20.6 mmol). The reaction mixture was stirred
for 2 h, then the solvent and the remaining TFA were removed under reduced
pressure. After being completely dried in a high vacuum, the crude material
was dissolved in DMF (3 mL) and treated with diisopropylethylamine (0.54 mL,
3.11 mmol) and methyl 2-(chloromethyl)oxazole-4-carboxylate (109 mg,
0.623 mmol). The mixture was stirred at 75 °C for 15 h. On completion of the
reaction (monitored by TLC), the solution was cooled to 75 °C and CH2Cl2/
water was added into the solution. The organic layer was separated and
washed with water and brine, dried over MgSO4, and concentrated. The crude
oil was purified by column chromatography (hexane/EtOAc = 1:4) to afford the
oxazole derivative 1h (215 mg, 71%) as a oil. 1H NMR (CDCl3, 300 MHz) d 8.21
(s, 1H), 7.37–7.34 (m, 2H), 7.14–7.03 (m, 4H), 6.95–6.89 (m, 2H), 4.57 (t,
J = 7.4 Hz, 1H), 3.89 (s, 3H), 3.70 (s, 2H), 3.56–3.53 (m, 2H), 3.38–3.35 (m, 2H),
2.94 (d, J = 7.5 Hz, 2H), 2.42–2.31 (m, 4H). 13C NMR (CDCl3, 75 MHz) d 169.1,
161.55 (1J = 243.4 Hz), 161.51, 161.46, 144.6, 143.0, 139.3 (4J = 3.2 Hz), 133.3,
131.7, 129.5, 129.3 (3J = 7.9 Hz), 120.4, 115.5 (2J = 21.2 Hz), 54.1, 52.7, 52.4,
52.3, 45.9, 45.5, 41.6, 38.7. HRMS (m/z): [M+H+] Calcd for C25H26BrFN3O4
530.1091, Found 530.1095.
1h proved to be the best analogue with an IC50 value of 0.86 lM.
In spite of difference between fluorescence-based assay and elec-
trophysiological assay, our filtering process using FDSS system is
necessary because compounds active in both methods could pro-
vide more reliable in vitro data to be eventually applied to
in vivo experiment. Besides, the preliminary evaluation procedure
to filter compounds allowed us to reduce excessive workload for
laborious patch-clamp assays.
Additionally, we investigated the IC50 values of the selected
compounds against hERG channel, which plays an important role
as a potential target for cardiac side effects (Table 3). In fact, we
found that compound 1h displayed twofold higher activity for T-
type over hERG channel (IC50 = 2.23 lM). Compared with IC50 val-
ues of mibefradil against T-type and hERG channels, 1h is not only
more potent than mibefradil, but also less effective to hERG inhibi-
tion. Therefore, compound 1h will be further investigated as a via-
ble T-type calcium channel blocker.
In summary, we have developed an efficient synthesis of the
3,3-diphenylpropanamides 1 and 2 which exhibit potential T-type
calcium channel blocking activity. In particular, the use of Mel-
drum’s acid as a coupling agent provided a practical method that
allows the preparation of propanoyl piperazines in good to excel-
lent yield. The in vitro biological evaluation of the title compounds
has led to the discovery of compound 1h, which is comparable to
mibefradil in terms of both potency and hERG channel inhibition.
These results suggest that the 3,3-diphenylpropanoyl piperazine
analogue 1h will be a potential lead compound to discover an
effective T-type calcium channel blocker.
Acknowledgment
Financial support for this research was provided by the Korea
Institute of Science and Technology (KIST).
21. Experimental procedure for FDSS6000 assay. HEK293 cells which stably express
both a1G and Kir2.1 subunits were grown in Dulbecco’s modified Eagle’s
medium supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U/
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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 4 Â 104 cells/well
and were used the next day for high-throughput screening (HTS) FDSS6000
assay. 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 the fluorescence-based FDSS6000
assay, a1G T-type Ca2+ channels were activated using high concentration of KCl
(70 mM) in 10 mM CaCl2 contained Hepes-buffered solution and the increase
in [Ca2+
] by KCl-induced depolarization was detected. During the whole
i
procedure, cells were washed using the BIO-TEK 96-well washer. All data were
collected and analyzed using FDSS6000 and related software (Hamamatsu,
Japan).
8. McGivern, J. G. Drug Discovery Today 2006, 11, 245.
9. Kim, H. S.; Kim, Y.; Doddareddy, M. R.; Seo, S. H.; Rhim, H.; Tae, J.; Pae, A. N.;
Choo, H.; Cho, Y. S. Bioorg. Med. Chem. Lett. 2007, 17, 476.

Y. Choi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 215–219
219
22. Experimental procedure for patch-clamp (electro-physiological recording). For the
recordings 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 contained (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
Germany).
/PULSEFIT software program (HEKA,