Synthesis and biological evaluation of 4-piperidinecarboxylate and 4-piperidinecyanide derivatives for T-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmcl.2011.07.087

To obtain selective and potent inhibitor for T-type calcium channel by ligand based drug design, 4-piperidinecarboxylate and 4-piperidinecyanide derivatives were prepared and evaluated for in vitro and in vivo activity against α_1G calcium channel. Among them, several compounds showed good T-type calcium channel inhibitory activity and minimal off-target activity over hERG channel (% inhibition at 10 μM = 61.85-71.99, hERG channel IC₅₀ = 1.57 ± 0.14-4.98 ± 0.36 μM). Selected compound 31a was evaluated on SNL model of neuropathic pain and showed inhibitory effect on mechanical allodynia.

Full text of DOI:10.1016/j.bmcl.2011.07.087

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5910–5915
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 4-piperidinecarboxylate
and 4-piperidinecyanide derivatives for T-type calcium channel blockers
Hyun Min Woo b, Yun Suk Lee b, Eun Joo Roh b, Seon Hee Seo b, Chi Man Song b, Hye Jin Chung b,
Ae Nim Pae b, Kye Jung Shin a,
⇑
a College of Pharmacy, The Catholic University of Korea, 43-1 Yeokgog-2-dong, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea
b Life/Health Division, Korea Institute of Science and Technology, 5 Wolsong-gil, Seongbuk-gu, Seoul 136-791, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 March 2011
Revised 7 July 2011
Accepted 23 July 2011
Available online 29 July 2011
To obtain selective and potent inhibitor for T-type calcium channel by ligand based drug design, 4-pip-
eridinecarboxylate and 4-piperidinecyanide derivatives were prepared and evaluated for in vitro and
in vivo activity against a1G calcium channel. Among them, several compounds showed good T-type cal-
cium channel inhibitory activity and minimal off-target activity over hERG channel (% inhibition at
10 lM = 61.85–71.99, hERG channel IC50 = 1.57 0.14–4.98 0.36 lM). Selected compound 31a was
evaluated on SNL model of neuropathic pain and showed inhibitory effect on mechanical allodynia.
Keywords:
T-type calcium channel
Neuropathic pain
Ó 2011 Elsevier Ltd. All rights reserved.
Calcium ion performs a significant role as an intracellular sec-
ond messenger and many physiological functions such as cell con-
traction, hormone secretion, neurotransmission, gene expression,
cell growth and division.1 Voltage-dependent calcium channels
(VDCC) are main calcium entry pathway in the cellular membrane
by change of membrane potential and play a significant physiolog-
ical role in excitability cells. According to electrophysiological and
pharmacological criteria, voltage-dependent calcium channels can
be divided into high voltage-activated (L, N, P, Q, R-type) and low
voltage-activated (T-type).2 But, on the basis of molecular cloning
has known as novel therapeutic targets, no potent and selective
T-type calcium channel blocker is available for clinical use until
now. Although dihydropyridine (DHP) compound, penfluridol (2),
was reported to have blocking activity, their clinical usage is lim-
ited because of low efficacy and selectivity.11,12 Recently, T-type
channel blockers with a piperidine scaffold were reported to have
good potency with minimal effects on L-type and hERG channels
and showed in vivo efficacy in epilepsy.14a,15 Based on this results,
1,4-substituted piperidine derivatives (3, Fig. 1) were designed and
prepared for T-type calcium channel blockers using 3D ligand
based pharmacophore model by common feature generation
approach (HipHop) implemented in CATALYST program.13
From the pharmacophore mapping data and our previous study,
3,5-disubstituted phenyl and pyrazole moiety (R3 groups) are ben-
eficial for potency, and N-alkyl group (R1) of the piperidine ring
were favorable for activity. Also in the point of R2 position, electron
withdrawing group (EWG), such as cayno and methyl ester group,
plays a crucial role on reducing the undesirable side effect of hERG
channel by lowering of the basicity of piperidine ring.14a,b (3, Fig. 1)
To obtain focused library with a suggested scaffold, we have
carried out the next reactions.
N-Boc-4-cyanopiperidine was prepared from commercially
available 4-cyanopiperidine (4), which was converted to the ethyl
ester (5) by treatment with LDA and ethyl chloroformate in THF at
ꢀ78 °C. The cyano group of 5 underwent reduction with PtO2 in
AcOH to provide 6. The resulting amino group was protected with
benzyloxycarbonyl (Cbz) group to give 7. The ethyl ester group of 7
was hydrolyzed with NaOH in THF–H2O under reflux condition to
provide acid compound, which was coupled with ammonia by
using EDCI and HOBt to give amide compound 8. Dehydration of
of the main pore-forming 10 different a1 subunits, they have been
classified in three families: CaV1.1–1.4 (L-type), CaV2.1–2.3 (N, P,
Q/R-type), CaV3.1–3.3 (T-type). T-type calcium channels are found
in CNS, peripheral tissues, heart, smooth muscle, kidney and endo-
crine tissue, and they have been proposed to be involved in cardiac
pace-making, blood pressure regulation,3 and the secretion of hor-
mones including aldosterone and insulin.4 Furthermore, T-type
calcium channels are identified in the thalamus and cortex where
they have a crucial role in the integration of neuronal firing for
thalamocortical signaling.5 Therefore, T-type calcium channels
are considered to participate in a variety of human diseases includ-
ing insomnia,6 neuropathic pain, epilepsy, certain types of cancer,
cardiac arrhythmia and hypertension.7,8
Mibefradil (1), which was developed for the treatment of hyper-
tension and angina pectoris, has a 10- to 30-fold selectivity for
T-type over L-type channel, but was withdrawn from the market
due to drug–drug interaction.9,10 Despite T-type calcium channels
⇑
Corresponding author. Tel.: +82 2 2164 4060; fax: +82 2 2164 4059.
E-mail address: kyejung@catholic.ac.kr (K.J. Shin).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.087

H. M. Woo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5910–5915
5911
O
F
R2
N
H
R3
O
O
OH
O
N
H
N
CF3
Cl
N
F
R1
N
N
R1=substituted benzyl, phenethyl,
F
2H-chromene-3-methyl
R2=cyano, methoxycarbonyl
R3=phenyl, pyrazolyl
3
mibefradil (1)
penfluridol (2)
Figure 1. T-type calcium channel blockers and 5-feature pharmacophore mapping of 28f (cyan: hydrophobic, green: hydrogen bond acceptor, red: positive ionizable).
the amide group of 8 with TFAA in the presence pyridine at 0 °C
afforded 9. Cbz protecting group of 9 was removed to the free
amine (10) with 10% Pd/C in MeOH. The ethyl ester (5) was con-
verted to methyl ester (11) with NaOMe in methanol. The cyano
group of 11 underwent reduction with PtO2 in AcOH to provide
12 (Scheme 1).
The aminomethyl group of compounds10, 12 were directly cou-
pled with 3,5-dimethoxybenzoyl chloride and 3,5-dichlorobenzoyl
chloride in CH2Cl2 to provide the amide compounds and then the
Boc group was removed to the free amine with 2 N HCl in EtOAc
to provide 21–24. Then, alkylation of secondary amine of piperi-
dine compounds 21–24 with benzyl halides a–g with NaI, TEA in
CH3CN under reflux condition afforded 26–29a–g. Also, com-
pounds 21–24 were coupled with aldehyde through reductive ami-
nation using NaBH(OAc)3 to give 26–29h (Scheme 4). Compounds
30a–h were synthesized by the same procedure as shown in
Scheme 4 (Scheme 5).
To obtain the R3 substituents, we purchased benzoic acid
derivatives, but pyrazole carboxylic acid was prepared as
follows. 2,4-Dioxoheptanoate (14), which was synthesized from
4-methyl-2-pentanone (13) and diethyl oxalate with NaOEt, was
converted into 1H-pyrazole 15 by the addition of phenyl hydrazine
in EtOH. And the ethyl ester group of 15 was hydrolyzed with NaOH
in THF–H2O under reflux condition to provide corresponding acid
compound 16 (Scheme 2).
In vitro screening of all the synthesized compounds was per-
formed with HEK293 cells expressed T-type calcium channels
(a1G) at 10 l
M concentration.16 From this preliminary data, the se-
For the synthesis of R2 substituent, we employed commercially
available benzyl halide and aldehyde except chromene moiety
which was easily synthesized by next reactions. Base-catalyzed
condensation reaction of salicylaldehyde (17) and 3-methyl-2-but-
enal with Na2CO3 in dioxane/H2O = 3:1 afforded compound 18,
which was treated with DIBAL-H to provide primary alcohol 19.
Then, chlorination of 19 with SOCl2 in CH2Cl2 gave compound 20
(Scheme 3).
lected compounds which showed above 50% blocking effect against
T-type calcium channel were evaluated through hERG channels by
whole-cell patch clamp method17 to get the selective lead com-
pound over T-type/hERG channel. Blockade of the hERG channel
can lead to a heart rhythm disorder known as long QT syndrome,
characterized by prolonged action potential of ventricular mus-
cle.18 In vitro biological data of the compounds were shown in
Tables 1–3 and mibefradil was used as reference compound.19
O
O
O
O
CN
Cbz
Cbz
NC
N
H
NH2
N
H
OEt
OEt
H2N
OEt
iv
v, vi
i , ii
iii
N
Boc
N
Boc
N
H
N
Boc
N
Boc
5
7
4
6
8
vii
ix
O
O
NC
Cbz
CN
CN
OMe
N
H
H2N
OMe
H2N
viii
x
N
N
N
N
Boc
Boc
Boc
Boc
12
10
9
11
Scheme 1. Reagents and conditions: (i) Boc2O, TEA, CH2Cl2, 0 °C to rt, 96%; (ii) ethyl chloroformate, LDA, THF, ꢀ78 °C to rt, 65%; (iii) H2, PtO2, AcOH, rt, 80%; (iv) benzyl
chloroformate, NaHCO3, CH2Cl2, 0 °C to rt, 92%; (v) NaOH, EtOH/H2O (1:1), reflux, 93%; (vi) EDCI, HOBt, DMF, rt, then 35% NH4OH, 94%; (vii) TFAA, pyridine, CH2Cl2, 0 °C to rt,
77%; (viii) H2, Pd/C, MeOH, rt, 83%; (ix) NaOMe, MeOH, rt, 95%; (x) H2, PtO2, AcOH, rt, 73%.

5912
H. M. Woo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5910–5915
O
O
O
O
O
N
N
(ii)
(i)
(iii)
O
HO
N
N
O
O
13
14
16
15
Scheme 2. Reagents and conditions: (i) diethyl oxalate, NaOEt, EtOH, 0 °C to rt, 81%; (ii) phenyl hydrazine, EtOH, 0 °C to rt, 70%; (iii) NaOH, EtOH/H2O (1:1), reflux, 87%.
O
O
(ii)
(i)
(iii)
Cl
OH
O
OH
O
O
17
20
18
19
Scheme 3. Reagents and conditions: (i) 3-methyl-2-butenal, Na2CO3, dioxane/H2O (3:1), 55 °C, sonication, 64%; (ii) DIBAL-H, CH2Cl2, ꢀ78 °C, 70%; (iii) SOCl2, CH2Cl2, rt, 87%.
O
R2
N
(ii)
R3
H
N
R1
O
R2
R2
26~29a-g
H2N
N
H
R3
(i)
N
Boc
N
O
H HCl
R2
N
H
(iii)
R3
10 , 12
21~24
N
26~29h
R1
=
OMe
MeO
CF3
OMe
b
a
c
d
F3C
O
CF3
e
f
g
Scheme 4. Reagents and conditions: (i) 3,5-disubstituted benzoyl chloride, TEA, CH2Cl2, 0 °C to rt, then 2 N HCl in EtOAc, 68–82%; (ii) R1 halides, TEA, NaI, CH3CN, 80 °C, 25–
89%; (iii) phenylacetaldehyde, NaBH(OAc)3 TEA, ClCH2CH2Cl, rt, 41–64%.
O
CN
N
(ii)
H
N
N
N
N
R1
O
CN
CN
H2N
N
H
(i)
30a-g
N
N
N
Boc
N
H HCl
O
CN
(iii)
N
H
10
25
N
N
30h
Scheme 5. Reagents and conditions: (i) compound 16, EDCI, HOBt, TEA, DMF, rt, then 2 N HCl in EtOAc; (ii) R1 halides, TEA, NaI, CH3CN, 80 °C, 41–69%; (iii)
phenylacetaldehyde, NaBH(OAc)3 TEA, ClCH2CH2Cl, rt, 65%.

H. M. Woo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5910–5915
5913
Table 1
In vitro activities of synthesized compound (1)
O
NC
N
R3
H
N
R1
Compound
R1
R3
% Inhibitiona (10
l
M)
hERGb IC50
(lM)
26a
26b
26c
26d
26e
26f
26g
26h
27a
27b
27c
27d
27e
27f
2-Methoxybenzyl
3-Methoxybenzyl
4-Methoxybenzyl
2-(Trifluoromethyl)benzyl
3-(Trifluoromethyl)benzyl
4-(Trifluoromethyl)benzyl
2,2-Dimethyl-2H-chromenyl
Phenethyl
2-Methoxybenzyl
3-Methoxybenzyl
4-Methoxybenzyl
2-(Trifluoromethyl)benzyl
3-(Trifluoromethyl)benzyl
4-(Trifluoromethyl)benzyl
2,2-Dimethyl-2H-chromenyl
Phenethyl
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dichloro
3,5-Dichloro
3,5-Dichloro
3,5-Dichloro
3,5-Dichloro
50.93
60.41
53.03
45.44
15.75
13.88
44.30
61.85
19.31
8.33
0.92 0.04
1.50 0.23
3.21 0.18
4.76 0.72
NT
NT
4.73 0.73
1.57 0.14
NT
NT
NT
NT
NT
NT
NT
16.11
9.99
8.76
3,5-Dichloro
3,5-Dichloro
3,5-Dichloro
8.70
27g
27h
Mibefradil
13.57
71.79
78.95
0.36 0.06
1.34 0.49
a
% Inhibition was obtained at 10
IC50 value ( SE) was determined from dose–response curve (n = 3).
l
M.
b
Table 2
In vitro activities of synthesized compound (2)
O
O
O
N
H
R3
N
R1
Compound
R1
R3
% Inhibitiona (10
l
M)
hERGb IC50
(l
M)
28a
28b
28c
28d
28e
28f
28g
28h
29a
29b
29c
29d
29e
29f
2-Methoxybenzyl
3-Methoxybenzyl
4-Methoxybenzyl
2-(Trifluoromethyl)benzyl
3-(Trifluoromethyl)benzyl
4-(Trifluoromethyl)benzyl
2,2-Dimethyl-2H-chromenyl
Phenethyl
2-Methoxybenzyl
3-Methoxybenzyl
4-Methoxybenzyl
2-(Trifluoromethyl)benzyl
3-(Trifluoromethyl)benzyl
4-(Trifluoromethyl)benzyl
2,2-Dimethyl-2H-chromenyl
Phenethyl
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dimethoxy
3,5-Dichloro
3,5-Dichloro
3,5-Dichloro
3,5-Dichloro
3,5-Dichloro
46.78
56.16
58.98
63.57
71.99
64.29
57.99
64.96
68.46
73.03
74.46
44.15
71.96
43.30
33.93
77.18
78.95
1.90 0.06
0.31 0.01
0.48 0.05
4.98 0.77
2.80 0.36
2.35 0.45
1.29 0.15
NT
0.16 0.03
0.36 0.07
0.13 0.01
0.46 0.01
0.89 0.11
0.29 0.05
3,5-Dichloro
3,5-Dichloro
3,5-Dichloro
29g
29h
Mibefradil
13.83 2.24
NT
1.34 0.49
a
% Inhibition was obtained at 10
IC50 value ( SE) was determined from dose–response curve (n = 3).
l
M.
b
As shown in Tables 1–3 on the N-1 position of piperidine (R1),
preliminary in vitro assay data, four compounds 26h and 28d–f
were chosen for the in vitro stability test in human hepatic micro-
some.20 According to the metabolic stability result of 26h and
28d–f, they were very unstable (Fig. 2a). The main reason was sup-
posed to be the hydrolysis of phenethyl and methyl ester group.
Thus, methyl ester group of 28f was converted to carboxylic acid
group (31a) with NaOH in THF–H2O under reflux condition, and
then the in vitro stability was re-evaluated. As expected, com-
pound 31a was more stable than parent compound 28f and mibe-
fradil (Fig. 2b).
the order of activity was generally phenethyl > benzyl > chrome-
nyl. The methyl ester derivatives on C-4 position of piperidine ring
showed better inhibitory activity than corresponding cyano deriv-
atives, especially in the case of the substituent of benzoyl amide
(R3) is di-chloro (Table 2). Contrary to our expectation based on
pharmacophore mapping study, there is less significant improve-
ment with inhibitory activity of pyrazole amide substituent
compared to benzoyl moiety. The inhibitory activity of pyrazole
amide is similar to that of benzoyl amide. Summing up every

5914
H. M. Woo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5910–5915
Table 3
In vitro activities of synthesized compound (3)
O
NC
N
H
N
N
N
R1
Compound
R1
% Inhibitiona (10
lM)
hERGb IC50
(l
M)
30a
30b
30c
30d
30e
30f
30g
30h
2-Methoxybenzyl
3-Methoxybenzyl
4-Methoxybenzyl
2-(Trifluoromethyl)benzyl
3-(Trifluoromethyl)benzyl
4-(Trifluoromethyl)benzyl
2,2-Dimethyl-2H-chromenyl
Phenethyl
50.93
60.41
53.03
45.44
15.75
13.88
44.30
61.85
78.95
0.92 0.04
1.50 0.23
3.21 0.18
4.76 0.72
NT
NT
4.73 0.73
1.57 0.14
1.34 0.49
Mibefradil
a
% Inhibition was obtained at 10 lM.
IC50 value ( SE) was determined from dose–response curve (n = 3).
b
100
100
80
60
40
20
0
Mibefradil
28f
Mibefradil
31a
26h
80
60
40
20
0
28e
28d
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Time (min)
Time (min)
(a)
(b)
O
O
OCH3
HO
N
H
N
HCl
OCH3
CF3
31a
Figure 2. In vitro metabolic stability of 26h, 28d–f, and 31a in human hepatic microsomes. Human liver microsomes (0.5 mg/ml) and 26h, 28d–f, and 31a (1
lM) in
potassium phosphate buffer (0.1 M, pH 7.4) were incubated at 37 °C. Reactions were initiated by the addition of b-NADPH (1.2 mM) and continued at 37 °C. After incubation
for 0, 5, 15, 30, and 60 min, the reaction was stopped by addition of acetonitrile with internal standard. The samples were analyzed using LC–MS/MS. Mibefradil was used as a
reference standard. The results are expressed as the percentage of drug remaining after incubation. Data represent the mean of duplicated experiments.
Compound 31a showed similar activity against T-type calcium
channel and better activity over hERG channel (IC50 of a1G
7.31 0.96, IC50 of hERG = 12.08 0.76) compared to 28f (IC50 of
1G = 4.18 0.51, IC50 of hERG = 2.35 0.45). T-type calcium
In summary, the piperidine derivatives designed and prepared
as T-type calcium channel blockers through previous SAR and
pharmacophore mapping study. Compound 31a was evaluated on
a rat model of neuropathic pain and it reduced mechanical allo-
dynia by oral administration. Further assay and optimization on
the piperidine derivatives may lead to development of potent
and selective T-type calcium channel blockers for the treatment
of disorders related to this channel.
=
a
channel blockers seem to be promising therapeutic agents for the
treatment of neuropathic pain, thus 31a was stepped into the
in vivo test for neuropathic pain model. Behavioral test for neuro-
pathic pain according to the spinal nerve ligation model21 (Chung’s
model) was done with 20 rats. After 14 days of surgical manipula-
tion, the experiment for effect on allodynia of 31a was performed.
Compound 31a showed suppression effect toward mechanical
allodynia and little inhibitory effect on cold allodynia ( Fig. 3),
however, 31a seems to have somewhat weak mechanical allodynia
effect in comparison with gabapentin.
Acknowledgments
This work was financially supported by Research Fund 2011 of
The Catholic University of Korea and Korea Institute of Science and
Technology (2E21610).

H. M. Woo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5910–5915
5915
A. Mechanical allodynia
B. Mechanical allodynia (MPE)
18
16
14
12
10
8
120
100
80
60
40
20
0
*
*
*
6
4
2
0
Pre
N14D
1h
3h
5h
Base
1h
3h
5h
Experimental time (day or hour)
Experimental time (hour)
C. Cold allodynia
120
D. Cold allodynia (MPE)
120
100
80
60
40
20
0
100
80
60
40
20
0
*
*
*
Pre
N14D
1h
3h
5h
Base
1h
3h
5h
Experimental time (day or hour)
Experimental time (hour)
Figure 3. Effect on mechanical allodynia (A and B) and cold allodynia (C and D) after oral administration of gabapentin (s, 100 mg/kg, n = 5) and 31a (d, 100 mg/kg, n = 5) to
neuropathic pain induced rats. Experimental time expressed as D for days after neuropathic injury (N) and h for hours after gabapentin or 31a administration, ⁄P <0.05
(gabapentin), |P <0.05 gabapentin versus 31a (unpaired t-test).
E.; Fox, S. V.; Kraus, R. L.; Doran, S. M.; Connolly, T. M.; Tang, C.; Ballard, J. E.;
Kuo, Y.; Adarayan, E. D.; Prueksaritanont, T.; Zrada, M. M.; Marino, M. J.;
References and notes
Graufelds, V. K.; DiLella, A. G.; Reynolds, I. J.; Vargas, H. M.; Bunting, P. B.;
1. Tsien, R. W.; Wheeler, D. B. Voltage-Gated Calcium Channels. In Calcium as a
Woltmann, R. F.; Magee, M. M.; Koblan, K. S.; Renger, J. J. J. Med. Chem. 2008, 51,
Cellular Regulator; Carafoli, E., Klee, C., Eds.; Oxford University Press, 1999; pp
6471.
171–199.
16. (a) Kim, T.; Choi, J.; Kim, S.; Kwon, O.; Nah, S. Y.; Han, Y. S.; Rhim, H. Biochem.
2. Perez-Reyes, E. Physiol. Rev. 2003, 83, 117.
Biophy. Res. Commun. 2004, 324, 401; (b) HEK293 cells which stably express
3. Ono, T.; Iijima, J. J. Pharmacol. Sci. 2005, 99, 197.
both a1G and Kir2.1 subunits were grown in Dulbecco’s modified Eagle’s
4. McGivern, L. G. CNS Neurol. Disord. Drug Targets. 2006, 5, 587.
medium supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U/
5. Cueni, L.; Canepari, M.; Adelman, J. P.; Luthi, A. Arch. Eur. J. Physiol. 2009, 457,
mL), streptomycin (100 lg/mL), geneticin (500 lg/mL), and puromycin (1 lg/
1161.
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 ꢁ 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
6. Crunelli, V.; Cope, D. W.; Hughes, S. W. Cell Calcium 2006, 40, 175.
7. Triggle, D. J. Biochem. Pharmacol. 2007, 74, 1.
8. Todorovic, S. M.; Jevtovic-Todorovic, V. C. N. S. Neurol. Disord.: Drug Targets.
2006, 5, 639.
9. Clozel, J. P.; Ertel, E. A.; Ertel, S. I. J. Hypertens. 1997, 15, S17.
10. Mullins, M. E.; Horowitz, B. Z.; Linden, D. H.; Smith, G. W.; Norton, R. L.; Stump,
J. J. Am. Med. Assoc. 1998, 280, 157.
11. Heady, T. N.; Gomora, J. C.; Macdonald, T. L.; Perez-Reyes, E. Jpn. J. Pharmacol.
2001, 85, 339.
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,
(70 mM) in 10 mM CaCl2 contained Hepes-buffered solution and the increase
in [Ca2+
by KCl-induced depolarization was detected. During the whole
a
1G T-type Ca2+ channels were activated using high concentration of KCl
]
i
12. Snutch, T. P.; David, L. S. Drug Dev. Res. 2006, 67, 404.
13. Doddareddy, M. R.; Choo, H.; Cho, Y. S.; Rhim, H.; Koh, H. Y.; Lee, J. H.; Jeong, S.
W.; Pae, A. N. Bioorg. Med. Chem. 2007, 15, 1091.
procedure, cells were washed using the BIO-TEK 96-well washer. All data were
collected and analyzed using FDSS6000 and related software (Hamamatsu,
Japan).
14. (a) Shipe, W. D.; Barrow, J. C.; Yang, Z. Q.; Lindsley, C. W.; Yang, F. V.; Schlegel,
K. A.; Shu, Y.; Rittle, K. E.; Bock, M. G.; Hartman, G. D.; Tang, C.; Ballard, J. E.;
Kuo, Y.; Adarayan, E. D.; Prueksaritanont, T.; Zrada, M. M.; Uebele, V. N.; Nuss,
C. E.; Connolly, T. M.; Doran, S. M.; Fox, S. V.; Kraus, R. L.; Marino, M. J.;
Graufelds, V. K.; Vargas, H. M.; Bunting, P. B.; Hasbun-Manning, M.; Evans, R.
M.; Koblan, K. S.; Renger, J. J. J. Med. Chem. 2008, 51, 3692; (b) Jamieson, C.;
Moir, E. M.; Rancovic, Z.; Wishart, G. J. Med. Chem. 2006, 49, 5029.
15. Yang, Z. Q.; Barrow, J. C.; Shipe, W. D.; Schlegel, K. A.; Shu, Y.; Yang, F. V.;
Lindsley, C. W.; Rittle, K. E.; Bock, M. G.; Hartman, G. D.; Uebele, V. N.; Nuss, C.
17. Choi, K. H.; Song, C.; Shin, D.; Park, S. Biochim. Biophys. Acta 2011, 1808, 1560.
18. De Bruin, M. L.; Pettersson, M.; Meyboom, R. H. B.; Hoes, A. W.; Leufkens, H. G.
M. Eur. Heart J. 2005, 26, 590.
19. Diaz, J. J.; Daniell, K.; Leitz, S. T.; Martin, R. L.; Su, Z.; McDermott, J. S. J.
Pharmacol. Toxicol. Methods 2004, 50, 187.
20. David, C. A.; Kevin, T. R.; Timothy, R. B. Optimization in Drug Disc. In vitro
Methods 2004, 151–162.
21. Kim, S. H.; Chung, J. M. Pain 1992, 50, 355.