Synthesis and T-type calcium channel-blocking effects of aryl(1,5-disubstituted-pyrazol-3-yl)methyl sulfonamides for neuropathic pain treatment

Article abstract of DOI:10.1016/j.ejmech.2016.07.032

A novel series of aryl(1,5-disubstituted pyrazol-3-yl)methyl sulfonamide derivatives was designed, synthesized, and evaluated for T-type calcium channel (α_1Gand α_1H) inhibitory activity. We identified several compounds, 9a, 9b, 9g, and 9h that displayed good T-type channel inhibitory potency with low hERG channel and CYP450 inhibition. Among them, 9a and 9b exhibited neuropathic pain alleviation effects in mechanical and cold allodynia induced in the SNL rat model. Compounds 9a and 9b displayed better efficacy than mibefradil and gabapentin against cold allodynia. In particular, compound 9a seemed to be valuable as shown fast acting performance in mechanical neuropathic pain model.

Full text of DOI:10.1016/j.ejmech.2016.07.032

European Journal of Medicinal Chemistry 123 (2016) 665e672
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and T-type calcium channel-blocking effects of
aryl(1,5-disubstituted-pyrazol-3-yl)methyl sulfonamides for
neuropathic pain treatment
,
, b, *
Jung Hyun Kim a, Gyochang Keum a b, Hesson Chung a, Ghilsoo Nam a
a Center for Neuro-Medicine, Brain Science Institute, Korea Institutes of Science and Technology (KIST), Seoul, 136-791, South Korea
b Department of Biological Chemistry, Korea University of Science and Technology (UST), Gajungro 217, Youseong-gu, Daejeon, 305-350, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of aryl(1,5-disubstituted pyrazol-3-yl)methyl sulfonamide derivatives was designed,
synthesized, and evaluated for T-type calcium channel (a1G and a1H) inhibitory activity. We identified
several compounds, 9a, 9b, 9g, and 9h that displayed good T-type channel inhibitory potency with low
hERG channel and CYP450 inhibition. Among them, 9a and 9b exhibited neuropathic pain alleviation
effects in mechanical and cold allodynia induced in the SNL rat model. Compounds 9a and 9b displayed
better efficacy than mibefradil and gabapentin against cold allodynia. In particular, compound 9a seemed
to be valuable as shown fast acting performance in mechanical neuropathic pain model.
© 2016 Elsevier Masson SAS. All rights reserved.
Received 31 March 2016
Received in revised form
14 July 2016
Accepted 18 July 2016
Available online 19 July 2016
Keywords:
T-type calcium channel inhibition
Aryl(1,5-disubstituted-pyrazol-3-yl)methyl
sulfonamide
Neuropathic pain
Allodynia
1. Introduction
neuropathies (DPN) [6]. T-type knock-out mice were shown to be
hyperalgesic to visceral pain, which is likely related to a T-type
Calcium homeostasis plays an important role in the pathogen-
esis of neurodegenerative disease, stroke, pain, and epilepsies [1].
Since calcium entry into cells via voltage-activated calcium chan-
nels (VACCs) was discovered through experiments on action po-
tential (AP) recordings in crayfish muscle fibers, biophysical and
pharmacological studies have identified five different subtypes of
high-voltage-activated (HVA) calcium channels e L, N, P, Q, and R e
and one low-voltage-activated channel (LVA), the T type [2e4]. The
discovery of specific blockers for different calcium channels types is
a focus in new drug development for neuronal calcium channel-
related diseases because each calcium channel subtypes contrib-
utes to the differing physiologies of neuronal cells.
T-type channels have been studied for pain treatment since
their discovery and characterization in dorsal root ganglion (DRG)
neurons [5], which implicates the T-type channel in sensitization to
pain responses (e.g., by enhancing the excitability of nociceptors, as
seen in animal models of type 1 and type 2 peripheral diabetic
calcium channel-dependent anti-nociceptive mechanism operating
in the thalamus [7]. T-type calcium channels have three different
subtypes, Cav3.1 (a1G), Cav3.2 (a1H), and Cav3.3 (a1I), which have
different biophysical functions; thus, inhibitors of T-type calcium
channels are an emerging therapeutic area for the treatment of
pain, especially neuropathic pain [8,9].
Over 26 million patients worldwide suffer from neuropathic
pain syndrome. Neuropathic pain is characterized by spontaneous
hypersensitive pain responses that can persist long after the orig-
inal nerve injury has healed. Although current pharmacological
treatments for neuropathic pain include combinations of agents
such as opioids, tricyclic antidepressants, anti-convulsant agents,
and non-steroidal anti-inflammatory (NSAID) analgesics, treatment
failure is high with these approaches, only providing ~30e50%
alleviation of pain in ~50% of patients [10]. Thus, the effective
treatment of neuropathic pain remains a great medical challenge.
Gabapentin (1-(aminomethyl)cyclohexene acetic acid) is
considered by physicians to be the gold standard treatment for
neuropathic pain, although its effectiveness may be limited in
some cases and varies among individuals [11]. Mibefradil, the first
selective T-type calcium channel blocker, was developed by
* Corresponding author. Center for Neuro-Medicine, Brain Science Institute,
Korea Institutes of Science and Technology (KIST), Seoul, 136-791, South Korea.
E-mail address: gsnam@kist.re.kr (G. Nam).
http://dx.doi.org/10.1016/j.ejmech.2016.07.032
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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J.H. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 665e672
researchers at Roche and exhibited antihyperalgesic effects in a
neuropathic pain spinal-nerve-ligation (SNL) rat model, although
it was marketed briefly as an antihypertensive agent, it was
withdrawn from market due to a drug-drug interaction concerns
[12,13]. Targeting the T-type calcium channel for the treatment of
neuropathic pain is attractive pathophysiologically due to the
strong connection, both mechanically and pharmacologically, be-
tween epilepsy and neuropathic pain, but no selective T-type
calcium channel blocker has been developed to date. Clinical
agents with T-type channel-blocking activity, such as the antiep-
ileptic drugs zonisamide, ethosuximide, and phenytoin, may pro-
vide important clues for the development of potent neuropathic
pain drugs. The synthetic peptide ziconotide, an N-type calcium
channel blocker, has been used clinically in chronic and severe
pain; however, its use is limited due to inconvenient treatment
options e such as intrathecal administration e and serious side
effects [14]. Thus, the development of orally active T-type calcium
channel inhibitors would have advantages for overcoming the
difficulties associated with current chronic and neuropathic pain
treatments. Zonisamide is a widely used broad-spectrum antiep-
ileptic, which is known to inhibit both Na channels and T-type
calcium channels [15]. The percentage of inhibition of the T-type
channel current in cultured neurons from rat cerebral cortex is
available diethyloxalate 1 with 4-methyl-2-pentanone or aceto-
phenone yielded a 1,3-diketoester compound bearing isopropyl 2
or phenyl 3, and ring cyclization with the corresponding hydrazine
to give 5-isopropyl-1-phenyl-pyrazolyl-3-ethyl ester 4 or 1-t-butyl-
5-phenyl-pyrazoyl-3-ethyl ester 5. The ester functionality of 4 was
transformed to aldehyde 6 by reduction with DIBAL-H, followed by
reaction with hydroxyl amine to give oxime derivative 7. The key
intermediate 5-isopropyl-1-phenyl-pyrazolyl-3-methylamine
8
was prepared by reduction of oxime derivative 7 with LAH. Sulfo-
nylation of the amine with arylsulfonyl chlorides having various
aromatic rings, such as phenyl, mono-substituted phenyl (3-fluoro,
3-chloro, 4-iodo, 4-methyl, 4-t-butyl), di-substituted phenyl (2-
fluoro-5-methyl, 2-methyl-3-chloro, 2,6-dichloro, 2,6-difluoro),
benzyl, phenethyl, naphthyl, and quinoyl rings, gave aryl
substituted 3-isopropyl-2-phenyl-pyrazol-5-yl)methyl sulfon-
amide derivatives 9ae9p.
In an attempt to understand the biological effects of the hy-
drophobic group according to the substituted position, we syn-
thesized pyrazolyl sulfonamides having a t-butyl group instead of a
phenyl group on the 2-N position, and phenyl or 4-fluoro phenyl
moieties on the 3-position, which were converted to the position of
the hydrophobic phenyl group. The (1-t-butyl-5-phenyl-pyrazol-3-
yl)methylamine 10 was prepared from the 1-t-butyl-5-phenyl-
pyrazoyl-3-ethyl ester 5 through a functional group transformation
with similar methods to compound 4. Next, sulfonylation of amines
derivative 10 with phenylsulfonyl chloride yielded 2N-t-butyl-3-
phenylsubatituted pyrazolyl methylsulfonamides 11.
~60% at 500
currents. Zonisamide also inhibits T-type currents in cultured
neuroblastoma cells (~40%, 50 M) [16]. Open-label case studies in
mM zonisamide, without inhibition of L-type calcium
m
the clinic have reported that zonisamide can be an effective
treatment for neuropathic pain [17].
Here, we describe the synthesis of a new series of pyrazolyl-
connected bezenesulfonamides (1) based on structures relevant
to known antiepileptics, such as zonisamide, ethosuximide, and
phenytoin, and the results of a biological evaluation of T-type cal-
cium channel-blocking activities in vitro and neuropathic pain
alleviating efficacy in vivo.
Zonisamide is composed of a benzoxazolidine core connected to
methyl sulfonamide, ethoximide consists of a cyclic diketoamide
with methyl and ethyl groups, and phenytoin was constructed with
a cyclic urea unit having two phenyl groups. Inspired in these
known chemical structures, we designed aryl sulfonamides with a
pyrazole ring bearing aromatic phenyl and aliphatic alkyl sub-
stituents by combining fragment of these antiepileptics (Fig. 1).
3. Results and discussion
In vitro inhibitory activities of the compounds synthesized were
evaluated against T-type calcium channels including a1G or a1H
using a fluorescence-based high-throughput screening (HTS) sys-
tem FDSS6000 assay and whole-cell patch-clamp methods. The
results for the (2-N-phenyl-3-isopropyl)pyrazolyl-5-methyl
substituted arylsulfonamide derivatives 9ae9p and 11 are sum-
marized in Table 1. Most of tested compounds showed similar
inhibitory potency against both T-calcium channel subtypes, a1G
and a1H. The results indicated that compounds 9g (R1 ¼ Bn) and 9h
(R1 ¼ (CH2)2Ph) showed the most activity against a1G (70.53%) and
a1H (70.53%), respectively.
Variation of the aryl substituent strongly affected the inhibitory
activity. The activity increased with methylene unit addition to the
phenyl group in R1. The inhibitory activity order was 9a (R1 ¼ Ph,
61.42%) < 9g (R1 ¼ CH2Ph, 65.69%) < 9h (R1 ¼ CH2CH2Ph, 70.53%).
Except for 9b (R1 ¼ 3-FPh, 64.10%), introducing any substituent on
the phenyl (9c, 9d, 9e, 9f, 9i, 9j, 9k, and 9l) showed lower activity
than the unsubstituted phenyl group, with the aryl groups showing
36e57% inhibition. Moreover, larger aromatic substituents, such as
naphthalene (9m, 9n) and quinoline (9o), showed lower potency in
the range of 19e29%, while the 1-methylnaphthyl-substituted 9p
almost lost activity (7%). Inhibitory activities of compounds 9i, 9k,
and 9o were higher against a1H than a1G. Compounds 9ae9p,
composed of aromatic R1 (Ph) and aliphatic R2 (iso-propyl)-bearing
pyrazole groups, and compound 11a e with a dispositioned
aliphatic R1 (t-butyl) and aromatic R2 (Ph) group e showed lower
inhibitory activity than 9a.
2. Chemistry
Synthesis of the final compounds was carried out according to
Scheme 1. A Claisen condensation reaction of commercially
In an attempt to determine the selectivity of other calcium
channels and the hERG channel, selected compounds showing
>60% in FDSS were evaluated for IC50 against two T-type channel
subtypes, the N-type channel and the hERG channel (Table 2).
The results indicated that most of the compounds tested
showing some inhibitory activity against the N-type calcium and
hERG channels. The inhibition of later is known to induce cyto-
toxicity. Among them, compound 9b was most active compound for
Fig. 1. Structure of antiepileptics with T-type channel blocking activity, and designed
molecules.

J.H. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 665e672
667
Scheme 1. Reagents and conditions: (a) R1COCH2Cl, NaOEt, EtOH, rt, yield: (b) R2hydrazine, EtOH, 0 ꢀC to rt, yield 85e87%, (c) DIBAL-H, DCM, ꢁ78 ꢀC, yield 86%, (d) hydroxylamine
HCl, TEA, DCM, rt, yield 97%; (e) LAH, DCM, 0 ꢀC to rt, 99%, and (f) ArSO2Cl, TEA, DCM, yield 67e91%.
the T-type calcium channel, showing 3.20 0.62 and 2.08 0.05
half inhibition concentrations against a1G and a1H, respectively, and
Table 1
also showing low inhibition potency against N-type
(IC50 ¼ 14.5 M) and hERG (IC50 ¼ 21 M).
Percent inhibition of aryl-N-((1-R1-5-R2-pyrazol-3-yl)methyl) sulfonamides against
the T-type (a1G and a1H) calcium channels.
m
m
Inhibitory activities of selected compounds against human cy-
tochrome P450 were evaluated to assess drug-drug interactions, and
microsomal stability was evaluated to compound stability (Table 3).
Recently, researchers have reported that CYP2D6 metabolizes 25% of
clinically used drugs with significant polymorphism. In particular,
several central nervous system and cardiovascular system-related
drugs are substantially metabolized by CYP2D6 [18]. CYP3A4 is
also involved in the metabolism of over 50% of clinical drugs. Thus,
these two subtypes (CYP2D6 and CYP3A4) play more important
roles in drug-drug interactions than CYP1A2 and CYP2C9 [19]. The
results presented show that most of the tested compound showed
no inhibition of CYP2D6, except 9h, and compounds 9a, 9b, and 9g
showed low inhibition of CYP3A4. Thus, 9a, 9b, and 9g would not be
expected to cause drug-drug interactions. Unfortunately, the
selected compounds have unsatisfactory human hepatic microsomal
stability except for compound 9h.
To identify neuropathic pain relief efficacy of our new T-type
calcium channel blocking compounds, 9a, 9b, 9g, and 9h, we
evaluated mechanical and cold allodynia in a rat SNL model
(Chung's model) [20].
Behavioral testing for neuropathic pain was performed with rats
at 14 days after surgical manipulation [21e23]. We orally admin-
istered 100 mg of the selected compound (9a, 9b, 9g, and 9h) to
each SNL rat and evaluated them in comparison with gabapentin-
and mibefradil-administered animals. Gabapentin is used to treat
neuropathic pain clinically, and mibefradil was the first T-type
calcium channel blocker; here, these agents were used as
references.
Compd.
Ar
R1
R3
FDSS %
inhibition
(10 m
M)a
a1G
a1H
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
11a
11b
Ph
3-FPh
3-ClPh
4-IPh
4-CH3Ph
4-C(CH3)3Ph
CH2Ph
CH2CH2Ph
2-F, 5-CH3Ph
2-CH3, 4-lPh
2,6-F2Ph
2,6-Cl2Ph
1-Naphthyl
2-Naphthyl
8-Quinoyl
CH2-1-Naphthyl
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
iso-butyl
iso-butyl
iso-butyl
iso-butyl
iso-butyl
iso-butyl
iso-butyl
iso-butyl
iso-butyl
iso-butyl
iso-butyl
iso-butyl
iso-butyl
iso-butyl
iso-butyl
iso-butyl
Ph
61.42
64.10
57.57
46.37
57.26
46.88
65.69
70.53
54.86
22.65
45.09
36.93
24.32
19.77
29.78
7.09
63.86
62.72
55.74
44.92
62.21
33.42
70.53
68.61
70.04
26.61
69.31
56.04
25.25
24.86
61.36
6.66
tert-butyl
tert-butyl
55.47
51.55
10.44
53.47
Ph
3-FPh
a
% inhibition value was obtained using FDSS6000 fluorescence-based high-
throughput screening assay on HEX293 cells.

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J.H. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 665e672
Table 2
In vitro activities of synthesized compounds 9 and 11 against T-type (a1G and a1H), N-type calcium, and hERG channels.
a
Compd.
T-type Ca2þ channel (IC50
)
N-type Ca2þ channel
% inhibitionb
hERG channel
IC50 M)
a1G
(
m
M)
a1H
(
m
M)
IC50
NDc
(
m
M)a
(m
9a
9b
9g
9h
5.65 0.50
3.20 0.62
12.71 2.31
12.36 0.36
5.80 0.61
2.08 0.05
9.01 1.46
14.60 2.11
38.21
55.60
22.31
42.18
21.00 2.92
14.50 3.86
6.55 0.55
8.73 1.70
14.50 3.87
NDc
NDc
a
IC50 values ( SD) were obtained from a dose-response curve using a patch clamp.
b
c
% inhibition was obtained using a patch clamp at 10
Not determined.
mM.
Table 3
Cytochrome P450 inhibition and microsomal stability of selected compounds.
Compd.
% Control of CYP-450 (10
1A2b
m
M)a
2D6c
HLMf stability
Remaining %
2C9d
3A4e
9a
9b
9g
9h
36.64
45.58
41.9
NT
207.58
204.06
115.86
45.23
38.4
30.4
34.39
45.23
69.59
80.10
137.81
38.69
13.88
1.72
1.27
88.15
a
Values represent the remaining % activities and the mean SD from triplicate experiments.
b
c
d
e
f
a
-naphthoflavon.
Quinidine.
Sulfaphenazol.
Ketoconazole.
HLM, human liver metabolic.
Fig. 2. Effects on mechanical allodynia (A and B) and cold allodynia (C and D) after oral administration of mibefradil (100 mg/kg, n ¼ 4), gabapentin (100 mg/kg, n ¼ 4), 9a (100 mg/
kg, n ¼ 4), 9b (100 mg/kg, n ¼ 4), 9g (100 mg/kg, n ¼ 4), and 9h (100 mg/kg, n ¼ 4) in neuropathic pain-induced rats. Experimental time expressed as days after neuropathic pain
injury (N) and hours after gabapentin, mibefradil, 9a, 9b, 9g, or 9h administration.
The in vivo pain relief results, including mechanical and cold
allodynia, of our new compounds are summarized in Fig. 2. The
result of each (see supplementary material) was represented to one
figure.
analgesic effect over the 3-folds higher than those of both gaba-
pentin and mibefradil at 1 h after administration. The efficacy of 9b,
exhibiting the highest in vitro T-type calcium inhibitory activity,
was increased gradually but the pain relieving efficacy showed
lower than reference compounds. The caveats of compound 9b
In mechanical allodynia, the compound 9a showed significant

J.H. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 665e672
669
Table 4
5. Experimental section
Pharmacokinetic parameters following intravenous (n
administration (10 mg/kg) of 9b to male rats.
¼
5) and oral (n
¼
5)
5.1. Chemistry
9b (mean SDa)
iv
5.1.1. General
Oral
All reagents and solvents were commercially available and were
used without further purification or drying. The reaction progress
was monitored on thin-layer chromatography (Merck, silica gel
60F254) and viewed with a UV lamp at 254 and 365 nm. Com-
pounds were purified by flash column chromatography (Merck,
230e400 mesh). 1H and 13C NMR FT-NMR spectra were recorded on
AUC0e∞
AUClast
Terminal half-life (min)
Cmax g/ml)
Tmax (min)
CL (ml/min/kg)
(
m
g min/ml)
120.98 14.48
120.68 24.45
55.66 4.96
e
10.09 3.96
9.82 3.62
66.80 21.90
0.28 0.20
26 (15e30)b
e
(
mg min/ml)
(
m
83.77 11.69
57.98 7,19
1.40
MRT (min)
Brain-to-plasma ratio (B/P) at 2 h
e
a Bruker Advance 300 or 400 MHz spectrometer. Chemical shifts (d)
0.19
F (%)
8.3
of protons and carbons are relative to trimethyl silyl (TMS) as an
internal standard at 0 ppm, and coupling constants (J) are reported
in Hz and presented as s (singlet), d (doublet), t (triplet) and (m,
multiplet). Mass spectra (MS) were measured with a Q-TOF SYNAPT
G2 (Waters MS Technologies, Manchester, UK).
Abbreviations: AUC0e∞, total area under the plasma concentrationetime curve from
time zero to infinity; AUClast, total area under the plasma concentrationetime curve
from time zero to the previous time; Cmax, peak plasma concentration; Tmax, time to
reach Cmax; CL, time-averaged total body clearance; MRT, mean residence time; F,
bioavailability.
a
SD: Standard deviation.
Median (range) for Tmax
b
5.1.2. (Z)-Ethyl 3-ethyl-2-hydroxy-6-methyl-4-oxohept-2-enoate
(2)
.
2-Methyl-4-heptanone (2.05 mL, 13.0 mmol) was added
at ꢁ78 ꢀC to a solution of lithium bis(trimethylsilyl)amide (1 M in
THF, 13.1 mL, 13.1 mmol) in diethyl ether (52 mL). The solution was
stirred for 45 min, and mixed with diethyl oxalate (1.85 mL,
13.7 mmol) for 1 h. Thereafter, the reaction mixture was warmed to
room temperature and stirred for an additional 18 h. It was acidified
with 1 N HCl, added with water, and extracted with diethyl ether.
The organic layer thus obtained was dried over magnesium sulfate,
and then concentrated in a water bath (65 ꢀC) only using a rotary
evaporator to afford the title compound; 1H NMR (400 MHz, CDCl3)
were that it had poor bioavailability (F ¼ 8.3) and brain-to-plasma
ratios (B/P ¼ 0.19) when administered orally (Table 4). The pain
alleviating effect of 9g was potent at 3 h but lost efficacy rapidly
within 5 h. The least active compound 9h in vitro did not showed
alleviating effect of mechanical allodynia (Fig. 2. A and B).
In cold allodynia, compound 9a and 9b having high in vitro po-
tency, showed higher pain relief efficacy than gabapentin, and
compound 9g and 9h having low in vitro potency exhibited almost
no pain reducing effect (Fig. 2. C and D). Based on these results, we
identified that the T-type calcium channel inhibitory effect in vitro of
aryl(1,5-disubstituted-pyrazol-3-yl)methyl sulfonamides was well
correlated on neuropathic pain alleviating efficacy in vivo, particu-
larly in cold allodynia. The most potent T-type calcium channel
blocking compound 9b needs to be further optimized to develop a
new drug candidate for the treatment of neuropathic pain.
d
6.35 (s, 1H), 4.46 (q, J ¼ 7.12 Hz, 2H), 2.35 (d, J ¼ 7.16 Hz, 2H), 2.18
(m, 1H), 1.38 (t, J ¼ 7.12Hz, 3H), 0.97 (d, J ¼ 6.61 Hz, 6H).
5.1.3. Ethyl(4-ethyl-5-isobutyl-1-phenyl-1H-pyrazole)-3-
carboxylate (4)
Phenyl hydrazine (1.28 mL, 13.0 mmol) was added to a solution
of
(Z)-ethyl(3-ethyl-2-hydroxy-6-methyl-4-oxohept-2-eno)ate
(2.97 g, 13.0 mmol) in ethanol (40 mL). One hour later, 1 N HCl was
added to the solution that was then stirred at 80 ꢀC for 3 h. After
removal of the solvent, the reaction mixture was added with water
and extracted with dichloromethane. The organic layer was dried
over magnesium sulfate, filtered in vacuo, and purified by column
chromatography (hexane:EtOAc ¼ 15:1 / 5:1) to afford 607 mg of
4. Conclusions
A novel series of arylsulfonamide derivatives bearing 1,5-
disubstituted pyrazol-3-yl methane were designed, inspired by the
chemical motifs of known antiepileptics possessing T-type calcium
inhibitory activity. The synthesis and structure activity relationships
(SAR) of this series of compounds in vitro and in neuropathic pain-
alleviating efficacy in vivo are reported in the current study. A
small unsubstituted phenyl having a longer methylene unit on the
Ar substituent displayed a positive effect on in vitro inhibitory ac-
tivity against both a1G and a1H calcium channels. Among the tested
compounds, we found that compounds 9a, 9b, 9g, and 9h exhibited
selective inhibition against T-type calcium channels versus N-type,
and showed low inhibition of hERG channels. Although three
compounds exhibited good CYP450 liability, especially against 2D6
and 3A4, their microsomal stabilities were very low. Nevertheless,
among selected compounds, 9a and 9b exhibited neuropathic pain
alleviation effects in mechanical and cold allodynia induced in the
SNL rat model. Compounds 9a and 9b displayed better efficacy than
mibefradil and gabapentin against cold allodynia. In particular,
compound 9a seemed to be valuable as shown fast acting perfor-
mance in mechanical pain. Thus, the aryl(1-phenyl-5-isopropyl-
pyrazol-3-yl)methyl sulfonamide derivatives appear to be good T-
type calcium blockers and could offer safe and effective oral
neuropathic pain treatments. Based on these results, we will
conduct further optimization studies with this class of compounds
to achieve development of new neuropathic pain drug candidates.
the title compound. 1H NMR (400 MHz, CDCl3)
d 7.46e7.39 (m, 5H),
6.76 (s, 1H), 4.42 (q, J ¼ 7.12 Hz, 2H), 2.51 (d, J ¼ 7.16 Hz, 2H),
1.87e1.79 (m, 1H), 1.41 (t, J ¼ 7.12 Hz, 3H), 0.86 (d, J ¼ 6.64 Hz, 6H).
5.1.4. Ethyl 1-(tert-butyl)-5-phenyl-1H-pyrazole-3-carboxylate (5)
Diethyloxalate (0.71 ml, 5.25 mmol) was added slowly at 0 ꢀC to
a solution of acetophenone (0.59 mL, 5.00 mmol) and NaOEt
(2.80 mL, 7.50 mmol) in ethanol (12 mL), and stirred for overnight.
It was acidified with 1 N HCl, added with water, and extracted with
dichloromethane. The organic layer was dried over magnesium
sulfate, and then concentrated in a water bath (65 ꢀC) only using a
rotary evaporator to afford ethyl 2,4-dioxo-4-phenylbutanoate (3,
552 mg). Without further purification, (Z)-ethyl 2-hydroxy-4-oxo-
4-phenylbut-2-enoate (86.3 mg, 0.392 mmol) was slowly added to
a solution of tert-butyl hydrazine hydrogen chloride (50.1 mg,
0.392 mmol) in ethanol (2.5 mL). The solution was stirred overnight
at room temperature. When the reaction was completed as
measured by TLC (hexane:EtOAc ¼ 6: 1), the reaction mixture was
concentrated in vacuo. After the extraction of the concentrate with
dichloromethane and water, the organic layer thus formed was
dried over anhydrous sodium sulfate, filtered, concentrated, and
purified by column chromatography (hexane:EtOAc ¼ 15: 1 / 6: 1)

670
J.H. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 665e672
to afford the title compound (59.8 mg, 86%). 1H NMR (300 MHz,
CDCl3)
(s, 9H), 1.43 (t, J ¼ 7.1 Hz, 3H).
13C NMR (75 MHz, CDCl3)
d 147.9, 144.3, 140.0, 139.5, 132.5, 129.1,
d
7.47e7.36 (m, 5H), 6.71 (s, 1H), 4.43 (q, J ¼ 7.1 Hz, 2H), 1.54
129.0,128.1127.2,125.7,104.9, 41.1, 35.0, 28.3, 22.3. HRMS [ESIþ] m/z
calcd for C20H23N3O2S [MþH]þ: 370.1511, found: 370.1600.
5.1.5. 4-Ethyl-5-isobutyl-1-phenyl-1H-pyrazole-3-carbaldehyde (6)
5.1.9. N-[(5-Isobutyl-1-phenyl-1H-pyrazol-3-yl)methyl]-3-
Dried
ethyl
4-ethyl-5-isobutyl-1-phenyl-1H-pyrazole-3-
fluorobenzenesulfonamide (9b)
carboxylate (454 mg, 1.51 mmol) was dissolved in dichloro-
methane (2.0 mL), and diisobutylaluminum hydride (1 M in hex-
ane, 4.53 mL, 4.53 mmol) was added at ꢁ78 ꢀC to the solution. It
was stirred for 1.5 h, and mixed with methanol and 1 N HCl. After
the temperature of the reaction mixture was elevated to room
temperature, it was added with water and extracted with
dichloromethane. The organic layer thus formed was dried over
magnesium sulfate, filtered in vacuo, and purified by column
chromatography (hexane:EtOAc ¼ 10:1) to afford the title com-
Triethylamine (121 m
L, 0.871 mmol) was added at 0 ꢀC to a so-
lution of (5-isobutyl-3-aminomethyl-1-phenyl)pyrazole (182 mg,
0.792 mmol) in dichloromethane (3.0 mL). The solution was stirred
for 5 min, and mixed with 3-fluorobenzenesulfonyl chloride
(111 mL, 0.832 mmol). Subsequently, the solution was warmed to
room temperature, and stirred for 1 h. Then, water and a saturated
sodium hydrogen carbonate solution were added to the solution
before extraction with dichloromethane. The organic layer thus
formed was dried over magnesium sulfate, concentrated in vacuo,
pound (332 mg, 85.8%). 1H NMR (400 MHz, CDCl3)
d
10.1 (s, 1H),
and
(hexane:EtOAc ¼ 3:1 / 2:1) to afford the title compound (262 mg,
85.5%). 1H NMR (400 MHz, CDCl3)
7.64e7.62 (m, 1H), 7.53e7.51
purified
by
column
chromatography
7.52e7.41 (m, 5H), 6.76 (s, 1H), 2.55 (d, J ¼ 7.17 Hz, 2H), 1.90e1.82
(m, 1H), 0.74 (d, J ¼ 6.64 Hz, 6H).
d
(m, 1H), 7.45e7.35 (m, 4H), 7.28e7.19 (m, 3H), 6.01 (s, 1H), 5.89 (t,
J ¼ 5.81 Hz, 1H), 4.18 (d, J ¼ 5.87 Hz, 2H), 2.41 (d, J ¼ 7.19 Hz, 2H),
1.76e1.69 (m, 1H), 0.80 (d, J ¼ 6.61 Hz, 6H); 13C NMR (100 MHz,
5.1.6. 4-Ethyl-3-(hydroxyamino)methyl-5-isobutyl-1-
phenylpyrazole (7)
Triethylamine (165
mL, 1.18 mmol) was added to a solution of
CDCl3) d 163.5, 161.0, 147.7, 144.4, 142.2, 142.1, 139.5, 130.7, 130.7,
hydroxylamine hydrogen chloride (82.5 mg, 1.18 mmol) in
dichloromethane (2.5 mL). After stirring for 5 min, the pH of the
solution was measured. A solution of dichloromethane in 4-ethyl-
129.1, 128.2, 125.6, 123.0, 123.0, 119.7, 119.5, 114.6, 114.4, 104.9, 41.0,
35.0, 28.3, 22.3. HRMS [ESIþ] m/z calcd for C20H22FN3O2S [MþH]þ:
388.1417, found: 388.1498.
5-isobutyl-1-phenyl-1H-pyrazole-3-carbaldehyde
(276
mg,
1.08 mmol) was added. The resulting reaction mixture was stirred
for 5 h, added with water, and extracted with dichloromethane. The
organic thus obtained was dried over magnesium sulfate, filtered,
concentrated in vacuo, and purified to afford the title compound
5.1.10. N-[(5-Isobutyl-1-phenyl-1H-pyrazol-3-yl)methyl]-3-
chlorobenzenesulfonamide (9c)
Triethylamine (122 m
L, 0.872 mmol) was added at 0 ꢀC to a so-
lution of (5-isobutyl-3-aminomethyl-1-phenyl)pyrazole (182 mg,
0.792 mmol) in dichloromethane (3.0 mL). The solution was stirred
for 5 min, and mixed with 3-chlorobenzenesulfonyl chloride
(284 mg, 97.0%). 1H NMR (400 MHz, CDCl3)
d 8.27 (s, 1H), 7.49e7.36
(m, 5H), 7.21 (s, 1H), 6.53 (s, 1H), 2.55 (d, J ¼ 7.16 Hz, 2H), 1.88e1.80
(m, 1H), 0.88 (d, J ¼ 6.60 Hz, 6H).
(117 mL, 0.832 mmol). Subsequently, the solution was warmed to
room temperature, and stirred for 1 h. Then, water and a saturated
sodium hydrogen carbonate solution were added to the solution
before extraction with dichloromethane. The organic layer thus
formed was dried over magnesium sulfate, concentrated in vacuo,
5.1.7. (4-Ethyl-5-isobutyl-1-phenyl-1H-pyrazol-3-yl)
methaneamine (8)
Dried
4-ethyl-3-(hydroxyamino)methyl-5-isobutyl-1-
phenylpyrazole (282 mg, 1.04 mmol) was dissolved in a mixed
solvent of diethyl ether (1.4 mL) and tetrahydrofuran (2.7 mL), and
lithium aluminium hydride (1 M in diethyl ether, 2.29 mL,
2.29 mmol) was added at -0 ꢀC to the solution. It was stirred for
30 min before for 30 min at room temperature. The temperature
was reduced again to 0 ꢀC to carefully add sodium sulfate hydrate to
the solution. The reaction mixture was filtered through celite and
sodium sulfate, concentrated in vacuo, and dried to afford the title
and purified by column chromatography (hexane:EtOAc
3:1 / 2:1) to afford the title compound (286 mg, 89.3%); 1H NMR
(400 MHz, CDCl3) 7.83e7.82 (m, 1H), 7.74e7.71 (m, 1H), 7.50e7.35
¼
d
(m, 5H), 7.27e7.24 (m, 2H), 6.01 (s, 1H), 5.70 (br, 1H), 4.20 (d,
J ¼ 5.86 Hz, 2H), 2.42 (d, J ¼ 7.18 Hz, 2H), 1.77e1.70 (m, 1H), 0.81 (d,
J ¼ 6.63 Hz, 6H); 13C NMR (100 MHz, CDCl3)
d 147.6, 144.4, 141.8,
139.5, 135.0, 132.5, 130.2, 129.1, 128.2, 127.3, 125.6, 125.3, 104.9, 41.1,
35.0, 28.3, 22.4.
compound (266 mg, 99.3%). 1H NMR (400 MHz, CDCl3)
d 7.48e7.36
(m, 5H), 6.14 (s, 1H), 3.91 (s, 2H), 2.50 (d, J ¼ 7.12 Hz, 2H), 2.48 (q,
5.1.11. N-[(5-Isobutyl-1-phenyl-1H-pyrazol-3-yl)methyl]-4-
J ¼ 7.57 Hz, 2H), 1.88e1.80 (m, 1H), 0.87 (d, J ¼ 6.64 Hz, 6H).
iodobenzenesulfonamide (9d)
Triethylamine (32.6 m
L, 0.234 mmol) was added at 0 ꢀC to a so-
5.1.8. N-[(5-Isobutyl-1-phenyl-1H-pyrazol-3-yl)methyl]
lution of (5-isobutyl-3-aminomethyl-1-phenyl)pyrazole (48.7 mg,
0.212 mmol) in dichloromethane (1.0 mL). The solution was stirred
for 5 min, and mixed with a solution of 4-iodobenzenesulfonyl
chloride (68.2 mg, 0.223 mmol) in dichloromethane. Subse-
quently, the solution was warmed to room temperature, and stirred
for 1 h. Then, water and a saturated sodium hydrogen carbonate
solution were added to the solution before extraction with
dichloromethane. The organic layer thus formed was dried over
magnesium sulfate, concentrated in vacuo, and purified by column
benzenesulfonamide (9a)
Triethylamine (194 m
L, 1.39 mmol) was added at 0 ꢀC to a so-
lution of (5-isobutyl-3-aminomethyl-1-phenyl)pyrazole (290 mg,
1.26 mmol) in dichloromethane (3.0 mL). The solution was stirred
for 5 min, and mixed with benzenesulfonyl chloride (170
1.33 mmol). Subsequently, the solution was warmed to room
temperature, and stirred for 1 h. Then, water and a saturated so-
dium hydrogen carbonate solution were added to the solution
before extraction with dichloromethane. The organic layer thus
obtained was dried over magnesium sulfate, concentrated in vacuo,
and purified by column chromatography (hexane:EtOAc ¼ 2:1) to
afford the title compound (419 mg, 85.6%). 1H NMR (300 MHz,
mL,
chromatography (hexane:EtOAc ¼ 3:1 / 2:1) to afford the title
1
compound (86.3 mg, 82.0%). H NMR (400 MHz, CDCl3)
d 7.76 (d,
J ¼ 8.16 Hz, 2H), 7.53 (d, J ¼ 8.24 Hz, 2H), 7.46e7.38 (m, 3H), 7.23 (d,
J ¼ 7.60 Hz, 2H), 5.95 (s, 1H), 5.72 (br, 1H), 4.18 (d, J ¼ 5.80 Hz, 2H),
2.40 (d, J ¼ 7.16 Hz, 2H), 1.75e1.68 (m, 1H), 0.81 (d, J ¼ 6.60 Hz, 6H);
CDCl3)
d
7.86 (d, J ¼ 7.97 Hz, 2H), 7.56e7.37 (m, 6H), 7.25 (d,
J ¼ 6.64 Hz, 2H), 6.01 (s, 1H), 5.59 (br, 1H), 4.18 (d, J ¼ 5.96 Hz, 2H),
13C NMR (100 MHz, CDCl3)
d 147.6, 144.4, 139.9, 139.5, 138.1, 129.2,
2.43 (d, J ¼ 7.17 Hz, 2H), 1.77e1.68 (m, 1H), 0.81 (d, J ¼ 6.62 Hz, 6H);
128.7, 128.2, 125.7, 104.8, 99.7, 41.0, 35.0, 28.3, 22.4.

J.H. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 665e672
671
5.1.12. N-[(5-Isobutyl-1-phenyl-1H-pyrazol-3-yl)methyl]-4-
methylbenzenesulfonamide (9e)
Triethylamine (38.4
L, 0.276 mmol) was added at 0 ꢀC to a
solution of (5-isobutyl-3-aminomethyl-1-phenyl)pyrazole
5.1.15. N-[(5-isobutyl-1-phenyl-1H-pyrazol-3-yl)methyl]-2-
phenylethanesulfonamide (9h)
Triethylamine (80.9
L, 0.580 mmol) was added at 0 ꢀC to a
m
m
solution of (5-isobutyl-3-aminomethyl-1-phenyl)pyrazole (121 mg,
0.528 mmol) in dichloromethane (1.5 mL). The solution was stirred
for 5 min, and mixed with a solution of 2-phenylethanesulfonyl
chloride (113 mg, 0.553 mmol) in dichloromethane. Subsequently,
the solution was warmed to room temperature, and stirred for 1 h.
Then, water and a saturated sodium hydrogen carbonate solution
were added to the solution before extraction with dichloro-
methane. The organic layer thus formed was dried over magnesium
sulfate, concentrated in vacuo, and purified by column chroma-
tography (hexane:EtOAc ¼ 2:1) to afford the title compound
(57.5 mg, 0.251 mmol) in dichloromethane (1.0 mL). The solution
was stirred for 5 min, and mixed with a solution of p-toluene-
sulfonyl chloride (50.5 mg, 0.263 mmol) in dichloromethane.
Subsequently, the solution was warmed to room temperature, and
stirred for 1 h. Then, water and a saturated sodium hydrogen car-
bonate solution were added to the solution before extraction with
dichloromethane. The organic layer thus formed was dried over
magnesium sulfate, concentrated in vacuo, and purified by column
chromatography (hexane:EtOAc ¼ 3:1 / 2:1) to afford the title
compound (87.7 mg, 91.2%). 1H NMR (400 MHz, CDCl3)
d
7.74 (d,
187 mg (89.3%). 1H NMR (300 MHz, CDCl3)
d 7.50e7.42 (m, 3H),
J ¼ 8.24 Hz, 2H), 7.44e7.37 (m, 3H), 7.27e7.24 (m, 4H), 6.00 (s, 1H),
5.43 (br, 1H), 4.16 (d, J ¼ 5.92 Hz, 2H), 2.42 (d, J ¼ 7.20 Hz, 2H), 2.39
(s, 3H), 1.76e1.69 (m, 1H), 0.81 (d, J ¼ 6.64 Hz, 6H); 13C NMR
7.34e7.24 (m, 5H), 7.13e7.10 (m, 2H), 6.22 (s, 1H), 5.40 (t,
J ¼ 5.94 Hz, 1H), 4.38 (d, J ¼ 5.97 Hz, 2H), 3.33e3.27 (m, 2H),
3.13e3.07 (m, 2H), 2.51 (d, J ¼ 7.20 Hz, 2H), 1.89e1.75 (m, 1H), 0.87
(100 MHz, CDCl3)
d
148.0, 144.3, 143.2, 139.6, 137.0, 129.6, 129.1,
(d, J ¼ 6.63 Hz, 6H); 13C NMR (100 MHz, CDCl3)
d 148.7, 144.6, 139.6,
128.1, 127.3, 125.6, 104.9, 41.1, 35.0, 28.3, 22.3, 21.5.
138.1, 129.2, 128.7, 128.3, 128.2, 126.8, 125.8, 105.0, 54.2, 40.9, 35.1,
29.8, 28.4, 22.4. HRMS [ESIþ] m/z calcd for C22H27N3O2S [MþH]þ:
398.1824, found: 398.1903.
5.1.13. N-[(5-Isobutyl-1-phenyl-1H-pyrazol-3-yl)methyl]-4-tert-
5.2. Biological evaluation
butylbenzenesulfonamide (9f)
Triethylamine (35.2
solution of (5-isobutyl-3-aminomethyl-1-phenyl)pyrazole
m
L, 0.252 mmol) at 0 ꢀC was added to a
5.2.1. In vitro evaluation of inhibitory activity against T-type
calcium channel
(52.6 mg, 0.229 mmol)indichloromethane (1.0 mL). The solution
was stirred for 5 min, and mixed with a solution of 4-tert-butyl-
benzenesulfonyl chloride (56.5 mg, 0.241 mmol) in dichloro-
methane. Subsequently, the solution was warmed to room
temperature, and stirred for 1 h. Then, water and a saturated so-
dium hydrogen carbonate solution were added to the solution
before extraction with dichloromethane. The organic layer thus
formed was dried over magnesium sulfate, concentrated in vacuo,
and purified by column chromatography (hexane:EtOAc
¼ 3:1 / 2:1) to afford the title compound (65.7 mg, 67.3%). 1H NMR
5.2.1.1. High throughput T-type calcium channel inhibitory assay.
T-type calcium channels (
HEK293 cells [24] and were grown in modified Eagle's medium
supplemented with puromycin (1 g/mL), streptomycin (100 mg/
a1G, a1H) were stably expressed in
m
mL), penicillin (100 U/mL), geneticin (500 mg/mL), and 10% (v/v)
fetal bovine serum at 37 ꢀC in a humid atmosphere of 95% air and
5% CO2. Cells were seeded in 96-well black-wall clear-bottom plates
at a density of 4 ꢂ 104 cells/well and were used the next day in a
HTS FDSS6000 assay [25]. Cells were incubated for 60 min at room
temperature with 5 mM 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). The increase in
[Ca2þ]i by KCl-induced depolarization was detected. During the
whole procedure, cells were washed using the BIO-TEK 96-well
washer. All data were collected and analyzed using the FDSS6000
and related software (Hamamatsu, Tokyo, Japan).
(400 MHz, CDCl3)
d
7.80 (d, J ¼ 8.52 Hz, 2H), 7.49 (d, J ¼ 8.52 Hz, 2H),
7.45e7.37 (m, 3H), 7.28e7.25 (m, 2H), 6.04 (s, 1H), 5.26 (br, 1H), 4.18
(d, J ¼ 5.88 Hz, 2H), 2.43 (d, J ¼ 7.16 Hz, 2H), 1.78e1.70 (m, 1H), 1.33
(s, 9H), 0.82 (d, J ¼ 6.64 Hz, 6H); 13C NMR (100 MHz, CDCl3)
d 156.3,
148.0, 144.3, 139.6, 136.8, 129.1, 128.1, 127.1, 126.0, 125.6, 104.9, 41.2,
35.1, 35.1, 31.1, 28.3, 22.4.
5.2.1.2. Electrophysiological recordings (whole-cell patch clamp
5.1.14. N-[(5-Isobutyl-1-phenyl-1H-pyrazol-3-yl)methyl]
assay). For recordings of T-type Ca2þ currents (
a1G, a1H), a stan-
benzylsulfonamide (9g)
Triethylamine (32.6
solution of (5-isobutyl-3-aminomethyl-1-phenyl)pyrazole
dard whole-cell patch-clamp method was used as described pre-
viously [26]. T-type Ca2þ currents were evoked every 15 s by a 50-
ms depolarizing voltage step from ꢁ100 mV to ꢁ30 mV. The
external solution contained (in mM) 140 NaCl, 2 CaCl2, 10 HEPES,
and 10 glucose (pH 7.4). The molar concentrations of test com-
m
L, 0.234 mmol) was added at 0 ꢀC to a
(48.7 mg, 0.212 mmol) in dichloromethane (1.5 mL). The solution
was stirred for 5 min, and mixed with a solution of phenyl-
methanesulfonyl chloride (43.0 mg, 0.223 mmol) in dichloro-
methane. Subsequently, the solution was warmed to room
temperature, and stirred for 1 h. Then, water and a saturated so-
dium hydrogen carbonate solution were added to the solution
before extraction with dichloromethane. The organic layer thus
formed was dried over magnesium sulfate, concentrated in vacuo,
and purified by column chromatography (hexane:EtOAc ¼ 3:1 /
2:1) to afford the title compound (64.2 mg, 78.9%). 1H NMR
pounds required to produce 50% inhibition of peak currents (IC50
)
were determined by fitting raw data into dose-response curves. The
current recordings were obtained using an EPC-9 amplifier and the
Pulse/Pulsefit software (HEKA, Elektronik, Lambrecht/Pfalz,
Germany).
5.2.2. Evaluation of inhibitory activity of hERG channels
The inhibitory activity of hERG channels by the synthesized
compounds was determined using CHO-K1 Tet-On hERG cells
purchased from IonGate Biosciences GmbH (Frankfurt, Germany).
After cell culture and preparation, an automated patch-clamp de-
vice, NPC-16 Patchliner (Nanion Technologies, Munich, Germany)
was used for whole-cell recordings. The hERG channel assay was
performed as described for the patch-clamp method [26]. Whole-
cell recordings were analyzed using the Patchmaster/Fitmaster
(400 MHz, CDCl3) d 7.49e7.45 (m, 2H), 7.42e7.38 (m, 1H), 7.36e7.32
(m, 2H), 7.26 (br, 5H), 6.17 (s, 1H), 4.97 (br, 1H), 4.25 (s, 2H), 4.19 (d,
J ¼ 5.88 Hz, 2H), 2.49 (d, J ¼ 7.16 Hz, 2H), 1.84e1.77 (m, 1H), 0.85 (d,
J ¼ 6.60 Hz, 6H); 13C NMR (100 MHz, CDCl3)
d 148.7, 144.6, 139.7,
130.8, 129.4, 129.2, 128.7, 128.6, 128.2, 125.7, 105.1, 59.1, 41.3, 35.1,
28.4, 22.4. HRMS [ESIþ] m/z calcd for C22H25N3O2S [MþH]þ:
384.1667, found: 384.1742.

672
J.H. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 665e672
(HEKA Elektronik), IGOR Pro (WaveMetrics Inc., Portland, OR, USA),
and GraphPad Prism 4 (GraphPad Software, Inc., La Jolla, CA, USA)
software.
The results of behavioral tests, including both mechanical and
cold allodynia, are expressed as % MPE. 100% MPE values near 100
indicate normal thresholds, whereas values near
allodynia.
0 indicate
5.2.3. Measurement of CYP450 inhibition
Inhibition of human CYP450 enzyme activities, including
CYP1A2, 2D6, 2C9, and 3A4, was measured using the Vivid CYP450
kit (Invitrogen, Madison, WI, USA) according to the manufacturer's
instructions. Briefly, the test compounds were diluted with water in
a 96-well plate and the Master Pre-Mix (450 BACULOSOMES Regent
and Regeneration system) were added. After incubating of the
mixture for 15 min at 37 ꢀC, the reaction was initiated by adding the
Vivid CYP450 substrates and NADPH buffer. A fluorescence plate
reader was used to measure the remaining enzyme activity. Posi-
tive controls were prepared by diluting solutions to 10 mM.
Acknowledgments
This research was supported by the KIST Intramural Program
(2E26650 and 2E26663). We thank Prof. Perez-Reyes (Department
of Pharmacology, University of Virginia) for providing HEK293/
Cav3.1 and Cav3.2 cells.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.07.032.
5.2.4. Metabolic stability in human liver microsomes
A metabolic stability assay of selected compound was per-
formed by incubating human liver microsomes (HLM, UltraPool
HLM 250 Mixed Gender Pooled Donor, Corning Gentest Co.,
Woburn, MA, USA) in the presence of NADPH (#44332000, Oriental
Yeast Co., Tokyo, Japan) as the cofactor at 37 ꢀC, as described pre-
viously. Briefly, human liver microsomes were incubated with a
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