Synthesis and diabetic neuropathic pain-alleviating effects of 2N-(pyrazol-3-yl)methylbenzo[d]isothiazole-1,1-dioxide derivatives

Article abstract of DOI:10.1016/j.bmc.2017.07.008

A novel series of fused-benzensulfonamide 2-N-(pyrazol-3-yl)methylbenzo[. d]isothiazole-1,1-dioxide derivatives was designed and synthesized as metabolically stable T-type calcium channel inhibitors. Several compounds, 9, 10, and 17, displayed potent T-type channel inhibitory activity. Among them, compounds 10 and 17 showed good metabolic stability in human liver microsomes, and low hERG channel and CYP450 inhibition. Compound 10 exhibited diabetic neuropathic pain-alleviating effects in a streptozotocin-induced peripheral diabetic neuropathy (PDN) model. The maximum efficacy of compound 10, which was 3-fold more potent than gabapentin, was observed at 1. h after administration, and co-administration of compound 10 with gabapentin showed a considerable synergic effect.

Full text of DOI:10.1016/j.bmc.2017.07.008

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and diabetic neuropathic pain-alleviating effects of
2N-(pyrazol-3-yl)methylbenzo[d]isothiazole-1,1-dioxide derivatives
Jin Ri Hong b,c, Young Jin Choi b,c, Gyochang Keum a,b, Ghilsoo Nam a,b,
⇑
a Center for Neuro-Medicine, Brain Science Institute, Korea Institutes of Science and Technology (KIST), Seoul 02792, Republic of Korea
b Division of Bio-Medical Sciences & Technology, KIST School, Korea University of Science and Technology (UST), Gajungro 217, Youseong-gu, Daejeon 305-350, Republic of 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 fused-benzensulfonamide 2-N-(pyrazol-3-yl)methylbenzo[d]isothiazole-1,1-dioxide
derivatives was designed and synthesized as metabolically stable T-type calcium channel inhibitors.
Several compounds, 9, 10, and 17, displayed potent T-type channel inhibitory activity. Among them, com-
pounds 10 and 17 showed good metabolic stability in human liver microsomes, and low hERG channel
and CYP450 inhibition. Compound 10 exhibited diabetic neuropathic pain-alleviating effects in a strep-
tozotocin-induced peripheral diabetic neuropathy (PDN) model. The maximum efficacy of compound
10, which was 3-fold more potent than gabapentin, was observed at 1 h after administration, and co-
administration of compound 10 with gabapentin showed a considerable synergic effect.
Ó 2017 Published by Elsevier Ltd.
Received 11 May 2017
Revised 4 July 2017
Accepted 6 July 2017
Available online xxxx
Keywords:
T-type calcium channel inhibition
Fused-benzsulfonamides
Neuropathic pain
Allodynia
1. Introduction
streptozotocin (STZ)-induced DPN.5 Therefore, the modulation of
T-type calcium channels could serve as an important target in
Peripheral neuropathy is one of the most common complica-
tions of early onset type 1 diabetes mellitus. Over 60% of diabetes
patients suffering from peripheral diabetic neuropathy (PDN) also
experience neuropathic pain.1 Current therapies for treating pain-
ful PDN are limited and associated with serious side effects and
addiction. Treatment of PDN is still based on pathogenic mecha-
nisms and symptomatic pain management, as well as clinically
recommended therapeutic agents such as antidepressants,
the treatment of painful PDN symptoms. This report describes
the development of potent 1H channel inhibitors for the treatment
of diabetic neuropathic pain (DNP).
a
T-type calcium channels are classified into three types, a1G, a1H,
and
a1I, based on molecular cloning of the a1 subunit. All three of
the cloned T-type calcium channels are present in the spinal dorsal
horn, with the a1G and a1H subtypes being most prominent in lam-
ina I.6 Studies of in situ hybridization and reserve-transcription
polymerase chain reaction (PCR) have shown that a1H is most
highly expressed in DRG neurons, while a1G and a1I are present
at lower levels.7 T-type calcium channels are important therapeu-
c-aminobutyric acid analogues, opioids, and topical agents. There
are currently only three Food and Drug Administration (FDA)-
approved PDN drugs: duloxetine (approved in 2004), pregabalin
(approved in 2004), and tapentadol (approved in 2012) (Fig. 1).2
Although the precise cellular mechanism of painful PDN is not
clearly understood, several recent studies have reported that the
tic targets in pain treatments. Intrathecal administration of a1H
-
specific, but not 1G- or 1I-specific, antisense oligonucleotides
a
a
has led to anti-nociceptive, and anti-allodynia effects in acute
and neuropathic pain models.8 We previously reported that aryl
(1,5-disubstituted-pyrazol-3-yl)methyl sulfonamides were T-type
calcium channel inhibitors.9 The most active candidate, 3-fluo-
rophenyl((1-phenyl-5-isopropyl)pyrazol-3-yl))sulfonamide,
showed good T-type calcium channel-blocking effects and good
pharmacokinetic properties, with the exception of microsomal sta-
bility, as well as potent anti-neuropathic pain effects.
a1H subtype of the T-type calcium channel plays an important role
in the hyper-excitability of small- and medium-sized dorsal root
ganglion (DRG) cells, resulting in painful PDN symptoms.3 The
selective T-type calcium channel blocker, (3b,5a,17b)-17-hydrox-
yestrane-3-carbonitrile (ECN), alleviates hyperalgesia in a dose-
dependent manner in diabetic ob/ob mice.4 In vivo silencing of
a1H in sensory neurons alleviates hyperalgesia in rat models with
In a continuing effort to develop metabolically stable, potent
a1H channel blockers, we have designed and synthesized a novel
series of fused-benzenesulfonamides substituted at the nitrogene
with a (1,5-disubstituted pyrazol-3-yl)methyl moiety. The sub-
stituent R1 and R2 on pyrazole ring have designed hydrophobic
⇑
Corresponding author at: Center for Neuro-Medicine, Brain Science Institute,
Korea Institutes of Science and Technology (KIST), Seoul 02792, Republic of Korea.
E-mail address: gsnam@kist.re.kr (G. Nam).
These authors made comparable contributions to this research.
c
http://dx.doi.org/10.1016/j.bmc.2017.07.008
0968-0896/Ó 2017 Published by Elsevier Ltd.
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2
J.R. Hong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 1. FDA-approved PDN drugs.
group such as phenyl, substituted phenyl and t-butyl group, and
bulky aliphatic groups such as i-butyl and t-butyl groups. We
describe herein the synthesis of 2N-(pyrazol-3-yl)methylbenzo[d]
isothiazole 1,1-dioxide derivatives and evaluate their a1H T-type
channel inhibitory activity, structure-activity relationships (SARs),
pharmaco-kinetic properties, and in vivo efficacy against STZ-
induced DNP in a rat model. The chemical structure of previous
candidate A and designed molecule in the current study are shown
in Fig. 2.
Fig. 2. Structure of designed molecules.
3. Results and discussion
The in vitro calcium channel inhibitory activities of all of the
2. Chemistry
synthetized compounds were screened using in a high throughput
screening (HTS) FDSS6000 method against CaV3.2 (a1H) calcium
Previously reported candidate compound A exhibited strong T-
type calcium channel inhibition and anti-neuropathic pain effects
with low metabolic stability (13.88% HLM).9
channels. IC50 values were determined for compounds showing
more than 60% inhibition.12 The results are summarized in Table 1.
The synthesized compounds displayed a broad range of inhibi-
tion (9.86–75.51%) of a1H T-type calcium channels. The activity of
fused-benzene sulfonamides 9–19 depended on the nature of the
R1 and R2 substituents on the pyrazole ring. The introduction of
hydrophobic groups, such as phenyl (9, 75.51%) and 4-fluorophenyl
(10, 71.57%) groups on R1, and an isopropyl group on R2, resulted in
high degrees of inhibition. The introduction of sterically hindered
aliphatic alkyl substituents, such as t-butyl (13–17) and i-propyl
(18, 19) groups on R1 and aromatic substituents, such as phenyl,
monosubstituted phenyl and cyclohexyl groups, as well as piperi-
dine derivatives containing phenyl groups on R2, resulted in low
degrees of inhibitory activity (9.86–23.06%). Compounds having
both alkyl substituents R1 and R2 displayed moderate activity
levels (17, 68.04%). For open-type benzenesulfonamides (A, 4 and
5, R1 = Ph, R2 = i-propyl), the substituent effects were similar to
those of fused-benzsufonamide derivatives. This result demon-
strates that R1 substituents having aromatic groups are more
potent than aliphatic groups for the inhibition of a1H T-type cal-
cium channels.
The HLM (human liver microsomal) stabilities of selected com-
pounds were evaluated and listed in Table 1. The methyl group
introduced compound 4 and 5, ring cyclized compound 9 of com-
pound A decreased HLM stability than compound A. Compound
10 (R1 = 4-FPh, R2 = i-Pro) and compound 17 (R1 = t-But, R2 = i-
Pro) significantly increased HLM stability.
To determine the pharmaceutical suitability of the synthesized
materials, compounds A (IC50 = 5.80 0.61), 5 (IC50 = 4.30 0.84), 9
(IC50 = 2.52 0.83), 10 (IC50=2.32 0.73) and 17 (IC50=3.69 0.60)
were evaluated for their inhibitory activity against human cyto-
chrome P450 and the hERG channel (Table 2).
Generally, chemical structural modification strategies have
applied to increase the metabolic stability of compounds.10
To overcome their inherently low metabolic stability, a methyl
group was introduced to previous candidate aryl(1,5-disubsti-
tuted-pyrazol-3-yl)methyl sulfonamides, and a fused methylene-
bridged ring was prepared between the phenyl and sulfonamide
groups, as in benzo[d]isothiazole-3(2H)-one-1,1-dioxide. Methyl
groups were added to the open ring skeletons of compounds 4
and 5 at the nitrogen of the sulfonamide and the 4-position of
pyrazole, respectively, as outlined in Scheme 1. N-Methylated sul-
fonamide 4 was prepared by sulfonylation of N-methylamine
derivatives 3a with phenyl sulfonyl chloride. The ester derivative
1a was reduced to an alcohol and subjected to azidation via the
Mitsunobu reaction (Merck method).11 to give the azide compound
2a. The azide group of compound 2a was reduced to an amine,
followed by monomethylation using triphenyl phosphine and
methyl iodide to give (5-isopropyl-1-phenyl)-3-pyrazolylmethyl-
N-methylamine 3a. Also, the tri-substituted pyrazoly(phenyl)
sulfonamide 5, possessing a methyl group at the C-4 position of
the pyrazole ring, was prepared by the coupling of (5-isopropyl-
4-methyl-1-phenyl-)pyrazol-3-methyl amine 3b with phenyl
sulfonyl chloride. The (pyrazol-3-yl)methylamine 3b was prepared
from (1-phenyl-5-isopropyl)pyrazol-3-acetate 1b by reduction of
the ester using DIBAL-H, followed by its transformation into
hydroxime and subsequent reduction with lithium aluminum
hydride in three steps.
Compounds 9–19, fused-benzenesulfonamide ring analogues
linked with an N-methylene group, were synthesized by the route
shown in Scheme 2. Reduction of pyrazol-3-yl ester 1 by LAH,
followed by bromination of the corresponding alcohol using
PBr3/PPh3, afforded 3-bromomethylpyrazole 6a–h. Nucleophilic
substitution of 6 with 2,3-dihydrobenzo[d]isothiazole 1,1-dioxide
8, prepared by the reduction of commercially available benzo[d]
isothiazole-3(2H)-one 1,1-dioxide 7, gave 2N-(pyrazol-5-yl)
methylbenzo[d]isothiazole 1,1-dioxide derivatives 9–19 as fused-
ring skeletons.
Compounds 9, 10 and 17 exhibited low inhibitory potency
against CYP2D6, which is known to induce the metabolism of sev-
eral central nervous system and cardiovascular system-related
drugs.13 All of the tested compounds displayed moderate levels
of inhibition of CYP3A4. Among them, compounds 10 (R1 = 4-FPh,
R2 = i-propyl), and 17 (R1 = t-butyl, R2 = i-propyl), both bearing
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J.R. Hong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Scheme 1. Reagents and conditions: (a) i) 1.0 M LAH in ether, THF, 0 °C to rt, 95%, ii) (PhO)2PON3, DBU, THF, for 1a; or i) DIBAL-H, DCM, ꢀ78 °C, yield 73%, ii)
hydroxylamineꢁHCl, TEA, DCM, rt, yield 50–90% for 1b; b) PPh3, CH3I, THF; c) 1.0 M LAH, THF, 0 °C to rt, 76–95%; (d) PhSO2Cl, K2CO3, DCM/DMF, rt, 30–61%.
Scheme 2. Reagents and conditions: (a) i) 1.0 M LAH in ether, THF, 0 °C to rt, 95%.; ii) PBr3, PPh3, DCM, 0 °C, 27–64%.;(b) 1.0 M LAH, THF, rt, 38%.; (c) K2CO3, DCM/DMF, rt, 30–
61%.
fused-benzenesulfonamides, showed relatively low inhibition of all
of the tested CYP enzymes. Thus, these two compounds would not
be expected to be disadvantaged by drug-drug interactions. Com-
pounds 10 and 17 showed high microsomal stability and low hERG
inhibition. Contrary to expectations, a methyl group was intro-
duced in N-position of sulfonamide (compound 4) and attached
at C4 position on pyrazole ring (compound 5) displayed lower sta-
bilities than that of compound A, which lacks a methyl group.
Compounds 9 and 10 were the most potent inhibitors of T-type
heat hypersensitivity tests. However, the efficacy of the anti-algetic
effect decreased at 3 h (Fig. 2). In contrast, gabapentin did not show
any pain-reducing activity 1 h after treatment. Instead, the activity
increased after 3 h and remained constant up to 5 h after the initial
mechanical allodynia. Against heat allodynia, gabapentin also
exhibited pain-reducing activity within 1 h, which persisted until
5 h after administration. Therefore, the anti-algetic effect of com-
pound 10 on DNP was faster-acting than that of gabapentin, but
the duration of activity was relatively short. Therefore, to investi-
gate any possible synergic effects of gabapentin and compound
10, compound 10 (50 mg/kg) and gabapentin (50 mg/kg) were
co-administrated orally, and their combined anti-nociceptive
effects on DNP were compared against a single administration of
each (Fig. 4).
Single low doses (50 mg/kg) of compound 10 did not exhibit
any inhibition of cold or mechanical allodynia at 1 h or 5 h,
although high doses (100 mg/kg) showed excellent anti-allodynia
effects after 1 h of administration. High doses of gabapentin exhib-
ited similar effects from 1 h after treatment. Co-administration of
compound 10 (50 mg/kg) with gabapentin (50 mg/kg) increased
the degree of pain alleviation more than the sum of individual
administrations, particularly at 3 h after treatment. The maximum
synergic effect against mechanical allodynia was observed at 3 h,
with a 3-fold increase of activity relative to that of a single dose
of gabapentin.
a1H calcium channels, having IC50 values (half maximum inhibition
concentrations) of 2.52 0.83 and 2.32 0.73 against 1H, respec-
a
tively, and also showing favorable pharmacological properties,
including low inhibition potency against CYP450 and hERG chan-
nels. Compound 10 showed high human liver microsomal stability
although the compound 9 having low HLM stability. These results
suggest that, relative to unsubstituted phenyl substituents, the 4-
fluorophenyl group at the R1 substituent of the pyrazole ring in
fused-benzenesulfonamide skeleton plays an important role in
determining metabolic stability.
Finally, we evaluated candidate compound 10 (R1 = 4-FPh,
R2 = i-butyl) for DNP alleviation in vivo. The pharmacokinetic data
of compound 10 are summarized in Table 3.
The efficacy of the selected compounds for alleviating DNP was
determined by behavioral tests of reduced hyperalgesia in an STZ-
induced DNP rat model.5 The anti-algetic effect of compound 10 on
DNP was examined and compared to that of gabapentin in a single
oral treatment of 100 mg/kg in each of five or six DNP rats. The
results are presented in Fig. 3.
4. Conclusions
The efficacy of compound 10 against DNP pain was highest
within 1 h after administration in both mechanical allodynia with
To develop T-type calcium channel blockers having greater
microsomal stability, a novel series of 2N-(pyrazol-5-yl)methyl-
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J.R. Hong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 1
Percent inhibition and IC50 values against T-type (
benzenesulfonamide derivatives.
a
1H) calcium channels, and human liver microsomal stabilities of substituted N-pyrazolylmethyl sulfoamides and their fused-
Compd.
R1
R2
a1H
HLMd Stability
Remaining%
% inhibition (10
l
M)a
IC50 (l
M)b
A
4
5
9
63.86
54.24
72.00
75.51
71.57
29.51
18.66
23.06
20.79
16.34
9.86
5.80 0.61
NDc
13.88
1.46
0.26
4.69
101.55
NDc
4.30 0.84
2.52 0.83
2.32 0.73
NDc
Ph
4-FPh
i-propyl
i-Propyl
i-Propyl
Ph
10
11
12
13
14
15
16
17
18
19
1,6-Cl2-Ph
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
i-Propyl
i-Propyl
NDc
NDc
4-FPh
NDc
NDc
4-CF3Ph
4-piperidylPh
4-cyclohexylPh
i-propyl
Ph
NDc
NDc
NDc
NDc
NDc
NDc
68.04
22.05
16.48
3.69 0.60
102.96
NDc
NDc
4-FPh
NDc
NDc
a
b
c
% inhibition was obtained using a FDSS6000 assay.
IC50 values ( SD) were obtained from a dose-response curve using a whole cell patch clamp.
Not determined.
HLM, human liver microsomal.
d
Table 2
In vitro activity of selected compounds against cytochrome P450 and hERG channels.
Compd.
% control of CYP-450 (10
1A2b
l
M)a
hERG channel
2D6c
2C9d
3A4e
IC50 (l
M)f
5
9
10
17
NTg
45.23
86.63
90.63
78.83
45.23
14.17
103.11
90.65
38.69
47.17
45.67
52.13
NTg
NTg
NTg
109.59
123.41
14.70 3.30
19.30 4.71
a
b
c
d
e
f
Values represent the remaining % activities and the mean SD from triplicate experiments.
-Naphthoflavone.
Quinidine.
Sulfaphenazol.
Ketoconazole.
a
IC50 values ( SD) were obtained from a dose-response curve using a patch clamp.
Not tested.
g
benzo[d]thiazole-1,1-dioxide derivatives, containing a fused-benz-
sulfonamide moiety, were designed and synthesized using known
methods. SARs of the synthesized compounds against the a1H T-
type calcium channel, and in vivo pain-alleviating efficacy against
DNP, are reported. The presence of the fused-benzsulfonamide
increased microsomal stability, although introducing a methyl
group at the amine or at the pyrazole ring decreased microsomal
stability.
activities, while substitution with aliphatic R1 (t-butyl) and
aromatic R2 groups including bulky-substituents resulted in low
potency. Pyrazole compounds with both aliphatic R1 and R2 sub-
stituents also showed high inhibitory potency against a1H T-type
calcium channels.
Compound 10 exhibited the highest pain-alleviating effect in
STZ-induced DNP rat models, as well as the most favorable phar-
macokinetic properties. Compound 10 exhibited a rapid onset of
activity against mechanical allodynia. Furthermore, co-administra-
tion of compound 10 with gabapentin at low doses displayed a
positive synergic effect in an in vivo DNP model. These data point
The nature of the substituent at the N1 and C5 positions of pyra-
zole, R1 and R2 respectively, plays a crucial role in determining the
in vitro a1H T-type calcium channel inhibitory activity. Compounds
having hydrophobic R1 (Ph and 4-FPh) and aliphatic R2 (i-propyl)
toward the importance of
ment of DNP.
a1H T-type calcium channels in the treat-
substituents at the pyrazole moiety showed high inhibitory
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J.R. Hong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Table 3
on analytical thin-layer chromatography (Merck, silica gel
60F254) with a UV lamp at 254 and 365 nm. Column chromatogra-
phy was used by silica gel (Merck, 230–400 mesh). 1H and 13C NMR
spectra were recorded on a Bruker Advance 300 or 400 MHz spec-
trometer. A Q-TOF SYNAPT G2 Mass spectrometry (Waters MS
Technologies, Manchester, UK) was used for measuring compound
mass.
Pharmacokinetic parameters following intravenous (n = 5) and oral (n = 4) adminis-
tration (10 mg/kg) of 10 to male rats.
10 (mean SDa)
iv
Oral
AUC0–1
AUClast
Terminal half-life (min)
Cmax g/ml)
Tmax (min)
CL (ml/min/kg)
MRT (min)
Brain-to-plasma ratio (B/P) at 2 h
(
l
g min/ml)
178.68 34.54
157,86 27.88
176.12 85.31
–
16.45 9.02
14.60 6.86
117.38 63.70
0.10 0.06
150 (120ꢂ240)b
–
(
lg min/ml)
(
l
5.1.2. Ethyl(4-ethyl-5-isobutyl-1-phenyl-1H-pyrazole)-3-carboxylate
(1a)
57.70 11.38
102.29 55.73
1.28
–
0.13
Phenyl hydrazine (911 lL, 9.27 mmol) was added to a solu-
tion of ethyl (Z)-(3-ethyl-2-hydroxy-6-methyl-4-oxohept-2-eno)ate
(1.86 g, 9.27 mmol) in ethanol. After stirring for 1 h at room
temperature, 1N HCl was added to the solution and stirred for
overnight. The reaction mixture was extracted with dichloro-
methane. The organic layer was dried over magnesium sulfate,
filtered in vacuo, and purified by column chromatography
(hexane:EtOAc = 10:1 then 1:1) to afford 1.95 g (77.4%) of
the title compound. 1H NMR (400 MHz, CDCl3) d 7.48–7.40 (m,
5H), 6.76 (s, 1H), 4.41 (q, J = 7.1 Hz, 2H), 2.51 (d, J = 7.2 Hz,
2H), 1.87–1.79 (m, 1H), 1.40 (t, J = 7.12 Hz, 3H), 0.86 (d, J =
6.6 Hz, 6H).
F (%)
9.2
Abbreviations: AUC0–1, total area under the plasma concentration–time curve from
time zero to infinity; AUClast, total area under the plasma concentration–time 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. Experimental section
5.1. Chemistry
5.1.3. Ethyl (5-isobutyl-4-methyl-1-phenyl-1H-pyrazole)-3-
carboxylate (1b)
Ethyl (Z)-2-hydroxy-3,6-dimethyl-4-oxohept-2-enoate (1.94 g,
9.06 mmol) was reacted with phenyl hydrazine (890 lL,
5.1.1. General
Commercially available solvents and reagents were used with-
out additional purification. The reaction progress was monitored
Fig. 3. Effects on mechanical allodynia (A and B) and heat hypersensitivity (C and D) of oral administration of gabapentin (s, 100 mg/kg, n = 6) and compound 10 (d, 100 mg/
kg, n = 5) to neuropathic pain-induced rats. Experimental time is expressed as D (days) after neuropathic injury (N) and h (hours) after gabapentin or compound 10
administration. *P < 0.05 (gabapentin), *P < 0.05 (compound 10) vs. pre-administration value (paired t-test), |P < 0.05 gabapentin vs. compound 10 unpaired t-test).
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6
J.R. Hong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 4. Effects on mechanical allodynia (A and B) and heat hypersensitivity (C and D) of oral administration of gabapentin (d, 50 mg/kg, n = 7), compound 10 ( , 50 mg/kg,
n = 6) and gabapentin + compound 10 ( , 50 mg/kg, n = 6) to neuropathic pain-induced rats. Experimental time is expressed as D (days) after neuropathic injury (N) and h
(hours) after gabapentin, compound 10 and gabapentin + compound 10 administration. *P < 0.05 (gabapentin, compound 10 and gabapentin + compound 10) vs. pre-
administration value (paired t-test), |P < 0.05 Co-treatment of gabapentin and compound 10 vs. gabapentin or compound 10 (unpaired t-test).
9.06 mmol) following same procedure of 1a. Final reaction mixture
was purified by column chromatography (hexane:EtOAc = 11: 1
then 6: 1) to afford the title compound (540 mg). 1H NMR
(400 MHz, CDCl3) d 7.46–7.38 (m, 5H), 4.42 (q, J = 7.1 Hz, 2H),
2.51 (d, J = 7.5 Hz, 2H), 2.30 (s, 3H), 1.60–1.52 (m, 1H), 1.41 (t,
J = 7.1 Hz, 3H), 0.74 (d, J = 6.7 Hz, 6H).
5.1.5. 3-(Hydroxyamino)methyl-5-isobutyl-4-methyl-1-
phenylpyrazole (2b)
Diisobutylaluminium hydride (1.0 M hexane, 2.88 mL) was
added to a solution of compound 1b (273 mg, 0.95 mmol) in
dichloromethane (1.0 mL) at ꢀ78 °C, and stirred for 1 h. the reac-
tion mixture was extracted with dichlomethane. The organic frac-
tion was dried over MgSO4, and purified by column
chromatography to give the corresponding aldehyde compound.
This aldehyde compound (194 mg, 0.80 mmol) was added to a
solution of hydroxylamine hydrochloride (61.2 mg, 0.88 mmol)
5.1.4. 3-Azidomethyl-5-isobutyl-1-phenyl-1H-pyrazole (2a)
Dried ethyl 4-ethyl-5-isobutyl-1-phenyl-1H-pyrazole-3-car-
boxylate (1.76 mg, 6.47 mmol) was dissolved in diethyl ether
(2.0 mL) and dichloromethane (2.0 mL), and lithium aluminum
hydride (1 M in hexane, 14.2 mL) was added at -0 °C to the solu-
tion. After the temperature of the reaction mixture was elevated
to room temperature, it was added with water and filtered out.
The reaction mixture was extracted with dichloromethane. The
organic layer was concentrated to give alcohol compound (1.48 g,
and triethyl amine (123 lL, 0.80 mmol) in dichloromethane, and
stirred for 4 h. The reaction mixture was extracted with dichloro-
methane and washed with brine. The organic layer was dried over
MgSO4, solvent was concentrated with reduced pressure to
afforded the title compound (196 mg, 95%). 1H NMR (400 MHz,
CDCl3) d 8.29 (s, 1H), 7.47–7.37 (m, 5H), 2.53 (d, J = 7.4 Hz, 2H),
2.21 (s, 3H), 1.68–1.60 (m, 1H), 0.74 (d, J = 6.7 Hz, 6H).
99.2%). Diphenyl phosphoryl azide (260
lL, 1.20 mmol) and 1,8-
diazabycyclo[5.4.0]undec-7-ene (179 L, 1.20 mmol) was added
l
to the reduced compound (231 mg, 1.00 mmol) in THF. After stir-
ring 16 h, the reaction mixture was diluted with toluene, and
washed with 5% HCl. The organic layer was dried over magnesium
sulfate, and solvent was removed by rotary evaporator to afford the
title compound (255 mg, 100%). 1H NMR (400 MHz, CDCl3) d 7.48–
7.38 (m, 5H), 6.23 (s, 1H), 4.39 (s, 2H), 2.52 (d, J = 7.3 Hz, 2H), 1.85–
1.78 (m, 1H), 0.87(d, J = 6.6 Hz, 6H).
5.1.6. 5-Isobutyl-1-phenyl-1H-pyrazol-3-yl)methaneamine (3a)
Polymer supported triphenyl phosphine (940 mg, 1.50 mmol)
was dissolved in THF (17 mL), and compound 2a (180 mg,
0.75 mmol) was added to the triphenyl phosphine solution, and
stirred for 4 h. Methyl iodide (141 lL, 2.26 mmol) was added to
the reaction mixture and reacted for overnight. The solid resin
was filtered and washed with dichloromethane, and the filtered
Please cite this article in press as: Hong J.R., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.07.008

J.R. Hong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
resin was pour into MeOH. Then KOH (2% MeOH, 28 mL) was
5.1.12. 3-Bromomethyl-5-isopropyl-1-(p-fluorophenyl)-1H-pyrazole
added to the reaction micture and stirred for 4 h at 65 °C. The solid
was filtered off and residual solution was extracted with dichloro-
methane, dried over Na2SO4 and solvent was removed to give com-
pound 3a (98.5 mg, 53.8%). 1H NMR (400 MHz, CDCl3) d 7.47–7.38
(m, 5H), 6.17 (s, 1H), 3.80 (s, 2H), 2.51 (s, 3H), 2.50 (d, J = 6.2 Hz,
2H), 1.88–1.79 (m, 1H), 0.85 (d, J = 6.2 Hz, 6H).
(6b)
Yield 83%; 1H NMR (300 MHz, CDCl3) d 7.43–7.31 (m, 2H), 7.22–
7.17 (m, 2H), 6.25 (s, 1H), 4.76 (d, J = 5.9 Hz, 2H), 2.51 (d,
J = 7.2 Hz), 1.98 (t, J = 5.9 Hz, 1H), 1.92–1.79 (m, 1H), 0.91 (d,
J = 6.6 Hz, 6H).
5.1.13. 3-Bromomethyl-5-isopropyl-1-(2,6-dichlophenyl)-1H-pyrazole
(6c)
5.1.7. 5-Isobutyl-4-methyl-1-phenyl-1H-pyrazol-3-yl)methaneamine
(3b)
Yield 99%; 1H NMR (300 MHz, CDCl3) d 7.52–7.38 (m, 3H), 6.28
(s, 1H), 4.79 (d, J = 6.1 Hz, 2H), 2.29 (d, J = 7.3 Hz, 2H), 1.98–1.85
(m, 2H), 0.95 (d, J = 6.6 Hz, 6H).
After dissolving the compound 2b (195 mg. 0.76 mmol) in ether
(1.0 mL) and THF (2.0 mL), lithium aluminium hydride (1 M in
ether 1.67 mmol) was added, and stirred for 30 min at 0 °C and
4 h at room temperature. Sodium sulfate hydrate was added care-
fully to the reaction mixture at 0 °C. The reaction mixture was fil-
tered through Celite and Na2SO4 and the mixture was concentrated
over reduced pressure to give amine product 3b (178 mg, 96.5%).
1H NMR (400 MHz, CDCl3) d 7.46–7.34 (m, 5H), 3.87 (s, 2H), 2.51
(d, J = 7.4 Hz, 2H), 2.04 (s, 3H), 1.88–1.55 (m, 1H), 0.75 (d,
J = 6.6 Hz, 6H).
5.1.14. 3-Bromomethyl-1-t-butyl-5-phenyl-1H-pyrazole (6d)
Yield 19%; 1H NMR (300 MHz, CDCl3) d 7.45–7.37 (m, 5H), 6.24
(s, 1H), 4.57 (s, 2H), 1.47 (s, 9H).
5.1.15. 3-Bromomethyl-1-t-butyl-5-(p-fluorophenyl)-1H-pyrazole
(6e)
Yield 98%; 1H NMR (300 MHz, CDCl3) d 7.37–7.33 (m, 2H), 7.12
(t, J = 8.6 Hz, 2H), 4.73 (d, J = 5.7 Hz, 2H), 2.03 (t, J = 5.7 Hz, 1H),
1.48 (s, 9H).
5.1.8. N-Methyl[(5-isobutyl-1-phenyl-1H-pyrazol-3-yl)methyl]
benzenesulfonamide (4)
5.1.16. 3-Bromomethyl-1-t-butyl-5-(p-trifluorophenyl)-1H-pyrazole
(6f)
Triethylamine (80.0
3a (95.3 mg, 0.39 mmol) in dichloromethane (3.0 mL) at 0 °C and
stirred for 5 min. Benzenesulfonyl chloride (52.6 L, 0.41 mmol)
lL, 0.43 mmol) was added to a solution of
1H NMR (300 MHz, CDCl3) d 7.70 (d, J = 8.0 Hz, 2H), 7.53 (d,
J = 8.0 Hz, 2H), 6.25 (s, 1H), 4.56 (s, 2H), 1.48 (s, 9H).
l
was added to the reaction mixture and stirred for 1 h at room tem-
perature. Then, water and a saturated NaHCO3 solution were added
to the solution before extraction with dichloromethane. The
organic layer was dried over magnesium sulfate, concentrated in
vacuo, and purified by column chromatography (hexane:
EtOAc = 3:1) to afford the title compound (78.8 mg, 52.5%). 1H
NMR (400 MHz, CDCl3) d 7.85–7.83 (m, 2H), 7.60–7.51 (m, 3H),
7.48–7.42 (m, 2H), 7.40–7.32 (m, 3H), 6.18 (s, 1H), 4.26 (s, 2H),
2.72 (s, 2H), 2.48 (d, J = 7.2 Hz, 2H), 1.83–1.76 (m, 1H), 0.85 (d,
J = 6.6 Hz, 6H); 13C NMR (100 MHz, CDCl3) d 147.7, 144.6, 139.7,
137.4, 132.6, 129.1, 129.0, 128.0 127.5, 125.6, 105.5, 47.9, 35.2,
34.6, 28.3, 22.4.
5.1.17. 3-Bromomethyl-1-t-butyl-5-isopropyl-1H-pyrazole (6g)
Yield 41%; 1H NMR (300 MHz, CDCl3) d 6.18 (s, 1H), 4.51 (s, 2H),
2.67 (d, J = 6.7 Hz, 2H), 2.10–1.95 (m, 1H), 1.65 (s, 9H), 1.03 (d,
J = 6.6 Hz, 6H).
5.1.18. 3-Bromomethyl-1-isopropyl-5-phenyl-1H-pyrazole (6h)
Yield 99%; 1H NMR (300 MHz, CDCl3) d 7.48–7.39 (m, 5H), 6.29
(s, 1H), 4.76 (d, J = 6.0 Hz, 2H), 3.92 (d, J = 7.6 Hz, 2H), 2.24–2.17 (m,
1H), 1.95 (t, J = 6.0 Hz, 1H), 0.79 (d, J = 6.8 Hz, 6H).
5.1.19. 1,2-Bezeneisothiazolin-1,1-dioxide (8)
Lithium aluminium hydride1 M in ether (4.09 mL) was added
slowly at 0 °C to a solution of o-sulfobenzimide (7, 554 mg,
3.02 mmol) in THF (8.0 mL), and stirred for 48 min. Subsequently,
sodium sulfate hydrate was added to the solution and filtered
through Celite. The organic layer was concentrated in reduced
pressure to afford the title compound (182 mg, 35.6%); 1H NMR
(400 MHz, CDCl3) d 7.85 (d, J = 7.7, 1H), 7.67 (dt, J = 7.5, 1.2 Hz,
1H), 7.58 (t, J = 7.4 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 4.74 (br, 1H),
4.59 (s, 2H).
5.1.9. N-Methyl[(5-Isobutyl-1-phenyl-1H-pyrazol-3-yl)methyl]
benzenesulfonamide (5)
Compound 3b (55.7 mg, 0.229 mmol) was reacted with ben-
zenxulsonyl chloride (30.7
lL, 0.24 mmol) following same proce-
dure of compound to give target compound
4
5
(77.1 mg,
87.8%).1H NMR (400 MHz, CDCl3) d 7.88–7.84 (m, 2H), 7.53–7.50
(m, 3H), 7.46–7.35 (m, 5H), 7.24–7.22 (m, 2H), 5.53 (bs, 1H), 4.12
(d, J = 5.7 Hz 2H), 2.44 (d, J = 7.4 Hz 2H), 1.94 (s, 3H), 1.58–1.50
(m, 1H), 0.69 (d, J = 6.6 Hz, 6H); 13C NMR (100 MHz, CDCl3) d
147.8, 141.1, 140.0, 139.7, 132.5, 129.1, 128.9, 128.0 127.1, 125.7,
113.1, 39.7, 33.2, 28.4, 22.2, 8.08.
5.2. General procedure for the synthesis of compound 9–19
Potassium carbonate was dissolved in DMF, and 1,2-
bezeneisothiazolin-1,1-dioxide (8) (1 equiv.) in dichloromethane
was added to the reaction solution, and stirred for overnight. Then,
the reaction mixture was filtered through Celite, and washed with
1 N HCl and saturated sodium hydrogen carbonate solution. Resid-
ual solution was dried over magnesium sulfate, and concentrated
in vacuo, and purified by column chromatography to afford the title
compound.
5.1.10. 3-Bromomethyl-5-R2-1-R1-1H-pyrazole (6)
General method. Phosphorous tribromide (531
lL, 5.65 mmol) was
added to
a
solution of (5-R2-3-hydroxymethyl-1-R1)pyrazole
(5.14 mmol) in dichloromethane (3.0 mL) at 0 °C, and stirred for
3 h at room temperature. Subsequently, the reaction mixture was
extracted with dichloromethane and washed with brine. The
organic layer was dried over magnesium sulfate and concentrated
in vacuo, and purified by column chromatography (hexane:
EtOAc = 1:1) to afford the title compound.
5.2.1. 2-[(5-isopropyl-1-phenyl-1H-pyrazol-3-yl)methyl]-2,3-
dihydrobenzo[d]isothiazole 1,1-dioxide (9)
5.1.11. 3-Bromomethyl-5-isopropyl-1-phenyl-1H-pyrazole (6a)
Yield 85%; 1H NMR (300 MHz, CDCl3) d 7.54–7.42 (m, 5H), 6.32
(s, 1H), 4.57 (s, 2H), 2.54 (d, J = 7.2 Hz, 2H), 1.91–1.82 (m, 1H), 0.91
(d, J = 6.6 Hz, 6H).
Yield; 85.5%; 1H NMR (400 MHz, CDCl3) d 7.81–7.79 (m, 2H),
7.58–7.33 (m, 8H), 6.36 (s, 2H), 4.54 (s, 2H), 4.39 (s, 2H), 2.50 (d,
J = 7.0 Hz, 2H), 1.84–1.77 (m, 1H), 0.84 (d, J = 6.6 Hz, 6H).; 13C
NMR (100 MHz, CDCl3) d 147.2, 144.7, 139.7, 135.1, 134.0, 132.6,
Please cite this article in press as: Hong J.R., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.07.008

8
J.R. Hong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
129.2, 129.0, 125.7, 124.6, 121.3, 106.0, 50.0, 41.3, 35.2, 28.3, 22.4.
HRMS [ESI+] m/z calcd for C21H23N3O2S [M + H]+: 382.1511, found:
382.1600.
131.1, 128.9, 124.5, 123.7, 121.4, 114.9, 109.0, 61.0, 50.0, 41.4,
31.2, 25.7, 24.3.
5.2.8. 2-[{1-(tert-Butyl)-5-(4-cyclohexylphenyl)-1H-pyrazol-3-yl}
methyl]-2,3-dihydrobenzo[d]isothiazole 1,1-dioxide (16)
5.2.2. 2-[(1–4-fluorophenyl)-5-isobutyl-1H-pyrazol-3-yl)methyl]-2,3-
dihydrobenzo[d]isothiazole1,1-dioxide (10)
Yield 31%;1H NMR (300 MHz, CDCl3) d 7.83 (d, J = 7.5 Hz, 1H),
7.63–7.50 (m, 2H), 7.40 (d, J = 7.5 Hz, 1H), 7.30–7.20 (m, 4H),
6.24 (s, 1H), 4.53 (s, 2H), 4.45 (s, 2H), 2.56 (brs, 1H), 1.92–1.77
(m, 5H), 1.48–1.27 (m, 14H).; 13C NMR (75 MHz, CDCl3) d 148.5,
144.3, 143.4, 135.4, 134.3, 132.5, 131.3, 130.3, 128.9, 126.2,
124.5, 121.4, 109.0, 61.1, 50.0, 44.3, 41.4, 34.4, 31.2, 26.9, 26.1.
Yield 79%; 1H NMR (300 MHz, CDCl3) d 7.83–7.49 (m, 3H), 7.41–
7.34 (m, 3H), 7.20–7.14 (m, 2H), 6.35 (s, 1H), 4.52 (s, 2H), 4.39 (s,
2H), 2.46 (d, J = 7.2 Hz, 2H), 1.87–1.73 (m, 1H), 0.84 (d, J = 6.6 Hz,
6H); 13C NMR (75 MHz, CDCl3) d 163.7, 160.4, 147.4, 144.8,
135.9, 135.1, 134.0, 132.7, 129.1, 127.7, 127.6, 124.6, 121.3,
116.2, 115.9, 106.1, 53.5, 50.0, 41.3, 35.1, 28.3, 22.4. HRMS [ESI+]
m/z calcd for C21H22FN3O2S [M+H]+: 400.1409, found: 400.1446.
5.2.9. 2-[{(1-t-butyl)-5-isobutyl-1H-pyrazol-3-yl-}methyl]2,3-
dihydrobenzo[d]isothiazol-1,1- dioxide (17)
Yield 79%; 1H NMR (300 MHz, CDCl3) d 7.81 (d, J = 6.6 Hz, 1H),
7.60–7.48 (m, 2H), 7.36 (d, J = 7.4 Hz), 6.18 (s, 1H), 4.45 (s, 2H),
4.34 (s, 2H), 2.64 (d, J = 7.1 Hz), 2H), 2.09–1.09 (m, 1H), 1.64 (s,
9H), 0.98 (d, J = 6.6 Hz, 6H); 13C NMR (75 MHz, CDCl3) d 143.6,
143.1, 135.4, 134.3, 132.5, 128.9, 124.5, 121.3, 106.7, 59.9, 49.9,
41.4, 27.3, 30.5, 28.4, 22,7.
5.2.3. 2-[1-(2,6-dichlorophenyl)-5-isobutyl-1H-pyrazol-3-yl)methyl]-
2,3-dihydrobenzo[d]isothiazole1,1-dioxide (11)
Yield; 74.0%; 1H NMR (300 MHz, CDCl3) d 7.84 (d, J = 7.7 Hz, 1H),
7.62–7.30 (m, 6H), 6.40 (s, 2H), 4.57 (s, 2H), 4.38 (s, 2H), 2.24 (d,
J = 7.3 Hz, 2H), 1.92–1.79 (m, 1H), 0.89 (d, J = 6.6 Hz, 6H).; 13C
NMR (75 MHz, CDCl3) d 148.3, 146.4, 135.4, 135.2, 135.1, 134.2,
131.0, 129.0, 128.8, 124.6, 121.4, 105.4, 49.6, 41.3, 34.8, 27.5, 22.5.
HRMS [ESI+] m/z calcd. for C18H27N3O2S [M + H]+: 362.1801,
found: 362.1850.
5.2.4. 2-[{1-(tert-Butyl)-5-phenyl-1H-pyrazol-3-yl}methyl]-2,3-
dihydrobenzo[d]isothiazole 1,1-dioxide (12)
5.2.10. 2-{(1-Isobutyl-5-phenyl-1H-pyrazol-3-yl)methyl}-2,3-
dihydrobenzo[d]isothiazole1,1-dioxide (18)
Yield 40%; 1H NMR (300 MHz, CDCl3) d 7.86 (d, J = 7.5 Hz, 1H),
7.65–7.52 (m, 2H), 7.43–7.35 (m, 2H), 6.27 (s, 1H), 4.54 (s, 2H),
4.47 (s, 2H), 1.49 (s, 9H).; 13C NMR (75 MHz, CDCl3) d 144.1,
143.5, 135.3, 134.3, 134.0, 132.5, 130.4, 129.0, 128.4, 127.8,
124.5, 121.5, 61.3, 50.0, 41.4, 31.2.
Yield 44%; 1H NMR (300 MHz, CDCl3) d 7.86–7.63 (m, 1H), 7.63–
7.51 (m, 2H), 7.49–7.37 (m, 6H), 6.42 (s, 1H), 4.56 (s, 2H), 4.41 (s,
2H), 3.96 (d, J = 7.5 Hz, 2H), 2.24–2.15 (m, 1H), 0.79 (d, J = 6.7 Hz,
6H).; 13C NMR (75 MHz, CDCl3) d 145.9, 145.6, 136.8, 135.2,
134.0, 133.0, 132.6, 130.8, 129.0, 128.7, 124.6, 121.4, 106.1, 56.8,
49.9, 41.3, 29.6, 19.8.
5.2.5. 2-[{1-(tert-Butyl)-5-(4-fluorophenyl)-1H-pyrazol-3-yl}methyl]-
2,3-dihydrobenzo[d]isothiazole 1,1-dioxide (13)
5.2.11. 2-[{5-(4-Fluorophenyl)-1-isobutyl-1H-pyrazol-3-yl}methyl]-
2,3-dihydrobenzo[d]isothiazole 1,1-dioxide (19)
Yield 42%; 1H NMR (300 MHz, CDCl3) d 7.86 (d, J = 7.6 Hz, 1H),
7.65–7.53 (m, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.35–7.29 (m, 2H),
7.13–7.06 (m, 2H), 6.27 (s, 1H), 4.53 (s, 2H), 4.46 (s, 2H), 1.48 (s,
9H).; 13C NMR (75 MHz, CDCl3) d 164.4, 162.3, 161.1, 143.6,
142.9, 135.3, 134.2, 132.6, 132.2, 132.1, 129.9, 129.0, 124.5,
121.5, 115.0, 114.7, 109.3, 61.3, 50.0, 41.3, 31.2.
Yield 62%; 1H NMR (300 MHz, CDCl3) d 7.61–7.52 (m, 2H), 7.46–
7.33 (m, 4H), 7.18–7.12 (m, 2H), 6.41 (s, 1H), 4.56 (s, 2H), 4.41 (s,
2H), 3.91 (d, J = 7.5 Hz, 2H), 2.26–2.08 (m, 1H), 0.80 (d, J = 6.7 Hz,
6H). 13C NMR (75 MHz, CDCl3) d 146.0, 144.5, 136.8, 135.2, 134.0,
133.0, 132.6, 131.0, 130.8, 129.2, 129.1, 124.8, 124.5, 121.4,
115.9, 115.6, 106.3, 56.7, 49.9, 45.7, 41.2, 29.6, 19.8.
5.2.6. 2-[{1-(tert-Butyl)-5-(4-trifluoromethylphenyl)-1H-pyrazol-3-
yl}methyl]-2,3-dihydrobenzo[d]isothiazole 1,1-dioxide (14)
Yield 39%; 1H NMR (300 MHz, CDCl3) d 7.85 (d, J = 7.7 Hz, 1H),
7.68–7.48 (m, 6H), 7.41 (d, J = 7.4 Hz, 1H), 6.29 (s, 1H), 4.54 (s,
2H), 4.47 (s, 2H), 1.49 (s, 9H).; 13C NMR (75 MHz, CDCl3) d 143.9,
142.4, 137.9, 135.3, 134.2, 132.6, 130.9, 130.8, 130.5, 129.0,
125.7, 124.8, 124.7, 124.5, 122.1, 121.5, 109.4, 61.5, 50.1, 41.3,
31.3.
5.3. Biological evaluation
5.3.1. In vitro evaluation of inhibitory activity against T-type calcium
channel
High throughput assay (HTS) and electrophysiological record-
ings (whole-cell patch clamp assay) for T-type calcium channel
inhibitory activity performed according to a previously described
protocol.9
5.2.7. 2-[{1-(tert-Butyl)-5-(4-piperidin-1-yl-phenyl)-1H-pyrazol-3-yl}
methyl]-2,3-dihydrobenzo[d]isothiazole 1,1-dioxide (15)
For FDSS binding assay: During the whole procedure, cells were
washed using the BIO-TEK 96-well washer. The stably expressed T-
type calcium channel in HEK293 cells was grown with puromycin
[1-(tert-Butyl)-5-{4-(piperidin-1-yl)phenyl}-1H-pyrazol-3-yl]
methanol (54.9 mg, 0.18 mmol) was reacted with tripheny phos-
phine (93.1 mg, 0.36 mmol) and carbontetrabromide (121 mg,
0.37 mmol) for 1 h. at 0 °C. Without purification, the reaction mix-
ture was added to compound 8 (27.3 mg, 0.16 mmol) in DMF, and
stirred for overnight. Then the reaction mixture was extracted with
ethyl acetate. The organic layer was dried over magnesium sulfate,
filtered, concentrated in reduced pressure, and purified by column
chromatography (Hex: EtOAc = 4: 1 then 1: 1) to give title com-
pound (15.2 mg, 20%).
(1 lg/mL), streptomycin (100 mg/mL), penicillin (100 U/mL),
geneticin (500 mg/mL), and 10% (v/v) fetal bovine serum in modi-
fied Eagle’s medium at 37 °C in a humid atmosphere of 95% air and
5% CO2. Prior to using in a HTS FDSS6000 assay, cells were seeded
in 96-well black-wall clear-bottom plates at a density of 4 ꢃ 104
cells/well. Cells were incubated for 60 min at room temperature
with 0.001% Pluronic F-127 in a HEPES-buffered solution com-
posed of (in mM) and 5 mM fluo3/AM: 20 HEPES, 13.8 glucose
(pH 7.4), 115 NaCl, 5.4 KCl, 0.8 MgCl2, 1.8 CaCl2. The increase in
[Ca2+]i by KCl-induced depolarization was detected. All data were
collected and analyzed using the FDSS6000 and related software
(Hamamatsu, Tokyo, Japan).
1H NMR (300 MHz, CDCl3) d 7.89 (d, J = 7.9 Hz, 1H), 7.64–7.52
(m, 2H), 7.40 (d, J = 7.1 Hz, 1H), 7.30–7.17 (m, 2H), 6.29 (d,
J = 8.7 Hz, 2H), 6.22 (s, 1H), 4.52 (s, 2H), 4.46 (s, 2H), 3.26–3.23
(m, 4H), 1.79–1.74 (m, 4H), 1.67–1.63 (m, 2H), 1.49 (s, 9H).; 13C
NMR (75 MHz, CDCl3) d 151.9, 144.4, 143.3, 135.4, 134.3, 132.5,
For IC50 (half inhibition concentration of peak current) was
determined, electrophysiological recordings (whole-cell patch
Please cite this article in press as: Hong J.R., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.07.008

J.R. Hong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
clamp assay) was performed by fitting raw data into dose-response
curves according to previously described method.14 A standard
whole-cell patch-clamp method was used. The current recordings
were obtained using an EPC-9 amplifier and the Pulse/Pulsefit soft-
ware (HEKA, Elektronik, Lambrecht/Pfalz, Germany). T-type Ca2+
currents were evoked every 15 s by a 50-ms depolarizing voltage
step from ꢀ100 mV to ꢀ30 mV.
behavioral test. Mechanical allodynia was measured by von Frey
according to previously reported methodology.16 The 6 rats were
treated orally with 100 mg/kg of gabapentin, and the 5 rats were
administrated orally compound 100 mg/kg, the tests were evalu-
ated at 1 h, 3 h, and 5 h. For mechanical allodynia tests,17 a von
Frey filament (Stoelting, Wood Dale, IL, USA) was touched five
times during every 3–4 s on the hind paw. For thermal hypersensi-
tivity, the hargreave test was performed by beam. The frequency of
paw withdrawal was expressed as a percentage [(No. of trials
accompanied by brisk foot withdrawal/total No. of trials] ꢃ 100].
The results of behavioral tests, including both mechanical and
thermal hypersensitivity, are expressed as % MPE. 100% MPE values
near 100 indicate normal thresholds, whereas values near 0 indi-
cate allodynia.
5.3.2. Evaluation of pharmacokinetic properties
The inhibitory activity of hERG channels and CYP450 enzyme,
and metabolic stability in human liver microsomes were deter-
mined by following previously described method.9 Briefly, hERG
channels inhibition of the synthesized compounds was determined
using CHO-K1 Tet-On hERG cells purchased from IonGate Bio-
sciences GmbH (Frankfurt, Germany), and IC50 was performed as
described for the patch-clamp method. Whole-cell recordings were
analyzed using the Patchmaster/Fitmaster (HEKA Elektronik), IGOR
Pro (WaveMetrics Inc., Portland, OR, USA), and GraphPad Prism 4
(GraphPad Software, Inc., La Jolla, CA, USA) software.
Inhibition of human CYP1A2, 2D6, 2C9, and 3A4 of tested com-
pound was measured using the Vivid CYP450 kit (Invitrogen, Madi-
son, WI, USA) according to the manufacturer’s instructions. Briefly,
the test compounds were diluted with water in a 96-well plate, fol-
lowed by adding with the Master Pre-Mix (450 BACULOSOMES
Regent and Regeneration system). The reaction was initiated by
adding the Vivid CYP450 substrates and NADPH buffer after incu-
bating of the mixture for 15 min at 37 °C. A fluorescence plate
reader was used to measure the remaining enzyme activity. Posi-
tive controls were prepared by diluting solutions to 10 mM.
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. Briefly, human
Acknowledgments
This research was supported by the KIST Intramural Program
(2E26850) Brain Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Science, ICT &
Future Planning (NRF-2016M3C7A1913845) and the Original Tech-
nology Research Program (NRF-2016M3C7A1904344). Thanks for
Dr. E. J. Lim and S. H. Seo for performing of in vivo efficacy test
and pharmacokinetic experiments.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.07.008.
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Please cite this article in press as: Hong J.R., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.07.008