Discovery of novel tetrahydroisoquinoline derivatives as orally active N-type calcium channel blockers with high selectivity for hERG potassium channels

Article abstract of DOI:10.1016/j.bmc.2014.10.020

N-type calcium channels represent a promising target for the treatment of neuropathic pain. The selective N-type calcium channel blocker ziconotide ameliorates severe chronic pain but has a narrow therapeutic window and requires intrathecal administration. We identified tetrahydroisoquinoline derivative 1a as a novel potent N-type calcium channel blocker. However, this compound also exhibited potent inhibitory activity against hERG channels. Structural optimizations led to identification of (1S)-(1-cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone ((S)-1h), which exhibited high selectivity for hERG channels while retaining potency for N-type calcium channel inhibition. (S)-1h went on to demonstrate in vivo efficacy as an orally available N-type calcium channel blocker in a rat spinal nerve ligation model of neuropathic pain.

Full text of DOI:10.1016/j.bmc.2014.10.020

Bioorganic & Medicinal Chemistry 22 (2014) 6899–6907
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel tetrahydroisoquinoline derivatives as orally active
N-type calcium channel blockers with high selectivity for hERG
potassium channels
a
a
a
a
Takashi Ogiyama a, , Makoto Inoue , Shugo Honda , Hiroyoshi Yamada , Toshihiro Watanabe ,
⇑
Takayasu Gotoh a, Tetsuo Kiso a, Akiko Koakutsu b, Shuichiro Kakimoto a, Jun-ichi Shishikura a
a Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
b Drug Discovery Research, Astellas Pharma Inc., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
N-type calcium channels represent a promising target for the treatment of neuropathic pain. The
selective N-type calcium channel blocker ziconotide ameliorates severe chronic pain but has a narrow
therapeutic window and requires intrathecal administration. We identified tetrahydroisoquinoline
derivative 1a as a novel potent N-type calcium channel blocker. However, this compound also exhibited
potent inhibitory activity against hERG channels. Structural optimizations led to identification of
(1S)-(1-cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone
((S)-1h), which exhibited high selectivity for hERG channels while retaining potency for N-type calcium
channel inhibition. (S)-1h went on to demonstrate in vivo efficacy as an orally available N-type calcium
channel blocker in a rat spinal nerve ligation model of neuropathic pain.
Received 9 September 2014
Revised 16 October 2014
Accepted 16 October 2014
Available online 22 October 2014
Keywords:
N-type calcium channel
Pain
hERG
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
ziconotide has been reported to cause substantial side effects, such
as nausea, dizziness, confusion and ataxia, which probably result
Neuropathic pain is characterized by abnormal pain sensation,
such as spontaneous pain, allodynia and hyperalgesia, and is
accompanied by dysesthesia and paresthesia. According to the
International Association for the Study of Pain, neuropathic pain
is defined as that caused by a lesion or disease of the somatosensory
nervous system.1 The prevalence of neuropathic pain is estimated
at 7–10% of the world’s population,2 and the unbearable pain
suffered by many of these patients reduces their quality of life.
Although studies have provided new insights into the mechanism
of neuropathic pain, it remains complicated and unclear. In addi-
tion, most of the key mediators are not specific for pain pathways
and play diverse physiological roles.3 Therefore, for 40–60% of
patients, current medications for neuropathic pain either lack of
efficacy or are intolerable due to adverse effects.4
Ziconotide is a potent and selective N-type calcium channel
blocker and has been used for treatment of severe chronic pain.5
This drug is a 25-amino acid synthetic conopeptide and requires
intrathecal administration via an implanted pump due to lack of
oral availability. Of note, intrathecal administration is associated
with spinal meningitis due to device-related infection,5 and
from the blockade of N-type calcium channels that are distributed
throughout the central nervous system (CNS). To reduce the risk of
serious CNS adverse effects, the United States Food and Drug
Administration (US FDA) and European Medicines Evaluation
Agency (EMEA) have only approved a slow dosage titration regi-
men.5 Although ziconotide is clinically effective in treating chronic
pain as an N-type calcium channel blocker, medical problems of
intrathecal administration and unwanted CNS side effects limit
its clinical use.
N-type calcium channels are localized in the neurons and
expressed at a high level in the presynaptic terminals of neurons.
In the spinal cord, these channels are involved in the transmission
of nociceptive information.6 N-type calcium channels have at least
three different conformational states: closed, open, and inactivated.
With prolonged excitation of sensory neurons, the proportion of
N-type calcium channels in inactivated state increases. Targeting
the inactivated state of N-type calcium channels can preferentially
suppress hyperexcitability of sensory neurons in chronic pain and
minimize impact on physiological signal transmission. Therefore,
the selective blockade of N-type calcium channels in an inactivated
state might provide a wider therapeutic window in pain than that of
ziconotide, which has little state dependency.5b,6 Consequently, an
orally active and selective N-type calcium channel blocker might
⇑
Corresponding author. Tel.: +81 29 863 6683; fax: +81 29 852 5387.
E-mail address: takashi.ogiyama@astellas.com (T. Ogiyama).
http://dx.doi.org/10.1016/j.bmc.2014.10.020
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

6900
T. Ogiyama et al. / Bioorg. Med. Chem. 22 (2014) 6899–6907
F
OMe
OMe
OMe
:
:
hydrophobic residue
N
N
N
H
basic nitrogen function
N
OMe
O
: hydrophobic residue
NP078585 (2)
1a
F
Figure 1. Structures of compound 1a and NP078585 (2).
represent a novel analgesic agent without the disadvantages of
ziconotide. Recent studies have focused on identifying small-
molecule N-type calcium channel blockers.7–9
2. Chemistry
The synthesis of the tetrahydroisoquinoline derivatives 1a–1k
is outlined in Scheme 1. Condensation of the tetrahydroisoquino-
line 412 with chloroacetyl chloride under Schotten–Baumann reac-
tion condition afforded the chloroacetamide 5. Substitution of the
chloroacetamide 5 with various amines 6a–6c, 6e, 6g–6j then gave
compounds 1a–1c, 1e, 1g–1j. Amine 6j was prepared as previously
described.13 Cleavage of methylether of compound 1a gave com-
pound 1d. Hydrolysis of the methoxycarbonyl 1e gave compound
1f. Optically active compound (S)-1h was synthesized from (S)-tet-
rahydroisoquinoline 414 and (R)-1h from (R)-tetrahydroisoquino-
line 4 in a manner similar to that described for compound 1h.
Acylation of tetrahydroisoquinoline 4 with Boc protected glycine,
followed by deprotection of Boc group afforded the amine 7. An
oxirane ring opening reaction between the amine 7 and the oxirane
815 yielded compound 1k.
At the outset of our N-type calcium channel blocker study, we
identified compound 1a as a potent small-molecule N-type cal-
cium channel blocker from our in-house compound collection.
The structure of compound 1a is characterized by a novel tetrahy-
droisoquinoline scaffold and has some structural similarity with
the N-type calcium channel blocker (NP078585 2) reported by
Zalicus scientists,9 with both containing a basic nitrogen function
linked with hydrophobic residues on both sides (Fig. 1).
Inhibitory activity against N-type calcium channels of com-
pound 1a is comparable to that of NP078585 (2). However, both
compound 1a and NP078585 (2) exhibited potent inhibitory activ-
ities against human Ether-a-go-go Related Gene (hERG) channels
(Table 1). Blockade of the hERG channels can give rise to a pro-
longed QT interval that is strongly associated with life-threatening
cardiac arrhythmias.10 The structural overlap of these two com-
pounds with general Class III antiarrhythmic agents,11 which is
represented by a phenyl ring linked to a basic nitrogen function
through a 1- to 4-atom-long chain, might play a critical role in
the compound’s high affinity for the hERG channels.
As represented by in vitro profile of compound 3, Zalicus scien-
tists demonstrated that conversion of the amine of NP078585 (2)
to an amide and modification of aryl substituents on the benzyl
moiety of NP078585 (2) significantly decreased hERG channel inhi-
bition while retaining highly potent blockade of N-type calcium
channels.9a However, our in-house evaluation revealed that these
two compounds had low aqueous solubility in the Japanese Phar-
macopoeia 2nd fluid for the dissolution test (JP2 pH 6.8). Further,
compound 3 also exhibited low aqueous solubility in the Japanese
Pharmacopoeia 1st fluid for the dissolution test (JP1 pH 1.2). In
contrast, compound 1a not only exhibited excellent solubility in
both JP1 and JP2 but also showed promising properties for further
optimization, such as low molecular weight and a simple structure.
We therefore conducted a hit-to-lead program to identify novel
compounds derived from 1a. Here, we report the discovery of novel
tetrahydroisoquinoline derivatives as orally active small-molecule
N-type calcium channel blockers with attenuated blockade of
hERG channels.
3. Results and discussion
Inhibitory activity against N-type calcium channels was evalu-
ated using a fluorometric imaging plate reader (FLIPR) calcium
imaging assay of IMR-32 human neuroblastoma cells. Experiments
were conducted in the presence of nitrendipine to block endoge-
nous L-type calcium channels. Inhibitory activity against hERG
channels was determined via an Rb efflux assay of Chinese hamster
ovary (CHO) cells.
We first investigated the effect of substituents on the benzene
moiety of compound 1a (Table 2). Removal of the methoxy group
(1b) slightly attenuated N-type calcium channel inhibition,
although potent inhibitory activity against hERG channels was
retained. Incorporation of an o-dichloro substituent (1c) into a ben-
zene moiety led to a further 2-fold decrease in N-type calcium
channel inhibition compared with compound 1b while attenuating
the blockade of hERG channels. Replacement of the methoxy
group (1a) with hydroxyl group (1d) resulted in a 5-fold decrease
in N-type calcium channel blockade. The inhibitory activity of
compound 1d against hERG channels was comparable to that of
compound 1b. Compound 1e, which has a p-methoxy carbonyl
group, exhibited weak inhibitory activity against N-type calcium
Table 1
In vitro activity, solubility and MW of compound 1a, NP078585 (2) and 3
Compound
N-type FLIPR IC50
(l
M)
hERG IC50
(l
M)
Aqueous solubility JP1/JP2 (
l
M)
Molecular weight
1a
0.60
1.1
8.3
3.4
>100/>100
>100/<1
406
539
NP078585 (2)
F
tBu
O
O
OMe
tBu
N
N
0.43
>100
<1/<1
619
3
F

T. Ogiyama et al. / Bioorg. Med. Chem. 22 (2014) 6899–6907
6901
NH2
R
6a-6c, 6e,
6g-6j
NH
N
N
a
b
R
Cl
N
H
O
O
4
5
1a-1j
a R = o-methoxyphenylmethyl
b R = phenylmethyl
g R = cyclohexyl
h R = 1-hydroxycyclohexyl
i R = 1-hydroxycyclopentyl
j R = 1-hydroxycyclohexylmethyl
c
c R = o-dichlorophenylmethyl
d R = o-hydroxyphenylmethyl
e R = p-methoxylcarbonylphenylmethyl
f R = p-carboxylphenylmethyl
d
O
8
OH
N
N
e, f
g
NH2
N
H
4
O
O
7
1k
Scheme 1. Reagents and conditions: (a) chloroacetyl chloride, AcOEt, satd NaHCO3, rt, 1 h; (b) iPr2NEt, CHCl3, 60 °C, 2 d; (c) BBr3, CH2Cl2, ꢀ78 °C to 0 °C, 23 h; (d) 1 M aq
NaOH, MeOH, rt, 22 h; (e) [(tert-butoxycarbonyl)amino]acetic acid, pivaloyl chloride, 4-methylmorpholine, CH2Cl2, rt, 14 h; (f) 4 M HCl/AcOEt, AcOEt, 50 °C, 5 h; and (g) 2-
propanol, reflux, 15 h.
Table 2
Effect of phenylethyl substitutions on N-type calcium channel and hERG channel potency
N
N
N
H
N
H
N-type FLIPR
hERG IC50
inhibition at 10
(
l
M) or %
N-type FLIPR
hERG IC50 (lM) or %
O
O
IC50
(l
M)
lM
IC50
(lM)
inhibition at 10 lM
Compound
Compound
1a
1b
0.60
1.3
8.3
1d
1e
3.0
2.3
54.4%
71.7%
OH
OMe
CO2Me
CO2H
57.8%
Cl
1c
2.4
54.7
1f
>10
9.0%
Cl
channels, but retained high potency for hERG channels. Introduc-
tion of p-carboxylic acid (1f) to the benzene moiety resulted in a
loss of N-type calcium inhibition and also reducing the potency
for hERG channels. Taken together, these results demonstrated
no correlation between inhibitory activity against hERG channels
and the polarity of substituents on the benzene moiety. Further,
maintaining the potency for N-type calcium channel inhibition
while reducing the inhibitory activity against hERG channels via
modification of substituents on the benzene moiety was shown
to be relatively difficult.
Based on findings from this preliminary study and due to the
structural basis of drugs binding to hERG channels, we next consid-
ered a more rational approach that did not involve the incorpora-
tion of substituents into the benzene moiety.
Regarding hERG channels, Mitchenson et al. reported that many
blockers of sodium and calcium channels bound to residues in the
S6 domains.16a These authors demonstrated that interactions
between the aromatic residues F656 and Y652 of the hERG S6
domain and the drug molecules were crucial for high-affinity bind-
ing.16 They further proposed the disruption of interactions with the
aromatic residues of F656 and Y652 to help minimize hERG chan-
nel inhibition.16b
Given these previous findings, we hypothesized that compound
1a bound to residues in the S6 domains, which is similar to the
action of other sodium and calcium channel blockers, to display
high affinity for hERG channels. Given that basic nitrogen atoms
of typical hERG blockers are considered to be involved in the
cation–
p interaction with aromatic residues or electrostatic
interactions within the pore regions,17 the conversion of the basic
nitrogen atom of compound 1a might therefore benefit the atten-
uation of hERG inhibition. However, this nitrogen atom was found
to be indispensable for good solubility.18 We therefore converted

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T. Ogiyama et al. / Bioorg. Med. Chem. 22 (2014) 6899–6907
Table 3
Effect of aliphatic ring substitutions on potency for N-type calcium channels and selectivity over hERG channels.
N
N
O
N-type FLIPR
hERG/N-type
selectivity ratio
N-type FLIPR
hERG/N-type
selectivity ratio
H
hERG IC50
(
lM)
Compound
hERG IC50 (lM)
IC50
(lM)
IC50
(lM)
Compound
1a
0.60
8.3
13.8
(S)-1h
(R)-1h
1.0
1.5
98
76
98.0
50.7
OMe
1g
1h
1.3
26.0
46.1
20.0
59.9
OH
0.77
OH
OH
1i
2.8
2.6
78.6
50.1
28.1
19.6
1k
OH
1j
1.7
NT
NT
NT: not tested.
Table 4
Inhibitory activities of compound 1a and (S)-1h against five major CYP isoforms
Compound CYP1A2 IC50 M) CYP2C9 IC50
(
l
(
lM)
CYP2C19 IC50
(
lM)
CYP2D6 IC50
(
lM)
CYP3A4*Preincubation time
0 min (%)
30 min (%)
N
N
H
26
4.8
3.0
0.37
65
53
OMe
O
O
1a
OH
N
N
H
>50
>50
>50
0.60
87
67
(S)-1h
*
Residual activities (%) of HLM were evaluated using midazolam as a probe substrate. Details are described in Section 5.
the phenyl moiety of compound 1a to non-aromatic rings to elim-
inate the p–p interaction with the aromatic residues, an approach
which allowed us to install a hydroxyl moiety on the aliphatic ring
to impair hydrophobic interactions (Table 3).
potent inhibitory activity against N-type calcium channels com-
pared to compound 1a. Neutral oxygen function, such as the meth-
oxy group of 1a and the hydroxyl group of 1h, is involved in the
enhancement of potency for N-type calcium inhibition (1a vs 1b
and 1g vs 1h). In contrast, addition of a phenolic hydroxyl group
decreased N-type calcium channel blockade (1b vs 1d).
We also examined the influence of the size of aliphatic rings and
the length of linking chain between the cyclohexyl ring and basic
nitrogen on compound 1h. Cyclopentyl (1i) and cycloheptyl (1k)
also exhibited weak inhibitory activity against hERG channels.
However, both compounds were less potent as N-type calcium
channel blocker than compound 1h. Introduction of a longer link-
ing chain (1j) resulted in an over 2-fold decrease in N-type calcium
channel inhibition compared with compound 1h.
First, we examined the bioisosteric conversion of the phenyl-
ethyl group of compound 1a to a cyclohexylmethyl group.19
Compound 1g exhibited 3-fold attenuation of inhibitory activity
against hERG channels compared to compound 1a. Although this
conversion resulted in a slight decrease in N-type calcium channel
inhibition, compound 1g exhibited improved selectivity over hERG
channel inhibition. Further, the cyclohexylmethyl group was
effective regarding N-type calcium potency as a bioisostere of the
phenylethyl group (1b vs 1g).
We next investigated the effect of introducing a hydroxyl moi-
ety to the cyclohexyl ring of compound 1g. As expected, inhibitory
activity against hERG channels of compound 1h was nearly two-
fold less than that of compound 1g. Further, compound 1h retained
The 1-hydroxycyclohexyl derivative 1h exhibited highly
potent blockade of N-type calcium channels and was optimal for
selectivity over hERG channel inhibition. We therefore prepared

T. Ogiyama et al. / Bioorg. Med. Chem. 22 (2014) 6899–6907
6903
A
B
1.2
1.0
0.8
0.6
0.4
0.2
0.0
300
250
**
200
*
*
150
100
##
50
0
Normal Vehicle
3
10
30
Vehicle
30
100
(S)-1h (mg/kg, po)
(S)-1h (mg/kg, po)
Figure 2. (A) Effect of compound (S)-1h on mechanical allodynia in SNL model in rats. Each bar represents the mean SEM withdrawal threshold of the hindpaw (n = 8).
##P < 0.005, statistically significant compared with normal group (Student’s t-test). ⁄P < 0.05 and ⁄⁄P < 0.005, statistically significant compared with vehicle-treated group
(Dunnett’s test). Closed columns, operated side paw in drug- and vehicle-treated group. Open column, non-operated side paw in vehicle-treated group. (B) Effect of
compound (S)-1h on motor coordination using an accelerating rotarod test in rats. Each symbol represents individual data. Dunnett’s test was used to test for statistical
significance of differences between drug- and vehicle-treated groups. No significant differences between these groups were noted.
and characterized both enantiomers of compound 1h. Both
enantiomers exhibited potent inhibitory activity against N-type
calcium channels20 and exhibited weak inhibitory activity against
hERG channels. Notably, (S)-1h exhibited higher selectivity over
hERG channels than (R)-1h and was selected for further
characterization.
5. Experimental section
5.1. Chemistry
5.1.1. General
All reactions were carried out using commercially available
reagents and solvents without further purification. Column
chromatography was performed using a silica gel cartridge Shoko
Lead compound (S)-1h was assessed for the inhibitory activities
against five major cytochrome P450 isoforms (CYP1A2, 2C9, 2C19,
2D6 and 3A4) (Table 4). Compared with compound 1a, (S)-1h
exhibited significantly improved the inhibitory activities against
all isoforms, particularly CYP2C9 and CYP2C19. Together, these
results prompted us to investigate the in vivo efficacy of (S)-1h.
The analgesic effect of (S)-1h was evaluated using an L5/L6
spinal nerve ligation (SNL) model of neuropathic pain in rats
(Fig. 2A).21 (S)-1h significantly reduced mechanical allodynia with
oral administration from 3 to 30 mg/kg. Oral administration of
(S)-1h at 30 mg/kg resulted in maximum reversal of allodynia.
The effect of (S)-1h on motor coordination was assessed using
an accelerating rotarod test in rats (Fig. 2B). (S)-1h did not decrease
the time on the rod at doses up to 100 mg/kg p.o., indicating that
(S)-1h exerted in vivo analgesic effect at 3–30 mg/kg without
affecting motor coordination.
Scientific SI series on a Shoko Scientific Purif-a
2. 1H NMR spectra
were recorded on a JNM-EX400 spectrometer. Chemical shifts are
expressed in d units (ppm) using tetramethylsilane as an internal
standard. Abbreviations used for the signal patterns are as follows:
s, singlet; d, doublet; t, triplet; m, multiplet; and br, broad. Mass
spectra were recorded on a JEOL LX-2000 or Waters ZQ-2000 mass
spectrometer. Elemental analysis was conducted using a Yanaco
MT-5 microanalyzer. HPLC analysis was performed using a Daicel
OD-H chiral column on a Hitachi HPLC system (L-7000 series),
equipped with a UV source (210 nm), utilizing hexane/ethanol/
diethylamine as an eluent. Specific rotations were measured using
a HORIBA SEPA-300 polarimeter.
5.1.2. 2-(Chloroacetyl)-1-cyclohexyl-1,2,3,4-tetrahydroiso-
quinoline (5)
State-dependent inhibitory activity of (S)-1h against N-type
calcium channels was evaluated using whole cell patch-clamp
recordings at two different membrane potentials in IMR-32 human
neuroblastoma cells. Under conditions favoring the inactivated
state (at ꢀ70 mV), (S)-1h inhibited N-type calcium channels with
To a solution of 1-cyclohexyl-1,2,3,4-tetrahydroisoquinoline
hydrochloride (4) (14.63 g) in ethyl acetate (90 mL) and saturated
aqueous sodium bicarbonate (85 mL) was added dropwise a
solution of chloroacetyl chloride (5.95 mL) in ethyl acetate (10 mL)
under 4 °C, and the reaction mixture was stirred at room tempera-
ture for 1 h. The reaction mixture was diluted with water, extracted
with ethyl acetate, dried over magnesium sulfate and then concen-
trated in vacuo. The residue was washed with cyclohexane to give
the title compound 5 (19.50 g, 98%) as a white solid. 1H NMR
(400 MHz, CDCl3): this compound exists as a pair of rotamers at
room temperature. d 0.94–1.30 (5H, m), 1.51–1.92 (6H, m),
2.87–3.13 (2H, m), 3.33–3.43 (minor rotamer, 2H, m), 3.72–3.88
(2H, m), 4.05–4.19 (2H, m), 4.23 (minor rotamer 1H, d, J = 12.0 Hz),
4.32 (minor rotamer, 2H, d, J = 8.0 Hz), 4.34–4.43 (minor rotamer,
1H, m), 5.27 (major rotamer, 1H, J = 8.0 Hz), 7.04–7.08 (minor
rotamer, 1H, m), 7.09–7.23 (4H, m); FAB MS m/z 292 [M+H]+.
an IC50 value of 1.7
inhibition (IC50 = 12.7
l
M, whereas (S)-1h exhibited weaker
lM) under resting state-biased condition
(at ꢀ90 mV). These results indicate that (S)-1h is an inactivated
state selective N-type calcium channel blocker. This property of
inactivated state selective inhibition of (S)-1h might allow the
compound to exert an in vivo analgesic effect without affecting
motor coordination.
4. Conclusion
Here, we identified (S)-1h as a novel small-molecule N-type cal-
cium channel blocker with significantly reduced inhibitory activity
against hERG channels from optimization study of 1a. Attenuation
of hERG inhibition was achieved via conversion to an aliphatic ring
5.1.3. 1-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[2-
(2-methoxyphenyl)ethyl]amino}ethanone oxalate (1a)
To a solution of compound 5 (2.47 g) in chloroform (50 mL) was
added N,N-diisopropylethylamine (3.77 mL) and [2-(2-methoxy-
phenyl)ethyl]amine (6.40 g) and the mixture was heated under
(elimination of
p–p interaction) and incorporation of a hydroxyl
moiety (impairment of hydrophobic interaction). (S)-1h alleviated
neuropathic pain in a rat SNL model at doses of 3–30 mg/kg p.o.
with no affect on motor coordination. Further structural optimiza-
tion will be reported in due course.

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T. Ogiyama et al. / Bioorg. Med. Chem. 22 (2014) 6899–6907
reflux for 2 days. The reaction mixture was diluted with water,
extracted with chloroform, washed with brine, dried over magne-
sium sulfate and then concentrated in vacuo. The resulting residue
was purified by silica gel column chromatography, eluting with
chloroform–ethanol–aqueous ammonia to give a colorless oil
(2.98 g). The oil was dissolved in ethyl acetate (20 mL) and ethanol
(3 mL). Oxalic acid (660 mg) was then added to the solution. The
precipitate was filtered off to obtain the title compound 1a as a
colorless solid (3.37 g, 80%). 1H NMR (400 MHz, DMSO-d6): this
compound exists as a pair of rotamers at room temperature. d
0.98–1.20 (5H, m) 1.46–1.77 (6H, m), 2.82–3.20 (5H, m), 3.28–
3.40 (minor rotamer, 1H, m), 3.52–3.72 (major rotamer, 2H, m),
3.78 (3H, s), 3.94–4.14 (1H, m), 4.16–4.26 (1H, m), 4.30 (minor rot-
amer, 1H, d, J = 10.0 Hz), 5.11 (major rotamer, 1H, d, J = 9.2 Hz),
6.90 (1H, t, J = 8.0 Hz), 6.99 (1H, d, J = 7.6 Hz), 7.08–7.28 (6H, m);
FAB MS m/z 407 [M+H]+. Anal. Calcd for C26H34N2O2ꢁC2H2O4: C,
67.72; H, 7.31; N, 5.64. Found: C, 67.44; H, 7.35; N, 5.62.
5.1.8. 1-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-
hydroxycyclohexyl)methyl]amino}ethanone oxalate (1h)
Compound 1h was synthesized using a protocol similar to that
for compound 1a. Compound 1h was obtained in 70% yield as a pale
brown solid. 1H NMR (400 MHz, DMSO-d6): this compound exists as
a pair of rotamers at room temperature. d 0.92–1.80 (21H, m), 2.74–
3.07 (5H, m), 3.31–3.41 (minor rotamer, 1H, m), 3.51–3.63 (major
rotamer, 1H, m), 3.63–3.73 (major rotamer, 1H, m), 3.73–3.81
(minor rotamer, 1H, m), 3.92–4.04 (2H, m), 4.12 (1H, d, J = 1.6 Hz),
4.15–4.26 (minor rotamer, 1H, m), 4.32 (minor rotamer, 1H,
J = 8.0 Hz), 5.11 (major rotamer, 1H, d, J = 8.0 Hz), 7.08–7.28 (4H,
m); FAB MS m/z 385 [M+H]+. Anal. Calcd for C24H36N2O2ꢁC2H2O4:
C, 65.80; H, 8.07; N, 5.90. Found: C, 65.45; H, 8.18; N, 5.85.
5.1.9. (1S)-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-
{[(1-hydroxycyclohexyl)methyl]amino}ethanone oxalate (S)-1h
Compound (S)-1h was synthesized using a protocol similar to
that for compound 1h, except that (1S)-cyclohexyl-1,2,3,4-tetrahy-
droisoquinoline14 was used as a starting material. Compound (S)-
1h was obtained in 69% yield as a colorless solid.
HPLC (Chiralpak OD-H [0.46 cm I.D. ꢂ 15 cm], hexane/ethanol/
diethylamine = 80:20:0.1 flow rate 0.5 ml/min, column temp:
40 °C, UV: 210 nm): retention time: ts = 8.34 min (major),
tr = 9.73 min (minor); 98.4% ee. FAB MS m/z 385 [M+H]+. Anal.
5.1.4. 1-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-[(2-
phenylethyl)amino]ethanone oxalate (1b)
Compound 1b was synthesized using a protocol similar to that
for compound 1a. Compound 1b was obtained in 57% yield as a col-
orless solid. 1H NMR (400 MHz, DMSO-d6): this compound exists as
a pair of rotamers at room temperature. d 0.96–1.22 (5H, m), 1.54–
1.68 (6H, m), 2.80–3.05 (5H, m), 3.06–3.24 (2H, m), 3.28–3.42
(minor rotamer, 1H, m), 3.54–3.74 (major rotamer, 2H, m), 4.02–
4.12 (1H, m), 4.16–4.26 (1H, m), 4.29 (minor rotamer, 1H, d,
J = 9.3 Hz), 5.12 (major rotamer, 1H, d, J = 9.7 Hz), 7.12–7.28 (7H,
m), 7.29–7.38 (2H, m); FAB MS m/z 377 [M+H]+.
Calcd for C24H36N2O2ꢁC2H2O4: C, 65.80; H, 8.07; N, 5.90. Found: C,
26
65.75; H, 8.09; N, 5.90. [
a
]
+10.8° (c 0.1, MeOH).
D
5.1.10. (1R)-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-
{[(1-hydroxycyclohexyl)methyl]amino}ethanone oxalate (R)-1h
Compound (R)-1h was synthesized using a protocol similar to
that for compound 1h, except that (1R)-cyclohexyl-1,2,3,4-tetrahy-
droisoquinoline14 was used as a starting material. Compound (R)-
1h was obtained in 57% yield as a colorless solid.
HPLC (Chiralpak OD-H [0.46 cm I.D. ꢂ 15 cm], hexane/ethanol/
diethylamine = 80:20:0.1 flow rate 0.5 ml/min, column temp:
40 °C, UV: 210 nm): retention time: tr = 9.73 min (major),
ts = 8.34 min (minor); 98.3% ee. FAB MS m/z 385 [M+H]+. Anal.
5.1.5. 1-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[2-
(2,6-dichlorophenyl)ethyl]amino}ethanone oxalate (1c)
Compound 1c was synthesized using a protocol similar to that
for compound 1a. Compound 1c was obtained in 82% yield as a col-
orless solid. 1H NMR (400 MHz, DMSO-d6): this compound exists as
a pair of rotamers at room temperature. d 0.90–1.37 (5H, m), 1.41–
1.85 (6H, m), 2.74–5.01 (11H, m), 5.13 (major rotamer, 1H, d,
J = 8.0 Hz), 7.09–7.26 (4H, m), 7.34 (1H, dd, J = 7.6, 7.6 Hz), 7.44–
7.54 (2H, m); FAB MS m/z 445 [M+H]+.
Calcd for C24H36N2O2ꢁC2H2O4: C, 65.80; H, 8.07; N, 5.90. Found: C,
26
65.70; H, 8.11; N, 5.88. [
a]
ꢀ13.6° (c 0.1, MeOH).
D
5.1.11. 1-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-
{[(1-hydroxycyclopentyl)methyl]amino}ethanone oxalate (1i)
Compound 1i was synthesized using a protocol similar to that
for compound 1a. Compound 1i was obtained in 78% yield as a col-
orless solid. 1H NMR (400 MHz, DMSO-d6): this compound exists as
a pair of rotamers at room temperature. d 0.96–1.26 (5H, m), 1.46–
1.82 (14H, m), 2.80–3.10 (4H, m), 3.21–4.66 (6H, m), 5.11 (major
rotamer 1H, d, J = 8.0 Hz), 7.14–7.23 (4H, m); FAB MS m/z 371
[M+H]+.
5.1.6. Methyl 4-(2-{[2-(1-cyclohexyl-3,4-dihydroisoquinolin-
2(1H)-yl)-2-oxoethyl]amino}ethyl)benzoate oxalate (1e)
Compound 1e was synthesized using a protocol similar to that
for compound 1a. Compound 1e was obtained in 82% yield as a col-
orless solid. 1H NMR (400 MHz, DMSO-d6): this compound exists as
a pair of rotamers at room temperature. d 0.95–1.26 (5H, m), 1.46–
1.78 (6H, m), 2.80–3.27 (5H, m), 3.29–3.40 (minor rotamer, 1H, m),
3.53–3.74 (major rotamer, 2H, m), 3.84 (3H, s), 3.96–4.13 (1H, m),
4.15–4.32 (1H, m), 5.11 (major rotamer, 1H, d, J = 9.6 Hz), 7.09–
7.26 (4H, m), 7.40 (2H, d, J = 8.0 Hz), 7.92 (2H, d, J = 8.4 Hz); FAB
MS m/z 435 [M+H]+.
5.1.12. 1-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[2-
(1-hydroxycyclohexyl)ethyl]amino}ethanone oxalate (1j)
Compound 1j was synthesized using a protocol similar to that
for compound 1a. Compound 1j was obtained in 48% yield as a col-
orless solid. 1H NMR (400 MHz, DMSO-d6): this compound exists as
a pair of rotamers at room temperature. d 0.92–1.84 (25H, m),
2.74–3.11 (4H, m), 3.30–3.40 (major rotamer, 1H, m), 3.53–3.73
(major rotamer, 2H, m), 3.98–4.12 (1H, m), 4.14–4.26 (1H, m),
4.32 (minor rotamer, 1H, J = 9.4 Hz), 5.11 (major rotamer, 1H, d,
J = 9.3 Hz), 7.08–7.23 (4H, m); FAB MS m/z 400 [M+2H]+.
5.1.7. 1-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-
[(cyclohexylmethyl)amino]ethanone fumarate (1g)
Compound 1g was synthesized using a protocol similar to that
for compound 1a. Compound 1g was obtained in 54% yield as a col-
orless solid. 1H NMR (400 MHz, DMSO-d6): this compound exists as
a pair of rotamers at room temperature. d 0.79–1.27 (10H, m),
1.43–1.79 (12H, m), 2.44–2.60 (3H, m), 2.75–3.06 (2H, m), 3.22–
3.35 (minor rotamer, 1H, m), 3.53–3.79 (3H, m), 3.84 (major rot-
amer, 1H, d, J = 18.4 Hz), 4.15–4.28 (minor rotamer, 1H, m), 4.41
(minor rotamer, 1H, d, J = 9.2 Hz), 5.12 (major rotamer, 1H, d,
J = 10.4 Hz), 6.51 (2H, s, fumaric acid), 7.01–7.32 (4H, m); FAB MS
m/z 369 [M+H]+.
5.1.13. 1-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[2-
(2-hydroxyphenyl)ethyl]amino}ethanone oxalate (1d)
To a solution of compound 1a (449 mg) in dichloromethane
(15 mL) was added boron tribromide (6.6 ml of 1 M dichloromethane

T. Ogiyama et al. / Bioorg. Med. Chem. 22 (2014) 6899–6907
6905
solution) at ꢀ78 °C. The reaction mixture was stirred at 0 °C for 23 h.
Then the reaction was quenched with saturated aqueous sodium
bicarbonate. The mixture was diluted with water, extracted with
chloroform, dried over magnesium sulfate and then concentrated
in vacuo. The resulting residue was purified using silica gel column
chromatography, eluting with chloroform–methanol to give a
brown foam (439 mg). The foam was dissolved in ethanol (5 mL).
Oxalic acid (109 mg) was added to the solution. Precipitate was fil-
tered off to obtain the title compound 1d as a colorless solid
(229 mg, 43%). 1H NMR (400 MHz, DMSO-d6): this compound exists
as a pair of rotamers at room temperature. d 0.94–1.24 (5H, m),
1.44–1.78 (6H, m), 2.73–3.03 (5H, m), 3.24–3.36 (minor rotamer,
1H, m), 3.55–3.70 (major rotamer, 2H, m), 3.75–3.87 (1H, m), 3.94
(major rotamer, 1H, d, J = 16.8 Hz), 4.16–4.26 (minor 1H, m), 4.36
(minor rotamer, 1H, d, J = 9.6 Hz), 5.12 (major rotamer, 1H, d,
J = 9.2 Hz), 6.70 (1H, t, J = 7.2 Hz), 6.77 (1H, d, J = 7.2 Hz), 7.03 (2H,
t, J = 7.6 Hz), 7.12–7.26 (4H, m); FAB MS m/z 393 [M+H]+.
rotamer, 1H, d, J = 8.0 Hz), 7.00–7.05 (minor rotamer, 1H, m), 7.07–
7.25 (4H, m); ESI MS m/z 273 [M+H]+.
5.1.16. 1-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-
{[(1-hydroxycycloheptyl)methyl]amino}ethanone oxalate (1k)
To a solution compound 7 (200 mg) in 2-propanol (2 mL) was
added compound 815 (139 mg) and it was refluxed for 15 h. The
reaction solvent was concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography, eluting with chloro-
form–methanol to afford a pale brown oil (89 mg). The oil was
dissolved in 2-propanol (3 mL). Oxalic acid (22 mg) was added to
the solution at 60 °C. The mixture was evaporated to give the title
compound 1k (72.2 mg, 20%) as a pale brown solid. 1H NMR
(400 MHz, DMSO-d6): this compound exists as a pair of rotamers
at room temperature. d 1.05–1.73 (23H, m), 2.76–3.06 (4H, m),
3.08–3.92 (2H, m), 4.01 (1H, d, J = 16.0 Hz), 4.08–4.26 (1H, m),
4.30 (1H, d, J = 8.0 Hz), 5.11 (major rotamer 1H, d, J = 8.0 Hz),
7.16–7.23 (4H, m); FAB MS m/z 399 [M+H]+.
5.1.14. 4-(2-{[2-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-
yl)-2-oxoethyl]amino}ethyl)benzoic acid (1f)
5.2. Pharmacology
To a solution of compound 1e (298 mg) in methanol (10 mL)
was added 1 M aqueous sodium hydroxide (3.5 mL). The reaction
mixture was stirred at room temperature for 22 h. Then, 1 M aque-
ous hydrochloric acid (3.5 mL) was added to the reaction mixture.
The reaction mixture was diluted with water, extracted with chlo-
roform, washed with brine, dried over magnesium sulfate and then
concentrated in vacuo. The residue was washed with diisopropyl
ether and 2-propanol to give the title compound 1f (227 mg,
95%) 1H NMR (400 MHz, DMSO-d6): this compound exists as a pair
of rotamers at room temperature. d 0.96–1.20 (5H, m), 1.46–1.76
(6H, m), 2.78–2.99 (5H, m), 3.22–3.32 (minor 1H, m), 3.52–3.84
(4H, m), 4.15–4.25 (minor rotamer, 1H, m), 4.38 (minor rotamer,
1H, d, J = 9.2 Hz), 5.12 (major rotamer, 1H, d, J = 9.6 Hz), 7.10–
7.23 (4H, m), 7.28–7.35 (2H, m), 7.86 (2H, d, J = 8.0 Hz); FAB MS
m/z 421 [M+H]+.
5.2.1. Determination of solubility
The first fluid for disintegration test in Japanese Pharmacopoeia,
pH 1.2 (JP1) and the second fluid for disintegration test in Japanese
Pharmacopoeia, pH 6.8 (JP2) were used as the aqueous buffer.
Small volumes of the compounds in DMSO were diluted to
130 lM by adding the aqueous buffer. After incubation at 25 °C
for 20 h, precipitates were separated by filtration, and solubility
of each filtrate was determined by HPLC analysis.
5.2.2. Evaluation of inhibitory activity against N-type calcium
channel using an in vitro FLIPR assay in IMR-32 human
neuroblastoma cells
Human neuroblastoma IMR-32 cells were cultured and differ-
entiated as previously described with some modifications.22 IMR-
32 cells were plated onto poly-L-lysine-coated 96-well assay plates
5.1.15. 2-(1-Cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-
oxoethanamine (7)
at a density of 6 ꢂ 104 cells/well and incubated overnight (37 °C,
5% CO2). Hank’s balanced salt solution without phenol red and con-
taining 20 mM HEPES and 0.5 mM probenecid was prepared on the
day of assay and used as assay buffer. Cells were incubated with
To
a solution of [(tert-butoxycarbonyl)amino]acetic acid
(1.54 g) and 4-methylmorpholine (2.2 mL) in methylene chloride
(20 mL), was added pivaloyl chloride (0.98 mL) under 5 °C. The
reaction mixture was stirred at room temperature for 30 min and
then 1-cyclohexyl-1,2,3,4-tetrahydroisoquinoline hydrochloride
(4) (1.00 g) was added to the reaction mixture at 4 °C. The reaction
mixture was stirred at room temperature for 14 h. The reaction
mixture was diluted with water. The mixture was extracted with
chloroform, washed with brine, dried over magnesium sulfate
and then concentrated in vacuo. The residue was purified by col-
umn chromatography, eluting with chloroform–methanol to give
100
at 37 °C for 1 h. Plates were washed with wash buffer to remove
loading buffer, and 100 L/well of the wash buffer was added.
lL of fluo-3 AM (Dojindo, Kumamoto, Japan) in assay buffer
l
Plates were then placed into a FLIPR system (Molecular Devices,
Sunnyvale, CA). Intracellular calcium concentration was measured
for 12 min. During fluorescence intensity monitoring, 50
the compound, diluted with assay buffer containing 1 M nitrendi-
pine, was added during the first 30 s, and 50 L/well KCl solution
lL/well of
l
l
(final concentration: 50 mM) at 10 min and 10 s after the start of
monitoring. The IC50 values were determined by in duplicate in
one experiment. And the IC50 values and 95% confidence intervals
were calculated using Sigmoid-Emax nonlinear regression analysis
with SAS software (Cary, NC, USA).
tert-butyl
[2-(1-cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-
oxoethyl]carbamate (1.49 g 100%) as a colorless oil; ESI MS m/z
373 [M+H]+. To a solution of tert-butyl[2-(1-cyclohexyl-3,4-dihy-
droisoquinolin-2(1H)-yl)-2-oxoethyl]carbamate (1.49 g) in ethyl
acetate (20 mL) was added 4 M hydrogen chloride in ethyl acetate
(3 mL) under 5 °C. The reaction mixture was stirred at 50 °C for 5 h.
Then, the reaction solvent was concentrated in vacuo. Saturated
aqueous sodium bicarbonate was added to the residue and
extracted with chloroform. The extract was washed with brine
and dried over magnesium sulfate. It was concentrated in vacuo
and the resulting residue was purified by silica gel column chroma-
tography, eluting with chloroform–methanol to give the title com-
pound 7 (1.09 g, 99%) as a pale yellow oil. 1H NMR (400 MHz,
DMSO-d6): this compound exists as a pair of rotamers at room
temperature. d 0.92–1.29 (5H, m), 1.50–1.96 (6H, m), 2.23 (2H, br
s), 2.82–3.12 (2H, m), 3.29–3.71 (4H, m), 4.42–4.51 (minor rot-
amer, 1H, m), 4.18 (minor rotamer, 1H, d, J = 12.0 Hz), 5.29 (major
5.2.3. Evaluation of hERG inhibition using Rb Efflux assay
CHO cells that stably expressed hERG channels cultured in D-
MEM containing 10% FBS, 1% PS, 1% Geneticin. Cell suspension
was diluted to 6 ꢂ 105 cells/ml. Cells were seeded into 96-well
plates and cultured in CO2 incubator for 24 h. Culture medium
was removed and cells were washed with wash buffer (130 mM
NaCl, 3 mM KCl, 0.6 mM MgCl2, 0.3 mM CaCl2, 10 mM HEPES,
10 mM
(130 mM NaCl, 3 mM RbCl, 0.6 mM MgCl2, 0.3 mM CaCl2, 10 mM
HEPES, 10 mM -glucose: pH 7.3) was added. Cells were incubated
D-glucose: pH 7.3). Thereafter 100 lL of Rb loading buffer
D
in CO2 incubator for 1.5 h. Excess Rb-containing media was
aspirated off and washed with wash buffer. Test compounds were

6906
T. Ogiyama et al. / Bioorg. Med. Chem. 22 (2014) 6899–6907
carried in high K buffer (83 mM NaCl, 50 mM KCl, 0.6 mM MgCl2,
0.3 mM CaCl2, 10 mM HEPES, 10 mM -glucose: pH 7.3), and then
100 L of the high K buffer was added to the plate. Cells were incu-
bated at room temperature for 10 min. Supernatants were col-
lected and transferred into another well (A). Cells were washed
5.2.5. Animal experiments
D
Male Sprague–Dawley rats (SLC, Hamamatsu, Japan) were used
for all in vivo experiments. Animals were group-housed and kept
on a 12-h light/dark cycle (lights on from 7:30 AM to 7:30 PM)
with free access to food and water. All animal experimental proce-
dures were approved by the Committee for Animal Experiments of
Astellas Pharma Inc. and conformed to the International Guiding
Principles for Biomedical Research Involving Animals (CIOMS)
and Guidelines for Proper Conduct of Animal Experiments (Science
Council of Japan, 2006). All efforts were made to minimize the
number of animals used and their suffering.
l
with the wash buffer and treated with 200 lL of lysis buffer (1%
Triton-X 100 in wash buffer) to give cell lysate (B). Rb content in
the supernatans (A) and the cell lysate (B) were determined using
an atomic absorption spectroscopy reader (ICR 8000, Aurora Bio-
sciences, Canada).
The efficacy of compound (S)-1h in neuropathic pain was eval-
uated in rat SNL model reported by Chung et al.21 Effects of com-
pound (S)-1h on motor coordination was assessed by rotarod
test. The solution of (S)-1h in 10% dimethyl sulfoxide and 10%
Cremophol was used in both test. Details of the experimental
procedures have been previously described.23
5.2.4. Measurement of CYP inhibition
The inhibitory activities of test compounds against CYP1A2,
2C9, 2C19 and 2D6 were determined using fluorescence-based
assay. Reaction mixtures containing recombinant human CYP pro-
tein, co-factors, fluorogenic substrate, test compound (0.8–50 lM)
and potassium phosphate buffer (pH 7.4) were prepared. Fluoro-
genic substrates and assay conditions for different CYP isoforms
are summarized in Table 5. Reactions were initiated by incubation
at 37 °C. Incubation times for each isoform was as follows: 15 min
(CYP1A2), 45 min (2C9), 30 min (2C19) and 30 min (2D6). After
incubation, the reactions were terminated by addition of stop solu-
tion (20% 0.5 M Tris base [2-amino-2-hydroxymethyl-1,3-propane-
diol], 80% acetonitrile). Fluorescence was measured to quantify the
metabolite formation, and IC50 determined.
For CYP3A4 inhibition assay, midazolam was used as a probe
substrate to monitor the changes in CYP3A4 activity during the
exposure to each test compound. 0.1 mg protein/mL reaction mix-
tures containing human liver microsomes (HLM), 1 mM NADPH,
0.1 mM EDTA, 100 mM Na+–K+ phosphate buffer (pH 7.4) and
5.2.6. The inhibitory effects of (S)-1h on N-type calcium
channels using the electrophysiological method
For electrical recording, differentiated IMR-32 cells were plated
on poly-D-lysine coated glass coverslips at a density of approxi-
mately 1.5 ꢂ 104 cells/cm2 and incubated for at least 3 h prior to
electrophysiological recording.24 Whole-cell patch clamp record-
ings were obtained from differentiated IMR-32 cells using an Axo-
patch 1D amplifier (Axon Instruments, Foster City, CA, USA).
Recording pipettes were constructed from borosilicate glasses
(Harvard Apparatus Ltd, Fircroft Way, Edenbridge, UK) with a mic-
ropipettes puller (MF-83, Narishige, Tokyo, Japan) and fire-pol-
ished in a microforge (PP-83, Narishige, Tokyo, Japan) to obtain
5
lM test compound were prepared and pre-incubated for 0 or
electrode resistances ranging from 4 to 6 M
X. Pipette solution
30 min at 37 °C. Reactions were initiated by the addition of 2
l
M
contained 125 mM CsCl, 2 mM MgCl2, 10 mM HEPES, 10 mM EGTA,
4 mM MgATP, and 4 mM phosphocreatine disodium, pH 7.3 with
CsOH. Internal cesium chloride was used to suppress potassium
currents. Cells were perfused continuously with standard bath
solution at room temperature (20–25 °C) containing 120 mM NaCl,
20 mM tetraethyl ammonium chloride, 3 mM KCl, 5 mM CaCl2,
2 mM MgCl2, 10 mM HEPES, and 10 mM glucose, pH 7.3 with
NaOH. To isolate barium currents for whole-cell recording, the
standard bath solution was replaced with a Ba2+-containing solu-
tion which contained 120 mM NaCl, 20 mM tetraethyl ammonium
chloride, 3 mM KCl, 10 mM BaCl2, 2 mM MgCl2, 10 mM HEPES,
of midazolam and incubated for additional 20 min before being
terminated by addition of 80% acetonitrile with internal standard.
Levels of the metabolite of midazolam, 10-hydroxymidazolam,
were measured using LC–MS/MS. Residual metabolic activities
for reversible (Eq. 1) and time-dependent (Eq. 2) inhibition were
calculated using the following equations:
ꢀ
%Residual Activity ¼ Activitycompound;0min Activityvehicle;0min ꢂ100
ð1Þ
10 mM glucose, 2 lM tetrodotoxin, and 1 lM nitrendipine, pH
À
ꢀ
Á
%ResidualActivity¼ Activitycompound;30min Activityvehicle;30min
7.3 with NaOH. Tetrodotoxin was used to block sodium currents
and nitrendipine to block L-type calcium currents, respectively.
Membrane currents were elicited by 100 ms voltage steps from
the holding potential (VH) of ꢀ70 or ꢀ90 mV to +10 mV. Test pulses
were delivered at 30-s intervals to allow recovery from inactiva-
tion. N-type calcium currents were isolated by subtracting
ꢀÀ
ꢀ
Á
Activitycompound;0min Activityvehicle;0min ꢂ100
ð2Þ
where Activitycompound, 0 min denotes activity obtained in the pres-
ence of compound and without pre-incubation, Activityvehicle, 0 min
denotes activity obtained in the absence of compound and without
pre-incubation, Activitycompound, 30 min denotes that obtained in the
presence of compound and with pre-incubation, and Activityvehicle,
ziconotide (1 lM)-insensitive currents.
A multiple-barrel perfusion system was employed to achieve a
rapid exchange of bath solutions. The barrels of the perfusion sys-
tem were directly connected to syringes containing the control and
test solutions. The flow rate of extracellular solutions was set at
0.5–1 mL/min. Data were filtered at 2 kHz with a 4-pole Bessel fil-
ter, digitized at 50 kHz, and stored on compact disk for off-line
analysis using a Digidata 1322A analog/digital interface along with
the pClamp 8.0 software (Axon Instruments).
denotes that obtained in the absence of compound and with
30 min
pre-incubation.
Table 5
Summary of assay conditions for different CYP450 enzymes
CYP isoform Protein conc. (pmol/mL) Substrate Substrate concd (lM)
Acknowledgments
1A2
2C9
2C19
2D6
0.06
1.16
0.60
1.88
CEC
MFC
CEC
5
75
25
1.5
We thank Dr. Tatsuya Niimi for his valuable comments regard-
ing the structural basis of drug binding to hERG channels. We also
thank Drs. Kenichi Onda, Shinya Nagashima and Susumu Watanuki
for their advice and careful reading of the manuscript. In addition,
AMMC
CEC, 3-cyano-7-ethoxycoumarin; MFC, 7-methoxy-4-trifluoromethylcoumarin;
AMMC, 3-[2-(N,N-diethyl-N-methyl amino)-ethyl]-7-methoxy-4-methylcoumarin.

T. Ogiyama et al. / Bioorg. Med. Chem. 22 (2014) 6899–6907
6907
Lett. 2013, 4, 1064; (h) Winters, M. P.; Subashinghe, N.; Wall, M.; Beck, E.;
Brandt, M. R.; Finley, M. F. A.; Liu, Y.; Lubin, M. L.; Neeper, M. P.; Qin, N.; Flores,
C. M.; Sui, Z. Bioorg. Med. Chem. Lett. 2014, 24, 2053; (i) Winters, M. P.;
Subashinghe, N.; Wall, M.; Beck, E.; Brandt, M. R.; Finley, M. F. A.; Liu, Y.; Lubin,
M. L.; Neeper, M. P.; Qin, N.; Flores, C. M.; Sui, Z. Bioorg. Med. Chem. Lett. 2014,
24, 2057.
we thank the staff of the Division of Analytical Science Laboratories
for conducting elemental analyses and spectral measurements.
References and notes
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