Structure-activity relationships of trimethoxybenzyl piperazine N-type calcium channel inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.04.054

We previously reported the small organic N-type calcium channel blocker NP078585 that while efficacious in animal models for pain, exhibited modest L-type calcium channel selectivity and substantial off-target inhibition against the hERG potassium channel

Full text of DOI:10.1016/j.bmcl.2012.04.054

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4153–4158
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationships of trimethoxybenzyl piperazine N-type
calcium channel inhibitors
Hassan Pajouhesh a, Zhong-Ping Feng a,b, , Lingyun Zhang a, Hossein Pajouhesh a, Xinpo Jiang a,à
,
Adam Hendricson a,§, Haiheng Dong a,–, Elizabeth Tringham a, Yanbing Ding a,k, Todd W. Vanderah c,
Frank Porreca c, Francesco Belardetti a, Gerald W. Zamponi b, Lester A. Mitscher d, Terrance P. Snutch e,
⇑
a Zalicus Pharmaceuticals Ltd, 301-2389 Health Sciences Mall, Vancouver, BC, Canada
b Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, Canada
c Department of Pharmacology, University of Arizona, Tucson, Arizona, USA
d Department of Medicinal Chemistry, University of Kansas, Lawrence, KS, USA
e Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 December 2011
Revised 9 April 2012
Accepted 10 April 2012
Available online 19 April 2012
We previously reported the small organic N-type calcium channel blocker NP078585 that while effica-
cious in animal models for pain, exhibited modest L-type calcium channel selectivity and substantial
off-target inhibition against the hERG potassium channel. Structure–activity studies to optimize
NP078585 preclinical properties resulted in compound 16, which maintained high potency for N-type
calcium channel blockade, and possessed excellent selectivity over the hERG (ꢀ120-fold) and L-type
(ꢀ3600-fold) channels. Compound 16 shows significant anti-hyperalgesic activity in the spinal nerve
ligation model of neuropathic pain and is also efficacious in the rat formalin model of inflammatory pain.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
N-Type calcium channel
Trimethoxybenzyl piperazine
Pain
hERG potassium channel
L-Type calcium channel
Calcium channels are the primary route of translating electrical
signals into biochemical events that underlie key processes such as
neurotransmitter release, cell excitability and gene expression.1
Calcium channels are classified into five pharmacological and
physiological subtypes: L-, N-, P/Q-, R- and T-types. Among these,
the N-, P/Q- and R-type channels have all been shown to play
key roles in neurotransmitter release.2 In particular, N-type cal-
cium channels are located at the presynaptic terminals of a subset
of spinal cord neurons where they mediate transmission of pain
signals from the periphery to the central nervous system. The block
of N-type channels with peptide agents has been shown to play a
significant role in the pathophysiological processes of inflamma-
tory and neuropathic pain.3
F
N
N
O
O
O
F
Figure 1. Chemical structure of NP078585.
Previously, we have reported NP078585 (Fig. 1) as an N-type
calcium channel blocker with good in vivo efficacy in the rat for-
malin model of inflammatory pain following oral administration.4
NP078585 is a potent (IC50 = 0.11
channel blocker with moderate selectivity over the L-type calcium
channel (IC50 = 2.8 M). A potential development concern is that
NP078585 also blocked the hERG potassium channel
(IC50 = 0.4 M).
lM) and CNS penetrant N-type
l
⇑
Corresponding author. Tel.: +1 604 822 6968; fax: +1 604 822 6470.
E-mail address: snutch@msl.ubc.ca (T.P. Snutch).
Present address: Department of Physiology and Biophysics, University of Toronto,
Toronto, Ontario, Canada.
l
In order to further optimize the development of orally available
N-type blockers, a major goal was to minimize hERG channel inhi-
bition compared to NP078585. In this regard, in the current study
we assessed; (i) modification of aryl substituents on the benzyl
moiety bound to the piperazine core of NP078585, (ii) conversion
of amines to amides, (iii) aliphatic substitutions on the piperazine
à
Present address: Genscript Technology Co., Ltd, 78 Shuangbaixiang, Xiaolingwei,
Xuanwu, Nanjing, Jiangsu, China.
§
Present address: Bristol-Myers Squibb, 345 Park Avenue, New York, USA.
Present address: WuXi AppTec Co. Ltd, 288 Fute Zhong Road, Shanghai, China.
Present address: Shanghai Chempartner Co. Ltd, Pudong New Area, Shanghai,
–
k
China.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.054

4154
H. Pajouhesh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4153–4158
iv
N-type affinities compared to the parent compound NP078585.
The 3,5-di-t-butyl analogs (compounds 11–12) also maintained
high affinity N-type inhibition with IC50 values of 0.14–0.3 M.
Compared to NP078585, the addition of a 4-methoxy substituent
(compound 10) slightly improved affinity (ꢀ0.09 M) and addi-
tionally dramatically improved selectivity both over the L-type
channel to greater than 900-fold (IC50 ꢀ81 M, not shown) and
the hERG channel by approximately 100-fold (IC50 ꢀ9 M). From
ii
ii
l
l
F
Y
X
N
N
l
R1
R2
l
R4
R4
i
this initial series, we determined that 3,5-substitution of the phe-
nyl ring was preferred. Further, the incorporation of a hydrogen
bond acceptor (–OCH3) as in the case of compound 10 increased
the selectivity for the N-type over the L-type and hERG targets.
In general, the amine-linked analogues exhibited higher affinity
for hERG as compared to the amide counterparts.
R3
F
iii
Figure 2. SAR studies focused on: (i) modification of R1. R2 and R3 substituents, (ii)
changing the amine into amide, (iii) aliphatic substitution on the piperazine core,
and (iv) replacement of piperiazine core with 3-aminomethyl-pyrrolidine.
Using aryl substituents (R1 = t-butyl, R2 = OCH3, R3 = t-butyl)
and an amide-linked functional group, we further explored substi-
tutions off the piperazine ring with aliphatic groups. Table 2 shows
that S-Me substitution was tolerated off the piperazine ring adja-
cent to the linker (compound 15) although resulted in high affinity
core, and (iv) replacement of the piperazine core with 3-amino-
methyl-pyrrolidine (Fig. 2).
Structure–activity relationship studies initially examined a
range of alternative substitution at the trimethoxybenzyl portion
of NP078585 with the aim of identifying a series with improved
in vitro channel selectivity profiles and with a reduction of hERG
inhibition. Compounds 1–14 were prepared from intermediates A
and B according to the general synthesis5 outlined in Scheme 1.
The structure–activity studies of this chemical series were
guided by a whole cell manual patch clamp electrophysiology as-
say for the N-type and L-type calcium channel with further assess-
ment for hERG activity.6 The replacement of R1 and R3 groups on
the benzyl region of parent compound NP078585 with a t-butyl
group (Table 1), in general resulted in maintaining good N-type
channel blocking affinity for most compounds. This region of the
molecule appears to be fairly accommodating, accepting a t-butyl
group in 3 and 5 positions being the most optimal substituents,
such as compounds 3–12, all of which showed better or similar
hERG channel blockade (IC50 ꢀ0.9
lM). Contrastingly, S-Me substi-
tution adjacent to the benzoyl linker (compound 16) resulted in
excellent N-type channel blockade and approximately 3600-fold
selectivity over the L-type channel. In addition, compound 16
maintained the favorable hERG channel block of the parent com-
pound 10 (IC50 = 4.9 lM). Due to its combined N-type affinity
and excellent selectivity profile compound 16 was progressed into
in vivo anti-nociception assessment.
Further optimization of NP078585 was attempted by replace-
ment of the piperazine core with 3-aminomethyl-pyrrolidine. We
hypothesized that 3-aminomethyl-pyrrolidine could be a potential
bioisostere for the piperazine core due to the relatively planar con-
formation of the pyrrolidine ring in combination with the flexible
3-aminomethyl group. To test this, we synthesized a series of com-
pounds containing amine and amide groups, in combination with a
F
O
N
N
R1
R2
R3
F
c
F
F
O
F
O
O
N
N
N
N
a
b
N
Boc
N
R1
R1
R2
R3
R2
R3
F
F
F
A
O
F
N
Boc
N
e
d
N
Boc
HN
N
N
R1
R1
R2
R3
R2
R3
F
B
Scheme 1. Reagents and conditions: (a) (i) TFA, CH2Cl2, (ii) benzoic acid analogs, EDC, DMAP, CH2Cl2; (b) LiAlH4. THF; (c) (i) LiAlH4. THF, (ii) TFA, CH2Cl2, (iii) benzoic acid
analogs, EDC, DMAP, CH2Cl2; (d) (i) benzoic acid analogs, EDC, DMAP,CH2Cl2, (ii) LiAlH4. THF; (e) (i) TFA, CH2Cl2, (ii) 6,6-bis(4-fluorophenyl)hexanoic acid, EDC, DMAP, CH2Cl2.

H. Pajouhesh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4153–4158
4155
Table 1
Blocking activities of compounds 1–14 against the N-type calcium channel and the hERG potassium channel
F
y
X
N
N
R1
R2
R3
R3
F
Compound
R1
R2
X
Y
N-type Est. IC50
(l
M)
hERG Est. IC50 (lM)
NP078585
1
2
3
4
5
6
7
OCH3
OCH3
OCH3
OCH3
OH
OH
OH
OH
OCH3
OCH3
OCH3
H
H
O
H
O
H
O
H
O
H
O
H
O
H
O
H
H
O
H
H
O
O
H
H
O
O
H
O
H
O
0.11 ( 0.03)
0.4 ( 0.2)
0.4
ND
ND
0.1
0.25
ND
ND
0.1
0.66
0.13
9.0
0.6 ( 0.39)
0.04 ( 0.03)
0.15 ( 0.08)
0.18 ( 0.12)
0.22 ( 0.11)
0.09 (0.04)
0.11 ( 0.02)
0.1 ( 0.03)
0.09 ( 0.05)
0.14 ( 0.08)
0.3 ( 0.25)
1.1 ( 1)
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
CF3
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
CF3
OH
OH
OCH3
OCH3
OCH3
OCH3
H
H
H
H
8
9
10
11
12
13
14
0.1
ND
ND
ND
CF3
CF3
0.51( 0.28)
Table 2
Blocking activities of compounds 15–16
Compound
Structure
N-type Est. IC50
(
lM)
L-type Est. IC50
(
lM)
hERG Est. IC50
(l
M)
F
O
O
N
N
15
0.01 (n = 2)
ND
0.9
O
F
F
O
O
N
N
16
0.04 ( 0.03)
144
4.9
O
F
3-aminomethyl-pyrrolidine scaffold with known structural
elements of NP078585 and compound 10.
20–21) did not improve activity concerning N-type blockade and
M) exhibited
further, the best of these (compound 20, IC50 ꢀ0.2
l
Initial efforts in this study focused on analogs wherein substitu-
tions on the aryl group were varied (Table 3). The synthesis of
these compounds is as described in Scheme 2 and is illustrative
of the general procedure. Briefly, readily available 1-benzyl-pyrr-
olidin-3-ylmethyl- carbamic acid tert-butyl ester was debenzylat-
ed via catalytic hydrogenation followed by coupling with
6,6-bis(4-fluorophenyl)hexanoic acid mediated by EDC and DMAP
to provide the desired intermediate. Subsequent deprotection of
the Boc group and reaction with the appropriate benzoic acid pro-
vided desired compounds. Reduction of the di-amide moiety with
LiAlH4 proceeded smoothly to afford di-amines compounds.7
Compounds (17–28) generally exhibited highly favorable N-
a poor hERG profile. Comparing amine-linked compounds 23–27
to their amide counterparts (compounds 17–21), in general the
di-amine linked analogues possessed higher affinity for both the
N-type calcium channel and the hERG potassium channel as com-
pared to their di-amide counterparts.
The stereogenic center introduced by the pyrrolidine ring ap-
peared to have little effect on N-type calcium channel activity with
the R:S IC50 values approaching unity (Table 4). These observations
would seem to be one of the many exceptions to Pfeiffer’s rule,
which states that the higher the activity of a eutomer, the higher
the separation in activities between eutomer and distomer.8
We further then focused on understanding the effect of replac-
ing one phenyl group on the benzhydryl with piperidine (Scheme
3). The introduction of the N-piperidinyl moiety (compounds 35–
37) resulted in analogs with reduced N-type affinity relative to
the corresponding phenyl analogs (see Table 5).
type channel inhibition (IC50 values ꢀ0.04–0.9
lM). Of these, com-
pounds 18–19 and 26–27 containing bulky substituents (t-butyl)
on the 3,5-position of the aryl group showed improved N-type
channel inhibition compared to NP078585. Of note, compound 19
with the highest N-type affinity also showed excellent selectivity
over the L-type calcium channel (ꢀ750-fold; not shown). Extending
the substitution of the phenyl ring to trifluoromethyl (compounds
Based on the encouraging results associated with compound 19,
we investigated whether swapping the ‘left hand’ and ‘right hand’
side moieties attached to the 3-amino(methyl) pyrrolidine core

4156
H. Pajouhesh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4153–4158
Table 3
Blocking activities of compounds 17–27 against the N-type calcium channel and the hERG potassium channel
R1
F
X
Y
R2
N
H
N
R3
F
Compound
R1
R2
R3
X
Y
N-type Est. IC50
(lM)
hERG Est. IC50 (lM)
17
18
19
20
21
22
23
24
25
26
27
H
t-Butyl
H
OCH3
H
H
O
O
O
O
O
O
O
O
H
H
H
O
O
O
O
O
H
H
H
H
H
H
0.08 ( 0.04)
0.11 ( 0.06)
0.04 (n = 2)
0.21 ( 0.15)
0.75 ( 1.24)
0.09 (n = 2)
0.28 ( 0.18)
0.15 (n = 2)
0.06 ( 0.04)
0.05 ( 0.03)
0.03 (n = 2)
0.82
2.8
1.9
0.7
ND
0.1
0.1
0.1
t-Butyl
t-Butyl
CF3
t-Butyl
t-Butyl
CF3
CF3
H
F
t-Butyl
OCH3
CF3
OCH3
t-Butyl
t-Butyl
OCH3
OCH3
H
OCH3
OH
t-Butyl
OCH3
CF3
OCH3
t-Butyl
t-Butyl
0.04
0.87
0.46
H
R2
O
O
O
O
O
O
HN
N
O
HN
HN
HN
a
F
F
b
c
F
F
N
N
4
N
H
O
O
4
17-24
d
e
R2
R2
O
HN
HN
F
F
F
N
4
N
4
28-31
25-27
F
Scheme 2. Reagents and conditions: (a) Pd/C, H2, MeOH; (b) 6,6-bis(4-fluorophenyl)hexanoic acid or 6-(1-methylpiperidin-4-yl)-6-phenylhexanoic acid, EDC, DMAP, CH2Cl2;
(c) (i) TFA, CH2Cl2, (ii) benzoic acid analogs, EDC, DMAP, CH2Cl2; (d) LiAlH4ꢁTHF; (e) (i) LiAlH4ꢁTHF, (ii) TFA, CH2Cl2, (iii) benzoic acid analogs, EDC, DMAP, CH2Cl2.
(Table 6). The change proved to be well tolerated and yielded one
analogue with excellent affinity for N-type calcium channel (com-
16 exhibited significant antinociceptive activity (ꢀ50–60% of
vehicle-treated group) in phase II and phase IIA of the formalin
response (Table 7).
pound 39, IC50 ꢀ0.01
lM). Unfortunately, these modifications did
not offer any advantages concerning hERG selectivity over com-
Compounds 10 and 16 were also evaluated for activity in the
Chung model of neuropathic pain10 with two behavioral endpoints
used: mechanical allodynia11 and thermal hyperalgesia.12 Animals
were administered a single 30 mg/kg oral dose of each compound
and were tested at 30 min intervals during a 2–3 h test period
beginning 30 min after test compound administration. One-way
ANOVA followed by the Bonferroni test were employed on the ori-
ginal raw data (not normalized) to establish the statistical signifi-
cance of the differences observed with the post SNL-baseline
value. Compounds 10 and 16 both demonstrated efficacious
pound 20.
Of the compounds synthesized and evaluated, compounds 10
and 16 exhibited the most promising overall in vitro target pro-
files; N-type blocking IC50s ꢀ0.04–0.09 uM and excellent selectiv-
ity over both the L-type calcium channel (ꢀ900- to 3600-fold)
and hERG potassium channel (ꢀ 100- to 122-fold). Reflective of
these combined properties, compounds 10 and 16 were selected
for in vivo assessment in the rat formalin model of inflammatory
pain.9 Upon intraperitoneal (ip) administration only compound

H. Pajouhesh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4153–4158
4157
Table 4
The effect of chirality for compounds 28–33 on the N-type and hERG blocking activities
R1
F
O
O
R2
N
H
N
R3
F
Compound
Stereochemistry
R1
R2
R3
N-type Est. IC50
(lM)
hERG Est. IC50 (lM)
28
29
30
31
32
33
R
S
R
S
R
S
t-Butyl
t-Butyl
t-Butyl
t-Butyl
CF3
H
H
OCH3
OCH3
H
t-Butyl
t-Butyl
t-Butyl
t-Butyl
CF3
0.16 ( 0.18)
0.12 ( 0.03)
0.15 ( 0.02)
0.36 ( 0.2)
0.1 ( 0.02)
0.09 ( 0.03)
5.0
2.0
5.0
2.0
0.75
0.8
CF3
H
CF3
O
Table 7
CN
Efficacy of compounds 10 and 16 in the rat formalin model of acute inflammatory
pain
b
a
+
CN
CHO
N
Compound
% Of vehicle response
Phase II
N
N
Phase I
Phase IIA
67
50 12⁄⁄
c
10
16
79
79
6
9
80
7
5
61 9⁄⁄⁄
Compounds, prepared in propylene glycol, were administered at a dose of 30 mg/kg
ip at 3 mL/kg 30 min prior to intraplantar injection of formalin into the hindpaw.
Data are the mean percent of vehicle effect (MPE) SEM, n = 5 animals tested for
compound 10 and n = 6 animals for compound 16. Data were analyzed for signifi-
cance using a one-way ANOVA to vehicle-treated group. ⁄⁄P <0.01; ⁄⁄⁄P <0.001
O
O
d
OH
OH
N
N
Scheme 3. Reagents and conditions: (a) NaOMe, MeOH; (b) Dibal, CH2Cl2; (c)
LiHMDS, THF; (d) Pd/C, H2, 50 Psi, MeOH.
inhibition of nerve ligation-induced thermal hyperalgesia (ranging
from ꢀ54% to 86% maximal antinociception; Table 8). Contrasting-
ly, only compound 16 exhibited discernible antiallodynic effects in
vivo. This may reflect distinct underlying pathophysiological
mechanisms contributing to these two pain behavioral endpoints
together with any as yet-to-be determined unique state-dependent
target and/or off-target activities related to compounds 10 and 16.
In summary, we have described a series of piperazine and 3-
amino(methyl)pyrrrolidine analogues, some of which display both
excellent potency as N-type calcium channel antagonists and pos-
sess favorable selectivity over the hERG potassium channel. Among
these, compound 16 exhibited good selectivity over the L-type cal-
cium channel and showed analgesic activity in both inflammatory
and neuropathic rat pain models. The development of N-type
calcium channel blockers for eventual clinical usage will require
optimization of multiple parameters, including high affinity and
selectivity for the N-type target, suitable pharmacokinetic charac-
Table 5
N-type calcium channel blocking activities for compounds 34–37
R1
O
O
N
R2
N
H
N
R3
Compound
R1
R2
R3
N-type Est. IC50 (lM)
34
35
36
37
OCH3
Isopropyl
CH3
OCH3
OCH3
OCH3
H
OCH3
Isopropyl
CH3
3.2 ( 2.8)
1.9 ( 2.2)
0.1 ( 0.02)
0.88 ( 0.33)
Cl
Cl
Table 6
N-type calcium channel and hERG potassium channel blocking activities for compounds 38–42
F
X
Y
R3
R2
N
H
N
R1
F
Compound
R1
R2
R3
X
Y
N-type Est. IC50
(l
M)
hERG Est. IC50 (lM)
38
39
40
41
42
OCH3
t-Butyl
OCH3
t-Butyl
t-Butyl
OCH3
OCH3
OCH3
OH
OCH3
t-Butyl
OCH3
t-Butyl
t-Butyl
O
O
H
H
H
O
O
H
H
H
0.84 ( 0.39)
1.9 ( 2.2)
0.06 ( 0.06)
0.004 (n = 2)
0.032
0.75
0.65
0.1
0.01
0.11
OCH3

4158
H. Pajouhesh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4153–4158
Table 8
Efficacy in the neuropathic SNL rat model for compounds 10 and 16
Compound
Tactile allodynia: maximum % antinociception at 60 min
Thermal hyperalgesia: maximum % antinociception at 60 min
10
16
1.9 1.4
54.2 12.9⁄⁄
85.8 14.1⁄⁄
31.7 6.9⁄⁄
Compounds, prepared in propylene glycol, were administered at a dose of 30 mg/kg po at 1 mL/kg after baseline assessment for neuropathic pain and behavioral assessments
were performed at 30 min intervals post-administration, up to 3 h. Data are the mean percent of maximal possible antinociception SEM; n = 5–6 animals. The following
formula was used to calculate the % anti-allodynic activity: 100% ꢄ [(paw withdrawal thresholds after drug treatment ꢂ postSNL baseline paw withdrawal thresholds)/
(preSNL baseline paw withdrawal thresholds ꢂ postSNL baseline paw withdrawal thresholds). The following formula was used to calculate the % anti-hyperalgesic activity:
100% ꢄ [(paw withdrawal latencies after drug treatment ꢂ postSNL baseline paw withdrawal latencies)/(preSNL baseline paw withdrawal latencies ꢂ postSNL baseline paw
withdrawal latencies). Data from the ipsilateral paw were also assessed by a one-way ANOVA-post-hoc for statistical significance. ⁄⁄P <0.01.
channel type. With
a few exceptions, each compound was examined for
teristics incorporating oral bioavailability and blood–brain barrier
penetration, and a wide therapeutic index against the cardiovascu-
lar, nervous and other systems. Similar to our previous reports,4,13
only a subset of high affinity in vitro N-type channel blockers in the
current study exhibited efficacy in rodent neuropathic and inflam-
matory pain models. An exact correlation between in vitro target
affinity and in vivo analgesia is generally lacking across many sim-
ilar studies, making direct compound-based predictive assess-
ments difficult and requiring empirical elucidation of efficacy. In
part, this may reflect a requirement for an optimal combination
of high affinity N-type channel blockade acting via a state- and/
or frequency-dependence mechanism and reflective of the under-
lying pathophysiology associated with specific pain states.14
blockade on 3–5 cells. Testing compounds for blockade of N-type and L-type
calcium channels was performed by whole-cell manual patch clamp analysis
using a near half-maximal concentration of compound applied to HEK293 cells
as per Ref.4. Pipettes (in the range of 2–4 M
X) were filled with an internal
solution containing 108 mM Cs-methanesulfonate, 4 mM MgCl2, 9 mM EGTA,
9 mM HEPES (pH 7.2 adjusted with TEA-OH) for N- and L-type channel currents
and 130 KCl, 1 MgCl2, 5 EGTA, 10 HEPES, 5 K2ATP (pH 7.2 with KOH) for hERG
K+ channel currents. Whole-cell barium currents were recorded using an
Axopatch 200B amplifier and an extracellular recording solution comprised of
5 mM BaCl2, 1 mM MgCl2, 10 mM HEPES, 40 mM TEA-Cl, 10 mM glucose,
97.5 mM CsCl (pH 7.2 adjusted with TEA-OH) for N- and L-type channel
currents and 137 NaCl, 4 KCl, 1.8 CaCl, 1 MgCl2, 10 HEPES, 10 glucose (pH 7.4
with NaOH) for hERG K+ channel currents. Data were filtered at 1 kHz using a
four pole Bessel filter and digitized at a sampling frequency of 2 kHz. Whole-
cell barium current inhibition was measured from a holding potential of
ꢂ80 mV to a test potential of +10 mV, applied at 0.2 Hz, or from a holding
potential of ꢂ80 mV to +1-mV following a 1 s prepulse to ꢂ60 mV, applied at
0.067 Hz for N-type channels. L-type channel currents were obtained from a
holding potential of ꢂ80 mV to a test potential of +10 mV, applied at 0.2 Hz. To
study compound effects on the hERG channel using manual patch clamp, the
peak of the slowly deactivating tail current was examined. A 2 s depolarization
to +60 mV from a holding potential of ꢂ80 mV, followed by a repolarization to
ꢂ50 mV was used to generate the hERG tail currents.
Acknowledgements
Work in the laboratories of T.P.S. and G.W.Z. is supported by the
Canadian Institutes of Health Research. T.P.S. is a Canada Research
Chair in Biotechnology and Genomics-Neurobiology. G.W.Z. is a
Scientist of the Alberta Heritage Foundation for Medical Research
and is also supported by a Canada Research Chair in Molecular
Neurobiology.
The estimated IC50 value was then calculated for all compounds tested in the
various electrophysiological assays such that estimated IC50 = [D]/((1/fr)-1)
where [D] is the drug concentration, and fr is the fraction of current remaining
after drug application. This analysis assumes that there is a 1:1 interaction
between the drug and the channel. The term ‘estimated IC50 value’ is utilized
since the determination of IC50 value via a single concentration point may be
slightly less precise than fits to entire concentration-dependent response
curves. For some compounds IC50 values were obtained using data obtained
from multiple concentrations and fitted with a logistic function where max is
the maximum current amplitude in the absence of compound, min is the
minimum current amplitude at steady-state inhibition with the compound and
nH is the Hill slope:
References and notes
1. (a) Barclay, J. W.; Morgan, A.; Burgoyne, R. D. Cell Calcium 2005, 38, 343; (b)
Zheng, X.; Bobich, J. A. Brain Res. Bull. 1998, 47, 117.
2. (a) Bowersox, S. S.; Valentino, K. L.; Luther, R. R. Drug News Perspect. 1994, 7,
261; (b) Bean, B. P. Annu. Rev. Physiol. 1989, 51, 367; (c) Hess, P. Annu. Rev.
Neurosci. 1990, 13, 337; (d) Nooney, J. M.; Lambert, R. C.; Feltz, A. Trends
Pharmacol. Sci. 1997, 18, 363.
3. (a) Sheng, Z. H.; Rettig, J.; Cook, T.; Catterall, W. A. Nature 1996, 379, 451; (b)
Saegusa, H.; Kurihara, T.; Zong, S.; Kazuno, A.; Matsuda, Y.; Nonaka, T.; Han, W.;
Toriyama, H.; Tanabe, T. EMBO J. 2001, 20, 2349; (c) Cox, B.; Denyer, J. C. Expert
Opin. Ther. Patents 1998, 8, 1237.
4. Zamponi, G. W.; Feng, Z. P.; Zhang, L.; Pajouhesh, Ho.; Ding, Y.; Belardetti, F.;
Pajouhesh, Ha.; Dolphin, D.; Mitscher, L. A.; Snutch, T. P. Bioorg. Med. Chem. Lett.
2009, 19, 6467.
"
#
max ꢂ min
ꢀ
ꢁ
nH
y ¼
þ min
½drugꢃ
1 þ
IC50
7. Pajouhesh, Ha.; Snutch, T.P.; Pajouhesh, Ho.; Ding, Y. U.S. Patent 20050014748
A1, 2005.
8. Pfeiffer, C. C. Science 1956, 124, 29.
9. (a) Dubuisson, D.; Dennis, S. G. Pain 1977, 4, 161; (b) Malmberg, A. B.; Yaksh, T.
L. J. Neurosci. 1994, 14, 4882.
10. Kim, S. H.; Chung, J. M. Pain 1992, 50, 355.
5. Pajouhesh, Ho..; Zamponi, G. W.; Pajouhesh, Ha..; Belardetti, F.; Snutch, T. P.
WO 2004/089922 and U.S. Patent 2004/0044004 A1.
6. The structure–activity analyses of the various chemical series were guided by
manual whole-cell patch clamp electrophysiological assay of rat neuronal N-
type (Bourinet, E.; Soong, T.-W.; Sutton, K.; Slaymaker, S.; Mathews, E.;
Monteil, A.; Zamponi, G. W.; Nargeot, J.; Snutch, T. P. Nat. Neurosci. 1999, 2, 407)
and L-type (Snutch, T. P.; Tomlinson, W. J.; Leonard, J. P.; Gilbert, M. M. Neuron
11. Chaplan, S. R.; Bach, F. W.; Pogrel, J. W.; Chung, J. M.; Yaksh, T. L. J. Neurosci.
Methods 1994, 53, 55.
12. Hargreaves, K.; Dubner, R.; Brown, F.; Flores, C.; Joris, J. Pain 1988, 32, 77.
13. Pajouhesh, Ho; Feng, Z.-P.; Ding, Y.; Zhang, L.; Pajouhesh, Ha; Morrison, J.-L.;
Belardetti, F.; Tringham, E.; Simonson, E.; Vanderah, T. W.; Porreca, F.;
Zamponi, G. W.; Mitscher, L. A.; Snutch, T. P. Bioorg. Med. Chem. Lett. 2010,
20, 1378.
1991, 7, 45) channels transiently expressed in HEK293 cells (N-type: a1B
/
CaV2.2 + 2d-1 + b1b subunits; L-type: 1C/CaV1.2 + 2d-1 + b1b subunits).
a
a
a
Compounds were also assayed against the hERG potassium channel stably
expressed in HEK293 cells. Whole cell barium currents were elicited from a
holding potential of ꢂ100 mV to the peak of current–voltage relation for each
14. Snutch, T. P. NeuroRx 2005, 2, 662.