Structure-activity relationships of diphenylpiperazine N-type calcium channel inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.01.008

A novel series of compounds derived from the previously reported N-type calcium channel blocker NP118809 (1-(4-benzhydrylpiperazin-1-yl)-3,3-diphenylpropan-1-one) is described. Extensive SAR studies resulted in compounds with IC₅₀ values in the range of 10-150 nM and selectivity over the L-type channels up to nearly 1200-fold. Orally administered compounds 5 and 21 exhibited both anti-allodynic and anti-hyperalgesic activity in the spinal nerve ligation model of neuropathic pain.

Full text of DOI:10.1016/j.bmcl.2010.01.008

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1378–1383
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationships of diphenylpiperazine N-type calcium
channel inhibitors
Hassan Pajouhesh a, Zhong-Ping Feng a,b, , Yanbing Ding a, Lingyun Zhang a, Hossein Pajouhesh a,
Jerrie-Lynn Morrison a, Francesco Belardetti a, Elizabeth Tringham a, Eric Simonson a, Todd W. Vanderah c,
Frank Porreca c, Gerald W. Zamponi b, Lester A. Mitscher d, Terrance P. Snutch e,
*
a Neuromed Pharmaceuticals Ltd, 301-2389 Health Sciences Mall, Vancouver, BC, Canada
b Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada
c Department of Pharmacology, University of Arizona, Tucson, AZ, 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:
A novel series of compounds derived from the previously reported N-type calcium channel blocker
NP118809 (1-(4-benzhydrylpiperazin-1-yl)-3,3-diphenylpropan-1-one) is described. Extensive SAR stud-
ies resulted in compounds with IC50 values in the range of 10–150 nM and selectivity over the L-type
channels up to nearly 1200-fold. Orally administered compounds 5 and 21 exhibited both anti-allodynic
and anti-hyperalgesic activity in the spinal nerve ligation model of neuropathic pain.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 24 September 2009
Revised 27 December 2009
Accepted 4 January 2010
Available online 7 January 2010
Keywords:
Calcium channel
N-type
L-type
Pain
Diphenylpiperazine
Numerous neuronal functions including neurotransmitter re-
lease, excitability and gene expression are highly dependent upon
of the activity of voltage-gated calcium channels. Of the L-, N- and
P/Q-types of native high voltage-gated calcium channels in neu-
rons, the N-type channel exhibits a variety of characteristics suit-
able for therapeutic interventions aimed at relieving chronic and
neuropathic pain conditions. In the dorsal horn of the spinal cord,
both US and European regulatory agencies have approved the use
of Prialt™ for the treatment of intractable pain, it must be deliv-
ered intrathecally and is not without adverse effects.4
A number of groups have focused on development of non-pep-
tidic based small molecule N-type calcium channel blockers aimed
at treating inflammatory and chronic pain.5 We recently reported
NP118809 (Fig. 1) as a potent N-type calcium channel blocker with
ꢀ110-fold selectivity over the L-type calcium channel and good
efficacy in a rodent model of inflammatory pain.6 As part of our ef-
fort to further optimize the development of orally available N-type
calcium channel blockers, we tested aryl substitutions on the benz-
hydryl moiety bound to the piperazine core of NP118809 (Table 1)
and also examined a bioisosteric series (Table 2). Subsequently, we
CaV2.2 (a1B) subunits encoding the N-type channel are concen-
trated at presynaptic terminals where they play a prominent role
in triggering the release of the pain-related transmitters glutamate,
CGRP and substance P.1 N-type channels are also a target for mod-
ulation by a number of G-protein coupled receptors including the
mu-opioid receptor-mediated inhibition that acts to attenuate
transmitter release via a G-protein-dependent mechanism.2 The
cone snail peptide,
x
-conotoxin-MVIIA (ZiconotideÒ, Prialt™),
reversibly blocks N-type channels, inhibits the release of substance
P and CGRP, and is highly efficacious towards alleviating inflamma-
tory and neuropathic pain in both animals and humans.3 While
O
N
N
* 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, University of Toronto, Toronto, ON,
Canada.
Figure 1. Structure of NP118809.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.008

H. Pajouhesh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1378–1383
1379
Table 1
N-type and L-type calcium channel blocking activities for compounds 1–20
R1
O
N
N
R2
Compound
R1
R2
N-type Est. IC50
(lM)
L-type Est. IC50
(
lM)
L-type FLIPR IC50 (lM)
NP118809
1
2
3
4 (Racemate)
5 (R-Isomer)
6 (S-Isomer)
7
H
H
H
H
H
H
H
H
H
H
H
H
H
0.11
0.07
0.11
0.30
0.11
0.05
0.14
0.04
0.09
0.38
0.31
0.25
0.24
0.51
0.17
0.21
0.22
>1
12.2
19.6
6.6
para-CF3
meta-CF3
ortho-CF3
para-Cl
para-Cl
para-Cl
meta-Cl
ortho-Cl
para-F
para-CH3
para-OCH3
para-OCH3
3,4,5-Tri-OCH3
3,5-Di-Cl
2,6-Di-Cl
2,3-Di-Cl
2,4-Di-Cl
3,4-Di-Cl
2,4-Di-CH3
3,5-Di-OCH3
6.1
1.8
10
7.4
6.9
6.8
6.2
>10
1.1
2.5
1.6
2.9
1.4
ꢁ10
ꢁ10
>10
ꢁ10
>10
9.6
—
4.3
8.5
106
8
9
10
11
12
13
14
15
16
17
18
19
20
p-OCH3
H
H
H
H
H
H
H
H
>1
0.23
0.21
The N-type and L-type IC50 columns indicate estimated IC50 values as determined by whole-cell patch clamp of transiently expressed calcium channel complexes. For the L-
type FLIPR-based assay IC50 values were calculated from an 8 point concentration-dependent response profile for each compound (0.003–10 M). In those instances wherein
the highest concentration (10 M) resulted in incomplete blockade we indicate >10: 10–50% inhibition at 10 M: less than 10% inhibition at 10 M.
M and ꢁ10
l
l
l
l
l
sequentially substituted heterocycles for the aryl group on the
benzhydryl moiety (Table 4).
the phenyl ring (Table 1). For example, compare compound 1,
where the trifluoromethyl is at the para-position with compounds
7 and 8 where the chlorine is at the meta- and ortho-positions,
respectively. In addition to possessing high N-type blocking activ-
ities, this series exhibited a high degree of selectivity over the neu-
ronal L-type calcium channel (ꢀ60–1178-fold).
The synthesis of compounds with the targeted substitution
changes is described in Scheme 1. The starting material diphenyl-
methanols were synthesized as described in the literature.7 Treat-
ment with thionyl chloride, followed by coupling with Boc-
piperazine, provided a benzhydrylpiperazine that was transformed
into product upon reaction with 3,3-diphenylpropionic acid and
diphenylmethyl isocyanate. Some experimental details have been
published in patent form.8
When matched pairs at the para-position are compared (Ta-
ble 1), the methyl- and methoxy-substituted phenyl compounds
(10 and 11) are consistently less potent N-type blockers than their
chloro- and trifluoromethyl counterparts (compounds 1 and 5).
These results suggest that electron-donating substituent groups
have lower overall affinity for the N-type channel compared to that
for compounds containing electron-withdrawing substituents.
As part of the structure–activity studies, the effects of di-substi-
tutions on a single phenyl ring were also explored (Table 1). Com-
pounds 14–16 illustrate that di-substitution with electron-
withdrawing groups at the ortho- and meta-positions of the phenyl
ring does not significantly affect N-type blocking affinity as com-
pared to the single substituent analogues. However, when one of
the electron-withdrawing substituents is at the para-position N-
type blocking affinity is lost (compounds 17 and 18).
The structure–activity analyses of the various chemical series
were guided by manual whole-cell patch clamp electrophysiologi-
cal assay of rat neuronal N-type9 and L-type10 channels expressed
in HEK cells (N-type:
a1B/CaV2.2 + a2d-1 + b1b subunits; L-type:
a
1C/CaV1.2 + 2d-1 + b1b subunits). Whole-cell barium currents
a
were elicited from a holding potential of À100 mV to the peak of
the current–voltage relation for each channel type.11 Each com-
pound was examined for inhibition on 3–5 cells. A FLIPR cell-based
functional assay also was used to characterize the block of L-type
voltage-dependent calcium channels in the partially inactivated
state.12 The basis of this assay is measurement in plate format of
calcium influx through a stably expressed L-type calcium channel
Compounds 21–27 (Table 2) represent the N,4-dibenzhydryl-
piperazine-1-carboxamide series (X@CO, Y@NH, Z@CH). The non-
substituted derivative (compound 21) showed a similar inhibitory
complex (a1C/CaV1.2 + a2d + b2a subunits) in response to potas-
sium-mediated depolarization. This cell line was also stably trans-
fected with the Kir2.3 inward rectifier K+ channel which allows for
changing the cell membrane potential by modulation of extracellu-
lar [K+]o containing buffer. Table 1 illustrates the structure–activity
trend in N-type inhibition for mono- versus di-substituted phenyl
on the diphenylmethylene. Compounds with a single electron-
withdrawing substituent on one of the phenyl groups are highly
potent N-type channel blockers (compounds 1–9). N-type blocking
affinity does not appear to depend upon either the exact nature of
the electron-withdrawing substituent (CF3, Cl, F) or its position on
activity for N-type calcium channels (est. IC50 = 0.15 lM) as that of
parent compound, NP118809. However, this compound had a
higher affinity for the L-type channel resulting in a comparatively
lower L-type:N-type selectivity ratio (ꢀ12-fold). The substitution
of a chlorine group at the 3-position of the benzhydryl moiety
(compound 23) led to a 2-fold decrease in N-type blocking activity
compared to compound 21, but a better overall L-type:N-type
selectivity (ꢀ53-fold). Further analogues with di-substitution at
the benzhydryl moiety did not show any overall improvement in

1380
H. Pajouhesh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1378–1383
Table 2
N-type and L-type calcium channel blocking activities for compounds 21–41
R1
X
Z
N
N
Y
R2
Compound
X
Y
Z
R1
R2
N-type Est. IC50
(l
M)
L-type Est. IC50
(l
M)
L-type
FLIPR
IC50 (lM)
NP118809
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CH
CH
CH
CH
CH
CH
CH
NH
NH
NH
NH
NH
NH
NH
NH
CH
CH
CH
CH
CH
CH
CH
CO
CO
CO
CO
CO
CO
CO
CH
CH
CH
CH
CH
CH
CH
CH
N
N
N
N
N
N
N
N
N
N
N
N
N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2-Cl
3-Cl
4-Cl
2,3-Di-Cl
2,4-Di-Cl
2,4-Di-Me
H
2-Cl
3-Cl
4-Cl
2,3-Di-Cl
2,4-Di-Cl
2,4-Di-Me
H
2-Cl
3-Cl
4-Cl
0.11
0.15
0.18
0.34
0.28
0.20
1.48
2.71
6.61
0.32
0.28
0.50
0.14
0.46
0.26
0.26
0.05
0.32
0.11
0.24
0.51
1.25
12.2
1.84
0.61
17.9
—
3.5
>10
>10
>10
ꢁ10
ꢁ10
>10
>10
>10
>10
>10
>10
ꢁ10
ꢁ10
1.6
1.1
1.4
2.9
7.9
8.6
3.8
1.03
37
38
39
40
2,3-Di-Cl
2,4-Di-Cl
2,4-Di-Me
41
N
The N-type and L-type IC50 columns indicate estimated IC50 values as determined by whole-cell patch clamp of transiently expressed calcium channel complexes. For the L-
type FLIPR-based assay IC50 values were calculated from an 8 point concentration-dependent response profile for each compound (0.003–10 M). In those instances wherein
the highest concentration (10 M and ꢁ10 M: less than 10% inhibition at 10 M.
M) resulted in incomplete blockade we indicate >10: ꢀ10–50% inhibition at 10
l
l
l
l
l
either N-type blocking affinity or in calcium channel selectivity
(compounds 25–27).
modifications (e.g., compound 35) did not seem to be highly dele-
terious. The information from this series (21–41) may provide in-
sight into the compound binding conformation at a site on the
channel where slight alterations in the linker can have dramatic ef-
fect on N-type calcium channel activity.
Replacement of the carboxamide in compounds 21 with the
ethanone group (X@CO, Y@CH, Z@N) (compound 28) showed a
dramatic 44-fold decrease in N-type channel blocking activity (Ta-
ble 2). Contrastingly, either mono- or di-substitution of one of the
phenyl groups on the benzhydryl did not significantly affect N-type
blocking affinity (compounds 29–34). Further exploration of the
structural activity relationship of derivatives focused around the
NP118809 linker moiety (X@CH, Y@CO, Z@N) resulted in com-
pounds with a generally good degree of N-type channel inhibition
(compounds 35–41). Unlike that for compound 28, these linker
In order to increase the aqueous solubility of the parent piper-
azine class, we replaced one aryl group (PhR1) of the benzhydryl
moiety with either an N-methylpiperidinyl or piperidinyl group
while retaining the second aryl group (PhR2; Table 4). The piperid-
inyl derivatives were synthesized from the corresponding phenyl
(pyridinyl-4-yl) methanol using methodology similar to Scheme 1.
The synthetic route used for the piperidinyl derivatives is pre-
sented in Scheme 2. As described in the literature,13 phenyl (piper-
idin-4-yl) methanol was prepared by activating with thionyl
chloride and then reacting with Boc-piperazine. The t-butoxycar-
bonyl protecting group of the product was cleaved with TFA. The
deprotected amine was coupled with 3,3-diphenylpropionic acid
to yield the target compounds (Scheme 2).
Table 3
Aqueous kinetic solubility
Compound
pH 7.4 (lg/ml)
NP118809
2
4
5
6
7
8
21
43
45
46
49
0.52
0.14
0.23
0.13
0.16
0.37
0.37
0.38
63.6
41.0
72.7
151
The activity of compounds from this series is presented in Ta-
ble 4. Compound 42, the 4-piperidinyl isomer with no substituent
on the nitrogen showed good N-type blocking activity (est.
IC50 = 0.06
lM) while the N-methylated analogue 43 was an order
of magnitude less potent (est. IC50 = 0.69
lM). Examining further
N-methylated piperidinyls (compounds 43–46) showed that com-
pounds 45 and 46 with an electron-withdrawing mono-substitu-
tion on one of the phenyl rings to be potent N-type blockers.
Eliminating the phenyl group while retaining a piperidinyl group
was detrimental towards maintaining N-type channel blocking
affinity (compounds 47–49; Table 4).
The thermodynamic (equilibrium) solubility was determined using methodology
based upon the OECD Water Solubility Method an LC/MS/MS system. Solubility was
calculated from plotting a calibration curve using regression analysis of standards
and interpolating the saturation solubility of the test compounds from the cali-
bration curve.15
Compounds 2, 4–8, 21 and 46 were subject to in vivo assay for
analgesic activity following oral dosing (Table 5). The compounds

H. Pajouhesh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1378–1383
1381
CHO
MgBr
OH
Cl
a
b
+
R1
R2
R1
R2
R1
R2
c
H
N
R1
N
O
d
N
N
R1
R2
e
g
R2
f
R1
R1
R1
O
O
N
N
N
H
N
N
N
N
N
N
O
R2
R2
R2
cmpd 35-41
cmpd 28-34
cmpd 21-27
Scheme 1. Reagents and conditions: (a) ether, reflux; (b) SOCl2, benzene, reflux; (c) anhydrous piperazine, K2CO3, KI, CH3CN, reflux; (d) 3,3-diphenylpropanoic acid, EDC,
DMAP, rt; (e) diphenylmethyl isocyanate, rt; (f) 2-(diphenylamino)acetic acid, EDC; (g) 2-bromo-N,N-diphenylacetamide, NaHCO3, CH3CN, reflux.
O
OH
O
Cl
O
OH
d
c
a
b
N
N
N
N
N
NBoc
O
Cl
NH
N
N
f
e
N
g
N
N
N
N
Scheme 2. Reagents: (a) SOCl2; (b) AlCl3, benzene; (c) NaBH4, CH3OH; (d) SOCl2; (e) 4-Boc-piperazine, K2CO3, CH3CN; (f) TFA, CH2Cl2; (g) 3,3-diphenylpropanoic acid or its
derivative, EDC, DMAP, CH2Cl2.
were tested at a single 30 mg/kg oral dose in the Chung model of
neuropathic pain with two behavioral endpoints used: mechanical
allodynia and thermal hyperalgesia.14 In both pain assessments,
animals were tested at multiple intervals during a 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 baseline value. Data
were normalized and expressed as means standard errors (SE).
Compounds 5–8 and 21 all showed inhibition of nerve ligation-in-
duced thermal hyperalgesia (ranging from ꢀ45% to 98% maximal
inhibition; Table 5). There did not appear to be a linear correlation
between in vitro blocking activity against the N-type channel and
analgesic effects in vivo (e.g., compounds 2 and 4). Further, only
two of the compounds, 5 and 21, exhibited significant anti-allo-
dynic effects in vivo. While compound 46 exhibited high N-type
channel affinity, selectivity over the L-type channel and improved
aqueous solubility compared to compounds 5 and 21, it did not

1382
H. Pajouhesh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1378–1383
Table 4
N-type and L-type calcium channel blocking activities for compounds 42–49
O
R3
N
N
R4
Compound
R3
R4
N-type IC50
(lM)
L-type Est. IC50
100
(lM)
L-type FLIPR IC50 (lM)
42
43
44
45
46
47
48
49
Phenyl
Phenyl
Phenyl
4-Cl-phenyl
4-F-phenyl
H
4-Piperidinyl
0.06
0.69
0.27
0.04
0.10
2.5
—
4-N-Methyl piperidinyl
3-N-Methyl piperidinyl
4-N-Methyl piperidinyl
4-N-Methyl piperidinyl
4-Piperidinyl
>10
9.3
3.1
—
11
30
ꢁ10
H
H
2-Piperidinyl
4-N-Methyl piperidinyl
2.7
2.4
—
ꢁ10
The N-type and L-type IC50 columns indicate estimated IC50 values as determined by whole-cell patch clamp of transiently expressed calcium channel complexes. For the L-
type FLIPR-based assay IC50 values were calculated from an 8 point concentration-dependent response profile for each compound (0.003–10 M). In those instances wherein
M: less than 10% inhibition at 10 M.
l
the highest concentration (10
lM) resulted in incomplete blockade we indicate >10: 10–50% inhibition at 10
lM and ꢁ10
l
l
Table 5
Efficacy of compounds in the rat spinal nerve ligation model following oral dosing
Compound
Oral dose (mg/kg)
Mechanical allodynia max % activity
at 60–90 min (mean SE)
Thermal hyperalgesia max % activity
at 60–90 min (mean SE)
NP118809
2
4
5
6
7
8
21
46
30
30
30
30
30
30
30
30
30
80.3 9.0 (p <0.001)
2.6 1.6 (p <0.001)
ND
40.2 15.9 (p <0.001)
3.1 6.3 (p <0.001)
27.6 3.0 (p <0.001)
27.6 3.0 (p <0.001)
80.9 13.3 (p <0.001)
ND
96.3 3.7 (p <0.001)
24.2 12.1 (p <0.001)
13.7 4.5 (p <0.001)
67.6 10.9 (p <0.001)
45.4 9.9 (p <0.001)
95.1 4.9 (p <0.01)
89.5 10.5 (p <0.001)
97.9 2.1 (p <0.01)
14.9 7.7 (p <0.001)
For administration compounds were dissolved in 3 ml/kg propylene glycol and compounds administered orally (n = 5–7 animals each). Thermal hyperalgesia and mechanical
hypersensitivity were measured at 30 min intervals following oral administration as described.14 The percentage responses noted reflect the maximum activity values from
baseline to the last time point examined (120 min). ND: not detected.
Table 6
Mean pharmacokinetic parameters in rat plasma following single oral or intravenous dosing for compounds 2, 7 and 21
Compound/route
Dose (mg/kg)
Cmax (nM)
Tmax (h)
AUC(0–24) (nM h)
t1/2 (h)
F (%)
Compd 2 intravenous
Compd 2 oral
Compd 7 intravenous
Compd 7 oral
Compd 21 intravenous
Compd 21 oral
2
10
2
10
2
10
2540
518
2549
582
2933
292
NA
5
NA
3
NA
2
1442
4670
1602
5305
2499
2785
5
4
8
6
11
6
NA
65
NA
66
NA
22
Data are from 2 to 3 animals each and presented as the mean.
AUC(0–24): area under the plasma concentration versus time curve from time zero to 24 h post-dose.
Cmax: the highest observable concentration.
Tmax: time to Cmax
.
t1/2: terminal elimination half-life.
F: oral bioavailability.
NA: not applicable.
show appreciable efficacy in either measure of analgesia in the SNL
model. Taken together, a subset of newly discovered orally admin-
istered high affinity N-type antagonists attenuate neuropathic pain
although N-type blocking affinity alone cannot predict in vivo
efficacy.
In order to ascertain whether general pharmacokinetic charac-
teristics could account in part for the differential in vivo affects
observed, representative compounds were selected for pro-
filing in rats (Table 6). Compound 2 was well absorbed orally
(Cmax ꢀ 275 ng/ml; bioavailability = 65%), albeit relatively slowly
(Tmax ꢀ 5 h) thus pain responses typically measured 1–3 h post-
dosing would likely not have reflected the optimal parameters
for assessing this particular agent. Comparatively, compound 7
was absorbed more rapidly (Tmax ꢀ 3 h) and exhibited good bio-
availability (F ꢀ 66%) and half-life (ꢀ6 h). Compound 21, which
exhibited the best overall pain efficacy profile across both
mechanical and thermal assessments, exhibited favorable Tmax
(ꢀ2 h) and elimination (t1/2 ꢀ 6 h) although bioavailability was
the lowest of the compounds examined (F ꢀ 22%). All three com-
pounds exhibit high affinity in vitro block of the N-type channel
target (IC50 values ꢀ 40–150 nM), good selectivity over the L-type
channel, plasma exposures that exceed the N-type target
(Cmax ꢀ 292–518 nM), similar aqueous solubility (Table 3) and
other favorable properties such as metabolic stability in rat plas-

H. Pajouhesh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1378–1383
1383
Oct. 2005.; (e) Pajouhesh, H.; Snutch, T. P.; Ding, Y.; Pajouhesh, H. WO 2006/
024160 A1, 9, March 2006.
ma (77–91% remaining at 60 min; not shown). In these regards,
and taken together with our previous report,6 it is becoming
evident that direct correlations cannot be reliably made between
in vitro N-type channel blocking affinity and efficacy in neuro-
pathic and inflammatory pain models. We hypothesize that a
combination of N-type affinity, physiochemical and pharmacoki-
netic properties together with the underlying mechanism of
N-type channel blockade by each compound (e.g., state- and fre-
quency-dependence) likely combine to determine efficacy in pain
states.
In summary, optimization and structure–activity relationship
studies on NP118809 have identified potent N-type calcium chan-
nel inhibitors with good selectivity against L-type calcium chan-
nels. These studies have clarified in vitro structure–activity
requirements for such agents. Compounds 5 and 21 are identified
as potent orally available N-type calcium channel blockers that
are ꢀ12–86-fold selective over L-type calcium channels and exhi-
bit mechanical and thermal analgesic activity in the rat spinal
nerve ligation model.
9. 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.
10. Snutch, T. P.; Tomlinson, W. J.; Leonard, J. P.; Gilbert, M. M. Neuron 1991, 7, 45.
11. Testing compounds for blockade of N-type and L-type calcium channels was
performed by whole-cell patch clamp analysis using a near half-maximal
concentration of compound applied to HEK cells transiently expressing each
particular type of cloned calcium channel complex (N-type: CaV
2.2/a1B + a2d-
1 + b1b subunits; L-type: CaV1.2/a1C 2d-1 + b1b subunits). Pipettes (in the
+ a
range of 2–4 M ) were filled with internal solution containing 108 mM Cs-
X
methanesulfonate, 4 mM MgCl2, 9 mM EGTA, 9 mM HEPES (pH 7.2 adjusted
with TEA-OH). 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 TEACl, 10 mM glucose, 97.5 mM
CsCl (pH 7.2 adjusted with TEA-OH). Data were filtered at 1 kHz using a 4 pole
Bessel filter and digitized at a sampling frequency of 2 kHz. Whole-cell barium
current inhibition was measured from a holding potential of À100 mV to a test
potential of +10 mV. The IC50 value was then calculated by rearranging the
Michaelis–Menton equation such that IC50 = [D]/((1/fr) À 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’ is utilized since the
determination of IC50 via a single concentration point may be slightly less
accurate than fits to entire concentration-dependent response curves.
12. For higher throughput FLIPR-based screening for L-type channel blockade, HEK
cells stably co-expressing the CaV1.2 L-type calcium channel complex and
Kir2.3 K+ channel were loaded with the fluorescent indicator dye, Fluo-4
(Invitrogen), and incubated for 45 min at 29 °C in 5% CO2. After removal of
Acknowledgements
excess Fluo-4 cells in buffer containing in mM: 110.5 NaCl, 10 HEPES, 10
glucose, 1 CaCl2, 30 mM KCl (pH adjusted to 7.4 with NaOH) compounds were
tested with an point concentration-dependent response profile (0.003–
10 M) generated by evoking calcium entry with addition of 130 mM KCl
stimulation buffer (in mM: 10.5 NaCl, 10 HEPES, 10 -glucose, 1 CaCl2, 130 KCl,
pH 7.4 adjusted with NaOH). A change in the Fluo-4 fluorescence signal was
assessed using FLIPR Tetra™ instrument (Molecular Devices, Sunnyvale, CA) for
3 min following the elevation of extracellular KCl using an illumination
wavelength of 470–495 nm with emissions recorded at 515–575 nm.
Concentration-dependent response curves were obtained by comparing the
fluorescence signal increase in the presence of compound to control substances
and fitted with a logistic function to obtain the concentration that inhibited
D-
We thank Molly Fee-Maki for helpful discussions concerning
the rat PK data. Work in the laboratories of T.P.S. and G.W.Z. is sup-
ported by the Canadian Institutes of Health Research. T.P.S. a Can-
ada 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.
8
l
D
References and notes
50% (IC50
) of the RLU signal using OriginPro v.7.5 software (OriginLab,
Northampton, MA). Given the nature of the L-type channel FLIPR assay, the
resulting IC50 values reflect blockade of channels in the partially inactivated
state. In comparison, data obtained from the L-type channel whole-cell patch
clamp analyses reflects open/closed state blockade. Selectivity ratios as stated
in the text derive from direct comparison of N-type and L-type whole-cell
patch clamp data.
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paw
withdrawal
thresholds)/(preSNL
baseline
paw
withdrawal
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withdrawal latencies after drug treatment À postSNL baseline paw withdrawal
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withdrawal latencies). All surgeries and behavioral testing were performed in
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the Animal Care and Use Committee of the University of Arizona.
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