Scaffold-based design and synthesis of potent N-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmcl.2009.09.008

The therapeutic agents flunarizine and lomerizine exhibit inhibitory activities against a variety of ion channels and neurotransmitter receptors. We have optimized their scaffolds to obtain more selective N-type calcium channel blockers. During this optim

Full text of DOI:10.1016/j.bmcl.2009.09.008

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6467–6472
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Scaffold-based design and synthesis of potent N-type calcium channel blockers
Gerald W. Zamponi a, Zhong-Ping Feng a, , Lingyun Zhang b, Hossein Pajouhesh b, Yanbing Ding b,
Francesco Belardetti b, Hassan Pajouhesh b, David Dolphin c, Lester A. Mitscher d, Terrance P. Snutch e,
*
a Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, Canada
b Neuromed Pharmaceuticals Ltd, 301-2389 Health Sciences Mall, Vancouver, BC, Canada
c Department of Chemistry, University of British Columbia, Vancouver, BC, Canada
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:
The therapeutic agents flunarizine and lomerizine exhibit inhibitory activities against a variety of ion
channels and neurotransmitter receptors. We have optimized their scaffolds to obtain more selective
N-type calcium channel blockers. During this optimization, we discovered NP118809 and NP078585,
two potent N-type calcium channel blockers which have good selectivity over L-type calcium channels.
Upon intraperitoneal administration both compounds exhibit analgesic activity in a rodent model of
inflammatory pain. NP118809 further exhibits a number of favorable preclinical characteristics as they
relate to overall pharmacokinetics and minimal off-target activity including the hERG potassium channel.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 31 December 2008
Revised 1 September 2009
Accepted 4 September 2009
Available online 11 September 2009
Keywords:
Calcium channel blocker
N-type
Pain
Diphenylmethylpiperazine
Voltage-gated ion channel blockers are attractive drug targets
for an expanding range of therapeutic indications.1 Voltage-depen-
dent calcium channels play a central role in the control of cellular
excitability and a number of calcium-dependent cellular functions,
including gene transcription and neurotransmitter release.2 Bio-
physical and pharmacological studies have identified four subtypes
of high voltage activated (HVA) calcium channels that are encoded
by a family of seven different a1 subunit proteins (Cav): L-type
(Cav1.1–Cav1.4), N-type (Cav2.2), P/Q-type (Cav2.1) and R-type
(Cav2.3) as well as low voltage activated calcium channels, called
T-type, encoded by three distinct a1 subunit proteins (Cav3.1–
Cav3.3).3 A subset of the HVA calcium channels are located at the
presynaptic termini of neurons, where they are directly involved
in the regulation of neurotransmitter release. Of particular interest,
the transmission of pain signals from periphery to the central ner-
vous system is mediated by N-type channels located at primary
afferent terminals in the dorsal horn of the spinal cord.4
ziconotideÒ), although the market for ZiconotideÒ is limited by
the requirement for intrathecal delivery.6
The objective of the current drug discovery program was the
identification of orally active, selective N-type blockers with an
acceptable therapeutic index aimed at the treatment of chronic/
inflammatory pain. Both flunarizine7 and lomerizine8 possess
diphenylmethylpiperazine moieties as the basic skeleton (Fig. 1)
and initial structure–activity relationship (SAR) investigations
indicated that this backbone contributed to calcium channel block-
ing activities. In the present report we investigated the structural
requirements for selective N-type calcium channel blockade
around the flunarizine and lomerizine backbones. Antagonist activ-
ity was measured in HEK293 cells transiently transfected with rat
F
F
Over the last decade, synthetic efforts have focused on develop-
ment of both peptidic and non-peptidic based small molecule N-
type calcium channel blockers for analgesia or neuroprotection.5
This work was encouraged by positive clinical observations for
the N-type channel blocking peptide drug, PrialtTM (SNX-111,
N
N
N
N
O
F
O
O
F
* 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,
Ontario, Canada.
Flunarizine
Lomerizine
Figure 1. Structures of flunarizine and lomerizine.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.008

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G. W. Zamponi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6467–6472
Table 3
brain calcium channel subunits cDNAs (N-type: a1B
+ a2d-1 + b1b
N-type and L-type calcium channel blocking activities for compounds 2a–2e
subunits; L-type: a1C 2d-1 + b1b subunits). After incubation for
+
a
24–72 h, whole cell current recordings were performed with bar-
ium as the charge carrier as previously described.9 Currents were
typically elicited from a holding potential of ꢀ100 mV to the peak
of current–voltage relation for each channel type. For each calcium
channel subtype each compound was examined for blockade by
patch clamp analysis on between 3 and 5 cells.
O
N
N
n
We began by testing the potency of flunarizine and lomerizine
against the cloned N-type calcium channel exogenously expressed
in HEK cells and found that they both potently inhibited functional
Compound
n
N-type
Est. IC50
L-type
Est. IC50 (lM)
L/N ratio
(l
M)
N-type channels (estimated IC50 values of 0.08 and 0.09 lM,
2a
0
1
2
3
4
>50
0.11
23
>50
50
NA
12.2
NA
NA
NA
—
111
—
—
—
respectively; Table 1). However, their inhibitory activities against
L-type channels was also high, providing only a 1.7–3.9-fold ratio
of selectivity over the two types of high voltage-activated calcium
channels (Table 1). A major direction of the SAR optimization was
to decrease L-type activity while maintaining the high affinity for
N-type calcium channel blockade.
2b (NP118809)
2c
2d
2e
We hypothesized that the benzhydrylpiperazine and trimeth-
oxybenzylpiperazine moieties, respectively, could be acting as
selectivity elements. With this in mind, we designed and synthe-
sized a series of compounds replacing the cinnamyl portion in flu-
narizine. We first examined the effect of the methylene spacer
between the piperazine and the benzhydryl group (Table 2). Fur-
ther, we investigated the effect on N-type calcium channel activity
and selectivity by changing the amine into an amide (Table 3). The
SAR studies of these chemical series were guided by the whole cell
patch clamp analysis of the functional blockade of N-type and L-
type channels expressed in HEK cells.
Table 3 shows that the introduction of an amide into the right
hand linker was also generally unfavorable compared to the parent
compounds.
A notable exception to this was compound 2b
(NP118809; see Scheme 1) with a total linker length of 3 carbons
(n = 1) and which exhibited approximately a 500-fold increased
N-type channel blockade compared to compounds in this series
with linkers either shorter (n = 0, 2a) or longer (n = 2–4, (2c–e).
Examination of the effect of NP118809 on functional L-type cal-
cium channel activity showed that this compound was approxi-
mately 111-fold more selective for N-type channels.
Table 2 shows that the benzhydrylpiperazine backbone with a
benzhydryl group on the right hand side resulted in compounds
with a significantly poorer degree of N-type blockade (compounds
1a–1e) compared with either parent compounds. Varying the
length of the linker between the benzhydryl and the piperazine
core resulted in a 10-fold range of N-type affinities suggesting that
linker length nonetheless contributed to N-type blocking affinity.
To probe the effect of bioisosteric replacement of carbon on the
NP118809 linker, we examined the bioisosteric potential of 3,3-
diphenylpropan-1-one bound to the piperazine core, while main-
taining the benzhydryl moiety on the opposite side of molecule
(Scheme 2). This modification allowed the maximum conservation
of the polarity and geometry of the NP118809 (Table 4). While
compound 3a maintained favorable inhibitory activity for N-type
channels (est. IC50 = 0.15
lM) compared to that of NP118809, the
N-type:L-type selectivity profile was significantly lower (12-fold).
Further exploration on the linker with compounds 3b–3c showed
a trend toward decreased N-type channel blocking affinity (Table 4).
Examination of a series of derivatives focused around the
lomerizine-based trimethoxybenzylpiperazine moiety resulted in
compounds with a generally high degree of N-type channel block-
ade. Unlike that for the flunarizine-based series, a longer linker to
the benzhydryl group (six carbons total length) did not seem to be
deleterious. Furthermore, there was little change in N-type affinity
regardless of whether an amide was included in the linker on
either side of the piperazine core (Tables 5 and 6). In particular,
compound 4a (NP078585; see Scheme 1) exhibited potent N-type
Table 1
Blocking activities of flunarizine and lomerizine against N-type and L-type calcium
channels
Compound
N-type
Est. IC50
L-type
Est. IC50 (lM)
L/N ratio
(lM)
Flunarizine
Lomerizine
0.08
0.09
0.31
0.15
3.9
1.7
Table 2
N-type calcium channel blocking activities for compounds 1a–1e
channel functional blockade (est. IC50 = 0.11 lM). Examination of
the effect of NP078585 on L-type calcium channels showed that
this compound was approximately 25-fold more selective for N-
type channels (Table 5).
Replacement of the trimethoxybenzyl with cinnamyl moiety
was studied without changing the chain linker lengths (Table 6).
Compound 5a had the closest N-type potency compared to
N
N
n
NP078585 (est. IC50 = 0.05 lM) but possessed a lower selectivity
ratio of L-type to N-type blockade (ꢁ7.8-fold). The other deriva-
tives in this series all exhibited sub-micromolar N-type channel
affinities but again with no improvement over NP078585 in the
L-type to N-type selectivity ratio (Table 6).
Compound
n
N-type
Est. IC50 (lM)
The two compounds demonstrating high affinity for the N-type
channel together with the best selectivity over L-type calcium
channels were selected for pharmacokinetic profiling. Examination
of pharmacokinetics and oral bioavailability showed that in rats
NP078585 was absorbed relatively rapidly (Tmax = 1.0 h), elimi-
1a
1b
1c
1d
1e
0
1
2
3
4
>50
5
6
20
50

G. W. Zamponi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6467–6472
6469
O
Diphenylpropionic acid,
EDC, CH2Cl2
N
N
N
NH
NP118809
F
Br
a
HO
P(Ph)3
b
c
HO
HO
Br
O
O
O
F
F
F
d
Boc
e
N
HO
N
O
O
F
F
O
F
O
O
F
O
O
f
O
N
N
N
O
N
O
F
F
NP078585
Scheme 1. Reagents and conditions: (a) PPh3, CH3CN, reflux; (b) LiHMDS, ꢀ78 °C, benzophenone; (c) MeOH, H2, 5% Pd/C, 50 psi; (d) Boc-piperazine, EDC; (e) 25% TFA, 3,4,5-
trimethoxybenzoylchloride, K2CO3; (f) LiAlH4, THF.
H
N
N
c
b
a
O
N
N
N
N
N
N
O
O
Cmpd 3b
N
N
N
H
cmpd 3c
Cmpd 3a
Scheme 2. Reagents and conditions: (a) diphenylmethyl isocyanate, rt; (b) 2-bromo-N,N-diphenylacetamide, NaHCO3, CH3CN, reflux;(c) 2-(diphenylamino)acetic acid, EDC, DMAP.

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G. W. Zamponi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6467–6472
Table 4
was suggestive of a high degree of protein binding and/or distribu-
tion outside of plasma.
N-type and L-type calcium channel blocking activities for compounds 3a–3c
Based upon their combined in vitro and pharmacokinetic prop-
erties NP078585 and NP118809 were subject to in vivo assay in
the rat formalin model inflammatory pain.10 Figure 2 shows that
upon intraperitoneal (ip) administration both NP118809 and
NP078585 (25 mg/kg) both exhibited significant analgesic activity
in the phase IIA portions of the rat formalin model.
X
Z
N
N
Y
In order to further refine the preclinical profiles of the two can-
didate molecules their off-target interactions with the hERG potas-
sium channel was examined by whole cell patch clamp analysis of
Compound
X
Y
Z
N-type
L-type
L/N ratio
HEK cells stably expressing hERG. A single 1 lM dose application of
Est. IC50
(
lM)
Est. IC50 (lM)
NP078585 was found to significantly block hERG currents
(98.7 0.2%, n = 3) suggesting the potential for cardiovascular lia-
bility for this agent. Contrastingly, an initial single test dose test
3a
3b
3c
CO
CO
CH
NH
CH
CO
CH
N
N
0.15
6.61
0.26
1.84
NA
NA
12
—
—
of NP118809 (1 lM) resulted in only partial blockade of hERG cur-
rents (24.8 0.3%, n = 3). Subsequently, a dose–response profile on
hERG tail currents was determined for NP118809. Figure 3 shows
the estimated IC50 of NP118809 for the hERG channel extrapolated
Table 5
N-type and L-type calcium channel blocking activities for compounds 4a–4c
from the concentration-dependent inhibition curve = 7.4 lM. As
such, NP118809 would not be considered a potent blocker of the
F
X
Y
hERG potassium channel.
N
N
Further examining NP118809 at a concentration of 10 lM for
off-target activity using a radioligand binding displacement screen
of 112 ion channel, receptor and other signaling targets (MDS Phar-
ma Services; http://discovery.mdsps.com) resulted in >50% dis-
placement of radioligand from only four targets: L-type calcium
O
O
O
F
channel, cannabinoid CB1,
l opiate receptor and voltage-gated so-
dium channel. Subsequent secondary screens for possible func-
tional interactions against these targets by NP118809 using
representative tissue assays (MDS Pharma Services) showed no
significant agonism or antagonism (>50%) at a higher test concen-
Compound
Y
X
N-type
Est. IC50
L-type
Est. IC50 (lM)
L/N ratio
(l
M)
4a (NP078585)
4b
4c
H
O
H
H
O
O
0.11
0.54
0.19
2.8
NA
NA
12
—
—
tration of 30
of NP118809 on L-type channel function in a guinea pig model of
atrial inotropy while we determined an IC50 ꢁ 12 M against the
lM. That there was no observed physiological effect
l
L-type channel by electrophysiological assessment is likely reflec-
tive of the highly state-dependent nature of NP118809 channel
blockade (data not shown). Voltage- and frequency-dependent
blockade would favor drug interaction with a subset of channels
under native conditions and are predicted to be important factors
towards maximizing the therapeutic window of ion channel block-
ers in vivo.11
nated moderately slowly (t1/2 = 4.5 h) and possessed an oral bio-
availability of 20% (Table 7). The mean volume of distribution for
NP078585 following intravenous administration (11,693 ml/kg)
was in excess of the calculated total body water in the rat suggest-
ing that NP078585 was widely distributed outside of plasma and/
or bound to plasma proteins. NP118809 exhibited both acceptable
absorption (Tmax = 2.3 h) and half-life (t1/2 = 2.1 h) characteristics
In summary, SAR studies around the flunarizine and lomerizine
diphenylmethylpiperazine backbones produced a series of high
and
a mean oral bioavailability of 30%. Similar to that for
NP078585, the volume of distribution of NP118809 (5787 ml/kg)
Table 6
N-type and L-type calcium channel blocking activities for compounds 5a–5h
X
Y
O
N
N
R
X
Compound
Y
X
R
N-type
L-type
L/N ratio
Est. IC50
(
lM)
Est. IC50 (lM)
5a
5b
5c
5d
5e
5f
H
H
H
H
O
H
O
O
H
p-F
H
H
H
H
H
H
H
H
H
0.05
0.09
0.12
0.19
0.09
0.19
0.13
0.15
0.39
0.12
0.33
NA
0.25
NA
8
1.3
2.8
—
2.8
—
p-Methoxy
p-Methoxy
1,3-Dioxole
1,3-Dioxole
p-Amino
5g
5h
0.74
0.83
5.7
5.5

G. W. Zamponi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6467–6472
6471
Table 7
Mean pharmacokinetic parameters in rat plasma following single oral or intravenous dosing for NP078585 and NP118809
Compound/route
Dose (mg/kg) Cmax (ng/ml)
Tmax (h)
AUC(0–8) (ng h/ml)
AUC(i) (ng h/ml) t1/2 (h)
Vdss (ml/kg)
CL/F (ml/h kg) F (%)
NA
NP078585 intravenous
NP078585 oral
NP118809 intravenous
NP118809 oral
2
462 (182)
173 (64)
771 (95)
235 (94)
NA
1.0 (0.0)
NA
725 (104)
753 (292)
715 (171)
975 (192)
1021 (269)
761 (169)
1136 (469)
5.0 (1.1)
4.5 (2.0)
3.3 (1.2) 5787 (2345)
11693 (1360) 2111 (449)
NA
10
2
10204 (2331)
2730 (680)
21 (5.5)
NA
10
2.3 (1.5) 999 (386)
2.1 (0.5)
NA
9868 (3969)
30 (12.3)
Data are presented as mean (standard error in brackets).
AUC(0–8): Area under the plasma concentration versus time curve from time zero to 8 h post-dose.
AUC(i): Area under the plasma concentration versus time curve from time zero to infinity.
Cmax: The highest observable concentration.
Tmax: Time to Cmax
.
CL/F: Plasma clearance.
Vdss: Apparent volume of distribution.
t1/2: Terminal elimination half-life.
F: Oral bioavailability.
NA: Not applicable.
900
800
700
600
500
400
300
200
100
0
***
900
800
700
600
500
400
300
200
100
0
PG
PG
PG
NP078585
NP078585
NP118809
*
N.S.
N.S.
N.S.
**
Phase I
Phase IIA
Phase IIB
Phase I
Phase IIA
Phase IIB
Figure 2. Efficacy of NP078585 and NP118809 in the rat formalin model of inflammatory pain. Compounds were administered at a dose of 25 mg/kg ip NP078585 n = 26
animals, NP118809 n = 24 animals. Statistical significance *p < 0.01; ***p < 0.001.
3 µM NP118809
5
0
5
0
5
0
2.5
2.0
1.5
1.0
0.5
0.0
100
75
50
25
0
0
400
time (s)
800
Estimated IC50 = 7.4
Estimated Hill slope
0.1 % DMSO
3 µM
NP118809
10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1
[NP118809] (M)
0
2
4
6
0
time (s)
Figure 3. Concentration-dependent inhibition of the hERG potassium channel by NP118809. (A) hERG tail currents recorded in the presence of 0.1% DMSO (control) and after
application of 3 M NP118809. Inset shows the rate of inhibition of the tail current by 3 M NP118809 from the same cell. (B) Concentration-dependent inhibition of the peak
hERG channel tail currents by NP118809. The concentration-dependent inhibition curve was generated by using the mean data obtained for 0.1, 1, 3 and 10 M NP118809
l
l
l
(solid line) and extrapolated to 100% inhibition (dash line). Data are the mean of 4 values standard error for each concentration applied. Data was analyzed and fitted using
OriginPro v.7 software. To obtain the IC50 and Hill slope coefficient (nH) the concentration–response curve was fitted with a logistic equation of:
2
3
1
drug
IC50
6
7
5
y ¼ max 1 ꢀ
ꢀ
ꢁ
4
nH
1 þ
affinity N-type calcium channel blockers (<200 nM IC50s) that exhi-
bit a significant degree of selectivity (25–111-fold) over the closely
related L-type channel. NP078585 and NP118809 were discovered
as N-type calcium channel antagonists that exhibit strong analge-
sic activity in an inflammatory rat model. Both compounds exhibit
suitable pharmacokinetic characteristics for animal testing
although NP118809 appears to be the more favorable preclinical
candidate based upon its lower off-target affinity to the hERG

6472
G. W. Zamponi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6467–6472
3. Snutch, T. P.; Peloquin, J.; Mathews, E.; McRory, J. Molecular Properties of
Voltage-Gated Calcium Channels. In Voltage-Gated Calcium; Zamponi, G., Ed.;
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Acknowledgments
Work in the laboratories of T.P.S. and G.W.Z. is supported by the
Canadian Institutes of Health Research. T.P.S. 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.
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