Subtype-selective nicotinic receptor antagonists: Potential as tobacco use cessation agents

Article abstract of DOI:10.1016/j.bmcl.2003.10.073

N-n-Alkylpicolinium and N,N′-alkyl-bis-picolinium analogues were assessed in nicotinic receptor (nAChR) assays. The most potent and subtype-selective analogue, N,N′-dodecyl-bis-picolinium bromide (bPiDDB), inhibited nAChRs mediating nicotine-evoked [³H]dopamine release (IC₅₀=5 nM; I_max of 60%), and did not interact with α4β2* or α7* nAChRs. bPiDDB represents the current lead compound for development as a tobacco use cessation agent.

Full text of DOI:10.1016/j.bmcl.2003.10.073

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1863–1867
Subtype-selective nicotinic receptor antagonists: potential as
tobacco use cessation agents
Linda P. Dwoskin,* Sangeetha P. Sumithran, Jun Zhu, A. Gabriela Deaciuc,
Joshua T. Ayers and Peter A. Crooks
Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA
Accepted 20 October 2003
Abstract—N-n-Alkylpicolinium and N,N0-alkyl-bis-picolinium analogues were assessed in nicotinic receptor (nAChR) assays.
The most potent and subtype-selective analogue, N,N0-dodecyl-bis-picolinium bromide (bPiDDB), inhibited nAChRs mediating
nicotine-evoked [3H]dopamine release (IC50=5 nM; Imax of 60%), and did not interact with a4b2* or a7* nAChRs. bPiDDB
represents the current lead compound for development as a tobacco use cessation agent.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Subtype assignment of native nAChRs mediating nico-
tine-evoked DA release has not been conclusively
established, but is based largely on inhibition of agonist-
induced response by subtype-selective antagonists. The
subtype selectivity of these antagonists has been defined
based on their ability to inhibit nAChR subtypes of
known subunit composition when expressed in cell
systems. Thus, for example, neuronal-bungarotoxin
(n-BTX) and a-conotoxin-MII (a-CTX MII) selectively
inhibit acetylcholine-induced electrophysiological respon-
ses in Xenopus oocytes expressing the a3b2 subtype.17,18
Moreover, n-BTX and a-CTX MII inhibit nicotine-
evoked [3H]DA overflow from striatal preparations,18ꢀ23
implicating a3b2*-containing nAChR subtypes as
mediating nicotine-stimulated DA release.
Several lines of evidence suggest that subtype-selective
nicotinic acetylcholine receptor (nAChR) antagonists
may be efficacious tobacco use cessation agents. Bupro-
pion, an antidepressant and nAChR antagonist that
inhibits nAChR subtypes mediating nicotine-evoked
dopamine (DA) release1 has some efficacy as a tobacco
use cessation agent.2ꢀ4 Also, mecamylamine, a non-
selective nAChR antagonist, shows some efficacy as a
tobacco use cessation agent, although its use is limited
by peripherally mediated autonomic side effects, mainly
constipation.5,6 Furthermore, the efficacy of nicotine
replacement therapy may be derived from its ability to
desensitize nAChRs, rather than due to its effects as a
nAChR agonist. Based on these observations and
hypotheses, we predict that subtype-selective nAChR
antagonists will be efficacious tobacco use cessation
agents with therapeutic advantages over currently avail-
able therapies.
Importantly, a-CTX MII inhibited only 50% of nico-
tine-evoked [3H]DA overflow, implicating the involve-
ment of at least two nAChR subtypes in this
response.22,24 These additional subtypes may contain
a4- and/or b4-subunits.20,22,25,26 Studies using b2 or a4
knockout mice also implicate b2- and a4-containing
nAChR subtypes in the mediation of DA release.21,27ꢀ29
Rat substantia nigra neurons express mRNA for a3, a4,
a5, a6, a7, b2, b3 and b4 subunits,30ꢀ34 such that mul-
tiple nAChR subtypes may be involved in nicotine-
evoked striatal DA release. Since DA-containing neu-
rons in the substantia nigra express high levels of a6 and
b3 mRNA,31,33,35,36 their combination with a3 and b2 in
the mediation of nicotine-evoked DA release is likely.
More recently, nAChR subtypes containing a6 subunits
Evidence suggests that the acute reinforcing actions of
drugs of abuse, including nicotine, may be mediated by
dopaminergic systems in the striatopallidal and extended
amygdala systems.7ꢀ9 Nicotine’s rewarding properties
are believed to result from activation of nAChRs loca-
ted presynaptically on cell bodies and terminals of DA
neuronal pathways, resulting in the release of DA.10ꢀ16
* Corresponding author. Tel.: +1-859-257-4743; fax: +1-859-257-
7564; e-mail: ldwoskin@uky.edu
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.073

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L. P. Dwoskin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1863–1867
have also been suggested to mediate nicotine-evoked
DA release,29,37ꢀ39 although multi-subunit combina-
tions may be involved.
High mM concentrations of nicotine have also been
shown to activate a7* nAChRs to stimulate [3H]DA
release, via an indirect mechanism, that is, stimulation
of glutamate release, which in turn indirectly stimulates
DA release.12,40 Glutamate receptor antagonists inhib-
ited ꢁ20–50% of nicotine-evoked [3H]DA release from
striatal slices, but not from synaptosomes,41 indicating
that local circuitry within the slice is sufficient to reveal
the indirect actions of nicotine on DA release.
The availability of novel compounds which selectively
antagonize specific nAChR subtypes, would be invalu-
able for unraveling the complexity of nicotine’s
response. Furthermore, such subtype-selective nAChR
antagonists may have potential as treatments for nico-
tine addiction.42ꢀ45 Pyridine N-n-alkylation of nicotine
affords N-n-alkylnicotinium analogues with alkyl chains
of C1ꢀ12. This structural modification converts nicotine
from an agonist into high affinity, subtype-selective
nAChR antagonists. N-n-Octylnicotinium iodide (NONI,
C8 analogue) competitively inhibits nicotine-evoked
[3H]DA overflow (IC50=0.62 mM), but does not inhibit
[3H]nicotine binding to rat brain membranes; while N-n-
decylnicotinium iodide (NDNI, C10 analogue) is a
competitive inhibitor of [3H]nicotine binding and nico-
tine-evoked 86Rb+ efflux, but does not inhibit nicotine-
evoked [3H]DA overflow from superfused striatal
slices.45,46 Thus, NONI and NDNI are selective and
relatively potent nAChR antagonists. N-n-Dodecyl-
nicotinium iodide (NDDNI, C12 analogue) has even
higher affinity at nAChR subtypes mediating nicotine-
evoked [3H]DA overflow, however selectivity of this
analogue for the nAChR subtype(s) mediating nicotine-
evoked DA release is diminished, relative to that for
NONI and NDNI.
Figure 1. Structures of N-n-alkylpicolinium (left) and N,N0-alkyl-
bis-picolinium (right) analogues.
priate n-alkyl iodide utilizing previously described con-
ditions.42 The N,N0-alkyl-bis-picolinium analogues
shown in Figure 1 (right) were prepared by reacting an
excess of 3-picoline with a variety of diiodo- or dibro-
moalkanes for 24 h in the absence of solvent. The
resulting solid was collected by filtration, dissolved in
water and the aqueous solution was washed with diethyl
ether (3ꢂ50 mL). The aqueous solution was then lyo-
philized to afford either a solid or viscous hygroscopic
1
oil. All compounds were characterized by H and 13C
NMR spectroscopy, mass spectroscopy and elemental
analysis.45
3. Biological assays
Male Sprague–Dawley rats (225–250 g) were obtained
from Harlan Industries (Indianapolis, IN) and housed
two per cage with free access to food and water in the
Division of Lab Animal Resources in the College of
Pharmacy at the University of Kentucky. All experi-
ments were carried out in accordance with the 1996 NIH
Guide for the Care and Use of Laboratory Animals and
were approved by the Institutional Animal Care and
Use Committee at the University of Kentucky.
The present report describes further optimization within
the N-n-alkylnicotinium series. The current structure–
activity approach is based on classical observations that
hexamethonium chloride and decamethonium bromide,
nAChR antagonists with bis-quaternary ammonium
structures, distinguish between neuromuscular and
ganglionic nAChR subtypes.47ꢀ52 Thus, in the current
study, nAChR subtype selectivity of the N-n-alkylpico-
linium and N,N0-alkyl-bis-picolinium series was deter-
mined. The latter series has afforded our most
promising lead candidate to date, that is, N,N0-dodecyl-
bis-picolinium bromide (bPiDDB, C12 analogue), which
inhibits nAChRs mediating nicotine-evoked [3H]DA
release with an IC50=5 nM and an Imax of 60%, but
does not inhibit a4b2* nAChRs or a7* nAChRs, as
determined in [3H]nicotine and [3H]methyllycaconitine
(MLA) binding assays, respectively.
[3H]DA release assays were performed according to
previously published methods,1,45,53 with minor modifi-
cations. Striatal slices (500 mm, 4–6 mg) were incubated
for 30 min in Krebs’ buffer (in mM: 118 NaCl, 4.7 KCl,
1.2 MgCl2, 1 NaH2PO4, 1.3 CaCl2, 11.1 glucose, 25
NaHCO3, 0.11 l-ascorbic acid and 0.004 disodium
EDTA, pH 7.4, saturated with 95% O2/5% CO2) in a
metabolic shaker at 34 ꢃC. Slices were incubated with
0.1 mM (final concentration) [3H]DA during the latter
30 min of the 60-min incubation period. Each slice was
transferred to a glass superfusion chamber maintained
at 34 ꢃCand superfused (1 mL/min) with Krebs’ buffer
containing nomifensine (10 mM) and pargyline (10 mM)
to inhibit [3H]DA reuptake and metabolism, respec-
tively, after release into the extracellular space, ensur-
ing that [3H]overflow primarily represents [3H]DA.54
Sample collection (5-min; 5 mL) began after 60 min
2. Chemistry
N-n-Alkylpicolinium analogues shown in Figure 1 (left)
were prepared by reacting 3-picoline with the appro-

L. P. Dwoskin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1863–1867
1865
of superfusion. The ability of N-n-alkylpicolinium and
4. Results and discussion
N,N0-alkyl-bis-picolinium analogues to evoke [3H]DA
release (that is, exhibit intrinsic activity) and to inhibit
nicotine-evoked [3H]DA release (that is, act as nAChR
antagonists) was determined. To establish that these
analogues act as nAChR antagonists, by definition,
inhibition of the response to nicotine must be observed
at analogue concentrations that do not evoke a
response. Thus, the ability of these analogues to elicit
intrinsic activity (evoke [3H]DA overflow) was deter-
mined. At the end of the experiment, each slice was
solubilized and [3H]content of the tissue determined.
Release during each min was normalized for total
[3H]content of the slice. Analogue-induced intrinsic
activity and inhibitory activity were determined using
slices from the same rat (repeated-measures design).
Table 1 shows the inhibitory activity of the N-n-alkyl-
picolinium analogues with carbon chain lengths of C8–
C12 in the nicotine-evoked [3H]DA overflow assay and
in the [3H]nicotine and [3H]MLA binding assays. This
series of analogues was relatively selective for the
nAChR subtype(s) mediating nicotine-evoked [3H]DA
overflow, in that, these analogues did not inhibit bind-
ing of either [3H]nicotine or [3H]MLA to rat brain
membranes, indicating low affinity for or no interaction
with a4b2* and a7* nAChRs.
In the nicotine-evoked [3H]DA overflow assay, NDDPiI
at concentrations of ꢄ1.0 mM evoked [3H]DA overflow,
and thus, exhibited intrinsic activity; whereas the C8 and
C10 analogues demonstrated no intrinsic activity up to
1.0 mM (data not shown). Importantly, as the carbon
chain length was increased the inhibitory potency at
nAChRs mediating nicotine-evoked [3H]DA overflow
increased with a rank order of NDDPiI >NDPiI
>NOPiI (Table 1). The most potent analogue in the
series was NDDPiI (IC50=30 nM). Furthermore,
NDDPiI inhibited nicotine-evoked [3H]DA overflow by
63%, indicating that this analogue may interact with a
single subtype of nAChR that mediates this effect of
nicotine.
Fractional release was calculated by dividing the total
tritium collected in each sample by the total tritium in
the tissue at the time of sample collection. The sum of
all the increases in [3H]DA fractional release resulting
from either exposure to analogue or nicotine equaled
‘total [3H]DA overflow’. ‘Overflow’, rather than
‘release’, is the more correct terminology because the
neurotransmitter measured is the net result of release
and reuptake. Typically, data were analyzed by weigh-
ted, least squares regression analysis of sigmoidal con-
centration–effect curves to obtain EC50 and IC50 values.
Table 2 illustrates the ability of a series of N,N0-bis-
alkylpicolinium analogues with carbon chain lengths of
C6–C12 to inhibit nicotine-evoked [3H]DA overflow and
the binding of [3H]nicotine and [3H]MLA to rat brain
Interaction of the analogues with nAChR subtypes
probed by [3H]nicotine binding (a4b2*) and [3H]MLA
binding (a7*) to rat brain membranes was determined
to assess nAChR subtype selectivity of the analogues.46
[3H]Nicotine and [3H]MLA binding assays were per-
formed using whole brain, excluding cortex and cere-
bellum. Whole brain was homogenized in 20 vol of ice-
cold buffer (in mM: 2 HEPES, 11.8 NaCl, 0.48 KCl,
0.25 CaCl2 and 0.12 MgSO4, pH 7.5). Homogenates were
centrifuged (25,000g, 15 min, 4 ꢃC). Pellets were resus-
pended in 20 vol of buffer and incubated at 37 ꢃC, for 10
min, cooled to 4 ꢃCand centrifuged (25,000 g, 15 min,
4 ꢃC). Pellets were resuspended and centrifuged again
using the same conditions. Final pellets were stored at
ꢀ70 ꢃCin assay buffer (in mM: 20 HEPES, 118 NaCl,
4.8 KCl, 2.5 CaCl2, and 1.2 MgSO4, pH 7.5). Upon use,
final pellets were resuspended in ꢁ20 vol assay buffer.
Samples (250 mL) contained 100–140 mg of membrane
protein, 3 nM [3H]nicotine or 3 nM [3H]MLA, and a
range of concentrations (0.1 mM–1 mM) of analogue in
assay buffer containing 50 mM Tris. Control was in the
absence of analogue. In [3H]nicotine and [3H]MLA
binding assays, nonspecific binding was determined in
the presence of 10 mM nicotine and 10 mM MLA,
respectively. Incubation proceeded for 60 min at room
temperature using 96-well plates and was terminated by
harvesting on Unifilter-96 GF/B filter plates, presoaked
in 0.5% polyethylenimine, using a Packard FiterMate
harvester. After washing 5 times with 350 mL ice-cold
assay buffer, filter plates were dried (60 min, 4 ꢃC), bot-
tom-sealed, and filled with Packard’s MicroScint 20
cocktail (40 mL/well). After 60 min, filter plates were
top-sealed, and radioactivity determined. Protein con-
centration was determined using BSA as the standard.55
Table 1. Inhibitory activity of N-n-alkylpicolinium iodide analogues
at native nAChRs
Compd
Nicotine-evoked
[3H]DA overflow
IC50 mMa
[3H]Nicotine
binding Ki mMb
[3H]MLA
binding Ki mMb
NOPiI
NNPiI
NDPiI
NUPiI
NDDPiI
1.0 (ꢅ0.09)
nd
na
62 (ꢅ17)
26 (ꢅ3.6)
na
na
na
na
na
na
0.3 (ꢅ0.05)
nd
0.03 (ꢅ0.02)
na
a Values are means of three to five independent experiments, standard
error is given in parentheses (nd=not determined).
b Values are means of four independent experiments, standard error is
given in parentheses (na=not active).
Table 2. Inhibitory activity of N,N0-bis-picolinium salts at native
nAChRs
Compd
Nicotine-evoked
[3H]DA overflow
(IC50 mM)a
[3H]Nicotine
[3H]MLA
binding (Ki mM)b binding (Ki mM)b
bPiHxI
bPiOI
bPiNB
bPiDI
bPiUB
bPiDDB
1.66 (ꢅ0.85)
0.01 (ꢅ0.009)
1.52 (ꢅ0.34)
0.03 (ꢅ0.01)
1.61 (ꢅ1.08)
0.005 (ꢅ0.003)
na
na
na
na
na
na
na
na
80 (ꢅ17)
na
69 (ꢅ29)
49 (ꢅ17)
a Values are means of four to six independent experiments, standard
error is given in parentheses.
b Values are means of four independent experiments, standard error is
given in parentheses (na=not active).

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membranes. None of the analogues in this series inhib-
ited either [3H]nicotine or [3H]MLA binding with high
affinity, indicating no affinity at a7* nAChRs and only
low affinity at a4b2* nAChRs.
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Furthermore, none of the analogues in this series
evoked [3H]DA overflow (data not shown), and thus,
did not exhibit intrinsic activity at subtypes mediating
nicotine-evoked DA release. Interestingly, the most
active analogues in this series are those with the longer
n-alkyl chain length and with an even number of carbon
atoms (Table 2). Moreover, bPiDDB (C12 analogue)
was the most potent (IC50=5 nM) inhibitor of the
nAChR subtype mediating nicotine-evoked [3H]DA
overflow. bPiDDB is also selective for the nAChR sub-
type mediating nicotine-evoked [3H]DA overflow, since
it has low or no affinity for the a4b2* and a7* nAChR
subtypes (Table 2). Thus, bPiDDB is a selective inhi-
bitor of the nAChR subtype that mediates nicotine-
evoked [3H]DA overflow. Furthermore, bPiDDB is
ꢁ320-fold more potent than DHbE in inhibiting nico-
tine-evoked [3H]DA overflow.45 Importantly, bPiDDB
and the other analogues in this series inhibited [3H]DA
overflow by a maximum of 60%, similar to the max-
imum inhibition observed for a-conotoxin-MII,22,24
suggesting that this small synthetic molecule (bPiDDB)
and the neurtoxic Conus peptide of higher molecular
weight may be acting at the same nAChR subtype to
inhibit nicotine-evoked DA release.
bPiDDB appears to be an excellent candidate for fur-
ther study, since it has 4–5 orders of magnitude higher
affinity for the nAChR subtype mediating nicotine-
evoked [3H]DA overflow compared to its affinity at
both a4b2* and a7* nAChRs. Thus, based on this pre-
clinical data, bPiDDB or related analogues have the
potential to diminish the rewarding effects produced by
nicotine self-administration, and as such may serve as
tobacco use cessation agents.
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Acknowledgements
This work was supported by grants from the National
Institute of Health (DA00399 and DA10934).
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