Bis-azaaromatic quaternary ammonium salts as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release: An investigation of binding conformation

Article abstract of DOI:10.1016/j.bmcl.2007.10.052

A series of conformationally restricted bis-azaaromatic quaternary ammonium salts (3 and 4) have been designed and synthesized in order to investigate the possible binding conformations of N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB; 2), a

Full text of DOI:10.1016/j.bmcl.2007.10.052

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6734–6738
Bis-azaaromatic quaternary ammonium salts as antagonists
at nicotinic receptors mediating nicotine-evoked dopamine
release: An investigation of binding conformation
Guangrong Zheng, Zhenfa Zhang, Marharyta Pivavarchyk, Agripina G. Deaciuc,
Linda P. Dwoskin and Peter A. Crooks*
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA
Received 17 August 2007; revised 15 October 2007; accepted 15 October 2007
Available online 18 October 2007
Abstract—A series of conformationally restricted bis-azaaromatic quaternary ammonium salts (3 and 4) have been designed and
synthesized in order to investigate the possible binding conformations of N,N0-dodecane-1,12-diyl-bis-3-picolinium dibromide
(bPiDDB; 2), a compound which potently inhibits neuronal nicotinic acetylcholine receptors (nAChRs) mediating nicotine-evoked
dopamine release. The preliminary structure–activity relationships of these new analogues suggest that bPiDDB binds in an
extended conformation at the nAChR binding site, and that flexibility of the linker may be important for its high potency in inhib-
iting nAChRs mediating nicotine-evoked dopamine release.
Ó 2007 Elsevier Ltd. All rights reserved.
Tobacco contains one of the most widely abused psy-
choactive substances in the world, that is, (S)-nicotine
(1; Fig. 1), which is believed to be primarily responsible
for tobacco dependence.1,2 Like many abused drugs,
nicotine addiction has been linked to the release of the
neurotransmitter, dopamine (DA).3–5 Nicotine-evoked
DA release, which is thought to be responsible for re-
ward, leading to addiction, is believed to result from
activation of presynaptic neuronal nicotinic acetylcho-
line receptors (nAChRs).6–12 Nicotine stimulates all
known nAChR subtypes13 and upon activation, these
receptors modulate the release of various neurotransmit-
ters.14–16 The subunit composition of nAChR subtypes
responsible for mediating nicotine-evoked DA release
has not been elucidated conclusively.17 In this regard,
subtype-selective nAChR antagonists, which inhibit nic-
otine-evoked DA release, may have potential as novel
treatments for nicotine addiction.18–21
nAChR subtype(s) mediating nicotine-evoked [3H]DA
release from superfused rat striatal slices in vitro
(IC50 = 5 nM), and did not interact at the ligand binding
site of either a4b2* or a7* nAChRs.22,23 In vivo microdi-
alysis studies demonstrated that pretreatment with
bPiDDB dose-dependently reduced the increase in
extracellular DA in rat nucleus accumbens produced
by acute or repeated nicotine treatment.24 In addition,
we have demonstrated that despite the cationic nature
and polarity of the molecule, bPiDDB is brain bioavail-
able, due to its facilitated transport via the blood–brain
barrier choline transporter.25 Moreover, behavioral
studies in rats showed that bPiDDB dose-dependently
decreased intravenous nicotine self-administration, but
not sucrose-maintained responding, suggesting a specific
inhibition of nicotine reward.26 Taken together,
bPiDDB and its analogues represent lead compounds
Previous work in our laboratories has led to the discov-
ery of N,N0-dodecane-1,12-diyl-bis-3-picolinium dibro-
mide (bPiDDB; 2; Fig. 1), which potently inhibited
CH3
2Br
N
CH3
N
N
N
Keywords: Nicotinic acetylcholine receptor; Quaternary ammonium;
Dopamine release; Nicotine addiction.
CH3
(S)-nicotine (1)
Figure 1. Structures of (S)-nicotine (1) and bPiDDB (2).
bPiDDB (2)
*
Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257
7585; e-mail: pcrooks@email.uky.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.052

G. Zheng et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6734–6738
6735
for the development of a new class of therapeutic agents
for the treatment of tobacco dependence.
The design of the model compounds incorporated a ben-
zene ring into the middle of the N-N linker unit, allow-
ing a variety of arrangements of the two methylene
linker units around the aromatic ring (i.e., 1,2-, 1,3-,
or 1,4-positions) (see 3a, 3b, 3c, respectively, Scheme
1), and thereby, constraining these molecules into an ‘ex-
tended’ or ‘angular’ geometry. In this respect, rigidifica-
tion has been used extensively in drug design to lock
ligands into a desired conformation or geometry with
the goal of increasing activity and selectivity of the ana-
logues. In the current series of compounds, a triple bond
also has been introduced into each of the linker units at-
tached to the central phenyl ring to provide further
restriction of conformational freedom and geometry
(4a, 4b, 4c, Scheme 1).
bPiDDB (2) contains two 3-picolinium head groups
connected via an N-N-12-methylene linker unit; thus,
it is a highly flexible, di-cationic molecule. A better
understanding of the potential binding conformation
of bPiDDB at nAChRs responsible for mediating nico-
tine-evoked DA release may provide some insight into
the nature of the pharmacophore requirements for opti-
mal inhibition of this receptor. Energy minimization cal-
culations reveal that the lowest energy conformation of
bPiDDB is one in which the N-N alkyl linker is fully
extended.27 However, the low energy conformations of
ligand molecules may not always reflect the conformation
in which the molecule binds to the receptor. Utilizing
this extended or linear low energy conformation, a
QSAR model utilizing a back-propagation artificial neu-
ral network approach has been constructed and has
been found to afford good predictivity for inhibition of
nicotine-evoked DA release.27 The hypothesis that
bPiDDB binds to nAChR subtype(s) mediating nico-
tine-evoked DA release in a ‘linear’ or ‘extended’ con-
formation is supported by experimentally determined
data23 showing that analogues with shorter N-N alkyl
linker units have decreased inhibitory potency compared
to bPiDDB. In the present study, we designed a series of
model compounds which mimic different binding con-
formations of bPiDDB to provide further information
on the active conformation of this novel nAChR
antagonist.
The requisite compoundsof general structure 3 and 4were
prepared by the route shown in Scheme 1. The synthesis
was initiated with Sonogashira coupling28 of 1,2-, 1,3-,
or 1,4-dibromobenzene with 4-pentyn-1-ol (for the 1,2-
and 1,3-isomers) or 3-butyn-1-ol (for 1,4-isomers) to af-
ford compound 5a (1,2-isomer), 5b (1,3-isomer), or 5c
(1,4-isomer). In the synthesis of compound 5a, the reac-
tion required a prolonged heating time (at 80 °C) for
6 days for completion. Compounds 5a–5c and their corre-
sponding Pd/C-catalyzed hydrogenation products were
transformed into dibromides 7a–7c and 6a–6c, respec-
tively, by bromination using PPh3/CBr4.29 Alkylation of
the corresponding azaaromatic free bases, including 2-
picoline, 3-picoline, 4-picoline, and nicotine, using the
above obtained dibromide 6a, 6b, 6c, 7a, 7b, or 7c, affor-
ded the corresponding bis-azaaromatic quaternary
ammonium compound 3a, 3b, 3c, 4a, 4b, or 4c, respec-
tively (Table 1). Among these analogues, the 1,2-isomers
in both the 3 and 4 series (3a and 4a, respectively) and 1,3-
isomers in series 4 (i.e., 4b) represent ‘angular’ conforma-
tions of bPiDDB, whereas the 1,3-isomers in series 3 (i.e.,
3b, see Fig. 2 for the difference between 3b and 4b) and the
1,4-isomers in both series 3 and 4 (i.e., 3c and 4c, respec-
tively) represent ‘extended’ conformations.
OH
( )n
Br
a
5a 1,2-; n = 2
5b 1,3-; n = 2
5c 1,4-; n = 1
Br
( )n
OH
b,c
c
Br
These analogues were evaluated for their ability to inhi-
bit nicotine-evoked [3H]DA release from superfused rat
striatal slices and to inhibit (S)-(ꢀ)-[3H]nicotine
([3H]NIC) binding (probing a4b2* nAChRs) and
[3H]methyllycaconitine ([3H]MLA) binding (probing
a7* nAChRs) to rat brain membranes. [3H]NIC and
[3H]MLA binding assays were performed using 3 and
2.5 nM concentrations of radioligand, respectively, and
10 lM NIC and 10 lM MLA to assess nonspecific bind-
ing to whole brain membranes.30 Analogues were evalu-
ated at a probe concentration of 100 nM. The amount of
inhibition is presented as a percentage of radioligand
binding in the absence of analogue (control, Table 1).
Analogue-induced inhibition of nicotine-evoked
[3H]DA release was determined using 10 lM nicotine
and 100 nM analogue.30 Inhibition is presented as a per-
centage of the response to nicotine under control condi-
tions (in the absence of analogue) and the values are
provided in Table 1.
( )n
Br
( )n
7a 1,2-; n = 2
6a 1,2-; n = 2
7b 1,3-; n = 2
( )n
6b 1,3-; n = 2
7c 1,4-; n = 1
6c 1,4-; n = 1
Br
( )n
Br
d
d
R
R
N
N
( )n
( )n
2Br
( )n
2Br
4a 1,2-; n = 2
4b 1,3-; n = 2
4c 1,4-; n = 1
N
R
( )n
N
3a 1,2-; n = 2
3b 1,3-; n = 2
3c 1,4-; n = 1
R
Scheme 1. Reagents and conditions: (a) 3-butyn-1-ol or 4-pentyn-1-ol,
Pd(PPh3)2Cl2, CuI, Et3N, 80 °C; (b) H2, 10% Pd/C, MeOH, 45 psi, rt;
(c) PPh3, CBr4, CH2Cl2, 0 °C; (d) azaaromatic compounds, 60 °C.
All of the analogues containing 2-, 3-, or 4-picolinium
head groups showed little or no affinity for either

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G. Zheng et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6734–6738
Table 1. Inhibition of [3H]NIC binding and [3H]MLA binding to rat brain membranes, and inhibition of nicotine-evoked [3H]DA release from
superfused rat striatal slices by bis-azaaromatic quaternary ammonium analogues
Compounda
Head group
[3H]NIC binding
(% inhibition at 100 nM)b
[3H]MLA binding
(% inhibition at 100 nM)b
Inhibition of nicotine-evoked
[3H]DA release
(% inhibition at 100 nM)b
bPiDDB
0
0
0
0
0
0
64
1,2-Isomers (3a)
3aa
3ab
7.6 4.3
8
3
5
3
N
N
0
0
0
0
5.4 3.4
3ac
3ad
3.1 3.1
11 5.5
11
3
N
71 2.0
59 16
N
CH3
N
N
N
1,3-Isomers (3b)
3ba
3bb
7.2 2.8
3.5 3.5
0
0
62
45
9
8
6.24 2.5
3.5 1.6
3bc
4.6 4.0
27 17
N
N
N
1,4-Isomers (3c)
3ca
3cb
3cc
6.30 4.21
4.33 2.19
1.10 0.89
0.16 0.16
7.41 4.07
7.73 2.41
40 22
44 19
32 16
N
N
3cd
3.66 2.39
5.3 5.3
17 12
N
CH3
1,2-Isomers (4a)
4aa
4ab
2.1 2.1
3.9 2.8
1.2 1.2
25
4
N
N
0
0
46 13
4ac
4ad
2.5 1.5
5.4 3.2
12
7
N
N
72 3.0
9.2 3.0
35 20
N
CH3

G. Zheng et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6734–6738
6737
Table 1 (continued)
Compounda
Head group
[3H]NIC binding
(% inhibition at 100 nM)b
[3H]MLA binding
(% inhibition at 100 nM)b
Inhibition of nicotine-evoked
[3H]DA release
(% inhibition at 100 nM)b
1,3-Isomers (4b)
4ba
4bb
1.9 0.7
0.5 0.4
0
0
35 15
N
N
8.0 3.0
6.4 3.5
2.6 1.6
6
4
4bc
4bd
10 5.6
3.1 1.7
20 12
18 10
N
N
CH3
N
N
1,4-Isomers (4c)
4cb
4cc
13.0 5.4
8.9 2.5
3.1 3.1
2.7 2.7
32 11
19 11
N
a The first letter in the compound designation indicates orientation around the central phenyl ring (i.e., a = 1,2; b = 1,3, and c = 1,4). The second
letter indicates the nature of the head group (i.e., a = 2-picolinium, b = 3-picolinium, c = 4-picolinum, and d = nicotinium).
b Each value represents data from at least three independent experiments, each performed in duplicate.
a4b2* or a7* nAChRs. However, some of the analogues
which contained nicotinium head groups, i.e., com-
pounds 3ad and 4ad, displayed considerable affinity at
a4b2*, which is consistent with the results obtained with
our previously reported mono-nicotinium and bis-nico-
tinium analogues.22,31
compared to the extended conformation in the simple
picolinium molecules.
Within the more conformationally rigid analogue series
(general structure 4), several differences in structure–
activity relationships were observed compared to those
in the more conformationally flexible 3 series. For exam-
ple, compound 4bb, a 3-picolinium analogue in the 1,3-
isomer series (4b), had significantly lower potency than
the two structurally related 3-picolinium analogues,
4ab in the 1,2-isomer series, and 4cb in the 1,4-isomer
series. Compound 4bb was also the least potent com-
pound in the 4 series. Of the 4-picolinium analogues in
the 4 series, inhibitory potency was similar between
the 1,4-analogue 4cc and the 1,2- and 1,3-analogues
4ac and 4bc.
Among the analogues of general structure 3 (Scheme 1),
the 1,2-isomers (3a), including compounds 3aa, 3ab, and
3ac, exhibited significantly lower potency for inhibition
of nicotine-evoked [3H]DA release than the 1,3- and
1,4-isomers (3b and 3c), including compounds 3ba,
3bb, 3bc, 3ca, 3cb, and 3cc. The 1,3- and 1,4-picolinium
analogues all had similar potency. The position of the
methyl group on the quaternary ammonium head group
did not significantly contribute to the inhibitory po-
tency. These results indicate that the active conforma-
tion for binding at nAChR subtypes mediating
nicotine-evoked DA release is suggested to be in a more
extended conformation, as reflected in the 1,3- and 1,4-
isomer series, rather than in the more angular conforma-
tion in the 1,2-isomer series. Of additional interest is the
observation that the 4-picolinium analogues in the 1,3-
and 1,4-series were the least potent compounds.
The transition from the more conformationally flexible
compounds of general structure 3 to the more conform-
ationally restricted compounds of structure 4 results
generally in a decrease in inhibitory potency of nico-
tine-evoked [3H]DA release. This finding supports the
interpretation that retaining the flexibility of the methy-
lene linker, such as is the case in bPiDDB, is important
for high inhibitory potency and optimal interaction with
the nAChR protein. From the structure–activity rela-
tionship of compounds of general structure 3, it is sug-
gested that bPiDDB binds in an ‘extended’
conformation, rather than an ‘angular’ conformation.
A possible explanation for the greater potency of the
more flexible analogues is that the binding site of the
nAChR subtype mediating nicotine-evoked DA release
Interestingly, in the 1,2- nicotinium compounds, 3ad and
4ad were more potent than the 1,4-compound 3cd and
the 1,3-compound 4bd which is inconsistent with what
was observed in the picolinium series. This suggests that
the addition of a second functionality in the head group,
that is, the basic pyrrolidino moiety, may promote
binding of a more angular molecular conformation

6738
G. Zheng et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6734–6738
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R
R
R
N
N
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3b
4b
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R
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Figure 2. Structures of compounds 3b and 4b.
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a ‘linear’ or ‘extended’
conformation of bPiDDB, nevertheless have lower po-
tency than their more flexible counterparts 3bb, 3bc,
3cb, and 3cc.
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In summary, a series of conformationally restricted bis-
azaaromatic quaternary ammonium analogues have been
designed and synthesized as structural models to probe
the possible binding conformation of the bPiDDB mole-
cule. From the ability of these analogues to inhibit nico-
tine-evoked DA release, the results suggest that
bPiDDB binds to the nAChR protein in an ‘extended’
conformation. However, flexibility of the methylene lin-
ker in the molecule is also important for high inhibitory
potency. Due to the limitation of the single concentration
assay utilized in the screening protocol, further studies are
needed to substantiate these conclusions. Nevertheless,
these findings may be of value in the further design of
bPiDDB analogues as nAChR antagonists.
24. Rahman, S.; Neugebauer, N. M.; Zhang, Z.; Crooks, P.
A.; Dwoskin, L. P.; Bardo, M. T. Neuropharmacology
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Acknowledgment
This research was supported by NIH Grant U19
DA17548.
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