Epibatidine analogues as selective ligands for the α xβ2-containing subtypes of nicotinic acetylcholine receptors

Article abstract of DOI:10.1016/j.bmcl.2005.06.039

A series of epibatidine analogues was synthesized and characterized in vitro. These compounds are high affinity ligands for the nicotinic acetylcholine receptors (nAChR). They display binding selectivity for the α_xβ₂ subtypes of nAChRs over the α_xβ₄ subtypes, and especially for the α₄β₂ and α₂β₂ subtypes. Furthermore, most of these new nicotinic compounds display little, if any, agonist activities at α₃β₄ nAChR. As a result they might become lead structures for the design and synthesis of highly selective ligands for nAChR subtypes containing the β₂ subunit.

Full text of DOI:10.1016/j.bmcl.2005.06.039

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4385–4388
Epibatidine analogues as selective ligands for the
axb2-containing subtypes of nicotinic acetylcholine receptors
a
c
a,b
Yiyun Huang,a,b, Zhihong Zhu, Yingxian Xiao and Marc Laruelle
*
aDepartment of Psychiatry, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
bDepartment of Radiology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
cDepartment of Pharmacology, Georgetown University School of Medicine, 3900 Reservoir Road, NW, Washington, DC 20057, USA
Received 3 May 2005; revised 8 June 2005; accepted 9 June 2005
Available online 21 July 2005
Abstract—A series of epibatidine analogues was synthesized and characterized in vitro. These compounds are high affinity ligands
for the nicotinic acetylcholine receptors (nAChR). They display binding selectivity for the axb2 subtypes of nAChRs over the axb4
subtypes, and especially for the a4b2 and a2b2 subtypes. Furthermore, most of these new nicotinic compounds display little, if any,
agonist activities at a3b4 nAChR. As a result they might become lead structures for the design and synthesis of highly selective
ligands for nAChR subtypes containing the b2 subunit.
Ó 2005 Elsevier Ltd. All rights reserved.
The neuronal nicotinic acetylcholine receptors (nAC-
hRs) belong to a family of ligand-gated ion channels
that are present in the mammalian central nervous
system (CNS) and peripheral nervous system (PNS).
These receptors mediate neurotransmission at autonom-
ic ganglia in the PNS and participate in the regulation of
neurotransmission in the CNS.1–4 In particular, nAC-
hRs appear to play an important role in cognition and
attention.5,6 Previous studies have found alterations of
nAChRs in a number of neurological and psychiatric
disorders such as schizophrenia, autism, AlzheimerÕs dis-
ease, ParkinsonÕs disease, and dementia with Lewy
bodies.7–13 As a result, nAChRs, in particular the a4b2
and a7 subtypes, have become the targets of drug devel-
opment for the treatment of cognitive and attention
impairment associated with these pathological states,
as well as mood and anxiety disorders.14–16 The avail-
ability of noninvasive in vivo imaging techniques such
as positron emission tomography (PET) and single-pho-
ton emission computed tomography (SPECT) will aid in
the understanding of nAChRs in neurological and psy-
chiatric disorders and in the development of therapeutic
treatments for these diseases. Up to date most available
PET and SPECT imaging agents have been nAChR
agonists, which are toxic when administered in high
doses.17 More recently, Carroll et al. reported the syn-
thesis of epibatidine-based nAChR antagonists that dis-
play much less toxicity.18–20 Our interest in the
development of PET imaging agents has prompted us
to investigate the utility of nAChR antagonists as candi-
date PET ligands that might provide a wider safety mar-
gin in imaging studies. In this paper, we report the
synthesis and in vitro characterization of epibatidine
analogues as high affinity nAChR ligands.
Synthesis of epibatidine analogues is depicted in Scheme
1. Compound 1, synthesized according to the procedures
of Carroll et al.18 underwent Suzuki coupling with a
variety of substituted phenylboronic acids to provide
compounds 2a–i in 78–94% yield. Compounds 2a–c
were then converted to compounds 3a–c in 36–43% yield
by reaction with sodium nitrite and copper (I) chloride.
Alternatively, reaction with sodium nitrite and tetraflu-
oroboronic acid afforded compounds 3d–k in 31–67%.
Reductive methylation of compound 3a with formalde-
hyde and sodium cyanoborohydride in turn provided
compound 4 in 69% yield.21 The structures of com-
1
pounds 3a–k and 4 were confirmed by H NMR and
MS. Conversion of these compounds to their respective
HCl salts was performed by addition of HCl in ether
and isolation of the resulting solids. These salts were
then used in pharmacological tests.
Keywords: Synthesis; Antagonist; Nicotinic receptors.
*
Candidate compounds were assayed for their affinities to
the nAChRs in competitive binding experiments in vitro
Corresponding author. Tel.: +1 212 543 6629; fax: +1 212 568
6171; e-mail: hh285@columbia.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.039

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Y. Huang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4385–4388
Scheme 1. Reagents and conditions: (a) arylboronic acid, Pd(OAc)2, (o-tolyl)3P, Na2CO3, DME; (b) 37% aq HCl, NaNO2, Cu(I)Cl or 48% aq HBF4,
NaNO2; (c) HCHO, NaBH3CN, AcOH, MeCN.
using rat nAChRs (a4b2, a4b4, a2b2, a2b4, a3b2, and a3b4
subtypes) stably expressed on transfected cell lines and
the radioligand [3H]epibatidine.22,23 In addition, com-
pounds were tested for their agonist activities on
86Rb+ efflux from KXa3b4R2 cells that stably express
functional rat a3b4 nicotinic receptors.22 Test results
are listed in Table 1.
Compound 3i, on the other hand, appears to possess
weak agonist activity (36.8% stimulation vs 100% for
(ꢀ)-nicotine at 100 lM). In summary, data from func-
tional assays indicate that compounds 3a–h, j–k, and 4
are nicotinic antagonists at the a3b4 subtype, while com-
pound 3i is a partial agonist.
Carroll et al.18,19 have reported the synthesis, binding
affinities at the ab nAChR in rat cortical membrane,
and antagonism of nicotine-elicited analgesia for com-
pounds 3a, d, g, and h. Binding affinities of these com-
pounds at the ab nAChR in rat cortical membrane are
similar to the affinities for the rat a4b2 and a2b2 subtypes
reported here. We have extended the observation of Car-
roll et al. by providing additional binding data of these
compounds for other nAChR subtypes, and by demon-
strating the binding selectivity of these compounds for
nAChR subtypes containing the b2 subunit. Further,
we obtained data indicating the antagonist nature of
these compounds at the a3b4 nAChR subtype, in addi-
tion to their reported antagonism of nicotine-elicited
analgesia that appears to be mediated by the a4b2 sub-
type.18,19 For these four compounds at least, it can be
stated that they are antagonists at both the a3b4 and
a4b2 nAChR subtypes. However, their functional activi-
ties at other nAChR subtypes remain to be elucidated.
Inspection of the binding data reveals that most com-
pounds display high affinities at the a4b2, a4b4, a2b2,
a2b4, and a3b2 subtypes of nAChR, with Ki values in
the subnanomolar to nanomolar range, while binding
affinities at the nAChR a3b4 subtype are generally lower
(Ki from 15.4 nM for compound 4 to 2940 nM for com-
pound 3k). Especially interesting is the binding selectivity
these compounds display among the nAChR subtypes. In
general, binding affinities for the axb2 subtypes are higher
than those for the axb4 subtypes. For example, the bind-
ing affinities of all compounds for the a4b2 subtype are
one order of magnitude higher than those for the a4b4
subtype, as well as for a2b2 over a2b4 subtype, while affin-
ities of the compounds for the a3b2 subtype are at least
two orders of magnitude higher than those for the a3b4
subtype. Among the axb2 combinations, all compounds
have equal affinities for both the a4b2 and a2b2 subtypes,
while affinities for the a3b2 subtype are 2–10 times lower.
The largest differences in binding affinities are between
the a4b2/a2b2 and a3b4 subtypes, with compound 3b dis-
playing the highest selectivity of 7397-fold for a4b2 over
a3b4 and 9750-fold for a2b2 over a3b4. Taken together,
it appears that these epibatidine analogues possess bind-
ing selectivity for the axb2 subtypes of nAChRs, and in
particular for the a4b2 and a2b2 subtypes. This same sub-
type selectivity has been noted in nAChR agonists among
the epibatidine analogues and pyridinyl ethers,24–26 but
not in the antagonists of Carroll et al.18–20
Examination of structure–activity relationship offers
several interesting observations. First, placement of a
chlorine instead of a fluorine at the 20-position of the
pyridine ring increases the binding affinity across recep-
tor subtypes. For example, the KiÕs of compound 3a
(with a 20-chlorine) are 17 (for a2b2 subtype) to 40 times
(for a3b4 subtype) lower than those of 3d (with a 20-fluo-
rine). Secondly, when chlorine is placed at the 20-posi-
tion of the pyridine ring, placement of a substituent at
the 4-position of the 30-phenyl ring maintains, but does
not increase the binding affinities of the parent com-
pound (compound 3a vs 3b–c). On the other hand, when
fluorine is present at the 20-position of the pyridine ring,
Results from functional assays indicate that all com-
pounds, with the exception of compound 3i, display neg-
ligible agonist activities at the nAChR a3b4 subunit.

Y. Huang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4385–4388
4387
Table 1. Pharmacological properties of new epibatidine analogues: comparison with (ꢀ)-nicotine and ( )-epibatidine
Compound
R1
R2
R3
R4
Ki (nM)a
Agonist effectb
% stimulation
a4b2
a4b4
a2b2
a2b4
a3b2
a3b4
(ꢀ)-Nicotinec
10
40
12
110
47
440
100
112
6.1
3.9
3.8
3.1
0.4
1.0
0.1
3.2
3.6
36.8
2.7
ꢀ0.7
( )-Epibatidinec
0.061
0.16
1.15
1.57
4.75
1.57
26.0
1.22
36.7
3.99
6.42
3.60
7.36
67.6
0.025
0.095
0.470
0.240
5.26
0.035
0.57
24.0
15.4
429
54.5
947
29.0
2400
3a
4
H
H
Cl
Cl
Cl
Cl
F
H
0.039
0.032
0.058
0.034
0.733
0.028
0.898
0.062
0.078
0.039
0.280
1.26
0.037
0.053
0.044
0.040
0.643
0.035
1.11
0.122
0.158
0.171
0.100
3.54
H
H
CH3
H
3b
3c
3d
3e
3f
3g
3h
3i
CH3
OCH3
H
H
H
H
0.862
H
H
16.6
0.541
31.3
CH3
H
H
F
H
0.078
9.27
CH3
H
F
H
Cl
F
F
H
0.064
0.079
0.066
0.245
1.54
4.12
6.12
3.67
6.99
0.339
0.504
0.351
2.31
256
405
H
F
H
CN
Cl
H
F
H
289
334
3j
3k
Cl
CH3
F
H
F
F
H
60.1
8.84
2940
a Competitive binding experiments were conducted as described previously.22 Ten concentrations of each compound were tested for their ability to
compete with [3H]epibatidine (0.5 nM) for binding sites in six stably transfected cell lines, each expressing one of the six defined receptor subtypes:
a2b2, a2b4, a3b2, a3b4, a4b2 and a4b4. Kd values (nM) for [3H]epibatidine used for calculating Ki values were 0.02 for a2b2, 0.08 for a2b4, 0.03 for
a3b2, 0.30 for a3b4, 0.04 for a4b2 and 0.09 for a4b4.23
b Functional properties of compounds were determined by measuring 86Rb+ efflux from KXa3b4R2 cells as described previously.22 The agonist
activity of a compound was determined by measuring stimulated 86Rb+ efflux in the presence of the compound (100 lM). Stimulated 86Rb+ efflux
by 100 lM of (ꢀ)-nicotine was defined as 100% of stimulation.
c Data for (ꢀ)-nicotine and ( )epibatidine were taken from the literature23,28 and are shown here for comparison.
placement of an additional substituent at the 4-position
of the 30-phenyl ring increases binding affinities by at
least one order of magnitude (compound 3d vs 3e and
3g–i). However, substitution at the 3-position of the
30-phenyl ring results in decreased affinities (compound
3d vs 3f). This later observation is in agreement with
the results of Carroll et al. who demonstrated a 2–5
times difference in KiÕs between the 3- and 4-substituted
compounds for the rat cortical nAChR ab subunits.19
Disubstitution on the 30-phenyl ring exerts a more
complex effect on affinities. For example, 3,4-dichloro
substitution increases the binding affinities (3j), while
the 3-methyl-4-fluoro substitution decreases them (3k).
Thirdly, the electronic nature of the substituent at the
4-position of the 30-phenyl ring has minimal effect on
the binding affinities (compare the KiÕs of compounds
3e, and 3g–i). Finally, N-methylation of the 7-azabicy-
clo[2.2.1]heptane moiety induces no significant changes
in binding affinities (3a vs 4), as has been demonstrated
elsewhere by other epibatidine analogues.27
selectivity for the axb2 subtypes of nicotinic acetylcho-
line receptor over the axb4 subtypes, and especially for
the a4b2 and a2b2 subtypes. Carroll et al. have reported
that similar compounds display low affinities for the a7
subtype of nACh receptors.19 Therefore, further struc-
ture–activity relationship studies of this class of com-
pounds might offer the opportunity for the discovery
of subtype selective nicotinic acetylcholine receptor
ligands.
Acknowledgments
This research was supported by the National Institutes
of Health (R21 MH66624-01), by the National Institute
of Mental Health through the Psychoactive Drug
Screening Program (Grant N01 MH32004), and by the
Lieber Center for Schizophrenia Research at Columbia
University.
In summary, a series of epibatidine analogues has been
synthesized. Pharmacological studies indicate that the
synthesized compounds bind with high affinities to all
nicotinic receptor subtypes tested, except the a3b4 sub-
type. In addition, these new compounds display binding
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