Neonicotinic analogues: Selective antagonists for α4β2 nicotinic acetylcholine receptors

Article abstract of DOI:10.1016/j.bmc.2013.03.024

Nicotine is an agonist of nicotinic acetylcholine receptors (nAChRs) that has been extensively used as a template for the synthesis of α4β2-preferring nAChRs. Here, we used the N-methyl-pyrrolidine moiety of nicotine to design and synthesise novel α4β2-preferring neonicotinic ligands. We increased the distance between the basic nitrogen and aromatic group of nicotine by introducing an ester functionality that also mimics acetylcholine (Fig. 2). Additionally, we introduced a benzyloxy group linked to the benzoyl moiety. Although the neonicotinic compounds fully inhibited binding of both [α-¹²⁵I]bungarotoxin to human α7 nAChRs and [³H]cytisine to human α4β2 nAChRs, they were markedly more potent at displacing radioligand binding to human α4β2 nAChRs than to α7 nAChRs. Functional assays showed that the neonicotinic compounds behave as antagonists at α4β2 and α4β2α5 nAChRs. Substitutions on the aromatic ring of the compounds produced compounds that displayed marked selectivity for α4β2 or α4β2α5 nAChRs. Docking of the compounds on homology models of the agonist binding site at the α4/β2 subunit interfaces of α4β2 nAChRs suggested the compounds inhibit function of this nAChR type by binding the agonist binding site.

Full text of DOI:10.1016/j.bmc.2013.03.024

Bioorganic & Medicinal Chemistry 21 (2013) 2687–2694
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Neonicotinic analogues: Selective antagonists for
acetylcholine receptors
a4b2 nicotinic
Manuel Faundez-Parraguez a, Nicolas Farias-Rabelo a, Juan Pablo Gonzalez-Gutierrez a,
Alvaro Etcheverry-Berrios a, Jans Alzate-Morales b, Francisco Adasme-Carreño b, Rodrigo Varas c,
Isabel Bermudez d, Patricio Iturriaga-Vasquez a,e,
⇑
a Department of Chemistry, Faculty of Sciences, University of Chile, Santiago, Chile
b Center for Bioinformatics and Molecular Simulation (CBSM), University of Talca, Talca, Chile
c Millennium Science Nucleus in Stress and Addiction and Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile
d Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, UK
e Interdisciplinary Center for Ionic Liquids, University of Chile, Santiago, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
Nicotine is an agonist of nicotinic acetylcholine receptors (nAChRs) that has been extensively used as a
Received 14 December 2012
Revised 8 March 2013
Accepted 16 March 2013
Available online 26 March 2013
template for the synthesis of
of nicotine to design and synthesise novel
tance between the basic nitrogen and aromatic group of nicotine by introducing an ester functionality
that also mimics acetylcholine (Fig. 2). Additionally, we introduced a benzyloxy group linked to the ben-
a
4b2-preferring nAChRs. Here, we used the N-methyl-pyrrolidine moiety
4b2-preferring neonicotinic ligands. We increased the dis-
a
zoyl moiety. Although the neonicotinic compounds fully inhibited binding of both [
to human 4b2 nAChRs, they were markedly more potent at
7 nAChRs and [3H]cytisine to human
displacing radioligand binding to human 4b2 nAChRs than to 7 nAChRs. Functional assays showed that
the neonicotinic compounds behave as antagonists at 4b2 and 4b2 5 nAChRs. Substitutions on the
aromatic ring of the compounds produced compounds that displayed marked selectivity for 4b2 or
5 nAChRs. Docking of the compounds on homology models of the agonist binding site at the
4/b2 subunit interfaces of 4b2 nAChRs suggested the compounds inhibit function of this nAChR type
a-
125I]bungarotoxin
Keywords:
a
a
Nicotinic acetylcholine receptors
Structure–activity relationships
Functional and affinities
Antagonism
a
a
a
a
a
a
a4b2a
a
a
by binding the agonist binding site.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
ubunit combinations yield functional nAChR that differ consider-
ably in their functional and pharmacological properties.11 The
most abundant form of heteromeric nAChR in the brain contains
Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-
gated ion channels1 expressed in the central and peripheral ner-
vous systems2 that respond to the neurotransmitter acetylcholine
(ACh)3 and exogenous compounds such as nicotine, cytisine and
epibatidine4,5 (Fig. 1). Neuronal nAChRs are predominantly located
in extra-synaptic regions, from where they modulate the release of
ACh and other neurotransmitters such as dopamine, GABA, gluta-
mate, noradrenalin and serotonin,6–8 which makes nAChR-signal-
ing one of the most important modulatory system in the nervous
system.8,9 Mammalian neuronal nAChRs assemble from combina-
a
4 and b2 subunits (
combine with other subunits such as
4b2 nAChRs bind nicotine with high affinity.6 They have been
a
4b2 nAChR). The
a
4 and b2 subunits also
a
5 to form
a
4b2 5 nAChRs.
a
a
implicated in nicotine self-administration, reward, nociception,
mood and cognition, and in diseases such as Alzheimer’s disease,
epilepsy, pain disorders and depression.6,10,12–14 Therefore, the
perceived validity of a4b2 nAChR as therapeutic targets has stim-
ulated the synthesis of a wide variety of novel compounds that
have added to the list of nicotinic drugs and pharmacological
tools.10,15–17
The crystal structure of a soluble ACh binding protein (AChBP)
from Aplysia californica and other molluscs18–20 has been used as
a template to build homology models of the agonist binding
domain of nAChRs.21 The agonist binding domain in nAChRs is a
hydrophobic pocket that lies at the interface between the extracel-
lular N-terminal domains of two adjacent subunits.4,10,21 In heter-
tions of nine
a
(a2–a
10) and three b (b2–b4) subunits.4 Some of
7 nAC-
these subunits form homo-pentameric receptors such as
hRs, whilst other subunits assemble into hetero-pentameric struc-
tures with various combinations of
a
a
and b subunits.10 Different
s-
⇑
Corresponding author. Tel.: +56 2 2978 7253; fax: +56 2 2271 3888.
E-mail address: iturriag@uchile.cl (P. Iturriaga-Vasquez).
omeric nAChRs,
a subunit contributes what is termed the principal
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.024

2688
M. Faundez-Parraguez et al. / Bioorg. Med. Chem. 21 (2013) 2687–2694
2. Results and discussion
O
N+
N
2.1. Synthesis
O
Thesynthesisofthe analogues was performedusingthe commer-
cial (S)-(À)-1-methyl-2-pyrrolidinemethanol as a template for the
pyrrolidine moiety of nicotine. Benzoyl chloride derivatives were
prepared using thionylchloride as the halogenating reagent, with
the exception of 6-chloronicotinoyl chloride, 4-bromo- and 4-chlo-
robenzoylchlorides that are commercially available from Aldrich.
The corresponding acids were mixed with thionylchloride using
dry THF as a solvent and refluxing for 24 h under N2 atmosphere.
Then, the solvent was evaporated under reduced pressure and the
acyl chloride obtained was used immediately to minimize its hydro-
lysis. The resulting acylchloride was dissolved in dry diethyl ether
with stirring and (S)-(À)-1-methyl-2-pyrrolidinemethanol was
added drop by drop to obtain the corresponding esters. To obtain
benzyloxy derivatives attachedto the corresponding 4-hydroxyben-
zoate or 6-hydroxynicotinates, we used a Fischer esterification
reaction to yield the corresponding methyl ester,27 followed by the
Williamson ether synthesis using benzylchloride and NaHCO3 in
N
Nicotine
Acetylcholine (ACh)
NH
NH
N
Cl
N
O
Cytisine
Epibatidine
Figure 1. Classical nicotinic agonists.
component (or positive face), whereas the non-
b4 in neuronal nAChRs and and d in the muscle nAChR
contribute the complementary component (or negative face). The
principal component comprises loop A, B and C, whereas the com-
plementary face consists of loops D, E and F.22 In the case of homo-
a subunit (b2 and
c, e
acetonitrile.28 Then
a basic Fischer hydrolysis produced the
meric nAChRs (e.g.,
a7 receptors), the a subunit contributes the
O-benzylated aromatic acid. This strategy was used to minimize
the problem posed by the insolubility of acids in the Williamsonsyn-
thesis, since in the presence of a base such as NaHCO3 a precipitate
forms, reducing the reaction yield considerably (Fig. 3).
primary and complementary faces of the agonist binding domain.
Loop C, which is positioned at the entrance of the agonist binding
domain, changes its conformation when an agonist or competitive
antagonist binds, and this appears to be an essential part of the
coupling mechanism that results in the opening of the ion chan-
nel.23 Highly conserved aromatic residues that are critical for the
binding of the neurotransmitter are located within loops A, B, C,
D and E. These residues are Y93 (loop A), W149 (loop B), Y190
and Y198 (loop C), W55 and W57 (loop D). (Numbering of residues
correspond to that of the Torpedo nAChR).22 Generally, the identity
of the hydrophobic residues on the principal component defines li-
gand affinity, whereas the residues contributed by the complemen-
tary face appear to determine ligand selectivity.24 Typically,
nicotinic agonists carry a positively charged nitrogen moiety that
is stabilized by electronegative interactions with the conserved
aromatic residues of the ligand binding site. Thus, the positive
To obtain the 3-aminobenzoyl derivative we used the classical
aromatic electrophilic nitration of methyl benzoate by a concen-
trated sulphuric–nitric acid mixture. This reaction produced the ni-
tro derivative in high yields, and the reaction product crystallized
in the reaction mixture. To obtain the 3-nitrobenzoyl ester of (S)-
(À)-1-methyl-2-pyrrolidinemethanol, the reaction was conducted
as mentioned above. The nitro group was reduced via catalytic
hydrogenation using 10% Pd/C in ethanol, which proved to be a fas-
ter and cleaner way of generating the aromatic amino group
(Fig. 4).
2.2. Biological evaluation
charged nitrogen moiety of ACh is stabilised by
p–cation interac-
2.2.1. Radioligand binding studies
tions with the electron rich aromatic side chain of the conserved
amino acids, specifically W149.25 Agonists also interact with other
residues within the agonist site through a number of electronega-
tive and hydrophobic interactions such as hydrogen bonding and
van der Waals interactions.20
First, we determined binding affinities (Ki) of the synthesised
neonicotinic compounds for human
nAChRs using respectively [3H]cytisine ([3H]cyt) and [125I]
garotoxin ( -BgTx) binding competition assays to evaluate the
a4b2 nAChRs and human
a7
a
-bun-
a
nAChR selectivity of the compounds. These radioligands are estab-
Guided by available information on how nicotinic ligands bind
nAChRs and the chemical structure of nicotine, we synthesised
simple molecules capable of interacting with the agonist binding
lished nicotinic ligands that bind the agonist site of, respectively
a
4b2 nACRs and
membrane homogenates prepared from SH-EP1-h
or SH-SY5Y-h 7 clonal cells. It has been shown previously that SH-
SY5Y-h 7 cells over-express human 7 nAChRs and that SH-EP-
4b2 cells express human
4b2 nAChRs.31–33 The latter cell line
is likely to express alternate forms of the 4b2 nAChR, namely
4b2)2 4 and ( -BgTx (1 0.2 nM)
4b2)2b2.34 The Kd for [125I]
binding to 4b2 nAChRs
7 nAChRs and [3H]cyt binding to
(0.43 0.08 nM) were comparable to previously published data.33
Figure 5 shows that specific binding of both [125I]
-BgTx to
nAChRs and [3H]cyt to
4b2 nAChRs was fully displaced by the
a
7 nAChRs.29,30 The assays were carried out on
a
4b2 clonal cells
site of
a4b2 nAChRs via
p–cation interactions. Here, we report
a
the synthesis of a series of nicotine derivatives that preserve the
N-methyl-pyrrolidine moiety of nicotine. We used as a lead mole-
a
a
ha
a
cule compound 1,
a
previously described neonicotinic com-
a
pound10,26 (Fig. 2). The neonicotinic compounds shown in Figure
2 were synthesised by esterification reactions with substituted
benzoic and nicotinic acid derivatives. We increased the distance
between the basic nitrogen and the aromatic group of nicotine
by introducing an ester functionality that also mimics acetylcho-
line (Fig. 2). Additionally, we introduced a benzyloxy group linked
to the benzoyl moiety. Subsequent radioligand binding and func-
tional assays in conjunction with homology modelling analysis
showed that introduction of an ester group between the pyrroli-
dine moiety and the aromatic ring of nicotine confers antagonist
(a
a
a
a
a
a
a
a7
a
synthesised compounds in a concentration-dependent and mono-
phasic manner. This pattern of inhibition strongly suggests that
the compounds bind the agonist site of the receptors. The Ki values
estimated for the affinity of the compounds for
a4b2 and a7
nAChRs are shown in Table 1. For comparison we include the affin-
properties to these ligands that selectively bind through
p–cation
ity of nicotine for both receptor types. All the compounds tested
interactions the agonist binding sites of 4b2 nAChRs. The com-
a
were significantly more potent at a4b2 nAChRs than at a7 nAChRs.
pounds also interact with the agonist binding site of
a7 nAChRs, al-
Despite the differences in affinity, the pattern of affinity change
beit with lower affinity.
obtained by incorporating various types of substituents into

M. Faundez-Parraguez et al. / Bioorg. Med. Chem. 21 (2013) 2687–2694
2689
O
O
N
N
O
O
X
X
N
1 X= H
4 X= Br
5 X= Cl
2 X= NO2
3 X= NH2
O
O
N
N
O
O
N
Cl
7
6
O
O
N
O
N
O
BnO
N
9
BnO
8
Bn = C6H5CH2
Figure 2. Simple neo-nicotinic analogues synthesised in this study.
O
O
O
C6H5CH2Cl/NaHCO3
AcCN/Reflux/72 hrs
MeOH/H2SO4
Reflux/ 5 days
OMe
OH
OMe
BnO
X
HO
X
HO
X
X= N or C
O
O
N
HO
SOCl2/THF
24 hrs /r.t.
KOH/H2O
Reflux/ 1 hr
Cl
OH
Et2O/ 24 hrs/ r.t.
BnO
X
BnO
O
X
8)
X= C (compound
N
X= N (compound 9)
O
BnO
X
Bn = C6H5CH2
Figure 3. Synthesis of benzyloxy derivatives compounds 8 and 9.
compound 1 was comparable in both receptor types, except for
compound 3. Thus, compound 8 showed the highest affinity in
2.2.2. Functional studies
Next, we determined the functional effects of the most potent
neonicotinic compounds (compounds 1, 3, 5 and 8) on 4b2n ACh-
the series with a Ki of 1.2 nM
a
4b2 nAChRs and of 1.1
l
M at
a7
a
nAChRs. In contrast, the pyridyl analogue 9 was markedly less po-
tent at both receptor types, suggesting that in this molecule the
presence of a pyridyl group leads to an unfavourable interaction
Rs expressed heterologously in Xenopus oocytes. The studies were
carried out using two-electrode voltage clamping procedures de-
scribed in the Section 4. We assayed the effects of the compounds
with the agonist binding site in both
a4b2 and
a
7 nAChRs. The
on three different subtypes of
a4b2 nAChRs, namely (
a
4b2)2b2,
presence of a nitro group (2) or a quinoline (7) moiety decreases
affinity in comparison to compound 1 at both receptor types. Addi-
tionally, we tested two 4-halobenzoate derivatives (compounds 4
and 5) and the 6-chlorinicotinate analogue 6. The binding affinities
(a4b2)2a4 and (a4b2)2a5 nAChRs. These a4b2 nAChRs display dif-
ferent functional sensitivity to nicotinic ligands,34–36 and they all
likely express in the brain.37 All of the compounds tested inhibited
the currents evoked by ACh at (
nAChRs. Estimated IC50 values are summarised in Table 2. For com-
parison, we include IC50 values estimated for the 4b2-preferring
competitive antagonist dihydro-b-erythroidine (DhbE) (Table 2).
Compounds 1, 3 and 5 displayed marked 4b2 nAChR subtype
selectivity. Thus, compounds 1 and 5 had higher inhibitory potency
at ( 4b2)2b2and ( 4b2)2 5 than at ( 4b2)2 4, whereas compound
3 showed marked preference for ( 4b2)2 4 and ( 4b2)2b2 nAChRs.
The latter compound is particularly interesting because it discrim-
inates between ( 4b2)2b2 and ( 4b2)2 5 nAChRs. In general,
a4b2)2b2, (a4b2)2a4 or (a4b2)2a5
at a4b2 nAChRs show that both 4-halobenzoate derivatives 4 and 5
bind at nanomolar concentrations (Ki 22.4 and 11.2 nM, respec-
tively), but the chloronicotinate derivative 6 had an almost tenfold
lower affinity compared with 5 (151 nM vs 11.2 nM) (Table 1). Of
a
a
all the compounds tested, 5 displayed high affinity at
(Ki 4.5 M) followed by 1 and 4 (Ki 6.7 M and Ki 10.5
tively) (Table 1). Taken together, the binding data demonstrate that
the synthesised compounds display higher selectivity for 4b2
nAChRs than for 7 nAChRs.
a7 nAChRs
l
l
lM respec-
a
a
a
a
a
a
a
a
a
a
a
a
a

2690
M. Faundez-Parraguez et al. / Bioorg. Med. Chem. 21 (2013) 2687–2694
O
O
O
H2SO4/HNO3
4° C/ 1 hr
KOH/H2O
OH
OMe
OMe
Reflux/ 1 hr
O
O
NO2
NO2
Et2O/ 24 hrs/ r.t.
N
N
SOCl2/THF
24 hrs /r.t.
O
Cl
HO
NO2
NO2
O
N
Pd/C 10% / EtOH
H2-70 psi / 3 hrs
O
NH2
Figure 4. Synthesis of 3-nitro and 3-amino derivatives compounds 2 and 3.
Figure 5. Effects of neonicotinic compounds on binding of [3H]cytisine to human
a
4b2 nAChRs and [125I]-
a-bungarotoxin to human
a7 nAChRs. Data points represent the
mean SEM of four experiments, each carried out in triplicate. The radioligand concentration in all displacement studies was 1 nM. The concentration-inhibition data was
analysed non-linearly as described in Section 4.
Table 1
Table 2
Comparisons of binding affinities of neonicotinoid compounds at human
nAChRs
a
7 and
a
4b2
Inhibitory potency of neonicotinoid compounds at
a
4b2 nAChRs
Compound
(
a
4b2)2b2 (
lM)
(
a
4b2)2
313 12
1.02 0.4
95
3.6 0.7
0.02
a
4 ( M) (a4b2)2a5 (lM)
l
Compound
a
4b2 IC50 (nM)
a
4b2 Ki (nM)
a
7 IC50
(lM)
a
7 Ki (
3.8
6.7
lM)
1
3
5
8
2.5 0.9
4.3 0.6
3.6 0.7
52.3
480
6.0
17.8 0.9
0.011
4
8
1
Nicotine
1.2
81.9
0.6
7.6
13.4
1
2
3
4
5
6
7
8
9
5
23.4
460
8.1
6
8
1
3
2
5
6
1
1
7
2
1611 12
464 17
N.E.
232
25.8 1.2
0.41
28.4
78.4
39.4
3
6
5
DhbE
22.4
11.2
151
217
1.2 0.3
45.5
21.1
8.9
58.5
97.3
4
5
7
6
10.5
2
The effects of the compounds were studied on (
nAChRs expressed heterologously in Xenopus oocytes as described elsewhere. The
inhibitory potency of the 4b2-preferring competitive antagonist dihydro-b-ery-
a4b2)2b2, (a4b2)2a4 and (a4b2)2a5
4.5 0.8
528 10
759
4.2 0.8
166
29.3
48.7
1.1 0.4
29.5
2
3
a
9
throidine (DhbE) is included for comparison.
2.3 0.6
59.1
7
4
3
3
Data represent the mean SEM of four experiments, each with triplicate samples.
For comparison we include equivalent Ki and IC50 estimates for nicotine. The
radioligand concentration in all displacement studies was 1 nM and the equilibrium
the inhibitory potency of compound 1 at (a4b2)2a4 nAChRs, bring-
ing it closer to that displayed at (
the aromatic ring of compound 1 at the 4 position (compound 5)
increased slightly the inhibitory potency at ( 4b2)2 4 nAChRs
(from 313 to 95 M), whereas at ( 4b2)2 5 nAChRs it increased
it by almost 10-fold without affecting potency at ( 4b2)2b2 nAC-
hRs. Of all compounds tested, compound 8, the bulkiest molecule
of the group tested, displayed the least 4b2 nAChR subtype selec-
tivity. Thus, even though compound 8 was more potent at inhibit-
ing ( 4b2)2 4 nAChRs (IC50 of 3.6 M), its inhibitory potency at
4b2)2b2 and ( 4b2)2 5 nAChRs decreased by only 5 and 7 times,
a4b2)2b2 nAChRs. Chlorination of
dissociation constants (Kd) used to estimate Ki values were 1 nM for [125I]
a-BgTx
7 nAChRs and 0.4 nM for the binding of [3H]cyt to human
a4b2
a
a
binding to human
a
nAChRs. Kd values were determined as indicated in the Section 4. N.E. no effects at
the highest concentration tested (1 mM).
l
a
a
a
a
(a4b2)2b2 and (a4b2)2a5 nAChRs display comparable sensitivity
for competitive nicotinic ligands36 (Table 2). Thus, introducing an
a
a
l
amino group at the meta position of the aromatic ring in compound
(a
a
a
1 (compound 3) decreases potency at (
10-fold without significantly affecting the inhibitory potency at
4b2)2b2 nAChRs. The presence of the amino group also increased
a
4b2)2
a
5 nAChRs by about
respectively (Table 2). Taken together, the findings suggest that a
positive nitrogen atom at the meta position in compound 1
(compound 3) increases selectivity for (a4b2)2b2 and (a4b2)2a4
(a

M. Faundez-Parraguez et al. / Bioorg. Med. Chem. 21 (2013) 2687–2694
2691
but decreases it for (
position 4 of the aromatic ring of compound 1 tend to decrease
4b2 receptor selectivity by increasing the inhibitory potency at
4b2)2 4 and ( 4b2)2 5 nAChRs closer to that displayed at
4b2)2b2 nAChRs. These results highlight interesting differences
a
4b2)2
a
5 nAChRs. In contrast, substitutions in
tent with the findings of the radioligand and functional assays that
suggest the synthesised nicotine derivatives inhibit the function of
a4b2 nAChRs by binding to the agonist binding site.
a
(a
a
a
a
(a
3. Conclusions
in the receptor subtype selectivity of the compounds tested, which
suggest that the ligand binding domain of ( 4b2)2b2 nAChRs is less
4b2)2 4 or ( 4b2)2
a
a
Herein we have combined radioligand binding assays, func-
tional characterisation and homology modelling to define the
mode of action and selectivity of novel neonicotinic compounds
discriminatory than its counterparts on (
a
a
a5
nAChRs.
at
strated that the synthesised compounds are
gands. The compounds fully displaced the binding of [125I]
to 4b2 nAChRs, suggesting that the
7 nAChRs and [3H]cyt to
compounds interact directly with the agonist binding site of these
nAChRs. This possibility is consistent with our docking data that
shows that the pyrrolidine moiety present in our molecules inter-
a
7 and
a
4b2 nAChRs. The radioligand binding assays demon-
4b2-preferring li-
-BgTx
2.3. Docking studies
a
a
The radioligand displacement studies suggested that the com-
pounds have higher affinity for the binding site of 4b2 nAChRs
than for that of 7 nAChRs. In an attempt to explain the differences
in binding affinity and functional potency of the neonicotinoid
compounds at 4b2 nAChRs, we performed docking calculations
with the compounds on homology models of the 4b2 nAChR ago-
nist binding site. The extracellular N-terminal ligand binding do-
main of the 4b2 nAChR was modeled using the X-ray structure
of the AChBP from Aplysia californica (Protein Data Bank codes
2BYN and 1UW6)19,21 as a template.
4b2 nAChR homology mod-
els were obtained in both the open and closed-channel states. All
the neonicotinoid compounds were docked into the
4(+)/b2(À)
interface centering the grid on 4W182. This residue is equivalent
to 1W149 on the muscle nAChR, whichs –cation interacts with
a
a
a
a
a
acts with the agonist binding site of
a4b2 nAChRs via p–cation
a
coupling. The subtype selectivity of our molecules appears to be
defined by the pyrrolidine moiety as it appears to be the case for
a
nicotine, which displays higher affinity for
7 nAChRs.16
Despite the submicromolar affinities shown by this pyrrolidine
a4b2 nAChRs than for
a
a
series, we found that just five of the compounds (1, 3, 4, 5 and 8)
tested have Ki values under 100 nM. With the exception of com-
pound 8 that contains a benzyloxy group at the para position of
the benzoate moiety, bulky compounds such as 7, 9, showed poor
affinities. The presence of the pyridine group in some molecules de-
creased affinity markedly, indicating an unfavourable interaction
with the protein for this kind of molecules, in contrast to nicotine.
The high affinity of compound 8, results from a favourable interac-
tion of the benzyloxy group in the aromatic cage of the binding site
a
a
a
p
the quaternary ammonium group of ACh25 affecting agonist bind-
ing affinity.38 The position of loop C in the open and close confor-
mations used to evaluate the binding energies of the ligand
dockings. The docking results indicate that small changes in the
nicotine structure such as addition of an ester link and other mod-
ifications in the aromatic ring lead to unfavourable interactions
with the agonist binding site. Thus, the docking energies predicted
do not favour interactions between such ligands and the agonist
binding site. However, when we performed the same docking anal-
ysis with loop C in the open conformation, all the neonicotinoid
compounds appear to dock into the agonist site with considerably
high energies (À8 to À12 kcal/mol) indicating higher affinities for
plus the p–cation interaction of the pyrrolidine-W182 couple. The
chlorine atom in compound 5 is better for affinity than a bromine
atom at the same position. The amino group at the meta position
in compound 3, increases affinity compared to 1, but in contrast a
nitro group, compound 2 decreases its affinity drastically.
All the compounds tested behaved as antagonists of
hRs. Although by comparison to the 4b2-preferring competitive
antagonist DhbE the synthesised neonicotinoids are moderate
inhibitors of 4b2 nAChRs, they display interesting 4b2 nAChR
stoichiometry-selectivity. Thus, for example, compounds 1 and 5
display preference for ( 4b2)2b2 and ( 4b2)2 5 nAChRs, whereas
compound shows marked preference for 4b2)2b2 and
4b2)2 4 nAChRs. This selectivity is intriguing because the ago-
nist site at the two 4/b2 subunit interfaces of these three receptor
types are structurally identical.39 Recently, however, it has been
shown that ( 4b2)2 4 are endowed with a third operational ago-
nist site at the 4/ 4 interface that is responsible for the signature
sensitivity for competitive and allosteric ligands of the ( 4b2)2
receptor isoform.39 Binding or lack of binding to this additional site
may explain the selectivity of the compounds for the ( 4b2)2
isoform. Little is known of the role of receptor specific subunit
interfaces in ( 4b2)2b2 or ( 4b2)2 5 but considering that the aro-
matic residues that contribute to /b agonist sites are highly con-
a4b2 nAC-
a
this conformational state of the
a4b2 nAChR. The binding modes
of the nicotine derivatives suggest that the principal interactions
stabilising binding of the compounds to the agonist binding site
are between the protonated nitrogen of the pyrrolidine moiety
and the W182 residue (Fig. 6). Our docking results are thus consis-
a
a
a
a
a
3
(a
(a
a
a
a
a
a
a
a
a4
a
a4
a
a
a
a
served in nAChRs subunits, it may be that the receptor selectivity
of the neonicotinic antagonists results from interactions with addi-
tional sites in the receptors. Further substitutions on the aromatic
ring of compound 1 are thus likely to lead to a better understand-
ing of the receptor selectivity and the structural relationships for
derivatives of compound 1 at the a4b2 nAChR family. In summary,
our results indicate that for this series of compounds there are
some structural requirements that confer selectivity for hetero-
meric nAChR subtypes rather than for the homomeric subtype.
Additionally, we found interesting information about the subtype
Figure 6. Superimposition of compounds 1, 3, 4 and 8 in the binding site of the
a4b2 nAChR. The image shows the
p–cation interaction between the ligands and
selectivity for (a4b2)2b2, (a4b2)2a4 and (a4b2)2a5 nAChRs.
a4W182 in the closed channel state conformation (loop C open).

2692
M. Faundez-Parraguez et al. / Bioorg. Med. Chem. 21 (2013) 2687–2694
response to ACh throughout the experiment, each co-application
was bracketed by an application of EC50 of ACh alone.
4. Materials and methods
4.1. Clonal cell lines
4.4. Homology modeling
The SH-SY5Y-h
acetylcholine receptors, was created as previously described31–33
and used to assay [125I]-
-BgTx binding. Membrane homogenates
for [3H]cyt binding studies were prepared from the SH-EP-h
4b2
clonal cell line, which express human
4b2 NAChRs.32,33 Cells were
a7 cell line, over-expressing human a7 nicotinic
The agonist binding domain of the
a4b2 nAChR was modeled
using as a template the crystal structures of the AChBP from Aplysia
californica in the close and open states (Protein Data Bank codes:
2BYN and 1UW6, respectively).19,21 Even though the sequence iden-
a
a
a
tity between AChBP and the subunits of the a4b2 nAChRs is only 18–
maintained in Dulbecco’s modified Eagle’s medium (Invitrogen,
20%, a similar fold and highly-conserved binding site residues make
UK) supplemented with 5% foetalcalfserum, 10% horseserum,
the AChBP structure suitable for modeling the agonist binding site
2 mM
2.5 g/mL amphotericin B and 0.4 mg/mL hygromycin. For SH-
EP-h 4b2 culture media, the hygromycin concentration was de-
creased to 130 g/mL, and zeocin (Invitrogen, UK) was added at
250 g/mL.
L-glutamine, 10 IU/mL penicillin, 10 lg/mL streptomycin,
of nAChRs. The sequences of human nAChR
a4 (P43681) and b2
l
a
(P17787) subunits were obtained from the ExPASy Server. Multiple
sequence alignment was performed using ClustalW40 and 200 mod-
l
els of each a4 and b2 nAChRs subunits were generated using Mod-
l
eller 8.41The overall average g factor for the best structures was
À0.20. This value is a good indicator for the quality of the models.
Each sub-unit was separately constructed and then relaxed by
1 nanosecond (ns) long molecular dynamics simulation with Des-
mond42,43 molecular dynamic software in a solvated SPC44 and neu-
tralized system. Receptor assembly was made by superposition, of
relaxed-subunit replicas, against the available AChBP crystal struc-
4.2. Ligand binding assays
Competition binding studies were performed on membrane
homogenates prepared from SH-SY5Y-h
a
7 clonal cell line or SH-
EP1-ha ]a-BgTx (PerkinElmer, UK)
4b2 clonal cell line, using [I125
or [3H]cyt (PerkinElmer, UK) respectively, as previously de-
scribed.33 Membrane homogenates were incubated at a final pro-
ture templates and following the (
generated homology models possess an explicit disulfide bond be-
tween 4 residues C225 and C226 and reproduce the amino acids
previously reported as implicated in agonist binding by nAChR.4,21
The whole 4b2 nAChR model was energy-minimized and equili-
a4b2)2b2 stoichiometry. All the
tein concentration of 30–50
of 500 L ([125I] -BgTx) or 250
nM: 120 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, 50 Tris, pH 7.0) for
120 min at room temperature (25 °C) with 1 nM [I125
-BgTx or
for 75 min at 4 °C with 1 nM [3H]cyt. For both binding assays,
10 M nicotine was used to define nonspecific binding. Bound
lg per assay tube in a final volume
l
a
lL ([3H]cyt) of binding saline (in
a
]
a
a
brated in explicit solvent, using long molecular dynamic (MD) sim-
ulations. All simulations were performed using the Desmond
molecular simulation package42,43 and OPLS 200544 all-atom force
field with explicit solvent and employing the single point charge
(SPC) water model. Detailed information about the system setup
and MD protocol followed in this study can be found in Muñoz et
al.45 Images were generated using PyMOL software. The best model
was energy-minimized in vacuo, using Desmond molecular dy-
namic software and the OPLS 2005 force field with explicit solvent.
l
and free fractions were separated by rapid filtration through What-
man GF/C filters pre-soaked in binding saline supplemented with
0.1% polyethyleneimine. Radioactivity was quantified by liquid
scintillation spectrometry.
4.3. Electrophysiological recordings
Concatenated (a4)2(2)3, (a4)3(b2)2 and a4b2a5 receptors were
expressed heterologously in Xenopus oocytes as previously de-
4.5. Docking analysis
scribed.34,35 The order of subunits in the concatenated receptors
was b2_
a4_b2_
a4_b2 for
(a
4)3(2)2, b2_
a
4_b2_
a
4_a4
for
Molecular docking of the nicotine analogues at the agonist
(a4)3(b2)2 and
a
5_b2_ 4_b2_
a
a
4 for 4b2 5 receptors. Oocytes
a
a
binding domain of the a4b2 homology models was investigated
were placed in a 0.1 mL recording chamber and perfused with
Ringer solution (in mM: NaCl 150, KCl 2.8, Hepes 10, CaCl2 1.8;
pH 7.2, adjusted with NaOH) at a rate of 15 mL/min. Oocytes were
impaled by two microelectrodes filled with 3 M KCl (0.3–2.0 MO)
and voltage-clamped at À60 mV using a Geneclamp 500B ampli-
fier. Typically current traces were filtered at 1 kHz during record-
ing and digitized at 1 kHz using the DigiData 1200 interface for
subsequent analysis using PClamp 8 software (Axon Instruments,
CA, USA). All experiments were carried out at room temperature.
Compounds were applied by gravity perfusion using a manually
activated valve. ACh concentration-response curves were obtained
by normalizing ACh-induced currents to the control responses in-
duced by 1 mM ACh (a near-maximum effective concentration at
using the Lamarckian genetic algorithm search method using soft-
ware AutoDock v4.0.46 The receptors were kept rigid, while full
flexibility was allowed for the ligands to translate/rotate. Polar
hydrogens were added to the receptors and Kollman-united atom
partial charges along with atomic solvation parameters were as-
signed to the individual protein atoms. The three-dimensional
structures of each ligand were generated using the SPARTAN’08
program and were then energy minimized. For each ligand, a rigid
root and rotatable bonds were assigned automatically. The non-po-
lar hydrogens were removed and the partial charges from these
were added to the carbons (Gasteiger charges). The atom type for
aromatic carbons was reassigned in order to use the AutoDock
4.0 aromatic carbon grid map. Docking was carried out using
60 Â 60 Â 60 grid points with a default spacing of 0.375 Å. The grid
was positioned to include the full ligand binding pocket in the cen-
(a4b2)2
4b2)2
a
4 nAChRs but a maximal concentration at (
ab2)2b2 or
(a
a
5 nAChRs.34–36 A minimum interval of 4 min was allowed
between ACh applications as this was found to be sufficient to
ensure reproducible recordings. The sensitivity of the receptors
to inhibition by antagonists was tested by first superfusing the
antagonist for 2 min and then co-applying it with an EC50 concen-
tral part of the
sampling around residue
a
4b2 subunit interfaces so as to allow extensive
4W182 (W143 in mature AChBP). With-
a
in this grid, the Lamarckian genetic search algorithm was used
with a population size of 150 individuals, calculated using 200 dif-
ferent runs (i.e. 200 dockings). Each run had two stop criteria, a
maximum of 1.5 Â 106 energy evaluations or a maximum of
50,000 generations, starting from a random position and confor-
mation; default parameters were used for the Lamarckian genetic
algorithm search.
tration of ACh (100 M for (
l
a4b2)2a4 nAChRs and 2 lM for
(a
b2)2b2 or ( 4b2)2a5 nAChRs). Antagonist concentration re-
a
sponse data were normalized to the appropriate ACh EC50 constant
responses to ACh were obtained before the co-application of ACh
and compound. To maintain on-going measurements of the control

M. Faundez-Parraguez et al. / Bioorg. Med. Chem. 21 (2013) 2687–2694
2693
4.6. Data analysis
to pH 8.0 and extracted with CH2Cl2. The hydrochloride salt was
obtained from acetone.
Concentration-response data for antagonists were fitted by
nonlinear regression (Prism 5.0, GraphPad, USA) to the equation
i = imax/[1+(IC50/x)nHill] wherein imax = maximal normalized current
response (in the absence of antagonist for inhibitory currents),
x = antagonist concentration, IC50 = antagonist concentration elicit-
ing half-maximal inhibition and nHill = Hill coefficient. Results are
presented as mean standard error of the mean (SEM) of at least
four separate experiments from at least two different batches of
oocytes. The same equation was used to estimate IC50 values for
inhibition of radioligand binding and the Ki value of the test com-
pounds was determined using the equation of Cheng and Prusoff:
Ki = IC50/1+[x]/KD, wherein x is the radioligand concentration and
KD the affinity binding constant. Statistical significance was as-
sessed using a two-tailed unpaired t-test, or one-way analysis of
variance (ANOVA). P values less than 0.05 were considered
significant.
5.1.4. ((S)-1-Methylpyrrolidin-2-yl)methyl benzoate (1)
Prepared in the similar way as described above in general pro-
cedures. (S)-(À)-1-Methyl-2-pyrrolidinylmethanol (4.0 mmol,
0.5 mL) was added to a solution of benzoyl chloride (4.0 mmol,
0.49 mL). Yield 816 mg (76%), mp 165.4–169.0 °C, 1H NMR
(400 MHz, D2O) d 7.28 (m, 2H), 6.93 (m, 2H), 4.13 (dd, J = 9.6,
J = 5.6 Hz, 1H), 3.95 (dd, J = 9.6, J = 5.4 Hz, 1H), 3.24 (m, 1H), 2.83
(m, 1H), 2.58 (s, 3H), 2.44 (m, 1H), 2.09 (m, 1H), 1.93 (m, 1H),
1.82 (m, 2H). 13C NMR (100 MHz, D2O) d 167.1, 133.1, 130.2,
129.9 (2C), 128.7 (2C), 65.4, 64.9, 57.9, 40.6, 28.1, 23.1. IR (KBr,
cmÀ1) 2978, 1720, 1279, 1112. Anal. Calcd for C13H18ClNO2: C,
61.05; H, 7.09; N, 5.48. Found: C, 61.35; H, 7.19; N, 5.53.
5.1.5. ((S)-1-Methylpyrrolidin-2-yl)methyl 3-nitrobenzoate (2)
Prepared in the similar way as described in general procedures.
(S)-(À)-1-Metil-2-pirrolidinilmetanol (4.0 mmol, 0.5 mL) was
added to
a solution of 3-nitrobenzoyl chloride (4.0 mmol,
5. Chemistry
783 mg). Yield 822 mg (65%), mp 171–174 °C, 1H NMR (400 MHz,
D2O) d 7.81 (d, J = 8.1 Hz, 1H), 7.75 (s, 1H), 7.42 (t, J = 8.2 Hz, 1H),
7.25 (d, J = 8.1 Hz, 1H), 4.05 (dd, J = 9.0, J = 5.4 Hz, 1H), 3.98 (dd,
J = 9.2, J = 5.3 Hz, 1H), 3.19 (m, 1H), 2.52 (s, 3H), 2.32 (m, 1H),
2.06 (m, 1H), 2.09 (m, 1H), 1.82 (m, 3H). 13C NMR (100 MHz,
D2O) d 168.9, 148.3, 136.2, 131.1, 129.6, 125.6, 124.8, 65.4, 64.9,
57.9, 40.6, 28.1, 23.1. IR (KBr, cmÀ1) 2980, 1715, 1550, 1350,
1266, 1108. Anal. Calcd for C13H17ClN2O4: C, 51.92; H, 5.70; N,
9.31. Found: C, 51.83; H, 5.61; N, 9.27.
Melting points are uncorrected and were determined with a
Reichert Galen III hot plate microscope. 1H NMR spectra were re-
corded using Bruker AMX 400 spectrometers at 400 MHz. Chemical
shifts are reported relative to TMS (d = 0.00) or HDO (d = 4.79) and
coupling constants (J) are given in Hz. IR spectra were recorded on
a Bruker Vector 22 spectrophotometer using KBr. The elemental
analyses for C, H, N were performed on a CE Instruments (model
EA 1108) analyzer. Reactions and product mixtures were routinely
monitored by thin-layer chromatography (TLC) on silica gel-pre-
coated F254 Merck plates, normally we use a mixture of CHCl3/
CH3OH as a mobile phase. All reagents and solvents were commer-
cially available and were used without further purification.
5.1.6. ((S)-1-Methylpyrrolidin-2-yl)methyl 3-aminobenzoate (3)
3-Aminobenzoate derivative was prepared by catalytic hydro-
genation of the corresponding ((S)-1-methylpyrrolidin-2-yl)-
methyl 3-nitrobenzoate (compound 2) (8.0 mmol, 2.0 g) using
Pd-C 10% in ethanol at 70 psi by 3 h. Yield 1.71 g (95%), mp 172–
176 °C, 1H NMR (400 MHz, D2O) d 7.31 (d, J = 8.3 Hz, 1H), 6.90 (d,
J = 8.3 Hz, 1H), 6.83 (d, J = 8.3 Hz, 1H), 6.81 (s, 1H), 4.33 (dd,
J = 11.3, J = 3.0 Hz, 1H), 4.15 (dd, J = 11.2, J = 5.9 Hz, 1H), 3.76 (m,
1H), 3.59 (m, 1H), 3.12 (m, 1H), 2.88 (s, 3H), 2.26 (m, 1H), 2.09
(m, 1H), 1.95 (m, 3H).13C NMR (100 MHz, D2O) d 163.9, 148.2,
131.2, 129.1, 120.6, 119.6, 114.3, 65.4, 64.9, 57.9, 40.6, 28.1, 23.1.
IR (KBr, cmÀ1) 3442, 3360, 2978, 1730, 1626,1285, 1270, 1115.
Anal. Calcd for C13H19ClN2O2: C, 57.67; H, 7.07; N, 10.35. Found:
C, 57.74; H, 7.12; N, 10.46.
5.1. General procedures
5.1.1. Synthesis of benzyloxy derivatives
6-Hydroxybenzoic acid and 6-hydroxynicotinic acid were previ-
ously transformed in their corresponding methyl esters, using
methanol–sulfuric acid under reflux by 5 days. Methyl esters were
benzylated using benzyl chloride in acetonitrile as solvent and
NaHCO3, the mixture was kept under reflux for 3 days. After this
time, the solvent was evaporated and the benzyloxy esters were
hydrolyzed in KOH by refluxing for 1 h. benzyloxy acids were puri-
fied by column chromatography using Silica gel 60 and a mobile
phase of CHCl3/CH3OH (90/10).
5.1.7. ((S)-1-Methylpyrrolidin-2-yl)methyl 4-bromobenzoate (4)
Prepared in the similar way as described above in general pro-
cedures. (S)-(À)-1-Methyl-2-pyrrolidinylmethanol (4.0 mmol,
0.5 mL) was added to a solution of 4-bromobenzoyl chloride
(4.0 mmol, 926 mg). Yield 981 mg (78%), mp 231.4–232, 1 °C, 1H
NMR (400 MHz, D2O) d 7.91 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.8 Hz,
2H), 4.67 (dd, J = 13.6, J = 2.9 Hz, 1H), 4.52 (dd, J = 13.5, J = 6.6 Hz,
1H), 3.90 (m, 1H), 3.75 (m, 1H), 3.26 (m, 1H), 2.98 (s, 3H), 2.37
(m, 1H), 2.23 (m, 1H), 1.89 (m, 2H). 13C NMR (100 MHz, D2O) d
166.0, 132.1 (2C), 131.6 (2C), 129.2, 127.4, 65.4, 64.9, 57.9, 40.6,
28.1, 23.1. IR (KBr, cmÀ1) 2980, 1729, 1281, 1115, 686. Anal. Calcd
for C13H17BrClNO2: C, 46.66; H, 5.12; N, 4.19. Found: C, 46.60; H,
5.05; N, 4.15.
5.1.2. Synthesis of benzoyl chlorides
The corresponding benzoic acids were converted into benzoyl
chlorides using thionylchloride as a halogenating agent, and the
reaction was performed with each of the corresponding benzoic
acids (6–10 mmol) and 5 mL of thionyl chloride using dry THF as
a solvent, under a nitrogen atmosphere. The mixture was main-
tained at room temperature for 24 h, and then the solvent, excess
reagents and remaining HCl and SO2 were evaporated under vac-
uum. The unpurified benzoyl chloride was used immediately for
the next reaction.
5.1.3. Synthesis of ((S)-1-methylpyrrolidin-2-yl)methyl
benzoates
5.1.8. ((S)-1-Methylpyrrolidin-2-yl)methyl 4-chlorobenzoate (5)
Prepared in the similar way as described above in general pro-
cedures. (S)-1-Methylpyrrolidin-2-yl)methanol (8.0 mmol, 1.0 mL)
was added to a solution of 4-chlorobenzoyl chloride (8.0 mmol,
1.1 mL). Yield 1.75 g (82%), mp 227.5–228.3 °C, 1H NMR
(400 MHz, D2O) d 7.81 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.6 Hz, 2H),
4.61 (dd, J = 13.1, J = 2.8 Hz, 1H), 4.42 (dd, J = 13.0, J = 6.5 Hz, 1H),
Benzoyl chlorides were dissolved in 50 mL of diethyl ether and
stirred at room temperature. One equivalent of (S)-1-methylpyrr-
olidin-2-ylmethanol (around 0.3-0.4 mL) in 30 mL of diethyl ether
was added drop by drop. The reaction mixture was kept at room
temperature with constant stirring for 24 h. Then, the solvent
was evaporated, the mixture was re-dissolved in water, adjusted

2694
M. Faundez-Parraguez et al. / Bioorg. Med. Chem. 21 (2013) 2687–2694
3.80 (m, 1H), 3.64 (m, 1H), 3.15 (m, 1H), 2.93 (s, 3H), 2.30 (m, 1H),
2.11 (m, 1H), 1.95 (m, 2H). 13C NMR (100 MHz, D2O) d 166.0, 138.6,
131.3 (2C), 128.8 (2C), 128.3, 65.4, 64.9, 57.9, 40.6, 28.1, 23.1. IR
1115. Anal. Calcd for C20H24ClNO3: C, 66.38; H, 6.69; N, 3.87.
Found: C, 66.56; H, 6.81; N, 3.95.
(KBr, cmÀ1
)
2995, 1737, 1277, 1108, 740. Anal. Calcd for
13H17Cl2NO2: C, 53.81; H, 5.90; N, 4.83. Found: C, 53,77; H,
5.93; N, 4.79.
Acknowledgments
C
This work was partially funded by FONDECYT Grants 1100542
and 11100177 and ICM Grant MSI 10-063-F NEDA, and supported
by Project ICM-P10-003-F CILIS, granted by Fondo de Innovación
para la Competitividad del Ministerio de Economía, Fomento y
Turismo, Chile.
5.1.9. ((S)-1-Methylpyrrolidin-2-yl)methyl 6-chloronicotinoate (6)
Prepared in the similar way as described above in general pro-
cedures. (S)-1-methylpyrrolidin-2-yl) methanol (3.0 mmol,
0.35 mL) was added in to a solution of 6-chloronicotinoyl chloride
(2.8 mmol, 500 mg). Yield 473 mg (65%), mp 169.3–171.5 °C, 1H
NMR (400 MHz, D2O) d 8.83 (d, J = 2.3 Hz, 1H), 8.27 (dd, J = 8.3,
J = 2.4 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 4.69 (dd, J = 13.1, J = 3.0 Hz,
1H), 4.49 (dd, J = 13.1, J = 6.2) Hz, 1H), 3.82 (m, 1H), 3.65 (m, 2H),
3.15 (m, 1H), 2.93 (s, 3H), 2.31 (m, 1H), 2.11 (m, 1H), 1.96 (m,
2H). 13C NMR (100 MHz, D2O) d 166.4, 151.2, 150.7, 139.1, 124.5,
122.6, 66.8, 65.1, 58.5, 41.7, 28.3, 23.2.IR (KBr, cmÀ1) 2975, 1737,
1677, 1270, 1118, 730. Anal. Calcd for C12H16Cl2N2O2: C, 49.50;
H, 5.54; N, 9.62. Found: C, 49.58; H, 5.59; N, 9.67.
References and notes
1. Le Novère, N.; Changeux, J. P. Nucleic Acids Res. 1999, 27, 340.
2. Zoli, M.; Le Novère, N.; Hill, J.; Changeux, J. P. J. Neurosci. 1995, 15, 1912.
3. Le Novère, N.; Corringer, P. J.; Changeux, J. P. J. Neurobiol. 2002, 53, 447.
4. Albuquerque, E. X.; Pereira, E. F. R.; Alkondon, M.; Rogers, S. W. Physiol. Rev.
2009, 89, 73.
5. Chavez-Noriega, L. E.; Crona, J. H.; Washburn, M. S.; Urrutia, A.; Elliott, K. J.;
Johnson, E. C. J. Pharmacol. Exp. Ther. 1997, 280, 346.
6. Paterson, D.; Nordberg, A. Prog. Neurobiol. 2000, 61, 75.
7. Sher, E.; Chen, Y.; Sharples, T. J.; Broad, L. M.; Benedetti, G.; Zwart, R.; McPhie,
G. I.; Pearson, K. H.; Baldwinson, T. Curr. Top. Med. Chem. 2004, 4, 283.
8. Wonnacott, S. Trends Neurosci. 1997, 20, 92.
5.1.10. ((S)-1-Methylpyrrolidin-2-yl)methyl 2-quinolinecarbo-
xylate (7)
9. Wonnacott, S. Trends Pharmacol. Sci. 1990, 11, 216.
10. Jensen, A.; Frølund, B.; Liljefors, T.; Krogsgaard-Larsen, P. J. Med. Chem. 2005, 48,
4705.
Prepared in the similar way as described above in general pro-
cedures. (S)-(À)-1-Methyl-2-pyrrolidinylmethanol (4.0 mmol,
0.5 mL) was added to a solution of 2-quinolinecarboxyl chloride
(4.0 mmol, 806 mg). Yield 790 mg (70%), mp 171–174 °C, 1H NMR
(400 MHz, D2O) d 9.40 (s, 1H), 9.21 (s, 1H), 8.25 (d, J = 8.2 Hz,
1H), 8.13 (d, J = 8.4 Hz, 1H), 7.96 (t, J = 8.2 Hz, J = 7.7 Hz, 1H), 7.75
(t, J = 7.9 Hz, J = 7.5 Hz, 1H), 4.05 (dd, J = 9.0, J = 5.4 Hz, 1H), 3.98
(dd, J = 9.2, J = 5.3 Hz, 1H), 3.19 (m, 1H), 2.52 (s, 3H), 2.32 (m,
1H), 2.06 (m, 1H), 2.09 (m, 1H), 1.82 (m, 3H). 13C NMR (100 MHz,
D2O) d 167.9, 148.8, 147.5, 138.8, 132.3, 129.7, 128.9, 127.8,
11. Gotti, C.; Moretti, M.; Gaimarri, A.; Zanardi, A.; Clementi, F.; Zoli, M. Biochem.
Pharmacol. 2007, 74, 1102.
12. Picciotto, M. R.; Zoli, M.; Rimondini, R.; Lena, C.; Marubio, L.; Pich, E.; Fuxe, K.;
Changeaux, J. P. Nature 1998, 391, 173.
13. Newhouse, P.; Singh, A.; Potter, A. Curr. Top. Med. Chem. 2004, 4, 267.
14. Shytle, R. D.; Silver, A.; Lukas, R. J.; Newman, M. B.; Sheehan, D. V.; Sanberg, P.
R. Mol. Psychiatry 2002, 7, 525.
15. Romanelli, M. N.; Gualtieri, F. Med. Res. Rev. 2003, 23, 393.
16. Wonnacott, S.; Barik, J. Tocris Sci. Rev. Ser. 2007, 6, 1.
17. Daly, J. W. Cell. Mol. Neurobiol. 2005, 25, 513.
18. Brejc, K.; Van Dijk, W. J.; Klaassen, R. V.; Schuurmans, M.; Van Der Oost, J.; Smit,
A. B.; Sixma, T. K. Nature 2001, 411, 269.
19. Celie, P. H. N.; Van Rossum-Fikkert, S. E.; Van Dijk, W. J.; Brejc, K.; Smit, A. B.;
Sixma, T. K. Neuron 2004, 41, 907.
127.5, 122.6, 65.7, 63.9, 55.6, 41.3, 29.1, 22.1. IR (KBr, cmÀ1
)
2995, 1745, 1683, 1278, 1128, 745. Anal. Calcd for C16H19ClN2O2:
C, 62.64; H, 6.24; N, 9.13. Found: C, 62.83; H, 6.61; N, 9.22.
20. Rucktooa, P.; Smit, A. B.; Sixma, T. K. Biochem. Pharmacol. 2009, 78, 777.
21. Hansen, S. B.; Sulzenbacher, G.; Huxford, T.; Marchot, P.; Taylor, P.; Bourne, Y.
Eur. Mol. Biol. Organ. J. 2005, 24, 3635.
22. Unwin, N. J. Mol. Biol. 2005, 346, 967.
23. Sine, S. M.; Engel, A. G. Nature 2006, 440, 448.
5.1.11. ((S)-1-Methylpyrrolidin-2-yl)methyl 4-benzyloxyben-
zoate (8)
24. Grady, S. R.; Drenan, R. M.; Breining, S. R.; Yohannes, D.; Wageman, C. R.;
Fedorov, N. B.; McKinney, S.; Whiteaker, P.; Bencherif, M.; Lester, H. A.; Marks,
M. J. Neuropharmacology 2010, 58, 1054.
25. Xiu, X.; Puskar, N. L.; Shanata, J. A.; Lester, H. A.; Dougherty, D. A. Nature 2009,
458, 534.
Prepared in the similar way as described above in general pro-
cedures. (S)-(À)-1-metil-2-pirrolidinilmetanol (3.0 mmol, 0.17 mL)
was added into
a solution of 4-benzyloxybenzoyl chloride
(3.0 mmol, 680 mg). Yield 628 mg (70%), mp 144.3–145.2 °C, 1H
NMR (400 MHz, D2O) d 7.51 (m, 2H), 7.42 (m, 3H), 7.08 (d,
J = 7.5 Hz, 2H), 6.8 (d, J = 7.2 Hz, 2H), 5.16 (s, 2H), 4.62 (d,
J = 2.8 Hz, 1H), 4.08 (d, J = 6.6 Hz, 1H), 3.78 (t, J = 4.2 Hz, 1H), 3.62
(t, 2H), 3.13 (m, 2H), 2.92 (s, 3H), 2.14 (m, 1H), 2.02 (m, 1H). 13C
NMR (100 MHz, D2O) d 166.0, 165.0, 141.2, 130.9 (2C), 129.0
(2C), 127.7, 127.2 (2C), 122.5, 114.2 (2C), 70.9, 65.4, 64.9, 57.9,
40.6, 28.1, 23.1. IR (KBr, cmÀ1) 2988, 1730, 1269, 1220, 1119. Anal.
Calcd for C20H24ClNO3: C, 66.38; H, 6.69; N, 3.87. Found: C, 66.56;
H, 6.81; N, 3.95.
26. Scriabine, A. CNS Drugs Rev. 1999, 5, 395.
27. Fischer, E.; Speier, A. Chemische Derichte 1895, 28, 3252.
28. Batcho, A.; Leimgruber, W. Org. Synth. 1990, 7, 34.
29. Anderson, D.; Arneric, S. Eur. J. Pharmacol. 1994, 253, 261.
30. Peng, X.; Katz, M.; Gerzanich, V.; Anand, R.; Lindstrom, J. Mol. Pharmacol. 1994,
45, 546.
31. Peng, J.; Fryer, J. D.; Hurst, R. S.; Schroeder, K. M.; George, A. A.; Morrissy, S.;
Groppi, V. E.; Leonard, S. S.; Lukas, R. J. J. Pharmacol. Exp. Ther. 2005, 313, 24.
32. Pacheco, M.; Pastoor, T.; Lukas, R.; Wecker, L. Neurochem. Res. 2001, 26, 683.
33. Houlihan, L. M.; Slater, Y.; Guerra, D. L.; Peng, J. H.; Kuo, Y. P.; Lukas, R. J.;
Cassels, B. K.; Bermudez, I. J. Neurochem. 2001, 78, 1029.
34. Moroni, M.; Zwart, R.; Sher, E.; Cassels, B. K.; Bermudez, I. Mol. Pharmacol. 2006,
70, 755.
35. Carbone, A. L.; Moroni, M.; Groot-Kormelink, P. J.; Bermudez, I. Br. J. Pharmacol.
2009, 156, 970.
5.1.12. ((S)-1-Methylpyrrolidin-2-yl)methyl 6-benzyloxynicoti-
noate (9)
36. Kuryatov, A.; Berrettini, W.; Lindstrom, J. Mol. Pharmacol. 2011, 79, 119.
37. Gotti, C.; Moretti, M.; Meinerz, N. M.; Clementi, F.; Gaimarri, A.; Collins, A. C.;
Marks, M. J. Mol. Pharmacol. 2008, 73, 1796.
Prepared in the similar way as described above in general pro-
cedures. (S)-(À)-1-metil-2-pirrolidinilmetanol (3.0 mmol, 0.17 mL)
was added into a solution of 4-benzyloxynicotinoyl chloride
(3.0 mmol, 680 mg.). Yield 628 mg (70%), mp 144.3–145.2 °C, 1H
NMR (400 MHz, D2O) d 7.51 (m, 2H), 7.42 (m, 3H), 7.08 (d,
J = 7.5 Hz, 2H), 6.8 (d, J = 7.2 Hz, 2H), 5.16 (s, 2H), 4.62 (d,
J = 2.8 Hz, 1H), 4.08 (d, J = 6.6 Hz, 1H), 3.78 (t, J = 4.2 Hz, 1H), 3.62
(t, 2H), 3.13 (m, 2H), 2.92 (s, 3H), 2.14 (m, 1H), 2.02 (m, 1H). 13C
NMR (100 MHz, D2O) d 166.0, 165.0, 141.2, 130.9 (2C), 129.0
(2C), 127.7, 127.2 (2C), 122.5, 114.2 (2C), 70.9, 65.4, 64.9, 57.9,
40.6, 28.1, 23.1. IR (KBr, cmÀ1) 2995, 1747, 1672, 1278, 1248,
38. Akk, G. J. Physiol. 2001, 535, 729.
39. Mazzaferro, S.; Benallegue, N.; Carbone, A.; Gasparri, F.; Vijayan, R.; Biggin, P.
C.; Moroni, M.; Bermudez, I. J. Biol. Chem. 2011, 286, 31043.
40. Thompson, J.; Higgins, D.; Gibson, T. Nucleic Acids Res. 1994, 22, 4673.
41. Sali, A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779.
42. Shaw, D. Desmond Molecular Dynamics System, v 3.0, New York, NY, 2011.
43. Maestro Desmond Interoprability Tools, v. 3.0, New York, NY, 2011.
44. Berendsen, H.; Postma, J.; Gunsteren, W.; Hermans, J. Intermolecular Forces
1981, 331, 331.
45. Muñoz, C.; Adasme, F.; Alzate-Morales, J. H.; Vergara-Jaque, A.; Kniess, T.;
Caballero, J. J. Mol. Graph. Model. 2012, 32, 39.
46. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.