Minimal structural changes determine full and partial nicotinic receptor agonist activity for nicotine analogues

Article abstract of DOI:10.3390/molecules24152684

Neuronal α4β2 nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels (LGIC) that have been implicated in nicotine addiction, reward, cognition, pain disorders, anxiety, and depression. Nicotine has been widely used as a template for the synthesis of ligands that prefer α4β2 nAChRs subtypes. The most important therapeutic use for α4β2 nAChRs is as replacement therapy for smoking cessation and withdrawal and the most successful therapeutic ligands are partial agonists. In this case, we use the N-methylpyrrolidine moiety of nicotine to design and synthesize new α4β2 nicotinic derivatives, coupling the pyrrolidine moiety to an aromatic group by introducing an ether-bonded functionality. Meta-substituted phenolic derivatives were used for these goals. Radioligand binding assays were performed on clonal cell lines of hα4β2 nAChR and two electrode voltage-clamp experiments were used for functional assays. Molecular docking was performed in the open state of the nAChR in order to rationalize the agonist activity shown by our compounds.

Full text of DOI:10.3390/molecules24152684

molecules
Article
Minimal Structural Changes Determine Full and
Partial Nicotinic Receptor Agonist Activity for
Nicotine Analogues
Juan Pablo Gonzalez-Gutierrez 1, Martin Hodar 2, Franco Viscarra 2, Pablo Paillali 2,
Nicolás Guerra-Díaz 1, Hernán Pessoa-Mahana 1, Juan José Hernández-Morantes 3
Horacio Pérez-Sánchez 4 , Isabel Bermúdez 5 , Miguel Reyes-Parada 6,7,* and
,
Patricio Iturriaga-Vásquez 2,
*
1
Departamento de Química Orgánica y Fisicoquímica, Facultad de Ciencias Químicas y Farmacéuticas,
Universidad de Chile, Santiago 8380494, Chile
Departamento de Ciencias Químicas y Recursos Naturales, Facultad de Ingeniería y Ciencias,
Universidad de la Frontera, Temuco 4811230, Chile
Facultad de Enfermería, Universidad Católica de Murcia, 30107 Murcia, Spain
Structural Bioinformatics and High Performance Computing Research Group (BIO-HPC),
Universidad Católica de Murcia, 30107 Murcia, Spain
2
3
4
5
6
School of Life sciences, Oxford-Brookes University, Oxford OX3 0BP, UK
Centro de Investigación Biomédica y Aplicada (CIBAP), Escuela de Medicina, Facultad de Ciencias Médicas,
Universidad de Santiago de Chile, Santiago 9170022, Chile
Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Sede Talca 3467987, Chile
Correspondence: miguel.reyes@usach.cl (M.R.-P.); patricio.iturriaga@ufrontera.cl (P.I.-V.)
7
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 13 May 2019; Accepted: 22 July 2019; Published: 24 July 2019
Abstract: Neuronal α4β2 nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels
(LGIC) that have been implicated in nicotine addiction, reward, cognition, pain disorders, anxiety,
and depression. Nicotine has been widely used as a template for the synthesis of ligands that prefer
α4β2 nAChRs subtypes. The most important therapeutic use for α4β2 nAChRs is as replacement
therapy for smoking cessation and withdrawal and the most successful therapeutic ligands are partial
agonists. In this case, we use the N-methylpyrrolidine moiety of nicotine to design and synthesize
new
an ether-bonded functionality. Meta-substituted phenolic derivatives were used for these goals.
Radioligand binding assays were performed on clonal cell lines of h 2 nAChR and two electrode
α4β2 nicotinic derivatives, coupling the pyrrolidine moiety to an aromatic group by introducing
α4β
voltage-clamp experiments were used for functional assays. Molecular docking was performed in the
open state of the nAChR in order to rationalize the agonist activity shown by our compounds.
Keywords: α4β2 nAChR; pyrrolidine ethers; partial agonist; docking
1. Introduction
Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels [
expressed in the central and peripheral nervous systems that respond to the neurotransmitter
acetylcholine (ACh) [ ] and exogenous compounds such as nicotine and cytisine (Figure 1) [ ].
Mammalian neuronal nAChRs assemble from combinations of nine − β4)
−α10) and three
subunits [ ]. The 2 nAChR contains two 2 pairs and a fifth subunit that can be 2 or 4, which
generates two alternate receptor isoforms known as the (α4β2)2β2 and the (α4β2)2α4 nAChRs [5].
1],
2
3,4
α
(α
2
β(β2
3
α
4
β
α
4
β
β
α
Molecules 2019, 24, 2684; doi:10.3390/molecules24152684
www.mdpi.com/journal/molecules

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Figure 1. Classical nAChRs agonists.
The binding of an agonist induces the opening of the ion channel, which allows a flow of cations
through the cell membrane, and, consequently, modulates the release of different neurotransmitters [ ].
nAChR are involved in different CNS functions and disorders such as cognition, analgesia, nicotine
addiction [ ], anxiety, depression, and attention-deficit hyperactivity disorder [ ] and constitute an
established target for drugs aiding smoking cessation [10] such as varenicline [11].
6
7,
8
9
The crystal structure of the ACh-binding protein (AChBP) from different species in the complex
with several ligands, as well as derived homology models, have revealed the structural characteristics
of the extracellular domain of nAChRs, as well as critical ligand-receptor interactions [12]. In addition,
the recent description of the X-ray structure of a near-intact α4β2 nAChR bound to nicotine [13,14] has
provided new insights regarding both the agonist binding site (the orthosteric site) and the structural
requirements of its ligands. In the neuronal nAChRs, the orthosteric binding site is located at the
interface formed between an subunit (
α
α3, α4, α7: the positive side) and a complementary subunit
(α
or : the negative side) [15,16]. The ACh binding site, at the subunit interfaces, is formed by loops
β
A, B, and C on the “principal face” and loops D, E, and F on the “complementary face” [17]. There
are several highly conserved aromatic amino acid residues at this binding site in different nAChRs
subtypes. It has been shown that a conserved tryptophan of loop B is a key residue required for ACh
and other agonists binding, as it stabilizes the ligand binding mode through a π-cation interaction [18].
Conformational changes of the C-loop located at the entrance of the agonist binding site occur when a
competitive agonist binds to the orthosteric site, which leads to coupling the ligand binding to the
opening of the ion channel [19].
Partial agonists of
α4β2 nAChRs such as cytisine or varenicline are currently used for smoking
cessation therapy [20 21]. The rationale behind this type of treatment is that these compounds might
,
help diminish withdrawal symptoms (acting as agonists), while reducing smoking satisfaction (acting
as antagonists). Despite their success, there is evidence that these compounds induce significant side
effects, which makes it necessary to develop novel drugs with better efficacy and safety profiles.
In this regard and based on previous theoretical, ethical, and experimental reports [22
have designed and synthesized a small series of known and novel compounds, based on the nicotine
structure, in order to modulate both their affinity and efficacy for 2 nAChRs. Thus, the pyridine
–24], we
α4β
moiety of nicotine was modified by moving the nitrogen atom outside of the ring, which either
maintains the amino group or changes it by a halogen atom such as chloro or bromo. This substructure
was linked to the N-methylpyrrolidine moiety of nicotine by an ether function (Figure 2).
Figure 2. Pyrrolidine phenyl ether derivatives used in this work (right) and nicotine structure (left).

Molecules 2019, 24, 2684
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Our design strategy considered that: (a) these molecules should still be able to establish the
-cation interaction, between the positively charged nitrogen of the N-methylpyrrolidine and the
π
conserved aromatic residue W156 present at the ligand binding site of the α4β2 nAChRs [16,25], and
(b) the aromatic moiety of our derivatives could interact with the aromatic cage into the binding site
and could form hydrogen or halogen bridge(s).
2. Results and Discussion
2.1. Synthesis
For the synthesis of these phenylpyrrolidine ether derivatives, the phenolic derivatives,
and (S)-(1-methylpyrrolidin-2-yl)methanol were obtained from usual commercial sources. As a
first step, (S)-2-(chloromethyl)-1-methylpyrrolidine was synthesized using thionyl chloride as a
halogenating agent in dry THF as solvent, at reflux for 24 hours under N2 atmosphere. After the
reaction was complete, the solvent was evaporated on a rotary evaporator under reduced pressure.
The (S)-2-(chloromethyl)-1-methylpyrrolidine obtained was used immediately. The phenolic derivatives
were mixed with (S)-2-(chloromethyl)-1-methylpyrrolidine, in dry acetonitrile as solvent, and refluxed
for 48 hours under N2 atmosphere (Figure 3). After the reaction was complete, the solvent was
evaporated on a rotary evaporator under reduced pressure and the mixture of the reaction was purified
using a combi-flash chromatograph. All compounds synthesized were obtained with a yield of 40% to
60% and the structures were confirmed by spectroscopy analysis.
In the specific case of the m-aminophenylpyrrolidine derivative, this was obtained from the
reaction of m-nitrophenol and (S)-2-(chloromethyl)-1-methylpyrrolidine under the conditions already
mentioned. The nitro group was then reduced via catalytic hydrogenation using 10% Pd/C as a catalyst
in dry ethanol to give the corresponding aromatic amino group.
N
N
a
OH
Cl
N
OH
O
N
b
Cl
R1
R1
Figure 3. Synthesis Scheme: (a) SOCl2, THF-dry, reflux, 24 hours, (b) (S)-2-(chloromethyl)-1-
methylpyrrolidine, phenolic derivatives, reflux 48 h, AcCN-dry.
2.2. Biological Evaluation
2.2.1. Competitive Radioligand Binding Studies
First, we determined the half maximal inhibitory concentration (IC50) of the synthesized molecules
for human
binds to the agonist (orthosteric) site of α4β2 nACRs [26]. The assays were carried out on membrane
homogenates prepared from SH-EP-h 2 clonal cells, which have been shown to stably express
human 2 nAChRs was fully displaced
2 nAChRs [27]. As shown in Figure 4, [3H]Cyt bound to
by the synthesized compounds in a concentration-dependent and mono-phasic manner.
α4β
2 nAChR using [3H]-cytisine ([3H-Cyt]) as a radioligand. It is well known that [3H-Cyt]
α4β
α
4
β
α4β

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100
50
0
1
2
3
4
-12
-10
-8
-6
-4
-2
Log C
Figure 4. Effects of phenylpyrrolidine derivatives on binding of [3H]cytisine to human
Data points represent the mean SEM of four experiments. Each experiment was carried out in triplicate.
α4β2 nAChRs.
±
The radioligand concentration in all displacement studies was 1 nM. The concentration-inhibition data
was analyzed non-linearly.
This pattern of radioligand binding inhibition indicates that the compounds bind to the agonist
site of the receptors. Compounds tested showed a high affinity at α4β2 nAChRs, and, in all cases,
[3H]Cyt was displaced with IC50 values in the nanomolar ranges. Compound 2 was the most potent
with an IC50 about 15 times lower than those of compounds 1, 3, and 4, which showed similar affinities
for the α4β2 nAChR (Table 1). These values are in agreement with the affinity previously reported for
the compounds 1 and 3 [22].
Table 1. Binding affinity (IC50), potency (EC50), and maximal current (Imax) of phenylpyrrolidine
derivatives at human
mean SEM of four experiments. Each experiment was done in triplicate. Assays were carried out on
membrane homogenates prepared from SH-EP-h 2 clonal cells and the radioligand concentration
in all displacement studies was 1 nM. In the case of potency (EC50) and maximal current responses
(Imax) determinations, values represent the mean SEM of four to five independent experiments.
Assays were carried out on human 2 nAChR expressed heterologously in Xenopus laevis oocytes.
α4β2 nAChR. In the case of affinity (IC50) determinations, data represent the
±
α4β
±
α4β
IC50 estimates for nicotine determined previously under the same conditions [28
comparative purposes.
,
29] were included for
Compound
α4β2 IC50 (nM)
α4β2 EC50 (µM)
Imax
Nicotine
1.2
10
1.00
1
2
3
4
204.0 ± 6.0
13.8 ± 0.5
221.0 ± 10.0
188.0 ± 7.0
68.8 ± 1.3
10.8 ± 0.7
63.3 ± 2.1
56.7 ± 1.7
0.54 ± 0.01
1.00 ± 0.01
0.29 ± 0.01
0.13 ± 0.01
As our results show, all compounds synthesized displaced [3H]Cyt from the agonist binding site of
the α4β2 nAChR, which indicates that the ether function of our pyrrolidine derivatives does not affect
their competitive binding properties. The inclusion of a substituent on the meta position of the aromatic
group affected differentially the binding affinity depending on the substituent. Thus, the halogenated
compounds (3 and 4) exhibited a similar affinity as compared to the unsubstituted derivative 1, whereas
the introduction of an amino group at this position produced a clear increase of affinity. This effect has
been reported with a hydroxyl analogue of compound 2, where the meta-hydroxyl position increases
its affinity [23]. This might be due to the amino group, with donor/acceptor properties, being able to
form hydrogen bridge(s) with some residues into the agonist binding site of the receptor.

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2.2.2. Functional Studies
The functional effects of our compounds were tested on
α
4β2 nAChR heterologously expressed
in Xenopus laevis oocytes. Experiments were carried out by using the two-electrode voltage clamp
procedure. The concentration-response data for our derivatives were best fitted to a single Hill
equation (Figure 5). All compounds tested evoked inward currents in oocytes expressing α4β2 nAChR,
which indicates that all of them are agonists. Nevertheless, the agonist properties of compounds
showed differences both in potency and maximal responses (Table 1). The most potent agonist at
the human
compound 1 (Table 1) and showed a potency similar to nicotine. The halogenated compounds 3 and
4 were equipotent as compared with compound 1. Compound 2 was a full agonist at human
α4β2 nicotinic receptor was compound 2, which was about seven-fold more potent than
α4β2
nAChR (100% of efficacy). In contrast, compounds 1, 3, and 4 showed partial agonist properties at
this receptor subtype (Table 1). Thus, the Imax of compound 1 was half of that of the full agonist
ACh whereas the halogenated derivatives (compounds 3 and 4) were partial agonists with maximal
responses that were, respectively, 29% and 13% of the ACh Imax. These results indicate that the
introduction of a halogen atom at the meta position of our derivatives does not increase potency while
dramatically and differentially decreasing the ability of the compounds to fully activate the receptors.
In contrast, the presence of an amino group at this position increases affinity and potency and confers
full agonist properties.
2
1
3
4
1.0
0.5
0.0
-7
-6
-5
-4
-3
Log C (Agonist)
Figure 5. Interaction of phenylpyrrolidine derivatives with human
α
4
β2 nicotinic receptors expressed
in Xenopus laevis oocytes. Whole cell currents in response to agonist application were measured by a
two-electrode voltage clamp. Concentration-response curves show activation of whole cell currents
by compounds 1, 2, 3, and 4. Each point represents data from four to five cells, normalized to the
amplitude of the maximal responses of ACh, which was stimulated by 1 mM of ACh. The data are
fitted to a single component Hill curve.
2.3. Molecular Docking Studies
Binding studies indicated that our phenylpyrrolidine derivatives bind at the agonist (orthosteric)
binding site of
affinity, functional potency, and maximal responses of our derivatives, the possible binding modes of
these compounds were analyzed by docking simulations on the 2 nAChR agonist binding site.
The extracellular N-terminal ligand binding domain of the receptor was obtained from the X-ray
structure of the human 2 nAChR (Protein Data Bank code 5KXI) [13] and used for the docking
studies. Phenylpyrrolidine derivatives were docked into the 4(+)/ 2( ) interface centering the grid
on 4W156. This residue is equivalent to 1W149 on the muscle nAChR, which forms a -cation
α4β2 nAChRs. Therefore, in an attempt to rationalize the differences in binding
α4β
α4β
α
β
−
α
α
π
interaction with the quaternary ammonium group of ACh and the protonated nitrogen of several
agonists [30]. Docking results indicate that small changes in the nicotine structure, such as the addition

Molecules 2019, 24, 2684
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of an ether linker between the pyrrolidine moiety and the aromatic part of the parent compound, does
not generate unfavorable interactions within the binding site. However, the introduction of different
substituents at the meta position of the aromatic moiety produces important changes for the binding
modes. Thus, the halogenated derivatives (compounds 3 and 4) docked into the agonist binding site in
a position allows establishing the classical π-cation interaction with the tryptophan (α4W156), and, at
the same time, generates an unfavorable (electronic repulsive) interaction between the bulky halogen
atom and the C-loop (Figure 6B). This binding mode correlates with the low potency and efficacy of
these derivatives. Additionally, the unsubstituted compound 1 shows the same binding mode of the
halogenated derivatives, but the absence of a bulky substituent into the aromatic group (i.e., chloro or
bromo atoms) allows the C-loop to keep closed. As compounds 1, 3, and 4 exhibit similar affinities, the
differences in the binding mode revealed by docking simulations might be related with the higher
agonist efficacy of 1 as compared with the halogenated derivatives. On the other hand, the meta-amino
derivative (compound 2) adopted a binding mode in which unfavorable interactions were not detected,
and that, besides the
amino group and the carbonyl group of asparagine (
the complementary 2 subunit (Figure 6A). These results align with the nicotinic profile of compound
2, which shows high binding affinity, considerable agonist potency, and full agonism.
π-cation interaction, exhibited additional hydrogen bonding between the drug
β
2N109) and phenylalanine ( 2F119) residues on
β
β
Figure 6.
agonist binding site of the
and 2F119. ( ) Binding mode of compound 4 (pink) into the binding site of the
images show the -cation interaction between the ligands and the 4W156 in the opened channel state
(
A
) Superimposed structures of compounds 1 (grey), and 2 (magenta) docked into the
2 nAChR. Compound 2 shows the additional interaction with 2N109
2 nAChR. The
α
4
β
β
β
B
α4β
π
α
conformation (C loop closed).
3. Materials and Methods
3.1. Clonal Cell Lines
Membrane homogenates for [3H]-Cyt binding studies were prepared from the SH-EP-h
clonal cell line, which express human 2 nAChRs. Cells were maintained in Dulbecco’s modified
Eagle’s medium (Invitrogen, UK) supplemented with 5% fetal serum, 10% horse serum, 2 mM
α4β2
α4β
l-glutamine, 10 IU/mL penicillin, 10
zeocin, and 0.13 mg/mL hygromycin.
µg/mL streptomycin, 2.5 µg/mL amphotericin-B, 0.25 mg/mL
3.2. Ligand Binding Assays
Competition binding studies were performed on membrane homogenates prepared SH-EP-h
α4β2
clonal cell line, using [3H]cyt (PerkinElmer, UK), as previously described [28]. Membrane homogenates
were incubated at a final protein concentration of 30 to 50
of binding saline (in nM: 120 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, 50 Tris, pH 7.0) for 75 min at 4 C
µ
g per assay tube in a final volume of 250 ◦
µL

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with 1 nM [3H]cyt. For this binding assay, 10
µM nicotine was used to define nonspecific binding.
Bound and free fractions were separated by rapid filtration through Whatman GF/C filters presoaked
in binding saline supplemented with 0.1% polyethyleneimine. Radioactivity was quantified by liquid
scintillation spectrometry.
3.3. Nicotinic Acetylcholine Receptors Expression in Xenopus laevis Oocytes
The α4β2 nAChRs (wild type) were expressed heterologously in defolliculated oocytes from
Xenopus laevis, which were dissected from adult female X. laevis frogs (Nasco, Fort Atkinson, WI,
USA). The Oxford Brookes University Animal Research Committee, in accordance with the guidelines
of the 1986 Scientific Procedures Act of the United Kingdom, approved the care and use of X.
laevis frogs in this study. A mixture of 1:1 of human of
α4 and β2 subunits for α4β2 subtype
cDNAs were injected into the nuclei of oocytes in a volume of 23 nL/oocyte by using a Nanoject
Automatic Oocyte Injector (Drummond Scientific, Broomall, PA, USA). After injection, oocytes were
◦
incubated at 17 C in OPS (NaCl, KCl, CaCl2, MgCl2, pH 7.4) supplemented with a mixture of
penicillin-streptomycin-anphotericin-B (10,000 IU/mL penicillin, 10 mg/mL streptomycin, and 25 µg
amphotericin-B/mL) and amikacin (100 µg/mL). Experiments were performed on oocytes 2 to 6 days
after injection.
3.4. Electrophysiological Recordings
Electrophysiological recording from oocyte post-injection was made at room temperature using a
standard two electrode voltage clamp technique with an automatic multichannel system (HiClamp,
Multichannel Systems, Reutlingen, Germany). Oocytes were impaled by two borosilicate capillary
glass (Harvard Instrument: 150 TF GC) microelectrodes filled with 3 M KCl (0.3–2.0 M
voltage-clamped at 60 mV. During recording, oocytes were perfused with OPS (oocytes perfusion
solution), as described in the manual of HiClamp. Agonist responses were normalized to the amplitude
of the responses induced by 1 mM ACh, which is the maximal ACh concentration at 2 nAChRs.
Ω) and
−
α4β
Between each successive ACh or compound application, the cell was perfused with OPS solution for
3 min to allow drug clearance and prevent receptor desensitization.
3.5. Molecular Docking Studies
Molecular docking of the phenylpyrrolidine derivatives at the agonist binding domain of the α4β2
(PDB code 5KXI; [13]) was investigated using the Lamarckian genetic algorithm search method using
software AutoDock v4.2. The receptor was 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, which were assigned to the individual protein atoms.
The three-dimensional structures of each ligand were generated using the SPARTAN’11 program and
were then their energy was minimized. For each ligand, a rigid root and rotatable bonds were assigned
automatically. The non-polar 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 v4.2 aromatic carbon grid map. Docking was carried out using 60
with a default spacing of 0.375 Å. The grid was positioned to include the full ligand binding pocket in
the central part of the 2 subunit interfaces in order to allow extensive sampling around residue
4W156. Within this grid, the Lamarckian genetic search algorithm was used and calculated using
×
60
×
60 grid points
α4β
α
100 different runs (i.e., 100 dockings).
4. Chemistry
NMR spectra were recorded using Bruker AMX 400 spectrometers. Chemical shifts are reported
relative to TMS (δ = 0.00) and coupling constants (J) are given in Hz. IR spectra were recorded
using Cary 630 FTIR-ATR (Agilent, Santa Clara, USA). The elemental analyses for C, H, and N were
performed on a CE Instruments (model EA 1108) analyzer (Waltham, MA, USA). Reactions and product

Molecules 2019, 24, 2684
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mixtures were routinely monitored by thin-layer chromatography (TLC) on silica gel-pre-coated F254
Merck plates (Darmstadt, Germany), using a mixture of CH3Cl/CH3OH as a mobile phase. All reagents
and solvents were commercially available and were used without further purification.
4.1. General Procedures
4.1.1. Synthesis of (S)-2-(chloromethyl)-1-Methylpyrrolidine
(S)-(1-methylpyrrolidin-2-yl) metanol were previously transformed in their corresponding
(S)-2-(chloromethyl)-1-methylpyrrolidine, using thionyl chloride in dry THF as a solvent and refluxed
under a nitrogen atmosphere. The mixture was maintained at room temperature for 24 h, and
then the solvent, excess reagents, and remaining HCl and SO2 were evaporated under vacuum.
The (S)-2-(chloromethyl)-1-methylpyrrolidine was used immediately for the next reaction.
4.1.2. Synthesis of (S)-1-methyl-2-(phenoxymethyl)pyrrolidine Derivatives
(S)-3-(chloromethyl)-1-methylpyrrolidine was dissolved in 50 mL of dry acetonitrile stirred at
room temperature. One equivalent of phenol derivatives in 30 mL of dry acetonitrile was added drop
by drop. The reaction mixture was kept at reflux with constant stirring for 48 h. Then, the solvent was
evaporated and the mixture was dissolved in water, adjusted to pH 8.0, extracted with CH3Cl, and
purified by chromatography using a combi flash chromatograph. The hydrochloride salt was obtained
from acetone.
4.2. (S)-1-methyl-2-(phenoxymethyl)pyrrolidine Ether (1)
Obtained as a light yellow oil in 56% yield, salt m.p. 175–178 ◦C.1H-RMN(DMSO-d6)
δ: 8.08 (d,
J = 7.8 Hz, 2H, 2-H y 6-H), 7.75 (t, J = 7.5 Hz, 1H, 4-H), 7.59 (t, J = 7.7 Hz, 2H, 3-H y 5-H), 4.76 (d, J = 2.9
Hz, 1H, 10-H), 4.58 (dd, J = 13.1, J = 6.7 Hz, 1H, 10-H), 3.95 (s, 1H, 20-H), 3.78 (s, 1H, 30-H), 3.29 (s, 1H,
30-H), 3.08 (s, 3H, 60-H), 2.45 (m, 1H, 50-Ha), 2.26 (m, 1H, 50-Hb), 2.12 (ddd, J = 31.3, 14.5, 7.1 Hz, 2H,
40-Ha y 40-Hb). 13C-RMN (DMSO-d6)
δ: 165.3 (1-C), 133.6 (4-C), 129.4 (2-C y 6-C), 129.1 (3-C), 128.7
(5-C), 66.0 (20-C), 62.5 (10-C), 56.1 (30-C), 39.5 (60-C), 26.6 (50-C), 21.7 (40-C). IR (cm−1) 3032, 2978, 1279,
1112 [22].
4.3. (S)-1-methyl-2-(30-aminophenoxymethyl)pyrrolidine Ether (2)
Obtained as a light brown oil in 41% yield, salt m.p. 198–200 ◦C. 1H-RMN (DMSO-d6)
δ: 7.90 (s,
1H, 2-H), 7.79 (d, J = 7.5 Hz, 1H, 6-H), 7.56 (dd, J = 13.3, 7.8 Hz, 1H, 4-H), 7.48 (t, J = 8.5 Hz, 1H, 5-H),
4.63 (s, 2H, 10-H), 3.81 (s, 1H, 20-H), 3.56 (s, 1H, 30-Ha), 3.13 (s, 1H, 30-Hb), 2.87 (s, 3H, 60-H), 2.24 (dt,
J = 13.8, 6.9 Hz, 1H, 50-Ha), 1.99 (m, 2H, 50-Hb y 40-Ha), 1.87 (dt, J = 20.7, 7.6 Hz, 1H, 40-Hb). 13C-RMN
(DMSO-d6) δ
: 167.9 (1-C), 162.6 (3-C), 132.4 (5-C), 127.17 (6-C), 122.7 (4-C), 117.8 (2-C), 68.9 (20-C), 64.2
(10-C), 58.8 (30-C), 42.2 (60-C), 27.7 (50-C), 23.6 (40-C). IR (cm−1) 3442, 3360, 3046, 2978, 1285, 1270, 1115.
Anal. Calcd for C12H19ClN2O: C, 59.38; H, 7.89; N, 11.54. Found: C, 58.74; H, 7.72; N, 11.46.
4.4. (S)-1-methyl-2-(30-Chlorophenoxymethyl)pyrrolidine Ether (3)
Obtained as a light yellow oil in 61% yield, salt m.p. 158–160 ◦C. 1H-RMN (DMSO-d6)
δ: 8.02
(s, 1H, 2-H), 7.77 (d, J = 7.8 Hz, 1H, 6-H), 7.59 (t, J = 7.8 Hz, 1H, 5-H), 7.45 (dd, J = 11.3, 6.9 Hz, 1H,
4-H), 4.63 (s, 2H, 10-H) 3.80 (s, 1H, 20-H), 3.58 (s, 1H, 30-H), 3.09 (d, J = 9.0 Hz, 1H, 30-H), 2.83 (s, 3H,
60-H), 2.25 (m, 1H, 50-H), 2.05 (m, 1H, 50-H), 1.92 (m, 2H, 40-H ). 13C-RMN (DMSO-d6)
δ: 166.8 (1-C),
135.3 (2-C), 135.2 (4-C), 135.2 (3-C), 131.9 (5-C), 129.40 (6-C), 70.80 (20-C), 59.74 (10-C), 57.83 (30-C), 41.33
(60-C), 26.98 (50-C), 22.95 (40-C). IR (cm-1) 3075, 2995, 1277, 1108, 740 [22].
4.5. (S)-1-methyl-2-(30-bromophenoxymethyl)pyrrolidine Ether (4)
Obtained as a light yellow oil in 52% yield, salt m.p. 147–1149 ◦C. 1H-RMN (DMSO-d6)
1H, 2-H), 7.93 (d, J = 7.7 Hz, 1H, 6-H), 7.82 (d, J = 7.9 Hz, 1H, 4-H), 7.47 (t, J = 7.8 Hz 1H, 5-H), 4.60
δ: 8.03 (s,

Molecules 2019, 24, 2684
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(dt, J = 12.7, 9.6 Hz, 2H, 10-H), 3.82 (s, 1H, 20-H), 3.56 (s, 1H, 30-Ha), 3.14 (s, 1H, 30-Hb), 2.90 (s, 3H,
60-H), 2.23 (m, 1H, 50-H), 2.03 (m, 1H, 50-H), 1.90 (m, 2H, 40-Ha y 40-Hb). 13C-RMN (DMSO-d6)
δ: 165.2
(1-C), 135.9 (4-C), 131.2 (5-C), 129.5 (2-C), 127.3 (6-C), 121.0 (3-C), 66.4 (20-C), 61.5 (20-C), 56.2 (30-C),
39.6 (60-C), 25.1 (50-C), 20.9 (40-C). IR (cm−1) 2980, 1281, 1115, 686. Anal. Calcd for C12H17BrClNO: C,
47.01; H, 5.59; N, 4.57. Found: C, 46.69; H, 5.25; N, 4.15.
5. Conclusions
Our results indicate that phenylpyrrolidine ether derivatives bind to the orthosteric site of the
α4β2 nAChR. Binding experiments show that all compounds synthesized fully displaced [3H]cyt from
its binding site with IC50s in the nanomolar range using the m-amino derivative (compound 2), which
shows the best affinity (IC50 =13 nM). Functional studies show that the introduction of a halogen atom
into the meta position of the aromatic ring dramatically and differentially decreases efficacy for the
agonist activity as compared with compounds 1 (the unsubstituted derivative) and 2 (the meta-amino
derivative). In addition, the meta-amino derivative was the most potent (EC50 =10 µM) agonist in this
series, was equipotent to nicotine, and was a full agonist. Notably, a whole range of efficacies could
be detected in this small series of compounds. Molecular docking studies indicate that differential
affinities, potencies, and efficacies of the compounds tested, might be related with their different
binding modes (yielding different interactions) at the agonist binding site of the α4β2 nAChR. Thus,
our data suggest some amino acid residues (and their corresponding three dimensional assemblies) at
the binding site, whose role, particularly in the modulation of efficacy of agonists, could be explored.
The ample spectrum of efficacies exhibited by this small series of compounds supports our idea
that subtle structural modifications can drastically modulate the nicotinic profile of agonists. It also
highlights the relevance of searching for novel compounds with varied pharmacodynamics, which
might be useful for treating patients with diverse characteristics. Further experiments, including
additional compounds, are necessary to confirm the hydrogen bonding effect and to evaluate the
therapeutic potential of the compounds described, particularly in the case of the partial agonists.
Author Contributions: P.I.-V. and M.R.-P. conceived and designed the experiments and wrote the manuscript.
J.P.G.-G., M.H., N.G.-D., F.V., and P.P. performed the experiments. I.B. discussed the manuscript with the help of
H.P.-M., J.J.H.-M., and H.P.-S. All authors read and approved the final manuscript.
Funding: The financial support for this research work, awarded by the National Foundation of Science and
Technology grants 1150615 (P.I.-V.), 1170662 (M.R.-P.) and 1170269 (H.P.-M.), is gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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