2421
derivatives (Sadek et al., 2012) and 1-(1-benzyl-1H-indol-3-yl)-
N,N,N-trimethylmethanaminium iodides (Pérez et al., 2013), and
the selective ␣42 nAChR antagonists, diazaspirocyclic compounds
(Strachan et al., 2012). Thus, an improved understanding of the
functional and structural interaction of selective ␣7 or ␣42 nAChR
antagonists is crucial to the development of safer ligands for differ-
ent therapeutic uses.
The nicotinoid pharmacophore spans two important moieties
well-characterized “aromatic box” of the nAChR; and a nitrogen-
containing heterocycle that form a hydrogen bond with the
complementary subunit in the receptor and is important for ago-
nist activity (Blum et al., 2010). The work by Crooks et al. (1995)
has led to the discovery of several nAChR antagonists which
potently inhibit selective nAChR subtypes. They showed that N-
n-alkylation of the pyridine nitrogen atom of nicotine with C1–C12
carbon chains converted nicotine from a potent agonist into potent
and subtype-selective nAChR antagonists. This suggests that com-
pounds with a center containing a positively charged nitrogen
as nAChR antagonists. Additionally, the bicycle quinuclidine (1-
azabicycle[2.2.2]octane) is frequently mentioned in the design and
synthesis of potent and selective ligands for the ␣7 nAChR sub-
type (Mazurov et al., 2006). Using this approach and looking for
potent and selective antagonists for the ␣7 nAChR subtype, a new
series of 2-(substituted benzyl)quinuclidines were designed and
their chemical identities characterized. Subsequently, the phar-
macological activity of each antagonist was determined by Ca2+
influx experiments on cell lines expressing human (h) ␣7 or h␣42
nAChRs, and the mechanisms of inhibition were differentiated by
radioligand binding assays. Finally, the binding site locations and
the structural components of these sites were characterized on each
receptor subtype by molecular docking and molecular dynamics
studies.
as base and solvent respectively; (ii) the alkyloxy aldehydes
were coupled with 3-quinuclidinone in a basic media to furnish
the corresponding (Z)-2-benzylidenequinuclidin-3-ones (6a–6k);
(iii) these ␣,-unsaturated carbonyl compounds were completely
reduced in two steps using H2, Pd/C and the Wolff-Kishner reac-
tion to produce the 2-(substituted benzyl)quinuclidines (7a–7k);
and finally, (iv) the quinuclidines were quaternized with methyl
iodide in acetone to obtain the N-methyl-2-(substituted ben-
zyl)quinuclidinium iodides (8a–8k).
2.2.2. Chemical characterization
Melting points were determined on a Reichert Galen III hot
plate microscope apparatus and are uncorrected. 1H NMR spectra
400 MHz, respectively. 13C NMR spectra were recorded on the same
instruments at 50 or 100 MHz. The results are detailed in Supple-
mental Information. Chemical shifts are reported in ı values (parts
per million, ppm) relative to an internal standard of tetramethyl-
silane in CDCl3 or DMSO-d6 and coupling constants (J) are given in
Hertz. Precoated silica gel 60 plates (Merck 60 F2540.2 mm) were
used for TLC. TLC spots were visualized by spraying with Dragen-
dorff’s reagent or by UV light at 254 nm.
2.2.3. Ca2+ influx measurements in cells containing different
AChR subtypes
Ca2+ influx assays were performed on HEK293-h␣42 and GH3-
h␣7 cells using the procedures previously described (Arias et al.,
2010a,b,c, 2011). Briefly, cells were seeded 48 h prior to the experi-
ment on black 96-well plates (Costar, New York, USA) at a density of
5 × 104 per well and incubated at 37 ◦C in a humidified atmosphere
(5% CO2/95% air). The medium was changed to 1% FBS in HEPES-
buffered salt solution (HBSS) (130 mM NaCl, 5.4 mM KCl, 2 mM
CaCl2, 0.8 mM MgSO4, 0.9 mM NaH2PO4, 25 mM glucose, 20 mM
HEPES, pH 7.4) 16–24 h before the experiment. On the day of the
experiment, the medium was removed by flicking the plates and
replaced with 100 L HBSS/1% BSA containing 2 M Fluo-4 (Molec-
ular Probes, Eugene, OR, USA) in the presence of 2.5 mM probenecid
(Sigma, Buchs, Switzerland). The cells were then incubated at 37 ◦C
in a humidified atmosphere (5% CO2/95% air) for 1 h. Plates were
flicked to remove excess of Fluo-4, washed twice with HBSS, and
then placed in the cell plate stage of the fluorimetric imaging plate
reader (Molecular Devices, Sunnyvale, CA, USA). To measure the
inhibitory activity of the compounds, each compound was added
at the concentrations indicated in the figures instead of the second
washing step, and pre-incubated for 5 min at RT. Thereafter, ( )-
epibatidine (0.1 M) was added from the agonist plate to the cell
plate using the 96-tip pipettor, and the fluorescence was recorded
for 3 min. For all measurements, a baseline consisting of 5 measure-
ments of 0.4 s each was recorded. The laser excitation and emission
wavelengths are 488 and 510 nm, at 1 W, and a CCD camera opening
of 0.4 s.
2. Material and methods
2.1. Material
[3H]Imipramine
(47.5 Ci/mmol)
and
[3H]cytisine
(35.6 Ci/mmol) were obtained from PerkinElmer Life Sciences Prod-
ucts, Inc. (Boston, MA, USA), and [3H]methyllycaconitine ([3H]MLA)
(100 Ci/mmol) was purchased from American Radiolabeled Chem-
icals Inc. (St. Louis, MO, USA). Imipramine hydrochloride and
carbamylcholine dichloride (CCh) were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). ( )-Epibatidine hydrochloride
was obtained from Tocris Bioscience (Ellisville, MO, USA). -
Bungarotoxin (-BTx) was obtained from Biotoxins Inc. (St. Cloud,
FL, USA).
The concentration–response data were curve-fitted, and the IC50
and Hill coefficient (nH) values were calculated by nonlinear least
squares analysis using the Prism software (GraphPad Software, San
Diego, CA).
2.2.4. Radioligand competition binding experiments
Radioligand experiments were performed using membranes
previously (Arias et al., 2010a,b,c, 2011). We first compared
the effect of novel ligands on [3H]cytisine binding to h␣42
nAChRs and on [3H]MLA binding to h␣7 nAChRs, using the
method previously developed in our laboratory (Arias et al., 2010a,
2011). To determine the interaction of these compounds with
the nAChR ion channels, additional [3H]imipramine competition
binding experiments were performed as described previously
2.2. Methods
2.2.1. Chemical synthesis
A
new series of 2-(substituted benzyl)quinuclidines and
N-methyl-2-(substituted benzyl)quinuclidinium iodides were syn-
thesized as follow: (i) the hydroxyl benzaldehydes (1–4) were
converted into the corresponding alkyloxy benzaldehydes (5a–5k)
by the classical Williamson methodology using K2CO3 and ethanol