Synthesis and characterization of [3H]-SN56, a novel radioligand for the σ1 receptor

Article abstract of DOI:10.1016/j.ejphar.2010.10.099

The study of the binding characteristics of σ ligands in vivo and in vitro requires radiolabeled probes with high affinity and selectivity. The radioligand presently used for in vitro studies of the σ₁ receptor, [³H](+)-pentazocine, has significant limitations; it is difficult to synthesize, has limited chemical stability, and can be problematic to obtain. Evaluation of a series of novel 2(3H)-benzothiazolone compounds revealed SN56 to have sub-nanomolar and preferential affinity for the σ₁ subtype, relative to σ₂ and non-sigma, binding sites. The goal of this study was to characterize the binding of [ ³H]-SN56 to σ₁ receptors isolated from rat brain. Standard in vitro binding techniques were utilized to 1) determine the specificity and affinity of binding to σ₁ receptors, 2) confirm that [³H]-SN56 labels sites previously identified as σ₁ by comparing binding to sites labeled by [ ³H](+)-pentazocine, and 3) characterize the kinetics of binding. The results indicate that [³H]-SN56 exhibits 1) specific, saturable, and reversible binding to the σ₁ receptor, with B_max = 340 ± 10 fmol/mg and K_d = 0.069 ± 0.0074 nM, 2) competitive displacement by classical sigma compounds, yielding σ₁ K_i values consistent with those reported in the literature, and 3) binding kinetics compatible with a 90 min incubation, and filtration for separation of free and bound radioligand. The results of these studies suggest that [³H]-SN56 may serve as a viable alternative to [³H](+)-pentazocine in radioligand binding assays.

Full text of DOI:10.1016/j.ejphar.2010.10.099

European Journal of Pharmacology 653 (2011) 1–7
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Molecular and Cellular Pharmacology
3
Synthesis and characterization of [ H]-SN56, a novel radioligand for the σ receptor
1
James A. Fishback a,b, Christophe Mesangeau , Jacques H. Poupaert ,
c
d
c
a,b,
Christopher R. McCurdy , Rae R. Matsumoto
a
⁎
Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506, USA
Department of Pharmacology, University of Mississippi, University, MS 38677, USA
Department of Medicinal Chemistry, University of Mississippi, University, MS 38677, USA
Universite Catholique de Louvain, Avenue Emmanuel Mounier 74, B-1200, Brussels, Belgium
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 May 2010
Received in revised form 30 September 2010
Accepted 6 October 2010
Available online 2 December 2010
The study of the binding characteristics of σ ligands in vivo and in vitro requires radiolabeled probes with high
3
affinity and selectivity. The radioligand presently used for in vitro studies of the σ
1
receptor, [ H](+)-
pentazocine, has significant limitations; it is difficult to synthesize, has limited chemical stability, and can be
problematic to obtain. Evaluation of a series of novel 2(3H)-benzothiazolone compounds revealed SN56 to
have sub-nanomolar and preferential affinity for the σ
The goal of this study was to characterize the binding of [ H]-SN56 to σ
Standard in vitro binding techniques were utilized to 1) determine the specificity and affinity of binding to σ
1
subtype, relative to σ
2
and non-sigma, binding sites.
3
1
receptors isolated from rat brain.
Keywords:
Radioligand binding assay
σ Receptor
1
3
1
receptors, 2) confirm that [ H]-SN56 labels sites previously identified as σ by comparing binding to sites
3
3
labeled by [ H](+)-pentazocine, and 3) characterize the kinetics of binding. The results indicate that [ H]-
SN56 exhibits 1) specific, saturable, and reversible binding to the σ receptor, with Bmax =340± 10 fmol/mg
and K =0.069± 0.0074 nM, 2) competitive displacement by classical sigma compounds, yielding σ values
1
d
1 i
K
consistent with those reported in the literature, and 3) binding kinetics compatible with a 90 min incubation,
3
and filtration for separation of free and bound radioligand. The results of these studies suggest that [ H]-SN56
3
may serve as a viable alternative to [ H](+)-pentazocine in radioligand binding assays.
©
2010 Elsevier B.V. All rights reserved.
1
. Introduction
efforts to identify novel selective compounds are ongoing. While the
receptor may also represent a feasible drug development target,
σ
2
The σ receptor was first identified as an opioid receptor subtype
further research in this area will require the discovery of additional
selective ligands for this subtype. The focus of the current work is
based on behavioral studies of morphine-like drugs in dogs (Martin
et al., 1976). Subsequent in vitro binding data revealed that this site
represents a new class of non-opioid receptor (Tam, 1983). To date,
therefore limited to the characterization of σ
1
binding.
DeCosta et al. (1989) first described [ H](+)-pentazocine, a highly
selective radioprobe for σ receptors. Subsequent studies demon-
3
two subtypes of σ receptors (σ
differences in ligand selectivity, tissue distribution, and molecular
characterization. The σ receptor has been cloned from multiple
species (Hanner et al., 1996; Kekuda et al., 1996; Mei and Pasternak,
001; Pan et al., 1998; Seth et al., 1997, 1998) and a significant
number of ligands with high affinity and selectivity for it are available.
The σ receptor is less well characterized; it has not been cloned, and
few specific ligands have been described.
The σ receptor is involved in numerous physiological processes
and disease states, and in vivo and in vitro studies indicate that
modulation of σ receptors using σ specific ligands can affect these
systems (Cobos et al., 2008; Guitart et al., 2004; Hashimoto and
Ishiwata, 2006; Maurice and Su, 2009). Consequently, the σ receptor
is recognized as a potential medication development target and
1
2
and σ ) have been identified based on
1
3
strated that [ H](+)-pentazocine labeled a single class of sites in
1
guinea-pig brain that correlated with the profile observed following
3
labeling with the prototypic σ
1
probe [ H](+)-3-PPP (Bowen et al.,
3
2
1993). [ H](+)-Pentazocine exhibited low levels of non-specific
binding and high affinity for σ receptors (K =4.8± 0.4 nM), with
N700 fold preference for the σ over the σ subtype (Bowen et al., 1993).
1
d
2
1
2
3
[ H](+)-Pentazocine does however exhibit shortcomings, including
poor chemical stability, that limit its usefulness in routine studies.
1
1
Efforts to design new σ specific ligands have produced a limited
1
number of radioprobes useful for exploring the pharmacology of the
3
σ
1
receptor. Unfortunately, like [ H](+)-pentazocine, each of the
proposed new radioprobes exhibit limtations. Therefore, we sought to
characterize the performance of SN56, a novel σ selective, 2(3H)-
benzothiazolone compound, as a tritiated radioligand for use in σ
competition binding experiments. SN56 exhibited sub-nanomolar
1
1
1
⁎
Corresponding author. West Virginia University, School of Pharmacy, P.O. Box 9500,
Morgantown, WV 26506, USA. Tel.: +1 304 293 1450; fax: +1 304 293 2576.
affinity (Kiσ1 =0.56 nM) and N1000 fold selectivity for the σ subtype
1
relative to σ and at least 350 times greater affinity for the σ receptor
1
E-mail address: rmatsumoto@hsc.wvu.edu (R.R. Matsumoto).
2
0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2010.10.099

2
J.A. Fishback et al. / European Journal of Pharmacology 653 (2011) 1–7
versus a battery of common receptors and transporters (Yous et al.,
005). These binding characteristics coupled with a simple and
was dissolved in ethyl acetate and the solvent was washed with water
and brine, dried and evaporated. The residue was recrystallized from
2
3
economical synthetic scheme suggested [ H]-SN56 may provide a
toluene/dioxane (2/1) to give 2.96 g (54%) of 6-propionylbenzo[d]
3
1
viable alternative to [ H](+)-pentazocine in competition binding
thiazol-2(3H)-one as a white solid. H NMR (DMSO-d
6
): δ 12.23 (br s,
studies of the σ
1
receptor.
1H), 8.20 (s, 1H), 7.87 (d, J=8.4 Hz, 1H), 7.17 (d, J=8.3 Hz, 1H), 2.97
q, J=7.1 Hz, 2H), 1.06 (t, J=7.1 Hz, 3H). 13C NMR (DMSO-d
): δ
198.64, 170.46, 140.13, 131.21, 126.74, 123.69, 123.04, 111.17, 30.93,
(
6
2
. Materials and methods
+
8
.22. MS (ESI) m/z 206 (M -1).
2
.1. Materials
2.2.2. 6-Propylbenzo[d]thiazol-2(3H)-one (2)
Reagents and starting materials for the synthesis of SN56 were
Triethylsilane (4.75 ml, 29.75 mmol) was added to a stirred solution
obtained from commercial suppliers and were used without purifica-
tion. Precoated silica gel GF Uniplates from Analtech (Newark, DE) were
used for thin-layer chromatography (TLC). Column chromatography
of 1 (2.5 g, 12.06 mmol) in trifluoroacetic acid (13 ml). The mixture was
stirred vigorously for 2 h at room temperature. The trifluoroacetic acid
was removed by evaporation and the residue was purified by
chromatography on a silica gel column using petroleum ether/ethyl
1
was performed on silica gel 60 (Sorbent Technologies, Atlanta, GA). H
and 13C NMR spectra were obtained on a Bruker APX400 (Billerica, MA)
at 400 and 100 MHz, respectively. High resolution mass spectra (HRMS)
were recorded on a Waters Micromass Q-Tof Micro mass spectrometer
with a lock spray source (Milford, MA). Mass spectra (MS) were
recorded on a Waters Acquity Ultra Performance LC (Milford, MA) with
ZQ detector in ESI mode. Elemental analysis (C, H, N) was performed on
a Perkin-Elmer CHN/SO Series II Analyzer (Waltham, MA). Chemical
names were generated using ChemDraw Ultra (CambridgeSoft, version
acetate (9:1) as the eluent to give 2.06 g (88%) of 6-propylbenzo[d]
1
6
thiazol-2(3H)-one as a white solid. H NMR (DMSO-d ): δ 11.76 (br s,
1H), 7.33 (s, 1H), 7.05 (d, J=8.1 Hz, 1H), 6.99 (d, J=8.1 Hz, 1H), 2.50
(t, J=7.4 Hz, 2H), 1.56–1.50 (m, 2H), 0.84 (t, J=7.3 Hz, 3H). 13C NMR
6
(DMSO-d ): δ 169.99, 136.68, 134.23, 126.57, 123.24, 122.12, 111.22,
+
36.87, 24.30, 13.54. MS (ESI) m/z 194 (M +1).
2.2.3. 4-Bromo-6-propylbenzo[d]thiazol-2(3H)-one (3)
1
0.0, Cambridge, MA).
3H](+)-Pentazocine (29 Ci/mmol) was purchased from Perkin-
Elmer (Boston, MS). (+)-Pentazocine, (−)-pentazocine, haloperidol,
,3-di-o-tolylguanidine (DTG), bovine serum albumin (BSA) fraction
Bromine (0.45 ml, 8.75 mmol) was added slowly to a solution of 2
(1.5 g, 7.76 mmol) in acetic acid (10 ml). The mixture was stirred 15 h
at room temperature, poured into water and extracted with ethyl
acetate (3×30 ml). The combined organic layers were washed with a
10% solution of potassium carbonate followed by brine. The solution
was dried over sodium sulfate and evaporated under vacuum. The
residue was purified by chromatography on a silica gel column using
petroleum ether/ethyl acetate (9:1) as the eluent to give 0.5 g (24%)
[
1
V, sucrose, sodium chloride, tris(hydroxymethyl)aminomethane, 1N
hydrochloric acid solution, and glacial acetic acid were purchased
from Sigma-Aldrich (St. Louis, MO). Bio–rad Protein Assay reagent
was purchased from Bio–rad (Hercules, CA). Ecoscint scintillation
fluid and Brandel GF/B filter papers, 2.25×12.25 in. were purchased
from Fisher Scientific (Pittsburgh, PA).
of 4-bromo-6-propylbenzo[d]thiazol-2(3 H)-one as a white solid.
1
H NMR (CDCl
t, J=7.6 Hz, 2H), 1.62 (m, 2H), 0.93 (t, J=7.2 Hz, 2H). C NMR
(CDCl ): δ 170.45, 139.35, 132.22, 129.34, 124.44, 121.24, 103.90,
7.37, 24.48, 13.54. MS (ESI) m/z 270 (M −1), 272 (M +1).
3
): δ 9.29 (s, 1H), 7.26 (s, 1H), 7.12 (s, 1H), 2.56
13
(
3
2
.2. Synthesis of [ H]-SN56
3
+
+
3
3
The design strategy for generating [ H]-SN56 involved replacing a
bromine atom on the aromatic ring of SN56 with a tritium atom
Fig. 1). The preparation of the brominated precursor 4 is described
2.2.4. 3-(2-(Azepan-1-yl)ethyl)-4-bromo-6-propylbenzo[d]thiazol-2
(3H)-one hydrochloride (4)
(
below. Compounds 1 and 2 were prepared according to previously
described procedures with minor modifications (Ucar et al., 1998;
Yous et al., 1994). Selective bromination of the 6-propylbenzo[d]
thiazol-2(3H)-one 2 at the C-4 position was effected with bromine in
acetic acid at room temperature. The bromo derivative was alkylated
with 2-(hexamethyleneimino)ethylchloride in the presence of potas-
sium carbonate in DMF to yield 4. Compound 4 was radiolabeled with
tritium (30 Ci/mmol) by AmBios Labs, Inc. (Newington, CT).
3
NaHCO (0.51 g, 6.09 mmol) and 2-(hexamethyleneimino)
ethylchloride hydrochloride (0.80 g, 4.06 mmol) were added, with
mechanical stirring, to a solution of 3 (0.55 g, 2.03 mmol) in
anhydrous DMF (15 ml). The reaction mixture was heated to 80 °C
for 1 h. After cooling, the mixture was poured into 80 ml of water,
extracted with ethyl acetate (3×60 ml), and the combined organic
layers were washed with brine and dried. The solvent was removed in
vacuo, and the residue was chromatographed on a silica gel column
using ethyl acetate/petroleum ether (4:6) as the eluent. 3-(2-
(azepan-1-yl)ethyl)-4-bromo-6-propylbenzo[d]thiazol-2(3H)-one
2
.2.1. 6-Propionylbenzo[d]thiazol-2(3H)-one (1)
Dimethylformamide (5.96 ml, 76.73 mmol) was added slowly to
was isolated as a hydrochloride salt (white solid, 0.44 g, 49%) by
1
aluminium chloride (35.5 g, 264.6 mmol) with vigorous stirring. After
5 min, 2-hydroxybenzothiazole (5.4 g, 40 mmol) was added and the
mixture was heated to 45 °C. After 15 min, propionyl chloride
3.46 ml, 39.7 mmol) was added and the reaction mixture was heated
to 85 °C for 3 h. The hot mixture was then poured on ice; the crude
product was collected by filtration, and washed with water. The solid
addition of HCl/dioxane. H NMR (DMSO-d
6
): δ 10.81 (br s, 1H), 7.60
1
(s, 1H), 7.46 (s, 1H), 4.72 (t, J=7.2 Hz, 2H), 3.47–3.24 (m, 6H), 2.56
(t, J=7.5 Hz, 2H), 1.86 (br s, 4H), 1.67–1.55 (m, 6H), 0.88
(
(t, J=7.2 Hz, 3H). 13C NMR (DMSO-d
6
): δ 170.54, 138.87, 132.87,
132.63, 125.62, 121.79, 103.79, 56.73, 55.89, 43.14, 37.04, 28.58,
27.22, 24.51, 13.81. Anal. calcd for C18 26 BrClN OS: C, 49.83; H, 6.04;
H
2
N, 6.46. Found: C, 50.06; H, 5.93; N, 6.47. HRMS calcd for C18
OSBr [M+H] 397.0949, found 397.0945.
H
26
N
2-
+
A synopsis of the synthetic route of the synthesis of the
brominated [ H]-SN56 precursor is provided in Fig. 2.
3
N
O
N
O
N
O
3
Br
H
N
S
N
S
N
S
2
.3. Membrane preparation
[3H]-SN56
Crude P rat brain homogenates were prepared from male, Sprague
2
SN56
4
Dawley rats (150–200 g) purchased from Harlan (Indianapolis, IN) as
described previously (Matsumoto et al., 1995). All procedures involving
3
Fig. 1. Structure of SN56, [ H]-SN56 and its bromo precursor 4.

J.A. Fishback et al. / European Journal of Pharmacology 653 (2011) 1–7
3
H
N
H
N
H
N
A
B
O
O
O
S
S
S
O
1
2
C
N
O
B r
B r
D
H
N
N
O
S
S
4
3
3
Fig. 2. Synthesis of brominated [ H]-SN56 precursor 4: A) propionyl chloride, AlCl
hydrochloride, K CO , DMF, 80 °C.
3
, 85 °C; B) (C
2 5
H )
3
SiH, CF
3
COOH, rt; C) Br
2
3
, CH COOH, rt; d) 2-(hexamethyleneimino)ethylchloride
2
3
live animals were performed as approved by the Institutional Animal
Care and Use Committee at the locations where the assays were
performed. Briefly, unanesthetized rats were sacrificed by decapitation;
brains minus cerebellum were harvested and maintained in ice cold
2.4.1. Association and dissociation assays
Association and dissociation studies were conducted to confirm
3
that the binding kinetics of [ H]-SN56 were appropriate for a 1–2 h
incubation and processing by filtration. Kinetic studies were
3
1
0 mM Tris–HCl/0.9% NaCl until processed. Tissues were homogenized
performed with 0.8 nM [ H]-SN56 and 100 μg membrane. For
with a Potter-Elvehjem homogenizer (5–10 strokes with motor driven
Teflon pestle) in ice-cold 10 mM Tris–sucrose buffer (0.32 M sucrose in
determination of association rates, samples were incubated for
times ranging from 5 min to 2 h prior to filtration. For determination
of dissociation rates, membranes were incubated for 120 min with
1
0 mM Tris–HCl, pH 7.4) using 10 ml buffer per gram of tissue×~3 g
3
tissue/batch. Homogenates from multiple batches were combined and
centrifuged for 10 min at 1000×g, at 4 °C. Supernatants were decanted,
combined and centrifuged for 15 min at 31,000×g, at 4 °C. To reduce
levels of bound endogenous ligand(s), the material from centrifugation
at 31,000×g was washed as follows: 1) pellets were re-suspended in
[ H]-SN56 prior to the addition of 100 μM (final concentration)
haloperidol, followed by filtration at times ranging from 30 min to 4 h
from the addition of haloperidol. The assays were performed in
duplicate and repeated three times.
1
0 mM Tris–HCl, pH 7.4 using 3 ml buffer per gram of tissue, 2) the
2.4.2. Saturation binding assays
resulting suspension was incubated for 30 min at 25 °C, 3) following
incubation, the suspension was centrifuged for 15 min at 31,000×g, at
d
For the determination of K and Bmax by saturation binding, ten
3
concentrations ranging from 0.01 to 0.8 nM of [ H]-SN56 were tested
per experiment with 100 μg membrane per sample. Non-specific
binding was determined by the addition of haloperidol, at a 100 μM
final concentration. Samples for the determination of total and non-
specific binding for each experiment were run concurrently and
filtered simultaneously.
4
°C. The resulting pellets were re-suspended in Tris–HCl, pH 7.4 buffer
at a final concentration of 1 g of tissue per 1.53 ml buffer. Tissue
preparations were aliquoted in 1 ml portions and stored at −80 °C. The
Bradford assay was used to quantitate protein concentration (Bradford,
1
976).
2.4.3. Competition binding assays
2
.4. Radioligand binding assays
i
For the determination of K for established σ ligands by
competition binding, aliquots of membrane were incubated with
[ H]-SN56 and varying concentrations of test ligands. The following
3
Initial optimization of assay conditions was performed to maximize
total binding and minimize non-specific binding. Parameters examined
included evaluation of the buffer composition and pH, ratio of
radioligand to membrane concentration, and determination of ligand
and ligand concentration for defining non-specific binding. The
following optimized conditions were used for subsequent studies
reported below: 0.5 ml final sample volume, 90 min sample incubation
at 25 °C, Tris–HCl pH8.0 (assaybuffer), and 10 μM haloperidol (to define
non-specific binding). Assay termination was effected by vacuum
filtration through glass fiber filters on a 24 position Brandel cell
harvester. Prior to use, filters were presoaked for 30 min in 0.5%
polyethyleneimine to reduce non-specific binding. Following the initial
filtration step, filters were washed in triplicate with 5 ml ice-cold
test compounds were evaluated: DTG, haloperidol, (+)-pentazocine,
(−)-pentazocine. For each test compound, 10 concentrations were
3
incubated with 0.7–0.8 nM [ H]-SN56 with 100 μg membrane per
sample. Non-specific binding was determined by the addition of
haloperidol, 100 μM final concentration. Samples for the determina-
tion of total and non-specific binding for each experiment were run
concurrently and filtered simultaneously.
2.5. Scintillation counting and data analysis
Following washing, filters were transferred to scintillation vials
and 5 ml scintillation cocktail was added. Filters were allowed to soak
in cocktail for a minimum of 10 h prior to counting.
1
0 mM Tris–HCl, pH 8. The conditions determined from the preliminary
studies were consistent with those reported in the literature for the
analysis of σ receptor binding using [ H](+)–pentazocine (Bowen
1
The data were analyzed using GraphPad Prism software (San
Diego, CA). Saturation binding data were fit using nonlinear
regression to a one site model. Association kinetics data were fit
3
et al., 1993; Hellewell et al., 1994; Matsumoto et al., 1996). It should be
3
noted that it was necessary to prepare [ H]-SN56 spiking solutions in
using linear regression of the plot of ln(B
B =radioligand bound at equilibrium, and B=radioligand bound at
e
e
−B/B ) versus time, where
1
mM HCl, to prevent non-specific binding of the radioligand to glass
e
and plasticware, which was problematic with solutions prepared in the
assay buffer. This requirement is not unprecedented and the small
amount of acid has no impact on the final pH of the assay sample
time t; the slope of the plot yielded kobs. The association rate constant
(k+1) was calculated using the pseudo first-order method from the
equation k+1 =(kobs −k−1)/[L], where [L]=radioligand concentra-
tion. Dissociation kinetics data were fit using linear regression of the
(Bylund and Toews, 1993).

4
J.A. Fishback et al. / European Journal of Pharmacology 653 (2011) 1–7
0 0
plot of ln(B/B ) versus time, where B =specific radioligand bound at
500
time of addition of haloperidol, and B=specific radioligand bound at
time t. For competition binding data, K values were calculated from
experimentally determined IC50 values using the Cheng–Prusoff
400
300
Total
i
Non-Specific
3
d
equation using the K for [ H]-SN56 determined from the saturation
binding experiments (0.069 nM).
2
1
00
00
0
3
. Results
3
.1. Basic binding parameters
0.0
0.2
0.4
0.6
0.8
[3
H]-SN56 (nM)
3
At near saturating conditions, non-specific binding of [ H]-SN56
3
remained constant in the presence of 25 to 200 μg membrane,
suggesting that the observed non-specific binding is due primarily to
radioligand binding to the filter. Total binding was linear from 50 to
Fig. 4. Saturation curve for [ H]-SN56 in rat brain membranes. Samples contained
00 μg membrane in a total volume of 0.5 ml. Data points represent the mean± SEM of
three independent determinations of duplicate samples at each concentration.
d
max =340± 10 fmol/mg, K =0.069± 0.007 nM and r =0.96.
1
2
B
2
00 μg membrane (Fig. 3).
4
. Discussion
Characterization of a series of novel 2(3H)-benzothiazolone
3
.2. Association and dissociation kinetics
From the association studies, kobs =0.080 min− and k+1=9.05×
1
compounds in σ receptor competition binding assays revealed SN56
(3-(2-(azepan-1-yl)ethyl)-6-propylbenzo[d]thiazol-2(3H)-one) to
7
−1
−1
−1
1
t
0 min
M
. From the dissociation studies, k−1=0.0076 min and
=91 min. The K calculated from k−1/k+1 was 0.084 nM. The low
dissociation rate permits the use of filtration for the separation of free from
bound radioligand (Bylund et al., 2004), while the association rate
supports 90 min incubations for attaining steady-state binding.
1
/2
d
have sub-nanomolar affinity and N1000 fold selectivity for the σ
subtype relative to σ (Yous et al., 2005). Binding of SN56 to non-σ
binding sites was tested with a battery of receptors and transporters
including, adrenergic α , adrenergic α , adrenergic β , adrenergic β
histamine H , histamine H , mu opioid, delta opioid, kappa opioid,
dopamine D , dopamine D , serotonin 5HT2a, serotonin 5HT , GABA
dopamine transporter, and serotonin transporters. Of the binding sites
tested, only α and H showed affinities greater than 1 μM
iα2 =205 nM, and KiH1 =311 nM respectively) (Yous et al., 2005);
however, the affinity of SN56 for the σ receptor is approximately 350
1
2
1
2
1
2
,
1
2
1
2
3
A
,
3
.3. Saturation binding
Fig. 4 shows the results of the saturation binding study of [ H]-SN56.
3
2
1
(
K
3
The binding affinity of [ H]-SN56 was K
represents a 70 fold higher affinity than reported for [ H](+)-
pentazocine (K =4.8 nM) (Bowen et al., 1993). Receptor density
max) as determined by saturation binding with [ H]-SN56 was 340±
0 fmol/mg.
d
=0.069± 0.007 nM which
3
1
times higher than its affinity for either of these receptors, indicating a
favorable selectivity profile for the development of a radioprobe for
use in radioligand binding studies.
d
3
(
B
1
3
In the present study, [ H]-SN56 exhibited N95% specific binding to
1 d
σ in rat brain membranes at concentrations up to 10 times the K .
3
3
.4. Competition binding
However, non-specific binding of [ H]-SN56 to the glass fiber filters
used to separate bound from free radioligand was 25–35% at 10 times
3
Tabulated values of K
i
s determined in this study using [ H]-SN56
d
the K concentration, resulting in a final specific binding signal of
3
3
versus values reported by Bowen et al. (1993) using [ H](+)-
pentazocine are shown in Table 1. In Fig. 5, a comparison of binding
profiles of the sites labeled by [ H]-SN56 versus [ H](+)-pentazocine
is shown in a correlation plot of K values obtained experimentally
65–75% of total observed binding. [ H]-SN56 exhibited saturable and
reversible binding to a single high affinity site in rat membranes with
3
3
3
a binding profile similar to that observed for [ H](+)-pentazocine.
The Bmax observed for [ H]-SN56 (340± 10 fmol/mg) was consistent
3
i
versus values reported by Bowen et al. (1993). For the group of
hallmark σ ligands tested, there was a significant correlation between
the affinities obtained using the novel versus conventional radioli-
gand. Of particular note is the higher affinity of (+)-pentazocine as
compared to (−)-pentazocine in the assays, a stereoselectivity
with the range of values reported in the literature for rat brain labeled
3
3
3
with [ H]-BHDP, [ H]-SA4503, or [ H](+)-pentazocine (Ishiwata
et al., 2003; Klouz et al., 2003). In addition, the K values of classical
i
3
σ compounds in competition binding assays against [ H]-SN56 were
consistent with those reported in the literature against the well
3
pattern that is consistent with binding to σ
1
receptors.
established σ
1
radioligand [ H](+)-pentazocine (Bowen et al., 1993).
3
The association and dissociation kinetics of [ H]-SN56 were also
1
00
shown to be amenable for filtration assays.
Total
3
Yous et al. (2005) reported a K
pentazocine in P
we obtained a K of 0.38 nM versus [ H](+)-pentazocine in P
i 2
i
of 0.56 nM for SN56 versus [ H](+)-
Non-Specific
2
membranes prepared from guinea pig brain. Similarly,
7
5
2
5
0
5
0
3
Table 1
Summary of data from competition binding experiments.
Compound
i
K (nM)
Literaturea
Experimental
0
50
100
150
200
DTG
Haloperidol
41.6± 8.8
3.48± 1.05
5.70± 1.04
77.0± 9.0
74.3± 14.9
1.9± 0.3
6.7± 1.2
Membrane (µg)
(
(
+)-Pentazocine
−6)-Pentazocine
3
Fig. 3. Total and non-specific binding of [ H]-SN56 to rat brain membranes. Samples
44.0± 1.2
3
contained 0.7 nM [ H]-SN56, in a total volume of 0.5 ml. Data points represent the mean
of three independent determinations of duplicate samples at each concentration.
a
Bowen et al., 1993.

J.A. Fishback et al. / European Journal of Pharmacology 653 (2011) 1–7
5
3
2
1
0
amounts from 5–50 μg in volumes from 50–1750 μl, and conditions
where the ratio of receptor concentration ([receptor]) to K were from
.42–~147. As the volume was decreased or the amount of membrane
d
0
4
d
was increased in these studies, the calculated K and Bmax increased.
When no corrections were made for radioligand depletion, with
[receptor]/K =0.42 (the lowest ratio tested, corresponding to 5 μg in
750 μl) the calculated K was approximately 2.3 times the “true”
value, and with [receptor]/K =1.25 the calculated K was approxi-
mately 2.8 times the true value. When the K s were recalculated,
1
d
2
1
d
d
d
d
taking into account radioligand depletion, the resulting values were
2.1 and 3.9 times the true value, respectively.
0
.5
1.5
3
log K (nM) [ H]-(+)-Pentazocine
i
The conditions chosen for our saturation studies utilized a
3
d
[receptor]/K of ~1. No corrections were made for radioligand
depletion because it was not possible to accurately assess what
portion of non-specific binding was due to binding to membrane
i
Fig. 5. Comparison of K values determined experimentally with [ H]-SN56 versus
3
2
values determined with [ H](+)-pentazocine by Bowen et al. (1993). r =0.89. 1)
haloperidol, 2) (+)-pentazocine, 3) (−)-pentazocine, 4) DTG.
1
versus binding to filter. Based on Carter's work, we might expect our
3
membranes prepared from rat brain (data not shown). These values are
5 fold higher than the affinity determined with saturation and kinetic
studies of [ H]-SN56 (~0.07 nMand ~0.08 nMrespectively). We suspect
that depletion of the non-labeled ligand results in an erroneously high
results to overestimate the K
d
of [ H]-SN56 by 2 to 3 fold.
~
Carter's studies for competition binding did not model the
approach we utilized, with [receptor]=~Kd[3H]-SN56 and concentra-
tion [ H]-SN56=~10 times Kd[3H]-SN56, so useful comparisons to their
3
3
value for the K
i
of SN56 as determined by competition binding.
i
data are not possible. However, as stated previously, the K values we
Systematic errors resulting from the use of high receptor
concentrations may also contribute to errors in both the determina-
derived for the σ ligands tested correlated well with values reported
in the literature.
tion of K
determination of K
are minimal, quantifiable, and in practice when [ H]-SN56 is used for
competition binding assays of σ ligands, our preliminary results
suggest they have no impact on our K determinations as compared to
d
from saturation and kinetic experiments, and in the
No significant difference in binding affinities was observed
3
3
i
s of un-labeled compounds. However, these errors
following labeling with [ H](+)-pentazocine versus [ H]-SN56 for
the ligands tested. A larger pool of compounds will need to be
screened to confirm that this relationship is maintained. However,
compounds that show significant differences in affinity following
labeling with the two different radioligands may provide valuable
insight into differences in the binding of σ ligands from different
chemical classes.
3
1
i
historical data for the compounds tested.
While most researchers are familiar with radioligand depletion due
to excessive receptor concentration, depletion of the un-labeled ligand
occurs when the affinity of the un-labeled compound greatly exceeds
the affinity of the labeled compound (Chang et al., 1975; Goldstein and
Barrett, 1987). Goldstein and Barrett (1987) used computer modeling to
3
[ H]-SN56 appears to provide similar binding characteristics
3
compared to [ H](+)-pentazocine with some noteworthy advan-
3
tages. Synthetically, [ H]-SN56 can be produced more easily and in
3
derive estimates of the error in the determination of K
i
of ligands
significantly higher yields than [ H](+)-pentazocine. The chemical
3
3
exhibiting higher affinities than the radioligands utilized in their
measurement; the authors projected that for an un-labeled ligand
with a true affinity 100 times greater than the radioligand (as in our
case), 10% radioligand depletion would result in an experimentally
stability of [ H]-SN56 is also expected to be greater than that of [ H]
(+)-pentazocine, which degrades over time, resulting in increased
background levels.
1
A number of radiolabeled σ ligands have been reported in the
determined K
d
~6 times higher than the true value. Thus, the ~5 fold
literature but none have been widely accepted as a replacement for
[ H](+)-pentazocine in competition binding studies. The two best
candidates, SA4503 (Matsuno et al., 1996) and BHDP (Klouz et al.,
2003), have been studied in rat brain membranes in tritiated and un-
labeled forms and exhibit approximately 100 fold higher affinity for
3
difference between the affinity of SN56, as determined by competition
binding, and the affinity of [ H]-SN56 determined with saturation and
kinetic studies may be explained by this phenomena.
Practical considerations dictated that we use relatively high receptor
concentrations; this introduces systematic error that is quantifiable and
within acceptable limits. When possible, experimental conditions for
binding experiments should be chosen so that the receptor concentra-
3
σ
1
versus σ
transporters (Klouz et al., 2003; Matsuno et al., 1996). However,
differences in relative expression of σ versus σ in disease states,
tissues, or cell lines may compromise the accurate analysis of σ
binding with these radioligands since their σ /σ selectivity ratios just
2
with no significant binding to other common receptors or
1
2
tion is less than 10% of the K
d
of the radioligand to minimize radioligand
1
depletion (Chang et al., 1975). However, with a radioligand with sub-
nanomolar affinity this would require multi-milliliter sample volumes.
Our experiments required 100 μg of tissue to obtain adequate signal for
precise detection. We chose 0.5 ml sample volumes because we intend
to adapt this method to a higher throughput 96-well method where
sample volumes are more limited than in test tube based binding
determinations. To ensure that N90% of added radioligand was “free”
1
2
meet the 100 fold difference generally accepted as the minimum
difference required for discriminating receptor subtypes. Because
3
[ H]-SN56 displays a N1000 fold higher affinity for σ
1
versus σ
2
,
changes in σ expression would not impact measurements performed
with this radioligand as much as with the other proposed alternatives.
In vitro binding studies with receptor specific radioligands have
historically been important in the discovery and characterization of
receptors, and continue to play a central role in the process of drug
discovery. Application of this technology to the σ receptor has
resulted in: 1) identification of σ receptors as a unique receptor type
(Su, 1982; Tam, 1983), 2) confirmation that σ receptors have at least
two subtypes (Bowen et al., 1989; Hellewell and Bowen, 1990), and 3)
determination of its anatomical distribution (Hellewell et al., 1994;
Jansen et al., 1991a,b; Walker et al., 1992). Radioligand binding
studies continue to play a primary role in selecting σ ligands for in vivo
testing because there are no widely accepted in vitro functional assays
(
unbound) in competition binding experiments run under these
3
conditions, we utilized high concentrations of [ H]-SN56. While these
conditions are not ideal, they are tolerated if required for detection and
the error in values obtained with the method are known and within an
acceptable range (Carter et al., 2007).
Carter et al. (2007) examined the effects of assay miniaturization
using the human muscarinic M3 receptor expressed in CHO cells. This
cell line expressed receptor at ~5 pmol/mg, and the novel radioligand
3
3
tested, [ H]-NMS (1-[N-methyl- H] scopolamine methyl chloride)
had an affinity of 0.42 pM. In saturation binding studies, receptor
concentrations were varied over a wide range, with membrane
1
for σ activation.

6
J.A. Fishback et al. / European Journal of Pharmacology 653 (2011) 1–7
Carter, C.M., Leighton-Davies, J.R., Charlton, S.J., 2007. Miniaturized receptor binding
In addition to its service in the initial characterization of the σ
receptor, radioligand binding data has contributed to the elucidation
of 1) structural elements that define high affinity σ ligands, and 2)
specific amino acids of the σ protein that are critical for selective,
high affinity binding of known ligands (Ablordeppey et al., 2000,
002; Glennon, 2005; Seth et al., 2001; Yamamoto et al., 1999).
assays: complications arising from ligand depletion. J. Biomol. Screen. 12, 255–266.
Chang, K.J., Jacobs, S., Cuatrecasas, P., 1975. Quantitative aspects of hormone–receptor
interactions of high affinity. Effect of receptor concentration and measurement of
dissociation constants of labeled and unlabeled hormones. Biochim. Biophys. Acta
1
1
406, 294–303.
Cobos, E.J., Entrena, J.M., Nieto, F.R., Cendan, C.M., Del Pozo, E., 2008. Pharmacology and
therapeutic potential of sigma1 receptor ligands. Curr. Neuropharmacol. 6, 344–366.
de Costa, B.R., Bowen, W.D., Hellewell, S.B., Walker, J.M., Thurkauf, A., Jacobson, A.E.,
Rice, K.C., 1989. Synthesis and evaluation of optically pure [3H]–(+)–pentazo-
cine, a highly potent and selective radioligand for sigma receptors. FEBS Lett. 251,
53–58.
de Costa, B., Radesca, L., Dominguez, C., Di Paolo, L., Bowen, W.D., 1992. Synthesis and
receptor binding properties of fluoro- and iodo-substituted high affinity sigma
receptor ligands: identification of potential PET and SPECT sigma receptor imaging
agents. J. Med. Chem. 35, 2221–2230.
2
Studies correlating structure with binding affinity are ongoing;
numerous examples of such labors have been reported in the
literature, with the majority of effort focused on identifying subtype
specific ligands (de Costa et al., 1992; Holl et al., 2009; Mesangeau
et al., 2008; Zampieri et al., 2009).
σ
1
Radioligands have also demonstrated utility in radio-imaging
studies; several examples of the successful use of the σ radioligand
C-SA4503] as a positron emission tomography (PET) imaging agent
1
Glennon, R.A., 2005. Pharmacophore identification for sigma-1 (σ ) receptor binding:
1
1
1
application of the “deconstruction−reconstruction−elaboration” approach. Mini
[
Rev. Med. Chem. 5, 927–940.
have been reported in recent years (Ishikawa et al., 2007; Rybczynska
Goldstein, A., Barrett, R.W., 1987. Ligand dissociation constants from competition binding
assays: errors associated with ligand depletion. Mol. Pharmacol. 31, 603–609.
Guitart, X., Codony, X., Monroy, X., 2004. Sigma receptors: biology and therapeutic
potential. Psychopharmacology (Berl.) 174, 301–319.
Hanner, M., Moebius, F.F., Flandorfer, A., Knaus, H.G., Striessnig, J., Kempner, E.,
Glossmann, H., 1996. Purification, molecular cloning, and expression of the
et al., 2009; Toyohara et al., 2009). Displacement of [11C]-SA4503 by
the σ
occupies σ
et al., 2007). Other studies showed reduced σ
defined brain regions of Alzheimer's and Parkinson's patients
Toyohara et al., 2009). Also, supporting a role for σ in cancer,
Rybczynska et al. (2009) reported that steroid hormones that are
known σ
ligands displace [11C]-SA4503 from tumors in rats
Rybczynska et al., 2009). Thus, these studies suggest σ radioligands
1
active antidepressant fluvoxamine confirmed that fluvoxamine
receptors in healthy human male volunteers (Ishikawa
receptor density in
1
1
1
mammalian sigma -binding site. Proc. Natl Acad. Sci. USA 93, 8072–8077.
Hashimoto, K., Ishiwata, K., 2006. Sigma receptor ligands: possible application as
therapeutic drugs and as radiopharmaceuticals. Curr. Pharm. Des. 12, 3857–3876.
Hellewell, S.B., Bowen, W.D., 1990. A sigma-like binding site in rat pheochromocytoma
(PC12) cells: decreased affinity for (+)-benzomorphans and lower molecular
weight suggest a different sigma receptor form from that of guinea pig brain. Brain
Res. 527, 244–253.
(
1
1
(
1
may hold significant diagnostic value for psychiatric diseases and
cancer.
Hellewell, S.B., Bruce, A., Feinstein, G., Orringer, J., Williams, W., Bowen, W.D., 1994. Rat
1 2
liver and kidney contain high densities of σ and σ receptors: characterization by
ligand binding and photoaffinity labeling. Eur. J. Pharmacol. 268, 9–18.
Holl, R., Schepmann, D., Frohlich, R., Grunert, R., Bednarski, P.J., Wunsch, B., 2009.
Dancing of the second aromatic residue around the 6, 8-diazabicyclo[3.2.2]nonane
framework: influence on sigma receptor affinity and cytotoxicity. J. Med. Chem. 52,
Future advances in the field of σ receptor therapeutic development
will require greater knowledge of the nature of the interaction of the σ
receptor with its ligands and protein binding partners. Additional
tools needed to further this knowledge include new subtype specific
agonist and antagonist ligands, radioligands, and other affinity labels
and probes. The development of other technologies, such as high
throughput methods for the determination of binding affinities, and in
vitro functional assays, will also hasten efforts to design and identify
new selective σ ligands with potential therapeutic value.
2126–2137.
Ishikawa, M., Ishiwata, K., Ishii, K., Kimura, Y., Sakata, M., Naganawa, M., Oda, K.,
Miyatake, R., Fujisaki, M., Shimizu, E., Shirayama, Y., Iyo, M., Hashimoto, K., 2007.
High occupancy of sigma-1 receptors in the human brain after single oral
administration of fluvoxamine:
a
positron emission tomography study using
1
1
[
C]SA4503. Biol. Psychiatry 62, 878–883.
Ishiwata, K., Kobayashi, T., Kawamura, K., Matsuno, K., 2003. Age-related changes of the
3
binding of [ H]SA4503 to sigma receptors in the rat brain. Ann. Nucl. Med. 17,
1
3
7
3–77.
In conclusion, the results of our studies suggest that [ H]-SN56
Jansen, K.L., Dragunow, M., Faull, R.L., Leslie, R.A., 1991a. Autoradiographic visualisation
possesses high affinity and selectivity for the σ
to be a viable alternative for [ H](+)-pentazocine in radioligand
binding assays. Further, because [ H]-SN56 has a N70 fold higher
affinity for the σ
1
receptor, and appears
3
3
3
of [ H]DTG binding to sigma receptors, [ H]TCP binding sites, and L-[ H]glutamate
binding to NMDA receptors in human cerebellum. Neurosci. Lett. 125, 143–146.
Jansen, K.L., Faull, R.L., Dragunow, M., Leslie, R.A., 1991b. Autoradiographic distribution
of sigma receptors in human neocortex, hippocampus, basal ganglia, cerebellum,
pineal and pituitary glands. Brain Res. 559, 172–177.
3
3
3
1
receptor than [ H](+)-pentazocine, competition
binding studies require less radioligand and membrane, resulting in
Kekuda, R., Prasad, P.D., Fei, Y.J., Leibach, F.H., Ganapathy, V., 1996. Cloning and
functional expression of the human type 1 sigma receptor (hSigmaR1). Biochem.
Biophys. Res. Commun. 229, 553–558.
significant efficiencies in resources when performing the assays. Thus,
3
[
H]-SN56 represents another valuable tool for the study of the σ
1
Klouz, A., Tillement, J.P., Boussard, M.F., Wierzbicki, M., Berezowski, V., Cecchelli, R.,
receptor and the development of σ
1
based therapeutics.
3
Labidalle, S., Onteniente, B., Morin, D., 2003. [ H]BHDP as a novel and selective
1
ligand for sigma receptors in liver mitochondria and brain synaptosomes of the
rat. FEBS Lett. 553, 157–162.
Acknowledgements
Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.E., Gilbert, P.E., 1976. The effects of
morphine- and nalorphine-like drugs in the nondependent and morphine-
dependent chronic spinal dog. J. Pharmacol. Exp. Ther. 197, 517–532.
Matsumoto, R.R., Bowen, W.D., Tom, M.A., Vo, V.N., Truong, D.D., De Costa, B.R., 1995.
Characterization of two novel σ receptor ligands: antidystonic effects in rats
suggest σ receptor antagonism. Eur. J. Pharmacol. 280, 301–310.
This study was supported by grants from the National Institute on
Drug Abuse (DA023205 and DA013978).
Matsumoto, R.R., Bowen, W.D., Walker, J.M., Patrick, S.L., Zambon, A.C., Vo, V.N., Truong,
D.D., De Costa, B.R., Rice, K.C., 1996. Dissociation of the motor effects of (+)-
References
pentazocine from binding to σ
1
sites. Eur. J. Pharmacol. 301, 31–40.
Ablordeppey, S.Y., Fischer, J.B., Glennon, R.A., 2000. Is a nitrogen atom an important
pharmacophoric element in sigma ligand binding? Bioorg. Med. Chem. 8,
Matsuno, K., Nakazawa, M., Okamoto, K., Kawashima, Y., Mita, S., 1996. Binding
properties of SA4503, a novel and selective σ
306, 271–279.
1
receptor agonist. Eur. J. Pharmacol.
2105–2111.
Ablordeppey, S.Y., Fischer, J.B., Law, H., Glennon, R.A., 2002. Probing the proposed
phenyl-A region of the sigma-1 receptor. Bioorg. Med. Chem. 10, 2759–2765.
Bowen, W.D., Hellewell, S.B., McGarry, K.A., 1989. Evidence for a multi-site model of the
rat brain σ receptor. Eur. J. Pharmacol. 163, 309–318.
Maurice, T., Su, T.P., 2009. The pharmacology of sigma-1 receptors. Pharmacol. Ther.
124, 195–206.
Mei, J., Pasternak, G.W., 2001. Molecular cloning and pharmacological characterization
1
of the rat sigma receptor. Biochem. Pharmacol. 62, 349–355.
3
Bowen, W.D., DeCosta, B.R., Hellewell, S.B., Walker, J.M., Rice, K.C., 1993. [ H]-(+)-
Mesangeau, C., Narayanan, S., Green, A.M., Shaikh, J., Kaushal, N., Viard, E., Xu, Y.T.,
Fishback, J.A., Poupaert, J.H., Matsumoto, R.R., McCurdy, C.R., 2008. Conversion of a
highly selective sigma-1 receptor-ligand to sigma-2 receptor preferring ligands
with anticocaine activity. J. Med. Chem. 51, 1482–1486.
Pentazocine: a potent and highly selective benzomorphan-based probe for sigma-1
receptors. Mol. Pharmacol. 1, 117–126.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein−dye binding. Anal. Biochem.
Pan, Y.X., Mei, J., Xu, J., Wan, B.L., Zuckerman, A., Pasternak, G.W., 1998. Cloning and
characterization of a mouse sigma1 receptor. J. Neurochem. 70, 2279–2285.
Rybczynska, A.A., Elsinga, P.H., Sijbesma, J.W., Ishiwata, K., de Jong, J.R., de Vries, E.F.,
Dierckx, R.A., van Waarde, A., 2009. Steroid hormones affect binding of the sigma
72, 248–254.
Bylund, D.B., Toews, M.L., 1993. Radioligand binding methods: practical guides and tips.
Am. J. Physiol. 265, 421–429.
Bylund, D.B., Deupree, J.D., Toews, M.L., 2004. Radioligand-binding methods for
membrane preparations and intact cells. Meth. Mol. Biol. 259, 1–28.
1
1
ligand C-SA4503 in tumour cells and tumour-bearing rats. Eur. J. Nucl. Med. Mol.
Imaging 36, 1167–1175.

J.A. Fishback et al. / European Journal of Pharmacology 653 (2011) 1–7
7
Seth, P., Leibach, F.H., Ganapathy, V., 1997. Cloning and structural analysis of the cDNA
and the gene encoding the murine type 1 sigma receptor. Biochem. Biophys. Res.
Commun. 241, 535–540.
Seth, P., Fei, Y.J., Li, H.W., Huang, W., Leibach, F.H., Ganapathy, V., 1998. Cloning and
functional characterization of a sigma receptor from rat brain. J. Neurochem. 70,
activity of 2(3H)-benzoxazolone and 2(3H)-benzothiazolone derivatives. J. Med.
Chem. 41, 1138–1145.
Walker, J.M., Bowen, W.D., Goldstein, S.R., Roberts, A.H., Patrick, S.L., Hohmann, A.G.,
DeCosta, B., 1992. Autoradiographic distribution of [3 H](+)-pentazocine and [3 H]
1, 3-di-o-tolylguanidine (DTG) binding sites in guinea pig brain: a comparative
study. Brain Res. 581, 33–38.
922–931.
Seth, P., Ganapathy, M.E., Conway, S.J., Bridges, C.D., Smith, S.B., Casellas, P., Ganapathy,
V., 2001. Expression pattern of the type 1 sigma receptor in the brain and identity of
critical anionic amino acid residues in the ligand-binding domain of the receptor.
Biochim. Biophys. Acta 1540, 59–67.
Yamamoto, H., Miura, R., Yamamoto, T., Shinohara, K., Watanabe, M., Okuyama, S.,
Nakazato, A., Nukada, T., 1999. Amino acid residues in the transmembrane domain
of the type 1 sigma receptor critical for ligand binding. FEBS Lett. 445, 19–22.
Yous, S., Poupaert, J.H., Lesieur, I., Depreux, P., Lesieur, D., 1994. AlCl3-DMF reagent in
the Friedel-Crafts reaction. Application to the acylation reaction of 2(3H)-
benzothiazolones. J. Org. Chem. 59, 1574–1576.
Yous, S., Wallez, V., Belloir, M., Caignard, D., McCurdy, C.R., 2005. Novel 2(3H)-
benzothiazolones as highly potent and selective sigma-1 receptor ligands. Med.
Chem. Res. 14, 158–168.
3
Su, T.P., 1982. Evidence for sigma opioid receptor: binding of [ H]SKF-10047 to etorphine-
inaccessible sites in guinea-pig brain. J. Pharmacol. Exp. Ther. 223, 284–290.
Tam, S.W., 1983. Naloxone-inaccessible sigma receptor in rat central nervous system.
Proc. Natl Acad. Sci. USA 80, 6703–6707.
1
Toyohara, J., Sakata, M., Ishiwata, K., 2009. Imaging of sigma receptors in the human
1
1
brain using PET and [ C]SA4503. Cent. Nerv. Syst. Agents Med. Chem. 9, 190–196.
Ucar, H., Van derpoorten, K., Cacciaguerra, S., Spampinato, S., Stables, J.P., Depovere, P.,
Isa, M., Masereel, B., Delarge, J., Poupaert, J.H., 1998. Synthesis and anticonvulsant
Zampieri, D., Grazia Mamolo, M., Laurini, E., Zanette, C., Florio, C., Collina, S., Rossi, D.,
Azzolina, O., Vio, L., 2009. Substituted benzo[d]oxazol-2(3H)-one derivatives with
1
preference for the sigma binding site. Eur. J. Med. Chem. 44, 124–130.