In vitro characterization of radioiodinated (+)-2-[4-(4-iodophenyl) piperidino]cyclohexanol [(+)-pIV] as a sigma-1 receptor ligand

Article abstract of DOI:10.1016/j.bmc.2004.11.029

We investigated the binding characteristics of a (+)-enantiomer of radioiodinated 2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-[ ¹²⁵I]pIV], radioiodinated at the para-position of the 4-phenylpiperidine moiety, to sigma receptors (σ-1, σ-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (+)-pIV (K_i = 1.30 nM) had more than 10 times higher affinity to the sigma-1 (σ-1) receptor than (+)-pentazocine (K_i = 19.9 nM) or haloperidol (K_i = 13.5 nM) known as sigma ligands. Also, the binding affinity of (+)-pIV for the σ-1 receptor (K_i = 1.30 nM), was about 16 times higher than the sigma-2 (σ-2) receptor (K_i = 20.4 nM). (+)-pIV (K_i = 1260 nM) had a much lower affinity for VAChT than (-)-vesamicol (K_i = 13.0 nM) or (-)-pIV (K_i = 412 nM). (+)-[¹²⁵I]pIV had low affinity for the dopamine, serotonin, adrenaline, and acetylcholine receptors. Furthermore, in a saturation binding study, (+)-[¹²⁵I]pIV exhibited a K_d of 6.96 nM with a B_max of 799 fmol/mg of protein. These results showed that (+)-pIV binds to the σ-1 receptor with greater affinity than sigma receptor ligands such as (+)-pentazocine or haloperidol, and that radioiodinated (+)-pIV is suitable as radiotracer for σ-1 receptor studies in vitro.

Full text of DOI:10.1016/j.bmc.2004.11.029

Bioorganic & Medicinal Chemistry 13 (2005) 1095–1099
In vitro characterization of radioiodinated (+)-2-[4-(4-iodophenyl)
piperidino]cyclohexanol [(+)-pIV] as a sigma-1 receptor ligand
Kazuhiro Shiba,* Kazuma Ogawa and Hirofumi Mori
Division of Tracer Kinetics, Advanced Science Research Center, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan
Received 13 September 2004; revised 15 November 2004; accepted 15 November 2004
Available online 8 December 2004
Abstract—We investigated the binding characteristics of a (+)-enantiomer of radioiodinated 2-[4-(4-iodophenyl)piperidino]cyclo-
hexanol [(+)-[125I]pIV], radioiodinated at the para-position of the 4-phenylpiperidine moiety, to sigma receptors (r-1, r-2) and
to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (+)-
pIV (Ki = 1.30 nM) had more than 10 times higher affinity to the sigma-1 (r-1) receptor than (+)-pentazocine (Ki = 19.9 nM) or halo-
peridol (Ki = 13.5 nM) known as sigma ligands. Also, the binding affinity of (+)-pIV for the r-1 receptor (Ki = 1.30 nM), was about
16 times higher than the sigma-2 (r-2) receptor (Ki = 20.4nM). (+)-pIV ( Ki = 1260 nM) had a much lower affinity for VAChT than
(ꢀ)-vesamicol (Ki = 13.0 nM) or (ꢀ)-pIV (Ki = 412 nM). (+)-[125I]pIV had low affinity for the dopamine, serotonin, adrenaline, and
acetylcholine receptors. Furthermore, in a saturation binding study, (+)-[125I]pIV exhibited a Kd of 6.96 nM with a Bmax of 799 fmol/
mg of protein. These results showed that (+)-pIV binds to the r-1 receptor with greater affinity than sigma receptor ligands such as
(+)-pentazocine or haloperidol, and that radioiodinated (+)-pIV is suitable as radiotracer for r-1 receptor studies in vitro.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
tively nonselective, such as haloperidol and 1,3-di-tolyl-
guanidine (DTG). The benzomorphans, such as N-
allylnormetazocine ((+)-SKF 10047) and (+)-pentazo-
cine are among the compounds, which show the highest
r-1/r-2 selectivity; however, these compounds also bind
to other neuroreceptors.15
It is known that sigma receptors are distributed at a
comparatively high density to the limbic system and cor-
tex that have a close relationship with schizophrenia.1–4
Sigma receptors specifically change in the limbic system
and a cortex in the brain after the death of a patient with
schizophrenia.5–7 Furthermore, it has been reported that
sigma receptors promote release of acetylcholine in the
cerebral cortex frontal lobe and hippocampus8 and that
learning/memory impairment is improved by sigma
receptors.9–12 Sigma receptors are thought to be closely
related to learning/memory mechanism. Thus, it is use-
ful to develop various sigma receptor ligands with which
to investigate the central nervous system (CNS).
In previous studies, we reported that the binding affinity
for acetylcholine transporter (VAChT) and sigma recep-
tors (r-1, r-2) of vesamicol varies with the position of
iodine introduced into the 4-phenylpiperidine moiety
of vesamicol.16,17 In this study, we synthesized the (+)-
enantiomer of radioiodinated 2-[4-(4-iodophenyl)piper-
idino]cyclohexanol [(+)-[125I]pIV] and investigated the
in vitro binding characteristics of (+)-[125I]pIV to r-1
receptor in the rat brain to evaluate the potential useful-
ness of (+)-[125I]pIV as radiotracer for r-1 receptor
studies in vitro.
Sigma receptors have been categorized into at least two
subtypes, termed as sigma-1(r-1) and sigma-2 (r-2).13
The r-1 receptor exhibits a high affinity for (+)-benzo-
morphans, whereas the r-2 receptor displays a low affin-
ity for (+)-benzomorphans.14 Most known r receptor
ligands are either selective for the r-1 receptor or are rela-
2. Material and methods
(ꢀ)-[3H]vesamicol (1.30 TBq/mmol), [3H]1,3-di-tolyl-
guanidine ([3H]DTG) (1.1 TBq/mmol), [3H]pentazocine
(1.0 TBq/mmol), and [125I]sodium iodide (644 GBq/mg)
were purchased.
Keywords: (+)-pIV; Vesamicol; Sigma receptors; Radioligand.
*
Corresponding author. Tel.: +81 76 265 2474; fax: +81 76 234
4245; e-mail: shiba@med.kanazawa-u.ac.jp
0968-0896/$ - see front matter ꢀ 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.11.029

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K. Shiba et al. / Bioorg. Med. Chem. 13 (2005) 1095–1099
2.1. Synthesis of (+)-p-iodovesamicol ((+)-pIV)
2.5. In vitro competitive binding study
The (+)-enantiomer of (+)-pIV was prepared using a
method described previously,16 but using the corre-
sponding enantiomer of vesamicol (Fig. 1). Briefly, race-
mic vesamicol was synthesized from the 4-phenylpridine
via a two-step reaction. The (+)-enantiomer of vesami-
col was provided from racemic vesamicol by recrystalliz-
ing the diastereoisomeric salts using (+)-di-p-toluoyl-D-
tartaric acid monohydrate. (+)-pIV was synthesized
from the (+)-enantiomer of vesamicol via a three-step
reaction.
2.5.1. VAChT binding. The assays were performed using
a method reported previously17 except that 514–717 lg
protein of rat cerebral membranes were used. Briefly,
10 nM (ꢀ)-[3H]vesamicol was used as a radioligand.
Various concentrations of (+)-pIV, enantiomers of
vesamicol or sigma ligands (from 10ꢀ10 to 10ꢀ5 M) were
used as subject compounds. The mixture was incubated
for 60 min at 37 ꢁC in the presence of 100 nM haloper-
idol to mask sigma receptors.
2.5.2. r-1 receptor binding. Rat cerebral membranes
(514–717 lg protein) were incubated in triplicate
with 3 nM (+)-[3H]pentazocine and various concentra-
tions of (+)-pIV, enantiomers of vesamicol or sigma
ligands (from 10ꢀ10 to 10ꢀ5 M) in 0.5 mL of 50 mM
Tris–HCl (pH 7.8) for 90 min at 37 ꢁC. Nonspecific bind-
ing was determined in the presence of 10 lM (+)-pentazo-
cine. The incubated samples were treated in the same
manner as described for the VAChT binding assays.
2.2. Characteristics of (+)-2-[4-(4-iodophenyl)piper-
idino]cyclohexanol[(+)-pIV]
Melting point: 162–164 ꢁC. NMR (CDCl3): d 1.19–1.25
(4H, m), 1.60–1.85 (8H, m), 2.12–2.15 (1H, m), 2.20–
2.25 (2H, m), 2.41–2.45 (1H, m), 2.71–2.75 (2H, m)
2.92–2.95 (1H, m), 3.37–3.41 (1H, m), 6.97 (2H, d,
J = 8.30 Hz), 7.61 (2H, d, J = 8.30 Hz). Mass spectrum
(m/e): 385 [M]+. Elemental analysis C17H24NOI; theo-
retical values C: 53.00, H: 6.28, N: 3.64; experimental
2.5.3. r-2 receptor binding. Rat liver membranes (150–
300 lg protein) were incubated in triplicate with 3 nM
[3H]DTG and various concentrations of (+)-pIV, enan-
tiomers of vesamicol or sigma ligands (from 10ꢀ10 to
10ꢀ5 M) in 0.5 mL of 50 mM Tris–HCl (pH 7.8) for
90 min at 37 ꢁC in the presence of 1 lM (+)-pentazocine
to mask r-1 sites. Nonspecific binding was determined
in the presence of 10 lM DTG and 1 lM (+)-pentazo-
cine. The incubated samples were treated in the same
manner as described for VAChT binding assays.
values C: 53.22, H: 6.44, N: 3.70. Specific rotation:
23
D
½aŠ ¼ ꢀ33:9, (c 0.555, CDCl3).
2.3. Radiolabeling
(+)-pIV was radioiodinated by solid-phase exchange
with [125I]NaI in the presence of ammonium sulfate as
previously reported.18
2.4. Tissue preparation
2.6. Saturation binding study
Animal experiments were carried out in compliance with
the Guidelines for the Care and Use of Laboratory Ani-
mals at the Takara-machi Campus of Kanazawa Uni-
versity. Rat brain membranes and rat liver membranes
were prepared from rat brains without a cerebellum
and liver from male Sprague-Dawley rats (250–300 g)
each using a method described previously.17
Rat cerebral membranes (514–717 lg protein) were
incubated in triplicate with various concentrations of
(+)-[125I]pIV (from 1 to 60 nM) at 37 ꢁC for 90 min.
The incubated samples were quickly diluted, passed
through glass-fiber filters (Whatman GF/B) and washed
twice with an ice-cold buffer. The level of bound (+)-
[
125I]pIV retained on the filter was measured in a gamma
scintillation counter (Aloka, ARC-600). Nonspecific
binding was determined in the presence of 10 lM of
unlabeled (+)-pIV. A saturation binding study in the
presence of 1 lM pentazocine was also done to mask
the r-1 receptor.
HO
1
NH
N
2.7. Inhibition of (+)-[125I]pIV binding to rat cerebral
membranes
2
3
( )-vesamicol
HO
N
HO
O2N
N
Rat cerebral membranes (514–717 lg protein) were
incubated in triplicate with 5 nM (+)-[125I]pIV and
various concentrations of (+)-pIV or reference com-
pounds (from 10ꢀ10 to 10ꢀ5 M) in 0.5 mL of 50 mM
Tris–HCl (pH 7.8) for 90 min at 37 ꢁC. The incubated
samples were treated in the same manner as described
for the saturation binding assay.
4
(+)-vesamicol
HO
N
HO
N
5
I
H2N
(+)-pIV
2.7.1. Data analysis. Ki values in inhibition studies and
Kd and Bmax values in saturation binding studies were
calculated with the ÔGraphpad PrismÕ computer pro-
gram (GraphPad Software, Inc. San Diego, USA).
Figure 1. Synthesis of (+)-pIV. 1. Cyclohexene oxide; 2. (+)-di-p-
toluoyl-D-tartaric acid; 3. HNO3, H2SO4; 4. Fe, HCL; 5. NaNO2,
HCL, KI.

K. Shiba et al. / Bioorg. Med. Chem. 13 (2005) 1095–1099
Table 1. Affinities (nM) of (+)-pIV and reference compounds for
1097
800
600
400
VAChT and sigma receptors (r-1, r-2)
VAChT
Sigma receptors
r-1
r-2
Ki (nM)a
Ki (nM)b
Ki (nM)c
(+)-pIV
1260 259
412 117
13.0 2.3
289 53
—
1.30 0.47
3.40 0.51
74.9 18.6
20.4 2.0
28.1 3.9
421 69
(ꢀ)-pIV
(ꢀ)-Vesamicol
(+)-Vesamicol
(+)-Pentazocine
DTG
31.8 11.4359 39
19.9 3.5
—
13.5 2.0
2680 162
22.5 4.2
110
—
—
Haloperidol
4
Ki values derived from IC50 values according to the equation,
Ki = IC50/(1 + C/Kd), where C is the concentration of the radioligand
and each Kd is the dissociation constant of the corresponding radio-
ligand ((ꢀ)-[3H]vesamicol to VAChT, [3H]pentazocine to r-1,
[3H]DTG to r-2).
200
Values are means SEM of three experiments.
a Kd = 7.40 nM.
b Kd = 19.9 nM.
c Kd = 22.3 nM.
0
0
10
20
30
40
(+)-[125
ntration (nM)
3. Results
I]pIV conce
Figure 2. Equilibrium saturation binding of (+)-[125I]pIV to rat
cerebral membranes. (n) Total binding; (d) specific binding; (j)
non-specific binding determined by the addition of 10 lM of (+)-pIV.
Kd (6.96 1.11 nM) and Bmax (799 44 fmol/mg of protein) values
were calculated with the ÔGraphpad PrismÕ computer program. Values
are means SEM of three experiments.
Preparation of the (+)-pIV from 4-phenylpyridine via a
six-step reaction produced overall yields of 21.2% (Fig.
1). Radiochemical yield, radiochemical purity and spe-
cific activity were estimated to be 40–75%, >95% and
600–1100 GBq/mmol, respectively.
Binding affinity of (+)-pIV and reference compounds to
sigma receptors (r-1, r-2) and VAChT binding sites are
shown in Table 1. pIV, as well as vesamicol, bound
enantioselectively to the sigma receptors (r-1, r-2) and
VAChT binding sites. The binding affinity of (+)-pIV
to r-1 and r-2 was greater than that of (ꢀ)-pIV. Also,
the binding affinity of (+)-pIV to VAChT binding sites
was less than that of (ꢀ)-pIV. (+)-pIV (Ki = 1.30 nM)
displayed more than 10 times greater affinity for the r-
1 receptor than (+)-pentazocine (Ki = 19.9 nM) or halo-
peridol (Ki = 13.5 nM), which are known as sigma
ligands. (+)-pIV displayed a very low affinity for
VAChT binding sites (Ki = 1260 nM).
Table 2. (+)-[125I]pIV binding parameters (Kd, Bmax) in rat brain
membranes
Pentazocine (1 lM)
In the absence
In the presence
Kd (nM)
Bmax (fmol/mg protein)
6.96 1.11
799 44
21.9 2.1
397 20
Values are means SEM of three experiments.
Table 3. Inhibition of (+)-[125I]pIV with various drugs in rat cerebral
membranes
Drugs
IC50 (nM)
The results of the saturation binding study of (+)-
(+)-pIV
6.31 1.19
71.1 28.7
18.2 6.0
[
[
125I]pIV are shown in Figure 2 and Table 2. (+)-
DTG
125I]pIV in the absence or presence of 1 lM pentazocine
Pentazocinea
Haloperidol
Spiperone
Ketanserin
QNB
exhibited a Kd of 6.96 or 21.9 nM with a Bmax of
799 fmol/mg of protein or 397 fmol/mg protein, respec-
tively. The Bmax of (+)-[125I]pIV to the r-1 receptor or
r-2 receptor is thought to be 402 fmol/mg protein or
397 fmol/mg protein, respectively.
12.6 5.3
5.53 · 103 1.21 · 103
1.64 · 104 2.30 · 103
9.78 · 103 0.88 · 103
>1.00 · 105
Noradrenaline
Values are means SEM of three experiments.
The results of the inhibition of (+)-[125I]pIV binding
with various reference compounds are shown in Table
3. IC50 values for spiperone, ketanserin, noradrenaline
and quinuclidinyl benzilate (QNB) exceeded 5 lM.
a The IC50 value exhibited a high affinity for pentazocine by the best fit
of a two site competition. The IC50 value of low affinity for pen-
tazocine was 2.40 · 103 nM.
position of the 4-phenylpiperidine moiety of (ꢀ)-vesami-
col, was suitable as a VAChT mapping agent.19 On
the other hand, Efange et al.20,22 and Custer et al.21 re-
ported that there were several vesamicol analogs show-
ing great affinity for sigma receptors. In the process of
developing a VAChT mapping agent, we found that
4. Discussion
Previously, we reported that radioiodinated (ꢀ)-o-
iodovesamicol [(ꢀ)-oIV], radioiodinated at the ortho-

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K. Shiba et al. / Bioorg. Med. Chem. 13 (2005) 1095–1099
both introduction of iodine into the 4-phenylpiperidine
moiety of vesamicol and a kind of optical isomer had
an influence on the binding affinity for VAChT and sig-
ma receptors (r-1, r-2). Namely, there is a possibility
that the introduction of iodine into the para-position
into the 4-phenylpiperidine moiety of (+)-enantiomer
of vesamicol causes its binding affinity for sigma recep-
tors to increase, and its binding affinity for VAChT
binding sites decrease. Therefore, we synthesized (+)-
In competitive inhibition studies using (+)-[125I]pIV (Ta-
ble 3), (+)-[125I]pIV exhibited a high affinity for r-1
receptor and a much lower affinity for the dopamine,
serotonin, noradrenaline, and acetylcholine receptors.
These results showed that (+)-[125I]pIV would be a radio-
ligand for r-1 receptor with a high affinity and
selectivity.
[
[
125I]pIV and evaluated the potential usefulness of (+)-
125I]pIV as a radiotracer for sigma receptor studies.
5. Conclusion
As we expected it, the dextrorotatory isomer (+)-pIV
(Ki = 1.30 nM [r-1], Ki = 20.4[ r-2]) had a higher affinity
for sigma receptors than not only the dextrorotatory iso-
mer (+)-vesamicol (Ki = 31.8 nM [r-1], Ki = 359 [r-2])
but also the levorotatory isomer (ꢀ)-pIV (Ki = 3.40 nM
[r-1], Ki = 28.1 [r-2]). On the other hand, (+)-pIV
(Ki = 1260 nM) displayed a much lower affinity for
VAChT than (ꢀ)-vesamicol (Ki = 13.0 nM) or (ꢀ)-pIV
(Ki = 412 nM).
Radioiodinated (+)-pIV exhibited a more than 10 times
higher affinity to the r-1 receptor than pentazocine and
haloperidol and the selectivity of (+)-pIV for the r-2
and r-1 receptors (r-2/r-1) was high (approximately
16). In conclusion, radioiodinated (+)-pIV has superior
potential as a radioligand for r-1 receptor studies
in vitro.
Acknowledgements
Furthermore, (+)-pIV displayed a more than 10 times
greater affinity for the r-1 receptor than superior r-1 li-
gands such as (+)-pentazocine and haloperidol. Though
(+)-pIV displayed a high affinity for the r-2 receptor
and the r-2 receptor binding affinity of (+)-pIV
(Ki = 20.4nM) was comparable to that of DTG
(Ki = 22.5 nM), the binding affinity of (+)-pIV to the
r-1 receptor (Ki = 1.30 nM) was about 16 times higher
than the r-2 receptor (Ki = 20.4nM). The selectivity of
(+)-pIV (approximately 16) for the r-2 and r-1 recep-
tors (r-2/r-1) was lower than that of (+)-pentazocine
(approximately 135). However, (+)-pentazocine is
thought to bind to other neuroreceptors.15 Further-
more, (+)-[125I]pIV is also expected to pass through
BBB and to accumulate in the rat brain significantly
in vivo, because racemic (+/ꢀ)-[125I]pIV was reported
to show significant accumulation (about 3% of the injec-
tion dose) with prolonged retention in the rat brain
previously.24 On the other hand, (+)-[3H]pentazocine
showed the low accumulation (about 0.30% dose of
the injection dose at 60 min post-injection) in the rat
brain (date not shown). Judging from the above, (+)-
This study was supported by a Grant-in-Aid for Scien-
tific Research (C 14570845) from the Japanese Ministry
of Education, Science, Sports and Culture.
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