Syntheses and in vitro evaluation of decalinvesamicol analogues as potential imaging probes for vesicular acetylcholine transporter (VAChT)

Article abstract of DOI:10.1016/j.bmc.2012.06.040

A series of vesamicol analogues, o-iodo-trans-decalinvesamicol (OIDV) or o-bromo-trans-decalinvesamicol (OBDV), were synthesized and their affinities to vesicular acetylcholine transporter (VAChT) and σ receptors (σ-1, σ-2) were evaluated by in vitro bind

Full text of DOI:10.1016/j.bmc.2012.06.040

Bioorganic & Medicinal Chemistry 20 (2012) 4936–4941
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Syntheses and in vitro evaluation of decalinvesamicol analogues as
potential imaging probes for vesicular acetylcholine transporter (VAChT)
Takashi Kozaka a, , Izumi Uno a, Yoji Kitamura a, , Daisuke Miwa a, Kazuma Ogawa b, Kazuhiro Shiba a,à
⇑
a Division of Tracer Kinetics, Advanced Science Research Center, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan
b Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of vesamicol analogues, o-iodo-trans-decalinvesamicol (OIDV) or o-bromo-trans-decalinvesami-
Received 15 May 2012
Revised 22 June 2012
Accepted 22 June 2012
Available online 29 June 2012
col (OBDV), were synthesized and their affinities to vesicular acetylcholine transporter (VAChT) and
receptors ( -1, -2) were evaluated by in vitro binding assays using rat cerebral or liver membranes.
OIDV and OBDV showed greater binding affinity to VAChT (Ki = 20.5 5.6 and 13.8 1.2 nM, respectively)
r
r
r
than did vesamicol (Ki = 33.9 18.1 nM) with low affinity to
r receptors. A saturation binding assay in rat
cerebral membranes revealed that [125I]OIDV had a single high affinity binding site with a Kd value of
1.73 nM and a Bmax value of 164.4 fmol/mg protein. [125I]OIDV revealed little competition with inhibitors,
Keywords:
Vesamicol analogues
Vesicular acetylcholine transporter
Imaging probe
which possessed specific affinity to each
r (r-1 and r-2), serotonin (5-HT1A and 5-HT2A), noradrenaline,
and muscarinic acetylcholine receptors. In addition, BBB penetration of [125I]OIDV was verified in in vivo.
The results of the binding studies indicated that OIDV and OBDV had great potential to be VAChT imaging
probes with high affinity and selectivity.
In vitro binding assay
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
et al. reported that trans-decalinvesamicol (DV) revealed a high
affinity for VAChT among various vesamicol analogues in in vitro
Cholinergic nerve disorder is one of the known pathophysiolo-
gies of Alzheimer’s disease (AD).1–3 In the dopamine neurons of
early Parkinson’s disease, it’s known that the presynaptic changes
represented by the dopamine transporter are bigger than the post-
synaptic changes represented by the dopamine D2 receptor.4–6 In
the cholinergic neurons of early AD, the presynaptic changes are
also more remarkable than the postsynaptic changes. The decrease
in the activity of choline acetyltransferase (ChAT),7 which synthe-
sizes acetylcholine (ACh), or vesicular acetylcholine transporter
(VAChT),8,9 which transports ACh into synaptic vesicles, are nota-
ble so that they are attractive targets of molecular imaging for
the purpose of early detection of AD with single-photon emission
computed tomography (SPECT) or positron emission tomography
(PET).10
and in vivo binding assays ( Fig. 1).24 Previously, we developed
(À)-o-iodovesamicol ((À)-oIV), which possessed high affinity for
VAChT and selectivity against
r-1 or r
-2 receptors.25–28 On the ba-
sis of these observations of DV and (À)-oIV, adequate vesamicol
analogues for VAChT imaging were expected with a trans-decalin
framework instead of the cyclohexane moiety and a halogen at
the ortho-position of the 4-phenylpiperidine moiety. In this paper,
we report the syntheses and evaluation of two newly designed
vesamicol analogues, o-iodo-trans-decalinvesamicol (OIDV) and
o-bromo-trans-decalinvesamicol (OBDV), in the development of a
useful VAChT imaging probe with higher affinity and selectivity.
2. Results
Vesamicol (2-(4-phenylpiperidin-1-yl)cyclohexanol), which is
known as a selective antagonist for VAChT in cholinergic neurons,
has attracted much attention as a potential VAChT imaging
2.1. Chemistry
4-(2-Bromophenyl)piperidine (3) was derived from o-bromo-
benzaldehyde (1) by four steps. (Scheme 1) coupling reaction of
3 with trans-decalin-2,3-oxide (4) furnished OBDV (5) in 57% yield.
A bromo substituent of 5 was replaced by a trimethylstannyl group
with Pd(PPh3)4 and hexamethylditin to obtain the key intermedi-
ate o-trimethylstannyl-trans-decalinvesamicol (OTDV) (6). Treat-
ment of 6 with I2 in CHCl3 at room temperature succeeded in the
introduction of an iodo substituent to give cold OIDV (7) in 80%
yield. DV was prepared according to Roger’s protocol.24
probe,11–22 but the additional affinity for
r
receptors
(r-1,
r
-2)23 is an obstacle to progress in this field. In 1989, Rogers
⇑
Corresponding author. Tel.: +81 76 265 2476; fax: +81 76 234 4245.
E-mail addresses: t-kozaka@staff.kanazawa-u.ac.jp (T. Kozaka), ykitamu@staff.
kanazawa-u.ac.jp (Y. Kitamura), shiba@med.kanazawa-u.ac.jp (K. Shiba).
Tel.: +81 76 265 2474; fax: +81 76 234 4245.
à
Tel.: +81 76 265 2470; fax: +81 76 234 4245.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.040

T. Kozaka et al. / Bioorg. Med. Chem. 20 (2012) 4936–4941
4937
HO
HO
H
N
I
HO
N
N
H
vesamicol
DV
(-)-oIV
Figure 1. Vesamicol and its analogues.
1) NH4OH
200 °C, 6 h
2) BH3•THF
1) ethyl acetoacetate
piperidine, EtOH
rt, overnight
Br
Br
O
CO2H
CO2H
THF, reflux, 20h
2) NaOH aq., EtOH
reflux, 3 h
77% (2 steps)
H
then HCl aq.
reflux, 20 h
62% (2 steps)
o-bromobenzaldehyde (1)
2
H
O
(4)
Br
Br
HO
N
Pd(PPh3)4
Me3SnSnMe3
H
H
trans-decalin-2,3-oxide
NH
EtOH, reflux, overnight
57%
toluene, Et3N
reflux, 24 h
65%
H
3
OBDV (5)
SnMe3
HO
I
HO
I2, CHCl3
H
H
rt, overnight
N
N
80%
H
H
OTDV (6)
OIDV (7)
Scheme 1. synthesis of OBDV, OTDV and OIDV.
2.2. In vitro binding assay
2.2.1. VAChT
The binding affinities (Ki values) of OBDV and OIDV to VAChT
were measured by displacement of a typical VAChT antagonist
(À)-[3H]vesamicol (dissociation constant: Kd = 7.40 nM) in rat
cerebral membranes using previously reported procedures.26 To
2.2.2.
To evaluate selectivity against VAChT, OBDV and OIDV were
screened for binding affinities to receptors using in vitro com-
petitive binding assays. The binding affinities of OBDV and OIDV
to the -1 receptor were measured by displacement of a -1
r-1 Receptor
r
r
r
receptor selective agonist (+)-[3H]pentazocine (Kd = 19.9 nM) in
rat cerebral membranes using previously reported procedures.26
DV, vesamicol, DTG, haloperidol, and non-radiolabeled pentazo-
cine were also studied as comparable standards. The results are
also summarized in Table 1. OBDV and OIDV (Ki = 150.7 62.9
and 241.8 98.9 nM, respectively) showed much lower affinity to
mask the
r receptors of the tissue, 1,3-di-o-tolylguanidine (DTG)
was added into each assay tube. DV and vesamicol were also stud-
ied as comparable standards. The percent inhibition against the
binding of (À)-[3H]vesamicol to VAChT and the Ki values were cal-
culated using GraphPad Prism v4 software. The inhibition constant
Ki values were calculated from IC50/(1 + C/Kd), where C is the
concentration of the radioligand. The results are summarized in
Table 1. Each binding affinity to VAChT of DV, OBDV, and OIDV
(Ki = 13.6 8.8, 13.8 1.2, and 20.5 5.6 nM, respectively) was
greater than that of vesamicol (Ki = 33.9 18.1 nM).
the
r-1 receptor than did vesamicol (Ki = 22.1 3.6 nM) or pentaz-
ocine (Ki = 12.1 5.0 nM).
2.2.3.
The binding affinities of OBDV and OIDV to the
were measured by displacement of a receptor selective agonist
[3H]DTG (Kd = 22.3 nM) in rat liver membranes using previously
reported procedures.26 To mask the
-1 receptor of the tissue, pen-
r-2 Receptor
r
-2 receptor
r
r
Table 1
tazocine was added to each assay tube. DV, vesamicol, pentazocine,
haloperidol, and non-radiolabeled DTG were also studied as com-
parable standards. The results are also summarized in Table 1.
OBDV and OIDV (Ki = 137.5 97.4 and 118.8 57.0 nM, respec-
In vitro binding assay of vesamicol analogues
Entry
Compounds
Ki (nM)
VAChT
r
-1
r-2
tively) showed lower affinity to the
r-2 receptor than did vesam-
1
2
3
4
5
6
7
OIDV
OBDV
DV
Vesamicol
DTG
20.5 5.6
13.8 1.2
13.6 8.8
33.9 18.1
—
241.8 98.9
150.7 62.9
74.1 39.9
22.1 3.6
131.1 39.2
12.1 5.0
118.8 57.0
137.5 97.4
68.3 25.5
86.7 35.7
31.7 3.6
icol (Ki = 86.7 35.7 nM) or DTG (Ki = 31.7 3.6 nM).
2.3. Radiolabeling
Pentazocine
Haloperidol
—
—
1880.8 953.9
51.6 14.6
Radioiodination of [125I]OIDV was achieved through the tin-
iodine exchange reaction from the precursor OTDV (6) with
3.5 0.8

4938
T. Kozaka et al. / Bioorg. Med. Chem. 20 (2012) 4936–4941
125I
HO
SnMe3
HO
[
125I]NaI
H
H
N
N
HCL
H
H2O2
H
OTDV
[
125I]OIDV
Scheme 2. Radiosynthesis of [125I] OIDV.
Table 2
In vitro binding assay of [125I]OIDV
Entry
Compounds
Ki (nM)
1
2
3
4
5
6
7
8
9
Pentazocine
DTG
Spiperone
Ketanserin
Noradrenalin
QNB
Vesamicol
OIDV
OBDV
>10,000
1134
1033
>10,000
>10,000
581.7
32.9
28.6
9.3
2.7. Brain uptake of [125I]OIDV in in vivo studies
Figure 2. Saturation curve and scatchard plot of [125I] OIDV.
To evaluate the brain uptake of [125I]OIDV, the biodistribution
studies were carried out using Sprague-Dawley rats (8 weeks, male,
250–300 g, n = 4). Data were calculated as percent of injected dose
per gram of the tissue (% ID/g). At 10 min postinjection, the accumu-
lation of [125I]OIDV in rat whole brain was 0.45 0.04% ID/g.
no-carrier-added [125I]NaI, HCl and H2O2. (Scheme 2) After purifi-
cation by HPLC, the radiochemical yield and purity of [125I]OIDV
were 80% (29.8 MBq) and 99%, respectively.
2.4. Partition coefficient determination
3. Discussion
The partition coefficient (logPo/w) of [125I]OIDV was determined
by the standard method with n-octanol and 0.1 M phosphate buf-
fer. The partition coefficient was calculated with the followed for-
mula: logPo/w = log10 (radioactivity in n-octanol layer/radioactivity
in aqueous layer). The logPo/w value of [125I]OIDV was 2.46 0.02.
Since (À)-vesamicol is known to possess a high affinity for
VAChT, its analogues are expected as potential VAChT probes. To
develop them with high affinity and selectivity, we have studied
various vesamicol analogues that have halogen at the ortho-,
meta-, or para-position of the 4-phenylpiperidine moiety. We
found that (À)-oIV and (À)-mIV have affinities for VAChT that were
as great as (À)-vesamicol, although (+)-oIV, (+)-mIV, and (+)-pIV
have low affinities for VAChT.26 The absolute configuration of the
vesamicol analogues must have a significant influence on the affin-
ity for VAChT. However, vesamicol was reported to possess addi-
2.5. Saturation binding assay of [125I]OIDV
To investigate specific binding of [125I]OIDV to rat cerebral
membranes, an in vitro saturation binding assay of [125I]OIDV
was performed with haloperidol to mask the
r receptors.
tional binding affinities for the
r
receptors (
r
-1,
r
-2).23 oIV and
The resulting saturation curve and Scatchard plot are shown in
Figure 2. The values of Kd and the maximum binding (Bmax) of
[
mIV also showed higher affinities for the
r-1 receptor than did
125I]OIDV, which were determined from the Scatchard plots with
(À)-vesamicol.29 To acquire high VAChT selectivity against
r
GraphPad Prism v4, were 1.73 nM and 164.4 fmol/mg of protein,
respectively. We had already studied an in vitro saturation binding
assay of (À)-[3H]vesamicol under almost the same conditions
using DTG instead of haloperidol.26 The Kd and Bmax values of
(À)-[3H]vesamicol were 7.40 nM and 163.7 fmol/mg of protein,
respectively.
receptors, further modification of the molecular structure, espe-
cially part of cyclohexane, was required. Roger et al synthesized
a number of vesamicol analogues, and examined their in vitro
binding assay only for VAChT.24 We guessed that DV, of which
the affinity is higher than that of (À)-vesamicol in Roger’s report,
might have potential as the new VAChT imaging agent with great
affinity and high selectivity. In fact, OIDV and OBDV, which have
the trans-decalin moiety, showed higher affinity and selectivity
for VAChT in in vitro than did vesamicol as standard. Since the
affinities of oIV and mIV for VAChT were slightly lower than that
of vesamicol in our previous report,30 OIDV and OBDV were sup-
posed to be more suitable for imaging probes of VAChT than oIV
or mIV. OIDV or OBDV revealed good affinity and selectivity even
as racemic compounds and we expected that the results would
be improved after optical resolution. Unfortunately, however, opti-
cal resolution of them is more difficult than that of vesamicol.
Experiments for optical resolution are now continuing.
2.6. Inhibition of [125I]OIDV binding to rat cerebral membranes
Displacement of [125I]OIDV binding was performed by various
ligands: pentazocine (a selective agonist for the
r-1 receptor),
DTG (an agonist for the -1 and -2 receptors), spiperone (an
r
r
antagonist for the serotonin 5-HT1A, 5-HT2A, and dopamine D2
receptors), ketanserin (a selective antagonist for the serotonin
5-HT2A receptor), noradrenalin (a ligand for the noradrenergic
receptor), QNB (an antagonist for the muscarinic acetylcholine
receptor), and vesamicol as a standard. The results are summarized
in Table 2. The obtained Ki values (nM) of pentazocine (>10,000),
DTG (1134), spiperone (1033), ketanserin (>10,000), noradrenalin
(>10,000), and QNB (581.7) were much higher than that of vesam-
icol (32.9) except for OIDV (28.6) and OBDV (9.3).
OBDV displayed the greatest affinity for VAChT and higher
selectivity against the
r-1 or r-2 receptors as far as the results of
in vitro binding assays, so it was expected to possess the potential
for being a great VAChT probe for PET after radiolabeling with

T. Kozaka et al. / Bioorg. Med. Chem. 20 (2012) 4936–4941
4939
76Br. In addition, OIDV also has the potential to be a great VAChT
probe for SPECT after radiolabeling with 123I and, clinically, it is ex-
pected to be more useful because of its ease of use and handling.
Consequently, OIDV radiolabeled with 125I was chosen for further
study of its characteristics. The typical tin-iodine exchange reaction
afforded [125I]OIDV in the sufficient radiochemical yield and purity
(80% and 99%, respectively) without any noticeable byproducts.
When formic acid was used instead of 0.1 N HCl in the radiolabeling
reaction, the radiochemical yield was just around 30%. In the case of
a stronger acid such as 1 N HCl, the radiochemical yield was 76%, but
most of the remaining precursor OTDV turned into DV as a byprod-
uct. The retention times of DV, [125I]OIDV, and OTDV in the adopted
HPLC condition were about 9, 12, and 18 min, respectively, so that
the ortho-position of the 4-phenylpiperidine moiety. These vesam-
icol analogues, OIDV and OBDV, showed high affinity and selectiv-
ity for VAChT in in vitro binding assay. [125I]OIDV was derived from
OTDV in high radiochemical yield and purity and revealed no
affinity for various receptors in rat cerebrum except for VAChT.
In addition, BBB penetration of [125I]OIDV was verified in in vivo.
From these results, [125I]OIDV is thought to be a superior radioli-
gand for VAChT in in vitro binding assay instead of (À)-[3H]vesam-
icol. OIDV and OBDV are also expected as potential superb VAChT
imaging agents after radiolabeling with 123I or 76Br. Further studies
are now underway to evaluate their in vivo characteristics, exam-
ine after optical resolution, and optimize their structure for PET
imaging.
[
125I]OIDV could be perfectly purified by HPLC.
An essential characteristic for in vivo imaging probes of the
5. Experimental
5.1. Syntheses
brain is to pass through the blood–brain barrier (BBB). Generally,
lipophilicity is one of the important factors in the transport
of chemical compounds through the BBB. Since [125I]OIDV exhib-
ited appropriate lipophilicity for BBB penetration (logPo/w
=
5.1.1. 4-(2-Bromophenyl)piperidine (3)
2.46 0.02), it was expected to show adequate brain uptake after
an intravenous injection.
A solution of o-bromobenzaldhyde (20.0 g, 108 mmol), ethyl
acetoacetate (27.3 mL, 216 mmol), and piperidine (2.14 mL,
21.6 mmol) in EtOH (77.2 mL) was stirred for 15 h at room temper-
ature. To the reaction solution was added 12 M NaOH (360 mL) and
EtOH (350 mL). The reaction mixture was refluxed for 4 h, cooled
to room temperature, and the organic solvents removed. The resi-
due was acidified with concd HCl (450 mL) in an ice bath, extracted
with AcOEt, washed with water and brine, and concentrated to
dryness. The residue was transferred to a glass filter and washed
with cold Et2O to obtain dicarboxylic acid 2 (23.9 g) as a white so-
lid without further purification.
The above 2 (23.9 g, 83.2 mmol) was dissolved in 28% ammonia
solution (280 mL) by heating, and then water was evaporated off
under reduced pressure. The resulting residue was heated to
200 °C to melt for 6 h, cooled to room temperature, extracted with
CH2Cl2, washed with Na2CO3 aq and brine, concentrated to dry-
ness, and washed with cold Et2O. BH3ÁTHF (1.0 M THF solution;
208 mL, 208 mmol) was added dropwise to a solution of the crude
cyclic imide in THF (166 mL) at 0 °C under Ar. The reaction mixture
was allowed to gradually reach room temperature and refluxed for
20 h. concd HCl aq (300 mL) was carefully added to the reaction
mixture at 0 °C. The reaction mixture was refluxed for an addi-
tional 20 h, neutralized with NaOH aq, extracted with AcOEt,
washed with water and brine, and concentrated to dryness. Sol-
vents were evaporated off. Et2O (1 L) was added to the residue at
0 °C. The resulting white precipitate was isolated by filtration
and washed with cold Et2O to obtain 3 (12.4 g, 48% from 1) as a
white solid without further purification: 1H NMR d 7.54–7.51 (m,
1H), 7.28–7.26 (m, 2H), 7.07–7.00 (m, 1H), 3.21–3.17 (m, 2H),
3.10 (tt, 1H, J = 3.7, 11.9 Hz), 3.10 (dt, 2H, J = 2.3, 11.9 Hz), 2.21–
2.19 (m, 1H), 1.87–1.83 (m, 2H), 1.65–1.53 (m, 2H); EI MS m/z
240 (M++1, 85.3), 241 (M+, 73.6).
Saturation binding assays of [125I]OIDV in rat cerebral mem-
branes were performed in order to investigate the binding property
with haloperidol for masking the
r receptors (r-1, r-2). The spe-
cific binding was determined by subtracting the nonspecific bind-
ing from the total binding in rat cerebral membranes at each
concentration of [125I]OIDV (0.1–10 nM). The resulting saturation
curve and the Scatchard plot are shown in Figure 2. A saturation
binding assay in rat cerebral membranes indicated that [125I]OIDV
had a single high affinity binding site. [125I]OIDV showed smaller
Kd (1.73 nM) to VAChT than did (À)-[3H]vesamicol (7.4 nM) or
(À)-[125I]oIV (17.4 nM).26 This means that the binding of [125I]OIDV
is stronger than vesamicol or (À)-oIV. Since the Bmax value of
[
125I]OIDV (164.4 fmol/mg protein) is almost same as the Bmax
value of (À)-[3H]vesamicol (163.7 fmol/mg protein), it is thought
that the binding site of OIDV is the same as vesamicol except for
r
receptors (r-1, r-2).
In the in vitro competitive binding assay of [125I]OIDV as a radi-
oligand, [125I]OIDV did not compete with pentazocine, DTG, spipe-
rone, ketanserin, noradrenalin, or QNB in rat cerebral membranes.
In other words, it was verified that [125I]OIDV possesses no affinity
to the
r (r-1 and r-2), serotonin (5-HT1A and 5-HT2A), dopamine
D2, noradrenaline, or muscarinic acetylcholine receptors in rat
cerebral membranes. In addition, the low Ki values of vesamicol
or OBDV indicated that they occupied the binding sites of
[
125I]OIDV. These results mean that the binding of [125I]OIDV with
rat cerebral membranes is specific for VAChT. DTG had just a weak
influence on the binding of [125I]OIDV in rat cerebral membranes,
although OIDV revealed middle affinity for
r-2 receptors in
in vitro binding assay using [3H]DTG and rat liver membranes.
We guess that it is because of the binding superiority of
[
125I]OIDV–VAChT in comparison with
r
-2 receptor and the differ-
ence of tissues, rat brain or liver. From these results, [125I]OIDV is
expected to bind to VAChT in brain preferentially, although -1
and -2 receptors present in rat brain widely.
5.1.2. o-Bromo-trans-decalinvesamicol (OBDV) (5)
r
A mixture of 3 (2.25 g, 9.36 mmol) and trans-decalin-2,3-
oxide (2.14 g, 14.0 mmol) in EtOH (9.4 mL) was refluxed for
20 h. The solvent was evaporated off. The residue was chromato-
graphed on silica gel with hexane-AcOEt (5:1) to give 5 (2.10 g,
57%) as a white solid: 1H NMR d 7.54–7.52 (m, 1H), 7.30–7.25
(m, 2H), 7.06–7.02 (m, 1H), 4.10–4.07 (m, 1H), 3.14–3.11 (m,
2H), 3.10 (tt, 1H, J = 3.4, 12.0 Hz), 2.33–2.30 (m, 1H), 2.21–2.16
(m, 1H), 2.12–2.07 (m, 1H), 1.92–1.19 (m, 17H), 1.03–0.91
(m, 2H); 13C NMR d 144.9, 132.8, 127.6, 127.5, 127.3, 124.5,
67.0, 64.7, 52.9, 49.4, 41.8, 36.7, 36.5, 35.9, 33.8, 33.7, 32.6,
32.5, 29.4, 26.6, 26.5; DART MS m/z 392 (M++1, 50.8), 394
(M++1, 47.3).
r
In in vivo studies of [125I]OIDV, the accumulation of [125I]OIDV
was detected in rat whole brain (0.45 0.04% ID/g) at 10 min
postinjection. We expected that [125I]OIDV passed through the
BBB. Further detailed in vivo evaluations of [125I]OIDV are now
continuing.
4. Conclusion
We have developed new VAChT imaging probes based on
vesamicol, which had the framework of DV and the halogen at

4940
T. Kozaka et al. / Bioorg. Med. Chem. 20 (2012) 4936–4941
5.1.3. o-Trimethylstannyl-trans-decalinvesamicol (OTDV) (6)
A mixture of 5 (42.1 mg, 0.107 mmol), Pd(PPh3)4 (7.5 mg,
6.44 Â 10À3 mmol) and hexamethylditin (87.9 mg, 0.268 mmol)
in anhydrous toluene (1.1 mL) was refluxed for 15 h under Ar.
The solvent was evaporated off. The residue was chromatographed
on silica gel with hexane-AcOEt (10:1) to give 6 (33.4 mg, 65%) as a
white solid: 1H NMR d 7.18–7.15 (m, 1H), 7.09–7.04 (m, 2H), 6.95–
6.90 (m, 1H), 3.86–3.81 (m, 1H), 2.94–2.86 (m, 2H), 2.15–0.65 (m,
23H), 0.06 (m, 9H); 13C NMR d 153.02, 141.65, 135.95, 128.80,
125.79, 125.65, 66.94, 64.65, 53.11, 49.85, 47.21, 36.62, 36.49,
35.85, 34.57, 34.54, 33.75, 33.62, 29.46, 26.55, 26.48, À8.22; DART
MS m/z 478 (M++1, 100).
to each ice-cold assay tube containing the above radioligand and
the displacing ligands at various concentrations (1.0 Â 10À10–10À5
M) in 50 mM Tris–HCl buffer (pH 7.8) in quadruplicate. After
the addition of the tissue in an ice bath, each reaction mixture
in tubes was incubated at 37 °C for 90 min, and then quenched
by cooling in an ice bath. Each sample was passed through
glass-microfiber filters (Whatman, GF/B), which were presoaked
in 0.3% polyethyleneimine, using a cell harvester. The filters
were washed with 50 mM Tris–HCl buffer (pH 7.8), and their
radioactivities were counted with a liquid scintillation counter
(Aloka, LSC-5100). Three independent experiments were carried
out.
5.1.4. o-Iodo-trans-decalinvesamicol (OIDV) (7)
5.3.3.
[3H]DTG (Kd = 22.3 nM) was used as a radioligand for the
receptor with pentazocine to mask the -1 receptor. Rat liver
membranes was added to each ice-cold assay tube containing
[3H]DTG, pentazocine (1.0
M), and the displacing ligands at vari-
r-2 Receptor
Iodine (405 mg, 1.60 mmol) was added to a reaction mixture of
6 (253 mg, 0.531 mmol) in CHCl3 (1.1 mL). The reaction mixture
was stirred for 24 h at the same temperature, quenched by addi-
tion of saturated aqueous Na2S2O3 and saturated aqueous NaHCO3,
extracted with CHCl3, washed with water and brine, dried, and
concentrated to dryness. The residue was chromatographed on sil-
ica gel with hexane-AcOEt (10:1) to give 7 (187 mg, 80%) as a white
solid: 1H NMR d 7.84–7.82 (m, 1H), 7.33–7.23 (m, 2H), 6.91–6.87
(m, 1H), 4.10–4.07 (m, 1H), 3.14–3.11 (m, 2H), 2.79 (tt, 1H,
J = 3.4, 12.0 Hz), 2.33–2.30 (m, 1H), 2.22–2.16 (m, 1H), 2.12–2.07
(m, 1H), 1.89–1.19 (m, 17H), 1.03–0.94 (m, 2H); 13C NMR d
151.8, 147.9, 139.6, 128.5, 127.9, 126.7, 67.02, 64.6, 52.8, 49.4,
47.0, 36.7, 36.5, 36.0, 33.9, 33.7, 32.9, 32.8, 29.4, 26.6, 26.5; DART
MS m/z 440 (M++1, 84.4).
r
-2
r
l
ous concentrations (1.0 Â 10À10–10À5 M) in 50 mM Tris–HCl buffer
(pH 7.8) in quadruplicate. After the addition of the tissue in an ice
bath, each reaction mixture in tubes was incubated at 37 °C for
90 min, and then quenched by cooling in an ice bath. Each sample
was passed through glass-microfiber filters (Whatman, GF/B),
which were presoaked in 0.3% polyethyleneimine, using a cell har-
vester. The filters were washed with 50 mM Tris–HCl buffer (pH
7.8), and their radioactivities were counted with a liquid scintilla-
tion counter (Aloka, LSC-5100). Three independent experiments
were carried out.
5.2. Preparation of rat cerebral and liver membranes
5.4. Radiolabeling
Animal experiments were performed in compliance with
the Guidelines for the Care and Use of Laboratory Animals at the
Takara-machi Campus of Kanazawa University. Sprague-Dawley
rat (8 weeks, male, 250–300 g) cerebrum or livers were homoge-
nized in ice-cold 0.32 M sucrose with a Teflon-glass homogenizer.
The homogenate was centrifuged at 1000g at 4 °C for 10 min. The
resulting precipitate was removed and the supernatant was centri-
fuged at 55000g at 4 °C for 1 h. Cerebral membranes needed
additional steps in that the resulting precipitate was suspended
in ice-cold water with a homogenizer and again centrifuged at
55000g at 4 °C for 1 h. Each resulting pellet of rat cerebrum or liver
was suspended in an ice-cold buffer (50 mM Tris–HCl/0.32 M su-
crose, pH 7.8) with a homogenizer.
To a solution of HCl (0.1 N, 50
l
L) and 6 (1 mg/mL, 50
l
L) in a
vial, [125I]NaI (carrier-free, 25
l
L, 37.1 MBq) and 3% H2O2 (50
lL)
were added. The reaction mixture was shaken at room tempera-
ture for 1 min, left for 20 min, quenched by addition of Na2S2O5
(200 mg/mL; 50
lL), turned basic by addition of NaOH (1 N,
50 L), diluted with H2O (5 mL), filtered through a Sep-Pak C18
l
(Waters, Light), which was pre-washed with EtOH (5 mL) and
H2O (15 mL). The Sep-Pak C18 containing [125I]OIDV was washed
with H2O (5 mL) to remove unreacted [125I]NaI or any salts.
[
125I]OIDV in the Sep-Pak C18 was eluted with EtOH (5 mL) and
concentrated to a 100-lL solution. The solution was then purified
by a reverse phase HPLC column (Zorbax-ODS RX-C18, 9.6 Â
250 mm) warmed to 40 °C. The retention time of [125I]OIDV was
about 12 min with the mobile phase consisting of 90:10:0.2 v/v/v
acetonitrile/H2O/monoethanolamine at a 4.0 mL/min flow rate.
The radiolabeling was repeated six times.
5.3. In vitro binding assay
5.3.1. VAChT
(À)-[3H]Vesamicol (Kd = 7.40 nM) was used as a radioligand for
the VAChT receptor. Rat cerebral membranes was added to each
5.5. Partition coefficient determination
ice-cold assay tube containing (À)-[3H]vesamicol, DTG (0.2
lM)
[
125I]OIDV (0.51 MBq) was added to a mixture of n-octanol
to mask the
r receptors and the displacing ligands at various con-
centrations (1.0 Â 10À10–10À5 M) in 50 mM Tris–HCl buffer (pH
7.8) in quadruplicate. After the addition of the tissue in an ice bath,
each reaction mixture in tubes was incubated at 37 °C for 60 min,
and then quenched by cooling in an ice bath. Each sample was
passed through glass-microfiber filters (Whatman, GF/F), which
were presoaked in 0.3% polyethyleneimine, using a cell harvester.
The filters were washed with 50 mM Tris–HCl buffer (pH 7.8)
and their radioactivities were counted with a liquid scintillation
counter (Aloka, LSC-5100). Three independent experiments were
carried out.
(15 mL) and 0.1 M phosphate buffer (pH 7.4; 15 mL) in a test tube
(n = 4). The test tube was vortexed for 5 min at room temperature,
followed by centrifugation for 10 min (2000g). The radioactivities
of four samples (1.0 mL) from each layer were measured with an
auto well gamma counter (Aloka, AccuFLEX c7010). The indepen-
dent experiments were repeated three times.
5.6. Saturation binding assay of [125I]OIDV
Saturation analysis was performed by incubation in various
concentrations of [125I]OIDV (0.1–10 nM) with rat cerebral mem-
branes (400 lg protein) and 100 nM haloperidol using 50 mM
Tris–HCl buffer (pH 7.8) for 1 h at 37 °C in quadruplicate. Each
sample was passed through glass-microfiber filters (Whatman,
5.3.2.
(+)-[3H]Pentazocine (Kd = 19.9 nM) was used as a specific radi-
oligand for the -1 receptor. Rat cerebral membranes was added
r-1 Receptor
r

T. Kozaka et al. / Bioorg. Med. Chem. 20 (2012) 4936–4941
4941
GF/F), which were presoaked in 0.3% polyethyleneimine, using a
References and notes
cell harvester. The filters were washed with 50 mM Tris–HCl buffer
(pH 7.8) and their radioactivities were counted with an auto well
1. Davies, P.; Maloney, A. J. Lancet 1976, 2, 1403.
2. Bartus, R. T.; Dean, R. L., 3rd; Beer, B.; Lippa, A. S. Science 1982, 217, 408.
3. Kasa, P.; Rakonczay, Z.; Gulya, K. Prog. Neurobiol. 1997, 52, 511.
4. Ishikawa, T.; Dhawan, V.; Kazumata, K.; Chaly, T.; Mandel, F.; Neumeyer, J.;
Margouleff, C.; Babchyck, B.; Zanzi, I.; Eidelberg, D. J. Nucl. Med. 1996, 37, 1760.
5. Seibyl, J. P.; Marek, K.; Sheff, K.; Zoghbi, S.; Baldwin, R. M.; Charney, D. S.; van
Dyck, C. H.; Innis, R. B. J. Nucl. Med. 1998, 39, 1500.
6. Ichise, M.; Kim, Y. J.; Ballinger, J. R.; Vines, D.; Erami, S. S.; Tanaka, F.; Lang, A. E.
Neurology 1999, 52, 1206.
gamma counter (Aloka, AccuFLEX
c7010). Nonspecific binding
was determined with non-radiolabeled OIDV (10
lM). Three inde-
pendent experiments were carried out.
5.7. In vitro binding assay of [125I]OIDV
7. Whitehouse, P. J.; Price, D. L.; Clark, A. W.; Coyle, J. T.; DeLong, M. R. Ann. Neurol.
1981, 10, 122.
8. Kuhl, D. E.; Minoshima, S.; Fessler, J. A.; Frey, K. A.; Foster, N. L.; Ficaro, E. P.;
Wieland, D. M.; Koeppe, R. A. Ann. Neurol. 1996, 40, 399.
9. Efange, S. M.; Garland, E. M.; Staley, J. K.; Khare, A. B.; Mash, D. C. Neurobiol.
Aging 1997, 18, 407.
10. Giboureau, N.; Som, I. M.; Boucher-Arnold, A.; Guilloteau, D.; Kassiou, M. Curr.
Top. Med. Chem. 2010, 10, 1569.
11. Parsons, S. M.; Bahr, B. A.; Rogers, G. A.; Clarkson, E. D.; Noremberg, K.; Hicks, B.
W. Prog. Brain Res. 1993, 98, 175.
12. Efange, S. M. FASEB J. 2000, 14, 2401.
13. Bando, K.; Naganuma, T.; Taguchi, K.; Ginoza, Y.; Tanaka, Y.; Koike, K.;
Takatoku, K. Synapse 2000, 38, 27.
14. Efange, S. M.; von Hohenberg, K.; Khare, A. B.; Tu, Z.; Mach, R. H.; Parsons, S. M.
Nucl. Med. Biol. 2000, 27, 749.
15. Keller, J. E.; Bravo, D. T.; Parsons, S. M. J. Neurochem. 2000, 74, 1739.
16. Grundstrom, E.; Gillberg, P. G.; Aquilonius, S. M. Acta Neurol. Scand. 2001, 103, 2.
17. Szymoszek, A.; Wenzel, B.; Scheunemann, M.; Steinbach, J.; Schuurmann, G. J.
Med. Chem. 2008, 51, 2128.
Rat cerebral membranes membranes was added to each
ice-cold assay tube containing 5 nM [125I]OIDV and each ligand
(pentazocine, DTG, spiperone, ketanserin, noradrenalin, QNB,
vesamicol, OIDV, or OBDV) at various concentrations (10À10–10À5 M)
in 50 mM Tris–HCl buffer (pH 7.8) in quadruplicate. Nonspecific
binding was determined at a concentration of 10 lM OIDV. After
the addition of the tissue, the tubes were removed from the ice,
and incubated at 37 °C for 1 h, quenched by cooling in an ice
bath. Each sample was passed through glass-microfiber filters
(Whatman, GF/F), which were presoaked in 0.3% polyethyleneim-
ine, using a cell harvester. The filters were washed with 50 mM
Tris–HCl buffer (pH 7.8), and their radioactivities were counted
with an auto well gamma counter (Aloka, AccuFLEX c7010).
5.8. Brain uptake of [125I]OIDV in in vivo studies
18. Khare, P.; White, A. R.; Parsons, S. M. Biochemistry 2009, 48, 8965.
19. Efange, S. M.; Khare, A. B.; von Hohenberg, K.; Mach, R. H.; Parsons, S. M.; Tu, Z.
J. Med. Chem. 2010, 53, 2825.
20. Khare, P.; Mulakaluri, A.; Parsons, S. M. J. Neurochem. 2010, 115, 984.
21. Kovac, M.; Mavel, S.; Deuther-Conrad, W.; Meheux, N.; Glockner, J.; Wenzel, B.;
Anderluh, M.; Brust, P.; Guilloteau, D.; Emond, P. Bioorg. Med. Chem. 2010, 18,
7659.
The [125I]OIDV (0.4 mL, 185 kBq) was injected intravenously via
the tail vein into Sprague-Dawley rats (8 weeks, male, 250–300 g,
n = 4). At 10 min postinjection, the rats were sacrificed. Their
whole brains were taken out and weighed. The radioactivity of
each sample was counted with an auto well gamma counter (Alo-
22. Wenzel, B.; Li, Y.; Kraus, W.; Sorger, D.; Sabri, O.; Brust, P.; Steinbach, J. Bioorg.
Med. Chem. 2012, 22, 2163.
23. Efange, S. M.; Mach, R. H.; Smith, C. R.; Khare, A. B.; Foulon, C.; Akella, S. K.;
Childers, S. R.; Parsons, S. M. Biochem. Pharmacol. 1995, 49, 791.
24. Rogers, G. A.; Parsons, S. M.; Anderson, D. C.; Nilsson, L. M.; Bahr, B. A.;
Kornreich, W. D.; Kaufman, R.; Jacobs, R. S.; Kirtman, B. J. Med. Chem. 1989, 32,
1217.
25. Shiba, K.; Mori, H.; Ichikawa, A.; Tonami, N. Life Sci. 1996, 1039, 59.
26. Shiba, K.; Yano, T.; Sato, W.; Mori, H.; Tonami, N. Life Sci. 2002, 71, 1591.
27. Shiba, K.; Mori, H.; Tonami, N. Ann. Nucl. Med. 2003, 17, 451.
28. Shiba, K.; Ogawa, K.; Mori, H. Bioorg. Med. Chem. 2005, 1095, 13.
29. Custers, F. G.; Leysen, J. E.; Stoof, J. C.; Herscheid, J. D. Eur. J. Pharmacol. 1997,
338, 177.
ka, AccuFLEX c7010). Data were calculated as percent of injected
dose per gram of the tissue (% ID/g).
Supplementary data
Supplementary data (HPLC charts of OBDV and OIDV) associ-
ated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2012.06.040. These data include
MOL files and InChiKeys of the most important compounds de-
scribed in this article.
30. Shiba, K.; Mori, H.; Matsuda, H.; Tsuji, S.; Kuji, I.; Sumiya, H.; Kinuya, K.;
Tonami, N.; Hisada, K.; Sumiyosi, T. Nucl. Med. Biol. 1995, 22, 205.