Syntheses and evaluation of a homologous series of aza-vesamicol as improved radioiodine-labeled probes for sigma-1 receptor imaging

Article abstract of DOI:10.1016/j.bmc.2019.03.054

Sigma-1 receptor imaging probes for determining the expression levels are desirable for diagnoses of various diseases and companion diagnoses of therapeutic agents targeting the sigma-1 receptor. In this study, we aimed to develop probes with higher affin

Full text of DOI:10.1016/j.bmc.2019.03.054

Accepted Manuscript
Syntheses and evaluation of a homologous series of aza-vesamicol as improved
radioiodine-labeled probes for sigma-1 receptor imaging
Kazuma Ogawa, Ryohei Masuda, Kenji Mishiro, Mengfei Wang, Takashi
Kozaka, Kazuhiro Shiba, Seigo Kinuya, Akira Odani
PII:
S0968-0896(18)31998-9
https://doi.org/10.1016/j.bmc.2019.03.054
BMC 14846
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 November 2018
27 March 2019
29 March 2019
Please cite this article as: Ogawa, K., Masuda, R., Mishiro, K., Wang, M., Kozaka, T., Shiba, K., Kinuya, S., Odani,
A., Syntheses and evaluation of a homologous series of aza-vesamicol as improved radioiodine-labeled probes for
sigma-1 receptor imaging, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.
2019.03.054
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Ogawa et al. 1/44
Syntheses and evaluation of a homologous series of aza-vesamicol as
improved radioiodine-labeled probes for sigma-1 receptor imaging
Kazuma Ogawaa,b*, Ryohei Masudab, Kenji Mishiroa, Mengfei Wangb,
Takashi Kozakac, Kazuhiro Shibac, Seigo Kinuyab, Akira Odanib
aInstitute for Frontier Science Initiative, Kanazawa University, Kakuma-
machi, Kanazawa, Ishikawa 920-1192, Japan;
bGraduate School of Pharmaceutical Sciences, Kanazawa University,
Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan;
cAdvanced Science Research Centre, Kanazawa University, Takara-machi,
Kanazawa, Ishikawa 920-8640, Japan;
*Corresponding Author:
Kazuma Ogawa
Telephone: 81-76-234-4460; Fax: 81-76-234-4459
E-mail: kogawa@p.kanazawa-u.ac.jp
Running Title: sigma-1 receptor imaging probe
Conflict of interest: The authors have declared that no conflict of interest.

Ogawa et al. 2/44
Abstract
Sigma-1 receptor imaging probes for determining the expression levels are
desirable for diagnoses of various diseases and companion diagnoses of
therapeutic agents targeting the sigma-1 receptor. In this study, we aimed to
develop probes with higher affinity for the sigma-1 receptor. For this
purpose, we synthesized and evaluated compounds, namely, vesamicol
derivatives, in which alkyl chains of varying chain length were introduced
between a piperazine ring and a benzene ring. The binding affinity of the
vesamicol derivatives for the sigma-1 receptor tended to increase
depending on the length of the alkyl chain between the benzene ring and
the piperazine ring. The sigma-1 receptor of 2-(4-(3-
phenylpropyl)piperazin-1-yl)cyclohexan-1-ol (5) (Ki = 5.8 nM) exhibited
the highest binding affinity; therefore, we introduced radioiodine into the
benzene ring in 5. The radioiodine labeled probe [125I]2-(4-(3-(4-
iodophenyl)propyl)piperazin-1-yl)cyclohexan-1-ol ([125I]10) showed high
accumulation in the sigma-1 receptor expressing DU-145 cells both in vitro
and in vivo. Co-injection of [125I]10 with an excess level of a sigma

Ogawa et al. 3/44
receptor ligand, haloperidol, resulted in a significant decrease in the tumor
accumulation in vitro and in vivo, indicating sigma receptor-mediated
tumor uptake. These results provide useful information for developing
sigma-1 receptor imaging probes.
Key Words: Sigma-1 receptor; Imaging; Probe; Radiolabel

Ogawa et al. 4/44
1. Introduction
Sigma receptors were originally regarded as a new subtype of the
opioid receptors by Martin et al. (1976).1 These receptors were
subsequently reclassified as original receptors with at least two subtypes,
sigma-1 and sigma-2.2 The sigma-1 receptor was cloned over 20 years
ago,3 and the sigma-2 receptor was cloned recently.4 The molecular size of
the sigma-1 receptor is 25.3 kDa, comprising 223 amino acids; this receptor
is located primarily on the endoplasmic reticulum (ER) membrane of the
cell. The sigma-1 receptor works to maintain cellular homeostasis as a
molecular chaperone5, 6 and is related to functions of the central nervous
system, including signal transduction, memory, recognition, and emotion.
Therefore, the expression level of the sigma-1 receptor is involved in
neurodegenerative diseases, such as Alzheimer's disease, Parkinson's
disease, and amyotrophic lateral sclerosis (ALS).7-9 Furthermore, it has
been reported that the sigma-1 receptor is highly expressed in various
cancer cells.10 This receptor may control the electrical plasticity of cancer
cells by driving ion channels to enhance their function, providing a suitable
environment for cancer cells.11 Therefore, sigma-1 receptor targeting

Ogawa et al. 5/44
agents, including sigma-1 receptor agonists for neurodegenerative diseases
and sigma-1 receptor antagonists for cancer therapy, may have potential for
use in therapy for these diseases.12, 13 With this background, the
determination of sigma-1 receptor expression levels in vivo should be
beneficial for the diagnosis and therapeutics treatment of diseases related to
the sigma-1 receptor and for the development of drugs targeting the sigma-
1 receptor.14 The nuclear medicine technique based on radiolabeled probes
is currently the only modality that can determine receptor expression levels
in vivo. Therefore, excellent sigma-1 imaging probes are desirable for
determining expression levels and, thus, may be useful for companion
diagnoses of therapeutic agents targeting this receptor.15
We previously developed radiolabeled compounds targeting the
sigma-1 receptor based on the vesamicol structure (Figure. 1a) as a lead
compound.16-21 In these studies, 2-[4-(4-iodophenyl)piperidino]
cyclohexanol (pIV, Figure. 1b) with an iodine at the para position of the
benzene ring in the vesamicol structure was shown to have a higher binding
affinity for the sigma-1 receptor compared with vesamicol and compounds
with an iodine at the meta or ortho position. Moreover, most reported

Ogawa et al. 6/44
sigma-1 receptor ligands have a cationic amine and an aromatic ring.22 In a
recent X-ray crystallography study, it was demonstrated that the sigma-1
receptor and its ligands bind via electrostatic interaction between 172Glu in
the sigma-1 receptor and a cationic amine in the sigma-1 receptor ligands.23
Additionally, it has been reported that the distance between the cationic
amine and hydrophobic sites influences the affinity for the sigma-1
receptor.22
In the structure of pIV, the piperidine ring and the iodobenzene sites
may correspond to the cationic amine and hydrophobic sites, respectively.
Thus, we hypothesized that the distance between the piperidine ring and the
iodobenzene may affect the affinity for the sigma-1 receptor. To develop
probes with a higher affinity for the sigma-1 receptor, we synthesized and
evaluated compounds in which the piperidine ring was transformed to a
piperazine ring for easy synthesis and alkyl chains of varying chain lengths
were introduced between the piperazine ring and the benzene ring (Scheme
1). From among the synthesized vesamicol derivatives, we selected a
compound with high affinity for the sigma-1 receptor. The compound was
radiolabeled by introducing radioiodine at the para position on the benzene

Ogawa et al. 7/44
ring because para was discovered to be the optimal position for enhancing
the affinity of the sigma-1 receptor in vesamicol analogs. The radiolabeled
compound was evaluated in vitro and in vivo. Although we are interested in
developing 123I (t1/2 = 12.3 h) labeled imaging probes for single photon
emission computed tomography (SPECT), 125I (t1/2 = 59.4 d) was used in
this study as an alternative radionuclide because of its long half-life.
(a)
(b)
I
HO
HO
N
N
Figure 1. Structures of (a) vesamicol and (b) pIV.
2. Materials and methods
2.1. General
Proton nuclear magnetic resonance (1H-NMR) spectra were
obtained with JEOL JNM-ECS400 (JEOL Ltd, Tokyo, Japan) for 400 MHz
and JNM-ECS600 (JEOL Ltd) for 600 MHz. Data for 1H NMR are
reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, qt = quintet, m = multiplet), coupling

Ogawa et al. 8/44
constant (Hz), and integration. Direct analysis in real time mass spectra
(DART-MS) and electrospray ionization mass spectra (ESI-MS) were
obtained with JEOL JMS-T100TD (JEOL Ltd). Optical rotations were
measured by a model SEPA-300 high-sensitive polarimeter (HORIBA,
Kyoto, Japan). [3H]1,3-o-Di-tolylguanidine ([3H]DTG) (1.1 TBq /mmol),
[3H]pentazocine (1.0 TBq/mmol), and [125I]Sodium iodide (644 GBq/mg)
were purchased from PerkinElmer (Waltham, MA, USA). TLC analyses
were performed with silica plates (Art 5553, Merck, Darmstadt, Germany).
SA4503 was kindly supplied by M’s Science (Kobe, Japan). DTG, (+)-
pentazocine, and haloperidol were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Other reagents were of reagent grade and used as
received.
2.1. Synthesis of compounds
Synthetic scheme is shown in Schemes 1 and 2.
2.1.1. trans-2-(4-Phenylpiperazin-1-yl)cyclohexan-1-ol (rac-1)
Phenylpiperazine (1.0 g, 6.2 mmol) and cyclohexene oxide (1.2 g,
12.3 mmol) in 3.5 mL of ethanol were refluxed for 22 h. After cooling,

Ogawa et al. 9/44
precipitates were washed with hexane to afford rac-1 (1.5 g, 92%) as a
colorless powder.
1H NMR (CDCl3): δ 1.24 (4H, m), 1.50-2.00 (3H, m), 2.10-2.45 (2H, m),
2.59 (2H, m), 2.89 (2H, m), 3.20 (4H, m), 3.41 (1H, m), 3.97 (1H, m),
6.86-6.94 (3H, m), 7.27 (2H, m).
HO
NH
1
1
(+)-
(a)
(b)
N
N
N
(–)-
1
rac-
HO
N
HO
N
(d)
(c)
NH
HN
N
n
HN
Br
n
2
3
4
5
6
(n= 1)
(n= 2)
(n= 3)
(n= 4)
(n = 1, 2, 3, 4)
Scheme 1. Syntheses of compounds (+)-1, (–)-1, and 3–6.
(a) cyclohexene oxide, ethanol; (b) di-p-toluoyl-D-tartaric acid, acetone; di-
p-toluoyl-L-tartaric acid, acetone; (c) cyclohexene oxide, H2O; (d) K2CO3,
acetonitrile.
2.1.2. (+)-trans-2-(4-phenylpiperazin-1-yl)cyclohexan-1-ol [(+)-(1)]

Ogawa et al. 10/44
(+)-Di-p-toluoyl-D-tartaric acid (700 mg, 5.4 mmol) was dissolved
in 10 mL of acetone, and then 1 (433 mg, 1.7 mmol) in 10 mL of acetone
was added dropwise to the solution at room temperature. A precipitate was
collected by filtration and the filtrate was used for preparation of (–)-
enantiomer of 1. The precipitate was dissolved in 2 M aqueous NaOH
solution and the solution was extracted using dichloromethane. The organic
phase was dried over sodium sulfate. After the dichloromethane was
removed in vacuo, (+)-1 (162 mg, 75%) was obtained as a colorless
powder.
1H NMR (400 MHz, CDCl3): δ 1.15‒1.39 (m, 4H), 1.68‒1.95 (m, 3H),
2.10‒2.20 (m, 1H), 2.21‒2.35 (m, 1H), 2.53‒2.65 (m, 2H), 2.83‒2.95 (m,
2H), 3.10‒3.38 (m, 4H), 3.35‒3.48 (m, 1H), 3.88‒4.20 (m, 1H), 6.87 (t, J =
7.3 Hz, 1H), 6.93 (d, J = 8.2 Hz, 2H, m), 7.27 (dd, J = 8.7, 7.3 Hz, 2H).
HRMS (DART) m/z calcd for C16H25N2O (M+H)+, 261.1967; found
261.1969.
Specific rotation:
= +16.5º (c = 0.14, methanol).
2.1.3. (–)-trans-2-(4-phenylpiperazin-1-yl)cyclohexan-1-ol [(–)-(1)]

Ogawa et al. 11/44
The solvent in the filtrate was removed in vacuo, and the residue
was dissolved in 2 M aqueous NaOH solution. The solution was extracted
using dichloromethane. The organic phase was dried over sodium sulfate.
The organic phase was concentrated in vacuo to afford a colorless solid.
(–)-(1) was prepared by the same procedure as (+)-1 using the residual
solid (433 mg, 1.7 mmol) and (−)-di-p-toluoyl-L-tartaric acid (700 mg, 5.4
mmol). (–)-(1) (210 mg) was obtained as a colorless powder.
1H NMR (400 MHz, CDCl3): δ 1.15‒1.39 (m, 4H), 1.68‒1.95 (m, 3H),
2.10‒2.20 (m, 1H), 2.21‒2.35 (m, 1H), 2.53‒2.65 (m, 2H), 2.83‒2.95 (m,
2H), 3.10‒3.38 (m, 4H), 3.35‒3.48 (m, 1H), 3.70‒4.15 (m, 1H), 6.87 (t, J =
7.3 Hz, 1H), 6.93 (d, J = 8.2 Hz, 2H, m), 7.27 (dd, J = 8.7, 7.3 Hz, 2H).
HRMS (DART) m/z calcd for C16H25N2O (M+H)+, 261.1967; found
261.1969.
Specific rotation:
= −15.0º (c = 0.14, methanol).
2.1.4. trans-2-(Piperazin-1-yl)cyclohexan-1-ol (2)
Piperazine (851 mg, 9.9 mmol) was dissolved in 100 mL of water,
and then cyclohexene oxide (970 mg, 9.9 mmol) in 100 mL of water was

Ogawa et al. 12/44
added dropwise to the solution. After stirring at room temperature for 15 h,
the solvent was removed in vacuo. The residue was purified by
chromatography on silica gel using chloroform-methanol (5:1) as the eluent
to obtain compound 2 (521 mg, 28%) as a colorless powder.
1H NMR (CDCl3): δ 1.10‒1.42 (m, 4H), 1.62‒1.90 (m, 3H), 2.05‒2.22 (m,
2H), 2.33‒2.45 (m, 2H), 2.65‒2.78 (m, 2H), 2.80‒2.98 (m, 4H), 3.30‒3.42
(m, 1H), 3.63‒3.76 (m, 1H).
2.1.5. trans-2-(4-Benzylpiperazin-1-yl)cyclohexan-1-ol (3)
Compound 2 (368 mg, 2.0 mmol) and benzyl bromide (342 mg, 2.0
mmol) were dissolved in 20 mL of acetonitrile and then potassium
carbonate (276 mg, 2.0 mmol) was added to the solution. After reflux for 1
h, potassium carbonate was removed by filtration. The solvent was
removed in vacuo. The residue was purified by chromatography on silica
gel using chloroform-methanol (19:1) as the eluent to obtain compound 3
(421 mg, 77%) as a colorless powder.
1H NMR (400 MHz, CDCl3): δ 1.10‒1.30 (4H, m), 1.65-1.85 (m, 3H),
2.05‒2.15 (m, 1H), 2.15-2.23 (m, 1H), 2.25‒2.65 (m, 6H), 2.68‒2.80, (m,

Ogawa et al. 13/44
2H), 3.30‒3.39 (m, 1H), 3.51 (s, 2H), 3.95‒4.05 (m, 1H), 7.25-7.30 (5H,
m).
HRMS (DART) m/z calcd for C17H27N2O (M+H)+, 275.2123; found
275.2132.
2.1.6. trans-2-(4-Phenethylpiperazin-1-yl)cyclohexan-1-ol (4)
Compound 2 (1.7 g, 9.3 mmol) and 2-phenethyl bromide (1.7 g, 9.3
mmol) were dissolved in 93 mL of acetonitrile and then potassium
carbonate (1.2 g, 9.3 mmol) was added to the solution. After reflux for 1 h,
potassium carbonate was removed by filtration. The solvent was removed
in vacuo. The residue was purified by chromatography on silica gel using
chloroform-methanol (20:1) as the eluent to obtain compound 4 (1.8 g,
70%) as a colorless powder.
1H NMR (600 MHz, CDCl3): δ 1.12‒1.25 (m, 4H), 1.65‒1.88 (m, 3H),
2.10‒2.15 (m, 1H), 2.15‒2.25 (m, 1H), 2.40‒2.75 (m, 8H), 2.75‒2.88 (4H,
m), 3.32‒3.42 (m, 1H), 3.88‒4.05 (m, 1H), 7.17‒7.23 (m, 3H), 7.28 (t, J =
7.6 Hz, 2H).

Ogawa et al. 14/44
HRMS (DART) m/z calcd for C17H27N2O (M+H)+, 289.2280; found
289.2284.
2.1.7. trans-2-(4-(3-Phenylpropyl)piperazin-1-yl)cyclohexan-1-ol (5)
Compound 2 (92 mg, 0.5 mmol) and 3-phenylpropyl bromide (100
mg, 0.5 mmol) were dissolved in 5 mL of acetonitrile and then potassium
carbonate (69 mg, 0.5 mmol) was added to the solution. After reflux for 1
h, potassium carbonate was removed by filtration. The solvent was
removed in vacuo. The residue was purified by chromatography on silica
gel using chloroform-methanol (25:1) as the eluent to obtain compound 5
(61 mg, 41%) as a colorless powder.
1H NMR (600 MHz, CDCl3): δ 1.11‒1.30 (m, 4H), 1.66‒1.87 (m, 5H),
2.08‒2.15 (m, 1H), 2.15‒2.23 (m, 1H), 2.30‒2.65 (m, 6H), 2.36 (t, J = 7.4
Hz, 2H), 2. (4H, m), 2.63 (t, J = 7.7 Hz, 2H), 2.72‒2.82 (m, 2H), 3.32‒3.40
(m, 1H), 3.90‒4.06 (m, 1H), 7.16‒7.22 (m, 3H), 7.28 (t, J = 7.6 Hz, 2H).
HRMS (DART) m/z calcd for C19H31N2O (M+H)+, 303.2436; found
303.2403.

Ogawa et al. 15/44
2.1.8. trans-2-(4-(4-Phenylbutyl)piperazin-1-yl)cyclohexan-1-ol (6)
Compound 2 (92 mg, 0.5 mmol) and 4-phenylbutyl bromide (106
mg, 0.5 mmol) were dissolved in 5 mL of acetonitrile and then potassium
carbonate (69 mg, 0.5 mmol) was added to the solution. After reflux for 1
h, potassium carbonate was removed by filtration. The solvent was
removed in vacuo. The residue was purified by chromatography on silica
gel using chloroform-methanol (50:1) as the eluent to obtain compound 6
(61 mg, 39%) as a colorless powder.
1H NMR (400 MHz, CDCl3): δ 1.10‒1.32 (m, 4H), 1.40‒1.85 (m, 7H),
2.08‒2.25 (m, 2H), 2.25‒2.65 (m, 8H), 2.63 (t, J = 7.6 Hz, 2H), 2.68‒2.83
(m, 2H), 3.30‒3.40 (m, 1H), 3.90‒4.10 (m, 1H), 7.15‒7.20 (m, 3H), 7.27 (t,
J = 7.9 Hz, 2H).
HRMS (DART) m/z calcd for C20H33N2O (M+H)+, 317.2593; found
317.2621.

Ogawa et al. 16/44
HO
HO
N
X
X
(e)
N
Br
N
9
(X = Br)
HN
7
8
2
(X = Br)
(X = I)
10
(X = I)
HO
N
HO
N
(f)
Br
Me3Sn
N
N
9
11
Scheme 2. Syntheses of compounds 9–11.
(e) K2CO3, acetonitrile; (f) hexamethylditin,
tetrakis(triphenylphosphine)palladium(0), toluene.
2.1.9. trans-1-(3-Bromopropyl)-4-bromobenzene (7)
Triphenylphosphine (140 mg, 534 µmol) was dissolved in 1 mL of
tetrahydrofuran (THF) at 0 ˚C, and then 3-(4-bromophenyl)propan-1-ol
(100 mg, 465 µmol) and tetrabromomethane (180 mg, 543 mmol) in 1 mL
of THF were added dropwise to the solution. After stirring for 4 h at room
temperature under a nitrogen atmosphere, the solvent was removed in
vacuo. The residue was purified by chromatography on silica gel using
chloroform as the eluent to obtain compound 7 (82 mg, 64%) as yellow oil.
1H NMR (400 MHz, CDCl3): δ 2.14 (qt, J = 7.0 Hz, 2H), 2.74 (t, J = 7.3
Hz, 2H), 3.39 (t, J = 6.6 Hz, 2H), 7.08 (d, J = 8.2 Hz, 2H), 7.41 (d, J = 8.2
Hz, 2H).

Ogawa et al. 17/44
2.1.10. 1-(3-Bromopropyl)-4-iodobenzene (8)
(3-Bromopropyl)benzene (100 mg, 0.5 mmol), iodine (50.7 mg,
0.2 mmol), and sodium iodate (20.8 mg, 0.1 mmol) were added to a
mixture of 500 µL of acetic acid and 50 µL of concentrated sulfuric acid.
After stirring for 18 h at 70 ˚C, the reaction solution was adjusted to pH 7-8
with saturated aqueous solution of sodium hydrogen carbonate, and was
extracted with chloroform. The organic layer was dried with Na2SO4, and
the solvent was removed in vacuo. The residue (8) was used in the next
reaction without further purification.
2.1.11. trans-2-(4-(3-(4-Bromophenyl)propyl)piperazin-1-yl)cyclohexan-1-
ol (9)
Compounds 2 (40 mg, 0.14 mmol) and 7 (27.6 mg, 0.15 mmol)
were dissolved in 1.5 mL of acetonitrile and then potassium carbonate
(20.7 mg, 0.15 mmol) was added to the solution. After reflux for 1 h,
potassium carbonate was removed by filtration. The solvent was removed
in vacuo. The residue was purified by chromatography on silica gel using

Ogawa et al. 18/44
chloroform-methanol (40:1) as the eluent to obtain compound 9 (52 mg,
94%) as a colorless powder.
1H NMR (400 MHz, CDCl3): δ 1.10‒1.32 (m, 4H), 1.65‒1.85 (m, 5H),
2.07‒2.25 (m, 2H), 2.35‒2.55 (m, 6H), 2.34 (t, J = 7.6 Hz, 2H),2.58 (t, J =
7.6 Hz, 2H), 2.70‒2.80 (m, 2H), 3.30‒3.40 (m, 1H), 3.90‒4.05 (m, 1H),
7.05 (d, J = 7.8 Hz, 2H), 7.39 (d, J = 7.8 Hz, 2H).
HRMS (DART) m/z calcd for C19H30BrN2O (M+H)+, 381.1542; found
381.1548.
2.1.12. trans-2-(4-(3-(4-Iodophenyl)propyl)piperazin-1-yl)cyclohexan-1-ol
(10)
Compounds 2 (5.7 mg, 30 µmol) and 8 (10 mg, 30 µmol) were
dissolved in 300 µL of acetonitrile and then potassium carbonate (4.1 mg,
30 µmol) was added to the solution. After reflux for 1 h, potassium
carbonate was removed by filtration. The solvent was removed in vacuo.
The residue was purified by reversed phase (RP)-HPLC performed with a
Cosmosil 5C18-MS column (20 × 250 mm; Nacalai Tesque, Kyoto, Japan)
at a flow rate of 12.0 mL/min with a gradient mobile phase of 80%

Ogawa et al. 19/44
methanol in water with 0.05% triethylamine to 100% methanol with 0.05%
triethylamine for 20 min. The fraction containing compound 10 was
determined by mass spectrometry, and collected. The solvent was removed
by lyophilization to yield compound 10 (4.0 mg, 17%) as a colorless
powder.
1H NMR (400 MHz, CDCl3): δ 1.10‒1.32 (m, 4H), 1.65‒1.85 (m, 5H),
2.08‒2.23 (m, 2H), 2.25‒2.65 (m, 6H), 2.34 (t, J = 7.8 Hz, 2H), 2.57 (t, J =
7.6 Hz, 2H), 2.65‒2.72 (m, 2H), 3.30‒3.40 (m, 1H), 3.80‒4.15 (m, 1H),
6.94 (d, J = 8.2 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H).
HRMS (DART) m/z calcd for C19H30IN2O (M+H)+, 429.1403; found
429.1399.
2.1.13. trans-2-(4-(3-(4-(Trimethylstannyl)phenyl)propyl)piperazin-1-
yl)cyclohexan-1-ol (11)
Compound 9 (38.1 mg, 0.10 mmol), hexamethylditin (81.9 mg, 0.25
mmol), and tetrakis(triphenylphosphine)palladium(0) (6.9 mg, 59.7 µmol)
were dissolved in toluene. The reaction mixture was refluxed for 12 h
under N2 atmosphere. The solvent was removed in vacuo. The residue was

Ogawa et al. 20/44
purified by chromatography on silica gel using chloroform as the eluent
and RP-HPLC performed with a Cosmosil 5C18-MS column (20 × 250 mm)
at a flow rate of 12.0 mL/min with a gradient mobile phase of 75%
methanol in water with 0.05% triethylamine to 85% methanol with 0.05%
triethylamine for 20 min. The fraction containing compound 11 was
determined by mass spectrometry, and collected. The solvent was removed
by lyophilization to yield compound 11 (35 mg, 75%) as clear oil.
HRMS (ESI) m/z calcd for C22H39N2OSn (M+H)+, 467.2084; found
467.2077.
2.2. In vitro competitive binding assay.
Samples of sigma-1 receptor and sigma-2 receptor for binding
experiments were prepared from rat brains without cerebellum and rat liver
in male Sprague-Dawley rats (200 g, Japan SLC, Inc., Hamamatsu, Japan)
using a method described previously.24, 25 A sigma-1 receptor binding assay
and a sigma-2 receptor binding assay for compounds (+)-1, (–)-1, 3-6, 10,
and reference compounds were performed by same methods described
previously.18

Ogawa et al. 21/44
2.3. Preparation of [125I]trans-2-(4-(3-(4-iodophenyl)propyl)piperazin-1-
yl)cyclohexan-1-ol ([125I]10)
[125I]10 was prepared by a standard iodination reaction of the
corresponding trimethylstannyl precursor. Briefly, 1 μL of [125I]NaI
solution was added to a reaction vial. Then, 100 μL of compound 11 (1
mg/mL in methanol), 15 μL of 10% acetic acid, and 20 μL of chroramine-T
aqueous solution (4 mg/mL) were added to the reaction vial. After standing
the reaction mixture at room temperature for 10 min, the reaction was
quenched by adding of NaHSO3 aqueous solution. The reaction mixture
was purified by RP-HPLC performed using a Cosmosil 5C18-MS-II column
(4.6 × 150 mm; Nacalai Tesque) with a flow rate of 1 mL/min with a
gradient mobile phase of 75% methanol in water with 0.05% triethylamine
to 85% methanol in water with 0.05% triethylamine for 20 min. The
column temperature was maintained at 40 °C.
2.4. Determination of partition coefficient
The partition coefficient of [125I]10 was measured as described

Ogawa et al. 22/44
previously.17 The partition coefficient was determined by calculating the
ratio of radioactivity in 1-octanol to that in the 0.1 M phosphate buffer (pH
7.4), and expressed as a common logarithm (log P).
2.5. Cellular uptake experiments in vitro
Cellular uptake experiments were performed as described
previously using a DU-145 prostate cancer cell lines (ATCC, Manassas,
VA).18 Briefly, the cells were seeded and pre-incubated on 6-well plates for
24 h, and incubated at 37 ºC in the culture medium without fetal bovine
serum containing [125I]10 (3.7 kBq/well) or (+)-[125I]pIV (3.7 kBq/well),
which was prepared by a method of a previous report,17 for different time
intervals (15, 30, 60, and 120 min). The cell uptake of [125I]10 or (+)-
[125I]pIV was also examined by incubation with 10 μM of haloperidol as a
blocking agent. After incubation, the cells were washed twice with ice-cold
PBS and were resolved by 1 M NaOH. The solutions were then collected
and the radioactivity was determined with an auto well gamma counter
(ARC-7010; Hitachi Medical, Ltd., Tokyo, Japan) and corrected for
background radiation. The radioactivity of each sample was normalized for

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the protein level, which was determined using a Protein Assay
Bicinchoninate Kit (Nacalai Tesque).
2.6. Biodistribution experiments of [125I]10 in tumor-bearing mice
The animal experimental protocols used were approved by the
Committee on Animal Experimentation of Kanazawa University.
Experiments with animals were conducted in accordance with the
Guidelines for the Care and Use of Laboratory Animals of Kanazawa
University. The animals were housed with free access to food and water at
23 ºC with a 12-h alternating light/dark schedule. Approximately 5 × 106
DU-145 cells were injected subcutaneously into the right dorsum of 4-
week-old male BALB/c nude mice (15-19 g, Japan SLC, Inc.). After 14-21
days post-inoculation, mice were intravenously administered 100 μL of
[125I]10 (37 kBq). At 1 and 24 h post-injection, the mice were sacrificed.
Tissues of interest were removed and weighed, and radioactivity counts
were determined with an auto well gamma counter.
2.7. Blocking studies in vivo

Ogawa et al. 24/44
For blocking studies, the DU-145 tumor-bearing mice were
intravenously administered 100 μL of [125I] 10 (37 kBq) solution with an
excess of an unlabeled sigma ligand, SA4503 (10 μmol/kg) or haloperidol
(10 μmol/kg). At 1 h post-injection, the mice were sacrificed and
biodistribution experiments were conducted as described above.
2.8. Statistical evaluation
All data were analyzed using GraphPad Prism 5.0 software (La
Jolla, CA, USA) and displayed as mean ± standard deviation (SD).
Significance for in vitro blocking studies was calculated using an unpaired
Student's t test. Significance among three groups for in vivo blocking
studies was calculated using a one-way analysis of variance (ANOVA)
followed by Tukey’s post hoc test. Results were considered statistically
significant at p < 0.05.
3. Results and discussion
3.1. Syntheses of compounds (+)-1, (–)-1, 3–6, reference compound 10,
precursor 11, and [125I]10

Ogawa et al. 25/44
The syntheses of compounds (+)-1, (–)-1, and 3–6 are outlined in
Scheme 1. Compounds (+)-1 and (–)-1 were synthesized by a previously
reported vesamicol synthesis method using phenylpiperazine as the starting
material instead of phenylpiperidine.26 Compounds 3–6 were synthesized
using coupling compound 2 with phenyl alkyl bromide. However, the
optical resolution of 3–6 by the same method as that applied for 1 was
unsuccessful. Herein, an in vitro binding assay and other experiments were
performed for the racemic compounds because these compounds are
sufficient to achieve the purpose of this study, which is to evaluate the
effect of the distance between the piperazine ring and the benzene ring.
The synthesis of the nonradioactive iodinated reference compound
10 and a labeling precursor 11 containing a trimethylstannyl group, which
was prepared via brominated compound 9, are outlined in Scheme 2. The
overall yields of reference compound 10 and precursor 11 were 5% and
12%, respectively. Radioiodination of [125I]10 was performed by the
standard electrophilic halogenation of the corresponding trimethylstannyl
precursor 11 with a radiochemical yield of 75%. After purification by RP-
HPLC, [125I]10 exhibited radiochemical purities exceeding 98%. The

Ogawa et al. 26/44
identity of [125I]10 was confirmed by comparing its retention time with that
of the corresponding nonradioactive iodinated reference compound 10 by
HPLC (Figure 2).
Figure 2. RP-HPLC chromatograms of (A) 10 and (B) [125I]10 after
purification. Conditions: A flow rate of 1 mL/min with a gradient mobile
phase of 75% methanol in water with 0.05% triethylamine to 85%
methanol in water with 0.05% triethylamine for 20 min.
3.2. In vitro competitive binding assay
Table 1 lists the binding affinities of synthesized compounds (+)-1,
(–)-1, 3–6, 10, and reference compounds for the sigma receptors (sigma-1
and sigma-2) determined by a competitive binding assay. The affinities of
compounds 3 and 4 for sigma-1 receptor were similar and the affinities

Ogawa et al. 27/44
were higher than those of compounds (+)-1 and (–)-1. Furthermore, the
affinities of compounds 5 and 6 for the sigma-1 receptor were similar and
the affinities were higher than those of compounds 3 and 4. Generally, the
binding affinities for the sigma-1 and sigma-2 receptors of the aza-
vesamicol homologs tended to increase as the length of the alkyl chain
increased between the benzene ring and the piperazine ring. Compound 5
showed the lowest Ki value (Ki = 5.8 nM) among compounds (+)-1, (–)-1,
and 3 – 6 and the affinity of compound 5 for the sigma-1 receptor was
higher than those of the reference compounds utilized as selective and non-
selective sigma-1 receptor ligands: (+)-pentazocine (Ki = 16.0 nM) and
haloperidol (Ki = 12.5 nM), respectively. Therefore, we introduced iodine
or radioiodine into the benzene ring in compound 5. The para position of
the phenyl group was selected as the introduction site because pIV (Figure
1b), a vesamicol analog with an iodine at the para position, had the highest
affinity for the sigma-1 receptor among vesamicol analogs in which iodine
was introduced at different positions of the phenyl group in our previous
studies [(+)-vesamicol: Ki = 74.9 nM, (+)-pIV: Ki = 1.3 nM].16, 24 We
expected the affinity for the sigma-1 receptor to increase because of the

Ogawa et al. 28/44
increased affinity resulting from the introduction of iodine into the benzene
ring in the case of vesamicol;16, 17 however, upon the introduction of iodine
to 5, the affinity for the sigma-1 receptor showed little change. This
unexpected result may be attributed to the large distance between the
benzene ring and piperazine ring. These results indicate that the optimal
halogen introduction position might not necessarily be the para position in
aza-vesamicol compounds.
Meanwhile, compound 10 showed low selectivity for the sigma-1
receptor against the sigma-2 receptor (Ki for sigma-1 = 8.7 nM and Ki for
sigma-2 = 22.3 nM, respectively). For receptor imaging in the brain,
selectivity is important. However, sigma-1 and sigma-2 dual receptor
imaging probes have also been studied, and their usefulness has been
evaluated for prostate cancer imaging.27 Thus, the affinity of compound 10
for the sigma receptors may be suitable for prostate cancer imaging.

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Table 1. Affinities (nM) of synthesized vesamicol derivatives and
reference compounds for sigma receptors.
Sigma-1 (Ki)
Sigma-2 (Ki)
328.0 ± 26.9
245.6 ± 45.3
723.0 ± 67.7
246.2 ± 69.9
50.4 ± 13.0
31.2 ± 4.9
(+)-1
85.3 ± 20.2
87.3 ± 9.0
33.2 ± 7.7
31.5 ± 4.2
5.8 ± 0.9
(–)-1
3
4
5
6
9.0 ± 1.7
10
8.7 ± 1.0
22.3 ± 3.5
(+)-Pentazocine
Haloperidol
16.0 ± 1.6
12.5 ± 0.9
2437.2 ± 313.4
81.5 ± 38.5
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 radioligand ([3H]pentazocine to
sigma-1 (Kd = 19.9 nM), [3H]DTG to sigma-2 (Kd = 22.3 nM)).
Values are means ± SEM of three experiments.
3.3. Partition coefficient
The partition coefficient was determined, and the log P value of
[125I]10 was found to be 2.54 ± 0.04 (mean ± SD for four samples). This
result indicates that the lipophilicity of [125I]10 is higher than that of (+)-
[125I]pIV, whose log P value is 2.08.17 The higher lipophilicity of [125I]10 is