Synthesis and biological evaluation of Tc-99m-cyclopentadienyltricarbonyl-technetium-labeled A-85380: An imaging probe for single-photon emission computed tomography investigation of nicotinic acetylcholine receptors in the brain

Article abstract of DOI:10.1016/j.bmc.2019.04.030

We have designed (S)-(5-(azetidin-2-ylmethoxy)pyridine-3-yl)methyl cyclopentadienyltricarbonyl technetium carboxylate ([^99mTc]CPTT–A–E)with high affinity for nicotinic acetylcholine receptors (nAChRs)using (2(S)-azetidinylmethoxy)-pyridine (A-85380)as the lead compound to develop a Tc-99m-cyclopentadienyltricarbonyl-technetium (^99mTc)-labeled nAChR imaging probe. Because technetium does not contain a stable isotope, cyclopentadienyltricarbonyl rhenium (CPTR)was synthesized by coordinating rhenium, which is a homologous element having the same coordination structure as technetium. Further, the binding affinity to nAChR was evaluated. CPTR–A–E exhibited a high binding affinity to nAChR (K_i = 0.55 nM). Through the radiosynthesis of [^99mTc]CPTT–A–E, an objective compound could be obtained with a radiochemical yield of 33% and a radiochemical purity of greater than 97%. In vitro autoradiographic study of the brain exhibited that the local nAChR density strongly correlated with the amount of [^99mTc]CPTT–A–E that was accumulated in each region of interest. Further, the in vivo evaluation of biodistribution revealed a higher accumulation of [^99mTc]CPTT–A–E in the thalamus (characterized by the high nAChR density)when compared with that in the cerebellum (characterized by the low nAChR density). Although additional studies will be necessary to improve the uptake of [^99mTc]CPTT–A–E to the brain, [^99mTc]CPTT–A–E met the basic requirements for nAChR imaging.

Full text of DOI:10.1016/j.bmc.2019.04.030

Accepted Manuscript
Synthesis and biological evaluation of Tc-99m-cyclopentadienyltricarbonyl-
technetium-labeled A-85380: An imaging probe for single-photon emission
computed tomography investigation of nicotinic acetylcholine receptors in the
brain
Hiroyuki Kimura, Masashi Ueda, Hidekazu Kawashima, Kenji Arimitsu,
Yusuke Yagi, Hideo Saji
PII:
S0968-0896(19)30206-8
https://doi.org/10.1016/j.bmc.2019.04.030
BMC 14884
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
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Revised Date:
Accepted Date:
12 February 2019
23 April 2019
25 April 2019
Please cite this article as: Kimura, H., Ueda, M., Kawashima, H., Arimitsu, K., Yagi, Y., Saji, H., Synthesis and
biological evaluation of Tc-99m-cyclopentadienyltricarbonyl-technetium-labeled A-85380: An imaging probe for
single-photon emission computed tomography investigation of nicotinic acetylcholine receptors in the brain,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.04.030
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Synthesis and biological evaluation of Tc-99m-cyclopentadienyltricarbonyl-
technetium-labeled A-85380: An imaging probe for single-photon emission computed
tomography investigation of nicotinic acetylcholine receptors in the brain
Hiroyuki Kimura1,2, Masashi Ueda1,3, Hidekazu Kawashima1,4, Kenji Arimitsu1,2, Yusuke
Yagi1,2, Hideo Saji1
1Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences,
Kyoto University, Kyoto, Japan
2Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University,
Kyoto, Japan
3Department of Biofunction Imaging Analysis, Graduate School of Medicine, Dentistry, and
Pharmaceutical Sciences, Okayama University, Okayama, Japan
4Radioisotope Research Center, Kyoto Pharmaceutical University, Kyoto, Japan
Address for correspondence:
Hideo Saji, PhD
Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences,
Kyoto University; 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
Tel: +81-75-753-4556, Fax: +81-75-753-4568
E-mail: hsaji@pharm.kyoto-u.ac.jp
Table of Abbreviation
nAChR
A-85380
CPTT
CPTR
DLT
Nicotinic acetylcholine receptor
3-(2(S)-azetidinylmethoxy)-pyridine
Cyclopentadienyltricarbonyl technetium
Cyclopentadienyltricarbonyl rhenium
Double ligand transfer
SPECT
HPLC
s.d.
Single-photon emission computed tomography
High-performance liquid chromatography
Standard deviation
1

Abstract
We
have
designed
(S)-(5-(azetidin-2-ylmethoxy)pyridine-3-yl)methyl
cyclopentadienyltricarbonyl technetium carboxylate ([99mTc]CPTT–A–E) with high affinity
for nicotinic acetylcholine receptors (nAChRs) using (2(S)-azetidinylmethoxy)-pyridine (A-
85380) as the lead compound to develop a Tc-99m-cyclopentadienyltricarbonyl-
technetium (99mTc)-labeled nAChR imaging probe. Because technetium does not contain a
stable isotope, cyclopentadienyltricarbonyl rhenium (CPTR) was synthesized by coordinating
rhenium, which is a homologous element having the same coordination structure as
technetium. Further, the binding affinity to nAChR was evaluated. CPTR–A–E exhibited a
high binding affinity to nAChR (Ki = 0.55 nM). Through the radiosynthesis of [99mTc]CPTT–
A–E, an objective compound could be obtained with a radiochemical yield of 33% and a
radiochemical purity of greater than 97%. In vitro autoradiographic study of the brain
exhibited that the local nAChR density strongly correlated with the amount of [99mTc]CPTT–
A–E that was accumulated in each region of interest. Further, the in vivo evaluation of
biodistribution revealed a higher accumulation of [99mTc]CPTT–A–E in the thalamus
(characterized by the high nAChR density) when compared with that in the cerebellum
(characterized by the low nAChR density). Although additional studies will be necessary to
improve the uptake of [99mTc]CPTT–A–E to the brain, [99mTc]CPTT–A–E met the basic
requirements for nAChR imaging.
Keywords: nicotinic acetylcholine receptors, single-photon emission computed tomography,
A-85380, cyclopentadienyltricarbonyl technetium, bifunctional radiopharmaceutical
2

1. Introduction
Recent studies have demonstrated that nicotinic acetylcholine receptors (nAChRs) in the
brain are involved in high brain functions (e.g., memory, learning, and cognition)1 and in the
progression of cranial nerve diseases such as the Alzheimer’s disease and Parkinson’s
disease.2,3,4 Therefore, a method for performing noninvasive measurement of the distribution
and density of nAChR in the human brain could provide useful information for diagnosing
various central nervous system disorders and for contributing to the development of
therapeutic drugs. Several imaging probes have been developed, and their usefulness has been
5–10
studied.
We intend to develop an nAChR imaging probe using Tc-99m-
cyclopentadienyltricarbonyl-technetium (99mTc) as the radionuclide for developing a clinically
versatile nAChR imaging probe. 99mTc is a radionuclide that releases radiation suitable for
single-photon emission computed tomography (SPECT). Because 99mTc is a generator-
producing nuclide, it can be prepared in the hospital at the time of use. Because of this high
clinical versatility, 99mTc is commonly used in clinical trials that require the use of a
radioactive element. However, 99mTc is a metallic element, and a chelating moiety is required
for introduction into an organic compound. Therefore, based on the concept of bifunctional
chelating reagents, we designed and synthesized
a
99mTc-labeled 3-(2(S)-
azetidinylmethoxy)pyridine (A-85380) derivative, which indicates high affinity for
nAChR.11,12 Cyclopentadienyltricarbonyl technetium (CPTT)13,14,15 was selected as the
chelating moiety because it is neutral in charge, it is small in size, and it forms a stable chelate
with 99mTc. (Figure 1) Further, cyclopentadienyltricarbonyl rhenium (CPTR) was synthesized
by coordinating rhenium, which is a homologous element with the same coordination
structure as that of technetium, because technetium does not contain any stable isotopes. We
further evaluated the potential of the 99mTc-labeled A-85380 derivative as an imaging probe in
vitro and in vivo.
nicotinic acetylcholine receptors
(nAChRs)
Conjugation
Brain
tissue
99mTc
Chelating moiety
Receptor binding moiety
O
N
H
CPTT : M = 99mTc
CPTR : M = Re
M
N
OC
CO
A-85380
CO
Figure 1. Schematic of a bifunctional radiopharmaceutical for developing a nAChR
imaging probe labeled with 99mTc
3

2. Results and Discussion
2.1. Chemistry
Previous reports of a structure–activity correlation have observed that the binding affinity
to nAChR was maintained even though a relatively large substituent (e.g., iodine atom and
benzene ring) is introduced at the 5-position of the pyridine ring of A-85380 (Figure 2).16,17,18
Based on this information, we have designed the A-85380 derivative as an (S)-(5-(azetidine-
2-ylmethoxy)pyridine-3-yl)methyl cyclopentadienyltricarbonyl technetium carboxylate
compound ([99mTc]CPTT–A–E).
O
O
O
5
O
N
N
H
H
N
99mTc
CO
N
OC
CO
A-85380
[
99mTc]CPTT-A-E
Figure 2. Drug design of the A-85380 derivative
2.1.1. Synthesis of the CPTR derivatives
The compound 4 was prepared for incorporating a chelating moiety in A-85380 (Scheme
1). The treatment of 1,1`-ferrocenedicarboxylic acid (1) with hydrogen chloride in methanol
yielded dimethyl ester 2, which was transformed to rhenium complex 3 by transmetalation
from ferrocene to rhenium based on the double ligand transfer (DLT) reaction. Ester 3 was
hydrolyzed under basic conditions to yield carboxylic acid 4. Compound 4 was further used
as the substrate for CPTR–A–E.
O
O
O
NH4ReO4
Cr(CO)6, CrCl3
OR
OR
2M NaOH aq.
1,4-dioxane
89%
OMe
OH
Fe
Re
MeOH
60%
Re
CO
4
OC
CO
OC
CO
CO
O
3
1
2
: R = H
: R = Me (87%)
HCl
/MeOH
Scheme 1. Synthesis of the CPTR derivatives
2.1.2.
Synthesis
of
(S)-(5-(azetidine-2-ylmethoxy)pyridine-3-yl)methyl
cyclopentadienyltricarbonyl rhenium carboxylate (CPTR–A–E)
L-azetidine-2-carboxylic acid 5 was reduced using borane tetrahydrofuran complex, which
was followed by the protection of the amino group with a tert-butoxycarbonyl group (Boc)
that yielded alcohol 6.19 The resulting compound 6 was further coupled with methyl 5-
hydroxynicotinate under the Mitsunobu reaction conditions to yield compound 7. The
4

reduction of 7 with lithium aluminum hydride yielded hydroxymethyl 8, which was
condensed with carboxylic acid 4 using 2-methyl-6-nitrobenzoic acid anhydride (MNBA) to
yield 9.20,21 Deprotection of the Boc group on 9 using trifluoroacetic acid provided CPTR–A–
E (Scheme 2).
HO
CO2Me
.
N
1) BH3 THF
THF, 90 oC
Ph3P, DEAD
OH
N
CO2H
N
H
THF
89%
2) (Boc)2O,1M aq.NaOH
1,4-dioxane-H2O
Boc
6
5
2 steps 97%
4
, MNBA
DMAP
LiAlH4
O
CO2Me
O
OH
N
N
THF, 0 oC
80%
CH2Cl2
68%
Boc
Boc
N
N
N
8
7
O
O
O
O
TFA
CH2Cl2
90%
O
O
N
N
H
Boc
Re
N
Re
OC
CO
CO
OC
CO
CO
9
CPTR-A-E
Scheme 2. Synthesis of CPTR–A–E
2.2. In vitro studies on CPTR–A–E binding to nAChR
We evaluated the binding affinity of CPTR–A–E to nAChR by examining the competition
of the A-85380 and nicotine with [3H]cytisine for various sites in rat cortical membranes.
Table 1 presents the Ki values determined from the IC50 values for the competitive binding
curves of the compounds. CPTR–A–E exhibited approximately equal binding affinity (Ki =
0.55 nM) to that of the lead compound A-85380. These results indicate that the current drug
design in which a chelation site is introduced at the 5-position of the pyridine ring is effective.
Table 1. Ki values for the inhibition of [3H]cytisine binding to nAChR
Ki (nM)
Nicotine
6.29 ± 4.62
0.19 ± 0.10
0.55 ± 0.2
A-85380
CPTR–A–E
The values are the mean ± s.d. of three independent determinations.
5

2.3. Radiosynthsis of [99mTc]CPTT–A–E
Because CPTR–A–E exhibited high affinity to nAChR, the labeling synthesis of
[
99mTc]CPTT–A–E was examined. First, ferrocene–A–E, which is the labeling precursor, was
synthesized (Figure S1). We attempted to directly label this compound via DLT reaction22,23
−
using 99mTcO4 . However, [99mTc]CPTT–A–E could not be obtained because ferrocene–A–E
was decomposed under severe reaction conditions (high temperature and high pressure)
(Scheme 3).
O
99mTcO4
-
O
Cr(CO)6, CrCl3
O
O
O
N
O
N
MeOH
180 oC, 45 min
Boc
N
Boc
99mTc
CO
Fe
N
OC
CO
Ferrocene-A-E
[
99mTc]CPTT-A-E
Scheme 3. Radiosynthesis of [99mTc]CPTT–A–E from ferrocene–A–E
Further, the radiosynthesis of [99mTc]CPTT–A–E was performed according to the
−
methodology used for synthesizing CPTR–A–E. Thus, compound 2 and 99mTcO4 reacted
24,25
under DLT reaction conditions to obtain 99mTc-labeled 10.
Further, compound 10 was
converted to carboxylic acid 11 by hydrolysis under basic conditions using an aqueous
sodium hydroxide solution, and carboxylic acid 11 was condensed with alcohol 8 to obtain 12
(Scheme 4). Another deprotection reaction was conducted using a HCl–MeOH solution, and
the reaction residue was purified using high-perfomance liquid chromatography (HPLC) to
yield the objective compound [99mTc]CPTT–A–E with a radiochemical yield of 33% and a
radiochemical purity of greater than 97% (Figure 3).
O
O
O
99mTcO4
-
8
, MNBA
DMAP
OMe
OMe
2M NaOH aq.
Cr(CO)6, CrCl3
OMe
OH
Fe
99mTc
CO
1,4-dioxane
45 oC, 15 min
CH2Cl2
rt, 30 min
MeOH
180 oC, 45 min
99mTc
CO
OC
CO
OC
CO
11
O
10
2
O
O
O
O
HCl-MeOH
O
O
N
Boc
N
60 oC, 15 min
H
N
99mTc
CO
N
99mTc
CO
OC
CO
OC
CO
12
[
99mTc]CPTT-A-E
Scheme 4. Radiosynthesis of [99mTc]CPTT–A–E
6

A) [99mTc]CPTT-A-E
17 min
B) CPTR-A-E
17.84 min
CPTR-A-E
[99mTc]CPTT-A-E
0
10
20
30
0
10
20 30
Time (min)
Time (min)
Figure 3. HPLC analysis of [99mTc]CPTT–A–E (A) and CPTR–A–E (B).
2.4. In vitro studies on [99mTc]CPTT–A–E binding selectivity to nAChR
The binding selectivity of [99mTc]CPTT–A–E to nAChR was evaluated based on the extent
of inhibition of various drugs for binding of [99mTc]CPTT–A–E to the crude cortical synaptic
membrane fraction. Table 2 presents the IC50 values for the competitive binding curves of the
compounds. Further, the binding of [99mTc]CPTT–A–E was inhibited by 5IA and epibatidine,
which exhibited an affinity to the α4β2 subtype of nAChR. However, the binding of
[
99mTc]CPTT–A–E was not inhibited by methyllycaconitine (MLA), which exhibited an
affinity to the α7 subtype of nAChR, or scopolamine, which exhibited an affinity to
muscarinic receptors. These results indicate that the binding of [99mTc]CPTT–A–E was
selective for the α4β2 subtype of nAChR.
Table 2. IC50 values for the inhibition of [99mTc]CPTT–A–E binding to the rat brain
membrane
Competitive Drugs
5IA
IC50 (μM)
140.4 ± 22.6
418.0 ± 32.2
>1000
Epibatidine
MLA
Scopolamine
>1000
Values are the mean ± s.d. of three independent determinations.
7

2.5. Local brain distribution of [99mTc]CPTT–A–E as determined by in vitro
autoradiography
The local distribution of [99mTc]CPTT–A–E in the brain was examined using in vitro
autoradiography. Figure 4 depicts the quantitative results with respect to the accumulation of
radioactivity in the regions of interest. The accumulation of [99mTc]CPTT–A–E was the largest
in the thalamus, where the nAChR receptor density was the highest. Further, the accumulation
of [99mTc]CPTT–A–E was lowest in the cerebellum, where the nAChR receptor density was
observed to be low.
400
350
300
250
200
150
100
50
0
Cortex
Striatum
Thalamus
Cerebellum
Figure 4. Binding of [99mTc]CPTT–A–E in various regions of the rat brain
Further, Figure 5 depicts the relation between the localization of nAChR26 in the rat brain
and the accumulation of [99mTc]CPTT–A–E in each region of interest. An extremely high
correlation was observed between the accumulation of [99mTc]CPTT–A–E and the nAChR
density (r2 = 0.94). These results indicate that the accumulation of the [99mTc]CPTT–A–E
radioactivity reflect the nAChR density.
8

Figure 5. Correlation between the [99mTc]CPTT–A–E binding and the nAChR density
2.6. In vivo biodistribution of [99mTc]CPTT–A–E
Table 3 presents the biodistribution of [99mTc]CPTT–A–E in mice. The accumulation of
[
99mTc]CPTT–A–E in liver and kidney was high immediately after administration, which
gradually cleared. The accumulated levels of [99mTc]CPTT–A–E in spleen, pancreas, lungs,
and heart were similarly high after administration and was further promptly excreted.
However, the accumulation in the stomach, which is an indicator of compound
decomposition, was low. These results indicate that [99mTc]CPTT–A–E is stable in vivo. The
thalamus-to-cerebellum ratio improved over time, with peak values (1.8) being observed 120
minutes after administration. As indicated by the results of in vitro autoradiography,
significantly higher accumulation was observed in the thalamus when compared with that
observed in the cortex and cerebellum (where the receptor density is low). However, the
difference in localization was not as apparent as that revealed by in vitro experiments,
suggesting the nonspecific binding of [99mTc]CPTT–A–E to nAChR.
9

Table 3. Biodistribution of [99mTc]CPTT–A–E in mice
Time after injection (min)
2
5
15
30
60
120
Liver
20.36 ± 3.58
6.10 ± 0.32
51.17 ± 4.29
6.38 ± 1.08
13.44 ± 1.12
3.53 ± 0.75
27.03 ± 1.77
16.08 ± 2.01
23.65 ± 2.74
6.68 ± 1.18
46.11 ± 4.62
5.18 ± 0.53
12.95 ± 1.45
4.26 ± 1.05
27.75 ± 5.34
9.51 ± 3.36
16.09 ± 2.28
3.83 ± 0.36
29.91 ±3.13
2.24 ± 0.77
5.18 ± 0.86
4.06 ± 1.71
11.9 ± 4.24
2.50 ± 0.43
15.26 ± 3.86
3.60 ± 0.97
23.60 ± 2.50
0.97 ± 0.30
2.45 ± 0.13
3.68 ± 3.39
6.04 ± 1.07
1.10 ± 0.47
17.37 ± 3.30
3.70 ± 0.42
22.30± 7.24
0.46 ± 0.38
0.98 ± 0.28
1.81 ± 1.48
4.69 ± 1.21
0.59 ± 0.63
19.32 ± 4.09
4.24 ± 0.81
18.94 ± 7.66
0.09 ± 0.04
0.29 ± 0.06
1.74 ± 1.52
1.08 ± 0.23
0.11 ± 0.06
Intestine
Kidney
Spleen
Pancreas
Stomach
Lung
Heart
Cortex
0.28 ± 0.01
0.34 ± 0.07
0.42 ± 0.05
0.20 ± 0.07
0.33 ± 0.13
0.34 ± 0.07
0.21 ± 0.04
0.31 ± 0.14
0.37 ± 0.08
0.15 ± 0.06
0.28 ± 0.05
0.21 ± 0.09
0.11 ± 0.04
0.27 ± 0.08
0.18 ± 0.05
0.06 ± 0.02
0.23 ± 0.12
0.13 ± 0.06
Thalamus
Cerebellum
Tha/Cer
0.80
0.97
0.83
1.33
1.55
1.80
aExpressed as % injected dose per gram. Each value is the mean ± s.d. for four mice.
2.7. Analysis of radioactivity in the brain
The levels of radioactivity in the brain were measured 120 minutes after the administration
of [99mTc]CPTT–A–E. Figure 6 depicts the levels of radioactivity in the brain as determined
by reverse-phase HPLC. The peak levels of radioactivity (66.1 ± 4.2%; n = 4) were observed
at the same retention time as that of the peak [99mTc]CPTT–A–E radioactivity. This result
indicates that [99mTc]CPTT–A–E is sufficiently stable in the brain.
Figure 6. RP–HPLC radioactivity of the brain homogenate 120 min after [99mTc]CPTT–
A–E administration (retention time: 17 min for [99mTc]CPTT–A–E)
10

3. Conclusion
The results presented in this study demonstrated that the [99mTc]CPTT–A–E, which was
designed as a bifunctional chelating reagent, had high affinity with nAChR in vitro. The
results of in vitro autoradiography denoted that the local levels of nAChR expression in the
brain correlated with the extent of [99mTc]CPTT–A–E accumulation in each region of interest.
The results of in vivo distribution analysis revealed higher accumulation of [99mTc]CPTT–A–
E in the thalamus when compared with that in the cerebellum (which has a low density of
nAChR). Although additional studies will be necessary to improve the uptake of
[
99mTc]CPTT–A–E to the brain, [99mTc]CPTT–A–E met the basic requirements for nAChR
imaging.
11

4. Materials and methods
4.1. General
Male ddY mice and SD rats were supplied by Japan SLC Co. Ltd (Hamamatsu, Japan). The
animal studies were conducted in accordance with the institutional guidelines, and all the
experimental procedures were approved by the Kyoto University Animal Care Committee.
1
The H and 13C NMR spectra were recorded using JEOL JNM-AL300 (JEOL, Tokyo,
Japan), Varian XL-300, and Varian GEMINI 2000/200 (Varian, California, USA) with CDCl3
and CD3OD as the solvents and with tetramethylsilane as the internal standard (Euriso-Top,
Saint-Aubin, France). The mass spectra (MS) were determined using a JMS-HX/HX110
model A and JEOL JMS–GC-mate mass spectrometer (JEOL). Further, specific rotations
were recorded on a p-1020 digital polarimeter (JASCO, Tokyo, Japan). Kiesel gel (70-230
mesh, Merk Art. 07734, Merck, Darmstadt, Germany) was used for open-column
chromatography, and Kieselgel 60 F-254 plates (Merck) were used for performing thin layer
chromatography (TLC). Preparative TLC (PTLC) was conducted using Kieselgel 60 F-254
plates (0.25 mm, Merck) or silica gel 60 F-254 plates (0.5 mm, Merk). JAI LC-908 (Japan
Analytical Industry, Tokyo, Japan) was used as recycle-HPLC, and JAIGEL 1H and JAIGEL
2H were used as GPC columns. (−)–3H–cytisine (1.48 TBq/mmol) was purchased from the
3
NEN Life Science Productions, Inc (Boston, MA, USA). The H radioactivity was measured
with a Tri-Carb 2500-TR Liquid Scintillation Analyzer (ParkinElmer. Inc., Massachusetts,
USA). BAS–SR (FUJIFILM, Tokyo, Japan) was used for autoradiography, and the images
were obtained by BAS5000 (FUJIFILM) and analyzed using a multi gauge device
(FUJIFILM). All the used chemicals were of reagent grade.
4.2. Chemistry
4.2.1. 1,1-bis(methoxycarbonyl)ferrocene (2)
Hydrogen chloride–methanol reagent (10%, 40 mL) was added to a solution of 1,1-
ferrocenedicarboxylic acid (1, 1.0 g, 3.65 mmol) in methanol (20 mL); this mixture was
stirred for 4 h at 60 °C. After the reaction was completed, the organic solvent was removed
under reduced pressure. The residue was purified by silica gel column chromatography (n-
hexane/ethyl acetate = 2/1) to yield compound 2 (957 mg, 87%) in the form of brownish-red
1
needle crystals: mp 115−116 °C; H NMR (300 MHz, CDCl3) δ: 4.82 (t, J = 1.84 Hz, 4H),
13
4.40 (t, J = 1.84 Hz, 4H), 3.79 (s, 6H); C-NMR (75 MHz, CDCl3) δ: 170.65 (2), 72.80 (2),
72.51 (4), 71.50 (4), 51.59 (2); IR (CHCl3) cm−1: 2953, 1715, 1468, 1285, 1196, 1147; FAB-
MS m/z: 302.0239 (Calcd for C14H14O3Fe: 302.0241); MS (FAB) m/z: 302 (M+, 100); Anal
Calcd for C14H14FeO4: C, 55.66; H, 4.67. Found: C, 55.66; H, 4.67.
4.2.2. Tricarbonyl(methoxycarbonylcyclopentadienyl)rhenium (3)
Ammonium perrhenate (335 mg, 1.2 mmol), chromium hexacarbonyl (410 mg, 1.8 mmol),
and chromium (III) chloride anhydrous (106 mg, 0.6 mmol) were added to a solution of
compound 2 (315 mg, 1.0 mmol) in methanol (1.5 mL). This mixture was stored at a constant
pressure in an airtight case (TVS-1 type, 5.0 cm3, Taiatsu Techno, Tokyo, Japan) at room
12

temperature, and the mixture was stirred for 1 h at 180 °C under argon atmospheric
conditions. After the reaction, the mixture was cooled and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography (n-
hexane/chloroform = 1/1) to yield 3 (245 mg, 60%), which formed a colorless crystal: mp
113−114 °C; 1H NMR (300 MHz, CDCl3) δ: 6.00 (t, J = 2.2 Hz, 2H), 5.37 (t, J = 2.2 Hz, 2H),
13
3.79 (s, 3H); C NMR (75 MHz, CDCl3) δ: 191.87 (3), 164.07, 88.78 (2), 88.09, 84.92 (2),
52.23. IR (CHCl3) cm−1: 2033, 1935, 1728, 1472, 1292, 1150; FAB-MS m/z: 393.9855 (Calcd
for C10H7O5Re: 393.9851); MS (FAB) m/z: 394 (M+, 38); Anal Calcd for C10H7: C, 30.53; H,
1.79. Found: C, 30.79; H, 1.77.
4.2.3. Tricarbonyl(carboxycyclopentadienyl)rhenium (4)
An aqueous solution of sodium hydroxide (2 M, 2.6 mL) was added to a solution of 3 (220
mg, 0.56 mmol) in 1,4-dioxane (0.9 mL), and the mixture was stirred for 5 h at room
temperature. After the reaction was completed, the pH was adjusted to 2.0 with 35%
hydrochloric acid, and the mixture was extracted using ethyl acetate. The organic layer was
dried over sodium sulfate and concentrated under reduced pressure to yield 4 (190 mg, 89%)
in the form of a white powder: 1H NMR (300 MHz, CD3OD) δ: 6.00 (t, J = 2.2 Hz, 2H), 5.45
(t, J = 2.2 Hz, 2H), 4.96 (br s, 1H); 13C-NMR (75 MHz, CDCl3) δ: 191.87 (3), 164.07, 88.78
(2), 88.09, 84.92 (2), 52.23; IR (CHCl3) cm−1: 3126, 2882, 2644, 2569, 2025, 1902, 1680,
1485, 1416, 1360, 1302, 1175; FAB-MS m/z: 379.9690 (Calcd for C9H5O5Re: 379.9695); MS
(FAB) m/z: 380 (M+, 13).
4.2.4. (S)-tert-Butyl 2-(hydroxymethyl)azetidine-1-carboxylate (6)
Borane tetrahydrofuran complex (1.0 M solution in tetrahydrofuran, 25.0 mL, 24.7 mmol)
was added to a solution of L-azetidine-2-carboxtlic acid (5, 1.0 g, 9.9 mmol) in
tetrahydrofuran (15.0 mL) under nitrogen atmospheric conditions. The resulting mixture was
stirred for 90 min under reflux. Afetr the reaction had cooled to room temperature, an aqueous
solution of potassium hydrogen sulfate (10%, 15.0 mL) was added to the mixture, which was
further refluxed for 15 min. The mixture was evaporated under reduced pressure to yield the
residue of (S)-azetidine-2-ylmethanol. An aqueous solution of sodium hydroxide (1 M, 20.0
mL) and di-tert-butyl dicarbonate (2.4 g, 11.9 mmol) were added to a solution of (S)-
azetidine-2-ylmethanol (862 mg, 9.9 mmol) in water (20.0 mL) and 1,4-dioxane (15.0 mL) at
0 °C. The mixture was then stirred for 24 h at room temperature. The reaction mixture was
poured into a saturated aqueous solution of sodium bicarbonate and extracted with
chloroform. The organic layer was dried over sodium sulfate and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography (n-hexane/ethyl
acetate = 1/1) to yield (–)-6 (1.79 g, 97%) in the form of a colorless oil: 1H NMR (300 MHz,
CDCl3) δ : 4.42 (br s, 1H), 3.91–3.73 (m, 4H), 2.32 (br s, 1H), 2.24–2.13 (m, 1H), 1.96 (brs,
1H), 1.45 (s, 9H); 13C NMR (75 MHz, CDCl3) δ: 157.1, 80.1, 66.4, 63.5, 46.6, 28.2 (3), 17.87;
IR (CHCl3) cm−1 : 3400, 3005, 2978, 1666, 1414, 1369; FAB-MS m/z: 188.1282 (Calcd for
C9H18NO3: 188.1287); MS (FAB) m/z:188 (M++H, 31); [α]D20 = –20.59 (c = 1.19, CHCl3).
13

4.2.5. (S)-methyl 5-((1-(tert-butoxycarbonyl)azetidine-2-yl)methoxy)nicotinate (7)
Triphenylphoshine (1052 mg, 4.01 mmol), 40% diethyl azodicarboxylate toluene solution
(698 mg, 4.01 mmol), and methyl 5-hydroxynicotinate (613 mg, 4.01 mmol) were added to a
solution of 6 (500 mg, 2.67 mmol) in tetrahydrofuran (18.0 mL) at 0 °C under nitrogen
atmospheric conditions. The resulting mixture was stirred for 10 min and for an additional 24
h at room temperature and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (petrolem ether/ethyl acetate = 1/1) to yield 7 (766 mg,
1
89%) in the form of a colorless oil: H NMR (300 MHz, CDCl3) δ: 8.83 (d, J = 1.3 Hz, 1H),
8.51 (d, J = 2.8 Hz, 1H), 7.81 (dd, J = 2.8, 1.3 Hz 1H), 4.58–4.51 (m, 1H), 4.41–4.36 (m, 1H),
4.17 (dd, J = 6.9, 2.9 Hz, 1H), 3.95 (s, 3H), 3.90 (t, J = 7.5 Hz, 2H), 2.41–2.34 (m, 1H), 2.31–
2.26 (m, 1H), 1.42 (s, 9H); 13C NMR (75 MHz, CDCl3) δ : 165.5, 155.9, 155.8, 142.9, 142.4,
126.3, 120.8, 79.5, 68.7, 59.8, 52.2, 46.9, 28.2 (3), 18.8; IR (CHCl3) cm−1 : 3007, 2978, 2934,
1724, 1686, 1589, 1394, 1367, 1302, 1151; FAB-MS m/z: 323.1611 (Calcd for C16H23N2O5:
323.1607); MS (FAB) m/z:323 (M++H, 62); [α]D20 = –60.28 (c = 1.01, CHCl3).
4.2.6.
(S)-tert-butyl
2-((5-(hydroxymethyl)pyridine-3-yloxy)methyl)azetidine-1-
carboxylate (8)
Lithium aluminum hydride (121 mg, 3.19 mmol) was added to a solution of 7 (858 mg,
2.66 mmol) in tetrahydrofuran (25.0 mL) at 0 °C under nitrogen atmospheric conditions, and
the mixture was stirred for 1 h. After the reaction had proceeded to completion, a saturated
aqueous sodium sulfate solution was added to the reaction mixture to quench an excess of
lithium aluminum hydride. The mixture was dried over sodium sulfate and concentrated under
reduced pressure. Further, the residue was purified by silica gel column chromatography
1
(chloroform/methanol = 20/1) to yield 8 (625 mg, 80%) in the form of a colorless oil: H
NMR (300 MHz, CDCl3) δ: 8.24 (d, J = 1.3 Hz, 1H), 8.17 (d, J = 2.8 Hz, 1H), 7.31 (dd, J =
2.8, 1.3 Hz 1H), 4.71 (s, 2H), 4.52–4.48 (m, 1H), 4.36–4.31 (m, 1H), 4.13 (dd, J = 9.9, 2.6
Hz, 1H), 3.89 (t, J = 7.7 Hz, 2H), 2.41-2.34 (m, 1H), 2.31-2.26 (m, 1H), 1.75 (brs, 1H), 1.41
13
(s, 9H); C NMR (75 MHz, CDCl3) δ : 156.2, 155.2, 140.5, 137.7, 136.9, 119.87, 79.77,
68.61, 61.93, 60.06, 47.14, 28.3 (3), 19.0; IR (CHCl3) cm−1 : 3348, 3009, 2976, 1688, 1591,
1406, 1367, 1290, 1150; FAB-MS m/z: 295.1653 (Calcd for C15H23N2O4: 295.1658); MS
(FAB) m/z: 295 (M++H, 100); [α]D20 = –68.52 (c = 1.08, CHCl3).
4.2.7.
(S)-tert-butyl
2-((5-((cyclopentadienyltricarbonyl
rhenium
carbonyloxy)methyl)pyridine-3-yloxy)methyl)azetidine-1-carboxylate (9)
Rhenium complex 4 (38 mg, 0.1 mmol), 4-dimethylaminopyridine (29 mg, 0.24 mmol),
and 2-methyl-6-nitrobenzoic anhydride (41 mg, 0.12 mmol) were added to a solution of 8 (29
mg, 0.1 mmol) in dichloromethane (1.5 mL) under nitrogen atmospheric conditions, and the
mixture was stirred for 24 h at room temperature. After the reaction had proceeded to
completion, a saturated aqueous sodium bicarbonate solution was added to the reaction, and
the mixture was extracted using chloroform. The organic layer was dried over sodium sulfate
14

and concentrated under reduced pressure. The residue was purified by silica gel column
chromatography (n-hexane/ethyl acetate = 1/1) to yield 9 (44 mg, 68%) in the form of a white
1
powder: H NMR (300 MHz, CDCl3) δ : 8.33 (d, J = 2.7 Hz, 1H), 8.24 (d, J = 1.3 Hz, 1H),
7.26 (dd, J = 2.7, 1.3 Hz 1H), 6.04 (t, J = 2.2 Hz, 2H), 5.38 (t, J = 2.2 Hz, 2H), 5.23 (s, 2H),
4.53–4.48 (m, 1H), 4.36–4.31 (m, 1H), 4.13(dd, J = 9.9, 2.7 Hz, 1H), 3.89 (t, J = 7.9 Hz, 2H),
2.41–2.34 (m, 1H), 2.31–2.26 (m, 1H), 1.41 (s, 9H); 13C NMR (75 MHz, CDCl3) δ : 191.7 (3),
163.4, 156.1, 155.1, 141.9, 138.6, 131.4, 120.9, 89.1 (2), 87.3, 85.1 (2), 79.7, 68.8, 64.2,
60.06, 47.14, 28.4 (3), 19.1; IR (CHCl3) cm−1 : 3348, 3009, 2980, 2033, 1935, 1726, 1690,
1406, 1367, 1283, 1138; FAB-MS m/z: 657.1243 (Calcd for C24H26N2O8Re: 657.1247); MS
(FAB) m/z: 657 (M++H, 76); [α]D20 = –33.11 (c = 1.07, CHCl3).
4.2.8. (S)-(5-(zzetidine-2-ylmethoxy)pyridine-3-yl)methyl cyclopentadienyltricarbonyl
rhenium carboxylate (CPTR–A–E)
Using a pipette, 10 drops of trifluoroacetic acid were added to a solution of 9 (10.5 mg,
0.02 mmol) in dichloromethane (0.1 mL) at 0 °C under nitrogen atmospheric conditions, and
the mixture was stirred for 20 min at room temperature. After the reaction was completed, the
solvent was eliminated under reduced pressure, and the saturated aqueous sodium bicarbonate
solution was added to the mixture before extraction with ethyl acetate. The organic layer was
dried over sodium sulfate and concentrated under reduced pressure. The residue was purified
by NH–silica gel column chromatography (chloroform/methanol = 20/1) to yield CPTR–A–E
(8 mg, 90%) in the form of a colorless oil: 1H NMR (200 MHz, CDCl3) δ : 8.31 (d, J = 2.7 Hz,
1H), 8.24 (d, J = 1.5 Hz, 1H), 7.24 (dd, J = 2.7, 1.5 Hz 1H), 6.04 (t, J = 2.6 Hz, 2H), 5.38 (t, J
= 2.6 Hz, 2H), 5.23 (s, 2H), 4.48–4.22 (m, 1H), 4.06–4.04 (m, 1H), 3.79–3.63 (m, 1H), 3.58–
3.43 (m, 2H), 2.61–2.38 (m, 1H), 2.36–2.20 (m, 1H), 1.92 (brs, 1H); IR (CHCl3) cm−1: 2361,
2341, 2033, 1946, 1728, 1283, 1138; FAB-MS m/z: 557.0717 (Calcd for C19H18N2O6Re:
557.0723); MS (FAB) m/z: 657 (M++H, 25).
4.3. In vitro studies on CPTR–A–E binding to nAChR
The affinity of CPTT–A–E to nAChR was measured as the displacement of (–)–
[3H]cytisine based on the preparation of rat cerebral cortical synaptosomal membranes, as
described by Saji et al.27 The cerebral cortexes of the SD male rats were homogenized in ice-
cold Tris-HCl buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, pH 7.4) using a Polytron
homogenizer. After the centrifugation of the homogenate (31,000 x g, 4 °C, 10 min), the
supernatant was removed; then, the residue was homogenized and centrifuged again. Tris-HCl
buffer was added to the residue to prepare the synaptosomal membrane homogenate sample
(10 mg protein/mL). The levels of protein in homogenate were measured by Lowry protein
assay: 100 µL of the synaptosomal membrane homogenate, [3H]cytisine (784 GBq/mmol, 5.0
nM, 100 µL), and each compound (10−12–10−4 M, 50 µL) were mixed and incubated at 4 °C
for 75 min. Each sample was filtered using a glass filter (Whatman GF/C). We soaked the
glass filters in 0.1% poly-L-lysine before use. The glass filters were washed thrice using 4 mL
of ice-cold Tris-HCl buffer. The levels of radioactivity of the filter were measured by the
15

liquid scintillation counter. Nonspecific binding was evaluated by adding 100 µM (−)–
nicotine. The inhibition curves and Ki values were calculated using GraphPad Prism
(GraphPad Software Inc, Ver 4.03). For the calculation of the inhibitor constant (Ki), the
value of 0.96 nmol/L obtained by Pabreza et al. was used as the dissociation constant (Kd) for
(−)–cytisine.
4.4. Radiosynthesis of [99mTc]CPTT–A–E
For the radiosynthesis of [99mTc]CPTT–A–E, the following modifications were made to the
−
procedure reported by Uehara et al.28 [99mTc]Pertechnetate (99mTcO4 ) (50 μL, 300 MBq),
chromium hexacarbonyl (5.0 mg), and chromium (III) chloride anhydrous (4.5 mg) were
added to a solution of 1,1-bis(methoxycarbonyl)ferrocene (2) in methanol (450 μL) under
argon atmospheric conditions. The resulting mixture was stirred for 45 min at 180 °C under
constant pressure in an airtight tight case (TVS-1 type, 5.0 cm3, Taiatsu Techno). The reaction
mixture was cooled to room temperature and filtered through a Sep-Pak® Plus Cartridge
(Silica) for the removal of impurities. The filtrate was concentrated under reduced pressure to
yield 10. Aqueous sodium hydroxide (2 M, 600 μL) was added to a solution of 10 in 1,4-
dioxane (200 μL). The resulting mixture was then stirred for 15 min at 45 °C. After the
adjustment of the reaction mixture to pH 2.0 by adding hydrochloric acid (35%, 120 μL), the
mixture was loaded on a Sep-Pak® Plus Cartridge (tC18) activated by acetonitrile (6.0 mL) and
water (6.0 mL) and was then eluted with water (3.0 mL) and acetonitrile (3.0 mL). The
organic eluate was concentrated to yield 11. Subsequently, 11, 4-dimethylaminopyridine (1.0
mg), and 2-methyl-6-nitrobenzoic anhydride (1.0 mg) were added to a solution of 8 (1.0 mg)
in dichloromethane (200 μL) under nitrogen atmospheric conditions, and the mixture was
stirred for 30 min at room temperature. After the reaction, the solvent was removed to yield
the crude 12 product. The hydrogen chloride–methanol reagent (10%, 50 μL) was added to a
solution of 12 in methanol (200 μL), and the mixture was stirred for 15 min at 60 °C. After
cooling, the reaction mixture was diluted with methanol (200 μL) and filtered through a 0.45-
μm filter. The filtrate was purified by reverse-phase HPLC on a Cosmosil 5C18 AR-II column
(10 × 250 mm, Nacalai, Kyoto, Japan) using phosphate buffer (pH 3.0)/methanol (60/40) as
the mobile phase at a flow rate of 2.0 mL/min to yield [99mTc]CPTT–A–E (99 MBq, retention
time = 17 min). The radiochemical purity of [99mTc]CPTT–A–E was evaluated by the same
reverse-phase HPLC system.
4.5. In vitro studies on [99mTc]CPTT–A–E binding selectivity to nAChR
We investigated the binding selectivity of [99mTc]CPTT–A–E to nAChR by inhibiting the
binding of [99mTc]CPTT–A–E to the synaptosomal membrane homogenate from SD rat
cerebral cortex with 5IA and epibatidine (α4β2 selective), (+)–methyllycaconitine (α7
selective), and (−)–scopolamine (muscarinic antagonist). Then, 100 μL of the synaptosomal
membrane homogenate (10 mg protein/mL), 100 μL of [99mTc]CPTT–A–E (15.7 kBq), and 5
µL of inhibitor solution were mixed with 50 mM Tris-HCl buffer (inhibitor; 10−10–10−2 M).
Each sample was incubated at 4 °C for 75 min and filtered with a glass filter (Whatman
16

GF/C). We soaked glass filters in 0.1% poly-L-lysine before use. The glass filters were
washed thrice using 4 mL of ice-cold Tris-HCl buffer. The level of radioactivity on the filter
was then measured by γ-counter. Nonspecific binding was evaluated by adding 100 µM (−)–
nicotine. The inhibition curves were elaborated, and the IC50 values were calculated using the
GraphPad Prism software (GraphPad Software Inc, Ver 4.03).
4.6. Local brain distribution of [99mTc]CPTT–A–E as determined by in vitro
autoradiography
In vitro autoradiography was performed on 20-µm coronal brain sections obtained from
male SD rats (300 g). The sections were thaw-mounted on precleaned silane-coated slides. The
brain sections were incubated with [99mTc]CPTT–A–E (10.1 MBq) in Tris-HCl buffer (pH
7.4) for 2 h at 4 °C. After washing with ice-cold Tris-HCl buffer (5.0 mL × 2) and ice-cold
water (1.0 mL × 1), the slides were placed on the Fuji Imaging Plates (BAS–SR, Fuji Photo
Film, Tokyo, Japan) for 24 h along with the calibrated 99mTc-labeled external standards.
Images were analyzed using a Bio Imaging Analyzer (BAS5000, Fuji Photo Film, Tokyo,
Japan) and the image gauge device (Fuji Photo Film, Tokyo, Japan).
4.7. In vivo biodistribution of [99mTc]CPTT–A–E
Biodistribution studies of [99mTc]CPTT–A–E were performed in seven-week-old male ddY
mice (20–25 g). [99mTc]CPTT–A–E (37 kBq) was administered to mice by tail vein injection.
Mice were sacrificed by exsanguination at 2, 5, 15, 30, 60, or 120 min after administration.
Blood and organs were removed and weighed, and the radioactivity was measured with an
auto-well gamma counter (Cobra Auto-Gamma Counter 5003, Packard). The levels of
radioactivity in tissue were expressed as percentages of injected dose per gram of tissue (%
ID/g).
4.8. Analysis of radioactivity in the brain
Male ddY mice (20–25 g) received 14 MBq [99mTc]CPTT–A–E by tail vein injection and
were decapitated 120 min after injection. Their brains were removed immediately and
homogenized in 1.0 ml of Tris-HCl buffer (pH 7.4) and 1.5 mL of acetonitrile. After
centrifugation at 1210 x g for 5 min at 4 °C, the precipitate was washed with 1.0 ml of
acetonitrile, and the wash was combined with the supernatant. The combined acetonitrile
extracts were evaporated and analyzed by HPLC on a COSMOSIL 5C18-AR-II device (4.6 ×
150 mm, Nacalai Tesque, Kyoto, Japan); further, they were eluted with the phosphate buffer
(pH 3.0) (MeOH = 60:40 at a flow rate of 1.0 mL/min; tR = 17 min for [99mTc]CPTT–A–E).
Acknowledgements
This work was partly supported by Practical Research for Innovative Cancer Control at the
Japan Agency for Medical Research and Development (AMED) and a Grant-in-Aid for
Scientific Research (A) from the Japan Society for the Promotion of Science, and a grant from
17

the Smoking Research Foundation.
Disclosure
The authors declare no competing financial interests.
18

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