Synthesis and biological evaluation of 18F labeled fluoro-oligo-ethoxylated 4-benzylpiperazine derivatives for sigma-1 receptor imaging

Article abstract of DOI:10.1016/j.bmc.2012.10.038

We report the synthesis and evaluation of a series of fluoro-oligo- ethoxylated 4-benzylpiperazine derivatives as potential σ₁ receptor ligands. In vitro competition binding assays showed that 1-(1,3-benzodioxol-5-ylmethyl)-4-(4-(2-fluoroethoxy)benzyl)piperazine (6) exhibits low nanomolar affinity for σ₁ receptors (K_i = 1.85 ± 1.59 nM) and high subtype selectivity (σ₂ receptor: K_i = 291 ± 111 nM; K_iσ₂/ K_iσ₁ = 157). [¹⁸F]6 was prepared in 30-50% isolated radiochemical yield, with radiochemical purity of >99% by HPLC analysis after purification, via nucleophilic ¹⁸F^- substitution of the corresponding tosylate precursor. The log D_{pH 7.4} value of [¹⁸F]6 was found to be 2.57 ± 0.10, which is within the range expected to give high brain uptake. Biodistribution studies in mice demonstrated relatively high concentration of radiotracers in organs known to contain σ₁ receptors, including the brain, lungs, kidneys, heart, and spleen. Administration of haloperidol 5 min prior to injection of [¹⁸F]6 significantly reduced the concentration of radiotracers in the above-mentioned organs. The accumulation of radiotracers in the bone was quite low suggesting that [¹⁸F]6 is relatively stable to in vivo defluorination. The ex vivo autoradiography in rat brain showed high accumulation of radiotracers in the brain areas known to possess high expression of σ₁ receptors. These findings suggest that [¹⁸F]6 is a suitable radiotracer for imaging σ₁ receptors with PET in vivo.

Full text of DOI:10.1016/j.bmc.2012.10.038

Bioorganic & Medicinal Chemistry 21 (2013) 215–222
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 18F labeled fluoro-oligo-ethoxylated
4-benzylpiperazine derivatives for sigma-1 receptor imaging
Xia Wang a, Yan Li a, Winnie Deuther-Conrad b, Fang Xie a, Xin Chen a, Meng-Chao Cui a, Xiao-Jun Zhang c,
Jin-Ming Zhang c, Jörg Steinbach b, Peter Brust b, Bo-Li Liu a, Hong-Mei Jia a,
⇑
a Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR China
b Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmacy/Department of Neuroradiopharmaceuticals, 04318 Leipzig, Germany
c Nuclear Medicine Department, Chinese PLA General Hospital, Beijing 100853, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and evaluation of a series of fluoro-oligo-ethoxylated 4-benzylpiperazine deriv-
atives as potential r1 receptor ligands. In vitro competition binding assays showed that 1-(1,3-ben-
zodioxol-5-ylmethyl)-4-(4-(2-fluoroethoxy)benzyl)piperazine (6) exhibits low nanomolar affinity for
Received 8 August 2012
Revised 11 October 2012
Accepted 23 October 2012
Available online 31 October 2012
r1 receptors (Ki = 1.85 1.59 nM) and high subtype selectivity (r2 receptor: Ki = 291 111 nM; Kir2
/
Ki
r
1 = 157). [18F]6 was prepared in 30–50% isolated radiochemical yield, with radiochemical purity of
>99% by HPLC analysis after purification, via nucleophilic 18FÀ substitution of the corresponding tosylate
precursor. The logDpH 7.4 value of [18F]6 was found to be 2.57 0.10, which is within the range expected
to give high brain uptake. Biodistribution studies in mice demonstrated relatively high concentration of
radiotracers in organs known to contain r1 receptors, including the brain, lungs, kidneys, heart, and
spleen. Administration of haloperidol 5 min prior to injection of [18F]6 significantly reduced the concen-
tration of radiotracers in the above-mentioned organs. The accumulation of radiotracers in the bone was
quite low suggesting that [18F]6 is relatively stable to in vivo defluorination. The ex vivo autoradiography
in rat brain showed high accumulation of radiotracers in the brain areas known to possess high expres-
sion of r1 receptors. These findings suggest that [18F]6 is a suitable radiotracer for imaging r1 receptors
with PET in vivo.
Keywords:
r1 Receptor
Fluoro-oligo-ethoxylated
4-Benzylpiperazine
F-18
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
eases, including brain disorders, tumors as well as heart
failure.3,6–9 Therefore, they represent potential targets for treat-
Sigma-1 (r1) receptors represent a distinct class of intracellular
ment of these diseases.3,6 Development of r1 receptor ligands
including agonists and antagonists could have wide therapeutic
applications, while development of specific radiotracers for
membrane proteins whose C-terminus possesses chaperone activ-
ity.1 They are predominantly expressed at the endoplasmic reticu-
lum (ER) in a variety of cells and organs. It has been reported that
r1 receptors translocate from the mitochondria-associated mem-
brane to the cell nucleus or cell membrane and regulate several
targets, including various ion channels, G-protein-coupled recep-
tors, lipids, and other signaling proteins by a direct protein–protein
interaction with another ER chaperone-binding immunoglobulin
protein/78 kDa glucose-regulated protein (BiP/GRP-78).1,2 It is
noteworthy that the chaperone activity can be manipulated by
synthetic or endogenous ligands or by cations such as Ca2+ in a
clear agonist-antagonist manner.1,3 More recent findings indicate
that r1 receptors are important regulators in cellular morphology
and neuronal plasticity.4,5 Although the molecular function of r1
receptors needs further elucidation, more and more evidence sug-
gests that the r1 receptors are linked to a number of human dis-
in vivo imaging of r1 receptors may provide useful diagnostic tools
for investigation of their pathophysiology.
Many potential radiotracers for imaging
r1 receptor expression
with PET and SPECT have also been reported in the past few years.
But until now, only a few tracers such as [11C]SA4503,10,11
[
18F]FPS,12 and [123I]TPCNE13 have been evaluated in human stud-
ies. However, [18F]FPS14 and [123I]TPCNE13 were found to have irre-
versible kinetics. Many receptor ligands have affinity for the
r
vesicular acetylcholine transporter (VAChT) and the emopamil
binding protein (EBP).15–18 SA4503 was reported to show affinities
for VAChT (Ki = 50.2 nM)15 and EBP (Ki = 1.72 nM).16 But
[
11C]SA4503 did not seem to bind to VAChT in the rat brain
in vivo.19 Recently, Toyohara et al. confirmed that the brain uptake
of [11C]SA4503 in mice was not blocked by high affinity EBP block-
ers tamoxifen (EBP Ki = 2.8 nM, Ki
zine (EBP Ki = 3.9 nM, Ki
1/KiEBP = 52).20 The newly developed
compound
18F]fluspidine showed weak affinity for VAChT
r1/KiEBP = 12) and trifluopera-
r
⇑
Corresponding author. Tel./fax: +86 10 58808891.
E-mail address: hmjia@bnu.edu.cn (H.-M. Jia).
[
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.038

216
X. Wang et al. / Bioorg. Med. Chem. 21 (2013) 215–222
(Ki = 1400 nM) and EBP (Ki = 211 nM).21 Selectivity toward these
2.2. Radiolabeling
binding sites should be taken into consideration when designing
new selective ligands, although the relationship between the
receptor and EBP has not been clearly elucidated.
r
The synthetic route of the tosylate precursors and the corre-
sponding 18F labeled radiotracers ([18F]6 and [18F]10) is given in
Scheme 2. Compound 11 and 13 were synthesized according to
the method reported in the literature.26,27 Compound 7 reacted
with compound 11 or 13 to provide the tosylate precursor 12
In our previous work, we have reported that [125I]BP-I showed
high binding affinity for r1 receptors and high selectivity to the
r2 receptor subtype in vivo.22 Since imaging with PET has many
advantages over SPECT such as higher detection sensitivity and
resolution, 18F labeled radiotracers for the neuroimaging of r1
receptors become more and more important with the increasing
number of PET centers.
or 14, respectively.
[
18F]Fluoride was transferred into the
Kryptofix 222/potassium carbonate solution, which is made up
by Kryptofix 222 and potassium carbonate in acetonitrile/water.
After the solvent was removed under a stream of nitrogen at
120 °C and the residue azeotropically dried three times with
1 mL of anhydrous acetonitrile, the tosylate precursor (12 or
14) dissolved in anhydrous acetonitrile was added to the reac-
tion tube. The reaction was carried out at 100 °C for 5 min.
After cooling to room temperature, the mixture was passed
Considering simple synthesis of 18F labeled radiotracers via
nucleophilic 18FÀ substitution of the corresponding tosylate pre-
cursor, we have synthesized three fluoro-oligo-ethoxylated 4-ben-
zylpiperazine ligands and measured their affinity for r1 receptors
by in vitro radioligand binding assays. Moreover, we synthesized
[
18F]6, determined its logD value, and evaluated its potential as a
through a Sep-Pak C18 cartridge to remove inorganic salts
putative PET tracer for imaging of r1 receptors by biodistribution
studies in mice and ex vivo autoradiography in rat.
including [18F]KF. The crude product was eluted and purified
by an isocratic semi-preparative radio-HPLC using a reverse-
phase column and mobile phase consisting of acetonitrile and
water (containing 10 mM NH4OAc) (80:20 v/v) at a flow rate
of 4 mL/min.
2. Results and discussion
2.1. Chemistry
In order to identify the radiotracer, [18F]6 and [18F]10 were co-
injected and co-eluted with the corresponding unlabeled com-
pounds, respectively. Their HPLC profiles using acetonitrile and
water (containing 10 mM NH4OAc) (80:20 v/v) at a flow rate of
1 mL/min are presented in Figure 1. The retention times of unla-
beled 6 and [18F]6 were observed to be 5.56 min and 5.60 min,
respectively. The retention times of unlabeled 10 and [18F]10 were
observed to be 5.23 min and 5.28 min, respectively. The difference
in retention time was in good agreement with the time lag, which
corresponds to the volume and flow rate within the distance be-
tween the UV and radioactivity detector of our HPLC system. After
purification by radio-HPLC, the radiochemical purity of the radio-
active products was higher than 99%. The isolated radiochemical
yields of [18F]6 and [18F]10 were 30–50% (n = 3) and 20–40%
(n = 3), respectively. The specific activity of the product was esti-
mated to be 1000 Ci/mmol.
The synthetic route of fluoro-ethoxylated 4-benzylpiperazine
derivatives is shown in Scheme 1. Compound 1 was synthesized
according to the method reported in the literature with minor
modification.23 p-Cresol reacted with 1-bromo-2-fluoroethane in-
stead of fluoroethyl tosylate to obtain compound 1. Compound 2,
3, 4 were synthesized based on the method reported in the litera-
ture.23–25 Alkylation of compound 5 with 2 provided compound 6.
Compound 5 reacted with 4-hydroxybenzaldehyde to get com-
pound 7. Compound 7 reacted with compound 4 or 2-(2-(2-chloro-
ethoxy)ethoxy)ethanol to provide compound 8 or compound 9,
respectively. Fluorination of Compound 9 with DAST (diethylamino
sulfur trifluoride) provided Compound 10, which was recrystal-
lized with the form of salt in methanol. The target compounds
(6, 8, and 10) were characterized by NMR, MS, and EA.
Scheme 1. Synthetic route of fluoro-ethoxylated 4-benzylpiperazine derivatives. Reagents and conditions: (a) 1-bromo-2-fluoroethane, K2CO3, acetone, 80 °C, 4 h; (b) NBS,
AIBN, CCl4, 90 °C, 3 h; (c) 4-toluene sulfonyl chloride, KOH, CH2Cl2, 0 °C to rt, 10 h; (d) TBAF, acetonitrile, 90 °C, 8 h; (e) 2, CH2Cl2, rt, 60 h; (f) (i) 4-hydroxybenzaldehyde, 1,2-
dichloroethane, rt, 2 h; (ii) NaBH(OAc)3, rt, 12 h; (g) 4, K2CO3, acetonitrile, 90 °C, 14 h; (h) 2-(2-(2-chloroethoxy)ethoxy)ethanol, DMF, K2CO3, 100 °C, 11 h; (i) (i) DAST, CHCl3,
À78 °C, 1 h; (ii) rt, 4 h; (j) ethanol, conc. HCl, rt, 1 h.

X. Wang et al. / Bioorg. Med. Chem. 21 (2013) 215–222
217
Scheme 2. Synthetic route of the tosylate precursors and the corresponding 18F labeled radiotracers. Reagents and conditions: (a) 7, K2CO3, CH3CN, 90 °C, 8 h; (b) 18FÀ,
Kryptofix 222, K2CO3, CH3CN, 100 °C, 5 min; (c) 4-toluene sulfonyl chloride, KOH, CH2Cl2, 0 °C to rt, 10 h.
Figure 1. (A) HPLC co-elution profiles of 6 and [18F]6, with retention time of 5.56 and 5.60 min, respectively. (B) HPLC co-elution profiles of 10 an [18F]10, with retention time
of 5.23 and 5.28 min, respectively.
Table 1
The apparent distribution coefficients of [18F]6 and [18F]10 were
Binding affinities of the fluoro-oligo-ethoxylated 4-benzylpiperazine derivatives
determined in a 1-octanol/0.05 mol LÀ1 sodium phosphate buffer
system at pH 7.4 as previously reported.28 At pH 7.4, the logD val-
ues of [18F]6 and [18F]10 were determined to be 2.57 0.10 and
2.22 0.06 (n = 3), respectively, which are within the range ex-
pected to give high brain uptake.
towards r1 and r2 receptorsa
Compound
Ki (r
1) (nM)
Ki (
r
2) (nM)
Ki(
r
2)/Ki(r1
)
6
8
1.85 1.59
40.7 22.8
505 120
0.85 0.91
0.80 0.14
4.95 1.74
291 111
666 106
1420 160
52.2 13.3
61.1 20.1
20.7 0.07
157
16.4
2.81
61.4
76.4
4.18
10
BP-Fb
BP-Ib
2.3. Biological studies
Haloperidol
2.3.1. In vitro radioligand competition studies
a
Values are means SD of three experiments performed in triplicate.
From Ref. 22.
b
r
1 and
r
2 receptor competition binding assays were performed
as previously reported.29 The
r
1 and 2 receptor affinities of fluoro-
r
oligo-ethoxylated 4-benzylpiperazine derivatives were determined
in competition experiments with the radioligands (+)-[3H]pentaz-
ocine (the mean working concentration: 2.0 nM) and [3H]ditolyl-
guanidine (the mean working concentration: 1.7 nM) using rat
brain and rat liver preparations, respectively. In vitro competition
binding assays showed that 6 exhibited low nanomolar affinity
for r1 receptors (Ki = 1.85 1.59 nM, Table 1) and high subtype
subtype selectivity. But substitution at the same position with in-
creased length of fluoro-oligo-ethoxylated group led to decreased
affinities for r1 receptors. With high affinity and high subtype
selectivity, compound 6 is a more promising r1 receptor ligand
compared to BP-F and BP-I.
2.3.2. Biodistribution and blocking studies in mice
selectivity
(r2 receptor: Ki = 291 111 nM; Kir2/Kir1 = 157,
In vivo biodistribution studies of [18F]6 and [18F]10 were
performed in male ICR mice. The values reflecting the uptake of
radiotracer in the organs of interest at 2, 15, 30, 60 and 120 min
after intravenous administration of [18F]6 or [18F]10 (0.1 mL,
Table 1). Compared to the previously reported BP derivatives, sub-
stituent at para-position of the benzyl moiety with small steric
group, such as F, I or OCH2CH2F, possessed low nanomolar affinity
for r1 receptors. Introducing OCH2CH2F group led to an increase of

218
X. Wang et al. / Bioorg. Med. Chem. 21 (2013) 215–222
Table 2
accumulation of radiotacer in the brain at 60 min p.i. which corre-
Biodistribution of [18F]6 in male ICR micea
sponded to values obtained with [18F]fluspidine.21 Moreover, the
concentration of radiotracer in organs known to possess r1 recep-
tors was significantly reduced with values of 47% determined in
heart, 52% in spleen, and 58% in lung. These data suggested that
Organ
2 min
15 min
30 min
60 min
120 min
Blood
Brain
Heart
Liver
Spleen
Lung
Kidney
Muscle
Stomachb
1.29 0.12 1.05 0.25 0.92 0.07 1.11 0.12 0.88 0.21
6.00 0.60 6.30 0.32 5.73 0.52 5.23 0.51 4.72 1.28
10.19 1.63 7.49 1.44 5.42 0.41 2.99 0.22 2.67 0.93
4.87 1.89 9.73 1.53 7.76 0.78 7.66 1.42 7.19 1.73
3.39 1.17 6.13 1.37 5.47 0.39 4.92 0.68 4.38 0.56
19.97 4.11 11.53 0.91 8.09 2.59 5.77 1.34 4.82 1.26
19.26 1.56 12.54 1.23 9.34 0.88 6.13 0.93 4.54 0.72
3.27 0.39 4.34 1.11 2.66 0.44 2.05 0.43 2.26 0.72
1.00 0.29 1.77 0.38 1.65 0.35 1.97 0.77 1.54 0.80
the binding of [18F]6 to
blocking studies of
(Table 5). No reduction of radiotracer accumulation in the above-
r
1 receptors was specific in vivo. The same
[
18F]10 was carried out for comparison
mentioned organs known to contain r1 receptors was found, which
was in accord with low affinity of [18F]10 for r1 receptors.
Small intestineb 7.47 0.72 7.94 1.97 8.35 1.29 8.00 1.77 8.57 5.71
Bone
Brain/blood
2.3.3. Ex vivo autoradiography
3.05 0.73 5.17 1.32 3.53 0.52 3.20 0.99 3.15 0.58
4.65 6.00 6.23 4.71 5.36
In order to investigate the radiotracer accumulation in different
brain regions, brain sections were evaluated by ex vivo autoradiog-
a
raphy at 60 min p.i. of [18F]6 (350
lCi/2 nmol) in rat (Fig. 2). The
Data are means of %ID/g of tissue SD, n = 5.
%ID/organ.
b
ex vivo autoradiography in rat brain showed high accumulation
of radiotracer in the brain areas known to possess high expression
of r1 receptors. For example, high accumulation of radiotracer was
4 lCi/0.02 nmol) are summarized in Table 2 and Table 3, respec-
tively. [18F]6 showed a high initial brain uptake, with slow
clearance and high brain-to-blood ratios. The radiotracer concen-
tration determined in the brain was highest at 15 min post-
injection (p.i.) with 6.30 0.32%ID/g, and slowly cleared thereafter
with 5.73 0.52%ID/g at 30 min, 5.23 0.51%ID/g at 60 min, and
4.72 1.28%ID/g at 120 min p.i. The values were higher than that
of [11C]SA4503 30 and [18F]fluspidine.21 In contrast to the high up-
take and retention in the brain, the blood radiotracer levels were
low with 1.05 0.25%ID/g at 15 min and 0.88 0.21%ID/g at
120 min p.i., resulting in high brain-to-blood ratios (6.00, 6.23,
4.71, and 5.36 at 15, 30, 60, and 120 min p.i., respectively).
Similar to what was found for [18F]fluspidine, high uptakes
(6.13–12.54%ID/g at 15 min p.i.) were observed in organs known
found in brain areas such as facial nucleus, cerebellum, cortex,
midbrain, hypothalamus, and hippocampus. Moderate accumula-
tion of radiotracer was found in the thalamus. Comparably low
accumulation of radiotracer was found in the striatum, nucleus
accumbens and olfactory bulb.
In order to confirm the specific binding of [18F]6 to
r1 receptors
ex vivo, the effects of preinjection of haloperidol (0.2 mL, 1.0
mg/kg) on the accumulation of radiotracer in the brain areas of
male SD rat were investigated. Administration of haloperidol
5 min prior to injection of
[
18F]6 significantly reduced the
concentration of radiotracer in the above-mentioned areas known
to possess high expression of r1 receptors. Therefore, the high
radioactive signals in corresponding distribution of r1 receptors
are specific.
to contain
r receptors, including the lungs, kidneys, heart, spleen
Currently, in vivo [11C]SA4503 imaging studies have shown that
and liver, followed by slow clearance over time. Different to what
was found for [18F]6, [18F]10 showed a high initial brain uptake,
with fast washout and low brain-to-blood ratios. Fast washout of
´
the density of r1 receptors was decreased in Parkinsons disease
(PD) and Alzheimer’s disease (AD) patients.10,11 This tracer could
be used as a probe of clinical studies in r1 receptors imaging in
the above brain diseases. However, use of [11C]SA4503 needs an
on-site cyclotron. Longer half-life 18F labelled ligand is more suit-
able for r1 receptors imaging. Unfortunately, there is lack of suit-
able 18F-labelled PET radiotracers for the neuroimaging of r1
receptors until now. The major problems of [18F]FPS, the only 18F
labeled r1 receptor radiotracer investigated in human studies,
are the irreversible kinetics and no suitable reference regions.14
In our previous work, based on the proposal that the possible
mechanisms of the treatment effect of sodium channel mediated
disease of BP-Br could be related to r1 receptors, we found that
BP derivatives possessed high affinity for r1 receptors. [125I]BP-I
showed specific binding to r1 receptors in vivo.22 Considering
radiotracer was also observed in the organs known to contain
receptors, which was in good agreement with low affinity of
18F]10 for r1 receptors. In addition, the accumulation of radio-
tracer in the bone at 120 min p.i. was quite low suggesting that
both
18F]6 and 18F]10 were relatively stable to in vivo
defluorination.
In order to verify the specific binding of [18F]6 to
r
[
[
[
r
receptors
in vivo, the effects of preinjection of haloperidol (0.1 mL, 1.0
mg/kg) on the biodistribution of radioactivity in various organs
of male ICR mice were examined. Either saline or haloperidol
was injected 5 min prior to the radiotracer injection. The results
of blocking studies at 60 min p.i. are given in Table 4. Of particular
interest was a significant reduction by 71% (p <0.001) of the
Table 3
Biodistribution of [18F]10 in male ICR micea
Organ
2 min
15 min
30 min
60 min
120 min
Blood
Brain
Heart
Liver
Spleen
Lung
Kidney
Muscle
Stomachb
Small intestineb
Bone
3.63 1.00
6.42 0.82
6.75 1.37
4.98 0.90
5.95 1.17
15.30 1.94
27.41 2.43
4.18 0.46
2.94 1.20
7.54 2.33
3.08 0.32
1.77
3.42 1.13
1.66 0.21
2.63 0.51
3.90 0.53
6.54 1.07
4.01 0.34
14.39 1.47
2.39 0.32
2.53 0.69
7.57 1.29
2.89 0.64
0.49
2.53 0.55
0.97 0.20
1.67 0.40
2.84 0.47
2.93 0.88
2.55 0.52
6.52 1.27
1.09 0.44
0.97 0.40
6.63 1.43
1.80 0.57
0.38
1.82 0.88
0.84 0.12
1.26 0.22
2.27 0.25
1.18 0.20
1.55 0.22
3.91 0.48
0.98 0.15
1.05 0.19
5.65 2.66
1.35 0.26
0.46
1.15 0.18
0.77 0.18
1.01 0.12
1.52 0.20
0.78 0.29
0.90 0.17
2.35 0.49
1.02 0.32
0.95 0.13
2.07 0.28
1.89 0.72
0.67
Brain/blood
a
Data are means of %ID/g of tissue SD, n = 5.
%ID/organ.
b

X. Wang et al. / Bioorg. Med. Chem. 21 (2013) 215–222
219
Table 4
mentioned areas, which confirmed that the high radioactive sig-
nals to the corresponding distribution of r1 receptors are specific.
Therefore, the encouraging results including high affinity and
selectivity towards r1 receptors, high brain uptake and specific
binding, as well as limited in vivo defluorination, warrant further
evaluation of [18F]6 as a putative tracer for imaging r1 receptors
with PET.
Effects of preinjection of haloperidol (0.1 mL, 1.0 mg/kg) 5 min prior to the injection
of [18F]6 on the biodistribution of radioactivity in male ICR micea
b
Organ
60 min
(Control)
60 min
(Blocking)
%
p
Blocking
Blood
Brain
Heart
Liver
Spleen
Lung
0.93 0.11
5.44 0.58
3.29 0.41
7.39 1.25
5.32 0.40
5.04 0.61
6.67 0.67
2.50 0.29
1.33 0.17
8.05 2.41
1.85 0.23
1.59 0.19
1.74 0.15
3.56 0.77
2.55 0.23
2.14 0.17
3.83 0.67
1.50 0.13
2.49 0.87
7.02 2.20
99
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.019
À71
À47
À52
À52
À58
À43
À40
87
As indicated in Introduction, many
r
receptor ligands have
affinity for the VAChT and the EBP.15–18 Although SA4503 was re-
ported to show affinities for VAChT15 and EBP,16
[
11C]SA4503 did
not seem to bind to VAChT19 and EBP20 in the rat brain in vivo.
Moreover, selectivity to the other neuroreceptors should be taken
into consideration when designing new selective ligands. For
Kidney
Muscle
Stomach
Small
c
À13
0.533
intestinec
example, Kawamura et al have demonstrated r1 receptor selectiv-
ity of [11C]SA4503 by treating with the other receptors selective li-
gands.30 For r1 receptor radiotracer applications in human brain,
appropriate brain kinetics and peripheral metabolism should also
be considered. The investigations including the selectivity to the
other neuroreceptors as well as the brain kinetics and peripheral
metabolism of [18F]6 are in progress.
bone
2.80 0.13
2.93 0.56
5
0.625
a
Data are means of %ID/g of tissue SD, n = 4–5.
p values for the control versus blocking group at 60 min p.i. calculated by
b
Student’s t test (independent, two-tailed).
c
%ID/organ.
Table 5
3. Conclusions
Effects of preinjection of haloperidol (0.1 mL, 1.0 mg/kg) 5 min prior to the injection
of [18F]10 on the biodistribution of radioactivity in male ICR micea
Fluoro-oligo-ethoxylated 4-benzylpiperazine derivative 6 has
been synthesized and evaluated as r1 receptor ligand with high
affinity and high subtype selectivity. [18F]6 has been obtained with
high radiochemical purity. The logD value of [18F]6 was within the
range expected to give excellent brain uptake. In biodistribution
studies [18F]6 was found to possess very high initial brain uptake
followed by slow clearance. The in vivo binding pattern of the tra-
cer was in good agreement with the known distribution of r1
receptors, and blocking studies confirmed high specific binding.
The ex vivo autoradiography in rat brain showed high accumula-
tion of radiotracer in the brain areas known to possess high expres-
sion of r1 receptors. These findings suggest further evaluation of
Organ
60 min
(Control)
60 min
(Blocking)
%
pb
Blocking
Blood
Brain
Heart
Liver
Spleen
Lung
1.25 0.11
0.75 0.12
1.22 0.26
2.01 0.17
0.95 0.39
1.07 0.15
3.22 0.76
0.90 0.18
1.08 0.43
5.01 1.74
2.16 0.31
1.13 0.13
1.62 0.34
2.57 0.24
1.51 0.28
1.87 0.23
3.60 0.55
1.39 0.38
1.17 0.34
7.57 1.69
73
51
33
28
59
75
12
54
8
<0.001
0.001
0.072
0.004
0.032
<0.001
0.385
0.029
0.730
0.062
Kidney
Muscle
Stomach
Small
c
51
intestinec
[
18F]6 as a suitable radiotracer for imaging r1 receptors with PET
Bone
1.17 0.39
1.60 0.24
37
0.091
a
in vivo.
Data are means of %ID/g of tissue SD, n = 4–5.
p values for the control versus blocking group at 60 min p.i. calculated by
b
Student’s t test (independent, two-tailed).
c
4. Experimental section
4.1. General information
%ID/organ.
higher detection sensitivity and resolution of imaging with PET
than that with SPECT, and simple synthesis of 18F labeled radiotra-
cers via nucleophilic 18FÀ substitution of the corresponding tosyl-
ate precursor, we synthesized and evaluated fluoro-oligo-
ethoxylated 4-benzylpiperazine ligands for r1 receptors. It is
encouraging that compound 6 exhibited low nanomolar affinity
All reagents and chemicals were purchased from commercial
suppliers and used without further purification unless otherwise
stated. Thin layer chromatography (TLC): Silica gel 60 F254 plates
(Merck). Flash column chromatography was conducted using silica
gel [45–75 lm]. The mobile phase used is reported in the experi-
for
r
1 receptors and higher subtype selectivity than BP-I (Table 1).
mental procedure for each compound. Melting point was recorded
on a SGW X-4 micro melting point apparatus (Shanghai precision
scientific instrument Co., LTD, China) and was uncorrected. 1H
NMR spectra were recorded on a Bruker Avance III (400 MHz)
NMR spectrometer. Chemical shift (d) are reported in ppm down-
field from tetramethylsilane and coupling constants (J) are re-
ported in Hertz (Hz).13C NMR spectra were recorded on a Bruker
Avance III (100 MHz) NMR spectrometer. MS spectra were ob-
tained by Quattro micro API ESI/MS (Waters, USA). Elemental anal-
yses (C, H, N) were obtained on an Elementar 240C (Perkin Elmer,
USA).
Generally speaking, proper lipophilicity of receptor ligand may be
responsible for high brain uptake and low non-specific binding.
The lipophilicity of [18F]6 (logDpH 7.4 = 2.57), which is lower than
that of [125I]BP-I (logDpH 7.4 = 2.98), is appropriate for a
r1 receptor
ligand. The radiotracer concentration determined in the brain after
intravenous administration of [18F]6 is higher than that of [125I]BP-
I. The brain uptake is also higher than that of [11C]SA4503
and
30
[
18F]fluspidine.21 In addition, the percentage of specific uptake in
the brain was 71% (60 min p.i.), which is comparable to that deter-
mined for [11C]SA4503 (about 70%),30 and higher than that of
[
125I]BP-I (63%)22 and [18F]fluspidine (62%).21 Third, the ex vivo
HPLC analyses were performed on a Waters 600 system (Waters
corporation, USA) equipped with Waters 2489 UV–VIS detector,
and Raytest Gabi NaI(Tl) scintillation detector (Raytest, Germany).
The sample was analyzed on a Agela Venusil MP C18 column
autoradiography at 1 h after intravenous injection of [18F]6 in rat
showed a high accumulation of radiotracer in brain areas known
to possess high expression of r1 receptors such as the facial nu-
cleus, cerebellum, cortex, midbrain, hypothalamus, and hippocam-
pus. Administration of haloperidol 5 min prior to injection of [18F]6
significantly reduced the concentration of radiotracer in the above-
(250 mm  4.6 mm, 5
lm) using acetonitrile and water (contain-
ing 10 mM NH4OAc) (80:20 v/v) as mobile phase at a flow rate of
1 mL/min. HPLC separation was carried out on a Shimadzu SCL-

220
X. Wang et al. / Bioorg. Med. Chem. 21 (2013) 215–222
Figure 2. Ex vivo autoradiograms of the rat coronal brain sections at 1 h after intravenous injection of [18F]6. (A) frontal cortex; (B) olfactory bulb; (C) cerebral cortex; (D)
nucleus accumbens; (E) striatum; (F) hippocampus; (G) thalamus; (H) hypothalamus; (I) midbrain; (J) cerebellum; (K) facial nucleus.
20 AVP HPLC system (Shimadzu Corporation, Japan) equipped with
a SPD-M20A UV–VIS detector operating at a wavelength of 254 nm,
extracted with CH2Cl2 (20 mL Â 3). The combined organic phases
were dried over Na2SO4 and concentrated under vacuum. The
crude product was purified by silica gel column chromatography
using ethyl acetate/petroleum ether (1/1, v/v) as the mobile phase
to afford 7 (Registry Number: 757195-64-5) as a white solid
(464.7 mg, 75%). ESI-MS, [M+H]+ (m/z = 327.4).
and a Bioscan Flow Count 3200 NaI/PMT
detector. The sample was separated on a Grace Allsphere ODS-2
m) using acetonitrile and water
c-radiation scintillation
column (250 mm  10 mm, 5
l
(containing 10 mM NH4OAc) (80:20 v/v) as mobile phase at a flow
rate of 4 mL/min.
Male ICR mice (5 weeks, 18–22 g) and male Spraque-Dawley
rats (9 weeks, 220 g) were purchased from Peking University
Health Science Center. All procedures of the animal experiments
were performed in compliance with relevant laws and institutional
guidelines. All of the animal experiments were approved by the
Institutional Animal Care and Use Committee of Beijing Normal
University.
4.4. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-(2-(2-
fluoroethoxy)ethoxy)benzyl)piperazine (8)
A mixture of 4 (58.1 mg, 0.22 mmol), 7 (92.0 mg, 0.28 mmol),
K2CO3 (30.61 mg, 0.22 mmol) and acetonitrile (5 mL) was heated
to 90 °C with stirring for 14 h. Then the reaction mixture was con-
centrated under vacuum and the crude product was dissolved in
CH2Cl2 (50 mL) and washed with water (50 mL). The organic layer
was dried over MgSO4 and concentrated under vacuum and puri-
fied by silica gel column chromatography using ethyl acetate/
petroleum ether/triethylamine (1/2/0.1, v/v/v) as the mobile phase
to afford 8 as a white solid (51.7 mg, 56%). 1H NMR(400 MHz,
CDCl3) d (ppm): 7.20 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H),
6.84 (s, 1H), 6.73 (s, 2H), 5.93 (s, 2H), 4.65 (t, J = 4.1 Hz, 1H), 4.53
(t, J = 4.1 Hz, 1H), 4.13 (t, J = 4.8 Hz, 2H), 3.88 (t, J = 4.8 Hz, 2H),
3.86 (t, J = 4.3 Hz, 1H), 3.78 (t, J = 4.1 Hz, 1H), 3.45 (s, 2H), 3.41 (s,
2H), 2.45 (br s, 8H). 13C NMR (100 MHz, CDCl3) d (ppm): 52.83,
52.89, 62.34, 62.70, 67.52, 69.98, 70.49, 70.69, 82.32, 84.00,
100.85, 107.83, 109.56, 114.37, 122.27, 130.25, 130.44, 131.99,
146.59, 147.61, 157.91. ESI-MS, [M+H]+ (m/z = 417.1). Mp: 80–
81 °C. Anal. Cacld for C23H29FN2O4: C 66.33, H 7.02, N 6.73. Found:
C 66.04, H 6.428, N 6.529.
4.2. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-(2-
fluoroethoxy)benzyl)piperazine (6)
1-Piperonylpiperazine (5, 35.0 mg, 0.16 mmol) was dissolved in
anhydrous CH2Cl2 (2 mL). Compound 2 (44.5 mg, 0.18 mmol) was
then added with stirring for over 60 h at room temperature. The
reaction mixture was concentrated under vacuum and purified
by silica gel column chromatography using ethyl acetate/petro-
leum ether/triethylamine (1/4/0.1, v/v/v) as the mobile phase to af-
ford 6 as a white solid (23.5 mg, 35%). 1H NMR (400 MHz, CDCl3) d
(ppm): 7.23 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 6.84 (s, 1H),
6.73 (s, 2H), 5.93 (s, 2H), 4.80 (t, J = 4.2 Hz, 1H), 4.69 (t, J = 4.2 Hz,
1H), 4.23 (t, J = 4.2 Hz, 1H), 4.16 (t, J = 4.2 Hz, 1H), 3.46 (s, 2H),
3.42 (s, 2H), 2.46 (br s, 8H). 13C NMR (100 MHz, CDCl3) d (ppm)
52.97, 62.37, 62.75, 67.04, 67.25, 81.10, 82.80, 100.83, 100.88,
107.81, 109.54, 114.34, 122.22, 130.44, 130.87, 132.13, 146.53,
147.58, 157.54. ESI-MS, [M+H]+ (m/z = 372.7). Mp: 98.4–99.6 °C.
Anal. Cacld for C21H25FN2O3: C 67.72, H 6.77, N 7.52. Found: C
67.63, H 6.875, N 7.215.
4.5. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-(2-(2-(2-
hydroxylethoxy)ethoxy)ethoxy)benzyl)piperazine (9)
Compound 7 (542.3 mg, 1.66 mmol) was dissolved in DMF
(6 mL), 2-(2-(2-chloroethoxy)ethoxy)ethanol (243 lL, 1.99 mmol)
4.3. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-
hydroxylbenzyl)piperazine (7)
and K2CO3 (688.9 mg, 4.98 mmol) were added. The reaction mix-
ture was heated to 100 °C with stirring for 11 h. Then the mixture
was concentrated under vacuum. The crude product was then dis-
solved in CHCl3 (30 mL), washed with water (30 mL), dried over
NaSO4, and purified by silica gel column chromatography using
ethyl acetate/petroleum ether (1/1, v/v) as the mobile phase to af-
ford 9 (280.0 mg, 37%). 1H NMR (400 MHz, CDCl3) d (ppm): 7.20 (d,
J = 8.5 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 6.84 (s, 1H), 6.73 (s, 2H), 5.93
(s, 2H), 4.12 (t, J = 4.8 Hz, 2H), 3.86 (t, J = 4.8 Hz, 2H), 3.73–3.69 (m,
6H), 3.63–3.60 (m, 2H), 3.46 (s, 2H), 3.42 (s, 2H), 2.46 (br s, 8H).
1-Piperonylpiperazine (5, 521.2 mg, 2.37 mmol) was dissolved
in 1,2-dichloroethane (15 mL), and 4-hydroxybenzaldehyde
(323.5 mg, 2.65 mmol) was added with stirring for 2 h at room
temperature. Then NaBH(OAc)3 (821.9 mg, 3.88 mmol) was added
into the reaction. This reaction mixture was stirred at room tem-
perature overnight. The mixture was washed with saturated NaH-
CO3 and concentrated under vacuum. The aqueous phase was

X. Wang et al. / Bioorg. Med. Chem. 21 (2013) 215–222
221
4.6. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-(2-(2-(2-
fluoroethoxy)ethoxy)ethoxy)benzyl)piperazine (10)
tal, using 18O(p,n)18F reaction. The radioactivity was collected on
the QMA cartridge (Waters) and the cartridge dried by airflow.
The 18F activity was then eluted using 1 mL of a Kryptofix 222/
potassium carbonate solution, which is made up of a mixture of
13 mg Kryptofix 222 and 1.1 mg potassium carbonate in acetoni-
trile/water (0.8 mL/0.2 mL). The solvent was removed under a
stream of nitrogen at 120 °C and the residue azeotropically dried
three times with 1 mL of anhydrous acetonitrile at 120 °C, followed
by adding 1 mL of the tosylate precursor 12 solution (1 mg/mL) in
anhydrous acetonitrile. The reaction mixture was kept at 100 °C for
5 min.
After cooling to room temperature, the mixture was passed
through a Sep-Pak C18 cartridge previously washed with 10 mL
ethanol and 10 mL water. After elution with 10 mL water, inor-
ganic salts including [18F]KF was separated from the product. The
crude product was recovered into a sample vial by slowly flushing
the cartridge with 2 mL of acetonitrile. The solvent was evaporated
by N2 and the residue was purified by an isocratic semi-preparative
radio-HPLC (Shimadzu SCL-20 AVP HPLC system, Grace Allsphere
Compound 9 (230.0 mg, 0.50 mmol) was dissolved in anhy-
drous CHCl3 (5 mL). DAST (132.5
l
L) was slowly added at À78 °C.
The mixture was stirring at À78 °C for 1 h and then at room tem-
perature for 4 h. Saturated NaHSO3 was added and was stirring
for 30 min. The aqueous phase was extracted with CHCl3
(20 mL Â 3). The combined organic phases were dried over MgSO4
and concentrated under vacuum. The crude product was purified
by silica gel column chromatography using ethyl acetate/petro-
leum ether/triethylamine (1/2/0.1, v/v/v) as the mobile phase to af-
ford 10 as a colorless oil (130.0 mg, 56%). Then the oil was
dissolved in ethanol (2 mL), concentrated hydrochloric acid was
added and the mixture was stirring for 1 h. The product was fil-
tered and recrystallized in methanol to afford 10Á2HCl as a white
crystal. 1H NMR (400 MHz, CDCl3) d (ppm): 7.20 (d, J = 8.4 Hz,
2H), 6.86 (s, 1H), 6.84 (s, 2H), 6.73 (s, 2H), 5.92 (s, 2H), 4.62 (t,
J = 4.2 Hz, 1H), 4.50 (t, J = 4.2 Hz, 1H), 4.12 (t, J = 4.9 Hz, 2H), 3.85
(t, J = 4.9 Hz, 2H), 3.80 (t, J = 4.2 Hz, 1H), 3.74–3.71 (m, 5H), 3.45
(s, 2H), 3.42 (s, 2H), 2.46 (br s, 8H). 13C NMR (100 MHz, CDCl3) d
(ppm): 52.89, 62.36, 62.72, 67.46, 69.85, 70.36, 70.56, 70.87,
82.30, 83.98, 100.84, 107.81, 109.57, 114.32, 122.24, 130.26,
130.39, 132.06, 146.55, 147.59, 157.94. ESI-MS, [M+H]+ (m/
z = 461.5). Anal. Cacld for C25H35Cl2FN2O5:C 56.29, H 6.61, N 5.25.
Found: C 56.34, H 6.696, N 4.795.
ODS-2, 5
l
m, 250 Â 10 mm; eluent: 80% CH3CN and 20% water
(containing 10 mM NH4OAc); flow: 4 mL/min). In order to identify
the radioactive product, the unlabeled 6 was co-injected and co-
eluted with the radioactive product (Waters 600 HPLC system,
Agela Venusil MP C18, 5
l
m, 250 mm  4.6 mm; eluent: 80%
CH3CN and 20% water (containing 10 mM NH4OAc); flow: 1
mL/min). To prepare a suitable solution of [18F]6, the eluted peak
corresponding to [18F]6 was collected, followed by removing of
the solvent. Solutions of [18F]6 in sterile saline containing no more
than 8% ethanol were used for animal studies. The same proce-
dures were applied to the synthesis, seperation and identification
of [18F]10.
4.7. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-(2-(4-
methylbenzenesulfonyloxy)ethoxy)benzyl)piperazine (12)
A mixture of 11 (1.04 g, 2.81 mmol), compound 7 (305.5 mg,
0.94 mmol), K2CO3 (132.8 mg, 0.96 mmol), and CH3CN (6 mL) was
heated to reflux for 8 h. The mixture was concentrated under vac-
uum and the residue was dissolved in CH2Cl2, washed with water,
dried over MgSO4 and concentrated under vacuum. The crude
product was purified by silica gel column chromatography and
recrystalized in ethanol to afford 12 (199.7 mg, 41%) as a white
crystal. 1H NMR (400 MHz, CDCl3) d (ppm): 7.82 (d, J = 8.3 Hz,
2H), 7.34 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 7.7 Hz, 2H), 6.84 (s, 1H),
6.73 (s, 3H), 6.71 (s, 1H), 5.93 (s, 2H), 4.36 (t, J = 4.8 Hz, 2H), 4.13
(t, J = 4.8 Hz, 2H), 3.44 (s, 2H), 3.42 (s, 2H), 2.45 (br s, 11H). ESI-
MS, [M+H]+ (m/z = 525.2). Mp: 95.2–96.6 °C.
4.10. Determination of logD value
The logD values of [18F]6 and [18F]10 were determined by mea-
suring the distribution of the radiotracer in
a 1-octanol/
0.05 mol LÀ1 sodium phosphate buffer system at pH 7.4. The two
phases were pre-saturated with each other. 1-octanol (3 mL) and
phosphate buffer (3 mL) were pipetted into a 10 mL plastic centri-
fuge tube. 100
18F]10 in ethanol was then added. The tube was vortexed for
5 min, followed by centrifugation for 5 min (3500 rpm, Anke
TDL80-2B, China). About 50 L of the 1-octanol layer was weighed
in a tared tube. The 1-octanol layer was removed and about 500
l lCi) or
L of a solution of HPLC-purified [18F]6 (20
[
l
4.8. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-(2-(2-(2-(4-meth-
ylbenzenesulfonyloxy)ethoxy)ethoxy)ethoxy)benzyl)
piperazine (14)
lL
of the buffer layer was weighed in a second tared tube. After add-
ing 0.50 mL buffer to the 1-octanol fraction and 0.05 mL of 1-octa-
nol to the aqueous fraction, activity in both tubes was measured in
A mixture of 13 (422.8 mg, 0.92 mmol), compound 7 (100.3 mg,
0.31 mmol), K2CO3 (51.3 mg, 0.37 mmol), and CH3CN (10 mL) was
heated to 90 °C with stirring for 8 h. The mixture was concentrated
under vacuum and the residue was dissolved in CH2Cl2, washed
with water, dried over MgSO4 and concentrated under vacuum.
The crude product was purified by silica gel column chromatogra-
phy using ethyl acetate/petroleum ether/triethylamine (1/1/0.1, v/
v/v) as the mobile phase to afford 14 as a colorless oil (83.9 mg,
43%). 1H NMR (400 MHz, CDCl3) d (ppm): 7.79 (d, J = 8.2 Hz, 2H),
7.32 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 6.4 Hz,
2H), 6.83 (s, 1H), 6.73 (s, 2H), 5.93 (s, 2H), 4.16 (t, J = 4.8 Hz, 2H),
4.09 (t, J = 4.8 Hz, 2H), 3.81 (t, J = 4.8 Hz, 2H), 3.69 (t, J = 4.8 Hz,
2H), 3.67–3.64 (m, 2H), 3.62–3.60 (m, 2H), 3.44 (s, 2H), 3.41 (s,
2H), 2.45–2.43 (m, 11H). ESI-MS, [M+H]+ (m/z = 613.2).
an automatic c-counter (Wallac 1470 Wizard, USA). Accurate vol-
umes of each counted phase were determined by weight and
known densities. The distribution coefficient was determined by
calculating the ratio of cpm/mL of 1-octanol layer to that of buffer
layer and expressed as logD. Samples from the 1-octanol layer
were re-distributed until consistent distribution coefficient values
were obtained. The measurement was carried out in triplicate and
repeated three times.
4.11. In vitro radioligand competition studies
r1 and r2 receptor competition binding assays were performed
as previously reported.29 Briefly, the r1 receptor assay was per-
formed using a rat brain membrane preparation as receptor mate-
rial and (+)-[3H]pentazocine (mean working concentration:
2.0 nM) as radioligand. The r2 receptor affinity was determined
using rat liver membrane preparation with the radioligand
[3H]ditolylguanidine (mean working concentration: 1.7 nM) in
4.9. Radiosynthesis of [18F]6 and [18F]10
[
18F]Fluoride was produced by the Sumitomo cyclotron HM-
20PS at Nuclear Medicine Department, Chinese PLA General Hospi-
the presence of 10 lM dextrallorphan to mask r1 binding sites.

222
X. Wang et al. / Bioorg. Med. Chem. 21 (2013) 215–222
Nonspecific binding was determined with 10 lM haloperidol. Ki
References and notes
values were calculated according to Cheng and Prusoff and repre-
sent data from at least three independent experiments, each per-
formed in triplicate. The results are given as mean standard
deviation (SD).
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p 60.05.
4.13. Ex vivo autoradiography
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Ex vivo autogradiography of the brain was carried out in male
Spraque-Dawley rats (9 weeks, 220 g), because of the large size
of the brain. The radioligand [18F]6 (350
lCi/2 nmol, 0.2 mL) was
intravenously injected into conscious rat via tail vein. For blocking
study, another conscious rat was injected with haloperidol (0.2 mL,
1.0 mg/kg) 5 min prior to radiotracer injection. The rat was killed
by cervical dislocation 1 h after injecton of the radioligand. The
brain was rapidly dissected, frozen, and coronally cut into 20 lm
thick sections using a cryotome (CM1900, Leica, Germany). The
brain sections were dried at room temperature and were exposed
to a phosphor plate (PerkinElmer, USA) for 2 h. Ex vivo autoradio-
graphic images were obtained using a phosphor imaging system
(CycloneÒPlus, PerkinElmer, USA).
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This work was supported by the National Natural Science Foun-
dation of China (No. 21071023), the Fundamental Research Funds
for the Central Universities, and Deutsche Forschungsgemeinschaft
(STE 601/10-3).