Novel cyclopentadienyl tricarbonyl 99mTc complexes containing 1-piperonylpiperazine moiety: Potential imaging probes for sigma-1 receptors

Article abstract of DOI:10.1021/jm5009488

We report the design, synthesis, and evaluation of a series of novel cyclopentadienyl tricarbonyl ^99mTc complexes as potent σ₁ receptor radioligands. Rhenium compounds 3-(4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl)propylcarbonylcyclopentadienyl tricarbonyl rhenium (10a) and 4-(4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl) butylcarbonylcyclopentadienyl tricarbonyl rhenium (10b) possessed high in vitro affinity for σ₁ receptors and moderate to high selectivity for σ₂ receptors and the vesicular acetylcholine transporter. Biodistribution studies in mice demonstrated high initial brain uptake for corresponding ^99mTc derivatives [^99mTc]23 and [ ^99mTc]24 of 2.94 and 2.13% injected dose (ID)/g, respectively, at 2 min postinjection. Pretreatment of haloperidol significantly reduced the radiotracer accumulation of [^99mTc]23 or [^99mTc]24 in the brain. Studies of the cellular uptake of [^99mTc]23 in C6 and DU145 tumor cells demonstrated a reduction of accumulation by incubation with haloperidol, 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine (SA4503), or 1,3-di-o-tolyl-guanidine (DTG). Furthermore, blocking studies in C6 glioma-bearing mice confirmed the specific binding of [^99mTc]23 to σ₁ receptors in the tumor.

Full text of DOI:10.1021/jm5009488

Article
pubs.acs.org/jmc
Novel Cyclopentadienyl Tricarbonyl 99mTc Complexes Containing
1‑Piperonylpiperazine Moiety: Potential Imaging Probes for Sigma‑1
Receptors
Xia Wang,† Dan Li,† Winnie Deuther-Conrad,‡ Jie Lu,† Ying Xie,§ Bing Jia,⊥ Mengchao Cui,†
Jorg Steinbach,‡ Peter Brust,‡ Boli Liu,† and Hongmei Jia*,†
̈
†Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal
University, Beijing 100875, China
‡Institute of Radiopharmaceutical Cancer Research/Department of Neuroradiopharmaceuticals, Helmholtz-Zentrum
Dresden-Rossendorf, 04318 Leipzig, Germany
§Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
⊥Department of Radiation Medicine, School of Basic Medical Sciences, Peking University, Beijing 100191, China
S
* Supporting Information
ABSTRACT: We report the design, synthesis, and evaluation
of a series of novel cyclopentadienyl tricarbonyl 99mTc
complexes as potent σ1 receptor radioligands. Rhenium
compounds 3-(4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl)-
propylcarbonylcyclopentadienyl tricarbonyl rhenium (10a)
and 4-(4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl)-
butylcarbonylcyclopentadienyl tricarbonyl rhenium (10b) pos-
sessed high in vitro affinity for σ1 receptors and moderate to
high selectivity for σ2 receptors and the vesicular acetylcholine
transporter. Biodistribution studies in mice demonstrated high
initial brain uptake for corresponding 99mTc derivatives [99mTc]
23 and [99mTc]24 of 2.94 and 2.13% injected dose (ID)/g,
respectively, at 2 min postinjection. Pretreatment of haloperidol
significantly reduced the radiotracer accumulation of [99mTc]23
or [99mTc]24 in the brain. Studies of the cellular uptake of [99mTc]23 in C6 and DU145 tumor cells demonstrated a reduction of
accumulation by incubation with haloperidol, 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine (SA4503), or 1,3-di-o-
tolyl-guanidine (DTG). Furthermore, blocking studies in C6 glioma-bearing mice confirmed the specific binding of [99mTc]23 to
σ1 receptors in the tumor.
Over the past decade, many PET and SPECT radiotracers for
σ1 receptors have been reported.17 However, no suitable
radiotracers for σ1 receptor imaging have been used in clinic to
date. Among the three radiotracers investigated thus far in
humans, [18F]1-(3-fluoropropyl)-4-((4-cyanophenoxy)methyl)-
piperidine ([18F]FPS) and (E)-[123I]1-(3-iodoallyl)-4-((4-
cyanophenoxy)methyl)piperidine ([123I]TPCNE) displayed
irreversible kinetics in the brain.18,19 [11C]1-(3,4-dimethox-
yphenethyl)-4-(3-phenylpropyl)piperazine ([11C]SA4503), the
first useful PET imaging agent, has been used for the evaluation
of σ1 receptor occupancy by therapeutic drugs in the human
brain.20−22 However, the use of [11C]SA4503 needs an on-site
cyclotron due to the short half-life of 11C (t1/2 = 20 min).
Currently, due to the widespread use of SPECT, the almost
ideal nuclear properties of 99mTc, and relatively low costs for
INTRODUCTION
■
The sigma-1 (σ1) receptor is a unique “ligand-operated receptor
chaperone”.1 It consists of 223 amino acids and is assumed to
contain two transmembrane segments.2,3 It is believed to be
involved in the regulation of various signal transduction
processes, endoplasmic reticulum (ER) stress, cellular redox
processes, cellular survival, and synaptogenesis in the central
nervous system (CNS).4−6 There is strong evidence for the
involvement of σ1 receptors in the pathophysiology of a
number of brain diseases, including drug addiction, depression,
Alzheimer’s disease (AD), and Parkinson’s disease (PD).4,5,7−11
Moreover, σ1 receptors are overexpressed in many human
tumors, and they play a significant role in cancer biology.12−16
Therefore, radiotracers with appropriate affinity and high
specificity for σ1 receptors could be useful in the early diagnosis
of various brain diseases and tumors using noninvasive imaging
techniques such as positron emission tomography (PET) and
single photon emission computed tomography (SPECT).
Received: June 22, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
SPECT imaging, 99mTc is the most widely used radionuclide,
employed in approximately 80% of all nuclear medicine
procedures. Thus, 99mTc-labeled radiotracers targeting σ1
receptors are expected to provide simple, convenient, and
low-cost diagnostic tools for the investigation of the σ1 receptor
status in a wide range of human diseases.
Recently, the neutral and lipophilic “piano stool” organo-
metallic core, [(Cp-R)M(CO)3] (M = 99mTc, Re), has become
particularly attractive due to its stability and compactness.30
Our previous work regarding σ2 receptor ligands demonstrated
that the replacement of the 1,2,3,4-tetrahydronaphthalene
group in 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydro-
naphthalen-1-yl)-propyl]piperazine (PB28) with the [(Cp-
R)M(CO)3] (M = 99mTc, Re) unit retained high affinity for
the target.28 Therefore, it seems reasonable to design novel
99mTc-labeled σ1 receptor-targeting radiotracers by using the
[(Cp-R)99mTc(CO)3] core. Previously, we reported the high
affinity (Ki(σ1) = 1.85 nM) and high subtype selectivity
(Ki(σ2)/Ki(σ1) = 157) of [18F]1-(1,3-benzodioxol-5-ylmethyl)-
4-(4-(2-fluoroethoxy)benzyl)piperazin ([18F]7) for σ1 recep-
tors in vitro as well as high initial brain uptake and specific
binding to σ1 receptors in vivo.31 In the present study, we used
[18F]7 as a lead compound to design novel [(Cp-R)M(CO)3]
(M = 99mTc, Re) complexes as potent σ1 receptor ligands
through an integrated approach. Our design concept is shown
in Figure 2. We replaced the benzyl moiety in compound 7
with the [(Cp-R)M(CO)3] unit and connected it to the 1-
piperonylpiperazine moiety via a proper linker. We introduced
a carbonyl or amide group to meet the requirement for an
electron-withdrawing group on the [(Cp-R)99mTc(CO)3]
substituent that would allow the double ligand transfer
(DLT) reaction to proceed efficiently for the synthesis of the
[(Cp-R)99mTc(CO)3] complex from the corresponding ferro-
cene precursor. In addition, the introduction of a carbonyl or
amide group could decrease the lipophilicity of the complex
and consequently decrease the nonspecific binding in vivo.
According to the pharmacophore model proposed by Glennon,
σ1 receptor ligands possess one basic nitrogen atom flanked by
two hydrophobic regions.32 This integrated strategy of
replacing a hydrophobic region with the lipophilic 99mTc-
chelate unit should minimize the change in molecular size and
might retain affinity for σ1 receptors. In this study, we report
the synthesis of novel [(Cp-R)M(CO)3] (M = 99mTc, Re)
complexes, and we evaluated them as potent σ1 receptor ligands
through biodistribution and blocking studies in mice, cellular
uptake studies in tumor cells, blocking studies in tumor-bearing
mice, and radiometabolite studies.
The basic requirements for a useful radiotracer for σ1
receptor imaging in the CNS include high binding affinity for
σ1 receptors and selectivity toward other potential targets, high
brain uptake, high specific binding to σ1 receptors, and low
nonspecific binding in vivo. If there is any radiometabolite in
the blood, it should not be able to cross the blood−brain
barrier. Unlike the 11C- or radiohalogen-labeled radiotracers,
99mTc-labeled imaging agents cannot be straightforwardly
derived from a suitable σ1 receptor ligand. 99mTc is a transition
metal and requires a chelate for complexation. A fundamental
challenge lies in the acceptable integration of 99mTc-chelate unit
into the σ1 receptor ligand. Moreover, the low brain uptake is
still the bottleneck in the development of 99mTc-labeled CNS
receptor radiotracers until now. To meet the above-mentioned
requirements for σ1 receptor radiotracers, three building blocks,
including a receptor-binding moiety, a small 99mTc-chelate unit,
and a suitable linker, need to be carefully considered for the
design of 99mTc-labeled radiotracers targeting σ1 receptors.23
The structures of 99mTc-labeled σ1 receptor radiotracers
developed recently are presented in Figure 1. Among these,
RESULTS
■
Chemistry. For the biological characterization of the 99mTc
complexes, we prepared the corresponding rhenium complexes
as shown in Scheme 1. Intermediates 8a−8c were synthesized
according to literature methods.28 Friedel−Crafts monoacyla-
tion of cyclopentadienyl tricarbonyl rhenium with bromide-
substituted anoyl chloride produced the corresponding
compounds 8a−8c in high yields. N-Alkylation of 1-
piperonylpiperazine (9) with 8a, 8b, and 8c provided 10a,
10b, and 10c, respectively.
Figure 1. Structures of 99mTc-labeled σ1 receptor radiotracers
developed recently.
no in vitro affinity data were reported for 3.24 Complexes 1,25
2,26 4,27 and 528 displayed micromolar affinity for σ1 receptors.
For compound 6, the subtype-independent binding affinity
estimated by [3H]1,3-di-o-tolyl-guanidine ([3H]DTG) displace-
ment studies was moderate (Ki = 42.7 nM).29 Thus, no
potential 99mTc-labeled σ1 receptor imaging agent has been
developed so far.
Compounds 12 and 13 were synthesized according to the
method reported previously.33 1-Piperonylpiperazine (9)
Figure 2. Design concept of novel [(Cp-R)M(CO)3] (M = 99mTc, Re) complexes as potent σ1 receptor ligands.
B
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthetic Routes for Rhenium Compounds 10a−10c and 16a−16c
a
Reagents and conditions: (a) anhydrous CH2Cl2, anhydrous AlCl3, 0 °C to rt, 71% for 8a, 75% for 8b, 87% for 8c; (b) toluene, KI, Et3N, 115 °C,
16% for 10a, 22% for 10b, 46% for 10c; (c) (i) anhydrous THF, −30 °C, (ii) 20% HCl, 14%; (d) Re2(CO)10, mesitylene, 190 °C, 95%; (e)
pentafluorophenyl trifluoroacetate, pyridine, DMF, N2, rt, 53%; (f) CH2Cl2, Et3N, rt, 43% for 14a, 74% for 14b, 70% for 14c; (g) THF, LiAlH4,
diethyl ether, 32% for 15a, 40% for 15b, 45% for 15c; (h) 13, anhydrous DMF, Et3N, rt, 40% for 16a, 92% for 16b, 61% for 16c.
reacted with bromine-substituted nitrile to provide the
corresponding 14a−14c. Reduction of 14a−14c using LiAlH4
afforded 15a−15c, respectively. The synthetic method for
compound 15a in this paper is different from that reported in
the literature.34 The activated ester 13 reacted with 15a, 15b, or
15c at room temperature to provide target complexes 16a, 16b,
or 16c, respectively.
receptors, thus giving them moderate subtype selectivity (9.70−
14.0) for σ1 receptors. The carbonyl-substituted complexes 10a,
10b, and 20 displayed very high σ1 binding affinities, with Ki
values ranging from 2.11 nM for 10a and 4.90 nM for 10b to
7.67 nM for 20. Moreover, these compounds displayed
moderate to low σ2 binding affinities. In particular, 10a
displayed comparable affinity and subtype selectivity for σ1
receptors to SA4503. 10b displayed comparable affinity and
even higher subtype selectivity (2-fold) for σ1 receptors to
SA4503. These results indicate that the nanomolar affinity and
moderate to high subtype selectivity for σ1 receptors could be
maintained by replacing the phenyl ring with a cyclopentadienyl
tricarbonyl rhenium unit or ferrocene (10a vs 7, 10b vs 7, 20 vs
7). However, if the number of carbon atoms between the
carbonyl group and the 1-piperonylpiperazine moiety was
increased to 5, decreased affinity and subtype selectivity for σ1
receptors was observed (10c vs10a, 10c vs10b), indicating that
the carbon linker length affected the affinity for σ1 receptors.
Considering the high affinity and subtype selectivity of
compounds 10a and 10b for σ1 receptors, both were further
tested for their affinity for VAChT. Compound 10a displayed
low affinity for VAChT (Ki = 1155 250 nM) and was thus
characterized by an excellent selectivity for σ1 receptors
(Ki(VAChT)/Ki(σ1) = 547). Complex 10b displayed moderate
The target compounds (10a−10c and 16a−16c) were
1
characterized by H NMR, 13C NMR, and high-resolution
mass spectrometry (HRMS) with the purities of ≥95% as
shown in the Supporting Information.
In Vitro Radioligand Competition Studies. The affinities
of the novel cyclopentadienyl tricarbonyl rhenium complexes
for the σ1 and σ2 receptors and for the vesicular acetylcholine
transporter (VAChT) were determined with radioligand
competition experiments as previously reported.27,35 The
binding assays used (+)-[3H]pentazocine for the σ1 receptors,
[3H]DTG in the presence of 10 μM dextrallorphan for the σ2
receptors, and (−)-[3H]vesamicol for VAChT. The results are
listed in Table 1. In general, cyclopentadienyl tricarbonyl
rhenium complexes and ferrocene precursors except for 10c
preferred to bind to σ1 receptors. The amide-substituted
complexes (16a−16c, 22) displayed moderate affinity for σ1
receptors with Ki values of 50.6−65.0 nM and low affinity for σ2
affinity for VAChT (Ki = 35.5
25.7 nM). To assess the
kinetics of the corresponding radiotracers, we prepared the
corresponding 99mTc-labeled complexes of 10a and 10b for
further evaluation.
Table 1. Binding Affinities of Cyclopentadienyl Tricarbonyl
Rhenium Complexes for σ1 and σ2 Receptors
a
Radiolabeling. The synthetic routes for the corresponding
ferrocene precursors were similar to those of the rhenium
complexes (Scheme 2). Acylation of ferrocene with 4-
bromobutanoyl chloride or 5-bromopentanoyl chloride pro-
vided compounds 17 and 18, respectively, according to the
method reported previously.36 Alkylation of 9 with 17 or 18
using triethylamine as a base yielded 20 or 21, respectively.
Ferrocenecarboxylic acid reacted with pentafluorophenyl
trifluoroacetate to obtain compound 19 in a synthesis different
from that reported in the literature.37 Condensation of 15a with
19 resulted in the ferrocene precursor 22.
compd
Ki (σ1) (nM)
Ki (σ2) (nM)
Ki(σ2)/Ki(σ1)
b
7
1.85 1.59
2.11 1.36
4.90 1.01
239 31.8
56.5 17.8
65.0 53.8
50.6 10.1
7.67 2.10
54.8 4.11
3.33 0.12
291 111
30.7 3.83
160 108
94.1 6.51
548 55.2
907 191
610 154
311 95.5
377 38.6
50.7 1.27
157
10a
10b
10c
16a
16b
16c
20
14.5
32.7
0.39
9.70
14.0
12.1
40.5
6.88
15.2
22
SA4503
The synthesis of [99mTc]23−25 was accomplished according
to published methods with minor modifications (Scheme
3).28,38 The formation of the desired 99mTc-labeled radiotracers
a
Values are means standard deviation (SD) of three experiments
performed in triplicate. From ref 31.
b
C
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 2. Synthetic Routes for Precursors 20−22
a
Reagents and conditions: (a) anhydrous CH2Cl2, anhydrous AlCl3, 0 °C to rt, 72% for 17, 79% for 18; (b) 9, toluene, KI, Et3N, 115 °C, 60% for 20,
62% for 21; (c) anhydrous DMF, pyridine, rt, 57%; (d) 15a, anhydrous DMF, Et3N, rt, 42%.
Scheme 3. Synthesis of [99mTc]23−25 from the
carbonyl group. However, the reverse-phase HPLC retention
a
times for 10a and 16 were 9.90 and 8.47 min (coinjection
HPLC profiles are shown in the Supporting Information),
respectively, indicating that the replacement of the carbonyl
group with an amide group decreased the lipophilicity of the
complex.
Corresponding Ferrocene Precursors
Biodistribution Studies in Mice and Blocking Studies.
Because 10a and 10b possess low nanomolar affinity for σ1
receptors, the kinetics of the corresponding [99mTc]23 and
[
99mTc]24 complexes were investigated in vivo by biodis-
tribution studies in male ICR mice. The respective data
obtained at 2, 15, 30, 60, 120, and 240 min postinjection in
various organs of interest are shown in Tables 2 and 3. [99mTc]
23 displayed high initial brain uptake (2.94 0.41% injected
dose (ID)/g at 2 min postinjection) with rather slow clearance
a
−
Reagents and conditions: (a) 99mTcO4 , Mn(CO)5Br, DMF, 140 °C,
−
50−60%; (b) 99mTcO4 , Mn(CO)5Br, DMF, 160 °C, 50−60%.
from the corresponding ferrocene precursors was accomplished
via the DLT reaction at 140 °C (or 160 °C) for 1 h. After
purification by semipreparative high performance liquid
chromatography (HPLC), [99mTc]23 (n = 6), [99mTc]24 (n =
3), and [99mTc]25 (n = 3) were obtained in approximately 50−
60% radiochemical yields (decay-corrected) with a radio-
chemical purity of >99%.
Evaluation of Radiolabeled Complexes. Lipophilicity.
The apparent distribution coefficients of 99mTc-labeled
complexes were determined by measuring their distribution
between 1-octanol and 0.05 mol·L−1 sodium phosphate buffer
at pH 7.4 as previously reported.39 The log D values of [99mTc]
(1.52
0.17% ID/g at 60 min postinjection) and relatively
high brain-to-blood ratios (3.03, 2.92, and 3.28 at 30, 60, and
240 min postinjection, respectively). The accumulation of
[
99mTc]23 in the brain peaked at 15 min postinjection with 3.25
0.38% ID/g. The lungs showed significantly high uptake at 2
min postinjection (35.08 8.01% ID/g), which considerably
decreased thereafter reaching 6.77 0.91% ID/g at 30 min
postinjection. The radiotracer accumulation in the thyroid was
very low at 240 min postinjection, indicating that no
pertechnetate was formed in vivo.
[
99mTc]24 also displayed high initial brain uptake (2.13
0.24% ID/g at 2 min postinjection). The blood clearance rate
of [99mTc]24 was faster than that of [99mTc]23, resulting in
higher brain-to-blood ratios (3.56, 4.37, 4.15, and 4.35 at 30, 60,
120, and 240 min postinjection, respectively). High initial
23−25 are 2.24
0.02, 2.68
0.01, and 3.09
0.02,
respectively (n = 3). It is interesting that by the shake-flask
method, the log D value of [99mTc]25 containing an amide
group is much higher than that of [99mTc]23 containing a
a
Table 2. Biodistribution of [99mTc]23 in Male ICR Mice
organ
blood
2 min
15 min
30 min
60 min
120 min
240 min
1.88 0.30
2.94 0.41
10.20 1.40
10.00 1.25
5.03 0.45
35.08 8.01
19.27 2.08
5.54 0.83
1.73 0.22
2.94 0.76
0.26 0.04
1.56
0.99 0.14
3.25 0.38
3.93 0.62
17.95 2.25
6.41 0.99
9.58 2.60
16.99 1.95
10.52 1.92
2.89 0.37
3.06 1.50
0.24 0.14
3.28
0.74 0.02
2.24 0.35
2.81 0.55
21.00 1.22
4.95 0.46
6.77 0.91
16.40 1.36
12.52 1.58
2.47 0.44
1.55 0.26
0.25 0.04
3.03
0.52 0.08
1.52 0.17
1.87 0.31
19.33 1.67
3.20 0.50
4.88 1.36
13.88 1.28
13.35 1.37
2.64 0.22
1.12 0.41
0.14 0.04
2.92
0.52 0.07
1.30 0.24
1.79 0.32
22.30 1.19
2.24 0.22
4.30 0.86
15.05 1.33
16.59 2.00
2.08 0.48
0.81 0.08
0.16 0.03
2.50
0.32 0.04
1.05 0.16
1.31 0.21
20.31 2.90
1.52 0.30
3.44 0.83
12.94 1.35
9.75 1.24
1.51 0.25
0.61 0.13
0.15 0.04
3.28
brain
heart
liver
spleen
lung
kidney
small intestine
b
b
stomach
muscle
b
thyroid
brain/blood
a
b
Data are expressed as percentage of injected dose per gram, mean SD, n = 5. Percentage of injected dose per organ.
D
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 3. Biodistribution of [99mTc]24 in Male ICR Mice
organ
blood
2 min
15 min
30 min
60 min
120 min
240 min
2.69 0.35
2.13 0.24
12.51 1.27
10.85 1.78
5.86 2.25
45.40 6.66
19.58 1.85
3.79 0.79
1.10 0.10
4.69 0.81
0.10 0.03
0.79
0.95 0.20
2.03 0.22
5.04 0.97
18.76 3.69
10.34 1.82
17.51 3.55
18.23 1.51
8.42 3.62
2.01 0.11
2.30 0.16
0.10 0.01
2.14
0.52 0.02
1.85 0.18
3.91 0.39
21.71 2.10
10.97 1.57
11.31 1.92
17.26 1.75
16.49 0.84
3.04 0.67
2.01 0.21
0.08 0.01
3.56
0.35 0.03
1.53 0.13
2.53 0.25
21.73 2.49
9.44 0.92
8.62 1.01
13.91 1.39
11.21 2.99
1.99 0.25
1.39 0.15
0.07 0.01
4.37
0.27 0.02
1.12 0.21
1.44 0.22
20.90 2.06
6.44 0.69
5.61 1.34
11.43 0.95
14.30 4.07
1.56 0.11
1.06 0.31
0.07 0.01
4.15
0.23 0.05
1.00 0.19
1.55 0.27
21.83 3.58
6.37 0.54
4.88 0.91
10.99 1.31
12.17 6.38
2.36 0.79
0.77 0.09
0.09 0.01
4.35
brain
heart
liver
spleen
lung
kidney
small intestine
b
b
stomach
muscle
b
thyroid
brain/blood
a
b
Data are expressed as percentage of injected dose per gram, mean SD, n = 5. Percentage of injected dose per organ.
Table 4. Effects of Preinjection of Haloperidol (0.1 mL, 1.0 mg/kg) 5 min Prior to Radiotracer Injection on the Biodistribution
a
of [99mTc]23 in Male ICR Mice
c
c
organ
blood
60 min (control)
60 min (blocking)
% blocking
p
120 min (control)
120 min (blocking)
% blocking
p
0.52 0.08
1.52 0.17
1.87 0.31
19.33 1.67
3.20 0.50
4.88 1.36
13.88 1.28
13.35 1.37
2.64 0.22
1.12 0.41
0.14 0.04
0.55 0.03
0.82 0.08
1.50 0.27
21.04 3.65
1.74 0.28
4.01 0.90
13.73 1.20
18.38 2.02
4.49 0.69
0.54 0.07
0.14 0.03
6
−46
−20
9
0.444
<0.001
0.076
0.366
<0.001
0.268
0.855
0.002
<0.001
0.015
0.931
0.52 0.07
1.30 0.24
1.79 0.32
22.30 1.19
2.24 0.22
4.30 0.86
15.05 1.33
16.59 2.00
2.08 0.48
0.81 0.08
0.16 0.03
0.52 0.05
0.80 0.17
1.26 0.23
23.73 2.44
1.26 0.33
3.39 0.92
14.60 2.29
16.29 3.02
2.75 1.00
0.34 0.28
0.16 0.04
0
−38
−30
6
0.942
0.005
0.017
0.271
<0.001
0.144
0.714
0.859
0.211
0.007
0.991
brain
heart
liver
spleen
lung
−46
−18
−1
38
−44
−21
−3
−2
32
kidney
small intestine
b
b
stomach
70
muscle
−52
0
−58
0
b
thyroid
a
b
c
Data are expressed as percentage of injected dose per gram, mean SD, n = 5. Percentage of injected dose per organ. p values for the control
versus blocking group at 60 and 120 min postinjection calculated by Student’s t test (independent, two-tailed).
uptake and fast clearance from the lungs were also observed for
99mTc]24 (45.40 6.66 and 11.31 1.92% ID/g at 2 and 30
min postinjection, respectively). The radiotracer accumulation
in the thyroid was very low at 240 min postinjection, indicating
that no pertechnetate was formed in vivo.
Table 5. Effects of Preinjection of Haloperidol (0.1 mL, 1.0
[
mg/kg) 5 min Prior to Radiotracer Injection on the
a
Biodistribution of [99mTc]24 in Male ICR Mice
60 min
60 min
%
c
organ
blood
(control)
(blocking)
blocking
p
To detect specific binding of [99mTc]23 in vivo, blocking
studies using haloperidol as the blocking agent were conducted.
Haloperidol (1.0 mg/kg) was injected 5 min prior to the
radiotracer injection. The blocking results at 60 and 120 min
postinjection are shown in Table 4. A significant reduction of
the radiotracer accumulation was observed in the brain (46%
and 39% at 60 and 120 min postinjection, respectively).
Moreover, radiotracer accumulation in the spleen, which is
known to possess high levels of σ1 receptors, was significantly
reduced by 46% (p < 0.001). These results demonstrated the
specific binding of [99mTc]23 to σ1 receptors in vivo. The
results obtained from the blocking studies with [99mTc]24 at 60
min postinjection are shown in Table 5. As radiotracer
accumulation in the brain was also significantly reduced
(43%, p < 0.001) by haloperidol pretreatment, the specific
binding of [99mTc]24 to σ1 receptors in the brain was
suggested.
0.35 0.03
1.53 0.13
2.53 0.25
21.73 2.49
9.44 0.92
8.62 1.01
13.91 1.39
11.21 2.99
1.99 0.25
1.39 0.15
0.07 0.01
0.39 0.03
0.87 0.08
2.14 0.31
22.52 2.25
10.45 2.14
8.94 1.50
13.21 1.70
13.85 5.08
3.65 0.71
1.43 0.24
0.10 0.01
11
−43
−15
4
0.034
<0.001
0.060
0.614
0.361
0.698
0.493
0.346
0.003
0.738
0.022
brain
heart
liver
spleen
lung
11
4
kidney
small intestine
−5
24
83
3
b
b
stomach
muscle
b
thyroid
43
a
Data are expressed as percentage of injected dose per gram, mean
b
c
SD, n = 5. Percentage of injected dose per organ. p values for the
control versus blocking group at 60 min postinjection calculated by
Student’s t test (independent, two-tailed).
and M6 were detected at retention times of 2.36, 3.78, 4.60,
6.30, 7.59, and 8.96 min, respectively. In plasma samples, the
parent radiotracer [99mTc]23 accounted for 48% of the total
activity. Two less lipophilic metabolites, M1 (39%) and M2
(13%), were detected at retention times of 3.83 and 4.49 min,
respectively. In brain samples, the parent compound [99mTc]23
accounted for 94% of the total activity, and only small amounts
In Vivo Stability of [99mTc]23. The metabolism of [99mTc]23
was investigated in brain, plasma, and liver samples from male
ICR mice at 15 min postinjection (33 MBq, 0.15 mL). The
HPLC chromatograms of [99mTc]23 are shown in Figure 3. In
the liver extract, 63% of the total radioactivity corresponded to
[
99mTc]23. Less lipophilic metabolites M5, M1, M2, M3, M4,
E
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. Analytical HPLC chromatograms of radioactive compounds in mouse plasma, brain, and liver samples at 15 min after intravenous injection
of [99mTc]23 (33 MBq, 0.15 mL).
of two less lipophilic radiometabolites were detected at
retention times of 6.30 min (M3, 2% of total activity) and
7.60 min (M4, 4% of total activity), respectively. It is interesting
that the small amount of the two less lipophilic radio-
metabolites detected in the brain are different from those in
plasma samples, suggesting that radiometabolites in the blood
do not enter the brain.
of the radiotracer remained at almost the same level (saline,
1.71 0.24% ID/g; cyclosporine A, 1.90 0.21% ID/g at 2
min postinjection). These data suggested that [99mTc]23 may
be a substrate for P-gp.
Uptake of [99mTc]23 in C6 and DU145 Tumor Cell Lines. In
recent years, different levels of σ1 receptor expression were
found in various tumor cell lines.13−15 For further character-
ization of the potential applications of [99mTc]23 for imaging σ1
receptors in tumors, in vitro cell uptake studies were performed
in C6 and DU145 cell lines according to the methods reported
in the literature.28
Effect of P-gp on the Brain Uptake of [99mTc]23 in Mice. It
is well-known that the blood−brain barrier expresses high levels
of permeability-glycoprotein (P-gp). The function of P-gp is
inversely related to the brain uptake of its substrate.40 To
identify whether [99mTc]23 is a substrate for P-gp at the
blood−brain barrier in vivo, biodistribution studies with
cyclosporine A, an inhibitor of P-gp, were performed. The
results are shown in Figure 4. Administration of cyclosporine A
60 min prior to [99mTc]23 injection considerably increased the
initial brain uptake of the radiotracer from 2.71 0.60 to 4.40
0.61% ID/g at 2 min postinjection. The blood accumulation
The uptake of [99mTc]23 in C6 glioma cells is shown in
Figure 5. An increase in the radiotracer uptake was observed
with longer incubation time. The percentage uptake was 2.98,
3.83, and 4.05% after incubation at room temperature for 15,
30, and 60 min, respectively. Blocking studies were conducted
with haloperidol, DTG, and SA4503 as blocking agents. The
blocking effects on [99mTc]23 uptake in C6 glioma cells are
Figure 4. Effect of P-gp on brain uptake of [99mTc]23 in mice.
Student’s t test (independent, two-tailed) was performed, p < 0.01 for
brain.
Figure 5. In vitro uptake of [99mTc]23 in C6 glioma cells.
F
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 6. In vitro blocking results of [99mTc]23 in tumor cells. (A) [99mTc]23 in C6 glioma cells with various inhibitors at different incubation times.
(B) [99mTc]23 in DU145 prostate cancer cells with different concentrations of SA4503 at 60 min.
Table 6. Effects of Preinjection of Haloperidol (1.0 mg/kg) or DTG (3 μmol/kg) 5 min Prior to Radiotracer Injection on the
a
Biodistribution of [99mTc]23 in C6 Glioma-Bearing Mice
c
d
c
d
organ
blood
control
haloperidol
% blocking
p
control
DTG
% blocking
p
0.34 0.04
1.37 0.11
1.90 0.30
14.09 1.25
3.82 0.46
5.32 1.67
15.86 0.49
16.66 2.20
3.70 1.00
0.63 0.13
2.18 0.37
3.47
0.37 0.02
0.68 0.13
1.00 0.04
15.21 1.90
2.02 0.06
3.22 0.75
15.80 3.01
17.66 2.41
4.21 0.73
0.38 0.06
1.16 0.10
3.05
10
−50
−47
8
0.158
<0.001
<0.001
0.320
<0.001
0.054
0.967
0.539
0.423
0.009
0.001
0.34 0.04
1.37 0.11
1.90 0.30
14.09 1.25
3.82 0.46
5.32 1.67
15.86 0.49
16.66 2.20
3.70 1.00
0.63 0.13
2.18 0.37
3.47
0.29 0.07
0.96 0.26
0.93 0.20
13.15 2.36
2.18 0.92
3.23 1.04
13.15 1.64
17.57 5.15
2.59 0.55
0.35 0.09
1.33 0.10
3.77
−14
−30
−51
−7
−43
−39
−17
5
0.227
0.015
<0.001
0.465
0.010
0.066
0.009
0.732
0.088
0.008
0.003
brain
heart
liver
spleen
−47
−40
0
lung
kidney
b
small intestine
stomach
muscle
6
14
−30
−44
−39
−39
−47
tumor
tumor/muscle
tumor/blood
6.39
3.10
6.39
4.55
a
b
c
Data are expressed as percentage of injected dose per gram, mean SD, n = 5. Percentage of injected dose per organ. %blocking = (blocking−
d
control)/control ×100%. p values for the control versus blocking group at 60 min postinjection calculated by Student’s t test (independent, two-
tailed).
time- and dose-dependent (Figure 6A). After a 60 min
coincubation with 2, 10, and 20 μM haloperidol, the uptake
was significantly reduced by 43%, 65%, and 69%, respectively. A
similar, strong reduction of cell uptake was found by treatment
with SA4503 at the same doses (31%, 42%, and 54%
inhibition), but the effects were considerably milder with
DTG (24−41% inhibition) due to the moderate affinity of
DTG for σ1 receptors. Among these blocking agents, SA4503
showed high affinity and selectivity for σ1 receptors. Thus,
blocking studies of [99mTc]23 in DU145 prostate cells were
performed with SA4503 as the blocking agent at 60 min.
Furthermore, a dose-dependent blocking effect of SA4503 on
A strong reduction of radiotracer accumulation was observed in
organs known to contain σ1 receptors, such as the heart, spleen,
and lung; this result is in good agreement with the results of
blocking studies in normal mice.
DISCUSSION
■
The σ1 receptor belongs to a unique family of proteins related
to a number of CNS diseases.4,5,11 PET or SPECT neuro-
imaging with radiotracers possessing appropriate affinity and
high specificity for σ1 receptors provides an important tool to
investigate the pathophysiology of CNS diseases.9,41 There are
some candidates, such as [11C]SA4503, that are available as
PET imaging agents for σ1 receptors. However, few 99mTc-
labeled radiotracers are suitable as SPECT imaging agents for
σ1 receptors. Formidable challenges, such as low brain uptake,
hinder the development of 99mTc-based CNS receptor imaging
agents. In our previous work, we found that 1-(1,3-
benzodioxol-5-ylmethyl)-4-(4-bromobenzyl)piperazine (BP-
Br) and its analogues, such as 1-(1,3-benzodioxol-5-ylmeth-
yl)-4-(4-iodobenzyl)piperazine (BP-I), possessed subnanomo-
lar affinity and high subtype selectivity for σ1 receptors.42 Later,
a 2-fluoroethoxy group was introduced at the para-position of
the benzyl moiety resulting in compound 7, which retained
nanomolar affinity and showed even higher subtype selectivity
for σ1 receptors. Moreover, the radioligand [18F]7 exhibited
high initial brain uptake and specific binding to σ1 receptors in
vivo.31 Recently, we reported a simpler method for synthesizing
[
99mTc]23 uptake in DU145 prostate cancer cells was shown
(Figure 6B). These data demonstrated that [99mTc]23 binds
specifically to σ1 receptors in C6 glioma cells and DU145
prostate cancer cells.
Blocking Studies in C6 Glioma-Bearing Mice. To
investigate the specific binding in tumors in vivo, blocking
studies of [99mTc]23 biodistribution in C6 glioma-bearing mice
were performed using haloperidol (1.0 mg/kg) and DTG (3
μmol/kg) as blocking agents. The blocking results are shown in
Table 6. Administration of haloperidol and DTG significantly
decreased the accumulation of [99mTc]23 in the tumors by 47%
and 39%, respectively, at 60 min, suggesting the specific binding
of [99mTc]23 to σ1 receptors in solid tumors. Similar to what
was found for biodistribution in normal mice, brain uptake was
also decreased by 50% with haloperidol and by 30% with DTG,
reflecting different affinities of these inhibitors for σ1 receptors.
G
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
[(Cp-R)M(CO)3] (M = 99mTc, Re) complexes under milder
reaction conditions and with higher yield compared to the
widely reported DLT reaction.28 The 99mTc-labeled selective σ2
receptor radioligand developed in our recent work showed
relatively high initial brain uptake and specific binding to the σ
receptors in brain tumors.28 These results encouraged us to
concentrate on the further exploration of 99mTc-labeled probes
for σ1 receptor imaging in vivo. In this current study, we used
[18F]7 as the lead compound to develop 99mTc-labeled
radiotracers for the SPECT imaging of the σ1 receptors using
an integrated approach. Because the linker may significantly
affect receptor binding and brain uptake, we replaced the benzyl
moiety carrying a [(Cp-R)M(CO)3] unit with an electron-
withdrawing group and connected the [(Cp-R)M(CO)3] unit
to a 1-piperonylpiperazine moiety via different carbon chain
lengths. The designed complexes still possess two basic
nitrogen atoms flanked by two hydrophobic regions in
accordance with the pharmacophore model proposed by
Glennon.32
To meet the first and possibly the most easily achieved
criterion, the rhenium surrogates were synthesized to examine
whether the complexes containing the [(Cp-R)M(CO)3] unit
retained binding affinity for σ1 receptors. In vitro binding assays
showed that compounds 10a and 10b exhibited comparable
low nanomolar affinity and comparable or improved subtype
selectivity for σ1 receptors relative to SA4503. Although several
rhenium complexes were developed as σ1 receptor ligands in
the past decades, few showed nanomolar affinities.24−29 The
retained high affinity and subtype selectivity of 10a and 10b for
σ1 receptors showed that the integrated strategy of assembling
the molecular structure of σ1 receptor ligands reported in this
paper was successful. Moreover, 10a displayed low affinity for
VAChT and thus high σ1/VAChT selectivity.
to the investigation of 99mTc-based CNS receptor ligands.
However, [99mTc]TRODAT-1 is the only 99mTc-based CNS
receptor radiotracer used in clinic, and it still suffers from
relatively low brain uptake.44 The low brain uptake remains the
bottleneck in the development of 99mTc-based CNS receptor
radiotracers, leading to decreasing interest in this laborious
field. The high accumulation of [99mTc]23 in the brain, together
with the nanomolar affinity for σ1 receptors, brightens the
future for 99mTc-based CNS receptor radiotracers.
To measure the specific binding of radiotracers to σ1
receptors in vivo, we used haloperidol, the most widely used
σ1 antagonist, as a blocking agent to perform the blocking
studies in vivo. Pretreatment of mice with haloperidol (1.0 mg/
kg) significantly reduced the accumulation of radiotracer in the
brain (46% and 43% reduction for [99mTc]23 and for [99mTc]
24, respectively, at 60 min postinjection). Although the
percentages of inhibition were lower than that of [18F]7
(71% reduction at 60 min postinjection), these data still
suggested that the accumulation of [99mTc]23 and [99mTc]24
could represent specific binding to σ1 receptors in vivo.
For the development of a brain radiotracer, it is very
important to investigate the metabolic profile of radioligands in
vivo. If radiometabolites could enter the brain, the binding
signals in the brain would be influenced. Therefore, the in vivo
metabolism of [99mTc]23 in the mouse brain, plasma, and liver
samples was studied. The results obtained at 15 min
postinjection showed that the parent compound [99mTc]23
was the main radioactive species present in the mouse brain
(94%). The two radiometabolites in the blood were different
from those in the brain, indicating no entry of radioactive
metabolites into the brain through blood.
Because of the high expression of P-gp at the blood−brain
barrier, the identification of a brain radiotracer as a substrate of
P-gp is currently an important issue.45 To further investigate
the identification of [99mTc]23 as a substrate of P-gp in vivo, we
performed biodistribution studies at 2 min postinjection of the
radiotracer using cyclosporine A as a blocking agent. P-gp
inhibition by cyclosporine A increased the brain uptake of
To find radiotracers with suitable kinetic profiles, we
prepared the corresponding 99mTc-labeled complexes [99mTc]
23 and [99mTc]24 of surrogates 10a and 10b for in vitro and in
vivo studies. Among the in vitro properties, suitable lipophilicity
is important for adequate blood−brain barrier permeability.
The log D values of [99mTc]23 and [99mTc]24 were within the
optimal range (2−3) for high brain uptake.43 In addition,
[
99mTc]23 approximately 1.6-fold without changing the radio-
tracer concentration in the blood, indicating that [99mTc]23 is
likely a substrate of P-gp. Thus, the effects of P-gp on the brain
uptake need to be considered for the evaluation of CNS
receptor-based radiotracers.
[
99mTc]23 and [99mTc]24 displayed high in vitro stability at
room temperature in saline.
Encouraged by the in vitro properties, we performed in vivo
biological experiments to evaluate the 99mTc-labeled complexes
as potential SPECT radiotracers. In biodistribution studies in
male mice, both [99mTc]23 and [99mTc]24 exhibited high initial
brain uptakes with 2.94 and 2.13% ID/g at 2 min postinjection,
respectively. Particularly for [99mTc]23, the radiotracer
accumulation in the brain peaked at 15 min postinjection
with 3.25% ID/g, which is higher than any of the previously
reported 99mTc-labeled complexes for σ1 receptor imaging.24
Over the past decades, considerable interest has been given to
the development of CNS receptor-based 99mTc probes due to
the ideal nuclear physical characteristics for routine nuclear
medicine diagnostics and ready availability of 99mTc. Because
technetium is a transition metal, a fundamental challenge exists
in the pharmacologically acceptable integration of the
technetium chelate unit into CNS receptor ligands. The
successful development of [2-[[2-[[[3-(4-chlorophenyl)-8-
methyl-8-azabicyclo[3.2.1]oct-2-yl]methyl](2-mercaptoethyl)-
amino]ethyl]amino]ethanethiolato-(3-)-N2,N2′,S2,S2′]oxo-
[1R-(exo-exo)]-[99mTc]technetium ([99mTc]TRODAT-1) for
imaging dopamine transporters in the brain brought optimism
In addition to the high density of σ1 receptors in the CNS,
high expression was also observed in many human tumor cell
lines.12,13,16,46 For example, the maximum specific binding
(Bmax) values of σ1 receptors in C6 glioma cell lines and in
prostate tumor cell lines from rats are approximately 980 and
1800 fmol/mg protein using radioligand (+)-[3H]-pentazocine,
respectively.13,46 Considering the relatively higher affinity for σ1
receptors and higher σ1/VAChT selectivity, [99mTc]23 was
selected for the evaluation of potential applications in tumor
imaging.
Haloperidol is generally accepted as a σ1 antagonist with high
affinity for σ receptors (Ki(σ1) = 4.95 nM, Ki(σ2) = 20.7 nM)31
and moderate to high affinity for dopamine D2, adrenergic, and
serotonin receptors.47 DTG has moderate affinity for σ
receptors and low affinities for other receptors. SA4503 is a
selective ligand with nanomolar affinity for σ1 receptors and low
or negligible affinity for 36 other receptors, ion channels and
components of second messenger systems.48 To investigate the
specific binding of [99mTc]23 to σ1 receptors in tumors, we first
performed cell uptake studies in C6 glioma cells by pretreat-
H
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(5-Bromopentylcarbonyl)cyclopentadienyl Tricarbonyl Rhenium
(8c). The procedure described for the synthesis of 8b was applied to 6-
bromohexanoyl chloride to afford 8c as a white solid (87%); mp 76.5−
78.5 °C. 1H NMR (400 MHz, CDCl3) δ 5.98 (t, J = 2.2 Hz, 2H), 5.40
(t, J = 2.2 Hz, 2H), 3.42 (t, J = 6.7 Hz, 2H), 2.61 (t, J = 7.2 Hz, 2H),
1.93−1.86 (m, 2H), 1.76−1.68 (m, 2H), 1.51−1.45 (m, 2H). ESI-MS,
[M + H]+ (m/z = 512.9).
ment with various concentrations of haloperidol, DTG, and
SA4503. The radiotracer uptake in C6 glioma cell lines was
significantly reduced in a time- and dose-dependent manner. In
addition, a higher affinity of competitors for σ receptors was
correlated with a stronger reduction of radiotracer accumu-
lation. Blocking studies of [99mTc]23 in DU145 prostate cancer
cells also displayed a reduction of radiotracer uptake in a dose-
dependent manner. These results demonstrated the specific
binding of [99mTc]23 to σ1 receptors in tumor cell lines. To
further determine the specific binding in solid tumors in vivo,
blocking studies in C6 glioma-bearing mice were performed.
Pretreatment with haloperidol or DTG resulted in 47% or 39%
reduction of radiotracer accumulation in tumors at 60 min
postinjection, respectively, reflecting specific binding of [99mTc]
23 to σ1 receptors in C6 glioma tumors.
3-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)-
propylcarbonylcyclopentadienyl Tricarbonyl Rhenium (10a). To a
solution of 8a (55.0 mg, 0.11 mmol) in 3 mL of toluene and 3 mL of
triethylamine, 1-piperonylpiperazine (9, 28.4 mg, 0.13 mmol) and KI
(9.1 mg, 0.05 mmol) were added successively. The mixture was heated
to 115 °C and stirred in the dark for 4 h. After cooling to room
temperature, the reaction mixture was concentrated under vacuum.
Purification by silica gel column chromatography (ethyl acetate/
petroleum ether/triethylamine = 1/10/1, v/v/v) afforded 10a as an
1
orange oil (11.2 mg, 16%). H NMR (400 MHz, CDCl3) δ 6.83 (s,
1H), 6.73 (s, 2H), 5.97 (t, J = 2.1 Hz, 2H), 5.93 (s, 2H), 5.38 (t, J =
2.1 Hz, 2H), 3.39 (s, 2H), 2.60 (t, J = 6.9 Hz, 2H), 2.42−2.33 (m,
10H), 1.90−1.83 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 194.69,
191.91, 147.58, 146.56, 131.91, 122.25, 109.54, 107.82, 100.84, 96.62,
87.73, 85.05, 62.69, 57.31, 52.96, 52.69, 36.56, 21.60. TOF-ES+-MS,
[M + H]+: m/z calcd for C24H26N2O6185Re 623.1321; found 623.1317.
4-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)-
butylcarbonylcyclopentadienyl Tricarbonyl Rhenium (10b). The
procedure described for the synthesis of 10a was applied to 8b to
CONCLUSION
■
In conclusion, the novel radiotracer [99mTc]23 possesses
advantageous characteristics such as nanomolar affinity for σ1
receptors, high brain uptake, and specific binding to σ1
receptors in the normal brain, tumor cell lines, and C6 glioma
tumors. This 99mTc-labeled probe warrants further evaluation as
a potential SPECT radiotracer for the investigation of σ1
receptor density in the CNS and as a putative imaging agent
for σ1 receptor expression in brain tumors. The encouraging
results obtained in this paper also indicate a advance in the
development of CNS receptor-based 99mTc-labeled probes.
Suitable 99mTc-based CNS receptor imaging agents may be
attainable if the [(Cp-R)M(CO)3] unit can be incorporated
into selective receptor ligands via an appropriate linker using
the integrated approach. Developments of new 99mTc-labeled
radiotracers with improved brain uptake and high specific
binding to σ1 receptors in vivo are in progress.
1
afford 10b as an orange oil (22%). H NMR (400 MHz, CDCl3) δ
6.85 (s, 1H), 6.74 (d, J = 0.8 Hz, 2H), 6.00 (t, J = 2.3 Hz, 2H), 5.93 (s,
2H), 5.38 (t, J = 2.3 Hz, 2H), 3.41 (s, 2H), 2.61 (t, J = 7.3 Hz, 2H),
2.57−2.45 (m, 8H), 2.35 (t, J = 7.5 Hz, 2H), 1.74−1.66 (m, 2H),
1.56−1.48 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 195.02, 191.85,
147.58, 146.53, 132.10, 122.22, 109.53, 107.81, 100.84, 96.12, 87.90,
85.13, 62.77, 57.98, 53.20, 52.95, 38.61, 26.17, 22.41. TOF-ES+-MS,
[M + H]+: m/z calcd for C25H28N2O6185Re 637.1477; found 637.1454.
5-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)-
pentylcarbonylcyclopentadienyl Tricarbonyl Rhenium (10c). The
procedure described for the synthesis of 10a was applied to 8c to
1
afford 10c as an orange oil (46%). H NMR (400 MHz, CDCl3) δ
EXPERIMENTAL SECTION
6.85 (s, 1H), 6.74 (s, 2H), 5.98 (t, J = 2.2 Hz, 2H), 5.94 (s, 2H), 5.40
(t, J = 2.2 Hz, 2H), 3.41 (s, 2H), 2.59 (t, J = 7.3 Hz, 2H), 2.46 (s, 8H),
2.34 (t, J = 7.3 Hz, 2H), 1.73−1.66 (m, 2H), 1.56−1.48 (m, 2H),
1.37−1.29 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 195.05, 191.80,
147.57, 146.52, 132.12, 122.20, 109.51, 107.79, 100.81, 96.20, 87.80,
85.12, 62.76, 58.42, 53.24, 52.93, 38.71, 27.08, 26.65, 24.25. TOF-ES+-
MS, [M + H]+: m/z calcd for C26H30N2O6185Re 651.1634; found
651.1633.
■
General Information. The reagents and chemicals, thin layer
chromatography (TLC), H NMR spectra, 13C NMR spectra, mass
1
spectrometry (MS), HPLC, and mice were obtained and handled very
similarly to previous work.31 These details are provided in the
Supporting Information. The HPLC method was used to determine
the purity of target compounds. All the final compounds were tested
with purity of more than 95% (HPLC profiles are shown in the
Supporting Information).
2-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)acetonitrile
(14a). To a solution of 9 (204.1 mg, 0.93 mmol) in 6 mL of CH2Cl2
and 3 mL of triethylamine, 2-bromoacetonitrile (154.5 mg, 1.29
mmol) was added. The reaction mixture was stirred at room
temperature overnight and then poured over water and extracted
with CH2Cl2. The organic layer was dried with anhydrous MgSO4,
filtered, concentrated under vacuum, and purified by silica gel column
chromatography using dichloromethane/methanol (100/1, v/v) as the
mobile phase to afford 14a as a light-yellow oil (103.0 mg, 43%).
Compared to the method in the literature,34 the reaction condition is
99mTc-pertechnetate was eluted from a commercial 99Mo−99mTc
generator obtained from Beijing Atomic High-Tech Co. Samples were
analyzed and separated on an Agela Venusil MP C18 column (250
mm × 4.6 mm, 5 μm) using acetonitrile with 0.1% trifluoroacetic acid
(TFA) and water with 0.1% TFA as the mobile phase at a flow rate of
1 mL/min.
All procedures related to 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.
1
relatively mild, but the yield is relatively low. H NMR (400 MHz,
CDCl3) δ 6.85 (s, 1H), 6.74 (s, 2H), 5.94 (s, 2H), 3.50 (s, 2H), 3.43
(s, 2H), 2.62 (t, J = 4.7 Hz, 4H), 2.50 (s, 4H). ESI [M + H]+ (m/z =
260.0).
Chemistry. (4-Bromobutylcarbonyl)cyclopentadienyl Tricarbon-
yl Rhenium (8b). Under N2, cyclopentadienyl tricarbonyl rhenium
(153.8 mg, 0.46 mmol) was added to a solution of 5-bromovaleryl
chloride (182.0 mg, 0.91 mmol) in dried CH2Cl2 (5 mL). The solution
was cooled to 0 °C, followed by the slow addition of dried aluminum
chloride (71.7 mg, 0.54 mmol). The mixture was warmed to room
temperature, stirred for 4 h, and poured over ice−H2O and 4 mL NH3·
H2O. The crude product was extracted with CH2Cl2, dried with
anhydrous MgSO4, and purified by silica gel column chromatography
(ethyl acetate/petroleum ether = 1/10, v/v) to afford 8b as a white oil
3-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)propanenitrile
(14b). The procedure described for the synthesis of 14a was applied to
1
3-bromopropionitrile to afford 14b as a light-yellow oil (74%). H
NMR (400 MHz, CDCl3) δ 6.84 (s, 1H), 6.74 (s, 2H), 5.93 (s, 2H),
3.42 (s, 2H), 2.70 (t, J = 7.1 Hz, 2H), 2.51−2.47 (m, 10H). ESI [M +
H]+ (m/z = 274.0).
4-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)butanenitrile
1
(14c). The procedure described for the synthesis of 14a was applied to
(172.6 mg, 75%). H NMR (400 MHz, CDCl3) δ 5.99 (t, J = 2.3 Hz,
1
4-bromobutyronitrile to afford 14c as a light-yellow oil (70%). H
2H), 5.41 (t, J = 2.3 Hz, 2H), 3.44 (t, J = 6.3 Hz, 2H), 2.64 (t, J = 6.8
Hz, 2H), 1.95−1.81 (m, 4H). ESI-MS, [M + H]+ (m/z = 498.7).
NMR (CDCl3, 400 MHz) δ 6.85 (s, 1H), 6.74 (d, J = 0.8 Hz, 2H),
I
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
5.93 (s, 2H), 3.42 (s, 2H), 2.47−2.39 (m, 12H), 1.85−1.77 (m, 2H).
ESI [M + H]+ (m/z = 288.7).
oacetate (610.4 mg, 2.18 mmol) were dissolved in 2 mL of anhydrous
DMF, and 100 μL of pyridine was added. The reaction mixture was
stirred at room temperature for 4 h. The mixture was diluted with 30
mL of ethyl acetate and washed with 1 M HCl, saturated NaHCO3,
and saturated NaCl. The organic layer was then dried with anhydrous
MgSO4, filtered, concentrated under vacuum, and purified by silica gel
column chromatography using petroleum ether as the mobile phase to
afford 19 as an orange solid (494.7 mg, 57%); mp 75.5−77.0 °C. H
NMR (400 MHz, CDCl3) δ 4.98 (t, J = 1.9 Hz, 2H), 4.59 (t, J = 1.9
Hz, 2H), 4.33 (s, 5H).
2-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)ethanamine
(15a). To a solution of LiAlH4 (94.5 mg, 2.49 mmol) in 10 mL of
anhydrous diethyl ether, the mixture of 14a (95.8 mg, 0.37 mmol) in 5
mL of anhydrous tetrahydrofuran (THF) was added dropwise at 0 °C.
The reaction mixture was stirred at room temperature overnight.
Then, the crude product was poured over ice−H2O and extracted by
ethyl acetate. The organic layer was dried by anhydrous MgSO4,
filtered, concentrated under vacuum, and purified by silica gel column
chromatography using dichloromethane/methanol/triethylamine (10/
1/1, v/v/v) as the mobile phase to afford 15a as a white solid (31.5
mg, 32%); mp 34.3−35.8 °C. Compared to the method in the
literature,34 the procedure seems more convenient, but the yield is
relatively low. 1H NMR (400 MHz, CDCl3) δ 6.85 (s, 1H), 6.74 (d, J
= 0.6 Hz, 2H), 5.93 (s, 2H), 3.41 (s, 2H), 2.80 (t, J = 6.2 Hz, 2H), 2.47
(s, 8H), 2.43 (t, J = 6.2 Hz, 2H), 1.91 (s, 2H). ESI [M + H]+ (m/z =
264.1).
1
3-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)-
propylcarbonylferrocene (20). To a solution of 17 (300.0 mg, 0.89
mmol) in 4 mL of toluene and 4 mL of triethylamine, 9 (65.4 mg, 0.30
mmol) and KI (19.7 mg, 0.12 mmol) were added. The mixture was
stirred at 115 °C in the dark for 4 h. After cooling to room
temperature, the reaction mixture was concentrated under vacuum and
purified by silica gel column chromatography using petroleum ether/
triethylamine (10/1, v/v) as the mobile phase to afford 20 as an
orange solid (84.5 mg, 60%); mp 82.1−84.1 °C. 1H NMR (400 MHz,
CDCl3) δ 6.84 (s, 1H), 6.73 (s, 2H), 5.93 (s, 2H), 4.78 (t, J = 1.7 Hz,
2H), 4.48 (t, J = 1.7 Hz, 2H), 4.19 (s, 5H), 3.41 (s, 2H), 2.75 (t, J =
7.2 Hz, 2H), 2.47 (s, 8H), 2.43 (t, J = 7.3 Hz, 2H), 1.94−1.86 (m,
2H). ESI−MS, [M + H]+ (m/z = 475.2).
4-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)-
butylcarbonylferrocene (21). The procedure described for the
synthesis of 20 was applied to 18 to afford 21 as an orange oil
(62%). 1H NMR (400 MHz, CDCl3) δ 6.84 (s, 1H), 6.73 (s, 2H), 5.93
(s, 2H), 4.78 (t, J = 1.8 Hz, 2H), 4.48 (t, J = 1.8 Hz, 2H), 4.19 (s, 5H),
3.41 (s, 2H), 2.72 (t, J = 7.3 Hz, 2H), 2.47 (s, 8H), 2.39 (t, J = 7.5 Hz,
2H), 1.76−1.68 (m, 2H), 1.61−1.53 (m, 2H). ESI−MS, [M + H]+
(m/z = 488.9).
2-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)-
ethylaminocarbonylferrocene (22). To a solution of 19 (74.9 mg,
0.19 mmol) and 15a (200.2 mg, 0.76 mmol) in anhydrous DMF, 200
μL of triethylamine was added. The reaction mixture was stirred at
room temperature overnight. The solvent was then removed, and
crude product was purified by silica gel column chromatography using
ethyl acetate/petroleum ether/triethylamine (10/10/1, v/v/v) as the
mobile phase to afford 22 as an orange solid (37.4 mg, 42%); mp
122.1−124.1 °C. 1H NMR (400 MHz, CDCl3) δ 6.84 (s, 1H), 6.75 (s,
2H), 5.95 (s, 2H), 4.81 (s, 2H), 4.35 (t, J = 1.8 Hz, 2H), 4.20 (s, 5H),
3.63 (s, 2H), 3.50 (s, 2H), 2.98−2.57 (m, 10H). ESI [M + H]+ (m/z =
476.5).
3-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)propan-1-amine
(15b). The procedure described for the synthesis of 15a was applied to
1
14b to afford 15b as a colorless oil (40%). H NMR (400 MHz,
CDCl3) δ 6.84 (s, 1H), 6.73 (s, 2H), 5.93 (s, 2H), 3.41 (s, 2H), 2.75
(t, J = 6.3 Hz, 2H), 2.47−2.38 (m, 10H), 2.14 (s, 2H), 2.68−1.61 (m,
2H). ESI [M + H]+ (m/z = 278.1).
4-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)butan-1-amine
(15c). The procedure described for the synthesis of 15a was applied to
14c to afford 15c as a colorless oil (45%). 1H NMR (400 MHz,
CDCl3) δ 6.85 (s, 1H), 6.74 (d, J = 0.8 Hz, 2H), 5.93 (s, 2H), 3.41 (s,
2H), 2.71 (t, J = 6.7 Hz, 2H), 2.46 (s, 8H), 2.34 (t, J = 7.4 Hz, 2H),
1.80 (s, 2H), 1.56−1.42 (m, 4H). ESI [M + H]+ (m/z = 292.0).
2-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)-
ethylaminocarbonylcyclopentadienyl Tricarbonyl Rhenium (16a).
Under N2, the solution of compound 15a (25.0 mg, 0.09 mmol) in 1
mL of anhydrous dimethylformamide (DMF) and 30 μL of
triethylamine was added to the solution of 13 (38.7 mg, 0.07
mmol) in 1 mL of anhydrous DMF dropwise. The reaction mixture
was stirred at room temperature for 4 h. Solvents were removed under
vacuum, and the crude product was purified by silica gel column
chromatography using ethyl acetate/hexane/triethylamine (10/10/1,
v/v/v) as the mobile phase to afford 16a as a light-orange oil (18.3 mg,
40%). 1H NMR (400 MHz, CDCl3) δ 6.84 (s, 1H), 6.74 (s, 2H), 5.94
(s, 2H), 5.92 (t, J = 2.2 Hz, 2H), 5.36 (t, J = 2.2 Hz, 2H), 3.46−3.41
(m, 4H), 2.59−2.50 (m, 10H). 13C NMR (100 MHz, CDCl3) δ
192.54, 162.22, 147.65, 146.65, 131.83, 122.19, 109.44, 107.86, 100.87,
95.24, 85.96, 84.59, 62.59, 56.07, 52.73, 52.71, 35.86. TOF-ES+-MS,
[M + H]+: m/z calcd for C23H25N3O6185Re 624.1273; found 624.1271.
3-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)-
propylaminocarbonylcyclopentadienyl Tricarbonyl Rhenium (16b).
The procedure described for the synthesis of 16a was applied to 15b
to afford 16b as a light-orange oil (92%). 1H NMR (400 MHz,
CDCl3) δ 7.90 (s, 1H), 6.83 (s, 1H), 6.76 (s, 1H), 6.74 (d, J = 0.9 Hz,
1H), 5.95 (s, 2H), 5.86 (t, J = 2.1 Hz, 2H), 5.34 (t, J = 2.2 Hz, 2H),
3.48 (s, 2H), 3.46−3.42 (m, 2H), 2.56−2.53 (m, 10H), 1.75−1.69 (m,
2H). 13C NMR (100 MHz, CDCl3) δ 192.69, 161.88, 147.62, 146.64,
131.35, 122.33, 109.57, 107.88, 100.90, 96.33, 85.30, 84.73, 62.60,
58.31, 53.47, 52.81, 40.68, 23.85. TOF-ES+-MS, [M + H]+: m/z calcd
for C24H27N3O6185Re 638.1430; found 638.1445.
In Vitro Radioligand Competition Studies. σ Receptor Binding
Assays. All the procedures for the radioligand competition studies
were previously described.27 Detailed procedures are shown in the
Supporting Information.
VAChT Binding Assays. Radioligand competition binding assays for
VAChT were performed according to literature,35 and detailed
procedures are provided in the Supporting Information.
Radiochemistry. The 99mTc-pertechnetate was eluted from a
commercial 99Mo−99mTc generator obtained from Beijing Atomic
High-Tech Co. The reactions were performed according to the
method in the literature.28,38 Detailed procedures are provided in the
Supporting Information.
Measurement of log D Values. The log D values of [99mTc]23−
25 were determined by measuring the distribution of the radiotracer
between 1-octanol and 0.05 mol·L−1 sodium phosphate buffer at pH
7.4 according to literature.31 Detailed procedures are provided in the
Supporting Information.
4-(4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl)-
butylaminocarbonylcyclopentadienyl Tricarbonyl Rhenium (16c).
The procedure described for the synthesis of 16a was applied to 15c to
afford 16c as a light-orange oil (61%). 1H NMR (400 MHz, CDCl3) δ
6.84 (s, 1H), 6.74 (s, 2H), 6.38 (t, J = 5.2 Hz, 1H), 5.94 (s, 2H), 5.92
(t, J = 2.2 Hz, 2H), 5.35 (t, J = 2.2 Hz, 2H), 3.41 (s, 2H), 3.36−3.32
(m, 2H), 2.48 (s, 8H), 2.38 (t, J = 6.6 Hz, 2H), 1.58−1.55 (m, 4H).
13C NMR (100 MHz, CDCl3) δ 192.60, 162.19, 147.62, 146.61,
131.93, 122.23, 109.49, 107.85, 100.87, 95.69, 85.73, 84.77, 62.68,
57.77, 53.18, 52.71, 39.49, 27.29, 24.03. TOF-ES+-MS, [M + H]+: m/z
calcd for C25H29N3O6185Re 652.1586; found 652.1580.
Pentafluorophenyl Ferrocenecarboxylate (19). Ferrocenecarbox-
ylic acid (501.5 mg, 2.18 mmol) and pentafluorophenyl trifluor-
Biodistribution Studies in Mice and Blocking Studies. All
animal experiments in ICR mice (n = 5, 4−5 weeks, 22−25 g) were
performed in compliance with the national laws related to the care and
experiments on laboratory animals. Biodistribution studies and
blocking studies of HPLC-purified [99mTc]23 or [99mTc]24 (370
kBq in 0.1 mL saline) were carried out based on the method reported
previously.31 Detailed procedures are shown in the Supporting
Information.
In Vivo Radiometabolic Stability of [99mTc]23. The in vivo
metabolism of [99mTc]23 (33 MBq, 0.15 mL) was studied in male ICR
J
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mice according to the previously reported method.49 Detailed
procedures are shown in the Supporting Information.
4 weeks of age. Animals were housed under standard conditions with
free access to food and water. All of the animal experiments adhered to
the principles of care and use of laboratory animals and were approved
by the Institutional Animal Care and Use Committee of Peking
University.
C6 glioma cells were routinely grown as monolayers. After
harvesting these cells, C6 glioma cells were diluted with serum-free
RPMI-1640 medium to achieve a cell concentration of 5 × 107/mL. A
100 μL cell suspension was implanted subcutaneously in the scapular
region of ICR mice (4−5 weeks old). The biodistribution studies were
performed 7−10 days after implantation when the tumor size was
approximately 0.2 cm3 (approximately 200 mg).
Blocking studies in C6 tumor-bearing mice (n = 5) were conducted
by injecting 1.0 mg/kg haloperidol (0.1 mL) or 3 μM DTG (0.1 mL)
5 min prior to [99mTc]23 injection. The animals were sacrificed at 60
min postinjection. Samples of the blood, brain, heart, liver, spleen,
lung, kidneys, muscle, and tumor were isolated and analyzed as
described above. Significant differences between control and blocking
groups were determined by Student’s t test (independent, two-tailed).
The criterion for significance was p ≤ 0.05.
Effect of P-gp on the Brain Uptake of [99mTc]23 in Mice. For
the investigation of the effect of P-gp on the brain uptake of [99mTc]
23, mice were injected via the tail vein with either saline (0.1 mL) or
cyclosporine A (0.1 mL, 50.0 mg/kg) 60 min prior to [99mTc]23
injection (370 kBq in 0.1 mL saline) according to the method in the
literature.50 Detailed information is provided in the Supporting
Information.
In Vitro Uptake of [99mTc]23 in C6 Glioma Cell Lines. C6
glioma cells (Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing, China)
were routinely grown as monolayers in RPMI-1640 medium (Macgene
Biotech Co., Ltd., Beijing, China) supplemented with 10% (v) heat-
inactivated fetal bovine serum (FBS) and antibiotics (100 U/mL
penicillin and 100 μg/mL streptomycin) in an atmosphere containing
5% CO2 at 37 °C.
Before each experiment, C6 glioma cells were seeded in the wells of
24-well plates. An equal number of cells (2 × 105 cells/well) was
dispensed in each well and incubated with 1 mL of RPMI 1640
medium supplemented with 10% FBS overnight at 37 °C to allow firm
adherence. For total binding experiments, 7.4 kBq of [99mTc]23 in 1
mL of RPMI-1640 medium (50 μL of [99mTc]23 in saline mixed with
0.95 mL of RPMI-1640 medium) was added to each well (in
triplicate). For blocking studies, various concentrations of different
competitors (2, 10, and 20 μM haloperidol; 2, 10, and 20 μM DTG;
and 2, 10, and 20 μM SA4503) in 1 mL of RPMI-1640 medium with
7.4 kBq of [99mTc]23 was added to each well (in triplicate). After 15,
30, or 60 min incubation at room temperature, the medium was
quickly removed. The cells were rinsed twice with ice-cold phosphate-
buffered saline containing 0.2% bovine serum albumin (BSA) and then
lysed with 1 mL of NaOH (1 M) at room temperature. The cell-
associated radioactivity was determined using a gamma counter
(Wallac 1470 Wizard, PerkinElmer, USA). The administered [99mTc]
23 (7.4 kBq) in each well was used as the administered tracer dose.
The cell uptake (%) of [99mTc]23 was calculated by the formula:
Uptake (%) = counts per minute (CPM) (in the cell suspension)/
CPM (administered tracer dose) × 100%.
ASSOCIATED CONTENT
■
S
* Supporting Information
General information and some parts of evaluation of the
radiotracers in the Experimental Section, purity of key target
compounds, the HPLC analysis of lipophilicity of rhenium
complexes 10a and 16a, and the HPLC coinjection profiles of
[
99mTc]23 and 10a, [99mTc]24 and 10b, [99mTc]25 and 16a.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +86-10-58808891. Fax: +86-10-58808891. E-mail:
hmjia@bnu.edu.cn.
The %blocking was calculated by [(radioactivity accumulation
under blocking condition) − (radioactivity accumulation under
control condition)]/(radioactivity accumulation under control con-
dition) × 100%. Significant differences between control and blocking
groups were determined by Student’s t test (independent, two-tailed).
The criterion for significance was p ≤ 0.05.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (no. 21071023).
In Vitro Uptake of [99mTc]23 in DU145 Prostate Cell Lines.
DU145 prostate cells (National Platform of Experimental Cell
Resources for Sci-Tech) were routinely grown as monolayers in
RPMI-1640 medium (Macgene Biotech Co., Ltd., Beijing, China)
supplemented with 10% (v) heat-inactivated FBS and 1% (v)
antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) in
an atmosphere containing 5% CO2 at 37 °C.
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; BSA, bovine serum albumin; CNS,
central nervous system; CPM, counts per minute; DLT, double
ligand transfer; DMF, dimethylformamide; DTG, 1,3-di-o-tolyl-
guanidine; FBS, fetal bovine serum; HPLC, high performance
liquid chromatography; ID, injected dose; PD, Parkinson’s
disease; PET, positron emission tomography; P-gp, perme-
ability-glycoprotein; rt, room temperature; SD, standard
deviation; SPECT, single photon emission computed tomog-
raphy; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC,
thin layer chromatography; VAChT, vesicular acetylcholine
transporter
The in vitro cell uptake experiments were performed with the
method described above. For total binding experiments, 7.4 kBq of
[
99mTc]23 was added in 1 mL of RPMI-1640 medium (50 μL of
radioligand in saline mixed with 0.95 mL RPMI-1640 medium) was
added to each well (in triplicate). For blocking studies, various
concentrations of SA4503 (10−11, 10−10, 10−9, 10−8, 10−7, 10−6, 10−5,
10−4, and 10−3 M) in 1 mL of RPMI-1640 medium with 7.4 kBq of
radioligand was added to each well (in triplicate). After 60 min
incubation at room temperature, the medium was quickly removed.
The cells were rinsed twice with ice-cold phosphate-buffered saline
containing 0.2% BSA and then lysed with 1 mL of NaOH (1 M) at
room temperature. The cell-associated radioactivity was measured
using a gamma counter (Wallac 1470 Wizard, PerkinElmer, USA). The
administered radioligand (7.4 kBq) in each well was used as the
administered tracer dose.
REFERENCES
■
(1) Hayashi, T.; Su, T.-P. Sigma-1 receptor chaperones at the ER-
mitochondrion interface regulate Ca2+ signaling and cell survival. Cell
2007, 131, 596−610.
(2) Hanner, M.; Moebius, F. F.; Flandorfer, A.; Knaus, H.-G.;
Striessnig, J.; Kempner, E.; Glossmann, H. Purification, molecular
cloning, and expression of the mammalian sigma1-binding site. Proc.
Natl. Acad. Sci. U. S. A. 1996, 93, 8072−8077.
The percentage of cell uptake (uptake (%)) and %blocking were
calculated as described above.
Blocking Studies in C6 Glioma Tumor-Bearing Mice. ICR
mice were purchased from Peking University Health Science Center at
K
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(3) Aydar, E.; Palmer, C. P.; Klyachko, V. A.; Jackson, M. B. The
sigma receptor as a ligand-regulated auxiliary potassium channel
subunit. Neuron 2002, 34, 399−410.
using [11C]SA4503. Int. J. Neuropsychopharmacol. 2009, 12, 1127−
1131.
(22) Sakata, M.; Kimura, Y.; Naganawa, M.; Oda, K.; Ishii, K.;
Chihara, K.; Ishiwata, K. Mapping of human cerebral sigma1 receptors
using positron emission tomography and [11C]SA4503. NeuroImage
2007, 35, 1−8.
(23) Johannsen, B.; Pietzsch, H.-J. Development of technetium-99m-
based CNS receptor ligands: have there been any advances? Eur. J.
Nucl. Med. Mol. Imaging 2002, 29, 263−275.
(24) Satpati, D.; Bapat, K.; Sarma, H. D.; Venkatesh, M.; Banerjee, S.
Synthesis, characterization and biological activity of 99mTc-labeled
piperidine analogues targeting sigma receptors. J. Labelled Compd.
Radiopharm. 2010, 53, 198−204.
(25) Friebe, M.; Mahmood, A.; Bolzati, C.; Drews, A.; Johannsen, B.;
Eisenhut, M.; Kraemer, D.; Davison, A.; Jones, A. G. [99mTc]-
oxotechnetium(V) complexes of amine-amide-dithiol chelates with
dialkylaminoalkyl substituents as potential diagnostic probes for
malignant melanoma. J. Med. Chem. 2001, 44, 3132−3140.
(26) Lu, J.; Kong, D.; Jia, H.; Deuther-Conrad, W.; Brust, P.; Wang,
X. Preparation and biological evaluation of 99mTcN-4-(cyclohexylpi-
perazin-1-yl)-dithioformate as a potential sigma receptor imaging
agent. J. Labelled Compd. Radiopharm. 2007, 50, 1200−1205.
(27) Fan, C.; Jia, H.; Deuther-Conrad, W.; Brust, P.; Steinbach, J.;
Liu, B. Novel 99mTc labeled σ receptor ligand as a potential tumor
imaging agent. Sci. China, Ser. B: Chem. 2006, 49, 169−176.
(28) Chen, X.; Cui, M. C.; Deuther-Conrad, W.; Tu, Y. F.; Ma, T.;
Xie, Y.; Jia, B.; Li, Y.; Xie, F.; Wang, X.; Steinbach, J.; Brust, P.; Liu, B.
L.; Jia, H. M. Synthesis and biological evaluation of a novel 99mTc
cyclopentadienyl tricarbonyl complex ([(Cp-R)99mTc(CO)3]) for
sigma-2 receptor tumor imaging. Bioorg. Med. Chem. Lett. 2012, 22,
6352−6357.
(4) Maurice, T.; Su, T.-P. The pharmacology of sigma-1 receptors.
Pharmacol. Ther. 2009, 124, 195−206.
(5) Hayashi, T.; Tsai, S.-Y.; Mori, T.; Fujimoto, M.; Su, T.-P.
Targeting ligand-operated chaperone sigma-1 receptors in the
treatment of neuropsychiatric disorders. Expert Opin. Ther. Targets
2011, 15, 557−577.
(6) Kourrich, S.; Su, T.-P.; Fujimoto, M.; Bonci, A. The sigma-1
receptor: roles in neuronal plasticity and disease. Trends Neurosci.
2012, 35, 762−771.
́
́ ́ ́ ́ ́ ́ ́ ́
, A.; Juhasz, A.; Laszlo, A.; Kalman, J., Jr.; Pakaski, M.;
(7) Feher
Kalman, J.; Janka, Z. Association between a variant of the sigma-1
́
́
receptor gene and Alzheimer’s disease. Neurosci. Lett. 2012, 517, 136−
139.
(8) Uchida, N.; Ujike, H.; Tanaka, Y.; Sakai, A.; Yamamoto, M.;
Fujisawa, Y.; Kanzaki, A.; Kuroda, S. A variant of the sigma receptor
type-1 gene is a protective factor for Alzheimer disease. Am. J. Geriatr.
Psychiatry 2005, 13, 1062−1066.
(9) Mishina, M.; Ishiwata, K.; Ishii, K.; Kitamura, S.; Kimura, Y.;
Kawamura, K.; Oda, K.; Sasaki, T.; Sakayori, O.; Hamamoto, M.
Function of sigma1 receptors in Parkinson’s disease. Acta Neurol.
Scand. 2005, 112, 103−107.
(10) Bermack, J. E.; Debonnel, G. The role of sigma receptors in
depression. J. Pharmacol. Sci. 2005, 97, 317−336.
(11) Fishback, J. A.; Robson, M. J.; Xu, Y.-T.; Matsumoto, R. R.
Sigma receptors: potential targets for a new class of antidepressant
drug. Pharmacol. Ther. 2010, 127, 271−282.
(12) Wang, B.; Rouzier, R.; Albarracin, C.; Sahin, A.; Wagner, P.;
Yang, Y.; Smith, T.; Bernstam, F. M.; Marcelo, A. C.; Hortobagyi, G.
Expression of sigma1 receptor in human breast cancer. Breast Cancer
Res. Treat. 2004, 87, 205−214.
(13) Vilner, B. J.; John, C. S.; Bowen, W. D. Sigma-1 and sigma-2
receptors are expressed in a wide variety of human and rodent tumor
cell lines. Cancer Res. 1995, 55, 408−413.
(14) van Waarde, A.; Rybczynska, A. A.; Ramakrishnan, N. K.;
Ishiwata, K.; Elsinga, P. H.; Dierckx, R. A.J.O. Sigma receptors in
oncology: therapeutic and diagnostic applications of sigma ligands.
Curr. Pharm. Des. 2010, 16, 3519−3537.
(15) Megalizzi, V.; Le Mercier, M.; Decaestecker, C. Sigma receptors
and their ligands in cancer biology: overview and new perspectives for
cancer therapy. Med. Res. Rev. 2012, 32, 410−427.
(16) Megalizzi, V.; Decaestecker, C.; Debeir, O.; Spiegl-Kreinecker,
S.; Berger, W.; Lefranc, F.; Kast, R. E.; Kiss, R. Screening of anti-
glioma effects induced by sigma-1 receptor ligands: potential new use
for old anti-psychiatric medicines. Eur. J. Cancer 2009, 45, 2893−2905.
(17) Brust, P.; Deuther-Conrad, W.; Lehmkuhl, K.; Jia, H.; Wuensch,
B. Molecular imaging of σ1 receptors in vivo: current status and
perspectives. Curr. Med. Chem. 2014, 21, 35−69.
(18) Waterhouse, R. N.; Chang, R. C.; Atuehene, N.; Collier, T. L. In
vitro and in vivo binding of neuroactive steroids to the sigma-1
receptor as measured with the positron emission tomography
radioligand [18F] FPS. Synapse 2007, 61, 540−546.
(19) Stone, J. M.; Årstad, E.; Erlandsson, K.; Waterhouse, R. N.; Ell,
P. J.; Pilowsky, L. S. [123I]TPCNEa novel SPET tracer for the
sigma-1 receptor: first human studies and in vivo haloperidol
challenge. Synapse 2006, 60, 109−117.
(20) Ishikawa, M.; Ishiwata, K.; Ishii, K.; Kimura, Y.; Sakata, M.;
Naganawa, M.; Oda, K.; Miyatake, R.; Fujisaki, M.; Shimizu, E.;
Shirayama, Y.; Iyo, M.; Hashimoto, K. High occupancy of sigma-1
receptors in the human brain after single oral administration of
fluvoxamine: a positron emission tomography study using [11C]-
SA4503. Biol. Psychiatry 2007, 62, 878−883.
(29) John, C. S.; Lim, B. B.; Geyer, B. C.; Vilner, B. J.; Bowen, W. D.
99mTc-labeled σ-receptor-binding complex: synthesis, characterization,
and specific binding to human ductal breast carcinoma (T47D) cells.
Bioconjugate Chem. 1997, 8, 304−309.
(30) Alberto, R.; Abram, U. 99mTc: labeling chemistry and labeled
compounds. In Handbook of Nuclear Chemistry, 2nd ed.; Ver
Nagy, S., Klencsar, Z., Lovas, R. G., Rosch, F., Eds.; Springer: New
York, 2011, Vol. 4; pp 2073−2120.
́
tes, A.,
́
̈
(31) Wang, X.; Li, Y.; Deuther-Conrad, W.; Xie, F.; Chen, X.; Cui,
M.-C.; Zhang, X.-J.; Zhang, J.-M.; Steinbach, J.; Brust, P.; Liu, B.-L.;
Jia, H.-M. Synthesis and biological evaluation of 18F labeled fluoro-
oligo-ethoxylated 4-benzylpiperazine derivatives for sigma-1 receptor
imaging. Bioorg. Med. Chem. 2013, 21, 215−222.
(32) Glennon, R. Pharmacophore identification for sigma-1 (σ1)
receptor binding: application of the “deconstruction−reconstruction−
elaboration” approach. Mini-Rev. Med. Chem. 2005, 5, 927−940.
(33) N’Dongo, H. P.; Liu, Y.; Can, D.; Schmutz, P.; Spingler, B.;
Alberto, R. Aqueous syntheses of [(Cp-R)M(CO)3] type complexes
(Cp = cyclopentadienyl, M = Mn, 99mTc, Re) with bioactive
functionalities. J. Organomet. Chem. 2009, 694, 981−987.
(34) Reynaud, P.; Brion, J.; Nguyentrixuong, E.; Davrinche, C.; Pieri,
F.; Arnouldguerin, M. Anti-atthythmic agents related to procainamide
II. Synthesis and pharmacological study. Eur. J. Med. Chem. 1989, 24,
427−434.
(35) Sorger, D.; Scheunemann, M.; Großmann, U.; Fischer, S.;
Vercouille, J.; Hiller, A.; Wenzel, B.; Roghani, A.; Schliebs, R.; Brust,
P.; Sabri, O.; Steinbach, J. A new 18F-labeled fluoroacetylmorpholino
derivative of vesamicol for neuroimaging of the vesicular acetylcholine
transporter. Nucl. Med. Biol. 2008, 35, 185−195.
(36) Cesati, R. R.; Tamagnan, G.; Baldwin, R. M.; Zoghbi, S. S.;
Innis, R. B.; Kula, N. S.; Baldessarini, R. J.; Katzenellenbogen, J. A.
Synthesis of cyclopentadienyltricarbonyl rhenium phenyltropanes by
double ligand transfer: organometallic ligands for the dopamine
transporter. Bioconjugate Chem. 2002, 13, 29−39.
̌
(21) Ishikawa, M.; Sakata, M.; Ishii, K.; Kimura, Y.; Oda, K.;
Toyohara, J.; Wu, J.; Ishiwata, K.; Iyo, M.; Hashimoto, K. High
occupancy of σ1 receptors in the human brain after single oral
administration of donepezil: a positron emission tomography study
(37) Step
̌
nick
̌
a, P.; Verníce
̌
k, M.; Schulz, J.; Císaro
̌
va,
́
I. Synthesis and
structural characterization of a sterically encumbered ferrocenecarbox-
amido diphosphine and its platinum(II) complex. J. Organomet. Chem.
2014, 755, 41−46.
L
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(38) Saidi, M.; Kothari, K.; Pillai, M.; Hassan, A.; Sarma, H.;
Chaudhari, P.; Unnikrishnan, T.; Korde, A.; Azzouz, Z. Cyclo-
pentadienyl technetium (99mTc) tricarbonyl piperidine conjugates:
biodistribution and imaging studies. J. Labelled Compd. Radiopharm.
2001, 44, 603−618.
(39) Chen, R.-Q.; Li, Y.; Zhang, Q.-Y.; Jia, H.-M.; Deuther-Conrad,
W.; Schepmann, D.; Steinbach, J.; Brust, P.; Wunsch, B.; Liu, B.-L.
̈
Synthesis and biological evaluation of a radioiodinated spiropiperidine
ligand as a potential σ1 receptor imaging agent. J. Labelled Compd.
Radiopharm. 2010, 53, 569−574.
(40) Kreisl, W. C.; Liow, J. S.; Kimura, N.; Seneca, N.; Zoghbi, S. S.;
Morse, C. L.; Herscovitch, P.; Pike, V. W.; Innis, R. B. P-glycoprotein
function at the blood−brain barrier in humans can be quantified with
the substrate radiotracer 11C-N-desmethyl-loperamide. J. Nucl. Med.
2010, 51, 559−566.
(41) Mishina, M.; Ohyama, M.; Ishii, K.; Kitamura, S.; Kimura, Y.;
Oda, K.; Kawamura, K.; Sasaki, T.; Kobayashi, S.; Katayama, Y.;
Ishiwata, K. Low density of sigma1 receptors in early Alzheimer’s
disease. Ann. Nucl. Med. 2008, 22, 151−156.
(42) Li, Z. J.; Ren, H. Y.; Cui, M. C.; Deuther-Conrad, W.; Tang, R.
K.; Steinbach, J.; Brust, P.; Liu, B. L.; Jia, H. M. Synthesis and
biological evaluation of novel 4-benzylpiperazine ligands for sigma-1
receptor imaging. Bioorg. Med. Chem. 2011, 19, 2911−2917.
(43) Patel, S.; Gibson, R. In vivo site-directed radiotracers: a mini-
review. Nucl. Med. Biol. 2008, 35, 805−815.
(44) Meegalla, S. K.; Plossl, K.; Kung, M.-P.; Chumpradit, S.;
̈
Stevenson, D. A.; Kushner, S. A.; McElgin, W. T.; Mozley, P. D.; Kung,
H. F. Synthesis and characterization of technetium-99m-labeled
tropanes as dopamine transporter-imaging agents. J. Med. Chem.
1997, 40, 9−17.
(45) Yamazaki, M.; Neway, W. E.; Ohe, T.; Chen, I.-W.; Rowe, J. F.;
Hochman, J. H.; Chiba, M.; Lin, J. H. In vitro substrate identification
studies for P-glycoprotein-mediated transport: species difference and
predictability of in vivo results. J. Pharmacol. Exp. Ther. 2001, 296,
723−735.
(46) John, C. S.; Vilner, B. J.; Geyer, B. C.; Moody, T.; Bowen, W. D.
Targeting sigma receptor-binding benzamides as in vivo diagnostic and
therapeutic agents for human prostate tumors. Cancer Res. 1999, 59,
4578−4583.
(47) Bowen, W. D.; Moses, E. L.; Tolentino, P. J.; Walker, J. M.
Metabolites of haloperidol display preferential activity at sigma
receptors compared to dopamine D-2 receptors. Eur. J. Pharmacol.
1990, 177, 111−118.
(48) Matsuno, K.; Nakazawa, M.; Okamoto, K.; Kawashima, Y.; Mita,
S. Binding properties of SA4503, a novel and selective σ1 receptor
agonist. Eur. J. Pharmacol. 1996, 306, 271−279.
(49) Li, Y.; Wang, X.; Zhang, J.; Deuther-Conrad, W.; Xie, F.; Zhang,
X.; Liu, J.; Qiao, J.; Cui, M.; Steinbach, J.; Brust, P.; Liu, B.; Jia, H.
Synthesis and evaluation of novel 18F-labeled spirocyclic piperidine
derivatives as σ1 receptor ligands for positron emission tomography
imaging. J. Med. Chem. 2013, 56, 3478−3491.
(50) Li, Z.; Cui, M.; Dai, J.; Wang, X.; Yu, P.; Yang, Y.; Jia, J.; Fu, H.;
Ono, M.; Jia, H.; Saji, H.; Liu, B. Novel cyclopentadienyl tricarbonyl
complexes of 99mTc mimicking chalcone as potential single-photon
emission computed tomography imaging probes for beta-amyloid
plaques in brain. J. Med. Chem. 2013, 56, 471−482.
M
dx.doi.org/10.1021/jm5009488 | J. Med. Chem. XXXX, XXX, XXX−XXX