99mTc-Cyclopentadienyl Tricarbonyl Chelate-Labeled Compounds as Selective Sigma-2 Receptor Ligands for Tumor Imaging

Article abstract of DOI:10.1021/acs.jmedchem.5b01378

We have designed and synthesized a series of cyclopentadienyl tricarbonyl rhenium complexes containing a 5,6-dimethoxyisoindoline or a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline pharmacophore as σ₂ receptor ligands. Rhenium compound 20a posse

Full text of DOI:10.1021/acs.jmedchem.5b01378

Article
pubs.acs.org/jmc
99mTc-Cyclopentadienyl Tricarbonyl Chelate-Labeled Compounds as
Selective Sigma‑2 Receptor Ligands for Tumor Imaging
Dan Li,†,⊥ Yuanyuan Chen,†,⊥ Xia Wang,† Winnie Deuther-Conrad,‡ Xin Chen,† Bing Jia,§
Chengyan Dong,∥ 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
§Medical and Healthy Analytical Center, Peking University, Beijing 100191, China
∥Interdisciplinary Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
S
* Supporting Information
ABSTRACT: We have designed and synthesized a series of cyclopentadienyl
tricarbonyl rhenium complexes containing a 5,6-dimethoxyisoindoline or a 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline pharmacophore as σ2 receptor
ligands. Rhenium compound 20a possessed low nanomolar σ2 receptor
affinity (Ki = 2.97 nM) and moderate subtype selectivity (10-fold). Moreover,
it showed high selectivity toward vesicular acetylcholine transporter (2374-
fold), dopamine D2L receptor, NMDA receptor, opiate receptor, dopamine
transporter, norepinephrine transporter, and serotonin transporter. Its
corresponding radiotracer [99mTc]20b showed high uptake in a time- and
dose-dependent manner in DU145 prostate cells and C6 glioma cells. In
addition, this tracer exhibited high tumor uptake (5.92% ID/g at 240 min) and
high tumor/blood and tumor/muscle ratios (21 and 16 at 240 min,
respectively) as well as specific binding to σ receptors in nude mice bearing
C6 glioma xenografts. Small animal SPECT/CT imaging of [99mTc]20b in the C6 glioma xenograft model demonstrated a clear
visualization of the tumor at 180 min after injection.
used radionuclide in clinical nuclear medicine application. With
the clinical implementation of quantitative SPECT in the
future,16 99mTc-labeled radiotracers with high affinity, high
selectivity, and specificity for σ2 receptors will provide a unique
tool for the early diagnosis of cancer and assessment of the
proliferative status of solid tumors. Over the past few decades, a
series of 99mTc-labeled radioligands targeting σ2 receptors have
been reported.17−22 However, there is a lack of ideal 99mTc-
labeled radiotracers for imaging σ2 receptors in human studies.
Therefore, the development of 99mTc-labeled σ2 receptor
radioligands is very attractive.
Previously, our structure−affinity relationship analyses in-
dicated the high importance of the σ2 preferring group to
improve the selectivity for σ2 receptors.23 Besides the 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline scaffold in ISO-1, the
5,6-dimethoxyisoindoline moiety was identified as a promising σ2
preferring group with less lipophilicity.24 Moreover, the [(Cp-
R)Tc(CO)3] unit can be incorporated into selective receptor
ligands without a significant change in the biological properties
via an appropriate linker.22,25 In the present study, our aim is to
INTRODUCTION
■
Uncontrolled cell proliferation is one of the hallmarks of cancer,
and its assessment will provide useful information to predict the
tumor aggressiveness and prognosis of cancer.1 The sigma-2 (σ2)
receptors are upregulated in a wide variety of human and rodent
tumor cells.2−6 Moreover, they showed an approximately 10-fold
higher expression in proliferating versus quiescent tumors.7−9
Significant effort has been dedicated to the validation of the σ2
receptor as a biomarker for the imaging of the proliferative status
of solid tumors.7−15 Currently, a promising radiotracer targeting
σ2 receptors, N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)butyl)-2-(2-[18F]fluoroethoxy)-5-methylbenzamide
([18F]ISO-1), is under clinical trials.10−12 Furthermore, a
significant correlation between the uptake of this radiotracer
and Ki-67 (the “gold standard” used in histological measure-
ments of cell proliferation in tumor surgical and biopsy
specimens) was observed in human studies.12 These findings
indicate that the σ2 receptor is an important biomarker for
determining the proliferative status of solid tumors using
positron emission tomography (PET).13−15
It is well-known that single photon emission computed
tomography (SPECT) is an extremely helpful, widely used, and
low cost clinical imaging modality. 99mTc is still the most widely
Received: September 5, 2015
© XXXX American Chemical Society
A
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Figure 1. Design concept of novel [(Cp-R)M(CO)3] (M = 99mTc, Re) complexes as potent σ2 receptor ligands.
a
Scheme 1. Synthetic Routes of the Rhenium Compounds
a
Reagents and conditions: (a) 33% HBr/HOAc, rt (20 h)−65 °C (1 h), 32%; (b) tritylamine, DIEA, DMF, 60 °C, 2 h, 77%; (c) TFA, CHCl3/
MeOH, 0 °C−rt, 1 h, 69%; (d) Br(CH2)mCN, Et3N, CH2Cl2, rt, 24 h, for 5−7, 67−73%, for 8−10, 59−70%; (e) LiAlH4, THF-Et2O, 0 °C−rt, 24 h,
for 11−13, 36−50%, for 14−16, 27−40%; (f) toluene, 3 or 4, KI, Et3N, 115 °C, 10% for 20a, 10% for 21a, 30% for 23a and 36% for 24a; (g)
acetonitrile, 3 or 4, KI, K2CO3, 90 °C, 41% for 22a and 51% for 25a; (h)11−16, Et3N, anhydrous DMF, rt, 4 h, 38−48% for 27a−29a, 74−83% for
30a−32a.
develop a 99mTc-labeled radioligand as a selective σ2 receptor
probe for tumor imaging with SPECT. The design concept is
shown in Figure 1. In the first approach, we directly incorporate
the [(Cp-R)M(CO)3] (M = Re, 99mTc) unit containing the
carbonyl group in compound 1 with the σ2 preferring group (5,6-
dimethoxyisoindoline moiety in compound 2 or 6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinoline moiety in ISO-1) via different
carbon chain lengths. In the second approach, the amide group
was connected to the [(Cp-R)M(CO)3] (M = Re, 99mTc) unit in
compound 1 first and then incorporated with a σ2 preferring
group. Both carbonyl and amide groups are electron-with-
drawing groups and would allow the double ligand transfer
(DLT) reaction for the efficient synthesis of the 99mTc-complex
from the corresponding ferrocene precursor. In addition,
different carbon chain lengths were used as the linker between
the [(Cp-R)M(CO)3] unit with an electron-withdrawing group
and the 5,6-dimethoxyisoindoline or 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline pharmacophore to find the optimal
radiotracer with appropriate interaction on σ2 receptors for in
vivo tumor imaging. Herein, we report the syntheses of novel
[(Cp-R)M(CO)3] (M = Re, 99mTc) complexes containing a 5,6-
dimethoxyisoindoline or 6,7-dimethoxy-1,2,3,4-tetrahydroiso-
quinoline motif, and we evaluated them as potential σ2 receptor
radioligands for tumor imaging in vivo.
RESULTS
■
Chemistry. For the characterization of 99mTc-labeled
compounds, the surrogates of the corresponding rhenium
complexes were prepared, and the synthetic routes are depicted
in Scheme 1. Intermediates 3,26 8−10,27 14−16,27 17−19,22 and
2628 were obtained according to the methods reported in the
literature. N-Alkylation of compound 3 or 4 with compounds
17−19 gave compounds 20a−25a (20a, 22a, 23a, and 25a),29
respectively, with yields of 10−51%. It needs to be mentioned
that compounds 22a and 25a could be obtained with higher
yields in acetonitrile with K2CO3 as base than in toluene with
B
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triethylamine. However, N-alkylation of compound 3 with
bromine-substituted nitrile, followed by reduction with LiAlH4
provided compounds 11−13, respectively, with yields of 36−
50%. The activated ester 26 reacted with the corresponding
amine analogues 11−16 in anhydrous DMF at room temper-
ature to afford the target rhenium analogues 27a−32a (27a, 28a,
and 29a).30 Complex 31a could be recrystallized by slow
evaporation of a mixture of methylene dichloride and hexane
solution to afford X-ray diffraction crystals.
To further confirm the chemical structure and examine the
binding model of 99mTc-labeled cyclopentadienyl tricarbonyl
complexes, the single X-ray crystal structure analysis of complex
31a was performed. Crystal structure data together with details of
the determinations are summarized in Tables S1−5. The crystal
structure with the atomic numbering scheme of compound 31a is
shown in Figure 2. The central rhenium atom is η5-coordinated
Table 1. Binding Affinities of Cyclopentadienyl Tricarbonyl
Rhenium Complexes and Ferrocene Precursors for σ1 and σ2
Receptors
a
compd
20a
Ki(σ1) (nM)
Ki(σ2) (nM)
Ki(σ1)/Ki(σ2)
30.1 11.3
6.67 0.36
5.99 1.72
121 10
2.96 0.15
17.4 2.2
10.2 4.2
20.9 0.2
7.42 1.11
13.9 7.5
508 132
444 242
22.6 0.5
35.0 10.1
41.0 10.5
127 48
10.2
0.4
21a
22a
23a
24a
25a
27a
28a
29a
30a
31a
32a
36
0.6
5.8
19.5 4.2
6.46 1.45
167 49
2.6
0.5
0.3
524
6
1.2
296 104
309 21
146 65
13.1
8.8
3.6
187
3
1.5
137 20
19.2 1.6
240 115
13.3 6.3
98 39
10.5 0.7
18.2 3.3
18.0 1.4
10.0 0.3
15.2 0.1
60.2 1.9
30.0 0.4
23.5 5.8
6.95 1.63
28.2 0.9
3.08 0.68
0.12
13.0
1.1
38
39
13.3
1.3
41
43
6.5
44
1300 305
181 25
155 27
330 25
102 15
4.69 2.36
17
21.6
6.0
45
46
6.6
b
c
ISO-1
ISO-1
47.5
3.6
c
siramesine
siramesine
siramesine
1.5
d
e
140
0.8
10.5 2.6
4.95 1.74
12.6 0.1
20.7 0.07
f
haloperidol
0.2
a
Values are the means
standard deviation (SD) of at least two
b c d
experiments performed in triplicate. From ref 10. From ref 23. IC50
value, from ref 31. From ref 32. From ref 33.
e
f
ISO-1. Similar to our previous findings, ferrocene precursors with
a carbonyl group displayed comparable affinity for σ2 receptors to
the corresponding rhenium complexes (36 vs 20a, 38 vs 22a, 39
vs 23a, and 41 vs 25a) and a little higher subtype selectivity.
Considering the low nanomolar affinity and subtype selectivity
of compound 20a for σ2 receptors, its affinities for the additional
receptors and transporters were further tested. Compound 20a
exhibited very low affinity for VAChT (Ki(VAChT) = 7026
163 nM) and was thus characterized by an excellent selectivity for
σ2 receptors (Ki(VAChT)/Ki(σ2) = 2374). Moreover, this ligand
also displayed low affinity for dopamine D2L receptors, NMDA
receptors, opiate receptors, dopamine transporter (DAT),
norepinephrine transporter (NET), and serotonin transporter
(SERT) as shown in Table S6.
Figure 2. Crystal structure of compound 31a.
to the cyclopentadienyl ring, and the coordination sphere is
completed by three carbonyl groups. The average bond lengths
of Re−C (Cp) and Re−C (CO) are 2.30 and 1.91 Å, respectively.
The bond angle of C−Re−C (between CO) is approximately
90°. The distance between the N atom and the center of the
aromatic ring of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
residue is 3.70 Å. The distance between the N atom and the
center of the Cp ring of the [CpM(CO)3] core is 5.05 Å.
In Vitro Radioligand Competition Studies. The radio-
ligand competition experiments were conducted as previously
reported.20 Typically, (+)-[3H]pentazocine was used for the σ1
receptors, and [3H]1,3-di-o-tolyl-guanidine ([3H]DTG) in the
presence of 10 μM dextrallorphan was used for the σ2 receptors.
The affinities of the rhenium complexes and ferrocene precursors
for σ1 and σ2 receptors are listed in Table 1. In general, the carbon
length of the linker displayed significant influence on the affinity
and subtype selectivity. Rhenium complexes with a carbonyl
group displayed higher affinities for σ2 receptors than those with
an amide group (20a−25a vs 27a−32a). Compound 20a and
24a possessed nanomolar affinity for σ2 receptors and moderate
subtype selectivity. Compounds 23a,29 29a,30 and 30a displayed
comparable affinity for σ2 receptors and subtype selectivity to
Radiolabeling. Considering the moderate to high affinity
and selectivity of the rhenium complexes 20a−25a and 29a−30a
for σ2 receptors, the corresponding 99mTc-labeled radiotracers
were prepared. The synthetic routes of the ferrocene precursors
were similar to those of the corresponding rhenium compounds
as shown in Scheme 2. The desired 99mTc-labeled radiotracers
were obtained via DLT reaction as shown in Scheme 3. After
purification by semipreparative high performance liquid
chromatography (HPLC), [99mTc]20b−25b ([99mTc]20b,
[
99mTc]22b, [99mTc]23b and [99mTc]25b),29 and [99mTc]29b−
30b ([99mTc]29b)30 were obtained with radiochemical yields of
13−67% and a radiochemical purity of >99%.
In Vitro Evaluation of the 99mTc-labeled Complexes.
Lipophilicity. For in vitro properties, the lipophilicity of the
C
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a
complexes with an amide group, the cellular association of
[
Scheme 2. Synthetic Routes for the Ferrocene Precursors
99mTc]29b and [99mTc]30b reached 4.91% and 6.89% at 45 min
and increased slightly afterward to 5.12% and 7.08% at 120 min,
respectively. In blocking studies, treatment with haloperidol and
ISO-1 significantly decreased the cellular association of [99mTc]
20b and [99mTc]23b in a dose-dependent manner, indicating the
specific binding of [99mTc]20b and [99mTc]23b to σ2 receptors in
DU145 cells. Moreover, treatment with various concentrations
of haloperidol led to decreased association of [99mTc]29b and
[
99mTc]30b in a dose-dependent manner. However, [99mTc]29b
showed a higher blocking percentage than [99mTc]30b (66% vs
47%) at the same concentration of haloperidol (10−4 M),
suggesting a higher specific binding of [99mTc]29b to σ receptors
in DU145 cells.
In Vitro Evaluation of the 99mTc-Labeled Complexes in
C6 Glioma Cells. C6 glioma tumor cells, with high expression of
σ2 receptors and low expression of σ1 receptors,3 were selected
for further evaluation of [99mTc]20b and [99mTc]29b. The results
are shown in Figure 4. High cellular association of [99mTc]20b
was observed in a time-dependent manner. The association
reached 25% after incubation for 120 min. Furthermore,
coincubation with different concentrations of ISO-1 led to
remarkably decreased association in a dose-dependent manner.
The cellular association was significantly reduced by about 80%
with 10−6 M of ISO-1, suggesting high specific binding of [99mTc]
20b to σ2 receptors in C6 glioma cells. However, much lower
uptake of [99mTc]29b (<1.49%) was observed. Treatment with
different concentrations of haloperidol also led to reduced
association of [99mTc]29b, but the blocking percentage was low
(37% with 10−4 M of haloperidol). In other words, [99mTc]20b
displayed higher association and higher specific binding to σ2
receptors than [99mTc]29b in C6 glioma cells.
a
Reagents and conditions: (a) toluene, 3 or 4, KI, Et3N, 115 °C, 16%
for 36, 19% for 37, 33% for 39 and 22% for 40; (b) acetonitrile, 3 or 4,
KI, K2CO3, 90 °C, 44% for 38 and 78% for 41; (c)13−16, Et3N,
anhydrous DMF, rt, 4 h, 65% for 43, 69% for 44, 63% for 45, 68% for
46.
radiotracer is an important factor in the prediction of its free
fraction in the plasma, the ability to cross the blood−brain barrier
(BBB) and nonspecific binding in vivo.34,35 The measurement of
the partition coefficients between 1-octanol and 0.05 M sodium
phosphate buffer at pH 7.4 was executed using a shake flask
method.22,36 The log D values of [99mTc]20b−25b and [99mTc]
29b−30b were 2.56 0.08,29 2.44 0.02, 2.56 0.08,29 2.61
0.05, 2.92 0.02, 2.60 0.06, 2.39 0.05,30 and 1.72 0.09,
respectively. The moderate lipophilicity of the above radiotracers
may lead to decreased nonspecific binding and enhanced specific
binding signal in vivo.
In Vitro Evaluation of the 99mTc-Labeled Complexes in
DU145 Prostate Cells. It has been reported that DU145
prostate tumor cells exhibit high expression of both σ1 and σ2
receptors.4 Considering the affinity and subtype selectivity for σ2
receptors, the binding assays of the radioligands [99mTc]20b,
Since compounds 21a and 24a had moderate to high affinity
for σ2 receptors, the binding assays of the corresponding [99mTc]
21b and [99mTc]24b were performed in C6 glioma cells. The
results are provided in Figure S3. The cellular association of
99mTc]21b and [99mTc]24b was 4.88% and 5.12%, respectively,
[
99mTc]23b, and [99mTc]29b−30b in DU145 prostate tumor
[
but treatment with 10−6 M of haloperidol only led to minimal
reduction of the cellular association, suggesting high nonspecific
binding of [99mTc]21b and [99mTc]24b in C6 glioma cells.
cells were performed. The results are summarized in Figure 3.
After incubation for 120 min, the cellular association of [99mTc]
20b and [99mTc]23b were 8.86% and 2.06%, respectively. For the
a
Scheme 3. Synthesis of the 99mTc-Labeled Complexes from the Ferrocene Precursors
a
−
Reagents and conditions: (a) 99mTcO4 , Mn(CO)5, DMF, 140 °C, 1 h, 13−32% for [99mTc]20b, 37−59% for [99mTc]21b, 25−33% for [99mTc]22b,
34−53% for [99mTc]23b, 44−62% for [99mTc]24b, 57−63% for [99mTc]25b; (b) 99mTcO4 , Mn(CO)5, DMF, 150 °C, 1 h, 36−67% for [99mTc]29b,
−
48−60% for [99mTc]30b.
D
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Figure 3. In vitro studies on cellular association of [99mTc]20b, [99mTc]23b, [99mTc]29b, and [99mTc]30b in DU145 prostate tumor cells.
Figure 4. In vitro studies on cellular association of [99mTc]20b and [99mTc]29b in C6 glioma cells.
a
Table 2. Biodistribution of [99mTc]20b in Male ICR Mice
organ
blood
2 min
15 min
30 min
60 min
120 min
240 min
1.82 0.18
2.57 0.31
11.81 1.58
4.76 1.29
3.43 0.89
29.38 4.97
14.46 1.08
4.07 0.70
0.96 0.09
2.42 0.78
0.20 0.03
0.81 0.07
2.70 0.18
3.25 0.22
10.03 0.61
7.46 0.81
9.61 2.58
15.48 1.92
11.05 1.77
2.42 0.74
2.16 0.22
0.17 0.05
0.71 0.07
2.08 0.22
2.07 0.23
12.51 1.88
7.15 1.44
7.14 2.44
11.78 1.04
11.69 3.19
2.67 0.74
1.74 0.20
0.17 0.03
0.57 0.11
1.06 0.17
1.31 0.21
13.41 0.71
4.67 0.38
3.77 0.80
8.92 1.17
14.01 2.20
1.46 0.40
1.40 0.18
0.09 0.02
0.43 0.07
0.47 0.08
0.83 0.14
16.14 1.73
2.76 0.58
2.19 0.69
6.62 0.99
15.08 1.87
1.56 0.77
0.78 0.23
0.12 0.01
0.31 0.04
0.17 0.04
0.43 0.07
17.33 3.11
1.24 0.36
1.47 0.53
6.11 1.21
12.94 2.36
1.23 0.26
0.24 0.11
0.08 0.03
brain
heart
liver
spleen
lung
kidney
small intestine
b
b
stomach
muscle
b
thyroid
a
b
Data are expressed as a percentage of injected dose per gram, means SD, n = 5. Percentage of injected dose per organ.
In Vivo Evaluation of the 99mTc-Labeled Complexes.
summarized in Tables 2−4, Figure 5, and Table S7 and Figure S4.
Both [99mTc]20b and [99mTc]23b exhibited high initial brain
uptake with 2.57 0.31% inject dose (ID)/g and 2.38 0.31%
ID/g at 2 min, respectively, and fast washout with 0.17 0.04%
ID/g and 0.09 0.01% ID/g, respectively, at 240 min. However,
Biodistribution and Blocking Studies in Male ICR Mice. To
investigate the kinetics of the radiotracers, we performed
biodistribution and blocking studies of [99mTc]20b, [99mTc]
23b, [99mTc]25b, and [99mTc]29b in ICR mice. The results are
E
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a
Table 3. Biodistribution of [99mTc]23b in Male ICR Mice
organ
blood
2 min
15 min
30 min
60 min
120 min
240 min
2.11 0.15
2.38 0.31
8.73 0.81
7.51 1.11
4.39 1.34
27.20 5.00
18.88 1.30
6.13 1.02
1.32 0.29
2.67 0.35
0.11 0.05
1.06 0.09
1.79 0.20
1.93 0.13
13.91 0.92
5.59 0.51
7.13 1.54
12.45 1.30
8.75 2.27
2.54 0.44
1.66 0.23
0.18 0.05
0.89 0.07
1.08 0.13
1.29 0.09
17.03 1.13
4.08 0.48
4.10 1.23
9.91 0.94
11.22 1.74
2.74 0.24
1.22 0.15
0.08 0.01
0.74 0.06
0.52 0.02
0.79 0.09
20.36 1.30
2.64 0.13
2.67 0.80
10.32 1.37
15.53 2.24
3.27 0.13
0.75 0.11
0.11 0.01
0.54 0.11
0.20 0.02
0.51 0.02
22.63 1.38
1.20 0.23
1.76 0.40
9.86 1.09
19.15 2.63
2.77 0.45
0.38 0.06
0.09 0.04
0.44 0.15
0.09 0.01
0.34 0.02
22.82 3.22
0.66 0.22
1.29 0.23
9.22 1.82
15.89 1.14
1.58 0.57
0.21 0.07
0.07 0.02
brain
heart
liver
spleen
lung
kidney
small intestine
b
b
stomach
muscle
b
thyroid
a
b
Data are expressed as the percentage of injected dose per gram, means SD, n = 5. Percentage of injected dose per organ.
a
Table 4. Biodistribution of [99mTc]29b in Male ICR Mice
organ
blood
2 min
15 min
30 min
60 min
120 min
240 min
2.04 0.29
0.24 0.06
11.22 1.20
9.27 1.69
5.08 1.42
31.54 3.28
27.45 4.91
6.00 1.31
1.27 0.32
4.12 0.61
0.11 0.02
0.74 0.06
0.21 0.02
3.27 0.20
18.56 2.62
7.32 0.65
12.11 2.08
17.68 3.19
13.15 1.09
2.59 0.47
2.02 0.16
0.08 0.02
0.53 0.03
0.16 0.02
2.27 0.24
24.43 1.60
4.50 0.64
8.93 1.88
14.62 0.61
10.62 2.49
2.58 1.16
1.44 0.21
0.14 0.03
0.43 0.05
0.11 0.02
1.74 0.13
27.13 2.13
1.91 0.58
7.63 2.40
12.14 1.81
11.92 3.25
2.17 0.79
0.98 0.13
0.09 0.01
0.41 0.04
0.11 0.01
1.45 0.20
27.98 2.32
1.21 0.18
4.17 0.42
10.21 0.51
22.56 3.75
2.32 0.92
0.68 0.10
0.06 0.01
0.34 0.03
0.10 0.01
1.01 0.16
28.45 3.32
0.78 0.12
4.03 0.54
8.79 1.04
17.64 4.15
1.46 0.41
0.41 0.03
0.05 0.03
brain
heart
liver
spleen
lung
kidney
small intestine
b
b
stomach
muscle
b
thyroid
a
b
Data are expressed as the percentage of injected dose per gram, means SD, n = 5. Percentage of injected dose per organ.
[
99mTc]25b exhibited lower brain uptake (1.05 0.06% ID/g)
radiotracer were performed in Balb/c nude mice bearing C6
glioma xenografts. The results are shown in Table 5 and Figure 6.
High accumulation of the radiotracer in the tumor was observed
with 5.41 0.91% ID/g at 120 min and 5.92 1.30% ID/g at
240 min. At the same time, low accumulation of the radiotracer in
blood and muscle were observed as well. Therefore, high tumor/
blood ratios and tumor/muscle ratios were obtained with 20.6
and 16.1 at 240 min, respectively. To further examine the specific
binding to σ receptors in the tumor, blocking studies with
haloperidol, ferrocene precursor 36, and siramesine as blocking
agents were performed. Pretreatment of haloperidol, 36, and
siramesine led to significant reduction of tumor accumulation by
33%, 62%, and 44% at 240 min, respectively, indicating the
specific binding of [99mTc]20b to σ receptors in the tumor. In
addition, a reduction of radiotracer in the organs known to
contain σ receptors such as the brain, heart, and lung were
observed, which is consistent with the biodistribution results in
normal mice.
and relatively slow washout with 0.32 0.04% ID/g at 240 min.
[
99mTc]20b, [99mTc]23b, and [99mTc]25b exhibited fast
clearance from blood and muscle. Low accumulation in the
blood of the above radiotracers was observed with 0.31 0.04%
ID/g, 0.44 0.15% ID/g, and 0.30 0.05% ID/g at 240 min,
respectively. Low accumulation in the muscle was also observed
with 0.24 0.11% ID/g, 0.21 0.07% ID/g, and 0.49 0.05%
ID/g at 240 min, respectively. In biodistribution studies of
[
99mTc]29b (Table 4), much lower brain uptake was observed
with 0.24 0.06% ID/g at 2 min, indicating that the amide group
had a negative effect on the potential of the radiotracer to cross
the BBB. Low accumulation of this radiotracer in blood and
muscle at 240 min was observed.
To examine the specific binding of the radiotracers to σ
receptors in vivo, blocking studies were carried out by
administration of haloperidol (1.0 mg/kg) 5 min prior to the
radiotracer administration. Pretreatment with haloperidol
significantly reduced the accumulation of [99mTc]20b, [99mTc]
23b, and [99mTc]29b in the brain by 43%, 45%, and 36%,
respectively, at 120 min after injection (Figure 5). Moreover,
remarkable reduction of radiotracers in the organs known to
express σ receptors was observed, indicating the specific binding
of [99mTc]20b, [99mTc]23b, and [99mTc]29b to σ receptors in
vivo. However, there is no significant reduction of radiotracer
accumulation observed in the brain for [99mTc]25b (Figure S4),
suggesting the high nonspecific binding of [99mTc]25b in vivo.
Biodistribution and Blocking Studies of [99mTc]20b in
Balb/c Nude Mice Bearing C6 Glioma Xenografts.
Encouraged by the optimal properties of [99mTc]20b in vitro
and in vivo, biodistribution and blocking studies of this
Small Animal SPECT/CT Imaging of [99mTc]20b. To
further confirm the potential applications of [99mTc]20b in
tumor imaging, small animal SPECT/CT scans of the C6 glioma
xenograft mouse model were performed using NanoScan
SPECT/CT. Representative transverse plane SPECT/CT
images at 180 min after injection of [99mTc]20b (22.2 MBq,
0.15 mL) are shown in Figure 7, and the coronal and sagittal
plane images are shown in the Figure S5. The solid tumor was
clearly visualized with high tumor-to-background contrast,
indicating that [99mTc]20b could determine the level of σ2
receptors in solid tumors in vivo.
In Vivo Radiometabolic Stability of [99mTc]20b. The
metabolic stability of [99mTc]20b was investigated in brain and
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Figure 6. Effects of pretreatment with haloperidol, 36, and siramesine
(0.1 mL, 1.0 mg/kg) on organ biodistribution of [99mTc]20b in Balb/c
nude mice bearing C6 glioma xenografts. Student’s t test (independent,
two-tailed) was performed, and p < 0.05 (except in the kidney with 36
and in the tumor with haloperidol at 240 min after intravenous
injection).
Figure 7. Representative transverse plane slices of NanoScan SPECT/
CT fusion images of [99mTc]20b (22.2 MBq, 0.15 mL) in male Balb/c
nude mouse bearing C6 glioma xenografts at 180 min after injection.
Isoflurane was used for anesthesia.
Figure 5. Effects of pretreatment with haloperidol (0.1 mL, 1.0 mg/kg)
on organ biodistribution of [99mTc]20b (A), [99mTc]23b (B), or [99mTc]
29b (C) in male ICR mice (n = 5). Student’s t test (independent, two-
tailed) was performed, and p < 0.05 (except for [99mTc]20b in the lungs
and kidney at 120 min after intravenous injection, [99mTc]23b in the
heart and lungs at 60 min after intravenous injection, and [99mTc]29b in
the spleen at 60 min after intravenous injection).
The parent radiotracer [99mTc]20b was the main radioactive
compound presented in the brain at both 15 and 30 min,
indicating that [99mTc]20b was very stable in the brain and that
there was no entry of radioactive metabolites into the brain. In
the liver samples, three main metabolites with retention times of
2.4 min (M1), 4.6 min (M2), and 5.4 min (M3), respectively,
were observed. Furthermore, the percentage of the parent
compound [99mTc]20b was decreased with time (31.8% and
16.1% at 15 and 30 min, respectively). The percentage of M1 was
significantly increased with 45.8% and 65.6% at 15 and 30 min,
respectively.
Table 5. Biodistribution of [99mTc]20b in Balb/c Nude Mice
a
Bearing C6 Glioma Xenografts
organ
blood
120 min
240 min
0.32 0.05
0.68 0.21
0.80 0.09
18.44 3.58
5.48 0.58
2.92 0.74
6.86 1.16
15.26 1.06
0.82 0.19
0.65 0.11
0.08 0.01
5.41 0.91
17.5 4.0
0.28 0.03
0.30 0.05
0.55 0.08
22.47 2.37
2.69 0.20
2.04 0.49
5.93 0.62
16.33 3.12
0.83 0.34
0.37 0.06
0.08 0.01
5.92 1.30
20.6 4.1
brain
heart
liver
spleen
lung
DISCUSSION
■
kidney
small intestine
A number of studies have demonstrated the high expression of σ2
receptors in many human tumors.2−4 The σ2 receptor has proved
to be a unique biomarker of proliferative status in solid
tumors.7−9 Therefore, the development of molecular probes
for imaging σ2 receptors will provide useful information on the
proliferative status of tumors in patients. Recently, [18F]ISO-1
was developed as a potential σ2 receptor PET radiotracer for
imaging the proliferative status of tumors.10−12 However, there is
still need for a σ2 receptor SPECT imaging radioligands in clinical
trials. 99mTc is the most common and convenient nuclide for
SPECT imaging in clinical use. Furthermore, 99mTc/188Re is
considered as the ideal “matched pair” of theragnostic radio-
nuclides. Development of the 99mTc-labeled radiotracer will
provide useful information on the synthesis and therapeutic
b
b
stomach
muscle
b
thyroid
tumor
tumor/blood
tumor/muscle
8.5 1.6
16.1 5.3
a
Data are expressed as the percentage of injected dose per gram,
b
means SD, n = 5. Percentage of injected dose per organ.
liver samples of ICR mice 15 and 30 min after the injection of the
radiotracer. The HPLC chromatograms are shown in Figure 8.
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Figure 8. Analytical HPLC chromatograms of radioactive compounds in mouse brain and liver samples after intravenous injection of [99mTc]20b (18.5
MBq, 0.15 mL). (A) Brain and liver samples at 15 min. (B) Brain and liver samples at 30 min. (C) [99mTc]20b.
doses of the corresponding 188Re-labeled radiopharmaceuticals.
Therefore, 99mTc-labeled radiotracers for σ2 receptors imaging
not only could be used in the early diagnosis of cancer but also
could provide useful information for the therapeutic tumor
medicine of 188Re-labeled radiopharmaceuticals. [99mTc] [N-[2-
((3′-N′-propyl-[3,3,1]aza-bicyclononan-3α-yl)(2″-methoxy-5-
methyl-phenylcarbamate)(2-mercapttoethyl)amino)acetyl]-2-
amino-ethanethiolato]technetium(V) oxide ([99mTc]47) was
the most potential σ2 receptor radiotracer ever reported with
moderate affinity for σ2 receptors (Ki = 22 nM) and high subtype
selectivity as well as visualization of tumor shape in planar gamma
imaging.18 However, no further evaluation, such as SPECT/CT
imaging, was reported since 2001. To the best of our knowledge,
only the radiotracers with high tumor uptakes and high tumor-to-
muscle ratios could have potential applications in the early
diagnosis of tumor. Because of the much higher expression of the
σ2 receptors in tumors and moderate to high expression of the σ1
receptors in the normal organs, development of highly subtype
selective 99mTc-labeled σ2 receptor radioligands with high tumor
uptakes and high tumor/background ratios will have wide
applications in imaging the proliferative status in solid tumors.
Our aim in this article is to develop novel 99mTc-labeled
radiotracers with high affinity for σ2 receptors and high selectivity
as well as clear visualization of solid tumors.
Enlightened by our previous results of 99mTc-labeled radio-
tracers with the [(Cp-R)Tc(CO)3] unit,22,25 we replaced the
aromatic ring of ISO-1 with a [(Cp-R)M(CO)3] unit with an
electron-withdrawing group and connected it to a 5,6-
dimethoxyisoindoline or 6,7-dimethoxy-1,2,3,4-tetrahydroiso-
quinoline scaffold via different carbon length linkers. The
designed complexes were in accordance with the σ2 receptor
ligand pharmacophore model proposed by Glennon with an
amine binding site flanked by two hydrophobic regions.37 It is
encouraging that complexes 20a, 23a, 29a, and 30a have high
affinity and subtype selectivity for σ2 receptors. Among these
ligands, compound 20a had low nanomolar affinity and higher
subtype selectivity than ISO-1. Moreover, it showed high
selectivity toward VAChT (2374-fold), dopamine D2L receptors,
NMDA receptors, opiate receptors, DAT, NET, and SERT.
H
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To detect the specific binding of the radiotracers to σ2
receptors, we selected DU145 human prostate tumor cells
(Bmax values of σ1 and σ2 receptors were 1800 and 1930 fmol/mg
protein, respectively)4 and C6 rat glioma cells (Bmax values of σ1
and σ2 receptors were 42 and 5507 fmol/mg protein,
respectively).3 In the in vitro binding assays in tumor cells,
20a possessed higher affinity and subtype selectivity for σ2
receptors. The corresponding radiotracer [99mTc]20b showed
high uptakes and specific binding to σ receptors in DU145
prostate cells and C6 glioma cells. Moreover, [99mTc]20b
exhibited high tumor uptake and high tumor/blood and tumor/
muscle ratios in nude mice bearing C6 glioma xenografts.
Furthermore, this radiotracer demonstrated specific binding to σ
receptors in the tumor. In particular, animal SPECT/CT imaging
studies with [99mTc]20b demonstrated a high uptake and clear
visualization of C6 glioma xenografts in nude mice. These
findings demonstrated that the further evaluation of [99mTc]20b
as a potential σ2 receptor probe for imaging the proliferative
status in brain tumors is worth doing.
[
99mTc]20b exhibited much higher cellular association and
comparable specific binding to σ2 receptors in DU145 cells than
[
99mTc]23b, which is in good agreement with the higher affinity
of the former compound. Coincubation with ISO-1 led to
significant reduction of cellular association in a dose-dependent
manner, indicating the specific binding of [99mTc]20b to σ2
receptors. [99mTc]29b exhibited comparable cellular association
and higher specific binding to σ receptors in DU145 cells than
[
99mTc]30b. Thus, it seemed that the 5,6-dimethoxyisoindoline
EXPERIMENTAL SECTION
■
moiety is a better σ2 preferring group than the 6,7-dimethoxy-
All reagents used in the synthesis were commercial products and were
used without further purification unless otherwise indicated. 99mTc-
pertechnetate was eluted from a commercial 99Mo−99mTc generator
obtained from Beijing Atomic High-tech Co. The 1H NMR spectra were
recorded on Bruker Avance III NMR (400 MHz) spectrometers in
CDCl3 solutions at room temperature with TMS as an internal standard.
Chemical shifts (δ) were reported in ppm values relative to the internal
TMS. Coupling constants (J) were reported in Hertz (Hz). Multiplicity
is defined by s (singlet), d (doublet), t (triplet), and m (multiplet). The
13C NMR spectra were recorded on Bruker Avance III NMR (100 MHz)
spectrometers. Mass spectra were acquired by Quattro micro API ESI/
MS (Waters, USA). High-resolution mass spectrometry (HRMS) was
performed on a LCT Premier XE ESI-TOF mass spectrometry
instrument (Waters, USA). X-ray crystallography data were collected
on a Bruker Smart APEX II diffractometer (Bruker Co., Germany). The
melting point (Mp) of solid compounds was tested on a WRX-4 micro
melting point apparatus (Shanghai yice instrument Co., LTD, China)
and was uncorrected. Reactions were monitored by TLC (TLC Silica gel
60 F254, Merck). Flash column chromatography was conducted using
silica gel (45−75 μm) from Qingdao Haiyang Chemical Co., Ltd. The
compounds were visualized by illumination with a short wavelength UV
lamp (λ = 254 nm). High performance liquid chromatography (HPLC)
separations and analyses were performed on a Waters 600 system
(Waters, corporation, USA) equipped with a Waters 2489 UV−VIS
detector and a Raytest Gabi NaI (Tl) scintillation detector (Raytest,
Germany) and on a Shimadzu SCL-20AVP system (Shimadzu
Corporation, Japan) equipped with a Bioscan Flow Count 3200 NaI/
PMT γ-radiation scintillation detector. 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 of the
final compounds were analyzed by HPLC with a purity of more than
95% (HPLC profiles are shown in the Supporting Information).
Male ICR mice (4−5 weeks, 22−25 g) were purchased from Vital
River Laboratory Animal Technology Co. Ltd. All procedures related to
animal experiments were performed in compliance with relevant laws
and institutional guidelines. All protocols requiring the use of mice were
approved by the animal care committee of Beijing Normal University.
Chemistry. 2-(5,6-Dimethoxyisoindolin-2-yl)acetonitrile (5).
Compound 3 (254.0 mg, 1.42 mmol) and 5 mL of triethylamine were
dissolved in 10 mL of CH2Cl2, followed by the addition of 2-
bromoacetonitrile (200.0 mg, 1.67 mmol). The reaction mixture was
stirred at room temperature for 24 h. The mixture was then washed with
H2O and extracted with CH2Cl2. The organic layer was dried over
MgSO4, filtered, and concentrated under vacuum. The residue was
purified by silica gel chromatography (dichloromethane/methanol =
100:1) to afford 5 (219.4 mg, 72%) as a white solid. Mp: 125.5−125.9
°C. 1H NMR (400 MHz, CDCl3) δ 6.76 (s, 2H), 4.07 (s, 4H), 3.87 (s,
6H), 3.81 (s, 2H). ESI-MS: [M + H]+ (m/z = 219.2).
1,2,3,4-tetrahydroisoquinoline moiety. In C6 glioma cells,
[
99mTc]20b displayed much higher cellular association and
higher specific binding to σ receptors than [99mTc]29b,
indicating that [99mTc]20b is the most potent radioligand in
this series and warrants further evaluation.
To investigate the kinetics and examine the specific binding of
the radiotracers in vivo, biodistribution studies of [99mTc]20b,
[
[
99mTc]23b, and [99mTc]29b were performed in male ICR mice.
99mTc]20b and [99mTc]23b displayed high initial brain uptake
with 2.57 and 2.38% ID/g at 2 min after injection, respectively.
[
99mTc]29b exhibited low brain uptake with 0.24% ID/g at 2 min
after injection, indicating that the amide and carbonyl group
showed different influences on the potential of the radiotracer to
cross the BBB. Interestingly, all the radiotracers mentioned
above exhibited fast clearance from the muscle and blood, which
is very important for tumor imaging agents. Pretreatment with
haloperidol significantly reduced the accumulation in the brain,
indicating the specific binding of [99mTc]20b, [99mTc]23b, and
[
99mTc]29b to σ receptors in vivo.
Finally, we selected the most potent radioligand [99mTc]20b
and evaluated its potential applications in tumor imaging. In
biodistribution studies in Balb/c nude mice bearing C6 glioma
xenografts, [99mTc]20b showed much higher tumor uptake
(5.41−5.92% ID/g) than [99mTc]47 (1.11−2.11% ID/g, 66
murine breast tumor)18 and [18F]ISO-1 (0.64−3.67% ID/g,
EMT-6 mouse mammary tumor).10 99mTc]20b also exhibited
[
fast clearance from the surrounding tissues. Thus, the tumor-to-
muscle ratios (8.5−16.1) are higher than those of [99mTc]47
(0.57−4.95) (16 vs 4.95 at 4 h).18 Moreover, the tumor-to-blood
ratios (17.5−20.6) are significantly higher than those of [99mTc]
47 (0.56−2.21)18 and [18F]ISO-1 (1.47−2.19).10 Pretreatment
with haloperidol, 36, and siramesine resulted in a remarkable
reduction of radiotracer accumulation in the tumors, indicating
the specific binding of [99mTc]20b to σ receptors in C6 glioma
tumors in vivo. Consistent with the high tumor uptakes and high
tumor-to-muscle ratios in biodistribution studies, the animal
SPECT/CT imaging of [99mTc]20b demonstrated a high uptake
and clear visualization of C6 glioma xenografts in nude mice at
180 min. These data suggest the potential use of [99mTc]20b for
SPECT imaging studies of brain tumors in patients.
CONCLUSIONS
■
By applying an integrated strategy, we have designed, prepared,
and evaluated a series of cyclopentadienyl tricarbonyl 99mTc/Re
complexes containing 5,6-dimethoxyisoindoline or 6,7-dime-
thoxy-1,2,3,4-tetrahydroisoquinoline moiety. Compared with
ISO-1, the rhenium complex of the corresponding radiotracer
3-(5,6-Dimethoxyisoindolin-2-yl)propanenitrile (6). The procedure
described for the synthesis of 5 was applied to compound 3 (251.2 mg,
1.40 mmol) and 3-bromopropanenitrile (223.7 mg, 1.67 mmol) to
afford 6 (215.5 mg, 67%) as a light brown solid. Mp: 137.1−137.5 °C.
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1H NMR (400 MHz, CDCl3) δ 6.75 (s, 2H), 4.03 (s, 4H), 3.86 (s, 6H),
3.12 (t, J = 7.0 Hz, 2H), 2.65 (t, J = 6.4 Hz, 2H). ESI-MS: [M + H]+ (m/z
= 233.2).
131.71, 105.88, 96.51, 87.90, 85.18, 59.26, 56.13, 55.93, 38.78, 28.72,
26.93, 24.31. HRMS (ESI-Tof), [M + H]+: m/z calcd. for
C24H27NO6185Re 610.1368; found 610.1362.
3-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-propylcarbo-
nylcyclopentadienyl Tricarbonyl Rhenium (23a). The procedure
described for the synthesis of 20a was applied to 17 (53.5 mg, 0.11
mmol) and 4 (32.0 mg, 0.16 mmol) to afford 23a as a yellow oil (19.7
mg, 30%). 1H NMR (400 MHz, CDCl3) δ 6.58 (s, 1H), 6.51 (s, 1H),
5.96 (t, J = 2.2 Hz, 2H), 5.34 (t, J = 2.2 Hz, 2H), 3.84 (s, 3H), 3.83 (s,
3H), 3.56 (s, 2H), 2.81−2.78 (m, 2H), 2.74−2.67 (m, 4H), 2.57 (t, J =
6.8 Hz, 2H), 2.03−1.96 (m, 2H). 13C NMR (125 MHz, CDCl3) δ
194.65, 191.90, 147.99, 147.58, 125.04, 111.31, 109.40, 96.15, 87.84,
85.25, 55.97, 55.95, 54.70, 50.23, 36.29, 29.71, 20.97. HRMS (ESI-Tof),
[M + H]+: m/z calcd. for C23H25NO6185Re 596.1212; found 596.1215.
4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butylcarbo-
nylcyclopentadienyl Tricarbonyl Rhenium (24a). The procedure
described for the synthesis of 20a was applied to 18 (60 mg, 0.12
mmol) and 4 (50.1 mg, 0.26 mmol) to afford 24a as a yellow oil (22 mg,
36%). 1H NMR (400 MHz, CDCl3) δ 6.59 (s, 1H), 6.52 (s, 1H), 6.00 (t,
J = 2.3 Hz, 2H), 5.33 (t, J = 2.3 Hz, 2H), 3.84 (s, 3H), 3.83 (s, 3H), 3.58
(s, 2H), 2.83 (t, J = 5.2 Hz, 2H), 2.74 (t, J = 5.4 Hz, 2H), 2.65 (t, J = 7.0
Hz, 2H), 2.56 (t, J = 7.0 Hz, 2H), 1.82−1.74 (m, 2H), 1.73−1.63 (m,
2H). 13C NMR (100 MHz, CDCl3) δ 195.10, 191.91, 147.59, 147.28,
126.65, 126.21, 111.46, 109.62, 96.18, 87.94, 85.08, 57.48, 55.96, 55.75,
50.91, 38.58, 29.71, 28.64, 26.29, 22.44. HRMS (ESI-Tof), [M + H]+:
m/z calcd. for C24H27NO6185Re 610.1368; found 610.1366.
5-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-pentylcarbo-
nylcyclopentadienyl Tricarbonyl Rhenium (25a). The procedure
described for the synthesis of 22a was applied to 19 (78.5 mg, 0.15
mmol) and 4 (45.2 mg, 0.23 mmol) to afford 25a as a colorless oil (48.6
mg, 51%). 1H NMR (400 MHz, CDCl3) δ 6.59 (s, 1H), 6.52 (s, 1H),
5.98 (t, J = 2.2 Hz, 2H), 5.38 (t, J = 2.2 Hz, 2H), 3.84 (s, 3H), 3.83 (s,
3H), 3.55 (s, 2H), 2.82 (t, J = 5.7 Hz, 2H), 2.70 (t, J = 5.8 Hz, 2H), 2.61
(t, J = 7.3 Hz, 2H), 2.51 (t, J = 7.5 Hz, 2H), 1.77−1.69 (m, 2H), 1.68−
1.59 (m, 2H), 1.45−1.37 (m, 2H). 13C NMR (125 MHz, CDCl3) δ
195.20, 191.88, 147.47, 147.17, 126.73, 126.25, 111.36, 109.50, 96.15,
87.89, 85.18, 58.14, 55.92, 55.91, 55.86, 51.10, 38.79, 28.72, 27.10, 27.04,
24.32. HRMS (ESI-Tof), [M + H]+: m/z calcd. for C25H29NO6185Re
624.1525; found 624.1519.
2-(5,6-Dimethoxyisoindolin-2-yl)-ethylaminocarbonylcyclopen-
tadienyl Tricarbonyl Rhenium (27a). Under N2, the solution of
compound 11 (60.0 mg, 0.27 mmol) in 1.5 mL of anhydrous DMF and
100 μL of triethylamine was added to the solution of compound 26
(98.2 mg, 0.18 mmol) in 1 mL of anhydrous DMF dropwise. The
reaction mixture was stirred at room temperature for 4 h. The solvent
was removed, washed with saturated sodium chloride, and extracted
with ethyl acetate. The organic layer was dried over anhydrous MgSO4,
filtered, and concentrated under vacuum. The residue was purified by
silica gel chromatography (ethyl ether/hexane/triethylamine = 10:5:1)
to afford 27a (49.9 mg, 48%) as a pale yellow solid. Mp: 165.1−167.1
°C. 1H NMR (400 MHz, CDCl3) δ 7.16 (br s, 1H), 6.76 (s, 2H), 6.04 (s,
2H), 5.34 (t, J = 2.2 Hz, 2H), 4.12 (s, 4H), 3.87 (s, 6H), 3.61 (q, J = 5.4
Hz, 2H), 3.07 (t, J = 5.3 Hz, 2H).13C NMR (100 MHz, CDCl3) δ
192.58, 162.28, 148.64, 131.10, 105.93, 95.13, 86.03, 84.66, 58.99, 56.15,
54.36, 38.12. HRMS (ESI-Tof), [M + H]+: m/z calcd for
C22H21N2O6185Re 583.1008, found 583.0995.
3-(5,6-Dimethoxyisoindolin-2-yl)-propylaminocarbonylcyclopen-
tadienyl Tricarbonyl Rhenium (28a). The procedure described for the
synthesis of 27a was applied to the compound 12 (40.0 mg, 0.17 mmol)
and compound 26 (60.0 mg, 0.11 mmol) to afford 28a (26.1 mg, 40%)
as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 9.05 (br s, 1H), 6.82
(s, 2H), 5.48 (t, J = 2.2 Hz, 2H), 5.10 (t, J = 2.2 Hz, 2H), 3.97 (s, 4H),
3.88 (s, 6H), 3.53 (q, J = 5.4 Hz, 2H), 3.03 (t, J = 5.4 Hz, 2H), 1.84−1.78
(m, 2H). HRMS (ESI-Tof), [M + H]+: m/z calcd for C22H24N2O6185Re
597.1164, found 597.1177.
4-(5,6-Dimethoxyisoindolin-2-yl)butanenitrile (7). The procedure
described for the synthesis of 5 was applied to 3 (254.5 mg, 1.42 mmol)
and 4-bromobutanenitrile (247.2 mg, 1.67 mmol) to afford 7 (254.9 mg,
73%) as a light brown solid. 1H NMR (400 MHz, CDCl3) δ 6.75 (s, 2H),
4.04 (s, 4H), 3.87 (s, 6H), 2.97 (t, J = 6.7 Hz, 2H), 2.55 (t, J = 7.1 Hz,
2H), 2.02 (t, J = 6.7 Hz, 2H). ESI-MS: [M + H]+ (m/z = 247.2).
2-(5,6-Dimethoxyisoindolin-2-yl)ethanamine (11). A solution of
LiAlH4 (145.7 mg, 3.83 mmol) in 10 mL of anhydrous diethyl ether was
cooled at 0 °C (ice bath), followed by the addition of a solution of 5
(210.0 mg, 0.96 mmol) in 5 mL of anhydrous THF. Then, the reaction
mixture was stirred at room temperature overnight. The reaction
mixture was quenched with ice−H2O and extracted with ethyl acetate.
The organic layer was dried over anhydrous MgSO4, filtered, and
concentrated under vacuum. The residue was purified by silica gel
chromatography (dichloromethane/methanol/triethylamine = 20:1:1)
1
to give 11 (97.2 mg, 46%) as a light yellow oil. H NMR (400 MHz,
CDCl3) δ 6.75 (s, 2H), 3.91 (s, 4H), 3.86 (s, 6H), 2.90−2.87 (m, 2H),
2.84−2.81 (m, 2H), 2.04 (br s, 2H). ESI-MS: [M + H]+ (m/z = 223.2).
3-(5,6-Dimethoxyisoindolin-2-yl)propan-1-amine (12). The pro-
cedure described for the synthesis of 11 was applied to LiAlH4 (130.5
mg, 3.44 mmol) and compound 6 (200.0 mg, 0.86 mmol) to afford 12
(73.0 mg, 36%) as a brown oil. 1H NMR (400 MHz, CDCl3) δ 6.75 (s,
2H), 4.07 (s, 4H), 3.83 (s, 6H), 3.15 (q, 2H, J = 7.3 Hz), 1.74−1.67 (m,
2H), 1.47−1.32 (m, 2H). ESI-MS: [M + H]+ (m/z = 237.2).
4-(5,6-Dimethoxyisoindolin-2-yl)butan-1-amine (13). The proce-
dure described for the synthesis of 11 was applied to LiAlH4 (129.0 mg,
3.40 mmol) and compound 7 (210.0 mg, 0.85 mmol) to afford 13 (92.4
mg, 50%) as a brown oil. 1H NMR (400 MHz, CDCl3) δ 6.73 (s, 2H),
4.81 (br s, 2H), 3.93 (s, 4H), 3.85 (s, 6H), 2.84−2.81 (m, 2H), 2.76 (m,
2H), 1.76−1.66 (m, 4H). ESI-MS: [M + H]+ (m/z = 251.2).
3-(5.6-Dimethoxyisoindolin-2-yl)-propylcarbonylcyclopentadien-
yl Tricarbonyl Rhenium (20a). Compound 3 (23.0 mg, 0.13 mmol) was
added to a solution of 17 (57.7 mg, 0.12 mmol) and KI (20.0 mg, 0.12
mmol) in 5 mL of toluene and 3 mL of triethylamine. The mixture was
refluxed at 115 °C for 5 h. After cooling to room temperature, the
solvent was evaporated in vacuum. The residue was purified by silica gel
chromatography (ethyl acetate/petroleum ether/triethylamine = 1:3:1)
to afford 20a (8.6 mg, 10%) as a dark oil. 1H NMR (400 MHz, CDCl3) δ
6.74 (s, 2H), 5.98 (t, J = 2.2 Hz, 2H), 5.37 (t, J = 2.2 Hz, 2H), 3.88 (s,
4H), 3.85 (s, 6H), 2.78−2.71 (m, 4H), 2.00−1.93 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ 194.85, 191.91, 148.53, 131.52, 105.99, 96.61,
87.69, 85.11, 59.04, 56.19, 54.98, 36.36, 23.51. HRMS (ESI-Tof), [M +
H]+: m/z calcd for C22H23NO6185Re 582.1055; found 582.1045.
4-(5.6-Dimethoxyisoindolin-2-yl)-butylcarbonylcyclopentadienyl
Tricarbonyl Rhenium (21a). The procedure described for the synthesis
of 20a was applied to 18 (70 mg, 0.14 mmol) and 3 (51.9 mg, 0.29
mmol) to afford 21a as a dark oil (12.8 mg, 10%). 1H NMR (400 MHz,
CDCl3) δ 6.74 (s, 2H), 5.99 (t, J = 2.3 Hz, 2H), 5.36 (t, J = 2.3 Hz, 2H),
3.94 (s, 4H), 3.85 (s, 6H), 2.79 (t, J = 7.2 Hz, 2H), 2.66 (t, J = 7.0 Hz,
2H), 1.84−1.77 (m, 2H), 1.70−1.62 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 194.74, 191.90, 148.66, 130.90, 105.95, 96.24, 87.82, 85.13,
59.10, 56.19, 55.93, 38.41, 29.70, 27.69, 22.00. HRMS (ESI-Tof), [M +
H]+: m/z calcd. for C23H25NO6185Re 596.1212; found 596.1219.
5-(5.6-Dimethoxyisoindolin-2-yl)-pentylcarbonylcyclopentadien-
yl Tricarbonyl Rhenium (22a). Compound 3 (31.9 mg, 0.18 mmol) was
added to a solution of 19 (60.0 mg, 0.12 mmol), KI (32.1 mg, 0.19
mmol), and K2CO3 (25.0 mg, 0.18 mmol) in 10 mL of anhydrous
acetonitrile. The mixture was stirred at 90 °C for 5 h. After cooling to
room temperature, the mixture was poured with cold water and
extracted with CH2Cl2. The organic layer was dried over anhydrous
MgSO4, filtered, and concentrated under vacuum. The residue was
purified by silica gel chromatography (ethyl acetate/petroleum ether/
1
triethylamine = 1:3:1) to afford 22a (29.8 mg, 41%) as a dark oil. H
4-(5,6-Dimethoxyisoindolin-2-yl)-butylaminocarbonylcyclopen-
tadienyl Tricarbonyl Rhenium (29a). The procedure described for the
synthesis of 27a was applied to compound 13 (40.0 mg, 0.17 mmol) and
compound 26 (81.8 mg, 0.15 mmol) to afford 29a (34.4 mg, 38%) as a
pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.14 (br s, 1H), 6.78 (s,
NMR (400 MHz, CDCl3) δ 6.74 (s, 2H), 5.99 (t, J = 2.2 Hz, 2H), 5.39
(t, J = 2.2 Hz, 2H), 3.89 (s, 4H), 3.86 (s, 6H), 2.72 (t, J = 7.4 Hz, 2H),
2.62 (t, J = 7.3 Hz, 2H), 1.78−1.71 (m, 2H), 1.66−1.58 (m, 2H), 1.48−
1.42 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 195.20, 191.88, 148.35,
J
DOI: 10.1021/acs.jmedchem.5b01378
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2H), 5.57 (t, J = 2.0 Hz, 2H), 5.11 (t, J = 2.2 Hz, 2H), 3.98 (s, 4H), 3.87
(s, 6H), 3.41 (q, J = 4.9 Hz, 2H), 2.85 (t, J = 5.4 Hz, 2H), 1.76−1.75 (m,
4H). 13C NMR (100 MHz, CDCl3) δ 192.95, 162.03, 148.79, 131.16,
106.02, 96.86, 84.99, 84.57, 59.19, 56.17, 55.96, 39.71, 27.71, 26.81.
HRMS (ESI-Tof), [M + H]+: m/z calcd for C23H26N2O6185Re 611.1321,
found 611.1323.
Hz, 2H), 4.48 (t, J = 1.9 Hz, 2H), 4.19 (s, 5H), 3.84 (s, 3H), 3.83 (s, 3H),
3.61 (s, 2H), 2.83 (t, J = 7.1 Hz, 2H), 2.77 (s, 2H), 2.63 (s, 2H), 2.04−
2.01 (m, 2H). ESI-MS: [M + H]+ (m/z = 448.7).
4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butylcarbo-
nylferrocene (40). The procedure described for the synthesis of 20a was
applied to 34 (102.6 mg, 0.29 mmol) and 4 (116.0 mg, 0.60 mmol) to
afford 40 as an orange oil (25.7 mg, 22%). 1H NMR (400 MHz, CDCl3)
δ 6.59 (s, 1H), 6.53 (s, 1H), 4.79 (t, J = 1.9 Hz, 2H), 4.49 (t, J = 1.9 Hz,
2H), 4.19 (s, 5H), 3.84 (s, 3H), 3.83 (s, 3H), 3.59 (s, 2H), 2.84 (t, J = 5.6
Hz, 2H), 2.78−2.73 (m, 4H), 2.57 (t, J = 7.4 Hz, 2H), 1.81−1.70 (m,
4H). ESI-MS: [M + H]+ (m/z = 461.9).
2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-ethylamino-
carbonylcyclopentadienyl Tricarbonyl Rhenium (30a). The proce-
dure described for the synthesis of 27a was applied to compound 14
(74.4 mg, 0.32 mmol) and compound 26 (85.0 mg, 0.16 mmol) to afford
1
30a as a light yellow solid (70.4 mg, 74%). Mp: 133.6−134.9 °C. H
NMR (400 MHz, CDCl3) δ 6.62 (s, 1H), 6.53 (s, 1H), 5.96 (s, 2H), 5.33
(t, J = 2.2 Hz, 2H), 3.85 (d, J = 6.3 Hz, 6H), 3.66 (s, 2H), 3.58 (q, J = 5.1
Hz, 2H), 2.90 (s, 4H), 2.81 (s, 2H). 13C NMR (100 MHz, CDCl3) δ
192.57, 162.19, 147.76, 147.41, 125.89, 125.83, 111.46, 109.54, 95.17,
85.98, 84.66, 56.09, 55.95, 55.93, 55.26, 50.64, 36.32, 28.40, 26.89.
HRMS (ESI-Tof), [M + H]+: m/z calcd for C22H24N2O6185Re 597.1164,
found 597.1176.
5-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-pentylcarbo-
nylferrocene (41). The procedure described for the synthesis of 22a was
applied to 35 (50.3 mg, 0.14 mmol) and 4 (34.1 mg, 0.18 mmol) to
afford 41 as an orange solid (152.8 mg, 78%). Mp: 118.6−120.8 °C. 1H
NMR (400 MHz, CDCl3) δ 6.59 (s, 1H), 6.53 (s, 1H), 4.78 (t, J = 1.9
Hz, 2H), 4.49 (t, J = 1.8 Hz, 2H), 4.20 (s, 5H), 3.84 (s, 3H), 3.83 (s, 3H),
2.84 (s, 2H), 2.73 (t, J = 7.4 Hz, 2H), 2.55 (s, 2H), 1.80−1.72 (m, 2H),
1.68 (s, 2H), 1.49−1.42 (m, 2H). ESI-MS: [M + H]+ (m/z = 476.2).
4-(5,6-Dimethoxyisoindolin-2-yl)-butylaminocarbonylferrocene
(43). Under N2, the solution of compound 13 (66.0 mg, 0.26 mmol) in
1.5 mL of anhydrous DMF and 100 μL of triethylamine was added to the
solution of compound 42 (102.9 mg, 0.26 mmol) in 1 mL of anhydrous
DMF dropwise. The reaction mixture was stirred at room temperature
for 4 h. The solvent was removed, then washed with saturated sodium
chloride, and extracted with ethyl acetate. The organic layer was dried
over anhydrous MgSO4, filtered, and concentrated under vacuum. The
residue was purified by silica gel chromatography (ethyl ether/hexane/
triethylamine = 10:5:1) to afford 43 (78.4 mg, 65%) as a yellow solid.
Mp: 119.6−120.4 °C. 1H NMR (400 MHz, CDCl3)δ 6.76 (s, 2H), 6.62
(br s, 1H), 4.61 (s, 2H), 4.25 (s, 2H), 4.16 (s, 5H), 4.00 (s, 4H), 3.87 (s,
6H), 3.44 (d, J = 5.4 Hz, 2H), 2.86 (s, 2H), 1.74−1.70 (m, 4H). 13C
NMR (100 MHz, CDCl3) δ 170.12, 148.68, 131.14, 130.90, 105.99,
70.13, 69.67, 68.07, 65.55, 59.20, 56.20, 55.62, 39.33, 27.72, 26.33.
HRMS (EI): m/z calcd for C25H31N2O3Fe [M + H]+463.1684, found
463.1686.
3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-propylamino-
carbonylcyclopentadienyl Tricarbonyl Rhenium (31a). The proce-
dure described for the synthesis of 27a was applied to compound 15
(70.2 mg, 0.28 mmol) and compound 26 (82.7 mg, 0.15 mmol) to afford
1
31a as a light yellow solid (67.6 mg, 74%). Mp: 123.6−124.5 °C. H
NMR (400 MHz, CDCl3) δ 6.65 (s, 1H), 6.59 (s, 1H), 5.39 (s, 2H), 5.01
(t, J = 2.0 Hz, 2H), 3.86 (d, J = 8.0 Hz, 6H), 3.66 (s, 2H), 3.51 (q, J = 5.2
Hz, 2H), 2.97 (t, J = 5.8 Hz, 2H), 2.86 (t, J = 5.5 Hz, 2H), 2.79 (t, J = 5.2
Hz, 2H), 1.83 (t, J = 4.7 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ
192.94, 161.69, 148.20, 147.62, 126.10, 125.90, 111.44, 109.89, 96.33,
84.93, 84.52, 56.08, 55.96, 55.42, 51.78, 41.31, 28.89, 23.86. HRMS
(ESI-Tof), [M + H]+: m/z calcd for C23H26N2O6185Re 611.1321, found
611.1323.
4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-butylamino-
carbonylcyclopentadienyl Tricarbonyl Rhenium (32a). The proce-
dure described for the synthesis of 27a was applied to compound 16
(89.0 mg, 0.34 mmol) and compound 26 (85.9 mg, 0.16 mmol) to afford
32a as a light yellow oil (81.9 mg, 83%). 1H NMR (400 MHz, CDCl3) δ
7.55 (br s, 1H), 6.63 (s, 1H), 6.56 (s, 1H), 5.58 (s, 2H), 5.07 (t, J = 2.2
Hz, 2H), 3.85 (d, J = 2.4 Hz, 6H), 3.65 (s, 2H), 3.41 (q, J = 5.6 Hz, 2H),
2.90 (t, J = 5.8 Hz, 2H), 2.80 (t, J = 5.6 Hz, 2H), 2.63 (t, J = 6.0 Hz, 2H),
1.79−1.68 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 192.92, 161.83,
147.83, 147.40, 126.26, 126.15, 111.49, 109.50, 96.02, 85.46, 84.55,
57.11, 56.66, 55.92, 50.01, 39.22, 28.52, 27.37, 24.69. HRMS (ESI-Tof),
[M + H]+: m/z calcd for C24H28N2O6185Re 625.1477, found 625.1483.
3-(5,6-Dimethoxyisoindolin-2-yl)-propylcarbonylferrocene (36).
The procedure described for the synthesis of 20a was applied to 33
(97.5 mg, 0.29 mmol) and 3 (75.9 mg, 0.42 mmol) to afford 36 as an
2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-ethylamino-
carbonylferrocene (44). The procedure described for the synthesis of
43 was applied to compound 14 (38.0 mg, 0.16 mmol) and compound
42 (53.8 mg, 0.14 mmol) to afford 44 (49.8 mg, 69%) as a light yellow
solid. Mp: 193.9−195.1 °C. 1H NMR (400 MHz, CDCl3) δ 6.62 (s, 1H),
6.55 (s, 1H), 6.48 (br s, 1H), 4.66 (s, 2H), 4.30 (t, J = 1.8 Hz, 2H), 4.15
(s, 5H), 3.84 (d, J = 9.8 Hz, 6H), 3.68 (s, 2H), 3.59 (q, J = 5.4 Hz, 2H),
2.89−2.84 (m, 4H), 2.78 (t, J = 5.6 Hz, 2H). 13C NMR (100 MHz,
CDCl3) δ 170.35, 147.68, 147.38, 126.12, 125.99, 111.43, 109.47, 70.24,
69.68, 68.18, 56.64, 55.96, 55.94, 55.36, 50.91, 36.19, 28.71. HRMS
(EI): m/z calcd for C24H29N2O3Fe [M + H]+ 449.1528, found 449.1520.
3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-propylamino-
carbonylferrocene (45). The procedure described for the synthesis of
43 was applied to compound 15 (100.0 mg, 0.40 mmol) and compound
42 (158.4 mg, 0.40 mmol) to afford 45 (116.6 mg, 63%) as an orange
1
orange solid (20.5 mg, 16%). Mp: 116.5−119.4 °C. H NMR (400
MHz, CDCl3) δ 6.75 (s, 2H), 4.81 (t, J = 1.7 Hz, 2H), 4.49 (t, J = 1.7 Hz,
2H), 4.20 (s, 5H), 3.94 (s, 4H), 3.86 (s, 6H), 2.88−2.80 (m, 4H), 2.04−
1.98 (m, 2H). ESI-MS: [M + H]+ (m/z = 434.6).
4-(5,6-Dimethoxyisoindolin-2-yl)-butylcarbonylferrocene (37).
The procedure described for the synthesis of 20a was applied to 34
(91.3 mg, 0.26 mmol) and 3 (107.5 mg, 0.60 mmol) to afford 37 as an
1
solid. Mp: 72.2−73.6 °C. H NMR (400 MHz, CDCl3) δ 7.73 (br s,
1H), 6.66 (s, 1H), 6.57 (s, 1H), 4.40 (s, 2H), 4.10 (s, 2H), 4.09 (s, 5H),
3.89 (s, 3H), 3.84 (s, 3H), 3.68 (s, 2H), 3.53 (q, J = 5.4 Hz, 2H), 2.94 (t, J
= 5.9 Hz, 2H), 2.86−2.84 (m, 2H), 2.77 (t, J = 5.6 Hz, 2H), 1.89−1.84
(m, 2H). 13C NMR (100 MHz, CDCl3) δ 170.02, 147.88, 147.42,
126.36, 126.04, 111.43, 109.62, 69.99, 69.54, 67.86, 58.13, 56.04, 55.94,
55.79, 51.54, 40.32, 28.83, 25.19. ESI-MS: [M + H]+ (m/z = 462.8).
4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-butylamino-
carbonylferrocene (46). The procedure described for the synthesis of
43 was applied to compound 16 (36.7 mg, 0.14 mmol) and compound
42 (63.0 mg, 0.16 mmol) to afford 46 (45.0 mg, 68%) as an orange solid.
Mp: 86.3−87.8 °C. 1H NMR (400 MHz, CDCl3) δ 6.61 (s, 1H), 6.52 (s,
1H), 4.59 (s, 2H), 4.25 (t, J = 1.8 Hz, 2H), 4.17 (s, 5H), 3.84 (d, J = 9.8
Hz, 6H), 3.62 (s, 2H), 3.43 (q, J = 6.2 Hz, 2H), 2.87 (d, J = 5.4 Hz, 2H),
2.79 (d, J = 5.1 Hz, 2H), 2.61 (t, J = 6.1 Hz, 2H), 1.76−1.68 (m, 4H). 13C
NMR (100 MHz, CDCl3) δ 170.04, 147.64, 147.29, 126.46, 126.17,
111.42, 109.56, 70.17, 69.66, 68.03, 57.52, 56.03, 55.94, 50.76, 39.31,
28.60, 27.79, 24.69. HRMS (ESI-Tof), [M + H]+: m/z calcd for
C26H33N2O3Fe 477.1841, found 477.1833.
1
orange solid (25.8 mg, 19%). Mp: 105.6−107.4 °C. H NMR (400
MHz, CDCl3) δ 6.74 (s, 2H), 4.81 (t, J = 1.9 Hz, 2H), 4.49 (t, J = 1.8 Hz,
2H), 4.20 (s, 5H), 3.91 (s, 4H), 3.86 (s, 6H), 2.79−2.75 (m, 4H), 1.85−
1.78 (m, 2H), 1.71−1.63 (m, 2H). ESI-MS: [M + H]+ (m/z = 447.9).
5-(5,6-Dimethoxyisoindolin-2-yl)-pentylcarbonylferrocene (38).
The procedure described for the synthesis of 22a was applied to 35
(49.3 mg, 0.14 mmol) and 3 (25.1 mg, 0.14 mmol) to afford 38 as an
1
orange solid (56.5 mg, 44%). Mp: 108.1−110.0 °C. H NMR (400
MHz, CDCl3) δ 6.75 (s, 2H), 4.78 (t, J = 1.9 Hz, 2H), 4.49 (t, J = 1.9 Hz,
2H), 4.20 (s, 5H), 3.95 (s, 4H), 3.86 (s, 6H), 2.79 (s, 2H), 2.74 (t, J = 7.4
Hz, 2H), 1.81−1.73 (m, 2H), 1.69 (s, 2H), 1.53−1.45 (m, 2H). ESI-MS:
[M + H]+ (m/z = 462.2).
3-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-propylcarbo-
nylferrocene (39). The procedure described for the synthesis of 20a was
applied to 33 (85.8 mg, 0.26 mmol) and 4 (62.1 mg, 0.32 mmol) to
afford 39 as an orange solid (34.3 mg, 33%). Mp: 114.1−116.1 °C. 1H
NMR (400 MHz, CDCl3) δ 6.60 (s, 1H), 6.53 (s, 1H), 4.79 (t, J = 1.9
K
DOI: 10.1021/acs.jmedchem.5b01378
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
X-ray Crystallography. All of the procedures for X-ray
crystallography were previously described.36 Detailed procedures are
described in the Supporting Information.
In Vitro Radioligand Competition Studies. σ Receptor Binding
Assays. All of the procedures for the radioligand competition studies
were previously described.20 Detailed procedures are described in the
Supporting Information.
VAChT Binding Assays. The determination of the affinity for VAChT
were conducted by a method in the literature.38 Detailed procedures are
described in the Supporting Information.
SMILES data (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-10-58808891. Fax: +86-10-58808891. E-mail:
hmjia@bnu.edu.cn.
■
Author Contributions
⊥D.L. and Y.C. contributed equally to this work.
Dopamine D2L Receptors, NMDA Receptors, Opiate Receptors,
DAT, NET, and SERT Binding Assay. The affinities of compound 20a for
the above receptors and transporters are presented in a percentage of
inhibition form. Detailed information is described in the Supporting
Information.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Radiochemistry. 99mTc-pertechnetate was eluted from a commer-
cial 99Mo−99mTc generator obtained from Beijing Atomic High-tech Co.
The reactions were performed according to the methods in the
literature.22,25,39 Detailed procedures are provided in the Supporting
Information.
We give special thanks to Professor Robert H. Mach for his
helpful suggestions and valuable comments on this manuscript.
We are grateful to Dr. Xuebing Deng (College of Chemistry,
Beijing Normal University) for his assistance with X-ray
diffraction. This work was supported by the National Natural
Science Foundation of China (No. 21471019).
Measurement of log D values. The log D values of [99mTc]20b−
25b and [99mTc]29b−30b were determined by measuring the
distribution of the radiotracer between 1-octanol and 0.05 M sodium
phosphate buffer at pH 7.4 according to the literature.22,25,36 Detailed
procedures are provided in the Supporting Information.
In Vitro Evaluation in DU145 Prostate Cells and C6 Glioma
Cells. The culture of cells and the in vitro cell uptake and blocking assays
were performed as previously reported.22,25 Detailed procedures are
provided in the Supporting Information.
Biodistribution and Blocking Studies in Mice. 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]20b, [99mTc]23b, [99mTc]25b, and [99mTc]29b
(370 kBq, 0.1 mL) were carried out based on a method reported
previously.22,25 Detailed procedures are provided in the Supporting
Information.
ABBREVIATIONS USED
■
CNS, central nervous system; DAT, dopamine transporter;
DLT, double ligand transfer; DMF, dimethylformamide; DTG,
1,3-di-o-tolyl-guanidine; [18F]ISO-1, N-(4-(6,7-dimethoxy-3,4-
dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-[18F]fluoroethoxy)-5-
methylbenzamide; FBS, fetal bovine serum; BBB, blood−brain
barrier; HPLC, high performance liquid chromatography; ID,
injected dose; NET, norepinephrine transporter; PET, positron
emission tomography; rt, room temperature; SD, standard
deviation; SERT, serotonin transporter; SPECT, single photon
emission computed tomography; TFA, trifluoroacetic acid;
THF, tetrahydrofuran; TLC, thin layer chromatography;
VAChT, vesicular acetylcholine transporter
Biodistribution and Blocking Studies of [99mTc]20b in Balb/c
Nude Mice Bearing C6 Glioma Xenografts. All animal experiments
in Balb/c nude mice (n = 4) were performed in compliance with the
national laws related to the care and experiments on laboratory animals.
Biodistribution studies and blocking studies of [99mTc]20b (370 kBq,
0.1 mL) were carried out based on a method reported previously.25
Detailed procedures are provided in the Supporting Information.
Small Animal NanoScan SPECT/CT Imaging of [99mTc]20b.
The detailed procedures of small animal imaging studies of [99mTc]20b
(22.2 MBq, 0.15 mL) in male Balb/c nude mice bearing C6 glioma
xenografts are provided in the Supporting Information.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01378.
■
S
General information and some parts of the evaluation of
the radiotracers in Experimental Section, purity of key
target compounds, the HPLC chromatograms of 20a−25a
and [99mTc]20b−25b, 29a−30a, and [99mTc]29b−30b,
X-ray crystallographic data for compound 31a, in vitro
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