Novel cyclopentadienyl tricarbonyl complexes of 99mTc mimicking chalcone as potential single-photon emission computed tomography imaging probes for β-amyloid plaques in brain

Article abstract of DOI:10.1021/jm3014184

Rhenium and technetium-99m cyclopentadienyl tricarbonyl complexes mimicking the chalcone structure were prepared. These complexes were proved to have affinity to β-amyloid (Aβ) in fluorescent staining on brain sections of Alzheimer's Disease (AD) patient and binding assay using Aβ _1-42 aggregates, with K_i values ranging from 899 to 108 nM as the extension of conjugated π system. In vitro autoradiograpy on sections of transgenic mouse brain confirmed the affinity of [^99mTc]5 (K _i = 108 nM). In biodistribution, all compounds showed good initial uptakes into the brain and fast blood clearance, while the decreasing of initial brain uptakes correspond to increasing of conjugation length, from 4.10 ± 0.38% ID/g ([^99mTc]3) to 1.11 ± 0.34% ID/g ([ ^99mTc]5). These small technetium-99m complexes (<500 Da) designed by an integrated approach provide encouraging evidence that development of a promising ^99mTc-labeled agent for imaging Aβ plaques in the brain may be feasible.

Full text of DOI:10.1021/jm3014184

Article
pubs.acs.org/jmc
Novel Cyclopentadienyl Tricarbonyl Complexes of 99mTc Mimicking
Chalcone as Potential Single-Photon Emission Computed
Tomography Imaging Probes for β‑Amyloid Plaques in Brain
Zijing Li,† Mengchao Cui,*,† Jiapei Dai,‡ Xuedan Wang,† Pingrong Yu,† Yanping Yang,† Jianhua Jia,†
Hualong Fu,† Masahiro Ono,§ Hongmei Jia,† Hideo Saji,§ and Boli Liu*,†
†Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.
R. China
‡Wuhan Institute for Neuroscience and Neuroengineering, South-Central University for Nationalities, Wuhan 430074, P. R. China
§Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida
Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
S
* Supporting Information
ABSTRACT: Rhenium and technetium-99m cyclopentadienyl
tricarbonyl complexes mimicking the chalcone structure were
prepared. These complexes were proved to have affinity to β-
amyloid (Aβ) in fluorescent staining on brain sections of
Alzheimer’s Disease (AD) patient and binding assay using Aβ1−42
aggregates, with Ki values ranging from 899 to 108 nM as the
extension of conjugated π system. In vitro autoradiograpy on
sections of transgenic mouse brain confirmed the affinity of
[
99mTc]5 (Ki = 108 nM). In biodistribution, all compounds
showed good initial uptakes into the brain and fast blood
clearance, while the decreasing of initial brain uptakes
correspond to increasing of conjugation length, from 4.10
0.38% ID/g ([99mTc]3) to 1.11 0.34% ID/g ([99mTc]5). These
small technetium-99m complexes (<500 Da) designed by an integrated approach provide encouraging evidence that
development of a promising 99mTc-labeled agent for imaging Aβ plaques in the brain may be feasible.
imaging agents have indicated that detecting Aβ plaques in the
living human brain by PET or SPECT may lead to
differentiation between AD patients and healthy humans.
However, the signal-to-noise ratio for plaque labeling of
INTRODUCTION
■
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder pathologically characterized by deposition of misfolded
β-amyloid (Aβ) peptides as senile plaques in the brain. Because
the deposition of Aβ plaques is an early event in the
development of AD, a validated biomarker of Aβ deposition
in the brain might prove useful to identify and follow
individuals at risk for AD and to assist in the evaluation of
new antiamyloid therapies currently under development.1−3
A number of groups have reported radiolabeled Aβ imaging
agents for positron emission tomography (PET) and single
photon emission computed tomography (SPECT) in clinical
trials such as [18F]FDDNP ([18F]-2-(1-(6-[(2-fluoroethyl)-
(methyl)amino]-2-naphthyl)ethylidene)malononitrile),4,5
[11C]PIB ([11C]-2-(4′-methylaminophenyl)-6-oxybenzothia-
zole),6,7 [11C]SB-13 ([11C]-4-N-methylamino-4′-hydroxystil-
[
123I]IMPY, which is the only SPECT Aβ imaging agents in
preclinical trail, was not robust in AD and healthy controls,
while it was believed that the in vivo instability and fast
metabolism of [123I]IMPY may ultimately lead to a decreased
signal. After that, there is no report of any Aβ imaging
candidate for SPECT moving into clinical trial.
However, the radionuclide technetium-99m is among the
most widely used isotopes in diagnostic nuclear medicine.
Readily produced by a 99 Mo/99mTc generator, essentially 24 h/
day, 99mTc emits medium γ-ray energy suitable for detection,
and its physical half-life is compatible with the biological
localization and residence time required for imaging. New
99mTc-labeled imaging agents for Aβ plaques will provide
simple, convenient and widespread SPECT-based imaging
methods for detecting and eventually quantifying Aβ plaques in
living brain tissue,17−19 whereas in the past 99mTc complexes
bene),8,9
[
11C]BF-227 ([11C]-(2-[fluoro]ethoxy)-
benzoxazole),10 [18F]BAY94−9172 ([18F]-4-(N-methylami-
no)-4′-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-stilbene),11
[18F]AV-45 ([18F]-(E)-4-(2-(6-(2-(2-(2-fluoroethoxy)ethoxy)-
ethoxy)pyridin-3-yl)vinyl)-N-methylaniline),12,13 and
[
123I]IMPY ([123I]-6-iodo-2-(4′-dimethylamino-)phenyl-
Received: September 29, 2012
Published: December 14, 2012
imidazo[1,2]pyridine).14−16 Recent reports using these amyloid
© 2012 American Chemical Society
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Figure 1. Chemical structures of reported 99mTc labeled Aβ imaging probes.
were preferentially applied as perfusion agents. A challenge now
lies in combining a 99mTc complex with a targeting molecule
such as a small central nervous system (CNS) receptor-binding
molecule.
volume expansion. This expansion twists the planar shape of
the binding agent, which is very important for a molecular
agent to fit into the planar gap on the Aβ plaques and also
increases the molecular weight greatly. Conversely, the
integrated approach strategy seems wiser but more difficult to
practice. Unfortunately, no clinical study of both kinds has been
reported because the low initial brain uptake is not sufficient for
SPECT imaging, even if they display high affinities to Aβ.
On the basis of the discovery so far, the best strategy to
design Aβ imaging agents is to find an small, lipophilic, 99mTc-
chelating core to substitute or mimic one part of the structure
of a binding agent by integrated approach in order to maintain
the planar shape of the ligand, minimize the molecular weight,
and maintain the ability to penetrate the BBB.
Kung et al. reported that the dopamine transporter imaging
agent [99mTc]TRODAT-120 is useful to detect the loss of
dopamine transporters in Parkinson’s disease. This is the first
example of a 99mTc imaging agent that can penetrate the
blood−brain barrier (BBB) via a simple diffusion mechanism
and localize at receptor binding sites in the CNS. On the basis
of this success, efforts were made to search for comparable
99mTc imaging agents that target binding sites on Aβ plaques in
the brain of AD patients. Several 99mTc-labeled imaging probes
have been developed (Figure 1). Two of them are based on an
integrated approach (the Congo Red derivative21 and the
biphenyl derivative22). The integrated approach involves the
replacement of part of a known high-affinity receptor ligand
with the requisite “unnatural” 99mTc chelate in such a way that
there are minimal changes in size, conformation, and receptor
binding affinity.23 The others are based on a bifunctional
approach (the chrysamine G derivative,24,25 the benzothiazole
aniline derivative,26−28 the chalcone derivative,29 the flavone
and aurone derivative,30 the benzoxazole derivative,31 the
DDNP derivative,32 etc.). The bifunctional approach leads to
As early as 1992, Wenzel reported a double ligand transfer
(DLT) reaction,33 which ultimately led to the formation of
[Cp99mTc(CO)3, Cp = cyclopentadienyl] starting from
99mTcO4 . Then the method of preparing [Cp99mTc(CO)3]
−
in water has been developed using sensitive organometallic
ligands.34 Apart from this feasibility consideration, the cyclo-
pentadienyl tricarbonyl ligand offers very attractive properties
as a ligand for radiopharmaceutical purposes. Its inherent
advantages are the small size, the low molecular weight, and the
high stability of half-sandwich configuration and the minimized
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a
steric interference with the receptor binding moiety of a labeled
biomolecule.35 The “piano stool” organometallic core [CpM-
(CO)3, M = Re(I), Tc(I)] is a neutral 18 electron species, with
high stability resulting from the low-spin d6 electron
configuration, further stabilized by the cyclopentadienyl and
tricarbonyl ligands. Although many highly stable chelate
systems have been developed for 99mTc, the small size of the
[CpM(CO)3] core is advantageous for maintaining biological
activity, particularly when labeling small molecules. The fact
that [CpM(CO)3] (M = Re and Tc) can be coupled to
biomolecules by classical organometallic methods without
affecting the bioactivity has been demonstrated by several
groups.34 Furthermore, [Cp99mTc(CO)3] complexes are highly
lipophilic, which makes them particularly promising as 99mTc-
labeled, BBB-crossing molecules. So, taking the configuration
and aromatic properties of [Cp99mTc(CO)3] core into
consideration, it is an excellent choice to substitute or mimic
a benzene ring of a ligand by an integrated approach.
Scheme 1
a
Reagents and conditions: (a) (1) NaH, 18-crown-6, dry THF, r.t., (2)
concentrated HCl, K2CO3 aq r.t.; (b) COCl2, AlCl3, CH2Cl2; (c)
NaOH, EtOH, r.t.
Ono et.al. reported in 2007 that chalcone derivatives,36
whose backbone structure is considered to be a promising
scaffold, showed excellent characteristics as new amyloid
imaging agents, such as high binding affinity to Aβ aggregates,
high uptake into the brain, and rapid clearance from the brain,
besides it can easily be formed by one-pot condensation
reaction.
first acetylated by acetyl chloride in ice bath to obtain
(acetylcyclopentadienyl)tricarbonylrhenium (2) at 95% yield.
This complex can react with three aromatic aldehydes
respectively through base-catalyzed Claisen condensation to
obtain final rhenium complexes (3, 4, and 5) of different π
conjugation length at yields above 90%. By the way, two
ferrocene complexes (6 and 7) were synthesized through the
same method as precursors for [99mTc]4 and [99mTc]5. All
these complexes were fully characterized by spectroscopic
methods. Complexes 3 and 4 could be recrystallized by slow
evaporation of an ethanol−CH2Cl2 solution to afford X-ray
quality crystals. The structures were elucidated and their
ORTEPs are given in Figures 3 and 4, with relevant
crystallographic data in Table 1.
After 125I-, 11C-, and 18F-labeled chalcone derivatives were
prepared and chalcone scaffold were proved promising
candidate as AD imaging agent,36−38 additionally, in 2010,
the same group reported synthesis of four chalcone derivatives
with monoamine−monoamide dithiol (MAMA) and bis-
amino-bis-thiol (BAT) selected as chelation ligands by
bifunctional approach. For the first time, 99mTc/Re complexes
as chalcone derivatives have been proposed as probes for the
detection of Aβ plaques in the brain.29 In their study, MAMA
and BAT were selected as chelation ligands to form an
electrically neutral complex with 99mTc, but these two big
chelating groups increase the molecular weight and also
decrease the affinities and initial brain uptakes as well.
Radiolabeling. To get the 99mTc-labeled cyclopentadienyl
tricarbonyl complexes [99mTc]3, [99mTc]4, and [99mTc]5, a two-
step sequential reaction (Scheme 2) has to be applied. In the
first step, we prepared [CH3COCp99mTc(CO)3] ([99mTc]2)
under 150 °C heat for 20 min through the DLT method with
an average radiochemical yield of 50% (no decay corrected).
Then [99mTc]2 went through a base-catalyzed Claisen
condensation with three corresponding aldehydes to yield
In the present study, we designed and synthesized novel
chalcone-mimic complexes by introducing [Cp99mTc(CO)3]
core to substitute a benzene ring (Figure 2) by an integrated
[
99mTc]3, [99mTc]4, and [99mTc]5. The final 99mTc-labeled
products were purified by radio high performance liquid
chromatography (HPLC), and the identity of [99mTc]3,
[
99mTc]4, and [99mTc]5 was verified by a comparison of the
retention time with that of the nonradioactive rhenium
compounds (Figures 5, 6, and 7). In the second step, the
radiochemical yields of [99mTc]3−5 were also about 50% (no
decay corrected), to achieve a 25% total yield with radio-
chemical purity of >95% after HPLC purification. We took this
two-step strategy because the direct DLT labeling method
under 150 °C would destroy the ferrocene precursor (6 and 7)
and give no products. Furthermore, this radiochemical
synthesis by Claisen condensation strategy was proved brilliant
in the following experiments because the reaction was not only
fast enough for 99mTc labeling but also ready to be applied to
label many different aldehyde or large molecules which contain
aldehyde groups with [Cp99mTc(CO)3] core at acceptable
yields.
Figure 2. Chemical structure of the designed 99mTc/Re labeled
[RCOCpM(CO)3] (M = 99mTc/Re) complexes.
approach in order to acquire good brain uptake while keeping
excellent affinities to Aβ plaques. Furthermore, the properties
of this series of 99mTc/Re complexes were studied as the
extension of conjugated π system.
RESULTS AND DISCUSSION
■
Chemistry. For characterization of the 99mTc complexes, we
have prepared the corresponding rhenium complexes (Scheme
1). To collect the three corresponding aromatic aldehydes, the
aromatic aldehyde 1 was prepared by Wittig reaction from (E)-
3-(4-(dimethylamino)phenyl)acrylaldehyde, which is commer-
cially available. (Cyclopentadienyl)tricarbonylrhenium was at
Biological Evaluation. In vitro fluorescent staining of Aβ
plaques in sections of brain tissue from AD patients and Tg
model mice (C57BL6, APPswe/PSEN1, 11 months old) were
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Figure 3. Molecular structure of 3 (thermal ellipsoids drawn at the 30% probability level).
Figure 4. Molecular structure of 4 (thermal ellipsoids drawn at the 30% probability level).
were observed in the brain section of an AD patient (Figure
8G). In contrast, no apparent labeling was observed in both
normal mouse and normal adult brain sections (Figure 8C,H)
stained by complex 4 (Figure 8F). The similar results of in vitro
fluorescent staining of Aβ plaques by complex 5 were showed
in Figure 9, while the fluorescent signal stained by complex 3
was weak (data not shown), which may be due to the low
affinity.
To quantitatively evaluate the binding affinities of these
chalcone-mimic complexes to Aβ1−42 aggregates, in vitro
inhibition assay was carried out in solutions with [125I]IMPY
as the competing radioligand according to conventional
methods. The three rhenium complexes (3, 4, and 5) inhibited
the binding of [125I]IMPY in a dose-dependent manner (Figure
10). With the result shown in Table 2, rhenium complexes of
different conjugation lengths showed moderate binding
affinities to Aβ1−42 aggregates (Ki = 899 78 nM for 3, Ki =
211 19 nM for 4, and Ki = 108 16 nM for 5), which are not
satisfactory but sufficiently high for Aβ aggregates comparable
to the value determined under the same assay system for IMPY
(Ki = 10.5 1.0 nM) and chalcone derivatives (with Ki ranged
from 2.9 to >10,000 nM as reported36). The crystal structures
elucidated in Figure 3 and Figure 4 implied that complex 4 is
less distorted than complex 3 because the dihedral angle
between the benzene plane (light-blue) and the Cp plane
(light-red) is 43.69° for complex 3, while the same dihedral
angle of complex 4 is 31.34°. So, we would like to blame the
Table 1. Summary of X-ray Crystallographic Data
3
4
formula sum
formula weight (g/mol)
crystal system
space group
a (Å)
C19H16NO4Re
508.53
C21H18NO4Re
534.56
triclinic
orthorhombic
Pca21 (29)
12.730(2)
P1 (2)
̅
5.9756(8)
11.8247(16)
13.6116(19)
66.46
b (Å)
6.3516(10)
12.730(2)
c (Å)
α (deg)
β (deg)
78.62
γ (deg)
80.76
cell volume (Å3)
860.82(82)
2
1845.45(52)
4
Z
calcd density (g/cm3)
RAll
1.96182
0.0379
1.92387
0.0669
Pearson code
formula type
Wyckoff sequence
aP82
oP180
NOP4Q16R19
i41
NOP4Q18R21
a45
carried out to evaluate the binding affinity of complexes 3, 4,
and 5 to Aβ plaques. As shown in Figure 8A,D, specific staining
of plaques were observed in the brain section of Tg mice for
complex 4. The presence and distribution of Aβ plaques was
consistent with the results of staining using thioflavin-S (a
common dye for staining of Aβ plaques) on the adajacent
section (Figure 8B,E). Furthermore, intense labeling of plaques
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a
Scheme 2
a
−
Reagents and conditions: (a) Mn(CO)5Br, 99mTcO4 , H2O, DMF, 150 °C, 20 min; (b) NaOH, EtOH, r.t.
Figure 7. HPLC profiles of 5 and [99mTc]5. HPLC conditions: Venusil
MP C18 column (Agela Technologies, 10 mm × 250 mm), CH3CN/
H2O = 80/20, 4 mL/min, UV, 254 nm.
Figure 5. HPLC profiles of 3 and [99mTc]3. HPLC conditions: Venusil
MP C18 column (Agela Technologies, 10 mm × 250 mm), CH3CN/
H2O = 70/30, 4 mL/min, UV, 254 nm.
extension weakens the influence of the three “CO” stools of the
[Cp99mTc(CO)3] core on the planar configuration. The affinity
of the two ferrocene complexes whose structures have no stools
still keep high affinity (Ki = 3.36 0.30 nM for 6, Ki = 5.08
1.74 nM for 7) also confirm our inference.
In binding assays using the aggregated Aβ1−42 peptides in
solution, we also confirmed that the Aβ1−42 aggregate-bound
radioactivities (%) were varied differently in the four 99mTc-
labeled complexes. In terms of Aβ1−42 aggregate-bound
radioactivity, the derivatives rank in the following order:
[
[
99mTc]5 (10.15%) > [99mTc]4 (3.87%) > [99mTc]3 (0.68%) >
99mTc]2 (0.32%) (Figure 11). Furthermore, the bound
radioactivities indicate that [99mTc]5 and [99mTc]4 occupied
the specific binding sites of Aβ1−42 aggregates, while [99mTc]3
and [99mTc]2 showed no remarkable binding to Aβ1−42
aggregates. This result suggests that the length of conjugated
π system played an important role in the binding of Aβ1−42
aggregates, which are also consistent with the binding affinities
of these chalcone-mimic complexes in the inhibition assay. The
results of blocking assay using excess of rhenium complexes 2−
5 showed that the specific binding of [99mTc]5 to Aβ1−42
aggregates was blocked about 80% by an excess of 5 (0.5 μM);
the binding of [99mTc]4 was blocked nearly a half by an excess
of 4 at 1.0 μM, while excess of complexes 3 and 2 at 1.0 μM
could not significantly block the binding radioactivity owing to
their lower affinities. These results confirmed that complex 5
displayed specific and high binding to Aβ1−42 aggregates.
Figure 6. HPLC profiles of 4 and [99mTc]4. HPLC conditions: Venusil
MP C18 column (Agela Technologies, 10 mm × 250 mm), CH3CN/
H2O = 70/30, 4 mL/min, UV, 254 nm.
decreasing of affinity for the three “CO” stools of the
[Cp99mTc(CO)3] core which distort the planar and flake-like
configuration of chalcone. Because the Ki values are decreasing
as the extension of conjugated π system, we can infer that the
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Figure 10. Inhibition curves for the binding of [125I]IMPY to Aβ1−42
aggregates.
Table 2. Inhibition Constants (Ki, nM) for Binding to
a
Aggregates of Aβ1‑42 versus [125I]IMPY
compd
Ki (nM)
compd
Ki (nM)
Figure 8. In vitro fluorescent staining of Aβ plaques by complex 4.
(A,D) complex 4 on brain section of a Tg model mouse (C57BL6,
APPswe/PSEN1, 11 months old, male); (B,E) the presence and
distribution of plaques on the sections were confirmed by fluorescence
staining using thioflavin-S on the adjacent section; (C) complex 4 on
brain section of a normal mouse as control; (F) thioflavin-S on the
adjacent brain section of the normal mouse; (G) complex 4 on brain
section of an AD patient (91 years old, male); (H) complex 4 on brain
section of a normal person (69 years old, female) as control.
3
4
5
899 78
211 19
108 16
6
3.36 0.30
5.08 1.74
11.5 2.5
7
IMPY
a
Measured in triplicate with results given as the mean SD.
was consistent with the results of fluorescent staining with
thioflavin-S. Although the binding affinity of [99mTc]5 to Aβ
aggregates was not potent (Ki = 108 16 nM), [99mTc]5 was
still able to label the plaques in sections of Tg mice.
The log D values (2.89 0.09 for [99mTc]3, 3.61 0.04 for
[
99mTc]4, and 3.45 0.09 for [99mTc]5, respectively) shown in
Table 3 indicate that complexes [99mTc]3, [99mTc]4, and
[
99mTc]5 have moderate lipophilicity suitable for brain imaging.
Biodistribution experiments in normal male ICR mice (5
weeks, male) were carried out to evaluate the ability of these
99mTc cyclopentadienyl tricarbonyl complexes of different
conjugation lengths ([99mTc]3, [99mTc]4, and [18F]5) to
penetrate the BBB and properties of clearance from the
brain. High initial brain uptake and high brain2 min/brain60 min
ratio in normal mouse brain are considered to be important as
pharmacokinetic indexes for selecting appropriate Aβ imaging
tracers. As shown in Tables 4, 5, and 6, [99mTc]3 with a short
conjugated π system displayed a very high initial brain uptake
(n = 1, 4.10 0.38% ID/g at 2 min) than that of [99mTc]4 and
[
99mTc]5 with a longer conjugated π system (n = 2, 2.30
0.27% ID/g at 2 min for [99mTc]4; n = 3, 1.11 0.34% ID/g at
2 min for [99mTc]5). What impressed us was that [99mTc]3
exhibits such high initial brain uptake barely not seen before for
99mTc-labeled receptor binding agent and that the decreasing
Figure 9. In vitro fluorescent staining of Aβ plaques by complex 5.
(A,D) complex 5 on brain section of a Tg model mouse (C57BL6,
APPswe/PSEN1, 11 months old, male); (B,E) the presence and
distribution of plaques on the sections were confirmed by fluorescence
staining using thioflavin-S on the adjacent section; (C) complex 5 on
brain section of a normal mouse as control; (F) thioflavin-S on the
adjacent brain section of the normal mouse; (G) complex 5 on brain
section of an AD patient (91 years old, male); (H) complex 5 on brain
section of a normal person (69 years old, female) as control.
a
of brain uptake is as sharp as the π system extension, where we
could not give a good explanation by now. Compared with the
99mTc-labeled chalcone derivatives (0.22, 0.78, 0.62, 1.48% ID/
g at 2 min) by bifunctional approach reported previously,29 the
initial brain uptakes of [99mTc]3 and [99mTc]4 are apparently
superior. Meanwhile, the brain2 min/brain60 min ratio of 8.20,
4.18, and 2.18 for [99mTc]3, [99mTc]4, and [99mTc]5 were from
good to acceptable. Furthermore, we can also conclude that
In vitro autoradiography studies of [99mTc]5 were performed
with sections from Tg mice (C57BL6, APPswe/PSEN1, 11
months old) and an age-matched control mice. As shown in
Figure 12, [99mTc]5 displayed good labeling of Aβ plaques in
the cortical regions of Tg mice, and the control case was clearly
void of any notable Aβ labeling. The distribution of Aβ plaques
[
99mTc]3, [99mTc]4, and [99mTc]5 are metabolized by liver and
small intestine, because the liver showed high uptakes with very
slow washout and the small intestine uptakes kept increasing
with time.
The biodistribution with permeability-glycoprotein 1 (PgP)
blocked by cyclosporin A (an immunosuppressant drug known
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Figure 11. Binding and blocking assay of [99mTc]2, [99mTc]3, [99mTc]4, and [99mTc]5 with Aβ1−42 aggregates. Values are the mean standard error
of the mean for tent experiments. Black columns represent the Aβ1−42 aggregate-bound radioactivities (%) of [99mTc]2−5. Gray columns represent
the Aβ1−42 aggregate-bound radioactivities (%) of [99mTc]2−5 blocked by complexes 2−4 (1.0 μM) and 5 (0.5 μM), respectively.
increased obviously. This result may reveal [99mTc]3−5 to be
substrates for the rodent PgP transporter.
CONCLUSIONS
■
Synthesis of organometallic complexes mimicking chalcone
structure with 99mTc cyclopentadienyl tricarbonyl core using a
two-step sequential reaction is described in this article. The first
step is to prepare the intermediate ([CH3COCp99mTc(CO)3])
through the DLT method, and the second step is base-catalyzed
Claisen condensation with appropriate aldehydes. We want to
emphasize that this pathway can probably be applied to label
other aldehydes with a [Cp99mTc(CO)3] core, especially
biomacromolecules, by avoiding the damage of 150 °C heat
to the aldehyde in one-step pathway. In vitro fluorescent
staining pictures of Aβ plaques on brain sections of patients
diagnosed with AD and Tg mouse were clear for complexes 4
Figure 12. (A) In vitro autoradiography of [99mTc]5 on a Tg model
mouse (C57BL6, APPswe/PSEN1, 11 months old, male). (B) The
and 5. The binding assay using Aβ1−42 aggregates indicated that
the Ki value ranges from 899 to 108 nM as the extension of
presence and distribution of plaques in the section A were confirmed
conjugated π system, among which complex 5 has the highest
by fluorescence staining using thioflavin-S on the same section with a
affinity. In vitro autoradiography on section of transgenic
filter set for GFP. (C) In vitro autoradiography of [99mTc]5 on a brain
mouse brain also confirmed the affinity of [99mTc]5 (Ki = 108
section of a normal mouse (C57BL6, 11 months old, male) as control.
(D) Fluorescence staining using thioflavin-S on the section C with a
filter set for GFP on a brain section of a normal mouse as control.
nM). In biodistribution, [99mTc]3 (4.10 0.38% ID/g at 2 min,
brain2 min/brain60 min ratio: 8.20) and [99mTc]4 (2.30 0.27%
ID/g at 2 min, brain2 min/brain60 min ratio: 4.18) showed
excellent initial uptakes and fast clearance in the brain, while
a
Table 3. log D Value of Compound [99mTc]3−5
[
99mTc]5 (1.11 0.34% ID/g at 2 min, brain2 min/brain60 min
ratio: 1.73) was also good as an 99mTc-labeled ligand for Aβ
imaging. Meanwhile, [99mTc]3−5 are probably substrates for
the rodent PgP transporter. Therefore, the pretreating BBB
abilities of these complexes are more remarkable with the PgP
blocked. These findings suggest that additional effort should be
made to explore why these kinds of complexes can penetrate
the BBB more efficiently than other 99mTc-labeled ligands,
which may lead to some new suggestions about how to design
99mTc-labeled CNS probes. In conclusion, these small
technetium-99m complexes (<500 Da) designed by an
integrated approach mimicking chalcone provide encouraging
compd
log D
[
[
[
99mTc]3
99mTc]4
99mTc]5
2.89 0.09
3.61 0.04
3.45 0.09
a
Measured in triplicate with results given as the mean SD.
to block PgP activity) further describes their brain penetration
abilities. The blood and brain uptakes at 2 min were measured,
and the results were shown in Table 7. After the PgP were
blocked by cyclosporin A, the brain uptakes of [99mTc]3−5
477
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Journal of Medicinal Chemistry
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a
Table 4. Biodistribution of [99mTc]3 in Male ICR Mice
time after injection
30 min
organ
blood
2 min
10 min
60 min
120 min
2.02 0.17
4.10 0.38
11.47 1.65
18.14 1.77
3.53 0.52
7.39 1.06
13.36 0.99
1.69 0.16
6.81 1.57
0.82 0.05
2.27 0.51
1.87 0.36
24.75 3.38
1.93 0.30
7.32 0.71
5.38 0.66
3.06 0.90
17.98 1.58
0.51 0.07
0.69 0.09
0.98 0.22
22.43 4.39
0.81 0.14
5.52 0.49
3.87 0.79
2.18 0.39
0.51 0.06
0.50 0.08
0.84 0.16
25.96 2.06
0.64 0.14
5.38 0.38
3.17 0.16
1.38 0.55
34.75 2.26
0.44 0.08
0.37 0.08
0.55 0.15
25.24 5.17
0.51 0.15
4.79 0.43
2.56 0.40
0.75 0.16
26.22 4.20
brain
heart
liver
spleen
lung
kidney
stomach
b
b
small intestine
37.71 7.94
a
b
Expressed as % injected dose per gram. Average for 5 mice standard deviation. Expressed as % injected dose per organ.
a
Table 5. Biodistribution of [99mTc]4 in Male ICR Mice
time after injection
30 min
organ
blood
2 min
10 min
60 min
120 min
3.80 0.71
2.30 0.27
12.94 2.16
32.64 4.34
4.50 0.91
9.08 2.20
15.59 1.89
1.11 0.24
5.27 0.46
1.25 0.17
1.85 0.25
3.58 0.51
28.81 4.57
4.34 0.92
6.18 1.59
6.56 1.01
1.28 0.33
15.12 5.32
0.98 0.49
0.93 0.09
1.92 0.26
34.32 5.35
2.84 0.90
4.03 0.83
5.56 0.94
1.86 0.60
0.64 0.15
0.55 0.08
1.36 0.13
35.93 4.25
1.25 0.23
3.29 1.45
4.31 0.83
1.90 0.22
34.63 4.72
0.55 0.07
0.49 0.11
0.92 0.12
29.59 5.35
1.00 0.16
2.74 0.38
3.82 0.68
1.38 1.63
29.94 5.47
brain
heart
liver
spleen
lung
kidney
stomach
b
b
small intestine
24.42 4.62
a
b
Expressed as % injected dose per gram. Average for 5 mice standard deviation. Expressed as % injected dose per organ.
a
Table 6. Biodistribution of [99mTc]5 in Male ICR Mice
time after injection
30 min
organ
blood
2 min
10 min
60 min
120 min
13.53 1.37
1.11 0.34
11.48 1.82
52.40 3.64
13.14 2.57
31.98 4.58
7.11 0.80
0.63 0.10
2.30 0.45
1.01 0.11
0.40 0.05
3.50 0.23
67.08 3.54
21.59 2.91
13.89 1.39
2.83 0.33
0.50 0.06
3.84 0.48
0.70 0.08
0.38 0.05
2.61 0.28
57.20 3.09
20.95 2.72
8.66 0.99
2.87 0.73
0.74 0.08
0.82 0.06
0.51 0.08
1.70 0.14
40.49 6.00
18.63 4.75
9.69 0.52
2.98 0.36
0.93 0.12
16.64 2.29
0.98 0.22
0.64 0.11
1.73 0.24
42.91 3.43
11.65 4.25
9.18 1.87
4.12 0.56
1.15 0.13
14.93 2.70
brain
heart
liver
spleen
lung
kidney
stomach
b
b
small intestine
9.85 0.72
a
b
Expressed as % injected dose per gram. Average for 5 mice standard deviation. Expressed as % injected dose per organ.
a
Table 7. Biodistribution of [99mTc]3−5 at 2 min with/without PgP Blocked by Cyclosporin A in Male ICR Mice
b
b
b
organ
[
99mTc]3
[
99mTc]3
[
99mTc]4
[
99mTc]4
[
99mTc]5
[
99mTc]5
blood
brain
2.02 0.17
4.10 0.38
4.20 0.37
6.34 0.81
3.80 0.71
2.30 0.27
4.24 0.19
3.68 0.07
13.53 1.37
1.11 0.34
13.07 1.01
1.64 0.17
a
b
Expressed as % injected dose per gram. Average for 5 mice standard deviation. Biodistribution of [99mTc]3-5 at 2 min with PgP blocked by
Cyclosporin A in male ICR mice
evidence that development of a 99mTc-labeled agent for imaging
Aβ plaques in the brain may be feasible.
TMS as an internal standard. Chemical shifts were reported as δ values
with respect to residual solvents. The 13C NMR spectra were obtained
at 100 MHz on Bruker spectrometer in CDCl3 at room temperature.
Chemical shifts were reported as δ values with respect to residual
solvents. The multiplicity is defined by s (singlet), d (doublet), t
(triplet), m (multiplet). Mass spectrometry was acquired under the
Surveyor MSQ Plus (ESI) (Waltham, MA, USA) instrument. X-ray
crystallography data were collected on a Bruker Smart APEX II
diffractometer (Bruker Co., Germany). Reactions were monitored by
TLC (TLC Silica gel 60 F254, Merck). Radiochemical purity was
determined by HPLC performed on a Shimadzu SCL-20 AVP
EXPERIMENTAL SECTION
■
General Information. All reagents used in the synthesis were
commercial products and were used without further purification unless
otherwise indicated. The 99mTc−pertechnetate was eluted from a
commercial 99Mo/99mTc generator which was obtained from Beijing
1
Atomic High-Tech Co. The H NMR spectra were obtained at 400
MHz on a Bruker spectrometer in CDCl3 at room temperature with
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equipped with a Bioscan Flow Count 3200 NaI/PMT γ-radiation
scintillation detector. Separations were achieved on a Venusil MP C18
column (Agela Technologies, 10 μm, 10 mm ×250 mm) eluted with a
binary gradient system at a 4.0 mL/min flow rate. Mobile phase A was
water, while mobile phase B was acetonitrile. Fluorescent observation
was performed by Axio Oberver Z1 (Zeiss, Germany) equipped with
DAPI (excitation, 405 nm) and GFP filter sets (excitation, 505 nm).
The purity of the synthesized key compounds was determined using
analytical HPLC and was found to be more than 95%. Normal ICR
mice (five weeks, male) were used for biodistribution experiments. All
protocols requiring the use of mice were approved by the animal care
committee of Beijing Normal University. Post-mortem brain tissues
from an autopsy-confirmed case of AD (91-year-old, male, 35 μm,
prefrontal cortex) and a control subject (69-year-old, female, 35 μm,
prefrontal cortex) were kindly gifted from Dr. Jiapei Dai, which were
obtained from The Netherlands Brain Bank (NBB) by autopsy.
Transgenic mice brain tissues (C57BL6, APPswe/PSEN1, 11 months
old, male) were purchased from Institute of Laboratory Animal
Sciences, Chinese Academy of Medical Sciences.
6.67 (d, J = 8.8 Hz, 2H), 6.77 (dd, J1 = 15.3 Hz, J2 = 11.3 Hz, 1H),
6.97 (d, J = 15.4 Hz, 1H), 7.39 (d, J = 8.8 Hz, 2H), 7.61 (dd, J1 = 14.6
Hz, J2 = 11.3 Hz, 1H). 13C NMR (CDCl3, 100 MHz): δ 40.2, 85.0,
87.7, 98.5, 111.9, 120.6, 121.8, 123.7, 129.1, 144.3, 146.2, 151.3, 183.8,
192.2. HRMS: m/z calcd for [C21H18NO4187Re + H]+ 536.0872; found
536.0859.
1-[(2E,4E,6E)-1-Oxo-7-(4-dimethylaminophenyl)-2,4,6-hepta-
trienyl)]-(cyclopentadienyl)tricarbonylrhenium (5). Complex 5 was
prepared following the procedure used for 3. The residue was purified
by silica gel chromatography (ethyl acetate/petroleum ether = 1:1, v/
1
v) to afford a red solid (yield 85%); mp 205.2−207.2 °C. H NMR
(CDCl3, 400 MHz): δ 3.00 (s, 6H), 5.40 (s, 2H), 6.04 (s, 2H), 6.32−
6.44 (m, 2H), 6.67 (d, J = 8.8 Hz, 2H), 6.72−6.74 (m, 2H), 6.82−6.88
(m, 1H), 7.34−7.40 (m, 2H), 7.53 (dd, J1 = 14.5 Hz, J2 = 11.8 Hz,
1H). 13C NMR (CDCl3, 100 MHz): δ 40.2, 85.1, 87.7, 98.3, 112.1,
121.5, 123.9, 124.7, 127.9, 128.8, 138.9, 145.1, 145.5, 150.8, 183.8,
192.1. HRMS: m/z calcd for [C23H20NO4187Re + H]+ 562.1028; found
562.1030.
1-[(2E,4E)-1-Oxo-5-(4-dimethylaminophenyl)-2,4-pentadienyl]-
ferrocene (6). Acetylferrocene (35 mg, 0.15 mmol), (E)-3-(4-
(dimethylamino)phenyl)acrylaldehyde (35 mg, 0.20 mmol), and
solid NaOH (40 mg, 1.0 mmol) were dissolved in anhydrous ethanol
(5 mL). The mixture was stirred at room temperature for 6 h. After
filtration, the solvent was removed under reduced pressure. The
residue was purified by silica gel chromatography (ethyl acetate/
petroleum ether = 1:1, v/v) to afford a dark-red solid (yield 90%). 1H
NMR (CDCl3, 400 MHz): δ 3.01 (s, 6H), 4.20 (s, 5H), 4.53 (s, 2H),
4.86 (s, 2H), 6.53−6.98 (m, 5H), 7.32−7.79 (m, 3H). MS: m/z calcd
for [C23H23FeNO4 + H]+ 386.1; found 386.5.
Chemistry. (2E,4E)-5-(4-(Dimethylamino)phenyl)penta-2,4-dien-
al (1). To a stirring solution of (E)-3-(4-(dimethylamino)phenyl)-
acrylaldehyde (526 mg, 3.0 mmol) in dry THF, (1,3-dioxolan-2-
yl)methyltriphenylphosphonium (1.55 g, 3.6 mmol) was added to
form a suspension at room temperature, followed by 18-crown-6 (79
mg, 0.3 mmol) and NaH (60% in paraffin wax, 200 mg, 5.0 mmol) in a
ice bath. The reaction was then stirred under room temperature for 2 h
until quenched by adding 2.0 mL of concentrated hydrochloric acid.
Thirty minutes later, the solution was neutralized with saturated
K2CO3 aqueous solution and extracted with ethyl acetate (3 × 50 mL).
The organic layer was dried over Na2SO4. After the solvent was
removed, the residue was purified by silica gel chromatography (ethyl
acetate/petroleum ether = 6:1, v/v) to afford the final products (yield
1-[(2E,4E,6E)-1-Oxo-7-(4-dimethylaminophenyl)-2,4,6-hepta-
trienyl)]-ferrocene (7). Complex 7 was prepared following the
procedure used for 6. The residue was purified by silica gel
chromatography (ethyl acetate/petroleum ether = 1:1, v/v) to afford
1
55%). H NMR (CDCl3, 400 MHz): δ 3.05 (s, 6H), 6.15−6.20 (m,
1
a brown solid (yield 88%). H NMR (CDCl3, 400 MHz): δ 3.01 (s,
1H), 6.51−6.57 (m, 1H), 6.68−6.96 (m, 3H), 7.22−7.46 (m, 3H),
9.55−9.60 (m, 1H). MS: m/z calcd for [C13H15NO + H]+ 202.1;
found 202.4.
6H), 4.20 (s, 5H), 4.54 (s, 2H), 4.85 (s, 2H), 6.53−6.50 (m, 1H), 6.56
(d, J = 14.8 Hz, 1H), 6.64−6.86 (m, 4H), 6.79−6.86(m, 1H), 7.33−
7.37 (m, 2H), 7.47−7.54(m, 1H). MS: m/z calcd for [C25H25FeNO4 +
H]+ 412.1; found 412.6.
X-ray Crystallography. Single-crystal X-ray diffraction measure-
ments were carried out on a Bruker Smart APEXII CCD
diffractometer at 150(2) K using graphite monochromated Mο Kα
radiation (λ = 0.71070 Å). An empirical absorption correction was
applied using the SADABS program.39 All structures were solved by
direct methods and refined by full-matrix least-squares on F2 using the
SHELXL-97 program package.40 All of the hydrogen atoms were
geometrically fixed using the riding model.
In Vitro Fluorescent Staining of Aβ Plaques in Transgenic
Mouse Brain Sections and Human Brain Materials. Paraffin-
embedded brain sections of Tg mouse (C57BL6, APPswe/PSEN1, 11
months old, male, 6 μm) were used for the fluorescent staining. The
brain sections were deparaffinized with 2 × 20 min washes in xylene, 2
× 5 min washes in 100% ethanol, 5 min washes in 90% ethanol/H2O,
5 min wash in 80% ethanol/H2O, 5 min wash in 60% ethanol/H2O,
and running tap water for 10 min and then incubated in PBS (0.2 M,
pH = 7.4) for 30 min. The sections of human prefrontal cortex (35
μm) were obtained from The Netherlands Brain Bank (NBB) by
autopsy, and the sections were stored at 0−4 °C in 50% glycerol
diluted with 0.05 M TBS before use.
The brain sections were incubated with 10% ethanol solution (1.0
μM) of 3, 4, and 5 for 10 min. The localization of plaques was
confirmed by staining with thioflavin-S (0.125%) on the adjacent
sections. Finally, the sections were washed with 50% ethanol and PBS
(0.2 M, pH = 7.4) for 10 min. Fluorescent observation was performed
by Axio Oberver Z1 (Zeiss, Germany) equipped with DAPI
(excitation, 405 nm) and GFP filter sets (excitation, 505 nm).
Binding Assay in Vitro Using Aβ Aggregates. The trifluoro-
acetic acid salt forms of peptides Aβ1−42 were purchased from
AnaSpec. Aggregation of peptides was carried out by gently dissolving
the peptide (0.25 mg/mL for Aβ1−42) in a buffer solution (pH = 7.4)
containing 10 mM potassium dihydrogen phosphate and 1 mM
(Acetylcyclopentadienyl)tricarbonylrhenium (2). To a stirring
solution of (cyclopentadienyl)tricarbonylrhenium (200 mg, 0.6
mmol) in dry CH2Cl2 in ice bath, acetyl chloride (94 mg, 1.2
mmol) was added dropwise. The reaction mixture was stirred for 30
min at 0 °C and 30 min at room temperature. After adding 50 mL of
water, the mixture was extracted with ethyl acetate (3 × 30 mL). The
organic layer was dried over Na2SO4. The solvent was removed to
1
afford the white solid products (yield 95%). H NMR (CDCl3, 400
MHz): δ 2.34 (s, 3H), 5.40 (t, J = 2.3 Hz, 2H), 5.98 (t, J = 2.3 Hz,
2H). MS: m/z calcd for [C10H7O4187Re + H]+ 379.0; found 379.1.
1-[(2E)-1-Oxo-3-(4-dimethylaminophenyl)-2-propenyl]-
(cyclopentadienyl)tricarbonylrhenium (3). Complex 2 (50 mg, 0.15
mmol), 4-(dimethylamino)benzaldehyde (30 mg, 0.2 mmol), and solid
NaOH (40 mg, 1.0 mmol) were dissolved in anhydrous ethanol (5
mL). The mixture was stirred at room temperature for 6 h until
stopping by adding 30 mL of water. After extraction with ethyl acetate,
the solvent was removed under reduced pressure. The residue was
purified by silica gel chromatography (ethyl acetate/petroleum ether =
1:1, v/v) to afford a yellow solid. The solid was crystallized from a
mixture of ethanol and dichloromethane as light-yellow crystals (yield
1
91%); mp 180.2−182.4 °C. H NMR (CDCl3, 400 MHz): δ 3.05 (s,
6H), 5.41 (t, J = 2.3 Hz, 2H), 6.09 (t, J = 2.3 Hz, 2H), 6.68 (d, J = 8.9
Hz, 2H), 6.73 (d, J = 15.3 Hz, 2H), 7.50 (d, J = 8.8 Hz, 1H), 7.78 (d, J
= 15.3 Hz, 1H). 13C NMR (CDCl3, 100 MHz): δ 40.1, 84.6, 87.8,
98.8, 111.8, 114.9, 122.0, 130.7, 145.6, 152.3, 183.8, 192.3. HRMS: m/
z calcd for [C19H16NO4187Re + H]+ 510.0715; found 510.0717.
1-[(2E,4E)-1-Oxo-5-(4-dimethylaminophenyl)-2,4-pentadienyl]-
(cyclopentadienyl)tricarbonylrhenium (4). Complex 4 was prepared
following the procedure used for 3. The residue was purified by silica
gel chromatography (ethyl acetate/petroleum ether = 1:1, v/v) to
afford an orange solid. The solid was crystallized from a mixture of
ethanol and dichloromethane as light-orange crystals (yield 87%); mp
217.6−218.4 °C. 1H NMR (CDCl3, 400 MHz): δ 3.02 (s, 6H), 5.40 (t,
J = 2.3 Hz, 2H), 6.04 (t, J = 2.3 Hz, 2H), 6.39 (d, J = 14.6 Hz, 1H),
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EDTA. The solutions were incubated at 37 °C for 42 h with gentle and
constant shaking. Inhibition experiments were carried out in 12 mm ×
75 mm borosilicate glass tubes according to procedures described
previously with some modification.17 Briefly, 100 μL of aggregated Aβ
fibrils (28 nM in the final assay mixture) was added to a mixture
containing 100 μL of radioligand ([125I]IMPY, 100000 cpm/100 μL),
100 μL of inhibitors (complexes 3, 4, or 5, 10−4 M to 10−10 M in
ethanol), and 700 μL of PBS (0.2 M, pH = 7.4) in a final volume of 1.0
mL. Nonspecific binding was defined in the presence of 1 μM IMPY.
The mixture was incubated for 2 h at 37 °C, and then the bound and
free radioactive fractions were separated by vacuum filtration through
borosilicate glass fiber filters (Whatman GF/B) using a Mp-48T cell
harvester (Brandel, Gaithersburg, MD). The radioactivity from filters
containing the bound 125I-ligand was measured in a γ-counter
(WALLAC/Wizard1470, USA) with 70% efficiency. Under the assay
conditions, the specifically bound fraction accounted for about 10% of
total radioactivity. The inhibitory concentration (IC50) was deter-
mined using Graph Pad Prism 4.0, and the inhibition constant (Ki)
was calculated using the Cheng−Prusoff inhibition constant equation:
Ki = IC50/(1 + [L]/Kd).41
expressed in terms of the percentage of the injected dose per gram (%
ID/g) of blood or organs.
The biodistribution with PgP blocked was conducted using male
ICR mice (5 weeks, male, n = 5) pretreated with Cyclosporin A
(manufactured by Nanjing Duly biotech Co., Ltd. USP grade). Each
mouse was injected via tail vein with 50 mg/kg (100 μL solution
consisting of 10% EtOH, 15% saline and 75% propylene glycol), 1 h
prior to administration of [99mTc]3−5.42 The blood and brain uptakes
at 2 min were measured the same way narrated before.
Partition Coefficient Determination. The determination of
partition coefficients of 99mTc-labeled complexes were performed
according to the procedure previously reported.19 A solution of 99mTc-
labeled complexes (1.5 MBq) was added to a premixed suspensions
containing 3.0 g n-octanol and 3.0 g PBS (0.05 M, pH = 7.4) in a test
tube. The test tube was vortexed for 3 min at room temperature,
followed by centrifugation for 5 min at 3000 rpm. Two samples from
the n-octanol (50 μL) and water (500 μL) layers were measured. The
partition coefficient was expressed as the logarithm of the ratio of the
count per gram from n-octanol versus PBS. Samples from the n-
octanol layer were repartitioned until consistent partition coefficient
values were obtained. The measurement was done in triplicate and
repeated three times.
Preparation of [99mTc]3, [99mTc]4, and [99mTc]5. To an orange
solution of 1.0 mg of acetylferrocene and 1.0 mg of Mn(CO)5Br in 1
−
mL of DMF, 1.0 mL of 99mTcO4 aqueous solution (10 mCi) were
ASSOCIATED CONTENT
* Supporting Information
added. The reaction mixture was kept at 150 °C for 20 min in a sealed
vial. After extraction with CH2Cl2 and water, inorganic salts were
separated from the product while the intermediate [99mTc]2 was saved
in CH2Cl2. After removing the CH2Cl2 under nitrogen gas, the residue
was dissolved again in 1.0 mL of absolute ethanol. The appropriate
aldehyde (3.0 mg) was added into the reaction as well as 0.5 mg
NaOH to catalyze the Claisen Condensation. The reaction was kept
under room temperature for 30 min. After extraction by CH2Cl2, the
solvent was evaporated under nitrogen gas and the residue was
dissolved in CH3CN and purified by radio-HPLC under conditions as
following: Venusil MP C18 column (Agela Technologies, 10 mm ×
250 mm), CH3CN/H2O = 70/30 for [99mTc]3, [99mTc]4, CH3CN/
H2O = 80/20 for [99mTc]5, 4 mL/min, UV = 254 nm.
■
S
Purities of key target compounds; HPLC profiles of 3, [99mTc]
3, 4, [99mTc]4, 5, [99mTc]5; 1H NMR spectra, 13C NMR spectra
and HRMS data of rhenium complexes; crystal parameters for
complex 3 and 4. 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:
cmc@bnu.edu.cn (M.C.); liuboli@bnu.edu.cn (B.L.).
■
Binding Assay Using Aβ Aggregates with [99mTc]2−5. The
binding assay was performed by mixing 100 μL of [99mTc]2−5
(100000 cpm/100 μL), 100 μL of Aβ1−42 aggregates (7.27 μg/mL),
and 800 μL of 10% ethanol in 12 mm × 75 mm borosilicate glass
tubes. The blocking assay was performed by conducting the binding
assay in the presence of excess of rhenium complexes 2−5 (1 μM for
2−4, 0.5 μM for 5) as blocking agents. After incubation for 2 h at
room temperature, the mixture was filtered through GF/B filters
(Whatman GF/B) using a Mp-48T cell harvester (Brandel,
Gaithersburg, MD). Filters containing the bound 99mTc-labeled form
were examined in an automatic γ-counter (Wallac 1470 Wizard, USA).
Autoradiography in Vitro Using Brain Sections of Human
and Transgenic Model Mouse. Paraffin-embedded brain sections of
Tg and control mice were deparaffinized with 2 × 20 min washes in
xylene, 2 × 5 min washes in 100% ethanol, a 5 min wash in 90%
ethanol/H2O, a 5 min wash in 80% ethanol/H2O, a 5 min wash in
60% ethanol/H2O, and a 10 min wash in running tap water and then
incubated in PBS (0.2 M, pH = 7.4) for 30 min. The sections were
incubated with [99mTc]5 (370 KBq/100 μL) for 1 h at room
temperature. They were then washed in 40% EtOH before being
rinsed with water for 1 min. After drying, the sections were exposed to
a phosphorus plate (Perkin-Elmer, USA) for 4 h. In vivo autoradio-
graphic images were obtained using a phosphor imaging system
(Cyclone, Packard). After autoradiographic examination, the same
mouse brain sections were stained by thioflavin-S to confirm the
presence of Aβ plaques. After drying, the fluorescent observation was
performed by Axio Oberver Z1 (Zeiss, Germany) equipped with DAPI
(excitation, 405 nm) and GFP filter sets (excitation, 505 nm).
Biodistribution Studies. A saline solution containing the HPLC-
purified 99mTc-labeled tracer (100 μL, 10% ethanol, 5 μCi) was
injected via tail vein of ICR mice (five weeks, male). The mice were
sacrificed exactly at 2, 10, 30, 60, and 120 min. Samples of blood and
organs of interest were removed, weighed, and counted in an
automatic γ-counter (Wallac 1470 Wizard, USA). The results were
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We present special thanks to Dr. Jin Liu (College of Life
Science, Beijing Normal University) for assistance in the in
vitro neuropathological staining and Dr. Xuebing Deng
(College of Chemistry, Beijing Normal University) for
assistance in the X-ray diffraction. This work was supported
by National Natural Science Foundation of China (grants
21201019, 20871021, and 31070961) and Research Fund for
the Doctoral Program of Higher Education of China
(20120003120013).
ABBREVIATIONS USED
■
Aβ, β-amyloid; AD, Alzheimer’s Disease; PET, positron
emission tomography; SPECT, single photon emission
computed tomography; CNS, central nervous system; BBB,
blood−brain barrier; DLT, double ligand transfer; Cp,
cyclopentadienyl; MAMA, monoamine-monoamide dithiol;
BAT, bis-amino-bis-thiol; HPLC, high performance liquid
chromatography; NBB, Netherlands Brain Bank; PgP, perme-
ability-glycoprotein 1
REFERENCES
■
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Journal of Medicinal Chemistry
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