2-Phenylbenzothiazole conjugated with cyclopentadienyl tricarbonyl [CpM(CO)3] (M = Re, 99mTc) complexes as potential imaging probes for β-amyloid plaques

Article abstract of DOI:10.1039/c5dt00023h

Technetium-99m-labeled cyclopentadienyl tricarbonyl complexes conjugated with the 2-phenylbenzothiazole binding motif were synthesized. The rhenium surrogates 20, 21, 22 and 23 were demonstrated to have moderate to high affinities for Aβ_1-42 aggregates with K_i values of 142, 76, 64 and 24 nM, respectively. During the fluorescence staining of brain sections of transgenic mice and patients with Alzheimer's disease, these rhenium complexes demonstrated perfect and intense labeling of Aβ plaques. Moreover, in in vitro autoradiography, ^99mTc-labeled complexes clearly detected β-amyloid plaques on sections of brain tissue from transgenic mice, which confirmed the sufficient affinity of these tracers for Aβ plaques. However, these compounds did not show desirable properties in vivo, especially showing poor brain uptake (below 0.5% ID g^-1), which will hinder the further development of these tracers as brain imaging agents. Nonetheless, it is encouraging that these ^99mTc-labeled complexes designed by a conjugate approach displayed sufficient affinities for Aβ plaques. This journal is

Full text of DOI:10.1039/c5dt00023h

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Journal Name
RSCPublishing
ARTICLE
2ꢀPhenylbenzothiazole
Conjugated
with
Cyclopentadienyl Tricarbonyl [CpM(CO)3] (M = Re,
99mTc) Complexes as Potential Imaging Probes for β‑
Amyloid Plaques
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012,
Accepted 00th January 2012
Jianhua Jiaa, Mengchao Cuia,*, Jiapei Daib and Boli Liua
DOI: 10.1039/x0xx00000x
Technetiumꢀ99mꢀlabeled cyclopentadienyl tricarbonyl complexes conjugated with the 2ꢀ
phenylbenzothiazole binding motif were synthesized. The rhenium surrogates 20, 21, 22 and 23 were
demonstrated to have moderate to high affinities for Aβ1ꢀ42 aggregates with Ki values of 142, 76, 64 and
24 nM, respectively. During the fluorescent staining of brain sections of transgenic mice and patients
with Alzheimer’s disease, these rhenium complexes demonstrated perfect and intense labeling of Aβ
plaques. Moreover, in in vitro autoradiography, 99mTcꢀlabeled complexes clearly detected βꢀamyloid
plaques on sections of brain tissue from transgenic mice, which confirmed the sufficient affinity of these
tracers for Aβ plaques. However, these compounds did not show desirable property in vivo, especially
the poor brain uptake (below 0.5% ID/g), which hindered the further development of these tracers as
brain imaging agents. Nonetheless, it is encouraging that these 99mTcꢀlabeled complexes designed by
conjugate approach displayed sufficient affinities for Aβ plaques.
www.rsc.org/
8
6, [18F]GE0677, and [18F]BAYꢀ94ꢀ91729, 10, which have been
Introduction
recently approved by FDA. However, these probes have
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder that leads to irreversible cognitive decline, memory
disadvantages such as their relatively short halfꢀlives, complicated
synthetic manipulations, intense reliance on the cyclotron and
expensive cost. 99mTcꢀlabeled Aβ probes for single photon emission
tomography (SPECT) could, given the easier accessibility of 99mTc
and its longer halfꢀlife compared to 18F, provide a more convenient
approach for the detection of AD. In the past few decades, some
small and neutral 99mTcꢀlabled probes for Aβ have been reported
2
impairment and behavioral changes.1, According to the World
Health Organization, AD affects over 36 million people worldwide
and will increasingly burden families and society with the ageing
society deepening. Autopsied brains of AD patients have shown that
senile plaques, composed of βꢀamyloid (Aβ) peptides, and
including derivatives of benzothiazole11 ꢀ 14, biphenyl15, flavone16, 17
,
neurofibrillary
tangles
(NFTs),
consisting
of
highly
chalcone18, 19, benzofuran20 and so on. Among them, 99mTcꢀlabeled 2ꢀ
phenylbenzothiazole derivatives (ThioflavinꢀT skeleton structure)
seemed to be very attractive. Several 99mTc chelation ligands, such as
bisꢀaminoꢀbisꢀthiol (BAT)11, monoamide monoamine (MAMA)11,
hydrazino nicotinic acid (HYNIC)12, mercaptoacetyltriglycine
(MAG3)12, iminodiacetic acid (IDA)12, picolylamine monoaceticacid
(PAMA)13 and bis(pyridinꢀ2ꢀylmethyl)amine (DPA)14, were adopted
in the 2ꢀphenylbenzothiazole derivatives (Figure 1). However, most
hyperphosphorylated tau proteins, are the two main
4
neuropathological features of AD.3, Based on targeting the two
types of abnormal proteins, some new methods for the nonꢀinvasive
diagnosis of early stage AD have been explored. Among them, the
detection of Aβ plaques in vivo by positron emission tomography
(PET) has obtained some success. Based on the structures of Congo
Red and ThioflavinꢀT, many 11Cꢀ and 18Fꢀlabeled imaging probes
have been investigated, such as the PET imaging agents [18F]AVꢀ455,
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of these 99mTcꢀlabeled probes displayed unfavorable binding affinity compared with an integrated approach. In the present study,
affinities to the Aβ plaques and limited blood brain barrier (BBB) we reported the synthesis and evaluation of 2ꢀphenylbenzothiazole
penetration; therefore, identifying a proper chelating ligand that can derivatives conjugated with [CpM(CO)3] (M = 99mTc, Re) by
not only retain a high affinity for the Aβ plaques but also possess different carbon linkers (Figure 2).
good brain penetration is pivotal in the design of 99mTcꢀlabeled brain
imaging agents.
Figure 2. Chemical structures of [CpM(CO)3] (M = Re, 99mTc)
complexes mimicking chalcone and conjugated with 2ꢀ
phenylbenzothiazoles.
Results and Discussion
Chemistry
The synthetic route of the ferrocene precursors and corresponding
rhenium complexes is shown in Scheme 1. The binding motifs of 2ꢀ
Figure 1. Chemical structures of the 99mTcꢀlabeled 2ꢀ
phenyl benzothiazole hydroxyl compounds (2 and 3) and brominated
phenylbenzothiazole derivatives previously reported as Aβ imaging
compounds (4, 5, 6 and 7) were prepared as previously reported in
the literature.14 The primary amines 12, 13, 14 and 15 were obtained
probes.
Cyclopentadiene organometallic complexes, as versatile and with acceptable yields though
a Gabriel reaction by the
typical entities, have been widely applied in radiometalꢀlabeled hydrazinolysis of the corresponding Nꢀalkylphthalimides (8, 9, 10
tracers, and cyclopentadiene was considered to be an ideal candidate and 11). The ferrocene precursors 16, 17, 18 and 19 with different
to stabilize complexes with the facꢀ[M(CO)3]+ (M = Re, 99mTc) lengths of carbon linkers were synthesized through a condensation
core.21 The cyclopentadienyl tricarbonyl ligand ([CpM(CO)3]) has reaction from the active ferrocene ester and the primary amines 12,
inherent advantages, including its low molecular weight, moderate 13, 14 and 15. Rhenium complexes 20, 21, 22 and 23, as nonꢀ
lipophility, small size, high stability of the halfꢀsandwich radioactive surrogates of the 99mTcꢀlabeled tracers, were obtained
configuration and minimal steric hindrance. [CpM(CO)3] could from the ferrocene precursors and (NEt4)2[Re(CO)3Br3] at high
conjugate with the target molecule though different carbon linkers temperature and high pressure in an autoclave with yields of
directly or mimic an aromatic group in relevant molecules. In approximately 20%. All of these complexes were fully characterized
summary, [Cp99mTc(CO)3] is considered to be a promising ligand for by spectroscopic methods. In addition, complex 23 could be
the design of 99mTcꢀlabeled brain receptor imaging agents. M. Saidi recrystallized to produce Xꢀray quality crystals by slow evaporation
et.al reported the complexes M(CO)3CpꢀCOOC5H9NꢀR (M = 99mTc, of a mixed methanol and methylene dichloride solution. The ORTEP
Re; R = Me, isopropyl) for the in vivo imaging of serotonin 5ꢀHT1A structure is illustrated in Figure 3, and the relevant crystallographic
receptors in the brain, and they displayed significant affinities for the data are shown in Table 1. Complex 23 crystallizes in the triclinic
target receptor as well as high brain uptakes (1.02% ID/g) in rats 20 space group Pꢀ1. The asymmetric unit contains one neutral complex
min after i.v. application.22 Our group has recently described three and one methylene dichloride solvent molecule. The central rhenium
99mTcꢀlabeled cyclopentadienyl tricarbonyl complexes mimicking atom is η5ꢀcoordinated to the cyclopentadienyl ring, and the
the chalcone structure for Aβ plaques in the brain by an integrated coordination sphere is completed by three carbonyl groups. The
approach. The initial brain uptakes improved significantly (4.10 ± geometry around rhenium is pseudoꢀoctahedral with average ReꢀCp
0.38% ID/g for the 99mTc complex with n = 1), while the binding carbon bond lengths of 2.30 Å, ReꢀCO carbon bond lengths of 1.92
affinity for Aβ was not well retained by this integrated approach.18 In Å and CꢀReꢀC (between CO) carbon bond angles of approximately
general, a conjugate approach has an advantage in the binding 90°. The target molecule 2ꢀphenylbenzothiazole skeleton retains a
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planar structure and forms a certain angle with the Cp plane. The decreases the spatial steric hindrance, therefore, the affinity of the
distance between the chelating group and the target molecule target molecule is not expected to be greatly affected.
Scheme 1. Synthesis of Re/99mTcꢀlabeled 2ꢀphenylbenzothiazoles. Reagents and conditions: a. perfluorophenyl 2,2,2ꢀtrifluoroacetate,
pyridine, DMF, r.t., 3 h; b. 1,3ꢀdibromo propane or 1,5ꢀdibromo pentane, K2CO3, CH3CN, 90 °C, 4 h; c. potassium phthalimide, DMF, 100
°C, overnight; d. hydrazine hydrate, EtOH, 100 °C, 2 h; e. 1, triethylamine, DMF, r.t., 4 h; f. [Re(CO)3Cl3][NEt4]2, HCl (1 M), DMF, 160 °C,
2 h; g. Mn(CO)5Br, Na[99mTcO4], H2O, DMF, 150 °C, 50 min.
Table 1. Summary of the Xꢀray crystallographic data for complex
23.
Formula sum
M (g/mol)
C29H29N3O5SRe·CH2Cl2
801.74 g/mol
Crystal system
Spaceꢀgroup
triclinic
Pꢀ1
a=7.8257(13) Å α=100.46(0)°
b=13.264(2) Å β=90.16(0)°
c=15.137(3) Å γ=101.81(0)°
a/b=0.5900; b/c=0.8763; c/a=1.9343
1511.15(119) Å3
Cell parameters
Cell ratio
Cell volume (Å3)
Z
Radiolabeling
The formation of 99mTcꢀlabeled cyclopentadienyl tricarbonyl
complexes is also depicted in Scheme 1, which is referred to as a
double ligand transfer (DLT) reaction from ferrocene precursors
according to the previously reported literature.23 The 99mTcꢀlabeled
products were obtained under heat at 150 °C for 50 min with an
average radiochemical yield of 50% (no decay correction), and the
final pure 99mTcꢀlabeled products (RCP > 98%, Figure 4 and Table
S1) were obtained by purification with RPꢀHPLC. To identify the
radioactive products, the retention times were compared between the
nonꢀradioactive rhenium surrogates and 99mTcꢀlabeled radiotracers
through coꢀinjection and coꢀelution. Each pair of retention times
differed by less than 1.5 min, which was in accordance with the time
delay caused by the distance between the UV and radioactivity
detectors.
4
Calc. density (g/cm3)
1.76189 g/cm3
RAll
0.0521
Pearson code
Formula type
aP144
NOP3Q5R30...
Wyckoff sequence
i72
Figure 3. Crystal structure of complex 23 with the thermal ellipsoids
drawn at the 30% probability level.
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complexes have affinities for Aβ plaques. Technetium and rhenium
in the same group tend to have a shared chemistry, as well as similar
structure and properties, therefore, 99mTcꢀlabeled complexes were
predicted to also have affinities for Aβ plaques.
Figure 4. HPLC profiles of the purified complexes (blue lines, UV
signals of the rhenium complexes; red lines, radioactivity signals of
the 99mTc complexes). HPLC conditions: Venusil MP C18 column
(Agela Technologies, 4.6 mm × 250 mm), CH3CN/H2O = 80/20, 1
mL/min, UV, 254 nm.
Biological Evaluation
Figure 5. Fluorescent staining with 20 (A), 21 (E), 22 (I) and 23 (M)
on the brain slices of Tg mice (C57BL6, APPswe/PSEN1, 12ꢀ
monthꢀold, female) and wild control mice (C57BL6, 12ꢀmonthꢀold,
female) (C, G, K and O). Adjacent sections were stained with
ThioflavinꢀS for comparison (B, F, J and N) (D, H, L and P).
(magnification: 5×, DAPI filter, EX 365 nm, EM 445 nm)
In Vitro Fluorescent Staining The rhenium complexes displayed
fluorescence emission properties in solution because of the
conjugated 2ꢀphenybenzothiazole structure. The spectroscopic
properties were shown in Table 2. Thus, neuropathological
fluorescent staining experiments with rhenium complexes were
implemented on sections of brain tissue from Tg mice (C57BL6,
APPswe/PSEN1, 12ꢀmonthꢀold, female) and an AD patient (91ꢀyearꢀ
old, male, temporal lobe) to confirm the affinity of the rhenium
complexes for Aβ plaques. As shown in Figure 5, they specifically
stained Aβ plaques on the brain sections of Tg mice (A, E, I and M),
and the results were in agreement with the staining of adjacent brain
sections (B, F, J and N) using ThioflavinꢀS (a histological dye for
the clinical detection of Aβ plaques). As expected, there was not
notable staining on the brain sections from normal mice with
rhenium complexes (C, G, K and O) as well as with ThioflavinꢀS (D,
H, L and P). Moreover, the Aβ plaques on the brain sections of the
AD patient were clearly labeled by these compounds (Figure 6, A, B,
C and D). More interestingly, NFTs and cerebrovascular amyloids
on the brain sections of the AD patient (Figure 6, E and F, complex
23) could also be stained by these rhenium complexes, which
indicated that they have affinities for tangles and cerebrovascular
amyloids. The staining results demonstrated that these rhenium
Figure 6. Fluorescent staining of 20 (A), 21 (B), 22 (C) and 23 (D, E
and F) on the brain slices of an AD patient (91ꢀyearꢀold, male,
temporal lobe). NFTs (E) and cerebrovascular amyloids (F) were
also stained by 23. (magnification: 10×, DAPI filter, EX 365 nm,
EM 445 nm)
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In Vitro Inhibition Assay Competitive binding assays were Figure 7. Inhibition curves of rhenium complexes for the binding of
performed to evaluate the affinity of these rhenium complexes for
125I]IMPY to Aβ1ꢀ42 aggregates.
Aβ1−42 aggregates using
6ꢀ[125I]iodoꢀ2ꢀ(4’ꢀN,Nꢀ
[
dimethylamino)phenylimidazo[1,2ꢀa]ꢀpyridine ([125I]IMPY) as the
competing radioligand. The binding of [125I]IMPY to Aβ1ꢀ42
aggregates was inhibited by these rhenium complexes in a doseꢀ
dependent manner, which manifested their affinities for Aβ1ꢀ42
aggregates (Figure 7). As shown in Table 2, rhenium complexes
showed moderate to high binding affinities for Aβ1−42 aggregates (Ki
= 24.0 ꢀ 142.6 nM). Complex 23, with a N,Nꢀdimethylamino group
and longer carbon linker, displayed the highest affinity compared
with the other three complexes. As shown in the crystal structure,
although the entire molecule is not in a planar configuration, the
affinity of the target molecule was wellꢀretained due to the action of
the carbon linker. The secondary Nꢀmethylamino analogues were
found to have weaker affinities than the tertiary N,Nꢀdimethylamino
analogues (20 vs. 22, 21 vs. 23), and the relatively longer carbon
linker is beneficial for retaining the high affinities of the target
molecules (20 vs. 21, 22 vs. 23), which were similar to the rules
previously reported.24 Compared to the reported 99mTcꢀlabeled 2ꢀ
phenylbenzothiazole analogues with other chelating groups, the
99mTcꢀlabeled cyclopentadienyl tricarbonyl benzothiazole analogues
based on the conjugate approach displayed slightly higher affinities
(Ki = 29.5 – 617 nM)25. In addition, they possessed better affinities
than the [CpRe(CO)3] complexes mimicking the chalcone structure,
which were designed according to the integrated approach (Ki = 108
– 899 nM)18. Additionally, the affinity of cold IMPY was also
evaluated using the same assay conditions for comparison, and
complex 23 displayed a comparable affinity to that of IMPY (Ki =
12.5 ± 2.8 nM).
Table 2. The binding data and spectroscopic properties (absorption,
excitation and emission wavelength) of rhenium complexes 20 – 23.
Compound
Ki (nM)a
142.6 ± 25.9
75.8 ± 36.3
64.1 ± 13.9
24.0 ± 8.1
12.5 ± 2.8
abs (nm)b
360.2
354.2
356.4
356.2
ꢀ
λex / λem (nm)b
364.0 / 421.0
364.0 / 421.0
364.0 / 424.0
367.0 / 425.0
ꢀ
20
21
22
23
IMPY
a The Ki values were determined in three independent experiments (n
= 3). b measured in ethanol (10 ꢀM).
In Vitro Autoradiography In vitro autoradiography studies could
visibly reflect the affinity of the radioꢀlabeled tracers for Aβ plaques
in brain tissue. In vitro autoradiography studies of the 99mTcꢀlabeled
radiotracers were performed on brain sections of Tg mice (C57BL6,
APPswe/PSEN1, 12ꢀmonthꢀold, female) and ageꢀmatched control
mice (C57BL6, 12ꢀmonthꢀold, female). As shown in Figure 8 (A, C,
E and G), many radioactive spots on the brain sections of Tg mice
were detected, with the distribution of the Aβ plaques being
identified by fluorescent staining with ThioflavinꢀS (Figure 8, B, D,
F and H). By comparison, no radioactive accumulation was observed
on the brain sections of the wild control mice (Figure S1). Although
the binding affinity of [99mTc]20 to Aβ1ꢀ42 aggregates was not
extremely high, the plaques in the brain sections of Tg mice was still
possible with [99mTc]20. In accord with the published results15,
[
99mTc]20 ꢀ 23 displayed negative labeling of Aβ plaques on brain
sections of AD patients, the reason for this may due to the different
structure or conformation of Aβ plaque between Tg mouse and AD
patient. However, as shown in Figure S2, they could label
cerebrovascular amyloids on the brain sections of the AD patient
intensely.
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“piano stool” organometallic core is small and neutral, the strategy
that incorporated it into the 2ꢀphenylbenzothiazole skeleton by an
amide bond is not successful. Additionally, these tracers are mainly
excreted by the liver and small intestine because a high accumulation
of radioactivity was found in liver, and the uptake in the small
intestine kept increasing after injection (Table S2).
Conclusions
Complexes
of
2ꢀphenylbenzothiazole
conjugated
with
[CpM(CO)3] (M = Re, 99mTc) were successfully synthesized for the
detection of Aβ plaques in AD brains. 99mTcꢀlabeled tracers with
high radiochemical purities have been prepared in satisfactory
radiochemical yields. In in vitro biological evaluations, these tracers
possessed optimal properties, and their sufficient binding affinities
for Aβ plaques and Aβ1−42 aggregates were proven by their in vitro
fluorescent staining of brain sections of Tg mice and an AD patient
as well as in an inhibition assay. Moreover, the high specific binding
of 99mTcꢀlabeld tracers to Aβ plaques was verified by in vitro
autoradiography on brain section of Tg mice. However, these tracers
do not fulfill the requirements for ideal brain imaging probes
because of their insufficient initial uptakes (0.26 ꢀ 0.5% ID/g) and
slow clearance (the brain2 min/brain60 min ratio is approximately 2 ꢀ 3)
in the brain. In summary, the preliminary results suggest that 99mTcꢀ
Figure 8. In vitro autoradiography of [99mTc]20 (A), [99mTc]21 (C),
[
99mTc]22 (E) and [99mTc]23 (G) on brain sections of Tg mice
(C57BL6, APPswe/PSEN1, 12ꢀmonthꢀold, female). The presence
and distribution of Aβ plaques were confirmed by fluorescence
staining using ThioflavinꢀS on the same sections (B, D, F and H).
(magnification: 5×, DAPI filter, EX 365 nm, EM 445 nm)
In Vivo Biodistribution in Normal Mice The biodistribution
experiments were carried out in normal mice to evaluate the
pharmacokinetics of the 99mTcꢀlabeled tracers in vivo, especially
their ability to penetrate the BBB and their rate of washout from
normal regions, which are regarded as important factors for Aβ
imaging probes. As shown in Table 3, [99mTc]20 ꢀ 23 displayed
unsatisfactory initial uptakes at 2 min postꢀinjection, which were
0.50, 0.36, 0.26 and 0.37% ID/g, respectively. In addition, they
possessed the property of limited clearance from the normal brain
cyclopentadienyl
tricarbonylꢀlabeled
2ꢀphenylbenzothiazole
complexes based on the conjugate approach have some potential to
detect Aβ plaques in the brains of Alzheimer’s patients. However,
some modifications are necessary to improve their abilities to cross
the BBB and accelerate their rates of washout by additional
structural refinements.
Instead of being used for radioꢀimaging for the detection of Aβ
plaques in brains, they may be used for diagnosing other diseases
that are related to abnormal Aβ deposits but have a low requirement
for BBB penetration, such as cerebral amyloid angiopathy and
cardiac amyloidosis. Moreover, it was reported that the
benzothiazole skeleton possesses highly selective potent antitumor
properties in vitro and in vivo; thus, the 99mTcꢀcyclopentadienyl
tricarbonylꢀlabeled 2ꢀphenylbenzothiazole complexes may be
developed as potential cancer radiopharmaceuticals.26, 27
(brain2 min/brain60
ratio is approximately 2 ꢀ 3). Although the
min
lipophilicity of these 99mTcꢀlabeled tracers is suitable for BBB
penetration, it does not offset the disadvantage that the molecular
weights of these tracers are large, and some of them even exceed
600, which is considered as the maximum limit of molecular weight
to cross the BBB. Furthermore, the amide bond in the linker, which
could form hydrogen bonds, has a negative effect on BBB
penetration. Although the [Cp99mTc (CO)3] ligand as a lipophilic
Table 3. Brain uptake of normal mice (ICR, 5 weeks, 22 ꢀ 25 g, male) after i.v. injection of [99mTc]20, [99mTc]21, [99mTc]22 and [99mTc]23.
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Brain uptake b
Compound
Log D a
2 min
10min
30min
60min
99mTc]20
99mTc]21
99mTc]22
99mTc]23
3.51 ± 0.14
3.17 ± 0.04
3.55 ± 0.10
3.26 ± 0.06
0.50 ± 0.10
0.36 ± 0.07
0.26 ± 0.04
0.37 ± 0.08
0.48 ± 0.09
0.34 ± 0.10
0.17 ± 0.02
0.12 ± 0.01
0.28 ± 0.10
0.29 ± 0.08
0.15 ± 0.01
0.11 ± 0.02
0.18 ± 0.07
0.19 ± 0.02
0.11 ± 0.02
0.14 ± 0.03
[
[
[
[
a Measured in triplicate with results given as the mean ± SD.
b Expressed as % injected dose per gram. Average for 5 mice ± standard deviation.
Axio Observer Z1 inverted fluorescence microscope (Zeiss,
Germany) equipped with a DAPI filter set (EX 365 nm, EM 445
nm). Normal mice (ICR, 5 weeks, 22 ꢀ 25 g, male) were used for the
biodistribution experiments. All protocols requiring the use of
animals were approved by the animal care committee of Beijing
Normal University. Postꢀmortem brain tissue from autopsyꢀ
Experimental Section
General Information All of the reagents used in the synthesis
were commercial products and were used without further
purification unless otherwise indicated. The HꢀNMR and 13CꢀNMR
1
spectra were obtained at 400 MHz on Bruker Avance Ⅲ NMR
spectrometers in CDCl3 and MeOD at room temperature with TMS confirmed AD patients (91ꢀyearꢀold, male, temporal lobe) (68ꢀyearꢀ
as an internal standard. Chemical shifts were reported as δ values old, female, frontal lobe) was obtained from the Chinese Brain Bank
relative to the internal TMS. Coupling constants were reported in Center (CBBC) by autopsy.
Hertz. Multiplicity is defined by s (singlet), d (doublet), t (triplet),
Chemistry
and
m (multiplet). Mass spectra were acquired using a
SurveyorMSQ Plus (ESI) (Waltham, MA, USA) instrument. The
radiochemical purity was determined by HPLC performed on a
Shimadzu SCLꢀ20 AVP system equipped with a SPDꢀ20A UV
detector (λ = 254 nm) and a Bioscan Flow Count 3200 NaI/PMT γꢀ
radiation scintillation detector. HPLC separations and analysis were
both achieved on a Venusil MP C18 reverse phase column (Agela
Technologies, 5 µm, 4.6 mm × 250 mm) eluted with an isocratic
system at a flow rate of 1.0 mL/min. Mobile phase A was water
Synthesis of perfluorophenyl ferrocenecarboxylate (1) To a
solution of ferrocenecarboxylic acid (231.5 mg, 1.00 mmol) in DMF
(1 mL), perfluorophenyl 2,2,2ꢀtrifluoroacetate (280.4 mg, 1.00
mmol) and pyridine (150 ꢀL) were added, and the solution was
stirred at r.t. for 3 h. After the completion of the reaction, the
mixture was diluted in ethyl acetate, and then it was washed by HCl
(0.1 M) three times and a 5% NaHCO3 solution one time. The
organic layer was collected and dried over anhydrous MgSO4. Then,
the organic solvent was removed in vacuum, and the residue was
purified by silica gel column chromatography (column
chromatography condition: petroleum ether/AcOEt = 50/1). Orange
while mobile phase
B was acetonitrile. The absorption and
fluorescence spectra were measured using UVꢀVis (UVꢀ3600,
Shimadzu,
5301PC,
Japan) and fluorescence spectrophotometers (RFꢀ
Shimadzu, Japan), respectively. Reactions were
1
solid, 658.6 mg, yield, 56.2 %. M.p. 60.3 – 61.3 °C. H NMR (400
monitored by TLC (Silica gel 60 F254 aluminum sheets, Merck) and
visualized by illumination with a short wavelength UV lamp (λ =
254 nm or 365 nm). Column chromatography purification was
performed on silica gel (54 ꢀ 74 ꢀm) from Qingdao Haiyang
Chemical Co., Ltd. Fluorescent observation was performed by the
MHz, CDCl3) δ 4.98 (d, J = 1.8 Hz, 2H), 4.59 (d, J = 1.9 Hz, 2H),
4.33 (s, 5H). MS (ES+): m/z calcd for [C17H9F5FeO2 ]+ 396.0; found
395.8.
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DOI: 10.1039/C5DT00023H
Compouds 4 ꢀ 7 were synthesized according to the previously
reported procedure.14
2ꢀ(5ꢀ((2ꢀ(4ꢀ(dimethylamino)phenyl)benzo[d]thiazolꢀ6ꢀ
yl)oxy)pentyl)isoindolineꢀ1,3ꢀdione (11) Yellow powder, 625.4 mg,
yield, 87.0 (column chromatography condition: petroleum
%
General Procedure for the Synthesis of Intermediates 8 ꢀ 11
A solution of 4 ꢀ 7 (1 equiv) and potassium 1,3ꢀdioxoisoindolinꢀ2ꢀ
ide (2 equiv) in DMF (10 mL), under a nitrogen atmosphere, was
heated at 100 °C overnight. After the completion of the reaction,
DMF was removed in vacuum at 110 °C, water (20 mL) was added
to the reactant after cooling, the resulting mixture was extracted by
dichloromethane (3 × 20 ml) and the organic layer was dried over
anhydrous MgSO4. The organic solvent was removed in vacuum,
ether/AcOEt/CH2Cl2 = 7/1/3). M.p. 152.5 – 153.1 °C. 1H NMR (400
MHz, CDCl3) δ 7.93 (d, J = 8.4 Hz, 2H), 7.88 – 7.81 (m, 3H), 7.71
(dd, J = 5.4, 3.1 Hz, 2H), 7.28 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.9,
2.4 Hz, 1H), 6.75 (d, J = 8.8 Hz, 2H), 4.01 (t, J = 6.4 Hz, 2H), 3.74
(t, J = 7.2 Hz, 2H), 3.05 (s, 6H), 1.91 – 1.84 (m, 2H), 1.79 – 1.75 (m,
2H), 1.60 – 1.52 (m, 2H). MS (ES+): m/z calcd for [C28H28N3O3S +
H]+ 486.2; found 485.9.
and then the residue was purified by silica gel column General Procedure for the Synthesis of Intermediates 12 ꢀ 15
chromatography.
A solution of 8 ꢀ 11 (1 equiv) and hydrazine hydrate (10 equiv) was
heated for 2 h at 100°C in EtOH (20 mL). After the yellow
suspension turned white, the solvent was removed in vacuum and
water was added. The resulting mixture was extracted by
dichloromethane (3 × 20 ml), and the organic layer was dried over
anhydrous MgSO4. The organic solvent was removed in vacuum,
and then the residue was purified by silica gel column
chromatography or recrystallization.
2ꢀ(3ꢀ((2ꢀ(4ꢀ(methylamino)phenyl)benzo[d]thiazolꢀ6ꢀ
yl)oxy)propyl)isoindolineꢀ1,3ꢀdione (8) Yellow powder, 206.9 mg,
yield, 54.9
% (column chromatography condition: petroleum
ether/AcOEt/CH2Cl2 = 7/1/3). M.p. 178.6 – 178.9 °C. 1H NMR (400
MHz, CDCl3) δ 7.87 (d, J = 8.7 Hz, 2H), 7.84 (dd, J = 5.4, 3.1 Hz,
2H), 7.81 (d, J = 8.9 Hz, 1H), 7.72 (dd, J = 5.4, 3.0 Hz, 2H), 7.23 (d,
J = 2.4 Hz, 1H), 6.90 (dd, J = 8.9, 2.5 Hz, 1H), 6.64 (d, J = 8.7 Hz,
2H), 4.09 (t, J = 6.0 Hz, 2H), 3.94 (t, J = 6.8 Hz, 2H), 2.91 (s, 3H),
2.26 – 2.20 (m, 2H). MS (ES+): m/z calcd for [C25H22N3O3S + H]+
444.1; found 443.9.
4ꢀ(6ꢀ(3ꢀaminopropoxy)benzo[d]thiazolꢀ2ꢀyl)ꢀNꢀmethylaniline
(12) White powder, 185 mg, yield, 65.6 % (recrystallization
1
condition: petroleum ether/AcOEt = 5/1). M.p. 107.3 – 108.1 °C. H
NMR (400 MHz, CDCl3) δ 7.88 – 7.84 (m, 3H), 7.32 (d, J = 2.4 Hz,
1H), 7.03 (dd, J = 8.9, 2.5 Hz, 1H), 6.64 (d, J = 8.7 Hz, 2H), 4.12 (t,
J = 6.1 Hz, 2H), 2.94 (t, J = 6.8 Hz, 2H), 2.91 (s, 3H), 2.00 – 1.94
(m, 2H). MS (ES+): m/z calcd for [C17H20N3OS + H]+ 314.1; found
314.1.
2ꢀ(5ꢀ((2ꢀ(4ꢀ(methylamino)phenyl)benzo[d]thiazolꢀ6ꢀ
yl)oxy)pentyl)isoindolineꢀ1,3ꢀdione (9) Yellow powder, yield,
249.9 mg, 62.4 % (column chromatography condition: petroleum
ether/AcOEt/CH2Cl2 = 8/1/3). M.p. 138.8 – 139.2 °C. 1H NMR (400
MHz, CDCl3) δ 7.88 (d, J = 8.4 Hz, 2H), 7.85 – 7.83 (m, 3H), 7.71
(dd, J = 5.4, 3.1 Hz, 2H), 7.28 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.9,
2.3 Hz, 1H), 6.65 (d, J = 8.6 Hz, 2H), 4.01 (t, J = 6.4 Hz, 2H), 3.74
(t, J = 7.2 Hz, 2H), 2.91 (s, 3H), 1.92 – 1.84 (m, 2H), 1.82 – 1.75 (m,
2H), 1.59 – 1.52 (m, 2H). MS (ES+): m/z calcd for [C27H26N3O3S +
H]+ 472.2; found 472.0.
4ꢀ(6ꢀ((5ꢀaminopentyl)oxy)benzo[d]thiazolꢀ2ꢀyl)ꢀN–methylaniline
(13) White powder, 206.9 mg, yield, 63.8
%
(column
chromatography condition: AcOEt/CH2Cl2/CH3OH = 5/5/3). M.p.
1
85.6 – 86.3 °C. H NMR (400 MHz, CDCl3) δ 7.93 – 7.78 (m, 3H),
7.30 (s, 1H), 7.02 (dd, J = 8.9, 2.0 Hz, 1H), 6.64 (d, J = 8.6 Hz, 2H),
4.02 (t, J = 6.4 Hz, 2H), 2.91 (s, 3H), 2.75 (t, J = 6.5 Hz, 2H), 1.88 –
1.81 (m, 2H), 1.63 – 1.43 (m, 4H). MS (ES+): m/z calcd for
[C19H24N3OS + H]+ 342.2; found 342.0.
2ꢀ(3ꢀ((2ꢀ(4ꢀ(dimethylamino)phenyl)benzo[d]thiazolꢀ6ꢀ
yl)oxy)propyl)isoindolineꢀ1,3ꢀdione (10) Yellow powder, 555.2
mg, yield, 54.6 % (column chromatography condition: petroleum
ether/AcOEt/CH2Cl2 = 8/1/3). M.p. 206.8 – 207.8 °C. 1H NMR (400
MHz, CDCl3) δ 7.93 (d, J = 8.3 Hz, 2H), 7.86 – 7.83 (m, 3H), 7.72
(dd, J = 5.4, 3.1 Hz, 2H), 7.23 (d, J = 2.4 Hz, 1H), 6.91 (dd, J = 8.9,
2.4 Hz, 1H), 6.76 (d, J = 8.6 Hz, 2H), 4.10 (t, J = 6.0 Hz, 2H), 3.94
(t, J = 6.8 Hz, 2H), 3.06 (s, 6H), 2.28 – 2.19 (m, 2H). MS (ES+): m/z
calcd for [C26H24N3O3S + H]+ 458.2; found 457.8.
4ꢀ(6ꢀ(3ꢀaminopropoxy)benzo[d]thiazolꢀ2ꢀyl)ꢀN,Nꢀdimethylaniline
(14) White powder, 175.0 mg, yield, 96.4 % (recrystallization
condition: petroleum ether/AcOEt = 5/1). M.p. 147.4 – 148.1 °C. 1H
NMR (400 MHz, CDCl3) δ 7.90 (d, J = 8.9 Hz, 2H), 7.85 (d, J = 8.9
Hz, 2H), 7.32 (d, J = 2.5 Hz, 1H), 7.03 (dd, J = 8.9, 2.5 Hz, 1H),
6.74 (d, J = 8.9 Hz, 2H), 4.12 (t, J = 6.1 Hz, 2H), 3.05 (s, 6H), 2.94
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(t, J = 6.7 Hz, 2H), 2.00 – 1.92 (m, 2H). MS (ES+): m/z calcd for ether/AcOEt/CH2Cl2 = 3/1/1). M.p. 219.4 – 220.1 °C. 1H NMR (400
[C18H22N3OS + H]+ 328.1; found 328.0.
MHz, CDCl3) δ 7.97 – 7.95 (m, 3H), 7.36 (d, J = 1.7 Hz, 1H), 7.10
(dd, J = 8.9, 2.2 Hz, 1H), 6.77 (d, J = 8.6 Hz, 2H), 6.14 (s, 2H), 4.67
(s, 2H), 4.35 (s, 2H), 4.22 – 4.15 (m, 7H), 3.68 – 3.60 (m, 2H), 3.07
4ꢀ(6ꢀ((5ꢀaminopentyl)oxy)benzo[d]thiazolꢀ2ꢀyl)ꢀN,Nꢀ
dimethylaniline (15) White powder, 366.4 mg, yield, 83.1 %
(s, 6H), 2.18
– 2.11 (m, 2H). MS (ES+): m/z calcd for
(column
chromatography
condition:
petroleum
[C29H30FeN3O2S + H]+ 540.1; found 539.9.
1
ether/CH2Cl2/CH3OH/Et3N = 30/10/1/1). M.p. 101.2 – 102.4 °C. H
NMR (400 MHz, CDCl3) δ 7.90 (d, J = 8.9 Hz, 2H), 7.85 (d, J = 8.9
Nꢀ(5ꢀ((2ꢀ(4ꢀ(dimethylamino)phenyl)benzo[d]thiazolꢀ6ꢀ
Hz, 1H), 7.30 (d, J = 2.4 Hz, 1H), 7.02 (dd, J = 8.9, 2.5 Hz, 1H), yl)oxy)pentyl)carboxamide)ꢀferrocene (19) Orange powder, 239.4
6.74 (d, J = 8.9 Hz, 2H), 4.03 (t, J = 6.4 Hz, 2H), 3.05 (s, 6H), 2.74 mg, yield, 84.4 % (column chromatography condition: petroleum
(t, J = 6.4 Hz, 2H), 1.88 – 1.80 (m, 2H), 1.56 – 1.52 (m, 4H). MS ether/AcOEt/CH2Cl2 = 3/1/1). M.p. 189.0 – 189.4 °C. 1H NMR (400
(ES+): m/z calcd for [C20H26N3OS + H]+ 356.2; found 356.1.
MHz, CDCl3) δ 7.91 (d, J = 8.6 Hz, 2H), 7.86 (d, J = 8.7 Hz, 1H),
7.30 (t, J = 7.0 Hz, 1H), 7.03 (dd, J = 8.9, 2.4 Hz, 1H), 6.75 (d, J =
8.8 Hz, 2H), 5.70 (s, 1H), 4.65 (s, 2H), 4.33 (s, 2H), 4.20 (s, 5H),
4.05 (t, J = 6.2 Hz, 2H), 3.45 – 3.40 (m, 2H), 3.05 (s, 6H), 1.91 –
1.87 (m, 2H), 1.71 – 1.66 (m, 2H), 1.65 – 1.58 (m, 2H). MS (ES+):
m/z calcd for [C31H34FeN3O2S + H]+ 568.2; found 568.0.
General Procedure for the Synthesis of Ferrocene Precursors 16
ꢀ 19
To a stirring solution of 11 ꢀ 15 (1 equiv) in anhydrous DMF,
compound 1 (1 equiv) and triethylamine (150 ꢀL) were added. The
resulting mixture was stirred for 4 h at room temperature, and then
the solvent was removed under reduced pressure. The residue was General Procedure for the Synthesis of Rhenium Complexes 20 –
purified by silica gel column chromatography.
23
A solution of [Re(CO)3Cl3][NEt4]2 (1.5 equiv) and 16 ꢀ 19 (1 equiv)
in DMF (2.5 ml) and 0.5 ml of HCl (1 M) were sealed in an
autoclave with a magnetic stir bar at 160 °C for 2 h. After cooling,
the reaction mixture was poured into a dichloromethane solution (10
ml). The solvent was removed in vacuum, and the crude product was
purified by column chromatography on silica gel.
Nꢀ(3ꢀ((2ꢀ(4ꢀ(methylamino)phenyl)benzo[d]thiazolꢀ6ꢀ
yl)oxy)propyl)carboxamide)ꢀferrocene (16) Orange powder, 189.2
mg, yield, 72.3 % (column chromatography condition: petroleum
ether/AcOEt/CH2Cl2 = 2/1/1). M.p. 227.7 – 229.2 °C. 1H NMR (400
MHz, CDCl3) δ 7.95 – 7.70 (m, 3H), 7.36 (d, J = 1.9 Hz, 1H), 7.09
(dd, J = 8.8, 1.8 Hz, 1H), 6.66 (d, J = 8.5 Hz, 2H), 6.16 (s, 1H), 4.67
(s, 2H), 4.35 (s, 2H), 4.21 – 4.13 (m, 7H), 3.66 – 3.61 (m, 2H), 2.92
Nꢀ(3ꢀ((2ꢀ(4ꢀ(methylamino)phenyl)benzo[d]thiazolꢀ6ꢀ
yl)oxy)propyl)carboxamide)ꢀ
(s, 3H), 2.19
[C28H28FeN3O2S + H]+ 526.1; found 526.0.
– 2.10 (m, 2H). MS (ES+): m/z calcd for
(cyclopentadienyl)tricarbonylrhenium (20) Light yellow powder,
18.3 mg, yield, 27.0
% (column chromatography condition:
petroleum ether /CH2Cl2 = 1/1 (containing 5% of Et3N)). M.p. 181.7
– 183.0 °C. 1H NMR (400 MHz, CDCl3) δ 7.90 – 7.86 (m, 3H), 7.31
(d, J = 1.8 Hz, 1H), 7.02 (dd, J = 8.9, 2.0 Hz, 1H), 6.65 (d, J = 8.5
Hz, 2H), 6.29 (s, 1H), 5.87 (s, 2H), 5.36 (s, 2H), 4.14 (t, J = 5.5 Hz,
2H), 3.62 – 3.58 (m, 2H), 2.92 (s, 3H), 2.12 – 2.01 (m, 2H). 13C
NMR (100 MHz, CDCl3) δ 192.44, 166.87, 162.23, 156.00, 151.36,
149.22, 135.94, 128.81, 122.95, 122.68, 115.30, 112.08, 105.38,
95.49, 85.38, 84.90, 67.31, 37.99, 30.37, 28.79, 26.93. TOF MS
(ES+): m/z calcd for [C26H23N3O5S185Re + H]+ 674.0888; found
674.0892.
Nꢀ(5ꢀ((2ꢀ(4ꢀ(methylamino)phenyl)benzo[d]thiazolꢀ6ꢀ
yl)oxy)pentyl)carboxamide)ꢀferrocene (17) Orange powder, 179.4
mg, yield, 54.0 % (column chromatography condition: petroleum
ether/AcOEt/CH2Cl2 = 2/1/1). M.p. 198.5 – 199.7 °C. 1H NMR (400
MHz, CDCl3) δ 7.91 – 7.89 (m, 3H), 7.30 (s, 1H), 7.03 (d, J = 8.7
Hz, 1H), 6.65 (d, J = 8.1 Hz, 2H), 5.72 (s, 1H), 4.65 (s, 2H), 4.33 (s,
2H), 4.20 (s, 5H), 4.05 (t, J = 6.0 Hz, 2H), 3.46 – 3.39 (m, 2H), 2.91
(s, 3H), 1.93 – 1.89 (m, 2H), 1.73 – 1.68 (m, 2H), 1.66 – 1.57 (m,
2H). MS (ES+): m/z calcd for [C30H32FeN3O2S + H]+ 554.2; found
554.0.
Nꢀ(5ꢀ((2ꢀ(4ꢀ(methylamino)phenyl)benzo[d]thiazolꢀ6ꢀ
yl)oxy)pentyl)carboxamide)ꢀ
Nꢀ(3ꢀ((2ꢀ(4ꢀ(dimethylamino)phenyl)benzo[d]thiazolꢀ6ꢀ
yl)oxy)propyl)carboxamide)ꢀferrocene (18) Orange powder, 22.5
mg, yield, 41.7 % (column chromatography condition: petroleum
(cyclopentadienyl)tricarbonylrhenium (21) Light yellow powder,
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18.5 mg, yield, 26.3
% (column chromatography condition: diffractometer at 100 K using graphite monochromated Mο Kα
petroleum ether /CH2Cl2 = 1/1 (containing 5% of Et3N)). M.p. 189.3 radiation (λ = 0.71073 Å). The structures were solved by direct
– 190.1 °C. 1H NMR (400 MHz, CDCl3) δ 7.91 – 7.89 (m, 3H), 7.28 methods and refined by a fullꢀmatrix leastꢀsquares on F2 using the
(s, 1H), 7.03 (d, J = 8.9 Hz, 1H), 6.66 (d, J = 8.5 Hz, 2H), 5.87 (s, SHELXLꢀ97 program package.28
2H), 5.75 (s, 1H), 5.35 (s, 2H), 4.02 (t, J = 6.1 Hz, 2H), 3.44 – 3.34
Preparation of [99mTc]20, [99mTc]21, [99mTc]22 and [99mTc]23
(m, 2H), 2.92 (s, 3H), 1.87 – 1.83 (m, 2H), 1.68 – 1.64 (m, 2H), 1.59
To a solution of ferrocene precursor (1.0 mg) and Mn(CO)5Br (1.0
mg) in DMF (0.7 mL), a Na[99mTcO4] aqueous solution (370 MBq,
167.00, 162.20, 156.77, 150.89, 134.81, 129.07, 122.47, 118.48,
– 1.53 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 192.48, 169.92,
1.0 mL) was added. The reaction mixture was heated at 150 °C for
115.72, 112.25, 105.23, 95.37, 85.67, 84.77, 68.38, 39.73, 30.40,
50 min in a sealed vial. After cooling, the mixture was extracted by
29.28, 28.84, 23.47. TOF MS (ES+): m/z calcd for
CH2Cl2, the organic solvent was collected and removed under a
[C28H27N3O5S185Re + H]+ 702.1201; found 702.1219.
stream of nitrogen gas, the residue was dissolved in CH3CN and it
was purified by radioꢀHPLC under the following conditions: Venusil
MP C18 column (Agela Technologies, 4.6 mm × 250 mm),
Nꢀ(3ꢀ((2ꢀ(4ꢀ(dimethylamino)phenyl)benzo[d]thiazolꢀ6ꢀ
yl)oxy)propyl)carboxamide)ꢀ
(cyclopentadienyl)tricarbonylrhenium (22) Light yellow powder, CH3CN/H2O = 65/35 for [99mTc]20, CH3CN/H2O = 70/30 for
24.7 mg, yield, 35.9 (column chromatography condition:
99mTc]21, [99mTc]22, and [99mTc]23, 1 mL/min, UV = 254 nm. The
%
[
petroleum ether /CH2Cl2 = 1/1 (containing 2% of Et3N)). M.p. 171.6 99mTcꢀlabeled tracers were confirmed by comparative HPLC using
1
– 172.1 °C. H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 8.8 Hz, 2H), the corresponding rhenium surrogates.
7.86 (d, J = 8.9 Hz, 1H), 7.30 (d, J = 2.4 Hz, 1H), 7.01 (dd, J = 8.9,
Partition Coefficient Determination The partition coefficients
of the 99mTcꢀlabeled complexes were determined according to the
previously reported procedure.18
2.5 Hz, 1H), 6.74 (d, J = 8.9 Hz, 2H), 6.34 (s, 1H), 5.86 (s, 2H), 5.36
– 5.35 (m, 2H), 4.13 (t, J = 5.6 Hz, 2H), 3.61 – 3.57 (m, 2H), 3.06 (s,
6H), 2.12 – 2.06 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 192.49,
166.98, 162.28, 156.05, 152.10, 148.88, 135.68, 128.66, 122.69,
In Vitro Fluorescent Staining The brain sections of Tg mice
121.22, 115.36, 111.81, 105.38, 95.48, 85.47, 84.92, 67.19, 40.21,
(C57BL6, APPswe/PSEN1, 12ꢀmonthꢀold, female) and the postꢀ
37.86, 28.79, 26.93. TOF MS (ES+): m/z calcd for
mortem brain tissues from an autopsyꢀconfirmed case of AD (91ꢀ
[C27H25N3O5S185Re + H]+ 688.1045; found 688.1060.
yearꢀold, male, temporal lobe) were deparaffinized with soaking in
xylene for 5 min and then 100% ethanol for 5 min. The brain
Nꢀ(5ꢀ((2ꢀ(4ꢀ(dimethylamino)phenyl)benzo[d]thiazolꢀ6ꢀ
sections were incubated with 25% ethanol solutions (1.0 ꢁM) of
yl)oxy)pentyl)carboxamide)ꢀ
rhenium complexes 20, 21, 22 and 23 for 10 min as well as
(cyclopentadienyl)tricarbonylrhenium (23) Light yellow powder,
ThioflavinꢀS (0.125%), which was used for comparison to stain the
23.2 mg, yield, 32.4
% (column chromatography condition:
adjacent sections. Then, the sections were washed by 40% ethanol
for 5 min. Fluorescent observation was carried out by an Axio
Oberver Z1 (Zeiss, Germany) equipped with DAPI filter set (EX 365
nm, EM 445 nm).
petroleum ether /CH2Cl2 = 1/1 (containing 2% of Et3N)). M.p. 149.1
– 150.1 °C. 1H NMR (400 MHz, CDCl3) δ 7.96 – 7.90 (m, 3H), 7.29
(s, 1H), 7.03 (d, J = 8.7 Hz, 1H), 6.76 (d, J = 8.4 Hz, 2H), 5.87 (s,
2H), 5.75 (s, 1H), 5.35 (s, 2H), 4.02 (t, J = 5.9 Hz, 2H), 3.44 – 3.36
(m, 2H), 3.06 (s, 6H), 1.89 – 1.82 (m, 2H), 1.69 – 1.65 (m, 2H), 1.62
– 1.52 (m, 2H). 13C NMR (100 MHz, MeOD) δ 194.23, 168.72,
164.76, 158.34, 153.79, 149.38, 136.65, 129.43, 123.05, 122.10,
116.90, 112.94, 106.20, 96.01, 87.77, 86.47, 70.81, 43.86, 40.48,
40.28, 30.20, 30.06, 24.53. TOF MS (ES+): m/z calcd for
[C29H29N3O5S185Re + H]+ 716.1358; found 716.1364.
In Vitro Inhibition Assay Inhibition experiments were performed
in borosilicate glass tubes (12 mm × 75 mm) according to previously
described procedures with some modifications.14 One hundred
microliters of Aβ aggregated fibrils (28 nM in the final assay
mixture) was added to a mixture that contained 100 ꢀL of
radioligand ([125I]IMPY, prepared as reported previously, 100000
cpm/100 ꢀL), 100 ꢀL of inhibitor (complexes 20, 21, 22 and 23, 10−4
M to 10−9.5 M in ethanol), 100 ꢀL of DMSO and 600 ꢀL of BSA (0.1
%, pH = 7.4) in a final volume of 1.0 mL. Then, incubation,
Xꢀray Crystallography Singleꢀcrystal Xꢀray diffraction
measurements were performed on a Bruker Smart APEXII CCD
10 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5DT00023H
separation and data processing were carried out as previously Electronic Supplementary Information (ESI) available: In vitro
reported.
and in vivo evaluations are presented including four figures and
two tables as described in the text, as well as spectrograms of all
synthesized complexes. See DOI: 10.1039/b000000x/
In Vitro Autoradiography Paraffinꢀembedded brain sections of
Tg (C57BL6, APPswe/PSEN1, 12ꢀmonthꢀold, female), control mice
(C57BL6, 12ꢀmonthꢀold, female) and an AD patient (68ꢀyearꢀold, 1. Karran, E.; Mercken, M.; De Strooper, B. The amyloid cascade
female, frontal lobe) were disposed according to the method in the in hypothesis for Alzheimer's disease: an appraisal for the development
vitro fluorescent staining experiment. Then, the sections were of therapeutics. Nat. Rev. Drug Discovery 2011, 10, 698ꢀ712.
incubated with [99mTc]20, [99mTc]21, [99mTc]22 and [99mTc]23, (3.7 × 2. Mucke, L. Neuroscience: Alzheimer's disease. Nature 2009, 461,
106 Bq/100 ꢁL) for 1 h at room temperature. They were washed with 895ꢀ897.
40% EtOH for 5 min. The sections were dried and placed in a 3. Hardy, J.; Selkoe, D. J. The amyloid hypothesis of Alzheimer's
phosphorus plate (PerkinꢀElmer, USA) for another 1 h. In vitro disease: progress and problems on the road to therapeutics. Science
autoradiographic images were visualized using a phosphor imaging 2002, 297, 353ꢀ356.
system (Cyclone, Packard). Thereafter, the distribution of Aβ 4. Binder, L. I.; GuillozetꢀBongaarts, A. L.; GarciaꢀSierra, F.; Berry,
plaques in the same brain sections was confirmed by staining with R. W. Tau, tangles, and Alzheimer's disease. Biochim. Biophys.
ThioflavinꢀS according to the method in the in vitro fluorescent Acta, Mol. Basis Dis. 2005, 1739, 216ꢀ223.
staining experiment.
5. Clark, C. M.; Schneider, J. A.; Bedell, B. J.; Beach, T. G.; Bilker,
W. B.; Mintun, M. A.; Pontecorvo, M. J.; Hefti, F.; Carpenter, A. P.;
Flitter, M. L. Use of florbetapirꢀPET for imaging βꢀamyloid
pathology. J. Am. Med. Assoc. 2011, 305, 275ꢀ283.
In Vivo Biodistribution Studies A saline solution containing
purified 99mTcꢀlabeled tracers (100 ꢀL, 10% ethanol, 185 KBq) was
injected via the tail vein of normal mice (ICR, 5 weeks, 22 ꢀ 25 g,
male). The mice were sacrificed at exact various time points postꢀ
injection (2, 10, 30 and 60 min). Samples of blood and interesting
organs were obtained, weighed, and the radioactivity was measured
with an automatic γꢀcounter.
6. Sperling, R. A.; Johnson, K. A.; Doraiswamy, P. M.; Reiman, E.
M.; Fleisher, A. S.; Sabbagh, M. N.; Sadowsky, C. H.; Carpenter,
A.; Davis, M. D.; Lu, M. Amyloid deposition detected with
florbetapir F 18 (18FꢀAVꢀ45) is related to lower episodic memory
performance in clinically normal older individuals. Neurobiol. Aging
2013, 34, 822ꢀ831.
Acknowledgements
7. Wong, D. F.; Moghekar, A. R.; Rigamonti, D.; Brašić, J. R.;
Rousset, O.; Willis, W.; Buckley, C.; Smith, A.; Gok, B.; Sherwin,
P. An in vivo evaluation of cerebral cortical amyloid with [18F]
flutemetamol using positron emission tomography compared with
parietal biopsy samples in living normal pressure hydrocephalus
patients. Mol. Imaging Biol. 2013, 15, 230ꢀ237.
The authors present special thanks to Dr. Jin Liu (College of Life
Science, Beijing Normal University) for assistance in the in vitro
fluorescent staining. This work was funded by the National Natural
Science Foundation of China (No. 21201019) and the Beijing
Municipal Natural Science Foundation (No. 7143180).
8. Wolk, D. A.; Grachev, I. D.; Buckley, C.; Kazi, H.; Grady, M. S.;
Trojanowski, J. Q.; Hamilton, R. H.; Sherwin, P.; McLain, R.;
Arnold, S. E. Association between in vivo fluorine 18ꢀlabeled
flutemetamol amyloid positron emission tomography imaging and in
vivo cerebral cortical histopathology. Arch. Neurol. 2011, 68, 1398ꢀ
1403.
Notes and references
a Key Laboratory of Radiopharmaceuticals, Ministry of Education,
College of Chemistry, Beijing Normal University, Beijing 100875, P.
R. China.
9. Barthel, H.; Sabri, O. Florbetaben to trace amyloidꢀβ in the
Alzheimer brain by means of PET. J. Alzheimers Dis. 2011, 26, 117ꢀ
121.
b
Wuhan Institute for Neuroscience and Neuroengineering, Southꢀ
Central University for Nationalities, Wuhan 430074, P. R. China.
10. FoderoꢀTavoletti, M. T.; Brockschnieder, D.; Villemagne, V. L.;
Martin, L.; Connor, A. R.; Thiele, A.; Berndt, M.; McLean, C. A.;
Krause, S.; Rowe, C. C. In vitro characterization of [18F]ꢀ
* Corresponding author, email: cmc@bnu.edu.cn
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 11

Dalton Transactions
Page 12 of 13
View Article Online
ARTICLE
Journal Name
DOI: 10.1039/C5DT00023H
florbetaben, an Aβ imaging radiotracer. Nucl. Med. Biol. 2012, 39, photon emission computed tomography imaging probes for βꢀ
1042ꢀ1048. amyloid plaques in Alzheimer’s brains. J. Med. Chem. 2012, 55,
11. Chen, X.; Yu, P.; Zhang, L.; Liu, B. Synthesis and biological 2279ꢀ2286.
evaluation of 99mTc, Reꢀmonoamineꢀmonoamide conjugated to 2ꢀ(4ꢀ 21. Bernard, J.; Ortner, K.; Spingler, B.; Pietzsch, HJ.; Alberto, R.
aminophenyl) benzothiazole as potential probes for βꢀamyloid Aqueous synthesis of derivatized cyclopentadienyl complexes of
plaques in the brain. Bioorg. Med. Chem. Lett. 2008, 18, 1442ꢀ1445.
technetium and rhenium directed toward radiopharmaceutical
12. Serdons, K.; Verduyckt, T.; Cleynhens, J.; Bormans, G.; application. Inorg. Chem. 2003, 42, 1014ꢀ1022.
Verbruggen, A. Development of 99mTcꢀthioflavinꢀT derivatives for 22. Saidi, M.; Seifert, S.; Kretzschmar, M.; Bergmann, R.; Pietzsch,
detection of systemic amyloidosis. J. Labelled Compd. Radiopharm. H.ꢀJ. Cyclopentadienyl tricarbonyl complexes of 99mTc for the in
2008, 51, 357ꢀ367.
vivo imaging of the serotonin 5ꢀHT1A receptor in the brain. J.
13. Sagnou, M.; Tzanopoulou, S.; Raptopoulou, C. P.; Psycharis, V.; Organomet. Chem. 2004, 689, 4739ꢀ4744.
Braband, H.; Alberto, R.; Pirmettis, I. C.; Papadopoulos, M.; 23. Saidi, M.; Kothari, K.; Pillai, M.; Hassan, A.; Sarma, H.;
Pelecanou, M. A phenylbenzothiazole conjugate with the tricarbonyl Chaudhari, P.; Unnikrishnan, T.; Korde, A.; Azzouz, Z.
facꢀ[M(I)(CO)3]+(M= Re, 99Tc, 99mTc) core for imaging of βꢀamyloid Cyclopentadienyl technetium
(
99mTc) tricarbonyl piperidine
plaques. Eur. J. Inorg. Chem. 2012, 2012, 4279ꢀ4286. conjugates: biodistribution and imaging studies. J. Labelled Compd.
14. Jia, J.; Cui, M.; Dai, J.; Wang, X.; Ding, Y.ꢀS.; Jia, H.; Liu, B. Radiopharm. 2001, 44, 603ꢀ618.
99mTcꢀlabeled benzothiazole and stilbene derivatives as imaging 24. Cui, M.; Wang, X.; Yu, P.; Zhang, J.; Li, Z.; Zhang, X.; Yang,
agents for Aβ plaques in cerebral amyloid angiopathy. Med. Chem. Y.; Ono, M.; Jia, H.; Saji, H. Synthesis and evaluation of novel 18F
Comm. 2014, 5, 153ꢀ158.
labeled 2ꢀpyridinylbenzoxazole and 2ꢀpyridinylbenzothiazole
15. Zhuang, Z.; Kung, M.; Hou, C.; Ploessl, K.; Kung, H. Biphenyls derivatives as ligands for positron emission tomography (PET)
labeled with technetium 99m for imaging betaꢀamyloid plaques in imaging of βꢀamyloid plaques. J. Med. Chem. 2012, 55, 9283ꢀ9296.
the brain. Nucl. Med. Biol. 2005, 32, 171ꢀ84.
25. Cui, M. Past and recent progress of molecular imaging probes for
16. Ono, M.; Ikeoka, R.; Watanabe, H.; Kimura, H.; Fuchigami, T.; βꢀAmyloid plaques in the brain. Curr. Med. Chem. 2014, 21, 82ꢀ112.
Haratake, M.; Saji, H.; Nakayama, M. 99mTc/Re complexes based on 26. Chua, M.ꢀS.; Kashiyama, E.; Bradshaw, T. D.; Stinson, S. F.;
flavone and aurone as SPECT probes for imaging cerebral βꢀamyloid Brantley, E.; Sausville, E. A.; Stevens, M. F. Role of CYP1A1 in
plaques. Bioorg. Med. Chem. Lett. 2010, 20, 5743ꢀ5748.
modulation of antitumor properties of the novel agent 2ꢀ(4ꢀaminoꢀ3ꢀ
17. Yang, Y.; Zhu, L.; Cui, M.; Tang, R.; Zhang, H. Preparation of methylphenyl) benzothiazole (DF 203, NSC 674495) in human
+
+
classical Re/99mTc(CO)3 and novel 99mTc(CO)2(NO)2 cores breast cancer cells. Cancer Res. 2000, 60, 5196ꢀ5203.
complexed with flavonol derivatives and their binding characteristics 27. Tzanopoulou, S.; Pirmettis, I. C.; Patsis, G.; ParavatouꢀPetsotas,
for Abeta (1ꢀ40) aggregates. Bioorg. Med. Chem. Lett. 2010, 20, M.; Livaniou, E.; Papadopoulos, M.; Pelecanou, M. Synthesis,
5337ꢀ5344.
characterization, and biological evaluation of M(I)(CO)3(NNO)
18. Li, Z.; Cui, M.; Dai, J.; Wang, X.; Yu, P.; Yang, Y.; Jia, J.; Fu, complexes (M = Re, 99mTc) conjugated to 2ꢀ(4ꢀaminophenyl)
H.; Ono, M.; Jia, H. Novel cyclopentadienyl tricarbonyl complexes benzothiazole as potential breast cancer radiopharmaceuticals. J.
of 99mTc mimicking chalcone as potential singleꢀphoton emission Med. Chem. 2006, 49, 5408ꢀ5410.
computed tomography imaging probes for βꢀAmyloid plaques in 28. Sheldrick, G. SHELXSꢀ97, program for Xꢀray crystal structure
brain. J. Med. Chem. 2013, 56, 471ꢀ482.
determination. Göttingen University, Germany 1997.
19. Ono, M.; Ikeoka, R.; Watanabe, H.; Kimura, H.; Fuchigami, T.;
Haratake, M.; Saji, H.; Nakayama, M. Synthesis and evaluation of
novel chalcone derivatives with 99mTc/Re complexes as potential
probes for detection of βꢀamyloid plaques. ACS Chem. Neurosci.
2010, 1, 598ꢀ607.
20. Cheng, Y.; Ono, M.; Kimura, H.; Ueda, M.; Saji, H.
Technetiumꢀ99m labeled pyridyl benzofuran derivatives as single
12 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5DT00023H
[CpM(CO)3] (M = Re, 99mTc) labeled 2ꢀPhenylbenzothiazoles designed by conjugate approach were
evaluated as SPECT tracers for Aβ plaques.
423x147mm (72 x 72 DPI)