99mTc-labeled dibenzylideneacetone derivatives as potential SPECT probes for in vivo imaging of β-amyloid plaque

Article abstract of DOI:10.1016/j.ejmech.2013.03.057

Four ^99mTc-labeled dibenzylideneacetone derivatives and corresponding rhenium complexes were successfully synthesized and biologically evaluated as potential imaging probes for Aβ plaques using SPECT. All rhenium complexes (5a-d) showed affinit

Full text of DOI:10.1016/j.ejmech.2013.03.057

European Journal of Medicinal Chemistry 64 (2013) 90e98
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
99mTc-labeled dibenzylideneacetone derivatives as potential SPECT
probes for in vivo imaging of b-amyloid plaque
*
Yanping Yang, Mengchao Cui , Bing Jin, Xuedan Wang, Zijing Li, Pingrong Yu, Jianhua Jia,
*
Hualong Fu, Hongmei Jia, Boli Liu
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR China
a r t i c l e i n f o
a b s t r a c t
Four 99mTc-labeled dibenzylideneacetone derivatives and corresponding rhenium complexes were suc-
Article history:
Received 22 February 2013
Received in revised form
25 March 2013
Accepted 27 March 2013
Available online 6 April 2013
cessfully synthesized and biologically evaluated as potential imaging probes for A
b plaques using SPECT.
All rhenium complexes (5aed) showed affinity for Ab(1e42) aggregates (Ki ¼ 13.6e120.9 nM), and
selectively stained the Ab plaques on brain sections of transgenic mice. Biodistribution in normal mice
revealed that [99mTc]5aed exhibited moderate initial uptake (0.31%e0.49% ID/g at 2 min) and reasonable
brain washout at 60 min post-injection. Although additional optimizations are still needed to facilitate
it’s penetration through BBB, the present results indicate that [99mTc]5a may be a potential SPECT probe
Keywords:
Alzheimer’s disease
for imaging Ab plaques in Alzheimer’s brains.
Ó 2013 Elsevier Masson SAS. All rights reserved.
b-Amyloid plaque
Dibenzylideneacetone
Technetium-99m
SPECT imaging
1. Introduction
noninvasive techniques such as positron emission tomography
(PET) or single photon emission computed tomography (SPECT).
Over recent years, a big variety of PET probes based on Congo Red
(CR) and Thioflavin-T (ThT) have been synthesized and evaluated for
Alzheimer’s disease (AD) is a neurodegenerative disorder char-
acterized by the devastating loss of memory and cognitive abilities,
and it will afflict about 63 million people by 2030, and 114 million
imaging
Ab
plaques. Among them,
[
11C]-4-N-methylamino-40-
11C]-2-(4-(methylamino)
by 2050 worldwide [1e3]. The deposition of extracellular
b-amy-
hydroxystilbene ([11C]SB-13) [9,10],
[
loid (A ) plaques and intracellular neurofibrillary tangles (NFTs)
b
phenyl)-6-hydroxybenzothiazol ([11C]PIB) [11,12], [18F]-4-(N-meth-
ylamino)-40-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-stilbene ([18F]
BAY94-9172) [13e15], [18F]-2-(3-fluoro-4-methyaminophenyl)ben-
zothiazol-6-ol ([18F]GE-067) [16] and [18F]-(E)-4-(2-(6-(2-(2-(2-
fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-N-methylaniline
([18F]AV-45) [17e19] have been evaluated clinically and demon-
containing highly phosphorylated tau protein in the brain have
been regarded as the dominant factors driving AD pathogenesis [4].
Currently, the diagnosis of AD primarily depends on clinical eval-
uation, patient history, structural and functional imaging of the
brain by computed tomography (CT) and magnetic resonance im-
aging (MRI) [5,6]. However, these methods are usually insufficient
and unreliable in the definite diagnosis of AD, especially for pa-
tients in early stage [7]. The final diagnosis of AD can be accom-
plished only by postmortem examination of brain tissues. As the
strated potential utilization. The imaging of Ab plaques in vivo with
PET has achieved remarkable progress so far, while the development
of SPECT tracers lags far behind. When compared with PET, SPECT
have several certain advantages including lower cost, widespread
availability, and the use of isotopes with longer half-lives. Hence
extraordinary efforts should be spent on the innovation of SPECT
accumulation of Ab plaques and NFTs in AD brains occurs 20e40
years before the onset of clinical symptoms [8], detecting their
changes in vivo may impressively facilitate early diagnosis and
monitoring the progression of AD. Therefore, there is an urgent
probes for Ab imaging.
Among the radioisotopes used in diagnostic imaging nuclear
need for in vivo imaging of A
b
plaques with the assistance of
medicine by SPECT, 99mTc (T1/2 ¼ 6.01 h,
g (89%): 141 keV) is the
most widely used and can even be regarded as a star radioisotope
due to the following aspects: it is readily produced by a commercial
99Mo/99mTc generator, besides, it’s moderate gamma-ray energy and
physical half-life extremely fulfill the SPECT imaging requirements
* Corresponding authors. Tel./fax: þ86 10 58808891.
E-mail addresses: cmc@bnu.edu.cn (M. Cui), liuboli@bnu.edu.cn (B. Liu).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.057

Y. Yang et al. / European Journal of Medicinal Chemistry 64 (2013) 90e98
91
by providing good biological localization and appropriate residence
time. Thus, new 99mTc-labeled A
imaging probes will provide
2. Results and discussion
b
simple, convenient, economical and widespread SPECT-based im-
aging methods for the early diagnosis of AD. In the past decade, a
great deal of research has been conducted in this field. Initially re-
ported 99mTc-labeled CR [20,21] and Chrysamine G derivatives
2.1. Chemistry
The synthesis of 99mTc/Re dibenzylideneacetone derivatives is
outlined in Scheme 1. The key step in the formation of the diben-
zylideneacetone skeleton was accomplished by base catalyzed
Claisen condensation reaction starting from (E)-4-(4-(dimethyla-
mino)phenyl)but-3-en-2-one and substituted benzaldehyde (2a or
2b). Claisen condensation afforded compound 3a and 3b in yields of
54.6% and 48.9%, respectively. Then, a BAT or MAMA chelating
group was conjugated with bromo-aliphatic compounds 3a or 3b
by reacting them with Tr-Boc-BAT or Tr-MAMA, which achieved
4aed in yields of 36.0%, 27.1%, 50.8% and 54.1%, respectively. The
structural and biochemical properties of 99mTc correlate well with
those of Re, thus Re complexes have been usually adopted as sur-
rogates for 99mTc complexes for structure identifications and bio-
logical evaluations [35,36]. After deprotection of the thiol groups
in 4aed by trifluoroacetic acid (TFA) and triethylsilane, the rhe-
nium complexes 5aed were prepared through a reaction with
(PPh3)2ReOCl3 in CH2Cl2 in 15.5e37.0% yields.
[22,23], which displayed high affinity for Ab aggregates in vitro,
failed to cross the bloodebrain barrier (BBB) for their high molecular
weight and charged character. Then further attempts were focused
on smaller, neutral, and more lipophilic 99mTc-labeled ligands. De-
rivatives of biphenyl [24], benzothiazole aniline [25e27], chalcone
[28], flavone, aurone [29], curcumin [30], benzofuran [31,32] and
benzoxazole [33] conjugated with monoamineemonoamide dithiol
(MAMA) and bis(aminoethanethiol) (BAT) chelating ligands (Fig. 1)
have been synthesized and evaluated as potential A
b imaging
probes, unfortunately, none of them showed specific binding to A
b
plaques in vivo due to either low affinity or low uptake in the brain.
Recently, based on the structure of curcumin, an extensive series
of dibenzylideneacetone derivatives developed as a novel molec-
ular scaffold for Ab imaging were reported [34]. Structureeactivity
relationships revealed that the para position on the phenyl ring of
dibenzylideneacetone showed extraordinarily high tolerance for
steric bulk substitutions. Increasing the size of the substituent has
2.2. In vitro binding studies using Ab(1e42) aggregates
negligible impact on the affinity for Ab aggregates, and even when
the ligand was substituted by a bulky trityloxy group also exhibited
high affinity (Ki ¼ 2.8 ꢀ 0.4 nM). This encouraging finding inspires
us that the introduction of a large 99mTc chelating structure prob-
ably won’t bring about a considerable change in the binding
properties.
The binding affinities of rhenium complexes 5aed for Ab(1e42)
aggregates were evaluated by competition binding assay using
[
[
125I]6-iodo-2-(40-dimethylamino)-phenyl-imidazo[1,2-a]pyridine,
125I]IMPY, as the competing radio-ligand according to conven-
tional methods [34]. IMPY was also tested using the same system
for comparison. As shown in Table 1, compounds with the MAMA
cheating ligand, 5c and 5d, exhibited moderate binding affinity to
In the present study, we designed four novel 99mTc-labeled
dibenzylideneacetone derivatives (Fig. 2) conjugated to MAMA
and BAT via a propoxy or pentyloxy spacer for Ab plaque imaging.
A
b(1e42) aggregates (Ki ¼ 120.9 and 59.1 nM, respectively), while
Hereon, we report the synthesis and preliminary biological
compounds with the BAT cheating ligand, 5a and 5b, displayed
high affinity (Ki ¼ 24.7 and 13.6 nM, respectively) which was
comparable to the value of IMPY (Ki ¼ 11.5 nM), indicating that they
hold sufficient affinity for Ab(1e42) aggregates in vitro. This result
evaluation of these novel 99mTc-labeled dibenzylideneacetone
derivatives as potential SPECT probes for imaging A
in AD.
b plaques
Fig. 1. Chemical structure of 99mTc-labeled A
b imaging probes reported previously.

92
Y. Yang et al. / European Journal of Medicinal Chemistry 64 (2013) 90e98
Fig. 2. Chemical structure of 99mTc-labeled dibenzylideneacetone derivatives reported in the present study.
demonstrates that the introduction of the bulky BAT chelating
group didn’t interfere with the binding to Ab(1e42) aggregates
observably. When considering the impact on affinity bring by the
length of spacers, the result indicated that the affinity of com-
pounds 5b and 5d, with longer pentyloxy spacer, was considerably
higher than that of compounds with propoxy spacer. As expected,
when the spacer getting longer, the interference of the large 99mTc
chelating structure on the binding property of targeting molecule
(dibenzylideneacetone) is smaller, and thus the affinity was
maintained preferably.
fluorescent staining with Thioflavin-S on the adjacent sections
(Fig. 3B, E, H and K). The results incontestably manifested that
these rhenium complexes could bind specifically to Ab plaques
with low background.
2.4. Radiolabeling
The novel radio-labeled ligands [99mTc]5aed were prepared by a
ligand exchange reaction employing the precursor 4aed and 99mTc-
glucoheptonate (99mTc-GH) in boiling water with radiochemical
yields of 31.8%, 43.8%, 47.9% and 37.7%, respectively. After purifica-
tion by high-performance liquid chromatography (HPLC), the
radiochemical purity of these radiotracers was higher than 95%. The
radiochemical identities of the 99mTc-labeled tracers were affirmed
by comparison of the retention times on HPLC column with that
of the corresponding Re complexes (Table 2, see Supporting
information). The retention times of [99mTc]5aed on HPLC column
were 10.45, 10.88, 15.18 and 15.36 min, and that of the corre-
sponding rhenium complexes 5aed were 8.97, 9.29 13.26 and
13.43 min, respectively. The approximation of retention times be-
tween 99mTc-labeled tracers and corresponding rhenium complexes
sufficiently suggested that the desired 99mTc-labeled dibenzylide-
neacetone derivatives were successfully generated.
2.3. In vitro fluorescent staining
In vitro fluorescent staining of Ab plaques on sections of brain
tissue from a transgenic (Tg) model mice (C57BL6, APPswe/
PSEN1, 11 months old) and an age-matched control mice were
conducted to confirm the specific and high binding affinity of
compounds 5aed to A
images revealed that these four rhenium complexes all displayed
excellent labeling of A plaques on the sections of Tg mice with
b plaques (Fig. 3). Fluorescent staining
b
low background (Fig. 3A, D, G and J), while there was no notable
staining in the age-matched control mice (Fig. 3C, F, I and L). The
distribution of A
b plaques was consistent with the results of
Scheme 1. Reagents and conditions: (a) 1,3-dibromopropane (or 1,5-dibromopentane), K2CO3, MeCN, 90 ꢁC; (b) NaOCH3, CH3CH2OH, r.t.; (c) Tr-Boc-BAT, K2CO3, MeCN, 90 ꢁC; (d) Tr-
MAMA, K2CO3, MeCN, 90 ꢁC; (e) (1) TFA, Et3SiH; (2) 99mTc-GH, 100 ꢁC; or (PPh3)ReOCl3, CH2Cl2, CH3OH, 90 ꢁC.

Y. Yang et al. / European Journal of Medicinal Chemistry 64 (2013) 90e98
93
Table 1
Table 2
Inhibition constants for the binding of [125I]IMPY to
HPLC retention times of 99mTc-labeled dibenzylideneacetone derivatives and cor-
A
b(1e42) aggregates.
responding Re complexes and log D values of [99mTc]5aed.
Compound
Ki (nM)a
99mTc compound Retention
time (min)
Re compound Retention
time (min) compoundc
Log D of 99mTc
5a
5b
5c
5d
24.7 ꢀ 6.1
13.6 ꢀ 7.8
120.9 ꢀ 4.3
59.1 ꢀ 24.0
11.5 ꢀ 2.5
[
[
[
[
99mTc]5aa
99mTc]5ba
99mTc]5cb
99mTc]5db
10.45
10.88
15.18
15.36
5a
5b
5c
5d
8.97
9.29
13.26
13.43
3.36 ꢀ 0.08
3.17 ꢀ 0.10
3.52 ꢀ 0.10
3.57 ꢀ 0.15
IMPY
a
a
Measured in triplicate with values given as the
Reversed-phase HPLC using a binary gradient system (acetonitrile/water:80%/
mean ꢀ SD.
20%) at a 1.0 mL/min flow rate.
b
Reversed-phase HPLC using a binary gradient system (acetonitrile/water:70%/
30%) at a 1.0 mL/min flow rate.
c
Measured in triplicate with values expressed as the mean ꢀ SD.
2.5. Partition coefficient determination
The log D values of [99mTc]5aed were 3.42 ꢀ 0.02, 3.17 ꢀ 0.04,
3.53 ꢀ 0.01, and 3.57 ꢀ 0.17, respectively (Table 2), which are all in
an optimal range (1e3.5) for BBB penetration, indicating that the
four tracers have appropriate lipophilicity suitable for brain
imaging.
2.6. In vivo biodistribution in normal mice
Biodistribution experiments in normal ICR mice (5 weeks, male)
were carried out to evaluate the pharmacokinetics of 99mTc-labeled
dibenzylideneacetone tracers in vivo (Table 3). A biodistribution
Fig. 3. In vitro fluorescent staining of A
G and J). The presence and distribution of A
5aed on brain sections of a normal mouse was also carried out as control (C, F, I and L).
b
plaques by compounds 5aed on brain sections of a Tg model mouse (C57BL6, APPswe/PSEN1, 11 months old, male) with a DAPI filter (A, D,
b
plaques on the adjacent sections were confirmed with Thioflavin-S with an AF filter (B, E, H and K). Fluorescence staining of compounds

94
Y. Yang et al. / European Journal of Medicinal Chemistry 64 (2013) 90e98
Table 3
successfully synthesized and biologically evaluated as SPECT im-
aging probes for A plaques. In vitro binding studies indicated that
rhenium complexes 5aed displayed moderate to high affinity for
b(1e42) aggregates varied from 13.6 to 120.9 nM. The high binding
affinity was further confirmed by fluorescent staining, and all the
dibenzylideneacetone derivatives specifically labeled A plaques
on the brain sections of Tg mice. Biodistribution in normal mice
revealed that
99mTc]5a exhibited moderate initial uptake
(0.49% ID/g at 2 min) and fast washout from the brain, with a
brain2min/brain60min ratio of 6.13. All in all, the preliminary results
suggest that the [99mTc]5a may be a potential SPECT probe for
imaging Ab plaques in Alzheimer’s brains. But additional chemical
refinements are earnestly required to further improve the pene-
Biodistribution of radioactivity after injection of [99mTc]5aed in normal ICR mice.a
b
Organ
2 min
10 min
30 min
60 min
99mTc]5a (log D ¼ 3.36 ꢀ 0.08)
A
[
Blood
6.36 ꢀ 1.19
0.49 ꢀ 0.08
10.23 ꢀ 1.20
26.69 ꢀ 4.08
3.03 ꢀ 0.61
17.66 ꢀ 3.05
21.45 ꢀ 2.66
3.78 ꢀ 0.66
1.10 ꢀ 0.18
5.77 ꢀ 0.72
1.61 ꢀ 0.18
0.23 ꢀ 0.04
2.43 ꢀ 0.20
26.22 ꢀ 0.91
1.67 ꢀ 0.37
6.05 ꢀ 1.82
13.2 ꢀ 2.81
2.74 ꢀ 0.61
1.64 ꢀ 0.54
12.02 ꢀ 1.41
0.71 ꢀ 0.09
0.12 ꢀ 0.03
0.86 ꢀ 0.15
17.57 ꢀ 1.42
0.95 ꢀ 0.23
1.44 ꢀ 0.56
6.96 ꢀ 1.76
1.09 ꢀ 0.37
2.55 ꢀ 0.78
25.04 ꢀ 4.70
0.61 ꢀ 0.07
0.08 ꢀ 0.01
0.53 ꢀ 0.06
12.58 ꢀ 3.90
0.55 ꢀ 0.14
0.89 ꢀ 0.14
5.97 ꢀ 1.16
0.52 ꢀ 0.11
1.53 ꢀ 0.13
54.01 ꢀ 6.54
Brain
Heart
Liver
Spleen
Lung
Kidney
Pancreas
Stomachb
Intestineb
b
[
[
99mTc]5b (log D ¼ 3.17 ꢀ 0.10)
Blood
7.21 ꢀ 1.38
0.47 ꢀ 0.11
29.79 ꢀ 5.78
77.67 ꢀ 18.54
16.64 ꢀ 2.62
11.87 ꢀ 2.56
12.33 ꢀ 3.20
4.21 ꢀ 0.76
1.56 ꢀ 0.75
5.97 ꢀ 1.22
1.25 ꢀ 0.08
0.27 ꢀ 0.03
11.72 ꢀ 1.75
56.22 ꢀ 9.89
9.08 ꢀ 1.25
3.84 ꢀ 1.09
7.93 ꢀ 1.24
4.80 ꢀ 0.82
3.72 ꢀ 0.79
25.67 ꢀ 6.08
0.80 ꢀ 0.05
0.19 ꢀ 0.03
10.12 ꢀ 3.29
52.99 ꢀ 4.78
5.68 ꢀ 1.53
2.42 ꢀ 0.57
5.02 ꢀ 0.27
2.74 ꢀ 0.87
5.09 ꢀ 2.15
54.31 ꢀ 6.14
0.52 ꢀ 0.05
0.12 ꢀ 0.02
6.63 ꢀ 1.32
43.84 ꢀ 6.36
4.06 ꢀ 0.71
1.75 ꢀ 0.16
3.41 ꢀ 0.48
1.44 ꢀ 0.21
1.57 ꢀ 0.60
60.14 ꢀ 16.90
tration through BBB.
Brain
Heart
Liver
Spleen
Lung
Kidney
Pancreas
Stomachb
Intestineb
4. Experimental
4.1. General information
All reagents used for chemical synthesis were commercial
products and were used without further purification. Na99mTcO4
was obtained from a commercial 99Mo/99mTc generator, Beijing
Atomic High-tech Co. All 1H NMR spectra were acquired at 400 MHz
on Bruker Avance III NMR spectrometers in CDCl3 solutions at room
temperature with trimethylsilyl (TMS) as an internal standard.
[
99mTc]5c (log D ¼ 3.52 ꢀ 0.10)
Blood
4.87 ꢀ 0.76
0.48 ꢀ 0.06
14.24 ꢀ 1.21
23.67 ꢀ 2.24
4.40 ꢀ 0.30
24.52 ꢀ 3.50
16.51 ꢀ 0.66
4.28 ꢀ 0.30
1.14 ꢀ 0.09
5.73 ꢀ 0.62
1.30 ꢀ 0.12
0.19 ꢀ 0.02
3.89 ꢀ 0.18
21.38 ꢀ 3.39
2.19 ꢀ 0.26
9.24 ꢀ 2.20
9.03 ꢀ 1.66
3.46 ꢀ 0.52
1.40 ꢀ 0.12
12.09 ꢀ 0.70
0.60 ꢀ 0.05
0.10 ꢀ 0.02
1.33 ꢀ 0.28
18.86 ꢀ 0.71
0.94 ꢀ 0.09
3.06 ꢀ 0.43
4.91 ꢀ 0.76
2.11 ꢀ 0.34
1.12 ꢀ 0.05
30.47 ꢀ 6.84
0.42 ꢀ 0.02
0.09 ꢀ 0.01
0.90 ꢀ 0.12
16.61 ꢀ 3.98
0.52 ꢀ 0.11
1.15 ꢀ 0.31
3.86 ꢀ 0.46
1.24 ꢀ 0.30
1.68 ꢀ 0.51
39.15 ꢀ 7.12
Brain
Heart
Liver
Spleen
Lung
Kidney
Pancreas
Stomachb
Intestineb
Chemical shifts were reported as
d values relative to the internal
TMS, and Coupling constants were reported in hertz. The multi-
plicity is defined by s (singlet), d (doublet), t (triplet), and m
(multiplet). Mass spectra were acquired with a micrOTOF-Q II in-
strument. Reactions were monitored by TLC (Silica gel 60 F254
aluminum sheets, Merck) and compounds were visualized by illu-
[
99mTc]5d (log D ¼ 3.57 ꢀ 0.15)
Blood
Brain
5.10 ꢀ 0.37
0.31 ꢀ 0.06
14.56 ꢀ 0.67
42.12 ꢀ 9.68
5.97 ꢀ 1.76
12.03 ꢀ 2.92
16.85 ꢀ 1.16
4.90 ꢀ 0.69
1.34 ꢀ 0.13
5.20 ꢀ 0.58
2.32 ꢀ 0.54
0.22 ꢀ 0.02
4.17 ꢀ 0.33
43.73 ꢀ 4.71
2.63 ꢀ 0.24
4.20 ꢀ 0.49
12.07 ꢀ 2.72
5.50 ꢀ 1.25
2.05 ꢀ 0.39
21.46 ꢀ 2.27
1.29 ꢀ 0.15
0.11 ꢀ 0.04
1.75 ꢀ 0.41
29.40 ꢀ 4.31
1.67 ꢀ 0.70
1.64 ꢀ 0.28
6.45 ꢀ 1.41
3.44 ꢀ 0.91
2.82 ꢀ 0.72
35.03 ꢀ 5.70
1.21 ꢀ 0.24
0.15 ꢀ 0.02
1.14 ꢀ 0.19
25.97 ꢀ 5.95
1.35 ꢀ 0.49
1.24 ꢀ 0.22
4.85 ꢀ 0.94
1.93 ꢀ 0.47
6.42 ꢀ 1.10
54.66 ꢀ 11.20
mination with a short wavelength Ultra-Violet lamp (
365 nm). Column chromatography purification were performed on
silica gel (54e74 m) from Qingdao Haiyang Chemical Co., Ltd.
HPLC was performed on a Shimadzu SCL-20 AVP (which is equip-
ped with a Bioscan Flow Count 3200 NaI/PMT -radiation scintil-
lation detector and a SPD-20A UV detector,
¼ 254 nm) and a
Venusil MP C18 reverse phase column (Agela Technologies, 5 m,
l
¼ 254 nm or
Heart
Liver
m
Spleen
Lung
Kidney
Pancreas
Stomachb
Intestineb
g
l
m
ID ¼ 4.6 mm, length ¼ 250 mm) eluted with a binary gradient
system at 1.0 mL/min flow rate. Mobile phase A was water, while
mobile phase B was acetonitrile. Fluorescent observation was per-
formed by the Axio Observer Z1 inverted fluorescence microscope
(Zeiss, Germany) equipped with a DAPI filter set (excitation,
405 nm) and AF488 filter set (excitation, 495 nm). Normal ICR mice
(five weeks, male) were used for biodistribution experiments.
Transgenic mice (C57BL6, APPswe/PSEN1, male, 11 months old),
used as an Alzheimer’s model, were purchased from the Institute of
Laboratory Animal Science, Chinese Academy of Medical Sciences.
All protocols requiring the use of mice were approved by the animal
care committee of Beijing Normal University. The purity of all key
compounds was proven to be more than 95% by HPLC.
a
Expressed as % injected dose per gram. Each value represents the mean ꢀ SD for
4e5 mice.
b
Expressed as % injected dose per organ.
study provides crucial information on penetration of the BBB and
radioactivity pharmacokinetics in vivo. [99mTc]5aed all showed the
highest initial uptake at 2 min post-injection, 0.49, 0.47, 0.48 and
0.31% ID/g, respectively. Although the values were comparable or
slightly lower than that of 99mTc-labeled tracers reported previ-
ously (0.2e1.80% ID/g) [24e26,28e32], [99mTc]5a and [99mTc]5b can
also expect excellent labeling of Ab plaques in vivo due to their high
affinities. The brain2min/brain60min ratio of [99mTc]5aec were 6.13,
3.92 and 5.33, respectively, which were superior than that of 99mTc-
labeled phenylbenzothiazole (2.06) [26], phenylbenzofuran (2.39)
[31], pyridylbenzofuran (2.28) [32], phenylbenzoxazole (3.24) [33]
and so on. This data undoubtedly indicated that all the four
tracers possessed good clearance property from the normal brain.
The accumulation of radioactivity was observed predominantly in
liver and kidney at the early stage, and then dropped gradually with
time, while continuous gastrointestinal accumulation of the ra-
diotracers resulted in a high intestine uptake at later time periods.
4.2. 4-(3-Bromopropoxy)benzaldehyde (2a)
To a solution of 1 (3.67 g, 30 mmol) in CH3CN (50 mL) was added
1,3-dibromopropane (9.10 g, 45 mmol) and K2CO3 (8.29 g,
60 mmol). The mixture was stirred at 90 ꢁC for 12 h, after cooling to
room temperature, solvent was removed by vacuum, and the res-
idue was extracted with CHCl3. The organic layer was dried over
MgSO4 and solvent was removed. The crude product was purified
by silica gel chromatography (petroleum ether/AcOEt ¼ 8/1, v/v) to
give 2a (4.41 g, 60.4%) as a colorless oil. 1H NMR (400 MHz, CDCl3)
3. Conclusion
In conclusion, four 99mTc-labeled dibenzylideneacetone de-
rivatives and their corresponding rhenium complexes were
d
9.89 (s, 1H, CHO), 7.84 (d, J ¼ 8.7 Hz, 2H, ArH), 7.01 (d, J ¼ 8.7 Hz,
2H, ArH), 4.20 (t, J ¼ 5.8 Hz, 2H, CH2), 3.61 (t, J ¼ 6.3 Hz, 2H, CH2),

Y. Yang et al. / European Journal of Medicinal Chemistry 64 (2013) 90e98
95
2.39e2.33 (m, 2H, CH2). HRMS (ESI): m/z calcd for C10H12BrO2
245.0000; found 245.0091 [M þ H]þ.
d 188.84, 160.89, 155.01, 151.94, 145.01, 144.83, 143.73, 141.96,
130.24,129.95, 129.64, 129.59, 127.91, 127.86, 126.70, 126.60, 123.63,
122.70, 121.11, 114.88, 111.90, 79.55, 77.38, 77.06, 76.74, 66.65, 53.40,
50.16, 47.21, 40.14, 30.05, 28.44, 27.22. HRMS (ESI): m/z calcd for
4.3. 4-(5-Bromopentyloxy)benzaldehyde (2b)
C
71H76N3O4S2 1098.5277; found 1098.5263 [M þ H]þ.
The procedure described above for the preparation of 2a was
employed to give 2b (6.61 g, 81.2%) as a colorless oil. 1H NMR
4.7. tert-Butyl 2-((5-(4-((1E,4E)-5-(4-(dimethylamino)phenyl)-3-
oxopenta-1,4-dienyl)phenoxy)pentyl)(2-(tritylthio)ethyl)amino)ethyl
(2-(tritylthio)ethyl)carbamate (4b)
(400 MHz, CDCl3)
d
9.88 (s, 1H, CHO), 7.83 (d, J ¼ 8.8 Hz, 2H, ArH),
6.99 (d, J ¼ 8.7 Hz, 2H, ArH), 4.06 (t, J ¼ 6.3 Hz, 2H, CH2), 3.45 (t,
J ¼ 6.7 Hz, 2H, CH2), 2.00e1.91 (m, 2H, CH2), 1.90e1.82 (m, 2H, CH2),
1.70e1.60 (m, 2H, CH2). HRMS (ESI): m/z calcd for C12H16BrO2
271.0334; found 271.0438 [M þ H]þ.
The procedure described above for the preparation of 4a was
employed to give 4b (211.0 mg, 27.1%) as yellow solid foams. 1H
NMR (400 MHz, CDCl3)
d
7.70 (d, J ¼ 15.8 Hz, 1H, trans eCH]CHe),
4.4. (1E,4E)-1-(4-(3-Bromopropoxy)phenyl)-5-(4-(dimethylamino)
phenyl)penta-1,4-dien-3-one (3a)
7.68 (d, J ¼ 15.8 Hz, 1H, trans eCH]CHe), 7.54 (d, J ¼ 7.7 Hz, 2H,
ArH), 7.52 (d, J ¼ 8.4 Hz, 2H, ArH), 7.41e7.39 (m, 13H, ArH), 7.24e
7.15 (m, 17H, ArH), 6.96 (d, J ¼ 15.8 Hz, 1H, trans eCH ¼ CHe), 6.89
(d, J ¼ 8.6 Hz, 2H), 6.87 (d, J ¼ 15.9 Hz, 1H, trans eCH ¼ CHe), 6.69
(d, J ¼ 8.8 Hz, 2H, ArH), 3.95 (t, J ¼ 6.4 Hz, 2H, CH2), 3.04 (s, 6H,
N(CH3)2), 2.98e2.79 (m, 4H, CH2), 2.39e2.17 (m, 10H, CH2), 1.77e
1.68 (m, 2H, CH2), 1.36 (s, 13H, CH2 and tert-Bu). 13C NMR (101 MHz,
A mixture of 2a (3.02 g, 12.4 mmol), (E)-4-(4-(dimethylamino)
phenyl)but-3-en-2-one (2.35 g, 12.4 mmol) and sodium methoxide
(0.95 g, 25.0 mmol) in ethanol (50 mL) was stirred at room tem-
perature overnight. The precipitate was collected by filtration and
then recrystallized from ethanol to give 3a (2.81 g, 54.6%) as a
CDCl3)
d 188.81, 160.93, 155.01, 151.95, 145.04, 144.86, 143.73,
yellow solid. 1H NMR (400 MHz, CDCl3)
d
7.70 (d, J ¼ 15.7 Hz, 1H,
141.89, 130.23, 129.96, 129.67, 127.91, 127.85, 126.69, 126.61, 123.69,
122.72, 121.11, 114.89, 111.91, 79.47, 77.39, 77.07, 76.75, 68.04, 66.68,
53.38, 47.09, 40.13, 29.94, 29.07, 28.46, 27.12, 23.72. HRMS (ESI): m/z
calcd for C73H80N3O4S2 1126.5590; found 1126.5538 [M þ H]þ.
trans eCH]CHe), 7.67 (d, J ¼ 15.8 Hz, 1H, trans eCH]CHe), 7.56
(d, J ¼ 8.7 Hz, 2H, ArH), 7.52 (d, J ¼ 8.8 Hz, 2H, ArH), 6.97 (d,
J ¼ 15.8 Hz, 1H, trans eCH]CHe), 6.93 (d, J ¼ 8.7 Hz, 2H, ArH), 6.87
(d, J ¼ 15.7 Hz, 1H, trans eCH]CHe), 6.69 (d, J ¼ 8.8 Hz, 2H, ArH),
4.15 (t, J ¼ 5.8 Hz, 2H, CH2), 3.61 (t, J ¼ 6.4 Hz, 2H, CH2), 3.04 (s, 6H,
N(CH3)2), 2.37e2.31 (m, J ¼ 6.1 Hz, 2H, CH2). HRMS (ESI): m/z calcd
for C22H25BrNO2 414.1069; found 414.0955 [M þ H]þ.
4.8. 2-((3-(4-((1E,4E)-5-(4-(Dimethylamino)phenyl)-3-oxopenta-1,4-
dienyl)phenoxy)propyl)(2-(tritylthio)ethyl)amino)-N-(2-(tritylthio)
ethyl)acetamide (4c)
4.5. (1E,4E)-1-(4-(5-Bromopentyloxy)phenyl)-5-(4-(dimethylamino)
phenyl)penta-1,4-dien-3-one (3b)
A mixture of 3b (414.3 mg, 1.0 mmol), N-(2-(tritylthio)ethyl)-2-
(2-(tritylthio)ethylamino)acetamide (622.8 mg, 1.0 mmol) and
K2CO3 (276.4 mg, 2.0 mmol) in acetonitrile (50 mL) was stirred at
90 ꢁC for 48 h. The solvent was removed by vacuum, and water was
added. The mixture was extracted with CHCl3. The organic layers
were combined and dried over Na2SO4 and evaporated dry. The
crude product was purified by silica gel chromatography (petro-
The procedure described above for the preparation of 3a was
employed to give 3b (2.22 g, 48.9%) as a yellow solid. 1H NMR
(400 MHz, CDCl3)
d
7.70 (d, J ¼ 15.7 Hz, 1H, trans eCH]CHe), 7.67
(d, J ¼ 15.8 Hz, 1H, trans eCH]CHe), 7.55 (d, J ¼ 8.7 Hz, 2H, ArH),
7.51 (d, J ¼ 8.8 Hz, 2H, ArH), 6.96 (d, J ¼ 15.8 Hz, 1H, trans eCH]
CHe), 6.90 (d, J ¼ 8.7 Hz, 2H, ArH), 6.87 (d, J ¼ 15.8 Hz, 1H, trans e
CH]CHe), 6.69 (d, J ¼ 8.8 Hz, 2H, ArH), 4.01 (t, J ¼ 6.3 Hz, 2H, CH2),
3.44 (t, J ¼ 6.7 Hz, 2H, CH2), 3.04 (s, 6H, N(CH3)2), 1.99e1.90 (m, 2H,
CH2), 1.88e1.79 (m, 2H, CH2), 1.64 (m, 2H, CH2). HRMS (ESI): m/z
calcd for C24H29BrNO2 444.1361; found 444.1333 [M þ H]þ.
leum ether/AcOEt ¼ 3/1, v/v) to give 4c (502.3 mg, 50.8%) as yellow
1
solid foams. H NMR (400 MHz, CDCl3)
d
7.71 (d, J ¼ 15.8 Hz, 1H,
trans eCH]CHe), 7.66 (d, J ¼ 15.8 Hz, 1H, trans eCH]CHe), 7.52
(d, J ¼ 8.8 Hz, 2H, ArH), 7.45 (d, J ¼ 8.5 Hz, 2H, ArH), 7.38e7.33 (m,
13H, ArH), 7.24e7.18 (m, 17H, ArH), 6.94 (d, J ¼ 15.8 Hz, 1H, trans e
CH]CHe), 6.88 (d, J ¼ 15.7 Hz, 1H, trans eCH]CHe), 6.82 (d,
J ¼ 8.7 Hz, 2H, ArH), 6.69 (d, J ¼ 8.9 Hz, 2H, ArH), 3.95 (t, J ¼ 5.9 Hz,
2H, CH2), 3.04 (s, 6H, N(CH3)2), 3.03e2.98 (m, 2H, CH2), 2.90 (s, 2H,
CH2), 2.50 (t, J ¼ 6.9 Hz, 2H, CH2), 2.40 (t, J ¼ 6.5 Hz, 2H, CH2), 2.35 (t,
J ¼ 6.3 Hz, 2H, CH2), 2.28 (t, J ¼ 6.5 Hz, 2H, CH2), 1.84e1.77 (m, 2H,
4.6. tert-Butyl 2-((3-(4-((1E,4E)-5-(4-(dimethylamino)phenyl)-3-oxo
penta-1,4-dienyl)phenoxy)propyl)(2-(tritylthio)ethyl)amino)ethyl(2-
(tritylthio)ethyl)carbamate (4a)
CH2). 13C NMR (101 MHz, CDCl3)
d 188.80, 170.96, 160.60, 151.96,
A mixture of 3a (294.2 mg, 0.71 mmol), tert-butyl 2-(tritylthio)
144.74, 143.81, 141.84, 130.26, 129.96, 129.56, 129.55, 127.94, 127.89,
126.74, 123.77, 122.66, 121.08, 114.85, 111.91, 77.41, 77.10, 76.78,
66.88, 66.78, 65.71, 58.47, 54.10, 51.19, 40.13, 38.06, 32.11, 30.09,
26.97. HRMS (ESI): m/z calcd for C66H66N3O3S2 1012.4546; found
1012.4502 [M þ H]þ.
ethyl(2-(2-(tritylthio)ethylamino)ethyl)carbamate
(539.2
mg,
0.71 mmol) and K2CO3 (193.6 mg, 1.42 mmol) in acetonitrile
(50 mL) was stirred at 90 ꢁC for 48 h. The solvent was removed by
vacuum, and water was added. The mixture was extracted with
CHCl3. The organic layers were combined and dried over anhydrous
Mg2SO4 and evaporated dry. The crude product was purified by
silica gel chromatography (petroleum ether/AcOEt ¼ 3/1, v/v) to
give 4a (272.5 mg, 36.0% yield) as yellow solid foams. 1H NMR
4.9. 2-((5-(4-((1E,4E)-5-(4-(Dimethylamino)phenyl)-3-oxopenta-
1,4-dienyl)phenoxy)pentyl)(2-(tritylthio)ethyl)amino)-N-(2-(trityl
thio)ethyl)acetamide (4d)
(400 MHz, CDCl3)
d
7.70 (d, J ¼ 15.6 Hz, 1H, trans eCH]CHe), 7.66
(d, J ¼ 15.6 Hz, 1H, trans eCH]CHe), 7.52 (d, J ¼ 8.8 Hz, 2H, ArH),
7.48 (d, J ¼ 8.6 Hz, 2H, ArH), 7.42e7.36 (m, 13H, ArH), 7.24e7.17 (m,
17H, ArH), 6.95 (d, J ¼ 15.8 Hz, 1H, trans eCH]CHe), 6.87 (d,
J ¼ 15.6 Hz, 1H, trans eCH]CHe), 6.84 (d, J ¼ 8.3 Hz, 2H, ArH), 6.70
(d, J ¼ 8.8 Hz, 2H, ArH), 3.93 (t, J ¼ 6.2 Hz, 2H, CH2), 3.04 (s, 6H,
N(CH3)2), 2.99e2.89 (m, 4H, CH2), 2.38e2.21 (m, 8H, CH2), 1.71 (s,
2H, CH2), 1.36 (s, 11H, CH2 and tert-Bu). 13C NMR (101 MHz, CDCl3)
The procedure described above for the preparation of 4c was
employed to give 4d (549.6 mg, 54.1%) as yellow solid foams. 1H
NMR (400 MHz, CDCl3)
d
7.70 (d, J ¼ 15.7 Hz, 1H, trans eCH]CHe),
7.67 (d, J ¼ 15.8 Hz, 1H, trans eCH]CHe), 7.52 (d, J ¼ 8.7 Hz, 2H,
ArH), 7.52 (d, J ¼ 8.9 Hz, 2H, ArH), 6.96 (d, J ¼ 15.8 Hz, 1H, trans e
CH]CHe), 6.87 (d, J ¼ 15.6 Hz, 1H, trans eCH]CHe), 6.85 (d,
J ¼ 8.6 Hz, 2H, ArH), 6.69 (d, J ¼ 8.8 Hz, 2H, ArH), 3.89 (t, J ¼ 6.3 Hz,

96
Y. Yang et al. / European Journal of Medicinal Chemistry 64 (2013) 90e98
2H, CH2), 3.04 (s, 6H, N(CH3)2), 3.01 (m, 2H, CH2), 2.85 (s, 2H, CH2),
4.12. 2-((3-(4-((1E,4E)-5-(4-(Dimethylamino)phenyl)-3-oxopenta-
1,4-dienyl)phenoxy)propyl)(2-mercaptoethyl)amino)-N-(2-mercapto
ethyl)acetamide-rhenium (V) oxide (5c)
2.44e2.34 (m, 4H, CH2), 2.33e2.22 (m, 4H, CH2), 1.74e1.66 (m, 2H,
CH2), 1.38 (s, 4H, CH2). 13C NMR (101 MHz, CDCl3)
d 188.82, 171.19,
160.82, 151.95, 144.77, 144.74, 143.79, 141.87, 130.25, 129.97, 129.56,
127.93, 127.77, 126.72, 123.71, 122.67, 121.07, 114.86, 111.90, 77.40,
77.08, 76.76, 67.86, 66.81, 66.74, 58.33, 54.72, 53.95, 40.14, 37.99,
32.07, 30.06, 29.01, 26.97, 23.79. HRMS (ESI): m/z calcd for
The procedure described above for the preparation of 5a was
employed to give 5c (57.4 mg, 15.5%) as an orange solid. m.p. 116.3e
117.5 ꢁC. 1H NMR (400 MHz, CDCl3)
d
7.71 (d, J ¼ 16.2 Hz, 1H, trans e
C
68H70N3O3S2 1040.4859; found 1040.4801 [M þ H]þ.
CH]CHe), 7.67 (d, J ¼ 16.6 Hz, 1H, trans eCH]CHe), 7.57 (d,
J ¼ 8.7 Hz, 2H, ArH), 7.52 (d, J ¼ 8.8 Hz, 2H, ArH), 6.98 (d, J ¼ 15.8 Hz,
1H, trans eCH]CHe), 6.91 (d, J ¼ 8.7 Hz, 2H, ArH), 6.87 (d,
J ¼ 15.8 Hz, 1H, trans eCH]CHe), 6.70 (d, J ¼ 8.7 Hz, 2H, ArH), 4.73
(d, J ¼ 16.4 Hz, 1H, CH2), 4.60 (dd, J ¼ 11.7, 5.5 Hz, 1H, CH2), 4.15 (d,
J ¼ 16.9 Hz, 2H, CH2), 4.12e4.07 (m, 2H, CH2), 3.85e3.75 (m, 1H,
CH2), 3.48e3.40 (m, 1H, CH2), 3.32e3.24 (m, 2H, CH2), 3.23e3.19
(m, 1H, CH2), 3.05 (s, 6H, N(CH3)2), 2.93e2.89 (m, 1H, CH2), 2.50e
2.41 (m, 1H, CH2), 2.37e2.24 (m, 3H, CH2). 13C NMR (101 MHz,
4.10. (1E,4E)-1-(4-(Dimethylamino)phenyl)-5-(4-(3-((2-mercapto
ethyl)(2-(2-mercaptoethylamino)ethyl)amino)propoxy)phenyl)
penta-1,4-dien-3-one-rhenium (V) oxide (5a)
A solution of 4a (157.8 mg, 0.15 mmol) in TFA (2 mL) was
stirred at room temperature for 5 min. Triethylsilane (60 mL) was
added and the mixture was stirred at 0 ꢁC for 10 min, then the
solvent was removed in vacuum. The residue was redissolved in a
mixed solvent (10 mL, CH2Cl2/CH3OH ¼ 10/1, v/v), (Ph3P)2ReOCl3
(122.3 mg, 0.15 mmol) and sodium acetate in methanol (1 M,
2 mL) were added. The reaction mixture was heated to reflux for
4 h. After cooling to room temperature, the solvent was evapo-
rated. Then water was added and the resulting mixture was
extracted by CH2Cl2 (3 ꢂ 15 mL), the combined organic layer was
dried over anhydrous MgSO4. After the solvent was removed, the
residue was purified by silica gel chromatography (CH2Cl2/
CH3OH ¼ 400/1, v/v) to give 5a (28.9 mg, 27.6%) as an orange solid.
CDCl3) d 188.67,186.86,159.57,155.59,151.94,143.95,141.30,130.24,
129.99, 128.53, 125.00, 124.30, 121.00, 114.77, 111.92, 77.31, 76.99,
76.68, 66.83, 64.54, 60.77, 59.88, 48.07, 40.15, 39.03, 32.20, 29.67,
26.40, 24.13, 23.41. HRMS (ESI): m/z calcd for C28H35N3O4ReS2
728.1626; found 728.1600 [M þ H]þ.
4.13. 2-((5-(4-((1E,4E)-5-(4-(Dimethylamino)phenyl)-3-oxopenta-
1,4-dienyl)phenoxy)pentyl)(2-mercaptoethyl)amino)-N-(2-mercapto
ethyl)acetamide-rhenium (V) oxide (5d)
The procedure described above for the preparation of 5a was
employed to give 5d (56.0 mg, 37.0%) as an orange solid. m.p.121.7e
m.p. 122.8e123.4 ꢁC. 1H NMR (400 MHz, CDCl3)
d 7.71 (d,
J ¼ 15.3 Hz, 1H, trans eCH]CHe), 7.67 (d, J ¼ 15.4 Hz, 1H, trans e
CH]CHe), 7.57 (d, J ¼ 8.6 Hz, 2H, ArH), 7.52 (d, J ¼ 8.7 Hz, 2H,
ArH), 6.98 (d, J ¼ 15.8 Hz, 1H, trans eCH]CHe), 6.90 (d, J ¼ 8.9 Hz,
2H, ArH), 6.87 (d, J ¼ 16.6 Hz, 1H, trans eCH]CHe), 6.69 (d,
J ¼ 8.7 Hz, 2H, ArH), 4.32e4.21 (m, 1H, CH2), 4.16 (dd, J ¼ 10.4,
6.2 Hz, 2H, CH2), 4.10 (s, 2H, CH2), 3.97e3.86 (m, 1H, CH2), 3.85e
3.74 (m, 2H, CH2), 3.45e3.35 (m, 2H, CH2), 3.32e3.24 (m, 1H, CH2),
3.04 (s, 6H, N(CH3)2), 2.83e2.74 (m, 1H, CH2), 2.35e2.31 (m, 2H,
CH2), 1.79 (td, J ¼ 11.9, 4.6 Hz, 2H, CH2), 0.90e0.78 (m, 2H, CH2).
122.3 ꢁC. 1H NMR (400 MHz, CDCl3)
d
7.70 (d, J ¼ 15.6 Hz,1H, trans e
CH]CHe), 7.67 (d, J ¼ 15.7 Hz, 1H, trans eCH]CHe), 7.56 (d,
J ¼ 8.5 Hz, 2H, ArH), 7.52 (d, J ¼ 8.7 Hz, 2H, ArH), 6.98 (d, J ¼ 15.9 Hz,
1H, trans eCH]CHe), 6.90 (d, J ¼ 8.8 Hz, 2H, ArH), 6.87 (d,
J ¼ 15.8 Hz, 1H, trans eCH]CHe), 6.69 (d, J ¼ 8.6 Hz, 2H, ArH), 4.66
(d, J ¼ 16.4 Hz, 1H, CH2), 4.58 (dd, J ¼ 11.5, 5.3 Hz, 1H, CH2), 4.12 (d,
J ¼ 16.2 Hz, 1H, CH2), 4.08e3.93 (m, 3H, CH2), 3.59e3.52 (m, 1H,
CH2), 3.41e3.33 (m, 1H, CH2), 3.27e3.17 (m, 3H, CH2), 3.04 (s, 6H,
N(CH3)2), 2.87 (dd, J ¼ 13.5, 4.1 Hz, 1H, CH2), 1.93e1.85 (m, 4H, CH2),
13C NMR (101 MHz, CDCl3)
d 188.79, 159.79, 151.98, 143.93, 141.43,
1.68e1.56 (m, 4H, CH2). 13C NMR (101 MHz, CDCl3)
d 188.77, 186.94,
130.29, 130.02, 128.57, 124.18, 122.66, 121.03, 114.80, 111.92, 77.31,
77.00, 76.68, 70.89, 65.48, 64.95, 62.67, 60.62, 59.14, 49.40, 40.11,
29.65, 23.41. HRMS (ESI): m/z calcd for C28H37N3O3ReS2 714.1834;
found 714.2107 [M þ H]þ.
160.45, 151.94, 143.85, 141.61, 130.23, 129.98, 128.17, 123.94, 122.73,
121.06, 114.80, 111.93, 77.30, 76.98, 76.67, 67.40, 67.00, 64.21, 63.68,
59.83, 47.91, 40.14, 38.95, 29.67, 28.71, 23.73, 23.55. HRMS (ESI): m/z
calcd for C30H39N3O4ReS2 756.1939; found 756.1945 [M þ H]þ.
4.11. (1E,4E)-1-(4-(Dimethylamino)phenyl)-5-(4-(5-((2-mercapto
ethyl)(2-(2-mercaptoethylamino)ethyl)amino)pentyloxy)phenyl)
penta-1,4-dien-3-one-rhenium (V) oxide (5b)
4.14. In vitro fluorescent staining using Tg mouse brain sections
The transgenic model mice (C57BL6, APPswe/PSEN1, 11 months
old) and age-matched control mice (C57BL6, 11 months old) were
The procedure described above for the preparation of 5a was
employed to give 5b (30.1 mg, 29.6%) as an orange solid. m.p.
used for the studies. Paraffin-embedded brain sections (10 mm
thick) were deparaffinized with 3 ꢂ 10 min washes in xylene,
2 ꢂ 5 min washes in 100% ethanol, 5 min wash in 80% ethanol/H2O,
5 min wash in 60% ethanol/H2O, and 10 min wash in running tap
water and then incubated in PBS (0.2 M, pH ¼ 7.4) for 30 min. The
154.5e155.3 ꢁC. 1H NMR (400 MHz, CDCl3)
d
7.70 (d, J ¼ 15.7 Hz,
1H, trans eCH]CHe), 7.67 (d, J ¼ 15.8 Hz, 1H, trans eCH]CHe),
7.56 (d, J ¼ 8.6 Hz, 2H, ArH), 7.51 (d, J ¼ 8.8 Hz, 2H, ArH), 6.97 (d,
J ¼ 15.8 Hz, 1H, trans eCH]CHe), 6.90 (d, J ¼ 8.7 Hz, 2H, ArH),
6.87 (d, J ¼ 15.8 Hz, 1H, trans eCH]CHe), 6.69 (d, J ¼ 8.8 Hz, 2H,
ArH), 4.18e4.09 (m, 2H, CH2), 4.02 (t, J ¼ 6.1 Hz, 2H, CH2), 3.89e
3.82 (m, 1H, CH2), 3.77 (dd, J ¼ 11.2, 5.2 Hz, 1H, CH2), 3.61e3.51
(m, 1H, CH2), 3.41e3.31 (m, 3H, CH2), 3.29e3.18 (m, 1H, CH2), 3.04
(s, 6H, N(CH3)2), 3.00e2.93 (m, 2H, CH2), 2.73 (dd, J ¼ 13.4, 3.1 Hz,
1H, CH2), 1.92e1.82 (m, 4H, CH2), 1.76e1.65 (m, 2H, CH2), 1.63e
brain sections were incubated with a 10% ethanol solution (1.0 mM)
of 5a, 5b, 5c and 5d for 10 min, respectively. The locations of pla-
ques were confirmed by staining with Thioflavin-S (0.125%) in
adjacent sections. Finally, the sections were washed with 40%
ethanol for 10 min. Fluorescent observation was performed by the
Axio Observer Z1 inverted fluorescence microscope (Zeiss, Ger-
many) equipped with DAPI (excitation, 405 nm) and AF (excitation,
495 nm) filter sets.
1.51 (m, 2H, CH2). 13C NMR (101 MHz, CDCl3)
d 188.78, 160.58,
151.96, 143.85, 141.67, 130.24, 129.97, 128.02, 123.86, 122.64,
121.01, 114.84, 111.89, 77.33, 77.01, 76.69, 70.92, 67.53, 65.48,
63.60, 62.75, 58.89, 49.14, 40.12, 40.00, 28.83, 23.76, 22.83. HRMS
(ESI): m/z calcd for C30H41N3O3ReS2 742.2147; found 742.2329
[M þ H]þ.
4.15. Binding assay in vitro using Ab aggregates
Peptides Ab(1e42) were purchased from Osaka Peptide Institute
(Osaka, Japan). Aggregation was carried out by gently dissolving the

Y. Yang et al. / European Journal of Medicinal Chemistry 64 (2013) 90e98
97
peptide [0.56 mg/mL for Ab(1e42)] in a buffer solution (pH ¼ 7.4)
containing 10 mM sodium phosphate and 1 mM EDTA. The solution
was incubated at 37 ꢁC for 42 h with gentle and constant shaking.
Inhibition experiments were carried out in 12 ꢂ 75 mm borosilicate
glass tubes according to procedures described previously with
(1.6 mL) and PBS (3.0 mL) were pipetted into it. The vortexing,
centrifuging, and counting protocols were repeated until consistent
distribution coefficient values were obtained.
4.18. Biodistribution in normal mice
some modification. Briefly, 100
the final assay mixture) was added into a mixture containing 100
of radioligand ([125I]IMPY,100,000 cpm/100
L),100 L of inhibitors
(5a, 5b, 5c or 5d, 10ꢃ4 to 10ꢃ8.5 M in ethanol), and 700
L of PBS
(0.2 M, pH ¼ 7.4) containing 0.5% BSA in a final volume of 1 mL.
Non-specific binding was defined in the presence of 1 M IMPY. The
mixture was incubated at room-temperature for 3 h, and then the
bound and free radioactive fractions were separated by vacuum
filtration through borosilicate glass fiberfilters (Whatman GF/B)
using an Mp-48T cell harvester (Brandel, Gaithersburg, MD). The
radioactivity of filters containing the bound 125I-ligand was
mL of Ab(1e42) aggregates (28 nM in
mL
A saline solution containing the HPLC-purified 99mTc-labled
tracer (100 mL, 10% ethanol, 185 kBq) was injected intravenously
into ICR mice (five weeks, male) via the tail vein. The mice (n ¼ 5 for
each time point) were sacrificed by decapitation exactly at 2, 10, 30,
and 60 min post-injection. Samples of blood and organs of interest
were removed, weighed and radioactivity was counted with an
automatic g-counter (Wallac 1470 Wizard, USA). The results were
expressed in terms of the percentage of the injected dose per gram
(%ID/g) of blood or organs.
m
m
m
m
measured in an automatic g-counter (WALLAC/Wizard1470, USA)
Acknowledgments
with 70% efficiency. Under the assay conditions, the specifically
bound fraction accounted for about 10% of total radioactivity. The
half-maximal inhibitory concentration (IC50) was determined from
displacement curves of three independent experiments using
GraphPad Prism 5.0, and the inhibition constant (Ki) was calculated
The authors present special thanks to Dr. Jin Liu (College of Life
Science, Beijing Normal University) for assistance in the in vitro
neuropathological staining. This work was funded by National
Natural Science Foundation of China (No: 21201019), Research Fund
for the Doctoral Program of Higher Education of China (No:
20120003120013) and Fundamental Research Funds for the Central
Universities (No: 2012LYB19).
using the Cheng-Prusoff inhibition constant equation: Ki ¼ IC50
/
(1 þ [L]/Kd), where [L] represents the concentration of [125I]IMPY
used in the assay and Kd is the dissociation constant of [125I]IMPY.
4.16. 99mTc labeling reaction and analysis by HPLC
Appendix A. Supplementary data
Na99mTcO4 solution (74 MBq, 200
reacted at room temperature for 10 min to give a 99mTc-GH solu-
tion. To a solution of precursor (4aed, 0.5 mg) in TFA (200 L) was
added triethylsilane (2 L), and the mixture was left at room tem-
mL) was added to a GH kit and
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.03.057.
m
m
References
perature for 10 min, then the solvents were removed under a
stream of nitrogen gas. The residue was redissolved in ethanol
[1] D.J. Selkoe, The origins of Alzheimer disease: A is for amyloid, JAMA 283
(2000) 1615e1617.
[2] D.J. Selkoe, Alzheimer’s disease: genes, proteins, and therapy, Physiol. Rev. 81
(2001) 741e766.
[3] A. Wimo, B. Winblad, H. Aguero-Torres, E. von Strauss, The magnitude of
dementia occurrence in the world, Alzheimer Dis. Assoc. Disord. 17 (2003)
63e67.
[4] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer’s disease:
progress and problems on the road to therapeutics, Science 297 (2002)
353e356.
[5] J.L. Cummings, Alzheimer’s disease, N. Engl. J. Med. 351 (2004) 56e67.
[6] B.C.P. Lee, M. Mintun, R.L. Buckner, J.C. Morris, Imaging of Alzheimer’s disease,
J. Neuroimaging 13 (2003) 199e214.
[7] V.L. Villemagne, C.C. Rowe, S. Macfarlane, K.E. Novakovic, C.L. Masters,
Imaginem oblivionis: the prospects of neuroimaging for early detection of
Alzheimer’s disease, J. Clin. Neurosci. 12 (2005) 221e230.
[8] H. Braak, E. Braak, Neuropathological stageing of Alzheimer-related changes,
Acta Neuropathol. 82 (1991) 239e259.
(200 m mL). The
L), and then mixed with the 99mTc-GH solution (200
reaction mixture was heated to 100 ꢁC for 10 min. After cooling to
room temperature, the product was extracted with CHCl3
(2 ꢂ 1 mL) and then the solvents were removed under a stream of
nitrogen gas. The residue was redissolved in 100
mL acetonitrile and
purified by HPLC on a Venusil MP C18 reverse phase column (5
mm,
4.6 ꢂ 250 mm) with a binary gradient system (acetonitrile/wa-
ter:70%/30%) at 1.0 mL/min flow rate to give [99mTc]5a, [99mTc]5b,
[
99mTc]5c and [99mTc]5d, respectively. The identity of [99mTc]5ae
d was verified by a comparison of the retention time with that of
the nonradioactive rhenium complexes from the HPLC profiles. The
four 99mTc-complexes were proven to show >95% radiochemical
purity by HPLC.
[9] M. Ono, A. Wilson, J. Nobrega, D. Westaway, P. Verhoeff, Z.-P. Zhuang, M.-
P. Kung, H.F. Kung, 11C-labeled stilbene derivatives as A
b-aggregate-specific
4.17. Partition coefficient determination
PET imaging agents for Alzheimer’s disease, Nucl. Med. Biol. 30 (2003)
565e571.
[10] N.P.L.G. Verhoeff, A.A. Wilson, S. Takeshita, L. Trop, D. Hussey, K. Singh,
H.F. Kung, M.-P. Kung, S. Houle, In-vivo imaging of Alzheimer disease [beta]-
amyloid with [11C]SB-13 PET, Am. J. Geriatr. Psychiatry 12 (2004) 584e595.
[11] W.E. Klunk, H. Engler, A. Nordberg, Y. Wang, G. Blomqvist, D.P. Holt,
M. Bergström, I. Savitcheva, G.-F. Huang, S. Estrada, B. Ausén, M.L. Debnath,
J. Barletta, J.C. Price, J. Sandell, B.J. Lopresti, A. Wall, P. Koivisto, G. Antoni,
C.A. Mathis, B. Långström, Imaging brain amyloid in Alzheimer’s disease with
Pittsburgh compound-B, Ann. Neurol. 55 (2004) 306e319.
The log D values of [99mTc]5a, [99mTc]5b, [99mTc]5c and [99mTc]5d
were determined by measuring the distribution of the radiotracer
between 1-octanol and PBS buffer at pH ¼ 7.4. The two phases were
pre-saturated with each other. 1-Octanol (3.6 mL) and PBS (3.0 mL)
were pipetted into a 10 mL plastic centrifuge tube containing 740 kBq
of 99mTc-labeled tracers. The mixture was vortexed for 5 min, fol-
lowed bycentrifugation for 5 min (3500 rpm, Anke TDL80-2B, China).
[12] C.A. Mathis, Y. Wang, D.P. Holt, G.-F. Huang, M.L. Debnath, W.E. Klunk, Syn-
thesis and evaluation of 11C-labeled 6-substituted 2-arylbenzothiazoles as
amyloid imaging agents, J. Med. Chem. 46 (2003) 2740e2754.
50
deposed in twotest tubes for counting. The amountof radioactivity in
each tube was measured with an automatic -counter (Wallac 1470
mL of the 1-octanol layer and 500 mL of the buffer layer were
[13] C.C. Rowe, U. Ackerman, W. Browne, R. Mulligan, K.L. Pike, G. O’Keefe,
H. Tochon-Danguy, G. Chan, S.U. Berlangieri, G. Jones, K.L. Dickinson-Rowe,
H.P. Kung, W. Zhang, M.P. Kung, D. Skovronsky, T. Dyrks, G. Holl, S. Krause,
M. Friebe, L. Lehman, S. Lindemann, L.M. Dinkelborg, C.L. Masters,
g
Wizard, USA). The measurement was carried out in triplicate and
repeated three times. The distribution coefficient was determined by
calculating the ratio of cpm/mL of 1-octanol phase versus that of PBS
phase and expressed as log D. Then 2.0 mL of the remaining 1-octanol
layer was transferred into a new centrifuge tube, and new 1-octanol
V.L. Villemagne, Imaging of amyloid
BAY94-9172, novel PET tracer: proof of mechanism, Lancet Neurol.
(2008) 129e135.
b
in Alzheimer’s disease with 18F-
a
7
[14] V.L. Villemagne, K. Ong, R.S. Mulligan, G. Holl, S. Pejoska, G. Jones, G. O’Keefe,
U. Ackerman, H. Tochon-Danguy, J.G. Chan, C.B. Reininger, L. Fels, B. Putz,

98
Y. Yang et al. / European Journal of Medicinal Chemistry 64 (2013) 90e98
B. Rohde, C.L. Masters, C.C. Rowe, Amyloid imaging with 18F-florbetaben in
Alzheimer disease and other dementias, J. Nucl. Med. 52 (2011) 1210e1217.
[15] M.T. Fodero-Tavoletti, D. Brockschnieder, V.L. Villemagne, L. Martin,
A.R. Connor, A. Thiele, M. Berndt, C.A. McLean, S. Krause, C.C. Rowe, C.L. Masters,
L. Dinkelborg, T. Dyrks, R. Cappai, In vitro characterization of [18F]-florbetaben,
phenylbenzothiazole conjugate as a potential in vivo tracer for visualization of
amyloid b, Bioorg. Med. Chem. Lett. 17 (2007) 6086e6090.
[26] X. Chen, P. Yu, L. Zhang, B. Liu, Synthesis and biological evaluation of 99mTc,
Re-monoamine-monoamide conjugated to 2-(4-aminophenyl)benzothiazole
as potential probes for b-amyloid plaques in the brain, Bioorg. Med. Chem.
an Ab
imaging radiotracer, Nucl. Med. Biol. 39 (2012) 1042e1048.
Lett. 18 (2008) 1442e1445.
[16] M. Koole, D.M. Lewis, C. Buckley, N. Nelissen, M. Vandenbulcke, D.J. Brooks,
R. Vandenberghe, K. Van Laere, Whole-body biodistribution and radiation
dosimetry of 18F-GE067: a radioligand for in vivo brain amyloid imaging,
J. Nucl. Med. 50 (2009) 818e822.
[17] K.J. Lin, W.C. Hsu, I.T. Hsiao, S.P. Wey, L.W. Jin, D. Skovronsky, Y.Y. Wai,
H.P. Chang, C.W. Lo, C.H. Yao, T.C. Yen, M.P. Kung, Whole-body biodistribution
and brain PET imaging with [18F]AV-45, a novel amyloid imaging agent-a pilot
study, Nucl. Med. Biol. 37 (2010) 497e508.
[27] K. Serdons, T. Verduyckt, J. Cleynhens, G. Bormans, A. Verbruggen, Develop-
ment of 99mTc-thioflavin-T derivatives for detection of systemic amyloidosis,
J. Labelled Comp. Radiopharm. 51 (2008) 357e367.
[28] M. Ono, R. Ikeoka, H. Watanabe, H. Kimura, T. Fuchigami, M. Haratake, H. Saji,
M. Nakayama, Synthesis and evaluation of novel chalcone derivatives with
99mTc/Re complexes as potential probes for detection of
b-amyloid plaques,
ACS Chem. Neurosci. 1 (2010) 598e607.
[29] M. Ono, R. Ikeoka, H. Watanabe, H. Kimura, T. Fuchigami, M. Haratake, H. Saji,
[18] S.R. Choi, G. Golding, Z. Zhuang, W. Zhang, N. Lim, F. Hefti, T.E. Benedum,
M. Nakayama, 99mTc/Re complexes based on flavone and aurone as SPECT
M.R. Kilbourn, D. Skovronsky, H.F. Kung, Preclinical properties of 18F-AV-45: a
probes for imaging cerebral b-amyloid plaques, Bioorg. Med. Chem. Lett. 20
PET agent for A
b
plaques in the brain, J. Nucl. Med. 50 (2009) 1887e1894.
(2010) 5743e5748.
[19] D.F. Wong, P.B. Rosenberg, Y. Zhou, A. Kumar, V. Raymont, H.T. Ravert,
[30] M. Sagnou, D. Benaki, C. Triantis, T. Tsotakos, V. Psycharis, C.P. Raptopoulou,
I. Pirmettis, M. Papadopoulos, M. Pelecanou, Curcumin as the OO bidentate
ligand in “2 þ 1” complexes with the [M(CO)3]þ (M ¼ Re, 99mTc) tricarbonyl
core for radiodiagnostic applications, Inorg. Chem. 50 (2011) 1295e1303.
[31] M. Ono, Y. Fuchi, T. Fuchigami, N. Kobashi, H. Kimura, M. Haratake, H. Saji,
M. Nakayama, Novel benzofurans with 99mTc complexes as probes for imaging
ꢀ ꢁ
R.F. Dannals, A. Nandi, J.R. Brasic, W. Ye, J. Hilton, C. Lyketsos, H.F. Kung,
A.D. Joshi, D.M. Skovronsky, M.J. Pontecorvo, Vivo imaging of amyloid depo-
sition in Alzheimer disease using the radioligand 18F-AV-45 (Florbetapir F 18),
J. Nucl. Med. 51 (2010) 913e920.
[20] H. Han, C.-G. Cho, P.T. Lansbury, Technetium complexes for the quantitation of
brain amyloid, J. Am. Chem. Soc. 118 (1996) 4506e4507.
cerebral b-amyloid plaques, ACS Med. Chem. Lett. 1 (2010) 443e447.
[21] W. Zhen, H. Han, M. Anguiano, C.A. Lemere, C.G. Cho, P.T. Lansbury, Synthesis
and amyloid binding properties of rhenium complexes: preliminary progress
toward a reagent for SPECT imaging of Alzheimer’s disease brain, J. Med.
Chem. 42 (1999) 2805e2815.
[32] Y. Cheng, M. Ono, H. Kimura, M. Ueda, H. Saji, Technetium-99m labeled
pyridyl benzofuran derivatives as single photon emission computed tomog-
raphy imaging probes for
b-amyloid plaques in Alzheimer’s brains, J. Med.
Chem. 55 (2012) 2279e2286.
[22] N.A. Dezutter, R.J. Dom, T.J. de Groot, G.M. Bormans, A.M. Verbruggen, 99mTc-
MAMA-chrysamine G, a probe for beta-amyloid protein of Alzheimer’s dis-
ease, Eur. J. Nucl. Med. 26 (1999) 1392e1399.
[33] X. Wang, M. Cui, P. Yu, Z. Li, Y. Yang, H. Jia, B. Liu, Synthesis and biological
evaluation of novel technetium-99m labeled phenylbenzoxazole derivatives
as potential imaging probes for
Chem. Lett. 22 (2012) 4327e4331.
[34] M. Cui, M. Ono, H. Kimura, B. Liu, H. Saji, Synthesis and structureeaffinity
b-amyloid plaques in brain, Bioorg. Med.
[23] N.A. Dezutter, T.J. de Groot, R.H. Busson, G.A. Janssen, A.M. Verbruggen,
Preparation of 99mTc-N2S2 conjugates of chrysamine G, potential probes for
the beta-amyloid protein of Alzheimer’s disease, J. Labelled Comp. Radio-
pharm. 42 (1999) 309e324.
relationships of novel dibenzylideneacetone derivatives as probes for b-am-
yloid plaques, J. Med. Chem. 54 (2011) 2225e2240.
[24] Z.-P. Zhuang, M.-P. Kung, C. Hou, K. Ploessl, H.F. Kung, Biphenyls labeled with
[35] S.S. Jurisson, J.D. Lydon, Potential technetium small molecule radiopharma-
ceuticals, Chem. Rev. 99 (1999) 2205e2218.
technetium 99m for imaging b-amyloid plaques in the brain, Nucl. Med. Biol.
32 (2005) 171e184.
[36] G. Tamagnan, R.M. Baldwin, N.S. Kula, R.J. Baldessarini, R.B. Innis, Cyclo-
pentadienyltricarbonylrheniumbenzazepines: synthesis and binding affinity,
Bioorg. Med. Chem. Lett. 10 (2000) 1113e1115.
[25] K. Serdons, T. Verduyckt, J. Cleynhens, C. Terwinghe, L. Mortelmans,
G. Bormans, A. Verbruggen, Synthesis and evaluation of
a
99mTc-BAT-