Preparation of classical Re/99mTc(CO)3+ and novel 99mTc(CO)2(NO)2+ cores complexed with flavonol derivatives and their binding characteristics for Aβ (1-40) aggregates

Article abstract of DOI:10.1016/j.bmcl.2010.04.026

Classical ^99mTc(CO)₃⁺ and novel ^99mTc(CO)₂(NO)²⁺ cores complexed with flavonol derivatives were prepared. Autoradiography of postmortem AD transgenic mice (Tg C57, APP, PS1 12-month-old) br

Full text of DOI:10.1016/j.bmcl.2010.04.026

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5337–5344
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Preparation of classical Re/99mTc(CO)3 and novel 99mTc(CO)2(NO)2+ cores
+
complexed with flavonol derivatives and their binding characteristics for
Ab(1–40) aggregates
*
Yang Yang, Lin Zhu, Mengchao Cui , Ruikun Tang, Huabei Zhang
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
+
Classical 99mTc(CO)3 and novel 99mTc(CO)2(NO)2+ cores complexed with flavonol derivatives were pre-
pared. Autoradiography of postmortem AD transgenic mice (Tg C57, APP, PS1 12-month-old) brain sec-
tion confirmed the binding property of [99mTc(CO)3+-3-OH-flavone]0 to Ab(1–40) aggregates, while the
novel 99mTc(CO)2(NO)2+ labeled compounds showed no binding sites in AD transgenic mice sections.
Article history:
Received 26 November 2009
Revised 28 January 2010
Accepted 9 April 2010
Available online 31 July 2010
Intravenous administration of
[
99mTc(CO)3+-3-OH-flavone]0 resulted in moderate brain uptake
(0.48 0.05%ID/g) at 5 min post-injection and slow clearance from the brain issues in 2 h post-injection
(120 min: 0.39 0.08%ID/g). Then an Ab(1–40)-receptor-targeted Re(CO)3+-3-OH-flavone, was prepared to
identify the structure of the technetium complex. UV–vis absorption and fluorescence emission proper-
ties have been studied at room temperature in order to determine the natures of the lowest electronically
excited states of Re(CO)3+-3-OH-flavone and the ligand. The fluorescent rhenium complex Re(CO)3+-3-
OH-flavone showed high affinity for Ab(1–40) aggregates in vitro by fluorescence spectra (dissociation con-
stant (Kd) = 11.16 nM). In conclusion, the results suggested that 99mTc(CO)3+-3-OH-flavone should be a
suitable candidate as Ab plaque SPECT imaging agent for AD.
Keywords:
Ab(1–40) aggregates
Re/99mTc(CO)3+-3-OH-flavone
99mTc(CO)2(NO)2+
Autoradiography
Kd
Fluorescence
UV-spectra
Biodistribution
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) caused 50–60% of all the dementia
cases, and it is a brain disorder associated with progressive mem-
ory loss and decrease of cognitive function.1 In the development
and progression of AD, the formation of the senile plaques are crit-
ical factors, and the senile plaques are extracellular deposits of
amyloid fibrils of b-amyloid (Ab) (Ab(1–40) and Ab(1–42)),2 the char-
acteristic of which was a typical b-sheet form.3,4 However, early
diagnosis of AD is often unreliable and the only definitive confir-
mation of AD is by postmortem histopathological examination of
amyloid deposits in the brain.5 Thus, the direct mapping of Ab
aggregates in the living brain by PET and SPECT Ab aggregates-spe-
cific imaging agents is an active research area. Many agents has
been attempted and several of them might be selected as the can-
didates for targeting Ab aggregates, such as 18F-FDDNP,6 11C-PIB,7
and 11C-SB-13.8 Currently, novel radioiodinated flavone derivatives
for imaging of b-amyloid plaques were reported by Masahiro
Ono,9,10 and the binding affinities of radioiodinated flavones were
from 13 to 77 nM. In addition, these compounds displayed high
brain uptakes at 2 min post-injection and rapid wash-out from
the brain at 30 min in normal mice. The reports suggested that
these flavone derivatives might be candidates for b-amyloid aggre-
gates imaging.
However, the development of 99mTc-labeled radioactive probes
for SPECT was lagging far behind. Technetium-99m labeled com-
pounds would be particularly useful in view of the favorable phys-
ical properties (t1/2 = 6 h, E = 140 keV), easy and widespread
c
availability, and low cost of the 99mTc nuclide as well.11 Since there
is no stable isotope of Tc available, the use of rhenium, as a VIIB
congener of technetium, may facilitate the development of techne-
tium-99m labeled complexes, because of the similar physical,
chemical and biodistribution properties of the two nuclides. More-
over, the methodologies developed to generate Re complexes are
generally applicable to generate their corresponding Tc analogs,
so rhenium complexes were often used as a non-radioactive alter-
native for the structural characterization of technetium complexes
and the determination of binding affinities of the Tc-radiotracers.12
Here to fore, no 99mTc-labeled compound has been successfully
applied for Ab aggregates imaging due to the low penetration into
blood–brain barrier (BBB), in spite of their high binding affinities
for Ab aggregates.13–16 However, the classical 99mTc(CO)3 and no-
+
vel 99mTc(CO)2(NO)2+ cores were helpful to design new radiophar-
maceuticals, and in our previous report, we have successfully
converted the 99mTc(CO)3 core into 99mTc(CO)2(NO)2+ with
+
[NO][BF4] in water and acetonitrile.17
Thus, in order to develop 99mTc labeled Ab plaques
imaging agents, we prepared novel Re/99mTc(CO)3+-complexed 3-
OH-flavone and 99mTc(CO)2(NO)2+ core labeled 6-OH-flavone
* Corresponding author. Tel.: +86 10 58802961.
E-mail address: hbzhang@bnu.edu.cn (H. Zhang).
0960-894X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.026

5338
Y. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5337–5344
(7-OH-flavone) derivatives. Moreover, since Klunk W. E.18 and H.
fluorescence spectra, there has been few report on the same meth-
LeVine III etc.19 reported the determination of the values of Kd by
od used to measure the values of Kd when Re(CO)3+-compounds
Figure 1. Reagents and conditions: Re(CO)5Br, KOH, H2O, reflux, 22 h (Re(CO)3+-3-OH-flavone); [99mTc(CO)3(H2O)]+, H2O, pH 11, 100 °C, 40 min (99mTc(CO)3+-3-OH-flavone);
(a) Ph3P, NBS, CH2Cl2; (b) 6-OH-flavone(7-OH-flavone), KOH, ethanol; (c) CF3COOH, CH2Cl2; (d) [99mTc(CO)3(H2O)]+, H2O, pH 11, 100 °C, 100 min; (e) NOBF4, H2O, pH 1, 100 °C,
15 min.

Y. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5337–5344
5339
binding to Ab aggregates. So the novelty of this research is to estab-
lish a credible method by fluorescence spectra in the measurement
of Kd of the Re(CO)3+-complexed 3-OH-flavone, and this method
was based on the factor that when Ab aggregates was added to
the solution of a small molecule, the quantum yield of fluorescence
of the molecule may be increased because the rotation of the rings
about one another upon transition to the excited state is inhabited
by the rigidity of the micro surrounding of the aggregates.20,21
the biological characterization of [99mTc(CO)3+-3-OH-flavone]0 in
normal mice was also performed to evaluate its potential as a tracer
for visualization of b-amyloid plaques.
The preparation routes of Re/99mTc(CO)3+-3-OH-flavone,
[
99mTc(CO)2(NO)2+-6-OH-flavone]0 and [99mTc(CO)2(NO)2+-7-OH-
flavone]0 are shown in Figure 1.
The compound Re(CO)3+-3-OH-flavone was obtained as a light
yellow solid (yield 78%) via a one-stage reaction,25a and the
In this work, classical 99mTc(CO)3 core labeled 3-OH-flavone
[
99mTc(CO)3+-3-OH-flavone]0 was prepared via
[
99mTc(CO)3-
+
and novel 99mTc(CO)2(NO)2+ core labeled 6-OH-flavone (7-OH-fla-
(H2O)3]+ moiety22 by direct labeling method,25c as outlined in
Figure 1. The chemical identities of Re(CO)3+-3-OH-flavone were
confirmed by NMR,26 MS-ESI+27 and Infrared Spectrum.28 The label-
ing yield of [99mTc(CO)3+-3-OH-flavone]0 detected by radio-TLC29
was higher than 95%, and the Rfs of 99mTc(CO)3+-3-OH-flavone and
vone) derivatives were prepared.
[
99mTc(CO)3+-3-OH-flavone]0
labeled the AD mice (Tg C57, APP, PS1 12-month-old) brain section
in comparison to the no binding sites in the normal mice section by
autoradiography, oppositely, the 99mTc(CO)2(NO)2+ complexes did
not label the AD mice brain section, so we did not synthesize the
Re(CO)2(NO)2+ complexes, but the Re(CO)3+-3-OH-flavone was syn-
thesized to identify the structure of the Tc-99m complex, and the
rhenium complex with affinity for Ab(1–40) aggregates in vitro was
determined to be 11.16 nM by fluorescence spectra in vitro. Then,
[
99mTc(CO)3(H2O)3]+ were 0.6–0.7 and 0–0.1, respectively (Table
1). The radio-chemical purity was higher than 92% by reversed
phase HPLC.30a Moreover, [99mTc(CO)3+-3-OH-flavone]0 was proved
to be neutral by paper electrophoresis31 (Table 3). The identity of the
radioactive complex was established by comparative HPLC30a
Table 1
The distributions of radioactivity of [99mTc(CO)3+-3-OH-flavone]0 (A) and [99mTc(CO)3(H2O)3]+ (B)
Rf
0
0.1
29,857
122,797
0.2
0.3
8045
35,401
0.4
0.5
0.6
0.7
0.8
8744
29,436
0.9
A
B
15,570
394,875
16,423
79,281
10,117
29,781
36,979
28,547
139,554
36,444
102,281
27,662
14,316
43,257
Figure 2. The HPLC chromatograms of [99mTc(CO)3(H2O)3]+ (A), 3-OH-flavone (B), [99mTc(CO)3+-3-OH-flavone]0 (C) and Re(CO)3+-3-OH-flavone (D).

5340
Y. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5337–5344
Table 2
Table 3
The distributions of radioactivity of
[
99mTc(CO)3(H2O)3]+ (A),
[
99mTc(CO)3+-6-OH-
Eletrophoresis pattern of [99mTc(CO)3+-3-OH-flavone]0 (A), [99mTc(CO)2(NO)2+-6-OH-
flavone]0 (B) and [99mTc(CO)2(NO)2+-7-OH-flavone]0 (C)
flavone]ꢀ (B), 99mTc(CO)3+-7-OH-flavone]ꢀ (C), 99mTc(CO)2(NO)2+-6-OH-flavone]0
[
[
(D) and [99mTc(CO)2(NO)2+-7-OH-flavone]0 (E)
Position
Cathode
Origin
Anode
Rf
0
0.1
95
103
129
1642
798
0.2
81
147
133
85
0.3
0.4
0.5
0.6
94
329
291
82
0.7
96
101
132
95
0.8
0.9
A
B
C
Radioactive counts
Radioactive counts
Radioactive counts
596
936
441
9673
11,875
9843
475
539
338
A
B
C
D
E
4578
98
103
92
98
103
97
87
87
96
80
99
91
644
782
78
116
92
101
91
104
91
77
112
101
78
278
149
84
101
81
85
87
(Fig. 2D) and 23.2 min (Fig. 2C), respectively, in comparison to the
ligand 3-OH-flavone (UV-HPLC,30a 16.9 min, Fig. 2B) and precursor
99mTc(CO)3(H2O)3]+ (radio-HPLC,30a 3.0 min, Fig. 2A), respectively.
studies using the corresponding rhenium complex as reference.
The retention times for Re(CO)3+-3-OH-flavone (UV) and
[
[
The retention time of [99mTc(CO)3+-3-OH-flavone]0 showed almost
the same to that of Re(CO)3+-3-OH-flavone, and these results
99mTc(CO)3+-3-OH-flavone]0 (radioactivity) on HPLC30a were 22.3
25000
A
B10000
20000
15000
10000
5000
0
8000
6000
4000
2000
0
-2000
0
5
10
15
20
25
0
5
10
15
20
25
30
35
retention time
retention time
800000
700000
600000
500000
400000
300000
200000
100000
0
700000
600000
500000
400000
300000
200000
100000
0
D
C
-100000
-100000
0
5
10
15
20
0
5
10
15
20
retention time
retention time
400000
350000
300000
250000
200000
150000
100000
50000
0
F
E
500000
400000
300000
200000
100000
0
-50000
0
5
10
15
20
5
10
15
20
0
retention time
retention time
Figure 3. The HPLC chromatograms of compound C (A), compound D (B), [99mTc(CO)3+-6-OH-flavone]ꢀ (C), [99mTc(CO)3+-7-OH-flavone]ꢀ (D), [99mTc(CO)2(NO)2+-6-OH-
flavone]0 (E) and [99mTc(CO)2(NO)2+-7-OH-flavone]0 (F).

Y. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5337–5344
5341
Figure 5. Room temperature fluorescence spectra (in ethanol solutions) of
Figure 4. UV–vis absorption spectra (in ethanol solutions) of the ligand (black line)
Re(CO)3+-3-OH-flavone (red line) and 3-OH-flavone (black line).
and Re(CO)3+-3-OH-flavone (red line).
demonstrated the same structure of Re(CO)3+-3-OH-flavone and
were higher than 92% according to the labeling methods in,25d
and the Rfs of [99mTc(CO)3+-6-OH-flavone]ꢀ, [99mTc(CO)3+-7-OH-fla-
vone]ꢀ, [99mTc(CO)2(NO)2+-6-OH-flavone]0 and [99mTc(CO)2(NO)2+-7
-OH-flavone]0 were 0.5–0.6, 0.5–0.6, 0.1 and 0.1, respectively
(Table 2). The radio-chemical purities of the Tc complexes were
all higher than 95% by reversed phase HPLC.30b Moreover,
[
99mTc(CO)3+-3-OH-flavone]0.
The preparation of compound A was according to23 and com-
pound B was prepared according to Ref. 24. Compound C and D
were synthesized successfully according to25b and the chemical
identities of C and D were confirmed by NMR,26 MS-ESI+27 and
Infrared Spectrum.28 The labeling yield of [99mTc(CO)3+-6-OH-fla-
vone]ꢀ, [99mTc(CO)3+-7-OH-flavone]ꢀ, [99mTc(CO)2(NO)2+-6-OH-fla-
vone]0 and [99mTc(CO)2(NO)2+-7-OH-flavone]0 detected by TLC29
[
99mTc(CO)2(NO)2+-6-OH-flavone]0 and [99mTc(CO)2(NO)2+-7-OH-
flavone]0 were proved to be neutral by paper electrophoresis31
(Table 3). The retention times for compound C, compound D,
100
A
B 0.0014
90
80
70
60
50
40
30
20
10
0
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000
-10
0.02
0.04
0.06
0.08
)
0.10
300
400
500
600
700
800
1/concentrations (nM-1
Emission wavelengths (nm)
C
2600
2400
2200
2000
1800
1600
1400
1200
5
10
15
20
25
30
35
40
45
50
Concentrations (nM)
Figure 6. A represents the fluorescence intensities of 45 nM Re(CO)3+-3-OH-flavonev (black line) and the complex incubated with Ab(1–40) aggregates (red line); B represents
the relationship of the increase of fluorescence intensities and concentrations of Re(CO)3+-3-OH-flavone incubated with Ab(1–40) aggregates; C represents ‘Lineweaver-Burk
plot’.

5342
Y. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5337–5344
Table 4
450 nm, different from that of the ligand 3-OH-flavone character-
Inhibition and Dissociation constants of compounds binding to aggregates of Ab(1-40)
ized by high-energy absorption bands at 270–420 nm. It can be
*
assigned as the
p?p absorption centred mostly in the ligand
Compounds
Binding affinities
References
3-OH-flavone (spin allowed S0?1IL transition) relative to the high-
er energy bands at 270–420 nm of the UV-absorbance, and the
spectral position of the 1IL bands were significantly red-shifted in
comparison to the free ligand 3-OH-flavone due to the coordina-
tion of the heavy metal Re(I) (tricarbonyl)+ core. The donating
character of the Re(CO)3+-3-OH-flavone lead to longer wavelengths
in comparison to the ligand and demonstrated MLCT absorption
band. In fact, the S0?1MLCT bands in the UV–vis spectra of the
investigated neutral complexes can be seen only as long wave-
length tails of the S0?1IL transitions. The Re(I) (tricarbonyl)+
complexed with 3-OH-flavone and the ligand under study were
fluorescent at room temperature. The fluorescence spectra re-
corded in ethanol at room temperature were shown in Figure 5.
The emission of Re(CO)3+-3-OH-flavone was blue-shifted in respect
to fluorescence of the corresponding 3-OH-flavone by about 60 nm
(from 540 to 480 nm). Moreover, 3-OH-flavone was characterized
by strong and weak fluorescence intensity at emission wavelength
of 410 and 540 nm, respectively. All the above mentioned findings
point to the MLCT character of the room temperature fluorescence
(i.e., spin-forbidden S0 3MLCT transition), supported also by the
big ‘Stokes shifts’ (several thousand cmꢀ1) between emission and
absorption maxima (Figs. 4 and 5).
Kd (nM)
Ki (nM)
Re(CO)3+-3-OH-flavone
11.16
—
9
9
9
9
10
10
10
10
11
19
20
6
22.6
13.2
29.0
72.5
32.0
17.5
8.7
9
12
[
99mTc(CO)3+-6-OH-flavone]ꢀ, [99mTc(CO)3+-7-OH-flavone]ꢀ, [99mTc
(CO)2(NO)2+-6-OH-flavone]0 and
[
99mTc(CO)2(NO)2+-7-OH-fla-
vone]0 on UV(radio)-HPLC30b were 11.0, 11.1, 2.2, 2.6, 5.0 and
5.3 min (Fig. 3), respectively.
The spectroscopic properties of the Re(CO)3+-3-OH-flavone have
been investigated by UV–vis absorption and fluorescence emission
properties.32 UV–vis absorption and fluorescence properties were
studied on 3-OH-flavone and Re(CO)3+-3-OH-flavone. The nature
of the auxiliary ligands (i.e., the water, three carbonyl groups and
the 3-OH-flavone molecule) were shown to profoundly influence
the photophysical behavior of the studied Re(CO)3+-3-OH-flavone.
Figure 4 shows UV–vis absorption spectra of the investigated spe-
cies measured in ethanol solution. The spectra of Re(CO)3+-3-OH-
flavone was characterized by strong absorption centred at
We used the similar method to that of the article published19,21
to carry on the binding assays between Re(CO)3+-3-OH-flavone and
Ab aggregates.33 When Ab aggregates was add to the solution of Re
Figure 7. Autoradiography of brain sections from AD mice (Tg C57, APP, PS1 12-month-old) (A) and normal mice (B) labeled with [99mTc(CO)3+-3-OH-flavone]0. A showed that
the Ab plaques were clearly labeled with the 99mTc tracer with low background labeling. [99mTc(CO)2(NO)2+-6-OH-flavone]0 (C) and [99mTc(CO)2(NO)2+-7-OH-flavone]0 (D)
showed the no binding sites in AD mice brain sections.

Y. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5337–5344
5343
Table 5
Biodistribution of [99mTc(CO)3+-3-OH-flavone]0 in normal micea
Tissues
5 min
15 min
30 min
60 min
120 min
Brain
Blood
Bone
Muscle
Kidneys
Heart
Spleen
Lung
0.48 0.05
18.90 3.44
5.54 2.59
3.38 0.29
17.46 0.13
8.53 2.05
6.65 0.77
16.95 1.45
9.38 0.87
0.43 0.05
11.09 1.23
4.89 0.68
2.38 0.35
12.95 1.82
5.40 0.12
4.99 0.66
11.06 3.19
6.32 0.58
0.49 0.22
11.50 2.89
5.76 1.82
2.66 0.13
13.76 1.07
5.27 0.77
4.97 0.86
11.37 1.67
8.65 0.91
0.44 0.08
8.25 1.23
3.92 0.11
1.88 0.49
12.11 1.89
3.43 0.23
4.40 1.00
9.97 1.34
7.67 0.97
0.39 0.08
7.51 0.18
3.73 0.38
1.66 0.48
11.20 1.77
3.16 0.40
4.37 1.17
8.90 0.60
7.25 0.28
Liver
a
Expressed as % of injected dose per gram. Each value represents the mean SD for 3–4 mice at each interval.
tracer, the fluorescence intensities were increased immediately at
the same emission wavelength (Fig. 6A). There was a rather good
linear relationship (Eq. 1) between the concentrations of
Re(CO)3+-3-OH-flavone (X) and the increase of fluorescence inten-
compound which could not pass through the blood–brain barrier
uneventfully, so the Tc molecule washed out slowly from the brain.
In conclusion, we successfully designed and prepared the neu-
tral iso-structural Re/99mTc(CO)3+-3-OH-flavone and the novel
99mTc(CO)2(NO)2+-complexes. UV–vis absorption and fluorescence
properties were studied on the photophysical behaviors of the li-
gand and Re(CO)3+-3-OH-flavone. In vitro binding affinity of
Re(CO)3+-3-OH-flavone for Ab(1–40) aggregates was up to
11.16 nM by fluorescence spectroscopy, superadd the encouraging
results of autoradiography, we concluded that [99mTc(CO)3+-3-OH-
flavone]0 might be a potential Alzheimer’s disease single photon
emission tomography imaging agent. However, the structural
modification of the coordination compound should be carried on
to improve the early brain uptake.
sities (
D
fluorescence intensities, Y) when Re(CO)3+-3-OH-flavone
of different concentrations were incubated with Ab(1–40) aggre-
gates, respectively (Fig. 6B).
Y ¼ 1007:6429 þ 31:83029 ꢁ X
ð1Þ
R: 0.97167, SD: 102.42378, P <0.0001, t-Value: 10.07012, F-Value:
101.40722.
Where Y is fluorescence intensities, X is concentrations, R is lin-
ear correlation coefficient, SD is standard deviation.
+
The linear relationship between the concentrations of Re(CO)3
-
3-OH-flavone (incubated with Ab(1–40) aggregates) and fluorescence
intensity was analysed using Lineweaver-Burk plot by Microcal
Origin 6.0 (Fig. 6C), obtaining (Eq. 2) as below:
Acknowledgments
This work was financially supported by the Natural Science
Foundation of China (No. 20671013); National Basic Research Pro-
gram of China (No. 2006CB500705).
Y ¼ 3:65643E ꢀ 4 þ 0:00408 ꢁ X
R: 0.93133, SD: 4.54129E-5, P: 7.6833E-4, t-Value: 6.26437, F-value:
ð2Þ
39.24234.
Where Y is
D
fluorescence intensitiesꢀ1, X is concentrationsꢀ1, R
References and notes
is linear correlation coefficient, SD is standard deviation.
According to the (Eq. 2), Kd should be the value of ꢀXꢀ1 when the
1. Cras, P. Acta. Neurol. Belg. 1998, 98, 186.
2. Kung, H. F.; Lee, C. W.; Zhuang, Z. P.; Kung, M. P.; Hou, C.; Plossl, K. J. Am. Chem.
Soc. 2001, 123, 12740.
3. Verdile, G.; Fuller, S.; Atwood, C. S.; Laws, S. M.; Gandy, S. E.; Martins, R. N.
Pharmacol. Res. 2004, 50, 397.
4. Pike, K. E.; Savage, G.; Villemagne, V. L.; Ng, S.; Moss, S. A.; Maruff, P.; Mathis, C.
A.; Klunk, W. E.; Masters, C. L.; Rowe, C. C. Brain 2007, 130, 2837.
5. Boss, M. A. Biochim. Biophys. Acta 2000, 1502, 188.
6. Small, G. W.; Kepe, V.; Ercoli, L. M.; Siddarth, P.; Bookheimer, S. Y.; Miller, K. J.;
Lavretsky, H.; Burggren, A. C.; Cole, G. M.; Vinters, H. V.; Thompson, P. M.;
Huang, S. C.; Satyamurthy, N.; Phelps, M. E.; Barrio, J. R. N. Eng. J. Med. 2006,
355, 2652.
7. Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y. M.; Blomqvist, G.; Holt, D. P.;
Bergstrom, M.; Savitcheva, I.; Huang, G. F.; Estrada, S.; Ausen, B.; Debnath, M.
L.; Barletta, J.; Price, J. C.; Sandell, J.; Lopresti, B. J.; Wall, A.; Koivisto, P.; Antoni,
G.; Mathis, C. A.; Langstrom, B. Ann. Neurol. 2004, 55, 306.
8. Ono, M.; Wilson, A.; Nobrega, J.; Westaway, D.; Verhoeff, P.; Zhuang, Z. P.;
Kung, M. P.; Kung, H. F. Nucl. Med. Biol. 2003, 30, 565.
9. Ono, M.; Yoshida, N.; Ishibashi, K.; Haratake, M.; Arano, Y.; Mori, H.; Nakayama,
M. J. Med. Chem. 2005, 48, 7253.
10. Ono, M.; Yoshifumi, M.; Ishibashi, K.; Haratake, M.; Nakayama, M. Bioorg. Med.
Chem. 2007, 15, 444.
11. Jurisson, S. S.; Lydon, J. D. Chem. Rev. 1999, 99, 2205.
12. Dilworth, J.; Parrot, S. Chem. Soc. Rev. 1998, 27, 43.
13. Zhen, W.; Han, H.; Anguiano, M.; Lemere, C. A.; Cho, C. G.; Lansbury, P. T. J. Med.
Chem. 1999, 42, 2805.
14. Dezutter, N. A.; De Groot, T. J.; Busson, R. H.; Janssen, G. A.; Verbruggen, A. M. J.
Labelled Compd. Radiopharm. 1999, 42, 309.
15. Dezutter, N. A.; De Groot, T. J.; Bormans, G. M.; Verbruggen, A. M.; Dom, R. J.
Eur. J. Nucl. Med. Mol. Imaging 1999, 26, 1392.
16. Serdons, K.; Verduyckt, T.; Cleynhens, J.; Terwinghe, C.; Mortelmans, L.;
Bormans, G.; Verbruggen, A. Bioorg. Med. Chem. Lett. 2007, 17, 6086.
17. Yang, Y.; Zhang, J. X.; Wang, J. J.; Zhu, L. J. Radioanal. Nucl. Chem. 2007, 273, 31.
18. Klunk, W. E.; Jacob, R. F.; Mason, R. P. Anal. Biochem. 1999, 266, 66.
19. LeVine, H., III Protein Sci. 1993, 2, 404.
20. Voropai, E. S.; Samtsov, M. P.; Kaplevskii, K. N.; Maskevich, A. A.; Stepuro, V. I.;
Povarova, b. O. I.; Kuznetsova, I. M.; Turoverov, K. K.; Fink, A. L.; Uverskiid, d.;
V.N. J. Appl. Spectrosc. 2003, 70, 868.
value of Y equalled zero, Thus, Kd = 0.00408 ꢁ (3.65643Eꢀ4)ꢀ1
=
11.16 nM. The Kd demonstrated that Re(CO)3+-3-OH-flavone
showed good binding affinity for Ab(1–40) aggregates, in comparison
to radio-iodide labeled flavone derivatives9,10 (Table 4). Although
there is no clear mechanism to explain the interaction of
small molecules and the Ab(1–40) aggregates, according to the re-
search published, we speculate that when Re-tracer inserted to
the aggregates, the rotation of the benzene ring in the molecule
might be inhabited by the rigidity of the microsurrounding of the
aggregates, and this inhibition resulted in a high quantum yield of
fluorescence, thus, the increase of the fluorescence intensities
occurred.
Autoradiography showed that
[
99mTc(CO)3+-3-OH-flavone]0
could label the plaques in the AD mice (Tg C57, APP, PS1
12-month-old) brain section with low background labeling in com-
parison to that of the normal mice34a (Fig. 7A and B), oppositely, the
two neutral 99mTc(CO)2(NO)2+-complexes did not label the AD mice
brain sections34b (Fig. 7C and D). These results may be explained
that the large-volume 99mTc(CO)2(NO)2+-complexes could not
insert into the Ab(1–40) aggregates. Then, [99mTc(CO)3+-3-OH-fla-
vone]0 was examined for biodistribution in normal mice35 (Table
5).
[
99mTc(CO)3+-3-OH-flavone]0 displayed moderate uptake
(0.48 0.05%ID/g) in the brain at 5 min post-injection, and it
displayed slow clearance from the normal brain issues
(0.39 0.08%ID/g) at 120 min post-injection. We speculate that
the retention of radioactivity in the brain from 5 to 120 min was
due to the coordinative H2O molecule substituted by some other
group in the brain, and the substitution resulted in a non-neutral

5344
Y. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5337–5344
21. Yang, Y.; Zhang, J. X.; Zhu, L.; Zhang, H. Bioinorg. Chem. Appl. 2009, 702730.
22. Alberto, R.; Schibli, R.; Egli, A.; Schubiger, A. P. J. Am. Chem. Soc. 1998, 120, 7987.
23. Yang, Y.; Zhu, L.; Zhang, H. Acta Crystallogr., Sect. E 2009, 65, 3167.
24. Williams, M. A.; Rapoport, H. J. Org. Chem. 1993, 58, 1151.
Compound C: 3381.0; 3056.1; 2961.8; 2925.6; 2855.1; 1683.2; 1634.7; 1437.8;
1376.6; 1184.0; 1120.3; 1045.1; 996.9; 879.8; 746.2; 722.6; 696.4 cmꢀ1
Compound D: 3444.6; 2924.1; 2849.5; 1733.3; 1630.0; 1459.4; 1430.5;
1375.3; 1252.7; 1091.4; 846.6; 776.9; 724.4 cmꢀ1
.
.
25. (a) To a aqueous solution of the equimolar mixtures of 3-OH-flavone precursor
(15 mg, 0.0625 mmol) and KOH (3.8 mg, 0.0678 mmol), Re(CO)5Br (24 mg,
0.0591 mmol) was added. The mixture was stirred for 5 min at rt, and then
heated at 100 °C for 22 h. After cooling to rt, the water was removed under
reduced pressure, and the crude product was washed by water for three times
to remove the reaction materials, and then the title compound Re(CO)3+-3-OH-
flavone was dried under rt and obtained.
29. Thin layer chromatography was used for the qualitative analysis of every
technetium complex. Thin layer chromatography experiments were performed
on a polyamide paper (10 ꢁ 0.4 cm) using CH3CN as a developing solvent. The
polyamide strips were left to dry, and the distribution of radioactivity on the
strip was determined using a 1470 Wizard™ Automatic Gamma Counter.
30. Reversed-phase high-pressure liquid chromatography (RP-HPLC) was
performed on an Alltech system with Alltech HPLC pump Model 626, LINEAR
UVIS-201, and BIOSCAN flow-counter. The analytic C-18 column
(4.6 ꢁ 250 mm, 5 mm particle size, Venusil MP-C18, Agela Technologies Inc.,
USA). The flow rate was 1 mL/min, A: pure water; B: pure acetonitrile.
(a) The HPLC analysis gradient was: 0.01–10.00 min, 100% A; 10.01–30.00 min,
from 50%A to 100% B; 30.00–40.00 min, 100%B.
(b) Synthesis of compound
C
(2,20-(2-(4-oxo-2-phenyl-4H-chromen-6-
yloxy)ethylazanediyl)diacetic acid): 6-OH-flavone (361 mg, 1.52 mmol), KOH
(85 mg, 1.52 mmol) and B (530 mg, 1.52 mmol) were mixed in 100 mL ethanol
and stirred at room temperature for 24 h. A white solid precipitated, the
mixture was filtered and the filtrates was evaporated off under reduced
pressure, and Si-60 was used for final purification: (petroleum ether/ethyl
acetate 2:1, Rf: 0.6), the product was obtained as a light yellow solid, then this
product (300 mg, 0.59 mmol) and CF3COOH (67 mg, 0.59 mmol) were mixed in
40 mL CH2Cl2 and stirred at room temperature for at least 3 days. The mixture
was then washed by saturated NaHCO3 solution, and the organic phase was
evaporated off under reduced pressure, the crude product was obtained as a
yellow oil, Si-60 was used for final purification: (petroleum ether/ethyl acetate
2:1, Rf: 0). In addition, the synthesis routes of compound D (2,20-(2-(4-oxo-2-
phenyl-4H-chromen-7-yloxy)ethylazanediyl)diacetic acid) was the same to
that of compound C, but 7-OH-flavone was used in stead of 6-OH-flavone.
(c) [99mTc(CO)3+-3-OH-flavone]0 was obtained by heating the aqueous solution
of 3-OH-flavone (1 mg/mL) and [99mTc(CO)3(H2O)3]+ moiety (10 mCi/mL) at pH
11 and 100 °C for 40 min.
(b) The HPLC analysis gradient was: 0.01–10.00 min, from 100%A to 100% B;
10.01–20.00 min, 100%B.
31. Paper electrophoresis experiments were performed on Xinhua 1#
filterpapers(13 ꢁ 1 cm) which were pre-treated with phosphate buffer (pH
7.4). The analyses were carried out using phosphate buffer (pH 7.4) at 150 V for
3 h. The developed paper strips were left to dry; and the distribution of
radioactivity on the strip was determined using a 1470 Wizard™ Automatic
Gamma Counter.
32. Emission spectra were obtained on
spectrometer.
a Varian Cary Eclipse fluorescence
33. Ab(1–40) aggregates were prepared according to the method published
previously18 and used immediately after preparation. Fresh solution (1
M,
0.5% DMSO/H2O) of Re(CO)3+-3-OH-flavone was diluted to obtain
final
concentration range of 15–40 nM in 500 L PBS (pH 7.4), respectively. Five
L solution
l
a
(d) [99mTc(CO)3+-6-OH-flavone]ꢀ ([99mTc(CO)3+-7-OH-flavone]ꢀ) were obtained
by heating the aqueous solution of compound C/compound D (0.5 mg/mL)
and [99mTc(CO)3(H2O)3]+ moiety (12 mCi/mL) at pH 11 and 100 °C for 100
l
micro litre solution of Ab(1–40) aggregates was added to every 500
l
of Re(CO)3+-3-OH-flavone, and incubated for at least 1 min at room
temperature before measuring the fluorescence intensity (slit-width: 5 nm;
Excitation wavelength was 450 nm, while emission wavelength was 487 nm
and a 455–850 nm scan range was performed). All the data were measured and
recorded for three times.
min. Neutral
[
99mTc(CO)2(NO)2+-6-OH-flavone]0 ([99mTc(CO)2(NO)2+-7-OH-
flavone]0) were obtained by heating the aqueous solution of 99mTc(CO)3+-6-
OH-flavone (99mTc(CO)3+-7-OH-flavone) (9 mCi/mL) and NOBF4 (0.2 mg) at pH 1
and 100 °C for 15 min.
26. NMR spectra were recorded using a Bruker-400 NMR spectrometer with TMS
as an internal standard. Coupling constants are reported in hertz. Multiplicity
is defined by s (singlet), d (doublet), t (triplet), br (broad), and m (multiplet).
Re(CO)3+-3-OH-flavone: 1H NMR (CDCl3, 400 MHz), d: 8.47 (d, J = 6.8 Hz, 1H),
8.28 (m, 2H), 7.78 (d, J = 7.6 Hz, 1H), 7.71 (m, 3H), 7.53 (m, 2H), 3.51 (s, 2H,
H2O-Re).
34. Film autoradiography: Brain sections from AD mice (Tg C57, APP, PS1 12-
month-old) and normal mice were obtained by freezing the brain in powdered
dry ice and cut into 20-
(a) The AD mice brain section and normal mice brain section were incubated
with [99mTc(CO)3+-3-OH-flavone]0 (7
Ci/1 mL) for 1.5 h at room temperature,
lm thick sections.
l
respectively. The sections were then dipped in saturated Li2CO3 in 40% EtOH
(two 2 min washes) and washed with 40% EtOH (one 2 min wash) followed by
rinsing with water for 30 s. After drying, the 99mTc(CO)3+-labeled sections were
exposed to the storage phosphor screen overnight.
Compound C: 1H NMR (CDCl3, 400 MHz): 7.68 (d, J = 8.4 Hz, 1H), 7.67 (d,
J = 5.2 Hz, 1H), 7.47 (m, 5H), 7.55 (s, 1H), 6.77 (s, 1H), 4.26 (t, J = 5.6 Hz, 2H),
3.57 (s, 4H), 3.23 (t, J = 5.6 Hz, 2H). 13C NMR (CDCl3, 400 MHz): d 177.96,
170.78, 163.41, 158.02, 132.17, 132.07, 128.60, 128.47, 126.21, 117.89, 114.93,
107.53, 101.08, 81.15, 68.40, 57.17, 52.96.
(b) Two different sections from the same AD mice were incubated with
[
99mTc(CO)2(NO)2+-6-OH-flavone]0 (8 Ci/1 mL)/[99mTc(CO)2(NO)2+-7-OH-fla-
l
Compound D: 1H NMR (CDCl3, 400 MHz): 7.90 (d, J = 4.4 Hz, 2H), 7.67 (dd,
J = 4.4 Hz and 5.2 Hz, 2H), 7.64 (d, J = 5.6 Hz, 1H), 7.52 (m, 2H), 7.46 (s, 1H), 7.45
(s, 1H), 6.81 (s, 1H), 4.23 (t, J = 5.2 Hz, 2H), 3.59 (s, 4H), 3.23 (t, J = 5.2 Hz, 2H).
13C NMR (CDCl3, 400 MHz): d 178.22, 170.65, 163.16, 156.23, 133.84, 133.64,
132.16, 128.70, 128.51, 128.45, 126.26, 123.88, 106.88, 81.10, 68.20, 56.91,
53.07.
vone]0 (9
lCi/1 mL) for 1 h at room temperature, respectively. The sections
were then dipped in saturated Li2CO3 in 40% EtOH (two 2 min washes) and
washed with 40% EtOH (one 2 min wash) followed by rinsing with water for
30 s. After drying, the two sections were exposed to the storage phosphor
screen overnight.
35. Biodistribution in normal mice: All animal biodistribution studies were carried
out in compliance with the national laws related to the conduct of animal
experimentation with our institutional guidelines and were approved by the
27. Mass spectra were obtained on Waters Micromass Quattro Micro TM tandem
quadrupole mass spectrometer (Micromass Manchester, UK) & LCT Premier XE
time-of-flight mass spectrometer (Micromass, Manchester, UK).
Animal Center of Peking University. A saline solution (100
lL) containing
Re(CO)3+-3-OH-flavone: MS-ESI+
[M+Na, 187Re]+.
(
187/185Re): m/z 550.9 [M+Na, 185Re]+, 552.8
radiolabeled agents (20 Ci) was injected directly into the tail vein of normal
l
Kunming mice (18–20 g, 3–4 animals per group). The mice were sacrificed at 5,
15, 30, 60 and 120 min post-injection and the tissues and organs of interest
(blood, heart, liver, spleen, lung, kidney, muscle, bone, brain) were collected,
wet weighed and counted in a 1470 Wizard™ Automatic Gamma Counter. The
percentage of injected dose per gram (%ID/g) for each sample was calculated by
comparing its activity with that of an appropriate aliquot of the injection
solution, the values are expressed as mean 1 standard deviation (SD).
Compound C: m/z 398.1 [M+H]+.
Compound D: m/z 788.6 [2 M+H]+, 398.0 [M+H]+.
28. Infrared spectra were measured on a Nicolet 360 Avatar instrument using a
potassium bromide (KBr) disk, scanning from 400 to 4000 cmꢀ1
.
Re(CO)3+-3-OH-flavone: 3448.2; 2014.1; 1884.6; 1845.6; 1593.4; 1538.6;
1492.8; 1421.7; 1206.0; 1158.1; 752.9; 684.6; 663.7 cmꢀ1
.