Novel (E)-5-styryl-2,2′-bithiophene derivatives as ligands for β-amyloid plaques

Article abstract of DOI:10.1016/j.ejmech.2011.04.015

In continuation of our investigation on the bithiophene structure as potential β-amyloid probes, a series of (E)-5-styryl-2,2′-bithiophene (SBTP) derivatives was designed and synthesized. In vitro binding showed that all of them displayed high binding affinities to Aβ_1-42 aggregates (K_i = 0.10-41.05 nM). Moreover, two radio-iodinated probes, [¹²⁵I]-(E)-5-(4-iodostyryl)-2,2′-bithiophene ([ ¹²⁵I]8) and [¹²⁵I]-(E)-5-iodo-5′-(4-methoxystyryl)- 2,2′-bithiophene ([¹²⁵I]31) were prepared. Both of them displayed specific labeling of Aβ plaques in the brain sections of AD model mice with low background. In vivo biodistribution in normal mice indicated that [¹²⁵I]8 exhibited high initial brain uptake (2.11% ID/g at 2 min) and rapid clearance (0.41% ID/g at 30 min). These preliminary results suggest that SBTP derivatives may be served as novel β-amyloid imaging probes.

Full text of DOI:10.1016/j.ejmech.2011.04.015

European Journal of Medicinal Chemistry 46 (2011) 2908e2916
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel (E)-5-styryl-2,20-bithiophene derivatives as ligands for
b-amyloid plaques
*
*
Mengchao Cui, Zijing Li, Ruikun Tang, Hongmei Jia , Boli Liu
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Xinjiekouwai Street 19#, Beijing 100875, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In continuation of our investigation on the bithiophene structure as potential b-amyloid probes, a series
of (E)-5-styryl-2,20-bithiophene (SBTP) derivatives was designed and synthesized. In vitro binding
showed that all of them displayed high binding affinities to Ab1e42 aggregates (Ki ¼ 0.10e41.05 nM).
Moreover, two radio-iodinated probes, [125I]-(E)-5-(4-iodostyryl)-2,20-bithiophene ([125I]8) and [125I]-
(E)-5-iodo-50-(4-methoxystyryl)-2,20-bithiophene ([125I]31) were prepared. Both of them displayed
Received 17 November 2010
Received in revised form
24 February 2011
Accepted 4 April 2011
Available online 12 April 2011
specific labeling of Ab plaques in the brain sections of AD model mice with low background. In vivo
biodistribution in normal mice indicated that [125I]8 exhibited high initial brain uptake (2.11% ID/g at
Keywords:
Alzheimer’s disease
2 min) and rapid clearance (0.41% ID/g at 30 min). These preliminary results suggest that SBTP deriva-
tives may be served as novel
b
-amyloid imaging probes.
Ó 2011 Elsevier Masson SAS. All rights reserved.
b-amyloid plaques
Imaging agent
Bithiophene
Binding assay
1. Introduction
ethoxy)-stilbene ([18F]BAY94-9172) [13] and [18F]-(E)-4-(2-(6-(2-(2-
(2-fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-N-methylaniline
([18F]AV-45) [14,15] have been evaluated in human studies. Some of
the candidates have already moved into Phase II, Phase III and even
finalization stages (such as Florbetapir, [18F]AV-45, is waiting for the
approval from FDA). On the other hand, to the best of our knowledge,
the only SPECT tracer tested in human studies is [123I]-6-iodo-2-(40-
dimethylamino-)phenyl-imidazo[1,2]pyridine ([123I]IMPY) [16e18].
However, some undesirable properties including high lipophilicity,
in vivo instability together with insufficient target-to-background
ratio may account for the failure of [123I]IMPY in clinical trials. After
Alzheimer’s disease (AD) is an irreversible neurodegenerative
disorder characterized by progressive decline in cognitive function of
brain and behavior. Histopathologically, b-amyloid plaques (Ab) and
neurofibrillary tangles (NFTs) are found in the brain of patients
suffering from AD [1]. Although the etiology of AD is not completely
understood, the most widelyaccepted theoryconcerning the etiology
of AD is the amyloid cascade hypothesis [2,3]. The clinical diagnosis of
this disease is made through the neurological observations and
history of patients, which is often difficult and unreliable. To facilitate
the early diagnosis of AD, functional imaging techniques such as
positron emission tomography (PET) and single photon emission
computed tomography (SPECT) have been employed. For this
that, there is no report of any Ab imaging candidate for SPECT moving
into clinical trial. For the routine clinical applications, imaging with
SPECT has some advantages over PET such as more widespread
availability, no need for an on-site cyclotron and lower cost. From this
purpose, a radionuclide-labeled probe that specifically binds to A
b
plaques in the brain may greatly facilitate the diagnosis of AD [4,5].
During the past decade, several attempts have been made to
point of view, there is an urgent need for developing A
agents for SPECT. In the past few years, lots of the research related to
imaging agents for SPECT have been reported. For example, Qu
et al. reported radio-iodinated aza-diphenylacetyllenes [19] and
styrylpyridines [20] as SPECT imaging agents for A plaque detection.
Ono et al. designed and synthesized radio-iodinated aurone deriva-
tives [21,22], chalcones and their related derivatives [23,24], as well
as 99mTc labeled flavonoids including chalcone [25], flavone and
b imaging
develop specific radiotracers for in vivo imaging A
b
plaques with PET
Ab
and SPECT. Most of the reported probes are based on the core
structure of Congo Red (CR), Thioflavin T (ThT) and DDNP. Several PET
tracers such as 2-(40-[11C]methylaminophenyl)-6-hydroxybenzoth-
b
iazole ([11C]PIB) [6,7],
[
18F]-2-(1-(2-(N-(2-fluoroethyl)-N-methyl-
amino)-naphthalene-6-yl)ethylidene)malononitrile
([18F]FDDNP)
([11C]SB-13)
[8e10],
[11,12],
4-N-[11C]methylamino-40-hydroxystilbene
aurone [26] as probes for SPECT imaging of A
In a search on novel A binding probes, Nesterov et al. first
proposed, synthesized and evaluated bithiophene type molecules
as fluorescent imaging markers for A plaques [27]. A bithiophene
molecule, NIAD-4 had been screened out as a simple fluorescent
b plaques.
[
18F]-4-(N-methylamino)-40-(2-(2-(2-fluoroethoxy)ethoxy)
b
b
* Corresponding authors. Tel./fax: þ86 10 58808891.
E-mail addresses: hmjia@bnu.edu.cn (H. Jia), liuboli@bnu.edu.cn (B. Liu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.015

M. Cui et al. / European Journal of Medicinal Chemistry 46 (2011) 2908e2916
2909
marker for optical imaging of amyloid plaques in brain (Fig. 1).
Later, Nilsson et al. identified that a class of luminescent conjugated
substituents [e.g., NO2 (9, 20)] and electron-donating substituents
[e.g., OCH3 (12, 23), N(CH3)2 (11, 21)] showed high affinities to A
b
polythiophenes (LCPs) with a striking specificity for A
shown in Fig. 1 [28e30]. In our previous work, a series of benzo-
thiazole derivatives based on the bithiophene structure (TZBP) was
b
plaques as
aggregates, especially compound 20 with a nitro group displayed the
highest affinity (Ki ¼ 0.10 nM).
In addition, the high affinities of compounds 14, 25 and 33
containing a bulk tert-butyl group indicated high tolerance for
steric bulk on the para position of phenyl ring. A long polyethylene
glycol chain, which is used to improve the pharmacokinetic prop-
erties of 18F labeled radiotracers [32], may be introduced at this
position to design new SBTP PET probes. More importantly, it is
evaluated for the imaging of A
played excellent binding affinity to A
b
plaques. These derivatives dis-
aggregates [31]. Inspired by
b
these successful results we decided to perform extensive research
on the ligands with bithiophene structure, which may play an
important role in maintaining the specific binding ability to A
b
plaques. Herein, we report a series of bithiophene derivatives
possible to design 99mTc labeled SBTP SPECT probes for A
b imaging.
combined with a vinylbenzene structure [(E)-5-styryl-2,20-bithio-
phene (SBTP)] as novel Ab imaging agents.
2.3. Radiochemistry
Due to the high binding affinities observed for iodinated ligands 8
(Ki ¼ 0.92 nM) and 31 (Ki ¼ 0.30 nM), these two ligands were chosen
for radiolabeling and further biological evaluations. The novel radio-
iodinated ligands [125I]8 and [125I]31 were prepared via an iododes-
tannylation reaction using hydrogen peroxide as an oxidant. The
reactionwasquenched withsaturatedNaHSO3. The resultingmixture
was purified by HPLC. The radioactive product was co-injected and
co-eluted with the corresponding nonradioactive compound. The
radio-iodinatedproductswereobtainedin39.8e46.3%radiochemical
yields with radiochemical purities of >98% after purification by HPLC.
It is anticipated that the specific activity of the no-carrier-added
preparation was comparable to that of [125I]NaI (2200 Ci/mmol).
Under the experimental conditions, the log D values of [125I]8 and
2. Results and discussion
2.1. Chemistry
The synthetic route of the SBTPderivativesisoutlinedin Scheme 1.
The key step was the base-catalyzed Wittig reaction between
substituted triphenyl phosphonium ylide and 50-substituted-2,
20-bithiophene-5-carbaldehydes. The substituted triphenyl phos-
phonium ylides reacted with 50-substituted-2,20-bithiophene-5-car-
baldehydes inthe presence ofCH3ONa under refluxinTHF to form the
target SBTP derivatives. The chemical yields of compounds 4e33
were ranged from 14% to 90%. Reduction of the nitro group of 9
provided the amino-substituted compound 10. The synthetic route of
the tributyltin precursors (34, 35) is shown in Scheme 2. The yields of
34 and 35 from the corresponding bromo compounds (7, 23) under
a bromo to tributyltin exchange reaction catalyzed by Pd(PPh3)4 were
21.7% and 39.3%, respectively. The tributyltin derivatives can be
readily used as starting materials to prepare the radio-iodinated
ligands.
[
125I]31 were 3.31 ꢀ0.10 and 3.07 ꢀ 0.08, respectively (measured by
a partition between 1-octanol and pH 7.4 phosphate buffer), which is
desirable for blood-brain barrier (BBB) penetration.
2.4. Neuropathological staining on brain sections of double
transgenic model mice
To confirm the specific binding of these SBTP derivatives to A
b
2.2. Binding studies using Ab aggregates in solution
plaques, in vitro neuropathological staining was performed on the
brain sections of a transgenic model mouse (C57, APP/PS1, 12
months). As shown in Fig. 2, compounds 11, 14, 20 and 21 distinc-
The affinity of these SBTP derivatives (4e33) for Ab1e42 aggre-
gates was determined by competition binding assay using [125I]
TZDM as radio-ligand. The Ki values shown in Table 1 suggest that all
of these new compounds inhibit the binding of [125I]TZDM in a dose-
dependent manner with a high binding affinity for Ab1e42 aggre-
gates (Ki < 10 nM) except for compound 10 (Ki ¼ 41.05 nM). The
affinity was increased by bromination or iodination of the 2 position
of bithiophene moiety (e.g., 4e5, 15e16, 26e27). Bromination or
iodination of the para position of phenyl ring also increased the
binding affinity. The tertiary N,N-dimethylamino analogs of 11 and
21 were found to have a higher affinity than the primary amino
analog 10, which is in accord with the previously limited data on
tertiary and primary amino analogs [5,23]. Methylation of the
hydroxy group improved the binding affinity [e.g., OH (22) versus
OCH3 (23)]. The ligands with both electron-withdrawing
tively stained A
(Fig. 2AeD). The similar pattern of A
b
plaques on the brain sections with low background
plaques was consistent with
b
that stained with thioflavin-S using the adjacent sections
(Fig. 2EeH). The results from neuropathological staining indicate
that the binding of these SBTP derivatives is specific for Ab plaques.
2.5. Autoradiography in vitro using brain sections of double
transgenic model mice
Next, the radio-iodinated probes [125I]8 and [125I]31 were inves-
tigated for their binding to A plaques by in vitro autoradiography in
b
the brain sections of a transgenic model mouse (C57, APP/PS1, 12
months). As shown in Fig. 3 autoradiographic images of [125I]8 and
Fig. 1. (A) Structures of [11C]PIB, [11C]SB-13, LCPs (tPTT) and TZBP; (B) Structure of the designed SBTP derivatives.

2910
M. Cui et al. / European Journal of Medicinal Chemistry 46 (2011) 2908e2916
Scheme 1. Synthesis of the SBTP derivatives (4e33). Reagents and conditions: (a) POCl3, DMF, 60 ꢁC; (b) AcOH, Br2, room temp; (2a) HgO, I2, toluene, room temp (2b); (c) PPh3,
toluene, reflux; (d) THF, CH3ONa, reflux; (e) SnCl2$2H2O, EtOH, HCl, reflux.
[
125I]31 showed excellent labeling of A
b
plaques in the cortex region
washout from the normal mouse brain [31]. Compared to [125I]TZBP-
PI, [125I]8 also possesses favorable invivo pharmacokinetics in normal
mice. It is worth mentioning that the introduction of a bithiophene
moiety and iodine atom into one molecule will largely increase the
lipophilicity of the molecule, which is detrimental to the in vivo bio-
ofthebrainsections, andnoremarkableaccumulationofradioactivity
was observed in the white matter (Fig. 3A and C). The same sections
were alsostainedwiththioflavin-SandthelocalizationsofAb plaques
were in accord with the results of autoradiography (Fig. 3B and D, red
arrows). These results demonstrate that [125I]8 and [125I]31 were
logical activities of this Ab imaging probe. Thus, further refinement in
specificforA
b
plaques,whichwasconsistentwithhighbindaffinityof
order to reduce the lipophilicity of this probe is needed.
compounds 8 and 31 to Ab1e42 aggregates.
3. Conclusion
2.6. Biodistribution experiments with normal mice in vivo
A new series of novel SBTP derivatives, was successfully designed
and synthesized asligands for A
b plaques. They displayed high invitro
To evaluate pharmacokinetic properties of the SBTP derivatives,
in vivo biodistribution experiments were performed in normal mice
with two radio-iodinated probes, [125I]8 and [125I]31. As shown in
Table 2, probe [125I]8 with a radio-iodine atom on the phenyl ring
displayed good BBB penetration with the initial brain uptake
binding affinities for A aggregates. The para position of the phenyl
b
ringwasfoundtohavehightoleranceforstericbulksubstituents. Two
radio-labeled probes [125I]8 and [125I]31 showed specific labeling of
Ab
plaques in the brain sections of ADmodel mice. Furthermore, [125I]
8 displayed favorable in vivo pharmacokinetics in normal mice. The
obtained results suggest that the novel SBTP derivatives may serve as
potential candidates for diagnosis of AD.
2.11 ꢀ0.23% ID/g at 2 min post-injection. Since there are no A
b
plaques in normal mice, the brain radioactivity concentration
decreased rapidly with 0.41 ꢀ0.06% ID/g at 30 min. However, the
radio-iodinated probe [125I]31 with a radio-iodine atom on the
thiophene ring displayed lower initial brain uptake (0.50 ꢀ 0.06%
ID/g at 2 min) and a relatively slow washout rate from the brain
with 0.39 ꢀ 0.09% ID/g at 30 min, suggesting the high non-specific
binding of this probe. In addition, compared with the brain uptake,
the initial blood uptake of the two probes was higher (12.1% ID/g for
4. Experimental
4.1. General information
All chemicals used in synthesis were commercial products and
[
125I]8 and 11.2% ID/g for [125I]31), which may due to the high lip-
were used without further purification unless otherwise indicated.
ophilicity (log D ¼ 3.31 and 3.07, respectively).
[
125I]NaI (2200 Ci/mmol) was obtained from PerkinElmer Life and
Previously, we reported an iodinated benzothiazole derivative
based on the bithiophene structure, [125I]-2-(2,20-bithiophen-5-yl)-
Analytical Sciences, USA. The double transgenic (APP/PS1) mouse
model was obtained from Institute of Laboratory Animal Science,
Chinese Academy of Medical Sciences and Comparative Medicine
Center of Peking Union Medical College, China.1H-NMR spectra were
6-iodobenzo[d]thiazole ([125I]TZBP-PI) as a potential A
b
imaging
agent. [125I]TZBP-PI showed high initial brain uptake and rapid
Scheme 2. Radiolabeling of ligands 8 and 31. Reagents and conditions: (a) (Bu3Sn)2, (PPh3)4Pd, toluene; (b) [125I]NaI, HCl (1 M), H2O2 (3%).

M. Cui et al. / European Journal of Medicinal Chemistry 46 (2011) 2908e2916
2911
Table 1
analysis was performed on a Shimadzu SCL-10 AVP equipped with
a Packard 500 TR series flow scintillation analyzer, conditions: Agi-
Inhibition constants (Ki, nM) for binding to aggregates of Ab1e42 versus [125I]TZDM.a
lent TC-C18 reverse-phase analytical column (5
m
m, ID ¼ 4.6 mm,
Compound
R1
R2
Ki (nM)
length ¼ 150 mm), 90/10 CH3CN/H2O, 1.0 mL/min, UV 254 nm.
Separations were achieved on a Alltech HPLC pump Model 626
equipped with LINEAR UVIS-201 and BIOSCAN flow-counter,
4
5
6
7
8
9
H
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
I
H
F
Cl
Br
4.78 ꢀ 1.01
4.06 ꢀ 0.32
0.74 ꢀ 0.12
0.99 ꢀ 0.24
0.92 ꢀ 0.08
0.56 ꢀ 0.13
41.05 ꢀ 3.65
0.73 ꢀ 0.27
1.48 ꢀ 0.19
1.15 ꢀ 0.34
0.73 ꢀ 0.05
1.32 ꢀ 0.06
0.81 ꢀ 0.16
1.02 ꢀ 0.34
0.22 ꢀ 0.03
0.51 ꢀ 0.11
0.10 ꢀ 0.02
0.36 ꢀ 0.08
3.60 ꢀ 0.79
0.72 ꢀ 0.31
0.45 ꢀ 0.22
0.95 ꢀ 0.17
0.24 ꢀ 0.09
0.55 ꢀ 0.19
1.10 ꢀ 0.07
0.19 ꢀ 0.02
0.63 ꢀ 0.28
0.30 ꢀ 0.05
0.80 ꢀ 0.03
1.58 ꢀ 0.24
conditions: Alltech C18 reverse-phase column (5
m
m, ID ¼ 4.6 mm,
I
length ¼ 250 mm) eluted with acetonitrile at a 2.0 mL/min flow rate.
All key compounds were proven by analytical HPLC analysis to show
ꢂ95% purity (Supporting information).
NO2
NH2
N(CH3)2
OCH3
CH3
t-Bu
H
F
Cl
Br
I
NO2
N(CH3)2
OH
OCH3
CH3
t-Bu
H
F
Cl
Br
I
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
4.2. 2,20-bithiophene-5-carbaldehyde (1)
Compound 1 was synthesized according to the literature with
some modification [33], POCl3 (2.30 g, 15.0 mmol) was added to
DMF (1.10 g, 15.0 mmol) at 0 ꢁC and the mixture was stirred for
15 min at 0 ꢁC. Then 2,20-bithiophene (2.00 g, 12.0 mmol) dissolved
in 1,2-dichloroethane (40 mL) was added drop-wise with stirring.
The reaction mixture was then heated for 2 h at 60 ꢁC. The organic
layer was washed with saturated NaHCO3 aqueous solution, and
dried with anhydrous Na2SO4. After the solvent was removed, the
residue was purified by column chromatography (petroleum ether/
AcOEt, 8/1) to give 1.47 g of 3 as a yellow solid (yield 63.1%).
I
I
I
I
I
I
I
4.3. 50-bromo-2,20-bithiophene-5-carbaldehyde (2a)
OCH3
CH3
t-Bu
To
a
stirring solution of 2,20-bithiophene-5-carbaldehyde
(1.94 g, 10.00 mmol) in CHCl3 (20 mL) at 0 ꢁC, was added bromine
(1.60 g, 10.00 mmol) dissolved in CHCl3 (20 mL). The mixture was
stirred at room temperature for 2 h. The organic layer was washed
with saturated NaHCO3 aqueous solution, and dried with anhy-
drous Na2SO4. Evaporation of the solvent under reduced pressure
gave 4 as a yellow solid (yield 86.3%). 1H NMR (400 MHz, CDCl3)
a
Measured in triplicate with results given as the mean ꢀ SD.
obtained at 400 MHz on Bruker NMR spectrometers in CDCl3 solu-
tion at room temperature with TMS as an internal standard.
Chemical shifts are reported as
d values relative to internal TMS.
Coupling constants are reported in Hertz. The multiplicity is defined
by s (singlet), d (doublet), t (triplet), and m (multiplet). Mass spectra
were acquired under Survey or MSQ Plus (ESI) instrument. HPLC
d
9.79 (s, 1H), 7.59 (d, J ¼ 4.0 Hz, 1H), 7.11 (d, J ¼ 3.9 Hz, 1H), 7.03
(d, J ¼ 3.8 Hz, 1H), 6.96 (d, J ¼ 3.8 Hz, 1H). MS(ESI): m/z calcd for
C9H5BrOS2 271.90; found 272.9 [M þ H]þ.
Fig. 2. Fluorescence staining of compounds 11, 14, 20 and 21 (A, B, C and D) on the sections from a transgenic model mouse (C57, APP/PS1, 10
plaques were confirmed by staining of the adjacent sections using thioflavin-S (E, F, G and H) with the filter set of GFP.
mm thick) with the filter set of GFP. The

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M. Cui et al. / European Journal of Medicinal Chemistry 46 (2011) 2908e2916
Fig. 3. In vitro labeling of brain sections from transgenic model mouse (C57, APP/PS1, 10
brain (A, C), while the white matter was clearly void of any notable labeling. The same sections were also stained with thioflavin-S (B, D) and the localizations of A
m
m thick) by autoradiography. [125I]8 and [125I]31 labeled the A
b
plaques in the cortex of the
plaques were in
b
accordance with the results of autoradiography (red arrows).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)
4.4. 50-iodo-2,20-bithiophene-5-carbaldehyde (2b)
solution of 2,20-bithiophene-5-carbaldehyde (1.94 g,
CDCl3)
d
9.87 (s, 1H), 7.67 (d, J ¼ 3.8 Hz, 1H), 7.23 (d, J ¼ 3.7 Hz, 1H),
7.19 (d, J ¼ 3.8 Hz, 1H), 7.02 (d, J ¼ 3.6 Hz, 1H). MS(ESI): m/z calcd for
To
a
C9H5IOS2 319.88; found 320.8 [M þ H]þ.
10.00 mmol) in toluene (20 mL) at 0 ꢁC, was added small portions of
mercury(II) oxide (2.23 g, 10.29 mmol, yellow powder) and iodine
(2.87 g, 11.32 mmol). The mixture was stirred at room temperature
for 8 h. The orange precipitate was filtered off and washed with ethyl
acetate. The filtrate and washings were combined and washed with
aqueous sodium thiosulfate and dried over magnesium sulfate.
Solvent was removed by rotary evaporation and the solid residue
was recrystallized from ethanol (2.37 g, 72%). 1H NMR (400 MHz,
4.5. General procedure for the synthesis of 4-substituted-
benzyltriphenyl phosphonium salts (3a-3k)
Triphenylphosphine (5 mmol) was added to a solution of
4-substituted-benzyl halide (5 mmol) in toluene (50 mL). The
mixture was heated to reflux for 6 h and then cooled to room
temperature. The precipitate was collected, recrystallized from
ethanol to give the products.
Table 2
4.5.1. Benzyltriphenylphosphonium bromide (3a)
Biodistribution of [125I]8 and [125I]31 in normal mice.a
White solid, yield 92%. 1H NMR (400 MHz, CDCl3)
d 7.86e7.47
[
125I]8 (log D ¼ 3.31 ꢀ 0.10)
(m, 15H), 7.20 (ddd, J ¼ 8.6, 4.0, 1.9 Hz, 1H), 7.15e7.03 (m, 4H), 5.35
(d, J ¼ 14.4 Hz, 2H).
Organ
2 min
15 min
30 min
60 min
120 min
Blood
Brain
Heart
Liver
Spleen
Lung
12.10 ꢀ 2.75 1.38 ꢀ 0.22 1.16 ꢀ 0.17 1.29 ꢀ 0.13 1.76 ꢀ 0.56
2.11 ꢀ 0.23 0.42 ꢀ 0.02 0.41 ꢀ 0.06 0.47 ꢀ 0.03 0.55 ꢀ 0.06
5.53 ꢀ 2.23 3.37 ꢀ 0.39 3.35 ꢀ 0.35 2.09 ꢀ 0.33 3.39 ꢀ 1.39
47.12 ꢀ 8.11 40.18 ꢀ 8.13 42.17 ꢀ 7.14 47.70 ꢀ 5.22 45.74 ꢀ 3.42
11.46 ꢀ 1.81 12.61 ꢀ 1.81 14.82 ꢀ 3.66 14.58 ꢀ 2.76 21.98 ꢀ 5.32
20.89 ꢀ 0.97 18.99 ꢀ 0.97 15.69 ꢀ 6.02 12.99 ꢀ 4.75 6.29 ꢀ 0.56
4.69 ꢀ 0.92 4.37 ꢀ 0.92 4.56 ꢀ 1.16 4.91 ꢀ 0.81 6.35 ꢀ 1.27
4.5.2. (4-fluorobenzyl)triphenylphosphonium bromide (3b)
White solid, yield 89%. 1H NMR (400 MHz, CDCl3)
d 7.84e7.52
(m, 15H), 7.22e7.07 (m, 2H), 6.78 (t, J ¼ 8.6 Hz, 2H), 5.53
(d, J ¼ 14.3 Hz, 2H).
Kidney
Stomachb 0.61 ꢀ 0.16 1.48 ꢀ 0.46 3.25 ꢀ 1.26 4.28 ꢀ 1.94 3.36 ꢀ 1.56
4.5.3. (4-chlorobenzyl)triphenylphosphonium chloride (3c)
White solid, yield 84%. 1H NMR (400 MHz, CDCl3)
d 7.91e7.48
Muscle
0.85 ꢀ 0.07 0.97 ꢀ 0.07 1.25 ꢀ 0.45 0.93 ꢀ 0.16 0.76 ꢀ 0.12
(m, 15H), 7.13 (d, J ¼ 6.7 Hz, 2H), 7.04 (d, J ¼ 8.2 Hz, 2H), 5.72 (d,
[
125I]31 (log D ¼ 3.07 ꢀ 0.08)
J ¼ 14.9 Hz, 2H).
Organ
Blood
Brain
Heart
Liver
Spleen
Lung
Kidney
2 min
15 min
30 min
60 min
120 min
11.21 ꢀ 0.66 7.99 ꢀ 1.68 6.18 ꢀ 0.60 6.70 ꢀ 0.91 6.85 ꢀ 0.57
0.50 ꢀ 0.06 0.44 ꢀ 0.08 0.39 ꢀ 0.09 0.50 ꢀ 0.06 0.59 ꢀ 0.07
5.08 ꢀ 0.77 6.25 ꢀ 1.70 5.55 ꢀ 0.68 5.33 ꢀ 0.61 4.18 ꢀ 0.52
45.73 ꢀ 3.78 46.86 ꢀ 4.61 26.91 ꢀ 3.27 15.74 ꢀ 2.06 11.00 ꢀ 2.41
9.14 ꢀ 2.11 11.20 ꢀ 3.29 9.40 ꢀ 1.02 6.38 ꢀ 0.84 7.21 ꢀ 4.43
26.17 ꢀ 3.11 16.37 ꢀ 3.42 12.95 ꢀ 1.73 10.45 ꢀ 0.75 7.08 ꢀ 2.90
4.82 ꢀ 0.45 6.61 ꢀ 1.43 5.92 ꢀ 0.86 6.42 ꢀ 0.98 6.33 ꢀ 0.59
4.5.4. (4-bromobenzyl)triphenylphosphonium bromide (3d)
White solid, yield 94%. 1H NMR (400 MHz, CDCl3)
d 7.88e7.43
(m,15H), 7.17 (d, J ¼ 7.8 Hz, 2H), 7.06 (dd, J ¼ 8.5, 2.6 Hz, 2H), 5.57 (d,
J ¼ 14.8 Hz, 2H).
Stomachb 0.39 ꢀ 0.18 2.60 ꢀ 0.51 5.11 ꢀ 2.17 8.90 ꢀ 3.10 20.22 ꢀ 5.59
4.5.5. (4-iodobenzyl)triphenylphosphonium bromide (3e)
White solid, yield 82%. 1H NMR (400 MHz, CDCl3)
d 7.74e7.53
Muscle
1.61 ꢀ 0.65 2.23 ꢀ 0.86 2.12 ꢀ 0.39 3.95 ꢀ 1.13 2.83 ꢀ 0.69
a
(m, 15H), 7.35 (d, J ¼ 7.6 Hz, 2H), 6.86 (dd, J ¼ 8.4, 2.5 Hz, 2H), 5.48
Expressed as % injected dose per gram. Average of 5 mice ꢀ standard deviation.
b
Expressed as % injected dose per organ.
(d, J ¼ 14.8 Hz, 2H).

M. Cui et al. / European Journal of Medicinal Chemistry 46 (2011) 2908e2916
2913
4.5.6. (4-nitrobenzyl)triphenylphosphonium chloride (3f)
Brown solid, yield 81%. 1H NMR (400 MHz, CDCl3)
4.6.4. (E)-5-(4-bromostyryl)-2,20-bithiophene (7)
Yellow solid, yield 83%. 1H NMR (400 MHz, CDCl3)
d
7.92e7.79
d
7.40 (d,
(m, 9H), 7.75 (dd, J ¼ 8.2, 6.7 Hz, 2H), 7.60 (td, J ¼ 7.8, 3.5 Hz, 6H),
J ¼ 8.5 Hz, 2H), 7.26 (d, J ¼ 8.5 Hz, 2H), 7.16 (dd, J ¼ 5.1, 1.1 Hz, 1H),
7.13 (dd, J ¼ 3.6, 1.1 Hz, 1H), 7.10 (d, J ¼ 16.1 Hz, 1H), 7.01 (d,
J ¼ 3.7 Hz, 1H), 6.96 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.91 (d, J ¼ 3.7 Hz, 1H),
6.74 (d, J ¼ 16.1 Hz, 1H). MS(EI): m/z calcd for C16H11BrS2 345.95;
found 346.14. m.p. 151.3e152.2 ꢁC.
7.46 (dd, J ¼ 8.7, 2.4 Hz, 2H), 6.09 (d, J ¼ 15.9 Hz, 2H).
4.5.7. (4-(dimethylamino)benzyl)triphenylphosphonium bromide
(3g)
White solid, yield 27%. 1H NMR (400 MHz, CDCl3)
d 7.92e7.49
(m, 15H), 6.90 (d, J ¼ 6.9 Hz, 2H), 6.54 (s, 2H), 5.14 (d, J ¼ 13.2 Hz,
4.6.5. (E)-5-(4-iodostyryl)-2,20-bithiophene (8)
2H), 2.89 (s, 6H).
Yellow solid, yield 60%. 1H NMR (400 MHz, CDCl3)
d 7.67 (d,
J ¼ 8.4 Hz, 2H), 7.24 (dd, J ¼ 5.1, 1.1 Hz, 1H), 7.21 (d, J ¼ 8.6 Hz, 2H),
7.20 (d, J ¼ 3.6 Hz, 1H), 7.18 (d, J ¼ 16.2 Hz, 1H), 7.08 (d, J ¼ 3.7 Hz,
1H), 7.04 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.98 (d, J ¼ 3.8 Hz, 1H), 6.79 (d,
J ¼ 16.1 Hz, 1H). MS(EI): m/z calcd for C16H11IS2 393.93; found
394.12. m.p. 160.7e161.3 ꢁC.
4.5.8. (4-hydroxybenzyl)triphenylphosphonium bromide (3h)
White solid, yield 18%. 1H NMR (400 MHz, DMSO)
d 7.96e7.60
(m, 15H), 6.74 (d, J ¼ 6.6 Hz, 2H), 6.58 (d, J ¼ 8.2 Hz, 2H), 5.00 (d,
J ¼ 14.8 Hz, 2H).
4.5.9. (4-methoxybenzyl)triphenylphosphonium bromide (3i)
4.6.6. (E)-5-(4-nitrostyryl)-2,20-bithiophene (9)
White solid, yield 88%. 1H NMR (400 MHz, CDCl3)
d
7.86e7.56
Dark red solid, yield 51%. 1H NMR (400 MHz, CDCl3)
d 8.22 (d,
(m, 15H), 7.02 (dd, J ¼ 8.8, 2.6 Hz, 2H), 6.65 (d, J ¼ 8.3 Hz, 2H), 5.33
J ¼ 8.5 Hz, 2H), 7.58 (d, J ¼ 8.5 Hz, 2H), 7.35 (d, J ¼ 16.0 Hz, 1H), 7.27
(d, J ¼ 4.5 Hz,1H), 7.24 (d, J ¼ 3.5 Hz,1H), 7.12 (d, J ¼ 3.6 Hz,1H), 7.08
(d, J ¼ 3.8 Hz, 1H), 7.06 (t, J ¼ 4.3 Hz, 1H), 6.90 (d, J ¼ 16.1 Hz, 1H).
MS(ESI): m/z calcd for C16H11NO2S2 313.02; found 313.9 (M þ Hþ).
m.p. 169.2e170.9 ꢁC.
(d, J ¼ 13.7 Hz, 2H), 3.72 (s, 3H).
4.5.10. (4-methylbenzyl)triphenylphosphonium chloride (3j)
White solid, yield 86%. 1H NMR (400 MHz, CDCl3)
d 7.88e7.48
(m, 15H), 6.94 (d, J ¼ 8.1 Hz, 2H), 6.90 (d, J ¼ 7.9 Hz, 2H), 5.37 (d,
J ¼ 14.2 Hz, 2H), 2.23 (s, 3H).
4.6.7. (E)-4-(2-(2,20-bithiophen-5-yl)vinyl)aniline (10)
Yellow solid, yield 27%. 1H NMR (400 MHz, CDCl3)
d 7.51 (d,
4.5.11. (4-tert-butylbenzyl)triphenylphosphonium bromide (3k)
J ¼ 8.2 Hz, 2H), 7.44 (d, J ¼ 4.8 Hz, 1H), 7.29 (d, J ¼ 16.1 Hz, 1H), 7.25
(d, J ¼ 2.1 Hz,1H), 7.27 (d, J ¼ 3.2 Hz,1H), 7.10 (d, J ¼ 8.1 Hz, 2H), 7.07
(d, J ¼ 3.4 Hz, 1H), 7.03 (t, J ¼ 4.1 Hz, 1H), 6.85 (d, J ¼ 16.1 Hz, 1H).
MS(EI): m/z calcd for C16H13NS2 283.05; found 283.25. m.p.
158.6e159.2 ꢁC.
White solid, yield 91%. 1H NMR (400 MHz, CDCl3)
d 7.91e7.44 (m,
15H), 7.14 (d, J ¼ 7.9 Hz, 2H), 7.01 (d, J ¼ 8.1 Hz, 2H), 5.33 (d,
J ¼ 14.0 Hz, 2H), 1.23 (s, 9H).
4.6. General procedure for the synthesis of SBTPs (4e9, 11e33)
4.6.8. (E)-4-(2-(2,20-bithiophen-5-yl)vinyl)-N,N-dimethylaniline (11)
To a stirring solution of 50-substituted-2,20-bithiophene-5-car-
baldehyde (1.00 mmol) and 4-substituted-benzyl halide (1.00 mmol)
in THF (50 mL), was added CH3ONa (1.00 mmol). The mixture was
then refluxedfor 2e6 h. Aftercooling, water (20 mL)wasadded tothe
reaction mixture. The resulting mixture was extracted by dichloro-
methane (3 times) and the organic layer was washed with brine and
dried over anhydrous MgSO4. The organic solvent was removed in
vacuum and the residue was purified by column chromatography.
Orange solid, yield 33%. 1H NMR (400 MHz, CDCl3)
d 7.29 (d,
J ¼ 8.4 Hz, 2H), 7.12 (d, J ¼ 5.1 Hz, 1H), 7.09 (d, J ¼ 3.4 Hz, 1H), 6.98
(d, J ¼ 3.6 Hz, 1H), 6.95 (d, J ¼ 3.9 Hz, 1H), 6.91 (d, J ¼ 15.9 Hz, 1H),
6.80 (d, J ¼ 3.6 Hz, 1H), 6.76 (d, J ¼ 16.0 Hz, 1H), 6.63 (d, J ¼ 8.2 Hz,
2H), 2.92 (s, 6H). MS(ESI): m/z calcd for C18H17NS2 311.08; found
311.9 [M þ H]þ.
4.6.9. (E)-5-(4-methoxylstyryl)-2,20-bithiophene (12)
Yellow solid, yield 86%. 1H NMR (400 MHz, CDCl3)
d 7.41 (d,
4.6.1. (E)-5-styryl-2,20-bithiophene (4)
J ¼ 8.7 Hz, 2H), 7.22 (dd, J ¼ 5.1, 1.0 Hz, 1H), 7.18 (dd, J ¼ 3.6, 1.0 Hz,
1H), 7.07 (d, J ¼ 3.6 Hz, 1H), 7.04 (d, J ¼ 3.5 Hz, 1H), 7.02 (d,
J ¼ 3.7 Hz, 1H), 6.93 (d, J ¼ 3.8 Hz, 1H), 6.90 (d, J ¼ 8.8 Hz, 2H), 6.86
(d, J ¼ 16.1 Hz, 1H), 3.84 (s, 3H). MS(EI): m/z calcd for C17H14OS2
298.05; found 298.21. m.p. 143.8 ꢁC.
Yellow solid, yield 85%. 1H NMR (400 MHz, CDCl3)
d 7.40 (d,
J ¼ 7.7 Hz, 2H), 7.28 (t, J ¼ 7.5 Hz, 2H), 7.20 (d, J ¼ 5.4 Hz, 1H), 7.15 (d,
J ¼ 5.3 Hz, 1H), 7.12 (d, J ¼ 4.4 Hz, 1H), 7.11(d, J ¼ 16.0 Hz, 1H), 7.01
(d, J ¼ 3.7 Hz, 1H), 6.96 (t, J ¼ 4.3 Hz, 1H), 6.90 (d, J ¼ 3.6 Hz, 1H),
6.82 (d, J ¼ 16.0 Hz, 1H). MS(EI): m/z calcd for C16H12S2 268.04;
found 268.25. m.p. 106.4e107.3 ꢁC.
4.6.10. (E)-5-(4-methylstyryl)-2,20-bithiophene (13)
Yellow solid, yield 78%. 1H NMR (400 MHz, CDCl3)
d 7.36 (d,
4.6.2. (E)-5-(4-fluorostyryl)-2,20-bithiophene (5)
J ¼ 7.6 Hz, 2H), 7.21 (d, J ¼ 5.0 Hz, 1H), 7.18 (d, J ¼ 6.8 Hz, 1H), 7.16 (d,
J ¼ 7.8 Hz, 2H), 7.13 (d, J ¼ 16.0 Hz, 1H), 7.07 (d, J ¼ 3.5 Hz, 1H), 7.03
(d, J ¼ 4.9 Hz, 1H), 6.94 (d, J ¼ 3.4 Hz, 1H), 6.87 (d, J ¼ 16.0 Hz, 1H),
2.35 (s, 3H). MS(EI): m/z calcd for C17H14S2 282.05; found 282.23.
m.p. 119.1e119.8 ꢁC.
Yellowsolid,yield87%.1HNMR(400 MHz,CDCl3)
d
7.36 (dd, J ¼ 7.1,
6.1 Hz, 2H), 7.15 (d, J ¼ 5.0 Hz, 1H), 7.12 (d, J ¼ 3.3 Hz, 1H), 7.01 (d,
J ¼ 16.2 Hz, 1H), 7.00 (d, J ¼ 3.9 Hz, 2H), 6.97 (s,1H), 6.95 (d, J ¼ 3.8 Hz,
1H), 6.88 (d, J ¼ 3.5 Hz,1H), 6.78 (d, J ¼ 15.8 Hz, 1H). MS(EI): m/z calcd
for C16H11FS2 286.03; found 285.9. m.p. 115.5e128.6 ꢁC.
4.6.11. (E)-5-(4-tert-butylstyryl)-2,20-bithiophene (14)
4.6.3. (E)-5-(4-chlorostyryl)-2,20-bithiophene (6)
Yellow solid, yield 86%. 1H NMR (400 MHz, CDCl3)
d 7.40 (d,
Yellow solid, yield 81%. 1H NMR (400 MHz, CDCl3)
d
7.32 (d,
J ¼ 8.6 Hz, 2H), 7.37 (d, J ¼ 8.6 Hz, 2H), 7.21 (d, J ¼ 5.1 Hz,1H), 7.18 (d,
J ¼ 3.6 Hz, 1H), 7.13 (d, J ¼ 16.0 Hz, 1H), 7.07 (d, J ¼ 3.7 Hz, 1H), 7.02
(dd, J ¼ 5.1, 3.7 Hz, 1H), 6.94 (d, J ¼ 3.7 Hz, 1H), 6.88 (d, J ¼ 16.1 Hz,
1H), 1.33 (s, 9H). MS(EI): m/z calcd for C20H20S2 324.10; found
324.31. m.p. 112.0e112.6 ꢁC.
J ¼ 8.3 Hz, 2H), 7.24 (d, J ¼ 8.2 Hz, 2H), 7.16 (d, J ¼ 5.1 Hz, 1H), 7.12 (d,
J ¼ 3.5 Hz,1H), 7.08 (d, J ¼ 16.0 Hz, 1H), 7.01 (d, J ¼ 3.6 Hz,1H), 6.96 (t,
J ¼ 4.3 Hz,1H), 6.90(d, J ¼ 3.6 Hz,1H), 6.76 (d, J ¼ 16.0 Hz,1H). MS(EI):
m/z calcd for C16H11ClS2 302.00; found 302.17. m.p. 137.5e139.8 ꢁC.

2914
M. Cui et al. / European Journal of Medicinal Chemistry 46 (2011) 2908e2916
4.6.12. (E)-5-bromo-50-styryl-2,20-bithiophene (15)
MS(EI): m/z calcd for C17H13BrS2 359.96; found 360.20. m.p.
188.1e188.9 ꢁC.
Yellow solid, yield 83%. 1H NMR (400 MHz, CDCl3)
d 7.46 (d,
J ¼ 7.4 Hz, 2H), 7.35 (t, J ¼ 7.2 Hz, 2H), 7.26 (d, J ¼ 13.3 Hz, 1H), 7.16
(d, J ¼ 16.1 Hz, 1H), 6.99 (d, J ¼ 11.4 Hz, 2H), 6.94 (d, J ¼ 10.3 Hz, 2H),
6.89 (d, J ¼ 16.9 Hz, 1H). MS(EI): m/z calcd for C16H11BrS2 345.95;
found 345.90. m.p. 138.2e139.9 ꢁC.
4.6.22. (E)-5-bromo-50-(4-tert-butylstyryl)-2,20-bithiophene (25)
Yellow solid, yield 85%. 1H NMR (400 MHz, CDCl3)
d 7.40 (d,
J ¼ 8.7 Hz, 2H), 7.37 (d, J ¼ 8.7 Hz, 2H), 7.12 (d, J ¼ 16.0 Hz, 1H), 7.00
(d, J ¼ 3.7 Hz, 1H), 6.97 (d, J ¼ 3.9 Hz, 1H), 6.93 (d, J ¼ 3.8 Hz, 1H),
6.91 (d, J ¼ 3.9 Hz, 1H), 6.88 (d, J ¼ 16.1 Hz, 1H), 1.33 (s, 9H). MS(EI):
m/z calcd for C20H19BrS2 402.01; found 402.22. m.p. 138.1 ꢁC.
4.6.13. (E)-5-bromo-50-(4-fluorostyryl)-2,20-bithiophene (16)
Yellow solid, yield 81%. 1H NMR (400 MHz, CDCl3)
d 7.43 (t,
J ¼ 6.3 Hz, 2H), 7.09e6.97 (m, 5H), 6.93 (d, J ¼ 4.0 Hz, 2H), 6.84 (d,
J ¼ 16.0 Hz, 1H). MS(EI): m/z calcd for C16H10BrFS2 363.94; found
364.13. m.p. 159.0e159.9 ꢁC.
4.6.23. (E)-5-iodo-50-styryl-2,20-bithiophene (26)
Yellow solid, yield 88%. H NMR (400 MHz, CDCl3)
d 7.46 (d,
J ¼ 7.7 Hz, 2H), 7.35 (t, J ¼ 7.5 Hz, 2H), 7.25 (d, J ¼ 16.0 Hz, 1H), 7.17
(d, J ¼ 5.8 Hz, 1H), 7.15 (d, J ¼ 6.4 Hz, 1H), 7.01 (d, J ¼ 3.7 Hz, 1H),
6.95 (d, J ¼ 3.7 Hz, 1H), 6.89 (d, J ¼ 16.0 Hz, 1H), 6.85 (d, J ¼ 3.7 Hz,
1H). MS(EI): m/z calcd for C16H11IS2 393.93; found 394.14. m.p.
160.3 ꢁC.
4.6.14. (E)-5-bromo-50-(4-chlorostyryl)-2,20-bithiophene (17)
Yellow solid, yield 60%. 1H NMR (400 MHz, CDCl3)
d 7.38 (d,
J ¼ 7.9 Hz, 2H), 7.31 (d, J ¼ 7.9 Hz, 2H), 7.13 (d, J ¼ 16.2 Hz, 1H), 6.99
(d, J ¼ 10.9 Hz, 2H), 6.94 (d, J ¼ 11.1 Hz, 2H), 6.83 (d, J ¼ 16.0 Hz, 1H).
MS(EI): m/z calcd for C16H10BrClS2 379.91; found 380.04. m.p.
187.3e188.8 ꢁC.
4.6.24. (E)-5-(4-fluorostyryl)-50-iodo-2,20-bithiophene (27)
Yellow solid, yield 86%. 1H NMR (400 MHz, CDCl3)
d 7.43 (d,
4.6.15. (E)-5-bromo-50-(4-bromostyryl)-2,20-bithiophene (18)
J ¼ 5.6 Hz,1H), 7.41 (d, J ¼ 5.6 Hz,1H), 7.16 (d, J ¼ 3.8 Hz,1H), 7.07 (d,
J ¼ 16.2 Hz, 1H), 7.05 (d, J ¼ 8.5 Hz, 2H), 7.01 (d, J ¼ 4.3 Hz, 1H), 6.94
(d, J ¼ 3.7 Hz, 1H), 6.85 (d, J ¼ 3.8 Hz, 1H), 6.84 (d, J ¼ 16.0 Hz, 1H).
MS(EI): m/z calcd for C16H10FIS2 411.93; found 412.11. m.p. 169.8 ꢁC.
Yellow solid, yield 86%. 1H NMR (400 MHz, CDCl3)
d 7.47 (d,
J ¼ 7.5 Hz, 2H), 7.32 (d, J ¼ 8.0 Hz, 2H), 7.15 (d, J ¼ 16.0 Hz, 1H), 6.99
(d, J ¼ 10.5 Hz, 2H), 6.95 (d, J ¼ 12.3 Hz, 2H), 6.81 (d, J ¼ 15.9 Hz,1H).
MS(EI): m/z calcd for C16H10Br2S2 423.86; found 424.06. m.p.
199.9e201.6 ꢁC.
4.6.25. (E)-5-(4-chlorostyryl)-50-iodo-2,20-bithiophene (28)
Yellow solid, yield 90%. 1H NMR (400 MHz, CDCl3)
d 7.38 (d,
4.6.16. (E)-5-bromo-50-(4-iodostyryl)-2,20-bithiophene (19)
J ¼ 7.8 Hz, 2H), 7.31 (d, J ¼ 8.3 Hz, 2H), 7.16 (d, J ¼ 3.7 Hz,1H), 7.13 (d,
J ¼ 16.0 Hz, 1H), 7.01 (d, J ¼ 3.7 Hz, 1H), 6.95 (d, J ¼ 3.6 Hz, 1H), 6.85
(d, J ¼ 3.7 Hz, 1H), 6.83 (d, J ¼ 16.2 Hz, 1H). MS(EI): m/z calcd for
C16H10ClIS2 427.90; found 428.08. m.p. 187.6 ꢁC.
Yellow solid, yield 79%. 1H NMR (400 MHz, CDCl3)
d 7.67 (d,
J ¼ 6.7 Hz, 2H), 7.19 (d, J ¼ 8.0 Hz, 2H), 7.14 (s, 1H), 7.05e6.91 (m,
4H), 6.79 (d, J ¼ 15.7 Hz, 1H). MS(EI): m/z calcd for C16H10BrIS2
471.85; found 472.06. m.p. 212.3e214.1 ꢁC.
4.6.26. (E)-5-(4-bromostyryl)-50-iodo-2,20-bithiophene (29)
4.6.17. (E)-5-bromo-50-(4-nitrostyryl)-2,20-bithiophene (20)
Yellow solid, yield 89%. 1H NMR (400 MHz, CDCl3)
d 7.46 (d,
Dark red solid, yield 45%. 1H NMR (400 MHz, CDCl3)
d
8.21 (d,
J ¼ 8.3 Hz, 2H), 7.32 (d, J ¼ 8.2 Hz, 2H), 7.16 (d, J ¼ 2.4 Hz,1H), 7.14 (d,
J ¼ 14.8 Hz, 1H), 7.01 (d, J ¼ 3.7 Hz, 1H), 6.96 (d, J ¼ 3.8 Hz, 1H), 6.85
(d, J ¼ 3.8 Hz, 1H), 6.81 (d, J ¼ 16.0 Hz, 1H). MS(EI): m/z calcd for
C16H10BrIS2 471.85; found 472.10. m.p. 179.7 ꢁC.
J ¼ 8.0 Hz, 2H), 7.57 (d, J ¼ 8.2 Hz, 2H), 7.32 (d, J ¼ 16.0 Hz, 1H), 7.05
(s, 2H), 6.98 (d, J ¼ 11.8 Hz, 2H), 6.90 (d, J ¼ 16.1 Hz, 1H). MS(EI): m/z
calcd for C16H10BrNO2S2 390.93; found 391.12. m.p. 202.2e203.7 ꢁC.
4.6.18. (E)-4-(2-(50-bromo-2,20-bithiophen-5-yl)vinyl)-N,N-
4.6.27. (E)-5-iodo-50-(4-iodostyryl)-2,20-bithiophene (30)
dimethylaniline (21)
Yellow solid, yield 77%. 1H NMR (400 MHz, DMSO-d6)
d 7.71 (d,
Yellow solid, yield 31%. 1H NMR (400 MHz, CDCl3)
d
7.38 (d,
J ¼ 8.2 Hz, 2H), 7.45 (d, J ¼ 16.2 Hz, 1H), 7.38 (d, J ¼ 8.3 Hz, 2H), 7.32
(d, J ¼ 3.7 Hz,1H), 7.24 (d, J ¼ 3.6 Hz,1H), 7.17 (d, J ¼ 3.6 Hz,1H), 7.05
(d, J ¼ 3.6 Hz, 1H), 6.90 (d, J ¼ 16.2 Hz, 1H). MS(EI): m/z calcd for
C16H10I2S2 519.83; found 519.97.
J ¼ 6.5 Hz, 2H), 7.00e6.96 (m, 3H), 6.90 (d, J ¼ 3.4 Hz, 1H), 6.84 (d,
J ¼ 16.0 Hz, 2H), 6.72 (s, 2H), 3.01 (s, 6H). MS(EI): m/z calcd for
C18H16BrNS2 388.99; found 389.18. m.p. 207.1e208.9 ꢁC.
4.6.19. (E)-4-(2-(50-bromo-2,20-bithiophen-5-yl)vinyl)phenol (22)
4.6.28. (E)-5-iodo-50-(4-methoxystyryl)-2,20-bithiophene (31)
Yellow solid, yield 14%. 1H NMR (400 MHz, CDCl3)
d
7.36 (d,
Yellow solid, yield 85%. 1H NMR (400 MHz, CDCl3)
d 7.40 (d,
J ¼ 8.5 Hz, 2H), 7.02 (d, J ¼ 16.5 Hz, 1H), 6.99 (d, J ¼ 4.8 Hz, 1H), 6.97
(d, J ¼ 3.9 Hz, 1H), 6.91 (d, J ¼ 2.7 Hz, 1H), 6.90 (d, J ¼ 3.4 Hz, 1H),
6.83 (d, J ¼ 16.3 Hz, 1H), 6.81 (d, J ¼ 8.5 Hz, 2H). MS(EI): m/z calcd
for C16H11BrOS2 361.94; found 362.18. m.p. 152.6 ꢁC.
J ¼ 8.6 Hz, 2H), 7.16 (d, J ¼ 3.8 Hz, 2H), 7.03 (d, J ¼ 16.1 Hz, 1H), 7.00
(d, J ¼ 3.7 Hz, 1H), 6.90 (d, J ¼ 2.6 Hz, 1H), 6.89 (d, J ¼ 8.4 Hz, 1H),
6.85 (d, J ¼ 15.8 Hz, 1H), 6.84 (d, J ¼ 3.9 Hz, 1H), 3.83 (s, 3H). MS(EI):
m/z calcd for C17H13IOS2 423.95; found 424.11. m.p. 199.5e201.6 ꢁC.
4.6.20. (E)-5-bromo-50-(4-methoxystyryl)-2,20-bithiophene (23)
4.6.29. (E)-5-iodo-50-(4-methylstyryl)-2,20-bithiophene (32)
Yellow solid, yield 86%. 1H NMR (400 MHz, CDCl3)
d
7.33 (d,
Yellow solid, yield 82%. 1H NMR (400 MHz, CDCl3)
d 7.36 (d,
J ¼ 8.3 Hz, 2H), 6.96 (d, J ¼ 16.0 Hz, 1H), 6.92 (d, J ¼ 3.6 Hz, 1H), 6.90
(d, J ¼ 3.8 Hz, 1H), 6.83 (s, 3H), 6.81 (s, 1H), 6.78 (d, J ¼ 16.2 Hz, 1H),
3.76 (s, 3H). MS(EI): m/z calcd for C17H13BrOS2 375.96; found 376.13.
m.p. 178.3e180.2 ꢁC.
J ¼ 7.8 Hz, 2H), 7.16e7.14 (m, 3H), 7.11 (d, J ¼ 16.0 Hz, 1H), 7.00 (d,
J ¼ 3.8 Hz, 1H), 6.92 (d, J ¼ 3.7 Hz, 1H), 6.87 (d, J ¼ 16.5 Hz, 1H), 6.84
(d, J ¼ 3.9 Hz, 1H), 2.35 (s, 3H). MS(EI): m/z calcd for C17H13IS2
407.95; found 408.14. m.p. 237.6e238.6 ꢁC.
4.6.21. (E)-5-bromo-50-(4-methylstyryl)-2,20-bithiophene (24)
4.6.30. (E)-5-(4-tert-butylstyryl)-50-iodo-2,20-bithiophene (33)
Yellow solid, yield 79%. 1H NMR (400 MHz, CDCl3)
d
7.36 (d,
Yellow solid, yield 88%. 1H NMR (400 MHz, CDCl3)
d 7.40 (d,
J ¼ 7.6 Hz, 2H), 7.16 (d, J ¼ 7.7 Hz, 2H), 7.11 (d, J ¼ 16.1 Hz, 1H), 6.98
J ¼ 8.7 Hz, 2H), 7.37 (d, J ¼ 8.7 Hz, 2H), 7.16 (d, J ¼ 3.8 Hz,1H), 7.12 (d,
(d, J ¼ 9.6 Hz, 2H), 6.92 (s, 2H), 6.87 (d, J ¼ 16.0 Hz, 1H), 2.35 (s, 3H).
J ¼ 16.0 Hz, 1H), 7.00 (d, J ¼ 3.7 Hz, 1H), 6.93 (d, J ¼ 3.8 Hz, 1H), 6.88

M. Cui et al. / European Journal of Medicinal Chemistry 46 (2011) 2908e2916
2915
(d, J ¼ 16.1 Hz, 1H), 6.84 (d, J ¼ 3.8 Hz, 1H), 1.33 (s, 9H). MS(EI): m/z
calcd for C20H19IS2 450.00; found 450.23. m.p. 152.4e154.0 ꢁC.
volume of 1 mL. Non-specific binding was defined in the presence of
100 nM TZDM. The mixture was incubated at 37 ꢁC for 2 h, then the
bound and free radioactivities were separated by vacuum filtration
4.7. (E)-(4-(2-([2,20-bithiophen]-5-yl)vinyl)phenyl)
through borosilicate glass fiber filters (0.1 mm, JiuDing, China) using
tributylstannane (34)
a ZT-II cell harvester (Shaoxing, China) followed by 3 ꢃ 4 mL washes
of cold PBS (0.2 M, pH ¼ 7.4) containing 10% ethanol at room
temperature. Filters containing the bound 125I ligand were counted
A mixture of 7 (123 mg, 0.5 mmol), (Bu3Sn)2 (580 mg, 1.0 mmol)
and (Ph3P)4Pd (57.8 mg, 0.05 mmol) in toluene (10 mL) was stirred
under reflux for 10 h. The solvent was removed, and the residue
was purified by silica gel chromatography to give 60.5 mg of
in a
g-counter (WALLAC/Wizard 1470, USA) with 70% counting
efficiency. The half maximal inhibitory concentration (IC50) values
were calculated using GraphPad Prism 4.0, and those for the inhi-
bition constant (Ki) were calculated using the ChengePrusoff equa-
tion: Ki ¼ IC50/(1 þ [L]/Kd) [36].
34.Yellow oil, yield 21.7%. 1H NMR (400 MHz, CDCL3)
d 7.46 (d,
J ¼ 8.0 Hz, 2H), 7.42 (d, J ¼ 8.1 Hz, 2H), 7.22 (dd, J ¼ 3.6, 1.2 Hz, 1H),
7.19 (dd, J ¼ 3.6, 1.1 Hz, 1H), 7.19 (d, J ¼ 16.0 Hz, 1H), 7.08 (d,
J ¼ 3.7 Hz, 1H), 7.03 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.96 (d, J ¼ 3.8 Hz, 1H),
6.88 (d, J ¼ 16.0 Hz, 1H), 1.84e0.57 (m, 27H). MS(EI): m/z calcd for
C20H19IS2 558.14; found 558.45.
4.11. In vitro fluorescent staining using brain sections of transgenic
model mouse
Paraffin-embedded brain sections of Tg-C57 mouse (10 mm)
4.8. (E)-tributyl(50-(4-methoxystyryl)-[2,20-bithiophen]-5-yl)
stannane (35)
were used for the autoradiography. The brain sections were
deparaffinized with 2 ꢃ 20 min washes in xylene, 2 ꢃ 5 min washes
in 100% ethanol, 5 min washes in 90% ethanol/H2O, 5 min washes in
80% ethanol/H2O, 5 min wash in 60% ethanol/H2O, running tap
water for 10 min, and then incubated in PBS (0.2 M, pH ¼ 7.4) for
30 min. The brain sections were incubated with 20% ethanol solu-
The reaction described for 34 was used, 115.5 mg of 35 was
obtained as a yellow oil, yield 39.3%. 1H NMR (400 MHz, CDCl3)
d
7.39 (d, J ¼ 8.8 Hz, 2H), 7.29 (d, J ¼ 3.4 Hz, 1H), 7.06 (d, J ¼ 3.7 Hz,
1H), 7.05 (d, J ¼ 3.9 Hz, 1H), 7.04 (d, J ¼ 16.4 Hz, 1H), 6.91 (d,
J ¼ 3.9 Hz, 1H), 6.88 (d, J ¼ 8.8 Hz, 2H), 6.84 (d, J ¼ 16.0 Hz, 1H), 3.83
(s, 3H), 1.74e0.78 (m, 27H). MS(EI): m/z calcd for C20H19IS2 588.15;
found 588.38.
tion (1
mM) of compounds for 10 min. The localization of plaques
was confirmed by staining with thioflavin-S (1
mM) on the adjacent
sections. Finally, the sections were washed with 50% ethanol and
PBS (0.2 M, pH ¼ 7.4) for 10 min. Fluorescent observation was per-
formed by the LSM 510 META (Zeiss, Germany) equipped with a LP
505 filter set (excitation, 405 nm; long-pass filter, 505 nm).
4.9. Iododestannylation reaction
The radio-iodinated ligands 8 and 31 were prepared from the
corresponding tributyltin derivatives by iododestannylation accord-
ingtoaproceduredescribedpreviouslywithsomemodifications[34].
4.12. Autoradiography in vitro using brain sections of transgenic
model mouse
Briefly, 50
derivative 100
activity 2200 Ci/mmol), and 100
reaction was allowed to proceed at room temperature for 15 min and
terminated by addition of 50 L saturated NaHSO3. After neutraliza-
m
L of H2O2 (3%) was added to a mixture of a tributyltin
L (1 mg/mL in EtOH), [125I]NaI (200
Ci, specific
L of 1 M HCl in a sealed vial. The
The brain sections mentioned above were incubated with [125I]8
m
m
or [125I]31 (5
mCi/100 mL) for 30 min at room temperature. The
m
sections were then washed with saturated Li2CO3 in 40% EtOH for
3 min and washed with 40% EtOH for 3 min, followed by rinsing
with water for 30 s. After drying, the 125I-labeled sections were
exposed to phosphorus film for 2 h then scanned with the phos-
phor imaging system (Cyclone, Packard) at the resolution of
600 dpi. The presence and localization of plaques was confirmed by
m
tion with sodium bicarbonate, the reaction mixture was extracted
with ethyl acetate (3 ꢃ 1 mL). The combined extracts were evapo-
rated to dryness. The residues were dissolved in 100 mL of EtOH and
purified by HPLC using a Alltech C18 reversed phase column
m) with the solvent of acetonitrile (100%) at a flow
the fluorescent staining with thioflavin-S (1 mM) on the same
sections using Stereo Discovery V12, excitation: 450e490 nm,
emission: 500e550 nm, (Zeiss, Germany).
(4.6 ꢃ 250 mm, 5
m
rate of 2.0 mL/min. Finally, the radiochemical identity of the radio-
labeled tracers were verified by co-injection and co-elution with
nonradioactive compound by HPLC (Agilent TC-C18, 4.6 ꢃ 150 mm,
4.13. Biodistribution experiments with normal mice
5
m
m, CH3CN/H2O ¼ 9/1 at the flow rate of 1.0 mL/min). The no-
carrier-added products were stored at ꢄ20 ꢁC up to 6 weeks for bio-
distribution experiments, binding assay, and autoradiography
studies.
Invivo biodistribution studies were performed in female KunMing
normal mice (average weight about 20 g) and approved by the
national laws related to the care and experiments on laboratory
animal. A saline solution (100 m mCi)
L) containing [125I]8 or [125I]31 (1
4.10. Binding assay in vitro using A
b
aggregates
was injected directly into the tail vein of mice. The mice were sacri-
ficedatvarioustimepointspost-injection. Theorgansofinterestwere
removed and weighed, and the radioactivity was counted with an
automatic g-counter (WALLAC/Wizard 1470, USA). The percentage
dose per gram of wet tissue was calculated by a comparison of the
tissue counts to suitably diluted aliquots of the injected material.
The trifluoro acetic acid salt forms of peptide Ab1e42 were
purchased from GL biochem (Shanghai, China). Aggregation of
peptides was carried out by gently dissolving the peptide (0.25
mg/mL) in a buffer solution (pH ¼ 7.4) containing 10 mM sodium
phosphate and 1 mM EDTA. The solutions were incubated at 37 ꢁC
for 42 h with gentle and constant shaking. Inhibition studies were
carried out in 12 ꢃ 75 mm borosilicate glass tubes according to the
procedure described previously with some modifications [31,34,35].
4.14. Partition coefficient determination
The partition coefficient of [125I]8 and [125I]31 was determined
according to the procedure previously reported with some modifi-
cations [37]. Five mCi of tracer was added to the premixed suspen-
50
were added to the mixture containing 100
TZDM) with appropriate concentration, 10
(10ꢄ5e10ꢄ10 M in EtOH) and 840
L PBS (0.2 M, pH ¼ 7.4) in a final
m
L aggregated Ab1e42 fibrils (28 nM in the final assay mixture)
L of radio-ligands ([125I]
of inhibitors
m
mL
sions containing 3 g of n-octanol and 3 g of PBS (0.05 M, pH ¼ 7.4) in
m
a test tube. The test tube was vortexed for 3 min at room

2916
M. Cui et al. / European Journal of Medicinal Chemistry 46 (2011) 2908e2916
[16] M.P. Kung, C. Hou, Z.P. Zhuang, B. Zhang, D. Skovronsky, J.Q. Trojanowski,
V.M. Lee, H.F. Kung, IMPY: an improved thioflavin-T derivative for in vivo
labeling of beta-amyloid plaques, Brain Res. 956 (2002) 202e210.
[17] Z.P. Zhuang, M.P. Kung, A. Wilson, C.W. Lee, K. Plossl, C. Hou, D.M. Holtzman,
H.F. Kung, Structure-activity relationship of imidazo[1,2-a]pyridines as
temperature, followed bycentrifugation for 5 min at 3500 rpm. Two
weighted samples from the n-octanol (100 L) and buffer (500 L)
m
m
layers were counted. The partition coefficient was expressed as the
logarithm of the ratio of the counts per gram from n-octanol versus
that of PBS. Samples from n-octanol layer were repartitioned until
consistent partitions of coefficient values were obtained. The
measurement was done in triplicate and repeated three times.
ligands for detecting b-amyloid plaques in the brain, J. Med. Chem. 46 (2003)
237e243.
[18] A.B. Newberg, N.A. Wintering, K. Plossl, J. Hochold, M.G. Stabin, M. Watson,
D. Skovronsky, C.M. Clark, M.P. Kung, H.F. Kung, Safety, biodistribution, and
dosimetry of 123I-IMPY: a novel amyloid plaque-imaging agent for the diag-
nosis of Alzheimer’s disease, J. Nucl. Med. 47 (2006) 748e754.
[19] W.C. Qu, M.P. Kung, C. Hou, L.W. Jinc, H.F. Kung, Radioiodinated aza-
diphenylacetylenes as potential SPECT imaging agents for b-amyloid plaque
detection, Bioorg. Med. Chem. Lett. 17 (2007) 3581e3584.
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probes for SPECT imaging of amyloid plaques, J. Med. Chem. 50 (2007)
2157e2165.
Acknowledgment
The authors thank Dr. Xiaoyan Zhang (College of Life Science,
Beijing Normal University) for assistance in the in vitro neuro-
pathological staining. This work was funded by NSFC (20871021)
and the Fundamental Research Funds for the Central Universities.
[21] M. Ono, Y. Maya, M. Haratake, K. Ito, H. Mori, M. Nakayama, Aurones serve as
probes of b-amyloid plaques in Alzheimer’s disease, Biochem. Biophys. Res.
Commun. 361 (2007) 116e121.
[22] Y. Maya, M. Ono, H. Watanabe, M. Haratake, H. Saji, M. Nakayama, Novel
Appendix. Supporting information
radioiodinated aurones as probes for SPECT imaging of b-amyloid plaques in
the brain, Bioconjug. Chem. 20 (2009) 95e101.
Supporting information related to this article can be found
online at doi:10.1016/j.ejmech.2011.04.015.
[23] M. Ono, M. Haratake, H. Mori, M. Nakayama, Novel chalcones as probes for
in vivo imaging of -amyloid plaques in Alzheimer’s brains, Bioorg. Med.
b
Chem. 15 (2007) 6802e6809.
[24] M. Ono, M. Hori, M. Haratake, T. Tomiyama, H. Mori, M. Nakayama, Structure-
activity relationship of chalcones and related derivatives as ligands for
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A
b