Homoisoflavonoids as potential imaging agents for β-amyloid plaques in Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2014.02.020

A series of homoisoflavonoids [(E)-3-benzylidenechroman-4-ones, 3a-l] as novel potential diagnostic imaging agents targeting β-amyloid (Aβ) plaques in Alzheimer's disease (AD) were synthesized and evaluated. In vitro binding studies using Aβ_1-40 aggregates with [ ¹²⁵I]IMPY as the reference ligand showed that these compounds demonstrated high to low binding affinities at the K_i values ranged from 9.10 to 432.03 nM, depending on the substitution of the phenyl ring. Fluorescent staining in vitro indicated that one compound with a N,N-dimethylamino group intensely stained Aβ plaques within brain sections of postmortem AD patients. Biodistribution studies in normal mice after i.v. injection of the radioiodinated homoisoflavonoid displayed good initial brain uptake (2.61% ID/g at 2 min postinjection) and rapid clearance from the brain (0.18% ID/g at 60 min), which is desirable for amyloid imaging agents. The results strongly suggest that these derivatives are worthy of further study and may be useful amyloid imaging agents for early detection of amyloid plaques in the brain of AD.

Full text of DOI:10.1016/j.ejmech.2014.02.020

European Journal of Medicinal Chemistry 76 (2014) 125e131
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Homoisoflavonoids as potential imaging agents for
in Alzheimer’s disease
b-amyloid plaques
,
a
b
a
a
Changsheng Gan a , Zhenzhen Zhao , Dou-Dou Nan , Binbin Yin , Jingyi Hu
*
a Engineering Research Center of Bio-process of Ministry of Education, Hefei University of Technology, Hefei, Anhui 230009, PR China
b CAS Key Laboratory of Brain Function and Disease, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of homoisoflavonoids [(E)-3-benzylidenechroman-4-ones, 3ael] as novel potential diagnostic
Received 7 November 2013
Received in revised form
31 December 2013
Accepted 8 February 2014
Available online 11 February 2014
imaging agents targeting
b-amyloid (Ab) plaques in Alzheimer’s disease (AD) were synthesized and
evaluated. In vitro binding studies using Ab1e40 aggregates with [125I]IMPY as the reference ligand
showed that these compounds demonstrated high to low binding affinities at the Ki values ranged from
9.10 to 432.03 nM, depending on the substitution of the phenyl ring. Fluorescent staining in vitro
indicated that one compound with a N,N-dimethylamino group intensely stained A
b plaques within
brain sections of postmortem AD patients. Biodistribution studies in normal mice after i.v. injection of
the radioiodinated homoisoflavonoid displayed good initial brain uptake (2.61% ID/g at 2 min post-
injection) and rapid clearance from the brain (0.18% ID/g at 60 min), which is desirable for amyloid
imaging agents. The results strongly suggest that these derivatives are worthy of further study and may
be useful amyloid imaging agents for early detection of amyloid plaques in the brain of AD.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Alzheimer’s disease
Amyloid
Imaging agent
Homoisoflavonoid
Binding assay
1. Introduction
and thioflavin-T, which have been used in fluorescent staining of
plaques and tangles in postmortem AD brain sections. Several
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder and a leading cause of dementia [1e3]. Clinical symptoms
of AD include cognitive decline, irreversible memory loss, disori-
entation, language impairment, etc. Although great progress has
been made towards understanding the disease mechanisms in the
last three decades, there remains no cure for AD. The current
medications, including donepezil, galantamine, rivastigmine and
memantine are primarily symptomatic treatments [4]. Early inter-
vention may delay the onset or progression of AD. It has been well
established that the major neuropathological characteristic of AD is
the presence of senile plaques (SPs), which are composed of
radioligands for positron emission tomography (PET) and single
photon emission computerized tomography (SPECT), including
[
11C]PIB
(2-(4-(methylamino)phenyl)-6-hydroxybenzothiazole,
Pittsburgh compound B) [7e9], [11C]SB-13 (4-N-methylamino-40-
hydroxystilbene) [10], [11C]AZD2184 (2-(6-(methylamino)pyridin-
3-yl)-1,3-benzothiazol-6-ol)
fluoroethyl)-methylamino)-2-naphthyl)ethylidene)malononitrile))
[12,13],
18F]GE067 (2-(30-fluoro-40-(methylamino)phenyl)-6-
hydroxybenzothiazole) [14],
18F]BAY94-9172 (4-(N-methyl-
amino)-40-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)stilbene)
[15],
18F]AV-45 ((E)-4-(2-(6-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)pyr-
idin-3-yl)vinyl)-N-methylbenzenamine) [16,17], and [123I]IMPY (2-
(40-dimethylaminophenyl)-6-iodoimidazo[1,2-
]pyridine) [18],
11C] BF-227 (2-(2-(2-Dimethylaminothiazol-5-yl)ethenyl)-6-(2-
[11],
[
18F]FDDNP
(2-(1-(6-(2-
[
[
[
b
-amyloid (Ab) protein aggregates, and neurofibrillary tangles
(NFTs), which are highly phosphorylated tau proteins [5,6]. The
amyloid cascade hypothesis indicates that the deposition of amy-
loid plaques constitutes a central and probably early event in the
a
[
fluoroethoxy)benzoxazole) [19], have been tested in clinical trials
pathogenesis of AD. Therefore, the monitoring of A
b
plaques would
and demonstrated the potential utility as in vivo imaging agents for
be beneficial for the diagnosis, staging, and treatment of AD.
The development of amyloid-specific imaging agents is gener-
ally based on conjugated dyes, such as Congo red, Chrysamine G,
A
b
plaque deposition in the living human brain (Fig. 1).
To date, [11C]PIB, the most widely studied amyloid probe, is
limited by the 20-min radioactive decay half-life of 11C for routine
clinical use. To overcome the limitation, there are now several
amyloid probes labeled with 18F (110-min radioactive decay half-
life) under clinical development, such as
[
18F]BAY94-9172,
* Corresponding author.
E-mail address: gancs7894@163.com (C. Gan).
18F-Florbetapir ([18F]AV-45), and [18F]30-F-PIB [20]. There are still
http://dx.doi.org/10.1016/j.ejmech.2014.02.020
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

126
C. Gan et al. / European Journal of Medicinal Chemistry 76 (2014) 125e131
Fig. 1. Chemical structures of representative amyloid imaging agents.
some limitations of these probes, such as the higher white matter
non-specific binding than [11C]PIB [21,22]. [123I]IMPY [18,23e26],
the first SPECT probe to be evaluated in human, displayed a poor
signal-to-noise ratio. Therefore, there is still a need to develop more
probes for the imaging of AD brain SPs.
It is well known flavonoid is one of the most important natural
products with a variety of physiological activities. Heo’s results
indicated that quercetin, one of the major flavonoids in some fruits
and vegetables, contributed significantly to the protective effects of
neuronal cells against oxidative stress-induced neurotoxicity from
AD [27]. And the effects of polyhydroxyflavones on the formation,
amyloid imaging agents. The results indicated that some com-
pounds, which can be further radiolabeled by C-11, F-18 or I-123
[40e42], may be developed into potential useful amyloid imaging
agents for in vivo detecting
2. Results and discussion
2.1. Chemistry
b-amyloid plaques in the brain of AD.
As shown in Scheme 1, the homoisoflavonoids were readily
prepared by direct condensation of chroman-4-one and different
benzaldehyde. These compounds except 3l were generally syn-
thesized by heating chroman-4-one with corresponding benzal-
dehyde in saturated phosphorous acid [38]. In the case of 3l,
chroman-4-one was reacted with the corresponding benzaldehyde
in HOAc and saturated hydrochloride acid at room temperature to
produce 3l [43]. Compound 3f was prepared by heating 3e in
ethanol under the catalysis of SnCl2$2H2O. The trans-configuration
of compounds 3ael was confirmed by the chemical shift of the
vinyl proton in 1H NMR. Because of the deshielding effect resulting
from the neighboring carbonyl group, the vinyl proton gave a
higher chemical shift in the trans-isomer (>7 ppm) than the cis-
isomer [44].
extension, and destabilization of A
in vitro [28]. These flavones could inhibit the formation of A
gregates, as well as destabilizing preformed A aggregates, and
b
aggregates were studied
b
ag-
b
some amyloid imaging agent based on the structure of flavone were
developed by Ono et al. [29,30].
Homoisoflavonoids constitute a small class of natural products
prevalently isolated from the bulbs, rhizomes, or roots of several
genera of Hyacinthaceae and Caesalpinioideae. Several natural and
synthetic homoisoflavonoids, like the related flavonoids, were
found to possess various biological properties such as antifungal
[31], antiviral [32], antimutagenic [33], antioxidant [34], anti-
allergic and antihistaminic [35], anti-inflammatory [36], protein
tyrosine kinase inhibitor activities [37], monoamine oxidase and
cholinesterase inhibitor activities [38,39]. In the present study, a
series of homoisoflavonoids as flavonoid derivatives or hybrids of
flavone and chalcone structure, were synthesized and evaluated as
The radioiodinated form of compound 3d was prepared from
the corresponding tributyltin precursor by an iododestannylation.
As shown in Scheme 2, the corresponding tributyltin derivative 3m
was synthesized from 3d by reacting with bis(tributyltin) and
Scheme 1. Synthesis of 3ae3l.

C. Gan et al. / European Journal of Medicinal Chemistry 76 (2014) 125e131
127
Scheme 2. Synthesis of 3m and [125I]3n.
Pd(PPh3)4 in Et3N at 80 ꢀC, and then reacted with [125I] sodium
iodide, hydrogen peroxide to produce radioiodinated product [125I]
3n [43].
visually investigating the binding ability of these compounds to the
senile plaques (SPs) in vitro. As shown in Fig. 2, almost all the SPs in
the brain section could be labeled by ligand 3d (B1), as confirmed
by staining with ThS (A1) in adjacent sections compared with the
blank control sections (C1). The details of the SPs staining were
clearly visualized by the fluorescence of 3d (B2). Unlike ThS (A2),
the NFTs couldn’t be labeled by 3d (A2, B2).
2.2. In vitro binding assay
The binding affinities (Ki) of the homoisoflavonoids for Ab1e40
aggregates were determined by competition binding assay with
125I]IMPY as radioligand, while IMPY was also screened using the
2.4. Radioiodination
[
same system for comparison. [125I]IMPY was prepared as described
previously [25]. The results were shown in Table 1.
The synthesis of the tributyltin precursors (3m) and corre-
sponding [125I]-labeled compounds 3n are shown in Scheme 2.
Standard iododestannylation reactions, using [125I] sodium iodide,
hydrogen peroxide and hydrochloric acid were conducted suc-
cessfully [25,43]. Purification of the radiolabeled compounds was
performed by HPLC (Column: Waters Symmetry C18, 4.6 ꢂ 250 mm,
In the compounds 3ae3l, when the substituents were changed,
the inhibition constant (Ki) values exhibited great variations. In the
case of para-substitution of the right phenyl ring, compound 3a
(R2 ¼ OCH3) and 3d (R2 ¼ NMe2) displayed a higher binding affinity
(Ki ¼ 9.98 ꢁ 0.81 nM, 9.10 ꢁ 1.11 nM, respectively) than the others,
which is slightly superior to that of IMPY (Ki ¼ 13.65 nM). However,
the replacement of these substituents with NO2 group resulted in a
steep decrease in the binding affinity (for 3e, Ki ¼ 107.60 nM)(E)-3-
benzylidene-6-bromo-chroman-4-one (3c) without any sub-
stituents on the right phenyl ring also showed poor affinity
(Ki ¼ 103.62 nM). These suggest that the phenyl rings should be
electron rich in order for these compounds to exhibit high binding
5
m
m; Mobile phase: MeOH/H2O ¼ 8/2; Flow rate ¼ 1.0 mL/min;
Bioscan Flow-Count FC-3100 Na/I PMT based radioactivity detec-
tor). The final product [125I]3n showed greater than 95% radio-
chemical purity with high specific activity (2200 Ci/mmol).
2.5. Biodistribution in normal mice
Biodistribution experiments were performed in normal mice in
order to evaluate the pharmacokinetic properties of these de-
rivatives. [125I]3n was injected intravenously into normal ICR mice
for biodistribution studies. As shown in Table 2, the initial brain
uptake was 2.61% ID/g at 2 min postinjection, indicating an efficient
BBB permeability, sufficient for brain imaging probe. In addition, it
displayed rapid clearance from the normal brain with 0.32% and
0.18% ID/g at 30 min and 60 min postinjection, respectively. The
ratio of 60 mine2 min brain uptake values for the ligand was 6.9%.
affinities, possibly due to their stronger pep stacking with amyloid
peptides. The introduction of Br at the ortho position of 3d resulted
in a slight reduction of binding affinity (for 3h, Ki ¼ 25.54 nM). From
the Ki values of 3ie3l, we can see that changing the substituent site
and substituent number can also affect the bind affinity (for 3i, 3j
and 3l, Ki ¼ 54.22, 73.46 and 39.19 nM, respectively), which are all
lower than that of 3a with only one para-substituted OCH3 group.
These results imply that the steric hindrance may lead to the
reduction of the binding properties.
3. Conclusions
2.3. AD brain tissue fluorescent staining
In conclusion, we have synthesized and evaluated a series of the
homoisoflavonoid derivatives as novel potential imaging probes for
-amyloid plaques in AD brain. In particular, compounds 3a and 3d
Since these homoisoflavonoids could emit fluorescence well, the
AD brain tissue fluorescent staining was further performed for
b
exhibited high binding affinities to amyloid plaques in the nano-
molar range and 3d could selectively label the amyloid plaques
within brain sections of postmortem AD patients by tissue fluo-
rescent staining. In the biodistribution studies using normal ICR
mice, [125I]3n displayed a good brain penetration and fast washout
from the brain, highly desirable characteristics for in vivo amyloid
imaging agents. Taken together, the present results suggest that the
homoisoflavonoid derivatives may be useful probes for detecting
amyloid plaques in the AD brain.
Table 1
Inhibition constants (Ki) of homoisoflavonoids on [125I]IMPY binding to Ab1e40
aggregates.
Compound
R1
R2
R3
Kia
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
H
H
H
H
H
Br
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H
N(CH3)2
NO2
NH2
Br
N(CH3)2
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
9.98 ꢁ 0.81
103.35 ꢁ 11.80
103.62 ꢁ 14.53
9.10 ꢁ 1.11
107.60 ꢁ 14.28
63.57 ꢁ 10.07
143.81 ꢁ 7.28
25.54 ꢁ 2.32
54.22 ꢁ 3.11
73.46 ꢁ 9.49
432.03 ꢁ 64.64
39.19 ꢁ 7.93
13.65 ꢁ 0.93
4. Experimental section
4.1. Materials and instruments
3j
3k
3l
OH
H
The reagents used in the syntheses were purchased from Alfa
Aesar, SigmaeAldrich and Sinopharm Chemical Reagent Co. Ltd and
were used without further purification unless otherwise indicated.
IMPY
a
Each value was determined in three separate experiments with duplicate
measurements each time.
[
125I]NaI (2200 Ci/mmol) was purchased from PerkinElmer Life and

128
C. Gan et al. / European Journal of Medicinal Chemistry 76 (2014) 125e131
Fig. 2. Three adjacent AD brain sections were stained (A: ThS, B: 3d, C: blank control). The hollow arrows indicate the amyloid plaques. The triangle arrows indicate the neuro-
fibrillary tangles. The bottom row was the magnified images of the boxed area in the top row. Bar ¼ 250 m.
m
Analytical Sciences (USA). The AD human paraffin brain sections
were obtained from the Netherlands brain bank (coordinator: Dr. I.
Huitinga). The NMR spectra were obtained on Agilent VNMRS-600
(600 MHz) for compound 3d, 3h and Bruker Avance-300 (300 MHz)
spectrometer for other compounds with TMS as the internal stan-
dard. Melting points were determined using capillary tubes with
YRT-3 apparatus. High-resolution MS were obtained on a Micro-
mass GCT mass spectrometer. FT-IR spectra were obtained on a
Thermo Scientific Nicolet 8700 system.
4.2.2. (E)-3-(4-Hydroxybenzylidene)-6-bromo-chroman-4-one
(3b)
Yellow solid, mp 223e224 ꢀC. Yield: 75%. 1H NMR (DMSO-d6)
d
7.92 (d, 1H, J ¼ 2.7 Hz, H-5), 7.74e7.71 (m, 2H, H-7, ]CHPh),
7.35 (d, 2H, J ¼ 8.7 Hz, H-20, H-60), 7.06 (d, 1H, J ¼ 8.7 Hz, H-8),
6.90 (d, 2H, J ¼ 8.7 Hz, H-30, H-50), 5.45 (s, 2H, H-2). 13C NMR
(CDCl3)
d 181.0, 160.1, 158.6, 138.8, 133.2, 131.3, 129.1, 127.5, 123.8,
79
121.2, 116.8, 115.4, 113.3, 68.2. HRMS: calcd for C
H
BrO3 [M]þ
16 11
81
329.9892, found 329.9896, calcd for C16
H
BrO3 [M]þ 331.9871,
11
found 331.9877. IR (KBr, cmꢃ1
828.
) g 3163, 1655, 1602, 1284, 1020,
4.2. Chemistry
Typical procedure for the synthesis of 3ae3e and 3ge3k: To a
stirring solution of substituted benzaldehyde (0.5 mmol) in 85%
phosphorous acid (8 mL) was added 6-bromochroman-4-one
(0.5 mmol). The mixture was stirred at 80 ꢀC for 8 h. After cool-
ing, the mixture was diluted with water (and made alkaline for the
preparation of compound 3d and 3h). The precipitate was sepa-
rated by vacuum filtration and washed with cold water and
recrystallized from EtOH or purified by silica gel chromatography
(6:1 petroleum ether/ethyl acetate).
4.2.3. (E)-3-Benzylidene-6-bromo-chroman-4-one (3c)
Gray solid, mp 143e144 ꢀC. Yield: 80%. 1H NMR (CDCl3)
d
8.12
(s, 1H, H-5), 7.88 (s, 1H, ]CHPh), 7.57e7.54 (m, 3H, H-7, H-20, H-40),
7.46e7.43 (m, 2H, H-30, H-50), 6.89e6.86 (m, 2H, H-8, H-40), 5.35 (s,
2H, H-2). 13C NMR (CDCl3)
d
181.1, 159.8, 138.7, 135.5, 132.7, 130.5,
129.2, 128.4, 124.0, 122.3, 120.4, 115.2, 113.0, 68.5. HRMS: calcd for
C
H H BrO2
BrO2 [M]þ 313.9942, found 313.9946, calcd for C16
16 11 11
79
81
[M]þ 315.9922, found 315.9928. IR (KBr, cmꢃ1
1283, 819.
) g 1675, 1607, 1472,
4.2.1. (E)-3-(4-Methoxybenzylidene)-6-bromo-chroman-4-one
4.2.4. (E)-3-(4-Dimethylamino-benzylidene)-6-bromo-chroman-4-
(3a)
Yellow solid, mp 155e156 ꢀC. Yield: 86%. 1H NMR (CDCl3)
d 8.12
one (3d)
Brown solid, mp 170e171 ꢀC. Yield: 83%. 1H NMR (CDCl3)
d 8.11
(s, 1H, H-5), 7.84 (s, 1H, ]CHPh), 7.55 (d, 1H, J ¼ 8.6 Hz, H-7), 7.29e
7.26 (m, 2H, H-20, H-60), 6.98 (d, 2H, J ¼ 7.9 Hz, H-30, H-50), 6.87 (d,
1H, J ¼ 8.6 Hz, H-8), 5.37 (s, 2H, H-2), 3.87 (s, 3H, OMe). 13C NMR
(d, 1H, J ¼ 2.4 Hz, H-5), 7.83 (s, 1H, ]CHPh), 7.52 (dd, 1H, J ¼ 2.4 Hz,
9.0 Hz, H-7), 7.25 (d, 2H, J ¼ 8.4 Hz, H-20, H-60), 6.85 (d, 1H,
J ¼ 9.0 Hz, H-8), 6.72 (d, 2H, J ¼ 8.4 Hz, H-30, H-50), 5.43 (s, 2H, H-2),
(CDCl3)
d 181.1, 161.1, 160.0, 138.4 (2C), 132.4, 130.5, 128.1, 127.0,
3.05 (s, 6H, NMe2). 13C NMR (CDCl3)
d 180.9, 159.9, 151.6, 139.4,
79
123.6, 120.1, 114.7, 114.6, 68.1, 55.6. HRMS: calcd for C
H BrO3
17 13
[M]þ 344.0048, found 344.0053, calcd for C17
H
BrO3 [M]þ
81
138.0, 133.1, 130.4, 125.2, 124.0, 122.2, 120.0, 114.5, 112.0, 68.6, 40.3.
13
79
HRMS: calcd for C
H
18 16
BrNO2 [M]þ 357.0364, found 357.0369, calcd
346.0028, found 346.0031. IR (KBr, cmꢃ1
1263, 1030, 819.
)
g 2955, 2835, 1661, 1600,
81
for C
H ) g
BrNO2 [M]þ 359.0344, found 359.0348. IR (KBr, cmꢃ1
18 16
2984, 1657, 1600, 1288, 822.
Table 2
Biodistribution in normal mice after intravenous injection of [125I]3n (% ID/g, n ¼ 5,
mean ꢁ SD).
4.2.5. (E)-3-(4-Nitrobenzylidene)-6-bromo-chroman-4-one (3e)
Yellow solid, mp 220e221 ꢀC. Yield: 92%. 1H NMR (CDCl3)
d 8.31
(d, 2H, J ¼ 8.7 Hz, H-30, H-50), 8.13 (d, 1H, J ¼ 1.5 Hz, H-5), 7.88 (s, 1H,
]CHPh), 7.60 (dd, 1H, J ¼ 1.5 Hz, 8.7 Hz, H-7), 7.47 (d, 2H, J ¼ 8.7 Hz,
H-20, H-60), 6.91 (d, 1H, J ¼ 8.7 Hz, H-8), 5.30 (s, 2H, H-2). 13C NMR
Organ
2 min
10 min
30 min
1 h
2 h
Blood
Brain
Heart
Liver
Spleen
Lung
8.35 ꢁ 1.46
2.61 ꢁ 0.51
4.46 ꢁ 1.13
13.57 ꢁ 1.84
2.54 ꢁ 0.23
9.33 ꢁ 1.76
3.87 ꢁ 0.94 2.38 ꢁ 1.33 1.22 ꢁ 0.47 1.05 ꢁ 0.46
0.83 ꢁ 0.05 0.32 ꢁ 0.05 0.18 ꢁ 0.02 0.10 ꢁ 0.02
1.87 ꢁ 0.21 0.87 ꢁ 0.16 0.49 ꢁ 0.02 0.36 ꢁ 0.06
4.71 ꢁ 0.33 2.07 ꢁ 0.34 1.23 ꢁ 0.10 1.05 ꢁ 0.26
1.27 ꢁ 0.14 0.70 ꢁ 0.16 0.40 ꢁ 0.07 0.35 ꢁ 0.04
4.87 ꢁ 0.54 2.29 ꢁ 0.70 0.99 ꢁ 0.12 0.73 ꢁ 0.18
(CDCl3)
d 180.8, 160.4, 140.8, 139.4, 135.6, 131.2, 130.9, 130.8, 130.8,
79
124.4, 123.6, 120.5, 115.4, 67.7. HRMS: calcd for C
H
16 10
BrNO4 [M]þ
81
358.9793, found 358.9795, calcd for C16
H
BrNO4 [M]þ 360.9773,
10
found 360.9779. IR (KBr, cmꢃ1
(1026), 823.
) g (3076), 1671, 1600, 1472, 1277,
Kidney 17.89 ꢁ 2.67 10.88 ꢁ 2.28 3.36 ꢁ 0.78 1.42 ꢁ 0.25 1.00 ꢁ 0.15

C. Gan et al. / European Journal of Medicinal Chemistry 76 (2014) 125e131
129
4.2.6. (E)-3-(4-Bromobenzylidene)-6-bromo-chroman-4-one (3g)
Gray solid, mp 162e163 ꢀC. Yield: 78%. 1H NMR (CDCl3)
8.12
residue was purified by silica gel chromatography (2:1 petroleum
ether/ethyl acetate) to give 3f as a pale brown solid. Yield: 52%. Mp
d
(d, 1H, J ¼ 2.4 Hz, H-5), 7.80 (s, 1H, ]CHPh), 7.61e7.53 (m, 3H, H-7,
186e187 ꢀC. 1H NMR (DMSO-d6)
d 7.95 (s, 1H, H-5), 7.88 (s, 1H, ]
H-30, H-50), 7.18 (d, 2H, J ¼ 8.4 Hz, H-20, H-60), 6.89 (d, 1H, J ¼ 8.7 Hz,
CHPh), 7.75e7.72 (m, 1H, H-7), 7.21 (d, 2H, J ¼ 8.4 Hz, H-20, H-60),
H-8), 5.30 (s, 2H, H-2). 13C NMR (CDCl3)
d 181.0, 160.2, 138.7, 137.6,
7.10e7.07 (m, 1H, H-8), 6.65 (d, 2H, J ¼ 8.4 Hz, H-30, H-50), 5.45 (s,
133.8, 133.2, 130.6, 124.9, 123.1, 122.5, 120.4, 115.3, 113.8, 68.1.
2H, H-2). 13C NMR (CDCl3)
d 180.9, 159.8, 148.5, 139.0, 138.7, 134.1,
HRMS: calcd for C16H10Br2O2 [M]þ 393.9027, found 393.9024. IR
130.8, 126.4, 124.0, 122.2, 120.8, 115.5, 113.7, 68.3. HRMS: calcd
79
(KBr, cmꢃ1
)
g
(3065), 1671, 1600, 1472, 1279, 1072, 823.
for
C
C H
BrNO2 [M]þ 329.0051, found 329.0058, calcd for
H
16 12
16 12
81
BrNO2 [M]þ 331.0031, found 331.0034. IR (KBr, cmꢃ1
) g 3383,
4.2.7. (E)-3-(3-Bromo-4-dimethylamino-benzylidene)-6-bromo-
2962, 1671, 1600, 1472, 1276, 1024, 816.
chroman-4-one (3h)
Yellow solid, mp 123e124 ꢀC. Yield: 71%. 1H NMR (CDCl3)
d
8.09
4.2.12. (E)-3-(3,5-Dimethoxylbenzylidene)-6-bromo-chroman-4-
one (3l)
(d, 1H, J ¼ 2.4 Hz, H-5), 7.74 (s, 1H, ]CHPh), 7.52 (dd, 1H, J ¼ 8.4 Hz,
2.4 Hz, H-7), 7.50 (s, 1H, H-20), 7.20 (d, 1H, J ¼ 8.4 Hz, H-60), 7.07
(d, 1H, J ¼ 8.4 Hz, H-50), 6.86 (d, 1H, J ¼ 8.4 Hz, H-8), 5.35 (s, 2H, H-
To the suspension of 6-bromo-chromosome 1 (1.35 mmol) in
acetic acid (2.1 mL) was added HCl (0.38 mL). After 10 min, 3, 5-
dimethoxyl benzaldehyde 2l (1.35 mmol) was added with stir-
ring. Then the mixture was stirred for 2 h at room temperature, and
placed for 2 d. It was treated with saturated aqueous Na2CO3 until
alkaline, and the precipitate was filtered to give a brown solid. The
crude product obtained was subjected to column chromatography
on silica gel (CH2Cl2/methanol ¼ 20/1) to give 3l. Yield: 53%. 1H
2), 2.89 (s, 6H, NMe2). 13C NMR (CDCl3)
d 180.9, 160.1, 153.4, 138.7,
136.9, 136.3, 130.7, 130.6, 129.3, 129.2, 123.6, 120.3, 120.2, 118.1,
114.8, 68.1, 44.1. HRMS: calcd for C18H15Br2NO2 [M]þ 436.9449,
found 436.9442. IR (KBr, cmꢃ1
819.
) g 2951, 2837, 1661, 1590, 1471, 1277,
4.2.8. (E)-3-(3,4-Dimethoxylbenzylidene)-6-bromo-chroman-4-
one (3i)
NMR (CDCl3) d 8.11 (s, 1H, H-5), 7.80 (s, 1H, ]CHPh), 7.56e7.54 (m,
1H, H-7), 6.87 (d, 1H, J ¼ 8.7 Hz, H-8), 6.51 (s, 1H, H-40), 6.42 (s, 2H,
Pale brown solid, mp 175e176 ꢀC. Yield: 65%. 1H NMR (CDCl3)
H-20, H-60), 5.33 (s, 2H, H-2), 3.84 (s, 6H, OMe). 13C NMR (CDCl3)
d
8.12 (s, 1H, H-5), 7.84 (s, 1H, ]CHPh), 7.54e7.52 (m, 1H, H-7),
6.93e6.86 (m, 4H, H-8, H-20, H-50, H-60), 5.39 (s, 2H, H-2), 3.94 (s,
3H, OMe), 3.91 (s, 3H, OMe). 13C NMR (CDCl3)
181.1, 160.0, 151.2,
150.5,138.5,132.7, 131.2, 128.3,127.8, 123.6, 120.3,119.6, 115.4,114.7,
d 181.3, 161.1, 160.3 (2C), 138.8, 138.7, 136.1, 130.7, 130.6, 123.5, 120.4,
79
114.8, 108.2 (2C), 101.8, 68.1, 55.8. HRMS: calcd for C
H
BrO4 [M]þ
18 15
81
d
374.0154, found 374.0150, calcd for C18H
BrO4 [M]þ 376.0133,
15
found 376.0128. IR (KBr, cmꢃ1
1274, 825.
) g 2960, 2935, 2837, 1670, 1597, 1474,
79
68.1, 55.6, 55.4. HRMS: calcd for C
H
18 15
BrO4 [M]þ 374.0154, found
81
374.0151, calcd for C
H
BrO4 [M]þ 376.0133, found 376.0128. IR
18 15
(KBr, cmꢃ1
)
g
2960, 2833, 1664, 1601, 1474, 1257, 817.
4.3. Iododestannylation reaction
4.2.9. (E)-3-(3-Methoxylbenzylidene)-6-bromo-chroman-4-one
(3j)
(E)-3-(4-Dimethylamino-benzylidene)-6-tributylstannyl-chro-
man-4-one (3m). A mixture of 3d (286 mg, 0.87 mmol), bis(-
tributyltin) (0.55 mL), and (Ph3P)4Pd (41 mg, 0.035 mmol) in
triethylamine (28 mL) was stirred at 90 ꢀC for 6 h. The solvent was
removed, and the residue was purified by silica gel chromatography
(6:1 hexane/ethyl acetate) to give 174 mg of 3m. Yield: 28%. 1H NMR
Pale brown solid, mp 108e109 ꢀC. Yield: 81%. 1H NMR (CDCl3)
d
8.12 (s, 1H, H-5), 7.85 (s, 1H, ]CHPh), 7.56 (d, 1H, J ¼ 8.7 Hz, H-7),
7.40e7.35 (m, 1H, H-50), 6.97 (d, 1H, J ¼ 8.4 Hz, H-60), 6.90e6.84 (m,
3H, H-20, H-40, H-8), 5.35 (s, 2H, H-2), 3.85 (s, 3H, OMe). 13C NMR
(CDCl3)
d
181.3, 160.4, 160.1, 138.8, 138.6, 135.8, 130.7, 130.6, 130.2,
(300 MHz, CDCl3) d 8.09 (s, 1H, H-5), 7.82 (s, 1H, ]CHPh), 7.56e7.53
123.6, 122.7, 120.4, 115.9, 115.6, 114.9, 68.1, 55.7. HRMS: calcd for
(m, 1H, H-7), 7.27e7.25 (d, 2H, J ¼ 8.4 Hz, H-20, H-60), 6.95e6.92 (m,
1H, H-8), 6.73e6.71 (d, 2H, J ¼ 8.4 Hz, H-30, H-50), 5.41 (s, 2H, H-2),
3.04 (s, 6H, NMe2), 1.55e1.51 (m, 6H, eCH2Sn), 1.35e1.32 (m, 6H, e
CH2CH2Sn), 1.09e1.06 (m, 6H, eCH2CH2CH2Sn), 0.90e0.88 (m, 9H,
CH3).
79
81
C
H H BrO3
BrO3 [M]þ 344.0048, found 344.0046, calcd for C17
17 13 13
[M]þ 346.0028, found 346.0025. IR (KBr, cmꢃ1
1271, 1062, 817.
) g 2986, 1675, 1618,
4.2.10. (E)-3-(3-Methoxyl-4-hydroxy-benzylidene)-6-bromo-
chroman-4-one (3k)
The radioiodinated form of compound 3d was prepared from
the corresponding tributyltin precursor 3m by iododestannylation
Pale brown solid, mp 115e116 ꢀC. Yield: 67%. 1H NMR (CDCl3)
reaction. Briefly, to initiate the reaction, 100
added to a mixture of the tributyltin derivative (100
EtOH), 1.5 mCi sodium [125I]iodide (specific activity 2200 Ci/mmol),
and 100 L of 1 N HCl in a sealed vial. The reaction was allowed to
m
L of H2O2 (3%) was
d
8.10 (s, 1H, H-5), 7.81 (s, 1H, ]CHPh), 7.54 (d, 1H, J ¼ 8.7 Hz, H-7),
m
g in 100 L of
m
6.98 (d,1H, J ¼ 8.1 Hz, H-60), 6.87e6.81 (m, 3H, H-20, H-50, H-8), 5.98
(br, 1H, OH), 5.37 (s, 2H, H-2), 3.98 (s, 3H, OMe). 13C NMR (CDCl3)
m
d
181.1, 160.3, 152.8, 146.3, 138.7, 138.4, 136.0, 131.2, 130.2, 123.7,
proceed at room temperature for 10 min and terminated by addi-
tion of NaHSO3. The reaction, after neutralization with sodium bi-
carbonate, was extracted with ethyl acetate. The extract was dried
by passing through an anhydrous Na2SO4 column and was then
blown to dryness with a stream of nitrogen gas. The radioiodinated
ligand was purified by HPLC (Column: Waters Symmetry C18,
122.8, 120.2, 116.1, 115.8, 115.0, 68.2, 55.9. HRMS: calcd for
79
81
C
H H BrO4
BrO4 [M]þ 359.9997, found 359.9992, calcd for C17
17 13 13
[M]þ 361.9977, found 361.9975. IR (KBr, cmꢃ1
1669, 1589, 1517, 1472, 1264, 1035, 821.
) g 3488, 2969, 2847,
4.2.11. (E)-3-(4-Aminobenzylidene)-6-bromo-chroman-4-one (3f)
Tin (II) chloride (3.08 mmol) was added to a solution of (E)-3-(4-
nitrobenzylidene)-6-bromo-chroman-4-one (3e) (0.61 mmol) in
ethanol (25 mL). The reaction mixture was refluxed under nitrogen
for 4 h and then cooled to room temperature. After ethanol was
evaporated, 1 M NaOH was added until the mixture became basic
(pH 8e9). After extraction with ethyl acetate (50 mL ꢂ 2), the
combined organic layers were washed with brine, dried over
anhydrous Na2SO4 and filtered. The solvent was removed, and the
4.6 ꢂ 250 mm, 5
m
m; Mobile phase: MeOH/H2O ¼ 8/2; Flow
rate ¼ 1.0 mL/min). The purified ligand was stored at ꢃ20 ꢀC for the
biodistribution study.
4.4. Biological evaluation
4.4.1. Binding assay in vitro using Ab aggregates
Inhibition experiments were carried out in 12 ꢂ 75 mm boro-
silicate glass tubes according to procedures described previously

130
C. Gan et al. / European Journal of Medicinal Chemistry 76 (2014) 125e131
with some modifications. Briefly, 100
mL of aggregated Ab1e40 fibrils
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