Novel Indanone Derivatives as Potential Imaging Probes for β-Amyloid Plaques in the Brain

Article abstract of DOI:10.1002/cbic.201200223

Molecular imaging probes to detect senile plaques (SPs) might help the early diagnosis of Alzheimer's disease (AD). In this study, a novel series of indanone derivatives were synthesized and characterized. In in vitro binding studies, compound 2e exhibited a K_i value of 16 nM with a human AD brain homogenate. Although they displayed relatively low affinities for 2i and 2j-with K_i values of 99 and 237 nM, respectively-the SPs in AD brain sections were positively stained by 2j. A method for in situ micro-autoradiography of AD brain was developed in this study and showed clear labeling of SPs by [¹²⁵I]2i and [¹²⁵I]2j. Both [¹²⁵I]2i and [¹²⁵I]2j had suitable lipophilicities and displayed high initial uptake and rapid clearance from the mouse brains. Furthermore, [¹²⁵I]2i and [¹²⁵I]2j were more stable in human brain homogenates than in mouse brain homogenates. These data suggest that such indanone derivatives might represent potential amyloid imaging agents for the detection of SPs in AD.

Full text of DOI:10.1002/cbic.201200223

DOI: 10.1002/cbic.201200223
Novel Indanone Derivatives as Potential Imaging Probes
for b-Amyloid Plaques in the Brain
Jin-Ping Qiao,[a] Chang-Sheng Gan,[b] Chen-Wei Wang,[a] Jin-Fang Ge,[a] Dou-Dou Nan,[a]
Jian Pan,[b] and Jiang-Ning Zhou*[a]
Molecular imaging probes to detect senile plaques (SPs) might
help the early diagnosis of Alzheimer’s disease (AD). In this
study, a novel series of indanone derivatives were synthesized
and characterized. In in vitro binding studies, compound 2e
exhibited a Ki value of 16 nm with a human AD brain homoge-
nate. Although they displayed relatively low affinities for 2i
and 2j—with Ki values of 99 and 237 nm, respectively—the
SPs in AD brain sections were positively stained by 2j. A
method for in situ micro-autoradiography of AD brain was
developed in this study and showed clear labeling of SPs by
125I]2i and [125I]2j. Both [125I]2i and [125I]2j had suitable lipophi-
[
licities and displayed high initial uptake and rapid clearance
from the mouse brains. Furthermore, [125I]2i and [125I]2j were
more stable in human brain homogenates than in mouse brain
homogenates. These data suggest that such indanone deriva-
tives might represent potential amyloid imaging agents for the
detection of SPs in AD.
Introduction
Alzheimer’s disease (AD), the most common form of dementia,
currently afflicts more than 35 million people worldwide, and
this number is predicted to increase to 106.2 million by
2050.[1,2] Although great progress towards understanding the
disease mechanisms has been made in the last three decades,
there remains no cure for AD. The medications currently in
use, including donepezil, galantamine, rivastigmine, and mem-
antine, are primarily symptomatic treatments.[3] Early interven-
tion might delay the onset or progression of AD. However,
early treatment depends on early disease diagnosis.[4] It has
been well established that the major neuropathological charac-
teristic of AD is the presence of senile plaques (SPs), which are
composed of b-amyloid (Ab) protein aggregates, and neuro-
fibrillary tangles (NFTs), which are highly phosphorylated tau
proteins. Noninvasive detection of SPs with the aid of positron
emission tomography (PET) and single-photon-emission com-
puted tomography (SPECT) imaging tracers has been proposed
as a useful tool for the early diagnosis of AD.[5,6]
matter-nonspecific binding higher than that of [11C]PIB.[24,25]
[
123I]IMPY (1),[26–30] the first SPECT probe to be evaluated in
humans, displayed a poor signal-to-noise ratio. There is there-
fore still a need to develop more probes for the imaging of AD
brain SPs.
With advances in the study of imaging probes for SPs, the
design features of an amyloid probe can be summarized as fol-
lows: the molecule should bear at least two phenyl (or hetero-
cyclic) rings and a linker, these two rings should be in conjuga-
tion with each other, and the molecule as a whole should be
neutral and have a hydrophobic, planarized (or easily planariza-
ble) p system.[31–34]
We searched the database of clinical drugs and found that
the scaffold of donepezil matches well with the design features
described above. As shown in Scheme 2, it is composed of
a heterocyclic ring (indanone group, large dashed-line circle),
a phenyl ring (small dashed-line circle), and a linker (piperidine
group, large dashed-line box). Because the piperidine group in
donepezil, as a flexible structure, is not a proper linker,
a double bond (small dashed-line box in indanone deriva-
tives 2) was introduced as a new linker to generate a conjugat-
ed, planarized p system.
A number of small molecules—such as derivatives of nap-
roxen, thioflavin T (ThT), and Congo Red (CR)—have been de-
veloped for imaging of SPs.[7] In the last few years, some of
these small molecules have been labeled with 11C, 18F, or 123I,
and have been evaluated in PET or SPECT studies in AD pa-
tients to test their abilities to detect SPs in vivo (Scheme 1).[8–11]
The most widely studied amyloid probe to date—
[11C]PIB[12,13]—is limited for routine clinical use by the radioac-
tive decay half-life of 11C (20 min). To overcome the limitation,
there are now several amyloid probes labeled with 18F
(110 min radioactive decay half-life) under clinical develop-
ment, such as [18F]BAY94-9172,[14–16] [18F]Florbetapir ([18F]AV-
45),[17–19] and [18F]3’-F-PIB.[20–23] Of these, [18F]Florbetapir could
become the first 18F-labeled amyloid probe approved by the
FDA. These probes still have some limitations, such as white-
[a] J.-P. Qiao, C.-W. Wang, J.-F. Ge, D.-D. Nan, Prof. J.-N. Zhou
CAS Key Laboratory of Brain Function and Disease
School of Life Sciences, University of Science and Technology of China
Room 729, Huangshan Road, Hefei, Anhui, 230027 (P. R. China)
E-mail: jnzhou@ustc.edu.cn
[b] Dr. C.-S. Gan, Prof. J. Pan
Engineering Research Center of Bio-process of Ministry of Education
Hefei University of Technology
Tunxi Road No.193, Hefei, Anhui, 230009 (P. R. China)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200223.
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Imaging Probes for Plaques
was prepared by heating 2c in ethanol with the catalyst
SnCl2·2H2O. As shown in Scheme 4, 5-hydroxyindan-1-one (3 f)
reacted with 4-(dimethylamino)benzaldehyde (4d) in the pres-
ence of HOAc and HCl to provide 2l. The E configurations of
compounds 2a–o were confirmed by the chemical shifts of
1
the vinyl protons in the H NMR spectra. Because of the de-
shielding effect resulting from the neighboring carbonyl
group, the vinyl proton would be expected to give a higher
chemical shift in the E isomer (>7 ppm) than in the Z.[36]
As shown in Scheme 5, 3-bromo-4-(dimethylamino)benzalde-
hyde (4 f) was prepared by procedures described previously in
the literature.[37] Compound 2m was prepared from 3a and 4 f
in a procedure similar to that described for compound 2a. As
shown in Scheme 6, compounds 2n and 2o were synthesized
from 2g and 2h, respectively, by treatment with bis(tributyltin)
and Pd(PPh3)4 in Et3N at 808C, and compound 2i was prepared
by treatment of 2n with iodine in THF at 08C.[38]
In vitro binding assay
Radioligand competition binding assays with human AD brain
homogenates and use of [125I]1 as the radioligand were per-
formed to determine the binding affinities of the synthesized
compounds.[29] The results are presented in Table 1.
In compounds 2a–f, when the substituents R and R1 were
kept constant as methoxy groups, similarly to the indanone-
like system in donepezil, while R2 was changed, the inhibition
constant (Ki) values exhibited large variations. Compound 2e
(R2 =NMe2) displayed a higher binding affinity (Ki =16Æ1 nm)
than the others. These results suggest that these compounds
likely bind to the SPs through the ThT binding site and likely
belong to the class of ThT-type ligands.[7] Next, R2 was kept
constant as NMe2 while R and R1 were changed, and the result-
ing compounds 2e (R=R1 =OMe) and 2k (R=H, R1 =OMe,
Ki =21Æ1 nm) displayed increased binding affinities. These re-
sults indicate the importance of the NMe2 and OMe groups in
the structures, perhaps because those moieties can form hy-
drogen bonds (or enter into other interactions) with Ab aggre-
gates more easily than the others.[7,39–41] Furthermore, from the
great affinity contrast between 2m (R3 =Br, Ki >1000 nm) and
2e (R3 =H), it can be inferred that the steric bulk or other
effect of bromine at the position ortho to NMe2 can lead to
unpredictable results. Unlike in the other ThT-type ligands
described in the literature,[7] the OMe group at the R1 (or R)
position appears to be important for binding of compound 2
derivatives to Ab aggregates.
Scheme 1. Chemical structures of clinically evaluated Ab-plaque imaging
probes.
Scheme 2. Design strategy for indanone derivatives.
A series of indanone derivatives were synthesized and their
biological activities were assayed. Their potentials as amyloid
imaging agents were also evaluated both in vitro and in
vivo.[35]
None of the compound 2 derivatives exhibited anti-cholines-
terase activity (either acetylcholinesterase (AchE) or butyrylcho-
linesterase (BChE); see the Supporting Information). This might
due to the replacement of the piperidine group by a double
bond (Scheme 2).[42] The piperidine group in donepezil had
been shown to be important or even essential for anti-cholin-
esterase activity. These results indicated that as potential SP
image probes, compounds 2 might have higher specificities
for SPs and fewer side effects (those of anti-cholinesterases,
such as diarrhea, nausea, and insomnia).
Results and Discussion
Chemistry
The synthesis of target compounds 2a–c, 2e–h, 2j, and 2k is
illustrated in Scheme 3. The readily available indanones 3a–
e and benzaldehydes 4a–e were treated with aqueous NaOH
solution (10%) in ethanol at room temperature. Compound 2d
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J.-N. Zhou et al.
imaging agents, ligands must have high binding af-
finities for Ab aggregates, preferably with Ki values
less than 20 nm.[27,44] The Ki values of ligands 2i and
2j might therefore not be low enough for them to
be viable in vivo agents for Ab imaging. Nevertheless,
the excellent fluorescence staining properties of
these two compounds are encouraging. We therefore
performed radioiodination and other experiments to
characterize these two ligands further.
Radioiodination
The syntheses of the tributyltin precursors (2n and
2o) and the corresponding [125I]-labeled compounds
2i and 2j are shown in Scheme 6. Standard iododes-
tannylation reactions with [125I] sodium iodide, hydro-
gen peroxide, and hydrochloric acid were conducted
successfully.[29] Purification of the radiolabeled com-
pounds was performed by HPLC, resulting in 20–30%
overall radiochemical yields of the isolated ligands.
The chemical identities of the [125I]-labeled ligands were con-
firmed by co-injection of the labeled compounds with authen-
tic reference standards and coelution of the two compounds
on HPLC (Figure 3). The final products—[125I]2i and [125I]2j—
were shown to have radiochemical purities greater than 95%
and high specific activities (2200 CimmolÀ1).
Scheme 3. Synthesis of 2a–h, 2j, and 2k.
Partition coefficient measurement
Scheme 4. Synthesis of 2l.
The partition coefficients (P) of the two radiolabeled ligands
[
125I]2i and [125I]2j were measured by conventional octanol/
buffer partitioning. For a PET or SPECT tracer, a lipophilicity
value, as measured by logP, in the range of 1 to 3 is desira-
ble.[44,45] The logP values of [125I]2i (2.80) and [125I]2j (2.57)
might be predictive of a favorable blood/brain-barrier (BBB)
permeability.
AD brain tissue fluorescent staining
The scaffold of compound 2 generally shows good fluores-
cence quantum yields,[43] so fluorescence staining was per-
formed for qualitative visual testing of the abilities of the li-
gands to bind SPs. As shown in Figure 1, almost all the SPs in
the brain section could be labeled by ligand 2j (C1), as con-
firmed by staining with CR (A1) and thioflavin S (ThS, B1) in ad-
jacent sections and comparison with the blank control sections
(D1). The details of the staining of the SPs were clearly visual-
ized by the fluorescence of 2j (C2). Interestingly, as shown in
Figure 2, the NFTs could also be labeled by 2j (C4), similarly to
the cases of CR (A4) and ThS (B4).
Biodistributions in normal mice
To evaluate the brain uptake and biodistribution of the com-
pound 2 derivatives further, in vivo biodistribution studies in
normal Institute of Cancer Research (ICR) mice were performed
with [125I]2i and [125I]2j (Table 2). The two radiolabeled ligands
each displayed efficient BBB permeability, reaching concentra-
tions of 2.74 and 4.58%, respectively, at 2 min postinjection.
The ratios of 60 min to 2 min brain uptake values for these
two ligands were 14.5 and 9.8%, respectively, which indicated
a fast clearance from normal
In the fluorescence staining experiments, both 2j and 2i
were shown to bind to the SPs rapidly (in less than 20 min). It
has been postulated that in order to be useful as in vivo SP
mouse brains.
Stability study
In vitro stability analysis was per-
formed to test the stabilities of
the ligands in mouse and
human tissues (Figure 4).[46] As
shown in Figure 4 (see also Fig-
Scheme 5. Synthesis of 2m.
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Imaging Probes for Plaques
trol brain homogenates, respec-
tively. Similarly, 46 and 32% of
[
125I]2j remained unchanged
after 30 min in mouse whole
blood and brain homogenate,
respectively. In human AD and
control brain homogenates, 98
and 98% of
changed after 60 min and 90
and 88% of
125I]2j was un-
[
125I]2j was un-
[
changed after 120 min, respec-
tively. The protein contents of
Scheme 6. Synthesis of 2n, 2o, 2i,[125I]2i, and [125I]2j. a) Pd(PPh3)4, (SnBu3)2, Et3N; b) for 2i: I2, THF; c) for [125I]2i,
[
125I]2j: Na125I, H2O2, HCl, EtOH.
the mouse brain (7.02 mgmLÀ1),
human AD brain (12.51 mgmLÀ1),
Table 1. Inhibition constants (Ki values) of indanone derivatives upon
binding of [125I]1 to amyloid plaques in AD brain homogenates.
and control brain (10.79 mgmLÀ1) homogenates were similar,
suggesting that [125I]2i and [125I]2j might be more stable in the
human brain than in the mouse brain.
Ligand
R
R1
R2
R3
Ki ÆSEM [nm][a]
2a
2b
2c
2d
2e
2 f
2g
2h
2i
2j
2k
2l
2m
1
OMe
OMe
OMe
OMe
OMe
OMe
H
Br
H
I
H
OMe
OMe
OMe
OMe
OMe
OMe
Br
H
I
H
OMe
OH
H
Br
NO2
NH2
NMe2
OMe
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
H
H
H
H
H
H
H
H
H
H
H
H
Br
>1000
285Æ30
>1000
>1000
16Æ1
In vitro autoradiography and in situ micro-autoradiography
Autoradiography (ARG) experiments were performed to test
the abilities of the radiolabeled ligands to bind SPs in human
brain tissues in vitro. As shown in Figure 5, [125I]2i (A2 and B2)
and [125I]2j (A3 and B3) labeled the SPs in the AD brain sec-
tions in similar manner to [125I]1 (A1 and B1). High concentra-
tions (2 mm) of cold 2i or 2j could inhibit no less than 70% of
the binding signals of [125I]2i and [125I]2j, respectively (data not
shown). In addition, only a few plaques were labeled in the
control sections (C1–C3, D1–D3). These ARG data suggested
high signal-to-noise ratios and low nonspecific binding of
111Æ6
613Æ69
70Æ2
99Æ5
237Æ105
21Æ1
H
OMe
>1000
>1000
OMe
5Æ1 (5.0Æ0.4)[b]
[a] Each value was determined in three separate experiments with dupli-
cate measurements each time. [b] According to the literature.[30]
[
125I]2i and [125I]2j.
To confirm the SP labeling details further, in situ micro-ARG
ure S1 in the Supporting Information), levels of [125I]2i were 65
and 13% unchanged after 30 min of incubation in mouse
whole blood and mouse brain homogenate, respectively. In
contrast, [125I]2i was 98 and 98% unchanged after 60 min and
87 and 90% unchanged after 120 min in human AD and con-
was performed with use of double labeling (radiolabeling and
ThS fluorescence staining) to localize SPs. To the best of our
knowledge, this was the first use of in situ micro-autoradiogra-
phy to show single SPs in an amyloid imaging study. As shown
in Figure 6, the accurate colocalization of ThS fluorescence and
Figure 1. Four adjacent AD brain sections (91-year-old female, Braak 5, temporal cortex, 6 mm thickness) were stained. A) CR. B) ThS. C) 2j. D) Blank control.
The three solid arrows indicate the landmarks. The hollow arrows indicate the amyloid plaques. The images in the bottom row are magnified images of the
boxed area in the top row. Scale bars=100 mm.
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J.-N. Zhou et al.
Figure 2. Four adjacent AD brain sections (91-year old female, Braak 5, temporal cortex, 6 mm thickness) were stained. A) CR. B) ThS. C) 2j. D) Blank control.
The two solid arrows indicate the landmarks. The hollow arrows indicate NFTs. The top three rows are the three channels (DAPI, CY3, GFP) of one section.
Images A4, B4, C4, and D4 are the magnified images of the boxed area in A2, B3, C3, and D3, respectively. Scale bars=100 mm.
silver particles (C1–C3) indicated that the ARG signals from the
labeled ligands represent SPs.
The Ki values of 2i and 2j—99 and 237 nm, respectively—
showed relatively low affinities for binding to the SPs. Howev-
er, the ARG result seemed to suggest that high binding affini-
ties appear to not be necessary for [125I]2i and [125I]2j to label
SPs, due to the high densities of the SPs in the human AD
brain.[44]
Conclusions
In conclusion, we have successfully designed and synthesized
a new series of indanone derivatives as potential probes for
the in vivo imaging of AD brain amyloid plaques. Of these,
compound 2j clearly stained SPs, and the radiolabeled com-
pounds [125I]2i and [125I]2j labeled the SPs with intense signals
comparable to those provided by [125I]1. In biodistribution
studies in normal ICR mice, [125I]2i and [125I]2j displayed good
brain penetration and rapid washout from the brain—highly
desirable characteristics for in vivo amyloid imaging agents. An
in vitro stability study showed that [125I]2i and [125I]2j were
more stable in brain homogenates from humans than in those
from mice. Whereas the binding affinities and lipophilicities of
the indanone derivatives were comparable to those of IMPY,
Figure 3. HPLC chromatograms of A) compound 2i, and D) compound 2j
(blue traces), and of B) radioligand [125I]2i, and E) radioligand [125I]2j (black
traces). Merged UV and g peaks of C) 2i and F) 2j. HPLC parameters: Waters
Symmetry C18 analytical column (4.6ꢁ250 mm, 5 mm); mobile phase:
MeOH/H2O 9:1; flow rate=1.0 mLminÀ1; UV detector at 260 nm. Retention
times: tR =8.4 min for 2i (UV), tR =8.5 min for [125I]2i (g), tR =8.2 min for 2j
(UV), tR =8.3 min for [125I]2j (g). The slight retention time differences be-
tween the radioactive g peaks and the UV peaks are due to the configura-
tions of the UV and radioactivity detectors.
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Imaging Probes for Plaques
Figure 4. In vitro stabilities of [125I]2i and [125I]2j in the ICR mouse whole blood, brain, and liver homogenate and in human AD and control brain homoge-
nates. The contents of the images are as follows: A)–H) [125I]2i, I)–P) [125I]2j, A) and I) purified [125I]2i and [125I]2j, B) and J) mouse brain homogenate 30 min, C)
and K) mouse whole blood 30 min, together with human AD brain homogenate E) and M) 60 min, and F) and N) 120 min, and human control brain homoge-
nate G) and O) 60 min, and H) and P) 120 min. D) and L) represent the statistical curves of [125I]2i and [125I]2j, respectively, in the mouse whole blood, liver,
and brain homogenate at 2 to 120 min after incubation. The percentage count in the box is the ratio of the unchanged radiolabeled compound after incuba-
tion. HPLC parameters: Waters Symmetry C18 analytical column (4.6ꢁ250 mm, 5 mm); mobile phase: MeOH/H2O 9:1; flow rate=1.0 mLminÀ1; UV detector at
260 nm. Retention times: tR =8.4 min for 2i (UV) and tR =8.5 min for [125I]2i (g), tR =8.2 min for 2j (UV) and tR =8.3 min for [125I]2j (g).
Table 2. Biodistributions of [125I]2i and [125I]2j (% IDgÀ1, n=5, meansÆSDs) in normal mice after intravenous injection, together with logP values.
Organ
2 min
10 min
30 min
1 h
2 h
6 h
24 h
[
125I]2i (logP=2.80)
blood
brain
heart
liver
spleen
lung
kidney
skin
muscle
bone
4.57Æ0.14
2.74Æ0.19
9.07Æ1.22
25.39Æ3.78
4.19Æ0.87
16.13Æ1.83
10.33Æ1.05
1.61Æ0.59
2.76Æ0.07
2.53Æ0.16
2.34Æ0.36
2.16Æ0.25
2.72Æ0.45
16.06Æ1.76
2.06Æ0.45
7.42Æ1.65
4.33Æ0.61
2.40Æ0.45
2.28Æ0.40
1.45Æ0.37
1.51Æ0.33
0.83Æ0.17
1.13Æ0.18
10.92Æ2.61
1.10Æ0.10
3.39Æ0.79
4.17Æ0.57
1.93Æ0.32
1.04Æ0.15
0.76Æ0.17
1.10Æ0.20
1.27Æ0.13
0.22Æ0.05
0.81Æ0.30
6.05Æ1.17
0.89Æ0.22
2.37Æ0.44
2.04Æ0.04
2.36Æ0.30
0.69Æ0.14
0.50Æ0.13
1.00Æ0.04
0.09Æ0.01
0.37Æ0.06
5.47Æ1.79
0.61Æ0.21
1.52Æ0.64
1.31Æ0.46
0.84Æ0.21
0.35Æ0.06
0.39Æ0.17
0.26Æ0.08
0.03Æ0.02
0.10Æ0.03
1.54Æ0.22
0.12Æ0.02
0.39Æ0.13
0.30Æ0.09
0.21Æ0.06
0.06Æ0.02
0.13Æ0.04
0.40Æ0.06
0.76Æ0.14
6.45Æ1.30
0.91Æ0.20
2.71Æ0.51
2.52Æ0.80
1.63Æ0.53
0.73Æ0.16
0.54Æ0.09
[
125I]2j (logP=2.57)
blood
brain
heart
liver
spleen
lung
kidney
skin
muscle
bone
11.31Æ1.96
4.58Æ0.42
10.64Æ1.35
28.12Æ3.29
5.73Æ0.43
16.15Æ2.15
15.12Æ2.10
4.24Æ0.96
4.57Æ0.68
4.20Æ0.65
9.39Æ1.63
3.35Æ0.38
6.20Æ1.40
26.34Æ3.89
4.98Æ0.66
11.33Æ2.94
14.12Æ3.92
5.33Æ1.20
3.07Æ0.43
3.36Æ0.54
3.95Æ0.44
0.75Æ0.08
2.29Æ0.42
14.84Æ1.68
1.64Æ0.04
4.45Æ0.67
10.98Æ3.47
3.6Æ0.81
2.99Æ0.31
0.45Æ0.02
1.48Æ0.12
11.18Æ1.90
1.41Æ0.16
2.85Æ0.44
8.50Æ1.15
2.58Æ0.38
1.47Æ0.46
1.29Æ0.19
2.31Æ0.41
0.32Æ0.04
1.21Æ0.17
9.12Æ1.72
1.10Æ0.13
2.52Æ0.16
6.63Æ2.25
1.81Æ0.44
1.03Æ0.38
0.75Æ0.25
1.15Æ0.16
0.15Æ0.02
0.68Æ0.09
7.30Æ1.18
0.76Æ0.09
1.78Æ0.18
2.24Æ0.45
1.22Æ0.32
0.63Æ0.17
0.46Æ0.09
0.84Æ0.26
0.09Æ0.02
0.39Æ0.07
2.40Æ0.46
0.57Æ0.07
0.84Æ0.11
0.80Æ0.17
0.43Æ0.13
0.20Æ0.03
0.32Æ0.07
1.25Æ0.20
1.48Æ0.16
Experimental Section
the more flexible design offers extra substituent groups for la-
beling with 11C or 18F, such as 2k or 2e.
Materials and instruments: The reagents used in the syntheses
were purchased from Alfa Aesar, Sigma–Aldrich, and Sinopharm
Chemical Reagent Co. Ltd and were used without further purifica-
tion unless otherwise indicated. Compound 1 was prepared as de-
Taken together, the primary results of this study provide
a structure scaffold for the design of compounds able to bind
to Ab with high affinities and suitable for development as in
vivo imaging agents for Ab plaques.
scribed previously.[29] 125I]NaI (2200 CimmolÀ1) was purchased from
[
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1657

J.-N. Zhou et al.
conducted overnight at room temperature. The precipi-
tate was filtered to produce a yellow solid, which was re-
crystallized from ethanol to give 2a (241 mg, 86%) as
1
a pale yellow solid. M.p. 184–1868C; H NMR (CDCl3): d=
7.66–7.64 (m, 2H; H-2’, H-6’), 7.59 (s, 1H; =CHPh), 7.46–
7.43 (m, 2H; H-3’, H-5’), 7.40–7.37 (m, 1H; H-4’), 7.33 (s,
1H; H-7), 6.98 (s, 1H; H-4), 4.00 (s, 3H; OCH3), 3.96 (s,
2H; H-3), 3.95 ppm (s, 3H; OCH3); 13C NMR (CDCl3): d=
193.3, 155.6, 149.8, 145.1, 135.7, 135.6, 132.6, 131.2,
130.7, 129.5, 129.2, 129.1, 129.0, 107.3, 105.2, 56.5, 56.3,
32.3 ppm; IR (KBr): n˜ =3002, 2938, 2835, 1688, 1631,
1587, 1501, 1305, 1255, 1223, 1128, 1095, 774 cmÀ1
;
HRMS: calcd for C18H16O3: 280.1099 [M]+; found:
280.1092; elemental analysis calcd (%) for C18H16O3: C
77.12, H 5.75; found: C 76.61, H 5.82.
(E)-2-(4-Bromobenzylidene)-5,6-dimethoxyindan-1-one
(2b): Compound 2b (294 mg, 82%) was prepared from
3a (192 mg, 1 mmol) and 4b (185 mg, 1 mmol), in a pro-
cedure similar to that described for compound 2a, as
1
a white solid. M.p. 178–1808C; H NMR (CDCl3): d=7.57–
7.55 (m, 2H; H-3’, H-5’), 7.49–7.47 (m, 3H; H-2’, H-6’, =
CHPh), 7.30 (s, 1H; H-7), 6.96 (s, 1H; H-4), 3.99 (s, 3H;
OCH3), 3.94 (s, 3H; OCH3), 3.89 ppm (s, 2H; H-3);
13C NMR (CDCl3): d=193.0, 155.7, 149.8, 144.9, 136.2,
134.6, 132.3, 132.2, 132.0, 131.9, 131.8, 131.1, 123.9,
107.2, 105.2, 56.5, 56.3, 32.3 ppm; IR (KBr): n˜ =3003,
2938, 2834, 1690, 1632, 1585, 1500, 1309, 1128, 1090,
1007, 796 cmÀ1; HRMS: calcd for C18H1579BrO3: 358.0205
[M]+; found: 358.0203; HRMS: calcd for C18H1581BrO3:
360.0184 [M]+; found: 360.0175; elemental analysis calcd
(%) for C18H15BrO3: C 60.18, H 4.21; found: C 60.40, H
4.21.
Figure 5. Autoradiography of human brain sections. The top two rows (A, B) contain sec-
tions of human AD (91-year old female, Braak 5, temporal cortex), the bottom two rows
(C, D) contain sections of human control brains (C: 72-year old female, Braak 0, hippo-
campus. D: 69-year old female, Braak 1, hippocampus). Columns 1–3 are sections labeled
with [125I]1,[125I]2i, and [125I]2j, respectively. In the AD brain sections, many SPs were la-
beled by [125I]2i (A2, B2) and [125I]2j (A3, B3), relative to those by [125I]1 (A1, B1). In the
control brain sections, just a few SPs were labeled by [125I]2i (C2, D2) and [125I]2j (C3,
D3), with images similar to those provided by [125I]1 (C1, D1). The solid arrows indicate
thick and abnormal silver particles due to bubbles or creases in the tissue sections.
(E)-5,6-Dimethoxy-2-(4-nitrobenzylidene)indan-1-one
(2c): Compound 2c (289 mg, 89%) was prepared from
3a (192 mg, 1.0 mmol) and 4c (151 mg, 1.0 mmol), in
a procedure similar to that described for compound 2a,
as a yellow solid. M.p. 211–2138C; 1H NMR (CDCl3): d=
8.31 (d, J=8.7 Hz, 2H; H-3’, H-5’), 7.79 (d, J=8.7 Hz, 2H;
H-2’, H-6’), 7.61 (s, 1H; =CHPh), 7.36 (s, 1H; H-7), 7.00 (s,
1H; H-4), 4.02 (s, 3H; OCH3), 4.01 (s, 2H; H-3), 3.96 ppm
Perkin–Elmer Life and Analytical Sciences (USA). The AD human
brain homogenates and paraffin brain sections were obtained from
the Netherlands brain bank (coordinator: Dr. Inge Huitinga); per-
mission was obtained for the use of the tissue and clinical informa-
tion for research purposes. The 1H NMR spectra were obtained
with a Bruker Avance-500 (500 MHz) spectrometer with TMS as the
internal standard. High-resolution MS were obtained with Micro-
mass GCT (EI-MS, for compounds 2a–j) and Thermo Electron Pro-
teomeX LTQ instruments (ESI-MS, for compounds 2k–m). Elemental
analyses were obtained with an Elementar Vario EL III system. FTIR
spectra were obtained with a Thermo Scientific Nicolet 8700
system. Melting points were determined by use of a YRT-3 appara-
tus and capillary tubes and were uncorrected. All key compounds
were found to be of ꢀ95% purity by elemental analysis. The HPLC
system consisted of a Waters 1525 binary high-pressure gradient
pump, a 2489 variable dual wavelength ultraviolet (UV) detector,
a Bioscan Flow-Count FC-3100 NaI PMT based radioactivity detec-
tor, and Hamilton manual glass injectors.
(s, 3H; OCH3); 13C NMR (CDCl3): d=192.3, 156.1, 150.0, 147.6, 144.8,
141.9, 139.3, 130.8, 129.4, 124.1, 107.2, 105.3, 56.4, 56.2, 32.1 ppm;
IR (KBr): n˜ =3077, 2944, 2866, 1682, 1631, 1516, 1502, 1341, 1310,
1095, 1002, 850 cmÀ1; HRMS: calcd for C18H15NO5: 325.0950 [M]+;
found: 325.0947; elemental analysis calcd (%) for C18H15NO5: C
66.46, H 4.65, N 4.31; found: C 65.60, H 4.53, N 4.24.
(E)-2-(4-Aminobenzylidene)-5,6-dimethoxyindan-1-one
(2d):
Tin(II) chloride dihydrate (695 mg, 3.08 mmol) was added to a solu-
tion of 5,6-dimethoxy-2-(4-nitrobenzylidene)indan-1-one (2c,
200 mg, 0.61 mmol) in ethanol (25 mL). The reaction mixture was
heated at reflux under nitrogen for 4 h and then allowed to cool
to room temperature. Ethanol was evaporated off, and NaOH (1m)
was added until the mixture became basic (pH 8–9). After extrac-
tion with ethyl acetate (50 mLꢁ2), the combined organic layers
were washed with brine, dried over anhydrous Na2SO4, and filtered.
The solvent was removed, and the residue was purified by silica
gel chromatography (petroleum ether/ethyl acetate 2:1) to pro-
duce 2d (92 mg, 56%) as a pale brown solid. M.p. 223–2258C;
1H NMR (CDCl3): d=7.52–7.48 (m, 3H; H-2’, H-6’, =CHPh), 7.34 (s,
1H; H-7), 6.97 (s, 1H; H-4), 6.74–6.71 (m, 2H; H-3’, H-5’), 3.99 (s,
3H; OCH3), 3.94 (s, 3H; OCH3), 3.91 ppm (s, 2H; H-3); 13C NMR
(CDCl3): d=193.5, 144.7, 133.2, 132.7, 115.3, 107.3, 105.1, 56.4, 56.3,
(E)-2-Benzylidene-5,6-dimethoxyindan-1-one (2a): Indanone 3a
(192 mg, 1.0 mmol) and benzaldehyde 4a (106 mg, 1.0 mmol) were
dissolved in ethanol (10 mL). Aqueous NaOH solution (10%) was
added to this solution dropwise with stirring. The reaction was
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Imaging Probes for Plaques
(E)-5-Bromo-2-[4-(dimethylamino)benzylidene]indan-1-
one (2g): Compound 2g (256 mg, 75%) was prepared
from 3b (211 mg, 1.0 mmol) and 4d (149 mg, 1.0 mmol),
in a procedure similar to that described for compound
2a, as a brown solid. M.p. 218–2208C; 1H NMR (CDCl3):
d=7.75–7.64 (m, 3H; H-7, H-6, H-4), 7.58–7.53 (m, 3H; =
CHPh, H-2’, H-6’), 6.74–6.71 (m, 2H; H-3’, H-5’), 3.95 (s,
2H; H-3), 3.05 ppm (s, 6H; NMe2); 13C NMR (CDCl3): d=
193.0, 151.3, 151.0, 137.7, 135.7, 132.9, 130.9, 129.2,
128.9, 128.6, 125.2, 122.8, 111.9, 40.0, 32.4 ppm; IR (KBr):
n˜ =2919, 1683, 1592, 1526, 1444, 1316, 1113, 960,
882 cmÀ1; HRMS: calcd for C18H1679BrNO: 341.0415 [M]+;
found: 341.0417; HRMS: calcd for C18H1681BrNO: 343.0395
[M]+; found: 343.0392; elemental analysis calcd (%) for
C18H16BrNO: C 63.17, H 4.71, N 4.09; found: C 61.69, H
4.54, N 3.94.
(E)-6-Bromo-2-[4-(dimethylamino)benzylidene]indan-1-
one (2h): Compound 2h (263 mg, 77%) was prepared
from 3c (211 mg, 1.0 mmol) and 4d (149 mg, 1.0 mmol),
in a procedure similar to that described for compound
2a, as a brown solid. M.p. 192–1948C; 1H NMR (CDCl3):
d=8.00 (s, 1H; H-7), 7.67–7.64 (m, 2H; H-5, =CHPh), 7.57
(d, J=8.8 Hz, 2H; H-2’, H-6’), 7.41 (d, J=8.0 Hz, 1H; H-4),
6.72 (d, J=8.8 Hz, 2H; H-3’, H-5’), 3.91 (s, 2H; H-3),
3.06 ppm (s, 6H; NMe2); 13C NMR (CDCl3): d=193.0,
151.5, 148.0, 140.9, 136.6, 136.2, 133.2, 129.4, 127.7,
127.2, 123.0, 121.8, 112.0, 40.3, 32.6 ppm; IR (KBr): n˜ =
2893, 1681, 1589, 1523, 1462, 1363, 1188, 1169, 1115,
810 cmÀ1; HRMS: calcd for C18H1679BrNO [M]+ 341.0415;
found: 341.0409; HRMS: calcd for C18H1681BrNO: 343.0395
Figure 6. Autoradiography of human AD brain sections (91-year old female, Braak 5, tem-
poral cortex). Columns 1–3: sections labeled with [125I]1,[125I]2i, and [125I]2j, respectively.
Rows A–C represent images from the ThS fluorescent staining and in situ micro-autora-
diography, as well as the merged images. The white solid arrows indicate the landmarks.
The black hollow arrows indicate SPs. The SPs are labeled by [125I]2i (B2) or [125I]2j (C2),
and the images are comparable to those labeled by [125I]1 (A2). Furthermore, the senile
plaques are co-labeled by ThS and merged with the silver particles very well (C1, C2, C3).
Scale bar=100 mm.
[M]+; found: 343.0387; elemental analysis calcd (%) for C18H16BrNO:
32.5 ppm; IR (KBr): n˜ =3427, 3348, 2929, 1671, 1578, 1499, 1304,
1130, 1099, 1004, 819 cmÀ1; HRMS: calcd for C18H17NO3: 295.1208
[M]+; found: 295.1201; elemental analysis calcd (%) for C18H17NO3:
C 73.20, H 5.80, N 4.74; found: C 72.12, H 5.95, N 4.53.
C 63.17, H 4.71, N 4.09; found: C 62.58, H 4.63, N 3.98.
(E)-2-[4-(Dimethylamino)benzylidene]-5-iodoindan-1-one (2i): A
solution of iodine (53 mg, 0.20 mmol) in THF (2 mL) was added
dropwise to a cooled (ice bath) solution of 2n (75 mg, 0.14 mmol)
in THF (3 mL). After the addition, the mixture was stirred at 08C for
1 h and the reaction was terminated by the addition of saturated
NaHSO3 solution (5 mL). The reaction mixture was extracted with
ethyl acetate (3ꢁ10 mL) after neutralization with saturated
NaHCO3 solution (5 mL). The combined organic layers were dried
over Na2SO4 and filtered. The solvent was removed, and the resi-
due was purified by silica gel chromatography (petroleum ether/
ethyl acetate 6:1) to produce 2i (42 mg, 77%) as a brown solid.
(E)-2-[4-(Dimethylamino)benzylidene]-5,6-dimethoxyindan-1-one
(2e): Compound 2e (258 mg, 80%) was prepared from 3a
(192 mg, 1.0 mmol) and 4d (149 mg, 1.0 mmol), in a procedure
similar to that described for compound 2a, as a light brown solid.
1
M.p. 206–2088C; H NMR (CDCl3): d=7.59–7.57 (m, 3H; H-2’, H-6’,
=CHPh), 7.34 (s, 1H; H-7), 6.98 (s, 1H; H-4), 6.75–6.73 (m, 2H; H-3’,
H-5’), 3.99 (s, 3H; OCH3), 3.95 (s, 3H; OCH3), 3.92 (s, 2H; H-3),
3.05 ppm (s, 6H; NMe2); 13C NMR (CDCl3): d=193.4, 154.6, 150.9,
149.3, 144.3, 133.4, 132.4, 131.6, 130.5, 123.3, 111.9, 107.1, 104.9,
56.2, 56.1, 40.1, 32.3 ppm; IR (KBr): n˜ =2923, 1676, 1596, 1524,
1303, 1122, 1001, 817 cmÀ1. HRMS: calcd for C20H21NO3: 323.1521
[M]+; found: 323.1513; elemental analysis calcd (%) for C20H21NO3:
C 74.28, H 6.55, N 4.33; found: C 72.33, H 6.42, N 4.63.
1
M.p. 203–2058C; H NMR (CDCl3): d=7.95 (s, 1H; H-4), 7.77 (m, 1H;
H-6), 7.66 (s, 1H; =CHPh), 7.62–7.58 (m, 3H; H-2’, H-6’, H-7), 6.78
(d, J=7.9 Hz, 2H; H-3’, H-5’), 3.96 (s, 2H; H-3), 3.07 ppm (s, 6H;
NMe2); 13C NMR (CDCl3): d=193.6, 151.1, 138.4, 137.0, 135.9, 135.5,
133.1, 125.5, 112.4, 101.9, 40.5, 32.4 ppm; IR (KBr): n˜ =2898, 1682,
1590, 1525, 1312, 1111, 959, 810 cmÀ1; HRMS: calcd for C18H16INO:
389.0277 [M]+; found: 389.0271; elemental analysis calcd (%) for
C18H16INO: C 55.54, H 4.14, N 3.60; found: C 52.86, H 3.96, N 3.42.
(E)-5,6-Dimethoxy-2-(4-methoxybenzylidene)indan-1-one
(2 f):
Compound 2 f (242 mg, 78%) was prepared from 3a (192 mg,
1.0 mmol) and 4e (136 mg, 1.0 mmol), in a procedure similar to
that described for compound 2a, as a yellow solid. M.p. 188–
1
(E)-2-[4-(Dimethylamino)benzylidene]-6-iodoindan-1-one
(2j):
1908C; H NMR (CDCl3): d=7.63–7.62 (d, J=8.6 Hz, 2H; H-2’, H-6’),
Compound 2j (295 mg, 76%) was prepared from 3d (258 mg,
1.0 mmol) and 4d (149 mg, 1 mmol), in a procedure similar to that
described for compound 2a, as a brown solid. M.p. 223–2258C;
1H NMR (CDCl3): d=8.20 (s, 1H; H-7), 7.85–7.83 (m, 1H; H-5), 7.63
(s, 1H; =CHPh), 7.55 (d, J=8.8 Hz, 2H; H-2’, H-6’), 7.30–7.26 (m,
1H; H-4), 6.71 (d, J=8.8 Hz, 2H; H-3’, H-5’), 3.88 (s, 2H; H-3),
3.05 ppm (s, 6H; NMe2); 13C NMR (CDCl3): d=192.9, 151.5, 148.7,
142.3, 141.1, 136.1, 133.3, 133.1, 129.1, 128.0, 123.0, 112.1, 92.9,
40.3, 32.6 ppm; IR (KBr): n˜ =2851, 1678, 1585, 1522, 1362, 1308,
1263, 1187, 1116, 809 cmÀ1; HRMS: calcd for C18H16INO: 389.0277
7.57 (s, 1H; =CHPh), 7.35 (s, 1H; H-7), 6.99–6.97 (m, 3H; H-3’, H-5’,
H-4), 3.99 (s, 3H; OCH3), 3.95–3.94 (m, 5H; OCH3, H-3), 3.87 ppm (s,
3H; OCH3); 13C NMR (CDCl3): d=193.3, 160.6, 155.2, 149.5, 144.7,
133.1, 132.3, 131.2, 128.3, 114.4, 107.2, 105.0, 56.3, 56.2, 55.4,
32.2 ppm; IR (KBr): n˜ =2941, 2834, 1685, 1605, 1504, 1299, 1258,
1091, 820 cmÀ1; HRMS: calcd for C12H18O4: 310.1205 [M]+; found:
310.1202; elemental analysis calcd (%) for C12H18O4: C 73.53, H
5.85; found: C 70.27, H 5.58.
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J.-N. Zhou et al.
[M]+; found: 389.0279; elemental analysis calcd (%) for C18H16INO:
C 55.54, H 4.14, N 3.60; found: C 52.71, H 4.07, N 3.36.
1.51 (m, 6H; SnBu3), 1.41–1.29 (m, 6H; SnBu3), 1.14–1.08 (m, 6H;
SnBu3), 0.92–0.90 ppm (t, J=7.2 Hz, 9H; SnBu3); IR (film): n˜ =2955,
2925, 1687, 1624, 1595, 1453, 1091, 882 cmÀ1; MS (ESI): m/z: 528.28
[M+H]+.
(E)-2-[4-(Dimethylamino)benzylidene]-5-methoxyindan-1-one
(2k): Compound 2k (249 mg, 85%) was prepared from 3e
(162 mg, 1.0 mmol) and 4d (149 mg, 1 mmol), in a procedure simi-
lar to that described for compound 2a, as a light brown solid. M.p.
(E)-2-[4-(Dimethylamino)benzylidene]-6-(tributylstannyl)indan-1-
one (2o): Compound 2o (106 mg, 33%) was prepared from 2h
(200 mg, 0.59 mmol), in a procedure similar to that described for
1
204–2058C; H NMR (CDCl3): d=7.83 (d, J=8.4 Hz, 1H; H-7), 7.59–
1
7.56 (m, 3H; H-2’, H-6’, =CHPh), 6.99 (m, 1H; H-4), 6.96–6.92 (dd,
J=8.4 Hz, 1.8 Hz, 1H; H-6), 6.74 (d, J=8.7 Hz, 2H; H-3’, H-5’), 3.95
(s, 2H; H-3), 3.90 (s, 3H; OCH3), 3.05 ppm (s, 6H; NMe2); 13C NMR
(CDCl3): d=193.0, 164.7, 152.2, 151.0, 133.7, 132.5, 132.1, 130.4,
125.7, 123.3, 114.8, 111.9, 109.7, 55.6, 40.1, 32.8 ppm; IR (KBr): n˜ =
3441, 2995, 2935, 1675, 1579, 1544, 1303, 1120 cmÀ1; HRMS: calcd
for C19H19NO2: 293.1416 [M+H]+; found: 294.1491; elemental analy-
sis calcd (%) for C19H19NO2: C 77.79, H 6.53, N 4.77; found: C 76.59,
H 6.56, N 4.73.
compound 2n, as a brown oil; H NMR (CDCl3): d=8.01 (s, 1H; H-
7), 7.66–7.64 (m, 2H; H-5, H-4), 7.61–7.58 (d, J=9.0 Hz, 2H; H-2’, H-
6’), 7.53 (s, 1H; =CHPh), 6.75–6.72 (d, J=9.0 Hz, 2H; H-3’, H-5’),
3.97 (s, 2H; H-3), 3.04 (s, 6H; NMe2), 1.58–1.49 (m, 6H; SnBu3),
1.37–1.30 (m, 6H; SnBu3), 1.13–1.07 (m, 6H; SnBu3), 0.91–0.89 ppm
(t, J=7.2 Hz, 9H; SnBu3); IR (film): n˜ =2925, 1735, 1686, 1596, 1524,
1458, 1109, 882 cmÀ1
.
Preparation of radioiodinated ligands: The radioiodinated ligands
125I]2i, [125I]2j, and [125I]1 were prepared through iododestannyla-
[
(E)-2-[4-(Dimethylamino)benzylidene]-5-hydroxyindan-1-one
(2l): Concentrated HCl (0.38 mL) was added to a suspension of 5-
hydroxyindan-1-one (3 f, 200 mg, 1.35 mmol) in acetic acid
(2.1 mL), followed after 10 min of stirring at room temperature by
4-(dimethylamino)benzaldehyde (4d, 201 mg, 1.35 mmol). The mix-
ture was stirred for 2 h at room temperature, allowed to stand for
2 days, and made basic with saturated aqueous Na2CO3. The result-
ing precipitate was collected and purified by column chromatogra-
phy on silica gel (CH2Cl2/methanol 20:1) to produce 2l (244 mg,
tion reactions from the corresponding tributyltin precursors (2n,
2o, and the tributyltin precursor of [125I]1) by the methods de-
scribed previously.[26,47,48] A solution of H2O2 (3%, w/v, 100 mL) was
added to a mixture of the tributyltin precursor solution (100 mg in
100 mL of EtOH), HCl (1n, 100 mL), and no-carrier-added [125I]NaI
solution (1–2 mCi, specific activity of 2200 CimmolÀ1) in a sealed
vial. The reaction was conducted at room temperature (258C) for
10 min and terminated by the addition of saturated NaHSO3 solu-
tion (200 mL). After neutralization with saturated NaHCO3 solution,
the reaction mixture was extracted with ethyl acetate (3ꢁ1 mL,
chromatographically pure). The combined organic extracts were
dried by blowing with a stream of nitrogen gas. The residue was
redissolved in MeOH (0.4 mL) and purified by reversed-phase HPLC
(column: Waters Symmetry C18, 4.6ꢁ250 mm, 5 mm; mobile
phase: MeOH/H2O 9:1 for [125I]2i and [125I]2j, or 8:2 for [125I]1; flow
rate=1.0 mLminÀ1). The fraction containing the labeled compound
was collected and checked for radiochemical purity by analytical
HPLC and for identity by co-injection with a reference standard.
The collected fraction was then dried by blowing with a stream of
nitrogen gas, and the residue was redissolved in EtOH (100%) and
stored at À208C until use. Prior to its use in the animal biodistribu-
tion studies, in vitro AD homogenate binding assays, partition co-
efficient determination, stability study, and autoradiography (ARG)
experiments, the radioligand was repurified by dilution with H2O
and passage through a Sep-Pak C18 column. After washing of the
column with H2O (2 mL) and EtOH (30%, 2 mL), the desired prod-
ucts were then eluted either with EtOH (90%, 1 mL, [125I]2i or
1
65%) as a brown solid. M.p. 252–2558C; H NMR ([D6]DMSO): d=
7.58–7.53 (m, 3H; H-7, H-2’, H-6’), 7.29 (s, 1H; =CHPh), 6.87 (s, 1H;
H-4), 6.80–6.75 (m, 3H; H-6, H-3’, H-5’), 3.88 (s, 2H; H-3), 3.63 (br,
1H; OH), 3.00 ppm (s, 6H; NMe2); 13C NMR ([D6]DMSO): d=190.9,
166.1, 152.5, 150.7, 132.1, 131.5, 131.3, 130.9, 128.4, 125.3, 122.7,
116.6, 112.1, 111.0, 40.1, 32.0 ppm; IR (KBr): n˜ =3151, 2892, 1666,
1577, 1526, 1296, 1189, 1091 cmÀ1; HRMS: calcd for C18H17NO2:
279.1259 [M+H]+; found: 280.1330; elemental analysis calcd (%)
for C18H17NO2: C 77.40, H 6.13, N 5.01; found: C 75.75, H 6.21, N
4.89.
(E)-2-[3-Bromo-4-(dimethylamino)benzylidene]-5,6-dimethoxyin-
dan-1-one (2m): Compound 2m (305 mg, 76%) was prepared
from 3a (192 mg, 1.0 mmol) and 3-bromo-4-(dimethylamino)ben-
zaldehyde (228 mg, 1.0 mmol), in a procedure similar to that de-
scribed for compound 2a, as a yellow solid. M.p. 157–1598C;
1H NMR (CDCl3): d=7.88 (d, J=1.8 Hz, 1H; H-2’), 7.53 (dd, J=
1.8 Hz, 8.4 Hz, 1H; H-6’), 7.48 (s, 1H; =CHPh), 7.34 (s, 1H; H-7), 7.09
(d, J=8.4 Hz, 1H; H-5’), 7.01 (s, 1H; H-4), 4.01 (s, 3H; OCH3), 3.96
(s, 3H; OCH3), 3.95 (s, 2H; H-3), 2.89 ppm (s, 6H; NMe2); 13C NMR
(CDCl3): d=192.9, 155.4, 152.6, 149.7, 144.6, 136.0, 135.7, 134.5,
131.1, 130.9 (2C), 130.7, 120.1, 118.2, 107.2, 105.1, 56.3, 56.2, 43.8,
32.0 ppm; IR (KBr): n˜ =3439, 2939, 2881, 1692, 1591, 1503, 1303,
1124 cmÀ1; HRMS: calcd for C20H2079BrNO3: 401.0627 [M+Na]+;
found: 424.0517; HRMS: calcd for C20H2081BrNO3: 403.0606 [M+Na]+
; found: 426.0497; elemental analysis calcd (%) for C20H20BrNO3: C
59.71, H 5.01, N 3.48; found: C 56.81, H 4.73, N 3.33.
[
125I]2j) or with EtOH (80%, 1 mL, [125I]1).
Preparation of brain tissue homogenates: Several frozen AD
brain tissues (stored at À808C) were thawed and placed on ice.
The gray and white matters were dissected, weighed, and homo-
genized in ice-cooled phosphate-buffered saline (PBS) solution
(0.05m, pH 7.4) at a concentration of approximately 100 mg wet
tissue per mL with the aid of a motor-driven homogenizer (Glas-
Col, USA, 30 rpm, 2 min). The homogenates were aliquoted into
1 mL portions and stored at À808C. For binding assays, the homo-
genates were thawed, diluted with ice-cold PBS solution (0.05m,
pH 7.4), and homogenized again with the aid of an ice-cold hand-
held glass homogenizer.
(E)-2-[4-(Dimethylamino)benzylidene]-5-(tributylstannyl)indan-1-
one (2n): A mixture of 2g (400 mg, 1.17 mmol), bis(tributyltin)
(4.0 g, 7.02 mmol), and Pd(PPh3)4 (135 mg, 0.12 mmol) in Et3N
(15 mL) was stirred at 808C under nitrogen for 16 h. The solvent
was removed, and the residue was purified by silica gel chroma-
tography (petroleum ether/ethyl acetate 8:1) to produce 2n
(176 mg, 27%) as a brown oil; 1H NMR (CDCl3): d=7.82 (d, J=
7.5 Hz, 1H; H-7), 7.68–7.64 (m, 2H; =CHPh, H-4), 7.62–7.59 (d, J=
9.0 Hz, 2H; H-2’, H-6’), 7.52 (d, J=7.5 Hz, 1H; H-6), 6.75–6.72 (d, J=
9.0 Hz, 2H; H-3’, H-5’), 3.99 (s, 2H; H-3), 3.04 (s, 6H; NMe2), 1.61–
In vitro binding assays with AD brain homogenates: Radioligand
competition binding assays were conducted in borosilicate glass
tubes (12ꢁ75 mm, VWR International, USA), by the method de-
scribed previously, with some modifications.[30,47,49] The AD brain
homogenates in PBS (0.05m, pH 7.4,100 mL), a solution of the
radioligand {[125I]1, 0.05 nm diluted with PBS/BSA [bovine serum al-
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Imaging Probes for Plaques
bumin (BSA, 0.1%) in PBS (0.05m, pH 7.4)], 100 mL}, a solution of
the inhibitor ligand [3ꢁ10À5–1ꢁ10À9 m, diluted in a solution con-
taining ethanol (9%) and DMSO (1%) in PBS/BSA, 100 mL], and
PBS/BSA (700 mL), for a final volume to 1 mL, were combined in
a glass tube. The nonspecific binding was defined in the presence
of unlabeled 1 (6ꢁ10À7 m final concentration, 100 mL) in the same
assay tubes. The mixture was incubated at 378C in a water bath
shaker (60 rpm) for 2 h. The bound and free radioactivity were sep-
arated by vacuum filtration through Whatman GF/B filters [im-
mersed with polyethylenimine (PEI, 1%) in PBS on ice] by use of
a ZT-II-12R cell harvester (Satellite Medical Equipment, Shaoxing,
China) and washed with ice-cold PBS solution (3ꢁ3 mL). The filters
containing the bound radioligand were counted in a gamma coun-
ter (USTC Zonkia GC-1200, Hefei, China) with a 65% counting effi-
ciency. The values for the half-maximum inhibition concentrations
(IC50) were computed from the displacement curves of three inde-
pendent experiments with use of GraphPad Prism 5. In addition,
the Ki values were calculated by use of the Cheng–Prusoff equa-
tion: Ki =IC50/(1+[L]/Kd),[50] where [L] is the concentration of radio-
ligand used in the assay and Kd is the dissociation constant of the
radioligand.
predetermined time points (2, 10, 30, 60, 120, 360, and 1440 min).
The organs of interest were removed and weighed, and the radio-
activity was counted with a gamma counter. The tissue radioactivi-
ty concentrations (% IDgÀ1
) were calculated by dividing the
sample counts per gram of tissue by the counts from 1% of the
initially injected dose (100-fold diluted aliquots of the injected
material).
Stability study: The in vitro stabilities of [125I]2i or [125I]2j were de-
termined by the procedures described in the literature, with minor
modifications.[46,53] ICR mice (18–25 g, SPF) were anesthetized by
peritoneal injection of pentobarbital sodium (50 mgkgÀ1). After
thoracotomy, blood samples were obtained by cardiac puncture
and mixed with heparin (Heaplink, Shenzhen, China). The mice
were then perfused with cold saline (0.9%, w/v, 40 mL) through
the exposed right atrium of the heart while the aorta was severed.
The excised brain and liver were homogenized with cold saline as
they cooled on ice with use of a motor-driven homogenizer
(30 rpm, 2 min). The human AD and control brain homogenates
were prepared similarly to the method described above for the re-
ceptor binding assay, with some modifications: the gray and white
matter were mixed together, the homogenate concentration was
higher than that in the binding assay, and saline (0.9%, w/v) was
used in place of the PBS. The protein concentrations of all the sam-
ples were determined by the bicinchoninic acid (BCA) method.[54]
AD brain tissue fluorescent staining: Brain tissues were obtained
from several autopsy-confirmed AD subjects. Adjacent tissue sec-
tions (6 mm thickness) were processed for staining by the methods
described in the literature, with some modifications.[30,47,51] Firstly,
the paraffinized brain sections were treated with washes in xylene
(2ꢁ10 min), washes in EtOH (100%, 2ꢁ10 min), sequential washes
in EtOH (95, 90, 80 and 70%, 5 min), and sequential rinses (5 min
each) in Milli-Q water and PBS (0.01m, pH 7.4). Secondly, the auto-
fluorescence was quenched as described previously.[47,52] The sec-
tions were blanched in potassium permanganate solution (0.25%)
for 20 min, washed in PBS, and treated with potassium metabisul-
fite (0.1%) and oxalic acid (0.1%) in PBS, followed by washing in
PBS. The quenched brain tissue sections were immersed in a solu-
tion of cold ligand [50 mm diluted in EtOH (30%) in PBS (0.1m),
20 min], ThS (1% in Milli-Q water, 5 min), or CR [0.5% in EtOH/H2O
(50%), 10 min]. Thirdly, the sections were differentiated with EtOH/
H2O (50%) for 10 min (cold ligand), EtOH/H2O (70%) for 10 min
(ThS), or KOH (0.2%) in EtOH/H2O (80%) for 10 min (CR). Finally,
the sections were washed in PBS (3ꢁ5 min) and sealed with glycer-
in/PBS (80%) and coverslips. The sections were stored at 48C in
the dark and viewed with an Olympus IX71 fluorescence micro-
scope (Olympus, Tokyo, Japan) and a SPOT digital camera (Diag-
nostic Instruments, USA).
The in vitro stabilities were determined by incubation of purified
[
125I]2i or [125I] 2j (10 mCi) in solutions of 50 mL mouse whole blood,
liver and brain or human AD and control brain homogenates at
378C for 2, 10, 30, 60 and 120 min. The proteins were precipitated
by addition of MeOH (200 mL), and the MeOH samples were count-
ed in a g-counter and centrifuged at 13000 rpm for 20 min. The su-
pernatant was analyzed by HPLC (under the same HPLC conditions
as described above in the section on the preparation of radioiodi-
nated ligands). The precipitates were measured for radioactivity
with a g-counter to allow calculation of the radioactivity that was
recovered from the MeOH.
AD brain sections autoradiography (film and in situ micro-auto-
radiography): The preparation procedures for ARG of tissue sec-
tions (6 mm thickness) were the same as those for the staining ex-
periments above. The tissue sections were incubated with solu-
tions of [125I]2i,[125I]2j, or [125I]1 [0.3 nm in PBS (0.05m, pH 7.4)] at
378C for 1 h. The sections labeled with [125I]1 were processed by
procedures described in the literature.[30] Sections labeled with
[
125I]2i or [125I]2j were washed with PBS for 30 s and differentiated
by washing with EtOH/H2O (70%) for 5 s and rinsing with Milli-Q
water for 1 min. After drying in a fume hood, all labeled sections
were affixed to X-ray films (Kodak BioMax MR) and exposed for dif-
ferent lengths of time. When optimal images were obtained, the
sections were dipped in an emulsion (Amersham Hypercoat Emul-
sions, RPN40, USA) for in situ micro-ARG. The exposure times were
adjusted to obtain optimal images. Finally, all sections were also
stained with ThS or CR, stored at 48C in the dark, and viewed by
fluorescence microscopy by the procedures described above.
Determination of partition coefficients: The partition coefficients
were measured by mixing ligand [125I]2i or [125I]2j with 3 g each of
octan-1-ol and PBS (0.1m, pH 7.4) in a test tube. The test tube was
vortexed for 3 min at room temperature, followed by centrifuga-
tion for 5 min. Several weighed samples (ca. 0.5 g each) drawn
from the octan-1-ol and buffer layers were counted in a gamma
counter. The partition coefficient was determined by calculating
the logarithm of the ratio of cpmgÀ1 of the octan-1-ol to that of
PBS. The sample from the octan-1-ol layer was repartitioned until
consistent values were obtained. The measurements were per-
formed in triplicate and repeated three times.
Acknowledgements
Organ biodistribution in normal mice: The animal experiments
were performed by the protocol approved by the ethics commit-
tee of the School of Life Science, University of Science and Technol-
ogy of China. A saline solution (0.9%, 100 mL) containing [125I]2i or
We thank Prof. Mei-Ping Kung, Prof. Yi-Yun Huang, Prof. Lin Zhu,
Dr. Ming-Qiang Zheng and Dr. Xin-Hong Duan for their technical
training, advice, and reviewing. We thank Dr. Shi-Cun Wang for
his kind assistance. This work was supported by grants from the
Nature Science Foundation of China (81030026) and the Ministry
[
125I]2j (2 mCi) was injected into the tail vein of a male ICR mouse
[average weight 20–30 g, fed in specific-pathogen-free (SPF)
cages]. The mice (n=5 for each time point) were euthanized at
ChemBioChem 2012, 13, 1652 – 1662
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www.chembiochem.org
1661

J.-N. Zhou et al.
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Keywords: Alzheimer’s disease
imaging agents · indanones · senile plaques
· amyloid beta-peptides ·
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Received: April 1, 2012
Published online on July 6, 2012
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