Novel anilinophthalimide derivatives as potential probes for β-amyloid plaque in the brain

Article abstract of DOI:10.1016/j.bmc.2009.12.023

A group of novel 4,5-dianilinophthalimide derivatives has been synthesized in this study for potential use as β-amyloid (Aβ) plaque probes. Staining of hippocampus tissue sections from Alzheimer's disease (AD) brain with the representative compound 9 indicated selective labeling of it to Aβ plaques. The binding affinity of radioiodinated [¹²⁵I]9 for AD brain homogenates was 0.21 nM (K_d), and of other derivatives ranged from 0.9 to 19.7 nM, except for N-methyl-4,5-dianilinophthalimide (K_i > 1000 nM). [¹²⁵I]9 possessed the optimal lipophilicity with Log P value of 2.16, and its in vivo biodistribution in normal mice exhibited excellent initial brain uptake (5.16% ID/g at 2 min after injection) and a fast washout rate (0.56% ID/g at 60 min). The encouraging results suggest that this novel derivative of [¹²³I]9 may have potential as an in vivo SPECT probe for detecting amyloid plaques in the brain.

Full text of DOI:10.1016/j.bmc.2009.12.023

Bioorganic & Medicinal Chemistry 18 (2010) 1337–1343
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel anilinophthalimide derivatives as potential probes for b-amyloid
plaque in the brain
Xin-Hong Duan a,b, Jin-Ping Qiao c, Yang Yang a, Meng-Chao Cui a, Jiang-Ning Zhou c, Bo-Li Liu a,
*
a Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, PR China
b College of Science, Beijing Forestry University, Beijing, PR China
c Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science & Technology of China, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of novel 4,5-dianilinophthalimide derivatives has been synthesized in this study for potential use
as b-amyloid (Ab) plaque probes. Staining of hippocampus tissue sections from Alzheimer’s disease (AD)
brain with the representative compound 9 indicated selective labeling of it to Ab plaques. The binding
affinity of radioiodinated [125I]9 for AD brain homogenates was 0.21 nM (Kd), and of other derivatives
ranged from 0.9 to 19.7 nM, except for N-methyl-4,5-dianilinophthalimide (Ki > 1000 nM). [125I]9 pos-
sessed the optimal lipophilicity with Log P value of 2.16, and its in vivo biodistribution in normal mice
exhibited excellent initial brain uptake (5.16% ID/g at 2 min after injection) and a fast washout rate
(0.56% ID/g at 60 min). The encouraging results suggest that this novel derivative of [123I]9 may have
potential as an in vivo SPECT probe for detecting amyloid plaques in the brain.
Received 30 September 2009
Revised 5 December 2009
Accepted 8 December 2009
Available online 28 December 2009
Keywords:
Alzheimer’s disease
b-Amyloid plaque
Imaging probe
Binding affinity
Brain uptake
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
evident that these two phenyl rings should be essential for a
probe’s affinity to Ab plaques; on the other hand, this linker may
One of the key pathological features in the Alzheimer’s disease
(AD) brain is the presence of abundant senile plaques (SP).1 The
plaques are composed of b-sheet-containing fibrils formed by the
aggregation of short b-amyloid (Ab) peptides.2,3 As Abs are at the
centre of pathogenesis of AD, non-invasive detection of them using
imaging probes will be a powerful strategy for early diagnosis of
dementia.4
serve to a great extent as an affinity-modifier based on its flexible
spatial arrangement. For example, it contributes to the probe mol-
ecule as a planar form entering the Ab plaque’s binding pockets
where binding will take place through the p–p interaction (inter-
molecular overlapping of p-orbitals) between the probe molecule
and the aromatic side chains of Ab such as Phe 19.8,15–17 This struc-
tural information may provide the basis for a rational probe design
effort centered on identifying the core structures, which could be
utilized to make potential Ab plaque probes.
Ab plaque imaging probes applicable for positron emission
tomography (PET) or single photon emission computed tomogra-
phy (SPECT) require two essential criteria including the high
binding affinity to Ab plaques and the adequate permeability of
blood–brain barrier (BBB).5 The consideration of in vitro binding
affinity as the first prerequisite to image plaques, the plaque-bind-
ing dyes of Congo red (CR) and Thioflavin T (ThT) were conse-
quently designed to develop several types of core structures for
Ab plaque imaging probes.6–8 Among them, [11C]PIB (Ki = 4.3 nM),9
11CH3
N
S
11CH3
HO
N
H
N
HO
H
[
11C]PIB
[
11C]SB-13
[
11C]SB-13 (Ki = 1.2 nM)10 and [123I]IMPY (Ki = 15.0 nM)11 have
been clinically utilized to in vivo detect SPs in the living AD
brain12–14 (Fig. 1). It is worth noting that all these three molecules
share similarity in core structure with the presence of two phenyl
rings (aromatic section) combined with a variety of linkers, such as
a thiazole ring in [11C]PIB and an ethylene group in [11C]SB-13. It is
NC
CN
123I
N
N
18F
N
N
[
123I]IMPY
[
18F]FDDNP
* Corresponding author. Tel./fax: +86 10 5880 8891.
E-mail address: liuboli@bnu.edu.cn (B.-L. Liu).
Figure 1. Chemical structures of Ab plaque imaging probes for clinical use.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.023

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X.-H. Duan et al. / Bioorg. Med. Chem. 18 (2010) 1337–1343
In fact, research has also been underway to target new core
ity of BBB, we proceeded to design a novel MAPH core by removal
of an aniline group from MDAPH (Scheme 1). There are several
reasons for such an optimized modification as Ab-plaque probe’s
core structure. Firstly, this remaining backbone with two phenyl
rings (aromatic section) retains MDAPH’s essential structural ele-
ments which seem to be responsible for the plaque-binding affin-
ity. Secondly, such core has only a NH group of linker, whose
conformational flexibility may obtain the best fit of the probe
molecules in the binding pocket so as to maximize the binding
affinity. Thirdly, it contains decreased molecular weight, which
may increase the extent of penetration of brain tissue. In addition,
its derivatives can be prepared via a simplified coupling reaction
scheme, which facilitates introduction of a wide variety of substit-
uents including –CH3, –Br, –I, –OH, –OCH3, and the chelate used
for further radiolabeling.
Because none of Ab plaque imaging probes has been reported so
far based on this type of core structure, we initially utilized the
derivative of N-methyl-4-(4-iodoanilino)phthalimide (MAPH-I) as
a representative by performing a tissue staining assay to test
whether the compound is capable of binding specifically to Ab pla-
ques. Once it was confirmed to possess the plaque-binding poten-
tial, we then proceeded to prepare other derivatives and the
radiolabeled tracer for further biological evaluation.
structures based on this rationale for Ab plaque imaging probes.
Several potent aromatic-based b-sheet inhibitors such as Naprox-
en,18,19 Flavone20 and Curcumin,21 were currently reported as no-
vel core structures of Ab plaque probes with their derivatives
exerting good Ab affinities.22–25 Naproxen,23 for example, based
on which inhibits and binds plaque (Ki = 5.7 nM), was designed
through substitutional-group modifications to be a PET radioligand
of [18F]FDDNP (Ki = 2.7 nM) for clinical use26 (Fig. 1).
4,5-Dianilinophthalimide (DAPH), a three-phenyl-ring-bearing
compound, is recently identified as a promising lead b-sheet
inhibitor because of its strong reversal of b-sheet formation
(IC50 ꢀ 15
l
m), as well as ability to destabilize preformed fibrils.27
Thus, such anti-aggregation action was believed to involve specific
Ab plaque-binding.15,16 This prompted us to apply it as a novel
core structure for Ab plaque imaging probes. As a matter of fact,
potential probes designed for detecting Ab plaques in vivo must
also cross the intact BBB. It is generally acceptable that an Ab
plaque imaging molecule with sufficient brain uptake has to be
relatively small (mol wt < 600), lipophilic (Log P = 2–3) and neu-
tral.8,28 However, DAPH was not expected to efficiently enter the
brain because of its ionizable group of the acidic phthalimide
NH. We thus hypothesized that its N-methylated analogue
(MDAPH) should be better BBB-permeable (as shown in Scheme
1). To develop better and more versatile core structure suitable
for providing Ab plaque probes with high affinity and permeabil-
In comparison with PET, SPECT is a more widely accessible and
cost-effective technique regardless of its limitations. Consequently,
we have a strong desire to develop SPECT probes labeled with 123I
O
O
O
H
H
N
H
Ph
Ph
Ph
Ph
N
N
N
NH
N
N
H
N
H
R
O
O
O
DAPH
MAPHs
MDAPH
Scheme 1. Design considerations of DAPH derivatives.
O
O
H
N
H
N
Ph
Ph
Ph
Ph
a
N
NH
N
H
N
H
O
O
DAPH
MDAPH
O
1
O
3
O
2
C
a
b
NH
N
N
O2N
O
O
O
O
O
H
N
d
N
N
+
R
I
H2N
R
O
O
5a-e
MAPHs
4
5a. R=H, 5b. R=CH3,
5c. R=Br, 5d. R=I,
5e. R=OCH3
6. R=H, 7. R=CH3, 8. R=Br,
9. R= I, 10. R=OCH3
O
O
H
N
H
e
N
N
N
HO
O
O
O
11
10
Scheme 2. Reagents and conditions: (a) CH3I, DMF, K2CO3, rt; (b) H2SO4, HNO3, 65 °C; (c) SnCl2, HCl, H2O, rt; (d) K2CO3, Cu, DMF, 130 °C; (e) BBr3, CH2Cl2, À70 °C.

X.-H. Duan et al. / Bioorg. Med. Chem. 18 (2010) 1337–1343
1339
(T1/2, 13 h, 159 keV) or 99mTc (T1/2, 6 h, 140 keV). Reported herein
are the synthesis and biological evaluations of a novel class of
DAPH-based compounds and especially, the radioiodinated deriva-
tive as a prospective SPECT tracer for imaging amyloid plaques in
the brain.
The choice of compounds 7 and 8 for additional studies showed
nearly the same staining pattern of SPs as 9 (data not shown).
2.3. In vitro binding studies
Encouraged by above promising results, we then quantified the
binding affinities of this class of compounds to Ab plaques by an
in vitro binding assay. The binding study of [125I]9 to homogenates
of cortex tissue from postmortem AD brain (Female, 91 years old,
Superior Temporalis Gyrus) was carried out. This ligand displayed
a saturable binding, and Scatchard analysis showed highly binding
affinity to AD brain homogenates with a Kd of 0.21 0.07 nM
(Fig. 4). Binding affinities (Ki) of MAPHs (compounds 6–11), DAPH
and MDAPH for AD brain homogenates in competition with [125I]9
were also evaluated (Table 1). All the MAPHs demonstrated excel-
lent affinity to Ab plaques with Ki values ranging from 0.9 to
19.7 nM. The derivative of N-methyl-4-anilinophthalimide (6)
without a substituent at 4-position on the aniline ring showed a
desirable affinity (7.6 nM). When introducing a higher hydropho-
bic substituent such as CH3 (7, Ki = 4.1 nM), Br (8, Ki = 3.8 nM) or
I group (9, Ki = 0.9 nM), a comparable increase in binding affinity
was observed. However, the presence of a lower hydrophobic
2. Results and discussion
2.1. Chemistry
MDAPH was achieved by the methylation of DAPH (Scheme 2),
and MAPHs (6–10) containing N-methyl-4-anilinophthalimide
core was synthesized through a coupling reaction between 4 and
the suitable commercially available p-substituted iodobenzenes
(5a–e). Intermediate 4 was prepared in three steps as outlined in
Scheme 2. Phthalimide 1 was first N-methylated to form N-meth-
ylphthalimide 2, and this compound was then treated by nitration
to yield N-methyl-4-nitrophthalimide 3. Finally, reduction of 3
with SnCl2 afforded the compound of N-methyl-4-aminophthali-
mide 4. The coupling was performed by reacting intermediate 4
with 5a–e in DMF at 130 °C in the presence of Cu powder and
K2CO3 (Ref. [29] with some modifications, Scheme 2). In this
reaction, the desired compounds 6–10 were achieved in yields
ranging from 7.8% to 27.8%. However, the coupling of p-OCH3-
substituted iodobenzenes with 4 yielded appreciable amounts of
disubstituted compounds, the desired products were formed only
in negligible amounts (7.8%). It is probably because this electron-
donating group OCH3 might increase the nucleofugacity of p-posi-
tion iodo group, and the unexpected disubstituted products were
greatly achieved. The free phenol derivative, 11, was obtained from
the corresponding 10 by a demethylation reaction using BBr3
(Scheme 2). The tributyltin derivative 12 was prepared from the
bromo precursor 8 using an exchange reaction catalyzed by Pd(0)
(10.8% yield), and its radioiodination was successfully carried out
by the standard iododestannylation reaction, using hydrogen per-
oxide as the oxidant (Scheme 3). The final HPLC-purified [125I]9
showed greater than 98% radiochemical purity with high specific
activities (ꢀ2000 Ci/mmol). Identification of the peak eluting at
7.30 min was done by comparing its retention time (tR) with that
of compound 9 at 7.05 min (Fig. 2).
2.2. Staining on AD human brain sections
To assess whether these novel DAPH-based derivatives inter-
acted with Ab plaques in vitro, the tissue stainingon AD humanbrain
sections (Female, 72 years old, hippocampus) was firstly utilized to
test their specificity for Ab plaques. As shown in Figure 3, the
pretreated brain section indeed showed lack of autofluorescence
(Fig. 3, A1, A2 or A3) while a positive staining can be observed when
stained with the representative compound 9. The staining with 9
was localized in Ab plaques in hippocampus tissue sections (Fig. 3,
B3), and moreover, relatively lower background staining was ob-
served. The identity of the plaques stained with 9 was confirmed
by staining serial sections with Thioflavin S (ThS) to Ab (Fig. 3, C1,
C2 and C3). In a qualitative way, the compound demonstrated its
specific affinity for Ab plaques, even in the complex milieu of human.
Figure 2. HPLC profiles of compound 9 (top) and [125I]9 (bottom). HPLC condition:
Agilent TC-C18 column (analytical 4.6 Â 150 mm) CH3OH/H2O = 8/2, 1 mL/min,
360 nm, tR = (UV) 7.05 min, (
c
) 7.30 min.
O
O
O
H
H
N
H
N
b
a
N
N
N
N
125I
Br
Bu3Sn
O
O
O
[
125I]9
8
12
Scheme 3. Reagents and conditions: (a) (Bu3Sn)2, Pd(PPh3)4, toluene, 110 °C; (b) 1—[125I]NaI, H2O2, HCl, rt; 2—NaOH.

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X.-H. Duan et al. / Bioorg. Med. Chem. 18 (2010) 1337–1343
Figure 3. Fluorescence micrographs of AD brain sections (6
obtained under similar incubatin conditions using a concentration of 50
l
m thick) blank (A1, A2 and A3), stained with 9 (B1, B2 and B3) and Thioflavin S (C1, C2 and C3). The images were
M. The pretreated brain section shows lack of autofluorescence on any channel of RFP (A1), GFP (A2)
and DAPI (A3) while a positive staining is observed when stained with compound 9 at the DAPI (B3) channel or ThS at the channel of RFP (C1), GFP (C2) and DAPI (C3). SPs
l
labeled with compound 9 are identical to staining in a serial section with ThS. Bar = 75 lm.
group such as OCH3 (10, Ki = 16.3 nM) or OH (11, Ki = 19.7 nM)
slightly decreased the affinity. The relative binding affinity order
for this class of derivatives was I > Br > CH3 > H > OCH3 > OH. In
this study, it was shown that binding affinity of the derivatives
to Ab plaques was related to their substituent’s hydrophobicity.
More importantly, the good binding affinity of [125I]MAPH-I to-
wards Ab plaques indicates that there is obviously a large degree
of bulk tolerance in the 4-substituent moiety for in vitro binding
to the Ab plaques. As a result, this [125I]9 could serve as a model
compound for 123I-labeled SPECT imaging studies.
tion),8,15–17 and therefore result in lack of binding competition.
Indeed, recent studies demonstrated that DAPH interacts with
the Ab42 fibrils by selectively targeting their intermolecular con-
tacts (the Phe stacking), while leaving particular intramolecular
contacts intact.31 That is to say, such a characteristic on Ab fibrils
seemed different from that of MAPHs on amyloid plaques.
In addition, the competition of CR and ThT to [125I]9 binding on
AD brain homogenates was examined with high Ki values (>1000
nM), indicating no binding competition. This finding suggests that
MAPHs did not share a same binding site with CR or ThT.
Surprisingly, despite definite interaction with Ab fibrils,27 DAPH
(and its derivative MDAPH) showed in this study no specific bind-
ing signal for amyloid plaques (both Ki values >1000 nM). Like
2.4. Biodistribution study
[
11C]PIB or [11C]SB-13, [125I]9 is a long, thin and flat molecule.8
One of the key prerequisites for an in vivo Ab plaque imaging
probe is its ability to cross the intact BBB after an iv injection. Actu-
ally, a relatively high (optimal) lipophilicity should be critical for a
probe’s initial brain permeability in addition to its neutral and
small properties.32 The lipophilicity of [125I]9 was 2.16 (measured
Comparatively, DAPH has a large asymmetric propeller-shape con-
formation.30 Therefore, an possibility may be that DAPH did not
match the tracer’s binding pockets where a thin and flat conforma-
tion should be capable of entering to bind (through
p–p interac-

X.-H. Duan et al. / Bioorg. Med. Chem. 18 (2010) 1337–1343
1341
affinity, desirable kinetic properties of high brain uptake and rapid
washout in the non-Ab plaque-containing areas. Therefore, [123I]9
is most likely to be successful as a probe for Ab plaques in the
brain. A more detailed biodistribution study using transgenic mice
containing an excess amount of Ab aggregate deposition in the
brain is currently under way.
160
140
120
100
80
3. Conclusion
In summary, MAPHs represent a novel class of probes for Ab
plaques. They bound efficiently to Ab plaques and showed high
in vitro binding affinity with Ki in the nM range. In particular,
[
125I]9, because of specific in vitro labeling of amyloid plaques,
and together with its excellent BBB permeability and fast washout,
is a promising candidate probe for SPECT imaging in the brain.
60
40
4. Experimental
0
3
6
9
12
15
18
Bound (pM)
4.1. General information
Figure 4. Scatchard plot of the binding of [125I]9 to the gray matter homogenates of
AD brain. This ligand displayed highly binding with a Kd of 0.21 0.07 nM.
Unless otherwise noted, starting materials were purchased from
Sigma–Aldrich and Alfa Asia organics and were used without fur-
ther purification. [125I]NaI (2200 Ci/mmol) was obtained from
PerkinElmer Life and Analytical Sciences, Japan. The AD human
brain homogenates and paraffin brain sections were obtained
through Hefei National Laboratory for Physical Sciences at Micro-
scale and School of Life Sciences, University of Science & Technol-
ogy of China, in cooperation with the Netherlands Brain Bank
(Coordinator, Dr. I. Huitinga). 1H NMR spectra were obtained on
Avance-500 Bruker (500 MHz) spectrometer with TMS as an inter-
nal standard. Mass spectra were obtained on a GC 2000/TRACE™
(EI-MS) and LCMS-2010 (ESI-MS). Elemental analyses were ob-
tained on a Elementar Vario EI.
Table 1
Inhibition constants (Ki) of DAPHsa and of PIB and IMPY
Compound
Ki (nM)a
Compound
Ki (nM)a
6
7
8
9
10
11
7.6 0.8
4.1 0.4
3.8 0.9
0.9 0.3
16.3 4.6
19.7 5.4
DAPH
MDAPH
CR
ThT
PIB
>1000
>1000
>1000
>1000
>1000
>1000
IMPY
a
Measured using amyloid plaques in AD brain homogenates and [125I]9 as the
ligand; each value represents the mean SEM of three experiments.
4.1.1. N-Methylphthalimide (2)
by a partition between 1-octanol and pH 7.4 phosphate buffer),
with a calculated Log P value (c Log P) of 3.73 (calculated using
ACD software package version 8.0),8 indicating a potentially BBB-
permeable ability. A biodistribution study in normal mice after
an iv injection showed that [125I]9 exhibited indeed an excellent
brain uptake of 5.16% ID/g at 2 min and a fast washout rate of
0.59% ID/g at 60 min (Table 2). Moreover, the blood levels of the
compound were relatively low throughout the time course mea-
sured (2.47–2.78% ID/g).
A good initial brain uptake combined with a rapid washout in
normal mouse brain (no Ab plaques for extra binding of the probes)
is a highly desirable property for Ab plaque-targeting probes. At
present, [123I]IMPY is the most promising SPECT tracer for clinically
detecting amyloid plaques because of its desirable properties: high
binding affinity for amyloid plaques (Ki = 15 nM) and fast kinetics
of high initial brain uptake (2.88% ID/organ at 2 min) and rapid
washout (0.21% ID/organ at 60 min) in normal brain. Similar to
the radioiodinated IMPY, [125I]9 also showed high in vitro binding
Phthalimide 1 (147 mg, 1 mmol) was added to a mixture of DMF
(15 mL) and K2CO3 (13.8 mg), then a solution of CH3I (213 mg,
1.5 mmol) in DMF was added dropwise to the resulting solution.
The solvent was removed after the mixture was stirred for 5 h at
room temperature. The residue was purified by recrystallization
with methanol to give 2 as a colorless needles (79 mg, 49.2%),
mp 132–133 °C.
4.1.2. N-Methyl-4-nitrophthalimide (3)
To a solution of 2 (161 mg, 1 mmol) dissolved in H2SO4 (0.4 mL),
0.2 mL HNO3 was added dropwise over 5 min. After 1 h, the reac-
tion mixture was poured into crushed ice and then filtrated. Com-
pound 3 (183 mg, 88.9% yield) was obtained as a pale yellow
needle after recrystallization with methanol, mp 175ꢀ176 °C.
4.1.3. N-Methyl-4-aminophthalimide (4)
Compound 3 (144 mg, 0.7 mmol) was added hydrochloric acid
(2 mL) and followed by SnCl2Á2H2O (400 mg). The mixture was stir-
red at room temperature until the white solid was largely formed.
The resulting residue was washed with water and purified by
recrystallization with ethanol to afford compound 4 (90.2 mg,
73.2%) as a yellow solid, mp 242–243 °C.
Table 2
Biodistribution in normal mice after iv injection of [125I]9 (% ID/g, avg of 4 mice SD)
and its partition coefficient
Organ
2 min
10 min
30 min
60 min
[
125I]9 (log P = 2.16 0.03)
4.1.4. General procedure for preparing DAPHs (6–10)
Blood
Heart
Lung
Kidney
Spleen
Liver
Brain
2.47 0.30
7.40 1.34
6.77 1.51
9.81 1.48
5.64 0.57
11.98 1.28
5.16 1.36
2.74 0.39
3.41 0.14
6.35 1.04
6.55 0.22
6.87 0.37
14.38 0.27
3.41 0.15
2.69 0.06
2.83 0.31
3.47 0.14
3.59 0.22
3.70 0.36
5.84 0.63
1.16 0.14
2.78 0.38
2.24 0.14
2.82 0.43
2.68 0.21
2.92 0.53
3.37 0.24
0.59 0.05
The suitable 4 (176 mg, 1 mmol), p-substituted-iodobenzene
5a–e (1.1 mmol), Cu powder (64 mg, 1 mmol) and K2CO3
(138 mg, 1 mmol) in 12 mL DMF was heated to 130 °C for about
6 h. The mixture was filtered and organic layer was concentrated
under vacuum. Crude materials were purified by column chroma-
tography on silica gel (petroleum ether/AcOEt, 80/20).

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X.-H. Duan et al. / Bioorg. Med. Chem. 18 (2010) 1337–1343
4.1.5. N-Methyl-4-anilinophthalimide (6)
7.67 (1H, d, J = 8.2 Hz), 7.48 (2H, d, J = 8.2 Hz), 7.41 (1H, s), 7.18
(2H, d, J = 8.1 Hz), 7.15 (1H, d, J = 8.2 Hz), 6.22 (1H, b, NH), 3.16
(3H, s), 1.58 (6H, m), 1.37 (6H, m), 1.09 (6H, m), 0.93 (9H, m).
ESI-MS: m/z (%) = 543 [MH+].
Yield 13.6%, mp 171À172 °C. 1H NMR (500 MHz, CDCl3, d ppm)
7.68 (1H, d, J = 8.2 Hz), 7.42 (1H, s), 7.39 (2H, d, J = 7.8 Hz), 7.22
(2H, d, J = 7.7 Hz), 7.17 (1H, t, J = 7.4 Hz), 7.13 (1H, d, J = 8.2 Hz),
6.27 (1H, b, NH), 3.16 (3H, s). EI-MS: m/z (%) = 252 [M+]. Anal. Calcd
for C15H12N2O2: C, 71.42; H, 4.79; N, 11.10. Found: C, 71.41; H,
4.86; N, 10.99.
4.2. N-methyl-4-(4-[125I]iodoanilino)phthalimide ([125I]9)
One-hundred microliters of H2O2 (3%) were added to a mixture
4.1.6. N-Methyl-4-(4-methylanilino)phthalimide (7)
of 12 (100
2200 Ci/mmol), and 100
was allowed to proceed at room temperature for 10 min and termi-
nated by addition of 100 L saturated NaHSO3 solution. After neu-
l
g/100
l
L EtOH), [125I]NaI (600
l
Ci, specific activity
Yield 17.2%, mp 195–196 °C. 1H NMR (500 MHz, CDCl3, d ppm)
7.65 (1H, d, J = 8.2 Hz), 7.31 (1H, s), 7.21 (2H, d, J = 8.0 Hz), 7.12
(2H, d, J = 7.6 Hz), 7.06 (1H, d, J = 7.6 Hz), 3.15 (3H, s), 2.37 (3H,
s). EI-MS: m/z (%) = 266 [M+]. Anal. Calcd for C16H14N2O2: C,
72.16; H, 5.30; N, 10.52. Found: C, 72.12; H, 5.43; N, 10.49.
lL of 1 N HCl in a sealed vial. The reaction
l
tralized with 1 N NaOH, the mixture was loaded on a small
preconditioned C-18 Sep-pak minicolumn and rinsed sequentially
with H2O and 30% EtOH solution. Then, rinsing of this column with
4.1.7. N-Methyl-4-(4-bromoanilino)phthalimide (8)
80% EtOH solution obtained the desired product of
(380 Ci). The radiochemical purity was checked by HPLC on a
[
125I]9
Yield 27.8%, mp 204–205 °C. 1H NMR (500 MHz, CDCl3, d ppm)
7.69 (1H, d, J = 8.1 Hz), 7.50 (2H, d, J = 8.3 Hz), 7.37 (1H, s), 7.13
(1H, d, J = 8.3 Hz), 7.10 (2H, d, J = 8.4 Hz), 6.21 (1H, b, NH), 3.17
(3H, s). EI-MS: m/z (%) = 332 [MH+]. Anal. Calcd for C15H12BrN2O2:
C, 54.40; H, 3.35; N, 8.46. Found: C, 54.19; H, 3.26; N, 8.89.
l
reversed-phase column (Agilent TC-C18 analytical column, 4.6 Â
150 mm, CH3OH/H2O 8/2 flow rate 1 mL/min). This no-carrier-
added product was stored at À20 °C for in vitro binding assay
and in vivo biodistribution study.
4.1.8. N-Methyl-4-(4-iodoanilino)phthalimide (9)
4.3. Tissue staining
Yield 25.5%, mp 212–213 °C. 1H NMR (500 MHz, CDCl3, d ppm)
7.70 (1H, d, J = 7.9 Hz), 7.69 (2H, d, J = 8.3 Hz), 7.38 (1H, s), 7.14
(1H, d, J = 8.1 Hz), 6.98 (2H, d, J = 8.4 Hz), 6.21 (1H, b, NH), 3.17
(3H, s). EI-MS: m/z (%) = 378 [MH+]. Anal. Calcd for C15H11IN2O2:
C, 47.64; H, 2.93; N, 7.41. Found: C, 47.70; H, 2.57; N, 6.93.
Postmortem brain tissues from an autopsyconfirmed case of AD
were used. 6 lm-thick tissue was processed for staining according
to previously described methods.33 Firstly, paraffin sections were
taken through two 10 min washes in xylene, two 10 min washes
in 100% ethanol, a 5 min sequential wash in 95%, 90%, 80% and
70% EtOH/H2O, and a 5 min wash under MQ water and then in
PBS (0.01 M, pH 7.4). Secondly, quenching of autofluorescence
was performed by the following method: the sections were
blanched in 0.05% potassium permanganate solution for 20 min.
They were then washed three times in PBS for 5 min and treated
with 0.2% potassium metabisulfite and 0.2% oxalic acid in PBS for
1 min, followed by washing three times in PBS for 5 min. Then,
4.1.9. N-Methyl-4-(4-methoxyanilino)phthalimide (10)
Yield 7.8%, mp 173–174 °C. 1H NMR (500 MHz, CDCl3, d ppm)
7.63 (1H, d, J = 8.2 Hz), 7.20 (1H, s), 7.17 (2H, d, J = 8.6 Hz), 6.95
(3H, d, J = 8.6 Hz), 6.07 (1H, b, NH), 3.86 (3H, s), 3.14 (3H, s). EI-
MS: m/z (%) = 282 [M+]. Anal. Calcd. for C16H14N2O3: C, 68.07; H,
5.00; N, 9.92. Found: C, 68.09; H, 5.12; N 9.78.
4.1.10. N-Methyl-4-(4-hydroxyanilino)phthalimide (11)
To a solution of 10 (341 mg, 1.21 mmol) in CH2Cl2 (15 mL)
cooled to À70 °C, BBr3 (12 mL, 1 M solution in CH2Cl2) was added
dropwise. The reaction mixture was then allowed to warm to room
temperature and stirred for 12 h. The precipitate was formed after
neutralization with 1 N NaOH, then filtered and washed with
water. The compound 11 was obtained as a yellow solid by recrys-
tallized with methanol (220 mg, 68.1% yield), mp 224–225 °C. 1H
NMR (500 MHz, CDCl3, d ppm) 7.61 (1H, d, J = 8.2 Hz), 7.17 (1H,
d, J = 8.0 Hz), 7.10 (2H, d, J = 8.6 Hz), 6.94 (1H, d, J = 8.0 Hz), 6.87
(2H, d, J = 8.6 Hz), 3.12 (3H, s). EI-MS: m/z (%) = 268 [M+]. Anal.
Calcd for C15H12N2O3: C, 67.16; H, 4.51; N, 10.44. Found: C,
67.41; H, 4.81; N, 10.47.
quenched tissues were immersed in the solution of 50 lM 9 or Thi-
oflavin S. To ensure the full solubilization of the compound 40%
EtOH in PBS was used. Finally, the sections were washed in 75%
EtOH/H2O for several seconds and PBS for 15 min. Fluorescent sec-
tions were viewed using an Olympus IX 71 fluorescence micro-
scope (Olympus, Tokyo) with the following filter sets: DAPI (U-
MNUA2, Excitation Spectral Region: Ultraviolet, narrow Excitation
Range, Dichromatic Mirror Cut-On Wavelength: 400 nm); GFP (U-
MWIBA2, Excitation Spectral Region: blue, wide Excitation Range,
Dichromatic Mirror Cut-On Wavelength: 505 nm); RFP (U-MWIG2,
Excitation Spectral Region: green, wide Excitation Range, Dichro-
matic Mirror Cut-On Wavelength: 565 nm).
4.4. Preparation of brain tissue homogenates
4.1.11. N-Methyl-4,5-dianilinophthalimide (MDAPH)
The same reaction described above to prepare 2 was used, and
5.9 mg of MDAPH was obtained in a 85.8% yield from DAPH
(6.6 mg, 0.02 mmol), mp192–193 °C. 1H NMR (500 MHz, CDCl3,
d ppm) 7.64 (2H, s), 7.37 (4H, t, J = 7.7 Hz), 7.07(6H, s), 5.89 (2H,
b, NH), 3.14 (3H, s). ESI-MS: m/z (%) = 344 [MH+]. Anal. Calcd for
C21H17N3O2: C, 73.45; H, 4.99; N, 12.24. Found: C, 73.42; H, 5.08;
N, 11.99.
AD brain homogenates were prepared from dissected gray and
white matters from pooled control and AD patients in phosphate
buffered saline (PBS, pH 7.4) at the concentration of approximately
100 mg wet tissue/mL (motor-driven glass homogenizer with set-
ting of 6 for 30 s). The homogenates were aliquoted into 1-mL por-
tions and stored at À80 °C.
4.5. In vitro binding assays with AD brain homogenates
4.1.12. N-Methyl-4-(4-tributylstannylanilino)phthalimide (12)
A mixture of 8 (33.1 mg, 0.1 mmol), bis(tributyltin) (290 mg,
0.5 mmol), and Pd(Ph3P)4 (12.0 mg, 0.01 mmol) in toluene (5 mL)
was stirred at 110 °C overnight. After removing the solvent in va-
cuo, the crude products were purified by column chromatography
(petroleum ether/AcOEt, 80/20) to give 12 as an orange-colored so-
lid with a yield of 10.8% (5.8 mg). 1H NMR (500 MHz, CDCl3, d ppm)
Binding studies were carried out in 12 Â 75 mm borosilicate
glass tubes according to the method described in Ref. 34 with some
modification. For saturation study, 100 lL AD brain homogenates
were added to a mixture containing 0.01–2.0 nM of [125I]9 in PBS
(pH 7.4; final volume of 1 mL). The competition binding assays
were performed by mixing 100 lL AD brain homogenates, 100 lL

X.-H. Duan et al. / Bioorg. Med. Chem. 18 (2010) 1337–1343
1343
0.04 nM [125I]9, 100
700
fined in the presence of 100
l
L of inhibitor (3 Â 10À6–1 Â 10À10 M) and
References and notes
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This work was funded by NSFC (20871021). We thank Professor
Duanzhi Yin of Shanghai Institute of Applied Physics and Ms. Xiao-
yan Zhang of College of Life Science for providing the equipment of
the Zeiss 510 META.
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