Synthesis and biological evaluation of radioiodinated quinacrine-based derivatives for SPECT imaging of Aβ plaques

Article abstract of DOI:10.1016/j.ejmech.2012.12.020

The aim of the present study was to characterize the binding property of quinacrine-based acridine derivatives for Aβ plaques and to evaluate this series of compounds as Aβ imaging probes. Quinacrine clearly stained Aβ plaques in the brain sections of Aβ

Full text of DOI:10.1016/j.ejmech.2012.12.020

European Journal of Medicinal Chemistry 60 (2013) 469e478
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of radioiodinated quinacrine-
based derivatives for SPECT imaging of Ab plaques
*
**
Takeshi Fuchigami , Nobuya Kobashi, Mamoru Haratake, Masao Kawasaki, Morio Nakayama
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of the present study was to characterize the binding property of quinacrine-based acridine
Received 10 July 2012
Received in revised form
29 October 2012
Accepted 8 December 2012
Available online 16 December 2012
derivatives for A
clearly stained A
b
plaques and to evaluate this series of compounds as A
b imaging probes. Quinacrine
b
plaques in the brain sections of A deposition model transgenic mice (Tg2576 mice).
b
Similarly, the quinacrine analog, 2-methoxy-9-(4-(dimethyl-1-methyl) -N-butyl) amino-6-iodo acridine
(5), labeled A
b
plaques in the brain slices of Tg2576 mice. In addition, [125I]5 showed modest affinity for
Ab(1e42) aggregates with a Kd value of 48 nM. Biodistribution studies using normal mice demonstrated
that [125I]5 displayed poor initial brain uptake. Next, 125I-labeled acridines without aliphatic amino
groups were synthesized and characterized. Similar to quinacrine and 5, these compounds could detect
Keywords:
Quinacrine
Acridine
Alzheimer’s disease
Amyloid plaques
125I
Single photon emission computed
tomography (SPECT)
A
b
plaques in the brain sections of Tg2576 mice. It should be noted that the acridines showed much
higher binding affinity for A
aggregates and greater in vivo blood brain barrier permeability than [125I]5.
Among them, 13 (6-Iodo-2-methoxy-9-methylaminoacridine) and 25 (2,9-Dimethoxy-6-iodo acridine)
b
exhibited high affinity for the Ab aggregates with Ki values of 14 and 29 nM, respectively. In the in vivo
studies, [125I]13 and [125I]25 showed excellent initial brain uptake (3.0 and 4.4% dose/g, respectively, at
2 min) with fast washout from the brain (0.33 and 0.37% dose/g, respectively, at 60 min). These acridine
derivatives are demonstrated to be promising SPECT imaging probes for amyloid in the living brain.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
of therapeutic interventions. Most of the amyloid beta (Ab) targeted
imaging agents have been developed by structural modification of
thioflavin-T or Congo Red. Among them, several ligands have been
confirmed to demonstrate the difference in uptake in the brain
between AD patients and control subjects in the clinical studies [7e
Several neurodegenerative disorders, such as Alzheimer’s
disease (AD) and prion disease, are called conformational diseases.
These disorders involve production of incorrectly conformed
proteins that lead to the formation of amyloid plaques with alter-
ation of normal cell function [1e3]. Even now, the definitive diag-
nosis of the illnesses still relies on histological confirmation of the
pathological features at postmortem examination [4e6]. In vivo
imaging of amyloid plaques in the brain is thus important to
monitor progression of these diseases and to evaluate the efficacy
12]. On the other hand, we have developed a number of Ab imaging
agents based on flavonoids, such as flavones [13,14], chalcones [15e
17], aurones [18,19], and styrylchromones [20], as promising posi-
tron emission tomography (PET) or single photon emission
computed tomography (SPECT) probes toward A
the difference in chemical structure from typical A
probes, several of these flavonoids showed excellent high affinity
for A aggregates, as well as specific detection of A plaques in the
model mice for AD. However, the clinical usefulness of these radi-
oligands for detection of A in human brain has not yet been
b
plaques. Despite
b
imaging
b
b
Abbreviations: AD, Alzheimer’s disease; Ab, amyloid beta; BBB, blood brain
barrier; BF-227, 2-(2-[2-dimethylaminothiazol-5-yl]ethenyl)-6-(2-[fluoro]ethoxy)
benzoxazole; Florbetapir, (E)-4-(2-(6-(2-(2-(2-[fluoro]ethoxy)ethoxy)ethoxy)pyr-
idin-3-yl)vinyl)-N-methyl benzenamine; IMPY, 2-(40-dimethylaminophenyl)-6-
iodo-imidazo[1,2-a]pyridine; PET, positron emission tomography; PrP, prion
b
confirmed. Most recently, Food and Drug Administration (FDA)
announced the approval of [18F]florbetapir {(E)-4-(2-(6-(2-(2-(2-
protein; SPECT, single photon emission computed tomography; PrPSc
insoluble PrP isoform.
, b-strand-rich
fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-N-methyl
zenamine} (AV-45/Amyvid) for PET imaging of the brain to estimate
plaque density in adults under evaluation for AD and other
ben-
* Corresponding author. Tel./fax: þ81 95 819 2443.
A
b
** Corresponding author. Tel./fax: þ81 95 819 2441.
causes of cognitive decline. On the other hand, there are currently
no SPECT ligands which are approved by FDA. SPECT imaging
E-mail addresses: t-fuchi@nagasaki-u.ac.jp (T. Fuchigami), morio@nagasaki-
u.ac.jp (M. Nakayama).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.020

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T. Fuchigami et al. / European Journal of Medicinal Chemistry 60 (2013) 469e478
probes could provide convenient and widespread diagnosis
methods for detecting A plaques in the living brain. Accordingly,
there is a clear need to develop clinically useful SPECT radioligands
targeting for A plaques.
Quinacrine (Fig. 1) has been used as a therapeutic agent for
malaria since the 1930’s. Recent researches have demonstrated that
To investigate the binding property of compound 5 for Ab pla-
b
ques in the Tg2576 transgenic mice brain, fluorescent staining was
performed as described above. As shown in Fig. 2D and E,
compound 5 labeled many amyloid plaques, in which the latter was
confirmed by thioflavin S staining. Conversely, no significant
staining of these compounds was observed in the brain sections of
age-matched wild-type mice (Fig. 2F). These results suggested that
quinacrine analog 5 could also recognize amyloid plaques by means
of fluorescent imaging.
b
quinacrine could inhibit amyloid plaque formation in a b-strand-
rich insoluble prion protein (PrP) isoform (PrPSc) infected cells [21e
23]. In addition, it has been reported that quinacrine could bind to
PrP segment aggregates [23]. Clinical investigations on treatment of
prion disease with quinacrine have also been performed [24,25].
To evaluate the binding property of [125I]5 to A
saturation assay was performed by using artificial A
b
aggregates,
peptide
b
Since A
several A
b
plaques form
b
-sheet-rich structure similar to PrPSc
,
aggregates. Firstly, the binding assays were conducted by using
filtration techniques as described in our previous paper [14].
However, the binding affinity of [125I]5 could not be determined by
the said methods because the tracer level of [125I]5 bound to the
glass filters could not be neglected. Therefore, centrifugation
method was used for the binding assay. A clear sigmoid saturation
b
imaging probes such as [125I]IMPY {2-(40-dimethylami-
nophenyl)-6-iodo-imidazo[1,2-a]pyridine} and [11C]BF-227 {2-(2-
[2-Dimethylaminothiazol-5-yl]ethenyl)-6-(2-[fluoro]ethoxy)ben-
zoxazole} have been applied to imaging of prion plaques [26e
29]. Since
a number of amyloid imaging probes could be
useful for the diagnosis of broad conformational diseases with
amyloid-like fibrils, we hypothesized that quinacrine could bind
curve of [125I]5 to A
b aggregates was then obtained. When the
saturation curve of [125I]5 was transformed to Scatchard plots,
to A
b
plaques and its radiolabeled derivatives have potential as
linear plots were obtained which suggested a single binding site of
A
b
imaging probes. In this paper, radioiodinated quinacrine-
[ b b
125I]5 on A aggregates (Fig. 3). The binding affinity of [125I]5 for A
related acridine derivatives were synthesized and evaluated in
order to discover new class of potential SPECT imaging agents
aggregates was modest (Kd ¼ 48.4 ꢀ 18.3 nM). The Bmax value of
[
125I]5 was estimated to be 0.21 ꢀ 0.03, which indicated that the
for in vivo detection of A
b
plaques.
complex between [125I]5 and A
b peptide was formed in a ratio of
1:5. Next, biodistribution study of [125I]5 was performed by using
normal mice. As shown in Table 1, [125I]5 showed poor initial brain
uptake (0.32% dose/g at 2 min after injection) which was lower than
in any other peripheral organs. In particular, an extremely high
accumulation of [125I]5 was observed in the lungs. Clinically useful
2. Results and discussion
2.1. Synthesis and evaluation of radioiodinated quinacrine analog
([125I]5)
A
b
probes require not only high affinity for A
initial brain uptake and rapid clearance by non-target regions. Since
125I]5 showed modest binding affinity for A
plaques it was sug-
gested that acridine derivatives could be useful A imaging probes.
However [125I]5 had inadequate binding affinity for A
. In addition,
125I]5 showed poor blood brain barrier (BBB) permeability and
b plaques but also high
Since there is no published report about the binding properties
[
b
of quinacrine for A
performed using brain slices from A
mice (Tg2576 mice). As shown in Fig. 2A and B, thioflavin S-positive
plaques were clearly visualized by fluorescence of quinacrine.
b
plaques, fluorescent imaging of quinacrine was
b
b
deposition model transgenic
b
[
A
b
high pulmonary accumulation. It is known that SPECT imaging
On the other hand, no significant fluorescence was detected in the
brain sections of wild-type mice (Fig. 2C).
agents with an aliphatic amino group such as [123I]N-isopropyl-p-
iodoamphetamine (IMP) and N-
u-(fluoropropyl)-2b-carbome-
Since quinacrine has been demonstrated to bind to A
b plaques,
thoxy-3
b
-(4-iodophenyl)tropane ([123I]FP-CIT) showed a consider-
a quinacrine analog, [125I]5, was synthesized and evaluated as an A
b
able amount of lung uptake [31,32]. These facts indicated that
structural modification of functional groups of [125I]5 such as
aliphatic amino groups at 9-position could change the pattern of
in vivo distribution. Accordingly, we designed radioiodinated acri-
dine derivatives (compound 12, 13, 14, 25, 26, and 27) with iodine
atom at the 6-position and aromatic amino or methoxy groups at
imaging agent. Compound 5 was synthesized as shown in Scheme
1. Coupling of 4-bromo-2-chlorobenzoic acid with p-anisidine by
UllmanneJourdan reaction [30] generated diphenylamine deriva-
tive 1 (81% yield). Compound 1 was reacted with POCl3 to give
acridine derivative 2 (69% yield), followed by condensation of
compound 2 and 4-amino-(1-diethylamino) pentane in phenol
under basic conditions which provided 6-bromo quinacrine
derivative 3 in a 29% yield. The tributyltin derivative 4 was prepared
from compound 3 using a bromo-to-tributyltin exchange reaction
catalyzed by Pd(0) in a 24% yield. Compound 4 was reacted with
iodine in CHCl3 to form the iodo derivative 5 (19% yield). Then, [125I]
5 was successfully synthesized from tributyltin precursor 4 by an
iododestannylation reaction using hydrogen peroxide as an oxidant
(radiochemical yield; 47%, radiochemical purity; >98%).
the 2- or 9-position as new Ab imaging probes.
2.2. Synthesis and evaluation of radioiodinated acridine analogs
The 9-amino-6-iodo acridine derivatives 12e14 were prepared
as shown in Scheme 2. Compound 2 was converted to 9-amino
acridine derivatives 6e8 using the above procedure for 3 in the
yield of 72%, 50%, and 71%, respectively. Tributyltin derivatives 9e
11 were synthesized from 6e8 by the same synthetic procedure
for 4 (32%, 50%, and 43% of yields, respectively). According to the
above method for 5, the target 6-iodo acridine derivatives 12e14
were prepared from compounds 9e11 in 47%, 49%, and 32% of
yields, respectively. The 9-methoxy-6-iodo acridine derivatives
25e27 were synthesized as shown in Scheme 3. Diphenylamine
derivatives 15, 16 and 6-bromo-acridine derivatives 17, 18 were
prepared using the same methods as described above. Conversion
of 9-chloro groups of compound 2, 17, and 18 to methoxy groups
were performed by sodium methoxide to give corresponding 19,
20, and 21 for yields of 44%, 100%, 44%, respectively. Tributyltin
derivatives 22e24 were synthesized from 19e21, respectively, by
Fig. 1. Chemical structure of quinacrine.

T. Fuchigami et al. / European Journal of Medicinal Chemistry 60 (2013) 469e478
471
Fig. 2. Neuropathological staining of quinacrine and quinacrine analog 5 (A and D) in 10
mm sections of Tg2576 mouse brain. Labeled plaques were confirmed by staining of the
adjacent sections with thioflavin S (B and E). Fluorescent staining of each compound in the age-matched control mouse brain sections (C and F) (20ꢁ magnification).
the same procedure as described above for yields of 47%, 44%, and
26%, respectively. The desired 6-iodo-acridine derivatives 25e27
were synthesized from corresponding tributyltin derivatives 22e
24 using the above methods for yields of 87%, 77%, and 67%,
respectively.
The binding affinity (Ki value) of non-radioactive acridine
derivatives for A aggregates was evaluated with displacement
studies of [125I]IMPY, which is the typical A
ligand, according to
the conventional procedures [35]. As shown in Table 2, quinacrine
analog 5 showed moderate affinity for A
aggregates (Ki ¼ 353 nM).
b
b
b
Next, radioiodinated acridine derivatives ([125I]12, [125I]13, [125I]
14, [125I]25, and [125I]26) were synthesized by an iododestannyla-
tion reaction of tributyltin derivatives (9, 10, 11, 22, and 23) with
hydrogen peroxide or chloramine T as the oxidants (Scheme 4).
Using HPLC systems, the radioligands were purified, and the
radiochemical qualities of these ligands were analyzed by co-
injection of non-radioactive compounds. The radioiodinated
products were obtained in radiochemical yields of 24e75% with
a radiochemical purity of >95%.
On the other hand, other acridine derivatives, except for 27, had
higher affinity than that of 5. Introduction of aromatic amino
groups (NMe2, NHMe, and NH2) at the 9-position of acridine skel-
eton increased the binding affinity (121 nM for 12, and 84 nM for
14, respectively). N-methyl amino analog 13 had the highest affinity
(14 nM) among the series of acridine derivatives in this study,
which is 25-fold higher affinity than 5 and comparable with that of
IMPY (8.0 nM). In addition, the compounds with methoxy group at
the 9-position of acridine backbone also exhibited high affinity for
Scheme 1. Preparation of [125I]5. Reagents and conditions: (a) p-anisidine, Cu(OAc)2eH2O, Et3N, dioxane, reflux, 5 h; (b) POCl3, dioxane, reflux, 6 h; (c) 4-Amino-(1-diethylamino)
pentane, PhOH, reflux, 4 h; (d) (SnBu3)2, Pd (PPh3)4, Et3N, dioxane, reflux 4 h, (e) I2, CHCl3, r.t., 10 min, (f) [125I]NaI, H2O2, HCl, EtOH, 3 min.

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T. Fuchigami et al. / European Journal of Medicinal Chemistry 60 (2013) 469e478
Table 1
Biodistribution of radioactivity after intravenous injection of 125I labeled acridines in
normal mice.a
Organ
Time after injection (min)
10
2
30
60
[
125I]5
Blood
Brain
Liver
4.21 (0.31)
0.32 (0.05)
8.15 (0.97)
10.23 (1.62)
1.89 (0.24)
6.14 (1.44)
73.08 (13.37)
1.18 (0.26)
4.76 (0.84)
15.53 (1.88)
1.85 (0.35)
0.73 (0.25)
0.52 (0.34)
0.33 (0.08)
9.70 (1.12)
13.58 (1.96)
2.55 (0.22)
9.38 (1.65)
59.55 (12.88)
1.33 (0.17)
5.85 (0.84)
13.09 (1.23)
0.24 (0.04)
11.02 (2.39)
13.93 (1.48)
4.40 (0.81)
10.88 (2.25)
35.64 (7.09)
1.48 (0.26)
7.67 (4.46)
7.00 (0.47)
0.28 (0.03)
11.61 (1.95)
12.19 (5.21)
5.59 (0.84)
11.17 (1.84)
25.58 (7.34)
1.24 (0.18)
6.32 (0.88)
3.96 (0.76)
Kidney
Intestine
Spleen
Lung
Stomachb
Pancreas
Heart
Fig. 3. Saturation curve and Scatchard plot of [125I]5 binding to A
b aggregates. In this
representative experiment, Kd value was 48.4 nM and Bmax was 0.21. Saturation
[
125I]12
analysis was performed using increasing concentrations of [125I]5 (4e500 nM). Non-
Blood
Brain
4.76 (0.62)
0.33 (0.05)
10.85 (1.29)
16.71 (2.00)
2.22 (0.38)
6.94 (1.17)
79.34 (12.10)
1.37 (0.20)
5.21 (0.71)
16.28 (1.79)
1.97 (0.14)
0.26 (0.03)
11.77 (2.14)
18.16 (2.37)
3.13 (0.30)
9.65 (1.14)
57.02 (8.19)
2.18 (0.37)
6.50 (0.56)
11.20 (0.72)
1.26 (0.36)
0.28 (0.14)
16.43 (4.74)
21.56 (9.26)
5.33 (0.97)
14.49 (2.60)
46.47 (21.25)
2.21 (0.50)
7.85 (3.96)
9.08 (3.28)
0.64 (0.08)
0.23 (0.03)
12.55 (1.50)
16.34 (1.93)
6.50 (1.26)
11.22 (2.78)
21.84 (3.95)
1.40 (1.52)
9.81 (5.32)
4.33 (1.36)
specific binding was determined with 10 mM of unlabeled 5.
Liver
Kidney
Intestine
Spleen
Lung
Ab aggregates (29 nM for 25, 30 nM for 26). On the other hand,
compound 27 showed poor binding competition (Ki > 10,000 nM),
which indicated that the electron donor group at the 9-position of
Stomachb
Pancreas
Heart
acridine derivatives had a crucial role in maintaining affinity for A
b
aggregates.
125I]13
In order to investigate the binding property of acridine deriva-
tives for A plaques in the Tg2576 mice brain, neuropathological
[
Blood
Brain
Liver
1.75 (0.34)
2.97 (0.16)
17.25 (2.59)
12.98 (2.27)
3.82 (0.50)
7.60 (1.75)
15.80 (2.86)
1.71 (0.45)
6.91 (0.81)
7.79 (0.88)
1.18 (0.09)
2.34 (0.27)
16.41 (1.17)
11.89 (0.87)
9.11 (1.05)
9.14 (1.10)
5.62 (0.95)
3.71 (0.60)
6.92 (1.02)
2.80 (0.19)
0.85 (0.08)
0.95 (0.15)
8.26 (1.27)
10.25 (1.35)
19.47 (3.60)
6.02 (0.62)
4.38 (0.86)
4.63 (0.41)
3.55 (0.78)
1.46 (0.32)
0.56 (0.20)
0.33 (0.06)
7.52 (0.97)
6.00 (0.71)
23.83 (2.61)
4.45 (1.06)
2.30 (1.21)
5.58 (1.21)
1.63 (0.18)
0.84 (0.09)
b
fluorescent staining with 13, 25, and 26 was performed as
described above. Fluorescence images of these acridine derivatives
(13, 25, and 26) in the brain slices of Tg2576 mice corresponded to
the labeling pattern by thioflavin S (Fig. 4A, B, D, E, G and H). By
contrast, wild-type mice brain displayed no significant accumula-
tion of the three acridines (Fig. 4C, F and I). These results indicated
Kidney
Intestine
Spleen
Lung
Stomachb
Pancreas
Heart
that these acridine derivatives could also detect Ab plaques.
Then, in vivo biodistribution studies of acridine derivatives
([125I]12, [125I]13, [125I]14, [125I]25, and [125I]26) were performed
using normal mice (Table 1). The 9-NMe2 acridine derivative [125I]
12 showed the same level of initial brain uptake with quinacrine
analog [125I]5. On the other hand, initial brain uptake of other
acridine derivatives (1.35e4.41% dose/g at 2 min) was higher than
that of [125I]5, probably due to the removal of the aliphatic amino
groups at 9-position from the acridine skeleton. In general, radio-
tracers with low molecular weight (<500 Da) and moderate lip-
ophilicity (log D7.4 values; 2.0e3.5) show optimal passive brain
entry under in vivo condition [33,34]. Since the molecular weights
of 125I-ligands in this study are all within 500 as shown in Table 2,
this factor might not be critical for BBB permeability. In this study,
[
125I]14
Blood
Brain
Liver
1.15 (0.13)
1.35 (0.17)
6.24 (0.67)
9.81 (1.41)
1.86 (0.28)
4.70 (0.61)
9.91 (1.82)
0.99 (0.22)
4.34 (0.71)
4.46 (0.72)
2.09 (0.56)
3.16 (0.33)
14.79 (1.55)
22.51 (2.85)
14.19 (1.25)
14.65 (2.68)
14.29 (3.65)
6.58 (4.30)
11.52 (1.81)
4.57 (0.66)
1.46 (0.47)
1.27 (0.49)
8.40 (2.41)
12.64 (4.09)
17.49 (6.26)
10.60 (2.55)
5.04 (0.94)
6.66 (2.34)
4.98 (1.38)
2.26 (0.77)
0.49 (0.10)
0.19 (0.05)
2.84 (0.94)
3.58 (0.79)
11.20 (3.35)
3.28 (1.04)
1.40 (0.40)
1.74 (0.88)
0.76 (0.18)
0.49 (0.13)
Kidney
Intestine
Spleen
Lung
Stomachb
Pancreas
Heart
[
125I]25
Blood
Brain
Liver
2.14 (0.41)
4.41 (0.46)
18.77 (1.83)
8.07 (0.30)
2.59 (0.30)
3.75 (0.70)
5.95 (0.56)
1.86 (0.38)
5.41 (1.06)
5.83 (0.55)
2.19 (0.21)
2.14 (0.19)
20.85 (3.05)
4.69 (0.53)
6.41 (0.90)
2.12 (0.24)
3.37 (0.73)
3.51 (0.62)
2.03 (0.64)
1.82 (0.19)
2.59 (0.41)
0.82 (0.12)
13.65 (3.62)
4.41 (0.61)
16.78 (5.01)
1.87 (0.53)
3.28 (0.81)
5.97 (1.31)
1.33 (0.31)
1.59 (0.47)
1.77 (0.28)
0.37 (0.02)
5.80 (0.87)
2.77 (0.36)
20.17 (4.27)
1.24 (0.21)
2.04 (0.33)
4.36 (0.61)
1.10 (0.13)
1.10 (0.16)
[
125I]12 with appropriate lipophilicity (log D7.4 ¼ 2.19) showed only
Kidney
Intestine
Spleen
Lung
poor brain uptake, while compound [125I]26 with slightly lower
lipophilicity than the optimal range (log D7.4 ¼ 1.81) showed high
BBB permeability. In view of log D7.4 values, there is no significant
correlation between lipophilicity and brain uptake in the case of
acridine derivatives in this study. Instead, accumulations of these
tracers in the lungs are likely to be associated with the degree of
brain uptake. Indeed, [125I]5 and [125I]12, which showed poor BBB
permeability, demonstrated a higher uptake in the lungs than other
acridine derivatives with high BBB permeability. It should be noted
Stomachb
Pancreas
Heart
[
125I]26
Blood
Brain
Liver
8.34 (1.41)
2.09 (0.53)
20.78 (1.42)
10.94 (2.12)
1.86 (0.28)
5.09 (0.83)
7.17 (2.84)
1.83 (2.01)
4.11 (0.82)
5.83 (1.21)
7.00 (1.04)
1.96 (0.27)
20.71 (3.70)
8.99 (0.86)
14.19 (1.25)
4.07 (0.64)
6.66 (1.27)
5.10 (2.89)
2.47 (0.36)
3.78 (0.41)
6.52 (1.47)
1.60 (0.56)
18.54 (2.99)
10.72 (1.93)
17.49 (6.26)
5.40 (1.19)
6.67 (0.98)
4.20 (2.33)
2.29 (0.58)
4.09 (1.38)
5.17 (1.50)
0.71 (0.14)
10.79 (2.66)
4.81 (0.99)
11.20 (3.35)
2.43 (0.54)
3.87 (0.59)
4.33 (1.70)
1.14 (0.38)
2.38 (0.57)
that [125I]13 and [125I]25 with high affinity for A
b aggregates
Kidney
Intestine
Spleen
Lung
exhibited excellent initial brain uptake (2.97 and 4.41% dose/g at
2 min, respectively) and fast washout from the brain tissue (0.33
and 0.37% dose/g at 60 min, respectively). To compare the washout
Stomachb
Pancreas
Heart
rate among Ab imaging probes, the brain2 min/brain60 min ratio is
often used as an index parameter. The brain2 min/brain60 min ratios
of [125I]13 and [125I]25 were 9.0 and 11.9, respectively. These values
Each value represents mean (SD) for 3e6 mice at each interval.
a
are comparable with clinically used Ab
probes, such as [125I]IMPY
Expressed as % injected dose per gram.
Expressed as % injected dose per organ.
b

T. Fuchigami et al. / European Journal of Medicinal Chemistry 60 (2013) 469e478
473
Scheme 2. Preparation of 9-amino-6-iodo acridine derivatives. Reagents and conditions: (a) R1-H, PhOH, dioxane, reflux, 3e6 h; (b) (SnBu3)2, Pd (PPh3)4, Et3N, dioxane, reflux 4 h,
(c) I2, CHCl3, r.t., 10 min.
(13.7) [35] and BF-227 (12.3) [27]. Accordingly, radioiodinated 13
and 25 are expected be promising SPECT probes for targeting A
plaques in the living brain. In addition, since quinacrine exhibits an
affinity for PrP segment aggregates [23], acridine derivatives could
be applied for the imaging of prion disease. It should be note that
that these acridine derivatives and quinacrine itself clearly stained
amyloid plaques in the brain section of Tg2576 mice. In bio-
distribution studies, [125I]13 and [125I]25 showed high initial brain
uptake and fast clearance from the brain. The washout level of these
b
tracers was comparable with Ab imaging probes in clinical use;
the binding affinity for A
b
aggregates and biodistribution pattern of
therefore, these acridine derivatives could be promising imaging
probes of cerebral amyloid plaques.
acridine derivatives were quite different depending on the
substituents attached at the 2- or 9-position of acridines. Further
structural modification of acridines could provide promising SPECT
or PET probes for amyloid imaging with optimal binding affinity
and pharmacokinetics.
4. Experimental
4.1. General information
3. Conclusion
All reagents were commercial products and used without
further purification unless otherwise indicated. Quinacrine dihy-
drochloride hydrate was purchase from Tokyo Chemical Industry
Co., Ltd. [125I]NaI was obtained by MP biomedicals (Costa Mesa, CA,
USA). 1H NMR spectra were obtained on a Varian Gemini 300
spectrometer with TMS as an internal standard. Mass spectra were
We developed radioiodinated quinacrine-based acridine deriv-
atives for SPECT imaging of A
aliphatic amino groups had higher binding affinity for A
gates than quinacrine analog 5. Fluorescent staining demonstrated
b
plaques. The acridines without
b
aggre-
Scheme 3. Preparation of 9-methoxy-6-iodo acridine derivatives. Reagents and conditions: (a) Cu(OAc)2eH2O, Et3N, dioxane, reflux, 4e5 h; (b) POCl3, dioxane, reflux, 5e6 h; (c)
NaOCH3, MeOH, THF, reflux, 2e6 h; (d) (SnBu3)2, Pd (PPh3)4, Et3N, dioxane, reflux 4e6 h, (e) I2, CHCl3, r.t., 10 min.

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T. Fuchigami et al. / European Journal of Medicinal Chemistry 60 (2013) 469e478
4.2.3. 6-Bromo-2-methoxy-9-(4-(dimethyl-1-methyl)-N-butyl)
aminoacridine (3)
2 (100 mg, 0.31 mmol) and 4-amino-(1-diethylamino)pentane
(1 mL) was dissolved in PhOH (8 mL) and heated to reflux for 4 h.
After cooling down to room temperature, 1 M NaOH (10 mL) and
exacted with CHCl3 three times. The combined organic layers were
dried with Na2SO4 and evaporated to dryness. The crude product
was chromatographed on silica gel with CHCl3/MeOH ¼ 1: 1 to give
3 (40 mg, 29%) as a yellow powder. 1H NMR (300 MHz CDCl3)
d 1.07
Scheme 4. Radiosynthesis of [125I]6-iodo acridine derivatives. Reagents and condi-
(t, J ¼ 7.2 Hz, 6H), 1.33 (d, J ¼ 6.3 Hz, 3H), 1.58e1.78 (m, 4H), 2.66e
2.79 (m, 6H), 3.98 (s, 3H), 4.05e4.07 (m, 1H), 7.32 (s, 1H), 7.40e7.46
(m, 2H), 7.98 (d, J ¼ 6.3 Hz, 1H), 8.03 (d, J ¼ 6.3 Hz, 1H), 8.29 (s, 1H).
MS (EI) m/z 443 (Mþ).
tions: (a) [125I]NaI, H2O2 or Chloramine-T, HCl, EtOH, 3 min.
obtained on JEOL IMS-DX or JMS-T100TD instruments. HPLC anal-
ysis was performed on a Shimadzu HPLC system (a LC-10AT pump
4.2.4. 2-Methoxy-9-(4-(dimethyl-1-methyl)-N-butyl)amino-6-
tributylstannylacridine (4)
with a SPD-10A UV detector,
l
¼ 254 nm). An automated gamma
counter with a NaI(Tl) detector (PerkinElmer, 2470 WIZARD2) was
used to measure radioactivity. All animals were supplied by Tagawa
Experimental Animal Laboratory (Nagasaki, Japan). The experi-
ments with animals were conducted in accordance with our insti-
tutional guidelines and were approved by Nagasaki University
Animal Care Committee.
A mixture of 3 (40 mg, 0.09 mmol), bis(tributyltin) (0.4 mL) and
Pd(PPh3)4 (20 mg, 0.017 mmol) in a mixed solvent (10 mL, 3:1
dioxane/triethylamine mixture) was stirred for 4 h under reflux.
The solvent was removed, and the residue was purified by silica gel
chromatography CHCl3/MeOH ¼ 1:1 to give 4 (14 mg, 24%)as
a yellow oil. 1H NMR (300 MHz CDCl3)
1.07e1.62 (m, 31H), 2.62e2.69 (m, 6H), 4.02 (s, 3H), 4.26 (s, 1H),
7.31e7.35 (m, 1H), 7.50e7.54 (m, 2H), 8.06e8.09 (m, 2H), 8.27 (s,
1H). MS (EI) m/z 656 (Mþ).
d
0.89 (t, 9H, J ¼ 7.2 Hz),
4.2. Chemistry
4.2.1. 4-Bromo-2-(4-methoxyanilino)benzoic acid (1)
4.2.5. 2-Methoxy-9-(4-(dimethyl-1-methyl)-N-butyl)amino-6-iodo
acridine (5)
To
a solution of 4-bromo-2-chlorobenzoic acid (705 mg,
3 mmol) and p-anisidine (369 mg, 3 mmol) in dioxane (8 mL) and
triethylamine (4 mL) was added (AcO)2 Cu$H2O (800 mg, 3 mmol).
The reaction mixture was stirred under reflux for 5 h. 1 M HCl was
added and following extraction with CHCl3, the organic phase was
dried over Na2SO4. The solvent was removed and the residue was
purified by silica gel chromatography (CHCl3:methanol ¼ 49:1) to
give 1 (785 mg, 81%) as a black powder. 1H NMR (300 MHz CDCl3)
To a solution of 4 (14 mg, 0.021 mmol) in CHCl3 (3 mL) was
added a solution of iodine in CHCl3 (1 mL, 0.25 M) at room
temperature. The mixture was stirred at room temperature for
10 min and a saturated NaHSO3 solution (5 mL) was added. The
mixture was stirred for 5 min and the organic phase was separated.
The aqueous layer was extracted with CHCl3 three times. The
combined organic layers were dried with Na2SO4 and evaporated to
dryness. The crude product was chromatographed on silica gel with
CHCl3/MeOH ¼ 1:1 to give 5 (2 mg, 19%) as a yellow powder. 1H
d
3.84 (s, 3H), 6.78 (d, 1H, J ¼ 8.4 Hz), 6.94 (d, 2H, J ¼ 8.4 Hz), 7.04 (s,
1H), 7.17 (d, 2H, J ¼ 12.6 Hz), 7.85 (d, 1H, J ¼ 8.7 Hz), 9.18 (s, 1H). MS
(DART) m/z 322, 324 (Mþ).
NMR (300 MHz CDCl3)
d
0.97 (t, 6 H, J ¼ 6.9 Hz), 1.28 (d, 3 H,
J ¼ 6.6 Hz), 1.61e1.73 (m, 4H), 2.40e2.51 (m, 6H), 3.98 (s, 3H), 4.00
4.2.2. 6-Bromo-9-chloro-2-methoxyacridine (2)
(s, 1H), 7.23 (s, 1H), 7.42e7.48 (m, 2H), 7.94 (d, 1 H, J ¼ 6.3 Hz), 8.02
To a solution of 1 (1.04 mg, 3.21 mmol) in dioxane (15 mL) was
added POCl3 (0.4 mL) at 0 ꢂC and the mixture was refluxed for 6 h.
After cooling down to 0 ꢂC, the reaction mixture was added 2 M
NaOH (40 mL) and then extracted with CHCl3 three times. The
combined organic layers were dried with Na2SO4 and evaporated to
dryness. The crude product was chromatographed on silica gel with
CHCl3/MeOH ¼ 49: 1 to give 2 (717 mg, 69%) as a light brown
(d, 1 H, J ¼ 6.3 Hz), 8.29 (s, 1H). 13C NMR (400 MHz CDCl3)
d 159.79,
153.02, 146.29, 145.77, 138.19, 132.48, 132.03, 128.60, 128.48, 125.33,
121.64, 117.65, 97.44, 58.71, 57.99, 46.10, 45.74, 28.28, 26.85, 17.82,
13.62. HRMS (EI) m/z: calcd for C23H30N3OI (Mþ) 492.1512, found
492.1456.
powder. 1H NMR (300 MHz CDCl3)
d 4.03 (s, 3H), 7.45 (d, 1 H,
4.2.6. 6-Bromo-9-dimethylamino-2-methoxyacridine (6)
To a solution of 2 (280 mg, 0.86 mmol) in DMSO (20 mL) was
added PhOH (1.13 mL, 12.9 mmol), 50% dimethylamine aqueous
solution (0.36 mL), and K2CO3 (118 mg, 0.86 mmol). The reaction
mixture was stirred under reflux for 5 h. After cooling down to
room temperature,1 M NaOH (10 mL) and exacted with CHCl3 three
times. The combined organic layers were dried with Na2SO4 and
evaporated to dryness. The crude product was chromatographed on
silica gel with CHCl3/MeOH ¼ 49: 1 to give 6 (204 mg, 72%) as
J ¼ 11.7 Hz), 7.66 (d, 1 H, J ¼ 9.3 Hz), 8.07 (d, 1 H, J ¼ 9.3 Hz), 8.22 (d,
2 H, J ¼ 9.3 Hz), 8.37 (s, 1H). MS (DART) m/z 321, 323 (Mþ).
Table 2
Inhibition constants (Ki) for A
b(1e42) aggregates, molecular weight, and experi-
mental log D7.4 values of acridine derivatives.
b
Compounds
Kia (nM)
Mw
log D7.4
5
353 ꢀ 81.5
121 ꢀ 80.2
13.8 ꢀ 10.8
83.5 ꢀ 30.7
28.9 ꢀ 7.97
39.2 ꢀ 18.1
>10000
491.4
378.2
364.2
350.2
365.2
378.2
335.1
1.11
2.19
2.14
2.13
1.81
2.20
12
13
14
25
26
27
IMPY
a yellow powder. 1H NMR (300 MHz CDCl3)
d 3.38 (s, 6H), 3.99 (s,
3H), 7.37 (s, 1H), 7.45 (d,1 H, J ¼ 9.6 Hz), 7.52 (d, 1 H, J ¼ 9.3 Hz), 8.05
(d, 2 H, J ¼ 9.0 Hz), 8.35 (s, 1H). MS (DART) m/z 331, 333 (Mþ).
4.2.7. 6-Bromo-2-methoxy-9-methylaminoacridine (7)
8.0 ꢀ 1.1
2 (37 mg, 0.11 mmol) and methylamine (0.5 mL) were dissolved
in PhOH (5 mL) and heated to reflux for 3 h. After cooling down to
room temperature, the reaction mixture was evaporated. The
residue was added 1 M NaOH (5 mL) and exacted with CHCl3 three
times. The combined organic layers were dried with Na2SO4 and
a
Inhibition constants (Ki) of acridine derivatives were determined using [125I]
IMPY as the ligand in A
b
(1e42) aggregates. Each value (mean ꢀ SEM) was deter-
mined by 3e6 independent experiments.
b
The partition coefficient between n-octanol and sodium phosphate buffer at
pH7.4 (log D7.4) was determined by conventional flask shaking method (n ¼ 3).

T. Fuchigami et al. / European Journal of Medicinal Chemistry 60 (2013) 469e478
475
Fig. 4. Neuropathological staining of acridine derivatives 13, 25, and 26 (A, D, and G) in 10
mm sections of Tg2576 mouse brain. Labeled plaques were confirmed by staining of the
adjacent sections with thioflavin S (B, E, and H). Fluorescent staining of each compound in the age-matched control mouse brain sections (C, F, and I) (20ꢁ magnification).
evaporated to dryness. The crude product was chromatographed on
4.2.11. 9-Amino-2-methoxy-6-tributylstannylacridine (11)
silica gel with CHCl3/MeOH ¼ 49: 1 to give 7 (18 mg, 50%) as
Using the above procedure for 4 starting from compound 8, the
a yellow powder. 1H NMR (300 MHz CDCl3)
d 3.50 (s, 3H), 3.97 (s,
title compound 11 (24 mg, 43%) was obtained as a yellow oil. 1H
3H), 7.21 (s, 1H), 7.43 (d, 2H, J ¼ 9.3 Hz), 7.98 (d, 2 H, J ¼ 9.0 Hz), 8.25
NMR (300 MHz CDCl3) d 0.86e1.61 (m, 27H), 3.97 (s, 3H), 7.05 (s,
(s, 1H). MS (DART) m/z 317, 319 (Mþ).
1H), 7.40 (d, J ¼ 9.3 Hz, 1H), 7.51 (d, 1 H, J ¼ 8.1 Hz), 7.82 (d, 1 H,
J ¼ 8.4 Hz), 8.01 (d, 1 H, J ¼ 9.3 Hz), 8.20 (s, 1H). MS (DART) m/z 515
(Mþ).
4.2.8. 9-Amino-6-bromo-2-methoxyacridine (8)
2 (90 mg, 0.28 mmol) and 28% ammonia solution (1.5 mL) were
dissolved in PhOH (5 mL) and heated to reflux for 4 h. After cooling
down to room temperature, the reaction mixture was evaporated.
The residue was added 1 M NaOH (10 mL) and exacted with CHCl3
three times. The combined organic layers were dried with Na2SO4
and evaporated to dryness. The crude product was chromato-
graphed on silica gel with CHCl3/MeOH ¼ 10: 1 to give 8 (60 mg,
4.2.12. 6-Iodo-9-dimethylamino-2-methoxyacridine (12)
Using the above procedure for 5 starting from compound 9, the
title compound 12 (13 mg, 47%) was obtained as a yellow powder.
1H NMR (300 MHz CDCl3)
d 3.34 (s, 6H), 3.99 (s, 3H), 7.37 (s, 1H),
7.45 (d, 1 H, J ¼ 9.3 Hz), 7.68 (d, 1 H, J ¼ 9.3 Hz), 7.90 (d, 1 H,
J ¼ 8.4 Hz), 8.05 (d, 1 H, J ¼ 9.3 Hz), 8.62 (s, 1H). 13C NMR (400 MHz
71%) as a yellow powder. 1H NMR (300 MHz CDCl3)
d
3.94 (s, 3H),
CDCl3) d 156.29, 154.24, 147.39, 144.72, 138.13, 135.11, 131.53, 130.84,
7.42e7.45 (m, 2H), 7.56 (s, 1H), 7.75 (d, 1 H, J ¼ 9.3 Hz), 7.98 (s, 1H),
128.99, 124.66, 123.87, 122.98, 100.90, 55.47, 45.05. HRMS (EI) m/z:
8.17 (d, 1 H, J ¼ 9.3 Hz). MS (DART) m/z 303, 305 (Mþ).
calcd for C16H16N2OI (Mþ) 379.0307, found 379.0261.
4.2.9. 9-Dimethylamino-2-methoxy-6-tributylstannylacridine (9)
Using the above procedure for 4 starting from compound 6, the
title compound 9 (107 mg, 32%) was obtained as a yellow oil. 1H
4.2.13. 6-Iodo-2-methoxy-9-methylaminoacridine (13)
Using the above procedure for 5 starting from compound 10, the
title compound 13 (5 mg, 49%) was obtained as a yellow powder. 1H
NMR (300 MHz CDCl3)
d
0.87e1.63 (m, 27H), 3.34 (s, 6H), 4.00 (s,
NMR (300 MHz CDCl3) d 3.53 (s, 3H), 3.92 (s, 3H), 7.28e7.38 (m, 2H),
3H), 7.41e7.43 (m, 2H), 7.56 (d, 1 H, J ¼ 8.4 Hz), 8.09e8.15 (m, 2H),
7.41 (s, 1H), 7.67 (d, 1 H, J ¼ 9.3 Hz), 7.88 (s, 1H), 8.19 (d, 1 H,
8.32 (s, 1H). MS (DART) m/z 543 (Mþ).
J ¼ 9.3 Hz). 13C NMR (400 MHz CDCl3)
d 156.71, 155.32, 150.04,
146.32, 141.11, 132.92, 131.72, 126.58, 125.23, 123.80, 120.56, 108.87,
100.73, 55.53, 44.96. HRMS (EI) m/z: calcd for C15H14N2OI (Mþ)
365.01509, found 365.0148.
4.2.10. 2-Methoxy-9-methylamino-6-tributylstannylacridine (10)
Using the above procedure for 4 starting from compound 7, the
title compound 10 (15 mg, 50%) was obtained as a yellow oil. 1H
NMR (300 MHz CDCl3)
d
0.86e1.59 (m, 27H), 3.50 (s, 3H), 3.97 (s,
4.2.14. 9-Amino-6-iodo-2-methoxyacridine (14)
3H), 7.31 (s, 1H), 7.48 (d, 2H, J ¼ 8.7 Hz), 8.03 (d, 2 H, J ¼ 5.4 Hz), 8.21
Using the above procedure for 5 starting from compound 11, the
(s, 1H). MS (DART) m/z 529 (Mþ).
title compound 14 (107 mg, 32%) was obtained as a yellow oil. 1H

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T. Fuchigami et al. / European Journal of Medicinal Chemistry 60 (2013) 469e478
NMR (300 MHz CD3OD)
d
3.96 (s, 3H), 7.51 (d, 1 H, J ¼ 9.3 Hz), 7.59
NMR (300 MHz CDCl3) d 0.87e1.63 (m, 27H), 4.00 (s, 3H), 4.21 (s,
(s, 1H), 7.67 (d, 1 H, J ¼ 9.0 Hz), 7.72 (d, 1 H, J ¼ 9.3 Hz), 8.02 (d, 1 H,
3H), 7.40 (s, 1H), 7.47 (d, 1 H, J ¼ 9.6 Hz), 7.63 (d, 1 H, J ¼ 8.4 Hz),
J ¼ 9.0 Hz), 8.21 (s, 1H). 13C NMR (400 MHz CDCl3)
d 159.18, 156.53,
8.11e8.18 (m, 1H), 8.34 (s, 1H). MS (DART) m/z 530 (Mþ).
150.97, 146.39, 133.90, 130.05, 129.53, 125.90, 123.50, 120.49,
102.60, 100.25, 95.55, 56.62. HRMS (EI) m/z: calcd for C14H12N2OI
(Mþ) 350.9994, found 349.9940.
4.2.23. 2-Dimethylamino-9-methoxy-6-tributylstannylacridine
(23)
Using the above procedure for 4 starting from compound 20, the
4.2.15. 4-Bromo-2-(4-dimethylaminoanilino) benzoic acid (15)
Using the above procedure for 1 starting from N,N-dimetyl-1,4-
phenylenediamine, the title compound 15 (741 mg, 44%) was ob-
title compound 23 (98 mg, 44%) was obtained as a yellow oil. 1H
NMR (300 MHz CDCl3)
d 0.87e1.62 (m, 27H), 3.15 (s, 6H), 4.19 (s,
3H), 7.09 (s, 1H), 7.54e7.59 (m, 1H), 8.09e8.13 (m, 2H), 8.29 (s, 1H).
tained as a dark green powder. 1H NMR (300 MHz CDCl3)
d 3.00 (s,
MS (DART) m/z 543 (Mþ).
6H), 6.76 (m, 1H), 7.09e7.26 (m, 2H), 7.62 (d, 2 H, J ¼ 8.1 Hz), 7.95 (d,
2 H, J ¼ 8.4 Hz). MS (DART) m/z 335, 337 (Mþ).
4.2.24. 9-Methoxy-6-tributylstannylacridine (24)
Using the above procedure for 4 starting from compound 21, the
title compound 24 (100 mg, 26%) was obtained as a white powder.
4.2.16. 2-Anilino-4-bromobenzoicacid (16)
Using the above procedure for 1 starting from aniline, the title
1H NMR (300 MHz CDCl3)
d 0.87e1.66 (m, 27H), 4.24 (s, 3H), 7.50 (t,
compound 16 (1.03 g, 70%) was obtained as a black powder. 1H NMR
1 H, J ¼ 7.5 Hz), 7.62 (d, 1 H, J ¼ 8.1 Hz), 7.76 (t, 1 H, J ¼ 7.5 Hz), 8.19e
(300 MHz CDCl3) d 6.91 (s, 1H), 7.17 (s, 1H), 7.40e7.61 (m, 6H), 7.74e
8.31 (m, 3H), 8.38 (s, 1H). MS (DART) m/z 500 (Mþ).
7.86 (m, 2H). MS (DART) m/z 292, 294 (Mþ).
4.2.25. 2,9-Dimethoxy-6-iodo acridine (25)
4.2.17. 6-Bromo-9-chloro-2-dimetylaminoacridine (17)
Using the above procedure for 5 starting from compound 22, the
title compound 25 (57 mg, 87%) was obtained as a light yellow
Using the above procedure for 2 starting from 15, the title
compound 17 (90 mg,12%) was obtained as a dark green powder. 1H
powder. 1H NMR (300 MHz CDCl3)
d 4.00 (s, 3H), 4.19 (s, 3H), 7.35 (s,
NMR (300 MHz CDCl3)
d
3.19 (s, 3H), 7.10 (s, 1H), 7.58e7.62 (m, 2H),
1H), 7.48 (d, 1 H, J ¼ 9.3 Hz), 7.74 (d, 1 H, J ¼ 9.0 Hz), 7.91 (d, 1 H,
8.03 (d, 1 H, J ¼ 9.9 Hz), 8.18 (d, 1 H, J ¼ 9.3 Hz), 8.33 (s, 1H). MS
J ¼ 9.0 Hz), 8.05 (d, 1 H, J ¼ 9.0 Hz), 8.64 (s, 1H). 13C NMR (400 MHz
(DART) m/z 335, 337 (Mþ).
CDCl3)
d 159.76, 157.26, 148.95, 147.92, 138.56, 133.78, 131.33,
126.09, 122.99, 121.09, 97.37, 95.68, 63.28, 55.61. HRMS (EI) m/z:
4.2.18. 6-Bromo-9-chloroacridine (18)
calcd for C15H13NO2I (Mþ) 365.9991, found 365.9940.
Using the above procedure for 2 starting from compound 16, the
title compound 18 (525 mg, 52%) was obtained as a light yellow
4.2.26. 2-Dimetylamino-9-methoxy-6-iodo acridine (26)
powder. 1H NMR (300 MHz CDCl3)
d
7.64e7.71 (m, 2H), 7.84 (t, 1 H,
Using the above procedure for 5 starting from compound 23, the
J ¼ 8.1 Hz), 8.19 (d, 1 H, J ¼ 8.7 Hz), 8.29 (d, 1 H, J ¼ 9.0 Hz), 8.40e
title compound 26 (47 mg, 77%) was obtained yellow powder. 1H
8.44 (m, 2H). MS (DART) m/z 292, 294 (Mþ).
NMR (300 MHz CDCl3) d 3.14 (s, 6H), 4.15 (s, 3H), 7.02 (s,1H), 7.58 (d,
1 H, J ¼ 9.6 Hz), 7.68 (d, 1 H, J ¼ 9.3 Hz), 7.87 (d, 1 H, J ¼ 9.0 Hz), 8.03
4.2.19. 6-Bromo-2,9-dimethoxyacridine (19)
(d, 1 H, J ¼ 9.6 Hz), 8.59 (s, 1H). 13C NMR (400 MHz CDCl3)
d 158.22,
To a solution of 2 (300 mg, 0.93 mmol) in a mixed solvent
(15 mL, 30:1 MeOH/THF) was added NaOCH3 methanol solution
(1.2 mL). The reaction mixture was stirred under reflux for 6 h. After
cooling down to room temperature, the reaction mixture was
evaporated. The residue was added satd. NaHCO3 solution (20 mL)
and exacted with CHCl3 three times. The combined organic layers
were dried with Na2SO4 and evaporated to dryness. The crude
product was chromatographed on silica gel with hexane/EtOAc ¼ 3:
1 to give 19 (130 mg, 44%) as a light yellow powder. 1H NMR
148.01, 147.60, 146.67, 138.50, 133.39, 130.53, 123.21, 122.96, 121.85,
119.43, 96.78, 94.27, 62.58, 40.65. HRMS (EI) m/z: calcd for
C16H16N2OI (Mþ) 379.0307, found 378.0261.
4.2.27. 6-Iodo-9-methoxyacridine (27)
Using the above procedure for 5 starting from compound 24, the
title compound 27 (45 mg, 67%) was obtained as a white powder. 1H
NMR (300 MHz CDCl3)
d
4.25 (s, 1H), 7.56 (t, 1 H, J ¼ 6.6 Hz), 7.74e
7.82 (m, 2H), 7.98 (d, 1 H, J ¼ 9.0 Hz), 8.18 (d, 1 H, J ¼ 8.7 Hz), 8.25 (d,
(300 MHz CDCl3)
d
4.00 (s, 3H), 4.20 (s, 3H), 7.37 (s, 1H), 7.49 (d, 1 H,
1 H, J ¼ 9.0 Hz), 8.69 (s, 1H). 13C NMR (400 MHz CDCl3)
d 162.18,
J ¼ 9.3 Hz), 7.60 (d, 1 H, J ¼ 9.3 Hz), 8.06e8.11 (m, 1H), 8.38 (s, 1H).
150.84, 150.67, 138.66, 133.73, 130.98, 129.78, 125.66, 123.48,
122.40, 120.38, 119.04, 97.47, 64.27. HRMS (EI) m/z: calcd for
C14H11NOI (Mþ) 335.9885, found 335.9845.
MS (DART) m/z 318, 319 (Mþ).
4.2.20. 6-Bromo-2-dimethylamino-9-methoxyacridine (20)
Using the above procedure for 19 starting from 17, the title
compound 20 (89 mg, quant) was obtained as a yellow powder. 1H
4.3. Radioiodination
NMR (300 MHz CDCl3)
d
3.15 (s, 6H), 4.17 (s, 3H), 7.04 (s, 1H), 7.51e
The radioiodinated forms of compounds 5, 12, 13, 14, 25 were
7.60 (m, 2H), 8.05 (d, 2 H, J ¼ 7.8 Hz), 8.33 (s, 1H). MS (DART) m/z
prepared from the corresponding tributyltin derivatives by iodo-
331, 333 (Mþ).
destannylation. In brief, 3% H2O2 (50
corresponding tributyltin derivative (50
(0.1e0.2 mCi, specific activity 2200 Ci/mmol), and 1 M HCl (50
m
L) was added to a mixture of
g/50
L EtOH), [125I]NaI
L)
m
m
4.2.21. 6-Bromo-9-methoxyacridine (21)
m
Using the above procedure for 19 starting from 18, the title
sealed vial. The reaction was allowed to proceed at room temper-
ature for 3 min and terminated by addition of NaHSO3. After alka-
compound 21 (371 mg, 44%) was obtained as a white powder. 1H
NMR (300 MHz CDCl3)
d
4.25 (s, 3H), 7.54e7.61 (m, 2H), 7.80 (t, 1 H,
lization with 200 mL of 2 M NaOH and extraction with ethyl acetate,
J ¼ 7.5 Hz), 8.14e8.28 (m, 2H), 8.4 (s, 1H). MS (DART) m/z 288, 290
the extract was dried by passing through an anhydrous Na2SO4
column and then blown dry with a stream of nitrogen gas. [125I]5
and [125I]12 were purified by HPLC on a Cosmosil C18 column with
an isocratic solvent of H2O/methanol/triethylamine (4000:6000:5),
(Mþ).
4.2.22. 2,9-Dimethoxy-6-tributylstannylacridine (22)
Using the above procedure for 4 starting from compound 19, the
[
125I]13 and [125I]14 were purified with H2O/methanol/triethyl-
title compound 22 (101 mg, 47%) was obtained as a brown oil. 1H
amine (5000:5000:5), and
[
125I]25 was purified with H2O/

T. Fuchigami et al. / European Journal of Medicinal Chemistry 60 (2013) 469e478
477
acetonitrile (3:7) at a flow rate of 1.0 mL/min, respectively. [125I]26
was prepared from the corresponding tributyltin derivatives by
iododestannylation. Briefly, Chloramine T (10 L, 10 mg/mL) was
added to a mixture of a tributyltin derivative (50 g/50 L EtOH),
125I]NaI (0.1e0.2 mCi, specific activity 2200 Ci/mmol), and 50
L of
4.6. Neuropathological staining of model mouse brain sections
m
The Tg2576 mice (female, 32-month-old) and wild-type mice
(female, 30-month-old) were used as the Alzheimer’s model and
control mice, respectively. After the mice were sacrificed by
decapitation, the brains were immediately removed and frozen in
powdered dry ice. The frozen blocks were sliced into serial sections,
m
m
[
m
1 M HCl in a sealed vial. The reaction was allowed to proceed at
room temperature for 3 min and terminated by addition of NaHSO3.
After alkalization with 200
mL of 2 M NaOH and extraction with
10
mm thick. Each slide was incubated with a 50% EtOH solution
ethyl acetate, the extract was dried by passing through an anhy-
drous Na2SO4 column and then blown dry with a stream of nitrogen
gas. The radioiodinated ligand was purified by HPLC on a Cosmosil
C18 column with an isocratic solvent of H2O/acetonitrile (3:7) at
a flow rate of 1.0 mL/min. Each 125I ligand was stable in an aqueous
solution at least 6 h.
(100
m
M) of quinacrine, 5, 13, 25, and 26 for 3e10 h. The sections
were washed in 50% EtOH or 50% DMSO for 5 min two times. The
fluorescence images were collected by BZ8100 (Keyence) using
a GFP-BP filter set (excitation, 470 nm; diachromic mirror, 495 nm;
longpass filter, 535 nm). Thereafter, the serial sections were also
stained with thioflavin S, a pathological dye commonly used for
staining Ab plaques in the brain, and examined using the micro-
4.4. Binding assays using the aggregated A
b
peptide in solution
scope in the same condition with that of acridines.
A solid form of A 42 was purchased from Peptide Institute
b
4.7. In vivo biodistribution in normal mice
(Osaka, Japan). Aggregation was carried out by gently dissolving the
peptide (0.25 mg/mL) in a buffer solution (pH 7.4) containing
10 mM sodium phosphate and 1 mM EDTA. The solution was
incubated at 37 ꢂC for 42 h with gentle and constant shaking.
A PBS solution (pH 7.4, 100
containing DMSO (30 L) was injected intravenously into the tail
m mCi)
L) of [125I]ligands (0.1e0.4
m
of ddY mice (5-week-old, 22e25 g). The mice were sacrificed at
various time points post-injection. The organs of interest were
removed and weighed, and radioactivity was measured with the
gamma counter.
For saturation binding assays,
concentration, 4e500 nM) was prepared by addition of nonradio-
active 5. Nonspecific binding was defined in the presence of 10
nonradioactive 5. The saturation assays were performed by mixing
an appropriate concentration of 100
L of [125I]5 in 10% EtOH,
0.25 mg/mL of A (1e42) aggregates, and 1.35 mL of 10% ethanol.
a solution of [
125I]5 (final
mM
m
Acknowledgments
b
After incubation for 2 h at room temperature, the mixture was
centrifuged at 16,000 rpm for 10 min at room temperature. The
Financial support was provided by a Grant-in-Aid for Scientific
Research (B) (Grant No. 21390348) from Japan Society for the
Promotion of Science (JSPS).
supernatant (500 mL) of each samples were measured by an auto-
matic gamma counter (PerkinElmer, 2470 WIZARD2) and bound/
free ratio of [125I]ligand was calculated. The dissociation constant
(Kd) and Bmax of compound 5 was determined by Scatchard analysis
using GraphPad Prism (GraphPad Software, San Diego, CA).
Binding assays by using filtration techniques were carried out as
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