Dibenzothiazoles as novel amyloid-imaging agents

Article abstract of DOI:10.1016/j.bmc.2006.11.022

Novel dibenzothiazole derivatives were synthesized and evaluated as amyloid-imaging agents. In vitro quantitative binding studies using AD brain tissue homogenates showed that the dibenzothiazole derivatives displayed high binding affinities with K_i values in the nanomolar range (6.8-36 nM). These derivatives are relatively lipophilic with partition coefficients (logP oct) in the range of 1.25-3.05. Preliminary structure-activity relationship studies indicated dibenzothiazole derivatives bearing electron-donating groups exhibited higher binding affinities than those bearing electron-withdrawing groups. A lead compound was selected for its high binding affinity and radiolabeled with [¹²⁵I] through direct radioiodination using sodium [¹²⁵I] iodide in the presence of Chloramine T. The radioligand (4-[2,6′]dibenzothiazolyl-2′-yl-2-[¹²⁵I]-phenylamine) displayed moderate lipophilicity (logP oct, 2.70), very good brain uptake (3.71 ± 0.63% ID/g at 2 min after iv injection in mice), and rapid washout from normal brains (0.78% and 0.43% ID/g at 30 and 60 min, respectively). These studies indicated that lipophilic dibenzothiazole derivatives represent a promising pharmacophore for the development of novel amyloid-imaging agents for potential application in Alzheimer's disease and related neurodegenerative disorders.

Full text of DOI:10.1016/j.bmc.2006.11.022

Bioorganic & Medicinal Chemistry 15 (2007) 2789–2796
Dibenzothiazoles as novel amyloid-imaging agents
Chunying Wu,a,b Jingjun Wei,b Kuanqiang Gaob and Yanming Wanga,b,
*
aDepartment of Chemistry and Radiology, Case Western Reserve University, Cleveland, OH 44106, USA
bCollege of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago,
Chicago, IL 60612, USA
Received 3 July 2006; revised 3 November 2006; accepted 13 November 2006
Available online 16 November 2006
Abstract—Novel dibenzothiazole derivatives were synthesized and evaluated as amyloid-imaging agents. In vitro quantitative bind-
ing studies using AD brain tissue homogenates showed that the dibenzothiazole derivatives displayed high binding affinities with Ki
values in the nanomolar range (6.8–36 nM). These derivatives are relatively lipophilic with partition coefficients (logP oct) in the
range of 1.25–3.05. Preliminary structure–activity relationship studies indicated dibenzothiazole derivatives bearing electron-donat-
ing groups exhibited higher binding affinities than those bearing electron-withdrawing groups. A lead compound was selected for its
high binding affinity and radiolabeled with [125I] through direct radioiodination using sodium [125I] iodide in the presence of Chlo-
ramine T. The radioligand (4-[2,60]dibenzothiazolyl-20-yl-2-[125I]-phenylamine) displayed moderate lipophilicity (logP oct, 2.70),
very good brain uptake (3.71 0.63% ID/g at 2 min after iv injection in mice), and rapid washout from normal brains (0.78%
and 0.43% ID/g at 30 and 60 min, respectively). These studies indicated that lipophilic dibenzothiazole derivatives represent a prom-
ising pharmacophore for the development of novel amyloid-imaging agents for potential application in Alzheimer’s disease and
related neurodegenerative disorders.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
agents that readily enter the brain and specifically target
at SP or NFTs have to be developed.
Alzheimer’s disease (AD) is a progressive and irrevers-
ible neurodegenerative disorder that is characteristic of
amyloid deposition in the forms of senile plaques (SPs)
and neurofibrillary tangles (NFTs).1–4 Both SP and
NFT accumulation have been suggested as early and
specific events in the pathogenesis of AD.5–8 Currently
postmortem histopathological examination of SP and
NFTs in the brain is still the only method for definitive
diagnosis. One of the major tasks in AD research is to
detect and quantify SP and NFT in living subjects, pref-
erably at early or even pre-symptomatic stages. Toward
this goal, nuclear imaging techniques such as positron
emission tomography (PET) and single photon emission
computed tomography (SPECT) have been employed.
When used in conjunction with trace amount radioli-
gands, PET and SPECT have the capacity to detect
and quantify amyloid deposition in vivo. As a prerequi-
site to using these imaging techniques, amyloid-imaging
To date, applications of PET and SPECT for amyloid-
imaging have been hampered by the lack of suitable
amyloid-imaging probes. Recent efforts have been
focused on small-molecule amyloid dyes used widely in
AD pathology. Such amyloid dyes include Congo Red
(CR), thioflavin T (ThT), and thioflavin S (ThS)
(Fig. 1). Because these histological dyes are either posi-
tively or negatively charged, they are incapable of pene-
trating the brain–blood barrier (BBB). This has led to
the development of neutral derivatives of the amyloid
dyes for potential in vivo imaging studies.
Several types of amyloid-imaging agents have thus been
synthesized and evaluated. Systematic modification of
CR resulted in a series of bisstyrylbenzene deriva-
tives.9–18 These bisstyrylbenzene derivatives exhibited
high binding affinity and specificity with improved brain
uptake. However, no lead compounds have been identi-
fied with in vivo pharmacokinetic profiles that meet a
series of strict requirements set for in vivo imaging.
Keywords: Amyloid-b; Alzheimer’s disease; Thioflavin S; PET; SPECT;
Imaging.
*
Further modification of CR also led to the design
and synthesis of a series of stilbene derivatives as
Corresponding author. Tel.: +1 216 844 3288; fax: +1 216 844
8062; e-mail: yanming.wang@case.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.022

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C. Wu et al. / Bioorg. Med. Chem. 15 (2007) 2789–2796
N+
NH2
H2N
S
N+
N
N
-O3S
S
N
S
N
N
N
N+
NaO3S
SO3Na
Congo Red (CR)
Thioflavin S (ThS)
(major component)
Thioflavin T (ThT)
Figure 1. Structures of histological amyloid dyes used as prototypes for the development of amyloid-imaging agents.
amyloid-imaging agents for either PET or SPECT
studies.19–25 Following appropriate radiolabeling, the
stilbene derivatives have been evaluated for in vivo
and in vitro binding properties to amyloid deposits
and pharmacokinetic profiles. Most of these stilbene
derivatives readily penetrated the BBB and selectively
bound to amyloid deposits at high affinities. These
studies have led to the identification of a lead com-
pound, termed [11C]SB-13 ([11C]-4-N-methylamino-40-
hydroxystilbene) (Fig. 2), that can be used for PET
amyloid-imaging in human subjects.20 In AD subjects,
[11C]SB-13 displayed an accumulate pattern that is
considered consistent with the previously reported
AD pathology. In contrast, little or no retention of
[11C]SB-13 was observed in age-matched control
subjects.20
in AD subjects. Conversely, PIB showed rapid entry and
clearance in all cortical gray matter of healthy control
subjects.
As an imaging agent for SPECT, a [123I]-labeled imidazo
[1,2-a] pyridine derivative, termed IMPY (6-iodo-2-(40-
dimethylamino-)phenyl-imidazo[1,2]pyridine) (Fig. 2),
has been identified and its pharmacological effects have
been evaluated in human subjects. Preliminary studies in
both AD and normal control subjects demonstrated that
IMPY is a safe radiotracer for clinical imaging studies.36
These studies paved the way for the potential use of
[
deposits in human subjects.
123I] IMPY in clinical SPECT imaging of amyloid
In addition, amyloid-imaging agents have also been
derived from other histological dyes such as acridine
orange,37,38 fluorene,39 and DDNP.40–42 In fact, the first
PET amyloid-imaging studies in human subjects were
carried out with a F-18-labeled DDNP analog termed
[18F] FDDNP ([18F]-2-(1-(2-(N- (2-fluoroethyl) -N-meth-
ylamino) naphthalene-6-yl) ethylidene) malononitrile)43
(Fig. 2). Clinical studies suggested that [18F]FDDNP’s
retention in amyloid deposit regions may be due to selec-
tive binding to both SPs and NFTs in the brain.
Another amyloid dye that has been extensively studied is
thioflavin T (ThT). ThT is a positively charged histolog-
ical dye for amyloid that cannot penetrate the BBB.26
Elimination of the positive charge has led to the
development of a series of benzothiazole and related het-
erocycles such as 2-aryl-substituted benzothiazole
derivatives,17,27–31 2-aryl-substituted benzooxazole de-
rivatives,32 2-aryl-substituted benzofuran,33 and imidazo
[1,2-a] pyridine derivatives.34,35 Most of these neutral,
lipophilic ThT analogs bind to amyloid fibrils with high
affinity and specificity. The in vivo pharmacokinetic pro-
files of the above heterocyclic compounds have been
extensively evaluated as potential amyloid-imaging
agents. Compared to neutral CR analogs, lipophilic
ThT analogs have even smaller molecular weights and
display a higher brain uptake. A lead compound, termed
PIB ([11C]-2-(4-(methylamino)phenyl)-6-hydroxybenzo-
thiazole) (Fig. 2), was thus identified for human studies.
Extensive clinical PET studies indicated that PIB readily
entered the brain and selectively bound to amyloid
deposits in AD subjects. PIB accumulation is predomi-
nant in the cortical areas known for amyloid deposition
In this work, we report a series of dibenzothiazole deriv-
atives as amyloid-imaging agents. The dibenzothiazole
pharmacophore is seen in several histological dyes such
as primuline44 and thioflavin S (ThS).45–47 Primuline
and ThS have been commonly used as viability stains
of starch in phytoplankton and a fluorescent stain for
amyloid, respectively. However, Primuline and ThS exist
as a mixture of several components. The major compo-
nent of these dyes contains two conjugated benzothiaz-
ole units.48 We thus designed and synthesized a series
of dibenzothiazole derivatives. Compared with primu-
line and ThS, these dibenzothiazole derivatives are lipo-
philic and readily enter the brain, making it possible for
HO
11CH3
S
HO
NH11CH3
N
H
N
SB-13
6-OH-BTA-1
CN
CN
N
18F
N(CH3)2
N
N
123I
FDDNP
IMPY
Figure 2. Structures of amyloid-imaging agents that have been evaluated in human subjects.

C. Wu et al. / Bioorg. Med. Chem. 15 (2007) 2789–2796
2791
potential in vivo amyloid-imaging agents. In vitro eval-
uations suggested that these dibenzothiazole derivatives
bound to amyloid deposits in AD brain homogenates
with high affinities. In vivo brain permeability studies
of selected compounds displayed high initial brain up-
take. These studies thus expand the current portfolio
of amyloid-imaging agents for potential clinical
applications.
methylated with methyliodide and K2CO3 in DMSO
to monomethylamino derivatives (14–16) and dimeth-
ylamino derivatives (17).
The synthesis of an iodinated compound 4-[2,60]dib-
enzothiazolyl-20-yl-2-iodo-phenylamine (18) and its
radiolabeling with 125I is described in Scheme 2. Thus,
the cold standard compound 18 was first synthesized
by treating 10 with ICl in AcOH at room temperature
for 18 h. Similarly, compound 10 was also used directly
as the precursor for radiolabeling. This synthesis of [125I]
18 was achieved through direct radioiodination using
sodium [125I] iodide in the presence of Chloramine T
(ChT). The reaction was monitored by HPLC and went
to completion after 3 h. The overall radiochemical yields
of [125I] 18 were 20–30% after HPLC purification. [125I]
18 was obtained with a radiochemical purity over 98%
and a specific activity near the theoretical limit
(80 TBq/mmol) based on the no-carrier added sodium
2. Results and discussion
2.1. Chemistry, synthesis, and radiolabeling
The synthesis of dibenzothiazole derivatives is described
in Scheme 1 starting from commercially available p-
aminobenzothiazole (1). As shown in Scheme 1, 2-amin-
obenzothiazole-6-carboxylic acid (2) was first prepared
from 1 based on previously reported procedures.49 Basic
hydrolysis of 2 followed by neutralizing in HCl and
ZnCl2 yielded the Zinc salt of 4-amino-3-mercaptoben-
zoic acid (3), which was coupled immediately with
p-nitrobenzoyl chloride to give 2-(4-nitro-phenyl)-ben-
zothiazole-6-carbolic acid (4) with 83% yield. The 6-car-
bolic acid of 4 was then converted into acyl chloride (5)
followed by coupling with a 5-substituted amino-
thiophenol to give 600-substitute-20-(4-nitro-phenyl)-
[2,60]dibenzothiazolyl (6–9). Reduction of 6–9 with
SnCl2 in ethanol afforded 600-substitute-20-([2,60]dibenzo-
thiazolyl-20-yl)-aniline (10–13), which can be further
[
125I] iodide. The radiochemical identity of [125I] 18
was verified by co-elution with the non-radioactive cold
standard 18 on HPLC profiles. [125I] 18 was stable en-
ough to be kept for up to 8 h at room temperature
and for up to 2 months in the refrigerator.
2.2. Partition coefficients
Based on the conventional octanol–water partition
measurement, the lipophilicity of [125I] 18 was deter-
mined in terms of partition coefficients (logP oct).
O
O
O
Zn
S
1. NaSCN/Br
S
2
KOH, H O
2
HO
HO
HO
NH2 .HCl
2. HCl
ZnCl
2
N
2
NH2
NH2
3
2
1
O
O
p-NO -PhCOCl
2
S
N
S
N
SOCl
2
HO
Cl
NO2
NO2
C H Cl
6
5
4
5
R
SH
NH
R
S
N
R
S
N
S
N
2
SnCl
2
S
NO2
NH2
EtOH, Con. HCl
N
10: R=-H
6: R=-H
11: R=-Cl
12: R=-F
13: R=-OCH3
7: R=-Cl
8: R=-F
9: R=-OCH3
R
S
R
S
CH I
3
S
S
+
NHCH3
N(CH3)2
N
N
K CO
2
3
N
N
14: R=-H
17: R=-F
15: R=-Cl
16: R=-F
Scheme 1. Synthesis of dibenzothiazole derivatives.

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C. Wu et al. / Bioorg. Med. Chem. 15 (2007) 2789–2796
Scheme 2. Synthesis and [125I] labeling of 18.
The logP oct of [125I] 18 was found at 2.70 and the
logP oct values of other dibenzothiazole derivatives
were then estimated based on coefficients determined
by Hansch and Leo50 As shown in Figure 3, the logP
oct values of these derivatives are between 1 and 3, a
range that has been previously proposed for optimal
brain uptake.51
aliquoted into 1-ml portions, and stored at ꢀ70 ꢁC for
future use.
As shown in Figure 3, the newly developed dibenzo-
thiazole derivatives competed effectively with [3H]PIB
binding site(s) on AD homogenates at high affinities,
the Ki values of compounds 10–18 are shown in Figure
3, which showed relatively high binding affinities in the
range of 6.8–36 nM. The results indicated that these dib-
enzothiazole derivatives bind to the same site as PIB
does on AD homogenates. According to the in vitro
binding assays, functional groups have moderate effects
on the binding affinity. Compounds containing electron-
donating groups showed slightly higher binding affinity
than those containing electron-withdrawing group. For
example, the Ki values decreased in the order of 12 (6-
F) > 11 (6-Cl) > 10 (6-H) > PIB (6-OH), consistent with
the order of increasing electron-donating capacity. In
addition, methylation of the amino group increased
the binding affinity. Thus, N,N-dimethylated derivative
(compound 17) and N-monomethylated derivatives
(compounds 14–16) displayed higher binding affinities
than that of the primary amino derivatives (compounds
10–13).
2.3. In vitro quantitative binding assay using AD brain
region homogenates
The binding affinities of these newly developed com-
pounds for b-amyloid were evaluated using AD brain
homogenates and tritiated PIB ([3H]PIB, Amersham),
a radioligand previously developed with a high affinity
for synthetic Ab aggregation (Ki = 4.3 nM).30 The post-
mortem brain tissues were obtained from well-defined
AD patients. The gray matter was then carefully sepa-
rated from white matter at autopsy and kept in
ꢀ70 ꢁC. The fresh frozen gray matter was then homog-
enized by milling it thoroughly in a mortar in the pres-
ence of liquid nitrogen. The homogenates were then
prepared in phosphate-buffered saline (PBS, pH 7.4) at
a concentration of approximately 400 mg tissue/ml,
Figure 3. Competitive binding assays of dibenzothiazole derivatives using [3H] PIB as the radioligand in AD brain tissue homogenates, and the
binding affinity (Ki) and lopophilicity (logP oct) of newly synthesized dibenzothiazole derivatives.

C. Wu et al. / Bioorg. Med. Chem. 15 (2007) 2789–2796
2793
Table 1. Brain uptake in mice (n = 3, %ID/g)
4.2. Synthesis of 2-aminobenzothiazole-6-carbolic acid
(2)49
Compound
125I]18
2 min
30 min
60 min
[
3.71 0.63
0.78 0.14
0.43 0.12
NaSCN (65 g, 0.8 mol) was added to a suspension of
commercially available 4-amino-benzoic acid (1, 100 g,
0.73 mol) in MeOH followed by the addition of Br2
(38 ml, 0.73 mol) in portions. The above solution was al-
lowed to cool to ꢀ10 ꢁC and stirred for 2 h while keep-
ing the inner temperature below ꢀ5 ꢁC. The precipitate
was then filtered and suspended in 350 ml of 1 M HCl.
The suspension was heated to reflux for 30 min. After
immediate filtration, 150 ml concd HCl was added to
the hot filtrate to give 70 g (yield 42%) of 2-amino-ben-
zothiazole-6-carboxylic acid (2) (as a white solid), which
was dried and used without further purification.
2.4. In vivo brain uptake in normal mice
For potential in vivo imaging studies, we radiolabeled
compound 10 with 125I for brain uptake studies. Follow-
ing a single iv injection of [125I] 18 (0.2 ml, 0.185 MBq),
the brain permeability was evaluated in normal mice.
The brain radioactivity concentration of [125I] 18 was
determined at 2, 30, and 60 min postinjection. As shown
in Table 1, [125I] 18 displayed rapid brain entry at early
time intervals. The initial brain uptake was 3.71 0.63%
ID/g at 2 min postinjection, a level that is considered for
potential clinical imaging studies. The brain radioactiv-
ity concentration decreased sharply to 0.78 0.14% ID/
g at 30 min and 0.43 0.12% ID/g at 60 min, with a 2-
to-30 min ratio of 5. These results indicate that the
non-specific binding of [125I] 18 was just as low as it rap-
idly clears from the normal mouse brain in the absence
of amyloid deposits.
4.3. Synthesis of Zinc salt of 4-amino-3-mercaptobenzoic
acid (3)
Under Argon, compound 2 (9.18 g, 40 mmol) was dis-
solved in a KOH solution (45 g KOH/45 ml water)
and heated to reflux for 3 h. After being cooled to room
temperature, the solution was neutralized by concd HCl
(50 ml). Then ZnCl2 in 25 ml of water was added slowly
while white solid precipitated out. The suspension was
acidified by AcOH. The solid was filtered, washed with
water, and dried in a vacuum to give 8.18 g (98 %) of
4-amino-3-mercaptobenzoic acid (3) as a white solid.
1H NMR (300 MHz, DMSO-d6) d12.02 (br, 2H), 7.90
(s, 1H), 7.63 (d, J = 7.0 Hz, 1H), 7.53 (s, 1H), 7.30 (d,
J = 8.0 Hz, 1H), 6.74 (d, J = 8.5 Hz, 1H), 6.55 (d,
J = 8.1 Hz, 1H), 6.24 (br, 2H), 5.66 (br, 2H).
3. Conclusion
In summary, we have developed a new type of amyloid-
imaging agent based on the dibenzothiazole pharmaco-
phore. These derivatives displayed high binding affinities
for AD brain homogenates. When labelled with 125I,
[
125I] 18 readily enters the brain at early time intervals
followed by rapid washout from the normal mouse
brain, indicating low non-specific binding. Further stud-
ies are under way to systematically evaluate these novel
amyloid-imaging agents for potential in vivo studies.
Once fully developed, these agents would allow for an
early detection and quantification of the amyloid load
in living subjects and facilitate efficacy in evaluating
anti-amyloid therapies currently being investigated. 52,53
4.4. Synthesis of 2-(40-nitrophenyl)-6-(benzothiazol-
yl)benzothiazole (4)
Compound 3 (8.18 g, 20 mmol) was suspended in pyri-
dine (50 ml) and heated to 80 ꢁC. p-Nitrobenzoyl chlo-
ride (7.95 g, 42.8 mmol) was added in portions to give
a clear solution, which was stirred for another hour.
After being cooled to room temperature, the precipitate
was filtered, washed with dilute hydrochloric acid and
water, and dried under a vacuum to afford 9.95 g
(83%) of 2-(40-nitrophenyl)-6-(benzothiazolyl)benzo-
thiazole (4), which was used directly without further
purification.
4. Experimental
4.1. General remarks
All chemicals were purchased from Sigma-Aldrich and
used without further purification. 1H NMR spectra were
obtained at 300 MHz on Bruker DPX-300 (QNP probe)
NMR spectrometers using 5 mm NMR tubes (Wilmad
528-PP) in CDCl3 or DMSO-d6 (Aldrich or Cambridge
Isotopes) solutions at room temperature. Chemical
shifts are reported as d values relative to internal
TMS. HR-ESIMS were acquired under the electron
spray ionization (ESI) condition. The radioactivities of
4.5. Synthesis of 2-(4-nitro-phenyl)-benzothiazole-6-car-
bonyl chloride (5)
Compound 4 (1.00 g, 3.3 mmol) was suspended in SOCl2
(5 ml) and heated to reflux for 1 h. Then excess SOCl2
was evaporated under reduced pressure to get 2-(4-
nitro-phenyl)-benzothiazole-6-carbonyl chloride (5),
which was used without further purification.
3
125I and H were calculated by the counts per minute
4.6. General synthesis of 600-substitute-20-(4-nitro-phenyl)-
[2,60]dibenzothiazolyl (6–9)
in a c counter (Cobra Packard model U5005) and a mul-
tiple-purpose scintillation counter (Beckman, LS 6500).
Radiochemical purity was determined by Hewlett Par-
kard high-pressure liquid chromatography (HPLC) sys-
tem equipped with UV and Bioscan flow count
detectors.
To a suspension of 5 in chlorobenzene (28 ml), 5-substi-
tuted aminothiophenol (2-aminothiaphenol (0.60 g,
4.8 mmol),
3.75 mmol), 2-amino-5-fluoro-benzenethiol (0.60 g,
2-amino-5-chloro-benzenethiol
(0.60 g,

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C. Wu et al. / Bioorg. Med. Chem. 15 (2007) 2789–2796
4.2 mmol), and 2-amino-5-methoxy-benzenethiol (0.65 g,
4.19 mmol)) were added, respectively. The obtained
mixtures were heated to reflux for 3 h. After being
cooled to room temperature, the solids were filtered
and dried under vacuum to give 6 (1.00 g, 79%), 7
(1.00 g, 73%), 8 (1.03 g, 76%), and 9 (1.02 g, 74%).
J = 8.8 Hz, 2H), 6.63 (s, 1H), 2.79 (s, 3H). HR-ESIMS:
m/zcalcd for C21H14ClN3S2 (M+H+): 408.0396, found
408.0382.
4.9. Synthesis of [4-(6-fluoro-[2,60]dibenzothiazolyl-20-yl)-
phenyl]-methylamine (16) and [4-(6-fluoro-[2,60]dibenzo-
thiazolyl-20-yl)-phenyl]-dimethylamine (17)
4.7. General synthesis of 4-(6-substitute-[2,60]dibenzo-
thiazolyl-20-yl)-phenylamine (10–13)
Under Argon, the compound 12 (0.50 g, 1.32 mmol) and
K2CO3 (1.10 g, 6 mmol) were suspended in DMSO
(15 ml) and MeI (0.17 ml, 2.78 mmol) was added. The
sealed vial was heated to 100 ꢁC and stirred for 31 h.
The solution was diluted with ethyl acetate and washed
with water and brine, dried on Na2SO4, concentrated,
and purified by column chromatogrpahy (hexane/ethyl
acetate = 4:1–2:1) to give compound 16 (58 mg, 22%)
and compound 17 (91 mg, 34%). Compound 16: 1H
NMR (400 MHz, DMSO-d6) d 8.38 (s, 1H), 7.95–8.21
(m, 4H), 7.87 (d, J = 7.3 Hz, 2H), 7.44 (d, J = 4.5 Hz,
1H), 6.69 (d, J = 7.6 Hz, 2H), 2.38 (s, 3H). HR-ESIMS:
m/z calcd for C21H14FN3S2 (M+H+): 392.0691, found
392.0680. Compound 17: 1H NMR (400 MHz,
DMSO-d6) d8.42 (s, 1H), 8.00–8.20 (m, 4H), 7.79 (d,
J = 7.3 Hz, 2H), 7.45 (d, J = 4.5 Hz, 1H), 6.70 (d,
J = 7.5 Hz, 2H), 2.44 (s, 6H). HR-ESIMS: m/z calcd
for C22H16FN3S2 (M+H+): 406.0848, found 406.0844.
To a suspension of 6–9 in concd HCl (13 ml), ethanol
(100 ml), and SnCl2 (2.00 g, 10.0 mmol) were added.
The suspension was heated to 80 ꢁC for 1 h. After being
cooled to room temperature; the solid was filtered;
washed with concentrated HCl, water, and dilute ammo-
nium; and dried in vacuum to give 10 (0.88 g, quant.),
1H NMR (300 MHz, DMSO-d6) d 8.82 (d, J = 1,5 Hz,
1H), 8.18 (d, J = 8.5 Hz, 2H), 8.08 (d, J = 8.0 Hz, 1H),
8.03 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.57
(t, J = 7.3 Hz, 1H), 7.48 (t, J = 7.3 Hz, 1H), 6.69 (d,
J = 8.5 Hz, 2H), 6.03 (s, 2H). HR-ESIMS: m/z calcd
for C20H14N3S2 (M+H+): 360.0629, found 360.0631.
Compound 11 (0.9 g, quant.), 1H NMR (300 MHz,
DMSO-d6) d 8.82 (s, 1H), 8.35 (s, 1H), 8.18 (d,
J = 4.4 Hz, 2H), 8.06 (t, J = 8.8 Hz, 2H), 7.81 (d,
J = 8.5 Hz, 2H), 7.60 (d, J = 4.3 Hz, 1H), 6.71 (d,
J = 8.5 Hz, 2H), 6.04 (s, 1H). HR-ESIMS: m/z calcd
for C20H12ClN3S2 (M+H+): 394.0239, found 394.0225.
Compound 12 (0.83 g, quant.), 1H NMR (400 MHz,
DMSO-d6) d 8.80 (d, J = 6.3 Hz, 1H), 8.01–8.18 (m,
6H), 7.83 (d, J = 7.4 Hz, 2H), 7.44 (d, J = 4.6 Hz, 1H),
6.71 (d, J = 7.7 Hz, 2H). HR-ESIMS: m/z calcd for
C20H12FN3S2 (M+H+): 378.0535, found 378.0523.
Compound 13 (1.03 g, quant), 1H NMR (400 MHz,
DMSO-d6) d 8.43 (d, J = 2.9 Hz, 1H), 7.98 (dd,
J = 6.9, 8.7 Hz, 2H), 7.80 (t, J = 8.6 Hz, 2H), 7.14 (m,
3H), 6.69 (d, J = 8.6 Hz, 2H), 6.02 (s, 2H), 3.87 (s,
3H). HR-ESIMS: m/z calcd for C21H15N3OS2
(M+H+): 390.0735, found 390.0728.
4.10. Synthesis of 4-[2,60]dibenzothiazolyl-20-yl-2-iodo-
phenylamine (18)
Under Argon, ICl (0.07 ml) was added dropwise to the
suspension of 10 (20 mg, 0.056 mM) in AcOH (10 ml).
The resulting mixture was sealed and stirred at room
temperature for 18 h. The reaction was quenched with
ethanol and the solvent was removed. The residue was
purified by preparative TLC to get 4-[2,60]dibenzothiaz-
olyl-20-yl-2-iodo-phenylamine (18, 10 mg, 38%) as
brown solid. 1H NMR (300 MHz, DMSO-d6) d 8.87
(s, 1H), 8.44 (s, 1H), 8.40 (d, J = 3.6 Hz, 1H), 8.32 (d,
J = 1.8 Hz, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.64 (s, 1H),
7.57 (t, J = 7.3 Hz, 1H), 7.48 (t, J = 7.3 Hz, 2H), 6.69
(d, J = 8.5 Hz, 1H), 6.10 (s, 1H). HR-ESIMS: m/z calcd
for C20H12IN3S2 (M+H+): 485.9596, found 485.9588.
4.8. General synthesis of [4-(6-substitute-[2,60]dibenzo-
thiazolyl-20-yl)-phenyl]-methylamine (14–15)
Under Argon, compounds 10–11 (1.30 mmol) and
K2CO3 (1.15 g, 8.34 mmol) were suspended in DMSO
(15 ml) followed by an addition of MeI (0.17 ml,
2.78 mmol). The sealed vial was heated to 100 ꢁC and
stirred for 31 h. The solution was diluted with ethyl ace-
tate and washed with water and brine, and dried over
Na2SO4. After evaporating the solvent, the crude prod-
uct was purified by flash column chromatography (hex-
4.11. Synthesis of 4-[2,60]dibenzothiazolyl-20-yl-2-125I -
phenylamine ([125I] 18)
To a solution of 10 (1 mg) in 1 ml acetic acid was added
sodium [125I]iodide (specific activity 83.05 TBq/mmol) in
0.01 M sodium hydroxide solution. Following the addi-
tion of 50 ll Chloramine T solution (ChT, 30 mg dis-
solved in 500 ll acetic acid), the reaction mixture was
stirred at room temperature for 3 h, and quenched with
200 l/L sodium hydrogensulfite (1 M) solution. The
mixture was diluted with 20 ml of water and adjusted
to pH 7–8 with saturated NaHCO3. The reaction mix-
ture was then loaded onto a Waters C-8 Sep-PakTM plus
cartridge. The Sep-Pak cartridge was washed with 10 ml
of water and dried with a rapid air bolus, and the radi-
oiodinated product was slowly eluted with 2 ml of meth-
anol. The solution was concentrated under nitrogen to
about 200 ll and the crude product was purified
1
ane/ethyl acetate = 4:1–2:1) to give 14 (36 mg, 7%). H
NMR (300 MHz, DMSO-d6) d8.83 (s, 1H), 8.18 (d,
J = 8.0 Hz, 2H), 8.08 (d, J = 8.0 Hz, 1H), 8.04 (d,
J = 8.5 Hz, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.55 (t,
J = 7.0 Hz, 1H), 7.46 (t, J = 7.0 Hz, 1H), 6.69 (d,
J = 8.6 Hz, 2H), 6.62 (d, J = 5.1 Hz, 1H), 2.78 (d,
J = 5.1 Hz, 3H), HR-ESIMS: m/z calcd for
C21H16N3S2 (M+H+): 374.0786, found 374.0797. Com-
pound 15 (100 mg, 19%). 1H NMR (300 MHz,
DMSO-d6) d 8.82 (s, 1H), 8.35 (s, 1H), 8.18 (d,
J = 4.3 Hz, 1H), 8.05 (t, J = 8.7 Hz, 2H), 7.81 (d,
J = 8.7 Hz, 2H), 7.61 (d, J = 4.4 Hz, 1H), 6.68 (d,
by HPLC using
a
Phenomenex C-18 column

C. Wu et al. / Bioorg. Med. Chem. 15 (2007) 2789–2796
2795
(250 · 4.6 mm, acetonitrile:TEA buffer (pH 7.5) = 85:15,
flow rate 1.0 ml/min, tR = 17.31 min). The desired frac-
tions were collected, diluted with 50 ml of water, and
loaded onto a water C-8 Sep-PakTM plus cartridge. After
being washed with another 10 ml of water and dried
with a rapid air bolus, the cartridge was eluted with
2 ml ethanol and dried under N2 to give the final prod-
uct [125I] 18 in overall 20–30% radiochemical yields with
radiochemical purities of >98% after purification by
HPLC.
paper using a Brandel M-24R cell harvest and rapidly
washed three times at room temperature with PBS.
The filters containing the bound radioactivity were
transferred to special vials containing 3 ml of universal
scintillation fluid. Vials were counted using Beckman
LS6500 multi-purpose scintillation counter. Specific
binding was estimated as the difference between total
and nonspecific binding. Under the assay conditions,
the specifically bound fraction was less than 15% of
the total radioactivity. The results were subjected to
nonlinear regression analysis using software GraphPad
Prism by which Kdand Ki values were calculated.
4.12. Partition coefficient determination
Partition coefficients were measured by mixing [125I] 18
(10 ll, RCP > 98%, approximately 50,0000 cpm) with
sodium phosphate buffer (PBS, 3 g, 0.1 M, pH 7.4)
and n-octanol (3 g, 3.65 ml) in test tubes. The tubes were
vortexed for 3 min (1 min 3·) at room temperature fol-
lowed by centrifugation at 3500 rpm for 5 min. Then
1 ml of buffer and 1 ml of n-octanol were taken out,
weighed, and counted. The partition coefficient was
determined by calculating the ratio of cpm/g of n-octa-
nol to that of PBS and expressed as logP oct = log
[cpm/g (n-octanol)/cpm/g(PBS)]. Another 2 ml from
the rest of n-octanol layer was taken out and reparti-
tioned in a tube previously containing 3 g PBS and
1.65 ml of n-octanol until consistent partitions of the
coefficient values were obtained. All assays were per-
formed in triplicate.
Acknowledgments
We thank Dr. Anil Gulati (University of Illinois at
Chicago) for assistance in the in vitro binding assays.
This work was supported in part by grants from the
Alzheimer’s Association, the Institute for the Study of
Aging (Y.W.), and the National Institute on Aging
(AG22048, Y.M.). This work was also supported by
National Natural Science Foundation of P.R. China
(30470496, C. Wu) and Natural Science Foundation of
Jiangsu Province, P.R. China (BK2004-423, C. Wu).
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