Aromatic radiofluorination and biological evaluation of 2-aryl-6-[ 18F]fluorobenzothiazoles as a potential positron emission tomography imaging probe for β-amyloid plaques

Article abstract of DOI:10.1016/j.bmc.2011.03.029

To develop agents for radionuclide imaging Aβ plaques in vivo, we prepared three fluorine-substituted analogs of arylbenzothiazole class; compound 2 has a high affinity for Aβ (K_i = 5.5 nM) and the specific binding to Aβ in fluorescent staining. In preparation for the synthesis of these arylbenzothiazole analogs in radiolabeled form as an Aβ plaques-specific positron emission tomography (PET) imaging probe, we investigated synthetic route suitable for its labeling with the short-lived PET radionuclide fluorine-18 (t_1/2 = 110 min) and diaryliodonium tosylate precursors (12, 13a-e and 14). 2-Aryl-6-[¹⁸F]fluorobenzothiazoles ([¹⁸F]1-3) were synthesized in efficiently short reaction times (40-60 min) with high radiochemical yields (19-40%), purities (>95%) and specific activities (85-118 GBq/μmol). Tissue distribution studies showed that high radioactivity of [¹⁸F]2 accumulated in the brain with rapid clearance in healthy mice. Radioactive metabolites were analyzed in brain samples of mice and corresponded to 81% of parent remained by 30 min after a tail-vein injection. These results suggest that [¹⁸F]2 is a promising probe for evaluation of Aβ plaques imaging in brain using PET.

Full text of DOI:10.1016/j.bmc.2011.03.029

Bioorganic & Medicinal Chemistry 19 (2011) 2980–2990
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Aromatic radiofluorination and biological evaluation of 2-aryl-6-[18F]
fluorobenzothiazoles as a potential positron emission tomography
imaging probe for b-amyloid plaques
Byung Chul Lee a,b, Ji Sun Kim a, Bom Sahn Kim c, Ji Yeon Son a, Soo Kyung Hong a, Hyun Soo Park a,
Byung Seok Moon a,b, Jae Ho Jung a, Jae Min Jeong b, Sang Eun Kim a,b,
⇑
a Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seoul 110-799, Republic of Korea
b Institute of Radiation Medicine, Seoul National University Medical Research Center, Seoul National University, Seoul 110-799, Republic of Korea
c Department of Nuclear Medicine, Ewha Womans University College of Medicine, Seoul 158-710, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
To develop agents for radionuclide imaging Ab plaques in vivo, we prepared three fluorine-substituted
analogs of arylbenzothiazole class; compound 2 has a high affinity for Ab (Ki = 5.5 nM) and the specific
binding to Ab in fluorescent staining. In preparation for the synthesis of these arylbenzothiazole analogs
in radiolabeled form as an Ab plaques-specific positron emission tomography (PET) imaging probe, we
investigated synthetic route suitable for its labeling with the short-lived PET radionuclide fluorine-18
(t1/2 = 110 min) and diaryliodonium tosylate precursors (12, 13a–e and 14). 2-Aryl-6-[18F]fluorobenzo-
thiazoles ([18F]1–3) were synthesized in efficiently short reaction times (40–60 min) with high radio-
Received 20 January 2011
Revised 11 March 2011
Accepted 12 March 2011
Available online 21 March 2011
Keywords:
b-Amyloid
Alzheimer’s disease
Diaryliodonium salt
PET
chemical yields (19–40%), purities (>95%) and specific activities (85–118 GBq/lmol). Tissue distribution
studies showed that high radioactivity of [18F]2 accumulated in the brain with rapid clearance in healthy
mice. Radioactive metabolites were analyzed in brain samples of mice and corresponded to 81% of parent
remained by 30 min after a tail-vein injection. These results suggest that [18F]2 is a promising probe for
evaluation of Ab plaques imaging in brain using PET.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
probe in order to develop noninvasive in vivo PET imaging for
AD. To this end, many specific ligands have been developed and
AD is a progressive and fatal neurodegenerative disease that
leads to brain disorder; half of the affected patients suffer from
dementia, cognitive impairment, and memory loss. The most seri-
ous risk factor of AD is that it becomes more pronounced with
increasing age of the patient, and this is a concern because of the
increasing life expectancy of populations worldwide; conse-
quently, there is an increasingly high incidence of AD in the older
population. Approximately 5% of those over 65 years are affected,
while over 20–30% of those over 80 years show signs of dementia.1
It has been reported that the main event in the pathogenesis of AD
is the formation and accumulation of aggregates of Ab peptides in
the brain.2,3 Therefore, Ab plaque, which seems to play an impor-
tant neuropathological role in AD, is mainly comprises an aggrega-
tion of the Ab peptide.4 An Ab plaques-specific imaging probe
would be useful for the diagnosis of AD at an early to moderate
stage, as well as the diagnosis of MCI and the monitoring of the
therapeutic efficacy AD treatment. There has been an ongoing
worldwide search for a clinically useful radiolabeled Ab plaque
evaluated for the imaging of Ab plaques. One of the lead structural
motifs, 2-(40-([11C]methylaminophenyl)-6-hydroxybenzothiazole
(known as [11C]PIB, Fig. 1) has already shown promising results
in clinical trial.5–7 Despite the promising clinical results in AD
patients, PIB remains suboptimal on account of its labeling with
the short-lived carbon-11 (half life = 20 min), which limits its
availability to centers equipped with an on-site cyclotron. To over-
come this limitation of [11C]PIB, due to the short half life of carbon-
11, many research groups tried to introduce fluorine-18 (half
life = 110 min) into their target compound. Thus far, the com-
pounds reported to have a high affinity for Ab plaque and which
contained fluorine-18 were fluorine-18 labeled BTA analogs
(GE-067 and 6-OH-40-FP-BTA),8–10 FDDNP, BAY94–9174, and
AV-45 (Fig. 1).11–13
Among these radioligands, FDDNP was known to bind to both
Ab plaques at different binding sites and in intracellular neurofi-
brillary tangles; it does not selectively measure
a specific
pathologic component of AD patients.11 The recently reported
fluoropegylated stilbene derivatives ([18F]BAY94-9172 and
AV-45) are currently under a phase II and phase III, respectively.
The most commonly studied and most promising of the reported
⇑
Corresponding author. Tel.: +82 31 787 7671; fax: +82 31 787 4018.
E-mail address: kse@snu.ac.kr (S.E. Kim).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.029

B. C. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2980–2990
2981
H
N
R1
Zheng et al. also described the preparation of 6-fluroine substi-
tuted benzothiazole analog as an imaging probe and showed their
compounds have higher binding affinities to Ab aggregates than
PIB.16 It was interesting to note that fluorine effectively replaced
hydroxyl group while continuing to manifest comparable activi-
ties, albeit with different properties, in numerous examples.17,18
Although 6-fluroine substituted benzothiazole analog showed high
binding affinity to Ab plaque, they failed fluorine-18 labeling in the
benzothiazole moiety of their compounds because the introduction
of fluorine-18 into benzothiazole ring was impossible by general
aromatic fluorination.
Gratifying, diaryliodonium salt precursors can be useful to label
fluorine-18 at aromatic position in aryl compounds, which have
proven difficult to label using the general aromatic fluorine-18
labeling methods in the previous works of our group19,20 and the
Pike group.21
N
S
N
S
F
HO
HO
R2
PIB: R1 = CH3, R2 = H
GE-067: R1 = CH3, R2 = F
6-OH-4'-FP-BTA
NC
CN
H3C
H
N
CH3
F
3
O
F
R
N
CH3
BAY94-9174: R = CH
AV-45 : R = N
FDDNP
Figure 1. Chemical structures of previously reported Ab plaque PET imaging
Therefore, we designed three 6-[18F]fluorine labeled BTA ana-
logs instead of the 6-hydroxy group in [11C]PIB and synthesized
various diaryliodonium tosylate precursors to allow aromatic fluo-
rine-18 labeling into the benzothiazole ring. In this investigation,
we prepared compounds 1–3 and [18F]1–3 using two approaches:
the first (Scheme 2) involved the preparation of authentic samples
of these 6-fluoro-substituted BTA analogs; the second was the syn-
thesis of precursors (diaryliodonium tosylate) and the radio-syn-
thetic procedure for BTA analogs in which fluorine-18 could be
introduced to the precursor at a very late step. Reported herein
are in vitro and in vivo evaluations of novel three 6-[18F]fluoro la-
beled BTA analogs for use as a prospective PET probe for Ab plaque
imaging in the brain.
probes.
compounds, members of the novel 2-aryl-6-hydroxybenzothiazole
class ([18F]GE-067, including PIB), showed a high initial brain up-
take, and a particularly high binding affinity to Ab plaque, and
are currently being used in clinical trials (phase III).
Tracing the history of the studies for the fluorine-18 labeled
compounds as an Ab plaque imaging probe, it is believed that
2-(4-aminophenyl)-benzothiazole (BTA) may have potential as a
parental compound and aromatic fluorine-18 labeling into the ben-
zothiazole moiety or aniline in BTA is essential for in vivo stability
and retaining its biological activities as PET imaging probe for Ab
plaque.
2. Results and discussion
2.1. Chemical synthesis
In order to achieve this, preliminary reports on fluorine-18
introduced at the ortho- or para-position in the right aromatic ring
of BTA analogs ([18F]GE-067 and 6-hydroxy-2-(40-[18F]fluoro-
phenyl)-benzothiazoles) were developed.10 In addition to, a previ-
ous article by Henriksen, et al., described the preparation of a
11C-labeled 5-fluorine substituted benzothiazole analog as an
imaging probe and mentioned the fluorine at 5-position metaboli-
cally stabilized BTA analog compare to [11C]PIB is known to be
rapidly metabolized to the C6-sulfonated derivatives.14,15 Recently,
We considered a number of approaches that would be suitable
for the preparation of 6-fluoro-substituted BTA analogs in fluorine-
18 labeled form that would have the high specific activity required
for a PET imaging probe. In our system, it was not clear whether
the benzothiazole substituent at the 6-position would provide
X
SH
(ii)
N
S
8: R = NO2, 57%
1: R = NH2, 74%
R
R
X = F
(iii)
NH2
F
2: R = NHCH3, 35%
3: R = N(CH3)2, 85%
4: X = F
5: X = Br
(i)
OHC
N
S
N
S
(v)
R
X =Br
R
Br
Bu3Sn
6: R = NO2
7: R = N(CH3)2
(ii), 10
(iv)
11a: R = NO2, 52%
11b: R = NCH3Boc, 48%
11c: R = N(CH3)2, 65%
9a: R = NO2, 95%
9b: R = NCH3Boc, 49%
9c: R = N(CH3)2, 21%
(vi)
N
R
S
I
+
-OTs
12: R = NO2, 38%
13a: R = NCH3Boc, 39%
14: R = N(CH3)2, 13%
Scheme 1. Reagents and conditions: (i) DMSO, 180 °C, 30 min; (ii) SnCl2, EtOH, 90 °C, 2 h; (iii) CH3I, K2CO3, DMSO, 100 °C, 16 h; (iv) (a) formaldehyde, MeOH, reflux, 2 h; (b)
NaCNBH3, AcOH, room temperature, 1.5 h; (c) Boc2O, THF, 85 °C, 12 h; (v) Sn2Bu6, Pd(0), THF, 85 °C, 8 h; (iv) Koser’s reagent (15a), CH3CN, room temperature, 12 h.

2982
B. C. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2980–2990
15a: Ar = phenyl
OTs
I
15b: Ar = p-anisole
15c: Ar = p-toluene
15d: Ar = 2-thiophenyl
15e: Ar = 3-thiophenyl
Ar
OH
N
N
S
(i)
NCH3Boc
NCH3Boc
Ar
S
I
Bu3Sn
+
-OTs
13b: Ar = p-anisole, 56%
11b
13c: Ar = p-toluene, 82%
13d: Ar = 2-thiophenyl, 57%
13e: Ar = 3-thiophenyl, 70%
Scheme 2. Reagents and conditions: (i) 15b–e, CH2Cl2, CH3CN, room temperature, 12 h.
Table 1
Reaction of aromatic radiofluorination with 12, 13a, and 14
Entry
Precursora
[
18F]FꢀX+
Solventc
MW or temp
Time
Yielde
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
14b
14
14
14
14
14
14
14
12
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
18F]FꢀCs+
DMF
130 °C
130 °C
100 W
130 °C
100 W
100 W
100 W
100 W
100 W
100 W
130 °C
130 °C
130 °C
130 °C
100 W
10 min
10 min
3 min
10 min
3 min
3 min
3 min
6 min
3 min
NPg
2.4%
26.7%
18F]FꢀCs+
MeCN
MeCN
MeCN
DMF
18F]FꢀCs+
18F]FꢀK+Kryptofix
18F]FꢀK+Kryptofix
18F]TBAF
5%
NPg
MeCNd
MeCN
MeCN
MeCN
MeCN
MeCN
MeCNd
MeCNd
MeCNd
MeCN
18.7%f
18F]TBAF
29%f
18F]TBAF
35.6 3.5% (n = 2)f
18F]FꢀK+Kryptofix
18F]TBAF
5%
12
6 min
35.3 5.7% (n = 4)f
9%
13a
13a
13a
13a
13a
18F]TBAF
10 min
10 min
10 min
15 min
6 min
18F]TBAF
19.3 7.7% (n = 5)f
6.4%
18F]FꢀK+Kryptofix
18F]FꢀK+Kryptofix
18F]TBAF
13.3%
24.4 5.5% (n = 14)f
a
b
c
d
e
f
Radiolabeling was carried out with a radical scavenger (TEMPO, 1 mg).
No TEMPO use.
Solvent (DMF-N0,N0-dimethylformamide or MeCN–acetonitrile, 300
lL) was used with H2O (10
l
L).
No water use.
Progress of the reaction (as shown Scheme 3) and yields were analyzed by radio-TLC (developing solvent: ethyl acetate/hexane = 40:60, v/v).
Yield of isolated pure compounds ([18F]1–3) by semipreparative column using HPLC (55:45 acetonitrile–water, 254 nm, 3 mL/min).
No product.
g
sufficient activation for fluoride ion substitution with general
nucleophilic aromatic substitution of a suitable leaving group.29,30
We were unable to effect fluoride ion substitution at the 6-position
with a nitro group. Curiously, we were also unable to prepare the
more reactive p-trimethylammonium precursor because of the
side compound (N-methylbenzothiazole) in the final methylation
step from the dimethylaniline precursor. The limitation of general
aromatic fluorine-18 labeling in our target compounds led us to
propose an alternative approach to aromatic fluorination. It is
known that diaryliodonium salts can be used as precursors for flu-
oroarenes,31,32 and their value corresponds well with our reported
fluorine-18 labeled PPARgamma ligand by iodonium salts in a pre-
vious work.20
The three 6-fluoro-substituted BTA analogs 1–3 were simply
synthesized from 2-amino-5-fluorobenzenethiol 4 using a little
modification of known methods (Scheme 1).23,33 6-Fluoro-substi-
tuted benzothiazol rings, 8 and 3 were synthesized directly with
2-amino-5-fluorobenzenethiol 4 and 4-nitrobenzaldehyde 6 or
4-(dimethylamino)-benzaldehyde 7, respectively, followed by
reduction of the nitro compound 8 with tin chloride to produce
the corresponding amine 1. This amine compound proceeded
N-monomethylation with iodomethane to produce the corre-
sponding monomethyl amine 2. A different approach was needed
for the synthesis of target compounds 1–3 in radiolabeled form.
The tributyltin analogs 11a–c can be readily prepared from the
bromo compounds 9a–c in the presence of bis(tributyl)tin and a
catalytic quantity of tetrakis(triphenylphosphine)palladium(0). At
a late stage in the synthesis, the fluorine-18 labeling step can rap-
idly and efficiently introduce fluorine-18 into appropriate precur-
sors that are available in radiolabeled form at high specific
activity because the fluorine-18 ion has a relatively short life (half
life = 110 min). Therefore, we designed a different radiolabeling
pathway in which one is a single step with direct aromatic fluo-
rine-18 labeling in the synthesis of [18F]3 and the other is a one-
pot two step with aromatic fluorine-18 labeling and reduction or
deprotection in the syntheses of [18F]1 and [18F]2. Therefore, the
tributyltin analogs were prepared in different functional groups:
dimethyl amine 14, nitro 12 and Boc-protected monomethyl amine
13a–e on the right aromatic ring.
To prepare iodonium tosylate precursors suitable for the intro-
duction of fluorine-18 ion into the BTA analogs, we reacted the tri-
butyltin compounds 11a–c with a commercially available Koser’s
reagent 15a and various hydroxy(tosyloxy)iodoarenes 15b–e.
The electron-rich hydroxy(tosyloxy)iodoarenes are known to be
unstable and to decompose violently at room temperature. Thus,
we prepared 15b–e according to the literature and use it immedi-
ately to prepare the diaryliodnium tosylate precursors 13b–e
under nitrogen atmosphere. Generally, the fluoride anion is

B. C. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2980–2990
2983
Table 2
The radiofluorination with diaryliodonium tosylates 13a–e and hydrolysisa
Table 3
Ki values of 2-aryl-6-fluorobenzothiazole analogs (1–3) and log P of 2-
aryl-6-[18F]fluorobenzothiazole analogs ([18F]1–3)
Entry
Precursor
Yieldb (decay-corrected yields)c
c
Compound
Kia (nM)
Log Poct/PBS
1
2
3
4
5
13a
13b
13c
13d
13e
23.5 7.7% (19.3%)c
18.9 8.3%
1
2
3
PIB
26.2
2.83 0.02
3.20 0.06
3.60 0.09
1.2d
26.5 8.1%
5.5 (0.2)b
5.9 (0.3)b
5.8 (1.6)b
34.3 6.2% (30.1%)c
60.4 5.6% (40.5%)c
a
a
Radiolabeling of precursors (2 mg) were carried out with a radical scavenger
(TEMPO, 1 mg), acetonitrile solvent without H2O at 130 °C for 10 min using
18F]TBAF. Deprotection was carried out using 3 M HCl in ethyl acetate (200
L) at
75 °C for 10 min.
Ki was measured by [125I]TZDM competition binding studies to a
precipitate of synthetic Ab1–42 peptide.
b
[
l
Previously published values16 shown here for comparison (Ki
was measured by radioligand [3H]-PIB competition binding studies
in human brain homogenate).
b
Progress of the reaction (as shown scheme 3) and yields were analyzed by
c
radio-TLC (developing solvent: ethyl acetate/hexane = 40:60, v/v) (n = 5).
Values are mean SD (n = 5).
Ref. 5.
c
d
Yields of isolated pure compound [18F]2 by semipreparative column using HPLC
(55:45 acetonitrile–water, 254 nm, 3 mL/min).
fluorine-18 anion attacked the more electron deficient ring, so that
when these diaryliodonium salt precursors bearing electron-
donating groups (4-MeO and 4-Me) and electron-rich heteroatom
aryl (2- or 3-thiophenyl) tend to undergo nucleophilic aromatic
fluorination on the contrary benzothiazole ring. These electron-
rich heteroaromatic ring effect had an impact on the radiofluorina-
tion of benzothiazole ring as shown in Table 2. The results indicate
that compounds, 13b and 13c did not work effectively compare to
compound 13a, while para-electron-donating groups substituted
compounds, 13d and 13e showed high aromatic radiochemical
yields of [18F]2 (Table 2, entries 4 and 5). Consequently, three fluo-
rine-18 labeled compounds [18F]1–3 were synthesized with de-
sired yields in an environment of radical degeneration,
microwave irradiation of 360 s or oil bath heating. On the 2-mg
scale of precursors in acetonitrile, the radiochemical yields of
expected to attack the more electron deficient ring in diaryliodoni-
um tosylate precursor, so that we have prepared various diaryliod-
onium salt precursors with the ‘dispensable ring’ is phenyl, or the
more electron rich rings; p-methoxyphenyl, p-methylphenyl, and
2- or 3-thiophenyl on 6-position of benzothiazole ring for [18F]2,
which might be superior to fluorine-18 labeling efficiency.
The aromatic radiofluorination between a diaryliodonium salt
and fluoride is dominated by an electron-rich heteroaromatic ring,
while the effects of base and solvent also play a role in increasing
the florine-18 labeling yield. Various conditions were explored for
the preparation of the three BTA analogs [18F]1–3 from different
diaryliodonium tosylate precursors 12, 13a–e and 14, whose prep-
aration has been previously described (Schemes 1 and 2). When the
precursor (14, 2 mg scale) was reacted with cesium [18F]fluoride
under general anhydrous radiofluorination conditions, aromatic
radiofluorination did not work (Table 1, entry 1). In condition of
acetonitrile for solvent or potassium [18F]fluoride Kryptofix 2.2.2
for the fluorine-18 salt form, the radiochemical yields were poor
(data not shown). Considering that these low radiochemical yields
might be due to the instability of iodonium tosylate, McEwen et al.
noted that diaryliodonium salts carried out radical-induced
decomposition in a base condition and generated aromatic hydro-
carbon.34 Therefore, we added a radical scavenger, 2,2,6,6-tetra-
[
18F]1–3 at end-of-synthesis (EOS) were 19–40%, which included
HPLC isolation and a total time of 40–60 min (Scheme 3). These
regioselectivity of diaryliodoniim salt precursors in benzothiazole
ring will be useful in introducing no-carrier-added fluorine-18 into
aromatic region of PET probes.
2.2. Determination of Ki-values
The three prepared 6-fluoro-substituted BTA analogs showed Ki
values in the range of 5.5–26.2 nM (PIB = 5.8 nM) on Ab1–42, indi-
cating that fluorine substituted compounds at the 6-position of
benzothiazole bind to the same site with good affinity (Table 3),
just as known BTA analogs do on AD homogenates despite the fact
that the fluorine ion has decreasing electron-donating capacity as
compared to a hydroxy group in PIB.36 Zheng et al. also synthesized
6-substituted benzothiazole anilines and evaluated their possibili-
ties as an amyloid-imaging probes using AD human brain homog-
enates (Table 3).14 Although the affinities of their compounds were
shown to be below 1 nM as measured using [3H]-PIB, they failed to
label at the 6-position of their compounds with radioisotopes.
methylpiperidine 1-oxyl (TEMPO) to the solvent (300 lL) and
found that the yields were significantly increased, except for
DMF solvent (entry 5).35 Microwave irradiation also successfully
improved the yields (entries 2 vs 3 and 11 vs 15), as compared
to an oil bath system. In the aqueous environment (addition of
10 lL of water), fluorine-18 labeling yield was increased more than
anhydrous condition in case of microwave irradiation condition for
360 s in 18–35% yield (entries 7–10 and 15). On the other hand, an
oil bath heating in the anhydrous acetonitrile at 130 °C for 10 min
gave higher yield than aqueous environment (entry 11 vs 12).
In order to understand the electron-rich heteroaromatic ring ef-
fect, we prepared five diaryliodonium salt precursors containing
the more electron rich rings; p-methoxyphenyl, p-methylphenyl,
and 2- or 3-thiophenyl, compare to phenyl ring on 6-position of
benzothiazole ring for [18F]2 (Scheme 2) and evaluated their aro-
matic radiofluorination using an oil bath heating. In general, the
2.3. In vitro fluorescent staining assay
The thioflavine-S dye fluorescence is widely used for the identi-
fication of amyloid fibrils in vitro. Our target compounds also have
N
N
(i) or (ii) or (iii)
19 -40% EOS yields
R
R'
Ar
I
18F
S
S
+
-OTs
[
[
[
18F]1: R' = NH2
12: R = NO2
13a-e: R = NCH3Boc
14: R = N(CH3)2
18F]2: R' = NHCH3
18F]3: R' = N(CH3)2
Scheme 3. Reagents and conditions: (i) (a) nBu4N[18F]F, CH3CN, TEMPO, M.W. (100 W), 6 min; (b) SnCl2, EtOH, 90 °C,10 min for [18F]1; (ii) (a) nBu4N[18F]F, CH3CN, TEMPO,
M.W. (100 W), 6 min or 130 °C, 10 min; (b) EtOAc/HCl (v/v = 3:1), 75 °C, 10 min for [18F]2; (iii) nBu4N[18F]F, CH3CN, TEMPO, M.W. (100 W), 6 min for [18F]3.

2984
B. C. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2980–2990
Figure 2. In vitro fluorescent staining of brain sections from double transgenic (APPswe/PS14E9, 23 months) and the wild-type mouse: confocal tile scan images—(A)
Thioflavine-S in a APPswe/PS14E9 mouse brain; (B) Thioflavine-S in a wild-type mouse brain; (C) compound 2 in a APPswe/PS14E9 mouse brain; (D) compound 2 in a wild-
type mouse brain.
similar structure, benzothiazole analog, to thioflavine-S and fluo-
resce strongly with excitation and emission. As expected, both thi-
oflavine-S and compound 2 clearly bind plaque using brain
sections of double transgenic mouse (Fig. 2 A and C). Other sets
of images (Fig. 2 B and D) indicate that compound 2 has specific
binding to amyloid plaque in mouse brain.
(at 2, 30, and 60 min, Table 4). For an ideal in vivo Ab plaque probe,
a high initial brain entry and low nonspecific binding in a normal
brain are necessary in order to increase the signal-to-noise ratio.
Table 4 lists the tracer uptake in the brain as the most important
terms of percent injected dose per gram of organ (% ID/g).
In a comparison of the three radiotracers [18F]1–3, the initial
brain uptake in the cortex of [18F]2 was the highest of the three
compounds (6.62 0.3% ID/g at 2 min post-injection), and its
radioactivity was rapidly washed out from the brain at 30 and
60 min post-injection (1.20 0.2 and 0.73 0.1% ID/g, respec-
tively). [18F]1 and [18F]3 showed an initial uptake in the cortex of
5.86 0.3 and 4.39 0.3% ID/g, respectively. In the measure of
brain clearance for the three radiotracers expressed as the ratio
of % ID/g in the cortex at 2 min over % ID in the cortex at 60 min,
2.4. Partition coefficient measurement
In order to measure the potential of compounds [18F]1–3 to
cross the blood–brain barrier (BBB) by passive diffusion, the log
of the partition coefficient (log Poct/PBS) values were distributed in
the range of 2.83–3.60 (Table 3). The results suggested that the
three compounds readily cross the BBB, and that [18F]1 and
[
18F]2, in particular, are within the optimal range (log P between
[
18F]2 showed a higher value (9.1) than [18F]1 (8.0) and [18F]3
1 and 3).
(2.7). The blood levels of [18F]1–3 were relatively low at all points
in time. Figure 3 shows the brain radiouptake of [18F]1–3 in ICR
mice in terms of percent injected dose per gram of organ normal-
ized to body weight (% ID-kg/g) to compare across animals of dif-
ferent size.
2.5. In vitro stability studies
Radiotracers [18F]1–3 were incubated in human serum at 37 °C
and analyzed by radio-TLC (data not shown). The results showed
that over 90% of the three radiotracers remained intact even after
120 min, demonstrating their high chemical stability.
Table 4
Tissue distribution of 2-aryl-6-[18F]fluorobenzothiazoles ([18F]1–3)
2.6. Analysis of metabolites
Radiotracer
Time
Tissue % ID/g SD
Cortex Cerebellum
Blood
Remnanta
Radiolabeled materials were extracted from ICR mice brain
homogenates with over 95% efficiency. When samples of brain
were analyzed by HPLC, ratios of peak area under metabolites to
[
[
[
18F]1
18F]2
18F]3
2 min
30 min
60 min
1.92 0.1
1.23 0.5
1.21 0.1
5.86 0.3
1.79 0.3
0.73 0.1
5.83 0.2
2.19 0.3
1.02 0.2
6.41 0.2
2.86 0.4
1.37 0.1
[
18F]2 were 5:95 at 5 min, 19:81 at 30 min, and 42:58 at 60 min
2 min
30 min
60 min
2.08 0.2
1.13 0.1
1.11 0.1
6.62 0.3
1.20 0.2
0.73 0.1
6.24 0.4
1.38 0.2
0.98 0.1
6.30 0.5
1.91 0.3
1.32 0.2
sample of the brain.
2.7. Biodistribution in normal mice
2 min
30 min
60 min
1.55 0.1
1.04 0.2
1.02 0.1
4.39 0.2
2.17 0.3
1.61 0.2
4.45 0.2
2.03 0.3
1.16 0.1
4.26 0.2
2.80 0.4
2.01 0.2
To measure their in vivo brain penetration and clearance, bio-
distribution studies of [18F]1–3 were performed in healthy male
ICR mice, and their blood, remnant, cortex and cerebellum regional
brain tissue uptakes were determined at different points in time
a
Remnant means the remaining brain tissues after extraction of cerebral cortex
and cerebellum.

B. C. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2980–2990
2985
Figure 3. Comparison of biodistribution data of 2-aryl-6-[18F]fluorobenzothiazoles ([18F]1–3) in ICR mice. The data are expressed as % ID-kg/g.
The highly lipophilic analog [18F]3, however, showed the poor-
est rate of brain entry and it elimination from the brain was slow,
despite its high binding affinity to Ab plaque.
Tissue distribution studies in healthy mice showed a good rate of
binding brain entry and rapid elimination from the brain. Judging
from these results, the new fluorine-18 labeled BTA analog,
The tendency toward uptake of [18F]1 and [18F]2 in the cerebel-
lum was similar to that they exhibited in the cortex, indicating that
these compounds exhibit low non-specific binding in the brain and
a rapid washout rate, which is a highly desirable combination of
properties for an Ab plaque-specific imaging probe. These results
then, taken together with other in vitro and in vivo results, showed
that [18F]1 and [18F]3 may be unsuitable for an Ab plaque imaging
probe because of their low binding affinity to Ab plaque, and the
poor rate of brain entry of the former and the slow washout of
the latter. On the basis of these results, [18F]2 stands out as a par-
ticularly promising compound because it exhibits good in vitro
binding affinity to Ab plaque, a high rate of brain uptake, and a fast
brain washout.
[
18F]2, may be a useful PET probe for Ab plaque imaging. Imaging
studies in the transgenic mouse model of AD are underway to pur-
sue the further development of [18F]2 as an amyloid-imaging
probe.
4. Experimental
4.1. Chemistry
Solvents and reagents were purchased from Sigma–Aldrich (St.
Louis, MO). 1H and 13C NMR were obtained on Varian Gemini-400
(Palo Alto, USA), JEOL Ltd-300 (Tokyo, Japan), and Bruker-300
instrument (Billerica, USA). Chemical shifts were reported in parts
per million (ppm, d units). Electron impact (EI) mass spectra were
obtained on a GC/MS QP5050A spectrometer (Shimadzu, Kyoto, Ja-
pan), and fast atom bombardment (FAB) mass spectra were ob-
tained on a JMS 700 (Jeol Ltd, Tokyo, Japan). Microwave-assisted
reactions were performed with model 520A Resonance Instru-
ments, Inc. (Skokie, IL). HPLC was carried out on a Thermo Separa-
3. Conclusion
In this study, we focused aromatic fluorine-18 labeling on the 6-
position of benzothiazole analogs in order to determine the practi-
cality of an diaryliodonium tosylate precursor, lipophilicity, in
terms of its binding affinity for Ab plaque as well as on the
in vivo biodistribution. Based on the radiosynthesis results re-
ported in our work, it is reasonable to conclude that the relatively
simple BTA analog, 2-(40-N-methylaminophenyl)-6-[18F]fluor-
obenzothiazole ([18F]2), can introduced fluorine-18 in the 6-posi-
tion with good radiochemical yields obtained with the
diaryliodonium tosylate precursor 13e; furthermore, it also shows
a good binding affinity to Ab aggregates by in vitro binding assay.
tion Products System (Fremont, USA) equipped with
semipreparative column (Waters, Xterra RP-18 column, 10
a
l,
7.9 ꢁ 300 mm) or an analytical column (Thermo, Hypresil Gold
C-18 silica gel 5 l,
l
, 4.6 ꢁ 250 mm and Hypersil silica gel 3
4.6 ꢁ 250 mm). Chromatography systems were fitted with a UV
detector (SpectraSystem UV3000 set at 254 nm; Thermo, Fremont,
USA) and a gamma-ray detector (Bioscan Flow-Count fitted with a
NaI(Tl) detector). Thin Layer Chromatography (TLC) was performed

2986
B. C. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2980–2990
using Merck F254 silica plates and analyzed on a Bioscan radio-TLC
scanner (Washington DC, USA). H218O was purchased from Taiyo
Nippon Sanso Corporation. 18F-Fluoride was produced at Seoul Na-
tional University Bundang Hospital by the 18O(p,n)18F reaction
through proton irradiation using the KOTRAN-13 cyclotron (Sam-
young Unitech Co., Ltd, Seoul, Korea). The syntheses of compounds
(2-amino-5-fluorobenzenthiol 4 and 2-amino-5-bromobenzenthiol
5) were prepared according to the literature, respectively.22,23 The
preparation of compound 15b–e was followed by the reported
methods.19,24,25
4.3. General procedure for the synthesis of 1 and 10
4.3.1. 2-(40-Aminophenyl)-6-fluorobenzothiazole (1)
To
a 8
solution of 2-(40-nitrophenyl)-6-fluorobenzenethiol
(1.6 g, 5.84 mmol) in ethanol (150 mL) was added tin(II) chloride
(4.92 g, 26.3 mmol). The reaction mixture was heated at 90 °C un-
der nitrogen gas for 2 h. Ethanol was removed by evaporator, and
the residue was dissolved in ethyl acetate (100 mL), the organic
solution was washed with saturated sodium bicarbonate (30 mL)
followed by water (30 mL) and dried over sodium sulfate. The
crude product was purified by flash chromatography (silica gel,
70:30 hexane–ethyl acetate) to give 1.05 g (74%) of the title com-
pound 1 as a yellow solid; mp = 201.2–201.5 °C; Analytical HPLC:
reverse phase (60:40 acetonitrile–H2O) k0 = 0.46, purity 98.37%;
normal phase (10:90 5% IPA in dichloromethane–hexane)
k0 = 5.39, purity 98.14%; 1H NMR (300 MHz, CDCl3) d 7.93–7.89
(m, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.52 (dd, J = 8.1, 2.4 Hz, 1H),
7.20–7.13 (m, 1H), 6.72 (d, J = 8.4 Hz, 2H), 4.01 (br s, 2H,
NH2);13C NMR (75 MHz, CDCl3) d 168.21 (d, J = 3.8 Hz), 160.06 (d,
J = 242.9 Hz), 150.87 (d, J = 1.8 Hz), 149.26, 135.52 (d, J = 11.1 Hz),
129.01, 123.67, 123.22 (d, J = 9.3 Hz), 114.76, 114.46 (d,
J = 24.0 Hz), 107.70 (d, J = 26.6 Hz); MS (CI) m/z 245 (M++H); Anal.
Calcd for C13H9FN2S: C, 63.92; H, 3.71; N, 11.47; S, 13.13. Found: C,
63.82; H, 3.71; N, 11.44; S, 13.24. Registry number: 328087-15-6.
4.2. General procedure for the synthesis of 8, 3, 9a, and 9c
4.2.1. 2-(40-Nitrophenyl)-6-fluorobenzothiazole (8)
To
a solution of 2-amino-5-fluorobenzenethiol 4 (300 mg,
2.09 mmol) in dimethylsulfoxide (3 mL) was added 4-nitrobenzal-
dehyde 6 (315 mg, 2.09 mmol). The reaction mixture was heated at
180 °C for 30 min. At the end of the reaction, the reaction mixture
was cooled to room temperature and poured into ice-water (6 mL).
The precipitate was filtered under reduced pressure. The filtrate
was purified by recrystallization from tetrahydrofuran (5 mL)-
methanol (150 mL) to give 325 mg (57%) of the title compound 8
as a yellow solid; mp = 201.2–201.5 °C; 1H NMR (300 MHz, CDCl3)
d 8.34 (d, J = 8.7 Hz, 2H), 8.21 (d, J = 8.7 Hz, 2H), 8.09–8.04 (m, 1H),
7.62 (dd, J = 8.1 2.4 Hz, 1H), 7.31–7.24 (m, 1H); 13C NMR (75 MHz,
CDCl3) d 162.00 (d, J = 246.6 Hz), 150.74 (d, J = 1.8 Hz), 149.02,
138.83, 136.50 (d, J = 11.2 Hz), 128.09, 124.97 (d, J = 9.2 Hz),
124.33, 115.78 (d, J = 24.8 Hz), 108.02 (d, J = 26.6 Hz); MS (EI) m/z
274 (M+); Anal. Calcd for C13H7FN2O2S: C, 56.93; H, 2.57; N,
10.21; S, 11.69. Found: C, 56.98; H, 2.60; N, 10.31; S, 11.69. Registry
number: 343975–46–2.
4.3.2. 2-(40-Aminophenyl)-6-bromobenzothiazole (10)
A
yellow solid; mp = 219.3–221.0 °C; 1H NMR (400 MHz,
DMSO-d6) d 8.23 (d, J = 1.8 Hz, 1H), 7.87 (d, J = 8.7 Hz, 2H), 7.82
(d, J = 8.7 Hz, 1H), 7.53 (dd, J = 8.4, 1.8 Hz, 1H), 6.73 (d, J = 8.7,
2.0 Hz, 2H), 4.03 (br s, 2H, NH2); MS (CI) m/z 306 (M++H); 13C
NMR (75 MHz, CDCl3) d 149.50, 149.48, 136.24, 129.48, 129.19,
123.92, 123.52, 123.43, 117.76, 117.73, 114.76; MS (FAB) m/z 307
(M++H, 81Br), 305 (M++H, 79Br) Anal. Calcd for C13H9BrN2S: C,
51.16; H, 2.97; N, 9.18; S, 10.51. Found: C, 51.04; H, 3.03; N,
9.03; S, 10.69. Registry number: 566169-97-9.
4.2.2. 2-(40-Dimethylaminophenyl)-6-fluorobenzothiazole (3)
A yellow solid; mp = 204.4–205.9 °C; Analytical HPLC: reverse
phase (70:30 acetonitrile–H2O) k0 = 2.01, purity 99.8%; normal
phase (10:90, 5% IPA in dichloromethane–hexane) k0 = 0.06, purity
99.11%; 1H NMR (300 MHz, CDCl3) d 7.92–7.87 (m, 3H), 7.50 (dd,
J = 8.1, 2.4 Hz, 1H), 7.19–7.12 (m, 1H), 6.73 (d, J = 9.0 Hz, 2H),
3.05 (s, 6H); 13C NMR (75 MHz, CDCl3) d 168.51 (d, J = 3.2 Hz),
159.92 (d, J = 242.3 Hz), 152.16, 151.04 (d, J = 1.88 Hz), 135.47 (d,
J = 11.2 Hz), 128.72, 122.95 (d, J = 9.3 Hz), 121.12, 114.29 (d,
J = 24.1 Hz), 111.67, 107.61 (d, J = 26.6 Hz), 40.11; MS (CI) m/z
273 (M++H); Anal. Calcd for C15H13FN2S: C, 66.15; H, 4.81; N,
10.29; S, 11.77. Found: C, 66.13; H, 4.85; N, 10.27; S, 11.77. Registry
number: 1071423–42–1.
4.3.3. 2-(40-Methylaminophenyl)-6-fluorobenzothiazole (2)
To a solution of 2-(40-aminophenyl)-6-fluorobenzenethiol 1
(300 mg, 1.23 mmol) in acetonitrile (15 mL) was added iodometh-
ane (153 lL, 2.46 mmol) and K2CO3 (1.7 g, 12.3 mmol. The reaction
mixture was refluxed for 4 h. After acetonitrile was removed by
evaporator and vacuum, the residue was dissolved in ethyl acetate
(50 mL), the organic solution was washed with saturated sodium
bicarbonate (30 mL) followed by water (30 mL) and dried over so-
dium sulfate. The crude product was purified by flash chromatog-
raphy (silica gel, 70:30 hexane–ethyl acetate) to give 111 mg (35%)
of 2-(40-methylaminophenyl)-6-fluorobenzothiazole 2 as a yellow
solid; mp = 153.5–154.5 °C; Analytical HPLC: reverse phase
(60:40 acetonitrile–H2O) k0 = 4.38, purity 99.67%; normal phase
(10:90 5% IPA in dichloromethane–hexane) k0 = 2.25, purity
98.58%; 1H NMR (300 MHz, CDCl3) d 7.92–7.86 (m, 3H), 7.51 (dd,
J = 8.1, 2.4 Hz, 1H), 7.20–7.12 (m, 1H), 6.63 (d, J = 8.4 Hz, 2H),
4.14 (br s, 1H, NH), 2.90 (s, 3H); 13C NMR (75 MHz, CDCl3) d
168.49 (d, J = 3.1 Hz), 159.96 (d, J = 242.3 Hz), 151.57, 150.97,
135.46 (d, J = 11.1 Hz), 128.95, 123.02 (d, J = 9.3 Hz), 122.25,
114.34 (d, J = 24.8 Hz), 112.01, 107.64 (d, J = 26.6 Hz), 30.26; MS
(CI) m/z 259 (M++H); Anal. Calcd for C14H11FN2S: C, 65.10; H,
4.29; N, 10.84; S, 12.41. Found: C, 65.13; H, 4.30; N, 10.86; S,
12.43. Registry number: 1071423-41-0.
4.2.3. 2-(40-Nitrophenyl)-6-bromobenzothiazole (9a)
A
yellow solid; mp = 192.4–193.0 °C; 1H NMR (300 MHz,
DMSO-d6)
d 8.55 (d, J = 2.1 Hz, 1H), 8.38 (dd, J = 9.3 Hz,
J = 9.0 Hz, 2H), 8.08 (d, J = 8.7 Hz, 1H), 7.76 (dd, J = 8.7 Hz,
J = 2.1, 1H); 13C NMR (100 MHz, DMSO-d6) d 165.87, 152.49,
148.92, 137.92, 137.02, 130.34, 128.51, 125.27, 124.98, 124.64,
123.58, 119.10; MS (EI) m/z 336 (M+, 81Br), 334 (M+, 79Br); Anal.
Calcd for C13H7BrN2O2S: C, 46.58; H, 2.11; N, 8.36; S, 9.57.
Found: C, 46.72; H, 2. 06; N, 8.38; S, 9.56. Registry number:
566169-96-8.
4.2.4. 2-(40-Dimethylaminophenyl)-6-bromobenzothiazole (9c)
A yellow solid; mp = 208.6–208.7 °C; 1H NMR (300 MHz, CDCl3)
d 7.96–7.91 (m, 3H), 7.81 (d, J = 8.7 Hz, 1H), 7.52 (dd, J = 8.7, 2.0 Hz,
1H), 6.74 (d, J = 9.0 Hz, 2H), 3.06 (s, 6H); 13C NMR (100 MHz, CDCl3)
d 169.46, 154.36, 153.49, 136.37, 129.52, 129.06, 123.99, 123.42,
120.96, 117.53, 111.80, 40.30; MS (EI) m/z 334 (M+, 81Br), 332
(M+, 79Br); Anal. Calcd for C15H13BrN2S: C, 54.06; H, 3.93; N, 8.41;
S, 9.62. Found: C, 54.05; H, 3.91; N, 8.45; S, 9.62. Registry number:
346691-88-1.
4.3.4. 2-(40-N-tert-Butyloxycarbonyl-methylaminophenyl)-6-
bromobenzothiazole (9b)
To a solution of 2-(40-N-aminophenyl)-6-bromobenzenethiol 10
(300 mg, 0.98 mmol) in methanol (20 mL) was added formalde-
hyde (220
lL, 2.96 mmol). The reaction mixture was refluxed for
1.5 h. Methanol was removed by evaporator and vacuum. The

B. C. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2980–2990
2987
residue was dissolved in methanol (100 mL) again, and was added
sodium cyanoborohydride (247 mg, 3.92 mmol) and acetic acid
(6 lL, pH 6) slowly. The reaction mixture was stirred at room tem-
0.88 (m, 9H); 13C NMR (100 MHz, CDCl3) d 168.44, 154.53,
152.36, 137.78, 135.00, 133.71, 129.08, 129.03, 121.97, 121.82,
111.91, 40.40, 29.21, 27.60, 13.89, 10.04; MS (EI) m/z 544 (M+);
Anal. Calcd for C27H40N2SSn: C, 59.68; H, 7.42; N, 5.16; S, 5.90.
Found: C, 59.65; H, 7.37; N, 5.22; S, 5.94. Registry number:
346691-92-7.
perature for 1.5 h. Methanol was removed by evaporator and the
residue was dissolved in ethyl acetate (50 mL), the organic solution
was washed with saturated sodium bicarbonate (30 mL) followed
by water (30 mL) and dried over sodium sulfate. The crude prod-
uct was purified by flash chromatography (silica gel, 70:30
hexane–ethyl acetate). To the obtained 2-(40-methylaminophe-
nyl)-6-bromobenzothiazole in tetrahydrofuran (15 mL) was added
di-tert-butyl-dicarbonate (217 mg, 0.96 mmol) at 0 °C. The reaction
mixture was refluxed for 12 h. At the end of the reaction, the reac-
tion mixture was cooled to room temperature and poured into ice-
water (20 mL) and ethyl acetate (40 mL). The organic solution was
washed with saturated sodium bicarbonate (30 mL) followed by
water (30 mL) and dried over sodium sulfate. The crude product
was purified by flash chromatography (silica gel, 80:20 hexane–
ethyl acetate) to give 200 mg (49%) of 2-(40-N-tert-butyloxycar-
bonyl-methylaminophenyl)-6-bromobenzothiazole 9b as a pale
yellow solid; mp = 144.5–145.2 °C; 1H NMR (300 MHz, CDCl3) d
8.02–8.00 (m, 3H), 7.89 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H),
7.40 (d, J = 8.1 Hz, 1H), 3.30 (s, 3H), 1.47 (s, 9H); 13C NMR
(75 MHz, CDCl3) d 167.88, 154.21, 153.01, 146.48, 136.63, 129.81,
129.57, 127.72, 125.21, 124.14, 118.60, 81.01, 67.95, 36.93,
28.29; MS (FAB) m/z 421 (M++H, 81Br), 419 (M++H, 79Br); Anal.
Calcd for C19H19BrN2O2S: C, 54.42; H, 4.57; N, 6.68; S, 7.65. Found:
C, 54.34; H, 4.56; N, 6.78; S, 7.62.
4.5. General procedure for the synthesis of 12, 13a–e and 14
4.5.1. 2-(40-Nitrophenyl)-6-iodo(phenyl)benzothiazole
iodonium tosylate (12)
To a solution of Koser’s reagent (69.5 mg, 0.17 mmol) in dichlo-
romethane (10 mL) was added 2-(40-nitrophenyl)-6-tribut-
ylstannylbenzothiazole 11a (95 mg, 0.17 mmol) under argon
atmosphere. The reaction mixture was stirred at room temperature
under argon atmosphere for 12 h. The solvent was evaporated
using a stream of nitrogen. The crude mixture was dissolved a
small amount of methanol (1.5 mL) and transferred to the centri-
fuge tube to which was added excess diethyl ether (20 mL). After
centrifuging, the collected oil was dried in vacuo to give 40 mg
(38%) of the title compound 12 as a yellow solid: mp = 216.6–
218.5 °C; 1H NMR (400 MHz, MeOH-d4) d 9.02 (d, J = 1.6 Hz, 1H),
8.43–8.37 (m, 5H), 8.31 (dd, J = 8.0, 2.0 Hz, 1H), 8.24–8.18 (m,
3H), 7.68 (d, J = 8.0 Hz, 2H), 7.54 (t, J = 8.0 Hz, 2H), 7.21 (d,
J = 8.0 Hz, 2H), 2.34 (s, 3H); 13C NMR (100 MHz, MeOH-d4) d
168.30, 154.33, 148.43, 140.77, 138.88, 136.77, 136.37, 133.74,
131.55, 131.04, 130.50, 128.69, 127.21, 127.03, 125.00, 124.18,
122.77, 113.89, 109.20, 18.54; MS (FAB) m/z 459 (M+ꢀOTs); HRMS
calcd for C19H12IN2O2S 458.9664, found 458.9671.
4.4. General procedure for the synthesis of 11a–c
4.4.1. 2-(40-Nitrophenyl)-6-tributylstannylbenzothiazole (11a)
To a mixture of 2-(40-nitrophenyl)-6-bromobenzothiazole 9a
(200 mg, 0.60 mmol) and tetrakis(triphenylphosphine)-palla-
dium(0) (69 mg, 0.06 mmol) in anhydrous tetrahydrofuran
4.5.2. 2-(40-N-tert-Butyloxycarbonyl-methylaminophenyl)-6-
iodo(phenyl)benzothiazole iodonium tosylate (13a)
A
yellow solid; mp = 156.2–156.9 °C; 1H NMR (400 MHz,
MeOH-d4) d 8.92 (d, J = 1.6 Hz, 1H), 8.26–8.20 (m, 3H), 8.13–8.06
(m, 3H), 7.70–7.63 (m, 3H), 7.56–7.47 (m, 4H), 7.19 (d, J = 8.0 Hz,
2H), 3.32 (s, 3H), 1.47 (s, 9H); 13C NMR (100 MHz, MeOH-d4) d
173.26, 157.30, 155.92, 148.67, 143.56, 141.63, 139.11, 136.40,
134.08, 133.74, 133.23, 131.13, 130.51, 129.78, 129.28, 127.67,
126.99, 126.95, 116.64, 110.86, 82.46, 37.45, 28.51, 21.30; MS
(FAB) m/z 543 (M+ꢀOTs); HRMS calcd for C25H24IN2O2S 543.0603,
found 543.0599.
(10 mL) was added and bistributyltin (658 lL, 1.31 mmol) in anhy-
drous tetrahydrofuran (10 mL) under argon gas at room tempera-
ture. The reaction mixture was heated at 85 °C for 8 h. At the end
of the reaction, the reaction mixture was cooled to room tempera-
ture and filtered by Celite. The crude product was purified by flash
chromatography (silica gel, 80:20 hexane–ethyl acetate) to give
170 mg (52%) of the title compound 11a as a pale yellow oil; 1H
NMR (300 MHz, CDCl3)
d 8.35 (d, J = 9.0 Hz, 2H), 8.27 (d,
J = 9.0 Hz, 2H), 8.09–8.03 (m, 2H), 7.62 (dd, J = 8.1, 0.4 Hz, 1H),
1.57–1.52 (m, 6H), 1.41–1.29 (m, 6H) 1.16–1.10 (m, 6H), 0.92–
0.85 (m, 9H); 13C NMR (100 MHz, CDCl3) d 164.39, 154.18,
149.17, 141.20, 139.63, 135.84, 134.52, 128.48, 128.42, 124.50,
123.37, 29.28, 27.57, 13.87, 10.13; MS (FAB) m/z 547 (M++H); Anal.
Calcd for C25H34N2O2SSn: C, 55.06; H, 6.28; N, 5.14; S, 5.88. Found:
C, 55.05; H, 6.28; N, 5.11; S, 5.88.
4.5.3. 2-(40-Dimethylaminophenyl)-6-iodo(phenyl)benzothia-
zole iodonium tosylate (14)
A
yellow solid; mp = 172.0–173.8 °C; 1H NMR (400 MHz,
MeOH-d4) d 8.81 (d, J = 2.0 Hz, 1H), 8.21–8.17 (m, 3H), 7.95–7.92
(m, 3H) 7.70–7.67 (m, 3H), 7.53 (t, J = 7.8 Hz, 2H), 7.20 (d,
J = 8.0 Hz, 2H), 6.82 (d, J = 8.8 Hz, 2H), 3.08 (s, 6H), 2.34 (s, 3H);
13C NMR (100 MHz, MeOH-d4) d 156.55, 153.76, 150.39, 140.98,
140.48, 135.07, 133.61, 132.78, 132.48, 131.99, 129.42, 129.24,
128.60, 125.75, 124.42, 116.27, 111.77, 108.07, 81.18, 39.10,
16.16; MS (FAB) m/z 457 (M+ꢀOTs); HRMS calcd for C21H18IN2S
457.0235, found 457.0246.
4.4.2. 2-(40-N-tert-Butyloxycarbonyl-methylaminophenyl)-6-
tributylstannylbenzothiazole (11b)
A yellow oil; 1H NMR (400 MHz, CDCl3) d 8.06–7.98 (m, 4H),
7.56 (d, J = 8.0 Hz, 1H), 7.38 (d, J = 8.8 Hz, 2H), 3.32 (s, 3H), 1.59–
1.53 (m, 6H), 1.40–1.31 (m, 6H), 1.14–1.10 (m, 6H), 0.94–0.88
(m, 9H); 13C NMR (75 MHz, CDCl3) d 166.81, 154.31, 154.02,
146.11, 139.05, 134.67, 133.78, 130.30, 129.04, 127.72, 125.24,
122.45, 36.98, 30.29, 29.07, 28.30, 27.36, 13.67, 9.82; MS (FAB)
m/z 631 (M++H); Anal. Calcd for C31H46N2O2SSn: C, 59.15; H,
7.37; N, 4.45; S, 5.09. Found: C, 59.13; H, 7.34; N, 4.45; S, 5.04.
4.5.4. 2-(40-N-tert-Butyloxycarbonyl-methylaminophenyl)-6-
iodo(40-methoxyphenyl)benzothiazole iodonium tosylate (13b)
A white solid; mp = 159.4–159.8 °C; 1H NMR (400 MHz, MeOH-
d4) d 8.87 (d, J = 1.6 Hz, 1H), 8.21 (dd, J = 8.8, 2.0 Hz, 1H), 8.14–8.10
(m, 4H), 8.08 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.49 (d,
J = 8.4 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 7.06 (d, J = 9.2 Hz, 2H),
3.83 (s, 3H), 3.31 (s, 3H), 2.35 (s, 3H), 1.48 (s, 9H); 13C NMR
(100 MHz, MeOH-d4) d 173.15, 164.60, 157.21, 155.93, 148.66,
141.64, 139.01, 138.53, 133.69, 130.63, 130.53, 129.79, 129.27,
127.00, 126.96, 126.84, 118.90, 111.44, 106.44, 105.10, 82.47,
56.35, 37.45, 28.51, 21.30; MS (FAB) m/z 573 (M+ꢀOTs); HRMS
calcd for C26H26IN2O2S 573.0709, found 573.0707.
4.4.3. 2-(40-Dimethylaminophenyl)-6-tributylstannylbenzo-
thiazole (11c)
A yellow oil; 1H NMR (400 MHz, CDCl3) d 7.98–7.92 (m, 4H),
7.50 (d, J = 7.6 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 3.06 (s, 6H),
1.58–1.52 (m, 6H), 1.39–1.32 (m, 6H), 1.12–1.08 (m, 6H), 0.91–

2988
B. C. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2980–2990
4.5.5. 2-(40-N-tert-Butyloxycarbonyl-methylaminophenyl)-6-
tively. After the ethanol was evaporated, radiotracer [18F]1 was
used for biological study. For the identification of the radio-prod-
uct, the collected HPLC fraction was matched with the authentic
compound. Specific activity at the end of synthesis was calculated
by relating radioactivity to the mass associated with the UV absor-
bance (254 nm) peak of authentic compound. Specific radioactivity
iodo(40-methylphenyl)benzothiazole iodonium tosylate (13c)
A
white solid; mp = 159.5–159.9 °C; 1H NMR (400 MHz,
MeOH-d4) d 8.90 (d, J = 1.6 Hz, 1H), 8.23 (dd, J = 8.8, 1.6 Hz, 1H),
8.12–8.07 (m, 5H), 7.68 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H),
7.35 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 3.31 (s, 3H), 2.39
(s, 3H), 2.35 (s, 3H), 1.48 (s, 9H); 13C NMR (100 MHz, MeOH-d4) d
173.21, 157.26, 155.93, 148.68, 143.56, 141.63, 139.06, 136.39,
133.95, 130.92, 130.88, 130.52, 129.78, 129.28, 127.00, 126.95,
126.88, 112.91, 111.00, 82.47, 37.45, 28.51, 21.35, 21.30; MS
(FAB) m/z 557 (M+ꢀOTs); HRMS calcd for C26H26IN2O2S 557.0760,
found 557.0762.
of [18F]1 (85 GBq/
lmol) was obtained after purification on analytic
HPLC column.
4.6.2. 2-(40-N-Methylaminophenyl)-6-[18F]fluorobenzothiazole
([18F]2)
The preparation of [18F]fluoride and the azeotropic distillations
were carried out according to [18F]1 experiment. Compound 13a–e
4.5.6. 2-(40-N-tert-Butyloxycarbonyl-methylaminophenyl)-6-
iodo(20-thiophenyl)benzothiazole iodonium tosylate (13d)
A pale yellow solid; mp = 146.8–147.2 °C; 1H NMR (400 MHz,
MeOH-d4) d 8.94 (d, J = 2.0 Hz, 1H), 8.27 (dd, J = 8.8, 2.0 Hz, 1H),
8.13–8.09 (m, 3H), 8.06 (dd, J = 4.0, 1.2 Hz, 1H), 7.89 (dd, J = 5.2,
1.2 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.8 Hz, 2H), 7.22
(d, J = 8.0 Hz, 2H), 7.18 (dd, J = 5.2, 4.0 Hz, 1H), 3.31 (s, 3H), 2.36
(s, 3H), 1.48 (s, 9H); 13C NMR (100 MHz, MeOH-d4) d 173.38,
157.28, 155.92, 148.69, 143.52, 142.15, 141.64, 138.97, 138.67,
133.47, 130.88, 130.52, 129.79, 129.29, 126.99, 126.94, 126.88,
113.86, 99.77, 82.47, 37.45, 28.51, 21.31; MS (FAB) m/z 549
(M+ꢀOTs); HRMS calcd for C23H22IN2O2S 549.0167, found
549.0167.
(2 mg, 2.9 lmol) in acetonitrile (300 lL, adding 10 lL of H2O and
1 mg of TEMPO) was added to the dried tetrabuthylammonium
fluoride in the reaction vial and reacted in the microwave equip-
ment with 100 W (180 sec. ꢁ 2) or heated in oil bath at 130 °C
for 10 min. The vial was cooled in an ice bath and the solvent
was removed under a gentle stream of nitrogen at 80 °C. 3 N HCl
in ethyl acetate (3:1 ethyl acetate–concd HCl, v/v, 250 lL) was
added to the crude reaction mixture and reacted at 75 °C for
10 min. After the reaction, the vial was cooled in an ice bath and
the solvent was removed under a gentle steam of nitrogen
at 75 °C. The reaction mixture was purified by HPLC at a flow
3 mL/min using a 45:55 mixture of H2O–acetonitrile, and [18F]2
was eluted at 17.5 min. Radiotracer [18F]2 collected from HPLC
was purified with Sep Pak cartridge with the help water (12 mL)
and ethanol (1 mL), respectively. Specific radioactivity of [18F]2
4.5.7. 2-(40-N-tert-Butyloxycarbonyl-methylaminophenyl)-6-
iodo(30-thiophenyl)benzothiazole iodonium tosylate (13e)
A pale yellow solid; mp = 128.2–130.2 °C; 1H NMR (400 MHz,
MeOH-d4) d 8.90 (d, J = 2.0 Hz, 1H), 8.54 (dd, J = 2.8, 1.2 Hz, 1H),
8.24 (dd, J = 2.0, 8.8 Hz, 1H), 8.12 (d, J = 8.4 Hz, 2H), 8.08 (d,
J = 8.8 Hz, 1H), 7.72–7.66 (m, 4H), 7.49 (d, J = 8.4 Hz, 2H), 7.21(d,
J = 8.4 Hz, 2H), 3.31 (s, 3H), 2.35 (s, 3H), 1.48 (s, 9H); 13C NMR
(100 MHz, MeOH-d4) d 173.21, 157.20, 155.92, 148.66, 143.55,
141.63, 139.02, 137.26, 133.78, 132.48, 131.81, 130.80, 130.52,
129.78, 129.28, 126.99, 126.94, 126.85, 111.80, 100.18, 82.46,
37.45, 28.52, 21.30; MS (FAB) m/z 549 (M+ꢀOTs); HRMS calcd for
(110 GBq/lmol) was obtained after purification on analytic HPLC
column.
4.6.3. 2-(40-N-Dimethylaminophenyl)-6-
[
18F]fluorobenzothiazole ([18F]3)
The preparation of [18F]fluoride and the azeotropic distillations
were carried out according to [18F]1 experiment. Compound 14
(2 mg, 3.3 lmol) in acetonitrile (300 lL, adding 10 lL of H2O and
1 mg of TEMPO) was added to the dried tetrabuthylammonium
fluoride in the reaction vial and reacted in the microwave equip-
ment with 100 W (180 sec. ꢁ 2) or heated in oil bath at 130 °C
for 10 min. The vial was cooled in an ice bath and the solvent
was removed under a gentle stream of nitrogen at 80 °C. The reac-
tion mixture was purified by HPLC at a flow 3 mL/min using a
40:3:57 mixture of 50 mM (NH4)H2PO4–tetrahydrofuran–acetoni-
trile, and [18F]3 was eluted at 15.7 min. Radiotracer [18F]3 collected
from HPLC was purified with Sep-Pak cartridge with the help water
(12 mL) and ethanol (1 mL), respectively. Specific radioactivity of
C23H22IN2O2S 549.0167, found 549.0161.
4.6. The radiosynthesis and purification of [18F]1–3
4.6.1. 2-(40-Aminophenyl)-6-[18F]fluorobenzothiazole ([18F]1)
[
18F]Fluoride was produced in a cyclotron by the 18O(p,n)18
F
reaction. A volume of 100–200
in water was added to a vacutainer containing n-Bu4NHCO3 (40%
aq 2.12 L, 2.76 mol). The azeotropic distillations were carried
out each time with 200 L aliquots of CH3CN at 85 °C under a
stream of nitrogen. Compound 12 (2 mg, 3.3 mol) in acetonitrile
(300 L, adding 10 L of H2O and 1 mg of TEMPO) was added to
l
L [18F]fluoride (18.5–370 MBq)
l
l
[
18F]3 (118 GBq/
HPLC column.
lmol) was obtained after purification on analytic
l
l
l
l
4.7. Ab binding affinity assay
the dried tetrabutylammonium fluoride in the reaction vial and re-
acted in the microwave equipment with 100 W (180 sec. ꢁ 2) or
heated in oil bath at 130 °C for 10 min. After the reaction, the vial
was cooled in an ice bath and the solvent was removed under a
gentle stream of nitrogen at 80 °C. The crude reaction mixture
was diluted with 2 mL of ethanol–tetrahydrofuran–ethyl acetate
(5:47.5:47.5, v/v), loaded into silica Sep-Pak and washed with
2 mL of the same solution again. The obtained organic solution
was removed under a gentle stream of nitrogen and added tin(II)
Aggregated Ab peptides were prepared using Ab1–42, and Ab
binding affinity was evaluated as published elsewhere.26,27 The so-
lid forms of peptides Ab1–42 was purchased from Bachem (King of
Prussia, PA). Aggregation of peptides was carried out by gently dis-
solving the peptide (0.5 mg/mL for Ab1–42) in a buffer solution (pH
7.4) containing 10 mM sodium phosphate and 1 mM EDTA. The
solutions were incubated for 42 h at 37 °C with gently shaking.
Aggregated Ab peptides were stored at ꢀ70 °C. For binding assay,
aggregated Ab (27.5 nM in the final assay mixture) were added
chloride (3.35 mg, 13 lmol) and EtOAc (200 lL). The mixture was
heated at 80 °C for 10 min. The solvent was removed with a gentle
stream of nitrogen. The reaction mixture was purified by HPLC at a
flow 3 mL/min using a 30:7:63 mixture of 50 mM (NH4)H2PO4–tet-
rahydrofuran–acetonitrile, and [18F]1 was eluted at 9.3 min. Radio-
tracer [18F]1 collected from HPLC was purified with C-18 Sep Pak
cartridge with the help water (12 mL) and ethanol (1 mL), respec-
to the mixture containing 50 l
L of radioligands ([125I]TZDM,
0.078–10 nM in 40% ethanol) and 10% ethanol in a final volume
of 1 mL for saturation studies. Nonspecific binding assay was de-
fined in the presence of 10
tube. For inhibition studies, 1 mL of the reaction mixture contained
50 L of inhibitors 1–3 (0.124–10,000 nM in 10% ethanol) and
lM thioflavin-T (M.W.: 318.87) per
l

B. C. Lee et al. / Bioorg. Med. Chem. 19 (2011) 2980–2990
2989
0.05 nM radioligand ([125I]TZDM) in 40% ethanol was incubated for
3 h at room temperature for the binding assay. The reaction mix-
ture was filtered through Whatman GF/B glass filters and washed
twice with 3 mL of 10% ethanol aliquots. The radioactivity retained
on the filter was counted by a gamma-counter. Under the assay
conditions, the percent of the specific binding fraction was less
than 20% of the total 125I radioactivity. The results of inhibition
and saturation experiments were subjected to non-linear regres-
sion analysis using PRISM software by which Kd (The Kd values of
mercial blender for 3 min and centrifuged 3500 rpm for 5 min at
4 °C. The resulting supernatant filtered through a 0.45 m GH Poly-
pro (GHP) membrane Disc filter. The samples were analyzed by
HPLC at a flow rate of 3 mL/min using a 70:30 mixture of acetoni-
trile and water as the eluants.
l
4.12. Biodistribution in mice
Brain distribution studies were measured as the followed meth-
od: ICR mice (male, 25–28 g, 5 mice per time point) were adminis-
trated of compounds ([18F]1–3, 1.1 MBq/mouse, respectively) in
0.2 mL of 10% ethanol-saline via a tail vein. Mice were sacrificed
by cervical dislocation at 2, 30, and 60 min post-injection. After
samples of blood, cortex, cerebellum, and remnant were removed,
there are weighed and counted with WizardÒ300 1480 automatic
gamma-counter (PerkinElmer, Turku, Finland). Data are expressed
as percentages of injected dose per gram of tissue (% ID/g) and nor-
malized to body weight (% ID-kg)/g. All animal experiments were
performed in compliance with the rules of the Seoul National Uni-
versity Bundang Hospital Laboratory Animal Care.
[
125I]TZDM in Ab1–42 aggregates were 0.45 0.032 nM) and Ki val-
ues of 1–3 were calculated.
Ki ¼ IC50=ð1 þ ðligandÞ=KdÞ
4.8. In vitro fluorescent staining assay
Brain sections were obtained from the transgenic mouse (APP-
swe/PS14E9, 25 months) and the wild-type mouse.28 After equili-
bration to ꢀ25 °C, 20
lm thickness of coronal sections were cut on
a Cryocut Microtome (CM3050S, LEICA), thaw-mounted onto silane
coated glass microscope slides (MUTO PURE CHEMICALS CO.),
dried in aeration room, and stored at room temperature until
use. To identify of Ab peptides deposits in the sections, fluores-
cence staining with Thioflavine-S, and compound 2 were per-
formed by the following steps: Staining with 0.0125% fluorescent
dye (Thioflavine-S or compound 2) in 40% ethanol-100 mM
phosphate buffered saline (PBS) for 3 min in the dark at room
temperature. After staining samples were rapidly washed in 50%
ethanol-PBS for 3 min, PBS for 1 min, and water for 5 min, respec-
tively. Staining samples were dried in the dark at room tempera-
ture. Fluorescent image of Ab deposits brain tissue were obtained
by Confocal Microscopy (LSM-700, Carl zeiss) using a FITC cube
with an excitation filter of 350–390 nm and emission filter of
530 15 nm.
Acknowledgments
We are grateful to Professor Y. J. Yoo (Gwangju Institute of
Science and Technology) for providing of brain sections which
obtained from the transgenic mouse (APPswe/PS14E9, 25 months)
and the wild-type mouse. This study was supported by grants from
the National Research Foundation of Korea (20100020379,
20100017508 (BAERI), 2010K-000817 (Brain Research Center of
the 21st Century Frontier Research Program) and KRF-2007-
313-E00385) funded by the Ministry of Education, Science and
Technology of Republic of Korea and the Seoul National University
Bundang Hospital Research Fund (03-2008-008).
References and notes
4.9. Partition coefficient determination
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