Synthesis and evaluation of 6-(3-[18F]fluoro-2-hydroxypropyl)-substituted 2-pyridylbenzothiophenes and 2-pyridylbenzothiazoles as potential PET tracers for imaging Aβ plaques

Article abstract of DOI:10.1016/j.bmc.2016.03.034

3-[¹⁸F]Fluoro-2-hydroxypropyl substituted compounds were synthesized and evaluated as novel ¹⁸F-labeled PET tracers for imaging Aβ plaque in a living brain. All compounds exhibited high binding affinities toward the synthetic Aβ

Full text of DOI:10.1016/j.bmc.2016.03.034

Accepted Manuscript
Synthesis and evaluation of 6-(3-[18F]fluoro-2-hydroxypropyl)-substituted 2-
pyridylbenzothiophenes and 2-pyridylbenzothiazoles as potential PET tracers
for imaging Aβ plaques
Byoung Se Lee, So Young Chu, Hye Rim Kwon, Chansoo Park, Uthaiwan
Sirion, Damian Brockschnieder, Thomas Dyrks, Seung Jun Oh, Jae Seung Kim,
Dae Yoon Chi
PII:
S0968-0896(16)30182-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.034
BMC 12883
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 January 2016
17 March 2016
18 March 2016
Please cite this article as: Lee, B.S., Chu, S.Y., Kwon, H.R., Park, C., Sirion, U., Brockschnieder, D., Dyrks, T.,
Oh, S.J., Kim, J.S., Chi, D.Y., Synthesis and evaluation of 6-(3-[18F]fluoro-2-hydroxypropyl)-substituted 2-
pyridylbenzothiophenes and 2-pyridylbenzothiazoles as potential PET tracers for imaging Aβ plaques, Bioorganic
& Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.03.034
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Synthesis and evaluation of 6-(3-[18F]fluoro-2-hydroxypropyl)-
substituted 2-pyridylbenzothiophenes and 2-pyridylbenzothiazoles as
potential PET tracers for imaging A plaques.
Byoung Se Lee a, So Young Chu a, Hye Rim Kwon b, Chansoo Park a, Uthaiwan Sirion b, †
Damian Brockschnieder c, Thomas Dyrks c, Seung Jun Oh d, Jae Seung Kim d, Dae Yoon Chia,b,*
a Research Institute of Labeling, FutureChem Co., Ltd, Seoul 138-736, Korea
b Department of Chemistry, Sogang University, Seoul 121-742, Korea
c Global Drug Discovery - Molecular Imaging, Bayer Healthcare AG, 13353 Berlin, Germany
d Department of Nuclear Medicine, Asan Medical Center, University of Ulsan, College of
Medicine, Seoul 138-736, Korea
* Corresponding author. Tel.: +82 2 715 2430; fax: +82 2 715 2411.
E-mail address: dychi@sogang.ac.kr.
† Current address: Department of Chemistry and Center for Innovation in Chemistry, Faculty of
Science, Burapha University, Sangsook, Chonburi 20131, Thailand.
Keywords: Alzheimer’s disease (AD), -Amyloid (A), Positron Emission Tomography (PET)
1

ABSTRACT
3-[18F]Fluoro-2-hydroxypropyl substituted compounds were synthesized and evaluated as novel
18F-labeled PET tracers for imaging plaque in a living brain. All compounds exhibited high
binding affinities toward the synthetic A1-42 aggregate and/or Alzheimer’s disease brain
homogenate. In the microPET study with normal mice, the 3-[F]fluoro-2-hydroxypropyl
substituted compounds resulted in fast brain washout by reducing the lipophilicities of the
compounds. Intriguingly, (S)-configured PET tracers, (S)-[F]1b and (S)-[F]1c, exhibited a 2.8
and 4.0-fold faster brain washout rate at a peak/30 min in the mouse brain than the corresponding
(R)-configured PET tracers despite there being no meaningful difference in binding affinities
toward  plaque. A further evaluation of (S)-[F]1c with healthy rhesus monkeys also revealed
excellent clearance from the frontal cortex with ratios of 7.0, 16.0, 30.0 and 49.0 at a peak/30,
60, 90, and 120 min, respectively. These results suggest that (S)-[F]1c may be a potential PET
tracer for imaging  plaque in a living brain.
1. Introduction
The accumulation of A plaque in the brain is believed to be the main origin of Alzheimer's
disease (AD), which is the leading cause of dementia, accounting for 60-70% of all cases.1 A
positron emitting tracer that binds to A plaque selectively offers an opportunity of the early
detection of AD,2 and will be useful for identifying new therapeutic drugs and monitoring the
11
treatment results as a surrogate marker.3 In 2002, C-labeled [11C]PIB (Pittsburgh compound B)
was developed for imaging A plaque in the brain of living subjects by modifying Thioflavin T,
which has been used as a fluorescent dye for staining A plaque in postmortem brains.4 [11C]PIB
2

exhibited good A plaque binding in a living brain with an appropriate brain accumulation and
washout ratio. On the other hand, it is of limited use in clinical trials because the C-11 isotope
has a half-life of only 20 min, which is too short to exhibit wide availability to AD patients from
a single automated production. In this regard, many researchers have searched for longer lived F-
18 (110 min half-life) labeled PET tracers for clinical use in the diagnosis of AD.5 As a result,
three F-18 labeled PET tracers have been approved by the U.S. Food and Drug Administration
(FDA) in 2012 (AmyvidTM, Eli Lilly),6 2013 (VizamylTM, GE Healthcare),7 and 2014 (NeuraceqTM,
Piramal)8 as A imaging agents and a secondary tool for the diagnosis of AD.
To be a good A imaging PET tracer, it should meet two essential criteria in the stage of
candidate discovery; selective binding toward A plaque with high affinity and rapid clearance
of non-specific binding from the brain.9 Most biaryl and conjugated biaryl compounds have good
binding affinities toward A plaque in vitro.10 On the other hand, many F-18 labeled biaryl
candidates have failed to meet the desired washout criterion in vivo. The nucleophilic aliphatic
[18F]fluorination of alkyl tosylate or mesylate precursors is a common way to label the molecule
of interest with F-18.11 The resulting [18F]fluoroalkyl substituents, however, play a key role
increasing the lipophilicity of the molecules, giving rise to poor pharmacokinetic properties as
well as undesirable non-specific interactions.
Our attempts have been made to find novel candidates suited to PET imaging of A deposition
considering these requirements. As an early our attempt (unpublished), 6-(3-
[18F]fluoropropyloxy)-2-(4-methylamino)phenyl
benzo[b]thiophene
([18F]FC028)
was
synthesized and evaluated by in vitro binding assay and in vivo biodistribution. Although it
showed an excellent binding affinity of 0.27 nM (Ki) to A aggregates compared to [11C]PIB
(0.78 nM), a biodistribution study with normal mice revealed low initial uptake (2.02 %ID at 2
3

min) and a poor washout effect (2.18 %ID at 30 min, and 3.14 %ID at 60 min), probably due to
its high lipophilicity (CLogP = 5.31). The desired LogP for BBB penetration is believed to range
from 1 to 3.12 Subsequently, an effort was made to reduce the lipophilicity of the compound for
better brain pharmacokinetics by modifying the core structure and functional groups. This paper
describes how the introduction of a hydroxyl group and nitrogen atoms to the molecule affects
the brain pharmacokinetics. More intriguingly, the large difference in brain pharmacokinetics
between (R)- and (S)-enantiomers is also described.
Figure 1. Structure modification and corresponding CLogP. The asterisk (*) indicates the
stereogenic carbon. The red letters mean polar modifiers.
2. Results and discussion
2.1. Chemistry
4

A biaryl compound, 2-phenylbenzo[b]thiophene was chosen as the starting molecule, and
the N-methylamino group on C4 position of the right ring was fixed. Two components, a
nitrogen atom and hydroxyl group, were considered as polar modifiers to reduce the lipophilicity
of the compounds (Figure 1). Nitrogen atoms were added to the phenyl and benzo[b]thiophene
rings to become pyridine and benzo[d]thiazole rings, respectively. The pyridine ring was
reported to reduce the level of non-specific white matter (no plaques region) binding
compared to a phenyl ring.13 The early developed F-18-labeled PET tracers (AmyvidTM,
VizamylTM, and [18F]NeuraceqTM 14) have considerable non-specific white matter uptake compared
to [11C]PIB.15 This white matter retention may limit the detection sensitivity of  plaque,
particularly in the prodromal phase of AD. The simple combination of pyridine and either
benzo[b]thiophene or benzo[d]thiazole rings gave 2-pyridyl-benzo[b]thiophene and
benzo[d]thiazole, as shown in Figure 1.
In addition, a hydroxyl group was introduced to the 3-fluoropropyl chain to reduce the
unwanted lipophilicity caused by the 3-fluoropropyl substituent. Based on the CLogP value,16 the
hydroxyl group might be effective in offsetting the additional lipophilicity of the 3-fluoropropyl
group. As a consequence of the structural modification, the CLogP value was found to decrease
by up to 2.91, which is suited to BBB penetration. Note that the secondary hydroxyl group
provides two enantiomers.
5

Scheme 1. Synthesis of 3-fluoro-2-hydroxypropyl substituted compounds. Reagents and
conditions: (a) Cs2CO3, DMF, 100 °C, 1 h; (b) Dowex 50WX2, 10% H2O/MeOH, 40 °C, 24 h;
(c) MsCl, DIPEA, CH2Cl2, -10 °C, 30 min; (d) DHP, PPTS, CH2Cl2, rt, 17 h; (e) TBAF, t-amyl
alcohol, 100 °C, 6 h; (f) 4 N HCl, 80 °C, 30 min.
Scheme 1 outlines the synthesis of 3-fluoro-2-hydroxypropyl substituted compounds 1a-1c.
The O-Alkylation of 6-hydroxy compounds 7a-7c with 2,3-isopropylideneglycerol
methanesulfonate (15) was carried out using Cs2CO3 at 80 °C overnight, in which compound 15
was a R/S racemate (see supplementary data for detail synthesis of 7a-7c). The selective
deprotection of the acetonide moiety was achieved using Dowex 50WX2 resin (hydrogen form)
with careful monitoring by TLC, affording 1,2-diol compounds 17a-17c.17 The primary alcohol
was treated with methanesulfonyl chloride (MsCl) and diisopropylethylamine (DIPEA), and the
secondary alcohol was then tetrahydropyranylated in the presence of pyridinium p-
6

toluenesulfonate (PPTS) to give compounds 19a-19c, respectively. The resulting N-Boc and O-
THP protected mesylate compounds 19a-19c were used as precursors for the synthesis of the F-
18 labeled radioligands. The cold authentic compounds 1a-1c were prepared as a racemate by the
nucleophilic displacement of the mesylate leaving group of compounds 19a-19c with tetra-n-
butylammonium fluoride (TBAF) in t-amyl alcohol solvent18 and the subsequent deprotection of
N-Boc and O-THP under acidic conditions. Enantiomerically pure compounds and mesylate
precursors were also prepared. They could be obtained from optically pure (S)- or (R)-2,3-
isopropylideneglycerol methanesulfonate (15, 99 ee% each) using the same procedure as Scheme
1. Synthesis with (S)-2,3-isopropylideneglycerol methanesulfonate ((S)-15) provided the (R)-
configured compound (R)-1, and synthesis with (R)-2,3-isopropylideneglycerol
methanesulfonate ((R)-15) provided the (S)-configured compound (S)-1. Figure 2 shows the
stereochemical structures, in which R and S indicate the absolute configuration on the hydroxyl
group-attached carbon.
Figure 2. (R)- and (S)-Configured compounds.
7

2.2. Binding assay
A binding study was performed in two ways using synthetic A1-42 protein aggregate and AD
brain homogenate. Cold PIB and florbetaben (NeuraceqTM, formerly, AV-1) were also examined
for comparison. A competition binding experiment was carried out first using synthetic A1-42
protein aggregate in the presence of 6-[125I]iodo-2-(4-dimethylaminophenyl)benzo[d]thiazole
([125I]TZDM, Kd = 0.13 nM),19 showing comparable binding affinities to PIB (Ki = 0.44 nM).
Consequently, 1a, 1b, and 1c showed 1.20, 0.49, and 0.16 nM binding affinities, respectively.
Subsequently, another binding study using AD brain homogenate was attempted for 1b, 1c, and
the corresponding pure enantiomers in terms of the IC50 value. The six compounds of this study
showed 13-15 nM binding with narrow differences as well as higher binding than florbetaben
(IC50 = 24 nM). This also indicated that the chirality of the 3-fluoro-2-hydroxypropyl substituent
has little effect on the binding to  plaque. As a consequence of these binding studies, 1b and
1c were found to have comparable affinity to PIB, and approximately 1.7-fold higher affinity
than florbetaben. The binding affinity of 1a was relatively low and more lipophilic than 1b and
1c. In this regard, 1a was excluded from further study.
8

Table 1
Binding affinities to A plaque.
1-42 fibril (Ki, nM)a
compound
Brain homogenate (IC50, nM)b
c
PIB
0.44
-
c
Florbetaben
1a
-
24
c
1.20
0.49
-
13
15
14
13
14
13
1b
c
-
(R)-1b
(S)-1b
1c
c
-
0.16
c
-
(R)-1c
c
-
(S)-1c
a performed at the Asan Medical Center.
b performed by Bayer Healthcare.
c not determined.
2.3. [F]Radiofluorination and biodistribution study
In vivo distribution studies were conducted with normal mice to obtain the brain uptake level
and brain washout abilities of the F-18 labeled compounds. In the first place, a
cerebrum/cerebellum kinetics study was carried out using microPET image acquisition and data
analysis, giving the time-activity curves. The [F]fluorinated PET tracers were prepared in a
9

two-step synthesis from the methanesulfonate precursors 19b and 19c. Nucleophilic
displacement with in situ prepared [F]TBAF in t-amyl alcohol solvent,19 and subsequent
deprotection with 1 N HCl afforded [F]1b and [F]1c in 17% and 40% decay-corrected
radiochemical yields (RCYs) after HPLC purification. Dynamic microPET images of the mice
were acquired for 70 min and the regions of interest (ROIs) were drawn in the cerebrum and
cerebellum brain, and measured as a percentage of the injected dose (%ID). For comparison,
[11C]PIB and [F]florbetaben were also synthesized using the procedures reported elsewhere,4,14
and microPET images were obtained under the same conditions. Table 2 summarizes the results
of the cerebrum kinetics using four PET tracers. The activity uptake into the brain reached the
peak within 1 min in all cases. The peak time of [11C]PIB was fastest at 18 seconds, and
[F]florbetaben showed a peak time of 35 seconds. The peak times of [F]1b and [F]1c were
30 and 45 seconds, respectively. The activity amounts in the brain at the peak time of [F]1b
and [F]1c were 22.4 and 11.1 %ID, respectively, which are higher than [11C]PIB (7.0 %ID).
After the peak time, [F]1b and [F]1c were washed out rapidly from the brain. The washout
ratios of the new PET tracers were 10.2 and 11.8 for [F]1b, and 11.1 and 13.9 for [F]1c at
peak/30 min and peak/60 min, respectively. Although those values were lower than [11C]PIB,
they were approximately 2.1-2.3 times higher than [F]florbetaben. In the case of [F]1b, a
biodistribution study was performed using normal mice (n = 3). The %ID/g values of 19
different organs or tissues were measured at 2, 5, 30, 60, and 240 min after being sacrificed (see
supplementary data, Table S1). The initial brain uptake was 4.55 %ID/g, and the ratios of 2
min/30 min and 2 min/60 min were 4.6 and 6.1, respectively. This result was consistent with
those of the microPET-cerebrum kinetics study (7.5 and 8.7, at 2 min/30 min and 2 min/60 min,
10

respectively), even though there was a slight difference, presumably due to the different
experimental methods.
Table 2
Mice cerebrum kinetics
Compound
ID%
Peak/
Peak/
30 min 60 min
Peak (sec) 2 min
30 min
0.4
60 min
0.3
[11C]PIB
7.0 (18)
4.1
17.5
4.9
23.3
5.3
[18F]Florbetaben
[18F]1b
17.6 (35)
22.4 (30)
11.1 (45)
15.8
16.5
6.5
3.6
3.3
2.2
1.9
10.2
11.1
11.8
13.9
[18F]1c
1.0
0.8
Further evaluation with enantiomerically pure F-18 labeled (R)- and (S)-PET tracers was
conducted by repeating the aforementioned process. A MicroPET study was performed with
normal mice to determine the cerebrum kinetics. The radioactivity changes in the ROI of the
brain were measured and time-activity curves in the cerebrum region were obtained (Figure 3).
Surprisingly, each enantiomer exhibited largely different brain washout results. Figure 3 shows
that the %ID of remaining activity of (R) are much higher than that of (S) forms in both [18F]1b
and [F]1c. Although the initial brain uptake of the (S)-PET tracers was slightly higher than the
(R)-PET tracers, as much as 21% and 13%, respectively, the washout abilities of the (S)-PET
tracers were superior to those of the (R)- PET tracers. The ratios of peak/30 min of (S)-PET
tracers was 2.8-fold ([F]1b) and 4.0-fold ([F]1c) higher than those of the corresponding (R)-
PET tracers. Moreover, the difference in the ratios at 60 min between the (S)-PET and (R)-PET
tracers increased up to 3.0 and 5.2 fold. A biodistribution study was performed with two
11

enantiomerically pure (R)-[F]1c and (S)-[F]1c to confirm the difference in the brain washout
abilities using the same procedure as that for the [F]1b racemate mentioned above. The initial
uptake of (S)-[F]1c at 2 min was 3.32 %ID/g, which is 11% higher than (R)-[F]1c. This is
consistent with the results of a microPET study. The rate of washout from the brain of (S)-
[F]1c was much larger than (R)-[F]1c, resulting in 6.1, 12.8, and 23.7 at 2 min/30 min, 2
min/60 min, and 2 min/120 min, respectively. These washout ratios of (S)-[F]1c were 2.8, 5.6,
and 6.5 times higher than those of (R)-[F]1c at 30 min, 60 min, and 120 min, respectively.
Therefore, the washout profile of the R/S racemates [18F]1b and [18F]1c was obtained as the
combined result of (R) and (S)-PET tracers.
Table 3
Mice cerebrum kinetics of (R)- and (S)-PET tracers
%ID
Peak/
peak/
Compound
Peak (sec) 2 min
30 min
4.0
60 min
3.8
30 min
60 min
(R)-[18F]1b
(S)-[18F]1b
(R)-[18F]1c
(S)-[18F]1c
14.3 (45)
17.3 (30)
19.0 (35)
21.4 (16)
13.5
13.5
9.2
3.6
3.8
1.7
1.5
10.2
6.1
11.5
5.6
3.1
3.4
10.9
0.88
0.74
24.3
28.9
12

25
20
15
10
5
(A)
(R)-[18F]1b
(S)-[18F]1b
0
0
15
30
45
60
Time (min)
25
20
15
10
5
(B)
(R)-[18F]1c
(S)-[18F]1c
0
0
15
30
45
60
Time (min)
Figure 3. Time-activity curves in normal mouse brain. (A) is for [F]1b, (B) is for [F]1c. Each
point is the average of repeated experiments (n = 2 for (R)-[F]1b, (S)-[F]1b, and (R)-[F]1c;
n = 4 for (S)-[F]1c).
In vitro autoradiograms in both AD patient and healthy brain sections were obtained with the
[F]1b racemate, (R)-[F]1c, and (S)-[F]1c. Although the internal regions of the brain (white
13

matter) exhibited little binding, the  plaque-rich regions of the outer brain (grey matter) were
stained well with F-18 labeled PET tracers (Figure 4). In the healthy brain, a binding image was
not observed thoroughly. This indicates that new F-18 labeled ligands bind specifically to the 
plaque-rich grey matter region of an AD brain.
Figure 4. In vitro autoradiograms of AD and healthy brain sections stained with F-18 labeled
PET tracers. (A) [F]1b, (B) (R)-[F]1c, (C) (S)-[F]1c, AD: Alzheimer’s disease brain, HC:
healthy control brain, 'blocked' indicates the use of excess cold substance.
Because (S)-[F]1c has been found to exhibit the best performance in mice brain
pharmacokinetics, it was chosen for further evaluation with healthy rhesus monkeys (n = 2). Two
PET images were acquired for 4.5-34 min, and 34-123 min. The left image in Figure 5(A) shows
the considerable brain uptake of (S)-[F]1c in the early period of the scan, whereas the right
image indicates that most of the activity has disappeared in monkey brain. The time-activity
curve provided clear evidence of the fast washout of (S)-[F]1c from the frontal cortex after
peak uptake at 1.83 min. The ratio of peak/30 min, 60 min, 90 min, and 120 min was 7.0, 16.0,
14

30.0, and 49, respectively. This washout result in monkeys is superior to most previously
reported F-18 labeled PET tracers for  imaging. The change in radioactivity in the cerebellum,
a reference region for non-specific binding, showed a similar curve pattern to the frontal cortex
(see supplementary data), indicating that there are very little non-specific interactions. The time-
activity curve in the white matter region revealed remarkable short retention compared to other
lipophilic -targeting PET tracers, which maintained long retention during scan time. The
washout ratio in the white matter was 2.7, 5.6, 9.6, and 16.5 at 30, 60, 90, 120 min, respectively,
compared to the peak time (2.33 min, SUV(%)=76.9). This is a similar washout pattern to the
grey matter region with a gentle slope.
Figure 5. PET study with a rhesus monkey. (A) (S)-[F]lc-PET image of the rhesus monkey;
transverse, sagittal, coronal (from top to bottom). The left image was acquired for 4.5-34 min
while the right image was acquired for 34-123 min. (B) Time- activity curve. FC: frontal cortex,
WM: white matter.
15

18
Thus far, there have been a few reports of the use of F-labeled stereoisomers for the diagnosis
of brain diseases, such as neuroinflammation (GE180, binding to translocator protein)20 and
Alzheimer’s disease (THK5351, binding to the tau protein)21. Each single stereoisomer was
found to have better brain pharmacokinetics than the opposite stereoisomer as well as the
racemate mixture. (S)-[18F]1c also showed better performance than (R)-[18F]1c and the racemate
for imaging A plaque. Further evaluation of (S)-[18F]1c and a comparison study of (R)- and (S)-
PET tracers in humans is being investigated.
3. Conclusion
The introduction of a 3-[F]fluoropropyl group to the molecules of interest has been a
common strategy in the development of F-18 labeled PET tracers until now. The 3-
[F]fluoropropyl group, however, plays a role in increasing the lipophilicity of the molecules,
resulting in poor pharmacokinetic properties as well as undesired non-specific interactions. To
reduce the lipophilicity of the molecules, a hydroxyl group and a nitrogen atom to 3-
[F]fluoropropyl and aromatic ring, respectively. In this regard, 3-[F]fluoro-2-hydroxypropyl
substituted benzo[b]thiophene, and benzo[d]thiazoles were synthesized and evaluated for the
imaging of  plaque in the brain. All compounds exhibited high binding affinities, and a
specific binding profile to  plaque, which was confirmed by in vitro autoradiography with
sections of postmortem AD and healthy brains, showing the specific staining of the outer AD
brain region. The R/S racemate compounds exhibited improved brain washout properties
compared to 3-[18F]fluoropropyl substituted molecules. Thereafter, the corresponding (R)- and
(S)-enantiomers were prepared and evaluated by an in vitro binding study and in vivo
16

pharmacokinetics study. The binding ability of the (R) and (S) compounds to the AD brain
homogenate was similar. Intriguingly, the (S)-configured compounds, (S)-[F]1b and (S)-
[F]1c, showed a significantly higher brain washout ratio (2.9 and 3.9-fold, respectively) than
the corresponding (R)-configured compounds, giving a peak/30 min ratio of 10.2 and 23.4,
respectively. Further evaluation of (S)-[F]1c with the PET imaging of healthy rhesus monkeys
resulted in rapid clearance from the frontal cortex region and moderate clearance in the white
matter region. A clinical PET study using (S)-[18F]1c is currently being investigated in South
Korea under IND approval by the Korea Ministry of Food and Drug Safety (MFDS). This study
is planned to be extended to humans to confirm the different brain pharmacokinetics between
(R)- and (S)-[18F]1c.
4. Experimental
4.1. Chemistry
4.1.1. General methods.
All chemicals were obtained from commercial suppliers and were used without further
purification unless otherwise stated. Analytical thin layer chromatography (TLC) was performed
with Merck silica gel F-254 glass-backed plates. Visualization on TLC was monitored by UV
light (254 nm and 365 nm) and phosphomolybdic acid indicator. Column chromatography was
carried out using Merck silica gel 60 (230-400 mesh) and eluting with ethyl acetate/hexanes or
1
methanol/dichloromethane. H and 13C NMR spectra were recorded using either a Varian
Gemini-2000 (200 MHz), 400MR (400 MHz), and an INOVA-500 (500 MHz) and calibrated
using residual undeuterated solvent or tetramethylsilane as an internal reference. Low resolution
mass spectra were obtained using Electrospray Ionization (ESI, Varian 500-MS) and Electron
Ionization (EI, Agilent-5973N). High resolution mass spectra were performed at Korea Basic
17

Science Institute (Daegu, Korea) using Jeol-JMS 700. Melting points were checked using
OptiMelt apparatus. Optical rotations were measured at the sodium D line with a 100 mm path
length cell by AUTOPOL I Automatic Polarimeter (Rudolph Research Analytical), and reported
as follows: concentration (g/100 mL), and solvent. High performance liquid
chromatography (HPLC) was conducted to determine racemate and pure (R)- and (S)-enantiomer
using ChiralPak IA column (Daicel) (4.6 mm x 250 mm). Varian 920LC was used for FC113
compounds under following conditions; isocratic, 0.1% diethylamine in MeOH (100%), 1.0
mL/min, 254 nm (retention time = 9.71 min (FC113R), 10.55 min (FC113S). Varian Prostar 210
was used for FC119 compounds under following conditions; isocratic, 0.1% diethylamine in
THF: 0.1% diethylamine in hexane (25:75), 1.0 mL/min, 254 nm (retention time = 17.6 min
(FC119R), 20.1 min (FC119S).
18
[18F]Fluoride was produced by the O(p,n)18F reaction using [18O]water and cyclotron (IBA
Cyclone 18/9, Belgium). The progress of [18F]fluorination and deprotection was monitored by
visualization of radioactivity on TLC using AR-2000 TLC Imaging Scanner (Bioscan). HPLC
analysis and purification were performed using Varian Prostar 210 and Nucleosil C18 column
(10 mm x 250 mm).
4.1.2.
2-[4-(N-Methyl-N-Boc-amino)phenyl]-6-[(2,2-dimethyl-1,3-dioxolan-4-
yl)methyloxy]benzo[b]thiophene (16a)
2-[4-(N-Methyl-N-Boc-amino)phenyl]-6-hydroxylbenzo[b]thiazole (7a, 200 mg, 0.563
mmol), (2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate (15, prepared by
methanesulfonylation of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol, 177 mg, 0.844 mmol), and
Cs2CO3 (550 mg, 1.69 mmol) were placed in a round-bottom flask, and then DMF (15 mL) was
added. The mixture was suspended at 100 °C for 1 h and cooled to room temperature. After the
addition of cold water, the aqueous phase was extracted with EtOAc (30 mL x 4), and the
18

combined organic phase was washed with 0.2 M NH4Cl aqueous solution (30 mL), brine (30 mL
x 2) and dried over Na2SO4, and then concentrated under reduced pressure. The crude product
was purified by flash column chromatography to give 16a (217 mg, 82%) as a white solid: m.p.
116 °C; 1H NMR (400 MHz, CDCl3) δ 1.42 (s, 3H), 1.48 (s, 9H), 1.49 (s, 3H), 3.29 (s, 3H), 3.94
(dd, J = 8.6, 5.8 Hz, 1H), 4.00 (dd, J = 9.4, 5.8 Hz, 1H), 4.13 (dd, J = 9.2, 5.6 Hz, 1H), 4.20 (dd,
J = 8.4, 6.4 Hz, 1H), 4.53 (quint, J = 5.8 Hz, 1H), 7.00 (dd, J = 8.8, 2.4 Hz, 1H), 7.28 (d, J = 8.8
Hz, 2H), 7.31 (d, J = 2.4 Hz, 1H), 7.42 (s, 1H), 7.60-7.65 (m, 3H); 13C NMR (100 MHz, CDCl3)
δ 25.4, 26.8, 28.3, 37.2, 66.8, 69.3, 74.0, 80.6, 105.8, 109.8, 114.8, 118.8, 124.2, 125.6, 126.3,
131.3, 135.1, 140.8, 141.3, 143.4, 154.6, 156.3. HRMS (FAB) m/z: Calcd for C26H31NO5S [M]+
469.1923. Found, 469.1920.
4.1.3.
2-[2-(N-Methyl-N-Boc-amino)pyridin-5-yl]-6-[(2,2-dimethyl-1,3-dioxolan-4-
yl)methyloxy]benzo[b]thiophene (16b)
White solid; yield (206 mg, 87%). m.p. 118 °C; 1H NMR (400 MHz, CDCl3) δ 1.42 (s, 3H),
1.48 (s, 3H), 1.55 (s, 9H), 3.44 (s, 3H), 3.93 (dd, J = 8.4, 6.0 Hz, 1H), 4.00 (dd, J = 9.4, 5.8 Hz,
1H), 4.12 (dd, J = 9.6, 5.2 Hz, 1H), 4.19 (dd, J = 8.4, 6.4 Hz, 1H), 4.52 (quint, J = 5.9 Hz, 1H),
7.00 (dd, J = 8.0, 2.4 Hz, 1H), 7.30 (d, J = 2.0 Hz, 1H), 7.41 (s, 1H), 7.64 (d, J = 8.8 Hz, 1H),
7.77 (d, J = 8.4 Hz, 1H), 7.85 (dd, J = 8.8, 2.4 Hz, 1H), 8.66 (d, J = 2.4 Hz, 1H); 13C NMR (100
MHz, CDCl3) δ 25.4, 26.8, 28.3, 34.2, 66.8, 69.2, 74.0, 81.4, 105.8, 109.8, 115.0, 118.6, 119.4,
124.3, 125.8, 134.2, 134.8, 137.9, 140.8, 144.5, 154.2, 154.4, 156.5. HRMS (FAB) m/z: Calcd
for C25H30N2O5S [M+H]+ 471.1954. Found, 471.1952.
4.1.4.
2-[2-(N-Methyl-N-Boc-amino)pyridin-5-yl]-6-[(2,2-dimethyl-1,3-dioxolan-4-
yl)methyloxy]benzo[d]thiazole (16c)
19

White solid; yield (388 mg, 92%). m.p. 154 °C; 1H NMR (400 MHz, CDCl3) δ 1.40 (s, 3H),
1.46 (s, 3H), 1.54 (s, 9H), 3.45 (s, 3H), 3.92 (dd, J = 8.6, 5.8 Hz, 1H), 4.00 (dd, J = 9.4, 5.8 Hz,
1H), 4.10 (dd, J = 9.4, 5.4 Hz, 1H), 4.18 (dd, J = 8.6, 6.2 Hz, 1H), 4.50 (quint, J = 5.8 Hz, 1H),
7.09 (dd, J = 8.8, 2.8 Hz, 1H), 7.34 (d, J = 2.8 Hz, 1H), 7.90 (dd, J = 8.8, 6.0 Hz, 2H), 8.22 (dd, J
13
= 8.8, 2.4 Hz, 1H), 8.94 (d, J = 2.6 Hz, 1H); C NMR (100 MHz, CDCl3) δ 25.3, 26.8, 28.3,
34.1, 66.7, 69.4, 73.9, 81.7, 105.1, 109.8, 116.1, 118.2, 123.6, 124.9, 135.1, 136.0, 146.0, 148.8,
154.0, 156.4, 156.8, 162.4. HRMS (FAB) m/z: Calcd for C24H29N3O5S [M+H]+ 472.1906.
Found, 472.1903.
4.1.5.
(17a)
2-[4-(N-Methyl-N-Boc-amino)phenyl]-6-(2,3-dihydroxypropoxy)benzo[b]thiophene
Dowex 50WX2 resin (hydrogen form, 250 mg) was added to a solution of 2-[4-(N-methyl-
N-Boc-amino)phenyl]-6-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyloxy]benzo[b]thiophene (16a,
200 mg, 0.426 mmol) in 10% H2O/MeOH (10 mL). The mixture was suspended at 40 °C for 24
h, and then filtered with a syringe filter equipped with polyethylene frit. After the filtered resin
was washed with MeOH, the combined organic solution was concentrated under reduced
pressure. Column chromatography was performed to give 17a (105 mg, 58%) as a white solid:
m.p. 167 °C; 1H NMR (400 MHz, MeOH-d4) δ1.46 (s, 9H), 3.25 (s, 3H), 3.67 (dd, J = 11.4, 5.4
Hz, 1H), 3.72 (dd, J = 11.4, 5.0 Hz, 1H), 3.99-4.05 (m, 2H), 4.12 (dd, J = 9.0, 3.8 Hz, 1H), 4.89
(br s, 2H), 7.02 (dd, J = 8.8, 2.4 Hz, 1H), 7.28 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 2.4 Hz, 1H), 7.53
13
(s, 1H), 7.65-7.68 (m, 3H); C NMR (100 MHz, MeOH-d4) δ27.2, 36.4, 62.8, 69.3, 70.4, 80.5,
105.3, 114.6, 118.9, 124.0, 125.8, 125.9, 131.9, 135.0, 140.5, 140.8, 143.1, 155.0, 157.0. HRMS
(FAB) m/z: Calcd for C23H27NO5S [M]+ 429.1610. Found, 429.1612.
4.1.6.
2-[2-(N-Methyl-N-Boc-amino)pyridin-5-yl]-6-(2,3-
dihydroxypropoxy)benzo[b]thiophene (17b)
20

White solid; yield (94 mg, 52%). m.p. 140 °C (decomposed); 1H NMR (200 MHz, CDCl3) δ
1.55 (s, 9H), 3.43 (s, 3H), 3.77 (dd, J = 11.4, 4.6 Hz, 1H), 3.87 (dd, J = 11.4, 3.6 Hz, 1H), 4.07-
4.15 (m, 3H), 6.98 (dd, J = 8.8, 2.2 Hz, 1H), 7.29 (d, J = 2.2 Hz, 1H), 7.39 (s, 1H), 7.62 (d, J =
8.8 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.84 (dd, J = 8.6, 2.4 Hz, 1H), 8.64 (d, J = 2.6 Hz, 1H);
13C NMR (100 MHz, CDCl3) δ 28.3, 34.2, 63.6, 69.6, 70.4, 81.5, 105.9, 114.8, 118.7, 119.4,
124.3, 125.9, 134.2, 134.9, 137.9, 140.9, 144.5, 154.3, 154.4, 156.4. HRMS (FAB) m/z: Calcd
for C22H26N2O5S [M+H]+ 431.1641. Found, 431.1644.
4.1.7.
2-[2-(N-Methyl-N-Boc-amino)pyridin-5-yl]-6-(2,3-
dihydroxypropoxy)benzo[d]thiazole (17c)
1
White solid; yield (188 mg, 58%). m.p. 181 °C; H NMR (400 MHz, DMSO-d6) δ 1.49 (s,
9H), 3.35 (s, 3H), 3.49 (t, J = 5.6 Hz, 2H), 3.82-3.89 (m, 1H), 3.97 (dd, J = 9.8, 6.2 Hz, 1H), 4.10
(dd, J = 9.8, 4.2 Hz, 1H), 4.74 (t, J = 5.6 Hz, 1H), 5.05 (d, J = 5.2 Hz, 1H), 7.14 (dd, J = 9.2, 2.4
Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H), 8.29 (dd, J =
13
9.2, 2.4 Hz, 1H), 8.94 (dd, J = 2.6, 0.8 Hz, 1H); C NMR (100 MHz, DMSO-d6) δ 28.2, 34.2,
63.1, 70.4, 70.7, 81.8, 105.9, 116.8, 118.6, 123.8, 124.9, 135.7, 136.3, 145.8, 148.2, 153.7, 156.2,
157.5, 161.8. HRMS (FAB) m/z: Calcd for C21H25N3O5S [M+H]+ 432.1593. Found, 432.1595.
4.1.8.
2-[4-(N-Methyl-N-Boc-amino)phenyl]-6-(3-methanesulfonyloxy-2-
hydroxypropoxy)benzo[b]thiophene (18a)
After
2-[4-(N-methyl-N-Boc-amino)phenyl]-6-(2,3-dihydroxypropoxy)benzo[b]thiophene
(17a, 180 mg, 0.419 mmol) and diisopropylethylamine (DIPEA, 0.088 mL, 0.503 mmol) were
dissolved in dichloromethane (13 mL) and cooled to -10 °C, MsCl (0.036 mL, 0.461 mmol) was
slowly added. The mixture was stirred at -10 °C for 30 min, and water (30 mL) was added. The
organic phase was separated, and the aqueous phase was extracted with dichloromethane (30 mL
21

x 4). The combined organic phase was dried over Na2SO4, and then concentrated under reduced
pressure. The crude product was purified by flash column chromatography to give 18a (103 mg,
1
48%) as a white solid: m.p. 155 °C; H NMR (200 MHz, CDCl3) δ 1.48 (s, 9H), 3.09 (s, 3H),
3.29 (s, 3H), 4.12 (d, J = 5.2 Hz, 2H), 4.30-4.34 (m, 1H), 4.43-4.47 (m, 2H), 6.97 (dd, J = 8.8,
2.6 Hz, 1H), 7.26-7.31 (m, 3H), 7.41 (s, 1H), 7.59-7.66 (m, 3H); 13C NMR (100 MHz, CDCl3) δ
28.3, 37.1, 37.6, 68.2, 69.3, 70.2, 80.7, 106.0, 114.6, 118.8, 124.3, 125.6, 126.3, 131.2, 135.4,
140.8, 141.7, 143.5, 154.6, 155.8. HRMS (FAB) m/z: Calcd for C24H29NO7S2 [M]+ 507.1385.
Found, 507.1386.
4.1.9.
2-[2-(N-Methyl-N-Boc-amino)pyridin-5-yl]-6-(3-methanesulfonyloxy-2-
hydroxypropoxy)benzo[b]thiophene (18b)
White solid; yield (67 mg, 62%). m.p. 146 °C; 1H NMR (400 MHz, CDCl3) δ 1.55 (s, 9H), 2.78
(br s, 1H), 3.10 (s, 3H), 3.44 (s, 3H), 4.15 (d, J = 5.2 Hz, 2H), 4.35 (m, 1H), 4.44 (dd, J = 11.2,
5.6 Hz, 1H), 4.50 (dd, J = 11.0, 4.2 Hz, 1H), 7.00 (dd, J = 8.8, 2.4 Hz, 1H), 7.32 (d, J = 1.6 Hz,
1H), 7.43 (s, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.86 (dd, J = 8.8, 2.8 Hz,
1H), 8.67 (d, J = 2.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 28.2, 34.2, 37.6, 68.3, 68.4, 70.2,
81.5, 106.0, 114.8, 118.7, 119.4, 124.5, 125.8, 134.3, 135.2, 138.3, 140.9, 144.5, 154.3, 154.5,
156.0. HRMS (FAB) m/z: Calcd for C23H28N2O7S2 [M+H]+ 509.1416. Found, 509.1412.
4.1.10.
2-[2-(N-Methyl-N-Boc-amino)pyridin-5-yl]-6-(3-methanesulfonyloxy-2-
hydroxypropoxy)benzo[d]thiazole (18c)
White solid; yield (430 mg, 77%). m.p. 134 °C; 1H NMR (200 MHz, CDCl3) δ 1.62 (s, 9H),
3.04 (d, J = 5.2 Hz, 1H), 3.17 (s, 3H), 3.53 (s, 3H), 4.21 (d, J = 4.6 Hz, 2H), 4.41-4.46 (m, 1H),
4.51-4.58 (m, 2H), 7.15 (dd, J = 9.1, 2.5 Hz, 1H), 7.42 (d, J = 2.2 Hz, 1H), 7.98 (d, J = 8.8 Hz,
1H), 8.00 (d, J = 8.8 Hz, 1H), 8.31 (dd, J = 8.8, 2.4 Hz, 1H), 9.02 (d, J = 2.2 Hz, 1H); 13C NMR
22

(50 MHz, CDCl3) δ 28.3, 34.1, 37.6, 68.2, 68.6, 70.1, 81.9, 105.3, 115.9, 118.3, 123.8, 124.8,
135.2, 136.1, 146.1, 149.1, 154.1, 156.2, 156.5, 162.8. HRMS (FAB) m/z: Calcd for
C22H27N3O7S2 [M+H]+ 510.1369. Found, 510.1367.
4.1.11. 2-[4-(N-Methyl-N-Boc-amino)phenyl]-6-[3-methanesulfonyloxy-2-(tetrahydropyran-
2-yloxy)propoxy]benzo[b]thiophene (19a)
Dihydropyran (30.7 L, 0.337 mmol) was added to a solution of 18a (100 mg, 0.197 mmol)
and pyridinium p-toluenesulfonate (10 mg, 0.039 mmol) in dichloromethane (3 mL). The
mixture was stirred at room temperature for 17 h. After the addition of water (10 mL), the
organic phase was separated, and the aqueous phase was extracted with dichloromethane (10 mL
x 3). The combined organic phase was dried over Na2SO4, and then concentrated under reduced
pressure. The crude product was purified by flash column chromatography to give 19a (110 mg,
94%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 1.48 (s, 9H), 1.56-1.63 (m, 4H), 1.76-1.85
(m, 2H), 3.05 (s, 1.5H), 3.07 (s, 1.5H), 3.29 (s, 3H), 3.52-3.58 (m, 1H), 3.94-3.98 (m, 1H), 4.13-
4.21 (m, 1.5H), 4.26-4.34 (m, 1.5H), 4.42 (dd, J = 11.2, 2.6 Hz, 0.5H), 4.51 (dd, J = 10.8, 4.4 Hz,
0.5H), 4.54-4.60 (m, 1H), 4.84-4.85 (m, 0.5H), 4.86-4.88 (m, 0.5H), 6.98 (dt, J = 8.4, 2.6 Hz,
1H), 7.29 (d, J = 8.8 Hz, 2H), 7.33 (dd, J = 8.4, 2.4 Hz, 1H), 7.42 (s, 1H), 7.61-7.66 (m, 3H); 13C
NMR (100 MHz, CDCl3) δ 19.5, 19.8, 25.2, 25.4, 28.4, 29.7, 30.5, 30.6, 30.7, 37.2, 37.4, 37.5,
37.6, 63.0, 66.8, 67.3, 68.3, 68.4, 69.0, 69.6, 70.2, 72.6, 73.0, 80.6, 94.7, 99.0, 99.3, 105.9, 106.1,
114.6, 114.7, 118.7, 118.8, 124.2, 124.3, 125.6, 126.2, 126.3, 131.2, 131.3, 135.2, 135.3, 135.4,
140.8, 141.4, 141.5, 141.8, 143.4, 143.5, 143.6, 154.6, 155.8, 156.0, 156.1. HRMS (FAB) m/z:
Calcd for C29H37NO8S2 [M]+ 591.1961. Found, 591.1963.
4.1.12.
2-[4-(N-Methyl-N-Boc-amino)pyridin-5-yl]-6-[3-methanesulfonyloxy-2-
(tetrahydropyran-2-yloxy)propoxy]benzo[b]thiophene (19b)
23

1
White solid; yield (103 mg, 75%). m.p. 70 °C; H NMR (400 MHz, CDCl3) δ 1.55 (s, 9H),
1.57-1.66 (m, 4H), 1.74-1.87 (m, 2H), 3.05 (s, 1.5H), 3.08 (s, 1.5H), 3.44 (s, 3H), 3.54-3.58 (m,
1H), 3.94-3.98 (m, 1H), 4.13-4.22 (m, 1.5H), 4.26-4.35 (m, 1.5H), 4.42 (dd, J = 11.0, 5.4 Hz,
0.5H), 4.51 (dd, J = 10.8, 4.4 Hz, 0.5H), 4.54-4.57 (m, 1H), 4.83-4.88 (m, 1H), 7.00 (dt, J = 8.8,
2.8 Hz, 1H), 7.34 (dd, J = 8.2, 2.2 Hz, 1H), 7.44 (s, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.8
13
Hz, 1H), 7.87 (dd, J = 8.8, 2.4 Hz, 1H), 8.68 (dd, J = 2.4, 0.6 Hz, 1H); C NMR (100 MHz,
CDCl3) δ 19.7, 25.4, 25.5, 28.5, 30.8, 30.9, 34.4, 37.6, 37.7, 63.1, 63.2, 67.0, 67.5, 69.2, 69.9,
72.8, 73.2, 81.7, 99.2, 99.6, 106.1, 106.2, 115.2, 118.9, 119.6, 124.6, 126.0, 126.1, 134.5, 135.1,
135.2, 138.2, 138.3, 141.1, 144.8, 154.5, 154.7, 156.4, 156.5. HRMS (FAB) m/z: Calcd for
C28H36N2O8S2 [M+H]+ 593.1991. Found, 593.1992.
4.1.13.
2-[4-(N-Methyl-N-Boc-amino)pyridin-5-yl]-6-[3-methanesulfonyloxy-2-
(tetrahydropyran-2-yloxy)propoxy]benzo[d]thiazole (19c)
1
White solid; yield (278 mg, 88%). m.p. 62 °C; H NMR (400 MHz, CDCl3) δ 1.56 (s, 9H),
1.51-1.65 (m, 4H), 1.72-1.85 (m, 2H), 3.06 (s, 1.5H), 3.08 (s, 1.5H), 3.48 (s, 3H), 3.54-3.58 (m,
1H), 3.93-3.98 (m, 1H), 4.14-4.23 (m, 1.5H), 4.27-4.35 (m, 1.5H), 4.43 (dd, J = 11.2, 5.2 Hz,
0.5H), 4.49-4.59 (m, 1.5H), 4.84-4.87 (m, 1H), 7.11 (dt, J = 9.2, 2.8 Hz, 1H), 7.40 (dd, J = 7.4,
2.6 Hz, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.94 (d, J = 9.2 Hz, 1H), 8.25 (dd, J = 8.8, 2.4 Hz, 1H),
8.97 (d, J = 2.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 19.5, 25.2, 28.2, 30.5, 30.6, 34.1, 37.4,
37.5, 62.9, 67.0, 67.5, 68.9, 69.6, 72.6, 72.9, 81.7, 99.1, 99.3, 105.1, 105.3, 116.0, 118.1, 123.7,
124.7, 124.8, 135.0, 136.1, 146.0, 148.8, 148.9, 154.0, 156.3, 156.4, 156.5, 162.4, 162.5. HRMS
(FAB) m/z: Calcd for C27H35N3O8S2 [M+H]+ 594.1944. Found, 594.1941.
4.1.14. 2-[4-(Methylamino)phenyl]-6-(3-fluoro-2-hydroxypropoxy)benzo[b]thiophene (1a)
24

19a (120 mg, 0.203 mmol) and tetra-n-butylammonium fluoride (TBAF; 63 mg, 0.243
mmol) were dissolved in t-amyl alcohol (3 mL) and stirred at 100 °C for 6 h. After the mixture
was cooled to room temperature, the solvent was removed under reduced pressure. Aqueous 4 N
HCl (0.55 mL) and THF (2 mL) were added to a reaction vessel, and the mixture was then stirred
at 80 °C for 30 min. After cooling to room temperature, water (5 mL) was added and the mixture
was then treated with saturated NaHCO3 aqueous solution until basic to litmus. The organic
compounds were extracted with EtOAc (5 mL x 4), and the combined organic phase was dried
over Na2SO4, and then concentrated under reduced pressure. The crude product was purified by
1
flash column chromatography to give 1a (65 mg, 81%) as a light brown solid: m.p. 171 °C; H
NMR (400 MHz, THF-d8) δ 2.79 (d, J = 5.2 Hz, 3H), 4.04-4.06 (m, 2H), 4.07-4.15 (m, 1H), 4.50
(dddd, J = 47.9, 23.6, 9.2, 4.8 Hz, 2H), 4.69 (d, J = 4.8 Hz, 1H), 5.17 (d, J = 4.8 Hz, 1H), 6.56
(d, J = 8.4 Hz, 2H), 6.92 (dd, J = 8.4, 2.4 Hz, 1H), 7.28 (s, 1H), 7.38 (d, J = 2.4 Hz, 1H), 7.45 (d,
J = 8.8 Hz, 2H), 7.54 (d, J = 8.8 Hz, 1H); 13C NMR (100 MHz, THF-d8) δ 30.3, 69.5 (d, J = 19
Hz), 69.8 (d, J = 7 Hz), 85.3 (d, J = 169 Hz), 106.6, 112.6, 115.1, 116.4, 123.4, 124.0, 127.7,
136.5, 140.9, 143.9, 151.0, 157.1. HRMS (FAB) m/z: Calcd for C18H18FNO2S [M]+ 331.1042.
Found, 331.1043.
4.1.15. 2-[4-(Methylamino)pyridin-5-yl]-6-(3-fluoro-2-hydroxypropoxy)benzo[b]thiophene
(1b)
White solid; yield (55 mg, 70%). m.p. 164 °C; 1H NMR (400 MHz, DMSO-d6) δ 2.81 (d, J
= 5.2 Hz, 3H), 3.99-4.04 (m, 2H), 4.05-4.12 (m, 1H), 4.51 (dddd, J = 47.6, 19.4, 9.6, 4.2 Hz,
2H), 5.50 (d, J = 4.8 Hz, 1H), 6.53 (d, J = 8.8 Hz, 1H), 6.84 (q, J = 4.7 Hz, 1H), 6.99 (dd, J =
8.8, 2.4 Hz, 1H), 7.50 (s, 1H), 7.53 (d, J = 2.0 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.73 (dd, J =
8.8, 2.4 Hz, 1H), 8.35 (d, J = 2.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 28.4, 68.1 (d, J =
25

19 Hz), 69.0 (d, J = 8 Hz), 84.9 (d, J = 167 Hz), 106.6, 108.3, 115.1, 117.1, 118.3, 124.1, 134.6,
135.2, 139.5, 139.7, 145.4, 156.3, 159.5. HRMS (FAB) m/z: Calcd for C17H17FN2O2S [M+H]+
333.1073. Found, 333.1071.
4.1.16.
(1c)
2-[4-(Methylamino)pyridin-5-yl]-6-(3-fluoro-2-hydroxypropoxy)benzo[d]thiazole
White solid; yield (35 mg, 78%). m.p. 180 °C; 1H NMR (400 MHz, DMSO-d6) δ 2.88 (d, J
= 4.8 Hz, 3H), 4.05-4.09 (m, 2H), 4.11-4.16 (m, 1H), 4.55 (dddd, J = 47.5, 18.6, 9.6, 4.5 Hz,
2H), 5.55 (d, J = 5.2 Hz, 1H), 6.59 (d, J = 8.8 Hz, 1H), 7.12 (dd, J = 8.8, 2.4 Hz, 1H), 7.25 (q, J
= 4.5 Hz, 1H), 7.67 (d, J = 2.8 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.98 (dd, J = 9.0, 2.6 Hz, 1H),
8.67 (d, J = 2.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 28.3, 68.6 (d, J = 19 Hz), 69.2 (d, J
= 7 Hz), 84.9 (d, J = 168 Hz), 106.1, 108.3, 116.1, 117.9, 122.9, 135.2, 135.4, 147.8, 148.6,
156.4, 160.9, 164.0. HRMS (FAB) m/z: Calcd for C16H16FN3O2S [M+H]+ 334.1026. Found,
334.1027.
4.1.17.
(R)-2-[4-(Methylamino)pyridin-5-yl]-6-(3-fluoro-2-
hydroxypropoxy)benzo[b]thiophene ((R)-1b)
(R)-1b was synthesized in the same manner as Scheme 1, starting from compound 7b and
(S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl
methanesulfonate
((S)-15,
prepared
by
methanesulfonylation of (R)-2,2-dimethyl-1,3-dioxolane-4-methanol (99% ee, Aldrich)).
=
-13.3º (1.5, THF); Chiral HPLC analysis, retention time = 10.15 min (>99 ee%).
4.1.18.
hydroxypropoxy)benzo[b]thiophene ((S)-1b)
(S)-1b was synthesized in the same manner as Scheme 1, starting from compound 7b and
(S)-2-[4-(Methylamino)pyridin-5-yl]-6-(3-fluoro-2-
(R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl
methanesulfonate
((R)-15,
prepared
by
26

methanesulfonylation of (S)-2,2-dimethyl-1,3-dioxolane-4-methanol (99% ee, Aldrich)).
=
+13.3º (1.5, THF); Chiral HPLC analysis, retention time = 11.18 min (>99 ee%)
4.1.19.
(R)-2-[4-(Methylamino)pyridin-5-yl]-6-(3-fluoro-2-
hydroxypropoxy)benzo[d]thiazole ((R)-1c)
(R)-1c was synthesized in the same manner as Scheme 1, starting from compound 7c and
(S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate ((S)-15, prepared by
methanesulfonylation of (R)-2,2-dimethyl-1,3-dioxolane-4-methanol (99% ee, Aldrich)).
=
-9.33º (1.5, THF); Chiral HPLC analysis, retention time = 17.3 min (>99 ee%)
4.1.20. (S)-2-[4-(Methylamino)pyridin-5-yl]-6-(3-fluoro-2-hydroxypropoxy)benzo[d]thiazole
((S)-1c)
(S)-1c was synthesized in the same manner as Scheme 1, starting from compound 7c and
(R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl
methanesulfonate
((R)-15,
prepared
by
methanesulfonylation of (S)-2,2-dimethyl-1,3-dioxolane-4-methanol (99% ee, Aldrich)).
=
+9.33º (1.5, THF); Chiral HPLC analysis, retention time = 20.6 min (>99 ee%)
4.2. In Vitro binding study
4.2.1. Method 1 (Synthetic Aprotein aggregate)
A peptide (A1-42, 1 mg, Bachem) was dissolved in 1 mL of DMSO, and diluted with
phosphate buffer (pH 7.4, 9 mL). Aprotein aggregate produced by incubating at 37 °C for
60 min were dispensed in 0.5 mL each and stored in a refrigerator at -80 °C. The Aprotein
aggregate solution (10 nM) was placed in a test tube and [125I]TZDM (6-[125I]iodo-2-(4-
dimethylaminophenyl)benzo[b]thiazole) (50 L, 0.046~5.9 pM) was added. The resulting
solution was diluted with 10% ethanol to be a total volume of 1 mL, and subjected to incubation
27

at room temperature for 3 h. Thereafter, [125I]TZDM bound to the Aprotein aggregate was
separated from unbound [125I]TZDM using a cell harvester (Brandel M-24R). Gamma radiation
was measured using a gamma counter, and then the dissociation constant Kd was calculated, in
which non-specific binding was tested using Thioflavin T (ThT, 2 M). As a result of that, Kd
value of [125I]TZDM was revealed to be 0.13 nM.
To a test tube, 10% ethanol (0.850 mL), Aprotein aggregate (50 L, 10 nM), the
respective unlabled ligand of this paper (50 L, 1 M) were added in sequence. After
[125I]TZDM (50 L, 0.05 nM) was added, the solution stood at room temperature for 3 h. Using a
cell harvester (Brandel M-24R), Aprotein aggregate binding [125I]TZDM was separated
from free [125I]TZDM. Each was measured by a gamma counter, in which non-specific binding
was tested using ThT (2 M).
4.2.2. Method 2 (Brain homogenate)
Brain homogenates were prepared by homogenizing dissected brain of AD patients in PBS
buffer solution (pH 7.4) at the concentration of 100 mg wet tissue/mL. Various concentrations of
unlabeled ligands of this paper were incubated with 100 mg/mL homogenate and tritiated ligand
at room temperature for 3 h. The tritiated ligand bound to homogenates was filtered through
Whatman CF/B filters using a cell harvester. The filters containing the bound tritiated ligand
were washed with PBS buffer and assayed for radioactivity amount in a gamma counter. The
result of inhibition experiments were converted to IC50 values through a nonlinear regression
analysis.
4.3. [18F]Radiosynthesis
4.3.1. Radiosynthesis of [18F]lb
28

[18F]Fluoride (40 mCi) ion produced by irradiation of [18O]H2O with cyclotron was trapped in
a Chromafix® (PS-HCO3) cartridge, and was released into a reactor by elution of a methanol
solution (0.7 mL) containing tetra-n-butylammonium bicarbonate (TBAHCO3, 0.08 mL). After
removing the solvent by azeotropic evaporation with acetonitrile (0.5 mL) at 100 °C while
passing nitrogen over the solution. A solution of precursor compound 19b (5 mg) dissolved in
tetrahydrofuran (0.05 mL) and t-amyl alcohol (1.0 mL) was added to the reactor. After the
reaction mixture was heated at 120 °C for 20 min, the solvent was removed by a gentle stream of
nitrogen under heating at 100 °C. 1 N HCl aqueous solution (0.5 mL) was added and stirred at
100 °C for 5 min. After cooling to room temperature, the reaction mixture was neutralized by the
addition of 2 N NaOH solution (0.25 mL). The crude solution was loaded onto a C18 Sep-Pak
cartridge (Waters), and washed with 5 mL of water, and then eluted with 2 mL of acetonitrile
into a vial. The acetonitrile solution was injected into HPLC equipped with a semipreparative
column. [18F]1b containing fraction was collected and diluted with 15 mL of water, which was
passed through a preconditioned C18 Sep-Pak cartridge. The final product was eluted with 1 mL
of ethanol. Radiochemical yield and specific activity of the final product, [18F]1c, were 16.95%
(decay-corrected) and 850 Ci/mmol, respectively. Total synthesis time was 65 min including
HPLC purification.
4.3.2. Radiosynthesis of [18F]1c
[18F]1c was prepared by the same procedure as the radiosynthesis of [18F]lb with 40 mCi of
[18F]fluoride ion. Radiochemical yield and specific activity were 40.17% (decay-corrected) and
>1200 Ci/mmol, respectively. Total synthesis time was 75 min including HPLC purification.
4.4. MicroPET image acquisition
29