Synthesis and evaluation of thioflavin-T analogs as potential imaging agents for amyloid plaques

Article abstract of DOI:10.1007/s00044-012-0414-2

Thioflavin-T (ThT) is a benzothiazole dye that exhibits enhanced fluorescence upon binding to beta-amyloid (Aβ) plaques and is commonly used in the diagnosis of Alzheimer's disease (AD). In this study, the ThT analogs were designed and synthesized, and screening was conducted for the detection of Aβ ₄₀ fibrils in vitro. Among these compounds, 2-(2′-methoxy-4′-methylaminophenyl)benzoxazole (2) meets two critical requirements for an imaging agent: high its higher fluorescent responsiveness and stronger binding affinity than those of ThT. This compound showed the highest binding affinity with a dissociation constant of 3.27 ± 0.29 μM, and selectively stained Aβ aggregated in SHSY5Y neuroblastoma cells. This finding demonstrates the compound's potential use as a brain-imaging agent for AD studies. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-012-0414-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0414-2
ORIGINAL RESEARCH
Synthesis and evaluation of thioflavin-T analogs as potential
imaging agents for amyloid plaques
•
Soon Jae Jung Yong Dae Park Jeong Hoon Park
•
•
•
Seung Dae Yang Min Goo Hur Kook Hyun Yu
•
Received: 19 September 2012 / Accepted: 11 December 2012
Ó Springer Science+Business Media New York 2012
Abstract Thioflavin-T (ThT) is a benzothiazole dye that
exhibits enhanced fluorescence upon binding to beta-
amyloid (Ab) plaques and is commonly used in the diag-
nosis of Alzheimer’s disease (AD). In this study, the ThT
analogs were designed and synthesized, and screening
was conducted for the detection of Ab40 fibrils in vitro.
Among these compounds, 2-(20-methoxy-40-methylamin-
ophenyl)benzoxazole (2) meets two critical requirements
for an imaging agent: high its higher fluorescent respon-
siveness and stronger binding affinity than those of ThT.
This compound showed the highest binding affinity with a
dissociation constant of 3.27 0.29 lM, and selectively
stained Ab aggregated in SHSY5Y neuroblastoma cells.
This finding demonstrates the compound’s potential use as
a brain-imaging agent for AD studies.
and Cotman, 1998). The accumulation of amyloid plaques
and neurofibrillary tangles in brain tissue is one of the
neuropathological hallmarks of AD (Luurtsema et al.,
2008). Amyloid plaques are composed of a tangle of reg-
ularly ordered fibrillar aggregates, known as amyloid
fibers. Beta-amyloid (Ab) is the main component of amy-
loid plaques. Ab is a peptide of 39–42 amino acids that is
processed from the amyloid precursor protein (Kang et al.,
1987; Lahiri and Maloney, 2010). The most common iso-
forms are Ab40 and Ab42; the shorter form is typically
produced by cleavage that occurs in the endoplasmic
reticulum while the longer form is produced by cleavage in
the trans-Golgi network (Hartmann et al., 1997). Current
drug research is mainly focused on decreasing Ab pro-
duction, blocking the formation of these plaques by pre-
venting Ab protofibril and fibril formation, and alleviating
the toxic effects of neuritic plaque deposition (Xia, 2003).
Therefore, assessing the degree of Ab deposition is an
important tool for AD diagnosis.
Keywords Alzheimer’s disease (AD) ꢀ
Amyloid plaques ꢀ Beta-amyloid peptide (Ab) ꢀ
Thioflavin-T (ThT)
Ab imaging pharmaceuticals have been developed with
the foundation of a dye chemical structure that is used for Ab
pathological examination. Several imaging agents, such as
the thioflavin-T derivative, [11C]PIB ([N-methyl-11C]-2-(40-
methylaminophenyl)-6-hydroxybenzothiazole), the stilbene
derivative [11C]SB-13 ([N-methyl-11C]-4-methylamino-40-
hydroxytrilben), and [18F]FDDNP (2-(1-{6-[(2-[18F]fluoro-
ethyl)(methyl)amino]2-naphthyl}ethylidene)malononitrile),
have been reported for use in the positron emission tomog-
raphy imaging of Ab plaques in AD patients (Mathis et al.,
2003). Among these agents, thioflavin-T (ThT) dye shows
enhanced fluorescence upon binding to amyloids in tissue
sections. Therefore, ThT dye fluorescence is widely used to
quantify the presence of Ab found in the brains of AD
patients (Hudson et al., 2009). In addition, fluorescent probes
can be utilized as a safer, faster, and less expensive
Introduction
Alzheimer’s disease (AD) is the most common form of
dementia in elderly people. AD is a general term used for a
loss of memory and other intellectual abilities that are
serious enough to interfere with daily activities (Berchtold
S. J. Jung ꢀ Y. D. Park ꢀ J. H. Park ꢀ S. D. Yang ꢀ M. G. Hur
Radiation Instrumentation Research Division, Korea Atomic
Energy Research Institute, Jeongeup 580-185, Republic of Korea
S. J. Jung ꢀ K. H. Yu (&)
Department of Chemistry, Dongguk University,
30 Pildong-ro 1-Gil, Jung-gu, Seoul 100-715, Republic of Korea
e-mail: yukook@dongguk.edu
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Med Chem Res
HRMS (FAB?) m/z: 240.09 (100), 241.09 (16). Anal. calcd.
for C14H12N2O2: 240.09: C, 69.99; H, 5.03; N, 11.66; O,
13.32. Found: C, 69.71; H, 5.01; N, 11.62;O, 13.28.
X
N
Fig. 1 Structure of thioflavin-T
analogs. Compounds reported in
this study include the following:
X = O, S; R = NH2, NHCH3,
N(CH3)2 (total of six ThT
analogs)
R
MeO
Synthesis of 2-(20-methoxy-40-
methylaminophenyl)benzoxazole (2)
alternative compared with radiolabeled probes for detecting
Ab plaques. In this study, we describe the optical properties
of ThT analogs, the structures of which are shown in Fig. 1
and their biological properties as fluorescent probes.
Sodium methoxide (27 mg, 0.5 mmol) and paraformalde-
hyde (13 mg, 0.5 mmol) were added to a solution of
compound 1 (0.12 g, 0.5 mmol) in methanol (10 mL), and
the mixture was refluxed for 2 h. The mixture was cooled
to 0 °C, sodium borohydride (19 mg, 0.5 mmol) was added
in portions, and the mixture was refluxed further for 1 h.
Next, the mixture was poured into ice water and extracted
with ethyl acetate. The combined extracts were dried over
sodium sulfate and filtered. The filtrate was concentrated to
dryness, and the residue was purified by column chroma-
tography to yield 2-(20-methoxy-40-methylaminophe-
nyl)benzothiazole (0.06 g, 28 %) as a light yellow solid.
Rf: 0.55 (EA:Hx = 1:8). m.p.: 146-148 °C. 1H NMR
(CDCl3) d 7.82 (d, J = 8.9 Hz, 1H), 7.62, (d, J = 8.0 Hz,
1H), 7.52 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H),
7.28 (t, J = 7.5 Hz, 1H), 6.36 (d, J = 8.9 Hz, 1H), 6.33 (d,
J = 2.6 Hz, 1H), 3.05 (s, 6H).13C NMR (CDCl3) d 164.00,
160.31, 154.33, 149.06, 140.72, 128.17, 124.59, 124.06,
118.35, 110.22, 104.65, 99.37, 98.65, 40.19. HRMS
(FAB?) m/z: 254.11 (100), 255.11 (17). Anal. calcd. for
C15H14N2O2: 240.09: C, 70.85; H, 5.55; N, 11.02; O,
12.58. Found: C, 70.57; H, 4.99; N, 10.98;O, 12.53.
Materials and methods
Chemistry
All commercial reagents and solvents were purchased from
Aldrich at analytical grade and used as received without
purification. The melting points were measured on a Mel-
Temp. 1H and 13C NMR spectra were recorded on a JEOL
ECA-500 NMR spectrometer, and chemical shift data for
the proton resonances were reported at a parts per million
scale with tetramethylsilane serving as an internal stan-
dard. Mass spectra were recorded on a JEOL JMS-
AX505WA mass spectrometer, and the compounds were
measured using the FAB method using m-nitrobenzyl
alcohol as a matrix at the National Center for Inter-
University Research Facilities. Gravity column chroma-
tography was performed on Merck silica gel 60 (70–230
mesh ASTM). Thin layer chromatography was performed
on Merck silica gel 60F-254 glass plates and visualized by
UV light.
Synthesis of 2-(20-methoxy-40-
dimethylaminophenyl)benzoxazole (3)
Synthesis of 2-(20-methoxy-40-aminophenyl)benzoxazole (1)
Methyl iodide (0.12 mL, 2 mmol) and K2CO3 (0.28 g,
2 mmol) were added to a solution of compound 1 (0.14 g,
0.58 mmol) in DMF (anhydrous, 3 mL). The reaction
mixture was heated at 140 °C for 4 h. Upon cooling to
room temperature, the reaction mixture was poured into
water and extracted with ethyl acetate (3 9 10 mL).
The organic layers were combined, and the solvent was
evaporated. The residue was purified by column chroma-
tography to yield 2-(20-methoxy-40-dimethylaminophenyl)
benzoxazole (60 mg, 39 %) as a light yellow solid. Rf: 0.09
A mixture of 2-aminophenol (0.54 g, 5 mmol) and 4-amino-
2-methoxybenzoic acid (0.83 g, 5 mmol) in polyphosphoric
acid (10 mL) was heated at 200 °C for 4 h. After cooling, the
reaction mixture was poured into 50 mL of 10 % sodium
carbonate. The crude product was extracted using ethyl
acetate and subsequently washed with brine. The ethyl ace-
tate portion was dried over sodium sulfate and filtered. The
filtrate was concentrated to dryness, and the residue was
purified using column chromatography to yield 2-(20-meth-
oxy-40-aminophenyl)benzoxazole (0.17 g, 14 %) as a light
yellow solid. Rf: 0.34 (EA:Hx = 1:8). m.p.: 117–
118 °C. 1H NMR (CDCl3) d 7.78 (d, J = 8.8 Hz, 1H), 7.62
(d, J = 7.5 Hz, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.31 (t,
J = 7.5 Hz, 1H), 7.27 (t, J = 7.5 Hz, 1H), 6.25 (s, 1H), 6.23
(d, J = 2.3 Hz, 1H), 2.90 (s, 3H).13C NMR (CDCl3) d
163.95, 160.87, 153.93, 149.01, 140.63, 128.31, 124.62,
124.13, 118.39, 110.23, 105.80, 100.20, 98.14, 30.26.
1
(EA:Hx = 1:8). m.p.: 62–63 °C. H NMR (CDCl3) d 7.82
(d, J = 8.9 Hz, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.52 (d,
J = 7.7 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.28 (t,
J = 7.8 Hz, 1H), 6.36 (dd, J = 8.9 Hz, J = 2.6 Hz, 1H),
6.33 (d, J = 2.6 Hz, 1H), 4.00 (s, 3H), 3.07 (s, 6H).13C
NMR (CDCl3) d 162.71, 160.14, 153.82, 150.05, 142.77,
132.16, 123.91, 123.85, 119.52, 110.00, 104.65, 103.85,
95.04, 56.04, 40.28. HRMS (FAB?) m/z: 268.13 (100),
269.13 (18). Anal. calcd. for C16H16N2O2: 268.12: C,
123

Med Chem Res
71.62; H, 6.01; N, 10.44; O, 11.93. Found: C, 71.55; H,
6.00; N, 10.43; O, 11.92.
271.09 (18). Anal. calcd. for C15H14N2OS: 270.08: C,
66.64; H, 5.22; N, 10.36; O, 5.92; S, 11.86. Found: C, 66.31;
H, 5.19; N, 10.31; O, 5.89; S, 11.80.
Synthesis of 2-(20-methoxy-40-aminophenyl)
benzothiazole (4)
Synthesis of 2-(20-methoxy-40-
dimethylaminophenyl)benzothiazole (6)
A mixture of 2-aminothiophenol (0.5 mL, 5 mmol) and
4-amino-2-methoxybenzoic acid (0.83 g, 5 mmol) in phos-
phorus oxychloride (5 mL) was heated at 100 °C for 4 h.
After cooling, the reaction mixture was carefully and slowly
poured into a beaker of distilled water while stirring. The pH
was adjusted to 7.0 by the addition of sodium hydroxide pel-
lets. The crude product was extracted using dichloromethane
and washed twice with distilled water. The combined extracts
were dried over sodium sulfate and filtered. The filtrate was
concentrated to dryness, and the residue was purified by col-
umn chromatography to yield yellow 2-(20-methoxy-40-ami-
nophenyl)benzothiazole (0.14 g, 11 %). Rf: 0.07
(EA:Hx = 1:8). m.p.: 146–148 °C. 1H NMR (CDCl3) d 8.31
(d, J = 8.6 Hz, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.86 (d,
J = 8.6 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.29 (t,
J = 8.0 Hz, 1H), 6.41 (dd, J = 8.6 Hz, J = 2.3 Hz, 1H),
6.29 (d, J = 2.0 Hz, 1H), 3.99 (s, 3H).13C NMR (CDCl3) d
163.92, 158.94, 152.35, 150.41, 135.59, 131.02, 125.72,
123.86, 122.08, 121.10, 113.17, 108.05, 97.60, 55.58. HRMS
(FAB?) m/z: 256.07 (100), 257.07 (17). Anal. calcd. for
C14H12N2OS: 256.07: C, 65.60; H, 4.72; N, 10.93; O, 6.24; S,
12.51. Found: C, 65.53; H, 4.72; N, 10.92; O, 6.23; S, 12.50.
A solution of compound 4 (0.13 g, 0.5 mmol) in tetrahy-
drofuran (5 mL) was slowly added to a stirred mixture of
37 % formaldehyde (0.5 mL, 5 mmol) and 4 M H2SO4
(0.4 mL, 1.6 mmol). Powdered iron (0.21 g, 4 mmol) was
added to the mixture, and the mixture was stirred
mechanically for 30 min. The iron precipitate was removed
by filtration and washed with ethyl acetate. The filtrate was
made strongly alkaline (pH [ 11) with 10 % NaOH and
was extracted with ethyl acetate. The combined ethyl
acetate extracts were dried over sodium sulfate and con-
centrated. The oily residue was subsequently purified with
column chromatography to yield yellow 2-(20-methoxy-40-
dimethylaminophenyl)benzothiazole (0.05 g, 35 %). Rf:
1
0.30 (EA:Hx = 1:8). m.p.: 95–96 °C. H NMR (CDCl3) d
8.36, (d, J = 8.9 Hz, 1H), 7.98 (d, J = 8.3 Hz, 1H), 7.85
(d, J = 7.8 Hz, 1H), 7.42 (t, J = 7.2 Hz, 1H), 7.27 (t,
J = 8.3 Hz, 1H), 6.45 (dd, J = 8.9 Hz, J = 2.3 Hz, 1H),
6.24 (d, J = 2.3 Hz, 1H), 4.04 (s, 3H), 3.07 (s, 6H).13C
NMR (CDCl3) d 164.15, 158.94, 153.36, 152.54, 135.55,
130.52, 125.61, 123.59, 121.89, 121.02, 111.10, 105.36,
94.65, 55.53, 40.35. HRMS (FAB?) m/z: 284.11 (100),
285.11 (19). Anal. calcd. for C16H16N2OS: 284.10: C,
67.58; H, 5.67; N, 9.85; O, 5.63; S, 11.28. Found: C, 67.38;
H, 5.65; N, 9.82; O, 5.61; S, 11.25.
Synthesis of 2-(20-methoxy-40-
methylaminophenyl)benzothiazole (5)
Sodium methoxide (27 mg, 0.5 mmol) and paraformalde-
hyde (13 mg, 0.5 mmol) were added to a solution of com-
pound 4 (0.13 g, 0.5 mmol) in methanol (10 mL), and the
mixture was subsequently refluxed for 2 h. The mixture was
cooled to 0 °C, sodium borohydride (19 mg, 0.5 mmol) was
added in portions, and the mixture was refluxed further for
1 h. Next, the mixture was poured into ice water and
extracted with ethyl acetate. The combined extracts were
dried over sodium sulfate and filtered. The filtrate was
concentrated to dryness, and the residue was purified by
column chromatography to yield orange 2-(20-methoxy-40-
methylaminophenyl)benzothiazole (0.06 g, 43 %). Rf: 0.16
Biological procedures
General method for Ab40 fibrils preparation
Purified Ab40 peptides were dissolved in phosphate-buf-
fered saline (PBS, pH 7.4) to obtain a final concentration of
200 lM. The solution was incubated by gentle shaking for
2 days at 37 °C. The formation of Ab40 fibrils was con-
firmed using a ThT assay (Klunka et al., 2001).
Fluorescence spectra measurement
1
(EA:Hx = 1:8). m.p.: 146–147 °C. H NMR (CDCl3) d
The excitation and emission kmax of each compound was
measured with a TECAN 200 Pro. The final concentrations
of 10–50 lM of the Ab40 fibrils in the PBS buffer solution
were used in the experiments. Generally, the kmax of the
emission was determined by scanning a fixed excitation
first, and the kmax of the excitation was determined using
a scanning emission spectrum with a fixed kem (Hong
et al., 2010).
8.33 (d, J = 8.8 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.85 (d,
J = 7.5 Hz, 1H), 7.42 (t, J = 7.7 Hz, 1H), 7.28 (t,
J = 8.0 Hz, 1H), 6.35 (dd, J = 8.6 Hz, J = 2.3 Hz, 1H),
6.18 (d, J = 2.0 Hz, 1H), 4.02 (s, 3H), 2.92 (s, 3H).13C
NMR (CDCl3) d 164.16, 159.16, 152.77, 152.44, 135.53,
130.80, 125.65, 123.66, 121.92, 121.05, 111.92, 105.77,
94.50, 55.54, 30.46. HRMS (FAB?) m/z: 270.09 (100),
123

Med Chem Res
X
N
NHCH3
O
OH
MeO
(ii)
XH
X
N
MeO
(i)
2 (X=O)
5 (X=S)
NH2
+
NH2
(iii)
MeO
NH2
X
N
1 (X=O)
4 (X=S)
N(CH3)2
MeO
3 (X=O)
6 (X=S)
Scheme 1 Synthesis of thioflavin-T analogs. Reagents and conditions: (i) X = O: polyphosphoric acid, 200 °C, 4 h/X = S: phosphorus
oxychloride, 100 °C, 4 h; (ii) NaOCH3, paraformaldehyde, NaBH4; (iii) X = O: CH3I/K2CO3/X = S: H2SO4, Fe, formaldehyde
Table 1 Fluorescence profile and Ab40 binding affinities (KD)
Compound
kex (nm)
kem (nm)
Fold (FAb/F0)a
KD (mean SD) (lM)b
Log Pcoct
1
2
3
4
5
6
a
340
340
340
350
350
350
400
410
410
410
430
440
23.3
36.1
6.4
6.03 0.47
3.27 0.29
17.56 0.38
34.33 1.53
7.33 0.58
12.05 0.67
3.8
3.7
4.7
4.8
4.7
4.6
3.3
4.2
5.3
Fold change values were calculated using the fluorescence emission intensity at kem of each compound (10 lM compound and 10 lM Ab40
aggregates were used for the measurement)
b
KD values were obtained from three independent replicates (N = 3)
c
The numbers in parentheses were log Poct values determined by conventional octanol–buffer partitioning
Measurement of binding affinity (KD)
Results and discussion
Ab40 fibrils of 10 lM concentration solutions were titrated
by a series concentration of the compounds 1, 2, 3, 4, 5,,
and 6. The dissociation constants (KD) were measured from
Chemistry
Compounds based on uncharged ThT cause an increase in
affinity for Ab fibrils as well as greater lipophilicity, and
therefore an easier crossing of the blood–brain barrier
(BBB) can be achieved (Yona et al., 2008). The synthesis of
the ThT analogs is shown in Scheme 1, producing the
desired products with a 11–43 % yield. 2-(20-Methoxy-40-
aminophenyl)benzoxazole (1) was synthesized by the con-
densation of 2-aminophenol and 4-amino-2-methoxyben-
zoic acid at 200 °C in polyphosphoric acid. Next, 2-(20-
methoxy-40-methylaminophenyl)benzoxazole (2) was
monomethylated using the deprotonating agent, sodium
methoxide, paraformaldehyde, and the reducing agent,
sodium borohydride, in methanol. Also, 2-(20-methoxy-40-
dimethylaminophenyl)benzoxazole (3) was synthesized
starting from compound 1, which was followed by dime-
thylation using methyl iodide and potassium carbonate.
the double reciprocal of the fluorescence maximum (Fmax
and the concentration of the probe (Sutharsan et al., 2010).
)
In vitro seeding
SHSY5Y neuroblastoma cell pellets were suspended in
2 % SDS in PBS, sonicated and diluted 1:40 in PBS with
0.02 % sodium azide, and loaded into Labtek 8-well
chambers to a final SDS concentration of 0.05 %. SHSY5Y
cells, which were incubated with or without (control) 1 lM
Ab42 for 3 days at 37 °C, were homogenized. Culture wells
were washed and incubated with 10 lM of compound 2
and 10 lM of ThT for 10 min, rinsed with 50 % ethanol
and water, and cover slipped for imaging. The images were
acquired by fluorescence microscopy (Hu et al., 2009).
123

Med Chem Res
0.1
Screening against Ab plaques
0.08
0.06
0.04
0.02
0
The six synthesized ThT analogs were tested with synthetic
Ab40 aggregates for their fluorescence response. The
aggregated fibrils of synthetic Ab40, and their methods of
utilization were previously reported (Klunka et al., 2001).
We evaluated the fluorescent properties of the six com-
pounds at 10 lM concentrations in PBS before and after
mixing with aggregated Ab peptide (20 lM, aggregated in
a PBS buffer for 2 days at 37 °C). Among the six tested
compounds, compounds 1 and 2 showed a greater than
20-fold increase at 10 lM Ab40 fibrils, and were further
tested for their binding constants against Ab40. The binding
constants (KD) were measured using 10 lM aggregated
Ab40 fibrils. The fluorescent intensity of each probe was
measured at concentrations of 1, 2, 5, and 10 lM mixed
with pre-aggregated Ab40 fibrils. The spectroscopic prop-
erties of the compounds are summarized in Table 1.
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
(concentration of probe)-1/[µM]-1
Fig. 2 Determination of the apparent binding constant (KD) of probes
compound 2 (filled triangle R2 = 0.97) and ThT (filled hexagon
R2 = 0.97) to pre-aggregated Ab fibrils
Subsequently, 2-(20-methoxy-40-aminophenyl)benzothia-
zole (4) was prepared through the conjugation of 2-amino-
thiophenol and 4-amino-2-methoxybenzoic acid in phos-
phorus oxychloride. Next, 2-(20-methoxy-40-methylamin-
ophenyl)benzothiazole (5) was monomethylated using a
deprotonating agent sodium methoxide, paraformaldehyde,
and a reducing agent, sodium borohydride, in methanol. In
addition, 2-(20-methoxy-40-dimethylaminophenyl)>benzo-
thiazole (6) was synthesized starting from compound 4,
which was followed by dimethylation using formaldehyde in
the presence of sulfuric acid and powdered iron.
Promisingly, compound 2 meets the following two crit-
ical requirements for a fluorescence imaging probe: high
fluorescence responsiveness (FAb/F0 = 36.1) and a strong
binding affinity (KD = 3.27 0.29 lM). Compared with
the conventional Ab probes, compound 2 exhibited stronger
bindings than ThT (FAb/F0 = 33.1, KD = 4.12 0.47) in
our measurement. A double reciprocal plot of the fluores-
cence intensity versus the concentration of probes in com-
pound 2 and ThT is shown in Fig. 2. The KD corresponds to
the -1/(x-intercept) of the linear regression. Table 1 also
Fig. 3 Cell extracts from Ab42
-
loaded cells seed the formation
of amyloid fibrils. Extracts from
control cells (grown in the
absence of Ab42) did not exhibit
compound 2 (a) or ThT staining
(c); Ab42-loaded cell extracts
developed compound 2
precipitates (b) which stained
for ThT (d)
123

Med Chem Res
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´
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Sutharsan J, Dakanali M, Capule CC, Haidekker MA, Yang J,
Theodorakis EA (2010) Rational design of amyloid binding
agents based on the molecular rotor motif. Chem Med Chem
5(1):56–60
Encouraged by these predominant properties, we further
tested the applicability of compound 2 to the fluorescence
imaging of intracellular aggregated Ab42 fibrils using
SHSY5Y cells. To determine the intracellular uptake
aggregation of Ab42, SHSY5Y cells, which were incubated
with or without 1 lM Ab42 for 3 days, were homogenized
and subsequently incubated with 10 lM of compound 2 for
10 min. Ab42-loaded cell extracts induced the appearance
of fluorescent precipitates that were visible by microscopy
(Fig. 3b), while extracts from cells grown in the absence of
Ab42 (Fig. 3a) did not induce the appearance of fluorescent
precipitates. These same precipitates were stained with
ThT suggesting the formation of fibrils (Fig. 3d). Again,
extracts from cells grown in the absence of Ab42 (Fig. 3c)
failed to develop fluorescent staining. These results suggest
that compound 2 can be a useful marker of intercellular
aggregated Ab.
Conclusions
We synthesized six thioflavin-T analogs of the Ab plaque
ligand, and we identified 2-(20-Methoxy-40-methyl-amino-
phenyl)benzoxazole (compound 2) which exhibited a
strong fluorescence response (FAb/F0 = 36.1) and binding
affinity (KD = 3.27 0.29 lM) to Ab40 aggregates and
SHSY5Y neuroblastoma cells. Therefore, compound 2 may
be a good alternative candidate as a fluorescence imaging
agent for studying Alzheimer’s disease.
´
´
Takacs-Novak K, Avdeef A (1996) Interlaboratory study of log P
determination by shake-flask and potentiometric methods.
J Pharm Biomed Anal 14:1405–1413
Xia W (2003) Amyloid inhibitors and Alzheimer’s disease. Curr Opin
Investig Drugs 4(1):55–59
Acknowledgments This research was supported by Nuclear R&D
Program through the Korean Ministry of Education, Science and
Technology.
`
Yona RL, Mazeres S, Faller P, Gras E (2008) Thioflavin derivatives
as markers for amyloid-b fibrils: insights into structural features
important for high-affinity binding. Chem Med Chem 3:63–66
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