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M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 7002–7007
4.1.2. N,N-Dimethyl-1,3-benzothiazol-6-amine (2)
2H), 7.85 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.50 (d,
J = 16.4 Hz, 1H), 7.38 (d, J = 16.4 Hz, 1H), 7.07 (d, J = 2.4 Hz, 1H),
6.96 (dd, J = 8.8, 2.4 Hz, 1H), 3.06 (s, 6H). MS m/z 309 [MH+].
A solution of 1 (1.47 g, 9.8 mmol) in THF (40 mL) was slowly
added to
a stirred mixture of 40% aqueous formaldehyde
(7.24 mL, 98 mmol) and 4 M H2SO4 (7.95 mL, 29.4 mL). Powdered
iron (4.36 g, 78.4 mL) was then added and the mixture was vigor-
ously stirred for 3 h. The precipitate of iron salts was removed by
filtration and washed with EtOAc (20 mL Â 2). The combined or-
ganic solutions were made strongly basic with 1 N NaOH (50 mL)
and extracted with EtOAc (50 mL Â 2). The combined EtOAc ex-
tracts were dried (Na2SO4) and the solvent was removed on a ro-
tary vacuum evaporator. The oily residue was purified by silica
4.1.7. 4-((E)-2-(6-(Dimethylamino)-1,3-benzothiazol-2-yl)
vinyl)benzylidene)malononitrile (PP-BTA-2, 7)
The same reaction as described above to prepare 5 was used,
and 45 mg of 7 was obtained in a 63.5% yield from 6. 1H NMR
(400 MHz, CDCl3) d 7.94 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.8 Hz, 1H),
7.73 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 16.4 Hz, 1H), 7.35
(d, J = 16.4 Hz, 1H), 7.08 (s, 1H), 6.97 (d, J = 10.0 Hz , 1H), 3.08 (s,
6H). MS m/z 357 [MH+]. Anal. Calcd for C21H16N4S: C, 70.76; H,
4.52; N, 15.72; S, 9.00. Found: C, 70.48; H, 4.57; N, 15.43; S, 8.99.
gel chromatography (hexane/EtOAc = 4:1) to give
2 (460 mg,
26.3%). 1H NMR (400 MHz, CDCl3)
d
8.67 (s, 1H), 7.95 (d,
J = 8.8 Hz, 1H), 7.15 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.8, 2.4 Hz,
1H), 3.04 (s, 6H). MS m/z 179 [MH+].
4.2. Fluorescence experiments
4.1.3. 6-(Dimethylamino)-1,3-benzothiazole-2-carbaldehyde (3)
To a vigorously stirred solution of n-BuLi (0.5 mL, 2.6 M in hex-
ane, 1.3 mmol) in THF (5.8 mL) at À78 °C under N2 was added
slowly a solution of 2 (220 mg, 1.23 mmol). The reaction mixture
was stirred, warmed to À50 °C and after 1 h cooled to À78 °C. To
the resulting solution of aryllithium salt was added slowly anhy-
drous DMF (0.38 mL). The solution was stirred for 2 h, poured into
H2O (9 mL), neutralized with an aqueous saturated solution of
NH4Cl and subsequently extracted with EtOAc (20 mL Â 2). The
combined extracts were dried over Na2SO4 and the solvent was re-
PP-BTA-1 and PP-BTA-2 were dissolved in 5% EtOH at 10 lM.
The fluorescence of PP-BTA-1 and PP-BTA-2 was measured with a
spectrophotometer (RF-1500, Shimadzu, Japan). For some mea-
surements, the spectra of PP-BTA-1 and PP-BTA-2 were determined
with or without Ab(1-42) aggregates (0, 5 and 10 lM).
4.3. Binding experiments using Ab(1-42) aggregates
A solid form of Ab(1-42) was purchased from Peptide Institute
(Osaka, Japan). Aggregation was carried out by gently dissolving
the peptide (0.25 mg/mL) in a buffer solution (pH 7.4) containing
10 mM sodium phosphate and 1 mM EDTA. The solution was incu-
bated at 37 °C for 42 h with gentle and constant shaking. Thiofla-
vin-T was used as the tracer for the competition binding
experiments. A mixture (3.6 mL of 10% EtOH) containing PP-BTA-
moved under vacuum to give
3
(255 mg, 97.3%). 1H NMR
(400 MHz, CDCl3) d 10.06 (s, 1H), 8.03 (d, J = 10.0 Hz, 1H), 7.07–
7.04 (m, 2H), 3.12 (s, 6H). MS m/z 207 [MH+].
4.1.4. ((6-(Dimethylamino)-1,3-benzothiazol-2-yl)methylene)-
malononitrile (PP-BTA-1, 4)
1, PP-BTA-2 and PIB (final concn 61.1 nMÀ5.48
l
M), thioflavin-T
A solution of 3 (124 mg, 0.6 mmol), malononitrile (60 mg,
0.9 mmol) and pyridine (0.12 mL) in 2-propanol (7.2 mL) was stir-
red and refluxed for 30 min. The mixture was poured into H2O
(20 mL) and extracted with CHCl3 (20 mL Â 3). The combined ex-
tracts were dried over Na2SO4 and the solvent was removed under
vacuum to give 4 (152 mg, 91.7%). 1H NMR (400 MHz, CDCl3) d 7.99
(s, 1H), 7.99 (d, J = 9.2 Hz, 1H), 7.08 (dd, J = 9.2, 2.4 Hz, 1H), 7.02 (d,
J = 2.4 Hz, 1H), 3.16 (s, 6H). MS m/z 255 [MH+]. Anal. Calcd for
C13H10N4S: C, 61.40; H, 3.96; N, 22.03; S, 12.61. Found: C, 61.34;
H, 3.84; N, 21.82; S, 12.64.
(final concn 3 M), and Ab(1-42) aggregates (final concn 10 l
l
g/
mL) was incubated at room temperature for 10 min. Fluorescence
intensity at an excitation wavelength of 445 nm was plotted, and
values for the apparent half-maximal inhibitory concentration
(IC50) were determined from a calibration curve of fluorescence
intensity at 478 nm in three independent experiments.
4.4. Staining of Ab plaques in Tg2576 mouse brain sections
Theexperimentswithanimalswereconductedinaccordancewith
our institutional guidelines and approved by the Kyoto University
Animal Care Committee. The Tg2576 transgenic mice (female, 27-
month-old) and wild-type mice (female, 27-month-old) were used
as the Alzheimer’s model and control mice, respectively. After the
miceweresacrificedbydecapitation, thebrainswereimmediatelyre-
moved and frozen in powdered dry ice. The frozen blocks were sliced
4.1.5. 4-((E)-2-(6-(Dimethylamino)-1,3-benzothiazol-2-yl)
vinyl)benzonitrile (5)
To
a solution of (4-cyanobenzyl)phosphonate (403.6 mg,
1.6 mmol) in MeOH (12.8 mL) was added NaOMe (0.632 mL). The
mixture was cooled in an ice bath, and stirred under reflux for
3 h after the addition of 3 (330 mg, 1.6 mmol). The solid that
formed in the reaction mixture was filtered to give 5 (385 mg,
78.8%). 1H NMR (400 MHz, CDCl3) d 7.84 (d, J = 9.6 Hz, 1H), 7.64
(dd, J = 21.2, 8.0 Hz, 4H), 7.45 (d, J = 16.4 Hz, 1H), 7.32 (d,
J = 16.4 Hz, 1H), 7.06 (d, J = 2.8 Hz, 1H), 6.95 (dd, J = 9.6, 2.8 Hz,
1H), 3.06 (s, 6H). MS m/z 306 [MH+].
into serial sections, 10
lm thick. Each slide was incubated with a 50%
EtOH solution (100 M) of PP-BTA-1 and PP-BTA-2 for 10 min. The
l
sectionswerewashedin50%EtOHfor1 mintwotimes,andexamined
using a microscope(Nikon Eclipse 80i) equippedwith a G-2Afilter set
(excitation, 510–560 nm; diachronic mirror, 575 nm; longpass filter,
470 nm) for PP-BTA-1, and a B-2A filter set (excitation, 450–480 nm;
diachronic mirror, 505 nm; longpass filter, 520 nm) for PP-BTA-2.
Thereafter, the serial sections were also stained with thioflavin S, a
pathological dye commonly used for staining Ab plaques in the brain,
andexaminedusingamicroscope(NikonEclipse80i)equippedwitha
BV-2A filter set (excitation, 400–440 nm; diachronic mirror, 455 nm;
longpass filter, 470 nm).
4.1.6. 4-((E)-2-(6-(Dimethylamino)-1,3-benzothiazol-2-yl)
vinyl)benzaldehyde (6)
To a solution of 5 (61 mg, 0.2 mmol) in THF (3.3 mL) was added
DIBAL-H (1 M in hexane, 0.5 mL) at À78 °C. The reaction mixture
was stirred at room temperature overnight. Thereafter, 10% acetic
acid (15 mL) was added and the mixture was extracted with CHCl3
(20 mL Â 2). After the organic layer was washed with saline, the
combined extracts were dried over Na2SO4. The residue was purified
by silica gel chromatography (hexane/EtOAc = 2:1) to give 6 (28 mg,
45.4%). 1H NMR (400 MHz, CDCl3) d 10.02 (s, 1H), 7.90 (d, J = 8.4 Hz,
4.5. Staining of Ab plaques in human AD brain sections
Postmortem brain tissues from an autopsy-confirmed case of AD
(73-year-old male) and a control subject (36-year-old male) were