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S.-Y. Chen et al. / Bioorg. Med. Chem. 19 (2011) 5596–5604
(d, J = 8.9 Hz, 4H), 3.70 (s, 4H), 3.29 (t, J = 8.0 Hz, 8H), 2.51 (t,
J = 8.0 Hz, 8H), 2.39 (s, 3H), 2.27 (s, 6H). 13C NMR (100 MHz, CDCl3)
d 185.69, 150.19, 135.19, 131.26, 129.39, 125.01, 113.68, 56.26,
53.82, 46.76, 45.09, 44.81. ESI-HRMS m/z: calcd for C30H39N5O
[M+H]+ 486.3233, found 486.3225.
7.7 Hz, 2H), 7.73 (d, J = 12.8 Hz, 2H), 7.61–7.44 (m, 5H), 7.35 (d,
J = 15.5 Hz, 2H), 6.85 (d, J = 11.4 Hz, 4H), 3.28 (t, J = 8.0 Hz, 8H),
2.51 (t, 8H), 2.29 (s, 6H). 13C NMR (100 MHz, CDCl3) d 188.91,
151.78, 144.88, 138.12, 130.94, 129.39, 127.82, 127.02, 124.05,
116.89, 113.73, 53.74, 46.57, 45.07. ESI-HRMS m/z: calcd for
C
34H38N4O2 [M+H]+ 535.3073, found 535.3068.
4.2.6. (1E,6E)-1,7-Bis(4-(4-methylpiperazin-1-yl)phenyl)hepta-
1,6-diene-3,5-dione (A6)
4.2.10. (2E,20E)-1,10-(1,4-Phenylene)bis(3-(4-(4-methylpipera-
zin-1-yl)phenyl)prop-2-en-1-one) (A10)
Compound A6 was prepared according to our previous Letter.36
Boric anhydride (87 mg, 1.25 mmol) was suspended in 10 mL
EtOAc in the presence of acetylacetone (251 mg, 2.5 mmol), and
the mixture was stirred for 3 h at 70 °C. After evaporation of the
solvent, the residue was washed with hexane and then, 5 mL
EtOAc, 4-(4-methylpiperazin-1-yl) benzaldehyde (1.02 g, 5 mmol),
and tributylborate (1.15 g, 5 mmol) were added, and the mixture
was stirred for further 30 min. Butylamine (18 mg, 0.25 mmol) dis-
solved in EtOAc was added drop wise. The mixture acted at 70 °C
for 24 h. Then 1 N HCl was added to adjust the pH to 5, and the
solution was heated for 30 min at 60 °C. EtOAc was used to extract
the product from the water layer. The compound A6 was purified
by recrystalization from EtOAc to yield yellow crystals (595 mg,
50%); mp: 214–216 °C. 1H NMR (400 MHz, CDCl3) d 7.52 (d,
J = 15.8 Hz, 2H), 7.41 (d, J = 13.2 Hz, 4H), 6.83 (d, J = 8.0 Hz, 4H),
6.39 (d, J = 15.7 Hz, 2H), 5.69 (s, 1H), 3.26 (t, J = 8.0 Hz, 8H), 2.51
(t, J = 8.0 Hz, 8H), 2.30 (s, 6H). 13C NMR (100 MHz, CDCl3) d
182.30, 151.24, 139.22, 128.60, 124.69, 119.60, 113.94, 100.11,
53.83, 46.82, 45.12. ESI-HRMS m/z: calcd for C29H34N4O2 [M+H]+
473.2917, found 473.2911.
Following the method described for production of A1, a red so-
lid A10 (184 mg, 69%) was obtained; mp: 224–226 °C. 1H NMR
(400 MHz, CDCl3) d 8.01 (s, 4H), 7.72 (d, J = 15.6 Hz, 2H), 7.50 (d,
J = 8.8 Hz, 4H), 7.30 (d, J = 15.6 Hz, 2H), 6.84 (d, J = 8.9 Hz, 4H),
3.30 (t, J = 8.0 Hz, 8H), 2.53 (t, J = 8.0 Hz, 8H), 2.31 (s, 6H). 13C
NMR (100 MHz, CDCl3) d 189.23, 151.80, 145.00, 140.66, 129.36,
127.45, 124.08, 113.79, 53.72, 46.55, 45.02. ESI-HRMS m/z: calcd
for C34H38N4O2 [M+H]+ 535.3073, found 535.3071.
4.2.11. (3E,5E)-3,5-Bis(4-(4-piperidin-1-yl)benzylidene)piper-
idin-4-one (B4)
Following the method described for production of A4, a red so-
lid B4 (174 mg, 32%) was obtained; mp: 208–210 °C. 1H NMR
(400 MHz, CDCl3) d 7.67 (s, 2H), 7.25 (d, J = 8.8 Hz, 4H), 6.83 (d,
J = 8.8 Hz, 4H), 4.10 (s, 4H), 3.26–3.17 (t, J = 8.0 Hz, 8H), 1.69–
1.50 (m, 12H). 13C NMR (100 MHz, CDCl3) d 186.47, 150.81,
135.03, 131.48, 130.55, 124.05, 113.65, 48.10, 47.22, 24.47, 23.32.
ESI-HRMS m/z: calcd for C29H35N3O [M+H]+ 442.2858, found
442.2864.
4.2.7. 1,3-Bis(4-(4-methylpiperazin-1-yl)styryl)benzene (A7)
The mixture of m-xylylene dibromide (625 mg, 2.5 mmol) and
triethyl phosphate (1 mL, 5 mmol) was refluxed at 120 °C for 2 h.
The reaction mixture was cooled to room temperature and then
eluted with THF (50 mL), adding 570 mg (5 mmol) potassium
tert-butoxide at 0 °C. After stirring for 20 min, 4-(4-methylpipera-
zin-1-yl)benzaldehyde (1.02 g, 5 mmol) was added and the reac-
tion mixture was stirred for 30 min at room temperature. After
slow addition of chilled water and stirred at room temperature
for 30 min, the mixture was extracted with 30 mL ethylacetate
twice. The combined organic layer was washed with saturated
NaHCO3 solution and brine, dried over MgSO4, and concentrated
to dryness to give a light yellow solid (199 mg, 17%); mp: 218–
4.3. Biological assay
4.3.1. ThT assay
Experiments were performed by incubating the peptides in
10 mM phosphate buffer (pH 7.4) at 37 °C for 48 h (final Ab1–42
20
centrations (2, 5, 10, 20, 50
were diluted to a final volume of 180
buffer (pH 8.5) containing 5 M Thioflavin T. Fluorescence signal
l
M) with and without the tested compounds at different con-
M). After incubation, the samples
L with 50 mM glycine-NaOH
l
l
l
was measured (excitation wavelength 450 nm, emission wave-
length 485 nm and slit widths set to 5 nm) on a monochromators
based multimode microplate reader (INFINITE M1000), adapted
for 96-well microtiter plates. The fluorescence intensities were re-
corded, and the percentage of inhibition on aggregation was calcu-
lated by the following expression: (1ꢁIFi/IFc) ꢀ 100% in which IFi
and IFc were the fluorescence intensities obtained for absorbance
in the presence and absence of inhibitors, respectively, after sub-
220 °C. 1H NMR (400 MHz, CDCl3)
d 7.51 (s, 1H), 7.37 (d,
J = 8.7 Hz, 4H), 7.30–7.20 (m, 3H), 7.01 (d, J = 16.3 Hz, 2H), 6.90
(d, J = 16.3 Hz, 2H), 6.85 (d, J = 8.8 Hz, 4H), 3.23 (t, J = 8.0 Hz, 8H),
2.59 (t, J = 8.0 Hz, 8H), 2.34 (s, 6H). 13C NMR (100 MHz, CDCl3) d
149.61, 137.13, 127.84, 127.75, 127.54, 126.48, 124.80, 123.86,
123.09, 114.80, 53.94, 47.60, 45.03. ESI-HRMS m/z: calcd for
tracting the background fluorescence of the 5
solution.
lM Thioflavin T
C
32H38N4 [M+H]+ 479.3175, found 479.3183.
4.3.2. CD assay
4.2.8. 2,6-Bis(4-(4-methylpiperazin-1-yl)styryl)pyridine (A8)
Following the method described for production of A7, a light
yellow solid A8 (102 mg, 8%) was obtained; mp: 212–214 °C. 1H
NMR (400 MHz, CDCl3) d 7.64–7.55 (m, 3H), 7.52 (d, J = 8.7 Hz,
4H), 7.20 (d, J = 16.1 Hz, 2H), 7.06 (d, J = 16.1 Hz, 2H), 6.92 (d, J =
8.8 Hz, 4H), 3.29 (t, J = 8.0 Hz, 8H), 2.60 (t, J = 8.0 Hz, 8H), 2.37 (s,
6H). 13C NMR (100 MHz, CDCl3) d 155.90, 151.02, 136.64, 132.45,
128.19, 128.06, 125.51, 119.30, 115.52, 54.98, 50.76, 48.44, 46.09.
Ab1–42 (20 lM) was mixed with and without 10 lM A4 in
10 mM sodium phosphate buffer (pH 7.4). All solutions were incu-
bated at 37 °C. CD spectra were obtained using a Jasco-810-150S
spectropolarimeter (Jasco, Japan). A quartz cell with 1 mm optical
path was used. Spectra were recorded at 25 °C between 190 and
260 nm with a bandwidth of 0.5 nm, a 3 s response time, and scan
speed of 10 nm/min. Background spectra and when applicable,
spectra of A4 were subtracted.
ESI-HRMS m/z: calcd for
C
31H37N5 [M+H]+ 480.3127, found
480.3121.
4.3.3. EM study
Ab1–42 peptide (Anaspec Inc.) was dissolved in 10 mM phos-
4.2.9. (2E,20E)-1,10-(1,3-Phenylene)bis(3-(4-(4-methylpiperazin-
1-yl)phenyl)prop-2-en-1-one) (A9)
Following the method described for production of A1, a yellow
solid A9 (154 mg, 57%) was obtained; mp: 216–218 °C. 1H NMR
(400 MHz, CDCl3) d 8.62–8.46 (m, 1H), 8.10 (dd, J = 15.6 Hz,
phate buffer (pH 7.4) at 4 °C to give an 80
was incubated in the presence and absence of A4 in 37 °C. The fi-
nal concentrations of and A4 were 40 M and 20 M, respec-
tively. At specified time points, aliquots of 10 L samples were
placed on carbon-coated copper/rhodium grid. After 1 min, the
lM solution. Ab1–42
l
l
l