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M. Cui et al. / Bioorg. Med. Chem. 19 (2011) 4148–4153
0.9 Hz, 2H), 4.25 (s, 2H). HRMS (EI): m/z calcd for C15H10BrNO2
314.9889; found 314.9894.
4.36 (s, 1H), 2.95 (s, 3H), 1.82–0.57 (m, 27H). MS (APCI): m/z calcd
for C28H39NO2Sn 541.20; found 542.30 (M+H+).
4.6. (4-Aminophenyl)(5-iodobenzofuran-2-yl)methanone (6)
4.12. (4-(Dimethylamino)phenyl)(5-(tributylstannyl)benzofu-
ran-2-yl)methanone (12)
The reaction described for 5 was used, and 6 was obtained in a
yield of 81%. 1H NMR (400 MHz, CD3OD) d 8.15 (d, J = 1.5 Hz, 1H),
7.94 (d, J = 8.8 Hz, 2H), 7.77 (dd, J = 8.7, 1.8 Hz, 1H), 7.51 (d,
J = 0.9 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 6.72 (d, J = 8.8 Hz, 2H),
4.85 (s, 2H). HRMS (EI): m/z calcd for C15H10INO2 362.9752; found
362.9756.
The reaction described for 11 was used, and 12 was obtained in
a yield of 29%. 1H NMR (400 MHz, CDCL3) d 8.11 (d, J = 8.8 Hz, 2H),
7.80 (s, 1H), 7.61 (d, J = 8.1 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.47
(s, 1H), 6.74 (d, J = 8.9 Hz, 2H), 3.10 (s, 6H), 1.69–0.76 (m, 27H).
MS (APCI): m/z calcd for C29H41NO2Sn 555.22; found 556.30
(M+H+).
4.7. (5-Bromobenzofuran-2-yl)(4-(methylamino)phenyl)meth-
anone (7)
4.13. Radiolabeling
To a solution of 5 (315 mg, 1 mmol) in acetone (15 mL) was
added CH3I (0.18 mL, 3 mmol) and anhydrous K2CO3 (138 mg,
1 mmol). The reaction mixture was stirred at room temperature
for 12 h and poured into water. The mixture was extracted with
ethyl acetate. The organic layers were combined and dried over
Na2SO4. Evaporation of the solvent afforded a residue, which was
purified by silica gel chromatography to give 108 mg of 7 (33%).
1H NMR (400 MHz, CDCL3) d 8.06 (d, J = 9.0 Hz, 2H), 7.85 (dd,
J = 1.9, 0.5 Hz, 1H), 7.55 (dd, J = 8.8, 1.9 Hz, 1H), 7.52–7.48 (m,
1H), 7.41 (d, J = 0.9 Hz, 1H), 6.65 (d, J = 8.9 Hz, 2H), 4.49 (s, 1H),
2.95 (s, 3H). HRMS (EI): m/z calcd for C16H12BrNO2 329.0042; found
329.0051.
The radioiodinated ligands [125I]8 and [125I]10 were prepared
from the corresponding tributyltin precursor through an iododest-
annylation reaction according to a procedure described previously
with some modifications.17 Briefly, 50
l
L of H2O2 (3%) was added
to a mixture of a tributyltin derivative (0.1 mg/100 L in ethanol),
200
Ci of sodium [125I]iodide (specific activity 2200 Ci/mmol),
and 100 L of 1 M HCl in a sealed vial. The reaction was allowed
to proceed at room temperature for 15 min and then quenched with
the addition of 50 L of a saturated NaHSO3 solution. The reaction
l
l
l
l
mixture was extracted with ethyl acetate (3 ꢀ 1 mL) after neutral-
ization with 10 mg of sodium bicarbonate. The combined extracts
were evaporated dry. The residues were dissolved in 100
lL of EtOH
and purified by HPLC using 5C18-AR-II analytical column
a
4.8. (5-Iodobenzofuran-2-yl)(4-(methylamino)phenyl)metha-
none (8)
(4.6 ꢀ 150 mm), CH3CN/H2O = 3/1, at a flow rate of 1.0 mL/min.
The desired fractions containing the product were evaporated dry
and redissolved in 100% ethanol. Finally, the radiochemical identity
of [125I]8 and [125I]10 was verified by co-injection with nonradioac-
tive compounds by HPLC. The final product was stored at ꢁ20 °C
until use for autoradiography and biodistribution experiments.
The reaction described for 7 was used, and 8 was obtained in a
yield of 41%. 1H NMR (400 MHz, CDCL3) d 8.03 (d, J = 1.8 Hz, 1H),
8.03 (d, J = 8.9 Hz, 2H), 7.70 (dd, J = 8.8, 1.2 Hz, 1H), 7.44–7.29 (m,
2H), 6.63 (d, J = 8.9 Hz, 2H), 2.92 (s, 3H). HRMS (EI): m/z calcd for
C16H12INO2 376.9904; found 376.9913.
4.14. Binding assay in vitro using Ab1–42 aggregates
4.9. (5-Bromobenzofuran-2-yl)(4-(dimethylamino)phenyl)meth
anone (9)
Inhibition experiments were carried out in 12 ꢀ 75 mm borosil-
icate glass tubes according to a procedure described previously
with some modifications.22 One hundred microliters of aggregated
Ab fibrils (60 nM in the final assay mixture) was added to a mixture
The reaction described for 7 was used, and 9 was obtained in a
yield of 26%.1H NMR (400 MHz, CDCL3) d 8.10 (d, J = 9.2 Hz, 2H),
7.85 (d, J = 1.7 Hz, 1H), 7.54 (dd, J = 8.8, 1.7 Hz, 1H), 7.50 (d,
J = 8.8 Hz, 1H), 7.41 (d, J = 0.8 Hz, 1H), 6.74 (d, J = 9.1 Hz, 2H),
3.11 (s, 6H). HRMS (EI): m/z calcd for C17H14BrNO2 343.0200; found
343.0207.
containing 100
concentration, 10
790 L of PBS (0.2 M, pH 7.4) in a final volume of 1 mL. Nonspecific
binding was defined in the presence of 1 M IMPY. The mixture
l
L of radioligand ([125I]IMPY) of the appropriate
l
L of inhibitor (10ꢁ5–10ꢁ10 M in ethanol), and
l
l
was incubated for 2 h at 37 °C with constant shaking, then the
bound and free radioactivity were separated by vacuum filtration
through borosilicate glass fiber filters (Whatman GF/B) using a cell
harvester (Brandel, M-24 Gaithersburg, MD, USA). Filters contain-
ing the bound 125I ligand were measured for radioactivity in a
4.10. (4-(Dimethylamino)phenyl)(5-iodobenzofuran-2-
yl)methanone (10)
The reaction described for 7 was used, and 10 was obtained in a
yield of 37%. 1H NMR (400 MHz, CDCL3) d 8.10 (d, J = 9.0 Hz, 2H),
8.05 (s, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.40 (d, J = 8.7 Hz, 1H), 7.39
(s, 1H), 6.74 (d, J = 9.1 Hz, 2H), 3.11 (s, 6H). HRMS (EI): m/z calcd
for C17H14INO2 391.0061; found 391.0069.
c-counter (WALLAC/Wizard 1470, USA) with 70% counting effi-
ciency. Under the assay conditions, the specifically bound fraction
accounted for about 10% of all the radioactivity. The half maximal
inhibitory concentration (IC50) was determined using GRAPHPAD PRISM
4.0, the inhibition constant (Ki) was calculated using the Cheng-
Prusoff equation: Ki = IC50/(1 + [L]/Kd).23
4.11. (4-(Methylamino)phenyl)(5-(tributylstannyl)benzofuran-
2-yl)methanone (11)
4.15. Autoradiography in vitro using AD transgenic mouse brain
sections
A mixture of 7 (165 mg, 0.5 mmol), (Bu3Sn)2 (0.5 mL), and
(Ph3P)4Pd (60 mg, 0.04 mmol) in a mixed solvent (10 mL, 4:1 diox-
ane/Et3N) was stirred under reflux for 10 h. The solvent was re-
moved, and the residue was purified by silica gel
chromatography to give 48 mg of 11 (18%). 1H NMR (400 MHz,
CDCL3) d 8.07 (d, J = 8.6 Hz, 2H), 7.79 (s, 1H), 7.61 (d, J = 8.1 Hz,
1H), 7.52 (d, J = 8.1 Hz, 1H), 7.47 (s, 1H), 6.64 (d, J = 8.6 Hz, 2H),
Paraffin-embedded brain sections of AD model mice (C57, APP/
PS1, 12 months) were used for autoradiography. The sections were
deparaffinized with 2 ꢀ 20 min washes in xylene; 2 ꢀ 5 min
washes in 100% ethanol; a 5 min wash in 90% ethanol/H2O; a
5 min wash in 80% ethanol/H2O; a 5 min wash in 60% ethanol/
H2O and a 10 min wash in running tap water, and then incubated