Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7543
11i (3.24 g, 82%) as white crystals, mp 80-83 °C. 1H NMR
(DMSO-d6) δ 1.15 (t, 3 H, J ) 7.3 Hz), 1.22-1.32 (m, 8 H), 1.46-
1.55 (m, 4 H), 2.25 (t, 2 H, J ) 7.4 Hz), 3.23 (app q, 2 H, J ) 6.6
Hz), 3.69 (s, 3 H), 3.81 (s, 6 H), 4.03 (q, 2 H, J ) 7.1 Hz), 7.15
(s, 2 H), 8.33 (t, 1 H, J ) 5.7 Hz); 13C NMR (DMSO-d6) δ 14.24,
24.57, 26.56, 28.51, 28.73, 28.74, 29.29, 33.64, 39.40, 56.13, 59.72,
60.17, 104.90, 130.04, 139.97, 152.65, 165.52, 172.99. Anal.
(C21H33NO6) C, H, N.
Acknowledgment. This work was supported by the Deutsche
Forschungsgemeinschaft, Graduiertenkolleg 677.
Supporting Information Available: Detailed experimental and
analytical data including spectral assignments for 1-8, 11a-h,
12a-f, 13a-h, 14a-f, 15a-h, 16a-f, and AS-1397; kinetic data
for inhibition of acetylcholinesterase from Electrophorus electricus
by compound 15h; and elemental analysis results. This material is
N-(9-Hydrazino-9-oxononyl)-3,4,5-trimethoxybenzamide (13i).
Ethyl 9-((3,4,5-trimethoxybenzoyl)amino)nonanoate 11i (1.98 g, 5.0
mmol) was added to a mixture of hydrazine hydrate (100%, 2.5
mL, 51.4 mmol) and absolute ethanol (20 mL), and the solution
was refluxed for about 24 h. The course of the reaction was followed
by TLC (toluene/acetone/methanol, 7:2:1). Once no more ester
could be detected, the solvent and excess hydrazine were removed
by evaporation. The crude product was recrystallized from ethanol
to obtain 13i (1.46 g, 77%) as white crystals, mp 133-136 °C. 1H
NMR (DMSO-d6) δ 1.17-1.33 (m, 8 H), 1.42-1.54 (m, 4 H), 1.98
(t, 2 H, J ) 7.4 Hz), 3.22 (app q, 2 H, J ) 6.6 Hz), 3.68 (s, 3 H),
3.81 (s, 6 H), 4.12 (bs, 2 H), 7.15 (s, 2 H), 8.36 (t, 1 H, J ) 5.5
Hz), 8.88 (s, 1 H); 13C NMR (DMSO-d6) δ 25.38, 26.67, 28.80,
28.87, 28.88, 29.38, 33.58, 39.46, 56.15, 60.22, 104.86, 130.06,
139.93, 152.69, 165.56, 171.76. Anal. (C19H31N3O5) C, H. N: calcd
11.02, found 10.39.
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3,4,5-Trimethoxy-N-(9-oxo-9-(2-(1,2,3,4-tetrahydroacridin-9-
yl)hydrazino)nonyl)benzamide (15i). 9-Chloro-1,2,3,4-tetrahy-
droacridine (8) (0.44 g, 2.0 mmol) and N-(9-hydrazino-9-oxononyl)-
3,4,5-trimethoxybenzamide (13i) (0.76 g, 2.0 mmol), dissolved in
absolute ethanol (20 mL), were heated to 140 °C for 24 h in a
sealed tube. Cooling to room temperature and evaporation yielded
a crude product that was taken up in ethanol (5 mL) and diluted
with water (20 mL). Addition of 1 M sodium hydroxide solution
(2.5 mL) liberated the base as an oily layer. After decantation, the
oily residue was suspended in ethyl acetate (25 mL) and shortly
refluxed until the final product precipitated. Compound 15i (0.61
1
g, 54%) was obtained as a yellow powder, mp 162-165 °C. H
NMR (DMSO-d6) δ 1.09-1.33 (m, 8 H), 1.38-1.56 (m, 4 H),
1.73-1.88 (m, 4 H), 2.07 (t, 2 H, J ) 7.1 Hz), 2.82 (bs, 2 H), 2.90
(bs, 2 H), 3.22 (app q, 2 H, J ) 6.2 Hz), 3.69 (s, 3 H), 3.81 (s, 6
H), 7.16 (s, 2 H), 7.30 (app t, 1 H, J ) 7.3 Hz), 7.51 (app t, 1 H,
J ) 7.4 Hz), 7.64 (s, 1 H), 7.70 (app d, 1 H, J ) 8.2 Hz), 8.31
(app d, 1 H, J ) 8.5 Hz), 8.34 (t, 1 H, J ) 4.7 Hz), 10.02 (s, 1 H);
13C NMR (DMSO-d6) δ 22.54, 22.71, 24.84, 25.02, 26.61, 28.65,
28.82, 29.34, 33.06, 33.78, 39.42, 56.15, 60.18, 104.91, 115.60,
119.12, 123.21, 123.44, 127.93, 128.41, 130.04, 139.98, 146.82,
148.83, 152.66, 158.13, 165.54, 172.06. Anal. (C32H42N4O5‚0.5H2O)
H, N. C: calcd 67.23, found 67.83.
Cholinesterase Inhibition. Acetylcholinesterase from Electro-
phorus electricus (G4, soluble tetramer) was purchased from Fluka
(Deisenhofen, Germany). Human acetylcholinesterase (recombinant,
expressed in HEK 293 cells, G2/G4) was from Sigma (Steinheim,
Germany) and butyrylcholinesterase (human plasma, G4) from Lee
Biosolutions (St. Louis, MO). Acetylcholinesterase from Torpedo
californica (recombinant, expressed in Pichia pastoris, G4) was a
kind gift from R. Gallitzendo¨rfer, Institute for Physiological
Chemistry, University of Bonn, Germany. Cholinesterase inhibition
was assayed spectrophotometrically at 412 nm in 100 mM sodium
phosphate buffer, 100 mM NaCl, pH 7.3. Acetyl- or butyrylthio-
choline (10 mM) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)
(7 mM) were dissolved in assay buffer. Stock solutions of the
inhibitors were prepared in a 1:1 mixture of acetonitrile and 0.1 M
HCl. IC50 values were determined in duplicate on a minimum of at
least four different inhibitor concentrations. Into a cuvette containing
825 µL of assay buffer, were added 50 µL of the DTNB solution,
55 µL of acetonitrile, 10 µL of an inhibitor solution, and 10 µL of
a cholinesterase solution (∼3 U/mL), and the mixture was
thoroughly mixed. After incubation for 15 min at 25 °C, the reaction
was initiated by adding 50 µL of the acetyl- or butyrylthiocholine
solution.
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