2016 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 10
Recanatini et al.
3.02-3.19 (m, 4H), 8.31-8.32 (m, 2H, Ar), 8.90 (s, 1H, Ar).
Anal. (C13H11ClN2O2) C, H, N.
(29.0), 91 (100). Anal. (C21H22N2) C, H, N. 14: δ 1.82-1.95 (m,
4H), 2.58-2.63 (m, 2H), 3.02-3.06 (m, 2H), 4.2 (br, 1H, NH),
4.63 (s, 2H), 7.28-7.97 (m, 8H, Ar); MS m/z (relative abun-
dance) 322 (M+, 3.1), 232 (30.3), 91 (100). Anal. (C20H19ClN2)
C, H, N. 16: δ 1.85-1.94 (m, 4H), 2.57-2.78 (m, 2H), 3.03-
3.14 (m, 2H), 4.65 (s, 2H), 7.35-8.83 (m, 8H, Ar); MS m/z
(relative abundance) 333 (M+, 14.6), 304 (6.7), 91 (100). Anal.
(C20H19N3O2) C, H, N. 17: δ 0.85-0.97 (m, 3H), 1.22-1.43 (m,
8H), 1.62-1.77 (m, 2H), 1.85-1.97 (m, 4H), 2.52 (s, 3H), 2.68-
2.73 (m, 2H), 3.03-3.07 (m, 2H), 3.46-3.51 (t, 2H), 7.38-7.88
(m, 3H, Ar); MS m/z (relative abundance) 310 (M+, 58.0), 225
(100), 211 (21.1). Anal. (C21H30N2) C, H, N. 18: δ 0.88-0.94
(m, 3H), 1.20-1.35 (m, 8H), 1.92-1.98 (m, 4H), 2.25 (s, 3H),
2.58-2.70 (m, 2H), 3.09-3.19 (m, 2H), 3.47-3.54 (t, 2H), 7.13-
7.90 (m, 3H, Ar); MS m/z (relative abundance) 310 (M+, 48.3),
225 (100), 211 (31.2). Anal. (C21H30N2) C, H, N. 19: δ 0.73-
0.93 (m, 3H), 1.12-1.48 (m, 8H), 1.52-1.78 (m, 2H), 1.82-
1.98 (m, 4H), 2.53-2.70 (m, 2H), 2.98-3.03 (m, 2H), 3.91 (br,
1H, NH), 7.30-7.95 (m, 3H, Ar); MS m/z (relative abundance)
330 (M+, 56.2), 245 (100), 231 (21.9). Anal. (C20H27ClN2) C, H,
N. 20: δ 0.86-0.92 (m, 3H), 1.21-1.47 (m, 8H), 1.61-1.74 (m,
2H), 1.89-2.01 (m, 4H), 2.66-2.73 (m, 2H), 3.01-3.11 (m, 2H),
3.47-3.64 (m, 2H), 8.03-8.80 (m, 3H, Ar); MS m/z (relative
abundance) 341 (M+, 48.5), 242 (53.6), 225 (100). Anal.
(C20H23N3O2) C, H, N. 21: δ 0.83-0.97 (m, 3H), 1.15-1.38 (m,
8H), 1.57-1.70 (m, 2H), 1.83-2.03 (m, 4H), 2.60-2.70 (m, 2H),
2.98-3.04 (m, 2H), 3.40-3.57 (t, 2H), 3.91 (s, 3H), 6.94-7.90
(m, 3H, Ar); MS m/z (relative abundance) 326 (M+, 100), 241
(71.6), 227 (26.5). Anal. (C21H30N2O) C, H, N. 22: δ 0.75-0.97
(m, 3H), 1.14-1.42 (m, 8H), 1.81-1.99 (m, 4H), 2.54-2.62 (m,
2H), 2.92-3.07 (m, 2H), 4.51 (br, 1H, NH), 7.29-7.98 (m, 2H,
Ar); MS m/z (relative abundance) 314 (M+, 51.6), 229 (100),
215 (16.5). Anal. (C20H27FN2) C, H, N.
In h ibition of ACh E. The method of Ellman et al.28 was
followed. The assay solution consisted of a 0.1 M phosphate
buffer, pH 8.0, with the addition of 340 µM 5,5′-dithiobis(2-
nitrobenzoic acid) (Ellman’s reagent), 0.035 unit/mL AChE
derived from human erythrocytes, and 550 µM acetylthiocho-
line iodide. The final assay volume was 1 mL. Test compounds
were added to the assay solution and preincubated with the
enzyme for 20 min, the addition of substrate following. Five
different concentrations of each inhibitor were used, to ob-
tain inhibition of AChE activity comprised between 20% and
80%.
Initial rate assays were performed at 37 °C with a J asco
V-350 double-beam spectrophotometer: the rate of increase
of the absorbance at 412 nm was followed for 5 min. Assays
were done with a blank containing all components except
AChE, to account for nonenzymatic reaction. The reaction
rates were compared and the percent inhibition due to the
presence of test compounds was calculated. Each concentration
was analyzed in triplicate. The percent inhibition of the
enzyme activity due to the presence of increasing test com-
pound concentration was calculated by the following expres-
sion: 100 - Vi/V0 × 100, where Vi is the rate calculated in the
presence of inhibitor and V0 is the enzyme activity. Inhibition
curves were obtained for each compound by plotting the
percent (%) inhibition vs the logarithm of inhibitor concentra-
tion in the assay solution. The linear regression parameters
were determined for each curve and the IC50 values extrapo-
lated.
6-Meth oxy-9-ch lor o-1,2,3,4-tetr a h yd r oa cr id in e (28): pu-
rified by flash chromatography, yield 55%; mp 90-92 °C
(ligroin); 1H NMR δ 1.80-1.99 (m, 4H), 2.85-3.09 (m, 4H),
3.88 (s, 3H), 7.07-8.03 (m, 3H, Ar). Anal. (C14H14ClNO) C, H,
N.
6-F lu or o-9-ch lor o-1,2,3,4-tetr a h yd r oa cr id in e (29): yield
65%; mp 75-77 °C (ligroin); 1H NMR δ 1.89-2.0 (m, 4H),
2.93-3.14 (m, 4H), 7.29-8.23 (m, 3H, Ar). Anal. (C13H11ClFN)
C, H, N.
6-Br om o-9-ch lor o-1,2,3,4-tetr a h yd r oa cr id in e (30): yield
60%; mp 73-74 °C (ligroin); 1H NMR δ 1.90-2.01 (m, 4H),
2.94-3.18 (m, 4H), 7.35-8.01 (m, 3H, Ar). Anal. (C13H11BrClN)
C, H, N.
6,7,9-Tr ich lor o-1,2,3,4-tetr a h yd r oa cr id in e (31): yield
60%; oil; 1H NMR δ 1.83-2.01 (m, 4H), 2.91-3.12 (m, 4H),
8.14 (s, 1H, Ar), 8.23 (s, 1H, Ar). Anal. (C13H10Cl3N) C, H, N.
P r ep a r a tion of Com p ou n d s 1-3, 6, 7, 10, a n d 45. A
mixture of selected 9-chloro-1,2,3,4-tetrahydroacridine (3.5
mmol) in phenol (3.12 g) was heated at 85-90 °C until an
homogeneous solution was obtained. The mixture was heated
at 125-130 °C for 4 h while a stream of NH3(g) was bubbled.
After cooling, ethyl acetate was added and the obtained
solution was treated with 10% NaOH solution, water and
dried. The solvent was removed under reduced pressure and
the residue was purified by flash chromatography (silica
gel 60, Merck, Darmstadt, Germany; ethyl acetate/methanol
9/1).
1H NMR (CDCl3) a n d Ma ss Sp ect r a for Com p ou n d s
1-3, 6, 7, 10, a n d 45. 1: δ 1.78-1.88 (m, 4H), 1.38 (s, 3H),
2.74-2.88 (m, 2H), 3.02-3.12 (m, 2H), 4.96 (br, 2H, NH2),
7.17-7.68 (m, 3H, Ar); MS m/z (relative abundance) 212 (M+,
10.1), 77 (51.4), 57 (100). Anal. (C14H16N2) C, H, N. 2: δ 1.63-
1.73 (m, 2H), 2.26 (s, 3H), 2.35-2.39 (m, 2H), 2.62-2.72 (m,
4H), 5.0 (br, 2H, NH2), 6.88-7.57 (m, 3H, Ar); MS m/z (relative
abundance) 212 (M+, 22.1), 149 (49.0), 57 (100). Anal. (C14H16N2)
C, H, N. 3: δ 1.65-1.85 (m, 4H), 2.35-2.43 (m, 2H), 2.75-
2.83 (m, 2H), 5.14 (br, 2H, NH2), 7.25-7.81 (m, 3H, Ar); MS
m/z (relative abundance) 232 (M+, 100), 92 (17.4), 63 (60.2).
Anal. (C13H13ClN2) C, H, N. 6: δ 1.86-1.95 (m, 4H), 2.51-
2.60 (m, 2H), 2.91-3.03 (m, 2H), 5.70 (br, 2H, NH2), 7.89-
8.73 (m, 3H, Ar); MS m/z (relative abundance) 243 (M+, 100),
215 (6.9), 197 (18.9). Anal. (C13H13N3O2) C, H, N. 7: δ 1.82-
2.01 (m, 4H), 2.50-2.61 (m, 2H), 2.92-3.02 (m, 2H), 3.91 (s,
3H), 4.75 (br, 2H, NH2), 6.95-7.62 (m, 3H, Ar); MS m/z
(relative abundance) 228 (M+, 2.9), 94 (100), 66 (48.0). Anal.
(C14H16N2O) C, H, N. 10: δ 1.91-2.08 (m, 4H), 2.55-2.68 (m,
2H), 2.98-3.08 (m, 2H), 4.66 (br, 2H, NH2), 7.79 (s, 1H, Ar),
8.01 (s, 1H, Ar); MS m/z (relative abundance) 266 (M+,
100), 250 (11.4), 238 (12.9). Anal. (C13H12Cl2N2) C, H, N. 45:
δ 1.85-2.01 (m, 4H), 2.48-2.59 (m, 2H), 2.97-3.01 (m, 2H),
5.93 (br, 2H, NH2), 7.29-7.91 (m, 3H, Ar); MS m/z (relative
abundance) 276 (M+, 100), 232 (17.1), 195 (11.3). Anal. (C13H13
BrN2) C, H, N.
-
P r ep a r a tion of Com p ou n d s 12-14 a n d 16-22. A mix-
ture of selected 9-chloro-1,2,3,4-tetrahydroacridine (3.5 mmol)
in phenol (3.12 g) was heated at 85-90 °C until an homoge-
neous solution was obtained. Heptylamine or benzylamine (7.7
mmol) was added and the mixture was heated at 125-130 °C
for 4 h. After cooling ethyl acetate was added and the obtained
solution was treated with 10% NaOH solution, water and
dried. The solvent was removed under reduced pressure and
the residue was purified by flash chromatography (silica gel
60, Merck, Darmstadt, Germany; ethyl acetate/methanol 9.5/
0.5).
1H NMR (CDCl3) a n d Ma ss Sp ect r a for Com p ou n d s
12-14 a n d 16-22. 12: δ 1.72-1.93 (m, 4H), 2.48 (s, 3H),
2.55-2.65 (m, 2H), 2.99-3.07 (m, 2H), 4.02 (br, 1H, NH), 4.57
(s, 2H), 7.25-7.88 (m, 8H, Ar); MS m/z (relative abundance)
302 (M+, 21.0), 218 (33.4), 91 (100). Anal. (C21H22N2) C, H, N.
13: δ 1.85-1.98 (m, 4H), 2.52 (s, 3H), 2.62-2.66 (m, 2H), 3.03-
3.09 (m, 2H), 4.15 (br, 1H, NH), 4.62 (d, 2H), 7.18-7.92 (m,
8H, Ar); MS m/z (relative abundance) 302 (M+, 13.4), 211
Acetylthiocholine iodide, 5,5′-dithiobis(2-nitrobenzoic acid),
and AChE (0.5 IU/mg) derived from human erythrocytes were
purchased from Sigma Chemical. Tacrine (9-amino-1,2,3,4-
tetrahydroacridine hydrochloride) was obtained from Aldrich
Italia. Buffer components and other chemicals were of the
highest purity commercially available.
QSAR a n d Molecu la r Mod elin g. The QSAR analysis was
performed by applying the classical Hansch approach and
using the C-QSAR program29 both to retrieve the substituent
constants and to calculate the regression equations.
The modeling of the inhibitors and the CoMFA procedures
were carried out by means of the SYBYL molecular modeling
package.30 Small molecule models were built by assembling