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S. D. SUKUMARAN ET AL.
(E)-2-(4-(3-(2-hydroxy-4,6-dimethoxyphenyl)-3-oxoprop-1-en-1-
J ¼ 8.7 Hz), 6.44 (1H, s), 4.49 (2H, t, J ¼ 4.5 Hz, –CH2–), 3.87, 3.86
(3H each, s, OCH3), 3.42 (2H, t, J ¼ 4.6 Hz, –CH2–), 3.33 (4H, brs,
–NCH2–), 2.09 (4H, brs, –CH2–); 13C NMR (150 MHz, CDCl3): d
191.43 (C¼O), 161.75, 159.37, 157.04, 143.84, 141.96, 130.49,
128.75, 118.74, 115.09, 112.03, 111.09, 100.85, 63.91 (–OCH2–),
57.03 (–CH2N–), 56.21 (–NCH2–), 54.55 (OCH3), 54.05 (OCH3), 23.27
(–CH2–). HRMS calc. for [C23H28NO5]þ ¼ [M þ H]þ ¼ 398.1967,
found ¼ 398.1968.
yl)phenyl)acetic acid (4h)
1
Yellow powders; yield 58%; mp 139.2–141.2 ꢀC. H NMR (400 MHz,
CDCl3): d 14.21 (1H, s, OH), 7.79 (1H, d, J ¼ 15.6 Hz, Hb), 7.68 (1H,
d, J ¼ 15.6 Hz, Ha), 7.48 (2H, d, J ¼ 8.0 Hz), 7.21 (2H, d, J ¼ 8.0 Hz),
6.02 (1H, d, J ¼ 2.2 Hz), 5.88 (1H, d, J ¼ 2.2 Hz), 3.83, 3.76 (3H each,
s, OCH3), 3.70 (2H, s, –CH2CO); 13C NMR (100 MHz, CDCl3): d 192.60
(C¼O), 169.01 (C¼O), 168.38, 166.26, 162.53, 141.93, 137.53,
134.15, 129.25, 128.68, 127.35, 106.37, 93.85, 91.29, 55.88 (OCH3),
55.58 (OCH3), 40.79 (–CH2CO). HRMS calc. for [C19H19O6]þ
[M þ H]þ ¼ 343.1182, found ¼ 343.1194.
¼
(E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(4-(2-(piperidin-1-yl)ethox-
y)phenyl)prop-2-en-1-one (8e)
Yellow needles; yield 69%; mp 164.3–166.7 ꢀC. 1H NMR (600 MHz,
CDCl3): d 14.30 (1H, s, OH), 7.73 (1H, d, J ¼ 15.6 Hz, Hb), 7.68 (1H,
d, J ¼ 15.6 Hz, Ha), 7.47 (2H, d, J ¼ 8.4 Hz), 6.85 (2H, d, J ¼ 8.4 Hz),
6.03 (1H, s), 5.89 (1H, s), 4.28 (2H, t, J ¼ 5.0 Hz, –CH2–), 3.85, 3.76
(3H each, s, OCH3), 2.98 (2H, t, J ¼ 5.0 Hz, –CH2–), 2.75 (4H, brs,
–NCH2–), 1.74 (4H, m, –CH2–), 1.48 (2H, brs, –CH2–); 13C NMR
(150 MHz, CDCl3): d 192.55 (C¼O), 168.36, 166.09, 162.47, 159.72,
142.13, 130.13, 128.92, 125.56, 114.98, 106.35, 93.83, 91.25, 64.61
(–OCH2–), 57.07 (–CH2N–), 55.87 (OCH3), 55.58 (–NCH2–), 54.60
(OCH3), 24.41 (–CH2–), 23.09 (–CH2–). HRMS calc. for [C24H30NO5]þ
¼ [M þ H]þ ¼ 412.2124, found ¼ 412.2131.
(E)-1-(2-hydroxy-4-methoxyphenyl)-3-(4-(2-(piperidin-1-yl)ethoxy)-
phenyl)prop-2-en-1-one (8a)
Yellow solid; yield 67%; mp 166.7–169.7 ꢀC. 1H NMR (600 MHz,
CDCl3 (90%) and MeOH-d4 (10%)): d 13.45 (1H, brs, OH), 7.76 (1H,
d, J ¼ 15.9 Hz, Hb), 7.74 (1H, d, J ¼ 8.0 Hz), 7.52 (2H, d, J ¼ 8.0 Hz),
7.37 (1H, d, J ¼ 15.4 Hz, Ha), 6.87 (1H, d, J ¼ 8.0 Hz), 6.40 (1H, d,
J ¼ 7.8 Hz), 6.39 (1H, s), 4.24 (2H, brt, –CH2–), 3.78 (3H, s, OCH3),
2.94 (2H, brt, –CH2–), 2.69 (4H, brs, –NCH2–), 1.70 (4H, brs, –CH2–),
1.46 (2H, brs,–CH2–); 13C NMR (150 MHz, CDCl3): d 191.97 (C¼O),
166.09, 165.97, 160.01, 143.98, 131.30, 130.35, 128.11, 118.27,
114.95, 114.02, 107.53, 100.96, 63.97 (–OCH2–), 56.70 (–CH2N–),
55.45 (–NCH2–), 54.37 (OCH3), 23.96 (–CH2–), 22.55 (–CH2–). HRMS
calc. for [C23H28NO4]þ ¼ [M þ H]þ ¼ 382.2018, found ¼ 382.2036.
(E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(4-(2-(pyrrolidin-1-yl)e-
thoxy)phenyl)prop-2-en-1-one (8f)
Yellow solid; yield 70%; mp 157.0–159.0 ꢀC. 1H NMR (600 MHz,
CDCl3): d 14.27 (1H, s, OH), 7.73 (1H, d, J ¼ 15.5 Hz, Hb), 7.67 (1H,
d, J ¼ 15.5 Hz, Ha), 7.48 (2H, d, J ¼ 8.6 Hz), 6.89 (2H, d, J ¼ 8.6 Hz),
6.03 (1H, d, J ¼ 2.3 Hz), 5.89 (1H, d, J ¼ 2.3 Hz), 4.50 (2H, t,
J ¼ 4.4 Hz, –CH2–), 3.85, 3.77 (3H each, s, OCH3), 3.45 (2H, t,
J ¼ 4.4 Hz, –CH2–), 3.35 (4H, brs, –NCH2–), 2.10 (4H, brs, –CH2–); 13C
NMR (150 MHz, CDCl3): d 192.52 (C¼O), 168.37, 166.17, 162.50,
158.75, 141.71, 130.18, 129.68, 126.10, 115.04, 106.35, 93.85, 91.28,
63.55 (–OCH2–), 55.89 (–CH2N–), 55.59 (OCH3), 54.54 (OCH3), 54.01
(–NCH2–), 23.26 (–CH2–). HRMS calc. for [C23H28NO5]þ ¼ [M þ H]þ
¼ 398.1967, found ¼ 398.1973.
(E)-1-(2-hydroxy-4-methoxyphenyl)-3-(4-(2-(pyrrolidin-1-yl)ethoxy)-
phenyl)prop-2-en-1-one (8b)
Yellow needles; yield 72%; mp 189.9–192.1 ꢀC. 1H NMR (600 MHz,
CDCl3): d 13.44 (1H, brs, OH), 7.77 (1H, d, J ¼ 15.0 Hz, Hb), 7.74 (1H,
d, J ¼ 9.0 Hz), 7.53 (2H, d, J ¼ 8.5 Hz), 7.38 (1H, d, J ¼ 15.4 Hz, Ha),
6.89 (2H, d, J ¼ 8.5 Hz), 6.42 (1H, dd, J ¼ 9.0, 2.0 Hz), 6.39 (1H, s),
4.32 (2H, t, J ¼ 5.0 Hz, –CH2–), 3.78 (3H, s, OCH3), 3.20 (2H, t,
J ¼ 5.0 Hz, –CH2–), 3.02 (4H, brs, –NCH2–), 1.95 (4H, brs, –CH2–); 13C
NMR (150 MHz, CDCl3): d 191.88 (C¼O), 166.14, 166.10, 159.90,
143.91, 131.26, 130.41, 128.33, 118.46, 115.06, 114.12, 107.62,
101.06, 64.79 (–OCH2–), 55.58 (–CH2N–), 54.64 (OCH3), 54.25
(–NCH2–), 23.26 (–CH2–). HRMS calc. for [C22H26NO4]þ ¼ [M þ H]þ
¼ 368.1862, found ¼ 368.1885.
Enzyme inhibition studies
To determine the inhibitory activity of the target compounds
towards cholinesterases, the AChE and BuChE inhibition assays
were performed in 96 well plates by the method of Ellman with
slight modifications16. First, sodium phosphate buffer (110 lL; pH
(E)-1-(2-hydroxy-4,5-dimethoxyphenyl)-3-(4-(2-(piperidin-1-yl)ethox-
y)phenyl)prop-2-en-1-one (8c)
Dark red needles; yield 61%; mp 145.0–146.2 ꢀC. 1H NMR 8.0) was added into each well followed by 20 lL of each test com-
(600 MHz, CDCl3): d 13.37 (1H, s, OH), 7.78 (1H, d, J ¼ 15.4 Hz, Hb), pound, 50 lL of 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB)
7.54 (2H, d, J ¼ 8.6 Hz), 7.32 (1H, d, J ¼ 15.5 Hz, Ha), 7.20 (1H, s), (0.126 mM) and 20 lL of AChE or BuChE (0.15 units per mL). Then,
6.88 (2H, d, J ¼ 8.6 Hz), 6.43 (1H, s), 4.38 (2H, t, J ¼ 3.6 Hz, –CH2–), the mixture was incubated for 20 min at 37 ꢀC. Subsequently, the
reaction was initiated by addition of 50 lL (0.120 mM) of the sub-
3.86, 3.85 (3H each, s, OCH3), 3.20 (2H, t, J ¼ 3.0 Hz, –CH2–), 2.99
(4H, brs, –NCH2–), 1.85 (4H, m, –CH2–), 1.54 (2H, brs, –CH2–); 13C
strate, acetylthiocholine (ATC) iodide or butyrylthiocholine (BTC)
iodide (depending on the enzyme). The hydrolysis of ATC or BTC
NMR (150 MHz, CDCl3): d 191.45 (C¼O), 161.73, 159.75, 157.04,
R
was monitored with an InfiniteV M200 PRO multimode reader
143.96, 141.76, 130.44, 128.43, 118.53, 115.08, 112.04, 111.17,
€
(Tecan Group Ltd., Mannedorf, Switzerland) by measuring the
100.84, 63.83 (–OCH2–), 57.05 (–CH2N–), 56.66 (–NCH2–), 56.19
(OCH3), 54.44 (OCH3), 23.61 (–CH2–), 22.51 (–CH2–). HRMS calc. for
[C24H30NO5]þ ¼ [M þ H]þ ¼ 412.2124, found ¼ 412.2130.
absorbance of the yellow 5-thio-2-nitrobenzoate anion at 412 nm,
every 30 s for 25 min. Donepezil, propidium iodide and tacrine
were used as standard inhibitors. Each assay was performed in
triplicate. The percentage of inhibition was calculated using the
following expression:
(E)-1-(2-hydroxy-4,5-dimethoxyphenyl)-3-(4-(2-(pyrrolidin-1-yl)e-
thoxy)phenyl)prop-2-en-1-one (8d)
ð
Þ
E–S =E ꢃ 100
Orange solid; yield 58%; mp 167.0–169.0 ꢀC. 1H NMR (600 MHz,
CDCl3): d 13.35 (1H, s, OH), 7.78 (1H, d, J ¼ 15.4 Hz, Hb), 7.55 (2H, where E is the activity of the enzyme without test compound and
d, J ¼ 8.7 Hz), 7.34 (1H, d, J ¼ 15.4 Hz, Ha), 7.19 (1H, s), 6.90 (2H, d, S is the activity of the enzyme with the test compound. IC50