708 W. Luo et al.
N1,N9-bis((7-methoxybenzo[d][1,3]dioxol-5-yl)methyl)
nonane-1,9-diamine (5g)
4H,−OCH2O–), 3.89 (s, 6H,−OCH3), 3.67 (s, 4H,−CH2–),
2.62 (t, J= 6.5 Hz, 4H,−CH2–), 1.82 (s, 2H,−CH2–), 1.71–
1.39 (m, 2H,−CH2–); 13C NMR (100 MHz, CDCl3) δ 148.79,
143.51, 135.02, 134.05, 107.36, 102.18, 101.29, 56.54, 53.96,
49.07, 27.81; purity: 99.9% by HPLC. HRMS (ESI): calcd
for (M + H)+ (C22H28N2O6) requires m/z 417.2026, found
417.2017.
Colorless oil. MS (ESI, m/z): [M + H]+ 487.2; 1H
NMR (400 MHz, CDCl3) δ 6.51 (s, 4H, Ar–H), 5.93 (s,
4H,−OCH2O–), 3.89 (s, 6H,−OCH3), 3.68 (s, 4H,−CH2–),
2.59 (t, J= 7.2 Hz, 4H,−CH2–), 1.67 (s, 2H,−CH2–), 1.56–
1.40 (m, 4H,−CH2–), 1.32–1.25 (s, 10H,−CH2–); 13C NMR
(100 MHz, CDCl3) δ 148.79, 143.51, 135.24, 134.02, 107.36,
102.21, 101.28, 56.55, 54.08, 49.34, 30.05, 29.52, 29.49,
27.34; purity: 99.7% by HPLC. HRMS (ESI): calcd for (M +
H)+ (C27H38N2O6) requires m/z 487.2808, found 487.2807.
N1,N5-bis((7-methoxybenzo[d][1,3]dioxol-5-yl)methyl)
pentane-1,5-diamine (5c)
Colorless oil. MS (ESI, m/z): [M + H]+ 431.2; 1H
NMR (400 MHz, CDCl3) δ 6.50 (s, 4H, Ar–H), 5.93 (s,
4H,−OCH2O–), 3.89 (s, 6H,−OCH3), 3.67 (s, 4H,−CH2–),
2.60 (t, J= 7.1 Hz, 4H,−CH2–), 1.57–1.46 (m, 4H,−CH2–),
1.45–1.32 (m, 4H,−CH2–); 13C NMR (100 MHz, CDCl3)
δ 148.79, 143.51, 135.27, 134.02, 107.34, 102.18, 101.28,
56.55, 54.11, 49.24, 30.00, 25.11; purity: 99.1% by HPLC.
HRMS (ESI): calcd for (M + H)+ (C23H30N2O6) requires m/z
431.2182, found 431.2178.
Pharmacology
All the synthesized compounds were screened for AChE
and BuChE inhibition activities. AChE (E.C. 3.1.1.7, from
electric eel), BuChE (E.C. 3.1.1.8, from equine serum),
5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent,
DTNB), butyrylthiocholine chloride and acetylthiocho-
line chloride (ATC) were purchased from Sigma–Aldrich
and rivastigmine hydrochloride standard was pur-
chased from Sunve (Shanghai) Pharmaceutical Co., Ltd.
Curcumin used in this work were synthesized and char-
acterized in our laboratory19.
N1,N6-bis((7-methoxybenzo[d][1,3]dioxol-5-yl)methyl)hexane-
1,6-diamine(5d)
Colorless oil. MS (ESI, m/z): [M + H]+ 445.1; 1H
NMR (400 MHz, CDCl3) δ 6.51 (s, 4H Ar–H), 5.93
(s, 4H,−OCH2O–), 3.89 (s, 6H,−OCH3), 3.67 (s,
4H,−CH2–), 2.60 (t, J = 7.2 Hz, 4H,−CH2–), 1.54–1.45
(m, 6H,−CH2–), 1.37–1.30 (m, 4H,−CH2–); 13C NMR
(100 MHz, CDCl3) δ 148.81, 143.53, 135.29, 134.04,
107.39, 102.19, 101.27, 56.57, 54.09, 49.28, 30.05,
27.30; purity: 100% by HPLC. HRMS (ESI): calcd for
(M + H)+ (C24H32N2O6) requires m/z 445.2339, found
445.2340.
Enzyme inhibition assays
Alltheassayswereunder0.1MKH2PO4/K2HPO4 buffer,pH
8.0, using a Shimadzu 2450 Spectrophotometer. Enzyme
solutions were prepared to give 2.0 units/mL in 2mL
aliquots. e assay medium contained phosphate buffer,
pH 8.0 (1 mL), 50 μL of 0.01 M DTNB, 10 μL of enzyme,
and 50 μL of 0.01 M substrate (ATC). e substrate was
added to the assay medium containing enzyme, buffer,
and DTNB with inhibitor after 15 min of incubation time.
e activity was determined by measuring the increase
in absorbance at 412 nm at 1 min intervals at 37°C.
Calculations were performed according to the method of
the equation in Ellman et al.20. In vitro BuChE assay use
the similar method described above.
Kinetic characterization of AChE was performed using
a reported method. Six different concentrations of sub-
strate were mixed in the 1 mL 0.1 M KH2PO4/K2HPO4 buf-
fer (pH 8.0), containing 50 μL of DTNB, 10 μL AChE, and
50 μL substrate. Test compound was added into the assay
solution and pre-incubated with the enzyme at 37°C for
15 min, followed by the addition of substrate. Kinetic
characterization of the hydrolysis of ATC catalyzed by
AChE was done spectrometrically at 412 nm. A parallel
control with no inhibitor in the mixture, allowed adjust-
ing activities to be measured at various times.
N1,N7-bis((7-methoxybenzo[d][1,3]dioxol-5-yl)methyl)
heptane-1,7-diamine (5e)
Colorless oil. MS (ESI, m/z): [M + H]+ 459.0; 1H
NMR (400 MHz, CDCl3) δ 6.51 (s, 4H, Ar–H), 5.94
(s, 4H,−OCH2O–), 3.90 (s, 6H,−OCH3), 3.68 (s,
4H,−CH2–), 2.59 (t, J = 7.2 Hz, 4H,−CH2–), 1.54–1.46
(m, 6H,−CH2–), 1.37–1.30 (m, 6H,−CH2–); 13C NMR
(100 MHz, CDCl3) δ 148.79, 143.52, 135.27, 134.03,
107.34, 102.20, 101.28, 56.56, 54.10, 49.33, 30.03, 29.46,
27.30; purity: 99.8% by HPLC. HRMS (ESI): calcd for
(M + H)+ (C25H34N2O6) requires m/z 459.2495, found
459.2493.
N1, N8-bis((7-methoxybenzo[d][1,3]dioxol-5-yl)methyl)octane-
1,8-diamine (5f)
Colorless oil. MS (ESI, m/z): [M + H]+ 473.1; 1H
NMR (400 MHz, CDCl3) δ 6.51 (s, 4H, Ar–H), 5.93 (s,
4H,−OCH2O–), 3.89 (s, 6H,−OCH3), 3.67 (s, 4H,−CH2–),
2.59 (t, J = 7.2 Hz, 4H,−CH2–), 1.61 (s, 2H,−CH2–),
1.54–1.44 (m, 4H,−CH2–), 1.33–1.25 (m, 8H,−CH2–); 13C
NMR (100 MHz, CDCl3) δ 148.77, 143.49, 135.24, 133.99,
107.32, 102.18, 101.26, 56.55, 56.48, 54.07, 49.32, 30.04,
29.47, 27.28; purity: 99.9% by HPLC. HRMS (ESI): calcd
for (M + H)+ (C26H36N2O6) requires m/z 473.2652, found
473.2652.
Inhibition of self-mediated Aβ aggregation
e thioflavin-T fluorescence method was used21.
Aβ42 peptide sodium (Anaspec Inc.) was dissolved in
phosphate buffer (pH 7.40, 0.01 M) to obtain a 20 μM
solution. Compounds were first prepared in dimethyl
sulfoxide (DMSO) to obtain a 10 mM solution. e final
concentration of Aβ42 and inhibitors was 20 μM. After
incubated in 37°C for 24 h, thioflavin-T (5 μM in 50 mM
Journal of Enzyme Inhibition and Medicinal Chemistry