Inhibitory evaluation of sulfonamide chalcones on β-secretase and acylcholinesterase

Article abstract of DOI:10.3390/molecules18010140

The action of β-secretase (BACE1) is strongly correlated with the onset of Alzheimer's disease (AD). Aminochalcone derivatives were examined for their ability to inhibit BACE1. Parent aminochalcones showed two digit micromolar IC50s against BACE1. Potency

Full text of DOI:10.3390/molecules18010140

Molecules 2013, 18, 140-153; doi:10.3390/molecules18010140
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Inhibitory Evaluation of Sulfonamide Chalcones on β-Secretase
and Acylcholinesterase
Jae Eun Kang 1,†, Jung Keun Cho 1,†, Marcus J. Curtis-Long 2, Hyung Won Ryu 1, Jin Hyo Kim 3,
Hye Jin Kim 1, Heung Joo Yuk 1, Dae Wook Kim 1 and Ki Hun Park 1,*
1
Division of Applied Life Science (BK21 Program), IALS, Gyeongsang National University,
Jinju 660-701, Korea
2
Graduate Program in Biochemistry and Biophysics, Brandeis University, 415 South Street,
Waltham, MA 02453, USA
3
Chemical Safety Division, National Academy of Agricultural Sciences, RDA, Suwon 441-701, Korea
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: khpark@gnu.ac.kr;
Tel.: +82-55-772-1965; Fax: +82-55-772-1969.
Received: 19 November 2012; in revised form: 16 December 2012 / Accepted: 18 December 2012 /
Published: 21 December 2012
Abstract: The action of β-secretase (BACE1) is strongly correlated with the onset of
Alzheimer’s disease (AD). Aminochalcone derivatives were examined for their ability to
inhibit BACE1. Parent aminochalcones showed two digit micromolar IC50s against
BACE1. Potency was enhanced 10-fold or more by introducing benzenesulfonyl derivatives
to the amino group: 1 (IC50 = 48.2 μM) versus 4a (IC50 = 1.44 μM) and 2 (IC50 = 17.7 μM)
versus 5a (IC50 = 0.21 μM). The activity was significantly influenced by position and
number of hydroxyl groups on the chalcone B-ring: 3,4-dihydroxy 5a (IC50 = 0.21 μM) >
4-hydroxy 4a (IC50 = 1.44 μM) > 2,4-dihydroxy 6 (IC50 = 3.60 μM) > 2,5-dihydroxy 7
(IC50 = 16.87 μM) > des hydroxy 4b (IC50 = 168.7 μM). Lineweaver-Burk and Dixon plots
and their secondary replots indicate that compound 5a was a mixed inhibitor with
reversible and time-dependent behavior. Potent BACE1 inhibitors 4a,c,f, 5a–c showed
moderate inhibition against two other enzymes implicated in AD pathogenesis,
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with IC50s ranging
between 56.1 ~ 95.8 μM and 19.5 ~ 79.0 μM, respectively.

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Keywords: sulfonamide chalcone; Alzheimer’s disease (AD); β-secretase;
acetylcholinesterase (AChE); butyrylcholinesterase (BChE); mixed inhibition
1. Introduction
As life expectancy grows, rendering elderly people more common in society, age related diseases
have become increasingly prevalent, so it is no surprise that incidences of dementia are consequentially
growing yearly. Alzheimer’s disease (AD) is the major cause of dementia (1 in 8 of people over 65 has
AD in the USA) [1]. This disease involves a progressive loss of neurons in the hippocampus and
cortex which leads to serious loss of global cognitive ability. AD manifests itself in the brain by loss of
dendrites and axons, myelin reduction, shrinkage and finally neuronal death [2]. At the biochemical
level, there is a heightened presence of extra neuronal aggregation of plaques composed of β-amyloid
(Aβ) peptide [3] and neurofibrillary tangles [4]. Aβ peptides are derived from a sequential proteolytic
cleavage of amyloid precursor protein (APP) by the aspartate proteases, β and γ-secretase. β-Secretase
(BACE1) was discovered by the four independent groups [5–8]. The Aβ hypothesis for AD started
from that endogenous BACE1 activity is increased in sporadic AD brain [9–11]. BACE1 is uniquely
able to process APP and thus form Aβ-peptides, because BACE1 knock-out mice are unable to form
Aβ peptides [12]. It has now been clearly established that overproduction of Aβ by BACE1 results in
toxic fibrils causing neurodegeneration.
Intriguingly, reduced AChE expression is another common feature of AD patients. However, higher
levels of AChE are observed around β-amyloid plaques [13]. Thus AChE is another good target for
AD treatment and it is generally accepted that AChE is associated with β-amyloid plaques. AChE
catalyzes the hydrolysis of the neurotransmitter acetylcholine into choline, silencing the signal which
is carried by acetylcholine [14]. There is redundancy in this system because a related enzyme, BChE
can replace AChE and hydrolyze acetylcholine. Because of their similar hydrolytic activities,
AChE/BChE overexpression around plaques can lead to reduced neurotransmitter levels, and thus it is
thought that AChE/BChE inhibition may alleviate some AD symptoms by prolonging the half lives
of neurotransmitters.
Previous work in designing inhibitors for BACE1 has centered on peptide-derived structures, which
act as transition state analogs based on the amino acid sequences at the cleavage site of APP by
BACE1 [15]. These species showed nanomolar IC50s against BACE1 or better, but their viability as
drug candidates is minimal because of their high hydrophilicity. This is because the inhibitor must
possess sufficient lipophilicity to traverse two lipid bilayers to reach BACE1, as it is localized in the
trans Golgi network/endosomal lumen [16]. We have previously reported the synthesis of a series of
sulfonamide chalcones and their biological applications. These species showed anti-tumorigenic
effects [17] as well as inhibitory activity against α-glucosidase [18]. Sulfonamide chalcones also acted
as ion channel blockers [19]. In this manuscript, we disclose that novel sulfonamide chalcone
derivatives can inhibit BACE1 at nanomolar levels. Their inhibitory mechanisms were assessed using
Lineweaver-Burk and Dixon plots. Importantly, the most potent BACE1 inhibitors were also shown to
have inhibitory activity against AChE and BChE, other key players in AD progression. Our aim was to

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find the structural features of chalcone with inhibitory activity for target enzymes (BACE1 and AChE).
We have studied the influence on target enzymes inhibitions of each of these elements; N-free
aminochalcone (1–2, Figure 1), N-acetyl aminochalcone (3, Figure 1), and N-sulfonamide chalcones
(4–7, Figure 1) that have different position and number of hydroxyl groups on the chalcone B-ring.
Figure 1. Chemical structures of chalcone derivatives.
O
O
O
O
OH
OH
O
H2N
OH
H2N
N
OH
H
1
2
3
O
OH
OH
O
O
R
S N
OH
OH
R
S N
H
O
H
O
4
6
5
7
O
OH
OH
O
OH
O
O
H3C
S N
H3C
S N
H
O
H
O
2. Results and Discussion
2.1. Chemistry
The typical synthetic procedure for sulfonamide chalcones 4–7 is described in Scheme 1 [11].
N-Sulfonylaminoacetophenone was easily prepared by treatment of aminoacetophenone with the
appropriate benzenesulfonyl chloride in the presence of pyridine. The sulfonamide chalcones 1–7 in
Table 1 were obtained by Claisen-Schmit condensation of the appropriate benzaldehyde and sulfonated
aminoacetophenone in presence of a catalytic amount of H2SO4.
Scheme 1. Synthesis of chalcone derivatives.
O
O
O
(i)
(ii)
O
O
R2
A
B
R1
S N
R1
S N
H
O
H
O
H2N
5a
Reagents and Conditions: (i) benzenesulfonylchloride derivative, pyridine, CH2Cl2, r.t;
(ii) benzaldhyde derivative, H2SO4, MeOH, reflux.

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Table 1. Inhibitory effects of compounds on BACE1 activities.
O
R1
R2
O
R3
S N
H
O
Compound
R1 R2 R3
IC50 (M) a
48.2 ± 1.2
17.7 ± 0.8
>200
Ki (M) b
43.8
10.5
NT
1
2
3
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
5d
5e
5f
6
H OH CH3
H H CH3
H OH H
1.44 ± 0.2
168.7 ± 2.4
6.28 ± 0.7
2.88 ± 0.5
79.3 ± 2.3
5.58 ± 1.4
107.4 ± 2.7
119.6 ± 3.2
0.21 ± 0.02
4.59 ± 1.5
0.62 ± 0.03
0.69 ± 0.04
101.3 ± 2.4
8.95 ± 1.0
3.60 ± 0.3
16.87 ± 0.8
0.56
NT
5.83
1.49
75.2
4.19
94.8
NT
H OH OH
H OH OCH3
H OH NH2
H OH NO2
H OH F
OH OH CH3
OH OH H
OH OH OH
OH OH NH2
OH OH NO2
OH OH F
0.07
3.78
0.56
0.69
92.4
10.8
1.89
11.7
7
a
All compounds were examined in a set of experiments repeated three times; Error is standard deviation.
b
IC50 values of compounds represent the concentration that caused 50% enzyme activity loss; Values of
inhibition constant.
2.2. Biological Activities
β-Secretase Inhibition Activities
The ability of compounds 1–7 to inhibit the cleavage of APP to Aβ was assessed using a fluorescent
resonance energy transfer (FRET) peptide cleavage assay [9]. In this assay, BACE1-catalyzed
proteolysis of a synthetic oligopeptide substituted with a fluorescent dye and a quencher leads to an
increase in fluorescence by breaking the tether between the dye and the quencher. The FRET
oligopeptide substrate, Rhodamine-EVNLDAEFK-Quencher, and recombinant human BACE1 were
purchased from RD System, Inc. (WI, USA) Morin was used as a positive control (IC50 = 12.3 μM). The
assay was carried out according to the supplied manual with small modifications. The concentration of
compound required to inhibit the enzyme 50% (IC50) values (μM) are presented in Table 1. These data
showed that most sulfonamide containing compounds are good inhibitors of BACE1. Sulfonamide
chalcones showed at least 10-fold greater potency than the des sulfonamido analogues (compounds 1
and 2). For example, sulfonamide chalcone 5a (IC50 = 0.21 μM) was 70-fold effective than the
analogue bearing an unsubstituted amine, 2 (IC50 = 17.7 μM). A similar trend was observed for

Molecules 2013, 18
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sulfonamide chalcone 4a (IC50 = 1.44 μM) and amino chalcone 1 (IC50 = 48.2 μM). The inhibition activity
was significantly influenced by position and number of hydroxyl groups on the chalcone B-ring: 3,4-
dihydroxy 5a (IC50 = 0.21 μM) > 4-hydroxy 4a (IC50 = 1.44 μM) > 2,4-dihydroxy 6 (IC50 = 3.60 μM) >
2,5-dihydroxy 7 (IC50 = 16.87 μM) > deshydroxy 4b (IC50 = 168.7 μM). Acylation and alkylation at
nitrogen resulted in considerable loss of potency (supplementary material). For example, acetoamide
chalcone 3 was inactive at concentrations as high as 200 μM.
The potency of these inhibitors was affected by subtle changes in structure. A general better
inhibition was observed when the benzenesulfonyl ring bears electron donating group (CH3, OH, NH2,
OCH3) in para position, but the inhibition was affected by electron donating potential as well as size of
functionality: 4d (OH, IC50 = 2.88 μM) versus 4e (R3=OCH3, IC50 = 79.3 μM). Consistent with this
trend, the strongly electron withdrawing groups, p-fluoro and p-nitro, were the least potent inhibitors
(IC50 = 119.6 and 107.4 μM respectively; Figure 2A). The 5-series, bearing a 3,4-dihydroxy group in
the B ring of the chalcone, was in general more potent than the corresponding 4-hydroxy derivatives.
Interestingly, the relationship between activity and substituent on the sulfonamide function held
steady. Smaller electron donating groups (CH3, OH, NH2) were more favored than larger species such
as OCH3 and electron withdrawing functions such as NO2.
2.3. Inhibitory Kinetics
We examined each sulfonamide compound 4a–5f for its inhibition of BACE1 (Figure 2). As the
concentrations of the inhibitors were augmented, the residual enzyme activity drastically diminished
(Figure 2A). The inhibition of BACE1 by compounds 4a (Ki = 0.56 μM) and 5a (Ki = 0.07 μM), the
most potent inhibitors, are illustrated in Figure 2, representatively. Raising the concentrations of the
inhibitors drastically lowered residual enzyme activity. Plots of residual enzyme activity versus
enzyme concentration at different concentrations of compound 5a gave a family of straight lines with a
y-axis intercept of 0; this indicates that 5a is a reversible inhibitor (Figure 2B). We then further
characterized the inhibitory mechanism of the synthesized chalcone derivatives. This was done using
both Lineweaver-Burk and Dixon plots. These double-reciprocal plots yield a family of lines with
different slopes and intercepts. They intersected to the left of the vertical axis and above the horizontal
axis, indicating that 5a is a mixed inhibitor (Figure 2D). The mixed behavior of 5a was also proved by
showing increasing concentrations of inhibitor resulted in increased Km values and diminished Vmax
The Ki of 5a was determined to be 70 nM (Table 1) using a Dixon plot (Figure 2F). Compound 4a
(Ki = 0.56 μM) also showed mixed type behavior with a similar kinetic pattern to 5a (Figure 2E,F).
The rest of tested compounds were also mixed inhibitors.
To further investigate the inhibition mechanism, time dependence of BACE1inhibition by
compound 5a was subsequently probed. The enzyme was preincubated with substrate (between 0 and
60 min) and the residual enzyme activity was measured by calculating the initial velocity (Vi) of the
hydrolysis (Figure 3B). Progress curves were continued to be run up for 3,600 s. These data showed
continued decrease in activity to the final steady state rate. Secondary replots of Vi/V0 after
preincubation with inhibitor compared to similar incubation times without inhibitor show that a
significant time dependence in inhibition is observed. Thus 5a is a slow binding inhibitor of this
enzyme (Figure 3D) [20].

Molecules 2013, 18
Figure 2. (A) Effect of compounds 4a–5f on the hydrolytic activity of BACE1. (B) The
145
hydrolytic activity of BACE1 as function of enzyme concentrations at different
concentrations of compound 5a. (C and F) Mechanistic analysis of the inhibition of
BACE1 by sulfonamide chalcones 4a and 5a. (C and D) Lineweaver-Burk plots for the
inhibition of compounds 4a and 5a on the hydrolytic activity of BACE1. (Inset) Km values
as a function of the concentrations of 4a and 5a. Dependence of the values of Vmax on the
concentration of 4a and 5a. (E and F) Dixon plots for the inhibition of compounds 4a and
5a on BACE1 catalyzed proteolysis of substrate.
100
80
60
40
20
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
4a
4b
4c
4d
4e
4f
A
C
E
B
D
F
4g
4h
5a
5b
5c
5d
5e
5f
0.01 0.1
1
10 100 1000 10000
0.05 0.10 0.15 0.20 0.25
E [Unit/mL]
[I, M]
60
50
40
30
20
10
0
0.30
0.25
0.20
0.15
0. 30
0. 25
0. 20
0 M
0 M
60
0.78 M
1.56 M
3.12 M
0.1 M
0.2 M
0.4 M
50
40
30
20
10
0
0. 15
0
1
2
3
4
0.0
0.1
0.2
0.3
0.4
5
4
8
6
3
2
1
4
2
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
0.0 0.1 0.2 0.3 0.4 0. 5
5a (M)
4a ( M)
-2
-1
0
1
2
3
-2
-1
0
1
2
3
1/[S]
1/[S]
60
50
40
30
20
10
0
375 nM
750 nM
1500 nM
375 nM
60
750 nM
1500 nM
50
40
30
20
10
0
-3 -2
-1
0
1
2
3
4
-0.2
0.0
0.2
0.4
[I, M]
[I, M]

Molecules 2013, 18
Figure 3. (A–C) Time-dependent inhibition of BACE1 in the presence of 0.1, 0.2, 0.4 μM
146
compound 5a; (D) Decrease in slopes of the lines of panel E as a function of time
(●, 0 μM; ○, 0.2 μM).
14
12
10
8
14
12
10
8
control
0 min
control
0 min
0.1 M
0.2 M
A
B
10 min
20 min
30 min
40 min
50 min
60 min
10 min
20 min
30 min
40 min
50 min
60 min
6
6
4
4
2
2
0
0
0
1000
2000
3000
0
1000
2000
3000
Time (s)
Time (s)
14
1.0
0.8
0.6
0.4
control
0 min
0.4 M
C
D
12
10
8
10 min
20 min
30 min
40 min
50 min
60 min
6
4
2
M
M
0
0
1000
2000
3000
0
10
20
30
40
50
60
Time (s)
t
/min
preinc
Since AChE and BChE have also been implicated in the onset of AD we were keen to see if our
compounds could inhibit these enzymes as well. Clearly an inhibitor capable of alleviating numerous
etiological factors simultaneously could have more benefit than a compound which was highly
selective for one enzyme. Our most potent BACE1 inhibitors 4a,c,f, 5a–c were thus assessed for
inhibition of AChE and BChE. As shown Table 2, many of our sulfonamide chalcones exhibited a
moderate degree of cholinesterase inhibition, with IC50s ranging from 56.1 ~ 95.8 μM for AChE and
19.5 ~ 79.0 μM for BChE. Further analysis showed all ChE inhibitors were reversible and exhibited
mixed inhibition kinetics (Table 2 and Supplementary Materials).
Table 2. Inhibitory effects of selected sulfonamide chalcones on the cholinesterases AChE
and BChE.
IC50a (M)
erythrocytes AChE
IC50a (M)
equine serum BChE
Compound
4a
Ki (M)b
Ki (M)b
56.1
40.4
47.4
25.3
4c
4f
5a
5b
5c
Eserin
83.3
95.8
75.9
75.4
57.4
0.15
80.3
61.5
55.8
57.2
47.6
NT c
75.0
34.3
19.5
79.0
24.7
3.7
72.2
19.0
9.8
63.5
26.6
NT
a
All compounds were examined in a set of experiments repeated three times; Error is standard deviation.
b
IC50 values of compounds represent the concentration that caused 50% enzyme activity loss; Values of
inhibition constant; c NT is not tested.

Molecules 2013, 18
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3. Experimental
3.1. General
Test chromatographic separations were carried out by Thin-layer chromatography (TLC) (E. Merck
Co., Darmstadt, Germany), using commercially available glass plate pre-coated with silica gel and
visualized under UV at 254 and 366 nm or with p-anisaldehyde staining reagent. Column
chromatography was carried out using 230–400 mesh silica gel (kieselgel 60, Merck). Melting points
were measured on a Thomas Scientific capillary melting point apparatus (Electrothermal 9300,
Swedesboro, NJ, USA) and are uncorrected. Infra red (IR) spectra were recorded on a Bruker IFS66
(Bruker, Karlsruhe, Germany) infrared Fourier transform spectrophotometer (KBr) and UV spectra
were measured on a Beckman DU650 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). 1H-
13
and C-NMR along with 2D-NMR data were obtained on a Bruker AM 500 (1H-NMR at 500 MHz,
13C-NMR at 125 MHz) spectrometer (Bruker, Karlsruhe, Germany) in CDCl3, acetone-d6, DMSO-d6,
and CD3OD. EIMS was obtained on a JEOL JMS-700 mass spectrometer (JEOL, Tokyo, Japan). All
the reagent grade chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
3.2. Synthesis of Chalcone Derivatives
To a solution of aminoacetophenone derivative (50 mmol) in methanol (100 mL) was added
benzaldehyde derivative (60 mmol) with a catalytic amount of H2SO4. The reaction mixture was
refluxed for 24 h and neutralized with 1.5% NaOH (50 mL). The mixture was extracted with EtOAc,
and dried over anhydrous Na2SO4. After evaporation of EtOAc layer, the residue was purified by silica
gel column chromatography eluting with hexane/EtOAc to give the pure chalcone derivatives.
1
4′-Amino-4-hydroxychalcone (1). m.p. 79–80 °C; IR (KBr): 3510, 1665 cm−1; H-NMR (300 MHz;
MeOD) δ 6.44 (2H, dd, J1 = 6.9, J2 = 1.9 Hz), 6.85 (2H, dd, J1 = 6.9, J2 = 1.8 Hz), 7.55 (3H, m), 7.68
(1H, d, J = 15.5 Hz), 7.91 (2H, dd, J1 = 7.9, J2 = 2.0 Hz); 13C-NMR (75 MHz; MeOD) δ 113.1, 115.5,
118.5, 126.5, 126.8, 130.1, 131.0, 143.4, 153.8, 159.7 and 189.1; EIMS m/z 239 [M+]; HREIMS m/z
239.0948 [M+] (calculated for C15H13NO2, 239.0946).
4′-Amino-3,4-dihydroxychalcone (2). m.p. 184–185 °C; IR (KBr): 3400, 1780 cm−1; 1H-NMR (300 MHz;
MeOD) δ 6.57 (2H, dd, J1 = 8.8, J2 = 1.9 Hz), 6.83 (2H, d, J = 9.0 Hz), 7.08 (1H, dd, J1 = 8.2, J2 = 2.0 Hz),
7.18 (1H, d, J = 2.0 Hz), 7.50 (1H, d, J = 15.4 Hz), 7.62 (1H, d, J = 15.5 Hz), 7.90 (2H, m); 13C-NMR
(75 MHz; MeOD) δ 113.8, 114.2, 115.2, 118.5, 121.8, 126.5, 127.4, 130.9, 143.7, 145.4, 148.1, 153.9
and 189.1; EIMS m/z 255 [M+]; HREIMS m/z 255.0896 [M+] (calculated for C15H13NO3, 255.0895).
4′-Acetamido-4-hydroxychalcone (3). m.p. 202 °C EIMS m/z 281 [M]+; HREIMS m/z 281.1052, (calcd
1
for C17H15NO3, 281.1052); H-NMR (300 MHz, acetone-d6)  2.05 (3H, s), 6.93 (2H, d, J = 8.7 Hz),
13
7.72 (4H, m), 7.81 (2H, d, J = 8.5 Hz), and 8.11 (2H, d, J = 8.7 Hz); C-NMR (75 MHz)  23.5,
115.8, 118.3, 118.7, 126.8, 129.5, 130.5, 133.3, 143.5, 159.9, 168.5, 175.7, and 187.4.; EIMS m/z 281
[M+]; HREIMS m/z 281.1053 [M+] (calculated for C15H13NO3, 281.1052).

Molecules 2013, 18
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4'-(4-Toluenesulfonamide)-4-hydroxychalcone (4a). m.p.: 105–107 °C; IR (KBr): 3520, 1675 cm−1;
1H-NMR (300 MHz; MeOD) δ 2.25 (3H, s), 6.82 (2H, d, J = 8.6 Hz), 7.23 (4H, m), 7.42 (1H, d,
J = 15.5 Hz), 7.51 (2H, d, J = 8.6 Hz), 7.66 (1H, d, J = 15.5 Hz), 7.73 (2H, d, J = 8.3 Hz), 7.90 (2H,
dd, J1 = 8.7, J2 = 2.0 Hz); 13C-NMR (75 MHz; MeOD) δ 20.1, 115.6, 118.0, 118.5, 126.4, 126.9,
129.4, 129.8, 130.5, 133.5, 136.6, 142.4, 144.1, 145.2, 160.2 and 189.7 (Found; C, 67.16; H, 4.87; N,
3.56; C22H19NO4S requires C, 67.14; H, 4.88; N, 3.57%).
1
4′-(4-Toluenesulfonamido)-chalcone (4b). m.p.: 160–161 °C; H-NMR (300 MHz; CDCl3) δ 2.38 (3H,
s), 7.25 (2H, d, J = 1.92 Hz), 7.28 (2H, s), 7.38 (1H, m), 7.42 (2H, m), 7.50 (1H, d, J = 15.7 Hz), 7.64
13
(1H, m), 7.78 (3H, m), 7.88 (1H, s), 7.94 (2H, d, J = 8.70 Hz); C-NMR (75 MHz; CDCl3) δ 21.6,
119.0, 121.5, 127.3, 128.5, 129.9, 130.2, 130.7, 134.0, 134.8, 135.8, 141.2, 144.5, 144.9, 189.1; EIMS
m/z 377 [M+]; HREIMS m/z 377.1083 [M+] (calculated for C22H19NO3S, 377.1086).
4′-(Benzenesulfonamide)-4-hydroxychalcone (4c). m.p. 206−208 °C; IR (KBr): 3535, 1655 cm−1;
1H-NMR (300 MHz; MeOD) δ 6.84 (2H, d, J = 8.6 Hz), 7.27 (2H, d, J = 8.7 Hz), 7.55 (6H, m), 7.71
(1H, d, J = 15.5 Hz), 7.88 (2H, d, J = 8.6 Hz), 7.96 (2H, d, J = 8.7 Hz); 13C-NMR (75 MHz; MeOD) δ
115.5, 117.9, 118.5, 126.3, 126.8, 128.4, 129.7, 130.5, 132.8, 133.6, 139.7, 142.3, 145.2, 160.3 and
189.6; EIMS m/z 379 [M+]; HREIMS m/z 379.0873 [M+] (calculated for C21H17NO4S, 379.0878).
4′-(4-Hydroxybenzenesulfonamide)-4-hydroxychalcone (4d). m.p. 102−103 °C; IR (KBr): 3525, 1670 cm−1;
1H-NMR (300 MHz; Acetone-d6) δ 6.94 (3H, m), 7.37 (1H, d, J = 8.7 Hz), 7.67 (3H, m), 7.77 (2H, d,
J = 8.8 Hz), 8.04 (2H, m), 8.70 (3H, s); 13C-NMR (75 MHz; Acetone-d6) δ 115.6, 115.8, 118.4, 118.5,
126.8, 129.6, 128.9, 130.3, 130.7, 133.7, 142.4, 144.0, 159.9, 161.5 and 187.6; EIMS m/z 395 [M+];
HREIMS m/z 395.0830 [M+] (calculated for C21H17NO5S, 395.0827).
4′-(4-Methoxybenzenesulfonamido)-4-hydroxychalcone (4e). m.p. 90−91 °C; IR (KBr): 3400, 1780 cm−1;
1H-NMR (300 MHz; MeOD) δ 3.84 (3H, s), 6.93 (2H, d, J = 1.9 Hz), 7.06 (2H, d, J = 2.1 Hz), 7.39
(2H, d, J = 1.9 Hz), 7.85 (4H, m), 8.05 (2H, d, J = 1.8 Hz), 8.07 (2H, d, J = 1.9 Hz); 13C-NMR (75 MHz;
MeOD) δ 55.2, 114.3, 115.9, 18.5, 126.8, 129.3, 129.9, 130.7, 131.3, 133.8, 142.3, 143.9, 159.9, 163.3
and 187.5; EIMS m/z 409 [M+]; HREIMS m/z 409.0985 [M+] (calculated for C22H19NO5S, 409.0984).
4′-(4-Aminobenzenesulfonamide)-4-hydroxychalcone (4f). m.p. 216−217 °C; IR (KBr): 3526, 1663 cm−1;
1H-NMR (300 MHz; Acetone-d6) δ 6.70 (2H, d, J = 8.7), 6.93 (2H, d, J = 8.6 Hz), 7.37 (2H, d, J = 8.7 Hz),
7.61 (2H, d, J = 8.8 Hz), 7.70 (4H, m) and 8.06 (2H, d, J = 8.7 Hz); 13C-NMR (75 MHz; Acetone-d6) δ
113.1, 115.9, 118.2, 118.6, 125.7, 126.9, 129.2, 129.8, 130.6, 133.4, 142.9, 143.9, 153.1, 159.9 and
187.6; EIMS m/z 394 [M+]; HREIMS m/z 394.0985 [M+] (calculated for C21H18N2O4S, 394.0987).
4′-(4-Nitrobenzenesulfonamide)-4-hydroxychalcone (4g). m.p. 136−138 °C; IR (KBr): 3513, 1656 cm−1;
1H-NMR (300 MHz; Acetone-d6) δ 6.92 (2H, d, J = 8.6 Hz), 7.42 (2H, d, J = 8.7 Hz), 7.68 (4H, m),
13
8.08 (2H, d, J = 8.7 Hz), 8.17 (2H, d, J = 8.9 Hz) and 8.41 (2H, d, J = 8.9 Hz); C-NMR (75 MHz;
Acetone-d6) δ 116.3, 118.7, 119.3, 125.3, 126.3, 128.8, 130.5, 131.4, 134.2, 141.7, 144.6, 145.1, 150.5,
160.6 and 187.9; EIMS m/z 424 [M+]; HREIMS m/z 424.0728 [M+] (calculated for C21H16N2O6S,
424.0729).

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4′-(4-Fluorobenzenesulfonamide)-4-hydroxychalcone (4h). m.p. 179−180 °C; IR (KBr): 3544, 1671 cm−1;
1H-NMR (300 MHz; MeOD) δ 6.84 (2H, d, J = 8.6 Hz), 7.26 (4H, m), 7.55 (3H, m), 7.72 (1H, d,
J = 15.5 Hz) and 7.94 (4H, m); 13C-NMR (75 MHz; MeOD) δ 115.5, 115.8, 116.1, 117.9, 118.7, 126.3,
129.7, 129.9, 130.5, 133.8, 135.8, 142.1, 145.2, 160.3 and 189.6; EIMS m/z 397 [M+]; HREIMS m/z
397.0782 [M+] (calculated for C21H16FNO4S, 397.0784).
4'-(4-Toluenesulfonamide)-3,4-dihydroxychalcone (5a). m.p.: 179–180 °C; IR (KBr): 3480, 1685 cm−1;
1H-NMR (300 MHz; MeOD+DMSO-d6) δ 2.29 (3H, s), 6.78 (1H, d, J = 8.2 Hz), 6.94 (1H, dd, J1 = 8.2,
J2 = 2.0 Hz), 7.10 (1H, d, J = 2.0 Hz), 7.19 (5H, m), 7.50 (1H, d, J = 15.2 Hz), 7.66 (2H, d, J = 8.2 Hz),
13
7.76 (2H, d, J = 8.6 Hz); C-NMR (75 MHz; MeOD+DMSO-d6) δ 21.4, 115.0, 115.9, 118.6, 122.0,
127.0, 129.6, 129.7, 133.6, 136.9, 142.2, 143.6, 145.0, 145.4, 148.3 and 188.7; EIMS m/z 409 [M+];
HREIMS m/z 409.0985 [M+] (calculated for C22H19NO5S, 409.0984).
4′-(Benzenesulfonamide)-3,4-dihydroxychalcone (5b). m.p. 206–208 °C; IR (KBr): 3535, 1655 cm−1;
1H-NMR (300 MHz; MeOD) δ 3.36 (3H, s), 6.82 (1H, d, J = 8.2 Hz), 7.02 (1H, d, J = 8.7 Hz), 7.09
(1H, d, J = 1.9 Hz), 7.27 (2H, d, J = 8.6 Hz), 7.46 (1H, d, J = 15.5 Hz), 7.56 (2H, m), 7.64 (1H, d,
J = 15.5 Hz), 7.87 (1H, d, J = 8.7 Hz), 7.95 (1H, d, J = 8.6 Hz); 13C-NMR (75 MHz; MeOD) δ 113.5,
116.8, 117.2, 120.4, 121.3, 127.3, 127.9, 129.1, 129.2, 130.7, 132.0, 139.7, 143.5, 145.2, 146.5, 147.2
and 189.7; EIMS m/z 395 [M+]; HREIMS m/z 395.0828 [M+] (calculated for C21H17NO5S, 395.0827).
4′-(4-Hydroxybenzenesulfonamide)-3,4-hydroxychalcone (5c). m.p. 102–103 °C; IR (KBr): 3525,
1
1670 cm−1; H-NMR (300 MHz; Acetone-d6) δ 6.65 (1H, m), 6.88 (2H, m), 7.64 (3H, m), 8.04 (2H, d,
J = 8.3 Hz); 13C NMR (75 MHz; Acetone-d6) δ 113.5, 115.8, 116.2, 116.8, 117.0, 117.2, 120.4, 121.3,
127.9, 128.7, 128.9, 130.7, 130.8, 132.3, 143.5, 145.2, 146.5, 147.2, 161.7, and 189.7; EIMS m/z 411
[M+]; HREIMS m/z 411.0778 [M+] (calculated for C21H17NO6S, 411.0777).
4′-(4-Aminobenzenesulfonamide)-3,4-dihydroxychalcone (5d). m.p. 216–217 °C; IR (KBr): 3526,
1
1663 cm−1; H-NMR (300 MHz; MeOD) δ 6.70 (2H, m), 6.91 (1H, m), 7.19 (1H, m), 7.36 (3H, m),
13
7.61 (4H, m), 8.05 (2H, m); C-NMR (75 MHz; MeOD) δ 113.1, 114.9, 115.5, 118.2, 118.7, 122.3,
125.7, 127.5, 129.2, 129.8, 133.4, 142.8, 144.2, 145.4, 148.0, 153.0 and 187.6; EIMS m/z 410 [M+];
HREIMS m/z 410.0937 [M+] (calculated for C21H16N2O5S, 410.0936).
4′-(4-Nitrobenzenesulfonamide)-3,4-dihydroxychalcone (5e). m.p. 136–138 °C; IR (KBr): 3513,
1656 cm−1; 1H-NMR (300 MHz; MeOD) δ 6.90 (1H, d, J = 8.2 Hz), 7.19 (1H, dd, J1 = 2.0, J2 = 1.9 Hz),
7.31 (1H, d, J = 1.7 Hz), 7.42 (2H, d, J = 8.7 Hz), 7.61 (2H, m), 8.07 (2H, d, J = 8.7 Hz), 8.17 (2H, d,
J = 8.9 Hz), 8.41 (2H, d, J = 8.9 Hz); 13C-NMR (75 MHz; MeOD) δ 114.9, 115.5, 118.6, 119.5, 122.3,
124.5, 127.4, 128.6, 129.9, 134.8, 141.1, 144.5, 145.1, 145.5, 148.1, 150.5 and 187.5; EIMS m/z 440
[M+]; HREIMS m/z 440.0679 [M+] (calculated for C21H16N2O7S, 440.0678).
4′-(4-Fluorobenzenesulfonamide)-3,4-dihydroxychalcone (5f). m.p. 136–138 °C; IR (KBr): 3513,
1656 cm−1; 1H-NMR (300 MHz; MeOD) δ 6.82 (1H, d, J = 8.2 Hz), 7.10 (1H, dd, J1 = 1.8, J2 = 1.7 Hz),
7.17 (1H, s), 7.27 (4H, m), 7.46 (1H, d, J = 15.5 Hz), 7.66 (1H, d, J = 15.4 Hz), 7.95 (4H, m);
13C-NMR (75 MHz; MeOD) δ 113.5, 115.8, 116.8, 117.2, 120.4, 121.3, 127.9, 128.9, 130.2, 130.7,

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135.3, 143.5, 145.2, 146.5, 147.2, 166.1 and 189.7; EIMS m/z 413 [M+]; HREIMS m/z 413.0734 [M+]
(calculated for C21H16FNO5S, 413.0733).
4'-(4-Toluenesulfonamide)-2,4-dihydroxychalcone (6). m.p.: 105–107 °C; IR (KBr): 3520, 1675 cm−1;
1H-NMR (300 MHz; MeOD) δ 2.36 (3H, s), 6.35 (1H, m), 6.39 (2H, m), 7.33 (5H, m), 7.78 (3H, m),
7.91 (2H, m); 13C-NMR (75 MHz; MeOD) δ 24.3, 103.5, 108.4, 109.1, 116.8, 121.3, 127.2,
127.9,129.2, 129.3, 130.7, 136.7, 141.6, 143.5, 145.3, 159.1, 159.7 and 189.7; EIMS m/z 409 [M+];
HREIMS m/z 409.0985 [M+] (calculated for C22H19NO5S, 409.0984).
4'-(4-Toluenesulfonamide)-2,5-dihydroxychalcone (7). m.p.: 105–107 °C; IR (KBr): 3520, 1675 cm−1;
1H-NMR (300 MHz; MeOD) δ 2.31 (3H, s), 6.76 (2H, d, J = 2.7 Hz), 7.08 (1H, d, J = 2.2 Hz), 7.26
(4H, m), 7.64 (1H, d, J = 15.9 Hz), 7.73 (2H, d, J = 8.3 Hz), 7.91 (2H, d, J = 8.7 Hz), 8.05 (1H, d,
13
J = 15.7 Hz); C-NMR (75 MHz; MeOD) δ 20.1, 113.7, 116.7, 118.4, 119.4, 120.7, 122.1, 126.9,
129.4, 129.8, 130.7, 133.5, 136.6, 140.7, 142.4, 144.1, 149.9, 150.8 and 190.2; EIMS m/z 409 [M+];
HREIMS m/z 409.0985 [M+] (calculated for C22H19NO5S, 409.0984).
3.3. Pharmacology
3.3.1. BACE1 Enzyme Assay
The assay was carried out according to the supplied manual with modifications. Briefly, a mixture
of 10 μL of assay buffer (50 mM sodium acetate, pH 4.5), 10 μL of BACE1 (1.0 U/mL), 10 μL of the
substrate (750 nM Rh-EVNLDAEFK-Quencher in 50 mM sodium acetate, pH 4.5), and 10 μL of
sample dissolved in 50 mM ammonium bicarbonate buffer (pH 7.8) was incubated for 60 min at room
temperature in the dark. The mixture was irradiated at 545 nm and the emission intensity at 590 nm
was recorded [21,22]. The inhibition ratio was obtained by the following equation:
Inhibition (%) = [1 − {(S−S0)/(C−C0)}] × 100
where C was the fluorescence of the control (enzyme, buffer, and substrate) after 60 min of incubation,
C0 was the fluorescence of control at zero time, S was the fluorescence of the tested samples (enzyme,
sample solution, and substrate) after incubation, and S0 was the fluorescence of the tested samples at
zero time. To allow for the quenching effect of the samples, the sample solution was added to the
reaction mixture C, and any reduction in fluorescence by the sample was then investigated. All data are
the mean of three experiments.
3.3.2. Slow and Time-Dependent Inhibitory Activity
Slow and time-dependent assays and progress curves were carried out using 1 U/mL unit BACE1,
and 10 μL substrate in 50 mM ammonium bicarbonate pH 7.8 at 37 °C. Enzyme activities were
measured continuously for 3,600 s spectrophotometrically. To determine the kinetic parameters
associated with time dependent inhibition of BACE1, progress curves for 3,600 s were obtained at one
inhibitor concentration using fixed substrate concentrations [23,24]. The data were analyzed using the
nonlinear regression program [Sigma Plot (SPCC Inc., Chicago, IL, USA)].

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3.3.3. Cholinesterase Inhibitory Activity
Cholinesterase was assayed according to standard procedures by following the hydrolysis of
acetylthiocholine iodide (AtCh) or butyrylthiocholine iodide (BtCh) by monitoring formation of
5-thio-2-nitrobenzoate from 5-5'-dithiobis(2-nitrobenzoic acid) spectrometrically at 412 nm [25–27].
The reaction mixture contained 100 mM sodium phosphate buffer (pH 8.0), 20 μL of test sample
solution and either 2 μL of human erythrocyte AChE (0.25 U/mL) or 2 μL of equine serum BChE
(0.05 U/mL) solution, which were mixed and incubated for 10 min at room temperature. Assays were
then initiated with the addition of 30 μL 5-5'-dithiobis(2-nitrobenzoic acid) (3.3 mM) and, respectively,
20 μL of AtCh (1 mM), and 7 μL of BtCh (1 mM). All assays were performed in triplicate.
Compounds showing the highest inhibitory activities were further characterized by determining the
concentration required to inhibit 50% of the enzyme activity under the assay conditions (defined as the
IC50 value). Kinetic parameters were determined using the Lineweaver-Burk double-reciprocal-plot
method at increasing concentration of substrates and inhibitors. The inhibitor eserine (Sigma-Aldrich,
St. Louis, MO, USA) was used in the assays for comparison.
3.3.4. Statistical Analysis
All the measurements were made in triplicate. The results were subject to variance analysis using
Sigma plot. Differences were considered significant at p < 0.05.
4. Conclusions
We set out to evaluate sulfonamide chalcones as inhibitors of an enzyme which is key in the
progression of AD, BACE1. Our work shows that these small molecules are actually very potent
inhibitors of this enzyme. Some of these compounds possess IC50s in the nanomolar range. Thus our
sulfonamide chalcones represent valuable lead compounds and could be useful in further
experimentation. Our own SAR studies have shown that electron donating groups are favored on the
sulfonamide function, whereas a 3,4-dihydroxy motif is preferred in the chalcone B ring. This
information can also serve for further optimization in the future.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/1/140/s1.
Acknowledgments
This work was carried out with the support of the Cooperative Research Program for Agriculture
Science & Technology Development (No. PJ00850601) and a grant from the Next-Generation
BioGreen 21 program (SSAC, No. PJ00810701), Rural Development Administration, Republic of
Korea. All students were supported by a scholarship from the BK21 program.

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References
1. 2012 Alzheimer’s Disease Facts and Figures. Available online: http://www.alz.org/downloads/
facts_figures_2012.pdf (accessed on 16 December 2012).
2. Sjöbeck, M.; Haglund, M.; Englund, E. Decreasing myelin density reflected increasing white
matter pathology in Alzheimer’s disease—A neuropathological study. Int. J. Geriatr. Psychiatry
2005, 20, 919–926.
3. Hardy, J.; Allsop, D. Amyloid deposition as the central event in the aetiology of Alzheimer’s
disease. Trends Pharmacol. Sci. 1991, 12, 383–388.
4. Lee, V.M.Y.; Goedert, M.; Trojanowski, J.Q. Neurodegenerative tauopathies. Annu. Rev. Neurosci.
2001, 24, 1121–1259.
5. Vassar, R.; Bennett, B.D.; Babu-Khan, S.; Kahn, S.; Mendiaz, E.A.; Denis, P.; Teplow, D.B.;
Ross, S.; Amarante, P.; Loeloff, R.; et al. β-Secretase cleavage of Alzheimer’s amyloid precursor
protein by the transmembrane aspartic protease BACE. Science 1999, 286, 735–741.
6. Sinha, S.; Anderson, J.P.; Barbour, R.; Basi, G.S.; Caccavello, R.; Davis, D.; Doan, M.; Dovey, H.F.;
Frigon, N.; Hong, J.; et al. Purification and cloning of amyloid precursor protein β-secretase from
human brain. Nature 1999, 402, 533–537.
7. Yan, R.; Bienkowski, M.J.; Shuck, M.E.; Miao, H.; Tory, M.C.; Pauley, A.M.; Brashler, J.R.;
Stratman, N.C.; Mathews, W.R.; Buhl, A.E.; et al. Membrane-anchored aspartyl protease with
Alzheimer’s disease β-secretase activity. Nature 1999, 402, 537–540.
8. Hussain, I.; Powell, D.; Howlett, D.R.; Tew, D.G.; Meek, T.D.; Chapman, C.; Gloger, I.S.;
Murphy, K.E.; Southan, C.D.; Ryan, D.M.; et al. Identification of a novel aspartic protease (Asp 2)
as β-secretase. Mol. Cell. Neurosci. 1999, 14, 419–427.
9. Yang, L.B.; Lindholm, K.; Yan, R.; Citron, M.; Xia, W.; Yang, X.L.; Beach, T.; Sue, L.; Wong, P.;
Price, D.; et al. Elevated β-secretase expression and enzymatic activity detected in sporadic
Alzheimer disease. Nat. Med. 2003, 9, 3–4.
10. Fukumoto, H.; Cheung, B.S.; Hyman, B.T.; Irizarry, M.C. β-Secretase protein and activity are
increased in the neocortex in Alzheimer disease. Arch. Neurol. 2002, 59, 1381–1389.
11. Holsinger, R.M.D.; McLean, C.A.; Beyreuther, K.; Masters, C.L.; Evin, G. Increased expression
of the amyloid precursor β-secretase in Alzheimer’s disease. Ann. Neurol. 2002, 51, 783–786.
12. McConlogue, L.; Buttini, M.; Anderson, J.P.; Brigham, E.F.; Chen, K.S.; Freedman, S.B.; Games, D.;
Johnson-Wood, J.; Lee, M.; Zeller, M.; et al. Partial reduction of BACE1 has dramatic effects on
Alzheimer plaque and synaptic pathology in APP transgenic Mice. J. Biol. Chem. 2007, 282,
26326–26334.
13. Garcia-Ayllon, M.S.; Small, D.H.; Avila, J.; Saez-Valero, J. Revisiting the role of
acetylcholinesterase in Alzheimer’s disease: cross-talk with P-tau and β-amyloid. Front. Mol.
Neurosci. 2011, 22, 1–9.
14. Arneric, S.P.; Holladay, M.; Williams, M. Neuronal nicotinic receptors: A perspective on two
decades of drug discovery research. Biochem. Pharmacol. 2007, 74, 1092–1101.
15. Asai, M.; Hattori, C.; Iwata, N.; Saido, T.C.; Sasagawa, N.; Szabo, B.; Hashimoto, Y.; Maruyama, K.;
Tanuma, S.; Kiso, Y.; et al. The novel β-secretase inhibitor KMI-429 reduces amyloid β peptide

Molecules 2013, 18
153
production in amyloid precursor protein transgenic and wild-type mice. J. Neurochem. 2006, 96,
533–540.
16. Vassar, R. Beta-secretase (BACE) as a drug target for Alzheimer’s disease. Drug Deliv. Rev. 2002,
54, 1589–1602.
17. Lee, S.A.; Ryu, H.W.; Kim, Y.M.; Choi, S.; Lee, M.J.; Kwak, T.K.; Kim, H.J.; Cho, M.; Park, K.H.;
Lee, J.W. Blockade of Four-Transmembrane L6 Family Member 5 (TM4SF5)-Mediated
Tumorigenicity in Hepatocytes by a Synthetic Chalcone Derivative. Hepatology 2009, 49,
1316–1325.
18. Seo, W.D.; Kim, J.H.; Kang, J.E.; Ryu, H.W.; Curtis-Long, M.J.; Lee, H.S.; Yang, M.S.; Park, K.H.
Sulfonamide chalcone as a new class of α-glucosidase inhibitors. Bioorg. Med. Chem. Lett. 2005,
15, 5514–5516.
19. Yarishkin, O.V.; Ryu, H.W.; Park, J.Y.; Yang, M.S.; Hong, S.G.; Park, K.H. Sulfonate chalcone
as new class voltage-dependent K+ channel blocker. Bioorg. Med. Chem. Lett. 2008, 18, 137–140.
20. Ryu, H.W.; Curtis-Long, M.J.; Jung, S.; Jeong, I.Y.; Kim, D.S.; Kang, K.Y.; Park, K.H.
Anticholinesterase potential of flavonols from paper mulberry (Broussonetia papyrifera) and their
kinetic studies. Food Chem. 2012, 132, 1244–1250.
21. Jeon, S.Y.; Bae, K.; Seong, Y.H.; Song, K.S. Green tea catechins as a BACE1 (β-Secretase)
inhibitor. Bioorg. Med. Chem. Lett. 2003, 13, 3905–3908.
22. Hwang, E.M.; Ryu, Y.B.; Kim, H.Y.; Kim, D.G.; Hong, S.G.; Lee, J.H.; Curtis-Long, M.J.;
Jeong, S.H.; Park, J.Y.; Park, K.H. BACE1 inhibitory effects of lavandulyl flavanones from
Sophora flavescens. Bioorg. Med. Chem. 2008, 16, 6669–6674.
23. Li, Y.Q.; Zhou, F.C.; Bian, J.S.; Shan, F. Comparative Evaluation of Quercetin, Isoquercetin and
Rutin as Inhibitors of α-Glucosidase. J. Agric. Food Chem. 2009, 57, 11463–11468.
24. Zhan, J.W.; Wang, Z.J.; Yan, Y.J.; Xiang, W.S. Comparative Studies on the Interaction of
Genistein, 8-Chlorogenistein, and 3',8-Dichlorogenistein with Bovine Serum Albumin. J. Agric.
Food Chem. 2011, 59, 7506–7513.
25. Ellman, G.L.; Courtney, K.D.; Andres, V.; Featherstone, R.M. A new and rapid colorimetric
determination of acetylcholinesterase actiity. Biochem. Pharmacol. 1961, 7, 88–95.
26. Gabrovska, K.; Marinov, I.; Godjevargova, T.; Portaccio, M.; Lepore, M.; Grano, V.; Diano, N.;
Mita, D.G. The influence of the support nature on the kinetics parameters, inhibition constants
and reactivation of immobilized acetylcholinesterase. Int. J. Biol. Macromol. 2008, 43, 339–345.
27. Kamal, M.A.; Klein, P.; Luo, W.; Li, Y.; Holloway, H.W.; Tweedie, D.; Greig, N.H. Kinetics of
human serum butyrylcholinesterase inhibition by a novel experimental Alzheimer therapeutic,
dihydrobenzodioxepine cymserine. Neurochem. Res. 2008, 33, 745–753.
Sample Availability: Samples of the compounds (1–7) are available from the authors.
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distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).