Synthesis and biological evaluation of helicid analogues as novel acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2007.03.018

A series of helicid analogues were prepared and evaluated in vitro for the cholinesterase (AChE and BuChE) inhibitory activities via UV spectroscopy. The results indicated that compounds 5, 6d and 8 exhibited potent AChE inhibitory activities with IC₅₀ values of 0.45 ± 0.02 μM, 0.49 ± 0.02 μM, and 0.20 ± 0.01 μM, respectively. High selectivity for AChE over BuChE was also observed. Kinetic study showed that the mechanism of AChE inhibition of compounds 5, 6d and 8 was all mixed-type.

Full text of DOI:10.1016/j.ejmech.2007.03.018

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 166e173
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological evaluation of helicid analogues as
novel acetylcholinesterase inhibitors
Huan Wen a, Chonglan Lin a, Ling Que a, Hui Ge a, Lin Ma a, Rihui Cao a,
Yiqian Wan a, Wenlie Peng b, Zihou Wang c, Huacan Song a,
*
a School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China
b School of Life Science, Sun Yat-sen University, Guangzhou 510275, China
c Centre for Drug Evaluation, State Drug Administration, Beijing 100050, China
Received 13 November 2006; received in revised form 12 March 2007; accepted 15 March 2007
Available online 5 April 2007
Abstract
A series of helicid analogues were prepared and evaluated in vitro for the cholinesterase (AChE and BuChE) inhibitory activities via UV
spectroscopy. The results indicated that compounds 5, 6d and 8 exhibited potent AChE inhibitory activities with IC50 values of
0.45 ꢀ 0.02 mM, 0.49 ꢀ 0.02 mM, and 0.20 ꢀ 0.01 mM, respectively. High selectivity for AChE over BuChE was also observed. Kinetic study
showed that the mechanism of AChE inhibition of compounds 5, 6d and 8 was all mixed-type.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Helicid analogues; 4-Hydroxybenzaldeyde; Cholinesterase inhibitor
1. Introduction
Recent studies focus on the complex nature of AD and
disclose the involvement of several other transmitter sys-
tems, such as serotonin (5-HT), noradrenalin (NA), dopa-
mine, histamine, excitatory amino acids and neuropeptides
[11]. Non-cholinergic neurotransmitter systems undergo se-
vere atrophy throughout the course of the disease. It is
also apparent that extensive neuropathology is common in
areas normally rich in 5-HT and NA, particularly in the
later stages of AD [12]. The multifunctional pathogenesis
of AD suggests that molecules with two or more mecha-
nisms of action, acting in a complementary manner, could
be more efficacious for AD patients. The present tendency
to develop new drugs of AD is to look for inhibitors of
AChE endowed with additional therapeutic effects, such as
anti-inflammatory and antioxidant agents, activation of other
neurotransmitter systems like GABA or monoaminergic sys-
tems [13,14].
Alzheimer’s disease (AD), a neurodegenerative disorder, is
one of the severe health problems of aged population [1]. A def-
icit in cholinergic neurotransmission was believed to be one of
the major causes of the memory impairments in AD patients
in the past decades [2,3]. The rational approach to treat AD is
to restore the acetylcholine (ACh) levels by inhibiting acetyl-
cholinesterase (AChE) with highly selective inhibitors [4].
Four AChE inhibitors (Fig. 1), tacrine, donepezil, galanthamine,
and rivastigmine, have been approved by FDA for the treatment
of AD [5,6]. Unfortunately, the potential effectiveness offered
by these inhibitors is often limited by the appearance of central
and peripheral side effects. Clinical studies have shown that tac-
rine has hepatotoxic liability [7], physostigmine has poor oral
activity [8], and rivastigmine has short half-life [9].
Natural helicid (1), one major active ingredient of Chinese
herb medicine, has been used clinically as antalgic and hyp-
notic for a long time in China, and no obvious side effect
has been reported [15,16]. Wang and his co-workers more
* Corresponding author. Tel.: þ86 20 8411 0918; fax: þ86 20 8411 2245.
E-mail address: yjhxhc@mail.sysu.edu.cn (H. Song).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.03.018

H. Wen et al. / European Journal of Medicinal Chemistry 43 (2008) 166e173
167
OH
N
NH2
O
CH3
CH3
N
O
H3C
N
H3CO
O
CH3
H3CO
H3CO
N
CH3
O
N
CH3
Galanthamine
Rivastigmine
Donepezil
Tacrine
Fig. 1. Structure of AChE inhibitors.
recently indicated that helicid could activate neurotransmitter
systems of 5-HT and NA in mice, resulting in an effective an-
tidepressant activity [17]. The aglycon of helicid, 4-hydroxy-
benzaldehyde, was reported as a potent antioxidant agent and
GABA transaminase inhibitor (IC50 ¼ 4.1 mg/mL) [18]. A
further study suggested that the aldehyde group and the hy-
droxyl group at C-4 are necessary for the actions [19]. Based
on our high throughput screening work, 4-hydroxybenzalde-
hyde also showed weak activity towards AChE, and its
IC50 value was 37.8 mM. This result prompted us to study
4-hydroxybenzaldehyde glycoside derivatives, analogues of
helicid, with the aim to explore new AChE inhibitors with
high activity, low toxicity and additional activation property
of other neurotransmitter systems like GABA, 5-HT and NA.
(BTC) in comparison with reference compound galanth-
amine-HBr, using the slightly modified method of Ellman
et al. [25].
According to the data in Table 1, the following results could
be found.
(1) Natural helicid (1) was not active up to 500 mM towards
AChE, whereas the synthetic helicid analogues 3a, 4c, 5,
6c, 6d, 7b, and 8 showed significant activities with IC50
values of less than 10 mM. Helicid analogues 5, 6d and
8, bearing several hydrophilic hydroxyl groups on the
sugar moiety, exhibited much higher AChE inhibitory ac-
tivity than the others, suggesting that the hydrogen atoms
of these hydroxyl groups might form hydrogen bond with
nitrogen or oxygen atoms of enzyme molecules. The sugar
moiety acted like a bridge to link the 4-formylphenoxy
moiety and enzyme molecules with these H-bonds, which
facilitated the 4-formylphenoxy moiety and enzyme mol-
ecules to interact and made the interaction stable.
(2) More interestingly, the configuration of sugar moiety
greatly affected the activity towards AChE. Compounds
2 and 3c were inactive towards AChE, similar to 1. How-
ever, compounds 3aeb and 3dee exhibited moderate to
high activities. It is indicated that the sugar moieties, glu-
cose, galactose, xylose and ribose were much preferable to
allose and lactose in inhibition of AChE.
(3) Compound 3a was 3-fold less active than unprotected gly-
coside 5 towards AChE, while 3e was 70-fold less active
than 8, which suggested that the introduction of the acetyl
or benzoyl groups on the sugar moieties led to a decrease
in AChE inhibitory effect. Benzoyl glycosides were less
potent than acetyl glycosides.
(4) The attachment of Schiff base moieties (4aed, 6aed and
7aed) did not effectively increase activity towards AChE.
Even the best one, compound 6d, was slightly less active
than 5.
2. Chemistry
In general, helicid analogues 3e8 were synthesized starting
from 4-hydroxybenzaldehyde, followed by glycosidation, de-
protection and condensation with amines (Schemes 2e4). In
the case of compound 2 (Scheme 1), it was obtained through
acetylation of helicid (1).
Five alpha glycosyl bromide compounds were firstly pre-
pared using the methods described elsewhere [20e22]. In
the presence of NaOH and tetrabutylammonium bromide
(TBAB), glycosyl bromide reacted with 4-hydroxybenzalde-
hyde to afford the corresponding protected phenyl glycosides
3aee. Beta phenyl glycoside could be obtained as a major
product using TBAB as phase-transfer catalyst [23,24]. Subse-
quent reaction of 3a and 3e with sodium methoxide in metha-
nol yielded the sugar-linked helicid analogues 5 and 8. Schiff
base derivatives 4aed, 6aed and 7aed were synthesized, re-
spectively, by the reaction of 3a, 3e and 5 with four amines
(thiosemicarbazide, semicarbazide, hydroxylamine and
methoxyamine).
(5) Regarding the inhibitory potency of BuChE, most heli-
cid analogues were not active, except benzoyl glycosides
3e and 7aed which showed moderate activities. Com-
pounds 5, 6d and 8 exhibited strong selectivity for
AChE over BuChE.
3. Biology
The inhibitory activities of the prepared compounds against
AChE and BuChE were investigated by determining the rate of
hydrolysis of acetylthiocholine (ATC) and butylthiocholine
OAc
OH
O
O
Ac2O,Py,
rt.,12h
AcO
O
CHO
O
CHO
HO
OAc
OH
AcO
HO
1
2
Scheme 1. Preparation of 2.

168
H. Wen et al. / European Journal of Medicinal Chemistry 43 (2008) 166e173
TBAB, NaOH,
CHO
R1O
CHO
HO
CHCl3/H2O,45°C , 6-16h
3
OAc
OAc
OAc
OAc
OAc
O
OAc
O
AcO
O
3b R1
=
3a R1 =
O
AcO
3c R1 =
AcO
O
AcO
OAc
O
AcO
OAc
OAc
OAc
O
BzO
3d R1 = BzO
3e R1 = BzO
BzO
OBz
OBz
Scheme 2. Preparation of 3aee.
We further examined the kinetics of inhibition of AChE by
doublets; m, multiplet. Mass spectra were recorded on
a Thermo Finnigan LCQ DECAXP ion trap mass spectrometer
or VS ZAB-HS spectrometer. IR spectra were recorded as po-
tassium bromide pellets on a Bruker Equinox 55 FT/IR spec-
trometer. Elemental analyses (C, H, N) were carried out on an
Elementary Vario EL series elemental analyzer and the results
were within ꢀ0.4%. Thin-layer chromatographies were done
on pre-coated silica gel 60 F254 plates (Merck). The spots
were visualized with UV light or iodine. All reagents were
used as received unless otherwise stated.
the potent derivatives 5, 6d and 8 (Fig. 2). The results demon-
strated that the mechanism of AChE inhibition of compounds
5, 6d and 8 was all mixed-type, while that of galanthamine-
HBr was competitive. Inhibition constant (Ki) was determined
from non-linear regression analysis by Dixon plot. Table 2
shows that the Ki values corresponding to the inhibition of
AChE with compounds 5, 6d, and 8 were 0.73 mM,
0.13 mM, and 0.51 mM, respectively, and the results were close
to that of galanthamine-HBr (Ki ¼ 0.45 mM) [26].
The following glycosyl bromide compounds were prepared
according to the standard literature procedure. 2,3,4,6-Tetra-
O-acetyl-a-D-glucopyranosyl bromide, white solid powder,
m.p. 87e89 ꢁC (lit. 88e89 ꢁC) [20]; 2,3,4,6-tetra-O-acetyl-a-
D-galapyranosyl bromide, white solid powder, m.p. 74e76 ꢁC
(lit. 75e76 ꢁC) [20]; 20,30,40,60,2,3,6-hepta-O-acetyl-a-D-lacto-
syl bromide, white solid powder, m.p. 122e124 ꢁC (lit. 123e
124 ꢁC) [20]; 2,3,4-tri-O-benzoyl-a-D-xylopyranosyl bromide,
white solid powder, m.p. 135e137 ꢁC (lit. 136e137 ꢁC) [21];
2,3,4-tri-O-benzoyl-a-D-ribopyranosyl bromide, white solid
powder, m.p. 162e165 ꢁC (lit. 164e166 ꢁC) [22].
4. Conclusion
In summary, a series of helicid analogues were synthesized
and evaluated as inhibitors of AChE. The most potent inhibi-
tor, compound 8, which presented an IC50 value of 0.20 mM on
electric eel AChE, was twice more active than galanthamine-
HBr. Our study suggested that helicid analogues might serve
as lead compounds for designing new potential AChE inhibi-
tors with high selectivity, low toxicity and additional pharma-
ceutical effects.
5. Experimental section
5.1.1. Synthesis of 4-formylphenyl (2,3,4,6-tetra-O-acetyl)-
b-D-allopyranoside (2)
5.1. Chemistry
To a solution of helicid (1) (1.0 g, 3.5 mmol) in 2 mL of an-
hydrous pyridine was added dropwise acetyl anhydride (2.5 g,
25 mmol). The mixture was stirred vigorously at room temper-
ature for 12 h, and then poured into 50 mL of ice water. After
30 min, 20 mL of dichloromethane was added. The organic
layer was separated and the aqueous layer was extracted twice
with dichloromethane. The combined organic phase was
washed with 1 M HCl and brine, dried with anhydrous
Melting points were determined with a WRS-1B digital
1
melting point apparatus and were uncorrected. H and 13C
NMR spectra were recorded on a Varian Mercury-Plus 300
NMR instrument (1H 300 MHz; 13C 75 MHz) in either
CDCl3 or DMSO-d6. Abbreviations for data quoted are s, sin-
glet; br s, broad singlet; d, doublet; t, triplet; dd, doublet of
-
N R2
CHO
b
CHO
OAc
OH
OAc
O
O
O
a
HO
HO
AcO
AcO
AcO
AcO
O
O
O
OH
OAc
3a
OAc
6a-d
5
b
6a R2 = NHCSNH2 6b R2 = NHCONH2
6c R2 = OH
-
N R2
6d R2 = OCH3
OAc
O
O
AcO
AcO
4a R2 = NHCSNH2 4b R2 = NHCONH2
4d R2 = OCH3
OAc
4a-d
4c R2 = OH
Scheme 3. Preparation of 4aed, 5 and 6aed. Reagents and conditions: (a) NaOMe, MeOH, rt, 3 h; (b) amine, EtOH, reflux or 45 ꢁC, 2e6 h.

H. Wen et al. / European Journal of Medicinal Chemistry 43 (2008) 166e173
169
-
CHO
a
N R2
CHO
O
O
O
BzO
HO
O
O
b
O
BzO
OBz
OH
OBz
OH
OBz
OBz
8
7a-d
3e
7a R2 = NHCSNH2
7c R2 =OH
7b R2 = NHCONH2
7d R2 =OCH3
Scheme 4. Preparation of 7aed and 8. Reagents and conditions: (a) NaOMe, MeOH, rt, 12 h; (b) amine, EtOH, reflux or 45 ꢁC, 2e6 h.
Na2SO4, and the solvent was removed under reduced pressure
to give light yellow solid. The crude product was recrystallized
in ethanol to yield white solid (2), yield 56%, m.p. 135e
136 ꢁC. IR (KBr, cmꢂ1) n: 1752, 1693, 1601, 1506, 1224,
as solution B). Then solutions A and B were simultaneously
dropped into the above mixture, while the system was stirred
vigorously at 45 ꢁC. After 6e18 h, the organic phase was sep-
arated and washed with NaOH (20 mL, 5%), brine, respec-
tively, dried with anhydrous Na2SO4, and then concentrated
under reduced pressure. The residue was purified by recrystal-
lization or column chromatography on silica gel to afford the
corresponding product.
1
1157, 1127, 1085, 914, 876; H NMR (CDCl3) d ppm: 9.91
(s, 1H, HeC]O), 7.85 (d, 2H, J ¼ 8.6 Hz, ArH), 7.13 (d,
2H, J ¼ 8.6 Hz, ArH), 5.75 (t, 1H, J ¼ 2.9 Hz, H-30), 5.48
(d, 1H, J ¼ 8.1 Hz, H-10), 5.19 (dd, 1H, J ¼ 3.0 Hz, 8.1 Hz,
H-20), 5.16 (dd, 1H, J ¼ 2.8 Hz, 9.9 Hz, H-40), 4.32e4.27
(m, 1H, H-50), 4.26e4.24 (m, 2H, H-60, H-60), 2.18, 2.08,
2.05, 2.04 (4 ꢃ s, 4 ꢃ 3H, 4 ꢃ CH3eC]O); 13C NMR
(CDCl3) d ppm: 191.2, 170.8, 170.6, 170.5, 169.8, 161.7,
132.3, 117.2, 99.0, 71.8, 71.1, 68.8, 67.2, 61.8, 21.2, 21.1,
21.0; ACPI-MS m/z (%): 470 (100) [M þ NH4þ]. Anal. C,
H for C21H24O11.
5.1.2.1. 4-Formylphenyl (2,3,4,6-tetra-O-acetyl)-b-D-glucopyr-
anoside (3a). White solid product, yield 54%, m.p. 141e
142 ꢁC (lit. 142e143 ꢁC) [27]. IR (KBr, cmꢂ1) n: 2963,
2746, 1750, 1737, 1692, 1601, 1222; 1H NMR (CDCl3)
d ppm: 9.90 (s, 1H, HeC]O), 7.83 (d, 2H, J ¼ 8.6 Hz,
ArH), 7.10 (d, 2H, J ¼ 8.6 Hz, ArH), 5.54e5.46 (m, 2H,
2 ꢃ CH), 5.17 (d, 1H, J ¼ 8.1 Hz, H-10), 5.14e5.10 (m, 1H,
CH), 4.25e4.09 (m, 3H, H-50, H-60, H-60), 2.19, 2.06, 2.02
(3 ꢃ s, 4 ꢃ 3H, 4 ꢃ CH3eC]O); ACPI-MS m/z (%): 470
(100) [M þ NHþ4 ].
5.1.2. Synthesis of compounds 3aee
General procedure. A mixture of tetrabutylammonium bro-
mide (4 mmol) in 1:1 waterechloroform (20 mL) was stirred
and heated to 45 ꢁC. Glycosyl bromide (0.02 mol) was dis-
solved in chloroform (20 mL, designated as solution A) and
4-hydroxybenzaldehyde (0.02 mol) was dissolved in 20 mL
of water containing 0.88 g (0.022 mol) of NaOH (designated
5.1.2.2. 4-Formylphenyl (2,3,4,6-tetra-O-acetyl)-b-D-galacto-
pyranoside (3b). White solid product, yield 59%, m.p. 121e
122 ꢁC (lit. 121.5e122.0 ꢁC) [28]. IR (KBr, cmꢂ1) n: 1753,
Table 1
AChE and BuChE inhibitory activities (IC50, mM) of prepared compounds
Compound
R1
IC50 ꢀ SE AChE (mM)
IC50 ꢀ SE BuChE (mM)
na
Selectivity for AChE/BuChE
1
naa
na
2
3a
na
na
1.50 ꢀ 0.22
45.42 ꢀ 4.8
na
3b
3c
na
na
3d
3e
34.96 ꢀ 4.1
14.04 ꢀ 1.64
21.47 ꢀ 3.0
142.58 ꢀ 20
9.15 ꢀ 1.15
15.09 ꢀ 2.8
0.45 ꢀ 0.02
22.89 ꢀ 3.1
120.70 ꢀ 20
4.79 ꢀ 0.61
0.49 ꢀ 0.03
46.43 ꢀ 5.2
7.07 ꢀ 1.05
12.55 ꢀ 2.5
3.08 ꢀ 0.54
0.20 ꢀ 0.01
0.55 ꢀ 0.02b
na
64.27 ꢀ 10
4.58
4a
4b
NHeCSeNH2
NHeCOeNH2
OH
na
na
4c
4d
na
na
OCH3
5
6a
na
na
NHeCSeNH2
NHeCOeNH2
OH
6b
6c
na
na
6d
7a
OCH3
na
NHeCSeNH2
NHeCOeNH2
OH
71.83 ꢀ 12
48.72 ꢀ 8.9
39.60 ꢀ 6.5
45.83 ꢀ 8.4
na
1.55
6.89
7b
7c
3.16
14.88
7d
8
Galanthamine-HBr
OCH3
15.24 ꢀ 3.1
27.70
a
na ¼ not active up to 500 mM.
b
IC50 values reported in the literature: 0.8e0.3 mM.

170
H. Wen et al. / European Journal of Medicinal Chemistry 43 (2008) 166e173
Fig. 2. Double-reciprocal plots of the inhibition kinetics of AChE by compounds 5, 6d and 8.
1694, 1602, 1507, 1371, 1230, 1077, 914, 835; 1H NMR
(CDCl3) d ppm: 9.90 (s, 1H, HeC]O), 7.84 (d, 2H,
J ¼ 8.6 Hz, ArH), 7.10 (d, 2H, J ¼ 8.5 Hz, ArH), 5.45 (dd,
1H, J ¼ 2.7 Hz, 10.3 Hz, H-30), 5.40 (d, 1H, J ¼ 3.5 Hz, H-
20), 5.10 (d, 1H, J ¼ 8.1 Hz, H-10), 5.08 (dd, 1H, J ¼ 3.3 Hz,
10.5 Hz, H-40), 4.25e4.10 (m, 3H, H-50, H-60, H-60), 2.19,
2.06, 2.02 (3 ꢃ s, 4 ꢃ 3H, 4 ꢃ CH3eC]O); ACPI-MS m/z
(%): 470 (100) [M þ NHþ4 ].
67.0, 62.4, 61.2, 21.2, 21.1, 21.0, 20.9; ESI-MS m/z (%):
763 (86) [M þ Na]. Anal. C, H for C33H40O19.
5.1.2.4. 4-Formylphenyl (2,3,4-tri-O-benzoyl)-b-D-xylopyrano-
side (3d). White solid product, yield 46%, m.p. 99 ꢁC. IR
(KBr, cmꢂ1) n: 1726, 1694, 1600, 1506, 1260, 1155, 1092,
1
1026, 831, 708; H NMR (CDCl3) d ppm: 9.90 (s, 1H, He
C]O), 8.10e7.97 (m, 5H, ArH), 7.85 (d, 2H, J ¼ 8.70 Hz,
ArH), 7.48e7.29 (m, 10H, ArH), 7.18 (d, 2H, J ¼ 8.0 Hz,
ArH), 5.73 (t, J ¼ 5.3 Hz, 1H, H-20), 5.65 (d, 1H,
J ¼ 3.5 Hz, H-10), 5.50 (dd, 1H, J ¼ 1.5 Hz, 5.2 Hz, H-30),
5.27 (dd, 1H, J ¼ 3.8 Hz, 8.5 Hz, H-40), 4.46 (dd, 1H,
J ¼ 9.5 Hz, 12.7 Hz, H-50), 3.90 (dd, 1H, J ¼ 8.4 Hz,
12.6 Hz, H-50); 13C NMR (CDCl3) d ppm: 191.2, 166.0,
165.6, 165.4, 161.5, 134.1, 132.4, 131.9, 130.4, 128.9,
117.0, 96.9, 69.0, 68.7, 68.3, 61.0; ACPI-MS m/z (%): 584
(100) [M þ NHþ4 ]. Anal. C, H for C33H26O9.
5.1.2.3. 4-Formylphenyl (20,30,40,60,2,3,6-hepta-O-acetyl)-b-D-
lactoside (3c). White solid product, yield 37%, m.p. 78e
79 ꢁC. IR (KBr, cmꢂ1) n: 1749, 1695, 1602, 1509, 1373,
1
1231, 1051, 902, 835; H NMR (CDCl3) d ppm: 9.90 (s, 1H,
HeC]O), 7.83 (d, 2H, J ¼ 8.7 Hz, ArH), 7.07 (d, 2H,
J ¼ 8.7 Hz, ArH), 5.36 (d, 1H, J ¼ 3.2 Hz, CH), 5.21e5.01
(m, 3H, 3 ꢃ CH), 4.99e4.95 (m, 1H, CH), 4.53e4.50 (m,
2H, 2 ꢃ CH), 4.17e4.05 (m, 4H, 4 ꢃ CH), 3.91e3.85 (m,
2H, 2 ꢃ CH), 2.17, 2.09, 2.08, 2.07, 1.98 (5 ꢃ s, 7 ꢃ 3H,
7 ꢃ CH3eC]O); 13C NMR (CDCl3) d ppm: 191.2, 170.7,
170.6, 170.5, 170.2, 170.0, 169.6, 161.7, 132.2, 117.1,
116.4, 101.6, 98.1, 73.4, 73.1, 71.7, 71.3, 71.2, 69.5, 69.1,
5.1.2.5. 4-Formylphenyl (2,3,4-tri-O-benzoyl)-b-D-ribopyrano-
side (3e). White solid product, yield 27%, m.p. 129e130 ꢁC.
IR (KBr, cmꢂ1) n: 1727, 1694, 1600, 1505, 1260, 1103, 1025,

H. Wen et al. / European Journal of Medicinal Chemistry 43 (2008) 166e173
171
Table 2
Inhibition constant and type of inhibition of selected compoundsa
3a (0.45 g, 1 mmol) in 15 mL of ethanol was added hydrox-
ylamine hydrochloride (0.07 g, 1 mmol) which had been ad-
justed with 2 M NaOH to pH ¼ 6e7. The mixture was
stirred for 2 h at 45 ꢁC. The precipitate was filtered off,
the solvent was removed under reduced pressure, and the
residue was washed with ether and recrystallized in ethanol
to give 4c. White powder, yield 78%, m.p. 161e162 ꢁC. IR
(KBr, cmꢂ1) n: 3454, 1752, 1687, 1376, 1224, 1099, 1054,
Compound
Ki ꢀ SE (mM)
Type of inhibition
5
0.73 ꢀ 0.02
0.51 ꢀ 0.01
0.13 ꢀ 0.004
0.45 ꢀ 0.01
Mixed
Mixed
8
6d
Mixed
Competitive
Galanthamine-HBr
a
Inhibition constants were calculated from kinetic data.
1
952; H NMR (CDCl3) d ppm: 8.07 (s, 1H, eCH]N), 7.50
1
859, 707; H NMR (CDCl3) d ppm: 9.83 (s, 1H, HeC]O),
7.96e7.94 (m, 4H, ArH), 7.80e7.77 (m, 4H, ArH), 7.49e
7.38 (m, 4H, ArH), 7.22e7.17 (m, 7H, ArH), 5.92 (t, 1H,
J ¼ 3.6 Hz, H-20), 5.86 (d, 1H, J ¼ 3.8 Hz, H-10), 5.65e5.63
(m, 1H, H-30), 5.62e5.60 (m, 1H, H-40), 4.22 (d, 1H,
J ¼ 1.8 Hz, 12.7 Hz, H-50), 4.09 (dd, 1H, J ¼ 1.8 Hz,
12.7 Hz, H-50); 13C NMR (CDCl3) d ppm: 191.3, 166.6,
166.4, 165.7, 161.0, 134.0, 132.4, 132.0, 128.9, 117.1, 96.9,
68.7, 67.7, 66.1, 62.7; ACPI-MS m/z (%): 584 (100)
[M þ NHþ4 ]. Anal. C, H for C33H26O9.
(d, J ¼ 8.6 Hz, 2H, ArH), 6.98 (d, 2H, J ¼ 8.6 Hz, ArH),
5.31e5.27 (m, 2H, 2 ꢃ CH), 5.20e5.10 (m, 2H, 2 ꢃ CH),
4.28 (dd, 1H, J ¼ 7.0 Hz, 12.3 Hz, CH), 4.17 (dd, 1H,
J ¼ 10.3 Hz, 12.3 Hz, CH), 3.91e3.86 (m, 1H, CH), 2.07,
2.06, 2.05, 2.04 (4 ꢃ s, 4 ꢃ 3H, 4 ꢃ CH3eC]O); ESI-MS
m/z (%): 468 (5) [M þ 1], 490 (100) [M þ Na]. Anal. C,
H, N for C21H25NO11.
5.1.3.4. 1-(4-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl) ben-
zylidene) methoxyamine (4d). The procedure was the same
as described in the preparation of 4c, starting from methoxy-
amine hydrochloride. White solid powder, yield 68%, m.p.
132 ꢁC. IR (KBr, cmꢂ1) n: 1748, 1608, 1509, 1236, 1046,
5.1.3. Synthesis of compounds 4aed
1
5.1.3.1. 1-(4-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl) ben-
zylidene) thiosemicarbazide (4a). A mixture of compound 3a
(0.45 g, 1 mmol), thiosemicarbazide (0.09 g, 1 mmol) in
15 mL of ethanol was refluxed for 3 h. After being cooled to
room temperature, the precipitate was filtered, washed with
ether, and recrystallized to get white powder (4a), yield 95%,
m.p. 136 ꢁC. IR (KBr, cmꢂ1) n: 3452, 1753, 1579, 1503, 1409,
917; H NMR (CDCl3) d ppm: 8.00 (s, 1H, eCH]N), 7.50
(d, 2H, J ¼ 8.6 Hz, ArH), 6.97 (d, 2H, J ¼ 8.6 Hz, ArH),
5.31e5.26 (m, 2H, 2 ꢃ CH), 5.19e5.09 (m, 2H, 2 ꢃ CH),
4.26 (dd, 1H, J ¼ 7.0 Hz, 12.3 Hz, CH), 4.17 (dd, 1H,
J ¼ 10.4 Hz, 12.4 Hz, CH), 3.95 (s, 3H, OCH3), 3.91e3.85
(m, 1H, CH), 2.08, 2.06, 2.05, 2.04 (4 ꢃ s, 4 ꢃ 3H,
4 ꢃ CH3eC]O); ESI-MS m/z (%): 482 (6) [M þ 1], 504
(100) [M þ Na]. Anal. C, H, N for C22H27NO11.
1
1226, 1092, 1046, 714; H NMR (DMSO-d6) d ppm: 9.94 (s,
1H, NeH), 7.86 (s, 1H, eCH]N), 7.59 (d, 2H, J ¼ 8.6 Hz,
ArH), 7.20 (s, 1H, NeH), 7.00 (d, 2H, J ¼ 8.6 Hz, ArH), 6.50
(s, 1H, NeH), 5.33e5.28 (m, 2H, 2 ꢃ CH), 5.21e5.14 (m,
2H, 2 ꢃ CH), 4.30 (dd, 1H, J ¼ 7.2 Hz, 12.3 Hz, CH), 4.18
(dd, 1H, J ¼ 10.3 Hz, 12.0 Hz, CH), 3.94e3.89 (m, 1H, CH),
2.09, 2.07, 2.06, 2.05 (4 ꢃ s, 4 ꢃ 3H, 4 ꢃ CH3eC]O); ESI-
MS m/z (%): 526 (13) [M þ 1], 548 (100) [M þ Na]. Anal. C,
H, N for C22H27N3O10S.
5.1.4. Synthesis of 4-formylphenyl-O-b-D-
glucopyranoside (5)
To a solution of compound 3a (4.52 g, 10 mmol) in 20 mL
of methanol was added 20 mL of methanol solution of
0.2 mol/L sodium methoxide. The mixture was stirred for
3 h at room temperature. The methanol was removed under
reduced pressure, and the residue was purified by chromatog-
raphy (MeOH/CHCl3, 1/9, v/v) to give pure 5. White powder,
yield 96%, m.p. 157e158 ꢁC (lit. 155e157 ꢁC) [29]. IR (KBr,
cmꢂ1) n: 3455, 2930, 1691, 1590, 1415, 1240, 1066, 841, 658;
1H NMR (DMSO-d6) d ppm: 9.87 (s, 1H, HeC]O), 7.86 (d,
2H, J ¼ 8.5 Hz, ArH), 7.19 (d, 2H, J ¼ 8.5 Hz, ArH), 5.43 (br
s, 1H, eOH), 5.16 (br s, 1H, eOH), 5.04 (d, 1H, J ¼ 6.9 Hz,
H-10), 4.60 (s, 1H, CH), 3.69 (d, 1H, J ¼ 10.9 Hz, CH), 3.45e
3.42 (m, 1H, CH), 3.32e3.25 (m, 2H, 2 ꢃ CH), 3.20 (dd, 1H,
J ¼ 8.5 Hz, 16.7 Hz, CH); ESI-MS m/z (%): 283 (26) [M ꢂ 1].
Anal. C, H for C13H16O7.
5.1.3.2. 1-(4-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl) ben-
zylidene) semicarbazide (4b). To a solution of compound 3a
(0.45 g, 1 mmol) in 15 mL of ethanol was added semicarbazide
hydrochloride (0.11 g, 1 mmol) which had been adjusted with
2M NaOH to pH ¼ 6e7. The mixture was refluxed for 6 h. After
being cooled, the precipitatewas filtered, washed with ether, and
recrystallized to get white powder 4b, yield 85%, m.p. 211e
212 ꢁC. IR (KBr, cmꢂ1) n: 3423, 1716, 1572, 1409, 1232,
1
1093, 1052, 709; H NMR (DMSO-d6) d ppm: 10.19 (s, 1H,
NeH), 7.79 (s, 1H, eCH]N), 7.67 (d, 2H, J ¼ 8.7 Hz, ArH),
6.98 (d, 2H, J ¼ 8.7 Hz, ArH), 6.45 (br s, 2H, NeH), 5.62 (d,
1H, J ¼ 8.0 Hz, H-10), 5.40 (t, 1H, J ¼ 9.5 Hz, CH), 5.08e
4.96 (m, 2H, 2 ꢃ CH), 4.25e4.04 (m, 3H, 3 ꢃ CH), 2.01, 1.97
(2 ꢃ s, 4 ꢃ 3H, 4 ꢃ CH3eC]O); ESI-MS m/z (%): 510 (3)
[M þ 1], 532 (100) [M þ Na]. Anal. C, H, N for C22H27N3O11.
5.1.5. Synthesis of compounds 6aed and 7aed
The procedure was the same as described in Section 5.1.3
(synthesis of 4aed).
5.1.5.1. 1-(4-(O-b-D-Glucopyranosyl) benzylidene) thiosemi-
carbazide (6a). Light orange powder, yield 76%, m.p.
212 ꢁC. IR (KBr, cmꢂ1) n: 3425, 1569, 1500, 1412, 1201,
1076, 956; 1H NMR (DMSO-d6) d ppm: 11.31 (s, 1H,
5.1.3.3. 1-(4-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl) ben-
zylidene) hydroxylamine (4c). To a solution of compound

172
H. Wen et al. / European Journal of Medicinal Chemistry 43 (2008) 166e173
NeH), 8.10 (s, 1H, NeH), 7.97 (s, 1H, eCH]N), 7.92 (s, 1H,
NeH), 7.71 (d, 2H, J ¼ 8.5 Hz, ArH), 7.01 (d, 2H, J ¼ 8.5 Hz,
ArH), 4.92 (d, 1H, J ¼ 7.0 Hz, H-10), 3.69e3.67 (m, 1H, CH),
3.46e3.43 (m, 1H, CH), 3.35e3.15 (m, 4H, 4 ꢃ CH); ESI-MS
m/z (%): 358 (5) [M þ 1], 380 (100) [M þ Na]. Anal. C, H, N
for C14H19N3O6S.
7.52 (m, 4H, ArH), 7.42e7.32 (m, 6H, ArH), 7.22 (d,
J ¼ 8.1 Hz, 2H, ArH), 6.45 (br s, 2H, NeH), 6.11 (t, 1H,
J ¼ 3.5 Hz, CH), 5.96 (d, 1H, J ¼ 5.1 Hz, CH), 5.68e5.60
(m, 2H, 2 ꢃ CH), 4.32 (d, 1H, J ¼ 12.6 Hz, CH), 4.13 (d,
1H, J ¼ 11.8 Hz, CH); FAB-MS m/z (%): 624 (5) [M þ 1],
646 (15) [M þ Na]. Anal. C, H, N for C34H29N3O9.
5.1.5.2. 1-(4-(O-b-D-Glucopyranosyl) benzylidene) semicarba-
zide (6b). Light yellow powder, yield 78%, m.p. 207e208 ꢁC.
IR (KBr, cmꢂ1) n: 3381, 2917, 1676, 1579, 1507, 1457, 1243,
5.1.5.7. 1-(4-(2,3,4-Tri-O-benzoyl-b-D-ribopyranosyl) benzyli-
dene) hydroxylamine (7c). Light orange foam-like solid, yield
88%, m.p. 107e108 ꢁC. IR (KBr, cmꢂ1) n: 3443, 1727, 1271,
1
1
1172, 1034, 926, 872; H NMR (DMSO-d6) d ppm: 10.09 (s,
1097, 711; H NMR (CDCl3) d ppm: 8.01 (s, 1H, eCH]N),
1H, NeH), 7.77 (s, 1H, eCH]N), 7.62 (d, 2H, J ¼ 8.5 Hz,
ArH), 7.00 (d, 2H, J ¼ 8.5 Hz, ArH), 6.42 (br s, 2H, NeH),
4.89 (d, 1H, J ¼ 7.0 Hz, H-10), 3.69e3.67 (m, 1H, CH),
3.48e3.42 (m, 1H, CH), 3.35e3.15 (m, 4H, 4 ꢃ CH); ESI-
MS m/z (%): 342 (3) [M þ 1], 366 (100) [M þ Na]. Anal. C,
H, N for C14H19N3O7.
7.96 (t, 4H, J ¼ 6.6 Hz, ArH), 7.80 (d, 2H, J ¼ 8.0 Hz, ArH),
7.49e7.39 (m, 5H, ArH), 7.23e7.16 (m, 6H, ArH), 7.06 (d,
2H, J ¼ 8.4 Hz, ArH), 5.93 (t, 1H, J ¼ 3.2 Hz, CH), 5.78 (d,
1H, J ¼ 4.6 Hz, CH), 5.66e5.60 (m, 2H, 2 ꢃ CH), 4.25 (d,
1H, J ¼ 12.9 Hz, CH), 4.07 (d, 1H, J ¼ 13.3 Hz, CH); ESI-
MS m/z (%): 582 (5) [M þ 1], 604 (100) [M þ Na]. Anal. C,
H, N for C33H27NO9.
5.1.5.3. 1-(4-(O-b-D-Glucopyranosyl) benzylidene) hydroxyl-
amine (6c). Light yellow powder, yield 80%, m.p. 162e
163 ꢁC. IR (KBr, cmꢂ1) n: 3384, 2913, 1606, 1513, 1242,
5.1.5.8. 1-(4-(2,3,4-Tri-O-benzoyl-b-D-ribopyranosyl) benzyli-
dene) methoxyamine (7d). Light yellow powder, yield 76%,
m.p. 78e79 ꢁC. IR (KBr, cmꢂ1) n: 1728, 1264, 1101, 1062,
1
1082, 965; H NMR (DMSO-d6) d ppm: 11.02 (br s, 1H, e
1
OH), 8.05 (s, 1H, eCH]N), 7.50 (d, 2H, J ¼ 8.5 Hz, ArH),
7.02 (d, 2H, J ¼ 8.5 Hz, ArH), 4.89 (d, 1H, J ¼ 7.0 Hz, H-10),
3.36e3.31 (m, 2H, 2 ꢃ CH), 3.29e3.23 (m, 2H, 2 ꢃ CH),
3.19e3.13 (m, 2H, 2 ꢃ CH); ESI-MS m/z (%): 300 (3)
[M þ 1], 322 (100) [M þ Na]. Anal. C, H, N for C13H17NO7.
712; H NMR (CDCl3) d ppm: 8.03e7.82 (m, 5H, eCH]N,
4 ꢃ ArH), 7.78 (d, 2H, J ¼ 7.9 Hz, ArH), 7.45e7.38 (m, 5H,
ArH), 7.22e7.18 (m, 6H, ArH), 7.04 (d, 2H, J ¼ 8.0 Hz,
ArH), 5.93e5.91 (m, 1H, CH), 5.76 (d, 1H, J ¼ 5.1 Hz, CH),
5.64e5.60 (m, 2H, 2 ꢃ CH), 4.23 (d, 1H, J ¼ 12.3 Hz, CH),
4.05 (d, 1H, J ¼ 12.7 Hz, CH), 3.86 (s, 3H, OCH3); ESI-MS
m/z (%): 596 (8) [M þ 1], 618 (100) [M þ Na]. Anal. C, H, N
for C34H29NO9.
5.1.5.4. 1-(4-(O-b-D-Glucopyranosyl) benzylidene) methoxy-
amine (6d). White powder, yield 68%, m.p. 103e104 ꢁC. IR
(KBr, cmꢂ1) n: 3355, 2930, 1608, 1509, 1236, 1046, 916;
1H NMR (DMSO-d6) d ppm: 8.14 (s, 1H, eCH]N), 7.52
(d, 2H, J ¼ 8.5 Hz, ArH), 7.03 (d, 2H, J ¼ 8.4 Hz, ArH),
4.91 (d, 1H, J ¼ 7.3 Hz, H-10), 3.84 (s, 3H, OCH3), 3.68 (br
s, 0.5H, eOH), 3.65 (br s, 0.5H, eOH), 3.47e3.41 (m, 1H,
CH), 3.35e3.17 (m, 5H, 5 ꢃ CH); ESI-MS m/z (%): 314 (8)
[M þ 1], 336 (100) [M þ Na]. Anal. C, H, N for C14H19NO7.
5.1.6. Synthesis of 4-formylphenyl-O-b-D-ribopyranoside (8)
The procedure was the same as described in Section 5.1.4
(synthesis of 5). Light yellow solid, yield 85%, m.p. 143e
144 ꢁC. IR (KBr, cmꢂ1) n: 3405, 1683, 1582, 1415, 1240,
1
1109, 1000, 825, 650; H NMR (DMSO-d6) d ppm: 9.85 (s,
1H, HeC]O), 7.84 (d, 2H, J ¼ 8.4 Hz, ArH), 7.17 (d, 2H,
J ¼ 8.5 Hz, ArH), 5.41 (d, 1H, J ¼ 5.2 Hz, H-10), 5.22 (br s,
1H, eOH), 5.03 (br s, 1H, eOH), 3.89 (s, 1H, CH), 3.69e
3.59 (m, 4H, 4 ꢃ CH); 13C NMR (DMSO-d6) d ppm: 192.5,
162.5, 133.0, 132.6, 131.4, 117.4, 116.8, 99.1, 71.0, 69.0,
68.6, 65.0; FAB-MS m/z (%): 255 (3) [M þ 1], 277 (15)
[M þ Na]. Anal. C, H for C12H14O6.
5.1.5.5. 1-(4-(2,3,4-Tri-O-benzoyl-b-D-ribopyranosyl) benzyli-
dene) thiosemicarbazide (7a). Light yellow powder, yield
88%, m.p. 155e156 ꢁC. IR (KBr, cmꢂ1) n: 3429, 3324,
1
1726, 1597, 1270, 1098, 711; H NMR (DMSO-d6) d ppm:
11.35 (s, 1H, NeH), 8.14 (s, 1H, NeH), 7.97 (s, 1H, e
CH]N), 7.94 (s, 1H, NeH), 7.88e7.84 (m, 6H, ArH), 7.75
(d, 2H, J ¼ 7.9 Hz, ArH), 7.67e7.58 (m, 3H, ArH), 7.50e
7.40 (m, 6H, ArH), 7.03 (d, 2H, J ¼ 8.0 Hz, ArH), 5.93 (t,
1H, J ¼ 3.5 Hz, CH), 5.78 (d, 1H, J ¼ 5.1 Hz, CH), 5.66e
5.60 (m, 2H, 2 ꢃ CH), 4.25 (d, 1H, J ¼ 12.9 Hz, CH), 4.07
(d, 1H, J ¼ 13.3 Hz, CH); ESI-MS m/z (%): 640 (4) [M þ 1],
662 (18) [M þ Na]. Anal. C, H, N for C34H29N3O8S.
5.2. Biological evaluation
5.2.1. Inhibition of AChE and BuChE
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric
eel), butylcholinesterase (BuChE, E.C. 3.1.1.8, from horse se-
rum), 5,50-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent,
DTNB), butylthiocholine chloride (BTC) and acetylthiocho-
line chloride (ATC) were purchased from SigmaeAldrich
(Steinheim, Germany). Galanthamine hydrobromide was ob-
tained from SigmaeAldrich (Sintra, Portugal).
5.1.5.6. 1-(4-(2,3,4-Tri-O-benzoyl-b-D-ribopyranosyl) benzyli-
dene) semicarbazide (7b). Light yellow powder, yield 86%,
m.p. 133e134 ꢁC. IR (KBr, cmꢂ1) n: 3476, 1728, 1687,
1
1266, 1101, 711; H NMR (DMSO-d6) d ppm: 10.18 (s, 1H,
NeH), 7.92 (d, 4H, J ¼ 7.8 Hz, ArH), 7.82 (s, 1H, e
The assay was performed as described in the following pro-
cedure [25]. Five different concentrations (normally in the
CH]N), 7.75 (dd, 4H, J ¼ 4.7 Hz, 13.3 Hz, ArH), 7.63e

H. Wen et al. / European Journal of Medicinal Chemistry 43 (2008) 166e173
173
range of 10ꢂ4e10ꢂ9 M) of each compound were measured at
412 nm for 1 min, each concentration in triplicate.
[3] G. Benzi, A. Moretti, Eur. J. Pharm. 346 (1998) 1e13.
[4] F. Gualtieri, S. Dei, D. Manetti, M.N. Romanelli, S. Secapecchi,
E. Teodori, Il Farmaco 50 (1995) 489e503.
For buffer preparation, 0.1 M dipotassium hydrogen phos-
phate was adjusted to pH ¼ 8.0 with 1 M potassium dihydrogen
phosphate. Enzyme solutions were prepared to give 2.5 units/
mL in 1.5 mL aliquots. Furthermore, 0.01 M DTNB solution,
0.075 M ATC and BTC solutions, respectively, were used. A cu-
vette containing 840 mL of phosphate buffer, 10 mL of the re-
spective enzyme, 50 mL of DTNB and 50 mL of the test
compound solution was allowed to pre-incubate for 15 min at
37 ꢁC, and the reaction was started by addition of 50 mL of the
substrate solution (ATC/BTC). The reaction was monitored by
measuring the absorbance at 412 nm. For the reference value,
50 mL of DMSO replaced the test compound solution. For deter-
mining the blank value, additionally 10 mL of buffer replaced
the enzyme solution.
[5] N.H.P. Allen, A. Burns, J. Psychopharmacol. 9 (1995) 43e56.
[6] P.T. Francis, A.M. Palmer, M. Snap, G.K. Wilcock, J Neurol. Neurosurg.
Psychiatry 66 (1999) 137e147.
[7] M. Galisteo, M. Rissel, O. Sergent, M. Chevanne, J. Cillard,
A. Guilouzo, D. Lagadic-Gossmann, J. Pharmacol. Exp. Ther. 294
(2000) 160e167.
[8] G.T. Grossberg, H.B. Stahelin, J.C. Messina, R. Anand, J. Veach, Int. J.
Geriatr. Psychiatry 15 (2000) 242e247.
[9] J. Kaur, M.Q. Zhang, Curr. Med. Chem. 7 (2000) 273e294.
[11] H.C. Dringenberg, Behav. Brain Res. 115 (2000) 235e249.
[12] J.J. Buccafusco, A.V. Terry Jr., J. Pharmacol. Exp. Ther. 295 (2000)
438e446.
[13] M.C. Carreiras, J.L. Marco, Curr. Pharm. Des. 10 (2004) 3167e3175.
[14] G. Ucar, N. Gokhan, A. Yesilada, A.A. Bilgin, Neurosci. Lett. 382 (2005)
327e331.
[15] J.Z. Sha, H.K. Mao, Yaoxuetongbao 22 (1987) 21e27.
[16] P. Liu, J. Li, D.Q. Na, J.M. Geng, S. Li, Zhongcaoyao 33 (2002) 238e
242.
5.2.2. Kinetic characterization of AChE inhibition
[17] Z. Wang, L.K. Hu, Z.P. Luo, Y.F. Li, N. Zhao, Z.H. Wang, Y.H. Wang,
Y.G. Chen, PCT Int. Appl., WO 2004054591, 2004.
[18] J.H. Ha, S.M. Shin, S.K. Lee, J.S. Kim, U.S. Shin, K. Huh, J.A. Kim,
C.S. Yong, N.J. Lee, D.U. Lee, Planta Med. 67 (2001) 877e880.
[19] J.H. Ha, D.U. Lee, J.T. Lee, J.S. Kim, C.S. Yong, J.A. Kim, J.S. Ha,
K. Huh, J. Ethnopharmacol. 73 (2000) 329e333.
[20] R.L. Whistler, M.L. Wolfrom, Methods in Carbohydrate Chemistry, vol.
II, Academic Press Inc., New York and London, 1963.
[21] H.G. Fletcher Jr., C.S. Hudson, J. Am. Chem. Soc. 69 (1947)
921e926.
Kinetic characterization of AChE was performed using a re-
ported method [30]. Five different concentrations of AChE and
inhibitors were mixed in the assay buffer (pH ¼ 8.0), contain-
ing 40 mM of 5,50-dithio-bis (2-nitrobenzoic acid), 0.035 units/
mL AChE, and 550 mM acetylthiocholine chloride. Test com-
pound 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
acetylthiocholine catalyzed by AChE was done spectrometri-
cally at 412 nm. A parallel control with no inhibitor in the
mixture, allowed adjusting activities to be measured at various
times.
[22] R.K. Ness, H.G. Fletcher Jr., C.S. Hudson, J. Am. Chem. Soc. 73 (1951)
959e964.
[23] D. Oulmi, P. Maillard, J.L. Guerquin-Kern, C. Huel, M. Momenteau, J.
Org. Chem. 60 (1995) 1554e1564.
[24] K. Jyothish, R.R. Avirah, D. Ramaiah, Org. Lett. 8 (2006) 111e114.
[25] G.L. Ellman, D.K. Courtney, V. Andres, R.M. Featherstone, Biochem.
Pharmacol. 7 (1961) 88e92.
[26] M.I. Choudhary, S.A. Nawaz, Z.U. Haq, M.K. Azim, M.N. Ghayre,
M.A. Lodhi, S. Jalil, A. Khalid, A. Ahmed, B.M. Rode, A.U. Rahman,
A.H. Gilani, V.U. Ahmad, Biochem. Biophys. Res. Commun. 332
(2005) 1171e1179.
Acknowledgements
The authors thank the Natural Science Foundation of
Guangdong Province, China (2004B30101007), and Kunming
Baker Norton Pharmaceutical Co. Ltd for financial support on
this study.
[27] C.Y. Bao, R. Lu, M. Jin, P.C. Xue, C.H. Tai, Y.Y. Zhao, G.F. Liu, Carbo-
hydr. Res. 339 (2004) 1311e1316.
[28] E.N. Olsufyeva, A.N. Tevyashova, Ivan D. Trestchalin,
M.N. Preobrazhenskaya, D. Platt, A. Klyosov, Carbohydr. Res. 338
(2003) 1359e1367.
References
[29] H.M. Liu, X.B. Yan, W. Li, C.H. Huang, Carbohydr. Res. 337 (2002)
1763e1767.
[30] J. Massoulie, S. Bon, I. Silman, Prog. Brain Res. 98 (1993) 139e146.
[1] E.K. Perry, Br. Med. Bull. 42 (1986) 63e69.
[2] R.T. Bartus, R.L. Dean, B. Beer, A.S. Lippa, Science 217 (1982)
408e414.