Design, synthesis and evaluation of flavonoid derivatives as potent AChE inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.07.072

A new series of flavonoid derivatives have been designed, synthesized and evaluated as potent AChE inhibitors. Most of them showed more potent inhibitory activities to AChE than rivastigmine. The most potent inhibitor isoflavone derivative 10d inhibit ACh

Full text of DOI:10.1016/j.bmc.2009.07.072

Bioorganic & Medicinal Chemistry 17 (2009) 6692–6698
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of flavonoid derivatives as potent
AChE inhibitors
Rong Sheng a, Xiao Lin a, Jing Zhang b, Kim Sun Chol c, Wenhai Huang a, Bo Yang b, Qiaojun He b,
Yongzhou Hu a,
*
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, School of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China
b Department of Pharmacology, School of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China
c Department of Chemistry, Pyongsong Medical University, Pyongsong, Democratic People’s Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of flavonoid derivatives have been designed, synthesized and evaluated as potent AChE
inhibitors. Most of them showed more potent inhibitory activities to AChE than rivastigmine. The most
potent inhibitor isoflavone derivative 10d inhibit AChE with a IC50 of 4 nM and showed high BChE/AChE
inhibition ratio (4575-fold), superior to donepezil (IC50 = 12 nM, 389-fold). Molecular docking studies
were also performed to explore the detailed interaction with AChE.
Received 11 June 2009
Revised 24 July 2009
Accepted 24 July 2009
Available online 3 August 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Flavonoid derivatives
Synthesis
AChE inhibitor
1. Introduction
lated to Alzheimer’s disease, such as neuro-protective effect,8 AChE
inhibitory activities,9 Ab fibril formation inhibitory activity,10
Since being introduced into clinical practice, acetylcholinester-
ase (AChE) inhibitors (tacrine, donepezil, galantamine, huperzine
A and rivastigmine) have been the standard approach to the symp-
tomatic treatment of Alzheimer’s disease (AD).1 In recent year,
mounting evidences proved that AChE may own its ‘nonclassical’
functions, that is, colocalizing with amyloid-b peptide (Ab) and
promoting amyloid fibril formation through its peripheral anionic
site (PAS).2 The crystallographic structure of AChE reveals that it
possesses two separate ligand binding sites—catalytic site and
peripheral anionic site (PAS). It implied that molecules that can
interact with both binding sites of AChE could not only inhibit
AChE, but prevent the aggregation of AChE with Ab. Therefore,
dual-site binding AChE inhibitors have been presented as a new
therapeutic strategic option for AD in recent years.3–5
H2O2-induced ROS formation reduction effect, and so on.11 In our
previous work, we have also designed and synthesized a series of
flavonoid derivatives by linking a benzyl piperidine moiety to the
flavonoid moiety through an oxygen atom or a OCH2 group. Most
of these compounds showed low micromolar inhibition to AChE.12
Modeling study of 1d (Fig. 1) with AChE indicated that ring B (phe-
nyl ring of phenoxy group) got close to the crucial amino acid Trp
279 in PAS, ring A (dimethoxyphenyl ring), which is designed to
bind to PAS, was away from Trp279.12 In this paper, we reported
the design, synthesis and evaluation of a series of new flavonoid
derivatives (6a–f, 7a–f, 8a–f, 10a–f) by replacing the 2-phenoxy
In our previous study, a series of indanone derivatives (1c,
Fig. 1) were synthesized and evaluated as dual-site binding AChE
inhibitors. These compounds contain an indanone moiety (from
donepezil, 1a, Fig. 1) and a dialkylbenzyl amine moiety (from riv-
astigmine, 1b, Fig. 1). The molecular docking study reveals that
these series of compounds can concurrently bind to both the active
and peripheral site.6,7 Flavonoids are well known natural com-
pounds possessing a broad range of pharmacological properties re-
* Corresponding author. Tel./fax: +86 571 8820 8460.
E-mail address: huyz@zju.edu.cn (Y. Hu).
Figure 1. The Structures of donepezil 1a, rivastigmine 1b, indanone derivatives 1c
and flavonoid derivative 1d.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.072

R. Sheng et al. / Bioorg. Med. Chem. 17 (2009) 6692–6698
6693
(or benzyl, benzylidiene)-indan-1-one moiety of 1c with different
flavonoids (chalcone, flavone, flavanone and isoflavone) and intro-
duction of various amino alkyl groups at the para- or meta-position
of ring B with the purpose to alter the binding mode of ring A, ring
B with AChE. In the designed compounds, the dimethoxyphenyl
(chalcone), dimethoxy-chroman-4-one (flavanone), and dime-
thoxy-4H-chromen-4-one (flavone, isoflavone) moiety, was ex-
pected to bind the PAS of AChE, and the nitrogen atom of benzyl
amino groups would interact with the catalytic center of AChE
As shown in Table 1, most of the tested compounds demon-
strated higher inhibitory activities against AChE than rivastigmine,
and seven of them (6d, 6e, 7d, 7f, 10d, 10e, 10f, IC50 = 0.070–
0.004 lM) exhibit similar activity to donepezil (IC50 = 0.012 lM).
The preliminary structure–activity relationships could be drawn
from the results as follows:
(1) The variation of flavonoid scaffold affected the AChE inhibi-
tory activities dramatically. Isoflavone derivatives, which
showed sub-micromolar or nanomolar AChE inhibitory
activities, were more potent than other series. For example,
through a cation–p interaction, which was presented in the dock-
ing study of indanone derivatives.6,7 Moreover, we described the
preliminary structure–activity relationship (SAR) and molecular
docking studies for the interactions of these flavonoid derivatives
with AChE.
compound 10d (IC50 = 0.004
inhibitory activity than that of donepezil (IC50 = 0.012
and compound 10e, 10f (IC50 = 0.070, 0.068 M, respec-
l
M) displayed potent AChE
lM),
l
tively) exhibited comparable inhibitory activities to donepe-
zil. The compounds of chalcone and flavone series
demonstrated sub-micromolar AChE inhibitory activities
except compound 6c and 7c, with IC50 values ranging from
2. Results and discussion
2.1. Chemistry
0.037 to 1.61 lM and 0.034 to 5.99 lM, respectively. The
flavanone series displayed weaker inhibitory activity than
others, most of them demonstrated low micromolar AChE
inhibitory activities.
The synthesis of flavonoid derivatives 6a–f, 7a–f, 8a–f, 10a–f
was outlined in Scheme 1. Acetylation of 1,4-benzoquinone 2 with
acetic anhydride in the presence of sulfuric acid, followed by
hydrolysis of the acetyl groups with p-toluenesulfonic acid (PTS)
and water yielded 2,4,5-trihydroxy acetophenone 3.13 Methylation
of 3 with dimethyl sulfate provided 2-hydroxy-4,5-dimethoxy ace-
tophenone 4.14 Condensation of 4 with alkylamino-methyl-benzal-
dehyde 5a–f7 in the presence of KOH afforded chalcone 6a–f.
Oxidation of 6a–f with iodine in DMSO furnished flavones 7a–f.15
Refluxing 6a–f with NaOAc in alcohol–H2O gave flavanones 8a–f.
For the preparation of isoflavone derivatives 10a–f, protection of
the hydroxyl groups in chalcones 6a–f with benzoyl group afforded
20-benzoyloxychalones 9a–f which were reacted with DIB/PTS and
then treated with NaOH to get target compounds.16
(2) From the IC50 values of the tested compounds, it appeared
that compounds with aminomethyl group substituted in
the para-position of ring B (i.e., 6b, 7d, 10d) were more
potent than those meta-position substituted ones (i.e., 6c,
7e, 10e), which was consistent with the SAR of previous syn-
thesized indanone derivatives.6,7
(3) The sort of aminomethyl group on ring B affected the activity
of AChE inhibition, compounds containing pyrrolidine or
piperidine group (i.e., 6d, 6e, 7f, 8f, 10d) showed higher
activity than those with methylethylamino or diethylamino
group (i.e., 6a, 6b, 7b, 8a, 10c), which indicated that a con-
formational constrained hydrophobic moiety would be
favorable in this position.
2.2. Biological activities
(4) Almost all the compounds showed very weak activity
against BChE, and several of them demonstrated higher
selectivity (i.e., 6d, 6e, 7f, 10d) for AChE over BChE than
donepezil. Especially, the most potent AChE inhibitors 10d
was 4575-fold more active inhibiting AChE than BuChE,
being much more selective than donepezil (389-fold).
The AChE and BuChE inhibitory activities of all the synthesized
flavonoid derivatives were tested according to the modified Ellman
method using rat cortex homogenate (AChE) and rat serum
(BuChE), with donepezil and rivastigmine as the reference
standard.17
2.3. Molecular docking
To gain insight into the molecular determinants that modulate
the inhibitory activity of these compounds, molecular docking sim-
ulations for 10c, 10d, 10e to TcAChE were performed using the
FLEXIDOCK program in Sybyl 6.9 software based on the X-ray crystal
structure of TcAChE-E2020 complex.18
As seen in Figure 2, compound 10d has a nice fit along the ac-
tive-site gorge of AChE, it can bind to the central and the peripheral
anionic site concurrently. The Figures 3a and 3b show that 10d
makes several principal interactions along the active-site gorge of
AChE with different mode in comparison with indanone deriva-
tives and donepezil.6,7 Near the top of the gorge (the PAS), besides
the expected hydrophobic interaction between dimethoxy-phenyl
moiety with Trp279, the dimethoxy-phenyl ring of isoflavone
formed classical p–p stacking with the phenyl ring of Tyr334, with
the distance of 2.8 Å, more over, the oxygen of one methoxy group
makes a hydrogen bond with the NH of indole ring in Trp279, with
the distance of 2.42 Å. Noteworthy, in the middle of the gorge, the
oxygen of carbonyl group of 10d formed a highly specific hydrogen
bond with OH group in Tyr70 with close distance of 1.46 Å, which
imply that it make some contribution on its high activity. Near the
bottom of the gorge (the central site), just as expected, the charged
Scheme 1. Synthesis of flavonoid derivatives 6, 7, 8, 10. Reagent and conditions (a)
Ac2O, H2SO4, H2O, 135 °C, 1 h; (b) PTS, H2O, reflux 1 h; (c) (CH3)2SO4, K2CO3,
acetone, reflux 1 h; (d) KOH, CH3OH, 50–55 °C, 48 h; (e) I2, DMSO, 80–85 °C, 24 h; (f)
NaOAc, CH3CH2OH–H2O, reflux 24 h; (g) PhCOCl, K2CO3, DMF, 60 °C, 4 h; (h) DIB,
PTS, CH3OH, rt 24 h; (i) NaOH, CH3OH–H2O, rt 24 h.

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R. Sheng et al. / Bioorg. Med. Chem. 17 (2009) 6692–6698
Table 1
Cholinesterase inhibitory activity and selectivity of flavonoid derivatives
Flavonoids scaffold
Position
–NR1R2
IC50
(lM)
Selectivity for AChEa
AChE
BChE
6a
6b
6c
6d
6e
6f
Para
Para
Meta
Para
Meta
Para
Methylethylamino
Diethylamino
0.126
0.103
1.61
0.037
0.047
0.122
>100
>100
>100
>100
77.3
>794
>970
>62.1
>2702
1645
>820
H3CO
H3CO
OH
O
Pyrrolidine-1-yl
Piperidine-1-yl
>100
7a
7b
7c
7d
7e
7f
Para
Para
Meta
Para
Meta
Para
Methylethylamino
Diethylamino
0.336
0.639
5.99
0.055
0.704
0.034
>100
64.6
>100
89.7
>100
75.2
>297
101
>16.7
1630
>142
2211
H3CO
H3CO
O
O
Pyrrolidine-1-yl
Piperidine-1-yl
8a
8b
8c
8d
8e
8f
Para
Para
Meta
Para
Meta
Para
Methylethylamino
Diethylamino
4.67
4.97
172
1.24
5.36
0.248
>100
>100
>100
68.2
>100
48.0
>21.4
>20.1
>0.58
55
>18.7
194
H3CO
H3CO
O
O
Pyrrolidine-1-yl
Piperidine-1-yl
10a
10b
10c
10d
10e
10f
Donepezil
Rivastigmine
Para
Para
Meta
Para
Meta
Para
Methylethylamino
Diethylamino
0.171
0.599
0.560
0.004
0.070
0.068
0.012
2.25
61.0
>100
>100
18.3
>100
>100
4.67
1.66
357
>167
>178
4575
>1428
>1471
389
H3CO
H3CO
O
O
Pyrrolidine-1-yl
Piperidine-1-yl
0.74
a
Selectivity for AChE is defined as IC50 (BuChE)/IC50 (AChE).
Figure 3a. Predicted binding model of compound 10c (carbon atoms depicted in
green) and 10d (carbon atoms depicted in yellow) to AChE. The carbon atoms in
several key residues in AChE are colored in blue, nitrogen and oxygen atoms are
colored in blue and red, respectively.
and make a close hydrogen band with it, while for 10c, it points
to the opposite direction, which result to the lost of hydrogen band,
and it could be account for its lower activity.
Figure 2. Compound 10d in the gorge of AChE.
The molecular docking (Fig. 3b) shows that although 10e has
similar bound conformation of 10d to AChE, especially has the
same hydrogen band with Tyr70 at same distance, the meta-posi-
tion substituted pyrrolidine methyl moiety causes some deviation.
At the PAS, it lost the hydrogen band with Trp279 because of the
different direction of methoxy moiety in comparison of 10d. All
these facts could explain its sub-micromolar AChE inhibitory activ-
nitrogen makes a cation–
p interaction with the phenyl ring of
Phe330, with the distance is 3.10 Å, and the pyrrolidine moiety also
display hydrophobic contacts with the indole group of Trp84.
Although there is only little difference between compounds 10c
and 10d, diethyl amino moiety in 10c and pyrrolidine moiety in
10d, the molecular docking (Fig. 3a) shows that there is obvious
difference in the conformation of dimethoxy-4H-chromen-4-one
between two compounds, the carbonyl in 10d is direct to Tyr70
ity, IC50 = 0.070 lM.

R. Sheng et al. / Bioorg. Med. Chem. 17 (2009) 6692–6698
6695
4.1.2. 2-Hydroxy-4,5-dimethoxy acetophenone (4)
To a refluxed solution of compound 3 (5.37 g, 32 mmol) and
potassium carbonate (13.25 g, 96 mmol) in 70 mL anhydrous ace-
tone, (CH3)2SO4 (6.64 mL, 70 mmol) was added in drop wise. The
mixture was stirred for another 1 h after addition, cooled to rt
and filtered. The filtrate was concentrated under reduced pressure
to get brown solid, which was recrystallized with 95% alcohol
twice to afford 3.67 g pale purple solid, yield 58.5%, mp 112–
115 °C. (lit.,14 114–116 °C).
4.1.3. General procedure for preparation of chalcone
derivatives 6a–6f
To a solution of potassium hydroxide (220 mg, 3.93 mmol) in
5 mL methanol and 0.22 mL H2O, compound 4 (1.02 mmol) and
substituted benzaldehyde 5 (1.02 mmol) were added in a portion.
The mixture was warmed to 50–55 °Cand stirred for 48 h, then,
the solvent was removed. H2O (15 mL) was added to the residue
and extracted with ethyl acetate (15 mL ꢀ 3). The combined organ-
ic layer was washed with brine, dried over anhydrous Na2SO4. The
solvent was evaporated to give crude product, which was purified
by silica gel column chromatography (eluent, petroleum ether/
EtOAc/Et3N = 40:10:1) to afford 6.
Figure 3b. Predicted binding model of compound 10e (carbon atoms depicted in
green) and 10d (carbon atoms depicted in yellow) to AChE. The carbon atoms in
several key residues in AChE are colored in blue, nitrogen and oxygen atoms are
colored in blue and red, respectively.
3. Conclusion
4.1.3.1. (E)-3-{4-[(Ethylmethylamino)-methyl]-phenyl}-1-(2-
hydroxy-4,5-dimethoxyphenyl)-prop-2-en-1-one (6a). Orange
In summary, based upon the SAR study of indanone derivatives
and the structural characteristic of flavonoids, a new series of fla-
vonoid derivatives were designed and synthesized as dual-site
binding AChE inhibitors and their inhibitory activities against AChE
and BChE were evaluated. The results indicate that isoflavone
derivatives were potent AChE inhibitors with higher BChE/AChE
selectivity, and the molecular Docking simulations of compounds
10c, 10d and 10e with TcAChE gave clear interpretation of the de-
tail interactions at the gorge of the AChE, and give some explana-
tion on SAR. According to the obtained results, we think
isoflavone skeleton would be a promising structural template for
the development of novel AChE inhibitors, and the preliminary
SAR and molecular modeling study results will shed light on the
further study.
solid, yield 57.8%, mp 74–75 °C; IR (KBr)
m 3583, 3060, 2968,
2937, 2835, 2789, 1675, 1635, 1612, 1504, 1265, 1158, 833 cmꢁ1
;
1H NMR (CDCl3) d 13.39 (s, 1H, OH), 7.88 (d, 1H, J = 15.6 Hz, H-3),
7.61 (d, 2H, J = 8.0 Hz, Ar–H), 7.49 (d, 1H, J = 15.2 Hz, H-2), 7.40
(d, 2H, J = 8.0 Hz, Ar–H), 7.27 (s, 1H, Ar–H), 6.52 (s, 1H, Ar–H),
3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.54 (s, 2H, benzylic-CH2),
2.45 (q, 2H, J = 7.2 Hz, CH2CH3), 2.22 (s, 3H, NCH3), 1.10 (t, 3H,
J = 7.2 Hz, CH2CH3); MS (ESI) m/z = 356.6 [M+H]+.
4.1.3.2. (E)-3-{4-[(Diethylamino)-methyl]-phenyl}-1-(2-hydro-
xy-4,5-dimethoxyphenyl)-prop-2-en-1-one (6b). Orange solid,
yield 46.6%, mp 84–86 °C; IR (KBr)
m
3584, 3057, 2966, 2873,
2786, 1671, 1635, 1582, 1510, 1250,1158, 832 cmꢁ1
;
1H NMR
(CDCl3) d 13.38 (s, 1H, OH), 7.86 (d, 1H, J = 15.2 Hz, H-3), 7.58 (d,
2H, J = 8.0 Hz, Ar–H), 7.46 (d, 1H, J = 15.6 Hz, H-2), 7.39 (d, 2H,
J = 8.0 Hz, Ar–H), 7.25 (s, 1H, Ar–H), 6.49 (s, 1H, Ar–H), 3.92 (s,
3H, OCH3), 3.90 (s, 3H, OCH3), 3.59 (s, 2H, benzylic-CH2), 2.49 (q,
4H, J =6.8 Hz, 2CH2CH3), 1.02 (t, 6H, J = 6.8 Hz, 2CH2CH3); MS
(ESI) m/z = 370.5 [M+H]+.
4. Experimental
4.1. Chemistry
All Reagents and solvents used were purchased from common
commercial of analytical grade. Melting points were recorded on
a B-540 Buchi melting-point apparatus and uncorrected, IR spectra
were recorded on a Brüker VECTOR 22 FTIR spectrophotometer. 1H
NMR spectra were recorded on a Brucker Advance DMX 400 MHz
spectrometer with TMS as the internal standard. Proton Chemical
shifts are expressed in parts per million (ppm) and coupling con-
stants in hertz. Mass spectra (ESI-MS, positive) were recorded on
an Esquire-LC-00075 spectrometer.
4.1.3.3. (E)-3-{3-[(Diethylamino)-methyl]-phenyl}-1-(2-hydro-
xy-4,5-dimethoxyphenyl)-prop-2-en-1-one (6c). Orange oil, yield
59.3%; IR (KBr)
m
3585, 3050, 2967, 2933, 2801, 1674, 1635, 1574,
1512, 1250, 1157, 841 cmꢁ1
;
1H NMR (CDCl3) d 13.40 (s, 1H, OH),
7.87 (d, 1H, J = 15.2 Hz, H-3), 7.64 (s, 1H, Ar–H), 7.50–7.53 (m, 2H,
H-2 and Ar–H), 7.36–7.39 (m, 2H, Ar–H), 7.27 (s, 1H, Ar–H), 6.50
(s, 1H, Ar–H), 3.92 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.60 (s, 2H, ben-
zylic-CH2), 2.52 (q, 4H, J = 7.2 Hz, 2CH2CH3), 1.04 (t, 6H, J = 7.2 Hz, 2
CH2CH3); MS (ESI) m/z = 370.5 [M+H]+.
4.1.1. 2,4,5-Trihydroxy acetophenone (3)
To a mixture of acetic anhydride (55.5 mL, 0.59 mol) and concd
H2SO4 (0.9 mL, 17 mmol), 1,4-benzoquinone (21 g, 0.194 mol) was
added in portions with stirring at 50 °C in 15 min. Then, the mix-
ture was heated to 135 °C and H2O (30 mL) was added in drop wise
with distilling off the formed acetic acid in the meanwhile. After
the addition, H2O (50 mL) and para-toluenesulfonic acid (PTS,
2.0 g, 11 mmol) were added to the residue. The resulting mixture
was refluxed for 1 h, and then it was cooled to rt and poured into
a beaker with 300 mL ice-water. The formed brown solid was fil-
tered and washed with ice-water, followed by recrystallization
with H2O to get 10.1 g pale yellow solid, yield 30.6%, mp 188–
190 °C (lit.,19 201 °C).
4.1.3.4. (E)-1-(2-Hydroxy-4,5-dimethoxyphenyl)-3-[4-(pyrroli-
din-1-ylmethyl)-phenyl]-prop-2-en-1-one (6d). Orange solid,
yield 52.5%, mp 104–108 °C; IR (KBr)
m
3585, 3054, 2964, 2876,
2785, 1676, 1636, 1576, 1507, 1267,1159, 836 cmꢁ1
;
1H NMR
(CDCl3) d 13.39 (s, 1H, OH), 7.87 (d, 1H, J = 15.2 Hz, H-3), 7.60 (d,
2H, J = 8.4 Hz, Ar–H), 7.47 (d, 1H, J = 15.2 Hz, H-2), 7.40 (d, 2H,
J = 8.4 Hz, Ar–H), 7.26 (s, 1H, Ar–H), 6.51 (s, 1H, Ar–H), 3.93 (s,
3H, OCH3), 3.91 (s, 3H, OCH3), 3.66 (s, 2H, benzylic-CH2), 2.53–
2.55 (m, 4H, pyrrolidine-CH2), 1.79–1.81 (m, 4H, pyrrolidine-
CH2); MS (ESI) m/z = 368.5 [M+H]+.

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R. Sheng et al. / Bioorg. Med. Chem. 17 (2009) 6692–6698
4.1.3.5. (E)-1-(2-Hydroxy-4,5-dimethoxyphenyl)-3-[3-(pyrrolidin-
1-ylmethyl)-phenyl]-prop-2-en-1-one (6e). Orange oil, yield
IR (KBr)
m
;
3053, 2926, 2800, 1630, 1598, 1506, 1431, 1262, 1080,
826 cmꢁ1
1H NMR (CDCl3) d 7.84 (d, 2H, J = 8.4 Hz, Ar–H), 7.56 (s,
53.4%; IR (KBr)
m
3584, 3066, 2962, 2786, 1635, 1574, 1512,
;
1H, H-5), 7.48 (d, 2H, J = 8.8 Hz, Ar–H), 7.00 (s, 1H, H-3), 6.78 (s, 1H,
H-8), 4.02 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 3.70 (s, 2H, benzylic-
CH2), 2.55–2.57 (m, 4H, pyrrolidine-CH2), 1.81–1.83 (m, 4H, pyrroli-
dine-CH2); MS (ESI) m/z = 366.5 [M+H]+.
1248, 1156, 844 cmꢁ1
1H NMR (CDCl3) d 13.40 (s, 1H, OH), 7.87
(d, 1H, J = 15.2 Hz, H-3), 7.69 (s, 1H, Ar–H), 7.52–7.56(m, 2H, H-2
and Ar–H), 7.35–7.39 (m, 2H, Ar–H), 7.28 (s, 1H, Ar–H), 6.50 (s,
1H, Ar–H), 3.93 (s, 6H, 2OCH3), 3.68 (s, 2H, benzylic-CH2), 2.53–
2.56 (m, 4H, pyrrolidine-CH2), 1.80–1.83 (m, 4H, pyrrolidine-
CH2); MS (ESI) m/z = 368.5[M+H]+.
4.1.4.5. 6,7-Dimethoxy-2-[3-(pyrrolidin-1-ylmethyl)-phenyl]-4H-
chromen-4-one (7e). Pale yellow solid, yield 35.0%, mp 128–130 °C;
IR (KBr)
m
3060, 2953, 2930, 2788, 1632, 1600, 1505, 1428, 1347, 1261,
4.1.3.6. (E)-1-(2-Hydroxy-4,5-dimethoxyphenyl)-3-[4-(piperidin-
1-ylmethyl)-phenyl]-prop-2-en-1-one (6f). Orange solid, yield
1079, 822 cmꢁ1
;
1H NMR (CDCl3) d 7.94 (s, 1H, Ar–H), 7.79 (d, 1H,
J = 8.0 Hz, Ar–H), 7.57 (s, 1H, H-5), 7.47–7.51 (m, 2H, Ar–H), 7.06 (s,
1H, H-3), 6.82 (s, 1H, H-8), 4.03 (s, 3H, OCH3), 4.00 (s, 3H, OCH3),
3.76 (s, 2H, benzylic-CH2), 2.62–2.65 (m, 4H, pyrrolidine-CH2), 1.83–
1.87 (m, 4H, pyrrolidine-CH2); MS (ESI) m/z = 366.5 [M+H]+.
55.0%, mp 118–120 °C; IR (KBr)
m
3585, 3057, 2926, 2852, 2787,
1636, 1574, 1510, 1442, 1248, 840 cmꢁ1
;
1H NMR (CDCl3) d 13.40
(s, 1H, OH), 7.88 (d, 1H, J = 15.6 Hz, H-3), 7.60 (d, 2H, J = 8.0 Hz,
Ar–H), 7.47 (d, 1H, J = 15.2 Hz, H-2), 7.39 (d, 2H, J = 8.0 Hz, Ar–H),
7.26 (s, 1H, Ar–H), 6.51 (s, 1H, Ar–H), 3.94 (s, 3H, OCH3), 3.92 (s,
3H, OCH3), 3.50 (s, 2H, benzylic-CH2), 2.39(m, 4H, piperidine-
NCH2), 1.56–1.61 (m, 4H, piperidine-CH2), 1.44–1.45 (m, 2H, piper-
idine-CH2); MS (ESI) m/z = 382.5 [M+H]+.
4.1.4.6. 6,7-Dimethoxy-2-[4-(piperidin-1-ylmethyl)-phenyl]-4H-
chromen-4-one (7f). Paleyellowsolid, yield 60.3%, mp135–137 °C;
IR (KBr)m3057,2934, 2853,2795, 1646,1603, 1509, 1434, 1272,1082,
840 cmꢁ1; 1H NMR (CDCl3) d 7.88 (d, 2H, J = 8.0 Hz, Ar–H), 7.58 (s, 1H,
H-5), 7.55 (d, 2H, J = 8.4 Hz, Ar–H), 7.01 (s, 1H, H-3), 6.80 (s, 1H, H-8),
4.03 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 3.71 (s, 2H, benzylic-CH2), 2.57–
2.60 (m, 4H, piperidine-CH2), 1.68–1.74 (m, 4H, piperidine-CH2),
1.51–1.52 (m, 2H, piperidine-CH2); MS (ESI) m/z = 380.5 [M+H]+.
4.1.4. General procedure for preparation of flavone derivatives
7a–7f
Compound 6 (0.23 mmol) and iodine (9.3 mg, 0.037 mmol) was
added to a solution of concd H2SO4 (0.042 mL) in DMSO (3 mL), the
mixture was warm to 80–85 °C and stirred for 24 h. When the
reaction was over (TLC analysis), a mixture of 1 N HCl/ethyl acetate
(20 mL:20 mL) was added at rt, the organic layer was discarded,
and concentrated ammonium hydroxide was added into the aque-
ous solution until it was clearly basic (pH > 9), the product was ex-
tracted with ethyl acetate (20 mL ꢀ 3). The extract was washed
with brine, and dried over anhydrous Na2SO4. The solvent was re-
moved under reduced pressure. The residue was purified by silica
gel column chromatography (eluent, petroleum ether/EtOAc/
Et3N = 20:10:1) to afford 7.
4.1.5. General procedure for preparation of flavanone derivatives
8a–8f
Compound
6 (0.11 mmol) and sodium acetate (163 mg,
1.99 mmol) was added to a solution of ethyl alcohol (2.2 mL) and
H2O (0.08 mL), the mixture was heated to reflux, and keep the tem-
perature for 24 h, the solvent was evaporated under vacuum,
10 mL H2O was added to the residue and extracted with ethyl ace-
tate (10 mL ꢀ 3), the combined organic layer was washed with 2 N
NaOH, brine, and dried over anhydrous Na2SO4. The solvent was re-
moved under reduced pressure and the residue was purified by sil-
ica gel column chromatography (eluent, petroleum ether/EtOAc/
Et3N = 40:10:1) to afford 8.
4.1.4.1. 6,7-Dimethoxy-2-{4-[(ethylmethylamino)-methyl)-phe-
nyl}-4H-chromen-4-one (7a). Pale yellow solid, yield 44.4%, mp
128–130 °C; IR (KBr)
m
3054, 2967, 2930, 2838, 2786, 1632, 1598,
4.1.5.1. 6,7-Dimethoxy-2-{4-[(ethylmethylamino)-methyl]-phe-
nyl}-chroman-4-one (8a). Pale yellow solid, yield 64.3%, mp 118–
1506, 1430, 1262, 1079, 826 cmꢁ1
;
1H NMR (CDCl3) d 7.85 (d, 2H,
J = 8.0 Hz, Ar–H), 7.57 (s, 1H, H-5), 7.47 (d, 2H, J = 8.4 Hz, Ar–H),
7.00 (s, 1H, H-3), 6.78 (s, 1H, H-8), 4.02 (s, 3H, OCH3), 3.99 (s, 3H,
OCH3), 3.56 (s, 2H, benzylic-CH2), 2.45 (q, 2H, J = 7.2 Hz, CH2CH3),
2.22 (s, 3H, NCH3), 1.10 (t, 3H, J = 7.2 Hz, CH2CH3); MS (ESI) m/
z = 354.4 [M+H]+.
119 °C; IR (KBr)
m
2965, 2834, 2778, 1672, 1614, 1503, 1470, 1441,
1264, 1056, 900, 829 cmꢁ1
;
1H NMR (CDCl3) d 7.42 (d, 2H,
J = 8.0 Hz, Ar–H), 7.38 (d, 2H, J = 8.0 Hz, Ar–H), 7.33 (s, 1H, H-5),
6.53 (s, 1H, H-8), 5.43 (dd, 1H, J1 = 13.6 Hz, J2 = 3.2 Hz, H-2), 3.91 (s,
3H, OCH3), 3.90 (s, 3H, OCH3), 3.51 (s, 2H, benzylic-CH2), 3.02 (dd,
1H, J1 = 16.8 Hz, J2 = 13.2 Hz, H-3a), 2.79 (dd, 1H, J1 = 16.8 Hz,
J2 = 3.2 Hz, H-3b), 2.43 (q, 2H, J = 7.2 Hz, CH2CH3), 2.21 (s, 3H,
NCH3), 1.09 (t, 3H, J = 7.2 Hz, CH2CH3); MS (ESI) m/z = 356.4 [M+H]+.
4.1.4.2. 2-[4-(Diethylamino-methyl)-phenyl]-6,7-dimethoxy-4H-
chromen-4-one (7b). Pale yellow solid, yield 55.0%, mp 125–
126 °C; IR (KBr)
m 3057, 2972, 2932, 2808, 1631, 1601, 1507, 1431,
1266, 1110, 836 cmꢁ1; 1H NMR (CDCl3) d 7.84 (d, 2H, J = 8.4 Hz, Ar–
H), 7.56 (s, 1H, H-5), 7.51 (d, 2H, J = 8.0 Hz, Ar–H), 7.00 (s, 1H, H-3),
6.78(s,1H, H-8),4.02(s,3H,OCH3),3.99(s,3H, OCH3),3.68(s,2H, ben-
zylic-CH2), 2.57 (q, 4H, J = 6.8 Hz, 2CH2CH3), 1.07 (t, 6H, J = 6.8 Hz,
2CH2CH3); MS (ESI) m/z = 368.5 [M+H]+.
4.1.5.2. 2-{4-[(Diethylamino)-methyl]-phenyl}-6,7-dimethoxy-
chroman-4-one (8b). Pale yellow solid, yield 54.5%, mp 120–
122 °C; IR (KBr)
m
2967, 2833, 2785, 2713, 1671, 1615, 1502,
1469, 1441, 1264, 1056, 845, 828 cmꢁ1
;
1H NMR (CDCl3) d 7.42–
7.46 (m, 4H, Ar–H), 7.33 (s, 1H, H-5), 6.53 (s, 1H, H-8), 5.43 (dd,
1H, J1 = 13.6 Hz, J2 = 3.2 Hz, H-2), 3.91 (s, 3H, OCH3), 3.90 (s, 3H,
OCH3), 3.61 (s, 2H, benzylic-CH2), 3.02 (dd, 1H, J1 = 17.2 Hz,
J2 = 13.6 Hz, H-3a), 2.79 (dd, 1H, J1 = 16.8 Hz, J2 = 3.2 Hz, H-3b),
2.52 (q, 4H, J = 7.2 Hz, 2CH2CH3), 1.04 (t, 6H, J = 7.2 Hz, 2CH2CH3);
MS (ESI) m/z = 370.5 [M+H]+.
4.1.4.3. 2-{3-[(Diethylamino)-methyl]-phenyl}-6,7-dimethoxy-
4H-chromen-4-one (7c). Pale yellow solid, yield 19.0%, mp 124–
127 °C; IR (KBr)
m 3066, 2967, 2929, 2804, 1635, 1603, 1507, 1430,
1354, 1263, 1082, 826, 699 cmꢁ1; 1H NMR (CDCl3) d 7.96 (s, 1H, Ar–
H), 7.78 (d, 1H, J = 7.6 Hz, Ar–H), 7.56 (s, 1H, H-5), 7.46–7.51 (m, 2H,
Ar–H), 7.06 (s, 1H, H-3), 6.81 (s, 1H, H-8), 4.03 (s, 3H, OCH3), 3.99 (s,
3H, OCH3),3.70(s, 2H,benzylic-CH2),2.59(q, 4H,J = 7.2 Hz,2CH2CH3),
1.08 (t, 6H, J = 7.2 Hz, 2CH2CH3); MS (ESI) m/z = 368.5 [M+H]+.
4.1.5.3. 2-{3-[(Diethylamino)-methyl]-phenyl}-6,7-dimethoxy-
chroman-4-one (8c). Pale yellow oil, yield 54.0%; IR (KBr)
m 2967,
2933, 2873, 2800, 1677, 1612, 1504, 1468, 1424, 1267, 1056, 870,
843 cmꢁ1; 1H NMR (CDCl3) d 7.43 (s, 1H, Ar–H), 7.33 (m, 3H, Ar–H),
7.29 (s, 1H, H-5), 6.50 (s, 1H, H-8), 5.40 (dd, 1H, J1 = 13.6 Hz,
J2 = 2.8 Hz, H-2), 3.87 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.57 (s,
4.1.4.4. 6,7-Dimethoxy-2-[4-(pyrrolidin-1-ylmethyl)-phenyl]-4H-
chromen-4-one (7d). Pale yellow solid, yield 33 %, mp 172–174 °C;

R. Sheng et al. / Bioorg. Med. Chem. 17 (2009) 6692–6698
6697
2H, benzylic-CH2), 2.98 (dd, 1H, J1 = 17.2 Hz, J2 = 13.6 Hz, H-3a),
2.76–2.81 (dd, 1H, J1 = 16.8 Hz, J2 = 2.8 Hz, H-3b), 2.48 (q, 4H,
J = 6.8 Hz, 2CH2CH3), 1.00 (t, 6H, J = 6.8 Hz, 2CH2CH3); MS (ESI) m/
z = 370.5 [M+H]+.
2.23 (s, 3H, NCH3), 1.10 (t, 3H, J = 7.2 Hz, CH2CH3); MS (ESI) m/
z = 354.4 [M+H]+.
4.1.6.2. 3-{4-[(Diethylamino)-methyl]-phenyl}-6,7-dimethoxy-
4H-chromen-4-one (10b). Pale yellow solid, yield 48.7%, mp
4.1.5.4. 6,7-Dimethoxy-2-[4-(pyrrolidin-1-ylmethyl)-phenyl]-ch-
roman-4-one(8d). Pale yellow solid, yield 57.5%, mp 125–126 °C;
121–123 °C; IR (KBr)
m
2964, 2933, 2869, 2796, 1636, 1600, 1508,
1473, 1420, 1273, 1045, 830 cmꢁ1
;
1H NMR (CDCl3) d 7.98 (s, 1H,
IR (KBr)
m
2961, 2876, 2809, 1667, 1611, 1503, 1469, 1421, 1262,
H-2), 7.64 (s, 1H, H-5), 7.52 (d, 2H, J = 8.0 Hz, Ar–H), 7.41 (d, 2H,
J = 8.0 Hz, Ar–H), 6.89 (s, 1H, H-8), 4.00 (s, 6H, 2OCH3), 3.63 (s,
2H, benzylic-CH2), 2.54 (q, 4H, J =7.2 Hz, 2CH2CH3), 1.06 (t, 6H,
J = 7.2 Hz, 2CH2CH3); MS (ESI) m/z = 368.5 [M+H]+.
1053, 861 cmꢁ1
;
1H NMR (CDCl3) d 7.44–7.48 (m, 4H, Ar–H), 7.33
(s, 1H, H-5), 6.53 (s, 1H, H-8), 5.43 (dd, 1H, J1 = 13.6 Hz,
J2 = 2.8 Hz, H-2), 3.91 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.69 (s,
2H, benzylic-CH2), 3.01 (dd, 1H, J1 = 16.8 Hz, J2 = 13.6 Hz, H-3a),
2.79 (dd, 1H, J1 = 17.2 Hz, J2 = 2.8 Hz, H-3b), 2.59–2.63 (m, 4H, pyr-
rolidine-CH2), 1.83–1.85 (m, 4H, pyrrolidine-CH2); MS (ESI) m/
z = 368.5 [M+H]+.
4.1.6.3. 3-{3-[(Diethylamino)-methyl]-phenyl}-6,7-dimethoxy-
4H-chromen-4-one (10c). Pale yellow oil, yield 33.5%; IR (KBr)
2965, 2926, 2853, 1638, 1606, 1506, 1471, 1432, 1272, 1057, 826,
801 cmꢁ1 1H NMR (CDCl3) d 8.18 (s, 1H, H-2), 7.78 (s, 1H, H-5),
m
;
4.1.5.5. 6,7-Dimethoxy-2-[3-(pyrrolidin-1-ylmethyl)-phenyl]-ch-
7.62 (m, 2H, Ar–H), 7.44–7.47 (m, 2H, Ar–H), 6.90 (s, 1H, H-8),
4.00 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 3.92 (s, 2H, benzylic-CH2),
2.85 (q, 4H, J = 6.8 Hz, 2CH2CH3), 1.25 (t, 6H, J = 6.8 Hz, 2CH2CH3).
MS (ESI) m/z = 368.5 [M+H]+.
roman-4-one (8e). Pale yellow oil, yield 52.8%; IR (KBr)
2875, 2833, 2787, 1678, 1612, 1503, 1468, 1267, 1056, 870,
843 cmꢁ1 1H NMR (CDCl3) d 7.44 (s, 1H, Ar–H), 7.32–7.37 (m, 3H,
m 2962,
;
Ar–H), 7.31 (s, 1H, H-5), 6.52 (s, 1H, H-8), 5.41 (dd, 1H, J1 = 13.6 Hz,
J2 = 2.8 Hz, H-2),3.89(s,3H,OCH3),3.88(s, 3H,OCH3),3.64(s, 2H, ben-
zylic-CH2), 2.99 (dd, 1H, J1 = 16.8 Hz, J2 = 13.6 Hz, H-3a), 2.78 (dd, 1H,
J1 = 17.2 Hz, J2 = 2.8 Hz, H-3b), 2.51–2.52 (m, 4H, pyrrolidine-CH2),
1.77–1.80 (m, 4H, pyrrolidine-CH2); MS (ESI) m/z = 368.5 [M+H]+.
4.1.6.4. 6,7-Dimethoxy-3-[4-(pyrrolidin-1-ylmethyl)-phenyl]-
4H-chromen-4-one (10d). Pale yellow solid, yield 54.0%, mp
126–128 °C; IR (KBr)
m
2952, 2926, 2855, 2784, 1629, 1599, 1507,
1462, 1431, 1276, 1047, 834 cmꢁ1
.
1H NMR (CDCl3) d 7.97 (s, 1H,
H-2), 7.64 (s, 1H, H-5), 7.53–7.55 (d, 2H, J = 8.0 Hz, H-20 and H-
60), 7.41–7.43 (d, 2H, J = 8.4 Hz, H-30 and H-50), 6.89 (s, 1H, H-8),
4.00 (s, 6H, 2 ꢀ OCH3), 3.67 (s, 2H, benzylic-CH2), 2.56 (m, 4H, pyr-
rolidine-CH2, H-200 and H-500), 1.81 (m, 4H, H-300 and H-400); MS
(ESI) m/z = 366.5 [M+H]+.
4.1.5.6. 6,7-Dimethoxy-2-[4-(piperidin-1-ylmethyl)-phenyl]-chro-
man-4-one (8f). Pale yellow solid, yield 77.8%, mp 137–140 °C;
IR (KBr)
m
2934, 2851, 2752, 1667, 1611, 1503, 1468, 1436, 1264,
1053, 835 cmꢁ1
;
1H NMR (CDCl3) d 7.43 (d, 2H, J = 8.0 Hz, Ar–H),
7.39 (d, 2H, J = 8.0 Hz, Ar–H), 7.34 (s, 1H, H-5), 6.54 (s, 1H, H-8),
5.43 (dd, 1H, J1 = 13.6 Hz, J2 = 3.2 Hz, H-2), 3.92 (s, 3H, OCH3),
3.91 (s, 3H, OCH3), 3.51 (s, 2H, benzylic-CH2), 3.03 (dd, 1H,
J1 = 16.8 Hz, J2 = 13.6 Hz, H-3a), 2.80 (dd, 1H, J1 = 16.8 Hz,
J2 = 3.2 Hz, H-3b), 2.40–2.43 (m, 4H, piperidine-CH2), 1.56–1.61
(m, 4H, piperidine-CH2), 1.45–1.46 (m, 2H, piperidine-CH2); MS
(ESI) m/z = 382.5 [M+H]+.
4.1.6.5. 6,7-Dimethoxy-3-[3-(pyrrolidin-1-ylmethyl)-phenyl]-
4H-chromen-4-one (10e). Pale yellow solid, yield 20.4%, mp
123–124 °C; IR (KBr): 2955, 2903, 2877, 2784, 1643, 1621, 1601,
1519, 1473, 1432, 1275, 1054, 820, 796 cmꢁ1 1H NMR (CDCl3) d
;
7.99 (s, 1H, H-2), 7.64 (s, 1H, H-5), 7.55 (s, 1H, H-20), 7.46 (d, 1H,
J = 7.2 Hz, Ar–H), 7.35–7.41 (m, 2H, Ar–H), 6.89 (s, 1H, H-8), 4.00
(s, 3H, OCH3), 3.99 (s, 3H, OCH3), 3.68 (s, 2H, benzylic-CH2), 2.55
(m, 4H, pyrrolidine-CH2), 1.79–1.80 (m, 4H, pyrrolidine-CH2); MS
(ESI) m/z = 366.5 [M+H]+.
4.1.6. General procedure for preparation of compounds 10a–10f
Compound 6 (0.16 mmol), anhydrous potassium carbonate
(110 mg, 0.40 mmol), and benzoyl chloride (75 mg, 0.53 mmol)
were mixed with anhydrous DMF (1.5 mL), and the mixture was
warmed to 60 °C and stirred for 4 h. H2O (20 mL) was added to
the mixture and extracted with ethyl acetate (20 mL ꢀ 3), the or-
ganic layer was washed with brine, dried over anhydrous Na2SO4.
The solvent was evaporated and the residue was purified by silica
gel column chromatography (eluent, petroleum ether/EtOAc/
Et3N = 40:10:1) to afford benzoate derivatives 9. To a solution of
9 (0.16 mmol) in methanol (3 mL), a mixture of iodosobenzene
diacetate (DIB, 105 mg, 0.33 mmol) and PTS (124 mg, 0.65 mmol)
in 2.5 mL methanol was added in dropwise, stirred for 24 h at.rt
Then, a solution of NaOH (41 mg, 1.02 mmol)in CH3OH–H2O
(6.8 mL: 1.4 mL) was added, and stirred for 24 h at rt. The solvent
was evaporated under vacuum and the residue was extracted with
ethyl acetate (20 mL ꢀ 3), washed with brine, and dried over anhy-
drous Na2SO4. The solvent was removed and the residue was puri-
fied by silica gel column chromatography (eluent, petroleum ether/
EtOAc/Et3N = 30:10:1) to afford 10.
4.1.6.6. 6,7-Dimethoxy-3-[4-(piperidin-1-ylmethyl)-phenyl]-4H-
chroman-4-one (10f). Pale yellow solid, yield 35.2%, mp 151–
152 °C; IR (KBr)
m
2935, 2854, 2761, 1636, 1599, 1508, 1456,
1423, 1271, 1044, 828 cmꢁ1
;
1H NMR (CDCl3) d 7.97 (s, 1H, H-2),
7.63 (s, 1H, H-5), 7.51 (d, 2H, J = 8.0 Hz, Ar–H), 7.38 (d, 2H,
J = 8.0 Hz, Ar–H), 6.89 (s, 1H, H-8), 4.00 (s, 3H, OCH3), 3.99 (s, 3H,
OCH3), 3.51 (s, 2H, benzylic-CH2), 2.40 (m, 4H, piperidine-CH2),
1.57–1.61 (m, 4H, piperidine-CH2), 1.43–1.44 (m, 2H, piperidine-
CH2); MS (ESI) m/z = 380.5 [M+H]+.
4.2. Pharmacology and molecular docking
4.2.1. AChE and BuChE inhibition assays
AChE and BuChE activities were measured by the spectrophoto-
metric method with slight modification, rat cortex homogenate
and rat serum were used as the resource of AChE and BuChE,
respectively. The brain homogenate was preincubated for 5 min
with tetraisopropyl pyrophosphoramido (isoOMPA, selective
inhibitor of BuChE, 0.04 mmol/L). For assay of AChE or BuChE activ-
4.1.6.1. 6,7-Dimethoxy-3-{4-[(ethylmethylamino)-methyl]-phe-
nyl}-4H-chromen-4-one (10a). Pale yellow solid, yield 22.8%, mp
ity, a reaction mixture of 200
dide 0.3 mmol/L or butyrylthiocholine iodide 0.4 mmol/L, sodium
phosphate buffer (0.1 mmol/L, pH 7.4) 100 l, homogenate or ser-
um 20 l and different concentrations of test compounds 20 l was
incubated at 37 °C for 15 min. The reaction was terminated by
adding 50 l 3% sodium lauryl sulface, then, 50
l 0.2% 5,50-
ll containing acetylthiocholine io-
102–104 °C; IR (KBr)
m
2968, 2936, 2837, 2789, 1635, 1605, 1507,
1471, 1432, 1273, 1048, 827 cmꢁ1
;
1H NMR (CDCl3) d 7.97 (s, 1H,
l
H-2), 7.64 (s, 1H, H-5), 7.53 (d, 2H, J = 8.0 Hz, Ar–H), 7.38 (d, 2H,
J = 8.0 Hz, Ar–H), 6.89 (s, 1H, H-8), 4.00 (s, 3H, OCH3), 3.99 (s, 3H,
OCH3), 3.54 (s, 2H, benzylic-CH2), 2.46 (q, 2H, J = 7.2 Hz, CH2CH3),
l
l
l
l

6698
R. Sheng et al. / Bioorg. Med. Chem. 17 (2009) 6692–6698
dithio-bis-(2-nitrobenzoic acid) was added to produce the yellow
anion of 5-thio-2-nitro-benzoic acid. The values of IC50 were cal-
culated by UV spectroscopy from the absorbance changes at
450 nm. Donepezil and rivastigmine were applied as positive
drugs. All samples were assayed in duplicate.
References and notes
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Andrisano, V.; Valenti, P.; Recanatini, M. J. Med. Chem. 2003, 46, 2279.
3. He, X. C.; Feng, S.; Wang, Z. F.; Shi, Y. F.; Zheng, S. X.; Xia, Y.; Jiang, H. L.; Tang, X.
C.; Bai, D. L. Bioorg. Med. Chem. 2007, 15, 1394.
4. Francesco, L.; Marco, C.; Orazio, N.; Leonardo, P.; Anna, C.; Angela, S.; Angelo, C.
Bioorg. Med. Chem. 2008, 16, 7450.
5. Pelayo, C.; Xavier, F.; Carles, G.; Tania, G.; Diego, M. T.; Michele, S.; Elisabet, V.;
Albert, B.; Victoria, M.; Antoni, C.; Merce, P.; Manuela, B.; Francesca, M.;
Vincenza, A.; Joan, E.; Monica, L.; Axel, B. C.; Javier, L. J. Med. Chem. 2008, 51,
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4.2.2. Molecular docking of compound 10d
Molecular docking studies were performed using FLEXIDOCK mod-
ule of Sybyl 6.9. The X-ray crystal structure of Tc-AChE/E2020
(donepezil) was used (PDB ID: 1EVE). A 10d-binding pocket was
defined to cover all residues within 3.0 Å of the ligand in the com-
plex. All bound waters and ligands were removed from the com-
plex, followed by adding hydrogen atoms. All the single bonds of
residue side chains inside the defined pocket and the ligand were
allowed to rotate on all single bonds. Kollman all-atom charges
were loaded to the protein, and Gasteiger–Huckel charges were
assigned to the ligand atoms. The structure optimization was per-
formed for 100,000 generations using a genetic algorithm and the
20 best scoring ligand–protein complexes were kept for further
analyses.
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Acknowledgments
This study was financially supported by the National Natural
Science Foundation of China (30572239), Health Bureau of
Zhejiang Province Foundation (No. WKJ2007-016).
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