Synthesis, biological evaluation and molecular modeling of aloe-emodin derivatives as new acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.01.015

A series of aloe-emodin derivatives were designed, synthesized and evaluated as acetylcholinesterase inhibitors. Most of the new prepared compounds showed remarkable acetylcholinesterase inhibitory activities. Among them, the compound 1-((4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) methyl) pyridin-1-ium chloride (C3) which has a pyridinium substituent possessed the best inhibitory activity of acetylcholinesterase (IC₅₀ = 0.09 μM). The docking study performed with AUTODOCK demonstrated that C3 could interact with the catalytic active site (CAS) and the peripheral anionic site (PAS) of acetylcholinesterase.

Full text of DOI:10.1016/j.bmc.2013.01.015

Bioorganic & Medicinal Chemistry 21 (2013) 1064–1073
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular modeling of
aloe-emodin derivatives as new acetylcholinesterase inhibitors
Da-Hua Shi a,b, Wei Huang a, Chao Li a, Ling-Ting Wang a, Shi-Fan Wang a,
⇑
a School of Ocean, Hainan University, Haikou 570228, People’s Republic of China
b School of Chemical Engineering, Huaihai Institute of Technology, Lianyungang 222005, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of aloe-emodin derivatives were designed, synthesized and evaluated as acetylcholinesterase
inhibitors. Most of the new prepared compounds showed remarkable acetylcholinesterase inhibitory
activities. Among them, the compound 1-((4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
methyl) pyridin-1-ium chloride (C3) which has a pyridinium substituent possessed the best inhibitory
Received 29 November 2012
Revised 4 January 2013
Accepted 5 January 2013
Available online 16 January 2013
activity of acetylcholinesterase (IC50 = 0.09 lM). The docking study performed with AUTODOCK demon-
strated that C3 could interact with the catalytic active site (CAS) and the peripheral anionic site (PAS) of
acetylcholinesterase.
Keywords:
Aloe-emodin derivatives
Acetylcholinesterase
Inhibitors
Ó 2013 Elsevier Ltd. All rights reserved.
Alzheimer’s disease
1. Introduction
weak AChE-inhibition activity when we screened the AChE inhibi-
tors from natural products. In search of new rationally designed
Alzheimer’s disease (AD) is a multifaceted neurodegenerative
disorder characterized at a molecular level by protein misfolding
and aggregation, oxidative stress, mitochondrial abnormalities,
and neuroinflammatory processes.1 Based on the cholinergic
hypothesis of memory dysfunction, the depletion of neurotrans-
mitter acetylcholine, and loss of associated synapses during the
progression of AD, some AChE inhibitors such as tacrine, donepezil,
rivastigmine and galantamine have been synthesized and devel-
oped for clinical practice to ameliorate the associated cognitive
symptoms.2 Though AChE inhibitors have become the mainstays
for treating AD,3 the nonselectivity of these drugs, poor bioavail-
ability, adverse cholinergic side effects in the periphery, narrow
therapeutic ranges and hepatotoxicity are some of the severe lim-
itations to their therapeutic success.4,5 It is necessary for other
studies on the AChE inhibitors derived from medicinal plants or
from designing and development of synthesis.
Natural products continue to provide useful drugs or templates
for the development of other compounds.6 Some natural products
with poor AChE-inhibition activities were designed and synthe-
sized as potent AChE inhibitors for the treatment of AD.7 Aloe-
emodin is the major anthraquinone in aloe plants and possesses
antibacterial, antiviral, hepatoprotective, anticancer, laxative and
anti-inflammation effects.8 We found that aloe-emodin possessed
AChE inhibitors for the treatment of AD, we designed and synthe-
sized a series of aloe-emodin derivatives with potent AChE inhibi-
tory activities.
2. Results and discussion
2.1. Chemistry
Three series of twenty-five aloe-emodin derivatives A2–A6, B1–
B9 and C1–C11 (Scheme 1–3) were synthesized starting from aloe-
emodin (A1). In series A (Scheme 1), the phenolic hydroxyl groups
of aloe-emodin were protected with methoxyl, acetyl, benzoyl or
tosyl in order to increase the lipid solubility of derivatives A2–
A6, so that those derivatives could be better through the blood
brain barrier. In series B (Scheme 2), the alcoholic hydroxyl of
aloe-emodin A1 and methylated aloe-emodin A2 were first respec-
tively oxidized into formyl group in anhydrous CH2Cl2 and car-
boxyl group in DMF–water (v:v = 50:1) mixed solution under the
catalytic action of pyridinium chlorochromate (PCC) to obtain the
intermediates B1, B2 and B7, B8. Then, the intermediates B1, B2
and B7, B8 were modified by the condensation or the acylation
with different amine, respectively. The results indicated that the
condensation of the intermediate B2 could give expected products
B3–B5 in a high yield, but except for compound B6, the condensa-
tion reaction of the intermediate B1 could not be conducted due to
the exposed phenolic hydroxyl group. The acylation of the
⇑
Corresponding author.
E-mail address: wangsf777@163.com (S.-F. Wang).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.015

D.-H. Shi et al. / Bioorg. Med. Chem. 21 (2013) 1064–1073
1065
O
OH
OTs O
OTs
A6
O
O
O
OAc
OH
OH
OAc O
OAc
OH
O
A1
OH
OCH3O
OCH3
A5
A2
O
O
O
OH
OBz
O
O
OBz O
OBz
OCH3O
OCH3
A4
A3
Scheme 1. Synthetic pathway for compounds A2–A6.
O
O
O
O
O
OH
R2
R3
OH
O
OH
OR1
O
OR1
OR1
OR1
A1
B7 B9
-
B1-B6
OAc
O
AcO
OAc
B6 R
B1 R1= H
R2= O
1= H R2=
OAc
N
B2 R1= CH3
B3 R1= CH3
B4 R1= CH3
B5 R1= CH3
R2= O
B7
B8
R1= H
R1= CH3
R3= OH
R3= OH
R2= NOH
R2= NNH2
R2= NC6H5
OAc
OAc
OAc
O
AcO
B9
R1= H R3=
N
H
Scheme 2. Synthetic pathway for compounds B1–B9.
O
O
O
OCH3O
O
Cl
OH
Cl
OH
O
OH
OH
O
O
OH
OCH3
C1
C2
A1
O
R
R
Cl
OCH3O
OCH3
OH
O
OH
OTs O
OTs
C3-C5
C6-C10
C11
N
C3 R=
C7 R=
C4 R=
N
Cl
C5
R=
N
C6
N
R=
Cl
Cl
Cl
Cl
N
N
C8
N
R=
N
N
N
C9 R=
N
C10 R=
Cl
Cl
Cl
Scheme 3. Synthetic pathway for compounds C1–C11.

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D.-H. Shi et al. / Bioorg. Med. Chem. 21 (2013) 1064–1073
Table 1
The acetylcholinesterase-inhibition activities of aloe-emodin and its derivatives (A1–A6, B1–B9, C1–C11)
Compd
Structures
Inhibition
Inhibit.a (%)
53.16 4.39
IC50 (lM)
O
OH
OH
A1
—
OH
O
O
OH
A2
A3
49.36 2.19
—
—
OCH3O
OCH3
O
O
O
<10
O
O
O
O
O
O
O
OH
O
A4
A5
60.27 8.56
—
—
O
OCH3O
O
OCH3
OAc
OAc
32.87 17.11
OAc
O
O
OH
A6
38.35 17.92
—
O
O
S
O
O
O
O
S
O
O
O
B1
B2
B3
B4
43.03 3.80
34.24 14.82
71.23 4.11
13.69 6.53
—
—
—
—
OH
O
O
O
OH
O
O
O
O
OH
N
O
O
O
O
NH2
N
N
O
O
O
O
B5
B6
35.61 4.75
<10
—
—
O
O
O

D.-H. Shi et al. / Bioorg. Med. Chem. 21 (2013) 1064–1073
1067
Table 1 (continued)
Compd
Structures
Inhibition
Inhibit.a (%)
IC50 (lM)
O
O
O
O
O
O
O
N
O
O
O
OH
OH
O
O
O
OH
OH
O
O
O
OH
OH
B7
B8
<10
—
—
O
O
69.86 6.28
O
O
O
O
O
O
B9
<10
—
N
H
O
O
O
O
O
OH
OH
O
O
OH
OH
O
Cl
Cl
C1
C2
C3
C4
C5
C6
27.39 7.11
52.05 6.28
101.51 1.26
93.15 2.37
82.22 3.55
76.71 6.28
—
O
O
—
OCH3O
O
OCH3
OH
Cl
N
0.09
3.76
OH
OH
OH
O
O
Cl
N
O
O
OH
Cl
N
25.38
O
O
OH
Cl
N
—
OCH3O
O
OCH3
OCH3
Cl
N
C7
C8
99.31 0.24
87.67 0.00
0.54
OCH3O
13.56
(continued on next page)

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D.-H. Shi et al. / Bioorg. Med. Chem. 21 (2013) 1064–1073
Table 1 (continued)
Compd
Structures
Inhibition
Inhibit.a (%)
IC50 (lM)
O
Cl
N
OCH3O
OCH3
O
O
Cl
N
N
N
C9
76.71 6.28
73.97 6.28
—
—
N
CH3O
O
OCH3
Cl
N
C10
OCH3O
OCH3
O
Cl
C11
<10
—
—
O
O
S
O
O
O
S
O
O
Tacrine
0.26
a
The inhibition activities of the compounds at the concentration of 100 lg/mL.
intermediate B7 with glucosamine derivative gives the conjugates
B9 in a high yield. In series C (Scheme 3), the alcoholic hydroxyl of
aloe-emodin A1 or methylated aloe-emodin A2 was first replaced
by chlorine atom and then the quaternary ammonium similar to
fragment of acetylcholine introduced into the molecule of aloe-
emodin give eight derivatives of aloe-emodin C3–C10, which were
expected to possess the cholinomimetic activity for the treatment
of AD.
mity with the reported results that a lot of potent natural AChE
inhibitors are alkaloids.11 Compound C3 possessed the best AChE
inhibitory activity with IC50 of 0.09 lM, which was better than
the positive control tacrine (IC50 = 0.26 lM) of the first drug used
for the treatment of AD.12 Compound C3 inhibited AChE with a
dose-dependent relationship (Fig. 1).
2.3. Kinetic characterization of AChE inhibition
2.2. In vitro inhibition studies of AChE
The Lineweaver–Burk plot (Fig. 2A) revealed that compound C3
was a mixed AChE inhibitor which was similar to that of tacrine.13
Double plot of the Lineweaver–Burk plot (Fig. 2B) showed that the
In our previous studies of the anti-AD agents, we found that
aloe-emodin could inhibit AChE with the inhibitory rat of 53.16%
Ki value of compound C3 against AChE was 0.279 lM. The result
at the concentration of 100
lg/mL. Aloe-emodin is the major
showed that compound C3 was able to interact with both CAS
anthraquinone in aloe plants and possesses antibacterial, antiviral,
hepatoprotective, anticancer, laxative and anti-inflammation ef-
fects.8 Considering the anti-inflammation effects and the AChE-
inhibition of aloe-emodin, we modified or optemized its structure
to find new and efficient AChE inhibitors with aloe-emodin as a
lead compound. To determine AChE inhibitory activities, com-
pounds were measured in vitro according to the modified Ellman’s
method9 using rat cortex homogenate (AChE).10 Tacrine was mea-
sured as a standard for the comparative purpose. The results were
summarized in Table 1. The series A and series B compounds
showed the weak AChE-inhibition activity. The derivatives that
the phenolic hydroxyl groups of aloe-emodin were protected with
methoxyl, acetyl, benzoyl or tosyl revealed weaker activities than
the mother compound. The results showed that the phenolic hy-
droxyl groups of the aloe-emodin played an important role in the
AChE-inhibition activity. In series C, the aloe-emodin derivatives
containing quaternary ammonium fragment C3–C10 showed po-
tent AChE-inhibition activities. These derivatives, which were de-
signed in view of the structure of cholinergic drugs, proved that
the quaternary ammonium groups are key groups and play an
important role in the inhibition against AChE. This was in confor-
and PAS of AChE.14
2.4. Molecular modeling study
The molecule docking of the compound C3 with AChE (PDB
No. 2CMF) showed that the compound C3 could interact with
120
100
80
60
40
20
0
0.0001
0.001
0.01
0.1
1
10
Concentration (μg/mL)
Figure 1. Dose-dependent inhibition of C3 against AChE. Values are means SD,
n = 3.

D.-H. Shi et al. / Bioorg. Med. Chem. 21 (2013) 1064–1073
1069
1800
1600
1400
1200
1000
800
32
27
22
1 7
1 2
7
A
B
600
400
200
2
0
-0. 01
-0. 005
0
0. 005
0. 01
0. 01 5
-3
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
[S-1 ], mM-1
[I], µM
Figure 2. Steady-state inhibition of AChE by C3. (A) Lineweaver–Burk plot of reciprocal of initial velocities versus reciprocal of five fixed acetylthiocholine iodide (ATC)
concentrations (50, 100, 200, 300 and 400 M) in the absence (ꢀ) and presence of C3 at 0.045 M (d), 0.090 M (N) and 0.18 0 M (j); (B) secondary plots of the Lineweaver–
Burk plot, slope versus various concentrations of C3.
l
l
l
l
Figure 3. A close view of the potential interaction between C3 and the binding site of AChE (PDB No. 2CMF). (A) Binding interactions of C3 with AChE. The most important
residues involved in the interactions with C3. The green compound was bis-tacrine; (B) interatomic contacts of the C3 and bis-tacrine (green) with the residues in the active
site of AChE.
both the CAS and PAS of AChE as that of bis-tacrine15 (Fig. 3)
3. Conclusion
which was also in agreement with the results of kinetic studies.
When C3 was docked with AChE, the 8-OH of the compound
could interact with HIS440 at the CAS of the AChE through the
hydrogen bond. This also proved the importance of the hydroxy
of aloe-emodin derivatives for the AChE-inhibition activity. The
pyridine ring of the compound could interact with the TRP279
A series of aloe-emodin derivatives were synthesized with
simple ways and high yields. The AChE inhibitory activities of
the derivatives were evaluated in vitro, and eight new AChE
inhibitors (C3–C10) were found more active than aloe-emodin.
The compound C3, which possessed the better AChE-inhibition
activity than tacrine, could interact with both the CAS and PAS
of AChE. This compound could be promising lead candidates as
AD therapeutics. Nowadays, drug discovery in AD is gradually
moving from the development of molecules with a single target
to the ‘multi-target-directed ligands’ (MTDLs). Considering the
affirmative effects of AChE inhibitors on AD, most MTDLs were
designed based on the acetylcholinesterase inhibition activity.17
In the further study, the anti-inflammatory activity of the aloe-
emodin derivatives will be tested. If the compound C3 also pos-
sessed the anti-inflammatory activity as the mother compound,
and TYR334 at the PAS through the
p–p interaction. The binding
of C3 was energetically favorable, with binding energy of ꢀ9.04
(Fig. 3).
The kinetic study and the molecule modeling showed that the
compound C3 could bind to the CAS and PAS at the same time. It
was reported that the Ab aggregation property of AChE could be
inhibited by non-competitive or mixed type of AChE inhibitors
which could interact with PAS of the AChE.16 Therefore, the com-
pound C3 might prevent the aggregation of Ab induced by AChE
based on its AChE-inhibition activity.

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D.-H. Shi et al. / Bioorg. Med. Chem. 21 (2013) 1064–1073
it will be a good multi-target-directed ligand for the treatment of
AD.
4.1.3. Synthesis of 4-((4,5-dimethoxy-9,10-dioxo-9,10-
dihydroanthracen-2-yl)methoxy)-4-oxobutanoic acid (A4)
Succinic anhydride (2.5 mmol, 0.25 g), and compound A2
(0.5 mmol, 0.15 g) was added into dry pyridine (10 mL). The mix-
ture was stirred at room temperature for 48 h, monitored by TLC.
After the completion of reaction, the reaction mixture was poured
into ice water. The pH value of the reaction mixture solution was
adjusted within the scope of 4–5, to give orange precipitate.
Recrystallization of the orange precipitate from ethanol gave com-
pound A4 as yellow tabular crystal, yield 73%; mp 109–113 °C; IR
mmax cmꢀ1: 3101, 2941, 1736, 1669, 1585, 1277, 1235, 750; 1H
NMR (CDCl3): d = 2.74 (t, 4H, J1 = 2.3 Hz, J2 = 3.1 Hz, 2CH2), 4.01
(s, 6H, 2CH3), 5.24 (s, 2H, CH2), 7.26 (s, 1H, H-C(1)), 7.30 (d, 1H,
J = 8.3 Hz, H-C(6)), 7.62 (s, 1H, H-C(3)), 7.79 (d, 1H, J = 7.8 Hz, H-
C(8)), 7.85 (dd, 1H, J1 = 8.3 Hz, J2 = 7.8 Hz, H-C(7)), 10.11 (s, 1H,
COOH); ESI-MS m/z: 399.11 [M+H]+.
4. Experimental part
4.1. Chemistry
Reaction and the resulted products were monitored by thin-
layer chromatography (TLC) on the self-made pre-coated silica
gel 60 F254 plates. Spots were detected by a UV lamp visualized
at 254 nm and/or 365 nm. Melting points (°C, uncorrected) were
determined on a XT4 MP apparatus (Taike Corp., Beijing, China).
The IR spectra were recorded on a Thermo iS10 IR spectrometer
(KBr pellets) and 1H NMR spectra were recorded in CDCl3 or
DMSO-d6 on a Bruker DPX400 spectrometer with TMS and solvent
signals allotted as internal standards. Splitting patterns are as fol-
lows: s, singlet; d, doublet; dd, double doublets; t, triplet; m, mul-
tiplet. Chemical shifts are reported in d (ppm) and coupling
constants are given in Hertz. Aloe-emodin (A1) were purchased
from Shanxi Sciphar Biotechnology Co. Ltd. (Shanxi, China) and
recrystallized from DMF, mp 225–227 °C; IR mmax cmꢀ1: 3131,
2367, 1672, 1628, 1572, 1400, 1288; 1H NMR (DMSO-d6):
d = 4.60 (s, 2H, CH2), 5.58 (s, 1H, HO-CH2), 7.21 (s, 1H, H-C(2)),
7.31 (d, 1H, J = 8.0 Hz, H-C(7)), 7.59 (s, 1H, H-C(4)), 7.62 (d, 1H,
J = 7.5 Hz, H-C(5)), 7.74 (dd, 1H, J1 = 7.5 Hz, J2 = 8.0 Hz, H-C(6)),
11.90 (s, 1H, HO-C(1)), 11.97 (s, 1H, HO-C(8)); ESI-MS m/z:
271.06 [M+H]+.
4.1.4. Synthesis of 3-(acetoxymethyl)-9,10-dioxo-9,10-
dihydroanthracene-1,8-diyl diacetate (A5)
Acetic anhydride (3 mL) was added into a solution of aloe-emo-
din A1 (1 mmol, 0.27 g) in dry pyridine (10 mL). The mixture was
stirred at 50 °C for 24 h, monitored by TLC. After the completion
of reaction, the reaction mixture was poured into ice water and
yellow solid was separated. Recrystallization of the yellow solid
from acetone gave compound A5 as pale yellow needle, yield
76%; mp 143–145 °C; IR mmax cmꢀ1: 3449, 2928, 2097, 2028,
1770, 1736, 1673, 1634, 1612, 1444, 1373, 1332, 1257; 1H NMR
(CDCl3): d = 2.20 (s, 3H, COCH3); 2.47 (s, 6H, 2COCH3); 5.23 (s,
2H, CH2); 7.39 (s, 1H, H-C(4)); 7.41 (d, 1H, J = 8.0 Hz, H-C(7));
7.78 (dd, 1H, J1 = 8.0 Hz, J2 = 7.9 Hz, H-C(6)); 8.20 (s, 1H, H-C(2));
8.23 (d, 1H, J = 7.9 Hz, H-C(5)); ESI-MS m/z: 397.09 [M+H]+.
Other reagents, all being of A.R. grade, were purchased from
Shanghai Chemical Reagent Company (Shanghai, China). All sol-
vents were distilled and dried before use.
4.1.1. Synthesis of 3-(hydroxymethyl)-1,8-
dimethoxyanthracene-9,10-dione (A2)
4.1.5. Synthesis of 3-(hydroxymethyl)-9,10-dioxo-9,10-
dihydroanthracene-1,8-diyl bis(4-methylbenzenesulfonate)
(A6)
Aloe-emodin (1 mmol, 0.27 g), dimethyl sulfate (10 mmol, 1 ml)
and potassium carbonate (10 mmol, 1.38 g) in 200 ml of dry ace-
tone were refluxed for 12 h at 56 °C till the reaction was accom-
plished. After the completion of reaction, the yielded mixture
was cooled to room temperature and filtered. The filtrate was dis-
tilled to afford a yellow solid. Recrystallization of the yellow solid
from acetone gave compound C11 as yellow needle, yield 65%; mp
223–225 °C; IR/cmꢀ1: 3129, 2989, 1662, 1591, 1400, 752; 1H NMR
(DMSO-d6): d = 3.91 (s, 6H, 2CH3), 4.64 (d, J = 5.7 Hz, 2H, CH2O),
5.54 (t, J = 5.7 Hz, 1H, OH), 7.21 (1H, s, H-C(2)), 7.30 (1H, d,
J = 8.2 Hz, H-C(7)), 7.58 (1H, s, H-C(4)), 7.61 (1H, d, J = 7.5 Hz, H-
C(5)), 7.74 (1H, dd, J1 = 8.2 Hz, J2 = 7.5 Hz, H-C(6)); ESI-MS m/z:
299.10 [M+H]+.
Tosyl chloride (2 mmol, 0.38 g), anhydrous K2CO3 (0.5 g) and
aloe-emodin A1 (1 mmol, 0.27 g) were added into dry acetone
(15 ml). The mixture was stirred at 60 °C for 6 h, monitored by
TLC. After the completion of reaction, the reaction mixture was
poured into ice water and pale yellow solid was separated. Recrys-
tallization of the pale yellow solid from acetone gave compound A6
as pale yellow granular crystals, yield 70%; mp 206–208 °C; IR mmax
cmꢀ1: 3482, 3071, 2924, 2363, 1925, 1683, 1597, 1493, 1448, 1365,
1319, 1250; 1H NMR (DMSO-d6): d = 2.39 (s, 6H, 2CH3); 4.66 (s, 2H,
CH2); 5.67 (s, 1H, OH); 7.42 (d, 1H, J = 8.0 Hz, H-C(7)), 7.43 (s, 1H,
H-C(4)), 7.70 (d, 1H, J = 8.0 Hz, H-C(2)), 7.57 (d, 4H, J = 8.0 Hz, H
on the ring of tosyl C(3, 5)), 7.88 (d, 4H, J = 8.0 Hz, H on the ring
of tosyl C(2, 6)), 8.07 (dd, 1H, J1 = 8.0 Hz, J2 = 8.0 Hz, H-C(6)), 8.13
(d, 1H, J = 8.0 Hz, H-C(5)); ESI-MS m/z: 579.08 [M+H]+.
4.1.2. Synthesis of 3-((benzoyloxy)methyl)-9,10-dioxo-9,10-
dihydroanthracene-1,8-diyl dibenzoate (A3)
Benzoyl chloride (2 mmol, 2 ml), anhydrous K2CO3 (0.5 g) and
aloe-emodin (0.5 mmol, 0.135 g) were added into dry acetone
(100 ml). The mixture was stirred at 60 °C for 24 h, monitored by
TLC. After the completion of reaction, the reaction mixture was
poured into ice water and pale yellow solid was separated. Recrys-
tallization of the pale yellow solid from acetone gave compound A3
4.1.6. 4,5-Dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-
carbaldehyde (B1)
0.33 g (1.50 mmol) of pyridinium chlorochromate (PCC) was
added in 200 ml of dichloromethane containing 0.27 g of com-
pound A1 (1.0 mmol). The yielded mixture was stirred at room
temperature 6 h till the reaction was accomplished. After the com-
pletion of reaction, the reaction mixture was washed with water in
the separatory funnel. The dichloromethane layer was separated
and the aqueous layer was extracted twice with dichloromethane.
The combined dichloromethane solutions were dried for eight
hours over anhydrous sodium sulfate and then the dichlorometh-
ane was distilled off to afford a yellow solid. Recrystallization of
the yellow solid from ethanol gave compound B1 as yellow needle,
yield 60%; mp 208–210 °C; IR mmax cmꢀ1: 3131, 1703, 1672, 1626,
1401, 1267; 1H NMR (DMSO-d6): d = 7.44 (d, 1H, J = 8.2 Hz, 1H, H-
C(6)), 7.78 (s, 1H, H-C(1)), 7.84 (s, 1H, H-C(3)), 7.87 (dd, 1H,
as pale yellow needle, yield 78%; mp 118-121 °C; IR mmax cmꢀ1
:
3434, 3086, 2924, 1733, 1676, 1599, 1449, 1339, 1314, 1276,
1249; 1H NMR (CDCl3): d = 5.51 (s, 2H, CH2), 7.25 (s, 1H, H-C(4));
7.29 (d, 2H, H on the ring of benzoyl C(3)), 7.45 (m, 4H, H on the
ring of benzoyl C(1, 8)), 7.50 (t, 1H, H on the ring of benzoyl
C(3)), 7.56 (d, 1H, J = 8.0 Hz, H-C(2)), 7.61 (dd, 1H, J1 = 8.0 Hz,
J2 = 8.0 Hz, H-C(6)), 7.78 (s, 1H, H-C(7)), 7.81 (d, 1H, J = 7.9 Hz, H-
C(5)), 8.02 (d, 4H, H on the ring of benzoyl C(1, 8)), 825 (d, 2H, H
on the ring of benzoyl C(3)); 831 (d, 2H, H on the ring of benzoyl
C(1, 8)); ESI-MS m/z: 583.14 [M+H]+.

D.-H. Shi et al. / Bioorg. Med. Chem. 21 (2013) 1064–1073
1071
J1 = 8.2 Hz, J2 = 7.5 Hz, H-C(7)), 8.15 (d, 1H, J = 7.5 Hz, H-C(8)),
4.1.12. 4,5-Dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-
carboxylic acid (B7)
10.13 (s, 1H, CHO), 11.94 (s, 2H, OH); ESI-MS m/z: 269.04 [M+H]+.
A mixture of compound A1 (1 mmol, 0.27 g), PCC (2 mmol,
0.45 g), DMF (100 ml) and 2 mL of water were stirred at room tem-
perature for 24 h, monitored by TLC. After the completion of reac-
tion, 100 mL of ice water was added under stirring to afford orange
precipitate. The orange precipitate was filtered, washed and dried.
Recrystallization of the orange precipitate from ethanol gave com-
pound B7 as brown acicular crystals, yield 62%; mp 310–313 °C; IR
4.1.7. 4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracene-2-
carbaldehyde (B2)
As described for B1, compound A2 was treated with PCC to give
compound B2. Recrystallization from acetone gave yellow needle,
yield 73%; mp 195–197 °C; IR mmax cmꢀ1: 3026, 2966, 2929,
2872, 2839, 1671, 1582, 1283; 1H NMR (DMSO-d6): d = 4.04 (s,
6H, 2CH3), 7.26 (s, 1H, H-C(1)), 7.34 (d, 1H, J = 8.2 Hz, H-C(6)),
7.68 (s, 1H, H-C(3)), 7.88 (d, 1H, J = 7.5 Hz, H-C(8)), 8.32 (dd, 1H,
J1 = 8.2 Hz, J2 = 7.5 Hz, H-C(7)), 10.13 (s, 1H, CHO); ESI-MS m/z:
297.07 [M+H]+.
m
max cmꢀ1: 3432, 3059, 2930, 1695, 1628, 1270, 1192, 757; 1H NMR
(DMSO-d6): d = 7.58 (d, 1H, J = 8.2 Hz, 1H, H-C(6)), 7.85 (s, 1H, H-
C(1)), 7.97 (dd, 1H, J1 = 8.2 Hz, J2 = 7.5 Hz, H-C(7)), 8.01 (s, 1H, H-
C(3)), 8.45 (d, 1H, J = 7.5 Hz, H-C(8)), 11.87 (s, 1H, HO-C(1)),
11.94 (s, 1H, HO-C(8)), 13.90 (s, 1H, COOH); ESI-MS m/z: 285.04
[M+H]+.
4.1.8. (E)-4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracene-2-
carbaldehyde oxime (B3)
0.11 g (1.5 mmol) of hydroxylamine hydrochloride and 0.28 g
(2.0 mmol) of anhydrous potassium carbonate were added into a
solution of compound B2 (0.296 g, 1.0 mmol) in absolute ethyl
alcohol (50 mL) under stirring at 65 °C for 0.5 h, monitored by
TLC. After the completion of reaction, the reaction mixture was fil-
tered to remove the solid of inorganic salt and the solvent was
evaporated at reduced pressure o afford a yellow solid. Recrystalli-
zation of the yellow solid from ethanol gave compound B3 as yel-
low needle, yield 65%; mp 247–249 °C; IR mmax cmꢀ1: 3315, 3059,
2929, 1659, 1587, 1283, 1237, 753; 1H NMR (DMSO-d6): d = 3.94
(s, 6H, 2CH3), 7.53 (s, 1H, H-C(1)), 7.66 (d, 1H, J = 8.2 Hz, H-C(6)),
7.69 (s, 1H, H-C(3)), 7.74 (d, 1H, J = 7.5 Hz, H-C(8)), 7.92 (dd, 1H,
J1 = 8.2 Hz, J2 = 7.5 Hz, H-C(7)), 8.31 (s, 1H, CH), 11.78 (s, 1H,
NOH); ESI-MS m/z: 312.09 [M+H]+.
4.1.13. 4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracene-2-
carboxylic acid (B8)
As described for B7, compound A2 was oxidized with PCC to
give compound B8 as yellow needle from ethanol, yield 64%; mp
293–296 °C; IR mmax cmꢀ1: 3501, 2926, 2589, 2363, 2027, 1728,
1672, 1651, 1588, 1468, 1444, 1410, 1334, 1281; 1H NMR
(DMSO-d6): d = 3.96 (s, 6H, 2CH3), 7.56 (d, 1H, J = 8.0 Hz, H-C(6)),
7.72 (s, 1H, H-C(1)), 7.75 (d, 1H, J = 8.0 Hz, H-C(8)), 7.91 (dd, 1H,
J1 = 8.0 Hz, J2 = 7.6 Hz, H-C(7)), 8.19 (s, 1H, H-C(3)), 13.90 (s, 1H,
COOH); ESI-MS m/z: 313.07 [M+H]+.
4.1.14. (3R,4S,5S,6R)-6-(Acetoxymethyl)-3-(4,5-dihydroxy-9,10-
dioxo-9,10-dihydroanthracene-2-carboxamido)tetrahydro-2H-
pyran-2,4,5-triyl triacetate (B9)
0.42 g (1.2 mmol) of acetylglucosamine and 0.25 g (1.2 mmol)
of dicyclohexylcarbodiimide (DCC) were added into a solution of
compound B2 (0.284 g, 1.0 mmol) in anhydrous DMF (50 mL) un-
der stirring at room temperature for 26 h, monitored by TLC. After
the completion of reaction, the reaction mixture was filtered to re-
move the precipitate. 2 mL of glacial acetic acid were slowly added
into the filtrate until no more precipitation was come out, standing
at 4 °C for 12 h. The precipitate was filtered and then 20 mL of
water was added into the filtrate under stirring to afford light yel-
low precipitate. The expected product was purified by column
chromatography with the acetic ether–cyclohexane mixture
(v:v = 4:5) to give compound B9 as pale yellow needle, yield 53%;
mp 239–242 °C; IR mmax cmꢀ1: 3388, 2937, 1750, 1627, 1543,
1278, 1221, 753; 1H NMR (CDCl3): d = 2.08–2.14 (m, 12H, 4COCH3),
3.96–5.84 (m, 7H, H-C (acetyl glucosamine)), 7.26 (d, 1H, J = 8.3 Hz,
H-C(6)), 7.30 (s, 1H, H-C(1)), 7.63 (dd, 1H, J1 = 8.0 Hz, J2 = 8.5 Hz, H-
C(7)), 7.65 (s, 1H, H-C(3)), 7.67 (d, 1H, J = 8.2 Hz, H-C(8)), 7.93 (s,
1H, NH), 11.73 (s, 1H, OH-5), 11.79 (s, 1H, OH-4); ESI-MS m/z:
614.15 [M+H]+.
4.1.9. (E)-3-(Hydrazonomethyl)-1,8-dimethoxyanthracene-9,10-
dione (B4)
As described for B3, compound B2 was treated with hydrazine
sulfate to give compound B4 as bright yellow needle from ethanol,
yield 74%; mp 280 °C (dec); IR m
max cmꢀ1: 3062, 2958, 1668, 1590,
1286, 1235, 748; 1H NMR (CDCl3): d = 3.54 (s, 2H, NH2), 4.03 (s, 6H,
2CH3), 7.33 (d, 1H; J = 8.1 Hz, H-C(7)), 7.67 (dd, 1H, dd, J1 = 8.1 Hz,
J2 = 7.8 Hz, H-C(6)), 7.86 (d, 1H, J = 7.4 Hz, H-C(5)), 7.95 (s, 1H, H-
C(2)), 8.16 (s, 1H, H-C(4)), 8.73 (s, 1H, CH@N); ESI-MS m/z:
311.10 [M+H]+.
4.1.10. (E)-1,8-Dimethoxy-3-((phenylimino)
methyl)anthracene-9,10-dione (B5)
As described for B3, compound B2 was treated with aniline to
give compound B5 as orange needle from ethanol, yield 70%; mp
225–228 °C; IR mmax cmꢀ1: 3092, 2927, 1666, 1586, 1277, 1235,
788; 1H NMR (CDCl3): d = 4.12 (s, 6H, 2CH3), 7.18–7.70 (m, 5H,
on the ring of phenyl), 7.65 (d, 1H; J = 8.1 Hz, H-C(7)), 7.71 (dd,
1H, dd, J1 = 8.1 Hz, J2 = 7.8 Hz, H-C(6)), 8.03 (d, 1H, J = 7.5 Hz, H-
C(5)), 8.18 (s, 1H, H-C(2)), 8.31 (s, 1H, H-C(4)), 8.57 (s, 1H, CH@N);
ESI-MS m/z: 372.12 [M+H]+.
4.1.15. 3-(Chloromethyl)-1,8-dihydroxyanthracene-9,10-dione
(C1)
2 ml of thionyl chloride was slowly added into a solution of
compound A1 (0.27 g, 1.0 mmol) in anhydrous DMF (20 mL) under
stirring at room temperature for 26 h, monitored by TLC. After the
completion of reaction, 60 mL of ice water was added under stir-
ring to afford orange precipitate. The orange precipitate was fil-
tered, washed and dried. Recrystallization of the orange
precipitate from ethanol gave compound C1 as yellow needle, yield
4.1.11. (3R,4S,5S,6R)-6-(Acetoxymethyl)-3-((E)-((4,5-dihydroxy-
9,10-dioxo-9,10-dihydroanthracen-2-
yl)methylene)amino)tetrahydro-2H-pyran-2,4,5-triyl triacetate
(B6)
As described for B3, compound B1 was treated with acetylated
glucosamine to give compound B6 as yellow needle from ethanol,
yield 69%; mp 231–233 °C; IR mmax cmꢀ1: 3031, 2853, 1749, 1624,
1253, 1220, 761; 1H NMR (CDCl3): d = 1.90–2.21 (m, 12H, 4COCH3),
3.57–5.98 (m, 7H, H-C (acetyl glucosamine)), 7.33 (d, 1H, J = 7.7 Hz,
H-C(6)), 7.67 (s, 1H, H-C(1)), 7.72 (dd, 1H, J1 = 7.8 Hz, J2 = 8.1 Hz, H-
C(7)), 7.87 (d, 1H, J = 8.1 Hz, H-C(8)), 8.12 (s, 1H, H-C(3)), 8.30 (s,
1H, CH@N), 12.01 (s, 1H, OH-5), 12.04 (s, 1H, OH-4); ESI-MS m/z:
598.16 [M+H]+.
55%; mp 177–178 °C; IR m
max cmꢀ1: 3426, 3041, 1672, 1627, 1569,
1478, 1454, 1422, 1381, 1276; 1H NMR (CDCl3): d = 4.62 (s, 2H,
CH2), 7.31 (s, 1H, H-C(4)), 7.35 (d, 1H, J = 8.0 Hz, H-C(7)), 7.71
(dd, 1H, J1 = 7.5 Hz, J2 = 8.0 Hz, H-C(6)), 7.85 (s, 1H, H-C(2)), 7.86
(d, 1H, J = 7.5 Hz, H-C(5), 12.04 (s, 1H, HO), 12.07 (s, 1H, HO);
ESI-MS m/z: 289.03 [M+H]+.

1072
D.-H. Shi et al. / Bioorg. Med. Chem. 21 (2013) 1064–1073
4.1.16. 3-(Chloromethyl)-1,8-dimethoxyanthracene-9,10-dione
(C2)
4.1.21. 1-(4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracen-2-
yl)-N,N,N-trimethylmethanaminium chloride(C7)
As described for C1, compound A2 was chloridized with thionyl
chloride to give compound C2 as yellow needle from acetone, yield
As described for C6, compound C2 was treated with trimethyl-
amine to give compound C7 as yellow needle, yield 74%; mp 219 °C
(dec); IR mmax cmꢀ1: 3009, 2962, 1661, 1591, 1282, 1235, 746; 1H
NMR (D2O): d = 3.20 (s, 9H, CH3), 3.90 (s, 6H, 2OCH3), 4.52 (s, 2H,
CH2), 7.30 (d, 1H, J = 7.5 Hz, H-C (6)), 7.38 (d, 1H, J = 8.3 Hz, H-C
(8)), 7.47 (s, 1H, H-C (3)), 7.57 (s, 1H, H-C (1)), 7.58 (dd, 1H,
J1 = 6.8 Hz, J2 = 9.3 Hz, H-C (7)); HRMS (ESI): Calcd for [MꢀCl]+
(C20H22NO4) m/z 340.1549, found 340.1560.
80%; mp 179–182 °C; IR m
max cmꢀ1: 3004, 2932, 1666, 1586, 1284,
1237, 712; 1H NMR (CDCl3): d = 4.01 (s, 3H, CH3), 4.04 (s, 3H, CH3),
4.65 (s, 2H, CH2), 7.30 (d, 1H, J = 8.3 Hz, H-C(7)), 7.34 (s, 1H, H-
C(2)), 7.65 (dd, 1H, J1 = 7.8 Hz, J2 = 8.3 Hz, H-C(6)), 7.83 (s, 1H, H-
C(4)), 7.84 (d, 1H, J = 7.8 Hz, H-C(5)); ESI-MS m/z: 317.06 [M+H]+.
4.1.17. 1-((4,5-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-
yl)methyl)pyridin-1-ium chloride (C3)
4.1.22. N-((4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracen-
2-yl)methyl)-N,N-dimethylbenzenaminium chloride (C8)
As described for C6, compound C2 was treated with N,N-
dimethylaniline to give compound C8 as yellow needle, yield
70%; mp 161 °C (dec); IR mmax cmꢀ1: 3005, 2963, 1674, 1585,
1285, 1241, 751; 1H NMR (D2O): d = 3.53 (s, 6H, 2NCH3), 3.74 (s,
6H, 2OCH3), 4.96 (s, 2H, CH2), 6.64 (s, 1H, H-C (1)), 6.81 (d, 1H,
J = 7.5 Hz, H-C (6)), 6.88 (s, 1H, H-C (3)), 7.12 (d, 1H, J = 8.1 Hz, H-
C (8)), 7.24 (dd, 1H, J1 = 8.3 Hz, J2 = 7.7 Hz, H-C (7)), 7.64–7.73 (m,
5H, H-C (benzene ring)); HRMS (ESI): Calcd for [MꢀCl]+
(C25H24NO4) m/z 402.1705, found 402.1699.
1 ml of dry pyridine was slowly added into a solution of com-
pound C1 (0.29 g, 1.0 mmol) in anhydrous CHCl3 (50 mL) under
stirring at room temperature. The reaction mixture was allowed
to stand at room temperature for 2 weeks and orange crystals were
formed at the bottom of the vessel, yield 60%; mp 235 °C (dec); IR
m
max cmꢀ1: 3418, 3059, 2955, 1629, 1563, 1280, 1214, 755; 1H NMR
(D2O): d = 5.92 (s, 2H, CH2), 7.19 (d, 1H, J = 8.4 Hz, H-C (6)), 7.24 (s,
1H, H-C (1)), 7.43 (d, 1H, J = 7.4 Hz, H-C (8)), 7.46 (s, 1H, H-C (3)),
7.62 (dd, 1H, J1 = 8.2 Hz, J2 = 7.8 Hz, H-C (7)), 8.20 (d, 2H,
J = 6.8 Hz, H-C (pyridyl-30,50)), 8.70 (dd, 1H, J1 = 7.9 Hz, J2 = 7.9 Hz,
H-C (pyridyl-40)), 9.02 (d, 2H, J = 5.6 Hz, H-C (pyridyl-20,60)), 11.81
(s, 2H, 2OH); HRMS (ESI): Calcd for [MꢀCl]+ (C20H14NO4) m/z
332.0943, found 332.0941.
4.1.23. N-((4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracen-
2-yl)methyl)-N,N-diethylethanaminium chloride (C9)
As described for C6, compound C2 was treated with urotropine
to give compound C9 as yellow needle, yield 71%; mp 198 °C (dec);
IR mmax cmꢀ1: 3099, 2984, 1667, 1587, 1285, 1234, 750; 1H NMR
(D2O): d = 3.93 (s, 3H, CH3-5), 3.99 (s, 3H, CH3-4), 4.20 (s, 2H,
CH2), 4.42 (s, 6H, 3NCH2N), 5.13 (s, 6H, 3NCH2N), 7.57 (d, 1H,
J = 8.4 Hz, H-C (6)), 7.62 (s, 1H, H-C (1)), 7.71 (d, 1H, J = 6.8 Hz, H-
C (8)), 7.79 (s, 1H, H-C (3)), 7.80 (dd, 1H, J1 = 6.6 Hz, J2 = 8.0 Hz,
H-C (7)); HRMS (ESI): Calcd for [MꢀCl]+ (C23H25N4O4) m/z
421.1876, found 421.1880.
4.1.18. 1-(4,5-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-
yl)-N,N,N-trimethylmethanaminium chloride (C4)
As described for C3, compound C1 was treated with trimethyl-
amine to give compound C4 as yellow needle, yield 65%; mp 230 °C
(dec); IR mmax cmꢀ1: 3413, 3022, 2957, 1629, 1477, 1285, 1200,
752; 1H NMR (D2O): d = 3.20 (s, 9H, CH3), 4.95 (s, 2H, CH2), 7.19
(d, 1H, J = 8.4 Hz, H-C (6)), 7.40 (s, 1H, H-C (3)), 7.44 (d, 1H,
J = 7.5 Hz, H-C (8)), 7.60 (s, 1H, H-C (1)), 7.64 (dd, 1H, J1 = 8.2 Hz,
J2 = 7.8 Hz, H-C (7)), 12.05 (s, 2H, 2OH); HRMS (ESI): Calcd for
[MꢀCl]+ (C18H18NO4) m/z 312.1256, found 312.1257.
4.1.24. 1-((4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracen-2-
yl)methyl)-1,3,5,7-tetraazaadamantan-1-ium chloride (C10)
As described for C6, compound C2 was treated with triethyl-
amine to give compound C10 as yellow needle, yield 73%; mp
204 °C (dec); IR mmax cmꢀ1: 3100, 2976, 1665, 1584, 1280, 1236,
786; 1H NMR (D2O): d = 1.45 (t, 9H, 3CH3), 3.31 (m, 6H, 3CH2),
3.94 (s, 3H, CH3-5), 3.95 (s, 3H, CH3-4), 4.46 (s, 2H, CH2), 7.33 (d,
1H, J = 7.4 Hz, H-C (6)), 7.42 (s, 1H, H-C (1)), 7.42 (d, 1H,
J = 8.4 Hz, H-C (8)), 7.53 (s, 1H, H-C (6)), 7.61 (dd, 1H, J1 = 7.9 Hz,
J2 = 8.2 Hz, H-C (7)); HRMS (ESI): Calcd for [MꢀCl]+ (C23H28NO4)
m/z 382.2018, found 382.2017.
4.1.19. N-((4,5-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-
yl)methyl)-N,N-dimethylbenzenaminium chloride (C5)
As described for C3, compound C1 was treated with N,N-
dimethylaniline to give compound C5 as orange needle, yield
63%; mp 182 °C (dec); IR mmax cmꢀ1: 3414, 3008, 2928, 1636,
1489, 1271, 1215, 746; 1H NMR (D2O): d = 3.52 (s, 6H, 2NCH3),
5.02 (s, 2H, CH2), 6.97 (s, 1H, H-C (1)), 7.05 (d, 1H, J = 8.4 Hz, H-C
(6)), 7.20 (s, 1H, H-C (3)), 7.28 (d, 1H, J = 7.4 Hz, H-C (8)), 7.35
(dd, 1H, J1 = 8.2 Hz, J2 = 7.8 Hz, H-C (7)), 7.70–8.53 (m, 5H, H-C
(benzene ring)), 12.05 (s, 2H, 2OH); HRMS (ESI): Calcd for [MꢀCl]+
(C23H20NO4) m/z 374.1392, found 374.1396.
4.1.25. Synthesis of 3-(chloromethyl)-9,10-dioxo-9,10-
dihydroanthracene-1,8-diyl bis(4-methylbenzenesulfonate)
(C11)
4.1.20. 1-((4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracen-2-
yl)methyl)pyridin-1-ium chloride(C6)
Tosyl chloride (2 mmol, 0.38 g), anhydrous K2CO3 (0.5 g) and
chloro-aloe emodin C1 (1 mmol, 0.29 g) were added into dry ace-
tone (20 ml). The mixture was stirred at 40 °C for 12 h, monitored
by TLC. After the completion of reaction, the reaction mixture was
poured into ice water and yellow solid was separated. Recrystalli-
zation of the yellow solid from acetone gave compound C11 as yel-
low flaky crystals, yield 74%; mp 169–170 °C; IR mmax cmꢀ1: 3444,
3072, 2921, 1934, 1685, 1597, 1493, 1449, 1420, 1379, 1356, 1260;
1H NMR (CDCl3): d = 2.43 (s, 6H, 2CH3), 4.63 (s, 2H, CH2), 7.27 (d,
1H, J = 8.0 Hz, H-C(7)), 7.32 (s, 1H, H-C(4)), 7.35 (d, 1H, J = 8.0 Hz,
H-C(2)), 7.71 (d, 4H, J = 8.0 Hz, H on the ring of tosyl C(3, 5)),
7.92 (d, 4H, J = 8.0 Hz, H on the ring of tosyl C(2, 6)), 8.19 (dd,
1H, J1 = 8.0 Hz, J2 = 8.0 Hz, H-C(6)), 8.20 (d, 1H, J = 8.0 Hz, H-C(5));
ESI-MS m/z: 597.04 [M+H]+.
1 ml of dry pyridine was slowly added into a solution of com-
pound C2 (0.32 g, 1.0 mmol) in anhydrous CHCl3 (50 mL) under
stirring at room temperature. The reaction mixture was allowed
to stand at room temperature for 2 weeks and yellow crystals were
formed at the bottom of the vessel, yield 73%; mp 168 °C (dec); IR
mmax cmꢀ1: 3219, 3047, 1663, 1587, 1277, 1239, 741; 1H NMR
(D2O): d = 3.94 (s, 3H, CH3-5), 3.96 (s, 3H, CH3-4), 5.93 (s, 2H,
CH2), 7.47 (d, 1H, J = 8.6 Hz, H-C (6)), 7.50 (d, 1H, J = 6.7 Hz, H-C
(8)), 7.52 (s, 1H, H-C (1)), 7.57 (s, 1H, H-C (3)), 7.68 (dd, 1H,
J1 = 8.1 Hz, J2 = 8.0 Hz, H-C (7)), 8.16 (d, 2H, J = 6.8 Hz, H-C (pyri-
dyl-30,50)), 8.66 (dd, 1H, J1 = 7.9 Hz, J2 = 7.9 Hz, H-C (pyridyl-40)),
8.99 (d, 2H, J = 5.6 Hz, H-C (pyridyl-20,60)); HRMS (ESI): Calcd for
[MꢀCl]+ (C22H18NO4) m/z 360.1236, found 360.11240.

D.-H. Shi et al. / Bioorg. Med. Chem. 21 (2013) 1064–1073
1073
4.2. Biology
docking. A population of random individuals (population size:
150), a maximum number of 2500,000 energy evaluations, a max-
imum number of generations of 27,000 was used. At the end of a
docking procedure (100 docking runs), the resulting positions were
clustered according to a root mean square criterion of 0.5 Å.
4.2.1. In vitro cholinesterase activity assay
AChE activities were measured through Ellman’s colorimetric
method with a slight modification.9 AChE was prepared from rat
cortex homogenate.10 In assays, 10
l
L of rat cortex homogenate
L of tested compounds and 140 L of
0.1 M PBS (pH 8.0) for 10 min in 96-well microplates before addi-
tion of 20 L of 3.33 mM DTNB solution and 20 L of 5.30 mM ATCI
was incubated with 10
l
l
Acknowledgments
l
l
The financial support of the Natural Science Foundation of P.R.
China (grated No: 21162006), the Project of Postgraduate Iinnova-
tion Research in Hainan Province (grated No: Hys2011-15) and
Postdoctoral Science Foundation of China (grated No:
2012M521659) are gratefully acknowledged.
solution. After the addition of DTNB and ATCI, the 96-well micro-
plates were read at 412 nm with a microplate reader (SPECTRA-
FLUOR, TECAN, Sunrise, Austria) for 15 min One triplicate sample
without inhibitors was always present to yield 100% of AChE activ-
ity. One triplicate sample with 20 lM tacrine was always present
to yield 100% AChE inhibition (to obviate the ChE-independent
substrate hydrolysis). The reaction rates were compared and the
percent inhibition due to the presence of tested compounds was
calculated. Tacrine was applied as a positive drug. All samples were
assayed in triplicate.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.015.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
4.2.2. Kinetic studies
Kinetic studies were performed using rat cortex homogenate as
AChE. Enzyme activities were determined at 25 °C using five con-
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The pdb structure of acetylcholinesterase (PDB No. 2CMF) was
obtained from the Protein Data Bank. The 3D structure of C3 was
constructed using ChemBioOffice 2008 and optimized according
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Docking studies were carried out using the AUTODOCK 4.2.3
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the enzyme. The resulting enzyme structure was used as an input
for the AUTOGRID program. AUTOGRID performed a precalculated
atomic affinity grid maps for each atom type in the ligand plus an
electrostatics map and a separate desolvation map present in the
substrate molecule. All maps were calculated with 0.375 Å spacing
between grid points. The centre of the grid box was placed at the
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Flexible ligand docking was performed for the compounds.
Docking calculations were carried out using the Lamarckian genet-
ic algorithm (LGA) and all parameters were the same for each