Design, synthesis and evaluation of novel 4-dimethylamine flavonoid derivatives as potential multi-functional anti-Alzheimer agents

Article abstract of DOI:10.1016/j.bmc.2013.09.061

A series of 4-dimethylamine flavonoid derivatives 5a-5r were designed, synthesized and evaluated as potential multi-functional anti-Alzheimer agents. The results showed that most of the synthesized compounds exhibited high acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity at the micromolar range (IC₅₀, 1.83-33.20 μM for AChE and 0.82-11.45 μM for BChE). A Lineweaver-Burk plot indicated a mixed-type inhibition for compound 5j with AChE, and molecular modeling study showed that 5j targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, the derivatives showed potent self-induced Aβ aggregation inhibitory activity at 20 μM with percentage from 25% to 48%. In addition, some compounds (5j-5q) showed potent oxygen radical absorbance capacity (ORAC) ranging from 1.5- to 2.6-fold of the Trolox value. These compounds should be further investigated as multi-potent agents for the treatment of Alzheimer's disease.

Full text of DOI:10.1016/j.bmc.2013.09.061

Bioorganic & Medicinal Chemistry 21 (2013) 7275–7282
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of novel 4-dimethylamine flavonoid
derivatives as potential multi-functional anti-Alzheimer agents
Wen Luo a, , Ya-Bin Su a,b, , Chen Hong , Run-Guo Tian , Lei-Peng Su , Yue-Qiao Wang , Yang Li ,
⇑
,
a
a
a
a
a
b
a,
⇑
Jun-Jie Yue , Chao-Jie Wang
a
Key Laboratory of Natural Medicine and Immuno-Engineering, Henan University, Kaifeng 475004, People’s Republic of China
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, People’s Republic of China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-dimethylamine flavonoid derivatives 5a–5r were designed, synthesized and evaluated as
potential multi-functional anti-Alzheimer agents. The results showed that most of the synthesized com-
pounds exhibited high acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity
at the micromolar range (IC50, 1.83–33.20 lM for AChE and 0.82–11.45 lM for BChE). A Lineweaver–Burk
plot indicated a mixed-type inhibition for compound 5j with AChE, and molecular modeling study
Received 7 August 2013
Revised 24 September 2013
Accepted 25 September 2013
Available online 4 October 2013
showed that 5j targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE.
Besides, the derivatives showed potent self-induced Ab aggregation inhibitory activity at 20 lM with per-
centage from 25% to 48%. In addition, some compounds (5j–5q) showed potent oxygen radical absor-
bance capacity (ORAC) ranging from 1.5- to 2.6-fold of the Trolox value. These compounds should be
further investigated as multi-potent agents for the treatment of Alzheimer’s disease.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Flavonoid derivatives
Anti-Alzheimer agent
Cholinesterase
Self-induced Ab aggregation
Antioxidation
1
. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative
able to hydrolyze acetylcholine, during the development of AD,
BChE activity increases by 40–90% in the temporal cortex and hip-
pocampus, while at the same time AChE activity decreases.1
Therefore, the concurrent inhibition of both AChE and BChE should
provide additional benefits in the treatment of AD.
1,12
brain disorder that is characterized by dementia, memory loss
and other cognitive impairments.1 The etiology of AD is unclear,
but there are common indicators that seem to play a significant
role in the disease, such as low levels of acetylcholine, b-amyloid
,2
The production and accumulation of Ab in the brain is generally
considered to be a central event in the pathogenesis of AD. Many
studies have shown that either the oligomeric or fibril form of Ab
is more toxic to neurons than the monomeric form.13 Therefore,
Ab aggregation is currently another potential target of AD.1
Oxidative stress has been strongly implicated in the pathophys-
3
–5
(
Ab) deposits,
s-protein aggregation and oxidative stress.
Current treatment of AD focuses on increasing neurotransmitter
4–16
acetylcholine levels at cerebral cortex synapses by inhibiting
acetylcholinesterase (AChE) activity with drugs such as tacrine,
donepezil, rivastigmine and galantamine.6 Unfortunately, AChE
inhibition alone is a palliative treatment and its clinical effective-
ness is still under debate.7 Due to the multi-pathogenesis of AD,
17
iology of neurodegenerative disorders such as AD. It has been
shown that oxidative damage caused by excessive reactive oxygen
18
species (ROS) is present in the brains of AD patients. Therefore
a
strategy called multi-target-directed ligands (MTDLs) has
designing antioxidants targeted towards ROS may offer an attrac-
tive therapeutic approach for AD.
recently emerged, this strategy is to develop novel anti-AD agents
with multiple potencies,8 including cholinesterase inhibition,
anti-oxidation, Ab deposit inhibition and so on, and the strategy
of combining these potencies in one structure was confirmed to
Flavonoids are a class of polyphenolic compounds present in
plants, including many that are commonly consumed by humans
1
9
such as fruits, vegetables, wine and tea. They express a wide
range of biological activities, such as antioxidant, anti-inflamma-
tory, neuroprotection, vasodilator, and inhibition of Ab aggrega-
tion. Flavonoids exhibit especially low toxicity and so they have
9
,10
be successful.
The two types of cholinesterase (ChE) found in the central
nervous system, AChE and butyrylcholinesterase (BChE), are both
2
0
been considered as leading anti-AD compounds. During recent
years, some research groups have attempted to optimize flavo-
noids’ chemical structures in order to develop new drugs to treat
⇑
Corresponding authors. Tel./fax: +86 0378 2864665.
E-mail addresses: luowen83@henu.edu.cn (W. Luo), wcjsxq@henu.edu.cn
21,22
AD (Fig. 1A and B).
(
C.-J. Wang).
These authors contributed equally to this work.
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.061

7
276
W. Luo et al. / Bioorg. Med. Chem. 21 (2013) 7275–7282
R
derivatives (Fig. 2C and D) display excellent binding affinities with
2
5,26
R
O
n
O
O
O
O
O
Ab during in vitro binding assays.
Therefore, in the present
study, we assessed a series of new N,N-dimethylated flavonoids
as multifunctional anti-AD agents. We hypothesized that these
flavonoids function as inhibitors of ChE, Ab self-induced aggrega-
tion and ROS. The flavonoids were designed by conjugating an
N,N-dimethylated flavonoid with a terminal amine using a long
chain linker. It is possible that the N,N-dimethylated flavonoids in-
hibit AChE activity through binding with the catalytic active site
(CAS) and the peripheral anionic site (PAS) of AChE, blocking Ab
self-aggregation and scavenging ROS.
A
B
O
multifunctional ChE inhibitor
ChE inhibitor
N
O
R
Y
1
4
R
2
O
18F
N
R5
X
R
R3
O
D
n
C
O
Aβ imaging agent
Aβ plaques probe
2
2
. Results and discussion
Figure 1. Flavonoids and N,N-dimethylated curcumin derivatives as new drugs or
imaging agents in the treatment of AD.
.1. Chemistry
Eighteen flavonoid derivatives (5a–5r) with linkers of varying
5
0
chain lengths were prepared as shown in Scheme 1. Condensation
of 1,3-benzenediol (1) with chloroacetonitrile catalyzed by ZnCl
non inhibitor
5j]=1.0uM
5j]=2.0uM
2
[
[
and HCl gas was followed by hydrolysis in water and produced ke-
tone in 87% yield. Treatment of compound 2 with 4-dimethylami-
40
[5j]=4.0uM
2
nobenzaldehyde in the presence of excess NaOH in H O/EtOH was
followed by acidification with aqueous HCl and resulted in the ex-
pected compound (3) in 92% yield. The alkylation of 3 with differ-
30
ent a, x-dibromoalkanes in acetone provided 4a–4e in satisfactory
yields. Finally, the reaction of 4a–4e with secondary amines pro-
2
0
duced the targets 5a–5r in 45–88% yields. The structures and puri-
1
13
ties of the target compounds were confirmed by H NMR, C NMR,
MS and elemental analysis.
10
2
.2. In vitro inhibition studies on AChE and BChE
0
-10
-5
0
5
10
15
20
To determine the potential efficacy of the target compounds
S] - mM -1
1
5a–5r for the treatment of AD, their ChE inhibitory activity was
[
2
7
determined by the Ellman method, with tacrine and rivastigmine
as reference compounds. The IC50 values for ChE inhibition and
their selectivity index are summarized in Table 1. The results
showed that most of the tested compounds (5a–5r) had significant
ChE inhibitory activity and selectivity for AChE. Compound 5j
(n = 8), with a diethylamino group, showed the most potent inhibi-
Figure 2. Lineweaver–Burk plot for the inhibition of acetylcholinesterase by
compound 5j.
Our research group has been involved in the synthesis of flavo-
noids and the development of ChE inhibitors for many years.
2
3,24
We recently found compounds containing a N,N-dimethylated
flavonoid moiety during routine screening for compounds with
biological activity. These compounds have significant antioxidant
activity and inhibit Ab self-induced aggregation. It has previously
been reported that N,N-dimethylated flavonoid and curcumin
tion for AChE with an IC50 value of 1.83
has pyrrolidine moiety and exhibited the strongest inhibition to
BChE with an IC50 value of 0.82 M.
Compounds with the same chain length were compared for
their ability to inhibit AChE and BChE. The AChE inhibition activity
lM. Compound 5o (n = 10)
l
N
HO
OH
HO
OH
O
a,b
c
O
O
OH
d
Cl
1
2
3
N
N
R1
N
e
O
O
O
Br
O
O
O
R2
n-2
n-2
4a-4e
5a-5r
n= 4, 6, 8, 10, 12
R1
N
=
N
N
N
N
O
N
R
2
Scheme 1. Synthesis of flavonoid derivatives. Reagents and conditions: (a) ClCH
2 2
CN, EtOEt, HCl, 0 °C; (b) HCl, H O, reflux; (c) 4-dimethylaminobenzaldehyde, 10% NaOH, rt,
EtOH; (d) dibromoalkane, K CO , acetone, reflux; (e) HNR , K CO , CH CN, 60 °C.
2
3
1
R
2
2
3
3

W. Luo et al. / Bioorg. Med. Chem. 21 (2013) 7275–7282
7277
Table 1
Inhibition of ChE activity, selectivity index and inhibition of Ab(1–42) self-induced aggregation
N
R
1
O
O
N
R2
n-2
O
a
b
Selectivity indexc
Ab(1–42)d inhibition
Compds
n
4
4
4
4
–NR
N
1
R
2
IC50 for AChE (
lM)
IC50 for AChE (
l
M)
5
5
5
5
a
b
c
2.24 ± 0.14
2.77 ± 0.02
3.04 ± 0.21
>50
7.88 ± 1.51
3.5
2.2
33.23 ± 1.44
36.56 ± 4.94
30.64 ± 0.65
44.67 ± 2.68
N
6.14 ± 0.27
N
6.12 ± 0.72
2.0
d
N
O
10.10 ± 1.32
<0.2
5
e
4
8.29 ± 2.11
>50
>6.0
39.13 ± 2.38
N
N
N
N
5
5
5
f
6
6
6
3.11 ± 0.15
9.18 ± 1.14
6.73 ± 0.18
4.61 ± 0.43
11.45 ± 1.90
5.52 ± 0.18
1.5
1.2
0.8
38.12 ± 5.67
32.61 ± 1.95
25.37 ± 5.92
g
h
5
i
6
7.85 ± 2.30
>50
6.4
46.55 ± 0.82
N
N
N
N
5
5
5
j
8
8
8
1.83 ± 0.15
1.99 ± 0.08
7.88 ± 1.38
5.01 ± 0.35
2.60 ± 0.03
8.72 ± 1.16
2.7
1.3
1.1
45.80 ± 2.77
42.15 ± 4.63
34.81 ± 2.16
k
l
5
m
8
23.99 ± 1.06
>50
2.1
39.52 ± 6.57
N
N
N
N
5
5
5
n
o
p
10
10
10
3.74 ± 0.10
2.26 ± 0.07
>50
2.31 ± 0.07
0.82 ± 0.04
2.42 ± 0.10
0.6
0.4
33.37 ± 3.79
38.60 ± 4.68
40.73 ± 1.79
<0.05
5
q
10
11.40 ± 1.22
>50
4.4
29.36 ± 5.71
48.13 ± 3.70
N
N
5
r
12
33.20 ± 2.06
>50
1.5
Rivastigmine
Tacrine
Curcumin
—
—
—
—
—
—
5.56 ± 1.50
0.219 ± 0.002
—
1.71 ± 0.03
0.047 ± 0.001
—
0.3
0.2
—
—
—
51.04 ± 3.51
a
b
c
AChE from electric eel; IC50, 50% inhibitor concentration (means ± SEM of three experiments).
BChE from equine serum; IC50, 50% inhibitor concentration (means ± SEM of three experiments).
Selectivity Index = IC50 (BChE)/IC50 (AChE).
d
The values are the mean ± SD of three independent experiments and the measurements were carried out in the presence of 20 lM compounds.
of compounds with a diethylamine or pyrrolidine group (5a, 5b, 5j,
length for inhibiting AChE and BChE was eight methylene groups
(5j, 5k) on the flavonoid compound and ten (5n, 5o, 5p) methylene
groups on the aminoalkyl substituted moiety compound. The var-
iation of chain length for the inhibitors had more influence on their
inhibition to AChE than BChE. This may be caused by conforma-
tional differences between these two enzymes. The active site of
BChE is wider than the active site of AChE and therefore, BChE is
able to accomodate inhibitors with variable linker lengths. The
length of the alkyl chain may also affect the selectivity for AChE
5
k, 5n, 5o) were more effective than compounds with a piperidine
or benzyl methylamine group (5c, 5e, 5l, 5m, 5p, 5q). The BChE
inhibition activity of compounds with an aminoalkyl substituted
group is not so obvious. Compounds with a benzyl methylamine
group (5e, 5i, 5m, 5q, 5r) exhibted the weakest inhibition activities
for both AChE and BChE, and they showed selectivity for AChE.
Compounds with the same aminoalkyl substituted group, but
different chain lengths were also compared. The optimal chain

7
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W. Luo et al. / Bioorg. Med. Chem. 21 (2013) 7275–7282
(
5
(
except compounds with a benzyl methylamine group 5e, 5i, 5m,
q and 5r). Compounds with four, six and eight methylene groups
5a, 5b, 5f, 5g, 5j, 5k) showed selectivity for AChE rather than the
compounds with ten methylene groups (5n, 5o, 5p).
3
2
1
0
2
.3. Kinetic characterization of AChE inhibition
The compound that appeared to be the most successful at inhib-
iting AChE was 5j. We further investigated 5j using graphical anal-
ysis of steady state inhibition data (Fig. 2). The Lineweaver–Burk
plots show an increasing slope (decreased Vmax) and an increasing
intercept (higher K
ing a mixed-type inhibition. Replots of the slope versus concentra-
tion of compound 5j gave an extimate of the inhibition constant, K
of 0.386 M. This suggests that compound 5j was able to bind both
m
) for higher inhibitor concentrations, indicat-
i
l
5j 5k 5l 5m 5n 5o 5p 5q Cur
CAS and PAS of AChE and supports the results of the molecular
modeling study.
Compounds
Figure 4. Oxygen radical absorbance capacity (ORAC) of compounds 5a–5r. Data
are expressed as ( mol Trolox)/( mol tested compound).
l
l
2
.4. Molecular modeling study
assay.30 The results suggest that these compounds prevent self-
In order to obtain more information about the binding interac-
tions between compound 5j and TcAChE (PDB code: 1ACJ), docking
studies were carried out using the AUTODOCK 4.0 package and Py-
MOL software as shown in Figure 3.2
The docking result demonstrates that compound 5j exhibits
multiple binding modes with AChE. In the 5j–TcAChE complex,
compound 5j occupied the entire enzymatic CAS, mid-gorge and
mediated Ab aggregation with percentages of inhibition ranging
from 25% to 48% at 20 lM. These are lower inhibition levels than
8,29
that of curcumin (51%; Table 1). The most potent compounds were
5d, 5i, 5j, 5k, 5p and 5r, with percentages of inhibition greater than
40%. However, the inhibitory potency of each derivative did not de-
pend on the chain length of the connecting linker. The current data
are not sufficient to establish a structure-activity relationship for
the activity toward anti-Ab aggregation, and further investigation
is required to determine the activity mechanism.
PAS. The charged nitrogen of diethylamine resulted in a cation–
interaction with Trp84 with a distance of 4.2 Å at the CAS. The fla-
vonoid moiety stacked against the Trp279 through stacking
p
p–p
with a distance of 3.8 Å at the PAS. The docking studies show that
our compounds exhibit a mixed type of inhibition, consistent with
our kinetic analysis result.
2.6. Studies of antioxidation activity
Since the crystal structure of BChE from equine serum has not
been reported and the sequence of equine BChE is highly similar
to human BChE, the crystal structure of HuBChE was used in the
docking study. Similar interactions were found in 5o in complex
The peroxyl radicals reduced by the selected compounds 5j–5q
were examined by using the ORAC (oxygen radical absorbance
3
1
capacity) assay,
(Fig. 4). The ability of compounds 5j–5q to scavenge radicals at
concentrations between 1–5 M was compared with that of the
with curcumin as a reference compound
with HuBChE (PDB code: 1POI), a cation–
p
interaction with
l
Trp82 was observed at the CAS.
highly potent compound Trolox and is expressed as their Trolox
equivalent. These compounds exhibited significant radical-capture
capacity. They showed potent peroxyl radical absorbance capaci-
ties ranging from 1.5 to 2.6 times the Trolox value. The 4-dimeth-
ylamine flavonoid moiety appears to be crucial to the compound’s
radical scavenging ability.
2
.5. Inhibition of self-mediated Ab(1–42) aggregation
The derivatives 5a–5r were tested for their ability to inhibit
self-mediated aggregation of Ab(1–42) using the thioflavin T
Figure 3. Docking models of compound–enzyme complex: (a) 5j–TcAChE complex; (b) 5o–HuBChE complex.

W. Luo et al. / Bioorg. Med. Chem. 21 (2013) 7275–7282
7279
3
. Conclusion
4.5. General procedures for the preparation of compound 5a–5r
In summary, a novel series of 4-dimethylamine flavonoid deriv-
To a solution of 4a–4e (0.5 mmol) in CH
different amines (1.0 mmol) and anhydrous K
After stirring at 60 °C for 8 h, the solvent was removed under vac-
uum, the mixture was diluted with CHCl and then washed with
water and brine. The organic layer was dried over Na SO , filtered,
concentrated in vacuo, and purified by column chromatography
with CHCl /MeOH/NH OH (30:1:0.5%) elution, and then crystal-
lized from ethanol.
3
CN (10 mL) was added
atives 5a–5r were synthesized and characterized. These synthetic
compounds exhibit high inhibitory potency for both AChE and
BChE, at levels similar to or better than those of Rivastigmine.
Compound 5j groups showed the best AChE inhibitory activity
and 5o exhibited the highest inhibitory effect for BChE. A kinetic
analysis and molecular modeling study indicated that compound
2
CO (2.5 mmol).
3
3
2
4
2
4
5
j was able to bind with both CAS and PAS of the AChE. Addition-
ally, these compounds exhibited potent inhibition of self-induced
Ab aggregation. Compounds 5j–5o also had significant capacity
to absorb peroxyl radicals. Our results indicate that this new type
of multifunctional flavonoid derivatives may provide a useful tem-
plate for the development of new anti-AD agents with multiple
potencies.
4.5.1. 7-(4-(Diethylamino)butoxy)-2-(4-(dimethylamino)
phenyl)-4H-chromen-4-one (5a)
Intermediate 4a was treated with diethylamine according to
general procedure to give the desired product 5a as yellow solid
1
(61% yield), mp 114–116 °C; H NMR (400 MHz, CDCl
3
) d 7.83 (d,
J = 8.8 Hz, 2H), 7.70 (d, J = 8.4 Hz, 1H), 6.84 (s, 1H), 6.79–6.71 (m,
H), 4.10 (t, J = 6.4 Hz, 2H), 3.07 (s, 3H), 2.60–2.48 (m, 6H), 1.86
dd, J = 14.5, 6.9 Hz, 2H), 1.83–1.64 (m, 2H), 1.05 (t, J = 7.1 Hz,
4
4
4
. Experimental section
(
1
3
6
1
9
3
H). C NMR (100 MHz, CDCl ) d 182.60, 167.69, 166.24, 151.09,
.1. Chemistry
45.90, 133.28, 125.39, 120.11, 115.42, 113.87, 112.13, 111.90,
6.85, 68.64, 58.33, 52.46, 46.81, 40.11, 27.08, 23.53, 18.48,
Melting points (mp) were determined using an X-6 hot stage
+
+
1
13
11.67. MS (ESI ): m/z: 409.1[M+H] . Elemental Anal. Calcd for
: C, 73.50; H, 7.90; N, 6.86. Found: C, 73.54; H, 7.90;
microscope and were not corrected. H and C NMR spectra were
recorded using TMS as the internal standard in CDCl with a Bruker
25 32 2 3
C H N O
3
N, 6.76.
AV-400 spectrometer at 400 MHz and 100 MHz, respectively. MS
spectra were recorded on a Shimadzu LCMS-2010A instrument
with an ESI mass selective detector. Elemental analyses were per-
formed on a Gmbe VarioEL Elemental Instrument. Flash column
chromatography was performed with silica gel (200–300 mesh)
purchased from Qingdao Haiyang Chemical Co. Ltd.
4
.5.2. 2-(4-(Dimethylamino)phenyl)-7-(4-(pyrrolidin-1-
yl)butoxy)-4H-chromen-4-one (5b)
Intermediate 4a was treated with pyrrolidine according to gen-
eral procedure to give the desired product 5b as yellow solid (56%
1
yield), mp 124–126 °C; H NMR (400 MHz, CDCl
3
) d 7.82 (d,
J = 8.8 Hz, 2H), 7.70 (d, J = 8.5 Hz, 1H), 6.84 (s, 1H), 6.79–6.69 (m,
4
.2. Synthesis of intermediate 2
4
1
1
1
2
H), 4.10 (t, J = 6.4 Hz, 2H), 3.07 (s, 6H), 2.53 (t, J = 7.1 Hz, 6H),
.91–1.70 (m, 8H). C NMR (100 MHz, CDCl ) d 182.56, 167.66,
3
1
3
To a mixture of resorcinol (4.37 g, 39.7 mmol) and chloroaceto-
66.21, 151.07, 145.88, 133.27, 125.34, 120.08, 115.41, 113.84,
12.13, 111.88, 96.84, 68.55, 56.02, 54.23, 40.09, 27.09, 25.47,
nitrile (2.5 mL, 39.7 mmol) in ether (50 mL) was added anhydrous
ZnCl (0.54 g, 3.96 mmol). The solution was cooled to 0 °C and HCl
2
+
+
3.42. MS (ESI ): m/z: 407.1[M+H] . Elemental Anal. Calcd for C25-
ꢀ0.4C OHꢀ0.1CHCl : C, 71.20; H, 7.50; N, 6.41. Found: C,
1.46; H, 7.23; N, 6.44.
was bubbled through the reaction for 2 h. The solution was left in
the cold-room overnight and HCl was bubbled again for 2 h. The
precipitated imine was filtered off and washed three times with
ether. The imine was dissolved in 100 mL of 1 N HCl and reflux
for 1 h. The solid was filtered off, washed three times with water
and dried under vacum to yield pure acetophenone 2 as a white so-
lid which was used without further purification. Yield 6.44 g (87%).
H
7
30
N
2
O
3
2
H
5
3
4
.5.3. 2-(4-(Dimethylamino)phenyl)-7-(4-(piperidin-1-
yl)butoxy)-4H-chromen-4-one (5c)
Intermediate 4a was treated with piperidine according to gen-
eral procedure to give the desired product 5c as yellow crystal
1
(
67% yield), mp 141–142 °C; H NMR (400 MHz, CDCl
3
) d 7.82 (d,
4
.3. Synthesis of intermediate 3
J = 8.9 Hz, 2H), 7.69 (d, J = 8.5 Hz, 1H), 6.84 (s, 1H), 6.79–6.68 (m,
4
1
H), 4.09 (t, J = 6.4 Hz, 2H), 3.07 (s, 6H), 2.65–2.19 (m, 6H), 1.88–
To a mixture of acetophenone 2 (3.96 g, 21.2 mmol) and 4-
dimethylaminobenzaldehyde (3.16 g, 21.2 mmol) in ethanol
50 mL) was added 10% sodium hydrate aqueous (30 mL), the mix-
.82 (m, 2H), 1.72–1.68 (m, 2H), 1.64–1.56 (m, 4H), 1.46 (d,
13
J = 4.6 Hz, 2H).
3
C NMR (100 MHz, CDCl ) d 182.50, 167.64,
(
1
1
2
66.19, 151.04, 145.85, 133.25, 125.29, 120.05, 115.38, 113.80,
12.12, 111.85, 96.82, 68.58, 58.93, 54.63, 40.06, 27.10, 26.04,
ture was stirred for 24 h at room temperature. The solution was
acidified with 1 N HCl and filtered, and the filter cake was recrystal-
lized from ethanol got a dark red solid 3 which was used without
+
+
4.50, 23.40. MS (ESI ): m/z: 421.2[M+H] . Elemental Anal. Calcd
: C, 74.26; H, 7.67; N, 6.66. Found: C, 74.20; H,
for C26
7
32 2 3
H N O
+
+
further purification. Yield 5.48 g (92%). MS (ESI ): m/z: 282.1[M+H] .
.93; N, 6.26.
4
4
.4. General procedures for the preparation of intermediate 4a–
e
4.5.4. 2-(4-(Dimethylamino)phenyl)-7-(4-morpholinobutoxy)-
4H-chromen-4-one (5d)
Intermediate 4a was treated with morpholine according to gen-
eral procedure to give the desired product 5d as orange solid (85%
To a solution of intermediate 3 (0.56 g, 2.0 mmol) and anhy-
1
drous K
2
CO
3
(1.38 g, 10.0 mmol) in acetone (10 mL),
a,x
-dibro-
yield), mp 157–159 °C; H NMR (400 MHz, CDCl ) d 7.82 (d,
3
moalkane (4.0 mmol) was added. After reflux for 10 h until the
starting material 3 disappeared, the solvent was removed under
vacuum, the residue was poured into water and extracted with
J = 8.9 Hz, 2H), 7.70 (d, J = 8.5 Hz, 1H), 6.84 (s, 1H), 6.79–6.74 (m,
2H), 6.73–6.71 (m, 2H), 4.10 (t, J = 6.4 Hz, 2H), 3.76–3.73 (m, 4H),
3.07 (s, 6H), 2.48–2.41 (m, 6H), 1.91–1.85 (m, 2H), 1.74–1.68 (m,
1
3
EtOAc, the solution was dried over Na
2
SO
4
and then concentrated,
2H). C NMR (100 MHz, CDCl ) d 182.53, 167.65, 166.13, 151.09,
3
the compounds 4a–4e were crystallized from ethanol which were
145.86, 133.28, 125.38, 120.07, 115.47, 113.88, 112.09, 111.89,
used without further purification.
96.84, 77.41, 77.09, 76.78, 68.46, 67.02, 58.44, 53.74, 40.10,

7
280
W. Luo et al. / Bioorg. Med. Chem. 21 (2013) 7275–7282
+
+
2
6.84, 22.98. MS (ESI ): m/z: 423.2[M+H] . Elemental Anal. Calcd
H N O
30 2 4
.21; N, 6.52.
4.5.9. 7-(6-(Benzyl(methyl)amino)hexyloxy)-2-(4-
(dimethylamino)phenyl)-4H-chromen-4-one (5i)
Intermediate 4b was treated with N-methyl-1-phenylmethan-
for C25 : C, 71.07; H, 7.16; N, 6.63. Found: C, 70.71; H,
7
amine according to general procedure to give the desired product
1
4
.5.5. 7-(4-(Benzyl(methyl)amino)butoxy)-2-(4-
5i as orange crystal (70% yield), mp 103–105 °C;
(400 MHz, CDCl
H NMR
(
dimethylamino)phenyl)-4H-chromen-4-one (5e)
3
) d 7.83 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 8.3 Hz, 1H),
Intermediate 4a was treated with N-methyl-1-phenylme-
thanamine according to general procedure to give the desired
7.33 (d, J = 4.3 Hz, 4H), 7.25 (d, J = 4.5 Hz, 1H), 6.84 (s, 1H), 6.78–
6.72 (m, 4H), 4.06 (t, J = 6.5 Hz, 2H), 3.50 (s, 2H), 3.07 (s, 6H),
product 5e as orange needle-like crystal (73% yield), mp 106–
2.47–2.32 (m, 2H), 2.21 (s, 3H), 1.90–1.81 (m, 2H), 1.65–1.54 (m,
2H), 1.52–1.39 (m, 4H). C NMR (100 MHz, CDCl ) d 182.57,
3
1
13
1
7
6
2
1
1
1
9
09 °C; H NMR (400 MHz, CDCl
3
) d 7.83 (d, J = 8.9 Hz, 2H),
.70 (d, J = 9.1 Hz, 1H), 7.34 (d, J = 4.4 Hz, 4H), 7.28 (s, 1H),
.85 (s, 1H), 6.77–6.68 (m, 4H), 4.06 (t, J = 6.4 Hz, 2H), 3.52 (s,
H), 3.07 (s, 6H), 2.46 (t, J = 7.1 Hz, 2H), 2.23 (s, 3H), 1.93–
167.68, 166.29, 151.06, 145.89, 139.25, 133.29, 129.08, 128.23,
126.92, 125.34, 120.08, 115.39, 113.86, 112.16, 111.89, 96.82,
68.75, 62.40, 57.37, 42.35, 40.11, 28.99, 27.37, 27.14, 25.94. MS
.83 (m, 2H), 1.76–1.68 (m, 2H). 13C NMR (100 MHz, CDCl
) d
+
+
3
36 2 3
(ESI ): m/z: 485.3[M+H] . Elemental Anal. Calcd for C31H N O :
82.55, 167.67, 166.24, 151.08, 145.90, 139.23, 133.29, 129.05,
28.26, 126.98, 125.32, 120.11, 115.41, 113.84, 112.15, 111.90,
6.87, 68.56, 62.42, 56.69, 42.26, 40.10, 26.74, 23.77. MS
C, 76.83; H, 7.49; N, 5.78. Found: C, 76.42; H, 7.47; N, 5.69.
4.5.10. 7-(8-(Diethylamino)octyloxy)-2-(4-
(dimethylamino)phenyl)-4H-chromen-4-one (5j)
+
+
(
ESI ): m/z: 457.2[M+H] . Elemental Anal. Calcd for
C
7
29
H
32
N
2
O
3
: C, 76.29; H, 7.06; N, 6.14. Found: C, 76.23; H,
Intermediate 4c was treated with diethylamine according to
.07; N, 6.09.
general procedure to give the desired product 5j as yellow solid
1
(
65% yield), mp 100–102 °C; H NMR (400 MHz, CDCl
3
) d 7.83 (d,
4
.5.6. 7-(6-(Diethylamino)hexyloxy)-2-(4-
J = 8.9 Hz, 2H), 7.70 (d, J = 8.4 Hz, 1H), 6.84 (s, 1H), 6.77–6.70 (m,
4H), 4.07 (t, J = 6.5 Hz, 2H), 3.07 (s, 6H), 2.57 (q, J = 7.1 Hz, 4H),
2.49–2.42 (m, 2H), 1.89–1.80 (m, 2H), 1.48 (d, J = 7.1 Hz, 4H),
(
dimethylamino)phenyl)-4H-chromen-4-one (5f)
Intermediate 4b was treated with diethylamine according to
1
3
general procedure to give the desired product 5f as yellow solid
1.37–1.31 (m, 8H), 1.05 (t, J = 7.2 Hz, 6H). C NMR (100 MHz,
CDCl ) d 182.58, 167.71, 166.32, 151.09, 145.92, 133.26, 125.37,
1
(
60% yield), mp 109–112 °C; H NMR (400 MHz, CDCl
3
) d 7.82 (d,
3
J = 9.0 Hz, 2H), 7.70 (d, J = 8.4 Hz, 1H), 6.84 (s, 1H), 6.78–6.69 (m,
120.16, 115.40, 113.79, 112.13, 111.91, 96.84, 68.81, 52.94, 46.86,
+
4
2
1
1
1
2
H), 4.07 (t, J = 6.5 Hz, 2H), 3.07 (s, 6H), 2.54 (q, J = 7.2 Hz, 4H),
40.10, 29.52, 29.30, 28.97, 27.63, 26.89, 25.93, 11.57. MS (ESI ):
+
.48–2.40 (m, 2H), 1.87–1.82 (m, 2H), 1.56–1.47 (m, 4H), 1.42–
m/z: 465.3[M+H] . Elemental Anal. Calcd for C29
40
H N
2
O
3
ꢀ0.4C
2 5-
H
.36 (m, 2H), 1.04 (t, J = 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl
) d
OH: C, 74.09; H, 8.85; N, 5.80. Found: C, 74.02; H, 8.68; N, 5.81.
3
82.56, 167.68, 166.28, 151.07, 145.90, 133.26, 125.35, 120.12,
15.40, 113.80, 112.13, 111.89, 96.83, 68.74, 52.85, 46.90, 40.10,
4.5.11. 2-(4-(Dimethylamino)phenyl)-7-(8-(pyrrolidin-1-
yl)octyloxy)-4H-chromen-4-one (5k)
+
+
8.98, 27.43, 26.99, 26.00, 11.69. MS (ESI ): m/z: 437.3[M+H] . Ele-
: C, 74.28; H, 8.31; N, 6.42.
mental Anal. Calcd for C27
H
36
N
2
O
3
Intermediate 4c was treated with pyrrolidine according to gen-
Found: C, 74.53; H, 8.33; N, 6.41.
eral procedure to give the desired product 5k as yellow solid (61%
1
3
yield), mp 89–91 °C; H NMR (400 MHz, CDCl ) d 7.80 (d, J = 8.9 Hz,
4
.5.7. 2-(4-(Dimethylamino)phenyl)-7-(6-(pyrrolidin-1-
2H), 7.67 (d, J = 8.5 Hz, 1H), 6.81 (s, 1H), 6.76–6.65 (m, 4H), 4.04 (t,
J = 6.5 Hz, 2H), 3.04 (s, 6H), 2.51 (s, 4H), 2.46–2.37 (m, 2H), 1.87–
yl)hexyloxy)-4H-chromen-4-one (5g)
1
3
Intermediate 4b was treated with pyrrolidine according to gen-
1.74 (m, 6H), 1.57–1.43 (m, 4H), 1.35 (s, 6H). C NMR (100 MHz,
CDCl ) d 182.53, 167.68, 166.31, 151.08, 145.90, 133.25, 125.33,
eral procedure to give the desired product 5g as orange solid (88%
yield), mp 139–141 °C; H NMR (400 MHz, CDCl
J = 8.9 Hz, 2H), 7.64 (d, J = 9.0 Hz, 1H), 6.77 (s, 1H), 6.67 (d,
J = 9.3 Hz, 4H), 3.99 (t, J = 6.5 Hz, 2H), 2.99 (s, 6H), 2.48 (d,
J = 6.4 Hz, 4H), 2.46–2.39 (m, 2H), 1.86–1.67 (m, 6H), 1.56–1.50
3
1
3
) d 7.75 (d,
120.14, 115.39, 113.77, 112.12, 111.90, 96.84, 68.80, 56.67, 54.24,
+
40.07, 29.48, 29.24, 29.01, 28.96, 27.64, 25.91, 23.40. MS (ESI ):
+
m/z: 463.3[M+H] . Elemental Anal. Calcd for C29
H
38
N
2
O
3
: C,
75.29; H, 8.28; N, 6.06. Found: C, 74.98; H, 8.28; N, 5.93.
13
(
(
1
6
m, 2H), 1.48–1.42 (m, 2H), 1.41–1.31 (m, 2H).
100 MHz, CDCl 182.47, 167.62, 166.24, 151.03, 145.83,
33.24, 125.26, 120.02, 115.34, 113.82, 112.12, 111.84, 96.78,
C NMR
3
)
d
4.5.12. 2-(4-(Dimethylamino)phenyl)-7-(8-(piperidin-1-
yl)octyloxy)-4H-chromen-4-one (5l)
+
8.70, 56.51, 54.23, 40.04, 28.91, 27.40, 25.94, 23.37. MS (ESI ):
Intermediate 4c was treated with piperidine according to gen-
+
m/z: 435.2[M+H] . Elemental Anal. Calcd for C27
34
H N
2
O
3
ꢀ0.4C
H
2 5-
eral procedure to give the desired product 5l as yellow solid (63%
1
OHꢀ0.1CHCl
3
: C, 72.07; H, 7.91; N, 6.03. Found: C, 72.55; H, 7.70;
yield), mp 130–132 °C; H NMR (400 MHz, CDCl
3
) d 7.83 (d,
N, 6.25.
J = 8.9 Hz, 2H), 7.70 (d, J = 8.3 Hz, 1H), 6.84 (s, 1H), 6.79–6.68 (m,
H), 4.07 (t, J = 6.5 Hz, 2H), 3.07 (s, 6H), 2.36 (d, J = 28.0 Hz, 4H),
2.29 (d, J = 7.9 Hz, 2H), 1.88–1.79 (m, 2H), 1.65–1.58 (m, 4H),
4
4
.5.8. 2-(4-(Dimethylamino)phenyl)-7-(6-(piperidin-1-
1
3
yl)hexyloxy)-4H-chromen-4-one (5h)
3
1.56–1.43 (m, 6H), 1.40–1.29 (m, 6H). C NMR (100 MHz, CDCl )
Intermediate 4b was treated with piperidine according to gen-
d 182.55, 167.69, 166.31, 151.08, 145.91, 133.25, 125.34, 120.15,
eral procedure to give the desired product 5h as orange crystal
115.39, 113.77, 112.12, 111.90, 96.84, 68.80, 59.66, 54.68, 40.09,
1
+
(
(
62% yield), mp 120–122 °C; H NMR (400 MHz, CDCl
d, J = 8.9 Hz, 2H), 7.67 (d, J = 8.3 Hz, 1H), 6.81 (s, 1H), 6.74–
3
) d 7.79
29.50, 29.25, 28.97, 27.68, 26.94, 26.00, 25.91, 24.51. MS (ESI ):
+
m/z: 477.3[M+H] . Elemental Anal. Calcd for C30
H
40
N
2
O
3
ꢀ0.4C
2 5-
H
6
6
.68 (m, 4H), 4.04 (t, J = 6.5 Hz, 2H), 3.04 (s, 6H), 2.36–2.26 (m,
OH: C, 74.72; H, 8.63; N, 5.66. Found: C, 74.97; H, 8.47; N, 5.76.
13
H), 1.87–1.78 (m, 2H), 1.64–1.32 (m, 12H).
C
NMR
(
100 MHz, CDCl
3
)
d
182.54, 167.65, 166.25, 151.03, 145.85,
4.5.13. 7-(8-(Benzyl(methyl)amino)octyloxy)-2-(4-
1
6
2
C
8
33.26, 125.30, 120.04, 115.35, 113.82, 112.13, 111.85, 96.78,
(dimethylamino)phenyl)-4H-chromen-4-one (5m)
8.71, 59.55, 54.69, 40.08, 28.93, 27.48, 26.93, 26.02, 25.97,
Intermediate 4c was treated with N-methyl-1-phenylmethan-
+
+
4.51. MS (ESI ): m/z: 449.3[M+H] . Elemental Anal. Calcd for
: C, 74.97; H, 8.09; N, 6.24. Found: C, 74.59; H,
.39; N, 6.37.
amine according to general procedure to give the desired product
1
28
H
36
N
2
O
3
5m as orange solid (46% yield), mp 80–82 °C; H NMR (400 MHz,
3
CDCl ) d 7.81 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.4 Hz, 1H), 7.31 (d,

W. Luo et al. / Bioorg. Med. Chem. 21 (2013) 7275–7282
7281
J = 4.4 Hz, 4H), 7.24 (d, J = 4.3 Hz, 1H), 6.83 (s, 1H), 6.76–6.71 (m,
H), 4.05 (t, J = 6.5 Hz, 2H), 3.48 (s, 2H), 3.05 (s, 6H), 2.45–2.29
m, 2H), 2.18 (s, 3H), 1.87–1.78 (m, 2H), 1.51–1.47 (m, 4H), 1.34
126.86, 125.39, 120.19, 115.41, 113.77, 112.13, 111.93, 96.86,
68.84, 62.34, 57.61, 42.30, 40.11, 29.59, 29.56, 29.52, 29.34,
4
(
(
+
+
28.99, 27.45, 25.97. MS (ESI ): m/z: 541.3[M+H] . Elemental Anal.
Calcd for C35 : C, 77.74; H, 8.20; N, 5.18. Found: C, 77.78;
H, 8.19; N, 5.13.
1
3
s, 6H). C NMR (100 MHz, CDCl
3
) d 182.57, 167.71, 166.33,
44 2 3
H N O
1
1
4
5
51.10, 145.93, 133.27, 129.11, 128.21, 126.93, 125.37, 120.16,
15.41, 113.79, 112.14, 111.92, 96.86, 68.82, 62.31, 57.50, 42.26,
+
0.10, 29.47, 29.31, 28.98, 27.35, 25.93. MS (ESI ): m/z:
4.5.18. 7-(12-(Benzyl(methyl)amino)dodecyloxy)-2-(4-
(dimethylamino)phenyl)-4H-chromen-4-one (5r)
+
13.4[M+H] . Elemental Anal. Calcd for C33
H
40
N
2
O
3
2 5
ꢀ0.4C H OH:
C, 76.44; H, 8.05; N, 5.27. Found: C, 76.46; H, 7.95; N, 5.27.
Intermediate 4e was treated with N-methyl-1-phenylmethan-
amine according to general procedure to give the desired product
1
4
.5.14. 7-(10-(Diethylamino)decyloxy)-2-(4-
dimethylamino)phenyl)-4H-chromen-4-one (5n)
Intermediate 4d was treated with diethylamine according to
general procedure to give the desired product 5n as yellow solid
5r as yellow crystal (45% yield), mp 66–68 °C; H NMR (400 MHz,
(
3
CDCl ) d 7.80 (d, J = 8.9 Hz, 2H), 7.67 (d, J = 8.6 Hz, 1H), 7.31 (d,
J = 4.4 Hz, 4H), 7.26–7.21 (m, 1H), 6.82 (s, 1H), 6.74–6.66 (m, 4H),
4.03 (t, J = 6.5 Hz, 2H), 3.47 (s, 2H), 3.04 (s, 6H), 2.39–2.31 (m,
2H), 2.17 (s, 3H), 1.87–1.77 (m, 2H), 1.55–1.41 (m, 4H), 1.38–
1
(
58% yield), mp 101–102 °C; H NMR (400 MHz, CDCl
3
) d 7.91–
1
3
7
4
2
.76 (m, 2H), 7.76–7.62 (m, 1H), 6.83 (s, 1H), 6.81–6.68 (m, 4H),
.06 (t, J = 6.6 Hz, 2H), 3.06 (s, 6H), 2.53 (q, J = 7.2 Hz, 4H), 2.43–
3
1.24 (m, 14H). C NMR (100 MHz, CDCl ) d 182.59, 167.70,
166.33, 151.07, 145.91, 139.25, 133.28, 129.10, 128.19, 126.88,
125.36, 120.12, 115.38, 113.83, 112.16, 111.90, 96.82, 68.85,
62.33, 57.63, 42.31, 40.12, 29.67, 29.65, 29.62, 29.60, 29.39,
.39 (m, 2H), 1.92–1.75 (m, 2H), 1.46–1.42 (m, 4H), 1.40–1.26
(
m, 10H), 1.03 (t, J = 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl
3
) d
+
+
1
1
2
82.56, 167.71, 166.33, 151.09, 145.93, 133.25, 125.36, 120.18,
15.41, 113.74, 112.12, 111.91, 96.85, 68.83, 53.03, 46.90, 40.09,
9.63, 29.55, 29.51, 29.33, 28.99, 27.73, 27.03, 25.96, 11.68. MS
29.01, 27.50, 27.46, 26.00. MS (ESI ): m/z: 569.4[M+H] . Elemental
Anal. Calcd for C37 : C, 78.13; H, 8.51; N, 4.93. Found: C,
48 2 3
H N O
78.09; H, 8.52; N, 4.85.
+
+
(
ESI ): m/z: 493.4[M+H] . Elemental Anal. Calcd for C31
44 2 3
H N O :
C, 75.57; H, 9.00; N, 5.69. Found: C, 75.25; H, 9.03; N, 5.54.
4.6. Biological activity
4
.5.15. 2-(4-(Dimethylamino)phenyl)-7-(10-(pyrrolidin-1-
4.6.1. Inhibition assays on AChE and BChE in vitro
yl)decyloxy)-4H-chromen-4-one (5o)
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel), butyr-
ylcholinesterase (BChE, E.C. 3.1.1.8, from equine serum), 5,5 -dithi-
0
Intermediate 4d was treated with pyrrolidine according to gen-
eral procedure to give the desired product 5o as yellow solid (47%
obis-(2-nitrobenzoic acid) (DTNB), acetylthiocholine chloride
(ATC), butylthiocholine chloride (BTC) were purchased from Sigma
Aldrich. Test compounds were dissolved in a minimum volume of
1
3
yield), mp 98–100 °C; H NMR (400 MHz, CDCl ) d 7.81 (d,
J = 8.9 Hz, 2H), 7.68 (d, J = 8.3 Hz, 1H), 6.82 (s, 1H), 6.76–6.69 (m,
H), 4.05 (t, J = 6.5 Hz, 2H), 3.05 (s, 6H), 2.54 (s, 4H), 2.48–2.42
m, 2H), 1.87–1.75 (m, 6H), 1.58–1.43 (m, 4H), 1.35–1.31 (m,
4
DMSO (1%) and then diluted in 0.1 M KH
8.0) to provide a final concentration range.
All the assays were under 0.1 M KH PO
2
PO
4
/K
2
HPO
4
buffer (pH
(
1
3
1
1
1
2
0H).
C NMR (100 MHz, CDCl
3
) d 182.58, 167.70, 166.32,
2
/K
4 2
HPO
4
buffer, pH 8.0,
51.07, 145.90, 133.26, 125.35, 120.12, 115.37, 113.81, 112.15,
11.89, 96.82, 68.82, 56.70, 54.24, 40.10, 29.57, 29.50, 29.32,
8.97, 28.93, 27.69, 25.95, 23.38. MS (ESI ): m/z: 491.3[M+H] . Ele-
ꢀ0.5C OH: C, 74.82; H, 8.83; N,
.45. Found: C, 74.88; H, 8.57; N, 5.71.
using a Shimadzu UV-2450 Spectrophotometer. Enzyme solutions
were prepared to give 2.0 units/mL in 2 mL aliquots. The assay
+
+
medium contained phosphate buffer, pH 8.0 (1 mL), 50
lL of
mental Anal. Calcd for C31
5
H
42
N
2
O
3
2
H
5
0.01 M DTNB, 10 L of enzyme, and 50 L of 0.01 M substrate
l
l
(ATC). The substrate was added to the assay medium containing
enzyme, buffer, and DTNB with inhibitor after 15 min of incubation
time. The activity was determined by measuring the increase in
absorbance at 412 nm at 1 min intervals at 37 °C. Calculations
were performed according to the method of the equation in Ellman
4
.5.16. 2-(4-(Dimethylamino)phenyl)-7-(10-(piperidin-1-
yl)decyloxy)-4H-chromen-4-one (5p)
Intermediate 4d was treated with piperidine according to gen-
2
7
eral procedure to give the desired product 5p as yellow solid
et al. In vitro BChE assay use the similar method described above.
Each concentration was assayed in triplicate.
1
(
52% yield), mp 105–107 °C; H NMR (400 MHz, CDCl
3
) d 7.83 (d,
J = 9.0 Hz, 2H), 7.70 (d, J = 8.4 Hz, 1H), 6.84 (s, 1H), 6.74 (m, 4H),
.07 (t, J = 6.5 Hz, 2H), 3.07 (s, 6H), 2.38 (s, 4H), 2.28 (d,
J = 8.1 Hz, 2H), 1.89–1.80 (m, 2H), 1.64–1.57 (m, 4H), 1.55–1.43
m, 7H), 1.33–1.27 (m, 10H). 13C NMR (100 MHz, CDCl
) d 182.61,
67.72, 166.34, 151.09, 145.93, 133.27, 125.39, 120.17, 115.40,
4
4.6.2. Kinetic characterization of AChE inhibition
Kinetic characterization of AChE was performed using a re-
23
(
3
ported method. Six different concentrations of substrate were
1
1
2
5
8
mixed in the 1 mL 0.1 M KH buffer (pH 8.0), contain-
2
PO
/K
4 2
HPO
4
13.79, 112.14, 111.92, 96.84, 68.83, 59.71, 54.68, 40.12, 29.60,
ing 50 L of DTNB, 10 L AChE, and 50 lL substrate. Test com-
l
l
+
9.51, 29.33, 28.98, 27.77, 26.96, 25.97, 24.50. MS (ESI ): m/z:
pound was added into the assay solution and pre-incubated with
the enzyme at 37 °C for 15 min, followed by the addition of sub-
strate. Kinetic characterization of the hydrolysis of ATC catalyzed
by AChE was done spectrometrically at 412 nm. A parallel control
with no inhibitor in the mixture, allowed adjusting activities to be
measured at various times.
+
05.3[M+H] . Elemental Anal. Calcd for C32
H
44
N
2
O
3
: C, 76.15; H,
.79; N, 5.55. Found: C, 76.29; H, 9.13; N, 5.31.
4
.5.17. 7-(10-(Benzyl(methyl)amino)decyloxy)-2-(4-
(
dimethylamino)phenyl)-4H-chromen-4-one (5q)
Intermediate 4d was treated with N-methyl-1-phenylmethan-
amine according to general procedure to give the desired product
4.6.3. Molecular modeling
1
5
q as yellow needle-like crystal (50% yield), mp 82–84 °C;
NMR (400 MHz, CDCl 7.83 (d, J = 8.9 Hz, 2H), 7.70 (d,
J = 8.7 Hz, 1H), 7.33 (d, J = 4.4 Hz, 4H), 7.27–7.24 (m, 1H), 6.84 (s,
H), 6.79–6.71 (m, 4H), 4.07 (t, J = 6.6 Hz, 2H), 3.49 (s, 2H), 3.07
s, 6H), 2.45–2.30 (m, 2H), 2.20 (s, 3H), 1.90–1.80 (m, 2H), 1.58–
.45 (m, 4H), 1.34 (m, J = 23.5 Hz, 10H). 13C NMR (100 MHz, CDCl
d 182.59, 167.72, 166.34, 151.10, 145.94, 133.26, 129.08, 128.18,
H
The crystal structure of the torpedo AChE (code ID: 1ACJ) and
the human BChE (code ID: 1POI) were obtained in the Protein Data
Bank after eliminating the inhibitor and water molecules. The 3D
structure of compounds 5j and 5o was built and performed geom-
etry optimization by molecular mechanics. Further preparation of
substrates included addition of Gasteiger charges, removal of
hydrogen atoms and addition of their atomic charges to skeleton
3
) d
1
(
1
3
)

7
282
W. Luo et al. / Bioorg. Med. Chem. 21 (2013) 7275–7282
atoms, and finally, assignment of proper atomic types. Autotors
was then used to define the rotatable bonds in the ligand.
(2). Net AUC = AUCantioxidant ꢂ AUCblank
; The ORAC-FL values
were calculated as following:
Docking studies were carried out using the AUTODOCK 4.0 prog-
rame using ADT, polar hydrogen atoms were added and Gasteiger
charges were assigned to the enzyme. The resulting enzyme struc-
ture was used as an input for the AUTOGRID program. AUTOGRID per-
formed a precalculated atomic affinity grid maps for each atom
type in the ligand plus an electrostatics map and a separate desolv-
ation map present in the substrate molecule. All maps were calcu-
lated with 0.375 Å spacing between grid points. The centre of the
grid box was placed at the bottom of the active site gorge (AChE
(3). [(AUCsample ꢂ AUCblank)/(AUCTrolox ꢂ AUCblank)] ꢁ [(concen-
tration of Trolox/concentration of sample)], and expressed as
Trolox equivalents by using the standard curve calculated
for each assay. Final results were in
lent/ M of pure compound.
lM of Trolox equiva-
l
Acknowledgements
[
2.781 64.383 67.971]; BChE [135.628 111.375 39.905]). The
We thank the Natural Science Foundation of China (Nos.
21172053, 21302041), the Postdoctoral Science Foundation of Chi-
na (No. 2012M521391), and the Postdoctoral Science Foundation
of Henan Province (No. BH2011043) for financial support of this
study.
dimensions of the active site box were set at 50 ꢁ 46 ꢁ 46 Å.
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
docking. To ensure the reliability of the results, the docking proce-
dures were repeated 10 independent times for the compounds and
the obtained orientations were analyzed.
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immediately placed into a Spectrafluor Plus plate reader (Tecan,
Switzerland), and the fluorescence was measured every 60 s for
9
0 min with exitation at 485 nm and emission at 535 nm. 6-Hydro-
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l
(
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l
least four independent runs were performed for each sample. Fluo-
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P
i¼90
i¼1
i
0
0
f =f where f is the initial fluorescence at
is the fluorescence at time i. The net AUC for
(
1). AUC ¼ 1 þ
0
min, and f
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a sample was calculated as following:
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