Design, synthesis and evaluation of novel tacrine-multialkoxybenzene hybrids as dual inhibitors for cholinesterases and amyloid beta aggregation

Article abstract of DOI:10.1016/j.bmc.2010.12.022

A new series of tacrine-multialkoxybenzene hybrids (9a-9n) were designed, synthesized and evaluated as dual inhibitors of cholinesterases (ChEs) and self-induced β-amyloid (Aβ) aggregation. All the synthesized compounds had high acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity with IC₅₀ values at the nanomolar range, which were much better than tacrine alone. A Lineweaver-Burk plot and molecular modeling study showed that these hybrids targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, compounds 9a-9f with methylenedioxybenzene moiety showed higher self-induced Aβ aggregation inhibitory activity than a reference compound, curcumin. These compounds could be selected as multi-potent agents for further investigation to treat AD.

Full text of DOI:10.1016/j.bmc.2010.12.022

Bioorganic & Medicinal Chemistry 19 (2011) 763–770
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of novel tacrine-multialkoxybenzene hybrids
as dual inhibitors for cholinesterases and amyloid beta aggregation
Wen Luo, Yan-Ping Li, Yan He, Shi-Liang Huang, Jia-Heng Tan, Tian-Miao Ou, Ding Li, Lian-Quan Gu,
⇑
Zhi-Shu Huang
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, 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 new series of tacrine-multialkoxybenzene hybrids (9a–9n) were designed, synthesized and evaluated
as dual inhibitors of cholinesterases (ChEs) and self-induced b-amyloid (Ab) aggregation. All the
synthesized compounds had high acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)
inhibitory activity with IC50 values at the nanomolar range, which were much better than tacrine alone.
A Lineweaver–Burk plot and molecular modeling study showed that these hybrids targeted both the
catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, compounds 9a–9f with
methylenedioxybenzene moiety showed higher self-induced Ab aggregation inhibitory activity than a
reference compound, curcumin. These compounds could be selected as multi-potent agents for further
investigation to treat AD.
Received 12 November 2010
Revised 5 December 2010
Accepted 6 December 2010
Available online 13 December 2010
Keywords:
Tacrine hybrids
Anti-Alzheimer agent
Acetylcholinesterase
Butyrylcholinesterase
Self-induced Ab aggregation
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
can be used for the treatment of diseases associated with
acetylcholine depletion, such as Alzheimer’s disease.9 On the
Alzheimer’s disease (AD) is a progressive neurodegenerative
brain disorder that is characterized by dementia, cognitive impair-
ment, and memory loss.1,2 Although many factors have been impli-
cated in AD, its etiology is not completely known. There are diverse
hallmarks such as low levels of acetylcholine, b-amyloid (Ab)
other hand, BuChE has been recently considered as a potential
target because it also plays an important role in regulating acetyl-
choline level.10 Therefore, the concurrent inhibition of both AChE
and BuChE should provide additional benefits in the treatment of
AD.
deposits and
s
-protein aggregation, which seem to play significant
The progressive deposition of Ab in the brain of AD patients is
generally considered to be fundamental to the development of
neurodegenerative pathology. The cell toxicity associated with Ab
fibril aggregation provides an explanation for the neuronal cell loss
found in AD patients.11,12 Therefore, Ab fibril aggregation in the
brain is currently another potential target for the treatment of
AD.13,14 Hence, several series of inhibitors including curcumin15,16
and benzofuran analogues17,18 have been developed, and found to
interfere with the self-induced aggregation of Ab.
Tacrine was the first approved ChEs inhibitor by the FDA for the
treatment of AD, although its side effects, the search for tacrine hy-
brids is still of interest. Recent years, many of studies focused on
the combined effects of ChEs inhibition for the enhancement of
the cholinergic neurotransmission and reduction of the Ab fibril
self-aggregation by conjugating tacrine with other active groups.
Based on this strategy, many of tacrine hybrids (A and B in Fig. 1)
were synthesised.19,20
roles in the disease.3–5
Current treatment of AD focuses on increasing cholinergic neu-
rotransmission in the brain through inhibiting cholinesterases
(ChEs) activity with drugs including tacrine, donepezil, rivastig-
mine and galantamine (Fig. 1). But clinical experience has shown
that ChEs inhibition alone is a palliative treatment, which does
not address AD’s etiology.6 Due to the multi-pathogenesis of AD,
one of the current strategies is to develop novel anti-Alzheimer
agents with multiple potencies,7 including compounds with
inhibitory activity for both ChEs and Ab self-aggregation, and the
strategy of combining these two properties in one structure was
confirmed to be successful.8
Two types of cholinesterase (ChE) were found in the central
nervous system, including acetylcholinesterase (AChE) and butyr-
ylcholinesterase (BuChE). AChE plays a crucial role in central and
peripheral nervous systems. Control of the AChE enzyme activity
Our research group has been involved in the development of
ChEs inhibitors as potential drugs of AD for many years.21–23
Recently, in our routine screening for compounds with inhibitory
activity towards Ab aggregation, it was found that compounds
⇑
Corresponding author. Tel./fax: +86 20 39943056.
E-mail address: ceshzs@mail.sysu.edu.cn (Z.-S. Huang).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.022

764
W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 763–770
Cl
O
O
R
HN
N
m
N
H
N
n
N
N
H
N
H
n
O
NH
Cl
A
B
O
O
OMe
OMe
OMe
R1
O
O
H
MeO
MeO
R2
R3
N
N
N
H
n
m
H
N
R4
MeO
OMe
Schizadrin derivatives
C
H3C
N
O
NH2
N
O
N
OCH3
N
H3CO
H3CO
O
O
H
N
HO
Tacrine
Galantamine
Donepezil
Rivastigmine
Figure 1. Chemical structures of tacrine, donepezil, rivastigmine, galantamine, schizadrin derivatives, and tacrine hybrids.
containing a methylenedioxybenzene moiety had significant activ-
ity for inhibiting Ab self-induced aggregation. In addition, it has
been reported that the schizadrin derivatives (Fig. 1) with a meth-
ylenedioxybenzene group show significant inhibition for the activ-
ity of AChE, and the methylenedioxybenzene group is important
through the structure–activity relationship studies.24 Moreover,
some studies have suggested that electron-rich aromatics would
bind to the peripheral binding site (C in Fig. 1), which have been
confirmed in a comparison of aromatics with differing electron
density.25–27 Therefore, in the present study, in order to further ex-
plore the anti-Alzheimer potential of the electron-rich aromatics, a
series of new inhibitors aimed at both ChEs and Ab self-induced
aggregation were designed by conjugating the methylenedioxy-
benzene or di- (or tri-) methoxybenzene moiety with tacrine using
a long chain linker. It is possible that the multialkoxybenzene inhi-
bit AChE activity through binding with PAS of AChE and block Ab
self-aggregation.
were obtained by the reaction of multialkoxy benzaldehyde
4a–4c with intermediates 8a–8f followed with reduction using
NaBH4 in MeOH.30
2.2. In vitro inhibition studies on AChE and BuChE
To determine the potential of the target compounds 9a–9n for
the treatment of AD, their ChEs inhibitory activity was determined
by the method of Ellman et al.31 The IC50 values for AChE and
BuChE inhibitions were summarized as shown in Table 1. The re-
sults showed that all the tested compounds (9a–9n) had significant
ChEs inhibitory activity and 1–11.5-fold of inhibition selectivity for
BuChE over AChE. Compound 9e, with a methylenedioxybenzene
group, showed the most potent inhibition for AChE with an IC50
value of 7.98 nM. Compound 9g, with trimethoxy substituted
benzene moiety, exhibited the strongest inhibition to BuChE with
an IC50 value of 2.59 nM.
With the same chain length, the inhibition activity of methylene-
dioxybenzene hybrids9c–9f for AChE was slightlybetterthan that of
trimethoxybenzene and dimethoxybenzene hybrids 9g–9n. In
contrast, the inhibition activity of 9c–9f for BuChE was slightly
weaker than that of 9g–9n. Compounds 9g–9j with three methoxy
groups on benzene ring were best inhibitors for BuChE. It is reason-
able that the binding pockets of BuChE is bigger than that of AChE
and this could allow bulky compounds to better fit inside the gorge
of BuChE.
The optimal chain length between two aromatic units deter-
mined experimentally for inhibiting AChE and BuChE were eight
(9e, 9i, 9m) and six (9c, 9g, 9k) methylene groups, respectively.
The variation of chain length for the inhibitors had more influence
on their inhibition to AChE rather than BuChE. The possible reason
is conformational differences between these two enzymes. BuChE
does not have a functional peripheral site, and the active site of
BuChE is wider than that of AChE. Therefore, BuChE had less
restriction to inhibitors with varying linker length.
2. Results and discussion
2.1. Chemistry
Fourteen tacrine-multialkoxybenzene hybrids (9a–9n) with
linkers of varying chain length were prepared as shown in
Scheme 1. Vanillin (1) was treated with bromine in CH3COOH to
generate 5-bromovanillin (2), which was heated with NaOH and
copper powder in water to give 3,4-dihydroxy-5-methoxybenzal-
dehyde (3). Methylenation of compound 3 with a reported proce-
dure afforded the aldehyde 4a in a yield of 72%.28 Compound 7
was synthesized from anthranilic acid (5) in 90% overall yield using
a previously reported method.29 Then, reaction of compound 7
with diaminoalkyl derivatives under reflux for 18 h in 1-pentanol
gave the intermediates 9-alkylaminotetrahydroacridines (8a–8f)
in good yields (60–70%). This step was carried out in the presence
of catalytic amount of KI. The final products, compounds 9a–9n

W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 763–770
765
CHO
Br
CHO
HO
HO
CHO
CHO
O
O
a
c
b
HO
HO
OMe
OMe
OMe
OMe
2
1
3
4a
HN
N
n-2
NH2
Cl
NH2
O
HOOC
d
f
e
O
N
H
6
N
5
7
8a-8f n = 4-9
R1
R2
R1
R2
CHO
HN
n-2
HN
N
n-2
N
H
NH2
+
g,h
R3
R3
N
4a
8a 8f
-
9a-9n
R1+R2=OCH2O, R3=OMe
4b R1=R2=R3=OMe
4c
R1=R2=OMe, R3=H
Scheme 1. Synthesis of tacrine-multialkoxybenzene hybrids. Reagents and conditions: (a) Br2, AcOH, rt; (b) NaOH, Cu, H2O, reflux; (c) K2CO3, CH2Br2, CuO, DMF, 140 °C;
(d) cyclohexanone, toluene, reflux; (e) POCl3, 120 °C; (f) diamine, KI, 1-pentanol, 160 °C; (g) methanol, rt; (h) NaBH4, methanol, rt.
Table 1
Inhibition of ChE activity, selectivity index and inhibition of Ab(1–42) self-induced aggregation
R1
HN
n-2
N
H
R2
R3
N
a
b
Compds
n
Structure of methylenedioxy or methoxyphenyl
group
IC50 for AChE
(nM)
IC50 for BuChE
(nM)
Selectivity
indexc
Ab(1–42) aggregation inhibitiond
(%)
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
Tacrine
Curcumin
4
5
6
7
8
9
6
7
8
9
6
7
8
9
—
—
R
R
R
R
R
R
R
R
R
R
R
R
R
R
1 + R2 = OCH2O; R3 = OMe
1 + R2 = OCH2O; R3 = OMe
1 + R2 = OCH2O; R3 = OMe
1 + R2 = OCH2O; R3 = OMe
1 + R2 = OCH2O; R3 = OMe
1 + R2 = OCH2O; R3 = OMe
1 = R2 = R3 = OMe
1 = R2 = R3 = OMe
1 = R2 = R3 = OMe
1 = R2 = R3 = OMe
113.02 1.67
78.55 0.28
20.52 0.03
18.43 0.15
7.98 0.12
18.66 0.17
22.41 0.21
17.29 1.15
9.77 0.49
20.19 0.20
30.92 2.72
24.03 0.33
11.35 0.19
25.10 2.96
192.6 2.0
—
46.29 3.2
29.83 0.03
5.19 0.20
9.73 0.06
7.94 0.02
11.36 0.21
2.59 0.14
3.55 0.15
3.38 0.25
4.73 0.36
2.68 0.17
5.51 0.10
5.57 0.83
5.67 0.32
27.08 1.16
—
2.4
2.6
4.0
1.9
1.0
1.6
8.7
4.9
2.9
4.3
11.5
4.4
2.0
4.4
7.1
—
63.81 3.22
63.10 13.65
67.13 5.57
65.01 9.85
65.16 1.84
68.49 3.63
—
—
—
—
—
—
—
—
—
1 = R2 = OMe; R3 = H
1 = R2 = OMe; R3 = H
1 = R2 = OMe; R3 = H
1 = R2 = OMe; R3 = H
—
—
51.77 3.33
a
b
c
AChE from electric eel; IC50, inhibitor concentration (means SEM of three experiments) for 50% inactivation of AChE.
BuChE from equine serum; IC50, inhibitor concentration (means SEM of three experiments) for 50% inactivation of BuChE.
Selectivity index = IC50 (AChE)/IC50 (BuChE).
d
Inhibition of self-mediated Ab(1–42) aggregation, the thioflavin-T fluorescence method was used, the mean SD of at least three independent experiments and the
M compounds.
measurements were carried out in the presence of 20
l
2.3. Kinetic characterization of AChE inhibition
2.4. Molecular modeling study
The inhibition of AChE by a representative compound 9e was
further investigated using graphical analysis of steady state inhibi-
tion data as shown in Figure 2. The Lineweaver–Burk plots showed
both increasing slopes and increasing intercepts for higher inhibi-
tor concentration. The pattern indicated a mixed-type inhibition,
which was similar to that of tacrine. This result showed that com-
pound 9e was able to bind both CAS and PAS of AChE which was
also in agreement with the results of molecular modeling studies.
With the aim of obtaining useful information about the binding
interactions between compound 9e and TcAChE (PDB code: 1ACJ), a
molecular modeling study was performed using the docking
program AUTODOCK 4.0 package with PyMOL program as shown in
Figure 3.32,33
The docking result demonstrated that all of the compounds
exhibited multiple binding modes with AChE. In the 9e–TcAChE
complex, compound 9e occupied the entire enzymatic CAS,

766
W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 763–770
activity (data not shown) were lower than that of curcumin. The cur-
rent data are not sufficient to establish structure-activity
relationship for the activity toward Ab aggregation, and further
25
20
15
10
5
non inhibitor
[9e] = 2.0 nM
[9e] = 4.0 nM
[9e] = 8.0 nM
a
investigation is required.
3. Conclusion
In summary, six new tacrine-methylenedioxybenzene hybrids
9a–9f, and eight new tacrine-trimethoxybenzene and tacrine-
dimethoxybenzene hybrids 9g–9n were synthesized and subjected
to biological evaluation. The results showed that these synthetic
compounds had high ChEs inhibitory potency and good selectivity
for BuChE over AChE, which were similar to or better than those of
tacrine. The methylenedioxy substituted hybrids (9c–9f) showed
higher inhibitory effects on AChE, and compound 9e with eight
methylene groups had best AChE inhibitory activity. The inhibition
kinetics of 9e was analyzed using Lineweaver–Burk plots, which
revealed that the compound was a mixed-type inhibitor, and could
bind to both the CAS and PAS of AChE which was also in agreement
with the results of molecular modeling studies. In addition, 9a–9f
exhibited higher self-induced Ab aggregation activity than curcu-
min. Our above results should shed light on the design and devel-
opment of new multi-potent anti-AD agents.
0
-10
-5
0
5
10
15
20
[S]-1 mM-1
Figure 2. Lineweaver–Burk plot for the inhibition of acetylcholinesterase by
compound 9e.
mid-gorge and PAS. The tacrine moiety was bound to CAS, displaying
a classic p–p stacking interaction between Trp84 and Phe330, with
the ring-to-ring distance being 3.4 Å and 3.8 Å, and the protonated
nitrogen atom of the quinoline ring establishes a hydrogen bond
(3.4 Å) with the carbonyl group of the main chain of His440. At the
PAS, the methylenedioxybenzene stacked against the Trp279
4. Experimental section
4.1. Chemistry
through p–pstacking with the distance of 3.8 Å. Similar interactions
were found in 9e in complex with HuBuChE (PDB code: 1POI).
1H and 13C NMR spectra were recorded using TMS as the internal
standard in CDCl3 with a Bruker BioSpin GmbH spectrometer at
400 MHz and 100 MHz, respectively. MS spectra were recorded on
a Shimadzu LCMS-2010A instrument with an ESI mass selective
detector. High resolution mass spectra (HRMS) were recorded on
Shimadzu LCMS-IT-TOF. Flash column chromatography was
performed with silica gel (200–300 mesh) purchased from Qingdao
Haiyang Chemical Co. Ltd. The purities of synthesized compounds
were confirmed to be higher than 95% by analytical HPLC performed
with a dual pump Shimadzu LC-20AB system equipped with a
2.5. Inhibition of self-mediated Ab(1–42) aggregation
The derivatives 9a–9f were tested for their ability to inhibit self-
mediated aggregation of Ab(1–42) by using a thioflavin T fluores-
cence method.34 Compared with the reference compound curcumin,
the results showed that compounds 9a–9f with methylenedioxy
group apparently prevented the self-mediated Ab aggregation with
percentages of inhibition ranging from 63% to 68%, which were high-
er than that of curcumin (51%) (Table 1 and Fig. 4). The most potent
compounds were 9c and 9f, and the inhibitory potency did not
depend on the chain length of the connecting linker. Compounds
9g and 9k have the same chain length with compound 9c, their
Ultimate XB-C18 column (4.6 Â 250 mm, 5
lm) and eluted with
methanol/water (35:65–45:55) containing 0.1% TFA at a flow rate
of 0.5 mL/min. Melting points (mp) were determined using an SRS-
OptiMelt automated melting point instrument without correction.
Figure 3. Docking models of compound-enzyme complex. Representations of compound 9e interacting with residues in the binding site of TcAChE and HuBuChE. The
compounds are rendered in green stick models, and the residues are rendered in orange sticks. Pictures are created with PyMOL.

W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 763–770
767
J = 8.4 Hz, 1H), 7.53 (t, J = 7.1 Hz, 1H), 7.33 (t, J = 7.1 Hz, 1H), 6.48
(d, J = 4.1 Hz, 2H), 5.92 (s, 2H), 4.07 (s, 1H), 3.86 (s, 3H), 3.65 (s,
2H), 3.48 (t, J = 7.0 Hz, 2H), 3.04 (t, J = 5.6 Hz, 2H), 2.68 (t,
J = 5.6 Hz, 2H), 2.63 (t, J = 6.9 Hz, 2H), 1.93–1.85 (m, 4H),
1.74–1.66 (m, 2H), 1.59 (m, 2H). 13C NMR (100 MHz, CDCl3) d
158.40, 150.70, 148.84, 147.37, 143.51, 135.00, 134.09, 128.62,
128.25, 123.59, 122.78, 120.22, 115.93, 107.45, 102.16, 101.29,
56.58, 54.01, 49.30, 48.78, 33.95, 29.50, 27.50, 24.86, 23.03,
22.75. Purity: 96.6% by HPLC, HRMS (ESI): calcd for (M+H)+
(C26H31N3O3) requires m/z 434.2444, found 434.2438.
90
80
70
60
50
40
30
20
10
0
4.4.2. N1-((7-Methoxybenzo[d][1,3]dioxol-5-yl)methyl)-N5-(1,2,
,4-tetrahydroacridin-9-yl)pentane-1,5-diamine (9b)
Intermediate 4a was treated with 8b according to general pro-
cedure to give the desired product 9b as light yellow oil (64%
yield). 1H NMR (400 MHz, CDCl3) d 7.98–7.84 (m, 2H), 7.52 (t,
J = 7.6 Hz, 1H), 7.33–7.28 (m, 1H), 6.49 (s, 2H), 5.90 (s, 2H), 3.86
(s, 3H), 3.65 (s, 2H), 3.45 (t, J = 7.2 Hz, 2H), 3.03 (d, J = 5.6 Hz,
2H), 2.66 (d, J = 5.3 Hz, 2H), 2.58 (t, J = 7.1 Hz, 2H), 1.94–1.79 (m,
4H), 1.74–1.58 (m, 2H), 1.51 (dt, J = 14.3, 7.1 Hz, 2H), 1.40 (dt,
J = 9.1, 6.9 Hz, 2H). 13C NMR (100 MHz, CDCl3) d 158.15, 150.81,
148.80, 147.09, 143.47, 134.64, 134.10, 128.30, 123.56, 122.81,
120.06, 115.69, 107.57, 102.21, 101.25, 56.55, 53.81, 49.22, 48.82,
33.70, 31.53, 29.54, 24.73, 24.61, 22.95, 22.65. Purity: 99.8% by
HPLC, HRMS (ESI): calcd for (MÀH)À (C27H33N3O3) requires m/z
446.2444, found 446.2447.
cur
9a
9b
9c
9d
9e
9f
Compounds
Figure 4. Inhibition of self-mediated Ab(1-42) aggregation by compounds 9a–9f
comparing with that of curcumin. The thioflavin-T fluorescence method was used,
and the measurements were carried out in the presence 20 lM compounds. The
mean SD values from three independent experiments were shown.
4.2. Synthesis of intermediate 4
Aldehyde 4b and 4c are available commercially. Compound 3
was synthesized from vanillin 1 which was reported in many pa-
pers. Intermediate 4a was prepared in the procedure described
below.
4.4.3. N1-((7-Methoxybenzo[d][1,3]dioxol-5-yl)methyl)-N6-(1,2,
3,4-tetrahydroacridin-9-yl)hexane-1,6-diamine (9c)
CuO (0.13 g), anhydrous K2CO3 (1.75 g) and CH2Br2 (1.25 g) were
added to a solution of compound 3 (1.0 g) in anhydrous DMF
(12.5 ml), and the mixture was heated at 140 °C for 4 h. The reaction
mixture was poured into water, and the product was extracted with
ether. The ethereal extract was washed successively with 2% HCl, 2%
NaOH and water. After drying over anhydrous K2CO3 and evaporat-
ing the solvent, light brown crystalline mass was obtained, which on
recrystallization from MeOH, afforded colorless pillars. MS (ESI) m/z:
181.1 (M+H)+. Yield: 72%, mp 129–130 °C (lit.35 mp 131–132 °C).
Intermediate 4a was treated with 8c according to general pro-
cedure to give the desired product 9c as light yellow oil (81% yield).
1H NMR (400 MHz, CDCl3) d 7.92 (dd, J = 13.8, 8.1 Hz, 2H), 7.52 (t,
J = 7.0 Hz, 1H), 7.31 (t, J = 7.1 Hz, 1H), 6.49 (d, J = 1.5 Hz, 2H), 5.90
(s, 2H), 3.86 (s, 3H), 3.64 (s, 2H), 3.44 (t, J = 7.2 Hz, 2H), 3.04 (d,
J = 6.1 Hz, 2H), 2.67 (t, J = 5.3 Hz, 2H), 2.56 (t, J = 7.2 Hz, 2H), 2.17
(s, 1H), 1.93–1.83 (m, 4H), 1.67–1.57 (m, 2H), 1.53–1.43 (m, 2H),
1.41–1.29 (m, 4H). 13C NMR (100 MHz, CDCl3) d 158.36, 150.68,
148.77, 147.44, 143.46, 135.18, 133.99, 128.67, 128.16, 123.50,
122.82, 120.22, 115.84, 107.38, 102.13, 101.23, 56.54, 54.04,
49.37, 49.12, 34.02, 31.68, 29.94, 27.08, 26.83, 24.76, 23.03,
22.77. Purity: 99.8% by HPLC, HRMS (ESI): calcd for (MÀH)À
(C28H35N3O3) requires m/z 460.2600, found 460.2600.
4.3. General procedures for the preparation of intermediate
8a–8f
9-Chloro-1,2,3,4-tetrahy-droacridine 7 (0.44 g, 2 mmol), dia-
mine (12 mmol), catalytic amount KI (0.05 g) and 1-pentanol
(5 mL) were combined and heated to reflux (160 °C) for 10 h. After
cooling to room temperature, the mixture was diluted with CH2Cl2
(50 mL) and then washed with 10% NaOH (1 Â 50 mL) and water
(2 Â 40 mL). The organic layer was dried over MgSO4, filtered, con-
centrated in vacuo, and purified by flash column chromatography
with CHCl3/MeOH/NH4OH (5:1:0.5%) elution.
4.4.4. N1-((7-Methoxybenzo[d][1,3]dioxol-5-yl)methyl)-N7-(1,2,
3,4-tetrahydroacridin-9-yl)heptane-1,7-diamine (9d)
Intermediate 4a was treated with 8d according to general pro-
cedure to give the desired product 9d as light yellow oil (81%
yield). 1H NMR (400 MHz, CDCl3) d 7.94 (dd, J = 12.4, 8.3 Hz, 2H),
7.54 (t, J = 7.6 Hz, 1H), 7.33 (t, J = 7.2 Hz, 1H), 6.51 (s, 2H), 5.93 (s,
2H), 3.89 (s, 3H), 3.69 (s, 2H), 3.63 (s, 1H), 3.49 (t, J = 7.2 Hz, 2H),
3.06 (s, 2H), 2.68 (s, 2H), 2.64–2.56 (m, 2H), 2.02 (s, 1H), 1.90 (t,
J = 3.1 Hz, 4H), 1.71–1.59 (m, 2H), 1.55–1.46 (m, 2H), 1.41–1.28
(m, 6H). 13C NMR (100 MHz, CDCl3) d 157.98, 151.09, 148.84,
146.84, 143.54, 134.24, 134.10, 128.50, 128.09, 123.64, 122.88,
119.93, 115.48, 107.72, 102.39, 101.33, 56.59, 53.62, 49.37, 48.79,
33.48, 31.65, 29.47, 29.19, 27.15, 26.82, 24.71, 22.96, 22.61. Purity:
99.3% by HPLC, HRMS (ESI): calcd for (MÀH)À (C29H37N3O3) re-
quires m/z 474.2757, found 474.2759.
4.4. General procedures for the preparation of compound 9a–9n
Aromatic aldehydes
4 (1 mmol) and intermediate 8a–8f
(1 mmol) were stirred in MeOH for 4 h and then were reduced di-
rectly by NaBH4 (4 mmol) at room temperature for 4 h. The solvent
was evaporated and the residue was poured into water and ex-
tracted with EtOAc, the solution was dried over MgSO4 and then
concentrated, the compounds were purified by flash chromatogra-
phy with CHCl3/MeOH/NH4OH (30:1:0.5%) elution.
4.4.5. N1-((7-Methoxybenzo[d][1,3]dioxol-5-yl)methyl)-N8-(1,2,
3,4-tetrahydroacridin-9-yl)octane-1,8-diamine (9e)
Intermediate 4a was treated with 8e according to general pro-
cedure to give the desired product 9e as light yellow oil (65% yield).
1H NMR (400 MHz, CDCl3) d 7.95 (d, J = 8.4 Hz, 1H), 7.91 (d,
J = 8.4 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 6.50
(s, 2H), 5.91 (s, 2H), 3.87 (s, 3H), 3.66 (s, 2H), 3.46 (t, J = 7.2 Hz,
4.4.1. N1-((7-Methoxybenzo[d][1,3]dioxol-5-yl)methyl)-N4-(1,2,
3,4-tetrahydroacridin-9-yl)butane-1,4-diamine (9a)
Intermediate 4a was treated with 8a according to general pro-
cedure to give the desired product 9a as light yellow oil (76% yield).
1H NMR (400 MHz, CDCl3) d 7.93 (d, J = 8.5 Hz, 1H), 7.90 (d,

768
W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 763–770
2H), 3.05 (s, 2H), 2.67 (s, 2H), 2.57 (t, J = 7.2 Hz, 2H), 1.94–1.83 (m,
4H), 1.62 (dd, J = 14.4, 7.3 Hz, 2H), 1.53–1.42 (m, 2H), 1.35–1.22 (m,
8H). 13C NMR (100 MHz, CDCl3) d 158.21, 150.83, 148.76, 147.26,
143.47, 135.07, 134.01, 128.46, 128.24, 123.50, 122.86, 120.11,
115.66, 107.43, 102.18, 101.23, 56.53, 53.98, 49.40, 49.21, 33.86,
31.69, 29.92, 29.37, 29.24, 27.19, 26.82, 24.72, 23.00, 22.71. Purity:
97.5% by HPLC, HRMS (ESI): calcd for (MÀH)À (C30H39N3O3)
requires m/z 488.2913, found 488.2918.
1.36–1.24 (m, 8H). 13C NMR (100 MHz, CDCl3) d 158.21, 153.14,
150.82, 147.27, 136.81, 136.17, 128.47, 128.24, 123.50, 122.86,
120.11, 115.67, 104.95, 60.76, 56.04, 54.31, 49.49, 49.40, 33.87,
31.68, 29.96, 29.39, 29.25, 27.22, 26.83, 24.72, 23.00, 22.71. Purity:
98.5% by HPLC, HRMS (ESI): calcd for (MÀH)À (C31H43N3O3)
requires m/z 504.3226, found 504.3226.
4.4.10. N1-(1,2,3,4-Tetrahydroacridin-9-yl)-N9-(3,4,5-trimethoxy-
benzyl)nonane-1,9-diamine (9j)
4.4.6. N1-((7-Methoxybenzo[d][1,3]dioxol-5-yl)methyl)-N9-(1,2,
3,4-tetrahydroacridin-9-yl)nonane-1,9-diamine (9f)
Aldehyde 4b was treated with 8f according to general proce-
dure to give the desired product 9j as light yellow oil (74% yield).
1H NMR (400 MHz, CDCl3) d 7.95 (d, J = 8.1 Hz, 1H), 7.90 (d,
J = 8.0 Hz, 1H), 7.52 (t, J = 7.1 Hz, 1H), 7.32 (s, 1H), 6.57 (s, 2H),
3.85 (s, 6H), 3.83 (s, 3H), 3.72 (s, 2H), 3.46 (t, J = 7.2 Hz, 2H), 3.05
(s, 2H), 2.68 (s, 2H), 2.65–2.59 (m, 2H), 1.90 (dd, J = 6.2, 3.1 Hz,
4H), 1.69–1.58 (m, 2H), 1.54–1.47 (m, 2H), 1.38–1.27 (m, 10H).
13C NMR (100 MHz, CDCl3) d 158.22, 153.15, 150.84, 147.26,
136.84, 135.95, 128.44, 128.23, 123.48, 122.86, 120.10, 115.63,
105.01, 60.76, 56.04, 54.20, 49.40, 33.82, 31.69, 29.87, 29.40,
29.38, 29.24, 27.27, 26.85, 24.71, 22.99, 22.70. Purity: 99.4% by
HPLC, HRMS (ESI): calcd for (MÀH)À (C32H45N3O3) requires m/z
518.3383, found 518.3389.
Intermediate 4a was treated with 8f according to general proce-
dure to give the desired product 9f as light yellow oil (63% yield).
1H NMR (400 MHz, CDCl3) d 7.95 (d, J = 8.4, 1H), 7.90 (d, J = 8.4,
1H), 7.53 (t, J = 7.3, 1H), 7.33 (d, J = 7.4, 1H), 6.50 (s, 2H), 5.92 (s,
2H), 3.88 (s, 3H), 3.67 (s, 2H), 3.47 (t, J = 7.2, 2H), 3.05 (s, 2H),
2.68 (s, 2H), 2.58 (t, J = 7.3, 2H), 1.94–1.83 (m, 4H), 1.69–1.59 (m,
2H), 1.48 (dd, J = 13.8, 7.0, 2H), 1.37–1.24 (m, 10H). 13C NMR
(100 MHz, CDCl3)
d 158.26, 150.85, 148.78, 147.29, 143.50,
134.87, 134.07, 128.49, 128.26, 123.51, 122.86, 120.12, 115.67,
107.48, 102.24, 101.27, 56.54, 53.92, 49.45, 49.16, 33.85, 31.72,
29.87, 29.41, 29.38, 29.26, 27.26, 26.87, 24.74, 23.02, 22.72. Purity:
98.9% by HPLC, HRMS (ESI): calcd for (MÀH)À (C31H41N3O3)
requires m/z 502.3070, found 502.3077.
4.4.11. N1-(3,4-Dimethoxybenzyl)-N6-(1,2,3,4-tetrahydroacridin-
9-yl)hexane-1,6-diamine (9k)
4.4.7. N1-(1,2,3,4-Tetrahydroacridin-9-yl)-N6-(3,4,5-trimethoxy-
benzyl)hexane-1,6-diamine (9g)
Aldehyde 4c was treated with 8c according to general proce-
dure to give the desired product 9k as light yellow oil (88% yield).
1H NMR (400 MHz, CDCl3) d 7.92 (dd, J = 12.5, 8.6 Hz, 2H), 7.51 (t,
J = 7.6 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 6.87 (s, 1H), 6.80 (q,
J = 8.3 Hz, 2H), 3.96 (s, 1H), 3.84 (d, J = 11.2 Hz, 6H), 3.69 (s, 2H),
3.44 (t, J = 6.9 Hz, 2H), 3.04 (s, 2H), 2.66 (s, 2H), 2.58 (t, J = 7.1 Hz,
2H), 2.51 (s, 1H), 1.88 (s, 4H), 1.61 (dd, J = 13.9, 7.0 Hz, 2H),
1.55–1.44 (m, 2H), 1.42–1.29 (m, 4H). 13C NMR (100 MHz, CDCl3)
d 158.33, 150.70, 148.91, 147.96, 147.39, 132.97, 128.59, 128.17,
123.49, 122.81, 120.19, 115.80, 111.44, 111.04, 55.87, 55.81,
53.80, 49.34, 49.21, 33.94, 31.65, 29.88, 27.07, 26.81, 24.74,
23.01, 22.73. Purity: 98.9% by HPLC, HRMS (ESI): calcd for
(MÀH)À (C28H37N3O2) requires m/z 446.2808, found 446.2811.
Aldehyde 4b was treated with 8c according to general proce-
dure to give the desired product 9g as light yellow oil (75% yield).
1H NMR (400 MHz, CDCl3) d 7.92 (dd, J = 16.0, 7.8 Hz, 2H), 7.52 (t,
J = 7.0 Hz, 1H), 7.31 (t, J = 7.0 Hz, 1H), 6.55 (s, 2H), 3.83 (d,
J = 4.3 Hz, 9H), 3.70 (s, 2H), 3.45 (t, J = 7.2 Hz, 2H), 3.04 (d,
J = 6.1 Hz, 2H), 2.67 (t, J = 5.3 Hz, 2H), 2.61 (t, J = 7.2 Hz, 2H), 2.30
(s, 1H), 1.93–1.84 (m, 4H), 1.69–1.58 (m, 2H), 1.57–1.45 (m, 2H),
1.42–1.32 (m, 4H). 13C NMR (100 MHz, CDCl3) d 158.35, 153.14,
150.69, 147.42, 136.78, 136.21, 128.64, 128.17, 123.50, 122.81,
120.20, 115.83, 104.90, 60.80, 56.07, 54.35, 49.36, 34.00, 31.68,
29.95, 27.10, 26.84, 24.75, 23.02, 22.75. Purity: 99.4% by HPLC,
HRMS (ESI): calcd for (MÀH)À (C29H39N3O3) requires m/z
476.2913, found 476.2919.
4.4.12. N1-(3,4-Dimethoxybenzyl)-N7-(1,2,3,4-tetrahydroacridin-
9-yl)heptane-1,7-diamine (9l)
4.4.8. N1-(1,2,3,4-Tetrahydroacridin-9-yl)-N7-(3,4,5-trimethoxy-
benzyl)heptane-1,7-diamine (9h)
Aldehyde 4c was treated with 8d according to general proce-
dure to give the desired product 9l as light yellow oil (65% yield).
1H NMR (400 MHz, CDCl3) d 7.93 (dd, J = 14.0, 8.5 Hz, 2H), 7.52 (t,
J = 7.6 Hz, 1H), 7.34–7.30 (m, 1H), 6.91 (s, 1H), 6.84 (d, J = 8.2 Hz,
1H), 6.79 (d, J = 8.1 Hz, 1H), 3.87 (s, 3H), 3.83 (s, 3H), 3.73 (s, 2H),
3.46 (t, J = 7.2 Hz, 2H), 3.05 (s, 2H), 2.67 (s, 2H), 2.61 (t, J = 7.3 Hz,
2H), 1.99 (s, 1H), 1.94–1.82 (m, 4H), 1.69–1.58 (m, 2H),
1.56–1.45 (m, 2H), 1.41–1.25 (m, 6H). 13C NMR (100 MHz, CDCl3)
d 158.11, 150.92, 148.97, 148.18, 147.06, 131.80, 128.34, 128.27,
123.55, 122.85, 120.52, 120.03, 115.59, 111.67, 111.06, 55.87,
55.83, 53.28, 49.34, 48.79, 33.66, 31.62, 29.32, 29.16, 27.13,
26.79, 24.71, 22.96, 22.65. Purity: 99.9% by HPLC, HRMS (ESI): calcd
for (MÀH)À (C29H39N3O2) requires m/z 460.2964, found 460.2971.
Aldehyde 4b was treated with 8d according to general proce-
dure to give the desired product 9h as light yellow oil (66% yield).
1H NMR (400 MHz, CDCl3) d 7.97 (d, J = 8.5 Hz, 1H), 7.92 (d,
J = 8.5 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.32 (t, J = 7.7 Hz, 1H), 6.61
(s, 2H), 5.06 (s, 1H), 3.84 (s, 6H), 3.82 (s, 3H), 3.77 (s, 2H), 3.50 (t,
J = 7.2 Hz, 2H), 3.04 (s, 2H), 2.65 (t, J = 7.3 Hz, 4H), 1.99 (s, 1H),
1.87 (s, 4H), 1.73–1.59 (m, 2H), 1.55 (s, 2H), 1.34 (d, J = 15.9 Hz,
6H). 13C NMR (100 MHz, CDCl3) d 157.13, 153.17, 151.53, 145.85,
137.17, 133.77, 128.78, 127.08, 123.67, 123.03, 119.42, 114.86,
105.53, 60.72, 56.05, 53.28, 49.09, 48.45, 32.78, 31.47, 29.04,
28.77, 27.00, 26.69, 24.57, 22.78, 22.33. Purity: 99.7% by HPLC,
HRMS (ESI): calcd for (MÀH)À (C30H41N3O3) requires m/z
490.3070, found 490.3074.
4.4.13. N1-(3,4-Dimethoxybenzyl)-N8-(1,2,3,4-tetrahydroacridin-
9-yl)octane-1,8-diamine (9m)
4.4.9. N1-(1,2,3,4-Tetrahydroacridin-9-yl)-N8-(3,4,5-trimethoxy-
benzyl)octane-1,8-diamine (9i)
Aldehyde 4c was treated with 8e according to general proce-
dure to give the desired product 9m as light yellow oil (69% yield).
1H NMR (400 MHz, CDCl3) d 7.94 (d, J = 8.4 Hz, 1H), 7.90 (d,
J = 8.4 Hz, 1H), 7.52 (t, J = 7.3 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 6.88
(d, J = 1.2 Hz, 1H), 6.84–6.76 (m, 2H), 3.87 (s, 3H), 3.83 (s, 3H),
3.71 (s, 2H), 3.45 (t, J = 7.2 Hz, 2H), 3.04 (d, J = 5.7 Hz, 2H), 2.67
(s, 2H), 2.60 (t, J = 7.2 Hz, 2H), 1.95–1.84 (m, 4H), 1.68–1.57 (m,
2H), 1.55–1.44 (m, 2H), 1.36–1.24 (m, 8H). 13C NMR (100 MHz,
CDCl3) d 158.27, 150.79, 148.93, 147.97, 147.34, 132.95, 128.54,
Aldehyde 4b was treated with 8e according to general proce-
dure to give the desired product 9i as light yellow oil (83% yield).
1H NMR (400 MHz, CDCl3) d 7.95 (d, J = 8.5 Hz, 1H), 7.91 (d,
J = 8.4 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.32 (t, J = 7.6 Hz, 1H), 6.56
(s, 2H), 3.85 (s, 6H), 3.83 (s, 3H), 3.71 (s, 2H), 3.45 (d, J = 7.2 Hz,
2H), 3.05 (s, 2H), 2.68 (s, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.94–1.84
(m, 4H), 1.69–1.58 (m, 2H), 1.49 (dd, J = 13.1, 6.4 Hz, 2H),

W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 763–770
769
128.21, 123.50, 122.85, 120.22, 120.15, 115.71, 111.46, 111.03,
55.87, 55.82, 53.77, 49.41, 49.33, 33.91, 31.69, 29.91, 29.38,
29.25, 27.21, 26.82, 24.74, 23.01, 22.73. Purity: 99.0% by HPLC,
HRMS (ESI): calcd for (MÀH)À (C30H41N3O2) requires m/z
474.3121, found 474.3123.
performed geometry optimization by molecular mechanics. Fur-
ther preparation of substrates included addition of Gasteiger
charges, removal of hydrogen atoms and addition of their atomic
charges to skeleton atoms, and finally, assignment of proper atom-
ic types. Autotors was then used to define the rotatable bonds in
the ligand.
4.4.14. N1-(3,4-Dimethoxybenzyl)-N9-(1,2,3,4-tetrahydroacridin-
9-yl)nonane-1,9-diamine (9n)
Docking studies were carried out using the AUTODOCK 4.0 pro-
gram. Using ADT, Polar hydrogen atoms were added to amino acid
residues and Gasteiger charges were assigned to all atoms of 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 electro-
statics map and a separate desolvation map present in the sub-
strate molecule. All maps were calculated 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 [2.781 64.383 67.971];
BuChE [112.0 20.0 40.0]). The dimensions of the active site box
were set at 50 Â 46 Â 46 Å.
Aldehyde 4c was treated with 8e according to general proce-
dure to give the desired product 9m as light yellow oil (70% yield).
1H NMR (400 MHz, CDCl3) d 7.95 (d, J = 7.9 Hz, 1H), 7.90 (d,
J = 7.8 Hz, 1H), 7.52 (t, J = 7.0 Hz, 1H), 7.32 (s, 1H), 6.89 (s, 1H),
6.83 (d, J = 8.1 Hz, 1H), 6.79 (d, J = 8.1 Hz, 1H), 3.87 (s, 3H), 3.84
(s, 3H), 3.71 (s, 2H), 3.45 (t, J = 7.2 Hz, 2H), 3.04 (d, J = 6.0 Hz,
2H), 2.68 (d, J = 5.4 Hz, 2H), 2.64–2.57 (m, 2H), 1.89 (dd, J = 6.3,
3.1 Hz, 4H), 1.68–1.57 (m, 2H), 1.49 (dd, J = 14.0, 7.1 Hz, 2H),
1.38–1.25 (m, 10H). 13C NMR (100 MHz, CDCl3) d 158.29, 150.79,
148.93, 148.00, 147.36, 132.77, 128.55, 128.20, 123.48, 122.84,
120.26, 120.15, 115.69, 111.49, 111.03, 55.87, 55.81, 53.68, 49.42,
49.26, 33.90, 31.70, 29.84, 29.40, 29.38, 29.24, 27.27, 26.85,
24.73, 23.01, 22.73. Purity: 99.5% by HPLC, HRMS (ESI): calcd for
(MÀH)À (C31H43N3O2) requires m/z 488.3277, found 488.3274.
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 each compound and
the obtained orientations were analyzed.
4.5. Biological activity
4.5.1. In vitro inhibition studies on AChE and BuChE
4.5.4. Inhibition of self-mediated Ab(1–42) aggregation
In order to investigate the self-mediated Ab(1–42) aggregation,
a
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel),
butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum),
5,5’-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), acet-
ylthiocholine chloride (ATC), butylthiocholine chloride (BTC), and
tarcine hydrochloride were purchased from Sigma Aldrich. Tacrine
and synthesized derivatives were dissolved in DMSO and then di-
luted in 0.1 M KH2PO4/K2HPO4 buffer (pH 8.0) to provide a final
concentration range.
thioflavin-T fluorescence assay was performed.34 Ab(1–42)
peptide (Anaspec Inc) was dissolved in phosphate buffer (pH
7.40, 0.01 M) to obtain a 20 M solution. Compounds were firstly
prepared in DMSO to obtain a 10 mM solution. The final concentra-
tion of Ab(1–42) and inhibitors were 20 M. After incubated in
37 °C for 48 h, thioflavin-T (5 M in 50 mM glycine–NaOH buffer,
pH 8.00) was added. Fluorescence was measured at 450 nm (kex
l
l
l
)
All the assays were under 0.1 M KH2PO4/K2HPO4 buffer, pH 8.0,
using a Shimadzu UV-2450 Spectrophotometer. Enzyme solutions
were prepared to give 2.0 units/mL in 2 mL aliquots. The assay
and 485 nm (kem). Each inhibitor was run in triplicate. The fluores-
cence intensities were recorded, and the percentage of inhibition
on aggregation was calculated by the following expression:
(1 À IFi/IFc) ⁄ 100% in which IFi and IFc were the fluorescence intensi-
ties obtained for absorbance in the presence and absence of inhibi-
tors, respectively, after subtracting the fluorescence of respective
blanks.
medium contained phosphate buffer, pH 8.0 (1 mL), 50
lL of
0.01 M DTNB, 10 L of enzyme, and 50 L of 0.01 M substrate
l
l
(acetylthiocholine chloride). 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 mea-
suring 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 et al.31 In vitro BuChE assay use the similar
method described above. Each concentration was assayed in
triplicate.
5. Conflict of interest
We declare that we have no conflict of interest.
Acknowledgements
We thank the Natural Science Foundation of China (Grants
U0832005, 90813011, 20772159, 81001400), and the Science
Foundation of Guangzhou (2009A1-E011-6) for financial support
of this study.
4.5.2. Kinetic characterization of AChE inhibition
Kinetic characterization of AChE was performed using a re-
ported method.21 Six different concentrations of substrate were
mixed in the 1 mL 0.1 M KH2PO4/K2HPO4 buffer (pH 8.0), contain-
ing 50 lL of DTNB, 10 lL AChE, and 50 lL substrate. Test com-
Supplementary data
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.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.022.
References and notes
1. Kung, H. F.; Lee, C. W.; Zhuang, Z. P.; Kung, M. P.; Hou, C.; Plössl, K. J. Am. Chem.
Soc. 2001, 123, 12740.
4.5.3. Molecular modelincg
The crystal structure of the torpedo acetylcholinesterase com-
plexed with tacrine (code ID: 1ACJ) and the human butyrylcholin-
esterase complexed with echothiophate (code ID: 1POI) were
obtained in the Protein Data Bank after eliminating the inhibitor
and water molecules. The 3D Structure of 9e was built and
2. Förstl, H.; Kurz, A. Eur. Arch. Psychiatry Clin. Neurosci. 1999, 249, 288.
3. Fernandez-Bachiller, M. I.; Perez, C.; Campillo, N. E.; Paez, J. A.; Gonzalez-
Munoz, G. C.; Usan, P.; Garcia-Palomero, E.; Lopez, M. G.; Villarroya, M.; Garcia,
A. G.; Martinez, A.; Rodriguez-Franco, M. I. ChemMedChem 2009, 4, 828.
4. Yankner, B. A.; Dawes, L. R.; Fisher, S.; Villa-Komaroff, L.; Oster-Granite, M. L.;
Neve, R. L. Science 1989, 245, 417.

770
W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 763–770
5. Masters, C. L.; Simms, G.; Weinman, N. A.; Multhaup, G.; McDonald, B. L.;
Andrisano, V.; Arce, M. P.; Rodriguez-Franco, M. I.; Huertas, O.; Dafni, T.; Luque,
F. J. J. Med. Chem. 2009, 52, 5365.
21. Tang, H.; Ning, F. X.; Wei, Y. B.; Huang, S. L.; Huang, Z. S.; Chan, A. S.; Gu, L. Q.
Bioorg. Med. Chem. Lett. 2007, 17, 3765.
22. Pan, L.; Tan, J. H.; Hou, J. Q.; Huang, S. L.; Gu, L. Q.; Huang, Z. S. Bioorg. Med.
Chem. Lett. 2008, 18, 3790.
23. Wang, B.; Mai, Y. C.; Li, Y.; Hou, J. Q.; Huang, S. L.; Ou, T. M.; Tan, J. H.; An, L. K.;
Li, D.; Gu, L. Q.; Huang, Z. S. Eur. J. Med. Chem. 2010, 45, 1415.
24. Hung, T. M.; Na, M.; Min, B. S.; Ngoc, T. M.; Lee, I.; Zhang, X.; Bae, K. Arch.
Pharm. Res. 2007, 30, 685.
Beyreuther, K. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 4245.
6. Cummings, J. L.; Doody, R.; Clark, C. Neurology 2007, 69, 1622.
7. Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini,
M.; Melchiorre, C. J. Med. Chem. 2008, 51, 347.
8. Rizzo, S.; Riviere, C.; Piazzi, L.; Bisi, A.; Gobbi, S.; Bartolini, M.; Andrisano, V.;
Morroni, F.; Tarozzi, A.; Monti, J. P.; Rampa, A. J. Med. Chem. 2008, 51, 2883.
9. Ibach, B.; Haen, E. Curr. Pharm. Des. 2004, 10, 231.
10. Mesulam, M. M.; Guillozet, A.; Shaw, P.; Levey, A.; Duysen, E. G.; Lockridge, O.
Neuroscience 2002, 110, 627.
11. Yankner, B. A.; Duffy, L. K.; Kirschner, D. A. Science 1990, 250, 279.
12. Lorenzo, A.; Yankner, B. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12243.
13. Thorsett, E. D.; Latimer, L. H. Curr. Opin. Chem. Biol. 2000, 4, 377.
14. Wolfe, M. S.; Citron, M.; Diehl, T. S.; Xia, W.; Donkor, I. O.; Selkoe, D. J. J. Med.
Chem. 1998, 41, 6.
15. Yang, F.; Lim, G. P.; Begum, A. N.; Ubeda, O. J.; Simmons, M. R.; Ambegaokar, S.
S.; Chen, P. P.; Kayed, R.; Glabe, C. G.; Frautschy, S. A.; Cole, G. M. J. Biol. Chem.
2005, 280, 5892.
25. Elsinghorst, P. W.; Tanarro, C. M.; Gutschow, M. J. Med. Chem. 2006, 49, 7540.
26. Ucar, G.; Gokhan, N.; Yesilada, A.; Bilgin, A. A. Neurosci. Lett. 2005, 382,
327.
27. Nadri, H.; Pirali-Hamedani, M.; Shekarchi, M.; Abdollahi, M.; Sheibani, V.;
Amanlou, M.; Shafiee, A.; Foroumadi, A. Bioorg. Med. Chem. 2010, 18, 6360.
28. Tomita, M.; Aoyagi, Y. Chem. Pharm. Bull. 1968, 16, 523.
29. Carlier, P. R.; Han, Y. F.; Chow, E. S.; Li, C. P.; Wang, H.; Lieu, T. X.; Wong, H. S.;
Pang, Y. P. Bioorg. Med. Chem. 1999, 7, 351.
16. Lim, G. P.; Chu, T.; Yang, F.; Beech, W.; Frautschy, S. A.; Cole, G. M. J. Neurosci.
2001, 21, 8370.
30. Zhou, J. J.; Huang, H.; Xie, S. Q.; Wang, Y. X.; Zhao, J.; Wang, C. J. Chin. Chem. Lett.
2008, 19, 99.
17. Howlett, D. R.; Perry, A. E.; Godfrey, F.; Swatton, J. E.; Jennings, K. H.;
Spitzfaden, C.; Wadsworth, H.; Wood, S. J.; Markwell, R. E. Biochem. J. 1999, 340,
283.
18. Ono, M.; Kung, M.-P.; Hou, C.; Kung, H. F. Nucl. Med. Biol. 2002, 29, 633.
19. Rosini, M.; Simoni, E.; Bartolini, M.; Cavalli, A.; Ceccarini, L.; Pascu, N.;
McClymont, D. W.; Tarozzi, A.; Bolognesi, M. L.; Minarini, A.; Tumiatti, V.;
Andrisano, V.; Mellor, I. R.; Melchiorre, C. J. Med. Chem. 2008, 51, 4381.
20. Camps, P.; Formosa, X.; Galdeano, C.; Munoz-Torrero, D.; Ramirez, L.; Gomez,
E.; Isambert, N.; Lavilla, R.; Badia, A.; Clos, M. V.; Bartolini, M.; Mancini, F.;
31. Ellman, G. L.; Courtney, K. D., Jr.; Andres, B.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
32. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
33. DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: San
Carlos, CA, 2002.
34. Bartolini, M.; Bertucci, C.; Bolognesi, M. L.; Cavalli, A.; Melchiorre, C.;
Andrisano, V. Chembiochem 2007, 8, 2152.
35. Schneiders, G. E.; Stevenson, R. J. Org. Chem. 1981, 46, 2969.