Synthesis and evaluation of heterobivalent tacrine derivatives as potential multi-functional anti-Alzheimer agents

Article abstract of DOI:10.1016/j.ejmech.2011.03.058

A new series of heterobivalent tacrine derivatives were designed, synthesized and evaluated as potential multi-functional anti-Alzheimer agents for their inhibitory activity on cholinesterases, antioxidant activity and self-induced β-amyloid (Aβ) aggregat

Full text of DOI:10.1016/j.ejmech.2011.03.058

European Journal of Medicinal Chemistry 46 (2011) 2609e2616
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of heterobivalent tacrine derivatives as potential
multi-functional anti-Alzheimer agents
*
Wen Luo, Yan-Ping Li, Yan He, Shi-Liang Huang, 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 heterobivalent tacrine derivatives were designed, synthesized and evaluated as potential
multi-functional anti-Alzheimer agents for their inhibitory activity on cholinesterases, antioxidant
Received 12 December 2010
Received in revised form
26 February 2011
Accepted 25 March 2011
Available online 3 April 2011
activity and self-induced
b-amyloid (Ab) aggregation. All these synthesized compounds had potent
inhibition activity on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) at nanomolar range.
A LineweavereBurk plot and molecular modeling study showed that these compounds targeted both the
catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. The compounds containing
hydroxyl group showed potent peroxyl radical absorbance activity. In addition, compound 5j exhibited
Keywords:
Tacrine
Alzheimer’s disease
Cholinesterase
Antioxidant
higher self-induced A
functional agent for further development for the treatment of AD.
Ó 2011 Elsevier Masson SAS. All rights reserved.
b aggregation inhibitory activity than curcumin, which could become a multi-
b
-Amyloid
1. Introduction
the aggregation of Ab and s-protein hyperphosphorylation. Many
evidences support that antioxidants could be able to attenuate the
syndrome of AD, and prevent the progression of the disease [7].
Thus, drugs that specifically scavenge oxygen radicals could be
useful for either the prevention or the treatment of AD [8].
Alzheimer’s disease (AD) is a progressive neurodegenerative
brain disorder that is characterized by dementia, cognitive
impairment, and memory loss [1]. It affects millions of elder
peoples, and the number of patients is expected to increase in the
next 20 years. Many factors have been found to be implicated in AD,
and its etiology and pathogenesis remain unclear. There are diverse
hallmarks such as low levels of acetylcholine, oxidative damage and
The progressive deposition of A
fundamental to the development of neurodegenerative pathology
[9]. There are two principal forms of A , and A 42 and A 40 are the
main components of the plaques in the brain tissue of AD patients
[10]. However, compared to relatively soluble A 40, A 42 is more
prone to aggregation and more neurotoxic. Therefore, the preven-
tion of A 42 formation or aggregation is currently another potential
b is generally considered to be
b
b
b
b
-amyloid (Ab) deposits, which seem to play significant roles in the
b
b
disease [2]. Current treatment of AD focuses on increasing cholin-
ergic neurotransmission in the brain by inhibiting cholinesterases
(ChEs) with medicines including donepezil, rivastigmine and gal-
antamine (Fig. 1) [3]. But clinical experience shows that inhibition
of ChEs is a palliative treatment, which does not address AD’s
etiology [4]. Due to the multi-pathogenesis of AD, one of the
current strategies is to develop novel anti-AD agents with multiple
potencies [5].
In recent years, more and more attention is paid to oxidative
stress for its role in the progression of AD [6]. An increasing
research has suggested that oxidative stress is involved in the early
stage of the pathogenesis and plays an important factor to initiate
b
method for treatment of AD [11,12], and some related inhibitors
including curcumin [13,14] and benzofuran analogs [15,16] have
been developed.
Tacrine (Fig. 1) is the first approved ChEs inhibitor by the FDA for
the treatment of AD. This medicine has some side effects, therefore,
the search for tacrine derivatives is still of interest. In recent years,
many studies have focused on the combined effects of ChEs inhi-
bition and scavenging oxygen radicals as well as reduction of the A
b
fibril self-aggregation by conjugating tacrine with other active
groups. Based on this strategy, many tacrine derivatives [17e20]
(AeD in Fig. 1) have been designed and synthesized.
Our research group has been involved in the development of
potential drugs for the treatment of AD for many years [21e23]. In
the present study, we are interested in the development of tacrine
derivatives as multi-functional agents that could possibly bind with
* Corresponding author. Tel.: þ8620 39949056; fax: þ8620 39943056.
E-mail address: ceshzs@mail.sysu.edu.cn (Z.-S. Huang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.058

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W. Luo et al. / European Journal of Medicinal Chemistry 46 (2011) 2609e2616
both the peripheral and catalytic site of acetylcholinesterase, and
also have antioxidation or anti-A aggregation activity. The tacrine
and many other reports [17,27] on bivalent inhibitors connecting
1,2,3,4-tetrahydroacridine moieties with an alkyl linker, a hepty-
lene or octylene has been found to be the optimal spacer, therefore,
compounds 5ie5p with octylene linker with various substituted
aromatic groups were designed. It has been well-known that
compounds containing pyridine group such as nicotine exhibited
b
derivatives were designed because of the following reasons. Firstly,
tacrine is a well-known ChEs inhibitor for the treatment of AD.
Next, it has been reported that compounds containing electron-rich
aromatic groups could possibly bind to the peripheral binding site
of acetylcholinesterase (E in Fig. 1) [24]. Besides, docking studies
have indicated that hydroxyl group on the benzene ring could form
hydrogen bonds with residues in the binding site of ChE [25]. In
addition, curcumin has a hydroxyl group on its benzene ring, which
has potent antioxidation activity. Other compounds with similar
structures might also have antioxidation activity. Here we report
our results on the synthesis and biological evaluation of a series of
new tacrine derivatives combined with aromatic groups as poten-
tial multi-functional agents for the treatment of AD.
anti-Ab aggregation and activity of scavenging free radicals [28,29],
therefore, compounds 5q and 5r were also synthesized.
Intermediate compound 3 was synthesized from anthranilic
acid in 90% overall yield with a previously reported procedure [30].
Then, reaction of compound 3 with alkylamine under reflux for 18 h
in 1-pentanol gave 9-alkylamino-tetra-hydroacridines 4ae4e in
good yields (60e70%). In our experiment, this step was modified
and carried out in the presence of catalytic amount of KI, and the
reaction time was shortened obviously with slightly increased
reaction yield. In the next step, the target compounds 5ae5q were
obtained by the reaction of aldehyde with intermediate 4 followed
with reduction using NaBH4 in methanol [31]. Compound 5r was
prepared in a solution of pyridine-4-carboxylic acid, N,N0-diiso-
propylcarbodiimide (DIC) and 4-dimethylaminopyridine (DMAP) in
dichloromethane at room temperature (Scheme 1).
2. Chemistry
Tacrine derivatives 5ae5r were prepared with a sequence of
reactions as shown in Scheme 1. Compounds 5ce5f and 5p were
reported in our previous work [26]. In our preliminary results [26]
Fig. 1. Chemical structures of donepezil, rivastigmine, galantamine, tacrine, and tacrine derivatives.

W. Luo et al. / European Journal of Medicinal Chemistry 46 (2011) 2609e2616
2611
Scheme 1. Synthetic pathways for tacrine derivatives 5ae5r. Reagents and conditions: (a) cyclohexanone, toluene, reflux; (b) POCl3, 120 ꢀC; (c) diamine, KI, 1-pentanol, 160 ꢀC; (d)
for 5ae5q: 1. ArCHO, methanol, rt, 2. NaBH4, methanol, rt, (e) for 5r: pyridine-4-carboxylic acid, DIC, DMAP, CH2Cl2, rt.
3. Results and discussion
Generally, most of these synthetic compounds showed good inhi-
bition selectivity for BuChE over AChE. Recently BuChE has been
considered as a potential drug target because it also regulates
acetylcholine levels, and concurrent inhibition of both ChEs has
been shown to provide additional benefits in the treatment of
Alzheimer’s disease [24].
Comparing compounds with the same dimethoxy group in
aromatic ring (5ae5f), compound 5e with a 8-methylene linker
showed the best inhibition for AChE. The inhibition activity of
3.1. Inhibition studies on AChE and BuChE
The inhibitory activity of our synthetic derivatives was evalu-
ated against AChE and BuChE with tacrine as a positive control
using the method of Ellman et al. [32]. The IC50 values and selec-
tivity index for the inhibition of AChE and BuChE were summarized
as shown in Table 1 (data of 5ce5f and 5p were taken from ref [26]).
Table 1
Inhibition of ChEs by target compounds, and their selectivity index and oxygen radical absorbance capacity (ORAC).
Selectivity indexc
Trolox [equiv]d
a
Compd
n
X
Y
R group
IC50 for AChE (nM)
IC50b for BuChE (nM)
5a
5b
4
5
6
7
8
9
6
6
8
8
8
8
8
8
8
8
8
8
e
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
O
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
e
R1 ¼ R4 ¼ H, R2 ¼ R3 ¼ OMe
R1 ¼ R4 ¼ H, R2 ¼ R3 ¼ OMe
R1 ¼ R4 ¼ H, R2 ¼ R3 ¼ OMe
R1 ¼ R4 ¼ H, R2 ¼ R3 ¼ OMe
R1 ¼ R4 ¼ H, R2 ¼ R3 ¼ OMe
R1 ¼ R4 ¼ H, R2 ¼ R3 ¼ OMe
R1 ¼ R2 ¼ R4 ¼ H, R3 ¼ OH
R1 ¼ R4 ¼ H, R2 ¼ OMe R3 ¼ OH
R1 ¼ R2 ¼ R4 ¼ H, R3 ¼ OH
R1 ¼ R4 ¼ H, R2 ¼ OMe, R3 ¼ OH
R1 ¼ H, R2 ¼ R4 ¼ OMe, R3 ¼ OH
R1 ¼ R3 ¼ R4 ¼ H, R2 ¼ OH
R1 ¼ OH, R2 ¼ R3 ¼ R4 ¼ H
R1 ¼ R2 ¼ R4 ¼ H, R3 ¼ OMe
R1 ¼ R4 ¼ H, R2þR3 ¼ OCH2O
R1 ¼ H, R2þR3 ¼ OCH2O, R4 ¼ OMe
e
365.2 ꢁ 7.8
104.4 ꢁ 0.2
30.9 ꢁ 2.7
24.0 ꢁ 0.3
11.35 ꢁ 0.19
25.1 ꢁ 3.0
25.6 ꢁ 0.9
43.6 ꢁ 1.2
7.48 ꢁ 0.39
4.55 ꢁ 0.13
4.61 ꢁ 0.21
15.1 ꢁ 0.2
12.5 ꢁ 0.4
15.3 ꢁ 0.2
6.14 ꢁ 1.04
7.98 ꢁ 0.12
4.70 ꢁ 0.30
38.5 ꢁ 2.4
193 ꢁ 2
37.2 ꢁ 0.6
24.4 ꢁ 0.8
2.68 ꢁ 0.17
5.51 ꢁ 0.10
5.57 ꢁ 0.83
5.67 ꢁ 0.32
7.50 ꢁ 0.02
9.28 ꢁ 0.24
5.31 ꢁ 0.13
3.41 ꢁ 0.17
4.08 ꢁ 0.24
4.91 ꢁ 0.30
10.2 ꢁ 0.3
4.82 ꢁ 0.41
3.44 ꢁ 0.02
7.94 ꢁ 0.02
4.66 ꢁ 0.44
38.7 ꢁ 2.7
27.1 ꢁ 1.2
9.8
4.3
11.5
4.4
2.0
4.4
3.4
4.7
1.4
1.3
1.1
3.1
1.2
3.2
1.8
1.0
1.0
1.0
7.1
0.20 ꢁ 0.02
0.31 ꢁ 0.03
0.42 ꢁ 0.01
0.13 ꢁ 0.02
0.11 ꢁ 0.01
0.22 ꢁ 0.03
2.7 ꢁ 0.2
5ce
5de
5ee
5fe
5g
5h
5i
5j
5k
5l
5m
5n
2.5 ꢁ 0.2
2.6 ꢁ 0.2
1.9 ꢁ 0.1
1.2 ꢁ 0.1
2.1 ꢁ 0.1
2.4 ꢁ 0.1
0.32 ꢁ 0.02
0.12 ꢁ 0.01
0.21 ꢁ 0.03
0.32 ꢁ 0.03
0.42 ꢁ 0.01
<0.01
5o
5pe
5q
5r
Tacrine
e
e
e
a
AChE from electric eel; IC50, inhibitor concentration (means ꢁ SEM of three experiments) for 50% inhibition of AChE.
BuChE from equine serum; IC50, inhibitor concentration (means ꢁ SEM of three experiments) for 50% inhibition of BuChE.
Selectivity Index ¼ IC50 (AChE)/IC50 (BuChE).
b
c
d
e
Data are expressed as (
mmol trolox)/(mmol tested compound).
Data of ChEs inhibition were taken from ref [26].

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W. Luo et al. / European Journal of Medicinal Chemistry 46 (2011) 2609e2616
methylene on the pyridine of compound 5q was replaced with an
non inhibitor
30
25
20
15
10
5
[5j] = 2 nM
[5j] = 4 nM
[5j] = 8 nM
amide group in compound 5r, and the resulting inhibition activity
of compound 5r for both ChEs was decreased for 10 times lower
than that of compound 5q.
3.2. Kinetic studies for the inhibition of AChE
The nature of AChE inhibition by the best inhibitor compound 5j
was studied through the graphical analysis of steady state inhibi-
tion data, as shown in Fig. 2 [23]. The LineweavereBurk plots
showed both increasing slopes and increasing intercepts at higher
inhibitor concentration. This pattern indicated a mixed-type inhi-
bition, which was similar to that of tacrine. This result showed that
compound 5j could bind to both CAS and PAS of AChE, which was
consistent with the results of molecular modeling studies.
0
-10
-5
0
5
10
15
20
-1
[S] mM
-1
3.3. Studies of antioxidation activity
Fig. 2. LineweavereBurk plot for the inhibition of acetylcholinesterase by compound 5j.
The peroxyl radicals reduced by the target compounds were
determined by using the ORAC (oxygen radical absorbance capacity)
assay [19], and the result was shown in Table 1. The activity of the
compounds to scavenge radicals was expressed as Trolox equiva-
lent, and their relative activity at concentrations between 1 and
compounds 5i and 5j for AChE was better than that of compounds
5g and 5h. Their inhibitory activity for BuChE was not significantly
affected by their chain length, which may be due to the confor-
mational flexibility of the enzyme, as reported previously [3,22].
With the same chain length (5ie5p), all of these compounds
exhibited more potent inhibition activity on both AChE and BuChE
than tacrine. Their IC50 values for AChE were less than 16 nM, and
their IC50 values for BuChE were less than 10 nM. Compound 5j was
found to have the best inhibitor for both ChEs with its IC50 values of
4.55 nM and 3.41 nM respectively, while the IC50 values of
compounds 5i and 5k were very close to those of compound 5j. It
seems that the hydroxyl group at the 40 position of the benzene ring
was beneficial for their activity. This was in agreement with our
results from molecular modeling studies, which indicated that this
hydroxyl group formed hydrogen bond with the residue in the
binding site of ChEs.
5
mM was compared with that of the highly potent compound
Trolox. Our result indicated that tacrine exhibited insignificant
radical-capture capacity, while the compounds containing hydroxyl
group showed potent peroxyl radical absorbance capacities ranging
from 1.2- to 2.7-fold of the trolox value. After connecting the
hydroxyl moiety to the tacrine, the resulting compounds showed
obvious antioxidation activity. However, compounds 5q and 5r
containing pyridine group showed relatively lower activity.
3.4. Molecular modeling
In order to understand the binding interactions between our
synthetic compounds and TcAChE (PDB code: 1ACJ), molecular
modeling study was carried out using docking program AUTODOCK
Compound 5q containing a pyridine group with a 8-methylene
linker also showed very potent activity for both ChEs. The
Fig. 3. Docking models of enzymeecompound complex. Compound 5j interacts 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 were generated with PyMOL. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

W. Luo et al. / European Journal of Medicinal Chemistry 46 (2011) 2609e2616
2613
inhibitory potency and good selectivity for BuChE over AChE.
Compound 5j exhibited the highest inhibitory effect for both ChEs.
The inhibition kinetic analysis and molecular modeling study
indicated that compound 5j had binding with both CAS and PAS of
the AChE. Besides, the compounds containing hydroxyl substitutive
group had significant peroxyl radical absorbance capacity. In
80
70
60
50
40
30
20
10
0
addition, compound 5j exhibited higher self-induced Ab aggrega-
tion inhibitory activity than curcumin. Our present results indicate
that this new type of multi-functional compounds may provide
a useful template for the development of new anti-AD agents with
multiple potencies.
5. Experimental section
5.1. Chemistry
curcumin
5a
5c
5j
5p
1H and 13C NMR spectra were recorded using TMS (tetramethyl
silicane) as the internal standard in CDCl3 with a Bruker BioSpin
GmbH spectrometer at 400 MHz and 100 MHz, respectively. High
resolution mass spectra (HRMS) were recorded on Shimadzu LCMS-
IT-TOF. Flash column chromatography was performed with silica
gel (200e300 mesh) purchased from Qingdao Haiyang Chemical
Co. Ltd. The purities of synthesized compounds were confirmed to
be higher than 95% by using analytical HPLC with a dual pump
Shimadzu LC-20AB system equipped with an Ultimate XB-C18
Compounds
Fig. 4. Inhibition of self-mediated A
5p comparing with that of curcumin. The measurements were carried out in the
presence of 20 compounds. The mean SD values from three independent
b(1e42) aggregation by compounds 5a, 5c, 5j, and
mM
ꢁ
experiments were shown. Data of 5p were taken from ref [26].
4.0 package with PyMOL program [33,34], and the result is shown
in Fig. 3. Our docking studies indicated that all of these compounds
exhibited multiple binding modes with AChE. In the complex of
TcAChE-5j, compound 5j occupied the entire enzymatic CAS, mid-
gorge and PAS. The tacrine moiety was bound to CAS with
column (4.6 ꢂ 250 mm, 5
mm) and eluted with methanol/water
(35:65 to 45:55) containing 0.1% TFA (trifluoroacetic acid) at a flow
rate of 0.5 mL/min.
a classic
pep stacking interaction between Trp84 and Phe330.
5.1.1. Synthesis of intermediate 4
Intermediate 4 was prepared according to the previous litera-
ture procedure in our lab [26].
Their ring-to-ring distance was between 3.4 Å and 3.8 Å, and the
protonated nitrogen atom of the quinoline ring had a hydrogen
bond (3.4 Å) interaction with the carbonyl group of His440. In the
PAS, a hydrogen bond was observed between the hydroxyl group
and Arg289 with a distance of 3.3 Å. These results may explain why
compound 5j had potent inhibition for AChE. The docking studies
showed that our compounds had a mixed type of inhibition, which
was consistent with our kinetic analysis result. Because the crystal
structure of BuChE from equine serum had not been reported, and
the sequences of BuChE from equine showed high homology with
that of human BuChE, so, the crystal structure of HuBuChE was used
in the docking study. The similar interactions were also observed in
5.1.2. General procedures for the preparation of compounds 5ae5q
Aromatic aldehydes (1 mmol) and intermediate 4 (1 mmol)
were stirred in MeOH for 4 h, and then were reduced through
reaction with NaBH4 (4 mmol) at room temperature for 4 h. The
solvent was evaporated, and the residue was poured into water and
extracted with ethyl acetate. The combined organic layers were
dried over MgSO4 and then concentrated. The resulting crude
product was purified by flash chromatography with chloroform/
methanol/ammonia as elution solvent. 5ce5f and 5p were
synthesized in previous literature in our lab [26].
the complex of HuBuChE-5j (enzyme PDB code: 1POI). A
pep
stacking interaction was found between tacrine and Trp82, and
a hydrogen bond was observed between the hydroxyl group and
Pro285 with a distance of 3.4 Å.
5.1.2.1. N1-(3,4-Dimethoxybenzyl)-N4-(1,2,3,4-tetrahydroacridin-9-yl)
butane-1,4-diamine (5a). Intermediate 4a was reacted with 3,
4-dimethoxybenzaldehyde following the general procedure to give
the desired product 5a as a yellow oil with a yield of 84%. 1H NMR
3.5. Inhibition of self-mediated Ab(1e42) aggregation
(400 MHz, CDCl3)
d
7.95 (d, J ¼ 7.8 Hz,1H), 7.90 (d, J ¼ 7.8 Hz,1H), 7.54
We further evaluated the activity of some selected compounds
to inhibit the self-mediated A (1e42) aggregation using thioflavin
T (ThT) assay [35]. Compared with the reference compound cur-
cumin, the results showed that compounds 5j apparently pre-
vented the self-mediated Ab aggregation with inhibition ratio of
71% (Fig. 4), which were higher than that of curcumin (50%). The
activity of compounds 5a and 5c was lower than that of curcumin.
The most potent compound was 5j. It has been hypothesized that
organic compounds with antioxidation activity could bind specifi-
(t, J ¼ 7.0 Hz, 1H), 7.33 (t, J ¼ 7.1 Hz, 1H), 6.88e6.77 (m, 3H), 4.11 (s,
1H), 3.86 (s, 6H), 3.72 (s, 2H), 3.50 (s, 2H), 3.06 (t, J ¼ 5.8 Hz, 2H),
2.73e2.63 (m, 4H), 1.96e1.86 (m, 4H), 1.76e1.68 (m, 2H), 1.66e1.57
b
(m, 2H). 13C NMR (100 MHz, CDCl3)
d 158.45, 150.71, 148.99, 148.06,
147.43, 132.97, 128.74, 128.29, 123.63, 122.78, 120.25, 120.17, 115.96,
111.37, 111.05, 55.88, 53.86, 49.38, 48.96, 34.04, 29.57, 27.58, 24.90,
23.07, 22.79. Purity: 98.9% by HPLC. HRMS (ESI): calcd for (M ꢃ H)ꢃ
(C26H36N2O6) 418.2495, found 418.2497.
cally to A
b or Ab fibril, but the mechanisms remain unknown [36].
5.1.2.2. N1-(3,4-Dimethoxybenzyl)-N5-(1,2,3,4-tetrahydroacridin-9-yl)
pentane-1,5-diamine (5b). Intermediate 4b was reacted with 3,
4-dimethoxybenzaldehyde following the general procedure to give
the desired product 5b as a yellow oil with a yield of 55%. 1H NMR
4. Conclusion
In summary, thirteen new heterobivalent tacrine derivatives
(5a, 5b, 5ge5o, 5q, 5r) were synthesized and characterized. Our
results showed that these synthetic compounds had high ChEs
(400 MHz, CDCl3)
7.57e7.49 (m, 1H), 7.37e7.29 (m, 1H), 6.87 (s, 1H), 6.84e6.75 (m, 2H),
3.87 (s, 3H), 3.85 (s, 3H), 3.71 (s, 2H), 3.47 (t, J ¼ 7.2 Hz, 2H), 3.04 (d,
d
7.94 (d, J ¼ 8.4 Hz, 1H), 7.90 (d, J ¼ 8.4 Hz, 1H),

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W. Luo et al. / European Journal of Medicinal Chemistry 46 (2011) 2609e2616
J ¼ 6.1 Hz, 2H), 2.70 (d, J ¼ 5.9 Hz, 2H), 2.62 (t, J ¼ 7.1 Hz, 2H), 2.25 (s,
1H), 1.95e1.85 (m, 4H), 1.72e1.60 (m, 2H), 1.54 (dt, J ¼ 14.1, 6.9 Hz,
29.38, 29.27, 27.24, 26.84, 24.76, 23.02, 22.71. Purity: 99.2% by HPLC.
HRMS (ESI): calcd for (MeH)ꢃ (C26H36N2O6) 460.2964, found
460.2950.
2H), 1.48e1.38 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 158.43, 150.70,
148.98, 148.05, 147.43, 132.97, 128.68, 128.24, 123.59, 122.77, 120.23,
120.20, 115.92, 111.46, 111.09, 55.92, 55.86, 53.82, 49.36, 49.15, 33.98,
31.64, 29.77, 24.80, 24.71, 23.03, 22.76. Purity: 98.5% by HPLC. HRMS
(ESI): calcd for (M ꢃ H)ꢃ (C26H36N2O6) 432.2651, found 432.2639.
5.1.2.7. 2,6-Dimethoxy-4-((8-(1,2,3,4-tetrahydroacridin-9-ylamino)
octylamino)methyl) phenol (5k). Intermediate 4e was reacted
with 4-hydroxy-3,5-dimethoxybenzaldehyde following the
general procedure to give the desired product 5k as a yellow oil
5.1.2.3. 4e((6-(1,2,3,4-Tetrahydroacridin-9-ylamino)hexylamino)me-
thyl)phenol (5g). Intermediate 4c was reacted with 4-hydroxy
-benzaldehyde following the general procedure to give the desired
product 5g as a yellow oil with a yield of 33%. 1H NMR (400 MHz,
with a yield of 47%. 1H NMR (400 MHz, CDCl3)
d
7.93 (dd, J ¼ 16.5,
8.4 Hz, 2H), 7.54 (t, J ¼ 7.6 Hz, 1H), 7.33 (t, J ¼ 7.5 Hz, 1H), 6.54 (s,
2H), 3.84 (s, 6H), 3.70 (s, 2H), 3.47 (t, J ¼ 7.2 Hz, 2H), 3.06 (s, 2H),
2.70 (s, 2H), 2.62 (t, J ¼ 7.3 Hz, 2H), 1.91 (t, J ¼ 3.2 Hz, 4H), 1.64 (dt,
J ¼ 14.6, 7.3 Hz, 2H), 1.50 (dd, J ¼ 13.6, 6.8 Hz, 2H), 1.39e1.24 (m,
CDCl3)
d
7.93 (t, J ¼ 9.1 Hz, 2H), 7.49 (t, J ¼ 7.3 Hz, 1H), 7.30 (t,
J ¼ 7.6 Hz, 1H), 7.07 (d, J ¼ 8.4 Hz, 2H), 6.77 (d, J ¼ 8.4 Hz, 2H), 5.98
(s, 1H), 4.09 (s, 1H), 3.66 (s, 2H), 3.47 (t, J ¼ 7.2 Hz, 2H), 3.04 (s, 2H),
2.72e2.51 (m, 4H), 1.86 (d, J ¼ 2.8 Hz, 4H), 1.69e1.56 (m, 2H),
1.57e1.45 (m, 2H), 1.32 (dd, J ¼ 6.3, 3.2 Hz, 4H). 13C NMR (100 MHz,
8H). 13C NMR (100 MHz, CDCl3)
d 158.37, 150.80, 147.40, 147.21,
133.86, 131.24, 128.68, 128.26, 123.56, 122.83, 120.20, 115.81,
104.89, 56.23, 54.37, 49.50, 49.45, 33.97, 31.75, 29.93, 29.43, 29.30,
27.28, 26.88, 24.78, 23.06, 22.78. Purity: 95.0% by HPLC. HRMS
(ESI): calcd for (MeH)ꢃ (C26H36N2O6) 490.3070, found 490.3046.
CDCl3)
d 157.98, 157.20, 151.33, 146.64, 129.66, 129.26, 128.70,
127.66, 123.70, 123.01, 119.84, 115.89, 115.30, 53.21, 49.28, 48.72,
33.11, 31.62, 29.30, 26.98, 26.71, 24.71, 22.91, 22.54. Purity: 99.4% by
HPLC. HRMS (ESI): calcd for (M ꢃ H)ꢃ (C26H36N2O6) 402.2545,
found 402.2529.
5.1.2.8. 3-((8-(1,2,3,4-Tetrahydroacridin-9-ylamino)octylamino)me-
thyl)phenol (5l). Intermediate 4e was reacted with 3-hydroxy
-benzaldehyde following the general procedure to give the
desired product 5l as a yellow oil with a yield of 44%. 1H NMR
5.1.2.4. 2-Methoxy-4-((6-(1,2,3,4-tetrahydroacridin-9-ylamino)hexyl
-amino)methyl)phenol (5h). Intermediate 4c was reacted with
4-hydroxy-3-methoxybenzaldehyde following the general proce-
dure to give the desired product 5h as a yellow oil with a yield of
(400 MHz, CDCl3)
d
7.84 (dd, J ¼ 11.3, 8.8, 2H), 7.38 (t, J ¼ 7.5, 1H),
7.20 (t, J ¼ 7.6, 1H), 6.99 (t, J ¼ 7.8, 1H), 6.70 (s, 1H), 6.63 (t, J ¼ 7.5,
2H), 5.48 (s, 1H), 3.97 (s, 1H), 3.56 (d, J ¼ 6.7, 2H), 3.38 (dd, J ¼ 12.0,
6.4, 2H), 2.95 (s, 2H), 2.55 (s, 2H), 2.46 (t, J ¼ 7.3, 2H), 1.75 (s, 4H),
1.59e1.43 (m, 2H), 1.41e1.28 (m, 2H), 1.26e1.09 (m, 8H). 13C NMR
42%. 1H NMR (400 MHz, CDCl3)
d
7.93 (t, J ¼ 7.6 Hz, 2H), 7.51 (t,
J ¼ 7.6 Hz, 1H), 7.31 (t, J ¼ 7.8 Hz, 1H), 6.83 (s, 1H), 6.79 (d,
J ¼ 8.0 Hz, 1H), 6.70 (dd, J ¼ 8.0, 1.2 Hz, 1H), 4.94 (s, 1H), 3.76 (s,
3H), 3.68 (s, 2H), 3.46 (t, J ¼ 7.2 Hz, 2H), 3.05 (s, 2H), 2.63 (dd,
J ¼ 17.2, 9.9 Hz, 4H), 1.88 (s, 4H), 1.69e1.58 (m, 2H), 1.57e1.47 (m,
(100 MHz, CDCl3) d 158.11, 158.08, 151.31, 146.83, 141.15, 129.49,
128.60, 127.79, 123.62, 123.05, 119.90, 118.90, 115.75, 115.28,
114.69, 53.71, 49.37, 49.02, 33.21, 31.74, 29.61, 29.33, 29.19, 27.14,
26.77, 24.72, 22.94, 22.59. Purity: 98.5% by HPLC. HRMS (ESI):
calcd for (MeH)ꢃ (C26H36N2O6) 430.2858, found 430.2845.
2H), 1.35 (d, J ¼ 3.1 Hz, 4H). 13C NMR (100 MHz, CDCl3)
d 158.23,
150.97, 147.44, 147.08, 145.66, 130.84, 128.40, 128.30, 123.61,
122.86, 121.00, 120.07, 115.65, 114.87, 111.45, 55.72, 53.77, 49.35,
48.99, 33.58, 31.66, 29.54, 27.06, 26.79, 24.76, 23.00, 22.68. Purity:
97.3% by HPLC. HRMS (ESI): calcd for (MeH)ꢃ (C26H36N2O6)
432.2651, found 432.2633.
5.1.2.9. 2-((8-(1,2,3,4-Tetrahydroacridin-9-ylamino)octylamino)met-
hyl)phenol (5m). Intermediate 4e was reacted with 2-hydroxy
-benzaldehyde following the general procedure to give the desired
product 5m as a yellow oil with a yield of 49%. 1H NMR (400 MHz,
CDCl3)
d
8.01e7.89 (m, 2H), 7.53 (t, J ¼ 7.5 Hz, 1H), 7.32 (t,
5.1.2.5. 4-((8-(1,2,3,4-Tetrahydroacridin-9-ylamino)octylamino)methyl)
phenol (5i). Intermediate 4e was reacted with 4-hydroxybenzal
-dehyde following the general procedure to give the desired product
J ¼ 7.6 Hz, 1H), 7.13 (t, J ¼ 7.5 Hz, 1H), 6.95 (d, J ¼ 7.2 Hz, 1H), 6.81
(d, J ¼ 7.9 Hz, 1H), 6.75 (t, J ¼ 7.2 Hz, 1H), 5.58 (s, 1H), 4.19 (s, 1H),
3.94 (s, 2H), 3.50 (t, J ¼ 7.1 Hz, 2H), 3.06 (s, 2H), 2.63 (dd, J ¼ 17.6,
10.5 Hz, 4H), 1.88 (s, 4H), 1.73e1.58 (m, 2H), 1.47 (d, J ¼ 6.3 Hz, 2H),
5i as a yellow oil with a yield of 83%. 1H NMR (400 MHz, CDCl3)
d 7.94
(dd, J ¼ 13.4, 8.4 Hz, 2H), 7.49 (t, J ¼ 7.2 Hz, 1H), 7.34e7.28 (m, 1H), 7.08
(d, J ¼ 8.4 Hz, 2H), 6.80 (d, J ¼ 8.4 Hz, 2H), 5.33 (s,1H), 4.10 (s,1H), 3.48
(s, 2H), 3.05 (s, 2H), 2.65 (s, 2H), 2.62e2.51 (m, 2H), 1.86 (s, 4H),
1.70e1.55 (m, 2H), 1.48 (s, 2H), 1.24 (dt, J ¼ 14.1, 6.8 Hz, 11H). 13C NMR
1.42e1.22 (m, 8H). 13C NMR (100 MHz, CDCl3)
d 158.31, 157.72,
151.26, 146.63, 128.63, 128.56, 128.26, 127.82, 123.68, 123.06,
122.66, 119.80, 118.88, 116.25, 115.33, 52.65, 49.35, 48.64, 33.44,
31.65, 29.48, 29.25, 29.19, 26.97, 26.79, 24.69, 22.94, 22.58. Purity:
97.0% by HPLC. HRMS (ESI): calcd for (MeH)ꢃ (C26H36N2O6)
430.2858, found 430.2841.
(100 MHz, CDCl3)
d 158.06, 157.02, 151.30, 146.79, 129.77, 129.56,
128.59, 127.68, 123.60, 123.04, 119.87, 115.82, 115.25, 57.51, 53.32,
49.37, 48.98, 33.15, 31.71, 29.54, 29.31, 29.21, 27.17, 26.79, 24.69, 22.91,
22.55, 18.34. Purity: 99.4% by HPLC. HRMS (ESI): calcd for (MeH)ꢃ
(C26H36N2O6) 430.2818, found 430.2837.
5.1.2.10. N1-(4-Methoxybenzyl)-N8-(1,2,3,4-tetrahydroacridin-9-yl)
octane-1,8-diamine (5n). Intermediate 4e was reacted with 4-
methoxybenzaldehyde following the general procedure to give the
desired product 5n as a yellow oil with a yield of 96%. 1H NMR
5.1.2.6. 2-Methoxy-4-((8-(1,2,3,4-tetrahydroacridin-9-ylamino)octy-
lamino)methyl)phenol (5j). Intermediate 4e was reacted with 4-
hydroxybenzaldehyde following the general procedure to give the
desired product 5j as a yellow oil with a yield of 49%. 1H NMR
(400 MHz, CDCl3)
d
7.92 (dd, J ¼ 14.2, 8.8 Hz, 2H), 7.51 (t, J ¼ 7.6 Hz,
1H), 7.31 (t, J ¼ 7.6 Hz,1H), 7.20 (d, J ¼ 8.6 Hz, 2H), 6.83 (d, J ¼ 8.7 Hz,
2H), 3.92 (s, 1H), 3.74 (s, 3H), 3.68 (s, 2H), 3.43 (t, J ¼ 7.2 Hz, 2H),
3.04 (d, J ¼ 6.1 Hz, 2H), 2.66 (t, J ¼ 5.4 Hz, 2H), 2.61e2.48 (m, 2H),
1.97e1.76 (m, 4H), 1.70e1.54 (m, 2H), 1.46 (dd, J ¼ 13.6, 6.8 Hz, 2H),
(400 MHz, CDCl3)
d
7.93 (dd, J ¼ 11.5, 8.7 Hz, 2H), 7.51 (t, J ¼ 7.6 Hz,
1H), 7.32 (t, J ¼ 7.6 Hz, 1H), 6.84 (s, 1H), 6.79 (d, J ¼ 8.0 Hz, 1H), 6.70
(d, J ¼ 8.0 Hz, 1H), 4.69 (s, 1H), 3.77 (s, 3H), 3.68 (s, 2H), 3.47 (t,
J ¼ 7.1 Hz, 2H), 3.06 (s, 2H), 2.68 (s, 2H), 2.61 (t, J ¼ 7.3 Hz, 2H), 1.88
(s, 4H), 1.62 (dd, J ¼ 14.3, 7.4 Hz, 2H), 1.49 (d, J ¼ 6.3 Hz, 2H), 1.32 (d,
1.36e1.13 (m, 8H). 13C NMR (100 MHz, CDCl3)
d 158.54, 158.38,
150.73, 147.50, 132.53, 129.26, 128.71, 128.17, 123.48, 122.88, 120.22,
115.77, 113.71, 55.19, 53.43, 49.45, 49.31, 34.05, 31.73, 29.98, 29.41,
29.27, 27.23, 26.85, 24.77, 23.05, 22.79. Purity: 98.2% by HPLC.
HRMS (ESI): calcd for (MeH)ꢃ (C26H36N2O6) 444.3015, found
444.2993.
J ¼ 29.4 Hz, 8H). 13C NMR (100 MHz, CDCl3)
d 158.31, 150.93, 147.42,
147.23, 145.56, 131.29, 128.44, 128.32, 123.55, 122.87, 120.89, 120.11,
115.64, 114.82, 111.37, 55.71, 53.91, 49.46, 49.28, 33.69, 31.75, 29.77,

W. Luo et al. / European Journal of Medicinal Chemistry 46 (2011) 2609e2616
2615
5.1.2.11. N1-(Benzo[d]
droacridin-9-yl)octane-1,8-diamine (5o). Intermediate 4e was
reacted with piperonal following the general procedure to give the
desired product 5o as a yellow oil with a yield of 72%. 1H NMR
[1,3]dioxol-5-ylmethyl)-N8-(1,2,3,4-tetrahy-
absorbance at 412 nm in 1 min intervals at 37 ꢀC. The calculations
were performed based on the method of Ellman et al. [32]. BuChE
assay followed a similar method to that described above.
(400 MHz, CDCl3)
d
7.84 (dd, J ¼ 12.6, 8.6 Hz, 2H), 7.42 (t, J ¼ 7.5 Hz,
5.3. Kinetic characterization of AChE inhibition
1H), 7.22 (t, J ¼ 7.6 Hz,1H), 6.72 (s,1H), 6.63 (s, 2H), 5.78 (s, 2H), 4.02
(s, 1H), 3.56 (s, 2H), 3.37 (t, J ¼ 7.2 Hz, 2H), 2.95 (s, 2H), 2.56 (s, 2H),
2.47 (t, J ¼ 7.2 Hz, 2H), 1.78 (s, 4H), 1.53 (dt, J ¼ 14.3, 7.1 Hz, 2H), 1.36
(d, J ¼ 6.2 Hz, 2H), 1.28e1.12 (m, 8H). 13C NMR (100 MHz, CDCl3)
Kinetic characterization of AChE was carried out following
a previously reported method [23]. Six different concentrations of
substrate were used, and the assays were carried out in 1 mL 0.1 M
d
157.85, 151.08, 147.62, 146.86, 146.44, 134.13, 128.45, 128.05,
KH2PO4/K2HPO4 buffer, pH 8.0, containing 50
mL of DTNB, 10 mL
123.58, 122.99, 121.22, 119.89, 115.42, 108.67, 108.00, 100.83, 53.70,
49.36, 49.12, 33.61, 31.66, 29.86, 29.37, 29.24, 27.18, 26.81, 24.70,
22.95, 22.62. Purity: 97.6% by HPLC. HRMS (ESI): calcd for (MeH)ꢃ
(C26H36N2O6) 429.2654, found 429.2636.
AChE, and 50 L substrate. The tested compound was added into
m
the assay solution and pre-incubated with the enzyme at 37 ꢀC for
15 min, followed by the addition of substrate. Kinetic character-
ization for the hydrolysis of ATC catalyzed by AChE was carried out
spectrometrically at 412 nm. The parallel control experiments were
carried out without inhibitor in the mixture.
5.1.2.12. N1-(Pyridin-4-ylmethyl)-N8-(1,2,3,4-tetrahydroacridin-9-yl)
octane-1,8-diamine (5q). Intermediate 4e was reacted with iso-
nicotinaldehyde following the general procedure to give the desired
product 5q as a yellow oil with a yield of 52%. 1H NMR (400 MHz,
5.4. Measurement of the antioxidation activity
CDCl3)
d
8.50 (d, J ¼ 6.0 Hz, 2H), 7.52 (t, J ¼ 7.6 Hz,1H), 7.34e7.29 (m,
The antioxidation activity was determined by using the oxygen
radical absorbance capacity e fluorescein (ORAC-FL) assay [19]. The
ORAC-assay measures antioxidant scavenging activity against per-
oxyl radical induced by 2,20-azobis(2-amidinopropane) dihydro-
chloride (AAPH) at 37 ꢀC.
1H), 7.23 (d, J ¼ 5.9 Hz, 2H), 3.76 (s, 2H), 3.47 (t, J ¼ 7.2 Hz, 2H), 3.05
(s, 2H), 2.67 (s, 2H), 2.57 (t, J ¼ 7.2 Hz, 2H), 1.88 (t, J ¼ 3.2 Hz, 4H),
1.71e1.57 (m, 2H), 1.47 (dd, J ¼ 13.7, 6.8 Hz, 2H), 1.40e1.16 (m, 8H).
13C NMR (100 MHz, CDCl3)
d 158.02, 150.94, 149.62, 149.59, 147.02,
128.31, 128.22, 123.51, 122.91, 119.99, 115.53, 52.62, 49.40, 49.33,
33.68, 31.64, 29.93, 29.33, 29.20, 27.09, 26.78, 24.70, 22.95, 22.63.
Purity: 99.4% by HPLC. HRMS (ESI): calcd for (MeH)ꢃ (C26H36N2O6)
415.2862, found 415.2847.
The reaction was carried out in 75 mM phosphate buffer (pH
7.4), and the final reaction mixture was 200
and fluorescein (120 L, 300 nM final concentration) were placed in
the wells of a black 96 well plate, and the mixture was incubated for
15 min at 37 ꢀC. Then AAPH (Aladdin, China) solution (60
L; 12 mM
mL. Antioxidant (20 mL)
m
m
5.1.3. Synthesis of N-(8-(1,2,3,4-tetrahydroacridin-9-ylamino)octyl)
isonicotinamide (5r)
final concentration) was added rapidly. The plate was immediately
placed into a Spectrafluor Plus plate reader (Tecan, Switzerland),
and the fluorescence was measured every 60 s for 180 min with
excitation at 485 nm and emission at 535 nm. 6-Hydroxy-2,5,7,
8-tetramethylchroman-2-carboxylic acid (Trolox, Sigma, Germany)
The mixture of pyridine-4-carboxylic acid (86 mg, 0.7 mmol),
intermediate 4e (230 mg, 0.7 mmol), N,N0-diisopropylcarbodiimide
(100 mg, 0.8 mmol), and 4-dimethylaminopyridine (37 mg,
0.3 mmol) were stirred overnight in CH2Cl2 at room temperature.
The resulting solution was filtered, and the filtrate was concen-
trated. The crude product was purified using flash chromatography
with chloroform/methanol/ammonia (40:1:0.5%) as elution solvent
to give the desired product 5r as a white solid with a yield of 50%
was used as a standard (1e5
mM, final concentration). A blank
(FL þ AAPH) using phosphate buffer instead of antioxidant and
Trolox calibration were carried out in each assay. The samples were
measured at different concentrations (1e5 mM), and at least four
independent runs were performed for each sample. Fluorescence
measurements were normalized based on the curve of the blank
(without antioxidant). From the normalized curves, the area under
the fluorescence decay curve (AUC) was calculated as:
(150 mg), m. p. 90e92 ꢀC, 1H NMR (400 MHz, CDCl3)
d 8.62 (d,
J ¼ 6.0 Hz, 2H), 7.96 (d, J ¼ 8.4 Hz,1H), 7.86 (d, J ¼ 8.4 Hz,1H), 7.51 (t,
J ¼ 7.6 Hz, 1H), 7.32 (s, 1H), 3.85 (s, 1H), 3.47 (t, J ¼ 7.2 Hz, 2H), 3.38
(dd, J ¼ 13.8, 6.6 Hz, 2H), 3.00 (s, 2H), 2.67 (s, 2H), 1.87 (t, J ¼ 3.0 Hz,
4H), 1.71e1.58 (m, 2H), 1.58e1.46 (m, 2H), 1.36e1.22 (m, 8H). 13C
P
(1) AUC ¼ 1 þ
i ¼ 90 fi=f 0 Where f0 is the initial fluorescence at
0 min, and fi is the fluorescence at time i. The net AUC for
a sample was calculated as following:
i ¼ 1
NMR (100 MHz, CDCl3)
d 165.64, 158.16, 150.99, 150.20, 147.14,
141.98, 128.36, 128.12, 123.55, 122.97, 121.14, 120.03, 115.57, 49.26,
40.14, 33.68, 31.62, 29.33, 29.03, 26.75, 26.68, 24.70, 22.95, 22.64.
Purity: 99.1% by HPLC. HRMS (ESI): calcd for (MeH)ꢃ (C26H36N2O6)
429.2654, found 429.2641.
(2) Net AUC ¼ AUCantioxidant ꢃ AUCblank. The ORAC-FL values were
calculated as following:
(3) [(AUCSample ꢃ AUCblank)/(AUCTrolox ꢃ AUCblank)]ꢂ [(concentration
of Trolox/concentration of sample)], and expressed as Trolox
equivalents by using the standard curve calculated for each assay.
5.2. Inhibition studies on AChE and BuChE
Final results were in
compound.
mM of Trolox equivalent/mM of pure
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel), butyr-
ylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum),
5,50-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB),
butylthiocholine chloride (BTC), acetylthiocholine chloride (ATC),
and tacrine hydrochloride were purchased from Sigma Aldrich.
All the assays were carried out in 0.1 M KH2PO4/K2HPO4 buffer,
pH 8.0, using a Shimadzu 2450 Spectrophotometer. Enzyme solu-
tions were prepared at 2.0 units/mL in 2 mL aliquots. The assay
5.5. Molecular modeling
The crystal structure of AChE complexed with tacrine (code ID:
1ACJ) and BuChE complexed with Echothiophate (code ID: 1POI)
were obtained in the Protein Data Bank after eliminating the
inhibitorand water molecules. The 3D Structure of compound 5j was
built, and its geometry optimization was performed with molecular
mechanics. Further preparation of the inhibitor included addition of
Gasteiger charges, removal of hydrogen atoms, and addition of their
atomic charges to skeleton atoms, and finally, assignment of proper
atomic types. Autotors was then used to define the rotatable bonds
medium (1 mL) contained phosphate buffer, pH 8.0, 50
mL of 0.01 M
DTNB, 10 L of enzyme, and 50 L of 0.01 M substrate (ATC). The
m
m
assay mixture containing enzyme, buffer, DTNB, and inhibitor was
incubated for 15 min, before the substrate was added to start the
reaction. The activity was determined by measuring the increase in

2616
W. Luo et al. / European Journal of Medicinal Chemistry 46 (2011) 2609e2616
[3] S. Rizzo, C. Riviere, L. Piazzi, A. Bisi, S. Gobbi, M. Bartolini, V. Andrisano,
F. Morroni, A. Tarozzi, J.P. Monti, A. Rampa, J. Med. Chem. 51 (2008)
2883e2886.
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C. Melchiorre, J. Med. Chem. 51 (2008) 347e372.
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Biophys. Acta 1502 (2000) 139e144.
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J. Neuropathol. Exp. Neurol. 65 (2006) 631e641.
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Rothenberg, A. Lerner, Expert Rev. Neurother 9 (2009) 1615e1621.
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R.L. Neve, Science 245 (1989) 417e420.
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311 (Pt 1) (1995) 1e16.
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41 (1998) 6e9.
[13] F. Yang, G.P. Lim, A.N. Begum, O.J. Ubeda, M.R. Simmons, S.S. Ambegaokar,
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280 (2005) 5892e5901.
in the ligand. Docking studies were carried out using the AUTODOCK
4.0 program. By 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 pre-
calculated atomic affinity grid maps for each atom type in the ligand,
plus an electrostatics map and a separatedesolvation map present in
the substrate molecule. All maps were calculated with 0.375 Å
spacing between grid points. The centerof 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]), and the dimensions of the active site box
were set at 50 ꢂ 46 ꢂ 46 Å.
Flexible ligand docking was performed for the compounds.
Docking calculations were carried out using the Lamarckian genetic
algorithm (LGA), and all parameters were the same for each dock-
ing. To ensure the reliability of the results, the docking procedures
were repeated independently for 10 times for each compound, and
the obtained orientations were analyzed.
[14] G.P. Lim, T. Chu, F. Yang, W. Beech, S.A. Frautschy, G.M. Cole, J. Neurosci. 21
(2001) 8370e8377.
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H. Wadsworth, S.J. Wood, R.E. Markwell, Biochem. J. 340 (Pt 1) (1999)
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5.6. Inhibition of self-mediated Ab42 aggregation
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J. Med. Chem. 52 (2009) 5365e5369.
The thioflavin-T fluorescence method was used. A
sodium (Anaspec Inc) was dissolved in 0.01 M phosphate buffer (pH
7.4) to obtain a 20 M solution. Compounds were firstly prepared in
DMSO (demethyl sulfoxide) at a concentration of 10 mM. The final
concentration of A 42 and inhibitors were 20 M. After incubated
in 37 ꢀC for 48 h, thioflavin-T (5
M in 50 mM glycineeNaOH buffer,
pH 8.0) was added. Fluorescence intensity was carried out (exci-
tation at 450 nm and emission at 485 nm). Each measurement was
run in triplicate. The fluorescence intensities were recorded, and
the percentage of inhibition on aggregation was calculated by using
the following equation: (1 ꢃ IFi/IFc)*100%, in which IFi and IFc were
the fluorescence intensities obtained for absorbance in the pres-
ence and absence of inhibitors, respectively, after subtracting the
fluorescence of respective blanks.
b42 peptide
m
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Conflict of interest
[25] Q. Xie, H. Wang, Z. Xia, M. Lu, W. Zhang, X. Wang, W. Fu, Y. Tang, W. Sheng,
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We declare that we have no conflict of interest.
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