Multifunctional tacrine-flavonoid hybrids with cholinergic, β-amyloid-reducing, and metal chelating properties for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2013.09.024

A new series of tacrine-flavonoid hybrids (13a-u) had been designed, synthesized, and evaluated as multifunctional cholinesterase (ChE) inhibitors against Alzheimer's disease (AD). In vitro studies showed that most of the molecules exhibited a significant ability to inhibit ChE and self-induced amyloid-β (Aβ_1-42) aggregation. Kinetic and molecular modeling studies also indicated compounds were mixed-type inhibitors, binding simultaneously to active, peripheral and mid-gorge sites of AChE. Particularly, compound 13k was found to be highly potent and showed a balanced inhibitory profile against ChE and self-induced Aβ_1-42 aggregation. Moreover, it also showed excellent metal chelating property and low cell toxicity. These results suggested that 13k might be an excellent multifunctional agent for AD treatment.

Full text of DOI:10.1016/j.ejmech.2013.09.024

European Journal of Medicinal Chemistry 69 (2013) 632e646
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Multifunctional tacrineeflavonoid hybrids with cholinergic,
-amyloid-reducing, and metal chelating properties for the treatment
b
of Alzheimer’s disease
Su-Yi Li a, Xiao-Bing Wang a, Sai-Sai Xie a, Neng Jiang a, Kelvin D.G. Wang a, He-Quan Yao b,
,
Hong-Bin Sun b, Ling-Yi Kong a
*
a State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang,
Nanjing 210009, People’s Republic of China
b Center for Drug Discovery, College of Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, 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 tacrineeflavonoid hybrids (13aeu) had been designed, synthesized, and evaluated
as multifunctional cholinesterase (ChE) inhibitors against Alzheimer’s disease (AD). In vitro studies
showed that most of the molecules exhibited a significant ability to inhibit ChE and self-induced amy-
Received 26 April 2013
Received in revised form
5 September 2013
Accepted 8 September 2013
Available online 21 September 2013
loid-b (Ab1e42) aggregation. Kinetic and molecular modeling studies also indicated compounds were
mixed-type inhibitors, binding simultaneously to active, peripheral and mid-gorge sites of AChE.
Particularly, compound 13k was found to be highly potent and showed a balanced inhibitory profile
against ChE and self-induced Ab1e42 aggregation. Moreover, it also showed excellent metal chelating
property and low cell toxicity. These results suggested that 13k might be an excellent multifunctional
agent for AD treatment.
Keywords:
Alzheimer’s disease
Flavonoid
Ó 2013 Elsevier Masson SAS. All rights reserved.
Tacrine
Cholinesterase
b-Amyloid aggregation
Metal chelator
ꢀ
1. Introduction
crystallographic structure of AChE reveals that it has a narrow 20 A
gorge, containing two binding sites: the catalytic active site (CAS) at
Alzheimer’s disease (AD), the most common form of adult onset
dementia, is a complex neurodegenerative process occurring in the
central nervous system (CNS), characterized by progressive cogni-
tive decline and memory loss [1,2]. Although the etiology of AD is
not fully known at present, several factors are considered to play
significant roles in the pathophysiology of AD. These include the
the bottom and the peripheral anionic site (PAS) near the entrance
of the gorge [8e10]. Hence, inhibitors that bind to either one or two
sites could inhibit the AChE. However, current studies indicated
that AChE could promote amyloid fibril formation by interaction
through the PAS of AChE, giving stable AChEeA
b complex, which
are more toxic than single A peptides alone [11]. For this reason,
b
deposits of aberrant proteins namely
oxidative stress, dyshomeostasis of biometals, and low levels of
acetylcholine (ACh) [3,4].
Current clinical therapy for AD patients is mainly based on the
cholinergic hypothesis [5,6]. It suggests that, the decline of ACh
level leads to cognitive and memory deficits, and sustaining or
recovering the cholinergic function is supposed to be clinically
beneficial. ACh can be degraded by two types of cholinesterases,
namely AChE and butyrylcholinesterase (BuChE) [7]. The
b
-amyloid (A
b
) and
s
-protein,
the dual binding inhibitors, which target both PAS and CAS, have
become more promising in AD treatment [12,13]; since they will
not only alleviate the cognitive deficit of AD patients by elevating
ACh levels but also act as disease-modifying agents delaying amy-
loid plaque formation [14]. In normal brain, AChE is more active
than BuChE and hydrolyzes about 80% of ACh. However, as AD
progresses, the activity of AChE decreases, while that of BuChE
significantly rises becoming a modulator to regulate ACh levels in
cholinergic neurons [15]. Consequently, both enzymes are useful
therapeutic targets for AD.
Converging lines of evidence suggest that progressive cerebral
deposition of A
b also plays an important role in the pathogenesis
* Corresponding author. Tel./fax: þ86 25 83271405.
and development of AD, as its accumulation may result in a cascade
E-mail addresses: cpu_lykong@126.com, lykong@jlonline.com (L.-Y. Kong).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.024

Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
633
Scheme 1. Synthesis of intermediates 4aem, 7aec. Reagents and conditions: (a) LiHMDS, THF, ꢀ78 ꢁC to r.t., 4 h; (b) Br(CH2)nBr (n ¼ 2e6), anhydrous K2CO3, acetone, reflux, 4 h; (c)
MeI, anhydrous K2CO3, acetone, reflux, 2 h; (d) SnCl2, EtOH, reflux, 1 h; (e) (CH2O)n, NaCNBH3, HOAc, r.t., 3 h; (f) DMF, CH3SO2Cl, BF3$Et2O, r.t. to 90 ꢁC; (g) Triethyl orthoformate, 70%
HClO4, ꢀ20 ꢁC to r.t., 8 h, then, H2O, 100 ꢁC, 1 h.
of biochemical events leading to neuronal dysfunction [16]. A
proteolytic fragment derived from the amyloid precursor protein
(APP), a transmembrane glycoprotein that is usually processed by
b
is a
acetylcholinesterase inhibitors (AChEIs) in general and the high
potency of tacrine in particular, this structure has been widely used
for application in hybrid or multitarget compounds in order to
obtain potent AChEIs with other pharmacological properties
[29,30]. For example, tacrineemelatonin hybrids and tacrinee
ferulic acid hybrids have been designed as potent ChEIs with
antioxidant properties [31], tacrinee4-oxo-4H-chromene hybrids
have been designed as multifunctional agents capable of inhibiting
the enzyme
small and soluble peptides [17]. In AD affected brain, amyloidogenic
pathway takes place involving the sequential action of -secretase
peptides,
42) amino acids in length. This longer
a-secretase to generate in physiological conditions
b
followed by
either 40 (A
g
b
-secretase to generate two predominant A
40) or 42 (A
b
b
form is more prone to aggregate into fibrils, and it makes up the
ChE and b-secretase [32], and tacrinee8-hydroxyquinoline hybrids
major component of amyloid plaques. Therefore, inhibitors of the
Ab42 aggregation may be effective in blocking the progression of
the pathology [18].
Recent studies indicate that another hypothesis, called metal
hypothesis, may contribute to AD pathology [19]. It is observed that
the level of metal ions (Fe, Cu, Zn) in AD patients is 3e7 folds higher
than that of healthy individuals [20]. The abnormal accumulation of
have been designed to exhibit neuroprotective, cholinergic, anti-
oxidant, and copper-complexing properties [33]. Flavonoids, a
group of naturally occurring compounds with low molecular
weight, have attracted increasingly widespread attention in
present-day society, since they possess a wide range of pharma-
cological properties related to a variety of neurological disorders,
such as AChE and BuChE inhibitory activities [34,35], Ab fibril for-
metals is able to accelerate the formation of A
b
aggregates and
mation inhibitory activity [36], free radical scavenging effect [37]
and metal-chelating ability [38]. Thus, the design and synthesis of
new effective flavonoid derivatives are an interesting strategy for
the research on anti-AD drugs.
Very recently, our group has reported the synthesis of tacrinee
coumarin hybrids as multifunctional ChEIs against AD [39].
Continuing with our research on various natural products with
potential application in the AD field. In this paper, flavonoid was
selected to hybridize with tacrine to design a series of new hybrids,
exhibiting multifunctional activities. Tacrine for the inhibition of
ChE through its binding to the CAS while flavonoid for its metal-
neurofibrillary tangles, which promote inflammation and activate
neurotoxic pathways, leading to dysfunction and death of brain
cells [21,22]. In addition, studies suggest that redox-active ions like
Cu2þ and Fe2þ are involved in the production of reactive oxygen
species (ROS) and oxidative stress which are critical for Ab neuro-
toxicity [23]. Therefore, modulation of such biometals in the brain
has been proposed as a potential therapeutic strategy for the
treatment of AD.
Due to the pathological complexity found in AD, a single drug
that acts on a specific target to produce the desired clinical effects
might not be suitable. Thus, multifunctional molecules with two or
more complementary biological activities may represent an
important advance for the treatment of the disease [24e26].
Herein, our aim is to obtain new multifunctional cholinesterase
inhibitors (ChEIs) endowed with addition properties such as
chelating, and Ab-lowering activities, as well as for its potential
interaction with the PAS due to its aromatic character. Since the
AChE-CAS is located at the bottom of a deep gorge, we considered
connecting tacrine and flavonoid fragments by piperazine side-
armed-alkane spacer. These flexible linkers could be lodged by
the enzyme cavity, allowing simultaneous interaction between the
heteroaromatic fragments and both the CAS and PAS of AChE.
Moreover, the linker fragment could establish additional contacts
by binding to the aromatic residues of the enzyme mild-gorge
lowering the Ab aggregation and chelating metals.
Tacrine, the most potent and clinically effective acetylcholines-
terase inhibitor (AChEI), was approved by FDA in 1993 [27]. How-
ever, it was withdrawn from the market due to its doseedependent
hepatotoxicity [28]. Because of the clinical effectiveness of
through cationep interactions [40,41].

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Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
In this paper we described the synthesis, pharmacological
fused and heterocyclic rings appeared in between 1445 and
1602 cmꢀ1. The characteristic strong bands appeared for C]O
stretching in flavonoid at 1631e1660 cmꢀ1. The 1H NMR spectra of
all the compounds showed signals for aromatic protons between
evaluation, and molecular modeling of a series of new tacrinee
flavonoid hybrids. The pharmacological evaluations of these new
compounds include AChE and BuChE inhibition, the kinetics of
enzyme inhibition, self-induced A
b
aggregation, and metal chela-
d
6.19e8.62 ppm and the eCH2eOe protons resonated as a triplet
between 4.08 and 4.12 ppm. The piperazine protons appeared at
2.50e2.80 ppm. In the 13C NMR spectra, the carbon resonance
frequencies of the C]O was at 177.0e182.5 ppm. The aromatic
carbons appeared at 93.3e165.1 ppm and the eCH2e groups
appeared at 16.9e66.8 ppm. Moreover, their ESI-MS spectra
tion. Finally, molecular modeling studies were performed to gain
insight into the binding mode and structureeactivity relationships
of new hybrid compounds.
d
d
d
d
d
2. Result and discussion
showed molecular ion [M þ H]þ peaks further confirming their
structure. The molecular formula and elemental composition were
confirmed by HRMS. All spectroscopic measurements confirmed
the structure and high purity of synthesized compounds.
2.1. Chemistry
The key intermediates (4aem, 7aec) were synthesized as
shown in Scheme 1. Compounds 3aef were obtained following a
described method [42] and then reacted with
a,
u
-dibromoalkanes
2.2. Cholinesterase inhibitory activity
in the presence of potassium carbonate to provide the key in-
termediates 4aef and 4hek. The methylation of 5-OH group of 4f
with methyl iodide and potassium carbonate afforded compound
4g. The free amino derivative 4l was readily prepared from 4k by
reduction with SnCl2 in ethanol under reflux condition [43]. Con-
version of 4l to the dimethylamino derivative 4m was achieved by
an efficient method with paraformaldehyde, sodium cyanobor-
ohydride, and acetic acid [44].
Commercially available 1b was treated with triethyl ortho-
formate and 70% perchloric acid, followed by hydrolysis in boiling
water to afford 5, which was then treated with dibromopropane to
afford 7a [45]. Compound 7a was methylated in presence of methyl
iodide and potassium carbonate in acetone under reflux condition
to gain 7b. Compound 6 was obtained from 1a underwent Vils-
meiereHaack formylation and cyclization, and then treated with
dibromopropane and potassium carbonate in acetone to afford 7c
[46].
On the other hand, compound 9 was synthesized from anthra-
nilic acid using a previous reported method [47]. The obtained
compound 9 was then treated with aminoethanol and 3-amino-1-
propano to afford 10a and 10b, respectively. Treatment of the 10
with 4-toluene sulfonyl chloride in the presence of triethylamine
provided 11, which were then treated with excess anhydrous
piperazine to give 12 (Scheme 2). The obtained compounds 12 were
finally reacted with corresponding flavonoid derivatives 4aem and
7aec to furnish desired compounds 13aeu (Scheme 3).
All synthesized compounds (13aeu) were characterized by IR,
1H NMR, 13C NMR and Mass spectroscopy including HRMS. The IR
spectra of compounds showed absorption bands of eNHe
stretching vibration at 3345e3445 cmꢀ1. Skeleton vibrations for
The inhibitory activity of hybrids 13aeu and relative compound
3a against AChE (from electric eel) and BuChE (from equine serum)
was measured according to the spectrophotometric method of
Ellman et al. [48]. For comparison purpose, tacrine and galanth-
amine were used as reference compounds. The IC50 values of all
tested compounds and their selectivity index for AChE over BuChE
were summarized in Table 1. From Table 1, it could be seen that all
new target compounds showed good inhibitory activity to both ChE
with IC50 values ranging from sub-micromolar to nanomolar.
Among the target compounds, 13t (IC50 ¼ 8.4 nM) showed the most
potent inhibitory activity for AChE, which was 31- and 318-times
stronger than those of the reference compounds tacrine
(IC50 ¼ 260 nM) and galanthamine (IC50 ¼ 2670 nM), respectively.
In contrast, 13u exhibited strongest inhibition to BuChE with IC50
value of 25.8 nM, which was 2- and 508-fold more potent than
those of tacrine (IC50
(IC50 ¼ 12,700 nM).
¼
50.0 nM) and galanthamine
From the IC50 value of compounds 13aej, it appeared that the
proper tether for the linker between the two anchoring groups,
tacrine and flavonoid, seemed to be six (m þ n ¼ 6), especially
m ¼ n ¼ 3. It also concluded that the variation of chain length for
the inhibitors had more influence on their inhibition of AChE than
BuChE. A possible reason is conformational difference between
these two enzymes. By contrast, BuChE does not have a function
peripheral site, and the active site of BuChE is wider than that of
AChE. Therefore, BuChE has less restriction to inhibitors with
varying linker length.
With the optimal length of the linker (m ¼ n ¼ 3) in hand, we
planned to introduce different substituents with varying electronic
Scheme 2. Synthesis of intermediates 12. Reagents and conditions: (h) POCl3, cyclohexanone, reflux, 4h; (i) NH2(CH2)mOH, m ¼ 2e3, 160 ꢁC, 8 h; (j) PTSC, TEA, DMAP, CH2Cl2, 0 ꢁC to
r.t., 4 h; (k) anhydrous piperazine, DMF, 80 ꢁC, 2 h.

Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
635
Scheme 3. Synthesis of tacrineeflavonoid hybrids 13aeu. Reagents and conditions: (l) intermediates 4aem, 7aec, anhydrous K2CO3, CH3CN, reflux, 8 h.
properties to the flavonoid. First of all, hydroxy and methoxy group
was introduced to the 5-position of flavone moiety producing 13ke
l that resulted in a slight increase in AChE inhibition, but no sig-
nificant influence on BuChE inhibition. Moreover, the inhibitory
activity of target compounds was sensitive to the substituents at 40-
position of flavone ring. As shown in Table 1, 13mep possessing an
electron-donating group at 40-position of the B ring in flavone
moiety showed a decreased AChE inhibitory activity, while 13qer
possessing an electron-drawing group showed an increased AChE
inhibitory activity. By contrast, both electron-donating group and
inhibition. These results implied that an electron-drawing group at
40-position of the B ring seemed beneficial to AChE inhibitory ac-
tivity, and made it more selective to AChE inhibitory activity.
To extend the series of the hybrids, compounds 13seu without
the B ring in flavone moiety were synthesized. Interestingly, it was
found that both AChE and BuChE inhibitory activities of these
compounds were increased more remarkably than 13g. For
example, the inhibitory activities of 13u for AChE and BuChE
(IC50 ¼ 10.3 nM for AChE; IC50 ¼ 25.8 nM for BuChE) were 15-fold
and 8-fold more potent, respectively, than those of 13g
(IC50 ¼ 153 nM for AChE; IC50 ¼ 208 nM for BuChE). It suggested
electron-drawing group showed
a slight decrease in BuChE
Table 1
Inhibition of ChEs activity and selectivity index of compounds.
Compound
R1
R2
R3
m
n
IC50 (nM)
AChEa
Selectivity indexc
BuChEb
3a
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
85,000 ꢂ 50
481 ꢂ 8
>100,000
520 ꢂ 8
311 ꢂ 15
197 ꢂ 7
258 ꢂ 10
289 ꢂ 12
266 ꢂ 8
208 ꢂ 5
317 ꢂ 15
423 ꢂ 7
414 ꢂ 8
558 ꢂ 6
360 ꢂ 8
573 ꢂ 14
655 ꢂ 8
685 ꢂ 5
845 ꢂ 10
994 ꢂ 10
460 ꢂ 8
45.0 ꢂ 3.0
35.0 ꢂ 0.5
25.8 ꢂ 0.8
50 ꢂ 3
>1.2
1.1
0.5
0.7
0.5
0.4
0.6
1.4
0.6
0.7
0.5
4.2
6.2
1.8
1.6
2.0
3.7
24.8
6.1
2.9
4.2
2.5
0.2
4.76
H
H
H
H
H
H
H
H
H
H
OH
CH3O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
3
4
5
6
2
3
4
5
6
3
3
3
3
3
3
3
3
3
3
3
624 ꢂ 14
268 ꢂ 12
568 ꢂ 12
717 ꢂ 15
416 ꢂ 12
153 ꢂ 8
547 ꢂ 15
594 ꢂ 13
848 ꢂ 15
133 ꢂ 5
13k
13l
57.7 ꢂ 4.5
316 ꢂ 15
417 ꢂ 30
336 ꢂ 12
230 ꢂ 6
13m
13n
13o
13p
13q
13r
13s
13t
13u
Tacrine
Galantamine
CH3
CH3O
NH2
N(CH3)2
Cl
40.1 ꢂ 0.3
75.0 ꢂ 2.5
15.4 ꢂ 0.5
8.4 ꢂ 0.8
10.3 ꢂ 0.5
260 ꢂ 8
H
NO2
OH
CH3O
H
2670 ꢂ 15
12700 ꢂ 2
a
Inhibitor concentration (mean ꢂ SEM of three experiments) required for 50% inactivation of AChE.
Inhibitor concentration (mean ꢂ SEM of three experiments) required for 50% inactivation of BuChE.
Selectivity index ¼ IC50 (BuChE)/IC50 (AChE).
b
c

636
Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
Table 2
Inhibition of Ab (1e42) self-induced aggregation of compounds.
Compound R1
R2
R3
m
n
A
b
(1e42)
Ab(1e42)
IC50
aggregation
(m
M)b
inhibition (%)a
3a
27.2 ꢂ 0.9
54.1 ꢂ 3.0
59.1 ꢂ 1.4
53.7 ꢂ 1.1
65.4 ꢂ 2.0
63.3 ꢂ 1.8
70.0 ꢂ 1.9
58.0 ꢂ 1.4
53.4 ꢂ 2.0
62.5 ꢂ 2.4
63.0 ꢂ 1.8
79.1 ꢂ 1.4
68.7 ꢂ 3.3
13.4 ꢂ 0.6
10.9 ꢂ 0.2
9.0 ꢂ 0.3
n.t.c
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
13q
13r
H
H
H
H
H
H
H
H
H
H
OH
CH3O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
3
4
5
6
2
3
4
5
6
3
3
3
3
3
3
3
3
3
3
3
16.8 ꢂ 2.1
13.7 ꢂ 2.3
16.4 ꢂ 1.6
12.8 ꢂ 0.7
14.2 ꢂ 1.2
9.8 ꢂ 1.1
14.9 ꢂ 2.0
17.3 ꢂ 2.1
13.2 ꢂ 1.9
13.5 ꢂ 0.4
6.5 ꢂ 1.9
9.6 ꢂ 0.7
n.t.c
CH3
CH3O
NH2
N(CH3)2
Cl
n.t.c
n.t.c
12.2 ꢂ 0.5
48.9 ꢂ 1.2
61.1 ꢂ 1.2
85.3 ꢂ 2.7
62.0 ꢂ 1.7
67.4 ꢂ 3.8
43.5 ꢂ 2.9
n.t.c
n.t.c
H
NO2
14.1 ꢂ 1.2
4.8 ꢂ 0.6
12.6 ꢂ 1.4
11.5 ꢂ 1.7
20.3 ꢂ 1.2
13s
13t
13u
Curcumin
OH
CH3O
H
Fig. 2. LineweavereBurk plots resulting from subvelocity curve of AChE activity with
different substrate concentrations (0.05e0.50 mM) in the absence and presence of 7.7,
15.4, 30.8 nM 13s. The Vmax and Km values shown in this table are the mean ꢂ SD of
three experiments.
a
Inhibition of Ab (1e42) self-induced aggregation, the thioflavin-T fluorescence
method was used, the mean ꢂ SD of at least three independent experiments and the
measurements were carried out in the presence of 20 M compounds the presence
of 20 M compounds.
m
compounds with inhibition rate greater than 50%. The result indi-
m
b
The IC50
experiments.
(m
M) values shown in this table are the mean
ꢂ
SD of three
cated that 13s (IC50 ¼ 4.8
mM) was the most potent inhibitor of Ab
(1e42) among the series of compounds featuring a hydroxyl group
at the 5 position of flavonoid moiety and without the B ring at the 2
position. From the inhibition values of compounds 13aej, it
appeared that the linker length didn’t play a role in determining the
c
n.t. means not tested.
that the high hydrophobicity and/or the steric hindrance of the
phenyl ring might be not favorable for ChE inhibition.
inhibition of A
b (1e42) self-aggregation, since no significant
change of the percentages of inhibition was observed along with
the lengthening the spacer length. In contrast, compounds 13mep
only showed the percentages of inhibition ranging from 9.0 to
13.4%, nearly 4-fold lower than that of the curcumin. These results
indicated that, similarly to ChE inhibition, an electron-donating
group at 40-position of flavone moiety might not favorable for in-
2.3. Inhibition of Ab (1e42) self-induced aggregation
All compounds tested their activities for both ChE inhibition
were also tested for their ability to inhibit A (1e42) self-induced
b
hibition of A
group or methoxy group at the 5 position of the A ring, exhibited
good inhibitory activities (13kel, 13set, from 4.8 to 12.6 M). These
results implied that an electron-donating group at the 5 position of
b (1e42) self-aggregation. Compounds with a hydroxyl
aggregation by using a thioflavin-T based fluorometric assay
[49,50]. Curcumin (Cur), a known active natural product for the
m
inhibition of A
b (1e42) self-aggregation, was used as reference
compound and the results were summarized in Table 2 and showed
in Fig. 1. From the results, it could be seen that most hybrids
A ring seemed to be beneficial to A
inhibitory activity.
b (1e42) self-aggregation
exhibited moderate-to-good potencies (9.0e85.3% at 20
compared to that of curcumin (43.5% at 20 M). The complete
doseeresponse curves were obtained by assaying those
mM)
m
2.4. Kinetic study of ChE inhibition
To gain information on the mechanism of inhibition, the potent
inhibitor 13s was s elected for kinetic studies because of its total
performance with regard to cholinesterase inhibition and self-
induced Ab (1e42) aggregation inhibition. The type of inhibition
was elucidated from the analysis of LineweavereBurk plots, which
were reciprocal rates versus reciprocal substrate concentrations
for the different inhibitor concentrations resulting from the sub-
strateevelocity curves for ChE. For AChE, the plot showed both
increased slopes (decreased Vmax, from 0.31 to 0.18) and intercepts
(higher Km, from 0.20 to 0.26) at increasing concentration of the
inhibitor (Fig. 2). This pattern indicated a mixed-type inhibition
and therefore revealed that compound 13s might be able to bind
to CAS as well as PAS of AChE. In contrast, a different plot for
BuChE was obtained, showing different Km (from 0.15 to 0.41) and
constant Vmax in different inhibitor concentrations (Fig. 3). This
Fig. 1. Inhibition of
comparing with that of curcumin. The thioflavin-T fluorescence method was used and
the measurements were carried out in the presence of 20 M test compound. The
mean ꢂ SD values from three independent experiments were shown.
Ab (1e42) self-induced aggregation by compounds (13aeu)
m
suggested
a
competitive inhibition, revealing that these

Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
637
Fig. 3. LineweavereBurk plots resulting from subvelocity curve of BuChE activity with
different substrate concentrations (0.05e0.50 mM) in the absence and presence of
22.5, 45.0, 90.0 nM 13s. The Vmax and Km values shown in this table are the mean ꢂ SD
of three experiments.
compounds compete for the same binding site (CAS) as the sub-
strate acetylcholine.
2.5. Molecular modeling studies
To further study the interaction mode of compound 13s for
AChE, molecular docking study was performed using software
package MOE 2008.10. The X-ray crystal structure of the TcAChE
complex with bis (7)etacrine (PDB ID 2CKM) was applied to build
the starting model of AChE. As showed in Fig. 4, tacrine moiety of
13s was bound to the CAS of AChE, it being stacked against the
phenyl ring of Phe 330 and the indole ring of Trp 84 with the ring-
Fig. 4. (a) 3D docking model of compound 13s with TcAChE. Atom colors: yellow e
carbon atoms of 13s, gray e carbon atoms of residues of TcAChE, dark blue e nitrogen
atoms, red e oxygen atoms. The dashed lines represent the interactions between the
protein and the ligand. (b) 2D schematic diagram of docking model of compound 13s
with TcAChE. The figure was prepared using the ligand interactions application in MOE.
(For interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)
ꢀ
to-ring distance of 3.44 and 3.68 A, respectively. The flavonoid ring
interacted with the indole ring of Trp 279 and Tyr70 of PAS via pep
ꢀ
stacking interactions with the distance of 4.24 and 5.05 A, respec-
tively. As we expected, the piperazine could bind to the mid-gorge
ꢀ
binding site of AChE through a catione
p
interaction (5.45 A) be-
tween its protonated nitrogen atom and Tyr 334 in the middle
gorge. All these results clearly indicated that compound 16s could
simultaneously bind to CAS, PAS and mid-gorge binding site of
AChE, thereby demonstrating rationality of our molecular design.
As known, flavonoids with free 5-hydroxyl and 4-keto groups
may use this 5-hydroxy-4-keto site for metal chelation. Compounds
13k and 13s may have metal-chelating ability since they all have
the 5-hydroxy-4-keto site. In addition, 13k and 13s showed potent
From this point of view, compound 13k could be more effective due
to its anti-Ab activity which was almost the same as activity of 13s
and had much higher IC50 values for ChE inhibition.
2.6. Metal chelating effect
inhibitory activities toward ChE and A
gregation. They had fine IC50 values in micromole range
(IC50 ¼ 6.5 M for 13k, IC50 ¼ 4.8 M for 13s) for inhibition of self-
aggregation of A , and the inhibitory activities of 13s for AChE and
BuChE (IC50 ¼ 15.4 nM for AChE; IC50 ¼ 45.0 nM for BuChE) were
8.6 and 12-fold more potent than those of 13k (IC50 ¼ 133 nM for
AChE; IC50 ¼ 558 nM for BuChE). Compound 13s might be more
potent than 13k due to their ChE inhibitory activity. However, we
wanted to achieve a balanced multitarget profile rather than just a
b
(1e42) self-induced ag-
To further study, the chelating effect of 13k for metals such as
Cu2þ and Fe2þ in methanol was studied by UVevis spectrometry
with wavelength ranging from 200 to 500 nm [51,52]. In Fig. 5a,
UVevis spectra of 13k at increasing Cu2þ concentrations were
shown as an example. The increase in absorbance, which could be
better estimated by an inspection of the differential spectra
(Fig. 5b), indicated that there was an interaction between Cu2þ and
m
m
b
13k. The similar behavior was also observed when using Fe2þ
.
These observations indicated that 13k could effectively chelate
Cu2þ and Fe2þ, and thereby could serve as metal chelator in treating
AD. The ratio of ligand/metal ion in the complex was investigated
by mixing the fix amount metal ion with increasing ligand; it was
highly potent ChE inhibitor. That IC50 values for ChE and Ab ag-
gregation inhibition in multifunctional compound were close
would be superior to compound with large difference of them.

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Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
Fig. 6. ALT and ASAT activity after the administration of tacrine and 13k. Values are
expressed as mean ꢂ SEM (n ¼ 8e9. t test, compared to control of the same time after
administration, p* ꢃ 0.05, p** ꢃ 0.01).
Fig. 5. (a) UVevis (200e500 nm) absorption spectra of 13k (25 mM) in methanol after
After being treated with tacrine,13k, the heparinized serum of mice
was obtained after different times and the levels of aspartate
aminotransferase (ASAT) and alanine aminotransferase (ALT) were
determined. In comparison to the control, tacrine caused significant
hepatotoxicity, as indicated by the increased activity of ASAT and
ALT. From the results, it appears that 13k may be safer than tacrine,
but not obviously (Fig. 6).
The antioxidant activities of 13aeu using a radical scavenging
assay (DPPH assay) had been tested. All the compounds showed
tiny antioxidant activities (at the concentration 2 mM, the activities
in % of compounds 13aeu were less than 36%). This explained the
weak hepatoprotective activity of 13aeu. In future studies, we
should introduce more free phenolic hydroxyl groups in the
structure to improve the antioxidant activity, and this might be
beneficial for the hepatoprotective activity.
addition of ascending amounts of CuCl2 (2e50 mmol/L). (b) The differential spectra due
to 13keCu2þ complex formation obtained by numerical subtraction from the above
spectra of those of Cu2þ and 13k at the corresponding concentrations.
possible to observe that the maximum intensity of difference
spectra was reach at 1:1 ratio, which was taken as an indication of
the stoichiometry of the complex.
2.7. SH-SY5Y neuroblastoma cell toxicity
On basis of the screening results above, the most potent
compound 13k was selected to further examine the potential
toxicity effect on the human neuroblastoma cell line SH-SY5Y
[33,53]. After exposing the cells to this compound for 24 h, the
cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium (MTT) assay. The result indicated that 13k
3. Conclusion
did not show significant effect on cell viability at 1e50
97.3 ꢂ 14.7%; 5 M: 96.5 ꢂ 17.5%; 10 M: 94.4 ꢂ 13.6%; 25
92.7 ꢂ 10.4%; 50
m
M (1
m
m
M:
M:
m
m
In conclusion, a series of tacrineeflavonoid hybrids had been
designed and synthesized as multifunctional ChEIs. Most of com-
pounds inhibited ChE in the nanomolar range in vitro effectively.
Kinetic and molecular modeling studies also indicated the com-
pounds were mixed-type inhibitors, binding simultaneously to
active, peripheral and mid-gorge sites of AChE. Most of the hybrids
exhibited higher percentages of inhibition than the reference
m
M: 90.1 ꢂ 8.5%). This suggested that 13k was
nontoxic to SH-SY5Y cells and might be a suitable multifunctional
ChE inhibitor for treating AD.
2.8. Hepatotoxicity studies
The main disadvantage of tacrine is the high hepatotoxicity
which can be prevented with free radical scavengers [54]. To
determine whether our compounds had hepatotoxicity in com-
parison to tacrine, 13k was selected for the assay with adult mice.
compound curcumin in inhibition of Ab (1e42) aggregation assay.
Among them, 13k and 13s showed moderate metal-chelating
ability since they had 5-hydroxy-4-keto site. The weaker ChE in-
hibition potencies and the concomitant nearly equipotent anti-

Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
639
amyloid activities of 13k with respect to 13s resulted in a more
balanced biological profile against both targets. Compound 13k
could be more effective, and it was selected as potent lead com-
pound for further study. In the cell toxicity assay, 13k showed
continuously until it cooled to room temperature. Anhydrous
diethyl ether (18 mL) was added to precipitate the intermediate
oxonium perchlorate salt, which was subsequently hydrolyzed in
hot water (30 mL) to provide 6 as a brown solid. Yield 65.0%, ESI-MS
m/z: 163.0 [M þ H]þ.
nontoxic to SH-SY5Y cell at 1e50 mM. Altogether, the multifunc-
tional effects of the new hybrids qualified them as potential anti-AD
drug candidates and 13k might be considered as a promising lead
compound for AD treatment.
4.1.4. General procedures for the preparation of intermediate 4aef,
4hek, 7a, and 7c
A mixture of 3aef/5e6 (5 mmol) with suitable
a,u-dibro-
4. Experimental section
moalkanes (50 mmol) and anhydrous K2CO3 (1.4 g, 10 mmol) in
acetone (15 mL) was refluxed under stirring for 4 h. After cooling,
the reaction mixture was filtered and the filtrate was evaporated
under reduced pressure. The obtained residue was purified by silica
gel chromatography with hexane/acetone (20:1) as eluent to give
compounds 4aef, 4hek, 7a, and 7c.
4.1. Chemistry
All chemicals (reagent grade) used were purchased from Sino-
pharm Chemical Reagent Co., Ltd. (China). Reaction progress was
monitored using analytical thin layer chromatography (TLC) on
precoated silica gel GF254 (Qingdao Haiyang Chemical Plant, Qing-
dao, China) plates and the spots were detected under UV light
(254 nm). Melting point was measured on an XT-4 micromelting
point instrument and uncorrected. IR (KBr-disc) spectra were
recorded by Bruker Tensor 27 spectrometer. 1H NMR and 13C NMR
spectra were measured on a Bruker ACF-500 spectrometer at 25 ꢁC
4.1.4.1. 7-(2-Bromoethoxy)-2-phenyl-4H-chromen-4-one
(4a).
Intermediate 3a was treated with 1,2-dibromoethane according to
the general procedure to give the desired product 4a as a light
yellow solid, yield 85.0%; 1H NMR (500 MHz, DMSO-d6)
d 8.10 (dd,
J ¼ 6.0, 2.0 Hz, 2H), 7.96 (d, J ¼ 8.5 Hz, 1H), 7.65e7.54 (m, 3H), 7.38
(d, J ¼ 2.5 Hz, 1H), 7.10 (dd, J ¼ 8.5, 2.0 Hz, 1H), 6.97 (s, 1H), 4.51 (t,
J ¼ 5.5 Hz, 2H), 3.88 (t, J ¼ 5.5 Hz, 2H); ESI-MS m/z: 345.0 [M þ H]þ.
and referenced to TMS. Chemical shifts are reported in ppm (d)
using the residual solvent line as internal standard. Splitting pat-
terns are designed as s, singlet; d, doublet; t, triplet; m, multiplet.
The purity of all compounds was confirmed to be higher than 95%
through analytical HPLC performed with Agilent 1200 HPLC System
(Supporting information, Table S1). Mass spectra were obtained on
a MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI-MS)
and a Mariner ESI-TOF spectrometer (HRESIMS), respectively. Col-
4.1.4.2. 7-(3-Bromopropoxy)-2-phenyl-4H-chromen-4-one
(4b).
Intermediate 3a was treated with 1,3-dibromopropane according to
the general procedure to give the desired product 4b as a light
yellow solid, yield 88.1%; 1H NMR (500 MHz, DMSO-d6)
d 8.11e8.09
(m, 2H), 7.97 (d, J ¼ 8.5 Hz,1H), 7.61e7.56 (m, 3H), 7.37 (d, J ¼ 2.0 Hz,
1H), 7.09 (dd, J ¼ 8.5, 2.0 Hz, 1H), 6.97 (s, 1H), 4.27 (t, J ¼ 6.0 Hz, 2H),
3.70 (t, J ¼ 6.5 Hz, 2H), 2.34e2.31 (m, 2H); ESI-MS m/z: 359.0
[M þ H]þ.
umn chromatography was performed on silica gel (90e150
mm;
Qingdao Marine Chemical Inc.)
4.1.1. General procedures for the preparation of intermediate 3aef
A solution of lithium bis(trimethyl)silyl amide (LiHMDS) in THF
(1 mol/L, 40 mmol) was added to a well-stirred solution of 1
(10 mmol) in THF under a nitrogen atmosphere at ꢀ78 ꢁC over
15 min. The reaction mixture was stirred at ꢀ78 ꢁC for 3 h and a
solution of the appropriate benzoyl chloride (10 mmol) in THF
(20 mL) was added over 10 min. Stirring was continued for 1 h
at ꢀ78 ꢁC and at room temperature for 4 h. Then the reaction
mixture was poured into a mixture of ice water (250 mL) and HCl
(10 mL). It was extracted with CH2Cl2 (3 ꢄ 25 mL) and the combined
extracts were dried over anhydrous Na2SO4. The solvent was
evaporated and the residue mixed with glacial acetic acid (50 mL)
and H2SO4 (0.25 mL) and heated at 100 ꢁC for 1 h. Water was added
and the product was filtered, washed with water and crystallized
from acetone/petrol ether.
4.1.4.3. 7-(4-Bromobutoxy)-2-phenyl-4H-chromen-4-one
(4c).
Intermediate 3a was treated with 1,4-dibromobutane according to
the general procedure to give the desired product 4c as a light
yellow solid, yield 85.5%; 1H NMR (500 MHz, DMSO-d6)
d 8.10e8.08
(m, 2H), 7.94 (d, J ¼ 8.5 Hz,1H), 7.61e7.56 (m, 3H), 7.33 (d, J ¼ 2.0 Hz,
1H), 7.06 (dd, J ¼ 8.5, 2.0 Hz, 1H), 6.96 (s, 1H), 4.19 (t, J ¼ 6.0 Hz, 2H),
3.63 (t, J ¼ 6.5 Hz, 2H), 2.01e1.97 (m, 2H),1.93e1.90 (m, 2H); ESI-MS
m/z: 373.0 [M þ H]þ.
4.1.4.4. 7-((5-Bromopentyl)oxy)-2-phenyl-4H-chromen-4-one (4d).
Intermediate 3a was treated with 1,5-dibromopentane according to
the general procedure to give the desired product 4d as a light
yellow solid, yield 78.0%; 1H NMR (500 MHz, DMSO-d6)
d 8.10 (m,
2H), 7.94 (d, J ¼ 8.5 Hz, 1H), 7.61e7.58 (m, 3H), 7.34 (d, J ¼ 2.0 Hz,
1H), 7.07 (dd, J ¼ 8.5, 2.0 Hz, 1H), 6.97 (s, 1H), 4.16 (t, J ¼ 6.5 Hz, 2H),
3.58 (t, J ¼ 6.5 Hz, 2H), 1.93e1.87 (m, 2H), 1.85e1.79 (m, 2H), 1.61e
1.54 (m, 2H); ESI-MS m/z: 387.0 [M þ H]þ.
4.1.2. 5,7-Dihydroxy-chromen-4-one (5)
BF3$Et2O (7.6 mL, 60 mmol) was added drop wise to a solution of
acetylphloroglucinol 1b (2.8 g,15 mmol) in anhydrous DMF (25 mL)
for 15 min. A solution of methanesulfonyl chloride (3.5 mL,
45 mmol) in anhydrous DMF (10 mL) was added to the mixture,
which was heated at 90 ꢁC for 3 h, cooled, and slowly poured into
ice-water (50 mL) under strong stirring. The crude product was
filtered off and washed with water to afford the dark yellow solid.
The crude product was purified by silica gel chromatography with
ethyl acetate and petroleum ether (1:3) to afford 5 as yellow solid.
Yield 91.0%, ESI-MS m/z: 179.0 [M þ H]þ.
4.1.4.5. 7-((6-Bromohexyl)oxy)-2-phenyl-4H-chromen-4-one
(4e).
Intermediate 3a was treated with 1,6-dibromohexane according to
the general procedure to give the desired product 4e as a light
yellow solid, yield 75.5%; 1H NMR (500 MHz, DMSO-d6)
d 8.10 (m,
2H), 7.94 (d, J ¼ 9.0, 2H), 7.61e7.56 (m, 3H), 7.33 (d, J ¼ 2.0 Hz, 1H),
7.06 (dd, J ¼ 9.0, 2.0 Hz, 1H), 6.97 (s, 1H), 4.15 (t, J ¼ 6.5 Hz, 2H), 3.54
(t, J ¼ 6.5 Hz, 2H),1.88e1.75 (m, 4H),1.52e1.40 (m, 4H); ESI-MS m/z:
401.1 [M þ H]þ.
4.1.4.6. 7-(3-Bromopropoxy)-5-hydroxy-2-phenyl-4H-chromen-4-
one (4f). Intermediate 3b was treated with 1,3-dibromopropane
according to the general procedure to give the desired product 4f
as a light yellow solid, yield 70.5%; 1H NMR (500 MHz, DMSO-d6)
4.1.3. 7-Hydroxychromone (6)
Perchloric acid (70%, 0.7 mL) was slowly added into a suspension
of 2,4-dihydroxyacetophenone 1a (1.0 g, 6.7 mmol) in triethyl
orthoformate (6 mL) with stirring. The mixture was stirred
d
12.82 (s, 1H), 8.17e8.12 (m, 2H), 7.67e7.60 (m, 3H), 7.07 (s, 1H),

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Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
6.88 (d, J ¼ 2.0 Hz, 1H), 6.44 (d, J ¼ 2.0 Hz, 1H), 4.25 (t, J ¼ 6.0 Hz,
2H), 3.70 (t, J ¼ 6.5 Hz, 2H), 2.33e2.30 (m, 2H); ESI-MS m/z: 375.0
[M þ H]þ.
general procedure to give the desired product 4g as a yellow solid,
yield 65.0%; 1H NMR (500 MHz, DMSO-d6)
8.06e8.04 (m, 2H),
7.61e7.53 (m, 3H), 6.92 (d, J ¼ 2.5 Hz, 1H), 6.78 (s, 1H), 6.54 (d,
J ¼ 2.5 Hz,1H), 4.25 (t, J ¼ 6.0 Hz, 2H), 3.84 (s, 3H), 3.70 (t, J ¼ 6.0 Hz,
2H), 2.33e2.30 (m, 2H); ESI-MS m/z: 389.0 [M þ H]þ.
d
4.1.4.7. 7-(3-Bromopropoxy)-2-(p-tolyl)-4H-chromen-4-one
(4h).
Intermediate 3c was treated with 1,3-dibromopropane according to
the general procedure to give the desired product 4h as a light
4.1.5.2. 7-(3-Bromopropoxy)-5-methoxy-4H-chromen-4-one
(7b).
yellow solid, yield 72.5%; 1H NMR (500 MHz, DMSO-d6)
d
8.01e7.99
Intermediate 7a was treated with MeI according to the general
procedure to give the desired product 7b as a yellow solid, yield
(m, 2H), 7.94 (d, J ¼ 8.5 Hz, 1H), 7.40e7.37 (m, 3H), 7.08 (dd, J ¼ 9.0,
2.5 Hz, 1H), 6.92 (s, 1H), 4.26 (t, J ¼ 6.0 Hz, 2H), 3.70 (t, J ¼ 6.5 Hz,
2H), 2.40 (s, 3H), 2.33e2.30 (m, 2H); ESI-MS m/z: 373.0 [M þ H]þ.
62.0%; 1H NMR (500 MHz, DMSO-d6)
d
7.60 (d, J ¼ 6.0 Hz, 1H), 6.44
(d, J ¼ 2.0 Hz,1H), 6.36 (d, J ¼ 2.0 Hz,1H), 6.18 (d, J ¼ 6.0 Hz,1H), 4.18
(t, J ¼ 5.5 Hz, 2H), 3.94 (s, 3H), 3.62 (t, J ¼ 6.5 Hz, 2H), 2.38e2.34 (m,
2H); ESI-MS m/z: 313.0 [M þ H]þ.
4.1.4.8. 7-(3-Bromopropoxy)-2-(4-methoxyphenyl)-4H-chromen-4-
one (4i). Intermediate 3d was treated with 1,3-dibromopropane
according to the general procedure to give the desired product 4i
as a light yellow solid, yield 75.0%; 1H NMR (500 MHz, DMSO-d6)
4.1.6. 2-(4-Aminophenyl)-7-(3-bromopropoxy)-4H-chromen-4-one
(4l)
d
8.08e8.06 (m, 2H), 7.94 (d, J ¼ 9.0 Hz, 1H), 7.36 (d, J ¼ 2.5 Hz, 1H),
A mixture of 4k (0.1 g, 0.25 mmol), SnCl2 (0.3 mg, 1.5 mmol), and
EtOH (7 mL) was stirred under reflux for 1 h. After the mixture was
cooled to room temperature, NaOH (1 M, 50 mL) was added until
the mixture became alkaline. After extraction with ethyl acetate,
the combined organic layers were washed with brine, dried over
anhydrous Na2SO4, and evaporated to give 4l as a yellow solid, yield
7.13e7.12 (m, 2H), 7.08 (dd, J ¼ 9.0, 2.5 Hz, 1H), 6.87 (s, 1H), 4.26 (t,
J ¼ 6.0 Hz, 2H), 3.87 (s, 3H), 3.71 (t, J ¼ 6.5 Hz, 2H), 2.36e2.28 (m,
2H); ESI-MS m/z: 389.0 [M þ H]þ.
4.1.4.9. 7-(3-Bromopropoxy)-2-(4-chlorophenyl)-4H-chromen-4-one
(4j). Intermediate 3e was treated with 1,3-dibromopropane ac-
cording to the general procedure to give the desired product 4j as a
light yellow solid, yield 82.3%; 1H NMR (500 MHz, DMSO-d6)
92.0%; 1H NMR (500 MHz, CDCl3 þ CD3OD)
7.77 (dd, J ¼ 7.0, 2.0 Hz,
d
2H), 7.36 (d, J ¼ 2.5 Hz, 1H), 7.00 (d, J ¼ 2.5 Hz, 1H), 6.80 (dd, J ¼ 7.0,
2.0 Hz, 2H), 6.65 (s, 1H), 4.26 (t, J ¼ 6.0 Hz, 2H), 3.65 (t, J ¼ 6.0 Hz,
2H), 2.43e2.38 (m, 2H); ESI-MS m/z: 374.0 [M þ H]þ.
d
8.15e8.13 (m, 2H), 7.97 (d, J ¼ 9.0 Hz, 1H), 7.67e7.65 (m, 2H), 7.39
(d, J ¼ 2.5 Hz, 1H), 7.09 (dd, J ¼ 9.0, 2.5 Hz, 1H), 7.01 (s, 1H), 4.27 (t,
J ¼ 6.0 Hz, 2H), 3.70 (t, J ¼ 6.5 Hz, 2H), 2.36e2.31 (m, 2H); ESI-MS m/
z: 393.0 [M þ H]þ.
4.1.7. 7-(3-Bromopropoxy)-2-(4-(dimethylamino)phenyl)-4H-
chromen-4-one (4m)
To a stirred mixture of 4l (90.0 mg, 0.24 mmol) and para-
formaldehyde (71.0 mg, 2.4 mmol) in AcOH (10 mL) was added
NaCNBH3 (74.5 mg, 1.2 mmol) in one portion at room temperature.
The resulting mixture was stirred at room temperature for 3 h, and
NaOH (1 M, 50 mL) was added followed by extraction with CH2Cl2.
The organic phase was dried over anhydrous Na2SO4. The solvent
was removed, and the residue was purified by silica gel chroma-
tography with hexane/ethyl acetate (4:1) to give 4m as a yellow
4.1.4.10. 7-(3-Bromopropoxy)-2-(4-nitrophenyl)-4H-chromen-4-one
(4k). Intermediate 3f was treated with 1,3-dibromopropane ac-
cording to the general procedure to give the desired product 4k as a
yellow solid, yield 76.5%; 1H NMR (500 MHz, DMSO-d6)
d 8.39e8.38
(m, 4H), 7.97 (d, J ¼ 9.0 Hz, 1H), 7.42 (d, J ¼ 2.5 Hz, 1H), 7.18 (s, 1H),
7.12 (dd, J ¼ 9.0, 2.5 Hz, 1H), 4.28 (t, J ¼ 6.0 Hz, 2H), 3.71 (t,
J ¼ 6.5 Hz, 2H), 2.35e2.32 (m, 2H); ESI-MS m/z: 404.0 [M þ H]þ.
solid, yield 81.3%; 1H NMR (500 MHz, CDCl3)
d 8.12e8.10 (m, 1H),
4.1.4.11. 7-(3-Bromopropoxy)-5-hydroxy-4H-chromen-4-one
(7a).
7.79 (dt, J ¼ 8.5, 3.0 Hz, 2H), 6.95e6.93 (m, 2H), 6.75 (dt, J ¼ 9.0,
3.0 Hz, 2H), 6.64 (s, 1H), 4.23 (t, J ¼ 6.0 Hz, 2H), 3.63 (t, J ¼ 6.5 Hz,
2H), 3.06 (s, 6H), 2.41e2.36 (m, 2H); ESI-MS m/z: 402.1 [M þ H]þ.
Intermediate 5 was treated with 1,3-dibromopropane according to
the general procedure to give the desired product 7a as a yellow
solid, yield 70.5%; 1H NMR (500 MHz, DMSO-d6)
d 12.70 (s,1H), 8.27
(d, J ¼ 6.0 Hz, 1H), 6.66 (d, J ¼ 2.5 Hz, 1H), 6.42 (d, J ¼ 2.5 Hz, 1H),
6.35 (d, J ¼ 6.0 Hz,1H), 4.20 (t, J ¼ 6.0 Hz, 2H), 3.66 (t, J ¼ 6.5 Hz, 2H),
2.29e2.24 (m, 2H); ESI-MS m/z: 299.0 [M þ H]þ.
4.1.8. General procedures for the preparation of intermediate 10a
and 10b
A mixture of 9 (10 mmol) and aminoethanol or 3-amino-1-
propano (60 mmol) was heated to 160 ꢁC for 8 h. When the reac-
tion was completed, it was diluted with CH2Cl2, washed with water,
followed by brine solution. The organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The
obtained residue was purified by silica gel chromatography with
CH2Cl2/MeOH (20:1) as eluent to give compounds 10a and 10b as
pale white solid.
4.1.4.12. 7-(3-Bromopropoxy)-4H-chromen-4-one
(7c).
Intermediate 6 was treated with 1,3-dibromopropane according to
the general procedure to give the desired product 7c as a yellow
solid, yield 82.5%; 1H NMR (500 MHz, DMSO-d6)
d
8.12 (d, J ¼ 9.0 Hz,
1H), 7.91 (d, J ¼ 6.0 Hz, 1H), 6.98 (dd, J ¼ 8.5, 2.0 Hz, 1H), 6.86 (d,
J ¼ 2.0 Hz,1H), 6.30 (d, J ¼ 6.0 Hz,1H), 4.21 (t, J ¼ 5.5 Hz, 2H), 3.62 (t,
J ¼ 6.5 Hz, 2H), 2.40e2.35 (m, 2H); ESI-MS m/z: 283.0 [M þ H]þ.
4.1.8.1. 2-((1,2,3,4-Tetrahydroacridin-9-yl)amino)ethanol
(10a).
4.1.5. General procedures for the preparation of intermediate 4g
and 7b
Intermediate 9 was treated with aminoethanol according to the
general procedure to give the desired product 10a as a pale white
A mixture of 4f/7a (5 mmol) with MeI (1.4 g, 10 mmol) and
anhydrous K2CO3 (1.4 g, 10 mmol) in acetone (15 mL) was refluxed
under stirring for 2 h. After cooling, the reaction mixture was
filtered and the filtrate was evaporated under reduced pressure.
The obtained residue was purified by silica gel chromatography
with hexane/acetone (20:1) as eluent to give compounds 4g and 7b.
solid, yield 81.0%; 1H NMR (500 MHz, CDCl3)
d
8.00 (d, J ¼ 8.5 Hz,
1H), 7.94 (d, J ¼ 8.5 Hz, 1H), 7.56 (t, J ¼ 7.5 Hz, 1H), 7.36 (t, J ¼ 6.0 Hz,
1H), 3.84 (t, J ¼ 5.5 Hz, 2H), 3.64 (br s, 2H), 3.07 (d, J ¼ 6.0 Hz, 2H),
2.77 (br s, 2H), 1.91 (t, J ¼ 3.5 Hz, 4H); ESI-MS m/z: 243.1 [M þ H]þ.
4.1.8.2. 3-((1,2,3,4-Tetrahydroacridin-9-yl)amino)propan-1-ol (10b).
Intermediate 9 was treated with 3-amino-1-propano according to
the general procedure to give the desired product 10b as a pale
4.1.5.1. 7-(3-Bromopropoxy)-5-methoxy-2-phenyl-4H-chromen-4-
one (4g). Intermediate 4f was treated with MeI according to the
white solid, yield 78.5%; 1H NMR (500 MHz, CDCl3)
d 8.02 (d,

Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
641
J ¼ 8.5 Hz, 1H), 7.95 (d, J ¼ 8.5 Hz, 1H), 7.56 (t, J ¼ 6.5 Hz, 1H), 7.34 (t,
J ¼ 6.5 Hz,1H), 3.94 (t, J ¼ 5.5 Hz, 2H), 3.72 (t, J ¼ 5.5 Hz, 2H), 3.05 (d,
J ¼ 6.0 Hz, 2H), 2.71 (d, J ¼ 5.5 Hz, 2H), 2.00e1.95 (m, 2H), 1.90e1.88
(m, 4H); ESI-MS m/z: 257.1 [M þ H]þ.
575.3 [M þ H]þ; HRMS: calcd for C36H39N4O3 [M þ H]þ, 575.3017,
found 575.3015.
4.1.10.2. 2-Phenyl-7-(3-(4-(2-((1,2,3,4-tetrahydroacridin-9-yl)
amino)ethyl)piperazin-1-yl)propoxy)-4H-chromen-4-one
(13b).
4.1.9. General procedures for the preparation of intermediate 12a
and 12b
Intermediate 12a was treated with 4b according to the general
procedure to give the desired product 13b as a pale white solid,
To a solution of 10 (5.0 mmol) and Et3N (6.0 mmol) in CH2Cl2
(10 mL) was drop wise added p-methyl benzene sulfonic chloride
(5.5 mmol) in CH2Cl2 (10 mL) at 0 ꢁC. The mixture was stirred for 2 h
at room temperature. When the reaction was completed, it was
diluted with CH2Cl2, washed with water, followed by brine solution.
The organic layer was dried over anhydrous Na2SO4 and concen-
trated under reduced pressure. The crude product 11 was dissolved
in DMF (40 mL) without purification. Anhydrous piperazine
(40 mmol) was added and stirred at 80 ꢁC for 2 h. After cooling, the
solvent was dissolvent in CH2Cl2 (40 mL) and then washed with
water (30 mL ꢄ 3). The organic phase was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The crude
product was purified by silica gel chromatography with CH2Cl2/
MeOH (20:1) as eluent to afford compound 12 as yellow oil.
yield 50.4%, m.p. 76e78 ꢁC; IR (KBr)
n
3345, 2935, 2812, 1640, 1569,
1498, 1447, 1374, 1162, 1088, 769, 681 cmꢀ1 1H NMR (500 MHz,
CDCl3):
;
d
8.14 (dd, J ¼ 6.0, 3.0 Hz, 1H), 8.05e8.00 (m, 2H), 7.92e7.90
(m, 2H), 7.57 (t, J ¼ 7.5 Hz, 1H), 7.53e7.50 (m, 3H), 7.35 (t, J ¼ 7.5 Hz,
1H), 7.00e6.99 (m, 2H), 6.77 (s, 1H), 4.17 (t, J ¼ 6.0 Hz, 2H), 3.62 (br
s, 2H), 3.11 (br s, 2H), 2.75 (br s, 2H), 2.64 (t, J ¼ 5.5 Hz, 2H), 2.61e
2.58 (m, 8H), 2.08e2.03 (m, 4H), 1.93 (t, J ¼ 3.5 Hz, 4H); 13C NMR
(125 MHz, CDCl3):
d 178.3, 164.1, 163.5, 158.5, 155.9, 151.5, 138.9,
132.4, 132.0, 131.8, 129.5, 127.8, 126.6, 124.2, 123.4, 121.1, 118.3, 115.1,
110.1, 108.1, 115.1, 110.1, 108.1, 101.5, 100.4, 67.3, 57.8, 55.3, 54.0, 53.1,
45.4, 30.1, 27.0, 25.2, 23.4, 23.0; ESI-MS m/z: 589.3 [M þ H]þ; HRMS:
calcd for C37H41N4O3 [M þ H]þ, 589.3173, found 589.3170.
4.1.10.3. 2-Phenyl-7-(4-(4-(2-((1,2,3,4-tetrahydroacridin-9-yl)
amino)ethyl)piperazin-1-yl)butoxy)-4H-chromen-4-one
(13c).
4.1.9.1. N-(2-(Piperazin-1-yl)ethyl)-1,2,3,4-tetrahydroacridin-9-
amine (12a). Intermediate 10a was treated with p-methyl benzene
sulfonic chloride to afford 11a followed by piperazine according to
the general procedure to give the desired product 12a as yellow
Intermediate 12a was treated with 4c according to the general
procedure to give the desired product 13c as a pale white solid,
yield 52.3%, m.p. 75e77 ꢁC; IR (KBr)
n
3347, 3056, 2932, 2810, 1639,
1603, 1499, 1447, 1374, 1248, 1161, 1088, 769, 689 cmꢀ1 1H NMR
(500 MHz, CDCl3):
;
oil, yield 58.5%; 1H NMR (500 MHz, CDCl3)
d
8.06 (d, J ¼ 8.5 Hz,
d
8.13 (d, J ¼ 8.5 Hz, 2H), 8.07 (d, J ¼ 8.5 Hz, 1H),
2H), 7.59 (t, J ¼ 7.0 Hz, 1H), 7.36 (t, J ¼ 8.0 Hz, 1H), 3.65 (br s, 2H),
3.14 (br s, 2H), 2.95 (t, J ¼ 5.0 Hz, 2H), 2.74 (br s, 2H), 2.63 (t,
J ¼ 6.0 Hz, 4H), 2.53e2.51 (m, 4H), 1.92e1.90 (m, 4H); ESI-MS m/z:
311.2 [M þ H]þ.
7.91e7.89 (m, 2H), 7.60 (t, J ¼ 7.5 Hz, 1H), 7.55e7.50 (m, 3H), 7.37 (t,
J ¼ 8.0 Hz, 1H), 6.99e6.96 (m, 2H), 6.76 (s, 1H), 4.12 (t, J ¼ 6.0 Hz,
2H), 3.69 (br s, 2H), 3.16 (br s, 2H), 2.71 (br s, 2H), 2.66 (t, J ¼ 5.5 Hz,
2H), 2.64e2.48 (m, 6H), 2.47 (t, J ¼ 7.5 Hz, 4H), 1.93e1.89 (m, 6H),
1.76e1.74 (m, 2H); 13C NMR (125 MHz, CDCl3):
d 178.0, 163.8, 163.2,
4.1.9.2. N-(3-(Piperazin-1-yl)propyl)-1,2,3,4-tetrahydroacridin-9-
amine (12b). Intermediate 10b was treated with p-methyl benzene
sulfonic chloride to afford 11b followed by piperazine according to
the general procedure to give the desired product 12b as yellow oil,
158.1, 156.1, 149.7, 138.9, 132.1, 131.6, 129.2, 127.2, 126.3, 123.3, 118.0,
114.8, 107.8, 101.2, 68.6, 58.2, 57.2, 53.6, 52.8, 44.8, 32.1, 29.8, 27.1,
24.5, 23.6, 22.9, 22.3; ESI-MS m/z: 603.3 [M þ H]þ; HRMS: calcd for
C
38H43N4O3 [M þ H]þ, 603.3330, found 603.3327.
yield 50.6%; 1H NMR (500 MHz, CDCl3)
d
8.05 (d, J ¼ 8.5 Hz, 1H),
8.00 (d, J ¼ 8.5 Hz, 1H), 7.56 (t, J ¼ 7.0 Hz, 1H), 7.34 (t, J ¼ 7.0 Hz, 1H),
3.66 (t, J ¼ 6.0 Hz, 2H), 3.10 (br s, 2H), 3.00e2.88 (m, 6H), 2.59e2.50
(m, 6H), 1.92 (t, J ¼ 3.0 Hz, 4H), 1.89e1.85 (m, 2H); ESI-MS m/z:
325.2 [M þ H]þ.
4.1.10.4. 2-Phenyl-7-((5-(4-(2-((1,2,3,4-tetrahydroacridin-9-yl)
amino)ethyl)piperazin-1-yl)pentyl)oxy)-4H-chromen-4-one (13d).
Intermediate 12a was treated with 4d according to the general
procedure to give the desired product 13d as a pale white solid,
yield 50.5%, m.p. 86e88 ꢁC; IR (KBr)
n
3419, 2931, 2811, 2353, 1641,
1602, 1518, 1447, 1374, 1248, 1161, 1087, 772, 676 cmꢀ1 1H NMR
(500 MHz, CDCl3):
8.27 (d, J ¼ 5.5 Hz, 1H), 8.13 (d, J ¼ 9.0 Hz, 1H),
4.1.10. General procedures for the preparation of compounds 13aeu
A mixture of 12aeb (2.5 mmol), anhydrous K2CO3 (2.5 mmol)
and the corresponding flavonoid derivatives 4aem or 7aec
(2.5 mmol) in dry acetonitrile was refluxed for 8 h. After cooling to
the room temperature, the mixture was filtered and the filtrate was
evaporated under vacuum. The obtained residue was purified by
silica gel chromatography with CH2Cl2/MeOH (15:1) as eluent to
yield target compounds 13aeu.
;
d
8.09 (d, J ¼ 8.5 Hz, 1H), 7.92e7.90 (m, 2H), 7.64 (t, J ¼ 8.0 Hz, 1H),
7.55e7.50 (m, 3H), 7.39 (t, J ¼ 8.0 Hz,1H), 6.99e6.95 (m, 2H), 6.77 (s,
1H), 4.10 (t, J ¼ 6.0 Hz, 2H), 3.79e3.74 (m, 2H), 3.24e3.23 (m, 2H),
2.70 (t, J ¼ 5.5 Hz, 2H), 2.69e2.66 (m, 2H), 2.65e2.45 (m, 6H), 2.45
(t, J ¼ 7.5 Hz, 2H), 2.07 (br s, 2H), 1.93e1.86 (m, 6H), 1.66e1.61 (m,
2H), 1.59e1.55 (m, 2H); 13C NMR (125 MHz, CDCl3):
d 177.9, 163.7,
163.1, 158.1, 155.9, 152.2, 139.5, 132.0, 131.9, 131.4, 129.0, 127.1, 126.2,
124.4, 123.3, 117.8, 115.7, 114.7, 110.2, 107.6, 101.0, 68.5, 58.4, 56.8,
53.4, 52.4, 44.3, 31.9, 28.9, 26.5, 24.0, 22.5, 21.9, 21.7; ESI-MS m/z:
617.3 [M þ H]þ; HRMS: calcd for C39H45N4O3 [M þ H]þ, 617.3486,
found 617.3488.
4.1.10.1. 2-Phenyl-7-(2-(4-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)
ethyl)piperazin-1-yl)ethoxy)-4H-chromen-4-one
(13a).
Intermediate 12a was treated with 4a according to the general
procedure to give the desired product 13a as a pale white solid,
yield 48.5%, m.p. 89e93 ꢁC; IR (KBr)
n
3424, 2935, 2814, 1636, 1602,
1499, 1447, 1375, 1260, 1163, 1088, 773, 676 cmꢀ1 1H NMR
(500 MHz, CDCl3):
;
4.1.10.5. 2-Phenyl-7-((6-(4-(2-((1,2,3,4-tetrahydroacridin-9-yl)
d
8.14 (d, J ¼ 8.5 Hz, 1H), 8.10 (d, J ¼ 9.0 Hz, 1H),
amino)ethyl)piperazin-1-yl)hexyl)oxy)-4H-chromen-4-one
(13e).
7.90 (dd, J ¼ 7.5, 2.0 Hz, 2H), 7.65 (t, J ¼ 7.5 Hz, 1H), 7.56e7.50 (m,
3H), 7.40 (t, J ¼ 7.5 Hz, 1H), 7.02e7.00 (m, 2H), 6.77 (s, 1H), 4.25 (t,
J ¼ 5.5 Hz, 2H), 3.81 (br s, 2H), 3.23 (br s, 2H), 2.92 (t, J ¼ 5.5 Hz, 2H),
2.72e2.63 (m, 12H), 1.93e1.92 (m, 4H); 13C NMR (125 MHz, CDCl3):
Intermediate 12a was treated with 4e according to the general
procedure to give the desired product 13e as a pale white solid,
yield 48.5%, m.p. 88e90 ꢁC; IR (KBr)
n
3398, 3064, 2934, 2809, 2313,
1640, 1603, 1498, 1374, 1248, 1161, 1088, 771, 689 cmꢀ1 1H NMR
(500 MHz, CDCl3):
;
d
177.8, 163.3, 163.1, 158.0, 155.8, 151.7, 139.0, 132.1, 131.9, 131.5,
d
8.50 (br s, 1H), 8.13 (d, J ¼ 8.5 Hz, 2H), 7.92e
129.1, 127.2, 126.2, 124.5, 123.4, 121.8, 118.1, 115.5, 114.6, 107.7, 101.3,
66.9, 56.9, 56.7, 53.9, 52.4, 44.2, 29.1, 24.0, 22.5, 21.6; ESI-MS m/z:
7.90 (m, 2H), 7.68 (t, J ¼ 8.0 Hz, 1H), 7.53e7.51 (m, 3H), 7.42 (t,
J ¼ 7.5 Hz, 1H), 6.99e6.96 (m, 2H), 6.77 (s, 1H), 4.09 (t, J ¼ 6.0 Hz,

642
Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
2H), 3.88 (br s, 2H), 3.31 (br s, 2H), 2.62e2.45 (m, 8H), 2.40 (t,
J ¼ 7.5 Hz, 2H), 1.96e1.90 (m, 4H), 1.90e1.85 (m, 2H), 1.60e1.51 (m,
procedure to give the desired product 13i as a pale white solid, yield
49.2%, m.p. 75e78 ꢁC; IR (KBr)
3420, 2934, 2810, 1639, 1602, 1499,
1446, 1373, 1248, 1159, 1087, 771, 680 cmꢀ1 1H NMR (500 MHz,
CDCl3):
7.63 (m, 1H), 7.53e7.50 (m, 3H), 7.37 (t, J ¼ 7.5 Hz, 1H), 6.99e6.96
(m, 2H), 6.77 (s,1H), 4.10 (t, J ¼ 6.0 Hz, 2H), 3.85 (br s, 2H), 3.23 (br s,
2H), 2.67e2.63 (m, 6H), 2.60e2.52 (m, 4H), 2.42 (t, J ¼ 7.5 Hz, 2H),
1.92e1.87 (m, 8H), 1.65e1.55 (m, 6H); 13C NMR (125 MHz, CDCl3):
n
6H), 1.46e1.40 (m, 2H); 13C NMR (125 MHz, CDCl3):
d
177.8, 163.7,
;
163.0, 158.1, 139.6, 132.2, 132.0, 131.4, 129.0, 127.1, 126.2, 125.0,
123.6, 121.2, 117.8, 115.2, 114.7, 109.7, 107.6, 101.0, 68.6, 58.5, 56.3,
53.6, 52.4, 49.0, 29.0, 27.2, 26.9, 26.0, 25.4, 23.7, 22.1, 21.1; ESI-MS
m/z: 631.4 [M þ H]þ; HRMS: calcd for C40H47N4O3 [M þ H]þ,
631.3643, found 631.3647.
d
8.29 (br s, 1H), 8.13 (d, J ¼ 9.0 Hz, 2H), 7.92e7.90 (m, 2H),
d
177.8, 163.7, 163.0, 158.0, 155.7, 152.0, 138.2, 132.3, 132.0, 131.4,
4.1.10.6. 2-Phenyl-7-(2-(4-(3-((1,2,3,4-tetrahydroacridin-9-yl)
129.0, 127.1, 126.2, 124.2, 123.7, 121.2, 117.8, 115.8, 114.7, 111.6, 107.6,
101.0, 68.6, 58.6, 58.2, 53.9, 49.9, 31.0, 28.9, 26.6, 26.2, 25.7, 24.0,
amino)propyl)piperazin-1-yl)ethoxy)-4H-chromen-4-one
(13f).
Intermediate 12b was treated with 4a according to the general
procedure to give the desired product 13f as a pale white solid,
22.4, 21.9; ESI-MS m/z: 631.3 [M
C
þ
H]þ; HRMS: calcd for
40H47N4O3 [M þ H]þ, 631.3643, found 631.3644.
yield 53.0%, m.p. 128e130 ꢁC; IR (KBr)
n
3398, 3056, 2934, 2812,
2320, 1631, 1597, 1523, 1447, 1375, 1249, 1164, 1088, 775, 676 cmꢀ1
1H NMR (500 MHz, CDCl3):
8.62 (d, J ¼ 8.5 Hz, 1H), 8.24 (d,
;
4.1.10.10. 2-Phenyl-7-((6-(4-(3-((1,2,3,4-tetrahydroacridin-9-yl)
amino)propyl)piperazin-1-yl)hexyl)oxy)-4H-chromen-4-one (13j).
Intermediate 12b was treated with 4e according to the general
procedure to give the desired product 13j as a pale white solid, yield
d
J ¼ 9.0 Hz, 1H), 8.14 (d, J ¼ 9.0 Hz, 1H), 7.91 (dd, J ¼ 7.5, 2.5 Hz, 2H),
7.69 (t, J ¼ 8.0 Hz, 1H), 7.56e7.54 (m, 3H), 7.42 (t, J ¼ 7.5 Hz, 1H),
7.05e6.97 (m, 2H), 6.77 (s, 1H), 4.25 (d, J ¼ 5.0 Hz, 2H), 4.14 (d,
J ¼ 5.0 Hz, 2H), 3.36 (t, J ¼ 6.0 Hz, 2H), 2.96e2.92 (m, 2H), 2.83e2.65
(m, 8H), 2.62 (d, J ¼ 6.5 Hz, 2H), 2.06e2.00 (m, 2H), 1.94 (d,
J ¼ 5.0 Hz, 2H), 1.88 (t, J ¼ 5.0 Hz, 2H), 1.73e1.59 (m, 2H); 13C NMR
52.0%, m.p. 104e106 ꢁC; IR (KBr)
n
3422, 2932, 2311, 1632, 1601,
1524, 1447, 1374, 1355, 1247, 1166, 1087, 772, 678 cmꢀ1 1H NMR
(500 MHz, CDCl3):
8.62 (d, J ¼ 8.0 Hz, 1H), 8.23 (d, J ¼ 8.5 Hz, 1H),
;
d
8.13 (d, J ¼ 8.5 Hz, 1H), 7.92e7.90 (m, 2H), 7.69 (t, J ¼ 7.5 Hz, 1H),
7.53e7.50 (m, 3H), 7.42 (t, J ¼ 7.0 Hz, 1H), 7.00e6.96 (m, 2H), 6.76 (s,
1H), 4.12e4.08 (m, 4H), 3.52 (br s, 2H), 2.75e2.74 (m, 6H), 2.61e
2.57 (m, 4H), 2.45 (br s, 2H), 1.98e1.93 (m, 4H), 1.89e1.85 (m, 4H),
1.60e1.52 (m, 6H), 1.48e1.43 (m, 2H); 13C NMR (125 MHz, CDCl3):
(125 MHz, CDCl3):
d 177.7, 163.2, 162.7, 158.0, 155.8, 151.3, 151.2,
139.0, 132.3, 131.9, 131.5, 129.1, 127.3, 126.2, 125.0, 124.3, 121.1, 118.2,
115.9, 114.6, 110.7, 107.7, 101.3, 66.8, 57.0, 56.5, 53.7, 52.8, 29.7, 28.4,
25.6, 25.2, 21.8, 20.8; ESI-MS m/z: 589.3 [M þ H]þ; HRMS: calcd for
C
37H41N4O3 [M þ H]þ, 589.3173, found 589.3176.
d 177.8,163.7,163.1,158.1,155.8,151.2,139.1,132.2,131.9,131.4,129.1,
127.1, 126.2, 124.9, 124.4, 121.1, 117.8, 115.9, 114.7, 107.6, 101.0, 68.6,
58.5, 53.6, 52.5, 49.9, 29.7, 28.9, 28.4, 27.1, 25.6, 25.4, 21.8, 20.8; ESI-
MS m/z: 645.4 [M þ H]þ; HRMS: calcd for C41H49N4O3 [M þ H]þ,
645.3799, found 645.3805.
4.1.10.7. 2-Phenyl-7-(3-(4-(3-((1,2,3,4-tetrahydroacridin-9-yl)
amino)propyl)piperazin-1-yl)propoxy)-4H-chromen-4-one (13g).
Intermediate 12b was treated with 4b according to the general
procedure to give the desired product 13g as a pale white solid,
yield 46.5%, m.p. 99e101 ꢁC; IR (KBr)
n
3422, 2936, 2814, 2319,1634,
1602, 1447, 1375, 1250, 1165, 1088, 771, 677 cmꢀ1 1H NMR
(500 MHz, CDCl3): 8.41 (br s, 1H), 8.17e8.13 (m, 2H), 7.92e7.90 (m,
4 .1.10 .11. 5 - H y d ro x y - 2 - p h e nyl - 7 - ( 3 - ( 4 - ( 3 - ( ( 1, 2 , 3 , 4 -
tetrahydroacridin-9-yl)amino)propyl)piperazin-1-yl)propoxy)-4H-
chromen-4-one (13k). Intermediate 12b was treated with 4f ac-
cording to the general procedure to give the desired product 13k as
;
d
2H), 7.66 (t, J ¼ 7.5 Hz,1H), 7.55e7.51 (m, 3H), 7.39 (t, J ¼ 8.0 Hz,1H),
7.01e6.99 (m, 2H), 6.77 (s,1H), 4.18 (t, J ¼ 6.0 Hz, 2H), 3.94 (br s, 2H),
3.28 (br s, 2H), 2.66 (m, 6H), 2.61e2.58 (m, 6H), 2.09e2.04 (m, 2H),
1.96e1.90 (m, 6H), 1.60 (br s, 2H); 13C NMR (125 MHz, CDCl3):
a light yellow solid, yield 45.0%, m.p. 84e86 ꢁC; IR (KBr)
n
3423,
2932, 2815, 2320, 1659, 1614, 1586, 1502, 1450, 1351, 1167, 767,
678 cmꢀ1; 1H NMR (500 MHz, CDCl3):
12.72 (s, 1H), 8.28 (br s, 1H),
d
d
177.8, 163.6, 163.1, 158.0, 154.5, 154.4, 131.9, 131.5, 131.0, 129.0,
8.13 (d, J ¼ 8.0 Hz, 1H), 7.89e7.88 (m, 2H), 7.63 (t, J ¼ 7.0 Hz, 1H),
7.57e7.48 (m, 3H), 7.37 (t, J ¼ 7.5 Hz, 1H), 6.67 (s, 1H), 6.51 (d,
J ¼ 2.0 Hz, 1H), 6.38 (d, J ¼ 2.0 Hz, 1H), 4.15e4.10 (m, 2H), 3.86 (br s,
2H), 3.22 (br s, 2H), 2.67e2.64 (m, 6H), 2.58e2.55 (m, 6H), 2.05e
2.00 (m, 2H), 1.93e1.92 (m, 6H), 1.26 (t, J ¼ 7.5 Hz, 2H); 13C NMR
128.4, 127.1, 126.2, 124.4, 124.0, 123.3, 117.9, 117.1, 114.7, 112.2, 107.6,
101.1, 66.8, 58.3, 54.9, 54.0, 52.9, 50.0, 29.9, 29.7, 26.6, 25.8, 25.7,
22.1, 21.4; ESI-MS m/z: 603.3 [M
þ
H]þ; HRMS: calcd for
C
38H43N4O3 [M þ H]þ, 603.3330, found 603.3331.
(125 MHz, CDCl3):
d 182.5, 165.1, 164.0, 162.3, 157.9, 156.1, 132.9,
4.1.10.8. 2-Phenyl-7-(4-(4-(3-((1,2,3,4-tetrahydroacridin-9-yl)
amino)propyl)piperazin-1-yl)butoxy)-4H-chromen-4-one (13h).
Intermediate 12b was treated with 4c according to the general
procedure to give the desired product 13h as a pale white solid,
131.8, 131.4, 129.1, 126.3, 124.2, 123.7, 105.9, 105.7, 98.6, 93.3, 66.8,
60.4, 58.2, 54.9, 53.9, 53.0, 49.8, 29.7, 26.6, 25.7, 22.4, 21.8; ESI-MS
m/z: 619.3 [M þ H]þ; HRMS: calcd for C38H43N4O4 [M þ H]þ,
619.3279, found 619.3282.
yield 50.2%, m.p. 165e166 ꢁC; IR (KBr)
n
3422, 3239, 2933, 2812,
2320, 1633, 1597, 1518, 1448, 1356, 1244, 1085, 785, 750 cmꢀ1 1H
NMR (500 MHz, CDCl3):
8.61 (d, J ¼ 8.5 Hz, 1H), 8.23 (d, J ¼ 8.5 Hz,
;
4 .1.10 .12 . 5 - M e t h o x y- 2 -p h e nyl - 7 - ( 3 - ( 4 - ( 3 - ( ( 1, 2 , 3 , 4 -
tetrahydroacridin-9-yl)amino)propyl)piperazin-1-yl)propoxy)-4H-
chromen-4-one (13l). Intermediate 12b was treated with 4g ac-
cording to the general procedure to give the desired product 13l as
d
1H), 8.13 (d, J ¼ 8.5 Hz, 1H), 7.91e7.89 (m, 2H), 7.68 (t, J ¼ 8.0 Hz,
1H), 7.54e7.51 (m, 3H), 7.41 (t, J ¼ 8.0 Hz, 1H), 6.99e6.96 (m, 2H),
6.76 (s, 1H), 4.15e4.12 (m, 4H), 3.45 (t, J ¼ 5.5 Hz, 2H), 2.73 (m, 6H),
2.62e2.60 (m, 6H), 2.51 (br s, 2H), 1.98 (br s, 2H), 1.96e1.88 (m, 6H),
a light yellow solid, yield 45.2%, m.p. 113e115 ꢁC; IR (KBr)
n
3423,
2931, 2814, 2311, 1643, 1605, 1523, 1449, 1348, 1164, 1118, 767,
693 cmꢀ1 1H NMR (500 MHz, CDCl3):
8.48 (br s, 1H), 8.25 (d,
1.77 (br s, 2H); 13C NMR (125 MHz, CDCl3):
d
177.8, 163.6, 163.0,
;
d
159.0, 155.8, 151.2, 139.1, 131.5, 129.1, 127.1, 126.2, 124.4, 121.0, 117.9,
115.9, 114.7, 110.9, 107.6, 101.1, 68.3, 58.1, 53.8, 52.6, 50.1, 28.4, 26.9,
25.7, 25.2, 23.3, 21.8, 20.8; ESI-MS m/z: 617.3 [M þ H]þ; HRMS: calcd
for C39H45N4O3 [M þ H]þ, 617.3486, found 617.3489.
J ¼ 8.5 Hz, 1H), 7.88e7.86 (m, 2H), 7.65 (t, J ¼ 7.0 Hz, 1H), 7.53e7.47
(m, 3H), 7.41 (t, J ¼ 8.0 Hz, 1H), 6.68 (s, 1H), 6.58 (s, 1H), 6.38 (d,
J ¼ 2.0 Hz, 1H), 4.16e4.10 (m, 4H), 3.96 (s, 3H), 3.28 (br s, 2H), 2.88e
2.75 (m, 6H), 2.69e2.61 (m, 6H), 1.91e1.86 (m, 4H), 1.60e1.56 (m,
4H), 1.26 (t, J ¼ 7.0 Hz, 2H); 13C NMR (125 MHz, CDCl3):
d 177.8,
4.1.10.9. 2-Phenyl-7-((5-(4-(3-((1,2,3,4-tetrahydroacridin-9-yl)
amino)propyl)piperazin-1-yl)pentyl)oxy)-4H-chromen-4-one (13i).
Intermediate 12b was treated with 4d according to the general
163.4, 163.3, 157.9, 155.8, 151.2, 142.1, 132.3, 131.6, 129.7, 129.1, 127.0,
126.1, 125.1, 124.4, 120.8, 118.0, 115.8, 114.5, 111.0, 107.0, 101.2, 66.5,
58.0, 54.7, 28.4, 26.3, 25.5, 21.8, 21.5, 20.7; ESI-MS m/z: 633.3

Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
643
[M þ H]þ; HRMS: calcd for C39H45N4O4 [M þ H]þ, 633.3435, found
6.94 (m, 2H), 6.75 (d, J ¼ 9.0 Hz, 2H), 6.63 (s, 1H), 4.16 (t, J ¼ 6.0 Hz,
2H), 3.75 (br s, 2H), 3.17e3.16 (m, 2H), 3.07 (s, 6H), 2.71e2.69 (m,
6H), 2.65e2.57 (m, 8H), 2.08e2.02 (m, 2H), 1.94e1.87 (m, 6H); 13C
633.3437.
4.1.10.13. 7-(3-(4-(3-((1,2,3,4-Tetrahydroacridin-9-yl)amino)propyl)
NMR (125 MHz, CDCl3) d 177.8, 163.9, 163.2, 157.9, 152.4, 127.6,
piperazin-1-yl)propoxy)-2-(p-tolyl)-4H-chromen-4-one
Intermediate 12b was treated with 4h according to the general
procedure to give the desired product 13m as a pale white solid,
(13m).
126.9, 123.9, 123.4, 118.6, 117.9, 114.0, 111.7, 104.4, 101.1, 100.0,
66.8, 57.9, 55.0, 53.8, 53.1, 49.4, 30.9, 29.7, 26.7, 25.7, 22.7, 22.3; ESI-
MS m/z: 646.4 [M þ H]þ; HRMS: calcd for C40H48N5O3 [M þ H]þ,
646.3752, found 646.3755.
yield 46.5%, m.p. 121e124 ꢁC; IR (KBr)
n
3425, 2937, 2815, 2309,
1629, 1597, 1521, 1441, 1373, 1251, 1167, 1088, 822, 760 cmꢀ1 1H
NMR (500 MHz, CDCl3):
8.51 (d, J ¼ 8.5 Hz, 1H), 8.23 (d, J ¼ 8.5 Hz,
;
d
4.1.10.17. 2-(4-Chlorophenyl)-7-(3-(4-(3-((1,2,3,4-tetrahydroacridin-
9-yl)amino)propyl)piperazin-1-yl)propoxy)-4H-chromen-4-one
(13q). Intermediate 12b was treated with 4j according to the
general procedure to give the desired product 13q as a light yellow
1H), 8.10e8.08 (m, 1H), 7.77 (d, J ¼ 8.5 Hz, 2H), 7.63 (t, J ¼ 7.5 Hz,
1H), 7.38 (t, J ¼ 7.5 Hz, 1H), 7.29 (d, J ¼ 8.0 Hz, 2H), 6.95e6.93 (m,
2H), 6.70 (s, 1H), 4.16e4.09 (m, 4H), 3.27 (br s, 2H), 2.90e2.70 (m,
6H), 2.65e2.59 (m, 6H), 2.41 (s, 3H), 2.08 (br s, 4H), 1.90e1.84 (m,
solid, yield 51.0%, m.p. 117e119 ꢁC; IR (KBr)
n
3445, 2926, 2872,
2822, 2311, 1638, 1600, 1490, 1440, 1261, 1088, 1035, 802, 756 cmꢀ1
1H NMR (500 MHz, DMSO-d6):
4H),1.23 (t, J ¼ 7.0 Hz, 2H); 13C NMR (125 MHz, CDCl3):
d
177.5,161.1,
;
160.7, 159.9, 155.8, 151.4, 132.25, 131.6, 131.2, 128.9, 127.4, 126.0,
125.3, 124.2, 115.9, 109.2, 96.5, 93.5, 66.2, 60.4, 56.5, 52.9, 46.6, 29.7,
29.7, 28.4, 25.6, 25.2, 21.9, 20.7; ESI-MS m/z: 617.3 [M þ H]þ; HRMS:
calcd for C39H45N4O3 [M þ H]þ, 617.3486, found 617.3484.
d
8.39 (d, J ¼ 8.5 Hz, 1H), 8.18 (d,
J ¼ 8.5 Hz, 2H), 7.99 (d, J ¼ 9.0 Hz, 1H), 7.85e7.80 (m, 2H), 7.70 (d,
J ¼ 8.5 Hz, 2H), 7.56e7.53 (m, 2H), 7.38 (d, J ¼ 2.5 Hz, 1H), 7.11 (dd,
J ¼ 8.5, 2.0 Hz, 1H), 7.06 (s, 1H), 4.24 (t, J ¼ 6.0 Hz, 2H), 3.88 (d,
J ¼ 4.5 Hz, 2H), 3.01 (br s, 2H), 2.72 (br s, 2H), 2.56e2.55 (m, 6H),
2.55e2.40 (m, 8H), 2.01e1.98 (m, 2H), 1.92e1.87 (m, 6H); 13C NMR
4.1.10.14. 2-(4-Methoxy phenyl)-7-(3-(4-(3-((1, 2, 3, 4-
tetrahydroacridin-9-yl)amino)propyl)piperazin-1-yl)propoxy)-4H-
chromen-4-one (13n). Intermediate 12b was treated with 4i ac-
cording to the general procedure to give the desired product 13n as
(125 MHz, DMSO-d6): d 176.6, 163.6, 161.3, 157.4, 153.0, 138.6, 136.7,
131.9, 130.4,129.4, 128.3,126.5,125.2,124.7, 117.3, 115.4,114.4, 112.4,
107.4,101.7, 67.1, 56.1, 54.4, 53.1, 52.7, 47.3, 29.5, 26.4, 26.1, 24.9, 21.9,
21.1; ESI-MS m/z: 637.3 [M þ H]þ; HRMS: calcd for C38H42ClN4O3
[M þ H]þ, 637.2940, found 637.2943.
a pale white solid, yield 50.5%, m.p. 79e81 ꢁC; IR (KBr)
n
3420, 2928,
2814, 2311, 1629, 1604, 1510, 1441, 1374, 1256, 1179, 1088, 830,
760 cmꢀ1; 1H NMR (500 MHz, DMSO-d6):
d
8.28 (d, J ¼ 8.5 Hz, 1H),
8.04 (d, J ¼ 9.0 Hz, 2H), 7.91 (d, J ¼ 9.0 Hz, 1H), 7.76 (d, J ¼ 8.5 Hz,
1H), 7.69 (t, J ¼ 7.0 Hz, 1H), 7.44 (t, J ¼ 8.0 Hz, 1H), 7.28 (d, J ¼ 2.5 Hz,
1H), 7.11 (d, J ¼ 8.5 Hz, 2H), 7.02 (dd, J ¼ 8.5, 2.0 Hz, 1H), 6.85 (s, 1H),
4.17 (t, J ¼ 6.0 Hz, 2H), 3.86 (s, 3H), 3.73e3.72 (m, 2H), 2.94 (br s,
2H), 2.67 (br s, 2H), 2.51e2.50 (m, 4H), 2.46e2.41 (m, 8H),1.95e1.90
4.1.10.18. 2-(4-Nitrophenyl)-7-(3-(4-(3-((1,2,3,4-tetrahydroacridin-
9-yl)amino)propyl)piperazin-1-yl)propoxy)-4H-chromen-4-one
(13r). Intermediate 12b was treated with 4k according to the
general procedure to give the desired product 13r as a dark yellow
solid, yield 50.5%, m.p. 146e148 ꢁC; IR (KBr)
n
3420, 2927, 2310,
1H NMR
8.42e8.39 (m, 2H), 8.29 (d, J ¼ 8.5 Hz,
(m, 2H), 1.83e1.79 (m, 6H); 13C NMR (125 MHz, DMSO-d6)
d
177.2,
1633, 1608, 1520, 1443, 1346, 1252, 1088, 852, 759 cmꢀ1
;
164.0, 163.2, 162.9, 158.3, 154.9, 154.0, 139.8, 131.1, 128.9, 127.0,
125.2, 124.8, 124.3, 118.7, 117.9, 115.7, 115.4, 114.2, 106.2, 102.3, 67.7,
56.8, 56.4, 55.1, 53.9, 53.5, 47.9, 31.6, 27.4, 26.8, 25.7, 22.9, 22.3; ESI-
MS m/z: 633.3 [M þ H]þ; HRMS: calcd for C39H45N4O4 [M þ H]þ,
633.3435, found 633.3440.
(500 MHz, CDCl3 þ CD3OD):
d
1H), 8.19e8.16 (m, 2H), 8.12 (d, J ¼ 8.5 Hz, 1H), 7.96 (d, J ¼ 7.5 Hz,
1H), 7.76 (t, J ¼ 7.5 Hz, 1H), 7.52e7.49 (m, 1H), 7.10e7.06 (m, 2H),
6.92 (s, 1H), 4.22 (t, J ¼ 6.0 Hz, 2H), 4.03 (t, J ¼ 6.0 Hz, 2H), 3.01 (t,
J ¼ 6.5 Hz, 2H), 2.70e2.68 (m, 6H), 2.66e2.64 (m, 8H), 2.14e2.09
(m, 2H), 2.03e1.95 (m, 6H); 13C NMR (125 MHz, CDCl3 þ CD3OD)
4.1.10.15. 2-(4-Aminophenyl)-7-(3-(4-(3-((1,2,3,4-tetrahydroacridin-
9-yl)amino)propyl)piperazin-1-yl)propoxy)-4H-chromen-4-one
(13o). Intermediate 12b was treated with 4l according to the
general procedure to give the desired product 13o as a yellow solid,
d 178.9, 164.7, 161.3, 158.5, 155.8, 152.1, 149.8, 140.6, 137.8, 132.4,
127.6, 127.3, 125.2, 125.1, 124.5, 121.3, 117.7, 116.9, 115.9, 112.4, 109.5,
101.4, 67.3, 57.6, 55.4, 53.6, 53.1, 29.4, 26.6, 26.4, 25.3, 22.2, 21.4. ESI-
MS m/z: 648.3 [M þ H]þ; HRMS: calcd for C38H42N5O5 [M þ H]þ,
648.3180, found 648.3186.
yield 54.0%, m.p. 96e98 ꢁC; IR (KBr)
n
3422, 2931, 2311, 1625, 1597,
1514, 1442, 1375, 1254, 1183, 832 cmꢀ1
;
1H NMR (500 MHz,
CDCl3 þ CD3OD):
d
8.33 (d, J ¼ 9.0 Hz, 1H), 8.06 (d, J ¼ 8.5 Hz, 1H),
4.1.10.19. 5-Hydroxy-7-(3-(4-(3-((1,2,3,4-tetrahydroacridin-9-yl)
8.02 (d, J ¼ 8.5 Hz, 1H), 7.80e7.76 (m, 1H), 7.75e7.31 (m, 2H), 7.53 (t,
J ¼ 7.5 Hz, 1H), 7.00e6.97 (m, 2H), 6.77 (d, J ¼ 8.5 Hz, 2H), 6.63 (s,
1H), 4.19 (t, J ¼ 6.0 Hz, 2H), 4.10 (t, J ¼ 6.5 Hz, 2H), 3.11 (t, J ¼ 6.0 Hz,
2H), 2.84e2.76 (m, 8H), 2.70e2.67 (m, 4H), 2.12 (br s, 4H), 2.01e
amino)propyl)piperazin-1-yl)propoxy)-4H-chromen-4-one
(13s).
Intermediate 12b was treated with 7a according to the general
procedure to give the desired product 13s as a light yellow solid,
yield 45.2%, m.p. 74e76 ꢁC; IR (KBr)
n
3446, 2926, 2310, 1660, 1569,
1502, 1454, 1262, 1162, 1108, 1034, 803, 759 cmꢀ1 1H NMR
(500 MHz, CDCl3):
1.93 (m, 4H); 13C NMR (125 MHz, CDCl3 þ CD3OD)
d
178.7, 164.8,
;
163.4, 157.9, 156.3, 150.9, 150.5, 138.6, 132.76, 127.9, 126.6, 125.3,
124.9, 119.9, 119.6, 117.4, 115.8, 114.5, 111.3, 103.8, 101.0, 100.0, 66.5,
54.7, 52.6, 51.9, 28.2, 25.9, 25.6, 24.8, 21.7, 20.6. ESI-MS m/z: 618.3
[M þ H]þ; HRMS: calcd for C40H48N5O3 [M þ H]þ, 618.3439, found
618.3440.
d
12,56 (s, 1H), 8.29 (d, J ¼ 3.5 Hz, 1H), 8.14 (d,
J ¼ 8.5 Hz, 1H), 7.74 (d, J ¼ 6.0 Hz, 1H), 7.62 (t, J ¼ 7.5 Hz, 1H), 7.39e
7.36 (m, 1H), 6.37 (dd, J ¼ 12.0, 2.0 Hz, 2H), 6.20 (d, J ¼ 6.0 Hz, 1H),
4.10 (t, J ¼ 6.5 Hz, 2H), 3.89 (t, J ¼ 5.5 Hz, 2H), 3.24e3.21 (m, 2H),
2.69e2.60 (m, 8H), 2.59e2.50 (m, 6H), 2.04e1.98 (m, 2H), 1.96e1.88
(m, 6H); 13C NMR (125 MHz, CDCl3):
d 181.8, 165.0, 162.4, 158.1,
4.1.10.16. 2-(4-(Dimethylamino)phenyl)-7-(3-(4-(3-((1,2,3,4-
tetrahydroacridin-9-yl)amino)propyl)piperazin-1-yl)propoxy)-4H-
chromen-4-one (13p). Intermediate 12b was treated with 4m ac-
cording to the general procedure to give the desired product 13p as
155.6, 153.7, 130.4, 124.2, 123.9, 111.4, 106.7, 98.6, 93.3, 66.8, 58.1,
54.9, 53.9, 53.0, 49.8, 30.9, 29.7, 26.5, 26.2, 25.7, 22.3, 21.7; ESI-MS
m/z: 543.3 [M þ H]þ; HRMS: calcd for C32H39N4O4 [M þ H]þ,
543.2966, found 543.2971.
a yellow solid, yield 49.5%, m.p. 90e92 ꢁC; IR (KBr)
n
3422, 2934,
2810, 2310, 1627, 1602, 1522, 1439, 1361, 1260, 1168, 1088, 815,
729 cmꢀ1; 1H NMR (500 MHz, CDCl3):
8.17e8.09 (m, 3H), 7.79 (d,
J ¼ 8.5 Hz, 2H), 7.59 (t, J ¼ 7.5 Hz, 1H), 7.36 (t, J ¼ 8.0 Hz, 1H), 6.96e
4.1.10.20. 5-Methoxy-7-(3-(4-(3-((1,2,3,4-tetrahydroacridin-9-yl)
d
amino)propyl)piperazin-1-yl)propoxy)-4H-chromen-4-one
(13t).
Intermediate 12b was treated with 7b according to the general

644
Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
procedure to give the desired product 13t as a light yellow solid,
yield 47.5%, m.p. 96e99 ꢁC; IR (KBr)
3420, 2929, 2310, 1648, 1603,
1521, 1455, 1279, 1159, 1107, 1078, 832, 762 cmꢀ1 1H NMR
(500 MHz, CDCl3)
8.54 (d, J ¼ 8.5 Hz, 1H), 8.25 (d, J ¼ 8.5 Hz, 1H),
using Ellman’s method [48]. Three concentrations of 13s were
selected for the studies: 30.8, 15.4 and 7.7 nM for the kinetic
analysis of AChE inhibition, and 90.0, 45.0, 22.5 nM for the kinetic
analysis of BuChE inhibition, respectively. The plots were assessed
by a weighted least-squares analysis that assumed the variance of
velocity (v) to be a constant percentage of v for the entire data set.
Slopes of these reciprocal plots were then plotted against the
concentration of 13s in a weighted analysis and Ki was determined
as the intercept on the negative x-axis. Data analysis was performed
with GraphPad Prism 4.03 software (GraphPad Software Inc.).
n
;
d
7.92e7.77 (m, 1H), 7.66 (ddd, J ¼ 8.5, 6.5, 1.0 Hz, 1H), 7.61 (d,
J ¼ 6.0 Hz, 1H), 7.41 (ddd, J ¼ 8.5, 6.5, 1.0 Hz, 1H), 6.45 (d, J ¼ 2.5 Hz,
1H), 6.36 (d, J ¼ 2.5 Hz,1H), 6.18 (d, J ¼ 6.0 Hz,1H), 4.12 (t, J ¼ 6.0 Hz,
4H), 3.93 (s, 3H), 3.31 (t, J ¼ 6.5 Hz, 2H), 2.64e2.56 (m, 4H), 2.63e
2.57 (m, 8H), 2.06e2.03 (m, 2H), 2.02e1.86 (m, 6H); 13C NMR
(125 MHz, CDCl3)
d 176.5, 163.4, 161.1, 160.1, 155.6, 152.6, 151.4,
139.5, 131.9, 124.8, 124.5, 121.3, 116.1, 114.8, 110.9, 110.4, 96.5, 93.5,
66.6, 58.3, 56.5, 54.9, 53.9, 52.8, 50.1, 28.7, 26.5, 25.7, 25.4, 21.8,
20.9; ESI-MS m/z: 557.3 [M þ H]þ; HRMS: calcd for C33H41N4O4
[M þ H]þ, 557.3122, found 557.3127.
4.4. Molecular modeling studies
Molecular modeling calculations and docking studies were
performed using Molecular Operating Environment (MOE) soft-
ware version 2008.10 (Chemical Computing Group, Montreal,
Canada). The X-ray crystallographic structure of AChE complexed
with bis(7)etacrine (PDB code 2CKM) was obtained from the Pro-
tein Data Bank. All water molecules in PDB files were removed and
hydrogen atoms were subsequently added to the protein. The
compound 13s was built using the builder interface of the MOE
program and energy minimized using MMFF94x force field. Then
the 13s was docked into the active site of the protein by the “Tri-
angle Matcher” method, which generated poses by aligning the
ligand triplet of atoms with the triplet of alpha spheres in cavities of
tight atomic packing. The Dock scoring in MOE software was done
using ASE scoring function and Forcefield was selected as the
refinement method. The best 10 poses of molecules were retained
and scored. After docking, the geometry of resulting complex was
studied using the MOE’s pose viewer utility.
4.1.10.21. 7-(3-(4-(3-((1,2,3,4-Tetrahydroacridin-9-yl)amino)propyl)
piperazin-1-yl)propoxy)-4H-chromen-4-one (13u).
Intermediate 12b was treated with 7c according to the general
procedure to give the desired product 13u as a light yellow solid,
yield 45.5%, m.p. 172e175 ꢁC; IR (KBr)
n
3409, 2927, 2810, 2309,
1636, 1590, 1521, 1442, 1264, 1228, 1033, 815, 759 cmꢀ1 1H NMR
(500 MHz, CDCl3)
8.51 (d, J ¼ 8.5 Hz, 1H), 8.22 (d, J ¼ 8.5 Hz, 1H),
;
d
8.10 (d, J ¼ 9.0 Hz, 1H), 7.78 (d, J ¼ 6.0 Hz, 1H), 7.70e7.54 (m, 2H),
7.40 (t, J ¼ 7.5 Hz, 1H), 6.97 (dd, J ¼ 9.0, 2.5 Hz, 1H), 6.86 (d,
J ¼ 2.0 Hz,1H), 6.27 (d, J ¼ 6.0 Hz,1H), 4.14 (t, J ¼ 6.0 Hz, 2H), 4.07 (s,
2H), 3.30 (t, J ¼ 6.0 Hz, 2H), 2.73e2.65 (m, 4H), 2.64e2.57 (m, 8H),
2.08e2.02 (m, 2H), 1.98e1.89 (m, 6H); 13C NMR (125 MHz, CDCl3)
d
176.9, 163.5, 158.3, 155.3, 154.9, 151.9, 140.2, 131.7, 127.2, 124.6,
124.4, 121.9, 118.8, 116.4, 112.9, 111.3, 101.1, 66.8, 58.4, 55.0, 54.0,
52.9 50.2, 29.7, 29.1, 26.6, 25.7, 25.5, 21.9, 21.0; ESI-MS m/z: 527.3
[M þ H]þ; HRMS: calcd for C32H39N4O3 [M þ H]þ, 527.3017, found
527.3018.
4.5. Inhibition of Ab (1e42) self-induced aggregation
4.2. In vitro inhibition studies on AChE and BuChE
Inhibition of A
oflavin T (ThT)-binding assay previously described by Bartolini et al.
[49,50] with little modifications. A (1e42) was dissolved in 100%
b (1e42) aggregation was measured using a Thi-
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel),
butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum), 5,50-
dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), S-butyr-
ylthiocholine iodide (BTCI), acetylthiocholine iodide (ATCI), and
tarcine hydrochloride were purchased from SigmaeAldrich. The
capacity of the test compounds (3a and 13aeu) to inhibit AChE and
BuChE activities was assessed by Ellman’s method. Stock solution of
test compounds was dissolved in a minimum volume of DMSO (1%)
and was diluted using the buffer solution (50 mM TriseHCl,
b
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to a concentration of 1 mg/
mL, sonicated in a water bath for 10 min, aliquoted into micro-
centrifuge tubes, dried under vacuum, and stored in ꢀ20 ꢁC. For the
inhibition of self-induced Ab(1e42) aggregation experiment, the
A
b
was diluted with 50 mM phosphate buffer (pH 7.4) to 50
before use. A mixture of the peptide (10 L, 25 M, final concen-
tration) with or without the tested compounds (10 L) at different
concentrations (2.5, 5, 10, 20, 40
M) were incubated at 37 ꢁC for
48 h. Blanks using 50 mM phosphate buffer (pH 7.4) instead of A
with or without inhibitors were also carried out. After incubation,
the samples were diluted to a final volume of 200 L with 50 mM
glycineeNaOH buffer (pH 8.0) containing thioflavin T (5 M). Then
mM
m
m
m
m
pH ¼ 8.0, 0.1 M NaCl, 0.02 M MgCl2$6H2O). In 96-well plates, 160
of 1.5 mM DTNB, 50 L of AChE (0.22 U/mL prepared in 50 mM Trise
HCl, pH ¼ 8.0, 0.1% w/v bovine serum albumin, BSA) or 50 L of
BuChE (0.12 U/mL prepared in 50 mM TriseHCl, pH ¼ 8.0, 0.1% w/v
BSA) were incubated with 10 L of various concentrations of test
compounds (0.001e100
M) at 37 ꢁC for 6 min followed by the
addition of the substrates (30 L) acetylthiocholine iodide (15 mM)
mL
b
m
m
m
m
m
the fluorescence intensities were measured on a SpectraMax M5
(Molecular Devices, Sunnyvale, CA, USA) multi-mode plate reader
with excitation and emission wavelengths at 446 nm and 490 nm,
respectively. Each inhibitor was examined in triplicate. The fluo-
rescence intensities were compared and the percent inhibition due
to the presence of the inhibitor was calculated by the following
formula: (1 ꢀ IFi/IFo) ꢄ 100% where IFi and IFo are the fluorescence
m
m
or S-butyrylthiocholine iodide (15 mM) and the absorbance was
measured at different time intervals (0, 60, 120, and 180 s) at a
wavelength of 405 nm. The concentration of compound producing
50% of enzyme activity inhibition (IC50) was calculated by nonlinear
regression analysis of the responseeconcentration (log) curve, us-
ing the Graph-Pad Prism program package (GraphPad Software;
San Diego, CA). Results are expressed as the mean ꢂ SEM of at least
three different experiments performed in triplicate.
intensities obtained for A
b (1e42) in the presence and in the
absence of inhibitors after subtracting the background,
respectively.
4.6. Spectrophotometric measurement of complex with Cu2þ and
Fe2þ
4.3. Kinetic analysis of ChE inhibition
To obtain of the mechanism of action 13s, reciprocal plots of 1/
velocity versus 1/[substrate] were constructed at different con-
centrations of the substrate thiocholine iodide (0.05e0.5 mM) by
The study of metal chelation was performed in methanol at
298 K using UVevis spectrophotometer (SHIMADZU UV-2450PC)
with wavelength ranging from 200 to 500 nm [51,52]. The

Su-Y. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 632e646
645
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by numerical subtraction of the spectra of the metal alone and the
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4.7. SH-SY5Y neuroblastoma cell toxicity
The toxicity effect of 13k on the human neuroblastoma cell line
SH-SY5Y cells was examined according to the previous methods
[33,53]. The SH-SY5Y cells were cultured in Eagle’s minimum
essential medium (EMEM)/ham’s F-12 (1:1) medium supple-
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m
L of MTT at 37 ꢁC for 4 h, living cells containing
L DMSO. The
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This research work was financially supported by Innovative
Research Team in University (IRT1193), A Project Funded by the
Priority Academic Program Development of Jiangsu Higher Edu-
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These data include MOL files and InChiKeys of the most
important compounds described in this article. Supplementary
data associated with this article can be found in the online version,
at http://dx.doi.org/10.1016/j.ejmech.2013.09.024. These data
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