Design, synthesis and evaluation of genistein-O-alkylbenzylamines as potential multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2014.02.045

A series of genistein derivatives with carbon spacer-linked alkylbenzylamines were designed, synthesized and tested as multifunctional agents for the treatment of Alzheimer's disease (AD). The results showed that most of these compounds exhibited good acetylcholinesterase (AChE) inhibitory activity, with moderate-to-good anti-oxidative activity. Specifically, compounds 10b, 19d and 25d exhibited significant inhibition of β-amyloid (Aβ) aggregation and exhibited metal chelating properties. In particular, 25d inhibited: self-induced Aβ_1-42 aggregation, Cu ²⁺-induced Aβ_1-42 aggregation, and human AChE-induced Aβ_1-40 aggregation by 35%, 77.8%, and 36.2%, respectively. Moreover, both kinetic analysis of AChE inhibition and the molecular modeling study suggested that 25d binds simultaneously to catalytic active site and peripheral anionic site of AChE. More importantly, compound 25d disassembled the well-structured Aβ fibrils generated by Cu²⁺-induced Aβ aggregation by 72.1%. Furthermore, the step-down passive avoidance test showed this compound significantly reversed scopolamine-induced memory deficit in mice. These results suggest that 25d may be a promising multifunctional agent for AD treatment.

Full text of DOI:10.1016/j.ejmech.2014.02.045

European Journal of Medicinal Chemistry 76 (2014) 314e331
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and evaluation of genistein-O-alkylbenzylamines as
potential multifunctional agents for the treatment of Alzheimer’s
disease
,
,
Xiaoming Qiang a 1, Zhipei Sang a 1, Wen Yuan a, Yan Li a, Qiang Liu a, Ping Bai a, Yikun Shi b,
Wei Ang a, Zhenghuai Tan b , Yong Deng
,
a,
*
*
a Department of Medicinal Chemistry, Key Laboratory of Drug Targeting and Drug Delivery System of the Education Ministry, West China School of
Pharmacy, Sichuan University, Chengdu 610041, China
b Institute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan Academy of Chinese Medicine Sciences, Chengdu 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of genistein derivatives with carbon spacer-linked alkylbenzylamines were designed, synthe-
sized and tested as multifunctional agents for the treatment of Alzheimer’s disease (AD). The results
showed that most of these compounds exhibited good acetylcholinesterase (AChE) inhibitory activity,
with moderate-to-good anti-oxidative activity. Specifically, compounds 10b, 19d and 25d exhibited
Received 26 November 2013
Received in revised form
9 February 2014
Accepted 16 February 2014
Available online 17 February 2014
significant inhibition of
b-amyloid (Ab) aggregation and exhibited metal chelating properties. In
particular, 25d inhibited: self-induced Ab1e42 aggregation, Cu2þ-induced Ab1e42 aggregation, and human
AChE-induced Ab1e40 aggregation by 35%, 77.8%, and 36.2%, respectively. Moreover, both kinetic analysis
of AChE inhibition and the molecular modeling study suggested that 25d binds simultaneously to cat-
alytic active site and peripheral anionic site of AChE. More importantly, compound 25d disassembled the
Keywords:
Alzheimer’s disease
Genistein-O-alkylbenzylamines
Multifunctional agents
Acetylcholinesterase inhibitors
Metal-chelating
well-structured Ab b aggregation by 72.1%. Furthermore, the step-
fibrils generated by Cu2þ-induced A
down passive avoidance test showed this compound significantly reversed scopolamine-induced
memory deficit in mice. These results suggest that 25d may be a promising multifunctional agent for
AD treatment.
Ab aggregation inhibitors
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
disequilibrium of biometals and b-amyloid (Ab) deposits have been
considered to play definitive roles in AD pathogenesis, and several
hypotheses based on these factors have been proposed to explain
the mechanism of AD pathogenesis [2]. The cholinergic hypothesis
for AD suggests that cognitive deterioration is mainly caused by low
levels of acetylcholine (ACh) because maintaining ACh levels (via
acetylcholinesterase [AChE] inhibitors) has been shown to alleviate
these symptoms [3]. AChE also accelerates the aggregation of am-
yloid fibrils, which is thought to involve the peripheral anionic site
(PAS) of AChE [4]. The ability for AChE inhibitors to simultaneously
bind to its catalytic and peripheral binding sites has thus become an
area of very active research [5]. Furthermore, several studies have
suggested that inhibition of a similar form of AChE, butyr-
ylcholinesterase (BuChE), is a desirable activity in anti-Alzheimer
compounds [6]. For example, rivastigmine, active on both enzymes,
is currently in use. However, serious inhibition of BuChE also may
contribute to the peripheral side effects of cholinesterase inhibitors,
such as the dual AChE and BuChE inhibitor, tacrine, which has
shown severe hepatotoxicity as well as other adverse effects [7].
Alzheimer’s disease (AD) is a complex neurodegenerative dis-
ease characterized by progressive and irreversible cognitive im-
pairments, and severe behavioral abnormalities, and ultimately
causing death [1]. Although many factors have been implicated in
AD over the last decades, its etiology is not still completely known.
Thus far, low levels of acetylcholine (ACh), oxidative stress, the
Abbreviations: AD, Alzheimer’s disease; AChE, acetylcholinesterase; BuChE,
butyrylcholinesterase; HuAChE, human AChE; EeAChE, Electrophorus electricus
AChE; Ab, b-amyloid peptide; CAS, catalytic active site; PAS, peripheral anionic site;
MOM, methoxymethyl ether; PBS, phosphate-buffered saline; DTNB, 5,50-dithiobis-
2-nitrobenzoic acid; PDB, Protein Data Bank; TcAChE, Torpedo californica AChE; ROS,
reactive oxygen species; ORAC-FL, Oxygen Radicals Absorbance Capacity by Fluo-
rescence; AAPH, 2,20-Azobis(amidinopropane) dihydrochloride; ThT, thioflavin T;
HEPES, 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid.
* Corresponding authors.
E-mail addresses: tanzhh616@163.com (Z. Tan), dengyong@scu.edu.cn (Y. Deng).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2014.02.045
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
315
Therefore, the development of AChE inhibitors for the treatment of
AD, with expectation of their fewer side effects, may be a more
suitable approach as a therapeutic strategy.
requirement for adequate AChE inhibition [27]. Therefore, we
aimed to use a multi-target-directed drug design strategy to
combine genistein with appropriate secondary amines using car-
bon spacers of different lengths. These new molecules may simul-
taneously possess a dual binding site for AChE inhibition, anti-
Increasing evidence supports the significant impact of oxidative
stress in the pathogenesis of AD [8]. Recent studies have indicated
that oxidative damage may promote the appearance of amyloid
plaques and neurofibrillary (NFT) tangles in AD [9]. Therefore,
drugs aimed at clearing or preventing the formation of the free
radicals may be useful for either the prevention or effective treat-
oxidative and metal chelating effects, and inhibit Ab aggregation.
In this study, a series of genistein-O-alkylbenzylamine de-
rivatives were designed, synthesized and evaluated for their bio-
logical activity, including inhibition of cholinesterase, antioxidant
ment of AD. Another hypothesis indicates that metal ions (Cu2þ
,
and metal chelating effects, inhibitory effects on Ab aggregation,
Fe2þ, Zn2þ and Al3þ) may play an important role in the pathogen-
esis of AD [10]. Metals have been observed to progressively accu-
mulate in AD patients during disease progression from moderate-
to-severe AD. The abnormal accumulation of metals is closely
and neuroprotective effects in the mouse scopolamine model of
memory impairment. The chemical design strategy for genistein
derivatives is depicted in Fig. 1.
associated with the formation of A
b plaques and neurofibrillary
tangles [11]. Abnormally high brain levels of redox-active metal
ions, like Cu2þ and Fe2þ, may contribute to the production of
reactive oxygen species (ROS), which promotes oxidative stress
thus contributing to AD pathogenesis [12]. Therefore, lowering the
concentration of brain metals by chelating metals represents an
additional rational approach for the treatment of AD.
According to the amyloid hypothesis, aggregation and deposi-
tion of Ab peptide is considered to be crucial for the pathogenesis of
AD because its accumulation may result in a cascade of biochemical
events leading to neuronal dysfunction [13]. Ab1e40 and Ab1e42 are
2. Results and discussion
2.1. Chemistry
The synthesis of genistein-O-alkylbenzylamine derivatives (8e
10, 18e20 and 24e26) occurred via the general pathway, for which
genistein was used as the starting material (Schemes 1e3). The 7-
OH group of genistein is more acidic than the 40-OH group [28].
However, the 40-phenolate or 40-OH group is a better nucleophile
than the 7-phenolate or 7-OH group, respectively. The hydrogen
bond-stabilized 5-OH group is the less nucleophilic group of gen-
istein [29]. The difference in reactivity thus allows for easy regio-
selectivity, for substitutions at 40-OH or/and 7-OH positions.
Synthesis of 7-O-modified genistein derivatives 8e10 is shown in
Scheme 1. Compounds 2e4 were the key intermediates, usually
prepared from alkylation of the 7-OH group by using haloalkanes in
the presence of a base, as previously described [30,31]. Thus, gen-
istein was alkylated with the equivalent amount of KOH in N,N-
dimethylformamide followed by the addition of excessive amounts
of 1,3-dibromopropane, 1,4-dibromobutane or 1,6-dibromohexane
furnished intermediates 2e4. Subsequently, compounds 2e4
were reacted with two equivalent secondary amines 7aed, anhy-
drous K2CO3 and the catalytic amount of KI in CH3CN, at 60e65 ꢀC.
The amines 7aed were synthesized by reductive amination of
methylamine or ethylamine with benzaldehydes in the presence of
NaBH4 [32]. This process produced the desired target compounds,
8aed, 9aed, and 10aed in moderate-to-good yields.
the main isoforms of Ab peptides. Although the amount of Ab1e42 is
only 10% of Ab1e40, Ab1e42 tends to aggregate more rapidly and
displays stronger neuronal toxicity than Ab1e40 [14]. Preventing the
formation and aggregation of A
for AD treatment.
b is a potential therapeutic strategy
Current treatments of AD focus on the symptomatic aspects of
its pathology. These drugs, which are currently approved by the
FDA, inhibit AChE to increase the level of ACh. These AChE in-
hibitors include: tacrine (now discontinued), donepezil, riva-
stigmine and galantamine. Alternatively, memantine has also been
used to antagonize N-methyl- -aspartate (NMDA) receptors to
D
prevent aberrant neuronal stimulation [15]. In clinical trials, how-
ever, the impact of these single target drugs on this disease has
been modest and transient because of the multifactorial nature of
AD. However, observational studies have suggested that combina-
tion treatments may increase the time before patients require
nursing home care [16]. Nevertheless, studies thus far have not
provided convincing evidence that these agents can prevent, halt,
or reverse the disease [15]. Therefore, despite the difficulties, the
development of agents that affect two or more biological activities
that correlate with those causing AD has drawn considerable
attention for their advancement in the treatment of AD [17e20].
The discovery of a lead compound with the potential to form part of
a multifunctional drug is a crucial step in the search for clinical
candidate for the treatment of AD.
For the synthesis of 40-O-modified genistein derivatives, 18e20,
the 7-OH group of genistein was protected (Scheme 2). Firstly,
genistein was reacted with KOH, and then chloromethyl methyl
ether (MOM-Cl) was added (dropwise) to produce compound 11, of
which the 7-OH group was protected by MOMCl [33]. Compound 11
was then reacted with excessive amounts of 1,3-dibromopropane,
1,4-dibromobutane or 1,6-dibromohexane in the presence of K2CO3
and the catalytic amount of KI in CH3CN at 65 ꢀC to obtain the in-
termediates, 12e14. In turn, these intermediates were reacted with
the corresponding compounds, 7aed, respectively, according to the
procedure adopted for 8aed to provide the key intermediates 15ae
d, 16aed and 17aed. Finally, HCl-mediated removal of methox-
ymethyl ether (MOM) protecting group gave the corresponding
target compounds, 18aed, 19aed and 20aed.
Genistein (40,5,7-trihydroxyisoflavone) is the most abundant
isoflavone in soybeans and also produced abundantly by red clover
containing plants (eg. Trifolium pratense), possessing a broad range
of pharmacological properties, such as estrogen activity, anti-
inflammatory, anti-oxidative and metal chelating effects, and
neuroprotective effects against Ab [21e25]. These results indicate
that genistein may be used as a starting compound in the design of
multifunctional drugs for the treatment of AD. However, the lack of
AChE inhibitory activity and low bioavailability of genistein re-
stricts its clinical activity as an anti-AD drug [26]. The 1-
benzylpiperidine fragment of donepezil has been shown as the
cholinesterase inhibitory pharmacophore [4]. Recently, many
studies have revealed compounds that bear an amine functional
group on the alkyl side chain consist of a lipophilic moiety and
often a tertiary amino group, which were found to be the key
The new compounds, 21e23, were the key intermediates for the
synthesis of 7,40-O-modified genistein derivatives, 24e26 (Scheme
3), which were prepared by treatment of genistein with 4 equiva-
lent of 1,3-dibromopropane, 1,4-dibromobutane or 1,6-
dibromohexane under reflux for 8e10 h in acetone. This step was
carried out in the presence of anhydrous K2CO3 and the catalytic
amount of KI. Because the 5-OH group was stabilized by the
hydrogen bond, the trisubstituted compounds were not produced
under these conditions. Subsequently, compounds 21e23 were

316
X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
Fig. 1. Design strategy for the genistein-O-alkylbenzylamine derivatives.
Scheme 1. Synthesis of 7-O-modified genistein derivatives 8e10. Reagents and conditions: (i) KOH, DMF, at room temperature (rt), for 5 h, then Br(CH2)nBr, at rt, for 24 h; (ii) MeNH2
or EtNH2, NaBH4, CH3OH, at rt, for 5e6 h; (iii) R1R2NH (7aed), K2CO3, KI, CH3CN, at 65 ꢀC, for 12e15 h.
Scheme 2. Synthesis of 40-O-modified genistein derivatives 18e20. Reagents and conditions: (i) KOH, DMF, for 1 h, then CH3OCH2Cl, at rt, for 12 h; (ii) Br(CH2)nBr, K2CO3, KI, CH3CN,
at 65 ꢀC, for 12e15 h; (iii) R1R2NH (7aed), K2CO3, KI, CH3CN, at 65 ꢀC, for 12e15 h; (iv) 10% HCl/EtOH, reflux for 3 h.

X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
317
Scheme 3. Synthesis of 7, 40-O-modified genistein derivatives 24e26. Reagents and conditions: (i) Br(CH2)nBr, K2CO3, KI, acetone, reflux for 8 h; (ii) N-benzylethylamine (7b) or N-(2-
methoxybenzyl)ethylamine (7d), K2CO3, KI, CH3CN, at 65 ꢀC, for 12e15 h.
reacted with N-benzylethylamine (7b) or N-(2-methoxybenzyl)
ethylamine (7d) to provide the final compounds, 24e26.
All new compounds were purified using recrystallization or
chromatography, and the analytical and spectroscopic data
confirmed their structures, as detailed in the experimental section.
activities against BuChE. AChE and BuChE activity was measured
from rat cortex homogenate and rat serum, respectively. Moreover,
the most potent and representative compounds were selected and
re-evaluated using Electrophorus electricus AChE (EeAChE) and hu-
man erythrocyte AChE (HuAChE). Donepezil was used as reference
compound.
The target compounds showed different amounts of inhibitory
activity to AChE with IC50 values ranging from the submicromolar to
micromolar range (Table 1). Compound 25d exhibited the highest
2.2. Pharmacology
2.2.1. Inhibition studies on AChE and BuChE
potency to inhibit AChE (IC50 ¼ 0.09 ꢁ 0.02
mM). The general trend
To study the multipotent profiles of the three novel series of
genistein derivatives (8e10aed, 18e20aed, 24d, 25b, 25d and
26d), they were first evaluated as inhibitors of AChE according to
Ellman’s method [34] but with some modifications. The selectivity
of the compounds was also tested by determining their inhibitory
for AChE inhibition was 7,40-O-modified > 7-O-modified >40-O-
modified genistein derivatives (with the only exception of 9a, which
was much less potent than 19a), and all synthetic derivatives
exhibited much higher inhibitory activity against AChE compared
Table 1
Inhibition of AChE and BuChE activity, and oxygen radical absorbance capacity (ORAC, Trolox Equivalents) by genistein derivatives, genistein and donepezil.
Compound
IC50
(
m
M) ꢁ SDa
BuChE inhibition (%)e
ORACf
Rat AChEb
EeAChEc
HuAChEd
8a
8b
8c
8d
9a
9b
9c
9d
36.29 ꢁ 2.28
1.42 ꢁ 0.07
13.55 ꢁ 1.08
0.47 ꢁ 0.04
54.50 ꢁ 7.70
2.42 ꢁ 0.13
5.55 ꢁ 1.04
0.83 ꢁ 0.09
4.24 ꢁ 0.20
0.35 ꢁ 0.01
3.19 ꢁ 0.08
0.53 ꢁ 0.05
49.25 ꢁ 3.49
42.83 ꢁ 3.78
37.43 ꢁ 1.05
16.01 ꢁ 0.80
3.79 ꢁ 0.59
12.80 ꢁ 1.41
5.31 ꢁ 0.54
1.45 ꢁ 0.09
3.54 ꢁ 0.24
38.45 ꢁ 1.07
5.62 ꢁ 0.79
1.69 ꢁ 0.10
0.21 ꢁ 0.03
0.90 ꢁ 0.06
0.09 ꢁ 0.02
0.51 ꢁ 0.08
n.a.h
NTg
NTg
7.4
0.89 ꢁ 0.08
1.12 ꢁ 0.06
1.05 ꢁ 0.10
1.02 ꢁ 0.15
1.00 ꢁ 0.17
2.40 ꢁ 0.11
1.56 ꢁ 0.10
1.06 ꢁ 0.04
0.65 ꢁ 0.02
1.20 ꢁ 0.06
2.70 ꢁ 0.12
0.56 ꢁ 0.01
0.46 ꢁ 0.01
0.58 ꢁ 0.07
0.62 ꢁ 0.02
0.64 ꢁ 0.02
0.32 ꢁ 0.03
0.60 ꢁ 0.03
1.10 ꢁ 0.08
1.10 ꢁ 0.05
0.30 ꢁ 0.01
0.29 ꢁ 0.01
0.40 ꢁ 0.02
0.38 ꢁ 0.01
0.49 ꢁ 0.03
0.26 ꢁ 0.01
0.30 ꢁ 0.01
0.13 ꢁ 0.01
5.00 ꢁ 0.32
NTg
NTg
NTg
9.9
NTg
NTg
n.a.h
n.a.h
8.9
NTg
NTg
NTg
NTg
NTg
NTg
10.4
n.a.h
7.7
NTg
NTg
NTg
NTg
10a
10b
10c
10d
18a
18b
18c
18d
19a
19b
19c
19d
20a
20b
20c
20d
24d
25b
25d
26d
Genistein
Donepezil
NTg
NTg
n.a.h
n.a.h
nah
4.98 ꢁ 0.38
5.80 ꢁ 0.65
NTg
NTg
NTg
NTg
n.a.h
n.a.h
11.6
n.a.h
17.6
10.8
n.a.h
9.7
NTg
NTg
NTg
NTg
NTg
NTg
NTg
NTg
NTg
NTg
NTg
NTg
NTg
NTg
15.90 ꢁ 2.10
3.88 ꢁ 0.64
6.7
7.7
NTg
NTg
NTg
NTg
n.a.h
n.a.h
n.a.h
12.5
10.3
20.0
11.6
n.a.h
NTg
NTg
NTg
NTg
0.16 ꢁ 0.04
NTg
NTg
NTg
0.14 ꢁ 0.003
3.79 ꢁ 0.17
n.a.h
0.12 ꢁ 0.01
0.35 ꢁ 0.03
NTg
n.a.h
0.011 ꢁ 0.001
0.015 ꢁ 0.002
IC50 ¼ 20.7 ꢁ 1.36
mM
a
IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and are the mean of 3 independent experiments, each performed in
triplicate (SD ¼ standard deviation).
b
From 5% rat cortex homogenate.
From Electrophorus electricus.
From human erythrocytes.
BuChE from rat serum was used and tested compounds were used at 50
c
d
e
m
m
M.
M of Trolox equivalent/
f
g
h
The mean ꢁ SD of the 3 independent experiments. Data are expressed as
m
M of tested compound.
M in the assay conditions.
NT ¼ not tested.
n.a. ¼ no active. Compounds defined “no active” means a percent inhibition of less than 5.0% at a concentration of 50
m

318
X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
with the original lead compound, genistein. The results showed that
the introduction of the O-alkylbenzylamine group to genistein
significantly increased the inhibitory activity. The length of the
linker and the benzylamine moiety were varied to optimize the
interaction of the inhibitors with both the catalytic and peripheral
binding sites of AChE. The potency of the derivatives to inhibit AChE
was related to the length of alkylene. In general, 7-O-modified
genistein derivatives with a 6-methylene linker between benzyl-
amine and 7-OH of genistein showed better inhibition of AChE than
those with a 3-methylene or 4-methlene chain. For example, com-
pounds 10aec with a linkerof 6 methylene groups weremorepotent
than compounds 8aec and 9aec. However, 40-O-modified de-
rivatives with a 4-methylene linker between benzylamine and 40-
OH of genistein showed better inhibition of AChE than those with a
3-methylene or 6-methylene chain. The 7,40-O-modified compound,
Fig. 2. Kinetic study on the mechanism of EeAChE inhibition by compound 25d.
Merged LineweavereBurk reciprocal plots of AChE initial velocity with increasing
substrate concentration (0.1e0.4 mM) in the absence or presence of 25d. Lines were
derived from a weighted least-squares analysis of data points.
25d, had an IC50 of 0.09 ꢁ 0.02
mM, and with a 4-methylene double
linkage, this compound displayed the most potent AChE inhibitory
activity. Compared with 25d, the activity of 24d (with a 3-methylene
result indicated that compound 25d was able to bind at both the
catalytic and peripheral site of AChE.
linker) was 2-fold lower (IC50 ¼ 0.21 ꢁ 0.03
26d (with 6-methylene linker) was 5-fold lower
(IC50 ¼ 0.51 ꢁ 0.08 M). Therefore, the alkyl chain with 4 or 6
mM), and the activity of
a
m
methylenes may have been beneficial for AChE inhibitory activity.
The results indicated that genistein-O-alkylbenzylamine derivatives
required side chains of a suitable length to bind to the AChE. The
results also revealed that the benzylamine moiety was crucial for
AChE inhibition. The substituents on the terminal side chain of ni-
trogen also played a significant role in the inhibitory activity. AChE
inhibitory activity was generally improved with compounds pos-
sessing an N-ethyl group (8ced, 9ced, 10c, 18ced, 19d, 20d)
compared with compounds possessing an N-methyl group (8aeb,
9aeb,10a,18aeb,19b, 20b). In contrast, AChE inhibitoryactivity was
ameliorated with compounds exhibiting an N-2-methoxybenzyl
group compared with compounds bearing an N-benzyl group
(except 10b). Compound 10b possessed an N-benzyl group but was
2.2.3. Molecular modeling study
To explore a possible interacting mode of the genistein de-
rivatives with TcAChE (PDB code: 1EVE), a molecular modeling
study was performed using the docking program, AutoDock 4.2
package with Discovery Studio 2.0 [9]. Results showed that
slightly more potent (IC50
¼
0.35
ꢁ
0.01 mM) than 10d
(IC50 ¼ 0.53 ꢁ 0.05 M), which had an N-2-methoxybenzyl group.
m
Taken together, the results suggested that N-(2-methoxybenzyl)
ethylamine may be the optimal substitution pattern for AChE inhi-
bition. Compounds 10b, 19d, and 25d displayed the most potent
inhibitoryactivity for rat AChE (IC50 ¼ 0.35 ꢁ 0.01
m
M,1.45 ꢁ 0.09
mM
and 0.09 ꢁ 0.02
m
M, respectively). Furthermore, they were re-
evaluated using EeAChE and HuAChE. Inhibition of EeAChE and
HuAChE by compounds 10b, 19d and 20d was generally less potent
than that of rat AChE (Table 1).
Almost all genistein-O-alkylbenzylamine derivatives (50
mM)
were inactive or weak on BuChE activity. This result showed that
these target compounds were potent AChE inhibitors with high
selectivity toward AChE. This selectivity profile may be a limitation,
but this also may be beneficial to diminish peripheral cholinergic
side effects and provide lower toxicity. For instance, severe side
effects of AChE inhibitors, such as tacrine, have been suggested to
be attributed to their poor selectivity [35].
Based on the above assay results, we next selected the most
potent AChE inhibitor, 25d, for kinetic analysis to investigate the
type of inhibition.
2.2.2. Kinetic studies for the inhibition of AChE
To gain further insight into the mechanism of action of this
family of compounds, a kinetics study was carried out with com-
pound 25d using EeAChE. Graphical analysis of the reciprocal
LineweavereBurk plots (Fig. 2) showed that with increasing con-
centrations of 25d, both the slopes (decreased Vmax) and intercepts
(higher Km) were increased. This pattern indicated a mixed-type
inhibition. Replots of the slope versus concentration of compound
Fig. 3. Representation of compound 25d (yellow stick) interacting with residues in the
binding site of TcAChE (PDB code: 1EVE), highlighting the protein residues that
participate in the main interactions with the inhibitor. Picture was generated with
Discovery Studio 2.0. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
25d gave an estimate of the inhibition constant (Ki ¼ 0.14
mM). This

X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
319
Fig. 4. (A) UV spectrum of compound 25d (37.5
m
M in methanol) alone or in the presence of CuCl2, FeSO4, ZnCl2 or AlCl3 (37.5
m
M for all metals in methanol). (B) Determination of
the stoichiometry of complex-Cu2þ by using the molar ratio method of titrating the methanol solution of compound 25d with ascending amounts of CuCl2. The final concentration
of tested compound was 37.5 m mM.
M, and the final concentration of Cu2þ ranged from 7.5 to 93.75
compound 25d occupied the entire enzymatic catalytic active site
(CAS), the mid-gorge sites and the peripheral anionic site (PAS), and
binds simultaneously to both the catalytic and peripheral site
(Fig. 3). In the TcAChEe25d complex, the 7-substituted terminal N-
(2-methoxybenzyl) ethylamine moiety of 25d was observed to bind
were thermally generated from 2,2-azobis-(amidinopropane)
dihydrochloride and reacted with fluorescein to form nonfluores-
cent products at 535 nm. The antioxidant capacity of genistein
derivatives was determined by their competition with fluorescein
in the radical capture, using a fluorescence microplate reader. Their
ability to scavenge radicals was provided as a Trolox (a vitamin E
to the CAS via a pep interaction with Trp84, and potentially induce
a hydrophobic interaction with residues Tyr130 and His440. The 4-
substituted terminal N-(2-methoxybenzyl) ethanamine moiety
occupied the PAS of AChE and exhibited a potential hydrophobic
interaction with residues Trp279 and Leu282. In addition, the long
chain of methylene and skeleton of genistein folded in a confor-
mation in the gorge that allowed them to interact with Gly335,
Phe290, Tyr334, Tyr70, Asp72, Tyr121 and Phe330 via the hydro-
phobic interaction. No direct hydrogen bond interaction was
observed between compound 25d and TcAChE. However, similar to
the donepezil complex, water-bridged hydrogen bonding may have
occurred under biological circumstances between compound 25d
and TcAChE. The simultaneous binding of 25d with the active gorge
and peripheral site of TcAChE provided an explanation for its highly
potent inhibitory activity for AChE and thus revealed a mixed-type
inhibition for this compound.
analog) equivalent, with their relative potency at 5 mM compared
with Trolox (Table 1). Genistein was also tested, with an ORAC-FL
value of 5.0 trolox equivalents. All of the target compounds
demonstrated weak to moderate antioxidant activity. Compounds
9b and 10c showed the most potent antioxidant activity of this
family with ORAC-FL values of 2.4 and 2.7 trolox equivalents,
respectively. The free 40-OH of genistein was found to be crucial to
the radical scavenging ability when the ORAC-FL values were
compared between the 7-O-modified derivatives (8aed, 9aed,
10aed) the 40-O-modified derivatives (18aed, 19aed, 20aed) and
the 7,40-O-modified derivatives (24d, 25b, 25d, 26d).
2.2.5. Studies of metal-chelating properties
The chelating ability of compounds 10b, 19d, and 25d for the
biologically relevant metal ions: Cu2þ, Fe2þ, Zn2þ, and Al3þ, was
studied by ultravioletevisible (UVevis) spectrometry. As an
example, a series of UVevis spectra of compound 25d is shown in
Fig. 4A (the spectra of compound 10b and 19d are given in
Supporting Information). In the absence of metal ions, the UVevis
spectrum of compound 25d showed the absorption maximum at
263 nm and a shoulder at 325 nm. When CuCl2 was added, a red
2.2.4. Evaluation of compounds for antioxidant activity via the
oxygen radical absorbance capacity by fluorescence (ORAC-FL)
method
The antioxidant activities of all the target compounds were
evaluated by following the ORAC-FL method [36]. Peroxyl radicals

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X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
shift in the maximum absorption from 263 nm to 275 nm and
325 nm to 380 nm occurred, indicating the formation of a 25de
Cu2þ complex. When AlCl3 was added, a slight shift in the
maximum absorption from 263 to 268 nm was observed, sug-
gesting that 25d can bind Al3þ. However, no significant shift was
seen with the addition of FeSO4 or ZnCl2. Similar results were
observed for compound 10b and 19d. The stoichiometry of the
25deCu2þ complex was determined using the molar ratio method,
by preparing solutions of compound 25d with increasing amounts
of CuCl2. The UV spectra were used to obtain the absorbance of the
25d complex and differing concentrations of CuCl2 at 380 nm. The
results showed that absorbance linearly increased initially and then
plateaued (Fig. 4B). The two straight lines intersected at a mole
fraction of 0.99, revealing a 1:1 stoichiometry for complex 25de
Fig. 5. Inhibition of self-induced Ab1e42 aggregation by compounds 10b, 19d, 25d or
curcumin. The thioflavin-T fluorescence method was used. Compounds 10b and 19d
were tested at 25 mM, and compound 25d was tested at 10, 25 and 50 mM.
Cu2þ
.
2.2.6. Effects on the A
A number of dual binding site AChE inhibitors have been found
to exhibit a significant inhibitory activity on A self-aggregation
[37]. Therefore, compounds 10b, 19d and 25d, which showed the
highest potency for AChE inhibition, were selected to assess their
ability to inhibit self-induced Ab1e42 aggregation via the thioflavin
T (ThT) fluorescence method [35,38]. Curcumin, a known active
b aggregation
aggregation by 36.2 ꢁ 0.8%, which was higher than that of done-
pezil (25.0 0.6%) (Table 2). Compared with donepezil
ꢁ
b
(25.0 ꢁ 0.6%), compounds 10b and 19d had slightly lower
inhibitory activity (18.7 ꢁ 0.31% and 22 ꢁ 1.1%, respectively) against
HuAChE-induced Ab1e40 aggregation, indicating that this new
family of genistein derivatives had higher affinity for CAS than for
PAS of AChE.
Cu2þ causes A
b
aggregation in solution, and the interaction of
natural product that inhibits self-induced A
as reference compound [39]. Compounds 10b, 19d and 25d (all at
25 M) were moderately potent with inhibition ratios of 34.3%,
24.6% and 35%, respectively (Table 2), compared with 25 M cur-
b aggregation, was used
peptides with Cu2þ is known to be pH dependent. Cu2þ-induced A
b
m
aggregation is more significant at pH 6.6 than pH 7.4 [41]. To
investigate the effects of compounds on Cu2þ-induced Ab1e42 ag-
gregation, we performed 2 individual studies (in weak acidic
buffered aqueous solution): (1) inhibitory activity of Cu2þ-induced
m
cumin (43.1%). The marketed AD drug, donepezil, did not show any
significant inhibitory activity under the same experimental condi-
tions (Table 2).
Ab1e42 aggregation by chelators and (2) disaggregation effects of
chelators on Cu2þ-induced Ab1e42 aggregates. ThT fluorescence
assay was used to identify the degree of Ab1e42 aggregation [42].
For the ThT fluorescence assay, curcumin was used as the reference
compound. Ab1e42 was first treated with one equivalent of Cu2þ for
2 min at room temperature and then incubated with or without the
testing compounds for 24 h at 37 ꢀC. Cu2þ accelerated the aggre-
gation of Ab1e42 (Fig. 6). In contrast, fluorescence was markedly
reduced by 72.1 ꢁ1.1%, 71.9 ꢁ 3.2%, or 77.8 ꢁ 4.0% when Ab1e42 was
treated with Cu2þ and compounds 10b, 19d, or 25d, respectively.
Donepezil did not show any significant inhibitory activity under the
same experimental conditions. These results suggested that com-
pounds 10b, 19d and 25d inhibited Cu2þ-induced Ab1e42 aggrega-
Compound 25d also showed a concentration-dependent inhib-
itory effect on self-induced Ab1e42 aggregation (Fig. 5).
A
b
deposition has been linked to AChE expression [4]. Further-
more, the PAS of AChE can bind to A , accelerating the formation of
amyloid fibrils. Therefore, inhibition of AChE, particularly the in-
hibition of PAS of AChE, may affect A aggregation. The kinetic
b
b
study showed that compounds 10b, 19d and 25d exhibited a mix-
type inhibition and were able to bind both the catalytic and pe-
ripheral site of AChE. Therefore, to further explore the dual action of
these compounds, we examined their capacity to inhibit HuAChE-
induced Ab1e40 via the ThT-based fluorometric assay [40]. Com-
pound 25d (100
mM) prevented HuAChE-induced
Ab1e40
tion by chelating with Cu2þ
. In particular, 25d significantly
inhibited Cu2þ-induced Ab1e42 aggregation in a concentration-
dependent manner (Fig. 6). For the disaggregation studies, com-
Table 2
Inhibition of self-induced Ab1e42 aggregation, Cu2þ-induced Ab1e42 aggregation, and
HuAChE-induced Ab1e40 aggregation by compounds 10b, 19d, 25d and reference
compounds.
pounds 10b, 19d or 25d were added to A
b fibrils, which were
Comp.
% inhibition of A
Self-inducedb,e
b
aggregationa
Cu2þ-inducedc,e
HuAChE-inducedd,e
10b
19d
25d
Curcumin
Donepezil
34.3 ꢁ 1.2
24.6 ꢁ 1.6
35.0 ꢁ 1.0
43.1 ꢁ 1.5
n.a.g
72.1 ꢁ 1.1
71.9 ꢁ 3.2
77.8 ꢁ 4.0
64.0 ꢁ 2.3
n.a.g
18.7 ꢁ 0.31
22.0 ꢁ 1.1
36.2 ꢁ 0.8
NTf
25.0 ꢁ 0.6
a
For Inhibition of A
b
aggregation, the thioflavin-T fluorescence method was used.
Inhibition of self-induced Ab1e42 aggregation (25 M) by tested inhibitors at
mM.
b
m
25
c
Inhibition of Cu2þ-induced Ab1e42 aggregation. The concentration of tested
compounds and Cu2þ was 25
m
M.
d
Inhibition of HuAChE-induced Ab1e40 aggregation. The concentration of tested
inhibitor and Ab1e40 was 100 and 230 mM, respectively, and the Ab1e40/AChE ratio
was equal to 100/1.
e
Data are presented as the mean ꢁ SD of 3 independent experiments.
f
NT ¼ not tested.
g
n.a. ¼ no active. Compounds defined “no active” meant that percent inhibition
Fig. 6. Inhibition of Cu2þ-induced Ab1e42 aggregation by compounds 10b, 19d, 25d and
Curcumin. The thioflavin-T fluorescence method was used.
was less than 5% at 25 mM.

X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
321
generated by reacting Ab1e42 with one equivalent of Cu2þ for 24 h at
37 ꢀC (Fig. 7). The ThT binding assay showed that 10b, 19d and 25d
compound 10b, 19d and 25d were found to bind Cu2þ and assemble
into metal complexes, which disassembled well-structured A fi-
brils as well as inhibit Cu2þ-induced A
aggregation. Under in vivo
conditions, mice treated with 10b, 19d or 25d displayed signifi-
cantly longer step-down latency times than the control group.
Taken together, among the synthesized compounds, compound
25d exhibited the most promising effects. This compound dis-
played the highest inhibition of AChE, exhibited good antioxidant
b
(all at 25
m
M) markedly disassembled A
b
fibrils by 36.1 ꢁ 1.3%
b
39.2 ꢁ 2.0%, and 72.1 ꢁ 2.8%, respectively (Fig. 7).
2.2.7. In vivo assay
Evaluation of the effect of genistein derivatives in animal models
of learning and memory impairment is lacking. Scopolamine in-
duces memory deficits and has been used extensively to evaluate
potential therapeutic agents for treating AD [43]. Therefore, we
investigated the protective effects of genistein derivatives against
scopolamine-induced memory deficits via the passive avoidance
task. The step-down latency of mice treated with scopolamine
alone (control group) was significantly (p < 0.05) shorter than that
of vehicle-treated mice (normal group). Treatment with com-
pounds 10b or 19d (2, 6 and 18 mg/kg), or 25d (3, 9 and 27 mg/kg)
increased the latency time in a dose-dependent manner. In
particular, the effect of compounds 19d (6 mg/kg) and 25d (9 mg/
kg) was comparable with that of donepezil (5 mg/kg) (see Fig. 8).
Compounds 10b, 19d and 25d inhibited AChE activity and reversed
scopolamine-induced memory deficit in the step-down passive
avoidance test. These results indicated that the synthesized genis-
tein-O-alkylbenzylamine derivatives may have reversed cognitive
deficit by increasing brain cholinergic activity due to the inhibition
of AChE.
and metal-chelating activities, significantly inhibited A
tion, induced a disaggregation of A
fibrils generated by Cu2þ
induced A aggregation, and reversed cognitive deficit, possibly by
b aggrega-
b
-
b
increasing brain cholinergic activity due to the inhibition of AChE.
Such multifunctional properties promote compound 25d as an
interesting candidate for further studies directed to the develop-
ment of novel multifunctional agents for the treatment of AD.
Further studies to evaluate 25d in vivo are in progress.
4. Experimental section
4.1. Chemistry
Melting points were recorded on YRT-3 melting-point apparatus
(China) and are uncorrected. 1H NMR and 13C NMR spectra were
recorded at room temperature in CDCl3 solutions using a Varian
INOVA 400 NMR spectrometer. Chemical shifts are reported in parts
per millions (ppm) relative to tetramethylsilane (TMS). Mass
spectra were recorded on Agilent-6210 TOF LCeMS Spectrometer.
Elemental analyses were performed on a Carlo Erba 1106 apparatus.
Genistein was commercially available and purchased from Nanjing
Zelang Medical Technology Co., Ltd. All commercially available and
anhydrous solvents were of the highest quality and were used
without further purification. All the reactions were monitored by
thin-layer chromatography (TLC) on silica gel GF254 plates from
Qingdao Haiyang Chemical Co. Ltd. (China), and then visualized in
an iodine chamber or with an UV lamp (254 nm). Where appro-
priate, crude products were purified by column chromatography
using silica gel (230e400 mesh) purchased from Qingdao Haiyang
Chemical Co. Ltd. (China).
3. Conclusion
In summary, 28 new genistein-O-alkylbenzylamine derivatives
(8e10aed, 18e20aed, 24d, 25b, 25d and 26d) have been designed,
synthesized and evaluated as novel multifunctional agents against
AD. Most of these compounds were potent in inhibiting AChE ac-
tivity and displayed high selectivity for AChE over BuChE. Com-
pounds 10b, 19d and 25d exhibited the highest potency for AChE
inhibition potency, particularly 25d (IC50 ¼ 0.09 ꢁ 0.02
mM). Both
the inhibition kinetic analysis and molecular modeling study sug-
gested that compound 25d showed a mixed-type inhibition,
binding to both CAS and PAS of AChE, and inducing a strong
inhibitory effect. Compounds 10b and 19d displayed antioxidant
activity, and compound 25d had relatively lower effects. Impor-
4.1.1. General procedure for the synthesis of 2e4 [30,31]
To a solution of genistein (6.0 g, 22.20 mmol) in DMF (50 mL),
powdered KOH (1.26 g, 22.20 mmol) was added. After the mixture
was stirred at room temperature for 1 h, the appropriate dibro-
moalkane derivative (26.0 mmol) and KI (296 mg, 1.78 mmol) were
added. Then the reaction mixture was stirred at room temperature
for 24 h, diluted with water (200 mL), treated with 10% HCl aqueous
solution until an acidic pH was reached. The solid formed was
collected by filtration, washed with water, dried, and purified by
flash chromatography on silica gel.
tantly, all 3 compounds moderately inhibited self-induced A
gregation, however, exhibited higher Cu2þ-induced A
aggregation
inhibitory activity than that of curcumin. Despite the small inhi-
bition of AChE-induced A aggregation by compound 25d, this
b ag-
b
b
compound proved to be more potent than donepezil. Moreover,
4.1.1.1. 7-(3-Bromopropoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-
chromen-4-one (2). It was synthesized from 1,3-dibromopropane.
Elution with CH2Cl2/acetone (100:1) afforded 2 as an almost white
solid; 57.5% yield; mp 124e126 ꢀC. (lit., [31] 124e126 ꢀC).
4.1.1.2. 7-(4-Bromobutoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-
chromen-4-one (3). It was synthesized from 1,4-dibromobutane.
Elution with CH2Cl2/acetone (100:1) afforded 3 as an almost white
solid; 67.8% yield; mp 130e132 ꢀC. (lit., [30] 130e131 ꢀC).
4.1.1.3. 7-((6-Bromohexyl)oxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-
chromen-4-one (4). It was synthesized from 1,6-dibromohexane.
Elution with CH2Cl2/acetone (100:1) afforded 4 as an almost white
solid; 53.5% yield; mp 124e126 ꢀC. (lit. [44] 121e123 ꢀC).
Fig. 7. The disaggregation of Cu2þ-induced Ab1e42 aggregation by compounds 10b,
19d, 25d and Curcumin. The measurements was carried out in the presence of 25
test compound. The thioflavin-T fluorescence method was used.
mM

322
X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
Fig. 8. Effects of compound 10b, 19d and 25d on scopolamine-induced memory deficit in the step-down passive avoidance test. Compounds 10b, 19d (both at 2, 6 and 18 mg/kg,
p.o.), 25d (3, 9, 27 mg/kg, p.o.), or donepezil (5 mg/kg, p.o.) were orally given 1 h before treatment with scopolamine. After 30 min, the mice were treated with scopolamine (3 mg/
kg, i.p.) and tested in the step-down passive avoidance. Values are expressed as the mean ꢁ SEM (n ¼ 10). #p < 0.05 vs normal group. *p < 0.05 and **p < 0.01 vs scopolamine-
treated control group.
4.1.2. General procedure for the synthesis of secondary amines (7ae
d)
J ¼ 2.0 Hz, 1H, 6-H), 4.04 (t, J ¼ 6.4 Hz, 2H, CH2O), 3.61 (s, 2H,
PhCH2), 2.63 (t, J ¼ 6.8 Hz, 2H, CH2N), 2.58 (t, J ¼ 6.8 Hz, 2H, CH2N),
1.97L1.94 (m, 2H, CH2), 1.07 (t, J ¼ 6.8 Hz, 3H, CH3); HR-ESI-MS:
Calcd. for C27H28NO5 [MþH]þ: 446.1967, found: 446.1972; Anal.
calcd. For (C27H27NO5): C, 72.79; H, 6.11; N, 3.14; found: C, 72.70; H,
6.02; N, 3.06.
Compounds 7aed were prepared as previously described [32].
4.1.3. General procedure for preparation of 7-O-modified genistein
derivatives (8aed, 9aed and 10aed)
To a mixture of the corresponding secondary amines 7ae
d (0.77 mmol), anhydrous K2CO3 (106.2 mg, 0.77 mmol) and KI
(8.6 mg, 0.052 mmol) in CH3CN (10 mL) were added the appropriate
intermediates 2e4 (0.60 mmol). The reaction mixture was warmed
to 60e65 ꢀC and stirred for 12e15 h under an argon atmosphere.
After complete reaction, the solvent was evaporated under reduced
pressure. Then water (30 mL) was added to the residue and the
mixture was extracted with dichloromethane (30 mL ꢂ 3). The
combined organic phases were washed with saturated aqueous
sodium chloride (30 mL), dried over sodium sulfate, and filtered.
The solvent was evaporated to dryness under reduced pressure. The
residue was purified on a silica gel chromatography using mixtures
of CH2Cl2/CH3OH as eluent, obtaining the corresponding 7-O-
modified genistein derivatives.
4 .1. 3 . 3 . 5 - H y d r o x y - 3 - ( 4 - h y d r o x y p h e n y l ) - 7 - ( 3 - ( ( 2 -
methoxybenzyl)(methyl)amino)propoxy)-4H-chromen-4-one
(8c).
It was obtained from N-(2-methoxybenzyl)-methylamine (7c) and
7-(3-bromopropoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-chro-
men-4-one (2) according to the general procedure. Elution with
CH2Cl2/MeOH ¼ 150:1 gave 8c as an almost solid; 54.4% yield; mp
119e122 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.82 (s,1H, 5-OH), 7.81 (s,
1H, 2-H), 7.35 (d, J ¼ 8.4 Hz, 2H, 20, 60-H), 7.33-7.24 (m, 2H, 2 ꢂ Are
H), 6.94e6.87 (m, 2H, 2 ꢂ AreH), 6.86 (d, J ¼ 8.4 Hz, 2H, 30, 50-H),
6.34(d, J ¼ 2.0 Hz, 1H, 8-H), 6.32 (d, J ¼ 2.0 Hz, 1H, 6-H), 4.07 (t,
J ¼ 6.4 Hz, 2H, CH2O), 3.81 (s, 3H, CH3O), 3.65 (s, 2H, PhCH2), 2.67 (t,
J ¼ 6.4 Hz, 2H, CH2N), 2.32 (s, 3H, CH3N), 2.09L2.07 (m, 2H, CH2);
HR-ESI-MS: Calcd. for
462.1920; Anal. calcd. For (C27H27NO6): C, 70.27; H, 5.90; N, 3.03;
found: C, 70.40; H, 5.88; N, 2.95.
C
27H28NO6 [MþH]þ: 462.1917, found:
4.1.3.1. 7-(3-(Benzyl(methyl)amino)propoxy)-5-hydroxy-3-(4-
hydroxyphenyl)-4H-chromen-4-one (8a). It was obtained from N-
benzylmethylamine (7a) and 7-(3-bromopropoxy)-5-hydroxy-3-
(4-hydroxyphenyl)-4H-chromen-4-one (2) according to the general
procedure. Elution with CH2Cl2/MeOH ¼ 200:1 gave 8a as an
almost white solid; 52.8% yield; mp 134e136 ꢀC; 1H NMR
4.1.3.4. 7-(3-(Ethyl(2-methoxybenzyl)amino)propoxy)-5-hydroxy-3-
(4-hydroxyphenyl)-4H-chromen-4-one (8d). It was obtained from
N-(2-methoxybenzyl)ethylamine (7d) and 7-(3-bromopropoxy)-5-
hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (2) according to
the general procedure. Elution with CH2Cl2/MeOH ¼ 150:1 gave 8d
as an almost solid; 60.2% yield; mp 127e129 ꢀC; 1H NMR (400 MHz,
(400 MHz, CDCl3)
d 12.81 (s, 1H, 5-OH), 7.83 (s, 1H, 2-H), 7.36 (d,
J ¼ 8.0 Hz, 2H, 20, 60-H), 7.31e7.25 (m, 5H, 5 ꢂ AreH), 6.85 (d,
J ¼ 8.0 Hz, 2H, 30, 50-H), 6.34 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.33 (d,
J ¼ 2.0 Hz, 1H, 6-H), 4.08 (t, J ¼ 6.0 Hz, 2H, CH2O), 3.56 (s, 2H,
PhCH2), 2.58 (t, J ¼ 6.8 Hz, 2H, CH2N), 2.26 (s, 3H, CH3N), 2.04e2.00
(m, 2H, CH2); HR-ESI-MS: Calcd. for C26H26NO5 [MþH]þ: 432.1811,
found: 432.1808; Anal. calcd. For (C26H25NO5): C, 72.37; H, 5.84; N,
3.25; found: C, 72.56; H, 5.80; N, 3.13.
CDCl3)
d
12.81 (s, 1H, 5-OH), 7.84 (s, 1H, 2-H), 7.38 (d, J ¼ 8.0 Hz, 2H,
20, 60-H), 7.37e7.36 (m, 1H, AreH), 7.24e7.20 (m, 1H, AreH), 6.93e
6.84 (m, 4H, 2 ꢂ AreH, 30, 50-H), 6.36 (d, J ¼ 2.0 Hz,1H, 8-H), 6.34 (d,
J ¼ 2.0 Hz, 1H, 6-H), 4.06 (t, J ¼ 6.4 Hz, 2H, CH2O), 3.81 (s, 3H, CH3O),
3.63 (s, 2H, PhCH2), 2.66 (t, J ¼ 6.8 Hz, 2H, CH2N), 2.57 (q, J ¼ 6.8 Hz,
2H, CH2N), 2.01L1.95 (m, 2H, CH2), 1.07 (t, J ¼ 6.8 Hz, 3H, CH3); HR-
ESI-MS: Calcd. for C28H30NO6 [MþH]þ: 476.2073, found: 476.2072;
Anal. calcd. For (C28H29NO6): C, 70.72; H, 6.15; N, 2.95; found: C,
70.83; H, 6.22; N, 3.09.
4.1.3.2. 7-(3-(Benzyl(ethyl)amino)propoxy)-5-hydroxy-3-(4-
hydroxyphenyl)-4H-chromen-4-one (8b). It was obtained from N-
benzylethylamine (7b) and 7-(3-bromopropoxy)-5-hydroxy-3-(4-
hydroxyphenyl)-4H-chromen-4-one (2) according to the general
procedure. Elution with CH2Cl2/MeOH ¼ 200:1 gave 8b as an
almost white solid; 56.4% yield; mp 100e102 ꢀC; 1H NMR
4.1.3.5. 7-(4-(Benzyl(methyl)amino)butoxy)-5-hydroxy-3-(4-
hydroxyphenyl)-4H-chromen-4-one (9a). It was obtained from N-
benzylmethylamine (7a) and 7-(4-bromobutoxy)-5-hydroxy-3-(4-
hydroxyphenyl)-4H-chromen-4-one (3) according to the general
procedure. Elution with CH2Cl2/MeOH ¼ 120:1 gave 9a as an almost
white solid; 79.5% yield; mp 158e160 ꢀC; 1H NMR (400 MHz,
(400 MHz, CDCl3)
d 12.81 (s, 1H, 5-OH), 7.85 (s, 1H, 2-H), 7.39 (d,
J ¼ 8.4 Hz, 2H, 20, 60-H), 7.34e7.21 (m, 5H, 5 ꢂ AreH), 6.88 (d,
J ¼ 8.4 Hz, 2H, 30, 50-H), 6.34 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.32 (d,

X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
323
CDCl3)
d
12.82 (s, 1H, 5-OH), 7.80 (s, 1H, 2-H), 7.36 (d, J ¼ 8.4 Hz, 2H,
J ¼ 6.4 Hz, 2H, CH2O), 3.57 (s, 2H, PhCH2), 2.43 (t, J ¼ 6.8 Hz, 2H,
CH2N), 2.24 (s, 3H, CH3N), 1.74e1.71 (m, 2H, CH2), 1.60e1.55 (m, 2H,
CH2), 1.42e1.36 (m, 4H, 2 ꢂ CH2); HR-ESI-MS: Calcd. for C29H32NO5
[MþH]þ: 474.2280, found: 474.2284; Anal. calcd. For (C29H31NO5):
C, 73.55; H, 6.60; N, 2.96; found: C, 73.52; H, 6.69; N, 3.16.
20, 60-H), 7.33e7.26 (m, 5H, 5 ꢂ AreH), 6.87 (d, J ¼ 8.4 Hz, 2H, 30, 50-
H), 6.32 (s, 2H, 8-H, 6-H), 3.97 (t, J ¼ 6.4 Hz, 2H, CH2O), 3.56 (s, 2H,
PhCH2), 2.48 (t, J ¼ 7.2 Hz, 2H, CH2N), 2.25 (s, 3H, CH3N), 1.85e1.80
(m, 2H, CH2), 1.75e1.70 (m, 2H, CH2); HR-ESI-MS: Calcd. for
C
27H28NO5 [MþH]þ: 446.1967, found: 446.1960; Anal. calcd. For
(C27H27NO5): C, 72.79; H, 6.11; N, 3.14; found: C, 72.58; H, 6.18; N,
3.05.
4.1.3.10. 7-((6-(Benzyl(ethyl)amino)hexyl)oxy)-5-hydroxy-3-(4-
hydroxyphenyl)-4H-chromen-4-one (10b). It was obtained from N-
benzylethylamine (7b) and 7-((6-bromohexyl)oxy)-5-hydroxy-3-
(4-hydroxyphenyl)-4H-chromen-4-one (4) according to the general
procedure. Elution with CH2Cl2/MeOH ¼ 100:1 gave 10b as an
almost white solid; 67.7% yield; mp 42e44 ꢀC; 1H NMR (400 MHz,
4.1.3.6. 7-(4-(Benzyl(ethyl)amino)butoxy)-5-hydroxy-3-(4-
hydroxyphenyl)-4H-chromen-4-one (9b). It was obtained from N-
benzylethylamine (7b) and 7-(4-bromobutoxy)-5-hydroxy-3-(4-
hydroxyphenyl)-4H-chromen-4-one (3) according to the general
procedure. Elution with CH2Cl2/MeOH ¼ 150:1 gave 9b as an almost
white solid; 61.0% yield; mp 109e111 ꢀC; 1H NMR (400 MHz, CDCl3)
CDCl3)
d
12.81 (s, 1H, 5-OH), 7.81 (s, 1H, 2-H), 7.38 (d, J ¼ 8.4 Hz, 2H,
20, 60-H), 7.37e7.26 (m, 5H, 5 ꢂ AreH), 6.87 (d, J ¼ 8.4 Hz, 2H, 30, 50-
H), 6.33 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.32 (d, J ¼ 2.0 Hz, 1H, 6-H), 3.94 (t,
J ¼ 6.4 Hz, 2H, CH2O), 3.64 (s, 2H, PhCH2), 2.58 (q, J ¼ 6.8 Hz, 2H,
CH2N), 2.48 (t, J ¼ 7.2 Hz, 2H, CH2N), 1.75e1.70 (m, 2H, CH2), 1.56e
1.53 (m, 2H, CH2), 1.41e1.34 (m, 4H, 2 ꢂ CH2), 1.08 (t, J ¼ 6.8 Hz, 3H,
d
12.81 (s, 1H, 5-OH), 7.81 (s, 1H, 2-H), 7.37e7.24 (m, 7H, 7 ꢂ AreH),
6.87 (d, J ¼ 8.0 Hz, 2H, 30, 50-H), 6.32 (s, 2H, 8-H, 6-H), 3.94 (t,
J ¼ 6.4 Hz, 2H, CH2O), 3.63 (s, 2H, PhCH2), 2.58 (q, J ¼ 6.8 Hz, 2H,
CH2N), 2.53 (t, J ¼ 6.8 Hz, 2H, CH2N), 1.84e1.77 (m, 2H, CH2), 1.69e
1.66 (m, 2H, CH2), 1.09 (t, J ¼ 6.8 Hz, 3H, CH3); HR-ESI-MS: Calcd. for
CH3); 13C NMR (100 MHz, CDCl
) d 180.8, 164.9, 162.3, 157.7, 157.1, 152.5,
3
137.5, 130.1, 129.4, 128.2, 127.2, 123.5, 121.8, 116.0, 105.9, 98.5, 92.5,
68.4, 57.4, 52.5, 46.6, 28.7, 27.0, 25.9, 25.6, 10.7; HR-ESI-MS: Calcd.
for C30H34NO5 [MþH]þ: 488.2437, found: 488.2429; Anal. calcd. For
(C30H33NO5): C, 73.90; H, 6.82; N, 2.87; found: C, 73.76; H, 6.90; N,
3.05.
C
28H30NO5 [MþH]þ: 460.2124, found: 460.2118; Anal. calcd. For
(C28H29NO5): C, 73.18; H, 6.36; N, 3.05; found: C, 73.15; H, 6.42; N,
3.00.
4 .1. 3 . 7 . 5 - H y d r o x y - 3 - ( 4 - h y d r o x y p h e n y l ) - 7 - ( 4 - ( ( 2 -
methoxybenzyl)(methyl)amino)butoxy)-4H-chromen-4-one
(9c).
4 .1. 3 .11. 5 - H y d ro x y - 3 - ( 4 - hy d r o x y p h e nyl ) - 7 - ( ( 6 - ( ( 2 -
methoxybenzyl)(methyl)amino)hexyl)oxy)-4H-chromen-4-one (10c).
It was obtained from N-(2-methoxybenzyl)-methylamine (7c) and
7-((6-bromohexyl)oxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-chro-
men-4-one (4) according to the general procedure. Elution with
CH2Cl2/MeOH ¼ 120:1 gave 10c as an almost white solid; 68.8%
It was obtained from N-(2-methoxybenzyl)-methylamine (7c) and
7-(4-bromobutoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-chro-
men-4-one (3) according to the general procedure. Elution with
CH2Cl2/MeOH ¼ 100:1 gave 9c as an almost white solid; 78.5%
yield; mp 123e125 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.82 (s, 1H, 5-
OH), 7.71 (s, 1H, 2-H), 7.36-7.27 (m, 4H, 4 ꢂ AreH), 6.97e6.87 (m,
2H, 2 ꢂ AreH), 6.86 (d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.27 (d, J ¼ 2.0 Hz,
1H, 8-H), 6.26 (d, J ¼ 2.0 Hz, 1H, 6-H), 3.90 (t, J ¼ 6.0 Hz, 2H, CH2O),
3.81 (s, 3H, CH3O), 3.74 (s, 2H, PhCH2), 2.62 (t, J ¼ 6.8 Hz, 2H, CH2N),
2.35 (s, 3H, CH3N), 1.80 (m, 4H, 2 ꢂ CH2); HR-ESI-MS: Calcd. for
yield; mp 45e47 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.80 (s, 1H, 5-
OH), 7.76 (s, 1H, 2-H), 7.38-7.26 (m, 4H, 4 ꢂ AreH), 6.97e6.87 (m,
4H, 4 ꢂ AreH), 6.26 (s, 2H, 8-H, 6-H), 3.85 (t, J ¼ 6.4 Hz, 2H, CH2O),
3.81(s, 3H, CH3O), 3.73 (s, 2H, PhCH2), 2.55 (t, J ¼ 7.2 Hz, 2H, CH2N),
2.33 (s, 3H, CH3N), 1.66e1.64 (m, 4H, 2 ꢂ CH2), 1.36 (s, 4H, 2 ꢂ CH2);
C
28H30NO6 [MþH]þ: 476.2073, found: 476.2071; Anal. calcd. For
HR-ESI-MS: Calcd. for
504.2378; Anal. calcd. For (C30H33NO6): C, 71.55; H, 6.61; N, 2.78;
found: C, 71.43; H, 6.73; N, 2.90.
C
30H34NO6 [MþH]þ: 504.2386, found:
(C28H29NO6): C, 70.72; H, 6.15; N, 2.95; found: C, 70.88; H, 6.11; N,
2.80.
4.1.3.8. 7-(4-(Ethyl(2-methoxybenzyl)amino)butoxy)-5-hydroxy-3-
(4-hydroxyphenyl)-4H-chromen-4-one (9d). It was obtained from
N-(2-methoxybenzyl)ethylamine (7d) and 7-(4-bromobutoxy)-5-
hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (3) according
to the general procedure. Elution with CH2Cl2/MeOH ¼ 120:1 gave
9d as an almost white solid; 81.7% yield; mp 45e48 ꢀC; 1H NMR
4.1.3.12. 7-((6-(Ethyl(2-methoxybenzyl)amino)hexyl)oxy)-5-
hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (10d). It was ob-
tained from N-(2-methoxybenzyl)ethylamine (7d) and 7-((6-
bromohexyl)oxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-
4-one (4) according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 100:1 gave 10d as an almost white solid; 79.2% yield; mp
(400 MHz, CDCl3)
d
12.81 (s, 1H, 5-OH), 7.74 (s, 1H, 2-H), 7.41 (d,
41e43 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.78 (s, 1H, 5-OH), 7.75 (s,
J ¼ 7.2 Hz, 1H, AreH), 7.32 (d, J ¼ 8.4 Hz, 2H, 20, 60-H), 7.28e7.24(m,
1H, AreH), 6.95 (t, J ¼ 7.2 Hz, 1H, AreH), 6.88e6.84 (m, 3H, AreH,
30, 50-H), 6.28 (s, 2H, 8-H, 6-H), 3.89 (t, J ¼ 6.4 Hz, 2H, CH2O), 3.81 (s,
3H, CH3O), 3.74 (s, 2H, PhCH2), 2.67e2.61 (m, 4H, 2 ꢂ CH2N),
1.78L1.72 (m, 4H, 2 ꢂ CH2), 1.14 (t, J ¼ 6.4 Hz, 3H, CH3); HR-ESI-MS:
Calcd. for C29H32NO6 [MþH]þ: 490.2230, found: 490.2236; Anal.
calcd. For (C29H31NO6): C, 71.15; H, 6.38; N, 2.86; found: C, 71.00; H,
6.46; N, 3.02.
1H, 2-H), 7.51 (d, J ¼ 7.2 Hz,1H, AreH), 7.37e7.31 (m, 3H, 3 ꢂ AreH),
7.01e6.90 (m, 4H, 4 ꢂ AreH), 6.21 (s, 2H, 8-H, 6-H), 3.96 (t,
J ¼ 6.4 Hz, 2H, CH2O), 3.85 (s, 3H, CH3O), 3.79 (s, 2H, PhCH2), 2.84
(brs, 2H, CH2N), 2.72 (brs, 2H, CH2N), 1.71 (brs, 2H, CH2), 1.62 (brs,
2H, CH2), 1.31e1.25 (m, 7H, 2 ꢂ CH2, CH3); HR-ESI-MS: Calcd. for
C
31H36NO6 [MþH]þ: 518.2543, found: 518.2540; Anal. calcd. For
(C31H35NO6): C, 71.93; H, 6.82; N, 2.71; found: C, 72.00; H, 6.87; N,
2.58.
4.1.3.9. 7-((6-(Benzyl(methyl)amino)hexyl)oxy)-5-hydroxy-3-(4-
hydroxyphenyl)-4H-chromen-4-one (10a). It was obtained from N-
benzylmethylamine (7a) and 7-((6-bromohexyl)oxy)-5-hydroxy-3-
(4-hydroxyphenyl)-4H-chromen-4-one (4) according to the general
procedure. Elution with CH2Cl2/MeOH ¼ 150:1 gave 10a as an
almost white solid; 57.0% yield; mp 110e111 ꢀC; 1H NMR (400 MHz,
4.1.4. 5-Hydroxy-3-(4-hydroxyphenyl)-7-(methoxymethoxy)-4H-
chromen-4-one (11)
Intermediate 11 was synthesized as previously described [33].
4.1.5. General procedure for the synthesis of 12e14
To a mixture of the intermediate 11 (1.00 g, 3.18 mmol), anhy-
drous K2CO3 (440 mg, 3.18 mmol) and KI (53 mg, 0.32 mmol) in
CH3CN (30 mL) the appropriate dibromoalkane derivative
(6.36 mmol) was added. The reaction mixture was warmed to 60e
CDCl3)
d
12.80 (s, 1H, 5-OH), 7.80 (s, 1H, 2-H), 7.38 (d, J ¼ 8.4 Hz, 2H,
20, 60-H), 7.33e7.26 (m, 5H, 5 ꢂ AreH), 6.86 (d, J ¼ 8.4 Hz, 2H, 30, 50-
H), 6.32 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.31 (d, J ¼ 2.0 Hz, 1H, 6-H), 3.92 (t,

324
X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
65 ꢀC and stirred for 12e15 h under an argon atmosphere. After
complete reaction, the solvent was evaporated under reduced
pressure. Water (30 mL) was added to the residue and the mixture
was extracted with dichloromethane (30 mL ꢂ 3). The combined
organic phases were washed with saturated aqueous sodium
chloride, dried over sodium sulfate, and filtered. The solvent was
evaporated to dryness under reduced pressure. The residue was
purified on a silica gel chromatography to give the intermediates
12e14.
15b as a light yellow oil; 73.6% yield; 1H NMR (400 MHz, CDCl3)
d
12.83 (s, 1H, 5-OH), 7.88 (s, 1H, 2-H), 7.44 (d, J ¼ 8.8 Hz, 2H, 20, 60-
H), 7.35e7.22 (m, 5H, 5 ꢂ AreH), 6.94 (d, J ¼ 8.8 Hz, 2H, 30, 50-H),
6.58 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.50 (d, J ¼ 2.0 Hz, 1H, 6-H), 5.24 (s, 2H,
OCH2O), 4.03 (t, J ¼ 6.4 Hz, 2H, CH2O), 3.62 (s, 2H, PhCH2), 3.50 (s,
3H, CH3O), 2.65 (t, J ¼ 6.8 Hz, 2H, CH2N), 2.56 (q, J ¼ 6.4 Hz, 2H,
CH2N), 1.99e1.96 (m, 2H, CH2), 1.07 (t, J ¼ 6.4 Hz, 3H, CH3).
4.1.6.3. 5-Hydroxy-3-(4-(3-((2-methoxybenzyl)(methyl)amino)pro-
poxy)phenyl)-7-(methoxy
methoxy)-4H-chromen-4-one
(15c).
4.1.5.1. 3-(4-(3-Bromopropoxy)phenyl)-5-hydroxy-7-(methox-
ymethoxy)-4H-chromen-4-one (12). It was synthesized from 1,3-
dibromopropane. Elution with CH2Cl2 afforded 12 as an almost
white solid; 62.1% yield; mp 98e100 ꢀC. (lit. [44] 94e97 ꢀC).
It was obtained from N-(2-methoxybenzyl)-methylamine (7c) and
3-(4-(3-bromopropoxy)phenyl)-5-hydroxy-7-(methoxymethoxy)-
4H-chromen-4-one (12) according to the general procedure.
Elution with CH2Cl2/MeOH ¼ 80:1 gave 15c as a light yellow oil;
86.7% yield; 1H NMR (400 MHz, CDCl3)
d 12.84 (s, 1H, 5-OH), 7.88 (s,
4.1.5.2. 3-(4-(4-Bromobutoxy)phenyl)-5-hydroxy-7-(methox-
ymethoxy)-4H-chromen-4-one (13). It was synthesized from 1,4-
dibromobutane. Elution with CH2Cl2 afforded 13 as an almost
white solid; 61.5% yield; mp 102e104 ꢀC. (lit. [44] 100e102 ꢀC).
1H, 2-H), 7.44 (d, J ¼ 8.4 Hz, 2H, 20, 60-H), 7.34 (d, J ¼ 7.2 Hz, 1H, Are
H), 7.26e7.24 (m, 1H, AreH), 6.97 (d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.92e
6.86 (m, 2H, 2 ꢂ AreH), 6.58 (d, J ¼ 1.6 Hz, 1H, 8-H), 6.50 (s,
J ¼ 1.6 Hz, 1H, 6-H), 5.24 (s, 2H, OCH2O), 4.08 (t, J ¼ 6.4 Hz, 2H,
CH2O), 3.84 (s, 3H, CH3O), 3.61 (s, 2H, PhCH2), 3.50 (s, 3H, CH3O),
2.66 (t, J ¼ 6.8 Hz, 2H, CH2N), 2.33 (s, 3H, CH3N), 2.09e2.07 (m, 2H,
CH2).
4.1.5.3. 3-(4-((6-Bromohexyl)oxy)phenyl)-5-hydroxy-7-(methox-
ymethoxy)-4H-chromen-4-one (14). It was synthesized from 1,6-
dibromohexane. Elution with CH2Cl2 afforded 14 as an almost
white solid; 52.1% yield; mp 79e80 ꢀC; 1H NMR (400 MHz, CDCl3)
4.1.6.4. 3-(4-(3-(Ethyl(2-methoxybenzyl)amino)propoxy)phenyl)-5-
hydroxy-7-(methoxymethoxy)-4H-chromen-4-one (15d). It was ob-
tained from N-(2-methoxybenzyl)-ethylamine (7d) and 3-(4-(3-
bromopropoxy)phenyl)-5-hydroxy-7-(methoxymethoxy)-4H-
chromen-4-one (12) according to the general procedure. Elution
with CH2Cl2/MeOH ¼ 60:1 gave 15d as a light yellow oil; 89.0%
d
12.83 (s, 1H, 5-OH), 7.88 (s, 1H, 2-H), 7.45 (d, J ¼ 8.4 Hz, 2H, 20, 60-
H), 6.97 (d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.58 (d, J ¼ 2.4 Hz, 1H, 8-H), 6.50
(d, J ¼ 2.4 Hz, 1H, 6-H), 5.24 (s, 2H, OCH2O), 4.00 (t, J ¼ 6.4 Hz, 2H,
CH2O), 3.50 (s, 3H, CH3O), 3.44 (t, J ¼ 6.4 Hz, 2H, CH2Br), 1.92e1.89
(m, 2H, CH2), 1.84e1.80 (m, 2H, CH2), 1.53e1.51 (m, 4H, 2 ꢂ CH2).
yield; 1H NMR (400 MHz, CDCl3)
d 12.84 (s, 1H, 5-OH), 7.88 (s, 1H, 2-
4.1.6. General procedure for preparation of 7-O-MOM-40-O-
modified genistein derivatives (15aed, 16aed and 17aed)
H), 7.45e7.42 (m, 3H, 3 ꢂ AreH), 7.23 (t, J ¼ 8.0 Hz, 1H, AreH),
6.95e6.91 (m, 3H, 3 ꢂ AreH), 6.86 (d, J ¼ 8.0 Hz, 1H, AreH), 6.58 (d,
J ¼ 1.6 Hz, 1H, 8-H), 6.50 (d, J ¼ 1.6 Hz, 1H, 6-H), 5.24(s, 2H, OCH2O),
4.05 (t, J ¼ 6.4 Hz, 2H, CH2O), 3.82 (s, 3H, CH3O), 3.70 (s, 2H, PhCH2),
3.50 (s, 3H, CH3O), 2.72 (brs, 2H, CH2N), 2.63 (brs, 2H, CH2N), 2.03
(brs, 2H, CH2), 1.11 (brs, 3H, CH3).
To a mixture of the corresponding secondary amines 7ae
d (0.77 mmol), anhydrous K2CO3 (106.2 mg, 0.77 mmol) and KI
(8.6 mg, 0.052 mmol) in CH3CN (10 mL) were added the appropriate
intermediates 12e14 (0.60 mmol). The reaction mixture was
warmed to 60e65 ꢀC and stirred for 12e15 h under an argon at-
mosphere. After complete reaction, the solvent was evaporated
under reduced pressure. Water (30 mL) was added to the residue
and the mixture was extracted with dichloromethane (30 mL ꢂ 3).
The combined organic phases were washed with saturated aqueous
sodium chloride (30 mL), dried over sodium sulfate, and filtered.
The solvent was evaporated to dryness under reduced pressure. The
residue was purified on a silica gel chromatography using mixture
of CH2Cl2/CH3OH as eluent, obtaining the corresponding 7-O-
MOM-40-O-modified genistein derivatives.
4.1.6.5. 3-(4-(4-(Benzyl(methyl)amino)butoxy)phenyl)-5-hydroxy-7-
(methoxymethoxy)-4H-chromen-4-one (16a). It was obtained from
N-benzylmethylamine (7a) and 3-(4-(4-bromobutoxy) phenyl)-5-
hydroxy-7-(methoxymethoxy)-4H-chromen-4-one (13) according
to the general procedure. Elution with CH2Cl2/MeOH ¼ 80:1 gave
16a as a light yellow oil; 81.6% yield; 1H NMR (400 MHz, CDCl3)
d
12.84 (s, 1H, 5-OH), 7.87 (s, 1H, 2-H), 7.44 (d, J ¼ 8.8 Hz, 2H, 20, 60-
H), 7.32e7.25 (m, 5H, 5 ꢂ AreH), 6.95 (d, J ¼ 8.8 Hz, 2H, 30, 50-H),
6.58 (d, J ¼ 2.4 Hz, 1H, 8-H), 6.50 (d, J ¼ 2.4 Hz, 1H, 6-H), 5.24 (s, 2H,
OCH2O), 3.99 (t, J ¼ 6.8 Hz, 2H, CH2O), 3.51 (s, 2H, PhCH2), 3.50 (s,
3H, CH3O), 2.45 (t, J ¼ 7.2 Hz, 2H, CH2N), 2.22 (s, 3H, CH3N), 1.85e
1.82 (m, 2H, CH2), 1.73e1.69 (m, 2H, CH2).
4.1.6.1. 3-(4-(3-(Benzyl(methyl)amino)propoxy)phenyl)-5-hydroxy-
7-(methoxymethoxy)-4H-chromen-4-one (15a). It was obtained
from N-benzylmethylamine (7a) and 3-(4-(3-bromopropoxy)
phenyl)-5-hydroxy-7-(methoxymethoxy)-4H-chromen-4-one (12)
according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 100:1 gave 15a as a light yellow oil; 61.9% yield; 1H
4.1.6.6. 3-(4-(4-(Benzyl(ethyl)amino)butoxy)phenyl)-5-hydroxy-7-
(methoxymethoxy)-4H-chromen-4-one (16b). It was obtained from
N-benzylethylamine (7b) and 3-(4-(4-bromobutoxy)phenyl)-5-hy-
droxy-7-(methoxymethoxy)-4H-chromen-4-one (13) according to
the general procedure. Elution with CH2Cl2/MeOH ¼ 60:1 gave 16b
NMR(400 MHz, CDCl3)
d 12.84 (s, 1H, 5-OH), 7.88 (s, 1H, 2-H), 7.44
(d, J ¼ 8.4 Hz, 2H, 20, 60-H), 7.31e7.24 (m, 5H, 5 ꢂ AreH), 6.96 (d,
J ¼ 8.4 Hz, 2H, 30, 50-H), 6.58 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.50 (d, J ¼ 2.
0 Hz, 1H, 6-H), 5.24 (s, 2H, OCH2O), 4.06 (t, J ¼ 6.4 Hz, 2H, CH2O),
3.53 (s, 2H, PhCH2), 3.50 (s, 3H, CH3O), 2.58 (t, J ¼ 6.8 Hz, 2H, CH2N),
2.24 (s, 3H, CH3N), 2.03e2.00 (m, 2H, CH2).
as a light yellow oil; 84.8% yield; 1H NMR (400 MHz, CDCl3)
d 12.84
(s,1H, 5-OH), 7.88 (s,1H, 2-H), 7.44 (d, J ¼ 8.4 Hz, 2H, 20, 60-H), 7.36e
7.24 (m, 5H, 5 ꢂ AreH), 6.94 (d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.58 (d,
J ¼ 2.4 Hz,1H, 8-H), 6.50 (d, J ¼ 2.4 Hz,1H, 6-H), 5.24 (s, 2H, OCH2O),
3.96 (t, J ¼ 6.4 Hz, 2H, CH2O), 3.59 (s, 2H, PhCH2), 3.50 (s, 3H, CH3O),
2.55e2.51 (m, 4H, 2 ꢂ CH2N), 1.83e1.78 (m, 2H, CH2), 1.68e1.67 (m,
2H, CH2), 1.06 (t, J ¼ 6.8 Hz, 3H, CH3).
4.1.6.2. 3-(4-(3-(Benzyl(ethyl)amino)propoxy)phenyl)-5-hydroxy-7-
(methoxymethoxy)-4H-chromen-4-one (15b). It was obtained from
N-benzylethylamine (7b) and 3-(4-(3-bromopropoxy) phenyl)-5-
hydroxy-7-(methoxymethoxy)-4H-chromen-4-one (12) according
to the general procedure. Elution with CH2Cl2/MeOH ¼ 80:1 gave
4.1.6.7. 5-Hydroxy-3-(4-(4-((2-methoxybenzyl)(methyl)amino)
butoxy)phenyl)-7-(methoxymethoxy)-4H-chromen-4-one
(16c).

X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
325
It was obtained from N-(2-methoxybenzyl)-methylamine (7c) and
3-(4-(4-bromobutoxy)phenyl)-5-hydroxy-7-(methoxymethoxy)-
4H-chromen-4-one (13) according to the general procedure.
Elution with CH2Cl2/MeOH ¼ 120:1 gave 16c as a light yellow oil;
J ¼ 2.0 Hz,1H, 8-H), 6.50 (d, J ¼ 2.0 Hz,1H, 6-H), 5.24 (s, 2H, OCH2O),
3.99 (t, J ¼ 6.4 Hz, 2H, CH2O), 3.82 (s, 3H, CH3O), 3.63 (s, 2H, PhCH2),
3.50 (s, 3H, CH3O), 2.49 (t, J ¼ 7.2 Hz, 2H, CH2N), 2.30 (s, 3H, CH3N),
1.82e1.79 (m, 2H, CH2), 1.64e1.63 (m, 2H, CH2), 1.51e1.46 (m, 2H,
CH2), 1.43e1.38 (m, 2H, CH2).
54.4% yield; 1H NMR (400 MHz, CDCl3)
d 12.83 (s, 1H, 5-OH), 7.88 (s,
1H, 2-H), 7.44 (d, J ¼ 8.4 Hz, 2H, 20, 60-H), 7.36 (d, J ¼ 7.2 Hz, 1H, Are
H), 7.26e7.23 (m, 1H, AreH), 6.97e6.87 (m, 4H, 4 ꢂ AreH), 6.58 (d,
J ¼ 2.0 Hz,1H, 8-H), 6.50 (d, J ¼ 2.0 Hz,1H, 6-H), 5.24 (s, 2H, OCH2O),
4.01 (t, J ¼ 6.4 Hz, 2H, CH2O), 3.83 (s, 3H, CH3O), 3.62 (s, 2H, PhCH2),
3.50(s, 3H, CH3O), 2.55 (t, J ¼ 7.2 Hz, 2H, CH2N), 2.30 (s, 3H, CH3N),
1.87e1.78 (m, 4H, 2 ꢂ CH2).
4.1.6.12. 3-(4-((6-(Ethyl(2-methoxybenzyl)amino)hexyl)oxy)phenyl)-
5-hydroxy-7-(methoxy methoxy)-4H-chromen-4-one (17d). It was
obtained from N-(2-methoxybenzyl)-ethylamine (7d) and 3-(4-((6-
bromohexyl)oxy)phenyl)-5-hydroxy-7-(methoxymethoxy)-4H-
chromen-4-one (14) according to the general procedure. Elution
with CH2Cl2/MeOH ¼ 50:1 gave 17d as a light yellow oil; 91.0%
4.1.6.8. 3-(4-(4-(Ethyl(2-methoxybenzyl)amino)butoxy)phenyl)-5-
hydroxy-7-(methoxymethoxy)-4H-chromen-4-one (16d). It was ob-
tained from N-(2-methoxybenzyl)-ethylamine (7d) and 3-(4-(4-
bromobutoxy)phenyl)-5-hydroxy-7-(methoxymethoxy)-4H-chro-
men-4-one (13) according to the general procedure. Elution with
CH2Cl2/MeOH ¼ 120:1 gave 16d as a light yellow oil; 52.6% yield; 1H
yield; 1H NMR (400 MHz, CDCl3)
d 12.84 (s, 1H, 5-OH), 7.88 (s, 1H, 2-
H), 7.45-7.43 (d, J ¼ 8.8 Hz, 3H, 3 ꢂ AreH), 7.22 (t, J ¼ 7.6 Hz,1H, Are
H), 6.97-6.92 (m, 3H, 2 ꢂ AreH), 6.86 (d, J ¼ 7.6 Hz, 1H, AreH), 6.58
(d, J ¼ 2.0 Hz, 1H, 8-H), 6.50 (d, J ¼ 2.0 Hz, 1H, 6-H), 5.24 (s, 2H,
OCH2O), 3.97 (t, J ¼ 6.4 Hz, 2H, CH2O), 3.82 (s, 3H, CH3O), 3.63 (s, 2H,
PhCH2), 3.50 (s, 3H, CH3O), 2.56 (brs, 2H, CH2N), 2.49 (brs, 2H,
CH2N), 1.81e1.75 (m, 2H, CH2), 1.50e1.48 (m, 2H, CH2), 1.48e1.43
(m, 2H, CH2), 1.39e1.34 (m, 2H, CH2), 1.07 (t, J ¼ 7.2 Hz, 3H, CH3).
NMR (400 MHz, CDCl3) d 12.83(s, 1H, 5-OH), 7.88 (s, 1H, 2-H), 7.47-
7.43 (m, 3H, 3 ꢂ AreH), 7.26e7.23 (m, 1H, AreH), 6.97e6.93 (m, 3H,
3 ꢂ AreH), 6.88 (d, J ¼ 8.0 Hz, 1H, AreH), 6.58 (d, J ¼ 2.0 Hz, 1H, 8-
H), 6.50 (d, J ¼ 2.0 Hz, 1H, 6-H), 5.22 (s, 2H, OCH2O), 3.98 (t,
J ¼ 6.0 Hz, 2H, CH2O), 3.83 (s, 3H, CH3O), 3.76 (s, 2H, PhCH2), 3.49 (s,
3H, CH3O), 2.68e2.64 (m, 4H, 2 ꢂ CH2N), 1.82e1.78 (m, 4H,
2 ꢂ CH2), 1.15 (brs, 3H, CH3).
4.1.7. General procedure for preparation of 40-O-modified genistein
derivatives (18aed, 19aed and 20aed)
7-O-MOM-40-O-modified genistein derivatives 15aed, 16aed or
17aed (1.0 mmol) were dissolved in ethanol (9 mL), and 10% HCl
solution (3 mL) was added. The reaction mixture was refluxed for
2e3 h under an argon atmosphere. After complete reaction, ethanol
was evaporated under reduced pressure. The residue was treated
with aqueous saturated NaHCO3 solution until an alkaline pH was
reached, and extracted with dichloromethane (20 mL ꢂ 3). The
combined organic phases were washed with saturated aqueous
sodium chloride, dried over sodium sulfate, and filtered. The sol-
vent was evaporated to dryness under reduced pressure. The res-
idue was purified on a silica gel chromatography using mixture of
CH2Cl2/CH3OH as eluent, obtaining the corresponding 40-O-modi-
fied genistein derivatives.
4.1.6.9. 3-(4-((6-(Benzyl(methyl)amino)hexyl)oxy)phenyl)-5-
hydroxy-7-(methoxymethoxy)-4H-chromen-4-one (17a). It was ob-
tained from N-benzylmethylamine (7a) and 3-(4-((6-bromohexyl)
oxy) phenyl)-5-hydroxy-7-(methoxymethoxy)-4H-chromen-4-one
(14) according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 60:1 gave 17a as a light yellow oil; 85.2% yield; 1H NMR
(400 MHz, CDCl3)
d 12.83 (s, 1H, 5-OH), 7.88 (s, 1H, 2-H), 7.44 (d,
J ¼ 8.8 Hz, 2H, 20, 60-H), 7.33-7.25 (m, 5H, 5 ꢂ AreH), 6.96 (d,
J ¼ 8.8 Hz, 2H, 30, 50-H), 6.58 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.50 (d,
J ¼ 2.0 Hz, 1H, 6-H), 5.24 (s, 2H, OCH2O), 3.98 (t, J ¼ 6.4 Hz, 2H,
CH2O), 3.52 (s, 2H, PhCH2), 3.50 (s, 3H, CH3O), 2.41 (t, J ¼ 7.2 Hz, 2H,
CH2N), 2.22 (s, 3H, CH3N), 1.82e1.78 (m, 2H, CH2), 1.59e1.56 (m, 2H,
CH2), 1.49e1.44 (m, 2H, CH2), 1.42e1.36 (m, 2H, CH2).
4.1.7.1. 3-(4-(3-(Benzyl(methyl)amino)propoxy)phenyl)-5,7-
dihydroxy-4H-chromen-4-one (18a). It was synthesized from 15a
according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 60:1 gave 18a as an almost white solid; 75.2% yield; mp
4.1.6.10. 3-(4-((6-(Benzyl(ethyl)amino)hexyl)oxy)phenyl)-5-
hydroxy-7-(methoxymethoxy)-4H-chromen-4-one (17b). It was ob-
tained from N-benzylethylamine (7b) and 3-(4-((6-bromohexyl)
oxy) phenyl)-5-hydroxy-7-(methoxymethoxy)-4H-chromen-4-one
(14) according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 60:1 gave 17b as a light yellow oil; 92.0% yield; 1H NMR
95e97 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.88 (s, 1H, 5-OH), 7.73 (s,
1H, 2-H), 7.33e7.28 (m, 7H, 7 ꢂ AreH), 6.83e6.81 (m, 2H, 30, 50-H),
6.19-6.18 (m, 2H, 8-H, 6-H), 4.02 (t, J ¼ 6.4 Hz, 2H, CH2O), 3.66 (s, 2H,
PhCH2), 2.70 (t, J ¼ 6.8 Hz, 2H, CH2N), 2.30 (s, 3H, CH3N), 2.06e2.03
(m, 2H, CH2); HR-ESI-MS: Calcd. for C26H26NO5 [MþH]þ: 432.1811,
found: 432.1816; Anal. calcd. For (C26H25NO5): C, 72.37; H, 5.84; N,
3.25; found: C, 72.45; H, 5.96; N, 3.16.
(400 MHz, CDCl3)
d 12.83 (s, 1H, 5-OH), 7.87 (s, 1H, 2-H), 7.44 (d,
J ¼ 8.8 Hz, 2H, 20, 60-H), 7.35L7.23 (m, 5H, 5 ꢂ AreH), 6.96 (d,
J ¼ 8.8 Hz, 2H, 30, 50-H), 6.57 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.50 (d,
J ¼ 2.0 Hz, 1H, 6-H), 5.23 (s, 2H, OCH2O), 3.97 (t, J ¼ 6.4 Hz, 2H,
CH2O), 3.59 (s, 2H, PhCH2), 3.49 (s, 3H, CH3O), 2.54 (q, J ¼ 6.8 Hz, 2H,
CH2N), 2.45 (t, J ¼ 6.8 Hz, 2H, CH2N), 1.80e1.76 (m, 2H, CH2), 1.54e
1.51 (m, 2H, CH2), 1.46e1.43 (m, 2H, CH2), 1.38e1.33 (m, 2H, CH2),
1.05 (t, J ¼ 6.8 Hz, 3H, CH3).
4.1.7.2. 3-(4-(3-(Benzyl(ethyl)amino)propoxy)phenyl)-5,7-
dihydroxy-4H-chromen-4-one (18b). It was synthesized from 15b
according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 60:1 gave 18b as an almost white solid; 78.7% yield; mp
59e61 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.89 (s, 1H, 5-OH), 7.75 (s,
1H, 2-H), 7.37e7.27 (m, 7H, 7 ꢂ AreH), 6.84 (d, J ¼ 8.0 Hz, 2H, 30, 50-
H), 6.25e6.20 (m, 2H, 8-H, 6-H), 4.00 (t, J ¼ 6.0 Hz, 2H, CH2O), 3.71
(s, 2H, PhCH2), 2.73 (t, J ¼ 6.8 Hz, 2H, CH2N), 2.64 (q, J ¼ 6.8 Hz, 2H,
CH2N), 2.04e1.98 (m, 2H, CH2), 1.11 (t, J ¼ 6.8 Hz, 3H, CH3); HR-ESI-
MS: Calcd. for C27H28NO5 [MþH]þ: 446.1967, found: 446.1966;
Anal. calcd. For (C27H27NO5): C, 72.79; H, 6.11; N, 3.14; found: C,
72.73; H, 6.20; N, 3.05.
4.1.6.11. 5-Hydroxy-3-(4-((6-((2-methoxybenzyl)(methyl)amino)
hexyl)oxy)phenyl)-7-(methoxy methoxy)-4H-chromen-4-one (17c).
It was obtained from N-(2-methoxybenzyl)-methylamine (7c) and
3-(4-((6-bromohexyl)oxy)phenyl)-5-hydroxy-7-(methox-
ymethoxy)-4H-chromen-4-one (14) according to the general pro-
cedure. Elution with CH2Cl2/MeOH ¼ 80:1 gave 17c as a light yellow
oil; 83.3% yield; 1H NMR(400 MHz, CDCl3)
d 12.83 (s,1H, 5-OH), 7.88
(s, 1H, 2-H), 7.44 (d, J ¼ 8.8 Hz, 2H, 20, 60-H), 7.37 (d, J ¼ 7.2 Hz, 1H,
AreH), 7.26e7.24 (m, 1H, AreH), 6.96 (d, J ¼ 8.8 Hz, 2H, 30, 50-H),
6.95e6.93 (m, 1H, AreH), 6.88 (d, J ¼ 8.8 Hz, 1H, AreH), 6.58 (d,
4.1.7.3. 5,7-Dihydroxy-3-(4-(3-((2-methoxybenzyl)(methyl)amino)
propoxy)phenyl)-4H-chromen-4-one (18c). It was synthesized from
15c according to the general procedure. Elution with CH2Cl2/

326
X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
MeOH ¼ 80:1 gave 18c as an almost white solid; 70.2% yield; mp
75e78 ꢀC; 1H NMR (400 MHz, CDCl3)
12.83 (s, 1H, 5-OH), 7.65 (s,
according to the general procedure. Elution with CH2Cl2/
d
MeOH ¼ 80:1 gave 19d as an almost white solid; 65.5% yield; mp
1H, 2-H), 7.32e7.26 (m, 4H, 4 ꢂ AreH), 6.93 (t, J ¼ 7.6 Hz,1H, AreH),
6.88 (d, J ¼ 8.8 Hz, 1H, AreH), 6.76 (d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.12
(brs, 1H, 8-H), 6.08 (brs, 1H, 6-H), 4.00 (t, J ¼ 6.0 Hz, 2H, CH2O), 3.78
(s, 5H, CH3O, PhCH2), 2.78 (t, J ¼ 6.8 Hz, 2H, CH2N), 2.35 (s, 3H,
CH3N), 2.09e2.06 (m, 2H, CH2); HR-ESI-MS: Calcd. for C27H28NO6
[MþH]þ: 462.1917, found: 462.1912; Anal. calcd. For (C27H27NO6): C,
70.27; H, 5.90; N, 3.03; found: C, 70.22; H, 5.93; N, 3.11.
60e63 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.81 (s, 1H, 5-OH), 7.56 (s,
1H, 2-H), 7.42 (d, J ¼ 7.6 Hz,1H, AreH), 7.33 (t, J ¼ 8.0 Hz, 1H, AreH),
7.28 (d, J ¼ 8.4 Hz, 2H, 20, 60-H), 6.98 (t, J ¼ 7.6 Hz,1H, AreH), 6.89 (d,
J ¼ 8.0 Hz, 1H, AreH), 6.69 (d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.20 (s, 1H, 8-
H), 6.13 (s, 1H, 6-H), 3.97 (s, 2H, CH2O), 3.82e3.77 (m, 5H, CH3O,
PhCH2), 2.86 (q, J ¼ 6.8 Hz, 2H, CH2N), 2.80 (t, J ¼ 7.6 Hz, 2H, CH2N),
1.88 (m, 2H, CH2), 1.78e1.74 (m, 2H, CH2), 1.27 (t, J ¼ 6.8 Hz, 3H,
CH3); 13C NMR (100 MHz, CDCl3)
d 180.1, 167.6, 162.4, 158.7, 158.2,
4.1.7.4. 3-(4-(3-(Ethyl(2-methoxybenzyl)amino)propoxy)phenyl)-
5,7-dihydroxy-4H-chromen-4-one (18d). It was synthesized from
15d according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 25:1 gave 18d as an almost white solid; 81.0% yield; mp
157.9, 152.2, 131.3, 130.5, 129.8, 129.3, 123.5, 123.2, 122.6, 120.4,
114.2, 110.5, 110.3, 104.2, 100.7, 94.8, 67.2, 55.2, 52.2, 51.0, 46.8, 26.8,
22.3, 10.2; HR-ESI-MS: Calcd. for C29H32NO6 [MþH]þ: 490.2230,
found: 490.2192; Anal. calcd. For (C29H31NO6): C, 71.15; H, 6.38; N,
2.86; found: C, 71.02; H, 6.45; N, 2.66.
61e63 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.84 (s, 1H, 5-OH), 7.68 (s,
1H, 2-H), 7.39e7.27 (m, 4H, 4 ꢂ AreH), 6.94 (t, J ¼ 7.2 Hz,1H, AreH),
6.87 (d, J ¼ 8.4 Hz,1H, AreH), 6.82 (d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.17 (s,
1H, 8-H), 6.16 (s, 1H, 6-H), 3.97 (t, J ¼ 6.0 Hz, 2H, CH2O), 3.80 (s, 5H,
CH3O, PhCH2), 2.80 (t, J ¼ 6.8 Hz, 2H, CH2N), 2.72 (q, J ¼ 7.2 Hz, 2H,
CH2N), 2.07e2.03 (m, 2H, CH2), 1.17 (t, J ¼ 7.2 Hz, 3H, CH3); HR-ESI-
MS: Calcd. for C28H30NO6 [MþH]þ: 476.2073, found: 476.2076;
Anal. calcd. For (C28H29NO6): C, 70.72; H, 6.15; N, 2.95; found: C,
70.66; H, 6.16; N, 2.88.
4.1.7.9. 3-(4-((6-(Benzyl(methyl)amino)hexyl)oxy)phenyl)-5,7-
dihydroxy-4H-chromen-4-one (20a). It was synthesized from 17a
according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 25:1 gave 20a as an almost white solid; 82.0% yield; mp
120e122 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.90 (s, 1H, 5-OH), 7.66
(s, 1H, 2-H), 7.34e7.32 (m, 7H, 7 ꢂ AreH), 6.85 (d, J ¼ 8.8 Hz, 2H, 30,
50-H), 6.23 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.20 (d, J ¼ 2.0 Hz, 1H, 6-H), 3.84
(t, J ¼ 6.4 Hz, 2H, CH2O), 3.69 (s, 2H, PhCH2), 2.55 (t, J ¼ 7.2 Hz, 2H,
CH2N), 2.30 (s, 3H, CH3N), 1.75e1.69 (m, 2H, CH2), 1.68e1.61 (m, 2H,
CH2), 1.44e1.38 (m, 4H, 2 ꢂ CH2); HR-ESI-MS: Calcd. for C29H32NO5
[MþH]þ: 474.2280, found: 474.2273; Anal. calcd. For (C29H31NO5):
C, 73.55; H, 6.60; N, 2.96; found: C, 73.42; H, 6.76; N, 2.78.
4.1.7.5. 3-(4-(4-(Benzyl(methyl)amino)butoxy)phenyl)-5,7-
dihydroxy-4H-chromen-4-one (19a). It was synthesized from 16a
according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 25:1 gave 19a as an almost white solid; 87.7% yield; mp
60e62 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.85 (s, 1H, 5-OH), 7.61 (s,
1H, 2-H), 7.34e7.26 (m, 7H, 7 ꢂ AreH), 6.71 (d, J ¼ 8.8 Hz, 2H, 30, 50-
H), 6.17 (s, 1H, 8-H), 6.11 (s, 1H, 6-H), 3.88 (t, J ¼ 5.6 Hz, 2H, CH2O),
3.71 (s, 2H, PhCH2), 2.63 (t, J ¼ 8.0 Hz, 2H, CH2N), 2.32 (s, 3H, CH3N),
1.86e1.78 (m, 4H, 2 ꢂ CH2); HR-ESI-MS: Calcd. for C27H28NO5
[MþH]þ: 446.1967, found: 446.1973; Anal. calcd. For (C27H27NO5):
C, 72.79; H, 6.11; N, 3.14; found: C, 72.90; H, 6.16; N, 2.95.
4.1.7.10. 3-(4-((6-(Benzyl(ethyl)amino)hexyl)oxy)phenyl)-5,7-
dihydroxy-4H-chromen-4-one (20b). It was synthesized from 17b
according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 25:1 gave 20b as an almost white solid; 82.4% yield; mp
47e50 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.90 (s, 1H, 5-OH), 7.68 (s,
1H, 2-H), 7.38e7.30 (m, 7H, 7 ꢂ AreH), 6.86 (d, J ¼ 8.4 Hz, 2H, 30, 50-
H), 6.24 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.21 (d, J ¼ 2.0 Hz, 1H, 6-H), 3.85 (t,
J ¼ 6.4 Hz, 2H, CH2O), 3.78 (s, 2H, PhCH2), 2.70 (q, J ¼ 7.2 Hz, 2H,
CH2N), 2.61 (t, J ¼ 7.2 Hz, 2H, CH2N), 1.74e1.68 (m, 2H, CH2), 1.67e
1.61 (m, 2H, CH2), 1.45e1.35 (m, 4H, 2 ꢂ CH2), 1.16 (t, J ¼ 7.2 Hz, 3H,
CH3); HR-ESI-MS: Calcd. for C30H34NO5 [MþH]þ: 488.2437, found:
488.2438; Anal. calcd. For (C30H33NO5): C, 73.90; H, 6.82; N, 2.87;
found: C, 73.76; H, 6.86; N, 2.99.
4.1.7.6. 3-(4-(4-(Benzyl(ethyl)amino)butoxy)phenyl)-5,7-dihydroxy-
4H-chromen-4-one (19b). It was synthesized from 16b according to
the general procedure. Elution with CH2Cl2/MeOH ¼ 25:1 gave 19b
as an almost white solid; 87.0% yield; mp 53e55 ꢀC; 1H NMR
(400 MHz, CDCl3)
d 12.85 (s, 1H, 5-OH), 7.64 (s, 1H, 2-H), 7.39e7.30
(m, 7H, 7 ꢂ AreH), 6.74 (d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.19 (s, 1H, 8-H),
6.15 (s, 1H, 6-H), 3.86 (t, J ¼ 5.2 Hz, 2H, CH2O), 3.79 (s, 2H, PhCH2),
2.72 (q, J ¼ 7.2 Hz, 2H, CH2N), 2.67 (t, J ¼ 7.6 Hz, 2H, CH2N), 1.79e
1.76 (m, 4H, 2 ꢂ CH2), 1.18 (t, J ¼ 7.2 Hz, 3H, CH3); HR-ESI-MS: Calcd.
for C28H30NO5 [MþH]þ: 460.2124, found: 460.2122; Anal. calcd. For
(C28H29NO5): C, 73.18; H, 6.36; N, 3.05; found: C, 73.06; H, 6.42; N,
2.90.
4.1.7.11. 5,7-Dihydroxy-3-(4-((6-((2-methoxybenzyl)(methyl)amino)
hexyl)oxy)phenyl)-4H-chromen-4-one (20c). It was synthesized
from 17c according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 25:1 gave 20c as an almost white solid; 87.2% yield; mp
59e61 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.87 (s, 1H, 5-OH), 7.62 (s,
1H, 2-H), 7.36 (t, J ¼ 7.6 Hz, 1H, AreH), 7.34 (d, J ¼ 8.0 Hz, 1H, AreH),
7.29 (d, J ¼ 8.4 Hz, 2H, 20, 60-H), 6.97 (t, J ¼ 7.6 Hz,1H, AreH), 6.91 (d,
J ¼ 8.0 Hz, 1H, AreH), 6.81 (d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.29 (d,
J ¼ 1.2 Hz, 1H, 8-H), 6.27 (d, J ¼ 1.2 Hz, 1H, 6-H), 3.93 (s, 2H, PhCH2),
3.82 (s, 3H, CH3O), 3.78 (t, J ¼ 6.4 Hz, 2H, CH2O), 2.74 (t, J ¼ 7.6 Hz,
2H, CH2N), 2.47 (s, 3H, CH3N), 1.75e1.68 (m, 4H, 2 ꢂ CH2), 1.43e1.38
(m, 4H, 2 ꢂ CH2); HR-ESI-MS: Calcd. for C30H34NO6 [MþH]þ:
504.2386, found: 504.2380; Anal. calcd. For (C30H33NO6): C, 71.55;
H, 6.61; N, 2.78; found: C, 71.46; H, 6.80; N, 3.01.
4.1.7.7. 5,7-Dihydroxy-3-(4-(4-((2-methoxybenzyl)(methyl)amino)
butoxy)phenyl)-4H-chromen-4-one (19c). It was synthesized from
16c according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 60:1 gave 19c as an almost white solid; 76.6% yield; mp
67e69 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.81 (s, 1H, 5-OH), 7.55 (s,
1H, 2-H), 7.36e7.33 (m, 2H, 2 ꢂ AreH), 7.25 (d, J ¼ 8.4 Hz, 2H, 20, 60-
H), 6.97 (t, J ¼ 7.6 Hz, 1H, AreH), 6.90 (d, J ¼ 8.4 Hz, 1H, AreH), 6.67
(d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.23 (s, 1H, 8-H), 6.15 (s, 1H, 6-H), 3.91 (s,
2H, CH2O), 3.82 (s, 5H, CH3O, PhCH2), 2.77 (t, J ¼ 7.6 Hz, 2H, CH2N),
2.59 (s, 3H, CH3N), 1.90e1.87 (m, 2H, CH2), 1.79e1.76 (m, 2H, CH2);
4.1.7.12. 3-(4-((6-(Ethyl(2-methoxybenzyl)amino)hexyl)oxy)phenyl)-
5,7-dihydroxy-4H-chromen-4-one (20d). It was synthesized from
17d according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 25:1 gave 20d as an almost white solid; 91.4% yield; mp
HR-ESI-MS: Calcd. for
476.2080; Anal. calcd. For (C28H29NO6): C, 70.72; H, 6.15; N, 2.95;
found: C, 70.80; H, 6.12; N, 3.03.
C
28H30NO6 [MþH]þ: 476.2073, found:
51e53 ꢀC; 1H NMR (400 MHz, CDCl3)
d 12.86 (s, 1H, 5-OH), 7.63 (s,
4.1.7.8. 3-(4-(4-(Ethyl(2-methoxybenzyl)amino)butoxy)phenyl)-5,7-
dihydroxy-4H-chromen-4-one (19d). It was synthesized from 16d
1H, 2-H), 7.40 (d, J ¼ 7.2 Hz,1H, AreH), 7.33e7.29 (m, 3H, 3 ꢂ AreH),
6.96 (t, J ¼ 7.2 Hz, 1H, AreH), 6.88 (d, J ¼ 8.0 Hz, 1H, AreH), 6.84 (d,

X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
327
J ¼ 8.8 Hz, 2H, 30, 50-H), 6.17 (s,1H, 8-H), 6.16 (s,1H, 6-H), 3.88 (s, 2H,
PhCH2), 3.83 (t, J ¼ 6.4 Hz, 2H, CH2O), 3.81 (s, 3H, CH3O), 2.80 (q,
J ¼ 7.2 Hz, 2H, CH2N), 2.69 (t, J ¼ 7.2 Hz, 2H, CH2N), 1.70e1.68 (m,
4H, 2 ꢂ CH2),1.37e1.34 (m, 4H, 2 ꢂ CH2),1.22 (t, J ¼ 7.2 Hz, 3H, CH3);
chromatography using mixtures of CH2Cl2/CH3OH as eluent,
obtaining the corresponding 7,40-O-modified genistein derivatives.
4.1.9.1. 7-(3-(Ethyl(2-methoxybenzyl)amino)propoxy)-3-(4-(3-
HR-ESI-MS: Calcd. for
C
31H36NO6 [MþH]þ: 518.2543, found:
(ethyl(2-methoxybenzyl)amino)
propoxy)phenyl)-5-hydroxy-4H-
518.2540; Anal. calcd. For (C31H35NO6): C, 71.93; H, 6.82; N, 2.71;
found: C, 71.79; H, 6.80; N, 2.58.
chromen-4-one (24d). It was obtained from N-(2-methoxybenzyl)-
ethylamine (7d) and 7-(3-bromopropoxy)-3-(4-(3-bromopropoxy)
phenyl)-5-hydroxy-4H-chromen-4-one (21) according to the gen-
eral procedure. Elution with CH2Cl2/MeOH ¼ 50:1 gave 24d as a
4.1.8. General procedure for synthesis of intermediates (21e23)
To a mixture of the genistein (300 mg, 1.11 mmol), anhydrous
K2CO3 (614 mg, 4.44 mmol) and KI (18 mg, 0.11 mmol) in acetone
(10.0 mL), the appropriate dibromoalkane derivative (4.44 mmol)
was added. The reaction mixture was stirred and refluxed for 8e
10 h under an argon atmosphere. After complete reaction, the
solvent was evaporated under reduced pressure. Water (30 mL)
was added to the residue and the mixture was extracted with
dichloromethane (30 mL ꢂ 3). The combined organic phases were
washed with saturated aqueous sodium chloride (30 mL), dried
over sodium sulfate, and filtered. The solvent was evaporated to
dryness under reduced pressure. The residue was purified on a
silica gel chromatography to give the intermediates 21e23.
light yellow oil; 56.0% yield; 1H NMR (400 MHz, CDCl3)
d 12.85 (s,
1H, 5-OH), 7.85 (s, 1H, 2-H), 7.44L7.38 (m, 4H, 4 ꢂ AreH), 7.21 (t,
J ¼ 7.2 Hz, 2H, 2 ꢂ AreH), 6.95e6.89 (m, 4H, 4 ꢂ AreH), 6.87e6.84
(m, 2H, 2 ꢂ AreH), 6.36 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.33 (d, J ¼ 2.0 Hz,
1H, 6-H), 4.09e4.03 (m, 4H, 2 ꢂ CH2O), 3.81 (s, 6H, 2 ꢂ CH3O), 3.63
(s, 4H, 2 ꢂ PhCH2), 2.66 (brs, 4H, 2 ꢂ CH2N), 2.58 (brs, 4H,
2 ꢂ CH2N), 1.99 (brs, 4H, 2 ꢂ CH2), 1.09e1.07 (m, 6H, 2 ꢂ CH3); HR-
ESI-MS: Calcd. for C41H49N2O7 [MþH]þ: 681.3540, found: 681.3535;
Anal. calcd. For (C41H48N2O7): C, 72.33; H, 7.11; N, 4.11; found: C,
72.12; H, 7.27; N, 3.95.
4.1.9.2. 7-(4-(benzyl(ethyl)amino)butoxy)-3-(4-(4-(benzyl(ethyl)
amino)butoxy)phenyl)-5-hydroxy-4H-chromen-4-one (25b). It was
obtained from N-benzylethylamine (7b) and 7-(4-bromobutoxy)-3-
(4-(4-bromobutoxy)phenyl)-5-hydroxy-4H-chromen-4-one (22)
according to the general procedure. Elution with CH2Cl2/
MeOH ¼ 30:1 gave 25b as a light yellow oil; 68.6% yield; 1H NMR
4.1.8.1. 7-(3-Bromopropoxy)-3-(4-(3-bromopropoxy)phenyl)-5-
hydroxy-4H-chromen-4-one (21). It was synthesized from 1,3-
dibromopropane. Elution with CH2Cl2 afforded 21 as an almost
white solid; yield 68.7%; mp 132e134 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
12.85 (s, 1H, 5-OH), 7.87 (s, 1H, 2-H), 7.46 (d, J ¼ 8.4 Hz, 2H,
(400 MHz, CDCl3) d 12.86 (s, 1H, 5-OH), 7.85 (s, 1H, 2-H), 7.44 (d,
20, 60-H), 6.99 (d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.42 (d, J ¼ 2.0 Hz,1H, 8-H),
6.38 (d, J ¼ 2.0 Hz, 1H, 6-H), 4.19e4.13 (m, 4H, 2 ꢂ CH2O), 3.63e3.59
(m, 4H, 2 ꢂ CH2Br), 2.38e2.31 (m, 4H, 2 ꢂ CH2).
J ¼ 8.8 Hz, 2H, 20, 60-H), 7.33-7.22 (m, 10H, 10 ꢂ AreH), 6.94 (d,
J ¼ 8.8 Hz, 2H, 30, 50-H), 6.36 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.34 (d,
J ¼ 2.0 Hz, 1H, 6-H), 3.99e3.94 (m, 4H, 2 ꢂ CH2O), 3.60 (s, 2H,
PhCH2), 3.58 (s, 2H, PhCH2), 2.57e2.48 (brs, 8H, 4 ꢂ CH2N), 1.84e
4.1.8.2. 7-(4-Bromobutoxy)-3-(4-(4-bromobutoxy)phenyl)-5-
hydroxy-4H-chromen-4-one (22). It was synthesized from 1,4-
dibromobutane. Elution with CH2Cl2 afforded 22 as an almost
white solid; yield 54.0%; mp 98e100 ꢀC; 1H NMR (400 MHz, CDCl3)
1.79 (m, 4H, 2 ꢂ CH2), 1.69e1.63 (brs, 4H, 2 ꢂ CH2), 1.06 (brs, 6H,
1
2 ꢂ CH3); 3C NMR (100 MHz, CDCl
) d 180.8, 165.0, 162.6, 159.2, 157.9,
3
152.6, 130.0, 128.9, 128.8, 128.1, 126.8, 123.6, 122.7, 114.6, 106.1, 98.5,
92.8, 68.3, 67.7, 58.0, 57.9, 52.5, 52.3, 47.2, 47.1, 26.9, 26.6, 23.3, 11.6,
11.5; HR-ESI-MS: Calcd. for C41H49N2O5 [MþH]þ: 649.3641, found:
649.3628; Anal. calcd. For (C41H48N2O5): C, 75.90; H, 7.46; N, 4.32;
found: C, 75.75; H, 7.45; N, 4.23.
d
12.85 (s, 1H, 5-OH), 7.86 (s, 1H, 2-H), 7.465 (d, J ¼ 8.4 Hz, 2H, 20, 60-
H), 6.96 (d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.39 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.36
(d, J ¼ 2.0 Hz, 1H, 6-H), 4.08e4.02 (m, 4H, 2 ꢂ CH2O), 3.52e3.49 (m,
4H, 2 ꢂ CH2Br), 2.12e2.02 (m, 4H, 2 ꢂ CH2), 2.00e1.94 (m, 4H,
2 ꢂ CH2).
4.1.9.3. 7-(4-(Ethyl(2-methoxybenzyl)amino)butoxy)-3-(4-(4-
(ethyl(2-methoxybenzyl)amino) butoxy)phenyl)-5-hydroxy-4H-chro-
men-4-one (25d). It was obtained from N-(2-methoxybenzyl)-
ethylamine (7d) and 7-(4-bromobutoxy)-3-(4-(4-bromobutoxy)
phenyl)-5-hydroxy-4H-chromen-4-one (22) according to the gen-
eral procedure. Elution with CH2Cl2/MeOH ¼ 30:1 gave 25d as a
4.1.8.3. 7-((6-Bromohexyl)oxy)-3-(4-((6-bromohexyl)oxy)phenyl)-5-
hydroxy-4H-chromen-4-one (23). It was synthesized from 1,6-
dibromohexane. Elution with CH2Cl2 afforded 23 as an almost
white solid; yield 75.5%; mp 97e109 ꢀC; 1H NMR (400 MHz, CDCl3)
d
12.85 (s, 1H, 5-OH), 7.86 (s, 1H, 2-H), 7.45 (d, J ¼ 8.4 Hz, 2H, 20, 60-
light yellow oil; 66.4% yield; 1H NMR (400 MHz, CDCl3)
d 12.85 (s,
H), 6.96 (d, J ¼ 8.4 Hz, 2H, 30, 50-H), 6.39 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.36
(d, J ¼ 2.0 Hz, 1H, 6-H), 4.04e3.98 (m, 4H, 2 ꢂ CH2O), 3.45e3.38 (m,
4H, 2 ꢂ CH2Br), 1.92e1.80 (m, 8H, 4 ꢂ CH2), 1.59e1.45 (m, 8H,
4 ꢂ CH2).
1H, 5-OH), 7.86 (s, 1H, 2-H), 7.45e7.43 (m, 4H, 4 ꢂ AreH), 7.25e7.23
(m, 2H, 2 ꢂ AreH), 6.95e6.93 (m, 4H, 4 ꢂ AreH), 6.87 (d, J ¼ 8.0 Hz,
2H, 30, 50-H), 6.36 (d, J ¼ 2.0 Hz, 1H, 8-H), 6.34 (d, J ¼ 2.0 Hz, 1H, 6-
H), 4.01e3.96 (m, 4H, 2 ꢂ CH2O), 3.83 (s, 6H, 2 ꢂ CH3O), 3.68 (s, 4H,
2 ꢂ PhCH2), 2.60 (brs, 8H, 4 ꢂ CH2N), 1.88e1.81 (m, 4H, 2 ꢂ CH2),
4.1.9. General procedure for preparation of 7,40-O-modified
genistein derivatives (24d, 25b, 25d and 26d)
1.73 (brs, 4H, 2 ꢂ CH2), 1.11 (brs, 6H, 2 ꢂ CH3); 13C NMR (100 MHz, CDCl
)
3
d
180.7, 164.8, 162.5, 159.0, 157.8, 157.7, 157.6, 152.5, 130.8, 130.5,
To
a
mixture of N-benzylethylamine (7b) or N-(2-
129.9, 128.5, 128.2, 123.4, 122.7, 120.4, 120.3, 114.5, 110.3, 110.2,
106.0, 98.5, 92.6, 68.2, 67.6, 55.3, 52.4, 50.9, 50.8, 47.5, 47.4, 26.9,
26.6, 22.9, 22.7, 11.2, 11.0; HR-ESI-MS: Calcd. for C43H53N2O7
[MþH]þ: 709.3753, found: 709.3754; Anal. calcd. For (C43H52N2O7):
C, 72.86; H, 7.39; N, 3.95; found: C, 72.67; H, 7.50; N, 3.82.
methoxybenzyl)-ethylamine (7d) (0.67 mmol), anhydrous K2CO3
(93 mg, 0.67 mmol) and KI (2.82 mg, 0.017 mmol) were added the
appropriate intermediates 21e23 (0.168 mmol). The reaction
mixture was warmed to 60e65 ꢀC and stirred for 12e15 h under an
argon atmosphere. After complete reaction, the solvent was evap-
orated under reduced pressure. Water (20 mL) was added to the
residue and the mixture was extracted with dichloromethane
(20 mL ꢂ 3). The combined organic phases were washed with
saturated aqueous sodium chloride (20 mL), dried over sodium
sulfate, and filtered. The solvent was evaporated to dryness under
4.1.9.4. 7-((6-(Ethyl(2-methoxybenzyl)amino)hexyl)oxy)-3-(4-((6-
(ethyl(2-methoxybenzyl)amino) hexyl)oxy)phenyl)-5-hydroxy-4H-
chromen-4-one (26d). It was obtained from N-(2-methoxybenzyl)
n-ethylamine
(7d)
and
7-((6-bromohexyl)oxy)-3-(4-((6-
bromohexyl)oxy) phenyl)-5-hydroxy-4H-chromen-4-one (23) ac-
reduced pressure. The residue was purified on
a
silica gel
cording to the general procedure. Elution with CH2Cl2/

328
X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
MeOH ¼ 50:1 gave 26d as a light yellow oil; 53.0% yield; 1H NMR
Structure of 25d was built and performed geometry optimization
by molecular mechanics. After addition of Gasteiger charges,
removal of hydrogen atoms, addition of their atomic charges to
skeleton atoms, and the assignment of proper atomic types, the
further preparation of the inhibitor was accomplished. Autotors
was then used to define the rotatable bonds in the ligands. Docking
studies were performed using the AUTODOCK 4.2 program. By
using Autodock Tools (ADT; version 1.5.6), polar hydrogen atoms
were added to amino acid residues, and Gasteiger charges were
assigned to all atoms of the enzyme. The resulting enzyme struc-
ture 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 separate des-
olvation map presented in the substrate molecule. All maps were
(400 MHz, CDCl3)
d 12.86 (s, 1H, 5-OH), 7.87 (s, 1H, 2-H), 7.71e7.67
(m, 2H, 2 ꢂ AreH), 7.45 (d, J ¼ 8.4 Hz, 2H, 20, 60-H), 7.37 (t, J ¼ 7.6 Hz,
2H, 2 ꢂ AreH), 7.03 (t, J ¼ 7.6 Hz, 2H, 2 ꢂ AreH), 6.97e6.92 (m, 4H,
4 ꢂ AreH), 6.39 (d, J ¼ 2.4 Hz, 1H, 8-H), 6.35 (d, J ¼ 2.4 Hz, 1H, 6-H),
4.15 (s, 4H, 2 ꢂ PhCH2), 4.02e3.96 (m, 4H, 2 ꢂ CH2O), 3.87 (s, 6H,
2 ꢂ CH3O), 2.99 (brs, 4H, 2 ꢂ CH2N), 2.87 (m, 4H, 2 ꢂ CH2N), 1.86e
1.76 (m, 8H, 4 ꢂ CH2),1.54e1.47 (m, 4H, 2 ꢂ CH2),1.45e1.34 (m,10H,
2 ꢂ CH3, 2 ꢂ CH2); HR-ESI-MS: Calcd. for C47H61N2O7 [MþH]þ:
765.4479, found: 765.4468; Anal. calcd. For (C47H60N2O7): C, 73.79;
H, 7.91; N, 3.66; found: C, 73.90; H, 7.75; N, 3.58.
4.2. Biological evaluation
ꢀ
4.2.1. Inhibition experiments of AChE and BuChE
calculated with 0.375 A spacing between grid points. The center of
To assess the inhibitory activity of the compounds toward AChE
or BuChE, we followed the spectrophotometric method of Ellman,
using AChE from 5% rat cortex homogenate or purified AChE from
both E. electricus (SigmaeAldrich Co.) and human erythrocytes
(SigmaeAldrich Co.) or BuChE from rat serum [33,45]. The brain
homogenate was preincubated for 5 min with tetraisopropyl
pyrophosphoramide (iso-OMPA, selective inhibitor of BuChE,
4.0 mmol/L) (SigmaeAldrich Co.) before use. For rat AChE or BuChE
the grid box was placed at the center of donepezil with coordinates
x ¼ 2.023, y ¼ 63.295, z ¼ 67.062. The dimensions of the active site
ꢀ
box were set at 50 ꢂ 50 ꢂ 50 A. Flexible ligand docking was per-
formed for the compounds. Each docked system was performed by
100 runs of the AUTODOCK search by the Lamarckian genetic al-
gorithm (LGA). Other than the referred parameters above, the other
parameters were accepted as default. A cluster analysis was per-
formed on the docking results using a root mean square (RMS)
tolerance of 1.0 and the lowest energy conformation of the highest
populated cluster was selected for analysis. Graphic manipulations
and visualizations were done by Autodock Tools or Discovery Stu-
dio 2.0 software.
inhibition assays, a reaction mixture (100
m
L) containing acetylth-
L) (J&K Scientific) or butyrylth-
L) (TCI Shanghai Development),
L), 5%
L) and different concentrations of
L) was incubated at 37 ꢀC for 15 min. Then
5,50-dithiobis-2-nitrobenzoic acid (DTNB, 0.2%, 30
L) (J&K Scien-
iocholine iodide (1 mmol/L, 30
iocholine iodide (1 mmol/L, 30
m
m
phosphate-buffered solution (0.1 mmol/L, pH ¼ 7.4, 40
homogenate or 25% serum (10
test compounds (20
m
m
m
4.2.4. Antioxidant activity assay
m
The antioxidant activity was determined by the oxygen radical
absorbance capacity fluorescein (ORAC-FL) method partially
modified by Fang Lei et al. [47]. 2,20-Azobis(amidinopropane)
dihydrochloride (AAPH) was purchased from Accela ChemBio Co.,
Ltd. 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid
(trolox), and fluorescein (FL) were purchased from TCI (Shanghai)
Development. All the assays were conducted with 75 mM phos-
tific) was added to produce the yellow anion of 5-thio-2-nitro-
benzoic acid. Changes in absorbance were detected at 405 nm in a
Varioskan Flash Multimode Reader (Thermo Scientific). For
E. electricus AChE and human AChE inhibition assay, EeAChE or
HuAChE (0.05 U/mL, final concentration) was used and the assay
was carried out in a phosphate buffer (0.01 mmol/L, pH ¼ 8.0).
Changes in absorbance were detected at 412 nm [34,46]. The other
procedure was the same as above. Compounds inhibiting AChE or
BuChE activity would reduce the color generation. Thus, IC50 values
were calculated as the concentration of compound that produces
50% AChE or BuChE activity inhibition. Donepezil was applied as
positive drug. All samples were assayed in triplicate.
phate buffer (pH 7.4), and the final reaction mixture was 200
mL.
Antioxidant (20 L) and fluorescein (120 L, 150 nM final concen-
m
m
tration) were placed in the wells of a black 96-well plate. The
mixture was pre-incubated for 15 min at 37 ꢀC, and then AAPH
solution (60 mL, 12 mM final concentration) was added rapidly us-
ing an autosampler. The plate was immediately placed in a Vari-
oskan Flash Multimode Reader (Thermo Scientific) and the
fluorescence recorded every minute for 90 min with excitation at
485 nm and emission at 535 nm. The plate was automatically
4.2.2. Kinetic characterization of AChE inhibition
Kinetic characterization of AChE inhibition was performed
based on a reported method using purified AChE from E. electricus
shaken prior to each reading. Trolox was used as standard (1e8 mM,
(EeAChE) [46]. The assay solution (100
phate buffer (pH 8.0), with the addition of 30
of 0.5 units/mL EeAChE, and 20 L of substrate (ATCh). Three
m
L) consists of 0.1 M phos-
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 concentration
mL of 0.2% DTNB, 10 mL
m
different concentrations of inhibitors were added to the assay so-
lution and pre-incubated for 15 min at 37 ꢀC with the EeAChE fol-
lowed by the addition of substrate in different concentrations.
Kinetic characterization of the hydrolysis of ATCh catalyzed by
EeAChE was done spectrometrically at 412 nm. The parallel control
experiments were performed without inhibitor in the assay. The
plots were assessed by a weighted least square analysis that
assumed the variance of 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 25d in a weighted analysis, and Ki was
determined as the intercept on the negative x-axis.
(1e10 mM). All the reaction mixture was prepared in duplicate, and
at least three independent assays were performed for each sample.
Antioxidant curves (fluorescence versus time) were normalized to
the curve of the blank in the same assay, and then the area under
the fluorescence decay curve (AUC) was calculated. The net AUC of a
sample was obtained by subtracting the AUC of the blank. ORAC-FL
values were expressed as Trolox equivalents by using the standard
curve calculated for each sample, where the ORAC-FL value of
Trolox was taken as 1, indicating the antioxidant potency of the
tested compounds.
4.2.5. Metal binding studies [9,42]
4.2.3. Molecular docking
The metal binding studies were carried out in a Shimadzu UV-
2450 spectrophotometer. To investigate the metal binding ability
of compound, the UV absorption of the tested compound 10b, 19d
or 25d, in the absence or presence of CuCl2, FeSO4, ZnCl2, and AlCl3,
The crystal structure of AChE complexed with donepezil (code
ID: 1EVE) was obtained from the Protein Data Bank after elimi-
nating the original inhibitors and water molecules. The 3D

X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
329
was recorded with wavelength ranging from 200 to 500 nm after
incubating for 30 min at room temperature. The final volume of
reaction mixture was 1 mL, and the final concentrations of tested
percent inhibition of the AChE-induced aggregation due to the
presence of the tested compound was calculated by the following
formula: 100 ꢃ IFi/(IFc*100), where IFi and IFc were the fluorescence
compound and metals were 37.5
mM. Numerical subtraction of the
intensities obtained for Ab plus AChE in the presence and in the
spectra of the metal alone and the compound alone from the
spectra of the mixture gave the difference UVevis spectra due to
complex formation. The molar ration method was performed to
determine the stoichiometry of the complex compound-metal by
titrating the methanol solution of tested compound with ascending
absence of inhibitors, respectively, minus the fluorescence in-
tensities due to the respectively blanks.
4.2.8. Effect of test compounds on metal-induced Ab1e42
aggregation and disaggregation experiments by ThT method
Solutions of Cu2þ were prepared from standards to concentra-
of CuCl2. The final concentration of tested compound was 37.5
m
M,
M.
and the final concentration of Cu2þ ranged from 7.5 to 93.75
m
tion of 75 mM using the HEPES buffer (20 mM, pH 6.6, 150 mM
The UV spectra was recorded and treated by numerical subtraction
of CuCl2 and tested compound at corresponding concentrations,
plotted versus the mole fraction of tested compound.
NaCl). For the inhibition of copper-induced Ab1e42 aggregation
assay [41,42], the Ab1e42 stock solution was diluted in HEPES buffer
(20 mM, pH 6.6, 150 mM NaCl). The mixture of the peptide (20
25 L, 25 M, final concen-
M, final concentration) and Cu2þ (20
tration), with or without the tested compound at different con-
centrations (20 L, 10e35 M, final concentration) was incubated at
37 ꢀC for 24 h. After incubation,190
L of 5 M thioflavin T in 50 mM
mL,
m
m
m
4.2.6. Determination of the inhibitory effect on the self-induced
Ab1e42 aggregation
m
m
In order to investigate the self-induced Ab1e42 aggregation, a
m
m
Thioflavin T-based flurometric assay was performed [35]. Thioflavin
T (Basic Yellow 1) was purchased from TCI (Shanghai) Develop-
ment. 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were purchased
glycineeNaOH buffer (pH 8.5) was added. The final concentration
of DMSO in each well was 12.5%. Each assay was run in triplicate.
The detection method was the same as that of self-induced Ab1e42
experiment.
from Energy Chemical.
b-Amyloid1e42 (Ab1e42), supplied as tri-
fluoroacetate salt, was purchased from ChinaPeptides Co., Ltd.
Briefly, Ab1e42 was dissolved in HFIP (1 mg/mL) and incubated for
24 h at room temperature, and solvent was evaporated. Then the
HFIP pretreated Ab1e42 was resolubilized in dry DMSO to a final
For the disaggregation of copper-induced A
the Ab1e42 stock solution was diluted in HEPES buffer (20 mM, pH
6.6, 150 mM NaCl). The mixture of the Ab1e42 (20 L, 25 M, final
concentration) with Cu2þ (20
L, 25 M, final concentration) was
L, 25 M, final
b fibrils experiment,
m
m
m
m
stock concentration of 200
m
M and was kept frozen at ꢃ80 ꢀC until
incubated 37 ꢀC for 24 h. The tested compound (20
m
m
use. Solutions of test compounds were prepared in DMSO in
2.5 mM for storage and diluted with phosphate buffer solution (pH
concentration) was then added and incubated at 37 ꢀC for another
24 h. To minimize evaporation effect the wells were sealed by a
7.4) before use. For the self-induced assay, Ab1e42 (20
final concentration) was incubated with 20 L of test compounds at
M in 50 mM
m
L, 25
m
M,
transparent heat-resistant plastic film. After incubation, 190
mL of
m
5 mM thioflavin T in 50 mM glycineeNaOH buffer (pH 8.5) was
different concentrations ranging from 10 to 50
m
added. The final concentration of DMSO in each well was 12.5%.
Each assay was run in triplicate. The detection method was the
same as above.
phosphate buffer solution (pH 7.4) at 37 ꢀC for 24 h. To minimize
evaporation effect the wells were sealed by a transparent heat-
resistant plastic film. After incubation, 160 mL of 5 mM thioflavin T
in 50 mM glycineeNaOH buffer (pH 8.5) was added. The final
concentration of DMSO in each well was 12.5%. Each assay was run
in triplicate. Fluorescence was measured on a Varioskan Flash
Multimode Reader (Thermo Scientific) with excitation and emis-
sion wavelengths at 446 nm and 490 nm, respectively. The fluo-
rescence intensities were compared and the percent inhibition due
to the presence of the inhibitor was calculated by the following
formula: 100 ꢃ IFi/(IFc*100), where IFi and IFc were the fluorescence
intensities obtained for Ab1e42 in the presence and in the absence of
inhibitors, respectively.
4.2.9. Step-down passive avoidance test
4.2.9.1. Materials and animals. Donepezil was purchased from Eisai
China Inc. Scopolamine was purchase from J&K Scientific. Scopol-
amine was dissolved in 0.9% saline. Donepezil and test compounds
were dissolved in vegetable oil, which was also used for the
negative control. Kunming mice at body weight of 18e22 g (six
weeks old, either gender) were supplied by the Center of Experi-
mental Animals of Sichuan Academy of Chinese Medicine Sciences
(eligibility certification no. SCXK-Sichuan 2008e19). Mice were
maintained under standard conditions with a 12 h:12 h lightedark
cycle, a temperature and humidity controlled environment with
access to food and water ad libitum. The mice were submitted to
behavioral tests one day after 7 days of treatment with compounds.
4.2.7. Inhibition of AChE-induced Ab1e40 aggregation
The thioflavin-T (ThT) fluorescence method was used as previ-
ously described [40]. Acetylcholinesterase from human erythro-
cytes was purchased from Sigma Co.
b
-Amyloid1e40 (Ab1e40),
4.2.9.2. Assay method. A modification of step-down passive
avoidance test was used to assess learning and memory in mice
[48,49]. The apparatus consisted of a grid floor with a wooden block
placed in the center. The block served as a shock free zone. The mice
underwent two separate trials: a training trial and a test trial 24 h
later. For training trial, mice were initially placed on the block and
were given an electrical foot shock (0.5 mA, 2 s) through the grid
floor on stepping down. We used a total of 120 mice in the passive
avoidance test with 10 mice were used per treatment. Compounds
10b and 19d (2, 6 and 18 mg/kg, p.o.) or 25d (3, 9, 27 mg/kg, p.o.) or
donepezil (5 mg/kg, p.o.) as a positive control were orally given 1 h
before each training trial. After 30 min, memory impairment was
induced by administering scopolamine (3 mg/kg, i.p.). Twenty-four
hours after the training trial, mice were placed on the block and the
time for the animal to step down was measured as latency time for
test trial. An upper cut-off time was set at 300 s.
supplied as trifluoroacetate salt, was purchased from ChinaPeptides
Co., Ltd. HFIP pre-treated Ab1e40 and tested compounds were dis-
solved in DMSO to obtain 2.3 mM and 1 mM solutions respectively.
For the AChE-induced assay, Aliquots of 2
bated for 24 h at room temperature in 0.215 mM sodium phosphate
buffer (pH 8.0) at a final concentration of 230 M. For co-
incubations experiments, 16 L of HuAChE (final concentration of
2.3 M, Ab1e40/AChE molar ration of 100:1) and AChE in the pres-
ence of 2 L of the tested inhibitor (final concentration 100 M) in
mL of Ab1e40 were incu-
m
m
m
m
m
0.215 M sodium phosphate buffer (pH 8.0) solutions were added.
Blanks containing Ab1e40 alone, human AChE alone, and Ab1e40
plus tested inhibitors in 0.215 sodium phosphate buffer (pH 8.0)
were prepared. After incubation, 180
mL of 5 mM thioflavin T in
50 mM glycineeNaOH buffer (pH 8.5) was added. Each assay was
run in triplicate. The detection method was the same as above. The

330
X. Qiang et al. / European Journal of Medicinal Chemistry 76 (2014) 314e331
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Acknowledgments
This work was supported in part by the Chinese National Natural
Science Foundation (20672077, 20872099), the Research Fund for
the Doctoral Program of Higher Education (20110181110079) and
the National Science and Technology Major Project on “Key New
Drug Creation and Manufacturing Program”(2013ZX09301304-
002).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.045.
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