Design, synthesis, and bioevaluation of benzamides: Novel acetylcholinesterase inhibitors with multi-functions on butylcholinesterase, Aβ aggregation, and β-secretase

Article abstract of DOI:10.1016/j.bmc.2012.09.016

Alzheimer's disease (AD) is a multifactorial syndrome with several target proteins contributing to its etiology. In this study, we conducted a structure-based design and successfully produced a series of new multi-site AChE inhibitors with a novel framewo

Full text of DOI:10.1016/j.bmc.2012.09.016

Bioorganic & Medicinal Chemistry 20 (2012) 6739–6750
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Bioorganic & Medicinal Chemistry
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Design, synthesis, and bioevaluation of benzamides: Novel
acetylcholinesterase inhibitors with multi-functions on butylcholinesterase,
Ab aggregation, and b-secretase
⇑
Da-Yong Peng , Qi Sun , Xiao-Lei Zhu , Hong-Yan Lin, Qiong Chen, Ning-Xi Yu, Wen-Chao Yang ,
Guang-Fu Yang
⇑
Key Laboratory of Pesticide & Chemical Biology, College of Chemistry, Ministry of Education, Central China Normal University, Wuhan 430079, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Alzheimer’s disease (AD) is a multifactorial syndrome with several target proteins contributing to its eti-
ology. In this study, we conducted a structure-based design and successfully produced a series of new
multi-site AChE inhibitors with a novel framework. Compound 2e, characterized by a central benzamide
moiety linked to an isoquinoline at one side and acetophenone at the other, was the most potent candi-
date with Ki of 6.47 nM against human AChE. Particularly, it showed simultaneous inhibitory effects
against BChE, Ab aggregation, and b-secretase. We therefore conclude that compound 2e is a very prom-
ising multi-function lead for the treatment of AD.
Received 6 August 2012
Revised 8 September 2012
Accepted 10 September 2012
Available online 17 September 2012
Keywords:
Acetylcholinesterase
Alzheimer’s disease
Benzamide
Ó 2012 Elsevier Ltd. All rights reserved.
Structure-based design
1. Introduction
the breakdown of ACh while the other approach strives to discover
small molecules to bind with ACh receptors as agonists.3,11–13 To
As the most common type of dementia, neurodegenerative
Alzheimer’s disease (AD) is one of the leading causes of death
worldwide. Despite decades of intensive study, the specific cause
of AD remains enigmatic.1 Most researchers support that several
pathological phenomena are closely associated with AD lesions,
such as a decrease of the neurotransmitter acetylcholine (ACh),
formation of amyloid b-protein (Ab) plaques, and abnormal post-
translational modifications of tau protein to yield neurofibrillary
tangles.2–7 During the last twenty years, much effort has been
aimed at developing strategies to reduce or clear the related AD
pathological phenomena.8,9 Based on the cholinergic hypothesis,
two major strategies have been developed to control the ACh
level.10 One approach focuses on discovery of small molecule
inhibitors as antagonists of acetylcholinesterase (AChE) that inhibit
block the formation of amyloid plaques, two methods have been
reported. One is the design of inhibitors of b-secretase or
c
-secretase to decrease the generation of Ab, or non-natural amino
acid or transition metal complexes to block Ab fibril formation.2,14
The other tactic is to design antibodies or catalytic antibodies to
bind and/or degrade Ab.14 In an effort to suppress the abnormal
posttranslational modifications, for example, phosphorylation of
tau protein, various attempts have been carried out, either to inhi-
bit serine/threonine kinases which function as phosphorylating
enzymes of tau protein, or to accelerate the dephosphorylation of
hyper-phosphorylated tau protein. Although some of these strate-
gies were invalidated due to severe side effects, several possible
candidates have been advanced to clinical trials.
Tacrine was the first AChE inhibitor permitted by the FDA to en-
ter the medical market. However, after a long period of using these
AChE inhibitors to treat mild or moderate AD in clinical practice, a
profile of adverse effects was gradually recognized, including in-
creased rates of syncope, bradycardia, acute liver toxicity, and hip
fracture in older adults. Most of the efforts in AChE inhibitor discov-
ery concentrated on structural modification of the existing drugs or
a combination of active substructures.15–21 While the resulting
compounds might suffer the risk of adverse effects due to the simi-
larities in the scaffold, though some of the modified compounds
showed much higher AChE-binding affinities.16,19,21 Therefore,
AChE inhibitors with novel scaffolds are urgently needed.
Abbreviations: AD, Alzheimer’s disease; AChE, acetylcholinesterase; BChE,
butylcholinesterase; ACh, acetylcholine; Ab, amyloid-b; CAS, catalytic active site;
PAS, peripheral active site; MD, molecular dynamic; SIE, solvated interaction
energy; ATC, acetylthiocholine; MTDLs, multi-target-directed ligands; ESI-MS,
electrospray ionisation mass spectrometry; DMF, dimethylformamide; DTNB, 50,
50-dithio-bis(2-nitrobenzoic acid); LGA, Lamarkian genetic algorithm; RESP,
restrained electrostatic potential.
⇑
Corresponding authors.
E-mail addresses: tomyang@mail.ccnu.edu.cn (W.-C. Yang), gfyang@mail.ccnu.
edu.cn (G.-F. Yang).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.016

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D.-Y. Peng et al. / Bioorg. Med. Chem. 20 (2012) 6739–6750
Figure 1. Molecular design strategy of lead compound 1a and structural optimization leading to the discovery of compound 2e. Left, the inhibitor-binding pocket of AChE
consists of three subsites: catalytic active site (CAS), peripheral active site (PAS), and a narrow channel between the CAS and PAS.
Three dimensional structures of AChE from different species,
including human, Electrophorus electricus, Torpedo californica, and
mouse, have been determined in the past decade.22,23 These struc-
tures revealed that there are two major subsites, the catalytic
active site (CAS) and the peripheral active site (PAS), in the
inhibitor-binding gorge-like pocket.16,24 Recently, a narrow chan-
nel between the CAS and PAS was also identified as an additional
binding site.25 In addition, the gorge-like pocket is very rich in aro-
matic residues, especially in the PAS and CAS, such as residues
Tyr72, Trp86, Tyr124, Trp286, Phe295, Tyr337, Tyr341 and
Trp439. Based on these observations and some typical publica-
tions,17,18,25,26 we proposed that an ideal AChE inhibitor should
be composed of three structural units: an aromatic ring as the head
to bind with the PAS, another aromatic ring as the tail to interact
with the CAS region, and a linker to connect the head and tail.
Taking into consideration that the indole moiety is an important
aromatic ring with a broad spectrum of biological activity, hence
we intentionally designed compound 1a by introducing a
p-hydroxybenzamide ring as a linker to connect the head and tail.
The present paper describes our step-by-step optimization of this
novel scaffold which culminated in development of compound
2e, characterized by a central benzamide moiety linked to an
isoquinoline at one side and acetophenone at the other, with a Ki
value of 6.47 nM against human AChE (Fig. 1). The inhibitory
effects of 2e on human butylcholinesterase (BChE), Ab aggregation
and b-secretase indicated that this compound could be a multi-
functional lead candidate for Alzheimer’s disease therapy.
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, 3-aminoethylindole reacted with
4-hydroxybenzoic acid to afford the key intermediate 4,
H2N
O
a
HO
N
NH
N
H
H
4
3
O
b
4
N
N
O
N
d
N
H
H
NH
5
Br
1a
7
c
4
O
N
N
d
N
H
N
O
NH
H
6
8
1b
Br
Scheme 1. Synthesis of compounds 1a and 1b. Reagents and conditions: (a) dicyclohexylcarbodiimide, p-hydroxybenzoic acid, THF, ice bath; (b), Br(CH2)4Br, KOH, DMF; (c)
Br(CH2)4Br, NaOH; TBAB, benzene; (d) K2CO3, acetone, reflux.

D.-Y. Peng et al. / Bioorg. Med. Chem. 20 (2012) 6739–6750
6741
O
HN
O
O
O
O
4
HO
a
b or c
Br
R
HN
HN
4
N
H
N
H
N
H
4
9
1 c-h
Scheme 2. Synthesis of compounds 1c–h. Reagents and conditions: (a) Br(CH2)4Br, K2CO3, acetone, reflux; (b) substituted phenol, K2CO3, acetone, reflux; (c) N,N-
dimethylaminopyridine or quinoline, CH3CN, reflux.
Br
O
n
O
AcO
HO
O
H
N
H
N
a, b, c
d
e
OH
2a-g
O
O
O
10
12
11
Scheme 3. Synthesis of compounds 2a–g. Reagents and conditions: (a) SOCl2, DMF, reflux; (b) o-aminoacetophenone, CH2Cl2, ice bath; (c) NaOH, MeOH/H2O, rt; (d)
Br(CH2)nBr, K2CO3, acetone, reflux; (e) quinoline, isoquinoline, or pyridine, CH3CN, reflux.
N-(2-(1H-indol-3-yl)ethyl)-4-hydroxybenzamide.
Meanwhile,
surface of the gorge, such as Trp286, Phe297, Phe295, Phe338,
Tyr124 and Tyr 72 in the PAS; Trp86, Tyr337 and Trp439 in the
CAS; and Tyr341 in the narrow linking channel. More aromatic res-
idues may indirectly affect the configuration of the active site
N-alkylation of indole or carbazole by 1,4-dibromobutane pro-
duced intermediates 7 and 8 according to the existing method,27,28
which were then reacted with 4 to afford compounds 1a–b.
Alternatively, as depicted in Scheme 2, compounds 1c–h were pre-
pared in moderate yields through a two-step procedure by using
N-(2-(1H-indol-3-yl)ethyl)-4-hydroxybenzamide 4 as the starting
material.29 Finally, compounds 2a–g were smoothly prepared in
good yields (75–85%) from 4-acetoxybenzoic acid by a multiple-
step synthetic route as displayed in Scheme 3. The structures of
all the target compounds were characterized by 1H NMR, 13C
NMR, elemental analysis, and MS spectra.
gorge.30,31 Optimization of the
p–p stacking interactions between
inhibitor and aromatic residues in the binding pocket can greatly
improve the binding affinity of inhibitors.32,33 Bearing this consid-
eration in mind, we initially designed a multi-site inhibitor which
can form favorable p–p stacking interactions in all three subsites of
the gorge. As a typical aromatic heterocyclic compound, the indole
ring was chosen as the moiety to bind both the PAS and CAS due to
its unique broad spectrum biological activities. To form a stacking
interaction in the channel area, a simple benzamide was intro-
duced to link the two indole rings, thus generating the compound
1a (Scheme 1). Corresponding to the three sites (PAS, linker or CAS)
of the active site gorge, the structure of this lead compound was
also divided into three parts designated as part A, part B and part
C (Fig. 1). When we assessed the inhibitory potency of 1a on
recombinant human AChE in comparison with tacrine and
2.2. Lead design and optimization
To obtain a lead compound that can fit well into the active site
and efficiently inhibit the AChE, we undertook a structural analy-
sis. A remarkable characteristic of the active site gorge is that more
than ten aromatic residues comprise a substantial portion of the
Table 1
Structures of compounds 1a–h and their inhibition constants (Ki) against human AChE
R
O
4
H
N
O
NH
Compound
Tacrine
R
Ki (nM)
Compound
R
Ki (nM)
O
O
O
O
—
32.12
1d
>10,000
Galanthamine
—
61.96
1e
>10,000
O
O
N
1a
1b
1c
>10,000
1437.00
>10,000
1f
>10,000
197.78
123.73
N
N
1g
1h
N
O
N
HN

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D.-Y. Peng et al. / Bioorg. Med. Chem. 20 (2012) 6739–6750
Figure 2. The simulated binding modes of four representative compounds. (A) Part A of compound 1a formed
p–p stacking interaction with Trp286 in PAS, while the part C of
compound 1a formed stacking interaction with Trp86 in CAS. (B) The potency of compound 1h is greatly improved due to the ionic interaction between the quinoline and
p–p
the surrounding negatively charged residues in PAS. (C) The potency of compound 2a is further improved due to the formation of the hydrogen bond (2.3 Å) between amide
oxygen of the bridge and Ser125. (D) compound 2e was obtained by replacing the four-methylene linker with a five-methylene linker, its potency was improved significantly
due to the formation of two hydrogen bonds (3.1 and 3.5 Å) between the carbonyl oxygen of acetophenone and Glu202, and a hydrogen bond (2.6 Å) between the carbonyl
oxygen of acetophenone and Gly121.
galanthamine (Table 1), the lead compound showed negligible
inhibition against human AChE. Molecular docking and molecular
dynamic (MD) simulations indicated that part A of 1a favorably
interacted with the PAS and part C interacted with the CAS. As indi-
cated in Figure 2A (AChE PDB ID, 1b41), the indole ring in part A of
1a stacked with Try286, the benzene ring of part B with Try337,
and the indole ring of part C formed a strong stacking interaction
with Trp86. However, compound 1a did not show high binding
respectively, about 70- and 100-fold better than 1a. The binding
model of compound 1h is illustrated in Figure 2B.
Hydrogen bonding makes
a great contribution to ligand-
receptor interactions, so we tried to introduce residues that would
serve as hydrogen bond acceptors into part C of the ligand with the
aim of forming new interactions in the CAS pocket in addition to
the
p–p stacking interactions. To avoid significantly changing the
binding conformation of compound 1h, we replaced the indole ring
of part C with acetophenone to yield compound 2a (Fig. 3).
Acetophenone was chosen here since it is a single aromatic ring
with a simple hydrogen bond acceptor. The potency of compound
2a was improved in a promising manner, displaying a Ki value of
53.34 nM against human AChE. The aim of introducing acetophe-
none was to form a hydrogen bond between the carbonyl oxygen
of acetophenone and the surrounding residues in the CAS.
However, as depicted in Figure 2C, the carbonyl oxygen of the
benzamide rather than that of the acetophenone formed a strong
hydrogen bond (2.3 Å) with Ser125. As expected, the binding con-
formation of compound 2a did not significantly change compared
to that of compound 1h.
affinity may because the
not optimized.
p-p stacking interactions in the PAS were
To enhance the stacking interaction between part A and PAS, we
designed compounds 1b–f, in which the part A indole ring was
replaced by a series of aromatic rings such as carbazole and benzo-
phenone (Table 1). Among these compounds, 1b displayed
improved potency with a Ki value of 1437.00 nM against human
AChE, whereas 1c–f showed no improvement in potency compared
to compound 1a.
We observed that the PAS region is also rich in negatively
charged residues. Generally, the ionic interactions are stronger
than the
p–p stacking interactions. We reasoned that if the part
A indole ring of 1a was replaced with a positively charged quater-
nary nitrogen, the resultant compounds 1g and 1h should bind
After analyzing the interaction between compound 2a and
AChE, we noticed that there are some important residues
surrounding the acetophenone of compound 2a, such as Gly121,
Glu202, Tyr337 and Tyr341. We speculated that adjusting the
length of the linker would permit the carbonyl oxygen of
with the PAS pocket through ionic interactions as well as
p–p
stacking. As expected, compounds 1g and 1h did show significantly
improved potency with Ki values of 197.78 and 123.73 nM,

D.-Y. Peng et al. / Bioorg. Med. Chem. 20 (2012) 6739–6750
6743
Br
N Br
N
O
N Br
4
5
6
O
O
O
NH O
O
NH O
O
NH O
N
O
Br
4O
2c
2b
Ki = 45.81 nM
2d
Ki = 44.66 nM
Ki = 9.41 nM
NH O
N
O
N Br
5O
N Br
Br
5
O
6
O
2a
Ki = 53.34 nM
NH O
NH O
2f
O
O
NH O
2g
2e
Ki = 6.47 nM
Ki = 81.37 nM
Ki = 47.25 nM
Figure 3. Structures of compounds 2a–g, and their inhibition constants (Ki) against human AChE.
Table 2
The detailed values and correlation between the experimental and calculated binding free energies (kcal/mol) of compounds 2a–g
-9.0
-9.2
y = 1.0758x + 1.2399
-9.4
R2 = 0.9257
-9.6
-9.8
-10.0
-10.2
-10.4
-10.6
-10.8
-11.0
-11.2-11.0-10.8-10.6-10.4-10.2-10.0 -9.8 -9.6 -9.4
Δ Gexp (kcal/mol)
Tacrine
2a
2b
2c
2d
2e
2f
2g
Ki (nM)
4Gexp
SIE
32.12
ꢀ10.18
ꢀ9.80
53.34
ꢀ9.88
ꢀ9.23
45.81
ꢀ9.97
ꢀ9.51
44.66
ꢀ9.99
ꢀ9.83
9.41
6.47
87.37
ꢀ9.59
ꢀ9.04
47.25
ꢀ9.96
ꢀ9.31
ꢀ10.91
ꢀ11.13
ꢀ10.37
ꢀ10.79
acetophenone to form hydrogen bonding interactions with these
residues. Therefore, we designed compounds 2b–g with linkers of
different lengths. We also substituted isoquinoline and pyridine
in part A. As a result, compounds 2b (Ki = 45.81 nM) and 2c
(Ki = 44.66 nM) showed slightly improved potency, but compound
2d, whose linker contained six methylene units, was found to ex-
hibit a much higher activity (Ki = 9.41 nM). Most promisingly, com-
pound 2e with a linker one carbon longer than in compound 2a
showed the highest inhibitory effects against human AChE with
Ki value of 6.47 nM, about 7-fold improved potency than 2a
(Ki = 53.34 nM). However, further extending the linker resulted in
the decrease of the potency. For example, compounds 2f
(Ki = 81.37 nM) and 2g (Ki = 47.25 nM), whose linkers respectively
contained five and six methylene units, showed much lower
potency than compound 2e. The lipophilicity and solubility of the
new compounds were affected during the above structural evolu-
tion. However no obvious relationships were identified between
these physicochemical properties and the practical inhibition
potencies, because some compounds with lower solvability
demonstrated better inhibition potency in our assay.
Compared with the original lead compound 1a, compound 2e
showed 1500-fold improved potency against human AChE. As
shown in Figure 2D based on molecular modeling, in addition to
the
p–p stacking interactions with Trp286 at PAS and Trp86 at

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D.-Y. Peng et al. / Bioorg. Med. Chem. 20 (2012) 6739–6750
Table 3
Inhibitory activities and selectivities of compounds 1g–h, 2a–g on human AChE and BChE
Compounds
Ki (nM)
Selectivity AChE/BChEa
Relative selectivityb
For AChE
For BChE
8.72 2.13
192.47 17.57
2360.00 331.00
97.00 10.20
166.00 10.90
56.00 6.70
Tacrine
Galanthamine
32.12 2.13
61.96 5.63
198.00 29.10
124.00 17.40
53.00 7.82
46.00 3.80
45.00 5.70
9.40 0.80
0.27
3.10
11.92
0.79
3.10
1.22
1.83
10.53
8.57
1.06
1.39
1.00
11.48
44.16
2.91
11.49
4.52
1g
1h
2a
2b
2c
2d
2e
2f
82.00 9.80
99.00 2.80
6.78
39.00
31.74
3.92
6.50 0.90
81.00 13.10
47.00 3.80
55.00 6.70
93.00 11.10
66.00 7.90
2g
5.15
a
Selectivity of inhibitor potency for AChE over BChE.
Relative selectivity of inhibitor potency for AChE over BChE compared with selectivity of tacrine against AChE over BChE.
b
Table 4
CAS, compound 2e formed four hydrogen bonds with its surround-
ing residues in the binding pocket. The amide oxygen of the bridge
formed a strong hydrogen bond with residue Ser125 with a
distance of 2.1 Å , while the carbonyl oxygen of acetophenone
formed two hydrogen bonds with Glu202 (3.1 and 3.5 Å) and a
hydrogen bond with Gly121 (2.6 Å). These hydrogen bond interac-
tions account for the molecular basis of the superior potency of 2e.
In order to quantitatively study the structure–activity relation-
ships of those new potent multi-site AChE inhibitors, solvated
interaction energy (SIE) calculations were performed based on
the molecular docking simulations, using tacrine as an internal
reference. The SIE were obtained by calculations and experimental
Ab aggregation and b-secretase inhibitory effects of compounds 2c–f
Targets
IC50 (lM)
Curcumin
2c
2d
2e
2f
Ab
10.4 0.8
5.7 1.2
71 3.2
>100
50 4.3
>100
79 4.7
85 10.1
>250
0.31 0.11
b-secretase
compound 2d decreased ꢁ38-fold compared to that of tacrine,
and it showed about ꢁ10-fold better inhibitory activity against hu-
man AChE than against BChE. Another interesting point is that
compound 2f showed almost same inhibitory activity against both
human AChE and BChE.
binding free energy (
DGexp) derived from the experimental data
(Table 2). The calculated SIE for the inhibitors ranged from ꢀ9.04
to ꢀ10.79 kcal/mol, while the
D
Gexp ranged from ꢀ9.59 to
ꢀ11.13 kcal/mol. Obviously, the order of the calculated SIE agreed
qualitatively with that of the experimental binding free energies.
More importantly, there was a good linear correlation (r2 = 0.93)
2.4. Inhibition of Ab aggregation
Recent explorations suggests that both AChE and BChE have been
associated with other non-traditional functions in addition to their
traditional regulation of ACh-mediated neurotransmission, includ-
ing modulatory effects on the Ab fibrillization, glial proliferation,
b-APP expression, tau protein phosphorylation, inflammatory pro-
cesses, and other pathological phenomena.35 Various AChE inhibi-
tors that interact with PAS of AChE have been demonstrated to
block in vitro Ab aggregation, reduce oxidative stress, and partially
relieve the hyper-phosphorylation of tau protein.21,36 Thus, AChE
inhibitors or ChE inhibitors may contribute to AD treatment in ways
other than those proposed by the cholinergic hypothesis. Discovery
of new AChE inhibitors or multi-target-directed ligands (MTDLs)
addressing distinct AD-relevant targets has been an active area in
AD research.7,16,18–20,37–41 We therefore tested the inhibitory activ-
ity of our newly synthesized multi-site AChE inhibitors against Ab
aggregation, using curcumin as a positive control.
The thioflavin T competition assay is a widely used fluorescent
method to detect amyloid structures, particularly in the presence
of AChE inhibitors. In parallel with the cholinesterase inhibition as-
say, all the new highly active AChE inhibitors were screened for
their inhibitory activity on Ab self-aggregation and tacrine was
used as another reference. Tacrine showed around 12% inhibition
rate in the crude screening, while some multi-site AChE inhibitors
also showed significant inhibitory activity against Ab self-
between the calculated SIE and the
DGexp, suggesting that our com-
putational protocol used to guide the structural optimization of
AChE inhibitors should be reliable.
2.3. Human BChE inhibition
In addition to AChE, BChE has now been recognized as another
target due to its relevance to the occurrence, symptoms, progres-
sion and responses to treatment in AD. Solid experimental
evidence has shown that the contribution of brain cholinesterase
activity from AChE is much higher than that of BChE.34 However,
the relative proportions of AChE to BChE are dependent on the
brain regions and the stage of disease progression in AD patients.35
In addition, clinical results have indicated that BChE activity
increases as AD progresses, which suggests that BChE may also
play an important role in cholinergic dysfunction, particularly at
the later stages of AD. In considering BChE as another important
target for AD treatment, the administration of inhibitors with
different AChE/BChE selectivity should be very useful as AD
progresses. Therefore, we screened all the newly synthesized com-
pounds against human BChE using tacrine and galanthamine as
references. For the candidates with better activity against human
AChE, their binding affinities for human BChE were assessed based
on the inhibitory kinetics with acetylthiocholine (ATC) as substrate
(Table 3). All the new compounds except 1g, with nanomolar level
activity, were less potent than tacrine for human BChE but more
active than galanthamine. According to the inhibitory kinetic
findings, the new cholinesterase inhibitors displayed various selec-
tivities on human AChE and BChE. Particularly, the selectivity of
aggregation. Based on screening with a single dosage of 100 lM,
compounds 2c–f with high inhibition percentages above 50% were
identified for full kinetic characterization and their detailed IC50
values are listed in Table 4. All these new compounds exhibited
limited activity toward Ab self-aggregation, which needs further
investigation in the future.

D.-Y. Peng et al. / Bioorg. Med. Chem. 20 (2012) 6739–6750
6745
2.5. Inhibition of b-secretase
The crude product was purified by silica column chromatography
using ethyl acetate/petroleum (V/V = 1/4) to give pure compounds
in a yield of 71% as a white solid. mp 173–175 °C. MS (EI) m/z:
280.21 (M)+. 1H NMR (600 MHz, DMSO-d6): d 10.79 (s, 1H), 9.92
(s, 1H), 8.34 (t, J = 5.4 Hz, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.58 (d,
J = 7.8 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.17 (s, 1H), 7.06 (t,
J = 7.2 Hz, 1H), 6.98 (t, J = 7.2 Hz, 1H), 6.79 (d, J = 8.4 Hz, 2H), 3.51
(dd, J = 13.8, 6.6 Hz, 2H), 2.92 (t, J = 7.8 Hz, 2H).
Due to its involvement in the proteolytic cleavage of amyloid
precursor protein (APP) to Ab, b-secretase has encouraged great
interest as a therapeutic target for disease-modifying agents in
AD.42–45 Some dual binding site AChE inhibitors such as tacrine
derivatives have been reported to exhibit b-secretase inhibitory
activity.46 Thus, the capability of newly synthesized compounds
was firstly evaluated at a single concentration (100 lM) against
b-secretase by fluorometric assay, followed by further kinetic
characterization of promising candidates if their inhibition rates
were no less than 50% percent at the screening concentration. As
displayed in Table 4, compound 2e showed modest potency
against b-secretase, whereas compound 2f revealed good potency
4.2. General synthesis of intermediate 7
To a stirred solution of 6.06 g (30 mmol) of 1,4-dibromobutane
in 50 mL of DMF was added 1.17 g (10 mmol) of indole and 0.57 g
(10 mmol) of ground KOH powder. The mixture was stirred over-
night. Water (100 mL) was added and the product was extracted
into ether (3 ꢂ 50 mL). The ether extracts were washed with
H2O, dried over MgSO4, and evaporated. The residue was chro-
matographed on silica gel using petroleum ether to give 1.8 g inter-
mediate 7 (oil). 1H NMR (600 MHz, DMSO-d6): d 7.55 (t, J = 9.6 Hz,
1H), 7.46–7.36 (m, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.14–7.04 (m, 1H),
7.00 (d, J = 6.0 Hz, 1H), 6.43 (d, J = 9.0 Hz, 1H). 4.21 (t, J = 6.0 Hz,
2H), 3.53 (t, J = 7.2 Hz, 2H), 1.93–1.81 (m, 2H), 1.79–1.67 (m, 2H).
against b-secretase with an IC50 value of 0.31 lM.
3. Conclusions
Tacrine and its derivatives have high inhibitory activity against
AChE, but their severe toxicity limited their clinical application. In
this work, non-tacrine based novel compounds as potent multi-site
AChE inhibitors were successfully designed by a combination of
computational simulations and experimental studies. In particular,
compound 2e, characterized by a central benzamide moiety linked
to an isoquinoline at one side and acetophenone at the other, was
identified as the most potent multi-site AChE inhibitor with a Ki
value of 6.47 nM, about 4-fold and 1500-fold higher potency than
tacrine and the original lead compound 1a, respectively. Moreover,
the inhibitory effects toward human BChE, Ab aggregation, and
b-secretase of some of the newly synthesized multi-site AChE
inhibitors were also evaluated. Interestingly, compound 2e also
showed simultaneously inhibitory effects against BChE, Ab aggre-
gation, and b-secretase. In addition, compound 2f, bearing a pyrid-
inium functionality as the head for the binding of PAS pocket, was
identified as an excellent b-secretase inhibitor with an IC50 value of
4.3. General synthesis of intermediate 8
A mixture of 59.1 g (237.7 mmol) of 1,4-dibromobutane, 1 g of
triethylbenzylammonium chloride, 5.16 g (30.86 mmol) of carba-
zole, 50 ml of aqueous 50% sodium hydroxide, and 50 mL benzene
was stirred at 40–50 °C for 6 h. The organic layer was separated,
and the aqueous layer was extracted three times with chloroform
(3 ꢂ 50 mL). The combined organic layer was washed three times
with water (3 ꢂ 50 mL) and dried over Na2SO4. The organic solvent
was distilled by vacuum distillation, and then chromatographed on
silica gel using petroleum ether to give 5.8 g compound 8. Yield
62%; mp 101–103 °C. 1H NMR (600 MHz, CDCl3): d 8.11 (d,
J = 7.8 Hz, 2H), 7.47 (t, J = 7.8 Hz, 2H), 7.41 (t, J = 8.4 Hz, 2H),
7.28–7.20 (m, 2H), 4.36 (t, J = 7.2 Hz, 2H), 3.38 (t, J = 6.6 Hz, 2H),
2.11–2.02 (m, 2H), 1.95–1.88 (m, 2H).
0.31 lM. In summary, due to the high potency towards not only
AChE, but also other important targets associated with AD therapy,
compound 2e could be a promising multifunctional lead for the
treatment of AD. For the requirement of entering the brain, further
structural improvement and ideal formulation of these novel
potent multi-functional inhibitors with lipophilic quaternary
ammonium will be needed.
4.4. General procedure for the synthesis of compounds 1a and
1b
Compound 4 (1 mmol), compound 7 or 8 (1.1 mmol), and potas-
sium carbonate (2.2 mmol) were mixed together and stirred in dry
acetone (50 mL) under reflux for 24 h. The solution was evaporated
in vacuum and purified by column chromatography using gradient
elution of petroleum ether/ethyl acetate (V/V = 2/3) to afford the
product as 1a or 1b.
4. Materials and methods
Unless otherwise noted, all chemical reagents were commer-
cially available and treated with standard methods before use.
Solvents were dried in a routine way and redistilled before use.
1H NMR and 1C NMR spectra were recorded on a VARIAN
Mercury-Plus 600 or 400 spectrometer in CDCl3 or DMSO-d6 with
TMS as the internal reference. Mass spectral data were obtained
on a ThermoFisher Mass platform DSQII by electrospray ionization
(ESI-MS). Elemental analyses were performed on a Vario EL III ele-
mentary analysis instrument. Melting points were taken on a Buchi
B-545 melting point apparatus and are uncorrected. All chemical
yields are unoptimized and generally represent the result of a
single experiment.
4.4.1. 4-(4-(1H-indol-1-yl)butoxy)-N-(2-(1H-indol-3-
yl)ethyl)benzamide (1a)
White solid; yield 34%; mp 57–59 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.80 (s, 1H), 8.43 (s, 1H), 7.80 (d, J = 8.4 Hz, 2H),
7.58 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 8.4 Hz,
1H), 7.39 (d, J = 2.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.17 (s, 1H),
7.13 (t, J = 7.8 Hz, 1H), 7.06 (t, J = 7.2 Hz, 1H), 7.01 (t, J = 7.2 Hz,
1H), 6.98 (d, J = 7.8 Hz, 1H), 6.95 (d, J = 8.4 Hz, 2H), 6.43 (s, 1H),
4.25 (t, J = 6.6 Hz, 2H), 4.02 (t, J = 6.0 Hz, 2H), 3.54–3.49 (m, 2H),
2.93 (t, J = 7.2 Hz, 2H), 1.93–1.88 (m, 2H), 1.72–1.65 (m, 2H). 13C
NMR (100 MHz, DMSO-d6): d 165.64, 160.77, 136.27, 135.66,
128.94, 128.56, 128.12, 127.32, 126.86, 122.58, 120.96, 120.44,
118.86, 118.34, 118.23, 113.86, 112.01, 111.39, 109.77, 100.47,
67.22, 45.11, 40.17, 26.55, 26.01, 25.33. MS (EI) m/z: 451.31 (M)+.
Anal. Calcd for C29H29N3O2: C, 77.13; H, 6.47; N, 9.31. Found: C,
77.04; H, 6.65; N, 9.35.
4.1. General procedure for N-(2-(1H-indol-3-yl)ethyl)
hydroxybenzamide 4
To
a mixture of p-hydroxybenzoic acid (6 mmol) in THF
(50 mL), dicyclohexylcarbodiimide (6 mmol) was added at 0 °C
with stirring for 1 h. Tryptamine (5 mmol) was then added and
stirred further for 24 h at room temperature, followed by filtration.

6746
D.-Y. Peng et al. / Bioorg. Med. Chem. 20 (2012) 6739–6750
4.4.2. N-(2-(1H-indol-3-yl)ethyl)-4-(4-(9H-carbazol-9-yl)butoxy)
benzamide (1b)
4.7.1. N-(2-acetylphenyl)-4-(4-bromobutoxy)benzamide (12a)
White solid; yield 85%; mp 84–86 °C. MS (EI) m/z: 389.06 (M)+.
1H NMR (600 MHz, DMSO-d6): d 12.33 (s, 1H), 8.67 (d, J = 8.4 Hz,
1H), 8.11 (d, J = 7.8 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.67 (t,
J = 7.8 Hz, 1H), 7.25 (t, J = 7.2 Hz, 1H), 7.14 (d, J = 8.4 Hz, 2H), 4.12
(t, J = 6.0 Hz, 2H), 3.63 (t, J = 6.6 Hz, 2H), 2.70 (s, 3H), 2.03–1.95
(m, 2H), 1.92–1.82 (m, 2H).
White solid; yield 41%; mp 131–132 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.81 (s, 1H), 8.45 (s, 1H), 8.16 (d, J = 7.8 Hz, 2H),
7.79 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 7.8 Hz,
1H), 7.46 (t, J = 7.8 Hz, 2H), 7.33 (d, J = 7.8 Hz, 1H), 7.20
(t, J = 7.2 Hz, 2H), 7.17 (s, 1H), 7.06 (t, J = 7.2 Hz, 1H), 7.01–6.96
(m, 1H), 6.95 (t, J = 7.8 Hz, 2H), 4.49 (t, J = 6.6 Hz, 2H), 4.03 (t,
J = 6.0 Hz, 2H), 3.61–3.51 (m, 2H), 2.92 (t, J = 7.2 Hz, 2H), 1.98–
1.89 (m, 2H), 1.80–1.73 (m, 2H). 13C NMR (100 MHz, DMSO-d6): d
165.65, 160.78, 139.97, 136.28, 128.95, 127.33, 126.87, 125.69,
122.59, 122.06, 120.95, 120.29, 118.70, 118.36, 118.24, 113.85,
112.02, 111.40, 109.26, 67.32, 41.87, 40.18 ,26.24, 25.34, 25.23.
MS (EI) m/z: 501.06 (M)+. Anal. Calcd for C33H31N3O2: C, 79.01; H,
6.23; N, 8.38. Found: C, 78.81; H, 6.36; N, 8.14.
4.7.2. N-(2-acetylphenyl)-4-((5-bromopentyl)oxy)benzamide
(12b)
White solid; yield, 83%. mp 92–94 °C. MS (EI) m/z: 403.19 (M)+.
1H NMR (600 MHz, DMSO-d6) d 12.30 (s, 1H), 10.27 (s, 1H), 8.68 (d,
J = 8.4 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.85 (d, J = 7.8 Hz, 2H), 7.66
(t, J = 7.8 Hz, 1H), 7.23 (t, J = 7.2 Hz, 1H), 6.94 (d, J = 8.4 Hz, 2H),
4.07 (t, J = 7.2 Hz, 2H), 3.61 (t, J = 6.0 Hz, 2H), 2.70 (s, 3H), 2.18–
2.08 (m, 2H), 1.88–1.79 (m, 2H), 1.56–1.48 (m, 2H).
4.5. General procedure for N-(2-(1H-indol-3-yl)ethyl)-(4-
bromobutoxy) benzamide 9
4.7.3. N-(2-acetylphenyl)-4-((6-bromohexyl)oxy)benzamide
(12c)
A
mixture of 1,4-dibromoalkane (30 mmol), compound
4
White solid; yield 83%; mp 92–94 °C. MS (EI) m/z: 403.19 (M)+.
1H NMR (600 MHz, DMSO-d6): d 12.33 (s, 1H), 8.67 (d, J = 8.4 Hz,
1H), 8.11 (d, J = 7.8 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.67 (t,
J = 7.8 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H), 7.13 (d, J = 8.4 Hz, 2H), 4.07
(t, J = 6.6 Hz, 2H), 3.55 (t, J = 6.6 Hz, 2H), 2.70 (s, 3H), 1.89–1.80
(m, 2H), 1.80–1.71 (m, 2H), 1.48–1.44 (m, 4H).
(10 mmol), potassium carbonate (20 mmol) and a catalytic amount
of KI in 30 mL of acetone was stirred at 65 °C for 3 days. When the
reaction was completed, as monitored by TLC detection, the solu-
tion was filtered. After solvent evaporation and purification by col-
umn chromatography using petroleum ether/CH2Cl2 (V/V = 1/0 to
2/3) for gradient elution, the final product was obtained in a yield
of 65% as a white solid. mp 111–113 °C. MS (EI) m/z: 414.25 (M)+.
1H NMR (600 MHz, DMSO-d6): d 10.81 (s, 1H), 8.46 (s, 1H), 7.82 (d,
J = 8.4 Hz, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.17
(s, 1H), 7.06 (t, J = 7.2 Hz, 1H), 7.01–6.95 (m, 3H), 4.06 (t, J = 6.0 Hz,
2H), 3.62 (t, J = 6.6 Hz, 2H), 3.52 (dd, J = 13.8, 6.6 Hz, 2H), 2.93 (t,
J = 7.2 Hz, 2H), 2.01–1.91(m, 2H), 1.89–1.81 (m, 2H).
4.8. General procedure for the syntheses of compounds 1c–h
and 2a–g
Compounds
9 (1 mmol), corresponding phenol derivatives
(1.1 mmol), and potassium carbonate (2.2 mmol) were mixed to-
gether and stirred in dry acetone (30 mL) under reflux for 24 h.
The solution was evaporated in vacuum and purified by column
chromatography using gradient elution of petroleum ether/ethyl
acetate (V/V = 2/3) to afford the products as 1c–f. Compounds 9
and 12a–c (1 mmol) and corresponding pyridine derivatives
(10 mmol) were mixed together and heated to reflux in dry aceto-
nitrile (30 mL) for 24 h. Then the solvent was evaporated in vac-
uum and the product was purified by column chromatography
using gradient elution of CH2Cl2/methanol (V/V = 10/3) to give
the products as 1g–h and 2a–g.
4.6. General procedure for 4-hydroxyl benzoyl aromatic amine
(11)
To a suspension of 4-acetoxybenzoic acid (50 mmol) and SOCl2
(20 ml), DMF (0.5 ml) was added dropwise as catalyst. After stir-
ring for a few minutes at room temperature, the solution became
clear and was then heated at reflux for 2 h. After evaporating under
reduced pressure, the solution was added dropwise to the mixture
of o-aminoacetophenone (45 mmol), NEt3 (100 mmol) and anhy-
drous CH2Cl2 (60 mL) in an ice bath. The reaction was continuously
stirred at room temperature overnight. The solid was obtained by
evaporation under reduced pressure and dissolved in MeOH/H2O
(V/V = 1/1, 100 ml). Later, the reaction mixture was stirred at room
temperature for another 2 h after addition of NaOH (100 mmol).
When the reaction was completed, as indicated by TLC detection,
the pH was adjusted to 6.0 with 1 M HCl. Finally, the precipitate
was filtered and washed by water, and then was redissolved in
EtOAc and evaporated under reduced pressure to give the product
in a yield of 81% as a white solid. mp 185–187 °C. MS (EI) m/z:
255.08 (M)+. 1H NMR (600 MHz, DMSO-d6): d 12.30 (s, 1H), 10.27
(s, 1H), 8.68 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.85 (d,
J = 7.8 Hz, 2H), 7.66 (t, J = 7.8 Hz, 1H), 7.23 (t, J = 7.2 Hz, 1H), 6.94
(d, J = 8.4 Hz, 2H), 2.70 (s, 3H).
4.8.1. N-(2-(1H-indol-3-yl)ethyl)-4-(4-((9H-carbazol-4-yl)oxy)
butoxy)benzamide(1c)
White solid; yield 28%; mp 160–162 °C; 1H NMR (600 MHz,
DMSO-d6): d 11.26 (s, 1H), 10.82 (s, 1H), 8.48 (t, J = 5.4 Hz, 1H),
8.14 (d, J = 7.8 Hz, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 7.8 Hz,
1H), 7.45 (d, J = 8.4 Hz, 1H), 7.36–7.26 (m, 3H), 7.18 (s, 1H), 7.11
(t, J = 7.2 Hz, 1H), 7.09–7.04 (m, 2H), 7.02 (d, J = 8.4 Hz, 2H), 6.98
(t, J = 7.2 Hz, 1H), 6.71 (d, J = 7.8 Hz, 1H), 4.28 (d, J = 5.4 Hz, 2H),
4.20 (d, J = 6.0 Hz, 2H), 3.54–3.49 (m, 2H), 2.94 (t, J = 7.2 Hz, 2H),
2.12–2.02 (m, 4H). 13C NMR (100 MHz, DMSO-d6): d 165.65,
160.83, 154.97, 141.13, 138.95, 136.27, 128.99, 127.33, 126.85,
126.54, 124.54, 122.60, 122.17, 121.77, 120.95, 118.66, 118.36,
118.25, 113.94, 112.02, 111.44, 110.45, 103.82, 100.40, 67.36,
67.10, 40.19, 25.73, 25.66, 25.36. MS (EI) m/z: 517.19 (M)+. Anal.
Calcd for C33H31N3O3: C, 76.57; H, 6.04; N, 8.12. Found: C, 76.41;
H, 6.11; N, 8.33.
4.7. General procedure for 4-(
aromatic amine 12
x-bromobutoxy) benzoyl
4.8.2. N-(2-(1H-indol-3-yl)ethyl)-4-(4-(3-benzoylphenoxy)
butoxy)benzamide (1d)
A mixture of
a,x-dibromoalkane (30 mmol), compound 11
(10 mmol), potassium carbonate (20 mmol) and a catalytic amount
of KI in 30 mL of acetone was stirred at 65 °C for 3 days. The precip-
itate was filtered and evaporated, and further purified by column
chromatography using gradient elution with petroleum ether/
EtOAc (V/V = 1/0 to 2/3) to give pure products as 12a–c.
White solid; yield 38%; mp 117–119 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.83 (s, 1H), 8.49 (s, 1H), 7.83 (d, J = 8.4 Hz, 2H),
7.75 (d, J = 7.2 Hz, 2H), 7.69 (t, J = 7.2 Hz, 1H), 7.61–7.64 (m, 3H),
7.48 (t, J = 7.8 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.29–7.24 (m, 3H),
7.18 (s, 1H), 7.07 (t, J = 7.2 Hz, 1H), 7.01–6.96 (m, 3H), 4.12 (t,

D.-Y. Peng et al. / Bioorg. Med. Chem. 20 (2012) 6739–6750
6747
J = 6.6 Hz, 2H), 3.55–3.49 (m, 2H), 3.17 (t, J = 5.4 Hz, 2H), 2.94 (t,
J = 7.2 Hz, 2H), 1.96–1.85 (m, 4H). 13C NMR (150 MHz, DMSO-d6):
d 195.60, 165.77, 160.88, 158.61, 138.43, 137.06, 136.34, 132.72,
129.71, 129.66, 129.04, 128.56, 127.39, 126.90, 122.63, 122.18,
121.00, 119.07, 118.41, 118.30, 114.80, 113.90, 112.09, 111.45,
67.40, 67.33, 40.27, 25.39, 25.37. MS (EI) m/z: 532.06 (M)+. Anal.
Calcd for C34H32N2O4: C, 76.67; H, 6.06; N, 5.26. Found: C, 76.70;
H, 5.77; N, 5.19.
118.30, 118.17, 113.88, 111.94, 111.36., 67.00, 56.94, 40.11,
26.34, 25.45, 25.28. MS (EI) m/z: 464.1 (M-Br)+. Anal. Calcd For
C30H30BrN3O2: C, 66.18; H, 5.55; N, 7.72. Found: C, 66.21; H,
5.30; N, 7.70.
4.8.7. 1-(4-(4-((2-acetylphenyl)carbamoyl)phenoxy)
butyl)quinolin-1-ium bromide (2a)
White solid; yield 79%; mp 167–169 °C. 1H NMR (600 MHz,
DMSO-d6):
d 12.35 (s, 1H),9.62(d, J = 8.4 Hz, 1H) , 9.32 (d,
4.8.3. N-(2-(1H-indol-3-yl)ethyl)-4-(4-(4-benzoylphenoxy)
butoxy)benzamide (1e)
J = 8.4 Hz, 1H), 8.70–8.59 (m,2H), 8.50 (t, J = 8.4 Hz, 1H), 8.36–
8.18 (m, 2H), 8.16–8.05 (m, 2H), 7.96–7.90 (m, 2H), 7.68 (t,
J = 7.8 Hz, 1H), 7.26 (t, J = 7.8 Hz, 1H), 7.15–7.10 (m, 2H), 5.17 (t,
J = 7.2 Hz, 2H), 4.16 (t, J = 7.2 Hz, 2H), 2.71 (s, 3H), 2.27–2.14 (m,
2H), 1.97–1.81 (m, 2H). 13C NMR (100 MHz, DMSO-d6): d 203.52,
164.37, 161.45, 140.27, 137.33, 136.89, 135.58, 134.66, 132.24,
129.67, 128.95, 127.17, 126.28, 125.82, 122.75, 122.13, 119.91,
118.95,114.61,67.20, 56.83, 28.75, 26.30, 25.37. MS (EI) m/z:
439.8 (M-Br)+. Anal. Calcd For C28H27BrN2O3: C, 64.74; H, 5.24; N,
5.39. Found: C, 64.85; H, 5.35; N, 5.17.
White solid; yield 31%; mp 134–136 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.82 (s, 1H), 8.47 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H),
7.75 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 7.8 Hz, 2H), 7.67–7.63 (m, 1H),
7.61–7.51 (m, 3H), 7.34 (d, J = 7.8 Hz, 1H), 7.17 (s, 1H), 7.10 (d,
J = 8.4 Hz, 2H), 7.09–7.04 (m, 1H), 7.03–6.94 (m, 3H), 4.17 (t,
J = 6.6 Hz 2H), 4.11 (t, J = 7.2 Hz,2H), 3.54–3.49 (m, 2H), 2.93 (t,
J = 7.2 Hz, 2H), 1.95–1.88 (m, 4H). 13C NMR (100 MHz, DMSO-d6):
d 194.43, 165.61, 162.39, 160.80,137.80, 136.25, 132.22,132.07,
129.26, 128.96, 128.44, 127.30, 126.85, 122.59, 120.92, 118.33,
118.22, 114.33, 113.89, 111.99, 111.38, 67.58, 67.27, 40.16, 25.32,
25.28. MS (EI) m/z: 532.26 (M)+. Anal. Calcd for C34H32N2O4: C,
76.67; H, 6.06; N, 5.26. Found: C, 76.70; H, 5.77; N, 5.19.
4.8.8. 2-(4-(4-((2-acetylphenyl)carbamoyl)phenoxy)
butyl)isoquinolin-2-ium bromide (2b)
White solid; yield 81%; mp 181–183 °C; 1H NMR (400 MHz,
DMSO-d6): d 12.30 (s, 1H), 10.32 (s, 1H), 8.94 (d, J = 6.6 Hz, 1H),
8.64 (d, J = 6.6 Hz, 2H), 8.51 (d, J = 8.4 Hz, 1H), 8.36 (d, J = 8.4 Hz,
1H), 8.24 (t, J = 7.8 Hz, 1H), 8.07 (t, J = 9.6 Hz, 2H), 7.88 (d,
J = 8.4 Hz, 2H), 7.64 (t, J = 7.8 Hz, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.10
(d, J = 8.4 Hz, 2H), 4.88 (t, J = 7.2 Hz, 2H), 4.13 (t, J = 6.0 Hz, 2H),
2.32–2.16 (m, 2H), 1.90–1.79 (m, 2H). 13C NMR (100 MHz,
DMSO-d6): d 203.56, 164.39, 161.47, 150.02, 140.04, 136.91,
136.73, 134.92, 134.69, 132.27, 131.06, 130.31, 128.96, 127.17,
126.33, 125.84, 122.82, 122.71, 119.95, 114.63, 67.18, 60.25,
28.76, 27.38, 25.18. MS (EI) m/z: 439.1 (M-Br)+. Anal. Calcd for
4.8.4. N-(2-(1H-indol-3-yl)ethyl)-4-(4-((9-oxo-9H-fluoren-2-yl)
oxy)butoxy)benzamide (1f)
Orange solid; yield 27%; mp 153–155 °C; 1H NMR (600 MHz,
DMSO-d6): d 10.82 (s, 1H), 8.47 (t, J = 5.4 Hz, 1H), 7.83 (d,
J = 8.4 Hz, 2H), 7.71–7.66 (m, 2H), 7.61–7.53 (m, 3H), 7.34 (d,
J = 8.4 Hz, 1H), 7.28 (t, J = 7.2 Hz, 1H), 7.20–7.12 (m, 3H), 7.07 (t,
J = 7.2 Hz, 1H), 7.03–6.94 (m, 3H), 4.15 (t, J = 7.2 Hz, 2H), 4.11 (t,
J = 6.6 Hz, 2H), 3.54–3.49 (m, 2H), 2.94 (t, J = 7.8 Hz, 2H), 1.94–
1.85 (m, 4H). 13C NMR (100 MHz, DMSO-d6): d 192.97, 165.72,
160.85, 159.89, 144.39, 136.31, 135.30, 135.06, 133.45, 128.99,
128.055, 127.35, 126.88, 123.85, 122.60, 122.20, 120.96, 120.63,
120.23, 118.27, 113.88, 112.06, 111.42, 109.78, 67.72, 67.27,
40.23, 25.39, 25.32. MS (EI) m/z: 530.18 (M)+. Anal. Calcd for
C28H27BrN2O3: C, 64.74; H, 5.24; N, 5.39. Found: C, 64.73; H,
5.26; N, 5.30.
4.8.9. 2-(5-(4-((2-acetylphenyl)carbamoyl)phenoxy)pentyl)
isoquinolin-2-ium bromide (2c)
C34H30N2O4: C, 76.96; H, 5.70; N, 5.28. Found: C, 76.66; H, 5.70;
N, 5.15.
White solid; yield 81%; mp 141–143 °C; 1H NMR (600 MHz,
DMSO-d6): d 12.33 (s, 1H), 10.14 (s, 1H), 8.84 (d, J = 6.6 Hz, 1H),
8.67 (d, J = 8.4 Hz, 1H), 8.62 (d, J = 6.6 Hz, 1H), 8.49 (d, J = 8.4 Hz,
1H), 8.36 (d, J = 7.8 Hz, 1H), 8.27 (t, J = 7.8 Hz, 1H), 8.12 (d,
J = 8.4 Hz, 1H), 8.08 (t, J = 7.8 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.68
(t, J = 7.8 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H), 7.10 (d, J = 8.4 Hz, 2H),
4.77 (t, J = 7.2 Hz, 2H), 4.10 (t, J = 6.0 Hz, 2H), 2.70 (s, 3H), 2.18–
2.08 (m, 2H), 1.88–1.79 (m, 2H), 1.56–1.48 (m, 2H). 13C NMR
(100 MHz, DMSO-d6): d 203.58, 164.43, 161.63, 149.93, 140.06,
136.88, 136.74, 134.93, 134.70, 132.28, 131.09, 130.29, 128.98,
127.22, 127.15, 126.22, 125.81, 122.81, 119.94, 114.58, 67.45,
60.45, 30.14, 28.77, 27.89, 22.06. MS (EI) m/z: 453.3 (M-Br)+ . Anal.
Calcd for C29H29BrN2O3: C, 65.29; H, 5.48; N, 5.25. Found: C, 65.28;
H, 5.38; N, 4.96.
4.8.5. 1-(4-(4-((2-(1H-indol-3-yl)ethyl)carbamoyl)phenoxy)
butyl)-4-(dimethylamino)pyridin-1-ium bromide(1g)
White solid; yield 79%; mp 101–103 °C. 1H NMR (600 MHz,
DMSO-d6): d 10.83 (s, 1H), 8.48 (s, 1H), 8.34 (d, J = 7.2 Hz, 2H),
7.83 (d, J = 8.5 Hz, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 8.0 Hz,
1H), 7.17 (s, 1H), 7.09–7.02 (m, 3H), 7.00–6.94 (m, 3H), 4.25 (t,
J = 7.2 Hz, 2H), 4.05 (t, J = 6.0 Hz, 2H), 3.55–3.49 (m, 2H), 3.18 (s,
6H), 2.93 (t, J = 9.6 Hz, 2H), 1.97–1.89 (m, 2H), 1.75–1.66 (m, 2H).
13C NMR (100 MHz, DMSO-d6): d 165.54, 160.58, 155.76, 141.94,
136.20, 128.93, 127.26, 122.54, 120.87, 118.30, 118.17, 113.88,
111.94, 111.36, 107.67, 66.95, 56.23, 40.28, 39.69, 27.10, 25.83,
25.13. MS (EI) m/z: 457.2 (M-Br)+. Anal. Calcd For C28H33BrN4O2:
C, 62.57; H, 6.19; N, 10.42. Found: C, 62.63 H, 5.96; N, 10.14.
4.8.10. 2-(6-(4-((2-acetylphenyl)carbamoyl)phenoxy)hexyl)
isoquinolin-2-ium bromide (2d)
4.8.6. 1-(4-(4-((2-(1H-indol-3-yl)ethyl)carbamoyl)phenoxy)
butyl)quinolin-1-ium bromide (1h)
White solid; yield 85%, mp 145–147 °C; 1H NMR (600 MHz,
DMSO-d6): d 12.35 (s, 1H), 10.16 (s, 1H), 8.85 (d, J = 7.2 Hz, 1H),
8.68 (d, J = 7.8 Hz, 1H), 8.62 (d, J = 6.6 Hz, 1H), 8.50 (d, J = 8.4 Hz,
1H), 8.36 (d, J = 8.4 Hz, 1H), 8.27 (t, J = 7.8 Hz, 1H), 8.13 (d,
J = 7.8 Hz, 1H), 8.09 (t, J = 7.8 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.68
(t, J = 7.8 Hz, 1H), 7.26 (t, J = 7.8 Hz, 1H), 7.10 (d, J = 8.4 Hz, 2H),
4.75 (t, J = 7.2 Hz, 2H), 4.07 (t, J = 6.6 Hz, 2H), 2.71 (s, 3H), 2.11–
2.02 (m, 2H), 1.80–1.73 (m, 2H), 1.54–1.47 (m, 2H), 1.45–1.39
(m, 2H). 13C NMR (100 MHz, DMSO-d6): d 203.55, 164.45, 161.68,
150.01, 140.05, 136.87, 136.71, 134.95, 134.66, 132.24, 131.05,
130.32, 129.01, 127.23, 127.18, 126.18, 125.81, 122.92, 122.72,
Orange solid; yield 53%; mp 78–80 °C. 1H NMR (600 MHz,
DMSO-d6): d 10.84 (s, 1H), 9.67 (d, J = 5.4 Hz, 1H), 9.30 (d,
J = 8.4 Hz, 1H), 8.66 (d, J = 9.0 Hz, 1H), 8.54–8.48 (m, 2H), 8.32–
8.16 (m, 2H), 8.12–7.99 (m, 1H), 7.87–7.78 (m, 2H), 7.57 (d,
J = 7.8 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.17 (s, 1H), 7.05 (t,
J = 7.8 Hz, 1H), 7.01–6.89 (m, 3H), 5.18 (t, J = 7.8 Hz, 2H), 4.08 (t,
J = 6.6 Hz, 2H), 3.56–3.50 (m, 2H), 2.94 (t, J = 7.2 Hz, 2H), 2.18–
2.09 (m, 2H), 1.93–1.86 (m, 2H). 13C NMR (150 MHz, DMSO-d6):
d 165.56, 160.58, 147.43, 137.40, 136.20, 135.62, 130.76, 129.85,
129.73, 128.95, 127.26, 126.89, 122.53, 122.16, 120.87, 118.92,

6748
D.-Y. Peng et al. / Bioorg. Med. Chem. 20 (2012) 6739–6750
120.02, 114.55, 67.65, 60.52, 30.41, 28.75, 28.20, 25.18, 24.93. MS
(EI) m/z: 467.0 (M-Br)+ . Anal. Calcd For C30H31BrN2O3: C, 65.81;
H, 5.71; N, 5.12. Found: C, 65.74; H, 5.99; N, 5.32.
30 °C, in 0.1 M potassium phosphate, pH 7.0. Each experiment
was repeated at least three times and the values were averaged.
The inhibition constant (Ki), the indication of an inhibitor’s po-
tency, was obtained from the Dixon plot of plotting 1/v against
concentration of inhibitor at certain concentrations of substrate.
Bovine serum albumin is usually added up to 0.5% of the total reac-
tion volume to reduce the coating of target enzyme during the
incubation. In our assay, no obvious effect from bovine serum albu-
min on the activity of the compounds has been found, which indi-
cates that the new compounds selectively inhibited the target
enzyme but not interacted with bovine serum albumin.
4.8.11. 1-(5-(4-((2-acetylphenyl)carbamoyl)phenoxy)
pentyl)quinolin-1-ium bromide (2e)
White solid; yield 81%; mp 133–135 °C; 1H NMR (600 MHz,
DMSO-d6): d 12.34 (s, 1H), 9.62 (d, J = 4.8 Hz, 1H), 9.32 (d,
J = 8.4 Hz, 1H), 8.67 (d, J = 8.4 Hz, 2H), 8.51 (d, J = 8.4 Hz, 1H),
8.33–8.25 (m, 1H), 8.21 (t, J = 6.6 Hz, 1H), 8.12 (d, J = 9.6 Hz, 1H),
8.07 (t, J = 7.8 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.68 (t, J = 7.2 Hz,
1H), 7.25 (t, J = 7.2 Hz, 1H), 7.10 (d, J = 8.4 Hz, 2H), 5.13 (t,
J = 7.2 Hz, 2H), 4.09 (t, J = 5.4 Hz, 2H), 2.71 (s, 3H), 2.11–2.04 (m,
2H), 1.86–1.79 (m, 2H), 1.62–1.55 (m, 2H). 13C NMR (100 MHz,
4.10. Self-mediated Ab (1–42) aggregation assay
DMSO-d6):
d
203.63, 164.47, 161.6, 149.64, 147.40, 140.08,
The thioflavin T based fluorometric assay was used here to
investigate the self-mediated Ab (1–42) aggregation.46,49–51 Ab
(1–42) samples (GL Biochem, Shanghai) were dissolved in DMSO
as a stable stock solution (Ab = 5 mM). Experiments were per-
formed by shaking the peptide in 50 mM phosphate buffer (pH
8.5) containing 100 mM NaCl at 37 °C for 10 h (final Ab concentra-
137.36, 135.65, 134.73, 132.31, 130.74, 129.84, 129.70, 129.02,
126.26, 122.85, 122.75, 122.14, 119.98, 118.99, 114.61, 67.55,
57.15, 29.23, 28.78, 27.97, 22.43. MS (EI) m/z: 453.3 (M-Br)+ . Anal.
Calcd for C29H29BrN2O3: C, 65.29; H, 5.48; N, 5.25. Found: C, 65.45;
H, 5.72; N, 5.18.
tion 100
concentrations. After incubation, 20
diluted to a final volume of 200 L with 50 mM glycine-NaOH buf-
fer (pH 8.5) containing 10 M thioflavin T. Then the measurement
of fluorescence intensity was carried out (kexc = 450 nm,
kem = 480 nm) by microplate reader (SpectraMax M5, Molecular
Devices) using black microwell plates (96 wells), and values at
the plateau were calculated after subtraction of the background
l
M) with and without the tested compound at various
4.8.12. 1-(5-(4-((2-acetylphenyl)carbamoyl)phenoxy)
pentyl)pyridin-1-ium bromide (2f)
l
L of the above solutions were
l
White solid; yield 85%; mp 180–182 °C; 1H NMR1H NMR
(600 MHz, DMSO-d6): d 12.35 (s, 1H), 9.13 (d, J = 6.0 Hz, 2H), 8.67
(d, J = 8.4 Hz, 1H), 8.62 (t, J = 7.8 Hz, 1H), 8.18 (t, J = 7.2 Hz, 2H),
8.13 (d, J = 7.2 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.68 (t, J = 7.8 Hz,
1H), 7.25 (t, J = 7.2 Hz, 1H), 7.12 (d, J = 8.4 Hz, 2H), 4.66 (t,
J = 7.2 Hz, 2H), 4.09 (t, J = 6.0 Hz, 2H), 2.71 (s, 3H), 2.06–1.96 (m,
2H), 1.86–1.76 (m, 2H), 1.50–1.42 (m, 2H). 13C NMR (100 MHz,
DMSO-d6): d 203.59, 164.46, 161.65, 145.45, 144.77, 140.06,
134.71, 132.28, 129.01, 128.06, 126.27, 122.89, 120.00, 114.62,
67.45, 60.46, 30.39, 28.77, 27.82, 21.97. MS (EI) m/z: 403.2 (M-
Br)+. Anal. Calcd for C25H27BrN2O3: C, 62.12; H, 5.63; N, 5.80.
Found: C, 62.03; H, 5.63; N, 5.58.
l
fluorescence of the 10 lM thioflavin T solution.
4.11. b-Secretase inhibition assay
b-Secretase in vitro assay was performed as described in the
following procedure by employing the b-secretase fluorescence
resonance energy transfer assay kit (P2985, PanVera) according
to the literature reports.39,42 The weakly fluorescent substrate be-
comes highly fluorescent once the enzymatic cleavage occurs and
the increase in fluorescence is linearly correlated with the rate of
proteolysis. The detailed procedures are as follows: various con-
4.8.13. 1-(6-(4-((2-
acetylphenyl)carbamoyl)phenoxy)hexyl)pyridin-1-ium
bromide (2g)
White solid; yield 75%; mp 78–79 °C; 1H NMR (600 MHz,
DMSO-d6): d 12.35 (s, 1H), 9.15 (d, J = 6.0 Hz, 2H), 8.67 (d,
J = 8.4 Hz, 1H), 8.62 (t, J = 7.8 Hz, 1H), 8.18 (t, J = 6.6 Hz, 2H), 8.12
(d, J = 7.8 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.68 (t, J = 7.8 Hz, 1H),
7.25 (t, J = 7.8 Hz, 1H), 7.12 (d, J = 8.4 Hz, 2H), 4.64 (t, J = 7.2 Hz,
2H), 4.07 (t, J = 6.6 Hz, 2H), 2.71 (s, 3H), 2.02–1.92 (m, 2H), 1.81–
1.70 (m, 2H), 1.52–1.44 (m, 2H), 1.43–1.31 (m, 2H). 13C NMR
(100 MHz, DMSO-d6): d 203.58, 164.53, 161.73, 145.47, 144.85,
140.04, 134.68, 132.24, 129.08, 128.07, 126.22, 123.08, 122.80,
120.11, 114.61, 67.65, 60.53, 30.72, 28.78, 28.23, 25.11, 24.88.
MS (EI) m/z: 417.9 (M-Br)+. Anal. Calcd for C26H29BrN2O3: C,
62.78; H, 5.88; N, 5.63. Found: C, 62.77; H, 6.03; N, 5.53.
centrations of test compound (5
priate amounts of enzyme (20
l
L) or DMSO (control), and appro-
l
L) were preincubated in 50 mM
sodium acetate (pH 4.5) at 25 °C. The substrate (150 nM Rhoda-
mine-EVNLDAEFK-quencher) was then added to start the reaction
and incubated for 60 min at the same temperature. The fluores-
cence signal was recorded at kem = 585 nm (kexc = 535 nm) by
microplate reader (Infinite F200 PRO, Tecan) using black microwell
(96 wells). The inhibition percentages corresponding to the pres-
ence of different concentrations of test compound was calculated
by the following equation: 100 ꢀ [(Fi/Fo) ꢂ 100], where Fi nd Fo
are the fluorescence intensities obtained for b-secretase in the
presence and absence of an inhibitor, respectively. The inhibition
curve was obtained by plotting the inhibition percentage versus
the logarithm of the inhibitor concentration in the reaction system.
The linear regression parameters were determined and the IC50
was extrapolated with Origin ver. 8.0 software.
4.9. AChE and BChE activity assay
Our assays on the in vitro inhibition of AChE and BChE were
measured by a modification of methods previously reported in
the literature.47,48 Recombinant human AChE and BChE obtained
from Sigma–Aldrich Company were dissolved in 0.1 M potassium
phosphate, pH 7.0. Acetylthiocholine (ATC) and 50,50-dithio-bis
(2-nitrobenzoic acid) (DTNB) purchased from Sigma–Aldrich Com-
pany were prepared in water as stock solutions of 0.01 M and fro-
zen at ꢀ20 °C. The reaction mixture in a total assay volume of
4.12. Molecular docking and solvated interaction energies (SIE)
calculation
Based on the crystal structure, the AutoDock 4.0 program was
applied to dock these inhibitors into the active site of human AChE,
and the Gasteiger charges were used for these inhibitors. To select
the best set of docking parameters and test the reliability of the
docking results, the title compounds were docked into the active
site. In the docking process, the Lamarckian genetic algorithm
(LGA)52 was used for the conformational search. Among a series
200 lL contained appropriate amounts of ATC, DTNB, 0.1 M potas-
sium phosphate buffer, inhibitor and enzyme. Enzymatic hydroly-
sis of ATC was monitored (microplate reader, BioTek) at 415 nm in
the presence or absence of various concentrations of inhibitor at

D.-Y. Peng et al. / Bioorg. Med. Chem. 20 (2012) 6739–6750
6749
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the HF/6-31G⁄ level. The energy minimization process was similar
to that used for modeling other systems previously, that is, first fix-
ing the backbone atoms of the protein, in order to relax the side
chains and the docked inhibitor molecule. Energy minimization
was then performed on the whole complex until the convergence
criterion of 0.001 kcal molꢀ1 Åꢀ1 was reached.
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The binding free energy calculations were performed by the SIE
method. Similar to MMPBSA/GBSA, SIE treats the protein–ligand
system in atomistic detail and solvation effects implicitly. The free
energy of binding between ligand and protein is computed by:
D
Gbind
ð
q
; Din;
a
;
c
; CÞ ¼
a
½
D
EVDW
SAð
þ
D
ECoulðDinÞ þ
D
GRF
ð ; DinÞ
q
25. Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini,
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þ
c
D
qÞꢃ þ C
ð1Þ
where
D
EVDW and
D
ECoul are the intermolecular van der Waals and
GRF (q,
Coulomb interaction energies between protein and ligand,
Din) is the difference in the reaction-field energy between the bound
D
and free state of the protein–ligand complex as calculated by solv-
ing the Poisson equation with BRI BEM,53,54 and
DSA(q) is the differ-
ence in molecular surface area between the bound and free state of
the protein. The five parameters in the above equation were fitted
to the absolute free energy of binding of 99 protein–ligand com-
plexes: The linear scaling factor
the AMBER99 force field, the dielectric constant inside the solute
Din, the coefficient for quantifying the free energy associated with
the difference in surface area upon protein–ligand binding, and the
prefactor that implicitly quantifies the loss of entropy upon bind-
ing, also known as entropy-enthalpy compensation, and a constant
C that includes protein-dependent contributions not explicitly
modeled by the SIE methodology, for example, the change in pro-
tein internal energy upon ligand binding. The default values of the
q of the van der Waals radii of
c
a
38. Camps, P.; Formosa, X.; Galdeano, C.; Munoz-Torrero, D.; Ramirez, L.; Gomez,
E.; Isambert, N.; Lavilla, R.; Badia, A.; Clos, M. V.; Bartolini, M.; Mancini, F.;
Andrisano, V.; Arce, M. P.; Rodriguez-Franco, M. I.; Huertas, O.; Dafni, T.; Luque,
F. J. J. Med. Chem. 2009, 52, 5365.
39. Piazzi, L.; Cavalli, A.; Colizzi, F.; Belluti, F.; Bartolini, M.; Mancini, F.; Recanatini,
M.; Andrisano, V.; Rampa, A. Bioorg. Med. Chem. Lett. 2008, 18, 423.
40. Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Melchiorre, C. Mini-Rev.
Med. Chem. 2008, 8, 960.
parameters are:
C = ꢀ2.89 kcal/mol, and
q
= 1.1, Din = 2.25,
= 0.1048.
c
= 0.0129 kcal/(mol Å2),
a
Acknowledgments
41. Cavalli, A.; Bolognesi, M. L.; Capsoni, S.; Andrisano, V.; Bartolini, M.; Margotti,
E.; Cattaneo, A.; Recanatini, M.; Melchiorre, C. Angew. Chem., Int. Ed. 2007, 46,
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The research was supported in part by the National Basic
Research Program of China (No. 2010CB126103), the NSFC (No.
20902034, 20925206 and 21102052) and the National Key
Technologies RD Program (2011BAE06B05).
43. Sankaranarayanan, S.; Price, E. A.; Wu, G.; Crouthamel, M. C.; Shi, X. P.;
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