Design, synthesis and biological evaluation of 4'-aminochalcone-rivastigmine hybrids as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2016.12.013

A series of 4'-aminochalcone-revastigmine hybrids were designed, synthesized and evaluated as multifunctional agents for the treatment of Alzheimer's disease. The results showed that most of these compounds exhibited good multifunctional activities. In particular, compound 6c displayed the best inhibitory potency on acetylcholinesterase (IC₅₀= 4.91 μM), and significant antioxidative activity with a value 2.83-fold of Trolox. The kinetic analysis of AChE inhibition revealed that 6c showed mixed-type inhibition, binding simultaneously to the catalytic active site and peripheral anionic site of AChE. In addition, 6c inhibited self-induced Aβ_1-42aggregation and Cu²⁺-induced Aβ_1-42aggregation by 89.5% and 79.7% at 25 μM respectively, as well as acted as a selective monoamine oxidase B inhibitor (IC₅₀= 0.29 μM) and a selective biometal chelator. Furthermore, 6c could cross the blood-brain barrier in vitro. Based on these results, Compound 6c could be considered as a very promising lead compound for Alzheimer's disease.

Full text of DOI:10.1016/j.bmc.2016.12.013

Accepted Manuscript
Design, synthesis and biological evaluation of 4′-aminochalcone-rivastigmine
hybrids as multifunctional agents for the treatment of Alzheimer's disease
Ganyuan Xiao, Yan Li, Xiaoming Qiang, Rui Xu, Yunxiaozhu Zheng,
Zhongcheng Cao, Li Luo, Xia Yang, Zhipei Sang, Fu Su, Yong Deng
PII:
S0968-0896(16)31071-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.12.013
BMC 13440
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 October 2016
3 December 2016
8 December 2016
Please cite this article as: Xiao, G., Li, Y., Qiang, X., Xu, R., Zheng, Y., Cao, Z., Luo, L., Yang, X., Sang, Z., Su,
F., Deng, Y., Design, synthesis and biological evaluation of 4′-aminochalcone-rivastigmine hybrids as
multifunctional agents for the treatment of Alzheimer's disease, Bioorganic & Medicinal Chemistry (2016), doi:
http://dx.doi.org/10.1016/j.bmc.2016.12.013
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis and biological evaluation of 4′-aminochalcone-rivastigmine
hybrids as multifunctional agents for the treatment of Alzheimer's disease
Ganyuan Xiaoa, Yan Lia, Xiaoming Qianga, Rui Xua, Yunxiaozhu Zhenga, Zhongcheng Caoa, Li Luoa,
Xia Yanga, Zhipei Sangb, Fu Sua,*, Yong Denga,*
aDepartment 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
bCollege of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang,
473061, China
*Corresponding Author.
Phone, +86-28-85501310; E-mail: sufu@scu.edu.cn (Fu Su)
Phone, +86-28-85503790; E-mail: dengyong@scu.edu.cn (Yong Deng)
1

Abstract
A series of 4′-aminochalcone-revastigmine hybrids were designed, synthesized and evaluated as
multifunctional agents for the treatment of Alzheimer’s disease. The results showed that most of
these compounds exhibited good multifunctional activities. In particular, compound 6c displayed the
best inhibitory potency on acetylcholinesterase (IC50 = 4.91 μM), and significant antioxidative
activity with a value 2.83-fold of Trolox. The kinetic analysis of AChE inhibition revealed that 6c
showed mixed-type inhibition, binding simultaneously to the catalytic active site and peripheral
anionic site of AChE. In addition, 6c inhibited self-induced Aβ1-42 aggregation and Cu2+-induced
Aβ1-42 aggregation by 89.5% and 79.7% at 25 μM respectively, as well as acted as a selective
monoamine oxidase B inhibitor (IC50 = 0.29 μM) and a selective biometal chelator. Furthermore, 6c
could cross the blood-brain barrier in vitro. Based on these results, Compound 6c could be
considered as a very promising lead compound for Alzheimer’s disease.
Keywords:
Alzheimer’s disease; Chalcone carbamate derivatives; Multifunctional agents; Acetylcholinesterase
inhibitors; Aβ aggregation inhibitors; Monoamine oxidase B inhibitors.
2

1. Introduction
Alzheimer’s disease (AD), featured with dementia, cognitive dysfunction and memory lapse, is
one of the most prevalent fatal neurodegenerative disorders among the elderly.1 Today, 47 million
people live with dementia worldwide, and this number is projected to increase to more than 131
million by 2050.2 Although the etiology of AD is not fully explicated, multiple factors, involving
deficits of acetylcholine (ACh), β-amyloid (Aβ) deposits, oxidative stress and dyshomeostasis of
biometals, seem to play crucial roles in the progression of the disease.3 Up to now,
acetylcholinesterase inhibitors (AChEIs) are the major and most developed class of drugs approved
for AD therapy, such as donepezil, rivastigime, galanthamine and Huperzine A.4 The cholinergic
hypothesis asserts that AChEIs could increase the levels of ACh in AD patients through the inhibition
of acetylcholinesterase (AChE), and, therefore, relieve some symptoms of AD patients. However,
AChEIs just show modest palliative clinical efficacy without underlying the pathogenic processes or
correcting the progressive neurodegeneration.5
Among the multiple factors of AD, Aβ plays a central role.6 The main isoforms of the Aβ
peptide are Aβ42 and Aβ40. Aβ40 is present in larger amounts in the brain, yet Aβ42 is more neurotoxic
and has a higher tendency to aggregate. A common concept of the amyloid cascade hypothesis is that
the aggregation of Aβ42 is the main initiating event that sets off the pathogenic cascade, ultimately
leading to neuronal loss and dementia.7 Additionally, excessive biometals such as Cu2+, Fe2+, Zn2+
and Al3+ have been found within the Aβ deposits in AD brains, and it is well known that interaction
of Aβ with redox-active metal ions contributes to the production of reactive oxygen species (ROS).8
Oxidative stress is one of the earliest events in the progress of AD, and it is shown to affect every
class of biological macromolecules, including nucleic acids, proteins, lipids, and carbohydrates.
Furthermore, recent studies have indicated that oxidative damage could promote the appearance of
amyloid plaques and neurofibrillar tangles in AD.9 Taken together, successful inhibition of Aβ
aggregation, modulation of biometals in the brain and protection of neuronal cells from oxidative
stress could potentially prevent AD.
Monoamine oxidase (MAO) is the enzyme that catalyzes the oxidative deamination of various
biogenic and xenobiotic amines.10 Studies have demonstrated that high expression levels of MAO-B
in neuronal tissue can increase the level of oxidative free radicals.11 Therefore, MAO inhibitors can
3

be acted as potential anti-AD drug candidates due to their capacity to inhibit oxidative stress.
Especially, selective MAO-B inhibitors, like selegiline and rasagiline, have been demonstrated to
significantly slow the disease progression and improve memory deficits in AD animal models.12
These observations suggest that selective MAO-B inhibitors may be of value for AD therapy.
Due to the multi-pathogenesis of AD, the development of agents that affect two or more
relevant targets has drawn considerable attention for their potential advancements in the treatment of
AD. In recent years, many multifunctional agents have been discovered with multiple potencies,
including cholinesterase (ChE) inhibition, Aβ deposit inhibition, antioxidative and metal chelating
properties.13-15 Natural products are rich and unexplored sources of novel leading compounds with
bioactive properties. One such natural scaffold abundant in edible plants is the chalcone
(1,3-diphenyl-2-propen-1-ones), which possesses a broad range of pharmacological activities related
to neurological disorders, such as anti-inflammatory, anti-oxidant, neuroprotective properties.15
Recently, many studies demonstrated that natural chalcone and synthetic analogs exhibited ChE and
MAO inhibitory activities, and they are more commonly used as Aβ imaging agents.16-18 These
results indicate that chalcone may be used as a leading compound in the design of multifunctional
drugs for the treatment of AD. Moreover, Masahiro Ono et al. reported that 18F-labeled chalcone with
a dimethylamino group at 4′-position (Figure 1, 1) showed a sufficient brain uptake with a good
binding capacity to Aβ plaques (Ki = 39.8 nM).19 Rivastigmine (Figure 1, 2), a carbamate compound,
is approved by the FDA as a ChE inhibitor for the treatment of AD. The carbamate moiety in
rivastigmine is the ChE inhibitory pharmacophore, which can covalently bind to the enzyme and
remain attached until it is slowly removed by hydrolysis.20,21 Therefore, we planned to use the
multitarget-directed ligand strategy to combine chalcone and rivastigmine, creating a series of
chalcone-rivastigmine hybrids (Figure 1), in the hope of acquiring novel molecules possessing more
potencies in various multifunctional activities.
In this study, a series of chalcone-revastigmine hybrids were designed, synthesized and
evaluated for their biological activity, including inhibition of cholinesterase, antioxidant and metal
chelating effects, inhibitory effects on Aβ aggregation and MAO and the ability to cross the
blood–brain barrier (BBB). The design strategy for chalcone-revastigmine hybrids is depicted in
Figure 1.
4

Figure 1. Design strategy for chalcone-revastigmine hybrids
2. Results and discussion
2.1. Chemistry
The synthetic routes for the preparation of 4′-aminochalcone-revastigmine hybrids were
depicted in Scheme 1. The chalcone scaffolds (3a-e) were the key intermediates, derived via the
aldol condensation of 2,4-dihydroxyacetophenone (2) with p-aminobenzaldehydes (1a-e) in the
anhydrous K2CO3 and DMF at 100 °C.22 N,N-disubstituted carbamoyl chlorides (5a-i) were prepared
by reaction of corresponding secondary amines (4a-i) with triphosgene in the presence of NaHCO3 in
anhydrous DCM.23 Subsequently, O-carbamoylation of the compounds 3a-e was accomplished by
the treatment with 5a-i in the presence of anhydrous K2CO3 and a catalytic amount of
4-dimethylaminopyridine (4-DMAP) in CH3CN at 40-45 °C to afford the desired target compounds
(6a-i, 7a, 7c-e, 8b-e) in moderate yields. These target compounds have not previously been reported
in the literature. Their structures were confirmed by 1H NMR, 13C NMR and ESI-MS.
5

Scheme 1. Synthesis of chalcone-revastigmine hybrids 6-8. Reagents and conditions: (i) K2CO3,
DMF, 100 °C, 10-12 h; (ii) triphosgene, NaHCO3, CH2Cl2, -10~0 °C, 6-8 h; (iii) R3R4NCOCl (5a-i),
4-DMAP, K2CO3,CH3CN, 40-45 °C, 5-10 h.
2.2. Pharmacology
2.2.1. AChE and BuChE Inhibition
The inhibitory activities of target compounds against AChE (from electric eel) and
butyrylcholinesterase (BuChE) (from rat serum) were measured according to the spectrophotometric
method of Ellman et al.24 The inhibitory activities of the target compounds were compared with the
parent compound 3a and the commercially available rivastigmine. The IC50 values for AChE and
BuChE inhibition were summarized in Table 1. These results revealed that the introduction of
carbamates increased the AChE inhibitory capacity, which was consistent with our design strategy.
Most of the target compounds displayed moderate AChE inhibitory activities with IC50 values in the
micromolar level, and demonstrated scarcely any BuChE inhibitory activities. The scarcity of BuChE
inhibitory activities may be a shortcoming, for BuChE plays a role in regulating the ACh level in the
process of AD.25 However, selective inhibition of AChE over BChE may include fewer side effects
associated with peripheral inhibition of cholinesterase, as BuChE is mainly localized in the
peripheral tissues.26 Initially, the hybrids were limited to one head group of dimethylamine at
4′-position and different carbamate tail groups at 4-position, to explore structure-activity relationship
(SAR). Compounds with cyclic amine groups (6c-6e) at 4-position showed better inhibitory activity
than those with non-cyclic amine groups (6a, 6b, 6f). Noticeably, compound 6c was the most potent
inhibitor with the IC50 value of 4.91 ± 0.15 μM, being 2-fold more potent than that of rivastigmine
6

(IC50 = 9.94 ± 0.83 μM). Meanwhile, compounds containing piperazine ring (6g, 6h) at 4-position
performed comparatively better, while the compound possessing the benzylpiperazine (6i) showed
relatively reduced AChE inhibitory activity. In addition, we decided to replace the dimethylamine
moiety with several aminoalkyl groups at 4′-position, remaining the optimal side chains pyrrolidine
carbamate fixed. Unfortunately, all of them (8b-8e) showed weaker inhibitory activity than 6c.
Compound 8e, containing a hydrophilic morpholine ring, showed significant reduced AChE
inhibitory activity, which implied that the hydrophobic interactions in this binding region of the
enzymes might be required for intensive inhibition. The results suggested electron-withdrawing
effects of oxygen atom might reduce the electronic density of the terminal nitrogen, thereby
impacting on protonation at physiological pH, which could diminish the cation–π interaction
between the terminal nitrogen and the catalytic active site (CAS) of AChE.27 Furthermore, we found
that introduction of carbamates simultaneously into 2- and 4-position of the chalcone structure
resulted in weak AChE inhibitors (7a, 7c-7e), which may result from a change in the overall shape of
the molecules, thereby making it difficult for them to align correctly in the gorge leading to the active
site.
Table 1. AChE and BuChE inhibitory activities, and oxygen radical absorbance capacity (ORAC,
Trolox equivalents) of chalcone-revastigmine hybrids and reference compound.
IC50 ± SDa (μM)
Compd.
3a
NR1R2
NR3R4
—
ORACd
2.00 ± 0.03
2.62 ± 0.05
EeAChEb
RatBuChEc
78.21 ± 2.04
n.a.e
32.62 ± 1.78
17.21 ± 1.21
n.a.
n.a.
6a
2.20 ± 0.02
6b
4.91 ± 0.15
6.90 ± 0.53
n.a.
n.a.
2.83 ± 0.05
2.51 ± 0.05
6c
6d
8.01 ± 0.71
n.a.
2.04 ± 0.04
6e
7

14.33 ± 0.80
11.94 ± 0.79
n.a.
n.a.
3.14 ± 0.05
2.08 ± 0.04
6f
6g
8.62 ± 0.81
21.43 ± 1.63
> 100
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
2.54 ± 0.02
3.51 ± 0.09
2.16 ± 0.04
2.45 ± 0.05
2.41 ± 0.03
1.85 ± 0.04
6h
6i
7a
7c
7d
7e
30.02 ± 2.29
32.44 ± 2.02
58.01 ± 2.94
n.a.
1.02 ± 0.02
8b
11.74 ± 0.92
n.a.
0.35 ± 0.01
1.20 ± 0.02
0.51 ± 0.01
NTf
8c
14.06 ± 1.10
10.62 ± 1.33
63.50 ± 1.93
9.94 ± 0.83
n.a.
8d
n.a.
8e
2.86 ± 0.22
Rivastigmine
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 intriplicate (SD = standard deviation).
b AChE from electric eel (EeAChE).
c BuChE from rat serum and tested compounds were used at 50 μM.
d The mean SD of the 3 independent experiments. Data are expressed as μM of Trolox equivalent/μM of tested
compound.
e n.a. = no active. Compounds defined “no active” means a percent inhibition of less than 10.0% at a
concentration of 50 μM in the assay conditions.
f NT = not tested.
2.2.2. Kinetic characterization of AChE inhibition
To gain further insight into the mechanism for this class of derivatives on AChE, a kinetics
study was carried out with compound 6c using EeAChE.28,29 Graphical analysis of the reciprocal
8

Lineweaver-Burk plots (Figure 2) showed both increasing slopes (decreased Vmax) and intercepts
(higher Km) at increasing concentration of the inhibitor. The intersection point of the
Lineweaver−Burk reciprocal plot fell in the second quadrant, which was indicative of mixed-type
inhibition. The result revealed that compound 6c was able to simultaneously bind to both CAS and
peripheral anionic site (PAS) of AChE.
Figure 2. Kinetic study on the mechanism of EeAChE inhibition by 6c. Overlaid Lineweaver–Burk
reciprocal plots of AChE initial velocity at increasing substrate concentrations (0.1–0.4 mM) in the
absence of inhibitor and in the presence of 6c are shown. Lines were derived from a weighted
least-squares analysis of data points.
2.2.3. Antioxidant activity
The antioxidant activities of the target compounds were measured by the oxygen radical
absorbance capacity assay using fluorescein (ORAC-FL).30 Trolox, a water-soluble vitamin E analog,
was used as a reference standard. The activities of tested compounds to scavenge radicals were
expressed as Trolox equivalent, and their relative activities at concentration of 5 μM were compared
with the highly potent compound Trolox. As shown in Table 1, all the compounds displayed
moderate to excellent antioxidant activity with ORAC-FL values in the range of 0.51–3.51 Trolox
equivalents. In particular, compounds 6c, 6f and 6i exhibited the most potent antioxidant activities
with ORAC-FL values of 2.83 ± 0.05, 3.14 ± 0.05 and 3.51 ± 0.09 Trolox equivalents respectively.
Generally, the 2- and 4-disubstituted homologues (7a, 7c-7e) were a little weaker than the
corresponding 2-unsubstituted homologues (6a, 6c-6e) in terms of antioxidant activity, which was
consistent with our previous research.31 The 2-OH of derivatives, which can form intramolecular
9

hydrogen, was beneficial to improve the radical scavenging ability to some degree.32,33 Furthermore,
all the 4′-dimethylamine chalcone-rivastigmine hybrids (6a-6i) showed decent antioxidant activity
and replacement of the 4′-dimethylamine with other aminoalkyl groups (8b-8e) presented a
considerable loss of antioxidant activity. From these results, it may be concluded that dimethylamine
substitution at 4′-position of chalcone is favorable for antioxidant potency.
2.2.4. Metal-chelating properties
Study revealed that the fragment of 2-hydroxy and ketone group in chalcone, which can form
intramolecular hydrogen, is responsible for the metal-chelating properties.34 To investigate the
metal-chelating properties of the series of hybrids, we selected 6c as a typical example. The
metal-chelating ability of 6c was investigated using UV-vis spectrometry.35 The results were shown
in Figure 3A. After CuCl2 was added to a solution of 6c, the maximum absorption wavelength
shifted from 441 nm to 482 nm, indicating the formation of 6c-Cu2+ complexes. Similar result was
obtained when AlCl3 was added: the peak in the electronic spectra of 6c at 441 nm shifted to 522 nm.
However, there was little change on the UV spectrum after adding FeSO4 and ZnCl2. Therefore, 6c
displayed selective metal chelation. The stoichiometry of the 6c-Cu2+ complex was determined using
the molar ratio method by preparing solutions of compound 6c with increasing amounts of CuCl2.
The UV spectra was used to obtain the absorbance of the complex of 6c and CuCl2 at different
concentrations at 474 nm. As shown in Figure 3B, the absorbance initially increased linearly and
then became stable. The two straight lines intersected at a mole fraction of 0.98, revealing a 1:1
stoichiometry for the complex.
Figure 3. A) UV spectra of compound 6c (37.5 μM in methanol) alone or in the presence of CuCl2,
ZnCl2, FeSO4 or AlCl3 (37.5 μM, in methanol). B) Determination of the stoichiometry of
10

complex-Cu2+ by using the molar ratio method of titrating the methanol solution of compound 6c
with ascending amounts of CuCl2. The final concentration of tested compound was 37.5 μM, and the
final concentration of Cu2+ ranged from 3.75 to 150 μM.
2.2.5. Effects on the Aβ aggregation
To investigate the activity of our synthetic compounds to inhibit the self- and Cu2+-induced
Aβ1−42 aggregation, the Thioflavin T (ThT) fluorescence assay was performed, with curcumin as the
reference compound.27,35,36 As shown in Table 2, 4-dimethylamino chalcone scaffold (3a) exhibited a
significant ability to inhibit Aβ1-42 aggregation and the introduction of various carbamate fragments
could further raise the value of inhibition ratio. These derivatives showed moderate to decent
inhibitory potencies of self- and Cu2+-induced Aβ1-42 aggregation (33.3-98.0% and 23.8-89.4% at 25
μM, respectively), compared with that of curcumin (41.3 ± 0.9% and 67.2 ± 1.3% at 25 μM,
respectively). The compounds with the most potent inhibition effects on self-induced Aβ1-42
aggregation were 6a, 6f, 6i, with respective inhibition ratio of 90.8 ± 1.8%, 98.0 ± 1.9% and 89.4 ±
1.4%, while the optimal Cu2+-induced Aβ1-42 aggregation inhibition properties were provided by 6f
(88.0 ± 2.4%), 6h (89.2 ± 1.9%) and 6i (94.2 ± 1.6%), respectively. Unlike the trend of AChE
inhibition, the different kinds of carbamate moieties had little effects on the Aβ1-42 aggregation
inhibitory activity, for the inhibition ratio of 6a-6i showed unconspicuous diversities. Noticeably, the
potencies of 4-O-modified derivatives (6a-6i) were much higher than those of 2, 4-O-bis-modified
derivatives (7a, 7c-7e). It revealed that exposure of hydroxy group at 2-position, which can form
hydrogen bond, is crucial for enhancing inhibition effect. The potencies of 8b-8e were relatively
weaker than that of 6c, indicating that 4′-dimethylamino group may intensify the interaction between
derivatives and Aβ1-42 protein.
Table 2 Inhibition of self- and Cu2+-induced Aβ1-42 aggregation, recombinant human MAO-A and -B
of chalcone-revastigmine hybrids and reference compounds.
%Inhibition of Aβ1-42 aggregationa
IC50 ± SD (μM)
Compd.
SIe
Self-inducedb, f
77.7 ± 1.6
90.8 ± 1.8
76.7 ± 2.0
89.5 ± 1.2
Cu2+-inducedc, f
MAO-Ad, f
MAO-Bd, f
0.31 ± 0.031
2.48 ± 0.045
3.50 ± 0.103
0.29 ± 0.021
60.1 ± 2.1
4.06 ± 0.084
10.20 ± 0.094
24.4 ± 1.4%
12.8 ± 1.1%
13.36
4.11
3a
6a
6b
6c
77.9 ± 1.9
49.0 ± 1.1
>0.87
>34.97
79.7 ± 1.9
11

70.2 ± 1.1
83.6 ± 1.4
98.0 ± 1.9
87.9 ± 1.8
83.2 ± 1.2
89.4 ± 1.4
34.7 ± 1.5
48.0 ± 0.8
38.0 ± 0.7
33.3 ± 1.2
67.8 ± 1.0
44.4 ± 1.8
57.2 ± 1.9
44.4 ± 0.8
41.3 ± 0.9
—
73.9 ± 1.7
79.7 ± 1.3
88.0 ± 2.4
75.5 ± 2.3
89.2 ± 1.9
94.2 ± 1.6
56.2 ± 1.1
26.3 ± 0.8
23.8 ± 0.6
41.4 ± 1.1
67.1 ± 1.2
48.9 ± 1.7
59.8 ± 2.1
48.9 ± 1.7
67.2 ± 1.3
—
27.8 ± 1.6%
34.6 ± 2.1%
25.9 ± 2.0%
49.5 ± 2.1%
42.5 ± 2.2%
42.1 ± 2.1%
34.6 ± 1.9%
25.2 ± 1.0%
17.0 ± 0.9%
15.0 ± 0.6%
21.0 ± 1.1%
15.2 ± 1.3%
19.6 ± 1.2%
15.2 ± 1.1%
—
31.1% ± 1.4%
42.3% ± 1.8%
2.10 ± 0.041
25.1% ± 1.2%
30.0% ± 2.1%
41.6% ± 2.7%
5.1% ± 1.1%
34.6% ± 2.1%
30.1% ± 1.9%
40.2% ± 1.7%
6.70 ± 0.201
0.71 ± 0.024
0.78 ± 0.032
0.71 ± 0.033
—
—
6d
—
6e
>4.76
—
6f
6g
—
6h
—
6i
—
7a
—
7c
7d
—
—
7e
>0.59
>14.07
>12.82
>14.07
—
8b
8c
8d
8e
Curcumin
Clorgyline
Rasagiline
Iproniazid
0.0027 ± 0.0006 4.19 ± 0.101
0.00064
17.21
1.31
—
—
1.42 ± 0.015
2.56 ± 0.023
0.083 ± 0.002
1.95 ± 0.074
—
—
a For inhibition of Aβ aggregation, the thioflavin-T fluorescence method was used.
b Inhibition of self-induced Aβ1-42 aggregation (25 μM) by tested inhibitors at 25 μM.
c Inhibition of Cu2+-induced Aβ1-42 aggregation. The concentration of tested compounds and Cu2+ were 25 μM.
d Percentages are the percent inhibition of MAO by tested compounds at 10 μM.
e SI = IC50 (MAO-A) / IC50 (MAO-B)
f The mean ± SD of the three independent experiments.
2.2.6. Recombinant human MAO-A and -B inhibition studies
To complete the study of the multi-target biological profile of the hybrid compounds, the
inhibitory activities against recombinant human MAO-A and -B were determined, with the
established MAOIs, clorgyline, rasagiline, and iproniazid, applied for comparative purposes.37-39 It
could be seen from the Table 2 that the parent compound 3a exhibited potent MAO-A and -B
inhibitory activity (IC50 = 4.06 ± 0.084 μM for MAO-A; IC50 = 0.31 ± 0.031 μM for MAO-B), and
the introduction of various carbamate moieties resulted in a dramatic drop of MAO-A inhibition. It
was apparent that most of the 4-O-modified hybrids could effectively inhibit MAO-B with IC50
ranging from micromolar to sub-micromolar, while no compound showed significantly inhibitory
activity for MAO-A, indicating that most of the hybrids are clearly selective MAO-B inhibitors.
Compounds 6a, 6b, 6f, bearing carbamates of non-cyclic amine groups at 4-O position, inhibited
MAO-B relatively potently and also showed moderate selectivity for MAO-B. Compounds
12

containing cyclic amine groups (6d, 6e, 6g-6i), demonstrated quite lower MAO-B inhibition
activities, with the exception of 6c, which was found to be the most potent MAO-B inhibitor (IC50 =
0.29 ± 0.021 μM). Compound 6c was also endowed with the highest selectivity towards MAO-B of
all, being more than 26.7 times and 2.0 times higher than that of Iproniazid and Rasagiline
respectively. Transforming dimethylamino at 4′-position to other aminoalkyl groups to yield 8b-8e
could lead to a slight loss of MAO-B inhibition potency compared with 6c, in line with the above
outcome of Aβ1-42 aggregation assays. The low potency of 7a, 7c-e suggested that carbamate group at
2-position was unprofitable.
2.2.7. Molecular docking study
To explore a possible interacting mode of the chalcone carbamate derivatives with TcAChE
(PDB code: 1EVE), a molecular modeling study was performed using the docking program,
AutoDock 4.2 package.9 The result was shown in Figure 4. The docking study showed that
compound 6c occupied the entire enzymatic CAS, the mid-gorge sites and the PAS, and could bind
simultaneously to both the catalytic and peripheral site. In the TcAChE-6c complex, the B ring of the
chalcone moiety in 6c bond to the PAS via the π-π interactions with Phe288, Phe331 and Phe290,
and also engaged in hydrophobic interactions with residues Ile287, Arg289, Leu282. In addition, the
α, β-unsaturated ketone in 6c folded in a conformation in the gorge that allowed it to interact with
Tyr334 via the hydrophobic interaction. The A ring of chalcone moiety could bind to the middle
gorge by establishing a π-π stacking interaction with the Phe330, and the 2-hydroxy at the chalcone
nucleus could bind to the Tyr121 via the intermolecular hydrogen bond. The substituted carbamate
fragment of 6c was observed to bind to the CAS via potential hydrophobic interactions with residues
Acn85, Asp72 and Gln69. The simultaneous binding of 6c with CAS, PAS and the active gorge of
TcAChE provides an explanation for its highly potent inhibitory activity for AChE and thus revealed
a mixed-type inhibition for this compound.
13

Figure 4. Hypothetical binding mode of compound 6c interacting with residues in the binding site of
TcAChE (1EVE). The protein residues that participate in the main interactions with the inhibitor are
labeled.
Furthermore, in order to evaluate the binding modes of this class of chalcone derivatives with
respect to human MAO-B, docking simulation was carried out using the docking program, AutoDock
4.2 package based on the X-ray crystal structures of human MAO-B (PDB code: 2V60).35 On the
basis of the in vitro inhibition results, we selected compound 6c, the most potent MAO-B inhibitor,
as a typical ligand for the evaluation. As shown in Figures 5 and 6, the dimethylamine group of
chalcone moiety was properly oriented to the flavin adenine dinucleotide (FAD) cofactor, adopting
parallel π-π interactions with Tyr398 and Tyr435. Furthermore, the B-ring of 6c adopted parallel π-π
interactions with Tyr326. The hydroxyl group at 2-position and carbonyl group at 1-position
interacted with Ile199 and Phe168 via two hydrogen bonds (Ile199=O • • • HO-6c; Phe168-OH • • •
O=6c). Besides, the carbamate group embedded into a hydrophobic pocket delimited by many
hydrophilic acids, such as Pro102, Leu88, Thr312, Thr201, Ile199, Tyr326.
14

Figure 5. Predicted positions of 6c into hMAO-B catalytic sites. Compound 6c and the FAD cofactor
were depicted using stick and space fill, respectively.
Figure 6. Representation of compound 6c docked into the binding site of hMAO-B, highlighting the
protein residues that form the main interactions with the inhibitor. Hydrogen-bonding interactions
between ligand and residues are shown with the green line.
2.2.8. In vitro blood-brain barrier permeation assay
Brain penetration is a major requirement for successful central nervous system (CNS) drugs. To
evaluate the brain penetration of 6c, we used a parallel artificial membrane permeation assay for the
blood-brain barrier (PAMPA-BBB), as recently described by Di et al.35,40,41 We compared the
15

permeability of 11 commercial drugs with reported values to validate the assay (Table 3). A plot of
the experimental data versus the reported values produced a good linear correlation, Pe (exp.) =
0.8792 × Pe (bibl.) − 0.0616 (R2 = 0.9555) (Figure 7). From this equation and considering the limit
established by Di et al. for blood−brain barrier permeation, we determined that compounds with
permeabilities above 3.46 × 10-6 cm/s could cross the blood−brain barrier (Table 4).41 Then, the
selected compound 6c was then tested in the PAMPA-BBB assay. The results presented in Table 5
showed that 6c could penetrate into the CNS and reach their biological targets located in the CNS.
Table 3. Permeability Pe (× 10-6 cm/s) in the PAMPA-BBB assay for 11 commercial drugs used in
the experiment validation.
Commercial drugs
Bibla
PBS/EtOH (70:30)b
Verapamil
Oxazepam
Diazepam
Clonidine
16
16.20 ± 0.20
9.20 ± 0.16
10.90 ± 0.32
5.50 ± 0.18
11.20 ± 0.29
15.50 ± 0.30
1.22 ± 0.04
0.55 ± 0.03
0.84 ± 0.02
0.33 ± 0.01
0.17 ± 0.005
10
16
5.3
13
Imipramine
Testosterone
Caffeine
17
1.3
0.9
2.5
0.1
0.12
Enoxacine
Piroxicam
Norfloxacin
Theophylline
a Taken from Ref.37
b Data are the mean ± SD of three independent experiments.
20
18
16
y = 0.8792x - 0.0616
14
12
10
8
R² = 0.9555
6
4
2
0
0
5
10
15
20
25
DescribedP e ( 10-6 cm/s)
×
Figure 7. Lineal correlation between experimental and reported permeability of commercial drugs
using the PAMPA-BBB assay. Pe (exp) = 0.8792 × Pe (bibl.) − 0.0616 (R2 = 0.9555).
16

Table 4. Ranges of permeability of PAMPA-BBB assays (Pe × 10-6 cm/s)
High BBB permeation predicted (CNS +)
Uncertain BBB permeation (CNS +/-)
Low BBB permeation predicted (CNS -)
Pe > 3.46
3.46 > Pe > 1.70
Pe < 1.70
Table 5. Permeability results Pe (× 10-6 cm/s) from the PAMPA-BBB assay for selected aurone
derivatives with their predicted penetration into the CNS.
Compd.a
Pe (× 10-6 cm/s)b
Prediction
CNS +
7.03 ± 0.19
6c
a
Compound 6c were dissolved in DMSO at 5 mg/mL and diluted with PBS/EtOH (70:30). The final
concentration of each compound was 100 μg/mL.
b Data are the mean ± SD of three independent experiments.
3. Conclusion
In conclusion, a series of 4′-aminochalcone-rivastigmine hybrids were designed, synthesized
and evaluated as multifunctional agents for the treatment of AD. Most of the synthesized compounds
showed potent AChE inhibitory activity and decent inhibitory activities of self- and Cu2+-induced
Aβ1–42 aggregation in vitro. Compound 6c demonstrated the best inhibitory activity toward AChE
with IC50 value of 4.91 ± 0.15 μM. The inhibition kinetic analysis suggested that compound 6c
showed a mixed-type inhibition, binding to both CAS and PAS of AChE. Compound 6c also
possessed excellent antioxidant activity (2.83 ± 0.05 Trolox equivalents), excellent inhibitory effects
on self-induced Aβ1-42 aggregation (89.5 ± 1.2%) and Cu2+-induced Aβ1-42 aggregation (79.7 ± 1.9%).
With regard to inhibition of MAO, most of target components displayed selective MAO-B inhibitory
activity, among which 6c presented decent IC50 values of 0.29 ± 0.021 μM towards MAO-B
inhibition. Moreover, 6c also possessed the prospective property of acting as a metal chelator and
permeating BBB. These properties highlighted that the compound 6c could be considered as a very
promising lead compound for Alzheimer’s disease.
4. Experimental section
4.1. Chemistry
17

1
Melting points were gauged on YRT-3 melting-point apparatus (China) without correction. H
NMR and 13C NMR spectra were recorded at room temperature in CDCl3 solutions with a Varian
INOVA spectrometer. Chemical shifts were expressed in parts per millions (ppm) relative to
tetramethylsilane (TMS). Mass spectra were recorded on Agilent-6210 TOF LC-MS Spectrometer.
The purity of all final compounds was determined by high-performance liquid chromatography
(HPLC) analysis to be over 97%. HPLC analysis was carried out on a Shimadzu LC-10Avp plus
system with the use of a Diamonsil C18 column (4.6 mm × 150 mm, 5 μM), the compounds eluted
with acetonitrile/water/glacial acetic acid, 40:60:1, at a flow rate of 1.0 mL/min). All reagents, unless
otherwise specified, were used as high grade commercial products and solvents were of analytical
grade. Reaction progress was 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). Crude products were purified by column chromatography using silica
gel (230-400 mesh) from Qingdao Haiyang Chemical Co. Ltd. (China).
4.1.1. General procedure for the synthesis of chalcones (3a-e)
Substituted benzaldehyde 1a-e (15 mol) was added portionwise to a solution of 2,4-dihydroxy
acetophenone 2 (1.5 g, 10 mol) and anhydrous K2CO3 (2.76 g, 20 mol) in DMF (15 mL), and the
mixture was refluxed for 10-12 h, cooled to room temperature, diluted with EtOAc, and basified with
10% NaOH to pH 9.0. The aqueous phase was separated and neutralized with glacial acetic acid to
pH 5.0. The precipitated solid was filtered, washed with cold water, dried in vacuum to give
compounds 3a-e as yellow solids, which were used without further purification.
4.1.2. General procedure for the synthesis of N,N-disubstituted carbamoyl chlorides (5a-i)
Compounds 5a-i were prepared as previously described.23
4.1.3. General procedure for the synthesis of 4′-amino chalcone-revastigmine hybrids (6a-i, 7a,
7c-e and 8b-e)
N,N-Disubstituted carbamoyl chlorides 5a-i (1.59 mmol) was added dropwise to a mixture of
anhydrous K2CO3 (124.52 mg, 0.90 mmol), 4-DMAP (5.0 mg, 0.039 mmol) and the appropriate
intermediates 3a-e (0.53 mmol) in 2 ml MeCN. The reaction mixture was warmed to 40-45 °C and
stirred for 5–10 h under an argon atmosphere to yield carbamylchalcone and 2,4-biscarbamyl
chalcone simultaneously. After the reaction was completed, the mixture was concentrated, diluted
18

with water (20 mL), and extracted with CH2Cl2 (20 mL × 3). The combined organic phases were
washed sequentially with saturated aqueous Na2CO3 (5 mL × 3), saturated aqueous NaCl (10 mL),
dried over anhydrous Na2SO4, and evaporated to dryness under reduced pressure. The crude product
was separated and purified by silica gel flash column chromatography using mixtures of petroleum
ether/acetone as eluent to obtain corresponding target compounds 6a-i, 7a, 7c-e, and 8b-e. The solid
of carbamylchalcone (6a-i, 8b-e) was further recrystallized with EtOH and dried in vacuo to
guarantee crystals of sufficient purity.
4.1.3.1. (E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-3-hydroxyphenyl dimethylcarbamate (6a)
Compound 3a and 5a were processed as above to afford product 6a. Elution with petroleum
ether/acetone = 3:1 and further recrystallization with EtOH gave 6a as a yellow solid, 98.2% HPLC
1
purity, mp 201.2-203.5 °C, yield 45.2%. H NMR (600 MHz, CDCl3) δ 13.43 (s, 1H), 7.91 (d, J =
9.6 Hz, 1H), 7.90 (d, J = 13.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 13.8 Hz, 1H), 6.76 (s,
1H), 6.75 (d, J = 9.6 Hz, 1H), 6.70 (d, J = 8.4 Hz, 2H), 3.10 (s, 3H ), 3.06 (s, 6H), 3.03 (s, 3H). MS
m/z: 355.2 [M+H]+.
4.1.3.2. (E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-1,3-phenylene bis(dimethylcarbamate)
(7a)
Compound 3a and 5a were processed as above to afford product 7a. Elution with petroleum
1
ether/acetone = 3:1 gave 7a as a yellow solid, 99.2% HPLC purity, mp 79.1-80.2 °C, yield 40.3%. H
NMR (400MHz, CDCl3) δ 7.63 (d, J = 9.2 Hz, 1H), 7.53 (d, J = 15.6 Hz, 1H), 7.45 (d, J = 8.8 Hz,
2H), 7.09 (s, 1H), 7.08 (d, J = 9.2 Hz , 1H), 6.93 (d, J = 15.6 Hz, 1H), 6.66 (d, J = 8.8 Hz, 2H), 3.09
(s, 3H), 3.03 (s, 6H), 3.02 (s, 3H), 2.96 (s, 3H), 2.90 (s, 3H). MS m/z: 426.2 [M+H]+.
4.1.3.3. (E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-3-hydroxyphenyl diethylcarbamate (6b)
Compound 3a and 5b were processed as above to afford product 6b. Elution with petroleum
ether/acetone = 2:1 and further recrystallization with EtOH gave 6b as a yellow solid, 98.0% HPLC
1
purity, mp 145.7-148.0 °C, yield 47.2%. H NMR (600 MHz, CDCl3) δ 13.42 (s, 1H), 7.91 (d, J =
15.6 Hz, 1H ),7.90 (d, J = 8.4 Hz, 1H ), 7.57 (d, J = 8.4 Hz, 2H), 7.38 (d, J= 15.6 Hz, 1H), 6.77 (s,
1H), 6.76 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 8.4 Hz, 2H), 3.44-3.81 (m, 4H), 3.06 (s, 6H), 1.24 (t, J =
6.6 Hz, 3H), 1.22 (t, J = 6.6 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 192.5, 165.0, 157.2, 153.1,
152.3, 146.4, 130.8 (2C), 130.3, 122.3, 117.6, 114.3, 112.5, 111.7(2C), 110.7, 42.3, 42.0, 40.1 (2C),
19

14.2, 13.3. MS m/z: 383.2 [M+H]+.
4.1.3.4. (E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-3-hydroxyphenyl pyrrolidine-1-
carboxylate (6c)
Compound 3a and 5c were processed as above to afford product 6c. Elution with petroleum
ether/acetone = 2:1 and further recrystallization with EtOH gave 6c as a yellow solid, 97.9% HPLC
1
purity, mp 168.5-170.1 °C, yield 42.5%. H NMR (600 MHz, CDCl3) δ 13.42 (s, 1H), 7.90 (d, J =
15.6 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 15.6 Hz, 1H), 6.79 (s,
1H), 6.78 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 8.4 Hz, 2H), 3.56 (t, J = 6.6 Hz, 2H), 3.50 (t, J = 6.6 Hz,
2H), 3.06 (s, 6H), 1.99-1.93 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 192.5, 165.1, 157.0, 152.3 (2C),
146.4, 130.8 (2C), 130.3, 122.3, 117.6, 114.3, 112.5, 111.8 (2C), 110.7, 46.5, 46.4, 40.1 (2C), 25.7,
24.9. MS m/z: 381.5 [M+H]+.
4.1.3.5.
(E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-1,3-phenylene
bis(pyrrolidine-1-
carboxylate) (7c)
Compound 3a and 5c were processed as above to afford product 7c. Elution with petroleum
1
ether/acetone = 2:1 gave 7c as a yellow solid, 98.3% HPLC purity, mp 89.6-90.7 °C, yield 41.2%. H
NMR (400MHz, CDCl3) δ 7.63 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 16.0 Hz, 1H), 7.45 (d, J = 8.4 Hz,
2H), 7.15 (s, 1H), 7.11 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 16.0 Hz, 1H), 6.66 (d, J = 8.4 Hz, 2H), 3.55 (t,
J = 5.6 Hz, 2H), 3.49 (t, J = 5.6 Hz, 2H), 3.43 (t, J = 5.6 Hz, 2H), 3.36 (t, J = 5.6 Hz, 2H), 1.99-1.75
13
(m, 8H). C NMR (150 MHz, CDCl3) δ 191.6, 153.6 (2C), 152.3 (2C), 149.7, 145.6, 130.2 (2C),
130.1 (2C), 121.3 (2C), 118.4, 117.0, 112.0 (2C), 46.4 (2C), 46.3 (2C), 40.3 (2C), 25.8, 25.6, 24.9,
24.8. MS m/z: 478.5 [M+H]+.
4.1.3.6. (E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-3-hydroxyphenyl piperidine-1-carboxylate
(6d)
Compound 3a and 5d were processed as above to afford product 6d. Elution with petroleum
ether/acetone = 3:1 and further recrystallization with EtOH gave 6d as a yellow solid, 98.5% HPLC
1
purity, mp 192.4-193.5 °C, yield 41.2%. H NMR (400 MHz, CDCl3) δ 13.42 (s, 1H), 7.91 (d, J =
14.8 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.8 Hz. 2H), 7.39 (d, J = 14.8 Hz, 1H), 6.76 (s,
13
1H), 6.71 (d, J = 8.8 Hz, 3H), 3.60 (s, 2H), 3.52 (s, 2H), 3.07 (s, 6H), 1.65 (s, 6H). C NMR (100
MHz, CDCl3) δ 192.5, 165.0, 157.2, 152.6, 152.3, 146.4, 130.8 (2C), 130.3, 122.3, 117.7, 114.3,
20

112.5, 111.8 (2C), 110.8, 45.7, 45.1, 40.1 (2C), 25.9, 25.5, 24.2. MS m/z: 395.3 [M+H]+.
4.1.3.7. (E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-1,3-phenylene bis(piperidine-1-
carboxylate) (7d)
Compound 3a and 5d were processed as above to afford product 7d. Elution with petroleum
ether/acetone = 3:1 gave 7d as a yellow solid, 97.3% HPLC purity, mp 79.2-80.7 °C, yield 41.9%. 1H
NMR (400 MHz, CDCl3) δ 7.62 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 16.0 Hz, 1H), 7.46 (d, J = 8.4 Hz,
2H), 7.08 (d, J = 8.4 Hz, 1H), 7.07 (s, 1H), 6.90 (d, J = 16.0 Hz, 1H), 6.68 (d, J = 8.4 Hz, 2H), 3.76
(s, 2H), 3.67 (s, 2H), 3.58 (s, 2H), 3.37 (s, 2H), 3.06 (s, 6H), 2.31 (s, 6H), 1.81 (s, 6H). MS m/z:
506.3 [M+H]+.
4.1.3.8.
(E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-3-hydroxyphenyl
morpholine-4-
carboxylate (6e)
Compound 3a and 5e were processed as above to afford product 6e. Elution with petroleum
ether/acetone = 3:1 and further recrystallization with EtOH gave 6e as a yellow solid, 97.9% HPLC
1
purity, mp 182.9-183.4 °C, yield 63.6%. H NMR (600 MHz, CDCl3) δ 13.45 (s, 1H), 7.92 (d, J =
16.2 Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 16.2 Hz, 1H), 6.77 (s,
1H), 6.74 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 8.4 Hz, 2H), 3.76 (s, 4H), 3.67 (s, 2H), 3.59 (s, 2H), 3.067
13
(s, 6H). C NMR (150 MHz, CDCl3) δ 192.5, 165.0, 156.7, 152.6, 152.3, 146.6, 130.9 (2C), 130.4,
122.2, 118.0, 114.2, 112.4, 111.8 (2C), 110.8, 66.6, 66.4, 45.0, 44.1, 40.1 (2C). MS m/z: 397.2
[M+H]+.
4.1.3.9.
(E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-1,3-phenylene
bis(morpholine-4-
carboxylate) (7e)
Compound 3a and 5e were processed as above to afford product 7e. Elution with petroleum
ether/acetone = 3:1 gave 7e as a yellow solid, 98.3% HPLC purity, mp 82.7-84.3 °C, yield 28.3%. 1H
NMR (600 HMz, CDCl3) 7.63 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 15.6 Hz, 1H), 7.45 (d, J = 8.4 Hz, 2H),
7.12 (s, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 15.6 Hz, 1H), 6.66 (d, J = 8.4 Hz, 2H), 3.75 (s,
13
4H), 3.67 (s, 2H), 3.58 (s, 2H), 3.51 (s, 6H), 3.45 (s, 2H), 3.04 (s, 6H). C NMR (100 Hz, CDCl3) δ
191.5, 153.1, 152.7, 152.6, 152.1, 149.4, 146.4, 130.5, 130.4 (2C), 130.3, 121.9, 120.7, 118.7, 117.0,
111.7 (2C), 66.6, 66.4 (2C), 66.3, 44.9 (2C), 44.1 (2C), 40.0 (2C). MS m/z: 510.2 [M+H]+.
4.1.3.10.
(E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-3-hydroxyphenyl
ethyl(methyl)
21

carbamate (6f)
Compound 3a and 5f were processed as above to afford product 6f. Elution with petroleum
ether/acetone = 3:1 and further recrystallization with EtOH gave 6F as a yellow solid, 98.5% HPLC
1
purity, mp 203.5-205.1 °C, yield 55.8%. H NMR (400 MHz, CDCl3) δ 13.44 (s, 1H), 7.91 (d, J =
14.8 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 14.8 Hz, 1H), 6.77 (s,
1H), 6.75 (d, J = 8.8 Hz, 1H), 6.70 (d, J = 8.4 Hz, 2H), 3.49-3.39 (m, 2H), 3.06 (s, 6H), 3.00 (s,
13
1.5H), 2.84 (s, 1.5H), 1.31-1.23 (m, 3H). C NMR (100 Hz, CDCl3) δ 192.6, 165.0, 157.1, 152.3
(2C), 146.4, 130.8 (2C), 130.3, 122.3, 117.7, 114.3, 112.5, 111.8 (2C), 110.8, 44.2 (2C), 40.1 (2C),
13.2. MS m/z: 369.2 [M+H]+.
4.1.3.11. (E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-3-hydroxyphenyl 4-methylpiperazine-1-
carboxylate (6g)
Compound 3a and 5g were processed as above to afford product 6g. Elution with petroleum
ether/acetone = 1:1 and further recrystallization with EtOH gave 6g as a yellow solid, 97.5% HPLC
1
purity, mp 157.5-158.7 °C, yield 59.6%. H NMR (400 MHz, CDCl3) δ 13.45 (s, 1H), 7.92 (d, J =
8.4 Hz, 1H), 7.91 (d, J = 14.4 Hz, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 14.4 Hz, 1H), 6.76 (s,
1H), 6.75 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 8.8 Hz, 2H), 3.72 (s, 2H), 3.63 (s, 2H), 3.07 (s, 6H), 2.51
(s, 4H), 2.38 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 192.5, 165.0, 156.8, 152.5, 152.3, 146.5, 130.9
(2C), 130.4, 122.2, 117.9, 114.2, 112.4, 111.8 (2C), 110.8, 54.6, 54.5, 46.0, 44.4, 43.7, 40.1 (2C). MS
m/z: 410.3 [M+H]+.
4.1.3.12. (E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-3-hydroxyphenyl 4-ethylpiperazine-1-
carboxylate (6h)
Compound 3a and 5h were processed as above to afford product 6h. Elution with petroleum
ether/acetone = 1:1 and further recrystallization with EtOH gave 6h as a yellow solid, 98.2% HPLC
1
purity, mp 189.2-191.3 °C, yield 53.2%. H NMR (400 MHz, CDCl3) δ 13.45 (s, 1H), 7.91 (d, J =
8.8 Hz, 1H), 7.90 (d, J = 15.2 Hz, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 15.2 Hz, 1H), 6.76 (d, J
= 8.8 Hz, 1H), 6.72 (d, J = 8.8 Hz, 2H), 6.69 (s, 1H), 3.69 (s, 2H), 3.61 (s, 2H), 3.06 (s, 6H), 2.52-
13
2.45 (m, 6H), 1.13 (t, J = 7.2 Hz, 3H). C NMR (150 MHz, CDCl3) δ 192.6, 165.0, 156.9, 152.5,
152.3, 146.5, 130.8 (2C), 130.4, 122.3, 117.8, 114.2, 112.4, 111.8 (2C), 110.8, 52.5, 52.3 (2C), 44.6,
44.0, 40.1 (2C), 12.0. MS m/z: 424.3 [M+H]+.
22

4.1.3.13. (E)-4-(3-(4-(dimethylamino)phenyl)acryloyl)-3-hydroxyphenyl 4-benzylpiperazine-1-
carboxylate (6i)
Compound 3a and 5i were processed as above to afford product 6i. Elution with petroleum
ether/acetone = 1:1 and further recrystallization with EtOH gave 6i as a yellow solid, 98.1% HPLC
1
purity, mp 132.1-133.5 °C, yield 52.9%. H NMR (400 MHz, CDCl3) δ 13.44 (s, 1H), 7.91 (d, J =
15.2 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 15.2 Hz, 1H), 7.35-7.28
(m, 5H), 6.76 (s, 1H), 6.74 (d, J = 8.8 Hz, 1H), 6.70 (d, J = 8.8 Hz, 2H), 3.67 (s, 2H), 3.57 (s, 4H),
3.07 (s, 6H), 2.51 (t, J = 6.4 Hz, 4H). 13C NMR (150 MHz, CDCl3) δ 192.5, 165.0, 156.9, 152.5,
152.3, 146.5, 130.8 (2C), 130.4, 129.2 (2C), 128.4 (2C), 127.4, 122.3, 117.8, 114.2, 112.4, 112.3,
111.8 (2C), 110.8, 62.9, 52.7, 52.5, 44.5, 43.9, 40.4 (2C). MS m/z: 486.3 [M+H]+.
4.1.3.14. (E)-4-(3-(4-(diethylamino)phenyl)acryloyl)-3-hydroxyphenyl pyrrolidine-1-
carboxylate (8b)
Compound 3b and 5c were processed as above to afford product 8b. Elution with petroleum
ether/acetone = 2:1 and further recrystallization with EtOH gave 8b as a yellow solid, 97.5% HPLC
1
purity, mp 132.0-133.6 °C, yield 45.2%. H NMR (400MHz, CDCl3) δ 13.50 (s, 1H), 7.90 (d, J = 9.6
Hz, 1H), 7.89 (d, J = 15.2 Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 15.2 Hz, 1H), 6.78 (d, J =
9.6 Hz, 1H), 6.77 (s, 1H), 6.66 (d, J = 8.8 Hz, 2H), 3.56 (t, J = 6.4 Hz, 2H), 3.49 (t, J = 6.4 Hz, 2H),
3.42 (q, J = 7.2 Hz, 4H), 1.99-1.92 (m, 4H), 1.21 (t, J = 7.2 Hz, 6H). 13C NMR (100MHz, CDCl3) δ
192.4, 165.0, 156.9, 151.9, 150.0, 146.4, 131.2, 130.3, 121.4, 117.6, 113.5, 112.4, 111.2 (2C), 110.6,
46.5, 46.4, 44.5 (2C), 25.7, 24.9, 12.5 (2C). MS m/z: 409.2 [M+H]+.
4.1.3.15. (E)-3-hydroxy-4-(3-(4-(pyrrolidin-1-yl)phenyl)acryloyl)phenyl pyrrolidine-1-
carboxylate (8c)
Compound 3c and 5c were processed as above to afford product 8c. Elution with petroleum
ether/acetone = 2:1 and further recrystallization with EtOH gave 8c as a yellow solid, 97.8% HPLC
1
purity, mp 154.8-155.4 °C, yield 56.9%. H NMR (400MHz, CDCl3) δ 13.50 (s, 1H), 7.91 (d, J =
14.4 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 14.4 Hz, 1H), 6.78 (s,
1H), 6.77 (d, J = 8.4 Hz, 1H), 6.56 (d, J = 8.4, 2H), 3.56 (t, J = 6.4, 2H), 3.49 (t, J = 6.4, 2H), 3.37 (t,
J = 6.4, 4H), 2.06-2.03 (m, 4H), 1.99-1.92 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 192.4, 165.0,
23

156.9, 149.9 (2C), 146.7, 131.0 (2C), 130.3, 121.7, 117.7, 113.5, 112.4, 111.7 (2C), 110.6, 47.5 (2C),
46.5, 46.4, 25.7, 25.4 (2C), 24.9. MS m/z: 407.3 [M+H]+.
4.1.3.16. (E)-3-hydroxy-4-(3-(4-(piperidin-1-yl)phenyl)acryloyl)phenyl pyrrolidine-1-
carboxylate (8d)
Compound 3d and 5c were processed as above to afford product 8d. Elution with petroleum
ether/acetone = 2:1 and further recrystallization with EtOH gave 8d as a yellow solid, 98.8% HPLC
1
purity, mp 145.7-148.0 °C, yield 47.2%. H NMR (400MHz, CDCl3) δ 13.26 (s, 1H), 7.80 (d, J = 9.2
Hz, 1H), 7.78 (d, J = 15.2 Hz, 1H), 7.45 (d, J = 9.2 Hz, 2H), 7.31 (d, J = 15.2 Hz, 1H), 6.79 (d, J =
9.2 Hz, 2H), 6.68 (d, J = 9.2 Hz, 1H), 6.67 (s, 1H), 3.46 (t, J = 6.8 Hz, 2H), 3.39 (t, J = 6.8 Hz, 2H),
13
3.24(t, J = 5.6 Hz, 4H), 1.89-1.82 (m, 4H), 1.59-1.56 (m, 6H). C NMR (100 MHz, CDCl3) δ 192.6,
165.0, 157.1, 153.3 (2C), 145.9, 130.7 (2C), 130.4, 123.8, 117.6, 115.2, 114.5, 112.5, 110.7 (2C),
48.8 (2C), 46.5, 46.4, 25.7, 25.4 (2C), 24.9, 24.3. MS m/z: 421.2 [M+H]+.
4.1.3.17. (E)-3-hydroxy-4-(3-(4-morpholinophenyl)acryloyl)phenyl pyrrolidine-1-carboxylate
(8e)
Compound 3e and 5c were processed as above to afford product 8e. Elution with petroleum
ether/acetone = 2:1 and further recrystallization with EtOH gave 8e as a yellow solid, 97.9% HPLC
purity, mp 154.8-155.4 °C, yield 56.9%. 1H NMR (600MHz, CDCl3) δ 13.27 (s, 1H), 7.92 (d, J = 8.4
Hz, 1H), 7.88 (d, J = 16.2 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 16.2 Hz, 1H), 7.37 (d, J =
8.4 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 7.8 Hz, 1H), 6.80 (s, 1H), 6.79 (d, J = 7.8 Hz, 1H),
3.87 (t, J = 4.8 Hz, 4H), 3.52 (t, J = 4.8 Hz, 4H), 3.29 (t, J = 4.8 Hz, 2H), 3.19 (t, J = 4.8 Hz, 2H),
1.99-1.92 (m, 4H). MS m/z: 423.2 [M+H]+.
4.2. Biological evaluation
4.2.1. In vitro inhibition of ChE
The assay followed the method of Ellman et al, using purified AChE from E. electricus
(Sigmae-Aldrich Co.) and BuChE from rat serum.24 For the measurement of E. electricus AChE
inhibition assays, acetylthiocholine iodide (1 mmol/L, 30 μL), phosphate-buffered solution (0.1
mmol/L, pH = 8.0, 40 μL), EeAChE (0.05 U/mL, final concentration, 10 μL) and different
concentrations of test compounds (20 μL) were added in 96-well plates successively. The mixture
24

was incubated at 37 °C for 15min. Then 5,5′-dithiobis-2-nitrobenzoic acid (DTNB, 0.2%, 30 μL) was
added to yield the yellow anion of 5-thio-2-nitrobenzoic acid. Activity was determined by measuring
the increase in absorbance at 412 nm, using Varioskan Flash Multimode Reader (Thermo Scientific).
For BuChE inhibition assays, butyrylthiocholine iodide (1 mmol/L, 30 μL), phosphate-buffered
solution (0.1 mmol/L, pH = 7.4, 40 μL), 25 % serum (10 μL) were used and changes in absorbance
were detected at 405 nm. The other procedures were the same as above. Each concentration was
assayed in triplicate. Blanks containing all components except AChE were carried out. The percent
inhibition was calculated by the following expression: (1 − Ai/Ac) × 100, where Ai and Ac are the
absorbance obtained for ChE in the presence and absence of inhibitors, respectively, after subtracting
the respective background. IC50 values were calculated as the concentration of compound that
produces more than 50% AChE or BuChE activity inhibition.
4.2.2. Kinetic characterization of AChE inhibition
Kinetic characterization of AChE inhibition was conducted based on an established method
using purified AChE from E. electricus (EeAChE) (Sigma–Aldrich Co.).28,29 The assay solution (100
μL) consists of 0.1 M phosphate buffer (pH 8.0), with the addition of 10 μL of 0.5 U/mL EeAChE,
30 μL of 0.2% DTNB and 20 μL of substrate ATCh (acetylthiocholine iodide). Three different
concentrations of inhibitors were added to the assay solution and preincubated for 15 min at 37 °C
with the EeAChE followed 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 ν to be a constant
percentage of ν for the entire data set. Slopes of these reciprocal plots were then plotted against the
concentration of the inhibitors in a weighted analysis.
4.2.3. Oxygen radical absorbance capacity (ORAC-FL) assay
The antioxidant activity was determined by the oxygen radical absorbance capacity-
fluorescein (ORAC-FL) assay.30 The ORAC-assay measured antioxidant scavenging activity against
peroxyl radical induced by 2,2′-Azobis(amidino propane) dihydrochloride (AAPH) at 37°C. The
reaction was carried out in 75 μL phosphate buffer (pH 7.4) and the final reaction mixture was 200
µL. Antioxidant (20 µL) and fluorescein (FL) (120 µL, 150 nM final concentration) were placed in
25

the wells of a black 96 well plate and the mixture was incubated for 15 min at 37°C. Then AAPH
solution (60 µL; 12 mM final concentration) was added rapidly. The plate was immediately placed
into a Varioskan Flash Multimode Reader (Thermo Scientific) and the fluorescence was measured
every 60 seconds for 90 minutes with exitation at 485 nm and emission at 535 nm.
6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was used as standard (1-8 µM,
final concentration). A blank (FL+AAPH) using phosphate buffer instead of antioxidant and trolox
calibration were carried out in each assay. The samples were measured at different concentrations
(1-10 µM). All reaction mixtures were prepared duplicate and at least three independent runs were
performed for each sample. Fluorescence measurements were normalized to the curve of the blank
(without antioxidant). From the normalized curves, the area under the fluorescence decay curve
(AUC) was calculated as: AUC = i=90 ( f
i
/ f ) , where f0 is the initial fluorescence reading at 0 min
o
åi=1
and fi is the fluorescence reading at time i. The net AUC was calculated by the expression AUCsample
− AUCblank. The ORAC-FL values were calculated: [(AUC – AUCblank)/(AUC trolox – AUC
sample
blank)]´[(concentration of trolox/concentration of sample)] and the ORAC-FL value of tested
compounds expressed as trolox equivalents.
4.2.4. Metal-chelating studies29
Compound 6c was tested as a metal chelator using difference UV−vis spectra, recorded with
a Varioskan Flash Multimode Reader (Thermo Scientific).The wavelength ranged from 200 to 600
nm. Numerical subtraction of the spectra of the metal (CuCl2, ZnCl2, AlCl3, and FeSO4) alone and
the compound alone from the spectra of the mixture gave the difference UV−vis spectra due to
complex formation, after incubating for 30 min at room temperature. The final concentrations of
tested compound and metals were 37.5 μM and the final volume of reaction mixture was 200 μL. The
molar ratio method was performed to determine the stoichiometry of the compound-Cu2+ by titrating
the methanol solution of compound 6c with ascending amounts of CuCl2. The UV spectra were
recorded and treated by numerical subtraction of CuCl2 and 6c at corresponding concentrations,
plotted versus the mole fraction of 6c.
4.2.5. In vitro inhibition of self and Cu2+-induced Aβ1-42 aggregation
Inhibition of Aβ1-42 aggregation was measured using a Thioflavin T (ThT)-binding assay.27,35,36
Experiments were performed by incubating Aβ1-42 dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol
26

(HFIP) (1 mg/mL) for 24 h at room temperature. Then, the solvent was evaporated. After incubation,
the HFIP pretreated Aβ1-42 were primarily diluted in dry DMSO to a concentration of 200 μM and
was kept at –80 °C, in order to remain a stable stock solution. Before use, solutions of test
compounds were prepared in DMSO in 2.5 mM for storage and diluted with phosphate buffer
solution (pH 7.4). For the self-induced Aβ1-42 aggregation assay, Aβ1-42 (20 μL, 25 μM, final
concentration) was incubated with 20 μL of test compounds (25 μM, final concentration) in 50 mM
phosphate buffer solution (pH 7.40) at 37 °C for 24 h. Next, 50 mM glycine-NaOH buffer (pH 8.5)
containing 160 μL of 5 μM ThT was added. Fluorescence signal was measured (λexc = 446 nm; λem
=
490 nm) on a Varioskan Flash Multimode Reader (Thermo Scientific). The percent inhibition of
aggregation was calculated using the following equation: (1-IFi/IFc) × 100, in which IFi and IFc are
the fluorescence intensities obtained for for absorbance in the presence and absence of inhibitors
after subtracting the background, respectively. The background activity was determined from wells
containing all components except Aβ1-42, which were replaced by a phosphate buffer solution (pH
7.40) and each assay was run in triplicate.
To study the effects of the Cu2+-induced Aβ1-42 aggregation, solutions of Cu2+ were prepared
from standards to final concentrations of 75 μM using the HEPES buffer at pH 6.6 (20 mM, 150 mM
NaCl). Aβ1−42 (20 μL, 25 μM,) was incubated with Cu2+ (20 μL, 25 μM) in HEPES buffer at pH 6.6,
without or with the tested compound (20 μL, 25 μM, final concentration). The incubation was
performed at 37 °C for 24 h. After incubation, 190 μL of 50 mM glycine−NaOH buffer (pH 8.5)
containing 5 μM thioflavin T was added. The detection was carried out as in the previous procedure
and each assay was run in triplicate.
4.2.6. In vitro inhibition of monoamine oxidase37-39
Recombinant human MAO-A and -B were obtained from commercial sources (Sigma–Aldrich
Co.). The enzymatic reactions were conducted in potassium phosphate buffer (100 mM, pH = 7.4,
made isotonic with KCl 20.2 mM) to a final volume of 500 μL, and contained kynuramine (45 μL for
MAO-A and 30 μL for MAO-B), various concentrations of the test inhibitors (0-100 μL) and lower
than 4% DMSO as cosolvent. The reactions were initiated by the addition of MAO-A or -B (7.5
μg/mL) and were subsequently incubated for 30 min at 37 °C. The reactions were terminated by the
addition of 400 μL NaOH (2 N) and 1000 μL water, centrifuged for 10 min at 16000 g and the
27

concentrations of 4-hydroxyquinoline in the supernatants were measured spectrofluorometrically
( λex= 310 nm; λem= 400 nm), using the Varioskan Flash Multimode Reader (Thermo Scientific). A
linear calibration curve was constructed by preparing samples containing 4-hydroxyquinoline
(0.047–1.56 μM) dissolved in 500 μL potassium phosphate buffer. Volumes of 400 μL NaOH (2 N)
and 1000 μL water were added to each calibration standard. The appropriate control samples were
included to confirm that the test inhibitors do not fluoresce or quench the fluorescence of
4-hydroxyquinoline under these assay conditions. IC50 values were estimated from sigmoidal
dose–response curves (graphs of the initial rate of kynuramine oxidation versus the logarithm of
inhibitor concentration). Each sigmoidal curve was constructed from six different inhibitor
concentrations spanning at least three orders of magnitude. Data analyses were carried out with the
Prism 5 software package (GraphPad) employing the one site competition model. The background
activity was determined from wells containing all components except the hMAO isoforms, which
were replaced by a sodium phosphate buffer solution. IC50 values were determined in triplicate and
are expressed as mean ± standard deviation (SD).
4.2.7. Docking study35
The crystal structures of AChE complexed with donepezil (code ID: 1EVE) and MAO-B
(code ID: 2V60) were obtained from the Protein Data Bank after eliminating the original inhibitors
and water molecules. First, in the PDB crystallographic structures any co-crystallized solvent and the
ligand were removed. Then, proper bonds, bond orders, hybridization and charges were assigned
using protein model tool in Discovery Studio, version 2.5, software package. CHARMm force field
was applied using the receptore ligand interactions tool in Discovery Studio, version 4.5, software
package. The ligand binding site was defined as 13.4 Å from the original ligand. Docking studies
were performed using Autodock 4.2 program and each docked system was performed by 30 runs of
the Autodock search by the Lamarckian genetic algorithm (LGA).
4.2.8. In vitro blood-brain barrier permeation assay
The blood-brain barrier penetration of compound 6c was evaluated using the parallel artificial
membrane permeation assay of the blood-brain barrier (PAMPA-BBB).35,40,41 The donor plate
(MATRNPS50) and the acceptor plate (PVDF membrane, pore size 0.45 μm, MAIPN4550) were
purchased from Millipore. Porcine brain lipid (PBL) was purchased from Avanti Polar Lipids and
28

commercial drugs were purchased from Sigma and Alfa Aesar. Filter PDVF membrane units
(diameter 25 mm, pore size 0.45 µm) from Pall Corporation were used to filter the samples.
Compound 6c was dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/EtOH (70:30) to a
final concentration of 100 μg/mL. Then the diluted compound solution (100 μg/mL, 350 μL) was
added to the donor wells. The acceptor wells were filled with 200 μL of PBS/EtOH (70:30) and the
filter membrane was coated with PBL in dodecane (selected empirically as 4 μL volume of 20 μg/mL
PBL in dodecane) and. The acceptor filter plate was carefully put on the donor plate to form a
sandwich (consisting of the aqueous donor with test compound on the bottom, lipid membrane in the
middle and the aqueous acceptor on the top), which was left undisturbed for 18 h at 25 °C. After
incubation, the donor and acceptor plates were separated carefully and the concentration of drug in
the donor and acceptor wells was determined using the Varioskan Flash Multimode Reader (Thermo
Scientific). Every sample was analyzed at ten wavelengths in four wells and in at least three
independent runs. Pe was calculated using the following expression:
Pe = – ln [1 – CA(t)/Cequilibrium]/[A × (1/VD + 1/VA) × t]
Cequilibrium = [CD(t) × VD + CA(t) × VA]/( VD + VA)
Where Pe is permeability in the unit of cm/s. A is effective filter area and t is the permeation time. VD
is the volume of donor well and VA is the volume of acceptor well. CA(t) is the compound
concentration in acceptor well at time t, and CD(t) is the compound concentration in donor well at
time t. Results are given as the mean ± SD. In the experiment, 11 quality control drugs of known
BBB permeability were included to validate the analysis set. A plot of the experimental data versus
literature values gave a strong linear correlation, Pe (exp.) = 0.8792 × Pe (bibl.)−0.0616 (R2 = 0.9555).
From this equation and taking into account the limit established by Di et al. for BBB permeation, we
determined that compounds with Pe values above 3.46 × 10-6 cm/s could cross the BBB.
Supplementary data
1
13
Supplementary data (the H NMR and C NMR spectra of target compounds) associated with this
article can be found, in the online version.
29