Design, synthesis and evaluation of novel dimethylamino chalcone-O-alkylamines derivatives as potential multifunctional agents against Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2021.113310

A novel series of dimethylamino chalcone-O-alkylamines derivatives was designed and synthesized as multifunctional agents for the treatment of AD. All the target compounds exhibited significant abilities to inhibit and disaggregate Aβ aggregation, and acted as potential selective AChE inhibitors, biometal chelators and selective MAO-B inhibitors. Among these compounds, compound TM-6 showed the greatest inhibitory activity against self-induced Aβ aggregation (IC50 = 0.88 μM) and well disaggregation ability toward self-induced Aβ aggregation (95.1%, 25 μM), the TEM images, molecular docking study and molecular dynamics simulations provided reasonable explanation for its high efficiency, and it was also found to be a remarkable antioxidant (ORAC-FL values of 2.1eq.), the best AChE inhibitor (IC50 = 0.13 μM) and MAO-B inhibitor (IC50 = 1.0 μM), as well as a good neuroprotectant. UV–visual spectrometry and ThT fluorescence assay revealed that compound TM-6 was not only a good biometal chelator by inhibiting Cu2+-induced Aβ aggregation (95.3%, 25 μM) but also could disassemble the well-structured Aβ fibrils (88.1%, 25 μM). Further, TM-6 could cross the blood-brain barrier (BBB) in vitro. More importantly, compound TM-6 did not show any acute toxicity in mice at doses of up to 1000 mg/kg and improved scopolamine-induced memory impairment. Taken together, these data indicated that TM-6, an excellent balanced multifunctional inhibitor, was a potential lead compound for the treatment of AD.

Full text of DOI:10.1016/j.ejmech.2021.113310

European Journal of Medicinal Chemistry 216 (2021) 113310
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and evaluation of novel dimethylamino chalcone-O-
alkylamines derivatives as potential multifunctional agents against
Alzheimer’s disease
, c,
Zhipei Sang a *, Qing Song c, Zhongcheng Cao c, Yong Deng c, Zhenghuai Tan d,
,
**
Li Zhang b
a College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang, 473061, China
b Department of Elderly Digestive, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu, 610072, China
c Department of Medicinal Chemistry, West China School of Pharmacy, Sichuan University, Chengdu, 610041, China
d Institute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan Academy of Chinese Medicine Sciences, Chengdu, 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of dimethylamino chalcone-O-alkylamines derivatives was designed and synthesized as
multifunctional agents for the treatment of AD. All the target compounds exhibited significant abilities to
Received 23 September 2020
Received in revised form
5 February 2021
Accepted 18 February 2021
Available online 23 February 2021
inhibit and disaggregate A
chelators and selective MAO-B inhibitors. Among these compounds, compound TM-6 showed the
greatest inhibitory activity against self-induced A M) and well disaggregation
aggregation (IC50 ¼ 0.88
ability toward self-induced A aggregation (95.1%, 25 M), the TEM images, molecular docking study and
molecular dynamics simulations provided reasonable explanation for its high efficiency, and it was also
found to be a remarkable antioxidant (ORAC-FL values of 2.1eq.), the best AChE inhibitor (IC50 ¼ 0.13 M)
and MAO-B inhibitor (IC50 ¼ 1.0 M), as well as a good neuroprotectant. UVevisual spectrometry and
ThT fluorescence assay revealed that compound TM-6 was not only a good biometal chelator by
inhibiting Cu2þ-induced A aggregation (95.3%, 25 M) but also could disassemble the well-structured
fibrils (88.1%, 25 M). Further, TM-6 could cross the blood-brain barrier (BBB) in vitro. More impor-
b aggregation, and acted as potential selective AChE inhibitors, biometal
b
m
b
m
Keywords:
m
Alzheimer’s disease
Pharmacophoric group
Multi-function agents
Blood-brain barrier permeability
Acute toxicity
m
b
m
A
b
m
tantly, compound TM-6 did not show any acute toxicity in mice at doses of up to 1000 mg/kg and
improved scopolamine-induced memory impairment. Taken together, these data indicated that TM-6, an
excellent balanced multifunctional inhibitor, was a potential lead compound for the treatment of AD.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
acetylcholinesterase (AChE) inhibitors (donepezil, rivastigmine and
galantamine) and one N-methyl-D-aspartate (NMDA) receptor
Alzheimer’s disease (AD) is firstly reported by the German
psychiatrist Alois Alzheimer in 1906. AD is a degenerative brain
disease and the most common form of dementia, and it is charac-
terized by deterioration of memory, language skills, and other
cognitive impairments [1]. According to the World Alzheimer Report
2018, there are over 50 million people live with dementia world-
wide and the number will be projected to 152 million in 2050,
revealing a new reported case each 3.2 s [2]. Currently, three
antagonist (menantine) approved anti-AD drugs are clinically
available. The long-term clinical trials show that these drugs only
presents modest symptomatic effects and cannot stop, prevent, or
reverse the progression of AD [3]. So, new anti-AD agents are
pursuing.
Up to now, the exact pathogenesis of AD are complex and
multifactorial [4,5]. The currently understood pathological and
biochemical manifestations include cholinergic, amyloid-
b (Ab),
tau, excitotoxicity, excess biometallic ions, oxidative stress, ApoE,
insulin resistance, etc, and the corresponding hypothesis have been
proposed and employed for the treatment of AD [4e6]. The amyloid
cascade hypothesis states that the production and accumulation of
* Corresponding author. College of Chemistry and Pharmaceutical Engineering,
Nanyang Normal University, Nanyang, 473061, China.
** Corresponding author.
E-mail addresses: sangzhipei@126.com (Z. Sang), ZL.gLx.gjq@163.com
(L. Zhang).
soluble amyloid oligomers of A
b
are a central event in the neuro-
oligomers leads to the
pathology of AD [7e9]. The soluble A
b
https://doi.org/10.1016/j.ejmech.2021.113310
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
damage and death of neurons, and further accelerates the spread of
tau-related neurofibrillary tangles, neuroinflammation, and
neuronal degeneration and death [10]. Recent phase III trials of the
the phenol hydroxyl, and this molecular feature extends the
planarity of the molecule, and almost always represents an
important requirement to achieve inhibition of Ab aggregation
anti-A
which failed to improve cognitive function in patients, suggest that
targeting of A alone might not be enough to prevent or slow AD
progression, as multiple mechanisms are involved in AD patho-
genesis and their relative contributions might vary at different
stages of the disease [11]. The cholinergic hypothesis states that the
levels of acetylcholine (ACh), a neurotransmitter responsible for the
learning and memory, are abnormally reduced by the high levels of
acetylcholinesterase (AChE) in the hippocampus and the neocortex
regions, which directly contributes to cognitive decline [12].
b
monoclonal antidodies solanezumab and bapineuzumab,
[36,37]. To further explore the structure-activity-relationship, two
novel series of dimethylamino chalcone-O-alkylamine derivatives
(Fig. 1) were designed by fusing multiple pharmacophore. And the
synthesized compounds were evaluated by inhibition and dis-
b
aggreation of Ab aggregation, antioxidant activity, metal chelation,
AChE inhibition and MAO-B inhibitory activity, as well as potential
blood-brain barrier (BBB) permeability and in vivo scopolamine-
induced memory impairment experiments.
2. Results and discussion
Moreover, AChE also induces the aggregation and deposition of A
fibrils by forming AChE-A complexes, leading to cognitive
dysfunction [13]. Thus, the elevation of ACh through AChE inhibi-
tion and the prevention of A aggregation are considered as the
b
b
2.1. Chemistry
b
The synthetic route for the dimethylamino chalcone-O-alkyl-
amine derivatives TM-1~TM-19 was shown in Scheme 1 and
Scheme 2. Firstly, the starting material 1 was treated with dimethyl
sulfate in acetone to obtain the intermediate 2, meanwhile, the key
intermediates 3a~3c were synthesized by adding excessive
amounts of 1,3-dibromopropane, 1,4-dibromobutane or 1,6-
dibromohexane to starting material 1 in anhydrous acetonitrile at
65 ꢀC. Then, the intermediates 3a~3c were reacted with secondary
amines 5a~5d, which were synthesized by reductive amination of
ethylamine with benzaldehydes in the presence of NaBH4, and
diethylamine, affording intermediates 6a~6e, 7a~7d and 8a~8d.
Finally, the products TM-1~TM-15 were obtained by the conden-
sation of intermediates 2, 6a~6e, 7a~7d or 8a~8d, with the appro-
priate benzaldehyde in ethanolic 30% KOH solution.
promising approaches to halting the progression of dementia.
In addition, excess of metal ions such as Cu2þ, Fe2þ, Zn2þ and
Al3þ have been found in A plaques of AD brains, Cu2þ and Zn2þ can
b
promote Ab aggregation, the abnormally high levels of redox-active
metal ions such as Cu2þ and Fe2þ in brain might contribute to the
overproduction of reactive oxygen species (ROS), oxidative stress
further promotes A
cals, and increases the generation of A
while, A could enter the mitochondria where it would increase the
b
toxicity through the production of free radi-
b
peptides [14,15]. Mean-
b
generation of ROS and further induce oxidative stress that damage
biological molecules such as proteins, DNA, and lipids [16]. There-
fore, metal chelators and antioxidant agents also served as a potent
therapeutic strategy for AD treatment.
Another important contributor to the progress in AD is the
catabolism of monoamine. Monoamine oxidases A and B (MAO-A
and MAO-B) are important membrane-bound flavoenzymes
responsible for the oxidative deamination of xenobiotic amies and
monoamine neurotransmitters such as dopamine (DA), serotonin
(5-HT), and noradrenaline (NE). The high levels of MAO-B are
observed in the temporal, parietal and frontal cortex of AD patients
[17]. Selective MAO-B inhibitors could improve learning and
memory deficits in animal models associated with AD and is
considered as a potential approach to treat AD, such as selective
MAO-B inhibitor rasagiline has been performed a phase II trial in
mild-to-moderate AD patients and presents trends to better per-
formance [18].
According to Scheme 2, commercially available p-hydroxy
benzaldehyde 9 was reacted with 1,4-dibromobutane in anhydrous
acetonitrile at 65 ꢀC to give the intermediate 10, which was treated
with secondary amines 5a and 5b to obtain the intermediates 11a
and 11b. The key intermediate 14 was synthesized through rear-
rangement of intermediate 13 which was obtained by the reaction
of commercially available 12 with acetylchloride in the presence of
triethylamine. Ultimately, the desired products TM-16~TM-19 were
synthesized as described according to the above procedure.
2.2. Biological activity
2.2.1. Effects on self-mediated Ab1ꢁ42 aggregation
Given that the complexity of AD and the involvement of multi-
systems in its progression. The multi-target-directed ligands
(MTDLs), hitting two or more AD-relevant complementary targets
and producing synergistic effect, have been recognized as the best
pharmacological option for halting the progression of AD [19e21].
Several promising MTDLs candidate drugs are now in the pipeline
and have completed phase I/II/III trials [22,23].
In order to evaluate the effects of the target compounds against
self-mediated Ab1ꢁ42 aggregation, we performed two individual
studies inhibitory activity and disaggregation potency on self-
induced Ab1ꢁ42 aggregation. Thioflavin T (ThT) fluorescence assay
were used to evaluate the degree of Ab1ꢁ42 aggregation [34,35].
Curcumin, a known active natural product for inhibition of self-
induced Ab aggregation, was used as reference compound.
Chalcone
(a-phenyl-b-benzoylethylene, I) are prominent
As shown in Table 1, almost all the target compounds exhibited
secondary-metabolite precursors of flavonoids and isoflavonoids in
plants. Chalcones and its derivatives are important bioactive mol-
ecules which exhibit significant biological activities including
radical-scavenging, anti-tumor, anti-inflammatory, MAO-B inhibi-
tion and neuroprotective properties [24,25]. Further, recent studies
reveal that 4-dimethylamino chalcone derivatives are widely
more potent inhibition potency for self-mediated Ab1ꢁ42 aggrega-
tion than curcumin (43.1% at 25 mM). In the primary experiments,
the precursor compound TM-1 (59.7%) exhibited low inhibitory
activity, and was suitably modified at the methoxy group with O-
alkylbenzylamines group, compounds TM-2~TM-13 was obtained.
As expected, these substituted groups seem to play an important
investigated to be as A
dimethylamino group is contribute to the strong binding affinity to
aggregates [26e28]. The 1-benzylpiperidine fragment of done-
pezil is the key pharmacophore of AChE inhibition and has been
widely applied to develop MTDLs [29e32], and based on this, the O-
alkylamine fragments were developed as AChE inhibition phar-
macophore [33e35]. Moreover, the carbonyl group of the synthe-
sized compounds could establish an intramolecular HB-bond with
b
aggregate imaging probes, it seems that the
role in the inhibition of Ab aggregation, all the goal compounds
exhibited more potent inhibitory activities than that of TM-1 and
curcumin, especially, compound TM-6, the [2-(CH3)2N]
PhCH2NC2H5 group was linked to the 4-dimethylamino chalcone
moiety by four-methylene spacers, showed the most potent inhi-
A
b
bition of A
b
aggregation (IC50 ¼ 0.88
mM), whereas the marketed
AD drug donepezil did not show any significant inhibitory activity
under the same experimental conditions. According to the
2

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Fig. 1. The design strategy for the dimethylamino chalcone-O-alkylamine derivatives.
screening data, the properties of the benzylamines groups in O-
alkylbenzylamines groups had significant effects on A aggregation
inhibitory activities, generally, the potency to inhibit A
tion were in the order, [2-(CH3)2N]PhCH2NC2H5
70.1% and 75.8% inhibitory activity at 25 mM, respectively. As
described above, the dimethylamino group at the 4-position of the
chalcone framework was beneficial to their inhibition activities of
b
b
aggrega-
(2-CH3O)
>
A
b1-42 aggregation.
PhCH2NC2H5 > [4-(CH3)2N]PhCH2NC2H5 > PhCH2NC2H5. More
Furthermore, compounds TM-2~TM-13 were selected to test the
interesting, replacing [2-(CH3)2N]PhCH2NC2H5 group of TM-5
disaggregation effects on self-induced Ab1ꢁ42 aggregation using
(IC50 ¼ 1.27
m
M) with N(C2H5)2 group to obtain compound TM-14
ThT binding assay [35]. As displayed in Table 1, compounds TM-
showed 64.1% inhibition at 25
mM. The results revealed that the
2~TM-13 could significantly decompose Ab fibrils with disaggre-
introduction of O-alkylbenzylamine groups (especially the strong
donor group at the 2 position of benzylamine group) could promote
the inhibitory activities of Ab1-42 aggregation, however, there was
no significant enhance after introducing the diethylin group.
Further, removal the dimethylamino group of chalcone derivative
TM-5 to obtain compound TM-15, giving the lowest inhibitory ac-
tivity (23.0%), which suggested that the dimethylamino group of
chalcone skeleton was critical to Ab1-42 aggregation inhibitory
activity.
To further determine the optimal position of dimethylamino
group on the chalcone skeleton, the synthesis of TM-16~TM-19 was
outlined in Scheme 2. According to the results, the corresponding
precursor compound TM-19 gave 36.1% inhibition activity
compared with TM-1 (59.7%). Replacing the methoxy group of TM-
19 with the dimethylamino group to get compound TM-18,
dramatically, gave 64.3% inhibition activity. Further, exchanging
gate ratio range from 73.7% to 99.7%. Compound TM-6 exhibited
good disaggregation ratio with 95.1%. These results showed that
TM-6 could inhibit and disaggregate self-induced
aggregation.
Ab1ꢁ42
2.2.2. Effects on abundance of Ab fibrils by compound TM-6
To complement the ThT binding assay, Ab1ꢁ42 aggregation was
also monitored by transmission electron microscopy (TEM) (Fig. 2)
[34,35]. The results displayed that the sample of Ab1ꢁ42 alone had
aggregated into Ab fibrils after 24 h of incubation, while only small
bulk aggregates were visible and no characteristic fibrils were
observed in the sample of in the presence of TM-6. The TEM results
were well consistent with the results of the ThT measurements,
which strongly proved that TM-6 could inhibit Ab1ꢁ42 fibrils
formation.
dimethylamino group of TM-3 (IC50 ¼ 1.48
(IC50 ¼ 0.88 M) with different O-alkylbenzylamine group, to
furnish the corresponding compounds TM-16 and TM-17, obtained
m
M) and TM-6
2.2.3. Molecular docking compound TM-6 with A
To further explore the binding modes of the active compound
TM-6 with A , a molecular docking experiment was performed
b
m
b
3

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Scheme 1. aReagents and conditions: (i) Me2SO4, acetone, refluxed, 1 h; (ii) Br(CH2)nBr, K2CO3, CH3CN, 65 ꢀC, 6e8 h; (iii) NaBH4, CH3OH, rt, 5e6 h; (iv) NHR1R2, K2CO3, CH3CN,
refluxed, 6e8 h. (v) 4-Dimethylaminobenzaldehyde or Benzaldehyde, 30%KOH, EtOH, 50 ꢀC, 72 h.
[38]. The structure of A
Data Bank (PDB: 1BA4). As shown in Fig. 3, compound TM-6 was
locked at the C-terminus hydrophobic area of A . The 2-hydroxy-
b
used was downloaded from the Protein
favorable for the binding of Ab and compound TM-6. This might
provide the molecular basis for interpreting the biological data.
b
acetophenone moiety acted as an important role, the carbonyl
group interacted with LYS16 and ASP1 through two intermolecular
hydrogen bonds, the hydroxyl could bind to ASP1 and ASP23 via
three intermolecular hydrogen bonds, and the benzene ring of 2-
hydroxy-acetophenone moiety could bind to PHE19 via one Pi-Pi
interaction. In addition, compound TM-6 formed the hydrophobic
interactions with the residues GLU3, LEU17, ASP1, LYS16, PHE19,
ASP23, PHE20, ALA2. These different interactions might be
2.2.4. Antioxidant activity in vitro
The antioxidant activities of the target compounds were eval-
uated by the ORAC-FL method (Oxygen Radicals Absorbance Ca-
pacity by Fluorescence) [36]. The vitamin E analogue Trolox was
used as a standard. All the compounds exhibited good ORAC-FL
values of 1.1e2.1 Trolox equivalents (Table 2). According to the
data, all the target compounds showed significant antioxidant ac-
tivity. Among which, compounds TM-5, TM-6 and TM-7 (ORAC-FL
4

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
a
Scheme 2. Reagents and conditions: (i) Br(CH2)4Br, K2CO3, CH3CN, 65 ꢀC, 6e8 h; (ii) NHR1R2, K2CO3, CH3CN, reflux, 6e8 h; (iii) CH3COCl, Et3N, CH2Cl2, -5 ꢀC ~ room temperature,
0.5 h; (iv) AlCl3, NaCl, 160 ꢀC, 2 h; (v) 11a~11b, 4-Dimethylaminobenzaldehyde or p-Anisaldehyde, 30%KOH, EtOH, 50 ꢀC, 72 h.
values of 2.1, 2.1, and 2.0 Trolox equivalents, respectively), in which
the [2-(CH3)2N]PhCH2NC2H5 group was linked to terminal of the
side chain, exhibited more potent antioxidant activities than other
analogues. It revealed that the hydroxy group of derivatives was
crucial to the radical scavenging capability, and the strong donor
group (e.g. 2-(dimethylamino)-N-ethyl-benzenemethanamine) on
the terminal of O-alkylamine group could contribute to the anti-
oxidant activity.
2~TM-13 were synthesized, and these compounds exhibited note-
worthy inhibitory activities for AChE. The properties of the ben-
zylamines groups in O-alkylbenzylamines groups indicated
significant effects on AChE inhibitory activities, the general trend
for AChE inhibition activity was that [2-(CH3)2N]PhCH2NC2H5 > (2-
CH3O)PhCH2NC2H5 > PhCH2NC2H5 > [4-(CH3)2N]PhCH2NC2H5. It
indicated that the donor groups at the 2-position of the N-ethyl-
benzenemethanamine could be contribute to the inhibitory po-
tency, conversely, substituent group at the 4-position had a slight
detrimental effect. Among these compounds, TM-6, with the [2-
(CH3)2N]PhCH2NC2H5 group and a linker of four-methylene chain,
exhibited the most potent inhibition of AChE with IC50 value of
2.2.5. AChE and BuChE inhibition effects
The inhibitory activities of AChE and BuChE toward compounds
TM-1~TM-19 were evaluated using the Ellman’s method [34].
RatAChE from rat cortex homogenate and electric eel, and ratBuChE
from rat serum. In the above experiments, donepezil was used as
reference compound.
0.13
mM. Interestingly, removal the dimethylamino group of TM-5
(IC50 ¼ 0.21
m
M) to get compound TM-15 (IC50 ¼ 0.31
m
M),
showed a slightly decrease inhibitory activity. Replacing benzyl-
amine group of TM-5 with the dimethylamine group to obtain
compound TM-14, exhibited a dramatically dropped inhibitory
As shown in Table 2, the target compounds showed different
amounts of AChE inhibitory activity with IC50 values ranging from
0.13
mM to superior 500
mM. As expected, the precursor compounds
potency with IC50 value of 2.25 mM. It revealed that the benzyl-
TM-1 (IC50 > 500
significant inhibition of AChE, moreover, replacing the methoxy
m
M) and TM-19 (IC50 > 500
m
M) exhibited no
amine groups promoted the more inhibitory potency than aliphatic
amines group, it might be that the benzylamine groups, especially,
group with the dimethylamino group on the compound TM-19 to
the
2-(dimethylamino)-N-ethyl-benzenemethanamine
group
obtain compound TM-18 (IC50 > 500
the preliminary experiments, to determine the optimal length of
the aliphatic linker and the substituent groups, compounds TM-
m
M), showed no any change. In
could contact closely the peripheral anionic site of the AChE. The
ethyl group on the terminal nitrogen atom of the side chain further
improved the inhibitory potency of AChE in accordance with our
5

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Table 1
Inhibition of Ab1-42 aggregation and disaggregation of Ab1-42 aggregation of donepezil, curcumin and chalcone derivatives TM-1~TM-19.
Compd.
n
NR1R2
Self-Induced Ab1-42 aggregationa
Inhibition IC50
(
m
M)b,d
Disaggregation (%)c,d
TM-1
TM-2
TM-3
TM-4
TM-5
TM-6
TM-7
TM-8
e
3
4
6
3
4
6
3
4
6
3
4
6
e
e
e
e
e
e
e
e
e
59.7 0.1%e,d
3.56 0.01
1.48 0.02
1.49 0.01
1.27 0.03
0.88 0.01
1.03 0.01
1.32 0.02
2.10 0.01
1.38 0.04
1.68 0.02
3.78 0.01
3.17 0.01
64.1 0.08%e,d
23.0 0.11%e,d
70.1 0.35%e,d
75.8 0.28%e,d
64.3 0.09%e,d
36.1 0.06%e,d
n.a.g
n.t.f
(2-CH3O)PhCH2NC2H5
(2-CH3O)PhCH2NC2H5
(2-CH3O)PhCH2NC2H5
[2-(CH3)2N]PhCH2NC2H5
[2-(CH3)2N]PhCH2NC2H5
[2-(CH3)2N]PhCH2NC2H5
[4-(CH3)2N]PhCH2NC2H5
[4-(CH3)2N]PhCH2NC2H5
[4-(CH3)2N]PhCH2NC2H5
PhCH2NC2H5
PhCH2NC2H5
PhCH2NC2H5
e
e
e
e
e
e
e
e
98.1 0.12
99.7 0.15
92.6 0.09
96.6 0.13
95.1 0.25
87.9 0.21
73.7 0.35
91.6 0.17
95.0 0.14
97.5 0.12
97.2 0.11
84.4 0.13
n.t.f
TM-9
TM-10
TM-11
TM-12
TM-13
TM-14
TM-15
TM-16
TM-17
TM-18
TM-19
Donepezil
Curcumin
n.t.f
n.t.f
n.t.f
n.t.f
n.t.f
n.t.f
43.1 0.031%e,d
n.t.f
a
Inhibition of Ab aggregation, the thioflavin-T fluorescence method was used.
b
The IC50
(m
M) values are calculated when the percentage inhibition of self-induced Ab1-42 aggregation (25
m
M) > 90%.
M.
c
d
e
f
Disaggregating of self-Induced Ab1-42 aggregation, the concentration of tested compounds and Ab1-42 were 25
Data are the mean SEM of three independent experiments.
m
Inhibition of self-Induced Ab1-42 aggregation, the concentration of tested compounds and Ab1-42 were 25
n.t. ¼ not tested.
mM.
g
n.a. ¼ no active. Compounds defined “no active” means percent inhibition less than 5.0% at a concentration of 25
mM.
Fig. 2. TEM images of Ab1ꢁ42 (25 mM) in the presence and absence of 25 mM compound TM-6 after 24 h of aggregation.
previous results [27]. As described above, compound TM-6 was the
most potent AChE inhibitor in this series with IC50 value of 0.13 M.
To further determine the optimal position of dimethylamino
group on the chalcone framework, compounds TM-3
of cholinesterase enzyme. It had been postulated that some severe
side effects of AChE inhibitors such as tacrine might be attributed to
their poor selectivity [35].
m
(IC50 ¼ 0.65
m
M) and TM-6 (IC50 ¼ 0.13
mM) which had better
2.2.6. Kinetic characterization of AChE inhibition
AChE inhibitory activities were chosen, we exchanged dimethyla-
To gain further insight into the mechanism for this family of
dimethylamino chalcone-O-alkylamines derivatives, compound
TM-6, the most potent AChE inhibitor, was selected to further ki-
netic study [34]. Graphical analysis of the reciprocal Lineweaver-
Burk plots (Fig. 4A) showed both increasing slopes (decreased
Vmax) and intercepts (higher Km) at increasing concentration of the
inhibitor. The intersection point of the LineweaverꢁBurk reciprocal
plot fell in the second quadrant, indicated a mixed-type inhibitory
type that was similar to that of donepezil. And the Ki of TM-6 was
mino group with the O-alkylbenzylamine group to give compounds
TM-16 (IC50 ¼ 0.71
mM) and TM-17 (IC50 ¼ 2.04
mM), respectively,
both of which exhibited slight weak inhibitory potency. It indicated
that the dimethylamino group at 4-position of chalcone framework
was a key role in AChE inhibitory activity. Furthermore, all the
target compounds were re-evaluated using electric eel AChE
(EeAChE). As shown in Table 2, in general, the inhibition activity of
EeAChE by compounds was somewhat less potent than that of rat
AChE.
0.50
mM (Fig. 4B), which was consistent with the measured IC50
For BuChE inhibition assay, almost all the target compounds
were proved to be poor activity on BuChE. This result showed that
these target compounds were potent AChE inhibitors with high
selectivity toward BuChE. This selectivity profile might provide
lesser degree of associated side effects due to peripheral inhibition
value.
2.2.7. Molecular modeling studies
In order to explore possible binding mechanism to AChE, a
further docking study was carried out for compound TM-6 using
6

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
observed to bind to the CAS via two Pi-Pi interactions with key
residue Trp286, the benzene ring of 2-hydroxy-acetophenone
moiety could interact with key residue Trp286 and Tyr341 through
one Pi-Pi interaction, respectively, and the CH2 interacted with
Trp86 via one sigma-Pi interaction. Moreover, the hydroxy group
and carbonyl group formed one intramolecular hydrogen bond and
the oxygen atom of the hydroxy group interacted with Tyr341 via
one intermolecular hydrogen bonding. In addition, hydrophobic
interactions could be observed between compound TM-6 and
Tyr341, Trp286, Tyr124, Tyr337 and Trp86.
2.2.8. Effect of TM-6 on hAChE-induced Ab1-40 aggregation
Studies showed that A
has been linked to AChE expression, and the PAS of AChE could bind
to the A and accelerated the formation of A fibrils [39]. So,
b deposition in the brain of AD patients
b
b
inhibiting hAChE-induced Ab1-40 aggregation were performed by
ThT fluorescence assay [35,40]. Based on the data in Table 3, com-
pound TM-6 obviously inhibited hAChE-induced Ab1-40 aggregation
with 70.7% inhibition rate, which was better than donepezil (18.6%).
2.2.9. Molecular dynamics (MD) simulations
To further explore the interactions between Ab1-42 or AChE and
TM-6, respectively, we performed a 10-ns MD simulation using
GROMACS package [41]. RMSD (root mean square deviation) values
were calculated for all frames in the MD simulation trajectory. As
displayed in Fig. 6, the Ab1-42-TM-6 complex quickly reached sta-
bility after 1 ns and the potential energy always went down, indi-
cating that the process was spontaneous. Moreover, the MD
simulation showed that there was no obvious separation between
the ligand TM-6 and Ab1-42 and there was no significant changes in
binding sites, revealing that the ligand TM-6 and Ab1-42 bond
firmly. Similarly, as presented in Fig. 7, the AChE-TM-6 complex
reached stability after 2 ns and the system always went down in
potential energy, showing the binding process was spontaneous.
Furthermore, the MD simulation indicated that there was no
obvious separation and no significant changes in binding sites be-
tween TM-6 and AChE, presenting that TM-6 firmly bond with
AChE. Therefore, the MD simulation exhibited that compound TM-6
firmly combined with Ab1-42 and AChE, respectively.
2.2.10. Metal-chelating properties of compound TM-6
The ability of compound TM-6 to chelate biometals such as
Cu2þ, Zn2þ, Al3þ and Fe2þ was studied by UVevisual spectrometry
[33,34]. As shown in Fig. 8, adding CuCl2 and AlCl3 to a solution of
TM-6, the maximum absorption wavelength shifted from 444 nm
to 460 nm and 468 nm, respectively, indicating the formation of
TM-6-Cu2þ and TM-6-Al3þ complexs. Conversely, the maximum
absorption at 444 nm exhibited no significant shift after the addi-
tion of FeSO4 and ZnCl2. Therefore, TM-6 had the selectivity for
metal chelation. It was reasonable that the hydroxyl and carbonyl
groups in the chalcone skeleton contributed to the chelation.
The stoichiometry of the TM-6-Cu2þ complex was determined
using molar ratio method by preparing solutions of compound TM-
6 with increasing amounts of CuCl2. The UV spectra were used to
obtain the absorbance of the complex of TM-6 and CuCl2 at
different concentrations at 460 nm. According to Fig. 9, the absor-
bance linearly increased at first and then tended to be stable. The
two straight lines intersected at a mole fraction of 1.05, revealing a
Fig. 3. Docking studies of compound TM-6 with Ab1-42 (PDB ID: 1BA4). (A) Cartoon
representations of compound TM-6 (green stick) interacting with Ab1-42. (B) Associa-
tion of TM-6 (green stick) and the C-terminus of obtained from docking calculations.
(C) 2D schematic diagram of docking model of compound TM-6 with Ab1-42
.
the docking program, AutoDock 4.2 package with Discovery Studio
2.5, based on the X-ray crystal structure of AChE (PDB code: 4EY7)
[33]. As shown in Fig. 5, the results showed that TM-6 occupied the
entire enzymatic catalytic site (CAS), the mid-gorge sites and the
peripheral site (PAS), and could simultaneously bind to both the
CAS and PAS thus providing an explanation for its potent inhibitory
activity against AChE. In the TM-6-AChE complex, the benzene ring
of 2-(dimethylamino)-N-ethyl-benzenemethanamine group was
1:1 stoichiometry for complex TM-6-Cu2þ
.
2.2.11. Effects on Cu2þ-mediated Ab1ꢁ42 aggregation
In order to investigate the effects of dimethylamino chalcone-O-
alkylamines derivatives on Cu2þ-induced Ab1-42 aggregation, we
also performed two individual studies: inhibitory activity and
disaggregation effects of compounds TM-2~TM-13 on Cu2þ
-
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Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Table 2
Oxygen Radical Absorbance Capacity (ORAC, Trolox Equivalents) and Inhibition of Cholinesterase and by Donepezil and chalcone derivatives.
Comp.
ORACa
IC50 SEMb
(
mM)
Selectivity indexf
RatAchEc
RatBuChEd
EeAChEe
TM-1
TM-2
TM-3
TM-4
TM-5
TM-6
TM-7
TM-8
1.2 0.02
1.5 0.02
1.4 0.01
1.4 0.02
2.1 0.03
2.1 0.04
2.0 0.02
1.3 0.02
1.5 0.01
1.4 0.01
1.6 0.02
1.5 0.03
1.4 0.02
1.2 0.01
1.3 0.02
1.6 0.02
1.5 0.02
1.7 0.02
1.5 0.02
NTg
>500
>500
>500
>500
>500
339 1.18
236 1.89
422 1.25
>500
68.9 0.85
>500
>500
202 1.61
>500
>500
>500
>500
>500
>500
>500
20.7 1.36
>500
e
0.63 0.02
0.65 0.03
0.56 0.05
0.21 0.04
0.13 0.01
0.31 0.03
2.47 0.03
1.35 0.01
1.37 0.05
1.44 0.01
1.39 0.02
4.00 0.06
2.25 0.02
0.31 0.05
0.71 0.02
2.04 0.01
>500
5.30 0.35
4.12 0.36
5.17 0.72
1.59 0.62
0.69 0.13
2.44 0.62
6.21 0.35
1.96 0.17
7.95 0.15
5.06 0.33
4.30 0.37
3.59 0.25
1.35 0.41
2.00 0.27
1.63 0.62
3.02 0.53
>500
>793.7
>769.2
>892.9
1614.3
1815.4
1361.3
202.4
51.0
>365.0
>347.2
145.3
>125.0
>222.2
>1612.9
>704.2
>245.1
e
TM-9
TM-10
TM-11
TM-12
TM-13
TM-14
TM-15
TM-16
TM-17
TM-18
TM-19
Donepezil
>500
0.015 0.002
>500
0.027 0.001
e
1380
a
Results are presented as the mean (n ¼ 3) SD and expressed as
m
mol of Trolox equivalent/
mmol of tested compound.
b
IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50%, and are expressed as the mean standard error of the mean of three
different experiments in quadruplicate.
c
From 5% rat cortex homogenate.
BuChE from rat serum.
From Electrophorus electricusc.
d
e
f
Selectivity Index ¼ IC50 (RatBuChE)/IC50 (RatAChE).
g
NT ¼ not tested.
inhibitory activity for Cu2þ-induced Ab1-42 aggregation, among
which, compound TM-6 (95.3%) could inhibit Cu2þ-induced Ab1-42
aggregation through chelating with Cu2þ. For the disaggregation
experiments, compounds TM-2~TM-13 effectively disassembled A
b
fibrils (Table 4), and the representative compound TM-6 indicated
good disaggregation potency (88.1%).
To complement the ThT binding assay, Cu2þ-induced Ab1-42
aggregation was also monitored by transmission electron micro-
scopy (TEM). For the inhibition of Cu2þ-induced Ab1-42 aggregation
experiments, more well-structured A
b
aggregates fibrils were
alone, and fewer A fibrils
observed in the presence of Cu2þ than A
b
b
were observed when compound TM-6 was added to the samples in
Fig. 10. For the disaggregation studies, the TEM images in Fig. 11
illustrated that much fewer A
adding compound TM-6, indicating that TM-6 was able to disas-
semble the well-structured A
fibrils from Cu2þ-induced Ab1-42
b aggregates were observed after
b
aggregation. Therefore, according to the above data, compound
TM-6 could be able to inhibit Cu2þ-induced Ab1-42 aggregation and
disassembling the well-structured Ab fibrils.
2.2.12. Inhibition of MAOs in vitro
To further study the multi-potent biological profiles of the target
compounds, the inhibitory activity against MAO-A and MAO-B
(recombinant human enzyme) was performed [42]. As shown in
Table 4, all the target compounds were significantly selective
hMAO-B inhibitors. Among these compounds, precursor compound
Fig. 4. (A) Lineweaver-Burk plot of TM-6 for AChE hydrolysis. (B) slope replot vs TM-6
concentration.
TM-1 (IC50 ¼ 0.21
mM) was the best MAO-B inhibitor, and the
selectivity index (SI) value was 36.3. In general, dimethylamino
group at 4-position of chalcone skeleton exhibited better inhibitory
activity than that at 40-position. The representative compound TM-
6 was a MAO-B inhibitor (IC50 ¼ 1.0 M; SI ¼ 18.4).
m
induced Ab1-42 aggregation by ThT binding assay [34,36]. Curcumin
was used as the reference compound. The Ab1-42 was first treated
with one equivalent of Cu2þ for 2 min at room temperature and
then incubated with or without the testing compounds for 24 h at
37 ꢀC. As shown in Table 3, all the compounds showed good
2.2.13. Molecular modeling study
In order to explore the hypothetical binding modes of the
compound TM-6, docking simulation was performed using the
8

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
docking program, AutoDock 4.2 package with Discovery Studio 2.5
based on the X-ray crystal structure of human MAO-B (PDB code:
2V60) [42]. As shown in Fig. 12, the dimethylamino moieties of
compound TM-6 was close to the FAD cofactor, the benzene ring of
the 2-hydroxy-acetophenone moiety adopted two Sigma-Pi in-
teractions with PHE168 and LEU171, respectively. The benzene ring
of N,N-dimethylaniline group bond to TYR435 via parallel Pi-Pi
interactions. In Fig. 3B, two intermolecular hydrogen bonds were
generated between the eOH of TM-6 with PHE168 and CYS172
(PHE168-
¼
O/HO-TM-6; CYS172eOH/O-TM-6). Meanwhile,
compound TM-6 interacted with many other amino acids, such as
TPR119, LEU164, PRO104, ILE316, LEU88, GLU84, TYR326, THR314,
GLN206, THR201, ILE199, PRO102, ILE198, SER200 and so on.
2.2.14. Neuroprotective effect on PC12 cell injury induced by H2O2
The neuroprotective ability of TM-6 against free radicals damage
was evaluated using MTT assay [33,36]. Firstly, the cytotoxicity of
TM-6 toward PC12 cells was tested. As shown in Fig. 13A, the
nontoxic concentration of TM-6 was below 50
evaluated the neuroprotective effects, after 100
m
M, and then we
m
M H2O2 exposure,
cell viability as determined by MTT reduction was obviously
decreased to 55.6% (P < 0.01 vs control), manifesting high sensi-
tivity to H2O2-induced injury. However, compound TM-6 showed
protective effects in a dose-dependent manner against H2O2-
induced PC12 cell injury compared with the reference compound
Trolox (100
mM) (Fig. 13B). At concentrations of 10.0 mM, compound
TM-6 exhibited significantly neuroprotective effect, and the cell
viability was 88.1%. When the concentration was reduced to 1.0
the cell viability decreased to 70.9%.
mM,
In addition, we further investigated the neuroprotective prop-
erty of TM-6 using lactate dehydrogenase (LDH) assay. The release
of LDH to the supernatant showed an increase of cell membrane
damage. Similar to the above assay neuroprotective effect on H2O2-
induced injury, as shown in Fig. 13C, the LDH activity dramatically
increased after exposure of PC12 cultures to H2O2. However, adding
TM-6 could significantly reduce the LDH release and protect
PC12 cells from H2O2-induced injury. Meanwhile, TM-6 was more
potent than Trolox. The above results clarified that compound TM-6
showed potent neuroprotective effect on H2O2-induced PC12 cell
injury.
2.2.15. In vitro BloodꢁBrain barrier permeation assay
Brain penetration is a major requirement for successful CNS
drugs. The parallel artificial membrane permeation assay of the
bloodꢁbrain barrier (PAMPA-BBB) was performed to evaluate the
BBB permeability of TM-6 [38,43]. We compared the permeability
of 11 commercial drugs with reported values to validate the assay.
The following ranges of permeability (Pe 10ꢁ6 cm/s) had been
determined in our previous work: Pe > 3.44 for compounds dis-
played high BBB permeation, Pe ꢂ 1.61 for compounds displayed
low BBB permeation, 3.44 ꢃ Pe > 1.61 for compounds displayed
uncertain BBB permeation. As shown in Table 5, TM-6 showed
13.37 ꢄ 10ꢁ6 cm/s permeability and could cross the BBB in vitro.
Fig. 5. (A) Representation of compound TM-6 (green stick) interacting with residues in
the binding site of AChE (PDB code: 4EY7). (B) 3D docking model of compound TM-6
(green stick) with AChE. (C) 2D schematic diagram of docking model of compound TM-
6 with AChE.
2.2.16. Acute toxicity
To investigate the safety profile of the dimethylamino chalcone-
O-alkylamines derivatives, the acute toxicity of TM-6 was evaluated
in Kunming mice at doses of 67.5, 125, 250, 500 and 1000 mg/kg
(n ¼ 10 per group) by oral administration. After administration of
compound TM-6, mice were observed continuously for the first 4 h
through 14 days for any abnormal behavior and mortality changes.
The results exhibited that the animals treated with compound TM-
6 did not show any acute toxicity or mortality immediately.
Therefore, compound TM-6 was proved to be nontoxic and well
tolerated at doses of up to 1000 mg/kg.
Table 3
Inhibition of hAChE-induced Ab1-40 aggregation by compounds 7d and donepezil.
Compound
% Inhibition of hAChE-induced Ab1-40 aggregationa
TM-6
Donepezil
70.7 0.61
20.1 0.38
a
Inhibition of human AChE-induced Ab1-40 aggregation, the concentration of TM-
M, respectively.
6 and Ab1-40 was 100 and 230
m
9

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Fig. 6. The MD simulation of TM-6 with Ab1-42
.
Effect of TM-6 on Scopolamine-Induced Amnesia Models. The step-
down passive avoidance task was employed to evaluate the effect of
TM-6 on scopolamine-induced memory impairment [34,36]. As
displayed in Fig. 14, the step-down latency of mice treated with
scopolamine alone (model group) was remarkably shorter than
that of vehicle-treated mice (untreated group, p < 0.05). Treated
with donepezil group (5 mg/kg) significantly increased latency
time (p < 0.01), indicating that donepezil clearly reversed the
cognitive deficit induced by scopolamine. Moreover, treated with
compound TM-6 (2.3, 6.8 and 20.4 mg/kg) increased the latency
time in a dose-dependent manner, and the medium dose group
(6.8 mg/kg) presented the longest latency time in the three doses,
while showed lower potency than donepezil. Thus, to some extent,
compound TM-6 could improve cognitive deficit induced by
scopolamine.
3. Conclusion
In conclusion, a novel series of dimethylamino chalcone-O-
alkylamines derivatives was synthesized as multifunctional agents
for the treatment of AD. All the target compounds showed amazing
inhibition of A
ted the greatest inhibitory activity against self-induced A
gation (IC50 ¼ 0.88 M), and well polymerized ability toward self-
induced A aggregation (95.1%, 25 M). Meanwhile, TM-6 could
inhibit Cu2þ-induced A
aggregation (95.3%, 25 M) and disas-
semble the well-structured A fibrils (88.1%, 25 M) by serving as a
selective metal chelator. In addition, TM-6 displayed the best AChE
M), both kinetic analysis of AChE
b
aggregation, particularly, compound TM-6 exhibi-
b
aggre-
m
b
m
b
m
b
m
inhibitory potency (IC50 ¼ 0.13
m
inhibition, molecular docking study and molecular dynamics sim-
ulations suggested that TM-6 binds simultaneously to the catalytic
10

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Fig. 7. The MD simulation of TM-6 with AChE.
active site and peripheral anionic site of AChE. Moreover, com-
pound TM-6 showed good MAO-B inhibitory activity
NMR and 13C NMR spectra were recorded using TMS as the internal
standard on a Varian INOVA at 400 and 100 MHz, respectively.
Coupling constants were given in hertz, Multiplicities are given as s
(singlet), brs (broad singlet), d (doublet), t (triplet), q (quadruplet),
and m (multiplet). Mass spectra were recorded on Agilent-6210 TOF
LC-MS Spectrometer. The purity of all final compounds was deter-
mined by high-performance liquid chromatography (HPLC) anal-
ysis to be over 96%. HPLC analysis was carried out on a Shimadzu
LC-10Avp plus system with the use of a Kromasil C18 column
(IC50 ¼ 1.0
mM). Significantly, compound TM-6 indicated remark-
able antioxidant activity (ORAC-FL values of 2.1eq.) and acted as a
neuroprotectant. Furthermore, TM-6 showed high BBB perme-
ability in vitro. Importantly, TM-6 was nontoxic at doses of up to
1000 mg/kg and it could improve scopolamine-induced memory
impairment. These results indicated that TM-6 was a potential lead
compound for the treatment of AD. Further structural refinements
are underway and will be reported in due course.
(4.6 mm ꢄ 250 mm, 5
mm).
4. Experimental section
4.1.1. 1-(2-Hydroxy-4-methoxyphenyl)ethanone (2)
To a solution of material 1 (10 mmol) and anhydrous K2CO3
(11 mmol) in dry acetone (15 mL) was added Me2SO4 (6 mmol). The
mixture was refluxed for 1 h under an argon atmosphere, after
cooling to room temperature, filtered and washed with acetone, the
solvents was concentrated in vacuum. The crude residue was
4.1. Chemistry
All materials were obtained from commercial suppliers. Melting
points were recorded on YRT-3 melting-point apparatus (China). 1H
11

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Fig. 8. The UV spectrum of compound TM-6 (37.5 mM, in methanol) alone or in the presence of CuCl2, ZnCl2, AlCl3 and FeSO4 (37.5 mM, in methanol).
solid, mp: 63.8e65.2 ꢀC, 60.5% yield. 1H NMR (400 MHz, CDCl3)
d
12.74 (s,1H), 7.63 (d, J ¼ 9.2 Hz,1H), 6.43 (dd, J1 ¼8.8 Hz, J2 ¼ 2.4 Hz,
1H), 6.40 (d, J ¼ 2.4 Hz,1H), 4.03 (t, J ¼ 6.0 Hz, 2H), 3.49 (t, J ¼ 6.4 Hz,
2H), 2.56 (s, 3H), 2.08e2.03 (m, 2H), 2.00e1.95 (m, 2H).
1-(4-(6-Bromohexyloxy)-2-hydroxyphenyl)ethanone (3c). The
starting material 1 was treated with 1,6-dibromohexane according
to the general procedure to give the desired product 3c as a white
solid, mp: 45.8e47.1 ꢀC, 50.6% yield. 1H NMR (400 MHz, CDCl3)
d
12.75 (s, 1H), 7.63 (d, J ¼ 8.8 Hz, 1H), 6.43 (dd, J1 ¼ 8.8 Hz,
J2 ¼ 2.4 Hz, 1H), 6.40 (d, J ¼ 2.4 Hz, 1H), 4.00 (t, J ¼ 6.0 Hz, 2H), 3.43
(t, J ¼ 6.8 Hz, 2H), 2.56 (s, 3H), 1.92e1.87 (m, 2H), 1.85e1.80 (m, 2H),
1.52e1.49 (m, 4H).
Fig. 9. Determination of the stoichiometry of complex-Cu2þ by using molar ratio
4.1.3. General procedure for the synthesis of 5a~5d
method through titrating the methanol solution of compound TM-6 with ascending
amounts of CuCl2. The final concentration of tested compound was 37.5
mM, and the
A solution of benzaldehydes 4a~4d (15 mmol) in methanol
(30 mL) followed by the addition of the ethylamine (15 mmol) and
Na2SO4 (15 mmol). The reaction mixture was stirred at room
temperature for 5e7 h, after which sodium borohydride (7.5 mmol)
was added in batches and the resulting mixture was further stirred
for another period of 2 h. The reaction was then quenched by the
addition of water (10 mL), the resulting inorganic precipitate was
filtered and washed with diethyl ether (20 mL). The organic layer
was separated and the aqueous part was further extracted with
diethyl ether (20 ml ꢄ 2). The combined ether extracts were dried
(K2CO3) and concentrated in vacuo to give N-ethyl secondary
amines in high purity.
final concentration of Cu2þ ranged from 3.75 to 112.5
m
M.
purified by column chromatography on silica gel (petroleum/
acetone as eluent) to give the desired product 2 as colorless oil,
68.1% yield. 1H NMR (400 MHz, CDCl3)
d 12.74 (s, 1H), 7.64 (d,
J ¼ 8.8 Hz, 1H), 6.46e6.43 (m, 2H), 4.15 (t, J ¼ 5.6 Hz, 2H), 3.60 (t,
J ¼ 6.4 Hz, 2H), 2.57 (s, 3H), 2.37e2.31 (m, 2H).
4.1.2. General procedure for the synthesis of 3a~3c
A mixture of starting material 1 (5 mmol), the corresponding
dibromoalkane (11 mmol), and anhydrous K2CO3 (5.5 mmol) in
CH3CN (10 mL) was heated at 65 ꢀC for 6e8 h under an argon at-
mosphere. The solvents were evaporated under a vacuum, water
(30 mL) was added, and the mixture was extracted with dichloro-
methane (30 mL ꢄ 2). The combined organic phases were washed
with saturated aqueous sodium chloride (30 mL), dried over so-
dium sulfate, and filtered. The solvent was evaporated under a
vacuum to give the crude product which was purified by column
chromatography on silica gel (petroleum/acetone as eluent) to
obtain the solid products 3a~3c.
N-Ethyl-2-methoxy-benzenemethanamine (5a). Colorless oil,
86.5% yield. 1H NMR (400 MHz, CDCl3)
d
7.24 (t, J ¼ 7.2 Hz, 2H), 6.91
(t, J ¼ 7.2 Hz, 1H), 6.86 (d, J ¼ 8.8 Hz, 1H), 3.84 (s, 3H), 3.80 (s, 2H),
2.68e2.62 (m, 2H), 1.13 (t, J ¼ 7.2 Hz, 3H).
2-(Dimethylamino)-N-ethyl-benzenemethanamine (5b). Yellow
oil, 64.0% yield.
4-(Dimethylamino)-N-ethyl-benzenemethanamine (5c). Yellow
oil, 66.7% yield. 1H NMR (400 MHz, CDCl3)
d 7.30e7.26 (m, 2H), 6.69
(d, J ¼ 8.0 Hz, 1H), 3.78 (s, 2H), 2.91 (s, 6H), 2.74 (q, J ¼ 6.8 Hz, 2H),
1.24 (t, J ¼ 7.2 Hz, 3H).
1-(4-(3-Bromopropoxy)-2-hydroxyphenyl)ethanone (3a). The
starting material 1 was treated with 1,3-dibromopropane according
to the general procedure to give the desired product 3a as a white
solid, mp: 74.5e75.6 ꢀC, 74.4% yield. 1H NMR (400 MHz, CDCl3)
N-Ethyl-benzenemethanamine (5d). Yellow oil, 78.3% yield. 1H
NMR (400 MHz, CDCl3) d 7.30e7.28 (m, 4H), 7.23e7.20 (m, 1H), 3.75
(s, 2H), 2.65 (q, J ¼ 6.8 Hz, 2H), 1.11 (t, J ¼ 7.2 Hz, 3H).
d
12.74 (s, 1H), 7.64 (d, J ¼ 8.8 Hz, 1H), 6.46e6.43 (m, 2H), 4.15 (t,
J ¼ 5.6 Hz, 2H), 3.60 (t, J ¼ 6.4 Hz, 2H), 2.57 (s, 3H), 2.37e2.31 (m, 2H).
1-(4-(4-Bromobutoxy)-2-hydroxyphenyl)ethanone (3b). The
starting material 1 was treated with 1,4-dibromobutane according
to the general procedure to give the desired product 3b as a white
4.1.4. General procedure for the synthesis of 6a-6e, 7a-7d and 8a-
8d
To a mixture of the intermediates 3a~3c (1.5 mmol), anhydrous
K2CO3 (1.6 mmol) in CH3CN (10 mL) was added the corresponding
12

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Table 4
Inhibition and disaggregation of Cu2þ-induced Ab1-42 aggregation of donepezil, curcumin and dimethylamino chalcone-O-alkylamines derivatives TM-1~TM-19, and inhibitory
activity of MAO isoforms (MAO-A and MAO-B) and selectivity index (SI) values of clorgyline, rasagiline, iproniazid and dimethylamino chalcone-O-alkylamines derivatives.
Comp.
Cu2þ-Induced Ab1-42 (%)a
IC50
(m
M) SDd
SIe
Inhibitionb
Disaggregationc
MAO-B
MAO-A
TM-1
TM-2
TM-3
TM-4
TM-5
TM-6
TM-7
TM-8
n.t.f
n.t.f
0.21 0.008
10.8 0.13
1.1 0.01
0.7 0.01
3.2 0.03
1.0 0.02
2.1 0.03
13.7 0.11
2.4 0.05
2.3 0.03
13.1 0.37
8.9 0.13
2.1 0.19
4.1 0.15
0.8 0.02
4.6 0.22
4.3 0.17
12.1 0.27
13.3 0.32
n.t.f
7.62 0.23
26.3 0.31
27.5 0.46
54.3 0.62
23.60 0.79
18.4 0.87
37.8 0.94
57.5 1.32
17.2 0.68
53.6 1.26
82.6 2.2
77.3 1.7
31.6 0.82
47.1 1.2
67.8 2.2
9.2 0.82
12.3 0.92
47.9 1.3
42.5 1.6
n.t.f
36.3
2.4
25.0
77.6
7.4
18.4
18.0
4.2
95.2 0.51
91.9 0.22
93.2 0.34
94.8 0.11
95.3 0.17
96.5 0.14
95.0 0.12
91.7 0.17
95.6 0.13
91.9 0.31
93.9 0.25
91.0 0.16
n.t.f
74.8 0.13
73.0 0.17
85.6 0.08
89.5 0.06
88.1 0.11
83.4 0.24
77.6 0.23
78.8 0.21
84.4 0.15
73.8 0.12
82.1 0.11
88.3 0.09
n.t.f
TM-9
7.2
23.3
6.3
TM-10
TM-11
TM-12
TM-13
TM-14
TM-15
TM-16
TM-17
TM-18
TM-19
Donepezil
Curcumin
Clorgyline
Rasagiline
Iproniazid
8.7
15.0
11.5
84.8
2.0
2.9
4.0
n.t.f
n.t.f
n.t.f
n.t.f
n.t.f
n.t.f
n.t.f
n.t.f
n.t.f
n.t.f
3.2
n.t.f
n.t.f
n.t.f
n.t.f
0.0001
20.9
4.1
81.0 0.21
56.5 0.21
n.t.f
n.t.f
n.t.f
n.t.f
20.8 0.27
0.0281 0.0068
1.35 0.02
0.0027 0.0001
0.587 0.038
5.48 0.03
n.t.f
n.t.f
n.t.f
n.t.f
a
Cu2þ-induced A
b aggregation, the thioflavin-T fluorescence method was used.
b
Inhibition of Cu2þ-induced Ab1-42 aggregation. The concentration of tested compounds and Cu2þ both were 25
mM.
c
d
e
f
Disaggregation of Cu2þ-induced Ab1-42 aggregation. The concentration of tested compounds and Cu2þ both were 25
All IC50 values shown in this table are the mean SEM from three experiments.
hMAO-B selectivity index ¼ IC50 (hMAO-A)/IC50 (hMAO-B).
mM.
n.t. ¼ not tested.
Fig. 10. Visualization of A
b
species from inhibition experiments: (Top) scheme of the inhibition of Cu2þ-induced Ab1-42 aggregation experiment. (A) results of the ThT binding assay.
(B) TEM images of samples.
13

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Fig. 11. Visualization of A
b
species from disaggregation experiments: (Top) scheme of the disaggregation of Cu2þ-induced Ab1-42 aggregation experiments. (A) results of the ThT
binding assay. (B) TEM images of samples.
secondary amines 5a~5d and diethylamine (1.5 mmol). The reac-
tion mixture was heated to 65 ꢀC for 6e8 h under an argon atmo-
sphere. After complete reaction, the solvent was concentrated in
vacuum. Then water (30 mL) was added to the residue and the
mixture was extracted with dichloromethane (30 mL ꢄ 2). The
combined organic phases were washed with saturated aqueous
NaCl (50 mL), dried over Na2SO4, and filtered. The solvent was
evaporated to dryness under reduced pressure. The residue was
purified on a silica gel chromatography using mixtures of petro-
leum/acetone as eluent to obtain the oil products 6a-6e, 7a-7d and
8a-8d.
(dimethylamino)-N-ethyl-benzenemethanamine according to the
general procedure to give the desired product 6c as light yellow oil,
70.9% yield. 1H NMR (400 MHz, CDCl3)
d 12.74 (s, 1H), 7.61 (d,
J ¼ 9.2 Hz, 1H), 7.19 (d, J ¼ 8.0 Hz, 2H), 6.69 (d, J ¼ 8.4 Hz, 1H),
6.41e6.37 (m, 2H), 4.02 (t, J ¼ 6.4 Hz, 2H), 3.57 (s, 2H), 2.92 (s, 6H),
2.63e2.57 (m, 4H), 2.56 (s, 3H), 1.99e1.97 (m, 2H), 1.10 (t, J ¼ 6.4 Hz,
3H).
1-(4-(3-(Benzyl(ethyl)amino)propoxy)-2-hydroxyphenyl)etha-
none (6d). Intermediate 3a was treated with N-ethyl-benzeneme-
thanamine according to the general procedure to give the desired
product 6d as light yellow oil, 69.1% yield. 1H NMR (400 MHz,
1-(4-(3-(Ethyl(2-methoxybenzyl)amino)propoxy)-2-
CDCl3)
d
12.75 (s, 1H), 7.61 (d, J ¼ 9.6 Hz, 1H), 7.33e7.22 (m, 5H),
hydroxyphenyl)ethanone (6a). Intermediate 3a was treated with N-
ethyl-2-methoxy-benzenemethanamine according to the general
procedure to give the desired product 6a as light yellow oil, 71.3%
6.39e6.37 (m, 2H), 4.03 (t, J ¼ 6.4 Hz, 2H), 3.61 (s, 2H), 2.63e2.60
(m, 2H), 2.59e2.57 (m, 2H), 2.56 (s, 3H), 1.97e1.94 (m, 2H), 1.06 (t,
J ¼ 6.4 Hz, 3H).
yield. 1H NMR (400 MHz, CDCl3)
d
12.75 (s, 1H), 7.61 (d, J ¼ 9.2 Hz,
1-(4-(3-(Diethylamino)propoxy)-2-hydroxyphenyl)ethanone
(6e). Intermediate 3a was treated with diethylamine according to
the general procedure to give the desired product 6e as light yellow
1H), 7.39 (d, J ¼ 6.8 Hz, 1H), 7.21 (t, J ¼ 7.6 Hz, 1H), 6.91 (t, J ¼ 7.6 Hz,
1H), 6.85 (d, J ¼ 8.0 Hz, 1H), 6.42e6.38 (m, 2H), 4.05 (t, J ¼ 6.4 Hz,
2H), 3.81 (s, 3H), 3.63 (s, 2H), 2.66e2.64 (m, 2H), 2.59e2.57 (m, 2H),
2.56 (s, 3H), 1.99e1.96 (m, 2H), 1.07 (t, J ¼ 6.4 Hz, 3H).
oil, 73.0% yield. 1H NMR (400 MHz, CDCl3)
d 12.70 (s, 1H), 7.63 (d,
J ¼ 8.8 Hz, 1H), 6.43 (dd, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, 1H), 6.38 (d,
J ¼ 2.4 Hz, 1H), 4.08 (t, J ¼ 6.0 Hz, 2H), 2.94e2.85 (m, 6H), 2.55 (s,
3H), 2.20e2.17 (m, 2H), 1.25 (t, J ¼ 7.2 Hz, 6H).
1-(4-(3-((2-(Dimethylamino)benzyl)(ethyl)amino)propoxy)-2-
hydroxyphenyl)ethanone (6b). Intermediate 3a was treated with 2-
(dimethylamino)-N-ethyl-benzenemethanamine according to the
general procedure to give the desired product 6b as light yellow oil,
1-(4-(4-(Ethyl(2-methoxybenzyl)amino)butoxy)-2-
hydroxyphenyl)ethanone (7a). Intermediate 3b was treated with N-
ethyl-2-methoxy-benzenemethanamine according to the general
procedure to give the desired product 7a as light yellow oil, 67.1%
71.7% yield. 1H NMR (400 MHz, CDCl3)
d 12.74 (s, 1H), 7.63 (d,
J ¼ 10.4 Hz, 1H), 7.61e7.58 (m, 1H), 7.21 (t, J ¼ 7.6 Hz, 1H), 7.09 (d,
J ¼ 7.6 Hz, 1H), 7.03 (t, J ¼ 7.6 Hz, 1H), 6.41e6.37 (m, 2H), 4.04 (t,
J ¼ 6.4 Hz, 2H), 3.68 (s, 2H), 2.67 (s, 6H), 2.66e2.58 (m, 4H), 2.56 (s,
3H), 1.99e1.97 (m, 2H), 1.08 (t, J ¼ 6.4 Hz, 3H).
yield.1H NMR (400 MHz, CDCl3)
d
12.74 (s,1H), 7.62 (d, J ¼ 9.2 Hz,1H),
7.49e7.47 (m,1H), 7.26 (t, J ¼ 7.2Hz,1H), 6.95(t, J ¼ 7.2Hz,1H), 6.87(d,
J ¼ 8.0 Hz,1H), 6.41 (dd, J1 ¼8.8 Hz, J2 ¼ 2.4 Hz,1H), 6.37 (d, J ¼ 2.4 Hz,
1H), 3.97 (t, J ¼ 5.6 Hz, 2H), 3.83(s, 3H), 3.74 (s, 2H), 2.72e2.58(m, 4H),
2.56 (s, 3H), 1.82e1.71 (m, 4H), 1.16 (t, J ¼ 6.4 Hz, 3H).
1-(4-(3-((4-(Dimethylamino)benzyl)(ethyl)amino)propoxy)-2-
hydroxyphenyl)ethanone (6c). Intermediate 3a was treated with 4-
14

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Fig. 12. (A) Representation of compound TM-6 (green stick) interacting with residues in the binding site of hMAO-B (PDB code: 2V60). (B) 3D docking model of compound TM-6
(green stick) with hMAO-B.
1-(4-(4-((2-(Dimethylamino)benzyl)(ethyl)amino)butoxy)-2-
hydroxyphenyl)ethanone (7b). Intermediate 3b was treated with 2-
(dimethylamino)-N-ethyl-benzenemethanamine according to the
general procedure to give the desired product 7b as light yellow oil,
1-(4-(4-(Benzyl(ethyl)amino)butoxy)-2-hydroxyphenyl)etha-
none (7d). Intermediate 3b was treated with N-ethyl-benzeneme-
thanamine according to the general procedure to give the desired
product 7d as light yellow oil, 77.8% yield. 1H NMR (400 MHz,
78.7% yield. 1H NMR (400 MHz, CDCl3)
d
12.73 (s, 1H), 7.74e7.72 (m,
CDCl3)
d
12.74 (s,1H), 7.61 (d, J ¼ 8.8 Hz,1H), 7.37e7.25 (m, 5H), 6.41
1H), 7.62 (d, J ¼ 8.8 Hz, 1H), 7.27e7.25 (m, 1H), 7.13 (d, J ¼ 8.4 Hz,
1H), 7.10 (t, J ¼ 6.8 Hz,1H), 6.40 (dd, J1 ¼8.8 Hz, J2 ¼ 2.4 Hz,1H), 6.36
(d, J ¼ 2.4 Hz, 1H), 3.96 (t, J ¼ 5.6 Hz, 2H), 3.94 (s, 2H), 2.74e2.66 (m,
4H), 3.65 (s, 6H), 2.56 (s, 3H), 1.82e1.78 (m, 4H), 1.19 (t, J ¼ 6.4 Hz,
3H).
(dd, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, 1H), 6.37 (d, J ¼ 2.4 Hz, 1H), 3.95 (t,
J ¼ 6.4 Hz, 2H), 3.66 (s, 2H), 2.62e2.57 (m, 4H), 2.56 (s, 3H),
1.83e1.77 (m, 2H), 1.72e1.69 (m, 2H), 1.11 (t, J ¼ 6.4 Hz, 3H).
1-(4-(6-(Ethyl(2-methoxybenzyl)amino)hexyloxy)-2-
hydroxyphenyl)ethanone (8a). Intermediate 3c was treated with N-
ethyl-2-methoxy-benzenemethanamine according to the general
procedure to give the desired product 8a as light yellow oil, 80.7%
1-(4-(4-((4-(Dimethylamino)benzyl)(ethyl)amino)butoxy)-2-
hydroxyphenyl)ethanone (7c). Intermediate 3b was treated with 4-
(dimethylamino)-N-ethyl-benzenemethanamine according to the
general procedure to give the desired product 7c as light yellow oil,
yield. 1H NMR (400 MHz, CDCl3)
d
12.75 (s, 1H), 7.63 (d, J ¼ 8.8 Hz,
1H), 7.59 (d, J ¼ 6.8 Hz, 1H), 7.31 (t, J ¼ 7.6 Hz, 1H), 7.00 (t, J ¼ 7.2 Hz,
1H), 6.91 (d, J ¼ 8.0 Hz, 1H), 6.43 (dd, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, 1H),
6.39 (d, J ¼ 2.0 Hz, 1H), 3.97 (t, J ¼ 6.4 Hz, 2H), 3.95 (s, 2H), 3.86 (s,
3H), 2.87e2.85 (m, 2H), 2.76e2.74 (m, 2H), 2.56 (s, 3H), 1.82e1.74
(m, 4H), 1.50e1.43 (m, 2H), 1.40e1.32 (m, 2H), 1.28 (t, J ¼ 6.4 Hz,
3H).
58.9% yield. 1H NMR (400 MHz, CDCl3)
d 12.74 (s, 1H), 7.62 (d,
J ¼ 8.8 Hz, 1H), 7.22 (d, J ¼ 8.4 Hz, 2H), 6.67 (d, J ¼ 8.4 Hz, 1H), 6.42
(dd, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, 2H), 3.96 (t, J ¼ 6.0 Hz, 2H), 3.66 (s, 2H),
2.93 (s, 6H), 2.72e2.59 (m, 4H), 2.56 (s, 3H), 1.82e1.78 (m, 4H), 1.16
(t, J ¼ 6.4 Hz, 3H).
15

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Fig. 13. (A) Cytotoxicity of TM-6. (B and C) Attenuation of H2O2-induced PC12 cell injury by compound TM-6. Cell viability was tested by measuring the MTT reduction. Three
independent experiments were carried out in triplicate. Data were expressed as mean SD and percentage of control value. ##p < 0.01 vs.control; **p < 0.01, *p < 0.05 vs H2O2
group.
1.49e1.41 (m, 2H), 1.39e1.33 (m, 2H), 1.15 (t, J ¼ 6.4 Hz, 3H).
Table 5
1-(4-(6-(Benzyl(ethyl)amino)hexyloxy)-2-hydroxyphenyl)etha-
Permeability Pe ( ꢄ 10ꢁ6 cm/s) in the PAMPA-BBB assay of the selected compound
none (8d). Intermediate 3c was treated with N-ethyl-benzeneme-
thanamine according to the general procedure to give the desired
product 8d as light yellow oil, 75.2% yield. 1H NMR (400 MHz,
TM-6 and commercial drugs, and their predictive penetration in the CNS.
Compound
Pe ( ꢄ 10ꢁ6 cm/s)
Prediction
TM-6
13.37 0.37
17.46 0.68
12.39 0.73
0.42 0.02
CNSþ
CNSþ
CNSþ
CNS-
CDCl3)
d
12.75 (s, 1H), 7.62 (d, J ¼ 8.8 Hz, 1H), 7.45e7.42 (m, 2H),
Diazepam
Verapamil
Norfloxacin
7.35e7.30 (m, 3H), 6.42 (d, J ¼ 8.8 Hz, 1H), 6.39 (s, 1H), 3.97 (t,
J ¼ 6.4 Hz, 2H), 3.77 (s, 2H), 2.73e2.69 (m, 2H), 2.63e2.59 (m, 2H),
2.55 (s, 3H), 1.79e1.74 (m, 2H), 1.67e1.62 (m, 2H), 1.46e1.40 (m,
2H), 1.38e1.34 (m, 2H), 1.20e1.17 (m, 3H).
1-(4-(6-((2-(Dimethylamino)benzyl)(ethyl)amino)hexyloxy)-2-
hydroxyphenyl)ethanone (8b). Intermediate 3c was treated with 2-
(dimethylamino)-N-ethyl-benzenemethanamine according to the
general procedure to give the desired product 8b as light yellow oil,
4-(4-Bromobutoxy)benzaldehyde (10). P-hydroxy benzaldehyde
9 was treated with 1,4-dibromobutane according to the general
procedure to give the desired product 16 as colourless oil, 55.1%
yield. 1H NMR (400 MHz, CDCl3)
d
9.88 (s, 1H), 7.83 (d, J ¼ 8.0 Hz,
71.9% yield. 1H NMR (400 MHz, CDCl3)
d 12.74 (s, 1H), 7.87e7.83 (m,
2H), 6.99 (d, J ¼ 8.4 Hz, 2H), 4.08 (t, J ¼ 6.0 Hz, 2H), 3.49 (t,
J ¼ 5.6 Hz, 2H), 2.12e2.04 (m, 2H), 2.02e1.97 (m, 2H).
4.15 General Procedure for the Synthesis of 11a-11b. Compounds
11a-1b was prepared according to the procedure of 6a-e, 7a-d and
8a-d.
1H), 7.63 (d, J ¼ 8.8 Hz, 1H), 7.28e7.26 (m, 1H), 7.17e7.13 (m, 2H),
6.42 (dd, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, 1H), 6.38 (d, J ¼ 2.4 Hz, 1H), 4.14 (s,
2H), 3.96 (t, J ¼ 6.4 Hz, 2H, 2.84e2.69 (m, 4H), 2.66 (s, 6H), 2.56 (s,
3H), 1.82e1.73 (m, 2H), 1.70e1.63 (m, 2H), 1.50e1.41 (m, 2H),
1.39e1.31 (m, 2H), 1.20 (t, J ¼ 6.4 Hz, 3H).
4-(4-(Ethyl(2-methoxybenzyl)amino)butoxy)benzaldehyde
(11a). Intermediate 10 was treated with N-ethyl-2-methoxy-ben-
zenemethanamine according to the general procedure to give the
product 11a as colorless oil, 89.5% yield. 1H NMR (400 MHz, CDCl3)
1-(4-(6-((4-(Dimethylamino)benzyl)(ethyl)amino)hexyloxy)-2-
hydroxyphenyl)ethanone (8c). Intermediate 3c was treated with 4-
(dimethylamino)-N-ethyl-benzenemethanamine according to the
general procedure to give the desired product 8c as light yellow oil,
d
9.88 (s, 1H), 7.81 (d, J ¼ 8.4 Hz, 2H), 7.43 (d, J ¼ 7.2 Hz, 1H), 7.22 (d,
74.6% yield. 1H NMR (400 MHz, CDCl3)
d 12.75 (s, 1H), 7.62 (d,
J ¼ 7.6 Hz,1H), 6.96 (d, J ¼ 8.8 Hz, 2H), 6.92 (t, J ¼ 7.2 Hz,1H), 6.85 (d,
J ¼ 8.0 Hz, 1H), 4.02 (t, J ¼ 6.4 Hz, 2H), 3.81 (s, 3H), 3.65 (s, 2H),
2.59e2.54 (m, 4H), 1.86e1.80 (m, 2H), 1.73e1.69 (m, 2H), 1.09 (t,
J ¼ 6.8 Hz, 3H).
J ¼ 8.8 Hz, 1H), 7.24 (d, J ¼ 8.0 Hz, 2H), 6.70 (d, J ¼ 8.4 Hz, 1H), 6.43
(dd, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, 1H), 6.39 (d, J ¼ 2.0 Hz, 1H), 3.97 (t,
J ¼ 6.4 Hz, 2H), 3.66 (s, 2H), 2.94 (s, 6H), 2.66e2.63 (m, 2H), 2.56 (s,
3H), 2.55e2.53 (m, 2H), 1.82e1.74 (m, 2H), 1.64e1.61 (m, 2H),
16

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Fig. 14. Effects of compound TM-6 on scopolamine-induced memory impairment. Values are expressed as the mean SEM (n ¼ 10). ##P < 0.01 vs vehicle-treated. *P < 0.05 and
**P < 0.01 vs model group.
4-(4-((2-(Dimethylamino)benzyl)(ethyl)amino)butoxy)benzal-
dehyde (11b). Intermediate 10 was treated with 2-(dimethyla-
mino)-N-ethyl-benzenemethanamine according to the general
procedure to give the desired product 11b as colorless oil, 75.2%
with saturated aqueous NaHCO3, saturated aqueous NaCl (30 mL),
dried over Na2SO4, and filtered. The solvent was evaporated in
vacuum, the crude product was purified on a silica gel chroma-
tography using mixtures of CH2Cl2/acetone as eluent to obtain the
product TM-1~TM-19.
yield. 1H NMR (400 MHz, CDCl3)
d
9.88 (s, 1H), 7.82 (d, J ¼ 8.8 Hz,
2H), 7.33e7.31 (m, 1H), 7.25e7.23 (m, 1H), 7.14e7.09 (m, 2H), 6.95
(d, J ¼ 8.8 Hz, 2H), 4.02 (t, J ¼ 6.8 Hz, 2H), 3.91 (s, 2H), 2.73e2.69 (m,
4H), 2.66 (s, 6H), 1.85e1.82 (m, 4H), 1.18 (t, J ¼ 6.8 Hz, 3H).
(E)-3-(4-(Dimethylamino)phenyl)-1-(2-hydroxy-4-
methoxyphenyl)-2-prop-1-one (TM-1). 4-Dimethylamine benzal-
dehyde was treated with intermediate 2 according to the general
procedure to give the desired product TM-1 as orange oil, 43.5%
3-(Dimethylamino)acetatephenol
(13).
Acetylchloride
(5.5 mmol) was added slowly to a mixture of the compound 12
(5 mmol), Et3N (6 mmol) in CH2Cl2 (10 mL) at ꢁ10 ꢀC. After 0.5 h,
the mixture was warmed to room temperature and stirred for 1 h.
Then water (20 mL) was added to the mixture and extracted with
dichloromethane (30 mL ꢄ 3). The combined organic phases were
washed with saturated aqueous NaHCO3, saturated aqueous NaCl
(30 mL), dried over Na2SO4, and filtered. The solvent was evapo-
rated to dryness under reduced pressure. The residue was purified
on a silica gel chromatography using mixtures of petroleum/
acetone as eluent to obtain the oil product 13.
yield. 1H NMR (400 MHz, CDCl3)
d
13.79 (s, 1H), 7.88 (d, J ¼ 15.2 Hz,
1H), 7.84 (d, J ¼ 9.2 Hz, 1H), 7.56 (d, J ¼ 8.8 Hz, 2H), 7.38 (d,
J ¼ 15.2 Hz, 1H), 6.70 (d, J ¼ 9.2 Hz, 2H), 6.49e6.45 (m, 2H), 3.85 (s,
3H), 3.05 (s, 6H). MS (ESI) m/z: 298.2 [M þ H]þ.
(E)-3-(4-(Dimethylamino)phenyl)-1-(4-(3-(ethyl(2-
methoxybenzyl)amino)propoxy)-2-hydroxyphenyl)prop-2-en-1-
one (TM-2). 4-Dimethylamine benzaldehyde was treated with in-
termediate 6a according to the general procedure to give the
desired product TM-2 as orange oil, 41.2% yield, 98.6% HPLC purity.
1H NMR (400 MHz, CDCl3)
d
13.77 (s, 1H), 7.88 (d, J ¼ 15.2 Hz, 1H),
1-(4-(Dimethylamino)-2-hydroxyphenyl)ethanone
(14).
A
7.83 (d, J ¼ 9.2 Hz, 1H), 7.56 (d, J ¼ 8.4 Hz, 2H), 7.53e7.48 (m, 1H),
7.38 (d, J ¼ 15.2 Hz, 1H),7.33e7.26 (m, 1H), 6.97 (t, J ¼ 7.2 Hz, 1H),
6.89 (d, J ¼ 8.4 Hz, 1H), 6.70 (d, J ¼ 8.4 Hz, 2H), 6.44e6.39 (m, 2H),
4.07 (t, J ¼ 6.0 Hz, 2H), 3.88 (s, 2H), 3.84 (s, 3H), 3.06 (s, 6H),
2.88e2.69 (m, 4H), 2.22e2.15 (m, 2H), 1.21 (t, J ¼ 6.8 Hz, 3H). MS
(ESI) m/z: 489.3 [M þ H]þ.
mixture of the compound 13 (10 mmol), NaCl (10 mmol), AlCl3
(30 mmol) was heated at 160 ꢀC for 2 h. After cooling to room
temperature, then water (10 mL) and 10% HCl (10 mL) were added
to the mixture, stirred for 1 h and extracted with ethyl acetate
(30 mL ꢄ 3). The combined organic phases were washed with
saturated aqueous NaHCO3 (30 mL), saturated aqueous NaCl
(30 mL), dried over Na2SO4 and filtered. The solvent was evaporated
to dryness under reduced pressure. The residue was recrystallized
from ethanol to obtain the yellow crystal plate product 14, mp:
(E)-3-(4-(Dimethylamino)phenyl)-1-(4-(4-(ethyl(2-
methoxybenzyl)amino)butoxy)-2-hydroxyphenyl)prop-2-en-1-
one (TM-3). 4-Dimethylamine benzaldehyde was treated with in-
termediate 7a according to the general procedure to give the
desired product TM-3 as orange oil, 31.6% yield, 98.1% HPLC purity.
121.3e122.1 ꢀC, 56.3% yield. 1H NMR (400 MHz, CDCl3)
d 12.88 (s,
1H), 7.53 (d, J ¼ 9.2 Hz, 1H), 6.21 (dd, J1 ¼ 9.2 Hz, J2 ¼ 2.4 Hz, 1H),
1H NMR (400 MHz, CDCl3)
d
13.78 (s, 1H), 7.88 (d, J ¼ 15.6 Hz, 1H),
6.09 (d, J ¼ 2.4 Hz, 1H), 3.05 (s, 6H), 2.49 (s, 3H).
7.83 (d, J ¼ 8.8 Hz, 1H), 7.57 (d, J ¼ 8.4 Hz, 2H), 7.56e7.52 (m, 1H),
7.38 (d, J ¼ 15.2 Hz, 1H), 7.34e7.26 (m, 1H), 6.98 (t, J ¼ 7.2 Hz, 1H),
6.89 (d, J ¼ 7.6 Hz, 1H), 6.70 (d, J ¼ 8.4 Hz, 2H), 6.46e6.40 (m, 2H),
4.00 (t, J ¼ 6.0 Hz, 2H), 3.98 (s, 2H), 3.84 (s, 3H), 3.06 (s, 6H),
4.16 General Procedure for the Synthesis of TM-1~TM-19. To a
mixture of acetophenone derivatives (1 mmol) in EtOH (3 mL) was
added the appropriate benzaldehyde (1 mmol). An amount of a 30%
aqueous KOH (4 mmol) solution was then slowly added dropwise to
the reaction. The mixture was stirred for 72 h at 50 ꢀC. After cooling
to room temperature, 10% HCl was added dropwise to the mixture
making pH ¼ 2, and then NaHCO3 powder was added adjusting
pH ¼ 8, diluted with H2O (10 mL), and extracted with dichloro-
methane (10 mL ꢄ 3). The combined organic phases were washed
2.86e2.65 (m, 4H), 1.94e1.78 (m, 4H), 1.23 (t, J ¼ 6.8 Hz, 3H). 13
C
NMR (100 MHz, CDCl3)
d 191.72, 166.28, 164.89, 157.76, 152.04,
145.34, 131.35, 130.90, 130.55 (2C), 129.23, 122.32, 120.63, 114.34,
114.20, 111.69 (2C), 110.43, 107.38, 101.51, 67.65, 55.35, 51.98, 50.61,
47.35, 40.02 (2C), 26.62, 22.21, 10.61. MS (ESI) m/z: 503.3 [M þ H]þ.
(E)-3-(4-(Dimethylamino)phenyl)-1-(4-((6-(ethyl(2-
17

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
methoxybenzyl)amino)hexyl)oxy)-2-hydroxyphenyl)prop-2-en-1-
one (TM-4). 4-Dimethylamine benzaldehyde was treated with in-
termediate 8a according to the general procedure to give the
desired product TM-4 as orange oil, 37.5% yield, 97.7% HPLC purity.
2.78e2.69 (m, 4H), 2.12e2.08 (m, 2H), 1.17 (t, J ¼ 7.2 Hz, 3H). 13C
NMR (100 MHz, CDCl3)
d 191.76, 166.31, 164.99, 152.06, 149.96,
145.30, 130.84 (2C), 130.56 (2C), 130.23, 122.42, 114.46, 114.21,
112.35 (2C), 111.74 (2C), 107.42, 101.60, 66.18, 57.06, 48.79, 47.09,
40.55 (2C), 40.06 (2C), 26.06, 11.14. MS (ESI) m/z: 502.3 [M þ H]þ.
(E)-1-(4-(4-((4-(Dimethylamino)benzyl)(ethyl)amino)butoxy)-
2-hydroxyphenyl)-3-(4-(dimethylamino)phenyl)prop-2-en-1-one
(TM-9). 4-Dimethylamine benzaldehyde was treated with inter-
mediate 7c according to the general procedure to give the desired
product TM-9 as orange oil, 30.8% yield, 97.8% HPLC purity. 1H NMR
1H NMR (400 MHz, CDCl3)
d13.79 (s, 1H), 7.87 (d, J ¼ 15.2 Hz, 1H),
7.83 (d, J ¼ 8.8 Hz, 1H), 7.56 (d, J ¼ 8.8 Hz, 2H), 7.53e7.46 (m, 1H),
7.38 (d, J ¼ 15.2 Hz, 1H), 7.30e7.24 (m, 1H), 6.96 (t, J ¼ 7.2 Hz, 1H),
6.87 (d, J ¼ 8.0 Hz, 1H), 6.70 (d, J ¼ 8.0 Hz, 2H), 6.48e6.41 (m, 2H),
3.98 (t, J ¼ 6.0 Hz, 2H), 3.88 (s, 3H), 3.78 (s, 2H), 3.05 (s, 6H),
2.73e2.52 (m, 4H), 1.82e1.74 (m, 2H), 1.66e1.58 (m, 2H), 1.50e1.45
(m, 2H), 1.39e1.32 (m, 2H), 1.17 (m, 3H). 13C NMR (100 MHz, CDCl3)
(400 MHz, CDCl3)
d
13.79 (s, 1H), 7.88 (d, J ¼ 15.2 Hz, 1H), 7.85 (d,
d
191.70, 166.32, 165.10, 157.70, 152.02, 145.26, 131.14, 130.85, 130.52
J ¼ 9.2 Hz, 1H), 7.57 (d, J ¼ 8.8 Hz, 2H), 7.38 (d, J ¼ 15.2 Hz, 1H), 7.32
(d, J ¼ 8.4 Hz, 2H), 6.70 (d, J ¼ 8.8 Hz, 2H), 6.63 (d, J ¼ 8.4 Hz, 2H),
6.45 (dd, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, 1H), 6.41 (d, J ¼ 2.0 Hz, 1H), 4.00 (t,
J ¼ 5.6 Hz, 2H), 3.98 (s, 2H), 3.06 (s, 6H), 2.98e2.81 (m, 4H), 2.93 (s,
(2C), 128.95, 122.34, 120.55, 114.38, 114.09, 111.68 (2C), 110.33,
107.54, 101.40, 68.00, 55.31, 52.54, 50.71, 47.30, 40.01 (2C), 28.80,
26.92, 25.67 (2C), 10.73. MS (ESI) m/z: 531.3 [M þ H]þ.
(E)-1-(4-(3-((2-(Dimethylamino)benzyl)(ethyl)amino)pro-
6H), 2.09e1.98 (m, 2H),1.88e1.80 (m, 2H),1.40 (t, J ¼ 7.2 Hz, 3H). 13
C
poxy)-2-hydroxyphenyl)-3-(4-(dimethylamino)phenyl)prop-2-en-
1-one (TM-5). 4-Dimethylamine benzaldehyde was treated with
intermediate 6b according to the general procedure to give the
desired product TM-5 as orange oil, 31.9% yield, 98.4% HPLC purity.
NMR (100 MHz, CDCl3) d 191.74, 166.22, 164.57, 152.09, 150.79,
145.49,131.61 (2C),130.97,130.58 (2C),122.26,114.36,114.22,112.14
(2C), 111.69 (2C), 107.23, 101.63, 66.97, 55.71, 50.26, 46.56, 40.11
(2C), 40.02 (2C), 26.25, 20.76, 9.47. MS (ESI) m/z: 516.4 [M þ H]þ.
(E)-1-(4-(6-((4-(Dimethylamino)benzyl)(ethyl)amino)hex-
yloxy)-2-hydroxyphenyl)-3-(4-(dimethylamino)phenyl)prop-2-
en-1-one (TM-10). 4-Dimethylamine benzaldehyde was treated
with intermediate 8c according to the general procedure to give the
desired product TM-10 as orange oil, 26.7% yield, 97.6% HPLC purity.
1H NMR (400 MHz, CDCl3)
d
13.78 (s, 1H), 7.88 (d, J ¼ 15.2 Hz, 1H),
7.82 (d, J ¼ 9.6 Hz, 1H), 7.56 (d, J ¼ 8.8 Hz, 2H), 7.38 (d, J ¼ 15.2 Hz,
1H), 7.26e7.19 (m, 1H), 7.14e7.09 (m, 1H), 6.70 (d, J ¼ 8.8 Hz, 2H),
6.43e6.40 (m, 2H), 4.05 (t, J ¼ 6.0 Hz, 2H), 3.75 (s, 2H), 3.06 (s, 6H),
2.82e2.54 (m, 4H), 2.67 (s, 6H), 2.09e2.00 (m, 2H), 1.15e1.11 (m,
3H). MS (ESI) m/z: 502.3 [M þ H]þ.
1H NMR (400 MHz, CDCl3)
d
13.79 (s, 1H), 7.87 (d, J ¼ 15.2 Hz, 1H),
(E)-1-(4-(4-((2-(Dimethylamino)benzyl)(ethyl)amino)butoxy)-
2-hydroxyphenyl)-3-(4-(dimethylamino)phenyl)prop-2-en-1-one
(TM-6). 4-Dimethylamine benzaldehyde was treated with inter-
mediate 7b according to the general procedure to give the desired
product TM-6 as orange oil, 33.2% yield, 98.7% HPLC purity. 1H NMR
7.83 (d, J ¼ 9.2 Hz, 1H), 7.56 (d, J ¼ 8.8 Hz, 2H), 7.38 (d, J ¼ 15.2 Hz,
1H), 7.35e7.31 (m, 2H), 6.69 (d, J ¼ 8.0 Hz, 4H), 6.45 (d, J ¼ 9.2 Hz,
1H), 6.42 (s, 1H), 4.00 (s, 2H), 3.98 (t, J ¼ 6.0 Hz, 2H), 3.05 (s, 6H),
2.98e2.90 (m, 2H), 2.96 (s, 6H), 2.88e2.78 (m, 2H), 1.86e1.75 (m,
4H), 1.51e1.46 (m, 2H), 1.42e1.39 (m, 2H), 1.38e1.35 (m, 3H). 13C
(400 MHz, CDCl3)
d
13.79 (s, 1H), 7.87 (d, J ¼ 15.2 Hz), 7.82 (d,
NMR (100 MHz, CDCl3) d 191.71, 166.28, 165.01, 152.04, 150.79,
J ¼ 8.8 Hz), 7.73e7.68 (m, 1H), 7.55 (d, J ¼ 8.8 Hz, 2H), 7.37 (d,
J ¼ 15.2 Hz, 1H), 7.23 (d, J ¼ 7.6 Hz, 1H), 7.10 (t, J ¼ 8.8 Hz, 1H), 6.69
(d, J ¼ 8.4 Hz, 2H), 6.44e6.39 (m, 2H), 3.97 (t, J ¼ 6.0 Hz, 2H), 3.83 (s,
2H), 3.05 (s, 6H), 2.72e2.58 (m, 4H), 2.67 (s, 6H), 1.80e1.75 (m, 4H),
145.32,131.60 (2C),130.89,130.54 (2C),122.30,114.33,114.12,112.17
(2C), 111.68 (2C), 107.50, 101.41, 66.79, 55.87, 50.82, 46.22, 40.17,
40.15, 40.02, 40.00, 28.63, 26.55, 25.41, 23.79. MS (ESI) m/z: 544.4
[M þ H]þ.
1.14 (t, J ¼ 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d
191.71, 166.30,
(E)-1-(4-(3-(Benzyl(ethyl)amino)propoxy)-2-hydroxyphenyl)-
164.98, 153.12, 152.02, 145.29, 130.86, 130.53 (2C), 128.96, 127.95,
123.93,119.10,114.37,114.16,111.69 (2C),107.44,101.47, 67.79, 52.33,
51.95, 47.38, 45.24 (2C), 40.08, 40.00, 26.65, 22.67,11.00. MS (ESI) m/
z: 516.4 [M þ H]þ.
3-(4-(dimethylamino)phenyl)prop-2-en-1-one
(TM-11).
4-
Dimethylamine benzaldehyde was treated with intermediate 6d
according to the general procedure to give the desired product TM-
11 as orange oil, 36.5% yield, 97.9% HPLC purity. 1H NMR (400 MHz,
(E)-1-(4-(6-((2-(Dimethylamino)benzyl)(ethyl)amino)hex-
yloxy)-2-hydroxyphenyl)-3-(4-(dimethylamino)phenyl)prop-2-
en-1-one (TM-7). 4-Dimethylamine benzaldehyde was treated with
intermediate 8b according to the general procedure to give the
desired product TM-7 as orange oil, 38.5% yield, 98.2% HPLC purity.
CDCl3)
d
13.79 (s, 1H), 7.88 (d, J ¼ 14.8 Hz, 1H), 7.82 (d, J ¼ 8.8 Hz,
1H), 7.57 (d, J ¼ 8.8 Hz, 2H), 7.38 (d, J ¼ 15.2 Hz, 1H), 7.36e7.26 (m,
5H), 6.70 (d, J ¼ 8.8 Hz, 2H), 6.43e6.38 (m, 2H), 4.06 (t, J ¼ 6.0 Hz,
2H), 3.59 (s, 2H), 3.06 (s, 6H), 2.68e2.52 (m, 4H), 1.98e1.93 (m, 2H),
1.07 (t, J ¼ 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d 191.80, 166.36,
1H NMR (400 MHz, CDCl3)
d
13.79 (s, 1H), 7.87 (d, J ¼ 15.2 Hz, 1H),
165.00, 152.09, 145.35, 130.87, 130.58 (2C), 129.10, 128.35, 127.29,
122.46, 114.50, 114.23, 111.76 (2C), 107.51, 101.55, 66.13, 57.84, 49.21,
47.33, 40.09 (2C), 26.23, 11.29. MS (ESI) m/z: 459.3 [M þ H]þ.
(E)-1-(4-(4-(Benzyl(ethyl)amino)butoxy)-2-hydroxyphenyl)-3-
7.83 (d, J ¼ 8.8 Hz, 2H), 7.38 (d, J ¼ 15.2 Hz, 1H), 7.27e7.24 (m, 1H),
7.15e7.10 (m, 1H), 7.00 (d, J ¼ 8.8 Hz, 2H), 6.46e6.39 (m, 2H), 3.98 (t,
J ¼ 6.0 Hz, 2H), 3.94 (s, 2H), 3.06 (s, 6H), 2.83e2.58 (m, 4H), 2.66 (s,
6H), 1.81e1.74 (m, 2H), 1.68e1.62 (m, 2H), 1.50e1.40 (m, 2H),
1.38e1.33 (m, 2H), 1.19e1.16 (m, 3H). 13C NMR (100 MHz, CDCl3)
(4-(dimethylamino)phenyl)prop-2-en-1-one
(TM-12).
4-
Dimethylamine benzaldehyde was treated with intermediate 7d
according to the general procedure to give the desired product TM-
12 as orange oil, 39.3% yield, 97.3% HPLC purity. 1H NMR (400 MHz,
d
191.73, 166.34, 165.11, 153.25, 152.04, 145.29, 130.87, 130.54 (2C),
128.97, 128.36, 123.76, 119.34, 114.43, 114.20, 111.71 (2C), 107.56,
101.42, 67.99, 52.43, 51.58, 47.19, 45.33 (2C), 40.11, 40.04, 28.80,
26.85, 25.65, 25.63, 11.02. MS (ESI) m/z: 544.4 [M þ H]þ.
CDCl3)
d
13.79 (s, 1H), 7.88 (d, J ¼ 15.2 Hz, 1H), 7.83 (d, J ¼ 8.8 Hz,
1H), 7.57 (d, J ¼ 8.8 Hz, 2H), 7.40e7.26 (m, 5H), 7.38 (d, J ¼ 15.2 Hz,
1H), 6.70 (d, J ¼ 9.2 Hz, 2H), 6.44 (dd, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, 1H),
6.41 (d, J ¼ 2.0 Hz, 1H), 3.98 (t, J ¼ 6.0 Hz, 2H), 3.64 (s, 2H), 3.06 (s,
(E)-1-(4-(3-((4-(Dimethylamino)benzyl)(ethyl)amino)pro-
poxy)-2-hydroxyphenyl)-3-(4-(dimethylamino)phenyl)prop-2-en-
1-one (TM-8). 4-Dimethylamine benzaldehyde was treated with
intermediate 6c according to the general procedure to give the
desired product TM-8 as orange oil, 32.7% yield, 98.6% HPLC purity.
6H), 2.73e2.52 (m, 4H),1.86e1.78 (m, 4H),1.16 (t, J ¼ 6.4 Hz, 3H). 13
C
NMR (100 MHz, CDCl3)
d 191.79, 166.35, 164.97, 152.08, 145.36,
130.91, 130.58 (2C), 129.41, 128.45, 127.62, 122.46, 114.46, 114.23,
111.75 (2C), 107.51, 101.52, 67.69, 57.44, 51.93, 47.07, 40.07 (2C),
26.59, 22.53, 10.90. MS (ESI) m/z: 473.3 [M þ H]þ.
1H NMR (400 MHz, CDCl3)
d
13.77 (s, 1H), 7.88 (d, J ¼ 15.2 Hz, 1H),
7.82 (d, J ¼ 9.6 Hz, 1H), 7.56 (d, J ¼ 8.8 Hz, 2H), 7.38 (d, J ¼ 15.2 Hz,
1H), 7.23 (d, J ¼ 8.0 Hz, 2H), 6.68 (t, J ¼ 8.8 Hz, 4H), 6.44e6.39 (m,
2H), 4.04 (t, J ¼ 6.0 Hz, 2H), 3.67 (s, 2H), 3.06 (s, 6H), 2.93 (s, 6H),
(E)-1-(4-(6-(Benzyl(ethyl)amino)hexyloxy)-2-hydroxyphenyl)-
3-(4-(dimethylamino)phenyl)prop-2-en-1-one
(TM-13).
4-
18

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
Dimethylamine benzaldehyde was treated with intermediate 8d
according to the general procedure to give the desired product TM-
13 as orange oil, 35.7% yield, 98.2% HPLC purity. 1H NMR (400 MHz,
(dimethylamino)phenyl)prop-2-en-1-one (TM-18). Intermediate
14 was treated with dimethylamine benzaldehyde according to the
general procedure to give the desired product TM-18 as orange oil,
CDCl3)
d
13.79 (s, 1H), 7.88 (d, J ¼ 15.2 Hz, 1H), 7.83 (d, J ¼ 8.8 Hz,
36.7% yield. 1H NMR (400 MHz, CDCl3)
d 13.93 (s, 1H), 7.81 (d,
1H), 7.56 (d, J ¼ 9.2 Hz, 2H), 7.46e7.26 (m, 5H), 7.38 (d, J ¼ 15.2 Hz,
1H), 6.70 (d, J ¼ 8.8 Hz, 2H), 6.48e6.42 (m, 2H), 3.99 (t, J ¼ 6.0 Hz,
2H), 3.68 (s, 2H), 3.06 (s, 6H), 2.81e2.52 (m, 4H), 1.83e1.75 (m, 2H),
1.70e1.60 (m, 2H), 1.49e1.42 (m, 2H), 1.39e1.27 (m, 2H), 1.19 (m,
J ¼ 15.2 Hz, 1H), 7.76 (d, J ¼ 9.2 Hz, 1H), 7.55 (d, J ¼ 8.8 Hz, 2H), 7.38
(d, J ¼ 15.2 Hz, 1H), 6.70 (d, J ¼ 8.8 Hz, 2H), 6.26 (dd, J1 ¼ 8.8 Hz,
J2 ¼ 2.4 Hz, 1H), 6.14 (d, J ¼ 2.4 Hz, 1H), 3.06 (s, 12H). MS (ESI) m/z:
311.2 [M þ H]þ.
3H). 13C NMR (100 MHz, CDCl3)
d
191.78, 166.39, 165.16, 152.08,
(E)-1-(4-(Dimethylamino)-2-hydroxyphenyl)-3-(4-
145.31, 130.89, 130.57 (2C), 129.45, 128.75, 127.61, 122.45, 114.50,
114.15, 111.75 (2C), 107.64, 101.44, 68.06, 57.44, 52.37, 47.01, 40.08
(2C), 28.85, 26.91, 25.71 (2C), 10.78. MS (ESI) m/z: 501.3 [M þ H]þ.
(E)-1-(4-(3-(Diethylamino)propoxy)-2-hydroxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one (TM-19). Intermediate 14 was
treated with anisaldehyde according to the general procedure to
give the desired product TM-19 as orange solid, mp:
169.3e171.0 ꢀC, 39.2% yield. 1H NMR (400 MHz, CDCl3)
d 13.68 (s,
(dimethylamino)phenyl)prop-2-en-1-one
(TM-14).
4-
1H), 7.80 (d, J ¼ 15.2 Hz, 1H), 7.74 (d, J ¼ 9.2 Hz, 1H), 7.59 (d,
J ¼ 8.8 Hz, 2H), 7.45 (d, J ¼ 15.2 Hz, 1H), 6.93 (d, J ¼ 8.8 Hz, 2H), 6.26
(dd, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, 1H), 6.14 (d, J ¼ 2.4 Hz, 1H), 3.85 (s, 3H),
3.07 (s, 6H). MS (ESI) m/z: 298.2 [M þ H]þ.
Dimethylamine benzaldehyde was treated with intermediate 6e
according to the general procedure to give the desired product TM-
14 as orange oil, 43.6% yield, 97.6% HPLC purity. 1H NMR (400 MHz,
CDCl3)
d
13.76 (s, 1H), 7.87 (d, J ¼ 15.6 Hz, 1H), 7.83 (d, J ¼ 8.8 Hz,
1H), 7.55 (d, J ¼ 8.8 Hz, 2H), 7.37 (d, J ¼ 15.6 Hz, 1H), 6.69 (d,
J ¼ 8.8 Hz, 2H), 6.46e6.43 (m, 2H), 4.07 (t, J ¼ 6.4 Hz, 2H), 3.72 (s,
2H), 3.05 (s, 6H), 2.80e2.70 (m, 6H), 2.09e2.04 (m, 2H), 1.14 (t,
4.2. Biological assays
4.2.1. Effect of test compounds on self-/Cu2þ-induced Ab1-42
aggregation and disaggregation experiments by ThT assay
The Thioflavin T-based fluorometric assay was performed to
J ¼ 6.8 Hz, 6H). 13C NMR (100 MHz, CDCl3)
d 191.82, 166.33, 164.82,
152.11, 145.43, 130.96, 130.59 (2C), 122.44, 114.45, 114.38, 111.76
(2C), 107.35, 101.64, 66.13, 49.08, 46.80 (2C), 40.09 (2C), 25.75, 10.72
(2C). MS (ESI) m/z: 397.2 [M þ H]þ.
investigate the effects on Ab1-42 aggregation. Amyloid-b1-42 (Ab1-42
)
was purchased from China Peptides Co., Ltd. Fluorescence was
measured by a Varioskan Flash Multimode Reader with excitation
and emission wavelengths at 446 nm and 490 nm, respectively.
Each assay was run in triplicate. The effects on self-induced and
Cu2þ-induced Ab1-42 aggregation experiments were performed
referenced our previous work [34,36].
(E)-1-(4-(3-((2-(Dimethylamino)benzyl)(ethyl)amino)pro-
poxy)-2-hydroxyphenyl)-3-phenylprop-2-en-1-one
(TM-15).
Benzaldehyde was treated with intermediate 6b according to the
general procedure to give the desired product TM-15 as orange oil,
46.2% yield, 97.5% HPLC purity. 1H NMR (400 MHz, CDCl3)
d 13.42 (s,
1H), 7.89 (d, J ¼ 15.2 Hz, 1H), 7.81 (d, J ¼ 9.2 Hz, 1H), 7.66e7.64 (m,
2H), 7.58 (d, J ¼ 15.2 Hz, 1H), 7.44e7.42 (m, 2H), 7.22e7.20 (m, 1H),
7.12e7.03 (m, 2H), 6.45e6.42 (m, 2H), 4.05 (t, J ¼ 6.4 Hz, 2H), 3.75 (s,
2H), 2.85e2.80 (m, 4H), 2.72 (s, 6H), 2.03e2.01 (m, 2H), 1.11 (t,
4.2.2. Antioxidant activity assay
The antioxidant activity was determined by the oxygen radical
absorbance capacity fluorescein (ORAC-FL) assay. The detailed
procedure referenced our previous work [36].
J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d 191.69, 166.56, 165.58,
153.08, 144.25, 134.71, 130.59, 130.48, 130.34, 128.92 (2C), 128.31,
127.73, 123.35, 120.26, 119.07, 113.95, 107.87, 101.58, 66.36, 52.30,
49.50, 47.63, 45.23 (2C), 26.13, 11.31. MS (ESI) m/z: 459.3 [M þ H]þ.
(E)-1-(4-(Dimethylamino)-2-hydroxyphenyl)-3-(4-(4-(ethyl(2-
methoxybenzyl)amino)butoxy)phenyl)prop-2-en-1-one (TM-16).
Intermediate 14 was treated with intermediate 11a according to the
general procedure to give the desired product TM-16 as orange oil,
4.2.3. Inhibition experiments of AChE and BuChE
AChE and BuChE inhibitory activities of the compounds were
measured by the Ellman assay. AChE from 5% rat cortex homoge-
nate (ratAChE) and electric eel (EeAChE) (Sigma-Aldrich Co.),
respectively, and BuChE from rat serum (ratBuChE). The ratAChE
and ratBuChE were obtained referenced our previous work. The
ChEs inhibition was carried out using a Varioskan Flash Multimode
Reader at 405 nm and 412 nm, respectively. The detailed procedure
was the same as described in our previous work [33,34].
40.3% yield, 98.1% HPLC purity. 1H NMR (400 MHz, CDCl3)
d 13.75 (s,
1H), 7.80 (d, J ¼ 15.6 Hz), 7.74 (d, J ¼ 9.2 Hz, 1H), 7.58 (d, J ¼ 8.8 Hz,
2H), 7.49 (d, J ¼ 7.2 Hz, 1H), 7.45 (d, J ¼ 15.6 Hz, 1H), 7.26 (t,
J ¼ 8.0 Hz, 1H), 6.95 (t, J ¼ 7.6 Hz, 1H), 6.90e6.84 (m, 3H), 6.26 (dd,
J1 ¼8.0 Hz, J2 ¼ 2.4 Hz,1H), 6.14 (d, J ¼ 2.4 Hz,1H), 4.00 (t, J ¼ 6.4 Hz,
2H), 3.83 (s, 3H), 3.79 (s, 2H), 3.07 (s, 6H), 2.68e2.66 (m, 4H),
1.82e1.79 (m, 4H), 1.17 (t, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3)
4.2.4. Kinetic characterization of AChE inhibition
Kinetic characterization of AChE inhibition was performed
based on a reported method using EeAChE at 412 nm [25]. The
detailed procedure could referenced our previous work [34].
d
189.91, 166.00, 160.70, 157.62, 155.59, 142.33, 131.03, 130.74,
129.90 (2C), 128.45, 128.39, 127.57, 120.34, 118.01, 114.68 (2C),
110.35, 110.23, 103.81, 97.84, 67.59, 55.21 (2C), 52.31, 50.69, 47.28,
39.76, 26.87, 26.64, 11.00. MS (ESI) m/z: 503.3 [M þ H]þ.
4.2.5. Metal binding studies
The metal binding ability of compound TM-6 was tested in a
Varioskan Flash Multimode Reader (Thermo Scientific) with
wavelength ranging from 200 to 600 nm. The tested metal ions
included CuCl2, FeSO4, ZnCl2, and AlCl3. The procedure referenced
our previous work [33,36].
(E)-1-(4-(Dimethylamino)-2-hydroxyphenyl)-3-(4-(4-((2-
(dimethylamino)benzyl)(ethyl)amino)butoxy)phenyl)prop-2-en-
1-one (TM-17). Intermediate 14 was treated with intermediate 11b
according to the general procedure to give the desired product TM-
17 as orange oil, 37.2% yield, 97.7% HPLC purity. 1H NMR (400 MHz,
CDCl3)
d
13.73 (s, 1H), 7.79 (d, J ¼ 15.6 Hz, 1H), 7.74 (d, J ¼ 9.6 Hz,
1H), 7.58 (d, J ¼ 8.0 Hz, 2H), 7.45 (d, J ¼ 15.6 Hz, 1H), 7.31e7.29 (m,
1H), 7.18e7.12 (m, 2H), 6.88 (d, J ¼ 7.6 Hz, 1H), 6.26 (d, J ¼ 8.0 Hz,
1H), 6.14 (s, 1H), 4.10 (s, 2H), 3.98 (t, J ¼ 6.4 Hz, 2H), 3.07 (s, 6H),
2.87e2.85 (m, 4H), 2.66 (s, 6H),1.92e1.79 (m, 4H),1.25 (t, J ¼ 6.8 Hz,
3H). MS (ESI) m/z: 516.3 [M þ H]þ.
4.2.6. HuMAO-A and huMAO-B inhibition studies
The MAOs inhibition was investigated by a Varioskan Flash
Multimode Reader with an excitation wavelength of 310 nm and an
emission wavelength of 400 nm. Recombinant human MAO-A and
-B were purchased from Sigma-Aldrich., and the detailed procedure
referenced our previous work [40].
(E)-1-(4-(Dimethylamino)-2-hydroxyphenyl)-3-(4-
19

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European Journal of Medicinal Chemistry 216 (2021) 113310
4.2.7. Neuroprotective effect
References
The neuroprotective effects were evaluated with 3-(4,5-
dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT)
assay and LDH assay. The detailed procedure referenced our pre-
vious work [33].
[1] R. Barnett, Alzheimer’s disease, Lancet 393 (10181) (2019) 1589.
[2] C. Patterson, World Alzheimer Report 2018dthe State of the Art of Dementia
Research: New Frontiers, Alzheimer’s Disease International (ADI), London,
2018, pp. 1e48.
[3] M. Citron, Alzheimer’s disease: strategies for disease modification, Nat. Rev.
Drug Discov. 9 (5) (2010) 387e398.
[4] P. Sharma, P. Srivastava, A. Seth, P.N. Tripathi, A.G. Banerjee, S.K. Shrivastava,
Comprehensive review of mechanisms of pathogenesis involved in Alz-
heimer’s disease and potential therapeutic strategies, Prog. Neurobiol. 174
(2019) 53e89.
[5] W. Jagust, Imaging the evolution and pathophysiology of Alzheimer disease,
Nat. Rev. Neurosci. 19 (11) (2018) 687e700.
[6] R. Anand, K.D. Gill, A.A. Mahdi, Therapeutics of Alzheimer’s disease: past,
present and future, Neuropharmacology 76 Pt (2014) 27e50.
[7] J.A. Hardy, G.A. Higgins, Alzheimer’s disease: the amyloid cascade hypothesis,
Science 256 (5054) (1992) 184e185.
[8] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer’s disease: progress
and problems on the road to therapeutics, Science 297 (5580) (2002)
353e356.
[9] S.H. Barage, K.D. Sonawane, Amyloid cascade hypothesis: pathogenesis and
therapeutic strategies in Alzheimer’s disease, Neuropeptides 52 (2015) 1e18.
[10] N.N. Nalivaeva, A.J. Turner, Targeting amyloid clearance in Alzheimer’s disease
as a therapeutic strategy, Br. J. Pharmacol. 176 (18) (2019) 3447e3463.
[11] Y.J. Wang, Alzheimer disease: lessons from immunotherapy for Alzheimer
disease, Nat. Rev. Neurol. 10 (4) (2014) 188e189.
4.2.8. In vitro BloodꢁBrain barrier permeation assay
The bloodꢁbrain barrier penetration of compounds was evalu-
ated using a parallel artificial membrane permeability assay
(PAMPA). The detailed procedure could referenced our previous
work [38].
4.2.9. Effects on scopolamine-induced memory impairment
Acute Toxicity. A total of 50 KM mice (22 days, 18e22 g) pur-
chased from the laboratory animal center of Sichuan Academy of
Chinese Medicine Sciences (Chengdu, China) were used to evaluate
the acute toxicity of compound TM-6. Mice were maintained with a
12 h light/dark cycle at 20e22 ꢀC with a relative humidity of
40e70%. Prior to each experiment, mice were fasted overnight and
allowed free access to water. Compound TM-6 (67.5, 125, 250, 500
and 1000 mg/kg) was given via oral administration according to the
divided experimental groups (n ¼ 10 per group). After the admin-
istration of the compounds, the mice were observed continuously
for the first 4 h for any abnormal behavior and mortality changes,
intermittently for the next 24 h, and occasionally thereafter for 14
days.
[12] P. Anand, B. Singh, A review on cholinesterase inhibitors for Alzheimer’s
disease, Arch Pharm. Res. (Seoul) 36 (4) (2013) 375e399.
[13] L.N. Hou, J.R. Xu, Q.N. Zhao, X.L. Gao, Y.Y. Cui, J. Xu, H. Wang, H.Z. Chen, A new
motif in the N-terminal of acetylcholinesterase triggers amyloid-beta aggre-
gation and deposition, CNS Neurosci. Ther. 20 (1) (2014) 59e66.
[14] P.M. Doraiswamy, A.E. Finefrock, Metals in our minds: therapeutic implica-
tions for neurodegenerative disorders, Lancet Neurol. 3 (7) (2004) 431e434.
[15] S.J. Dixon, B.R. Stockwell, The role of iron and reactive oxygen species in cell
death, Nat. Chem. Biol. 10 (1) (2014) 9e17.
Assay Method. A step-down passive avoidance test was used to
assess the effects [33,36]. The apparatus consisted of a grid floor
with a wooden block placed in the center. The block served as a
shock free zone. The mice underwent two separate trials: a training
trial and a test trial 24 h later. For training trial, mice were initially
placed on the block and were given an electrical foot shock (0.5 mA,
2s) through the grid floor on stepping down. We used a total of 90
mice in the passive avoidance test with 10 mice were used per
treatment. Compound TM-6 (2.3, 6.8 and 20.4 mg/kg, p.o.) or
donepezil (5.0 mg/kg, p.o.) as a positive control were orally given
1 h before each training trial. After 30 min, memory impairment
was induced by administering scopolamine (3 mg/kg, i.p.). Twenty-
four hours after the training trial, mice were placed on the block
and the time for the animal to step down was measured as latency
time for test trial. An upper cut-off time was set at 300 s.
Statistical analysis. The results are expressed as the mean SD
of at least three independent experiments. Data were subjected to
Student’s test or one-way analysis of variance (ANOVA) followed by
Dunnett’s test.
[16] C. Cheignon, M. Tomas, D. Bonnefont-Rousselot, P. Faller, C. Hureau, F. Collin,
Oxidative stress and the amyloid beta peptide in Alzheimer’s disease, Redox
Biol 14 (2018) 450e464.
[17] S. Schedin-Weiss, M. Inoue, L. Hromadkova, Y. Teranishi, N.G. Yamamoto,
B. Wiehager, N. Bogdanovic, B. Winblad, A. Sandebring-Matton, S. Frykman,
L.O. Tjernberg, Monoamine oxidase B is elevated in Alzheimer disease neu-
rons, is associated with g-secretase and regulates neuronal amyloid b-peptide
levels, Alzheimer’s Res. Ther. 9 (1) (2017) 57.
[18] https://www.alzforum.org/therapeutics/rasagiline.
[19] A. Cavalli, M.L. Bolognesi, A. Minarini, M. Rosini, V. Tumiatti, M. Recanatini,
C. Melchiorre, Multi-target-directed ligands to combat neurodegenerative
diseases, J. Med. Chem. 51 (3) (2008) 347e372.
[20] P. Zhang, S. Xu, Z. Zhu, J. Xu, Multi-target design strategies for the improved
treatment of Alzheimer’s disease, Eur. J. Med. Chem. 176 (2019) 228e247.
[21] M. de Freitas Silva, K.S.T. Dias, V.S. Gontijo, C.J.C. Ortiz, C. Viegas Jr., Multi-
target directed drugs as a modern approach for drug design towards Alz-
heimer’s disease: an update, Curr. Med. Chem. 25 (29) (2018) 3491e3525.
[22] http://integrity.thomson-pharma.com.
[23] J. Cummings, G. Lee, T. Mortsdorf, A. Ritter, K. Zhong, Alzheimer’s disease drug
development pipeline, Alzheimers Dement (N Y). 3 (3) (2017) 367e384, 2017.
[24] C. Zhuang, W. Zhang, C. Sheng, W. Zhang, C. Xing, Z. Miao, Chalcone: a priv-
ileged structure in medicinal chemistry, Chem. Rev. 117 (12) (2017)
7762e7810.
[25] X. Zhang, K.P. Rakesh, S.N.A. Bukhari, M. Balakrishna, H.M. Manukumar,
H.L. Qin, Multi-targetable chalcone analogs to treat deadly Alzheimer’s dis-
ease: current view and upcoming advice, Bioorg. Chem. 80 (2018) 86e93.
[26] T. Fuchigami, Y. Yamashita, M. Haratake, M. Ono, S. Yoshida, M. Nakayama,
Synthesis and evaluation of ethyleneoxylated and allyloxylated chalcone de-
rivatives for imaging of amyloid beta plaques by SPECT, Bioorg. Med. Chem. 22
(2014) 2622e2628.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
[27] M. Ono, R. Watanabe, H. Kawashima, Y. Cheng, H. Kimura, H. Watanabe,
M. Haratake, H. Saji, M. Nakayama, Fluoro-pegylated chalcones as positron
emission tomography probes for in vivo imaging of beta-amyloid plaques in
Alzheimer’s disease, J. Med. Chem. 52 (2009) 6394e6401.
[28] Y. Yang, M. Cui, B. Jin, X. Wang, Z. Li, P. Yu, J. Jia, H. Fu, H. Jia, B. Liu, (99m)Tc-
labeled dibenzylideneacetone derivatives as potential SPECT probes for
Acknowledgments
in vivo imaging of b-amyloid plaque, Eur. J. Med. Chem. 64 (2013) 90e98.
[29] Q. Li, S. He, Y. Chen, F. Feng, W. Qu, H. Sun, Donepezil-based multi-functional
cholinesterase inhibitors for treatment of Alzheimer’s disease, Eur. J. Med.
Chem. 158 (2018) 463e477.
[30] M. Shidore, J. Machhi, K. Shingala, P. Murumkar, M.K. Sharma, N. Agrawal,
A. Tripathi, Z. Parikh, P. Pillai, M.R. Yadav, Benzylpiperidine-linked diary-
lthiazoles as potential anti-Alzheimer’s agents: synthesis and biological
evaluation, J. Med. Chem. 59 (12) (2016) 5823e5846.
[31] P. Sharma, A. Tripathi, P.N. Tripathi, S.S. Singh, S.P. Singh, S.K. Shrivastava,
Novel molecular hybrids of N-benzylpiperidine and 1,3,4-oxadiazole as mul-
titargeted therapeutics to treat Alzheimer’s disease, ACS Chem. Neurosci. 10
(10) (2019) 4361e4384.
This work was supported by funding from the China Scholarship
Council (NO. 201808410456) and the Key Scientific Research Project
of Colleges and Universities in Henan Province (NO. 20A350006).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113310.
20

Z. Sang, Q. Song, Z. Cao et al.
European Journal of Medicinal Chemistry 216 (2021) 113310
[32] P. Sharma, A. Tripathi, P.N. Tripathi, S.K. Prajapati, A. Seth, M.K. Tripathi,
P. Srivastava, V. Tiwari, S. Krishnamurthy, S.K. Shrivastava, Design and
development of multitarget-directed N-Benzylpiperidine analogs as potential
candidates for the treatment of Alzheimer’s disease, Eur. J. Med. Chem. 167
(2019) 510e524.
[33] Z. Sang, K. Wang, J. Shi, W. Liu, X. Cheng, G. Zhu, Y. Wang, Y. Zhao, Z. Qiao,
A. Wu, Z. Tan, The development of advanced structural framework as multi-
target-directed ligands for the treatment of Alzheimer’s disease, Eur. J. Med.
Chem. 192 (2020) 112180.
[34] Z. Sang, X. Qiang, Y. Li, W. Yuan, Q. Liu, Y. Shi, W. Ang, Y. Luo, Z. Tan, Y. Deng,
Design, synthesis and evaluation of scutellarein-O-alkylamines as multifunc-
tional agents for the treatment of Alzheimer’s disease, Eur. J. Med. Chem. 94
(2015) 348e366.
(2018) 1544.
[38] Z. Sang, K. Wang, P. Bai, A. Wu, J. Shi, W. Liu, G. Zhu, Y. Wang, Y. Lan, Z. Chen,
Y. Zhao, Z. Qiao, C. Wang, Z. Tan, Design, synthesis and biological evaluation of
novel O-carbamoyl ferulamide derivatives as multi-target-directed ligands for
the treatment of Alzheimer’s disease, Eur. J. Med. Chem. 194 (2020) 112265.
[39] L.N. Hou, J.R. Xu, Q.N. Zhao, X.L. Gao, Y.Y. Cui, J. Xu, H. Wang, H.Z. Chen, A new
motif in the N-terminal of acetylcholinesterase triggers amyloid-b aggrega-
tion and deposition, CNS Neurosci. Ther. 20 (1) (2014) 59e66.
[40] P. Mishra, P. Sharma, P.N. Tripathi, S.K. Gupta, P. Srivastava, A. Seth,
A. Tripathi, S. Krishnamurthy, S.K. Shrivastava, Design and development of
1,3,4-oxadiazole derivatives as potential inhibitors of acetylcholinesterase to
ameliorate scopolamine-induced cognitive dysfunctions, Bioorg. Chem. 89
(2019) 103025.
[35] X. Qiang, Z. Sang, W. Yuan, Y. Li, Q. Liu, P. Bai, Y. Shi, W. Ang, Z. Tan, Y. Deng,
Design, synthesis and evaluation of genistein-O-alkylbenzylamines as po-
tential multifunctional agents for the treatment of Alzheimer’s disease, Eur. J.
Med. Chem. 76 (2014) 314e331.
[41] A. Zhou, J. Hu, L. Wang, G. Zhong, J. Pan, Z. Wu, A. Hui, Combined 3D-QSAR,
molecular docking, and molecular dynamics study of tacrine derivatives as
potential acetylcholinesterase (AChE) inhibitors of Alzheimer’s disease, J. Mol.
Model. 21 (10) (2015) 277.
[36] Z. Sang, K. Wang, J. Shi, X. Cheng, G. Zhu, R. Wei, Q. Ma, L. Yu, Y. Zhao, Z. Tan,
W. Liu, Apigenin-rivastigmine hybrids as multi-target-directed ligands for the
treatment of Alzheimer’s disease, Eur. J. Med. Chem. 187 (2020) 111958.
[37] R. Purgatorio, M. de Candia, A. De Palma, F. De Santis, L. Pisani, F. Campagna,
S. Cellamare, C.D. Altomare, M. Catto, Insights into structure-activity re-
lationships of 3-arylhydrazonoindolin-2-one derivatives for their multitarget
activity on beta-amyloid aggregation and neurotoxicity, Molecules 23 (7)
[42] Z. Sang, K. Wang, P. Zhang, J. Shi, W. Liu, Z. Tan, Design, synthesis, in-silico and
biological evaluation of novel chalcone derivatives as multi-function agents
for the treatment of Alzheimer’s disease, Eur. J. Med. Chem. 180 (2019)
238e252.
[43] L. Di, E.H. Kerns, K. Fan, O.J. McConnell, G.T. Carter, High throughput artificial
membrane permeability assay for blood-brain barrier, Eur. J. Med. Chem. 38
(2003) 223.
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