Design, synthesis, and in vitro evaluation of 4-aminoalkyl-1(2H)-phthalazinones as potential multifunctional anti-Alzheimer's disease agents

Article abstract of DOI:10.1016/j.bioorg.2021.104895

A series of 4-aminoalkyl-1(2H)-phthalazinone derivatives was designed and synthesized as potential multifunctional agents for Alzheimer's disease (AD) treatment. In vitro biological assay results demonstrated that most synthesized compounds exhibited sign

Full text of DOI:10.1016/j.bioorg.2021.104895

Bioorganic Chemistry 111 (2021) 104895
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, and in vitro evaluation of 4-aminoalkyl-1
(2H)-phthalazinones as potential multifunctional anti-Alzheimer’s
disease agents
Chanyuan Ye1, Rui Xu1, Zhongcheng Cao, Qing Song, Guangjun Yu, Yichun Shi, Zhuoling Liu,
Xiuxiu Liu*, Yong Deng*
Department of Medicinal Chemistry, Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering
Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu
610041, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of 4-aminoalkyl-1(2H)-phthalazinone derivatives was designed and synthesized as potential multi-
functional agents for Alzheimer’s disease (AD) treatment. In vitro biological assay results demonstrated that most
synthesized compounds exhibited significant AChE inhibition, moderate to high MAOs inhibitory potencies and
good anti-platelet aggregation abilities. Among them, compound 15b exhibited the highest inhibitory potencies
Alzheimer’s disease
4-(aminoalkyl)-1(2H)-phthalazinone
derivatives
Multifunctional agents
Acetylcholinesterase inhibitors
Monoamine oxidases inhibitors
Aβ aggregation inhibitors
Anti-neuroinflammation
towards MAO-B and MAO-A (IC50 = 0.7 µM and 6.4 µM respectively), moderate inhibition towards AChE (IC50
=
8.2 µM), and good activities against self- and Cu2+-induced Aβ1–42 aggregation and platelet aggregation.
Moreover, 15b also displayed antioxidant capacity, neuroprotective potency, anti-neuroinflammation and BBB
permeability. These excellent results indicated that compound 15b could be worthy of further studies to be
considered as a promising multifunctional candidate for the treatment of AD.
1. Introduction
comprising two types: acetylcholinesterase (AChE) and butyr-
ylcholinesterase (BuChE), can affect the activity of cholinergic neurons
Alzheimer disease (AD), the most common neurodegenerative dis-
ease, is the main cause of dementia. This slowly progressive disease is
age-related and mainly affects people over 65 years old, and is charac-
terised by memory and orientation loss, impaired judgement, language
disturbances and behavioural changes [1,2]. It is estimated that there
are over 50 million people living with dementia globally, a figure set to
increase to 152 million by 2050, which has led to considerable impli-
cations along with the colossal financial burden on the families and
society [3]. Although the exact cause of AD is still unclear, several
pathological hallmarks such as β-amyloid (Aβ) deposition, tau protein
hyperphosphorylation and accumulation, decline in acetylcholine (ACh)
levels, oxidative stress, biometal dyshomeostasis, astrogliosis and
neuronal dystrophy are thought to play vital roles in the onset and
progression of the disease [4].
through hydrolyzing the acetylcholine (ACh). In addition, cholinergic
can also regulate the synthesis and release of nerve growth factor (NFG)
in the cerebral cortex and hippocampus, which further lead to the
deposition of Aβ. Accordingly, cholinesterase inhibitors have been used
to reduce the pathological ACh hydrolysis and the Aβ toxicity, thereby
achieving the effect of AD threatment [4–6]. Several acetylcholines-
terase inhibitors (AChEIs) approved by FDA, including donepezil, riva-
stigmine and galantamine, have been demonstrated to enhance
cholinergic levels and improve cognitive dysfunction to some extent [7].
Therefore, inhibition towards AChE has been an attractive treatment
strategy for AD. However, the above marketed AChEIs, unfortunately,
can not change the pathological process and reverse the condition of AD
due to its complex etiology, and more single-target drugs in the clinical
research stage have been reported failures one after another [8]. As a
consequence, more researches have paid attention to applying “multi-
target-directed ligands” (MTDLs) to AD treatment [9].
The decrease of cholinergic transmission is considered to cause a
series of clinical manifestations, mainly cognitive and memory
dysfunction. Cholinesterase (ChE), a ubiquitous serine hydrolase
Monoamine oxidase (MAO), mitochondrial membrane flavoenzyme
* Corresponding authors.
E-mail addresses: liuxiuxiu@scu.edu.cn (X. Liu), dengyong@scu.edu.cn (Y. Deng).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104895
Received 8 December 2020; Received in revised form 21 February 2021; Accepted 4 April 2021
Available online 8 April 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
existing in two different isoforms (MAO-A and -B), is responsible for
oxidative deamination of major xenobiotic amines and monoamine
neurotransmitters to regulate their concentration and metabolization in
the brain and peripheral tissues [4,10]. It is noteworthy that the MAO-B
expression was increased in the cerebral cortex and the hippocampus
regions of AD patients, and activation of the MAO-B was also associated
with disruption of cholinergic neurons, formation of Aβ plaques, and
NFTs, further contributing to the etiology of AD [11]. In addition, it is
known that MAO-A inhibitors have been used as antidepressant agents,
which may be helpful to relieve depressive symptoms in AD patients
[12]. Moreover, the overexpression of MAOs in the nerve tissue facili-
tates the generation of excessive H2O2 and toxic free radicals, which
enhance the oxidative stress and further produce reactive oxygen species
(ROS), thereby causing the death of neurons in the brain. These pro-
cesses play an important role in neurodegenerative diseases, such as AD
and Parkinson disease (PD) [13]. Therefore, drugs with inhibitory po-
tencies towards MAOs (especially MAO-B), antioxidant properties and
neuroprotective potencies may have a positive effect on the treatment of
AD [14].
for AD. In addition, some evidence indicated that a large amount of APP
and a complete enzyme system that can hydrolyze APP to Aβ were found
in platelets [18]. Activated platelets are the main source of Aβ in the
blood. Simultaneously, Aβ deposited on the blood vessel wall also acti-
vates platelets in turn, thereby leading to a vicious cycle. Moreover,
excessive Aβ can damage blood vessels, which may further cause the
deposition of Aβ in the brain and cerebral blood vessels, ultimately
promoting the occurrence and development of AD [19]. According to
these, inhibiting platelet aggregation in brain tissue may be helpful for
preventing AD and changing its course.
Besides, brain tissue damage caused by neuroinflammation, an
important pathological change in AD patients, is mainly related to
microglia, astrocytes and inflammatory factors [20,21]. Microglia acti-
vated by Aβ stimulation is considered to produce a large number of
neurotoxic substances such as inflammatory factors and free radicals,
including tumor necrosis factor (TNF-α), interferon γ (INF-γ), ROS and so
on. These neurotoxic substances induce neuroinflammation, neurons
damage and the production of complement components, leading to
autoimmune reactions, which in turn cause cognitive dysfunction and
Aβ are derived from β-amyloid precursor protein (APP), a trans-
membrane glycoprotein, via the proteolytic functions of β-secretase
(BACE1) and γ-secretase [15]. Noteworthy, Aβ (especially Aβ1-42) are
prone to aggregate into β sheet conformations in the form of higher-
order oligomers, protofibrils, and fibrils, which have been broadly
examined in AD brain. Deposition of Aβ initiated a cascade of
biochemical processes of AD pathogenesis, thereby leading to severe
neurodegeneration or neuronal death [15–17]. Therefore, inhibition
and clearance of Aβ1-42 aggregation may be a potent therapeutic strategy
neuron loss. In addition, TNF-α and INF-γ can also reduce the content of
insulin degrading enzymes, the key enzymes for Aβ degradation, thereby
increasing Aβ deposition [22,23]. The above findings suggest that anti-
inflammatory drugs may be promising candidates for AD treatment.
It is reported that phthalazinone derivatives showed a wide range of
biological activities such as AChE inhibition, Aβ1-42 aggregation inhi-
bition, anti-platelet aggregation, and other potencies including anti-
oxidant, anti-inflammatory, anti-depressant and protective effect on
PC12 injury induced by H2O2 with low hepatotoxicity [24–26], which
Fig. 1. Design strategy for 4-aminoalkyl-1(2H)-phthalazinone derivatives.
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C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
suggested that such derivatives may play an important role in AD
therapy. In addition, our group had reported a series of phthalide alkyl
tertiary amine derivatives (Fig. 1) with MTDLs’ profiles [27]. These
derivatives showed excellent AChE inhibition potencies (IC50 values
described in Scheme 3. The commercial 4,5-dimethoxyphthalic acid
(17) was used as starting material. The intramolecular dehydration of 17
gave 4,5-dimethoxyphthalic anhydride (18), then reacted with ethyl 2-
(triphenylphosphinoidene)acetate to provide compound 19 via wittig
reaction [36,37]. Synthesis of 20 was carried out using 19 and hydrazine
hydrate as reactants [38], and subsequently reduced with sodium
borohydride in the presence of LiCl to give intermediate 21 [39], which
then reacted with thionyl chloride to obtain the chlorinated product 22
[40]. Finally, compound 22 was condensed with the corresponding
secondary amines using tetrabutylammonium bromide (TBAB) as phase
transfer catalyst in acetonitrile to provide the target products 23a and
23c. The standard techniques including 1H NMR, 13C NMR and ESI-MS
were used to characterize the chemical structure of all target products.
And the purity of all the target compounds was determined by high-
performance liquid chromatography (HPLC) analysis to be over 97.0%.
ranging from 2.66 nM to 24.9
μM) and good anti-oxidant activities
expressed as ORAC values from 0.31 to 3.64-fold of Trolox. Unfortu-
nately, this series of derivatives only exhibited weak Aβ1-42 aggregation
inhibition and low MAOs inhibitory potencies. Therefore, based on our
previously reported scaffold (Fig. 1, phthalide alkyl tertiary amine de-
rivatives), we replaced the phthalide nucleus with phthalazinone moiety
(Fig. 1, 1(2H)-phthalazinone) to optimize their multi-target anti-AD
potentials. To this end, eighteen candidates of 4-aminoalkyl-1(2H)-
phthalazinone derivatives containing carbon chains of different length
and terminal tertiary amines were designed, synthesized and expected to
possess excellent inhibition of AChE, enhanced inhibition of MAOs,
Aβ
aggregation and platelet aggregation, biological activities of
1–42
antioxidant, anti-inflammatory and neuroprotective, and proper BBB
permeability simultaneously as balanced and multifunctional agents for
AD therapy.
2.2. Pharmacology
2.2.1. AChE and BuChE inhibition
2. Results and discussion
The 4-aminoalkyl-1(2H)-phthalazinone derivatives 15, 16 and 23
were examined by modified Ellman’s method for their in vitro inhibitory
potencies against AChE (from electric eel, EeAChE) and BuChE (from rat
serum, RatBuChE), and their potencies were compared to the well-
known AChE inhibitors, donepezil and rivastigmine [41]. In order to
clarify the effect of phthalazinone nucleus on ChE inhibitory potency,
the key intermediates 13a, 13c, 13e, 13f and 13i were also evaluated for
their ChE inhibitory potencies. The ChE inhibitory results were
expressed as IC50 values or percent inhibition rates, which were pre-
sented in Table 1.
2.1. Chemistry
The synthetic route of intermediates 3a, 3b and 7 is shown in Scheme
1. Commercially available α,ω-diols 1a or 1b was reacted with benzoyl
chloride to obtain
α,ω-diol benzoic acid monoesters 2a or 2b [28].
Treating compounds 2a or 2b with 2,4,6-trichloro-1,3,5-triazine and
DMSO in the presence of triethylamine afforded the corresponding
benzoyloxyalkyl aldehyde 3a or 3b [29]. In addition, the reaction of 5,6-
dimethoxyphthalide (4) with N-bromosuccinimide, using AIBN as
initiator, afforded the compound 5, which then was hydrolyzed to
provide 6 [30]. Then, compound 6 was reacted with P(OEt)3 in the
presence of Py⋅HClO4 to give the intermediate 7 [31].
All synthesized target compounds showed potent and selective
inhibitory potencies towards AChE over BuChE. Especially, the activities
of 16a, 16b, 16c, 16d and 16e are particularly excellent with the IC50
values as 0.10 μM, 0.16 μM, 0.47 μM, 0.68 μM and 0.45 μM, respectively.
As the intermediates 3a, 3b and 7 in hand, the designed compounds
were obtained according to Scheme 2. Compound 3a or 3b was
condensed with 7 via the Horner-Wadsworth-Emmons reaction, using
NaOH as base to yield the corresponding intermediate 8a or 8b [32],
then followed by aminolysis reaction to get 9a or 9b [33]. Subsequently,
compound 9a or 9b was hydrolyzed with K2CO3 in a mixed solution of
methanol and water to obtain alkyl alcohol 10a or 10b, which was then
reacted with benzenesulfonylchloride to provide the benzenesulfonyl
ester 11a or 11b, respectively. Then, substitution of these benzene-
sulfonyl esters with a series of secondary amines 12a-j in the presence of
K2CO3 gave the corresponding key intermediates 13a-j or 14a-f [34].
Finally, the target products 15a-j and 16a-f were obtained by the re-
action of these key intermediates with hydrazine hydrate in ethanol
[35].
Although they showed lower inhibitory effect than donepezil (IC50
0.078 M), most of the synthesized compounds were still more excellent
than rivastigmine (IC50 = 25.81 M) for AChE inhibition. Through the
=
μ
μ
structure–activity relationship (SAR) analysis, we could find that there
are three important factors affecting the inhibition of AChE, including
the presence of phthalazinone moiety, the length of the methylene side
chain and terminal tertiary amine species. As shown in Table 1,
compared with the compounds 15a-j, 16a-f, 23a and 23c, the in-
termediates 13a-f without the phthalazinone moiety showed much
lower or almost no AChE inhibitory potency. It indicated that the
presence of phthalazinone moiety was crucial for the inhibition of AChE,
which was consistant with our design strategy. Besides, the activities of
the compounds with six methylene moieties as the side chains (16a-f)
exhibited higher than that of the compounds containing four (15a-j),
and compound 23a or 23c with two methylene moieties exhibited
lowest AChE inhibition when it comes to the same terminal tertiary
As for target compounds 23a and 23c, the synthetic method is
Scheme 1. Synthesis of intermediates 3a, 3b and 7. Reagents and conditions: (i) PhCOCl, Et3N, THF, 50 ◦C for 3 h, yield 86.0%(2a), 92.3%(2b); (ii) 2,4,6-tri-
chloro-1,3,5-triazine, Et3N, DMSO, THF, ꢀ 30 ◦C for 2 h then rt for 2 h, yield 85.2%(3a), 90.5%(3b); (iii) NBS, AIBN, PhCl, 70 ◦C for 3 h, yield 78.2%; (iv) H2O,
reflux for 2 h, yield 65.8%; (v) P(OEt)3, Py⋅HClO4, CHCl3, rt for overnight, yield 80.5%.
3

C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
Scheme 2. Synthesis of compounds 15a-j and
16a-f. Reagents and conditions: (i) NaOH,
CH2Cl2, rt for 3 h; (ii) HN(CH3)2⋅HCl, Et3N, EtOH,
reflux for 3 h, yield 76.5%(9a), 75.4%(9b) (two-
steps); (iii) K2CO3, CH3OH, H2O, rt for 15 h, yield
81.3%(10a), 78.6%(10b); (iv)benzenesulfonly-
chloride, Et3N, CH2Cl2, rt for overnight, yield
89.4%(11a), 92.0%(11b); (v) NHR1R2 (12a-j),
K2CO3, CH3CN, 50 ◦C for 5 ~ 7 h, yield 62.2 ~
73.1%; (vi) N2H4⋅H2O, EtOH, reflux for 8 ~ 10 h,
yield 50.1 ~ 62.2%.
Scheme 3. Synthesis of compounds 23a and 23c. Reagents and conditions: (i) Ac2O, THF, reflux for 4 h, yield 94.8%; (ii) Toluene, 80 ◦C for 3 h, yield 89.0%; (iii)
N2H4⋅H2O, EtOH, reflux for 3 h, yield 83.0%; (iv) NaBH4, LiCl, EtOH, THF, rt for overnight, yield 80.1%; (v) SOCl2, DMF, CHCl3, reflux for 10 h, yield 89.2%; (vi)
NHR1R2, K2CO3, TBAB, CH3CN, reflux for 10 h, yield 62.1 ~ 63.2%.
amine species. Thus, it could be speculated that in the compound 23a or
23c, the length of two methylenes side chain is too short to bind to the
catalytic active site (CAS) and the peripheral anionic site (PAS) of the
enzyme simultaneously. In addition, based on the displayed data, we
could find that compounds with benzylamine side chains (15a-f)
showed higher activities against AChE than those with the aliphatic
amines side chains (15 g-j) in general. Besides, it seems that derivatives
having N-(2-dimethylamino)benzyl group moiety (15e, 16e) showed
higher anti-AChE activities in comparison to those having N-(4-dime-
thylamino)benzyl group (15f, 16f). Furthermore, the anti-AChE activ-
ities of tested compounds slightly increased when the N-ethyl group
connected to the end of the carbon chain (15b, 16b, 16d) was replaced
by N-methyl structure (15a, 16a, 16c). Concerning BuChE inhibition,
the inhibitory effect of the synthesized compounds was weaker than
their corresponding anti-AChE potency, showing selective inhibition
towards AChE over BuChE.. In conclusion, the ChE inhibition assay re-
sults indicated that our designed 4-aminoalkyl-1(2H)-phthalazinone
derivatives may be a potent AChE inhibitor and beneficial for the
compounds’ multi-target and balanced anti-AD function.
2.2.2. Kinetic study for the inhibition of AChE
In order to investigate the mechanism of AChE inhibition, compound
15b was used for a kinetic study with EeAChE as a substrate and
Lineweaver-Burk curves were showed as Fig. 2 [42]. According to the
results, it could be observed that both slopes and vertical intercepts
increased, which means that Km values decreased and Vmax increased at
an increasing concentration of 15b, and simultaneously, the curves
intersected in the second quadrant of the coordinate axis. Thus, the
above results indicated that compound 15b might be a mixed-type in-
hibitor of AChE and it could bind to both the CAS and PAS sites of AChE.
In addition, a plot of the slope versus the inhibitor’s concentration
afforded the inhibition constant Ki for compound 15b as 4.06 μM.
2.2.3. Molecular modeling study
The results above at the enzyme level were in accordance with our
design strategy, which prompted us to further investigate the potential
binding modes of the ligand 15b with its target acetylcholinesterase
(Fig. 3A). The reported X-ray crystal structure of TcAChE (PDB code:
1EVE) was chosen for our molecular modeling study using the docking
program, AutoDock 4.2 package. And the docking result compared with
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C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
Table 1
In vitro inhibition of EeAChE, RatBuChE and MAOs by 4-aminoalkyl-1(2H)-phthalazinone derivatives 15, 16, 23, intermediates 13 and reference compounds.
Compd.
n
NR1R2
IC50
EeAChEc
(
μ
M) ± SDa or % Inhibitionb
RatBuChEd
MAO-A
12.1%
MAO-B
25.9%
13a
13c
3
3
8.0%
n.a.e
6.4%
n.a.e
n.a.e
17.7%
6.1%
12.4%
8.5%
13e
3
6.9%
13f
3
n.a.e
6.8%
26.0%
10.4%
13i
3
3
n.a.e
n.a.e
18.4%
21.9%
19.6%
25.0%
15a
8.0 ± 0.2
9.6%
15b
15c
3
3
8.2 ± 0.2
7.4 ± 0.3
8.9%
6.6%
6.4 ± 0.2
0.7 ± 0.1
18.0%
10.5%
15d
15e
15f
3
3
3
7.3 ± 0.1
9.0 ± 0.2
12.6 ± 0.2
7.8%
5.4%
n.a.e
23.5%
21.0%
37.7%
32.2%
17.9%
3.6 ± 0.2
15 g
15 h
3
3
26.3%
25.4%
n.a.e
n.a.e
19.8%
15.6%
45.8%
37.5%
15i
15j
16a
3
3
5
10.4%
n.a.e
n.a.e
14.9%
13.7%
9.8%
28.5%
32.8%
5.0 ± 0.1
38.9%
0.1 ± 0.1
14.0%
16b
16c
5
5
0.2 ± 0.1
0.5 ± 0.1
31.1%
9.2%
21.3%
22.3%
36.2%
48.3%
16d
16e
5
5
0.7 ± 0.1
0.5 ± 0.1
11.1%
15.9%
21.5%
25.9%
46.2%
34.1%
16f
23a
23c
5
1
1
6.3 ± 0.1
28.1%
n.a.e
n.a.e
n.a.e
24.7%
6.9%
5.5%
32.5%
15.7%
34.8%
32.3%
Donepezil
Rivastigmine
Clorgyline
Rasagiline
Iproniazid
—
—
—
—
—
—
—
—
—
—
0.08 ± 0.001
25.8 ± 1.2
NTf
22.6 ± 0.1
8.2 ± 0.1
NTf
NTf
NTf
NTf
NTf
0.003 ± 0.0005
2.6 ± 0.02
0.8 ± 0.01
2.4 ± 0.1
0.01 ± 0.0007
0.98 ± 0.03
NTf
NTf
NTf
NTf
a
IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and are the mean of three independent experiments, each
performed in triplicate. Data were expressed as mean ± SD (SD = standard deviation).
b
Percentages are the percent inhibition of ChE or MAO by tested compounds at 10
AChE from Electrophorus electricus.
μM.
c
d
e
f
BuChE from rat serum.
n.a. = no active. Compounds defined “no active” means that percent inhibition is<5.0% at a concentration of 10
NT = not tested.
μM in the assay conditions.
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Bioorganic Chemistry 111 (2021) 104895
PAS sites of AChE as a dual-site AChE inhibitor. In addition, the similar
binding modes with TcAChE could be observed from the conformational
overlap of the compound 15b and donepezil (Fig. 3B), which also
confirmed our previous experimental results. Besides, according to the
docking result of compound 16b with AChE (Fig. 4), we could find that
the 2-position amide of 16b also interacted with Phe331 via a strong
hydrogen bond. Moreover, the phthalazinone moiety and 5,6-dimethox-
yphenyl structure of 16b was observed to form parallel π-π stacking
interaction with Tyr334, and 16b also interacted with more amino acids
such as Phe288, Ser286 and Gln69. Furthermore, the combination of
compound 16b (estimated bingding energy = -11.37 kcal/mol) with
AChE was more compact than that of 15b (estimated binding energy =
-10.73 kcal/mol). In other words, compound 16b occupied the CAS site,
the active site of the “cavity” and the PAS site of the enzyme more firmly,
which verified our previous result that the AChE inhibition of the
compounds with six methylene side chain were better than those of
compounds with four.
Fig. 2. Kinetic study on the mechanism of EeAChE inhibition by compound
15b. Merged Lineweaver-Burk reciprocal plots of AChE initial velocity with
increasing substrate concentration (0.1–0.4 mM) in the absence or presence of
15b. Lines were derived from a weighted least-squares analysis of data points.
2.2.4. Inhibition of MAOs in vitro
To further evaluate the multifunctional biological profile of 4-ami-
noalkyl-1(2H)-phthalazinone derivatives, the recombinant human
MAO-A and MAO-B were used to messure the MAOs inhibitory potencies
of our designed derivatives with clorgyline, rasagiline and iproniazid as
the positive controls [43]. The screening results were expressed as IC50
the donepezil molecule was shown in Fig. 3B. In addition, in order to
interpret and verify our result that the inhibition towards AChE was
affected by the length of the carbon chain mentioned in section 2.2.1, we
also conducted a docking experiment of compound 16b. The results
were shown in Fig. 4. The molecular docking results provided a
reasonable explanation for the excellent AChE inhibitory potency of 4-
aminoalkyl-1(2H)-phthalazinone derivatives. As shown in Fig. 3A,
compound 15b bound perfectly to AChE and occupied the entire enzy-
matic CAS, the mid-gorge site and the PAS. In the TcAChE-15b complex,
various of intermolecular interactions were observed. The 2-position
amide of compound 15b interacted with Phe331 via a strong intermo-
lecular hydrogen bond. Simultanously, the phthalazinone moiety and N-
values or percent inhibition by tested compounds at 10 μM. As shown in
Table 1, all the designed derivatives exhibited MAOs inhibitory po-
tencies of varying intensity. In particular, compound 15b elicited
outstanding and representative inhibitory potency towards both MAO-B
(IC50 = 0.7
μ
M) and MAO-A (IC50 = 6.4
μ
M), and the MAO-B inhibitory
M)
M). In addition, compounds 15f and 15j
also exhibited excellent MAO-B inhibitory potencies, with IC50 values as
3.6 M and 5.0 M, respectively. Besides, we could find that compounds
effect was superior to the positive controls clorgyline (IC50 = 2.43
and iproniazid (IC50 = 0.98
μ
μ
μ
μ
benzylethylamine moiety of 15b formed parallel π-π stacking interaction
with N-ethylbenzyl moiety (15b, 15d) exhibited higher MAOs inhibition
than those with N-methylbenzyl moiety (15a, 15c) when it comes to
four methylene moieties as the side chains, while there is no obvious law
of the same kind when compounds with six methylene moieties were
tested. Moreover, it is worth noting that the target derivatives (15a-j,
16a-f, 23a, 23c) exhibited generally higher activities than the in-
termediates without the phthalazinone nuclear (13a-e) and also the lead
with Trp279 and Tyr70, respectively. Besides, hydrophobic bonds could
be observed between 4-position methylene side chain moiety and
various residues including Tyr121, Tyr334 and Phe330. Furthermore,
compounds 15b also exhibited a potential hydrophobic interaction with
residues Ile287, Gly335, Leu332, Asp72, Ser81, Trp84, and Asn85. The
docking results was consistant with the kinetic study, which further
verified that compound 15b could simultaneously bind to the CAS and
Fig. 3. Hypothetical binding mode (A) of compound 15b interacting with residues in the binding site of TcAChE (PDB: 1EVE) and overlap (B) of compounds 15b
(yellow stick) and donepezil (green stick) interacting with residues in the binding site of TcAChE (PDB: 1EVE). The protein residues that participate in the main
interactions with the inhibitor are labeled. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
6

C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
Fig. 4. Hypothetical binding mode (A) of compound 16b interacting with residues in the binding site of TcAChE (PDB: 1EVE) and overlap (B) of compounds 16b (red
stick) and donepezil (green stick) interacting with residues in the binding site of TcAChE (PDB: 1EVE). The protein residues that participate in the main interactions
with the inhibitor are labeled. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
phthalide alkyl tertiary amine derivatives (Fig. 1, percent inhibition
ranging from 1.09% to 38.8%) for inhibition of MAOs [27], which
indicated that the phthalazinone moiety was beneficial to the
improvement of MAOs inhibition.
Cys323, Val210, Ser209, Ile325, Ile335, Ile480, Leu337, Phe208,
Gln215, Met350, Phe352, Tyr444 and so on. As shown in Fig. 5B, the
phthalazinone nucleus of 15b was also close to the coenzyme factor FAD
of MAO-B, and two intermolecular hydrogen bonds with residues
Cys172 and Tyr407 could be observed. In addition, π-π stacking inter-
2.2.5. Molecular modeling studies of MAOs
action existed between ligand 15b and residue Tyr407. Besides, it can be
seen that 15b also interacted with other residues such as Leu164,
Leu167, Leu171, Leu328, Trp119, Pro102, Phe168, Phe343, Ile198,
Ile199, Ile316, Gln206, Tyr60, Tyr326, Tyr398 and so on. Comparing
Fig. 5A with Fig. 5B comprehensively, we can conclude that both MAO-
A and MAO-B could be combined with 15b very well, even if their
combination methods were somewhat different. Indeed, it could be
calculated by simulation that the binding energy of 15b with MAO-A
and MAO-B were ꢀ 9.87 kcal/mol and ꢀ 11.76 kcal/mol, respectively.
In particular, according to the docking result of compound 15a with
MAO-B and overlap of compound 15a and 15b interacting with residues
in the binding site of MAO-B (Supplementary Fig. S3 A/B), we could
find that the phthalazinone nuclear of 15a interacted with Cys172 via a
To further study the hypothetical interactions with both isoforms of
human MAOs, the most potent compound 15b was chosen for docking
simulations using the X-ray crystal structures of human MAO-A (PDB
code: 2Z5X) and MAO-B (PDB code: 2V60). In MAO-A docking simula-
tion (Fig. 5A), the phthalazinone moiety of compound 15b was close to
the coenzyme factor FAD and formed a parallel π-π stacking interaction
with the residue Tyr69. In addition, the 2-amide of compound 15b could
interact with residues Asn181 and Ile207 simultaneously to form two
intermolecular hydrogen bonds, and the same interaction also existed
between the 4-methoxy group of the ligand and residue Tyr407.
Furthermore, it’s worth noting that compound 15b could interact with
many other residues including Leu97, Val93, Gly110, Thr211, Ala111,
Fig. 5. Representation of compound 15b docked into the binding sites of MAO-A (A) and -B (B), highlighting the protein residues that participate in the main
interactions with the inhibitor. Ligand 15b and FAD are displayed in yellow and red, respectively. Hydrogen-bonds are shown with the green dotted lines. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
7

C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
hydrogen bond, which means that 15b has one more intermolecular
hydrogen bond with residue Tyr407 of MAO-B than 15a. Moreover, the
combination of compound 15b with MAO-B (estimated bingding energy
= -11.79 kcal/mol) was more compact than that of 15a (estimated
binding energy = -10.38 kcal/mol), which proves that the the type of N-
substituent on 15b may be important for MAO-B inhibitory potency. In
summary, the above data and analysis of the docking results interpreted
these two facts well that compound 15b exhibited excellent MAOs
inhibitory potency and the inhibition towards MAO-B was stronger than
MAO-A, which further confirmed the reliability of our study on the in-
hibition of MAOs in vitro.
Table 2
Inhibition of platelet aggregation and self-induced Aβ1-42 aggregation, disag-
gregation of self-induced Aβ1-42 aggregation, oxygen radical absorbance ca-
pacity (ORAC, Trolox equivalents) by 4-aminoalkyl-1(2H)-phthalazinone
derivatives
15,
16,
23
and
reference
compounds.
Compd.
% Inhibition of
platelet
% Inhibition of
Aβ1-42
% Disaggregation
of Aβ1-42
ORACf,g
2.2.6. Inhibition of self-induced Aβ1-42 aggregation
Aβ
and Aβ1–42, the two primary isoforms of Aβ peptide, play
1–40
aggregationa,f
aggregationb,c,f
aggregationd,e,f
different roles in the pathogenesis of AD. Between them, Aβ1–42 is more
prone to self-aggregation, thus forming oligomers with higher neuro-
toxicity than Aβ1–40 even if the amount of Aβ1–42 is much lower than Aβ1-
40 [44]. Therefore, Aβ1–42 was selected to assess inhibitory effect of self-
induced Aβ1-42 aggregation by our designed derivatives using the Thi-
oflavin T (ThT) fluorescence assay, with curcumin as the positive con-
trol. The evaluation results were shown in Table 2. In general, most of
the tested compounds exhibited moderate inhibitory percentages of self-
15a
15b
15c
15d
15e
15f
16.1 ± 0.1
14.0 ± 0.2
14.8 ± 0.1
14.5 ± 0.2
15.6 ± 0.1
16.1 ± 0.1
9.8 ± 0.1
15.1 ± 0.2
13.9 ± 0.2
7.4 ± 0.1
11.9 ± 0.1
9.2 ± 0.1
10.9 ± 0.1
9.2 ± 0.1
5.7 ± 0.1
9.9 ± 0.1
20.3 ± 0.2
18.6 ± 0.3
6.1 ± 0.1
23.0 ± 0.1
31.7 ± 0.1
29.1 ± 0.1
34.6 ± 0.2
27.9 ± 0.3
29.5 ± 0.3
31.4 ± 0.1
20.9 ± 0.2
22.3 ± 0.2
28.5 ± 0.3
34.6 ± 0.3
36.2 ± 0.3
33.2 ± 0.3
27.2 ± 0.2
24.5 ± 0.2
30.3 ± 0.2
26.8 ± 0.2
26.5 ± 0.3
NTh
13.3 ± 0.1
21.9 ± 0.1
22.2 ± 0.1
NTh
0.36 ±
0.02
0.49 ±
0.05
0.37 ±
0.01
0.48 ±
0.04
NTh
0.67 ±
0.03
induced Aβ1-42 aggregation, ranging from 20.9% to 36.2% at 25
particular, compounds 15d, 16a, 16b and 16c provided better inhibi-
tory activities (34.6%, 34.6%, 36.2% and 33.2% at 25 M, respectively),
which were stronger than the lead phthalide alkyl tertiary amine de-
rivatives (Fig. 1, percent inhibition ranging from 5% to 30% at 25 M)
M). However,
μM. In
NTh
3.64 ±
0.06
μ
15 g
15 h
15i
NTh
0.32 ±
0.05
NTh
0.42 ±
0.09
μ
[27], but slightly weaker than curcumin (38.1% at 25
μ
NTh
0.42 ±
0.01
almost no significant change of inhibition rates could be observed when
the length of the carbon chain or the tertiary amine structural fragments
varied, which means that no obvious SAR existed for this series of
compounds on the inhibition of self-induced Aβ1ꢀ 42 aggregation.
15j
NTh
0.51 ±
0.05
16a
16b
16c
16d
16e
16f
16.6 ± 0.1
23.2 ± 0.1
24.6 ± 0.2
NTh
0.42 ±
0.01
0.45 ±
0.05
2.2.7. Disaggregation of self-induced Aβ1ꢀ 42 aggregation fibrils
We chose some relatively high-activity target compounds as the
representatives to investigate their disaggregation potency of self-
induced Aβ1ꢀ 42 aggregation fibrils. As shown in Table 2, all the tested
0.43 ±
0.01
0.45 ±
0.03
compounds (25
μM) manifested low to moderate inhibition rates
NTh
0.55 ±
0.02
ranging from 12.0% to 24.6%, compared with the reference compound
curcumin (24.3% at 25 M). In particular, compounds 16b and 16c
exhibited better disaggregation potency (23.2% and 24.6% at 25 M,
respectively), equivalent to the positive control curcumin (24.3% at 25
μ
NTh
3.16 ±
0.08
μ
23a
23c
dl-NBP
12.0 ± 0.2
18.6 ± 0.2
NTh
0.31 ±
0.02
μ
M). However, there was also no obvious SAR could be found among
0.32 ±
0.02
these tested compounds on the disaggregation potency of self-induced
Aβ1ꢀ 42 fibrils, and their further dose-dependent experiment did not be
conducted since the low disaggregation potency.
0.21 ±
0.01
aspirin
curcumin
Compoud
I
9.4 ± 0.1
NTh
NTh
NTh
NTh
38.1 ± 0.1
—
24.3 ± 0.2
—
NTh
2.2.8. Inhibition of platelet aggregation
5.6 ± 0.08
—
Several of literatures have reported that phthalazinone derivatives
showed excellent inhibition of platelet aggregation, which was benefi-
cial to the treatment of AD [45,46]. Therefore, the inhibitory potencies
of platelet aggregation of our designed 4-aminoalkyl-1(2H)-phthalazi-
none derivatives were assessed by the classic turbidimetric method,
using aspirin and n-butylphthalide (dl-NBP) as the positive controls. In
addition, the anti-platelet aggregation potency of compound I, the
typical compound of n-butyphthalide tertiary amines derivatives
(Fig. 1), was also tested as a control for SAR analysis. According to the
experimental results shown in Table 2, we could find that all the syn-
thesized target compounds exhibited better inhibitory potencies of
platelet aggregation ranging from 5.7% to 20.3%, compared with
a
Platelet from rat serum. Tested compounds were used at 33 μM;
b
For inhibition of self-induced Aβ aggregation, the thioflavin-T fluorescence
method was used;
c
Inhibition of self-induced Aβ1-42 aggregation by tested inhibitors at 25 μM;
d
For disaggregation of self-induced Aβ aggregation, the thioflavin-T fluores-
cence method was used;
e
Disaggregation of self-induced Aβ1-42 aggregation by tested inhibitors at 25
μ
M;
f
The mean ± SD of the three independent experiments.
g
h
Data are expressed as μM of Trolox equivalent/μM of tested compound;
NT = not tested.
compound I (5.6% at 33 μM), which indicated that the phthalazinone
(20.3%, 18.6%, respectively) than others. Unfortunately, there was no
obvious difference for inhibition of platelet aggregation could be
observed among these tested compounds, which means that no more
SAR existed among them on anti-platelet aggregation potency.
moiety was crucial for the anti-platelet aggregation potency of this new
series of derivatives. Besides, most of the target compounds displayed
good inhibitory potency, superior to the positive controls aspirin (9.4%
at 33
μM) and butylphthalide (6.1% at 33 μM). Among these com-
pounds, 23a and 23c possessed relatively stronger inhibitory ability
8

C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
2.2.9. In vitro antioxidant potency assay
cells with H2O2 (150
μ
M) decreased to 46% after incubated for 24 h.
To further explore the multi-target biological potency of 4-amino-
alkyl-1(2H)-phthalazinone derivatives, we used the well-established
ORAC-FL method (oxygen radical absorbance capacity by fluorescein)
with a water-soluble vitamin E analog (Trolox) as a standard to assess
their abilities of scavenging radicals, equivalent to antioxidant activ-
ities. The evaluation results were expressed as Trolox equivalent and
shown in Table 2. According to the displayed data, all the tested de-
rivatives exhibited moderate to strong antioxidant activities with the
ORAC values ranging from 0.31 to 3.6-fold of Trolox, superior to dl-NBP
(ORAC value of 0.21 Trolox equivalent). Among them, the antioxidant
potencies of 15f and 16f were particularly prominent with ORAC values
as 3.6 and 3.2-fold Trolox equivalents, respectively, which suggested
that the introduction of N-(4-dimethylaminobenzyl)ethylamine (12f) to
the end of the linker was beneficial to improving the antioxidant po-
tencies of the compounds. In addition, we could find that derivatives
with N-4-dimethylamino group moiety (15f, 16f) showed higher anti-
oxidant activities in comparison to those having N-2-dimethylamino
group (15e, 16e), through which we could conclude that the position of
dimethylamine substituent attached to the benzene ring is a crucial
factor affecting the antioxidant capacities of tested compounds. How-
ever, almost no obvious SAR could be observed when the length of
carbon chains and tertiary amine substituents species changed, which
indicated that these two factors may have no significant influence on the
antioxidant capacity of tested compounds.
However, when compounds 15b, 16b, 15f or 16f, at the concentration
of 1.0 μM, was added to the cells with H2O2 respectively, the cell via-
bilities correspondingly increased to 64.3%, 63.9%, 73.8% or 72.4%.
Furthermore, when the concentrations of the above compounds were
increased to 10
μM, the cell viabilities further increased to 75.7%,
75.5%, 85.2% and 83.8%, respectively. Testing results clearly showed
that the cell viability markedly increased when pretreated with
increasingly concentrations of tested compounds, which further indi-
cated that our designed compounds could effectively attenuate the
damage of PC12 cells induced by H2O2, thereby exerting excellent
neuroprotective activity.
2.2.11. In vitro anti-neuroinflammatory potency evaluation
As reported in the literature, neuroinflammation is closely related to
the development of AD. The activation of microglia, as the main hall-
mark of neuroinflammation, leads to the release of various neurotoxic
substances such as inflammatory cytokines and free radicals, thereby
activating the immune inflammatory response and producing neuro-
toxicity [48–50]. Therefore, inhibition of the release of inflammatory
cytokines and free radicals may play an important role in AD treatment.
In this assay, 15b and 16b were selected as the representative com-
pounds to investigate their anti-neuroinflammatory activities, which
included the evaluation of interference induced by the cytotoxic effect of
compound itself and the measurement on the production of inflamma-
tory mediators NO and TNF-α in LPS-induced BV-2 microglial cells.
2.2.10. Neuroprotective potency against H2O2-induced cell injury in PC12
cells
2.2.11.1. Effect of compounds on survival rate of BV-2 cells. The cyto-
toxicities of tested compounds were assessed by MTT assay [51]. As
shown in Fig. 7, in the presence of different concentrations of 15b or 16b
According to the above collected data, compounds 15b and 16b
exhibited excellent and balanced multi-target activities for AD. Besides,
compounds 15f and 16f showed the strongest antioxidant potencies,
suggesting that they may be potential neuroprotective agents. Thus,
these four representative compounds were selected as the most prom-
ising candidates of this small series for AD treatment, and worthy to be
explored for their neuroprotective activities. It is reported that hydrogen
peroxide (H2O2) is a metabolite in the body and a ROS that plays a key
role in inducing PC12 cell damage [47]. Thus, the neuroprotection po-
tentials of tested compounds at different concentrations (1 and 10 µM)
were evaluated by cell viability assay with PC12 cells in the presence of
H2O2 containing 10% calf serum. Prior to the assessment of neuro-
protective properties, PC12 cells were treated with 15b, 16b, 15f and
(0.5, 2.5 and 10.0 μM), the cell viability for BV-2 cells did not change
significantly with or without lipopolysaccharide (LPS) as a stimulus,
which indicated that both the tested compounds and LPS had no toxic
effect on BV-2 cells.
2.2.11.2. Evaluation of NO and TNF-α in LPS-stimulated BV-2 cells. In-
hibition of LPS-induced NO production was evaluated by Griess reaction
method [52]. First, we evaluated the effect of different concentrations of
tested compounds (0.5, 2.5 and 10.0 μM) on the release of NO in BV-2
cells without LPS. As shown in Fig. 8, the release volume of NO did
not change significantly in the presence of tested compounds, which
suggested that compounds themselves had no effect on the release of NO
in BV-2 cells. Then a markedly increased production of NO could be
16f at 25 μM concentration. Cell viabilities of 97.5%, 99.0%, 97.3% and
98.6%, respectively, indicated that these compounds were not cytotoxic
at the concentrations that were further used for neuroprotection assay
The subsequent measurement results were depicted in Fig. 6. Compared
to the control group without treating H2O2, the cell viabilities of PC12
observed when exposed to LPS (1
μ
g/mL). Noteworthy, pre-treatment
with tested compounds (0.5, 2.5 and 10.0
μ
M) led to a markedly
reduction of LPS-induced NO production, which expressed as the
percent inhibition of 30.2%, 45.8% and 59.6%, respectively, for com-
pound 15b. In addition, the inhibition rates of representative compound
16b were 24.8%, 41.4% and 50.5% at the above three concentrations
respectively. To further investigate the effects of tested compounds on
LPS-induced TNF-
nosorbent assay (ELISA) was used to evaluate their ability of suppressing
TNF- levels [53]. Firstly, we measured the effect of tested compounds
on the TNF- production without the treatment of LPS. As displayed in
Fig. 9, the release volume of TNF- had no significantly change in test
group compared with the control group, thus the compounds themselves
had no effect on it. Then, we could find a markedly increase in TNF-
production when the cells were exposed to LPS (1 g/mL) without tested
compounds. Obviously, the compounds 15b and 16b, in the test group,
exhibited effective inhibitory potencies towards TNF- production in
α production in BV-2 cells, the enzyme-linked immu-
α
α
α
α
μ
α
LPS-stimulated BV-2 cells in a concentration dependent way. The inhi-
bition rates of 15b at three concentrations of 0.5, 2.5 and 10.0 µM were
25.4%, 49.5% and 72.1%, respectively. The inhibition rates of 16b at the
concentrations of 0.5, 2.5 and 10.0 µM were 22.3%, 47.8% and 69.4%,
respectively. In conclusion, both the representative compounds 15b and
Fig. 6. Effects of compounds 15b, 16b, 15f and 16f on H2O2-induced PC12
cells injury (n = 3, mean ± SD; ###p < 0.001 versus control, **p < 0.01 versus
H2O2, ***p < 0.001 versus H2O2).
9

C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
Fig. 7. Effects of compounds 15b and 16b without LPS. (A) or with LPS (1.0
μg/mL); (B) on the cell viability of microglia BV-2 cells. Cell viability was determined by
MTT assay. The data are expressed as the mean ± SD from three independent experiments.
Table 3
Permeability results Pe (×10-6 cm/s) from the PAMPA-BBB assay for selected 4-
(aminoalkyl)-1(2H)-phthalazinone derivatives with their predicted penetration
into the CNS.
Compd.a
Pe (×10-6 cm/s)b
Prediction
15b
16b
4.29 ± 0.32
CNS +
CNS +
10.55 ± 0.40
a
Compounds were dissolved in DMSO and diluted with PBS/EtOH (70:30).
The final concentration of each compound was 100
μ
g/mL.
b
Data are the mean ± SD of three independent experiments.
carried out the parallel artificial membrane permeation assay of the
blood–brain barrier (PAMPA-BBB) reported by Di et al. to evaluate the
BBB permeability of our designed derivatives [54]. Based on the above
pharmacological test results, 15b and 16b were selected as the repre-
sentative compounds to assess their permeability. First, the permeability
of 11 commercial drugs were detected and compared with the literature
value to validate the assay (Supplementary Table S1). A plot of
experimental data versus the literature values obtained a good linear
correlation [49], Pe (exp.) = 0.9163 × Pe (bibl.) ꢀ 0.2247 (R2 = 0.9558)
(Supplementary Fig. S1). Then, according to the correlation equation
and the evaluation conditions established by Di, we defined that com-
pounds with Pe values higher than 3.44 × 10 -6 cm/s could across the
BBB. As shown in Table 3, the Pe values of compounds 15b and 16b
were 4.29 × 10-6 cm/s and 10.55 × 10ꢀ 6 cm/s, respectively, which
indicated that compounds 15b and 16b could penetrate the BBB and
reach the CNS. In addition, the longer the methylene linkers of the tested
compounds were, the better permeability the compounds exhibited. In
summary, these measured results about BBB permeability suggested that
this series of compounds may be drugable and of good clinical
applicability.
Fig. 8. Effects of compounds 15b and 16b on NO release in BV-2 cells and LPS-
stimulated BV-2 cells (n = 3, mean ± SD; ###p < 0.001 versus control, **p <
0.01, ***p < 0.001 versus LPS).
3. Conclusion
In summary, our study presented aimed to developing new small
molecule ligands endowed with multi-target biological activities against
AD. A series of 4-aminoalkyl-1(2H)-phthalazinone derivatives were
designed, synthesized and evaluated as multifunctional agents for AD
therapy. Overall, in vitro assays demonstrated that most of the synthe-
sized compounds, compared with the lead phthalide alkyl tertiary amine
Fig. 9. Effects of compounds 15b and 16b on TNF-α release in LPS-stimulated
BV-2 cells (n = 3, mean ± SD; ###p < 0.001 versus control, *p < 0.05, ***p <
0.001 versus LPS).
16b inhibited the release of NO and TNF-α in LPS-induced BV-2 cells,
indicating that they exhibited good anti-neuroinflammatory activities in
vitro and could be beneficial to the treatment of AD.
derivatives, possessed enhanced inhibition of MAOs, self- and Cu2+
-
induced Aβ1–42 aggregation and platelet aggregation, almost equivalent
antioxidant activities and slightly decreased AChE inhibitory effects,
which indicated that this new series of derivatives have more balanced
and effective multi-target potencies against AD, consist with our ex-
pectations. Among these synthesized derivatives, compound 15b was
2.2.12. In vitro blood–brain barrier permeation assay
Penetrating the blood–brain barrier (BBB), followed by reaching the
central nervous system (CNS), is crucial to determine the efficacy of
drugs and development value as candidate anti-AD drugs. Thus, we
10

C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
demonstrated as a potent and balanced multi-target candidate for AD.
The kinetic analysis and molecular modeling study suggested 15b was a
mixed-type AChE inhibitor (IC50 = 8.24 µM), and could bind to both CAS
and PAS of AChE. In addition, 15b also exhibited highest inhibition
potency towards MAO-B and MAO-A (IC50 = 0.72 µM and 6.41 µM
4.1.3. Synthesis of 3-bromo-5,6-dimethoxyisobenzofuran-1(3H)-one (5)
To a solution of 5,6-dimethoxyisobenzofuran-1(3H)-one (4) (2 g,
10.30 mmol) and AIBN (100 mg, 0.60 mmol) in dry chlorobenzene (40
mL), under an argon atmosphere and heated to 85 ◦C, was added NBS
(2.02 g, 11.33 mmol) slowly in portions. After the addition, the mixture
was kept at 85 ◦C for 3 h. The mixture was cooled to room temperature,
filtered and washed with dry chlorobenzene three times, concentrated
under reduced pressure to afford the crude compound 5 as a brown oil;
yield 78.2%.
respectively), good inhibition of self- and Cu2+-induced Aβ
aggre-
1-42
gation, platelet aggregation and neuroinflammation, effective antioxi-
dant activity and neuroprotective potency. Moreover, 15b manifested
the appropriate BBB permeability to enter the brain. Taken together,
these outstanding and comprehensive properties qualified compound
15b as a promising multi-target candidate for AD therapy, worthy of
further studies.
4.1.4. Synthesis of 3-hydroxy-5,6-dimethoxyisobenzofuran-1(3H)-one (6)
A mixture of compound 5 (2 g, 7.32 mmol) and H2O (20 mL) was
refluxed for 2 h. After completion of the reaction as indicated by TLC,
the white solid crude was filtered from the reaction mixture, washed
with aqueous 10% solution of HCl two times, and dried to yield white
solid product 6; yield 65.8%; mp 173.5 ~ 175.6 ◦C; (Lit. [55] 173.0 ~
176.0 ◦C).
4. Experimental section
4.1. General chemistry
All reagents and solvents, unless otherwise noted, were purchased
from commercial sources and used without further purification. The 1H
NMR and 13C NMR spectra were recorded in CDCl3 or DMSO‑d6 using
TMS as the internal standard on a Varian INOVA spectrometer at 25 ◦C.
Coupling constants are given in Hz. Mass spectra were recorded on
Agilent-6210 TOF LC-MS Spectrometer. And melting points were
measured on YRT-3 melting-point apparatus (China). The purity of all
the target compounds was confirmed through HPLC analysis on a Shi-
madzu LC-10Avp plus system by a Kromasil C 18 column (4.6 mm × 250
4.1.5. Synthesis of (5,6-Dimethoxyisobenzofuran-1(3H)-one-3-yl)
phosphonic acid diethylester (7)
Compound 6 (2.5 g, 11 mmol) and triethyl phosphate (3.65 g, 22
mmol) were dissolved in CHCl3 (30 mL), and then Py⋅HClO4 was added
◦
at 0 C. The resulting mixture stirred at room temperature overnight.
After that, to the reaction mixture dichloromethane (30 mL) was added,
washed with H2O (4 × 20 mL) and brine (2 × 20 mL), dried over sodium
sulfate and filtered, concentrated under reduced pressure. The obtained
residue was recrystallized from ether to afford the desired compound 7
as a light yellow solid; yield 80.5%; mp 120.1 ~ 122.3 ◦C; 1H NMR (600
MHz, CDCl3) δ 7.32 (s, 1H) , 7.18 (s, 1H), 5.59 (d, J = 10.8 Hz, 1H),
4.34–4.29 (m, 2H), 4.04–4.02 (m, 1H), 4.00 (s, 3H), 3.96 (s, 3H),
3.88–3.83 (m, 1H), 1.43 (t, J = 7.2 Hz, 3H), 1.11 (t, J = 7.2 Hz, 3H).
mm, 5
μm). The mobile phase was a mixture of methanol and water and
the injection volume was 20
μ
L. Separation was performed under a
constant temperature of 37 ◦C and the effluent was monitored at a UV
absorption wavelength of 254 nm with a flow rate of 1.0 mL/min. In
addition, reaction progress was monitored by thin-layer chromatog-
raphy (TLC) using silica gel GF 254 plates from Qingdao Haiyang
Chemical Co. Ltd. (China). The spots were visualized under a UV lamp or
in iodine chamber. Column chromatography was performed using silica
gel (230–400 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd.
(China).
4.1.6. General procedure for the synthesis of 8a-b
To a solution of compound 7 (1 equiv.) and corresponding inter-
mediate 3a or 3b (1.2 equiv.) in dichloromethane was added ethanol
solution of NaOH (1.2 equiv.). The resulting mixture stirred at room
temperature for 2 h, to the mixture was added dichloromethane, washed
with H2O and brine, dried over sodium sulfate, filtered and concentrated
under reduced pressure to provide the crude product 8a or 8b.
4.1.1. General procedure for the synthesis of α, ω-diol benzoic acid
monoesters (2a or 2b)
A mixture of corresponding
α,ω-diols 1a or 1b (5.0 equiv.) and
triethylamine (1.2 equiv.) in THF was stirred at room temperature for
10 min. After that, the reaction mixture was cooled to 0 ◦C, and then a
solution of benzoyl chloride (1.0 equiv.) in THF was added dropwise.
4.1.7. General procedure for the synthesis of 9a-b
Compound 8a or 8b (1 equiv.), dimethylamine hydrochloride (3.0
equiv.) and triethylamine (3.0 equiv.) were dissolved in ethanol and
refluxed for 3 h under an argon atmosphere. After the reaction was
finished, the solvent was evaporated under reduced pressure. Then H2O
was added to the residue and the mixture was extracted with dichloro-
methane three times. The combined organic phases were washed with
brine, dried over sodium sulfate, filtered and evaporated to dryness to
afford the corresponding crude product 9a or 9b.
◦
The reaction mixture was stirred at 50 C for an additional 3 h. Upon
completion of the reaction, water was added to the mixture and then
extracted with ethyl acetate. The combined organic phases were washed
with saturated aqueous solution of NaHCO3 and brine, dried over
anhydrous Na2SO4, filtered and evaporated to dryness to afford crude
intermediate 2a or 2b.
4.1.2. General procedure for the synthesis of benzoyloxyalkyl aldehydes
(3a or 3b)
4.1.8. General procedure for the synthesis of 10a-b
To a mixture of compound 9a or 9b (1 equiv.) in CH3OH/H2O (5:1)
was added K2CO3 (2 equiv.). The mixture was stirred at room temper-
ature for 15 h. The organic solvent was evaporated under vacuum and
H2O was added when TLC verified the disappearance of the precursor.
Then, the resulting mixture was extracted with dichloromethane three
times. The combined organic phases were washed with brine, dried over
sodium sulfate, filtered and concentrated under reduced pressure to give
corresponding intermediates 10a (yield 81.3%) or 10b (yield 78.6%).
Cyanuric chloride (1.5 equiv.) was dissolved in THF at room tem-
perature. When the solution cooled to about ꢀ 30 ◦C, a solution of DMSO
(6 equiv.) in tetrahydrofuran was added slowly and stirred for 30 min.
Then to the mixture intermediate 2a or 2b (1.0 equiv.) in THF was added
and stirred for another 30 min, followed by triethylamine and the re-
action mixture stirred at ꢀ 30 ◦C for 20 min. Later, the solution was
allowed to warm up to room temperature and kept for 3 h and then
concentrated in vacuum. To the residue 5% aqueous HCl was added,
extracted with ethyl acetate, washed with saturated aqueous solution of
NaHCO3 and brine, dried over sodium sulfate and filtered, concentrated
under reduced pressure to give the crude compund 3a or 3b; yield 85.2%
(3a) and 90.5% (3b).
4.1.9. General procedure for the synthesis of 11a-b
Triethylamine (1.2 equiv.) was added to a solution of intermediate
10a or 10b (1.0 eq.) in dichloromethane. The mixture was cooled to
0–5 ◦C, followed by adding a solution of benzoyl chloride (1.2 equiv.) in
dichloromethane and the resulting mixture continued to stir in the ice
bath for 5 min. Later, the solution was allowed to warm up to room
11

C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
temperature and kept overnight. To the resulting reaction mixture was
added H2O and extracted with dichloromethane three times. Then the
combined organic phases were washed with saturated aqueous solution
of NaHCO3 and brine, dried over anhydrous sodium sulfate and evap-
orated under reduced pressure to afford the corresponding crude prod-
ucts, which were purified on a silica gel chromatography to obtain
compound 11a or 11b.
4.1.11.3. 2-[5-(N-ethyl-N-(2-dimethylaminobenzyl)amino)pentanoyl]-
4,5-dimethoxy-N,N-dimethyl benzamide (13e). Synthesized from com-
pound 11a and N-(2-dimethylaminobenzyl)ethylamine 12e via general
procedure. Purified by column chromatography (petroleum ether/
acetone = 10/1, v/v) to afford intermediate 13e as a light yellow oil;
yield 62.2%; 1H NMR (600 MHz, CDCl3) δ 7.83 (d, J = 7.2 Hz, 1H), 7.36
(t, J = 7.2 Hz, 1H), 7.32 (s, 1H), 7.22 (d, J = 7.2 Hz, 1H), 7.18 (t, J = 7.2
Hz, 1H), 6.74 (s, 1H), 4.26 (s, 2H), 3.98 (s, 3H), 3.93 (s, 3H), 3.12 (s,
2H), 2.99–2.90 (m, 6H), 2.76 (s, 2H), 2.63 (s, 6H), 1.98–1.85 (m, 2H),
1.73–1.69 (m, 2H), 1.36–1.30 (m, 5H).
4.1.9.1. 5-[(2-N,N-Dimethylcarbamoyl-4,5-dimethoxy)phenyl]-5-oxo-
benzenesulfonic acid pentylester (11a). Synthesized from compound 10a
via general procedure. Purified by column chromatography (petroleum
ether/acetone = 20/1, v/v) to afford intermediate 11a as a white solid;
yield 89.4%; mp 127.5 ~ 128.6 ◦C; 1H NMR (400 MHz, CDCl3) δ 7.90 (d,
J = 7.6z, 2H), 7.66 (t, J = 7.6 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.28 (s,
1H) , 6.75 (s, 1H), 4.08 (t, J = 6.4 Hz, 2H), 3.96 (s, 3H), 3.93 (s, 3H),
3.12 (s, 3H), 2.88 (t, J = 7.2 Hz, 2H), 2.75 (s, 3H), 1.76–1.75 (m, 4H).
4.1.11.4. 2-[5-(N-ethyl-N-(4-dimethylaminobenzyl)amino)pentanoyl]-
4,5-dimethoxy-N,N-dimethyl benzamide (13f). Synthesized from com-
pound 11a and N-(4-dimethylaminobenzyl)ethylamine 12f via general
procedure. Purified by column chromatography (petroleum ether/
acetone = 10/1, v/v) to afford intermediate 13f as a light yellow oil;
yield 65.2%; 1H NMR (600 MHz, CDCl3) δ 7.32–7.30 (m, 2H), 7.29 (s,
1H), 6.75 (s, 1H), 6.68 (d, J = 8.4 Hz, 2H), 3.97 (s, 3H), 3.96 (s, 2H),
3.93 (s, 3H), 3.13 (s, 3H), 2.96 (t, 2H), 2.92 (s, 6H), 2.84–2.80 (m, 2H),
2.80–2.77 (t, 2H), 2.77 (s, 3H), 1.92–1.82 (m, 2H), 1.74–1.70 (m, 2H),
1.34–1.28 (m, 3H).
4.1.9.2. 7-[(2-N,N-dimethylcarbamoyl-4,5-dimethoxy)phenyl]-7-oxo-
benzenesulfonic acid heptylester (11b). Synthesized from compound 10b
via general procedure. Purified by column chromatography (petroleum
ether/acetone = 20/1, v/v) to afford intermediate 11b as a light yellow
oil; yield 92.0%; 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 7.6 Hz, 2H),
7.66 (t, J = 7.6 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.29 (s, 1H) , 6.76 (s,
1H), 4.05 (t, J = 6.4 Hz, 2H), 3.96 (s, 3H), 3.93 (s, 3H), 3.13 (s, 3H), 2.85
(t, J = 7.2 Hz, 2H), 2.76 (s, 3H), 1.70–1.64 (m, 4H), 1.35–1.33 (m, 4H).
4.1.11.5. 2-(5-morpholinovaleryl)-4,5-dimethoxy-N,N-dimethylbenzamide
(13i). Synthesized from compound 11a and morpholine 12i via general
procedure. Purified by column chromatography (petroleum ether/
acetone = 10/1, v/v) to afford intermediate 13i as a light yellow oil;
yield 73.1%; 1H NMR (600 MHz, CDCl3) δ 7.30 (s, 1H), 6.76 (s, 1H), 3.96
(s, 3H), 3.93 (s, 3H), 3.77–3.68 (m, 4H), 3.13 (s, 3H), 2.92 (t, J = 7.2 Hz,
2H), 2.76 (s, 3H), 2.51–2.44 (m, 4H), 2.39 (t, J = 7.2 Hz, 2H), 1.76–1.73
(m, 2H), 1.60–1.58 (m, 2H).
4.1.10. General procedure for the synthesis of secondary amines (12a-j)
Compounds 12a-j (colorless or yellow oil, yield 73.6 ~ 93.8%) were
prepared as previously reported by Kumpaty et al. [56].
4.1.11. General procedure for the synthesis of derivatives (13–14)
The derivative 13 or 14 was synthesized in acetonitrile at 50 ◦C for
5–7 h by the reaction of intermediate 11a or 11b (1.0 equiv.) with
secondary amines(1.0 equiv.) in presence of potassium carbonate (1.7
equiv.). Then H2O was added to the reaction solution to obtain a
mixture, which was extracted with ethyl acetate three times. The com-
bined organic phases were washed with water two times and 10%
aqueous HCl four times to give a combined acidic water layer, of which
the pH was adjusted to 13 with 10% aqueous NaOH. Subsequently, the
resulting mixture was extracted with dichloromethane three times, the
combined organic phases were washed by brine, dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuum to give the crude
product which was purified by flash chromatography on silica gel to
afford the desired compound 13 or 14.
4.1.12. General procedure for the synthesis of 4-(aminoalkyl)-1(2H)-
phthalazinone derivatives 15–16
Compound 13 or 14 and hydrazine hydrate were dissolved in ethanol
and refluxed for 8 ~ 10 h under an argon atmosphere. After the reaction
checked by TLC was finished, the solvent was evaporated under reduced
pressure, and the obtained residue was purified by preparative thin-
layer chromatography to produce target compound 15 or 16.
4.1.12.1. 4-[4-(N-methyl-N-benzylamino)butyl]-6,7-dimethoxy-1(2H)-
phthalazinone (15a). Synthesized from compound 13a via general pro-
cedure. Purified by preparative thin-layer chromatography using ethyl
acetate/methanol (15:1, v/v) as the eluent to produce compound 15a as
a light yellow oil; yield 61.1%; 1H NMR (400 MHz, CDCl3) δ 10.80 (s,
1H), 7.82 (s, 1H), 7.32–7.24 (m, 5H), 7.08 (s, 1H), 4.05 (s, 3H), 4.01 (s,
3H), 3.55 (s, 2H), 2.92 (t, J = 7.2 Hz, 2H), 2.50 (t, J = 7.2 Hz, 2H), 2.24
(s, 3H), 1.89–1.81 (m, 2H), 1.75–1.69 (m, 2H). 13C NMR (150 MHz,
CDCl3) δ 160.5, 153.7, 152.2, 146.4, 138.1, 129.2 (2C), 128.2 (2C),
127.1, 125.7, 122.7, 106.6, 104.6, 61.9, 56.7, 56.4, 56.2, 41.9, 31.8,
26.7, 25.1. ESI-MS m/z: 382.2 [M + H]+.
4.1.11.1. 2-[5-(N-methyl-N-benzylamino)pentanoyl]-4,5-dimethoxy-N,N-
dimethylbenzamide (13a). Synthesized from compound 11a and N-
benzylmethylamine 12a via general procedure. Purified by column
chromatography (petroleum ether/acetone = 10/1, v/v) to afford in-
termediate 13a as a light yellow oil; yield 71.1%; 1H NMR (400 MHz,
CDCl3) δ 7.35–7.26 (m, 6H), 6.76 (s, 1H), 3.96 (s, 3H), 3.93 (s, 3H), 3.58
(s, 2H), 3.13 (s, 3H), 2.90 (t, J = 7.2 Hz, 2H), 2.76 (s, 3H), 2.49 (t, J =
7.2 Hz, 2H), 2.26 (s, 3H), 1.77–1.70 (m, 2H), 1.69–1.63(m, 2H).
4.1.12.2. 4-[4-(N-ethyl-N-benzylamino)butyl]-6,7-dimethoxy-1(2H)-
phthalazinone (15b). Synthesized from compound 13b via general pro-
cedure. Purified by preparative thin-layer chromatography using ethyl
acetate/methanol (15:1, v/v) as the eluent to produce compound 15b as
a light yellow oil; yield 62.2%; 1H NMR (400 MHz, CDCl3) δ 11.00 (s,
1H), 7.83 (s, 1H), 7.33–7.19 (m, 5H), 7.06 (s, 1H), 4.05 (s, 3H), 4.00 (s,
3H), 3.62 (s, 2H), 2.89 (t, J = 7.2 Hz, 2H), 2.57–2.50 (m, 4H), 1.85–1.77
(m, 2H), 1.69–1.63 (m, 2H), 1.06 (t, J = 7.2 Hz, 3H). 13C NMR (150
MHz, CDCl3) δ 160.6, 153.7, 152.3, 146.6, 139.1, 128.9 (2C), 128.1
(2C), 126.8, 125.2, 122.8, 106.6, 104.6, 57.8, 56.5, 56.4, 52.6, 47.2,
31.9, 26.5, 25.3, 11.5. ESI-MS m/z: 396.2 [M + H]+.
4.1.11.2. 5-(N-methyl-N-(2-methoxybenzyl)amino)pentanoyl]-4,5-dime-
thoxy-N,N-dimethylbenzamde (13c). Synthesized from compound 11a
and N-(2-methoxybenzyl)methylamine 12c via general procedure. Pu-
rified by column chromatography (petroleum ether/acetone = 10/1, v/
v) to afford intermediate 13c as a light yellow oil; yield 71.5%; 1H NMR
(400 MHz, CDCl3) δ 7.41 (d, J = 7.2 Hz, 1H), 7.30 (s, 1H), 7.28 (t, J =
7.2 Hz, 1H), 6.95 (t, J = 7.2 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.75 (s,
1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.84 (s, 3H), 3.77 (s, 2H), 3.13 (s, 3H),
2.94 (t, J = 7.2 Hz, 2H), 2.76 (s, 3H), 2.63 (t, J = 7.2 Hz, 2H), 2.37 (s,
3H), 1.79–1.65 (m, 4H).
4.1.12.3. 4-[4-(N-methyl-N-(2-methoxybenzyl)amino)butyl]-6,7-dime-
thoxy-1(2H)-phthalazinne (15c). Synthesized from compound 13c via
12

C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
general procedure. Purified by preparative thin-layer chromatography
using ethyl acetate/methanol (15:1, v/v) as the eluent to produce
compound 15c as a light yellow oil; yield 61.5%; 1H NMR (400 MHz,
CDCl3) δ 10.75 (s, 1H), 7.81 (s, 1H), 7.40 (d, J = 7.2 Hz, 1H), 7.29 (t, J =
7.2 Hz, 1H), 7.10 (s, 1H), 6.94 (t, J = 7.2 Hz, 1H), 6.88 (d, J = 8.4 Hz,
1H), 4.05 (s, 3H), 4.02 (s, 3H), 3.81 (s, 3H), 3.79 (s, 2H), 2.95 (t, J = 7.2
12.37 (s, 1H), 7.62 (s, 1H), 7.28 (s, 1H), 3.99 (s, 3H), 3.94 (s, 3H),
3.12–3.01 (m, 6H), 2.99–2.94 (m, 2H), 1.82–1.72 (m, 4H), 1.23–1.19
(m, 6H). 13C NMR (150 MHz, DMSO‑d6) δ 159.5, 153.7, 152.4, 145.2,
124.8, 122.6, 106.3, 106.0, 56.6, 56.3, 50.9, 46.5 (2C), 31.0, 24.6, 23.3,
9.0 (2C) . ESI-MS m/z: 334.2 [M + H]+.
Hz, 2H), 2.69 (t, J = 7.2 Hz, 2H), 2.39 (s, 3H), 1.86–1.69 (m, 4H). 13
C
4.1.12.9. 4-(4-morpholinylbutyl)-6,7-dimethoxy-1(2H)-phthalazinone
(15i). Synthesized from compound 13i via general procedure. Purified
by preparative thin-layer chromatography using ethyl acetate/methanol
(15:1, v/v) as the eluent to produce compound 15i as a white solid; mp
192.8 ~ 194.1 ◦C; yield 50.1%; 1H NMR (400 MHz, CDCl3) δ 10.74 (s,
1H), 7.81 (s, 1H), 7.08 (s, 1H), 4.05 (s, 3H), 4.04 (s, 3H), 3.81–3.75 (m,
4H), 2.95 (t, J = 7.2 Hz, 2H), 2.57–2.52 (m, 6H), 1.89–1.77 (m, 2H),
1.69–1.61 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 160.4, 153.7, 152.3,
146.2, 125.1, 122.8, 106.6, 104.6, 66.7, 58.5, 56.5, 56.4, 56.3, 56.2,
53.6, 31.8, 26.0, 25.1. ESI-MS m/z: 348.3 [M + H]+.
NMR (150 MHz, CDCl3) δ 160.5, 157.9, 153.7, 152.3, 146.3, 131.6 (2C),
129.3, 125.1, 122.6, 120.5, 110.5, 106.5, 104.6, 56.5, 56.4, 56.3, 55.3,
54.6, 41.3, 31.6, 25.9, 24.9. ESI-MS m/z: 412.2 [M + H]+.
4.1.12.4. 4-[4-(N-ethyl-N-(2-methoxybenzyl)amino)butyl]-6,7-dime-
thoxy-1(2H)-phthalazinone (15d). Synthesized from compound 13d via
general procedure. Purified by preparative thin-layer chromatography
using ethyl acetate/methanol (15:1, v/v) as the eluent to produce
compound 15d as a light yellow oil; yield 59.5%; 1H NMR (400 MHz,
CDCl3) δ 10.89 (s, 1H), 7.82 (s, 1H), 7.48 (d, J = 6.8 Hz, 1H), 7.26 (t, J =
6.8 Hz, 1H), 7.08 (s, 1H), 6.93 (t, J = 7.2 Hz, 1H), 6.86 (d, J = 8.0 Hz,
1H), 4.05 (s, 3H), 4.01 (s, 3H), 3.81 (s, 2H), 3.80 (s, 3H), 2.92 (t, J = 7.2
4.1.12.10. 4-[4-(4-Methylpiperazin-1-yl)butyl]-6,7-dimethoxy-1(2H)-
phthalazinone (15j). Synthesized from compound 14j via general pro-
cedure. Purified by preparative thin-layer chromatography using ethyl
acetate/methanol (15:1, v/v) as the eluent to produce compound 15j as
a white solid; mp 108.7 ~ 109.9 ◦C; yield 51.2%; 1H NMR (400 MHz,
CDCl3) δ 11.15 (s, 1H), 7.81 (s, 1H), 7.08 (s, 1H), 4.05 (s, 3H), 4.02 (s,
3H), 2.93 (t, J = 7.6 Hz, 2H), 2.72–2.50 (m, 8H), 2.48 (t, J = 7.6 Hz, 2H),
2.34 (s, 3H), 1.86–1.79 (m, 2H), 1.70–1.66 (m, 2H). 13C NMR (150 MHz,
CDCl3) δ 160.3, 153.7, 152.3, 146.2, 125.1, 122.8, 106.6, 104.5, 58.0,
56.4, 56.3, 54.6 (2C), 52.7 (2C), 45.7, 31.8, 26.3, 25.2. ESI-MS m/z:
361.2 [M + H]+.
Hz, 2H), 2.79–2.61 (m, 4H), 1.92–1.69 (m, 4H), 1.25–1.10 (m, 3H). 13
C
NMR (150 MHz, CDCl3) δ 160.3, 157.8, 153.8, 152.3, 146.3, 131.1 (2C),
128.9, 125.1, 122.7, 120.5, 110.4, 106.6, 104.6, 56.4, 56.3, 55.3, 52.5,
50.8, 47.4, 31.7, 25.7, 25.0, 10.7. ESI-MS m/z: 426.2 [M + H]+.
4.1.12.5. 4-[4-(N-ethyl-N-(2-dimethylaminobenzyl)amino)butyl]-6,7-
dimethoxy-1(2H)-phthalazinone (15e). Synthesized from compound 13e
via general procedure. Purified by preparative thin-layer chromatog-
raphy using ethyl acetate/methanol (15:1, v/v) as the eluent to produce
compound 15e as a light yellow oil; yield 52.2%; 1H NMR (400 MHz,
CDCl3) δ 10.87 (s, 1H), 7.82–7.75 (m, 2H), 7.29 (t, J = 7.2 Hz, 1H),
7.17–7.08 (m, 3H), 4.06 (s, 2H), 4.05 (s, 6H), 2.94 (t, J = 7.2 Hz, 2H),
2.90–2.78 (m, 2H), 2.65 (s, 6H), 1.95–1.78 (m, 3H), 1.32–1.15 (m, 4H).
13C NMR (150 MHz, CDCl3) δ 160.3, 153.9, 153.6, 152.4, 146.0, 131.3,
129.2, 125.1, 124.2, 122.6, 119.8, 106.6, 104.6, 56.4, 52.3, 51.3, 47.1,
45.1, 31.4, 29.6, 29.3, 24.9, 24.6, 9.7. ESI-MS m/z: 438.8 [M + H]+.
4.1.12.11. 4-[6-(N-methyl-N-benzylamino)hexyl]-6,7-dimethoxy-1(2H)-
phthalazinone (16a). Synthesized from compound 14a via general pro-
cedure. Purified by preparative thin-layer chromatography using ethyl
acetate/methanol (15:1, v/v) as the eluent to produce compound 16a as
a light yellow oil; yield 61.8%; 1H NMR (600 MHz, CDCl3) δ 10.75 (s,
1H), 7.83 (s, 1H), 7.32–7.25 (m, 5H), 7.09 (s, 1H), 4.05 (s, 3H), 4.04 (s,
3H), 3.52 (s, 2H), 2.90 (t, J = 7.2 Hz, 2H), 2.41 (t, J = 7.2 Hz, 2H), 2.21
4.1.12.6. 4-[4-(N-ethyl-N-(4-dimethylaminobenzyl)amino)butyl]-6,7-
dimethoxy-1(2H)-phthalazinone (15f). Synthesized from compound 13f
via general procedure. Purified by preparative thin-layer chromatog-
raphy using ethyl acetate/methanol (15:1, v/v) as the eluent to produce
(s, 3H), 1.82–1.77 (m, 2H), 1.60–1.55 (m, 2H), 1.48–1.39 (m, 4H). 13
C
NMR (150 MHz, CDCl3) δ 160.4, 153.8, 152.3, 146.7, 138.1, 129.2 (2C),
128.2 (2C), 127.1, 125.2, 122.9, 106.7, 104.7, 62.2, 57.2, 56.5, 56.3,
42.0, 32.1, 29.3, 27.6, 27.2, 27.1. ESI-MS m/z: 410.2 [M + H]+.
1
compound 15f as a light yellow oil; yield 52.2%; H NMR (400 MHz,
CDCl3) δ 10.75 (s, 1H), 7.81 (s, 1H), 7.23 (d, J = 8.4 Hz, 2H), 7.08 (s,
1H), 6.66 (d, J = 8.4 Hz, 2H), 4.05 (s, 3H), 4.03 (s, 3H), 3.74 (s, 2H),
2.93 (s, 6H), 2.92 (t, J = 7.2 Hz, 2H), 2.80–2.61 (m, 4H), 1.86–1.79 (m,
4H), 1.22–1.18 (m, 3H). 13C NMR (150 MHz, CDCl3) δ 160.4, 153.7,
152.3, 150.2, 146.1, 130.7 (2C), 128.1, 125.1, 122.6, 112.2 (2C), 106.5,
104.5, 56.5, 56.4, 56.3, 51.6, 46.6, 40.4 (2C), 31.5, 25.2, 24.8, 10.4. ESI-
MS m/z: 439.1 [M + H]+.
4.1.12.12. 4-[6-(N-ethyl-N-benzylamino)hexyl]-6,7-dimethoxy-1(2H)-
phthalazinone (16b). Synthesized from compound 14b via general pro-
cedure. Purified by preparative thin-layer chromatography using ethyl
acetate/methanol (15:1, v/v) as the eluent to produce compound 16b as
a light yellow oil; yield 60.2%; 1H NMR (400 MHz, CDCl3) δ 10.77 (s,
1H), 7.83 (s, 1H), 7.35–7.21 (m, 5H), 7.08 (s, 1H), 4.05 (s, 3H), 4.03 (s,
3H), 3.60 (s, 2H), 2.89 (t, J = 7.6 Hz, 2H), 2.54 (q, J = 7.2 Hz, 2H), 2.46
(t, J = 7.2 Hz, 2H), 1.82–1.74 (m, 2H), 1.57–1.51 (m, 2H), 1.47–1.28 (m,
4H), 1.06 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 160.2, 153.7,
152.3, 146.7, 138.2, 128.9 (2C), 128.1 (2C), 126.8, 125.1, 122.8, 106.7,
104.7, 57.8, 56.4, 56.2, 52.8, 47.1, 32.0, 29.2, 27.5, 27.1, 26.6, 11.3.
ESI-MS m/z: 424.3 [M + H]+.
4.1.12.7. 4-(4-piperidylbutyl)-6,7-dimethoxy-1(2H)-phthalazinone
(15
g). Synthesized from compound 13 g via general procedure. Purified by
preparative thin-layer chromatography using ethyl acetate/methanol
(15:1, v/v) as the eluent to produce compound 15 g as a white solid; mp
83.1 ~ 84.1 ◦C; yield 52.4%; 1H NMR (400 MHz, CDCl3) δ 11.19 (s, 1H),
7.79 (s, 1H), 7.08 (s, 1H), 4.06 (s, 3H), 4.05 (s, 3H), 2.98–2.92 (m, 2H),
2.78–2.67 (m, 6H), 2.00–1.84 (m, 8H), 1.71–1.55 (m, 2H). 13C NMR
(150 MHz, DMSO‑d6) δ159.5, 153.6, 152.3, 145.5, 124.8, 122.6, 106.2,
105.9, 57.7, 56.4, 56.2, 53.7 (2C), 31.3, 25.2, 25.1, 24.8 (2C), 23.6. ESI-
MS m/z: 346.2 [M + H]+.
4.1.12.13. 4-[6-(N-methyl-N-(2-methoxybenzyl)amino)hexyl]-6,7-dime-
thoxy-1(2H)-phthalazinone (16c). Synthesized from compound 14c via
general procedure. Purified by preparative thin-layer chromatography
using ethyl acetate/methanol (15:1, v/v) as the eluent to produce
1
compound 16c as a light yellow oil; yield 59.5%; H NMR (400 MHz,
CDCl3) δ 10.86 (s, 1H), 7.82 (s, 1H), 7.40 (d, J = 7.2 Hz, 1H), 7.28 (t, J =
7.2 Hz, 1H), 7.10 (s, 1H), 6.95 (t, J = 7.2 Hz, 1H), 6.88 (d, J = 8.0 Hz,
1H), 4.05 (s, 3H), 4.00 (s, 3H), 3.83 (s, 3H), 3.76 (s, 2H), 2.90 (t, J = 7.2
Hz, 2H), 2.61–2.55 (m, 2H), 2.37 (s, 3H), 1.86–1.78 (m, 2H), 1.77–1.62
(m, 2H), 1.55–1.36 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 160.3, 157.9,
153.8, 152.3, 146.6, 131.5 (2C), 129.3, 125.2, 122.8, 120.5, 110.5,
4.1.12.8. 4-(4-Diethylaminobutyl)-6,7-dimethoxy-1(2H)-phthalazinone
(15 h). Synthesized from compound 13 h via general procedure. Puri-
fied by preparative thin-layer chromatography using ethyl acetate/
methanol (15:1, v/v) as the eluent to produce compound 15 h as a white
solid; mp 59.4 ~ 60.5 ◦C; yield 51.5%; 1H NMR (400 MHz, DMSO‑d6) δ
13

C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
106.7, 104.7, 56.9, 56.5, 56.3, 55.4, 54.6, 41.3, 32.0, 29.0, 27.3, 27.0,
dimethoxyisobenzofuran-3- one-1-ylidene) ethyl acetate 212 ~ 214 ◦C).
26.1. ESI-MS m/z: 439.8 [M + H]+.
4.1.15. Synthesis of 2-(6,7-dimethoxy-1(2H)-phthalazinone-4-yl)ethyl
acetate (20)
4.1.12.14. 4-[6-(N-ethyl-N-(2-methoxybenzyl)amino)hexyl]-6,7-dime-
thoxy-1(2H)-phthalazinone (16d). Synthesized from compound 14d via
general procedure. Purified by preparative thin-layer chromatography
using ethyl acetate/methanol (15:1, v/v) as the eluent to produce
compound 16d as a light yellow oil; yield 59.8%; 1H NMR (400 MHz,
CDCl3) δ 10.85 (s, 1H), 7.83 (s, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.28 (t, J =
7.6 Hz, 1H), 7.09 (s, 1H), 6.98 (t, J = 7.2 Hz, 1H), 6.89 (d, J = 8.4 Hz,
1H), 4.05 (s, 3H), 4.02 (s, 3H), 3.95 (s, 2H), 3.84 (s, 3H), 2.90 (t, J = 7.2
Hz, 2H), 2.87–2.79 (m, 2H), 2.78–2.71 (m, 2H), 1.82–1.73 (m, 4H),
1.47–1.38 (m, 4H), 1.25–1.20 (m, 3H). 13C NMR (150 MHz, CDCl3) δ
160.3, 157.8, 153.8, 152.3, 146.5, 131.8 (2C), 128.9, 125.2, 122.7,
120.8, 110.5, 106.6, 104.7, 56.4, 56.3, 55.4, 52.3, 50.5, 47.1, 31.9, 28.9,
27.2, 27.0, 25.0, 10.2. ESI-MS m/z: 454.2 [M + H]+.
(E/Z)-2-(5,6-dimethoxyisobenzofuran-1-one-3-ylidene) ethyl acetate
(19) (360 mg, 1.3 mmol) and hydrazine hydrate (0.16 mL, 2.6 mmol)
were resolved in ethanol (3 mL) and refluxed for 3 h. After the reaction
was finished, the solvent was evaporated under reduced pressure, and
the obtained residue was purified by column chromatography to afford
intermediate 20 as a white solid; yield 83%; mp 183.1 ~ 186.0 ◦C; 1H
NMR (400 MHz, CDCl3) δ 10.68 (s, 1H), 7.82 (s, 1H), 7.10 (s, 1H), 4.19
(q, J = 7.2 Hz, 2H), 4.05 (s, 3H), 4.02 (s, 3H), 3.94 (s, 2H), 1.28–1.22 (m,
3H).
4.1.16. Synthesis of 4-(2-hydroxyethyl)-6,7-dimethoxy-1(2H)-
phthalazinone (21)
To a solution of intermediate 20 (180 mg, 0.65 mmol) in ethanol (2
mL) and tetrahydrofuran (0.5 mL) was added sodium borohydride (100
mg, 2.6 mmol) and lithium chloride (112 mg, 2.6 mmol) at 0–5 ◦C,
stirred and kept for 30 min. The mixture was warmed up to room tem-
perature and stirred overnight. Then, the pH of the reaction mixture was
adjusted to 4 with aqueous 10% solution of HCl, stirred at room tem-
perature for 30 min and filtered to give the residue, which was recrys-
tallized to afford product 21 as a white solid; yield 80.1%; mp > 220 ◦C.
4.1.12.15. 4-[6-(N-ethyl-N-(2-dimethylaminobenzyl)amino)hexyl]-6,7-
dimethoxy-1(2H)-phthalazin one (16e). Synthesized from compound
14e via general procedure. Purified by preparative thin-layer chroma-
tography using ethyl acetate/methanol (15:1, v/v) as the eluent to
produce compound 16e as a light yellow oil; yield 52.8%; 1H NMR (400
MHz, CDCl3) δ 10.49 (s, 1H), 7.88 (d, J = 7.2 Hz, 1H), 7.81 (s, 1H), 7.34
(t, J = 7.2 Hz, 1H), 7.23–7.14 (m, 2H), 7.09 (s, 1H), 4.28 (s, 2H), 4.07 (s,
3H), 4.05 (s, 3H), 3.08–2.87 (m, 6H), 2.65 (s, 6H), 1.83–1.45 (m, 4H),
1.44–1.28 (m, 7H). 13C NMR (150 MHz, CDCl3) δ 160.1, 153.9, 153.6,
152.4, 146.3, 132.0 (2C), 130.4, 125.2, 125.0, 122.7, 120.4, 106.7,
104.7, 56.5, 51.8, 50.6, 46.7, 45.6 (2C), 31.7, 29.7, 28.7, 26.9, 26.7,
23.6, 8.8. ESI-MS m/z: 467.3 [M + H]+.
4.1.17. Synthesis of 4-(2-chloroethyl)-6,7-dimethoxy-1(2H)-
phthalazinone (22)
Thionyl chloride (0.03 mL, 0.48 mmol) was added to the solution of
compound 21 in N,N-dimethylformamide (1 drop) and chloroform (2
mL) and refluxed for 10 h. After the reaction was finished, the solvent of
the mixture was evaporated in vacuum to give the crude product, which
was then recrystallized with ethanol to yield compound 22 as a light
yellow solid; yield 89.2%; mp > 220 ◦C; 1H NMR (600 MHz, DMSO‑d6) δ
12.45 (s, 1H), 7.62 (s, 1H), 7.30 (s, 1H), 4.04 (t, J = 6.6 Hz, 2H), 3.99 (s,
3H), 3.94 (s, 3H), 3.24 (t, J = 6.0 Hz, 2H).
4.1.12.16. 4-[6-(N-ethyl-N-(4-dimethylaminobenzyl)amino)hexyl]-6,7-
dimethoxy-1(2H)-phthalazin one (16f). Synthesized from compound 14f
via general procedure. Purified by preparative thin-layer chromatog-
raphy using ethyl acetate/methanol (15:1, v/v) as the eluent to produce
1
compound 16f as a light yellow oil; yield 52.2%; H NMR (400 MHz,
CDCl3) δ 10.50 (s, 1H), 7.82 (s, 1H), 7.27 (d, J = 8.4 Hz, 2H), 7.09 (s,
1H), 6.69 (d, J = 8.4 Hz, 2H), 4.06 (s, 3H), 4.05 (s, 3H), 3.78 (s, 2H),
2.98 (s, 6H), 2.89 (t, J = 7.2 Hz, 2H), 2.80–2.71 (m, 2H), 2.69–2.61 (m,
2H), 1.81–1.67 (m, 4H), 1.46–1.38 (m, 4H), 1.25–1.22 (m, 3H). 13C
NMR (150 MHz, CDCl3) δ 160.0, 153.7, 152.3, 150.3, 146.5, 130.9 (2C),
128.1, 125.1, 122.7, 112.3 (2C), 106.6, 104.7, 56.4, 56.3, 51.7, 46.4,
40.4 (2C), 31.8, 29.6, 28.9, 27.1, 26.9, 25.1, 10.2. ESI-MS m/z: 467.3
[M + H]+.
4.1.18. General procedure for the synthesis of 4-(aminoalkyl)-1(2H)-
phthalazinone derivatives (23a or 23c)
The target compound 23a or 23c was synthesized in acetonitrile
under reflux condition for 10 h by the reaction of compound 22 (1.0
equiv.) with the corresponding secondary amine 12a or 12c (1.5 equiv.)
in presence of potassium carbonate (1.5 equiv.) and a catalytic amount
of tetrabutylammonium bromide (TBAB). After the reaction was
finished, the solvent was evaporated under reduced pressure. Then H2O
was added to the residue and the mixture was extracted with dichloro-
methane. The combined organic phases were washed with brine, dried
over sodium sulfate, filtered and concentrated under reduced pressure to
obtain the crude product, which was then purified by preparative thin-
layer chromatography to yield compound 23a or 23c.
4.1.13. Synthesis of 5,6-dimethoxyphthalic anhydride (18)
In a 10 mL round bottom flask, 4,5-dimethoxyphthalic acid (17)
(600 mg, 2.65 mmol) and acetic anhydride (1 mL, 10.61 mmol) were
dissolved in tetrahydrofuran(3 mL) and refluxed for 5 h. To remove
tetrahydrofuran and acetic anhydride of the mixture, it was evaporated
under reduced pressure. Then petroleum ether (5 mL) was added to the
residue, refluxed for 5 min and filtered while hot. The resulting residue
was washed with an appropriate amount of petroleum ether, and dried
in vacuum to obtain compound 18 as a white solid; yield 94.8%; mp
178.2 ~ 179.6 ◦C. (Lit.[57] 179 ~ 181 ◦C)
4.1.18.1. 4-[2-(N-methyl-N-benzylamino)ethyl]-6,7-dimethoxy-1(2H)-
phthalazinone (23a). Synthesized from compound 22 and N-benzylme-
thylamine (12a) via general procedure. Purified by preparative thin-
layer chromatography using ethyl acetate/methanol (15:1, v/v) as the
eluent to produce compound 23a as a light yellow oil; yield 62.1%; 1H
NMR (400 MHz, CDCl3) δ 10.39 (s, 1H), 7.79 (s, 1H), 7.30–7.26 (m, 5H),
7.04 (s, 1H), 4.04 (s, 3H), 3.91 (s, 3H), 3.66 (s, 2H),3.15 (t, J = 7.2 Hz,
2H), 2.88 (t, J = 7.2 Hz, 2H), 2.43 (s, 3H). ESI-MS m/z: 354.2 [M + H]+.
4.1.14. Synthesis of (E/Z)-2-(5,6-dimethoxyisobenzofuran-1-one-3-
ylidene) ethyl acetate (19)
To a solution of compound 18 (168 mg, 0.48 mmol) in toluene (2 mL)
was added ethyl 2-(triphenylphosphinoidene)acetate (100 mg, 0.48
mmol) prepared as the literature reported [38] and refluxed for 3 h.
Upon completion and cooling to room temperature, the solvent was
evaporated under reduced pressure, and the obtained residue was pu-
rified by column chromatography to afford intermediate 19 as a white
4.1.18.2. 4-[2-(N-methyl-N-(2-methoxybenzyl)amino)ethyl]-6,7-dime-
thoxy-1(2H)-phthalazinone (23c). Synthesized from compound 22 and
N-benzylmethylamine (12c) via general procedure. Purified by prepar-
ative thin-layer chromatography using ethyl acetate/methanol (15:1, v/
v) as the eluent to produce compound 23c as a light yellow oil; yield
63.2%; 1H NMR (400 MHz, CDCl3) δ 11.25 (s, 1H), 7.81 (s, 1H),
◦
solid; yield 89%; mp 155.1 ~ 158.0 C (Lit.[58] (E)-2-(5,6-dimethoxy
isobenzofuran-3-one-1-ylidene) ethyl acetate 168 ~ 170 ◦C; (Z)-2-(5,6-
14

C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
7.33–7.23 (m, 2H), 7.16 (s, 1H), 6.92–6.85 (m, 2H), 4.05 (s, 3H), 3.95 (s,
3H), 3.82 (s, 3H), 3.74 (s, 2H), 3.25 (t, J = 7.6 Hz, 2H), 2.96 (t, J = 7.6
Hz, 2H), 2.46 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 160.5, 157.8, 153.9,
152.4, 145.2, 131.1 (2C), 128.8, 125.2, 122.7, 120.4, 110.5, 106.6,
104.9, 56.5, 56.4, 55.6, 55.4, 55.2, 42.3, 30.3. ESI-MS m/z: 383.8 [M +
H]+.
Å. The AUTODOCK4.2 program package was used for molecular docking
experiments, and the docking calculation was performed by 100 runs
using the Lamarckian genetic algorithm (LGA) of the AUTODOCK. Apart
from the mentioned parameters above, others were as defaulted.
Furthermore, a cluster analysis of docking results was carried out using a
root mean square (RMS) tolerance of 1.0, followed by recording and
analyzing the optimal cluster docking results and comprehensive se-
lection. Graphic manipulations and visualizations were done by Auto-
dock Tools or Discovery Studio 2.5 software.
4.2. Biological evaluation
4.2.1. Inhibition experiments of AChE and BuChE
4.2.4. In vitro inhibition of MAO [59]
The inhibitory potencies of synthesized compounds on AChE and
BuChE were evaluated by the modified Ellman’s method [41]. Purified
AChE derived from Electrophorus electricus (Sigma-Aldrich Co.) and
BuChE from rat serum. For the determination of AChE inhibition assay,
The MAO inhibition assay were carried out using the recombinant
human MAO-A and MAO-B purchased from commercial sources (Sigma
Co.), pre-aliquoted and stored at ꢀ 80 ◦C. Kynurenamine was used as the
substrate of MAO, since kynuramine could be oxidized by monoamine
oxidase (MAO) to produce 4-hydroxyquinoline, which could further
produce fluorescence under alkaline conditions. Thus, the inhibitory
potency of the tested compounds towards MAO was measured by
detecting its fluorescence value. First, the tested compounds were dis-
solved with DMSO and diluted with phosphate buffer (100 mM, pH =
7.40, containing 20.2 mM KCl) before use. Then, to the reaction plate
thioacetylcholine iodide (1 mmol/L, 30
tion (0.1 mmol/L, pH = 8.0, 40
tested compounds solution (20
(0.05 U/mL, 10 L) were added to the 96-well plate. After shaking for 1
μ
L), phosphate-buffered solu-
L), different concentrations of the
L, DMSO content<1%) and EeAChE
μ
μ
μ
min, incubation for 15 min at 37 ◦C, then 5,5′-dithiobis-2-nitrobenzoic
acid (DTNB, 0.2%, 30 µL) (J&K Scientific) was added to this mixed so-
lution. The absorbance (OD value) of each well was measured at 412 nm
with a Varioskan Flash Multimode Reader (Thermo Scientific). With
regard to the BuChE inhibition assay, almost the same method as above
was used excepting the differences that butyrylthiocholine iodide (1
mmol/L, 30 µL) as a substrate, a phosphate-buffered solution (0.1
mmol/L, pH = 7.4) and 25% rat serum supernatant (10 µL) were used in
this assay, and the change in absorbance of each well was measured at
405 nm. In addition, the IC50 values represented the concentration of
inhibitor that produced 50% enzyme activity inhibition. donepezil and
Tacrine were used as the positive controls and the results obtained were
the average values after performing at least three parallel experiments,
expressed as mean ± SD.
was added a final volume of 500
of tested compounds (0–100 mol, 100
mL, 300 L) and kynuramine solution (225
μ
L containing different concentrations
L), MAO-A solution (12.5 g/
M, 100 L), with the final
g/mL and 45 M,
μ
μ
μ
μ
μ
μ
concentrations of MAO-A and kynuramine being 7.5
μ
μ
respectively, and the DMSO content being less than 4%. The obtained
◦
mixed solution was incubated for 30 min at 37 C, then quenched by
NaOH solution (2 mol/L, 400 μL) and distilled water (1000 μL), followed
by centrifuging for 10 min at 16,000 g. After that, the supernatant was
extracted and measured the fluorescence intensity using a Varioskan
Flash Multimode Reader at excitation and emission wavelengths of 310
nm and 400 nm, respectively. IC50 values were obtained with excitation
wavelengthgmoidal dose–response curves (graphs of the initial rate of
kynuramine oxidation versus the logarithm of inhibitor concentration)
using GraphPad Prism software and expressed as mean ± SD. The
drawing of sigmoidal curve required selecting at least six different
concentrations of compounds with three orders of magnitude. Each
group of experiments were performed independently and repeated for at
least three times. In addition, the measurement method of inhibitor
activities against MAO-B for tested compounds was almost the same as
that against MAO-A, excepting that the concentration of kynuramine
4.2.2. Kinetic study for the inhibition of AChE
The kinetic characterization of the compound for the inhibition of
AChE was carried out according to the reported method [42].To the
solution containing phosphate-buffered solution (20
μ
L, pH = 8.0),
EeAChE (0.5 U/mL, 10 μL), DTNB (30 μL, 0.2%) in 96-well plate was
added three different concentrations of tested compounds. After shaking
and mixing, the mixed solution was incubated at 37 ◦C for 15 min,
followed by the addition of substrate (ATCh) in different concentrations,
and then the OD values were measured and recorded at a wavelength of
412 nm. The parallel control experiments was performed without in-
hibitor.The resulting graph was analyzed by a weighted least square
analysis. Furthermore, the slopes of Lineweaver-Burk curves were
plotted against the concentrations of 15b in a weighted analysis to
obtain a linear graph and the intercept on the negative x-axis was the Ki
for the inhibition of AChE.
added was changed to 150 μM.
4.2.5. Molecular modeling study of MAO
The crystal structure of hMAO-A (PDB: 2Z5X) and hMAO-B (PDB:
2V60) were used to study the molecular docking with tested compound
15b [60]. After performing a series of pretreatments including the
removal of the original ligand and water molecular and the addition of
polar hydrogen onto both protein and coenzyme factor, AUTODOCK 4.2
program was used to perform the docking studies. Furthermore, each
group of docking experiment was calculated by Lamarck Genetic Algo-
rithm (LGA) for 100 times. Finally, a cluster analysis was performed
based on the docking results, with a root mean square (RMS) tolerance
of 1.0. Noteworthy, the lowest docking-energy conformation of the
highest populated cluster was comprehensively selected for the result
analysis. Graphic manipulations and visualizations were performed by
using Autodock Tools or Discovery Studio 2.5 software.
4.2.3. Molecular modeling study
The crystal structure of TcAChE complex with donepezil (PDB: 1EVE)
was used to study the molecular docking between compound 15b and
AChE. First, deleting the ligand donepezil to obtain the receptor TcAChE
required for the docking. Then, the visualization software Autodock
Tools (ADT, version 1.5.6) was used for the setting of docking parame-
ters as well as performing the pretreatment of protein and small mo-
lecular, which included two aspects: (1) Adding polar hydrogen to the
amino acid residues of the protein, and loading Gasteiger charges on
each atom of the protein molecular. (2) Charging the small molecular,
calculating and setting freely rotating keys. In addition, the obtained
protein structure was calculated, using AUTOGRID, to obtain the atomic
affinity grid maps for each atom type in the ligand, which included
desolvation and electrostatics. All the maps were calculated with 0.375
Å spacing between grid points., and the grid point was placed at the
bottom of the enzyme activity (coordinates: x = 2.023, y = 63.295, z =
67.062). The dimensions of the active site box were set at 60 × 60 × 60
4.2.6. Inhibition of self-induced Aβ1-42 aggregation
Thioflavin-T assay was used to measure the inhibitory potencies of
tested compounds towards self-induced Aβ1-42 aggregation [61,62]. The
commercial Aβ
trifluoroacetate was first dissolved in hexa-
1-42
fluoroisopropanol (TFIP) and incubated at room temperature for 24 h.
Then, the solvent was evaporated under reduced pressure, the residue
was further dissolved in DMSO to obtain the Aβ1-42 solution and stored
at ꢀ 80 ◦C, and the frozen stock solution above was diluted by potassium
15

C. Ye et al.
Bioorganic Chemistry 111 (2021) 104895
phosphate buffer (50 mM, pH = 7.4) to be at the concentration of 50
μ
M
excitation and emission wavelengths of 485 nm and 535 nm, respec-
tively. Finally, the area AUC under the fluorescence decay curve was
calculated by the instrument, and the net AUC was obtained by sub-
tracting the AUC of the blank. Trolox (6-Hydroxy-2,5,7,8-tetramethyl-
before use. To a 96-well plate was added three different samples
including the mixture of Aβ1-42 solution (50
compounds (50 M, 20 L), Aβ1-42 solution (50
potassium phosphate buffer (50 mM, pH = 7.4, 20
2%) and a blank control of potassium phosphate buffer (50 mM, pH =
7.4, 20 L, DMSO content 2%) plus potassium phosphate buffer (50 mM,
pH = 7.4, 20 L, DMSO content 25%). After addition and mixing, the
samples above were placed in a thermostat and incubated at 37 ◦C for
24 h. Then, to each-well sample was added a solution (5 M, 160 L) of
μ
M, 20
M, 20
L, DMSO content
μ
L) and tested
μ
μ
μ
μL) diluted by
μ
chromane-2-carboxylic acid) was used as standard (1–8 μM, final
concentration). The blank control consisting of fluorescein (FL), potas-
sium phosphate buffer and AAPH were performed in each assay. The
antioxidant activities of the compounds were expressed as Trolox
equivalents by using the standard curve calculated for each sample. All
the reaction mixture was prepared in duplicate, and each set of experi-
ment was performed independently at least three times.
μ
μ
μ
μ
Thioflavin T in Glycine-NaOH buffer (50 mM, pH = 8.5), followed by
shaking and scanning with a Varioskan Flash Multimode Reader for 5
min at excitation and emission wavelengths of 446 nm and 490 nm,
respectively. The fluorescence value of each sample was recorded.
Finally, the inhibition rate of the compounds was calculated as the
formula: 100-(Ifi-IF0)/(Ifc-IF0) × 100, in which the Ifi and Ifc repre-
sented the fluorescence value obtained from Aβ1ꢀ 42 in the presence and
in the absence of inhibitors, respectively, and the IFO represented the
fluorescence value of the sample with only potassium phosphate buffer.
In this assay, curcumin was used as a positive control and each com-
pound was tested in three wells in parallel with each concentration.
4.2.10. Neuroprotective activity against H2O2-induced cell injury in PC12
cells [24,66,67]
The neuroprotection assay was conducted using PC12 cells con-
taining 10% calf serum. First, PC-12 cells were prepared in DMEM and
seeded into the 96-well plate at a density of 1.5 × 105 cells/mL and
incubated in a constant temperature incubator containing 5% CO2 at
37 ◦C for 24 h. After that, to the administration group was added
different concentrations (1 and 10 µM) of the tested compounds and
incubated for 2 h, followed by adding H2O2 (150 µM) a inducer of PC12
cell injury and incubated for 24 h. Then, to the cultured PC12 cells was
4.2.7. Disaggregation of self-induced Aβ1-42 aggregation fibrils
added MTT solution (0.5 mg/mL, 100 μL) to measure the cell viability
For the disaggregation of self-induced Aβ1-42 fibrils experiment, Aβ
and cultured at 37 ◦C for another 2 h. Finally, DMSO (100 µL) was added
into each well to dissolve the precipitated formazan and the optical
density (OD) was meassured at 490 nm with a microplate reader. Results
were adjusted considering OD measured in the blank. Each essay was
performed independently at different concentrations in triplicate.
1-
42 solution (50
μ
M, 20
μ
L) and potassium phosphate buffer (20
μ
L, pH =
7.4, DMSO content 25%) were first added to the 96-well plate and
incubated at 37 ◦C for 24 h to fully aggregate the Aβ1-42. Then, to the
above sample was added the tested compounds (50
μM, 20
μ
L), the
◦
resulting mixture was further incubated at 37 C for 24 h. Finally, to
each-well sample was added a solution (5
μM, 160
μL) of Thioflavin T in
4.2.11. In vitro anti-neuroinflammatory activity evaluation [51–53]
The anti-neuroinflammatory activity of tested compounds was
evaluated through measuring their in vitro cytotoxicity and inhibition of
Glycine-NaOH buffer (50 mM, pH = 8.5). The final concentration of
both Aβ1-42 and the tested compounds in each well were 25 μM. Each
assay was run in triplicate. The detection method was the same as that of
LPS-induced NO and TNF-α production. Dulbecco’s modified eagle
self-induced Aβ1–42 experiment.
medium (DMEM) was purchased from Gibco. Murine microglia cell line
(BV-2) obtained from ATCC was cultured in DMEM and incubated at
37 ◦C in a humidified incubator supplemented with 5% CO2. The
inducing reagent lipopolysaccharide (LPS) was purchased from Sigma,
4.2.8. Inhibition of platelet aggregation
The inhibitory potencies of the tested compounds towards platelet
aggregation were evaluated by the classic turbidimetric method [63,64].
First, the rats were anesthetized with chloral hydrate, and the blood was
collected from their abdominal aorta, followed by anticoagulation per-
formed with a 3.8% sodium citrate saline solution. Then, the obtained
blood was centrifuged at 800 r/min for 10 min, the supernatant was
carefully collected to give the desired platelet-rich plasma PRP. After
that, the remaining part was further centrifuged at 3000 r/min for 15
min, and collected the supernatant to obtain the platelet-poor plasma
PPP. Finally, the PRP concentration was adjusted with PPP so that the
rat TNF-α ELISA kit were obtained from Abcam, and nitric oxide kit was
purchased from Nanjing Jiancheng Bioengineering Institute. All the
tested compounds were dissolved in DMSO and diluted with PBS buffer
to afford various concentrations (0.5, 2.5 and 10.0 µM) of sample diluent
before use.
In vitro cytotoxicity, meassured by the MTT assay, was assessed
through evaluating the effect of compounds on cell viability. First, BV-2
cells were seeded in DMEM (100 μL) and cultured in 96-well cell culture
microplate and incubated in a humidified incubator containing 5% CO2
platelet count was 6–7.5 × 104/
μ
L to obtain the required plasma. After
L) and the tested
L) were added to a test cup, and incubated in
the platelet aggregation apparatus at 37 ◦C for 5 min. Then, to the
mixture above was added the induction reagent ADP solution (300 M,
at 37 ◦C for 24 h. Then, 10
μL of tested samples (0.5, 2.5 and 10.0 µM)
preparing anticoagulated blood, the plasma (270
μ
were added to cells in triplicate wells and a blank control group was set,
followed by incubation for another 30 min. Subsequently, the treated
compounds (0.5 mM, 20
μ
cells were exposed to LPS (1.0 μg/mL, 10 μL) and continued the culture
μ
for 24 h, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide) solution (0.5 mg/mL, 10 L), dissolved with PBS
buffer, was added to each well and incubated for 4 h at 37 ◦C. Finally,
10
μ
L), and the maximum aggregation rate was tested and recorded
μ
within 5 min. The measurement was performed using physiological sa-
line (containing 20% DMSO) instead of the tested compounds as a
control group.
the crystals obtained were dissolved in DMSO (200 μL) and OD values
were measured at 490 nm using a spectrophotometer. The results were
recorded and expressed as the percent cell viability compared to the
control group.
4.2.9. In vitro antioxidant activity assay
The well-established ORAC-FL method (oxygen radical absorbance
capacity by fluorescein) were used to assess the antioxidant activities of
Griess reagent system, following the manufacturers’ protocols, was
used to evaluate the inhibition of LPS-induced NO production. The
methods of culturing BV-2 cells, setting blank control and adding tested
compounds and LPS were the same as those in cytotoxicity assay. After
that, the cell culture supernatants and different concentrations of NaNO2
as a standard were added to a 96-well plate since the NaNO2 concen-
tration in the supernatant could act as an indicator of NO production.
Then, to each well was added the same volume of Griess reagent and
incubated at room temperature for 10 min. Finally, the absorbance was
the tested compounds [65]. First, the compounds solution (10 μM or 50
μ
M, 20 μL) and fluorescein solution (250 μM, 120 μL) were added to the
96-well plate. After mixing, the mixture was incubated at 37 ◦C for 15
min and then added 2,2′-Azobisisobutyronitrile dihydrochloride
(AAPH) solution (60 μL, 12 mM final concentration) rapidly by the
automatic sampler of a Varioskan Flash Multimode Reader, followed by
shaking for 30 s and measuring the fluorescence values for 90 min at
16

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Bioorganic Chemistry 111 (2021) 104895
´
´
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enzyme-linked immunosorbent assay (ELISA). The methods of BV-2 cells
culture, adding different concentration of tested compounds (10 L) and
LPS (1.0 g/mL, 10 L) and setting blank control group were the same as
above in cytotoxicity assay. Then, the supernatant of the cell culture
μ
μ
μ
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solution (50
μ
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TNF-
kit.
α was measured by ELISA according to the instructions of the ELISA
4.2.12. In vitro blood–brain barrier permeation assay
The parallel artificial membrane permeation assay of the blood–-
brain barrier (PAMPA-BBB) reported by Di et al. was performed to
meassure the BBB permeability of the selected compounds 15b and 16b
[54]. First, the porcine brain lipid (PBL) was dissolved in dodecane (20
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lipophilic filter membrane of the receptor hole to simulate the biofilm.
After standing for 5 min, the evenly infiltrated hydrophobic film
changed from white to translucent. Then, to the donor microplate was
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added the tested compounds (100
μ
g/mL, 350
μL), and the acceptor
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microplate was filled with 300
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pholipid membrane could touch the donor fluid, thus forming a
sandwich-like structure with the donor solution at the bottom. After
that, the sandwich-like structure was placed in a humidified environ-
◦
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adding the solution above (150 μL) in the acceptor and donor wells to
the quartz 96-well plate, respectively, scanning at 200–600 nm (step =
10 nm) with the Varioskan Flash Multimode Reader (Thermo Scientific),
and finally recording the OD values. Each essay was performed inde-
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Declaration of Competing Interest
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The authors declare that they have no known competing financial
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the work reported in this paper.
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Acknowledgments
This work was supported by Chinese National Natural Science
Foundation (22077091), Sichuan Science and Technology Program
(2020YFS0067) and the Fundamental Research Funds for the Central
Universities.
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