Design, synthesis, and biological evaluation of novel xanthone-alkylbenzylamine hybrids as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2021.113154

In this study, a series of multifunctional hybrids against Alzheimer's disease were designed and obtained by conjugating the pharmacophores of xanthone and alkylbenzylamine through the alkyl linker. Biological activity results demonstrated that compound 4j was the most potent and balanced dual ChEs inhibitor with IC₅₀ values 0.85 μM and 0.59 μM for eeAChE and eqBuChE, respectively. Kinetic analysis and docking study indicated that compound 4j was a mixed-type inhibitor for both AChE and BuChE. Additionally, it exhibited good abilities to penetrate BBB, scavenge free radicals (4.6 trolox equivalent) and selectively chelate with Cu²⁺ and Al³⁺ at a 1:1.4 ligand/metal molar ratio. Importantly, after assessments of cytotoxic and acute toxicity, we found compound 4j could improve memory function of scopolamine-induced amnesia mice. Hence, the compound 4j can be considered as a promising lead compound for further investigation in the treatment of AD.

Full text of DOI:10.1016/j.ejmech.2021.113154

European Journal of Medicinal Chemistry 213 (2021) 113154
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis, and biological evaluation of novel xanthone-
alkylbenzylamine hybrids as multifunctional agents for the treatment
of Alzheimer’s disease
Zhipeng Zhang a, Jie Guo a, Maojun Cheng a, Weixin Zhou a, Yang Wan a, Rikang Wang a,
,
, *
Yuanying Fang a, Yi Jin a, Jing Liu b **, Sai-Sai Xie a
a National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Traditional Chinese Medicine,
Nanchang, 330006, PR China
b School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang, 330006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a series of multifunctional hybrids against Alzheimer’s disease were designed and obtained
by conjugating the pharmacophores of xanthone and alkylbenzylamine through the alkyl linker. Bio-
logical activity results demonstrated that compound 4j was the most potent and balanced dual ChEs
Received 4 November 2020
Received in revised form
15 December 2020
Accepted 31 December 2020
Available online 11 January 2021
inhibitor with IC50 values 0.85 mM and 0.59 mM for eeAChE and eqBuChE, respectively. Kinetic analysis
and docking study indicated that compound 4j was a mixed-type inhibitor for both AChE and BuChE.
Additionally, it exhibited good abilities to penetrate BBB, scavenge free radicals (4.6 trolox equivalent)
and selectively chelate with Cu2þ and Al3þ at a 1:1.4 ligand/metal molar ratio. Importantly, after as-
sessments of cytotoxic and acute toxicity, we found compound 4j could improve memory function of
scopolamine-induced amnesia mice. Hence, the compound 4j can be considered as a promising lead
compound for further investigation in the treatment of AD.
Keywords:
Alzheimer’s disease
Xanthone
Cholinesterase
Antioxidant
© 2021 Elsevier Masson SAS. All rights reserved.
Metal chelator
1. Introduction
cholinergic neurons and the associated loss of cholinergic neuro-
transmission in the cerebral cortex are responsible for the deteri-
Alzheimer’s disease (AD), one of the most severe and prevalent
neurodegenerative diseases, affects approximately 46 million
people worldwide and incurs a huge economic loss on families and
societies [1]. According to the World Alzheimer Report, the popu-
lation of AD will be increased up to 66 million by 2030 and 115
million by 2050 [2]. Clinical evidence demonstrate that AD is a
complex disease characterized by progressive memory loss, severe
behavioral abnormalities, cognitive impairment and ultimately
death. Although the etiology of AD is not completely known,
oration of cognitive function observed in the brain of AD patients
[4]. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)
are the two types of cholinesterases (ChEs) for hydrosis of ACh in
brain. The enzymatic cavity of AChE is characterized by a nearly
20 Å deep narrow gorge, which is composed of two binding sites:
the catalytic active site (CAS) at the bottom and the peripheral
anionic site (PAS) near the entrance of the gorge [5]. BuChE also has
a similar structure of the cavity. However, compared to the BuChE,
the AChE accounts for approximately 80% of ACh hydrolysis in
normal brains, so it has received much attention in past decades,
and many approved drugs are AChE inhibitors [6]. Additionally,
apart from the function of hydrolysis of ACh, studies show that
AChE can accelerate amyloid fibril formation through its peripheral
several factors including deficits of acetylcholine (ACh),
b-amyloid
(A ) deposits, oxidative stress, dyshomeostasis of biometals and
b
neuroinflammation, have been appointed to play crucial roles in AD
onset and progression [3].
The cholinergic hypothesis proposes that the degeneration of
anionic site (PAS), producing stable AChE-Ab complexes, which
have more neurotoxicity than single A peptide [7]. Thus, dual
b
binding site inhibitors that interact with both CAS and PAS appear
to be more beneficial for AD treatment. Recent years, inhibition of
BuChE is also becoming the focus on AD treatment, because a
growing body of evidence has indicated that, with disease
* Corresponding author.
** Corresponding author.
E-mail addresses: liujing860828@163.com (J. Liu), xiesaisainanchang@hotmail.
com (S.-S. Xie).
https://doi.org/10.1016/j.ejmech.2021.113154
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
progression, the activity of AChE decreases to 10e15% of normal
values in certain brain regions, while BuChE as a compensating
enzyme is maintained at the normal or even higher level [8]. Hence,
designing ChE inhibitors that simultaneously block both AChE and
BuChE may lead to additional benefits for the treatment of AD.
Oxidative stress is put as one of the most important causes of
Alzheimer’s Disease [9]. Reactive oxygen species (ROS) including
superoxide anion radical (Oꢀ2ꢁ), hydroxyl radical (ꢀOH) and
peroxide (O22ꢁ), and reactive nitrogen species (RNS) including per-
oxynitrite anion (ONOOꢁ) and nitric oxide (NOꢀ) are primary oxi-
dants [10]. Under normal physiological conditions, the level of
oxidants formation is in equilibrium with the antioxidant capacity,
while the production of oxidants overwhelms the cellular antioxi-
dant capacity in AD [11]. The excessive generation of ROS damage
the biological molecules, such as DNA, proteins and lipids, leading
to the death of neurons. Therefore, engaging in accelerating the free
radical scavenging is beneficial for the prevention of AD.
could inhibit both AChE and BuChE simultaneously. Furthermore, in
order to get optimal linker length for balanced inhibition of AChE
and BuChE, compounds with varied linker length were synthesized
firstly. And once the optimal length was determined, various
alkylbenzylamine groups were changed for SAR studies.
Here, a series of novel hybrids assembled by the xanthone and
alkylbenzylamine fragments were synthesized and evaluated for
their biological activity, including inhibition of cholinesterases,
antioxidant and metal chelating effects, and the ability to cross the
blood-brain barrier.
2. Results and discussion
2.1. Chemistry
The synthetic scheme for the synthesis of the compounds 4a-n
and 5a-f are shown in Schemes 1 and 2. Initially, condensation of
hydroxybenzoic acid with phloroglucinol in the presence of anhy-
drous zinc chloride and phosphoryl chloride afforded 1,3-
dihydroxy-9H-xanthen-9-one (2). Considering the 1-hydroxyl of
compound 2 may form intramolecular hydrogen bond with 9-
carbonyl in a manner of stable six-member ring, it is less reactive
in alkylation than 3-hydroxyl [20]. Thus, 3-hydroxyl group of
compound 2 was directly bromoalkylated in the presence of po-
tassium carbonate to lead to a series of 1-hydroxy-3-O-bro-
moalkylxanthones (3a-f, n ¼ 3e8). Then, the compounds 3a-f were
reacted with corresponding secondary amines to give the target
compounds 4a-n. Finally, to further extent the structure-activity
relationships, 4j and 4n were also selected to treat with the cor-
responding isocyanate to produce the additional target compounds
5a-f.
Metal ions are one of the necessary substances for all organisms
and play an irreplaceable physiological role in the metabolic pro-
cess of organisms, but high levels of biometal ions are closely
implicated in the pathogenesis of AD [12]. Studies have found that
the level of Cu2þ, Zn2þ, Fe3þ, Al3þ and other metal ions in the senile
plaques in the brains of AD patients is higher than the background
concentration of metal ions in the brains of normal people [13].
Furthermore, it was reported that the redox-active metal ions, like
Cu2þ, interact with A
b to produce ROS through Fenton-like re-
actions, thus leading to the oxidative stress and ultimately neuronal
death [14]. Studies also have demonstrated that excessive Al3þ
could not only inhibit AChE non-competitively, promote the for-
mation of neurofibrillary tangles but also damage the dynamic
balance of serum metal ions and facilitate oxidative stress, thereby
triggering AD [15]. Accordingly, adjustment of such biometals in the
brain may be also a promising strategy for treating AD.
2.2. In vitro AChE and BuChE inhibitory activity
Up to now, only four AChE inhibitors (rivastigmine, galantamine,
donepezil, tacrine) have been approved by the US Food and Drug
Administration (FDA) for the treatment of AD in clinic. However,
they can only provide temporary and incomplete symptomatic
relief. Considering the multifactorial pathogenesis of AD as
mentioned above, we focused on multifunctional agents, which
could simultaneously inhibit ChEs, chelate metals and exert anti-
oxidant effect.
The inhibitory activities of all synthesized hybrids towards
electric eel acetylcholinesterase (AChE) and equine serum butyr-
ylcholinesterase (BuChE) were evaluated using modified Ellman’s
method [21]. Inhibitory potencies are reported in Table 1 as IC50
(m
M) or, for poorly active compounds, as the percentage of inhibi-
tion at 100 M. As can be seen, most of the target compounds
m
showed both AChE and BuChE inhibitory potency with IC50 values
ranging from submicromolar to micromolar. Of these compounds,
compounds 4j and 4n exhibited the most potent inhibitory activity
Natural products play a highly significant role in drug discovery
and development processes. Xanthone (Fig. 1), chemical name is
dibenzo-
g
-pyrone, has been reported to display the abilities of
for AChE (IC50 ¼ 0.85 0.043
compounds 4e, 4h and 4j showed the most potent inhibitory ac-
tivity for BuChE (IC50 ¼ 0.50 0.005 M, 0.79 0.08 M,
0.59 0.007 M, respectively).
To investigate the influence of the length of the carbon chain
linker (n ¼ 3e8) between the xanthone and the benzylamine
moiety, compounds 4a-l were prepared. In general, the derivatives
with a three-carbon atom linker showed weak enzyme inhibition.
For example, compound 4a (AChE inhibitory activity:
mM, 0.75 0.07 mM, respectively), and
anticancer, antibacteria, antioxidant and antineuroinflammation
[16]. Recently, it was reported that xanthone derivatives could
effectively inhibit AChE, and some xanthone derivatives also pre-
sented other biological activities related to AD, such as free radicals
scavenging and metal ions chelation [17]. Therefore, it is advisable
to design multifunctional molecules on basis of the xanthone
scaffold. On the other hand, alkylbenzylamine is a versatile phar-
macophore with a wide range of biological activity profiles [18]. In
recent years, it has been intensively exploited to design ChE in-
hibitors and has been viewed as the key fragment for ChE inhibition
[19]. Given the fact above, in present study, we attempted to con-
nect xanthone with alkylbenzylamine through different length of
the carbon chain linker to design novel multifunctional compounds
for the treatment of AD (Fig. 1). The xanthone was utilized to
interact with the PAS of AChE and entrance cavity of BuChE.
Meanwhile, a hydroxyl was introduced to its 1-position, which
could form intramolecular hydrogen bond with 9-carbonyl in a
manner of stable six-member ring, thereby producing the abilities
of scavenging free radicals and chelating metal ions. The alkyl-
benzylamine was employed to bind to CAS of AChE and acyl binding
pocket of BuChE, which ensured that the designed compounds
m
m
m
IC50 ¼ 59.85 0.14
m
M) and 4g (AChE inhibition rate at 100
m
m
M:
M)
34.30 0.021%; BuChE inhibitory activity: IC50 ¼ 32.26 0.11
both performed poor inhibitory activities. Elongation of the spacer
to four methylene groups as seen for compounds 4b and 4h led to a
significant increase of inhibitory potency. For example, compared
with 4a (n ¼ 3), the AChE inhibitory activity of 4b (n ¼ 4) increased
by about 8.8-fold (IC50 ¼ 6.79 0.09
mM), and compared with 4g
(n ¼ 3), the BuChE inhibitory activity of 4h (n ¼ 4) increased by
almost 41-fold (IC50 ¼ 0.79 0.08
mM).
When the linker length was extended from four to seven carbon
atoms, although the tendency of the inhibitory potency was not
obvious, compounds containing the linker length with even num-
ber of carbon atoms seemed better than the corresponding
2

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
Fig. 1. Design strategy of the xanthone derivatives.
Scheme 1. Synthesis of xanthone derivatives 4a-n. Reagents and conditions: (i) ZnCl2/POCl3, Salicylic acid, 1,3,5-trihydroxybenzene, 75 ꢂC, 2 h; (ii) Br(CH2)nBr, K2CO3, DMF, r.t., 6 h;
(iii) For 4a-f: 3-((methylamino)methyl)phenol, CH3CN, NaI, TEA, reflux, 9 h; for 4g-l: 4-((methylamino)methyl)phenol, CH3CN, NaI, TEA, reflux, 10 h; for 4m: 4-Methoxy-N-
methylbenzylamine, CH3CN, NaI, TEA, reflux, 9 h; for 4n: 4-((ethylamino)methyl)phenol, CH3CN, NaI, TEA, reflux, 12 h.
Scheme 2. Synthesis of xanthone derivatives 5a-f. Reagents and conditions: (i) DMF, RNCO, TEA, N2 atmosphere, r.t., 36e48 h.
3

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
Table 1
Thus, a series of new compounds 5a-f were synthesized. Unfortu-
nately, the activities against AChE and BuChE were not improved,
but reduced. In general, compounds possessing N-ethyl substitu-
tion at R1 were more active than those with N-methyl substitution,
and compounds containing the chloroethyl (5b, 5e) at R4 per-
formed relatively better than those with the ethyl and cyclohexyl
substitutions, which suggested that electron-withdrawing effect of
chlorine atom might be positive.
Above all, the results demonstrated that compound 4j was the
most optimal compound as a dual inhibition agent for AChE and
BuChE, which implied the H-bond effect between the terminal
hydroxyl and CAS of ChEs was required.
Inhibition of eeAChE and eqBuChE by compounds 4a-n and 5a-f.
Compd.
n
R1
R2
R3
R4
IC50
(100
(
m
m
M)
M) or inhibition rate %
eeAChEa
eqBuChEb
4a
4b
4c
4d
4e
4f
4g
4h
4i
3
4
5
6
7
8
3
4
5
6
7
8
6
6
6
6
6
6
6
6
Me OH
Me OH
Me OH
Me OH
Me OH
Me OH
Me
Me
Me
Me
Me
Me
Me
Et
Me
Me
Me
Et
Et
Et
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OMe
OH
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
Et
59.85 0.14
6.79 0.09
80.79 0.18
2.68 0.10
8.24 0.06
42.60 0.017% 6.14 0.02
34.30 0.021% 32.26 0.11
1.79 0.08
1.60 0.12
0.85 0.043
7.43 0.03
2.96 0.07
8.94 0.09
24.24 0.14
22.33 0.12
0.50 0.005
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.79 0.08
3.87 0.04
0.59 0.007
1.82 0.08
4j
4k
4l
2.3. In vitro antioxidant activity assay
42.60 0.015% 9.53 0.05
4m
4n
5a
5b
5c
5d
5e
5f
55.02 0.17
0.75 0.07
9.69 0.15
9.37 0.06
1.70 0.09
24.00 0.74%
1.74 0.14
40.10 0.21%
1.15 0.13
1.03 0.04
1.15 0.03
Free radicals-induced oxidative stress is believed to be condu-
cive to the progression of AD [23]. Thus, each compound was pre-
liminarily determined for scavenging activity by the DPPH
(diphenyl-1-picrylhydrazyl) radical scavenging method and ABTS
(2, 20-azino-bis(3-ethylbenzthiazoline-6-sulfonicacid)) radical
scavenging method [24,25]. Trolox, a water-soluble vitamin E
analog, was used as a reference standard. From Table 2, it could be
seen that the range of the variation of the free radicals scavenging
activity is wide. Among these compounds, although less potent
than trolox, compound 4c exhibited the most potent activity. In
general, the length of methylene chain affected the antioxidant
activities. For example, the abilities of compounds 4a-f and 4g-l
(n ¼ 3, 4, 5, 6, 7, 8) to capture free radicals were decreased as the
length of the alkyl chain were increased. Moreover, the position of
the hydroxyl group of the terminal benzene ring also affected the
activity. The compounds (4a-f) with the hydroxyl group at 3-
position showed more effective activities than compounds (4g-l)
with that at 4-position, which was opposite with the ChEs inhibi-
tory abilities. Strikingly, replacement of the hydroxyl group on the
terminal benzene ring by other groups, affording compounds 5a-f,
led to an obvious decline in potency of scavenging the DPPH and
ABTS free radicals.
The oxygen radical absorbance capacity assay (ORAC-FL) was
also performed to evaluate the antioxidant activity of each com-
pound. As shown in Table 2, the tested compounds displayed poor
to excellent antioxidant abilities that expressed as a trolox equiv-
alent. Among them, compounds 4a-d, 4h, 4j, 4n, 5e exhibited the
excellent free radical quenching abilities (ORAC＞1). Especially
compound 4b (ORAC ¼ 6.11) exerted optimal radical scavenging
activity and is close to the well-known strong free radical scav-
enging agent, quercetin (ORAC ¼ 6.55). Similarly, the structure-
activity relationships obtained by the ORAC method were basi-
cally consistent with the trend from DPPH and ABTS methods.
However, compound 4b, rather than 4c, presented the most potent
antioxidant activity determined by the ORAC method, which may
probably be attributed to the fact that DPPH and ABTS are both in
fact a kind of nitrogen radicals while AAPH is the peroxyl radical
inducer [26].
e
(CH2)2Cl 1.02 0.21
Cy
Et
(CH2)2Cl 1.90 0.05
Cy 22.23 0.13
e
141.9 0.22
2.41 0.04
e
e
e
a
AChE from electric eel (EeAChE). All values are expressed as mean SD from
three independent experiments.
b
BuChE from equine serum (eqBuChE). All values are expressed as mean SD
from three independent experiments.
compounds containing the linker length with odd number of car-
bon atoms (AChE inhibitory activity: 4b (n ¼ 4) > 4c (n ¼ 5), 4d
(n ¼ 6) > 4e (n ¼ 7), 4j (n ¼ 6) > 4k (n ¼ 7); BuChE inhibitory
activity: 4b (n ¼ 4) > 4c (n ¼ 5), 4h (n ¼ 4) > 4i (n ¼ 5), 4j
(n ¼ 6) > 4k (n ¼ 7)). Further elongating the linker length to eight
carbon atoms led to a sharp drop in the both ChEs inhibitory ac-
tivity of all compounds. For example, the AChE inhibitory activity of
4e (n
IC50 ¼ 7.43 0.03
100 M. The BuChE inhibitory activity of 4e and 4k respectively
decreased by 12-fold (4e: IC50 ¼ 0.50 0.005 M vs 4f: n ¼ 8,
IC50 ¼ 6.14 0.02 M) and 5.3-fold (4k: IC50 ¼ 1.82 0.08 M vs 4l:
n ¼ 8, IC50 ¼ 9.53 0.05 M).
¼
7, IC50
¼
8.24
0.06
mM) and 4k (n
¼
7,
mM) both decreased to 42.6% inhibition rate at
m
m
m
m
m
Additionally, it also revealed that the position of the substituent
of the terminal benzene ring affected the activities. Most of com-
pounds 4g-l possessing a hydroxyl substituent at 4-position of the
benzene ring usually displayed better activities than corresponding
those 4a-f with a hydroxyl substituent at 3-position (AChE inhibi-
tory activity: 4h > 4b, 4i > 4c, 4j > 4d, 4k > 4e; BuChE inhibitory
activity: 4h > 4b, 4i > 4c, 4j > 4d). Among them, compound 4j,
bearing a hydroxyl group at 4-position of the terminal benzene ring
and a six-methylene linker, exhibited the optimal and balanced
inhibition activity against ChEs. Therefore, we decided to select 4j
for further optimation.
Compounds 4m and 4n were obtained by modification at ben-
zylamine moiety in 4j for exploring the SARs. Noticeably, compared
with compound 4j, when maintaining the methyl group at R1, the
installation of a methoxy group at 4-position of the benzene ring
dramatically decreased AChE and BuChE inhibitory activity, which
2.4. In vitro blood-brain barrier permeation assay
made the IC50 values change from 0.85
mM to 55.02 mM (4m) for
Blood-brain barrier (BBB) permeability is the major requirement
for drugs acting at targets inside of the central nervous system [27].
Thus, the parallel artificial membrane permeability assay was per-
formed to predict the BBB permeation of the target compounds
[28,29]. First, the permeabilities of 9 commercial drugs with their
reported values were used to validate the assay in Table 3. And a
plot of experimental data versus the bibliographic values gave a
good linear correlation: Pe (exp.) ¼ 0.6703 ꢃ Pe (bibl.) þ 0.6571
(R2 ¼ 0.9623). It could be seen from the Table 4 that, with exception
AChE and 0.59 M to 9.37 M (4m) for BuChE. In contrast, when
m
m
maintaining the hydroxyl group at R3, the introduction of an ethyl
group at R1 slightly enhanced the AChE inhibitory activity (4n:
IC50 ¼ 0.75 0.07
m
M), but reduced BuChE inhibitory activity (4n:
IC50 ¼ 1.70 0.09
mM).
According to the previous studies, the carbamate group tend to
be transferred onto the serine-OH in the CAS, which enhances the
binding affinity between the target compound and enzyme [22].
4

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
Table 2
DPPH and ABTS scavenging activities and Oxygen Radical Absorbance Capacity by Fluorescence (ORAC-FL, Trolox Equiv) of the synthesized compounds.
Compd.
n
R1
R2
R3
R4
IC50
(
mM) or inhibition (%)
ORACc
DPPHa
ABTSb
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
5a
5b
5c
3
4
5
6
7
8
3
4
5
6
7
8
6
6
6
6
6
6
6
6
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
Me
Me
Me
Et
Et
Et
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OMe
OH
e
e
31.4 0.52%
521.9 0.72
456.9 0.68
708.2 0.77
719 0.69
2387 0.96
544.9 0.80
648.7 0.81
1074 0.93
1280 0.92
1306 0.85
1751 0.87
25.9 0.29%
25.6 0.30%
36.8 0.44%
35.1 0.43%
48.9 0.53%
23.3 0.26%
33.4 0.43%
40.3 0.51%
15.5 0.13
e
46.95 0.63
32.48 0.55
30.25 0.48
32.04 0.50
52.68 0.61
34.57 0.49
56.45 0.65
66.97 0.68
33.52 0.32
41.64 0.33
20.94 0.25%
33.75 0.41
58.77 0.55
60.10 0.58
33.89 0.41%
37.98 0.47%
14.51 0.16%
27.61 0.27%
70.60 0.67
41.00 0.35
13.8 0.14
e
4.78 0.08
6.11 0.12
1.04 0.04
2.39 0.03
0.42 0.01
0.035 0.01
0.79 0.02
3.34 0.05
0.44 0.02
4.60 0.09
0.05 0.01
<0.01
e
e
e
e
e
e
e
e
e
e
e
e
<0.01
e
1.69 0.07
0.26 0.03
0.43 0.02
0.093 0.01
0.92 0.04
2.31 0.08
0.35 0.02
e
Et
e
(CH2)2Cl
Cy
Et
(CH2)2Cl
Cy
e
5d
5e
5f
Trolox
Quercetin
e
e
H
e
6.55 0.11
a
The 50% inhibitory concentration of DPPH or percent inhibition with inhibitor at 1 mM (means SD of three experiments).
b
c
The 50% inhibitory concentration of ABTS or percent inhibition with inhibitor at 100
mM (means SD of three experiments).
Data are expressed as M of Trolox equivalent/
m
m
M of tested compound and are mean (n ¼ 3) SD.
of the compounds 4f and 5b, which exhibited questionable BBB
permeability (CNS ), most of the target compounds have Pe values
above the threshold (Pe ¼ 3.33 ꢃ 10ꢁ6 cm/s), indicating that they
have great BBB permeability (CNSþ) and enough drug exposure in
the central nervous system.
2.6. Molecular docking of AChE and BuChE by 4j
To gain insight into the interaction of compound 4j with the
active site residues of AChE and BuChE, a computational study was
performed using the docking program by the X-ray crystal struc-
tures of huAChE (PDB code: 4EY7), eeAChE (PDB code: 2CKM) and
huBuChE (PDB code: 1P0I).
2.5. Kinetic study of the AChE and BuChE inhibitions by compound
4j
In the huAChE-4j complex, compound 4j occupied the entire
huAChE enzymatic catalytic site (CAS), the mid-gorge site and the
To better understanding of the inhibition mechanism, kinetic
studies of compound 4j as a dual AChE and BuChE inhibitor were
performed. As shown in Fig. 2A and C, the Lineweaver-Burk double
reciprocal plots revealed increasing slopes (decreased Vmax) and
intercepts (higher Km) with higher inhibitor concentrations and
intersected in the second quadrant. This pattern indicated that 4j
was a mixed-type inhibitor for both AChE and BuChE. Replots of the
slope versus concentrations were utilized to obtain the inhibition
Table 4
Permeability Pe ( ꢃ 10ꢁ6 cm/s) in the PAMPA-BBB assay for all compounds and their
predicted penetration into CNS.
Compd.
Pe ( ꢃ 10ꢁ6 cm/s)a
predictoinb
4a
4b
4c
4d
4e
4f
4g
4h
4i
6.09 0.33
12.87 0.62
11.03 0.55
15.17 0.48
9.27 0.19
3.20 0.12
11.94 0.23
16.91 0.33
11.50 0.45
5.29 0.21
4.39 0.13
10.03 0.17
6.82 0.15
5.95 0.14
5.09 0.09
2.33 0.11
4.61 0.12
7.38 0.36
5.02 0.21
7.36 0.18
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNS
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNS
constants (Ki), which were estimated to be 1.4 mM and 0.98 mM for
AChE and BuChE respectively based on Fig. 2B and D.
Table 3
4j
4k
4l
Permeability pe ( ꢃ 10ꢁ6 cm/s) in the PAMPA-BBB assay for 9 commercial drugs in
the experiment validation.
4m
4n
5a
5b
5c
5d
5e
5f
Commercial drugs
Bibliographya
Experimentb
Testosterone
Estradiol
Progesterone
Chlorpromazine
Caffeine
Corticosterone
Hydrocortisone
Atenolol
17.0
12.0
9.3
6.5
1.3
5.1
1.9
0.8
0.1
10.86 0.46
9.38 0.35
7.53 0.25
6.20 0.12
1.33 0.03
4.06 0.05
1.71 0.11
0.22 0.02
0.83 0.05
CNSþ
CNSþ
CNSþ
CNSþ
a
Experimental data are expressed as mean
SD from three independent ex-
periments, using PBS: EtOH (70:30) as solvent.
Theophylline
b
Compounds with permeabilities Pe > 3.33 ꢃ 10ꢁ6 cm/s could cross the BBB by
passive diffusion (CNSþ). Compounds with Pe < 2.00 ꢃ 10ꢁ6 cm/s could not cross the
BBB (CNS－), and compounds with 2.00 ꢃ 10ꢁ6 cm/s < Pe < 3.33 ꢃ 10ꢁ6 cm/s
indicate uncertain BBB permeation (CNS ).
a
Taken from Ref. [30].
b
Experimental data are expressed as mean
periments, using PBS: EtOH (70:30) as solvent.
SD from three independent ex-
5

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
Fig. 2. Lineweaver-Burk plot of 4j for AChE hydrolysis (A) and slope replot vs 4j concentration (B). Lineweaver-Burk plot of 4j for BuChE hydrolysis (C) and slope replot vs 4j
concentration (D).
peripheral anionic site (PAS) (Fig. 3A and C). The xanthone moiety
of 4j was oriented towards the PAS of huAChE, via a face-to-face
phenyl rings respectively from the xanthone and Tyr332. In the
middle gorge, the alkylene linker folded in a conformation in gorge
that provided optimum flexibility to the terminal phenyl group to
insert deep within the active-site gorge. The terminal benzene ring
and its hydroxyl group bound to Trp231 and Leu286 of the acyl
binding pocket (Trp 231, Leu 286, Val 288) at the bottom of the
aromatic
p-p stacking interaction between its phenyl ring and
the indole ring from Trp286 at PAS. Besides, the carbonyl oxygen of
xanthone moiety generated a hydrogen bond with the Arg296,
which further enhanced the binding ability to PAS. The flexible
alkylene linker extended the alkylbenzylamine deep into CAS of
gorge via p-p interactions and hydrogen bond respectively [33]. In
huAChE through aromatic
p-p
stacking interactions with the
addition, the charged nitrogen of alkylbenzylamine interacted with
phenyl ring from Trp86. Moreover, the hydroxyl group of the
alkylbenzylamine formed a hydrogen bond with Glu202 at the
anionic subsite. Also, compound 4j was modeled into the structure
of eeAChE (Fig. 3B and D). The docking pose of compound 4j in the
eeAChE was similar to that in the huAChE. The xanthone moiety of
4j interacted with the Trp279 and Tyr70 at PAS of eeAChE and the
alkylbenzylamine of 4j interacted with the Trp84 and Glu199 at CAS
of eeAChE, which was broadly in line with the results in huAChE.
The AChE and BuChE share more than 50% homological amino
acid sequences [31,32]. Despite the similarity between two en-
zymes, the presence of a similar PAS in BuChE is still under dis-
cussion [22]. In contrast to the aromatic amino acids forming the
PAS in AChE, several relatively small aliphatic amino acids endow
BuChE with a wider pocket in the comparable area of BuChE,
whereas only Asp70 and Tyr332 are conserved.
His438 residue of the catalytic triad (Ser 198, Glu 325, His 438) via
cation-p interaction, which is pivotal for huBuChE inhibition [34].
The observed docking studies between the ligand and residues
provided the reasonable explain for mixed-type inhibition model of
kinetic studies, which agreed with the dual binding site mode.
2.7. Metal-chelating properties
Research showed that the fragment of 2-hydroxyl and 9-
carbonyl group in the xanthone, which can form intramolecular
hydrogen bond in a manner of stable six-member ring, is respon-
sible for the metal chelation ability. Based on the above studies, we
selected the compound 4j as representative to investigate its abil-
ities to chelate biometals such as Naþ, Mg2þ, Fe2þ, Zn2þ, Ca2þ, Al3þ
and Cu2þ, using UVevis and fluorescence spectroscopy. As shown
in Fig. 5A, the specific fluorescence emission peak of 4j can be
observed at 300 nm (lex ¼ 232 nm). After NaCl, MgCl2, FeCl2, ZnCl2
and CaCl2 were added to the solution of 4j, the fluorescence
In the huBuChE-4j complex, xanthone, the bulkier moiety of 4j,
located near the entrance cavity of huBuChE (Fig. 4A and B). This
pose was stabilized by a
p-p stacking interaction between two
6

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
Fig. 3. Molecule docking results: A) and C) were 3D and 2D docking models of compound 4j (yellow stick) with huAChE (PDB code: 4EY7) complex generated with MOE; B) and D)
were 3D and 2D docking models of compound 4j (yellow stick) with eeAChE (PDB code: 2CKM) complex generated with MOE. (For interpretation of the references to color in this
figure legend, the reader is referred to the Web version of this article.)
Fig. 4. Molecule docking results: A) and B) were 3D and 2D docking models of compound 4j (yellow stick) with huBuChE (PDB code: 1P0I) complex generated with MOE. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
intensity of the emission peaks decreased variously, while the
AlCl3 and CuCl2. These phenomena are indicative of a possible
fluorescence intensity obviously increased with the addition of
interaction between these biometals and ligand. Also, a UVevis
7

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
Fig. 5. (A) Fluorescence spectra of 4j (50
mM in methanol/water) alone or in the presence of NaCl, MgCl2, FeCl2, ZnCl2, CaCl2, AlCl3 and CuCl2 (1 equivalent in methanol/water). (B) UV
absorbance spectra of 4j (50uM in methanol/water) alone or in the presence of NaCl, MgCl2, FeCl2, ZnCl2, CaCl2, AlCl3 and CuCl2(1 equivalent in methanol/water). (C) Determination
of the stoichiometry of the 4j-Cu2þ complex by the molar ratio method. (D) Determination of the stoichiometry of the 4j-Al3þ complex by the molar ratio method.
spectroscopy assay was performed to further evaluate the metal-
chelating properties of 4j. As shown in Fig. 5B, the absorption
peak at the original 308 nm shift to 315 nm with the intensity
decreasing moderately and the spectra trough at the 270 nm moved
to 283 nm after the addition of AlCl3. Meanwhile, new optical band
was detected at approximately 400 nm. Similarly, when CuCl2 was
added, a red shift from 270 nm to 283 nm with the intensity
increasing significantly and new optical band was also detected at
approximately 400 nm. There were only vague optical shift and
slight fluctuation of maximum absorption peak after the addition of
the other biometals. These results demonstrated that compound 4j
could selectively chelate with Cu2þ and Al3þ. To further confirm the
metal-chelating properties of 4j, the stoichiometries of the 4j-Cu2þ
and 4j-Al3þ complexes were determined using molar ratio method.
Following the absorption at 400 nm, a series of UVevis spectra
were collected of 4j titrated with ascending amounts of CuCl2 and
AlCl3 respectively as shown in Fig. 5C and D. It can be seen from the
embedded figures, the absorbance linearly increased initially and
then became stable. For the 4j-Cu2þ and 4j-Al3þ complexes, the two
sets of straight lines both intersected at a 1.4:1 Cu2þ/ligand and
Al3þ/ligand molar ratio.
the neural cells (PC12 and BV-2), hepatic cells (HepG2) and car-
diomyocytes (H9C2) by the Cell Counting Kit-8 (CCK-8) [35].
Tacrine was used as a positive control. From the results shown in
Fig. 6, 4j did not show modified cell viability on two neural cell lines
less than 12.5
higher than that of 4j on neural cell lines, which up to the con-
centration of 25 M. Although 4j presented the more than 50% cell
toxicity on cells H9C2 at 25 M or higher concentrations, it exerted
no obvious toxicity below the 12.5 M. Therefore, compound 4j has
mM. And the toxic dose of 4j on cells HepG2 was
m
m
m
a medium therapeutic safety range and it is necessary to further
evaluate the safety in vivo.
2.9. Acute toxicity assay
Forty KM mice (18e22 g, n ¼ 10 per group, half male and half
female) that are common closed colony mice and most widely used
in biomedical research in China were randomly allocated into 4
groups [36]. The tested compound 4j was delivered to mice by oral
administration at four doses of 0, 625, 1250 and 2500 mg/kg.
During the treatment period, no death and abnormal behaviors
(emesis-like or diarrhea behavior) were observed and the body
weights gradually enhanced (Fig. 7). Moreover, the mean body
weight of the mice in the compound group presented no significant
differences with those in the control group. Furthermore, all mice
were sacrificed on the 14th day after drug administration, and no
significant histological abnormal changes were detected in the
2.8. In vitro cytotoxicity evaluation
To investigate the safety of the compound 4j, its 5 different
concentrations (6.25, 12.5, 25, 50 and 100 mM) were tested using
8

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
Fig. 6. Cytotoxicity of compound 4j on neural cells (PC12 and BV-2), hepatic cells (HepG2) and cardiomyocytes (H9C2) over coincubation for 24 h. Tacrine was used as a positive
control.
2.10. Cognitive and memory improvement test
Considering the good ChEs inhibitory activity and safety of 4j,
we further investigated its effect on memory impairment induced
by scopolamine (3 mg/kg, i.p.) in mice via the step-down test [38].
Donepezil (10 mg/kg) was used as a positive control. Compound 4j
and donepezil were orally administered into the KM mice 1 h
before each training trial [39]. As shown in Fig. 9, compared with
the control group, treatment with scopolamine led to a much
shorter step-down latency time (250.7 vs 65.3 s, p < 0.05), and the
number of errors was significantly increased (0.3 vs 2.3, p < 0.001),
which indicated the successful establishment of the memory
impairment mice model. After treatment with 4j, the latency and
number of the errors were reversed in a dose-dependent manner.
Moreover, high (100 mg/kg) and medium (50 mg/kg) dose groups
could prolong the step-down latency and exhibited a comparable
activity to donepezil group. And high dose group showed fewer
numbers of errors, but medium and low (25 mg/kg) dose groups
displayed a worse effect. As a conclusion, compound 4j improved
Fig. 7. The mean daily body weight profile of mice in different groups during the
therapy period (n ¼ 10).
cognitive deficit by increasing brain cholinergic level.
2.11. Theoretical prediction of the ADME properties
heart, liver, lung, kidney and brain of the mice at the maximum
tested dosage 2500 mg/kg by hematoxylin and eosin (HE) staining
(Fig. 8) [37]. Overall, compound 4j demonstrated no acute toxicity
and was well tolerated at doses up to 2500 mg/kg.
To investigate the drug-like profiles of 4j, the ADME properties
were estimated using SwissADME web tool. As shown in Table 5,
compound 4j fulfilled the requirements of Lipinski’s and Veber’s
9

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
Fig. 8. Histological analysis of heart, liver, lung, kidney, and brain for the acute toxicity studies of compound 4j at dosage of 2500 mg/kg in mice; 200 mm indicate the scale bar of
images (HE staining). Representative images of HE-stained liver, heart, lung, kidney and brain for each group are shown.
Fig. 9. Effects of compound 4j on scopolamine-induced memory deficit in the step-down passive avoidance test. (A) latency time, (B) number of errors. Values are expressed as the
mean SEM. n ¼ 6 mice from three independent experiments; #p < 0.05, ###p < 0.001 versus control group; *p < 0.05, **p < 0.01 versus model group.
rules, and its structure did not contain any characteristic structural
elements of pan-assay interference compounds (PAINS), suggesting
that target compound 4j presented drug-likeness properties.
safety of the compound 4j, we found it no acute toxicity and it was
well tolerated at doses up to 2500 mg/kg. Most importantly, the
step-down test showed 4j dose-dependently reversed
scopolamine-induced memory deficit. All of these results made
compound 4j a promising candidate in the development of anti-AD
drugs.
3. Conclusion
In summary, a series of xanthone-alkylbenzylamine hybrids
were designed, synthesized and evaluated as multifunctional
agents for the treatment of Alzheimer’s disease. Most of the target
compounds displayed moderate to good ChEs inhibitory activities.
Among them, compound 4j exhibited both AChE and BuChE sub-
4. Experimental section
4.1. Chemistry
micromolar inhibitory potency with IC50 values 0.85
mM and
All chemical reagents and solvents were purchased from com-
mercial suppliers and used without further purification. Reaction
progress was monitored by thin-layer chromatography (TLC) on
silica gel GF254 plates from Qingdao Haiyang Chemical Co. Ltd.
(China), and then visualized by UV light (254 nm). Chromato-
graphic separation were performed on silica gel (100e200 mesh)
from Qingdao Haiyang Chemical Co. Ltd. (China). Melting points
were determined in glass capillary tubes on an XT-4 micro melting
point apparatus (China) and are uncorrected. 1H NMR spectra
(600 MHz) and 13C NMR spectra (151 MHz) were recorded on a
Bruker ACF-600 spectrometer at room temperature using CDCl3 or
0.59 M. The kinetic analysis revealed that 4j showed mixed-type
m
inhibition, which was consistent with the docking studies. In
addition, compound 4j exerted significant metal-chelating ability
with Cu2þ and Al3þ at a 1:1.4 ligand/Cu2þ and ligand/Al3þ molar
ratio and free radicals scavenging capacity (4.6 trolox equivalent).
The PAMPA assay showed that 4j could cross the BBB and interact at
targets inside of the central nervous system. To investigate the
Table 5
Drug-likeness properties of selected compound.
DMSO‑d6 as the solvent. Chemical shifts (d) are reported in parts per
Compd. MWa
nrotbb nONc nOHNHd tPSAe ilogPf PAINS alerts
447.52 10 83.14 4.50
MW: molecular weight.
million (ppm) relative to tetramethylsilane (TMS), and spin multi-
plicities are given as s (singlet), d (doublet), t (triplet), m (multiplet)
or br (broad). The purity of these compounds was determined by
analytical HPLC performed on a Waters ACQUITY Arc HPLC system
equipped with a 2998 PDA detector. (Column: Agilent XDB-C18,
4j
6
2
0
a
b
c
d
e
f
nrotb: number of rotatable bonds.
nON: number of hydrogen acceptors.
nOHNH: number of hydrogen donors.
tPSA: total polar surface area.
5
m
m particle size, 4.6 mm ꢃ 250 mm; mobile phase: A ¼ CH3OH,
ilog P: octanol/water partition coefficent.
B ¼ H2O (0.1% acetic acid), isocratic elution, A ¼ 75%, B ¼ 25%; flow
10

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
rate ¼ 1 mL/min;
l
¼ 254 nm; 10
m
L injection). All target com-
4H), 1.53e1.50 (m, 4H), 1.45e1.42 (m, 2H).
pounds possessed purity higher than 95%. High resolution mass
spectra were conducted on an AB Sciex Triple TOF 5600 spec-
trometer (HR-ESI-MS).
4.3.6. 3-((8-bromooctyl)oxy)-1-hydroxy-9H-xanthen-9-one (3f)
Yield 78%; yellow solid; m.p. 99e101 ꢂC; 1H NMR (600 MHz,
CDCl3)
d
12.88 (s, 1H), 8.27 (dd, J ¼ 7.8, 1.8 Hz, 1H), 7.75e7.72 (m,
4.2. Synthesis of 1,3-dihydroxy-9H-xanthen-9-one (2)
1H), 7.45 (dd, J ¼ 8.4, 0.6 Hz, 1H), 7.41e7.38 (m, 1H), 6.45 (d,
J ¼ 2.4 Hz,1H), 6.36 (d, J ¼ 2.4 Hz,1H), 4.07 (t, J ¼ 6.6 Hz, 2H), 3.44 (t,
J ¼ 6.6 Hz, 2H), 1.92e1.82 (m, 4H), 1.51e1.46 (m, 4H), 1.43e1.38 (m,
4H).
To a 50-mL flask, 17 mL phosphorus oxychloride (POCl3) and
anhydrous zinc chloride (14.8 g, 0.11 mol) were added. The sus-
pension was stirred at 75 ꢂC until ZnCl2 was completely dissolved
into phosphorus oxychloride. Then salicylic acids (7.24 mmol) and
1,3,5-trihydroxybenzene (7.96 mmol) were added, respectively, and
the mixture was heated at 75 ꢂC for 2 h. After the mixture was
cooled down to r. t., the mixture was pulled into ice water stirring
for 10 min and extracted with ethyl acetate (25 mL ꢃ 3). The ethyl
acetate layer was collected, dried over anhydrous sodium sulfate,
filtered and concentrated under reduced pressure to give the crude
product 2. The residue was purified on a silica gel chromatography
using petroleum ether/ethyl acetate (25:3 to 5:2) as eluent.
4.4. General procedure for the synthesis of 1-hydroxyl-3-
aminoalkoxyxanthen derivatives (4a-n)
To a mixture of sodium iodide (77 mg, 0.51 mmol), appropriate
intermediates 3a-f (0.51 mmol) and the corresponding secondary
amines (0.61 mmol) in acetonitrile (10 mL), triethylamine
(0.77 mmol) was added. The reaction mixture was refluxed for
9e12 h. Upon completion of the reaction, the mixture was filtered
when hot and concentrated under reduced pressure to give the
crude product. The residue was purified by silica gel chromatog-
raphy to give compounds 4a-n, respectively.
4.3. General procedure for the synthesis of (3a-f)
To a mixture of compound 2 (200 mg, 0.88 mmol) and anhy-
drous potassium carbonate (242 mg, 1.75 mmol) in DMF (5 mL),
appropriate dibromoalkane derivative (22 mmol) was added. The
reaction mixture was allowed to stir for 6 h at room temperature
until the starting material disappeared. Then the mixture was
filtered and concentrated under reduced pressure to give the crude
product. The residue was purified by silica gel chromatography
using petroleum ether/ethyl acetate (30:1) as eluent to afford the
desired products 3a-f.
4.4.1. 1-Hydroxy-3-(3-((3-hydroxybenzyl) (methyl)amino)
propoxy)-9H-xanthen-9-one (4a)
It was prepared from 3-(3-bromopropoxy)-1-hydroxy-9H-
xanthen-9-one (3a) and 3-((methylamino)methyl)phenol accord-
ing to the general procedure, then purified on a silica gel chroma-
tography eluted with dichloromethane/methanol (25:1) to obtain
the pure product 4a. 69% yield; yellow solid; m.p. 145e147 ꢂC; 1H
NMR (600 MHz, DMSO)
d 12.81 (s, 1H), 9.72 (s, 1H), 8.17 (d,
J ¼ 7.8 Hz, 1H), 7.90 (t, J ¼ 8.4 Hz, 1H), 7.63 (d, J ¼ 8.4 Hz, 1H), 7.51 (t,
J ¼ 7.8 Hz,1H), 7.27 (s,1H), 6.89 (d, J ¼ 28.2 Hz, 3H), 6.66 (s,1H), 6.40
(s, 1H), 4.36 (s, 1H), 4.22 (s, 3H), 3.18 (s, 2H), 2.72 (s, 3H), 2.18 (s, 2H).
4.3.1. 3-(3-bromopropoxy)-1-hydroxy-9H-xanthen-9-one (3a)
Yield 79%; yellow solid; m.p. 123e125 ꢂC; 1H NMR (600 MHz,
13C NMR (151 MHz, DMSO)
d 180.68, 165.99, 163.08, 158.09, 157.78,
DMSO)
d
12.80 (d, J ¼ 2.4 Hz, 1H), 8.16 (dd, J ¼ 7.8, 1.2 Hz, 1H),
155.96, 136.54 (2C), 130.41, 125.82, 125.15, 121.94, 120.33, 118.27,
117.00, 103.74 (2C), 98.01, 93.79, 66.31, 59.10, 55.41, 52.51, 27.02.
HRMS: calcd for C24H24NO5 [MþH]þ 406.1649, found 406.1604.
HPLC purity, 99.15%.
7.90e7.87 (m, 1H), 7.62 (d, J ¼ 8.4 Hz,1H), 7.51e7.49 (m,1H), 6.68 (d,
J ¼ 1.8 Hz,1H), 6.43 (d, J ¼ 2.4 Hz,1H), 4.24 (t, J ¼ 6.0 Hz, 2H), 3.68 (t,
J ¼ 6.6 Hz, 2H), 2.39e2.16 (m, 2H).
4.3.2. 3-(4-bromobutoxy)-1-hydroxy-9H-xanthen-9-one (3b)
Yield 80%; yellow solid; m.p. 118e120 ꢂC;1H NMR (600 MHz,
4.4.2. 1-Hydroxy-3-(4-((3-hydroxybenzyl) (methyl)amino)butoxy)-
9H-xanthen-9-one (4b)
DMSO)
d
12.79 (s, 1H), 8.15 (dd, J ¼ 7.8, 1.8 Hz, 1H), 7.90e7.87 (m,
It was prepared from 3-(4-bromobutoxy)-1-hydroxy-9H-
xanthen-9-one (3b) and 3-((methylamino)methyl)phenol accord-
ing to the general procedure, then purified on a silica gel chroma-
tography eluted with dichloromethane/methanol (25:1) to obtain
the pure product 4b. 71% yield; yellow solid; m.p. 157e161 ꢂC; 1H
1H), 7.61 (d, J ¼ 8.4 Hz, 1H), 7.49 (d, J ¼ 7.8 Hz, 1H), 6.64 (d,
J ¼ 2.0 Hz, 1H), 6.40 (d, J ¼ 1.8 Hz, 1H), 4.17 (t, J ¼ 6.0 Hz, 2H), 3.74 (t,
J ¼ 6.6 Hz, 2H), 2.04e1.93 (m, 2H), 1.91e1.80 (m, 2H).
4.3.3. 3-((5-bromopentyl)oxy)-1-hydroxy-9H-xanthen-9-one (3c)
Yield 82%; yellow solid; m.p. 124e126 ꢂC;1H NMR (600 MHz,
NMR (600 MHz, DMSO)
d
12.82 (s, 1H), 9.72 (s, 1H), 8.17 (dd, J ¼ 7.8,
1.8 Hz, 1H), 7.92e7.89 (m, 1H), 7.63 (d, J ¼ 8.4 Hz, 1H), 7.53e7.50 (m,
1H), 7.26 (t, J ¼ 7.8 Hz, 1H), 6.93e6.91 (m, 2H), 6.86 (dd, J ¼ 8.4,
1.8 Hz, 1H), 6.66 (d, J ¼ 2.4 Hz, 1H), 6.42 (d, J ¼ 2.4 Hz, 1H), 4.33 (d,
J ¼ 12.0 Hz, 1H), 4.17 (t, J ¼ 6.0 Hz, 3H), 3.21 (s, 1H), 3.10e3.09 (m,
1H), 2.69 (s, 3H), 1.88e1.77 (m, 4H). 13C NMR (151 MHz, DMSO)
DMSO)
d
12.81 (s, 1H), 8.17 (d, J ¼ 7.8 Hz, 1H), 7.90 (t, J ¼ 7.2 Hz, 1H),
7.63 (d, J ¼ 8.4 Hz, 1H), 7.51 (t, J ¼ 7.2 Hz, 1H), 6.66 (s, 1H), 6.42 (s,
1H), 4.15 (t, J ¼ 6.0 Hz, 2H), 3.58 (t, J ¼ 6.6 Hz, 2H), 1.89 (t, J ¼ 7.2 Hz,
2H), 1.79 (t, J ¼ 7.2 Hz, 2H), 1.55 (t, J ¼ 6.6 Hz, 2H).
d
180.60, 166.36, 163.07, 157.92, 157.77, 155.93, 136.44, 130.04 (2C),
4.3.4. 3-((6-bromohexyl)oxy)-1-hydroxy-9H-xanthen-9-one (3d)
Yield 80%; yellow solid; m.p. 134e135 ꢂC; 1H NMR (600 MHz,
125.78, 125.07 (2C), 120.31, 118.23 (2C), 103.57 (2C), 97.95, 93.72,
68.64, 48.94, 30.57, 29.47, 26.20, 17.69. HRMS: calcd for C25H26NO5
[MþH]þ 420.1805, found 420.1757. HPLC purity, 98.94%.
DMSO)
d
12.81 (s, 1H), 8.17 (d, J ¼ 12.0 Hz, 1H), 7.90 (t, J ¼ 6.0 Hz,
1H), 7.63 (d, J ¼ 6.0 Hz, 1H), 7.51 (t, J ¼ 6.0 Hz, 1H), 6.67 (s, 1H), 6.42
(s, 1H), 4.14 (t, J ¼ 6.0 Hz, 2H), 3.56 (t, J ¼ 6.0 Hz, 2H), 1.84 (d,
J ¼ 6.0 Hz, 2H), 1.76 (s, 2H), 1.46 (s, 4H).
4.4.3. 1-Hydroxy-3-((5-((3-hydroxybenzyl) (methyl)amino)pentyl)
oxy)-9H-xanthen-9-one (4c)
It was prepared from 3-((5-bromopentyl)oxy)-1-hydroxy-9H-
xanthen-9-one (3c) and 3-((methylamino)methyl)phenol accord-
ing to the general procedure, then purified on a silica gel chroma-
tography eluted with dichloromethane/methanol (25:1) to obtain
the pure product 4c. 65% yield; yellow solid; m.p. 200e202 ꢂC; 1H
4.3.5. 3-((7-bromoheptyl)oxy)-1-hydroxy-9H-xanthen-9-one (3e)
Yield 81%; yellow solid; m.p. 114e115 ꢂC; 1H NMR (600 MHz,
CDCl3)
d
12.88 (s, 1H), 8.27 (d, J ¼ 7.8 Hz, 1H), 7.73 (t, J ¼ 7.8 Hz, 1H),
7.45 (d, J ¼ 8.4 Hz, 1H), 7.40 (t, J ¼ 7.2 Hz, 1H), 6.44 (s, 1H), 6.36 (s,
1H), 4.07 (t, J ¼ 6.0 Hz, 2H), 3.45 (t, J ¼ 7.2 Hz, 2H), 1.93e1.83 (m,
NMR (600 MHz, DMSO)
d
12.81 (s, 1H), 9.73 (s, 1H), 8.17 (dd, J ¼ 8.4,
11

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
1.8 Hz, 1H), 7.91e7.88 (m, 1H), 7.63 (d, J ¼ 8.3 Hz, 1H), 7.52e7.50 (m,
1H), 7.27 (t, J ¼ 7.8 Hz, 1H), 6.93e6.86 (m, 3H), 6.66 (d, J ¼ 2.4 Hz,
1H), 6.42 (d, J ¼ 2.4 Hz, 1H), 4.32 (d, J ¼ 12.0 Hz, 1H), 4.15 (t,
J ¼ 6.3 Hz, 3H), 3.13e3.09 (m, 2H), 2.65 (s, 3H), 1.81e1.74 (m, 4H),
4.4.7. 1-Hydroxy-3-(3-((4-hydroxybenzyl) (methyl)amino)
propoxy)-9H-xanthen-9-one (4g)
It was prepared from 3-(3-bromopropoxy)-1-hydroxy-9H-
xanthen-9-one (3a) and 4-((methylamino)methyl)phenol accord-
ing to the general procedure, then purified on a silica gel chroma-
tography eluted with dichloromethane/methanol (25:1) to obtain
the pure product 4g. 61% yield; yellow solid; m.p. 178e180 ꢂC; 1H
1.47e1.42 (m, 2H). 13C NMR (151 MHz, DMSO)
d 180.61, 166.44,
163.09, 158.08, 157.79, 155.94, 136.47, 131.77, 130.44, 125.79, 125.10,
121.98, 120.32, 118.26, 118.23, 116.98, 103.57, 97.94, 93.73, 68.72,
58.94, 55.21, 46.22, 28.23, 23.40, 23.01. HRMS: calcd for C26H28NO5
[MþH]þ 434.1962, found 434.1902. HPLC purity, 99.15%.
NMR (600 MHz, DMSO)
d 12.82 (s, 1H), 9.81 (s, 1H), 8.17 (d,
J ¼ 8.4 Hz, 1H), 7.92e7.89 (m, 1H), 7.64 (d, J ¼ 8.4 Hz, 1H), 7.52 (d,
J ¼ 7.2 Hz, 1H), 7.33 (d, J ¼ 7.8 Hz, 2H), 6.84 (d, J ¼ 9.0 Hz, 2H), 6.66
(s, 1H), 6.41 (s, 1H), 4.32 (s, 1H), 4.22 (d, J ¼ 6.0 Hz, 3H), 3.29 (s, 2H),
4.4.4. 1-Hydroxy-3-((6-((3-hydroxybenzyl) (methyl)amino)hexyl)
oxy)-9H-xanthen-9-one (4d)
2.70 (s, 3H), 2.18 (s, 2H). 13C NMR (151 MHz, DMSO)
d 180.67, 165.95,
It was prepared from 3-((6-bromohexyl)oxy)-1-hydroxy-9H-
xanthen-9-one (3d) and 3-((methylamino)methyl)phenol accord-
ing to the general procedure, then purified on a silica gel chroma-
tography eluted with dichloromethane/methanol (25:1) to obtain
the pure product 4d. 67% yield; yellow solid; m.p. 106e110 ꢂC; 1H
163.07, 158.97, 157.77, 155.95, 136.54, 133.21 (2C), 125.81, 125.15
(2C), 120.31 (2C), 118.27, 116.01, 103.74, 98.00, 93.79, 66.27, 58.79,
52.02, 46.23, 23.85. HRMS: calcd for C24H24NO5 [MþH]þ 406.1649,
found 406.1608. HPLC purity, 98.68%.
NMR (600 MHz, DMSO)
d
12.81 (s, J ¼ 7.8, 1.8 Hz, 1H), 9.72 (s, 1H),
4.4.8. 1-Hydroxy-3-(4-((4-hydroxybenzyl) (methyl)amino)butoxy)-
9H-xanthen-9-one (4h)
8.16 (dd, J ¼ 7.9, 1.6 Hz, 1H), 7.91e7.88 (m, 1H), 7.62 (d, J ¼ 8.4 Hz,
1H), 7.52e7.49 (m, 1H), 7.27 (t, J ¼ 7.8 Hz, 1H), 6.92e6.86 (m, 3H),
6.65 (d, J ¼ 1.8 Hz, 1H), 6.41 (d, J ¼ 2.4 Hz, 1H), 4.31 (d, J ¼ 11.4 Hz,
1H), 4.14 (t, J ¼ 6.0 Hz, 3H), 3.12e3.09 (m, 1H), 3.00 (s, 1H), 2.67 (s,
3H), 1.79e1.69 (m, 4H), 1.47e1.43 (m, 2H), 1.36e1.34 (m, 2H). 13C
It was prepared from 3-(4-bromobutoxy)-1-hydroxy-9H-
xanthen-9-one (3b) and 4-((methylamino)methyl)phenol accord-
ing to the general procedure, then purified on a silica gel chroma-
tography eluted with dichloromethane/methanol (25:1) to obtain
the pure product 4h. 59% yield; yellow solid; m.p. 150e153 ꢂC; 1H
NMR (151 MHz, DMSO)
d 180.60, 166.50, 163.09, 158.07, 157.80,
155.94, 136.46, 131.81, 130.44, 125.79, 125.09, 121.95, 120.32, 118.23,
116.96, 103.55 (2C), 97.92, 93.70, 68.94, 58.93, 55.25, 46.21, 28.52,
26.07, 25.38, 23.66. HRMS: calcd for C27H30NO5 [MþH]þ 448.2118,
found 448.2050. HPLC purity, 99.62%.
NMR (600 MHz, DMSO)
d
12.82 (s, 1H), 9.80 (s, 1H), 8.17 (dd, J ¼ 7.8,
1.2 Hz, 1H), 7.92e7.89 (m, 1H), 7.64 (d, J ¼ 8.4 Hz, 1H), 7.52 (d,
J ¼ 7.2 Hz, 1H), 7.32 (d, J ¼ 8.4 Hz, 2H), 6.82 (d, J ¼ 8.4 Hz, 2H), 6.67
(d, J ¼ 2.4 Hz, 1H), 6.43 (d, J ¼ 1.8 Hz, 1H), 4.28 (d, J ¼ 12.0 Hz, 1H),
4.18e4.14 (m, 3H), 3.18 (s, 1H), 3.04 (s, 1H), 2.66 (s, 3H), 1.87e1.77
4.4.5. 1-Hydroxy-3-((7-((3-hydroxybenzyl) (methyl)amino)heptyl)
oxy)-9H-xanthen-9- one (4e)
(m, 4H). 13C NMR (151 MHz, DMSO)
d 180.64, 166.26, 163.09, 158.96,
157.79, 155.95, 136.50, 133.23 (3C), 125.80, 125.13, 120.33, 118.25,
115.99 (2C), 103.63, 97.98, 93.78, 68.39, 58.63, 54.47, 46.21, 25.91,
20.85. HRMS: calcd for
It was prepared from 3-((7-bromoheptyl)oxy)-1-hydroxy-9H-
xanthen-9-one (3e) and 3-((methylamino)methyl)phenol accord-
ing to the general procedure, then purified on a silica gel chroma-
tography eluted with dichloromethane/methanol (25:1) to obtain
the pure product 4e. 68% yield; yellow solid; m.p. 121e124 ꢂC; 1H
C
25H26NO5 [MþH]þ 420.1805, found
420.1746. HPLC purity, 95.12%.
4.4.9. 1-Hydroxy-3-((5-((4-hydroxybenzyl) (methyl)amino)pentyl)
oxy)-9H-xanthen-9-one (4i)
NMR (600 MHz, DMSO)
d 12.80 (s, 1H), 9.72 (s, 1H), 8.16 (d,
J ¼ 7.8 Hz, 1H), 7.89 (t, J ¼ 8.4 Hz, 1H), 7.62 (d, J ¼ 8.4 Hz, 1H), 7.50 (t,
J ¼ 7.8 Hz,1H), 7.26 (t, J ¼ 7.2 Hz,1H), 6.91e6.85 (m, 3H), 6.65 (s,1H),
6.40 (s, 1H), 4.29 (s, 1H),4.13 (d, J ¼ 6.0 Hz, 3H), 3.04 (d, J ¼ 54.6 Hz,
2H), 2.66 (s, 3H), 1.42 (d, J ¼ 6.6 Hz, 2H), 1.37e1.22 (m, 8H). 13C NMR
It was prepared from 3-((5-bromopentyl)oxy)-1-hydroxy-9H-
xanthen-9-one (3c) and 4-((methylamino)methyl)phenol accord-
ing to the general procedure, then purified on a silica gel chroma-
tography eluted with dichloromethane/methanol (25:1) to obtain
the pure product 4i. 63% yield; yellow solid; m.p. 221e223 ꢂC; 1H
(151 MHz, DMSO)
d 180.60, 166.53, 163.08, 158.07, 157.79, 155.93,
136.45, 131.83, 130.43, 125.78, 125.08, 121.92, 120.31, 118.23, 116.93,
103.53 (2C), 97.91, 93.69, 69.04, 58.96, 55.32, 48.94, 28.68, 28.59,
26.34, 25.62, 23.68. HRMS: calcd for C28H32NO5 [MþH]þ 462.2275,
found 462.2183. HPLC purity, 98.12%.
NMR (600 MHz, DMSO)
d
12.81 (s, 1H), 9.81 (s, 1H), 8.16 (dd, J ¼ 7.8,
1.8 Hz, 1H), 7.91e7.88 (m, 1H), 7.62 (d, J ¼ 8.4 Hz, 1H), 7.51 (d,
J ¼ 7.2 Hz, 1H), 7.32 (d, J ¼ 8.4 Hz, 2H), 6.83 (d, J ¼ 8.4 Hz, 2H), 6.66
(d, J ¼ 2.4 Hz, 1H), 6.41 (d, J ¼ 1.8 Hz, 1H), 4.28 (d, J ¼ 12.6 Hz, 1H),
4.15 (d, J ¼ 6.0 Hz, 2H), 4.11 (d, J ¼ 9.0 Hz,1H), 3.10 (d, J ¼ 4.8 Hz,1H),
4.4.6. 1-Hydroxy-3-((8-((3-hydroxybenzyl) (methyl)amino)octyl)
oxy)-9H-xanthen-9-one (4f)
2.98 (s, 1H), 2.65 (s, 3H), 1.80e1.73 (m, 4H), 1.46e1.42 (m, 2H). 13
C
NMR (151 MHz, DMSO)
d 180.61, 166.44, 163.08, 158.95, 157.79,
It was prepared from 3-((8-bromooctyl)oxy)-1-hydroxy-9H-
xanthen-9-one (3f) and 3-((methylamino)methyl)phenol accord-
ing to the general procedure, then purified on a silica gel chroma-
tography eluted with dichloromethane/methanol (25:1) to obtain
the pure product 4f. 65% yield; yellow solid; m.p. 124e127 ꢂC; 1H
155.93, 136.47, 133.20 (3C), 125.79, 125.09, 120.31, 118.23, 116.01
(2C), 103.56, 97.93, 93.72, 68.73, 58.62, 54.72, 38.97, 28.23, 23.45,
23.03. HRMS: calcd for
C
26H28NO5 [MþH]þ 434.1962, found
434.1897. HPLC purity, 98.18%.
NMR (600 MHz, DMSO)
d
12.81 (s, 1H), 9.72 (s, 1H), 8.16 (d,
4.4.10. 1-Hydroxy-3-((6-((4-hydroxybenzyl) (methyl)amino)hexyl)
oxy)-9H-xanthen-9-one (4j)
J ¼ 6.0 Hz, 1H), 7.89 (t, J ¼ 8.4 Hz, 1H), 7.62 (d, J ¼ 7.8 Hz, 1H),
7.52e7.49 (m, 1H), 7.27 (t, J ¼ 7.8 Hz, 1H), 6.91e6.85 (m, 3H), 6.65 (d,
J ¼ 1.8 Hz, 1H), 6.40 (d, J ¼ 1.2 Hz, 1H), 4.29 (s, 1H), 4.13 (t, J ¼ 6.0 Hz,
3H), 3.08 (s, 1H), 2.98 (s, 1H), 2.66 (s, 3H), 1.77e1.67 (m, 4H),
1.43e1.40 (m, 2H), 1.33e1.30 (m, 6H). 13C NMR (151 MHz, DMSO)
It was prepared from 3-((6-bromohexyl)oxy)-1-hydroxy-9H-
xanthen-9-one (3d) and 4-((methylamino)methyl)phenol accord-
ing to the general procedure, then purified on a silica gel chroma-
tography eluted with dichloromethane/methanol (25:1) to obtain
the pure product 4j. 70% yield; yellow solid; m.p. 113e116 ꢂC; 1H
d
180.59, 166.54, 163.08, 158.07, 157.79, 155.93, 136.44, 131.81,
130.43, 125.78, 125.07, 121.93, 120.32, 118.23, 116.94, 103.52 (2C),
97.91, 93.69, 69.07 (2C), 58.92, 55.29, 28.88, 28.86, 28.78, 26.35,
25.75, 23.69. HRMS: calcd for C29H34NO5 [MþH]þ 476.2431, found
476.2363. HPLC purity, 98.56%.
NMR (600 MHz, DMSO)
d
12.81 (s, 1H), 9.80 (s, 1H), 8.17 (dd, J ¼ 7.8,
1.8 Hz, 1H), 7.91e7.88 (m, 1H), 7.63 (d, J ¼ 8.4 Hz, 1H), 7.51 (d,
J ¼ 7.8 Hz, 1H), 7.31 (d, J ¼ 8.4 Hz, 2H), 6.83 (d, J ¼ 8.4 Hz, 2H), 6.66
(d, J ¼ 2.4 Hz, 1H), 6.41 (d, J ¼ 1.8 Hz, 1H), 4.27 (d, J ¼ 12.6 Hz, 1H),
12

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
4.15e4.10 (m, 3H), 3.09 (s, 1H), 2.95 (s, 1H), 2.65 (s, 3H), 1.78e1.68
(m, 4H), 1.47e1.42 (m, 2H), 1.37e1.33 (m, 2H). 13C NMR (151 MHz,
4.4.14. 3-((6-(ethyl(4-hydroxybenzyl)amino)hexyl)oxy)-1-
hydroxy-9H-xanthen-9-one (4n)
DMSO)
d
180.61, 166.51, 163.09, 158.94, 157.80, 155.94, 136.47, 133.18
It was prepared from 3-((6-bromohexyl)oxy)-1-hydroxy-9H-
xanthen-9-one (3d) and 4-((ethylamino)methyl)phenol according
to the general procedure, then purified on a silica gel chromatog-
raphy eluted with dichloromethane/methanol (25:1) to obtain the
pure product 4n. 74% yield; yellow solid; m.p. 74e75 ꢂC; 1H NMR
(3C), 125.79, 125.09, 120.32, 118.24, 116.01 (2C), 103.55, 97.93, 93.71,
68.94, 58.62, 54.79, 46.21, 28.52, 26.09, 25.37, 23.71. HRMS: calcd
for C27H30NO5 [MþH]þ 448.2118, found 448.2060. HPLC purity,
99.44%.
(600 MHz, DMSO)
d
12.74 (s, 1H), 9.26 (s, 1H), 8.15 (d, J ¼ 7.8 Hz,1H),
7.88 (t, J ¼ 7.8 Hz, 1H), 7.62 (d, J ¼ 8.4 Hz, 1H), 7.50 (t, J ¼ 7.2 Hz, 1H),
7.07 (d, J ¼ 8.4 Hz, 2H), 6.68 (d, J ¼ 8.4 Hz, 2H), 6.63 (s, 1H), 6.39 (s,
1H), 4.10 (t, J ¼ 6.6 Hz, 2H), 2.42e2.39 (m, 2H), 2.34 (t, J ¼ 7.2 Hz,
2H), 1.71 (t, J ¼ 7.2 Hz, 2H), 1.42 (t, J ¼ 7.2 Hz, 2H), 1.38 (t, J ¼ 7.8 Hz,
2H), 1.30 (t, J ¼ 6.6 Hz, 4H), 0.95 (t, J ¼ 7.2 Hz, 3H). 13C NMR
4.4.11. 1-Hydroxy-3-((7-((4-hydroxybenzyl) (methyl)amino)heptyl)
oxy)-9H-xanthen-9-one (4k)
It was prepared from 3-((7-bromoheptyl)oxy)-1-hydroxy-9H-
xanthen-9-one (3e) and 4-((methylamino)methyl)phenol accord-
ing to the general procedure, then purified on a silica gel chroma-
tography eluted with dichloromethane/methanol (25:1) to obtain
the pure product 4k. 75% yield; yellow solid; m.p. 128e130 ꢂC; 1H
(151 MHz, DMSO)
d 180.58, 166.56, 163.08, 157.79, 156.52, 155.93,
136.40, 130.13 (3C), 125.76, 125.03, 120.32, 118.23, 115.25, 103.51
(2C), 97.93, 93.67, 69.05, 57.28, 52.52, 46.84, 28.83, 26.97, 25.69,
22.56, 11.98. HRMS: calcd for C28H32NO5 [MþH]þ 462.2275, found
462.2198. HPLC purity, 97.95%.
NMR (600 MHz, DMSO)
d
12.81 (s, 1H), 9.79 (s, 1H), 8.17 (dd, J ¼ 7.8,
1.8 Hz, 1H), 7.91e7.89 (m, 1H), 7.63 (d, J ¼ 8.4 Hz, 1H), 7.52e7.50 (m,
1H), 7.31 (d, J ¼ 8.4 Hz, 2H), 6.83 (d, J ¼ 8.4 Hz, 2H), 6.66 (d,
J ¼ 1.8 Hz, 1H), 6.41 (d, J ¼ 1.8 Hz, 1H), 4.26 (d, J ¼ 13.2 Hz, 1H), 4.14
(t, J ¼ 6.6 Hz, 3H), 3.00e2.95 (m, 2H), 2.64 (s, 3H),1.77e1.70 (m, 4H),
1.44e1.41 (m, 2H), 1.37e1.35 (m, 2H), 1.23 (t, J ¼ 4.8 Hz, 2H). 13C
4.5. General procedure for the synthesis of carbamates (5a-f)
NMR (151 MHz, DMSO)
d 180.62, 166.55, 163.09, 158.94, 157.82,
A mixture of the selected 1-hydroxyl-3-aminoalkoxyxanthen
derivative (0.22 mmol), the selected isocyanate (1.1 mmol) and
triethylamine (0.43 mmol) in dry DMF, under nitrogen atmosphere,
was stirred at room temperature for 36e48 h. Upon completion of
the reaction, the mixture was pulled into water stirring for minutes
and extracted with ethyl acetate (10 ml ꢃ 3). The ethyl acetate layer
was collected, dried over anhydrous sodium sulfate, filtered and
concentrated under reduced pressure to give the compounds 5a-f.
155.94, 136.48, 133.19 (3C), 125.10, 120.40, 120.33, 118.25, 116.00
(2C), 103.55, 97.94, 93.72, 69.06, 58.59, 54.83, 53.15, 28.68, 28.57,
26.33, 25.61, 25.51. HRMS: calcd for C28H31NO5 [MþH]þ 462.2275,
found 462.2193. HPLC purity, 96.15%.
4.4.12. 1-Hydroxy-3-((8-((4-hydroxybenzyl) (methyl)amino)octyl)
oxy)-9H-xanthen-9-one (4l)
It was prepared from 3-((8-bromooctyl)oxy)-1-hydroxy-9H-
xanthen-9-one (3f) and 4-((methylamino)methyl)phenol accord-
ing to the general procedure, then purified on a silica gel chroma-
tography eluted with dichloromethane/methanol (25:1) to obtain
the pure product 4l. 81% yield; yellow solid; m.p. 125e127 ꢂC; 1H
4.5.1. 4-(((6-((1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)hexyl)
(methyl)amino)methyl) phenyl ethylcarbamate (5a)
Using the previous procedure and starting from 4j and iso-
cyanatoethane, 5a was obtained. 48% yield; yellow solid; m.p.
NMR (600 MHz, DMSO)
d
12.81 (s, 1H), 9.80 (s, 1H), 8.16 (dd, J ¼ 7.8,
103e106 ꢂC; 1H NMR (600 MHz, CDCl3)
d 12.88 (s, 1H), 8.26 (dd,
1.8 Hz, 1H), 7.91e7.88 (m, 1H), 7.62 (d, J ¼ 8.4 Hz, 1H), 7.51 (d,
J ¼ 7.8 Hz, 1H), 7.30 (d, J ¼ 8.4 Hz, 2H), 6.83 (d, J ¼ 8.4 Hz, 2H), 6.65
(d, J ¼ 1.8 Hz, 1H), 6.40 (d, J ¼ 2.4 Hz, 1H), 4.25 (d, J ¼ 12.6 Hz, 1H),
4.14e4.10 (m, 3H), 3.06 (s, 1H), 2.94 (s, 1H), 2.64 (s, 3H), 1.77e1.72
(m, 2H), 1.68e1.64 (m, 2H), 1.43e1.39 (m, 2H), 1.35e1.30 (m, 6H).
J ¼ 7.8, 1.8 Hz, 1H), 7.74e7.71 (m, 1H), 7.45 (d, J ¼ 8.4 Hz, 1H),
7.40e7.38 (m, 1H), 7.30 (d, J ¼ 8.4 Hz, 2H), 7.07 (d, J ¼ 8.4 Hz, 2H),
6.44 (d, J ¼ 1.8 Hz, 1H), 6.36 (d, J ¼ 2.4 Hz, 1H), 5.19 (s, 1H), 4.04 (t,
J ¼ 6.6 Hz, 2H), 3.47 (s, 2H), 3.34e3.29 (m, 2H), 2.37 (t, J ¼ 7.8 Hz,
2H), 2.21 (s, 3H), 1.83e1.81 (m, 2H), 1.58e1.53 (m, 2H), 1.47e1.45
(m, 2H), 1.42e1.39 (m, 2H), 1.24e1.21 (m, 3H). 13C NMR (151 MHz,
13C NMR (151 MHz, DMSO)
d 180.62, 166.56, 163.10, 158.95, 157.81,
155.95, 136.47, 133.18 (3C), 125.79, 125.09, 120.33, 118.25, 116.01
(2C), 103.54, 97.93, 93.71, 69.08, 58.62, 54.83, 46.22, 28.88, 28.85,
28.78, 26.36, 25.74, 23.74. HRMS: calcd for C29H34NO5 [MþH]þ
476.2431, found 476.2357. HPLC purity, 97.20%.
CDCl3)
d 180.82, 166.41, 163.46, 158.46, 157.75, 156.06, 149.93,
135.00, 129.82 (3C), 125.84, 123.99, 121.30, 120.63, 117.60, 103.81
(2C), 97.50, 93.28, 68.68, 61.78, 57.00, 42.26, 36.12, 28.91, 27.19,
26.98, 25.79, 15.11. HRMS: calcd for C30H35N2O6 [MþH]þ 519.2489,
found 519.2392. HPLC purity, 98.81%.
4.4.13. 1-Hydroxy-3-((6-((4-methoxybenzyl) (methyl)amino)hexyl)
oxy)-9H-xanthen-9-one (4m)
It was prepared from 3-((6-bromohexyl)oxy)-1-hydroxy-9H-
xanthen-9-one (3d) and 1-(4-methoxyphenyl)-N-methylmethan-
amine according to the general procedure, then purified on a silica
gel chromatography eluted with dichloromethane/methanol (25:1)
to obtain the pure product 4m. 69% yield; yellow solid; m.p.
4.5.2. 4-(((6-((1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)hexyl)
(methyl)amino)methyl) phenyl (2-chloroethyl)carbamate (5b)
Using the previous procedure and starting from 4j and 1-chloro-
2-isocyanatoethane, 5b was obtained. 45% yield; yellow solid; m.p.
121e124 ꢂC; 1H NMR (600 MHz, CDCl3)
d 12.88 (br s, 1H), 8.27 (dd,
70e72 ꢂC; 1H NMR (600 MHz, CDCl3)
d
12.88 (s, 1H), 8.27 (dd,
J ¼ 7.8, 1.2 Hz, 1H), 7.74e7.71 (m, 1H), 7.45 (d, J ¼ 8.4 Hz, 1H),
J ¼ 8.4, 1.8 Hz, 1H), 7.74e7.71 (m, 1H), 7.44 (d, J ¼ 8.4 Hz, 1H),
7.41e7.38 (m, 1H), 7.25 (d, J ¼ 8.4 Hz, 2H), 6.88 (d, J ¼ 9.0 Hz, 2H),
6.44 (d, J ¼ 2.4 Hz, 1H), 6.36 (d, J ¼ 2.4 Hz, 1H), 4.06 (t, J ¼ 6.0 Hz,
2H), 3.82 (s, 3H), 3.49 (s, 2H), 2.42 (t, J ¼ 7.2 Hz, 2H), 2.23 (s, 3H),
1.86e1.82 (m, 2H), 1.62e1.57 (m, 2H), 1.52e1.47 (m, 2H), 1.43e1.40
7.41e7.36 (m, 3H), 7.12 (d, J ¼ 8.4 Hz, 2H), 6.44 (d, J ¼ 2.4 Hz, 1H),
6.36 (d, J ¼ 1.8 Hz, 1H), 5.09 (s, 1H), 4.05 (d, J ¼ 6.0 Hz, 2H),
d 3.70 (t,
J ¼ 5.4 Hz, 2H), 3.65e3.62 (m, 3H), 3.56 (t, J ¼ 6.0 Hz, 1H),2.47 (t,
J ¼ 7.2 Hz, 2H), 2.29 (s, 3H), 1.85e1.80 (m, 2H), 1.65e1.60 (m, 2H),
1.50e1.45 (m, 2H), 1.43e1.39 (m, 2H). 13C NMR (151 MHz, CDCl3)
(m, 2H). 13C NMR (151 MHz, CDCl3)
d
180.78, 166.34, 163.52, 158.73,
d 180.80, 166.35, 163.47, 157.74, 157.37, 156.05, 150.11, 134.99, 130.27
157.74, 156.04, 134.96, 130.38 (3C), 125.87, 123.98, 120.66, 117.58,
113.61, 103.83 (2C), 97.46, 93.24, 68.62, 61.51, 56.96, 55.25, 41.93,
28.91, 27.09, 27.05, 25.86. HRMS: calcd for C28H32NO5 [MþH]þ
462.2275, found 462.2187. HPLC purity, 98.81%.
(3C), 125.86, 123.99, 121.44, 120.64, 117.60, 103.83 (2C), 97.49, 93.27,
68.60, 61.36, 56.68, 43.89, 42.99, 41.84, 28.87, 26.89, 26.66, 25.74.
HRMS: calcd for C30H34ClN2O6 [MþH]þ 553.2099, found 553.1986.
HPLC purity, 97.28%.
13

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
4.5.3. 4-(((6-((1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)hexyl)
(methyl)amino)methyl) phenyl cyclohexylcarbamate (5c)
163.49, 157.73, 156.76, 156.06, 149.82, 134.96, 129.99, 129.57, 129.41,
125.86, 123.97, 121.21, 120.66, 117.60, 115.08, 103.81, 97.50, 93.29,
68.69, 57.49, 52.75, 50.10, 47.25, 33.96 (2C), 28.93, 27.00 (2C), 26.85,
25.62, 24.95 (2C), 11.84. HRMS: calcd for C35H43N2O6 [MþH]þ
587.3115, found 587.2982. HPLC purity, 99.56%.
Using the previous procedure and starting from 4j and iso-
cyanatocyclohexane, 5c was obtained. 44% yield; yellow solid; m.p.
126e128 ꢂC; 1H NMR (600 MHz, CDCl3)
d 12.89 (s, 1H), 8.27 (dd,
J ¼ 7.8, 1.2 Hz, 1H), 7.74e7.71 (m, 1H), 7.45 (d, J ¼ 8.4 Hz, 1H),
7.41e7.38 (m, 1H), 7.30 (d, J ¼ 8.4 Hz, 2H), 7.09 (d, J ¼ 8.4 Hz, 2H),
6.44 (d, J ¼ 2.4 Hz, 1H), 6.37 (d, J ¼ 1.8 Hz, 1H), 4.97 (d, J ¼ 7.8 Hz,
1H), 4.05 (t, J ¼ 6.0 Hz, 2H), 3.59e3.54 (m, 1H), 3.49 (s, 2H), 2.38 (t,
J ¼ 7.2 Hz, 2H), 2.22 (s, 3H), 2.02 (d, J ¼ 9.0 Hz, 2H), 1.85e1.73 (m,
8H), 1.65e1.53 (m, 4H), 1.48e1.46 (m, 2H), 1.42e1.40 (m, 2H). 13C
5. Biological evaluation
5.1. Inhibitory activities of AChE and BuChE
NMR (151 MHz, CDCl3)
d 180.79, 166.39, 163.49, 157.73, 156.05,
The inhibition activities of target compounds 4a-n and 5a-f
against ChEs were determined according to Ellman’s method.
Acetylcholinesterase (AChE) from electric eel (eeAChE), butylcho-
linesterase (BuChE) from equine serum (eqBuChE), acetylthiocho-
line iodide (ATCI), S-butylthiocholine iodide (BTCI) and 5,5-
dithiobis-(2-nitrobenzoicacid) (DTNB) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). For AChE inhibition assay, the
target compound was dissolved in DMSO firstly and then diluted to
different concentrations with Tris-HCl buffer solution (50 mM,
pH ¼ 8.0, 0.1 M NaCl, 0.02 M MgCl2$H2O) (DMSO < 0.01%). The
experimental protocol was performed on 96-well plate. For each
153.77, 150.01, 134.96, 129.83 (3C), 125.86, 123.97, 121.30, 120.65,
117.59, 103.82 (2C), 97.50, 93.27, 68.67, 61.75, 56.96, 50.11, 42.23,
33.26 (2C), 28.92, 27.16, 27.00, 25.81, 25.46, 24.76 (2C). HRMS: calcd
for C34H40N2O6 [MþH]þ 573.2959, found 573.2837. HPLC purity,
95.57%.
4.5.4. 4-((ethyl(6-((1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)hexyl)
amino)methyl) phenyl ethylcarbamate (5d)
Using the previous procedure and starting from 4n and iso-
cyanatoethane, 5d was obtained. 45% yield; yellow solid; m.p.
85e88 ꢂC; 1H NMR (600 MHz, CDCl3)
d 12.89 (s, 1H), 8.27 (dd,
test well, 160
eeAChE) and 10
pound were added, and then the mixture was incubated at 37 ꢂC for
5 min. After that, 30 L of ATCI (15 mM) as substrate was quickly
mL of DTNB (1.5 mM), 50
mL of AChE (0.22 U/mL
J ¼ 7.8, 1.2 Hz, 1H), 7.74e7.72 (m, 1H), 7.45 (d, J ¼ 7.8 Hz, 1H), 7.39 (d,
J ¼ 7.8 Hz, 1H), 7.32 (d, J ¼ 8.4 Hz, 2H), 7.07 (d, J ¼ 7.8 Hz, 2H), 6.44
(d, J ¼ 2.4 Hz, 1H), 6.37 (d, J ¼ 2.4 Hz, 1H), 5.08 (s, 1H), 4.03 (d,
J ¼ 6.6 Hz, 2H), 3.55 (s, 2H), 3.33e3.31 (m, 2H), 2.55e2.51 (m, 2H),
2.44 (t, J ¼ 7.2 Hz, 2H), 1.81 (d, J ¼ 6.6 Hz, 2H), 1.51 (t, J ¼ 7.8 Hz, 2H),
1.45e1.42 (m, 2H), 1.39e1.37 (m, 2H), 1.23 (t, J ¼ 7.2 Hz, 3H), 1.05 (d,
mL of corresponding concentration of tested com-
m
added and the absorbance was determined with a wavelength of
405 nm by a UV plate reader at different time intervals (0, 60, 120,
and 180 s). For BuChE inhibition assay, the procedure was similar
with the method described above. The inhibition activity of tested
compound was reported with IC50 value that was calculated as
concentration of the compound that produced 50% enzyme activity
inhibition. The results were expressed as mean SD of three in-
dependent experiments. Data analysis was performed using Graph
Pad Prism 4.03 software (San Diego, CA, USA).
J ¼ 7.2 Hz, 3H). 13C NMR (151 MHz, CDCl3)
d 180.80, 166.42, 163.48,
157.73, 156.06, 154.62, 149.78, 134.97 (2C), 129.58 (2C), 125.86,
123.97, 121.22 (2C), 120.65, 117.59, 103.81, 97.50, 93.29, 68.69, 57.50,
52.75, 47.27, 36.12, 28.93, 26.99 (2C), 25.75, 15.14, 11.72. HRMS:
calcd for C31H37N2O6 [MþH]þ 533.2646, found 533.2527. HPLC
purity, 97.43%.
4.5.5. 4-((ethyl(6-((1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)hexyl)
amino)methyl) phenyl (2-chloroethyl)carbamate (5e)
5.2. Free radical scavenging activity on DPPH
Using the previous procedure and starting from 4n and 1-
chloro-2-isocyanatoethane, 5e was obtained. 45% yield; yellow
The DPPH (1,1-diphenyl-2-picrylhydrazyl radical) assay mea-
sures the hydrogen donation ability of the antioxidant to convert
the stable DPPH free radical into 1,1-diphenyl-2-(2,4,6-
trinitrophenyl)-hydrazine. This can be evaluated by measuring
the percent decrease in absorbance of the solution at 517 nm, which
is accompanied by a change of color from deep-violet to light-
yellow, after the radical reaction with products to be tested. DPPH
was dissolved in MeOH to make 10 mM stock solution, which was
diluted to 0.1 mM as the test sample. The target compound was
dissolved in DMSO to make 10 mM stock solution, which was
diluted to five concentrations as test samples. Trolox was used as a
standard. The experimental protocol was performed on 96-well
solid; m.p. 66e68 ꢂC; 1H NMR (600 MHz, CDCl3)
d 12.89 (br s, 1H),
8.26 (dd, J ¼ 7.8, 1.8 Hz, 1H), 7.74e7.71 (m, 1H), 7.45 (d, J ¼ 8.4 Hz,
1H), 7.40e7.38 (m, 3H), 7.10 (d, J ¼ 8.4 Hz, 2H), 6.44 (d, J ¼ 2.4 Hz,
1H), 6.36 (d, J ¼ 2.4 Hz, 1H), 5.15 (s, 1H),4.03 (d, J ¼ 6.0 Hz, 2H), 3.70
(t, J ¼ 5.4 Hz, 2H), 3.64e3.62 (m, 3H), 3.56 (t, J ¼ 6.0 Hz, 1H), 2.61 (s,
2H), 2.51 (s, 2H), 1.82e1.80 (m, 2H), 1.57 (s, 2H), 1.47e1.42 (m, 2H),
1.40e1.37 (m, 2H), 1.11 (s, 3H). 13C NMR (151 MHz, CDCl3)
d 180.82,
166.38, 163.48, 157.74, 157.45, 156.06, 154.63, 135.00 (2C), 130.01
(2C), 125.87, 124.00, 121.35, 120.65, 117.60 (2C), 103.83, 97.50, 93.29,
68.63, 64.33, 57.24, 52.51, 43.00, 42.14, 28.89, 26.91 (2C), 25.70,
11.30. HRMS: calcd for C31H36ClN2O6 [MþH]þ 567.2256, found
567.2134. HPLC purity, 96.68%.
plate. For each test well, 100
mL of tested compound, 100 mL of
DPPH was added, then was shook and left to incubate at room
temperature in the dark for 30 min. Afterwards, the absorbance was
determined with a microplate reader, three determinations in
parallel, at 517 nm. The percentages of DPPH free radicals were
calculated using Equation:
4.5.6. 4-((ethyl(6-((1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)hexyl)
amino)methyl) phenyl cyclohexylcarbamate (5f)
Using the previous procedure and starting from 4n and iso-
cyanatocyclohexane, 5f was obtained. 43% yield; yellow solid; m.p.
80e83 ꢂC; 1H NMR (600 MHz, CDCl3)
d 12.89 (s, 1H), 8.27 (dd,
J ¼ 8.4, 1.8 Hz, 1H), 7.74e7.72 (m, 1H), 7.45 (d, J ¼ 8.4 Hz, 1H),
7.41e7.38 (m, 1H), 7.33 (t, J ¼ 7.8 Hz, 2H), 7.07 (d, J ¼ 7.8 Hz, 2H),
6.44 (d, J ¼ 2.4 Hz, 1H), 6.37 (d, J ¼ 2.4 Hz, 1H), 4.98 (d, J ¼ 7.8 Hz,
1H), 4.03 (t, J ¼ 6.6 Hz, 2H), 3.53 (s, 1H), 3.50 (s, 2H), 2.55e2.51 (m,
2H), 2.45e2.43 (m, 2H), 1.82e1.79 (m, 2H), 1.65e1.60 (m, 4H),
1.54e1.49 (m, 2H), 1.38e1.35 (m, 6H), 1.23 (d, J ¼ 10.2 Hz, 3H),
DPPH , free , radicals , ð%Þ , ¼ , ½A0 , ꢁ , ðA1 , ꢁ , A2Þꢄ=A0 ,
ꢃ ,100%
where A0 is the absorbance of the DPPH radical methanol solution,
A1 is the absorbance of the sample (DPPH þ compounds), A2 is the
absorbance of the sample (compounds) alone.
1.07e1.04 (m, 4H). 13C NMR (151 MHz, CDCl3)
d 180.80, 166.41,
14

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
5.3. ABTS radical cation scavenging activity assay
were dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/
EtOH (7:3) to achieve a concentration of 100 mg/mL, 200 mL of which
The ABTS radical scavenging capacities of target compounds
were measured using a Total Antioxidant Capacity Assay Kit ac-
cording to the manufacture’s instructions. The ABTS radical cation
(ABTSþ) was produced by reacting an aqueous solution of ABTS and
oxidant in a ratio of 1:1 (v/v) respectively. The ABTS working so-
lutions were kept to react completely in the dark for 12e16 h, then
diluted to an absorbance of 1.4 0.1 at 405 nm with ethanol. To test
free radical scavenging effects, compounds and trolox were
was added to the donor wells. The acceptor filter plate was carefully
placed on the donor plate to form a sandwich, which was left un-
disturbed for 18 h at 25 ꢂC. After incubation, the donor plate was
carefully removed and the concentration of compound in the
acceptor wells was determined using a UV plate reader (Spec-
traMax Plus 384, Molecular Devices, Sunnyvale, CA, USA). Every
sample was analyzed at five wavelengths, in four wells, in at least
three independent runs, and the results are given as the
mean standard deviation. In each experiment, 9 quality control
standards of known BBB permeability were included to validate the
analysis set. Pe was calculated by the following expression: Pe ¼ {-
VdVa/[(Vd þ Va)At]}ln (1-drugacceptor/drugequilibrium), where Vd is the
volume of donor well, Va is volume in acceptor well, A is the filter
area, t is the permeation time, drug acceptor is the absorbance
obtained in the acceptor well and drug equilibrium is the theo-
retical equilibrium absorbance. A good linear correlation was ob-
tained by plotting the experimental data against the bibliographic
values: Pe (exp.) ¼ 0.6703 Pe (bibl.) - 0.6571 (R2 ¼ 0.9623).
adjusted with ethanol solution to final concentrations of 0e100
Serial dilutions of the test sample (10 L) were combined with the
ABTS working solution (200 L) in a 96-wellmicrotitre plate. After
mM.
m
m
2e6 min in the dark, the absorbance was measured at 405 nm in a
microplate reader. Trolox was used as a standard compound.
5.4. Oxygen radical absorbance capacity (ORAC-FL) assay
The antioxidant potency was determined by the oxygen radical
absorbance capacity-fluorescein (ORAC-FL) assay with slight
modification [40]. 2,20-Azobis(2-methylpropionamidine) dihydro-
chloride (AAPH) was purchased from Sigma-Aldrich Chemical Co.,
Fluorescein (FL) and 6-hydroxy-2,5,7,8-tetramethylchromane-2-
carboxylic acid (Trolox) were purchased from TCI (Shanghai)
Development. All the assays were carried out in 10 mM phosphate
5.6. Kinetic studies of enzyme inhibition
Compound 4j was selected for kinetic studies of AChE and
BuChE inhibition and performed similar to enzyme inhibition
assay. The experiment was performed at four varied concentrations
buffer (pH ¼ 7.4), and the final reaction mixture was 200
mL. The
of inhibitor 4j (0, 0.5, 1, 2 mM for AChE and BuChE), and evaluated
tested compound (20 L) and FL (120 L, 150 nM final concentra-
m
m
against all the six concentrations of substrates (0.05, 0.075, 0.1, 0.15,
0.2, 0.5 mM for ATCI and BTCI). To obtain estimates of the inhibition
type, reciprocal plots of 1/V versus 1/[S] were constructed by using
reported method with minor modifications. The Dixon plots were
generated, and respective Ki values were determined. The assay
was conducted in triplicate.
tion) were placed in the wells of a black 96-well plate and the
mixture was pre-incubated for 5 min at 37 ꢂC. Then AAPH solution
(60
mL, 12 mM final concentration) was added rapidly using an
autosampler and the fluorescence was recorded every 5 min for
90 min with excitation at 485 nm and emission at 538 nm. Trolox
was used as standard (0.3125e5 mM, final concentration). A blank
(FL þ AAPH) using phosphate buffer instead of antioxidant and
trolox calibration were carried out in each assay. The samples were
measured at different concentration. Antioxidant curves (fluores-
cence versus time) were normalized to the curve of the blank in the
same assay (Figure S1), and the area under the fluorescence decay
curve (AUC) was calculated using following equation:
5.7. Molecular docking
The crystal structures of the huAChE, eeAChE and huBuChE en-
zymes were retrieved from the Protein Data Bank (PDB), under the
accession codes 4EY7, 2CKM and 1P0I, respectively. Molecular
modeling simulation were carried out using the Chemical
Computing Group’s Molecular Operating Environment (MOE)
software (Montreal, Canada, version 2015.10). The protein was
energy minimized and 3D protonated using the structure prepa-
ration module of MOE. Ligand file for the molecular docking studies
were prepared in MOE and were followed by energy optimization
at a standard MMFF94 force field level, with a 0.01 kcal/mol energy
gradient convergence criterion. Then, the optimized geometry of
ligand was saved in a molecular database file and docked into the
active site of the protein using the MOE-Dock program. All poses
generated with docking were analyzed and the best-scored pose
with the lowest binding energy for each compound was selected for
further investigation of interactions with the corresponding
enzyme.
AUC, ¼ ,5, ꢃ ,ðf0, þ ,f1, þ ,:::, þ ,fnꢁ1, þ ,fnÞ, ꢁ ,2:5,
ꢃ ,ðf0, þ ,fnÞ
where f0 is the initial fluorescence reading at 0 min and fn is the
fluorescence reading at time n.
The net AUC of a sample was obtained by subtracting the AUC of
the blank. ORAC-FL values were expressed as Trolox equivalents by
using the standard curve calculated for each sample, where the
ORAC-FL value of Trolox was taken as 1, indicating the antioxidant
potency of the tested compounds.
5.5. PAMPA-BBB penetration assay
The ability of the test compound penetrating into the brain was
conducted by a parallel artificial membrane permeation assay
(PAMPA) for the blood-brain barrier (BBB) based on previous work
of Di et al. Porcine brain lipid (PBL) and dodecane were purchased
from Avanti Polar Lipids and Sigma-Aldrich respectively. The donor
96-well filter microplate with a PVDF membrane (pore size
5.8. Metal-chelating study
The chelating studies were performed with a fluorescence
spectrophotometer and an UVevis spectrophotometer. Stock so-
lutions (1 mM) of the test compound 4j was prepared by dissolving
the sample in DMSO and diluting with a mixture of methanol-
water (1/1) to the final concentration of 1 mM. Stock solutions
(10 mM) of the cations salts (NaCl, MgCl2, FeCl2, ZnCl2, CaCl2, AlCl3
and CuCl2) were prepared in deionized water. The absorption
0.45
mM) and acceptor indented 96-well microplate were pur-
chased from Millipore. The 96-well UV plate (COSTAR) was from
Corning Incorporated. The acceptor 96-well microplate was filled
with 300
impregnated with 4
m
L of PBS/EtOH (7: 3), and the filter membrane was
spectra of ligand (4j, 50
presence of cations salts (50
m
M, final concentration) alone or in the
M, final concentration) for 30 min
mL of PBL in dodecane (20 mg/mL). Compounds
m
15

Z. Zhang, J. Guo, M. Cheng et al.
European Journal of Medicinal Chemistry 213 (2021) 113154
were recorded at room temperature. For the stoichiometry of the
Outstanding Youth Talents in Jiangxi province [Grant No.
20192BCB23018]; the Program of Natural Science Foundation of
Jiangxi province of China [Grant No. 20192ACBL21034,
20202BAB216042]; the 1050 Youth Talent Project of Jiangxi Uni-
versity of Traditional Chinese Medicine [Grant No.1141900616]; the
Open Project Program of Guangxi Key Laboratory of Brain and
Cognitive Neuroscience, Guilin Medical University [Grant No.
GKLBCN-20200108-07]; Nanchang Double-Hundred Talents Proj-
ect [Grant No.20200729]; Foundation of Jiangxi Educational Com-
mittee [Grant No. GJJ190673].
4j-Cu2þ and 4j-Al3þ complexes, a fixed amount of 4j (50
mM) was
mixed with ascending amounts of CuCl2 and AlCl3 respectively. The
UVevis spectra were examined to investigate the ratio of ligand/
metals in the complexes.
5.9. Cell culture and toxicity
All cells were obtained from the Cell Bank of the Chinese
Academy of Sciences (Shanghai, China). All cells were maintained in
high-glucose DMEM supplemented with 10% fetal bovine serum,
100 U/mL penicillin, and 100 U/mL streptomycin in a humidified
atmosphere of 5% CO2 at 37 ꢂC. Cells were subcultured in 96-well
plates at a seeding density of 10,000 cells per well and allowed to
adhere and grow. When cells reached the required confluence, they
were placed into serum-free medium and treated with compounds.
After 24 h, the survival of cells was determined by CCK-8 assay
according to the manufacture’s instructions. Results are expressed
as the mean SD of three independent experiments.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113154.
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Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgement
This work was supported by National Natural Science Founda-
tion of China [No. 81760622, 21807052]; Support Program for
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