2-(3-Hydroxybenzyl)benzo[d]isothiazol-3(2H)-one Mannich base derivatives as potential multifunctional anti-Alzheimer’s agents

Article abstract of DOI:10.1007/s00044-021-02725-6

A series of 2-(3-hydroxybenzyl)benzo[d]isothiazol-3(2H)-one Mannich base derivatives were designed as potential multifunctional agents against Alzheimer’s disease. The twelve derivatives were synthesized and evaluated with various biological activities. I

Full text of DOI:10.1007/s00044-021-02725-6

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-021-02725-6
ORIGINAL RESEARCH
2-(3-Hydroxybenzyl)benzo[d]isothiazol-3(2H)-one Mannich base
derivatives as potential multifunctional anti-Alzheimer’s agents
Yuxi He1 Ganyuan Xiao1 Guangjun Yu1 Qing Song1 Heng Zhang1 Zhuoling Liu1 Zhenghuai Tan2
Yong Deng1
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Received: 11 January 2021 / Accepted: 31 March 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
A series of 2-(3-hydroxybenzyl)benzo[d]isothiazol-3(2H)-one Mannich base derivatives were designed as potential
multifunctional agents against Alzheimer’s disease. The twelve derivatives were synthesized and evaluated with various
biological activities. In vitro, biological assays showed that most of the target compounds are selective AChE inhibitors and
good antioxidants. Among them, compounds 6d and 13d are representative agents exhibiting significant selective AChE
inhibition (IC50 = 1.09 µM and 2.01 µM, respectively), potent antioxidant activity, moderate inhibition of self-induced
Aβ1–42 aggregation, selective metal-chelating activity with Fe2+ and Cu2+, excellent neuroprotective effect on H2O2-induced
PC12 cell injury and good BBB permeability. Moreover, the step-down passive avoidance test demonstrated that 6d and 13d
can improve the scopolamine-induced cognitive deficit in mice. The above results suggested that compounds 6d and 13d can
be envisioned as multifunctional lead compounds for further development of drugs against AD.
Graphical Abstract
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Keywords Alzheimer’s disease 2-(3-Hydroxybenzyl)benzo[d]isothiazol-3(2H)-one Mannich base
Multifunctional anti-Alzheimer’s agents
These authors contributed equally: Yuxi He, Ganyuan Xiao
Introduction
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1007/s00044-
021-02725-6.
Alzheimer’s disease (AD) is a common chronic neuro-
degenerative disease, characterized by progressive mem-
ory impairment, cognitive dysfunction, personality
change, and language impairment [1], which has become a
serious social health problem [2]. The currently approved
drugs against AD by FDA include acetylcholinesterase
inhibitors (donepezil, rivastigmine, galantamine) and N-
methyl-D-aspartate receptor antagonist (memantine),
which can only alleviate the symptoms but cannot ter-
minate or reverse the progression of AD [3]. Though the
researches on the pathogenesis of AD have been delivered
for decades, the exact etiology of AD remains to be
* Yong Deng
dengyong@scu.edu.cn
1
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
2
Institute of Traditional Chinese Medicine Pharmacology and
Toxicology, Sichuan Academy of Chinese Medicine Sciences,
Chengdu 610041, China

Medicinal Chemistry Research
elucidated. There are several hypotheses proposed to
explain the cause of AD, such as the cholinergic
hypothesis, the toxic β-amyloid protein (Aβ) hypothesis,
the oxidative stress hypothesis, and so on [4].
on AD drug development are difficult and most potential
agents ended up in failure [17, 18]. In recent years, the
researchers developed some strategies for AD including
cocktail therapy, patient stratification, precision medicine,
and drugs of multiple targets [19]. The “multi-target-
directed ligands (MTDLs)” strategy was proposed to
develop new drugs for AD treatment because the effects of
the current single-target anti-AD drugs are limited due to
the complex pathogenesis of AD [9]. The multi-target
drugs can simultaneously activate or inhibit multiple tar-
gets involved in AD to exert a synergistic effect with fewer
side effects [20]. As reported, the typical drugs (tacrine,
donepezil, rivastigmine, memantine) for AD treatment and
some natural products (resveratrol, curcumin, coumarin,
etc.) with specific pharmacological activities were gen-
erally used as lead compounds for multifunctional hybrids
[21]. Based on the “MTDLs” strategy, many multi-
functional agents have been designed, synthesized, and
tested in biological assays.
Our team has been engaged in the researches of multi-
target drugs against AD for a long time, and the studies have
shown that some compounds exhibit good multi-target
activities in vitro experiments, and some compounds also
perform well in animal assays [22, 23]. As reported, sulfo-
namide and its derivatives are traditional antibacterial com-
pounds, which may be beneficial to AD treatment with a
variety of biological activities [24]. Our previous work also
confirmed it and reported a series of benzo[d]isothiazol-3
(2H)-one-N-alkyl benzylamine derivatives (Fig. 1A) on the
basis of sulfonamide derivatives, which are potential multi-
functional agents against AD with good AChE and Aβ
aggregation inhibitory activities, but weak antioxidant
capacity and no metal-chelating potency [25]. In addition,
some studies suggested that Mannich base derivatives are
extensively effective in anti-inflammation, antioxidant, and
adjusting the homeostasis of metal ions [26], with relatively
high bioavailability and water solubility. Besides, It was
reported that the compounds containing phenolic hydroxyl
Mannich bases (Fig. 1B) possess significant AChE inhibitory,
antioxidant, and metal-chelating activities [27, 28]. Thus, the
Mannich base unit can be a potent pharmacophore to improve
the antioxidant capacity and metal chelating potency.
The cholinergic hypothesis is widely accepted, which
proposes that the degeneration of cholinergic neurons and
the loss of acetylcholine (ACh) contribute to cognitive
dysfunction [5]. ACh is an important cholinergic neuro-
transmitter, which can be hydrolyzed by acetylcholinester-
ase (AChE) and butyrylcholinesterase (BuChE) [6]. AChE
mainly exists in the blood and nerve synapses, while
BuChE mainly distributes in peripheral tissues, which
means intense inhibition of BuChE may cause potential
peripheral side effects. Therefore, selective AChE inhibitors
can elevate the cholinergic level in the brains of AD patients
with little peripheral cholinergic side effects [7].
It is well-known that Aβ aggregation is one of the
neuropathological hallmarks of AD, and Aβ1–42 is far more
dangerous than Aβ1–40 because it is easier to aggregate and
form the amyloid plaques. More importantly, Aβ aggre-
gation has a tight correlation with other etiologies related
to AD such as cholinergic activity, oxidative stress, and
neuroinflammation [8]. AChE can accelerate the aggre-
gation of Aβ through the peripheral anion site (PAS)
binding to Aβ, and Aβ can also modulate the cholinergic
system conversely. Besides, the accumulation of Aβ pla-
ques is closely related to the negative feedback mechan-
isms of oxidative stress [8]. Overall, reducing Aβ1–42
aggregation can improve cholinergic function and cogni-
tive ability to a certain extent [9].
Oxidative stress refers to the excessive production of
highly active molecules in the body, such as reactive oxy-
gen species (ROS) and reactive nitrogen species (RNS),
which is a typical pathological hallmark of AD [10]. The
abnormally increased ROS and RNS not only cause the
functional loss of many antioxidant defense enzymes but
also damage lipids and nucleic acids [11]. It was reported
that oxidative stress is closely related to the transition metal
ions dyshomeostasis [12, 13]. Studies indicated that the
concentrations of transition metals (Zn2+, Cu2+, Fe2+, etc.)
are relatively high in the brains of AD patients [14], and
those transition metal ions play a key role in the accumu-
lation of Aβ and the formation of senile plaques [13].
Moreover, some synthetic small-molecule metal chelators
were proved to be effective in reducing oxidative stress and
neurotoxicity mediated by metal ions [15]. Therefore,
controlling oxidative stress and regulating the metal ions
homeostasis provide a new strategy for the treatment of AD
and other neurodegenerative diseases [16].
In order to overcome the drawbacks of the benzo[d]iso-
thiazol-3(2H)-one-N-alkylbenzylamine derivatives, in this
paper, a series of 2-(3-hydroxybenzyl)benzo[d]isothiazol-3
(2H)-one Mannich base derivatives (Fig. 1C) were designed,
synthesized and expected to possess enhanced inhibition of
AChE and Aβ1–42 aggregation, good antioxidant activity,
selective metal-chelating property, and proper BBB perme-
ability. So these compounds can interact with multiple bio-
chemical pathways, which related to the pathogenesis of AD,
to produce a synergistic effect.
Up to now, the clinical drugs can only alleviate the
symptoms of AD but cannot cure the disease, so it is
urgent to develop new anti-AD drugs. But the researches

Medicinal Chemistry Research
Fig. 1 Chemical structures of benzo[d]isothiazol-3(2H)-one-N-alkylbenzylamines (A), phenolic Mannich base derivatives (B), and designed 2-(3-
hydroxybenzyl)benzo[d]isothiazol-3(2H)-one Mannich base derivatives (C)
Scheme 1 Synthesis of 2-(3-hydroxybenzyl)benzo[d]isothiazol-3(2H)-
one Mannich bases 6a-j. Reagents and conditions: (i) HONH2·HCl,
AcONa, EtOH, at r.t. for 6 h; (ii) 10% Pd-C, H2, EtOH, at r.t. for 10 h;
(iii) Py, CH2Cl2, at r.t. for 48 h; (iv) (HCHO)n, HNR1R2 (a-j), EtOH,
reflux for 24–48 h
Results and discussion
dimethoxybenzo[d]isothiazol-3(2H)-one-1,1-dioxide (10)
[25] under alkaline condition by N-alkylation gave the key
intermediate 11. Then, the MOM group of product 11 was
removed using 10% HCl to yield 2-(3-hydroxybenzyl)-5,6-
dimethoxybenzo[d]isothiazol-3(2H)-one-1,1- dioxide (12).
Mannich reactions of compound 12 with paraformaldehyde
and the secondary amines HNR1R2 (c, d) gave the 4′-sub-
stituted Mannich base derivatives (13c, 13d, respectively).
Chemistry
The synthetic route of target compounds 6a-j is outlined in
Scheme 1. The starting material m-hydroxybenzaldehyde
(1) was reacted with hydroxylamine hydrochloride to afford
the 3-hydroxybenzaldehyde oxime (2) [29, 30], then cata-
lytic hydrogenation was performed in the presence of 10%
Pd/C to obtain m-hydroxybenzylamine (3) [31]. The key
intermediate 2-(3-hydroxy benzyl)-5,6-dimethoxybenzo[d]
isothiazol-3(2H)-one (5) was synthesized by the reaction of
the 2-(chlorocarbonyl)phenyl hypochlorothioite derivative
(4) [25] and compound 3. Finally, Mannich reactions of
compound 5 with paraformaldehyde and different second-
ary amines HNR1R2 (a-j) gave the 4′-substituted Mannich
base derivatives 6a-j.
For target compounds 13c and 13d, the synthetic route
is showed in Scheme 2. m-Hydroxybenzaldehyde (1) was
treated with chloromethyl methyl ether (MOMCl) in presence
of NaH to yield the MOM-protected compound 7, followed
by reduction of aldehyde group with NaBH4 in methanol to
obtain the MOM-protected benzyl alcohol (8). Treated
compound 8 with Ph3P/NBS to generate the brominated
product 9 in good yield, and compound 9 reacted with 5,6-
1
The key intermediate 5 was characterized by H NMR,
and all the synthetic target compounds were characterized by
1H NMR, ESI-MS, and elemental analysis, and most of them
1
were further characterized by 13C NMR. In the H NMR
spectrum of the intermediate 5, the proton of hydroxyl
showed no signal due to the replacement by deuterium in
CDCl3. The aromatic hydrogens of the A ring appear at about
7.24 and 6.78 ppm. The aromatic hydrogens of the B ring
appear at about 7.21, 7.18–7.06, 6.94, and 6.86 ppm. The
signal at about 4.9 ppm is the methylene attached to the C
ring, and the two singlets are the signals of methoxy (δ =
3.92, 3.72 ppm). In summary, the structure of intermediate 5
1
was confirmed by H NMR data. The core of the target
compounds (6a-6j, 13c, 13d) is the same as the intermediate
5, so do the analytical methods. Besides, the aminomethyl
fragments of all the target compounds were at 4′-position,
which can be explained by the regioselectivity for the

Medicinal Chemistry Research
Scheme 2 Synthesis of 2-(3-hydroxybenzyl)benzo[d]isothiazol-3(2H)-
one-1,1-dioxide Mannich bases 13c and 13d. Reagents and conditions:
(i) MOMCl, NaH, THF, at r.t. for 2 h; (ii) NaBH4, CH3OH, at r.t. for
2 h; (iii) Ph3P, NBS, CH2Cl2, at r.t. for 30 min; (iv) K2CO3, DMF, at
r.t. for 12 h; (v) 10% HCl, THF, reflux for 5 h; (vi) (HCHO)n, HNR1R2
(c, d), EtOH, reflux for 24–48 h
Mannich reaction [32]. The ortho-substituted products of
phenolic hydroxyl were obtained exclusively due to the
directing effect of the hydroxyl and the hydrogen bonds
between the nitrogen and the ortho-hydroxyl group, which
form a stabilized six-membered ring [33]. Besides, the steric
hindrance makes it difficult for aminoalkylation at 2′-position.
And the 1H NMR signals of the aromatic hydrogens in the B
ring of target compounds also showed no triplet, demon-
strating that the aminoalkylation did not occur at the 2′-posi-
tion. The ESI-MS, elemental analysis, and 13C NMR spectra
have further confirmed the structure of the target compounds.
and nitrogen atoms to the 4-position of piperidine ring
(morpholinyl or substituted piperazinyl) is not beneficial for
the AChE inhibition based on the data of 6e-6h. And the
mechanism may be the electron-withdrawing effect of
nitrogen or oxygen atom, decreasing the electrostatic
interaction between the terminal nitrogen and the catalytic
active site (CAS) of AChE [36]. Moreover, there is no
obvious difference in AChE inhibitory activity between the
compounds containing cyclic aliphatic amines and those
with chain aliphatic amines. Compounds 13c/13d exhibit
comparable AChE inhibition with compounds 6c/6d, which
indicated that the oxidation of sulfur atoms at benzo[d]
isothiazol-3(2H)- one has little effect on the AChE inhibi-
tion. On the other hand, this series of compounds exhibit
weak RatBuChE inhibitory activity with inhibition rates
range from 7.7% to 46.5% at 50 μM, which suggests that
those compounds are relatively high selective AChE inhi-
bitors with little associated peripheral side effects.
Biological evaluation
In vitro inhibition of AChE and BuChE
The modified Ellman method [34, 35] was applied to
measure the AChE and BuChE inhibitory activities of the
target compounds (6a-j, 13c, 13d), with donepezil and riv-
astigmine used as the positive control compounds. As
shown in Table 1, the IC50 values of EeAChE inhibitory
activities almost range from 1.09 μM to 54.0 μM. The
intermediate 5 is weak in inhibiting AChE (IC50 > 100 μM),
while the AChE inhibitory activities of target compounds
with Mannich bases extensively increase, which means the
introduction of Mannish base fragments into target com-
pounds is critically important to AChE inhibitory effect.
Among the target compounds, compound 6d displays the
best AChE inhibition (IC50 = 1.09 μM), indicating that the
piperidine fragment is favorable to AChE inhibitory activ-
ity. It also can be observed that the introduction of oxygen
Kinetic study for the inhibition of AChE
To further clarify the inhibitory mechanism of target com-
pounds with AChE, compound 6d, the best AChE inhibitor
among these Mannich base derivatives, was chosen for the
kinetic study with EeAChE [37]. In the graph of the reci-
procal Lineweaver–Burk plots (Fig. 2), the slope (decreased
Vmax) and the longitudinal intercept (higher Km) increase with
the concentrations of the compounds increase, and these
straight lines intersect at the third quadrant. The above results
indicate that compound 6d is a mixed-type inhibitor of AChE,
binding to the CAS and PAS of AChE simultaneously.

Medicinal Chemistry Research
Table 1 AChE and BuChE
inhibitory activities, and oxygen
radical absorbance capacity
(ORAC, Trolox equivalents) of
the target compounds and
control compounds
Comp.
5
NR1R2
/
IC50 (μM) SD EeAChE
% Inhibition RatBuChEa
ORACb
>100
12.0 0.28%
0.96 0.03
6a
6b
6c
6d
6e
6f
13.70 0.03
>100
14.6 0.30%
8.0 0.17%
22.7 0.38%
22.8 0.35%
16.2 0.27%
7.7 0.15%
14.1 0.29%
25.0 0.40%
24.2 0.38%
1.31 0.05
1.31 0.06
1.74 0.06
1.68 0.07
1.41 0.07
2.03 0.09
1.65 0.08
1.49 0.02
0.97 0.03
5.70 0.04
1.09 0.02
17.70 0.05
7.80 0.04
54.00 0.10
22.80 0.05
37.90 0.06
6g
6h
6i
6j
16.10 0.05
5.50 0.04
46.5 0.80%
23.2 0.41%
1.11 0.07
2.44 0.13
13c
13d
2.01 0.01
0.023 0.001
9.94 0.83
22.5 0.50%
2.68 0.12
not tested
not tested
Donepezil
Rivastigmine
/
/
IC50 = 21.4 0.097 μM
IC50 = 2.86 0.22 μM
aBuChE from rat serum was used and tested compounds were used at 50 μM.
bThe mean SD of the three independent experiments. Data are expressed as μM of Trolox equivalent/μM of
tested compound.
Molecular modeling study of AChE
docking results suggest that compounds 6d and 13d can
simultaneously bind to the CAS and PAS of AChE tightly,
which means they are mixed-type AChE inhibitors. Speci-
fically, in the complex of compound 6d and TcAChE, the
5,6-dimethoxybenzo [d]isothiazol-3(2H)-one combines
with the PAS of AChE via the hydrophobic interaction with
To investigate the potential binding modes of promising
compounds 6d and 13d with TcAChE structure (PDB code:
1EVE), the molecular docking study was performed using
AutoDock 4.2 package [38]. As shown in Fig. 3, the

Medicinal Chemistry Research
Ile287, Gly335, Phe288, and the benzene (A ring, Fig. 1)
interacts with Phe330 through π–π stacking. Besides, the
terminal N-benzylpiperidine group of 6d occupies the CAS
of AChE via the hydrophobic interaction with Trp279,
Trp84, Tyr70. And the nitrogen atom of piperidine and
3-hydroxyl interact with Ser122 and Asp72 through the
hydrogen bond, respectively.
Similarly, in the 13d-TcAChE complex, the 5,6-dime-
thoxybenzo[d]isothiazol-3(2H)-one 1,1-dioxide binds to the
PAS of AChE via establishing a hydrophobic interaction
with amino acid residues, such as Gly335, Ile287, Phe290,
Trp279. Besides, the benzene (A ring, Fig. 1) interacts with
Phe331 through π–π stacking, and the oxygen atom of the
sulfone interacts with Tyr334 and Tyr121 through an
intermolecular hydrogen bond. As for the binding mode of
the terminal N-benzylpiperidine moiety of 13d with AChE,
it mainly interacts with Trp84, Tyr70 through hydrophobic
interaction, almost the same as 6d. Besides, the binding
energy of 6d/13d combined with AChE is −11.13 kcal/mol
and −11.16 kcal/mol, respectively. Moreover, the AChE
binding energy of compound 6i (Supplementary Material
Fig. S1) is −10.26 kcal/mol, higher than that of 6d and 13d,
which may explain its worse AChE inhibitory effect.
Antioxidant activity assay
The antioxidant activities of the target compounds were
determined by the oxygen radical absorbance capacity
assay (ORAC-FL) [39], with 6-hydroxy-2,5,7,8-tetra-
methyl-chroman-2-carboxylic acid (Trolox) used as a
standard and the ORAC-FL values characterized as the
Trolox equivalents. As indicated in Table 1, all compounds
exhibit good antioxidant activity with the ORAC-FL values
of 0.97–2.68 Trolox equivalents, which are more active
than the N-alkyl benzylamine derivatives (Fig. 1a), the vast
majority of ORAC-FL values varies in 0.16–0.91 Trolox
equivalents, except for two compounds with 2.14, 1.92
Trolox equivalents [25]. It can be observed that the inter-
mediate 5 displays moderate antioxidant potency (0.96
Trolox equivalents), while the antioxidant capacities of the
target compound endowed with phenolic hydroxyl Man-
nich base fragment dramatically increase, which is
Fig. 2 Kinetic study on the mechanism of EeAChE inhibition by
compound 6d. Overlaid Lineweaver–Burk reciprocal plots of AChE
initial velocity at the substrate concentrations (0, 1.0, 2.5, 5.0 μM) are
shown. Lines were derived from a weighted least-squares analysis of
data points
Fig. 3 Docking models of the complexes of 6d and 13d with TcAChE
(PDB code: 1EVE). (A) 6d-TcAChE complex. (B) 13d-TcAChE
complex. Compounds (colored by atom type) interacting with residues
in the binding site of TcAChE, highlighting the protein residues that
participate in the main interactions with the inhibitor

Medicinal Chemistry Research
consistent with our design strategy. The antioxidant activ-
ities of the compounds with benzylamines in Mannich
moiety are generally poorer than the compounds with ali-
phatic amines, and the target compounds with cyclic
alkanes
and 13d bearing the piperidine group possess moderate
self-induced Aβ1–42 aggregation inhibitory activities (25.0%
and 29.9% at 25 µM, respectively).
Metal-chelating studies
(6c-j) are slightly more active than those with chain alkanes
(6a, 6b). Interestingly, after oxidizing sulfur atoms to sul-
fone at benzo[d]isothiazol, the antioxidant activities of the
obtained compounds 13c and 13d significantly increase.
Within the series, compound 13d displays the best anti-
oxidant activity (2.68 Trolox equivalents), which may be
due to the free radical scavenging effect of the phenolic
hydroxyl Mannich base fragments and the potential anti-
oxidant activity of saccharin [40].
Based on the above biological evaluating results, 6d is the
most promising compound with excellent multiple functions.
To measure the metal-chelating ability of 6d, such as Cu2+
,
Fe2+, Al3+, Zn2+, the UV–Vis spectroscopy assay was
performed [43]. First, we measured metal ions alone, and
there was no absorption peak and also no significant dif-
ference in absorbance compared with MeOH (Supplemen-
tary Material Fig. S3). As depicted in Fig. 5, compound 6d
had an obvious absorption peak at 240 nm. After the addi-
tion of CuCl2 and FeSO4, the maximum absorption of
compound 6d at 240 nm dramatically increases, and the
maximum absorption wavelengths shift from 240 nm to
238 nm and 235 nm, respectively. However, there is little
change in the curve after the addition of AlCl3 and ZnCl2
compared with the blank group. The metal-chelating study
results indicated that compound 6d can selectively chelate
with Cu2+ and Fe2+, and the UV–Vis spectra of compound
13d showed the similar phenomenon (Supplementary
Material Fig. S4). Whereas lead compounds (Fig. 1A)
without α-phenolic hydroxyl Mannich base fragment had no
metal-chelating property [25], which means the designing
goal was achieved.
Inhibition of self-induced Aβ1–42 aggregation
The classic thioflavin T (ThT) fluorescence method was
used to evaluate the inhibitory activity of self-induced
Aβ1–42 aggregation among the target compounds, with
curcumin as a control compound and the test concentration
at 25 μM [41]. The data summarized in Fig. 4 showed that
the designed target compounds are endowed with weak to
moderate inhibition of Aβ1–42 aggregation (1.1–29.9% at
25 µM) compared with the reference compound curcumin
(41.30% at 25 µM). In general, the compounds with aro-
matic amines in Mannich moiety exhibit more active inhi-
bitory capacity than those bearing the aliphatic amines,
which may be contributed to the aromatic structure is
conducive to the hydrophobic interaction with Aβ1–42 [42].
After the introduction of nitrogen or oxygen atom at the 4-
position of piperidine ring, the inhibition of Aβ1–42 aggre-
gation decreases, which may be due to the decrease of
electron density of nitrogen atom to make it unfavorable to
the combination with Aβ1–42. Particularly, compounds 6d
Neuroprotective activity of H2O2-induced PC12 cell injury
The neuroprotective capacities of the Mannich base deri-
vatives were evaluated by the H2O2-mediated oxidative
damage model of PC12 cell (Fig. 6) [36]. It is manifested
that the survival rate of PC12 cells treated with 150 μM
Fig. 5 UV–Vis spectra of compound 6d (37.5 μM in methanol) alone
or in the presence of CuCl2, ZnCl2, FeSO4 or AlCl3 (37.5 μM in
methanol)
Fig. 4 In vitro inhibition of self-induced Aβ1–42 aggregation of target
compounds and control compound

Medicinal Chemistry Research
Table 2 Permeability Pe (×10−6 cm/s) in the PAMPA-BBB assay of
the selected compounds 6d and 13d and their predictive penetration in
the CNS
Compounda
Pe (×10−6 cm/s)b
Prediction
6d
13.5 0.21
3.5 0.06
CNS+
CNS+
13d
aCompounds 6d and 13d were dissolved in DMSO at 5 mg/mL and
diluted with PBS/EtOH (70:30). The final concentration of the
compounds was 100 μg/mL.
bValues are expressed as the mean SD of three independent
experiments.
Fig. 6 Effects of donepezil and compounds 6d, 13d on H2O2-induced
PC12 cell injury (n = 3, mean SD; ###p < 0.0001 versus control,
*p < 0.05 versus model, **p < 0.01 versus model)
throughput, low cost, and reproducibility in predicting
passive blood-brain barrier penetration [47]. Thus, a parallel
artificial membrane permeation assay of the blood-brain
barrier (PAMPA-BBB) model was performed to evaluate
the BBB permeability of the representative compounds 6d
and 13d.
The assay validation was carried out by comparing the
experimental and reported permeability values of eleven
commercial drugs (Supplementary Material Table S2.1 and
Fig. S2), thereby we concluded that the compounds with
Pe value superior to 3.44 × 10−6 cm/s could cross the BBB
(Supplementary Material Table S2.2). As indicated in Table 2,
the permeability values of compounds 6d and 13d are
13.5 × 10−6 cm/s and 3.5 × 10−6 cm/s, respectively, both can
reach the CNS through the blood-brain barrier.
H2O2 (model group) was 42.11%, indicating that H2O2 can
induce PC12 cell injury significantly. When donepezil and
the representative compounds 6d/13d were added at a
concentration of 1.0 μM, the viability of PC12 was 47.60%,
60.47%, and 65.65%. Moreover, the viability of PC12
increased to 49.85%, 73.42%, 83.56%, respectively at the
concentration of 10.0 μM. The above results demonstrated
that the test compounds 6d and 13d possess a better pro-
tective effect on the oxidative injury of PC12 cells induced
by H2O2 compared with donepezil. Interestingly, compound
13d bearing sulfone group exhibits better neuroprotective
effect than compound 6d with the sulfur atom, which is
consistent with the results of ORAC-FL assay.
In vivo step-down passive avoidance test
In vitro blood-brain barrier permeation assay
To further evaluate the effects of compounds 6d and 13d
on ameliorating memory impairment induced by scopo-
lamine, the modified step-down passive avoidance test was
carried out [22, 23], with donepezil as the positive drug.
Obviously, the latency time of the scopolamine-treated
group was significantly (p < 0.05) shorter than that of the
blank group, and the number of shocks dramatically
increased (p < 0.01), which means the mice models were
established successfully. As depicted in Fig. 7, intragastric
treatment of 6d (2.2, 6.5, 19.5 mg/kg) and 13d (2.3, 7.0,
21.0 mg/kg) increased the latency time and decreased
the electric shocking. Moreover, the effects of 6d and 13d
showed a dose-dependent manner. In particular, the
latency times of the groups of high dose test compounds
are just slightly lower than the positive compound done-
pezil (5.0 mg/kg), which means that compounds 6d and
13d probably display comparable effects with donepezil.
Overall, the representative compounds 6d and 13d can
ameliorate the memory impairment and improve the cog-
nitive deficit in the scopolamine-treated mice to some
extent, which may be attributed to their potent inhibition
of AChE.
Good brain permeability is a prerequisite for anti-AD drugs
to target the central nervous system (CNS) [22]. It has been
demonstrated that the parallel artificial membrane perme-
ability assay (PAMPA) can identify compounds appro-
priately as either BBB permeable (CNS+) or non-
permeable (CNS−) [44], and the PAMPA models can be
divided into four species. First, the PAMPA-DS (double
sink) model is suitable for testing gastrointestinal absorption
data in an environment of different pH. And the relation-
ships between permeability and Log BB obtained by the
PAMPA-DS model and the PAMPA-BBB-UWL (unstirred
water layer) method were characterized by very poor cor-
relations [45]. Thus, both of them cannot meet the needs
of the BBB permeability test in this work. Besides, the
PAMPA-BLM model using a black lipid membrane and
PAMPA-BBB method with a porcine polar brain lipid, both
can predict the passive diffusion of compounds reasonably
well based on the established classification ranges, but the
latter is more accurate [46]. More importantly, the PAMPA-
BBB assay possess the advantages of high success, high

Medicinal Chemistry Research
Experimental section
Chemistry
All reagents and solvents, unless otherwise noted, were
purchased from commercial sources and used without fur-
ther purification. All reactions progress was monitored by
thin-layer chromatography (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). Melting points of solid
compounds were measured on the YRT-3 melting-point
apparatus (China) and the thermometer was not calibrated.
The nuclear magnetic resonance spectrums were recorded in
CDCl3 with a Bruker AC-E400 nuclear magnetic resonance
instrument at 25 °C with TMS as an internal standard. The
mass spectra data were recorded with Agilent-6210 TOF
LC-MS spectrometer. The elemental analysis was per-
formed with Vario EL Cube.
Fig. 7 Effects of compounds 6d and 13d on scopolamine-induced
memory deficit in the step-down passive avoidance test. Compound 6d
(2.2, 6.5, and 19.5 mg/kg, i.g.), 13d (2.3, 7.0, and 21.0 mg/kg, i.g.) or
donepezil (5.0 mg/kg, i.g.) were given to the mice. 30 min later, sco-
polamine (3.0 mg/kg, i.p.) was given, and the passive avoidance
training was conducted after another 30 min. Twenty-four hours after
the training trial, donepezil/6d/13d were administrated intragastrically
to the mice again, and the mice were placed on the platform to perform
the test trail. Values are expressed as the mean SEM (n = 8).
#p < 0.05 versus normal group. *p < 0.05 and **p < 0.01 versus
scopolamine-treated control group
Synthesis of 3-hydroxybenzaldehyde oxime (2) [29, 30]
Conclusion
m-Hydroxybenzaldehyde (2.00 g, 16.38 mmol), hydro-
xylamine hydrochloride (1.37 g, 19.65 mmol) and sodium
acetate (1.74 g, 21.29 mmol) were added to 20 mL etha-
nol in sequence, and kept stirring for 6 h at room tem-
perature. Then concentrated under reduced pressure and
the residue was extracted with anhydrous ether (15 mL)
three times, washed with saturated brine and dried with
anhydrous Na2SO4, finally filtered to provide the filtrate.
The filtrate was evaporated under reduced pressure to
remove the solvent and obtain product 2 as a colorless oil,
yield 96.3%.
Based on the “MTDLs” strategy, a series of novel
2-(3-hydroxybenzyl)-benzo[d]isothiazol-3(2H)-one Man-
nich base derivatives were designed, synthesized, and
evaluated with various biological activities. The phar-
macological results showed that most of compounds
possessed potent antioxidant potency and selective AChE
inhibition. Among them, the representative compounds
6d and 13d are significant selective AChE inhibitors
(IC50 = 1.09 μM and 2.01 μM, respectively), which can
bind to both CAS and PAS of AChE simultaneously.
They also exhibited moderate inhibitory effects on self-
induced Aβ1–42 aggregation (25.0% and 29.9% at 25 µM,
respectively). Besides, compounds 6d and 13d displayed
significant antioxidant capacity, excellent neuroprotective
effect on H2O2-induced PC-12 cell injury and BBB per-
meability. Besides, compound 6d and 13d could selec-
tively chelate with Cu2+ and Fe2+ due to the introduction
of α-phenolic hydroxyl Mannich base fragment, which
validates our design strategy. Moreover, the in vivo study
indicated that mice treated with 6d or 13d in high dose
group significantly reversed scopolamine-induced mem-
ory deficit, which was almost as good as donepezil
(5.0 mg/kg, i.g.) in the step-down passive avoidance test.
Based on the above assays, compounds 6d and 13d could
be envisioned as multifunctional lead compounds for the
development of drugs for the prevention and treatment
of AD.
Synthesis of m-hydroxybenzylamine (3)
Compound 2 (2.00 g, 14.58 mmol) was dissolved in 30 mL
ethanol, then 10% Pd–C (0.20 g) was added in the solution
and kept stirred for 10 h at room temperature. After com-
pletion of the reaction as indicated by TLC, the suspension
was filtered to obtain the filtrate, then concentrated under
reduced pressure to obtain m-hydroxybenzylamine (3) as an
off-white solid, yield 95.0%.
Synthesis of 2-(3-hydroxybenzyl)-5,6-dimethoxybenzo[d]
isothiazol-3(2H)-one (5)
To a solution of compound 4 (2.00 g, 7.49 mmol) in 20 mL
dichloromethane was added dropwise m-hydroxybenzylamine
(0.84 g, 6.82 mmol) in 10 mL dichloromethane, then pyridine

Medicinal Chemistry Research
(1.0 mL, 13.60 mmol) was added to the mixture and kept
stirring at room temperature for 48 h. After the reaction
was completed, the solution was concentrated under
reduced pressure. To the residue water (15 mL) was added,
extracted with dichloromethane (20 mL) three times, washed
with brine, dried over anhydrous Na2SO4, filtered, con-
centrated under reduced pressure, and the crude was purified
by silica gel column chromatography (petroleum ether/acet-
one/acetic acid = 3:1:0.03 v/v) to produce the product 5 as a
white solid, yield 65.0%, melting point was higher than
1H, Ar–H), 6.76 (s, 1H, Ar–H), 6.71 (d, J = 7.6 Hz, 1H,
Ar–H), 4.92 (s, 2H, NCH2Ar), 3.90 (s, 6H, 2× OCH3), 3.62
(s, 2H, NCH2Ar), 2.69 (m, 4H, 2× NCH2), 1.15 (m, 6H, 2×
CH3); ESI-MS m/z: 403.2 [M + H]+; Anal. Calcd. for
C21H26N2O4S: C, 62.66; H, 6.51; N, 6.96; S, 7.97. Found:
C, 62.75; H, 6.55; N, 6.90; S, 8.12.
2-(3-hydroxy-4-(pyrrolidin-1-ylmethyl)benzyl)-5,6-
dimethoxybenzo[d]isothiazol-3(2H)-one (6c) Synthesized
from paraformaldehyde, tetrahydropyrrole and compound 5
via general procedure. Purified to produce compound 6c,
yield 49.2%, mp 90.2–91.7 °C; 1H NMR (400 MHz,
CDCl3) δ 7.38 (s, 1H, Ar–H), 7.10 (d, J = 7.6 Hz, 1H,
Ar–H), 6.88 (s, 2H, Ar–H), 6.77 (d, J = 7.6 Hz, 1H, Ar–H),
4.95 (s, 2H, NCH2Ar), 3.94 (s, 5H, OCH3, NCH2Ar), 3.90
(s, 3H, OCH3), 2.82 (brs, 4H, 2× NCH2), 1.91 (brs, 4H, 2×
CH2); 13C NMR (100 MHz, CDCl3) δ 165.4, 157.9, 153.6,
148.6, 137.8, 134.0, 129.7, 120.7, 119.0, 116.7, 116.0,
106.9, 101.5, 56.6, 56.3, 56.2, 53.2 (2 C), 47.3, 23.4(2 C);
ESI-MS m/z: 401.1 [M + H]+; Anal. Calcd. for
C21H24N2O4S: C, 62.98; H, 6.04; N, 6.99; S, 8.01. Found:
C, 62.81; H, 6.02; N, 7.09; S, 8.18.
1
230 °C. H NMR (400 MHz, CDCl3) δ 7.24(s, 1H, Ar–H),
7.21 (d, J = 8.0 Hz, 1H, Ar–H), 7.18–7.06 (brs, 1H, Ar–H),
6.94 (s, 1H, Ar–H), 6.86 (d, J = 8.0 Hz, 1H, Ar–H), 6.78 (s,
1H, Ar–H), 4.99 (s, 2H, NCH2Ar), 3.92 (s, 3H, OCH3), 3.72
(s, 3H, OCH3).
Synthesis of 2-(3-hydroxybenzyl)benzo[d]isothiazol-3(2H)-
one Mannich base derivatives (6a-j)
Different secondary amines a-j (0.77 mmol) and paraf-
ormaldehyde (0.069 g, 0.77 mmol) were dissolved in etha-
nol, then slowly heated to reflux and kept stirring for 1 h.
Compound 5 (0.12 g, 0.39 mmol) was added to the solution
and continued refluxing for 24–48 h. After reaction was
completed, the solvent was removed and the water was
added. The mixture was extracted with 10 mL dichlor-
omethane three times. The organic phase was separated,
washed with brine, dried on anhydrous Na2SO4, and eva-
porated under reduced pressure. The pure target products
(6a-j) were obtained by silica gel column chromatography.
2-(3-hydroxy-4-(piperidin-1-ylmethyl)benzyl)-5,6-dimethox-
ybenzo[d]isothiazol-3(2H)-one (6d) Synthesized from par-
aformaldehyde, piperidine and compound 5 via general
procedure. Purified to produce compound 6d, yield 53.8%,
1
mp 95.3–96.1 °C; H NMR (400 MHz, CDCl3) δ 7.44 (s,
1H, Ar–H), 6.96 (d, J = 7.6 Hz, 1H, Ar–H), 6.88 (s, 1H,
Ar–H), 6.80 (s, 1H, Ar–H), 6.75 (d, J = 7.6 Hz, 1H, Ar–H),
4.95 (s, 2H, NCH2Ar), 3.95 (s, 3H, OCH3), 3.94 (s, 3H,
OCH3), 3.69 (s, 2H, NCH2Ar), 2.64–2.54 (brs, 4H, 2×
NCH2), 1.67–1.49 (m, 4H, 2× CH2), 1.25 (brs, 2H, CH2);
13C NMR (100 MHz, CDCl3) δ 165.3, 158.2, 153.5, 148.6,
137.2, 134.0, 129.2, 121.0, 118.7, 116.9, 115.9, 107.0,
101.4, 61.2, 56.3, 56.2, 53.7 (2C), 47.3, 25.5 (2C), 23.7;
ESI-MS m/z: 415.2 [M + H]+; Anal. Calcd. for
C22H26N2O4S: C, 63.75; H, 6.32; N, 6.76; S, 7.74. Found:
C, 63.82; H, 6.30; N, 6.90; S, 7.63.
2-(4-((dimethylamino)methyl)-3-hydroxybenzyl)-5,6-
dimethoxybenzo[d]isothiazol-3(2H)-one (6a) Synthesized
from paraformaldehyde, dimethylamine hydrochloride and
compound 5 via general procedure. Purified to give com-
pound 6a as a white solid, yield 63.0%, mp 83.2–84.5 °C;
1H NMR (400 MHz, CDCl3) δ 7.44 (s, 1H, Ar–H), 6.96 (d,
J = 7.6 Hz, 1H, Ar–H), 6.87 (s, 1H, Ar–H), 6.80 (s, 1H,
Ar–H), 6.75 (d, J = 7.6 Hz, 1H, Ar–H), 4.96 (s, 2H,
NCH2Ar), 3.94 (s, 6H, 2× OCH3), 3.66 (s, 2H, NCH2Ar),
2.35 (s, 6H, 2× NCH3); 13C NMR (100 MHz, CDCl3) δ
165.4, 158.3, 153.5, 148.6, 137.3, 134.0, 129.0, 121.3,
118.7, 116.9, 115.9, 107.0, 101.4, 62.1, 56.3, 56.2, 47.3,
44.3 (2C); ESI-MS m/z 375.2 [M + H]+; Anal. Calcd. for
C19H22N2O4S: C, 60.94; H, 5.92; N, 7.48; S, 8.56. Found:
C, 60.80; H, 6.03; N, 7.42; S, 8.38.
2-(3-hydroxy-4-(morpholinomethyl)benzyl)-5,6-dimethoxy-
benzo[d]isothiazol-3(2H)-one (6e) Synthesized from par-
aformaldehyde, morpholine and compound 5 via general
procedure. Purified to produce compound 6e, yield 35.7%,
1
mp 98.5–99.8 °C; H NMR (400 MHz, CDCl3) δ 7.44 (s,
1H, Ar–H), 6.98 (d, J = 8.0 Hz, 1H, Ar–H), 6.88 (s, 1H,
Ar–H), 6.80 (s, 1H, Ar–H), 6.77 (d, J = 8.0 Hz, 1H,
Ar–H), 4.98 (s, 2H, NCH2Ar), 3.94 (s, 3H, OCH3), 3.91
(s, 3H, OCH3), 3.75 (brs, 4H, 2× OCH2), 3.64 (s, 2H,
NCH2Ar), 2.58 (brs, 4H, 2× NCH2); ESI-MS m/z: 417.1
[M + H]+; Anal. Calcd. for C21H24N2O5S: C, 60.56; H,
5.81; N, 6.73; S, 7.70. Found: C, 60.40; H, 5.87; N, 6.65;
S, 7.82.
2-(4-((diethylamino)methyl)-3-hydroxybenzyl)-5,6-
dimethoxybenzo[d]isothiazol-3(2H)-one (6b) Synthesized
from paraformaldehyde, diethylamine, and compound 5 via
general procedure. Purified to produce compound 6b, yield
1
42.4%, mp 92.3–94.7 °C; H NMR (400 MHz, CDCl3) δ
7.40 (s, 1H, Ar–H), 6.92 (d, J = 7.6 Hz, 1H, Ar–H), 6.81 (s,

Medicinal Chemistry Research
2-(3-hydroxy-4-((4-methylpiperazin-1-yl)methyl)benzyl)-
5,6-dimethoxybenzo[d]isothiazol- 3(2H)-one (6f) Synthe-
sized from paraformaldehyde, N-methylpiperazine and
compound 5 via general procedure. Purified to produce
general procedure. Purified to produce compound 6i,
yield 76.2%, mp 156.8–158.0 °C; H NMR (400 MHz,
1
CDCl3) δ 7.44 (s, 1H, Ar–H), 7.29 (t, J = 7.2 Hz, 1H,
Ar–H), 7.21 (d, J = 7.2 Hz, 1H, Ar–H), 6.97 (d, J =
7.6 Hz, 1H, Ar–H), 6.93–6.88 (m, 2H, Ar–H), 6.86 (s,
1H, Ar–H), 6.80 (s, 1H, Ar–H), 6.74 (d, J = 7.6 Hz, 1H,
Ar–H), 4.95 (s, 2H, NCH2Ar), 3.94 (s, 3H, OCH3), 3.93
(s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.75 (s, 2H,
NCH2Ar), 3.68 (s, 2H, NCH2Ar), 2.21 (s, 3H, NCH3);
13C NMR (100 MHz, CDCl3) δ 165.3, 158.2, 158.1,
153.5, 148.5, 137.1, 134.0, 131.4, 129.4, 129.0, 124.4,
122.0, 120.2, 118.7, 117.0, 115.9, 110.5, 107.0, 101.4,
60.0, 56.9, 56.3, 56.2, 55.1, 47.3, 40.8; ESI-MS m/z:
481.1 [M + H]+; Anal. Calcd. for C26H28N2O5S: C,
64.98; H, 5.87; N, 5.83; S, 6.67. Found: C, 65.12; H,
5.80; N, 5.91; S, 6.56.
1
compound 6f, yield 71.9%, mp 99.2–100.8 °C; H NMR
(400 MHz, CDCl3) δ 7.45 (s, 1H, Ar–H), 6.94 (d, J =
7.2 Hz, 1H, Ar–H), 6.87 (s, 1H, Ar–H), 6.77 (s, 1H, Ar–H),
6.74 (d, J = 7.2 Hz, 1H, Ar–H), 4.95 (s, 2H, NCH2Ar), 3.95
(s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.68 (s, 2H, NCH2Ar),
2.62–2.17 (m, 8H, 4× NCH2), 2.30 (s, 3H, NCH3); ESI-MS
m/z: 430.0 [M + H]+; Anal. Calcd. for C22H27N3O4S: C,
61.52; H, 6.34; N, 9.78; S, 7.47. Found: C, 61.65; H, 6.30;
N, 9.95; S, 7.36.
2-(4-((4-ethylpiperazin-1-yl)methyl)-3-hydroxybenzyl)-5,6-
dimethoxybenzo[d]isothiazol- 3(2H)-one (6g) Synthesized
from paraformaldehyde, N-ethylpiperazine and com-
pound 5 via general procedure. Purified to produce
compound 6g, yield 78.2%, mp 123.6–124.9 °C; 1H NMR
(400 MHz, CDCl3) δ 7.45 (s, 1H, Ar–H), 6.96 (d, J =
7.6 Hz, 1H, Ar–H), 6.88 (s, 1H, Ar–H), 6.78 (s, 1H,
Ar–H), 6.76 (d, J = 7.6 Hz, 1H, Ar–H), 4.96 (s, 2H,
NCH2Ar), 3.96 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.70
(s, 2H, NCH2Ar), 2.64 (brs, 6H, 3× NCH2), 2.50–2.40 (m,
4H, 2× NCH2), 1.11 (t, J = 7.2 Hz, 3H, CH3); 13C NMR
(100 MHz, CDCl3) δ 165.3, 157.9, 153.5, 148.5, 137.1,
133.9, 128.9, 120.9, 118.8, 116.8, 115.7, 107.0, 101.4,
60.9, 56.2, 56.1, 52.3(2C), 52.2(2C), 52.0, 47.2, 11.7;
ESI-MS m/z: 444.0 [M + H]+; Anal. Calcd. for
C23H29N3O4S: C, 62.28; H, 6.59; N, 9.47; S, 7.23. Found:
C, 62.15; H, 6.60; N, 9.59; S, 7.30.
2-(4-(((2-(dimethylamino)benzyl)(ethyl)amino)methyl)-3-
hydroxybenzyl)-5,6-dimethoxy
benzo[d]isothiazol-3(2H)-
one (6j) Synthesized from paraformaldehyde, N,N-
dimethyl-2-((ethylamino)methyl)aniline and compound
5 via general procedure. Purified to produce compound
6j, yield 75.3%, mp 168.1–169.7 °C; 1H NMR
(400 MHz, CDCl3) δ 7.45 (s, 1H, Ar–H), 7.34 (d, J =
7.2 Hz, 1H, Ar–H), 7.24 (t, J = 7.2 Hz, 1H, Ar–H), 7.14
(d, J = 7.2 Hz, 1H, Ar–H), 7.07 (t, J = 7.2 Hz, 1H,
Ar–H), 6.95 (d, J = 7.6 Hz, 1H, Ar–H), 6.87 (s, 1H,
Ar–H), 6.79 (s, 1H, Ar–H), 6.73 (d, J = 7.6 Hz, 1H,
Ar–H), 4.94 (s, 2H, NCH2Ar), 3.95 (s, 3H, OCH3), 3.93
(s, 3H, OCH3), 3.76 (s, 2H, NCH2Ar), 3.74 (s, 2H,
NCH2Ar), 2.65 (s, 6H, 2× NCH3), 2.53 (q, J = 7.2 Hz,
2H, NCH2), 1.11 (t, J = 7.2 Hz, 3H, CH3); 13C NMR
(100 MHz, CDCl3) δ 165.2, 158.1, 153.4, 153.3, 148.4,
136.8, 133.8, 131.4, 130.5, 128.9, 128.2, 123.5, 122.0,
119.6, 118.5, 116.8, 115.6, 106.8, 101.4, 56.6, 56.2,
56.1, 52.6, 47.2, 46.5, 45.1 (2 C), 10.6; ESI-MS m/z:
508.2 [M + H]+; Anal. Calcd. for C28H33N3O4S: C,
66.25; H, 6.55; N, 8.28; S, 6.32. Found: C, 66.10; H,
6.60; N, 8.42; S, 6.22.
2-(4-((4-benzylpiperazin-1-yl)methyl)-3-hydroxybenzyl)-5,6-
dimethoxybenzo[d]isothiazol- 3(2H)-one (6h) Synthesized
from paraformaldehyde, N-benzylpiperazine and compound
5 via general procedure. Purified to produce compound 6h,
yield 69.3%, mp 138.6–139.1 °C; 1H NMR (400 MHz,
CDCl3) δ 7.45 (s, 1H, Ar–H), 7.34–7.25 (m, 5H, Ar–H),
6.94 (d, J = 7.6 Hz, 1H, Ar–H), 6.88 (s, 1H, Ar–H), 6.77 (s,
1H, Ar–H), 6.75 (d, J = 7.6 Hz, 1H, Ar–H), 4.95 (s, 2H,
NCH2Ar), 3.95 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.69 (s,
2H, NCH2Ar), 3.54 (s, 2H, NCH2Ar), 2.58 (brs, 8H, 4×
NCH2); 13C NMR (100 MHz, CDCl3) δ 165.3, 158.0, 153.5,
148.5, 137.3, 137.2, 133.9, 129.2 (2C), 129.0, 128.3 (2C),
127.2, 120.9, 118.8, 116.9, 115.7, 107.0, 101.4, 62.7,60.9,
56.3, 56.2, 53.7 (2C), 52.3(2C), 47.3; ESI-MS m/z: 506.2
[M + H]+; Anal. Calcd. for C28H31N3O4S: C, 66.51; H, 6.18;
N, 8.31; S, 6.34. Found: C, 66.40; H, 6.25; N, 8.22; S, 6.20.
Synthesis of 3-(methoxymethoxy)benzaldehyde (7)
Sixty percent NaH (0.55 g, 13.70 mmol), m-hydro-
xybenzaldehyde (0.84 g, 6.84 mmol) were added to 10 mL
THF and kept stirring for 1 h at room temperature. Chlor-
omethyl methyl ether (1.10 g, 13.70 mmol) was dropped
slowly and continued stirring for 4 h. After the reaction was
completed, the mixture was quenched with 20 mL water and
extracted with ethyl acetate (20 mL) three times. The
organic layer was washed with 5% NaOH and brine, dried
on anhydrous Na2SO4, and the solvent evaporated under
reduced pressure to obtain product 7 as a yellow oil,
yield 85.1%.
2-(3-hydroxy-4-(((2-methoxybenzyl)(methyl)amino)methyl)
benzyl)-5,6-dimethoxybenzo[d] isothiazol-3(2H)-one (6i)
Synthesized from paraformaldehyde, 1-(2-methox-
yphenyl)-N-methylmethanamine and compound 5 via

Medicinal Chemistry Research
Synthesis of (3-(methoxymethoxy)phenyl)methanol (8)
the compound 12 as a white solid, yield 78.0%, mp
100.3–102.1 °C.
NaBH4 (0.21 g, 3.00 mmol) was added slowly to the solu-
tion of compound 7 (0.90 g, 5.40 mmol) in 10 mL CH3OH
under an ice bath. The suspension was kept stirring at room
temperature for 2 h until TLC showed the disappearance of
the starting material. The suspension was quenched by 10%
HCl, concentrated under reduced pressure, and extracted
with dichloromethane (20 mL) three times. The extract was
washed with brine, dried over anhydrous Na2SO4, filtered,
concentrated to produce the compound 8 as light-yellow oil,
yield 90.3%.
Synthesis of 2-(3-hydroxybenzyl)-5,6-dimethoxybenzo[d]
isothiazol-3(2H)-one-1,1-dioxide Mannich base derivatives
(13c, 13d)
Different secondary amines (c or d) (0.77 mmol) and
paraformaldehyde (0.069 g, 0.77 mmol) were dissolved in
ethanol, slowly heated to reflux for 1 h under argon pro-
tection. Compound 12 (0.14 g, 0.39 mmol) was added to
the solution and continued refluxing for 24–48 h. Then the
solvent was removed under reduced pressure and the
residue was diluted with 10 mL water, extracted with
dichloromethane (10 mL) three times, washed with brine.
After drying over Na2SO4, dichloromethane was removed,
and the residue was purified by silica gel column
chromatography.
Synthesis of 1-(bromomethyl)-3-(methoxymethoxy)benzene
(9)
To a stirring solution of compound 8 (0.50 g, 2.97 mmol) in
5 mL dry dichloromethane were added Ph3P (1.67 g,
4.46 mmol) and NBS (0.79 g, 4.46 mmol) at 0 °C, then kept
stirring at room temperature for 30 min. After completion of
the reaction, 15 mL water was added and the mixture was
extracted with dichloromethane (15 mL) three times. The
organic layer was washed with 10% NaOH and brine
sequentially, dried over anhydrous Na2SO4, filtered, and
concentrated to obtain the product 9 as a brown solid,
yield 81.2%.
2-(3-hydroxy-4-(pyrrolidin-1-ylmethyl)benzyl)-5,6-dimethoxy-
benzo[d] isothiazol-3(2H)-one-1,1-dioxide (13c) Synthe-
sized from paraformaldehyde, tetrahydropyrrole and
compound 12 via general procedure. Purified to produce
compound 13c as a white solid, yield 59.6%, mp
1
110.2–112.3 °C; H NMR (400 MHz, CDCl3) δ 7.41 (s,
1H, Ar–H), 7.31 (s, 1H, Ar–H), 7.02 (d, J = 8.0 Hz, 1H,
Ar–H), 6.99 (s, 1H, Ar–H), 6.90 (d, J = 8.0 Hz, 1H,
Ar–H), 4.81 (s, 2H, NCH2Ar), 4.02 (s, 3H, OCH3), 4.00
(s, 3H, OCH3), 3.87 (s, 2H, NCH2Ar), 2.77 (brs, 4H, 2×
NCH2), 1.90 (brs, 4H, 2× CH2); HRMS (ESI+) m/z calcd
for C21H25N2O6S 433.1433, found 433.1420 [M + H]+;
Anal. Calcd. for C21H24N2O6S: C, 58.32; H, 5.59; N,
6.48; S, 7.41. Found: C, 58.38; H, 5.52; N, 6.61; S, 7.35.
Synthesis of 5,6-dimethoxy-2-(3-(methoxymethoxy)benzyl)
benzo[d]isothiazol-3(2H)-one-1,1-dioxide (11)
Compound 9 (300 mg, 1.24 mmol), compound 10 (0.43 g,
1.86 mmol), and anhydrous K2CO3 (0.26 g, 1.86 mmol)
were added to 3 mL DMF and stirred at room temperature
for 12 h until the reaction was completed. Then 10 mL
water was poured into the mixture and extracted with ethyl
acetate (10 mL) three times, the obtained organic phase was
washed with brine, dried with anhydrous Na2SO4, filtered.
The concentrated filtrate was purified by silica gel column
chromatography (petroleum ether/acetone = 5:1 v/v) to
obtain the intermediate 11 as a white solid, yield 89.3%, mp
123.1–123.9 °C.
2-(3-hydroxy-4-(piperidin-1-ylmethyl)benzyl)-5,6-dimethox-
ybenzo[d] isothiazol-3(2H)-one-1,1-dioxide (13d) Synthe-
sized from paraformaldehyde, piperidine and compound
12 via general procedure. Purified to produce compound
13d as a white solid, yield 60.7%, mp 120.2–120.9 °C;
1H NMR (400 MHz, CDCl3) δ 7.41 (s, 1H, Ar–H), 7.31
(s, 1H, Ar–H), 6.94–6.92 (m, 2H, Ar–H), 6.87 (d, J =
7.6 Hz, 1H, Ar–H), 4.81 (s, 2H, NCH2Ar), 4.02 (s, 3H,
OCH3), 4.00 (s, 3H, OCH3), 3.64 (s, 2H, NCH2Ar),
2.64–2.49 (brs, 4H, 2× NCH2), 1.63–1.61 (m, 4H, 2×
CH2), 1.49 (brs, 2H, CH2); 13C NMR (100 MHz, CDCl3)
δ 159.1, 158.2, 154.5, 153.9, 135.4, 128.7, 121.3, 120.7,
118.7, 115.9, 109.6, 105.8, 102.5, 61.7, 56.8, 56.7, 53.8
(2 C), 42.3, 25.7 (2 C), 23.9; HRMS (ESI+) m/z calcd
for C22H27N2O6S 447.1590, found 447.1592 [M + H]+;
Anal. Calcd. for C22H26N2O6S: C, 59.18; H, 5.87; N,
6.27; S, 7.18. Found: C, 59.02; H, 5.90; N, 6.35;
S, 7.30.
Synthesis of 2-(3-hydroxybenzyl)-5,6-dimethoxybenzo[d]
isothiazol-3(2H)-one-1,1-dioxide (12)
To a solution of compound 11 (0.30 g, 0.76 mmol) in 2 mL
THF was added 10% HCl (1 mL) and refluxed for 5 h. The
mixture was quenched by 5 mL water and extracted with
ethyl acetate (10 mL) three times. The organic layer was
washed with brine, dried with anhydrous Na2SO4, finally
filtered. The volatile was then removed, and the crude was
purified by silica gel column chromatography to obtain

Medicinal Chemistry Research
Biological evaluation
setting the pretreatment parameters for proteins and small
molecules. The pretreatments for proteins and small mole-
cules include: (1) the polar hydrogen was added to these
amino acid residues in the protein, and Gasteiger charge
was loaded on each atom of the protein molecule; (2) charge
calculation was performed on small molecules, and the
rotate chemical bonds were set. Then the obtained protein
structure was calculated by Autogrid for each type of atom
on the ligand, including desolvation and electrostatics. The
spacing of each grid point was 0.375 Å, and the grid point
was placed at the bottom of the enzyme activity (coordi-
nates x = 2.023, y = 63.295, z = 67.062). The dimensions
of the grid box were set at 50 Å × 50 Å × 50 Å containing
the residues of the active pocket. Lamarck genetic algorithm
(LGA) was applied for docking calculation with the energy
evaluation coefficient was 2,500,000, the random individual
group was 100, and the remaining parameters were as
default. Cluster analysis was applied on the condition that
the root means square (RMS) of the docking results was
within 1.0. Meanwhile, the optimal cluster docking results
of each sample was recorded and analyzed.
In vitro inhibition of AChE and BuChE
The AChE and BuChE inhibition of the target compounds
were assessed by the modified Ellman assay [34, 35], with
donepezil and rivastigmine as the positive drugs, purified
AChE from E. electricus, and BuChE from rat serum.
30 μL acetylthiocholine iodide (1 mmol/L), 40 μL
phosphate-buffered solution (PBS, pH = 8.0), 20 μL solu-
tions of test compounds at different concentrations, 10 μL
EeAChE (the final concentration is 0.05 U/mL) were added
to the 96-well plates in order. The mixture was incubated at
37 °C for 15 min, then 30 μL solution of 5,5′-dithiobis-(2-
nitrobenzoic acid) (DTNB, 0.2%) was added to each well
for color development. A multifunctional Varioskan Flash
Multimode Reader (Thermo Scientific) was used to mea-
sure the absorbance value (OD) of the mixture in each well
at 412 nm. The procedures of the BuChE inhibitory assay
were almost the same as above except PBS (0.1 mmol/L,
pH = 7.4, 40 µL), 25% rat serum supernatant (10 µL) and
butyrylthiocholine iodide (1 mmol/L, 30 µL) were used,
and the absorbance was measured at 405 nm. All samples
were tested in triplicate.
Antioxidant activity assay
To evaluate the antioxidant potency of target compounds
was performed the oxygen radical absorbance capacity-
fluorescein (ORAC-FL) assay [39], To the black 96-well
plates were added 20 μL sample solution (50 μmol/L or
10 μmol/L) and 120 μL fluorescein (FL) solution orderly,
6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid
(Trolox) used as a standard (1–8 μM, final concentration)
and the blank group was the mixture of FL, PBS, 2,2′-
azobis(amidinopropane) dihydrochloride (AAPH). After
incubating at 37 °C for 15 min, 60 μL of AAPH solution
was added and the mixture was immediately placed in the
Varioskan Flash Multimode Reader instrument for con-
tinuous measurement for 1.5 h, detecting the fluorescence
values of the compounds at 485 nm excitation and the
535 nm emission. ORAC-FL values were calculated with
the calculation formula: [(AUCSample−AUCblank)/(AUCTrolox
−AUCblank)] × [(concentration of Trolox/concentration of
sample)], AUC represents the area under the fluorescence
decay curve, the experimental results were expressed as the
equivalent of Trolox. Each experiment was repeated in tri-
ple independently.
Kinetic characterization of AChE inhibition
The enzyme kinetic test was conducted by the established
method [37], with purified AChE from E. electricus
(EeAChE, Sigma-Aldrich Co). 20 μL sample solutions of
different concentrations (DMSO was below 1%), 20 μL
PBS (pH = 8.0), and 10 μL EeAChE solution (0.5 U/mL)
were added to the 96-well plates in order, followed by 30 μL
DTNB solution (0.2%). After the mixture was mixed,
incubated at 37 °C for 15 min. In total, 20 μL thioace-
tylcholine of different concentrations (0.1–0.4 mM, the final
concentration) was added, and the mixed solution was
immediately measured and recorded for the absorbance at
412 nm within 10 min. The Lineweaver-Burk kinetic curves
of the sample on AChE were obtained by plotting 1/V to 1/
S, which can reveal the inhibiting mode between the test
compound and AChE.
Molecular modeling study
Autodock 4.2 program was used to study the interaction
mechanisms of the promising compounds with AChE based
on the previous reports [38]. The crystal structure of the
complex, TcAChE and donepezil (PDB code: 1EVE), was
downloaded from the PDB database, then the protein was
obtained by deleting the ligand molecule donepezil. The
Autodock Tools (ADT; version 1.5.6) was applied for
graphic processing and visualization of docking study by
Inhibition of self-induced Aβ1–42 aggregation
A thioflavin T fluorometric assay was performed to
determine the inhibitory Aβ aggregation activity of the
synthesized compounds [41, 48]. Firstly, 1 mg Aβ1–42 tri-
fluoroacetate was dissolved in 1 ml HFIP at room tem-
perature, then sonicated, incubated for 24 h at room

Medicinal Chemistry Research
temperature. After the HFIP was evaporated under reduced
pressure, the residue was dissolved in DMSO to prepare a
200 μM Aβ1–42 solution, which was stored at −80 °C and
diluted to 50 μM before use. The peptide (20 µL, 25 µM,
final concentration) was incubated with or without the test
compounds (20 µL, 25 µM, final concentration) at 37 °C
for 24 h in 50 mM PBS (pH 7.4), while the blank group
with PBS (pH 7.4) instead of Aβ1–42. Then, 160 μL of
glycine-NaOH buffer solution (pH = 8.5) containing 5 μM
ThT was added, and the mixture was shaken quickly,
immediately scanned and recorded under a Varioskan
Flash Multimode Reader at the excitation wavelength of
446 nm and emission wavelength of 490 nm. The inhibi-
tion rate was calculated by the formula: 100−(IFi − IF0)/
(IFc − IF0) × 100, IFi represents the fluorescence value of
Aβ1–42 with tested samples, IFc represents the fluorescence
value of Aβ1–42 with PBS, IF0 represents the fluorescence
value of PBS only. Curcumin was used as the positive
compound for the assay, and each experiment was repe-
ated in triple independently.
5-diphenyltetrazolium bromide (MTT, 100 μL, 5 mg/mL)
solution was added for living cell staining, then solubilized
in DMSO (100 μL/well). The absorbance of each well was
measured under the test wavelength at 490 nm. All results
were tested three times and expressed as the percentage of
control group.
In vitro blood-brain barrier permeation assay
The parallel artificial membrane PAMPA-BBB was applied
to evaluate the BBB penetration of the representative
compounds [22, 23]. The porcine brain lipid (PBL) was
dissolved in n-dodecane (20 mg/mL), of which 4 μL was
dropped on the lipophilic filter membrane and stayed for
5 min to simulate the biofilm. 350 μL sample solution
(100 μg/mL) was added to the donor microplate, and 200 μL
PBS/EtOH (7:3) solution was added to the acceptor pool.
The mixture was incubated at 25 °C for 18 h, then 150 μL
solution of the donor microplate/acceptor well were
removed to the 96-well plates. The solution was scanned
and the OD values were recorded at 200–600 nm. Each
experiment was repeated three times and the concentrations
of the compounds in the donor/acceptor well were calcu-
lated. The effective permeability Pe (cm s-1) was calculated
by the following formula:
Metal-chelating studies
A Varioskan Flash Multimode Reader (Thermo Scientific)
was used to determine the metal-chelating effect of com-
pounds according to the method reported previously [43].
The UV absorption spectra were recorded at the range of
200 nm to 600 nm after incubating for 30 min in methanol at
room temperature, with the tested compounds alone or in
the presence of CuCl2, ZnCl2, AlCl3, and FeSO4. The final
concentration of the test samples and the metal ions was
37.5 μM, and the final volume of the reaction solution was
1 mL. The displacement of the maximum absorption peak
and the change of the maximum absorption peak intensity
were observed and recorded after the addition of metal ions.
Â
Ã
Pe ¼ Àln 1 À CAðtÞ=Cequilibrium =½A  ð1=VD þ 1=VAÞ Â tŠ
Cequilibrium ¼ ½CDðtÞ Â VD þ CAðtÞ Â VAŠ=ðVD þ VAÞ
Among them, A represents the area of artificial phos-
pholipid membrane (A = 0.28 cm2), VD represents the
volume of the donor pool (mL), VA represents the volume of
the acceptor pool (mL), t represents the penetrating time (s),
CA(t) represents the concentration of the acceptor pool at
time of t, CD(t) represents the concentration of the donor
pool at the time of t.
Neuroprotective activity of H2O2-induced PC12 cell injury
To determine the neuroprotective effect of the selected
compounds was applied the method reported previously
[36]. The PC12 cells were incubated in DMEM supple-
mented with 10% bovine calf serum (Gibco), at a 37 °C
atmosphere containing 5% CO2. First, PC12 cells were
sub-cultured in a 96-well flat-bottom plate at a density of
105 cells/mL and grown for 24 h. Then the administration
group was treated with different concentrations of selected
test compounds (1 μM or 10 μM), while the control group
was added only PBS (10 μL/well). Subsequently, 10 μL
H2O2 (150 μM) was added to the administration group and
the culture medium was used instead of serum-free and red-
free DMEM medium. After continuous culturing at 37 °C
for another 24 h, the viability rate of PC12 cells was mea-
sured by MTT assay. 3-(4,5-dimethylthiazol-2-yl)-2,
Step-down passive avoidance test
The step-down passive avoidance test was used to assess the
learning and memory capacity in mice. Experimental ani-
mals were Kunming mice at a bodyweight of 18–22 g
(6 weeks old, male and female in half) supplied by the
Center of Experimental Animals of Sichuan Academy of
Chinese Medicine Sciences (eligibility certification no.
SCXK-Sichuan 2018–19). The mice were housed in an
environment with a temperature of 24 2 °C and humidity
of 50–60%, in random groups of six per cage. Commercial
chow diet and water were provided ad libitum, and the mice
were kept under a 12-light-dark cycle. All mice were kept to
adapt to the laboratory environment for at least one week
before the tests. Experimental mice were randomly

Medicinal Chemistry Research
distributed into nine groups, with each group eight mice: a
blank group, a model group, a positive control group treated
with donepezil (5.0 mg/kg), and six test groups treated with
different doses of compound 6d (2.2, 6.5, 19.5 mg/kg) and
13d (2.3, 7.0, 21.0 mg/kg), respectively. The solution of test
compounds or the equal volume of 0.5% CMC-Na solution
were given by intragastric administration (i.g.), once a day
for 7 days consecutively.
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These mice were submitted to behavioral tests after one
week of treatment with tested compounds. The experi-
mental apparatus was composed of a grid floor with a
small platform at the center, which is a zone freed by
electric shock. The step-down passive avoidance test
includes the training trial and the test trial. On the 6th day,
the donepezil and the target compounds 6d and 13d were
administrated orally, then the scopolamine (3.0 mg/kg,
i.p.) was administered to each group except the blank
group after 30 min. After another 30 min, mice were
initially placed on the platform, and were given an elec-
trical shock at feet (36 V) if they stepped down on the grid
floor. Each trial was conducted for 5 min, and the time to
step down firstly was recorded as latency time for normal
learning performance. Twenty-four hours after the training
trial, the donepezil and 6d and 13d were administrated
intragastrically to the mice again, and the mice were
placed on the platform to perform the test trail. The latency
time and the number of shocks received within 5 min were
recorded. All values were expressed as mean SEM.
Differences between groups were examined for statistical
significance using one-way ANOVA with Student’s t-test.
Statistical significance was set at p < 0.05.
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Acknowledgements This work was supported by Chinese National
Natural Science Foundation (22077091), Sichuan Science and Tech-
nology Program (2020YFS0067) and the Fundamental Research
Funds for the Central Universities.
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Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Ethical approval All applicable international, national, and/or institu-
tional guidelines for the care and use of animals were followed.
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trials in early (predementia) AD. Neurology. 2011;76:280–6.
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