Design, synthesis and biological evaluation of Schiff’s base derivatives as multifunctional agents for the treatment of Alzheimer’s disease

Article abstract of DOI:10.1007/s00044-020-02666-6

A series of Schiff’s base derivatives was rationally designed, synthesized, and evaluated as multi-function agents for the treatment of Alzheimer’s disease (AD). The results revealed that compound 3b was a novel multifunctional agent. It acted as a highly selective monoamine oxidase-B inhibitor (IC₅₀ = 8.4 nM), which was explained by the docking study. Compound 3b also was an antioxidant agent (2.3 eq) and could significantly inhibit self-induced Aβ_1-42 aggregation (31.8%). Meanwhile, compound 3b was a selective metal chelator and could inhibit Cu²⁺-induced Aβ_1-42 aggregation (62.3%). Furthermore, compound 3b presented good neuroprotective effects on H₂O₂-induced PC12 cell injury. More importantly, compound demonstrated good blood brain barrier permeability and druglike properties. Therefore, compound 3b, a promising multi-targeted active molecule, offers an attractive starting point for further study in the drug-discovery process against AD.[Figure not available: see fulltext.].

Full text of DOI:10.1007/s00044-020-02666-6

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02666-6
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of Schiff’s base
derivatives as multifunctional agents for the treatment of
Alzheimer’s disease
Jian Shi1 Yi Zhou1 Keren Wang1 Qinge Ma2 Rongrui Wei2 Qingfeng Li3 Yiyang Zhao1 Zhanpin Qiao1
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Shuang Liu1 Yumin Leng4 Wenmin Liu1 Zhipei Sang1
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Received: 26 August 2020 / Accepted: 5 November 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A series of Schiff’s base derivatives was rationally designed, synthesized, and evaluated as multi-function agents for the
treatment of Alzheimer’s disease (AD). The results revealed that compound 3b was a novel multifunctional agent. It acted as
a highly selective monoamine oxidase-B inhibitor (IC50 = 8.4 nM), which was explained by the docking study. Compound
3b also was an antioxidant agent (2.3 eq) and could significantly inhibit self-induced Aβ1-42 aggregation (31.8%).
Meanwhile, compound 3b was a selective metal chelator and could inhibit Cu2+-induced Aβ1-42 aggregation (62.3%).
Furthermore, compound 3b presented good neuroprotective effects on H2O2-induced PC12 cell injury. More importantly,
compound demonstrated good blood brain barrier permeability and druglike properties. Therefore, compound 3b, a
promising multi-targeted active molecule, offers an attractive starting point for further study in the drug-discovery process
against AD.
These authors contributed equally: Jian Shi, Yi Zhou
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02666-6) contains supplementary
material, which is available to authorized users.
* Yumin Leng
2
State Key Laboratory of Innovative Drugs and High Efficiency
Energy Saving and Consumption Reduction Pharmaceutical
Equipment, Jiangxi University of Traditional Chinese Medicine,
Nanchang 330004, China
yumin_leng@126.com
* Wenmin Liu
liuwm1969@163.com
3
4
* Zhipei Sang
Foreign Languages School, Nanyang Normal University,
Nanyang 473061, China
sangzhipei@126.com
College of Physics and Electronic Engineering, Nanyang Normal
University, Nanyang 473061, China
1
College of Chemistry and Pharmaceutical Engineering, Nanyang
Normal University, Nanyang 473061, China

Medicinal Chemistry Research
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Keywords Alzheimer’s disease Schiff’s base derivatives Multifunctional agents Blood barrier permeability Drug-like
property
Introduction
The excess MAO-B produces hydroxyl radicals, which
could increase the former of Aβ plaques [12]. Recently,
selective inhibition of MAO-B has been acted as a potential
approach for treating AD. Rasagiline is a selective MAO-B
inhibitor, which has been approved for treating symptoms
of Parkinson’s disease [13]. Rasagiline has been performed
a phase 2 trial in people with mild-to-moderate AD, and it
significantly improves on the Quality of Life-AD test, and
displays trends to better performance https://www.alzforum.
org/therapeutics/rasagiline.
Schiff’s base is a privileged skeleton, the Schiff’s base
derivatives as multifunctional agents have been widely
investigated for the treatment of AD, such as selective
MAO-B inhibitor, cholinesterase inhibitors, anti-
oxidants, and modulators of Cu2+-mediated Aβ aggre-
gation [14–18]. Moreover, Clioquinol is a zinc and
copper chelator, which would prevent Aβ accumulation
and reduce brain amyloid deposition. The phase 2 trial of
Clioquinol in patients with moderate to severe AD pre-
sents a beneficial cognitive https://www.alzforum.org/
therapeutics/clioquinol. Herein, the chelation fragment
of Clioquinol is fused into Schiff’s base skeleton based
on the MTDLs strategy to obtain a novel series of
Schiff’s base derivatives (Fig. 1), hoping these deriva-
tives could be promising multifunctional agents posses-
sing Aβ inhibitory activity, selective MAO-B inhibitory
potency, antioxidant activity, metal chelation property,
and anti-inflammation activity.
Alzheimer’s disease (AD) is a neurodegenerative disease,
which is the most common form of dementia, characterized
by memory disorders, behavioral disorders, and other cog-
nitive impairments. It is estimated that more than 50 million
people suffering from dementia globally nowadays and the
figure will be 152 million by 2050 [1]. A heavy economic
burden has been brought by the enormous expense to the
family of patients, which may trigger serious socio-
economic problems [1]. Numerous efforts have been made
in exploring the mechanism of AD, and four acet-
ylcholinesterase inhibitors (such as tacrine, donepezil,
galantamine, and rivastigmine) and one N-methyl-D-aspar-
tate receptor antagonist (memantine) had been approved by
the food and drug administration for the treatment of AD
patients. During the period of long-term clinical use, these
drugs are effective in controlling the AD symptoms to a
certain extent, but they cannot stop, prevent, or reverse the
progression of AD [2].
Due to the multifactorial pathogenesis of AD, the mul-
tifunctional agents, possessing two or more complementary
biological activities, have been regarded as the best phar-
macological option [3–5]. In addition, several multitarget-
directed ligand strategy (MTDLs) candidate drugs now
reach the testing stage in clinical trials [6, 7].
The amyloid hypothesis states that accumulation of
amyloid-β (Aβ) is the triggering event in the pathogenesis
of AD and it is prone to aggregate into oligomers, which
leads to the damage and death of neurons. In addition,
accumulation of Aβ in the brain triggers the spread of tau-
related neurofibrillary tangles, neuroinflammation, and
neuronal degeneration and death, causing AD [8, 9]. Thus,
inhibiting accumulation of Aβ oligomers in the brain will be
beneficial in managing AD.
Results and discussion
Chemistry
The synthetic of 17 Schiff’s base derivatives were depicted in
Scheme 1. Briefly, the excessive amounts of benzaldehyde
derivatives (benzaldehyde, 1a; salicylaldehyde, 1b; 4-
hydroxy-3-methoxybenzaldehyde, 1c; 2-bromobenzaldehyde,
1d; 4-methoxybenzaldehyde, 1e; 2-pyridinecarboxaldehyde,
1f; 3-pyridinecarboxaldehyde, 1g; 4-pyridinecarboxaldehyde,
1h) as starting materials were dissolved in anhydrous ethanol,
acetic acid was added dropwise to the solution as a catalyst.
And then, hydrazine (2a), ethylenediamine (2b), diethylene-
triamine (2c), and N,N-di(2-aminoethyl)ethylenediamine (2d),
respectively, was added to the mixture under reflux for
2~6 h. Finally, the target compounds 3a-f, 4a-g, 5a-b, and
6a-b were obtained by crystallization, and the derivatives
Metal ion hypothesis states that high levels and dysre-
gulation of metal ions (such as Cu2+, Fe2+, and Zn2+) are
observed in the AD brains, accelerating formation of
amyloid plaques as well as neurotoxic oxidative processes.
Cu2+ and Zn2+ induce the generation of toxic Aβ oligo-
mers by binding to Aβ peptides, and the redox-active
metals, Cu(I/II) and Fe(II/III), generate cytotoxic reactive
oxygen species and cause neuronal damage. So, biometal
chealtors would be a potential therapeutic strategy for
treating AD [10, 11].
Increasing evidence suggests that levels of monoamine
oxidase-B (MAO-B) activity could increase up to threefold
in the temporal, parietal, and frontal cortex of AD patients.
1
were characterized by H NMR, 13C NMR, and IR.

Medicinal Chemistry Research
Fig. 1 The design strategy of
Schiff’s base derivatives
N
Ar
NH2NH2
Ar
N
3a-3f
2a
N
Ar
NH2
Ar
N
N
NH2
(i)
2b
4a-4g
Ar CHO
+
H
N
1a~1h
NH
Ar
N
N
Ar
NH2
NH2
5a-5b
2c
Ar
N
Ar
N
N
N
CH
Ar
NH
NH2
NH2
NH
2d
6a-6b
Br
OCH3
OH
N
N
Ar =
OCH3
HO
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Scheme 1 Synthesis of target compounds 3a-f, 4a-g, 5a-b, and 6a-b. Reagents and conditions: (i) ArCHO(1a~1h), anhydrous ethanol, reflux,
2–6 h
Biological activity
in Table 1, most of these derivatives showed much better
MAO-B inhibitory activities than towards MAO-A, indi-
cating that these compounds were selective MAO-B
inhibitors. Generally, the primary amine skeleton and the
aromatic formaldehyde significantly influenced the MAO-
B inhibitory activity. When the primary amine skeleton
was hydrazine, the target compounds 3a-f exhibited
better inhibitory activity of MAO-B than other derivatives
Inhibition of monoamine oxidases
All the synthesized Schiff’s base derivatives were eval-
uated for their inhibitory activity against human MAO-A
and MAO-B by a fluorimetric method [19]. Rasagiline was
used as the positive control. The results were summarized

Medicinal Chemistry Research
Table 1 In vitro IC50 values of test compounds toward hMAO-A, hMAO-B and inhibition of self-/Cu2+-induced Aβ1-42 aggregation
IC50 (μM)a
MAO-A(%)b
% inhibition of Aβ1-42 aggregatione
Compd
Ar
ORACd
MAO-B
Self-inducedf
Cu2+-inducedg
18.5 0.67
23.1 0.69
33.3 2.6
36.9 1.3
12.4 0.79
16.4 1.1
6.5 0.37
0.3 0.12
13.0 0.47
30.6 0.87
0.4 0.01
23.5 1.7
31.8 1.3
46.2 2.3
24.3 1.5
20.1 1.3
14.5 0.69
20.9 1.1
37.8 2.5
28.9 2.1
—
3a
3b
3c
3d
3e
3f
0.0084 0.0006 2.3 0.05
62.3 2.89
28.9 1.6
14.0 0.48
15.4 0.59
34.2 0.93
30.4 2.1
n.a.c
—
—
—
—
—
—
—
2.6 0.08
0.6 0.03
0.5 0.02
0.5 0.03
0.3 0.02
2.5 0.08
2.7 0.07
4a
4b
4c
n.a.c
7.3 0.66
n.a.c
—
0.3 0.02
16.5 0.92
4d
5.6 0.38
0.4 0.01
2.4 0.39
27.5 1.7
13.8 0.86
12.2 0.63
3.6 0.14
54.6 2.6
11.9 0.41
53.5 2.6
n.a.c
21.1 1.2
21.4 0.68
28.6 0.89
13.1 0.78
13.2 1.1
20.6 0.88
24.1 1.3
—
—
—
—
—
—
—
0.6 0.03
0.6 0.01
0.5 0.05
2.7 0.09
2.8 0.07
2.9 0.06
2.7 0.08
4e
4f
4g
5a
5b
6a
6b
55.7 3.7
n.a.c
n.a.c
0.029 0.003
—
—
—
—
rasagiline
curcumin
0.59 0.04 μM
—
38.1 2.6
67.4 4.16
aIC50: 50% inhibitory concentration(means SD of three experiments)
bResults are expressed as μM of Trolox equivalent/μM of tested compound
cFor inhibition of Aβ aggregation, the thioflavin-T fluorescence method was used, data are presented as the mean SEM of three independent
experiments
dMAO-A inhibition rate at the test concentration of 12.5 μM
eInhibition of self-induced Aβ1-42 aggregation, the concentration of tested compounds and Aβ1-42 were 25 μM
fInhibition of Cu2+-induced Aβ1-42 aggregation produced by tested compounds at 25 μM
gn.a. = no active. Compounds defined “no active” means that inhibition is less than 5.0% at a concentration of 50 μM in the assay condition
(Table 1), particularly, compound 3b showed the best
potency for inhibition of MAO-B with an IC50 value of
8.4 nM, which was better than the reference compound
rasagiline (IC50 = 29 nM), and the selective index toward
MAO-B of 3b was higher than rasagiline. In addition,
the structure-activity-relationship also displayed that ben-
zaldehyde fragment (3a) showed moderate MAO-B
inhibitory activity (IC50 = 30.6 μM), when replacing ben-
zaldehyde fragment with salicylaldehyde fragment to get
compound 3b, the MAO-B inhibitory activity sharply
increased to 8.4 nM, maybe the 2-hydroxy moiety of
salicylaldehyde served as a critical role in MAO-B inhi-
bitory potency. When replacing benzaldehyde fragment of
3a with 4-hydroxy-3-methoxybenzaldehydes fragment
to get compound 3c, the MAO-B inhibitory activity did
not show obvious change. Subsequently, when replacing
benzaldehyde fragment of 3a with 2-pyridinecarbox-
aldehyde,
3-pyridinecarboxaldehyde,
and
4-pyr-
idinecarboxaldehyde, respectively, to obtain compounds
3d, 3e, and 3f, respectively, the IC50 values of MAO-B
inhibitory activities were 14.0 μM, 15.4 μM and 34.2 μM,
respectively, indicating that the 2-pyridinecarboxaldehyde

Medicinal Chemistry Research
(ORAC-FL) method using Trolox (vitamin E analog) as a
referenced compound [21]. As displayed in Table 1, all the
target compounds displayed moderate to good antioxidant
potency with ORAC value of 0.3–2.9 eq. Generally
speaking, the derivatives (3b, 3c, 4b, 4c, 5a, 5b, 6a, 6b)
with hydroxyl group showed better antioxidant activity than
other groups, revealing that the hydroxyl group of target
compounds was crucial to the radical scavenging capability
and could contribute to the antioxidant activities. Moreover,
the data showed that other benzaldehyde fragment without
hydroxyl group did not show significant influence on the
antioxidant activity.
Fig. 2 Compound 3b (green stick) interacting with residues in the
binding site of huMAO-B (PDB code: 2V60)
Inhibition of self-induced Aβ1−42 aggregation
and 3-pyridinecarboxaldehydefragment would be bene-
ficial for the MAO-B inhibition. Furthermore, the MAO-B
inhibitory activity significantly decreased as the length of
linker increased, such as 3b < 4b, 5a, and 6a; 3c < 4c, 5b,
and 6b, implying that the the more long linker was harmful
to the MAO-B inhibitory potency. In short, compound 3b
presented the best selective MAO-B inhibitory potency
and was worth in-depth study.
All the target compounds were selected to test the self-
induced Aβ1−42 aggregation inhibitory activity by thioflavin
T (ThT) fluorescence assay using curcumin as reference
compound [22]. According to the results in Table 1, all the
derivatives showed moderate to good inhibitory potency
towards self-induced Aβ1−42 aggregation compared with
curcumin (38.1%). Among these derivatives, compound 3c
(46.2%) showed the best inhibitory potency against self-
induced Aβ1−42 aggregation, which was better than curcu-
min. Moreover, the length of linker and the aromatic for-
maldehyde remarkably influenced the inhibitory activity,
generally, the inhibition rate declined as the length of linker
increased. In addition, the electron-donating group (such as
OH group) indicated better inhibition rate than other groups,
such as compounds 3b, 3c, 4b, and 4c. Especially, com-
pound 3b displayed good inhibition rate with 31.8%, which
was similar with curcumin.
Molecular modeling study of MAO-B
To further explore the binding mode of 3b with MAO-B, a
molecular docking was performed with the X-ray crystal
structures of human MAO-B (PDB code: 2V60) [20]. In 3b-
MAO-B complex (Fig. 2), the O atom of hydroxyl group in
one side of 3b interacted with key amino acid Thr 201 via
one intermolecular hydrogen bonding, the H atom of
hydroxyl group in one side of 3b formed one intermolecular
hydrogen bonding with key amino acid Glu84, and the H
atom of hydroxyl group interacted with N atom of 3b via
intramolecular hydrogen bonding interaction. Moreover, the
hydroxyl group in another side of 3b interacted with key
amino acid Thr201 via two intermolecular hydrogen
bonding interactions. Meanwhile, the N atom in one side of
3b interacted with key amino acid Thr201 via one inter-
molecular hydrogen bonding, and the N atom in another
side of 3b interacted with key amino acid Thr201 and
Thr314 via two intermolecular hydrogen bonding interac-
tions. Besides, there were some hydrophobic interactions
could be observed between the ligand 3b and amino acids
Pro102, Leu88, and Leu328 in MAO-B. Thus, the observed
interactions provided rational explanation for the high
MAO-B inhibitory activity toward 3b.
Metal-chelating properties of 3b
The UV-visual spectrometry was applied to test the chela-
tion ability of compound 3b using Cu2+, Fe2+, Zn2+ and
Al3+ [21, 22]. As presented in Fig. 3, when adding Cu2+
,
the characteristic peak of 3b presented a red shift from
354 nm to 398 nm, while, when treating with Fe2+, Zn2+
,
and Al3+, respectively, the electronic spectra did not show
obvious shift, revealing that compound 3b was a selective
metal chelator. Furthermore, the molar ratio method was
employed to assess the stoichiometry of the 3b-Cu2+
complex at 398 nm. As exhibited in Fig. 4, the two straight
lines intersected at a mole fraction of 1.98, displaying a
1:2 stoichiometry for the 3b-Cu2+ complex.
Effects on Cu2+-induced Aβ1−42 aggregation
Antioxidant activity assay
According to the above data, compound 3b was a selective
Cu2+ chelator, in order to further assess the inhibition effect
of 3b on Cu2+-induced Aβ1-42 aggregation, ThT binding
The antioxidant activity of target compounds were eval-
uated by oxygen radical absorbance capacity fluorescein

Medicinal Chemistry Research
Under the same experiments condition, when treating with
10 μM compounds 3b, 3c, and 4b, the cell viabilities
increased to 74.3, 69.4, and 81.8%, respectively. When the
concentration increased to 100 μM, the cell viabilities were
79.1, 60.9, and 84.3%, respectively. Therefore, the data
showed that compounds 3b, 3c and 4b showed better
neuroprotective effect than VE. In addition, neuroprotective
effect of compounds 3b, 3c, and 4b were also evaluated
through LDH assay [21]. As displayed in Fig. 6b, 100 μM
H2O2 was exposed to the PC12 cell, the LDH vitality
suddenly increased to 785.5 (p < 0.01) compared with nor-
mal group (674.7), when treating with 100 μM VE, the
LDH vitality decreased to 693.1. When treating with 10 μM
compounds 3b, 3c, and 4b, the LDH vitality declined to
590.4, 579.6 and 724.1, respectively. When the concentra-
tion increased to 100 μM, the vitality were 570.6, 659.8, and
681.3. In summary, the data reveled that compounds 3b, 3c,
and 4b displayed potential protective effect against H2O2-
induced PC12 cell injury.
Fig. 3 The UV spectrum of compound 3b (37.5 μM, in methanol)
alone or in the presence of CuCl2, FeSO4, ZnCl2. and AlCl3 (37.5 μM,
in methanol)
In vitro blood-brain barrier permeation assay
The parallel artificial membrane permeation assay
(PAMPA) of the blood-brain barrier was applied to evaluate
the BBB permeability of 3b [23, 24]. 11 commercial drugs
were selected to validate the assay (Table S1), Pe(exp) =
0.9163Pe(bibl.) − 0.2247 (R2 = 0.9558) (Fig. S1), was
obtained by experimental data versus the reported values.
We determined that compounds with permeability above
3.44 × 10−6 cm/s could cross the blood-brain barrier based
on the limit established by Di et al. (Table S2). The mea-
sured data were shown in Table 2, compound 3b showed
13.29 × 10−6 cm/s permeability, indicating good BBB per-
meability in vitro.
Fig. 4 Determination of the stoichiometry of complex-Cu2+ by using
molar ratio method through titrating the methanol solution of com-
pound 3d with ascending amounts of CuCl2. The final concentration of
tested compound was 37.5 μM, and the final concentration of Cu2+
ranged from 3.75 to 375 μM
assay was performed [22]. The result from Table 1 showed
that compound 3b significantly inhibit Cu2+-induced
Aβ1-42 aggregation (62.3%), which was similar with the
positive compound curcumin (67.4%). Moreover, trans-
mission electron microscopy (Fig. 5) was used to observe
the Cu2+-induced Aβ1-42 aggregation experiments, which
was consistent with the ThT assay.
Theoretical evaluation of ADME properties
The druglike properties of 3b were evaluated using
Molinspiration property program, the items including log P,
topological polar surface area (TPSA), the number of
hydrogen-bond acceptors, and the number of hydrogen-
bond donors [25]. As presented in Tables 3, 3b complied
with the Lipinski’s rule of five, which could be a potent
candidate for the treatment of AD.
Neuroprotective effects on H2O2-induced PC12 cell injury
Compounds 3b, 3c, and 4b were selected to test the
neuroprotective effects against H2O2-induced PC12 cell
injury by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(MTT) assays and lactate dehydrogenase (LDH) assay using
Vitamine E (VE) as the positive compound [21]. As dis-
played in Fig. 6a, when PC12 cells were treated with
100 μM H2O2, the cell viability sharply decreased to 57.8%
(p < 0.01) compared with the normal group. When treating
with 100 μM VE, the cell viability increased to 67.7%.
Conclusion
In summary, a series of Schiff’s base derivatives was
rationally designed and synthesized, the biological activity
of these derivatives were evaluated by MAO-A/MAO-B
inhibition, antioxidant activity, Aβ1-42 aggregation effects,
metal chelation property and neuroprotective effect.

Medicinal Chemistry Research
Aβ (0h)
Aβ + Cu2+ (48h)
Aβ + Cu2+ + curcumin (48h)
Aβ + Cu2+ + 3b (48h)
Fig. 5 TEM images of Aβ species from inhibition experiments
Fig. 6 a The cell viability (%) of compounds 3b, 3c, and 4b on H2O2-
induced PC12 cell injury by MTT assay. b The LDH vitality of
compounds 3b, 3c, and 4b on H2O2-induced PC12 cell injury. Three
independent experiments were carried out in triplicate. Data were
expressed as mean SD and percentage of control value. ##p < 0.01 vs
control; **p < 0.01, *p < 0.05 vs H2O2 group
The results revealed that compound 3b showed the best
MAO-B inhibitory activity with IC50 value of 8.4 nM,
which was confirmed by the docking study. Compound 3b
also displayed significant antioxidant activity (ORAC = 2.3
eq) and remarkably inhibited self-induced Aβ1-42 aggrega-
tion with 31.8% inhibition rate. In addition, compound 3b
was a metal chelator and could inhibit Cu2+-induced Aβ1-42
aggregation (62.3%). Moreover, compound 3b showed
good neuroprotective effects on H2O2-induced PC12 cell
injury. Further, compound 3b could cross BBB in vitro and
Table 2 Permeability Pe (×10−6 cm/s) of compound 3b and its
predictive penetration in the CNS
Compounda
Pe (×10−6 cm/s)b
Prediction
3b
13.29 1.23
CNS+
aCompound 3b was dissolved in DMSO at 5 mg/mL and diluted with
PBS/EtOH (70:30). The final concentration of compounds was 100 μg/
mL
bValues are expressed as the mean SD of three independent
experiments

Medicinal Chemistry Research
Table 3 Theoretical prediction
of the ADME properties of
compound 3b
Comp.a
Log P MW
TPSA (Å2) n-ON n-OHNH nviolations nrotb Volume (Å3)
Lipinski’s rule of five ≤ 5
3b 3.09
≤ 500
≤ 10 ≤ 5
≤ 10
240.26 65.18
4
2
0
3
218.01
MW molecular weight, TPSA topological polar surface area, n-ON number of hydrogen acceptors, n-OHNH
number of hydrogen bond donors
aThe data were determined with the Molinspiration calculation software
exhibited good drug-like properties. Therefore, compound
3b could be a promising multifunctional agent for the
treatment of AD. Further studies to evaluate compound 3b
in vivo and to develop structural refinements are in progress
and will be reported in due course.
DMSO-d6): 161.9, 134.3, 131.8, 129.4, 128.8. IR (KBr,
cm−1) 1622.2 (C=N), 1574.0, 1488.6, 1445.8. MS (ESI) m/
+
z: 209.1 [M+H]
.
2,2’-((1E,1’E)-hydrazine-1,2-diylidenebis(methanylylidene))
diphenol (3b) Bright yellow solid, 71.08% yield, mp:
196.4–197.2 °C, 99.0% purity. 1H NMR (400 MHz, CDCl3)
δ 11.40 (s, 2H), 8.72 (s, 2H), 7.40–7.35 (m, 4H), 7.04 (d,
J = 8.4 Hz, 2H), 6.98 (t, J = 7.6 Hz, 2H).13C NMR
(100 MHz, DMSO-d6): 163.3, 159.1, 133.7, 131.3, 120.1,
Experimental
Chemistry
118.7, 117.0. IR (KBr, cm−1) 3434.6(O-H), 1624.1(C=N),
+
General information
1573.6, 1487.1, 1387.6. MS (ESI) m/z: 241.1 [M+H]
.
All materials were obtained from commercial sources and
utilized without further purification unless otherwise indi-
cated. Melting points were measured on X-5 precision
micro melting point apparatus (China). 1H NMR (400 MHz)
and 13C NMR (100 MHz) spectra were recorded on a
Varian INOVA spectrometer and referenced to Tetra-
methylsilane (TMS), using CDCl3 or DMSO-d6 as solvents.
Chemical shifts (δ) are reported in ppm. Splitting patterns
are designated as s, single; d, doublet; dd, double-doublet; t,
triplet; m, multiplet. MS spectra data were obtained on an
Agilent-6210 TOF LC-MS spectrometer.
4,4’-((1E,1’E)-hydrazine-1,2-diylidenebis(methanylylidene))
bis(2-methoxyphenol) (3c) Light yellow solid, 73.63%
yield, mp 178.4–179.6 °C, 98.6% purity. 1H NMR
(400 MHz, CDCl3) δ 8.58 (s, 2H), 7.54 (d, J = 1.6 Hz, 2H),
7.21 (dd, J1 = 6.4 Hz, J2 = 1.6 Hz, 2H), 6.97 (d, J = 8.4 Hz,
2H), 5.99 (s, 2H), 3.99 (s, 6H). 13C NMR (100 MHz,
DMSO-d6): 161.1, 150.4, 148.5, 126.0, 123.9, 115.0, 110.6,
56.0. IR (KBr, cm−1) 3480.4 (O-H), 2933.5 (C-H), 1623.9
(C=N), 1600.6, 1540.0, 1459.0. MS (ESI) m/z: 301.1
[M+H]+.
(1E,2E)-1,2-bis(pyridin-2-ylmethylene)hydrazine (3d) Yel-
low solid, 74.38% yield, mp 172.4–173.6 °C, 98.6% purity.
1H NMR (400 MHz, CDCl3) δ9.03 (s, 2 H, 2 × =CH),
8.21–7.26 (m, 8H, 2 × C6H4). IR (KBr, cm−1) 1611.2
(C=N), 1586.1, 1554.5, 1463.1, 1434.3. MS (ESI) m/z:
211.1 [M+H]+.
General procedure for the preparation of Schiff’s base
derivatives 3a-f, 4a-g, 5a-b, and 6a-b
The excessive amounts of benzaldehyde derivatives 1a~h (2-
3 mmol) were dissolved in anhydrous ethanol (8 mL), acetic
acid was added two or three drops to the solution as a cat-
alyst. Finally, hydrazine (2a), ethylenediamine (2b), diethy-
lenetriamine (2c), or triethylenetetramine (2d) (1 mmol) was
added to the aforementioned solution, respectively. The
reaction mixture was stirred under reflux condition for 6 h
monitored by TLC. After reaction finished, the solvent was
concentrated under reduced pressure. The crude product was
purified by crystallization from anhydrous ethanol to give the
target compounds 3a-f, 4a-g, 5a-b, and 6a-b.
(1E,2E)-1,2-bis(pyridin-3-ylmethylene)hydrazine (3e) Yel-
low solid, 75.38% yield, mp 145.5–146.6 °C, 98.4% purity.
1H NMR (400 MHz, CDCl3) δ 8.74 (dd, J1 = 4.0 Hz, J2 =
0.8 Hz, 2H), 8.70 (s, 2H), 8.13 (d, J = 8.0 Hz, 2H), 7.81 (dt,
J1 = 7.6 Hz, J2 = 1.2 Hz, 2H), 7.39 (dd, J1 = 4.0 Hz, J2 =
0.8 Hz, 1H), 7.37 (dd, J1 = 4.0 Hz, J2 = 0.8 Hz, 1H). 13C
NMR (100 MHz, DMSO-d6): 161.6, 152.6, 150.4, 137.6,
126.2, 122.2. IR (KBr, cm−1) 1628.9 (C=N), 1583.0,
1463.4, 1437.5. MS (ESI) m/z: 211.1 [M+H]+.
1,2-di((E)-benzylidene)hydrazine (3a) Bright yellow solid,
1
65.71% yield, mp: 94.2–94.8 °C, 98.7% purity. H NMR
(1E,2E)-1,2-bis(pyridin-4-ylmethylene)hydrazine (3f) Yel-
low solid, 61.35% yield, mp 145.7–146.0 °C, 98.7% purity.
1H NMR (400 MHz, CDCl3) δ 8.99 (s, 2 H), 8.71 (d, J =
8.0 Hz, 2H), 8.69 (s, 2H), 8.23 (d, J = 8.0 Hz, 2H), 7.43 (d,
(400 MHz, CDCl3) δ 8.68 (s, 2H, 2 × =CH), 7.86 (d, J =
2.0 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 2.0 Hz,
4H), 7.45 (d, J = 1.6 Hz, 2H). 13C NMR (100 MHz,

Medicinal Chemistry Research
J = 4.8 Hz, 1H), 7.41 (d, J = 4.8 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6): 160.1, 152.5, 150.4, 135.3, 129.9,
124.6. IR (KBr, cm−1) 1627.1 (C=N), 1582.8, 1470.2,
1418.0. MS (ESI) m/z: 211.1 [M+H]+.
C6H4), 3.91 (t, 4H, 2 × CH2), 3.81 (s, 6H, 2 × OCH3). IR
(KBr, cm−1) 3440 (O-H), 2969, 2919 (C-H), 1641(C=N),
1604, 1509, 1457, 1308 (C-N), 1282, 1020 (C-O-C). 239.1
[M+H]+.
(1E,1’E)-N,N’-(ethane-1,2-diyl)bis(1-phenylmethanimine)
(1E,1’E)-N,N’-(ethane-1,2-diyl)bis(1-(pyridin-4-yl)methani-
mine) (4g) Yellow solid, 49.6% yield, 98.7% purity. H
NMR (400 MHz, CDCl3) δ 8.62 (s, 2H, 2 × CH = ),
8.42–7.29 (m, 8H, 2 × C6H4), 4.06(s, 4H, 2 × CH2). 13C
NMR (100 MHz, DMSO-d6): 163.5, 154.5, 149.8, 137.3,
125.5, 120.9. MS (ESI) m/z: 239.1 [M+H]+.
1
(4a) Bright
yellow
solid,
77.46%
yield,
mp
180.4–182.4 °C, 98.6% purity. 1H NMR (400 MHz, CDCl3)
δ 8.76 (dd, J1 = 3.6 Hz, J2 = 1.2 Hz, 4H), 8.58 (s, 2H), 7.71
(dd, J1 = 3.2 Hz, J2 = 1.2 Hz, 4H). 13C NMR (100 MHz,
DMSO-d6): 160.1, 151.0, 140.9, 122.5. IR (KBr, cm−1
)
1627.5 (C=N), 1594.5, 1551.7, 1417.5. MS (ESI) m/z:
237.1 [M+H]+.
2,2’-((1E,1’E)-((azanediylbis(ethane-2,1-diyl))bis(azanylyli-
dene))bis(methanylylidene))diphenol (5a) Yellow solid,
40.3% yield, 97.6% purity. H NMR (400 MHz, CDCl3) δ
1
2,2’-((1E,1’E)-(ethane-1,2-diylbis(azanylylidene))bis(metha-
nylylidene))diphenol (4b) Bright yellow solid, 70.4%
yield, 98.3% purity. 1H NMR (400 MHz, CDCl3) δ 8.29 (s,
2H, 2 =CH), 7.70–7.68 (m, 4H, C6H5), 7.40–7.36 (m, 4H,
C6H5), 3.98 (s, 4H, 2 × CH2). 13C NMR (100 MHz, DMSO-
d6): 161.6, 129.9, 129.4, 114.5, 61.5, 55.7. MS (ESI) m/z:
269.1 [M+H]+.
8.83(s, 2 H, 2 × CH = ), 8.64–7.33 (m, 8H, 2 × C6H4), 4.02
(s, 4H, 2 × CH2). MS (ESI) m/z: 312.2 [M+H]+.
4,4’-((1E,1’E)-((azanediylbis(ethane-2,1-diyl))bis(azanylyli-
dene))bis(methanylylidene))bis(2-methoxyphenol) (5b)
Yellow solid, 36.8% yield, 97.8% purity. 1H NMR
(400 MHz, CDCl3) δ 8.67–8.69 (m, 4H, 2 × CH =+C6H4),
8.29 (t, 2H, C6H4), 7.58–7.55 (m, 4H, C6H4), 4.06 (q, 4H,
2 × CH2). 13C NMR (100 MHz, DMSO-d6): 161.4, 150.8,
143.0, 122.2, 61.0. MS (ESI) m/z: 372.2 [M+H]+.
4,4’-((1E,1’E)-(ethane-1,2-diylbis(azanylylidene))bis(metha-
nylylidene))bis(2-methoxyphenol) (4c) Bright yellow
solid, 68.8% yield, mp 124.5–125.0 °C, 97.8% purity. H
1
NMR (400 MHz, CDCl3)δ 13.23 (s, 2H, 2 × OH), 8.36 (s,
2H, 2 × CH = ), 7.23–7.30 (m, 4H, C6H4), 6.86–6.95 (m,
4H, C6H4), 3.94 (s, 4H, 2 × CH2). 13C NMR (100 MHz,
DMSO-d6): 167.4, 161.0, 132.8, 132.1, 119.1, 119.0, 116.9,
59.2. IR (KBr, cm−1) 3438 (O-H), 2928 (C-H), 1634
(C=N), 1578, 1497, 1459, 1416, 1284 (C-N), 1202, 1194
(C-O-C). MS (ESI) m/z: 329.1 [M+H]+.
2,2’-((1E,1’E)-(((2-(2-hydroxyphenyl)imidazolidine-1,3-diyl)
bis(ethane-2,1-diyl))bis(azanylylidene))bis(methanylyli-
dene))diphenol (6a) Bright yellow solid, 33.02% yield,
1
97.6% purity. H NMR (400 MHz, CDCl3) δ 13.12–13.27
(m, 1H, OH), 8.25 (s, 1H, CH = ), 6.79–7.31 (m, 9H, 2 ×
C6H4, CH = ), 3.42–3.83 (m, 5H, 2 × CH2+OH), 2.95~2.98
(m, 1H, NH), 2.66–2.69 (m, 4H, 2 × CH2). IR (KBr, cm−1
)
(1E,1’E)-N,N’-(ethane-1,2-diyl)bis(1-(2-bromophenyl)metha-
nimine) (4d) Bright yellow solid, 60.21% yield, 98.0%
purity. 1H NMR (400 MHz, DMSO) δ 9.54 (s, 2H, 2 × OH),
8.19 (s, 2H, 2 × CH = ), 7.32–6.70 (m, 6H, 2 × C6H3), 3.78
(s, 10H, 2 × CH2+2 × OCH3). 13C NMR (100 MHz,
DMSO-d6): 161.9, 148.3, 128.4, 128.4, 123.1, 115.7,
110.40, 61.4, 55.9. MS (ESI) m/z: 393.0 [M+H]+.
3427(O-H), 2860, 2822(C-H), 1631(-CH=N), 1580, 1495,
1456, 1414.9, 1261(C-N), 1206, 1116 (C-O-C). MS (ESI)
m/z: 459.2 [M+H]+.
4,4’-((1E,1’E)-(((2-(4-hydroxy-3-methoxyphenyl)imidazoli-
dine-1,3-diyl)bis(ethane-2,1-diyl))bis(azanylylidene))bis
(methanylylidene))bis(2-methoxyphenol)
(6b) Yellow
1
solid, 68.48% yield, mp 103.2–104.0 °C, 97.7% purity. H
NMR (400 MHz, CDCl3) δ 13.24 (s, 2H), 10.70 (s, 1H), 8.25
(s, 2H), 7.29 (dt, J1 = 6.8 Hz, J2 = 2.0 Hz, 2H), 7.25 (dd,
J1 = 6.4 Hz, J2 = 0.8 Hz, 1H), 7.21 (dd, J1 = 6.0 Hz, J2 =
1.6 Hz, 2H), 7.00 (dd, J1 = 6.0 Hz, J2 = 1.6 Hz, 1H), 6.93 (d,
J = 8.0 Hz, 2H), 6.86 (dt, J1 = 6.8 Hz, J2 = 0.8 Hz, 2H), 6.82
(dd, J1 = 3.6 Hz, J2 = 0.8 Hz, 1H), 6.82 (dd, J1 = 3.6 Hz,
J2 = 0.8 Hz, 1H), 6.80 (dd, J1 = 4.4 Hz, J2 = 0.8 Hz, 1H),
3.83 (s, 1H), 3.60 (t, J = 6.4 Hz, 4H), 3.45–3.41 (m, 2H),
2.99 (t, J = 6.4 Hz, 1H), 2.95 (t, J = 6.4 Hz, 1H), 2.71–2.62
(m, 4H). IR (KBr, cm−1) 3439 (O-H), 2856、2802 (C-H),
1632 (C=N), 1581, 1494, 1460, 1415, 1372, 1312(C-N),
1206, 1033 (C-O-C). MS (ESI) m/z: 549.3 [M+H]+.
(1E,1’E)-N,N’-(ethane-1,2-diyl)bis(1-(4-methoxyphenyl)
methanimine) (4e) Bright yellow solid, 56.27% yield,
1
98.3% purity. H NMR (400 MHz, CDCl3) δ 8.65 (s, 2H,
2 × CH = ), 8.0–7.23 (m, 8H, 2 × C6H4), 4.03(s, 4H, 2 ×
CH2). 13C NMR (100 MHz, DMSO-d6): 161.1, 134.5,
133.5, 132.9, 129.0, 128.4, 124.6, 60.9. MS (ESI) m/z:
297.2 [M+H]+.
(1E,1’E)-N,N’-(ethane-1,2-diyl)bis(1-(pyridin-2-yl)methani-
mine) (4f) Yellow solid, 62% yield, mp 153.2–153.5 °C,
1
98.5% purity. H NMR (400 MHz, CDCl3)δ 8.20 (s, 2H,
2 × CH = ), 7.64–7.62 (m, 4H, C6H4), 6.90–6.88 (m, 4H,

Medicinal Chemistry Research
Biological activity
In vitro blood-brain barrier permeation assay
Human MAO-A and MAO-B inhibition studies
The blood-brain barrier penetration of compounds was
evaluated using the PAMPA [23, 24]. Commercial drugs
were purchased from Sigma and Alfa Aesar. Porcine brain
lipid was obtained from Avanti Polar Lipids. The donor
microplate (PVDF membrane, pore size 0.45 mm) and
acceptor microplate were both from Millipore. The 96-well
UV plate (COSTAR) was from Corning Incorporated.
According to the detailed procedure in our previous work,
we concluded that compounds with Pe values above 3.44 ×
10−6 cm/s could cross the blood-brain barrier.
Recombinant human MAO-A and -B were purchased from
Sigma-Aldrich, all the test compounds were prepared in
DMSO (2.5 mM) and diluted with potassium phosphate
buffer (100 mM, pH 7.40, containing KCl 20.2 mM).
Rasagiline was used as the positive compound. Kynuramine
was used as MAOs substrate and the final concentration was
45 μM for MAO-A and 30 μM for MAO-B. The detailed
procedure referenced our previous work [19, 20].
Acknowledgements This work was supported by funding from the
China Scholarship Council (NO.201808410456). International Train-
ing of High-level Talents in Henan Province in 2019 (NO.17) (Henan
Foreign Experts Bureau), the Key Scientific Research Project of
Colleges and Universities in Henan Province (NO.20A350006), and
the Special Project of Nanyang Normal University (SYKF2018081,
2020STP006, 2020QN036 and 2020QN045).
Molecular modeling studies
Docking was employed to identify the potential binding of
compound 3b to MAO-B. The crystal structure of huMAO-
B (PDB code: 2V60) was obtained from the Protein Data
Bank after removing the original inhibitors and water
molecules [19, 20]. The 3D Structure of 3b was built and
performed geometry optimization by molecular mechanics.
Docking studies were performed using the AUTODOCK
4.2.6 program and each docked system was performed by
100 runs of the AUTODOCK search by the Lamarckian
genetic algorithm. A cluster analysis was performed on the
docking results using a root mean square tolerance of 1.0
and the lowest energy conformation of the highest popu-
lated cluster was selected for analysis. Graphic manipula-
tions and visualizations were done by Autodock Tools or
Discovery Studio 2.5 software [19, 20].
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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