Pyridoxine-resveratrol hybrids as novel inhibitors of MAO-B with antioxidant and neuroprotective activities for the treatment of Parkinson's disease

Article abstract of DOI:10.1016/j.bioorg.2020.103707

A series of pyridoxine-resveratrol hybrids were designed and synthesized as monoamine oxidase B inhibitors for the treatment of Parkinson's disease. Most of them exhibited potent inhibitory activities on MAO-B with high selectivity. Specifically, compounds 12a, 12g and 12l showed the most excellent inhibition to hMAO-B with the IC₅₀ values of 0.01 μM, 0.01 μM and 0.02 μM, respectively. Further reversibility study demonstrated that 12a and 12l were reversible and 12g was irreversible MAO-B inhibitors. Molecular docking studies of MAO revealed the binding mode and high selectivity of these compounds with MAO-B. In addition, these three representative compounds also exhibited low cytotoxicity and excellent neuroprotective effect in the test on H₂O₂-induced PC-12 cell injury. Moreover, 12a, 12g and 12l showed good antioxidant activities and high blood-brain barrier permeability. Overall, all of these results highlighted 12a, 12g and 12l were potential and excellent MAO-B inhibitors for PD treatment.

Full text of DOI:10.1016/j.bioorg.2020.103707

Journal Pre-proofs
Pyridoxine-resveratrol hybrids as novel inhibitors of MAO-B with antioxidant
and neuroprotective activities for the treatment of Parkinson’s disease
Wei Li, Xia Yang, Qing Song, Zhongcheng Cao, Yichun Shi, Yong Deng, Li
Zhang
PII:
S0045-2068(19)32220-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.103707
YBIOO 103707
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
25 December 2019
14 February 2020
26 February 2020
Please cite this article as: W. Li, X. Yang, Q. Song, Z. Cao, Y. Shi, Y. Deng, L. Zhang, Pyridoxine-resveratrol
hybrids as novel inhibitors of MAO-B with antioxidant and neuroprotective activities for the treatment of
Parkinson’s disease, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103707
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Pyridoxine-resveratrol hybrids as novel inhibitors of MAO-B with antioxidant
and neuroprotective activities for the treatment of Parkinson’s disease
Wei Lia,1, Xia Yanga,1, Qing Songa, Zhongcheng Caoa, Yichun Shia, Yong Denga,*, Li Zhangb,*
aDepartment 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 611041, China
bDepartment of Elderly Digestive, Sichuan Academy of Medical Sciences & Sichuan Provincial
People’s Hospital, Chengdu,610072, China
1 These authors contributed equally to this work.
*Corresponding Author.
E-mail: dengyong@scu.edu.cn (Yong Deng), ZL.gLx.gjq@163.com (Li Zhang)
1

Abstract
A series of pyridoxine-resveratrol hybrids were designed and synthesized as monoamine oxidase B
inhibitors for the treatment of Parkinson’s disease. Most of them exhibited potent inhibitory activities
on MAO-B with high selectivity. Specifically, compounds 12a, 12g and 12l showed the most
excellent inhibition to hMAO-B with the IC50 values of 0.01 μM, 0.01 μM and 0.02 μM, respectively.
Further reversibility study demonstrated that 12a and 12l were reversible and 12g was irreversible
MAO-B inhibitors. Molecular docking studies of MAO revealed the binding mode and high
selectivity of these compounds with MAO-B. In addition, these three representative compounds also
exhibited low cytotoxicity and excellent neuroprotective effect in the test on H2O2-induced PC-12
cell injury. Moreover, 12a, 12g and 12l showed good antioxidant activities and high blood-brain
barrier permeability. Overall, all of these results highlighted 12a, 12g and 12l were potential and
excellent MAO-B inhibitors for PD treatment.
Keywords:
Parkinson’s disease; Pyridoxine-resveratrol hybrids; MAO-B inhibitors; Reversibility; Antioxidant;
Neuroprotective agents.
2

1. Introduction
Parkinson’s disease (PD) is the world’s second common age-associated and progressive
neurodegenerative disease. It is characterized by motor behavioral abnormalities (resting tremors,
bradykinesia, postural instability and rigidity), and accompanied the non-motor symptoms consisting
neuropsychiatric symptoms (depression, cognitive dysfunctions and dementia), sleep disturbance
(insomnia, rapid eye movement disorders, vivid dreaming), autonomic symptoms (bladder disorders,
orthostatic hypotension) and anosmia [1-3]. With the increase of average life expectancy, PD has
become a severe social and economic problem. About 0.3% of the general population and 1-3% of
the population over the age of 65 are affected by PD. Unfortunately, the number of people who suffer
from PD is going to rise from 8.7 to 9.3 million by 2030 [4, 5]. The main pathologies of PD include
the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta of the
midbrain, the presence of lewy bodies mainly formed by fibrillar α-synuclein and the mitochondrial
dysfunction in CNS [6-8]. Recent reports indicated that oxidative stress and inflammation also
participate in the disease pathogenesis [9-11].
Monoamine oxidase B (MAO-B) with flavin adenine dinucleotide (FAD) as the cofactor, is
present in the outer mitochondrial membrane [12]. It is mainly utilized to catalyze the oxidative
deamination (e.g., dopamine, tyramine, benzylamine, phenylethamine), which participates in
Fenton-type reactions with Fe (II) to generate reactive oxygen species [12]. Some evidence suggests
that MAO-B activity increases in the human brain with age, and elderly PD patients have been
proved to show a high MAO-B catalytic rate in their brain. This probably reduces the central
dopamine (DA) supply and increases the production of hydrogen peroxide and aldehyde species,
which may cause the neurodegeneration associated with PD [12, 13]. So drugs with inhibition of
MAO-B are necessary in PD therapy.
It is widely believed that the pathology of PD are associated with oxidative stress [1, 9, 11].
Oxidative stress mainly comes from the overproduction of reactive oxygen species (ROS) such as
hydroxyl radical (OH) and lipid peroxide radical (ROO-) [14]. In human body, many enzymatic
reactions and metabolic events have been considered as the major endogenous sources of ROS [9].
When ROS productions surpass the antioxidant capacity of a cell, it will lead to oxidative stress,
cause irreversible damage to cellular macromolecules and can ultimately result in cell death [12].
Therefore, protection of neuronal cells from oxidative stress is a potential therapeutic strategy for the
3

treatment of PD.
The existing therapies for PD include dopamine pathway, anticholinergics and deep brain
stimulation. All currently approved therapies to increase striatal dopamine levels include levodopa,
dopamine agonists, MAO-B inhibitors (such as rasagiline, selegiline, safinamide and so on),
catechol-O-methyltransferase inhibitors and amantadine [15-17]. Unfortunately, these therapies do
not represent a long-term solution, and there is no disease-modify treatment for PD [6]. In order to
achieve better therapeutic effect, multiple medication therapies and multiple compound medications
both have been applied. However, the hidden drawbacks include complex ADMET properties and
possible drug-drug interactions leading to severe side effects. A multifunctional compound that can
exert multiple bioactivities may be an optimum choice [18, 19].
Resvertrol (RSV) is a polyphenol presented in a variety of plants, such as grapes, peanuts,
berries and so on. In recent years, RSV has been revealed to possess a variety of beneficial properties
which include anti-oxidant, anti-inflammatory, anti-apoptosis, anti-steatotic, anti-proliferative and
neuroprotective properties [20-23]. Some studies also showed that RSV derivatives had a character
of MAO-B inhibition [24]. Pyridoxine (vitamin B6) is considered to be an enzymatic cofactor for at
least 140 enzymes [25]. In addition to its regulatory role, pyridoxine has also been shown to be an
antioxidant. It can inhibit the production of radicals and serve as quenchers for single oxygen [26,
27]. Because of their versatile properties which are beneficial for the treatment of neurodegenerative
diseases, in our previous works, we designed and synthesized a series of pyridoxine-resvertrol
hybrids Mannich base derivatives and found the intermediate bearing ketal-protected six-membered
ring demonstrated excellent MAO-B inhibitory activity. According to the docking results, the
isopropylidene group on the pyridine ring of the intermediate occupies the inlet cavity of the
monoamine oxidase and stably binds with a series of amino acid residues through hydrophobic
interaction [28]. Therefore, we suspect that the ketal-protected six-membered ring on the pyridine
moiety is essential for its MAO-B inhibitory activity. In addition, these hybrids Mannich bases also
showed good antioxidant activity due to the presence of one or more free hydroxyl groups [28].
However, this also brings two disadvantages: first, the presence of hydroxyl group increases the
polarity of the compounds, making them low liposolubility and not good for passing the blood-brain
barrier; second, the compounds are easily metabolized and excreted in the body. Therefore, in this
paper, we reduced the number of free hydroxyl groups by retaining the ketal-protected six-membered
4

ring on the pyridine moiety and introducing different substituent on the benzene ring in order to
improve their lipophilicity and the permeability of the blood-brain barrier, so that it can exert
MAO-B inhibition in the central nervous system and provide a new study direction for the treatment
of PD. Through the above considerations, a new series of pyridoxine-resvertrol hybrid derivatives
were designed, synthesized and evaluated for their biological activities including inhibition of
MAOs, reversibility study of hMAO-B inhibition, molecular docking studies, anti-oxidative
activities, neuroprotective activity and the ability to cross the blood-brain barrier. The design strategy
for pyridoxine-resveratrol hybrid derivatives is depicted in Fig. 1.
Low lipophilicity
Low BBB permeability
Improve lipophilicity and
the permeability of BBB
Occupies the inlet
cavity of the MAO-B
OH
H3C
O
4'
H3C
O
HO
HO
R1
R
N
3'
R2
A M
R = 4'- ~
H3C
N
H3C
N
-B D
R = 3'
,
Pyridoxine-resveratrol hybrids
Pyridoxine-resveratrol Mannich bases
CH
CH3
CH3
N
N
CH3
OCH3
N
CH3
Cl
R
=
OH
CH3
CH3
A
B
C
D
E
F
G
CH
CH
N
N
N
O
N
N CH3
N
N C2H5
N
H
I
J
K
L
M
Figure 1. Design strategy for the pyridoxine-resveratrol hybrid derivatives.
2. Results and discussion
2.1. Chemistry
The synthetic pathways toward the target compounds were summarized in Scheme 1 and
Scheme 2. The 4-(methoxymethoxy)benzaldehydethe (2) was synthesized by using
p-hydroxybenzaldehyde (1) as starting material, and the para-substituted benzaldehyde 2-1g was
prepared from aniline (3), through N-propargylation, N-methylation and Vilsmeier formylation
reaction with DMF/POCl3 [29]. For the preparation of 2-1h, compound 3 underwent
N-dipropargylation reaction to afford dipropargyl-substituted aniline (6), and then formylation by
Vilsmeier reaction. The other para-substituted benzaldehydes 2-1i~m were obtained by the reaction
5

of 4-fluorobenzaldehyde (7) with corresponding secondary amine [30].
The designed compounds were obtained by using the pyridoxine as starting material. In the first
step, commercially available pyridoxine hydrochloride (8) was isopropylidene protected at the 3- and
4-hydroxy groups based on published procedures [31]. Chlorination of the hydroxymethyl on the
5-position gave intermediate 10. 10 reacted with triethyl phosphite through Arbuzov reaction to give
compound 11, which was then subjected to a Wittig-Horner reaction with meta- or para-substituted
benzaldehyde to yield target compounds 12b-12m and 13a-13b. However, 12a was prepared through
different synthetic route. Firstly, 11 reacted with 4-(methoxymethoxy)benzaldehyde (2) to obtain 14,
and then subjected to hydrolysis reaction to afford 15. Finally, the target compound 12a was obtained
through isopropylidene protection reaction of 15. All target compounds were not reported previously
and characterized by 1H NMR, 13C NMR and MS.
CHO
CHO
CH3
N
CH3
N
CH
iv
H
N
CH
iii
CH
NH2
i
ii
OHC
OMOM
OH
2-1g
5
3
4
2
1
v
CHO
CHO
CH
CH
CH
CH
vi
N
N
iv
F
R
OHC
I~M
R =
2-1i~m
6
2-1h
7
Scheme 1. Synthesis of para-substituted benzaldehydes (2, 2-1g-m). Reagents and conditions: (i)
p-hydroxybenzaldehyde, chlormethyl methyl ether, K2CO3, acetone, reflux for 4 h; (ii) 1.5 eq.
propargyl bromide, K2CO3, DMF, at r.t. for 5 h; (iii) (CH3)2SO4, K2CO3, acetone, reflux for 4 h; (iv)
o
POCl3, DMF, at 30 C for 3 h; (v) 2.5 eq. propargyl bromide, K2CO3, DMF, at r.t. for 5 h; (vi)
secondary amine, K2CO3, DMF, at 100 oC for 4 -6 h.
6

H3C
H3C
O
H3C
H3C
O
CH2OH
CH2OH
ii
i
iii
HO
O
CH2Cl
HCl
O
CH2OH
HCl
·
·
H3C
N
H3C
N
H3C
N
8
9
10
H3C
H3C
O
CHO
H3C
H3C
O
O
P
iv
R
O
OEt
OEt
+
O
R
H3C
N
H3C
N
11
2-1b-m
2-2a-b
B~M
R = 4'-
B,D
R = 3'-
12b-m,
13a,
B~M
R = 4'-
R = 3'-
R = 3'-
B
D
v
13b,
H3C
H3C
O
OH
H3C
H3C
O
O
OCH3
vi
HO
OH
O
O
vii
HO
H3C
N
H3C
N
H3C
N
14
15
12a
Scheme 2. Synthesis of pyridoxine-resveratrol hybrids. Reagents and conditions: (i) conc. H2SO4,
acetone, at r.t. for overnight; (ii) SOCl2, toluene, reflux for 30 min; (iii) P(OEt)3, reflux for 4 h; (iv)
o
para- or meta-substituted benzaldehydes (2-1b~m, 2-2a~b), NaH, dry THF, at 0 C for 4-7 h; (v)
4-(methoxymethoxy)benzaldehyde, NaH, dry THF, at 0 oC for 5 h; (vi) 10% HCl, THF, at 70 oC for
3 h; (vii) conc. H2SO4, acetone, at r.t. for 2 h.
2.2. Pharmacology
2.2.1. Recombinant human MAO-A and MAO -B inhibition studies
All of the synthesized compounds were evaluated for their inhibitory activities against
recombinant human MAO-A and MAO-B, and use clorgyline, rasagiline and iproniazid as reference
compounds. The corresponding IC50 values were shown in Table 1.
The screening results of the target compounds demonstrated that all tested compounds showed
weak MAO-A inhibitory activities (percent inhibition < 45% at 10 μM). However, most compounds
exhibited significant MAO-B inhibitory activities with IC50 values ranging from 35.87 μM to 0.01
μM. The results revealed that all pyridoxine-resveratrol hybrids were selective MAO-B inhibitors. A
plenty of evidences showed that selective inhibition of MAO-B was beneficial for the treatment of
PD because the inhibition of MAO-A might cause adverse effects (e.g. cheese reaction) [32, 33].
Among these compounds, 12a, 12g and 12l had excellent MAO-B inhibitory activities with the IC50
7

value of 0.01 μM, 0.01 μM and 0.02 μM, respectively, which were more effective than that of the
positive control drugs rasagiline (IC50 = 0.0437 μM) and iproniazid (IC50 = 4.32 μM). In summary,
12a, 12g and 12l were excellent MAO-B inhibitors.
Compared the MAO-B inhibitory activities of the target compounds (12d, 13b), we found that
compounds (12d) containing para-position substituent group performed better in inhibiting MAO-B
than the one possessing meta-position substituent group (13b). The result revealed that the
introduction of different substituents on para-position might be more helpful for improving MAO-B
inhibitory activities. In addition, the assay results of the compound 12a and 12d showed that MAO-B
inhibitory activities were significantly decreased after the hydroxyl group was methylated. It
revealed that decreasing the electron-donating ability of substituent group would reduce MAO-B
inhibitory activities. And compared the MAO-B inhibitory activities of the target compounds 12g
and 12h, when the substituent group was changed from N-methyl-N-propargylamine to
N,N-dipropargylamine, the MAO-B inhibitory activities was greatly reduced. The result showed that
introducing another propargyl would lead to a dramatic decrease in MAO-B inhibition. In addition,
compared the compounds 12e and 12f, we found that extending the carbon chain was not beneficial
for inhibiting the activities of MAO-B. Also, we found that compound (12i) with cyclic amine group
showed better MAO-B inhibitory activities than 12f which was substituted by chain amine group. It
reflected that compounds with cyclic amine groups might have better potential on MAO-B inhibition
than compounds with chain amine groups.
Table 1 The structures of compound 12a-m, 13a-b and in vitro inhibition of MAO and oxygen
radical absorbance capacity (ORAC, Trolox equivalents) by pyridoxine-resveratrol hybrids and
reference compounds.
H3C
H3C
O
4'
R
3'
O
H3C
N
R
MAO-A
inhibition(%) a
MAO-B
IC50 (μM) b
Comp.
R
ORAC c
position
OH
12a
12b
4´
24.1 ± 1.38
0.01 ± 0.003
n.a.d
2.89 ± 0.12
N.T.e
4´
n.a.d
n.a.d
Cl
12c
4´
11.35 ± 0.13
N.T.e
CH3
8

OCH3
12d
12e
4´
4´
21.3 ± 0.58
18.3 ± 0.12
35.87 ± 0.33
0.68 ± 0.02
N.T.e
CH3
N
CH3
2.87 ± 0.08
CH3
N
12f
12g
12h
4´
4´
4´
8.0 ± 0.43
28.0 ± 3.17
14.7 ± 1.02
27.29 ± 1.70
0.01 ± 0.005
10.98 ± 0.28
1.98 ± 0.15
2.53 ± 0.07
N.T.e
CH3
CH3
N
CH
CH
N
CH
12i
12j
4´
4´
4´
4´
4´
3´
3´
—
—
—
N
N
43.2 ± 1.21
10.5 ± 0.64
0.65 ± 0.02
17.83 ± 1.88
1.62 ± 0.07
0.02 ± 0.005
23.40 ± 1.02
n.a.d
2.26 ± 0.12
N.T.e
N
O
12k
21.9 ± 0.91
2.30 ± 0.10
2.43 ± 0.09
N.T.e
N
N
CH3
C2H5
12l
12.0 ± 0.22
N
N
12m
11.4 ± 0.18
n.a.d
N.T.e
Cl
13a
13b
n.a.d
n.a.d
N.T.e
OCH3
—
Clorgiline
Rasagiline
Iproniazid
0.0079 ± 0.0002 μM f
0.712 ± 0.021 μM f
1.37 ± 0.043 μM f
8.85 ± 0.201
0.0437 ± 0.002
4.32 ± 0.174
N.T.e
—
N.T.e
—
N.T.e
a
Percentages are the percent inhibition of MAO by tested compounds at 10 μM. Data were expressed as mean ±
SD.
b IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and are the mean
of 3 independent experiments, each performed in triplicate. Data were expressed as mean ± SD.
c The mean ± SD of the 3 independent experiments. Data are expressed as μM of Trolox equivalent/μM of tested
compound.
d
n.a. = no active. Compounds defined “no active” means a percent inhibition of less than 5.0% at a concentration
of 10 μM under the assay conditions.
e N.T. = not tested.
f IC50 values of the compounds
2.2.2. Reversibility study of hMAO-B inhibition
In order to further understand the binding mode of the target compounds with MAO-B, the
reversibility of MAO-B inhibition by 12a, 12g and 12l was investigated by measuring the recovery
of MAO-B activities after dialysis of enzyme-inhibitor mixtures [34]. 12a, 12g and 12l at a
concentration of 4 × IC50 were preincubated with MAO-B for a period of 30 min and subsequently
the mixtures were dialyzed for 24 h respectively. As negative and positive controls, MAO-B was also
9

preincubated in the absence of inhibitor, presence of a concentration equal to 4 × IC50 of the
irreversible inhibitor rasagiline (MAO-B IC50 = 0.014 μM) and reversible inhibitor safinamide
(MAO-B IC50 = 0.039 μM). For comparison, undialyzed mixtures of MAO-B-12a, MAO-B-12g,
MAO-B-12l, MAO-B-rasagiline and MAO-B-safinamide were treated under the same conditions.
The results were shown in Fig. 2, the catalytic activities of MAO-B inhibited by rasagiline and
safinamide was restored to 5.6% and 88.5% of the control value (recorded in the absence of inhibitor)
after 24 h dialysis. In contrast, the MAO-B activities in undialyzed mixtures were 1.7% and 37.2% of
the control value. The results suggested that the dialysis model which was used to study the
reversibility of hMAO-B inhibition was successful. The catalytic activity of MAO-B inhibited by the
three target compounds was restored to 90.6%, 3.2% and 90.1% after 24 h dialysis. Meanwhile, the
MAO-B activities in undialyzed mixtures were 36.8%, 1.5% and 38.1%. These data showed that 12a
and 12l were reversible inhibitors, while 12g interacted with MAO-B irreversibly. And the results
were indicated that the different binding modes of these representative compounds might come from
their different chemical structures. The chemical structure of 12g was similar to rasagiline and both
of them all contained the propargylamine group. There has been reported that rasagiline and its
analogue R-M4CPAI occupied the active site cavity of MAO-B and reacted with the flavin forming
an irreversible covalent adduct with the N5 atom of the cofactor (Fig. 3) [35]. Therefore, the
irreversible inhibitor 12g may have the similar binding mode of rasagiline with MAO-B.
Figure 2 Reversibility of the inhibition of MAO-B by 12a, 12g and 12l. MAO-B and 12a, 12g, 12l,
rasagiline, safinamide (at 4 × IC50) were preincubated for 30 min, dialyzed for 24 h and the residual
enzyme activities was measured (12a, 12g, 12l-dialysis). MAO-B was similarly preincubated in the
absence (no-inhibitor) and presence of the irreversible inhibitor rasagiline (rasagiline-dialysis),
reversible inhibitor safinamide (safinamide-dialysis) and dialyzed. For comparison, the residual
10

MAO-B activities of undialyzed mixtures of MAO-B with 12a, 12g, 12l, rasagiline and safinamide
was also shown. The values are given as mean ± standard deviation (SD) of triplicate determinations.
CH
4
2
3
5
1
H3C
H3C
6
O
N
9
8
CH3
7
N 10
O
11
O
12
H3C
CH
N
H3C
N
O
13
O
12g
CH3
4a
5a
N
CH3
4
6
5
HN3
7
8
10
2
1
N
9
9a
10a
O
N
CH2
Cys
CH
N
R
HN
Rasagiline
H3C
Rasagiline-flavocyanine adduct
R-M4CPAI
Figure 3 Chemical formula of rasagiline-related inhibitors. Irreversible inhibitors: R-M4CPAI,
rasagiline, 12g and rasagiline that form a covalent adduct with the flavin.
2.2.3. Molecular modeling study of MAO
To explore the possible interacting mode of the pyridoxine-resveratrol hybrids with MAOs,
molecular modeling study was performed with respect to both isoforms of human MAOs. In view of
the above results, we selected the most potent compound 12a to dock with MAOs based on the X-ray
crystal structures of human MAO-A (PDB code: 2Z5X) and MAO-B (PDB code: 2V60) [36]. To
further elucidate why 12g and 12l displayed the similar inhibitory activities and selectivities of
MAO-B as 12a, docking studies of 12g and 12l with MAOs were also performed (Supplementary
Material Figure S1). Molecular modeling study was performed using the docking program,
AutoDock 4.2 package. The docking results of 12a were shown in Fig. 4 and the estimated binding
energies of compounds 12a, 12g and 12l with MAOs were shown in Table 2.
As shown in Fig. 4A, a hydrogen bond was formed between the hydroxyl group on the benzene
ring of compound 12a with Ala121. The compound 12a-MAO-A complex was stabilized by
hydrophobic interactions with Asn181, Cys323, Ile180, Ile207, Ile325, Ile335, Leu97, Phe108,
Phe208, Phe352, Yyr69, Tyr407, Tyr444, Val210. As can be seen from Fig. 4B, the sterically
hindered isopropylidene group in the pyridine ring occupied the inlet cavity of MAO-B and
interacted with a series of hydrophobic residues to stabilize the complex structure of the docking
compounds with MAO-B, such as Gln206, Ile199, Leu171, Pro102, Pro104, Tyr534, and so on. The
benzene ring moiety of compound 12a and MAO-B formed a complex by the π-π stacking effect
with Tyr398. In addition, a hydrogen bond was observed between the hydroxyl group and Tyr 435.
11

Besides, the nitrogen atom on the pyridine ring formed a hydrogen bond with Tyr326. The binding
energies of target compound 12a with MAO-A and MAO-B were -9.44 kcal/mol and
-11.65kcal/mol.
From the Table 2, we can see the reversible MAO-B inhibitors 12a and 12g showed similar low
MAO-B binding energies but high MAO-A binding energies, which reasonably explained their
excellent and selective MAO-B inhibitory activities. However, just one hydrogen bond was observed
in 12g-MAO-B complex (Figure S1), while there were two in 12a-MAO-B complex. Therefore, 12a
exhibited stronger interaction with MAO-B than 12g, which gave the reason why the MAO-B
inhibitory activity of 12a was greater than that of 12g. As for the irreversible MAO-B inhibitor 12l,
its N-methyl-N-propargylamine moiety is closed to the enzymatic cofactor FAD, indicating this
moiety may form covalent bond subsequently [35]. Overall, the docking results generally showed the
binding modes of pyridoxine-resveratrol hybrids, and explained the reason of their good and
selective MAO-B inhibition.
Figure 4 Docking models of the representative compounds with MAO-A (PDB code: 2Z5X) and -B (PDB code:
2V60). (A) 12a-MAO-A complex. (B) 12a-MAO-B complex. Compound (colored by atom type) interacting with
residues in the binding site of MAO-A and -B, highlighting the protein residues that participate in the main
interactions with the inhibitors.
Table 2 The binding energies of compound 12a, 12g, 12l with MAO-A and MAO-B.
Estimated binding energies (kcal/mol)b
Comp.a
MAO-A
-9.44
MAO-B
-11.65
-12.22
-12.86
12a
12g
12l
-7.69
-4.09
12

a
Representative compounds docked with MAOs based on the X-ray crystal structures of human MAO-A (PDB
code: 2Z5X) and MAO-B (PDB code: 2V60). b The data got from AutoDock 4.2 package.
2.2.4. Antioxidant activity
The antioxidant activities of the pyridoxine-resveratrol hybrids were evaluated by following the
oxygen radical absorbance capacity assay that uses fluorescein (ORAC-FL) [37]. The results were
displayed as Trolox (a water-soluble vitamin E analogue, which was used as a standard) equivalent.
As shown in Table 1, the selected representative compounds all showed strong antioxidant activities,
and the antioxidant capacity index was between 1.98 and 2.89 Trolox equivalents. Compound 12a
showed the most potent antioxidant activities with an ORAC-FL value of 2.89 Trolox equivalents.
And the representative compounds 12g, 12l had almost the same antioxidant capacity (2.53 and 2.43
Trolox equivalents). In addition, the length of chain amine groups may influence the antioxidant
activities. 12e bearing dimethylamino group showed more potent radical scavenge ability than 12f
bearing diethylamino group, indicating the increase of carbon chain length may not be beneficial for
the antioxidant activities. The antioxidant activities of all tested representative compounds which
contained different substituted amines showed no obvious difference.
2.2.5. In vitro blood-brain barrier permeation assay
For a successful CNS drug, it must have the ability to cross the blood brain barrier (BBB) easily
and penetrate into the brain. To predict the brain penetration of the targeted compounds, 12a, 12g
and 12l were selected to be representative compounds. At the same time, the parallel artificial
membrane permeation assay of the blood-brain barrier (PAMPA-BBB) was performed as reported
[38]. First, we compared the permeability of 11 commercial drugs with reported values to validate
the assay. A plot of experimental data versus reported values gave a good linear correlation, Pe (exp.)
= 0.9163 × Pe (bibl.) − 0.2247 (r2 = 0.9558). From this equation and in view of the limit established
by Di et al. for BBB permeation, it can be concluded that compounds with Pe values overtopping
3.44 × 11-6 cm/s could cross the blood-brain barrier. The results in Table 3 demonstrated that 12a,
12g and 12l could penetrate into the CNS with good BBB permeability. It reflected that these
representative compounds might become potential drugs of the central nervous system diseases. And
the results were consistent with our design strategy.
13

Table 3 Permeability results Pe (× 10-6 cm/s) from the PAMPA-BBB assay for 12a, 12g and 12l with
the predicted penetration into the CNS.
Compoundsa
Pe (× 11-6 cm/s)b
6.15 ± 0.35
Prediction
CNS +
12a
12g
12l
7.12 ± 0.41
CNS +
6.69 ± 0.77
CNS +
a
Representative compounds 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.
b Data are the mean ± SD of three independent experiments.
2.2.6 Neuroprotective effect on H2O2-induced PC-12 cell injury
To examine the cytotoxicity of the representative compounds 12a, 12g and 12l, PC-12 cells
were incubated with 12a, 12g, 12l at three different concentrations (10, 50 and 100 μM), and the cell
viability was tested using the MTT assay [39]. The active mitochondria of living cells can cleave
MTT to produce formazan, the amount of formazan was a direct reflection of the living cell number.
As shown in Fig.5, compounds 12a, 12g and 12l showed no influence on the cell viability at the
concentration of 10 μM. However, when the concentration was increased to 50 μM, 12a, 12g and 12l
induced a decrease of cell viability (83%, 85% and 82% of the control value). These results revealed
that these representative compounds had a wide therapeutic safety range up to 10 μM and showed
cytotoxicity at 50 μM. For the further neuroprotective effect evaluation, as shown in Fig.6, cell
viability remarkably decreased to 58% of the control value after exposure PC-12 cells to 100 μM
H2O2, suggesting that PC-12 cells were highly sensitive to H2O2. However, when the cells were
preincubated with compounds 12a, 12g and 12l (1 μM, 10 μM, 50 μM), H2O2-induced cell toxicity
significantly attenuated. The three compounds at 10 μM resulted in about 20% recovery from
H2O2-induced cell injury without cytotoxicity and showed concentration dependency. And their
neuroprotective effect at 10 μM can be similar to or better than the neuroprotective effect of Trolox
(100 μM). The results showed that these representative compounds had great neuroprotective effect
on H2O2-induced PC-12 cell injury. In addition, the reduced cell viability at 50 μM of 12a, 12g, 12l
might be the comprehensive impact result of the neuroprotective effect and cytotoxicity.
14

Figure 5 Cell viability was assessed by measuring the MTT reduction. Three independent
experiments were carried out in triplicate. Data were expressed as mean ± SD and percentage of the
Cont. value. * p < 0.05 vs. Cont. group.
Figure 6 Neuroprotective effects of 12a, 12g and 12l toward PC-12 cells. Cell viability was assessed
by measuring the MTT reduction. Three independent experiments were carried out in triplicate. Data
were expressed as mean ± SD and percentage of the control value. ## p < 0.01 vs control; ** p < 0.01
vs model group (exposure PC-12 cells to H2O2 and without adding any compound).
3. Conclusion
A series of pyridoxine-resveratrol hybrids were designed, synthesized and evaluated as MAO-B
inhibitors for the treatment of PD. In vitro assays demonstrated that most of them exerted high
selective and reversibility inhibitory potency on MAO-B, some of them also performed good
antioxidant activities. Among all the target compounds, 12a, 12g and 12l exhibited significant
antioxidant activities and excellent inhibitory potency for MAO-B with the IC50 values of 0.01 μM,
0.01 μM and 0.02 μM. In addition, these three representative compounds also showed high BBB
permeability. Furthermore, neuroprotective effect of the three compounds on H2O2-induced PC-12
15

cell injury showed that they had a wide therapeutic safety range and great neuroprotective effects.
These properties highlighted that compounds 12a, 12g and 12l might become promising and
excellent MAO-B inhibitors for the treatment of PD.
4. Experimental section
4.1. Chemistry
Unless otherwise noted, all of the materials and reagents were obtained commercially and used
without further purification. All the air sensitive reactions were performed under argon, and all the
reactions were monitored by thin-layer chromatography (TLC) on silica gel GF254 plates from
Qingdao Haiyang Chemical Co. Ltd. (China), and spots were visualized in an iodine chamber or with
an UV light (254 nm). Column chromatography was performed on silica gel (230-400 mesh)
purchased from Qingdao Haiyang Chemical Co. Ltd (China). Melting points (uncorrected) were
1
13
recorded on YRT-3 melting-point apparatus (China). H NMR and C NMR spectra were recorded
using TMS (Tetramethylsilicane) as the internal standard at the temperature 25 °C in CDCl3 with a
Varian INOVA 400 NMR spectrometer. Chemical shifts are reported in parts per millions (ppm)
relative to TMS and the coupling constants in Hz. Mass spectra were recorded on Agilent-6211 TOF
LC-MS Spectrometer.
4.1.1 4-(methoxymethoxy)benzaldehyde (2)
p-Hydroxybenzaldehyde (1, 1.00 g, 8.19 mmol), acetone (9 mL) and anhydrous K2CO3 (2.26 g,
16.38 mmol) were added to the reaction bottle. And the mixture was stirred at room temperature for
15 min. Then a solution of chlormethyl methyl ether (0.75mL, 9.83 mmol) in acetone (3.5 mL) was
added dropwise. The mixture was refluxed for 4 h under argon. After the reaction was completed,
acetone was removed under reduced pressure. The residue was diluted with water (50 mL), and
extracted with ethyl acetate (25 mL × 3). The combined organic phases were washed with brine,
dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain compound
2 as colorless oil, which was used without further purification.
4.1.2 4-(methyl(prop-2-yn-1-yl)amino)benzaldehyde (2-1g)
Aniline (3, 0.30 mL, 3.36 mmol), DMF (2.5 mL) and anhydrous K2CO3 (697 mg, 5.04 mmol)
were added to the reaction bottle. And the mixture was stirred at room temperature under argon for
30 min. Then a solution of propargyl bromide (0.40 mL, 5.04 mmol) in DMF (1.5 mL) was slowly
dropped into the mixture. The mixture was continued to react at room temperature for 5 h under
argon. Then the mixture was poured into ice water and extracted with ethyl acetate (15 mL × 3). The
16

combined organic phases were washed with brine, dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure to afford crude product. After purification by thin-layer
chromatography on silica gel, the pure compound 4 (231 mg, 52.3%) was obtained as a yellow oil.
The prepared propargylaniline (4, 228 mg, 1.74 mmol), anhydrous K2CO3 (360 mg, 2.61 mmol)
and dry acetone (15 mL) were added to the reaction bottle. The mixture was stirred at room
temperature for 30 min, and dimethyl sulfate (0.20 mL, 2.10 mmol) was dropped into the mixture.
After the addition was completed, the mixture was refluxed for 4 h. Then the mixture was poured
into ice water and extracted with ethyl acetate (15 mL × 3). The combined organic phases were
washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure
to obtain compound 5 (230 mg) as a yellow oil, which was used without further purification.
The obtained methylpropargylaniline (5, 153 mg, 1.16 mmol), phosphorus oxychloride (0.12
mL, 1.16 mmol) and DMF (5.0 mL) were added to the dry reaction bottle. The mixture was stirred at
30 oC for 3 h under argon. Then the reaction mixture was basified with saturated aqueous solution of
Na2CO3 and extracted with ethyl acetate (15 mL × 3). The combined organic phases were washed
with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to get
crude product 2-1g. Without purification, 2-1g was directly used for further reaction.
4.1.3 4-(di(prop-2-yn-1-yl)amino)benzaldehyde (2-1h)
In the preparation of propargylidene aniline, the ratio of aniline to propargylidene bromide was
1:2.5, and the other reaction conditions were the same as that of propargylidene aniline. The obtained
N,N-di(prop-2-yn-1-yl)aniline (6, 51 mg, 0.30 mmol), phosphorus oxychloride (0.03 mL, 0.30 mmol)
and DMF (2.5 mL) were added to a dry reaction bottle and reacted at 30 oC for 3 h under argon. Then
the mixture was basified with saturated aqueous solution of Na2CO3 and extracted with ethyl acetate
(15 mL × 3). The combined organic phases were washed with the brine, dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure to obtain crude product 2-1h, which can
be directly used for further reaction without purification.
4.1.4. General procedure for the synthesis of 2-1i-m
p-Fluorobenzaldehyde (0.86 mL, 8.06 mmol), corresponding secondary amines (9.67 mmol),
anhydrous K2CO3 (2.23 g, 16.12 mmol) and DMF (10 mL) were added to the reaction bottle. The
o
mixture was stirred at 100 C for 4-6 h under argon. After the reaction was completed, the mixture
was poured into ice water and the corresponding solid was precipitated. The solid was filtered and
17

washed with ice water. And then the solid was collected and dried at room temperature to obtain the
corresponding substituted benzaldehydes (2-1i-m).
4.1.4.1 Preparation of compound 2i-m by general procedure
4-(pyrrolidin-1-yl)benzaldehyde (2-1i)
A light yellow solid, 68.0% yield; mp: 77.4-79.8 oC (lit.78-80 oC) [40].
4-(piperidin-1-yl)benzaldehyde (2-1j)
A light yellow solid, 64.0% yield; mp: 61.7-63.9 oC (lit.65-66 oC) [41].
4-morpholinobenzaldehyde (2-1k)
A white solid, 72.0% yield; mp: 62,8-64,4 oC (lit.62-63 oC) [40].
4-(4-methylpiperazin-1-yl)benzaldehyde (2-1l)
A light yellow solid, 81.0% yield; mp: 58.0-60.7 oC (lit.58-60 oC) [42].
4-(4-ethylpiperazin-1-yl)benzaldehyde (2-1m)
A yellow oil, 61.3% yield.
4.1.5 Synthesis of (2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)methanol (9)
To a mixture of pyridoxine hydrochloride (8, 5.00 g, 24.31 mmol) in acetone (100 mL) was
added concentrated sulfuric acid (10 mL) dropwise at room temperature. The mixture was stirred at
room temperature for 24 h. Then the mixture was basified with saturated aqueous solution of Na2CO3
and the organic solvent was evaporated under reduced pressure. The residue was dissolved in
dichloromethane (40 mL × 3), then the mixture was washed with water and brine, dried over
anhydrous Na2SO4, filtered and concentrated under reduced pressure to provide crude product. The
cude product was recrystallized from ethyl acetate to give compound 9 (3.12 g, 61.3%) as a white
crystal. mp: 108-109 oC (lit.113-115 oC) [43].
4.1.6. Synthesis of 5-(chloromethyl)-2,2,8-trimethyl-4H-[1,3]dioxino [4,5-c]pyridine (10)
Compound 9 (3.00 g, 14.39 mmol) was dissolved in toluene (50 mL) and thionyl chloride (2.20
mL, 30.24 mmol) in toluene (10 mL) was added dropwise. The mixture was refluxed for 40 min.
Then the mixture was directly filtered and washed by toluene to afford a brown-gray solid compound
10 (2.75 g, yield 84.0%). mp: 181-184 oC (lit.185-187 oC) [44].
4.1.7 Synthesis of diethyl(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)methyl)phosphonate
(11)
A mixture of compound 10 (2.47 g, 9.35 mmol) and P(OEt)3 (18 mL) was refluxed for 4 h under
18

argon. Then the mixture was purified by column chromatography on silica gel using a mixture of
petroleum ether/ethyl acetate (1:1) as eluent to give intermediate 11 (2.83 g, yield 92.0%) as a light
yellow oil.
4.1.8 Synthesis of (E)-5-(4-(methoxymethoxy)styryl)-2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine
(14)
To a mixture of NaH (146 mg, 6.08 mmol) in THF (2 mL) was added a solution of intermediate
11 (500 mg, 1.52 mmol) in THF (4 mL) at 0 oC and the mixture was stirred for 40 min under argon.
Then a solution of 4-(methoxymethoxy)benzaldehyde (253 mg, 1.52 mmol) in THF (1 mL) was
added dropwise, and the mixture was stirred for another 5 h under argon. The mixture was quenched
with 3 mol/L HCl and basified with saturated aqueous solution of Na2CO3. Then the mixture was
extracted with ethyl acetate (15 mL × 3). The combined organic phases were washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to provide crude
product. The crude product was purified on silica gel chromatography using mixtures of petroleum
ether/ethyl acetate (2:1) as eluent to afford compound 14 (292 mg, yield 56.3%) as a colorless oil.
4.1.9 Synthesis of (E)-4-(hydroxymethyl)-5-(4-hydroxystyryl)-2-methylpyridin-3-ol (15)
The intermediate 14 (509 mg, 1.49 mmol) was dissolved in THF (8 mL), and an aqueous
solution of HCl (10%, 8 mL) was added. The mixture was refluxed for 3 h under argon. Then THF
was removed under reduced pressure. The residue was basified with saturated aqueous solution of
Na2CO3 and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed
with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to
provide crude product. The crude product was purified on silica chromatography using mixtures of
petroleum ether/ethyl acetate (2:1) as eluent to afford 15 (337 mg, yield 87.8%) as a light yellow oil.
4.1.10 Synthesis of (E)-4-(2-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)vinyl)phenol (12a)
The intermediate 15 (49 mg, 0.19 mmol) was dissolved in acetone (1 mL) and concentrated
sulfuric acid (0.1 mL) was added dropwise at room temperature. The mixture was stirred at room
temperature for 3 h under argon. Then the mixture was basified with saturated aqueous solution of
Na2CO3 and extracted with ethyl acetate (15 mL × 3). The combined organic phases were washed
with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to
provide crude product. The crude product was purified on silica gel chromatography using mixtures
1
of petroleum ether/ethyl acetate (3:2) as eluent to afford 12a as light yellow oil. yield 29.4%; H
19

NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 7.37 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 16.0 Hz, 1H), 6.89 (d,
J = 8.4 Hz, 2H), 6.66 (d, J = 16.0 Hz, 1H), 4.92 (s, 2H), 2.45 (s, 3H), 1.59 (s, 6H); MS (ESI) m/z:
298.0 [M+H]+.
4.1.11 General procedure for the synthesis of 12b-m
To a mixture of NaH (146 mg, 6.08 mmol) in THF (2 mL) was added a solution of intermediate
11 (500 mg, 1.52 mmol) in THF (4 mL) at 0 oC, and the mixture was stirred for 40 min under argon.
And a solution of corresponding substituted benzaldehydes (2-1b-m, 1.52 mmol) in THF (1 mL) was
added dropwise. Then the mixture was stirred for another 4-7 h under argon. After the reaction was
completed, the mixture was quenched with 3 mol/L HCl and basified with saturated aqueous solution
of Na2CO3. Then the mixture was extracted with ethyl acetate (15 mL × 3) and the combined organic
phases were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure to provide crude product, the crude product was purified on silica chromatography
to afford corresponding target compounds 12b-m.
4.1.11.1 (E)-5-(4-chlorostyryl)-2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine (12b)
Compound 12b was synthesized from 11 and 2-1b according to the general procedure. After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (5:2) as eluent, the
1
pure product 12b was obtained as a light yellow solid, 55.6% yield; mp 136.3-138.2 °C; H NMR
(400 MHz, CDCl3) δ 8.26 (s, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 16.0
13
Hz, 1H), 6.82 (d, J = 16.0 Hz, 1H), 4.91 (s, 2H), 2.43 (s, 3H), 1.57 (s, 6H); C NMR (100 MHz,
CDCl3) δ 146.8, 145.6, 137.4, 135.1, 133.8, 130.7, 128.9 (2C), 127.8 (2C), 126.9, 123.6, 121.6, 99.5,
59.1, 24.6 (2C), 18.5.
4.1.11.2 (E)-2,2,8-trimethyl-5-(4-methylstyryl)-4H-[1,3]dioxino[4,5-c]pyridine (12c)
(
,
t
t
H
-
N
t
)
-
]
i
,
p
i
Compound 12c was synthesized from 11 and 2-1c according to the general procedure. After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (5:2) as eluent, the
pure product 12c was obtained as a colorless oil, 49.6% yield; 1H NMR (400 MHz, CDCl3) δ 8.27 (s,
1H,), 7.39 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 16.0, 1H), 6.80 (d, J = 16.0 Hz,
13
1H), 4.91 (s, 2H), 2.42 (s, 3H), 2.37(s, 3H), 1.57 (s, 6H); C NMR (100 MHz, CDCl3) δ 146.3,
145.5, 138.2, 137.3, 133.9, 132.0, 129.4(2C), 127.4, 126.5(2C), 123.5, 120.0, 99.4, 59.2, 24.6(2C),
21.2, 18.4.
4.1.11.3.(E)-5-(4-methoxystyryl)-2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine (12d)
20

(
(
o
r
,
e
-
o
4
y
Compound 12d was synthesized from 11 and 2-1d according to the general procedure. After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (3:1) as eluent, the
1
pure product 12d was obtained as a yellow oil, 31.7% yield; H NMR (400 MHz, CDCl3) δ 8.26 (s,
1H,), 7.43 (d, J = 8.0 Hz, 2H), 6.97 (d, J = 16.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 2H), 6.70 (d, J = 16.0
Hz, 1H), 4.91 (s, 2H), 3.83 (s, 3H), 2.42 (s, 3H), 1.57 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 159.7,
146.0, 145.5, 137.2, 131.6, 129.4, 127.9 (2C), 127.5, 123.4, 128.7, 124.1 (2C), 99.4, 59.1, 55.2, 24.6
(2C), 18.4.
4.1.11.4
(E)-N,N-dimethyl-4-(2-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)vinyl)aniline
(12e)
Compound 12e was synthesized from 11 and 2-1e according to the general procedure. After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (5:2) as eluent, the
pure product 12e was obtained as a light yellow oil, 45.3% yield; 1H NMR (400 MHz, CDCl3) δ 8.26
(s, 1H), 7.39 (d, J = 9.2 Hz, 2H), 6.96 (d, J = 16.0 Hz, 1H), 6.71(d, J = 9.2 Hz, 2H), 6.62 (d, J = 16.0
Hz, 1H), 4.91 (s, 2H), 3.00 (s, 6H), 2.42 (s, 3H), 1.57 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 150.4,
145.3, 136.9, 132.3, 129.8, 128.1, 127.8 (2C), 124.9, 123.3, 126.2, 122.2 (2C), 99.4, 59.3, 40.3 (2C),
24.7 (2C), 18.3; MS (ESI) m/z: 325.2 [M+H]+.
4.1.11.5 (E)-N,N-diethyl-4-(2-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)vinyl)aniline (12f)
Compound 12f was synthesized from 11 and 2-1f according to the general procedure. After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (3:2) as eluent, the
pure product 12f was obtained as a light yellow oil, 53.2% yield; 1H NMR (400 MHz, CDCl3) δ 8.25
(s, 1H), 7.36 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 16.0 Hz, 1H), 6.66 (d, J = 8.8 Hz, 2H), 6.58 (d, J = 16.0
Hz, 1H), 4.90 (s, 2H), 3.39 (q, J = 7.2 Hz, 4H), 2.41 (s, 3H), 1.57 (s, 6H), 1.18 (t, J = 7.2 Hz, 6H);
13C NMR (100 MHz, CDCl3) δ 147.8, 145.6, 145.2, 136.9, 132.3, 128.3, 128.1 (2C), 123.9, 123.2,
125.6, 121.5 (2C), 99.3, 59.3, 44.4 (2C), 24.7 (2C), 18.4, 12.6 (2C); MS (ESI) m/z: 353.0 [M+H]+.
4.1.11.6 (E)-N-methyl-N-(prop-2-yn-1-yl)-4-(2-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)
vinyl)aniline (12g)
Compound 12g was synthesized from 11 and 2-1g according to the general procedure After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (3:1) as eluent, the
1
pure product 12g was obtained as a yellow oil, 36.9% yield; H NMR (400 MHz, CDCl3) δ 8.26 (s,
1H,), 7.42 (d, J=8.4 Hz, 2H), 6.96 (d, J=16.0 Hz, 1H), 6.84 (d, J=8.4 Hz, 2H), 6.66 (d, J=16.0 Hz,
21

1H), 4.91 (s, 2H), 4.09 (d, J=2.0 Hz, 2H), 3.02(s, 3H), 2.42 (s, 3H), 2.20 (t, J=2.0 Hz, 1H), 1.57 (s,
13
6H); C NMR (100 MHz, CDCl3) δ 148.6, 146.1, 142.2, 137.8, 132.0, 129.8, 127.8 (2C), 126.7,
123.6, 127.3, 123.9 (2C), 99.4, 78.9, 72.1, 59.3, 42.2, 38.5, 24.7 (2C), 18.4; MS (ESI) m/z: 349.1
[M+H]+.
4.1.11.7 (E)-N,N-di(prop-2-yn-1-yl)-4-(2-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)vinyl)
aniline (12h)
Compound 12h was synthesized from 11 and 2-1h according to the general procedure. After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (3:1) as eluent, the
pure product 12h was obtained as a light yellow oil, 40.2% yield; 1H NMR (400 MHz, CDCl3) δ 8.28
(s, 1H), 7.44 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 16.0 Hz, 1H), 6.95 (d, J = 8.8 Hz, 2H), 6.66 (d, J = 16.0
Hz, 1H), 4.94 (s, 2H), 4.17 (d, J = 2.0 Hz, 4H), 2.50 (s, 3H), 2.77 (t, J = 2.0 Hz, 2H), 1.59 (s, 6H);
13C NMR (100 MHz, CDCl3) δ 147.8, 146.3, 144.3, 134.5, 133.1, 128.8, 127.9 (2C), 127.4, 125.4,
126.6, 125.1 (2C), 110.0, 78.8 (2C), 72.8 (2C), 59.2, 40.3 (2C), 24.6 (2C), 17.1; MS (ESI) m/z: 373.2
[M+H]+.
4.1.11.8 (E)-2,2,8-trimethyl-5-(4-(pyrrolidin-1-yl)styryl)-4H-[1,3]dioxino[4,5-c]pyridine (12i)
(
-
y
y
-
y
1
x
,
r
Compound 12i was synthesized from 11 and 2-1i according to the general procedure After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (1:1) as eluent, the
1
pure product 12i was obtained as a yellow oil, 44.6% yield; H NMR (400 MHz, CDCl3) δ 8.26 (s,
1H), 7.38 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 16.0 Hz, 1H), 6.58 (d, J = 8.4 Hz, 2H), 6.55 (d, J = 16 Hz,
1H), 4.91 (s, 2H), 4.33 (s, 4H), 2.42 (s, 3H), 2.02 (s, 4H), 1.57 (s, 6H); 13C NMR (100 MHz, CDCl3)
δ 148.0, 146.4, 145.0, 136.7, 132.7, 128.4, 128.0 (2C), 124.0, 125.3, 121.7 (2C), 99.4, 59.3, 47.6
(2C), 25.4 (2C), 24.7 (2C), 18.2; MS (ESI) m/z: 351.2 [M+H]+.
4.1.11.9 (E)-2,2,8-trimethyl-5-(4-(piperidin-1-yl)styryl)-4H-[1,3]dioxino[4,5-c]pyridine (12j）
Compound 12j was synthesized from 11 and 2-1j according to the general procedure After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (7:2) as eluent, the
pure product 12j was obtained as a light yellow oil, 38.5% yield; 1H NMR (400 MHz, CDCl3) δ 8.25
(s, 1H), 7.36 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 16.0 Hz, 1H), 6.90(d, J = 8.4 Hz, 2H), 6.66 (d, J = 16.0
Hz, 1H), 4.91 (s, 2H), 3.22 (t, J = 5.2 Hz, 4H), 2.42 (s, 3H), 1.71 (t, J = 5.2 Hz, 4H), 1.63-1.60 (m,
13
2H,), 1.57 (s, 6H); C NMR (100 MHz, CDCl3) δ 151.8, 145.7, 145.5, 137.1, 132.0, 127.9, 127.6
(2C), 127.1, 123.3, 127.3, 125.8 (2C), 99.4, 59.3, 48.9 (2C), 25.6 (2C), 24.7 (2C), 24.3, 18.4; MS
22

(ESI) m/z: 365.1 [M+H]+.
4.1.11.10 (E)-2,2,8-trimethyl-5-(4-morpholinostyryl)-4H-[1,3]dioxino[4,5-c]pyridine (12k）
Compound 12k was synthesized from 11 and 2-1k according to the general procedure. After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (2:1) as eluent, the
1
pure product 12k was obtained as a yellow oil, 40.3% yield; H NMR (400 MHz, CDCl3) δ 8.26 (s,
1H,), 7.42 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 16.0 Hz, 1H), 6.90(d, J = 8.4 Hz, 2H), 6.69 (d, J = 16.0
Hz, 1H), 4.91 (s, 2H), 3.87 (t, J = 4.4 Hz, 4H), 3.21 (t, J = 4.4 Hz, 4H), 2.42 (s, 3H), 1.57 (s, 6H); 13C
NMR (100 MHz, CDCl3) δ 151.1, 145.9, 145.5, 137.2, 131.7, 128.2, 127.7 (2C), 123.3, 128.1, 125.3
(2C), 118.8, 99.4, 66.7 (2C), 59.2, 48.7 (2C), 24.6 (2C), 18.4; MS (ESI) m/z: 367.0 [M+H]+.
4.1.11.11
(E)-2,2,8-trimethyl-5-(4-(4-methylpiperazin-1-yl)styryl)-4H-[1,3]dioxino[4,5-c]pyridine
(12l）
Compound 12l was synthesized from 11 and 2-1l according to the general procedure. After
purification by chromatography on silica gel using petroleum ether/acetone (2:1) as eluent, the pure
1
product 12l was obtained as a light yellow oil, 55.7% yield; H NMR (400 MHz, CDCl3) δ 8.25 (s,
1H), 7.40 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 16.0 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 6.67 (d, J = 16.0
Hz, 1H), 4.91 (s, CH2), 3.28 (t, J = 4.8 Hz, 4H), 2.60 (t, J = 4.8 Hz, 4H), 2.41 (s, 3H), 2.37 (s, 3H),
13
1.57 (s, 6H); C NMR (100 MHz, CDCl3) δ 151.0, 145.8, 145.5, 137.2, 131.8, 127.9, 127.7, 127.6
(2C), 123.3, 127.8, 125.5 (2C), 99.3, 59.2, 54.8 (2C), 48.4 (2C), 46.0, 24.6 (2C), 18.4; MS (ESI) m/z:
380.2 [M+H]+.
4.1.11.12
(E)-5-(4-(4-ethylpiperazin-1-yl)styryl)-2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine
(12m）
Compound 12m was synthesized from 11 and 2-1m according to the general procedure. After
purification by chromatography on silica gel using dichloromethane/methanol (30:1) as eluent, the
pure product 12m was obtained as a yellow oil, 24.0% yield; 1H NMR (400 MHz, CDCl3) δ 8.25 (s,
1H), 7.41 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 16.0 Hz, 1H), 6.92 (d, J = 8.4 Hz, 2H), 6.68 (d, J = 16.0
Hz, 1H), 4.91 (s, 2H), 3.31 (t, J = 4.8 Hz, 4H), 2.67 (t, J = 4.8 Hz, 4H), 2.53 (q, J = 7.2 Hz, 2H), 2.41
13
(s, 3H), 1.57 (s, 6H), 1.17 (t, J=7.2 Hz, 3H); C NMR (100 MHz, CDCl3) δ 151.0, 145.8, 145.5,
137.2, 131.8, 127.9, 127.7, 127.6 (2C), 123.3, 127.9, 125.6 (2C), 99.4, 59.2, 52.5 (2C), 52.3, 48.3
(2C), 24.6 (2C), 18.4, 12.7; MS (ESI) m/z: 394.2 [M+H]+.
4.1.12 General procedure for the synthesis of 13a-b
23

13a-b was synthesized from mata-substituted benzaldehyde according to the general method in
4.1.11 of this article.
4.1.12.2 (E)-5-(3-chlorostyryl)-2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine (13a)
Compound 13a was synthesized from 11 and 2-2a according to the general procedure. After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (3:1) as eluent, the
o
1
pure product 13a was obtained as a yellow solid, 61.3% yield; mp: 129.8-131.3 C; H NMR (400
MHz, CDCl3) δ 8.26 (s, 1H), 7.44 (s, 1H), 7.36-7.25 (m, 3H), 6.95 (d, J = 16.0 Hz, 1H), 6.86 (d, J =
13
16.0 Hz, 1H), 4.92 (s, 2H), 2.43 (s, 3H), 1.57 (s, 6H); C NMR (100 MHz, CDCl3) δ 147.0, 145.6,
138.5, 137.4, 134.6, 130.5, 129.9, 128.0, 126.7, 126.3, 124.9, 123.7, 122.4, 99.5, 59.0, 24.6 (2C),
18.5.
4.1.12.3 (E)-5-(3-methoxystyryl)-2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine (13b)
Compound 13b was synthesized from 11 and 2-2b according to the general procedure. After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (3:1) as eluent, the
pure product 13b was obtained as a light yellow oil, 38.0% yield; 1H NMR (400 MHz, CDCl3) δ 8.27
(s, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.09 (d, J = 8.0 Hz, 1H), 7.01 (s, 1H), 6.98 (d, J = 16.0 Hz, 1H),
6.85 (d, J = 8.0 Hz, 1H), 6.84 (d, J = 16.0 Hz, 1H), 4.91 (s, 2H), 3.84 (s, 3H), 2.43 (s, 3H), 1.57 (s,
6H); 13C NMR (100 MHz, CDCl3) δ 159.8, 146.6, 145.5, 138.1, 137.4, 132.0, 129.7, 127.1, 123.6,
121.3, 129.2, 123.7, 122.0, 99.4, 59.1, 55.2, 24.6 (2C), 18.4.
4.2. Biological evaluation
4.2.1. In vitro inhibition of MAO activity
To further study the biological profile of the target compounds, the inhibitory activity against
MAO-A and MAO-B was determined [45]. MAO-A and -B were obtained from commercial sources
(Sigma Co.), pre-aliquoted and stored at -80 °C. The target compounds were dissolved in DMSO
(2.5 mL) and diluted with potassium phosphate buffer (100 mM, pH = 7.40, containing KCl 20.2
mM) before use. The enzymatic reactions were conducted in potassium phosphate buffer (pH = 7.4,
made isotonic with KCl) to a final volume of 500 μL and contained kynuramine, various
concentrations of the test compounds and 4% DMSO as cosolvent. The addition of MAO-A or -B
(7.5 μg/mL) is a trigger of the reaction, and then the mixture was incubated for 30 min at 37 °C. The
reactions were ended by the addition of 400 μL NaOH (2 mol/L) and 1000 μL water and then
centrifuged at 16000 g for 10 min. The rates of MAO catalysis can be conveniently determined by
24

measuring the formation of 4-hydroxyquinoline via fluorescence spectrophotometry at excitation and
emission wavelengths of 310 nm and 400 nm, respectively. Samples containing 4-hydroxyquinoline
(0.047-1.56 μM) dissolved in 500 μL potassium phosphate buffer were prepared to give a linear
calibration curve. IC50 values were estimated from sigmoidal dose-response curves (graphs of the
initial rate of kynuramine oxidation versus the logarithm of inhibitor concentration) and were
determined in triplicate and are expressed as mean ± standard deviation (SD). Each measurement
was run in triplicate and each reaction was repeated for at least three times.
4.2.2. Reversibility study of hMAO-B inhibition
The reversibility of the MAO-B inhibition by 12a, 12g, 12l was investigated by dialysis [34]
employing dialysis bag (Baoke Scientific, USA) with a molecular weight cut-off of 10000, a
flattening width of 24 mm (0.45 mL/cm) and a length of about 8-10 cm. MAO-B (0.03 mg/mL) and
12a, 12g, 12l, at a concentration equal to 4 × IC50, were preincubated for 30 min at 37 °C. These
reactions were conducted in potassium phosphate buffer (100 mM, pH 7.4) containing 5% sucrose
and the final volume was 0.6 mL. As negative and positive controls, MAO-B was also preincubated
in the absence of inhibitor, presence of a concentration equal to 4 × IC50 of the irreversible inhibitor,
rasagiline (MAO-B IC50 = 0.014 μm) and reversible inhibitor safinamide (MAO-B IC50 = 0.039 μm).
The reactions were subsequently dialyzed at 4 °C in 200 mL outer buffer (100 mM potassium
phosphate, pH 7.4, 5.0% sucrose). The outer buffer was replaced with fresh buffer at 3 h and 7 h
after the start of dialysis. After dialysis was started for 24 h, the reactions were diluted twofold with
the addition of kynuramine (dissolved in potassium phosphate buffer, 100 mM, pH 7.4, made
isotonic with KCl), and the residual MAO-B activities were measured as described above (the part of
4.2.1). The final concentration of kynuramine in these reactions was 50 μM while the final inhibitor
concentrations were equal to twofold of their IC50 values for the inhibition of MAO-B. For
comparison, undialyzed mixtures of MAO-B-12a, MAO-B-12g, MAO-B-12l, MAO-B-rasagiline
and MAO-B-safinamide were maintained at 4 °C over the same time period. These reactions were
carried out in triplicate and the residual enzyme catalytic rates were expressed as mean ± SD.
4.2.3. Antioxidant activity
The antioxidant activity was determined by an oxygen radical absorbance capacity assay that
uses fluorescein (ORAC-FL), based on the method previously described [37]. 2,2′-Azo-bis
(amidinopropane)dihydrochloride (AAPH) was purchased from Accela ChemBio Co. Ltd.
25

6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxyic acid (Trolox) and fluorescein (FL) were
purchased from TCI (Shanghai) Development. All the assays were conducted in the wells of a black
96-well plate containing 75 mM phosphate buffer (pH = 7.4), antioxidant (20 μL) and fluorescein
(120 μL, 150 nM final concentration). The mixture was incubated for 15 min at 37 °C, and then
AAPH solution (60 μL, 12 mM final concentration) was added rapidly by an autosampler. The plate
was placed in a Varioskan Flash Multimode Reader (Thermo Scientific) immediately, and the
fluorescence recorded every minute for 90 min with excitation at 485 nm and emission at 535 nm.
The plate was automatically shaken prior to each reading. Trolox, a vitamin E analogue, was used as
standard (1-8 μM, final concentration). A blank (FL + AAPH) using phosphate buffer instead of
antioxidant and Trolox calibration were carried out in each assay. The samples were performed at
different concentration (1-10 μM). All the reaction mixture was prepared in duplicate, and at least
three independent assays were performed for each sample. Antioxidant curves (fluorescence versus
time) were normalized to the curve of the blank in the same assay, and then the area under the
fluorescence decay curve (AUC) was calculated. The net AUC of a sample was obtained by
subtracting the AUC of the blank. ORAC-FL values were expressed as Trolox equivalents by using
the standard curve calculated for each sample, where the ORAC-FL value of Trolox was taken as
1.0, indicating the antioxidant potency of the tested compounds.
4.2.4. In vitro blood-brain barrier permeation assay
To evaluate the in vitro blood-brain barrier permeation assay, the method described by Di et al.
was used with some modifications [38]. Commercial drugs were purchased from Sigma and Alfa
Aesar. Porcine brain lipid (PBL) was obtained from Avanti Polar Lipids. The donor plate
(MATRNPS50) and the acceptor plate (PVDF membrane, pore size is 0.45 μm, MAIPN4550) were
both from Millipore. Filter PDVF membrane units (diameter 25 mm, pore size 0.45 µm) from Pall
Corporation were used to filter the samples. Test compounds were dissolved in DMSO at 5 mg/mL
and diluted 50-fold in PBS/EtOH (70:30) to a final concentration of 100 μg/mL. Then 350 μL of the
diluted compound solution (100 μg/mL) were added to the donor wells. The acceptor wells were
filled with 200 μL of PBS/EtOH (70:30). The filter membrane was coated with PBL in dodecane
(selected empirically as 4 μL volume of 20 μg/mL PBL in dodecane). The acceptor filter plate was
carefully put on the donor plate to form a sandwich (consisting of the aqueous donor with test
compound on the bottom, lipid membrane in the middle and the aqueous acceptor on the top), which
26

was left undisturbed for 18 h at 25 °C. After incubation, the donor plate was removed and the
concentration of the compounds in the acceptor and donor wells was determined using the Varioskan
Flash Multimode Reader (Thermo Scientific). Permeability rates (Pe) was calculated using the
following expression: Pe = –ln [1 – CA(t)/Cequilibrium]/[A × (1/VD + 1/VA) × t] Cequilibrium = [CD(t) × VD
+ CA(t) × VA]/(VD + VA), where A is the filter area, t is the permeation time, VD is the volume of
donor well, VA is the volume in the acceptor well, CA(t) is the compound concentration in acceptor
well at time t, and CD(t) is the compound concentration in donor well at time t. Eleven quality control
standards of known BBB permeability were used to monitor the consistency of each experiment.
Every sample was analyzed at ten wavelengths in four wells and in at least three independent runs.
The results were given as the mean ± standard deviation.
4.2.5 Neuroprotective effect on H2O2-induced PC-12 cell injury
The neuroprotective effect of the representative compounds on H2O2-induced PC-12 cell injury
was determined using the classical MTT assay [39]. For the cell viability assay, briefly, PC-12 cells
were seeded into 96-well plates at a density of 1 × 105 cells per mL in DMEM medium (GIBCO)
supplemented with 10% heat-inactivated bovine calf serum (Hyclone). After incubation overnight,
the medium was replaced with fresh DMEM medium without calf serum and phenol red. And tested
compounds at various concentrations were added and incubated with the cells for further 2 h. Then,
H2O2 (100 mM) was added and the cells were further incubated for 24 h. At the end of the assay, the
cells were incubated with 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) for 4 h at 37 ℃. The supernatants were carefully removed, and 150 mL DMSO was added to
each well. The absorbance at 570 nm of the formazan was determined using a Varioskan Flash
Multimode Reader (Thermo Scientifific). Data were expressed as mean ± SD. To determine the
cytotoxicity of the compound, tested compound was incubated with PC-12 cells for 48 h without the
addition of H2O2, and other procedures were similar to the above mentioned.
4.2.6. Molecular modeling study of MAO
The simulation systems were built based on the X-ray crystal structures of hMAO-A (PDB code:
2Z5X) and hMAO-B (PDB code: 2V60), both are from the Protein Data Bank. The original ligands
and water molecules were removed and hydrogen atoms were added to both proteins and cofactors.
Pretreatment of small molecules involved performing charge calculations and setting rotable bonds.
Then docking study was performed using the AUTODOCK 4.2. The center of the grid box was
27

placed at the center of original ligand and the dimensions of the active site box were set at 60 × 60 ×
60 Å. Each docked system was executed 100 times of the autodock search by using the Lamarckian
genetic algorithm (LGA). Based on the docking results, the root mean square (rms) tolerance of 1.0
was used for clustering analysis, and the lowest energy form of the cluster with the highest filling
density was selected for analysis. Graphic processing and visualization were accomplished by
Autodock Tools 1.5.6 or Discovery Studio 2.5 software.
Acknowledgments
This work was supported by Sichuan Science and Technology Program (2018SZ0014,
2019YFS0088) and the Fundamental Research Funds for the Central Universities.
Appendix A. Supplementary Material
The supplementary data associated with this article can be found in the online version.
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