Discovery of coumarin Mannich base derivatives as multifunctional agents against monoamine oxidase B and neuroinflammation for the treatment of Parkinson's disease

Article abstract of DOI:10.1016/j.ejmech.2019.04.016

Due to the complexity of the pathogenesis of Parkinson's disease (PD), multimodal treatment may achieve better results. In this study, a series of coumarin Mannich base derivatives were designed and synthesized as multifunctional agents for PD treatment. Among the derivatives, 3-(3-(dimethylamino)propanoyl)-7-hydroxy-5-methyl- 2H-chromen-2-one hydrochloride (24) exhibited the most potent and selective hMAO-B inhibitory activity, and anti-inflammatory and neuroprotective effects in the in vitro studies. It significantly attenuated PD-associated behavioural deficits in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Furthermore, preliminary mechanistic studies indicated that 24 could selectively inhibit MAO-B activity, decrease the neuroinflammatory process, and protect tyrosine hydroxylase-immunopositive dopaminergic neurons. These results suggest that 24 is a promising multifunctional agent for effective therapy for PD.

Full text of DOI:10.1016/j.ejmech.2019.04.016

Accepted Manuscript
Discovery of coumarin Mannich base derivatives as multifunctional agents against
monoamine oxidase B and neuroinflammation for the treatment of Parkinson's
disease
Deng Tao, Yue Wang, Xiu-Qi Bao, Bei-Bei Yang, Fan Gao, Lin Wang, Dan Zhang, Li
Li
PII:
S0223-5234(19)30322-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.016
EJMECH 11251
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 January 2019
Revised Date: 8 April 2019
Accepted Date: 8 April 2019
Please cite this article as: D. Tao, Y. Wang, X.-Q. Bao, B.-B. Yang, F. Gao, L. Wang, D. Zhang, L. Li,
Discovery of coumarin Mannich base derivatives as multifunctional agents against monoamine oxidase
B and neuroinflammation for the treatment of Parkinson's disease, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.04.016.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Discovery of coumarin Mannich base derivatives as multifunctional
agents against monoamine oxidase B and neuroinflammation for the
treatment of Parkinson’s disease
#
#
*
Deng Tao , Yue Wang , Xiu-Qi Bao, Bei-Bei Yang, Fan Gao, Lin Wang, Dan Zhang ,
*
Li Li
Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation,
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union
Medical College, Beijing 100050, China
Graphical Abstract

ACCEPTED MANUSCRIPT
Discovery of coumarin Mannich base derivatives as multifunctional
agents against monoamine oxidase B and neuroinflammation for the
treatment of Parkinson’s disease
#
#
*
Deng Tao , Yue Wang , Xiu-Qi Bao, Bei-Bei Yang, Fan Gao, Lin Wang, Dan Zhang , Li
*
Li
Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, State Key
Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica,
Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
Abstract
Due to the complexity of the pathogenesis of Parkinson’s disease (PD), multimodal
treatment may achieve better results. In this study, a series of coumarin Mannich base
derivatives were designed and synthesized as multifunctional agents for PD treatment.
Among
the
derivatives,
3-(3-(dimethylamino)propanoyl)-7-hydroxy-5-methyl-
2
H-chromen-2-one hydrochloride (24) exhibited the most potent and selective hMAO-B
inhibitory activity, and anti-inflammatory and neuroprotective effects in the in vitro
studies. It significantly attenuated PD-associated behavioural deficits in a
1
-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD.
Furthermore, preliminary mechanistic studies indicated that 24 could selectively inhibit
MAO-B activity, decrease the neuroinflammatory process, and protect tyrosine
hydroxylase-immunopositive dopaminergic neurons. These results suggest that 24 is a
promising multifunctional agent for effective therapy for PD.
Keywords
#
D. Tao and Y. Wang contributed equally to this work.
*
Corresponding authors. Tel.: +86-10-63165247, e-mail: annaleelin@imm.ac.cn (L. Li);
Tel: +86-10-63165203, e-mail: danzhang@imm.ac.cn (D. Zhang).

ACCEPTED MANUSCRIPT
Parkinson’s disease; Multifunctional compounds; Coumarin Mannich base; MAO-B
inhibitors; Anti-inflammatory effect
1
. Introduction
Parkinson’s disease (PD) is a neurodegenerative disease characterized by movement
disorders that leads to disability in millions of elderly individuals worldwide [1]. Several
hypotheses have been proposed regarding its pathogenesis, such as mitochondrial
dysfunction, oxidative stress, neuroinflammation, and α-synuclein aggregation [1-3]. The
occurrence of oxidative stress in PD contributes to the degeneration of dopaminergic
neurons, which has a substantial role in the progression of the disease [4-5].
Monoamine oxidase B (MAO-B) catalyses the oxidative deamination of dopamine to
yield hydroperoxide, which participates in Fenton-type reactions with Fe(II) to generate
reactive oxygen species [6]. MAO-B is a valid target for PD treatment, and several
monoamine oxidase B inhibitors (MAO-BIs) have been introduced, including selegiline,
rasagiline and safinamide (Figure 1). Neuroinflammation is a salient feature of
neurodegenerative diseases, including PD, and can exert deleterious effects on neuronal
cells through the generation of reactive oxygen and nitrogen species [7-9].
Neuroinflammatory pathways have also been suggested as potential therapeutic targets
for PD [10].
Figure 1. MAO-BIs approved for the treatment of Parkinson's disease.
Current therapies for PD include levodopa, dopamine agonists and MAO-BIs, which
provide effective symptomatic control by balancing the equilibrium between

ACCEPTED MANUSCRIPT
acetylcholine and dopamine [11-12]. In addition, many clinical trials have been
implemented to explore disease-modifying therapies for PD. However, most of these
trials did not meet the criteria for significant improvement, such as coenzyme Q10
(CoQ10) and PF-06669571, which could reduce oxidative stress in PD and act as a
dopamine D1 receptor partial agonist, respectively [13-14]. Thus, a combination
approach may be more effective in managing PD symptoms and ultimately slowing or
stopping disease progression [12, 15].
For a combination approach to PD treatment, both multiple medication therapies and
multiple compound medications have been applied. However, the underlying defects
include complex ADMET properties and possible drug-drug interactions leading to
severe side effects [16-17]. A multifunctional compound that can exert multiple
bioactivities may be an optimal choice [18-20]. Therefore, developing MAO-BIs with
anti-neuroinflammatory and neuroprotective bioactivities may be desirable to slow or halt
the progression of PD.
To investigate new molecular entities for the treatment of PD, we explore natural
products, which are important sources of novel pharmacologically active agents and have
led to the discovery of many new chemical entities [21-22]. Coumarins are potential and
valuable resources for the prevention and therapy of central nervous system diseases
[23-24]. These agents can display a broad range of biological activities, including
antidepressant [25], antioxidative [26], and MAO-B inhibitory activities [27].
Figure 2. Structures of select 3-substituted coumarins as MAO-BIs.

ACCEPTED MANUSCRIPT
Substitutions at position C3 of the coumarin nucleus have been reported to modulate
MAO-B inhibitory activity and MAO-A/MAO-B inhibitory selectivity [28]. Compounds
1
-6 (Figure 2) are representative 3-substituted coumarins that display potent and selective
activities against MAO-B [29-32]. Coumarin is also a potential nucleus for the
development of anti-inflammatory drugs [33,34]. In particular, 7-hydroxycoumarins,
which are metabolites of coumarins, may show even more potent anti-inflammatory
activity [35,36]. Therefore, we selected coumarin as a scaffold to identify compounds
with good bioactivities for PD treatment in this study.
The introduction of Mannich bases may improve hydrophilic properties and enhance
various biological activities [37,38]. Therefore, a Mannich base was introduced at
position C3 on the coumarin ring through a Mannich reaction between the acetyl group
and various amines to achieve both selective MAO-B inhibition and anti-inflammatory
activities [39]. Thus, we designed and synthesized Mannich bases of 3-acetyl-7-hydroxyl
coumarins to explore their bioactivities in association with PD.
2
2
. Results and discussion
.1 Chemistry
The synthetic routes for intermediates 7-10 and target compounds 11-27 are shown in
Scheme 1. Knoevenagel reaction and Pechmann condensation were separately applied to
synthesize intermediates 7 and 10. Starting from compound 7, intermediates 8 and 9 were
obtained through Williamson synthesis and a bromination reaction using dimethyl sulfate
and N-bromosuccinimide (NBS), respectively.
a
Scheme 1. Synthetic routes to intermediates 7-10 and target compounds 11-27

ACCEPTED MANUSCRIPT
a
Reagents and conditions: (a) piperidine, CH COOH, ethanol, r.t., 5 h; (b) dimethyl
3
sulfate, K CO , acetone, reflux, 1 h; (c) NBS, tetrahydrofuran, reflux, 1 h; (d) C H ONa,
2
3
2
5
ethanol, reflux, 5 h; (e) PFA, secondary amines or heterocyclic amines, ethanol,
concentrated hydrochloric acid, reflux, 2 h.
All the target compounds were synthesized through a Mannich reaction, which was
carried out in ethanol refluxed at 78 °C and pH 2-3. Mixing paraformaldehyde (PFA) and
secondary amines in ethanol first could improve the productivity because the mechanism
involves the formation of a carbonium ion from the amine and formaldehyde [40]. In
addition, the feed sequence and reactant ratio were optimized for the Mannich reaction
(Table S1). The results indicated that ten equivalents of PFA and secondary amines

ACCEPTED MANUSCRIPT
relative to the coumarin intermediates was the most suitable ratio.
2
.2 Inhibitory activity against MAO enzymes in vitro
First, all the synthesized compounds were evaluated for their inhibitory activities
®
against recombinant human MAO-A and MAO-B enzymes using an Amplex Red MAO
Assay Kit. The corresponding IC50 values and selectivity indices (SI, SI = IC50 (MAO-A)/
IC (MAO-B)) are shown in Table 1.
5
0
Table 1. MAO-A and MAO-B inhibitory activities of the target compounds and their
selectivity indexes for MAO-B
a
IC50 (µM)
b
Compd.
SI
hMAO-B
71.38 ± 21.55
94.02 ± 8.90
> 200
hMAO-A
109.57 ± 5.05
89.95 ± 18.34
> 200
1
1
1.53
0.96
-
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
> 200
> 200
-
151.10 ± 22.75
41.11 ± 4.93
21.10 ± 7.46
20.12 ± 1.51
18.11 ± 3.33
4.62 ± 3.15
> 200
110.70 ± 6.37
108.20 ± 5.23
7.63 ± 1.12
12.18 ± 2.02
53.12 ± 6.87
2.46 ± 1.54
> 200
0.73
2.63
0.36
0.61
2.93
0.53
-
14.08 ± 1.23
> 200
34.09 ± 13.37
155.15 ± 16.48
> 200
2.42
-
3.66 ± 1.64
35.98 ± 2.78
119.30 ± 7.07
> 100
2.74
1.14
98.49 ± 0.92
136.10 ± 24.47

ACCEPTED MANUSCRIPT
2
7
43.67 ± 5.28
173.40 ± 23.05
0.75 ± 0.22
3.97
Rasagiline
0.0084 ± 0.0071
88.47
a
b
The results are expressed as the mean ± SD of three experiments.
The selectivity index for MAO-B is defined as IC50(MAO-A)/IC50(MAO-B).
The screening results of the target compounds showed that a methoxy group at the C7
position (17, 18, 19 and 20) significantly enhanced MAO-B and MAO-A inhibitory
activities (Table 1), which is consistent with previous reports [41,42]. The IC and SI
5
0
values indicate that 20 is a good nonselective MAO inhibitor (IC50 = 4.62 µM, SI = 0.53).
Considering the effect of a Mannich base introduced at the C3 position, we found that a
dimethylamino group (11, 17 and 24) might be a key molecular determinant to inhibit
MAO-B activity. In addition, a methyl group at the C5 position (24, 25, 26 and 27) could
improve MAO-B inhibitory activity compared with the counterparts 11, 15, 14 and 13.
The identification of compound 24 as a highly selective MAO-B inhibitor (IC50 = 3.66
µM, SI > 100) was encouraging.
2
.3 Molecular docking simulation
The enzyme assays indicate 20 and 24 as promising MAO-B inhibitors, although they
display different activities against MAO-A. For further insight, the binding modes of 20
and 24 in hMAO-B and hMAO-A were explored using molecular docking. Protein Data
Bank (PDB) crystallographic structures (PDB entry: 4A79 and 2C64) were adopted as
receptor models of hMAO-B and hMAO-A, respectively. The studies were carried out
using the Flexible Docking protocol implemented in Discovery Studio 2016 (DS2016)
[43]. The optimal configuration was selected according to the CDOCKER energy. The
docking results generated were directly loaded and analysed with DS2016.

ACCEPTED MANUSCRIPT
Figure 3. Molecular docking studies of 24 in MAO-B (PDB code: 4A79) and MAO-A
(PDB code: 2C64). (A) Two-dimensional (2D) and three-dimensional (3D) diagrams of
interactions between compound 24 and MAO-B. Ligand 24 and receptor MAO-B are
coloured green and light blue, respectively. For the FAD and residues, oxygen atoms are
coloured red, nitrogen atoms are coloured blue and sulfur atoms are coloured yellow.
Conventional hydrogen bonds are shown as green dashed lines, π-π stacking interactions
as red dashed lines, and π-alkyl interactions as purple dashed lines. (B) 2D and 3D
diagrams of interactions between compound 24 and MAO-A. The MAO-A receptor is
coloured light blue.
Figure 3A and Figure S1A show the positions of 20 and 24 in the binding site of
MAO-B. The coumarin moiety of both compounds binds the substrate cavity near the
flavinadenine dinucleotide (FAD) cofactor and the aromatic sandwich defined by Tyr398
and Tyr435, which is consistent with previous docking studies [30,44-45]. In addition, the
interaction between MAO-B and these two ligands included marked stabilization owing
to hydrogen bonds (HBs) between the oxygen atoms of the lactone ring and Cys172

ACCEPTED MANUSCRIPT
[30,45-46]. Meanwhile, the coumarin nucleus of 24 is more stable than that of 20 in the
substrate cavity because of van der Waals interactions with Tyr398, Tyr435, Phe343,
Tyr60, Ile198 and Leu171. These interactions may explain the significant inhibitory
activities of 20 and 24 against MAO-B.
The docking results of 20 and 24 in complex with MAO-A (Figure 3B and Figure S1B)
showed that the coumarin moiety was stabilized in the entrance cavity compared to the
substrate cavity, similar to the results in MAO-B. The morpholine group at the C3
position of 20 is close to the flavin ring of the FAD cofactor and may interact with the
flavin ring through HB and van der Waals interactions. However, only π-alkyl
interactions occur between 24 and the binding site of MAO-A, which may lead to the
striking difference in the selective inhibition of MAO-B and MAO-A by compounds 20
and 24.
2
.4 Inhibition of NO production
The inhibitory activities of the synthesized compounds against nitric oxide (NO)
release in lipopolysaccharide (LPS)-challenged BV2 cells were detected to evaluate the
anti-inflammatory effects. Most compounds (11-16, 18, 20, and 24-26) were found to
possess good inhibitory activities against inflammation (Table 2), verifying the original
design concept of the target compounds. In addition, two bromide atoms at the C6 and C8
positions (21-23) could impair anti-inflammatory activity. Notably, compounds 11, 13, 15,
1
8, 24, 25 and 26 exhibited stronger inhibitory activities than the positive control drug
curcumin and are thus promising compounds with potent anti-inflammatory effect.
Table 2. NO release inhibitory activities and neuroprotective activities of the target
compounds
a
a
IC50 (µM)
EC50 (µM)
Compd.
b
c
Inhibition of NO production
Cell viability
Cell viability
11
0.56 ± 0.08
> 100
-

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
2.67 ± 0.35
0.61 ± 0.10
2.13 ± 0.41
0.01 ± 0.01
3.64 ± 0.52
> 100
13.04 ± 2.1
-
-
> 100
45.41 ± 5.6
> 100
> 100
1.82 ± 0.22
> 100
> 100
> 100
-
22.89 ± 2.1
> 100
-
0.02 ± 0.01
> 100
-
-
8.61 ± 0.92
> 100
> 100
-
> 100
> 100
75.06 ± 6.1
19.69 ± 1.7
> 100
83.66 ± 6.5
-
24.55 ± 3.51
> 100
-
-
0.22 ± 0.01
0.28 ± 0.03
0.11 ± 0.01
> 100
25.70 ± 3.3
> 100
> 100
> 100
-
Curcumin
1.58 ± 0.21
-
a
b
The results are expressed as the mean ± SD of three experiments.
Cells were damaged by L-Glu. The cellular viability of the L-Glu group was 53% of that
of the control group.
c
Cells were damaged by H O The cellular viability of the H O group was 60% of that
2
2.
2
2
of the control group.
2
.5 Neuroprotection against neuronal injury
MAO-BIs may reportedly have a neuroprotective effect that can modify or slow
disease progression in PD models [47-49]. The present study investigated the
neuroprotective effects of synthesized compounds against neuronal injury caused by

ACCEPTED MANUSCRIPT
L-glutamic acid (L-Glu) and
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays.
The EC values representing neuroprotection are shown in Table 2. Compounds 12, 15
H
2
O
2
.
Cellular viability was detected by
3
5
0
and 24 displayed moderate neuroprotective effects on SH-SY5Y cells challenged by
L-Glu, with EC50 values of 13.04, 22.89 and 25.70 µM, respectively. Meanwhile,
compounds 16 and 24 exhibited good neuroprotective activities in H O -damaged
2
2
SH-SY5Y cells, with EC values of 1.82 and 19.69 µM, respectively. Compounds 17, 18,
5
0
1
9 and 20, with a methoxy group at the C7 position, were not found to protect cells
against damage from L-Glu or H O . Based on these results, 24 was identified as the
2
2
2 2
most potent compound that could promote cellular viability in both L-Glu and H O
injury cell models. Therefore, 24 is a promising compound with good neuroprotective
effects.
2
.6 In vivo efficacy of compound 24 on a PD mouse model
Considering that compound 24 showed both good selective MAO-B inhibitory and
anti-inflammatory activities as well as neuroprotective effects in vitro, we further
evaluated the efficacy of compound 24 in a subacute PD mouse model induced by
1
-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Mice were treated with
compound 24 (50 mg/kg) and selegiline (10 mg/kg) 30 min before MPTP injection.
The rotarod test results showed that the latency of the mice in the MPTP-treated group
significantly decreased compared to that in the control group, suggesting that MPTP
induced behavioural disorder. As we expected, treatment with compound 24 (50 mg/kg)
markedly improved the motor behaviour dysfunction of the mice by increasing their
latency, with an effect equivalent to that of selegiline treatment.
To evaluate the efficacy of compound 24 in more detail, the doses of compound 24
were reduced to 1, 5, and 25 mg/kg. Compared to MPTP-treated mice, the latency of
compound 24-treated mice increased in a dose-dependent manner. Notably, treatment

ACCEPTED MANUSCRIPT
with 25 mg/kg compound 24 significantly increased the latency of the mice. These results
revealed that oral administration of compound 24 could alleviate MPTP-induced motor
dysfunction.
Dopaminergic neurons located in the substantia nigra project to the striatum and other
parts of the basal ganglia and play crucial roles in motor control. Tyrosine hydroxylase
(TH) is an enzyme involved in the synthesis of dopamine in substantia nigra pars
compacta (SNpc) dopaminergic neurons. To determine whether the recovery of motor
ability caused by compound 24 was due to the protection of dopaminergic neurons, the
number of TH-positive dopaminergic neurons was measured by immunohistochemistry.
Figure 4. In vivo effects of compound 24 on the MPTP-induced PD mouse model. To

ACCEPTED MANUSCRIPT
induce the PD model, mice were continuously injected with MPTP for 7 days. Compound
2
4 and selegiline were administered 30 min before MPTP injection, and administration
continued for another 5 days. (A) The effect of compound 24 (50 mg/kg) on the rotarod
test results (n = 15). (B) The effects of different doses of compound 24 (1, 5, and 25
mg/kg) on the rotarod test results (n = 15). (C) The effect of compound 24 on TH-positive
neurons. Representative sections of the SNpc from four mice are shown.
TH-immunopositive cells in the SNpc were counted using Image-Pro Plus 6.0 (Media
Cybernetics, MD, USA). (D) Western blot assay of TH expression. A representative
immunoblot from four mice is shown. Data from the rotarod test are expressed as the
#
#
###
mean ± SEM, and other data are expressed as the mean ± SD; P < 0.01 and
P <
*
**
0
.001 versus control mice; P < 0.05 and P < 0.01 versus MPTP-injected mice.
The substantia nigra TH-positive neurons in the control group exhibited intense
staining, while these neurons in the MPTP-treated group were lightly stained and sparsely
distributed (Figure 4). Compared to the control, MPTP significantly reduced the number
of TH-positive neurons in the SNpc. Compared to MPTP treatment, compound 24 and
selegiline treatment increased the number of TH-positive neurons. Moreover, the
expression of TH in the mouse midbrain decreased markedly in MPTP-injected mice as
measured by Western blotting, while 24 and selegiline treatment significantly increased
TH expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a
housekeeping gene and is generally constant in the same cell or tissue, was used as an
internal control for calculating TH expression. Notably, TH expression was higher in
compound 24-treated mice than in selegiline-treated mice, suggesting that compound 24
had potential efficacy for improving dopaminergic neuron function in the MPTP-induced
subacute PD mouse model.
2
.7 Inhibitory activity of compound 24 against MAO enzymes ex vivo

ACCEPTED MANUSCRIPT
To further verify the inhibitory activity of compound 24 against MAO enzymes ex vivo,
we examined the MAO-B and MAO-A activities in the midbrain of PD mice induced by
MPTP using an Amplex® Red MAO Assay Kit. Compared with MPTP treatment alone,
2
4 (50 mg/kg) treatment markedly suppressed MAO-B activity, whereas MAO-A activity
did not significantly change, suggesting that 24 exhibited a selective MAO-B inhibitory
effect ex vivo (Figure 5). Compound 24 showed strong and selective MAO-B inhibitory
activity based on our ex vitro studies, which may have contributed to the improvement in
motor dysfunction in the MPTP-treated PD mice.
Figure 5. Inhibitory effect of compound 24 on MAO activity ex vivo. (A). Compound 24
(50 mg/kg) treatment inhibited MAO-B activity in MPTP-treated mice. (B). Compound
2
4 (50 mg/kg) treatment did not significantly change MAO-A activity in MPTP-treated
#
##
*
mice. The data are expressed as the mean ± SD; P < 0.001 versus control mice; P <
0
.05 versus MPTP-injected mice; results from three mice are shown.
2
.8 In vivo inhibition of COX-2 and iNOS expression
Cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) are important
regulatory molecules and are highly expressed during the inflammatory response. High
expression levels of iNOS and COX-2 can promote the overproduction of inflammatory
mediator NO and prostaglandin E2 (PGE2) in activated microglia, respectively. To
further evaluate the anti-inflammatory activity of compound 24 in vivo, we tested iNOS
and COX-2 expression in the midbrains of PD mice by Western blot analysis (Figure 6).

ACCEPTED MANUSCRIPT
Figure 6. Compound 24 treatment decreased COX-2 and iNOS expression in the
midbrains of mice. (A) Western blot assay of COX-2 expression in the midbrains of mice
treated with MPTP. A representative immunoblot from four mice is shown. (B) Western
blot assay of iNOS expression in the midbrains of mice treated with MPTP. A
#
#
*
representative immunoblot from four mice is shown. P < 0.01 versus control mice; P <
*
*
0
.05 and P < 0.01 versus MPTP-injected mice.
Compared to control mice, the MPTP-treated mice exhibited significantly increased
COX-2 and iNOS expression in the midbrain, demonstrating that inflammation occurred
in the brains of MPTP-induced PD mice. Additionally, treatment with 50 mg/kg
compound 24 markedly suppressed COX-2 and iNOS expression, suggesting that 24
prevented the neuroinflammatory response in mice. Our present results revealed that
compound 24 exhibited a good anti-inflammatory effect in vivo, which was closely
associated with the neuroprotective effect of 24 on dopaminergic neurons.
3
. Conclusions
In summary, seventeen novel coumarin Mannich base derivatives were designed and
synthesized as multifunctional agents against PD. SAR analysis of these compounds was

ACCEPTED MANUSCRIPT
performed, and a potential candidate for PD treatment was identified. In vitro
experiments indicated that compound 24 is a highly selective MAO-B inhibitor with good
anti-inflammatory and neuroprotective effects. In the MPTP-induced mouse model of PD,
2
4 significantly attenuated PD-associated behavioural deficits and protected
TH-immunopositive dopaminergic neurons. Further mechanistic studies demonstrated
that 24 selectively inhibited MAO-B activity and decreased the inflammatory response in
the midbrains of PD mice.
4
4
. Experimental Section
.1 Chemistry
All reagents were obtained from commercial sources and used without further
1
13
purification. With tetramethylsilane (TMS) as an internal standard, H NMR and
C
NMR spectra were recorded on a Joel ECZ-400S NMR system using DMSO-d as a
6
deuterated solvent. HR-ESIMS data were collected on an Agilent 6520 Accurate-Mass
Q-TOF LC/MS spectrometer. Thin-layer chromatography (TLC) was conducted with
glass precoated with silica gel GF254 (Qingdao Marine Chemical Inc., China) to monitor
the reactions. Melting points were measured by a Yanaco melting point apparatus.
4
.1.1 3-Acetyl-7-hydroxyl-2H-chromen-2-one (7)
Ethyl acetoacetate (3.95 g, 30.0 mmol) was added to a solution of
,4-dihydroxybenzaldehyde (3.45 g, 25.0 mmol) in ethanol (40 mL). Then,
2
hexahydropyridine (2.13 g, 25.0 mmol) dissolved in ethanol (3 mL) was dropped slowly
into the mixture, followed by the addition of acetic acid (0.10 mL). The reaction mixture
was stirred at room temperature for 5 h. Then, the solvent was removed in vacuo.
Subsequently, acetone (15 mL) was added, and a filtration operation was conducted. The
resulting residue was purified to obtain a light yellow crystalline solid (4.60 g, 90%, mp
1
2
8
38-240 °C) by recrystallization using 95% ethanol. H NMR (400 MHz, DMSO-d
6
): δ
.37 (s, 1H), 7.50 (d, J = 8.8 Hz, 1H), 6.49 (dd, J = 8.8, 2.0 Hz, 1H), 6.28 (dd, J = 2.0, 0.8

ACCEPTED MANUSCRIPT
Hz, 1H), 2.48 (s, 3H).
.1.2 3-Acetyl-7-methoxy-2H-chromen-2-one (8)
Anhydrous potassium carbonate (0.71 g, 50.0 mmol) and dimethyl sulfate (4.65 g, 38.0
4
mmol) were added to a stirred suspension of 7 (5.10 g, 25.0 mmol) in acetone (20 mL).
The reaction mixture was refluxed at 57 °C for 1 h, followed by immediate filtration. The
solvent was removed in vacuo. Subsequently, the resulting residue was washed two or
three times with water and dried at 100 °C for 4 h, resulting in a faintly yellow powder
1
(
4.70 g, 86%, mp 150-153 °C). H NMR (400 MHz, DMSO-d ): δ 8.64 (s, 1H), 7.88 (d, J
6
=
8.8 Hz, 1H), 7.07 (d, J = 2.8 Hz, 1H), 7.02 (dd, J = 8.8, 2.8 Hz, 1H), 3.90 (s, 3H), 2.56
(s, 3H).
4
.1.3 3-Acetyl-7-hydroxyl-6,8-dibromo-2H-chromen-2-one (9)
NBS (1.78 g, 10.0 mmol) was added to a stirred suspension of 7 (1.02 g, 5.0 mmol) in
tetrahydrofuran (20 mL), and the reaction mixture was refluxed at 67 °C for 2 h. After
cooling, a filtration operation was conducted. The resulting residue was washed with
1
acetone to obtain a yellow solid (1.50 g, 82%, mp 108-110 °C). H NMR (400 MHz,
DMSO-d
.1.4 3-Acetyl-5-methyl-7-hydroxyl-2H-chromen-2-one (10)
Sodium (0.34 g, 14.8 mmol) was added to ethanol (20 mL), and the reaction mixture
was stirred until the sodium was dissolved completely. Then, 3,5-dihydroxybenzaldehyde
1.81 g, 14.5 mmol) and 2-ethoxymethylene-3-oxobutanoic acid ethyl ester (2.72 g, 14.5
6
): δ 8.56 (s, 1H), 8.23 (s, 1H), 2.55 (s, 3H).
4
(
mmol) were added. The mixture was stirred at 50 °C for 1.5 h. After cooling, the solution
was filtered to obtain a brick red solid. The resulting residue was dissolved in water,
which was then acidified to pH 2-3 with concentrated hydrochloric acid. Subsequently, a
filtration operation was conducted to obtain an orange solid, which was then washed two
or three times with water and dried at 100 °C for 8 h to obtain an orange powder (1.30 g,
1
4
1%, mp 218-220 °C). H NMR (400 MHz, DMSO-d
6
): δ 11.06 (s, 1H, D
2
O
exchangeable), 8.52 (s, 1H), 6.70 (d, J=2.0 Hz, 1H), 6.58 (d, J=2.0 Hz, 1H), 2.55 (s, 3H),

ACCEPTED MANUSCRIPT
2
4
.48 (s, 3H).
.1.5 General procedure for preparation of the target compounds 11-27
Secondary alkyl amine (20.0 mmol, 10 equiv) was added to a stirred suspension of
PFA (0.61 g, 20.0 mmol, 10 equiv) in ethanol (25 mL), and the pH of the solution was
adjusted to 2-3 with concentrated hydrochloric acid. After 2 h of refluxing at 78 °C,
coumarin intermediate (2.0 mmol, 1 equiv) was added, and the reaction mixture was
refluxed at 78 °C for 10-30 h with TLC to monitor it. Subsequently, the mixture was
cooled to room temperature, and filtration was conducted to obtain a yellow solid. The
resulting residue was washed three times with ethanol or methanol to obtain a pure
product.
4
.1.5.1 3-(3-(Dimethylamino)propanoyl)-7-hydroxy-2H-chromen-2-one
11)
hydrochloride
(
1
Yellow solid, yield: 82.8%, mp: 236-238 °C. H NMR (400 MHz, DMSO-d ): δ 8.69 (s,
6
1
3
H), 7.83 (d, J = 8.8 Hz, 1H), 6.91 (dd, J = 8.8, 2.4 Hz, 1H), 6.83 (d, J = 2.4 Hz, 1H),
1
3
.52 (t, J = 7.2 Hz, 2H), 3.36 (t, J = 7.2 Hz, 2H), 2.77 (s, 6H). C NMR (100 MHz,
DMSO-d
6
): δ 193.7, 164.8, 159.0, 157.4, 148.7, 133.0, 118.1, 114.5, 110.7, 101.8, 51.8,
+
+
4
2
4
2.4, 36.6. HR-ESIMS calcd m/z for [M-HCl+H] C H NO 262.1074, found
14 16 4
62.1072.
.1.5.2 3-(3-(Diethylamino)propanoyl)-7-hydroxy-2H-chromen-2-one hydrochloride (12)
1
Yellow solid, yield: 77.9%, mp 194-197 °C. H NMR (400 MHz, DMSO-d
6
): δ 11.41
(
brs, 1H, D
2
2
O exchangeable), 10.11 (brs, 1H, D O exchangeable), 8.70 (s, 1H), 7.84 (d, J
=
8.8 Hz, 1H), 6.91 (dd, J = 8.8, 2.0 Hz, 1H), 6.83 (d, J = 2.0 Hz, 1H), 3.52 (t, J = 7.2 Hz,
1
3
2
H), 3.35 (t, J = 7.2 Hz, 2H), 3.13 (q, J = 7.2 Hz, 4H), 1.23 (t, J = 7.2 Hz, 6H). C NMR
(
100 MHz, DMSO-d ): δ 193.6, 164.8, 159.0, 157.3, 148.6, 132.9, 118.1, 114.5, 110.7,
6
+
+
1
4
01.8, 46.6, 46.1, 36.2, 8.5. HR-ESIMS calcd m/z for [M-HCl+H] C16H20NO 290.1387,
found 290.1382.
.1.5.3 7-Hydroxy-3-(3-(pyrrolidin-1-yl)propanoyl)-2H-chromen-2-one
4
hydrochloride

ACCEPTED MANUSCRIPT
(13)
1
Yellow solid, yield: 71%, mp 228-230 °C. H-NMR (400 MHz, DMSO-d ): δ 11.35
6
(
brs, 1H, D O exchangeable), 10.31 (brs, 1H, D O exchangeable), 8.69 (s, 1H), 7.84 (d, J
2
2
=
8.8 Hz, 1H), 6.90 (dd, J = 8.8, 2.0 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 3.52 (t, J = 6.4 Hz,
1
3
4
H), 3.43 (t, J = 6.8 Hz, 2H), 3.04 (brs, 2H), 1.77-2.09 (m, 4H). C-NMR (100 MHz,
DMSO-d
6
): δ 193.6, 164.7, 159.0, 157.4, 148.6, 133.0, 118.1, 114.5, 110.8, 101.8, 53.3,
+
+
4
2
4
9.0, 37.9, 22.7. HR-ESIMS calcd for [M-HCl+H] C H NO 288.1230, found
16 18 4
88.1230.
.1.5.4 7-Hydroxy-3-(3-(piperidin-1-yl)propanoyl)-2H-chromen-2-one
14)
Yellow solid, yield: 80%, mp 244 °C decomposed. H NMR (400 MHz, DMSO-d
1.28 (brs, 1H, D O exchangeable), 9.51 (brs, 1H, D O exchangeable), 8.70 (s, 1H), 7.84
hydrochloride
(
1
6
): δ
1
2
2
(d, J = 8.8 Hz, 1H), 6.90 (dd, J = 8.8, 2.0 Hz, 1H), 6.81 (d, J = 2.0 Hz, 1H), 3.54 (t, J =
7
.2 Hz, 2H), 3.33-3.47 (m, 4H), 2.90-3.04 (brs, 2H), 1.59-1.99 (m, 5H), 1.39 (brs, 1H).
1
3
C NMR (100 MHz, DMSO-d
10.8, 101.8, 52.5, 51.1, 36.4, 22.6, 21.2. HR-ESIMS calcd for [M-HCl+H] C17
02.1387, found 302.1378.
6
): δ 193.7, 164.7, 159.0, 157.4, 148.7, 133.0, 118.2, 114.5,
+
+
1
3
4
H20NO
4
.1.5.5 7-Hydroxy-3-(3-(morpholino)propanoyl)-2H-chromen-2-one hydrochloride (15)
1
Yellow solid, yield: 84%, mp 238 °C decomposed. H NMR (400 MHz, DMSO-d ): δ
6
1
7
3
3
1
2 2
1.33 (brs, 1H, D O exchangeable), 10.55 (brs, 1H, D O exchangeable), 8.70 (s, 1H),
.84 (d, J = 8.8 Hz, 1H), 6.90 (dd, J = 8.8, 2.0 Hz, 1H), 6.81 (d, J = 2.0 Hz, 1H),
.99-4.03 (m, 2H), 3.67-3.91 (m, 2H), 3.57 (t, J = 6.8 Hz, 2H), 3.42 (t, J = 6.4 Hz, 4H),
1
3
.10 (brs, 2H). C NMR (100 MHz, DMSO-d ): δ 193.6, 164.7, 159.0, 157.4, 148.7,
6
33.0, 118.1, 114.5, 110.8, 101.8, 63.3, 51.4, 51.2, 36.1. HR-ESIMS calcd for
+
+
[
5
M-HCl+H] C16H18NO 304.1179, found 304.1177.
4
.1.5.6 3-(3-(4-Benzylpiperazin-1-yl)propanoyl)-7-hydroxy-2H-chromen-2-one
hydrochloride (16)

ACCEPTED MANUSCRIPT
1
Yellow solid, yield: 21%, mp 209-211 °C. H NMR (400 MHz, DMSO-d
6
): δ 11.37
(
brs, 1H, D O exchangeable), 8.69 (s, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.61 (brs, 2H),
2
7
2
1
3
4
.42-7.51 (m, 3H), 6.90 (dd, J = 8.6, 2.0 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 4.17-4.59 (brs,
1
3
H), 3.21-4.05 (m, 12H). C NMR (100 MHz, DMSO-d ): δ 193.4, 164.8, 159.0, 157.4,
6
48.8, 133.0, 131.3, 129.5, 128.8, 118.0, 114.5, 110.8, 109.5, 101.9, 58.5, 50.9, 48.4, 47.7,
+
+
6.3. HR-ESIMS calcd for [M-HCl+H] C23
.1.5.7 3-(3-(Dimethylamino)propanoyl)-7-methoxy-2H-chromen-2-one hydrochloride
17)
Yellow solid, yield: 60%, mp 200-201 °C. H NMR (400 MHz, DMSO-d ): δ 10.35
25 2 4
H N O 393.1809, found 393.1806.
(
1
6
(
brs, 1H, D
2
O exchangeable), 8.75 (s, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 2.4 Hz,
1
H), 7.05 (dd, J = 8.8, 2.4 Hz, 1H), 3.91 (s, 3H), 3.55 (t, J = 7.2 Hz, 2H), 3.37 (t, J = 7.2
1
3
Hz, 2H), 2.78 (s, 6H). C NMR (100 MHz, DMSO-d ): δ 193.7, 165.2, 158.8, 157.2,
6
1
48.4, 132.4, 119.3, 113.7, 111.8, 100.3, 56.4, 51.7, 42.3, 36.6. HR-ESIMS calcd
+
+
[
M-HCl+H] for C H NO 276.1230, found 276.1226.
15 18 4
4
.1.5.8 7-Methoxy-3-(3-(piperidin-1-yl)propanoyl)-2H-chromen-2-one
18)
Yellow solid, yield: 45%, mp 182-184 °C. H NMR (400 MHz, DMSO-d ): δ 10.28
hydrochloride
(
1
6
(
brs, 1H, D O exchangeable), 8.76 (s, 1H), 7.94 (d, J = 8.8 Hz, 1H), 7.12 (d, J = 2.0 Hz,
2
1
4
H), 7.06 (dd, J = 8.8, 2.0 Hz, 1H), 3.92 (s, 3H), 3.60 (t, J = 7.2 Hz, 2H), 3.30-3.46 (m,
1
3
6
H), 2.90 (brs, 2H), 1.65-1.85 (m, 5H), 1.39 (brs, 1H). C NMR (100 MHz, DMSO-d ):
δ 193.7, 165.2, 158.8, 157.2, 148.4, 132.5, 119.3, 113.8, 111.8, 100.3, 56.4, 52.3, 50.9,
+
+
3
3
4
6.3, 22.5, 21.3. HR-ESIMS calcd for [M-HCl+H] C H NO 316.1543, found
18 22 4
16.1540.
.1.5.9 7-Methoxy-3-(3-(pyrrolidin-1-yl)propanoyl)-2H-chromen-2-one
19)
Yellow solid, yield: 50.5%, mp 177-181 °C. H NMR (400 MHz, DMSO-d
brs, 1H, D O exchangeable), 8.74 (s, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 2.4 Hz,
hydrochloride
(
1
6
): δ 10.61
(
2

ACCEPTED MANUSCRIPT
1
1
1
H), 7.05 (dd, J = 8.8, 2.4 Hz, 1H), 3.91 (s, 3H), 3.40-3.58 (m, 6H), 3.03 (brs, 2H),
1
3
.91-2.07 (m, 4H). C NMR (100 MHz, DMSO-d ): δ 193.7, 165.2, 158.8, 157.2, 148.4,
6
32.5, 119.3, 113.7, 111.8, 100.3, 56.4, 53.1, 48.8, 37.9, 22.7. HR-ESIMS calcd for
+
+
[
M-HCl+H] C H NO 302.1387, found 302.1382.
17 20 4
4
.1.5.10 7-Methoxy-3-(3-morpholinopropanoyl)-2H-chromen-2-one hydrochloride (20)
1
Yellow solid, yield: 54%, mp 172-175 °C. H NMR (400 MHz, DMSO-d
6
): δ 10.90
(
brs, 1H, D O exchangeable), 8.75 (s, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 2.4 Hz,
2
1
3
H), 7.05 (dd, J = 8.8, 2.4 Hz, 1H), 3.89-3.99 (overlapped, 5H), 3.78 (t, J = 12.0 Hz, 2H),
1
3
.60 (t, J = 7.0 Hz, 2H), 3.38-3.47 (m, 4H), 3.10 (dd, J = 12.0, 7.0 Hz, 2H). C NMR
): δ 193.6, 165.2, 158.8, 157.2, 148.4, 132.4, 119.3, 113.7, 111.8,
(100 MHz, DMSO-d
6
+
+
1
3
4
00.3, 63.2, 56.4, 51.3, 51.0, 36.0. HR-ESIMS calcd for [M-HCl+H] C17
H20NO
5
18.1336, found 318.1331.
.1.5.11 6,8-Dibromo-3-(3-(dimethylamino)propanoyl)-7-hydroxy-2H-chromen-2-one
hydrochloride (21)
1
Yellow solid, yield: 65%, mp 260 °C decomposed. H NMR (400 MHz, DMSO-d
6
): δ
1
3
1
2
0.14 (brs, 1H, D O exchangeable), 8.61 (s, 1H), 8.23 (s, 1H), 3.51 (t, J = 7.2 Hz, 2H),
1
3
.38 (t, J = 7.2 Hz, 2H), 2.78 (d, J = 4.8 Hz, 6H). C NMR (100 MHz, DMSO-d ): δ
6
93.3, 158.8, 158.4, 153.3, 147.0, 133.1, 118.0, 111.9, 109.3, 98.7, 51.8, 42.4, 36.6.
+
+
HR-ESIMS calcd [M-HCl+H] for C H Br NO 419.9264, found 419.9261.
1
4
14
2
4
4
.1.5.12 6,8-Dibromo-7-hydroxy-3-(3-morpholinopropanoyl)-2H-chromen-2-one
hydrochloride (22)
1
Yellow solid, yield: 64.5%, mp 230-231 °C decomposed. H NMR (400 MHz,
DMSO-d ): δ 10.60 (brs, 1H, D O exchangeable), 8.61 (s, 1H), 8.22 (s, 1H), 3.95 (t, J =
6
2
1
3
1
2.0 Hz, 2H), 3.75 (t, J = 12.0 Hz, 2H), 3.55 (t, J = 7.2 Hz, 2H), 3.38-3.47 (m, 4H),
1
3
.05-3.16 (brs, 2H). C NMR (100 MHz, DMSO-d
6
): δ 193.1, 158.7, 158.4, 153.3, 147.1,
+
33.1, 117.9, 111.9, 109.2, 98.7, 63.3, 51.4, 51.1, 36.0. HR-ESIMS calcd [M-HCl+H] for
+
C H Br NO 461.9369, found 461.9366.
16
16
2
5

ACCEPTED MANUSCRIPT
4
.1.5.13 6,8-Dibromo-7-hydroxy-3-(3-(pyrrolidin-1-yl)propanoyl)-2H-chromen-2-one
hydrochloride (23)
1
Yellow solid, yield: 62%, mp 220-222 °C decomposed. H NMR (400 MHz,
DMSO-d ): δ 10.31 (brs, 1H, D O exchangeable), 8.60 (s, 1H), 8.23 (s, 1H), 3.39-3.56 (m,
6
2
1
3
6
1
H), 2.98-3.10 (m, 2H), 1.90-2.06 (m, 4H); C-NMR (100 MHz, DMSO-d
6
): δ 193.2,
58.7, 158.4, 153.3, 147.1, 133.1, 118.0, 111.9, 109.2, 98.7, 53.2, 48.9, 37.8, 22.7.
+
+
HR-ESIMS calcd [M-HCl+H] for C H Br NO 445.9420, found 445.9415.
1
6
16
2
4
4
.1.5.14 3-(3-(Dimethylamino)propanoyl)-7-hydroxy-5-methyl-2H-chromen-2-one
hydrochloride (24)
1
Yellow solid, yield: 77%, mp 235-238 °C. H NMR (400 MHz, DMSO-d
6
): δ 11.41
O exchangeable), 8.61 (s, 1H), 6.78 (d, J
2.0 Hz, 1H), 6.68 (d, J = 2.0 Hz, 1H), 3.54 (t, J = 7.2 Hz, 2H), 3.37 (t, J = 7.2 Hz, 2H),
(
2 2
brs, 1H, D O exchangeable), 10.32 (brs, 1H, D
=
1
3
2
1
.78 (s, 6H), 2.51 (overlapped, 3H). C NMR (100 MHz, DMSO-d ): δ 193.7, 164.5,
6
58.9, 158.2, 145.2, 141.6, 117.1, 115.5, 109.8, 100.0, 51.8, 42.2, 36.6, 18.0. HR-ESIMS
+
+
4
calcd [M-HCl+H] for C15H18NO 276.1230, found 276.1229.
4
.1.5.15 7-Hydroxy-5-methyl-3-(3-(morpholino)propanoyl)-2H-chromen-2-one
hydrochloride (25)
1
Yellow solid, yield: 86%, mp 239-241 °C decomposed. H NMR (400 MHz,
DMSO-d ): δ 11.34 (brs, 1H, D O exchangeable), 10.79 (brs, 1H, D O exchangeable),
6
2
2
8
3
2
1
.62 (s, 1H), 6.77 (d, J = 2.0 Hz, 1H), 6.67 (d, J = 2.0 Hz, 1H), 3.94 (t, J = 12.0 Hz, 2H),
.77 (t, J = 12.0 Hz, 2H), 3.59 (t, J = 7.6 Hz, 2H), 3.42 (t, J = 7.6 Hz, 4H), 3.01-3.19 (brs,
1
3
H), 2.51 (overlapped, 3H). C NMR (100 MHz, DMSO-d ): δ 193.6, 164.5, 158.9,
6
58.2, 145.2, 141.6, 117.1, 115.5, 109.8, 100.0, 63.3, 51.3, 51.1, 36.0, 18.0. HR-ESIMS
+
+
calcd for [M-HCl+H] C H NO 318.1336, found 318.1331.
1
7
20
5
4
.1.5.16 7-Hydroxy-5-methyl-3-(3-(piperidin-1-yl)propanoyl)-2H-chromen-2-one
hydrochloride (26)
1
Yellow solid, yield: 84%, mp 227-230 °C. H NMR (400 MHz, DMSO-d ): δ 11.34
6

ACCEPTED MANUSCRIPT
(brs, 1H, D
2
O exchangeable), 10.09 (brs, 1H, D
2
O exchangeable), 8.61 (s, 1H), 6.77 (s,
1
1
1
H), 6.68 (s, 1H), 3.58 (t, J = 7.2 Hz, 2H), 3.34-3.49 (m, 4H), 2.92-3.01 (brs, 2H),
1
3
.60-1.85 (m, 5H), 1.30-1.45 (brs, 1H). C NMR (100 MHz, DMSO-d ): δ 193.7, 164.5,
6
59.0, 158.2, 145.2, 141.6, 117.1, 115.5, 109.8, 100.0, 52.4, 51.0, 36.3, 22.5, 21.2, 18.0.
+
+
4
HR-ESIMS calcd for [M-HCl+H] C18H22NO 316.1543, found 316.1541.
4
.1.5.17 7-Hydroxy-5-methyl-3-(3-(pyrrolidin-1-yl)propanoyl)-2H-chromen-2-one
hydrochloride (27)
1
Yellow solid, yield: 95%, mp 230-231 °C decomposed. H NMR (400 MHz,
DMSO-d ): δ 11.33 (brs, 1H, D O exchangeable), 10.41 (brs, 1H, D O exchangeable),
6
2
2
8
2
.61 (s, 1H), 6.77 (d, J = 2.0 Hz, 1H), 6.67 (d, J = 2.0 Hz, 1H), 3.40-3.57 (m, 6H),
1
3
.96-3.12 (brs, 2H), 2.51 (overlapped, 3H), 1.91-2.06 (m, 4H). C NMR (100 MHz,
DMSO-d ): δ 193.7, 164.5, 159.0, 158.2, 145.2, 141.6, 117.1, 115.5, 109.8, 100.0, 53.3,
6
+
+
4
3
4
9.0, 37.9, 22.7, 18.0. HR-ESIMS calcd for [M-HCl+H] C H NO 302.1387, found
17 20 4
02.1384.
.2 BV-2 cell culture, treatment and NO assay
BV-2 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Solarbio, Beijing,
China) containing 10% foetal bovine serum (FBS, Sijiqing, Hangzhou, China), 100 U/ml
penicillin, and 100 µg/mL streptomycin in a 5% CO atmosphere at 37 °C. Cells were
2
4
plated into 96-well plates at a density of 1.5 × 10 cells/well. After 24 h, different
concentrations of the screened compounds and the positive control drug curcumin were
added to the plates. After 1 h, the cells were challenged with LPS (500 ng/mL), and
incubation was continued for a further 24 h. Finally, one hundred microlitres of each
supernatant was collected, and the NO concentration was detected using Griess reagent.
The OD value was measured at 540 nm on a microplate reader. We calculated the
-
concentration of NO
2
to reflect the concentration of NO.
4
.3 SH-SY5Y cell culture, treatment and MTT reduction assay
SH-SY5Y cells were grown in DMEM supplemented with 10% FBS, 100 U/mL

ACCEPTED MANUSCRIPT
penicillin, and 100 µg/mL streptomycin in a 5% CO
2
humidified atmosphere at 37 °C.
3
Cells were plated into 96-well plates at a density of 8×10 cells/well. After 24 h, the test
compounds were added to the plates, and after 1 h, L-glutamic acid / H O was added to
2
2
achieve a final concentration of 160 mM / 400 µM. After another 24 h, the medium in
each well was removed and replaced with 100 µL of MTT solution (0.5 mg/mL). After 4
h, the solution was discarded, and 150 µL of DMSO was added to each well. Finally, the
absorbance was measured at a wavelength of 570 nm on a microplate reader.
4
.4 Animals, drug administration and development of the PD model
Ten-week-old male C57BL/6 mice weighing 23-25 g were supplied by the Animal
Center of the Chinese Academy of Medical Sciences and housed under the following
standard conditions: constant temperature (24 ± 1 °C), humidity (relative, 60%) and a 12
h light/dark cycle. All experimental procedures were performed in accordance with the
instructions of the Beijing Municipal Ethics Committee for the Care and Use of
Laboratory Animals.
Before experimentation, all mice were allowed to acclimate to the conditions described
above for 3 days, and mice exhibiting uncoordinated movement according to the rotarod
test were removed. Then, the subacute PD mouse model was prepared according to
previously described methods [50]. Mice were injected with MPTP⋅HCl (Sigma, Aldrich,
USA; 30 mg/kg) for 7 consecutive days. Compound 24 (1, 5, 25 mg/kg or 50 mg/kg) and
selegiline (10 mg/kg) suspended in 0.5% CMC-Na were administered 30 min before
MPTP injection for 7 days, and administration continued for the next 5 days after the last
injection of MPTP. The rotarod test was conducted on the 12th day of experimentation.
After behavioural tests, four mice in each group were perfused with PFA for TH
immunohistochemistry, and fresh striatum and midbrain tissues were isolated for the
measurement of TH, iNOS, COX-2 and other biomarkers.
4
.5 Rotarod test
The rotarod test is widely used to demonstrate animals’ balance and coordination on a

ACCEPTED MANUSCRIPT
self-propelled rotating beam. In this study, the rotarod test was performed as previously
described [50]. Briefly, animals were positioned on the rotarod and then tested on the
revolving rod for up to 120 s at 30 r.p.m. The time until each mouse first fell off the rod
was automatically recorded by the rotarod and reported as the latency. For each animal,
the experiment was repeated two times, and the average time was calculated.
4
.6 MAO inhibitory studies in vitro
The MAO inhibitory activity of compound 24 was determined according to the
instructions supplied in an Amplex® Red MAO Assay Kit (Invitrogen, CA, USA). One
hundred microlitres of compound 24 at varying concentrations was incubated in black
opaque 96-well microplates with hMAO-B (Sigma, Aldrich, USA; 0.0075 mg/mL) or
hMAO-A (Sigma, Aldrich, USA; 0.0011 mg/mL) for 60 min at 37 °C. Enzymatic
reactions were started by the addition of 100 µL of a solution containing 200 µM Amplex
red, 2ꢀU/mL horseradish peroxidase and 2 mM p-tyramine in 0.25 mM sodium phosphate
solution. After 30 min, the fluorescence production of resorufin was measured at 560/590
nm (excitation: 560ꢀnm, emission: 590ꢀnm) in a spectrophotometric plate reader to reflect
the activity of MAO-A / MAO-B.
4
.7 MAO inhibitory studies ex vivo
Compound 24 (50 mg/kg) was administered (i.g.) to subacute PD mice for in vivo
MAO inhibitory studies. The concentration of midbrain homogenates was adjusted to 1
mg/mL. Then, 100 µL of midbrain homogenates and an equal volume of the mix solution
were added for 30-min incubation at 37 °C. The remaining steps were the same as those
for MAO enzyme measurement in our in vitro studies.
4
.8 Histochemical analysis
In this study, immune-histochemical analysis was performed as previously described
[51]. Mice were anaesthetized with 1% pentobarbital sodium (50 mg/kg, i.p.), perfused
transcardially with 0.9% saline, and then fixed by perfusion with 4% PFA in 0.1 M
phosphate buffer. Isolated brains were postfixed in 4% PFA containing 30% sucrose

ACCEPTED MANUSCRIPT
solution and then cut into 40-µm sections using a freezing microtome. Coronal sections
through the substantia nigra were processed for TH immunohistochemistry and incubated
with the TH antibody (ABclonal, Wuhan, China). Then, labelling was visualized using
0
.04% hydrogen peroxidase and 0.05% DAB. Finally, the sections were observed by light
microscopy. The number of TH-immunopositive cells was calculated using Image-Pro
Plus 6.0 (Media Cybernetics, MD, USA).
4
.9 Western blot assay
The midbrain tissues of the mice were lysed using nondenaturing lysis buffer, and
immunoblotting was performed as previously described [52]. Protein homogenates were
subjected to Western blot analysis using antibodies against TH (ABclonal, Wuhan,
China), COX-2 (Abcam, MA, USA) and iNOS (Abcam, MA, USA). The blots were
visualized by incubating the membranes with ECLꢀPlus reagents (Yeasen, Shanghai,
China) and developed with an LAS 4000 chemiluminescence system (Fujifilm, Tokyo,
Japan). The densities of the bands were determined using Gel-Pro Analyzer 4.0 software
(Media Cybernetics, MD, USA).
4
.10 Statistical analysis
The data are expressed as the mean ± SD. Statistical analysis was carried out using
one-way ANOVA. P-values less than 0.05 were considered statistically significant.
Supporting Information
Optimization of the Mannich reaction, diagrams of the interactions of 20 with MAO-A
and MAO-B, and the NMR and HR-ESIMS spectra for compounds 7-27 are included.
This material is available free of charge via the Internet at https://.
Acknowledgements
We thank Dr. Qi Gao for his help with the synthesis process. This work was financially
supported by the CAMS Innovation Fund for Medical Sciences (CIFMS, No.

ACCEPTED MANUSCRIPT
2
016-I2M-3-009), the National Sciences Foundation of China (81630097, 81773718), the
National Major Scientific and Technological Special Project (2018ZX09711-001-005,
018ZX09711-003-020, 2018ZX09711-008-005), and the CAMS Fundamental Research
2
Funds for the Central Universities (2018RC350002).
References
1
2
3
4
. W. Poewe, K. Seppi, C.M. Tanner, G.M. Halliday, P. Brundin, J. Volkmann, A.E.
Schrag, A.E. Lang, Parkinson disease, Nat. Rev. Dis. Prim. 3 (2017) 17013.
. Z.T. Kincses, L. Vecsei, Pharmacological therapy in Parkinson’s disease: focus on
neuroprotection, CNS Neurosci. Ther. 17 (2011) 345-367.
. H.E. Moon, S.H. Paek, Mitochondrial dysfunction in Parkinson's disease, Exp.
Neurobiol. 24 (2015) 103-116.
. L.F. Burbulla, P. Song, J.R. Mazzulli, E. Zampese, Y.C. Wong, S. Jeon, D.P. Santos, J.
Blanz, C.D. Obermaier, C. Strojny, J.N. Savas, E. Kiskinis, X. Zhuang, R. Krüger,
D.J. Surmeier, D. Krainc, Dopamine oxidation mediates mitochondrial and
lysosomal dysfunction in Parkinson’s disease, Science 357 (2017) 1255-1261.
. T. Jiang, Q. Sun, S. Chen, Oxidative stress: A major pathogenesis and potential
therapeutic target of antioxidative agents in Parkinson’s disease and Alzheimer’s
disease, Prog. Neurobiol. 147 (2016) 1-19.
5
6
7
. D.N. Hauser, T.G. Hastings, Mitochondrial dysfunction and oxidative stress in
Parkinson's disease and monogenic parkinsonism, Neubiol. Dis. 51 (2013) 35-42.
. A. De Virgilio, A. Greco, G. Fabbrini, M. Inghilleri, M.I. Rizzo, A. Gallo, M. Conte,
C. Rosato, M.C. Appiani, M. de Vincentiis, Parkinson's disease: autoimmunity and
neuroinflammation, Autoimmun. Rev. 15 (2016) 1005-1011.
8
. K. Kaur, J.S. Gill, P.K. Bansal, R. Deshmukh, Neuroinflammation - a major cause for
striatal dopaminergic degeneration in Parkinson's disease, J. Neurol. Sci. 381 (2017)
3
08-314.

ACCEPTED MANUSCRIPT
9
. J.M. Taylor, B.S. Main, P.J. Crack, Neuroinflammation and oxidative stress:
co-conspirators in the pathology of Parkinson’s disease, Neurochem. Int. 62 (2013)
8
03-819.
1
1
1
1
1
0. Q. Wang, Y. Liu, J. Zhou, Neuroinflammation in Parkinson’s disease and its potential
as therapeutic target, Transl. Neurodegener. 4 (2015) 19.
1. A. AlDakheel, L.V. Kalia, A.E. Lang, Pathogenesis-targeted, disease-modifying
therapies in Parkinson disease, Neurotherapeutics 11 (2014) 6-23.
2. J.M. Ellis, M.J. Fell, Current approaches to the treatment of Parkinson’s disease,
Bioorg. Med. Chem. Lett. 27 (2017) 4247-4255.
3. M. Spindler, M.F. Beal, C. Henchcliffe, Coenzyme Q10 effects in neurodegenerative
disease, Neuropsych. Dis. Treat. 5 (2009) 597-610.
4. R. Gurrell, S. Duvvuri, P. Sun, N. DeMartinis, A phase I study of the safety,
tolerability, pharmacokinetics, and pharmacodynamics of the novel dopamine D1
receptor partial agonist, PF-06669571, in subjects with idiopathic Parkinson’s
disease, Clin. Drug Invest. 38 (2018) 509-517.
1
1
1
5. A.E. Lang, A.J. Espay, Disease modification in Parkinson’s disease: current
approaches, challenges, and future considerations, Mov. Disord. 33 (2018) 660-677.
6. R. Morphy, Z. Rankovic, Designed multiple ligands. An emerging drug discovery
paradigm, J. Med. Chem. 48 (2005) 6523-6543.
7. A. Cavalli, M.L. Bolognesi, A. Minarini, M. Rosini, V. Tumiatti, M. Recanatini, C.
Melchiorre, Multi-target-directed ligands to combat neurodegenerative diseases, J.
Med. Chem. 51 (2008) 347-372.
1
1
8. M.B.H. Youdim, J.J. Buccafusco, Multi-functional drugs for various CNS targets in
the treatment of neurodegenerative disorders, Trends Pharmacol. Sci. 26 (2005)
2
7-35.
9. Y. Bansal, O. Silakari, Multifunctional compounds: smart molecules for
multifactorial diseases, Eur. J. Med. Chem. 76 (2014) 31-42.