Neuroprotective effects of benzyloxy substituted small molecule monoamine oxidase B inhibitors in Parkinson's disease

Article abstract of DOI:10.1016/j.bmc.2016.09.050

The benzyloxy substituted small molecules are well-known highly potent monoamine oxidase B inhibitors, but their therapeutic potential against Parkinson's disease have not been investigated in detail. In this paper, a series of representative benzyloxy substituted derivatives were synthesized and evaluated for MAO-A/B inhibition. In addition, their neuroprotective effects were investigated in 6-OHDA- and rotenone-treated PC12 cells. It was observed that most of the compounds exhibited a marked increase in survival of PC12 cells which treated with the neurotoxins. Among them, 13 exhibited remarkable and balanced neuroprotective potency. The protective effects of 13 against neurotoxins-induced apoptosis were confirmed with flow cytometry and staining methods. Furthermore, 13 also showed good BBB permeability and low toxicity according to in vitro BBB prediction and in vivo acute toxicity test. The results indicated that 13 is an effective and promising candidate to be further developed as disease-modifying drug for Parkinson's disease therapy.

Full text of DOI:10.1016/j.bmc.2016.09.050

Accepted Manuscript
Neuroprotective effects of benzyloxy substituted small molecule monoamine
oxidase B inhibitors in Parkinson’s disease
Zhimin Wang, Jiajia Wu, Xuelian Yang, Pei Cai, Qiaohong Liu, Kelvin D.G.
Wang, Lingyi Kong, Xiaobing Wang
PII:
S0968-0896(16)30768-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.050
BMC 13303
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 August 2016
19 September 2016
20 September 2016
Please cite this article as: Wang, Z., Wu, J., Yang, X., Cai, P., Liu, Q., Wang, K.D.G., Kong, L., Wang, X.,
Neuroprotective effects of benzyloxy substituted small molecule monoamine oxidase B inhibitors in Parkinson’s
disease, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.09.050
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Neuroprotective effects of benzyloxy substituted small molecule
monoamine oxidase B inhibitors in Parkinson’s disease
1
1
Zhimin Wang , Jiajia Wu , Xuelian Yang, Pei Cai, Qiaohong Liu, Kelvin D. G.
Wang, Lingyi Kong *, and Xiaobing Wang *
State Key Laboratory of Natural Medicines, Department of Natural Medicinal
Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009,
People’s Republic of China
*Corresponding Authors: Tel/Fax: +86-25-8327-1405. E-mail: cpu_lykong@126.com;
xbwang@cpu.edu.cn.
1 These authors contributed equally.
Abstract
The benzyloxy substituted small molecules are well-known highly potent monoamine
oxidase B inhibitors, but their therapeutic potential against Parkinson’s disease have
not been investigated in detail. In this paper, a series of representative benzyloxy
substituted derivatives were synthesized and evaluated for MAO-A/B inhibition. In
addition, their neuroprotective effects were investigated in 6-OHDA- and
rotenone-treated PC12 cells. It was observed that most of the compounds exhibited a
marked increase in survival of PC12 cells which treated with the neurotoxins. Among
them, 13 exhibited remarkable and balanced neuroprotective potency. The protective
effects of 13 against neurotoxins-induced apoptosis were confirmed with flow
cytometry and staining methods. Furthermore, 13 also showed good BBB
permeability and low toxicity according to in vitro BBB prediction and in vivo acute
toxicity test. The results indicated that 13 is an effective and promising candidate to
be further developed as disease-modifying drug for Parkinson’s disease therapy.
Keywords:
Parkinson’s disease, Monoamine oxidase, BBB permeability, Apoptosis,
Neuroprotection
1

1. Introduction
Parkinson’s disease (PD) is a degenerative disorder of the central nervous system
(CNS) which characterized by a progressive loss of pigmented dopaminergic neurons
in the substantia nigra pars compacta 1, 2. The cornerstone of PD therapy is aimed at
enhancing central dopamine levels with its direct metabolic precursor, L-3,
3
4-dihydroxyphenylalanine (L-DOPA) . To enhance the therapeutic efficacy of
L-DOPA and reduce the occurrence of dopamine-associated side effects, the drug is
frequently administered in combination with the inhibitors that reduce the metabolism
of dopamine, such as catechol-O-methyl transferase (COMT) and monoamine oxidase
type B (MAO-B) inhibitors 4, 5. Thus, inhibition of MAO-B leads to enhancement of
dopaminergic neurotransmission, which considered useful in the early PD therapy 6, 7
.
In the previous discovery of MAO-B inhibitors, propargylamine derivatives have
been shown as selective and potent MAO-B inhibitory activities, and those
compounds have been further examined neuroprotective capabilities against
neurodegeneration by preventing apoptosis 8-12. Among them, selegiline and rasagiline
were successfully approved by the U. S. FDA for the treatment of PD. Both selegiline
and rasagiline are used in early PD as monotherapy and adjunctive therapy to
levodopa and dopamine agonists in later stages of PD. However, the clinical trials
were unsuccessful to shown a clear neuroprotective action, which led to the limited
usage of selegiline and rasagiline as disease-modifying drugs 13. Therefore, the
development of novel MAO-B inhibitors with potent inhibitory potency, definite
therapeutic action and fewer side effects is still necessary. In fact, various benzyloxy
substituted molecules as new type of excellent MAO-B inhibitors have been highly
described by Petzer’s group and other researchers in recent years, such as
acetophenones, indoles, quinolinones, coumarins, chromones, chromanones,
a-tetralone and phthalide analogues 14-21. Although these benzyloxy substituted small
molecules have been identified as highly potent, selective, and irreversible MAO-B
inhibitors, but their therapeutic potentials against Parkinson’s disease have not been
investigated in detail. It is necessary to evaluate the neuroprotective activity and
2

molecular mechanism of these derivatives in cells model.
For this purpose, we had synthesized a series of representative benzyloxy
substituted compounds with different scaffolds (Fig 1) and their MAO-A/B inhibitory
activities had also been evaluated. In addition, these compounds were also tested for
neuroprotective effects with PC12 cells model treated by 6-OHDA and rotenone,
respectively. The neuroprotective effects of the preferred compounds against
neurotoxins-induced apoptosis were confirmed with flow cytometry and staining
method. To investigate drug-like properties, the blood-brain barrier (BBB)
permeability, ADMET properties, and toxicity were also evaluated by in vitro and in
vivo methods.
Fig 1.
3

2. Results and Discussion
2.1. Chemistry
These representative benzyloxy substituted derivatives (1-31) were efficiently
synthesized along with the pathway shown in Fig 2. The starting materials (various
17,
skeletons, see Supporting Information for the synthesis of key intermediates S1-4)
19, 21 reacted with the appropriate benzyl bromides in the presence of K2CO3 to give
the target compounds in moderate to good yields.
Fig 2.
2.2.Inhibition of MAOs
According to the reported assay with rasagiline and iproniazid 22-24, the MAO
inhibitory activities of compounds 1-31 were explored by measuring the effects on the
production of hydrogen peroxide from p-tyramine. As a screening result shown in
Table 1, most of the tested compounds with C4- or C3-halogen substituted benzyloxy
side chains, were selective inhibitors toward MAO-B with IC50 values in the
4

nanomolar range. The MAO-A inhibition of the tested compounds was weak with no
apparent structure-activity relationship (SAR) existence. It seems that compounds
bearing acetophenone and chromanone skeletons drastically decreased in MAO-A
inhibition. The corresponding inhibitory activities of the reported compounds were
listed in the Table S1 (Supporting Information).
5

Table 1. Inhibition of human MAO-A and MAO-B activities and neuroprotective
effects of compounds 1-31.
Compounds
MAO-A
IC50 (μM) a
MAO-B
IC50 (nM) a
Selectivity
Index b
% survival of PC12 cells c
6-OHDA
rotenone
>100
88.43 ± 6.90
39.45 ± 5.26
53.68 ± 2.4
>100
15.48 ± 1.34
25.32 ± 4.65
10.52 ± 1.04
92.41 ± 3.47
41.38 ± 6.00
28.42 ± 5.35
25.12 ± 3.50
42.75 ± 3.18
96.51 ± 4.56
56.89 ± 1.31
13.25 ± 2.24
52.93 ± 0.28
12.34 ± 1.62
42.05 ± 2.48
12.85 ± 5.38
39.21 ± 2.94
12.55 ± 1.68
7.21 ± 0.81
43.42 ± 8.25
63.22 ± 3.54
27.82 ± 1.35
25.31 ± 8.21
15.62 ± 4.29
8.35 ± 1.01
13.87 ± 3.14
6.41 ± 2.73
14.27 ± 1.66
8.17 ± 1.78
5.03 ± 0.81
6.48 ± 1.37
4.25 ± 1.40
>6460
3492
3750
581
115.53 ± 6.57
123.30 ± 4.28
88.59 ± 4.20
95.77 ± 5.08
1
2
131.78 ± 3.52 109.05 ± 10.79
109.39 ± 3.96 105.04 ± 10.46
133.41 ± 9.23 106.30 ± 11.51
3
4
>2417
1526
1913
593
5
43.38 ± 2.15
48.05 ± 3.92
25.34 ± 7.91
>100
143.64 ± 6.80
134.00 ± 10.97
98.31 ± 5.73
93.34 ± 2.66
6
7
144.11 ± 10.59 105.07 ± 10.26
115.85 ± 9.35 108.75 ± 10.80
104.54 ± 8.08 113.99 ± 10.01
8
>1036
>1758
>7547
1682
>8104
>2378
>7782
>2550
23
9
>100
10
>100
133.45 ± 5.85
141.51 ± 6.71
134.19 ± 6.76
143.35 ± 3.79
111.85 ± 2.09
101.33 ± 6.58
126.38 ± 6.92
111.02 ± 1.89
11
89.05 ± 3.9
>100
12
13
>100
14
>100
148.17 ± 10.30 111.47 ± 4.05
15
>100
143.14 ± 7.83
139.15 ± 8.24
126.78 ± 3.69
117.17 ± 5.87
124.17 ± 8.29
122.61 ± 8.42
122.00 ± 3.61
114.67 ± 4.41
116.04 ± 4.69
108.32 ± 4.12
95.92 ± 5.40
109.51 ± 0.71
101.38 ± 1.73
115.37 ± 1.82
110.30 ± 6.00
114.67 ± 4.26
106.33 ± 5.61
113.67 ± 2.27
16
1.29 ± 0.51
0.34 ± 0.04
23.59 ± 7.21
18.52 ± 1.14
18.94 ± 4.07
4.52 ± 0.73
13.59 ± 3.21
1.33 ± 0.48
3.61 ± 1.01
9.24 ± 2.37
>100
17
47
18
543
19
293
20
681
21
179
22
870
23
159
24
260
137.19 ± 10.90 113.35 ± 4.58
25
1441
>7008
6018
>19881
1045
>23529
0.84
124.63 ± 6.70
121.21 ± 1.52
111.71 ± 2.99
129.93 ± 9.72
114.33 ± 4.81
137.63 ± 3.53
-
111.05 ± 5.29
110.21 ± 7.08
133.52 ± 3.24
115.42 ± 7.47
119.33 ± 7.84
116.67 ± 8.01
-
26
27
49.17 ± 2.73
>100
28
29
6.77 ± 1.18
>100
30
31
7.57 ± 0.43 8.96 ± 0.38 μM
iproniazid
control
rasagiline
-
-
-
99.33 ± 1.86
134.52 ± 9.09
98.33 ± 3.38
109.03 ± 6.77
50.71 ± 3.19
7.87 ± 1.25
6443
a
b
IC50: 50% inhibitory concentration (means ± SEM of three experiments).
Selectivity Index = IC50 (MAO-A)/IC50 (MAO-B). c Survival data are expressed as the
percentage of 6-OHDA- and rotenone-treated cells. All data are the means ± SEM of
at least five values measured in two independent plates.
6

2.3. Cytotoxicity and neuroprotection assays in PC12 cells
To investigate the safety index of these potent MAO-B inhibitors, all the compounds
1-31 were selected for cytotoxicity assay in neuroblastoma cells (PC12). After
incubating the cells with the compounds for different times (24 and 72 h), the viability
was
investigated
by
the
MTT
[3-(4,
5-dimethyl-2-thiazolyl)-2,
25
5-diphenyl-2H-tetrazolium bromide] assay . As shown in the Fig 3A and Table S2
(Supporting Information), the results revealed that compounds 17-28 displayed low
toxicities at higher concentration (100 μM), while most of the compounds at
concentration of 100 and 20 μM did not shown significant neurotoxicity. This result
suggested these compounds could be used for further development.
Fig 3.
To gain insight into the therapeutic potentials of these compounds, the
neuroprotective capacity was also determined by MTT assay in PC12 cells. PC12
cells were chosen for this purpose due to the similarity of dopaminergic neurons, and
different types of neurotoxins were used to increase the predictive value of the model
for clinical neuroprotection 12. Two parallel experiments were performed with
6-hydroxydopamine- (6-OHDA) and rotenone-treated PC12 cells 26, 27. According to
28
the method described by Zheng et al. with slight modification, rasagiline was used
as positive control in these experiments. Cells were incubated with 6-OHDA (40 μM)
7

for 24 h, and tested compounds and rasagiline were applied in a single 20 μM
concentration for 1 h prior to 6-OHDA application. The MTT assay was used to assess
cell viability. In the similar manner, rotenone (1 μM) was used to assess the ability of
the tested compounds (20 μM) to facilitate cell survival.
As the results were summarized in Table 1 and Fig 3B, rasagiline exhibited
significant protective effect (39%) in 20 μM concentration in 6-OHDA-treated PC12
cell (Table 1) which was consistent with literature data 12, 26. Among the tested
compounds, compounds 5-8, 12, 14-17, 25 and 31 were protective against
6-OHDA-induced cell death (>30% protection), while only compounds 13 and 28
shown >20% protection against rotenone-induced cell death. Especially, compound 15
showed the highest increase of 48% in the survival of 6-OHDA-treated cells.
Compound 28 had the best neuroprotection (34% increased) in rotenone-induced cell
death. It is worth mentioning that, compound 13, a benzyloxy substituted chromanone
hMAO-B inhibitor, was the only compound which shown effective and balanced
neuroprotective activities in both 6-OHDA- and rotenone-treated PC12 cells in a
concentration-dependent manner (Fig 4).
Fig 4.
2.4.Effects of compound 13 on both 6-OHDA- and rotenone-induced apoptosis in
PC12 cells
Compound 13 was chosen to investigate the protective effects against
neurotoxins-induced apoptosis not only because of no neurotoxicity, but also because
8

of its remarkable and balanced activities against both 6-OHDA- and rotenone-induced
cell death. Apoptotic cells were quantified with 7-AAD and Annexin V APC dual
staining method using flow cytometry 29. The Annexin V APC (-)/7-AAD (-)
population consisted primarily of normal healthy cells, while Annexin V APC
(+)/7-AAD (-) cells were considered to be in the early stage of apoptosis, and Annexin
V APC (+)/7-AAD (+) cells were those considered to be in the necrosis/late apoptosis
stage.
Phase-contrast images (Fig S1A and B) showed that cell viability significantly
decreased when cells were treated with 40 M 6-OHDA and rotenone for 24 hours,
respectively, compared with those of control. When PC12 cells were exposed to 5-40
M compound 13 in the presence of 40 M 6-OHDA, or 1 M rotenone for 24 hours,
the co-treated cells showed a significant increase in cell viability in a concentration
dependent manner, compared to those treated with neurotoxins-treated alone. As
shown in Fig 5, apoptotic cells percentage of 6-OHDA group were significantly
increased compared to control group. In contrast, the rates of apoptotic cells were
reduced when 6-OHDA-treated PC12 cells were pretreated with 5, 10, 20, 40 μM of
compound 13. As the result in Fig 6, PC12 cells death of rotenone-treated group was
appeared due to the severe neurotoxicity. Similarly, compound 13 significantly
decreased the cell death rate in a concentration dependent manner.
9

Fig 5.
10

Fig 6.
Nuclear morphology characteristic of apoptosis and neuroprotection of 13 were
further investigated using DNA staining with Hoechst 33258 30. Condensed or
fragmented nuclei were characterized as apoptotic nuclei. Treatment with 20 M
compound 13 decreased the number of apoptotic nuclei (Fig S2). These results
showed that 13 significantly decreased the percentage of apoptotic nuclei when
compared to 6-OHDA- and rotenone-treated groups, which indicated that 13 have
neuroprotective capability against neurodegeneration by preventing apoptosis.
2.5. Compound 13 reduced 6-OHDA- and rotenone-induced intracellular ROS
accumulation
11

The oxidative stress caused by ROS is partly responsible for a wide variety of cellular
damage and is regarded as a key initiator of neurotoxins-induced apoptotic 31. As
shown in Fig 7, when PC12 cells were exposed to 40 M 6-OHDA, or 1 M rotenone
for 24 h, the intracellular ROS increased obviously by using the DCFH-DA probe.
While treated with 13 at the concentrations of 5, 10 and 20 μM，the level of ROS
reduced in a concentration-independent manner. This suggested that 6-OHDA and
rotenone could induce ROS production and 13 effectively reduce neurotoxins-induced
intracellular ROS accumulation.
Fig 7.
2.6. Theoretical evaluation of physical properties and ADMET
The compounds with higher activity (5-8, 12-17, 25 and 31) were evaluated for the
32
ADMET properties in silico by the Accelrys Discovery Studio (Table 2) . All the
compounds showed ADMET properties similar to drug rasagiline. Compared with the
standard drug, these compounds were predicted to have fine absorption, high blood
12

brain barrier (BBB) level and good solubility. In addition, the physical properties of
the selected compounds were followed Lipinski’s rule of five 33: molecular weight
(MW) less than 500, the number of hydrogen bond donor atoms (HBD) less than 5,
the number of hydrogen bond acceptor atoms (HBA) less than 10, the calculated
logarithm of the octanol-water partition coefficient (Clog P) less than 5, and the small
polar surface area less than 90 Å2. However, these compounds showed a vast
probability of hepatotoxicity (>0.92). The results indicated these compounds have
anti-Pakinson’s disease potentials which satisfied possible BBB penetration and
containing drug-like properties. However, the compounds need to be further
optimized for the pharmacokinetics properties.
13

Table 2. Physical properties and ADMET prediction of the selected compounds.
Compounds
MW a
Clog P a
HBA a
HBD a
PSAa
Log BB a
Absorption
Level b
Solubility
Level b
Hepatotoxicity
Probability b
PPB
Level b
BBB
Level b
260.26
260.26
276.72
276.72
278.71
272.28
288.73
300.33
316.09
270.30
274.70
332.20
316.30
171.24
≤450
3.898
3.898
4.468
4.468
4.017
3.656
4.226
4.694
5.264
4.256
3.452
4.002
4.677
2.344
≤5.0
2
2
1
1
46.53
46.53
46.53
46.53
26.3
0.722496
0.722496
0.809136
0.809136
0.740584
0.685712
0.772352
0.843488
0.930128
0.776912
0.654704
0.738304
0.840904
0.664332
≥-1.0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-
3
3
3
3
2
2
2
2
2
2
2
2
2
3
-
1
1
1
1
1
1
1
1
1
1
1
1
1
0
-
0
0
0
0
0
2
2
0
1
2
2
2
1
2
-
2
2
2
2
1
1
1
1
1
0
1
1
0
1
-
5
6
2
1
7
2
1
8
12
2
0
3
0
35.53
35.53
35.53
35.53
26.3
13
3
0
14
3
0
15
3
0
16
2
0
17
3
0
35.53
38.33
35.53
12.03
≤90
25
2
1
28
3
0
31
0
1
rasagiline
Rules
≤10
≤5
a
MW: molecular weight; C log P: calculated logarithm of the octanol-water partition coefficient; HBA: hydrogen-bond acceptor atoms; HBD:
b
hydrogen-bond donor atoms; PSA: polar surface area; log BB = 0.0148 × PSA + 0.152 × Clog P + 0.13. Absorption level (0 = good and 3 =
very poor); Solubility level (0 = extremely low and 3 = good); Hepatotoxicity (0 = nontoxic and 1 = toxic); PPB, Plasma protein binding (0 =
PPB < 90% and 2 = PPB > 95%); BBB level (0 = very high and 3 = low).
14

2.7. In vitro BBB permeation assay
To determine whether the promising compounds could cross BBB besides the
calculated results of log BB and in silicon prediction, we also used a parallel artificial
membrane permeation assay for BBB (PAMPA-BBB), which was described by Di et
al. 34, 35 Assay validation was performed by comparing experimental permeability of 9
commercial drugs with reported values (Table S4). A plot of experimental data versus
bibliographic values gave a good linear correlation: Pe (exp.) = 1.0976 Pe (bibl.) -
0.346 (R2= 0.9537) (Fig S3). From the equation and taking into account the limits
established by Di et al. for BBB permeation. The Pe values of these selected
compounds are summarized in Table 3. It can be indicated that compounds 5, 6, 8, 13,
15, 17 and 28 shown higher Pe values than 4.04 were able to cross the BBB.
Table 3. Permeability results (Pe × 10-6 cm s-1) from the PAMPA-BBB assay for
selected compounds with their predicted penetration into the CNS.
Compounds Permeability (Pe × 10-6 cm s-1) a Prediction b
15.9 ± 0.6
13.5 ± 0.4
12.7 ± 0.2
14.3 ± 0.4
9.71 ± 0.52
13.2 ± 0.7
8.89 ± 0.37
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
5
6
8
13
15
17
28
a Data are the mean ± SEM of three independent experiments; b Pe (10−6 cm s−1) > 4.5
for high BBB permeation (CNS+), Pe (10−6 cm s−1) < 1.85 for low BBB permeation
(CNS−), and 4.04 > Pe (10−6 cm s−1) > 1.85 for uncertain BBB permeation (CNS±).
2.8. Acute toxicity test in vivo
According to the results of in silicon prediction, most of the compounds showed a
probability of hepatotoxicity. Therefore, it is necessary to test the in vivo toxicity of
the most promising compound 13. The procedure for acute toxicity study followed
similar protocols from the reported studies 36, 37. After administration of the compound
13 (2,000 mg/kg), mice were monitored continuously for the first 4 h for any
abnormal behavior and mortality changes, intermittently for the next 24 h, and
occasionally thereafter for 14 days to monitor the onset of any delayed effects. During
the experimental period, no acute toxicity, such as mortality, or significant abnormal
15

changes in water or food consumption or body weight reduction were observed (Fig
8). Furthermore, all mice were sacrificed on the 14th day after drug administration.
This indicated that 13 was nontoxic and tolerated at doses up to 2,000 mg/kg.
Fig 8.
2.9. Statistical analysis
Statistical analyses were performed with one-way ANOVA test followed by a post hoc
analysis (Tukey’s multiple comparison test) using GraphPad Prism 5 Software for
Windows (GraphPad Software, Inc., San Diego, CA, USA). All values were presented
as mean ± standard error of the mean (mean ± SEM) for each group. < 0.05 was
considered statistical significant.
3. Conclusions
In conclusion, this study showed that most of the representative benzyloxy substituted
derivatives 5-8, 12-17, 25, 28 and 31 not only possessed selective and potent MAO-B
inhibitory activities, but also exhibited neuroprotective properties in 6-OHDA- and
rotenone-treated PC12 cells. Additionally, the selected compounds displayed no
significant cytotoxicity, fine oral absorption and BBB permeability when evaluated
for ADMET properties in silico. Moreover, the most promising compound 13
exhibited neuroprotective effects via reducing intracellular ROS and preventing
neurotoxins-induced apoptosis. Furthermore, 13 showed good BBB permeability and
16

low toxicity according to in vitro BBB prediction and in vivo acute toxicity test.
Based on the results, compound 13 and other benzyloxy substituted MAO-B
inhibitors could be used for the further development of promising drug candidates for
the therapy of Parkinson’s disease.
4. Methods
4.1. Chemistry
All common reagents and solvents were obtained from commercial suppliers and used
without further purification. Reaction progress was monitored using analytical thin
layer chromatography (TLC) on precoated silica gel GF254 plates (Qingdao Haiyang
Chemical Plant, Qingdao, China) plates and the spots were detected under UV light
(254 nm). Column chromatography was performed on silica gel (90-150 μm; Qingdao
Marine Chemical Inc.) Melting points were measured on an XT-4 micromelting point
instrument and uncorrected. NMR spectra were measured on a Bruker ACF-500
spectrometer at 25 °C and referenced to TMS. Chemical shifts are reported in ppm (δ)
using the residual solvent line as internal standard. Mass spectra were obtained on a
MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI-MS) and a Mariner
ESI-TOF spectrometer (HRESI-MS), respectively.
4.1.1. General procedure for the preparation of the benzyloxy substituted
derivatives (1-31)
The procedures for the preparation of the key intermediates (S1-4) were summarized
in supporting information. The starting material (1.0 mmol) was suspended in
acetonitrile (20 mL) containing K2CO3 (2.0 mmol). The reaction was treated with an
appropriately substituted arylalkyl bromide (1.2 mmol) and heated under reflux for 8
h. The reaction progress was monitored using silica gel TLC with Petroleum
ether/EtOAc as mobile phase. Upon completion, the acetonitrile was evaporated in
vacuo and the mixture was then poured into water, which was extracted with 3 × 50
mL of EtOAc, washed with brine, dried over anhydrous Na2SO4 and purified by
chromatography (PE/EA, 50:1, 20:1, 10:1) on silica gel.
1-(4-((4-fluorobenzyl)oxy)phenyl)ethanone (1). Yield 83%, white solid, m.p. 75-77 °C;
17

Purity 97.5%; 1H NMR (500 MHz, DMSO-d6) δ 7.95 (d, J = 8.8 Hz, 2H), 7.54 (dd, J
= 8.4, 5.7 Hz, 2H), 7.25 (t, J = 8.8 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 5.21 (s, 2H),
2.53 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 196.72, 162.56, 161.39, 133.26,
130.93, 130.93, 130.68, 130.54, 130.48, 115.87, 115.70, 115.15, 115.15, 69.29, 26.84.
ESI-MS m/z: 244.9 [M+H]+; HRMS (ESI) m/z 245.0970 [M+H]+ (calcd for 245.0972,
C15H14FO2).
1-(4-((3-chlorobenzyl)oxy)phenyl)ethanone (2). Yield 89%, white solid, m.p. 79-81 °C;
1
Purity 98.6%; H NMR (500 MHz, DMSO-d6) δ 7.96 (d, J = 8.8 Hz, 2H), 7.55 (s,
1H), 7.49 - 7.39 (m, 3H), 7.14 (d, J = 8.8 Hz, 2H), 5.25 (s, 2H), 2.53 (s, 3H); ESI-MS
m/z: 260.9 [M+H]+; 13C NMR (125 MHz, DMSO-d6) δ 196.73, 162.39, 139.61,
133.67, 130.95, 130.95, 130.89, 130.78, 128.39, 127.84, 126.69, 115.16, 115.16,
69.05, 26.85. HRMS (ESI) m/z 261.0679 [M+H]+ (calcd for 261.0677, C15H14ClO2).
1-(3-((4-chlorobenzyl)oxy)phenyl)ethanone (3). Yield 82%, light yellow oil; Purity
1
99.3%; H NMR (500 MHz, DMSO-d6) δ 7.59 (d, J = 7.7 Hz, 1H), 7.55 (d, J = 2.0
Hz, 1H), 7.54 - 7.43 (m, 5H), 7.30 (dd, J = 8.0, 2.2 Hz, 1H), 5.20 (s, 2H), 2.59 (s, 3H);
13C NMR (125 MHz, DMSO-d6) δ 198.11, 158.83, 138.83, 136.37, 132.98, 130.39,
129.97, 129.97, 128.95, 128.95, 121.55, 120.41, 114.28, 69.13, 27.29. ESI-MS m/z:
260.9 [M+H]+; HRMS (ESI) m/z 261.0676 [M+H]+ (calcd for 261.0677, C15H14ClO2).
1-(3-((4-bromobenzyl)oxy)phenyl)ethanone (4). Yield 90%, light yellow oil; Purity
1
99.7%; H NMR (500 MHz, DMSO-d6) δ 7.60 (dd, J = 14.3, 8.0 Hz, 3H), 7.54 (s,
1H), 7.51 - 7.40 (m, 3H), 7.30 (dd, J = 8.2, 1.9 Hz, 1H), 5.19 (s, 2H), 2.59 (s, 3H); 13C
NMR (125 MHz, DMSO-d6) δ 198.10, 158.81, 138.83, 136.80, 131.87, 131.87,
130.39, 130.27, 130.27, 121.56, 121.49, 120.41, 114.28, 69.16, 27.29. ESI-MS m/z:
306.9 [M+H]+; HRMS (ESI) m/z 326.9990 [M+Na]+ (calcd for 326.9991,
C15H13BrNaO2).
1-(4-((3-fluorobenzyl)oxy)-2-hydroxyphenyl)ethanone (5). Yield 89%, white solid,
m.p. 103-104 °C; Purity 98.5%; 1H NMR (500 MHz, DMSO-d6) δ 12.61 (s, 1H), 7.88
(d, J = 8.9 Hz, 1H), 7.54 – 7.39 (m, 1H), 7.30 (dd, J = 7.8, 4.7 Hz, 2H), 7.19 (td, J =
8.6, 1.9 Hz, 1H), 6.64 (dd, J = 8.9, 2.4 Hz, 1H), 6.58 (d, J = 2.4 Hz, 1H), 5.24 (s, 2H),
18

2.59 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 202.37, 165.04, 164.75, 163.75,
162.12, 138.41, 138.36, 132.39, 130.28, 130.23, 122.73, 122.71, 115.21, 115.07,
114.29, 114.19, 114.15, 108.02, 101.83, 101.81, 69.27, 69.25, 26.51. ESI-MS m/z:
260.9 [M+H]+; HRMS (ESI) m/z 261.0922 [M+H]+ (calcd for 261.0921, C15H14FO3).
1-(4-((4-fluorobenzyl)oxy)-2-hydroxyphenyl)ethanone (6). Yield 85%, white solid,
m.p. 111-112 °C; Purity 98.5%; 1H NMR (500 MHz, DMSO-d6) δ 12.62 (s, 1H), 7.87
(d, J = 8.9 Hz, 1H), 7.53 (dd, J = 8.4, 5.7 Hz, 2H), 7.25 (t, J = 8.9 Hz, 2H), 6.62 (dd, J
= 8.9, 2.4 Hz, 1H), 6.58 (d, J = 2.4 Hz, 1H), 5.19 (s, 2H), 2.58 (s, 3H); 13C NMR (125
MHz, DMSO-d6) δ 201.19, 165.13, 164.92, 163.45, 161.81, 132.36, 131.60, 131.58,
129.45, 129.40, 115.70, 115.56, 114.12, 108.07, 101.78, 101.77, 69.49, 26.34.
ESI-MS m/z: 260.9 [M+H]+; HRMS (ESI) m/z 261.0916 [M+H]+ (calcd for 261.0922,
C15H14FO3).
1-(4-((3-chlorobenzyl)oxy)-2-hydroxyphenyl)ethanone (7). Yield 83%, white solid,
m.p. 128-129 °C; Purity 99.6%; 1H NMR (500 MHz, DMSO-d6) δ 12.61 (s, 1H), 7.88
(d, J = 8.9 Hz, 1H), 7.54 (s, 1H), 7.50 – 7.34 (m, 3H), 6.64 (dd, J = 8.9, 2.4 Hz, 1H),
6.58 (d, J = 2.4 Hz, 1H), 5.24 (s, 2H), 2.59 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
δ 202.63, 165.11, 164.74, 137.88, 134.61, 132.40, 129.96, 128.41, 127.41, 125.35,
114.22, 108.02, 101.82, 69.24, 26.25. ESI-MS m/z: 274.9 [M-H]-; HRMS (ESI) m/z
275.0479 [M-H]- (calcd for 275.0480, C15H12ClO3).
1-(4-((4-chlorobenzyl)oxy)-2-hydroxyphenyl)ethanone (8). Yield 88%, white solid,
m.p. 102-103 °C; Purity 100%;1H NMR (500 MHz, DMSO-d6) δ 12.61 (s, 1H), 7.87
(d, J = 8.9 Hz, 1H), 7.58 – 7.39 (m, 4H), 6.62 (dd, J = 8.9, 2.4 Hz, 1H), 6.57 (d, J =
13
2.4 Hz, 1H), 5.22 (s, 2H), 2.58 (s, 3H); C NMR (125 MHz, DMSO-d6) δ 202.60,
165.10, 164.80, 134.32, 134.11, 132.37, 128.86, 128.79, 114.16, 108.01, 101.80,
69.34, 26.24. ESI-MS m/z: 274.9 [M+H]+; HRMS (ESI) m/z 275.0482 [M-H]- (calcd
for 275.0480, C15H12ClO3).
1-(2-fluoro-4-((3-fluorobenzyl)oxy)phenyl)ethanone (9). Yield 93%, light yellow solid,
m.p. 104-105 °C; Purity 99.4%; 1H NMR (500 MHz, DMSO-d6) δ 7.83 (t, J = 8.9 Hz,
1H), 7.48 (dd, J = 14.2, 7.7 Hz, 1H), 7.32 (d, J = 7.8 Hz, 2H), 7.20 (dd, J = 9.1, 7.2
19

Hz, 1H), 7.05 (dd, J = 13.5, 2.2 Hz, 1H), 6.99 (dd, J = 8.8, 2.3 Hz, 1H), 5.27 (s, 2H),
13
2.53 (s, 3H); C NMR (500 MHz, DMSO-d6) δ 193.5, 163.8, 163.1, 161.1, 138.9,
131.7, 130.5, 123.6, 118.4, 114.9, 114.2, 111.7, 102.9, 69.1, 30.6. ESI-MS m/z: 262.9
[M+H]+; HRMS (ESI) m/z 263.0879 [M+H]+ (calcd for 263.0878, C15H13F2O2).
1-(2-fluoro-4-((4-fluorobenzyl)oxy)phenyl)ethanone (10). Yield 87%, light yellow
solid, m.p. 69-71 °C; Purity 99.2%; 1H NMR (500 MHz, DMSO-d6) δ 7.83 (t, J = 8.9
Hz, 1H), 7.54 (dd, J = 8.3, 5.7 Hz, 2H), 7.25 (t, J = 8.8 Hz, 2H), 7.04 (dd, J = 13.6,
1.1 Hz, 1H), 6.98 (dd, J = 8.8, 2.1 Hz, 1H), 5.22 (s, 2H), 2.53 (s, 3H); 13C NMR (125
MHz, DMSO-d6) δ 193.5, 163.8, 162.8, 160.9, 132.2, 131.7, 130.2, 130.2, 118.3,
115.4, 115.4, 111.7, 102.8, 69.3, 30.6. ESI-MS m/z: 262.9 [M+H]+; HRMS (ESI) m/z
263.0877 [M+H]+ (calcd for 263.0878, C15H13F2O2).
1-(2-fluoro-4-((3-chlorobenzyl)oxy)phenyl)ethanone (11). Yield 93%, light yellow
solid, m.p. 70-72 °C; Purity 99.9%; 1H NMR (500 MHz, DMSO-d6) δ 7.83 (t, J = 8.9
Hz, 1H), 7.56 (s, 1H), 7.51 – 7.38 (m, 3H), 7.05 (d, J = 13.5 Hz, 1H), 7.00 (dd, J = 8.8,
2.0 Hz, 1H), 5.26 (s, 2H), 2.53 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 193.6,
163.8, 161.8, 138.5, 133.1, 131.7, 130.4, 128.0, 127.4, 126.3, 118.4, 111.7, 102.9,
69.0, 30.6. ESI-MS m/z: 278.9 [M+H]+; HRMS (ESI) m/z 301.0400 [M+Na]+ (calcd
for 301.0402, C15H12ClFNaO2).
1-(2-fluoro-4-((4-chlorobenzyl)oxy)phenyl)ethanone (12). Yield 85%, light yellow
solid, m.p. 74-75 °C; Purity 100%; 1H NMR (500 MHz, DMSO-d6) δ 7.83 (t, J = 8.9
Hz, 1H), 7.57 – 7.42 (m, 4H), 7.04 (dd, J = 13.5, 1.6 Hz, 1H), 6.98 (dd, J = 8.8, 1.9
13
Hz, 1H), 5.24 (s, 2H), 2.53 (s, 3H); C NMR (125 MHz, DMSO-d6) δ 193.5, 163.8,
161.8, 135.0, 132.7, 131.7, 129.6, 129.6, 128.4, 128.4, 118.3, 111.7, 102.9, 69.1, 30.7.
ESI-MS m/z: 278.9 [M+H]+; HRMS (ESI) m/z 301.0403 [M+Na]+ (calcd for 301.0402,
C15H12ClFNaO2).
7-((4-Fluorobenzyl)oxy)chroman-4-one (13). Yield 91%, colorless solid, m.p.
125-127 °C; Purity 99.9%; 1H NMR (500 MHz, DMSO-d6) δ 7.69 (d, J = 8.8 Hz, 1H),
7.50 (dd, J = 8.5, 5.6 Hz, 2H), 7.23 (t, J = 8.8 Hz, 2H), 6.70 (dd, J = 8.8, 2.3 Hz, 1H),
6.63 (d, J = 2.3 Hz, 1H), 5.16 (s, 2H), 4.51 (t, J = 6.4 Hz, 2H), 2.71 (t, J = 6.4 Hz, 2H);
20

13C NMR (125 MHz, DMSO-d6) δ 189.86, 164.25, 163.27, 162.79, 132.47, 132.45,
130.01, 129.95, 128.12, 115.33, 115.16, 110.09, 101.78, 68.93, 67.04, 36.82. ESI-MS
m/z: 272.9 [M+H]+; HRMS (ESI) m/z 567.1586 [2M+Na]+ (calcd for 567.1590,
C32H26NaO6F2).
7-((4-Chlorobenzyl)oxy)chroman-4-one (14). Yield 89%, colorless solid, m.p.
139-140 °C; Purity 99.6%; 1H NMR (500 MHz, DMSO-d6) δ 7.69 (d, J = 8.8 Hz, 1H),
7.47 (s, 4H), 6.70 (dd, J = 8.8, 2.4 Hz, 1H), 6.62 (d, J = 2.4 Hz, 1H), 5.18 (s, 2H),
4.50 (t, J = 6.4 Hz, 2H), 2.70 (t, J = 6.4 Hz, 2H); 13C NMR (125 MHz, DMSO-d6) δ
190.44, 164.71, 163.82, 135.86, 133.15, 130.05, 130.05, 129.01, 129.01, 128.71,
115.62, 110.66, 102.38, 69.33, 67.61, 37.37. ESI-MS m/z: 288.9 [M+H]+; HRMS (ESI)
m/z 311.0446 [M+Na]+ (calcd for 311.0445, C16H13ClNaO3).
7-((4-fluorobenzyl)oxy)-2,2-dimethylchroman-4-one (15). Yield 87%, yellow solid,
1
m.p. 91-92 °C; Purity 98.6%; H NMR (500 MHz, DMSO-d6) δ 7.66 (d, J = 8.7 Hz,
1H), 7.50 (dd, J = 8.5, 5.6 Hz, 2H), 7.23 (t, J = 8.8 Hz, 2H), 6.66 (dd, J = 8.8, 2.3 Hz,
13
1H), 6.58 (d, J = 2.3 Hz, 1H), 5.15 (s, 2H), 2.71 (s, 2H), 1.39 (s, 6H); C NMR (125
MHz, DMSO-d6) δ 190.78, 165.18, 163.48, 161.88, 133.09, 130.61, 130.54, 128.10,
115.89, 115.72, 114.29, 110.12, 102.65, 80.16, 69.47, 48.26, 26.62, 26.62. ESI-MS
m/z: 300.9 [M+H]+; HRMS (ESI) m/z 623.2210 [2M+Na]+ (calcd for 623.2216,
C36H34F2NaO6).
7-((4-chlorobenzyl)oxy)-2,2-dimethylchroman-4-one (16). Yield 85%, colorless solid,
m.p. 130-131 °C; Purity 99.5%; 1H NMR (500 MHz, DMSO-d6) δ 7.66 (d, J = 8.7 Hz,
1H), 7.46 (s, 4H), 6.66 (dd, J = 8.7, 2.3 Hz, 1H), 6.57 (d, J = 2.3 Hz, 1H), 5.17 (s, 2H),
2.70 (s, 2H), 1.38 (s, 6H); 13C NMR (125 MHz, DMSO-d6) δ 190.79, 165.08, 161.86,
135.91, 133.14, 130.07, 130.07, 129.00, 129.00, 128.12, 114.40, 110.13, 102.67,
80.18, 69.31, 48.24, 26.62, 26.62. ESI-MS m/z: 317.1 [M+H]+; HRMS (ESI) m/z
655.1621 [2M+Na]+ (calcd for 655.1625, C36H34Cl2NaO6).
6-((4-fluorobenzyl)oxy)-3,4-dihydronaphthalen-1(2H)-one (17). Yield 87%, yellow
solid, m.p. 83-85 °C; Purity 98.8%; 1H NMR (500 MHz, DMSO-d6) δ 7.85 (d, J = 9.4
Hz, 1H), 7.53 (dd, J = 8.1, 5.8 Hz, 2H), 7.25 (t, J = 8.8 Hz, 2H), 6.98 (d, J = 5.4 Hz,
21

2H), 5.19 (s, 2H), 2.92 (t, J = 5.9 Hz, 2H), 2.58 – 2.53 (m, 2H), 2.14 – 1.92 (m, 2H);
13C NMR (125 MHz, DMSO-d6) δ 196.48, 162.57, 147.62, 133.25, 133.23, 130.53,
130.46, 129.18, 126.48, 115.86, 115.69, 114.31, 114.10, 69.23, 38.86, 29.80, 23.42.
ESI-MS m/z: 271.0 [M+H]+; HRMS (ESI) m/z 293.0947 [M+Na]+ (calcd for
293.0948, C17H15FNaO2).
6-((4-chlorobenzyl)oxy)-3,4-dihydronaphthalen-1(2H)-one (18). Yield 81%, yellow
solid, m.p. 97-99 °C; Purity 99.0%; 1H NMR (500 MHz, DMSO-d6) δ 7.93 – 7.77 (m,
1H), 7.62 - 7.40 (m, 4H), 6.98 (d, J = 1.8 Hz, 2H), 5.21 (s, 2H), 2.92 (t, J = 5.8 Hz,
13
2H), 2.58 - 2.53 (m, 2H), 2.11 - 1.89 (m, 2H); C NMR (125 MHz, DMSO-d6) δ
196.49, 162.48, 147.63, 136.08, 133.08, 130.01, 129.20, 128.97, 126.54, 114.32,
114.13, 69.09, 38.86, 29.80, 23.42. ESI-MS m/z: 287.0 [M+H]+; HRMS (ESI) m/z
309.0650 [M+Na]+ (calcd for 309.0653, C17H15ClNaO2).
7-((3-chlorobenzyl)oxy)-4H-chromen-4-one (19). Yield 81%, white solid, m.p.
136-138 °C; Purity 96.5%; 1H NMR (500 MHz, DMSO-d6) δ 8.24 (d, J = 6.0 Hz, 1H),
7.98 (d, J = 8.9 Hz, 1H), 7.58 (s, 1H), 7.46 (dd, J = 12.6, 5.4 Hz, 3H), 7.25 (d, J = 2.1
13
Hz, 1H), 7.17 (dd, J = 8.9, 2.2 Hz, 1H), 6.29 (d, J = 6.0 Hz, 1H), 5.30 (s, 2H); C
NMR (125 MHz, DMSO-d6) δ 176.08, 162.98, 158.13, 156.95, 139.16, 133.69,
130.94, 128.54, 128.01, 127.01, 126.85, 118.87, 115.53, 112.69, 102.44, 69.58.
ESI-MS m/z: 286.9 [M+H]+; HRMS (ESI) m/z 309.0287 [M+Na]+ (calcd for
309.0289, C16H11ClNaO3).
7-((4-fluorobenzyl)oxy)-4H-chromen-4-one (20). Yield 86%, white solid, m.p.
121-123 °C; Purity 99.0%;1H NMR (500 MHz, DMSO-d6) δ 8.23 (d, J = 6.0 Hz, 1H),
7.97 (d, J = 8.9 Hz, 1H), 7.56 (dd, J = 8.0, 5.9 Hz, 2H), 7.34 – 7.21 (m, 3H), 7.15 (dd,
13
J = 8.9, 1.9 Hz, 1H), 6.29 (d, J = 6.0 Hz, 1H), 5.26 (s, 2H); C NMR (125 MHz,
DMSO-d6) δ 176.09, 163.13, 161.46, 158.16, 156.93, 132.85, 130.75, 130.68, 126.95,
118.77, 115.93, 115.76, 115.57, 112.68, 102.37, 69.84. ESI-MS m/z: 270.9 [M+H]+;
HRMS (ESI) m/z 293.0583 [M+Na]+ (calcd for 293.0584, C6H11FNaO3).
7-((4-(trifluoromethyl)benzyl)oxy)-4H-chromen-4-one (21). Yield 80%, white solid,
m.p. 159-161 °C; Purity 99.9%;1H NMR (500 MHz, DMSO-d6) δ 8.24 (d, J = 6.0 Hz,
22

1H), 7.99 (d, J = 8.9 Hz, 1H), 7.80 (d, J = 8.1 Hz, 2H), 7.73 (d, J = 8.1 Hz, 2H), 7.26
(d, J = 2.2 Hz, 1H), 7.18 (dd, J = 8.9, 2.3 Hz, 1H), 6.30 (d, J = 6.0 Hz, 1H), 5.41 (s,
2H); 13C NMR (125 MHz, DMSO-d6) δ 176.08, 162.93, 158.14, 156.97, 141.47,
128.69, 127.05, 125.92, 125.89, 123.58, 121.86, 118.92, 115.53, 112.70, 102.48,
69.59. ESI-MS m/z: 321.0 [M+H]+; HRMS (ESI) m/z 343.0553 [M+Na]+ (calcd for
343.0552, C17H11F3NaO3).
6-((4-fluorobenzyl)oxy)-4H-chromen-4-one (22). Yield 45%, colorless solid, m.p.
139-141 °C; Purity 99.8%; 1H NMR (500 MHz, DMSO-d6) δ 7.83 (d, J = 5.9 Hz, 1H),
7.67 - 7.59 (m, 1H), 7.46 - 7.37 (m, 3H), 7.30 (dt, J = 9.2, 2.2 Hz, 1H), 7.07 (t, J = 8.4
13
Hz, 2H), 6.31 (d, J = 6.0 Hz, 1H), 5.08 (s, 2H); C NMR (125 MHz, DMSO-d6) δ
176.83, 163.14, 158.09, 154.78, 138.26, 132.53, 128.39, 127.62, 127.17, 118.83,
114.99, 112.88, 101.39, 70.15. ESI-MS m/z: 270.9 [M+H]+; HRMS (ESI) m/z
271.0694 [M+H]+ (calcd for 271.0692, C16H12FO3).
6-((4-chlorobenzyl)oxy)-4H-chromen-4-one (23). Yield 65%, colorless solid, m.p.
169-171 °C; Purity 99.7%;1H NMR (500 MHz, DMSO-d6) δ 7.82 (d, J = 5.9 Hz, 1H),
7.68 - 7.59 (m, 1H), 7.48 - 7.26 (m, 6H), 6.35 - 6.24 (m, 1H), 5.08 (s, 2H); 13C NMR
(125 MHz, DMSO-d6) δ 175.94, 162.46, 157.64, 156.58, 135.55, 131.43, 130.04,
126.48, 121.32, 118.28, 115.12, 112.19, 101.81, 69.56. ESI-MS m/z: 286.9 [M+H]+;
HRMS (ESI) m/z 287.0395 [M+H]+ (calcd for 287.0397, C16H12ClO3).
5-((4-fluorobenzyl)oxy)isobenzofuran-1(3H)-one (24). Yield 85%, colorless solid, m.p.
133-134 °C; Purity 99.8%;1H NMR (500 MHz, DMSO-d6) δ 7.55 (dd, J = 11.4, 5.0
Hz, 3H), 7.26 (t, J = 8.9 Hz, 2H), 6.93 (d, J = 1.9 Hz, 1H), 6.79 (dd, J = 8.6, 2.0 Hz,
13
1H), 5.23 (s, 2H), 4.79 (s, 2H); C NMR (125 MHz, DMSO-d6) δ 171.08, 159.49,
139.32, 134.53, 134.21, 128.99, 128.87, 127.15, 123.71, 123.12, 108.71, 69.72, 69.53.
ESI-MS m/z: 259.0 [M+H]+; HRMS (ESI) m/z 281.0586 [M+Na]+ (calcd for
281.0584, C15H11FNaO3).
5-((4-chlorobenzyl)oxy)isobenzofuran-1(3H)-one (25). Yield 75%, colorless solid,
m.p. 130-131 °C; Purity 99.4%;1H NMR (500 MHz, DMSO-d6) δ 7.56 (d, J = 8.6 Hz,
1H), 7.51 (q, J = 8.6 Hz, 4H), 6.92 (d, J = 1.6 Hz, 1H), 6.80 (dd, J = 8.6, 1.9 Hz, 1H),
23

5.26 (s, 2H), 4.79 (s, 2H); 13C NMR (125 MHz, DMSO-d6) δ 171.13, 159.55, 139.27,
131.86, 129.49, 127.12, 123.76, 123.14, 115.78, 115.67, 108.66, 69.85, 69.52.
ESI-MS m/z: 274.9 [M+H]+; HRMS (ESI) m/z 297.0286 [M+Na]+ (calcd for
297.0289, C15H11ClNaO3).
5-((4-(trifluoromethyl)benzyl)oxy)isobenzofuran-1(3H)-one (26). Yield 85%, colorless
solid, m.p. 127-128 °C; Purity 98.7%;1H NMR (500 MHz, DMSO-d6) δ 7.76 (dd, J =
45.4, 7.9 Hz, 4H), 7.58 (d, J = 8.6 Hz, 1H), 6.94 (s, 1H), 6.88 – 6.75 (m, 1H), 5.38 (s,
2H), 4.79 (s, 2H); 13C NMR (125 MHz, DMSO-d6) δ 171.03, 159.35, 140.09, 139.40,
130.38, 127.45, 127.27, 125.73, 124.95, 123.62, 123.21, 108.65, 69.58, 69.54.
ESI-MS m/z: 309.0 [M+H]+; HRMS (ESI) m/z 331.0556 [M+Na]+ (calcd for
331.0552, C16H11F3NaO3).
7-((3-chlorobenzyl)oxy)-3,4-dihydroquinolin-2(1H)-one (27). Yield 68%, white solid,
m.p. 158-160 °C; Purity 98.8%;1H NMR (500 MHz, DMSO-d6) δ 10.01 (s, 1H), 7.64
(s, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.45 (d, J = 7.6 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H),
7.08 (d, J = 8.2 Hz, 1H), 6.58 (dd, J = 8.2, 2.1 Hz, 1H), 6.53 (d, J = 1.9 Hz, 1H), 5.07
(s, 2H), 2.80 (t, J = 7.5 Hz, 2H), 2.43 (t, J = 7.5 Hz, 2H); 13C NMR (125 MHz,
DMSO-d6) δ 170.72, 157.81, 140.47, 139.79, 131.12, 131.09, 130.55, 128.93, 126.95,
122.16, 116.59, 108.35, 102.65, 68.77, 31.19, 24.52. ESI-MS m/z: 288.0 [M+H]+;
HRMS (ESI) m/z 288.0785 [M+H]+ (calcd for 288.0786, C16H15ClNO2).
7-((3-bromobenzyl)oxy)-3,4-dihydroquinolin-2(1H)-one (28). Yield 76%, white solid,
m.p. 125-127 °C; Purity 98.7%;1H NMR (500 MHz, DMSO-d6) δ 10.01 (s, 1H), 7.50
(s, 1H), 7.47 – 7.36 (m, 3H), 7.08 (d, J = 8.3 Hz, 1H), 6.58 (dd, J = 8.2, 2.3 Hz, 1H),
6.53 (d, J = 2.2 Hz, 1H), 5.07 (s, 2H), 2.80 (t, J = 7.5 Hz, 2H), 2.43 (t, J = 7.5 Hz, 2H);
13C NMR (125 MHz, DMSO-d6) δ 170.73, 157.81, 140.22, 139.79, 133.60, 130.83,
128.93, 128.18, 127.67, 126.55, 116.59, 108.35, 102.65, 68.83, 31.19, 24.51. ESI-MS
m/z: 332.0 [M+H]+; HRMS (ESI) m/z 332.0280 [M+H]+ (calcd for 332.0281,
C16H15BrNO2).
7-((4-fluorobenzyl)oxy)-3,4-dimethyl-2H-chromen-2-one (29). Yield 81%, light
yellow solid, m.p. 139-141 °C; Purity 99.8%;1H NMR (500 MHz, DMSO-d6) δ 7.68
24

(d, J = 8.5 Hz, 1H), 7.18 (t, J = 7.7 Hz, 1H), 7.00 – 6.96 (m, 2H), 6.87 – 6.83 (m, 2H),
6.70 (dd, J = 8.5, 1.6 Hz, 1H), 5.15 (s, 2H), 2.34 (s, 3H), 2.06 (s, 3H); 13C NMR (125
MHz, DMSO-d6) δ 163.78, 161.29, 160.74, 156.49, 147.18, 142.56, 130.57, 129.73,
129.07, 125.82, 119.57, 115.46, 113.54, 104.09, 69.87, 20.13, 18.50. ESI-MS m/z:
299.0 [M+H]+; HRMS (ESI) m/z 321.0895 [M+Na]+ (calcd for 321.0897,
C18H15FNaO3).
7-((4-chlorobenzyl)oxy)-3,4-dimethyl-2H-chromen-2-one (30). Yield 75%, light
yellow solid, m.p. 135-137 °C; Purity 99.6%;1H NMR (500 MHz, DMSO-d6) δ 7.69
(d, J = 8.9 Hz, 1H), 7.42 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 8.1 Hz, 2H), 7.11 – 6.99 (m,
1H), 6.94 (dd, J = 8.9 Hz, J = 2.8 Hz, 1H), 5.13 (s, 2H), 2.37 (s, 3H), 2.06 (s, 3H); 13C
NMR (125 MHz, DMSO-d6) δ 166.52, 160.31, 154.82, 147.23, 140.25, 134.87,
130.79, 133.24, 130.18, 129.73, 129.07, 125.82, 119.57, 113.54, 104.12, 70.81, 20.09,
18.45. ESI-MS m/z: 315.0 [M+H]+; HRMS (ESI) m/z 337.0601 [M+Na]+ (calcd for
337.0602, C18H15ClNaO3).
7-((3,4-difluorobenzyl)oxy)-3,4-dimethyl-2H-chromen-2-one (31). Yield 73%, light
yellow solid, m.p. 152-155 °C; Purity 98.7%;1H NMR (500 MHz, DMSO-d6) δ 7.48
(d, J = 8.8 Hz, 1H), 7.09 – 7.03 (m, 2H), 6.91 – 6.88 (m, 1H), 6.88 (dd, J = 8.9, 2.8
13
Hz, 1H), 6.81 (d, J = 2.3 Hz, 1H), 5.16 (s, 2H), 2.35 (s, 3H), 2.16 (s, 3H); C NMR
(125 MHz, DMSO-d6) δ 164.67, 160.34, 154.82, 150.11, 147.23, 146.38, 139.75,
134.87, 130.79, 133.24, 130.18, 129.89, 129.12, 125.45, 118.35, 113.65, 104.09,
70.21, 21.13, 18.67. ESI-MS m/z: 317.0 [M+H]+; HRMS (ESI) m/z 339.0805
[M+Na]+ (calcd for 339.0806, C36H34Cl2NaO6).
4.2. MAO inhibition 22-24
Human MAO-A and MAO-B were purchased from Sigma-Aldrich. Briefly, 0.1 mL of
sodium phosphate buffer (0.05 mM, pH 7.4) containing the tested drugs at various
concentrations and adequate amounts of recombinant MAO-A or MAO-B required
and adjusted to obtain in our experimental conditions the same reaction velocity, i.e.,
to oxidize (in the control group) the same concentration of substrate: 165 pmol of
p-tyramine/min (MAO-A: 1.1 μg protein; specific activity: 150 nmol of p-tyramine
25

oxidized to p-hydroxyphenylacetaldehyde/min/mg protein; MAO-B: 7.5 μg protein;
specific activity: 22 nmol of p-tyramine transformed/min/mg protein) were incubated
for 15 min at 37 °C in a flat-black-bottom 96-well microtest plate placed in a dark
fluorimeter chamber. After this incubation period, the reaction was started by adding
200 μM (final concentrations) Amplex Red reagent, 1 U/mL horseradish peroxidase,
and 1 mM p-tyramine. The production of H2O2 and consequently, of resorufin, was
quantified at 37 °C in a SpectraMax Paradigm (Molecular Devices, Sunnyvale, CA)
muti-mode detection platform reader based on the fluorescence generated (excitation,
545 nm; emission, 590 nm). The specific fluorescence emission was calculated after
subtraction of the background activity. The background activity was determined from
wells containing all components except the MAO isoforms, which were replaced by a
sodium phosphate buffer solution (0.05 mM, pH 7.4). The percent inhibition was
calculated by the following expression: (1 - IFi/IFc) × 100% in which IFi and IFc are
the fluorescence intensities obtained for MAOs in the presence and absence of
inhibitors after subtracting the respective background.
4.3. Cell viability and neuroprotection activity assay
PC12 cells (rat pheochromocytoma) was obtained from the Cell Bank of the Chinese
Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium
containing 10% (v/v) foetal bovine serum, 100 U penicillin/mL and 100 mg
streptomycin/mL under 5% CO2 at 37 °C. The culture media were changed every
other day. Cells were subcultured in 96-well plates at a seeding density of 5 × 103
cells/well and allowed to adhere and grow. When cells reached the required
confluence, they were placed into serum-free medium and treated with compounds
1-31 (100 μM and 20 μM). Twenty-four hours (and seventy-two hours) later the
survival of cells was determined by MTT assay. Briefly, after incubation with 20 μL
of MTT at 37 °C for 4 h, living cells containing MTT formazan crystals were
solubilized in 150 μL DMSO-D6. The absorbance of each well was measured using a
microculture plate reader with a test wavelength of 570 nm and a reference
wavelength of 630 nm. Results are expressed as the mean ± SD of three independent
26

experiments.
PC12 cells were seeded at 5 × 103 cells/well in 96-well plates for neuroprotection
activity assay. After 24 h, the medium was removed and replaced with the tested
compounds (20 μM) at 37 °C and incubated for another 24 h. Rasagiline was used as
the control with the same concentration of 20 μM. Then, the cells were exposed to
6-OHDA(40 μM) and Rotenone (1 μM) respectively, and incubated at 37 °C for 24 h
before assayed with MTT. PC12 cells were cultured without test compound or
neurotoxins as control groups and the results were expressed by percentage of control.
Results are expressed as the mean ± SEM of three independent experiments.
4.4. Annexin V APC/7-AAD double staining to detect apoptosis 29
Cells growing in the logarithmic phase were trypsinized and seeded into a 6-well plate.
The corresponding drug containing medium was added (100, 20 or 4 μg/ml) after the
cells were attached to the plate and negative control group was included at the same
time. After treatment with the drug for 24 h, 0.25% trypsin (without EDTA) was used
to trypsinize and gather the cells. The cells were washed twice with
phosphate-buffered saline (PBS) (centrifugation at 2,000 rpm, 5 min), and 2 × 105
cells were collected. The cells were then resuspended in 500 μl of binding buffer.
After 5 μl of Annexin V APC was added and mixed well, 5 μl of 7-AAD was added
and mixed well. The reaction was performed at room temperature for 5-15 min in the
dark, and a flow cytometer (FACSCalibur; Becton-Dickinson, USA) was used to
detect apoptosis.
4.5. Hoechst 33258 staining assay 30
For Hoechst 33258 staining assay, PC12 cells were stained with Hoechst 33258 for 30
min. The cells were plated on the glass slide and covered with cover slip, then
observed using a fluorescent inverted microscope (Nikon ti-s, Japan).
4.6. Evaluation of intracellular ROS 31
The level of intracellular ROS was measured by using the ROS-sensitive dye, 2,
7-dichloro-fluorescein diacetate (DCFH-DA), as a probe. In brief, PC12 cells were
seeded in six-well plates at 2 × 105 cells/well, culturing in the presence or absence of
27

various concentrations of tested samples for additional 24 h, and then washed three
times and incubated with final concentration of 10 mM DCFH-DA for 30 min at
37 °C in the dark. After incubation, cells were washed three times and harvested in
free-serum medium. The fluorescence of 2, 7-dichlorofluorescein (DCF) was detected
by flow cytometry (488 nm excitation and 525 nm emission filters) using BD Accuri
C6 flow cytometer (Becton & Dickinson Company, Franklin Lakes, NJ, USA). Data
were processed by using cell quest software (Becton & Dickinson Company, Franklin
Lakes, NJ).
4.7. In vitro BBB permeation assay 33
Commercial drugs were purchased from Sigma and Alfa Aesar. The porcine brain
lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate (PVDF
membrane, pore size 0.45 mm) and the acceptor microplate were both from Millipore.
The 96-well UV plate (COSTAR@) was from Corning Incorporated. The acceptor
96-well microplate was filled with 300 μL of PBS/EtOH (7:3), and the filter
membrane was impregnated with 10 μL of PBL in dodecane (20 mg/mL). Compounds
were dissolved in DMSO-D6 at 5 mg/mL and diluted 50-fold in PBS/EtOH (7:3) to
achieve a concentration of 100 mg/mL, 200 μL of which was added to the donor wells.
The acceptor filter plate was carefully placed on the donor plate to form a sandwich,
which was left undisturbed for 16 h at 25 °C. After incubation, the donor plate was
carefully removed and the concentration of compound in the acceptor wells was
determined using a UV plate reader (Flexsta-tion@ 3). Every sample was analyzed at
five wavelengths, in four wells, in at least three independent runs, and the results are
given as the mean ± SEM. In each experiment, 9 quality control standards of known
BBB permeability were included to validate the analysis set.
4.8. Animals and acute toxicity test 35, 36
Twenty Kunming mice (20 ± 2 g) were purchased from Laboratory Animal Research
Center, Nanjing University (Nanjing, China). Animals were housed in stainless steel
cages by sex in a ventilated animal room. Room temperature was maintained at
25 ± 2 °C, relative humidity at 50 ± 10%, and a 12 h light/dark cycle. Distilled water
28

and sterilized food for mice were available ad libitum. They were acclimated to this
environment for 5 days prior to dosing. All procedures were approved by the China
Pharmaceutical University Animal Care and Use Committee (IACUC) and were in
compliance with the National Institute of Health (NIH) guidelines.
Animals were randomly divided into two groups: control group and experimental
group (2000 mg/kg, n=10 per group). Before treatment, animals were fasted overnight.
Compound 13 was suspended in 0.5% carboxymethyl cellulose sodium (CMC-Na)
salt solution and orally administered according to the divided groups. Food and water
were provided later. After administration of 13, the mice were observed continuously
for the first 4 h for any abnormal behavior and mortality changes, intermittently for
the next 24 h, and occasionally thereafter for 14 days for the onset of any delayed
effects. All animals were sacrificed after being anaesthetized by ether on the 14th day
after drug administration.
Acknowledgments
The research work was financially supported by the Project of National Natural
Sciences Foundation of China (81573313), the Program for Changjiang Scholars and
Innovative Research Team in University (PCSIRT-IRT_15R63), and the Priority
Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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