Neuroprotective effects of benzyloxy substituted small molecule [626931]

Neuroprotective effects of benzyloxy substituted small molecule
monoamine oxidase B inhibitors in Parkinson’s disease
Zhimin Wangy, Jiajia Wuy, Xuelian Yang, Pei Cai, Qiaohong Liu, Kelvin D. G. Wang, Lingyi Kong⇑,
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
article info
Article history:Received 8 August 2016Revised 19 September 2016Accepted 20 September 2016Available online 21 September 2016
Keywords:
Parkinson’s disease
Monoamine oxidase
BBB permeabilityApoptosisNeuroprotectionabstract
The benzyloxy substituted small molecules are well-known highly potent monoamine oxidase B inhibi-
tors, 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 evaluatedfor MAO-A/B inhibition. In addition, their neuroprotective effects were investigated in 6-OHDA- and rote-none-treated PC12 cells. It was observed that most of the compounds exhibited a marked increase in sur-
vival of PC12 cells which treated with the neurotoxins. Among them, 13exhibited remarkable and
balanced neuroprotective potency. The protective effects of 13against neurotoxins-induced apoptosis
were confirmed with flow cytometry and staining methods. Furthermore, 13also showed good BBB per-
meability and low toxicity according to in vitro BBB prediction and in vivo acute toxicity test. The results
indicated that 13is an effective and promising candidate to be further developed as disease-modifying
drug for Parkinson’s disease therapy.
/C2112016 Elsevier Ltd. All rights reserved.
1. Introduction
Parkinson’s disease (PD) is a degenerative disorder of the cen-
tral nervous system (CNS) which characterized by a progressive
loss of pigmented dopaminergic neurons in the substantia nigra
pars compacta.1,2The cornerstone of PD therapy is aimed at
enhancing central dopamine levels with its direct metabolic pre-
cursor, L-3,4-dihydroxyphenylalanine ( L-DOPA).3To enhance the
therapeutic efficacy of L-DOPA and reduce the occurrence of dopa-
mine-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.4,5Thus, inhibition
of MAO-B leads to enhancement of dopaminergic neurotransmis-
sion, 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 inhi-
bitory activities, and those compounds have been further exam-
ined neuroprotective capabilities against neurodegeneration by
preventing apoptosis.8–12Among them, selegiline and rasagilinewere 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.13Therefore, 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.14–21Although 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 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,
http://dx.doi.org/10.1016/j.bmc.2016.09.050
0968-0896/ /C2112016 Elsevier Ltd. All rights reserved.⇑Corresponding authors. Tel./fax: +86 25 8327 1405.
E-mail addresses: cpu_lykong@126.com (L. Kong), xbwang@cpu.edu.cn
(X. Wang).
yThese authors contributed equally.Bioorganic & Medicinal Chemistry 24 (2016) 5929–5940
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.else vier.com/locate/bmc

respectively. The neuroprotective effects of the preferred com-
pounds 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.
2. Results and discussion
2.1. Chemistry
These representative benzyloxy substituted derivatives ( 1–31)
were efficiently synthesized along with the pathway shown inFig. 2 . The starting materials (various skeletons, see Supporting
information for the synthesis of key intermediates S1–4)
17,19,21
reacted with the appropriate benzyl bromides in the presence of
K2CO3to give the target compounds in moderate to good yields.
2.2. Inhibition of MAOs
According to the reported assay with rasagiline and iproni-
azid22–24, the MAO inhibitory activities of compounds 1–31were
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 substitutedbenzyloxy side chains, were selective inhibitors toward MAO-B
with IC 50values in the 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) .
2.3. Cytotoxicity and neuroprotection assays in PC12 cells
To investigate the safety index of these potent MAO-B inhibitors,
all the compounds 1–31were 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)-2,5-
diphenyl-2 H-tetrazolium bromide] assay.25As shown in the Fig-
ure 3 A and Table S2 (Supporting information) , the results revealed
that compounds 17–28displayed low toxicities at higher concentra-
tion (100 lM), while most of the compounds at concentration of 100
and 20 lM did not shown significant neurotoxicity. This result sug-
gested these compounds could be used for further development.
To gain insight into the therapeutic potentials of these com-
pounds, the neuroprotective capacity was also determined by
MTT assay in PC12 cells. PC12 cells were chosen for this purpose
Figure 1. Structures of the benzyloxy substituted MAO-B inhibitors 1–31.5930 Z. Wang et al. / Bioorg. Med. Chem. 24 (2016) 5929–5940

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.12Two parallel experiments
were performed with 6-hydroxydopamine- (6-OHDA) and rote-
none-treated PC12 cells.26,27According to the method described
by Zheng et al.28with slight modification, rasagiline was used as
positive control in these experiments. Cells were incubated with
6-OHDA (40 lM) for 24 h, and tested compounds and rasagiline
were applied in a single 20 lM concentration for 1 h prior to 6-
OHDA application. The MTT assay was used to assess cell viability.
In the similar manner, rotenone (1 lM) was used to assess the abil-
ity of the tested compounds (20 lM) to facilitate cell survival.
As the results were summarized in Table 1 andFigure 3 B, rasag-
iline exhibited significant protective effect (39%) in 20 lM concen-
tration in 6-OHDA-treated PC12 cell ( Table 1 ) which was
consistent with literature data.12,26Among the tested compounds,
compounds 5–8,12,14–17,25and 31were protective against
6-OHDA-induced cell death (>30% protection), while only
compounds 13and 28shown >20% protection against rotenone-
induced cell death. Especially, compound 15showed the highest
increase of 48% in the survival of 6-OHDA-treated cells. Compound
28had 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 neuroprotec-
tive activities in both 6-OHDA- and rotenone-treated PC12 cells in
a concentration-dependent manner ( Fig. 4 ).
2.4. Effects of compound 13 on both 6-OHDA- and rotenone-
induced apoptosis in PC12 cells
Compound 13was chosen to investigate the protective effects
against neurotoxins-induced apoptosis not only because of noneurotoxicity, but also because 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 cytometry29. The Annexin V
APC ( /C0)/7-AAD ( /C0) population consisted primarily of normal
healthy cells, while Annexin V APC (+)/7-AAD ( /C0) cells were con-
sidered 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 ( Figs. S1A and B ) showed that cell
viability significantly decreased when cells were treated with
40lM 6-OHDA and rotenone for 24 h, respectively, compared with
those of control. When PC12 cells were exposed to 5–40 lM com-
pound 13in the presence of 40 lM 6-OHDA, or 1 lM rotenone for
24 h, the co-treated cells showed a significant increase in cell via-
bility in a concentration dependent manner, compared to those
treated with neurotoxins-treated alone. As shown in Figure 5 ,
apoptotic cells percentage of 6-OHDA group were significantly
increased compared to control group. In contrast, the rates of apop-
totic cells were reduced when 6-OHDA-treated PC12 cells were
pretreated with 5, 10, 20, 40 lM of compound 13. As the result
inFigure 6 , PC12 cells death of rotenone-treated group was
appeared due to the severe neurotoxicity. Similarly, compound
13significantly decreased the cell death rate in a concentration
dependent manner.
Nuclear morphology characteristic of apoptosis and neuro-
protection of 13were further investigated using DNA staining
with Hoechst 33258.30Condensed or fragmented nuclei were
characterized as apoptotic nuclei. Treatment with 20 lM com-
pound 13decreased the number of apoptotic nuclei ( Fig. S2 ).
These results showed that 13significantly decreased the per-
centage of apoptotic nuclei when compared to 6-OHDA- and
rotenone-treated groups, which indicated that 13 have
Figure 2. Synthesis of the benzyloxy substituted derivatives 1–31. Reagents and conditions: (a) H 2, Pd/C, MeOH/EtOAc, 24 h; (b) H 2SO4/NaNO 2, 50% H 2SO4, 125 /C176C;
(c) CF 3SO3H, 75–80 /C176C, 1.5 h; (d) ZnCl 2,5 0/C176C, 2 h; (e) dioxane, concd H 2SO4,9 0/C176C, 4 h; (f) K 2CO3, acetonitrile, reflux, 8 h.Z. Wang et al. / Bioorg. Med. Chem. 24 (2016) 5929–5940 5931

neuroprotective capability against neurodegeneration by pre-
venting apoptosis.
2.5. Compound 13 reduced 6-OHDA- and rotenone-induced
intracellular ROS accumulation
The oxidative stress caused by ROS is partly responsible for
a wide variety of cellular damage and is regarded as a key ini-
tiator of neurotoxins-induced apoptotic.31As shown in Figure 7 ,
when PC12 cells were exposed to 40 lM 6-OHDA, or 1 lM
rotenone for 24 h, the intracellular ROS increased obviously
by using the DCFH-DA probe. While treated with 13at the
concentrations of 5, 10 and 20 lM, 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.
2.6. Theoretical evaluation of physical properties and ADMET
The compounds with higher activity ( 5–8,12–17,25and 31)
were evaluated for the ADMET properties in silico by the Accelrys
Discovery Studio ( Table 2 ).32All the compounds showed ADMET
properties similar to drug rasagiline. Compared with the standard
drug, these compounds were predicted to have fine absorption,
high blood brain barrier (BBB) level and good solubility. In
addition, the physical properties of the selected compounds werefollowed Lipinski’s rule of five:33molecular 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
(ClogP) 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-Parkinson’s disease potentials which satisfied possible
BBB penetration and containing drug-like properties. However,the compounds need to be further optimized for the pharmacoki-
netics properties.
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 silico prediction,
we also used a parallel artificial membrane permeation assay for
BBB (PAMPA-BBB), which was described by Di et al.
34,35Assay val-
idation 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.) /C00.346 ( R2= 0.9537)
(Fig. S3 ). From the equation and taking into account the limits
established by Di et al. for BBB permeation. The Pevalues of these
selected compounds are summarized in Table 3 . It can be indicated
that compounds 5,6,8,13,15,17and28shown higher Pevalues
than 4.04 were able to cross the BBB.Table 1
Inhibition of human MAO-A and MAO-B activities and neuroprotective effects of compounds 1–31
Compounds MAO-A IC 50a(lM) MAO-B IC 50a(nM) Selectivity indexb% survival of PC12 cellsc
6-OHDA Rotenone
1 >100 15.48 ± 1.34 >6460 115.53 ± 6.57 88.59 ± 4.20
2 88.43 ± 6.90 25.32 ± 4.65 3492 123.30 ± 4.28 95.77 ± 5.08
3 39.45 ± 5.26 10.52 ± 1.04 3750 131.78 ± 3.52 109.05 ± 10.79
4 53.68 ± 2.4 92.41 ± 3.47 581 109.39 ± 3.96 105.04 ± 10.46
5 >100 41.38 ± 6.00 >2417 133.41 ± 9.23 106.30 ± 11.51
6 43.38 ± 2.15 28.42 ± 5.35 1526 143.64 ± 6.80 98.31 ± 5.73
7 48.05 ± 3.92 25.12 ± 3.50 1913 134.00 ± 10.97 93.34 ± 2.66
8 25.34 ± 7.91 42.75 ± 3.18 593 144.11 ± 10.59 105.07 ± 10.26
9 >100 96.51 ± 4.56 >1036 115.85 ± 9.35 108.75 ± 10.80
10 >100 56.89 ± 1.31 >1758 104.54 ± 8.08 113.99 ± 10.01
11 >100 13.25 ± 2.24 >7547 133.45 ± 5.85 111.85 ± 2.09
12 89.05 ± 3.9 52.93 ± 0.28 1682 141.51 ± 6.71 101.33 ± 6.58
13 >100 12.34 ± 1.62 >8104 134.19 ± 6.76 126.38 ± 6.92
14 >100 42.05 ± 2.48 >2378 143.35 ± 3.79 111.02 ± 1.89
15 >100 12.85 ± 5.38 >7782 148.17 ± 10.30 111.47 ± 4.05
16 >100 39.21 ± 2.94 >2550 143.14 ± 7.83 108.32 ± 4.12
17 1.29 ± 0.51 12.55 ± 1.68 23 139.15 ± 8.24 95.92 ± 5.40
18 0.34 ± 0.04 7.21 ± 0.81 47 126.78 ± 3.69 109.51 ± 0.71
19 23.59 ± 7.21 43.42 ± 8.25 543 117.17 ± 5.87 101.38 ± 1.73
20 18.52 ± 1.14 63.22 ± 3.54 293 124.17 ± 8.29 115.37 ± 1.82
21 18.94 ± 4.07 27.82 ± 1.35 681 122.61 ± 8.42 110.30 ± 6.00
22 4.52 ± 0.73 25.31 ± 8.21 179 122.00 ± 3.61 114.67 ± 4.26
23 13.59 ± 3.21 15.62 ± 4.29 870 114.67 ± 4.41 106.33 ± 5.61
24 1.33 ± 0.48 8.35 ± 1.01 159 116.04 ± 4.69 113.67 ± 2.27
25 3.61 ± 1.01 13.87 ± 3.14 260 137.19 ± 10.90 113.35 ± 4.58
26 9.24 ± 2.37 6.41 ± 2.73 1441 124.63 ± 6.70 111.05 ± 5.29
27 >100 14.27 ± 1.66 >7008 121.21 ± 1.52 110.21 ± 7.08
28 49.17 ± 2.73 8.17 ± 1.78 6018 111.71 ± 2.99 133.52 ± 3.24
29 >100 5.03 ± 0.81 >19881 129.93 ± 9.72 115.42 ± 7.47
30 6.77 ± 1.18 6.48 ± 1.37 1045 114.33 ± 4.81 119.33 ± 7.84
31 >100 4.25 ± 1.40 >23529 137.63 ± 3.53 116.67 ± 8.01
Iproniazid 7.57 ± 0.43 8.96 ± 0.38 lM 0.84 — —
Control — — — 99.33 ± 1.86 98.33 ± 3.38Rasagiline 50.71 ± 3.19 7.87 ± 1.25 6443 134.52 ± 9.09 109.03 ± 6.77
aIC50: 50% inhibitory concentration (means ± SEM of three experiments).
bSelectivity Index = IC 50(MAO-A)/IC 50(MAO-B).
cSurvival 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 i ndependent
plates.5932 Z. Wang et al. / Bioorg. Med. Chem. 24 (2016) 5929–5940

2.8. Acute toxicity test in vivo
According to the results of in silico prediction, most of the com-
pounds showed a probability of hepatotoxicity. Therefore, it is nec-
essary to test the in vivo toxicity of the most promising compound
13. The procedure for acute toxicity study followed similar proto-
cols from the reported studies.36,37After administration of the
compound 13(2000 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 signif-
icant abnormal changes in water or food consumption or bodyweight reduction were observed ( Fig. 8 ). Furthermore, all mice
were sacrificed on the 14th day after drug administration. This
indicated that 13was nontoxic and tolerated at doses up to
2000 mg/kg.
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 Soft-
ware, Inc., San Diego, CA, USA). All values were presented as
mean ± standard error of the mean (mean ± SEM) for each group.
P<0.05 was considered statistical significant.
Figure 3. Cytotoxicity and neuroprotective effects of the tested compounds 1–31. Survival data are expressed as the percentage of 6-OHDA- and rotenone-treated cells.
Symbols represent significant changes from 6-OHDA- and rotenone-treated PC12 cells (*P<0.05;**P<0.01), respectively. All data are the means ± SEM of at least five values
measured in two independent plates.
Figure 4. Neuroprotective effects of compound 13against 6-OHDA- and rotenone-induced toxicity in PC12 cells. Rasagiline was used as the reference compound. Results are
expressed as percent viability compared to cells not treated with neurotoxins. All data are the means ± SEM of at least five values measured in two indepe ndent plates
(*P<0.05;**P<0.01).Z. Wang et al. / Bioorg. Med. Chem. 24 (2016) 5929–5940 5933

3. Conclusions
In conclusion, this study showed that most of the representa-
tive benzyloxy substituted derivatives 5–8,12–17,25,28and 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 com-
pound 13exhibited neuroprotective effects via reducing intra-
cellular ROS and preventing neurotoxins-induced apoptosis.
Furthermore, 13showed good BBB permeability and low toxicity
according to in vitro BBB prediction and in vivo acute toxicity
test. Based on the results, compound 13and 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 com-
mercial suppliers and used without further purification. Reaction
progress was monitored using analytical thin layer chromatogra-
phy (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 lM; 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 /C176C and referenced to TMS. Chemical shifts
are reported in ppm ( d) 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.
Figure 5. Effects of compound 13 on 6-OHDA-induced apoptosis in PC12 cells. Symbols represent significant changes (**P<0.01;***P<0.001).5934 Z. Wang et al. / Bioorg. Med. Chem. 24 (2016) 5929–5940

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) con-
taining K 2CO3(2.0 mmol). The reaction was treated with an appro-
priately substituted arylalkyl bromide (1.2 mmol) and heated
under reflux for 8 h. The reaction progress was monitored using sil-
ica gel TLC with Petroleum ether/EtOAc as mobile phase. Upon
completion, the acetonitrile was evaporated in vacuo and the mix-
ture was then poured into water, which was extracted with
3/C250 mL of EtOAc, washed with brine, dried over anhydrous
Na2SO4and purified by chromatography (PE/EA, 50:1, 20:1, 10:1)
onsilica gel .
4.1.1.1. 1-(4-((4-Fluorobenzyl)oxy)phenyl)ethanone (1). Yield
83%, white solid, mp 75–77 /C176C; Purity 97.5%;1H NMR (500 MHz,
DMSO- d6)d7.95 (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)d196.72, 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/z245.0970 [M+H]+(calcd for 245.0972, C 15H14FO2).
4.1.1.2. 1-(4-((3-Chlorobenzyl)oxy)phenyl)ethanone (2). Yield
89%, white solid, mp 79–81 /C176C; Purity 98.6%;1H NMR (500 MHz,
DMSO- d6)d7.96 (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)d196.73,
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, C 15H14ClO 2).
4.1.1.3. 1-(3-((4-Chlorobenzyl)oxy)phenyl)ethanone (3). Yield
82%, light yellow oil; Purity 99.3%;1H NMR (500 MHz, DMSO- d6)
d7.59 (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)d198.11, 158.83, 138.83, 136.37,
132.98, 130.39, 129.97, 129.97, 128.95, 128.95, 121.55, 120.41,
Figure 6. Effects of compound 13 on rotenone-induced apoptosis in PC12 cells. Symbols represent significant changes (**P<0.01;***P<0.001).Z. Wang et al. / Bioorg. Med. Chem. 24 (2016) 5929–5940 5935

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, C 15H14ClO 2).
4.1.1.4. 1-(3-((4-Bromobenzyl)oxy)phenyl)ethanone (4). Yield
90%, light yellow oil; Purity 99.7%;1H NMR (500 MHz, DMSO- d6)
d7.60 (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)d198.10, 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, C 15H13BrNaO 2).
4.1.1.5. 1-(4-((3-Fluorobenzyl)oxy)-2-hydroxyphenyl)ethanone
(5). Yield 89%, white solid, mp 103–104 /C176C; Purity 98.5%;1H
NMR (500 MHz, DMSO- d6)d12.61 (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,
Figure 7. Effect of 13 against ROS generation was measured by DCFH-DA staining and analyzed by flow cytometry. Analysis of ROS production is presented as the mean
fluorescence intensity (MFI). (*P<0.05;**P<0.01;***P<0.001.)
Table 2
Physical properties and ADMET prediction of the selected compounds
Compounds MWaCLogPaHBAaHBDaPSAaLogBBaAbsorption levelbSolubility levelbHepatotoxicity probabilitybPPBLEVELbBBB levelb
5 260.26 3.898 2 1 46.53 0.722496 0 3 1 0 2
6 260.26 3.898 2 1 46.53 0.722496 0 3 1 0 2
7 276.72 4.468 2 1 46.53 0.809136 0 3 1 0 2
8 276.72 4.468 2 1 46.53 0.809136 0 3 1 0 2
12 278.71 4.017 2 0 26.3 0.740584 0 2 1 0 1
13 272.28 3.656 3 0 35.53 0.685712 0 2 1 2 1
14 288.73 4.226 3 0 35.53 0.772352 0 2 1 2 1
15 300.33 4.694 3 0 35.53 0.843488 0 2 1 0 1
16 316.09 5.264 3 0 35.53 0.930128 0 2 1 1 1
17 270.30 4.256 2 0 26.3 0.776912 0 2 1 2 0
25 274.70 3.452 3 0 35.53 0.654704 0 2 1 2 1
28 332.20 4.002 2 1 38.33 0.738304 0 2 1 2 1
31 316.30 4.677 3 0 35.53 0.840904 0 2 1 1 0
Rasagiline 171.24 2.344 0 1 12.03 0.664332 0 3 0 2 1Rules 6450 65.0 610 65 690P/C01.0 — — — — —
aMW: molecular weight; ClogP: calculated logarithm of the octanol–water partition coefficient; HBA: hydrogen-bond acceptor atoms; HBD: hydrogen-bond donor atoms;
PSA: polar surface area; logBB = 0.0148 /C2PSA + 0.152 /C2ClogP+ 0.13.
bAbsorption level (0 = good and 3 = very poor); solubility level (0 = extremely low and 3 = good); hepatotoxicity (0 = nontoxic and 1 = toxic); PPB, plasma pr otein binding
(0 = PPB < 90% and 2 = PPB > 95%); BBB level (0 = very high and 3 = low).5936 Z. Wang et al. / Bioorg. Med. Chem. 24 (2016) 5929–5940

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), 2.59 (s, 3H);13C NMR (125 MHz, DMSO- d6)d202.37,
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/z261.0922 [M+H]+(calcd for 261.0921,
C15H14FO3).
4.1.1.6. 1-(4-((4-Fluorobenzyl)oxy)-2-hydroxyphenyl)ethanone
(6). Yield 85%, white solid, mp 111–112 /C176C; Purity 98.5%;1H
NMR (500 MHz, DMSO- d6)d12.62 (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)d201.19, 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/z261.0916 [M+H]+(calcd for
261.0922, C 15H14FO3).
4.1.1.7. 1-(4-((3-Chlorobenzyl)oxy)-2-hydroxyphenyl)ethanone
(7). Yield 83%, white solid, mp 128–129 /C176C; Purity 99.6%;1H
NMR (500 MHz, DMSO- d6)d12.61 (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)d202.63, 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 /C0H]/C0; HRMS (ESI) m/z275.0479
[M/C0H]/C0(calcd for 275.0480, C 15H12ClO 3).4.1.1.8. 1-(4-((4-Chlorobenzyl)oxy)-2-hydroxyphenyl)ethanone
(8). Yield 88%, white solid, mp 102–103 /C176C; Purity 100%;1H
NMR (500 MHz, DMSO- d6)d12.61 (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= 2.4 Hz, 1H), 5.22 (s, 2H), 2.58 (s, 3H);13C NMR (125 MHz,
DMSO- d6)d202.60, 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/z275.0482 [M /C0H]/C0(calcd for
275.0480, C 15H12ClO 3).
4.1.1.9. 1-(2-Fluoro-4-((3-fluorobenzyl)oxy)phenyl)ethanone
(9). Yield 93%, light yellow solid, mp 104–105 /C176C; Purity 99.4%;
1H NMR (500 MHz, DMSO- d6)d7.83 (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 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), 2.53 (s, 3H);13C NMR (500 MHz, DMSO- d6)d
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/z263.0879 [M+H]+(calcd for 263.0878, C 15H13F2O2).
4.1.1.10. 1-(2-Fluoro-4-((4-fluorobenzyl)oxy)phenyl)ethanone
(10). Yield 87%, light yellow solid, mp 69–71 /C176C; Purity 99.2%;
1H NMR (500 MHz, DMSO- d6)d7.83 (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)d193.5, 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/z263.0877
[M+H]+(calcd for 263.0878, C 15H13F2O2).
4.1.1.11. 1-(2-Fluoro-4-((3-chlorobenzyl)oxy)phenyl)ethanone
(11). Yield 93%, light yellow solid, mp 70–72 /C176C; Purity 99.9%;
1H NMR (500 MHz, DMSO- d6)d7.83 (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)d193.6, 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/z301.0400 [M+Na]+(calcd for 301.0402,
C15H12ClFNaO 2).
4.1.1.12. 1-(2-Fluoro-4-((4-chlorobenzyl)oxy)phenyl)ethanone
(12). Yield 85%, light yellow solid, mp 74–75 /C176C; Purity 100%;
1H NMR (500 MHz, DMSO- d6)d7.83 (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 Hz,
1H), 5.24 (s, 2H), 2.53 (s, 3H);13C NMR (125 MHz, DMSO- d6)d
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/z301.0403 [M+Na]+(calcd for 301.0402,
C15H12ClFNaO 2).
4.1.1.13. 7-((4-Fluorobenzyl)oxy)chroman-4-one (13). Yield
91%, colorless solid, mp 125–127 /C176C; Purity 99.9%;1HN M R
(500 MHz, DMSO- d6)d7.69 (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);13C NMR (125 MHz, DMSO- d6)d189.86,
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/z567.1586 [2M+Na]+(calcd for
567.1590, C 32H26NaO 6F2).
4.1.1.14. 7-((4-Chlorobenzyl)oxy)chroman-4-one (14). Yield
89%, colorless solid, mp 139–140 /C176C; Purity 99.6%;1HN M R
(500 MHz, DMSO- d6)d7.69 (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,Table 3
Permeability results ( Pe/C210/C06cm s/C01) from the PAMPA-BBB assay for selected
compounds with their predicted penetration into the CNS
Compounds Permeability ( Pe/C210/C06cm s/C01)aPredictionb
5 15.9 ± 0.6 CNS+
6 13.5 ± 0.4 CNS+
8 12.7 ± 0.2 CNS+
13 14.3 ± 0.4 CNS+
15 9.71 ± 0.52 CNS+
17 13.2 ± 0.7 CNS+
28 8.89 ± 0.37 CNS+
aData are the mean ± SEM of three independent experiments.
bPe(10/C06cm s/C01) > 4.5 for high BBB permeation (CNS+), Pe(10/C06cm s/C01) < 1.85
for low BBB permeation (CNS /C0), and 4.04 > Pe(10/C06cm s/C01) > 1.85 for uncertain
BBB permeation (CNS±).
Figure 8. Effects on body weight of mice fed 13 in the acute toxicity test.Z. Wang et al. / Bioorg. Med. Chem. 24 (2016) 5929–5940 5937

DMSO- d6)d190.44, 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/z311.0446 [M+Na]+
(calcd for 311.0445, C 16H13ClNaO 3).
4.1.1.15. 7-((4-Fluorobenzyl)oxy)-2,2-dimethylchroman-4-one
(15). Yield 87%, yellow solid, mp 91–92 /C176C; Purity 98.6%;1H
NMR (500 MHz, DMSO- d6)d7.66 (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,
1H), 6.58 (d, J= 2.3 Hz, 1H), 5.15 (s, 2H), 2.71 (s, 2H), 1.39 (s,
6H);13C NMR (125 MHz, DMSO- d6)d190.78, 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/z623.2210 [2M+Na]+(calcd for
623.2216, C 36H34F2NaO 6).
4.1.1.16. 7-((4-Chlorobenzyl)oxy)-2,2-dimethylchroman-4-one
(16). Yield 85%, colorless solid, mp 130–131 /C176C; Purity 99.5%;1H
NMR (500 MHz, DMSO- d6)d7.66 (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)d190.79,
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/z655.1621 [2M+Na]+
(calcd for 655.1625, C 36H34Cl2NaO 6).
4.1.1.17. 6-((4-Fluorobenzyl)oxy)-3,4-dihydronaphthalen-1
(2H)-one (17). Yield 87%, yellow solid, mp 83–85 /C176C; Purity
98.8%;1H NMR (500 MHz, DMSO- d6)d7.85 (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, 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)d196.48,
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/z293.0947 [M+Na]+(calcd for
293.0948, C 17H15FNaO 2).
4.1.1.18. 6-((4-Chlorobenzyl)oxy)-3,4-dihydronaphthalen-1
(2H)-one (18). Yield 81%, yellow solid, mp 97–99 /C176C; Purity
99.0%;1H NMR (500 MHz, DMSO- d6)d7.93–7.77 (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, 2H), 2.58–2.53 (m, 2H), 2.11–1.89 (m, 2H);13C NMR
(125 MHz, DMSO- d6)d196.49, 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, C 17H15ClNaO 2).
4.1.1.19. 7-((3-Chlorobenzyl)oxy)-4 H-chromen-4-one (19). Yield
81%, white solid, mp 136–138 /C176C; Purity 96.5%;1H NMR (500 MHz,
DMSO- d6)d8.24 (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 Hz, 1H), 7.17 (dd,
J= 8.9, 2.2 Hz, 1H), 6.29 (d, J= 6.0 Hz, 1H), 5.30 (s, 2H);13CN M R
(125 MHz, DMSO- d6)d176.08, 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, C 16H11ClNaO 3).
4.1.1.20. 7-((4-Fluorobenzyl)oxy)-4 H-chromen-4-one (20). Yield
86%, white solid, mp 121–123 /C176C; Purity 99.0%;1H NMR (500 MHz,
DMSO- d6)d8.23 (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, J= 8.9, 1.9 Hz,
1H), 6.29 (d, J= 6.0 Hz, 1H), 5.26 (s, 2H);13C NMR (125 MHz,
DMSO- d6)d176.09, 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/z293.0583
[M+Na]+(calcd for 293.0584, C 6H11FNaO 3).4.1.1.21. 7-((4-(Trifluoromethyl)benzyl)oxy)-4 H-chromen-4-
one (21). Yield 80%, white solid, mp 159–161 /C176C; Purity 99.9%;
1H NMR (500 MHz, DMSO- d6)d8.24 (d, J= 6.0 Hz, 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)d
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/z343.0553 [M+Na]+
(calcd for 343.0552, C 17H11F3NaO 3).
4.1.1.22. 6-((4-Fluorobenzyl)oxy)-4 H-chromen-4-one (22). Yield
45%, colorless solid, mp 139–141 /C176C; Purity 99.8%;1HN M R
(500 MHz, DMSO- d6)d7.83 (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 Hz,
2H), 6.31 (d, J= 6 . 0 H z ,1 H ) ,5 . 0 8( s ,2 H ) ;13C NMR (125 MHz,
DMSO- d6)d176.83, 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/z271.0694 [M+H]+(calcd for
271.0692, C 16H12FO3).
4.1.1.23. 6-((4-Chlorobenzyl)oxy)-4 H-chromen-4-one (23). Yield
65%, colorless solid, mp 169–171 /C176C; Purity 99.7%;1HN M R
(500 MHz, DMSO- d6)d7.82 (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);13CN M R
(125 MHz, DMSO- d6)d175.94, 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/z287.0395
[M+H]+(calcd for 287.0397, C 16H12ClO 3).
4.1.1.24. 5-((4-Fluorobenzyl)oxy)isobenzofuran-1(3 H)-one
(24). Yield 85%, colorless solid, mp 133–134 /C176C; Purity 99.8%;1H
NMR (500 MHz, DMSO- d6)d7.55 (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,
1H), 5.23 (s, 2H), 4.79 (s, 2H);13C NMR (125 MHz, DMSO- d6)d
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/z281.0586 [M+Na]+(calcd for 281.0584,
C15H11FNaO 3).
4.1.1.25. 5-((4-Chlorobenzyl)oxy)isobenzofuran-1(3 H)-one
(25). Yield 75%, colorless solid, mp 130–131 /C176C; Purity 99.4%;1H
NMR (500 MHz, DMSO- d6)d7.56 (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), 5.26 (s, 2H), 4.79 (s, 2H);13C NMR (125 MHz, DMSO- d6)d
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/z297.0286 [M+Na]+(calcd for 297.0289,
C15H11ClNaO 3).
4.1.1.26. 5-((4-(Trifluoromethyl)benzyl)oxy)isobenzofuran-1
(3H)-one (26). Yield 85%, colorless solid, mp 127–128 /C176C; Purity
98.7%;1H NMR (500 MHz, DMSO- d6)d7.76 (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)d171.03,
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/z331.0556 [M+Na]+(calcd for 331.0552,
C16H11F3NaO 3).
4.1.1.27. 7-((3-Chlorobenzyl)oxy)-3,4-dihydroquinolin-2(1 H)-
one (27). Yield 68%, white solid, mp 158–160 /C176C; Purity 98.8%;
1H NMR (500 MHz, DMSO- d6)d10.01 (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,5938 Z. Wang et al. / Bioorg. Med. Chem. 24 (2016) 5929–5940

J= 7.5 Hz, 2H);13C NMR (125 MHz, DMSO- d6)d170.72, 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/z288.0785 [M+H]+(calcd for 288.0786,
C16H15ClNO 2).
4.1.1.28. 7-((3-Bromobenzyl)oxy)-3,4-dihydroquinolin-2(1 H)-
one (28). Yield 76%, white solid, mp 125–127 /C176C; Purity 98.7%;
1H NMR (500 MHz, DMSO- d6)d10.01 (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)d170.73,
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/z332.0280 [M+H]+(calcd for
332.0281, C 16H15BrNO 2).
4.1.1.29. 7-((4-Fluorobenzyl)oxy)-3,4-dimethyl-2 H-chromen-2-
one (29). Yield 81%, light yellow solid, mp 139–141 /C176C; Purity
99.8%;1H NMR (500 MHz, DMSO- d6)d7.68 (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)d163.78, 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/z321.0895 [M+Na]+(calcd for 321.0897,
C18H15FNaO 3).
4.1.1.30. 7-((4-Chlorobenzyl)oxy)-3,4-dimethyl-2 H-chromen-2-
one (30). Yield 75%, light yellow solid, mp 135–137 /C176C; Purity
99.6%;1H NMR (500 MHz, DMSO- d6)d7.69 (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)d166.52, 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/z337.0601 [M+Na]+(calcd for
337.0602, C 18H15ClNaO 3).
4.1.1.31. 7-((3,4-Difluorobenzyl)oxy)-3,4-dimethyl-2 H-chro-
men-2-one (31). Yield 73%, light yellow solid, mp 152–155 /C176C;
Purity 98.7%;1H NMR (500 MHz, DMSO- d6)d7.48 (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 Hz, 1H), 6.81 (d, J= 2.3 Hz, 1H), 5.16 (s, 2H), 2.35 (s, 3H), 2.16
(s, 3H);13C NMR (125 MHz, DMSO- d6)d164.67, 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/z339.0805 [M
+Na]+(calcd for 339.0806, C 36H34Cl2NaO 6).
4.2. MAO inhibition22–24
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 ade-
quate 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:
1.1lg protein; specific activity: 150 nmol of p-tyramine oxidized
top-hydroxyphenylacetaldehyde/min/mg protein; MAO-B: 7.5 lg
protein; specific activity: 22 nmol of p-tyramine transformed/
min/mg protein) were incubated for 15 min at 37 /C176C 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 lM (final concentrations) Amplex Red reagent, 1 U/mLhorseradish peroxidase, and 1 mM p-tyramine. The production of
H2O2and consequently, of resorufin, was quantified at 37 /C176Ci na
SpectraMax Paradigm (Molecular Devices, Sunnyvale, CA) multi-
mode detection platform reader based on the fluorescence gener-
ated (excitation, 545 nm; emission, 590 nm). The specific fluores-
cence 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 /C0IFi/IFc) /C2100% in which IFi and IFc are the fluo-
rescence 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%
CO2at 37 /C176C. The culture media were changed every other day.
Cells were subcultured in 96-well plates at a seeding density of
5/C2103cells/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(100lM and 20 lM).
Twenty-four hours (and seventy-two hours) later the survival of
cells was determined by MTT assay. Briefly, after incubation with
20lL of MTT at 37 /C176C for 4 h, living cells containing MTT formazan
crystals were solubilized in 150 lL 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 indepen-
dent experiments.
PC12 cells were seeded at 5 /C2103cells/well in 96-well plates
for neuroprotection activity assay. After 24 h, the medium was
removed and replaced with the tested compounds (20 lM) at
37/C176C and incubated for another 24 h. Rasagiline was used as the
control with the same concentration of 20 lM. Then, the cells were
exposed to 6-OHDA (40 lM) and Rotenone (1 lM) respectively,
and incubated at 37 /C176C for 24 h before assayed with MTT. PC12
cells were cultured without test compound or neurotoxins as con-
trol groups and the results were expressed by percentage of con-
trol. Results are expressed as the mean ± SEM of three
independent experiments.
4.4. Annexin V APC/7-AAD double staining to detect apoptosis29
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 lg/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% tryp-
sin (without EDTA) was used to trypsinize and gather the cells. The
cells were washed twice with phosphate-buffered saline (PBS)
(centrifugation at 2000 rpm, 5 min), and 2 /C2105cells were col-
lected. The cells were then resuspended in 500 ll of binding buffer.
After 5 ll of Annexin V APC was added and mixed well, 5 llo f
7-AAD was added and mixed well. The reaction was performed
at room temperature for 5–15 min in the dark, and a flow cytome-
ter (FACSCalibur; Becton–Dickinson, USA) was used to detect
apoptosis.
4.5. Hoechst 33258 staining assay30
For Hoechst 33258 staining assay, PC12 cells were stained with
Hoechst 33258 for 30 min. The cells were plated on the glass slideZ. Wang et al. / Bioorg. Med. Chem. 24 (2016) 5929–5940 5939

and covered with cover slip, then observed using a fluorescent
inverted microscope (Nikon ti-s, Japan).
4.6. Evaluation of intracellular ROS31
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/C2105cells/well, culturing in the presence or absence of 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 /C176C in the dark. After incubation,
cells were washed three times and harvested in free-serum med-
ium. The fluorescence of 2,7-dichlorofluorescein (DCF) was
detected by flow cytometry (488 nm excitation and 525 nm emis-sion 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 lL of PBS/
EtOH (7:3), and the filter membrane was impregnated with 10 lL
of PBL in dodecane (20 mg/mL). Compounds were dissolved in
DMSO- d6at 5 mg/mL and diluted 50-fold in PBS/EtOH (7:3) to
achieve a concentration of 100 mg/mL, 200 lL 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 /C176C. After incubation, the donor plate was carefully
removed and the concentration of compound in the acceptor wells
was determined using a UV plate reader (Flexstation@ 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 test35,36
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 at25 ± 2 /C176C, relative humidity at 50 ± 10%, and a 12 h light/dark cycle.
Distilled water and sterilized food for mice were available ad libi-
tum. 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 13was sus-
pended 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 ofany delayed effects. All animals were sacrificed after being anaes-
thetized 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 Pro-
gram Development of Jiangsu Higher Education Institutions
(PAPD).
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.09.050 .
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