Synthesis and biological evaluation of aryloxyacetamide derivatives as neuroprotective agents

Article abstract of DOI:10.1016/j.bmcl.2016.03.094

A series of new aryloxyacetamide derivatives 10a-s and 14a-m are designed and synthesized. Their protective activities against the glutamate-induced cell death were investigated in differentiated rat pheochromocytoma cells (PC12 cells). Most compounds exhibited neuroprotective effects, especially for 10m, 10r, 14b and 14c, which showed potential protection of PC12 cells at three doses (0.1, 1.0, 10 μM). MTT assay, Hoechst 33342/PI double staining, and high content screening (HCS) revealed that pretreatment of the cells with 10m, 10r, 14b and 14c has significantly decreased the extent of cell apoptosis in a dose-dependent manner. The results of western blot analysis demonstrated these compounds suppressed apoptosis of glutamate-induced PC12 cells via caspase-3 pathway. These compounds can be lead compounds for further discovery of neuroprotective agents for treating cerebral ischemic stroke. Basic structure-activity relationships are also presented.

Full text of DOI:10.1016/j.bmcl.2016.03.094

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of aryloxyacetamide derivatives
as neuroprotective agents
Yan Zhong a,y, Yi Xu b,y, Ai-Xia Zhang b, Xiao-Feng Li b, Zhao-Ying Xu b, Ping Li c, Bin Wu b,
⇑
a School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China
b School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
c School of Basic Medical Sciences, Nanjing Medical University, Nanjing 210029, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new aryloxyacetamide derivatives 10a–s and 14a–m are designed and synthesized. Their
protective activities against the glutamate-induced cell death were investigated in differentiated rat
pheochromocytoma cells (PC12 cells). Most compounds exhibited neuroprotective effects, especially
for 10m, 10r, 14b and 14c, which showed potential protection of PC12 cells at three doses (0.1, 1.0,
Received 22 December 2015
Revised 16 March 2016
Accepted 25 March 2016
Available online xxxx
10 lM). MTT assay, Hoechst 33342/PI double staining, and high content screening (HCS) revealed that
pretreatment of the cells with 10m, 10r, 14b and 14c has significantly decreased the extent of cell
apoptosis in a dose-dependent manner. The results of western blot analysis demonstrated these
compounds suppressed apoptosis of glutamate-induced PC12 cells via caspase-3 pathway. These
compounds can be lead compounds for further discovery of neuroprotective agents for treating cerebral
ischemic stroke. Basic structure–activity relationships are also presented.
Keywords:
Aryloxyacetamide
Neuroprotective activity
Cerebral ischemic stroke
Caspase-3
Ó 2016 Published by Elsevier Ltd.
Stroke is the second commonest cause of death and leading
cause of adult disability worldwide. Over two-thirds of stroke
deaths worldwide are in developing countries.1,2 In China, stroke
is the second cause for mortality in all diseases. Particularly,
ischemic strokes account for 60–80% of these strokes.3 The patho-
logical mechanisms of ischemic strokes are complex and not fully
understood, but it is generally accepted that a pathological release
of glutamate from neurons plays a central role in mediating
subsequent neuronal cell injury and death.4 Excessive excitatory
transmission can be transformed into an implement of neuronal
destruction resulting in CNS disorder, such as cerebral ischemia,
hypoxia, autoimmune, Alzheimer’s, Parkinson’s diseases, and
so on.5
So far, treatment options for stroke-related brain damage
are very limited. Most pharmacological agents have focused on
mechanisms that occur in acute stage of stroke, such as restoration
of blood flow with antithrombosis and thrombolytic therapy, or
reducing the effects of ischemia by neuroprotective therapy.
Unfortunately, thrombolytic therapy can only be given to highly
selected patients.6 Therefore, there is an urgent need for effective
neuroprotective agents to treat stroke-related brain damage.
Natural products are the single most productive source of
lead molecules for development as clinically useful drugs for
human disorders.7 It has been reported that the cinnamon
extract has vasodilative, antithrombotic, anti-ulcerous, and antial-
lergic action.8 It is believed that the extracts (aqueous and/or
organic solvent extraction) would provide different compositions
of phytochemicals (such as cinnamic acid, cinnamaldehyde, proan-
thocyanidins), and these are responsible for the above effect.9
Encouraged by these observations and in continuation of our
ongoing research program, we designed and synthesized several
cinnamide derivatives to explore their neuroprotective proper-
ties.10 We found that cinnamide scaffold often affords neuropro-
tective compounds (Fig. 1). Especially the compound NY-308 (1),
which has the (E)-p-methoxycinnamoyl moiety, exhibited good
neuroprotection in vitro PC12 cells and in vivo rat focal cerebral
ischemic animal model.10,11 Structurally, the (E)-p-methoxycin-
namoyl moiety in NY-308 was believed to play a very important
role in its activity. To further elucidate the structure–activity
relationship, we designed and synthesized a novel series of
phenoxyacetamide derivatives using –OCH2CO– connecting bridge
as the surrogate replacing –CH@CHCO– moiety of cinnamide.
Furthermore, we replaced diphenylmethylpiperazine of NY-308
with benzylpiperazine and changed substituent groups of benzene
rings to find new chemical entities with better neuroprotective
activity (Fig. 2).
⇑
Corresponding author. Tel./fax: +86 025 8686 8467.
E-mail address: wubin@njmu.edu.cn (B. Wu).
These authors contributed equally to this work.
y
http://dx.doi.org/10.1016/j.bmcl.2016.03.094
0960-894X/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Zhong, Y.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.094

2
Y. Zhong et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
O
N
O
F
N
R2
R1
N
N
O
R3
NY-308
F
Figure 1. Structures of new cinnamide derivatives.
O
F
N
N
O
NY-308
F
O
O
O
O
N
N
B
R3
R1
R2
R1
A
A
N
N
B
R2
䊠
䊡
Figure 2. Structure exploration strategy based on compound NY-308 (1).
Therefore, a total of 32 target compounds were designed and
investigating neuroprotection on impairment induced by glutamine
(Glu) in differentiated PC12 cells,12,13 as evaluated by MTT
assay.14–16 The results are showed in Table 1. The vast majority
of compounds tested exhibited protection of PC12 cells against
glutamate-induced cell death, indicating that its bioactivity
remained after introduction of the –O–CH2–CO– connecting bridge
into structures. Potency and toxicity were highly sensitive to struc-
tural variations. Remarkably, compounds 10j, 10k, 10m, 10r, 14b,
14c, 14f, 14i, and 14j showed good neuroprotective activity for
synthesized. Their in vitro neuroprotective activities in the cell
injury induced by glutamate, apoptosis assays, and the inhibition
of caspase-3 were evaluated. Herein, the synthesis and preliminary
biological evaluation of these compounds were reported.
Synthetic route for substituted diphenylmethylpiperazine
analogs 10a–s was depicted in Scheme 1. Commercially available
substituted aromatic phenols 2a–g were transformed into the
desired intermediates 3a–g, via reacting with ethyl chloroacetate
in acetonitrile. Subsequent hydrolysis of 3a–f with 10% sodium
hydroxide gave carboxylic acids 4a–f, which were chloridized with
oxalyl chloride to give acyl chlorides 5a–f. The synthesis of other
key intermediates 9a–d commenced with the reduction reactions
of benzophenones 6a–d under sodium borohydride. Subsequent
chlorination of benzhydrols 7a–d with SOCl2, followed by alkyla-
tion with anhydrous piperazine, afforded diphenylmethylpiperazi-
nes 9a–d. The target compounds 10a–s were finally obtained by
acylation of various substituted diphenylmethylpiperazines with
the corresponding acyl chloride under mild conditions (at RT in
acetone, and with triethylamine as base).
all three test concentrations (0.1, 1.0, 10
From Table 1, it was found that the cumulative addition of the
compounds 10m, 10r, 14b, and 14c (0.1–10 M) caused concentra-
tion-dependent neuroprotective effects with the maximal effect
observed at 10 M. Compounds 10j, 10k, 14d, and 14j showed a
pattern of increased protection with increasing concentrations
(0.1–1 M) in terms of cell protection. Compounds 10n, 14f, 14h
and 14k were observed to have the highest protection at the
lowest concentrations of 0.1 M (cell protection: 45.45%, 35.28%,
lM) (protection >20%).
l
l
l
l
53.34% and 39.86%, respectively). Compounds 10a, 10b, 10f, 10g,
10i, 10l, and 14e were observed to have the highest protection at
The second series of benzylpiperazine compounds 14a–m were
conveniently synthesized as outlined in Scheme 2. Substituted
aryloxy acetyl piperazine intermediates 11a–g, which were
obtained by aminolysis of substituted ethyl aryloxyacetates 3a–g
with anhydrous piperazine, were used to react with benzyl
bromides 13a–d to directly provide the target compounds 14a–m.
The key intermediates benzyl bromides 13a–d, were prepared by
free-radical bromination of commercially available substituted
toluenes 12a–d with little excess of N-bromosuccinimide (NBS).
In order to study the potential neuroprotective activities of
the title compounds, a preliminary screening was performed
the highest concentrations of 10 lM (cell protection: 25.64%,
30.24%, 21.17%, 56.98%, 80.50%, 50.34% and 25.10%, respectively).
Though many of derivatives showed interesting neuroprotective
effects, unfortunately, some of them possess unexpected cytotoxi-
city toward PC12 cells. Moreover, some compounds have limited
solubility in cell culture medium, which complicates interpretation
of these negative results.
Derivatives of I and II were substituted with different functional
groups to study of the substituent variability influence on the
biological activity and find new chemical entities with better
neuroprotective activity. Based on lipophilic and electronic
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Y. Zhong et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
O
O
O
OH
a
O
O
O
O
OH
Cl
c
b
R1
R1
R1
R1
3a-g
2a-g
4a-f
5a-f
H
N
g
O
OH
Cl
N
d
e
R2
R2
f
R2
R2
R2
R2
R2
R2
8a-d
9a-d
7a-d
6a-d
O
O
N
R2
R1
N
R2
10a-s
Scheme 1. Reagents and conditions: (a) ClCH2COOEt, K2CO3, CH3CN; (b) 10% NaOH, EtOH; (c) oxalyl chloride, CH2Cl2; (d) NaBH4, EtOH; (e) SOCl2, CH2Cl2; (f) anhydrous
piperazine, cyclohexane; (g) TEA, acetone.
O
O
O
O
O
N
a
R1
R1
O
NH
Br
O
11a-g
3a-g
c
N
R3
R1
N
b
R3
R3
14a-m
13a-d
12a-d
Scheme 2. Reagents and conditions: (a) anhydrous piperazine, CHCl3; (b) NBS, AIBN, CCl4, h.v.; (c) TEA, CH3CN.
attributes, these substituents were considered and selected, such
as lipophilic electron-withdrawing groups F, Cl and Br; hydrophilic
electron-donating groups OCH3; lipophilic electron-withdrawing
groups COOCH3; and lipophilic electron-releasing groups CH3. For
series I, compounds with 2-substituent of A moiety were more
active than those with 4-substitutent, while 4-substituent of A
moiety exhibited more neuroprotective effect than 2-substituent
for series II. The general structure–activity relationship of these
compounds showed that the derivatives having electron-releasing
groups were more active than those having electron-withdrawing
groups. Among the electron-donating groups, hydrophilic methoxy
derivatives were more active than the lipophilic methyl derivatives
(cell protection: 10a > 10c, 10f > 10h, 14j > 14e, and 14i > 14d). On
the other hand, for the different isoelectronic halogens, the lipophilic
chloro substituent at the 4-position of A ring was more potent and
less neurotoxic than the corresponding bromo-substituted
derivatives (cell protection: 10p > 10s, 14k > 14m). Similarly, the
lipophilic F-substituent at the 4-position of B ring was more potent
and less neurotoxic than the corresponding bromo-substituted
derivatives (cell protection: 10i > 10l). It was worthwhile to note
that the compound 10r exhibited promising neuroprotective effect
of increased protection with increasing concentrations (0.1–10 lM)
in terms of cell protection. On the contrary, the similar trend was
not observed for the analogue 10a–d. Perhaps it was because the
rigidity of molecule prevented binding with the receptors.
For further investigation, apoptosis assays and western blot
analysis were performed to determine their mechanism of action,
at three concentration of 0.1, 1.0 and 10 lM, against glutamate-
induced cell death. According to the in vitro biological activities,
the compounds 10m, 10r, 14b and 14c were selected and further
studied.
Apoptosis after cerebral ischemia is one of the major pathways
that leads to the process of cell death.17 Therefore, we investigated
the anti-apoptotic effects of the compounds 10m, 10r, 14b and 14c
by Hoechst 33342/PI double staining assay using high content
screening system.18,19 Quantitative analysis of the stained cells
indicated that pretreatment of the cells with 10m, 10r, 14b or
14c has significantly decreased the extent of cell apoptosis in a
dose-dependent manner. As shown in Table 2, after glutamate
insult for 24 h, 43.97% of cultured cells showed typical characteris-
tics of apoptosis. However, under pretreatments of 10m, 10r, 14b
and 14c at three doses (0.1, 1.0 and 10 lM respectively), the
(cell protection: 77.12% at 10
0.1 M, respectively), although it includes bromo substituent.
It is interesting to note that both compounds 14b and 14c pos-
sessing naphthyl moieties attached to the A system show a pattern
l
M, 47.58% at 1.0
l
M, and 26.80% at
apoptotic percentages were significantly reduced (Table 2).
Another important finding, which confirmed the protective
effect of these derivatives, was the data obtained from western blot
analysis of procaspase-3 and caspase-3. As shown in Figure 3,
l
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Y. Zhong et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
Neuroprotective effects of all target compounds against glutamate-induced neurotoxicity in PC12 cells
O
O
O
O
N
N
R2
R3
R1
R1
N
N
R2
10a-s
14a-m
Compd
R1
R2
R3
Cell viability (%)
1.0
0.1
lM
l
M
10 lM
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
R1-phenyl = 2-naphthyl
R1-phenyl = 2-naphthyl
R1-phenyl = 2-naphthyl
R1-phenyl = 2-naphthyl
2-Methyl
2-Methyl
2-Methyl
2-Methyl
4-Methoxy
4-Methoxy
4-Methoxy
4-Methoxy
2-Methoxy
2-Methoxy
2-Methoxy
4-Chloro
4-Chloro
4-Bromo
4-Bromo
4-Methyl
R1-phenyl = 2-naphthyl
R1-phenyl = 2-naphthyl
2-Methyl
2-Methyl
2-Methyl
4-Methoxy
2-Methyl
2-Methoxy
2-Methoxy
4-Chloro
4-Bromo
4-Bromo
4-Methoxy
4-Bromo
4-Methyl
4-Fluoro
4-Bromo
4-Methoxy
4-Fluoro
4-Methyl
4-Fluoro
4-Methyl
4-Methoxy
4-Bromo
4-Fluoro
4-Bromo
4-Methyl
4-Bromo
4-Methyl
4-Fluoro
4-Bromo
2.55
4.06
5.42
7.87
25.64
30.24
4.75
5.63
10.28
a
a
a
a
a
a
2.45
7.92
3.24
3.10
21.67
59.44
3.52
3.75
8.70
4.24
5.26
42.28
80.67
7.06
21.17
56.98
11.32
80.50
47.50
56.66
50.34
79.67
6.67
32.03
45.45
45.89
12.82
a
a
a
—
—
—
4.22
5.81
26.80
8.01
16.74
9.20
9.14
47.58
77.12
a
a
a
10s
—
—
—
—
—
—
a
a
a
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
NY-308
Edaravone
2-Methoxycarbonyl
2-Methoxycarbonyl
2-Ethoxy
4-Bromo
2-Methoxycarbonyl
4-Ethoxy
2-Methoxycarbonyl
2-Ethoxy
4-Bromo
2-Methoxycarbonyl
2-Methoxycarbonyl
4-Bromo
25.55
29.77
10.25
6.39
37.44
45.70
22.14
9.55
49.50
59.58
20.80
25.10
28.93
35.28
30.65
a
a
a
—
—
—
53.34
38.00
23.20
39.86
11.90
41.00
32.82
5.15
8.52
20.76
23.06
6.36
a
a
a
—
—
—
—
—
—
a
a
a
2-Methoxycarbonyl
71.01
49.66
77.75
33.04b
a
No activity.
Edaravone—90 lM.
b
(active) caspase-3 were weaker compared to glutamate-treated
cells. The compounds 10m and 14b significantly inhibited
the expression of both procaspase-3 and caspase-3 induced by
glutamate in a dose-dependent manner. The procaspase-3 level
was not depressed significantly by compounds 10r and 14c, while,
10r and 14c significantly inhibited the expression of caspase-3 in a
dose-dependent manner. The above results demonstrated these
compounds inhibited apoptosis of glutamate-induced PC12 cells
via caspase-3 pathway.
In summary, we synthesized a series of novel aryloxyacetamide
derivatives and studied their protective activities against the
glutamate-induced cell death in differentiated PC12 cells. Most
target compounds displayed protective effects on the cell
viability against the damage caused by glutamate, especially
for 10m, 10r, 14b and 14c, which had significant potency and effi-
ciently inhibited glutamate induced cell apoptosis via caspase-3
pathway. The present findings provided a preliminary pharmaco-
logical basis to exploit preventive or therapeutic strategies for
stroke and other neurological insults.
Table 2
Effect of some aryloxyacetamide on apoptosis of PC12 cells induced by glutamate
Compd
Rate of apoptotic cells (%)
1.0
0.1
lM
l
M
10 lM
Control
Glutamate
10m
10r
14b
19.27 2.00
43.97 2.69a
42.28 3.47
41.83 3.84
35.67 3.54b
35.09 3.64b
31.04 3.11b
32.18 3.12b
29.27 3.33b
23.73 3.03b
19.18 3.64b
24.65 3.18b
21.81 3.38b
16.21 2.04b
14c
a
Significantly different from control group (p <0.05).
Significantly different from glutamate group (p <0.05).
b
glutamate induced the appearance of cleaved active caspase-3,
showing the involvement of caspase-3 in glutamate-induced cell
death in PC12 neurons. In the cells pretreated with 10m, 10r,
14b or 14c at three doses, bands of both procaspase and cleaved
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Y. Zhong et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Figure 3. Caspase-3 activity in PC12 cells pretreated with aryloxyacetamide derivatives.
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