Design, synthesis and biological evaluation of novel dicarbonylalkyl piperazine derivatives as neuroprotective agents

Article abstract of DOI:10.1016/j.cclet.2015.11.002

In the search of novel neuroprotective agents with higher potency than our previously identified anti-ischemic stroke drug candidate 1, a series of novel dicarbonyl piperazine derivatives were synthesized and evaluated on their neuroprotective activity via oxygen-glucose deprivation test in the neuron-like PC12 cells, hypoxia tolerance model in mice and focal cerebral ischemia model in rats. The result obtained indicated that compounds 7f, 7k and 7o, exhibited neuroprotective activity. Particularly, compound 7o containing 2,5-dimethylpiperazin moiety, showed prolonged life time of mice and reduced cerebral infarction of rats, which provided a potential candidate for the development of neuroprotective agents.

Full text of DOI:10.1016/j.cclet.2015.11.002

G Model
CCLET 3476 1–4
Chinese Chemical Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
1
2
Original article
3
4
Design, synthesis and biological evaluation of novel dicarbonylalkyl
piperazine derivatives as neuroprotective agents
a,b,1
c,1
a,b
a,b
5
6
Q1 Wen-Ya Wang
, Cheng-Wu Shen , Zhi-Jie Weng , Tie-Chuang Wang
,
a,b
a,b
a,b,
Chuang Zhang , Xun-Qi Jin , Jian-Qi Li
*
a
7
8
9
Novel Technology Center of Pharmaceutical Chemistry, Shanghai Institute of Pharmaceutical Industry, Shanghai 201203, China
Shanghai Engineering Research Center of Pharmaceutical Process, Shanghai 201203, China
b
c
Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China
A R T I C L E I N F O
A B S T R A C T
Article history:
In the search of novel neuroprotective agents with higher potency than our previously identified anti-
ischemic stroke drug candidate 1, a series of novel dicarbonyl piperazine derivatives were synthesized
and evaluated on their neuroprotective activity via oxygen–glucose deprivation test in the neuron-like
PC12 cells, hypoxia tolerance model in mice and focal cerebral ischemia model in rats. The result
obtained indicated that compounds 7f, 7k and 7o, exhibited neuroprotective activity. Particularly,
compound 7o containing 2,5-dimethylpiperazin moiety, showed prolonged life time of mice and
reduced cerebral infarction of rats, which provided a potential candidate for the development of
neuroprotective agents.
Received 14 July 2015
Received in revised form 17 August 2015
Accepted 21 October 2015
Available online xxx
Keywords:
Dicarbonylalkyl piperazine
Neuroprotective
Ischemic stroke
ß 2015 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
1
1
0
1
1. Introduction
in 2005 and 2002, respectively. They exhibited neuroprotective 29
effects and antithrombotic activity in cerebral infarction and acute 30
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Stroke has been the second-leading cause of death and a main
cause of neurological disability in the world over the past decade
[1]. In particular, ischemic stroke, which accounts for more than
80% of all strokes, carries a high morbidity and mortality.
Furthermore, strokes are likely to significantly increase in
prevalence given its associated risk factors, such as obesity,
diabetes, and hypertension [2,3]. However, available drugs are very
limited, and some of them suffer from poor efficacy and high
toxicity. Clinically, intravenous thrombolysis and mechanical
thrombectomy are current strategies for treating acute ischemic
stroke [4], and tissue plasminogen activator (rt-PA) is the only
FDA-approved drug for thrombolysis treatment. Unfortunately, it
is used in less than 5% of patients due to its narrow therapeutic
window and complexity of administration [5]. Edaravone [6–9]
and Butylphthalide [10–14] (Fig. 1) were approved by CFDA for
acute treatment as neurointervention therapy and cerebral
microcirculation improvement drug in ischemic stroke patients
stroke, but the potency is limited unless administered with other 31
antistroke drugs. Most critically, the penumbra (a peripheral zone 32
of focal cerebral ischemia that is damaged but potentially 33
reversible) transfers to irreversible damage within a few hours 34
[15,16]. Hence, there is an emergent need for development of novel 35
effective neuroprotective agents.
36
Previous studies have showed that aralkyl dicarbonyl pipera- 37
zine compound 1 (Fenazinel, Fig. 1) exhibited potent anti-ischemic 38
stroke activity as neuroprotective therapy, and was developed into 39
clinical trials [17–21]. In order to investigate the structure–activity 40
relationships (SARs), a series of novel dicarbonyl piperazine 41
derivatives (Fig. 2) were synthesized and evaluated on their 42
neuroprotective activity via oxygen–glucose deprivation test in 43
neuron-like PC12 cells, a hypoxia tolerance model in mice, and a 44
focal cerebral ischemia model in rats.
. Experimental
.1. Chemistry
45
2
46
47
2
*
Corresponding author at: Shanghai Institute of Pharmaceutical Industry, Novel
Q2
Technology Center of Pharmaceutical Chemistry, Shanghai 201203, China.
All the starting materials were obtained from commercial 48
sources and used directly without further purification. All the 49
E-mail address: lijianqb@126.com (J.-Q. Li).
1
These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.cclet.2015.11.002
1
001-8417/ß 2015 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: W.-Y. Wang, et al., Design, synthesis and biological evaluation of novel dicarbonylalkyl piperazine
derivatives as neuroprotective agents, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.11.002

G Model
CCLET 3476 1–4
2
W.-Y. Wang et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
The general synthetic routes of compounds 7a–t are outlined in
Schemes 1 and 2. Compounds 2a–c were acylated by chloro-
substituted acyl chloride in CHCl to give compounds 3a–f.
Intermediates 5a-i were obtained by alkylation of 4a and b with
60
61
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63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
3
3
6
a–f in EtOH via refluxing, which was subsequently alkylated with
a–e in acetone at presence of K CO to afford the targeted
2
3
Fig. 1. Chemical structures of representative anti-ischemic stroke agents and the
lead compound 1.
compounds 7a–r in 79–87% yield. Compounds 7s and t were
synthesized via two alkylation steps. 2,5-Dimethylpiperazine (8)
was alkylated with 6d-e in presence of KOH and CTBA with molar
ratio of 3.5:1:1:0.01, and then alkylated with 3a and b in MeOH at
presence of K CO to afford 7s and t in 80–85% yield. Structures of
2 3
targeted compounds 7a–t are illustrated in Table 1.
All the compounds 7a–t were evaluated on their neuroprotec-
tive activity via in vitro (oxygen–glucose deprivation test in PC12
cells) and in vivo assays (hypoxia tolerance model in mice and focal
cerebral ischemia model in rats).
50
51
52
53
54
55
56
57
58
59
reactions were monitored by TLC analysis, carried out on silica gel
plates HSGF254 (Yantai Jiangyou Chemical, China). Melting points
(uncorrected) were determined on a Shanghai Jingmi WRR
apparatus. H NMR spectra were recorded on a Varian INOVA-
400 NMR spectrometer, using TMS as an internal standard and
1
DMSO as solvents. Chemical shifts (
d values) and coupling
constants (J values) are given in ppm and Hz, respectively. ESI
mass spectra were performed on a Finning-MAT 212 spectrometer.
Silica gel column chromatography was performed with Silica gel
60G (Qingdao Haiyang Chemical, China).
The detailed synthetic procedure, in vitro and in vivo biological
evaluations, and identification data of target compounds 7a–t,
1
characterized by H NMR and mass spectrometry techniques are
shown in the Supporting information.
Fig. 2. Design rational of the targeted compounds 7a–t.
R1
HN
NH HCl
R4
O
R4
O
4
a-b
a
R5
R1
R5
Cl
Cl
+
H N
Cl
N
2
m2
m3
m
2 H
3a-f
3a: m = 0, m = 1, R = H, R = H
m3
b
1a-c
2a-c
1
1
1
a: m = 0, R = H
2a: m = 1, R = H
4a: R = H
1
4b: R = CH
2
4
4
4
3
3
3
5
5
5
2
3
3
4
4
5
5
b: m = 0, R = Me
2b: m = 1, R = OMe
3b: m = 0, m = 1, R = H, R = OMe
2
2
2
3
c: m = 1, R =H
2c: m = 2, R =H
3c: m = 0, m = 2, R = H, R = H
2
2 3 4 5
3
3
3
d: m = 1, m = 1, R = H, R = H
2 3 4 5
e: m = 0, m = 1, R = Me, R = H
2
3
4
4
5
5
f: m = 0, m = 1, R = Me, R = OMe
2
3
O
R2
R1
O
R2
R4
O
N
Cl
m1
R5
R1
R4
O
N
N
R3
m
m3
6a-e
c
m1
2 H
R5
HN
N
N
R3
m2
m3
R1
H
6
a: m = 0, R = H, R = H
1 2 3
HCl
R1
7a-r
6b: m = 0, R = H, R = OMe
1
2
3
5a-i
6c: m1 = 1, R2 = H, R3 = H
6
6
d: m = 0, R = Me, R = H
1 2 3
e: m = 0, R = Me, R = OMe
1
2
3
3
Scheme 1. Synthetic route of compounds 7a–r. Reagents and conditions: (a) CHCl , 0 8C, 2 h, 60–75%; (b) EtOH, reflux, 1.5 h, 75–85%; (c) K
2
CO
3
, Acetone, reflux, 12 h, 79–87%.
Please cite this article in press as: W.-Y. Wang, et al., Design, synthesis and biological evaluation of novel dicarbonylalkyl piperazine
derivatives as neuroprotective agents, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.11.002

G Model
CCLET 3476 1–4
W.-Y. Wang et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
3
2 3
Scheme 2. Synthetic route of compounds 7s and t. Reagents and conditions: (a) KOH, CTBA, EtOH, reflux, 1 h, 80%; (b) K CO , MeOH, reflux, 12 h, 80–85%.
8
0
3. Results and discussion
4-phenyl as compounds 7a, 7c, 7f and 7g exhibited higher 95
survival rate than the lead compound 1, while methylene 96
insertion led to a decrease or lose of survival rate as the data 97
revealed for compounds 7b, 7d and 7e. Compounds 7o 98
containing 2,5-dimethylpiperazin moiety displayed higher 99
survival rate than compound 1. The above compounds showed 100
85–97% viable rate at three levels of concentrations, suggesting 101
potential neuroprotective activity by means of relieving OGD 102
81
82
83
84
85
86
87
88
89
90
91
92
93
94
The design rationale for the targeted compounds is depicted in
Fig. 2. Methyl, methylene or methoxy were introduced to
methylene, piperazine, middle of actetyl and piperazine-N, as
well as both terminal phenyl of the lead compound 1, respectively.
Twenty new compounds 7a–t (Schemes 1 and 2) were synthesized
and characterized.
In vitro oxygen–glucose deprivation (OGD) test data was shown
in Table 2. The MTT assays indicated that treatment with OGD
for 2 h effectively inhibited the growth of PC12 cells, and the
survival rate was 72.35%. Compared to the OGD for 2 h treated
group, most of the targeted compounds plus OGD for 2 h treated
group could significantly increase the survival rate in a dose-
dependent manner and the rate was higher than that of OGD
group. Methyl or methoxy introduction in methylene or
damage.
103
In vivo neuroprotective activity data in hypoxia tolerance 104
model in mice and focal cerebral ischemia model in rats was 105
shown in Tables 3 and 4. Hypoxia tolerance assay showed that 106
compounds 7a, 7f, 7g, 7k and 7o could significantly prolong the 107
survival time of mice under hypoxic condition at dose of 10 108
and 40 mg/kg than the control group, and were comparable with 109
lead compound 1. As focal cerebral ischemia assay, the ratio of 110
cerebral infarction in brain after ischemia for 24 h was 111
investigated. Compounds 7f, 7k, and 7o could significantly 112
reduce the ratio of cerebral infarction (5.43–5.99%) compared 113
with the control group (10.28%) and then improve the 114
neurological function. In particular, compound 7o showed 115
excellent anti-stroke activity, which is a potential candidate for 116
Table 1
Structures of the targeted compounds of 7a–t
the development of neuroprotective agents.
117
Table 2
In vitro neuroprotective activity in oxygen–glucose deprivation test in PC12 cells.
*
Compd. Control OGD
Survival rate (%)
.1 mol/L
0
m
1
m
mol/L
10 mmol/L
.
7
7
a
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
72.35 ꢀ 0.30 82.84 ꢀ 4.41
72.35 ꢀ 0.30 77.08 ꢀ 0.78
72.35 ꢀ 0.30 86.69 ꢀ 2.28
88.11 ꢀ 11.14 92.84 ꢀ 1.09
Compd.
R
1
R
2
R
3
R
4
R
5
m
1
m
2
m
3
b
81.42 ꢀ 0.27
94.08 ꢀ 6.38
84.01 ꢀ 13.30
90.05 ꢀ 8.07
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
H
Me
H
H
H
H
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
7c
7d
7e
7f
H
H
H
H
72.35 ꢀ 0.30 73.41 ꢀ 19.77 75.16 ꢀ 32.60 79.24 ꢀ 39.53
H
H
OMe
H
H
H
72.35 ꢀ 0.30 78.81 ꢀ 3.27
72.35 ꢀ 0.30 93.76 ꢀ 2.28
86.53 ꢀ 1.12
97.01 ꢀ 6.38
72.24 ꢀ 1.19
97.46 ꢀ 8.07
H
H
H
H
H
H
H
H
H
7g
7h
7i
72.35 ꢀ 0.30 94.37 ꢀ 12.64 85.65 ꢀ 13.19 88.41 ꢀ 10.04
H
H
H
H
OMe
H
72.35 ꢀ 0.30 78.73 ꢀ 7.04
72.35 ꢀ 0.30 80.95 ꢀ 3.54
72.35 ꢀ 0.30 73.84 ꢀ 0.92
72.35 ꢀ 0.30 76.85 ꢀ 7.55
72.35 ꢀ 0.30 71.17 ꢀ 0.92
83.21 ꢀ 7.44
78.25 ꢀ 0.93
77.28 ꢀ 3.34
79.50 ꢀ 7.88
76.30 ꢀ 3.34
76.71 ꢀ 6.86
60.43 ꢀ 28.35
81.24 ꢀ 12.16
80.91 ꢀ 4.23
86.09 ꢀ 12.16
g
h
i
H
H
H
Me
H
H
Me
Me
H
H
OMe
OMe
OMe
H
7j
H
OMe
OMe
H
H
7k
7l
j
H
H
k
l
H
Me
Me
Me
Me
H
Me
Me
Me
Me
H
7m
7n
7o
7p
7q
7r
7s
7t
72.35 ꢀ 0.30 93.07 ꢀ 13.26 95.72 ꢀ 29.55 91.50 ꢀ 17.38
H
H
OMe
H
72.35 ꢀ 0.30 78.65 ꢀ 4.25
72.35 ꢀ 0.30 85.66 ꢀ 5.48
81.19 ꢀ 8.66
89.33 ꢀ 9.95
79.56 ꢀ 7.95
m
n
o
p
q
r
H
OMe
OMe
H
87.61 ꢀ 14.53
H
OMe
H
72.35 ꢀ 0.30 77.89 ꢀ 12.30 70.34 ꢀ 23.41 62.81 ꢀ 19.14
Me
Me
Me
Me
Me
Me
72.35 ꢀ 0.30 67.76 ꢀ 7.56
64.44 ꢀ 21.77 65.76 ꢀ 7.75
H
H
H
OMe
OMe
H
72.35 ꢀ 0.30 76.21 ꢀ 49.95 77.08 ꢀ 56.53 60.84 ꢀ 25.32
72.35 ꢀ 0.30 74.67 ꢀ 12.30 67.03 ꢀ 23.41 58.18 ꢀ 19.14
H
OMe
H
H
H
H
72.35 ꢀ 0.30 78.73 ꢀ 7.04
72.35 ꢀ 0.30 79.69 ꢀ 7.13
83.39 ꢀ 7.46
79.78 ꢀ 7.14
88.04 ꢀ 7.87
81.54 ꢀ 7.30
s
Me
Me
H
H
H
Lead 1 100
t
OMe
H
OMe
*
p < 0.05 vs. control group.
Please cite this article in press as: W.-Y. Wang, et al., Design, synthesis and biological evaluation of novel dicarbonylalkyl piperazine
derivatives as neuroprotective agents, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.11.002

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W.-Y. Wang et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
Table 3
Appendix A. Supplementary data
132
In vivo neuroprotective activity in hypoxia tolerance model in mice.
Compd.
n
Survival time (s)
0 mg/kg
Supplementary material related to this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.cclet.2015.11.002.
133
134
1
40 mg/kg
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
1244 ꢀ 248
1206ꢀ 238
1205ꢀ 219
1112 ꢀ 123
1187 ꢀ 141
1411 ꢀ 177
1297 ꢀ 195
1265 ꢀ 179
1186 ꢀ 142
1107ꢀ 117
1202 ꢀ 175
1230 ꢀ 137
1137 ꢀ 112
1037 ꢀ 82
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Dose (mg/kg)
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a
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2.5
2.5
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3
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3
3
3
11.31 ꢀ 2.34
5.43 ꢀ 3.33
9.57 ꢀ 2.39
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118
4. Conclusion
119
120
121
122
123
124
125
126
127
In summary, a series of novel dicarbonyl piperazine derivatives
were designed, synthesized, and evaluated on their neuroprotec-
tive activity. The SARs revealed that methyl or methoxy mono-
introduction was favorable for neuroprotective activity, while
methylene introduction was not. It is worth mentioning that
compound 7o, containing a 2,5-dimethylpiperazin moiety, showed
good anti-ischemic stroke activity in vitro and in vivo, which
provided a good starting point for a potential candidate for the
development of neuroprotective agents.
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Acknowledgment
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This work was supported by Shanghai Natural Science
Foundation (no. 12ZR1450100).
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Please cite this article in press as: W.-Y. Wang, et al., Design, synthesis and biological evaluation of novel dicarbonylalkyl piperazine
derivatives as neuroprotective agents, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.11.002