Novel multi-functional nitrones for treatment of ischemic stroke

Article abstract of DOI:10.1016/j.bmc.2012.04.016

Ischemic stroke resulting from obstruction of blood vessels is an enormous public health problem with urgent need for effective therapy. The co-administration of thrombolytic/antiplatelet agent and neuroprotective agent improves therapeutic efficacy and agent possessing both thrombolytic/ antiplatelet and antiradical activities provides a promising strategy for the treatment of ischemic stroke. We have previously reported a novel compound, namely TBN, possessing both antiplatelet and antiradical activities, showed significant neuroprotective effect in a rat stroke model. We herein report synthesis of a series of new pyrazine derivatives, and evaluation of their biological activities. Their mechanisms of action were also investigated. Among these new derivatives, compound 21, armed with two nitrone moieties, showed the greatest neuroprotective effects in vitro and in vivo. Compound 21 significantly inhibited ADP-induced platelet aggregation. In a cell free antiradical assay, compound 21 was the most effective agent in scavenging the three most damaging radicals, namely ^.OH, O₂^.- and ONOO ^-.

Full text of DOI:10.1016/j.bmc.2012.04.016

Bioorganic & Medicinal Chemistry 20 (2012) 3939–3945
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel multi-functional nitrones for treatment of ischemic stroke
⇑
Yewei Sun, Gaoxiao Zhang, Zaijun Zhang, Pei Yu, Haijing Zhong, Jing Du, Yuqiang Wang
Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine, Jinan University College of Pharmacy,
Guangzhou 510632, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Ischemic stroke resulting from obstruction of blood vessels is an enormous public health problem with
urgent need for effective therapy. The co-administration of thrombolytic/antiplatelet agent and neuro-
protective agent improves therapeutic efficacy and agent possessing both thrombolytic/antiplatelet
and antiradical activities provides a promising strategy for the treatment of ischemic stroke. We have
previously reported a novel compound, namely TBN, possessing both antiplatelet and antiradical
activities, showed significant neuroprotective effect in a rat stroke model. We herein report synthesis
of a series of new pyrazine derivatives, and evaluation of their biological activities. Their mechanisms
of action were also investigated. Among these new derivatives, compound 21, armed with two nitrone
moieties, showed the greatest neuroprotective effects in vitro and in vivo. Compound 21 significantly
inhibited ADP-induced platelet aggregation. In a cell free antiradical assay, compound 21 was the most
Received 8 February 2012
Revised 5 April 2012
Accepted 6 April 2012
Available online 21 April 2012
Keywords:
Chemical synthesis
Antioxidant
Antiplatelet aggregant
Ischemic stroke
Middle cerebral artery occlusion
Å
effective agent in scavenging the three most damaging radicals, namely OH, O2 and ONOOꢁ.
ꢀꢁ
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Nitrones were originally developed as free radical-trapping
agents in free radical chemistry. Two decades later, nitrones were
Ischemic stroke is one of the major causes of death behind heart
diseases and cancer, and is the leading cause of adult disability in
the developed countries. There are approximately 795,000 Ameri-
cans each year suffering a new or recurrent stroke, killing more than
137,000 people. It is estimated that Americans will pay about $50
billion annually for stroke-related medical costs and disability.1
The large numbers of stroke patients and great economic expenses
have imposed great burden to society.
Occlusion of cerebral vessels by blood thrombus is the main
cause of ischemic stroke, which subsequently leads to a cascade
of damaging biologic events, including the release of excitatory
amino acid, calcium influx, neuroinflammation and over produc-
tion of free radicals.2–5 There are increasing evidences showing
that free radicals play a crucial role in the damaging effects caused
by stroke.5–7 Thus, agent that either lyses blood thrombus/inhibits
platelet aggregation or scavenges free radicals or combination of
these two effects can be useful for the treatment of stroke.8–10
In China, Ligusticum wallichii Franchat (Chuan Xiong) and its
main active component, 2,3,5,6-tetramethylpyrazine (TMP), have
been used for treatment of ischemic stroke for many years.11 The
precise mechanism(s) of action of TMP has/have not been com-
pletely understood. TMP inhibits platelet aggregation,12,13 lyses
blood clots,12 blocks calcium entry14,15 and scavenges free radi-
cals.16,17 TMP is neuroprotective in animal stroke models.18
found to protect biological systems from oxidative stress. Nitrones
had been tested as therapeutic agents for neural and systemic
dysfunctions including atherosclerosis, stroke, and Alzheimer0s
disease.19–21 For example, N-tert-butyl-a-phenylnitrone (PBN),
the most widely investigated nitrone, was shown to ameliorate
ischemic brain damage in a rodent model.22,23 The nitrone NXY-
059 (disodium 4-[(tert-butylimino)methyl] benzene-1,3-disulfo-
nate N-oxide) was shown to significantly reduce infarct volumes
in animal stroke models.24 Although the clinical results of NXY-
059 are disappointing, the concept of using neuroprotective agents
for stroke therapy remains viable.
For an anti-stroke therapy to be most effective, it should
ideally accomplish two things. Firstly, it should dissolve the
thrombus to restore blood flow or should inhibit thrombus
formation; that is it should have a thrombolytic/antiplatelet
function. Secondly, it should reduce the impact of biochemical
changes in the brain brought on by the stroke.25,26 Our current
research seeks optimal combinations of both thrombolytic/
antiplatelet and neuroprotective agents to prevent cell death
and damage during and after ischemia and reperfusion. We have
previously reported a multifunctional compound, TBN, a conju-
gate of TMP and a nitrone moiety. In our previous work, we found
that TBN showed significant neuroprotective effect by scavenging
free radicals and directly removing blood thrombus.27,28 Based on
the significant anti-stroke effect of TBN, we had synthesized a
new series of multi-functional nitrone derivatives, and evaluated
their biologic activities.
⇑
Corresponding author. Tel.: +86 20 8522 8481; fax: +86 20 8522 4451.
E-mail address: yuqiangwang2001@yahoo.com (Y. Wang).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.016

3940
Y. Sun et al. / Bioorg. Med. Chem. 20 (2012) 3939–3945
N
N
N
percentage of viable neurons after only t-BHP treated cells was
49.4%. TMP offered weak neuroprotective effect against t-BHP
induced cell damage up to 1000 M (58.4%). In sharp contrast,
N
N
b
a
N
O
O
N
1
l
compound 16, the TMP dimer amine, and compound 21, bearing
2
3
two nitrone moieties, afforded greater neuroprotective effect at
the concentrations of 100 lM and 1000 lM. At 1000 lM,
80.6 6.0% of cells treated with compound 21 survived while
75.5 3.0% of cells treated with compound 16 survived. Com-
pounds 3 and 9 were less potent while compounds 13 and 19
had no effects compared with the group treated with t-BHP.
Scheme 1. Synthesis of compound 3. Reagents and conditions: (a) SeO2, 84 °C, 2 h,
70%; (b) N-tert-butyl-hydroxylamine, reflux, 4 h, 35%.
2. Results
2.1. Drug design and chemical synthesis
2.3. Neuroprotective effects in rat transient middle cerebral
artery occlusion model
Our goal is to design compounds possessing both the antiplate-
let/thrombolytic and free radical-scavenging activities. In addition,
the compounds must penetrate the blood–brain barrier (BBB) read-
ily, an important requirement for a stroke drug. Furthermore, the
compound must be non-toxic at therapeutic doses. With these
points in mind, we designed and synthesized a series of new
TMP analogues, most of which were armed with nitrone moi-
ety(ies). Compounds 1, 4, 10, 14 and 17 were chosen as parent
compounds for their antiplatelet activities, since inhibition of
platelet aggregation and thrombus formation is crucial for stroke
treatments.29 In addition, compound 14 shows thrombolytic effect
in a vivo study at a dose of 2 mg/kg.12 The purpose of the structure
modification is to provide these compounds with free radical-
scavenging effects, meanwhile, their antiplatelet activities can be
retained. Since the nitrone moiety is the main functional group
in scavenging free radicals, and it is therefore possible that a com-
pound armed with two nitrone moieties will be more potent in
scavenging free radicals than those with only one. For this reason,
compound 21, bearing two nitrone moieties, was synthesized.
The new compounds (compounds 3, 9, 19 and 21) were synthe-
sized through condensation of the corresponding aldehyde and
N-tert-butyl-hydroxylamine (Schemes 1, 2, 5 and 6)30 or oxidized
by Na2WO4/H2O2 from the corresponding secondary amine
(compound 13, Scheme 3).31 The aldehyde intermediates were
synthesized by oxidation of alcohols using activated manganese
dioxide or directly oxidized by selenium dioxide.32 The corre-
sponding secondary amines were synthesized through condensa-
tion of the bromo-substituted precursors and tert-butylamine.
In synthesis of compound 9, the starting parent 4 was first
transformed to compound 7, which was then oxidized by activated
manganese dioxide to produce compound 8. Without further
purification, compound 8 was allowed to react with N-tert-butyl-
hydroxylamine to afford the target compound 9.
After demonstrating that compound 21 showed the strongest
neuroprotective effect in vitro, we then examined its neuroprotec-
tive effect in vivo. The neuroprotective effect of compound 21 was
investigated in a rat transient middle cerebral artery occlusion (t-
MCAO) model. The model was established according to a published
method with modifications.34 Compound 21 was administered (ip)
2 h after MCAO, and the animals were sacrificed after another 22 h
reperfusion. The brain infarct size was determined by TTC staining
24 h after MCAO. Image-analysis of the brains showed that com-
pound 21 at a dose of 110 mg/kg reduced the total infarct volume
by 65.0% compared to the vehicle-treated control. The data demon-
strated that compound 21 significantly protected brain tissues
against ischemic damage compared to the control group
(Fig. 2A). Rats received compound 21 treatment had lower neuro-
logical scores (Fig. 2B) (compound 21: n = 13; control: n = 13).
2.4. Effect on ADP-induced platelet aggregation
To investigate whether these new compounds retained anti-
platelet activity, their effects on inhibiting platelet aggregation
were studied. The turbidimetric method with a platelet-aggregom-
eter was applied in the measurement.35 Since aspirin was a golden
standard in inhibiting platelet aggregation and TMP was effective
in inhibiting platelet aggregation, aspirin and TMP were used as
positive controls (Fig. 3). At 2 mM, the maximum percentage of
platelet aggregation treated with aspirin, TMP, compound 3, 9,
16 and 21 were 26.6 4.7%, 42.9 9.2%, 33.8 2.5%, 48.0 12.4%,
44.3 5.5% and 37.7 4.0%, respectively; while that of the sample
treated with saline was 57.3 8.3%. The results demonstrated that
compound 21 was more effective than TMP in inhibiting platelet
aggregation, but less effective than aspirin.
In synthesis of compounds 3, 19 and 21, the formyl aldehyde
intermediates were prepared through direct oxidation by selenium
dioxide from their corresponding parent compounds.
2.5. Free radical-scavenging capacity in vitro
In preparation of compound 13, the parent 10 was transformed
to compound 11 in the presence of N-bromosuccinimide and ben-
zoyl peroxide. Compound 11 was then treated with tert-butyl-
amine to produce intermediate 12, which was then oxidized by
Na2WO4/H2O2 to afford the target compound 13.
Compound 16 was synthesized by treatment of excess
2-(bromomethyl)-3,5,6-trimethylpyrazine (compound 15) with
tert-butylamine (Scheme 4).
Results in Figure 4A showed that all compounds especially com-
pound 16 were highly effective in scavenging the ÅOH radical (100%
at 1000 lM). Compounds 3, 9 and 21 showed significant activity for
removing O2ꢀꢁ, but compounds 16, TMP and PBN were less potent
(Fig. 4B). Compound 21 potently reduced the amount of ONOOꢁ
by 31.9% at a concentration of 1 lM and 99.4% at 1000 lM. Com-
pounds 13, 16, 19 and TMP had no effect. The effect of compounds
3, 9, and PBN were moderate (Fig. 4C). Compounds 9, 16 and 21
remarkably decreased the free radical DPPH; while compounds 3,
13, 19, TMP and PBN were only marginally effective (Fig. 4D). These
results demonstrated that compound 21 had significant activities in
removing all of the three most detrimental radicals.
2.2. Protective effect on t-BHP induced cerebellar granule cell
injury
The neuroprotective activities of the new compounds were
investigated using rat cerebellar granule neurons (CGNs) exposed
to tert-butyl-hydroperoxide (t-BHP) as an oxidant (the final con-
3. Discussion
centration of t-BHP is 50 lM). Preparation of rat cerebellar granule
neurons was performed according to a published method.33 TMP
was used as a positive control in the experiment (Fig. 1). The basal
Stroke occurs as a result of an obstruction by blood clots within
blood vessels supplying blood to the brain. Since thrombosis plays

Y. Sun et al. / Bioorg. Med. Chem. 20 (2012) 3939–3945
3941
N
N
N
N
N
c
a
b
O
N
O
O
4
5
6
N
N
N
N
e
d
N
O
O
OH
N
N
7
8
9
Scheme 2. Synthesis of compound 9. Reagents and conditions: (a) AcOH, 30% H2O2, 70 °C, 10 h; (b) AC2O, reflux, 12 h; (c) 20% NaOH, 5 h, 45% from 4; (d) MnO2, C2H5OH,
reflux, 12 h; (e) N-tert-butyl-hydroxylamine, reflux, 8 h, 30% from 7.
N
N
N
N
N
N
N
N
b
a
c
Br
NH
N
10
11
12
13
O
Scheme 3. Synthesis of compound 13. Reagents and conditions: (a) NBS, benzoyl peroxide, 70 °C, 10 h, 40%; (b) tert-butylamine, 4 h, 100%; (c) Na2WO4, 30% H2O2, 2 h, 65.2%.
N
N
N
N
reperfusion. Neuroprotective agents such as free radical scavengers
have been developed and tested for this purpose in animal models
or in clinic.37,38
N
N
N
b
a
N
Br
N
The ÅOH, O2 and ONOOꢁ are the three most detrimental active
ꢀꢁ
species in human body. The ÅOH is a very active radical with a short
life. It can quickly react with cell membranes or other cell compo-
14
15
16
Scheme 4. Synthesis of compound 16. Reagents and conditions: (a) NBS, benzoyl
peroxide, 70 °C, 10 h, 52%; (b) tert-butylamine, 2 h, 42.5%.
ꢀꢁ
nents to cause cell damage. The O2 is the culprit of many other
Å
radicals. It can react with H2O2 to form OH through the Fenton
reaction or react with nitric oxide (ÅNO) to afford ONOOꢁ, which
is about 1000 times more destructive than H2O2. Therefore, it is
important for a stroke drug to be able to neutralize all of these det-
rimental radicals. DPPH is a dark-colored crystalline powder com-
posed of stable free-radical molecules. It has been used widely in
laboratory as an agent to test a compound’s antiradical activity.
TMP is reported to scavenge ÅOH, O2ꢀꢁ and ÅNO,39,40 but its ability
N
N
N
N
N
b
a
O
N
O
N
17
18
19
Scheme 5. Synthesis of compound 19. Reagents and conditions: (a) SeO2, 84 °C, 2 h,
75.2%; (b) N-tert-butyl-hydroxylamine, reflux, 4 h, 75.2%.
toward ONOOꢁ is not reported. Nitrone is effective in scavenging
ÅNO, ÅOH and O2
.
41,42 There is only indirect evidence showing that
ꢀꢁ
nitrone might inhibit ONOOꢁ formation by scavenging O2ꢀꢁ, but its
direct activity in scavenging ONOOꢁ was not performed in the re-
port.43 In the ÅOH assay, the introduction of a nitrone group in com-
pounds 3, 9 and 21 did not significantly improve their anti-ÅOH
an important role in the occurrence of ischemic stroke, drugs that
interfere with thrombus formation such as anticoagulants and
antiplatelet aggregants are commonly employed in the manage-
ment of patients with cerebrovascular disease. Considerable evi-
dence supports the use of antiplatelet aggregants in stroke
prevention and treatment.36,37 In our present work, the newly syn-
thesized nitrone compounds 3, 9, 21 and compound 16 retained
the parent TMP0s antiplatelet activities. The introduction of nitrone
moiety(ies) did not significantly affect their antiplatelet activity
(the percentages of platelet aggregation of the compounds 1, 4
and 14 were 43.1%, 43.1% and 46.5%).29
activity. While, in the O2 and ONOOꢁ assays, the nitrone com-
ꢀꢁ
pounds 3, 9 and 21 were more potent comparing with TMP. These
results demonstrate that the nitrone group is essential for their
activities against O2 and ONOOꢁ. This conclusion is further con-
ꢀꢁ
firmed by the fact that compound 16, bearing no nitrone, had no
activities against O2 and ONOOꢁ, and compound 21, bearing
ꢀꢁ
ꢀꢁ
two nitrone groups, was the most powerful against O2 and
ONOOꢁ.
In the region of the brain affected by ischemia, neuronal cells in
the ischemic core may die but those in the other parts (penumbra)
may be salvaged. Thrombolytic/antiplatelet agents are not in-
tended to salvage injured neurons in the penumbra for their pow-
erlessness in scavenging the active radical species produced by
Structure–activity relationship analysis of the new compounds
suggest that firstly, introduction of a nitrone group to TMP and
other pyrazines retains antiplatelet activity; Secondly, a nitrone
group increases antiradical activity, and especially accounts for
the anti-superoxide and anti-peroxynitrite activities.
O
N
N
N
N
b
N
a
O
N
O
O
N
N
14
20
21
Scheme 6. Synthesis of compound 21. Reagents and conditions: (a) SeO2, 84 °C, 2 h, 35.6%; (b) N-tert-butyl-hydroxylamine, reflux, 4 h, 86.2%.

3942
Y. Sun et al. / Bioorg. Med. Chem. 20 (2012) 3939–3945
Figure 1. Protective effect against t-BHP-induced cell damage. The results are expressed as the percentage of that of the untreated cells. The basal percentage of viable
neurons after only t-BHP treated cells was 49.4%. At the concentration of 1000 M, the percentage of viable neurons was: TMP 58.4%, compound 3 64.6%, compound 9 66.0%,
l
compound 13 54.8%, compound 16 75.5%, compound 19 53.9%, compound 21 80.6%. Data was performed via one-way analysis of variance, with subsequent individual
comparisons performed by Scheffe0s test. Data were expressed as means SD of three independent experiments. ⁄P <0.05 compared to t-BHP group.
5. Experimental
5.1. Chemistry
Melting points were determined with an electro-thermal melt-
ing point apparatus (Electro-thermal 9100). 1H NMR spectra were
recorded at ambient temperature on a 300 MHz spectrometer (AV-
300, Bruker) in CDCl3 or DMSO-d6. The chemical shifts values were
expressed in ppm relative to tetramethylsilane as an internal stan-
dard. Electrospray ionization mass spectra (ESI-MS) was obtained
in the positive ion detection mode on a Finnigan LCQ Advantage
MAX mass spectrometer (Applied Biosystems, 4000 Q TRAP). Ele-
mental analysis was performed at the experimental center of Jinan
University, Guangzhou, China, and the results were within 0.4% of
the theoretical values unless otherwise noted. High resolution
mass spectrometry was performed in an Agilent 6210 ESI/TOF
mass spectrometer. Column chromatography was carried out using
ordinary silica gel (200–300 mesh). Most chemicals and solvents
were analytical grade and were used without further purification.
Figure 2. (A) Protective effect in the rat t-MCAO model. Rats were subjected to 2 h
ischemia followed by another 22 h of reperfusion. The percentage of infarct volume
in contralateral hemisphere was calculated. Compound 21: 110 mg/kg; control
group received the same volume of saline. Compound 21: n = 13; control: n = 13.
Data was performed via one-way analysis of variance, with subsequent individual
comparisons performed by Scheffe0s test. ⁄P <0.05 compared to the control. (B)
Neurological recovery score in animals treated with compound 21. The neurological
deficit scores were analyzed by Mann–Whitney nonparametric test, and the
difference was considered significant at P <0.05.
5.1.1. 3-Ethylpyrazine-2-carbaldehyde (2)
Compound 2 was synthesized according to a report by Thomas
and Nowak.44
5.1.2. 2-[[(1,1-Dimethylethyl)oxidoimino]methyl]-3-ethylpyr
azine (3)
To compound 2 (1.3 g, 0.01 mol) in ethanol (200 mL) was added
N-tert-butyl-hydroxylamine (1 g, 0.011 mol), and the solution was
refluxed for 2 h. Another portion of N-tert-butyl-hydroxylamine
(1 g, 0.011 mol) was then added, and the solution was refluxed un-
til compound 2 was completely transformed. Solvent was removed
in vacuo. The crude product was purified by column chromatogra-
phy, eluting with ethyl acetate/petroleum ether (1:1, v/v), to pro-
duce compound 3 (brown liquid, 0.7 g, 35% yield). 1H NMR
(CDCl3): 1.21 (t, 3H), 1.54 (s, 9H), 2.74 (q, 2H), 8.06 (s, 1H), 8.51
(s, 1H), 8.56 (d, 1H). ESI-MS: 208 [M+H]+.
Figure 3. Prevention of platelet aggregation. Rabbit platelet suspensions were pre-
incubated with each compound (2 mM) at 37 °C for 8 min followed by the addition
of ADP (8 lM). The extent of aggregation was expressed as the percentage of the
control (in the absence of drugs). Data was performed via one-way analysis of
variance, with subsequent individual comparisons performed by Scheffe0s test. Data
were expressed as means SD of four independent experiments. ⁄P <0.05 compared
to saline treated group.
5.1.3 3-Propylpyrazin-2-yl-methanol (7) Compound 7 was
prepared according a published method.45
4. Conclusion
5.1.4. 2-[[(1,1-Dimethylethyl)oxidoimino]methyl]-3-propyl
pyrazine (9)
New pyrazine derivatives were synthesized. Among the new
compounds, compound 21 showed significant neuroprotective ef-
fect in cultured cerebellar granule neurons and in rat MCAO model.
Compound 21 was effective in inhibiting ADP-induced platelet
aggregation and was the most powerful compound in scavenging
Compound 7 (0.9 g, 0.006 mol) in ethanol (200 mL) was oxi-
dized at 84 °C for 12 h by activated MnO2 (5 g, 0.06 mol) to afford
compound 8. Without further purification, to the compound 8 in
ethanol (200 mL) was added N-tert-butyl-hydroxylamine (1 g,
0.011 mol), and the solution was refluxed for 2 h. Another portion
of N-tert-butyl-hydroxylamine (1 g, 0.011 mol) was then added,
and the solution was refluxed until compound 8 was completely
reacted. Solvent was removed in vacuo. The crude product was
Å
the three most damaging radicals to brain tissues, including OH,
O2 and ONOOꢁ. Compound 21 was a promising multi-functional
ꢀꢁ
agent for the treatment of ischemic stroke.

Y. Sun et al. / Bioorg. Med. Chem. 20 (2012) 3939–3945
3943
Figure 4. Free radical-scavenging effects. (A) Activity against ÅOH; (B) activity against O2ꢀꢁ, (C) activity against ONOOꢁ, (D) activity against DPPH. TMP and PBN were used as
positive controls. Results were the mean of three independent experiments.
purified by column chromatography, eluting with ethyl acetate/
petroleum ether (1:1, v/v), to produce compound 9 (brown liquid,
0.4 g, 30% yield, two steps). 1H NMR (CDCl3): 0.87 (t, 3H), 1.49 (s,
9H), 1.67 (m, 2H), 2.73 (t, 2H), 7.77 (s, 1H), 8.40 (dd, 2H). ESI-
MS: 222 [M+H]+, 244 [M+Na]+. HRMS (ESI, m/z): calcd for
5.1.8. 2-Methyl-N,N-bis((3,5,6-trimethylpyrazin-2-
yl)methyl)propan-2-amine (16)
To compound 15 (1.5 g, 0.007 mol) was added tert-butylamine
(0.26 g, 0.0035 mol) and the mixture was stirred at room temper-
ature for 5 h. After that, the reaction mixture was extracted with
ethyl acetate (100 mL) and dried with Na2SO4. The solvent was re-
moved in vacuo and the crude was further purified by column
chromatography (petroleum ether/ethyl acetate 1:4) to provide
compound 16 as a white solid (10.6 g, 42.5% yield), mp: 106–
108 °C. 1H NMR (CDCl3): 1H NMR (CDCl3): 1.25 (s, 9H), 2.30 (s,
6H), 2.35 (s, 6H), 2.39 (s, 6H), 3.86 (s, 4H). ESI-MS: 342 [M+H]+,
364 [M+Na]+. Anal. (C20H31N5ꢀ2.5 H2O) C, H, N.
C12H20N3O 221.14848, found 221.14879.
5.1.5. 5-Bromomethyl-quinoxaline (11)
Compound 11 was synthesized as described by Berg et al.46
5.1.6. 5-[(1,1-Dimethylethyl)oxidoimino]methylquinoxaline
(13)
To compound 11 (1.8, 0.008 mol) was added excess tert-butyl-
amine and the reaction mixture was stirred at room temperature
for 4 h until compound 11 was completely transformed to com-
pound 12. The excess tert-butylamine was removed in vacuo.
Without further purification, to compound 13 in 20 mL methanol
was added 646 mg Na2WO4ꢀ2H2O, 4 mL 30% H2O2 and the reaction
mixture was stirred for another 2 h at room temperature. After
that, the mixture was filtered, and solvent removed in vacuo. To
the residue was added saturated Na2S2O3 solution (8 mL). The
product was extracted with ethyl acetate (3 ꢂ 25 mL), and the
solution was dried with Na2SO4. Solvent was removed in vacuo
and the product was purified by column chromatography (petro-
leum ether/ethyl acetate 4:1) to afford compound 13 as a red solid
(1.2 g, 65.2% yield), mp: 84–86 °C. 1H NMR (CDCl3): 1.69 (s, 9H),
7.83 (dd, 1H), 8.10 (dd, 1H), 8.80 (d, 1H), 8.87 (d, 1H), 9.19 (s,
1H), 9.96 (dd, 1H). ESI-MS: 230 [M+H]+. Anal. (C13H15N3O) C, H, N.
5.1.9. 2-Quinoxalinecarbaldehyde (18)
Compound 18 was synthesized as described by Dale et al.47
5.1.10. 2-[(1,1-Dimethylethyl)oxidoimino]methylquinoxaline
(19)
Compound 18 (3.2 g, 0.02 mol) was dissolved in 250 mL etha-
nol. To the solution was added freshly prepared N-tert-butyl-
hydroxylamine (2.67 g, 0.03 mol). The reaction was allowed to pro-
ceed for 4 h. After the workup, solvent was removed in vacuo and
the crude product was purified by column chromatography (petro-
leum ether/ethyl acetate 4:1) to afford compound 19 as a red solid,
(3.49 g, 75.2% yield), mp: 88–89 °C. 1H NMR (CDCl3): 1.70 (s, 9H),
7.77 (m, 2H), 8.03 (m, 2H), 8.14 (s, 1H), 10.49 (s, 1H). ESI-MS:
230 [M+H]+. Anal. (C13H15N3O) C, H, N.
5.1.7. 2-(Bromomethyl)-3,5,6-trimethylpyrazine (15)
5.1.11. 3,6-Dimethylpyrazine-2,5-dicarbaldehyde (20)
Compound 20 was synthesized as described by Schumann and
Luo.48
Comopund 15 was synthesized according to
a published
method.27

3944
Y. Sun et al. / Bioorg. Med. Chem. 20 (2012) 3939–3945
5.1.12. 2,5-[[(1,1-Dimethylethyl)oxidoimino]methyl]-3,6-
trimethylpyrazine (21)
5.3.2. Evaluation of infarct size by TTC staining
Rats were re-anesthetized by 10% chloral hydrate and were
killed by decapitation. Brain was quickly removed and chilled in
the fridge for about 30 min. Six 2-mm thick coronal slices were
cut with a tissue slicer, beginning 1 mm posterior to the anterior
pole. Then the slices were immersed in PBS solution containing
0.5% 2,3,5-triphenyl-tetrazolium chloride (TTC) (Sigma) at 37 °C
for 30 min and fixed by immersion in 4.0% phosphate-buffered for-
malin solution. The sections were scanned with a digital camera
(Sony W220, Japan) and the areas of infarction were quantified
using an image analyzing system (Osiris 4 software, developed
by Université de Genève, Switzerland). The infarct area in each
slice was calculated by subtracting the normal ipsilateral area from
that of the contralateral hemisphere to reduce errors due to cere-
bral edema. The total infarct volume was determined by summing
up the infarct volume of the six sections and is presented as a per-
centage of the volume of the contralateral hemisphere.49,50
Compound 20 (1.2 g, 0.007 mol) was dissolved in 200 mL etha-
nol and then N-tert-butyl-hydroxylamine (2.49 g, 0.028 mol) was
added. The reaction was allowed to continue for 4 h at 84 °C. After
that, solvent was removed in vacuo and the crude product was
purified by column chromatography (petroleum ether/ethyl ace-
tate 1:1) to provide compound 21 as a yellow solid (1.9 g, 86.2%
yield), mp: 198–201 °C. 1H NMR (CDCl3): 1.61 (s, 18H), 2.48 (s,
3H), 2.50 (s, 3H), 7.83 (s, 2H). ESI-MS: 307 [M+H]+, 329 [M+Na]+.
Anal. (C16H26N4O2) C, H, N.
5.2. In vitro neuron protection assay
Cerebellar granule neurons were prepared and cultured accord-
ing to a published method.33 Cerebellar granule neurons were
placed into 96-well cell culture plates and were cultured for 7 days
at 37 °C under 5% CO2. Drugs at different concentrations were
added, and the cells were incubated at 37 °C for 30 min. t-BHP
5.4. Platelet aggregation assay
was then added (the final concentration of t-BHP was 50 lM),
and the cells were incubated for 4 h at 37 °C under 5% CO2. A solu-
tion of 3-(4,5-dimethylthiazol-2-ly)-2,5-diphenyl-tetrazoliun bro-
mide was added, and the cells were incubated for another 4 h
before DMSO was added. After the crystals were completely dis-
solved, the absorbance was read at 570 nm with a spectrophotom-
eter (Bio-Rad Model 680, Japan). The results were expressed as the
percentage of the control group.
Rat platelet suspensions were prepared from New Zealand
rabbits as previously described.12 Briefly, rabbits were fasted over-
night with free access to tap water before blood collection. Blood
was withdraw from arteria auricularis and mixed with sodium
citrate (9:1, v:v). Platelet rich plasma (PRP) was obtained by centri-
fugation of the blood at 800 rpm/min for 20 min. The supernatant
was collected to afford PRP. The platelet poor plasma (PPP) was ob-
tained by a further centrifugation at 3000 rpm/min for 10 min.
Drugs (the final drug concentration was 2 mM) were added into
PRP suspension (0.29 mL) and pre-warmed at 37 °C for 8 min. After
5.3. Neuroprotective effect in rat transient-MCAO model
5.3.1. Animal preparation for transient-MCAO model
that, ADP (the final concentration was 8 lM) was added to induce
Experimental protocols were approved by the ethical commit-
tee and conformed to internationally accept ethical standards
(Guide for the Care and Use of Laboratory Animals. NIH Publication
86-23, revised 1985). Adult male Sprague–Dawley rats (Guang-
dong Province Animal Center) weighing 260–310 g (approximate
3 month old) were used. Animals were housed in groups of five
in plastic cages under controlled temperature (20–22 °C), humidity
(40 60%), ventilation and lighting (12 h light/dark cycle) and were
allowed free access to food and water.
aggregation. The reaction was allowed to proceed for 5 min. Light
transmission through the platelet suspension was monitored by a
Platelet-Aggregometer (Sc-2000 Platelet-Aggregometer, Beijing)
to measure platelet aggregation. Maximum change in light trans-
mission was used as the aggregation endpoint for potency
comparisons.
5.5. Free radical scavenging capacity in vitro
The t-MCAO model was made according to a published method
with modification.34 Briefly, rats were anesthetized with 10% chlo-
ral hydrate (400 mg/kg, ip). A neck incision was made and the right
common carotid artery (CCA) was carefully closed by a ligature.
The occipital branch of the external carotid artery (ECA) was li-
gated and the internal carotid artery (ICA) was isolated. ICA was
temporarily closed by a microvascular clip and a small incision
5.5.1. Determination of hydroxyl radical-scavenging activity
The newly synthesized compounds0 ÅOH-scavenging activity
was determined in a cell-free assay following a published proce-
dure.51 The assay was dependent upon a compound0s ability to in-
Å
hibit the OH-dependent bleaching of para-nitrosodimethylaniline
(p-NDA). p-NDA was prepared at 1.6 mM in 50 mM NaCl solution.
ÅOH was generated using the Fenton reaction (Fe2+/H2O2). FeSO4
was dissolved in double-distilled water to afford a final concentra-
tion of 4.0 mM while H2O2 solution (7.3 mM) was freshly prepared
from a 30% stock solution. The compounds were dissolved in dou-
ble-distilled water and the final concentrations were 125, 250, 500
was made on CCA. A poly-L-lysine-coated nylon suture (0.36 mm
in diameter) was inserted into the CCA and was advanced into
ICA until MCA was occluded, about a distance of approximately
18–20 mm from the bifurcation of CCA. A ligature was tied around
the ICA, and then the incisions were closed. The nylon suture was
withdrawn after 2 h to achieve reperfusion.
and 1000 lM. The bleaching of p-NDA was monitored as the loss in
absorbance at 440 nm for 100 s. TMP and PBN were used as posi-
tive controls. The clearance was calculated as follows: Clearance
(%) = 1ꢁ[(A0–A100)/A0] ꢂ 100, where AC(0) was the absorbance at
t = 0 s and A(100) was the absorbance at t = 100 s.
During the experiments, body temperature (rectal temperature)
was monitored and adjusted with heating lamps and a heated
operating mat if the temperature fluctuated beyond predetermined
limits of 37 1 °C. Rats were divided into drug treated and vehicle
(control) treated groups. In order to ensure the reliability of the re-
sults, only rats with neurological scores of 2 or 3 were used in the
experiments, and each group had a large number of animals. The
failure rate of MCAO model was about 3% and the mean survival
rate of the rats enrolled in the experiments after 24 h was about
90%. Neurological scores were measured 2 h after transient MCAO
according to a published method.34 After surgery, animals were al-
lowed to recover in a warm (27 3 °C), quiet environment and gi-
ven free access to food and water.
5.5.2. Determination of superoxide anion-scavenging activity
A published procedure was used to determine these new com-
pounds0 ability to scavenge O2 radicals.52 A solution in a glass
ꢀꢁ
cuvette containing 1.565 mL Tris–HCl buffer (pH 8.2), 0.174 mL
pyrogallic acid (0.16 mmol/L) and 1.461 mL testing sample (the
final concentrations were 125, 250, 500 and 1000 lM) was pre-
pared. The solution was mixed thoroughly and absorbance was
recorded continuously for 300 s at 320 nm on a UV 22PC ultraviolet
spectrophotometer. The autoxidation rate of pyrogallic acid was

Y. Sun et al. / Bioorg. Med. Chem. 20 (2012) 3939–3945
3945
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duces blue light upon oxidation.53 SIN-1 was used to generate
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ONOOꢁ. Luminol was dissolved in 5% NaOH (20
l
L) and was di-
luted to 1 mM with 0.1 M PBS buffer (pH 7.4). SIN-1 was dissolved
in 0.1 M PBS, giving a final concentration of 100 M for this exper-
iment. The solution contained 350 L PBS, 50 L luminol, 50
each compound (the final concentration 1, 10, 100 and 1000
or PBS (control) and 50 L SIN-1 which was added last to initiate
l
l
l
lL
M)
l
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l
the reaction. The reaction was allowed to continue for 1250 s.
Chemiluminescence was recorded per 100 s. The clearance at peak
height represented the compounds0 ability to trap ONOOꢁ. The
clearance was calculated according to the formula: Clearance
(%) = [(Ac–Asample)/Ac] ꢂ 100.
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Acknowledgments
This work was partially supported by grants from the China
‘11.5’ Innovative Drug Project, the Natural Science Fund of China
(30772642 to Y.W.) and the Science and Technology Plans of
Guangzhou City (2006Z3-E4071 to P.Y.) as well as the ‘211’ of Jinan
University.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.016.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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