Design, synthesis, and biological evaluation of novel tetramethylpyrazine derivatives as potential neuroprotective agents

Article abstract of DOI:10.1248/cpb.c16-00699

Oxidative stress plays a crucial role in neurological diseases, resulting in excessive production of reactive oxygen species, mitochondrial dysfunction and cell death. In this work, we designed and synthesized a series of tetramethylpyrazine (TMP) derivatives and investigated their abilities for scavenging free radicals and preventing against oxidative stress-induced neuronal damage in vitro. Among them, compound 22a, consisted of TMP, caffeic acid and a nitrone group, showed potent radical-scavenging activity. Compound 22a had broad neuroprotective effects, including rescuing iodoacetic acid-induced neuronal loss, preventing from tert-butylhydroperoxide (t-BHP)-induced neuronal injury. Compound 22a exerted its neuroprotective effect against t-BHP injury via activation of the phosphatidyl inositol 3-kinase (PI3K)/Akt signaling pathway. Furthermore, in a rat model of permanent middle cerebral artery occlusion, compound 22a significantly improved neurological deficits, and alleviated the infarct area and brain edema. In conclusion, our results suggest that compound 22a could be a potential neuroprotective agent for the treatment of neurological disease, particularly ischemic stroke.

Full text of DOI:10.1248/cpb.c16-00699

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c16-00699
Advance Publication
October 14, 2016
Chem. Pharm. Bull.
Regular Article
Design, Synthesis, and Biological Evaluation of Novel Tetramethylpyrazine Derivatives
as Potential Neuroprotective Agents
Haiyun Chen, Guolian Tan, Jie Cao, Gaoxiao Zhang, Peng Yi, Pei Yu, Yewei Sun,*
Zaijun Zhang,* 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
*Corresponding author. Yewei Sun and Zaijun Zhang.
E-mail address: yxy0723@163.com (Y. Sun), zaijunzhang@163.com (Z. Zhang)
Ⓒ 2016 The Pharmaceutical Society of Japan

Abstract
Oxidative stress plays a crucial role in neurological diseases, resulting in excessive
production of reactive oxygen species, mitochondrial dysfunction and cell death. In this work,
we designed and synthesized a series of tetramethylpyrazine derivatives and investigated
their abilities for scavenging free radicals and preventing against oxidative stress-induced
neuronal damage in vitro. Among them, compound 22a, consisted of TMP, caffeic acid and a
nitrone group, showed potent radical-scavenging activity. Compound 22a had broad
neuroprotective effects, including rescuing iodoacetic acid-induced neuronal loss, preventing
from t-BHP-induced neuronal injury. Compound 22a exerted its neuroprotective effect
against t-BHP injury via activation of the PI3K/Akt signaling pathway. Furthermore, in a rat
model of permanent middle cerebral artery occlusion, compound 22a significantly improved
neurological deficits, and alleviated the infract area and brain edema. In conclusion, our
results suggest that compound 22a could be a potential neuroprotective agent for the
treatment of neurological disease, particularly ischemic stroke.
Keyword
Tetramethylpyrazine, nitrones, caffeic acid, free radical, neuroprotection, permanent middle
cerebral artery occlusion.
Chemical and Pharmaceutical Bulletin Advance Publication

1. Introduction
Ischemic stroke, resulting mainly from the interruption of cerebral blood flow, is the second
cause of morbidity and mortality worldwide. Many neuroprotective drug candidates have
been tested in preclinical and clinical studies, unfortunately, none have succeeded clinically.
In the past few decades, great progresses have been made in understanding the
pathophysiology of ischemic stroke. A cascade of biochemical events involved in ischemic
stroke produces profound cellular changes, including a rapid decrease in ATP content,
disruption of various ion channels, calcium overload, glutamate release, acidosis and edema.1)
Many of these changes are associated with excessive production of free radicals. Free radicals
including reactive oxygen species (ROS) and reactive nitrogen radicals (RNS) cause
mitochondrial dysfunction and subsequent cellular apoptosis.2) Therefore, antioxidants could
be developed as neuroprotective therapies against neuronal damage because of their
capability to combat free radicals.
Tetramethylpyrazine (TMP, Fig. 1) is a biologically active component of the traditional
Chinese medicine Chuanxiong (Ligusticum wallichii Franchat), which is widely used in
China for the treatment of cardio- and cerebro-vascular diseases clinically.3,4) Previous
research found that TMP had therapeutic effects in neurodegenerative diseases, such as
Parkinson’s disease (PD), Alzheimer’s disease (AD), and ischemic stroke.5-8) TMP’s precise
mechanisms of neuroprotection are not completely understood, but it has been proven to be
linked to its ability in maintaining mitochondrial function, scavenging free radicals,
increasing transcription of redox proteins and protecting antioxidant enzyme.8,9) In addition,
TMP has superior capability to enter the brain.10) Using a microdialysis method, Tsai et al.
demonstrated that TMP effectively penetrated the blood-brain barrier (BBB), yielding a
progressively higher brain/blood ratio at 10 to 120 min following intravenous
administration.11)
Chemical and Pharmaceutical Bulletin Advance Publication

Nitrones are a class of free radical-trapping agent and have been proven their therapeutic
potential for providing beneficial effects in a number of diseases including stroke, AD and
PD.12) PBN (N-tert-butyl-α-phenylnitrone) and NXY-059 are two representative nitrone
compounds. PBN reduced brain infarction in a rat model of middle cerebral artery occlusion
(MCAO).13) NXY-059 had exhibited promising therapeutic effects when evaluated in various
animal stroke models but failed its second phase III clinical trial because of its poor ability to
cross the BBB.14) Previously, we have reported a TMP nitrone conjugate, named TBN, which
readily crossed the BBB and showed potent free radical scavenging ability, protecting
neurons and rat suffering ischemic stroke and PD.15,16)
Caffeic acid (Fig. 1), a hydroxyl derivative of cinnamic acid bearing a phenolic moiety, is
belonging to the family of the phenyl propanoids.17) Caffeic acid is abundant in nature, has
versatile pharmacological activities, including anti-oxidative stress, anti-inflammatory,
anti-cancer, and anti-viral activities.18) Caffeic acid and its ester derivatives showed an
excellent neuroprotection and therapeutic effects in various neurological diseases.19) The
pharmacokinetics and tissue distribution study revealed that caffeic acid had weak BBB
permeability and presented a low brain distribution proportion.20)
Traditional drug development approach, one drug one target, is proven unsuccessful in
treating complex brain disorders that involve multiple pathogenic factors.21) A relatively
simple strategy for developing novel multi-functional drugs starts from structural
combination/modification of compounds with some desirable biological properties, may lead
to novel molecule with multiple properties derived from the original compounds, even
possessing unique properties that are distinct from the original compounds.22) Since all of the
three pharmacologically active compounds/functional moieties, i.e., TMP, cafferic acid and
nitrone, have versatile functions beneficial to neurological diseases, we reason that
compounds combing one or more of these moieties may afford new compounds with much
Chemical and Pharmaceutical Bulletin Advance Publication

improved efficacy. In addition, because of the superior capability penetrating BBB, TMP
could be used as a carrier to deliver nitrone/caffeic acid into the brain. Thus, in this work, we
designed and synthesized two series of novel TMP derivatives: (1) TMP moiety conjugated
with another TMP molecule or caffeic acid; (2) TMP-caffeic acid derivatives conjugated with
the potent radical scavenging nitrone moiety. Moreover, their free radical scavenging
activities against ROS and RNS were tested, and their neuroprotective effects were also
evaluated in oxidative stress damaged PC12 cells and in a rat model of permanent middle
cerebral artery occlusion (p-MCAO).
2. Results and Discussion
2.1. Rational drug design and synthesis
Previous structure-activity relationship studies revealed that the pyrazine ring of TMP might
principally determine its pharmacodynamics and the methyl groups might primarily govern
its pharmacokinetics and toxicity.23) Substitutions of the methyl group apparently improved
drug efficacy and prolonged action time.24) Our previous study also demonstrated that the
derivative of two TMP molecules linked via a tert-butyl amine group exhibited enhanced
efficacy in preventing cells against oxidative damage and in scavenging hydroxyl radicals.25)
Our goal is to design a compound possessing multi-functions necessary for effective
treatment of neurological diseases. In addition, the compound must penetrate the BBB readily,
an important requirement for central nervous system drugs. Furthermore, the compound must
be non-toxic at therapeutic doses. With these criteria in mind, based on the previous findings
and by virtue of properties of TMP, caffeic acid and nitrone, in this work, we introduce
another TMP or caffeic acid molecule(s) to TMP moiety via an ester bond or an ether bond
(compounds 4, 6, 8a-8b, 12a-12b, Fig. 2). Since the nitrone moiety has great potential to
scavenging free radicals, to further enhance the anti-radical activities, the nitrone moiety(ies)
was (were) introduced into the new compounds to obtain compounds 20a-20b and 22a-22b
Chemical and Pharmaceutical Bulletin Advance Publication

(Fig. 2).
Compound 4 was obtained by treatment of compound 2 with compound 3 in a solution of
NaHCO3 (Chart 1). Compounds 2 and 3 were synthesized as previously reported.26,25) The
preparation of compounds 6 was depicted in Chart 2. TMP was oxidized to afford compound
5, which was then treated with compound 3 to yield compound 6.
Synthesis of compounds 8a-b was showed in Chart 3. The commercially available caffeic
acid (7a) was treated with acetic anhydride to afford compound 7b. Compounds 7a and 7b
were treated with compound 3 in the presence of NaHCO3 to afford compounds 8a and 8b,
respectively.
Synthesis of compounds 12a and 12b was showed in Chart 4. TMP (1) was oxidized by
SeO2 to afford compound 9, which was reduced to compound 10 by treatment with NaBH4.
Compound 10 was brominated in the presence of PBr3 to afford compound 11. Compound 11
was reacted with compound 7a or 7b to produce compound 12a or 12b.
The synthesis of compounds 20a and 20b was showed in Chart 5. The key intermediates 18a
and 18b were prepared by treatment of compound 17 with compound 16a or 16b. Compound
17 was obtained by treating compound 9 with ethylene glycol catalyzed by p-toluenesulfonic
acid. The protective group of compounds 18a and 18b was cleaved in a solution of
concentrated sulfuric acid, water and tetrahydrofuran to afford compounds 19a-19b, which
were then reacted with tert-butyl-hydroxylamine to produce the target compounds 20a-20b.
Synthesis of compounds 22a and 22b was showed in Chart 6. Compounds 7a and 7b were
treated with compound 14 in the presence of NaHCO3 to afford compounds 21a and 21b.
Compounds 21a and 21b were converted into compounds 22a and 22b using a similar
method to that as described for the synthesis of compounds 20a and 20b.
2.2. Free radical scavenging activity in vitro
A growing body of evidence suggests that free radicals play a pivotal role in cellular and
Chemical and Pharmaceutical Bulletin Advance Publication

tissue injury during numerous diseases, including cardiovascular27) and neurodegenerative
diseases.28) Free radical scavengers ameliorated cell damage caused by reactive oxygen
species during the development of diseases.19,29) Because TMP, caffeic acid and nitrone group
all have anti-free radical activity,9,12,30) we first investigated the new compounds’ activity to
•-
scavenge DPPH•, •OH, O2 and ONOO- in vitro. As shown in Figs. 3A-D, all the new
compounds showed a radical clearance activity in a dose-dependent manner and the
TMP-caffeic acid derivatives were more potent than TMP dimer derivatives. There is a
positive correlation between free radical scavenging activity and the numbers of the phenolic
hydroxyl and nitrone groups. As shown in Figs. 3A and 3D, compound 12a showed the best
activity in scavenging DPPH• and ONOO-, while 8a exhibit potent scavenging activity of
•OH and O2•- radicals (8a>22a>12a). However, TMP had weak activities on clearance for the
tested radicals. TMP was oxidized to TMP-1, 4-(N,N´)-dioxide in the presence of H2O2 at 70
o
oC24) or catalyzed by horseradish peroxidase at 37 C.9) The direct free radical scavenging
ability of TMP is weak. The mechanism of anti-oxidative activity and neuroprotection effects
of TMP is mostly related to its role in maintaining mitochondrial function, regulating cellular
Ca2+ homeostasis, enhancing the expression of anti-oxidant proteins and suppressing the ROS
generation in vivo.9,31) Although the anti-oxidant activity of caffeic acid derivatives had been
reported, the free cinnamic acid had no activity on free radical scavenging. However, caffeic
acid with phenolic hydroxyl group on the benzene ring of cinnamic acid, can scavenge free
radicals directly.32) The phenolic hydroxyl group is essential for free radical scavenging
activity, and the 3-hydroxyl group is more important than the 4-hydroxyl group.30)
2.3. Protective effects of new compounds against chemical ischemic insult induced by
iodoacetic acid in cultured PC12 cells
Iodoacetic acid (IAA) is an irreversible inhibitor of the glycolytic enzyme glyceraldehyde
3-phosphate dehydrogenase. It inhibits glycolysis and triggers cellular energy metabolism
Chemical and Pharmaceutical Bulletin Advance Publication

deficits and induces cell injury quickly in vitro.33) IAA was usually applied to induce
chemical ischemia as a model of neuronal ischemic/hypoxic injury in vitro.33,34) We evaluated
the cytoprotective effects of TMP derivatives against IAA-induced ischemic injury in PC12
cells. As shown in Fig. 4A, there was no cytotoxicity up to 10 μM when cells were incubated
for 24 h with all compounds except compound 12b. Compounds 4, 6, 8a, 20a-b, and 22a-b
were not toxic even up to 100 μM. Exposure to 30 μM IAA for 2 h induced approximate 50%
reduction of cell viability. All the compounds showed good cytoprotective effect (Fig. 4B).
Compounds 8a-b, 12a, 20a-b and 22a-b significantly protected PC12 cells from IAA injury
at a concentration of 0.1-10 μM. Although the exact mechanism of IAA toxicity is unclear,35)
neural cells exposed to IAA present with an increased production of ROS,36) leading to
mitochondrial stress and DNA damage.37,38) Since TMP can maintain mitochondrial function
and suppresses ROS generation,9) TMP was found to significantly prevented PC12 cells
against IAA-induced neurotoxicity at 1-100 μM. However, TMP was less potent than 8a-b,
12a and 22a-b (Fig. 4B). By comparison, the compounds that combing TMP with other
anti-oxidant and radical scavenging moieties were more potent than TMP and TMP dimer
compounds in protecting cells against IAA injury. Moreover, compound 22a exhibited more
potent cytoprotection and was less cytotoxic than other derivatives. Therefore, compound 22a
was selected for further investigation.
2.4. Compound 22a effectively prevents t-BHP-induced neurotoxicity
We have previously reported that t-BHP, an oxidizing agent, produced
a
concentration-dependent decrease of cell viability in PC12 cells.33) In the present experiments,
exposure to 60 μM t-BHP for 24 h induced about 50% reduction of cell viability in PC12
cells. As shown in Fig. 5A, compound 22a concentration-dependently prevented
t-BHP-induced toxicity and the maximum protection reached 96.75±2.55%. Consistently,
pretreatment with 22a significantly inhibited t-BHP-induced LDH release (Fig. 5B). The
Chemical and Pharmaceutical Bulletin Advance Publication

parent TMP at 100 μM had no obvious protection against t-BHP-induced PC12 cells injury.
To further characterize the neuroprotective effects of 22a against neurotoxicity induced by
t-BHP, the impact of 22a on mitochondria membrane potential and intracellular ROS was
evaluated. As shown in Fig. 5C, compound 22a significantly prevented the decrease in
mitochondrial membrane potential. Moreover, pretreatment of PC12 cells with 22a (1-100
μM) significantly prevented the increase of intracellular ROS induced by t-BHP in PC12 cells
(Fig. 5D). Also, TMP at 100 μM had mild effects on the decrease of mitochondrial membrane
potential and the ROS overproduction. Therefore, our result suggests that the neuroprotective
effect of 22a against t-BHP neurotoxicity is partially attributed to maintaining mitochondrial
membrane potential and inhibiting the ROS overproduction.
2.5. Compound 22a blocks t-BHP-induced apoptosis in PC12 cells
To verify weather apoptosis is involved in 60 µM t-BHP induced neuronal injury in PC12
cells, Hoechst 33342 staining was performed. As observed from phase contrast fluorescence
microscopy (Fig. 6A), most nuclei in the control displayed uniform distribution of chromatin.
Intense Hoechst-stained nuclei (chromatin condensation), indicating cells apoptosis, were not
frequently observed in control group. However, apoptotic nuclei were observed with
increased frequency in t-BHP-stimulated cells compared to control cells. Pretreatment with
22a significantly reversed the cell numbers of nuclear condensation (Fig. 6B). Furthermore,
Western blot assay showed that pretreatment of 22a reversed the t-BHP-induced
down-regulation of Bcl-2 expression and up-regulation of Bax expression, two
apoptosis-related proteins. The Bcl-2/Bax ratio was increased to 124.6±2.4% of control by
pretreatment of 22a (Figs. 6C and 6D).
2.6. Effect of compound 22a on the PI3K/Akt/GSK3β signaling pathway
In order to elucidate the signaling pathways involved in the protective effects of 22a against
oxidative stress-induced cell injury, we assessed the relationship between cell viability and
Chemical and Pharmaceutical Bulletin Advance Publication

regulation of PI3K/Akt/GSK3β. PC12 cells were pretreated with Akt inhibitor IV (1 μM) and
PI3K inhibitor LY294002 (1 μM) for 30 min prior to the addition of compound 22a for 2 h
incubation, and were then exposed to 60 μM t-BHP. Pretreatment with these inhibitors could
significantly attenuate the cytoprotective effect of 22a (Fig. 7A), suggesting that the
cytoprotective effect of 22a was dependent on activation of PI3K/Akt pathway. Furthermore,
Western blot assay showed that pretreatment of 22a reversed the t-BHP-induced
down-regulation of Akt and GSK3β phosphorylation (Figs. 7B-E). When the cells were
exposed to the PI3K inhibitor, LY294002 (1 μM), 30 min prior to 22a treatment, 22a-induced
phosphorylation of Akt and GSK3β were almost completely suppressed by LY294002 (Figs.
7B-E). Previous studies have demonstrated that activation of Akt promoted cell survival and
provided protection against oxidative stress-induced neurotoxicity via the PI3K/Akt/GSK3β
pathway.39,40) In our present study, exposure to t-BHP significantly down-regulated
phosphorylation of Akt and GSK3β in PC12 cells. However, 22a pretreatment reversed
down-regulation of phosphorylated Akt and GSK3β stimulated by t-BHP. Moreover, PI3K
inhibitor LY29004 completely suppressed upregulation of phosphorylated Akt and GSK3β,
and abolished the neuroprotection conferred by 22a pretreatment. Collectively, our results
suggest that activation of PI3K/Akt/GSK3β pathway is involved in the neuroprotective effect
of 22a against t-BHP-induced PC12 cell injury.
2.7. Effect of compound 22a on neurological deficits, cerebral infarction and brain
edema in a rat model of p-MCAO
Since compound 22a conferred potent radical scavenging and cytoprotective activities in
vitro, we further evaluated its effects in a rat model of p-MCAO. Neurological deficits may
persist for months or years post-stroke in those affected individuals who survived in initial
cerebrovascular insult.41) As one of the most commonly used stroke model, the rat MCAO
model produces a replicable and reliable lesion both in the cortex and striatum, and results in
Chemical and Pharmaceutical Bulletin Advance Publication

both motor and some cognitive deficits.42) As shown in Fig. 8A, all rats except sham-operated
ones presented neurological deficit. However, the rats received 30 mg/kg of 22a by
intraperitoneal administration at 3 and 6 h after p-MCAO exhibited significantly
improvement in neurological deficits at 24 h after p-MCAO injury compared with the model
group.
The size of the infarction after an ischemic insult is regarded as the most straight forward
indicator of the effectiveness of a neuroprotective agent because it shows the severity of brain
damage. To observe the neuroprotective efficacy of 22a against p-MCAO injury, the infract
area and brain edema were also evaluated (Figs. 8B and 8C). Infract area of p-MCAO model
group was 62.1±6.2%, while the infract area was decreased to 49.1±8.9% in the 22a treated
group. In addition, the brain edema of vehicle group was 17.1±3.2% and that of the 22a
treated group was reduced to 11.5±5.4%. These data revealed that 22a significantly reduced
the infarct volume and brain edema compared to p-MCAO rat. The reduced brain infarction
and edema would contribute the neurobehavioral improvement exerted by 22a.
It has been reported that oxidative stress is an early and foremost pathological factor of
MCAO-induced brain damage, which leads to deteriorating functional outcomes.43)
Numerous neuroprotective agents with anti-oxidant activity have been tested on stroke
animal models and in clinical stroke patients.44) One of the principle features of an effective
stroke neuroprotective agent is its ability to penetrate the BBB to reach the site of action.
TMP’s superior ability to enter the brain was documented as early as 500 years ago in the
landmark traditional Chinese medicine literature.10) Compound 22a consisted of TMP, caffeic
acid and a nitrone group, thus, it is highly likely that 22a can readily penetrate the BBB to
exert potent anti-free radical activity in the experimental animal stroke model. In further
studies, we will investigate the ability of compound 22a in penetrating the BBB and report
the result in due course.
Chemical and Pharmaceutical Bulletin Advance Publication

Taken from all the result, compounds 22a consisted of TMP, caffeic acid and nitrone group
exhibited much more potent radical-scavenging activity and neuroprotective effect in vitro
and in cerebral ischemic stroke in vivo. In free radicals scavenging assay, compound 22a was
effective in scavenging different types of free radicals, especially for peroxynitrite. We think
that the improved peroxynitrite scavenging activity of compound 22a is primarily due to the
introduction of nitrone group since TMP showed very weak activity in scavenging
peroxynitrite in the current and our previous studies.45) It should be noted that compound
22a’s free radical-scavenging activity and neuroprotective effect in oxidative-induced
neurotoxicity are much more potent than the parent compound TMP. However, TMP moiety
has super ability to cross BBB, thus it can deliver caffeic acid and nitrone to the brain tissue,
conferring neuroprotection in vivo. Therefore, the excellent bioactivities of compounds 22a
in vitro and in vivo are possible combinational effects of TMP, caffeic acid and nirtone.
3. Conclusion
In this study, we have designed and synthesized novel TMP derivatives. Among them,
compound 22a, TMP conjugated with caffeic acid and tert-butyl nitrone, showed potent free
radical scavenging activity and neuroprotective effect against oxidative injury in vitro and
cerebral ischemic stroke in vivo. Based on these findings, compound 22a may be a promising
drug candidate for treating neurodegenerative disorders, including ischemic stroke in
particular. Therefore, the pharmacokinetics, toxicity profile, and the BBB penetrating
property of 22a warrant further evaluation.
Experimental Section
The details of the experimental procedures are provided in the supplementary materials,
which can be found as attachment.
Chemical and Pharmaceutical Bulletin Advance Publication

Acknowledgments
This work was supported in part by grants from the Natural Science Fund of China
(81502908), the Natural Science Foundation of Guangdong Province (2014A030310174,
2016A030313107), the Science and Technology Project of Guangdong Province
(2013A022100030, 2015A020211019, 2015B020232011 and 2016A020217013), as well as
the Key Laboratory Project of Guangzhou (151800010).
Conflict of Interest
The authors declare no conflict of interest.
Supplementary Materials
The online version of this article contains supplementary materials.
Chemical and Pharmaceutical Bulletin Advance Publication

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Chemical and Pharmaceutical Bulletin Advance Publication

Figure and Chart legends
Fig. 1. Structures of TMP, TBN and caffeic acid.
Fig. 2. Structures of new compounds.
Fig. 3. Free radical scavenging effects. (A) Activity against DPPH•. (B) Activity against
•-
•OH. (C) Activity against O2 (D) Activity against ONOO-.
Fig. 4. Neurorescue effect of new compounds on IAA-induced PC12 cell injury. (A)
Effect of new compounds on viability of PC12 cells (24 h treatment). Cell viability was
measured by the MTT assay after 24 h incubation with different concentrations of compounds.
(B) New compounds rescued IAA-induced PC12 cell injury. PC12 cells were exposed to IAA
(30 μM) for 2 h, and the culture medium was then removed and replaced with fresh culture
medium contained new compounds with different concentration. Cell viability was measured
after 24 h by the MTT assay. ###P<0.001 versus control; ***P<0.001 versus IAA
alone-treated cells.
Fig. 5. Compound 22a prevents t-BHP-induced neurotoxicity. PC12 cells were
pre-incubated with 22a or TMP at the indicated concentrations for 2 h, and were then
exposed to 60 μM t-BHP. (A) Compound 22a prevented t-BHP-induced neuronal death in
PC12 cells. (B) Compound 22a attenuated t-BHP-induced LDH release in PC12 cells. LDH
release was measured at 24 h after t-BHP challenge. (C) Compound 22a prevented
t-BHP-induced decrease in mitochondrial membrane potential in PC12 cells. (D) Compound
22a attenuated t-BHP-induced increase of ROS in PC12 cells. Data were expressed the mean
± SEM from three independent experiments; ###P<0.001 versus control group; **P<0.01 and
***P<0.001versus t-BHP group.
Fig. 6. Compound 22a blocks t-BHP-induced cell apoptosis in PC12 cells. PC12 cells
were pre-incubated with 22a or TMP at the indicated concentrations for 2 h, and were then
exposed to 60 μM t-BHP. (A) Compound 22a attenuated t-BHP-induced increase of pyknotic
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nuclei in PC12 cells. (B) Statistical analysis of the number of pykonitic nuclei. The number
of pyknotic nuclei with condensed chromatin were counted from representative Hoechst
staining photomicrographs and represented as a percentage of the total number of nuclei
counted. (C) Representative blots of the apoptosis-related protein Bcl-2 and Bax. (D)
Densitometric analysis of the protein expression of Bcl-2 and Bax. Data were expressed the
mean ± SEM from three independent experiments; ###P<0.001 versus control group;
*P<0.05, **P<0.01 and ***P<0.001versus t-BHP group.
Fig. 7. Involvement of PI3K/Akt/GSK3β pathway in cytoprotection exerted by 22a in
PC12 cells. (A) Pretreatment with the LY294002 (LY29) and Akt inhibitor IV (Akt IV-i)
abolished the protective effect of 22a against t-BHP-induced PC12 cells damage. PC12 cells
were pretreated with PI3K inhibitor LY294002 (1 μM) and Akt inhibitor IV (1 μM) for 30
min prior to the addition of compound 22a for another 2 h co-incubation, and then followed
by exposure to 60 μM t-BHP. Cell viability was measured at 24 h post t-BHP challenge by
using the MTT assay. (B) Representative blots of p-Akt and Akt expression. (C)
Densitometric analysis of the protein expression of p-Akt and Akt. (D) Representative blots
of p-GSK3β and GSK3β expression. (E) Densitometric analysis of the protein expression of
p-GSK3β and GSK3β. Data were expressed the mean ± SEM from three independent
experiments. ###P<0.001 and ##P<0.01 versus control group; *P<0.05, **P<0.01 and
***P<0.001versus t-BHP group.
Fig. 8. Therapeutic effect of 22a in a rat model of p-MCAO. (A) Compound 22a alleviated
neurological deficits. (B) Compound 22a attenuated infract areas. (C) Compound 22a
reduced brain edema. Data were expressed the mean ± SEM, n=6 rats/group. *P<0.05 versus
model group.
Chart 1. Synthesis of compound 4. Reagents and conditions: (a) KMnO4, H2O, 50 oC, 10 h;
(b) NBS, benzoyl peroxide, r.t., overnight; (c) NaHCO3, DMF, r.t., 2 h.
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Chart 2. Synthesis of compound 6. Reagents and conditions: (a) 1. H2O2, AcOH, 70 ºC, 8 h;
2. Ac2O, 125 ºC, 3 h; 3. NaOH, r.t., 5 h; (b) 1. NaH, THF, r.t.; 2. compound 3, DMF, r.t., 3 h.
Chart 3. Synthesis of compounds 8a-8b. Reagents and conditions: (a) DMAP, Ac2O, r.t.,
overnight; (b) compound 7a for 8a, or compound 7b for 8b, NaHCO3, DMF, r.t., 2 h.
Chart 4. Synthesis of compounds 12a-12b. Reagents and conditions: (a) SeO2, dioxane, 110
oC, 6 h; (b) NaBH4, (CH2Cl)2, r.t., 3 h; (c) PBr3, DCM, 0 oC, 3 h; (d) compound 7a for 12a or
compound 7b for 12b, NaHCO3, DMF, r.t., 3 h.
Chart 5. Synthesis of compounds 20a-20b. Reagents and conditions: (a) NaBH(OCOCH3)3,
(CH2Cl)2, r.t., 4 h; (b) PBr3, CH2Cl2, 0 oC, 3 h; (c) HOCH2CH2OH, toluene, TsOH, 80 oC, 3 h;
o
(d) (CH3CH2O)3OP, toluene, 110 oC, overnight; (e) HOCH2CH2OH, toluene, TsOH, 80 C, 3
h; (f) compound 16a or 16b, NaOCH3, CH2Cl2, r.t., 2 h; (g) con.HCl/H2O/THF=2/6/7, r.t., 2 h;
(h) tert-butylhydroxylamine, CH3CH2OH, r.t., 4 h.
Chart 6. Synthesis of compounds 22a-22b. Reagents and conditions: (a) compound 14,
NaHCO3, DMF, r.t., 3 h; (b) tert-butylhydroxylamine, CH3COCH3, r.t., 4 h.
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Fig.1．
Fig. 2.
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Fig. 3.
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Fig. 4.
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Fig. 5.
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Fig. 6.
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Fig. 7.
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Fig. 8.
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Chart 1.
Chart 2.
Chart 3.
Chart 4.
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Chart 5.
Chart 6.
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