Design, synthesis, and evaluation of NDGA analogues as potential anti-ischemic stroke agents

Article abstract of DOI:10.1016/j.ejmech.2017.09.028

Exogenous supplementation of antioxidants with ROS scavenging activity would be a potential therapy to cerebral ischemia-reperfusion injury in stroke. In the present study, a series of NDGA analogues with attenuation oxidative stress by directly scavenging ROS and indirectly through keap1/Nrf2/ARE pathway activation were designed and synthesized. All analogues were found to effectively remove ROS directly by DPPH radical scavenging assay, and compound 3a conferred potent protection from the oxidative injury in PC12 cells via promoting Nrf2 to translocate into nucleus and increasing expression of heme oxygenase-1(HO-1), where strongly reduced intracellular ROS level indirectly. More importantly, 3a significantly reduced brain infarction after cerebral ischemia-reperfusion injury in rats subjected to transient middle cerebral artery occlusion (MCAO). Overall, our findings shown compound 3a could serve as a promising compound for the treatment of stroke.

Full text of DOI:10.1016/j.ejmech.2017.09.028

Accepted Manuscript
Design, synthesis, and evaluation of NDGA analogues as potential anti-ischemic
stroke agents
Lili Huang, Jiabing Wang, Liping Chen, Min Zhu, Shoubiao Wu, Shenghui Chu,
Yuantie Zheng, Ziliang Fan, Jiafeng Zhang, Wulan Li, Dahui Chen, Xiufei Yang, Sicen
Wang, Peihong Qiu, Jianzhang Wu
PII:
S0223-5234(17)30742-0
10.1016/j.ejmech.2017.09.028
EJMECH 9743
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 August 2017
Revised Date: 5 September 2017
Accepted Date: 15 September 2017
Please cite this article as: L. Huang, J. Wang, L. Chen, M. Zhu, S. Wu, S. Chu, Y. Zheng, Z. Fan, J.
Zhang, W. Li, D. Chen, X. Yang, S. Wang, P. Qiu, J. Wu, Design, synthesis, and evaluation of NDGA
analogues as potential anti-ischemic stroke agents, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2017.09.028.
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ACCEPTED MANUSCRIPT
Graphical abstract:

ACCEPTED MANUSCRIPT
Design, Synthesis, and Evaluation of NDGA Analogues as Potential
Anti-Ischemic Stroke Agents
Lili Huangab1, Jiabing Wangab1, Liping Chena1, Min Zhua1, Shoubiao Wuca, Shenghui Chua, Yuantie
Zhenga, Ziliang Fana, Jiafeng Zhanga, Wulan Liad, Dahui Chena, Xiufei Yangc, Sicen Wange, Peihong
Qiua1*, Jianzhang Wuab*
(aChemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou Medical
Universtiy, Wenzhou, Zhejiang 325035, China)
(bWenzhou Biomedical Innovation Center, Wenzhou University and Wenzhou Medical University,
Wenzhou, Zhejiang 325035, China)
(cDepartment of Pharmacy，Wenzhou Central Hospital，Wenzhou，Zhejiang，325000，China)
(dCollege of Information Science and Computer Engineering, The First Clinical Medical College,
Wenzhou Medical University, Wenzhou, Zhejiang 325035, China)
(eSchool of Pharmacy, Xi’an Jiaotong University, Xi’an, Shanxi, 710049, China)
1 These authors contribute to this work equally.
*Correspondence authors.
Address: Chemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou Medical
Universtiy, Wenzhou, Zhejiang 325035, China. Tel./Tex: +86 577 86689984: (Peihong Qiu).
wjzwzmc@126.com (Peihong Qiu)
Address: Chemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou Medical
Universtiy, Wenzhou, Zhejiang 325035, China. Tel./Tex: +86 577 86689984: (Jianzhang Wu).
wjzwzmu@163.com (Jianzhang Wu)
Abstract
Exogenous supplementation of antioxidants with ROS scavenging activity would be a
potential therapy to cerebral ischemia-reperfusion injury in stroke. In the present study, a
series of NDGA analogues with attenuation oxidative stress by directly scavenging ROS
and indirectly through keap1/Nrf2/ARE pathway activation were designed and
synthesized. All analogues were found to effectively remove ROS directly by DPPH
radical scavenging assay, and compound 3a conferred potent protection from the oxidative
injury in PC12 cells via promoting Nrf2 to translocate into nucleus and increasing
expression of heme oxygenase-1(HO-1), where strongly reduced intracellular ROS level
indirectly. More importantly, 3a significantly reduced brain infarction after cerebral
ischemia-reperfusion injury in rats subjected to transient middle cerebral artery occlusion

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(MCAO). Overall, our findings shown compound 3a could serve as a promising
compound for the treatment of stroke.
Key words: Synthesis; ROS; Keap1/Nrf2/ARE pathway; Anti-ischemic stroke agent
1. Introduction
Stroke, a common cause of morbidity and mortality across the world, is the fourth-leading
cause of death in the United States[1,2]. According to a recent study, ischemic stroke
accounts for 87% of all strokes[3]. Currently, the treatment of ischemic stroke is used for
thrombolytic therapy in generally, which brings the second brain injury, namely, cerebral
ischemia-reperfusion injury[4-6]. Oxidative stress has been considered as the most
important reason among a series of mechanisms related to the pathogenesis of cerebral
ischemia-reperfusion injury. Because oxidative stress can accelerate reactive oxygen
species (ROS) generation, which exacerbate ischemic brain damage extremely[7]. On the
other hand, overproduction of ROS can not be neutralized in ischemic condition because
the antioxidant defense system in ischemic tissues is interrupted[8]. Hence, exogenous
supplementation of antioxidants with ROS scavenging activity would be a potential
therapy to cerebral ischemia-reperfusion injury. At present, there are two broad categories
of antioxidants: (1)The antioxidants with scavenging reactive oxygen species directly, like
edaravone, resveratrol, carnosic acid, rosmarinic acid and so on[9-10]. (2)The antioxidants
with scavenging reactive oxygen species indirectly, namely activating cellular endogenous
antioxidant signaling pathways and promoting the transcription of a broad range of
cytoprotective genes to remove ROS[11], where Keap1/Nrf2/ARE is one of the important
antioxidant signaling pathways, such as TBHQ, xanthohumol, quercetin, etc[12-13].
Despite these two types of antioxidants have been reported in many studies, in addition to
edaravone and other very few antioxidants, many antioxidants failed to put into clinical to
treatment of cerebral ischemia-reperfusion injury. Hence in this sense, the development of
novel antioxidant agents with ROS scavenging activity will have the big rise space and be
of importance in treating cerebral ischemia-reperfusion injury.
Natural products and their synthetic analogues have been shown to be invaluable
resources in drug discovery due to various great bioactivities and less side effects[14,15].
For example, Nordihydroguaiaretic Acid (NDGA) or 4,4'-(2,3-Dimethylbutane-1,4-
diyl)dibenzene-1,2-diol is naturally occurring antioxidant lignan isolated from creosote
bush, Larrea tridentate, which has shown to have promising applications in the treatment
of multiple diseases[16-21], like cardiovascular, immune and neurological systems, cancer,
etc. Besides, many studies have indicated that NDGA has important antioxidant properties,

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such as becoming a ROS scavenger and an activators of cellular intrinsic keap1/Nrf2/ARE
antioxidant pathway[22,23], which showed it could be a promising antioxidants for drug
discovery. Curcumin, is polyphenol yellow pigment in dietary spice turmeric from
Curcuma longa, contains α,β-unsaturated ketone moiety(Michael acceptors) that is able to
modify cysteine thiols present in Keap1, which could active Nrf2 signaling pathway, and
further sensitized the transcription of phase II genes and these genes encode enzymes
(HO-1, SODs, GCLC, etc.) to remove ROS indirectly[24,25]. Accordingly, we
hypothesize that hybrids of NDGA and α,β-unsaturated ketone scaffold may have potent
antioxidant activity against cerebral ischemia-reperfusion injury. Hence, in this study, a
series of hybrids of NDGA and α,β-unsaturated ketone scaffold were designed and
synthesized, and investigated the underlying antioxidative mechanism in a neuronlike rat
pheochromocytoma cell line, PC12. In addition, 3a was found to attenuate brain injury
after transient middle cerebral artery occlusion (MCAO) in rats.
OH
HO
OH
HO
Curcumin
NDGA
indirectly
directly
Keap1
Nrf2
transcriptional pathway
Antioxidantive defense
enzymes
ROS
Nrf2
Figure 1. Design of NDGA derivatives with attenuation oxidative stress by directly scavenging ROS
and indirectly through keap1/Nrf2/ARE pathway activation.
2. Results and discussion
2.1. Chemistry
The synthetic pathway adopted to synthesize the NDGA derivatives were as described as
in Figure 2. All compounds(3a-3m) were obtained in reaction of appropriate
ketone(1a-1m) with 3,4-dihydroxybenzaldehyde(2) at room temperature under HCl gas.
All compounds were purified by column chromatography and were obtained in medium
yields (30%-70%) after purification. The products were characterized by analysis and
comparison of their spectral data including high performance liquid chromatography-mass
1
spectrometry (HPLC-MS) and H-NMR. These characteristic data including color, yield,

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1
melting points, HPLC-MS and H-NMR spectrum of compounds were presented in
chemistry synthetic section.
Comp.
3a
X
CH2
CH2
n1
1
1
0
1
1
1
1
n2
0
1
0
1
1
1
1
Comp.
3h
X
n1
1
n2
1
S
3b
3i
NCH2CH2Ph
NCOOCH2Ph
NCOOCH3
NCH2Ph
1
1
a
3c
/
3j
1
1
3d
NCH2CH2CH3
3k
1
1
3e
NCH3
NH
O
3l
1
1
NCHCH2CH2
1
1
3f
3m
3g
Figure 2. Synthesis and structures of NDGA derivatives. Synthetic conditions: (I) HCl gas, EtOH,
room temperature. a “/” represents none.
2.1 Scavenging free radicals in vitro
Due to NDGA and its derivatives belong to phenolic compounds, and thus we first
investigated their antioxidant activity in vitro[26]. 2,2-Diphenyl-1-picrylhydrazyl (DPPH)
radical scavenging activity assay is considered as a valid and easy assay to evaluate the
radical-scavenging activity of antioxidants, because DPPH radicals are more stable and
easier to handle than oxygen free radicals[27,28]. In this experiment, the DPPH radical
scavenging activities of NDGA derivatives 3a-3m were determined. As shown in Figure 3,
all analogues were found to effectively scavenge DPPH radical via chemical ways, and
showed the radical scavenging activity similar to NDGA. Meanwhile, we found that A1,
the methylated derivative of 3a, can not scavenge free radicals directly (Figure S1 in the
supporting information). In all, all NDGA derivatives can act as direct radical scavengers.
Figure 3. DPPH radical scavenging rate of NDGA derivatives (20 mg/L). Data are expressed as the
mean ± SD, n = 3.

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2.2 Protection of NDGA analogues from H2O2-induced PC12 cells damage
Hydrogen peroxide(H2O2), an endogenous cellular signaling molecule, has been widely
used as an inducer of oxidative stress in many cellular models[29]. And treatment with
compounds for 24 h followed by H2O2 insult generally as an experimental method for
studying antioxidant activity was used in this test[30-33]. Additionally, we chose
H2O2-induced PC12 cell damage model to evaluate the cytoprotection of NDGA
analogues. As shown in Figure 4A, pre-incubation with these compounds for 24 h, only 3a,
3f and 3g presented good protection of PC12 cells. Notably, compounds pre-incubation for
1 h, all analogues and NDGA showed valuable protection against oxidative damage in
PC12 cells induced by H2O2 insult (Figure 4B), while A1 exhibited no protection (Figure
S2 in the supporting information). The reason why these compounds pre-incubation for 1
h has potent protection may be that they bear four phenolic hydroxyl groups, which make
them remove reactive oxygen species directly. In addition, only a few analogues
pre-incubation for 24 h showed protection against H2O2 insult, it may be due to their effect
of activating antioxidant signal pathway. Moreover, 3a, 3f and 3g were found to have
better advantageous cytoprotection at both time points. Furthermore, the cytotoxicity of
the three compounds toward the PC12 cells at 5 µM after incubation time were determined
by the MTT assay. As shown in figure 4C, when the PC12 cells were treated with drugs
for 24 h, there is not apparent toxicity of the tested compounds; while treatment with for
72 h, three compounds and NDGA exhibited a degree of toxicity (Figure 4D). Based on
the above results
compounds.
, 3a exhibited lower cytotoxicity and better protection among all
A
C
24h
24h
B
D
1h
72h

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Figure 4. Compounds’cytoprotection on PC12 cells in H2O2 damage model(A,B) and cytotoxicity
screening of three active compounds in PC12 cells (C,D). PC12 cells were pretreated for 24 h (A) or 1h
(B) with NDGA and its derivatives 3a-3m (5 µM), then another 24 h exposure in H2O2 (400 µM),
finally determined by the MTT assay. The viability of untreated cells is defined as 100%. The cells
were treated with the 5 µM of NDGA analogues (3a, 3f, 3g) and NDGA for 24 h (C) or 72 h (D), and
the cytotoxicity of compounds was determined by the MTT assay. Data are expressed as the mean ±
SD, n = 3. ##P < 0.01, #P < 0.05 vs DMSO, **P < 0.01, *P < 0.05 vs H2O2. one way ANOVA, followed
by Tukey’s multiple comparison test.
2.4 Protection of PC12 cells from H2O2-induced damage by 3a.
As compound 3a exhibited the better protection, this compound was selected for the
further study. As shown in Figure 5A, B, PC12 cells treated with H2O2 only showed about
40% cell death compared with the control group. However, when the cells were treated
with 3a for 1 h or 24 h followed by H2O2 insult, the population of viable cells increased in
a dose-dependent manner. Malondialdehyde(MDA), a byproduct of polyunsaturated fatty
acid peroxidation caused by ROS, is regarded as a significant biomarker of oxidative
stress[34,35]. As shown in Figure 5C and D, pretreatment of PC12 cells with 3a for 1 h or
24 h significantly reduced the MDA in a dose-dependent manner. Stimulation of the cells
with H2O2 could produced a burst of ROS in the cells, so we then examined whether 3a
could prevent ROS accumulation in PC12 cells. As illustrated in Figure 5E, F, it is found
that pretreatment of the cells with 3a for 1 h or 24 h remarkably reduced the ROS
accumulation. Moreover, prevention of ROS accumulation in PC12 cells by compound 3a
might account for their cytoprotection against oxidative injury. Taken together, compound
3a can significantly protect PC12 cells from H2O2-induced cell injury.

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Figure 5. Protection of compound 3a by scavenging ROS and reducing MDA against H2O2-induced
PC12 cell damage. (A,B) 3a protected PC12 cells from H2O2-induced cell injury. PC12 cells were
pretreated with 3a in different doses (2.5, 5, 7.5 µM) for 1 h or 24 h, then treated with 400 µM H2O2 for
24 h, and determined by the MTT assay. (C,D) 3a reduced the MDA induced by H2O2 in PC12 cells.
PC12 cells were pre-incubated with 3a at 2.5, 5, 7.5 µM, NDGA (5 µM) or TBHQ (5 µM) for 1 h or 24
h, then treated with 700 µM H2O2 for 16 h, finally determined by the manufacturer’s instructions. (E,F)
3a decreased ROS level in H2O2 treated PC12 cells. PC12 cells were pre-treated with 5 µM of 3a,
NDGA or TBHQ, then treated with H2O2 for 3 h, and the ROS level was measured by flow cytometry.
Data are expressed as the mean ± SD, n = 3. ###P < 0.001, ##P < 0.01, #P < 0.005 vs DMSO, **P < 0.01,
*P < 0.05 vs H2O2, one way ANOVA, followed by Tukey’s multiple comparison test.
2.5 3a activated the Nrf2 signaling pathway
Promoting Nrf2 to translocate into nucleus by antioxidants has been considered as the
mechanism of preventing PC12 cells from H2O2-induced damage[36,37]. Therefore
immunofluorescence assay is used to examined whether 3a could promote Nrf2 to
accumulate in nuclei. As shown in Figure 6A, the blue staining and the red staining
represent nuclei and Nrf2, respectively. Compared with the blank control group, 3a-treated
group showed strong red fluorescent light in the nucleus, and it revealed that Nrf2 entered
and concentrated in the nucleus after treatment by 3a. The similar phenomenon occurred
in positive group both NDGA and TBHQ. The data supported that 3a can induce Nrf2
translocation into the nucleus.
Next, we examined the effect of 3a on the expression of HO-1 in PC12 cell. A
dose-dependent manner could be observed for 3a to enhance the expression of HO-1
(Figure 6B), indicating the potent Nrf2-ARE inductivity of 3a at a cell-based level.
Moreover, 3a at 10 µM exhibited even more stronger promoting effect than that of NDGA
and TBHQ. To investigate whether the expression of HO-1 caused by the 3a is responsible
for the cytoprotective effects against H2O2-derived oxidative cell death, its specific
inhibitor, ZnPP was utilized in this study[38]. PC12 cells were treated by ZnPP (15 µM) in
for 1 h before the addition of 3a. As shown in Figure 6C, it was obvious that ZnPP had no
significant adverse effect on cell viability in H2O2-induced model group, while 3a
increased the cell viability. However, when ZnPP and 3a were applied together, the
cytoprotection of 3a was suppressed. This result showed that 3a executed its anti-oxidative
activities through, at least partly, activating HO-1 protein expression. In all, these results
showed that 3a induced the Nrf2 translocation into the nucleus and further activated HO-1
expression to exhibit an inspirable protection against H2O2 induced PC12 cell injury.

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Figure 6. Activating the Nrf2 signaling pathway by 3a. (A) The nucleus translocation of Nrf2 caused
by 3a. PC12 cells were incubated with 3a, NDGA and TBHQ at 5 µM for 6 h, and then stained with
Nrf2 antibody and DAPI. (B) Elevating the expression level of protein HO-1 by 3a. PC12 cells were
incubated with 3a at 2.5, 5, 10 µM , NDGA or TBHQ at 10 µM for 24 h, and the HO-1 was determined
by western blotting experiment. Data are expressed as the mean ± SD, n = 3. **P < 0.01, *P < 0.05 vs
DMSO, one way ANOVA, followed by Tukey’s multiple comparison test. (C) ZnPP diminished the
protected effect of 3a on H2O2 induced cell damage. PC12 cells were incubated with HO-1 inhibitor
ZnPP for 1 h, then treated with 3a at 5 µM for 24 h. Finally MTT assay measured the OD values in 490
#
nm. Data are expressed as the mean ± SD, n = 3. P < 0.1, *P < 0.05, one way ANOVA, followed by
Tukey’s multiple comparison test.
2.6 3a attenuated brain injury after cerebral ischemia-reperfusion injury in rats subjected
to transient middle cerebral artery occlusion (MCAO)
We further evaluated for the neuroprotective activity of 3a in vivo in a rat model of
transient focal cerebral ischemia by intraluminal occlusion of the middle cerebral artery
(MCAO), which has considered to the most common reason for inducing I/R-related brain
injury in the clinic[39]. The efficacy of 3a was evaluated by preinjection into lateral
ventricles. The infarct size of individual rats were evaluated by the 2,3,5-
triphenyltetrazolium chloride (TTC) staining. As shown in Figure 7A and B, there were no
obvious infarction damage in the sham group, while the infarct area in the model group
and vehicle group of rats significantly increased, as shown in the white region of rat brain
sections. However, intracerebroventricular administration of 3a significantly reduced the
infarct sizes of I/R rats. The treatment with edaravone also showed a protective effect on

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infarction damage. Furthermore, the effect of 3a was more pronounced than that of
edaravone. Additionally, treatment with 3a and edaravone markedly reduced the
neuroloigic scoring in the brains from ischemic rats (Figure 7C). These results suggested
the potential of 3a for the treatment of ischemic brain damage.
Figure 7. Effect of 3a on middle cerebral artery occlusion(MCAO)-induced focal cerebral ischemia in
rats. (A) Representative images of TTC-stained brain slices. The corresponding (B) infarct areas and (C)
neuroloigical score levels in brain tissues. Cerebral infarction in sham-operated (sham) or MACO
reperfusion rats from a representative animal that received normal saline (NS), vehicle, 0.2 mg/kg 3a or
ED(edaravone) by intraventricular injection. Data are expressed as the mean ± SD, n ≥ 6. ###P < 0.001
vs sham-operated group, ***P < 0.001, **P < 0.01, *P < 0.05 vs vehicle-treated group. one way
ANOVA, followed by Tukey’s multiple comparison test.

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3. Conclusion
It is well known that generation of excessive ROS during reperfusion contribute to brain
injury associated with stroke. Administration of anti-ischemic stroke agent with ROS
scavenging activity at the time of reperfusion could potentially decrease the risk, which
may a valid neuroprotective strategy. The case of edaravone, an effective free radical
scavenger approved for the treatment of patients with acute stroke, proves the viability of
this approach. Also, researches showed that many small molecules could activate Nrf2
pathway to confer protection against ROS and prevent the ischemia-reperfusion
injury[40-42]. Hence, exogenous supplementation of agents with attenuation oxidative
stress by directly scavenging ROS and indirectly through keap1/Nrf2/ARE pathway
activation would be a potential therapy to cerebral ischemia-reperfusion injury.
In this study, a series of hybrids of NDGA and α,β-unsaturated ketone scaffold were
designed and synthesized. All analogues can effectively scavenge DPPH radicals. Then
their cytoprotection in H2O2 induced PC12 cells injury model was tested. Notably, after
pre-incubation with compounds for 1 h, all analogues showed valuable cytoprotection
against oxidative damage in PC12 cells; and after pre-incubation for 24 h, compounds 3a,
3f and 3g also displayed excellent cytoprotection. Among them, compound 3a was
screened out to exhibit low cytotoxicity and an inspirable protection against H2O2 induced
PC12 cell injury in a concentration-dependent manner. 3a can remarkably remove the
ROS accumulation, and reduce the MDA in a dose-dependent manner. Furthermore, 3a
can induce Nrf2 nuclear translocation and hence upregulate HO-1 protein expression,
conferring protection of PC12 cells against H2O2 insult. Meanwhile, using a rat model of
ischemic stroke, also showed that preinjection of 3a can significantly decrease the infarct
area and neuroloigical scoring in the ischemic brain.
In summary, our work presented a series of novel NDGA analogus with antioxidant
activity. 3a may confer protection for PC12 cells against oxidative insults through double
mechanisms, including scavenging free radicals directly and activating the Nrf2
anti-oxidative signaling pathway at the same time, and serve as a promising compound for
the treatment of ischemia-reperfusion injury in the stroke.
4. Experimenta
l
4.1. Chemistry
All chemical reagents were obtained commercially from Sigma-Aldrich(St Louis,
Missouri, USA), Aladdin (Shanghai, China) and used without further purification unless
otherwise noted. Reactions were monitored by TLC using silica gel GF254, the

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chromatograms were conducted on silica gel (200-300 mesh) and observed under UV light
at 254 and 365 nm. Melting points (mp) are uncorrected and were measured in open
capillary tubes on a Fisher-Johns melting apparatus. Mass spectra (MS) were recorded on
1
an Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA). H NMR spectra were obtained
from 600 MHz spectrometer (Bruker Corporation, Switzerland) with TMS as an internal
standard. The chemical data of compounds are presented as follows:
4.1.1
(2Z,5E)-2,5-bis(3,4-dihydroxybenzylidene)cyclopentanone (3a): Green powder, 53.6%
1
yield, mp>300°C. (Lit.[43] mp>300°C). H-NMR (600MHz, d-DMSO), δ: 9.563 (s, 2H,
3-OH×2), 9.211 (s, 2H, 4-OH×2), 7.238 (s, 2H, Ar-CH=C×2), 7.113 (d, J=1.8Hz, 2H,
Ar-H2×2), 7.006 (dd, J=1.8Hz, J=8.4Hz, 2H, Ar-H6×2), 6.834 (d, J=8.4Hz, 2H, Ar-H5×2),
3.001 (s, 4H, CH2-O-CH2). LC-MS m/z: 325.10(M+H)+, calcd for C19H16O5: 324.10.
4.1.2
(2Z,6E)-2,6-bis(3,4-dihydroxybenzylidene)cyclohexanone (3b): Orange powder, 45.7%
1
yield, mp 242.7-243.8°C. (Lit.[43] 244-246°C). H-NMR (600MHz, d-DMSO), δ: 9.438
(s, 2H, 3-OH×2), 9.131 (s, 2H, 4-OH×2), 7.446 (s, 2H, Ar-CH=C×2), 6.980 (d, J=1.8Hz,
2H, Ar-H2×2), 6.873 (dd, J=1.8Hz, J=8.4Hz, 2H, Ar-H6×2), 6.799 (d, J=8.4Hz, 2H,
Ar-H5×2), 2.845 (t, J=4.8Hz, 4H, CH2-C-CH2), 1.725 (t, J=4.8Hz, 2H, C-CH2-C). LC-MS
m/z: 339.18(M+H)+, calcd for C20H18O5: 338.12.
4.1.3
(1E,4E)-1,5-bis(3,4-dihydroxyphenyl)penta-1,4-dien-3-one (3c): Green powder, 58.9%
1
yield, mp242.7-245.4°C(Lit.[43] 221-223°C). H-NMR (600MHz, d-DMSO), δ: 9.646 (s,
2H, 3-OH×2), 9.171 (s, 2H, 4-OH×2), 7.568(d, J=16.2Hz, 2H, Ar-CH=C×2), 7.155 (d,
J=1.8Hz, 2H, Ar-H2×2), 7.083(dd, J=1.8Hz, J=1.8Hz, 2H, CO-CH=C×2), 7.002(d,
J=15.6Hz, 2H, Ar-H5×2), 6.800(d, J=8.4Hz, 2H, Ar-H6×2). LC-MS m/z: 299.11(M+H)+,
calcd for C17H14O5: 298.08.
4.1.4
(3E,5E)-3,5-bis(3,4-dihydroxybenzylidene)-1-propylpiperidin-4-one (3d): Yellow
1
powder, 58.9% yield, mp 232.2-233.8°C. H-NMR (600MHz, d-DMSO), δ: 9.877 (s, 2H,
3-OH×2), 9.439 (s, 2H, 4-OH×2), 7.719 (s, 2H, Ar-CH=C×2), 6.942 (s, 2H, Ar-H2×2),
6.902 (d, J=18.6Hz, 4H, Ar-H5×2, Ar-H6×2), 4.576 (d, J=37.2Hz, 4H, CH2-N-CH2),
3.513-3.479 (m, 2H, N-CH2), 1.698 (d, J=7.2Hz, 2H, N-C-CH2), 0.895 (t, J=14.4Hz, 3H,
CH3). LC-MS m/z: 382.17(M+H)+, calcd for C22H23NO5: 381.16.
4.1.5
(3E,5E)-3,5-bis(3,4-dihydroxybenzylidene)-1-methylpiperidin-4-one (3e): Drown
powder, 51.3% yield, mp>300°C. (Lit.[44] mp>300°C). 1H-NMR (600MHz, d-DMSO), δ:
9.886 (s, 2H, 3-OH×2), 9.403 (s, 2H, 4-OH×2), 7.708 (s, 2H, Ar-CH=C×2), 6.965 (d,
J=1.2Hz, 2H, Ar-H2×2), 6.900-6.879(m, 4H, Ar-H5×2, Ar-H6×2), 4.609(d, J=44.4Hz, 4H,

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CH2-N-CH2), 3.009 (s, 3H, CH3). LC-MS m/z: 354.17(M+H)+, calcd for C20H19NO5:
353.13.
4.1.6
(3E,5E)-3,5-bis(3,4-dihydroxybenzylidene)piperidin-4-one (3f): Yellow-green powder,
49.7% yield, mp>300°C. (Lit.[43] mp>300°C). 1H-NMR (600MHz, d-DMSO), δ: 9.832 (s,
2H, 3-OH×2), 9.687 (d, J=19.8Hz, 1H, NH), 9.425 (s, 2H, 4-OH×2), 7.690 (s, 2H,
Ar-CH=C×2), 6.937 (s, 2H, Ar-H2×2), 6.901 (d, J=8.4Hz, 2H, Ar-H6×2), 6.872 (d,
J=8.4Hz, 2H, Ar-H5×2), 4.428 (s, 4H, CH2×2). LC-MS m/z: 340.04(M+H)+, calcd for
C19H17NO5: 339.11.
4.1.7
(3E,5E)-3,5-bis(3,4-dihydroxybenzylidene)dihydro-2H-pyran-4(3H)-one (3g): Green
1
powder, 50.8% yield, mp 262.4-264.7°C. (Lit.[43] mp>300°C). H-NMR (600MHz,
d-DMSO), δ: 9.563 (s, 2H, 3-OH×2), 9.241 (s, 2H, 4-OH×2), 7.461 (s, 2H, Ar-CH=C×2),
6.807 (d, J=7.8Hz, 2H, Ar-H6×2), 6.805 (s, 2H, Ar-H2×2), 6.756 (d, J=8.4Hz, 2H,
Ar-H5×2), 4.836 (s, 4H, CH2-O-CH2). LC-MS m/z: 341.10(M+H)+, calcd for C19H16O6:
340.19.
4.1.8
(3Z,5Z)-3,5-bis(3,4-dihydroxybenzylidene)dihydro-2H-thiopyran-4(3H)-one
(3h):
1
Drown powder, 43.9% yield, mp 223.3-224.6°C. (Lit.[43] 223-225°C). H-NMR
(600MHz, d-DMSO), δ: 7.436 (s, 2H, Ar-CH=C×2), 6.941 (d, J=1.8Hz, 2H, Ar-H2×2),
6.857 (dd, J=8.4Hz, J=1.8Hz, 2H, Ar-H6×2), 6.808 (d, J=7.8Hz, 2H, Ar-H5×2), 3.946 (s,
4H, CH2-S-CH2). LC-MS m/z: 356.98(M+H)+, calcd for C19H16O5S: 356.07.
4.1.9
(3E,5E)-3,5-bis(3,4-dihydroxybenzylidene)-1-phenethylpiperidin-4-one (3i): Green
1
powder, 58.7% yield, mp150.8-152.8°C. H-NMR (600MHz, d-DMSO), δ: 9.886 (s, 2H,
3-OH×2), 9.462 (s, 2H, 4-OH×2), 7.719 (s, 2H, Ar-CH=C×2), 7.327 (t, J=4.8Hz, 4H,
Ar-H6’, Ar-H2’, Ar-H3’, Ar-H5’), 7.262-7.239 (m, 1H, Ar-H4’), 7.021 (s, 2H, Ar-H2×2),
6.904-6.871 (m, 4H, Ar-H5×2, Ar-H6×2), 4.657 (d, J=4.8Hz, 4H, CH2-N-CH2), 3.587 (s,
2H, CH2×2), 3.099 (t, J=16.2Hz, 2H, CH2). LC-MS m/z: 444.17(M+H)+, calcd for
C27H25NO5: 443.17.
4.1.10
(3E,5E)-benzyl 3,5-bis(3,4-dihydroxybenzylidene)-4-oxopiperidine-1-carboxylate (3j):
Green powder, 50.8% yield, mp250.4-251.5°C. 1H-NMR (600MHz, d-DMSO), δ: 9.646 (s,
4H, 3-OH×2), 9.285 (s, 2H, 4-OH×2), 7.541 (s, 2H, Ar-CH=C×2), 7.242 (d, J=1.8Hz, 2H,
Ar-H2’, Ar-H6’), 7.231 (t, J=5.4Hz, 1H, Ar-H4’), 7.079 (t, J=7.2Hz, 2H, Ar-H3’, Ar-H5’),
6.974 (s, 2H, Ar-H2×2), 6.857 (d, J=7.8Hz, 4H, Ar-H5×2, Ar-H6×2), 5.017 (s, 2H, CH2),
4.795(d, J=30.6Hz, 4H, CH2-N-CH2). LC-MS m/z: 474.25(M+H)+, calcd for C27H23NO7:
473.15.

ACCEPTED MANUSCRIPT
4.1.11
(3E,5E)-ethyl3,5-bis(3,4-dihydroxybenzylidene)-4-oxopiperidine-1-carboxylate(3k):
1
Orange powder, 43.9% yield, mp280.5-283.6°C. H-NMR (600MHz, d-DMSO), δ: 9.640
(s, 2H, 3-OH×2), 9.276 (s, 2H, 4-OH×2), 7.510 (s, 2H, Ar-CH=C×2), 6.939 (s, 2H,
Ar-H2×2), 6.881-6.843 (m, 4H, Ar-H5×2, Ar-H6×2), 4.734 (s, 4H, CH2-N-CH2),
4.002-3.966 (m, 2H, CH2), 1.050 (t, J=13.8Hz, 3H, CH3). LC-MS m/z: 412.06(M+H)+,
calcd for C22H21NO7: 411.13.
4.1.12
(3E,5E)-1-benzyl-3,5-bis(3,4-dihydroxybenzylidene)piperidin-4-one (3l): Yellow-green
1
powder, 50.8% yield, mp158.4-160.2°C. H-NMR (600MHz, d-DMSO), δ: 9.868 (s, 2H,
3-OH×2), 9.408 (s, 2H, 4-OH×2), 7.737 (s, 2H, Ar-CH=C×2), 7.554 (d, J=6.6Hz, 2H,
Ar-H2’, Ar-H6’), 7.386-7.344 (m, 3H, Ar-H3’, Ar-H4’, Ar-H5’), 6.9005 (d, J=2.4Hz, 2H,
Ar-H2×2), 6.868 (d, J=8.4Hz, 2H, Ar-H6×2), 6.816 (dd, J=1.8Hz, J=1.8Hz, 2H, Ar-H5×2),
4.512 (s, 6H, CH2×3). LC-MS m/z: 430.17(M+H)+, calcd for C26H23NO5: 429.16.
4.1.13
(3E,5E)-1-cyclopropyl-3,5-bis(3,4-dihydroxybenzylidene)piperidin-4-one
(3m):
Orange powder, 50.8% yield, mp 247.0-249.3°C. 1H-NMR (600MHz, d-DMSO), δ: 9.884
(s, 2H, 3-OH×2), 9.456 (s, 2H, 4-OH×2), 7.716 (s, 2H, Ar-CH=C×2), 6.967 (s, 2H,
Ar-H2’×2), 6.921-6.891 (m, 4H, Ar-H5’×2, Ar-H6’×2), 4.605 (s, 4H, CH2-N-CH2), 1.190 (t,
J=15.6Hz, 1H, CH), 0.483 (d, J=3.0Hz, 4H, CH2×2). LC-MS m/z: 380.16(M+H)+, calcd
for C22H21NO5: 379.14.
4.2. General procedure for synthesis of NDGA analogues 3a-3m
A mixture of 3,4-dihydroxybenzaldehyde (2, 10 mmol) and appropriate ketone (5 mmol
1a: cyclopentanone, 1b: cyclohexanone, 1c: acetone, 1d: 1-propylpiperidin-4-one, 1e:
1-methylpiperidin-4-one,
1g:
dihydro-2H-pyran-4(3H)-one,
1h:
dihydro
-2H-thiopyran-4(3H)-one, 1i: 1-phenethylpiperidin-4-one, 1j: benzyl 4-oxopiperidine
-1-carboxylate, 1k: ethyl 4-oxopiperidine-1-carboxylate, 1l: 1-benzylpiperidin-4-one, 1m:
1-cyclopropylpiperidin-4-one)was stirred at room temperature. HCl (gas) was bubbled
into the solution to catalyze the reaction. Completion of the reaction was monitored by
thin layer chromatography. 4-Piperidone hydrochloride hydrate (1f) and
3,4-dihydroxybenzaldehyde were dissolved in the mixture solvent of ethanol and water
(10:1) different with others. The crude mixture was cooled and poured into cold water (20
mL) to precipitate the product. The filter residue was purified by silica gel
chromatography using hexane and ethyl acetate gradient to obtain desired products 3a-3m.
4.3 Biological evaluation
4.3.1. DPPH radical scavenging assay

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DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of NDGA analogues
was measured as described by Ikram Dib et al[45] with slight modifications. The diluted
NDGA analogues solution (20 mg/mL) were prepared in ethanol. 0.15 mM DPPH•
solution was prepared in ethanol. A volume of 120 µL of DPPH• solution (0.15 mM) was
added to 80 µL NDGA analogues solution(Ai). The control was 80 µL NDGA analogues
solutions diluted in 120 µL ethanol solution(Aj). A blank preparation was obtained by
adding 120 µL of DPPH• solution to 80 µL of ethanol solution(Ac). These solution
mixtures were incubated for 30 min at 25 °C, then after, absorbance was measured at 517
nm. Measurements were carried out in triplicate for each experiment. Antioxidant activity
was calculated using the equation: % scavenging=[1-(Ai -Aj)/Ac] ×100.
4.3.2. Cell Culture
PC12 cells, a rat pheochromocytoma cell line, were provided by the Cell Storage Center
of Wuhan University (Wuhan, China). Cells were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) with 10 % fetal bovine serum(FBS), (containing 100 U/mL penicillin
and 100 U/ mL streptomycin), and incubated at 37 °C with 5 % CO2.
4.3.3. MTT assay
MTT assay was utilized to measure PC12 cells cytotoxicity and viability. PC12 cells
(5×103 cells/well) were plated in 96-well plates and allowed to attach overnight. After
appropriate treatment, The cells were treated with MTT solution (5 mg/mL) for 4 h at
37 °C. The formazan crystals were dissolved in 120 µL DMSO and the optical density
(OD) was measured using a Microplate Reader at 490 nm. The cell viability was given in
a percentage of the OD value of the control cultures. In vitro cell cytotoxicity assay, the
cell viability was determined after treated with NDGA analogues and NDGA for 24 h or
72 h. In vitro antioxidant activities, the cells were pre-treated with NDGA analouges and
NDGA for 1 h or 24 h, H2O2 was added for an additional 24 h.
4.3.4. Intracellular ROS analysis
PC12 cells were seeded in 6-well plates at a density of 3×105/mL and allowed to attach
overnight. After 1 h or 24 h of pre-incubation with compounds, H2O2 was added for 3 h.
Then, cells were incubated with DCFH-DA (10 mM) at 37 °C for 30 min. Then cells were
digested with enzyme and rinsed with PBS, and re-suspended in 500 mL PBS. After
filtration, the suspension was analyzed by Flow Cytometry .
4.3.5. Measurement of MDA

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PC12 cells were incubated with 3a and NDGA for 1 h or 24 h. Then another 16 h exposure
in H2O2. The supernatants were collected through centrifuge at 1600×g for 10 min at 4 °C.
The supernatants were removed to measure the levels of MDA according to the
manufacturer’s instructions.
4.3.6. Immunostaining of Nrf2
Cells were cultured in a 6-well plates with clean-glass cover slips. After treatment, the
cells were fixed by 4% paraformaldehyde for 20 min at 4 °C. The cells were then washed
three times with PBS and incubated in 1% Triton X-100 for 15 min and 1% BSA for 1 h at
room temperature. Cells were then incubated overnight at 4 °C on addition of anti-Nrf2
primary antibody solution (1:300 in 3% BSA). After rewashed in PBS, the cells were
allowed to react with PE-labeled secondary antibody (1:300 in 3% BSA) for 1h in a dark
room and counterstained 4,6-diamidino-2-phenylindole dihydrochloride(DAPI) for 5 min.
Images were captured under the fluorescence microscope.
4.3.7. Western Blot Analysis
PC12 cells treated with 3a, NDGA and TBHQ for 24 h and lysed with ice-cold RIPA lysis
buffer. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamidegel
(SDS-PAGE) and then transferred onto a Polyvinylidene Fluoride (PVDF) membrane
blocking with 5% skim milk. The blots were incubated with antibodies against HO-1
(1:300), GADPH (1:1000) overnight at 4 °C and were washed three times with 1×TBST.
Then, the blots were incubated for 1 h at room temperature with a 1:1000 dilution of
horseradish peroxidase-labeled anti-rabbit IgG and washed three times with 1×TBST.
Target proteins were detected by Image J software.
4.3.8. Focal Cerebral Ischemia
Animals:Male Sprague-Dawley rats (250-280g) were obtained from the Shanghai Slaccas
Lab Animal Co., Ltd. The 35 adult rats were randomly divided into five groups (n = 7): (A)
sham control; (B) normal saline (NS) group; (C) vehicle (polymer:DMSO:distilled water
at 50mg:1mL:3mL)+MCAO; (D) 0.2 mg/kg 3a+MCAO; (E) 0.2 mg/kg edaravone
+MCAO. Rats were administered by intracerebroventricular injection with the
isovolumetric normal saline, vehicle, 3a nanoparticles and edaravone nanoparticles 2 h
before MCAO. All animal experiments and care were performed according to the Guide
for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC,
1996).

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Preparation of 3a/edaravone-nanoparticles: the 3a/edaravone-loaded nanoparticles were
prepared as described previously[46]. The concentration of the 3a(3a nanoparticles) and
edaravone (edaravone nanoparticles) were 0.2 mg/kg.
MCAO:Male Sprague-Dawley rats (250-280g) were used male as the object of middlecere
bral artery occlusion (MCAO)method. Firstly, animals were anesthetized with 10% chloral
hydrate (0.35mL/100kg; intraperitoneally). After disinfection with 75% alcohol, making a
appropriate incision on the neck. Then, the right common carotid artery (CCA), external
carotid artery (ECA), and internal carotid artery (ICA) were exposed and separated. ICA
and CCA were clamped with artery clips temporarily. Cut an incision on ECA, insert
thread, until we feel resistance to stop (about 1.8 cm). The thread fixed and the incision
stitched. After 2 h of MCAO, the rats achieved reperfusion for 72 h.
4.3.9. Neurological deficit score and TTC staining.
Neurological score was determined 72 h after MCAO using Longa’s method[47]. Briefly,
the test as follows: 0: no deficit; 1: forelimb weakness and torso turning to the ipsilateral
side when held by the tail; 2: circling to the affected side when held by the tail on the
bench; 3: unable to bear weight on the affected side or spontaneous circling to the affected
side; 4: no spontaneous locomotor activity or barrel rolling. For TTC staining, the brains
were quickly removed from rats and was cut into slices. Five the slices were bathed in the
TTC solution at 37 °C for 30 minutes. The TTC-stained brain slices were photographed
with a digital camera. The infarct area was calculated by Image-Pro plus.
4.3.10. Statistical analysis
Data were expressed as mean ± standard deviation of three independent experiments.
Statistical comparisons of the results were made using analysis of variance (ANOVA).
Significant differences were established at P < 0.05.
Acknowledgement
Thank you for the nano-dosage form of the compound produced by Professor Yingzheng
Zhao of wenzhou medical university. This work was supported by the National Natural
Science Foundation of China (Grant Nos. 81402839 and 81272462), the ZheJiang
Province Natural Science Fund of China (Grant Nos. LY17H160059). The Opening
Project of Zhejiang Provincial Top Key Discipline of Pharmaceutical Sciences.

ACCEPTED MANUSCRIPT
References
[1] E.H. Lo, T. Dalkara, M.A. Moskowitz, Mechanisms, challenges and opportunities in stroke, Nature
reviews. Neuroscience, 4 (2003) 399-415.
[2] G.A. Donnan, M. Fisher, M. Macleod, S.M. Davis, Stroke, Lancet, 371 (2008) 1612-1623.
[3] G. Tsivgoulis, A.V. Alexandrov, Timeframe for thrombolysis in acute ischaemic stroke, Lancet, 372
(2008) 1275-1276.
[4] C.A. Molina, Reperfusion therapies for acute ischemic stroke: current pharmacological and
mechanical approaches, Stroke, 42 (2011) S16-19.
[5] C.A. Molina, J. Alvarez-Sabin, Recanalization and reperfusion therapies for acute ischemic stroke,
Cerebrovascular diseases, 27 Suppl 1 (2009) 162-167.
[6] G.J. del Zoppo, B.R. Copeland, K. Anderchek, W. Hacke, J.A. Koziol, Hemorrhagic transformation
following tissue plasminogen activator in experimental cerebral infarction, Stroke, 21 (1990)
596-601.
[7] E. Candelario-Jalil, Injury and repair mechanisms in ischemic stroke: considerations for the
development of novel neurotherapeutics, Current opinion in investigational drugs, 10 (2009)
644-654.
[8] J. Wu, J. Ling, X. Wang, T. Li, J. Liu, Y. Lai, H. Ji, S. Peng, J. Tian, Y. Zhang, Discovery of a
potential anti-ischemic stroke agent: 3-pentylbenzo[c]thiophen-1(3H)-one, Journal of medicinal
chemistry, 55 (2012) 7173-7181.
[9] A. Perez-Gonzalez, A. Galano, OH radical scavenging activity of Edaravone: mechanism and
kinetics, The journal of physical chemistry. B, 115 (2011) 1306-1314.
[10] N.A. Kelsey, H.M. Wilkins, D.A. Linseman, Nutraceutical antioxidants as novel neuroprotective
agents, Molecules, 15 (2010) 7792-7814.
[11] R. Zhang, M. Xu, Y. Wang, F. Xie, G. Zhang, X. Qin, Nrf2-a Promising Therapeutic Target for
Defensing Against Oxidative Stress in Stroke, Molecular neurobiology, (2016) 1-12.
[12] K.L. Cheung, S. Yu, Z. Pan, J. Ma, T.Y. Wu, A.N. Kong, tBHQ-induced HO-1 expression is
mediated by calcium through regulation of Nrf2 binding to enhancer and polymerase II to
promoter region of HO-1, Chemical research in toxicology, 24 (2011) 670-676.
[13] J. Yao, B. Zhang, C. Ge, S. Peng, J. Fang, Xanthohumol, a polyphenol chalcone present in hops,
activating Nrf2 enzymes to confer protection against oxidative damage in PC12 cells, Journal of
agricultural and food chemistry, 63 (2015) 1521-1531.
[14] D.G.I. Kingston, Modern natural products drug discovery and its relevance to biodiversity
conservation, Journal of natural products, 74 (2011) 496-511.
[15] B.B. Mishra, V.K. Tiwari, Natural products: an evolving role in future drug discovery, European
journal of medicinal chemistry, 46 (2011) 4769-4807.
[16] Q. Chen, Nordihydroguaiaretic acid analogues: their chemical synthesis and biological activities,
Current topics in medicinal chemistry, 9 (2009) 1636-1659.
[17] S. Arteaga, A. Andrade-Cetto, R. Cardenas, Larrea tridentata (Creosote bush), an abundant plant of
Mexican and US-American deserts and its metabolite nordihydroguaiaretic acid, Journal of
ethnopharmacology, 98 (2005) 231-239.
[18] J. Gnabre, R. Bates, R.C. Huang, Creosote bush lignans for human disease treatment and
prevention: Perspectives on combination therapy, Journal of traditional and complementary
medicine, 5 (2015) 119-126.

ACCEPTED MANUSCRIPT
[19] J.M. Lu, J. Nurko, S.M. Weakley, J. Jiang, P. Kougias, P.H. Lin, Q. Yao, C. Chen, Molecular
mechanisms and clinical applications of nordihydroguaiaretic acid (NDGA) and its derivatives: an
update, Medical science monitor : international medical journal of experimental and clinical
research, 16 (2010) RA93-100.
[20] I. Asiamah, H.L. Hodgson, K. Maloney, K.J. Allen, E.S. Krol, Ring substitution influences
oxidative cyclisation and reactive metabolite formation of nordihydroguaiaretic acid analogues,
Bioorganic & medicinal chemistry, 23 (2015) 7007-7014.
[21] J.M. Lu, J. Nurko, S.M. Weakley, J. Jiang, P. Kougias, P.H. Lin, Q. Yao, C. Chen, Molecular
mechanisms and clinical applications of nordihydroguaiaretic acid (NDGA) and its derivatives: an
update, Medical science monitor : international medical journal of experimental and clinical
research, 16 (2010) RA93-100.
[22] E. Floriano-Sanchez, C. Villanueva, O.N. Medina-Campos, D. Rocha, D.J. Sanchez-Gonzalez, N.
Cardenas-Rodriguez, J. Pedraza-Chaverri, Nordihydroguaiaretic acid is a potent in vitro scavenger
of peroxynitrite, singlet oxygen, hydroxyl radical, superoxide anion and hypochlorous acid and
prevents in vivo ozone-induced tyrosine nitration in lungs, Free radical research, 40 (2006)
523-533.
[23] S. Guzman-Beltran, S. Espada, M. Orozco-Ibarra, J. Pedraza-Chaverri, A. Cuadrado,
Nordihydroguaiaretic acid activates the antioxidant pathway Nrf2/HO-1 and protects cerebellar
granule neurons against oxidative stress, Neuroscience letters, 447 (2008) 167-171.
[24] S. Magesh, Y. Chen, L. Hu, Small molecule modulators of Keap1-Nrf2-ARE pathway as potential
preventive and therapeutic agents, Medicinal research reviews, 32 (2012) 687-726.
[25] K.A. Jung, M.K. Kwak, The Nrf2 system as a potential target for the development of indirect
antioxidants, Molecules, 15 (2010) 7266-7291.
[26] P.C. Trippier, K. Jansen Labby, D.D. Hawker, J.J. Mataka, R.B. Silverman, Target- and
mechanism-based therapeutics for neurodegenerative diseases: strength in numbers, Journal of
medicinal chemistry, 56 (2013) 3121-3147.
[27] Z.Z. Jiao, S. Yue, H.X. Sun, T.Y. Jin, H.N. Wang, R.X. Zhu, L. Xiang, Indoline Amide Glucosides
from Portulaca oleracea: Isolation, Structure, and DPPH Radical Scavenging Activity, Journal of
natural products, 78 (2015) 2588-2597.
[28] A. Kicel, M. Wolbis, Phenolic content and DPPH radical scavenging activity of the flowers and
leaves of Trifolium repens, Natural product communications, 8 (2013) 99-102.
[29] E. Nagababu, J.G. Mohanty, J.S. Friedman, J.M. Rifkind, Role of peroxiredoxin-2 in protecting
RBCs from hydrogen peroxide-induced oxidative stress, Free radical research, 47 (2013) 164-171.
[30] S. Peng, B. Zhang, X. Meng, J. Yao, J. Fang, Synthesis of piperlongumine analogues and
discovery of nuclear factor erythroid 2-related factor 2 (Nrf2) activators as potential
neuroprotective agents, Journal of medicinal chemistry, 58 (2015) 5242-5255.
[31] O.J. Olatunji, H. Chen, Y. Zhou, Neuroprotective effect of trans-N-caffeoyltyramine from Lycium
chinense against H2O2 induced cytotoxicity in PC12 cells by attenuating oxidative stress,
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 93 (2017) 895-902.
[32] S. Peng, Y. Hou, J. Yao, J. Fang, Activation of Nrf2-driven antioxidant enzymes by cardamonin
confers neuroprotection of PC12 cells against oxidative damage, Food & function, 8 (2017)
997-1007.
[33] G.S. Cao, S.X. Li, Y. Wang, Y.Q. Xu, Y.N. Lv, J.P. Kou, B.Y. Yu, A combination of four effective
components derived from Sheng-mai san attenuates hydrogen peroxide-induced injury in PC12

ACCEPTED MANUSCRIPT
cells through inhibiting Akt and MAPK signaling pathways, Chinese journal of natural medicines,
14 (2016) 508-517.
[34] J. Chen, L. Zeng, T. Xia, S. Li, T. Yan, S. Wu, G. Qiu, Z. Liu, Toward a biomarker of oxidative
stress: a fluorescent probe for exogenous and endogenous malondialdehyde in living cells,
Analytical chemistry, 87 (2015) 8052-8056.
[35] M. Aleman, R. Bou, A. Tres, J. Polo, R. Codony, F. Guardiola, Oxidative stability of a heme
iron-fortified bakery product: Effectiveness of ascorbyl palmitate and co-spray-drying of heme
iron with calcium caseinate, Food chemistry, 196 (2016) 567-576.
[36] K.A. Jung, M.K. Kwak, The Nrf2 system as a potential target for the development of indirect
antioxidants, Molecules, 15 (2010) 7266-7291.
[37] M.C. Lu, J.A. Ji, Z.Y. Jiang, Q.D. You, The Keap1-Nrf2-ARE Pathway As a Potential Preventive
and Therapeutic Target: An Update, Medicinal research reviews, 36 (2016) 924-963.
[38] J. Wu, J. Ren, S. Yao, J. Wang, L. Huang, P. Zhou, D. Yun, Q. Xu, S. Wu, Z. Wang, P. Qiu, Novel
antioxidants' synthesis and their anti-oxidative activity through activating Nrf2 signaling pathway,
Bioorganic & medicinal chemistry letters, 27 (2017) 1616-1619.
[39] J. Wu, J. Ling, X. Wang, T. Li, J. Liu, Y. Lai, H. Ji, S. Peng, J. Tian, Y. Zhang, Discovery of a
potential anti-ischemic stroke agent: 3-pentylbenzo[c]thiophen-1(3H)-one, Journal of medicinal
chemistry, 55 (2012) 7173-7181.
[40] L. Liang, C. Gao, M. Luo, W. Wang, C. Zhao, Y. Zu, T. Efferth, Y. Fu, Dihydroquercetin (DHQ)
induced HO-1 and NQO1 expression against oxidative stress through the Nrf2-dependent
antioxidant pathway, Journal of agricultural and food chemistry, 61 (2013) 2755-2761.
[41] H.C. Bertrand, M. Schaap, L. Baird, N.D. Georgakopoulos, A. Fowkes, C. Thiollier, H. Kachi, A.T.
Dinkova-Kostova, G. Wells, Design, Synthesis, and Evaluation of Triazole Derivatives That
Induce Nrf2 Dependent Gene Products and Inhibit the Keap1-Nrf2 Protein-Protein Interaction,
Journal of medicinal chemistry, 58 (2015) 7186-7194.
[42] M. Agrawal, V. Kumar, A.K. Singh, M.P. Kashyap, V.K. Khanna, M.A. Siddiqui, A.B. Pant,
trans-Resveratrol protects ischemic PC12 Cells by inhibiting the hypoxia associated transcription
factors and increasing the levels of antioxidant defense enzymes, ACS chemical neuroscience, 4
(2013) 285-294.
[43] M. Artico, R. Di Santo, R. Costi, E. Novellino, G. Greco, S. Massa, E. Tramontano, M.E.
Marongiu, A. De Montis, P. La Colla, Geometrically and conformationally restrained cinnamoyl
compounds as inhibitors of HIV-1 integrase: synthesis, biological evaluation, and molecular
modeling, Journal of medicinal chemistry, 41 (1998) 3948-3960.
[44] R. Costi, R.D. Santo, M. Artico, S. Massa, R. Ragno, R. Loddo, M. La Colla, E. Tramontano, P. La
Colla, A. Pani, 2,6-Bis(3,4,5-trihydroxybenzylydene) derivatives of cyclohexanone: novel potent
HIV-1 integrase inhibitors that prevent HIV-1 multiplication in cell-based assays, Bioorganic &
medicinal chemistry, 12 (2004) 199-215.
[45] I. Dib, M.L. Fauconnier, M. Sindic, F. Belmekki, A. Assaidi, M. Berrabah, H. Mekhfi, M. Aziz, A.
Legssyer, M. Bnouham, A. Ziyyat, Chemical composition, vasorelaxant, antioxidant and
antiplatelet effects of essential oil of Artemisia campestris L. from Oriental Morocco, BMC
complementary and alternative medicine, 17 (2017) 82.
[46] H.L. Xu, K.L. Mao, C.T. Lu, Z.L. Fan, J.J. Yang, J. Xu, P.P. Chen, D.L. ZhuGe, B.X. Shen, B.H.
Jin, J. Xiao, Y.Z. Zhao, An injectable acellular matrix scaffold with absorbable permeable

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nanoparticles improves the therapeutic effects of docetaxel on glioblastoma, Biomaterials, 107
(2016) 44-60.
[47] X. Li, K.K. Blizzard, Z. Zeng, A.C. DeVries, P.D. Hurn, L.D. McCullough, Chronic behavioral
testing after focal ischemia in the mouse: functional recovery and the effects of gender,
Experimental neurology, 187 (2004) 94-104.

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Highlights:
1. A series of NDGA derivatives containing α,β-unsaturated ketone
scaffold were designed and synthesized base combination principles.
2. Compound 3a could confer protection of PC12 cells against H2O2
insult by directly scavenging ROS and indirectly through
keap1/Nrf2/ARE pathway activation.
3. The effect of 3a was more pronounced than that of edaravone after
MCAO in rats.