Novel antioxidants’ synthesis and their anti-oxidative activity through activating Nrf2 signaling pathway

Article abstract of DOI:10.1016/j.bmcl.2017.02.006

Novel structure compounds (WS) containing 3,4,5-trimethoxyphenyl and acyl pyrazole were designed and synthesized based combination principles. Among them, WS13 was screened out to possess desirable anti-oxidative activity in vitro. Cell survival assay and apoptosis experiment in H₂O₂ induced PC12 cells injury model all showed that its cytoprotection exhibited a concentration-effect manner. WS13 at 10?μM could remove ROS with equal effiency to edaravone. Further, it clearly activated Nrf2 nuclear translocation and upregulated GCLC mRNA transcription and protein expression in dose-dependent manner, and its cytoprotection was reversed by GCLC protein inhibitor. In total, WS13 with further promotion can serve as Nrf2-GCLC activator in anti-oxidative therapy.

Full text of DOI:10.1016/j.bmcl.2017.02.006

Accepted Manuscript
Novel antioxidants’ synthesis and their anti-oxidative activity through activating
Nrf2 signaling pathway
Jianzhang Wu, Jiye Ren, Song Yao, Jiabing Wang, Lili Huang, Peng Zhou, Di
Yun, Qing Xu, Shoubiao Wu, Zhankun Wang, Peihong Qiu
PII:
S0960-894X(17)30124-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.006
BMCL 24678
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 September 2016
31 January 2017
2 February 2017
Please cite this article as: Wu, J., Ren, J., Yao, S., Wang, J., Huang, L., Zhou, P., Yun, D., Xu, Q., Wu, S., Wang,
Z., Qiu, P., Novel antioxidants’ synthesis and their anti-oxidative activity through activating Nrf2 signaling pathway,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.006
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Novel antioxidants’ synthesis and their anti-oxidative activity
through activating Nrf2 signaling pathway
Jianzhang Wua1, Jiye Rena1, Song Yaoa1, Jiabing Wanga, Lili Huanga, Peng Zhoub, Di
Yuna, Qing Xuc, Shoubiao Wua, Zhankun Wangd,a, Peihong Qiua*
a Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou
Medical University, Wenzhou, Zhejiang, 325035, China
b Department of Clinical Pharmacy，Taizhou Hospital，Zhejiang，Linhai，317000,
China
c College of Chemistry and Materials Engineering, Wenzhou University , Wenzhou,
Zhejiang 325035, China
d Institute of Sports Science, Wenzhou Medical University, Wenzhou, Zhejiang, 325035,
China
(1 These authors contribute to this work equally)
Abstract:
Novel structure compounds (WS) containing 3,4,5-trimethoxyphenyl and acyl
pyrazole were designed and synthesized based combination principles. Among them,
WS13 was screened out to possess desirable anti-oxidative activity in vitro. Cell
survival assay and apoptosis experiment in H2O2 induced PC12 cells injury model all
showed that its cytoprotection exhibited a concentration-effect manner. WS13 at 10
µM could remove ROS with equal effiency to edaravone. Further, it clearly activated
Nrf2 nuclear translocation and upregulated GCLC mRNA transcription and protein
expression in dose-dependent manner, and its cytoprotection was reversed by GCLC
protein inhibitor. In total, WS13 with further promotion can serve as Nrf2-GCLC
activator in anti-oxidative therapy.
Key words: Antioxidant; Nrf2; GCLC; PC12
Acute or chronic accumulation of oxidant in cells from external and internal is
extremely harmful to human health.1 Through damaging cell lipids, protein and
nucleic acids, oxidative stress is closely associated with numerous disease such like
cancer, Alzheimer and chronic heart failure.2-4 Human bodies are equipped with multi
layers of anti-oxidative mechanisms, which can clean up internal oxidants in
physiological condition. Once internal oxidants were over-loaded or attacked by
external stimulation, cells resulted in oxidative stresses or inflammatory reactions.5-8
At molecule level, the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant
response element (ARE) pathway has drawn much attentions in recent years because
it plays important roles in the oxidative stress related physiological processes.9-12 And

the pathway is generally considered to be composed of Keap1, cis-antioxidant
response element, and downstream phase II detoxifying enzymes and anti-oxidant
enzymes coding genes. Glutamate-cysteine ligase catalytic subunit (GCLC), part of
glutamate cysteine ligase (GCL) which limiting the rate of synthesizing glutathione
(GSH) and regulated by Nrf2/ARE signaling pathway, is one of the most general
indices used to indicate the activation of Nrf2-ARE pathway. The pathway activation
usually provides cells with enhanced capacity in eliminating reactive oxygen species
(ROS) and in coping with oxidative stress. But up to now, few anti-oxidative targeted
agents have been approved for clinic therapy. So many researchers focus on
developing novel Nrf2-ARE signaling modulators.
Figure 1. Synthetic routes and compounds’ structures. Synthetic conditions: (I) sol. cyclohexane; cat. TsOH; temp.
90 oC; reflux 5-6 h. (II) sol. alcohol; temp. 78 oC; reflux 0.5-1.5 h. (III) reagent HCl (aq); temp. room temperature;
stir to pH ~2. (IV) sol. alcohol; temp. 78 oC; reflux 2 h. (V) sol. THF; reagent Et3N; temp. 0 oC; stir 2 h.

Evidences show that 3,4,5-trimethoxy benzyl may be a novel Nrf2-ARE
activator.13-17 For instance, Jianguo Fang reported that piperlongumine analogues
which containing 3,4,5-trimethoxy benzyl can serve as Nrf2 activator.13 Further,
Roberto Motterlini found that there were structure-activity relationships between
methoxyl chalcones and its abilities inducing heme oxygenase-1 (HO-1) protein.14
When its aromatic rings’ hydrogens were replaced by 3,4,5-trimethoxy, the inducing
activities peaked. Azhar Ariffin also analyzed the correlation of new hydrazone
compounds bearing a 3,4,5-trimethoxybenzyl moiety and their antioxidant activities.15
And there are other literatures reported some novel natural products containing
3,4,5-trimethoxybenzyl having anti-oxidant property.16,17 So it’s believed that
3,4,5-trimethoxybenzyl is beneficial for activating Nrf2-ARE signaling pathway. Acyl
pyrazoles were rarely investigated as synthetic modules, whereas some compounds
showed anticancer, antioxidant or anti-inflammatory activities.18-21 Further, acyl
pyrazoles were kind of similar to edaravone which can eliminate free radicals and
intracellular ROS.22 Therefore, based on combination principles, a series of novel
compounds containing 3,4,5-trimethoxyphenyl and acyl pyrazole moieties were
designed and synthesized. Then their cytoprotection in H2O2 induced PC12 cells
injury model was tested, as well their abilities to clear ROS and activate the
Nrf2-ARE signaling pathway. The synthetic routes and all compounds’ structures
were displayed in Figure 1.
Figure 2. Compounds’ cytoprotection on PC12 cells in H2O2 damage model. PC12 cells were pretreated for 24 h
with WS compounds (10 µM), tBHQ (10 µM), or ED (Edaravone) (10 µM), then another 24h exposure in H2O2
(440 µM), finally added MTT and measured the OD value in 490nm. Values are means SEM (n = 3 or 4). *p <
0.01 vs H2O2, one way ANOVA, followed by Tukey's multiple comparison test.

Figure 3. WS13 resisted the H2O2 induced damage in PC12 cells. (A): WS13 raised PC12 cell viability
dose-dependently in H2O2 induced cell injury model. PC12 cells were pre-treated with WS13 in different doses
(2.5, 5, 10 µM) for 24 h and then treated with H2O2 for 24 h. The cell viability was measured by MTT assay.
Values are means SEM (n = 3 or 4). **P < 0.01, ***P < 0.001 vs H2O2, one way ANOVA, followed by Tukey's
multiple comparison test. (B): WS13 pre-incubation decreased ROS level in H2O2 treated PC12 cells. PC12 cells
were pre-treated with 10 µM of WS13, ED (Edaravone), or tBHQ; then treated with H2O2 for 5 h, and the ROS
level was measured by flow cytometry. (C): WS13 protected PC12 cells from apoptosis in H2O2 induced cell
damage model. PC12 cells were pre-treated with tBHQ (10 µM), Edaravone (10 µM) or WS13 (2.5, 5, 10 µM) for
24 h, and then followed by another 24 h exposed in H2O2 (440 µM). The apoptosis was detected with flow
cytometry. (C-a) DMSO; (C-b) H2O2; (C-c) tBHQ; (C-d) Edaravone; (C-e-g) WS13 at 2.5 µM, 5 µM and 10 µM.
Pre-incubation with these compounds for 24 hours, only WS13 presented a
valuable protection against oxidative damage in PC12 cells induced by H2O2 (Figure
2). Cell viability in WS13 treatment group was significantly higher than that in model
group. Then, the concentration-effect relationship of WS13 was also investigated as
showed in Figure 3A. When WS13 was applied at 2.5, 5 and 10 µM level, a gradually
improved protection against oxidative damage in PC12 cells induced by H2O2 was
observed. Annexin V-FITC/PI double staining test with flow cytometry (Figure 3C)
also showed that WS13 performed protective effect at a dose of 2.5, 5 and 10 µM in
dose-dependent manner. Further, WS13 at 10 µM significantly eliminated
endocellular ROS level with the same effiency of Edaravone (Figure 3B).
Hypothetically, compounds WS13 may execute its cytoprotection through
scavenging free radicals directly and activating the anti-oxidative signaling pathway
at the same time. But DPPH free radical scavenging experiment indicated WS13
showed about half fold lower effect than edaravone or tBHQ (Figure S1), which
mean that WS13 may execute the anti-oxidant activities partly through clearing free
radical directly but mainly through activating anti-oxidative signaling pathway.
Since WS13 didn’t mainly work directly on free radical, its ability to activate the
anti-oxidative signaling pathway was investigated. As showed in Figure 4,
immunofluorescence experiment revealed that Nrf2 nuclear translocation was
activated by WS13 at a dose of 10 µM.

Figure 4. WS13 activated Nrf2 nuclear-translocation. PC12 cells were incubated with tBHQ, ED or WS13 at dose
of 10 µM for 6 h, and then stained with Nrf2 antibody and DAPI.
RT-PCR and western-blotting experiments all indicated that GCLC protein coding
genes in the Nrf2-ARE signaling pathway was activated. As exhibited in Figure 5A,
GCLC mRNA level was up-regulated by WS13 at 2.5, 5 and 10 µM level. Further,
western-blotting experiments also revealed that WS13 up-regulated GCLC protein
expression in PC12 cells and followed a concentration-dependent manner (Figure
5B).
Figure 5. WS13 increased the expression of GCLC. (A): WS13 induced the mRNA expression of GCLC. PC12
cells were incubated with WS13 in different doses for 24 h and then evaluated the mRNA level of GCLC by
RT-PCR experiment. Values are means SEM (n = 3 or 4). *p < 0.05 vs control, one way ANOVA, followed by
Tukey's multiple comparison test. (B): WS13 induced the expression of GCLC. PC12 cells were incubated with
WS13 in 2.5, 5, 10 µM and tBHQ in 10 µM for 24 h, and the GCLC was determined by Western-blotting
experiment and representative result was present. Values are means SEM (n = 3 or 4). *p < 0.1, **p < 0.01 vs
DMSO, one way ANOVA, followed by Tukey's multiple comparison test. (C): BSO diminished the protected effect
of WS13 on H2O2 induced cell damage. PC12 cells were incubated with GCLC inhibitor BSO for 2 h, then treated
with WS13, tBHQ or ED in 10 µM for 24 h. Finally MTT assay measured the OD values in 490 nm. Values are

means SEM (n = 3 or 4). *p < 0.1; #p < 0.1 vs H2O2, one way ANOVA, followed by Tukey's multiple comparison
test.
To make sure if WS13 executes anti-oxidative protection through activating GCLC
protein expression, its specific enzyme inhibitor namely BSO is applied to this study.
As showed in Figure 5C, it’s obvious that BSO had no influence on cell viability in
H2O2 induced model group, while WS13 applied alone raised the cell viability. But,
when BSO and WS13 were applied together, the cytoprotection was constrained. This
result showed that WS13 executed its anti-oxidative activities through, at least partly,
activating GCLC protein expression.
In summary, seventeen novel compounds containing 3,4,5-trimethoxyphenyl and
acyl pyrazole moieties were designed and synthesized, and WS13 was screened out to
exhibit an inspirable protection against H2O2 induced PC12 cell injury in
concentration-dependent manner. Mechanism study showed that through activating
Nrf2 pathway, upregulating GCLC mRNA transcription and protein expression,
WS13 eliminate ROS with equal effiency to edaravone and executed its
cytoprotection. However, including WS13, the activity of these compounds still need
to be promoted and further research of activity in vivo is required.
Acknowledgment
The work was supported by National Natural Science Foundation of China
(81272462), Zhejiang Province Natural Science Funding of China (LY17H160059,
LY13H300005), and the Opening Project of Zhejiang Provincial Top Key Discipline
of Pharmaceutical Sciences.
Supplementary data
Supplementary data (complete structures and characterization information of all the
compounds) associated with this article can be found in the online version.
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Graphical abstract