Design and Green Synthesis of Piperlongumine Analogs and Their Antioxidant Activity against Cerebral Ischemia-Reperfusion Injury

Article abstract of DOI:10.1021/acschemneuro.9b00402

The supplementation of exogenous antioxidants to scavenge excessive reactive oxygen species (ROS) is an effective treatment for cerebral ischemia-reperfusion injury (CIRI) in stroke. Piperlongumine (PL), a natural alkaloid, has a great potential as a neur

Full text of DOI:10.1021/acschemneuro.9b00402

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Article
Design, Green Synthesis of Piperlongumine Analogs and Their
Antioxidant Activity against Cerebral Ischemia-reperfusion Injury
Ge Li, Yuantie Zheng, Jiali Yao, Linya Hu, Qunpeng Liu, Furong Ke, Weixiao Feng, Ya
Zhao, Pengcheng Yan, Wenfei He, Hui Deng, Peihong Qiu, Wulan Li, and Jianzhang Wu
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00402 • Publication Date (Web): 06 Sep 2019
Downloaded from pubs.acs.org on September 6, 2019
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Design, Green Synthesis of Piperlongumine Analogs and Their
Antioxidant Activity against Cerebral Ischemia-Reperfusion Injury
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Ge Lia, Yuantie Zhenga, Jiali Yaoa, Linya Hua, Qunpeng Liuad, Furong Kea, Weixiao Fengab, Ya
Zhaoca, Pencheng Yana, Wenfei Hea, Hui Dengc, Peihong Qiua, Wulan Liba*, Jianzhang Wua*
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(aChemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical
Universtiy, Wenzhou, Zhejiang 325035, China)
(bThe First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325035, China)
(cDepartment of Periodontics, Hospital & School of Stomatology, Wenzhou Medical University,
No. 373 West Xueyuan Road, Wenzhou, Zhejiang 325035, China)
(dCollege of Chemistry and Materials Engineering, Wenzhou University, Wenzhou, Zhejiang
325035, China)
*Correspondence authors.
Address: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou
Medical Universtiy, Wenzhou, Zhejiang 325035, China.
Tel./Tex: +86 577 86689984
E-mail: wjzwzmu@163.com
Address: The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325035,
China.
Tel./Tex: +86 577 86689916
lwlwzmu@163.com
Disclosures/Conflicts of Interest: None declared
Abstract: The supplementation of exogenous antioxidants to scavenge excessive reactive
oxygen species (ROS) is an effective treatment for cerebral ischemia reperfusion injury
(CIRI). Piperlongumine (PL) is a kind of natural product which has a great potential as
neuroprotective agents, but it also has obvious neurotoxicity. Moreover, its neuroprotective
effects remain to be improved. In this study, we designed a series of novel PL analogs by
hybridizing the screened low-toxic diketene skeleton with antioxidant effect and the
3,4,5-trimethoxyphenyl group, which may increase the antioxidant activity of PL. The
intermediate was synthesized by a novel green synthesis method, and 34 compounds were
obtained. All the compounds without obvious cytotoxicity have remarkable antioxidant
effects, especially compared with diketene skeletons and PL. The cytoprotection of the
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active compound decreased significantly by reduction of the carbon-carbon double bonds
of the Michael acceptor in the diketene skeleton. More importantly, further study revealed
that compound A9 which has the best activity can confer protection for cells against
oxidative stress and attenuate brain injury in vivo. Overall, this study provided a promising
drug candidate for the treatment of CIRI and guide the further development of drug
research in oxidative stress-mediated diseases.
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Keywords: Piperlongumine; Michael acceptor; Green synthesis; Antioxidation; Oxidative
stress; Neuroprotection; Cerebral ischemia-reperfusion injury
1. Introduction
Oxidative stress is increasingly considered as primary causes of neurodegenerative
diseases (NDDs) [1-2]. Under pathological conditions, a large number of reactive oxygen
species (ROS) were induced by oxidative stress, and the accumulation of excessive ROS
can create a state of redox imbalance leading to brain damage [3-4]. Ischemic stroke, one of
NDDs, is characterized by the high mortality and disability rate in clinical practice across
the world [5]. At present, reperfusion is a major therapy to reverse brain damage after
ischemic stroke [6-7], but this treatment could cause secondary cellular and tissue damage
that is called cerebral ischemia reperfusion injury (CIRI) [8]. The supplementation of
exogenous antioxidants is a promising therapy for the treatment of CIRI. However, there
are few approved neuroprotective agents were approved in the clinical therapy [9]. Hence, it
is of enormous significance to develop new therapeutic antioxidants with high efficacy and
low toxicity.
As a major type of natural products, alkaloids are a group of main sources for drug
development. Piperlongumine (PL), a naturally occurring alkaloid from Piper longum L.,
possesses extensive pharmacological activities such as anti-cancer, anti-inflammatory,
anti-angiogenic, anti-plate aggregation, and anti-fungal properties [10-12]. Previous studies
have indicated that PL possesses the neuroprotective effect [13-14]. However, its activity and
toxicity of PL still need to be further improved. Another study found that the olefinic bond
in the α, β-unsaturated amide structure of PL analogs is essential for the cytoprotection in
neural cells [15]. Nevertheless, some reports indicate that the α, β-unsaturated ketone
structure is a kind of Michael acceptor, which can result in toxicity [16]. To sum up, the
above results suggest that both the cytoprotection and cytotoxicity of PL may be concerned
with its α, β-unsaturated ketone structure in the molecules of PL. Meanwhile ， some
compounds (shown in Figure 1B) which contain the α, β-unsaturated ketone structure
including two olefinic bonds (namely diketene structure) have excellent protective effects.
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[17-20]. While the structure-activity relationship (SAR) between the activity or toxicity of
these compounds’ skeletons and the types of linking ketones in the diketene skeleton were
not clear yet. In our previous work, it was found that the activity and toxicity vary
significantly from compounds with the same benzene ring substituents but the different
diketene skeletons [21]. It is suggested that the protective activity and toxicity of these
compounds may be strongly associated with the different type of linking ketones in the
diketene skeletons. Thus, we conducted the SAR research on various diketene skeletons.
As shown in Figure 1C, on the one hand, compounds a (the linking ketone is
cyclopentanone) and e (the linking ketone is pyrone) all showed low cytotoxicity compared
to others. On the other hand, only a (2,5-(dibenzylidene)-cyclopentan-1-one) could display
obvious antioxidant effect against H2O2 damage in PC12 cells. Moreover, accumulated
evidences have confirmed that the compounds (shown in Figure 1A) containing a 3,4,5-
trimethoxyphenyl group can be proposed as potential antioxidants [22-24]. Although PL has
this structural unit, there are no studies evaluating the relevance of it with the antioxidant
activity. In order to reduce the cytotoxicity and improve antioxidant activity of PL, a series
of novel PL analogs whose structure are 2-(substituted benzylidene)-5-(3,4,5-trimethoxy
benzylidene) cyclopentan-1-one (Figure 1D) were designed by hybridizing skeleton a and
the 3,4,5-trimethoxyphenyl group. In brief, this research adopted a greener system for
synthesizing intermediates using L-proline as the catalyst to obtain a series of PL analogs,
and evaluated their antioxidant activities against CIRI.
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Figure 1. The design of PL analogs (A) The antioxidant compounds bearing a 3,4,5-trimethoxy benzyl
moiety (B) The protective agents with the α, β-unsaturated ketone structure which including two olefinic
bonds (C) The cytoprotection and cytotoxicity of various diketene skeletons (D) The structure of
asymmetric PL analogs
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2. RESULTS AND DISCUSSION
2.1 Chemistry
At present, the intermediate’s synthesis methods of asymmetric analogues have the
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disadvantages of cumbersome synthesis steps, environmentally unfriendly and low yield
[25-27]. For example, the synthesis of intermediate (E)-2-(benzylidene) cyclopentanone
,
[23]
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(E)-2-(benzylidene) pyrone [25-26] or (E)-2-(benzylidene) cyclohexanone [27] consist of three
steps. ketones are generally protected by morpholine and exposed a single α-H to generate
Claisen-Schmidt reaction with aromatic aldehydes. The imine immediate product of this
reaction is hydrolyzed under acidic conditions to form the (E)-2-(benzylidene) cycloketone
intermediates [23]. Another example is the intermediate (E)-4-phenylbutyl-3-ene-2-ketone,
which is synthesized by Claisen-Schmidt reaction. Since strong bases such as NaOH are
common catalysts in Claisen-Schmidt reaction [28], the equipment can be corroded and a lot
of solid waste were produced. All these above are not in conformity with the concept of
Green Chemistry. Hence, it is necessary to find a simple, green and highly efficient
synthetic method for asymmetric analogs [29]. We then established a reaction condition
taking L-proline as a catalyst for one-step synthesis of intermediate (E)-2-(benzylidene)
cyclopentanone (Scheme 1). Compared with the previously reported methods, this
synthetic method is not only simpler and easier to perform, but also more environment-
friendly. To be more specific, A and B series compounds were synthesized by the reaction
of the intermediate and substituted benzaldehyde. C and D series compounds were
synthesized through hydrogen reduction. All reactions were monitored on the silica gel thin
layer chromatography and all compounds were purified by column chromatography. These
compounds were obtained in good yields after purification, and their structures were
illustrated in Table 1. The products were characterized by analysis and comparison of their
1H-NMR and MS spectroscopic data. These characteristic data including color, yield,
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melting points, MS and H-NMR spectra of compounds were presented in chemistry
synthetic section. Before being used for the biological experiments, all compounds were
purified using re-crystallization or column chromatography.
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Scheme 1. The general route to produce the asymmetric 1,5-diaryl(substituted)penta-1,4-diene-3-one
analogs. Reagents and conditions: (I): a: L-proline, anhydrous dimethylsulfoxide, room temperature; b:
Con. HCl, room temperature; (II): 40% NaOH or HCl gas, room temperature; (III): H2, Pb/C, 12 h ,
room temperature; (IV): H2, Pb/C, 24 h , room temperature.
Table 1. The asymmetric 1,5-diaryl(substituted)penta-1,4-diene-3-one analogs
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Comp.
A1
R1
R2
Comp.
A18
A19
A20
A21
A22
A23
A24
A25
A26
R1
R2
2,5-OCH3
2-Br
3,4,5-OCH3
3,4,5-OCH3
2,4-Cl
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
A2
4-N(CH3)2
A3
3,4,5-OCH3 3-OCH3, 4-OH
4-Cl
A4
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
4-OCH3
2-OH
4-OH
3-OH
3-F
3,4-Cl
A5
3,4-F
A6
3,4,5-OCH3
3,4-OCH3
2,3-Cl
A7
A8
A9
3,4,5-OCH3 2,4,6-OCH3
2-F, 5-OCH3
4
N
A10
3,4,5-OCH3 3-OH, 4-OCH3
A27
3,4,5-OCH3
A11
A12
3,4,5-OCH3
3,4,5-OCH3
2,4-OCH3
2-OCH3
A28
A29
3,4,5-OCH3 3,5-OCH3, 4-OH
3,4,5-OCH3
2,6-Cl
4
N
A13
A14
3,4,5-OCH3
3,4,5-OCH3
A30
A31
3,4,5-OCH3
4-Br
O
4
N
3,4,5-OCH3
3,5-Cl
A15
A16
A17
3,4,5-OCH3
3,4,5-OCH3
3,4,5-OCH3
2,3-OCH3
2-CF3
A32
A33
B9
3,4,5-OCH3
3,4,5-OCH3
/
3-OCH3
3,5-OCH3
3,4,5-OCH3
3,4-OH
2.2 The cytotoxicity screening and protection of compounds in PC12 cells
In general, ideal antioxidant agents have lower toxicity, so the cytotoxicity of
compounds toward the PC12 cells were determined by the MTT assay. As shown in Figure
2A, it is noticeable that most compounds (25 compounds) displayed no prominent toxicity
to PC12 cells in comparison with the control group. The other 11 compounds, while
showing certain toxicity in PC12 cells, were still much less toxic than PL. In addition, A1,
A13, A14, A27 and A28 were showed distinct toxicity toward the cells, but the cell
viability of each treatment group was still more than 60 %. Only A2 and A10 were two
most toxic compounds, but results in a considerably higher survival rate than the PL-
treatment group.
Hydrogen peroxide (H2O2) is an uncharged redox sensing and redox signaling
molecule, and can generate highly reactive free radicals immediately, which is able to
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cause deleterious effects to lipid, proteins and nucleic acid, and ultimately lead to many
pathophysiological conditions [30]. Therefore, the cytoprotection against the H2O2-induced
oxidative damage in PC12 cells was evaluated for the PL analogs. As shown in Figure 2B,
the survival rate of PC12 cells treated with H2O2 was about half of the control group
(DMSO). However, when the cells were pretreated by the tested compounds for 24 h prior
to H2O2 insult, 24 compounds (10 μM) improved the survival rate to 70-80%, of which
about half provided more protection than compound a. Among these compounds, A3, A5,
A7, A9, A11, A18, A21 and A23 showed the same efficient antioxidant effect as TBHQ
(tert-butylhydroquinone, a Nrf2 activator). In contrast, compounds A2, A10, A13, A14,
A27 and A28 could not significantly protect PC12 cells from H2O2-induced cell injury,
which might relate to their toxic effects. PL showed no protective effect in this assay.
Besides, compounds A15, A16 and A17 also could not greatly reduce the cell injury
because of the low cytotoxicity. Taken together, as long as the analogs are not toxic or less
toxic, they can display cytoprotection activity.
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Figure 2. The cytotoxicity screening and cytoprotection of compounds in PC12 cells. (A) Cells were
plated in 96-well plates for 24 h and subsequently treated with 10 μM compounds for 24 h. The cell
viability was determined by the MTT assay. (B) PC12 cells were pretreated with the compounds for 24 h
(10 μM), and then exposed to H2O2 (450 μM) for another 24 h. The effect of compounds on cell viability
measured by MTT assay and the viability of untreated cells is defined as 100%. Data are expressed as
the mean ± SD, n = 3. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 vs DMSO, ####p < 0.0001,
###p < 0.001, ##p < 0.01, #p < 0.05 vs H2O2.
To get insight in the SAR and to evaluate the effects of various substituents on the
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bioactivity, the SAR of 33 analogs was further analyzed. Interesting, results showed that
analogs [A4 (4-methoxy), A12 (2-methoxy) A11 (2,4-dimethoxy) and A9 (2,4,6-
trimethoxy)] with increasing number of methoxy substitutions on phenyl ring showed
enhanced cytoprotection. Additionally, the similar phenomenon occurred in analogs [A32
(3-methoxy), A24 (3,4-dimethoxy) A33 (3,5-dimethoxy) and A23 (3,4,5-trimethoxy)]. It
was found that except A15 (2,3-dimethoxy) with high level of toxicity cytotoxicity, the
same sort of phenomenon was observed among A12 (2-methoxy), A11 (2,4-dimethoxy)
and A18 (2,5-dimethoxy). Nevertheless, there is opposite phenomenon in the benzene ring
with hydroxy moiety. The analogs with one hydroxyl group [A6 (4-hydroxy) or A7
(3-hydroxy)] exhibited stronger antioxidant activity than that containing two hydroxyl
groups [A17 (3,4-dihydroxy)]. Furthermore, compared with electron withdrawing group at
4-position [compounds A20 (4-Cl) and A30 (4-Br)], analogs with electron withdrawing
group at 2-position [A16 (2-CF3) and A19 (2-Br)] displayed weaker cytoprotection. In
summary, the substitution of methoxy group can reinforce the protective effect, and the
position of electron-withdrawing substitution on a ring could affect the activity,
respectively.
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To explore the importance of 3,4,5-trimethoxy and olefinic double bonds, the most
active compound A9 was selected for structural modification. As shown in Figure 2B, the
3,4,5-tridemethoxy derivative (B9), showed weaker cytoprotective activity compared with
A9. In the meanwhile, compounds 1 and 2, two reduction products of the carbon-carbon
double bonds in A9, also exhibited weaker protection in comparison with A9. These results
further confirmed that the double bonds of Michael acceptor and 3,4,5-trimethoxy
(highlighted in Figure 3) may be essential to strengthen the protective effect.
Figure 3. SAR analysis of PL analogs
2.3 Protective effect of A9 in different cellular injury models
A9 was selected for further studies owing to its the better protection and lower toxicity.
Besides H2O2-induced PC12 cell damage model, the other three cellular models including
glutamate-, glucose- and LPS-induced cellular PC12 cell damage model were employed for
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further evaluating the antioxidant effect of A9 in vitro. The proper concentrations of the
glutamate, glucose or LPS were determined under the guidance of literature material [31-33]
.
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As shown in Figure 4, when the cells were treated with A9 for 24 h prior to different insults,
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the cell viabilities were increased in a dose-dependent manner in each model.
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Figure 4. A9 Protects PC12 Cells in different cellular injury model. Cells were preincubated with
different concentrations of A9 for 24 h before treatment with 450 μM H2O2 (A), 18 mM glutamate (B),
45 μg/ml LPS (D) for 24 h or 140 mM glucose (C) for 48 h. The survival cells were determined by MTT
assay and the viability of untreated cells is defined as 100%. Data are expressed as the mean ± SD, n = 3.
****p < 0.0001, **p < 0.01vs DMSO, ##p < 0.01, #p < 0.05 vs H2O2.
2.4 The cytoprotection of A9 in rat primary astrocytes and cortical neurons
Since A9 was verified to have significant cytoprotective effect, its antioxidant activity
was further investigated in rat primary cells. It is crucial that astrocytes provide
[34-35]
antioxidants and support the survival of neurons in the central nervous system
.
Therefore, the cytoprotection against H2O2-induced rat primary astrocytes and cortical
neurons cell damage was evaluated for A9. As shown in Figure 5, after treatment with A9
for 24 h prior to H2O2 insult, the population of viable cells was increased remarkably.
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Figure 5. A9 protected rat primary astrocytes (A) and cortical neurons (B) from H2O2-induced cell
injury in a dose-dependent manner. The cell viability was determined by the MTT assay. Data are
expressed as the mean ± SD, n = 3. ***p < 0.001, **p < 0.01vs DMSO, ##p < 0.01, #p < 0.05 vs H2O2.
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2.5 The antioxidative protection of A9 against H2O2-induced oxidative stress injury
The antioxidant activity of A9 (2 μM) against H2O2-induced oxidative damage was
further studied. A9 promoted colony formation of PC12 cells in H2O2-induced oxidative
damage model (Figure 6A). The apoptosis rate increased after treatment with H2O2, but A9
resisted H2O2-induced apoptosis in PC12 cells (Figure 6B). ROS can induce the excessive
accumulation of malondialdehyde (MDA), lactate dehydrogenase (LDH) and so on. The
level of intracellular ROS was elevated by H2O2, and A9 significantly suppressed the
formation of ROS (Figure 6C). In addition, as shown in Figure 6D, the level of MDA in the
A9 group was dramatically lower than that in the control group, suggesting that A9 blocked
MDA accumulation in PC12 cells, and the treatment of cells with H2O2 led to LDH release,
which was significantly decreased by a post-treatment of the cells with A9 (Figure 6E).
Above all, A9 protected PC12 cells from H2O2-induced cell injury by inhibiting cell
apoptosis, scavenging ROS, decreasing LDH release and reducing MDA.
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Figure 6. The antioxidant effects of A9 (2ꢀμM) against H2O2-induced oxidative damage in PC12 cells.
(A) A9 promoted colony formation of PC12 cells in H2O2 damage model. Cells were incubated by A9
for 24 h, and then incubation with 100 μM H2O2 for 6 days. Representative photographs of the colony
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formation assay. (B, C, D) PC12 cells were incubated with A9 for 24 h before exposure in H2O2 (1 mM)
for 6 h (B, D) or 2h (C). The rate of cell apoptosis (B), the level of intracellular ROS (C) and the MDA
levels (D) were determined according to the manufacturer’s instructions. (E) Effect of A9 on the LDH
release from PC12 cells after 450 μM H2O2 treatment for 24 h. TBHQ (2ꢀμM) was used as the positive
control. Data are expressed as the mean ± SD, n = 3. ****p < 0.0001, *p < 0.05 vs DMSO, ####p < 0.001,
##p < 0.01, #p < 0.05 vs H2O2.
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2.6 The antioxidant effect of A9 by activating Nrf2 signaling pathway
The Nrf2 (nuclear factor erythroid 2-related factor 2) transcription factor plays a
critical role in cellular oxidative stress response [36-38]. Hence, to investigated whether the
antioxidant effect of A9 is dependent on the activation of Nrf2 signaling pathway, Nrf2
siRNA or control siRNA was transfected into PC12 cells using Lipofectamine 2000 reagent,
and the cytoprotective effects of A9 against oxidative stress injury toward different cells
were evaluated. The results indicated that down-regulation of Nrf2 expression dramatically
decreased the cytoprotective effect of A9 (Figure 7A and B). Besides, A9 enhanced the
expression of HO-1 (heme oxygenase-1) protein, while its cytoprotection was reversed by
the HO-1 protein inhibitor ZnPP [39] (Figure 7C and D). Over all, A9 can activate the Nrf2
signaling pathway to protect PC12 cells against H2O2-derived oxidative cell death.
Figure 7. A9 protects PC12 cells from H2O2-induced cell injury by activating Nrf2 signaling pathway.
(A, B) Down-regulation of Nrf2 expression by siRNA diminished the protective effect of A9 on H2O2
induced cell damage. PC12 cells were transfected with si Nrf2 or negative control siRNA (NC) for 48 h
and the transfected cells were collected. The cells were pretreated with 2ꢀμM A9 followed by H2O2
treatment for 24ꢀh. The survival cells were represented by the colony formation assay and determined by
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MTT assay. (C) A9 increased the levels of HO-1 protein. PC12 cells were incubated with A9 (1, 2 and 5
μM) for 24 h. Next, the proteins were extracted and analyzed by western blot assay. (D) HO-1 inhibitor
ZnPP decreased the protection of A9 in H2O2 damage model. PC12 cells were pretreated with ZnPP for
1 h, and then treated with the 2ꢀμM A9 for 24 h followed by another 24 h H2O2 (450 μM) exposure.
TBHQ was used as the positive control. Data are expressed as the mean ± SD, n = 3. ****p < 0.0001,
***p < 0.001, *p < 0.05 vs DMSO, ##p < 0.01 vs H2O2, &&p < 0.01.
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2.7 Preventative therapies of A9 against CIRI
Transient transcranial middle cerebral artery occlusion (MCAO) model is a common
method to evaluate ischemic stroke in vivo [40-41]. The neuroprotective effect of A9 was
further investigated using a MCAO model. At 72 h after reperfusion, the neurological
deficit was evaluated on the basis of neurologic score and the infarct area was measuring
using TTC staining. The treatment with A9 showed a protective effect on infarction
damage (Figure 8). Pre-treatment of A9 reduced not only the infracted brain areas but also
corresponding neurological score.
Figure 8. Protective effect of A9 after MCAO in rats. (A) Representative photographs of brain slices
with TTC staining of each group. (B) Quantitative analysis of the infracted brain regions. (C) The
corresponding neurological 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 (the ratio
of DMSO and NS was 1:100) and 0.15 mg/kg A9 by intraventricular injection. Data are expressed as the
mean ± SD, n = 6. ****p < 0.0001 vs sham-operated group, ##p < 0.01, p < 0.05 vs vehicle-treated
#
group.
3. DISCUSSION
Natural products play an important part in the discovery of small molecule drugs, and
more and more attention has been paid to the biological functions of PL extracted from
long pepper [42-44]. In this study, a series of new PL analogs have been synthesized, and a
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potent compound with the neuroprotective effects against CIRI was identified.
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PL can cause a prominent toxic effect on nerve cells, though, it has wide prospects of
application as neuroprotective agent. To minimize the cytotoxicity of PL, we have
introduced a diketene structure based on the diketene skeletons of cytoprotective agents.
Although these agents (shown in Figure 1B) have different diketene skeletons, all of them
can protect the tissue or cells from damage. For example, compound B82 derived from
acetone demonstrated cytoprotection abilities in a dose-dependent manner [17]. The
piperid-4-one- containing F36 dose-dependently prevented NF-κB and ERK activation in
the cells and attenuated the septic death in mice [18]. The new MD2 specific inhibitor
L48H37 whose central linker is a N-ethyl-4-piperidin-4-one significantly improved
survival and protected lung injury in mice [19]. Pyranone-derived compound MAC 17
effectively inhibited the LPS-stimulated cytokine secretion from macrophages, and
protected against acute lung injury and sepsis [20]. Then we discovered that the activity and
toxicity of the diketene skeletons may be related to the different type of intermediate join
ketone. We conducted subsequent studies to examine the various diketene skeletons and
later discovered 2,5-di((E)-benzylidene) cyclopentan-1-one (skeleton a) with low toxicity
and antioxidant ability.
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To investigate the effects of 3,4,5-trimethoxybenzyl and α,β-unsaturated ketone
structures on the antioxidant activity of PL, we used the selected skeleton a to combine
with the 3,4,5-trimethoxyphenyl group of PL to obtain a series of PL analogs, namely
2-((E)-substituted
benzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-1-one
analogs. To gain insight into the structure-activity relationship of the synthesized PL
analogs, the toxicity and activity of a number of compounds were tested. The results
showed that any the synthesized PL analogs without obvious cytotoxicity have better
antioxidant function than the diketene skeleton and PL. When the 3,4,5-trimethoxy group
was remove from the structure of the optimal compound A9, its activity was greatly
reduced, suggesting that the 3,4,5-trimethoxy moiety in the benzene ring in PL could
increase the antioxidative effect. In addition, the cell viability also notably decreased when
the carbon-carbon double bonds of A9 were reduced by hydrogen, indicating that the
diketene skeleton of PL analogs is important for the ability of antioxidation. In short, it was
concluded that the methoxy groups and olefinic double bonds of PL analogs may be critical
for its neuroprotective effects.
The pathophysiological mechanism of CIRI is quite complicated, which includes
oxidative stress, glutamate excitotoxicity, inflammation and so on [45-47]. In this study, four
cellular models (H2O2-, glucose-, glutamate- and LPS-induced damage models) were
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selected to study the antioxidative protection of the optimal compound A9. As a result, it
was found that the active compound A9 could dose-dependently enhance the cell viability
in each damage model. Subsequently, the cytoprotective antioxidant effect of A9 was
confirmed in rat primary astrocytes. In addition, A9 was identified as a potent Nrf2
activator, which could reduce the levels of intracellular ROS and MDA, and inhibit LDH
release and apoptosis to protect cells from oxidative damage. More interestingly, A9
showed a favorable neuroprotection against ischemic brain injury.
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4. CONCLUSIONS
In summary, a series of new PL analogs were designed by the combination of active
diketene skeleton and the 3,4,5-trimethoxybenzyl moiety which could enhance the
antioxidant property of PL. The synthesis was accomplished by a green synthetic method of
taking L-proline as a catalyst for one-step synthesis of intermediates. The vast majority of
PL analogs obtained exhibited stronger antioxidant activity than the diketene skeleton
(skeleton a), and the most active compound A9 was revealed to be an effective Nrf2
activator with lower cytotoxicity. A9 can not only activate the Nrf2-dependent
cytoprotective pathway and up-regulate the expression of antioxidant proteins, but also
remarkably reduce the infarcted brain areas of rats subjected to CIRI. Taken together, the
PL analogs obtained by a green synthetic strategy demonstrated neuroprotective
antioxidant effects and might have the potential to treat brain disorders. A9 as a promising
anti-CIRI lead compound, its targets and underlying molecular mechanism remain to be
clarified.
5. Experimental Section
5.1 General
Commercially available starting materials and reagents were purchased from
Sigma-Aldrich (St Louis, Missouri, USA), Aladdin (Shanghai, China) and used without
further purification. All reactions were monitored by thin-layer chromatography TLC using
silica gel GF254. The chromatograms were conducted on silica gel (200-300 mesh) and
observed under UV light at 254 and 365 nm. Moreover, melting points (Mp) were
determined in open capillary tubes on a Fisher-Johns melting apparatus and uncorrected.
Mass spectra (MS) were recorded at the Agilent 1100 LC-MS (Agilent, Palo Alto, CA,
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USA). H NMR spectra were acquired on a 500 or 600 MHz spectrometer (Bruker
Corporation, Switzerland) using CDCl3 or DMSO-d6 as solvent. Tetramethylsilane (TMS)
was used as the internal standard. Coupling constants (J) are expressed in Hz, and splitting
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patterns are described as follows: s = singlet; d = doublet; t = triplet; m = multiplet; dd =
doublet of doublets; dt = doublet of triplets.
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5.2 Synthetic procedures
5.2.1 General synthetic procedure for the synthesis of intermediate
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3,4,5-trimethoxybenzaldehyde (0.196 g, 1.0 mmol, 1.0 equiv.), cyclopentanone (0.421
g, 5.0 mmol, 5.0 equiv.) and L-proline (0.23 g, 0.2 mmol, 0.2 equiv.) were dissolved in
anhydrous dimethylsulfoxide (2 mL) and the mixture was stirred for 24 h at room
temperature. Then, add 5 drops concentrated hydrochloric acid was added to the solution
and continuously stirred for 2 h at room temperature. The completion of the reaction was
monitored using TLC analysis. When the starting material was completely consumed, H2O
(10 mL) was added to the reacted mixture and extracted by ethyl acetate (15 mL, 3 times).
The organic layer was dried with anhydrous MgSO4, concentrated and evaporated in vacuo.
Finally, the crude product was purified by silica gel chromatography. Ethyl acetate and
petroleum ether were used as mobile phases.
5.2.2 General synthetic procedure for the synthesis of A and B series compounds
A mixture of the intermediate (0.26 g, 1.0 mmol, 1.0 equiv.) and the corresponding
aldehyde (1.2 mmol, 1.2 equiv.) in anhydrous EtOH was stirred at room temperature for 5
min. Then NaOH or HCl (gas) was added into the solution as catalyst to accelerate the
reaction. The reaction mixture was stirred at room temperature until raw material was
totally consumed (usually 6-12 h). Completion of the reaction was monitored by thin layer
chromatography using ethyl acetate/hexanes as the solvent system. After the reaction
completed, the crude product was filtered out and crystallized with hot ethanol or purified
by using silica gel chromatography.
5.2.3 General synthetic procedure for the synthesis of 1 and 2
A9 (0.44 g, 1.0 mmol, 1.0 equiv.) was dissolved in ethanol-ethyl acetate (5 mL/1mL),
and 10% palladium on charocoal was stirred under hydrogen for 12 h or 24 h at room
temperature. The catalyst was removed by filtration and the solvent was evaporated. The
residue was purified on a silica gel column using petroleum ether/ethyl acetate as eluent to
yield the 1 or 2 as colourless oil.
The spectral data of compounds are listed as follows:
2-((E)-2,4-dichlorobenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-
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1-one (A1): Yellow power, 61.9% yield, mp 202.3~203.5 °C. H-NMR (600 MHz,
d-DMSO), δ: 7.846 (s, 1H, β-H), 7.555-7.501 (m, 3H, Ar-H3’, Ar-H5’, Ar-H6’), 7.325 (d,
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J=7.2 Hz, 1H, α-H), 6.852 (s, 2H, Ar-H2, Ar-H6), 3.931 (s, 9H, 3-OCH3, 5-OCH3, 4-OCH3),
3.125 (s, 2H, CH2), 3.031 (s, 2H, CH2). LC-MS m/z: 419.17 (M+H)+, calcd for
C22H20Cl2O4: 418.07.
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2-((E)-4-(dimethylamino)benzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclop
entan-1-one (A2): Orange power, 65.6% yield, mp 201.3~202.4 °C. H-NMR (600 MHz,
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d-DMSO), δ: 7.604 (s, 1H, β-H), 7.557 (d, J=8.4 Hz, 2H, Ar-H6’, Ar-H2’), 7.448 (s, 1H,
α-H), 6.868 (s, 2H, Ar-H2, Ar-H6), 6.757 (d, J=9.0 Hz, 2H, Ar-H3’, Ar-H5’), 6.930 (d, J=6.6
Hz, 9H, 3-OCH3, 5-OCH3, 4-OCH3), 3.118 (s, 4H, CH2CH2), 3.070 (s, 6H, CH3NCH3).
LC-MS m/z: 394.05 (M+H)+, calcd for C24H27NO4: 393.19.
2-((E)-4-hydroxy-3-methoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cycl
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opentan-1-one (A3): Orange power, 68.6% yield, mp 114.8~115.5 °C. H-NMR (600
MHz, d-DMSO), δ: 9.703 (s, 1H, 4’-OH), 7.371 (dt, J=9.6 Hz, J=10.2 Hz, 2H, β-H, α-H),
7.245 (d, J=1.8 Hz, 1H, Ar-H6’), 7.157 (dd, J=1.8 Hz, J=8.4 Hz, 1H, Ar-H2’), 6.983 (s, 2H,
Ar-H2, Ar-H6), 6.878 (d, J=8.4 Hz, 1H, Ar-H5’), 3.827 (d, J=1.8 Hz, 9H, 3-OCH3, 5-OCH3,
3’-OCH3), 3.702 (s, 3H, 4-OCH3), 3.129-3.111 (m, 2H, CH2), 3.076-3.052 (m, 2H, CH2).
LC-MS m/z: 396.16 (M+H)+, calcd for C23H24O6: 397.17.
2-((E)-4-methoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-1-
one (A4): Yellow power, 69.8% yield, mp 186.5~189.2 °C. 1H-NMR (600 MHz, CDCl3), δ:
7.594 (d, J=8.4 Hz, 2H, Ar-H2’, Ar-H6’), 7.553 (t, J=4.2 Hz, 1H, β-H), 7.525 (s, 1H, α-H),
7.000 (d, J=9.0 Hz, 2H, Ar-H3’, Ar-H5’), 6.870 (s, 2H, Ar-H2, Ar-H6), 3.933 (d, J=5.4 Hz,
9H, 3-OCH3, 5-OCH3, 4’-OCH3), 3.886 (s, 3H, 4-OCH3), 3.153-3.113 (m, 4H, CH2CH2).
LC-MS m/z: 381.16 (M+H)+, calcd for C23H24O5: 380.16.
2-((E)-2-hydroxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-1-
one (A5): Yellow power, 68.7% yield, mp 123.6~125.9 °C. 1H-NMR (600 MHz, CDCl3), δ:
9.904 (s, 1H, 2’-OH), 7.530 (s, 1H, β-H), 7.317 (t, J=2.4 Hz, 2H, α-H, Ar-H6’), 6.855 (s,
3H, Ar-H2, Ar-H6, Ar-H5’), 6.713 (s, 1H, Ar-H4’), 6.642 (s, 1H, Ar-H3’), 3.893 (s, 9H,
3-OCH3, 4-OCH3, 5-OCH3), 3.150 (s, 2H, CH2), 3.012-2.984 (m, 2H, CH2). LC-MS m/z:
367.26 (M+H)+, calcd for C22H22O5: 366.15.
3-((E)-4-hydroxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-1-
one (A6): Brown power, 77.3% yield, mp 232.5~233.1 °C. 1H-NMR (600 MHz, d-DMSO),
δ: 10.104 (s, 1H, 4’-OH), 7.558 (d, J=8.4 Hz, 2H, Ar-H2’, Ar-H6’), 7.384 (d, J=2.4 Hz, 2H,
β-H, α-H), 7.033 (s, 2H, Ar-H2, Ar-H6), 6.891 (d, J=8.4 Hz, 2H, Ar-H3’, Ar-H5’), 3.850 (s,
6H, 3-OCH3, 5-OCH3), 3.723 (s, 3H, 4-OCH3), 3.138-3.123 (m, 2H, CH2), 3.048-3.033 (m,
2H, CH2). LC-MS m/z: 367.15 (M+H)+, calcd for C22H22O5: 366.15.
2-((E)-3-hydroxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-1-
one (A7): Yellow power, 78.9% yield, mp 123.6~125.9 °C. 1H-NMR (600 MHz, d-DMSO),
δ: 9.639 (s, 1H, 3’-OH), 7.401 (s, 1H, β-H), 7.324 (s, 1H, α-H), 7.273 (t, J=7.8 Hz, 1H,
Ar-H5’), 7.096 (d, J=7.2 Hz, 1H, Ar-H6’), 7.061 (s, 1H, Ar-H4’), 6.996 (s, 2H, Ar-H2,
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Ar-H6), 6.836-6.821 (m, 1H, Ar-H2’), 3.831 (s, 6H, 3-OCH3, 5-OCH3), 3.705 (s, 3H,
4-OCH3), 3.126 (d, J=6.0 Hz, 2H, CH2), 3.050 (d, J=5.4 Hz, 2H, CH2). LC-MS m/z:
367.26 (M+H)+, calcd for C22H22O5: 366.15.
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2-((E)-3-fluorobenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-1-on
e (A8): Yellow power, 69.9% yield, mp 140.4~141.5 °C. H-NMR (600 MHz, CDCl3), δ:
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7.555 (s, 3H, β-H, α-H, Ar-H6’), 7.447-7.410 (m, 2H, Ar-H5’, Ar-H4’), 7.105 (td, J=1.2 Hz,
J=7.2 Hz, 1H, Ar-H2’), 6.868 (s, 2H, Ar-H2, Ar-H6), 3.934 (d, J=2.4 Hz, 9H, 3-OCH3,
5-OCH3, 4-OCH3), 3.148 (s, 4H, CH2CH2). LC-MS m/z: 369.11 (M+H)+, calcd for
C22H21FO4: 368.14.
2-((E)-2,4,6-trimethoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopen
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tan-1-one (A9): Yellow power, 72.3% yield, mp 163.3~164.0 °C. H-NMR (600 MHz,
CDCl3), δ: 7.698 (s, 1H, β-H), 7.475 (s, 1H, α-H), 6.851 (s, 2H, Ar-H2, Ar-H6), 6.178 (s,
2H, Ar-H3’, Ar-H5’), 3.921 (s, 6H, 2’-OCH3, 6’-OCH3), 3.860 (s, 9H, 3-OCH3, 5-OCH3,
4’-OCH3), 3.806 (s, 3H, 4-OCH3), 3.055-2.988 (m, 4H, CH2CH2). LC-MS m/z: 441.24
(M+H)+, calcd for C25H28O7: 440.18.
2-((E)-3-hydroxy-4-methoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cycl
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opentan-1-one (A10): Yellow power, 73.8% yield, mp 190.1~191.3 °C. H-NMR (600
MHz, d-DMSO), δ: 9.299 (s, 1H, 3’-OH), 7.388 (s, 1H, β-H), 7.325 (s, 1H, α-H), 7.148 (d,
J=7.2 Hz, 2H, Ar-H2’, Ar-H6’), 7.042 (d, J=9.0 Hz, 1H, Ar-H5’), 7.006 (s, 2H, Ar-H2,
Ar-H6), 3.852 (s, 6H, 3-OCH3, 5-OCH3), 3.836 (s, 3H, 4’-OCH3), 3.724 (s, 3H, 4-OCH3),
3.146-3.135 (m, 2H, CH2), 3.039-3.029 (m, 2H, CH2). LC-MS m/z: 397.23 (M+H)+, calcd
for C23H24O6: 396.16.
2-((E)-2,4-dimethoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopenta
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n-1-one (A11): Yellow power, 68.2% yield, mp 164.0~165.8 °C. H-NMR (600 MHz,
CDCl3), δ: 8.027 (s, 1H, Ar-H6’), 7.541 (d, J=8.4 Hz, 1H, β-H), 7.509 (s, 1H, α-H), 6.862 (s,
2H, Ar-H2, Ar-H6), 6.584-6.506 (m, 2H, Ar-H3’, Ar-H5’), 3.933-3.914 (m, 12H, 2’-OCH3,
4’-OCH3, 3-OCH3, 5-OCH3), 3.900 (s, 3H, 4-OCH3), 3.118-3.060 (m, 4H, CH2CH2).
LC-MS m/z: 411.15 (M+H)+, calcd for C24H26O6: 410.17.
2-((E)-2-methoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-1-
one (A12): Yellow power, 71.3% yield, mp 117.2~118.3 °C. 1H-NMR (600 MHz, CDCl3),
δ: 8.046 (s, 1H, Ar-H4’), 7.558 (t, J=7.2 Hz, 2H, β-H, Ar-H6’), 7.401-7.374 (m, 1H, α-H),
7.032 (t, J=7.8 Hz, 1H, Ar-H3’), 6.968 (d, J=7.8 Hz, 1H, Ar-H5’), 6.866 (s, 2H, Ar-H2,
Ar-H6), 3.935 (s, 6H, 3-OCH3, 5-OCH3), 3.927 (s, 3H, 2’-OCH3), 3.918 (s, 3H, 4-OCH3),
3.104 (s, 4H, CH2CH2). LC-MS m/z: 381.16 (M+H)+, calcd for C23H24O5: 380.16.
2-((E)-4-(piperidin-1-yl)benzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclope
ntan-1-one (A13): Orange powder, 64.8% yield, mp 171.2~171.7 °C. 1H-NMR (600 MHz,
CDCl3), δ: 7.585 (s, 1H, Ar-H2’), 7.542 (d, J=9.0 Hz, 2H, β-H, α-H), 7.497 (s, 1H, Ar-H6’),
6.948 (d, J=9.0 Hz, 2H, Ar-H3’, Ar-H5’), 6.869 (s, 2H, Ar-H2, Ar-H6), 3.930 (d, J=6.6 Hz,
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9H, 3-OCH3, 5-OCH3, 4-OCH3), 3.349 (t, J=10.2 Hz, 4H, CH2NCH2), 3.123 (s, 4H,
CH2CH2), 0.901 (t, J=7.8 Hz, 6H, CH2CH2CH2). LC-MS m/z: 433.96 (M+H)+, calcd for
C27H31NO4: 433.23.
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2-((E)-4-morpholinobenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopenta
n-1-one (A14): Orange powder, 56.4% yield, mp 191.4~192.5 °C. H-NMR (600 MHz,
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CDCl3), δ: 7.563 (d, J=9.0 Hz, 2H, Ar-H2’, Ar-H6’), 7.531 (d, J=7.2 Hz, 4H, β-H, α-H,
Ar-H2, Ar-H6), 7.404-7.318 (m, 8H, Ar-H3’, Ar-H5’, 3-OCH3, 5-OCH3), 7.009 (t, J=6.9 Hz,
4H, CH2OCH2), 6.947 (d, J=9.0 Hz, 3H, 4-OCH3), 3.891 (t, J=4.8 Hz, 4H, CH2NCH2),
3.295 (t, J=4.8 Hz, 4H, CH2CH2). LC-MS m/z: 436.20 (M+H)+, calcd for C26H29NO5:
435.20.
2-((E)-2,3-dimethoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopenta
1
n-1-one (A15): Yellow powder, 58.6% yield, mp 150.5~151.3 °C. H-NMR (600 MHz,
CDCl3), δ: 7.957 (s, 1H, β-H), 7.545 (d, J=1.2 Hz, 1H, α-H), 7.196 (d, J=7.2 Hz, 1H,
Ar-H6’), 7.134 (t, J=16.2 Hz, 1H, Ar-H5’), 6.997-6.946 (m, 1H, Ar-H4’), 6.867 (s, 2H,
Ar-H2, Ar-H6), 3.973-3.911 (m, 12H, 2’-OCH3, 3’-OCH3, 3-OCH3, 5-OCH3), 3.897 (s, 3H,
4-OCH3), 3.107-2.915 (m, 4H, CH2CH2). LC-MS m/z: 411.20 (M+H)+, calcd for C24H26O6:
410.17.
2-((E)-2-(trifluoromethyl)benzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclop
entan-1-one (A16): Yellow powder, 67.5% yield, mp 157.3~157.9 °C. 1H-NMR (600 MHz,
CDCl3), δ: 7.871 (d, J=2.4 Hz, 1H, β-H), 7.772 (d, J=7.8 Hz, 1H, Ar-H3’), 7.622 (d, J=4.2
Hz, 2H, Ar-H5’, α-H), 7.578-7.544 (m, 1H, Ar-H6’), 7.499-7.432 (m, 1H, Ar-H4’),
6.856-6.792 (m, 2H, Ar-H2, Ar-H6), 3.931 (s, 9H, 3-OCH3, 5-OCH3, 4-OCH3), 3.105-3.007
(m, 4H, CH2CH2). LC-MS m/z: 419.11 (M+H)+, calcd for C23H21F3O4: 418.14.
2-((E)-3,4-dihydroxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopenta
1
n-1-one (A17): Orange powder, 65.7% yield, mp 184.1~185.5 °C. H-NMR (600 MHz,
d-DMSO), δ: 9.652 (s, 1H, 3’-OH), 9.262 (s, 1H, 4’-OH), 7.374 (s, 1H, β-H), 7.301 (s, 1H,
α-H), 7.127 (d, J=1.8 Hz, 1H, Ar-H2’), 7.034 (dd, J=1.2 Hz, J=6.6 Hz, 1H, Ar-H6’), 7.003
(s, 2H, Ar-H2, Ar-H6), 6.855 (d, J=7.8 Hz, 1H, Ar-H5’), 3.852 (s, 6H, 3-OCH3, 5-OCH3),
3.723 (s, 3H, 4-OCH3), 3.142-3.012 (m, 4H, CH2CH2). LC-MS m/z: 383.14 (M+H)+, calcd
for C22H22O6: 382.14.
2-((E)-2,5-dimethoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopenta
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n-1-one (A18): Yellow powder, 65.6% yield, mp 122.4~123.5 °C. H-NMR (600 MHz,
CDCl3), δ: 8.002 (s, 1H, β-H), 7.536 (s, 1H, α-H), 7.131 (d, J=3.0 Hz, 1H, Ar-H3’), 6.938
(dd, J=3.0 Hz, J=9.0 Hz, 1H, Ar-H6’), 6.900 (d, J=9.0 Hz, 1H, Ar-H4’), 6.865 (s, 2H, Ar-H2,
Ar-H6), 3.931 (d, J=3.6 Hz, 9H, 3-OCH3, 5-OCH3, 5’-OCH3), 3.874 (s, 3H, 2’-OCH3),
3.836 (s, 3H, 4-OCH3), 3.114 (s, 4H, CH2CH2). LC-MS m/z: 411.20 (M+H)+, calcd for
C24H26O6: 410.17.
2-((E)-2-bromobenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-1-o
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ne (A19): Yellow powder, 72.6% yield, mp 165.2~166.0 °C. 1H-NMR (600 MHz, CDCl3),
δ: 7.864 (s, 1H, β-H), 7.691 (d, J=7.8 Hz, 1H, Ar-H2’), 7.574 (t, J=2.4 Hz, 2H, α-H, Ar-H5’),
7.384 (d, J=7.8 Hz, 1H, Ar-H4’), 7.254-7.207 (m, 1H, Ar-H6’), 6.863 (s, 2H, Ar-H2, Ar-H6),
3.928 (d, J=9.0 Hz, 9H, 3-OCH3, 5-OCH3, 4-OCH3), 3.118-3.010 (s, 4H, CH2CH2).
LC-MS m/z: 429.05 (M+H)+, calcd for C22H21BrO4: 428.06.
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2-((E)-4-chlorobenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-1-on
e (A20): Yellow powder, 80.2% yield, mp 181.2~182.7 °C. 1H-NMR (600 MHz, CDCl3), δ:
7.557-7.539 (m, 4H, Ar-H2’, Ar-H6’, Ar-H3’, Ar-H5’), 7.435 (d, J=8.4 Hz, 2H, β-H, α-H),
6.869 (s, 2H, Ar-H2, Ar-H6), 3.935 (d, J=2.4 Hz, 9H, 3-OCH3, 5-OCH3, 4-OCH3),
3.170-3.110 (m, 4H, CH2CH2). LC-MS m/z: 385.12 (M+H)+, calcd for C22H21ClO4:
384.11.
2-((E)-3,4-dichlorobenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-
1
1-one (A21): Yellow powder, 67.2% yield, mp 153.8~154.3 °C. H-NMR (600 MHz,
CDCl3), δ: 7.692 (d, J=1.2 Hz, 1H, Ar-H6’), 7.564-7.518 (m, 2H, β-H, α-H), 7.485 (s, 1H,
Ar-H5’), 7.430 (dd, J=1.8 Hz, J=6.6 Hz, 1H, Ar-H2’), 6.871 (s, 2H, Ar-H2, Ar-H6), 3.938 (d,
J=1.8 Hz, 9H, 3-OCH3, 5-OCH3, 4-OCH3), 3.184-2.975 (m, 4H, CH2CH2). LC-MS m/z:
419.11 (M+H)+, calcd for C22H20Cl2O4: 418.07.
2-((E)-3,4-difluorobenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-
1
1-one (A22): Yellow powder, 63.5% yield, mp 169.0~170.1 °C. H-NMR (600 MHz,
CDCl3), δ: 7.549 (d, J=7.2 Hz, 1H, Ar-H6’), 7.501 (s, 1H, β-H), 7.447-7.394 (m 1H, α-H),
7.340 (s, 1H, Ar-H5’), 7.245 (t, J=18.0 Hz, 1H, Ar-H2’), 6.867 (s, 2H, Ar-H2, Ar-H6), 3.936
(d, J=3.0 Hz, 9H, 3-OCH3, 5-OCH3, 4-OCH3), 3.168-3.106 (m, 4H, CH2CH2). LC-MS m/z:
387.16 (M+H)+, calcd for C22H20F2O4: 386.13.
2-((E)-3,4,5-trimethoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopen
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tan-1-one (A23): Yellow powder, 69.8% yield, mp 203.1~203.4 °C. H-NMR (600 MHz,
CDCl3), δ: 7.553 (d, J=9.0 Hz, 2H, β-H, α-H), 6.871 (s, 4H, Ar-H2×2, Ar-H6×2), 3.932 (d,
J=1.8 Hz, 18H, 3-OCH3×2, 5-OCH3×2, 4-OCH3×2), 3.167 (s, 4H, CH2CH2). LC-MS m/z:
441.11 (M+H)+, calcd for C25H28O7: 440.18.
2-((E)-3,4-dimethoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopenta
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n-1-one (A24): Yellow powder, 59.9% yield, mp 168.3~169.5 °C. H-NMR (600 MHz,
CDCl3), δ: 7.557 (t, J=15.9 Hz, 2H, β-H, α-H), 7.265-7.208 (m, 1H, Ar-H6’), 7.159 (d,
J=1.2 Hz, 1H, Ar-H2’), 6.969 (d, J=8.4 Hz, 1H, Ar-H5’), 6.870 (s, 2H, Ar-H2, Ar-H6),
3.994-3.884 (m, 15H, 3-OCH3, 5-OCH3, 4-OCH3, 3’-OCH3, 4’-OCH3), 3.152 (s, 4H,
CH2CH2). LC-MS m/z: 411.13 (M+H)+, calcd for C25H26O6: 410.17.
2-((E)-2,3-dichlorobenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-
1
1-one (A25): Yellow power, 63.8% yield, mp 181.6~182.1 °C. H-NMR (600 MHz,
CDCl3), δ: 7.862 (s, 1H, β-H), 7.553 (s, 1H, Ar-H4’), 7.479-7.446 (m, 2H, Ar-H5’, Ar-H6’),
7.250 (s, 1H, α-H), 6.836 (s, 2H, Ar-H2, Ar-H6), 3.911 (s, 9H, 3-OCH3, 5-OCH3, 4-OCH3),
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3.092 (d, J=5.4 Hz, 2H, CH2), 3.008 (d, J=4.8 Hz, 2H, CH2). LC-MS m/z: 419.17 (M+H)+,
calcd for C22H20Cl2O4: 418.07.
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2-((E)-2-fluoro-5-methoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclo
pentan-1-one (A26): Yellow powder, 70.3% yield, mp 144.5~145.4 °C. H-NMR (600
MHz, CDCl3), δ: 7.774 (s, 1H, β-H), 7.554 (s, 1H, α-H), 7.109-7.093 (m, 1H, Ar-H3’),
7.070 (d, J=9.0 Hz, 1H, Ar-H4’), 6.921-6.895 (m, 1H, Ar-H6’), 6.865 (s, 2H, Ar-H2, Ar-H6),
3.934 (d, J=1.8 Hz, 9H, 3-OCH3, 5-OCH3, 5’-OCH3), 3.848 (s, 3H, 4-OCH3), 3.152-3.090
(m, 4H, CH2CH2). LC-MS m/z: 399.21 (M+H)+, calcd for C23H23FO5: 398.15.
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2-((E)-4-(pyrrolidin-1-yl)benzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclop
entan-1-one (A27): Orange powder, 61.8% yield, mp 221.4~222.3 °C. 1H-NMR (600 MHz,
CDCl3), δ: 7.608 (s, 1H, β-H), 7.547 (d, J=7.8 Hz, 2H, Ar-H2’, Ar-H6’), 7.477 (s, 1H, α-H),
6.865 (s, 2H, Ar-H2, Ar-H6), 6.612 (d, J=7.8 Hz, 2H, Ar-H4’, Ar-H5’), 3.927 (d, J=6.0 Hz,
9H, 3-OCH3, 5-OCH3, 4-OCH3), 3.380 (s, 4H, CH2NCH2), 3.109 (s, 4H, CH2CH2), 2.055
(s, 4H, CH2CH2). LC-MS m/z: 420.19 (M+H)+, calcd for C26H29NO4: 419.21.
2-((E)-4-hydroxy-3,5-dimethoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)
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cyclopentan-1-one (A28): Brown powder, 66.4% yield, mp 194.5~196.0 °C. H-NMR
(600 MHz, d-DMSO), δ: 9.122 (s, 1H, 4’-OH), 7.402 (d, J=12.6 Hz, 2H, β-H, α-H), 7.007
(d, J=5.4 Hz, 4H, Ar-H2, Ar-H6, Ar-H2’, Ar-H6’), 3.845 (d, J=7.2 Hz, 12H, 3-OCH3,
5-OCH3, 3’-OCH3, 5’-OCH3), 3.724 (s, 3H, 4-OCH3), 3.145 (s, 4H, CH2CH2). LC-MS m/z:
427.18 (M+H)+, calcd for C24H26O7: 426.17.
2-((E)-2,6-dichlorobenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-
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1-one (A29): Yellow powder, 68.5% yield, mp 164.7~164.9 °C. H-NMR (600 MHz,
CDCl3), δ: 7.544 (s, 1H, β-H), 7.480 (s, 1H, Ar-H4’), 7.366 (d, J=7.8 Hz, 2H, Ar-H3’,
Ar-H5’), 7.230 (t, J=7.8 Hz, 1H, α-H), 6.832 (s, 2H, Ar-H2, Ar-H6), 3.905 (d, J=3.0 Hz, 9H,
3-OCH3, 5-OCH3, 4-OCH3), 3.059-3.030 (m, 2H, CH2), 2.715-2.686 (m, 2H, CH2). LC-MS
m/z: 419.05 (M+H)+, calcd for C22H20Cl2O4: 418.07.
2-((E)-4-bromobenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-1-o
ne (A30): Yellow powder, 66.3% yield, mp 189.9~192.8 °C. 1H-NMR (600 MHz, CDCl3),
δ: 7.570 (d, J=8.4 Hz, 2H, Ar-H3’, Ar-H5’), 7.523 (d, J=9.0 Hz, 2H, Ar-H2’, Ar-H6’), 7.451
(d, J=8.4 Hz, 2H, β-H, α-H), 6.845 (s, 2H, Ar-H2, Ar-H6), 3.912 (d, J=2.4 Hz, 9H, 3-OCH3,
5-OCH3, 4-OCH3), 3.147-3.049 (m, 4H, CH2CH2). LC-MS m/z: 429.14 (M+H)+, calcd for
C22H21BrO4: 428.06.
2-((E)-3,5-dichlorobenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-
1
1-one (A31): Yellow powder, 65.7% yield, mp 184.3~187.1 °C. H-NMR (600 MHz,
CDCl3), δ: 7.564 (s, 1H, Ar-H4’), 7.459 (s, 2H, β-H, α-H), 7.440 (s, 1H, Ar-H2’), 7.384 (s,
1H, Ar-H6’), 6.869 (d, J=3.6 Hz, 2H, Ar-H2, Ar-H6), 3.936 (s, 9H, 3-OCH3, 5-OCH3,
4-OCH3), 3.181-3.120 (m, 4H, CH2CH2). LC-MS m/z: 419.06 (M+H)+, calcd for
C22H20Cl2O4: 418.07.
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2-((E)-3-methoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopentan-1-
one (A32): Yellow powder, 69.6% yield, mp 133.8~136.7 °C. 1H-NMR (600 MHz, CDCl3),
δ: 7.546 (s, 2H, β-H, α-H), 7.363 (t, J=7.8 Hz, 1H, Ar-H5’), 7.203 (d, J=7.8 Hz, 1H, Ar-H6’),
7.126 (s, 1H, Ar-H2’), 6.944 (dd, J=1.8 Hz, J=8.4 Hz, 1H, Ar-H4’), 6.848 (s, 2H, Ar-H2,
Ar-H6), 3.912 (d, J=3.6 Hz, 9H, 3-OCH3, 5-OCH3, 3’-OCH3), 3.855 (s, 3H, 4-OCH3),
3.133 (s, 4H, CH2CH2). LC-MS m/z: 381.18 (M+H)+, calcd for C23H24O5: 380.16.
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2-((E)-3,5-dimethoxybenzylidene)-5-((E)-3,4,5-trimethoxybenzylidene)cyclopenta
n-1-one (A33): Yellow powder, 63.6% yield, mp 125.8~128.4 °C. H-NMR (600 MHz,
CDCl3), δ: 7.544 (d, J=5.4 Hz, 2H, β-H, α-H), 6.870 (s, 2H, Ar-H2, Ar-H6), 6.773 (d, J=1.8
Hz, 2H, Ar-H2’, Ar-H6’), 6.536 (s, 1H, Ar-H4’), 3.933 (d, J=3.6 Hz, 9H, 3-OCH3, 5-OCH3,
4-OCH3), 3.858 (s, 6H, 3’-OCH3, 5’-OCH3), 3.152 (t, J=10.2 Hz, 4H, CH2CH2). LC-MS
m/z: 411.15 (M+H)+, calcd for C24H26O6: 410.17.
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(B9):
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Orange powder, 78.2% yield, mp 128.5~129.9 °C. H-NMR (600 MHz, d-DMSO), δ:
7.636 (d, J=7.2 Hz, 2H, β-H, α-H), 7.473-7.434 (m, 3H, Ar-H3, Ar-H4, Ar-H5), 7.409-7.387
(m, 2H, Ar-H2’, Ar-H6’), 6.282 (s, 2H, Ar-H2, Ar-H6), 3.821 (s, 3H, 4-OCH3), 3.802 (s, 6H,
3-OCH3, 5-OCH3), 2.971-2.814 (m, 4H, CH2CH2). LC-MS m/z: 351.15 (M+H)+, calcd for
C22H22O4: 350.15.
2-((E)-2,4,6-trimethoxybenzyl)-5-(3,4,5-trimethoxybenzylidene))cyclopentan-1-on
e (1): Oil, 68.5% yield. 1H-NMR (500 MHz, CDCl3), δ: 7.538 (s, 1H, Ar-CH=C), 6.431 (s,
2H, Ar-H2, Ar-H6), 6.133 (s, 2H, Ar-H3’, Ar-H5’), 3.855 (s, 3H, 4’-OCH3), 3.846 (s, 6H,
2’-OCH3, 6’-OCH3), 3.827 (s, 4H, CH2CH2), 3.819 (s, 6H, 3-OCH3, 5-OCH3), 3.804 (s, 3H,
4-OCH3), 3.768 (s, 3H, CH2CH). LC-MS m/z: 443.06 (M+H)+, calcd for C25H30O7: 442.20.
2-(2,4,6-trimethoxybenzyl)-5-(3,4,5-trimethoxybenzyl)cyclopentan-1-one (2): Oil,
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45.3% yield. H-NMR (500 MHz, CDCl3), δ: 6.431 (s, 2H, Ar-H2, Ar-H6), 6.133 (s, 2H,
Ar-H3’, Ar-H5’), 3.836-3.855 (m, 9H, 3-OCH3, 5-OCH3, 4-OCH3), 3.827 (s, 3H, 4’-OCH3),
3.819 (s, 6H, 2’-OCH3, 6’-OCH3), 3.799-3.808 (m, 5H, CH2CHCH2), 3.761-3.780 (m, 5H,
CH2CHCH2). LC-MS m/z: 445.06 (M+H)+, calcd for C25H32O7: 444.20.
5.3 Pharmacology
5.3.1 Cell culture
The rat pheochromocytoma cell line (PC12) was purchased from the Cell Storage
Center of Wuhan University (Wuhan, China). Primary astrocytes were obtained from the
cerebral cortices of Sprague-Dawley (SD) rat pups and cortical neurons from rat fetuses
(embryonic day 16–17) were prepared as described by previous authors [48-49]. Cells were
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cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Eggenstein, Germany)
containing 10% fetal bovine serum (FBS; Gibco) and antibiotics (1% penicillin–
streptomycin; Gibco). The cells were incubated at a temperature of 37°C under a 5% CO2
atmosphere in a humidified incubator (Thermo Fisher Scientific, Massachusetts, USA).
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5.3.2 MTT assay
PC12 cells were seeded in a 96-well plate (NEST Biotechnology Co. LTD., Wuxi,
China) at a density of 5×103 cells/well and incubated overnight. The cells were exposed to
H2O2, glucose, glutamate or LPS after treatment with the compounds for 24 h. Then 20 µL
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, thiazolyl blue (MTT; 0.5
mg/mL; Sigma, St Louis, Missouri, USA) was added to the culture medium and incubated
at 37 °C for 4 h in the dark. Subsequently, the medium was removed carefully and the
crystals of formazan were dissolved sufficiently in 120 μL dimethyl sulfoxide (DMSO;
Sigma). The optical density (OD) was measured at 490 nm by a microplate reader (Thermo
Fisher Scientific, Massachusetts wavelength, USA) and the cell viability was expressed as
OD value of the experiment group / OD value of the control group × 100%. The
experiment was repeated three times.
5.3.3 Cell colony formation assay
PC12 cells were seeded into 12-well plates (NEST) at a density of 1×103 cells/well.
After 24 h, cells were treated with the compounds for 24 h, and then exposed to 100 μM
H2O2. The plates were incubated at 37°C in 5% CO2 for 6 days. Next, the colony cells were
fixed with 4% paraformaldehyde for 40 min and stained with crystal violet for 20 min. The
plates were then washed with PBS and dried prior to taking photos.
5.3.4 LDH assay
The amount of LDH released into the medium was determined by using an assay kit
according to the manufacturer’s protocol from the LDH release cytotoxicity detection kit
(Beyotime Institute of Biotechnology, Haimen, China). The changes in absorbance were
determined at 490 nm by a microplate reader (Thermo Fisher Scientific). LDH leakage was
expressed as the percentage (%) of the total LDH (extracellular + intracellular) activity.
5.3.5 Lipid peroxidation assay
PC12 cells (3×105 cells/well) were seeded into 6-well plates (NEST). After cells
adhered to the wells overnight, cells were incubated with the compounds for 24 h followed
by adding 1 mM H2O2 to stimulate cells for 6 h. The malondialdehyde (MDA) level was
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measured by an assay kit (Beyotime Institute of Biotechnology, Nanjing, China) according
to the manufacturer's instructions. The absorbance was read at a wavelength of 532 nm.
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5.3.6 Cell apoptosis assay
PC12 cells (3×105 cells/well) were seeded into 6-well plates overnight and then
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treated with the compounds for 24 h followed by another 6 h H2O2 (1 mM) exposure. The
rate of cell apoptosis was measured by using an Annexin V-FITC and propidium iodide (PI)
double staining kit (BD Biosciences Clontech, San Jose, CA, USA). The procedures were
exactly followed according to the manufacturer’s instructions and the cell apoptosis was
detected by flow cytometry (Becton Dickinson, USA). The results were analyzed by using
the Flow Jo analysis program.
5.3.7 ROS assay
Intracellular Reactive Oxygen Species (ROS) generation was measured by
dichlorodihydr- ofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology,
Shanghai, China). In brief, PC12 cells were seeded into 6-well plates at a density of 3×105
per well overnight. Subsequently, cells were pretreated with the compounds for 24 h,
followed by another 2 h H2O2 (1 mM) exposure. After incubation with 1 μL DCFH-DA (10
μM) for 30 min at 37 °C in the dark, the cells were collected, washed and resuspended in
PBS. The intracellular ROS level was quantified by flow cytometry and analyzed with
Flow Jo software.
5.3.8 Western blot analysis
PC12 cells were seeded into 6-well culture plates at a density of 3×105 cells/ml in
6-well plates and incubated for 48 h at 37 °C. The treated cells were collected and lysed
with RIPA lysis buffer (Boster Biological Technology Co. Ltd., Wuhan, China). Then an
amount of 80 µg of the protein samples was separated by 10% sodium dodecyl
sulfate−polyacrylamide gel (SDS-PAGE) electrophoresis and transferred to a
Polyvinylidene Fluoride (PVDF) membrane (Millipore, Massachusetts, USA). The
membranes were incubated with the primary antibodies (sc-10789, Santa Cruz
Biotechnology, 1:500) and anti-β-actin (AP0060, Bioworld Technology, 1:3000) overnight
at 4°C after blocked with 5% non-fat milk at room temperature for 90 min. The next day,
the membranes were washed with 1×TBST (Tris-buffered saline, pH 7.6, containing 0.05%
Tween 20) three times and incubated with secondary antibody anti-rabbit IgG (1: 1000) for
1 h at room temperature. The blots were imaged by ChemiDoc XRS+system (Bio-Rad,
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Hercules, CA), and quantified with Image J software (NIH, Bethesda, MD).
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5.3.9 Transfection assay
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The short interfering RNA (siRNA) sequences (sense: 5′-GGGUAAGUCGAGAA
GUGUUTT-3′, antisense: 5′-AACACUUCUCGACUUACCCTT-3′) targeting Nrf2 was
chemically synthesized by Shanghai GenePharma Company. The Nrf2 siRNA or control
siRNA was transfected into PC12 cells using Lipofectamine 2000 reagent (Invitrogen,
Carlsbad, CA, USA) according to the manufacturer's instructions. The fresh cell culture
medium containing FBS was added after 10ꢀh of transfection. Following 48 h knockdown
of the Nrf2 expression, the transfected cells were collected and used in further analysis as
described in the figure legends.
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5.3.10 Experimental animals
Adult male Sprague-Dawley (SD) rats (250-280 g) were obtained from the Beijing
Vital River Laboratory Animal Technology Co., Ltd. The rats were fed with a standard
laboratory diet and housed under a 12/12 h dark/light cycle in a temperature-controlled
room (24 °C). All animal experiments were performed in compliance with the National
Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No.
8023, revised 1978) and approved by the Wenzhou Medical University Animal Policy and
Welfare Committee.
5.3.11 MCAO model
Focal cerebral ischemia reperfusion model was induced by transient middle cerebral
artery occlusion (MCAO) as previously described [50-51]. Briefly, the SD rats were randomly
allocated to treatment groups. All surgical procedures were performed after the
intraperitoneal injection of 10% chloral hydrate (0.35mL/100 kg; Sinopharm Chemical
Reagent Co., Ltd., Shanghai, China) and then an incision was made on the neck to expose
the common carotid artery (CCA), the right external carotid artery (ECA) and the internal
carotid artery (ICA). A nylon suture was inserted along the cut to the ICA until it occluded
the middle cerebral artery (about 18 mm). The suture was pulled out for reperfusion after 2
h of ischemia and the incision was sutured closed. Moreover, the rats were fixated in a
stereotaxic apparatus with their head secured firmly in place by ear bars for
intracerebroventricular injection. At 2 h before MCAO, the rats received the drugs in a
volume of 10 μL using the 25 μL microsyringe and the needle was kept in place for 1 min
to prevent backflow after injection. The sham-operated group underwent the same surgical
procedure without insertion of the suture.
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5.3.12 TTC staining and neurological deficit score
The 2,3,5-triphenyltetrazolium chloride (TTC; Sigma, St. Louis, MO, USA) staining
was used to determine the infarct area. The rats were sacrificed by decapitation at 72 h after
completion of reperfusion and thereafter the brains were quickly collected and immediately
sliced into sections of 2 mm thickness. Subsequently, the brain slices were stained with the
2% TTC at 37 °C for about 30 minutes in the dark and fixed with 4% paraformaldehyde
overnight. Infarct sizes were quantified using Image J software (National Institutes of
Health, Bethesda, MD, USA). Besides, the neurological score was assessed before animal
euthanasia and scoring of each rat was performed within 3 min. Five types of motor
neurological findings as follows: 0, no deficits; 1, having difficulty in fully extending the
contralateral forelimb; 2, unable to extend the contralateral forelimb; 3, circling to the
contralateral side; 4, decreasing level of consciousness; 5, falling to the contralateral side.
The higher the neurological score, the more severe the impairment of motor motion.
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5.3.13 Statistical analysis
All experiments were repeated more than three times. The results were expressed as
means ± standard deviation (SEMs). Statistics were analyzed by using the Student’s t-test
or one-way analysis of variance for multiple comparisons in GraphPad Pro (GraphPad, San
Diego, CA, USA). P values less than 0.05 (P <0.05) were considered as significance.
Supporting Information:
Supplemental Figure S1-S33: LC‒MS and 1H NMR spectra of Compound A1-A33.
Supplemental Figure S34: LC‒MS and 1H NMR spectra of Compound B9.
Supplemental Figure S35-S36: LC‒MS and 1H NMR spectra of Compound 1 and
Compound 2.
Author Contributions
Ge Li conducted the biological experiments and prepared the manuscript. Yuantie
Zheng and Linya Hu helped the animal experiments. Jiali Yao oversaw and designed the
chemistry. Furong Ke and Qunpeng Liu synthesized compounds. Weixiao Feng and Ya
Zhao helped to analyze the data. Pencheng Yan, Wenfei He and Hui Deng helped to revise
the manuscript. Peihong Qiu helped to identify structures. Jianzhang Wu and Wulan Li
designed the study, synthesized compounds and drafted the manuscript.
Acknowledgement
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We are grateful to Professor Li Lin of Wenzhou Medical Universtiy for her help. This
work was supported by the ZheJiang Province Natural Science Fund of China (Grant Nos.
Y19B020043, LY17H160059), and Natural Science Foundation of Wenzhou City (Grant
Nos. Y20170158). The Opening Project of Zhejiang Provincial Top Key Discipline of
Pharmaceutical Sciences.
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Graphical abstract：
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