Design, green synthesis, antioxidant activity screening, and evaluation of protective effect on cerebral ischemia reperfusion injury of novel monoenone monocarbonyl curcumin analogs

Article abstract of DOI:10.1016/j.bioorg.2021.105080

Antioxidants with high efficacy and low toxicity have the potential to treat cerebral ischemia reperfusion injury (CIRI). Dienone monocarbonyl curcumin analogs (DMCA) capable of overcoming the instability and pharmacokinetic defects of curcumin possess notable antioxidant activity but are found to be significantly toxic. In this study, a novel skeleton of the monoenone monocarbonyl curcumin analogue sAc possessing reduced toxicity and improved stability was designed on the basis of the DMCA skeleton. Moreover, 32 sAc analogs were obtained by applying a green, simple, and economical synthetic method. Multiple sAc analogs with an antioxidant protective effect in PC12 cells were screened using an H₂O₂-induced oxidative stress damage model, and quantitative evaluation of structure–activity relationship (QSAR) model with regression coefficient of R² = 0.918921 was built through random forest algorithm (RF). Among these compounds, the optimally active compound sAc15 elicited a potent protective effect on cell growth of PC12 cells by effectively eliminating ROS generation in response to oxidative stress injury by activating the Nrf2/HO-1 antioxidant signaling pathway. In addition, sAc15 exhibited good protection against CIRI in the mice middle cerebral artery occlusion (MCAO) model. In this paper, we provide a novel class of antioxidants and a potential compound for stroke treatment.

Full text of DOI:10.1016/j.bioorg.2021.105080

Bioorganic Chemistry 114 (2021) 105080
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, green synthesis, antioxidant activity screening, and evaluation of
protective effect on cerebral ischemia reperfusion injury of novel
monoenone monocarbonyl curcumin analogs
a
,
b
,
1
b
,
1
b
,
1
b
,
1
c
,
1
b
Wenfei He
, Jingsong Wang , Qiling Jin , Jiafeng Zhang , Yugang Liu , Zewu Jin ,
c
b
c
a
d
c
c,*
Hua Wang , Linya Hu , Lu Zhu , Mengya Shen , Lili Huang , Shengwei Huang , Wulan Li
,
c,
*
a,b,*
Qichuan Zhuge , Jianzhang Wu
a
Department of Gynecology and Obstetrics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000,
China
b
Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
c
d
Department of Pharmacy, Ningbo Medical Centre Lihuili Hospital, Ningbo, Zhejiang 315041, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Antioxidants with high efficacy and low toxicity have the potential to treat cerebral ischemia reperfusion injury
Monoenone monocarbonyl curcumin analogs
Michael acceptor
(
CIRI). Dienone monocarbonyl curcumin analogs (DMCA) capable of overcoming the instability and pharma-
cokinetic defects of curcumin possess notable antioxidant activity but are found to be significantly toxic. In this
study, a novel skeleton of the monoenone monocarbonyl curcumin analogue sAc possessing reduced toxicity and
improved stability was designed on the basis of the DMCA skeleton. Moreover, 32 sAc analogs were obtained by
applying a green, simple, and economical synthetic method. Multiple sAc analogs with an antioxidant protective
Toxicity
Antioxidant activity
Cerebral ischemia reperfusion injury
Stroke
2 2
effect in PC12 cells were screened using an H O -induced oxidative stress damage model, and quantitative
2
evaluation of structure–activity relationship (QSAR) model with regression coefficient of R = 0.918921 was
built through random forest algorithm (RF). Among these compounds, the optimally active compound sAc15
elicited a potent protective effect on cell growth of PC12 cells by effectively eliminating ROS generation in
response to oxidative stress injury by activating the Nrf2/HO-1 antioxidant signaling pathway. In addition,
sAc15 exhibited good protection against CIRI in the mice middle cerebral artery occlusion (MCAO) model. In this
paper, we provide a novel class of antioxidants and a potential compound for stroke treatment.
1
. Introduction
main pathogenic mechanism that occurs at the outset [6–8]. Antioxi-
dants have been widely used to treat CIRI by removing the reactive
Stroke, a major cerebrovascular disease worldwide, is associated
with high morbidity, disability, and mortality in people aged >40 years
1]. Stroke is divided into two main categories: ischemic and hemor-
oxygen species (ROS) generated by oxidative stress through activation of
the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant
signaling pathway. Indeed, this is a reportedly effective therapeutic
strategy for CIRI [9,10]. Although intensive studies and even clinical
trials have been conducted in this area, very few drugs capable of acti-
vating the antioxidant signaling pathway have been authorized for
clinical therapy [11]. Therefore, research into powerful and low-toxicity
antioxidants for therapy against CIRI is urgently needed.
[
rhagic. Ischemic stroke accounts for ~87% of all strokes [2]. At present,
thrombolytic therapy to recover blood reperfusion is considered to be
the most efficacious clinical strategy for treating stroke [3,4]. However,
reperfusion can also aggravate brain-tissue injury, a process called
“
cerebral ischemia–reperfusion injury” (CIRI) [5].
Among the various mechanisms underlying CIRI, oxidative stress is a
Currently, leading compounds derived from natural products have
*
Corresponding authors at: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035,
China (W. Li, J. Wu).
E-mail addresses: lwlwzmu@163.com (W. Li), qc.zhuge@wmu.edu.cn (Q. Zhuge), wjzwzmu@163.com (J. Wu).
1
These authors contribute to this work equally.
https://doi.org/10.1016/j.bioorg.2021.105080
Received 13 March 2021; Received in revised form 7 June 2021; Accepted 8 June 2021
Available online 16 June 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

W. He et al.
Bioorganic Chemistry 114 (2021) 105080
emerged as biotherapeutic sources for the discovery and development of
drugs [12–14]. Curcumin, an active and almost non-toxic small-mole-
cule product extracted from the rhizomes of Curcuma longa, exhibits
strong antioxidant activity by activating the Kelch-like ECH-associating
protein 1/Nrf2-antioxidant response element (Keap1/Nrf2/ARE) anti-
oxidant signaling pathway [15–19]. More than 100 curcumin-related
clinical trials have been launched (https://clinicaltrials.gov), but sig-
nificant progress has not been made. This situation can be explained (at
least in part) by the poor stability, low bioavailability, and pharmaco-
kinetic deficiencies caused by an unstable β-diketone moiety in the
curcumin skeleton [20–24]. Consequently, much attention has been
focused on improving the stability of curcumin while retaining or
increasing its biological activities [25–27].
activities and contain a Michael acceptor in the skeleton, but are nearly
non-toxic [35]. The reason for this phenomenon may be that the Michael
acceptor in the skeleton of chalcones comprises only one double bond,
whereas that in DMCAs contains two double bonds. Based on this
analysis, it was hypothesized that the toxicity of DMCAs might be
reduced by removing an olefinic double bond in the skeleton.
Thus, in the present study, a new scaffold was created by removing
an olefinic double bond and a benzene ring from the skeleton of the
DMCA (Ac). That is, a novel scaffold of a monoenone monocarbonyl
curcumin analog (sAc) was designed (Fig. 1A). A cytotoxicity assay
revealed that sAc (10 µM) did not elicit obvious growth inhibition of
PC12 cells compared with the significant inhibitory effect of curcumin
2 2
and the DMCA skeleton Ac. Meanwhile, in a model of H O -induced
The
α
,β-unsaturated ketone moiety (a natural Michael acceptor) is a
oxidative-stress damage, sAc could significantly increase the viability of
PC12 cells, whereas curcumin and Ac could not (Fig. 1C, D). In addition,
the results of stability determination in phosphate-buffered saline
showed that, with the prolongation of time, the OD value of curcumin
(Cur) and its skeleton (Cur-S) decreased sharply, and that of the DMCA
skeleton Ac also decreased notably, suggesting that they were degraded
as a result of structural instability. In contrast, the OD value of sAc
showed no variation, indicating its exceptional stability (Fig. 1B).
Overall, the new skeleton sAc effectively reduced toxicity, improved
stability and, more importantly, greatly enhanced antioxidant
protection.
common introduction group in drug molecules [28] and pharmacophore
that plays a pivotal role in the antioxidant activity of curcumin [28,29].
Thus, the scaffold of an
α,β-unsaturated ketone has always been retained
during the structural modification of curcumin [25,30,31]. By removing
a single carbonyl group from the unstable β-diketone moiety in the
curcumin skeleton, dienone monocarbonyl curcumin analogs (DMCAs)
were obtained (Ac in Fig. 1A). This change has greatly improved the
stability and pharmacokinetic properties of curcumin while maintaining
its original antioxidant activity [32]. However, in subsequent studies,
monocarbonyl curcumin analogs showed a certain degree of toxicity
[
30,33].
In addition, some reports have indicated that the presence of the
In our study, a series of analogs bearing the novel skeleton sAc were
designed and synthesized by applying an environmentally friendly
(“green”) method [36,37]. In addition, their antioxidant activity and
protective effects upon CIRI were evaluated.
Michael acceptor in molecules often resulted in distinct toxicity simul-
taneous with exhibition of antioxidant activity [34]. However, it has
been shown in our previous study that chalcones (which are present in
various fruits and vegetables) possess a broad range of pharmacological
Fig. 1. The design of monoenone monocarbonyl curcumin analogs (A) The design process from the parent compound to the monoenone monocarbonyl curcumin
analogs skeleton (B) Structural stability comparison of the curcumin (Cur.), curcumin skeleton (Cur-S), diketene monocarbonyl curcumin analogs skeleton (Ac) and
monoenone monocarbonyl curcumin analogs skeleton (sAc) (C, D) The cytoprotection and cytotoxicity of curcumin and its different modified skeletons. Cells were
incubated with the 10
Cur., Cur-S, Ac and sAc (10
viability of untreated cells (with 60% DMSO) defined as 100%. Data are expressed as the mean ± SD, n = 3.
μ
M of Cur., Cur-S, Ac and sAc for 42 h (C), and the cytotoxicity of skeletons was determined by the MTT assay. PC12 cells were pre-treated with
μ
M) for 18 h and then exposed to H (640 M) for 24 h (D). The protection of compounds finally determined by the MTT assay and the
2
O
2
μ
#
###
##
****
p < 0.0001,
p < 0.01 vs DMSO,
p < 0.0001,
*
*
2 2
pꢀ0.01 vs H O .
2

W. He et al.
Bioorganic Chemistry 114 (2021) 105080
2
. Results and discussion
Table 1
The monoenone monocarbonyl curcumin analogs.
2
.1. Green synthesis
Comp.
R in ring
Yields
Comp.
R in ring
Yields
sAc1
H
70%
66%
68%
71%
65%
67%
64%
61%
67%
59%
57%
57%
50%
48%
45%
52%
sAc17
sAc18
sAc19
sAc20
sAc21
sAc22
sAc23
sAc24
sAc25
sAc26
sAc27
sAc28
sAc29
sAc30
sAc31
sAc32
3-OCH
3
, 4-OH
, 4-OH
56%
44%
40%
57%
60%
51%
55%
56%
61%
57%
62%
61%
58%
54%
51%
55%
We used proline as a catalyst to synthesize the monoketene mono-
sAc2
2-OCH
3-OCH3
4-OCH
3
3,5-OCH
3-CHO
3-F
3
carbonyl curcumin analog sAc, which takes acetone as the bridge ketone
Scheme 1). All compounds (sAc1–sAc32) were obtained in reactions of
sAc3
(
sAc4
3
sAc5
2,3-OCH
2,4-OCH
2,5-OCH
3,4-OCH
3,5-OCH
3
3
3
3
3
4-F
acetone with the appropriate aldehyde at room temperature. All com-
pounds were purified by column chromatography and obtained in high
yields (40%–71%) after purification (Table 1). This method compen-
sates for the shortcomings of traditional synthetic methods (e.g.,
complicated steps, harsh reaction conditions, and low yield), simplifies
the reaction steps, improves yields, reduces pollution, and realizes green
synthesis.
sAc6
3,4-F
3,5-F
3-Cl
sAc7
sAc8
sAc9
4-Cl
sAc10
sAc11
sAc12
sAc13
sAc14
sAc15
sAc16
2,4,5-OCH
2,4,6-OCH
3,4,5-OCH
3-OH
3
3
3
3,5-Cl
4-Br
4-N(CH
3 2
)
2 5 2
4-N(C H )
4-OH
4-Piperidinyl
4-Morpholin
3,4-OH
2
.2. Cytotoxicity screening and protective effect of curcumin derivatives
a
3-OH, 4-OCH
3
/
on PC12 cells and structure–activity relationship (SAR)
An adrenal pheochromocytoma cell line from rats (PC12) exhibits
the general characteristics of neuroendocrine cells and is used widely in
neurophysiological and neuropharmacological research [39]. The
cytotoxic activity of monoketene monocarbonyl curcumin analogs was
first evaluated by analyzing their effects on the growth of PC12 cells, in
direct comparison with the parent natural products and tertiary butyl-
hydroquinone (TBHQ), a well-known activator of Nrf2. None of the
compounds (sAc1–sAc32) displayed significant cytotoxicity to PC12
cells compared with curcumin or piperlongumine (PL) control groups
comparable to dihydroxy.
2
.3. Quantitative evaluation of structure–activity relationship (QSAR)
QSAR is widely used in drug development as a means to guide drug
design. In recent years, QSAR based on machine learning has been
widely used, among which random forest algorithm (RF) is one of the
effective machine learning algorithms for building QSAR model [38]. RF
is to generate multiple different sub data sets by sampling the total data
set, and train a decision tree for each sub data set. After that, the pre-
diction results of each decision tree are combined as the final prediction
results. In this study, QSAR model was built through RF based on the
corresponding data of cell viability after oxidative damage. As shown in
the Fig. 3, the abscissa represents the cell-survival rate, and the ordinate
represents the value calculated by the QSAR. Regression coefficient of
(
Fig. 2A). Furthermore, the curcumin derivatives sAc(1–7), sAc12,
sAc15, sAc(18–20), sAc23, sAc25, sAc30 and sAc31 showed even less
cytotoxicity than TBHQ.
Uncontrolled ROS production exceeds the clearance capacity of the
endogenous defense system. This action causes excessive ROS accumu-
lation and, ultimately, results in secondary oxidative stress and
apoptosis in neurons [40]. Hydrogen peroxide (H
ROS, is often used to induce oxidative damage and apoptosis of neurons
in vitro [41,42]. Therefore, a model of H -induced oxidative damage
2 2
O ), an important
2
R = 0.918921 with excellent fitting result, and the data distribution of
the QSAR with uniform has a certain guidance for the follow-up
2 2
O
research. sAc15 (blue) is noted at the upper right of the Fig. 3.
was used to assess the cytoprotective activity of compounds. Cell
viability after oxidative damage was ~55% that of the solvent [dimethyl
sulfoxide (DMSO)] group (Fig. 2B). In most groups pre-incubated with
compounds, the cell-survival rate improved to 70%–80%. The protective
activity of sAc15 and nineteen other compounds (sAc(1–5), sAc7, sAc9,
sAc14, sAc(16–24), sAc27 and sAc32) was even better than that of
TBHQ. It should be noted that the cell-survival rates of curcumin and PL
2 2
2.4. sAc15 inhibited H O -induced cell damage to PC12 cells
Considering the toxicity of compounds (sAc1–sAc32) and their
protective effect on PC12 cells, compound sAc15 was selected as the
active compound for further research. Compound sAc15 contains two
phenolic hydroxyl groups, which could directly and indirectly scavenge
free radicals through antioxidant pathways, and exert excellent dual
2 2
groups were lower than that of the H O group, which may be related to
their own toxicity.
Based on the cell-survival rates of sAc1-sAc32 (at 10 µM), a struc-
ture–activity relationship (SAR) was analyzed. The 3,4-dihydroxy cur-
cumin derivatives (sAc15) showed superior protective effects in Fig. 2B
and Table 1. With the decrease of the hydroxyl groups on the benzene
ring, the antioxidant protection of the analogs [sAc13 (3-hydroxy),
sAc14 (4-hydroxy)] weakened. Meanwhile, in contrast to the hydroxy,
the antioxidant activity of the analogs containing one methoxy group
antioxidant activities [8]. sAc15 protected cells damaged by H
induced oxidation and promoted colony formation, further confirming
the cytoprotective effect of sAc15. The damage induced by H
2 2
O -
2 2
O
severely inhibited the colony formation of PC12 cells (Fig. 4A), but this
effect was reversed by sAc15 at very low concentrations. Indeed, the
colony-formation effect of sAc15 was far superior to that of the positive
control drug TBHQ (2.5 µM). In addition, cells with different concen-
trations of sAc15 were pre-incubated to evaluate its antioxidant activity
in vitro. After treatment with sAc15, cell-survival rates increased with
increasing doses in the range of 0.625–20 µM (Fig. 4B). Thus, the pro-
tective effect of sAc15 clearly exceeded that of TBHQ at 20 µM.
[
sAc2 (2-methoxy) or sAc4 (4-methoxy)] was stronger than that of the
two or more methoxy groups [sAc6 (2,4-dihydroxy) and sAc11 (2,4,6-
trimethoxy)]. Consequently, dihydroxy and monomethoxy groups can
enhance the antioxidant activity, but that of monomethoxy could be not
Scheme 1. Synthesis and structures of monoenone monocarbonyl curcumin analogs. Synthetic conditions: (I) L-proline, DMSO, room temperature.
3

W. He et al.
Bioorganic Chemistry 114 (2021) 105080
Fig. 2. Compounds’ cytotoxicity screening on PC12 cells (A) and its cytoprotection on PC12 cells in H
2
O
2
damage model (B). Cells were treated with the 10 M of
μ
curcumin analogs (sAc1-sAc32), TBHQ, PL and curcumin for 42 h (A), and the cytotoxicity of compounds was determined by the MTT assay. PC12 cells were pre-
incubated with curcumin and its derivatives sAc1-sAc32 (10
μ
M) for 18 h and then subjected to H
2
O
2
(640 M) for 24 h. The protection of compounds finally
μ
#
###
determined by the MTT assay and the viability of untreated cells (with 60% DMSO) is defined as 100%. Data are expressed as the mean ± SD, n = 3.
p < 0.0001
*
***
***
**
vs DMSO,
2 2
pꢀ0.0001, pꢀ0.001, p < 0.01, *p < 0.05 vs H O .
level was measured by dichloro-dihydro-fluorescein diacetate (DCFH-
DA) assay. ROS levels in PC12 cells exposed to H were significantly
2 2
O
increased compared with those in the DMSO group (Fig. 4D, E). Pre-
treatment with sAc15 for 18 h eliminated intracellular ROS in a dose-
dependent manner, and the fluorescence intensity of sAc15 at 5 µM
was similar to that of TBHQ at the same concentration.
2
.5. sAc15 increased HO-1 protein expression and exerted antioxidant
properties by activating the Nrf2 signaling pathway
Compound sAc15 exhibited excellent antioxidant properties, so its
mechanism of action was further explored. Nrf2 is a “master” regulator
of oxidative damage to cells [8]. Therefore, an immunofluorescence
assay was used to ascertain if sAc15 could promote Nrf2 accumulation
in nuclei. Blue and red staining represent nuclei and Nrf2, respectively,
in Fig. 5A. Compared with the DMSO group, red fluorescence appeared
in cell nuclei of the sAc15 treatment group, and was stronger than that
of the positive control group (TBHQ). These results suggested that sAc15
could promote Nrf2 accumulation in nuclei. During oxidative stress,
Nrf2 is activated to induce downstream expression of heme oxygenase-1
Fig. 3. QSAR model of sAc15 and related on the cell-survival rate. The QSAR
model of cell-survival rate of PC12 cells was constructed by RF, which was
based on the molecular descriptors of physical and chemical properties and
molecular structure.
(
HO-1) and other antioxidant enzymes, which protect cells through anti-
inflammatory and anti-oxidation mechanisms, and apoptosis regulation
44]. Next, the effect of sAc15 on expression of HO-1 protein in PC12
cells was evaluated. Increased expression of the antioxidant enzyme HO-
was observed in cells incubated with sAc15; moreover, with
[
Imbalance in the production and clearance of ROS leads to oxidative
stress, which will destroy proteins, enzymes, lipids and other cellular
components, and form the lipid-peroxidation product malondialdehyde
1
increasing concentrations of sAc15, higher expression of HO-1 protein
(
MDA). MDA test results shown in Fig. 4C reveal that MDA contents in
the H -damaged group were appreciably higher than that in the sol-
vent group. After pre-incubation with sAc15 for 18 h, the MDA content
decreased significantly. When cells are stimulated by H
, a “burst” of
ROS occurs [43]. Compound sAc15 had an inhibitory effect on excessive
ROS generation in cells caused by H -induced oxidation. The ROS
was documented (Fig. 5B). To further confirm the protective effect of
2 2
O
2 2
compound sAc15 on cells damaged by H O -induced oxidative stress,
expression of Nrf2 protein was downregulated by transfecting Nrf2 small
interfering (si)RNA into PC12 cells (Fig. 5D). The protection elicited by
sAc15 against oxidative-stress damage was evaluated for cells in Nrf2
siRNA and control siRNA groups. Downregulation of Nrf2 expression
2 2
O
2 2
O
4

W. He et al.
Bioorganic Chemistry 114 (2021) 105080
Fig. 4. Protective effects of sAc15 against H
2
O
2
-induced oxidative stress damage in PC12 cells. (A) Compound sAc15 promoted the colony formation of PC12 cells
after exposed to H
2
O
2
. PC12 cells were pretreated with sAc15 (0.625, 1.25 and 2.5
μ
M) and TBHQ (2.5
-induced cell damage in a dose-dependent manner. PC12 cells were pre-incubated with TBHQ (20
M) for 18 h. Then, cells were stimulated with H (650 М) for 24 h. The MTT assay was used to detect the
M) and TBHQ (5 M) for
2 2
O (1 mM) for 6 h (C) or 2 h (D, E). The MDA levels (C) and the level of intracellular ROS (D, E) were determined according to the
μ
M) for 18 h, and then incubated with H
2
O
2
(200
μ
M) for 8
days. (B) Compound sAc15 protected PC12 cells from H
sAc15 in different doses (0.625, 1.25, 2.5, 5, 10 and 20
2
O
2
μ
M) and
μ
2
O
2
μ
cell viability and the viability of untreated cells (with 60% DMSO) is defined as 100%. PC12 cells were incubated with sAc15 (1, 5 and 10
8 h, then treated with H
manufacturer’s instructions. Data are expressed as the mean ± SD, n = 3.
.05 vs H
μ
μ
1
#
###
###
****
***
**
p < 0.0001,
p < 0.001 vs DMSO,
p < 0.0001, p < 0.001, p < 0.01, *p <
0
2 2
O .
inhibited the protective effect of sAc15 (Fig. 5C). Thus, sAc15 could
protect PC12 cells from oxidative damage by targeting Nrf2 to enhance
HO-1 expression.
had a good neuroprotective effect against cerebral ischemia.
3. Conclusions
2
.6. sAc15 alleviated brain injury after ischemia–reperfusion in mice
In view of the excellent cytoprotective effect of compound sAc15 in
In the current study, a green, simple, and economical synthetic
method using edible proline as a catalyst was adopted. This method is
suitable for synthesis of monoenone monocarbonyl curcumin analogs
with either cyclopentanone or acetone as the bridged ketone, and
different substituent groups on the benzene ring.
vitro, the neuroprotective effect of sAc15 in vivo was investigated further
using mice. Middle cerebral artery occlusion (MCAO), a common model
of focal cerebral ischemia, is widely used to study the pathogenesis of
cerebral ischemia and screen for therapeutic drugs because its patho-
genesis is similar to that of ischemic stroke in humans [45]. Butylph-
thalide (NBP) is widely used in patients with acute ischemic stroke, the
neuroprotective effects of compound sAc15 and NBP on mice were
assessed by intraperitoneal injection before MCAO. Brain slices from the
sham-operated group had no infarcted tissue, whereas the prevalence of
infarction in the model group brain tissue was ~ 30% (Fig. 6A–C). There
was no statistically significant difference in cerebral infarction rates
between the vehicle group and the model group, indicating that the
solvent itself had no neuroprotective effect. Areas of infarction in the
administration group (sAc15 and NBP) were reduced markedly
compared with those in model or solvent groups, and neurological
scores were remarkably improved. Hence, the active compound sAc15
To improve the stability of curcumin and retain its pharmacological
activities, extensive studies have explored a large number of obtained
derivatives. Among them, DMCAs are considered promising derivatives
that can overcome the defects of curcumin. In the present study, a series
of monoenone monocarbonyl curcumin analogs with better prospects
for drug development were identified and found to have greater stabil-
ity, lower toxicity, and stronger antioxidant activity compared with
DMCAs.
The vast majority of the 32 analogs did not generate prominent
toxicity in PC12 cells at 10 µM. Moreover, various sAc derivatives with
distinguished antioxidant protective effects were identified by screening
2 2
with a model of H O -induced oxidation damage in PC12 cells. Among
these analogs, the optimally active compound sAc15, which exhibited
far stronger activity than the positive drug TBHQ, was acquired. Our
5

W. He et al.
Bioorganic Chemistry 114 (2021) 105080
Fig. 5. Compound sAc15 protects PC12 cells against oxidative stress damage by activating the Nrf2 signaling pathway. (A) Compound sAc15 induced Nrf2 nuclei
translocation in PC12 cells. After incubation with sAc15 (10 M) and TBHQ (5 M) for 6 h, Nrf2 antibody was incubated and DAPI staining was used. The nuclei
translocation images were taken under a fluorescence microscope. (B) Compound sAc15 up-regulated the protein expression of HO-1 in PC12 cells. The cells were
incubated with sAc15 (1, 5 and 10 M) and TBHQ (5 M) for 18 h, and the level of HO-1 protein was measured by western blot assay. (C, D) The cytoprotection of
sAc15 weakened after down-regulated the level of Nrf2 protein by siRNA. PC12 cells were transfected respectively with Nrf2 siRNA (si-Nrf2) and control siRNA (NC),
pretreated with sAc15 (0.625 M) for 18 h, and then exposed to H (650 M) for another 24 h. The cell viability was determined by MTT assay and the protein
μ
μ
μ
μ
μ
2
O
2
μ
#
###
##
#
&
levels were analyzed by western blot assay. Data are expressed as the mean ± SD, n = 3.
2 2
p < 0.0001, pꢀ0.01, pꢀ0.05 vs DMSO, *p < 0.05 vs H O , pꢀ0.05
vs NC.
results revealed that sAc15 could significantly increase the viability of
PC12 cells damaged by H
, even at 0.625 µM. Moreover, sAc15 could
dramatically promote colony formation, suppress MDA production, and
reduce intracellular ROS accumulation in PC12 cells damaged by H
4. Experimental
2 2
O
4.1. Chemistry
2 2
O -
induced oxidative stress. Furthermore, sAc15 markedly activated the
Nrf2 signaling pathway and promoted Nrf2 translocation into nuclei to
upregulate expression of the antioxidant protein HO-1, whereas its
protective antioxidant effect on cells almost disappeared when Nrf2
expression was silenced by siRNA. In addition, 2,3,5-triphenyltetrazo-
lium chloride staining of MCAO model mice showed that sAc15 could
effectively decrease the area of cerebral infarction.
Chemical materials and reagents were commercially purchased from
Sigma Aldrich (St Louis, Missouri, USA), Aladdin (Shanghai, China), and
no further purification was carried out. Reaction progress was moni-
tored with thin-layer chromatography (TLC) making of silica gel GF254.
The chromatograms were performed on silica gel (200–300 mesh) and
observed under UV light. In addition, melting points (mp) were
measured with open capillary tubes on a Fisher-Johns melting apparatus
without correction. Mass spectra (MS) were acquired on the Agilent
In summary, a group of stable, low-toxicity curcumin analogs with
outstanding antioxidant activity were synthesized by an environmen-
tally friendly and economical method, and screened. The optimally
active compound sAc15 could effectively scavenge ROS and MDA
generated from oxidative-stress damage by activating the Nrf2/HO-1
antioxidant signaling pathway, and exhibited excellent antioxidant ac-
tivity in vitro. Moreover, sAc15 showed a strong protective effect against
CIRI in vivo. Overall, our findings show that sAc15 could emerge as a
novel antioxidant candidate drug, as well as a promising chemothera-
peutic agent, for the further development of CIRI treatment.
1
1100 LC-MS device (Agilent, Palo Alto, CA, USA). H NMR spectra were
recorded on the 600 MHz spectrometer (Bruker Corporation,
Switzerland) with tetramethylsilane (TMS) as an internal standard
3 6
substance and CDCl or DMSO-d as solvent. Coupling constants (J) are
expressed in Hz, and splitting patterns are explained as follows: s =
singlet; d = doublet; t = triplet; m = multiplet; dd = doublet of doublets;
dt = doublet of triplets.
The spectral data of compounds are listed as follows:
4
.1.1. (E)-4-phenylbut-3-en-2-one (sAc1)
Yellow powder, 70% yield, mp 34.8–36.0 ꢀ [35-36 ꢀ,[46]]. H NMR
1
2
(
600 MHz, CDCl
3
), δ: 7.558–7.542 (m, 2H, Ar-H , β-H), 7.520 (d, J =
6 3 4 5
1
6.2 Hz, 1H, Ar-H ), 7.409–7.398 (m, 3H, Ar-H , Ar-H , Ar-H ), 6.725
-H), 2.390 (s, 3H, CH
). LC-MS m/z: 147.08 (M +
(
d, J = 16.2 Hz, 1H,
α
3
6

W. He et al.
Bioorganic Chemistry 114 (2021) 105080
Fig. 6. Compound sAc15 showed pre-protective effect on mice brain tissue after MCAO. (A) Representative images of TTC-stained brain sections from different
experimental groups. (B) Infarct areas (%) levels and neurological score (C) of each group. The sham-operated (Sham) mice or MCAO reperfusion mice received
normal saline (NS), vehicle (DMSO: cremophor: normal saline = 1: 19: 80), sAc15 (10 or 20 mg/kg) and BNP (10 mg/kg) respectively by intraperitoneal injection.
#
###
****
***
Data are expressed as the mean ± SD, n ≥ 6.
p < 0.0001 represent the statistical difference between NS and sham-operated group,
pꢀ0.0001, p < 0.001,
*
p < 0.05 represent the statistical difference between vehicle-treated and the medicated group.
+
4
5
H) , calcd for C10
H
10O: 146.07.
(d, J = 2.4 Hz, 2H, Ar-H , Ar-H ), 6.815 (d, J = 16.8 Hz, 1H,
s, 3H, 2-OCH ), 3.762 (s, 3H, 3-OCH ), 2.317 (s, 3H, CH ). LC-MS m/z:
: 206.09.
α
-H), 3.814
(
3
+
3
3
4
.1.2. (E)-4-(2-methoxyphenyl)but-3-en-2-one (sAc2)
Yellow powder, 66% yield, mp 43.9–45.7 ꢀ [liquid,[46]]. 1H NMR
600 MHz, DMSO-d
), δ: 7.770 (d, J = 16.2 Hz, 1H, β-H), 7.682 (t, J =
207.10 (M+H) , calcd for C12
14 3
H O
(
6
4.1.6. (E)-4-(2,4-dimethoxyphenyl)but-3-en-2-one (sAc6)
6
4
White powder, 67% yield, mp 56.1–57.2 ꢀ [58.5-59.5 ꢀ,[47]]. 1H
6
1
α
.6 Hz, 1H, Ar-H ), 7.425–7.396 (m, 1H, Ar-H ), 7.086 (d, J = 8.4 Hz,
3
5
6
H, Ar-H ), 6.983 (t, J = 7.2 Hz, 1H, Ar-H ), 6.821 (d, J = 16.8 Hz, 1H,
NMR (600 MHz, CDCl
3
), δ: 7.806 (d, J = 16.2 Hz, 1H, Ar-H ), 7.487 (d, J
5
-H), 3.859 (s, 3H, 2-OCH
3
), 2.293 (s, 3H, CH
: 176.08.
3
). LC-MS m/z: 177.09 (M
= 9.0 Hz, 1H, β-H), 6.675 (d, J = 16.2 Hz, 1H, Ar-H ), 6.517 (dd, J = 2.4
+
3
+
H) , calcd for C11
H
12
O
2
Hz, J = 6.6 Hz, 1H, Ar-H ), 6.455 (d, J = 2.4 Hz, 1H,
α
-H), 3.877 (s, 3H,
2
-OCH
3
), 3.844 (s, 3H, 4-OCH
3
), 2.360 (s, 3H, CH
3
). LC-MS m/z: 207.10
+
4
.1.3. (E)-4-(3-methoxyphenyl)but-3-en-2-one (sAc3)
(M + H) , calcd for C12
H O : 206.09.
14 3
1
Orange oil, 68% yield [liquid,[46]]. H NMR (600 MHz, DMSO-d
6
),
5
δ: 7.578 (d, J = 16.2 Hz, 1H, β-H), 7.330 (t, J = 8.4 Hz, 1H, Ar-H ), 7.264
4.1.7. (E)-4-(2,5-dimethoxyphenyl)but-3-en-2-one (sAc7)
Yellow powder, 64% yield, mp 41.2–42.1 ꢀ [49-51 ꢀ,[48]]. H NMR
(600 MHz, CDCl
), δ: 7.859 (d, J = 16.2 Hz, 1H, β-H), 7.076 (d, J = 3.0
2
6
4
1
(
(
d, J = 7.2 Hz, 2H, Ar-H , Ar-H ), 6.983 (t, J = 7.8 Hz, 1H, Ar-H ), 6.814
d, J = 16.8 Hz, 1H,
α
-H), 3.780 (s, 3H, 3-OCH
3
), 2.317 (s, 3H, CH
: 176.08.
3
).
3
+
3
6
LC-MS m/z: 177.09 (M + H) , calcd for C11
H
12
O
2
Hz, 1H, Ar-H ), 6.931 (dd, J = 3.0 Hz, J = 6.0 Hz, 1H, Ar-H ), 6.859 (d, J
4
=
9.0 Hz, 1H, Ar-H ), 6.713 (d, J = 16.2 Hz, 1H,
α
-H), 3.855 (s, 3H,
4
.1.4. (E)-4-(4-methoxyphenyl)but-3-en-2-one (sAc4)
Yellow powder, 71% yield, mp 67.4–68.2 ꢀ [75-78 ꢀ,[46]]. H NMR
2-OCH
3
), 3.791 (s, 3H, 5-OCH
3
), 2.391 (s, 3H, CH
3
). LC-MS m/z: 207.10
1
+
(M + H) , calcd for C12
H O : 206.09.
14 3
2
6
(
600 MHz, CDCl
3
), δ: 7.505–7.463 (m, 3H, Ar-H , Ar-H , β-H), 6.917 (d,
3 5
J = 8.4 Hz, 2H, Ar-H , Ar-H ), 6.608 (t, J = 7.2 Hz, 1H,
α
-H), 3.838 (d, J
4.1.8. (E)-4-(3,4-dimethoxyphenyl)but-3-en-2-one (sAc8)
Yellow powder, 61% yield, mp 78.1–79.9 ꢀ [81-82 ꢀ,[49]]. H NMR
(600 MHz, DMSO-d
), δ: 7.541 (d, J = 16.2 Hz, 1H, β-H), 7.305 (d, J =
1
=
7.2 Hz, 3H, 4-OCH
3
), 2.354 (d, J = 7.2 Hz, 3H, CH
3
). LC-MS m/z:
+
1
77.09 (M + H) , calcd for C11
H
12
O
2
: 176.08.
6
2
5
1
.2 Hz, 1H, Ar-H ), 7.236 (dd, J = 1.2 Hz, J = 7.2 Hz, 1H, Ar-H ), 6.986
6
4
.1.5. (E)-4-(2,3-dimethoxyphenyl)but-3-en-2-one (sAc5)
(d, J = 8.4 Hz, 1H, Ar-H ), 6.720 (d, J = 16.2 Hz, 1H,
α
-H), 3.785 (d, J =
1
Yellow oil, 65% yield. H NMR (600 MHz, DMSO-d
6
), δ: 7.724 (d, J
6.0 Hz, 6H, 3-OCH
3
, 4-OCH
3
), 2.287 (s, 3H, CH
3
). LC-MS m/z: 207.10
6
+
=
16.2 Hz, 1H, β-H), 7.315 (dd, J = 1.8 Hz, J = 4.8 Hz, 1H, Ar-H ), 7.114
(M + H) , calcd for C12
H O : 206.09.
14 3
7

W. He et al.
Bioorganic Chemistry 114 (2021) 105080
4
.1.9. (E)-4-(3,5-dimethoxyphenyl)but-3-en-2-one (sAc9)
White powder, 67% yield, mp 68.5–69.4 ꢀ [70-72 ꢀ,[50]]. H NMR
600 MHz, DMSO-d
), δ: 7.529 (d, J = 16.2 Hz, 1H, β-H), 6.873 (d, J =
4.1.18. (E)-4-(4-hydroxy-3,5-dimethoxyphenyl)but-3-en-2-one (sAc18)
1
Beige powder, 44% yield, mp 131.2–132.5 ꢀ [132.5-134.5 ꢀ,[55]].
1
(
6
H NMR (600 MHz, CDCl
3
), δ: 7.429 (d, J = 16.2 Hz, 1H, β-H), 6.794 (s,
2
6
2
6
1
.8 Hz, 2H, Ar-H , Ar-H ), 6.820 (d, J = 16.8 Hz, 1H,
α
-H), 6.542 (s, 1H,
), 2.310 (s, 3H, CH ). LC-MS m/z:
: 206.09.
2H, Ar-H , Ar-H ), 6.597 (d, J = 16.2 Hz, 1H,
3-OCH , 5-OCH ), 2.369 (s, 3H, -CH
calcd for C12 : 222.09.
α
-H), 3.926 (s, 6H,
4
+
Ar-H ), 3.761 (s, 6H, 3-OCH
3
, 5-OCH
3
3
3
3
α
3
). LC-MS m/z: 223.09 (M + H) ,
+
2
07.10 (M + H) , calcd for C12
H
14
O
3
14 4
H O
4
.1.10. (E)-4-(2,4,5-trimethoxyphenyl)but-3-en-2-one (sAc10)
4.1.19. (E)-3-(3-oxobut-1-en-1-yl)benzaldehyde (sAc19)
White powder, 59% yield, mp 94.5–95.0 ꢀ [106-108 ꢀ,[51]]. 1H
White powder, 40% yield, mp 47.9–48.8 ꢀ. H NMR (600 MHz,
1
6
NMR (600 MHz, CDCl
3
), δ: 7.732 (d, J = 16.2 Hz, 1H, β-H), 7.225 (s, 1H,
CDCl
3
), δ: 10.052 (s, 1H, 3-CHO), 8.048 (t, J = 1.8 Hz, 1H, Ar-H ), 7.905
3
5
4
α
-H), 6.734 (t, J = 16.2 Hz, 2H, Ar-H , Ar-H ), 3.839 (d, J = 11.4 Hz, 6H,
(dt, J = 7.8 Hz, J = 7.2 Hz, 1H, Ar-H ), 7.798 (dd, J = 1.2 Hz, J = 6.6 Hz,
2 5
2
-OCH
3
, 5-OCH
3
), 3.733 (s, 3H, 4-OCH
3
), 2.251 (s, 3H, CH
: 236.10.
3
). LC-MS
1H, β-H), 7.601–7.547 (m, 2H, Ar-H , Ar-H ), 6.809 (d, J = 16.2 Hz, 1H,
+
+
m/z: 237.11 (M + H) , calcd for C13
H
16
O
4
α
-H), 2.406 (s, 3H, CH
3
). LC-MS m/z: 175.07 (M + H) , calcd for
11 10 2
C H O : 174.07.
4
.1.11. (E)-4-(2,4,6-trimethoxyphenyl)but-3-en-2-one (sAc11)
Yellow powder, 57% yield, mp 116.8–117.6 ꢀ [116.8-117.6ꢀ,[51]].
H NMR (600 MHz, CDCl
), δ: 7.937 (d, J = 16.8 Hz, 1H, β-H), 7.063 (d,
4.1.20. (E)-4-(3-fluorophenyl)but-3-en-2-one (sAc20)
1
1
3
Yellow oil, 57% yield [liquid,[46]]. H NMR (600 MHz, DMSO-d
6
), δ:
5
3
5
6
J = 16.8 Hz, 1H,
α
-H), 6.115 (s, 2H, Ar-H , Ar-H ), 3.858 (d, J = 15.6 Hz,
, 4-OCH , 6-OCH ), 2.343 (s, 3H, CH ). LC-MS m/z: 237.11
: 236.10.
7.604 (t, J = 4.2 Hz, 2H, β-H, Ar-H ), 7.537 (d, J = 7.2 Hz, 1H, Ar-H ),
4
2
9
H, 2-OCH
3
3
3
3
7.481–7.444 (m, 1H, Ar-H ), 7.266–7.235 (m, 1H, Ar-H ), 6.863 (d, J =
+
+
(
M + H) , calcd for C13
H
16
O
4
16.2 Hz, 1H,
calcd for C10
α
-H), 2.322 (s, 3H, CH
3
). LC-MS m/z: 165.07 (M + H) ,
9
H FO: 164.06.
4
.1.12. (E)-4-(3,4,5-trimethoxyphenyl)but-3-en-2-one (sAc12)
White powder, 57% yield, mp 87.0–88.1 ꢀ [84-85 ꢀ,[52]]. H NMR
600 MHz, CDCl
), δ: 7.433 (d, J = 16.2 Hz, 1H, β-H), 6.775 (s, 2H,
-H), 3.893 (d, J = 4.2 Hz, 9H,
), 2.384 (s, 3H, CH
). LC-MS m/z: 237.11 (M +
: 236.10.
1
4.1.21. (E)-4-(4-fluorophenyl)but-3-en-2-one (sAc21)
1
(
3
Yellow oil, 60% yield [38-40 ꢀ,[46]]. H NMR (600 MHz, DMSO-d
6
),
2
6
3
5
Ar-H , Ar-H ), 6.631 (d, J = 16.2 Hz, 1H,
α
δ: 7.788–7.764 (m, 2H, Ar-H , Ar-H ), 7.613 (d, J = 16.8 Hz, 1H, β-H),
2 6
3
-OCH
3
, 4-OCH
3
, 5-OCH
3
3
7.261 (t, J = 9.0 Hz, 2H, Ar-H , Ar-H ), 6.762 (d, J = 16.2 Hz, 1H,
α
-H),
+
+
H) , calcd for C13
H
16
O
4
2.312 (s, 3H, CH
3
). LC-MS m/z: 165.07 (M + H) , calcd for C10
H
9
FO:
1
64.06.
4
.1.13. (E)-4-(3-hydroxyphenyl)but-3-en-2-one (sAc13)
White powder, 50% yield, mp 91.3–92.1 ꢀ [94-95 ꢀ,[53]]. H NMR
600 MHz, DMSO-d
), δ: 9.654 (s, 1H, 3-OH), 7.508 (d, J = 16.2 Hz, 1H,
β-H), 7.214 (t, J = 7.8 Hz, 1H, Ar-H ), 7.110 (d, J = 7.8 Hz, 1H, Ar-H ),
1
4.1.22. (E)-4-(3,4-difluorophenyl)but-3-en-2-one (sAc22)
1
(
6
Yellow oil, 51% yield [39 ꢀ,[56]]. H NMR (600 MHz, DMSO-d
6
), δ:
5
6
5
6
7.893–7.860 (m, 1H, β-H), 7.578 (d, J = 16.8 Hz, 2H, Ar-H , Ar-H ),
4
2
2
7
.025 (s, 1H, Ar-H ), 6.819 (dd, J = 1.2 Hz, J = 6.6 Hz, 1H, Ar-H ), 6.665
d, J = 16.2 Hz, 1H, -H), 2.304 (s, 3H, CH ). LC-MS m/z: 163.07 (M +
: 162.07.
7.491 (d, J = 10.2 Hz, 1H, Ar-H ), 6.829 (d, J = 16.2 Hz, 1H,
α
-H), 2.310
+
(
α
3
(s, 3H, CH
3
). LC-MS m/z: 183.06 (M + H) , calcd for C10
H F O: 182.05.
8 2
+
10 2
H) , calcd for C10H O
4
.1.23. (E)-4-(3,5-difluorophenyl)but-3-en-2-one (sAc23)
1
4
.1.14. (E)-4-(4-hydroxyphenyl)but-3-en-2-one (sAc14)
Yellow oil, 55% yield [58 ꢀ,[56]]. H NMR (600 MHz, DMSO-d
6
), δ:
6
2
Yellow powder, 48% yield, mp 83.2–85.5 ꢀ [102.1-104.8 ꢀ,[54]].
7.572 (d, J = 16.2 Hz, 1H, β-H), 7.501 (d, J = 6.6 Hz, 2H, Ar-H , Ar-H ),
1
4
H NMR (600 MHz, CDCl
3
), δ: 7.495 (d, J = 16.2 Hz, 1H, β-H), 7.453 (dt,
7.312–7.282 (m, 1H,
3H, CH
α
-H), 6.928 (d, J = 16.2 Hz, 1H, Ar-H ), 2.319 (s,
2
6
+
J = 8.4 Hz, J = 9.6 Hz, 2H, Ar-H , Ar-H ), 6.889 (dd, J = 1.8 Hz, J = 4.8
3
). LC-MS m/z: 183.06 (M + H) , calcd for C10
8 2
H F O: 182.05.
3
5
Hz, 2H, Ar-H , Ar-H ), 6.609 (d, J = 16.2 Hz, 1H,
α
10
-H), 2.383 (s, 3H,
: 162.07.
+
CH
3
). LC-MS m/z: 163.07 (M + H) , calcd for C10
H
O
2
4.1.24. (E)-4-(3-chlorophenyl)but-3-en-2-one (sAc24)
1
Yellow oil, 56% yield [36 ꢀ,[57]]. H NMR (600 MHz, DMSO-d
6
), δ:
6
4
.1.15. (E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one (sAc15)
Brown powder, 45% yield, mp 162.8–164.0 ꢀ [173-175 ꢀ,[53]]. H
NMR (600 MHz, DMSO-d
), δ: 9.587 (s, 1H, 3-OH), 9.159 (s, 1H, 4-OH),
.432 (d, J = 16.2 Hz, 1H, β-H), 7.042 (d, J = 1.8 Hz, 1H, Ar-H ), 6.990
7.812 (s, 1H, β-H), 7.671 (d, J = 7.2 Hz, 1H, Ar-H ), 7.587 (d, J = 16.2
1
5 2 4
Hz, 1H, Ar-H ), 7.477–7.430 (m, 2H, Ar-H , Ar-H ), 6.880 (d, J = 16.2
+
6
Hz, 1H,
α-H), 2.319 (s, 3H, CH
3
). LC-MS m/z: 181.04 (M + H) , calcd for
2
7
10 9
C H ClO: 180.03.
5
6
(
dd, J = 1.8 Hz, J = 6.6 Hz, 1H, Ar-H ), 6.758 (d, J = 8.4 Hz, 1H, Ar-H ),
6
.462 (d, J = 16.2 Hz, 1H,
α
-H), 2.258 (s, 3H, CH
3
). LC-MS m/z: 179.07
4.1.25. (E)-4-(4-chlorophenyl)but-3-en-2-one (sAc25)
White powder, 61% yield, mp 51.6–52.2 ꢀ [63-65 ꢀ,[46]]. H NMR
+
1
(
M + H) , calcd for C10
10 3
H O : 178.06.
2
6
(
600 MHz, CDCl
3
), δ: 7.485–7.448 (m, 3H, Ar-H , Ar-H , β-H),
3
5
4
.1.16. (E)-4-(3-hydroxy-4-methoxyphenyl)but-3-en-2-one (sAc16)
7.387–7.365 (m, 2H, Ar-H , Ar-H ), 6.689 (d, J = 16.2 Hz, 1H,
α-H),
1
+
Beige powder, 52% yield, mp 84.0–85.5 ꢀ [74-76 ꢀ,[53]]. H NMR
2.382 (s, 3H, CH
180.03.
3
). LC-MS m/z: 181.04 (M + H) , calcd for C10
H
9
ClO:
(
600 MHz, CDCl
3
), δ: 7.428 (d, J = 16.2 Hz, 1H, β-H), 7.155 (d, J = 2.4
2
5
Hz, 1H, Ar-H ), 7.059 (dd, J = 2.4 Hz, J = 6.0 Hz, 1H, Ar-H ), 6.861 (d, J
6
=
7.8 Hz, 1H, Ar-H ), 6.588 (d, J = 16.2 Hz, 1H,
-OCH ), 2.358 (s, 3H, CH
: 192.08.
α
-H), 3.936 (s, 3H,
4.1.26. (E)-4-(3,5-dichlorophenyl)but-3-en-2-one (sAc26)
+
White powder, 57% yield, mp 72.0–72.5 ꢀ. 1H NMR (600 MHz,
4
3
3
). LC-MS m/z: 193.08 (M + H) , calcd for
4
C
11
H
12
O
3
CDCl
3
), δ: 7.407 (d, J = 1.8 Hz, 2H, β-H, Ar-H ), 7.367 (t, J = 1.8 Hz, 2H,
2
6
Ar-H , Ar-H ), 6.700 (d, J = 16.2 Hz, 1H,
α
-H), 2.379 (s, 3H, CH
3
). LC-
+
4
.1.17. (E)-4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one (sAc17)
MS m/z: 215.99 (M + H) , calcd for C10
H
8
2
Cl O: 214.00.
Beige powder, 56% yield, mp 115.9–117.8 ꢀ [125.3-126.6 ꢀ,[54]].
1
H NMR (600 MHz, CDCl
3
), δ: 7.451 (d, J = 16.2 Hz, 1H, β-H), 7.093
4.1.27. (E)-4-(4-bromophenyl)but-3-en-2-one (sAc27)
White powder, 62% yield, mp 74.9–76.4 ꢀ [46-48 ꢀ,[46]]. H NMR
2
5
1
(
dd, J = 1.8 Hz, J = 6.6 Hz, 1H, Ar-H ), 7.060 (d, J = 1.8 Hz, 1H, Ar-H ),
6
3
5
6
.935 (d, J = 8.4 Hz, 1H,
α
-H), 6.589 (d, J = 16.2 Hz, 1H, Ar-H ), 3.937
(600 MHz, CDCl
3
), δ: 7.532 (d, J = 8.4 Hz, 2H, Ar-H , Ar-H ), 7.442 (d, J
+
2 6
(
s, 3H, 3-OCH
3
), 2.368 (s, 3H, CH
3
). LC-MS m/z: 193.08 (M + H) , calcd
= 16.2 Hz, 1H, β-H), 7.405 (d, J = 8.4 Hz, 2H, Ar-H , Ar-H ), 6.700 (d, J
+
for C11
H
12
O
3
: 192.08.
= 16.2 Hz, 1H,
α
-H), 2.376 (s, 3H, CH
3
). LC-MS m/z: 224.99 (M + H) ,
8

W. He et al.
Bioorganic Chemistry 114 (2021) 105080
calcd for C10
H
9
BrO: 223.98.
evaluated by ultraviolet spectrophotometry.
4
.1.28. (E)-4-(4-(dimethylamino)phenyl)but-3-en-2-one (sAc28)
4.4. Biological evaluation
1
Yellow powder, 61% yield, mp 129.7–130.7 ꢀ [135-137 ꢀ,[58]]. H
2
6
NMR (600 MHz, CDCl
3
), δ: 7.453 (t, J = 16.8 Hz, 3H, Ar-H , Ar-H , β-H),
4.4.1. Cell culture
3
5
6
.678 (d, J = 8.4 Hz, 2H, Ar-H , Ar-H ), 6.546 (d, J = 16.8 Hz, 1H,
α
-H),
An adrenal pheochromocytoma cell line from rats (PC12 ) were
purchased from Wuhan University Cell Storage Center (Wuhan, China).
The cell lines were cultured in 1 × DMEM (4.5 g/L D-glucose, Gibco,
Eggenstein, Germany) complemented with 10% fetal bovine serum
(FBS, Gibco) and 1% penicillin/streptomycin. The cells were maintained
3
.031 (s, 6H, 4-N(CH
3
)
2
), 2.340 (s, 3H, CH
3
). LC-MS m/z: 190.12 (M +
+
H) , calcd for C12
H
15NO: 189.12.
4
.1.29. (E)-4-(4-(diethylamino)phenyl)but-3-en-2-one (sAc29)
1
◦
Yellow oil, 58% yield. H NMR (600 MHz, DMSO-d
6
), δ: 7.480–7.451
at 37 C constant temperature incubators with 5% CO (Thermo Fisher
2
2
6
3
5
(
m, 3H, Ar-H , Ar-H , β-H), 6.658 (d, J = 9.0 Hz, 2H, Ar-H , Ar-H ),
Scientific, Massachusetts, USA).
6
.485 (d, J = 16.2 Hz, 1H,
α
-H), 3.388–3.353 (m, 4H, N(CH
), 1.086 (t, J = 6.6 Hz, 6H, C(CH ). LC-MS m/z: 218.15 (M +
19NO: 217.15.
2 2
) ), 2.235 (s,
3
H, CH
3
3
)
2
4.4.2. MTT assay
+
H) , calcd for C14
H
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
(
MTT; Sigma, St Louis, Missouri, USA) assay also called MTT colorimetry
4
.1.30. (E)-4-(4-(piperidin-1-yl)phenyl)but-3-en-2-one (sAc30)
was used to detect the toxicity and the cytoprotection of compounds. In
Yellow powder, 54% yield, mp 98.4–99.4 ꢀ. 1H NMR (600 MHz,
3
brief, approximate 5 × 10 PC12 cells were plated onto each well of 96-
2
6
DMSO-d
6
), δ: 7.490 (t, J = 8.4 Hz, 3H, Ar-H , Ar-H , β-H), 6.913 (d, J =
well plates (NEST Biotechnology Co. LTD., Wuxi, China) and incubated
3
5
◦
9
.0 Hz, 2H, Ar-H , Ar-H ), 6.545 (d, J = 16.2 Hz, 1H,
α
-H), 3.031 (s, 4H,
at 37 C in 5% CO for the night. Then, the compounds dissolved in 60%
2
N(CH
2
)
2
), 2.250 (s, 3H, CH
3
), 1.557 (s, 6H, CH
19NO: 229.15.
2
CH
2
CH ). LC-MS m/z:
2
DMSO was added to incubate with cells for 18 h, and H O was added to
2
2
+
2
30.15 (M + H) , calcd for C15
H
stimulate for 24 h or not. 20
μ
L MTT (0.5 mg/mL) was added and
◦
incubated with cells at 37 C for 4 h shielded from light. The liquid was
4
.1.31. (E)-4-(4-morpholinophenyl)but-3-en-2-one (sAc31)
then carefully removed and DMSO (120 L) was added to each well.
μ
1
Yellow powder, 51% yield, mp 131.7–132.8 ꢀ. H NMR (600 MHz,
Finally, the absorbency at 490 nm was measured using a Microplate
Reader (Bio-Rad, USA). Every test repeated at least three times.
2
6
CDCl
3
), δ: 7.466–7.415 (m, 3H, Ar-H , Ar-H , β-H), 6.884 (t, J = 7.8 Hz,
3 5
2
H, Ar-H , Ar-H ), 6.603–6.524 (m, 1H,
α
-H), 3.841 (d, J = 30.0 Hz, 4H,
), 2.351–2.298 (m, 3H, CH ).
17NO : 231.13.
O(CH
2
)
2
), 3.257–3.206 (m, 4H, N(CH
2
)
2
3
4.4.3. Cell colony formation assay
+
LC-MS m/z: 232.13 (M + H) , calcd for C14
H
2
The cell colony formation assay is used to assess the proliferation
capacity of cells at very low densities. 2000 individual PC12 cells were
evenly seeded into 6-well plates and cultured for night. Cells were pre-
treated with sAc15 and TBHQ for 18 h, and the cells were incubated
4
.1.32. (E)-4-(naphthalen-2-yl)but-3-en-2-one (sAc32)
White powder, 55% yield, mp 97.0–98.3 ꢀ [103-104 ꢀ,[59]]. 1H
3
NMR (600 MHz, CDCl
3
), δ: 7.965 (s, 1H, Ar-H ), 7.878–7.836 (m, 3H,
with H O (200 M) continuously for 8 days. Finally, cell colonies were
μ
2
2
2
6
7
8
Ar-H , Ar-H , Ar-H ), 7.698–7.672 (m, 2H, Ar-H , β-H), 7.539–7.519 (m,
2
fixed with 4% paraformaldehyde for 30 min and the fixed colony cells
were stained using crystal violet for 30 min before dried to be taken
photographs.
4
5
H, Ar-H , Ar-H ), 6.842 (d, J = 16.2 Hz, 1H,
α
-H), 2.431 (s, 3H, CH
3
).
+
LC-MS m/z: 197.09 (M + H) , calcd for C14
H12O: 196.09.
4
.2. General procedure for synthesis of sAc15 analogs
4.4.4. MDA assay
5
PC12 cells were seeded on 6-well plates at 3 × 10 /well for 24 h, then
Taking a reaction flask as reaction vessel, 1 mmol of substituted
incubated with compound sAc15 and TBHQ for 18 h, and then stimu-
benzaldehyde and 5 mmol of acetone were dissolved in anhydrous
dimethyl sulfoxide (DMSO). Then, 0.2 mmol of L-proline was added to
the reaction system as a catalyst, and the mixture was stirred at room
temperature overnight. Next, 2–3 drops of condensed HCl were added to
the solution, and the reaction was unceasingly stirred at room temper-
ature for 2–3 h. Taking thin layer chromatography (TLC) as a monitoring
way, when the endpoint of the reaction was arrived, the mixture was
extracted with ethyl acetate/saturated sodium chloride aqueous solu-
tion (3 × 20 mL). The organic layer was enriched and dried with
lated with H O (1 mM) for 6 h. The protein supernatant was collected
2
2
and reacted with thiobarbituric acid (TBA) to measure the level of MDA.
The relative MDA content was calculated strictly according to the
method provided by the reagent manufacturer (Beyotime Biotech,
China).
4.4.5. Reactive oxygen species assay
The fluorescent probe dichloro-dihydro-fluorescein diacetate
(DCFH-DA; Beyotime Biotech, Shanghai, China) was utilized to detect
intracellular reactive oxygen species (ROS) formation. The experimental
procedures are strictly carried out according to manufacturer’s recom-
2 4
anhydrous Na SO . And a crude product was obtained by concentrated
and evaporated the organic layer in vacuo. At last, the pure product was
got on a silica gel column and ethyl acetate and petroleum ether were
used as eluent.
5
mendations (Beyotime Biotech). Briefly, 3 × 10 PC12 cells were plated
in 6-well plates, allowed to attach for 24 h and then cells were treated
2 2
with sAc15 for 18 h and H O (1 mM) was carried out for another 2 h.
4
.3. Stability test
Then cells were incubated with DCFH-DA at a terminal concentration of
◦
1
0
μM at 37 C for 30 min in the dark. The images were made in a
The certain mass of compound was completely dissolved in a certain
fluorescence microscope (Nikon).
volume of dimethyl sulfoxide (DMSO) to get a solution with a concen-
tration of 1 mM, then the concentration of the solution was diluted to 30
4.4.6. Immunofluorescence assay
5
μ
M with phosphate buffer (PBS, PH = 7.4). The optical density (OD) of
PC12 cells were seeded on 6-well plates at 2 × 10 /well, incubated
with sAc15 and TBHQ for 6 h after 24 h, fixed with 4% para-
formaldehyde for 20 min, washed with PBS for three times, then treated
with 1% Triton X100 (Beyotime Biotech, China) for 15 min, washed with
PBS for three times, then sealed with 2% BSA at room temperature for 1
h, and then added Nrf2 antibody (sc-13032, Santa Cruz biotechnology,
USA, 1: 200). The next day, washed with PBS for 3 times, and then
the diluted solution at a wavelength of 200 to 800 nm was measured by
UV–Visible spectrophotometer. Taking 5 min as a time interval and 5 nm
as a wavelength interval, every single diluted sample solution was tested
for 30 min each time. Then three times independent experiments were
conducted to each sample solution and the absorption curve was ob-
tained by data analysis. Finally, the stability of compounds can be
9

W. He et al.
Bioorganic Chemistry 114 (2021) 105080
incubated with fluorescent mouse antibody (sc-13032, Goat anti mouse
loco-motor activity, loss of consciousness. For 2,3,5-triphenyltetrazo-
lium chloride (TTC; Sigma, St. Louis, MO, USA) staining, the brain tis-
◦
IgG PE, Santa Cruz biotechnology, USA, 1: 300) in 37 C incubator for 1
◦
h. Washed with PBS for 3 times, and then incubated with DAPI (Beyo-
time Biotech, China) in dark for 8 min. After sealing with neutral resin,
the cells photographed under the microscope (Nikon, Japan).
sue was taken out and frozen at ꢀ 20 C for 20 min, and then placed in a
brain slice mold and cut into 1 mm continuous sections. After that, the
◦
brain slices were stained with 2% of TTC at 37 C shielded from light
until the normal sections were rosy red and the infarcted tissues were
white and were fixed with 4% paraformaldehyde. In the end, after brain
tissues were completely fixed and the brain slices were photographed,
and the infarct size was measured by Image J software (National In-
stitutes of Health, Bethesda, MD, USA).
4
.4.7. Western blot assay
Western blot assay was used to detect the level of protein expression
in cells. At 18 h post incubation with compounds, cells were harvested
for western blot. Total protein was extracted with ice-cold RIPA lysis
buffer and then was separated by sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS-PAGE). The separated protein was
subsequently transferred to a Polyvinylidene Fluoride (PVDF) mem-
brane (Millipore, Massachusetts, USA), which were blocked at room
temperature with 5% skim milk for 1 h and then probed overnight using
primary antibodies (HO-1, sc-10789, Santa Cruz Biotechnology, 1:500;
4.4.12. Quantitative structure–activity relationships analysis
This modeling is based on Python language and SCI learn machine
learning library. First of all, we use rdkit to calculate the molecular
descriptors of small molecules, which is a basic requirement for building
QSAR model. The results include 208 dimensional 2D molecular de-
scriptors, such as molecular weight, topological polarity surface area,
lipid water partition coefficient, etc. In these descriptors, the descriptors
whose contents are equal and undifferentiated are deleted. After
screening, 123 dimensional molecular descriptors were retained. The
molecular descriptors are input into the random forest, and the QSAR
model is obtained by computer training. In this modeling process, the
default number of sub decision trees is 800.
◦
anti-GADPH, sc-47724, Santa Cruz Biotechnology, 1:2000) at 4 C. Next
day, the PVDF membranes were incubated with secondary antibodies for
9
0 min at room temperature. The protein bands were imaged by a
ChemiDoc XRS + system (Bio-Rad, Hercules, CA, USA) and measured
using Image J software (National Institute of Health, Bethesda, MD,
USA).
4
.4.8. Transfection assay
The short interfering RNA (siRNA) targeting Nrf2 was provided by
4.4.13. Statistical analysis
′
Shanghai GenePharma Company (siRNA sequence, sense: 5 -GGUUCA-
The datum was presented as means ± SEM. The statistical differences
were analyzed by the unpaired t test or ordinary one-way ANOVE. P
values <0.05 (P < 0.05) were considered as significant differences.
′
′
GUGACUCGGAAAUTT-3 ,
antisense:
5 -AUUUCCGAGUCACU-
′
GAACCTT-3 ). For the process of transfection, PC12 cells were plated in
culture dishes (1 million per dish) and transfection with Nrf2 siRNA was
performed using the Lipofectamine™ 2000 Transfection reagent (Invi-
trogen, Carlsbad, CA, USA) according to the manufacturer’s recom-
mendations. During transfection, cells were incubated with the
starvation medium to improve the transfection efficiency. 10 h after
transfection, the medium containing fetal bovine serum was used to
replace the original starvation medium and the transfected cells could be
cultured for further experiments.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
4
.4.9. Experimental animals
This work was supported by Zhejiang Province Natural Science Fund
of China (Grant Nos. LY19B020012, LQ20H090004), National Natural
Science Foundation of China (82003755), National Undergraduate
All male C57BL/6 mice used for animal experiments were obtained
from the Zhejiang Muke Biotechnology Co., Ltd. All mice were fed
during a period of adaptive breeding until the weight of the mice
reached 23–26 g. All animal experiments are followed the principles of
the National Institutes of Health guide for the care and use of Laboratory
animals (NIH Publications No.8023, revised 1978).
Training
Programs
for
Innovation
and
Entrepreneurship
(201910343026, 201810343025), International (Regional) Cooperation
and Exchange Projects of the National Natural Science Foundation of
China (81820108011), and the Key Project of Science Technology
Department of Zhejiang Province (2017C03027)
4
.4.10. MCAO-induced cerebral ischemia/reperfusion injury model
We thank Arshad Makhdum, PhD, from Liwen Bianji, Edanz Group
China (www.liwenbianji.cn/ac), for editing the English text of a draft of
this manuscript.
All the animals were divided into 6 groups evenly, and the enter-
ocoelia was injected with normal saline, solvent (DMSO: cremophor:
normal saline = 1: 19: 80), sAc15 and butylphthalide (NBP) separately
at 1 h before MCAO. Firstly, male B6 mice were narcotized by injecting
Appendix A. Supplementary material
1
% sodium pentobarbital (50 mg/kg) into their peritoneal cavities, and
placed them in the supine position. Secondly, the neck was cut and
exposed the right common carotid artery (CCA), external carotid artery
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105080.
(
ECA) and the internal carotid artery (ICA). Thirdly, ECA and its small
vessels were ligated with thin wires, ICA and CCA were temporarily
closed with arterial forceps. In the end, a small incision was made on the
ECA ~ 2 mm away from the CCA branch to insert the occlusion line to
block the middle cerebral artery. 1 h after MCAO, the nylon mono-
filament was removed and reperfusion for 48 h. The sham operation
group performed the same operation but was not inserted the suture.
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