Discovery of coumarin-derived imino sulfonates as a novel class of potential cardioprotective agents

Article abstract of DOI:10.1016/j.ejmech.2019.111779

The burst of reactive oxygen species (ROS) contributes to and exacerbates cardiac injury. Exogenous supplementation of antioxidants or upregulation of endogenous antioxidant defense genes should be the potential therapies for cardiovascular disease. Sixteen coumarin-derived imino sulfonates compounds were synthesized with the ability of attenuating oxidative stress directly by reducing intracellular ROS level via promoting Nrf2 pathway. The cell-based assays showed that most of the compounds had significant protective activity against H₂O₂-induced oxidative injury in H9c2 cells. Compound 5h with the highest activity and low cytotoxicity was demonstrated to remarkably remove the intracellular ROS accumulation by activating expressions of Nrf2 and its downstream antioxidant proteins (ie. HO-1 and NQO1), indicating a novel promising antioxidant and Nrf2 activator. Overall, these findings demonstrated that compound 5h could serve as a potential cardioprotective agent. Moreover, our study features developing new antioxidants and Nrf2 activators by introducing a sulfonyl group and nitrogen atom to the α,β-unsaturated carbonyl entity in coumarin, rather than adding new functional groups or active fragments to coumarin itself.

Full text of DOI:10.1016/j.ejmech.2019.111779

Journal Pre-proof
Discovery of coumarin-derived imino sulfonates as a novel class of potential
cardioprotective agents
Bo Wei, Jing Zhou, Jia-Jia Xu, Jing Cui, Feng-Feng Ping, Jing-Jing Ling, Ya-Jing
Chen
PII:
S0223-5234(19)30931-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.111779
EJMECH 111779
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 August 2019
Revised Date: 24 September 2019
Accepted Date: 9 October 2019
Please cite this article as: B. Wei, J. Zhou, J.-J. Xu, J. Cui, F.-F. Ping, J.-J. Ling, Y.-J. Chen, Discovery
of coumarin-derived imino sulfonates as a novel class of potential cardioprotective agents, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111779.
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2019 Published by Elsevier Masson SAS.

Cl
N
S
O
sulfonyl group
imino group
O
O
O
O
Coumarin
Imino sulfonate 5h
5h presents potential cardioprotective
activity and low cytotoxicity with the
ability of attenuating oxidative stress
directly by reducing intracellular ROS
level via promoting Nrf2 pathway.

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Discovery of coumarin-derived imino sulfonates as a novel class of
potential cardioprotective agents
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Bo Wei , Jing Zhou , Jia-Jia Xu , Jing Cui , Feng-Feng Ping , Jing-Jing Ling , Ya-Jing
a,*
Chen
a
School of Pharmaceutical Sciences, Key Laboratory of Advanced Drug Preparation
Technologies, Ministry of Education of China; Co-innovation Center of Henan
Province for New Drug R & D and Preclinical Safety, Zhengzhou University, 100
Science Avenue, Zhengzhou 450001, Henan, P.R. China
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Wuxi People’s Hospital affiliated to Nanjing Medical University, Wuxi 214023, P.R.
China
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Wuxi children’s hospital, Wuxi 214023, P.R. China
*
Corresponding author. School of Pharmaceutical Sciences, Zhengzhou University,
100 Science Avenue, Zhengzhou 450001, Henan, P.R. China
E-mail address: chenyj@zzu.edu.cn (Y.-J. Chen).
Abbreviations
Con, control; CVD, cardiovascular disease; DHE, dihydroethidium; LDH, lactate
dehydrogenase; LUT, luteolin; ROS, reactive oxygen species; Nuclear factor
erythroid 2 related factor 2 (Nrf2); heme oxygenase-1 (HO-1), NAD(P)H quinone
oxidoreductase 1 (NQO1).
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Abstract
The burst of reactive oxygen species (ROS) contributes to and exacerbates cardiac
injury. Exogenous supplementation of antioxidants or upregulation of endogenous
antioxidant defense genes should be the potential therapies for cardiovascular disease.
Sixteen coumarin-derived imino sulfonates compounds were synthesized with the
ability of attenuating oxidative stress directly by reducing intracellular ROS level via
promoting Nrf2 pathway. The cell-based assays showed that most of the compounds
had significant protective activity against H O -induced oxidative injury in H9c2 cells.
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Compound 5h with the highest activity and low cytotoxicity was demonstrated to
remarkably remove the intracellular ROS accumulation by activating expressions of
Nrf2 and its downstream antioxidant proteins (ie. HO-1 and NQO1), indicating a
novel promising antioxidant and Nrf2 activator. Overall, these findings demonstrated
that compound 5h could serve as a potential cardioprotective agent. Moreover, our
study features developing new antioxidants and Nrf2 activators by introducing a
sulfonyl group and nitrogen atom to the α,β-unsaturated carbonyl entity in coumarin,
rather than adding new functional groups or active fragments to coumarin itself.
Keywords:
Cardiovascular disease; Coumarin; Imino sulfonates; Cardioprotective agents;
Oxidative stress; Nrf2
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1. Introduction
Cardiovascular disease (CVD) is one of the critical diseases seriously endangering
human health [1]. Accumulated evidences have demonstrated that the burst of reactive
oxygen species (ROS) induced by oxidative stress extremely exacerbate structural and
functional alterations in the heart tissue in cardiovascular disease, including
myocardial ischemia/reperfusion injury, cardiac hypertrophy and heart failure [2-9].
Thus, exogenous supplementation of antioxidants and upregulation of endogenous
antioxidant defense genes should be the potential therapies for cardiovascular diseases
[10-12].
Nuclear factor erythroid 2 related factor 2 (Nrf2) is a transcription factor that
regulates oxidative stress by inducing expression of antioxidant enzyme genes, such
as heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1),
glutamate-cysteine ligase (GCL), and several members of the glutathione
S-transferase family [13]. Notably, Nrf2 is closely associated with cancer,
neurodegenerative diseases, especially for cardiovascular diseases [14-17]. Our
previous study has also proved that activation of Nrf2 signaling could upregulate the
damaged cardiac endogenous antioxidant system and decrease cardiac injury in rats
[11,12,18]. Therefore, development of antioxidants and Nrf2 activators may display
significant advantages against cardiovascular disease [19,20].
Kumar reported that a chalcone derivative (1) was a potent activator of the Nrf2
signaling pathway [21] (Fig. 1). Park found that the introducing of a vinyl sulfonyl
group (2) to the α,β-unsaturated carbonyl entity of chalcone 1 led to higher activity to
activate Keap1-Nrf2 signaling pathway and subsequent dose-dependent induction of
Nrf2-dependent antioxidant enzymes expression (HO-1, NQO1, GCLC, and GLCM)
[22,23]. In addition, nitrogen heterocycles are among the most significant structural
components of pharmaceuticals, and 59% of unique small-molecule drugs in U.S.
Food and Drug Administration-approved drugs contain a nitrogen heterocycle [24-29].
Optimized vinyl sulfone (3) by introducing nitrogen heterocycle showed superior
effect on Nrf2 activation in cell-based assays compared to compound 2 [30]. These
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results indicate that sulfonyl group and nitrogen heterocycle are benefit for activating
Nrf2 signaling pathway.
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Fig. 1. Superior effect on Nrf2 activation by introducing sulfonyl group and nitrogen atom to
chalcone.
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Coumarins, a wide class of natural and synthetic compounds, have attracted
considerable attention in recent years due to its diverse pharmacological actions, such
as neuroprotective [31-35], antifungal [36] and anticancer [37] activities. Some
coumarin derivatives have also been proven to be excellent antioxidants and Nrf2
activators [38-43].
Traditional methods: adding new functional groups or active fragments to coumarin
OH
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OH
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Ph
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Oxypeucedanin
Imino derivative of coumarin
(A)
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Coumarin
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Quadricyclic coumarin
Amino derivative of coumarin
This work: introducing a sulfonyl group and nitrogen atom to the unsaturated carbonyl entity in coumarin
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Scheme 1. Previous reports and our design for developing new antioxidants or Nrf2 activators
based on the structure of coumarin.
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Traditional methods for developing new coumarin-derived active compounds rely
on adding new functional groups or active fragments to coumarin (Scheme 1A).
Inspired by Park’s work, what we did innovatively in this study was that we
introduced a sulfonyl group and nitrogen atom to the α,β-unsaturated carbonyl entity
in coumarin to generate coumarin-derived imino sulfonates 5, and the potential
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cardioprotective activities of these synthesized compounds were further evaluated
(Scheme 1B).
Initially, we screened the derivatives for their potential capacity to protect H9c2
cardiomyocyte cells from oxidative stress injury induced by hydrogen peroxide
(H O ). We found that the introducing of a sulfonyl group and imino group indeed led
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to the potent activities. The compound with highest activity was demonstrated to
activate Nrf2 signaling pathway, thus further inducing the expressions of
Nrf2-dependent endogenous antioxidant proteins (ie. HO-1 and NQO1).
2. Results and discussion
2.1 Chemistry
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As illustrated in Scheme 2, sixteen coumarin-derived imino sulfonates (5) were
prepared based on a standard protocol [44]. Briefly, the reaction of chlorosulfonyl
isocyanate with formic acid generate chlorosulfonamide. Next, target compounds 5
were synthesized by cyclization reaction of various salicylaldehydes and
chlorosulfonamide.
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Scheme 2. Synthesis of compounds 5.
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2.2 Biological screenings
Primarily, all target compounds 5 were screened for their protective activity against
oxidative stress induced by H O in H9c2 cells, a pivotal model for potential
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cardioprotective agents screening [45].
2.2.1 Protective activity against H O -induced H9c2 cells damage and inherent
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toxicity
In order to explore the potential cardioprotective activity of the target compounds,
sixteen coumarin-derived imino sulfonates (5) were evaluated against oxidative stress
injury induced by H O in H9c2 cells by MTT assay. To demonstrate the
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structure-activity relationship, different substituents were introduced to
coumarin-derived imino sulfonates.
As depicted in Fig. 2A, most of analogues, except 5o, were found to effectively
increased the cell viability in H9c2 subjected to H O .
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Fig. 2. Compounds' cytoprotection on H9c2 cells in H O damage model (A) and cytotoxicity
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evaluation of three targeted compounds (5f, 5g and 5h) at different concentrations in H9c2
cells (B-D). H9c2 cells were pretreated for 24 h with LUT or coumarin-derived imino
sulfonates 5 at 20 µM, respectively, followed by 2 h exposure in H O (250 µM). The
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cytotoxicity of compounds was determined by the MTT assay. H9c2 cells were pretreated for
24 h with 5f, 5g or 5h at 0.1, 1, 10, 100 µM, respectively, Three individual experiments were
performed for each group. The data was expressed as the Mean ± SD. ##P < 0.01, #P < 0.05
vs Control, **P < 0.01, *P < 0.05 vs H O . one way ANOVA, followed by Tukey's multiple
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comparison test.
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All electron-donating group substituted compounds (5d, 5e, 5i, 5j, 5k, 5n, 5o, 5p)
are unfavorable for the activity comparing to 5a.
Comparison of fluoride substituted compounds (5b, 5g, 5l) (5, 7, 8-position on
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benzene ring) demonstrated that substitutes on 5- and 8- position are unfavorable for
the activity.
Comparison of chloride substituted compounds (5c, 5h, 5m) (6, 7, 8-positionon
benzene ring) further demonstrated that substitute on 8-position is unfavorable for the
activity.
Notably, for 5- and 8- substituted compounds, neither electron withdrawing group
nor electron donating group is unfavorable for the activity.
For 6- and 7- substituted compounds, electron-withdrawing group substituted
compounds (5c, 5f, 5g, 5h) led to increased activities. However, electron-donating
group substituted compounds (5d, 5e, 5i, 5j, 5k) led to obvious decreased activities
comparing to 5a. Moreover, substituted on 7- position is more favorable than
substituted on 6- position (5c vs 5h).
Among the sixteen compounds, 5f, 5g and 5h showed the cardioprotective action
similar or even better to that of LUT. In addition, we found that 5f, 5g and 5h at up to
100 µM did not exhibit any significant cytotoxicity in H9c2 cells, suggesting the low
cytotoxicity of these compounds (Fig. 2B-D).
2.2.2 Protection of H9c2 cells from H O -induced damage by 5f, 5g or 5h
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To further explore the exact compound possessing cardioprotective activity,
different concentrations of three targeted compounds (5f, 5g and 5h) were conducted
in H9c2 suffered from H O2 injury, respectively. As showed in Fig. 3A,
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pre-incubation with these compounds for 24 h, 2.5 µM of 5f, 5g and 5h exhibited
none cardioprotective action, as well as LUT. At 5 µM, only 5h significantly
increased the cell viability, as compared to the H O group (Fig. 3B). Moreover, 5h at
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10 µM were found to have better advantageous cytoprotection, relative to that in cells
preincubated with 5f, 5g or LUT (Fig. 3C).
Meanwhile, the release rate of LDH is an important index to monitor the cellular
membrane integrity [10,46]. Our study revealed that treatment with H O caused
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dramatically increased LDH release rate, compared to the control group, indicating
the loss of cell membrane integrity and necrotic cell death caused by oxidative stress.
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Pretreatment with compound 5h at 10 µM could significantly decreased such index
(Fig. 3D), compared to the H O treated cells. However, administration with
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compounds 5f and 5g showed no significant difference compared to those in H O
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group, respectively. Moreover, 5f, 5g and 5h at 20 µM could markedly decrease the
LDH release rate (Fig. 3E). Notably, 5h (20 µM) exhibited the most excellent activity
in declining LDH release rate in H9c2 against H O induced injury.
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Fig. 3. Compound 5h exhibited the excellent cytoprotective activity in H9c2 against
H O -induced cell damage. H9c2 cells were pretreated with 5f, 5g, 5h and LUT at different
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concentrations (2.5, 5, 10 µM) for 24 h, respectively, then treated with 250 µM H O for 2 h,
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and determined by the MTT assay (A-C). The viability of untreated cells is defined as 100%.
H9c2 cells were pre-incubated with 5f, 5g, 5h or LUT at 10, 20 µΜ for 24 h, respectively,
then treated with 250 µM H O for 2 h, thus the LDH release rate was determined (D and E).
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Three individual experiments were performed for each group. The data was expressed as the
Mean ± SD. ##P < 0.01, #P < 0.05 vs Control, **P < 0.01, *P < 0.05 vs H O . one way
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ANOVA, followed by Tukey's multiple comparison test.
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From the bioactivity data of compounds 5f, 5g and 5h, the results revealed that 5h
had the most valuable cellular protective activity for cardiomyocytes. Given the fact
that coumarin derivatives have been demonstrated to possess positive cardiovascular
effects, which are not only based on their anti-oxidant action, but also based on the
anti-inflammatory effects, regulation of the intracellular calcium or nitric oxide (NO)
level, vasorelaxant properties and so on [47-50]. Therefore, in order to investigate the
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mechanism of this series of compounds, compound 5h with the highest potency was
further evaluated for the possibility of signaling pathways in its cardioprotective
effect.
Due to the important role of oxidative stress, inflammation, and overload of
calcium in cardiovascular diseases (CVD), we have conducted an experiment to
analyse the possible mechanisms underlying the potential cardioprotective effect of
compound 5h. Specifically, transcription factor Nrf2 is the master regulator of the
antioxidative enzyme genes expression [51], and activation of Nrf2 has been
considered to be the crucial therapy against oxidative stress in CVD. NF-κB, another
important transcription factor, mainly mediates the inflammatory responses, and
downregulation of NF-κB could significantly inhibit the inflammation in CVD [52].
In addition, prior animal and human studies have documented that decreased
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expression of sarcoplasmic reticulum Ca -ATPase 2 (SERCA2), a major cardiac
calcium cycling protein ameliorating the overload of calcium, as a primary defect
found in CVD [53,54]. Importantly, increasing the activity of SERCA2α in patients
with moderate to severe heart failure improves their cardiac function, disease status
and quality of life [55,56].
Hence, the protein expressions of Nrf2, NF-κB p65 in nucleus, along with SERCA2
in cytoplasm were measured by Western blotting, as Fig. 4 depicted. Our data showed
that H O treatment induced a robust increase in the expression of NF-κB and Nrf2 in
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nucleus. The levels of SERCA2 were significantly decreased in H O group,
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compared to the control cells. By contrast, pretreatment with compound 5h further
stimulated protein expression of Nrf2, suggest a hyperactivation of the Nrf2 pathway.
However, 5h administration only showed a trend to reverse the abnormally increased
NF-κB expression and declined SERCA2 level induced by H O , and no statistical
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difference was found. Collectively, these results indeed elucidated that compound 5h
reduced the myocardial injury induced by H O mainly through its antioxidant
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activity.
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Fig. 4. Possible mechanisms underlying the protective effect of compound 5h. H9c2 cells
were pretreated with 5h (20 µM) for 24 h, then treated with 250 µM H O for 2 h. The protein
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expressions of Nrf2, NF-κB p65 and SERCA2 were determined by Western Blotting (A-D).
Cells treated with none compound were served as the Control (Con). Protein expression levels
were normalised against levels of Lamin B1 or GAPDH, which was used as a loading control
for nuclear and cytoplasmic protein samples, respectively. The data was expressed as the
Mean ± SD (n=3). ##P < 0.01, #P < 0.05 vs Con, **P < 0.01, *P < 0.05 vs H O . one way
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ANOVA, followed by Tukey's multiple comparison test.
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2.2.3 Compound 5h scavenged the cellular ROS by activating the Nrf2 signaling
pathway
The burst of oxidants, such as reactive oxygen species (ROS), has been
demonstrated to participate in breaking down the cell membrane integrity, which
cause the cardiac injury [10,11]. Stimulation of cardiomyocytes with H O led to the
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cellular ROS accumulation. Thus, we further examined whether compound 5h could
decline the ROS production, and explored the mechanisms underlying such action. As
illustrated in Fig. 5A-B, pretreatment with compound 5h (10 µM) for 24 h
dramatically reduced the cellular ROS accumulation, suggesting that scanvenging
ROS by 5h may contribute to its cytoprotective activity against oxidative injury
induced by H O .
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The NF-E2-related factor 2 (Nrf2) transcription factor pathway has been
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demonstrated to restore cellular redox homeostasis by increasing endogenous
antioxidant response element-mediated expression of phase II and antioxidant
enzymes, such as NAD(P)H quinone oxidoreductase-1 (NQO1) and heme
oxygenase-1 (HO-1). Importantly, Nrf2 closely correlates with cardiac protection
against myocardial injury. Therefore, the Nrf2 pathway has been considered as a
novel therapeutic target for cardiovascular diseases, including myocardial infarction,
myocardial ischemia/reperfusion and myocardial hypertrophy [11,57]. In the present
study, we discovered that 5h could dose-dependently induce the Nrf2 translocation
into the nucleus, with a corresponding upregulated Nrf2-dependent proteins, such as
HO-1 and NQO1 (Fig. 5C-F). Additionally, 5h at 10 µM showed even more stronger
promoting effect on Nrf2 pathway than that of LUT.
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Fig. 5. Protection of compound 5h by scavenging ROS via activating the Nrf2 signaling
pathway. H9c2 cells were pretreated with 5h (10 µM) for 24 h, then treated with 250 µM
H O for 2 h, and intracellular reactive oxygen species (ROS) generation was measured by the
DHE assay (200×, bar=400µm) (A and B). H9c2 cells were treated with 5h (0.5, 1, 2, 5, 10
µM) or LUT (10 µM) for 24 h, respectively. Then the protein expressions of Nrf2, HO-1and
NQO1 were determined by Western Blotting (C-F). Cells treated with none compound were
served as the Control (Con). Protein expression levels were normalised against levels of
Lamin B1 or GAPDH, which was used as a loading control for nuclear and cytoplasmic
protein samples, respectively. The data was expressed as the Mean ± SD (n=3). ##P < 0.01,
#P < 0.05 vs Con, one way ANOVA, followed by Tukey's multiple comparison test.
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3. Conclusion
In this study, a series of coumarin-derived imino sulfonates were synthesized by
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introducing a sulfonyl group and nitrogen atom to the α,β-unsaturated carbonyl entity
in coumarin. The cytoprotection of all these analogues was detected in H O induced
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H9c2 cells injury model. Most synthesized compounds displayed potent
cardioprotective activities at 20 µM. Structure-activity relationships indicated that
compounds with different substituents on benzene ring have great influence on their
biological activities. Notably, after pre-incubation with compounds for 24 h,
compounds 5f, 5g and 5h displayed valuable cytoprotection against oxidative damage
in H9c2 cells. Among them, compound 5h was screened out to exhibit low
cytotoxicity and an inspirable protection against oxidative stress in myocytes in a
concentration dependent manner. Consistent with its cytoprotective activity, 5h also
exhibited potent anti-oxidative activity by removing the cellular ROS accumulation,
thus reducing the loss of cellular membrane integrity as demonstrated by LDH release
rate. In addition, mechanism of action studies confirmed that 5h may confer its
protection for H9c2 cells against oxidative insults through inducing the nuclear
translocation of Nrf2, thus upregulating the expression of endogenous antioxidant
proteins HO-1 and NQO1.
In summary, our work presented a series of coumarin-derived imino sulfonates with
antioxidant activity, and 5h represents a novel therapeutic agent with potential for
treating cardiovascular diseases. Our present study may prove important for the future
design of coumarin-derived structurally related Nrf2 activators.
4. Experimental
4.1. Chemistry
All reactions were performed in oven-dried glassware with magnetic stirring.
Unless otherwise stated, all reagents were purchased from commercial suppliers and
used without further purification. All solvents were purified and dried according to
standard methods prior to use. Organic solutions were concentrated under reduced
pressure on a rotary evaporator or an oil pump. Reactions were monitored through
thin layer chromatography (TLC) on silica gel-precoated glass plates. Subsequent to
elution, plates were visualized using UV radiation at 254 nm and by staining with
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94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
aqueous potassium permanganate or ethanolic phosphomolybdic acid solution. Flash
column chromatography was performed using silica gel (300-400 mesh). Nuclear
Magnetic Resonance (NMR) spectras were acquired on a Varian Mercury 400
1
13
19
operating at 400, 100 and 376 MHz for H, C and F, respectively. Chemical shifts
1
are reported in δ ppm referenced to an internal SiMe standard for H NMR,
4
1
3
1
chloroform-d (δ 77.16) for C NMR. Datas for H NMR are recorded as follows:
chemical shift (δ ppm), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
13
multiplet; br s, broad), coupling constant (Hz) and integration. Datas for C NMR
1
9
and F NMR are reported in terms of chemical shift (δ, ppm). High-resolution mass
spectra (HRMS) were recorded on a Thermo Q-Exactive Spectrometer (ESI source).
4.1.1. General procedure for the preparation of imino sulfonates (5) [44]
Anhydrous formic acid (40.0 mmol, 1.5 mL, 1 eq) was added dropwise to neat
o
chlorosulfonyl isocyanate (40.0 mmol, 3.5 mL, 1 eq) at 0 C with rapid stirring.
Vigorous gas evolution was observed during the addition process. The resulting
viscous suspension was stirred at room temperature until gas evolution ceased (12 h).
The resulting colorless solid was used in the following step immediately and stored
under -20 °C.
o
To a solution of salicylaldehyde (15.0 mmol) in DMA (100 mL) at 0 C was
carefully added freshly prepared ClSO NH (4.6 g, 40.0 mmol) in small portions, and
2
2
the resulting solution was stirred for 12 h at room temperature. The reaction was
quenched carefully with ice-cold water (100 mL), and the mixture was transferred to a
separating funnel containing CH Cl (200 mL). The aqueous layer was separated and
2
2
extracted with CH Cl (3 × 50 mL), and the combined organic layers were washed
2
2
with saturated NaHCO solution (100 mL), dried (MgSO ), filtered through a short
3
4
pad of silica using CH Cl as eluent and concentrated in vacuo. Imino sulfonates 5
2
2
were isolated by flash chromatography (PE/EtOAc) on silica gel.
1
4.1.1.1 benzo[e][1,2,3]oxathiazine 2,2-dioxide (5a). H NMR (400 MHz, CDCl ) δ
3
8.68 (s, 1H), 7.79-7.74 (m, 1H), 7.70-7.68 (m, 1H), 7.46-7.42 (m, 1H), 7.30 (d, J =
1
3
8.4 Hz, 1H) ppm. C NMR (100 MHz, CDCl ) δ 167.9, 154.3, 137.8, 131.0, 126.3,
3
1
3

—
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
118.7, 115.5 ppm. HRMS (ESI): m/z Exact mass calcd. for C H NO S [M+OCH ] :
8 8 4 3
214.0180, found: 214.0160.
4.1.1.2 5-fluorobenzo[e][1,2,3]oxathiazine 2,2-dioxide (5b). H NMR (400 MHz,
1
1
3
CDCl ) δ 8.96 (s, 1H), 7.78-7.72 (m, 1H), 7.15-7.11 (m, 2H) ppm; C NMR (100
3
MHz, CDCl ) δ 161.9 (d, J = 6.0 Hz), 161.1 (d, J = 261.9 Hz), 154.7 (d, J = 3.0 Hz),
3
139.0 (d, J = 11.0 Hz), 114.6 (d, J = 4.0 Hz), 112.8 (d, J = 20.0 Hz), 105.9 (d, J = 17.0
—
Hz) ppm. HRMS (ESI): m/z Exact mass calcd. for C H FNO S [M+OCH ] :
8
7
4
3
232.0085, found: 232.0065.
1
4.1.1.3 6-chlorobenzo[e][1,2,3]oxathiazine 2,2-dioxide (5c). H NMR (400 MHz,
CDCl ) δ 8.64 (s, 1H), 7.72-7.67 (m, 2H), 7.29-7.26 (m, 1H) ppm. HRMS (ESI): m/z
3
—
Exact mass calcd. for C H ClNO S [M+OCH ] : 247.9790, found: 247.9769.
8
7
4
3
1
4.1.1.4 6-methoxybenzo[e][1,2,3]oxathiazine 2,2-dioxide (5d). H NMR (400 MHz,
CDCl ) δ 8.63 (s, 1H), 7.30-7.27 (m, 1H), 7.23 (d, J = 9.2 Hz, 1H), 7.09 (d, J = 2.8 Hz,
3
1H), 3.88 (s, 3H) ppm. HRMS (ESI): m/z Exact mass calcd. for C H NO S
9
10
5
—
[M+OCH ] : 244.0285, found: 244.0265.
3
1
4.1.1.5 6-methylbenzo[e][1,2,3]oxathiazine 2,2-dioxide (5e). H NMR (400 MHz,
CDCl ) δ 8.62 (s, 1H), 7.55 (dd, J = 8.8 Hz, J = 1.8 Hz, 1H), 7.46 (d, J = 1.6 Hz, 1H),
3
13
7.19 (d, J = 8.4 Hz, 1H), 2.45 (s, 3H) ppm. C NMR (100 MHz, CDCl ) δ 167.9,
3
152.3, 138.6, 136.5, 130.8, 118.4, 115.3, 20.8 ppm. HRMS (ESI): m/z Exact mass
—
calcd. for C H NO S [M+OCH ] : 228.0336, found: 228.0316.
9
10
4
3
1
4.1.1.6 6-bromobenzo[e][1,2,3]oxathiazine 2,2-dioxide (5f). H NMR (400 MHz,
1
3
CDCl ) δ 8.63 (s, 1H), 7.86-7.82 (m, 2H), 7.21 (d, J = 8.8 Hz, 1H) ppm. C NMR
3
(100 MHz, CDCl ) δ 166.4, 153.3, 140.4, 133.1, 120.6, 118.8, 116.6 ppm. HRMS
3
—
(ESI): m/z Exact mass calcd. for C H BrNO S [M+OCH ] : 291.9285, found:
8
7
4
3
291.9264.
1
4.1.1.7 7-fluorobenzo[e][1,2,3]oxathiazine 2,2-dioxide (5g). H NMR (400 MHz,
CDCl ) δ 8.63 (s, 1H), 7.74-7.71 (m, 1H), 7.17-7.12 (m, 1H), 7.04-7.02 (m,1H) ppm.
3
1
3
C NMR (100 MHz, CDCl ) δ 167.7 (d, J = 262.5 Hz, 1C), 166.8, 156.3 (d, J = 13.7
3
Hz, 1C), 133.4 (d, J = 11.3 Hz, 1C), 114.5 (d, J = 22.7 Hz, 1C), 112.4 (d, J = 2.8 Hz,
1
4

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
1C), 107.0 (d, J = 25.9 Hz, 1C) ppm. HRMS (ESI): m/z Exact mass calcd. for
—
C H FNO S [M+OCH ] : 232.0085, found: 232.0065.
8
7
4
3
1
4.1.1.8 7-chlorobenzo[e][1,2,3]oxathiazine 2,2-dioxide (5h). H NMR (400 MHz,
CDCl ) δ 8.64 (s, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.41 (dd, J = 8.4 Hz, J = 1.6 Hz, 1H),
3
1
3
7.33 (d, J = 1.6 Hz, 1H) ppm. C NMR (100 MHz, CDCl ) δ 166.9, 154.8, 144.2,
3
131.8, 127.0, 119.3, 113.9 ppm. HRMS (ESI): m/z Exact mass calcd. for
—
C H ClNO S [M+OCH ] : 247.9790, found: 247.9769.
8
7
4
3
1
4.1.1.9 7-methoxybenzo[e][1,2,3]oxathiazine 2,2-dioxide (5i). H NMR (400 MHz,
CDCl ) δ 8.51 (s, 1H), 7.57 (d, J = 8.8 Hz, 1H), 6.90 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H),
3
13
6.73 (d, J = 2.4 Hz, 1H), 3.94 (s, 3H) ppm. C NMR (100 MHz, CDCl ) δ 167.4,
3
166.9, 157.0, 132.6, 113.7, 109.3, 103.0, 56.6 ppm. HRMS (ESI): m/z Exact mass
—
calcd. for C H NO S [M+OCH ] : 244.0285, found: 244.0265.
9
10
5
3
1
4.1.1.10 7-methylbenzo[e][1,2,3]oxathiazine 2,2-dioxide (5j). H NMR (400 MHz,
CDCl ) δ 8.61 (s, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.10 (s, 1H),
3
—
2.51 (s, 3H) ppm. HRMS (ESI): m/z Exact mass calcd. for C H NO S [M+OCH ] :
9
10
4
3
228.0336, found: 228.0315.
1
4.1.1.11 7-(diethylamino)benzo[e][1,2,3]oxathiazine 2,2-dioxide (5k). H NMR
(400 MHz, CDCl ) δ 8.26 (s, 1H), 7.35 (d, J = 8.8 Hz, 1H), 6.54-6.52 (m, 1H),
3
13
6.33-6.32 (m, 1H), 3.47 (q, J = 7.2 Hz, 4H), 1.25 (t, J = 7.2 Hz, 6H) ppm. C NMR
(100 MHz, CDCl ) δ 165.4, 157.7, 154.7, 132.8, 109.1, 105.1, 98.7, 45.5, 12.5 ppm.
3
+
HRMS (ESI): m/z Exact mass calcd. for C H N O S [M+H] : 255.0798, found:
11
15
2
3
255.0744.
1
4.1.1.12 8-fluorobenzo[e][1,2,3]oxathiazine 2,2-dioxide (5l). H NMR (400 MHz,
CDCl ) δ 8.70 (d, J = 1.2 Hz, 1H), 7.59-7.54 (m, 1H), 7.51-7.48 (m, 1H),7.42-7.37 (m,
3
—
1H) ppm. HRMS (ESI): m/z Exact mass calcd. for C H FNO S [M+OCH ] :
8
7
4
3
232.0085, found: 232.0064.
1
4.1.1.13 8-chlorobenzo[e][1,2,3]oxathiazine 2,2-dioxide (5m). H NMR (400 MHz,
CDCl ) δ 8.67 (s, 1H), 7.82-7.79 (m, 1H), 7.62-7.60 (m, 1H), 7.41-7.37 (m, 1H) ppm.
3
—
HRMS (ESI): m/z Exact mass calcd. for C H ClNO S [M+OCH ] : 247.9790, found:
8
7
4
3
1
5

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
247.9770.
1
4.1.1.14 8-methoxybenzo[e][1,2,3]oxathiazine 2,2-dioxide (5n). H NMR (400
MHz, CDCl ) δ 8.65 (s, 1H), 7.37-7.30 (m, 2H), 7.25-7.23 (m, 1H), 3.97 (s, 3H) ppm.
3
—
HRMS (ESI): m/z Exact mass calcd. for C H NO S [M+OCH ] : 244.0285, found:
9
10
5
3
244.0265.
1
4.1.1.15 8-(tert-butyl)benzo[e][1,2,3]oxathiazine 2,2-dioxide (5o). H NMR (400
MHz, CDCl ) δ 8.65 (s, 1H), 7.75 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 7.52 (dd, J = 7.6
3
Hz, J = 1.6 Hz, 1H), 7.38-7.34 (m, 1H), 1.48 (s, 9H) ppm. HRMS (ESI): m/z Exact
—
mass calcd. for C H NO S [M+OCH ] : 270.0806, found: 270.0785.
1
2
16
4
3
1
4.1.1.16 naphtho[1,2-e][1,2,3]oxathiazine 3,3-dioxide (5p). H NMR (400 MHz,
CDCl ) δ 10.0 (s, 1H), 8.70 (d, J = 8.4 Hz, 1H), 8.55 (d, J = 9.2 Hz, 1H), 8.17 (d, J =
3
8.0 Hz, 1H), 7.90-7.86 (m, 1H), 7.76-7.72 (m, 1H), 7.67 (d, J = 9.2 Hz, 1H) ppm.
—
HRMS (ESI): m/z Exact mass calcd. for C H NO S [M+OCH ] : 264.0336, found:
1
2
10
4
3
264.0316.
4.2 Biological assays
4.2.1. Cell culture
TM
H9c2 cell line (CRL-1446 ) was provided by the American Type Culture
Collection (ATCC, Shanghai, China). Cells were cultured as previously described [7].
Briefly, cells were cultured in Dulbecco's Modified Eagle's Medium: Nutrient Mixture
F-12 (DMEM/F12) with 10% fetal bovine serum (FBS) containing 100 U/mL
penicillin and 100 U/mL streptomycin. Cells were incubated at 37 °C with 5% CO2,
and were synchronized by serum starvation before stimulated with hydrogen peroxide
(H O ) or the compounds being tested.
2
2
4.2.2. Cardioprotection assay against H O and cardiotoxicity assay in H9c2 cells
2
2
Compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma Aldrich,
Shanghai, China) and diluted with medium.Generally, 80% confluent cells were
3
seeded 5×10 cells/well in 96-well plates and were allowed to attach overnight.
For the cardiotoxicity assay, previous medium was removed, and 0.1, 1, 10 and 100
µM of the compounds was added to the wells, respectively. 0.01% DMSO in cell
1
6

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4
4
4
4
4
4
4
4
4
4
4
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4
4
4
4
4
4
4
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
culture media served as control. Cells was incubated with compounds for 24 h.
For the cardioprotection assay, cells were subjected to 250 µM H O for 2h to
2
2
induce the myocardial injury. Different concentrations of respective compounds were
added to the cells 24 h prior to hydrogen peroxide treatment. Luteolin (LUT) (Sigma
Aldrich, Shanghai, China) was served as positive control [10].
4.2.3. MTT assay
MTT assay was utilized to determine H9c2 cells cytotoxicity and viability. After
appropriate treatment, Cells were treated with MTT solution (4 mg/mL in PBS) for 4
h at 37 °C. The formazan crystals were dissolved in DMSO and the optical density
(OD) was examined at 490 nm using a BioTek plate reader. The cell viability was
given in a percentage of the OD value of the control group. In vitro cardiotoxicity or
cardioprotection assay, the cell viability was determined after treated with the
compounds being tested for 24 h.
4.2.4. Determination of intracellular H O -induced ROS production in H9c2 cells
2
2
5
H9c2 cells were seeded in 6-well plates (2.5×10 cells/well) and allowed to attach
overnight. After 24 h of preincubation with compound 5h (10 µM), H O was added
2
2
for 2 h. Then, cells were incubated with non-fluorescent dihydroethidium (DHE, 5
µM) at 37 °C for 30 min. Ethidium fluorescence (excitation at 488 nm, emission at
525 nm) was monitored by fluorescence microscopy (Nikon Eclipse Ti-S, Nikon Ltd,
Japan).
4.2.5. Measurement of LDH release rate
After appropriate treatment, the LDH release rate was measured by the cytotoxicity
detection kit, according to the manufacturer's instructions (Jiancheng Bioengineering
Institute, Nanjing, China) and quantified by absorbance at 490 nm.
4.2.6. Western blot analysis
H9c2 cells were treated with compound 5h (0.5, 1, 2, 5, 10 µM) or LUT (10 µM)
for 24 h, and were lysed with a commercial kit to extract cytoplasmic and nuclear
proteins (KeyGEN BioTECH, Nanjing, China). Proteins were separated by 10%
sodium dodecyl sulfate-polyacrylamidegel (SDS-PAGE) and then transferred to
17

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4
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4
4
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4
4
4
4
4
4
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
Polyvinylidene Fluoride (PVDF) membranes (Millipore Corporation, MA, USA).
Blots were blocked with 5% nonfat dry milk-TBS-0.1% Tween 20 for 2 h. Primary
antibodies were incubated overnight at 4 °C. Thereafter, all blots were washed three
times with 1×TBST and were incubated with a horseradish peroxidase-conjugated
secondary anti-rabbit antibody (1:5000; Cell Signaling Technology Co., Ltd, MN,
USA) for 2 h. Immunoreactivities of target proteins were detected by a gel imaging
system (Protein Simple, Santa Clara, California, USA), and were analysed by Image J
software. The primary antibodies used were polyclonal antibodies against Nrf2
(1:1000; Proteintech Biotech, Wuhan, China), NF-κB p65 (1:1000; Cell Signaling
Technology Co., Ltd, MN, USA), SERCA2 (1:1000; Cell Signaling Technology Co.,
Ltd, MN, USA), HO-1 (1:1000; Cell Signaling Technology Co., Ltd, MN, USA) and
NQO1 (1:1000; Cell Signaling Technology Co., Ltd, MN, USA) . Protein expression
levels were normalised against levels of Lamin B1 (1:1000, Bioworld Technology, St.
Louis Park, MN, USA) or GAPDH (1:1000, Bioworld Technology, St. Louis Park,
MN, USA), which were used as a loading control.
4.3. Statistical analysis
Results were expressed as mean ± SD of three independent experiments. One-way
ANOVA followed by Tukey’s post hoc test were used for multi-group comparison
(GraphPad Prizm 5.0). Significant differences were established at P < 0.05.
Conflicts of interest
The authors declare no competing interest.
Acknowledgements
This work was supported by the Education Department of Henan Province (Grant
Nos. 17A150021), the National Natural Science Foundation of China (Grant Nos.
81603114 and 81874330), China Postdoctoral Science Foundation (Grant Nos.
2016M592301 and 2019M652596), the Initiation funds for postdoctoral research
projects in Henan Province (Grant Nos. 1901003), and the Jiangsu Youth Medical
Talents (QNRC2016194 and QNRC2016195).
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87
Appendix A. Supplementary data
Copies of the NMR spectras for all compounds.
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25

Highlights
•
•
Sixteen coumarin-derived imino sulfonates were synthesized and characterized.
Most compounds displayed potent cardioprotective activities with low
cytotoxicity.
•
5h attenuated the oxidative stress via promoting Nrf2 pathway in cardiomyocytes.