Synthesis and biological evaluation of bifendate derivatives bearing acrylamide moiety as novel antioxidant agents

Article abstract of DOI:10.1016/j.ejmech.2018.11.003

Oxidative stress plays a significant role in the pathogenesis of various human diseases. In this study, a series of bifendate derivatives bearing acrylamide moiety were synthesized and evaluated as anti-oxidant agents. Biological evaluation indicated that

Full text of DOI:10.1016/j.ejmech.2018.11.003

Accepted Manuscript
Synthesis and biological evaluation of bifendate derivatives bearing acrylamide
moiety as novel antioxidant agents
Xiaoke Gu, Yanfei Jiang, Jing Chen, Yinpeng Zhang, Mingyu Guan, Xin Li, Qingqing
Zhou, Qian Lu, Jingying Qiu, Xiaoxing Yin
PII:
S0223-5234(18)30951-6
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.003
EJMECH 10860
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 October 2018
Accepted Date: 1 November 2018
Please cite this article as: X. Gu, Y. Jiang, J. Chen, Y. Zhang, M. Guan, X. Li, Q. Zhou, Q. Lu, J. Qiu,
X. Yin, Synthesis and biological evaluation of bifendate derivatives bearing acrylamide moiety as novel
antioxidant agents, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.11.003.
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ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of bifendate derivatives bearing acrylamide moiety as
novel antioxidant agents
Xiaoke Gu a, 1, , Yanfei Jiang a, 1, Jing Chen a, Yinpeng Zhang a, Mingyu Guan a, Xin Li a, Qingqing Zhou a, Qian Lu
a, Jingying Qiua,*, Xiaoxing Yin a,
aJiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Jiangsu Center for the Collaboration and
Innovation of Cancer Biotherapy, Xuzhou Medical University, Xuzhou 221004, People’s Republic of China
Graphical Abstract
Compound 6a and 6e exhibited significant anti-oxidant activities via activating Nrf2 signaling.
Corresponding authors. Tel.: +86-516-83372005; fax: +86-516-83372005; e-mail: yinxx@xzhmu.edu.cn (X. Y.)
Tel.: +86-516-83262137; fax: +86-516-83262137; e-mail: gu_xk@xzhmu.edu.cn (X. G.)
Tel.: +86-516-83262138; fax: +86-516-83262138; e-mail: jingyqiu@126.com (J. Q.)
1 These two authors contributed equally to this work.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of bifendate derivatives bearing acrylamide moiety as novel
antioxidant agents
Xiaoke Gu a, 1, , Yanfei Jiang a, 1, Jing Chen a, Yinpeng Zhang a, Mingyu Guan a, Xin Li a, Qingqing Zhou a, Qian Lu a, Jingying
Qiua,*, Xiaoxing Yin a,
aJiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Jiangsu Center for the Collaboration and
Innovation of Cancer Biotherapy, Xuzhou Medical University, Xuzhou 221004, People’s Republic of China
Abstract:
Oxidative stress plays a significant role in the pathogenesis of various human diseases. In this study, a
series of bifendate derivatives bearing acrylamide moiety were synthesized and evaluated as anti-oxidant
agents. Biological evaluation indicated that compounds 6a and 6e displayed more potent cytoprotective
effect against H2O2-induced HBZY-1 mesangial cells death than lead compound bifendate and positive
control resveratrol and sulforaphane. Preliminary anti-oxidant mechanism studies showed that compound
6e could diminish the ROS accumulation by dose- and time-dependently activating Nrf2 and increasing the
expression of downstream detoxification enzymes NQO-1, HO-1, GCLM and GCLC at protein and mRNA
levels, thus displaying potent anti-oxidant activity. Interestingly, the Nrf2 activating effect of 6e is achieved,
at least partly, in Michael acceptor and Keap1-dependent manners. These results, together with the low
intrinsic cytotoxicity, suggested that compound 6e might be a promising lead for the development of novel
anti-oxidant agents to prevent diseases induced by oxidative stress.
Keywords: oxidative stress; bifendate derivatives; Nrf2; anti-oxidant agents.
1. Introduction
Oxidative stress, an imbalance between the production of reactive oxygen species (ROS) and antioxidant
defenses [1, 2], plays a significant role in the pathogenesis of various human diseases like cancer,
atherosclerosis, aging, diabetes and its complications such as diabetic nephropathy [3-5]. Consequently,
Corresponding authors. Tel.: +86-516-83372005; fax: +86-516-83372005; e-mail: yinxx@xzhmu.edu.cn (X. Y.)
Tel.: +86-516-83262137; fax: +86-516-83262137; e-mail: gu_xk@xzhmu.edu.cn (X. G.)
Tel.: +86-516-83262138; fax: +86-516-83262138; e-mail: jingyqiu@126.com (J. Q.)
1 These two authors contributed equally to this work.
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discovery of novel antioxidants to prevent diseases induced by oxidative stress is an active research field in
medicinal chemistry [3, 6].
A variety of factors are associated with the cellular response to oxidative stress. Among them, nuclear
factor-erythroid 2-related factor 2 (Nrf2) pathway is considered as the most significant regulators in the cell
to protect against oxidative stress. As a nuclear transcription factor, Nrf2 controls the expression of a
battery of defensive genes whose protein products are involved in the elimination of reactive oxidants and
electrophilic agents through conjugative reactions, thus enhancing the cellular antioxidant capacity [7-9].
Accordingly, activation of Nrf2 may have therapeutic potential in disease associated with oxidative stress
[10]. Actually, plenty of Nrf2-activating compounds with diverse structure have been identified, including
various natural products such as resveratrol, butein, curcumin, piperlongumine, caffeic acid and A.
frutescens constituents (Figure 1) [11-14]. Notably, many of these compound possessed a α,β-unsaturated
carbonyl scaffold, which could covalently modify the cysteine residues in Keap1, resulting in Nrf2
disassociation from Keap1, thereby causing it stabilization and translocation to the nucleus [10].
Figure 1. Natural products with Nrf2-activating effect
Recently, it has been demonstrated that Schisandra chinensis extracts including Schisandrin A,
Schisandrol B and Schizandrin C also exert multi-pharmaceutical bioactivities by activation of the Nrf2
mediated defense response [15-21]. In our previous studies, bifendate, an analog of Schizandrin C and its
derivatives were found to have multiple bioactivities [22, 23]. Interestingly, recent investigations
demonstrated that the bioactivity of bifendate was related to its anti-oxidant properties [24-26]. With these
in mind, and in view of the crucial role of α,β-unsaturated carbonyl moiety (such as acrylamide moiety) in
many natural products for their anti-oxidant activities [12, 14], we hypothesized that bifendate scaffold
hybridized with acrylamide moiety might enhance the anti-oxidant activities of bifendate. Therefore, a
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series of bifendate derivatives possessing acrylamide moiety (3a-g) was firstly synthesized. In addition,
considering that introduction of nitrile group to the molecule could enhance anti-oxidant activity of Nrf2
activators [27, 28], we next prepared bifendate-acrylamide hybrids (6a-g) containing a nitrile substituent in
the α,β-unsaturated carbonyl moiety. Thus, fourteen target compounds (Figure 2) were synthesized, and
their anti-oxidant activities and preliminary anti-oxidant mechanism were subsequently evaluated in this
context.
Figure 2. Chemical structures of bifendate and the target compounds (3a-g and 6a-g)
2. Chemistry
The synthetic route of target compounds 3a-g was depicted in Scheme 1. The key intermediate 1 was
prepared starting from bifendate by six steps according to our previous reported method [29]. Treatment of
1 with malonic acid through Knoevenagel condensation reaction afforded intermediate 2. Subsequently,
compound 2 was directly condensed with various amines in the presence of EDCI and DMAP to afford the
target compounds 3a-g, respectively. The synthetic routes of the target compounds 6a-g are shown in
Scheme 2. Briefly, treatment of 1 with cyanoacetic acid through Knoevenagel condensation reaction
afforded intermediate 4 according to previous reported method [30]. And then compound 4 was treated
with oxalyl chloride to result in acyl chloride 5. Finally, directly condensation of 5 with various amines led
to the target compounds 6a-g, respectively. Additionally, in order to study the relationship between
structures and anti-oxidant effect, compounds 9a-c were prepared by replacement of sixalkoxyl biphenyl
moiety in 6a with alkoxyl phenyl moiety, respectively.
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Scheme 1. Synthetic route of the target compounds 3a-g. Reagents and conditions: a) malonic acid,
pyridine, DMF, 120°C, 6 h; b) amines, DMAP, EDCI, DCM, r.t, 4~6 h.
Scheme 2. Synthetic route of the target compounds 6a-g and 9a-c. a) cyanoacetic acid, pyridine, DMF,
70°C, 6 h; b) oxalyl chloride, DCM, ice cooled, 0.5 h; c) amines, 1,4-dioxane, 90°C, 3~6 h; d) cyanoacetic
acid, KF, EtOH, 60°C, 15 h; e) oxalyl chloride, DCM, ice cooled, 0.5 h; f) 2-methoxyaniline, 1,4-dioxane,
r.t, 3 h.
3. Results and discussion
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3.1. Biological evaluation
3.1.1 ytoprotective effect of the target compounds
C
Firstly, we determined the intrinsic cytotoxicity of the target compounds against HBZY-1 mesangial
cells by MTT assay, and found that the target compounds displayed low intrinsic cytotoxicity since their
IC50 values were all above 200 µM. Subsequently, the H2O2-induced oxidative death of HBZY-1 mesangial
cells was employed as a model to evaluate the cytoprotective effect of the target compounds. Specifically,
the HBZY-1 mesangial cells were preincubated with or without the target compounds at a nontoxic
concentration (6 µM) for 2 h, followed by the treatment with 600 µM H2O2 for another 24 h. Then, the cell
viability was determined by MTT assay. As shown in Figure 3A, the viability of cells treated H2O2 was
significantly reduced to 62.1% of control, indicating that H2O2 could significantly attenuate the viability of
HBZY-1 mesangial cells. Notably, the cell viability in bifedate-pretreated group at the high dose level
(Figure 3A: red bar, 100 µM) was 89.8% of control, indicating that bifendate exhibited potential
cytoprotective effect against H2O2-induced HBZY-1 mesangial cells death. Sadly, the cytoprective effect of
bifendate was dramatically reduced with the decrease of concentration, for the viability was only 66.1% of
control at the dose of 6 µM. In sharp contrast, the viability of cells preincubated with the target compounds
was still high at the same dose (6 µM). For instance, the viability of cells treated with 3a, 6a, 6e and 6f
were 90.6%, 99.2%, 97.2% and 95.3% of control, respectively, suggesting that these compounds were more
potent anti-oxidant agents. More excitingly, the cytoprotective effect of these target compounds was much
better than that of positive controls resveratrol and sulforaphane under our experimental conditions.
Considering that 6a and 6e were the most potent cytoprotective agents among the target compounds, their
cytoprotective activity at different concentrations was subsequently determined by MTT assay. As shown
in Fig. 3B, pretreatment of the cells with 6a and 6e (1.5, 3 and 6 µM) could dose-dependently protected the
cells from oxidative death.
To verify the impact of the bifendate moiety on the cytoprotective activity of the target compounds, we
replaced sixalkoxyl biphenyl moiety in 6a with alkoxyl phenyl moiety to produce compounds 9a-c
(Scheme 2). Interestingly, the cytoprotective activity of compound 9a-c was dramatically weakened relative
to 6a (Figure 3A). In addition, when the acrylamide moiety was reduced, the cytoprotective activity was
also remarkably reduced (Data was shown in the Supplementary Material), suggesting that both the
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sixalkoxyl biphenyl and acrylamide moiety were crucial for the cytoprotective effect of the target
compounds.
In view of the fact that the compound 6e are less toxic than 6a (Data was shown in the Supplementary
Material), we subsequently selected compound 6e to study the preliminary antioxidant mechanism.
Figure 3. Cytoprotective effect of the target compounds against H2O2-induced HBZY-1 mesangial
cells death. Briefly, HBZY-1 mesangial cells were pretreated with or without the target compounds (Figure
3A: 6 µM; Figure 3B: 1.5, 3 and 6 µM) for 2 h, followed by incubation with 600 µM H2O2 for another 24 h.
Then, the cell viability was determined by MTT assay. Bifendate (Bif) was used as the lead compound
control. Resveratrol (Res) and sulforaphane (Sul) were selected as the positive controls. All data represent
##
the mean ± SD of three independent experiments. P < 0.01 of H2O2-treated group vs. the vehicle control
group (Ctrl); **P < 0.01 of compound group vs. the H2O2-treated group.
3.1.2 Effect of compound 6e on ROS accumulation induced by H2O2
It is well-known that H2O2 can be metabolized to free radicals, ultimately leading to oxidative stress [31].
Therefore, the extent of ROS generation induced by H2O2 was determined by flow cytometry using
DCFH-DA [32]. As shown in Figure 4, the DCF fluorescence intensity in HBZY-1 mesangial cells treated
with H2O2 (600 µM) is 7.6-fold to the control (Figure 4, red bar), indicating that H2O2 could significantly
increase the intracellular ROS levels. Interestingly, the intracellular DCF fluorescence intensity was
dramatically reduced after preincubating with compound 6e, suggesting that 6e could dose-dependently
diminish the ROS accumulation in HBZY-1 mesangial cells.
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Figure 4. Effect of compound 6e (6.0 µM) on ROS accumulation induced by H2O2. Resveratrol (Res) was
selected as the positive control. All values are denoted as means ± SD from three independent batches of
cells. ##P < 0.01 of H2O2 group vs. the vehicle group (red bar); **P < 0.01 of compound group vs. the
H2O2-stimulated group.
3.1.3 Effect of compound 6e on Nrf2 activation
As mentioned above, Nrf2 pathway is considered as the most significant regulators in cells to protect
against oxidative stress. Therefore, it was of great interest to determine whether compound 6e exhibits the
ability to activate Nrf2 signaling, thus displaying potent anti-oxidant activity. Human embryonic kidney
293 (HEK293) cells transfected with the Nrf2/ARE luciferase reporter was employed as an evaluation
model [33, 34]. Tert-Butylhydroquinone (t-BHQ) was selected as a positive control. As shown in Figure 5,
the intracellular luciferase activity in blank control (Crl) group was relative weak. As was expected, the
relative luciferase activity was significantly increased when cells was treated with t-BHQ, a known Nrf2
activator. Notably, compound 6e could also dose-dependently increase the intracellular luciferase activity,
suggesting that 6e possessed potential Nrf2 activation effect.
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Figure 5. Compound 6e activates the Nrf2 signaling. HEK293 cells were transfected with the
ARE-dependent luciferase reporter plasmid, and subsequently treated in triplicate with 6e at the indicated
concentrations (1.5, 3 and 6 µM) for 16 h. The luciferase activity was measured to assess the Nrf2 activity.
t-BHQ was selected as a positive control. All values are denoted as means ± SD from three independent
batches of cells. **P < 0.01 vs. blank control group (Ctrl).
3.1.4 Effect of compound 6e on the expression of Nrf2 and its downstream detoxification enzymes
Generally, under activation condition, Nrf2 detaches from Keap1, translocates into the nucleus, and
activates its downstream detoxification enzymes to combat oxidative and electrophilic stress. Therefore, we
next tested the effect of 6e on the expression of Nrf2 and its downstream detoxification enzymes using
Western blot and RT-PCR, respectively.
As shown in Figure 6A and B, treatment with compound 6e, the ratios of nuclear to cytosolic Nrf2 in
cells was significantly increased in a dose and time dependent manners, to a peak value at 12 h. As was
expected, compound 6e could dose- and time-dependently increase the expression of downstream
detoxification enzymes of Nrf2 such as NQO-1, HO-1, GCLM and GCLC at protein levels (Figure 6C and
D) and mRNA (Figure 7). Notably, compound 6e was a more effective Nrf2 activator than positive control
resveratrol. These results suggested that the anti-oxidant activity of the target compound might be related to
activating the Nrf2 signaling.
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Figure 6. Compound 6e could dose (A and C) and time (B and D) dependently increase the expression
of nuclear and cytosolic Nrf2, NQO-1, HO-1, GCLM and GCLC at protein levels. Briefly, HBZY-1
mesangial cells were incubated with the various concentrations test compounds for indicated time points,
then the cells were harvested and lysed. The target proteins were subsequently determined by Western blot
assay. Resveratrol (Res) was used as a positive control.
Figure 7. Compound 6e can dose (A-D) and time (E-H) dependently increase the relative mRNA
levels of NQO-1, HO-1, GCLM and GCLC in a dose-dependent manner. Briefly, HBZY-1 mesangial cells
were cultured without or with indicated concentrations of target compound for 12 h. Total RNA was
extracted and then reverse transcripted from mRNA to cDNA using the RT-PCR kit. Resveratrol (Res) was
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used as a positive control. All values are denoted as means ± SD from three independent batches of cells.
*P < 0.05, **P < 0.01 vs. control group (Ctrl) or 0 h group.
3.1.5 Effects of TRG, ZnPP and BSO on the cytoprotection induced by compound 6e
To further ascertain the anti-oxidant activity of compound 6e against HBZY-1 mesangial cells depends
on activating Nrf2 signaling, we next determined the cytoprotective effect of compound 6e in the presence
of trigonelline (TRG, an Nrf2 inhibitor), Zinc protoporphyrin IX (ZnPP, a HO-1 inhibitor) or
L-buthionine-(S,R)-sulfoximine (BSO, a GCL inhibitor) by MTT assay, respectively. As shown in Figure 8,
H2O2 could significantly attenuate the viability of HBZY-1 mesangial cells, and 6e exhibited significant
cytoprotective effect against H2O2-induced HBZY-1 mesangial cells death. Notably, such cytoprotective
effect was remarkably attenuated after preincubating with non-toxic concentrations of TRG, ZnPP and BSO,
respectively. These results clearly indicated that compound 6e alleviated H2O2-induced death of HBZY-1
mesangial cells via activating Nrf2 signaling.
Figure 8. Effects of TRG (A), ZnPP (B) and BSO (C) on the cytoprotection of compound 6e. The
cytotoxicity was determined by the MTT assay. The cells were preincubated with TRG, ZnPP or BSO for 2
h before the addition of the test compound 6e. All values are denoted as means ± SD from three
independent batches of cells. **P < 0.01 vs. H2O2 group; ##P < 0.01 vs. compound 6e + H2O2 group.
3.1.6 Compound 6e activated Nrf2 in a Keap1-dependent manner
It is known that Michael acceptors can directly conjugate with the active cysteine residues of Keap1,
leading to the activation of Nrf2, and such conjugation reaction can be quenched by dithiothreitol (DTT), a
Michael acceptor attack-abolishing agent. To confirm whether the Nrf2 activating effect of compound 6e is
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achieved in Michael acceptor and Keap1-dependent manners, we subsequently examined the effect of
compound 6e on the expression of Nrf2 and Keap1 coupled with DTT by Western blot assay. Interestingly,
when cells were pretreated with DTT, the increased Nrf2 expression induced by compound 6e can be
reversed, indicating that compound 6e activated the expression of Nrf2, at least partly, via a Michael
acceptor dependent manner. Moreover, the decreased Keap1 expression in cells treated by compound 6e
was also partly reversed by DTT, suggesting that the Nrf2 activation is also Keap1-dependent.
Figure 9. Compound 6e triggers Nrf2 activation via disrupting Keap1-Nrf2 complex. The expression of the
target proteins was determined by Western blot assay as mentioned above. In the case of inhibition, the
cells were pretreated with DTT (400 µM) for 1 h before the addition of the test compound 6e.
3.2. Structure-Activity Relationships
Based on the cytoprotective results (Figure 3), the structure-activity relationships (SARs) of target
compounds on anti-oxidant activities can be summarized as follows: compounds with the acrylamide
moiety exhibited more potent cytoprotective effect than lead compound bifendate and positive control
resveratrol. Interestingly, introducing a nitrile substituent in the α,β-unsaturated carbonyl moiety could
significantly increase the intensity of cytoprotection (6a-g vs. 3a-g, respectively). Notably, the substitute at
nitrogen atom of acrylamide moiety significantly affected the intensity of cytoprotection. Compound with a
benzyl group at nitrogen atom showed better cytoprotection, and introduction of alkyl chain between
nitrogen atom and benzene ring could significantly attenuate cytoprotective activity (3a vs. 3d; 6a vs. 6d).
In addition, the position and number of methoxy group in benzene ring linking with nitrogen atom also
affected the cytoprotective activity. Briefly, compound with one methoxy group displayed most potent
cytoprotective effect (3a, 3e vs. 3c, 3g; 6a, 6e vs. 6c, 6g), and ortho-methoxy group showed greater benefit
for the cytoprotection than para-methoxy group (3a vs. 3e; 6a vs. 6e). One plausible explanation is that
introduction of methoxy group might affect the electron density of the Michael acceptor moiety, leading to
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the changes of the affinity with the active cysteine residues of Keap1 and consequent cytoprotective effect.
However, the precise SARs remain further investigation when more derivatives of this kind will be
available in near future.
4. Conclusions
In summary, fourteen novel bifendate derivatives containing acrylamide moiety were synthesized and
evaluated as anti-oxidant agents. Most of the target compounds exhibited more potent cytoprotective effect
against H2O2-induced HBZY-1 mesangial cells death than lead compound bifendate and positive control
resveratrol and sulforaphane. Among them, compounds 6a and 6e were the most potent ones. Notably,
preliminary anti-oxidant mechanism studies found that compound 6e could impair the ROS accumulation
by activating Nrf2 and increasing the expression of downstream detoxification enzymes at mRNA and
protein levels, thus displaying potent anti-oxidant activity. Interestingly, such activating effect is achieved
partly in Michael acceptor and Keap1-dependent manners. These results, together with the low intrinsic
cytotoxicity, indicated that 6e might be a promising lead for the development of novel anti-oxidant agents
to prevent diseases induced by oxidative stress.
5. Experimental protocols
5.1. Chemical analysis
All of the synthesized compounds were purified by column chromatography on silica gel 60 (200–300
mesh) or thin layer chromatography (TLC) on silica gel 60 F254 plates (250 mm; Qingdao Ocean Chemical
Company, China). Melting points of individual compounds were measured on a YRT-3 melting point
apparatus in an uncorrected manner. Subsequently, the structure of the target compounds were analyzed by
1
IR (Thermofisher Nicolet is10), HNMR and 13C NMR (JEOL, 400 MHz), and MS (Agilent 1290/6460
LC/MSD spectrometer) routinely. Compound 1 was synthesized as previously described. All solvents were
reagent grade and, when necessary, were purified and dried by standards methods.
5.2. General procedure for the preparation of 3a-g
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A solution of compound 1 (1 g), malonic acid (1.6 g, 6 eq) and pyridine (420 µL, 2 eq) in DMF was
stirred at room temperature for 1 h, then the reaction mixture was stirred at 120 °C for 6 h. Then 1N HCl
was added to the reaction solution (pH= 2~3), and extracted with EtOAc. The organic layer was washed
with brine, dried with anhydrous sodium sulfate, filtered and evaporated in vacuum. The crude product was
purified by column chromatography (petroleum ether: ethyl acetate = 5:3 v/v) to yield the compound 2,
which was then reacted with various amines in the presence of EDCI and DMAP in CH2Cl2 to give
corresponding target compounds, respectively.
5.2.1 methyl (E)-7,7'-dimethoxy-5'-(3-((2-methoxyphenyl)amino)-3-oxoprop-1-en-1-yl)-[4,4'-bibenzo[d]
[1,3]dioxole]-5-carboxylate (3a)
The title compound was obtained starting from 2 and 2-methoxyaniline. As a white solid, yield: 63.8%.
1
m.p. 215.9-217.4 °C; H NMR (CDCl3, 400 MHz) δ 8.43 (s, 1H, -NH-), 7.88 (s, 1H, Ar-H), 7.46 (d, J =
15.3 Hz, 1H, =CH-), 7.40 (s, 1H, Ar-H), 7.07 – 7.01 (m, 1H, Ar-H), 6.98 (d, J = 4.7 Hz, 1H, Ar-H), 6.94 (d,
J = 7.7 Hz, 1H, Ar-H), 6.88 (d, J = 8.0 Hz, 1H, Ar-H), 6.39 (d, J = 15.3 Hz, 1H, =CH-), 6.02 (s, 2H, -CH2-),
5.97 (s, 2H, -CH2-), 4.00 (s, 3H, -OCH3), 3.98 (s, 3H, -OCH3), 3.90 (s, 3H, -OCH3), 3.67 (s, 3H, -OCH3);
13C NMR (CDCl3, 100 MHz) δ 166.16, 163.82, 147.88, 147.07, 143.42, 143.07, 139.49, 138.57, 136.40,
128.05, 127.97, 124.36, 123.73, 121.25 (2C, 2 × Ar-C), 120.83, 119.87, 111.94, 111.89, 110.06, 109.95,
105.41, 102.59, 102.13, 56.75, 56.64, 55.79, 52.11; IR (KBr, cm-1): ν 3357.84, 3001.03, 2947.03, 2902.67,
2840.95, 1716.53, 1672.17, 1637.45, 1596.95, 1529.45, 1419.51, 1323.08, 1251.72, 1195.78, 1172.64,
1101.28, 1045.35, 933.48, 754.12; ESI-MS (m/z): 558.0 ([M + Na]+). HRMS (ESI-TOF): calculated for
([M + Na]+) 558.1376, found 558.1368, PPM error 1.4.
5.2.2 methyl (E)-5'-(3-((3,4-dimethoxyphenethyl)amino)-3-oxoprop-1-en-1-yl)-7,7'-dimethoxy-[4,4'-bibenzo
[d][1,3]dioxole]-5-carboxylate (3b)
The title compound was obtained starting from 2 and 3,4-dimethoxyphenylethylamine. As a white solid,
1
yield: 50.0%. m.p. 98.8-99.7 °C; H NMR (CDCl3, 400 MHz) δ 7.41 (s, 1H, -NH-), 7.37 (d, J = 15.4 Hz,
1H, =CH-), 6.89 (s, 1H, Ar-H), 6.83 (d, J = 9.0 Hz, 1H, Ar-H), 6.78 – 6.70 (m, 1H, Ar-H), 6.73 (s, 1H,
Ar-H), 6.13 (d, J = 15.5 Hz, 1H, =CH-), 6.02 (dd, J = 6.2, 1.5 Hz, 2H, -CH2-), 5.97 (s, 2H, -CH2-), 5.53 (s,
1H, Ar-H), 4.00 (s, 3H, -OCH3), 3.96 (s, 3H, -OCH3), 3.89 (s, 3H, -OCH3), 3.88 (s, 3H, -OCH3), 3.67 (s,
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3H, -OCH3), 3.59 – 3.54 (m, 2H, -CH2-), 2.80 (t, J = 7.4 Hz, 2H, -CH2-); C NMR (CDCl3, 100 MHz) δ
166.21, 165.91, 149.12, 147.80, 147.77, 147.04, 143.33, 143.03, 138.68, 138.58, 136.19, 131.41, 128.07,
124.34, 120.72, 120.15, 111.97, 111.82, 111.68, 111.40, 110.13, 105.45, 102.58, 102.09, 56.76, 56.58, 56.01,
55.97, 52.11, 40.95, 35.32; IR (KBr, cm-1): ν 3373.27, 3001.03, 2941.24, 2908.45, 2839.02, 1720.39,
1635.52, 1515.94, 1433.01, 1417.58, 1350.08, 1323.08, 1259.43, 1238.21, 1191.93, 1172.64, 1159.14,
1099.35, 1043.42, 929.63, 757.97; ESI-MS (m/z): 616.0 ([M + Na]+). HRMS (ESI-TOF): calculated for
([M + Na]+) 616.1795, found 616.1791, PPM error 0.6.
5.2.3 methyl (E)-7,7'-dimethoxy-5'-(3-oxo-3-((3,4,5-trimethoxyphenyl)amino)prop-1-en-1-yl)-[4,4'-bibenzo
[d][1,3]dioxole]-5-carboxylate (3c)
The title compound was obtained starting from 2 and 3,4,5-trimethoxyphenylamine. As a white solid,
1
yield: 53.1%. m.p 185.3-186.9 °C; H NMR (CDCl3, 400 MHz) δ 7.46 (s, 1H, -NH-), 7.43 (s, 1H, =CH-),
7.37 (s, 1H, Ar-H), 6.87 (s, 2H, 2 × Ar-H), 6.27 (d, J = 15.2 Hz, 1H, =CH-), 5.99 (d, J = 6.1 Hz, 2H, -CH2-),
5.88 (d, J = 11.4 Hz, 2H, -CH2-), 3.96 (s, 3H, -OCH3), 3.89 (s, 3H, -OCH3), 3.80 (s, 6H, 2 × -OCH3), 3.79
(s, 3H, -OCH3), 3.68 (s, 3H, -OCH3); 13C NMR (CDCl3, 100 MHz) δ 166.38, 163.97, 153.29 (2C, 2 ×
Ar-C), 147.95, 147.07, 143.33, 143.08, 139.32, 138.74, 136.53, 134.73, 134.49, 127.86, 124.04, 120.57,
111.98, 111.78, 110.12, 105.69, 102.63, 102.13, 97.19, 61.06, 56.76, 56.52, 56.14, 56.14, 52.22; IR (KBr,
cm-1): ν 3369.41, 3006.82, 2948.96, 2910.38, 2840.95, 1714.60, 1635.52, 1552.59, 1510.16, 1415.65,
1352.01, 1323.08, 1247.86, 1190.00, 1166.85, 1130.21, 1101.28, 1041.49, 1000.99, 983.63, 931.55;
ESI-MS (m/z): 618.0 ([M + Na]+). HRMS (ESI-TOF): calculated for ([M + Na]+) 618.1588, found
618.1585, PPM error 0.5.
5.2.4 methyl (E)-7,7'-dimethoxy-5'-(3-((4-methoxybenzyl)amino)-3-oxoprop-1-en-1-yl)-[4,4'-bibenzo[d]
[1,3]dioxole]-5-carboxylate (3d)
The title compound was obtained starting from 2 and 4-methoxybenzylamine. As a white solid, yield:
1
57.5%. m.p. 97.8-98.4 °C; H NMR (CDCl3, 400 MHz) δ 7.39 (s, 1H, -NH-), 7.36 (s, 1H, =CH-), 7.20 (s,
1H, Ar-H), 7.18 (s, 1H, Ar-H), 6.85 (s, 2H, 2 × Ar-H), 6.83 (s, 1H, Ar-H), 6.16 (d, J = 14.7 Hz, 1H, =CH-),
5.99 (s, 2H, -CH2-), 5.93 (s, 2H, -CH2-), 5.77 (s, 1H, Ar-H), 4.41 (s, 2H, -CH2-), 3.95 (s, 3H, -OCH3), 3.91
14

ACCEPTED MANUSCRIPT
(s, 3H, -OCH3), 3.77 (s, 3H, -OCH3), 3.64 (s, 3H, -OCH3); 13C NMR (CDCl3, 100 MHz) δ 166.23, 165.69,
159.12, 147.81, 147.03, 143.33, 143.03, 138.91, 138.59, 136.18, 130.44, 129.33 (2C, 2 × Ar-C), 128.13,
124.35, 120.20, 114.15 (2C, 2 × Ar-C), 111.82, 111.66, 110.13, 105.51, 102.58, 102.08, 56.75, 56.54, 55.40,
52.12, 43.33; IR (KBr, cm-1): ν 3290.33, 2948.96, 2916.17, 1720.39, 1635.52, 1514.02, 1433.01, 1417.58,
1352.01, 1323.08, 1247.86, 1172.64, 1099.35, 1043.42, 968.20, 929.63, 823.55; ESI-MS (m/z): 572.0 ([M
+ Na]+). HRMS (ESI-TOF): calculated for ([M + Na]+) 572.153, found 572.1525, PPM error 1.7.
5.2.5
methyl
(E)-7,7'-dimethoxy-5'-(3-((4-methoxyphenyl)amino)-3-oxoprop-1-en-1-yl)-[4,4'-bibenzo[d][1,3]
dioxole]-5-carboxylate (3e)
The title compound was obtained starting from 2 and 4-methoxyphenylamine. As a white solid, yield:
36.1%. m.p. 213.9-214.5 °C; 1H NMR (CDCl3, 400 MHz) δ 7.48 (s, 1H, -NH-), 7.47 (s, 1H, =CH-), 7.43 (s,
1H, Ar-H), 7.39 (s, 1H, Ar-H), 7.31 (s, 1H, Ar-H), 6.91 (s, 1H, Ar-H), 6.86 (s, 1H, Ar-H), 6.83 (s, 1H, Ar-H),
6.29 (d, J = 15.3 Hz, 1H, =CH-), 6.01 (dd, J = 6.4, 1.1 Hz, 2H, -CH2-), 5.92 (dd, J = 10.9, 1.3 Hz, 2H,
-CH2-), 3.98 (s, 3H, -OCH3), 3.93 (s, 3H, -OCH3), 3.79 (s, 3H, -OCH3), 3.68 (s, 3H, -OCH3); 13C NMR
(DMSO-d6, 100 MHz) δ 166.06, 163.55, 155.73, 148.07, 147.16, 143.45, 142.98, 138.35, 137.49, 136.07,
133.03, 128.40, 124.85, 122.22, 120.99 (2C, 2 × Ar-C), 114.42 (2C, 2 × Ar-C), 111.66, 111.41, 110.00,
105.14, 102.95, 102.33, 56.97, 56.69, 55.66, 52.48; IR (KBr, cm-1): ν 3274.90, 2948.96, 2902.67, 1718.46,
1635.52, 1512.09, 1444.58, 1413.72, 1350.08, 1245.93, 1174.57, 1101.28, 1043.42, 929.63, 829.33, 756.04.
ESI-MS (m/z): 558.0 ([M + Na]+). HRMS (ESI-TOF): calculated for ([M + Na]+) 558.1376, found
558.1367, PPM error 1.6.
5.2.6
methyl
(E)-5'-(3-(butylamino)-3-oxoprop-1-en-1-yl)-7,7'-dimethoxy-[4,4'-bibenzo[d][1,3]dioxole]-5-carboxylate
(3f)
The title compound was obtained starting from 2 and n-butylamine. As a white solid, yield: 29.3%. m.p.
162.6-163.7 °C; 1H NMR (CDCl3, 400 MHz) δ 7.37 (s, 1H, -NH), 7.31 (d, J = 15.5 Hz, 1H, =CH-), 6.87 (s,
1H, Ar-H), 6.14 (d, J = 15.5 Hz, 1H, =CH-), 5.99 (d, J = 3.0 Hz, 2H, -CH2-), 5.94 (s, 2H, -CH2-), 5.48 (s,
15

ACCEPTED MANUSCRIPT
1H, Ar-H), 3.96 (s, 3H, -OCH3), 3.94 (s, 3H, -OCH3), 3.64 (s, 3H, -OCH3), 3.32 – 3.27 (m, 2H, -CH2-),
1.53 – 1.44 (m, 2H, -CH2-), 1.38 – 1.29 (m, 2H, -CH2-), 0.91 (t, J = 7.3 Hz, 3H, -CH3); 13C NMR (CDCl3,
100 MHz) δ 166.28, 165.91, 147.80, 147.02, 143.33, 143.01, 138.58, 138.35, 136.09, 128.22, 124.39,
120.51, 111.79, 111.59, 110.16, 105.42, 102.57, 102.06, 56.76, 56.54, 52.11, 39.46, 31.81, 20.17, 13.84; IR
(KBr, cm-1): ν 3245.97, 2954.74, 2894.95, 1722.31, 1635.52, 1589.23, 1483.16, 1431.08, 1415.65, 1350.08,
1321.15, 1247.86, 1174.57, 1101.28, 1041.49, 983.63, 927.70; ESI-MS (m/z): 508.1 ([M + Na]+). HRMS
(ESI-TOF): calculated for ([M + Na]+) 508.1584, found 508.1578, PPM error 1.2.
5.2.7 methyl (E)-5'-(3-((3,4-dimethoxyphenyl)amino)-3-oxoprop-1-en-1-yl)-7,7'-dimethoxy-[4,4'-bibenzo[d]
[1,3]dioxole]-5-carboxylate (3g)
The title compound was obtained starting from 2 and 3,4-dimethoxyphenylamine. As a white solid, yield:
1
59.3%. m.p. 222.6-223.5 °C; H NMR (CDCl3, 400 MHz) δ7.49 (s, 1H, -NH-), 7.46 (d, J = 16.3 Hz, 1H,
=CH-), 7.39 (s, 1H, Ar-H), 7.32 (s, 1H, Ar-H), 6.92 (s, 1H, Ar-H), 6.86 (d, J = 8.6 Hz, 1H, Ar-H), 6.79 (d, J
= 8.6 Hz, 1H, Ar-H), 6.30 (d, J = 15.5 Hz, 1H, =CH-), 6.03 – 5.97 (m, 2H, -CH2-), 5.95 – 5.91 (m, 2H,
-CH2-), 3.98 (s, 3H, -OCH3), 3.94 (s, 3H, -OCH3), 3.87 (s, 3H, -OCH3), 3.86 (s, 3H, -OCH3), 3.68 (s, 3H,
-OCH3); 13C NMR (CDCl3, 100 MHz) δ 166.36, 163.81, 149.00, 147.91, 147.04, 145.65, 143.34, 143.06,
139.02, 138.71, 136.41, 132.28, 127.95, 124.08, 120.70, 111.92, 111.73, 111.28 (2C, 2 × Ar-C), 110.13,
105.55, 104.49, 102.62, 102.12, 56.75, 56.47, 56.17, 55.94, 52.20; IR (KBr, cm-1): ν 3433.06, 2952.81,
2904.60, 1716.53, 1635.52, 1608.52, 1514.02, 1444.58, 1434.94, 1413.72, 1317.29, 1259.43, 1240.14,
1174.57, 1132.14, 1103.21, 1039.56, 970.13, 842.83; ESI-MS (m/z): 566.1 ([M + H]+). HRMS (ESI-TOF):
calculated for ([M + Na]+) 588.1482, found 588.1481, PPM error 0.2.
5.3. General procedure for the preparation of compound 4
A solution of compound 1 (5 g), cyanoacetic acid (6.4 g, 6 eq) and pyridine (2 mL, 2 eq) in DMF was
stirred at room temperature for 1 h, then the reaction mixture was stirred at 70 °C for 6 h. Then water was
added to the reaction solution, and extracted with EtOAc. The organic layer was washed with brine, dried
with anhydrous sodium sulfate, filtered and evaporated in vacuum. The crude product was purified by
column chromatography (petroleum ether: ethyl acetate = 5:3 v/v) to yield the compound 4.
16

ACCEPTED MANUSCRIPT
(E)-2-cyano-3-(7,7'-dimethoxy-5'-(methoxycarbonyl)-[4,4'-bibenzo[d][1,3]dioxol]-5-yl)acrylic acid (4)
As a yellow solid, yield: 51.2%. 1H NMR (CDCl3, 400 MHz) δ 8.04 (s, 1H, Ar-H), 7.99 (s, 1H, Ar-H),
7.41 (s, 1H, Ar-H), 6.10 – 5.92 (m, 4H, 2 × -CH2-), 5.29 (s, 1H, -OH), 4.00 (s, 6H, 2 × -OCH3), 3.68 (s, 3H,
-OCH3); 13C NMR (CDCl3, 100 MHz) δ 168.07, 166.09, 154.25, 148.04, 147.14, 143.54, 143.49, 139.97,
138.49, 124.41, 124.15, 115.95, 115.19, 112.09, 108.75, 108.25, 102.86, 102.71, 99.31, 56.77, 56.63, 52.35;
IR: 3014.53, 2950.89, 2906.53, 2844.81, 2221.84, 1710.74, 1679.88, 1635.52, 1566.09, 1433.01, 1415.65,
1355.86, 1309.58, 1269.07, 1174.57, 1105.14, 1039.56, 931.55. HRMS (ESI-TOF): calculated for ([M +
Na]+) 478.0750, found 478.0737, PPM error 2.7.
5.4. General procedure for the preparation of 6a-g
To a solution of 4 (2 g, 4.4 mmol) in dry dichloromethane (50 mL) was added oxalyl chloride (0.82 g,
0.56 mL, 6.6 mmol) at 0-5°C. The mixture was stirred for 30 min and then evaporated under a reduced
pressure to obtain the crude product 5, which was used in the next step without further purification.
To a stirred solution of freshly prepared acid chloride 5 (200 mg, 0.44 mmol) in 10 mL of dry dioxane,
various amines in dioxane was added droply. The reaction mixture was stirred at 90°C for 3-6 h, then the
reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed
with 1N HCl, brine, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure.
The resulting crude product was purified by flash chromatography on silica gel column (petroleum ether:
ethyl acetate = 5:1 ~ 1:2 v/v) to give the title compounds 6a-g, respectively.
5.4.1
methyl
(E)-5'-(2-cyano-3-((2-methoxyphenyl)amino)-3-oxoprop-1-en-1-yl)-7,7'-dimethoxy-[4,4'-bibenzo[d][1,3]di
oxole]-5-carboxylate (6a)
The title compound was obtained starting from 5 and 2-methoxyaniline. As a yellow solid, yield:
1
77.4%. m.p. 211.6-211.8 °C; H NMR (CDCl3, 400 MHz, ) δ 8.80 (s, 1H, -NH-), 8.31 (d, J = 8.0 Hz, 1H,
=CH-), 8.18 (s, 1H, Ar-H), 7.86 (s, 1H, Ar-H), 7.41 (s, 1H, Ar-H), 7.10 (t, J = 7.5 Hz, 1H, Ar-H), 6.97 (d, J
= 7.7 Hz, 1H, Ar-H), 6.91 (d, J = 8.0 Hz, 1H, Ar-H), 6.03 (s, 2H, -CH2-), 5.99 (s, 2H, -CH2-), 4.03 (s, 3H,
17

ACCEPTED MANUSCRIPT
-OCH3), 3.99 (s, 3H, -OCH3), 3.94 (s, 3H, -OCH3), 3.68 (s, 3H, -OCH3); 13C NMR (CDCl3, 100 MHz) δ
166.08, 158.24, 151.1, 151.12, 148.56, 148.04, 147.15, 143.39 (2C, 2 × Ar-C), 138.84, 138.57, 127.01,
124.97, 124.85, 124.34, 121.13, 120.02, 117.73, 114.53, 112.08, 110.16, 108.72, 107.95, 102.80, 102.67
(2C, 2 × -CH2-), 56.74, 56.55, 56.05, 52.23; IR (KBr, cm-1): ν 3398.34, 2948.96, 2898.81, 2200.63, 1716.53,
1677.95, 1635.52, 1602.74, 1564.16, 1529.45, 1355.86, 1299.93, 1288.36, 1193.85, 1103.21, 1047.27,
931.55, 756.04; ESI-MS (m/z): 561.1 ([M + H]+). HRMS (ESI-TOF): calculated for ([M - H]-) 559.1353,
found 559.1436, PPM error 1.3.
5.4.2
methyl
(E)-5'-(2-cyano-3-((3,4-dimethoxyphenethyl)amino)-3-oxoprop-1-en-1-yl)-7,7'-dimethoxy-[4,4'-bibenzo[d][
1,3]dioxole]-5-carboxylate (6b)
The title compound was obtained starting from 5 and 3,4-dimethoxyphenylethylamine. As a white solid,
yield: 79.8%. m.p. 138.3-139.7 °C; 1H NMR (CDCl3, 400 MHz) δ 8.07 (s, 1H, -NH-), 7.76 (s, 1H, =CH-),
7.38 (s, 1H, Ar-H), 6.81 (d, J = 8.0 Hz, 1H, Ar-H), 6.74 (s, 1H, Ar-H), 6.71 (d, J = 5.2 Hz, 1H, Ar-H), 6.34
(s, 1H, Ar-H), 5.99 (s, 2H, -CH2-), 5.94 (s, 2H, -CH2-), 3.98 (s, 3H, -OCH3), 3.96 (s, 3H, -OCH3), 3.86 (s,
3H, -OCH3), 3.85 (s, 3H, -OCH3), 3.65 (s, 3H, -OCH3), 3.60 – 3.47 (m, 2H, -CH2-), 2.79 (t, J = 6.9 Hz, 2H,
-CH2-); 13C NMR (CDCl3, 100 MHz) δ 166.05, 160.56, 150.70, 149.23, 147.99, 147.94, 147.12, 143.36,
143.32, 138.72, 138.56, 130.82, 124.88, 124.32, 120.72, 117.83, 114.45, 112.09, 111.86, 111.56, 108.75,
107.85, 102.62 (2C, 2 × -CH2-), 101.93, 56.73, 56.56, 56.00, 55.95, 52.18, 41.94, 35.26; IR (KBr, cm-1): ν
3365.55, 2948.96, 2920.03, 2208.34, 1722.31, 1668.31, 1637.45, 1517.87, 1417.58, 1353.94, 1261.36,
1193.85, 1174.57, 1103.21, 1041.49, 927.70; ESI-MS (m/z): 641.1 ([M + Na]+). HRMS (ESI-TOF):
calculated for ([M + Na]+) 641.1748, found 641.1743, PPM error 0.8.
5.4.3
methyl
(E)-5'-(2-cyano-3-oxo-3-((3,4,5-trimethoxyphenyl)amino)prop-1-en-1-yl)-7,7'-dimethoxy-[4,4'-bibenzo[d][
1,3]dioxole]-5-carboxylate (6c)
The title compound was obtained starting from 5 and 3,4,5-trimethoxyphenylamine. As a yellow solid,
yield: 58.8%. m.p. 206.9-207.1 °C; 1H NMR (CDCl3, 400 MHz, ) δ 8.17 (s, 1H, -NH), 7.84 (s, 1H, =CH-),
18

ACCEPTED MANUSCRIPT
7.82 (s, 1H, Ar-H), 7.40 (s, 1H, Ar-H), 6.84 (s, 2H, 2 × Ar-H), 6.02 (s, 2H, -CH2-), 5.98 (s, 2H, -CH2-), 4.02
(s, 3H, -OCH3), 3.98 (s, 3H, -OCH3), 3.85 (s, 6H, 2 × -OCH3), 3.81 (s, 3H, -OCH3), 3.67 (s, 3H, -OCH3);
13C NMR (CDCl3, 100 MHz) δ 166.06, 158.50, 153.51 (2C, 2 × Ar-C), 151.67, 148.05, 147.24, 143.41 (2C,
2 × Ar-C), 139.08, 138.60, 135.56, 133.08, 124.86, 124.43, 117.93, 114.65, 112.16, 108.66, 108.15, 102.67
(2C, 2 × -CH2-), 102.04, 98.16 (2C, 2 × Ar-C), 61.06, 60.46, 56.78, 56.66, 56.32, 52.21; IR (KBr, cm-1): ν
3406.05, 2958.60, 2939.31, 2837.09, 2204.48, 1716.53, 1685.67, 1610.45, 1508.23, 1452.30, 1415.65,
1357.79, 1325.01, 1301.86, 1245.93, 1195.78, 1176.50, 1128.28, 1103.21, 1043.42, 1002.92; ESI-MS (m/z):
643.1 ([M + Na]+). HRMS (ESI-TOF): calculated for ([M + Na]+) 643.1540, found 643.1536, PPM error
0.6.
5.4.4
methyl
(E)-5'-(2-cyano-3-((4-methoxybenzyl)amino)-3-oxoprop-1-en-1-yl)-7,7'-dimethoxy-[4,4'-bibenzo[d][1,3]dio
xole]-5-carboxylate (6d)
The title compound was obtained starting from 5 and 4-methoxybenzylamine. As a yellow solid, yield:
39.6%. m.p. 159.8-160.3 °C; 1H NMR (CDCl3, 400 MHz) δ 8.12 (s, 1H, -NH), 7.76 (s, 1H, =CH-), 7.39 (s,
1H, Ar-H), 7.22 (s, 1H, Ar-H), 7.20 (s, 1H, Ar-H), 6.87 (s, 1H, Ar-H), 6.85 (s, 1H, Ar-H), 6.53 (s, 1H, Ar-H),
5.98 (d, J = 15.6 Hz, 4H, 2 × -CH2-), 4.45 (d, J = 4.9 Hz, 2H, -CH2-), 3.98 (s, 3H, -OCH3), 3.97 (s, 3H,
-OCH3), 3.78 (s, 3H, -OCH3), 3.66 (s, 3H, -OCH3); 13C NMR (CDCl3, 100 MHz) δ 166.07, 160.46, 159.36,
151.06, 148.00, 147.13, 143.33, 138.73, 138.58, 129.34 (2C, 2 × Ar-C), 129.14, 124.94, 124.37, 117.85,
114.45, 114.32 (2C, 2 × Ar-C), 113.74, 112.14, 108.75, 107.94, 102.60, 101.96, 56.75, 56.58, 55.41, 55.36,
52.18, 44.05; IR (KBr, cm-1): ν 3373.27, 3006.82, 2941.24, 2908.45, 2210.27, 1712.67, 1674.10, 1635.52,
1514.02, 1417.58, 1353.94, 1326.93, 1249.79, 1193.85, 1174.57, 1103.21, 1041.49, 927.70, 831.26;
ESI-MS (m/z): 597.1 ([M + Na]+). HRMS (ESI-TOF): calculated for ([M + Na]+) 597.1485, found
597.1473, PPM error 2.0.
5.4.5
methyl
(E)-5'-(2-cyano-3-((4-methoxyphenyl)amino)-3-oxoprop-1-en-1-yl)-7,7'-dimethoxy-[4,4'-bibenzo[d][1,3]di
oxole]-5-carboxylate (6e)
19

ACCEPTED MANUSCRIPT
The title compound was obtained starting from 5 and 4-methoxyphenylamine. As a yellow solid, yield:
63.2%. m.p. 185.1-185.4 °C; 1H NMR (CDCl3, 400 MHz) δ8.18 (s, 1H, -NH), 7.87 (s, 1H, =CH-), 7.82 (s,
1H, Ar-H), 7.45 (s, 1H, Ar-H), 7.43 (s, 1H, Ar-H), 7.39 (s, 1H, Ar-H), 6.87 (s, 1H, Ar-H), 6.85 (s, 1H,
Ar-H), 6.01 (s, 2H, -CH2-), 5.97 (s, 2H, -CH2-), 4.00 (s, 3H, -OCH3), 3.96 (s, 3H, -OCH3), 3.78 (s, 3H,
-OCH3), 3.67 (s, 3H, -OCH3); 13C NMR (CDCl3, 100 MHz) δ 166.07, 158.58, 157.21, 151.36, 148.03,
147.19, 143.39, 138.95, 138.58, 130.05, 124.91, 124.35, 122.41 (2C, 2 × Ar-C), 117.96, 114.63, 114.35 (3C,
3 × Ar-C), 112.15, 108.69, 108.01, 102.65 (2C, 2 × -CH2-), 102.04, 56.74, 56.61, 55.58, 52.21; IR (KBr,
cm-1): ν 3375.20, 3001.03, 2937.38, 2906.53, 2839.02, 2206.41, 1716.53, 1674.10, 1635.52, 1600.81,
1537.16, 1512.09, 1440.73, 1415.65, 1357.79, 1317.29, 1301.86, 1238.21, 1195.78, 1176.50, 1135.99,
1103.21, 1039.56, 929.63, 829.33; ESI-MS (m/z): 583.1 ([M + Na]+). HRMS (ESI-TOF): calculated for
([M + Na]+) 583.1329, found 583.1329, PPM error 0.1.
5.4.6 methyl (E)-5'-(3-(butylamino)-2-cyano-3-oxoprop-1-en-1-yl)-7,7'-dimethoxy-[4,4'-bibenzo[d][1,3]
dioxole]-5-carboxylate (6f)
The title compound was obtained starting from 5 and n-butylamine. As a white solid, yield: 28.4%. m.p.
145.9-147.3 °C; 1H NMR (CDCl3, 400 MHz) δ 8.09 (s, 1H, -NH-), 7.78 (s, 1H, =CH-), 7.40 (s, 1H, Ar-H),
6.27 (s, 1H, Ar-H), 6.02 (s, 2H, -CH2-), 5.97 (s, 2H, -CH2-), 4.00 (s, 3H, -OCH3), 3.97 (s, 3H, -OCH3), 3.67
(s, 3H, -OCH3), 3.40 – 3.30 (m, 2H, -CH2-), 1.56 – 1.50 (m, 2H, -CH2-), 1.42 – 1.31 (m, 2H, -CH2-), 0.93 (t,
J = 7.3 Hz, 3H, -CH3); 13C NMR (CDCl3, 100 MHz) δ 166.06, 160.50, 150.67, 147.97, 147.12, 143.35,
143.32, 138.63, 138.57, 124.97, 124.34, 117.98, 114.38, 112.12, 108.78, 107.86, 102.61, 102.58, 102.14,
56.73, 56.57, 52.16, 40.32, 31.54, 20.12, 13.78; IR (KBr, cm-1): ν 3373.27, 3010.67, 2958.60, 2939.31,
2885.31, 2206.41, 1724.24, 1674.10, 1637.45, 1575.73, 1525.59, 1481.23, 1454.23, 1417.58, 1352.01,
1326.93, 1307.65, 1244.00, 1191.93, 1174.57, 1159.14, 1103.21, 1039.56, 925.77, 831.26, 757.97; ESI-MS
(m/z): 533.1 ([M + Na]+). HRMS (ESI-TOF): calculated for ([M + Na]+) 533.1536, found 533.1532, PPM
error 0.8.
20

ACCEPTED MANUSCRIPT
5.4.7
methyl
(E)-5'-(2-cyano-3-((3,4-dimethoxyphenyl)amino)-3-oxoprop-1-en-1-yl)-7,7'-dimethoxy-[4,4'-bibenzo[d][1,3
]dioxole]-5-carboxylate (6g)
The title compound was obtained starting from 5 and 3,4-dimethoxyphenylamine. As a white solid, yield:
1
64.1%. m.p. >240 °C; H NMR (CDCl3, 400 MHz) δ 8.17 (s, 1H, -NH), 7.87 (s, 1H, =CH-), 7.81 (s, 1H,
Ar-H), 7.39 (s, 1H, Ar-H), 7.27 (d, J = 2.4 Hz, 1H, Ar-H), 6.98 (dd, J = 8.6, 2.4 Hz, 1H, Ar-H), 6.81 (d, J =
8.7 Hz, 1H, Ar-H), 6.01 (s, 2H, -CH2-), 5.98 (s, 2H, -CH2-), 4.00 (s, 3H, -OCH3), 3.96 (s, 3H, -OCH3), 3.87
(s, 3H, -OCH3), 3.85 (s, 3H, -OCH3), 3.67 (s, 3H, -OCH3); 13C NMR (CDCl3, 100 MHz) δ 166.07, 158.48,
151.42, 149.21, 148.04, 147.20, 146.68, 143.40 (2C, 2 × Ar-C), 138.97, 138.59, 130.52, 124.91, 124.38,
117.97, 114.61, 112.68, 112.13, 111.32, 108.68, 108.05, 105.19, 102.67 (2C, 2 × -CH2-), 102.11, 56.76,
56.63, 56.18, 56.11, 52.21; IR (KBr, cm-1): ν 3384.84, 3001.03, 2952.81, 2910.38, 2833.24, 2208.34,
1718.46, 1672.17, 1635.52, 1535.23, 1514.02, 1433.01, 1417.58, 1357.79, 1323.08, 1234.36, 1220.86,
1191.93, 1172.64, 1103.21, 1041.49, 966.27, 931.55; ESI-MS (m/z): 613.1 ([M + Na]+). HRMS (ESI-TOF):
calculated for ([M + Na]+) 613.1435, found 613.1429, PPM error 0.9.
5.5. General procedure for the preparation of 9a-g
A solution of various substituted benzaldehyde (200 mg), cyanoacetic acid (2 eq) and KF in EtOH (1 mL)
was stirred at room temperature for 10 min, then the reaction mixture was stirred at 60 °C for 15 h. Then
water was added to the reaction solution, and extracted with EtOAc. The organic layer was washed with 1N
HCl, brine, dried with anhydrous sodium sulfate, filtered and evaporated in vacuum. The crude product was
recrystallized (EtOH/H2O) to yield compound 7a-c, which was dissolved in dry dichloromethane (5 mL)
and then oxalyl chloride (1.5 eq) was added droply at 0-5°C. The mixture was stirred for 30 min and then
evaporated under a reduced pressure to obtain the crude product 8a-c, which was dissolved in dry dioxane
(2 mL). Then, 2-methoxyaniline was added, and the reaction mixture was stirred at room temperature for 3
h. Finally, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer
was washed with 1N HCl, brine, dried over anhydrous sodium sulfate, filtered, and evaporated under
reduced pressure. The resulting crude product was recrystallized (EtOH/H2O) to yield the title compounds
9a-c, respectively.
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5.5.1 (E)-2-cyano-N-(2-methoxyphenyl)-3-(4-methoxyphenyl)acrylamide (9a)
The title compound was obtained starting from 2-methoxybenzaldehyde and 2-methoxyaniline. As a
yellow solid, yield: 42.2%. m.p. 192.5-193.2 °C; 1H NMR (CDCl3, 400 MHz) δ 8.81 (s, 1H, -NH), 8.41 (d,
J = 8.0 Hz, 1H, =CH-), 8.32 (s, 1H, Ar-H), 7.99 (s, 1H, Ar-H), 7.97 (s, 1H, Ar-H), 7.11 (t, J = 7.8 Hz, 1H,
Ar-H), 7.00 (d, J = 6.4 Hz, 2H, 2 × Ar-H), 6.98 (d, J = 3.4 Hz, 1H, Ar-H), 6.92 (d, J = 8.1 Hz, 1H, Ar-H),
3.94 (s, 3H, -OCH3), 3.88 (s, 3H, -OCH3); 13C NMR (CDCl3, 100 MHz) δ 163.53, 158.54, 152.70, 148.59,
133.34, 133.34, 127.14, 124.79, 124.79, 121.17, 120.10, 117.52, 114.84, 114.84, 114.84, 110.15, 101.23,
56.07, 55.70; IR (KBr, cm-1): ν 3386.77, 2210.27, 1668.31, 1577.66, 1533.30, 1512.09, 1487.01, 1458.08,
1255.57, 1220.86, 1118.64, 1027.99, 829.33, 740.61, 619.11, 522.67. ESI-MS (m/z): 309.1 ([M + H]+).
5.5.2 (E)-2-cyano-3-(3,4-dimethoxyphenyl)-N-(2-methoxyphenyl)acrylamide (9b)
The title compound was obtained starting from 3,4-dimethoxybenzaldehyde and 2-methoxyaniline. As a
yellow solid, yield: 42.7%. m.p. >240 °C; 1H NMR (CDCl3, 400 MHz) δ 8.82 (s, 1H, -NH-), 8.42 (dd, J =
8.0, 1.6 Hz, 1H, =CH-), 8.32 (s, 1H, Ar-H), 7.75 (d, J = 2.1 Hz, 1H, Ar-H), 7.52 (dd, J = 8.4, 2.2 Hz, 1H,
Ar-H), 7.16 – 7.09 (m, 1H, Ar-H), 7.04 – 6.99 (m, 1H, Ar-H), 6.97 (d, J = 8.4 Hz, 1H, Ar-H), 6.94 (dd, J =
8.1, 1.3 Hz, 1H, Ar-H), 3.98 (s, 3H, -OCH3), 3.98 (s, 3H, -OCH3), 3.96 (s, 3H, -OCH3); 13C NMR (CDCl3,
100 MHz) δ 158.46, 153.39, 152.99, 149.32, 148.58, 127.34, 127.13, 125.02, 124.81, 121.19, 120.10,
117.70, 111.63, 111.11, 110.16, 101.33, 56.23, 56.08, 56.07; IR (KBr, cm-1): ν 3382.91, 2360.71, 2198.70,
1683.74, 1602.74, 1587.31, 1573.81, 1539.09, 1512.09, 1460.01, 1436.87, 1425.30, 1280.65, 1255.57,
1164.92, 1024.13, 738.69, 630.68; ESI-MS (m/z): 361.1 ([M + Na]+). HRMS (ESI-TOF): calculated for
([M + Na]+) 361.1165, found 361.1157, PPM error 2.2.
5.5.3 (E)-2-cyano-N-(2-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)acrylamide (9c)
The title compound was obtained starting from 3,4,5-trimethoxybenzaldehyde and 2-methoxyaniline. As
a yellow solid, yield: 27.8%. m.p. >240 °C; 1H NMR (CDCl3, 400 MHz) δ 8.84 (s, 1H, -NH-), 8.41 (dd, J =
8.0, 1.6 Hz, 1H, =CH-), 8.31 (s, 1H, Ar-H), 7.29 (s, 1H, Ar-H), 7.16 – 7.11 (m, 1H, Ar-H), 7.04 – 7.00 (m,
1H, Ar-H), 6.94 (dd, J = 8.2, 1.2 Hz, 1H, Ar-H), 3.96 (s, 3H, -OCH3), 3.96 (s, 3H, -OCH3), 3.94 (s, 6H, 2 ×
-OCH3). 13C NMR (CDCl3, 100 MHz) δ 157.99, 153.36 (2C, 2 × Ar-C), 153.12, 148.57, 142.38, 127.06,
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126.98, 124.95, 121.17, 120.09, 117.35, 110.16, 108.26 (2C, 2 × Ar-C), 103.17, 61.19, 56.32 (2C, 2 ×
-OCH3), 56.06; IR (KBr, cm-1): ν 3392.55, 2839.02, 2206.41, 1677.95, 1602.74, 1577.66, 1537.16, 1506.30,
1463.87, 1330.79, 1255.57, 1159.14, 1132.14, 999.06, 835.12, 752.19, 642.25. ESI-MS (m/z): 369.1 ([M +
H]+). HRMS (ESI-TOF): calculated for ([M + Na]+) 391.1270, found 391.1264, PPM error 1.5.
5.7. Biological assays
5.7.1. Cell viability assay
The cytotoxicity was determined by the MTT assay. Briefly, HBZY-1 mesangial cells (1×104 cells/well)
were seeded in 96-well plates in RPMI-1640 and cultured for 24 h. The exponentially growing cells were
incubated with various concentrations of compounds for 2 h at 37°C (5% CO2, 95% humidity), then H2O2
(600 µM) was added and continue to incubate 37°C (5% CO2, 95% humidity) for further 24 h. Then 20 µL
MTT (5 mg/mL) in fresh medium was added to each well and incubated for further 4 h. DMSO (150 µL)
was added to dissolve formazan crystals, and the absorbance was analyzed in a multiwall-plate reader
(BioTek ELx800) at 490 nm. In the case of inhibition, the cells were preincubated with TRG (100 or 200
nM), ZnPP (3 or 6 µM) or BSO (10 or 20 µM) for 2 h before the addition of the test compounds.
5.7.2 Determination of the ROS levels in HBZY-1 mesangial cells
HBZY-1 mesangial cells (1×105 cells/well) were incubated with indicated concentrations of test
compounds (1.5, 3 or 6 µM) or control (DMSO) for 12 h followed by exposing cells to 600 µM H2O2 for 3
h. Then the cells were collected, stained with DCFHA-DA (3 mM) for 30 min at 37°C in the dark, and
washed with PBS. The relative fluorescent intensities of FITC were analyzed immediately using a FACS
Calibur flowcytometer (Becton-Dickinson, San Jose, CA, USA). ROS levels of treatment groups were
normalized to the relative control group.
5.7.3 Nrf2 luciferase reporter assay
The activation effect of the tested compounds on Nrf2 was investigated by Nrf2 luciferase reporter assay
as described previously [33, 34]. Briefly, HEK293 cells transiently transfected with Nrf2/ARE luciferase
reporter plasmid (Qiagen, Germany) and Renilla luciferase plasmid cultured in 96-well plate (1×104
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cells/well), then the cells were stimulated with indicated concentrations (1.5, 3.0 and 6.0 µM) of the target
compounds or positive control t-BHQ (20 µM) for 16 h. After the cells were harvested, Renilla luciferase
activities were determined using dual-luciferase reporter assay system (Promega, USA). Nrf2 activity was
expressed by normalizing luciferase activity to the Renilla luciferase activity within the same sample.
5.7.4 Western blot assay
HBZY-1 mesangial cells (1×105 cells/well) were incubated with the various concentrations test
compounds for indicated time points. The cells were harvested and lysed. The cell lysates (30 µg) were
separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12% gel) and
transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% non-fat dry
milk in TBST buffer, the target proteins were probed with appropriate specific antibody overnight at 4°C,
respectively. Subsequently, the membranes were washed with TBST buffer and incubated with horseradish
peroxidase (HRP)–conjugated secondary antibodies. After being washes with TBST buffer, the
protein-antibody complex was visualized using the enhanced Phototope TM-HRP Detection Kit (Cell
Signaling, USA) and exposed to Kodak medical X-ray processor (Kodak, USA). In the case of inhibition,
the cells were pretreated with DTT (400 µM) for 1 h before the addition of the test compounds.
5.7.5 RT-PCR analysis
HBZY-1 mesangial cells (1×105 cells/well) were seeded in 6-well plates and then cultured without or
with indicated concentrations of target compounds for 12 h. Total RNA was extracted and then reverse
transcripted from mRNA to cDNA using the RT-PCR kit (Promega, WI, USA). The PCR profile was as
follows: 10 min at 95 °C, followed by 30 cycles of 30 s at 95 °C and 1 min at 60 °C. The standard curve
and data analysis were produced using Bio-Rad iQ5 software. The relative mRNA level treated with
inhibitors was expressed as fold change of the control (in the presence of 0.1% DMSO).
Acknowledgements
The work was financially supported by National Natural Science Foundation of China (Grant No.
81402789 and 81703359), Natural Science Foundation of Jiangsu Province (Grant No. BK20171179 and
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BK20181151), the Foundation for Jiangsu Six Talent Peaks Program (Grant No. 2017-YY-039), Jiangsu
Provincial 333 High-level Talents Cultivation Project and the Science and Technology Project of Xuzhou
City (Grant No. KC16SG254).
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