Design, synthesis and biological evaluations of diverse Michael acceptor-based phenazine hybrid molecules as TrxR1 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.104736

A series of novel phenazine derivatives (1~27) containing the Michael acceptor scaffolds were designed and synthesized in this study. Some compounds exhibited selective cytotoxicity against Bel-7402 cancer cell line in vitro, in which compound 26 were found to have the best antiproliferative activity. Meanwhile, compound 26 showed no obvious cell toxicity against human normal liver epithelial L02 cells, which means this compound possessed a better safety potential. In the following research, compound 26 was verified to inhibit TrxR1 enzyme activity, ultimately resulting in cellular molecular mechanism events of apoptosis including growth of intracellular ROS level, depletion of reduced Trx1, liberation of ASK1 and up-regulation of p38, respectively. Together, all these evidences implicated that compound 26 acted as the TrxR1 inhibitor against Bel-7402 cells, and could activate apoptosis through the ROS-Trx-ASK1-p38 pathway.

Full text of DOI:10.1016/j.bioorg.2021.104736

Bioorganic Chemistry 109 (2021) 104736
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluations of diverse Michael
acceptor-based phenazine hybrid molecules as TrxR1 inhibitors
,
b
,
,
Yucheng Zhonga 1, Jing Liuc 1, Xiangyu Chengc, Hao Zhangc, Chunhua Zhangc, Zhuolu Xiac,
Zhongxi Wuc, Lu Zhangc, Yuting Zhengc, Zhanyu Gaoc, Zhidong Jiangc, Zhixiang Wanga
,
,
c
Dechun Huanga c, Yuanyuan Lua *, Feng Jianga
,
,
b,
,c,*
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
b School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
c School of Engineering, China Pharmaceutical University, Nanjing 210009, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel phenazine derivatives (1~27) containing the Michael acceptor scaffolds were designed and
synthesized in this study. Some compounds exhibited selective cytotoxicity against Bel-7402 cancer cell line in
vitro, in which compound 26 were found to have the best antiproliferative activity. Meanwhile, compound 26
showed no obvious cell toxicity against human normal liver epithelial L02 cells, which means this compound
possessed a better safety potential. In the following research, compound 26 was verified to inhibit TrxR1 enzyme
activity, ultimately resulting in cellular molecular mechanism events of apoptosis including growth of intra-
cellular ROS level, depletion of reduced Trx1, liberation of ASK1 and up-regulation of p38, respectively.
Together, all these evidences implicated that compound 26 acted as the TrxR1 inhibitor against Bel-7402 cells,
and could activate apoptosis through the ROS-Trx-ASK1-p38 pathway.
Phenazines
Michael receptor
Antiproliferative activity
TrxR1 inhibitor
1. Introduction
derivative N-(2-hydroxyphenyl)-2-phenazinamine (A) (Fig. 1), isolated
from a marine actinomycete BM-17, which showed high cytotoxicity
Phenazines, a large group of natural or synthetic nitrogen-containing
heterocyclic compounds, have been reported to be associated with
anticancer activities since 1959. Phenazine compounds are a kind of
secondary metabolites of microorganisms with redox activity [1]. The
mammalian Thioredoxin reductase (TrxR) proteins, existing mainly as
the cytosolic TrxR1 and mitochondrial TrxR2, are seleno-flavoenzymes
containing a penultimate C-terminal selenocysteine (Sec) residue, and
it is a NADPH-dependent dimer selenoenzyme containing Flavin
Adenine Dinucleotide (FAD) domain, belonging to pyridine nucleotide-
disulfide oxidoreductase family members [2]. TrxR1 has also been found
to be overexpressed in a variety of cancer cells [3]. TrxR1 is necessary to
convert Trx1 from the oxidized states to the reduced forms, improving
the viability of cells under oxidative stress [4], which is especially
important for cancer cells with high redox levels. The abnormal function
of TrxR/Trx enzyme is related to a variety of pathological conditions,
including cancer, inflammation and neurodegenerative diseases [5].
In previous study [6], we have discovered a natural phenazine
against several cancer cells. Subsequently we synthesized a series of 2-
phenazinamine derivatives, in which compound B (Fig. 1) was found
to possess good cytotoxic activities against K562 and HepG2 cancer cells
[7]. Furthermore, based on the molecular hybridization strategy, we
designed, synthesized and screened several phenazine hybrid molecules
as antitumor agents such as C [8], D [9], E [10] and F [11] (Fig. 1).
Among of them, compound D was found to show the most potential than
positive control drug against HepG2 cancer cells both in vivo and in vitro
[9]. Moreover, our previous studies have proven that the phenazine
hybrid molecule D played the significant role in anti-tumor activities
through inhibiting TrxR1 pathway [12].
A variety of TrxR1 enzyme inhibitors have been reported to selec-
tively inhibit the growth of cancer cells [3,13–15]. Recently, Fang et al
[16], synthesized a series of Michael acceptor-based 1,4-naphthoqui-
none derivatives and discovered compound G (Fig. 1) as a potent cyto-
toxic agent with a submicromolar half maximal inhibitory concentration
to HL-60 cells. The cellular action of compound G is related to its ability
* Corresponding authors.
E-mail addresses: luyy@cpu.edu.cn (Y. Lu), jiangfeng@cpu.edu.cn (F. Jiang).
1
Y.Z. and J.L. contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104736
Received 22 December 2020; Received in revised form 23 January 2021; Accepted 4 February 2021
Available online 19 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

Y. Zhong et al.
Bioorganic Chemistry 109 (2021) 104736
to target the selenoenzyme TrxR. It could induce ROS production and
promote oxidative stress-mediated apoptosis in HL-60 cells. Michael
acceptor-based scaffolds have been widely used as an effective template
in medicinal chemistry for drug discovery. Michael acceptors (i.e., al-
kenes attached to electron-withdrawing groups such as ester, carbonyl,
cyano, nitro and sulfonyl group) containing an electrophile are generally
biologically active, which were involved in the regulation of many
signaling pathways in cells. Chalcone, a potential Michael acceptor,
diaminophenazine reacted with diverse acryloyl chlorides to get com-
pounds 5~23, respectively, as shown in Schemes 2–6. Furthermore, we
used 1,4-phenylenediamine as the starting material to synthesize 6-
methoxy-2-aminophenazine following the modified method in the
literature [21], which subsequently reacted with diverse acryloyl chlo-
rides to get compounds 24~27 (see Scheme 7).
2.2. Cell proliferation and TrxR1 inhibitory activities of compounds
containing an α,β-unsaturated carbonyl scaffold, can readily form co-
1~27
valent bonds with the sulfhydryl of cysteine or other thiols to obtain the
Michael adducts, which may play an important role in the biological
activities [17]. When combining Michael acceptor-based scaffolds and
natural compound skeletons to produce novel hybrid molecules, it will
greatly increase the probability of discovering new drug leads with
clinical potentials against various diseases [18–20].
MTT assay was applied to preliminarily screen the synthesized
phenazine compounds in which several compounds with strong cyto-
toxicity against cancer cells were detected. By comparing the cytotox-
icity of different compounds on various cells including five kinds of
cancer cell lines and one kind of normal liver cell line (Table 1), sur-
prisingly, four compounds 19, 20, 26 and 27 with certain significances
in cytotoxicity were found, which are relatively more specific to liver
cancer cells than the other cells. Specifically, compound 19 and 20
The above results inspired us that the phenazine derivative D
inhibited TrxR1 mainly through combining to Sec residues of TrxR1,
which was further evidenced by docking results between D and TrxR1
[12]. To continue the investigation of phenazine analogues as potential
TrxR1 inhibitors, in the present study, we synthesized a series of novel
phenazine derivatives which were designed as hybrid molecules of
phenazine and Michael acceptor-based scaffolds. Here, we reported the
facile construction of a structurally diverse compound library for a
better investigation of the antitumor activity, TrxR1 inhibitory activity
and mechanism of action of these novel compounds.
displayed good cytotoxicity against HuH-7 cells (IC50 = 6.1 and 9.6
μМ,
respectively) and Bel-7402 cells (IC50 = 8.2 and 12.4 М, respectively).
μ
Meanwhile, compound 19 displayed better cytotoxicity against MCF-7
cells. Compound 27 displayed commensurate cytotoxicity than the
positive drug Cisplatin. In particular, compared with Cisplatin, com-
pound 26 displayed better or commensurate cytotoxicity against three
kinds of liver cancer cells (HepG2, HuH-7 and Bel-7402 cells) rather
than human breast cancer cell MCF-7 and human gastric cancer cell
SGC-7901, and meanwhile, it displayed lower cytotoxicity against one
kind of normal liver cells (L02 cells). Hence, it could be said that com-
pound 26 showed better selectivity and possessed good targeting ability
to liver cancer cells.
2. Results and discussion
2.1. Chemistry
According to the procedure described in the literatures, we synthe-
sized a series of important phenazine precursors including 1-methoxy-
All compounds were designed as hybrid molecules of α,β-unsaturated
carbonyl scaffold and diverse phenazine precursors including 1-hydrox-
yphenazine, 2-amino-3-hydroxyphenazine, 2,3-dihydroxyphenazine, 1-
aminophenazine, 2-aminophenazine and 2,3-diaminophenazine, while
comparing the relationship of compounds’ structure and cytotoxicity,
we could conclude that 2-amino substitution on the phenazine core
plays a particularly critical role to improve compounds’ cytotoxicity.
Furthermore, when the strong electron-withdrawing group including
fluoro or trifluoromethyl substituent group was attached to
phenazine
[21],
2-amino-3-hydroxyphenazine
[22],
2,3-
dihydroxyphenazine [23], 1-aminophenazine [24], 2-aminophenazine
[7] and 2,3-diaminophenazine [22]. After treated with BBr3, 1-methox-
yphenazine was transformed to 1-hydroxyphenazine, which subse-
quently reacted with diverse acryloyl chlorides to get compounds 1~4
(Scheme 1). Similarly, 2-amino-3-hydroxyphenazine, 2,3-dihydrox-
yphenazine,
1-aminophenazine,
2-aminophenazine
and
2,3-
Fig. 1. Structures of compounds A-G.
2

Y. Zhong et al.
Bioorganic Chemistry 109 (2021) 104736
Scheme 1. Synthetic routes of compounds 1~4. Reagents and conditions: (a)Pd2(dba)3, rac-BINAP, NaOtBu, PhMe, 110 ◦C; (b) NaBH4, NaOtBu, EtOH, 60 ◦C; (c)
BBr3, DCM; (d) Pyridine, DCM.
Scheme 2. Synthetic route of compounds 5~8.
Scheme 3. Synthetic routes of compounds 9~11. Reagents and conditions: (a) H2O, 100 ◦C; (b) Pyridine, DCM.
Scheme 4. Synthetic routes of compounds 12~15. Reagents and conditions: (a) Cu powder, CuCl, N-ethylmorpholine, 2,3-butanediol; (b) NaBH4, NaOH, H2O; (c)
DPPA, TEA; (d) H2O; (e) Pyridine, DCM.
α
,β-unsaturated carbonyl scaffold, compounds’ antiproliferative activity
batch of compounds, displayed the best inhibitory level of TrxR1
enzyme activity (EC50 = 21.4 М). Therefore, compound 26 was
selected for the follow-up research.
was significantly improved, just like compounds 19, 20, 26 and 27.
In addition, the TrxR1 inhibitory activities of related compounds
were further evaluated in free cells assays. The results indicated that the
compounds’ inhibitory activities of TrxR1 were congruously associated
with the pace of cytotoxicities. Moreover, compound 26, one of the best
μ
3

Y. Zhong et al.
Bioorganic Chemistry 109 (2021) 104736
Scheme 5. Synthetic routes of compounds 16~20. Reagents and conditions: (a) Pd/C, H2; (b) PhNO2, MgSO4; (c) Pyridine, DCM.
Scheme 6. Synthetic routes of compounds 21~23.
Scheme 7. Synthetic routes of compounds 24~27. Reagents and conditions: (a) (Boc)2O; (b) Pd2(dba)3, rac-BINAP, NaOtBu, PhMe, 110 ◦C; (c) NaBH4, NaOtBu,
EtOH, 60 ◦C; (d) TFA, DCM; (e) Pyridine, DCM.
2.3. Compound 26 will increase the level of reactive oxygen species
of Trx1 in cells by inhibiting TrxR1 enzyme activity. Secondly, the ratio
of GSH/GSSG was measured in cells at different time after treated the
same doses of compound 26 (Fig. 2c). It was found that at 2 h, the ratio
of GSH decreased significantly compared with the control group, and
then gradually returned to the original level.
through inhibiting TrxR1 in Bel-7402 cells
Inhibition of TrxR1 deeply affected the intracellular redox system
balance and increased reactive oxygen species (ROS) [25]. And the
cellular H2O2 level within a certain period of time after administrations
were detected for which H2O2 is a primary component of cellular ROS
[5,26]. After adding compound 26, the H2O2 level increased signifi-
cantly with time compared to the control group (Fig. 2a).
In combination with the above time-course experiments, it could be
supposed that the ROS produced by the cancer cells was mainly elimi-
nated by the reduced Trx1, and the H2O2 content did not increase
sharply in a short period of time. If TrxR1 enzyme activity was inhibited
by compound 26, the ROS could not be eliminated by the oxidized Trx1
and gradually accumulated in the cell [27]. Interestingly, the H2O2
content was still increasing after the intracellular reduced glutathione
returned to the original level, which indicated that compound 26 will
not affect the glutathione system.
The following time-course experiments were tested to figure out the
relationship between TrxR1, GSH, and H2O2. Firstly, non-reductive
western blotting was performed on thioredoxin redox status (Fig. 2b).
It was found that Trx1 in the reduced state decreased significantly from
0.5 h after the administration, and then increased to a certain extent, but
never returned to the previous level. Meanwhile, the Trx1 in the
oxidized state showed a trend of increasing with the administration
time, and a high level has been maintained after 2 h of administration.
The above results showed that compound 26 could affect the redox level
4

Y. Zhong et al.
Bioorganic Chemistry 109 (2021) 104736
cleaved-caspase-3/9 which indicated that compound 26 played a dose-
dependent role in the process of caspase-3/9 activation and Bax/Bcl-2
ratio increase. Thus, these data demonstrated that compound 26 could
play an initial role in apoptosis by activating multiple caspase cascades.
Many cell stress-induced apoptosis involving p38 affects the Bcl-2
protein family which includes Bax proteins that promote apoptosis
and Bcl-2 proteins that resist apoptosis [28,29]. Bax protein causes
altering in the permeability of mitochondrial membranes which causes
the release of cytochrome c into the cytoplasm [30], where it forms a
cytochrome c/Apaf-1/caspase-9 apoptotic complex and activates
caspase-3 [31]. At the same time, the accumulation of ROS destroys
redox homeostasis and alters the permeability of the mitochondrial
membrane, ultimately stimulating the release of cytochrome c and
inducing apoptosis [32], and Zhang [2] reported that Trx1 was
demonstrated to directly inhibit cell apoptosis by catalyzing the S-
nitrosylation of pro-caspase 3 and caspase 3.
Table 1
Six kinds of cells (HepG2, HuH-7, Bel-7402, L02, MCF-7, SGC-7901) were co-
cultured with different concentrations of different compounds for 48 h, and
then cell viability was determined by MTT method and IC50 value was
calculated.
No.
IC50 values* (
μ
M)
EC50
values
M)
*
HepG2
HuH-
7
Bel-
L02
MCF-
7
SGC-
7901
(
μ
7402
1
2
>50
>50
>50
33.0
± 2.1
35.1
± 0.8
>50
39.1
± 2.5
>50
>50
>50
>50
>50
>50
>50
39.6
± 1.8
>50
39.1
± 0.6
25.5
± 2.6
>50
26.7
± 2.0
>50
>50
>50
>50
>50
>50
>50
>50
39.2
± 1.5
>50
>50
>50
35.5 ±
4.2
>50
29.7
± 0.7
>50
25.9
± 1.6
>50
>50
>50
>50
>50
>50
>50
26.6
± 4.8
>50
3
4
>50
31.4 ±
5.7
>50
>50
>50
>50
>50
>50
5
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
38.4
± 0.5
>50
>50
>50
>50
>50
>50
>50
>50
>50
6
7
8
2.5. Compound 26 activates the ASK1-p38-MAPK pathway
9
10
11
12
The western blot was operated for detecting the ROS-related protein
status after treated with compound 26 (Fig. 4a). It has been reported
that Trx1 is identified as a negative regulator of the ASK1-JNK/p38
pathway [4,33]. The reduced Trx1 could bind to ASK-1 and inactive
ASK1, thereby blocking the pro-apoptotic activity of ASK-1. The
oxidized Trx1 will dissociate from ASK1, thereby converting the inactive
form of ASK1 into active kinase [34]. There are two main key proteins,
P38 and JNK [31], in downstream of the ASK1-mediated MAPK
signaling pathway. The results showed that the ratio of phosphorylated
ASK1 and p38 were up-regulated significantly comparing to the control
group, respectively. However, the ratio of phosphorylated JNK did not
show significant differences between administration and control groups
(Fig. 4b).
13
37.2 ±
5.9
>50
>50
>50
14
15
16
17
18
>50
>50
>50
>50
>50
10.7
± 0.8
6.14
±
>50
>50
>50
>50
16.5
± 1.5
8.24
±
>50
>50
>50
>50
49.1
± 4.1
18.6
± 1.1
>50
>50
>50
>50
20.9
± 1.4
9.8 ±
0.7
>50
>50
>50
>50
20.9
± 1.1
9.60
±
>50
>50
>40
>50
>50
>50
>50
>50
18.8 ±
2.7
19
20.6 ±
2.9
25.0 ±
3.8
0.36
9.62
±
0.27
12.4
± 0.6
0.62
12.5
± 1.0
20
19.3 ±
28.5
27.8
28.1 ±
3.2
± 0.7
± 2.2
4.3
0.36
>50
>50
Altogether, compound 26 inhibited TrxR1 enzyme activity,
increased the level of intracellular ROS and reduced the ratio of reduced
Trx1, releasing ASK1 and activating the ASK1 pathway. Thus, 26 could
activate cell apoptosis through the ROS-Trx-ASK1-p38 pathway. At the
same time, apoptosis sometimes triggers p38 activation through sec-
ondary pathways, such as the production of ROS which is also a
recognized activator of p38 MAPK signaling pathway [35,36].
21
22
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
31.5 ±
4.1
23
24
25
26
>50
>50
>50
>50
>50
>50
35.9 ±
13.2
27.2 ±
2.5
12.5
± 1.0
30.6
± 4.4
7.43
±
34.2
± 0.6
>50
36.4
± 1.9
>50
16.3
± 3.3
>50
35.8
24.4 ±
2.6
± 3.3
39.4
32.4 ±
4.0
24.9 ±
4.5
± 1.8
37.4
3. Conclusions
8.25 ±
0.78
10.3
>50
17.9
21.4 ±
11.1
± 0.4
± 1.5
± 1.7
0.57
27.1
± 2.5
4.42
±
In this study, a series of novel phenazine derivatives (1~27) con-
taining the Michael acceptor scaffolds were designed and synthesized, in
which some compounds exhibited selective cytotoxicity against Bel-
7402 cancer cell lines in vitro. In particular, compound 26 showed bet-
27
9.97 ±
1.01
>50
>50
43.2
>50
30.0 ±
4.1
± 7.2
12.9
Cisplatin
10.9 ±
1.6
12.3
11.0
4.8 ±
–
± 0.7
± 0.4
± 1.9
0.4
0.37
ter selectivity for liver cancer cells (IC50 = 10.3
comparing to MCF-7 (IC50 = 17.9
M) cancer cell lines. Meanwhile, compound 26 showed no obvious cell
toxicity against human normal liver epithelial L02 cells (IC50 > 50 M),
μ
M for Bel-7402)
μ
M) and SGC-7901 (IC50 = 37.4
“–” not detected.
*
μ
Data is expressed as the mean ± SD of at least three parallel test results.
μ
which means this compound possessed a better safety potential. In the
following research, compound 26, the best antiproliferative activity in
this series, was verified to inhibit TrxR1 enzyme activity, ultimately
resulting in cellular molecular mechanism events of apoptosis including
growth of intracellular ROS, loss of reduced Trx1, liberation of ASK1 and
up-regulation of p38, respectively. Together, all these evidences sug-
gested that compound 26 acted as the TrxR1 inhibitor against Bel-7402
cancer cell line, and could activate the cells apoptosis through the ROS-
Trx-ASK1-p38 pathway. The further optimization of the structure and
activity of this series of compounds is being studied in our laboratory.
2.4. Compound 26 induces apoptosis by activating multiple caspase
cascades
The apoptosis of the Bel-7402 cells caused by compound 26 was
subsequently detected by flow cytometry. After treatment for 24 h with
26, the Bel-7402 cells induced a significant dose-dependent apoptosis
(Fig. 3a, b), compared with the control group. The ratios of viable cells
under the treatment with doses from 10 to 40 μM were significantly
diminished, while the apoptosis ratio was significantly increased in a
dose-dependent manner.
In order to further understand the potential mechanism of apoptosis
triggered by compound 26, we performed western blot analysis to
examine the expression of several apoptosis-related proteins. As shown
in Fig. 3b and c, compound 26 treatments resulted in a decrease in the
expression of Bcl-2, an increase in the expression level of Bax and
4. Experimental section
4.1. General
All commercially available chemicals and solvents were of analytical
5

Y. Zhong et al.
Bioorganic Chemistry 109 (2021) 104736
Fig. 2. Compound 26 induced the level of ROS through TrxR1-Trx1 system in Bel-7402 cancer cells. (a) 16
μ
M compound 26 or the same concentration of DMSO (as
a solvent control) were performed to treat the cells, record the ROS (H2O2) levels of the cells at different times. (b) After treating the cells with 16 μM compound 26
for the corresponding time, perform non-reducing immunoblotting with the whole cell lysate, used Trx1 antibody as a probe and β-actin as control, loading 15 μg
proteins in each lane, and perform quantitative processing on the results and calculate the ratio of reduced Trx1 to oxidized Trx1. (c) Treated the cells with 16 μM
Compound 26, and recorded the GSH/GSSG ratio of the cells at different times (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Compared with the
control group).
reagent grade and were used without further purification unless other-
wise specified. Column chromatography was carried out on silica gel
(100–300 mesh). TLC was conducted on silica gel 250 µm, GF254 plates
with short-wavelength UV light for visualization. 1H NMR and 13C NMR
spectroscopic measurements were performed on the Bruker AV-300 or
AV-500 NMR spectrometer, using TMS as internal references at 298 K,
respectively. High-resolution mass spectra (HRMS) were recorded on an
Agilent 6520B UPLC-Q-TOF mass spectrometer (Agilent Technologies,
Santa Clara, CA, USA). All of the products were identified by 1H NMR,
13C NMR and HRMS spectrometry.
mL). Diverse acryloyl chlorides (1.5–8 mmol) diluted in CH2Cl2 (30 mL)
then added dropwise to the above solution. The mixture was stirred
under room temperature for 1–5 h, as monitored by TLC until reaction
completed, and then 100 mL of CH2Cl2 was added, followed by washing
with dilute 2 N hydrochloric acid, saturated sodium bicarbonate and
water, respectively. Separating the organic layer, the solvent was
removed by distillation. The residue was purified by flash chromatog-
raphy on silica gel using petroleum ether/ethyl acetate (20/1–3/1, v/v),
and then recrystallized with anhydrous ethanol to give the compounds
1~4, respectively.
4.3.1. Phenazin-1-yl acrylate 1
4.2. General procedure for the synthesis of diverse phenazine precursors
Yield: 41%; Yellow solid; 1H NMR (500 MHz, CDCl3) δ 8.33 (d, J =
8.4 Hz, 1H), 8.28–8.23 (m, 2H), 7.90–7.83 (m, 5H),7.80 (t, J = 7.8 Hz,
1H), 7.27 (d, J = 7.2 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 151.7,
144.2, 143.8, 141.2, 134.7, 133.2, 131.8, 130.8, 130.5, 129.7, 129.2,
127.9, 127.6, 119.9, 108.9. HR ESI-MS [M+H]+ m/z = 251.0817 (calcd
for C15H11N2O2, 251.0821).
1-methoxyphenazine, 2-amino-3-hydroxyphenazine, 2,3-dihydrox-
yphenazine, 1-aminophenazine, 2-aminophenazine and 2,3-diamino-
phenazine were prepared according to the methods described in the
literature [21], [22], [23], [24], [7] and [22], respectively. After treated
with
BBr3,
1-methoxyphenazine
was
transformed
to
1-
hydroxyphenazine.
4.3.2. Phenazin-1-yl cinnamate 2
Yield: 47%; Reddish brown solid; 1H NMR (300 MHz, CDCl3) δ
8.27–8.19 (m, 3H), 8.04 (d, J = 16.0 Hz, 1H), 7.89–7.79 (m, 3H),
4.3. General procedure for the synthesis of compounds 1~4
7.70–7.65 (m, 3H), 7.48–7.46 (m, 3H), 6.95 (d, J = 16.0 Hz, 1H); 13
C
Compounds 1~4 were prepared according to the procedure
described as below. 1-hydroxyphenazine (196 mg, 1 mmol) was dis-
solved in the solution of dry CH2Cl2 (20 mL) and anhydrous pyridine (1
NMR (75 MHz, CDCl3) δ 165.5, 147.2, 144.0, 143.6, 142.8, 137.5, 135.6,
134.3, 131.0, 130.8, 130.6, 130.3, 129.5, 129.5, 129.0, 129.0, 128.4,
6

Y. Zhong et al.
Bioorganic Chemistry 109 (2021) 104736
Fig. 3. Compound 26 induced caspases-depended apoptosis in Bel-7402 cancer cells. (a) The flow cytometry was applied to detect apoptosis induced by compound
26, and calculate the percentage of apoptotic cells. (b) (c) Perform immunoblotting on the whole cell lysate and probe with Bcl-2, Bax, caspase-3/9, cleaved-caspase-
3/9 antibodies, and β-actin as loading control, 15
μ
g proteins were loaded in each lane to quantify the results. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p <
0.0001 vs control group).
Fig. 4. Compound 26 induced cell apoptosis
through ASK-1/P38 pathway in Bel-7402 cancer
cells. (a) Compound 26 was incubated with Bel-
7402 cells for 48 h in accordance with the con-
centration gradient, and whole cell lysates were
applied for immunoblotting, β-actin as a loading
control, and load 10 μg proteins in each lane to
calculate the ratio and quantify the results (b).
The means and SD of three independent experi-
ments were performed in triplicate are shown. (*
p < 0.05, ** p < 0.01, *** p < 0.001, **** p <
0.0001 vs the control group).
7

Y. Zhong et al.
Bioorganic Chemistry 109 (2021) 104736
128.4, 127.8, 121.5, 116.9. HR ESI-MS [M+H]+ m/z = 327.1141 (calcd
4.5.1. Phenazine-2,3-diyl diacrylate 9
1
for C21H15N2O2, 327.1134).
Yield: 61%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
8.72 (s, 2H), 8.25 (dd, J = 6.6, 3.4 Hz, 2H), 7.92 (dd, J = 6.6, 3.4 Hz,
2H), 6.58 (dd, J = 17.0, 10.0 Hz, 2H), 6.44 (dd, J = 17.0, 2.1 Hz, 2H),
5.83 (dd, J = 10.0, 2.1 Hz, 2H); 13C NMR (75 MHz, DMSO‑d6) δ 165.4,
165.4, 144.7, 144.7, 142.8, 142.8, 141.9, 141.9, 136.1, 136.1, 131.2,
131.2, 129.7, 129.7, 126.8, 126.8, 120.6, 120.6. HR ESI-MS [M+H]+ m/
z = 321.0881 (calcd for C18H13N2O4, 321.0875).
4.3.3. Phenazin-1-yl (E)-3-(3-(trifluoromethyl)phenyl)acrylate 3
Yield: 57%; Yellow solid; 1H NMR (500 MHz, CDCl3) δ 8.34 (d, J =
8.5 Hz, 1H), 8.29 (t, J = 8.6 Hz, 2H), 8.07 (d, J = 16.0 Hz, 1H), 7.95 (s,
1H), 7.92–7.85 (m, 4H), 7.74 (d, J = 7.8 Hz, 1H), 7.70 (d, J = 7.2 Hz,
1H), 7.62 (t, J = 7.8 Hz, 1H), 7.03 (d, J = 16.0 Hz, 1H); 13C NMR (125
MHz, CDCl3) δ 164.9, 147.0, 145.4, 143.2, 142.9, 137.4, 135.0, 131.8,
131.5, 131.4, 130.9, 130.2, 130.0, 129.6, 129.0, 127.4, 127.2, 127.1,
125.0, 125.0, 121.7, 119.0. HR ESI-MS [M+H]+ m/z = 395.1020 (calcd
for C22H14F3N2O2, 395.1007).
4.5.2. Phenazine-2,3-diyl (2E,2′E)-bis(but-2-enoate) 10
1
Yield: 65%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
8.74 (s, 2H), 8.22 (dd, J = 6.7, 3.4 Hz, 2H), 7.89 (dd, J = 6.7, 3.4 Hz,
2H), 7.03–6.94 (m, 2H), 6.43 (dd, J = 15.3, 1.8 Hz, 2H), 1.97 (dd, J =
6.9, 1.8 Hz, 6H); 13C NMR (75 MHz, DMSO‑d6) δ 165.6, 165.6, 144.5,
144.5, 142.6, 142.6, 141.7, 141.7, 136.5, 136.5, 131.7, 131.7, 129.4,
129.4, 126.5, 126.5, 120.4, 120.4, 18.3, 18.3. HR ESI-MS [M+H]+ m/z
= 349.1182 (calcd for C20H17N2O4, 349.1188).
4.3.4. Phenazin-1-yl (E)-3-(4-fluorophenyl)acrylate 4
Yield: 49%; Reddish brown solid; 1H NMR (300 MHz, CDCl3) δ 8.30
(d, J = 4.2 Hz, 1H), 8.25 (d, J = 4.2 Hz, 2H), 7.99 (d, J = 8.0 Hz, 1H),
7.90–7.82 (m, 3H), 7.68–7.66 (m, 3H), 7.15 (t, J = 4.2 Hz, 2H), 6.86 (d,
J = 8.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 165.9, 165.4, 147.1, 145.9,
143.9, 143.5, 143.1, 137.4, 131.1, 130.7, 130.4, 130.3, 130.2, 129.6,
129.4, 127.8, 121.5, 116.7, 116.7, 116.4, 116.1. HR ESI-MS [M+H]+ m/
z = 345.1047 (calcd for C21H14FN2O2, 345.1039).
4.5.3. Phenazine-2,3-diyl bis(3-methylbut-2-enoate) 11
1
Yield: 59%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
8.75 (s, 2H), 8.19 (dd, J = 6.7, 3.3 Hz, 2H), 7.90 (dd, J = 6.7, 3.3 Hz,
2H), 6.58 (s, 2H), 2.21 (s, 6H), 1.97 (s, 6H); 13C NMR (75 MHz,
DMSO‑d6) δ 164.7, 164.7, 145.1, 145.1, 142.5, 142.5, 141.4, 141.4,
136.8, 136.8, 132.2, 132.2, 129.7, 129.7, 126.3, 126.3, 116.8, 116.8,
26.8, 26.8, 19.3, 19.3. HR ESI-MS [M+H]+ m/z = 377.1509 (calcd for
4.4. General procedure for the synthesis of compounds 5~8
Compounds 5~8 were prepared with the similar method of prepar-
ing compounds 1~4. 2-amino-3-hydroxyphenazine was used in place of
1-hydroxyphenazine as the reactant to get compounds 5~8.
C22H21N2O4, 377.1501).
4.6. General procedure for the synthesis of compounds 12~15
4.4.1. 3-aminophenazin-2-yl acrylate 5
1
Yield: 61%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
Compounds 12~15 were prepared with the similar method of pre-
paring compounds 1~4. 1-aminophenazine was used in place of 1-
hydroxyphenazine as the reactant to get compounds 12~15.
7.92 (s, 2H), 7.59 (s, 2H), 7.01–6.95 (m, 2H), 6.31 (s, 2H), 6.52 (dd, J =
17.0, 10.0 Hz, 1H), 6.38 (dd, J = 17.0, 2.0 Hz, 1H), 5.86 (dd, J = 10.0,
2.0 Hz, 1H); 13C NMR (75 MHz, DMSO‑d6) δ 164.3, 146.5, 144.2, 142.2,
141.3, 140.4, 134.6, 132.1, 130.8, 130.0, 128.2, 127.1, 126.3, 125.3,
110.5. HR ESI-MS [M+H]+ m/z = 266.0937 (calcd for C15H12N3O2,
266.0930).
4.6.1. N-(phenazin-1-yl)acrylamide 12
Yield: 73%; Yellow solid; 1H NMR (500 MHz, CDCl3) δ 9.95 (s, 1H),
8.93 (d, J = 7.5 Hz, 1H), 8.29–8.25 (m, 2H), 7.96 (d, J = 8.7 Hz, 1H),
7.89–7.85 (m, 3H), 6.60 (d, J = 5.6 Hz, 2H), 5.93 (t, J = 5.7 Hz, 1H); 13C
NMR (125 MHz, CDCl3) δ 163.8, 143.8, 143.1, 142.2, 134.7, 134.0,
131.7, 131.5, 130.8, 130.7, 129.6, 129.2, 128.1, 123.4, 116.4. HR ESI-
MS [M+H]+ m/z = 250.0992 (calcd for C15H12N3O, 250.0980).
4.4.2. 3-aminophenazin-2-yl (E)-but-2-enoate 6
1
Yield: 65%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
7.94 (s, 2H), 7.57 (s, 2H), 7.03–6.98 (m, 2H), 6.33 (s, 2H), 6.07–6.03 (m,
1H), 5.85 (dd, J = 15.2, 1.7 Hz, 1H), 1.95 (dd, J = 6.9, 1.4 Hz, 3H); 13
C
NMR (75 MHz, DMSO‑d6) δ 164.6, 146.4, 144.6, 142.5, 141.2, 140.6,
134.3, 132.5, 130.6, 130.1, 128.5, 127.2, 126.5, 125.1, 114.6, 18.3. HR
ESI-MS [M+H]+ m/z = 280.1074 (calcd for C16H14N3O2, 280.1086).
4.6.2. N-(phenazin-1-yl)cinnamamide 13
Yield: 54%; Yellow solid; 1H NMR (500 MHz, CDCl3) δ 10.00 (s, 1H),
8.99 (d, J = 7.5 Hz, 1H), 8.32 (dt, J = 6.6, 3.4 Hz, 2H), 7.98 (d, J = 8.8
Hz, 1H), 7.92–7.88(m, 4H), 7.69 (d, J = 6.9 Hz, 2H), 7.48–7.43 (m, 3H),
6.90 (d, J = 15.6 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 164.2, 143.7,
143.1, 142,8, 142.8, 141.2, 134.7, 134.3, 131.9, 130.8, 130.7, 130.1,
129.6, 129.2, 128.9, 128.9, 128.1, 128.1, 123.2, 121.1, 116.4. HR ESI-
MS [M+H]+ m/z = 326.1287 (calcd for C21H16N3O, 326.1293).
4.4.3. 3-aminophenazin-2-yl 3-methylbut-2-enoate 7
1
Yield: 62%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
7.92 (s, 2H), 7.56 (s, 2H), 7.00–6.96 (m, 2H), 6.30 (s, 2H), 6.55 (s, 1H),
2.16 (s, 3H), 1.93 (s, 3H); 13C NMR (75 MHz, DMSO‑d6) δ 164.6, 146.4,
144.6, 142.5, 141.2, 140.6, 134.3, 132.5, 130.6, 130.1, 128.5, 127.2,
126.5, 125.1, 114.6, 26.5, 19.1. HR ESI-MS [M+H]+ m/z = 294.1252
(calcd for C17H16N3O2, 294.1243).
4.6.3. (E)-N-(phenazin-1-yl)-3-(3-(trifluoromethyl)phenyl)acrylamide 14
Yield: 65%; Yellow solid; 1H NMR (500 MHz, CDCl3) δ 10.04 (s, 1H),
8.99 (d, J = 7.4 Hz, 1H), 8.35–8.31 (m, 2H), 7.99 (d, J = 8.8 Hz, 1H),
7.94 (d, J = 5.2 Hz, 1H), 7.93–7.87 (m, 4H), 7.83 (d, J = 7.6 Hz, 1H),
7.69 (d, J = 7.6 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 6.97 (d, J = 15.5 Hz,
1H); 13C NMR (125 MHz, CDCl3) δ 163.6, 143.8, 143.1, 141.2, 141.1,
135.2, 134.7, 134.1, 131.8, 131.5, 130.9, 130.8, 129.6, 129.5, 129.2,
126.5, 126.5, 124.3, 124.3, 123.5, 123.0, 116.5. HR ESI-MS [M+H]+ m/
z = 394.1178 (calcd for C22H15F3N3O, 394.1167).
4.4.4. 3-aminophenazin-2-yl methacrylate 8
1
Yield: 71%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
7.89 (s, 2H), 7.60 (s, 2H), 7.01–6.97 (m, 2H), 6.35 (s, 2H), 6.44 (d, J =
11.2 Hz, 1H), 6.21 (d, J = 10.2 Hz, 1H), 2.03 (s, 3H); 13C NMR (75 MHz,
DMSO‑d6) δ 165.3, 146.8, 144.3, 143.2, 141.9, 140.8, 135.3, 133.4,
131.2, 130.4, 128.8, 127.5, 126.2, 125.7, 112.6, 18.3. HR ESI-MS
[M+H]+ m/z = 280.1093 (calcd for C16H14N3O2, 280.1086).
4.6.4. (E)-3-(4-fluorophenyl)-N-(phenazin-1-yl)acrylamide 15
4.5. General procedure for the synthesis of compounds 9~11
Yield: 69%; Yellow solid; 1H NMR (500 MHz, CDCl3) δ 9.98 (s, 1H),
8.98 (d, J = 7.3 Hz, 1H), 8.33–8.29 (m, 2H), 7.97 (d, J = 8.5 Hz, 1H),
7.92–7.84 (m, 4H), 7.67 (dd, J = 8.4, 5.5 Hz, 2H), 7.15 (t, J = 8.5 Hz,
2H), 6.82 (d, J = 15.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 164.8,
164.1, 162.8, 143.8, 143.1, 141.6, 141.2, 134.7, 134.2, 131.8, 130.8,
Compounds 9~11 were prepared with the similar method of pre-
paring compounds 1~4. 2,3-dihydroxyphenazine was used in place of 1-
hydroxyphenazine as the reactant to get compounds 9~11.
8

Y. Zhong et al.
Bioorganic Chemistry 109 (2021) 104736
130.7, 130.0, 130.0, 129.6, 129.2, 123.26, 120.8, 116.4, 116.2, 116.0.
2H), 5.92 (dd, J = 10.0, 2.2 Hz, 2H); 13C NMR (75 MHz, DMSO‑d6) δ
165.8, 165.8, 144.7, 144.7, 142.5, 142.5, 141.7, 141.7, 136.1, 136.1,
131.4, 131.4, 129.5, 129.5, 126.8, 126.8, 120.4, 120.4. HR ESI-MS
[M+H]+ m/z = 319.1190 (calcd for C18H15N4O2, 319.1195).
HR ESI-MS [M+H]+ m/z = 344.1187 (calcd for C21H15FN3O, 344.1199).
4.7. General procedure for the synthesis of compounds 16~20
4.8.2. (2E,2′E)-N,N’-(phenazine-2,3-diyl)bis(but-2-enamide) 22
Compounds 16~20 were prepared with the similar method of pre-
paring compounds 1~4. 2-aminophenazine was used in place of 1-
hydroxyphenazine as the reactant to get compounds 16~20.
1
Yield: 30%; Red powder; H NMR (300 MHz, DMSO‑d6) δ 9.88 (s,
2H), 8.67 (s, 2H), 8.20 (dd, J = 6.7, 3.4 Hz, 2H), 7.90 (dd, J = 6.7, 3.4
Hz, 2H), 7.01–6.89 (m, 2H), 6.39 (dd, J = 15.3, 1.8 Hz, 2H), 1.94 (dd, J
= 6.9, 1.8 Hz, 6H); 13C NMR (75 MHz, DMSO‑d6) δ 164.9, 164.9, 143.1,
143.1, 141.6, 141.6, 141.4, 141.4, 135.0, 135.0, 130.7, 130.7, 129.5,
129.5, 126.4, 126.4, 120.4, 120.4, 18.1, 18.1. HR ESI-MS [M+H]+ m/z
= 347.1509 (calcd for C20H19N4O2, 347.1508).
4.7.1. N-(phenazin-2-yl)acrylamide 16
1
Yield: 41%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
10.79 (s, 1H), 8.81 (d, J = 2.2 Hz, 1H), 8.23 (m, 3H), 8.02 (dd, J = 9.4,
2.3 Hz, 1H),7.97–7.87 (m, 2H), 6.57 (dd, J = 17.0, 10.0 Hz, 1H), 6.40
(dd, J = 17.0, 2.0 Hz, 1H), 5.90 (dd, J = 10.0, 2.0 Hz, 1H); 13C NMR (75
MHz, DMSO‑d6) δ 164.5, 144.3, 143.7, 142.4, 141.0, 141.0, 131.9,
131.4, 130.4, 130.4, 129.8, 129.4, 128.6, 126.8, 114.7. HR ESI-MS
[M+H]+ m/z = 250.0972 (calcd for C15H12N3O1, 250.0980).
4.8.3. N,N’-(phenazine-2,3-diyl)bis(3-methylbut-2-enamide) 23
1
Yield: 41%; Red powder; H NMR (300 MHz, DMSO‑d6) δ 9.85 (s,
2H), 8.73 (s, 2H), 8.22 (dd, J = 6.8, 3.4 Hz, 2H), 7.92 (dd, J = 6.8, 3.4
Hz, 2H), 6.56 (s, 2H), 2.24 (s, 6H), 1.98 (s, 6H); 13C NMR (75 MHz,
DMSO‑d6) δ 165.2, 165.2, 145.4, 145.4, 143.3, 143.3, 141.7, 141.7,
136.5, 136.5, 132.6, 132.6, 129.7, 129.7, 126.5, 126.5, 116.6, 116.6,
26.7, 26.7, 19.5, 19.5. HR ESI-MS [M+H]+ m/z = 375.1828 (calcd for
4.7.2. (E)-N-(phenazin-2-yl)but-2-enamide 17
1
Yield: 38%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
10.59 (s, 1H), 8.77 (d, J = 2.2 Hz, 1H), 8.23–8.18 (m, 3H), 8.00 (dd, J =
9.4, 2.3 Hz, 1H), 7.95–7.86 (m, 2H), 6.99–6.89 (m, 1H), 6.25 (dd, J =
15.2, 1.7 Hz, 1H), 1.93 (dd, J = 6.9, 1.4 Hz, 3H); 13C NMR (75 MHz,
DMSO‑d6) δ 164.8, 144.3, 143.6, 142.3, 141.9, 141.9, 141.3, 140.9,
131.3, 130.3, 129.7, 129.4, 126.9, 126.0, 114.3, 18.1. HR ESI-MS
[M+H]+ m/z = 264.1144 (calcd for C16H14N3O, 264.1137).
C22H23N4O2, 375.1821).
4.9. General procedure for the synthesis of compounds 24~27
1,4-phenylenediamine was used as the starting material to synthesize
6-methoxy-2-aminophenazine following the modified method in the
literature [21], which subsequently reacted with diverse acryloyl chlo-
rides to prepare compounds 24~27 with the similar method of pre-
paring compounds 1~4.
4.7.3. N-(phenazin-2-yl)cinnamamide 18
1
Yield: 43%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
10.86 (s, 1H), 8.85 (s, 1H), 8.26–8.20 (m, 3H), 8.05–7.99 (m, 1H),
7.91–7.85 (m, 2H), 7.72–7.65 (m, 3H), 7.27–7.20 (m, 2H), 7.23 (d, J =
7.2 Hz, 1H), 6.96 (d, J = 15.6 Hz, 1H); 13C NMR (75 MHz, DMSO‑d6) δ
165.6, 155.4, 144.7, 144.1, 141.7, 141.2, 139.8, 135.8, 134.4, 131.5,
130.6, 129.3, 129.0, 128.5, 126.7, 122.5, 120.2, 116.4, 115.4, 114.1,
107.6. HR ESI-MS [M+H]+ m/z = 326.1283 (calcd for C21H16N3O,
326.1293).
4.9.1. N-(6-methoxyphenazin-2-yl)acrylamide 24
Yield: 41%; Orange solid; 1H NMR (500 MHz, DMSO‑d6) δ 10.76 (s,
1H), 8.75 (s, 1H), 8.24 (d, J = 9.3 Hz, 1H), 7.99 (dd, J = 9.3, 2.2 Hz, 1H),
7.84–7.80 (m, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.21 (d, J = 7.4 Hz, 1H),
6.56 (dd, J = 16.9, 10.1 Hz, 1H), 6.40 (d, J = 17.0 Hz, 1H), 5.93–5.85
(m, 1H), 4.06 (s, 3H); 13C NMR (125 MHz, DMSO‑d6) δ 164.6, 155.6,
144.4, 144.1, 141.2, 139.6, 135.6, 131.9, 131.7, 130.8, 128.7, 126.3,
120.7, 114.5, 107.1, 56.4. HR ESI-MS [M+H]+ m/z = 280.1079 (calcd
for C16H14N3O2, 280.1086).
4.7.4. (E)-N-(phenazin-2-yl)-3-(3-(trifluoromethyl)phenyl)acrylamide 19
1
Yield: 44%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
10.9 (s, 1H), 8.87 (d, J = 2.1 Hz, 1H), 8.27–8.21 (m, 3H), 8.06–8.05 (m,
2H), 8.02–7.97 (m, 2H), 7.96–7.90 (m, 2H), 7.82–7.80 (m, 1H), 7.73 (t,
J = 7.7 Hz, 1H), 7.08 (d, J = 15.8 Hz, 1H); 13C NMR (75 MHz, DMSO‑d6)
δ 164.6, 144.4, 143.7, 142.4, 141.2, 141.1, 140.0, 136.2, 132.2, 131.5,
130.7, 130.6, 130.5, 130.2, 130.1, 129.8, 129.4, 126.8, 124.7, 124.7,
124.4, 114.6. HR ESI-MS [M+H]+ m/z = 394.1175 (calcd for
4.9.2. N-(6-methoxyphenazin-2-yl)cinnamamide 25
1
Yield: 42%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
10.83 (s, 1H), 8.80 (d, J = 2.0 Hz, 1H), 8.25 (d, J = 9.4 Hz, 1H), 8.01 (dd,
J = 9.4, 2.2 Hz, 1H), 7.85–7.80 (m, 1H), 7.75–7.68 (m, 4H), 7.50–7.44
(m, 3H), 7.21 (d, J = 7.2 Hz, 1H), 6.94 (d, J = 15.7 Hz, 1H), 4.06 (s, 3H);
13C NMR (75 MHz, DMSO‑d6) δ 165.0, 155.6, 144.5, 144.3, 141.9,
141.5, 139.7, 135.6, 135.0, 131.7, 130.8, 130.6, 129.6, 128.4, 126.3,
122.2, 120.8, 116.1, 115.1, 114.3, 107.1, 56.5. HR ESI-MS [M+H]+ m/z
= 356.1395 (calcd for C22H18N3O2, 356.1399).
C
22H15F3N3O, 394.1167).
4.7.5. (E)-3-(4-fluorophenyl)-N-(phenazin-2-yl)acrylamide 20
1
Yield: 37%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
10.83 (s, 1H), 8.83 (s, 1H), 8.23–8.19 (m, 3H), 8.03 (dd, J = 9.4, 1.7 Hz,
1H), 7.90 (m, 2H), 7.77–7.70 (m, 3H), 7.30 (t, J = 8.7 Hz, 2H), 6.88 (d, J
= 15.7 Hz, 1H); 13C NMR (75 MHz, DMSO‑d6) δ 164.9, 144.4, 143.7,
142.4, 141.3, 141.0, 140.7, 131.7, 131.7, 131.4, 130.7, 130.6, 130.5,
130.4, 129.8, 129.4, 126.9, 122.0, 116.6, 116.5, 114.4. HR ESI-MS
[M+H]+ m/z = 344.1208 (calcd for C21H15FN3O, 344.1199).
4.9.3. (E)-N-(6-methoxyphenazin-2-yl)-3-(3-(trifluoromethyl)phenyl)
acrylamide 26
1
Yield: 45%; Reddish brown solid; H NMR (500 MHz, DMSO‑d6) δ
10.91 (s, 1H), 8.82 (d, J = 2.3 Hz, 1H), 8.27 (d, J = 9.3 Hz, 1H), 8.06 (s,
1H), 8.01 (dd, J = 9.2, 2.4 Hz, 2H), 7.85–7.80 (m, 3H), 7.75–7.71 (m,
4.8. General procedure for the synthesis of compounds 21~23
2H), 7.23 (d, J = 7.7 Hz, 1H), 7.07 (d, J = 9.7 Hz, 1H), 4.07 (s, 3H); 13
C
NMR (125 MHz, DMSO‑d6) δ 164.5, 155.6, 144.5, 144.2, 141.4, 140.0,
139.7, 136.2, 135.6, 132.2, 131.7, 130.9, 130.7, 126.8, 126.7, 126.2,
124.7, 124.6, 124.4, 120.8, 114.4, 107.1, 56.5. HR ESI-MS [M+H]+ m/z
= 424.1288 (calcd for C23H17F3N3O2, 424.1273).
Compounds 21~23 were prepared with the similar method of pre-
paring compounds 1~4. 2,3-diaminophenazine was used in place of 1-
hydroxyphenazine as the reactant to get compounds 21~23.
4.8.1. N,N’-(phenazine-2,3-diyl)diacrylamide 21
4.9.4. (E)-3-(4-fluorophenyl)-N-(6-methoxyphenazin-2-yl)acrylamide 27
1
1
Yield: 47%; Red powder; H NMR (300 MHz, DMSO‑d6) δ 9.85 (s,
Yield: 42%; Reddish brown solid; H NMR (300 MHz, DMSO‑d6) δ
2H), 8.71 (s, 2H), 8.24 (dd, J = 6.7, 3.3 Hz, 2H), 7.90 (dd, J = 6.7, 3.3
Hz, 2H), 6.59 (dd, J = 17.0, 10.0 Hz, 2H), 6.43 (dd, J = 17.0, 2.2 Hz,
10.84 (s, 1H), 8.81 (d, J = 2.2 Hz, 1H), 8.27 (d, J = 9.3 Hz, 1H), 8.01 (dd,
J = 9.4, 2.4 Hz, 1H), 7.87–7.72 (m, 5H), 7.33 (t, J = 9.0 Hz, 2H), 7.23
9

Y. Zhong et al.
Bioorganic Chemistry 109 (2021) 104736
(dd, J = 7.7, 1.2 Hz, 1H), 6.89 (d, J = 15.8 Hz, 1H), 4.07 (s, 3H); 13
C
4.13. Flow cytometry analysis
NMR (75 MHz, DMSO‑d6) δ 164.9, 155.7, 144.5, 144.3, 141.5, 140.7,
139.7, 135.6, 131.7, 130.9, 130.7, 130.6, 130.1, 126.3, 122.1, 122.0,
120.8, 116.7, 116.5, 114.3, 107.1, 56.5. HR ESI-MS [M+H]+ m/z =
374.1308 (calcd for C22H17FN3O2, 374.1305).
Using flow cytometry (BD FACS Accuri C6) to determine and analyze
the apoptosis of the tested cells. Collect cells treated with different
concentrations of compound 26 for 24 h, and detect the apoptosis of
cells treated with compound 26 according to the steps of Annexin V-PI
Apoptosis Detection Kit (Cat. # Y6002, US EVERBRIGHT). FACS anal-
ysis was performed to determine the cell cycle distribution and the
percentage of apoptotic cells.
4.10. Cell culture and reagents
MTT (Cat.# S19063, YuanyeBio, Shanghai, China), DMSO (Cat.#
30072418, SCR, Cina), RIPA (Cat.# P0013B) (Beyotime, China), Dual
Color Prestained Protin Marker (Cat.# WJ101) (EpiZyme, China).
Antibody against Bcl-2 (Cat.# AF6139), Bax (Cat.# AF0120), Caspase 9
(Cat.# AF6348), Cleaved-Caspase 3 (Asp 175) (Cat.# AF7022), Ask1
(Cat.# AF6477), Phospho-Ask1 (Thr838) (Cat.# AF8096) was from
Affinity. Antibody against Caspase-3 (Cat.# LTO6560), Cleaved-Caspase
9 (D330) (Cat.# LTO3100C), JNK1/2/3 (Cat.# LTO9342), Phospho-
JNK1/2/3 (T183/Y185) (Cat.# LTO7510P), P38 (Cat.# LTO3153),
Phospho-P38 (Cat.# LTO8380P) was from Litho. Antibody against
Thioredoxin (Cat.# 14999–1-AP) from proteinech.
4.14. Hydrogen peroxide assay
Use a Hydrogen Peroxide Assay Kit (Cat.# S0038, Beyotime) to
detect the concentration of hydrogen peroxide of the cell. Bel-7402 cells
were seeded into six-well plates at a density of 2 × 105 cells per well and
incubated for 24 h. Subsequently, the corresponding concentration of
the compounds were diluted with medium, and added to the six-well
plate at the corresponding time, cultured in a 5% CO2 incubator at
37 ◦C. The cells were collected and centrifuged at 3500 rpm for 5 min at
4
◦C. After discarding the supernatant, 150
μL of hydrogen peroxide
Cells were cultured in medium with 10% (v/v) fetal bovine serum
(FBS) (Gibco, Grand Island, NY, USA), 100 µg/ml streptomycin, and 100
detection lysate per 1 million cells were added to the lysate, then ho-
mogenized thoroughly to break and lyse the cells and centrifuged at
◦
units/ml penicillin. in a 5% CO2 incubator at 37 C. The human liver
◦
12,000 rpm at 4 C for 5 min to take the supernatant for subsequent
cancer cell lines, HepG2 (Cat.# KG020) cell line was obtained from
ATCC, KeyGEN BioTECH, China. HuH-7 (Cat.# FH05946), Bel-7402
(Cat.# FH98464) and MCF-7 cell line purchased from FuHeng Cell
Center, Shanghai, China. Human gastric cancer cell lines SGC-7901 was
obtained from China academia Sinica Cell repository (Shanghai). HuH-7
and MCF-7 cells preserved were cultured in high glucose DMEM medium
(KeyGEN BioTECH, China). HepG2 cells preserved were cultured in
MEM medium (KeyGEN BioTECH, China). Bel-7402, SCG-7901 and L02
cells preserved were cultured in 1640 medium (KeyGEN BioTECH,
China).
determination. The supernatant of cultured cells were used directly for
subsequent determinations. A 96-well plate was used for detection. Add
50
μ
L of the sample to be tested or standard to each well, and then add
100
μ
L of hydrogen peroxide detection reagent (containing Fe2+ and
xylenol orange), mix well and place at room temperature for 30 min, and
then immediately determine at 560 nm. The concentration of hydrogen
peroxide was calculated in the sample according to the standard curve.
4.15. GSH and GSSH assay
Use GSH and GSSH Assay Kit (Cat.# S0053, Beyotime) to detect the
content of GSH and GSSH inside the cell. BEL-7402 cells were seeded
into six-well plates at a density of 2 × 105 cells per well and incubated
for 24 h. Subsequently, the compound was diluted with the medium to
the corresponding concentration, and added to the six-well plate at the
corresponding time, and the cells were cultured at 37 ◦C in a 5% CO2
incubator. At the same time, prepare test reagents according to the in-
structions of the kit. After the corresponding co-cultivation time, collect
the cells, add the protein removal reagent M solution according to the
ratio of 1:3 (v/v) and vortex. After two quick freezing and thawing with
liquid nitrogen and 37 ◦C water baths, centrifuging to obtain the total
glutathione determination samples (supernatant). To prepare GSSG test
4.11. Cytotoxic activity in vitro
The cytotoxic activities of compounds in vitro were measured using
the 3-(4,5-Dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT)
assay. Cells in the form of cell suspension were placed in 96-well plates
(4 × 103 cells per well) and grown overnight at 37 ◦C in a 5% CO2
incubator. The tested compounds were dissolved in DMSO and then
added to the wells to achieve final concentrations ranging from 5 to 40
μ
M after serial dilution. Blank controls were prepared by addition of
culture medium (200 μL). The solvent control group was added with
medium containing the corresponding concentration of DMSO. The
plates were incubated at 37 ◦C in a 5% CO2 incubator for 48 h. At the end
samples, add 20
μL of diluted GSH removal auxiliary solution per 100 μL
of the treatment, 20 μL MTT solutions (5 mg/mL) were added to each
samples, and then add GSH removal working solution according to the
well and incubated for 4 h at 37 ◦C, then 150
μL DMSO was added to
proportion of 4 μL per 100 μL samples for vortexing and settled sample at
solubilize the MTT formazan after removing the original medium,
respectively. The optical density of each well was measured with a
microplate reader at a wavelength of 490 nm. Each experiment was
repeated at least three times to obtain the mean values. Five different
tumor cell lines and one normal hepatocyte are the subjects of this study:
HepG2, HuH-7, Bel-7402, MCF-7, SCG-7901 and L02.
25 ◦C for 60 min. Subsequently, the total glutathione and GSSG content
were determined according to the instructions, and the GSH content was
calculated according to the measurement results.
4.16. Western blot analysis
Bel-7402 cells were seeded into six-well plates at a density of 2 × 105
cells per well and incubated for 24 h. Subsequently, the compounds were
diluted with the medium to the corresponding concentration, and
incubated with the cells at 37 ◦C in a 5% CO2 incubator. The cells were
collected and after centrifugation at 3000 rpm at 4 ◦C, and the cells were
lysed with RIPA added with PMSF and a phosphatase inhibitor. After
being lysed on ice for 30 min, the samples were centrifuged at 4 ◦C and
12,000 rpm for 15 min. The supernatant contained protein. The protein
concentration of the supernatant was determined by BCA assay. Deter-
mine the loading amount according to the BCA results, so that the
protein loading amount of each lane of sodium dodecyl sulfa-
te–polyacrylamide gel electrophoresis (SDS-PAGE) is equal. Then add
4.12. Detection of TrxR1 enzyme activities
The TrxR1 enzyme activity kit (Cat.# BC1155, Solarbio) was applied
to perform TrxR1 enzyme activity detection. The Bel-7402 cells were
directly sonicated (300 w, ultrasound 3 s, interval 7 s, total time 3 min)
before administration, the total protein was extracted in the cell by
centrifuge (12,000 rpm, 15 min). Then, the different concentrations of
the compounds and the protein supernatants were incubated in 37 ◦C for
30 min. After the incubation, the subsequent enzyme activity tests were
carried out according to the instructions, and the enzyme activities re-
sults of the different doses calculate EC50
.
10

Y. Zhong et al.
Bioorganic Chemistry 109 (2021) 104736
¨
[4] X. Cheng, P. Holenya, S. Can, H. Alborzinia, R. Rubbiani, I. Ott, S. Wolfl, Mol.
loading buffer to the sample, which contains (for reducing SDS-PAGE) or
without (for non-reducing SDS-PAGE) reducing agents such as DDT. Gels
were then transferred to PVDF membranes and blocked with Protein
Free Rapid Blocking Buffer for 30 min. Then the membranes were
incubated with primary antibodies at 1:1000 dilutions in 5% BSA
overnight at 4 ◦C. After 12 h, the strips were washed three times with
TBST for a total of 30 min. After that, the secondary antibody was
conjugated in a 1:5000 dilution at room temperature for 45 min, and
then washed with TBST 3 times, each for 10 min. Levels of a β-actin, used
as loading control were also quantified to confirm equal protein loading
on the gels.
Cancer 13 (2014) 1.
[5] H. Sies, D.P. Jones, Nat. Rev. Mol. Cell Biol. 21 (2020) 363.
[6] X. Gao, Y. Lu, Y. Xing, Y. Ma, J. Lu, W. Bao, Y. Wang, T. Xi, Microbiol. Res. 167
(2012) 616.
[7] X. Gao, Y. Lu, L. Fang, X. Fang, Y. Xing, S. Gou, T. Xi, Eur. J. Med. Chem. 69 (2013)
1.
[8] J. Gao, M. Chen, X. Tong, H. Zhu, H. Yan, D. Liu, W. Li, S. Qi, D. Xiao, Y. Wang,
Y. Lu, F. Jiang, Comb. Chem. High Throughput Screen. 18 (2015) 960.
[9] Y. Lu, Y. Yan, L. Wang, X. Wang, J. Gao, T. Xi, Z. Wang, F. Jiang, Eur. J. Med.
Chem. 127 (2017) 928.
[10] Y. Lu, L. Wang, X. Wang, T. Xi, J. Liao, Z. Wang, F. Jiang, Eur. J. Med. Chem. 135
(2017) 125.
[11] M. Zhang, S. Gu, G. Liu, C. Li, H. Xu, Z. Wu, B. Ye, Y. Lu, D. Huang, Z. Wang,
F. Jiang, Comb. Chem. High Throughput Screen. 22 (2019) 35.
[12] J. Liao, L. Wang, Z. Wu, Z. Wang, J. Chen, Y. Zhong, F. Jiang, Y. Lu, J. Enzyme
Inhib. Med. Chem. 34 (2019) 1158.
Declaration of Competing Interest
[13] J. Chen, Y. Zhang, X. Jie, J. She, G. Dongye, Y. Zhong, Y. Deng, J. Wang, B. Guo,
L. Chen, J. Inorg. Biochem. 193 (2019) 112.
[14] S. Busker, W. Qian, M. Haraldsson, B. Espinosa, L. Johansson, S. Attarha, I.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
´
´
Kolosenko, J. Liu, M. Dagnell, D. Grander, E.S.J. Arner, K. Pokrovskaja Tamm, B.D.
G. Page, Sci. Adv. 6 (2020) eaax7945.
[15] Y. Zhang, J. Zhou, Q. Ye, K. Zeng, J. Pan, L. Chen, Y. Wang, B. Yang, Q. He, J. Gao,
S. Zeng, L. Yu, Toxicol. Appl. Pharmacol. 401 (2020) 115079.
[16] J. Zhang, Y. Liu, D. Shi, G. Hu, B. Zhang, X. Li, R. Liu, X. Han, X. Yao, J. Fang, Eur.
J. Med. Chem. 140 (2017) 435.
Acknowledgements
[17] C. Zhuang, W. Zhang, C. Sheng, W. Zhang, C. Xing, Z. Miao, Chem. Rev. 117 (2017)
7762.
The study received financial support from the National Science and
Technology Major Projects for “Major New Drugs Innovation and
Development” (grant No. 2019ZX09201001-004-001), the Project Pro-
gram of National Natural Science Foundation of China (Grant No.
81872757), the Project Program of Natural Science Foundation of
Jiangsu Province of China (Grant No. BK20201329), the Fundamental
Research Funds for the Central Universities (grant No. 2632018ZD01),
Innovation and Entrepreneurship Training Program for Undergraduate
(No. 201910316220 and No. 202010316245), the Priority Academic
Program Development of Jiangsu Higher Education Institution and Qing
Lan Project.
[18] M. Decker, Curr. Med. Chem. 18 (2011) 1464.
[19] K. Nepali, S. Sharma, M. Sharma, P.M. Bedi, K.L. Dhar, Eur. J. Med. Chem. 77
(2014) 422.
[20] Shaveta, S. Mishra, P. Singh, Eur. J. Med. Chem. 124 (2016) 500.
[21] A.T. Garrison, Y. Abouelhassan, D. Kallifidas, H. Tan, Y.S. Kim, S. Jin, H. Luesch, R.
W. Huigens III, J. Med. Chem. 61 (2018) 3962.
[22] A.A. El Bahnasawy, M.F. El Ahwany, Phosphorus, sulfur, and silicon and the
related elements, 182 (2007) 1937.
[23] C. Seillan, H. Brisset, O. Siri, Org. Lett. 10 (2008) 4013.
[24] N.G. Paciaroni, N.V. Borrero, J.R. Rocca, R.W. Huigens III, Res. Rev.: J. Med. Org.
Chem. 2 (2015) 67.
[25] T. Liu, J. Zhang, X. Han, J. Xu, Y. Wu, J. Fang, Free Radical Biol. Med. 135 (2019)
216.
[26] M. del P.S. Idelchik, U. Begley, T.J. Begley, J.A. Melendez, Semin. Cancer Biol. 47
(2017) 57.
Appendix A. Supplementary material
[27] V.R. Pannala, R.K. Dash, Free Radical Biol. Med. 78 (2015) 42.
[28] K. Hui, Y. Yang, K. Shi, H. Luo, J. Duan, J. An, P. Wu, Y. Ci, L. Shi, C. Xu, Cancer
Lett. 354 (2014) 189.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104736.
[29] Y. Xiang, W. Ye, C. Huang, B. Lou, J. Zhang, D. Yu, X. Huang, B. Chen, M. Zhou,
Biochem. Biophys. Res. Commun. 487 (2017) 820.
[30] G.M. Jose, M. Raghavankutty, G.M. Kurup, Process Biochem. 68 (2018) 197.
[31] M. Bian, R. Fan, S. Zhao, W. Liu, J. Med. Chem. 62 (2019) 7309.
[32] S.A. Ansari, U.R. Pendurthi, L.V.M. Rao, Blood Adv. 1 (2017) 2399.
References
´
[33] E.S.J. Arner, A. Holmgren, Semin. Cancer Biol. 16 (2006) 420.
´
[1] N. Guttenberger, W. Blankenfeldt, R. Breinbauer, Bioorg. Med. Chem. 25 (2017)
6149.
[34] V. Branco, L. Coppo, S. Sola, J. Lu, C.M.P. Rodrigues, A. Holmgren, C. Carvalho,
Redox Biol. 13 (2017) 278.
´
[2] J. Zhang, X. Li, X. Han, R. Liu, J. Fang, Trends Pharmacol. Sci. 38 (2017) 794.
[3] L. Xie, Z. Luo, Z. Zhao, T. Chen, J. Med. Chem. 60 (2017) 202.
[35] E.F. Wagner, A.R. Nebreda, Nat. Rev. Cancer 9 (2009) 537.
[36] A.G. Barata, T.P. Dick, Redox Biol. 28 (2020), 101340.
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