Design, synthesis and biological evaluation of anilide (dicarboxylic acid) shikonin esters as antitumor agents through targeting PI3K/Akt/mTOR signaling pathway

Article abstract of DOI:10.1016/j.bioorg.2021.104872

Triple-negative breast cancer (TNBC) has an unfavorable prognosis attribute to its low differentiation, rapid proliferation and high distant metastasis rate. PI3K/Akt/mTOR as an intracellular signaling pathway plays a key role in the cell proliferation, m

Full text of DOI:10.1016/j.bioorg.2021.104872

Bioorganic Chemistry 111 (2021) 104872
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of anilide (dicarboxylic acid)
shikonin esters as antitumor agents through targeting PI3K/Akt/mTOR
signaling pathway
,
,
,c,
Yingying Maa 1, Xiaorong Yangb 1, Hongwei Hana 1, Zhongling Wena, Minkai Yanga,
Yahan Zhanga, Jiangyan Fua, Xuan Wanga, Tongming Yinc, Guihua Lua c, Jinliang Qia
,
,
,c
,
c,
,
c,
,c,*
Hongyan Lina *, Xiaoming Wanga *, Yonghua Yanga
a State Key Laboratory of Pharmaceutical Biotechnology, Institute of Plant Molecular Biology, School of Life Sciences, Nanjing University, Nanjing 210023, China
b School of Biology and Geography Science, Yili Normal University, Yining 835000, China
c Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Triple-negative breast cancer (TNBC) has an unfavorable prognosis attribute to its low differentiation, rapid
proliferation and high distant metastasis rate. PI3K/Akt/mTOR as an intracellular signaling pathway plays a key
role in the cell proliferation, migration, invasion, metabolism and regeneration. In this work, we designed and
synthesized a series of anilide (dicarboxylic acid) shikonin esters targeting PI3K/Akt/mTOR signaling pathway,
and assessed their antitumor effects. Through three rounds of screening by computer-aided drug design method
(CADD), we preliminarily obtained sixteen novel anilide (dicarboxylic acid) shikonin esters and identified them
as excellent compounds. CCK-8 assay results demonstrated that compound M9 exhibited better antiproliferative
activities against MDA-MB-231, A549 and HeLa cell lines than shikonin (SK), especially for MDA-MB-231 (M9:
TNBC
PI3K/Akt/mTOR
Anilide (dicarboxylic acid) shikonin esters
Antitumor
3D-QSAR
IC50 = 4.52 ± 0.28 μM; SK: IC50 = 7.62 ± 0.26 μM). Moreover, the antiproliferative activity of M9 was better
than that of paclitaxel. Further pharmacological studies showed that M9 could induce apoptosis of MDA-MB-231
cells and arrest the cell cycle in G2/M phase. M9 also inhibited the migration of MDA-MB-231 cells by inhibiting
Wnt/β-catenin signaling pathway. In addition, western blot results showed that M9 could inhibit cell prolifer-
ation and migration by down-regulating PI3K/Akt/mTOR signaling pathway. Finally, a three-dimensional
quantitative structure–activity relationship (3D-QSAR) model was also constructed to provide a basis for
further development of shikonin derivatives as potential antitumor drugs through structure–activity relationship
analysis. To sum up, M9 could be a potential candidate for TNBC therapy.
1. Introduction
paclitaxel and Olaparib. Although there are various chemotherapy
regimens, 35% of TNBC patients still have recurrence and metastasis
Breast cancer is a common malignant tumor in women across the
world. A recent epidemiological clinical study reported that the preva-
lence of breast cancer is over 10% [1,2]. Triple-negative breast cancer
(TNBC) is a subtype of breast cancer characterized by negative expres-
sion of estrogen receptor (ER), progesterone receptor (PR) and human
epidermal growth factor receptor 2 (HER2 or EGFR). More threaten-
ingly, about 30%-40% of TNBC can develop into metastatic breast
cancer. Amongst, visceral metastasis is widespread, especially in the
lung and brain [3,4]. Currently, the main clinical drugs for TNBC are
[5]. There are some targeted drugs for TNBC in preclinical research and
clinical trials, but most of them have not achieved the desired effect and
have no apparent improvement in patients’ Overall survival. This could
be correlated with different subtypes of TNBC and the high heteroge-
neity of tumor cell signaling pathways, which suggests that we need to
explore more potential targets for TNBC. Therefore, it is still a significant
challenge for humans to discover more effective TNBC therapy.
Numerous studies indicate that the PI3K/Akt/mTOR signaling
pathway is abnormally activated in patients with TNBC [6]. It is
* Corresponding authors at: State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210023, China (Y. Yang, X. Wang, H. Lin).
E-mail addresses: linhy@nju.edu.cn (H. Lin), wangxm07@nju.edu.cn (X. Wang), yangyh@nju.edu.cn (Y. Yang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104872
Received 16 December 2020; Received in revised form 24 March 2021; Accepted 24 March 2021
Available online 29 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

Y. Ma et al.
Bioorganic Chemistry 111 (2021) 104872
commonly present in cells and is involved in the complex regulation of
cell senescence, angiogenesis, energy metabolism and glucose meta-
bolism, and is closely related to many other signaling pathways. Over-
activation of this pathway can promote cell proliferation and abnormal
differentiation, thereby mediating the generation, metastasis and inva-
sion of tumors. Thus, it is crucial for the occurrence, development and
prognosis of tumors. In this pathway, Akt is a core member of PI3K/Akt/
mTOR signaling pathway, and its’ over-activation can lead to tumori-
genesis, metastasis and the generation of drug resistance [7].
2. Results and discussion
2.1. Preliminary calculation
Before synthesis, CADD method was used for three rounds of struc-
tural screening. Molecular docking was performed to calculate the
binding energy (CDOCKERINTERACTION ENERGY in CDOCKER pro-
tocol in Discovery Studio 3.5) between all target compounds and Akt
(PDB code: 3CQW) complex structure. In the first round, 33 compounds
were evaluated to screen out the backbone. Then, the length of the in-
termediate bridge of shikonin derivatives was changed in the first round
of screening to carry out the second round of screening. Next, the sub-
stituents on the benzene ring were changed to provide 15 options for the
third round of screening. Totally, >2000 compounds were recalculated
during the process of virtual screening and 16 compounds were selected
for further study. (Supporting Information).
Shikonin and its derivatives commonly possess antibacterial, anti-
tumor, antiviral, anti-inflammatory and other pharmacological effects
[8–12]. Previous studies indicate that shikonin and its derivatives can
induce apoptosis in various cancer cell lines through inhibiting the
PI3K/Akt/mTOR pathway [13–18]. In addition, some other studies have
shown that shikonin derivatives such as SYUNZ-16 [19] and DMAKO-05
[20] can inhibit Akt activity and induce apoptosis, thereby inhibiting the
occurrence and development of tumor. In this study, we first used a
computer-aided drug design (CADD) approach to screen out 16 potential
candidates for targeting the PI3K/Akt/mTOR signaling pathway. Based
on the CADD results, we performed further biochemical and pharma-
ceutical analysis to evaluate the 16 novel anilide (dicarboxylic acid)
shikonin esters as potent antitumor agents for TNBC through regulating
PI3K/Akt/mTOR signaling pathway. Finally, we use 3D-QSAR software
(discovery studio 3.5, Accelrys, Co. Ltd) to provides reference for the
structural optimization of shikonin derivatives that inhibit the prolif-
eration of MDA-MB-231 cells in the subsequent experiments.
In terms of the binding energy, M9 (ꢀ 49.0427 kcal/mol) was the best
one amongst the 16 target compounds and its binding energy was much
lower than SK (ꢀ 28.163 kcal/mol) (Fig. S1, Supporting Information). In
order to better understand the potential of M9, we listed all the amino
acid residues that M9 interacted with Akt (PDB code: 3CQW), and the
results were shown in Fig. 1. In this binding model, M9 can stably bind to
the active cavity of Akt by forming the hydrogen bond with GLU278.
Other weak interactions, such as Pi-Anion force, carbon-hydrogen bond,
van der Waals and Pi-alkyl interactions which were formed with
HIS354, GLU314, LFU347, PRO313, LYS276, TYR315, LEU277,
Fig. 1. The binding mode analysis of M9 and SK (PDB Code: 3CQW). (PDB code: 3CQW). (A) Two-dimensional image of M9 interacting with amino acid residues in
the active cavity of Akt. (B) Three-dimensional image of M9 inserted into the Akt binding site. (C) Two-dimensional image of SK interacting with amino acid residues
in the active cavity of Akt. (D) Three-dimensional image of SK inserted into the Akt binding site. Carbon atoms are color gray (2D image) or blue (3D image), oxygen
atoms are color red, hydrogen atoms are color white. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)
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Y. Ma et al.
Bioorganic Chemistry 111 (2021) 104872
PHF236, GLU341 etc. In comparison, SK (the positive control) formed
two hydrogen bonds with the amino acid residues (LYS276 and GLU314)
of Akt, but the binding remained unstable because its structure is too
small to match the active cavity.
Table 1
Chemical structures of M1-M16.
2.2. Chemistry
Synthesis routes of the anilide (dicarboxylic acid) shikonin esters
M1-M16 were shown in Scheme 1. All of the target compounds were
reported for the first time here, and their structures were fully charac-
terized by HRMS, 1H NMR, 13C NMR and melting test. The results were
completely consistent with the structure described. (Supporting
Information).
Compounds
n
R1
R2
R3
R4
R5
R6
M1
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
H
H
H
H
H
H
H
Ph
M2
F
H
F
M3
F
H
H
M4
H
CF3
H
H
2.3. Biological activities
M5
H
Cl
CH3
H
M6
H
H
M7
CH3
H
H
2.3.1. M9 significantly inhibited cancer cell proliferation
M8
H
H
Cell Counting Kit-8 (CCK-8) assay were conducted for the anti-
proliferative activities by anilide (dicarboxylic acid) shikonin esters
M1-M16 against five human cancer cell lines, including two human
breast cancer cell lines (MCF-7 and MDA-MB-231), one human cervical
cancer cell line (HeLa), one human colon cancer cell line (HCT-8), one
human lung adenocarcinoma cell line (A549), and one human normal
breast epithelial cell line (MCF-10A). Results shown in Table 2 indicated
that all target compounds exhibited good anti-proliferative activity
against the five kinds of cancer cell lines, and some of them even showed
better inhibitory activity than SK while avoiding MCF-10A, the non-
cancer cell line.
M9
H
H
H
M10
M11
M12
M13
M14
M15
M16
F
H
F
F
H
H
H
CF3
H
H
H
Cl
CH3
H
H
H
CH3
H
H
H
H
Scheme 1. The synthesis routes of compounds M1-M16. (a) Triethylamine, CH2Cl2, room temperature; (b) DCC, DMAP, CH2Cl2, ice bath.
3

Y. Ma et al.
Bioorganic Chemistry 111 (2021) 104872
Table 2
Inhibition of cell proliferation against five human cancer cell lines (MDA-MB-231, MCF-7, A549, HCT-8, HeLa) and one human normal breast epithelial cell line (MCF-
10A) by M1-M16, SK and PTX.
Compounds
IC50 ± SD (µM)
MDA-MB-231 MDA231
MCF-7
A549
HCT-8
Hela
MCF-10A
M1
16.34 ± 2.12
30.72 ± 2.5
6.21 ± 1.68
25.94 ± 1.83
9.82 ± 2.17
5.91 ± 1.47
12.04 ± 1.05
11.83 ± 1.07
4.52 ± 0.28
38.31 ± 4.31
5.13 ± 0.41
9.12 ± 1.17
22.9 ± 2.12
10.54 ± 1.44
18.19 ± 0.91
15.19 ± 1.49
7.62 ± 0.26
＞100
19.55 ± 1.79
35.6 ± 1.07
21.18 ± 1.68
41.84 ± 2.3
15.52 ± 0.21
48.14 ± 2.19
11.98 ± 0.95
24.02 ± 2.71
15.83 ± 1.97
10.79 ± 1.15
14.71 ± 0.71
17.13 ± 1.27
14.28 ± 1.61
13.22 ± 1.52
15.98 ± 1.13
16.48 ± 1.66
15.09 ± 0.81
17.72 ± 2.11
13.97 ± 1.12
21.9 ± 1.59
9.2 ± 1.66
15.92 ± 2.15
42.45 ± 4.51
11.35 ± 1.04
24.06 ± 3.21
15.35 ± 0.69
12.83 ± 1.09
20.66 ± 1.21
18.5 ± 1.56
83.55 ± 6.59
88.07 ± 6.83
78.36 ± 6.4
52.36 ± 4.95
61.31 ± 3.67
55.62 ± 4.42
88.18 ± 8.45
61.08 ± 3.12
>100
M2
M3
20.15 ± 1.83
36.43 ± 1.37
18.01 ± 1.22
18.26 ± 0.32
23.34 ± 0.46
18.63 ± 1.14
12.21 ± 0.41
22.08 ± 1.42
11.28 ± 0.81
17.17 ± 1.51
26.65 ± 2.25
19.3 ± 1.14
7.51 ± 1.04
28.81 ± 1.19
28.12 ± 1.15
12.1 ± 0.73
16.13 ± 0.32
13.47 ± 1.18
6.45 ± 1.11
21.08 ± 1.23
7.23 ± 1.25
11.13 ± 1.47
15.25 ± 1.71
17.99 ± 1.64
12.01 ± 0.21
18.57 ± 0.14
7.73 ± 0.61
0.00008 ± 0.00002
M4
M5
M6
M7
M8
M9
8.77 ± 1.22
M10
M11
M12
M13
M14
M15
M16
SK
8.91 ± 1.25
>100
9.12 ± 0.87
98.35 ± 7.62
84.12 ± 6.46
54.65 ± 5.15
60.11 ± 3.44
90.28 ± 5.13
58.52 ± 4.31
20.94 ± 1.1
＞100
18.91 ± 1.28
17.09 ± 0.19
13.52 ± 1.28
17.56 ± 1.84
26.02 ± 1.17
11.07 ± 0.17
0.06219 ± 0.0134
30.32 ± 1.21
25.15 ± 0.79
9.32 ± 0.17
PTX
0.00416 ± 0.00066
＞100
Fig. 2. M9 can induce MDA-MB-231 cell apoptosis. (A) The effect of M9 on cell apoptosis detect by flow cytometry. (B) Statistic analysis of the apoptotic cells detect
by flow cytometry. (C) Western blot analysis of the effect of M9 on the expression of apoptosis-related proteins. (D) and (E) The relative band intensity was analyzed
by Image J software. The GAPDH band was used as the loading control. The images shown are representative of three repeated experiments. Data shown are mean ±
SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
4

Y. Ma et al.
Bioorganic Chemistry 111 (2021) 104872
More specifically, M3 (IC50 = 6.21 ± 1.68
1.47 M), M9 (IC50 = 4.52 ± 0.28 M) and M11 (IC50 = 5.13 ± 0.41
performed better anti-proliferation activities than SK (IC50 = 7.62 ±
0.26 M) and PTX (IC50 > 100 M) in MDA-MB-231 cells. For A549 cells,
M3 (IC50 = 7.51 ± 1.04 M), M9 (IC50 = 6.45 ± 1.11 M) and M11 (IC50
= 7.23 ± 1.25 M), displayed better anti-proliferation effect than other
derivatives and SK (IC50 = 7.73 ± 0.61 M). For HeLa cell line, M9 (IC50
= 8.77 ± 1.22 M), M10 (IC50 = 8.91 ± 1.25 M) and M11 (IC50 = 9.12
± 0.87 M), showed better anti-proliferation activities than SK (IC50
11.07 ± 0.17
μ
M), M6 (IC50 = 5.91 ±
were pre-treated with various dosage of M9 (0, 1, 2, 4 μM) and SK (4 μM)
μ
μ
μM)
for 24 h. Then, using flow cytometer with apoptotic markers to analyze
cells for changes in vitro. As shown in Fig. 2 (A and B), the percentage of
apoptotic cells in the M9 treated groups increased in a dosage-dependent
manner. In addition, M9 can induce more significant cell apoptosis than
SK at the same concentration. Further western blot results figure out that
M9 could significantly decrease the expression level of the anti-
apoptotic proteins (Bcl-2, caspase-3), but increased Cytochrome C
(Cyt-C), cleaved-PARP and cleaved-caspase-3 expression in MDA-MB-
231 cell line (Fig. 2C, D and E).
μ
μ
μ
μ
μ
μ
μ
μ
μ
=
μ
M). For MCF-7 and HCT-8 cells, their antiproliferative
effect were also significant but slight weaker than that of SK. Moreover,
the antiproliferative effect of all target compounds on MCF-10A were
obviously weaker than that of cancer cell lines.
2.3.3. Effect of M9 on MDA-MB-231 cell cycle arrest
Next, we assessed the effects of M9 on cell cycle distribution in MDA-
MB-231 cells by flow cytometry with SK as a positive control. As shown
in Fig. 3 (A, B), M9 caused more and more cell accumulation in S and G2/
M phases in a dosage-dependent manner. To be specific, about 59.15%
Based on the above results, we conjectured that anilide (dicarboxylic
acid) shikonin esters intervention could improve the anti-proliferation
activities of SK against the cancer cell lines but remarkably reduce its
cytotoxicity towards the non-cancer cell line. In order to clarify their
potential anticancer mechanism, we chose M9 on MDA-MB-231 cell for
further investigation.
of cells were arrested in S and G2/M phases after exposure to 4 μM M9
for 24 h, while about 54.5% cells were arrested at S and G2/M phases
after been exposure to 4 μM SK. Moreover, there was no significant time-
dependent effect of M9 on MDA-MB-231 cell cycle arrest. Therefore, we
speculated that M9 could not induce cell apoptosis through arresting cell
cycle.
2.3.2. M9 can induce apoptosis of MDA-MB-231 cell in a dosage-
dependent manner
To further assess the effect of M9 on cell viability, we carried out the
apoptosis assay with SK as a positive control. Firstly, MDA-MB-231 cells
Fig. 3. Effects of M9 and SK on cell cycle distribution of MDA-MB-231 cells. (A) Cell cycle distribution analysis of MDA-MB-231 cells treated with variably dosed M9
and 4 μM SK detect by flow cytometry (G1 phase is color green, S phase is color yellow and G2/M phase is color blue). (B) Statistic analysis of the dosage-dependent
assay. (C) Cell cycle distribution analysis of MDA-MB-231 cells after treated with M9 and SK for different time course detect by flow cytometry. (D) Statistic analysis
of the time-dependent assay. The images shown are representative of three repeated experiments. Data shown are mean ± SD of three repeated experiments. (*P <
0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)
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Y. Ma et al.
Bioorganic Chemistry 111 (2021) 104872
2.3.4. Effect of M9 on cell migration of MDA-MB-231 cells
demonstrated that cells incubated in M9 showed lower clone formation
rate and smaller clone size than the control group and SK-treated group
cells. Moreover, the clone formation inhibitory effect of M9 on MDA-
MB-231 cells displayed a dosage-dependent manner. Such results were
consistent with the above CCK-8 assay.
TNBC is an invasive ductal carcinoma, which is commonly associated
with high heterogeneity, low differentiation and rapid proliferation and
diffusion [21]. In order to verify the effect of M9 on the migration of
MDA-MB-231 cells, we performed wound healing and trans-well assay.
As shown in Fig. 4 (A, C), the control group cells gradually occupied the
cell-free space in the channel with the passage of time, and the cell
channel almost completely healed after 48 h. However, after M9 treat-
ment, the cell scratch healing was significantly slow down in a dosage-
dependent manner. Trans-well assay results also indicated that M9 could
effectively inhibit cell migration and its effect was much better than SK.
Wnt/β-catenin signaling pathway plays an important role in many
cellular processes, such as cell proliferation and migration. Based on
these, we further studied the effect of M9 on Wnt/β-catenin signaling
pathway in MDA-MB-231 cells. Western blot analysis results showed
that the expression of β-Catenin and Wnt3a protein was decreased in a
dose-dependent manner in MDA-MB-231 cells after being treated with
M9 (Fig. 4 D and E). These results suggest that M9 may inhibit the
migration of MDA-MB-231 cells by regulating Wnt/β-catenin signaling
pathway.
2.4. 3d-Qsar analysis
We used 20 compounds, including compounds 1c, 3c and 4c [27]
reported in our previous study, for 3D-QSAR study [28]. As shown in
Table S1, all test compounds were divided into training set and testing
set. The value of R2 shown in Fig. 6 demonstrated that the model had
predictive ability.
In the electrostatic map, the red contour line indicates that the
antiproliferative activity is expected to increase in the region of high
electron density (negative charge); the blue contour line indicates that
the antiproliferative activity is expected to increase in the region of low
electron density (partial positive charge). Similarly, the steric map
shows green contour lines, which indicate areas where large steric bulk
is expected to increase activity, and yellow contour lines indicate areas
where small space volume is predicted to decrease activity. The results
demonstrated that the compounds with low negative charge and small
group of benzene ring had strong anti-proliferative activity Fig. 7. Based
on the above data, this model provides reference for the structural
optimization of shikonin derivatives that inhibit the proliferation of
MDA-MB-231 cells in the subsequent experiments.
2.3.5. Effect of M9 on the expression of PI3K/Akt/mTOR in MDA-MB-231
cells
The PI3K/Akt/mTOR signaling pathway are involved in multiple
biological processes of cancer progression like cell proliferation,
migration and metastasis, etc. [22–24]. As shown in Fig. 5 (A and B), the
expression of PI3K, Akt, p-Akt and mTOR was down-regulated in a dose-
dependent manner after being treated with M9. Further, we evaluated
the effect of M9 on cell proliferation in MDA-MB-231 cells through a
colony formation assay [25,26]. The results shown in Fig. 5 (C)
3. Conclusion
PI3K/Akt/mTOR signaling pathway plays an important role in the
Fig. 4. Effects of M9 on MDA-MB-231 cell migration. (A) Cell migration was detected by wound scratch assay. The representative images of cell migration in the
wound scrape model at 0, 24 and 48 h (100 × ). (B) The representative images of trans-well assay at 24 h (100 × ). (C) Statistical analysis of the wound scrape assay.
(D) Western blot analysis of the effect of M9 on the expression Wnt and β-catenin. (E) Relative band intensity was analyzed by Image J software. The GAPDH band
was used as the loading control. All images shown are representative of three repeated experiments. Data shown are mean ± SD of three repeated experiments. (*P <
0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
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Y. Ma et al.
Bioorganic Chemistry 111 (2021) 104872
Fig. 5. M9 inhibited MDA-MB-231 cells prolif-
eration by down-regulating the PI3K/Akt/mTOR
signaling pathway. (A) Western blot analysis of
the effect of M9 on the expression some key
proteins in PI3K/Akt/mTOR signaling pathway.
(B) Relative band intensity was analyzed by
Image
J software. The GAPDH band acts as
loading control. (C) Colony formation of MDA-
MB-231 cells after 7 days of treatment with
different concentrations of M9. The images
shown are representative of three repeated ex-
periments. Data shown are mean ± SD of three
repeated experiments. (**P < 0.01, ***P < 0.001,
****P < 0.0001).
Fig. 6. The experimental and predicted anti-proliferative activity curves of training set and test set.
Fig.7. (A) 3D-QSAR model coefficients on electrostatic potential grids. (B) 3D-QSAR model coefficients on van der Waals grids. The blue and green sections represent
positive coefficients; the red and yellow sections represent negative coefficients. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
7

Y. Ma et al.
Bioorganic Chemistry 111 (2021) 104872
research of antitumor drugs [29]. In this study, the pharmacophore
structure of aniline was introduced into the shikonin framework, and a
series of anilide (dicarboxylic acid) shikonin ester were obtained. The
antiproliferative activity showed that M9 had the best inhibitory activity
against MDA-MB-231 cells. Western blot results showed that M9 facili-
tate the expression of some pro-apoptotic proteins, such as cleaved-
caspase-3, cytochrome C, and cleaved-PARP. Further studies have
shown that M9 can cause cell cycle arrest, but the effect is not signifi-
cant. Moreover, M9 could negatively regulate the PI3K/Akt/mTOR
signaling pathway and eventually lead to dose-dependent apoptosis in
MDA-MB-231 cells. Meanwhile, the inhibition of migration of MDA-MB-
231 cell may effectively reduce the possibility of distal metastasis. The
wound scrape and Trans-well assay suggested that M9 might further
inhibit the migration of MDA-MB-231 cells through the inhibition of
Wnt/β-catenin signaling pathway. Molecular docking results demon-
strated that M9 could bind well to the active site of Akt (PDB code:
3CQW) by forming various binding interactions, thus promoting its
antitumor effect. Therefore, we can speculate that M9 may have great
potential in the development of anti-breast cancer drugs by targeting
PI3K/AKT/mTOR signaling pathway.
for 6 h and monitored by thin layer chromatography (TLC). When the
reaction is completed, the pure intermediate B1-B16 was obtained by
filtered and recrystallization.
4.3. General procedure for the synthesis of compounds M1-M16
Compounds M1-M16 (0.2 mmol) was dissolved in anhydrous
dichloromethane (20 mL) at room temperature. Then, 0.4 mmol dicy-
clohexylcarbodiimide (DCC) and 0.05 mmol 4-dimethylaminopyridine
(DMAP) was added and stirred for 0.5 h. After that, 0.2 mmol of shi-
konin was added to the reaction mixture and stirred in the ice bath for
over 4 h. The solvent was then removed by rotary evaporation and the
residue was purified by silica gel column chromatography (with ethyl
acetate: petroleum ether, the specific proportion is shown in Table S2) to
get target compounds (Scheme 1). Chemical structures of the target
compounds (M1-M16) shown in Table 1.
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 4-oxo-4-(phenylamino) butanoate (M1)
Red powders, yield 53%. Mp: 79.1–80.3 ◦C. 1H NMR (600 MHz,
CDCl3) δ 12.58 (d, J = 4.6 Hz, 1H), 12.42 (d, J = 4.7 Hz, 1H), 7.49 (t, J =
7.8 Hz, 3H), 7.41 (t, J = 7.5 Hz, 1H), 7.29 (dd, J = 7.3, 5.3 Hz, 3H), 7.17
(s, 1H), 7.05 (s, 1H), 6.09–6.00 (m, 1H), 5.11 (t, J = 7.2 Hz, 1H),
2.86–2.80 (m, 2H), 2.70–2.64 (m, 2H), 2.63–2.57 (m, 1H), 2.49 (dt, J =
14.8, 7.3 Hz, 1H), 1.65 (d, J = 17.4 Hz, 3H), 1.56 (s, 3H). 13C NMR (151
MHz, CDCl3) δ 177.77 (s), 176.26 (s), 172.44 (s), 169.54 (s), 168.03 (s),
167.50 (s), 148.13 (s), 138.17 (s), 132.82 (s), 131.64 (s), 129.24 (s),
128.97 (s), 128.71 (s), 126.47 (s), 124.29 (s), 119.66 (s), 117.48 (s),
111.81–111.80 (m), 111.58 (s), 70.04 (s), 33.87 (s), 28.43 (s), 25.57 (s),
24.90 (s), 17.97 (s). HRESIMS [M+H] + m/z 463.1588 (Calcd for
4. Methods
4.1. Materials and measurements
All chemical reagents or solvents used to synthesize compounds are
analytical grade and purchased from Nanjing Chemical CO. Ltd (China)
and Sigma-Aladdin (St. Louis, USA). All the 1H NMR and 13C NMR
spectra were recorded on a Bruker DPX 600 model spectrometer in d6-
DMSO or CDCl3. The chemical shifts (δ) for 1H NMR and 13C NMR
spectra were reported in ppm(δ), with TMS as an internal standard. The
ESI-MS spectra were obtained on a Mariner Biospectrometry Worksta-
tion (ESI-TOF) mass spectrometer (4600 UPLC/Triple). The melting
point (uncorrected) was detected by using a XT4 MP Apparatus (Taike
Corp., Beijing, China). Column chromatography was conducted by using
silica gel (200–300 mesh) and eluting with ethyl acetate, dichloro-
methane and petroleum ether (bp 30–60 ◦C). Thin layer chromatog-
raphy (TLC) was carried out on glass-backed silica gel plates (Silica Gel
60 Å GF254) and visualized in UV light (λ 254 nm).
C
26H25NO7 463.1631).
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl
oxobutanoate (M2)
4-((2,4-difluorophenyl)
amino)-4-
Red powders, yield 72%. Mp: 57.8–58.7 ◦C. 1H NMR (600 MHz,
CDCl3) δ 12.58 (s, 1H), 12.43 (s, 1H), 7.44 (d, J = 9.7 Hz, 2H), 7.21–7.15
(m, 3H), 7.05 (s, 2H), 6.07–6.01 (m, 1H), 5.14–5.09 (m, 1H), 2.86–2.83
(m, 2H), 2.72 (dd, J = 10.6, 5.9 Hz, 2H), 2.53–2.47 (m, 2H), 1.67 (d, J =
6.3 Hz, 3H), 1.57 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 177.83 (s),
176.31 (s), 175.24 (s), 171.77 (s), 169.35 (s), 167.77 (s), 163.06 (dd, J
= 250.5, 12 Hz), 157.69 (dd, J = 253.5, 12 Hz), 147.64 (s), 136.24 (s),
132.99 (s), 132.78 (s), 131.64 (s), 130.20 (d, J = 10.5 Hz), 117.49 (s),
112.08 (dd, J = 22.5, 3 Hz), 111.78 (s), 105.31 (dd, J = 25.5, 22.5 Hz),
70.10 (s), 33.75 (s), 32.76 (s), 28.52 (s), 24.85 (s), 17.95 (s).HRESIMS
[M+H] + m/z 499.1393 (Calcd for C26H23F2NO7 499.1443).
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 4-((2-fluorophenyl) amino)-4-oxobutanoate
(M3)
PTX was purchased from Sigma-Aldrich (St. Louis, USA). RIPA lysis
buffers (P0013B) was purchased from Beyotime (Shanghai, China).
PVDF membranes were purchased from Thermo Scientific. Protease
inhibitor cocktail I (C0001) and phosphatase inhibitor cocktail I (C0002)
were purchased from Target Mol (Boston, USA). BCA protein assay kit
(#23227) was purchased from Pierce (Rockford, IL, USA). Anti-
Cytochrome C (10993-1-AP) was purchased from Proteintech (Wuhan,
China); anti-wnt3a (ab219412) was purchased from Abcom. (Cam-
bridge, UK). Anti-β-Catenin (66379-2-Ig), anti-GAPDH (60004-1-Ig),
anti-PARP (66520-1-Ig), anti-Bcl-2 (60178-1-Ig), anti-PI3K (67121-1-
Ig), anti-Akt (60203-2-Ig), anti-p-Akt (66444-Ig), anti-mTOR (66888-1-
Ig), the secondary antibody goat anti-mouse IgG (H + L) (SA00001-1),
goat anti-rabbit IgG (H + L) (SA00001-2) were purchased from Pro-
teintech (Wuhan, China). Anti-Caspase-3 (WL02117), anti-cleaved cas-
pase-3 (WL03089) were purchased from Wanlei Biotech Co. Ltd
(Shenyang, China). CCK-8, Cell cycle, Annexin V-FITC cell apoptosis
assay kits and ECL detection kit were purchased from YEASEN Biotech
Co. Ltd (Shanghai, China).
Red powders, yield 68%. Mp: 66.5–68.1 ◦C. 1H NMR (600 MHz,
CDCl3) δ 12.58 (s, 1H), 12.43 (s, 1H), 8.29 (t, J = 7.8 Hz, 1H), 7.54 (s,
1H), 7.20–7.15 (m, 2H), 7.13–7.00 (m, 4H), 6.09–6.01 (m, 1H), 5.12 (t,
J = 7.2 Hz, 1H), 2.85 (t, J = 6.4 Hz, 2H), 2.73 (dd, J = 13.0, 6.8 Hz, 2H),
2.62 (dd, J = 12.2, 6.1 Hz, 1H), 2.50 (dt, J = 14.7, 7.3 Hz, 1H), 1.65 (d, J
= 26.8 Hz, 3H), 1.55 (d, J = 21.3 Hz, 3H). 13C NMR(151 MHz, CDCl3) δ
177.96 (s), 176.46 (s), 171.76 (s), 169.34 (s), 167.67 (s), 167.15 (s),
152.20 (d, J = 243 Hz), 147.71 (s), 136.25 (s), 132.95 (s), 132.75 (s),
131.70 (s), 124.58 (d, J = 3.6 Hz), 124.31 (d, J = 7.6 Hz), 121.65 (s),
117.52 (s), 114.72 (d, J = 19 Hz), 111.70 (d, J = 33 Hz), 70.08 (s), 32.78
(s), 31.96 (s), 29.31 (s), 25.74 (s), 17.97 (s). HRESIMS [M+H] + m/z
481.1492 (Calcd for C26H24FNO7 481.1537).
4.2. General procedure for the synthesis of compounds B1-B16
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 4-oxo-4-((3-(trifluoromethyl) phenyl) amino)
butanoate (M4)
All the compounds were listed in Table 1 and their synthesis routes
were outlined in Scheme 1. Aniline (1 mol) and succinic anhydride OR
glutaric anhydride (1 mol), glutaric anhydride (1 mol) were put into a
50 mL conical flask. Then, add 20 mL of anhydrous dichloromethane to
make them dissolved. The appropriate amount of triethylamine was
used as catalyst. The reaction system was stirred at room temperature
Red powders, yield 65%. Mp: 71.2–72.5 ◦C. 1H NMR (600 MHz,
CDCl3) δ 12.60 (d, J = 18.6 Hz, 1H), 12.41 (s, 1H), 7.53 (d, J = 8.0 Hz,
4H), 7.33 (d, J = 7.2 Hz, 1H), 7.20–7.14 (m, 2H), 7.06 (s, 1H), 6.08–6.02
(m, 1H), 5.11 (t, J = 7.0 Hz, 1H), 2.86–2.78 (m, 3H), 2.71 (s, 2H),
8

Y. Ma et al.
Bioorganic Chemistry 111 (2021) 104872
2.53–2.46 (m, 1H), 1.66 (s, 3H), 1.57 (s, 3H). 13C NMR (151 MHz,
CDCl3) δ 177.46 (s), 175.70 (s), 172.07 (s), 169.70 (s), 167.89 (s),
147.51 (s), 138.40 (s), 136.33 (s), 133.03 (d, J = 37.5 Hz), 132.38 (s),
131.58 (d, J = 7.5 Hz), 129.77 (s), 129.46 (s), 124.46 (q, J = 279 Hz),
120.81–120.60 (m), 117.46 (s), 116.54–116.23 (m), 111.80 (s), 111.56
(s), 70.16 (s), 33.71 (s), 30.75 (s), 28.41 (s), 24.84 (s), 17.97 (s). HRE-
SIMS [M+H] + m/z 531.1467(Calcd for C27H24F3NO7 531.1505).
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 4-((4-chlorophenyl) amino)-4-oxobutanoate
(M5)
13C NMR (151 MHz, CDCl3) δ 177.54 (s), 175.99 (s), 172.28 (s), 170.19
(s), 168.11 (s), 167.58 (s), 148.01 (s), 137.77 (s), 136.28 (s), 133.14 (s),
132.99 (s), 131.26 (s), 128.99 (s), 124.29 (s), 119.74 (s), 117.59 (s),
111.80 (s), 111.56 (s), 69.64 (s), 36.21 (s), 33.09 (s), 32.84 (s), 25.98 (s),
20.89 (s), 17.98 (s). HRESIMS [M+H] + m/z 477.1746 (Calcd for
C27H27NO7 477.1788).
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl
oxopentanoate (M10)
5-((2,4-difluorophenyl)
amino)-5-
Red powders, yield 71%. Mp: 61.8–62.6 ◦C. 1H NMR (600 MHz,
CDCl3) δ 12.62–12.56 (m, 1H), 12.45–12.37 (m, 1H), 8.26–8.17 (m, 1H),
7.34 (s, 1H), 7.22–7.14 (m, 2H), 7.00 (s, 1H), 6.90–6.82 (m, 2H), 6.05
(dd, J = 6.9, 4.8 Hz, 1H), 5.13 (dd, J = 18.0, 10.8 Hz, 1H), 2.58–2.51 (m,
2H), 2.51–2.44 (m, 3H), 2.18 (s, 1H), 2.13–2.04 (m, 2H), 1.67 (d, J =
15.5 Hz, 3H), 1.58 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 177.49 (s),
175.96 (s), 172.02 (s), 170.29 (s), 162.78 (dd, J = 249, 12 Hz), 157.87
(dd, J = 250.5, 12 Hz), 147.94 (s), 136.26 (s), 133.15 (s), 132.98 (s),
131.30 (s), 131.15 (d, J = 10.5 Hz), 117.61 (s), 111.80 (dd, J = 22.5, 3
Hz), 111.81 (s), 111.56 (s), 104.93 (dd, J = 27, 24 Hz), 69.60 (s), 35.95
(s), 32.80 (s), 25.77 (s), 25.58 (s), 20.47 (s), 17.97 (s). HRESIMS [M+H]
+ m/z 513.1557 (Calcd for C27H25F2NO7 513.1599).
Red powders, yield 58%. Mp: 74.2–75.4 ◦C. 1H NMR (600 MHz,
CDCl3) δ 12.56 (d, J = 18.7 Hz, 1H), 12.42 (s, 1H), 7.69 (s, 1H), 7.45 (dd,
J = 8.7, 1.9 Hz, 3H), 7.23 (s, 1H), 7.20–7.15 (m, 2H), 7.04 (s, 1H),
6.08–6.01 (m, 1H), 5.11 (t, J = 7.1 Hz, 1H), 2.86–2.79 (m, 2H),
2.71–2.64 (m, 2H), 2.49 (dt, J = 14.8, 7.3 Hz, 2H), 1.66 (s, 3H), 1.56 (s,
3H). 13C NMR (151 MHz, CDCl3) δ 177.54 (s), 175.99 (s), 172.28 (s),
170.19 (s), 168.11 (s), 167.58 (s), 148.01 (s), 137.77 (s), 136.28 (s),
133.14 (s), 132.99 (s), 131.26 (s), 128.99 (s), 124.29 (s), 119.74 (s),
117.59 (s), 111.80 (s), 111.56 (s), 69.64 (s), 36.21 (s), 33.09 (s), 32.84
(s), 25.98 (s), 20.89 (s), 17.98 (s). HRESIMS [M+H] + m/z 497.1200
(Calcd for C26H24ClNO7 497.1241)
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 4-oxo-4-(p-tolylamino) butanoate (M6)
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 5-((2-fluorophenyl) amino)-5-oxopentanoate
(M11)
Red powders, yield 55%. Mp: 98.7–100.2 C. 1H NMR (600 MHz,
◦
CDCl3) δ 12.58 (s, 1H), 12.42 (s, 1H), 7.39 (d, J = 17.2 Hz, 1H), 7.35 (d,
J = 8.0 Hz, 1H), 7.19–7.13 (m, 3H), 7.11–7.02 (m, 3H), 6.08–6.01 (m,
1H), 5.11 (t, J = 6.6 Hz, 1H), 2.90 (s, 3H), 2.70–2.58 (m, 3H), 2.29 (s,
3H), 1.66 (s, 3H), 1.56 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 177.81 (s),
176.41 (s), 172.03 (s), 169.13 (s), 167.78 (s), 167.26 (s), 147.69 (s),
136.28 (s), 131.66 (s), 129.91 (s), 129.44 (s), 126.28 (s), 119.75 (s),
117.49 (s), 111.80 (s), 111.57 (s), 70.00 (s), 33.76 (s), 32.80 (s), 28.41
(s), 24.85 (s), 21.22 (s), 17.97 (s). HRESIMS [M+H] + m/z 477.1744
(Calcd for C27H27NO7 477.1788).
Red powders, yield 67%. Mp: 67.5–68.8 ◦C. 1H NMR (600 MHz,
CDCl3) δ 12.60 (d, J = 8.8 Hz, 1H), 12.42 (s, 1H), 8.29 (t, J = 7.8 Hz,
1H), 7.44 (s, 1H), 7.22–7.15 (m, 2H), 7.14–6.96 (m, 4H), 6.05 (dd, J =
6.8, 4.8 Hz, 1H), 5.15–5.08 (m, 1H), 2.68–2.43 (m, 7H), 2.12–2.06 (m,
2H), 1.73–1.64 (m, 3H), 1.56 (d, J = 20.7 Hz, 3H). 13C NMR (151 MHz,
CDCl3) δ 177.57 (s), 176.05 (s), 172.03 (s), 170.26 (s), 168.08 (s),
167.55 (s), 152.34 (d, J = 241.5 Hz), 147.96 (s), 136.27 (s), 133.12 (s),
132.94 (s), 131.35 (s), 124.58 (d, J = 3.5 Hz), 124.38 (d, J = 7.6 Hz),
121.80 (s), 117.61 (s), 114.80 (d, J = 19.5 Hz), 111.70 (d, J = 36 Hz),
69.59 (s), 36.17 (s), 33.06 (s), 32.86 (s), 25.77 (s), 20.48 (s), 17.97 (s).
HRESIMS [M+H] + m/z 495.1646 (Calcd for C27H26FNO7 495.1693).
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 5-oxo-5-((3-(trifluoromethyl) phenyl) amino)
pentanoate (M12)
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 4-oxo-4-(o-tolylamino)butanoate (M7)
Red powders, yield 61%. Mp: 80.6–81.4 ◦C. 1H NMR (600 MHz,
CDCl3) δ 12.59 (s, 1H), 12.41 (s, 1H), 7.78 (dd, J = 29.8, 7.6 Hz, 2H),
7.33 (ddd, J = 21.9, 15.1, 7.5 Hz, 1H), 7.22–7.12 (m, 3H), 7.10–7.03 (m,
2H), 6.11–6.01 (m, 1H), 5.11 (t, J = 7.2 Hz, 1H), 2.91–2.82 (m, 2H),
2.71 (t, J = 6.5 Hz, 2H), 2.65–2.59 (m, 1H), 2.53–2.46 (m, 1H),
2.24–2.12 (m, 3H), 1.67 (s, 3H), 1.57 (s, 3H). 13C NMR (151 MHz,
CDCl3) δ 177.58 (s), 176.21 (s), 172.12 (s), 169.46 (s), 167.98 (s),
167.45 (s), 156.82 (s), 147.62 (s), 136.31 (s), 135.54 (s), 133.08 (s),
132.84 (s), 130.41 (s), 129.63 (s), 127.93 (s), 125.16 (s), 122.95 (s),
117.45 (s), 111.79 (s), 111.55 (s), 70.01 (s), 33.87 (s), 32.79 (s), 25.49
(s), 24.90 (s), 17.95 (s), 17.74 (s). HRESIMS [M+H] + m/z 477.1743
(Calcd for C27H27NO7 477.1788).
Red powders, yield 63%. Mp: 44.7–45.6 ◦C. 1H NMR (600 MHz,
CDCl3) δ 12.60 (d, J = 8.8 Hz, 1H), 12.42 (s, 1H), 8.29 (t, J = 7.8 Hz,
1H), 7.44 (s, 1H), 7.22–7.15 (m, 2H), 7.14–6.96 (m, 4H), 6.05 (dd, J =
6.8, 4.8 Hz, 1H), 5.15–5.08 (m, 1H), 2.68–2.43 (m, 6H), 2.12–2.06 (m,
2H), 1.73–1.64 (m, 3H), 1.56 (d, J = 20.7 Hz, 4H). 13C NMR (151 MHz,
CDCl3) δ 177.39 (s), 175.82 (s), 172.31 (s), 170.47 (s), 168.31 (s),
167.78 (s), 147.94 (s), 138.36 (s), 136.34 (s), 133.16 (d, J = 20.5 Hz),
131.19 (d), 129.77 (s), 129.52 (s), 123.82 (q,
J = 273 Hz),
120.94–120.70 (m), 117.55 (s), 116.55–116.24 (m), 111.81 (s), 111.57
(s), 69.75 (s), 36.12 (s), 33.02 (s), 32.83 (s), 25.78 (s), 20.57 (s), 17.98
(s). HRESIMS [M+H] + m/z 545.1622 (Calcd for C28H26F3NO7
545.1661)
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 4-(diphenylamino)-4-oxobutanoate (M8)
Red oil, yield 51%. 1H NMR (600 MHz, CDCl3) δ 12.58 (s, 1H), 12.44
(s, 1H), 7.50 (s, 1H), 7.43 (d, J = 6.5 Hz, 3H), 7.35 (d, J = 12.7 Hz, 4H),
7.21–7.16 (m, 4H), 7.03 (s, 1H), 6.07–5.94 (m, 1H), 5.11 (t, J = 7.1 Hz,
1H), 2.78–2.71 (m, 2H), 2.66 (d, J = 6.6 Hz, 2H), 2.55 (t, J = 6.6 Hz,
2H), 1.66 (s, 3H), 1.55 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 177.54 (s),
175.99 (s), 172.28 (s), 170.19 (s), 168.11 (s), 167.58 (s), 148.01 (s),
137.77 (s), 136.28 (s), 133.14 (s), 132.99 (s), 131.26 (s), 128.99 (s),
124.29 (s), 119.74 (s), 117.59 (s), 111.80 (s), 111.56 (d, J = 4.3 Hz),
69.64 (s), 36.21 (s), 33.09 (s), 32.84 (s), 25.98 (s), 20.89 (s), 17.98 (s).
HRESIMS [M+H] + m/z 539.1936 (Calcd for C32H29NO7 539.1944).
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 5-oxo-5-(phenylamino) pentanoate (M9)
Red powders, yield 55%. Mp: 46.2–47.1 ◦C. 1H NMR (600 MHz,
CDCl3) δ 12.60 (s, 1H), 12.42 (s, 1H), 7.51 (d, J = 7.9 Hz, 2H), 7.41 (dd,
J = 22.0, 12.6 Hz, 1H), 7.30 (t, J = 7.7 Hz, 2H), 7.20–7.16 (m, 2H), 7.09
(t, J = 6.9 Hz, 1H), 6.99 (s, 1H), 6.09–6.00 (m, 1H), 5.12 (t, J = 6.7 Hz,
1H), 2.65–2.39 (m, 6H), 2.12–2.04 (m, 2H), 1.68 (s, 3H), 1.58 (s, 3H).
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 5-((4-chlorophenyl) amino)-5-oxopentanoate
(M13)
Red powders, yield 60%. Mp: 65.8–67.1 ◦C. 1H NMR (600 MHz,
CDCl3) δ 12.60 (s, 1H), 12.42 (s, 1H), 7.57–7.40 (m, 4H), 7.20 (d, J =
10.0 Hz, 2H), 7.05–6.95 (m, 2H), 6.06–6.01 (m, 1H), 5.09 (dd, J = 28.9,
22.1 Hz, 2H), 2.88–2.75 (m, 2H), 2.57–2.50 (m, 2H), 2.43 (dd, J = 16.5,
7.4 Hz, 2H), 2.07 (dd, J = 14.2, 7.1 Hz, 2H), 1.67 (d, J = 13.2 Hz, 3H),
1.58 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 177.62 (s), 176.21 (s), 172.33
(s), 170.61 (s), 168.42 (s), 157.46 (s), 147.96 (s), 136.33 (s), 133.22 (s),
133.08 (s), 131.21 (s), 129.84 (s), 129.54 (s), 128.98 (s), 120.94 (s),
117.56 (s), 111.86 (s), 111.56 (s), 69.70 (s), 36.16 (s), 33.80 (s), 32.82
(s), 25.54 (s), 24.87 (s), 17.98 (s). HRESIMS [M+H] + m/z 511.1353
(Calcd for C27H26ClNO7 511.1398)
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 5-oxo-5-(p-tolylamino) pentanoate (M14)
9

Y. Ma et al.
Bioorganic Chemistry 111 (2021) 104872
Red powders, yield 53%. Mp: 83.5–84.1 ◦C. 1H NMR (600 MHz,
concentration, and each experiment was repeated three times. The IC50
value was defined as a concentration that caused 50% loss of cell
viability, which was calculated by Origin 7.5.
CDCl3) δ 12.60 (s, 1H), 12.42 (d, J = 4.9 Hz, 1H), 7.39 (d, J = 8.3 Hz,
2H), 7.27 (d, J = 8.8 Hz, 1H), 7.21–7.15 (m, 2H), 7.15–7.07 (m, 2H),
7.01–6.95 (m, 1H), 6.04 (dd, J = 6.9, 4.8 Hz, 1H), 5.11 (t, J = 7.1 Hz,
1H), 2.56–2.45 (m, 3H), 2.45–2.34 (m, 3H), 2.30 (s, 2H), 2.14–1.99 (m,
3H), 1.68 (s, 3H), 1.59 (d, J = 12.6 Hz, 3H). 13C NMR (151 MHz, CDCl3)
δ 177.63 (s), 176.09 (s), 172.28 (s), 170.05 (s), 168.05 (s), 167.52 (s),
148.04 (s), 136.28 (s), 135.20 (s), 133.94 (s), 133.11 (s), 132.95 (s),
131.29 (s), 129.47 (s), 119.85 (s), 117.60 (s), 111.82 (s), 111.58 (s),
69.62 (s), 36.17 (s), 33.12 (s), 32.85 (s), 25.78 (s), 20.86 (s), 20.72 (s),
17.98 (s). HRESIMS [M+H] + m/z 491.1903 (Calcd for C28H29NO7
491.1944).
4.6. Cell cycle analysis
Targeted cells (MDA-MB-231) cells were seeded in 6-well plates (5.0
× 104 cells/well) and incubated at 37 ◦C in the 5% CO2 incubator for 12
h. Then, the exponential cells were exposed to different concentrations
of M9 (0, 1, 2, 4 μM) and 4 μM SK for 24 h. For the time-dependent assay,
the adherent cells were pre-treated with 2 μM M9 and SK for 0, 24, 36,
and 48 h. The cells were centrifuged with (2000 rpm, 4 ◦C, 10 min) and
fixed in 70% cooled ethanol at ꢀ 20◦ C for overnight. Whereafter, cells
were re-suspended in binding buffer containing 0.1 mg/mL RNase A and
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 5-oxo-5-(o-tolylamino) pentanoate (M15)
Red powders, yield 59%. Mp: 58.2–59.2 ◦C. 1H NMR (600 MHz,
CDCl3) δ 12.60 (s, 1H), 12.41 (s, 1H), 7.78 (d, J = 7.9 Hz, 1H), 7.24–7.15
(m, 4H), 7.14–7.04 (m, 2H), 7.00 (s, 1H), 6.10–6.02 (m, 1H), 5.11 (d, J
= 6.5 Hz, 1H), 2.59 (ddd, J = 19.6, 14.0, 7.8 Hz, 3H), 2.52–2.44 (m, 3H),
2.24 (d, J = 16.0 Hz, 3H), 2.11 (dd, J = 19.9, 12.9 Hz, 2H), 1.69 (s, 3H),
1.58 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 177.20 (s), 175.68 (s), 171.91
(s), 169.98 (s), 167.88 (s), 167.35 (s), 147.66 (s), 136.00 (s), 135.20 (s),
132.87 (s), 132.69 (s), 131.01 (s), 130.21 (s), 126.46 (s), 125.01 (s),
122.95 (s), 117.31 (s), 111.52 (s), 111.28 (s), 69.29 (s), 35.73 (s), 33.56
(s), 32.58 (s), 24.61 (s), 20.55 (s), 17.69 (s), 17.54 (s). HRESIMS [M+H]
+ m/z 491.1904 (Calcd for C28H29NO7 491.1944).
5 μg/mL propidium iodide (PI). Flow cytometry (BD FACS Calibur, USA)
was used to determine the DNA content of cells for cell cycle distribution
analysis, at least 10,000 events were plotted for each sample. The cell
percentages of G0/G1, S and G2/M phases was measured by Flowjo7.6
software.
4.7. Flow cytometric analysis of apoptosis
Cell apoptosis was measured by using Annexin V-FITC/PI double
staining.3Briefly, MDA-MB-231 cells were seeded in 6-well plates with
5.0 × 10 cells /well, cultured overnight at 37◦ C in 5% CO2 incubator,
(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 5-(diphenylamino)-5-oxopentanoate (M16)
Red oil, yield 53%. 1H NMR (600 MHz, CDCl3) δ 12.58 (s, 1H), 12.42
(d, J = 4.8 Hz, 1H), 7.49–7.41 (m, 2H), 7.40–7.33 (m, 5H), 7.21–7.16
(m, 3H), 7.08 (d, J = 7.8 Hz, 1H), 6.93 (d, J = 10.9 Hz, 2H), 6.04–5.93
(m, 1H), 5.07 (t, J = 7.2 Hz, 1H), 2.86–2.76 (m, 2H), 2.46 (dt, J = 15.9,
7.4 Hz, 3H), 2.34 (dt, J = 27.7, 7.0 Hz, 3H), 1.66 (s, 3H), 1.55 (s, 3H).
13C NMR (151 MHz, CDCl3) δ 178.20 (s), 176.73 (s), 173.21 (s), 172.30
(s), 167.39 (s), 166.86 (s), 148.21 (s), 142.70 (s), 142.62 (s), 136.10 (s),
132.86 (s), 132.67 (s), 131.48 (s), 129.79 (s), 128.97 (s), 126.43 (s),
117.62 (s), 111.79 (s), 111.55 (s), 69.29 (s), 34.06 (s), 33.34 (s), 32.83
(s), 25.77 (s), 20.72 (s), 17.95 (s). HRESIMS [M+H] + m/z 553.2081
(Calcd for C33H31NO7 553.2101.
and then treated with M9 (0, 1, 2, 4 μM) and 4 μM SK for 24 h in the
dose-dependent test. Cells were harvested and washed twice with PBS
and then stained with Annexin V-FITC (5 L) and PI (5 L) in the binding
μ
μ
buffer for 15–20 min at room temperature away from light. Finally,
using BD Accuri C6 Flowjo Cytometer (BD, USA) to measure the
apoptotic cells. Data analysis was performed by using Flowjo 7.6 soft-
ware. The percentage of cells distributed in the right quadrant (Annexin
V + ) was used for statistics analysis using Graphpad prism 8.
4.8. Wound-healing assay
MDA-MB-231 cells were seeded in 6-well culture plates with 2 × 105
cells/cm2 and were cultured overnight. When the cell density is above
90%, the monolayer cells were scraped using microtubule tips to form
scratches. Next, cells were washed with PBS three times, and incubated
in serum-free medium with different concentrations of M9, SK and PTX.
The wound healing was observed with microscopy (NIKON, Japan), and
the scratches were photographed at 0, 24 and 48 h. Then, NIH Image J
image analysis software was used to delineate the wound areas for
analysis.
4.4. Cell lines and culture conditions
The cell lines used in our work were human breast cancer cell lines
(MCF-7, MDA-MB-231), human cervical cancer cell line (HeLa), human
colon cancer cell line (HCT-8), human lung adenocarcinoma cell line
(A549), and human normal breast epithelial cell line (MCF-10A). All cell
lines were obtained from the State Key Laboratory of Pharmaceutical
Biotechnology, Nanjing University. Amongst, MCF-7, MDA-MB-231,
HeLa and A549 cell lines were cultured in DMEM/high glucose sup-
plemented with 10% fetal bovine serum and 1% penicillin/strepto-
mycin. HCT-8 cell line was maintained in RPMI medium 1640 basic
supplemented with 10% fetal bovine serum and 1% penicillin/strepto-
mycin. MCF-10A cell line was maintained in MEpiCM. All cells were
incubated at 37 ◦C in a humidified atmosphere containing 5% CO2.
4.9. Trans-well assay
MDA-MB-231 cells were pretreated with M9, SK and PTX for 24 h
and were then seeded in serum-free media on the upper side of a trans-
well plate. Next, cells were allowed to migrate towards media containing
10% of FBS (in the lower side) for 24 h. After the incubation period, cells
on the lower side of the membrane were fixed with methanol for 10 min
and were then stained with 1% crystal violet for 5 min. After that, cells
were washed with PBS for 3 to 5 times. Finally, the stained cells were
observed under microscope (five random fields at × 100 magnification).
4.5. cck-8 assay
The anti-proliferation assay was performed by using CCK-8 assay.
Cells were cultured in medium to log phase and then digested, centri-
fuged and resuspended in fresh medium. Then, they were seeded into 96
well plates (104 cells / well) and incubated overnight at 37◦ C in 5% CO2
to make them reattach. Subsequently, cells were treated by the target
4.10. Colony formation assay
MDA-MB-231 cells were treated with M9, SK and PTX for 24 h. Cells
were then collected and diluted into 1 × 102 cells/mL cell suspension
with complete medium. 1 mL of cell suspension was added into each
well of 12 well plate, and then grew at 37◦ C in 5% CO2 atmosphere for 7
days. The medium was replaced every 2–3 days. The culture was
terminated until obvious clones appeared in the dish. Washing the
clones thrice with PBS and fixed them with 4% paraformaldehyde for
compounds at increasing concentrations (0.1, 1, 10 and 100 μM) for
another 24 h. Then, the cell viability was detected in CCK-8 assay and
operated in strict accordance with the manufacturer instructions. Using
ELISA reader (EPOCH, BioTek, USA) to read the absorbance (OD450).
The positive drug was PTX [30]. There are three repeated wells for each
10

Y. Ma et al.
Bioorganic Chemistry 111 (2021) 104872
[2] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer
statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36
cancers in 185 countries, CA Cancer J. Clin. 68 (6) (2018) 394–424.
[3] B.D. Lehmann, J.A. Bauer, X. Chen, M.E. Sanders, A.B. Chakravarthy, Y. Shyr, J.
A. Pietenpol, Identification of human triple-negative breast cancer subtypes and
preclinical models for selection of targeted therapies, J. Clin. Invest. 121 (7) (2011)
2750–2767.
15–20 min. After that, staining the clones with 1% crystal violet dye for
30 min and then washing them for 3 to 5 times with PBS. Finally, the 12-
well plate was placed upside down to drain the PBS. The cells were
observed under a microscope. Each experiment was repeated at least
three times.
[4] W.D. Foulkes, I.E. Smith, J.S. Reis, Triple-negative breast cancer, New Engl. J. Med.
363 (20) (2010) 1938–1948.
4.11. Western bolt analysis
[5] E.A. Rakha, M.E. El-Sayed, A.R. Green, A.H.S. Lee, J.F. Robertson, I.O. Ellis,
Prognostic markers in triple-negative breast cancer, Cancer 109 (1) (2007) 25–32.
[6] K. Gelmon, R. Dent, J.R. Mackey, K. Laing, D. McLeod, S. Verma, Targeting triple-
negative breast cancer: optimising therapeutic outcomes, Ann. Oncol. 23 (9)
(2012) 2223–2234.
MDA-MB-231 cells were lysed with RIPA lysis buffer containing 1%
protease and phosphatase inhibitor cocktail I (Target Mol, USA) to
extract the total proteins. Then, using BCA protein assay kit to determine
[7] P.F. McAuliffe, F. Meric-Bernstam, G.B. Mills, A.M. Gonzalez-Angulo, Deciphering
the role of PI3K/Akt/mTOR pathway in breast cancer biology and pathogenesis,
Clin. Breast Cancer 10 (2010) S59–S65.
protein concentration. About 60 μg of total protein was separated by
10% SDS- polyacrylamide gels and then transferred to PVDF membrane.
Membranes were then sealed with 5% skim milk solution for 1 h and
were incubated with diluted primary antibody (1:1000 dilution) at 4◦ C
and shaken gently overnight. After washing with TBST for 30 min
(change TBST every 5 min), membranes were incubated with secondary
antibodies (1:5000–1:10000 dilution) for another 1 h at room temper-
ature. Bands were developed by super ECL detection reagent and pho-
tographed by Tanon 5200 digital camera (Tanon, China). Finally, Image
J software (National Institutes of Health, BetheSEMa, Maryland, USA)
was used to analyze the results, and all target proteins were standardized
by reference to GAPDH.
[8] X.Y. Zhang, Y. Liu, Y.P. Jia, Q. Zhao, W.Y. Wang, Z. Zhang, W. Li, L.C. Sun,
Ameliorative effect of acetylshikonin on cigarette smoke induced lung
inflammation in mice, Lat. Am. J. Pharm. 38 (9) (2019) 1862–1873.
[9] X. Chen, L. Yang, N. Zhang, J.A. Turpin, R.W. Buckheit, C. Osterling, J.
J. Oppenheim, O.M.Z. Howard, Shikonin, a component of Chinese herbal medicine,
inhibits chemokine receptor function and suppresses human immunodeficiency
virus type 1, Antimicrob. Agents Ch. 47 (9) (2003) 2810–2816.
[10] X.Y. Huang, H.L. Fu, H.Q. Tang, Z.Q. Yin, W. Zhang, G. Shu, L.Z. Yin, L. Zhao, X.
R. Yan, J.C. Lin, Optimization extraction of Shikonin using ultrasound-assisted
response surface methodology and antibacterial studies, Evid-Based Compl. Alt.
2020 (2020) 1–10.
[11] N. Guo, R. Miao, X. Gao, D. Huang, Z. Hu, N. Ji, Y. Nan, F. Jiang, X. Gou, Shikonin
inhibits proliferation and induces apoptosis in glioma cells via downregulation of
CD147, Mol. Med. Rep. 19 (5) (2019) 4335–4343.
[12] J.C. Boulos, M. Rahama, M.F. Hegazy, T. Efferth, Shikonin derivatives for cancer
prevention and therapy, Cancer Lett. 459 (2019) 248–267.
4.12. Docking simulation
[13] B. Wiench, Y.R. Chen, M. Paulsen, R. Hamm, S. Schroder, N.S. Yang, T. Efferth,
Integration of different “-omics” technologies identifies inhibition of the IGF1R-
Akt-mTOR signaling cascade involved in the cytotoxic effect of Shikonin against
Leukemia cells, Evid-Based Compl. Alt. (2013) 1–11.
The crystal structures of protein complexes were retrieved from the
RCSB protein database. Compound molecules bind to Akt (PDB Code:
3cqw) by DS-CDOCKER, a graphical user interface in Discovery Studio
3.5. All binding water and ligands were removed from the protein. After
molecular docking, the interaction types between the ligand-based
pharmacophore model and the docking protein were analyzed.
[14] F. Ni, X.B. Huang, Z.Z. Chen, W.B. Qian, X.M. Tong, Shikonin exerts antitumor
activity in Burkitt’s lymphoma by inhibiting C-MYC and PI3K/AKT/mTOR
pathway and acts synergistically with doxorubicin, Sci. Rep.-Uk 8 (1) (2018) 3317.
[15] B.Y. Li, Z.G. Yuan, J. Jiang, Y.Q. Rao, Anti-tumor activity of Shikonin against
afatinib resistant non-small cell lung cancer via negative regulation of PI3K/Akt
signaling pathway, Biosci. Rep. 38 (6) (2018).
[16] Y. Chen, T.T. Wang, J. Du, Y.C. Li, X. Wang, Y. Zhou, X.X. Yu, W.M. Fan, Q.J. Zhu,
X.M. Tong, Y. Wang, The critical role of PTEN/PI3K/AKT signaling pathway in
Shikonin-induced apoptosis and proliferation inhibition of chronic myeloid
leukemia, Cell. Physiol. Biochem. 47 (3) (2018) 981–993.
4.13. 3D-QSAR
3D-QSAR is a reliable method to better understand the SAR spectra of
synthetic compounds. All compounds were divided into two parts:
training set and test set. The anti-proliferative activity of the compounds
was used for QSAR analysis. The reliability of QSAR model could be
evaluated by cross-validation correlation coefficient.
[17] Y.Z. Zhu, Y. Zhong, X. Long, Z. Zhu, Y. Zhou, H. Ye, X.B. Zeng, X.B. Zheng,
Deoxyshikonin isolated from Arnebia euchroma inhibits colorectal cancer by
down-regulating the PI3K/Akt/mTOR pathway, Pharm. Biol. 57 (1) (2019)
412–423.
[18] S. Jiang, H.T. Wang, Y. Guo, Z. Liu, W. Song, Acetylshikonin inhibits the migration
and invasion of A375 cells by reversing EMT process via the PI3K/Akt/mTOR
pathway, Biotechnol. Biotec. Eq. 33 (1) (2019) 699–706.
[19] R. Deng, J. Tang, B.F. Xie, G.K. Feng, Y.H. Huang, Z.C. Liu, X.F. Zhu, SYUNZ-16, a
newly synthesized alkannin derivative, induces tumor cells apoptosis and
suppresses tumor growth through inhibition of PKB/AKT kinase activity and
blockade of AKT/FOXO signal pathway, Int. J. Cancer 127 (1) (2010) 220–229.
[20] Y.Y. Yang, H.Q. He, J.H. Cui, Y.J. Nie, Y.X. Wu, R. Wang, G. Wang, J.N. Zheng, R.
D. Ye, Q. Wu, S.S. Li, F. Qian, Shikonin derivative DMAKO-05 inhibits akt signal
activation and melanoma proliferation, Chem. Biol. Drug Des. 87 (6) (2016)
895–904.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[21] A. Eng, V. McCormack, I.d.-S.-S.J.P. medicine, Receptor-defined subtypes of breast
cancer in indigenous populations in africa: a systematic review and meta-analysis,
PLOS Med. 11 (9) (2014).
Acknowledgements
[22] B. Roy, A.K. Pattanaik, J. Das, S.K. Bhutia, B. Behera, P. Singh, T.K. Maiti, Role of
PI3K/Akt/mTOR and MEK/ERK pathway in Concanavalin A induced autophagy in
HeLa cells, Chem. Biol. Interact. 210 (2014) 96–102.
This research was supported by the National Natural Science Foun-
dation of China (NSFC) (U1903201, 21702100, 21907051), the Program
for Changjiang Scholars and Innovative Research Team in University
(IRT_14R27), and the Natural Science Foundation of Jiangsu Bureau of
Science and Technology (BK20191254), and the Fundamental Research
Funds for the Central Universities (020814380140, 020814380151).
[23] S. Kumar, S.K. Guru, A.S. Pathania, S. Manda, A. Kumar, S.B. Bharate, R.
A. Vishwakarma, F. Malik, S. Bhushan, Fascaplysin induces caspase mediated
crosstalk between apoptosis and autophagy through the inhibition of PI3K/AKT/
mTOR signaling cascade in human leukemia HL-60 cells, J. Cell. Biochem. 116 (6)
(2015) 985–997.
[24] S. Ebrahimi, M. Hosseini, S. Shahidsales, M. Maftouh, G.A. Ferns, M. Ghayour-
Mobarhan, S.M. Hassanian, A. Avan, Targeting the Akt/PI3K signaling pathway as
a potential therapeutic strategy for the treatment of pancreatic cancer, Curr. Med.
Chem. 24 (13) (2017) 1321–1331.
Appendix A. Supplementary material
[25] X. Cheng, J.B. Shi, H. Liu, L.Z. Chen, Y. Wang, W.J. Tang, X.H. Liu, Discovery of (4-
bromophenyl)(3-hydroxy-4-methoxyphenyl) methanone through upregulating
hTERT induces cell apoptosis and ERS, Cell Death Dis. 8 (2017) e3016.
[26] Y. Ke, J.J. Liang, R.J. Hou, M.M. Li, L.F. Zhao, W. Wang, Y. Liu, H. Xie, R.H. Yang,
T.X. Hu, J.Y. Wang, H.M. Liu, Synthesis and biological evaluation of novel Jiyuan
Oridonin A-1,2,3-triazole-azole derivatives as antiproliferative agents, Eur. J. Med.
Chem. 157 (2018) 1249–1263.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104872.
References
[1] S.M. Albeshan, M.G. Mackey, S.Z. Hossain, A.A. Alfuraih, P.C. Brennan, Breast
cancer epidemiology in gulf cooperation council countries: a regional and
international comparison, Clin. Breast Cancer 18 (3) (2018) E381–E392.
11

Y. Ma et al.
Bioorganic Chemistry 111 (2021) 104872
[27] H.Y. Lin, H.W. Han, W.X. Sun, Y.S. Yang, C.Y. Tang, G.H. Lu, J.L. Qi, X.M. Wang, Y.
H. Yang, Design and characterization of alpha-lipoic acyl shikonin ester twin drugs
as tubulin and PDK1 dual inhibitors, Eur. J. Med. Chem. 144 (2018) 137–150.
[28] W.X. Sun, H.W. Han, M.K. Yang, Z.L. Wen, Y.S. Wang, J.Y. Fu, Y.T. Lu, M.Y. Wang,
J.X. Bao, G.H. Lu, J.L. Qi, X.M. Wang, H.Y. Lin, Y.H. Yang, Design, synthesis and
biological evaluation of benzoylacrylic acid shikonin ester derivatives as
irreversible dual inhibitors of tubulin and EGFR, Bioorg. Med. Chem. 27 (23)
(2019) 115153.
[29] S. Fattahi, F. Amjadi-Moheb, R. Tabaripour, G.H. Ashrafi, H. Akhavan-Niaki, PI3K/
AKT/mTOR signaling in gastric cancer: epigenetics and beyond, Life Sci. 262
(2020) 118513.
[30] P. Chowdhury, P.K.B. Nagesh, E. Hatami, S. Wagh, N. Dan, M.K. Tripathi, S. Khan,
B.B. Hafeez, B. Meibohm, S.C. Chauhan, M. Jaggi, M.M. Yallapu, Tannic acid-
inspired paclitaxel nanoparticles for enhanced anticancer effects in breast cancer
cells, J. Colloid Interface Sci. 535 (2019) 133–148.
12