Design, Synthesis, Molecular Docking, and Biological Evaluation of New Emodin Anthraquinone Derivatives as Potential Antitumor Substances

Article abstract of DOI:10.1002/cbdv.202000328

The emodin anthraquinone derivatives are generally used in traditional Chinese medicine due to their various pharmacological activities. In the present study, a series of emodin anthraquinone derivatives have been designed and synthesized, among which 1,3-dihydroxy-6,8-dimethoxyanthracene-9,10-dione is a natural compound that has been synthesized for the very first time, and 1,3-dimethoxy-5,8-dimethylanthracene-9,10-dione is a compound that has never been reported earlier. Interestingly, while total seven of these compounds showed neuraminidase inhibitory activity in influenza virus with inhibition rate more than 50 %, specific four compounds exhibited significant inhibition of tumor cell proliferation. The further results demonstrate that 1,3-dimethoxy-5,8-dimethylanthracene-9,10-dione showed the best anticancer activity among all the synthesized compounds by inducing highest apoptosis rate to HCT116 cancer cells and arresting their G0/G1 cell cycle phase, through elevation of intracellular level of reactive oxygen species (ROS). Moreover, the binding of 1,3-dimethoxy-5,8-dimethylanthracene-9,10-dione with BSA protein has thoroughly been investigated. Altogether, this study suggests the neuraminidase inhibitory activity and antitumor potential of the new emodin anthraquinone derivatives.

Full text of DOI:10.1002/cbdv.202000328

Title: Design, synthesis, molecular docking, and biological evaluation
of new emodin anthraquinone derivatives as potential antitumor
substances
Authors: Yuying Li, Fang Guo, Tinggui Chen, Liwei Zhang, Zhuanhua
Wang, Qiang Su, and Liheng Feng
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Biodiversity 10.1002/cbdv.202000328
Link to VoR: https://doi.org/10.1002/cbdv.202000328

Chemistry & Biodiversity
10.1002/cbdv.202000328
Design, synthesis, molecular docking, and biological evaluation of
new emodin anthraquinone derivatives as potential antitumor
substances
*
Yuying Li , Fang Guo, Tinggui Chen, Liwei Zhang, Zhuanhua Wang, Qiang Su,
Liheng Feng
Key Laboratory for Chemical Biology and Molecular Engineering of Ministry of Education,
Institute of Biotechnology, Shanxi University, Taiyuan, 030006, China
Corresponding author.
E-mail address: lyy9030@sxu.edu.cn (Y. Li)
Abstract
The emodin anthraquinone derivatives are generally used in traditional Chinese medicine due to
their various pharmacological activities. In the present study, a series of emodin anthraquinone
derivatives have been designed and synthesized among which 1,3-Dihydroxy-6,8-dimethoxy
-9,10-anthraquinone (11b) is a natural compound that has been synthesized for the very first time,
and 1,4-Dimethyl-6,8-dimethoxy-9,10-anthraquinone (11c) is a compound that has never been
reported earlier. Interestingly, while total seven of these compounds showed neuraminidase
inhibitory activity in influenza virus with inhibition rate more than 50%, specific four compounds
exhibited significant inhibition of tumor cell proliferation. The further results demonstrate that 11c
showed the best anticancer activity among all the as synthesized compounds by inducing highest
apoptosis rate to HCT116 cancer cells and arresting their G0/G1 cell cycle phase, through
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10.1002/cbdv.202000328
elevation of intracellular level of reactive oxygen species (ROS). Moreover, the binding of 11c
with BSA protein has thoroughly been investigated. Altogether, this study suggests the
neuraminidase inhibitory activity and antitumor potential of the new emodin anthraquinone
derivatives.
Keywords: emodin anthraquinone derivatives, synthesis, Anti influenza virus, antitumor
properties, molecular docking
Introduction
Since ancient ages, many medicinally important plants have been employed for the treatment of
various diseases, throughout the world especially in Asia and Africa. The pharmacological
activities of some of these plants have been well documented. [1] Various medicinal plants such as
Rheum, Salamander, and Rhamnus are the key sources of different bioactive molecules, including
many emodin anthraquinone derivatives such as emodin, rhein, aloe-emodin and emodin methyl
ether. [2] Another emodin derivative (1,3,8-trihydroxy-6-methyl-anthraquinone) is found to be the
active ingredient of Chinese traditional rhubarb, which has various pharmacological effects
including antibacterial, antiviral, anti-inflammatory, liver-protective, and anti-tumor activities. [3-10]
Therefore, the research on emodin derivatives has recently attracted great attention to the scientists.
To comprehend the structure-activity relationship, several research groups synthesized different
emodin anthraquinone derivatives employing naturally extracted emodin as raw material and
investigated their potential activities. For instance, Steffi K and research group used halogenated
emodin as a raw material to fabricate emodin derivatives, which were capable of inhibiting ATP
citrate lyase. [11] Similarly, Yang K and team developed a series of lead compounds with antitumor
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Chemistry & Biodiversity
10.1002/cbdv.202000328
activity employing emodin as a raw material. [12]
Recent studies have reported that emodin can induce apoptosis, cell migration inhibition and
cell cycle arrest in a variety of tumor cells (lung and liver cancer). [12-14] Although emodin
derivatives are present in many plants, their practical applications for cancer therapy are
associated with several factors such as their low content in plants, complicated extraction
process, and limitation of active screening. [1] Therefore, it is urgently required to focus on the
total synthesis of emodin derivatives along with the modification of their structure to find novel
anticancer drugs with better efficacy and less toxicity.
In the present study, a series of emodin anthraquinone derivatives has been synthesized by
combining the principle and mechanism of antitumor drugs with the structure-activity relationship
of anthraquinone derivatives. After through biological screening, several anthraquinone
compounds have been found to exert neuraminidase inhibitory activity in influenza virus and
antitumor activity toward HCT116 colon cancer cells. Among these anthraquinone compounds,
the natural product 11b (1,3-dihydroxy-6,8-dimethoxy-9,10-anthraquinone) has been synthesized
for the very first time, and compound 11c (1,4-dimethyl-6,8-dimethoxy-9,10-anthraquinone) has
not earlier been reported. As the compound 11c exhibited the best better anticancer activity among
all the as synthesized compounds toward HCT116 cells, 11c was employed for subsequent
mechanistic studies. The results demonstrated that 11c treatment could enhance the level of
intracellular reactive oxygen species (ROS) in HCT116 cells, leading to induction of apoptosis
and cell cycle arrest in G0/G1 phase. Furthermore, the binding of 11c with BSA protein has been
studied employing fluorescence titration and CD spectroscopy, followed by molecular docking.
Overall, this study suggests that the emodin anthraquinone derivatives could be beneficial for the
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Chemistry & Biodiversity
10.1002/cbdv.202000328
treatment of different virus mediated diseases and cancers in near future.
Result and Discussion
Chemistry
Synthesis of a series of anthraquinone derivatives
To date, anthraquinone derivatives can be synthesized mainly through two steps of Friedel-crafts
reaction and cyclization process. [15,16] Mugunthu RD and co-workers reported the synthesis of
anthraquinone derivatives in the molten state of aluminum trichloride and sodium chloride (2:1),
using phthalic anhydride and substituted benzene derivatives as the raw materials. [17] The above
synthetic methods have several disadvantages including harsh reaction conditions, long reaction
time, as well as low yield. To overcome these limitations, in the present study, anthraquinone
derivatives (3a-3h) have been synthesized employing microwave assisted one step process, using
phthalic anhydride and substituted benzene derivatives as the raw materials, and AlCl /PPA or
3
3
AlCl /H
2
SO
4
as catalysts (Scheme 1). The obtained compounds have thoroughly been
1
characterized by different spectroscopic techniques including UV, IR, H NMR and EI-MS.
Scheme 1. Synthesis of a series of anthraquinone derivatives
Synthesis of emodin anthraquinone derivatives
To synthesize emodin anthraquinone derivatives, the raw material methyl 3,5-dimethoxybenzoate
was first reacted with chloral hydrate. Further 3,5-dimethoxyphthalic anhydride was synthesized
employing a series of reactions such as alkylation, transesterification, hydrolysis, and
decarboxylation. A series of emodin anthraquinone derivatives (11a-11b) were then synthesized
through the interaction of 3,5-dimethoxyphthalic anhydride and different substituted benzenes
under microwave assisted heating, where AlCl
3
/H
2
SO was used as a catalyst. The corresponding
4
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Chemistry & Biodiversity
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synthetic route is presented in Scheme 2.
During synthesis of the anthraquinone derivative 11a, a conventional heating involving two
steps was used at the beginning. At first, Friedel-Crafts reaction was performed using CS , CCl
2
4
,
and CH
Cl
2 2
as solvents. The effect of CS was found to be better with higher yield as compared to
2
the other solvents. However, during cyclization, it was found that the use of concentrated sulfuric
acid/boric acid, oleum/boric acid or polyphosphoric acid (PPA) led to higher reaction temperature,
which was not easy to control. According to published literature, when the reaction temperature is
higher than 100 °C, the oxymethyl group can be cleaved to form a hydroxyl group. [18] Therefore,
during the synthesis of compound 11a, all of its methoxy groups were cleaved to form
1,3,5,8-Tetrahydroxy-2-methyl-anthraquinone with low yield. However, after the microwave
reaction, the methoxy cleavage of the latter two compounds became very weak, and hence the
yield was also increased. Nonetheless, the amplitude was not large, which could be attributed to
the influence of the phthalic anhydride substituent.
Scheme 2.
Synthesis of 3,5-Dimethoxy-phthalic anhydride and emodin anthraquinone
derivatives.
Synthesis of a series of amide anthraquinone derivatives
As show in Scheme 3, a series of new amide anthraquinone derivatives have been synthesized. The
compound 13 (1-Nitro-2-methyl-anthraquinone) was first fabricated by nitration reaction
employing 2-methylanthraquinone as a raw material, followed by reduction process to obtain the
compound 14 (1-amino-2-methylanthraquinone). Finally the compounds 15a and 15b were
obtained by acylation. In addition, compound 16 (1-Nitro-2-carboxy-anthraquinone) was
synthesized by oxidation of Na
2
Cr
2
O , and then the nitro group at position 1 was converted to
7
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Chemistry & Biodiversity
10.1002/cbdv.202000328
amino group through reduction to obtain the compound 17.
Scheme 3. Synthesis of anthraquinone derivatives
Biology
Biological activity of anthraquinone derivatives
The anthraquinone derivatives have thoroughly been screened to evaluate their neuraminidase
inhibitory activity in influenza virus (NA). To check this activity, the compounds were allowed to
interact with a fluorescent substrate MUNANA under the catalysis of influenza virus
neuraminidase, and the decrease in fluorescence intensity of the metabolite was measured at 450
nm. Table 1, demonstrates that the compounds 11a, 11c, 11d, 11c, 11e, 11f, and 17 could
efficiently inhibit the neuraminidase activity with inhibition rate more than 50%. Among these
seven compounds, 11a exerted the highest neuraminidase inhibitory activity, with an IC50 value of
96.1 μM. Aloin, (10-β-D-Glucopyranosyl-1,8-dihydroxy-3-(hydroxymethyl)-9(10H)anthracenone),
has good anti-influenza virus activity in vivo and in vitro, and its anti-influenza virus mechanism
is also the same as inhibition of neuraminidase, IC50 value is 100 μM. Compared with aloin, 11a
has similar activity, which proves that 11a has relatively strong anti-influenza virus activity. [19]
Further the structural analysis indicated that the difference between the compounds 11c and
3
c was only the methoxy modification at the R position in 11c. However, these modification could
enhance the neuraminidase inhibitory effect of 11c as observed by the lower IC50 value of 11c
IC50 = 108.1 μM) as compared to that of 3c (IC50=157.2 μM). Contrastingly, the structural
difference between 3e and 3f was the methyl substitution at R position in 3f, which led to the
(
2
higher IC50 value of 3e (IC50 = 129.1 μM) than that of 3f (IC50 = 100.0 μM), with enhanced
activity. In addition, compound 17 with amino and carboxyl groups at the positions of R1 and R2,
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Chemistry & Biodiversity
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respectively exhibited enhanced neuraminidase inhibitory activity. Therefore, it can be suggested
that the structural modification on R1 and R2 can increase the inhibitory activity of these
compounds to neuraminidase. However, the structural modification of the compounds on R was
not conducive to increase their inhibitory activity.
Table 1. Neuraminidase inhibitory activity of anthraquinone derivatives.
Compound
R
R1
R2
R3
R4
H
Inhibition
ratio (%)
67.86
IC50
(μM)
96.1
1
1a
1c
1d
OH
OCH
OCH
H
OH
CH
H
3
OH
H
1
3
3
CH
3
CH
H
3
50.55
108.1
113.1
157.2
129.1
100.0
108.1
1
OH
H
H
61.26
3
3
c
e
CH
OH
OH
3
H
H
CH
H
3
53.80
H
H
OH
OH
H
61.49
3f
H
CH
3
H
75.06
17
H
NH
2
COOH
H
61.06
These synthesized anthraquinone derivatives have also been screened to check their
antitumor potential, besides the evaluation of their inhibitory activity to neuraminidase. The
anthraquinone derivatives have been administered to eight different kinds of tumor cells (HepG2,
EC9706, SW480, QBC-939, HeLa, SGC-7901, MCF-7, and HCT116) and one type of normal
intestinal epithelial cells (FHC), followed by determination of cell viability using MTT reagent.
After thorough screening, it was found that the compounds 11a-11d inhibited the proliferation of
tumor cells without exerting any cytotoxicity to the normal cells (IC50>1000 μM), suggesting their
potential antitumor activities (Table 2). The result exhibits that the compound 11a had the
strongest inhibitory effect on EC9706 cell proliferation with IC50 value of 172.8 μM and SI value
of 6.7. Contrastingly, the compound 11b demonstrated its strongest anti-proliferative effect toward
HepG2 cells with IC50 value of 144 μM and SI value of 6.6. While the compound 11c significantly
inhibited the proliferation of HCT116 cells with IC50 value of 108.1 μM and SI value of 10.9, the
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compound 11d exhibited the strongest anti-proliferative effect toward HepG2 cells with IC50 value
of 241.5 μM and and SI value of 2.9. Nonetheless, the antitumor effect of 11d was not as good as
the other three compounds. In addition, it is reported in the literature that the IC50 value of rhein
for inhibiting SW480 cell proliferation is 130 μM. [20] 11c inhibited the proliferation of this cell
with an IC50 value of 118.9 μM. The activity of 11c is significantly enhanced compared to rhein.
The compound 11c was found to induce the highest antitumor effect as compared to the other
compounds to most of the cancer cells, especially toward HCT116 cells (IC50=108.1 μM).
Therefore, the further mechanistic studies were performed with the compound 11c to explicate the
mechanism of anticancer activities of anthraquinone derivatives in HCT116 cells.
Table 2. Emodin anthraquinone derivatives inhibit tumor cell proliferation.
HepG2
EC9706
SW480
QBC-939
HeLa
SGC-7901
MCF-7
HCT116
SI
1a 181.1 6.4 172.8 6.7 185.8 6.3 242.2 4.8 222.8 5.2 260.4 4.5 193.3 6.0 189.6 6.1
1b 144.0 6.6 225.7 4.2 202.7 4.7 257.0 3.7 167.0 5.6 203.7 4.3 153.7 6.1 182.0 5.2
1c 115.9 10.2 111.5 10.6 118.9 9.9 238.2 5.0 120.9 9.8 204.4 5.8 135.1 8.8 108.1 10.9
1d 241.5 2.9 244.4 2.9 261.3 2.7 282.4 2.5 265.5 2.7 305.6 2.3 257.0 2.7 267.6 2.6
IC50[b]
SI IC50[b]
SI IC50[b] SI IC50[b] SI IC50[b] SI IC50[b] SI IC50[b] SI IC50[b]
1
1
1
1
[a] Use normal intestinal epithelial cells (FHC) as a control
[b] IC50 value unit is micromolar (μM).
Cell viability in HCT116 and FHC cells administered with the compound 11c
Previous studies demonstrated that emodin can inhibit tumor cell proliferation. However, the
antitumor potential of the compound 11c has not earlier been reported. [21] In the present study, the
cell viability assay was performed employing MTT reagent in both cancerous (HCT116) as well as
non-cancerous (FHC) cells, incubated with the compound 11c, relative molecular mass 296, (0, 25,
50, 100 μg/mL) for 24 h. Figure 1,A 1,B demonstrate that the compound 11c significantly
inhibited the proliferation of HCT116 cells as compared to the untreated control experiment in a
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concentration dependent manner. However, the compound 11c treatment could not alter the cell
viability of FHC cells significantly in comparison with the untreated control experiment. These
results suggest the potential antitumor activity of this anthraquinone derivative.
Figure 1. Cell viability assay in HCT116 and FHC cells using MTT reagent. The effect of 11c
treatment (0, 25, 50, 100 μg/mL, 24 h) on the viability of A) HCT116 cells and B) FHC cells.
*
p <0.05, ** p <0.01 as compared to the control group were considered as significantly different.
Apoptosis analysis in HCT116 cells using flow cytometry and western blot analysis
Apoptosis, referring to a method of programmed cell death, controls the occurrence and
development of tumor cells. Earlier study demonstrated that the emodin anthraquinone compounds
can induce apoptosis to tumor cells leading to cell death. [22] Therefore, after detecting the efficient
anti-proliferative effect of 11c toward HCT116 cells, further mechanistic studies was carried out to
comprehend whether the apoptosis process was involved in 11c-induced cancer cell death. Figure
2
,A 2,B (flow cytometry data) exhibits that the compound 11c treatment significantly induced
apoptosis in HCT116 cells in a dose dependent fashion (apoptotic cell: 20.4% for 25 μg/mL dose;
6.7% for 50 μg/mL dose) as compared to the control experiment (apoptotic cell: 4.8%). To
3
confirm this result, western blot analysis was further performed in HCT116 cells treated with
compound 11c for checking the expression of apoptosis-related proteins including Bax, Bcl-2,
Cleaved caspase 3, and Cleaved caspase 9. As shown in Figure 2,C 2,D, while 11c treatment
upregulated the expression of apoptotic protein Bax, it could downregulate the expression of
anti-apoptotic protein Bcl-2 in a dose dependent manner as compared to the untreated control
experiment. Moreover, 11c treatment also led to dose dependent upregulation of the expressions of
Cleaved caspase 3 and Cleaved caspase 9 in comparison with the untreated control cells. The
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western blot analysis clearly suggests that the compound 11c induced cancer cell death through
apoptosis.
Figure 2. The compound 11c induces apoptosis to HCT116 cells in a dose-dependent manner. A)
Flow cytometry was used to detect the apoptosis of HCT116 cells incubated with 11c (0, 25, 50
μg/mL, 24 h). B) According to the results of flow cytometry, the apoptotic histogram was prepared.
The experiment was repeated thrice. * p <0.05 and ** p <0.01 as compared to control group were
considered as significantly different C) Western Blot was performed in HCT116 cells treated with
compound 11c (0, 25, 50 μg/mL, 24 h) to check the expression of apoptosis-related proteins. D) A
grayscale analysis was performed based on the results of the western blot to obtain a histogram.
The experiment was repeated thrice. * p <0.05 and ** p <0.01 as compared to control group were
considered as significantly different.
Cell cycle analysis in HCT116 cells using flow cytometry
To explicate further mechanism of cancer cell death induced by compound 11c, cell cycle analysis
was performed in HCT116 cells using flow cytometry. The result exhibits that 11c treatment led to
increase the cell population in G0/G1 phase in a dose dependent fashion (cell population: 55.39%
for 25 μg/mL dose; 60.31% for 50μg/mL dose) with subsequent decrease of cell population in G2/
M and S phases as compared to the untreated control cells (Figure 3,A 3,B). This study suggests
that 11c treatment could arrest the G0/G1 cell cycle phase of HCT116 cancer cells, leading to
apoptosis process.
Figure 3. The treatment of compound 11c led to cell cycle arrest in G0/G1 phase of HCT116 cells.
A) HCT116 cell cycle arrest was detected by flow cytometry upon treatment with the compound
11c (25-50 μg/mL, 24 h). B) Histogram analysis showing the cell distribution in different phases
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of cell cycle. The experiment was repeated thrice. * p <0.05 and ** p <0.01 as compared to
control group were considered as significantly different.
Determination of intracellular ROS level in HCT116 cells
The earlier literature reported that excessive production of intracellular ROS causes oxidative
stress, leading to apoptosis induction to a variety of tumor cells, including colon cancer cells. [23]
In addition, previous reports exhibited that emodin can reverse the apoptosis of gastric cancer cells
through addition of ROS scavengers. [24] Therefore, in the present study, the production of
intracellular ROS was investigated in HCT116 cells upon treatment with the compound 11c.
Figure 4,A 4,B depict that 11c treatment enhanced the number of DCFH-DA positive cells in a
dose dependent manner (829.44 for 25 μg/mL; 1174.48 for 50 μg/mL) as compared to the control
experiment. This result indicates that 11c can induce the intracellular production of ROS in
HCT116 cells, which could be one of plausible mechanisms behind the antitumor activity of the
anthraquinone derivative.
Figure 4. The treatment of 11c enhances the ROS level in HCT116 cells. A) Determination of
ROS production in HCT116 cells treated with 11c (0, 25, 50 μg/mL, 24 h) using flow cytometry. B)
A corresponding bar graph based on the fluorescence values. The experiment was repeated thrice.
*
p <0.05 and ** p <0.01 as compared to control group were considered as significantly different.
Interaction between 11c and BSA
Fluorescence spectroscopy, possessing higher accuracy and specificity as compared to UV-visible
spectroscopy, is an important method to study the interaction of biological macromolecules with
various organic small molecules, ions and inorganic compounds.[25] Common serum albumins
include human serum albumin and bovine serum albumin, which are involved in various
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Chemistry & Biodiversity
10.1002/cbdv.202000328
transportation and regulation of the body system. [26] The interaction of a compound with BSA can
enhance its solubility and dispersibility during blood transport. [27] Based on the importance of
BSA in pharmacokinetics, the interaction between the compound 11c and BSA was further studied
using fluorescence titration, which would be helpful to better understand the role of emodin-BSA
binding complexes for the in vivo applications of emodin anthraquinone derivatives.
It is well known that the endogenous fluorescence of BSA is formed by a combination of
fluorescence from tyrosine, tryptophan, as well as phenylalanine. [28] Keeping the concentration of
BSA unchanged, the same volume of 11c solution with different concentrations, was added in
order, followed by scanning the fluorescence spectrum of the mixed solution system sequentially.
As shown in Figure 5,A, with increase in concentration of 11c, the fluorescence intensity of the
solution system was regularly quenched and the emission peaks exhibited a slight red shift. This
result proves that with the addition of 11c, the microenvironment of BSA was altered significantly
leading to reduce its hydrophobicity, disintegrate its internal hydrophobic structure, and enhance
the degree of peptide chain extension. It can be speculated that the compound 11c could easily
enter into the hydrophobic cavity of BSA and further interact with the hydrophobic amino acid
residues. 11c could approach the hydrophobic cavity through electrostatic attraction, which might
affect the conformational change of BSA, leading to quenching of the endogenous fluorescence of
BSA.
Fluorescence quenching could be of two types namely, dynamic quenching and static
quenching. It was initially assumed that 11c mediated BSA fluorescence quenching is involved
dynamic quenching. The Stern-Volmer equation for dynamic quenching is:
F
0
/F=1+K
q
τ
0
[Q]=1+ksv[Q] [29]
[1]
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In the formula, F and F refer to the fluorescence intensities in absence and presence of the
0
quencher Q. Kq is the rate constant of the bimolecular quenching process and τ is the average life
0
of the biological macromolecules in absence of the quencher. ksv refer to the dynamic quenching
constant.
Using S0-F to [Q] as a Sern-Volmer plot, the results are presented in Figure 5,B. Based on the
slope of the fitted straight line, the quenching constant ksv of 11c to BSA was calculated to be 1.1
4
×
10 L/mol, and the linear correlation coefficient was R = 0.9982. Previous reports demonstrated
-8
that the fluorescence lifetime of biological macromolecules is about 10 s, and the maximum
diffusion collision quenching rate constant of different quenchers for biological macromolecules is
1
0
2
1
.0 × 10 L / (mol • s). The 11c-BSA fluorescence quenching rate constant was found to be 1.1 ×
012 L / (mol • s), which is much larger than the abovementioned maximum diffusion collision
quenching rate constant. This result indicates that the quenching of BSA fluorescence by 11c is a
static quenching.
In the serum albumin protein molecule, tryptophan, tyrosine and phenylalanine residues have
strong absorption peaks at 230-310 nm. Therefore, CD spectra was employed to study the
conformational changes of serum albumin upon interacting with the compound 11c. As shown in
Figure 5,C, BSA exerted a negative Cotton effect at 223 nm, which was contributed by the alpha
helix structure in the BSA. After the addition of 11c, the negative Cotton effect of BSA was
weakened. However, the shape of the CD spectrum did not alter and a red shift was observed. This
phenomenon could be attributed to the hydrophobic effect of the compound or the increased
β-sheet structure. This result suggests that the combination of 11c and BSA caused a significant
impact on the secondary structure of BSA. The compound 11c could destroy the α-helix structure
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Chemistry & Biodiversity
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of BSA through hydrophobic interaction as well as hydrogen bonding interaction, leading to a
decrease in the α-helix content in the structure of BSA.
Figure 5. Interaction between the compound 11c and BSA A) Compound 11c quenched the
fluorescence of BSA. CBSA=1.0×10-6 mol/L；C11c=1.0×10-4 mol/L; T=297K B) Stern-Volmer
fluorescence quenching curve of 11c versus BSA system. C) The effect of 11c on the CD spectrum
-6
-4
of BSA. CBSA=1.0×10 mol/L；C11c=1.0×10 mol/L; T=298.15K
Molecular docking studies
Molecular docking is one of the most useful tools for studying the interaction between proteins
and organic molecules. Molecular docking studies can easily confirm the results of previous
experiments.[30] Therefore, docking analysis was performed employing AutoDock 4.2.6 software
to investigate the interaction between compound 11c and BSA protein. As the binding site of 11c
and BSA was previously unknown, the compound 11c was first put into the crystal structure of
BSA to allow free binding, and it was observed that 11c could finally bind to the cavity on the A
chain of BSA (Figure 6). In addition, the hydrogen bonding interaction between 11c and BSA was
also analyzed. As shown in Figure 6,B, using Automatic the total score was 1.9599, and three
hydrogen bonds were formed by tightly binding with amino acid residues ARG435 and LYS431.
Subsequently, the hydrophobicity of 11c was explored through molecular docking as shown in
Figure 6,B. Here, the blue color referred to the hydrophilic site of BSA, while the green color
indicated its hydrophobic site. It can be clearly observed that the combination of 11c with the
hydrophobic cavity of BSA can interact with hydrophobic amino acid residues, suggesting that 11c
is hydrophobic in nature. This data corroborates with the earlier fluorescent titration results that
the compound 11c could alter the microenvironment especially the hydrophobicity of BSA. The
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secondary structure of 11c and BSA was further analyzed and presented in Figure 6,D. The result
exhibits that the compound 11c entered into the position of α-helix of the A chain in BSA. From
the results of CD spectra, it can be clearly observed that the combination of 11c and BSA caused
the conformational changes in of BSA. This phenomenon led to the destruction of α-helix
structure of BSA through hydrogen bonding interaction and hydrophobic interaction, which is in
agreement with the docking result.
Figure 6. Molecular docking between 11c and BSA molecules. A) Binding of 11c with BSA. B)
11c forms hydrogen bonds with BSA. C) 11c can bind to BSA hydrophobic site. D) Binding of 11c
with secondary structure of BSA.
Conclusion
In conclusion, we designed and synthesized a series of new emodin anthraquinone derivatives,
followed by their characterizations. After thorough biological screening, it was observed that
seven anthraquinone compounds could inhibit the neuraminidase activity of influenza virus with
inhibition rate more than 50%. Moreover, four of these compounds especially 11c inhibited the
proliferation of cancer cells significantly without showing any cytotoxicity to normal cells,
suggesting their potential antitumor activity. The mechanistic studies showed that the compound
11c treatment can elevate the intracellular level of ROS in HCT116 cells, leading to induction of
apoptosis and cell cycle arrest in the G0/G1 phase. In addition, the binding studies using
fluorescence titration and CD spectroscopy exhibited that the combination of 11c and BSA caused
a significant impact on the secondary structure of BSA. The compound 11c could destroy the
α-helix structure of BSA through hydrophobic interactions and hydrogen bonding interactions,
which corroborate with the molecular docking analysis. Overall, this study offers the basis for
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Chemistry & Biodiversity
10.1002/cbdv.202000328
further development of anthraquinone derivatives for various biological applications.
Experimental Section
Materials
The synthesis and characterizations were performed employing chemical microwave reactor
Nanjing Lingjiang Technology, China), IR spectrum (Perkin elmer, Japan), UV-2000 (UNICO,
(
Shanghai), and DRX-300 (300Hz) nuclear magnetic resonance instrument (Bruker Biospin,
Switzerland). All the reagents used in the experiments were purchased and utilized directly
without further purification. The deuterated CDCl , DMSO and tetramethylsilane were procured
3
from Energy Chemical, China. GF 254 silica gel plate was purchased from Shandong Qingdao
Chemical Industry, China. While RPMI-1640 medium was obtained from HyClone, USA, fetal
bovine serum (FBS) was procured from Sangon Biotechnology, Shanghai. MTT was purchased
from Sigma, USA. While Annexin V-FITC assay kit was obtained from Pharmingen-Becton
Dickinson, USA, the Cell Cycle Detection Kit was procured from KeyGen Biotech, Nanjing. The
primary antibodies (e.g. Bcl-2, Bax, Cleaved caspase-3, and Cleaved caspase-9), and ROS
detection kit were purchased from Beyotime Biotechnology, China. Horseradish
peroxidase-labeled goat anti-rabbit IgG secondary antibody, and β-actin antibody primary
antibody were procured from Bioworld, USA. While, ECL luminescence assay kit was obtained
from Engreen Biosystem, China, BCA protein quantification kit was purchased from Minbio,
china. PVDF membrane was procured from Millipore, Germany. Bovine serum albumin was
purchased from Solarbio, Beijing.
Methods
Screening of anthraquinone derivatives for the neuraminidase inhibitory activity in influenza virus.
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Chemistry & Biodiversity
10.1002/cbdv.202000328
A certain concentration of the test compounds and influenza virus neuraminidase (NA) were
suspended in reaction buffer (pH 6.5), and fluorescent substrate
’-4-methylumbelliferyl-α-N-acetylneuraminate (MUNANA) was added into the reaction system,
a
a
2
followed by incubation for 60 min, at 37˚C. The reaction was then stopped by adding a reaction
stop solution. Next, the fluorescence intensity was measured at an excitation wavelength of 360
nm and an emission wavelength of 450 nm. It is to be mentioned here that the fluorescence
intensity of the reaction system can directly reflect the enzyme activity. Therefore the NA
inhibitory activity of the test compounds can be calculated from the decrease in fluorescence
intensity.
Cell culture. Human colon cancer cell line HCT116, normal intestinal epithelial cell line FHC,
human cervical cancer cell line HeLa, human breast cancer cell line MCF-7, human bile duct
cancer cell line QBC-939, human liver cancer cell line HepG2, human gastric cancer cell line
SGC-7901 and human colorectal cancer cell line SW480 were purchased from Cell Bank of the
Chinese Academy of Sciences (Shanghai, China). The human esophageal cancer cell line EC9706
was obtained by Prof. Ming Rong Wang, Chinese Academy Medical of Sciences from Cancer
Hospital Institute. While EC9706, HeLa and SW480 cells were cultured in RPMI 1640 medium
containing 10% (v/v) heat-inactivated fetal calf serum (FCS), MCF-7, QBC-939, SGC-7901 and
HepG2 cells were cultured in DMEM medium containing 10% FBS, 100 U/mL penicillin and 100
U/mL streptomycin. The above cells were cultured inside a humidified cell incubator with 5%
CO
2
at 37 °C.
Preparation of stock solutions. The test compounds were soluble in DMSO. Therefore, they
-4
were dissolved in DMSO at a concentration of 10 mol/L to prepare the respective stock solutions,
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Chemistry & Biodiversity
10.1002/cbdv.202000328
which were further diluted to the required concentration with the corresponding cell culture
medium for all the experiments.
5
Cell viability test. The cells were seeded at a density of 1 × 10 cells/mL in a volume of 100
μL media into each well of 96-well microtiter plate. After adherence, the cells were incubated with
the test compounds (25-100 μg/mL) for 24h. The cells were then incubated with 20 μL of MTT
solution (5 mg/mL) at 37 °C for another 4 h. After removing the culture medium and containing
MTT solution, 150 μL of DMSO was added to each well and the plate was placed on a shaker at
room temperature for around 5 min. The absorbance was measured using a microplate reader at
490 nm. The average absorbance of three parallel experiments was calculated using the following
formula:
Percent inhibition = 1- (average experimental absorbance/average control absorbance) × 100% [2]
Annexin V-FITC/PI staining assay. Six-well tissue culture plates were seeded with 1 × 104
HCT116 cells per well. After adherence, the cells were incubated with the compound 11c (25-50
μg/mL) for 24 h. The apoptosis analysis was then performed employing Annexin V-FITC/PI
apoptosis detection kit. At first, the cells were resuspended in 500 μL of binding buffer containing
5
μL of annexin V-FITC and 1 μL of PI, and incubated for 30 min in the dark at room temperature.
The number and distribution of apoptotic cells were then counted using flow cytometry analysis
FACSCalibur).
(
Cell cycle analysis. In brief, HCT116 cells were seeded into 6-well tissue culture plates and
after adherence, the cells were incubated with the compound 11c (25-50 μg/mL) for 24h. The cells
were then fixed with 0.7 mL of 70% ethanol at 4 °C for overnight. Next, the fixed cells were
centrifuged and washed with PBS for 30 min at room temperature under dark condition. After that,
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Chemistry & Biodiversity
10.1002/cbdv.202000328
the cells were stained with 100 μL PI (1 mM) containing 50 μL RNase A (1 mM) subjected to
flow cytometer for cell cycle analysis.
Measurement of intracellular ROS. The intracellular ROS level in HCT116 cells were
measured through flow cytometry, employing 2', 7'-Dichlorofluorescein-diacetate (DCFH-DA)
4
reagent. The cells (1×10 cells per well) were first seeded into six-well tissue culture plates. After
adherence, the cells were treated with the compound for respective time duration, followed by
incubation with 1 mL of DCFH-DA(10 μM) for 30 min at 37 °C under dark condition. The stained
cells were then washed thrice with PBS and further kept in 300 μL of PBS. The fluorescence
intensity was measured using a flow cytometer (FACSCalibur) with 488/525 nm
excitation/emission filter.
Western blot analysis. HCT116 cells were incubated with the compound 11c for 24 h. The
cells were then washed with cold PBS thrice and incubated with lysis buffer for 30 min on ice.
After that, the cells were centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant cell
lysate was collected. The protein concentration of the obtained cell lysate was measured using a
BCA protein quantification kit (Beyotime Institute of Biotechnology, Shanghai). The total protein
of 60 μg was separated by 10% SDS-PAGE, followed by transfer to a PVDF membrane. The
membrane was then blocked with 5% skim milk in 0.1% Tris buffer containing 0.05% Tween-20
(TBST) for 1 h at room temperature. After that, PVDF membrane was incubated with primary
antibodies (anti-Bax, Bcl-2, cleaved caspase 9 and cleaved caspase 3) at 4 °C for overnight. The
membrane was washed with TBST thrice, followed by incubation with HRP-conjugated
secondary antibody for 1 h at room temperature. After washing the membrane again with TBST
thrice, the protein bands on the membrane were detected employing an enhanced
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Chemistry & Biodiversity
10.1002/cbdv.202000328
chemiluminescence detection kit (Engreen) and exposed to X-ray film.
Docking analysis. The crystal structure of BSA with a resolution of 2.47Å (4F5S) was
obtained from the Protein Data Bank (PDB) database. The water molecules in the target protein
were removed using SYBYL-2.0X software. It was saved in mol2 format, and the processed BSA
was hydrogenated and charged in AutoDuck Tools software. It was then molecularly docked with
the compound 11c. The affinity of BSA and 11c was analyzed through scoring and hydrogen
bonding.
-6
Fluorescence spectroscopy. 2 mL of BSA (1.0 × 10 mol/L) solution was taken into 1 cm
-4
quartz cuvette and 2 μL of 10 mol/L 11c compound solution was poured into it. The solution
mixture was shaken well and kept for 5 min without disturbing. Fluorescence spectra of BSA and
compound solutions were measured at room temperature. While the fluorescence excitation
wavelength was fixed at λex = 251 nm, the emission wavelength was set to λem = 344 nm. The
scanning speed was 250 nm/min, and the slit was 5 nm.
-6
CD spectrometry. 2 mL of BSA (1.0×10 mol/L) solution was taken in 1 cm sample cell and
-4
2
μL of 10 mol/L 11c compound solution was poured into it, followed by shaking the well mix
the solutions well. CD spectra of BSA and 11c were measured at room temperature with scanning
speed 1 nm/min and scanning range 210-250 nm.
Acknowledgments
This work was supported by the Key projects of Shanxi Province (No. 201903D321095) and
Transformation of Scientific and Technological Achievements Programs of Higher Education
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Chemistry & Biodiversity
10.1002/cbdv.202000328
Institutions in Shanxi (TSTAP). We thank Prof. Daxiong Han from Medical College Xiamen
University for software support. We would also like to thank Mr. Du Guanhua, Mr. Liu Ailin, and
Mr. Hu for their assistance in the activity testing of the National Drug Screening Center of the
Institute of Materia Medica, Chinese Academy of Medical Sciences.
Author Contribution Statement
Q. S. and L. F. performed the synthetic experiments and analyzed the data. F. G. and T. C.
performed the biological experiments and analyzed the data. Y. L. and F. G. designed the
experiments. F. G., Y. L., L.Z., Z. W. wrote/corrected the article.
Conflict of interest
The authors declare they have no conflict of interest.
Appendix A. Supplementary data
Supplementary data associated with this article can be found.
References
[1]X. Dong, Y. Zeng, Y. Liu , L. You, X. Yin, J. Fu, J. Ni, 'Aloe-emodin: A Review of its
Pharmacology, Toxicity and Pharmacokinetics', Phytother. Res. 2020, 34, 270-281.
[2]B. A. Monisha, N. Kumar, A. B. Tiku, 'Emodin and Its Role in Chronic Diseases', Adv. Exp.
Med. Biol. 2016，928，47-73.
[3]X. Hou, W. Wei, Y. Fan, J. Zhang, N. Zhu, H. Hong, C. Wang, 'Study on synthesis and
bioactivity of biotinylated emodin', Appl. Microbiol. Biotechnol. 2017，13, 5259-5266.
[4]L. Li, X. Song, Z. Yin, R. Jia, Z. Li, X. Zhou, Y. Zou, L. Li, Yin L, G. Yue, et al., 'The
antibacterial activity and action mechanism of emodin from Polygonum cuspidatum against
Haemophilus parasuis in vitro', Microbiol. Res. 2016, 186, 139-145.
This article is protected by copyright. All rights reserved.

Chemistry & Biodiversity
10.1002/cbdv.202000328
[5]S. W. Li, T. C. Yang, C. C. Lai, S. H. Huang, J. M. Liao, L. Wan, Y. J. Lin, C. W. Lin,
'
Antiviral activity of aloe-emodin against influenza A virus via galectin-3 up-regulation', Eur. J.
Pharmacol. 2014, 738, 125-32.
[6]J. W. Han, D. W. Shim, W. Y. Shin, K. H. Heo, S. B. Kwak, E. J. Sim, J. H. Jeong , T. B.
Kang, K. H. Lee, 'Anti-inflammatory effect of emodin via attenuation of NLRP3 inflammasome
activation', Int. J. Mol. Sci. 2015, 16, 8102-9.
[
7]E. H. Lee, S. Y. Baek, J. Y. Park, Y. W. Kim, 'Emodin in Rheum undulatum inhibits oxidative
stress in the liver via AMPK with Hippo/Yap signalling pathway', Pharm. Microbiol. 2020，58，
33-341.
3
[8]G. Dai, K. Ding, Q. Cao, T. Xu, F. He, S. Liu, W, Ju 'Emodin suppresses growth and invasion
colorectal cancer cells by inhibiting VEGFR2', Eur. J. Pharmacol. 2019, 15, 172525.
[
9]Y. Ding, P. Liu, Z. L. Chen, S. J. Zhang, Y. Q. Wang, X. Cai, L. Luo, X. Zhou, L. Zhao,
Emodin Attenuates Lipopolysaccharide-Induced Acute Liver Injury via Inhibiting the TLR4
Signaling Pathway in vitro and in vivo', Front. Pharmacol. 2018, 9, 962.
'
[
10]S. F. Chen, Z. Y. Zhang, J. L. Zhang, 'Emodin enhances antitumor effect of paclitaxel on
human non-small-cell lung cancer cells in vitro and in vivo', Drug Des. Dev. Ther. 2019, 13,
145-1153.
1
[11]K. Steffi, Koerner, J. Hanai, S. Bai, F. E. Jernigan, M. Oki, C. Komaba, E. Shuto, V. P.
Sukhatme, L. Sun，'Design and synthesis of emodin derivatives as novel inhibitors of ATP-citrate
lyase', Eur. J. Med. Chem. 2017, 126, 920-928.
[
12]K. Yang, M. Jin, Z. Quan, H. Piao, 'Design and Synthesis of Novel Anti-Proliferative Emodin
Derivatives and Studies on their Cell Cycle Arrest, Apoptosis Pathway and Migration', Molecules.
019, 24, 884.
2
[13]X. P. Pan，C. Wang，Y. Li，L. Zhu，T. Zhang, 'Protective autophagy induced by physcion
suppresses hepatocellular carcinoma cell metastasis by inactivating the JAK2/STAT3 Axis', Life
Sci. 2018, 214, 124-135.
[
14]N. Yang, C. Li, H. Li, M. Liu, X. J. Cai, F. J. Cao, Y. B. Feng, M. L. Li, X. B. Wang,
Emodin Induced SREBP1-Dependent and SREBP1-Independent Apoptosis in Hepatocellular
Carcinoma Cells', Front. Pharmacol. 2019, 10, 709.
'
[15]L. Mahesh, Patil, B. B. Hanumant,D. E. Ponde, V. H. Deshpande, 'Total synthesis of
This article is protected by copyright. All rights reserved.

Chemistry & Biodiversity
10.1002/cbdv.202000328
(±)-brasiliquinone. Tetrahedron', Tetrahedron. 2002, 58, 6615-6620.
[16]S. Yeap, M. N. Akhtar, K. L. Lim, N. Abu, W. Y. Ho, S. Zareen, K. Roohani, H. Ky, S. W.
Tan, N. Lajis, N. B. Alitheen, 'Synthesis of an anthraquinone derivative (DHAQC) and
its effect on induction of G2/M arrest and apoptosis in breast cancer MCF-7 cell line', Drug Des.
Devel Ther. 2015, 17, 983-992.
[
17]M. R. Dhanganjeyan, Y. P. MilevP, M. A. Kron, M. G. Nair, 'Synthesis and activity of
substituted anthraquinones against a human filarial parasite, Brugia malayi', J. Med. Chem. 2005,
8, 2822-2830.
4
[18]G. D. Graves, R. Adams, 'Trihydroxy-methyl-anthraquinones. Journal of the American
Chemical Society', J. Am. Chem. Soc. 1923, 45, 2439-2455.
[19]C. T. Huang, C. Y. Huang, Y. C. Hseih, C. H. Chang, A. B. Velu, Y. C. He, Y. L. Huang, T. A.
Chen, T. C. Chen, C. Y. Lin, Y. C. Lin, S. R. Shih, A. Dutta. 'Effect of aloin on viral neuraminidase
and hemagglutinin-specific T cell immunity in acute influenza', Phytomedicine. 2019, 64, 152904.
[
20]S. W. Tsang, Z. X. Bian, 'Anti‐fibrotic and Anti‐tumorigenic Effects of Rhein, a Natural
Anthraquinone Derivative, in Mammalian Stellate and Carcinoma Cells', Phytotherapy Research.
015, 29, 407-414.
2
[21]Q. Z. Ma, Y. Q. Ding, Z. Q. Wu , Y. Li, 'Antitumor effects of emodin in CACO-2 human colon
carcinoma cells are mediated via apoptosis, cell cycle arrest and downregulation of PI3K/AKT
signalling pathway', J BUON. 2018, 23, 587-591.
[
22]G. Pistritto, D. Trisciuoglio, C. Ceci, A. Garufi, G. D'Orazi, 'Apoptosis as anticancer
mechanism: function and dysfunction of its modulators and targeted therapeutic strategies', Aging.
016, 8, 603-619.
2
[23]J. Y. Jang, Y. J. Kang, B. Sung, M. J. Kim, C. Park, D. Kang, H. R. Moon, H. Y. Chung, N. D.
Kim, 'a novel topoisomerase iota inhibitor, induces cell cycle arrest and apoptosis via a
rosdependent DNA damage signaling pathway in ags human gastric cancer cells',
Molecules. 2018, 24, 96.
[24]Y. Z. Jin, H. N. Sun, Y. Liu, D. H. Lee, J. S. Kim, S. U. Kim, B. Y. Jiao, Y. H. Han, M. H. Jin,
G. N. Shen, D. S. Lee, T. Kwon, D. Y. Xu, Y. Jin. 'Peroxiredoxin V Inhibits Emodin-induced
Gastric Cancer Cell Apoptosis via the ROS/Bcl2 Pathway', In Vivo. 2019, 33, 1183-1192.
[
25]J. Stanicova, V. Verebova, J. Benes. In Vivo. 'Interaction of a Potential Anticancer Agent
Hypericin and its Model Compound Emodin with DNA and Bovine Serum Albumin', In Vivo.
018, 32, 1063-1070.
2
This article is protected by copyright. All rights reserved.

Chemistry & Biodiversity
10.1002/cbdv.202000328
[
26]J. Esfahlan, V. Panahi-Azar, S. Sajedi, 'Spectroscopic and moleculardocking studies on the
interaction between N-acetyl cysteine and bovineserum albumin', Biopolymers. 2015, 103,
38-645.
6
[27]M. K. Siddiqi, P. Alam, S. K. Chaturvedi, S. Nusrat, M. R. Ajmal, A. S. Abdelhameed, R. H.
Khan, 'Investigating the site selective binding of busulfan to human serum albumin: biophysical
and molecular docking approaches', Int. J. Biol. Macromol. 2018, 107, 1414-1421.
[28]P. K. Suresh, D. Naik, N. Shah, R. Rajasekaran, 'Phenytoin-Bovine serum albumin
interactions-modeling plasma protein-drug binding: A multi-spectroscopy and in silico-based
correlation', Spectrochim. Acta, Part A. 2018, 193, 523-527.
[29]P. L. Koen, Kuijpers, C. Bottecchia, D. Cambié, D. Koen, N. J. König, T. Noël, 'A Fully
Automated Continuous‐Flow Platform for Fluorescence Quenching Studies and Stern–Volmer
Analysis', Angew. Chem. Int. Ed. Engl. 2018, 57, 11278-11272.
[
30]D. Raghav, S. Mahanty, K. Rathinasamy, 'Biochemical and toxicological investigation of
karanjin, a bio-pesticide isolated from Pongamia seed oil', Pestic. Biochem. Physiol. 2019, 157,
08-121.
1
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10.1002/cbdv.202000328
Scheme 1. Synthesis of a series of anthraquinone derivatives
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Chemistry & Biodiversity
10.1002/cbdv.202000328
Scheme 2.
Synthesis of 3,5-Dimethoxy-phthalic anhydride and emodin anthraquinone
derivatives
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Chemistry & Biodiversity
10.1002/cbdv.202000328
Scheme 3. Synthesis of anthraquinone derivatives
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Chemistry & Biodiversity
10.1002/cbdv.202000328
Figure 1. Effect of 11c compound treatment on the viability of HCT116 and FHC cells. A) The
effect of 11c treatment (0, 25, 50, 100 μg/mL, 24 h) on the viability of HCT116 cells was detected
using MTT reagent. B) The effect of 11c treatment (0, 25, 50, 100 μg/mL, 24 h) on the viability of
FHC cells was detected employing MTT reagent. Significant difference (* p <0.05, ** p <0.01),
compared with control group.
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Chemistry & Biodiversity
10.1002/cbdv.202000328
Figure 2. The compound 11c induces apoptosis to HCT116 cells in a dose-dependent manner. A)
Flow cytometry was used to detect the apoptosis of HCT116 cells after treatment with 11c (0, 25,
0 μg/mL, 24 h). B) According to the results of flow cytometry, the apoptotic histogram was
5
prepared. The experiment was repeated thrice with significant differences (* p <0.05, ** p <0.01).
C) Western Blot was performed in HCT116 cells treated with compound 11c (0, 25, 50 μg/mL, 24
h) to check the expression of apoptosis-related proteins. D) A grayscale analysis was performed
based on the results of the western blot to obtain a histogram. The experiment was repeated thrice
with significant differences (* p <0.05, ** p <0.01), compared with control group.
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