Synthesis, antitumor activity, and molecular docking of (?)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin conjugates

Article abstract of DOI:10.1080/10286020.2020.1786066

Two new (?)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin conjugates (7 and 8) were synthesized and evaluated for biological activity. Compound 8 showed highly potent anticancer activity against A-549 cell line with IC₅₀ of 2.16 ± 1.02

Full text of DOI:10.1080/10286020.2020.1786066

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Synthesis, antitumor activity, and molecular
docking of (−)-epigallocatechin-3-gallate-4β-
triazolopodophyllotoxin conjugates
Cheng-Ting Zi, Liu Yang, Yue Hu, Pan Zhang, Han Tang, Bang-Lei Zhang, Xiao-
Jing Shen, Qing-Hua Kong, Ya Wang, Xuan-Jun Wang & Jun Sheng
To cite this article: Cheng-Ting Zi, Liu Yang, Yue Hu, Pan Zhang, Han Tang, Bang-Lei
Zhang, Xiao-Jing Shen, Qing-Hua Kong, Ya Wang, Xuan-Jun Wang & Jun Sheng (2020):
Synthesis, antitumor activity, and molecular docking of (−)-epigallocatechin-3-gallate-4β-
triazolopodophyllotoxin conjugates, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2020.1786066
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2020.1786066
Synthesis, antitumor activity, and molecular docking
of (ꢀ)-epigallocatechin-3-gallate-4b-
triazolopodophyllotoxin conjugates
c
a
a
a
Cheng-Ting Zia,b , Liu Yang , Yue Hu , Pan Zhang , Han Tang , Bang-Lei
Zhanga, Xiao-Jing Shenb, Qing-Hua Kongc, Ya Wanga,b, Xuan-Jun Wanga,b and
Jun Shenga
ꢁ
ꢁ
aKey Laboratory of Pu-er Tea Science, Ministry of Education, Yunnan Agricultural University,
b
Kunming 650201, China; College of Science, Yunnan Agricultural University, Kunming 650201,
c
China; State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming
Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
ABSTRACT
ARTICLE HISTORY
Received 12 February 2020
Accepted 17 June 2020
Two new (ꢀ)-epigallocatechin-3-gallate-4b-triazolopodophyllotoxin
conjugates (7 and 8) were synthesized and evaluated for bio-
logical activity. Compound 8 showed highly potent anticancer
activity against A-549 cell line with IC50 of 2.16 1.02 lM, which
displayed the highest selectivity index value (SI ¼ 14.5) in A-549
cells. Molecular docking indicated that compound 8 could bind
with the active site of Top-II. Therefore, compound 8 might be a
promising candidate for further development.
KEYWORDS
Podophyllotixin; EGCG; click
reaction; antitumor activity;
molecular docking
CONTACT Ya Wang
shengj@ynau.edu.cn
wangya9188@126.com; Xuan-Jun Wang
Key Laboratory of Pu-er Tea Science, Ministry of Education, Yunnan Agricultural University,
wangxuanjun@gmail.com; Jun Sheng
Kunming 650201, China
ꢁ
The authors Cheng-Ting Zi and Liu Yang contributed equally to the work.
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2020.1786066.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
C.-T. ZI ET AL.
1. Introduction
Cancer has been reported as a leading cause of death in many countries of the world
in the 21st century, especially in less developed countries [1, 2]. Chemotherapy is the
primary choice in cancer treatment, and development of targeted anticancer drugs,
increase of bioavailability and decrease of toxicity are the key topics which are cur-
rently being studied [3]. DNA topoisomerases (Tops) enzymes relax helical supercoil-
ing regenerated during transcription, replication, recombination and chromatin
remodeling is one of the most effective and successful drug target in recent years [4,
5]. Topos enzymes are classified as topoisomerase I (Top-I) and topoisomerase II
(Top-II), Top-II is a more vital nuclear enzyme involved in various DNA processed
and a major target for many drugs currently used than Top-I in cancer chemotherapy
[6, 7]. Thus, many researchers in medicinal chemistry recently focused on developing
Top-II-selective inhibitors as anticancer agents.
The natural lignan podophyllotoxin (PPT, 1, Figure 1) is extracted from the roots
of Podophyllotoxin peltatum and shows cytotoxic activity against a various cancer cells
[8, 9]. However, lack tumor selectivity and high toxicity of PPT has limited its appli-
cation as a drug in cancer chemotherapy [10]. Many derivatives of PPT that are
potent inhibitors have been synthesized and tested as antitumor agents. Interestingly,
etoposide (VP-16, 2, Figure 1) is reported as a clinical Top-II inhibitor which indi-
cates that the substitution at C-4 position on the C-ring of PPT may be responsible
for the Top-II inhibition [11, 12], and the active site of Top-II protein and etoposide
was shown in Figure S1 (Supplementary material). Although VP-16 is currently in
use, many side effects like low blood pressure, hair loss, diarrhea, etc were accompa-
nied with this drug [13]. Hence, development of new drugs with fewer side effects is
highly desirable.
(ꢀ)-Epigallocatechin-3-gallate (EGCG, 3, Figure 1) is the most abundant catechin
(accounting for approximately 50% of total catechins) and has been reported to have
stronger pharmacological activities [14, 15]. Interestingly, it was found that EGCG
was the most potent to inhibit dose-dependently the Top-II catalytic activity [16].
However, the use of EGCG is often hindered by problems such as easy oxidation,
ready degradation in aqueous solutions and the poor intestinal absorbance. In our
Figure 1. Chemical structures of podophyllotixin (PPT, 1), etoposide (VP-16, 2) and (-)-epigallocate-
chin-3-gallate (EGCG, 3).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Scheme 1. Synthesis of (ꢀ)-epigallocatechin-3-gallate-4b-triazolopodophyllotoxin conjugates 7 and
8. Reagents and reaction conditions: (a) NaH, DMF, 0 ꢃC, propargyl bromide, then, reflux, overnight,
30%; (b) MeSO3H, NaI, CH2Cl2, then, H2O-Acetone, BaCO3, rt. 90–92%; (c) NaN3-TFA, CHCl3, 55–60%;
t-
(d) CuSO4ꢂ5H2O, sodium ascorbate, THF, BuOH: H2O (1: 1), 4 h, rt, 80–82%.
previous studies, we found that 1,2,3-trizole-EGCG derivatives showed high anti-
cancer activities and improved the stability of the EGCG scaffold [17, 18].
Dual target inhibitors would likely show high cytotoxicity and more selectivity
than the one. The aim of this research was interested in the development of com-
pounds that can act on the Top-II by conjugating derivatives of the podophyllotoxin
with an analogue of EGCG. In this paper, we have synthesized two (ꢀ)-epigallocate-
chin-3-gallate-4b-triazolopodophyllotoxin conjugates by click reaction, and evaluated
for their cytotoxic activity against a panel of five human cancer cell lines HL-60 (leu-
kemia), SMMC-7721 (hepatoma), A-549 (lung cancer), MCF-7 (breast cancer) and
SW480 (colon cancer) in vitro by MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenylte-
trazolium bromide) assay. Further studies including selectivity of active compounds
against cancer cells and the normal human cell line (BEAS-2B), and molecular dock-
ing were performed.
2. Results and discussion
The synthesis of the novel (ꢀ)-epigallocatechin-3-gallate-4b-triazolopodophyllotoxin
conjugates 7 and 8 was performed according to the reaction pathways illustrated in
Scheme 1. PPT and EGCG were used as starting materials. 4b-Azido-4-deoxypodo-
phyllotoxin (4) and 4b-azido-4-deoxy-40-demethypodophyllotoxin (5) were synthe-
sized according to the literature procedures [19, 20]. (ꢀ)-Epigallocatechin-3-gallate-1-
propyne (3) was prepared by the treatment of EGCG with sodium hydride (NaH)
and propargyl bromide to afford the terminal alkyne (6) in 30% yield. Then, 4b-
azido-podophyllotoxins 4 and 5 were allowed to react with the above terminal alkyne
t-
(6) in the presence of CuSO4ꢂ5H2O, sodium ascorbate in THF and BtOH-H2O (1:1)
at room temperature for 4 h to give (ꢀ)-epigallocatechin-3-gallate-4b-triazolopodo-
phyllotoxin conjugates 7 and 8 in 80–82% yields. All of synthesized compounds were
1
characterized by H-NMR, 13C-NMR, ESI-MS and HRESI-MS analytical data.

4
C.-T. ZI ET AL.
Table 1. In vitro anticancer activity (IC50, lM) of (ꢀ)-epigallocatechin-3-gallate-4b-triazolopodo-
phyllotoxin conjugates 7 and 8a.
IC50 (lM)
Compd.
HL-60
A-549
MCF-7
SW480
SMMC-7721
BEAS-2B
7
8
17.16 0.29
22.76 1.95
<0.064
>40
0.766 0.03
7.31 1.83
2.16 1.02
<0.064
>40
4.72 0.35
13.37 1.15
12.84 1.28
<0.064
>40
14.20 0.93
11.84 1.92
8.96 1.11
<0.064
>40
10.20 1.20
20.67 1.00
14.63 0.06
<0.064
>40
1.93 0.43
26.25 0.16
30.87 0.28
NTb
PPT (1)
EGCG (2)
DDP
NTb
12.86 0.25
aValue are means of three independent experiments;
bNT ¼ not tested.
Table 2. Selectivity of the cytotoxicity of (ꢀ)-epigallocatechin-3-gallate -4b-triazolopodophyllo-
toxin conjugates 7 and 8.
Selectivity index (SIa)
Compd.
HL-60
A-549
MCF-7
SW480
SMMC-7721
7
8
DDP
1.5
1.4
16.8
3.6
14.5
2.7
2.0
2.4
0.9
2.2
3.9
1.3
1.3
2.1
6.7
aSelectivity index (SI) ¼ IC50 of the compound in BEAS-2B cell line/IC50 of the compound in cancer cell line.
The anti-proliferative activity of (ꢀ)-epigallocatechin-3-gallate-4b-triazolopodo-
phyllotoxin conjugates against HL-60, SMMC-7721, A-549, MCF-7 and SW480 cell
lines was screened in vitro by MTT assay. PPT, EGCG and cisplatin were used as
positive controls. The compounds were tested in concentration range of
2.16–30.87 mM and the calculated IC50 values (concentration of drug inhibiting 50%
cell growth) were reported in Table 1. As shown in Table 1, compounds 7 and 8
showed better growth inhibition in five human cancer cells, and 8 exhibited the most
potent anti-proliferative activity against A-549 cells with IC50 value of 2.16 1.02 mM.
To evaluate the degree of selectivity of 7 and 8, the growth inhibitory effect on a
normal human bronchial epithelial cell line (BEAS-2B) was tested (Table 1), and
selectivity index (SI) values of cytotoxic drugs and DDP were measured (Table 2). As
shown in Table 2, compounds 7 and 8 have SI value ranging from 1.3 to 14.5 in all
five cancer cell lines tested. Importantly, compound 8 displayed higher selectivity
than DDP in three of the five cancer cells, and 8 showed the highest selectivity index
value (SI ¼ 14.5) in A-549 cell line. These data suggest that the highest potency com-
pound 8 (IC50 ¼ 2.16 1.02 mmM) is significantly more cytotoxic to the cancer cell
lines as compared with the normal cell line.
To further investigate the potential binding between Top-II and the compounds,
the molecular docking was performed. In the docking results, it was observed that
the vdW þ Hbond þ desolv energy for 7, 8 and etoposide were ꢀ21.16 kcal/mol,
ꢀ21.77 kcal/mol and ꢀ17.75 kcal/mol, respectively. It was also observed that the elec-
trostatic energy for compound 7 is ꢀ0.03 kcal/mol, which is equal with compound 8.
The internal energy and inhibition constant were also calculated and shown in
Table S1 (Supplementary material), while compounds 7 and 8 have the potential to
binding Top-II. Furthermore, the inhibition constants calculated for compounds 7, 8
and etoposide were 4190 nM, 1490 nM and 1220 nM, respectively, which indicated
that compounds 7 and 8 might have considerable Top-II inhibition ability. The

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 2. Binding conformations of the analogs at the active site of Top-II. A. Proposed binding
modes of etoposide (in green), PPT (in yellow), EGCG (in red), 7 (in magental) and 8 (in orange) in
the active site of TOP-II (PDB ID: 3QX3); B and C. Compounds 7 and 8 docked into the binding site
of TOP-II: The protein is shown as cartoon; ligand and the key residues are shown as cartoon sticks
(ligand color: C green, N blue, O red and polar hydrogen); hydrogen bonds are shown as yellow
dotted lines; hydrophobic interactions are shown as black dotted lines; salt bridges were shown as
purple dotted lines.
molecules in complex with Top-II were analyzed (Figure 2). As shown in Figure 2A,
the 3 D binding modes of the compounds PPT, EGCG, 7 and 8 at the etoposide
binding site of Top-II, compounds 7 and 8 could be perfectly docked in the site of
Top-II. As the result presented in Figure 2B and C, it000can be easily seen that two
00
hydrogen bonds [C5 –OH···NH2 (ARG-503)] and [C13 –CH2O···NH2 (GLN-778)],
and the E ring of PPT lead to hydrophobic interactions with the aromatic residue
GLN-778 in 7/T0o000p-II docking complex, while four hydrogen bonds [C7–OH···NH2
0000
(GLN-778)], [C3 -OCH3· ·· NH2 (GLN-778)], [C3 –OCH3· · ·NH2 (ARG-802)], and
0000
[C3 –OCH3···NH (ARG-802)], the E ring of PPT leads to hydrophobic interactions
with the aromatic residue GLN-778, and one salt bridge [C ¼ O···NH2-C(NH)¼NH
(ARG-503)] are found in 8/Top-II docking complex, respectively. The presence of
more hydrogen bonds and salt bridge in compound 8 seems to be the key factor for
its high activity.
3. Experimental
3.1. General experimental procedures
(ꢀ)-Epigallocatechin-3-gallate and podophyllotoxin were purchased from Chengdu
Proifa Technology Development Co., Ltd (Chengdu, China); 3-(4,5-dimethyl-thiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St.
Louis, MO, USA). Dichloromethane and acetonitrile were distilled over calcium
hydride. All reagents were commercially available and used without further purifica-
tion unless indicated otherwise. Melting points were measured by an X-4 melting
point apparatus and are uncorrected. MS data were obtained in the ESI mode on API
Qstar Pulsar instrument; HRMS data were obtained in the ESI mode on LCMS-IT-
1
TOF (Shimadzu, Kyoto, Japan); H-NMR and 13C-NMR spectra were recorded on
Bruker AVANCE III 500 MHz, or 600 MHz (Bruker BioSpin GmbH, Rheinstetten,

6
C.-T. ZI ET AL.
Germany) instruments, using tetramethylsilane (TMS) as an internal standard: chem-
ical shifts (d) are given in ppm, coupling constants (J) in Hz. Column chromatog-
raphy (CC) was performed over silica gel (200–300 mesh; Qingdao Makall Group
CO., LTD; Qingdao; China). All reactions were monitored by thin-layer chromatog-
raphy (TLC), which was visualized by ultraviolet light (254 nm) and sprayed with 5%
H2SO4 in EtOH, followed by heating.
3.2. Synthesis of (2)-epigallocatechin-3-gallate-1-propyne (6)
(-)-Epigallocatechin-3-gallate (458 mg, 1.0 mmol) was dissolved in N,N-dimethylfor-
mamide (DMF) (5 ml), then sodium hydride (60 mg, 1.5 mmol) was added at 0 ꢃC
under nitrogen and the mixture was stirred at room temperature for 0.5 h. Propargyl
bromide (0.1 ml, 1 mmol) was quickly added and the reaction was stirred at 80 ꢃC for
12 h. After cooling, the mixture was concentrated under vacuum and the resulting
residue was purified by silica gel chromatography with CHCl3/CH3OH, (9:1!4:1) to
00
1
afford the product 6 (137 mg, 30%). H-NMR (CD3OD, 500 MHz) d 6.90 (s, 2H, C2 -
00
0
0
H, C6 -H), 6.50 (s, 2H, C2 -H, C6 -H), 5.96 (s, 2H, C6-H, C8-H), 5.53 (brs, 1H, C3-H),
4.97 (s, 1H, C2-H), 4.78 (d, 2H, J ¼ 2.4 Hz, OCH2), 3.29 (t, 1H, J ¼ 1.6 Hz, CꢄCH),
2.96 (dd, 1H, J ¼ 4.6, 12.0 Hz, C4-Ha), 2.85 (dd, 1H, J ¼ 4.6, 12.0 Hz, C4-Hb); 13C-
NMR (CD3OD, 125 MHz) d 167.0 (C ¼ O), 157.9 (C-7), 157.8 (C-9), 157.2 (C-5),
151.9 (C-30, C-50), 146.7 (C-300, C-500), 138.4 (C-400), 133.8 (C-40), 130.7 (C-10), 127.1
(C-100), 110.1 (C-200, C-600), 106.8 (C-20, C-60), 99.3 (C-10), 96.5 (C-8), 95.9 (C-6), 80.4
(C-2), 79.5 (CꢄCH), 78.5 (CꢄCH), 76.7 (C-3), 60.0 (OCH2), 26.8 (C-4); ESIMS: m/z
495 [M – H]–.
3.3. General procedure for the synthesis of (2)-epigallocatechin-3-gallate -4b-
triazolopodophyllotoxin conjugates (7 and 8)
To a solution of 4b-azido-podophyllotoxins 4/5 (0.1 mmol) and (ꢀ)-epigallocatechin-
t-
3-gallate-1-propyne (6) (0.1 mmol) in THF (1.0 ml) and BtOH-H2O (1.0 ml, 1:1) at
room temperature, copper (II) sulfate pentahydrate (0.1 mmol) and sodium ascorbate
(0.05 mmol) were added and the reaction mixture was stirred for 4 h. Removal of the
solvents gave a residue which was chromatographed on silica gel (CHCl3:CH3OH ¼
9:1) to afford the product.
3.3.1.
ether (7)
(ꢀ)-Epigallocatechin-3-gallate-[4b-(1,2,3-triazol-1-yl-4-deoxypodophyllotoxin)]
1
White amorphous powder; yield 82%;0 m.p. 192–194 ꢃC; H-NMR (CD3OD, 500 MHz)
0
000
000
d 7.47 (s, 1H, C14-H), 6.86 (s, 2H, C2 , C6 -H), 6.59 (s, 1H, C5 -H), 6.57 (s, 1H, C8 -
0000
0000
0
0
H), 6.50 (s, 2H, C2 , C6 -H), 6.40 (s, 2H, C2 , C6 -H), 6.38-6.39 (m, 2H, C6-H, C8-
000
000
H), 6.18 (d, 1H, J ¼ 4.9 Hz, C4 -H), 5.90-5.97 (m, 4H, C3-H, C2-H, C13 -CH2), 5.22-
000
000
5.24 (m, 2H, C12-CH2), 4.76 (d, 1H, J ¼ 4.8 Hz, C1 -H), 4.18-4.20 (m, 2H, C11 -CH2),
000
0
0
000
3.74 (s, 3H, C4 -OCH3), 3.72 (s, 6H, C3 , C5 -OCH3), 2.98-3.01 (m, 1H, C3 -H), 2.95-
000
2.96 (m, 1H, C4-Ha), 2.85-2.87 (m, 2H, C2 -H, C4-Hb); 13C-NMR (CD3OD,
125 MHz) d 175.8 (C-12000), 166.9 (C-11), 164.9 (C-5), 157.9 (C-7), 157.8 (C-9), 153.9

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
(C-30000, 50000), 151.9 (C-3000, 5000), 150.6 (C-6000), 149.3 (C-7000), 146.7 (C-300, 500), 142.3
(C-10300), 136.7 (C-400), 134.7 (C-10000), 131.3 (C-40000), 130.7 (C-40), 127.3 (C-10), 126.6
(C-9 ), 126.5 (C-10000), 119.4 (C-14), 115.9 (C-5000), 115.3 (C-8000), 111.1 (C-100), 110.2
(C-200, 600), 109.5 (C-20, 60), 106.8 (C-200, 600), 103.3 (C-13000), 100.1 (C-10), 96.5 (C-8),
95.9 (C-6), 79.9 (C-12), 78.5 (C-2), 70.4 (C-4000), 68.9 (C-3000), 67.5 (C-11), 61.1 (40000-
OCH3), 56.6 (30000, 50000-OCH3), 44.8 (C-1000), 42.4 (C-2000), 38.6 (C-3000), 29.3 (C-4);
ESIMS: m/z 958 [M þ Na]þ.
3.3.2. (ꢀ)-Epigallocatechin-3-gallate-[4b-(1,2,3-triazol-1-yl)-4-deoxy-4’-demethylpo-
dophyllotoxin] ether (8)
1
White amorphous powder; yield 80%; m.p. 200–202 ꢃC; H-NMR (CD3OD, 500 MHz)
000
0
0
d 7.47 (s, 1H, C14-H), 6.96 (s, 1H, C5 -H), 6.86 (s, 2H, C2 , C6 -H), 6.74-6.78 (s, 1H,
000
0000
0000
0
0
C8 -H), 6.58 (s, 2H, C2 , C6 -H), 6.50 (s, 2H, C2 , C6 -H), 6.37 (s, 2H, C6-H, C8-H),
000
000
6.18 (d, 1H, J ¼ 4.9 Hz, C4 -H) , 5.90-5.97 (m, 2H, C3-H, C2-H, C13 -CH2), 5.22-5.24
000
000
(m, 2H, C12-CH2), 4.76 (d, 1H, J ¼ 4.8 Hz, C1 -H), 4.17-4.20 (m, 4H, C11 -CH2), 3.72
0
0
000
(s, 6H, C3 , C5 -OCH3), 2.98-3.00 (m, 1H, C3 -H), 2.96-2.98 (m, 1H, C4-Ha), 2.84-
000
2.86 (m, 1H, C2 -H), 2.85 (dd, 1H, J ¼ 4.6, 12.0 Hz, C4-Hb); 13C-NMR (CD3OD,
125 MHz) d 176.1 (C-12000), 165.0 (C-11), 162.1 (C-5), 157.9 (C-7), 157.4 (C-9), 151.9
(C-3000, 5000), 148.7 (C-30, 50), 146.7 (C-300, 500), 146.1 (C-6000, C-7000), 144.5 (C-13), 142.0
(C-400), 132.3 (C-10000), 131.3 (C-40000), 130.8 (C-40), 127.3 (C-10), 126.6 (C-9000), 126.5
(C-10000), 119.4 (C-14), 115.9 (C-5000), 115.3 (C-8000), 111.1 (C-100), 110.2 (C-200, 600),
109.4 (C-20, 60), 106.8 (C-20000, 60000), 103.2 (C-13000), 100.1 (C-10), 96.4 (C-8), 95.9 (C-
6), 79.9 (C-12), 78.5 (C-2), 70.4 (C-4000), 68.9 (C-3), 67.5 (C-11000), 56.8 (30000, 50000-
OCH3), 44.7 (C-1000), 42.6 (C-2000), 38.6 (C-3000), 29.3 (C-4); ESIMS: m/z
944 [M þ Na]þ.
3.4. Cytotoxicity assay
The cytotoxicity of all compounds against HL-60, A-549, MCF-7, SW480, and
SMMC-7721 cell lines was measured using MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-
diphenyltetrazolium bromide] method [21]. Adherent cells (100 ll) were seeded into
each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug
addition, while suspended cells were seeded just before drug addition, both with an
initial density of 1 ꢅ 105 cells/ml in 100 ll of medium. Each tumor cell line was
exposed to the test compound at various concentrations in triplicate for 48 h. After
the incubation, MTT (100 lg) was added to each well, and the incubation continued
for 4 h at 37 ꢃC. The cells were lysed with SDS (200 ll) after removal of 100 ll of
medium. The optical density of lysate was measured at 595 nm in a 96-well microtiter
plate reader (Bio-Rad 680). IC50 values were calculated by Reed and
Muench’s method.
3.5. Molecular modeling
The crystal structure of Top-II (code ID: 3QX3) [22] was obtained in Protein Data
Bank. PyMol 2.3 software was used to the preparation of ligand and receptor.

8
C.-T. ZI ET AL.
Autodock Tools v1.56 was used for grid and docking according to the literature [23].
Docking parameters were set as the defaults values, except “The Number of GA
Runs” was set to 50 and maximum number of evals (medium) was set to 5,000,000
on AutoGrid v4.2.6 and AutoDock v4.2.6. Docking conformations were classified into
different clusters by binding energy, and the cluster with the lowest binding energy
was selected. In the selected cluster, conformations with the lowest binding energy
and RMSD (<2.0 Å) were finally chosen to analyze the receptor-ligand interaction.
Etoposide was chosen as control ligand.
Acknowledgements
The authors are grateful to the staffs of the analytical group at the State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese
Academy of Sciences, for measuring the spectral data.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by grants from National Nature Science Foundation of
China (21602196, 31960075); the Science and Technology Project of Yunnan province
(2017ZF003, 2017FD084, and 2017FG001-046) and Yunnan Agricultural University Natural
Science Foundation for Young Scientists and Postgraduates (2015ZR08).
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