Novel perbutyrylated glucose derivatives of (–)-epigallocatechin-3-gallate inhibit cancer cells proliferation by decreasing phosphorylation of the egfr: Synthesis, cytotoxicity, and molecular docking

Article abstract of DOI:10.3390/molecules26144361

Lung cancer is one of the most commonly occurring cancer mortality worldwide. The epidermal growth factor receptor (EGFR) plays an important role in cellular functions and has become the new promising target. Natural products and their derivatives with various structures, unique biological activities, and specific selectivity have served as lead compounds for EGFR. D-glucose and EGCG were used as starting materials. A series of glucoside derivatives of EGCG (7–12) were synthesized and evaluated for their in vitro anticancer activity against five human cancer cell lines, including HL-60, SMMC-7721, A-549, MCF-7, and SW480. In addition, we investigated the structure-activity relationship and physicochemical property–activity relationship of EGCG derivatives. Compounds 11 and 12 showed better growth inhibition than others in four cancer cell lines (HL-60, SMMC-7721, A-549, and MCF), with IC₅₀ values in the range of 22.90–37.87 μM. Compounds 11 and 12 decreased phosphorylation of EGFR and downstream signaling protein, which also have more hydrophobic interactions than EGCG by docking study. The most active compounds 11 and 12, both having perbutyrylated glucose residue, we found that perbutyrylation of the glucose residue leads to increased cytotoxic activity and suggested that their potential as anticancer agents for further development.

Full text of DOI:10.3390/molecules26144361

molecules
Article
Novel Perbutyrylated Glucose Derivatives of
(–)-Epigallocatechin-3-Gallate Inhibit Cancer Cells Proliferation
by Decreasing Phosphorylation of the EGFR: Synthesis,
Cytotoxicity, and Molecular Docking
Ya Wang 1,2,†, Xiao-Jing Shen 3,†, Fa-Wu Su 4,† , Yin-Rong Xie 1,2, Li-Xia Wang 1,2, Ning Zhang 1,2, Yi-Long Wu 1,2
,
Yun Niu 1,2, Dong-Ying Zhang 1, Cheng-Ting Zi 1,
* *
, Xuan-Jun Wang 1,* and Jun Sheng 1,2,
1
Key Laboratory of Pu-er Tea Science, Ministry of Education, College of Science, Yunnan Agricultural
University, Kunming 650201, China; wangya9188@126.com (Y.W.); xyr351527080@163.com (Y.-R.X.);
wanglixia0315@163.com (L.-X.W.); zn1193594574@163.com (N.Z.); ynau_wuyl@126.com (Y.-L.W.);
Niuyun0429@163.com (Y.N.); Zhangdongying365@163.com (D.-Y.Z.)
College of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, China
Party Committee of Organ, Yunnan Agricultural University, Kunming 650201, China; sxj243@sina.com
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural
University, Kunming 650201, China; su_faw@126.com
2
3
4
*
Correspondence: 2015055@ynau.edu.cn (C.-T.Z.); Jwang@ynau.edu.cn (X.-J.W.); shengj@ynau.edu.cn (J.S.);
Tel.: +86-159-1257-9655 (C.-T.Z. & X.-J.W. & J.S.); Fax: +86-871-6522-3261 (C.-T.Z. & X.-J.W. & J.S.)
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
†
Citation: Wang, Y.; Shen, X.-J.; Su,
F.-W.; Xie, Y.-R.; Wang, L.-X.; Zhang,
N.; Wu, Y.-L.; Niu, Y.; Zhang, D.-Y.; Zi,
C.-T.; et al. Novel Perbutyrylated
Glucose Derivatives of
Abstract: Lung cancer is one of the most commonly occurring cancer mortality worldwide. The
epidermal growth factor receptor (EGFR) plays an important role in cellular functions and has
become the new promising target. Natural products and their derivatives with various structures,
unique biological activities, and specific selectivity have served as lead compounds for EGFR. D-
glucose and EGCG were used as starting materials. A series of glucoside derivatives of EGCG
(–)-Epigallocatechin-3-Gallate Inhibit
Cancer Cells Proliferation by
Decreasing Phosphorylation of the
EGFR: Synthesis, Cytotoxicity, and
Molecular Docking. Molecules 2021,
26, 4361. https://doi.org/10.3390/
molecules26144361
(7–12) were synthesized and evaluated for their in vitro anticancer activity against five human cancer
cell lines, including HL-60, SMMC-7721, A-549, MCF-7, and SW480. In addition, we investigated
the structure-activity relationship and physicochemical property–activity relationship of EGCG
derivatives. Compounds 11 and 12 showed better growth inhibition than others in four cancer
cell lines (HL-60, SMMC-7721, A-549, and MCF), with IC50 values in the range of 22.90–37.87 µM.
Compounds 11 and 12 decreased phosphorylation of EGFR and downstream signaling protein, which
also have more hydrophobic interactions than EGCG by docking study. The most active compounds
11 and 12, both having perbutyrylated glucose residue, we found that perbutyrylation of the glucose
residue leads to increased cytotoxic activity and suggested that their potential as anticancer agents
for further development.
Academic Editor: Ricardo Calhelha
Received: 5 May 2021
Accepted: 14 July 2021
Published: 19 July 2021
Keywords: EGCG; synthesis; cytotoxicity; EGFR; molecular docking
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Lung cancer is one of the most commonly occurring cancer mortality worldwide,
accounting for 20% of all cancer-related deaths [1]. Approximately 75–85% of lung cancers
are non-small-cell lung cancer (NSCLC), including lung adenocarcinoma, squamous cell
carcinoma (SCC), and large cell carcinoma (LCC) histological subtypes, and are typically
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
diagnosed at advanced stages [
mutations has led to the development of molecular targeted therapies for patients with
NSCLC [ ]. The epidermal growth factor receptor (EGFR) plays an important role in cellular
functions, and approximately 50% of lung cancer patients possess EGFR overactivated [ ].
Thus, EGFR tyrosine kinase has become an attractive therapeutic target for NSCLC [6–8].
2,3]. Recently, the identification of key oncogenic-driver
4
5
Molecules 2021, 26, 4361. https://doi.org/10.3390/molecules26144361
https://www.mdpi.com/journal/molecules

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EGFR tyrosine kinase inhibitors (TKI), such as gefitinib and erlotinib are used in
clinical therapy to treat NSCLC [
led to resistance to these EGFR TKIs [13], and new drugs have been developed, such
as afatinib [10 14] and osimertinib [15]. Natural products and their derivatives with
9–12]. However, the T790M mutation of EGFR has
,
various structures, unique biological activities, and specific selectivity have served as lead
compounds for EGFR. For example, aeroplysinin selectively inhibits EGFR [16]; shikonin
preferentially inhibits cell proliferation by inhibiting EGFR-signaling [17].
Green tea (Gamellia sinensis leaves) is an extremely popular drink in the world, and
many studies show the positive effects of green tea on cancer, including lung cancer [18,19].
It is generally agreed that tea catechins were first isolated from green tea, making it a
promising chemopreventive agent [20]. The major catechins in green tea leaves are (-)-
epicatechin (EC,
(-)-epigallocatechin-3-gallate (EGCG,
most studied due to its abundance, constituting 50–80% of total catechins content in green
tea [21], and has been reported to have stronger physiological activities than others [22 24].
1
), (-)-epicatechin-3-gallate (ECG,
2), (-)-epigallocatechin (EGC, 3), and
4
) (Figure 1). Among these catechins, EGCG is the
–
Several studies have indicated that treatment with EGCG inhibits tumor incidence such
as skin, lung, liver, breast, prostate, and stomach [25]. There is considerable evidence that
EGCG inhibits tumorigenesis, signal transduction pathway, cell invasion, angiogenesis, and
metastasis [26
of EGFR and human epidermal growth factor receptor 2 (HER2) in human lung cancer
cells [31 32], and EGCG inhibits the tyrosine kinase activity of EGFR in human A431
–30]. Notably, previous studies reported that EGCG inhibits the activation
,
epidermoid carcinoma cells [33].
Figure 1. Structures of (-)-epicatechin (EC, 1), (-)-epicatechin-3-gallate (ECG, 2), (-)-epigallocatechin
(EGC, 3), and (-)-epigallocatechin-3-gallate (EGCG, 4).
However, the use of EGCG often has limitations such as easy oxidation [34], low
bioavailability [35], and easy hydrolyses by bacterial and possibly host esterases [36]; thus,
attempts to use EGCG in the treatment of human neoplasia have been mostly unsuccessful.
To obtain more potent analogs and overcome these problems, many EGCG derivatives have
been synthesized, including methyl-protected EGCG [37], acetyl-protected EGCG, [38],
and EGCG monoester derivatives [39], and most of them exhibited more potent activity
than EGCG. Recently, the glycoconjugates of small molecule anticancer drugs have be-
come an attractive strategy in order to improve drug efficacy and pharmacokinetics, in
addition to reducing side effects [40–42]. In our previous study, we thus reported that
the synthesized glucosylated EGCG derivatives exhibited enhanced cytotoxicity and were
more stable [5,43].

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Since butyrate is a well-known histone deacetylase (HDAC) inhibitor, and its anti-
cancer effect shows promising therapeutic potential [44], acetyl-protected EGCG analogs
exhibited more activity than EGCG [37]. In this study, we synthesized a series of glucoside
derivatives of EGCG (7–12) and evaluated for their in vitro anticancer activity against five
human cancer cell lines, including HL-60 (leukemia), SMMC-7721 (hepatoma), A-549 (lung
cancer), MCF-7 (breast cancer), and SW480 (colon cancer). In addition, Western blotting
and molecular docking analyses of these compounds were studied and correlated with
their anticancer activity.
2. Results and Discussion
2.1. Chemistry
The synthesis of the (-)-epigallocatechin-3-gallate glucoside derivatives
performed according to the reaction pathways illustrated in Schemes 1 and 2. D-glucose
and EGCG were used as starting materials. 2,3,4,6-Tetra-O-butyryl- -D-glucopyranosyl
bromide was prepared using the method reported in the literature [45]. EGCG ( ) was
allowed to react with the above compound in the presence of K2CO3 in acetone at
55 ◦C for 12 h to obtain peracetyl EGCG glucoside derivatives
and in 25–30% yields
Scheme 1). Then, compounds and
were treated with KOH solution in CH3OH at 0 ◦C
for 72 h to yield EGCG glucoside derivatives and 10 in 52–55% yields [27]. Finally, the
and 10 was buturylated with butyric anhydride in pyridine
7–12 was
α
6
4
6
7
8
(
7
8
9
free glucose o◦f compounds
9
solution at 0 C for 12 h to yield perbutyryl EGCG glucoside derivatives 11 and 12 in
80–82% yields (Scheme 2).
Scheme 1. Synthesis of compounds
CH2Cl2, 0 C, 8 h, 70%; (c) K2CO3, acetone, 55 C, 12 h, 25–30%.
7
and 8. R◦eagents and conditions: (a) Ac2O, AcONa, 100 ◦C, 20 min, ~99%; (b) HBr,
◦
Scheme 2. Synthesis of compounds
72 h, 52–55%; (b) (CH3CH2CH2CO)2O, pyridine, 0 C, 12 h, 80–82%.
9
–
12. Reagents and conditions: (a) KOH solution (0.5 M in CH3OH), CH3OH, 0 ◦C,
◦
1
All the synthesized compounds were characterized by H-NMR, 13C-NMR, elec-
trospray ionization mass spectrometry (ESI-MS), and high-resolution mass spectrometry
(HRESI-MS). In the 1H-NMR spectra, the formation of the D-glucose residue was confirmed
by the resonance of C1000-H/C10000-H signal (
δ 4.54–4.97 ppm) with the coupling constant
0000
of the anomeric proton of D-glucose residue (J1000/J2 = 7.6 Hz–7.8 Hz) (Table S1). In our
early study, the coupling constant of the anomeric proton of the glucose residue (J1−2) is
typically <4.0 Hz for the α–linkage and >7.6 Hz for the β–linkage [46].

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The chemical structures of compounds were further confirmed by 2D-NMR spectra
data. Based on the HMBC data (Figure 2), the strong coupling occurred between C10000-H (
δ
4.97 ppm) and C-4” (137.3 ppm) of the D ring from the HMBC of compound
coupling occurred between C10000-H (
4.54 ppm) and C-4” ( 138.5 ppm) of the D ring and
also between C1000-H (
4.67 ppm) of the other glucosyl residue and C-4 (
B ring from the HMBC of compound
. The chemical shift for the proton at C4-H exhibited
coupling with C-2, C-3; C20-H, C6”-H occurred with C-2 and C2”-H, C6”-H occurred with
C-11; and C2-H occurred with C-11 in both compounds
and . These results indicated
that the C-4”) had attached to the EGCG scaffold
-peracetylated glucoside (H-10000-H
in compound , and two C-4”)
-peracetylated glucosides (H-1000-H C-40 and H-10000-H
7. Similarly,
δ
δ
0
δ
δ 137.9 ppm) of the
8
7
8
β
→
7
β
→
→
had attached in compound 8.
Figure 2. HMBC correlations in compounds 7 and 8.
2.2. Evaluation of Anticancer Activity
All EGCG derivatives
cancer cell lines, including HL-60 (leukemia), SMMC-7721 (hepatoma), A-549 (lung cancer),
MCF-7 (breast cancer), and SW480 (colon cancer). EGCG ( ) and cisplatin were taken
7–12 were evaluated for their cytotoxicity against five human
4
as positive control compounds. The screening procedure was based on the standard 3-
(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method [47]. Their
activities were expressed by the IC50 value (concentration of drug inhibiting 50% cell
growth), and the data are presented in Table 1.
Table 1. In vitro anticancer activity (IC50
7–12.
,
µM) of glucoside derivatives (-)-epigallocatechin-3-gallate
IC50 (µM)
Com-
pounds
SMMC-
7721
HL-60
A-549
MCF-7
SW480
BEAS-2B
4
7
8
9
10
11
12
>40.0
29.5 ± 0.10
28.2 ± 0.06
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
NT
NT
NT
NT
NT
>40.0
>40.0
38.5 ± 0.08
34.4 ± 0.43 36.1 ± 0.11
39.6 ± 0.02
>40.0
>40.0
>40.0
>40.0
28.4 ± 1.00 37.9 ± 0.12 32.1 ± 0.10 28.7 ± 0.38
22.9 ± 0.23 30.4 ± 1.00 24.4 ± 0.31 26.4 ± 0.12 39.7 ± 0.01
0.8 ± 0.03 4.7 ± 0.35 1.9 ± 0.43 14.2 + 0.93
Cisplatin
NT: not tested.
10.2 ± 1.20 12.9 ± 0.25
As shown in Table 1, the majority of glucoside derivatives of EGCG display weak
cytotoxicity against all five cancer cell lines, and most of them show higher potency than the
control EGCG. Among the glucoside derivatives of EGCG, compounds 11 and 12 showed
better growth inhibition than others in four cancer cell lines (HL-60, SMMC-7721, A-549,
and MCF), with IC50 values in the range of 22.90–37.87
µM. Interestingly, all glucoside
derivatives of EGCG showed good cytotoxicity against A-549 cells except compound 10

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(having IC50 > 40
µM). Previously, we reported that the cytotoxic activity of glucosylated
EGCG derivatives with a free glucose residue mostly shows weak activity against breast
cancer (MCF and MDA-MB-231 cells) [27]. However, our findings indicate that EGCG
derivatives with a perbutyrylated glucose residue (11 and 12) lead to increased activity
rather than a free glucose residue (11 vs. 9 and 12 vs. 10).
Cancer chemotherapy is often associated with low/non-selectivity of cancer drugs,
which attack cancer cells and normal cells, leading to serious side effects. EGCG derivatives
11 and 12 were tested for their growth inhibitory effects on normal human bronchial
epithelial cells (BEAS-2B) (Table 1). The IC50 values of compounds 11 and 12 (all having
IC50 > 40 µM), indicated that glucoside derivatives of EGCG showed moderated selectivity
toward cancer cells.
2.3. EGCG Derivatives Inhibited Phosphorylation of EGFR and Downstream Signaling Protein in
A-549 Cells
To investigate whether the effects of EGCG derivatives 11 and 12 might involve EGFR
signaling, we assessed the expression of several key regulators that function within the
EGFR signaling pathway. The results showed that treatment with EGCG slightly exhibited
P-EGFR, P-AKT, and P-Erk (Figure 3A). Compounds 11 and 12 further exhibited the
phosphorylation of EGFR and downstream signaling protein, while the total protein level
of EGFR remained unchanged (Figure 3B,C). As shown in Figure 3D,E, it can be observed
that EGCG, 11 and 12 can reduce the expression of phosphorylated EGFR, ERK, and AKT
proteins. In summary, EGFR signaling may play a significant role in the effects of EGCG
derivatives 11 and 12 in A-549 cells.
2.4. Molecular Docking Analysis
To further investigate the potential binding between EGFR and the compounds,
molecular docking was performed. The binding modes of compounds 4, 11, and 12 at
the ATP-binding pocket of EGFR (PDB code: 2ITY) were shown in Figure 4. As shown in
Figure 4, compounds 11 and 12 could be docked into the active site of EGFR, the butyryl
group occupies the ATP-binding pocket. The detailed docking results were presented
in Table S2. It was observed that the binding energies between EGFR and glucoside
derivatives of EGCG 11 and 12 are weak, compared with EGCG. The docking energy
for compounds
respectively. The complex structures for compounds 11 and 12 were presented in Figure 5
The key residues were labeled and the important molecular interaction including the
hydrogen bonds (black dotted lines), hydrophobic interactions (red dotted lines), -stacking
4,
11, and 12 were
−
5.18 kcal/mol, 0.47 kcal/mol, and 1.81 kcal/mol,
.
π
(gray dotted lines), and salt bridges (yellow dotted lines) were summarized in Table 2.
As shown in Figure 5, the molecules in complex with EGFR were analyzed in Figure 5A,B
.
It was observed that there are three hydrophobic interactions between compound 11 and
three residues of EGFR (PHE723, ALA743, LEU844), five hydrogen bonds between 11 and
three residues of EGFR (LEU718, MET793, ASN842), and two salt bridges between 11 and
two residues of EGFR (LYS745, ARG841). There are seven hydrophobic interactions be-
tween compound 12 and seven residues of EGFR (ALA722, PHE723, VAL726, LYS745,
MET793, ARG841, and VAL876), three hydrogen bonds between 12 and three residues of
EGFR (GLU758, ASP855, and VAL876), one π-stacking and a salt bridge between 12 and the
residue of EGFR (LYS745 and ARG841, respectively). Similarly, EGCG was docked into the
EGFR are shown in Figure S1, which result in ten hydrogen bonds, one hydrophobic inter-
action, one
π-stacking and one salt bridge, respectively. The presence of more hydrophobic
interactions in compounds 11 and 12 seems the key factor for their high activity.

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Figure 3. Effects of EGCG and its derivatives 11 and 12 on the EGCG signaling pathway in A-549 cells: (
with various concentrations of EGCG (20, 40, 60 M); ( ) A-549 cells treated with various concentrations of compound 11
(20, 40, 60 M); ( ) A-549 cells treated with various concentrations of compound 12 (20, 40, 60 M); ( ) EGCG, compound
11 and compound 12 P-EGFR quantitative analysis chart; ( ) EGCG, compound 11, and compound 12 P-AKT quantitative
analysis chart; ( ) EGCG, compound 11, and compound 12 P-ERK quantitative analysis chart. The expression levels of the
proteins were determined by Western blotting after 24 h with -tubulin as the loading control. Data represent the average of
three independent experiments (mean ± SEM). ** p < 0.005, *** p < 0.001 vs. the control.
A) A-549 cells treated
µ
B
µ
C
µ
D
E
F
β
Figure 4. Proposed binding modes of the small molecules at the site of EGFR: binding modes of
EGCG (in red), 11 (in blue), and 12 (in yellow) in the active site of EGFR (PDB code: 2ITY) by overview
and zoom-in of ATP-binding pocket.

Molecules 2021, 26, 4361
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Figure 5. The details of the binding between EGFR and compounds 11
(A) and 12 (B): the protein is shown as lines; ligand
and the key residues are shown as sticks (ligand colors: C cyan, N blue, O red, and H green); hydrogen bonds are shown as
black dotted lines; hydrophobic interactions are shown as red dotted lines;
bridges were shown as yellow dotted lines.
π-stacking is shown as gray dotted lines; salt
Table 2. The residues for molecular interaction between EGFR and compounds 4, 11 and 12.
Hydrogen
Bonds
Molecules
Hydrophobic Interactions
π-Stacking
—
Salt Bridges
LEU718,
MET793 ASN842
LYS745,
ARG841
11
12
PHE723, ALA743, LEU844
ALA722, PHE723, VAL726,
LYS745, MET793, ARG841,
VAL876
GLU758,
ASP855, VAL876
LYS745
ARG841
3. Experimental
3.1. Materials and Methods
D-glucose and n-butyric anhydride were purchased from Aladdin Chemical Co.,
Ltd. (Guangzhou, China); (-)-epigallocatechin-3-gallate was obtained from Chengdu
Proifa Technology Development Co., Ltd. (Chengdu, China); boron trifluoride etherate
was obtained from J&K Chemical Technology Co., Ltd. (Beijing China); 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich
(St. Louis, MO, USA). All reagents were commercially available and used without further
purification unless indicated otherwise. The melting points were measured by an X-4
melting point apparatus and were uncorrected. Optical rotations were obtained with a
Jasco P-1020 Automatic Digital Polariscope MS data were obtained in the ESI mode on
API Qstar Pulsar instrument; HRMS data were obtained in the ESI mode on LCMS-IT-TOF
(Shimadzu, Kyoto, Japan); 1H-NMR and 13C-NMR spectra were recorded on Bruker DRX-
500 (Bruker BioSpin GmbH, Rheinstetten, Germany) instruments, using tetramethylsilane
(TMS) as an internal standard: chemical shifts (δ) are given in ppm, coupling constants (J) in
Hz, the solvent signals were used as references (CD3OD: δC = 49.0 ppm; residual CH3OH
in CD3OD: δH = 4.78 ppm). Column chromatography (CC): silica gel (200–300 mesh;
Qingdao Makall Group CO., LTD; Qingdao; China). All reaction was monitored using
thin-layer chromatography (TLC) on silica gel plates, which was visualized by ultraviolet
light (254 nm) and/or 10% phosphomolybdic acid/EtOH. The human cancer cell lines
(HL-60, SMMC-7721, A-549, MCF-7, and SW480) were obtained from a Shanghai cell bank
in China.

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3.2. Chemistry
General procedure for the synthesis of 2,3,4,6-tetra-O-acetyl-
epigallocatechin-3-gallates (7 and 8)
β-D-glucopyranosyl (-)-
A solution of EGCG (1.8 g, 4 mmol) was dissolved in acetone (5 mL), and potassium
carbonate (K2CO3, 1.1 g, 8 mmol) was added with stirring 30 min, and 2,3,4,6-tetra-O-acetyl-
◦
α
-D-glucopyranosyl bromide
6
(1.6 g, 4 mmol) was added with heating and stirring at 55 C
for 12 h. The mixture was cooled and filtered, the filtrate was concentrated under vacuum,
and the residue was purified by column chromatography in silica gel (CHCl3/CH3OH,
15:1 → 9:1) to afford the major product.
0000 0000 0000 0000
[4”-O-(2 ,3 ,4 ,6 -tetra-O-acetyl-
β
-D-glucopyranosyl)]-(-)-epigallocatechin-3-gallate (
7).
White powder; yield 25%; mp. 100 ◦C to 101 ◦C; 1H-NMR (CD3OD, 500 MHz)
δ
6.91
(s, 2H, C2”, C6”-H), 6.49 (s, 2H, C20, C60-H), 5.95 (s, 2H, C6-H, C8-H), 5.54 (brs, 1H, C2-H),
5.34 (t, 1H, J = 9.0 Hz), 5.24–5.22 (m, 1H, C3-H), 5.21 (m, 1H), 5.09 (t, 1H, J = 9.0 Hz), 4.97
(d, 1H, J = 9.0 Hz, C10000-H), 4.28 (dd, 1H, J = 5.4 Hz, 12.4 Hz), 4.04 (dd, 1H, J = 2.4 Hz,
12.4 Hz), 3.88–3.84 (m, 1H), 2.98 (dd, 1H, J = 4.6 Hz, 17.6 Hz, C4-Ha), 2.86–2.83 (m, 1H,
C4-Hb), 2.04–1.99 (m, 12H, 4
×
OCH3); 13C-NMR (CD3OD, 125 MHz)0δ 172.5, 171.6, 171.6,
0
171.3, 166.8 (C=O), 157.9 (C-5), 157.8 (C-9), 157.2 (C-7), 151.7 (C-3 , C-5 ), 146.7 (C-3”, C-5”),
137.3 (C-4”), 133.7 (C-40), 130.7 (C-10), 128.2 (C-1”), 110.1 (C-2”, C-6”), 106.7 (C-20, C-60),
102.7 (C-10000), 99.2 (C-10), 96.5 (C-6), 95.8 (C-8), 78.4 (C-2), 74.1, 73.2, 72.9, 70.4 (C-3), 69.7,
62.9 (C-60000), 26.8 (C-4), 20.8 (OCH3), 20.6 (OCH3), 20.5 (OCH3), 20.5 (OCH3); ESIMS: m/z
811 [M + Na]+.
[40-O-(2000,3000,4000,6000-tetra-O-acetyl- -D-glucopyranosyl)-4”-O-(20000,30000,40000,60000-tetra-O-
β
acetyl-β-D-glucopyranosyl)]-(-)-epigallocatechin-3-gallate (8)
White powder; yield 30%; mp. 108 ◦C to 110 ◦C; 1H-NMR (CD3OD, 600 MHz)
δ 6.92
(s, 2H, C2”, C6”-H), 6.58 (s, 2H, C20, C60-H), 5.98 (s, 1H, C3-H), 5.96 (s, 2H, C6-H, C8-H), 5.55
(brs, 1H, C2-H), 5.04 (s, 2H), 4.67 (d, 1H, J = 9.5 Hz, C10000-H), 4.54 (d, 1H, J = 9.0 Hz, C1000-H),
4.41–4.36 (m, 2H), 4.28–4.24 (m, 2H), 3.59–3.55 (m, 2H), 3.51–3.40 (m, 2H), 3.36–3.31 (m,
2H), 3.00 (dd, 1H, J = 2.4 Hz, 12.6 Hz, C4-Ha), 2.89–2.86 (m, 1H, C4-Hb), 2.08–2.00 (m, 24H,
8× OCH3); 13C-NMR (CD3OD, 150 MHz)
δ
173.1, 173.1, 173.1,0173.1, 173.0, 173.0, 173.0,
0
173.0, 166.9 (C=O), 158.0 (C-5), 157.9 (C-9), 157.0 (C-7), 151.6 (C-3 , C-5 ), 151.3 (C-3”, C-5”),
138.5 (C-4”), 137.9 (C-40), 134.2 (C-10), 128.5 (C-1”), 110.4 (C-2”, C-6”), 107.3 (C-10000), 107.1
(C-20, C-60), 107.0 (C-1000), 99.3 (C-6, C-8), 96.8 (C-10), 78.2 (C-2), 77.6, 77.5, 76.2, 76.1, 75.1,
75.0, 71.6, 71.5, 70.7 (C-3), 64.6 (C-6000), 64.5 (C-60000), 26.8 (C-4), 20.9 (OCH3), 20.9 (OCH3),
20.9 (OCH3), 20.9 (OCH3), 20.8 (OCH3), 20.8 (OCH3), 20.8 (OCH3), 20.8 (OCH3); ESIMS:
m/z 1141 [M + Na]+.
General procedure for the synthesis of
gallates (9 and 10)
To a solution of 2,3,4,6-tetra-O-acetyl-
β
-D-glucopyranosyl (-)-epigallocatechin-3-
β
-D-glucopyranosyl (-)-epigallocatechin-3-gall-
ates 7/8 (0.1 mmol) in CH3OH (2 mL), a potassium hydroxide solution (0.1 mmol, dis-
solved in CH3OH) was added. The mixture was stirred at 0 ◦C for 72 h and then neutral-
ized with Dowex 50WX4-400 ion-exchange resin to pH = 7. The solvent was evaporated
under vacuum, and the residue was purified by column chromatography in silica gel
(CHCl3/CH3OH, 4:1) to afford the product.
[4”-O-(β-D-glucopyranosyl)]-(-)-epigallocatechin-3-gallate (9)
White powder; yield 55%; mp. 118 ◦C to 120 ◦C; 1H-NMR (CD3OD, 400 MHz)
δ 6.90
(s, 2H, C2”, C6”-H), 6.59 (s, 2H, C20, C60-H), 6.02 (d, 1H, J = 2.2 Hz, C6-H), 6.00 (d, 1H,
J = 2.2 Hz, C8-H), 5.73 (brs, 1H, C3-H), 5.12 (s, 1H, C2-H), 4.69 (d, 1H, J = 9.0 Hz, C10000-H),
3.81–3.78 (m, 1H), 3.74–3.72 (m, 1H), 3.49–3.41 (m, 4H), 3.03–3.01 (m, 1H, C4-Ha), 2.95–2.92
(m, 1H, C4-Hb); 13C-NMR (CD3OD, 100 MHz)
δ 166.4 (C=O), 158.2 (C-7), 157.9 (C-5), 157.8
(C-9), 151.6 (C-30, C-50), 146.7 (C-3”, C-5”), 138.6 (C-4”), 133.7 (C-40), 130.7 (C-10), 127.6
(C-1”), 110.2 (C-2”, C-6”), 108.2 (C-10000), 106.9 (C-20, C-60), 98.3 (C-10), 97.1 (C-6), 95.7 (C-8),
78.4, 77.6, 76.3, 75.1, 70.6, 68.6 (C-2), 61.8 (C-60000), 26.5 (C-4); ESIMS: m/z 619 [M−H]–.

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0
[4 -O-(
β-D-glucopyranosyl)-4”-O-(
β-D-glucopyranosyl)]-(-)-epigallocatechin-3-gallate (10
)
White powder; yield 52%; mp. 125 ◦C to 126 ◦C; 1H-NMR (CD3OD, 400 MHz)
δ
6.92
(s, 2H, C2”, C6”-H), 6.57 (s, 2H, C20, C60-H), 5.98 (d, 1H, J = 2.3 Hz, C6-H), 5.96 (d, 1H,
J = 2.3 Hz, C8-H), 5.55 (brs, 1H, C3-H), 5.11 (s, 1H, C2-H), 4.67 (d, 1H, J = 9.5 Hz, C10000-H),
4.54 (d, 1H, J = 9.5 Hz, C1000-H), 3.83 (m, 6H), 3.48–3.41 (m, 6H), 2.99–2.89 (dd, 1H, J = 2.4 Hz
,
12.6 Hz, C4-Ha), 2.89–2.86 (m, 1H, C4-Hb); 13C-NMR (CD3OD, 100 MHz)
δ 166.8 (C=O),
157.9 (C-5), 157.8 (C-9), 156.9 (C-7), 151.5 (C-30, C-50), 151.2 (C-3”, C-5”), 138.4 (C-4”), 137.6
(C-10), 134.1 (C-40), 128.3 (C-1”), 110.3 (C-2”, C-6”), 107.8 (C-10000), 107.0 (C-1000), 106.9 (C-20,
C-60), 99.1 (C-10), 96.6 (C-695.8 (C-8), 78.5, 78.4, 78.4, 78.3, 78.1 (C-3), 77.5, 77.5, 75.1, 75.1,
70.6, 70.5, 70.4 (C-2), 61.8 (C-60000), 61.8 (C-6000), 26.7 (C-4); ESIMS: m/z 781 [M−H]–.
General procedure for the synthesis of 2,3,4,6-tetra-O-butyryl-
β-D-glucopyranosyl
(-)-epigallocatechin-3-gallates (11 and 12)
To a solution of β-D-glucopyranosyl (-)-epigallocatechin-3-gallates 9/10 (0.05 mmol)
in pyridine (1 mL), ◦butyric anhydride (0.1 mL, 0.5 mmol) was added, and the stirring
was continued at 0 C for 12 h. The reaction mixture was diluted with CH2Cl2 (5 mL)
and washed with aqueous saturated NaHCO3 solution, the organic layer was then dried
with Na2SO4, and the solvent was evaporated under vacuum; the residue was purified by
column chromatography in silica gel (CHCl3/CH3OH, 15:1) to afford the product.
0000 0000 0000 0000
[4”-O-(2 ,3 ,4 ,6 -tetra-O-butyryl-
β
-D-glucopyranosyl)]-(-)-epigallocatechin-3-gallate (11
)
White powder; yield 82%; mp. 92 ◦C to 94 ◦C; 1H-NMR (CD3OD 3, 400 MHz)
δ
6.91
(s, 2H, C2”, C6”-H), 6.48 (s, 2H, C20, C60-H), 5.94 (s, 2H, C6-H, C8-H), 5.54 (brs, 1H, C2-H),
5.31 (m, 1H), 5.24–5.22 (m, 1H, C3-H), 5.21 (m, 1H), 5.10 (t, 1H, J = 9.0 Hz), 4.97 (d, 1H ,
J = 9.0 Hz, C10000-H), 4.29 (dd, 1H, J = 5.4 Hz, 12.4 Hz), 4.06–4.03 (m, 1H), 3.88–3.84 (m,
1H), 2.98 (dd, 1H, J = 4.6 Hz, 17.6 Hz, C4-Ha), 2.86–2.83 (m, 1H, C4-Hb), 2.21–2.18 (m, 8H,
4× COCH2), 1.63–1.55 (m, 8H, 4
×
CH2CH3), 0.94–0.88 (m, 12 H, 4
×
CH2CH3); 13C-NMR
(CD3OD, 100 MHz) 172.4, 171.6, 171.6, 171.2, 166.8 (C=O), 157.9 (C-5), 157.8 (C-9), 157.1
δ
(C-7), 151.6 (C-30, C-50), 146.7 (C-3”, C-5”), 137.2 (C-4”), 133.7 (C-40), 130.7 (C-10), 128.1
(C-1”), 110.0 (C-2”, C-6”), 106.6 (C-20, C-60), 102.7 (C-10000), 99.2 (C-10), 96.5 (C-6), 95.8 (C-8),
78.2 (C-2), 74.0, 73.1, 72.9, 70.4 (C-2), 69.7, 62.8 (C-60000), 35.9 (COCH2), 35.8 (COCH2), 35.7
(COCH2), 35.6 (COCH2), 26.7 (C-4), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.1 (CH2CH3), 18.1
(CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3); ESIMS: m/z
923 [M + Na]+.
0
000 000 000 000
0000 0000 0000 0000
[4 -O-(2 ,3 ,4 ,6 -tetra-O-butyryl-
yryl-β-D-glucopyranosyl)]-(-)-epigallocatechin-3-gallate (12)
β-D-glucopyranosyl)-4”–(2 ,3 ,4 ,6 -tetra-O-but-
◦
◦
White powder; yield: 80%; mp. 95 C to 97 C; 1H-NMR (CD3OD, 600 MHz)
δ 6.92 (s,
2H, C2”, C6”-H), 6.58 (s, 2H, C20, C60-H), 5.98 (s, 1H, C3-H), 5.96 (s, 2H, C6-H, C8-H), 5.55
(brs, 1H, C2-H), 5.03 (s, 2H), 4.68 (d, 1H, J = 9.5 Hz, C10000-H), 4.56 (d, 1H, J = 9.5 Hz, C1000-H),
4.42–4.38 (m, 2H), 4.28–4.26 (m, 2H), 3.60–3.56 (m, 2H), 3.50–3.40 (m, 2H), 3.36–3.32 (m,
2H), 3.01 (dd, 1H, J = 2.4 Hz, 12.6 Hz, C4-Ha), 2.88–2.86 (m, 1H, C4-Hb), 2.60–2.58 (m, 4H,
2
×
COCH2), 2.35–2.20 (m, 12H, 6
(m, 12H, 6 CH2CH3), 1.04–0.93 (m, 6H, 2
13C-NMR (CD3OD, 150 MHz)
×
COCH2), 1.62–1.60 (m, 4H, 2
×
CH2CH3), 1.58–1.56
×
×
CH2CH3), 0.91–0.93 (m, 18H, 6× CH2CH3);
δ
173.1, 173.1, 173.1, 173.1, 173.0, 173.0, 173.0, 173.0, 166.9
(C=O), 158.0 (C-05), 157.9 (C-9), 157.0 (C-7), 151.6 (C-30, C-50), 151.3 (C-3”, C-5”), 138.4
0
0000
000
(C-4”), 137.9 (C-4 ), 134.1 (C-1 ), 128.5 (C-1”), 110.3 (C-2”, C-6”), 107.8 (C-1 ), 107.1 (C-1 ),
107.0 (C-20, C-60), 99.2 (C-6), 99.2 (C-8), 95.9 (C-10), 78.2 (C-2), 77.5, 77.5, 76.1, 76.1, 75.0,
74.9, 71.5, 71.5, 70.6 (C-3), 64.5 (C-60000), 64.5 (C-6000), 35.9 (COCH2), 35.8 (COCH2), 35.8
(COCH2), 35.7 (COCH2), 35.7 (COCH2), 35.7 (COCH2), 35.6 (COCH2), 35.6 (COCH2), 26.8
(C-4), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.1
(CH2CH3), 18.1 (CH2CH3), 18.1 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3),
13.5 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.4 (CH2CH3), 13.4 (CH2CH3); ESIMS:
m/z 1365 [M + Na]+.

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3.3. Cytotoxicity Assay
The effects of the (-)-epigallocatechin-3-gallate glucoside derivatives on the survival
of cancer cells were determined using the MTT assay. Five human cancer cell lines (HL-60,
SMMC-7721, A-549, MCF-7, and SW480) were used in the cytotoxicity assay. All the cancer
cells were seeded in 96-well plates and then each tumor cell line was exposed to the test com-
pound at various concentrations in triplicate for 48 h. After the in◦cubation, MTT (100
µg)
was added to each well, and the incubation continued for 4 h at 37 C. After removal of the
culture medium, the produced MTT formazan crystals were dissolved with 150 µL DMSO
and measured at 492 nm using a microplate reader. The percentage of inhibition was cal-
culated as follows: inhibition ratio (IR, %) = (1 − OD(sample)/OD(control)) × 100%. The
experiments were carried out in triplicate, and the IC50 (the concentration of drug that
inhibits cell growth by 50%) values were determined.
3.4. Western Blotting
A-549 cells were lysed in RIPA buffer containing PMSF (protease and phosphatase
inhibitors) and quantified via BCA protein assay. Proteins separated on 8% SDS–PAGE
electrophoresis and then blotted onto polyvinyl difluoride membranes. After the mem-
branes were blocked with BSA for 1 h, the expression of various proteins was detected
using primary and secondary antibodies conjugated with horseradish peroxidase. HRP
was detected using the Prolight HRP Chemiluminescent Kit (Tiangen Biotech, Beijing,
China) and FluorChem E Synthem (ProteinSimple, Santa Clara, CA, USA).
3.5. Docking Studies
The X-ray crystal structure of EGCG (PDB code: 2ITY) was retrieved from Protein
Data Bank (www.rcsb.org (accessed on 17 February 2020)). Ligand docking was carried
out by applying the Lamarckian Genetic Algorithm implemented in AutoDock 4.2. The
modeled EGCG structure was employed as the receptor to docking with these small
molecules. DiscoveryStudio 4.0 software was used for the preparation of ligand and
receptor. Autodock Tools 1.5.6 was used for grid and docking according to the literature [48
]
The grid size was set to 60 Å, 60 Å, and 60 Å along the X-, Y- and Z-axis to recognize the
binding site. The default 0.375 Å spacing was adopted for the grid box. The number of GA
runs was set to 50, and the maximum number of evals (medium) was set to 5,000,000 on
AutoGrid 4.2.6 and AutoDock 4.2.6. In the selected cluster, conformations with the lowest
binding energy and RMSD (<2.0 Å) were finally chosen to analyze the receptor–ligand
interaction. Other miscellaneous parameters were assigned to the default values obtained
from the AutoDock 4.2.6.
4. Conclusions
In conclusion, we synthesized a series of glucoside derivatives of EGCG (7–12) and
evaluated for their in vitro anticancer activity against five human cancer cell lines, including
HL-60, SMMC-7721, A-549, MCF-7, and SW480. All these derivatives display different
levels of anticancer activity, which can be affected by the nature of substituents on the
glucose residue. Among them, compounds 11 and 12 showed better growth inhibition than
others in four cancer cell lines (HL-60, SMMC-7721, A-549, and MCF), with IC50 values in
the range of 22.90–37.87 µM. EGCG derivatives 11 and 12 also display moderate selectivity
toward cancer cells over normal human bronchial epithelial cells (BEAS-2B). The docking
study indicated that the presence of more hydrophobic interactions in compounds 11 and
12. In addition, compounds 11 and 12 decreased phosphorylation of EGFR and downstream
signaling protein. The most active compounds 11 and 12, both having perbutyrylated
glucose residue, led us to conclude that perbutyrylation of the glucose residue causes
increased cytotoxic activity, which is indicative of their potential as anticancer agents for
further development.

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Supplementary Materials: The following are available online, Table S1: The characteristic 1H-
NMR and 13C-NMR of compounds
7
–
12, Table S2: The energies of the binding between EGFR
11, and 12, Figure S1: The details of the binding between EGFR and EGCG ( ),
Figures S2–S17: Spectra of compound 6–12.
and compounds
4,
4
Author Contributions: C.-T.Z., X.-J.W., and J.S. conceived and designed the experiments; Y.W.,
X.-J.S., F.-W.S., Y.-R.X., L.-X.W., N.Z., Y.-L.W., Y.N., D.-Y.Z. and C.-T.Z. performed the experiments;
C.-T.Z., Y.W., X.-J.S. and F.-W.S. analyzed the data. D.-Y.Z., C.-T.Z., X.-J.W. and J.S. contributed
reagents/materials/analysis tools; C.-T.Z., Y.W., X.-J.S. and F.-W.S. wrote the manuscript. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Nature Science Foundation of China (Nos.
31960075, 21602196, and 31760226); the Yunnan Provincial Science and Technology Department
(Nos. 2017ZF003, 2017FD084, and 2017FG001-046); the Yunnan Provincial Key Programs of Yun-
nan Eco-friendly Food International Cooperation Research Center Project (Nos. 2019ZG00904 and
2019ZG00909).
Institutional Review Board Statement: Not Applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors thank the staff of the analytical group of the State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy
of Sciences, for measurements of all spectra.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Compounds
7 and 8 (Scheme 1) are available from the authors. Compounds
9–12 (Scheme 2) are only synthesized on demand.
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