Synthesis, molecular docking and biological evaluation of Glycyrrhizin analogs as anticancer agents targeting EGFR

Article abstract of DOI:10.3390/molecules19056368

Glycyrrhizin (GA) analogs in the form of 3-glucuronides and 18-epimers were synthesized and their anticancer activities were evaluated. Alkaline isomerization of monoglucuronides is reported. In vitro and in vivo studies showed that glycyrrhetinic acid monoglucuronides (GAMGs) displayed higher anticancer activities than those of bisglucuronide GA analogs, while anticancer activity of the 18α-epimer was superior to that of the 18βepimer. 18α-GAMG was firstly nicely bound to epidermal growth factor receptor (EGFR) via six hydrogen bonds and one charge interaction, and the docking calculation proved the correlation between anticancer activities and EGFR inhibitory activities. Highly active 18α-GAMG is thus of interest for the further studies as a potential anticancer agent.

Full text of DOI:10.3390/molecules19056368

Molecules 2014, 19, 6368-6381; doi:10.3390/molecules19056368
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Molecular Docking and Biological Evaluation of
Glycyrrhizin Analogs as Anticancer Agents Targeting EGFR
1
2,
1
1
1,2
Yong-An Yang , Wen-Jian Tang *, Xin Zhang , Ji-Wen Yuan , Xin-Hua Liu and
1
,
Hai-Liang Zhu *
1
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,
China; E-Mails: yangyan73@163.com (Y.-A.Y.); prczhangxin@163.com (X.Z.);
3
63718480@163.com (J.-W.Y.); xhliuhx@163.com (X.-H.L.)
2
School of Pharmacy, Anhui Medical University, Hefei 230032, China
*
Authors to whom correspondence should be addressed; E-Mails: tangwjster@gmail.com (W.-J.T.);
zhuhl@nju.edu.cn (H.-L.Z.); Tel./Fax: +86-551-6516-1115 (W.-J.T.).
Received: 10 April 2014; in revised form: 4 May 2014 / Accepted: 14 May 2014 /
Published: 19 May 2014
Abstract: Glycyrrhizin (GA) analogs in the form of 3-glucuronides and 18-epimers were
synthesized and their anticancer activities were evaluated. Alkaline isomerization of
monoglucuronides is reported. In vitro and in vivo studies showed that glycyrrhetinic acid
monoglucuronides (GAMGs) displayed higher anticancer activities than those of
bisglucuronide GA analogs, while anticancer activity of the 18α-epimer was superior to that
of the 18β-epimer. 18α-GAMG was firstly nicely bound to epidermal growth factor receptor
(EGFR) via six hydrogen bonds and one charge interaction, and the docking calculation
proved the correlation between anticancer activities and EGFR inhibitory activities. Highly
active 18α-GAMG is thus of interest for the further studies as a potential anticancer agent.
Keywords: glycyrrhizin; anticancer activity; EGFR; 18-epimer; molecular docking
1
. Introduction
The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that defines a family
of tyrosine kinase receptors (TKRs) including ErbB2/HER2, ErbB3/HER3 and ErbB4/HER4 [1,2]. As a
cell surface protein that binds to epidermal growth factor, its binding to a ligand induces receptor
dimerization and tyrosine autophosphorylation and leads to cell proliferation, of which altered activity
has been implicated in the development and growth of many tumors [3]. EGFR is highly expressed in

Molecules 2014, 19
6369
adult hepatocytes and the EGFR family plays a central hepatoprotective and pro-regenerative role in the
liver [4,5]. Mice lacking EGFR or heparin-binding EGF show delayed regeneration after partial
hepatectomy (PH), which demonstrates that EGFR is a critical regulator of hepatocyte proliferation during
liver regeneration [6,7]. The treatment of hepatocellular carcinoma (HCC) cells with EGFR-specific
tyrosine kinase inhibitors or neutralizing antibodies induces cell cycle arrest and apoptosis and increases
chemosensitivity [8,9]. Hence, EGFR has long been an attractive candidate as anticancer drug target.
Over the past 30 years, much effort has been directed at developing anticancer agents that can interfere
with EGFR activity, such as, monoclonal antibodies and small-molecule inhibitors.
Natural products play a major role in drug discovery, and nearly half of the new drugs introduced into
the market over the past two decades are natural products or their derivatives [10]. The roots and
rhizomes of licorice (Glycyrrhiza) species have long been used worldwide as a herbal medicine and
natural sweetener. Glycyrrhizin (Glycyrrhizic acid, GA, 18β-GA), the major bioactive compound in
licorice, is developed as a drug with multi-pharmacological effects such as anti-inflammation, antivirus,
anti-tumor, and immuno-modulating properties, among others [11‒13]. GA has been used in Japan for
more than 60 years as a treatment for chronic hepatitis C, thus long-term administration was effective in
preventing hepatic cirrhosis and HCC [14‒16]. GA exhibits hepatoprotective activity by decreasing
serum liver enzyme levels and improving tissue pathology in hepatitis patients, while in vitro studies
showed that its anticancer activity is achieved by inhibiting abnormal cell proliferation, tumor formation
and growth [17‒20].
GA is a conjugate of an 18β-H-oleanane-type aglycone and two glucuronic acids at the C-3 position,
and it could be transformed into 18β-glycyrrhetinic acid monoglucuronide (18β-GAMG) by removing
one terminal glucuronic acid [21,22]. Compared to GA, 18β-GAMG showed similar (or stronger)
pharmacological activities, such as antitumor, antivirus, anti-allergic, and anti-inflammatory
activities [23,24]. 18α-Glycyrrhizin (18α-GA), a D/E-trans-epimer, was prepared by alkaline isomerization
of 18β-GA [25]. 18α-GA also showed similar anti-inflammatory and anticancer activity [26,27].
Researches showed that GA analogs are primary hepatocyte mitogens that bind to EGFRs and
subsequently stimulate the receptor tyrosine kinase mitogenactivated protein kinase pathway to induce
hepatocyte DNA synthesis and proliferation [28]. Herein, we prepared glycyrrhizin analogs (Figure 1)
and further evaluated their anticancer activities in vitro and in vivo.
Figure 1. Glycyrrhizin analogs with 3-glucuronides and 18-epimers.
O
O
HO
H
HO
H
O
H
O
H
H
H
HOOC
HO
HO
HOOC
HO
HO
O
O
O
O
OR
OR
1
8β-GAMG: R = H
8β-GA: R = glucuronide
18α-GAMG: R = H
18α-GA: R = glucuronide
1

Molecules 2014, 19
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Based on the EGFR complex structure (PDB Code 1M17) [29], computer-generated docking
molecular models of GA analogs were analyzed using the Discovery Studio 3.5, and we also initiated an
effort to leverage molecular modeling in combination with available data to study the effect of structure
of glycyrrhizin on anticancer activity.
2
. Results and Discussion
GA (18β-GA) was provided by Jiangsu Tian Sheng Pharmaceutical Co. Ltd., Jiangsu, China.
Aspergillus sp Ts-1, a kind of β-glucuronidase, selectively hydrolyzed the terminal–glucuronyl linkage
of 18β-GA to produce 18β-GAMG in 54% yield. 18α-GA and 18α-GAMG were respectively
synthesized in yields of 63% and 71% after recrystallization from 18β-GA and 18β-GAMG by alkaline
1
3
isomerization. The isomerization reaction was monitored by C-NMR spectroscopy, and the structure
1
13
of 18α-GAMG was elucidated by comparison with H-NMR and C-NMR data of 18β-GAMG [30]
(
see Supplementary Information).
Anti-proliferative activities of glycyrrhizin analogs and erlotinib against the HepG2 (hepatocellular
carcinoma), HeLa (cervix of uterus adenocarcinoma) and A549 (lung carcinoma), were evaluated by
CCK8 dye assays. The results, summarized in Table 1, revealed that four glycyrrhizin analogs
exhibited significant antitumor activities. Two monoglucuronide compounds, 18α-GAMG and
1
8β-GAMG, exhibited more significant antitumor activities than those with bisglucuronide GAs, while
the antitumor activity of the 18α-epimer was superior to that of 18β-epimer for identical cell lines.
Among the four analogs, 18α-GAMG displayed the most potent activity, with IC values of 6.67, 7.43
5
0
and 15.76 μM against HepG2, Hela and A549, respectively.
Table 1. In vitro anticancer activities (IC50, μM) of title compounds against human tumor cell lines.
a
IC50, (μM)
Compd.
b
b
b
HepG2
6.67
HeLa
7.43
A549
1
8α-GAMG
8β-GAMG
15.76
21.55
41.57
51.92
0.13
1
33.60
54.24
63.59
0.12
8.39
1
8α-GA
8β-GA
15.13
18.93
0.20
1
Erlotinib
a
Antiproliferation activity was measured using the CCK-8 assay. Values are the average of three independent
b
experiments run in triplicate. Variation was generally 5%–10%; Cancer cells kindly supplied by State Key
Laboratory of Pharmaceutical Biotechnology, Nanjing University.
To generate data concerning the broad spectrum potential of these compounds in Table 2, the IC50
values of synthesized compounds against EGFR enzymes are summarized in Table 2. Reference data for
erlotinib had also been included for comparison with the compounds reported in this study. For the
majority of the compounds, we found that compound 18α-GAMG, with an IC of 0.028 μM, was a
50
better inhibitor than the positive control erlotinib with an IC50 of 0.030 μM, suggesting that, at least in
part, inhibition of proliferation of the these lines may be the result of EGFR inhibition.

Molecules 2014, 19
6371
Table 2. Data of the in vitro EGFR (IC , μM) enzyme inhibition assay of the synthesized compounds.
5
0
a
a
Compd.
EGFR (IC50, μM)
0.028
Compd. EGFR (IC50, μM)
1
8α-GAMG
8β-GAMG
18β-GA
0.092
0.030
1
0.069
Erlotinib
1
8α-GA
0.081
a
Minimum cytotoxic concentration required to cause a microscopically detectable alteration of normal cell
morphology.
In order to gain more understanding of the structure–activity relationships observed at the EGFR,
molecular docking of the most potent inhibitor 18α-GAMG and EGFR was performed on the binding
model based on the EGFR complex structure (PDB Code 1M17) using the Discovery Studio 3.5
software [29]. The docking calculation of the analogs was depicted in Table 3, and as shown in Table 3,
all analogs had nice binding affinity to EGFR and four analogs' -EDOCKER_ INTERACTION_
ENERGY had the same trend as the anti-proliferative activities, which further proved the correlation
between the anti-proliferative activities and EGFR inhibitory activities of the analogs.
Table 3. -EDOCKER_INTERACTION_ENERGY of title compounds and 1M17.
Compd.
-EDOCKER_INTERACTION_ENERG ΔG (kcal/mol)
1
8α-GAMG
8β-GAMG
72.0274
66.9106
58.7009
58.6731
44.3732
1
1
8α-GA
8β-GA
1
Erlotinib
In the result of molecular docking, 18α-GAMG showed maximum -EDOCKER_
INTERACTION_ENERGY, which suggested it was mostly easy to bind to EGFR. The 2D and 3D
binding models of 18α-GAMG with EGFR are depicted in Figure 2. The amino acid residues which had
interactions with EGFR as well as bond lengths were labeled. In the binding models, 18α-GAMG was
nicely bound to EGFR via six hydrogen bonds with ASP831 (angle = 120.49°, distance = 2.14 Å),
GLU738 (angle = 142.28°, distance = 2.2 Å), THR766 (three bonds: angle = 140.28°, distance = 2.1 Å;
angle = 117.6°, distance = 2.3 Å; angle = 152.94°, distance = 2.0 Å) and LYS721 (angle = 179.02°,
distance = 1.7 Å). In addition, compound 18α-GAMG was also nicely bound to EGFR via one charge
interaction. The end group of LYS692 formed one charge interaction with a carboxyl which
strengthened the binding affinity, leading to the increased anticancer activities of 18α-GAMG. Besides,
the hydrogens of LYS692, LYS692 and PRO770 formed three hydrogen bonds interaction with the
amino group nitrogen atom of 18β-GAMG (angle H-NLYS692 O35 = 151.7°, distance = 1.98 Å, angle H
LYS692 O36 = 123.6°, distance = 2.47 Å, angle H96 O PRO770 = 113.4°, distance = 2.22 Å). Furthermore,
compound 18β-GAMG was also nicely bound to EGFR via three charge interactions. The end group of
LYS692, LYS704 and LYS721 respectively formed three charge interactions with two carboxyls. These
molecular docking results, along with the biological assay data, suggest that compound 18α-GAMG
possesses higher anticancer activity than 18β-GAMG, which will help us carry out structure
optimization based on computer-aided design.

Molecules 2014, 19
6372
Recently, 18β-GA has been recognized as a hepatoprotective high-mobility group protein 1 (HMGB1)
inhibitor, which binds directly to both HMG boxes in HMGB1 and attenuates HMGB1-induced
hepatocyte apoptosis, thus leading to induce hepatocyte DNA synthesis and proliferation [31,32]. As it
is, 18β-GA induced hepatocyte proliferation, while we got the opposite results in cancer cells. The
binding moiety of 18α-GAMG with EGFR was mainly the glucuronide unit, but GA could inhibit
HMGB1 by binding of its triterpene ring directly to the two HMG boxes. These results showed that the
protein targets and molecular pathways affected by GA may be complicated and heterogeneous.
Figure 2. (a) 2D molecular docking modeling of 18α-GAMG with 1M17. (b) 3D model of
the interaction between 18α-GAMG and 1M17 site. (c) 2D molecular docking modeling of
18β-GAMG with 1M17. (d) 3D model of the interaction between 18β-GAMG and 1M17 site.

Molecules 2014, 19
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Figure 2. Cont.
To further verify the inhibitory effect of glycyrrhizin analogs on the growth of tumor cells in vivo,
sarcoma cells S180, hepatoma cells HepG2 and Ehrlich ascites cells EAC were selected to evaluate
in vivo antitumor effects. The inhibitory effects of glycyrrhizin analogs on the growth of the
transplanted S180 or HepG2 carcinoma are presented in Table 4 and Figure 3. The results revealed that
glycyrrhizin analogs significantly decreased the tumor weights of S180 and HepG2 tumor-bearing mice.
The inhibitory rates of GAMGs were higher than those of GAs, while the inhibitory rate of the
1
1
8α-epimer was higher than that of corresponding 18β-epimer. The most potent activity was showed by
8α-GAMG with inhibitory rates 39.8% and 49.7% for S180 and HepG2 tumor-bearing mice at the
dosage of 60 mg/kg/day, respectively.
EAC tumor-bearing mice were observed for mean survival time. The effect of glycyrrhizin analogs
on percentage increases in life span was calculated on the basis of mortality of the experimental mice.
Survival response of untreated EAC-bearing mice died within 16.4 days (Table 5). A similar
phenomenon was observed: mice administered monoglucuronide and 18α-epimer displayed longer
survival times. The 18α-GAMG group was observed to enhance the survival rate to 45.4%.

Molecules 2014, 19
6374
Based on in vitro and in vivo experiments, followed by molecular docking, we here demonstrated that
the protein target Epidermal Growth Factor Receptor (EGFR) was also sensitive to four glycyrrhizin
analogs in three types of carcinoma cells, indicative of their potential anticancer activity as the EGFR
inhibitors. The result was significant and intriguing, but further studies needs to be provided to
systematically elucidate the direct correlation between the glycyrrhizin analogs and the EGFR target,
which would reveal the new mechanism of glycyrrhizin action.
Table 4. Antitumor effects of glycyrrhizin analogs against tumor growth on the S180 and
a
HepG2 xenograft mice.
Animal number
Body weight (g)
Beginning End
19.80 ± 1.32 23.97 ± 2.23
Tumor weight Inhibition rate
Models
Groups
(
End, n)
10
(g)
(%)
Control
1.91 ± 0.29
1
8α-GAMG
9
19.40 ± 1.07 25.25 ± 1.80 1.15 ± 0.50 **
19.80 ± 1.39 25.02 ± 2.58 1.25 ± 0.19 **
20.00 ± 1.49 25.20 ± 1.11 1.29 ± 0.47 **
19.70 ± 1.25 24.48 ± 2.37 1.33 ± 0.67 **
39.8
34.6
32.5
30.4
S180
18β-GAMG
10
1
8α-GA
8β-GA
10
1
9
Control
10
19.4 ± 1.35 25.05 ± 1.89
1.95 ± 0.22
1
8α-GAMG
10
19.6 ± 1.51 27.02 ± 2.10 0.98 ± 0.43 **
19.00 ± 0.94 27.8 ± 1.57 1.20 ± 0.35 **
20.1 ± 1.45 26.08 ± 1.26 1.22 ± 0.46 **
19.4 ± 1.08 26.94 ± 2.05 1.26 ± 0.65 **
49.7
38.4
37.4
35.4
HepG2 18β-GAMG
10
1
8α-GA
8β-GA
10
1
10
a
Mice were inoculated with S180 or HepG2 subcutaneously into the right front armpit and randomly
divided into five test groups. The mice were daily treated by 18α-GAMG, 18β-GAMG, 18α-GA, 18β-GA
60 mg/kg/day), or normal saline (NS, 10 mL/kg) by oral gavage for ten consecutive days. Data were analyzed
(
using SPSS11.0. Significant difference between each treatment and the control are shown as P < 0.05 (*) and
P < 0.01 (**).
Figure 3. Solid tumors from S180 (above) and HepG2 (below) tumor-bearing mice. A:
1
8α-GAMG; B: 18β-GAMG; C: 18α-GA; D: 18β-GA; M: Normal saline.

Molecules 2014, 19
6375
Figure 3. Cont.
Table 5. Effects of glycyrrhizin analogs against the survival of EAC-bearing mice.
a
Groups
Control
Animal number (n) Body weight (g) Survival time (d) Survival rate (%)
10
10
10
10
10
19.0 ± 1.25
19.5 ± 1.27
19.0 ± 0.82
19.4 ± 0.97
19.4 ± 1.27
16.40 ± 2.07
23.85 ± 5.41*
21.05 ± 4.65*
19.75 ± 3.08*
19.40 ± 3.77*
1
8α-GAMG
8β-GAMG
45.4
28.4
20.4
18.3
1
1
8α-GA
8β-GA
1
a
Time denoted by number of days. P < 0.05 (*)
3
3
3
. Experimental Section
.1. Synthesis of Glycyrrhizin Analogs
.1.1. General Methods
Aspergillus sp. Ts-1 was isolated from soil collected in Kashi of the Xinjiang Uygur Autonomous
Region (China) and selectively hydrolyzed the terminal–glucuronyl linkage of 18β-GA to yield
glycyrrhetic acid 3-O-mono-β-D-glucuronide (18β-GAMG). Its subculture and 18β-GA were provided
by Jiangsu Tian Sheng Pharmaceutical Co. Ltd. (Nanjing, China). All materials were obtained from
commercial suppliers, were of analytical reagent grade and used without further purification. Melting
points were uncorrected. Silica gel (200–300 mesh, Huanghai, Qingdao, China). TLC: pre-coated silica
gel F254 plates. Optical rotations: polar 3002 polarimeter. NMR spectra: Bruker AV NMR spectrometer
1
13
(
H: 500 or 300 and C: 125 or 75 MHz), the residual solvent peaks used as an internal standard, J in Hz.
TOF- HR MS: Agilent 1260-6221 TOF LC/MS.
3
.1.2. Preparation of 18β-GAMG from 18β-GA via Biotransformation
D
13
1
8β-GA: white powder. Mp 234−236 °C; [α]20 = +52 (c = 1.0, MeOH); C-NMR (75 MHz,
+
DMSO-d ): Table S1. TOF-HRMS: m/z [M + Na] calcd for C H NaO : 845.3930; found: 845.3935.
6
42 62
16

Molecules 2014, 19
6376
Aspergillus sp. Ts-1 on glucose yeast agar slant was inoculated into a 250 mL Erlenmeyer flask
containing 100 mL of seed medium consisting of 1.0 g glucose, 0.2 g yeast, 1.0 g agar, 0.1 g KH PO4
2
4
and 0.025g MgSO in distilled water (pH 7.0). The culture media were sterilized at 121 °C for 20 min
and the fermentation was carried out at 30 °C on a rotary shaker at 200 rpm. After 24 h of inoculation,
3
3
0 mL sterilized medium was inoculated into a 1,000 mL Erlenmeyer flask containing
00 mL pre-culture sample consisting of 15 g GA, 0.30 g KH PO , 3.0 g urea and 0.24 g MgSO in
2
4
4
distilled water and the pH value was adjusted to 6.0. The culture media were sterilized at 121 °C for
2
0 min and the fermentation was carried out at 30 °C on a rotary shaker at 250 rpm.
After 72 h of inoculation, the culture solution was filtered and the filtrate was extracted with ethyl
acetate. The extract was concentrated under the reduced pressure. The residue (14.5 g) was applied to a
silica gel column (800 g, 5.0 × 100 cm) and eluted with CHCl –MeOH in a gradient manner from 100:1
3
to 1:1. By TLC analysis, fractions I–IX was obtained. Fractions VI–VIII was concentrated in vacuo and
recrystallization from aqueous MeOH to give 18β-GAMG (6.35 g, 54% yield) as a white crystalline
D
1
powder. Mp 237−239 °C; [α]20 = +91 (c = 1.0, MeOH); H-NMR (500 MHz, DMSO-d ) δ (ppm):
6
0
.76 (s, 3H, 24-CH ), 0.77 (s, 3H, 28-CH ), 0.99 (s, 3H, 23-CH ), 1.06 (s, 2 × 3H, 25-CH , 26-CH ), 1.10
3
3
3
3
3
(
s, 3H, 29-CH
3
), 1.34 (s, 3H, 27-CH
3
), 2.34 (s, 1H, 9-H), 3.01 (m, 1H, 4'-H), 3.08 (dd, 1H, J
1
= 4.8 Hz,
J
2
= 11.2 Hz, 3-H), 3.15 (t, 1H, J = 9.0 Hz, 3'-H), 3.30 (m, 1H, overlapped, 2'-H), 3.58 (d, 1H, J = 9.7 Hz,
1
3
5
6
'-H), 4.25 (d, 1H, J = 7.8 Hz, 1'-H), 5.40 (s, 1H, 12-H); C-NMR (125 MHz, DMSO-d ): Table S1.
+
TOF-HRMS: m/z [M + Na] calcd for C36
H54NaO10: 669.3609; found: 669.3608.
3
.1.3. General Procedure of Alkaline Isomerization of the 18β-isomer to the 18α-isomer
A solution of 18β-isomer (6.0 mmol) in 5.0 M NaOH solution (100 mL) was heated and stirred for
1
2 h at 90 °C. After the reaction mixture was cooled to <5 °C, the pH was adjusted to 2.5 with
concentrated HCl. After 12 h, the mixture was filtrated, washed with water, dried. The product
18α-isomer) was obtained by crystallization from ethanol/EtOAc. (Scheme S1 and Figure S1).
8α-GA: According to the above procedure, diammonium 18α-GA (3.17 g, 63% yield) was obtained
(
1
D
from 18β-GA (5.00 g) as a white crystalline powder. Mp 211−216 °C; [α]20 = +20 (c = 1.0, MeOH);
1
H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 0.65 (s, 3H, 28-CH
3
), 0.73 (s, 3H, 24-CH
3
), 0.95 (s, 3H, 23-CH
3
),
1
.04 (s, 3H, 26-CH ), 1.10 (s, 3H, 25-CH
3
3
), 1.16 (s, 3H, 29-CH
3
), 1.33 (s, 3H, 27-CH
3
), 4.31 (d, 1H,
1
3
J = 7.3 Hz, 1'-H), 4.49 (d, 1H, J = 7.6 Hz, 1''-H), 5.33 (s, 1H, 12-H); C-NMR (75 MHz, DMSO-d
6
):
+
Table S1. TOF-HRMS: m/z [M + Na] calcd for C42
H62NaO16: 845.3930; found: 845.3938.
1
8α-GAMG: 18α-GAMG (2.83 g, 71% yield) was obtained from 18β-GAMG (4.00 g) as a white
D
1
crystalline powder. Mp 229−231 °C; [α]20 = +24 (c = 1.0, MeOH); H-NMR (300 MHz, DMSO-d
δ (ppm): 0.65 (s, 3H, 28-CH ), 0.77 (s, 3H, 24-CH ), 0.92 (s, 3H, 23-CH ), 0.98 (s, 3H, 25-CH ), 1.04
s, 3H, 26-CH ), 1.16 (s, 3H, 29-CH ), 1.33 (s, 3H, 27-CH ), 2.27 (overlapped, 9-H), 3.01 (t, 1H, J = 8.4 Hz,
'-H), 3.07 (dd, 1H, J = 6.5 Hz, J = 9.7 Hz, 3-H), 3.15 (t, 1H, J = 9.0 Hz, 3'-H), 3.30 (t, 1H, J = 9.8 Hz,
6
)
3
3
3
3
(
3
3
3
4
2
1
2
1
3
'-H), 3.58 (d, 1H, J = 9.7 Hz, 5'-H), 4.24 (d, 1H, J = 7.8 Hz, 1'-H), 5.33 (s, 1H, 12-H); C-NMR
+
(
75 MHz, DMSO-d ): Table S1. TOF-HRMS: m/z [M + Na] calcd for C H NaO : 669.3609;
6
36 54
10
found: 669.3600.

Molecules 2014, 19
.2. Biological Assay of in Vitro Anticancer Activities
CCK8 is much more convenient and helpful than MTT for analyzing cell proliferation, because it can
6377
3
be reduced to soluble formazan by dehydrogenase in mitochondria and has little toxicity to cells. Cell
proliferation was determined using CCK8 dye (BeyotimeInst Biotech, Shanghai, China) according to
3
manufacturer’s instructions. Briefly, 1–5 × 10 cells per well were seeded in a 96-well plate, grown at 37 °C
for 12 h, Subsequently, cells were treated with compounds at increasing concentrations in the presence
of 10% FBS for 24 or 48 h. After 10 µL CCK8 dye was added to each well, cells were incubated at 37 °C
for 1–2 h and Plates were read in a Victor-V multilabel counter (Perkin-Elmer, Waltham, MA, USA)
using the default europium detection protocol. Percent inhibition or IC values of compounds were
5
0
calculated by comparison with DMSO-treated control wells.
3
.3. General Procedure for Preparation, Purification of EGFR, and Inhibitory Assay
A 1.6 kb cDNA encoded for the EGFR cytoplasmic domain (EGFR-CD, amino acids 645–1186)
were cloned into baculoviral expression vectors pBlueBacHis2B and pFASTBacHTc (Huakang
Company, Changsha, China), separately. A sequence that encodes (His) was located at the 5′ upstream
6
to the EGFR sequences. Sf-9 cells were infected for 3 days for protein expression. Sf-9 cell pellets were
solubilized at 0 °C in a buffer at pH 7.4 containing 50 mM HEPES, 10 mM NaCl, 1% Triton, 10 µM
ammonium molybdate, 100 µM sodium vanadate, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL
pepstatin, and 16 µg/mL benzamidine HCl for 20 min followed by 20 min centrifugation. Crude extract
supernatant was passed through an equilibrated Ni-NTA superflow packed column and washed with
1
0 mM and then 100 mM imidazole to remove nonspecifically bound material. Histidine tagged proteins
were eluted with 250 and 500 mM imidazole and dialyzed against 50 mM NaCl, 20 mM HEPES, 10%
glycerol, and 1 µg/mL each of aprotinin, leupeptin and pepstatin for 2 h. The entire purification
procedure was performed at 4 °C or on ice [29].
EGFR kinase assays were set up to assess the level of autophosphorylation based on
DELFIA/Time-Resolved Fluorometry. All compounds were dissolved in 100% DMSO and diluted to
the appropriate concentrations with 25 mM HEPES at pH 7.4. In each well, 10 µL compound was
incubated with 10 µL (5 ng for EGFR) recombinant enzyme (1:80 dilution in 100 mM HEPES) for
1
0 min at room temperature. Then, 10 µL of 5 mM buffer (containing 20 mM HEPES, 2 mM MnCl
2
,
1
00 µM Na VO and 1 mM DTT) and 20 µL of 0.1 mM ATP–50 mM MgCl were added for 1 h.
3
4
2
Positive and negative controls were included in each plate by incubation of enzyme with or without
ATP–MgCl . At the end of incubation, liquid was aspirated, and plates were washed three times with
2
wash buffer. A 75 µL (400 ng) sample of europium labeled anti-phosphotyrosine antibody was added to
each well for another 1 h of incubation. After washing, enhancement solution was added and the signal
was detected by Victor (Wallac Inc., Gaithersburg, MD, USA) with excitation at 340 nm and emission at
6
15 nm. The percentage of auto-phosphorylation inhibition by the compounds was calculated using the
following formula: 100% − [(negative control)/(positive control − negative control)]. The IC50 was
obtained from curves of percentage inhibition with eight concentrations of compound. As the
contaminants in the enzyme preparation are fairly low, the majority of the signal detected by the
anti-phosphotyrosine antibody is from EGFR.

Molecules 2014, 19
6378
3
.4. Evaluation of the in Vivo Antitumor Activities
3.4.1. Animals and Cell Lines
Kunming mice (SPF, male or female, 20 ± 2 g) were purchased from the experimental animal center
of China Pharmaceutical University. Animals were housed in a temperature (22 ± 2 °C) and relatively
humidity (50%)-controlled room on a 12 h light/dark cycle, given free access to food and water, and
acclimatized for at least one week prior to use. All the animal experiments were performed in
accordance with the Regulations of the Experimental Animal Administration issued by the State
Committee of Science and Technology of China.
Cell lines used for evaluation of the in vivo antitumor activity in this study included three tumor cell
lines, namely S180 (sarcoma tumer cell line), HepG2 (liver carcinoma cell line), EAC (Ehrlich ascites
carcinoma cell line). All of cell lines were purchased by the Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, and the cells were cultured in RPMI-1640 medium, which was
supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 U/mL
streptomycin and cultured in an atmosphere of 5% CO at 37 °C. Cells were collected for the
2
experiments in the logarithmic growth phase.
To establish the tumor-bearing mouse model, the cell lines were harvested and inoculated
subcutaneously into the right armpit region of the mice. On the 7th day, the tumor ascrites were obtained
and washed with sterile PBS. Under sterile condition, the tumor ascrites were diluted with sterile nomal
1
0
saline to 1 × 10 /L cell suspension. Tumor ascites were maintained in vivo in mice by transplantation of
6
0
.2 mL of ascites (2 × 10 cells) from the infected mice to the non-infected mice.
3
.4.2. In Vivo Tumor Xenograft Model
Each Kunming mouse (male or female, weight 20 ± 2 g) were inoculated with seven-day-old ascrite
6
(
0.2 mL, 2 × 10 cells) subcutaneously into the right front armpit. 24 h after implantation of tumor cells,
the mice were randomly divided into five test groups with 10 mice per group. Each mouse was weighed
immediately after inoculation. The mice were treated by oral gavage with test samples (60 mg/kg/day) or
normal saline (NS, 10 mL/kg) for ten days once daily. On day 11, the mice were sacrificed via cervical
dislocation, and the mouse and tumor were excised and weighed for evaluating the tumor growth
inhibition. The tumor inhibitory rate was calculated by the following formula:

Wcontrol Wtreated

Tumor inhibitory rate rate (%)  
100%
(
1)



Wcontrol

where Wcontrol and Wtreated were the average tumor weights of the control and treated mice, respectively.
EAC tumor-bearing mice were observed for mean survival time. The effect of glycyrrhizin analogs on
percentage increases in life span was calculated on the basis of mortality of the experimental mice:
ΣSurvival time (days) of each mouse in a group
Mean survival time 
(
(
2)
3)
Total number of mice
MST of treated group
%ILS 
 100
MST of control group

Molecules 2014, 19
. Conclusions
In summary, we synthesized glycyrrhizin analogs by glucuronidase biotransformation and alkaline
6379
4
isomerization, and evaluated their biological activities in vitro and in vivo. Anticancer activities of
monoglucuronide GAMGs were higher than those of bisglucuronide GAs, while 1the 8α-epimer showed
better activity than the 18β-epimer. Among them, 18α-GAMG displayed the most potent in vitro
activity, with IC values of 6.67, 7.43 and 15.76 μM against HepG2, Hela and A549, and in vivo
5
0
activity with inhibitory rates 39.8% and 49.7% for S180 and HepG2 tumor-bearing mice, respectively,
and it significantly enhanced the survival rate of EAC tumor-bearing mice to 45.4%. The docking
calculations showed that four analogs had better binding affinity to EGFR than the reference compound
erlotinib and their binding energy had the same trend as anticancer activities, which further proved the
correlation between anticancer activities and EGFR inhibitory activities of these compounds. In the
binding model, high active compound 18α-GAMG was nicely bound to EGFR via six hydrogen bonds
with ASP831, GLU738, THR766 (three bonds), LYS721 and one charge interaction, leading to the
increased anticancer activities of 18α-GAMG. Therefore, 18α-GAMG is of interest for further studies as
a potential anticancer agent. Further structural optimization of 18α-GAMG is ongoing using a variety of
rational design strategies [33].
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/5/6368/s1.
Acknowledgments
The authors wish to thank the National Natural Science Foundation of China (Nos. 21272008,
2
0802003), Science and Technological Fund of Anhui Province for Outstanding Youth (1408085J04).
Author Contributions
YongAn Yang and HaiLiang Zhu designed research; YongAn Yang, WenJian Tang, Xin Zhang,
JiWen Yuan and XinHua Liu performed research and analyzed the data; YongAn Yang and
WenJian Tang wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the four compounds are available from the authors.
©
2014 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 license
http://creativecommons.org/licenses/by/3.0/).
(