Design, preparation and studies regarding cytotoxic properties of glycyrrhetinic acid derivatives

Article abstract of DOI:10.1248/bpb.b19-00615

Glycyrrhetinic acid (GA) is a natural product with certain antitumor activity. In order to enhance the cytotoxicity, a total of eighteen derivatives of GA were designed and synthesized. Their cytotoxicity against MDA-MB-231cells (human breast cancer cells) and HeLa cells (human cervical cancer cells), were evaluated by the MTT method (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide). The results indicated that these target compounds have a wide molar activity range and some of them show better activity than the commercial drugs gefitinib and doxorubicin. Compound 6g induces apoptosis of 7, 10 and 44% of MDA-MB-231 cells at 5, 10, and 20μM, respectively.

Full text of DOI:10.1248/bpb.b19-00615

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Biol. Pharm. Bull. 43, 102–109 (2020)
Vol. 43, No. 1
Regular Article
Design, Preparation and Studies Regarding Cytotoxic Properties of
Glycyrrhetinic Acid Derivatives
Qing-Xuan Zheng,a,# Rui Wang,b,# Yan Xu,a Chang-Xin He,a Cai-Yun Zhao,a Zhi-Fang Wang,a
,a
Rui Zhang,a Wim Dehaen,b Hui-Jing Li,c and Qi-Yong Huai*
b
a Marine College, Shandong University; Weihai 264209, China: Molecular Design and Synthesis, Department
c
of Chemistry, KU Leuven; Celestijnenlaan 200F, B-3001 Leuven, Belgium: and School of Marine Science and
Technology, Harbin Institute of Technology at Weihai; Weihai 264209, China.
Received July 24, 2019; accepted September 4, 2019
Glycyrrhetinic acid (GA) is a natural product with certain antitumor activity. In order to enhance the
cytotoxicity, a total of eighteen derivatives of GA were designed and synthesized. Their cytotoxicity against
MDA-MB-231cells (human breast cancer cells) and HeLa cells (human cervical cancer cells), were evaluated
by the MTT method (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide). The results indicated
that these target compounds have a wide molar activity range and some of them show better activity than
the commercial drugs gefitinib and doxorubicin. Compound 6g induces apoptosis of 7, 10 and 44% of MDA-
MB-231 cells at 5, 10, and 20µM, respectively.
Key words glycyrrhetinic acid; cytotoxicity; MDA-MB-231; apoptosis
glycyrrhetinates, 8a–8c and 3-fluorobenzoyl benzyl glycyr-
rhetinates, 9a–9e. We tested the cytotoxicity against human
INTRODUCTION
Cancer, a type of diseases caused by abnormal cell growth, breast cancer, MDA-MB-231 cell lines and human cervical
has been the major reason of death around the world. Accord- cancer HeLa cell lines of all synthetic compounds, 6a–6g, 7a–
ing to data from WHO, there are about 9.6 million cancer 7c, 8a–8c and 9a–9e, Charts 1, 2 and 3, respectively, by the
deaths, accounting for 1/6 of the global death toll, and most 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
cancer deaths come from low- or middle-income countries in (MTT), method, with gefitinib and doxorubicin as standards.
2018.1) Due to the very high mortality, researchers are paying
increasing attention to the development of new drugs which
have a better selectivity and activity. Compared with the
construction of new skeletons of compounds, modification of
RESULTS AND DISCUSSION
Chemistry: Design and Synthesis In this work, a total
natural compounds may be seen as a more efficient and rapid of eighteen derivatives of GA were designed and synthesized.
way to reach these goals.2)
The overall route of synthesis of targeted compounds is shown
Glycyrrhetinic acid (GA, see Fig. 1), which is isolated from in Charts 1–3, respectively. The target compounds, 6a–6g and
Glycyrrhiza uralensis Fisch, Glycyrrhiza inflata Batalin and intermediates, 7a–7c, were synthesized starting from GA and
G. glabra L. and is the main biologically active component of the corresponding benzyl halides using K2CO3 base in N,N-
these plants, and GA derivatives show a variety of pharmaceu- dimethylformamide (DMF).
tical effects, for example, anticancer,3–7) anti-oxidation,8) anti-
4-Benzyloxy benzyl bromides, the benzyl halides for the
inflammatory,9,10) antiviral11–14) and anti-ulcer15) properties, etc. preparation of compounds 6a–6g, were synthesized following
In our previous work, we have reported on antitumor activity three steps, including Williamson etherification using K2CO3
of derivatives of GA, and some of these compounds showed in DMF,20) reduction of aldehydes with NaBH4 in methanol21)
better anti-tumor activity than GA itself and Gefitinib.16–18)
(MeOH) and bromination of alcohols using PBr3 in ethyl
However, one of the limiting factors for the application of ether22,23) (Et2O). In the Williamson reactions, an excess of
glycyrrhetinic acid is that its biological activity is too low. hydroxy benzaldehyde, is used involving easy purification by
It has been reported19) that the esterification of C30 carboxyl removal of the excess with base, such as K2CO3. Similarly,
groups could increase the antitumor activity of glycyrrhetinic a slight excess of sodium borohydride and phosphorus tri-
acid. Benzyl glycyrrhetinate, which was obtained from GA bromide allows complete conversion of the substrate for the
and benzyl bromide, has a better activity than ethyl glycyr- reduction and bromination reactions, respectively, without
rhetinate, which was synthesized by GA and ethyl bromide. column chromatography. We used dichloromethane (DCM)
Hence, we would like to enhance the lipophilicity of GA by and diethyl ether as solvents for the bromination reactions
introduction 4-(arylmethyl)benzyloxy glycyrrhetinates and ex- respectively and crude product could be used directly in the
pect that the activity would be increased. Compounds 6a–6g next step when Et2O was used as solent. Compound 8 was
were designed and synthesized according to this idea. To study prepared from compound 7 under the action of Dess–Martin
the effect of the C3 hydroxyl groups on the antitumor activity Periodinane in DCM. Acylation reactions of compound 7 were
of glycyrrhetinic acid, we designed 3-oxo-(substituted)benzyl performed using 4-dimethylaminopyridine (DMAP) base in
DCM at room temperature,24) overnight. Another method for
# These authors contributed equally to this work.
synthesis of compound 9 was to use pyridine as both solvent
*To whom correspondence should be addressed. e-mail: huaiqy01@163.com
© 2020 The Pharmaceutical Society of Japan

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Fig. 1. The Structure of 18β-GA, 1a, Benzyl Glycyrrhetinate 1b, Ethyl Glycyrrhetinate, 1c
Reagents and conditions: a) appropriate halogenated hydrocarbons, K2CO3, DMF, room temperature (r.t.), overnight; b) NaBH4, MeOH, 0–5°C for 30min and r.t. for
3–4h; c) PBr3, Et2O, 0–5°C for 30min and r.t. for 3–4h, d) GA, K2CO3, DMF, r.t., overnight.
Chart 1. The Synthetic Route of 4-(Arylmethyl)benzyloxy Glycyrrhetinates
Reagents and conditions: e) appropriate halogenated hydrocarbons, K2CO3, DMF, r.t., overnight; f) Dess-Martin peroxidation, DCM, r.t, 3–4h.
Chart 2. The Synthetic Route of 3-Oxo-(substituted)benzyl Glycyrrhetinate
Reagents and conditions: g) appreciate halogenated hydrocarbons, K2CO3, DMF, r.t., overnight; h) appropriate benzoyl chloride, 4-dimethylaminopyridine, DCM, 0–5°C
for 30min and r.t. overnight.
Chart 3. The Synthetic Route of 3-Fluorobenzoyl Benzyl Glycyrrhetinate
and base.25)
MTT method. All target compounds had a cytotoxicity with a
Antitumor Activity The IC50 values of the compounds wide molar range. 2″-Fluorobenzyloxy-3′-ethoxybenzyl glycyr-
against two cancer cell lines, human breast cancer cells, rhetinate (6g) and 3-oxo-2′,6′-dichlorobenzyl glycyrrhetinate
MDA-MB-231, and human cervical cancer cells, HeLa, are (8b) have a better activity against HeLa cells, with IC50 1.14
shown in Table 1. All data were determined in vitro with the and 2.04µM, respectively, than doxorubicin and gefitinib.

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Table 1. Inhibitory Effects of Target Compounds on the Growth of HeLa
Cells and MDA-MB-231 Cells
IC50 (µM)a)
Compound
MDA-MB-231
HeLa
>100
>100
>100
6a
>100
>100
>100
57.35
52.64
51.99
17.01
9.56
6b
6c
6d
30.75
6e
>100
>100
6f
6g
1.14
7a
22.16
10.24
17.53
7b
>100
52.64
>100
>100
>100
>100
60.54
56.82
>100
17.03
>100
10.38
4.78
7c
8a
>100
2.04
>100
>100
63.92
58.46
>100
34.48
>100
8b
8c
9a
9b
9c
9d
Fig. 2. Apoptosis of MDA-MB-231 Cells Was Detected by Annexin
V/7-AAD Assay after Coincubation with Various Concentrations of 6g
(Drug Concentrations Were 0, 5, 10 and 20µM) for 24h
9e
GA26,27)
Gefitinib
Doxorubicin
6.78
2.40
by compound 8b. Compared to benzyl glycyrrhetinates, com-
pounds 7a, 7b and 7c, benzyl fluorobenzoyl glycyrrhetinates,
compounds 9a, 9b, 9c, 9d and 9e, have a decreasing activity
a)
IC50 is 50% inhibitory concentration.
Compounds 6d, 7a, 7b, 7c, 9b, 9c and 9e have a moderate against both MDA-MB-231 cells and HeLa cells. This result
activity against HeLa cells, with values of IC50 from 22.16 demonstrated that the hydroxy, and perhaps the active hydro-
to 63.92µM. For MDA-MB-231 cells, only compound 7a has gen, at C3 position is a key group or fragment.
a better activity than gefitinib, but the activity is lower than
doxorubicin. Compounds 6d, 6e, 6f, 6g, 7c, 9b, 9c and 9e
have a moderate activity, with values of IC50 from 17.01 to
60.54µM.
CONCLUSION
In this work, a total of eighteen derivatives of GA were
Apoptosis Induced by Compound 6g Apoptosis is one designed and synthesized and the cytotoxicity against human
of the mechanisms of cell death and is a common response breast cancer cells, MDA-MB-231, and human cervical cancer
to anticancer agents. For a more in-depth study of whether cells, HeLa, was detected by the MTT method. The result
the anti MDA-MB-231 activity of compound 6g is related to indicated that 2″-fluorobenzyloxy-3′ ethoxybenzyl glycyr-
apoptosis, we detected the apoptosis rate by the Annexin V/7- rhetinate (6g) and 3-oxo-2′,6′-dichlorobenzyl glycyrrhetinate
AAD (7-aminoactinomycin D, a kind of dyeing agent used (8b) have a better activity against HeLa cells, with IC50 1.14
for apoptosis detection) double staining method (Fig. 2). The and 2.04µM, respectively, than doxorubicin and gefitinib.
result indicated that compound 6g, induces apoptosis in 7, 10, Compound 7a has a better activity than gefitinib, but less than
and 44% of MDA-MB-231 cells at 5, 10, and 20µM, respec- doxorubicin. Half of these compounds can be studied as lead
tively, after treatment of different concentrations (0µM as compounds for each cell line. Compound 6g induces apoptosis
control, 5, 10 and 20µM).
of MDA-MB-231 in 7, 10, and 44% of MDA-MB-231 cells at
Structure–Activity Relationship According to this study, 5, 10, and 20µM, respectively.
an introduction of halogenated benzyl, compounds 7a and 7c,
led an increasing of cytotoxicity for both MDA-MB-231 cells
and HeLa cells. Compound 7b, which has a 2,4-dichloroben-
zyl fragment, shows a good cytotoxicity and selectivity over
HeLa cells. The results illustrated that halogenated benzyl
glycyrrhetinates show a good cytotoxicity to MDA-MB-231
MATERIALS AND METHODS
Chemistry
General Information
All organic reagents were purchased from Aladdin (Shang-
cells and HeLa cells. As we assumed, the newly synthesized hai, China), Energy-e (Shanghai, China), Macklin (Shanghai,
4-(arylmethyl)benzyloxy glycyrrhetinates, compounds 6d, China) and Ehsy (Shanghai, China) and used without further
6e, 6f and 6g, will also show good cytotoxicity than glycyr- purification. All of the inorganic reagents were bought from
rhetinic acid. Among them, compound 6g shows the best Xinyue Chemical and Glass Co., Ltd. (Weihai, China). DCM,
cytotoxicity to both MDA-MB-231 cells and HeLa cells and Et2O, n-hexane and MeOH were obtained from Fuyu Chemi-
selectivity over HeLa cells. The oxidation of the C3 hydroxyl cal Co., Ltd. (Tianjin, China) via dealer Haituo Chemical Co.,
groups, compounds 8a and 8c, reduces the antitumor activity Ltd. (Weihai, China). Silica gel (200–300 mesh) for column
of glycyrrhetinic acid except for the inhibition of HeLa cells chromatography and silica gel plates (GF254) were purchased

Vol. 43, No. 1 (2020)
Biol. Pharm. Bull.
105
from Hailang Chemical Co., Ltd. (Qingdao, China). All the by column chromatography, with silica gel as the stationary
reactions were detected on silica gel plates (GF254) under UV phase and eluting with n-hexane:EtOAc, 1:1, v/v.
1
light, 254nm. H-NMR (400MHz) and 13C-NMR (101 MHz)
General Method for the Synthesis of 9
spectra were recorded on a Bruker-400 instrument (Bruker;
A solution of 7 (1mmol), DMAP (0.18g, 1.5mmol) and
Fallanden, Switzerland) at 25°C with tetramethylsilane (TMS) the corresponding benzoyl chloride (1mmol) in dried DCM
as an internal standard and CDCl3 as solvents. Mass spectra (20mL) was stirred in a 50mL round bottom flask at 30°C
were measured on an LTQ Orbitrap mass spectrometer (Ther- overnight. Saturated brine (20mL) was added after the reac-
mo Fisher Scientific, U.S.A.).
tion was finished and the organic layer was washed with a
General Method for the Synthesis of 3
solution of sodium bicarbonate (5% (w/w) 30mL×2). Then
A mixture of 2 (10mmol), the corresponding halogenated the solvent was removed in vacuo to furnish target compound
hydrocarbons (8mmol), K2CO3 (2.07g, 15mmol) in DMF 9 and the crude products were purified by column chromatog-
(5mL) was stirred in a 50mL round bottom flask at 25°C for raphy, with n-hexane:EtOAc, from 8:1 to 4:1 (v/v).
8–12h. Saturated brine was added when the reaction was over,
and the mixture was extracted with DCM (25mL×2). The
combined organic layers were washed with a saturated solu- (6a)
tion of potassium carbonate, until the spot of 2 was invisible
Characterization of Target Compounds
4-((2,4-Dichlorobenzyl)oxy)-3-methoxybenzyl Glycyrrhetinate
1
White solid. Yield (Y)=68%. H-NMR (400MHz, CDCl3)
on the TLC plate, and with water, and dried with anhydrous δ: 7.51 (d, J=8.3Hz, 1H), 7.40 (d, J=2.1Hz, 1H), 7.25 (dd,
sodium sulfate. Solvent was removed in vacuo to furnish a J=8.4, 2.1Hz, 1H), 6.93 (d, J=1.8Hz, 1H), 6.89 (dd, J=8.2,
white solid and the product can be used for the next step with- 1.8Hz, 1H), 6.84 (d, J=8.2Hz, 1H), 5.56 (s, 1H), 5.20 (s,
out further purification.
2H), 5.08 (dd, J=26.3, 12.0Hz, 2H), 3.91 (s, 3H), 3.22 (dd,
J=10.6, 5.6Hz, 1H), 2.78 (dt, J=13.2, 3.3Hz, 1H), 2.32 (s,
General Method for the Synthesis of 4
A mixture of 3 (8mmol) in MeOH (35mL) was cooled 1H), 2.06–1.94 (m, 3H), 1.92 (dd, J=13.5, 2.7Hz, 1H), 1.80
down to 0°C and then NaBH4 (0.45g, 12mmol) was added in (td, J=13.5, 4.2Hz, 1H), 1.71–1.54 (m, 5H), 1.51 (s, 2H), 1.43
ten portions. The mixture was stirred at 0°C for 30min and (d, J=13.0Hz, 2H), 1.38 (d, J=4.3Hz, 1H), 1.34 (s, 3H),
at room temperature for 3–4h. An equal volume of saturated 1.27 (ddd, J=8.7, 5.9, 2.0Hz, 2H), 1.20 (d, J=12.5Hz, 1H),
brine was added into the mixture when the reaction was over. 1.14 (s, 3H), 1.13 (s, 3H), 1.10 (s, 3H), 1.00 (s, 3H), 0.95 (dd,
MeOH was removed in vacuo and the resulting mixture was J=12.8, 4.5Hz, 1H), 0.80 (s, 3H), 0.71 (s, 3H), 0.68 (s, 1H).
extracted by EtOAc (25mL×2). The combined organic layers 13C-NMR (101MHz, CDCl3) δ: 200.07, 176.26, 169.08, 149.65,
were washed with dilute hydrochloric acid (30mL×2) and 147.67, 133.95, 133.47, 132.89, 129.95, 129.52, 129.10, 128.48,
water (30mL×2), dried with anhydrous sodium sulfate and 127.34, 121.26, 113.96, 112.38, 78.76, 77.24, 67.68, 66.11, 61.81,
the solvent removed in vacuo to give a white solid.
General Method for the Synthesis of 5
56.09, 54.94, 48.21, 45.33, 43.98, 43.17, 41.11, 39.14, 37.66,
37.08, 32.75, 31.75, 31.20, 28.47, 28.24, 28.10, 27.31, 26.46,
A solution of 4 (8mmol) in Et2O (30mL) was cooled to 26.40, 23.38, 18.68, 17.49, 16.37, 15.59. High resolution (HR)
0°C and then a solution of PBr3 (1.13mL, 12mmol) in Et2O MS electrospray ionization (ESI+): Calculated for C45H59Cl2O6
(10mL) was added slowly in a dropwise manner, about one (M+H)+: 765.3689, found: 765.3707.
drop per second. The mixture was stirred at 0°C for 3–4h and
the reaction was monitored by TLC. After the reaction was (6b)
completed, ice saturated sodium bicarbonate was added in a
4-((2-Chlorobenzyl)oxy)-3,5-dimethylbenzyl Glycyrrhetinate
1
White solid. Y=72%. H-NMR (400MHz, CDCl3) δ: 7.72
dropwise manner, and the organic layer was extracted from (dd, J=7.5, 1.5Hz, 1H), 7.39 (dd, J=7.8, 1.4Hz, 1H), 7.33 (tt,
the system and washed with saturated sodium bicarbonate and J=3.9, 1.9Hz, 1H), 7.29 (dd, J=7.6, 1.8Hz, 1H), 7.05 (s, 2H),
water/ice, dried with anhydrous sodium sulfate and the solvent 5.51 (s, 1H), 5.19 (d, J=11.9Hz, 1H), 4.94 (d, J=11.9Hz, 1H),
was removed in vacuo to give a white or yellow solid.
General Method for the Synthesis of 8
4.89 (s, 2H), 3.22 (dd, J=10.6, 5.6Hz, 1H), 2.78 (dt, J=13.4,
3.3Hz, 1H), 2.32 (s, 7H), 2.07–1.88 (m, 4H), 1.80 (td, J=13.6,
A
mixture of 7 (1mmol) and Dess–Martin reagent 4.4Hz, 1H), 1.68–1.54 (m, 5H), 1.52–1.36 (m, 5H), 1.34 (s,
(1.5mmol) in DCM (25mL) was stirred at 25°C for about 3H), 1.29 (t, J=7.4Hz, 2H), 1.16 (s, 3H), 1.12 (s, 3H), 1.09
30min. Then, the mixture was washed with a saturated solu- (s, 3H), 1.00 (s, 3H), 0.97–0.93 (m, 1H), 0.80 (s, 3H), 0.73 (s,
tion of sodium bicarbonate (25mL×2) and water (25mL×2). 3H), 0.69 (d, J=10.4Hz, 1H); 13C-NMR (101MHz, CDCl3) δ:
The organic layer was dried with anhydrous sodium sulfate 200.00, 176.30, 169.04, 155.67, 135.49, 132.59, 131.90, 131.48,
and the solvent was removed in vacuo to give the compound. 129.26, 129.17, 129.09, 128.92, 128.46, 126.97, 78.78, 77.24,
Crude products were purified bycolumn chromatography, with 70.75, 65.97, 61.78, 54.94, 48.15, 45.29, 43.94, 43.17, 41.15,
silica gel as the stationary phase and eluting with n-hexane: 39.14, 37.64, 37.06, 32.74, 31.74, 31.25, 28.42, 28.24, 28.10,
EtOAc, 1:1, v/v.
27.32, 26.48, 26.41, 23.35, 18.69, 17.48, 16.38, 16.36, 15.59.
General Method for the Synthesis of 6 and 7
HRMS (ESI+): Calculated for C46H62ClO5 (M+H)+: 729.4286,
A mixture of GA (1mmol), 5 or appropriate halogenated found: 729.4307.
hydrocarbon (1mmol), K2CO3 (0.21g, 1.5mmol) in DMF
(5mL) was stirred in a 50mL round bottom flask at 25°C for (6c)
8–12h. Saturated brine was added when the reaction was over,
4-((2,4-Dichlorobenzyl)oxy)-3-ethoxybenzyl Glycyrrhetinate
White solid. Y=81%. 1H-NMR (400MHz, CDCl3) δ:
and the mixture was extracted with DCM (25mL×2). The 7.33 (d, J=7.9Hz, 2H), 7.21 (dd, J=8.5, 7.5Hz, 1H), 7.05
combined organic layers were washed with water and dried (d, J=8.5Hz, 1H), 6.94–6.88 (m, 2H), 5.61 (s, 1H), 5.32 (s,
with anhydrous sodium sulfate. The solvent was removed in 2H), 5.13–5.03 (m, 2H), 4.10 (dq, J=17.5, 7.1Hz, 3H), 3.22
vacuo to furnish a white solid and the product was purified (dd, J=10.7, 5.6Hz, 1H), 2.79 (dt, J=13.3, 3.2Hz, 1H), 2.30

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(d, J=15.0Hz, 1H), 2.07–1.96 (m, 4H), 1.91 (dd, J=13.2, 3H), 1.28–1.24 (m, 1H), 1.12 (s, 3H), 1.02 (s, 3H), 0.99 (s, 1H).
10.4Hz, 1H), 1.80 (td, J=13.5, 4.3Hz, 1H), 1.63 (qd, J=13.3, 13C-NMR (101MHz, CDCl3) δ: 200.07, 176.27, 169.12, 149.26,
7.3Hz, 5H), 1.52–1.43 (m, 2H), 1.40 (t, J=7.0Hz, 4H), 147.99, 130.66, 130.62, 130.17, 128.50, 127.60, 127.56, 121.27,
1.35 (s, 3H), 1.34–1.29 (m, 1H), 1.29–1.22 (m, 2H), 1.19 (t, 118.99, 118.74, 115.02, 114.00, 78.77, 77.25, 66.10, 64.70, 64.66,
J=7.9Hz, 1H), 1.14 (d, J=4.6Hz, 6H), 1.11 (s, 3H), 1.00 64.62, 61.80, 54.93, 48.17, 45.34, 43.98, 43.17, 41.10, 39.14,
(s, 3H), 0.96 (dd, J=12.4, 4.3Hz, 1H), 0.80 (s, 3H), 0.72 (s, 37.65, 37.08, 32.74, 31.74, 31.20, 28.46, 28.24, 28.10, 27.31,
3H), 0.66 (d, J=13.4Hz, 1H). 13C-NMR (101MHz, CDCl3) δ: 26.45, 26.39, 23.39, 18.66, 17.48, 16.37, 15.59. HRMS (ESI+):
200.04, 176.27, 169.10, 150.13, 148.71, 137.21, 132.53, 130.77, Calculated for C46H61BrFO5 (M+H)+: 807.3630, found:
130.24, 128.55, 128.39, 121.45, 117.37, 114.83, 78.77, 77.24, 809.3735 (M+2+H)+.
67.31, 66.12, 64.99, 61.80, 54.95, 48.13, 45.34, 43.98, 43.18,
41.13, 39.14, 37.67, 37.09, 32.76, 31.75, 31.22, 28.46, 28.25, (6g)
28.11, 27.32, 26.46, 26.42, 23.39, 18.67, 17.49, 16.37, 15.59,
4-((2-Fluorobenzyl)oxy)-3-methoxybenzyl Glycyrrhetinate
1
White solid. Y=85%. H-NMR (400MHz, CDCl3) δ: 7.54
14.90. HRMS (ESI+): Calculated for C46H61Cl2O6 (M+H)+: (dt, J=7.5, 3.8Hz, 1H), 7.32–7.24 (m, 1H), 7.13 (t, J=7.2Hz,
779.3845, found: 779.3865.
4-((2,6-Dichlorobenzyl)oxy)-3,5-dimethylbenzyl J=8.2, 1.7Hz, 1H), 5.59 (s, 1H), 5.20 (s, 2H), 5.10–5.02 (m,
Glycyrrhetinate (6d) 2H), 4.10 (q, J=7.0Hz, 2H), 3.22 (dd, J=10.7, 5.6Hz, 1H),
1H), 7.10–7.03 (m, 1H), 6.92 (dd, J=4.9, 3.2Hz, 2H), 6.88 (dd,
1
White solid. Y=75%. H-NMR (400MHz, CDCl3) δ: 7.36 2.79 (dt, J=13.3, 3.3Hz, 1H), 2.32 (s, 1H), 2.08–1.96 (m, 3H),
(d, J=8.0Hz, 2H), 7.24 (dd, J=8.6, 7.4Hz, 1H), 7.02 (s, 2H), 1.91 (dt, J=20.6, 10.9Hz, 1H), 1.80 (td, J=13.5, 4.2Hz, 1H),
5.56 (s, 1H), 5.19–5.12 (m, 3H), 4.97 (d, J=11.9Hz, 1H), 3.23 1.72–1.54 (m, 7H), 1.50–1.37 (m, 5H), 1.35 (s, 3H), 1.31 (dd,
(dd, J=10.7, 5.6Hz, 1H), 2.80 (dt, J=13.3, 3.3Hz, 1H), 2.34 J=8.1, 5.1Hz, 1H), 1.28–1.22 (m, 2H), 1.18 (t, J=8.3Hz, 1H),
(s, 6H), 2.03 (dd, J=20.3, 7.4Hz, 3H), 1.97–1.90 (m, 1H), 1.13 (d, J=1.1Hz, 6H), 1.10 (s, 3H), 1.00 (s, 3H), 0.95 (dd,
1.81 (td, J=13.6, 4.3Hz, 1H), 1.64 (dt, J=19.6, 8.5Hz, 5H), J=12.4, 4.2Hz, 1H), 0.80 (s, 3H), 0.71 (s, 3H), 0.68 (s, 1H).
1.52 (s, 1H), 1.48–1.39 (m, 3H), 1.36 (s, 3H), 1.33–1.24 (m, 13C-NMR (101MHz, CDCl3) δ: 200.07, 176.29, 169.14, 149.23,
3H), 1.17 (s, 3H), 1.16 (s, 1H), 1.13 (s, 3H), 1.11 (s, 3H), 1.01 148.32, 129.86, 129.54, 129.50, 129.44, 129.36, 128.52, 124.20,
(s, 4H), 0.99–0.95 (m, 1H), 0.81 (s, 3H), 0.74 (s, 3H), 0.70 124.16, 121.33, 115.26, 115.05, 114.86, 114.10, 78.76, 77.26,
(d, J=10.5Hz, 1H). 13C-NMR (101MHz, CDCl3) δ: 199.99, 66.15, 64.69, 61.80, 54.93, 48.14, 45.34, 43.98, 43.17, 41.10,
176.30, 169.07, 155.53, 137.09, 132.96, 131.80, 131.67, 130.30, 39.14, 37.67, 37.08, 32.75, 31.74, 31.20, 28.46, 28.25, 28.11,
129.15, 128.59, 128.50, 78.76, 77.37, 77.26, 77.05, 76.74, 68.63, 27.31, 26.45, 26.40, 23.39, 18.66, 17.49, 16.37, 15.60. HRMS
65.94, 61.78, 54.94, 48.10, 45.30, 43.93, 43.17, 41.12, 39.14, (ESI+): Calculated for C46H62FO6 (M+H)+: 729.4530, found:
37.64, 37.07, 32.75, 31.73, 31.25, 28.45, 28.25, 28.11, 27.32, 729.4551.
26.47, 26.42, 23.37, 18.69, 17.49, 16.78, 16.36, 15.60. HRMS
(ESI+): Calculated for C46H61Cl2O5 (M+H)+: 763.3896, found:
763.3907.
2′,4′-Dichlorobenzyl Glycyrrhetinate (7a)
White solid. Y=90%. H-NMR (400MHz, CDCl3) δ: 7.42
(d, J=2.0Hz, 1H), 7.38 (d, J=8.3Hz, 1H), 7.28 (dd, J=8.3,
1
4 -((2,4 -Dichlorobenzyl)oxy)-3,5-dimethylbenzyl 2.0Hz, 1H), 5.60 (s, 1H), 5.19 (s, 2H), 4.09 (dd, J=19.4,
Glycyrrhetinate (6e) 12.3Hz, 1H), 3.21 (dt, J=24.7, 12.4Hz, 1H), 2.83–2.70 (m,
1
White solid. Y=71%. H-NMR (400MHz, CDCl3) δ: 7.67 1H), 2.33 (s, 1H), 2.12–1.88 (m, 7H), 1.81 (tt, J=17.5, 8.8Hz,
(d, J=8.4Hz, 1H), 7.41 (d, J=2Hz, 1H), 7.32 (dd, J=2Hz, 1H), 1.71–1.53 (m, 5H), 1.43 (dd, J=23.7, 11.8Hz, 3H), 1.36
8.4Hz, 1H),7.05 (s, 2H), 5.48 (s, 1H), 5.30 (s, 1H), 5.20 (d, (s, 3H), 1.31 (t, J=5.2Hz, 1H), 1.28–1.23 (m, 3H), 1.18 (s,
J=12Hz, 1H), 4.92 (d, J=12Hz, 1H), 4.84 (s, 2H), 3.22 3H), 1.12 (d, J=6.3Hz, 6H), 1.00 (s, 3H), 0.95 (dd, J=13.0,
(q, J=5.6Hz, 10.8Hz, 1H), 2.80–2.75 (m, 1H), 2.29 (s, 6H), 4.1Hz, 1H), 0.81 (s, 3H), 0.77 (s, 3H), 0.70 (d, J=11.3Hz,
2.05–1.89 (m, 5H), 1.83–1.76 (m, 1H), 1.66–1.52 (m, 7H), 1H). 13C-NMR (101MHz, CDCl3) δ: 200.10, 175.94, 168.94,
1.52–1.36 (m, 3H), 1.34 (s, 3H), 1,32–1.24 (m, 3H), 1.16 (s, 134.78, 134.43, 132.27, 130.92, 129.45, 128.53, 127.25, 78.53,
3H), 1.11 (s, 3H), 1.09 (s, 3H), 1.00 (s, 3H), 0.80 (s, 3H), 0.73 63.09, 61.76, 60.36, 54.88, 48.15, 45.32, 44.12, 43.14, 40.95,
(s, 3H), 0.68 (d, J=10.4Hz, 1H); 13C-NMR (101MHz, CDCl3) 39.12, 37.65, 37.02, 32.71, 31.76, 31.08, 28.44, 28.32, 28.10,
δ: 200.03, 176.29, 169.08, 155.49, 134.19, 134.00, 133.11, 27.21, 26.39, 26.34, 23.36, 21.02, 18.63, 17.45, 15.63. HRMS
132.05, 131.38, 129.84, 129.21, 129.06, 128.41, 127.29, 78.76, (ESI+): Calculated for C37H50Cl2O6 (M+H)+: 629.3164, found:
77.25, 70.14, 65.92, 61.78, 54.92, 48.18, 45.29, 43.94, 43.16, 629.3173.
41.14, 39.13, 39.11, 37.63, 37.05, 32.73, 31.74, 31.24, 28.42,
28.23, 28.10, 27.30, 26.47, 26.40, 23.35, 18.67, 17.48, 16.37,
2′,6′-Dichlorobenzyl Glycyrrhetinate (7b)
1
White solid. Y=88%. H-NMR (400MHz, CDCl3) δ: 7.38
15.59. HRMS (ESI+): Calculated for C46H61Cl2O5 (M+H)+: (d, J=8.2Hz, 2H), 7.30–7.26 (m, 1H), 5.48–5.41 (m, 2H), 5.34
763.3896, found: 763.3911.
4-((4-Bromo-2-f luorobenzyl)oxy)-3-ethoxybenzyl 1H), 2.76 (dt, J=13.3, 3.3Hz, 1H), 2.29 (s, 1H), 2.20–2.07 (m,
Glycyrrhetinate (6f) 1H), 2.06–1.94 (m, 3H), 1.92–1.75 (m, 2H), 1.66–1.55 (m, 6H),
(d, J=11.6Hz, 1H), 5.30 (s, 1H), 3.19 (dt, J=35.7, 17.9Hz,
1
White solid. Y=75%. H-NMR (CDCl3, 400MHz) δ: 7.74 1.38 (dd, J=11.7, 9.3Hz, 4H), 1.32 (s, 3H), 1.26 (t, J=7.1Hz,
(t, J=8.4Hz, 1H), 7.61–7.55 (m, 3H), 7.20 (s, 2H), 5.88 (s, 1H), 1.17 (s, 3H), 1.12 (d, J=5.9Hz, 6H), 0.99 (s, 3H), 0.93
1H), 5.46 (s, 2H), 5.38 (q, J=24Hz, 12Hz, 2H), 4.40 (q, (dd, J=13.1, 4.6Hz, 1H), 0.80 (s, 3H), 0.67 (t, J=9.2Hz, 1H).
J=12Hz, 8Hz, 2H), 3.56–3.51 (m, 1H), 3.12–3.07 (m, 1H), 13C-NMR (101MHz, CDCl3) δ: 200.16, 176.11, 169.02, 136.85,
2.63 (s, 1H), 2.36–2.28 (m, 3H), 2.25–2.20 (m, 1H), 2.14–2.07 131.47, 130.73, 128.52, 128.47, 78.73, 61.76, 61.37, 60.41,
(m, 1H), 2.02–1.87 (m, 6H), 1.77–1.72 (m, 5H), 1.70–1.68 54.90, 48.25, 45.33, 44.31, 43.13, 40.94, 39.13, 37.66, 37.04,
(m, 1H), 1.66 (s, 3H), 1.64–1.61 (m, 1H), 1.59–1.57 (m, 2H), 32.75, 31.81, 31.14, 28.44, 28.38, 28.10, 27.28, 26.48, 26.38,
1.55–1.49 (m, 1H), 1.44 (d, J=4Hz, 6H), 1.41 (s, 3H), 1.31 (s, 23.30, 18.67, 17.48, 16.38, 15.59. HRMS (ESI+): Calculated for

Vol. 43, No. 1 (2020)
Biol. Pharm. Bull.
107
C37H50Cl2O6 (M+H)+: 629.3164, found: 629.3180.
2′-Chlorobenzyl Glycyrrhetinate (7c)
55.41, 48.21, 47.78, 45.19, 44.17, 43.29, 41.08, 39.78, 37.66,
36.68, 34.23, 32.12, 31.82, 31.16, 28.48, 28.38, 26.52, 26.41,
White solid. Y=75%. 1H-NMR (400MHz, CDCl3) δ: 23.30, 21.43, 18.80, 18.52, 15.68. HRMS (ESI+): Calculated for
7.48–7.37 (m, 2H), 7.32–7.29 (m, 1H), 7.26 (d, J=5.4Hz, 2H), C37H50ClO4 (M+H)+: 593.3398, found: 593.3408.
5.53 (d, J=14.9Hz, 1H), 5.34–5.13 (m, 2H), 3.22 (dd, J=10.7,
5.6Hz, 1H), 2.78 (dt, J=13.4, 3.4Hz, 1H), 2.30 (d, J=16.3Hz,
3-(2′-Fluorobenzoyl)-2″-chlorobenzyl Glycyrrhetinate (9a)
White solid. Y=34%. H-NMR (400MHz, CDCl3) δ: 7.94
1
1H), 2.13–2.01 (m, 3H), 1.99–1.90 (m, 1H), 1.81 (td, J=13.6, (t, J=7.0Hz, 1H), 7.54–7.46 (m, 1H), 7.46–7.38 (m, 2H),
4.4Hz, 1H), 1.71–1.51 (m, 6H), 1.50–1.36 (m, 3H), 1.35 (s, 7.33–7.28 (m, 2H), 7.20 (t, J=7.6Hz, 1H), 7.16–7.09 (m, 1H),
3H), 1.35–1.22 (m, 3H), 1.19 (s, 3H), 1.13 (s, 3H), 1.11 (s, 3H), 5.56 (s, 1H), 5.33–5.14 (m, 2H), 2.85 (d, J=13.7Hz, 1H),
1.00 (s, 3H), 0.98–0.89 (m, 1H), 0.80 (s, 3H), 0.76 (s, 3H), 0.69 2.38 (s, 1H), 2.17–1.92 (m, 4H), 1.82 (dd, J=15.5, 8.3Hz,
(d, J=10.3Hz, 1H). 13C-NMR (101MHz, CDCl3) δ: 200.18, 3H), 1.72–1.57 (m, 3H), 1.45 (dt, J=27.9, 8.4Hz, 3H), 1.37
176.09, 169.01, 133.84, 133.69, 130.19, 129.75, 129.69, 128.56, (s, 3H), 1.36–1.23 (m, 5H), 1.20 (s, 6H), 1.13 (s, 3H), 1.01
126.98, 78.77, 77.24, 63.80, 61.79, 54.92, 48.19, 45.36, 44.17, (s, 3H), 0.98 (s, 3H), 0.87 (d, J=11.0Hz, 2H), 0.77 (s, 3H).
43.17, 41.03, 39.14, 37.67, 37.06, 32.76, 31.81, 31.17, 28.44, 13C-NMR (101MHz, CDCl3) δ: 199.96, 176.02, 169.02, 133.78,
28.41, 28.10, 27.30, 26.47, 26.40, 23.37, 18.67, 17.49, 16.39, 133.65, 132.06, 130.14, 129.70, 129.64, 128.48, 126.94, 123.86,
15.59. HRMS (ESI+): Calculated for C37H52ClO4 (M+H)+: 123.83, 119.29, 117.04, 116.82, 81.99, 63.75, 61.65, 55.01, 48.18,
595.3554, found: 595.3560.
45.36, 44.14, 43.16, 40.98, 38.79, 38.26, 37.63, 36.93, 32.67,
31.78, 31.14, 29.67, 28.41, 28.37, 28.10, 26.43, 26.37, 23.51,
3-Oxo-2′,4′-dichlorobenzyl Glycyrrhetinate (8a)
1
White solid. Y=70%. H-NMR (400MHz, CDCl3) δ: 7.44 23.32, 18.66, 17.39, 16.83, 16.43. HRMS (ESI+): Calculated for
(d, J=2.0Hz, 1H), 7.37 (d, J=8.3Hz, 1H), 7.27 (s, 1H), 5.64 C44H55ClFO5 (M+H)+: 717.3717, found: 717.3731.
(s, 1H), 5.19 (s, 2H), 2.96 (ddd, J=13.5, 7.0, 4.0Hz, 1H), 2.64
(ddd, J=15.8, 11.2, 7.1Hz, 1H), 2.44–2.31 (m, 2H), 2.12 (dd, (9b)
J=13.4, 3.3Hz, 1H), 2.08–1.90 (m, 3H), 1.84 (td, J=13.6,
3-(3′-Fluorobenzoyl)-2″,6″-dichlorobenzyl Glycyrrhetinate
1
White solid. Y=40%. H-NMR (400MHz, CDCl3) δ: 7.83
4.3Hz, 1H), 1.74–1.50 (m, 5H), 1.49–1.39 (m, 3H), 1.36 (s, (d, J=7.7Hz, 1H), 7.70 (d, J=8.3Hz, 1H), 7.45–7.34 (m, 3H),
3H), 1.31 (d, J=6.5Hz, 2H), 1.27 (s, 3H), 1.21 (d, J=8.6Hz, 7.28 (d, J=11.1Hz, 1H), 7.23 (s, 1H), 5.48–5.42 (m, 2H), 5.34
1H), 1.18 (s, 3H), 1.16 (s, 3H), 1.09 (s, 3H), 1.06 (s, 3H), 1.05 (d, J=11.6Hz, 1H), 4.75 (dd, J=11.5, 5.0Hz, 1H), 2.83 (d,
(s, 1H), 0.79 (s, 3H), 13C-NMR (101MHz, CDCl3) δ: 217.22, J=13.4Hz, 1H), 2.35 (s, 1H), 2.13 (d, J=9.6Hz, 1H), 2.01
199.38, 175.99, 169.43, 134.93, 134.60, 132.33, 131.04, 129.57, (d, J=13.2Hz, 2H), 1.91–1.65 (m, 5H), 1.62 (d, J=13.5Hz,
128.53, 127.28, 77.24, 63.20, 61.07, 55.43, 48.23, 47.79, 45.21, 2H), 1.55 (s, 2H), 1.45 (dd, J=27.2, 15.3Hz, 4H), 1.34 (s, 3H),
44.18, 43.31, 41.06, 39.79, 37.68, 36.70, 34.23, 32.13, 31.83, 1.25 (s, 1H), 1.19 (d, J=7.7Hz, 6H), 1.14 (s, 3H), 1.03 (s, 3H),
31.13, 28.52, 28.36, 26.49, 26.39, 23.33, 21.43, 18.80, 18.53, 0.97 (d, J=5.6Hz, 2H), 0.94 (s, 3H), 0.86 (d, J=11.6Hz, 1H),
15.68. HRMS (ESI+): Calculated for C37H49Cl2O4 (M+H)+: 0.81 (s, 3H). 13C-NMR (101MHz, cdcl3) δ: 199.92, 176.07,
627.3008, found: 627.3026.
169.08, 136.83, 131.44, 130.68, 129.95, 129.87, 128.48, 128.39,
125.21, 119.87, 116.48, 116.25, 81.76, 77.19, 61.60, 61.35, 55.01,
3-Oxo-2′,6′-dichlorobenzyl Glycyrrhetinate (8b)
1
White solid. Y=75%. H-NMR (400MHz, CDCl3) δ: 7.41– 48.26, 45.33, 44.29, 43.13, 40.90, 38.74, 38.38, 37.63, 36.89,
7.36 (m, 2H), 7.30–7.28 (m, 1H), 5.45 (d, J=11.7Hz, 1H), 32.65, 31.79, 31.12, 28.42, 28.34, 28.18, 26.46, 26.35, 23.53,
5.34 (d, J=11.6Hz, 1H), 2.93 (ddd, J=13.5, 7.0, 4.0Hz, 1H), 23.24, 18.66, 17.37, 16.94, 16.41. HRMS (ESI+): Calculated for
2.62 (ddd, J=15.9, 11.1, 7.1Hz, 1H), 2.40–2.30 (m, 2H), 2.15 C44H54Cl2FO5 (M+H)+: 751.3327, found: 751.3340.
(dd, J=13.3, 3.2Hz, 1H), 2.07–1.96 (m, 2H), 1.92–1.77 (m, 2H),
1.74–1.48 (m, 5H), 1.49–1.35 (m, 4H), 1.33 (s, 3H), 1.32–1.28 Glycyrrhetinate (9c)
(m, 2H), 1.26 (s, 3H), 1.21 (s, 1H), 1.17 (d, J=8.6Hz, 6H),
3- (2′, 5′-Dif luoroben zoyl) -2 ″,6″- d ich loroben z yl
White solid. Y=40%. 1H-NMR (400MHz, CDCl3) δ:
1.09 (s, 3H), 1.06 (s, 3H), 1.03–0.99 (m, 1H), 0.82 (s, 3H); 7.60 (dd, J=10.1, 6.8Hz, 1H), 7.38 (d, J=8.0Hz, 2H), 7.28
13C-NMR (101MHz, CDCl3) δ: 217.26, 199.40, 176.07, 169.53, (d, J=8.4Hz, 1H), 7.19 (dd, J=10.0, 6.4Hz, 1H), 7.10 (td,
136.87, 131.47, 130.74, 128.52, 128.38, 77.25, 61.40, 61.01, J=9.3, 4.1Hz, 1H), 5.48–5.41 (m, 2H), 5.34 (d, J=11.7Hz,
55.40, 48.30, 47.77, 45.17, 44.31, 43.26, 41.00, 39.77, 37.65, 1H), 4.76 (dd, J=10.7, 5.6Hz, 1H), 2.83 (d, J=13.6Hz, 1H),
36.66, 34.21, 32.12, 31.83, 31.13, 28.48, 28.36, 26.54, 26.41, 2.34 (s, 1H), 2.13 (d, J=10.3Hz, 1H), 2.01 (d, J=13.0Hz,
23.23, 21.41, 18.80, 18.53, 15.69. HRMS (ESI+): Calculated for 2H), 1.84 (dd, J=36.5, 10.0Hz, 4H), 1.72–1.51 (m, 5H),
C37H49Cl2O4 (M+H)+: 627.3008, found: 627.3020.
3-Oxo-2′-chlorobenzyl Glycyrrhetinate (8c)
1.50–1.38 (m, 3H), 1.34 (s, 3H), 1.25 (t, J=16.3Hz, 2H), 1.19
(d, J=3.8Hz, 6H), 1.13 (s, 3H), 1.07 (d, J=18.1Hz, 1H), 1.00
White solid. Y=72%. 1H-NMR (400MHz, CDCl3) δ: (s, 3H), 0.96 (s, 3H), 0.85 (d, J=11.5Hz, 1H), 0.81 (s, 3H).
7.46–7.39 (m, 2H), 7.33–7.28 (m, 2H), 7.27 (s, 1H), 5.59 (s, 13C-NMR (101MHz, CDCl3) δ: 199.93, 176.07, 169.09, 163.15,
1H), 5.24 (dd, J=46.4, 12.8Hz, 2H), 2.95 (ddd, J=13.4, 7.0, 159.18, 156.73, 156.65, 136.82, 131.43, 130.69, 128.48, 128.39,
4.0Hz, 1H), 2.63 (ddd, J=15.9, 11.2, 7.1Hz, 1H), 2.42 (s, 82.55, 77.19, 61.60, 61.35, 54.97, 48.26, 45.32, 44.29, 43.12,
1H), 2.36 (ddd, J=15.8, 6.4, 4.1Hz, 1H), 2.12 (dd, J=13.4, 40.89, 38.74, 38.24, 37.63, 36.88, 32.65, 31.79, 31.11, 29.68,
3.6Hz, 1H), 2.08–1.91 (m, 3H), 1.83 (td, J=13.6, 4.2Hz, 1H), 28.42, 28.35, 28.09, 26.46, 26.35, 23.45, 23.25, 18.66, 17.38,
1.73–1.50 (m, 4H), 1.43 (ddd, J=17.6, 9.6, 4.8Hz, 2H), 1.36 (s, 16.81, 16.43. HRMS (ESI+): Calculated for C44H53Cl2F2O5
3H), 1.35–1.28 (m, 3H), 1.27 (s, 3H), 1.25–1.20 (m, 1H), 1.20 (M+H)+: 769.3233, found: 769.3232.
(s, 3H), 1.15 (s, 3H), 1.09 (d, J=7.6Hz, 3H), 1.07 (s, 3H), 1.03
(dd, J=13.7, 2.0Hz, 1H), 0.78 (s, 3H). 13C-NMR (101MHz, (9d)
CDCl3) δ: 217.25, 199.41, 176.04, 169.51, 133.86, 133.67,
3-(4′-Fluorobenzoyl)-2″,4″-dichlorobenzyl Glycyrrhetinate
1
White solid. Y=45%. H-NMR (400MHz, CDCl3) δ: 8.05
130.22, 129.78, 129.70, 128.46, 126.98, 77.25, 63.82, 61.03, (dd, J=8.5, 5.6Hz, 2H), 7.44 (d, J=1.7Hz, 1H), 7.37 (d,

108
Biol. Pharm. Bull.
Vol. 43, No. 1 (2020)
J=8.3Hz, 1H), 7.29 (d, J=1.7Hz, 1H), 7.11 (t, J=8.6Hz, tal Research Funds for the Innovation and Entrepreneurship
2H), 5.62 (s, 1H), 5.19 (s, 2H), 4.75 (dd, J=11.3, 4.7Hz, 1H), Foundation from Huancui District of Wehai City, the project of
2.85 (d, J=13.7Hz, 1H), 2.39 (s, 1H), 2.16–1.91 (m, 4H), 1.81 Weihai Science and Technology Bureau (No. 1070432121708),
(dd, J=24.3, 8.4Hz, 4H), 1.62 (dd, J=32.1, 18.4Hz, 3H), the Fundamental Research Funds for the Central Universities
1.52–1.40 (m, 3H), 1.38 (s, 3H), 1.30 (dd, J=27.3, 12.7Hz, (HIT.NSRIF.201701) and the National Natural Science Foun-
3H), 1.20 (d, J=9.9Hz, 6H), 1.14 (s, 3H), 1.03 (s, 3H), dation of China (Nos. 21672046 and 21372054).
1.02–0.98 (m, 1H), 0.94 (s, 3H), 0.88 (d, J=11.3Hz, 1H),
0.78 (s, 3H). 13C-NMR (101MHz, CDCl3) δ: 199.93, 175.98,
Conflict of Interest The authors declare no conflict of
168.98, 165.29, 134.86, 132.31, 132.04, 131.95, 130.95, 129.52, interest.
128.55, 127.24, 115.51, 115.30, 81.41, 77.19, 63.14, 61.67, 55.04,
48.21, 45.36, 44.16, 43.18, 40.95, 38.77, 38.39, 37.65, 36.93,
Supplementary Materials The online version of this ar-
32.66, 31.79, 31.11, 28.45, 28.34, 28.19, 26.41, 26.36, 23.60, ticle contains supplementary materials.
23.34, 18.66, 17.37, 16.96, 16.41. HRMS (ESI+): Calculated for
C44H54Cl2FO5 (M+H)+: 751.3327, found: 751.3341.
REFERENCES
3-(2′-Fluorobenzoyl)-2″,6″-Dichlorobenzyl Glycyrrhetinate
1) World Health Organization. “Cancer—Key Facts, 2018.”: ‹http://
(9e)
1
www.who.int/ news-room/fact-sheets/detail/cancer›, 2018.
2) Dias DA, Urban S, Roessner U. A historical overview of natural
products in drug discovery. Metabolites, 2, 303–336 (2012).
3) Logashenko EB, Salomatina OV, Markov AV, Korchagina DV, Sal-
akhutdinov NF, Tolstikov GA, Vlassov VV, Zenkova MA. Synthesis
and pro-apoptotic activity of novel glycyrrhetinic acid derivatives.
ChemBioChem, 12, 784–794 (2011).
4) Schwarz S, Siewert B, Xavier NM, Jesus AR, Rauter AP, Csuk
RA. “natural” approach: Synthesis and cytoxicity of monodesmo-
sidic glycyrrhetinic acid glycosides. Eur. J. Med. Chem., 72, 78–83
(2014).
5) Chadalapaka G, Jutooru I, McAlees A, Stefanac T, Safe S. Struc-
ture-dependent inhibition of bladder and pancreatic cancer cell
growth by 2-substituted glycyrrhetinic and ursolic acid derivatives.
Bioorg. Med. Chem. Lett., 18, 2633–2639 (2008).
6) Parida PK, Sau A, Ghosh T, Jana K, Biswas K, Raha S, Misra
AK. Synthesis and evaluation of triazole linked glycosylated
18β-glycyrrhetinic acid derivatives as anticancer agents. Bioorg.
Med. Chem. Lett., 24, 3865–3868 (2014).
White solid. Y=35%. H-NMR (400MHz, CDCl3) δ: 7.93
(t, J=7.3Hz, 1H), 7.50 (dd, J=12.6, 7.0Hz, 1H), 7.38 (d, J=
8.0Hz, 2H), 7.28 (d, J=8.4Hz, 1H), 7.20 (t, J=7.6Hz, 1H),
7.15–7.09 (m, 1H), 5.50–5.38 (m, 2H), 5.34 (d, J=11.6Hz,
1H), 4.76 (dd, J=10.0, 6.4Hz, 1H), 2.82 (d, J=13.6Hz, 1H),
2.35 (s, 1H), 2.13 (d, J=10.1Hz, 1H), 2.01 (d, J=12.9Hz,
2H), 1.93–1.76 (m, 4H), 1.72–1.54 (m, 4H), 1.44 (dt, J=26.5,
11.0Hz, 4H), 1.34 (s, 3H), 1.31–1.22 (m, 2H), 1.19 (d, J=4.6Hz,
6H), 1.13 (s, 3H), 1.06 (d, J=5.1Hz, 1H), 0.99 (d, J=14.8Hz,
6H), 0.85 (d, J=11.3Hz, 1H), 0.81 (s, 3H). 13C-NMR (101
MHz, CDCl3) δ: 199.96, 176.06, 169.04, 136.81, 134.12, 132.05,
131.43, 130.68, 128.48, 128.39, 123.86, 123.82, 117.04, 116.82,
81.99, 77.21, 61.62, 61.34, 54.99, 48.26, 45.33, 44.29, 43.12,
40.89, 38.78, 38.25, 37.63, 36.90, 32.67, 31.79, 31.11, 28.41,
28.34, 28.09, 26.45, 26.35, 23.49, 23.25, 18.66, 17.38, 16.83,
16.43. HRMS (ESI+): Calculated for C44H54Cl2FO5 (M+H)+:
751.3327, found: 751.3337.
In-Vitro Antitumor Assays MDA-MB-231 cells and
HeLa cells in logarithmic growth phase were inoculated in
96-well plates, with a cell density of 3–4×103/well. The cells
were incubated in a 5% CO2, 37°C cell culture. When the cells
had adhered, the test compound was added at the specified
concentration. The same concentration of dimethyl sulfoxide,
DMSO, was used as the negative control. After 48h of incuba-
tion, 20µL MTT (5mg/mL) was added to each well, and the
culture was continued for 4h. After aspirating the supernatant
with a pump, 150µL of DMSO was added and the OD of each
well was measured at 570nm using a microplate reader. IC50
values were calculated using IC50 software (Prism 5.0).
7) Liu Y, Qian K, Wang CY, Chen CH, Yang X, Lee KH. Synthesis
and biological evaluation of novel spin labeled 18β-glycyrrhetinic
acid derivatives. Bioorg. Med. Chem. Lett., 22, 7530–7533 (2012).
8) Maitraie D, Hung CF, Tu HY, Liou YT, Wei BL, Yang SC, Wang JP,
Lin CN. Synthesis, anti-inflammatory, and antioxidant activities of
18β-glycyrrhetinic acid derivatives as chemical mediators and xan-
thine oxidase inhibitors. Bioorg. Med. Chem., 17, 2785–2792 (2009).
9) Radwan MO, Ismail MAH, El-Mekkawy S, Ismail NSM, Hanna
AG. Synthesis and biological activity of new 18β-glycyrrhetinic acid
derivatives. Arab. J. Chem., 9, 390–399 (2016).
10) Inoue H, Mori T, Shibata S, Koshihara Y. Inhibitory effect of glyc-
yrrhetinic acid derivatives on arachidonic acid-induced mouse ear
oedema. J. Pharm. Pharmacol., 40, 272–277 (1988).
Apoptosis Test The apoptosis rate of MDA-MB-231 cells 11) Zígolo MA, Salinas M, Alche L, Baldessari A, Linares GG. Che-
moenzymatic synthesis of new derivatives of glycyrrhetinic acid
with antiviral activity. molecular docking study. Bioorg. Chem., 78,
210–219 (2018).
was examined by double staining with Annexin V-fluorescein
isothiocyanate (FITC) and propidium iodide (PI). Cells were
seeded at a density of 5×104 cells/mL in 4-well plates (Nest,
12) Lin JC, Cherng JM, Hung MS, Baltina LA, Baltina L, Kondratenko
Wuxi, China). After 24h incubation, the cells were treated
with 250µM PQ for 24h, and pre-treated with VE and various
concentrations (0, 5, 10, 20µM) of 6g for 3h. After treatment
with the indicated drugs, cells were washed twice with cold
R. Inhibitory effects of some derivatives of glycyrrhizic acid
against Epstein–Barr virus infection: structure–activity relation-
ships. Antiviral Res., 79, 6–11 (2008).
13) Wang LJ, Geng CA, Ma YB, Huang XY, Luo J, Chen H, Zhang
PBS and resuspended in 4°C binding buffer (Annexin V-FITC
kit) containing 5µL Annexin V-FITC stock for 15min in the
dark at 4°C; then, 10µL PI was added to each tube for another
5min, and then analysed using flow cytometry. The Annexin
V+cells were considered apoptotic cells, the percentage of
which was calculated by WinMDI 2.8 (J. Trotter, 1993–1998).
XM, Chen JJ. Synthesis, biological evaluation and structure–activ-
ity relationships of glycyrrhetinic acid derivatives as novel anti-
hepatitis B virus agents. Bioorg. Med. Chem. Lett., 22, 3473–3479
(2012).
14) Tsuji M, Sriwilaijaroen N, Inoue H, Miki K, Kinoshita K, Koyama
K, Furuhata K, Suzuki Y, Takahashi K. Synthesis and anti-influ-
enza virus evaluation of triterpene–sialic acid conjugates. Bioorg.
Med. Chem., 26, 17–24 (2018).
Acknowledgments Financial support from the Fundamen-

Vol. 43, No. 1 (2020)
Biol. Pharm. Bull.
109
15) Farina C, Pinza M, Pifferi G. Synthesis and anti-ulcer activity of
glifehrin A. J. Med. Chem., 60, 1000–1017 (2017).
new derivatives of glycyrrhetic, oleanolic and ursolic acids. Farma- 22) Mao J, Zhang SQ, Shi BF, Bao W. Palladium (0)-catalyzed cy-
co, 53, 22–32 (1998).
clopropanation of benzyl bromides via C (sp3)-H bond activation.
Chem. Commun. (Camb.), 50, 3692–3694 (2014).
23) Pollice R, Bot M, Kobylianskii IJ, Shenderovich I, Chen P. At-
tenuation of London dispersion in dichloromethane solutions. J. Am.
Chem. Soc., 139, 13126–13140 (2017).
24) Gaspa S, Porcheddu A, De Luca L. Metal-free firect oxidation of
aldehydes to esters using TCCA. Org. Lett., 17, 3666–3669 (2015).
25) Altmann PJ, Pöthig A. A pH-dependent, mechanically interlocked
switch: organometallic [2] rotaxane vs. organic [3] rotaxane. Angew.
Chem. Int. Ed. Engl., 56, 15733–15736 (2017).
26) Liu X, Li BL, Zhang Z, et al. Synthesis and discovery novel anti-
cancer stem cells compounds derived from the natural triterpenoic
acids. J. Med. Chem., 61, 10814–10833 (2018).
16) Li Y, Feng L, Song ZF, Li HB, Huai QY. Synthesis and anticancer
activities of glycyrrhetinic acid derivatives. Molecules, 21, 747–752
(2016).
17) Wang R, Zheng QX, Wang W, Feng L, Li HJ, Huai QY. Design and
synthesis of new anticancer glycyrrhetinic acids and oleanolic acids.
Biol. Pharm. Bull., 40, 703–710 (2017).
18) Wang R, Li Y, Huai XD, Zheng QX, Wang W, Li HJ, Huai QY.
Design and preparation of derivatives of oleanolic and glycyrrhe-
tinic acids with cytotoxic properties. Drug Des. Dev. Ther., 12,
1321–1336 (2018).
19) Schwarz S, Csuk R. Synthesis and antitumour activity of glycyrrhe-
tinic acid derivatives. Bioorg. Med. Chem., 18, 7458–7474 (2010).
20) Bering L, Jeyakumar K, Antonchick AP. Metal-free C-O bond
functionalization: catalytic intramolecular and intermolecular ben-
zylation of arenes. Org. Lett., 20, 3911–3914 (2018).
27) Zhao CH, Zhang CL, Shi JJ, Hou XJ, Feng B, Zhao LX. Design,
synthesis, and biofunctional evaluation of novel pentacyclic triter-
penes bearing O-[4-(1-piperazinyl)-4-oxo-butyryl moiety as antipro-
liferative agents. Bioorg. Med. Chem. Lett., 25, 4500–4504 (2015).
21) Steadman VA, Pettit SB, Poullennec KG, et al. Discovery of potent
cyclophilin inhibitors based on the structural simplification of san-