Synthesis of novel heterocyclic ring-fused 18β-glycyrrhetinic acid derivatives with antitumor and antimetastatic activity

Article abstract of DOI:10.1111/cbdd.12308

Glycyrrhetinic acid (GA) is one of the most important triterpenoic acids shows many pharmacological effects, especially antitumor activity. GA triggers apoptosis in various tumor cell lines. However, the antitumor activity of GA is weak, thus the synthesis of new synthetic analogs with enhanced potency is needed. By introducing various five-member fused heterocyclic rings at C-2 and C-3 positions, 18 novel GA derivatives were obtained. These compounds were evaluated for their inhibitory activity against the growth of eight different tumor cell lines using a SRB assay. The most active compound 37 showed IC ₅₀ between 5.19 and 11.72 μm, which was about 11-fold more potent than the lead compound GA. An apoptotic effect of GA and 37 was determined using flow cytometry and trypan blue exclusion assays. We also demonstrated here for the first time that GA and the synthetic derivatives exhibited inhibitory effect on migration of the tested tumor cells, especially 37 which was about 20-fold more potent than GA on antimetastatic activity.

Full text of DOI:10.1111/cbdd.12308

Chem Biol Drug Des 2014
Research Article
Synthesis of Novel Heterocyclic Ring-Fused
18b-Glycyrrhetinic Acid Derivatives with Antitumor and
Antimetastatic Activity
1
2
1
Cheng Gao , Fu-Jun Dai , Hai-Wei Cui ,
important classes of natural products occurring and have
been studied intensively for their diverse biological and
pharmacological activities (2–5). Glycyrrhetinic acid (GA,
Scheme 1), a natural pentacyclic triterpene acid, is known
to show many biological effects including anti-inflamma-
tory, antiviral, and hepatoprotective (6). Recently, GA
attracted the focus of research interests not only for it can
be accessed abundantly from the roots of licorice [up to
2
2
2
Shi-Hong Peng , Yuan He , Xue Wang ,
Zheng-Fang Yi * and Wen-Wei Qiu
2
,
1,3,
*
1
Department of Chemistry, East China Normal University,
Shanghai 200062, China
Shanghai Key Laboratory of Regulatory Biology, Institute
of Biomedical Sciences and School of Life Sciences, East
China Normal University, Shanghai 200241, China
Shanghai Engineering Research Center for Molecular
Therapeutics and New Drug Development, East China
Normal University, Shanghai 200062, China
Corresponding authors: Zheng-Fang Yi,
2
2
4% (7,8)], but also for its triggering apoptosis on tumor
3
cells (9–12). However, GA has only weak inhibitory effect
against various tumor cell lines (IC50 is approximately
8
0 lM), and some structural modifications have been pre-
*
yizhengfang@gmail.com, Wen-Wei Qiu,
wwqiu@chem.ecnu.edu.cn
viously performed (13–20). We also become interested in
producing more active GA analogs. Recently, we and
other groups have found that introduction of the nitrogen-
containing heterocyclic rings, especially five-membered
heterocyclic rings to the pentacyclic triterpenoids, such as
maslinic acid and betulinic acid, can improve the biological
activities (21–24). Thus, to obtain more potent anticancer
analogs, we designed and synthesized a series of novel
five-membered heterocyclic ring-fused GA derivatives at
C-2 and C-3 positions, and their inhibitory activity against
the growth of 8 different tumor cell lines was evaluated
using a SRB assay. GA and its derivative were screened
by flow cytometry and trypan blue exclusion assays to
determine the apoptotic behavior, and their inhibitory effect
on tumor cell migration was tested for the first time.
Glycyrrhetinic acid (GA) is one of the most important
triterpenoic acids shows many pharmacological
effects, especially antitumor activity. GA triggers apop-
tosis in various tumor cell lines. However, the antitu-
mor activity of GA is weak, thus the synthesis of new
synthetic analogs with enhanced potency is needed.
By introducing various five-member fused heterocyclic
rings at C-2 and C-3 positions, 18 novel GA derivatives
were obtained. These compounds were evaluated for
their inhibitory activity against the growth of eight dif-
ferent tumor cell lines using a SRB assay. The most
active compound 37 showed IC50 between 5.19 and
1
1.72 lM, which was about 11-fold more potent than
the lead compound GA. An apoptotic effect of GA and
7 was determined using flow cytometry and trypan
3
blue exclusion assays. We also demonstrated here for
the first time that GA and the synthetic derivatives
exhibited inhibitory effect on migration of the tested
tumor cells, especially 37 which was about 20-fold
more potent than GA on antimetastatic activity.
Methods and Materials
Chemistry
All reagents and chemicals were purchased from commer-
cial suppliers and used without further purification unless
otherwise stated. When needed, the reactions were car-
ried out in flame or oven-dried glassware under a positive
Key words: antitumor, apoptosis, glycyrrhetinic acid, migra-
tion, triterpenoic acids
pressure of dry N . Column chromatography was per-
2
formed on silica gel (QinDao, 200–300 mesh) using the
indicated eluents. Thin-layer chromatography was carried
out on silica gel plates (QinDao) with a layer thickness of
Received 14 January 2014 and accepted for publication 12
February 2014
0
.25 mm. Melting points were determined using the MEL-
1
TEMP 3.0 apparatus and are uncorrected. H (300, 400,
and 500 MHz) and C (100 and 125 MHz) NMR spectra
were recorded on Varian Mercury-300, Bruker AM-400,
13
Natural products have been used to treat human disease
for thousands of years and play an increasingly significant
role in drug discovery and development process in recent
years (1). Pentacyclic triterpenoids are one of the most
3 6
and Bruker AV-500 spectrometer with CDCl or DMSO-d
as solvent and tetramethylsilane (TMS) as the internal
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12308
1

Gao et al.
COOR'
COOH
COOH
COOH
COOR'
O
O
O
H
H
a
H
d
O
HO
O
GA
1
6 R'= -Me
7
R'= -Bn
b
b
COOH
COOR'
O
O
O
R
O
R
R
O
H
H
c
H
O
O
HN
N
4
5
R= -H
R= -CF3
2 R= -H
3 R= -CF3
8
9
R= -H
R= -CF
R'= -Me
R'= -Me
R'= -Bn
3
1
1
0 R= -H
c
1 R= -COOEt R'= -Bn
O
R
H
HN
N
1
1
1
1
2 R= -H
3 R= -CF
4 R= -H
R'= -Me
R'= -Me
R'= -Bn
3
5 R= -COOEt R'= -Bn
Scheme 1: Reagents and conditions: (a) Jone’s reagent, THF, 0 °C, 1 h, (92%); (b) RCOOEt (R = -H, -CF
rt, 8 h (91% for 2, 82% for 3, 86% for 8, and 74% for 9); (c) NH NH O, AcOH, rt, 12 h (88% for 4, 78% for 5, 92% for 12, 68% for
ÁH
3; 88% and 69% for 14 and 15, respectively, over two steps). (d) MeI or BnBr, K CO , DMF, rt, 2 h (87% for 6 and 92% for 7).
3
, -COOEt), NaH (60%), THF,
2
2
2
1
2
3
standard. All chemical shift values were reported in units
of d (ppm). The following abbreviations were used to indi-
cate the peak multiplicity: s = singlet; d = doublet; t = trip-
let; m = multiplet; br = broad. High-resolution mass data
were obtained on a Bruker micrOTOF-Q II spectrometer.
1.39 (s, 3H), 1.35 (s, 3H), 1.29 (s, 3H), 1.18 (s, 3H), 1.15
13
(s, 3H), 1.08 (s, 3H), 0.83 (s, 3H). C NMR (100 MHz,
CDCl3) 199.60, 176.98, 169.99, 163.80, 128.67,
d
127.92, 118.21, 59.93, 53.29, 51.89, 48.47, 45.31, 44.13,
43.46, 41.32, 41.09, 37.89, 37.86, 36.67, 34.91, 31.95,
3
1
1.53, 31.22, 28.69, 28.39, 26.65, 26.51, 24.73, 23.35,
8.65, 18.28, 15.48. ESI-HRMS (m/z) [M + Na] calcd for
+
Preparation of compound 26
C H N NaO S, 607.3182, found 607.3160.
33 48 2 5
To a solution of 12 (203 mg, 0.4 mmol) and DMAP
(49 mg, 0.4 mmol) in dry DCM (20 mL) was added mesyl
chloride (0.15 mL, 2.0 mmol) under N . The reaction mix-
2
Preparation of compound 27
ture was stirred for 12 h at 23 °C and then concentrated.
To a solution of 12 (150 mg, 0.3 mmol) and DMAP
The residue was purified by column chromatography
(36 mg, 0.3 mmol) in dry DCM (20 mL) was added Ac O
2
(
petroleum ether/AcOEt, 4/1 v/v) to give 26 as a white
2
(151 mg, 1.5 mmol) under N . The reaction mixture was
1
solid (168 mg, 73%); mp. 245.0–247.0 °C.
(
J = 15.6 Hz, 1H), 3.70 (s, 3H), 3.22 (s, 3H), 2.52 (s, 1H),
H
NMR
stirred for 12 h at 23 °C and then concentrated. The resi-
due was purified by column chromatography (petroleum
ether/AcOEt, 5/1 v/v) to give 27 as a white solid, (148 mg,
400 MHz, CDCl ) d 7.63 (s, 1H), 5.74 (s, 1H), 3.83 (d,
3
2
Chem Biol Drug Des 2014

Novel GA Derivatives with Antitumor Activity
1
9
7
(
1%); mp. 225.0–227.0 °C. H NMR (400 MHz, CDCl
3
) d
J = 7.4, 4.2 Hz, 2H), 2.67 (t, J = 11.8 Hz, 2H), 2.53
(s, 1H), 1.45 (s,9H), 1.39 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H),
1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H).
.85 (s, 1H), 5.73 (s, 1H), 3.82 (d, J = 15.7 Hz, 1H), 3.70
s, 3H), 2.63 (s, 3H), 2.53 (s, 1H), 1.39 (s, 3H), 1.33 (s,
3
0
1
5
3
2
1
H), 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H),
13
.83 (s, 3H).
3
C NMR (100 MHz, CDCl ) d 199.61,
76.95, 169.79, 169.48, 162.24, 128.69, 124.78, 119.32,
9.92, 53.29, 51.85, 48.43, 45.30, 44.10, 43.42, 41.29,
7.94, 37.84, 36.91, 34.71, 31.95, 31.93, 31.56, 31.20,
8.66, 28.36, 26.63, 26.50, 24.75, 23.33, 21.66, 18.67,
Compound 32
By a similar procedure described for 28, 32 was obtained
as a white solid (237 mg, 78%). H NMR (400 MHz,
1
3
CDCl ) d 7.82 (s, 1H), 5.73 (s, 1H), 4.19 (d, J = 7.1 Hz,
+
8.25, 15.46. ESI-HRMS (m/z) [M + Na] calcd for
1H), 4.00 (d, J = 13.4 Hz, 1H), 3.83 (d, J = 15.8 Hz, 1H),
3.70 (s, 4H), 3.15 (d, J = 10.4 Hz, 1H), 2.86 (t,
J = 11.1 Hz, 1H), 2.52 (s, 1H),1.45 (s, 9H), 1.39 (s, 3H),
C H N NaO , 571.3512, found 571.3541.
3
4
48
2
4
1
.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08
Preparation of compounds 29 and 33–37
(s, 3H), 0.83 (s, 3H).
Compound 28
To
a
solution of N-Boc-piperidine-3-carboxylic acid
Compound 33
(275 mg, 1.2 mmol) in dry DCM (20 mL) was added N,N’-
To a solution of 28 (159 mg, 0.21 mmol) in Et O (5 mL)
2
carbonyldiimidazole (194 mg, 1.2 mmol). The reaction
mixture was stirred for 0.5 h at 23 °C, and then 12
was added boron trifluoride etherate (2 mL). The reaction
mixture was stirred for 0.5 h at 23 °C and then poured
into ice water (20 mL). The precipitate was filtered and
(203 mg, 0.4 mmol) and DMAP (146 mg, 1.2 mmol) was
added. The reaction mixture was stirred for 3 h at 23 °C
and then concentrated. The residue was purified by col-
umn chromatography (petroleum ether/AcOEt, 5/1 v/v) to
washed with Et
solid (104 mg, 80%); mp. 218.0–221.0 °C.
(400 MHz, CDCl ) d 7.81 (s, 1H), 5.73 (s, 1H), 3.83 (d,
2
O (5 mL), then dried to give 33 as a white
1
H
NMR
3
1
give 28 as a white solid (213 mg, 76%). H NMR
J = 15.8 Hz, 1H), 3.75–3.63 (m, 4H), 3.43 (d, J = 13.1 Hz,
1H), 3.23 (d, J = 11.4 Hz, 1H), 3.09 (d, J = 10.3 Hz, 1H),
2.87 (s, 1H), 2.52 (s, 1H), 1.39 (s, 3H), 1.33 (s, 3H), 1.26 (s,
(
(
(
3
400 MHz, CDCl ) d 7.82 (s, 1H), 5.73 (s, 1H), 4.19
d, J = 7.1 Hz, 1H), 4.00 (d, J = 13.4 Hz, 1H), 3.83
d, J = 15.8 Hz, 1H), 3.70 (s, 4H), 3.15 (d, J = 10.4 Hz,
13
3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H).
C
1H), 2.86 (t, J = 11.1 Hz, 1H), 2.52 (s, 1H), 1.43(s,9H),
1.39 (s, 3H), 1.33, 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H),
1.09 (s, 3H), 0.83 (s, 3H).
NMR (100 MHz, CDCl ) d 199.80, 177.07, 171.90, 170.09,
163.19, 128.68, 125.18, 119.90, 59.92, 53.21, 51.91,
48.47, 45.36, 44.15, 43.48, 41.30, 37.97, 37.88, 36.88,
3
3
2
6.69, 34.78, 31.96, 31.54, 31.24, 28.70, 28.40, 26.66,
6.53, 25.15, 24.95, 24.80, 23.37, 18.66, 18.29, 15.56,
+
Compound 29
14.43. ESI-HRMS (m/z) [M + Na] calcd for C38
H
55
N
3
4
NaO ,
By a similar procedure described for 28, 29 was obtained
as a white solid (206 mg, 73%). H NMR (400 MHz,
640.4090, found 640.4119.
1
CDCl ) d 7.83 (s, 1H), 5.73 (s, 1H), 4.15 (d, J = 12.6 Hz,
3
2
(
1
(
H), 3.83 (d, J = 15.7 Hz, 1H), 3.78–3.65 (m, 4H), 2.90
t, J = 11.8 Hz, 2H), 2.52 (s, 1H), 1.46(s, 9H), 1.39 (s, 3H),
.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08
s, 3H), 0.83 (s, 3H).
Compound 34
By a similar procedure described for 33, 34 was obtained
1
as a white solid (99 mg, 77%); mp. 227.0–231.0 °C.
NMR (500 MHz, CDCl ) d 7.82 (s, 1H), 5.73 (s, 1H), 3.82
d, J = 15.8 Hz, 1H), 3.76 (s, 1H), 3.70 (s, 3H), 3.25 (s,
H), 2.86 (t, J = 11.8 Hz, 2H), 2.52 (s, 1H), 1.39 (s, 3H),
1.33 (s, 3H), 1.26 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.07
H
3
(
2
Compound 30
By a similar procedure described for 28, 30 was obtained
as a white solid (228 mg, 78%). H NMR (400 MHz,
CDCl
13
(s, 3H), 0.83 (s, 3H).
3
C NMR (100 MHz, CDCl ) d
1
199.85, 177.10, 171.75, 170.16, 163.25, 128.75, 125.28,
120.00, 59.97, 53.28, 51.97, 48.53, 45.41, 44.19, 44.15,
43.53, 41.36, 38.02, 37.93, 36.94, 36.59, 34.84, 32.01,
31.60, 31.29, 28.74, 28.45, 26.71, 26.59, 25.18, 25.01,
24.85, 23.41, 18.72, 18.34, 15.61, 14.47. ESI-HRMS
3
) d 7.85 (s, 1H), 5.73 (s, 1H), 4.07 (d, J = 13.5 Hz,
2
2
1
H), 3.82 (d, J = 15.7 Hz, 1H), 3.70 (s, 3H), 2.95-3.07 (m,
H), 2.75 (t, J = 12.0 Hz, 2H), 2.52 (s, 1H), 1.45(s,9H),
.39 (s, 3H), 1.33 (s, 3H), 1.26 (s, 3H), 1.18 (s, 3H), 1.15
+
(s, 3H), 1.08 (s, 3H), 0.83 (s, 3H).
(m/z) [M + Na] calcd for C H N NaO , 640.4090, found
38
55
3
4
6
40.4085.
Compound 31
By a similar procedure described for 28, 31 was obtained
as a white solid (235 mg, 79%). H NMR (400 MHz,
Compound 35
By a similar procedure described for 33, 35 was obtained
as a white solid (90 mg, 68%); mp. 219.0–220.0 °C. H
1
1
CDCl
3
) d 7.85 (s, 1H), 5.73 (s, 1H), 4.08 (d, J = 13.3 Hz,
2H), 3.82 (d, J = 15.7 Hz, 1H), 3.70 (s, 3H), 3.10 (td,
NMR (400 MHz, CDCl ) d 7.82 (s, 1H), 5.74 (s, 1H), 4.17
3
Chem Biol Drug Des 2014
3

Gao et al.
s, 1H), 3.85 (d, J = 16.0 Hz, 1H), 3.70 (d, J = 5.7 Hz,
(
10% FBS, penicillin (100 units/mL), and streptomycin (100
3
1
H), 3.60 (d, J = 15.4 Hz, 2H), 3.42 (s, 2H), 2.53 (s, 1H),
units/mL). All cells were incubated at 37 °C and 5% CO
2
incubator.
.40 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.16
13
(
s, 3H), 1.07 (s, 3H), 0.84 (s, 3H). C NMR (100 MHz,
CDCl 199.76, 177.08, 171.67, 169.95, 162.52,
28.74, 124.95, 119.50, 59.98, 53.28, 51.93, 48.50,
3
) d
1
4
3
2
1
6
Viability assay
5.37, 45.33, 44.18, 43.49, 41.34, 38.00, 37.91, 36.95,
4.79, 33.16, 31.99, 31.60, 31.26, 30.70, 30.51, 28.72,
8.67, 28.44, 26.69, 26.57, 24.85, 23.39, 18.71, 18.32,
5.57. ESI-HRMS (m/z) [M + H] calcd for C39
32.4427, found 632.4408.
The cell viability of eight tumor cell lines in the presence
of our synthetic compounds was determined by SRB
(Sigma Aldrich, St.Louis, MO, USA) assay (14) In brief,
cells were seeded into 96-well plates at the appropriate
cell densities to prevent confluence during the experi-
ment. After incubation for 24 h, the cells were treated
with various concentrations of the compound (0–50 lM)
for 96 h. Control cells were exposed to DMSO at a con-
centration equivalent to that of the compound-treated
cells, and the final dilution of DMSO solvent used in this
assay never exceeded 0.4%. After treatment for 96 h, the
supernatant medium was removed from the 96-well
plates, and the cells were fixed by 50% TCA at 4 °C.
After fixation for 1 h, the plates were washed by water for
five times. The plates were allowed to dry using hair dryer
followed by being dyed with 100 lL of 0.4% SRB for
10 min. After dying, the plates were washed by 1% ace-
tic acid to remove the dye and allowed to dry using hair
dryer. One hundred microliter of 10 mM Tris base solution
was added to each well, and absorbance was examined
using a 96-well plate reader. The IC50 was calculated
using GraphPad Software (San Diego, CA, USA).
+
H
58
N
3
O
4
,
Compound 36
By a similar procedure described for 33, 36 was obtained
1
as a white solid (95 mg, 71%); mp. 210.0–211.0 °C.
NMR (400 MHz, CDCl ) d 7.84 (s, 1H), 5.73 (s, 1H), 3.82
d, J = 15.8 Hz, 1H), 3.71 (s, 3H), 3.56 (d, J = 12.4 Hz,
H
3
(
2
3
0
1
5
4
3
2
H), 3.13–2.93 (m, 4H), 2.53 (s, 1H), 1.40 (s, 3H), 1.33 (s,
H), 1.27 (s, 3H), 1.19 (s, 3H), 1.16 (s, 3H), 1.08 (s, 3H),
1
3
.83 (s, 3H).
3
C NMR (100 MHz, CDCl ) d 199.70,
77.01, 171.85, 169.89, 162.24, 128.67, 124.82, 119.20,
9.93, 53.24, 51.86, 48.44, 45.31, 45.09, 44.11, 43.43,
1.28, 37.94, 37.84, 36.90, 35.19, 34.72, 33.64, 33.55,
1.93, 31.57, 31.20, 28.70, 28.67, 28.37, 26.62, 26.50,
4.79, 23.34, 21.48, 18.65, 18.26, 15.50. ESI-HRMS (m/
+
z) [M + H] calcd for C H N O , 646.4584, found
40 60 3 4
6
46.4573.
Compound 37
By a similar procedure described for 33, 37 was obtained
Trypan blue exclusion assay
Apoptosis of mouse CT-26 colorectal cancer cells was
assessed by trypan blue exclusion assay (25). In short,
cells were plated onto 6-well plates and were exposed
to GA and 37 for 24 h. After treatment with various
doses of the compounds for 24 h, all cells including
the supernatant were collected and centrifuged to col-
lect the viable, apoptotic, and dead cells. The cells
were suspended in serum-free medium. Equal volume
of trypan blue and cell suspension were mixed and
analyzed by an inverted microscope (Olympus, Tokyo,
Japan). The colorless cells were considered as viable
cells, and the colored cells were considered as apopto-
tic and dead cells.
1
as a white solid (92 mg, 67%); mp. 201.0–204.0 °C.
H
NMR (400 MHz, CDCl ) d 7.84 (s, 1H), 5.73 (s, 1H), 3.82
3
(
(
d, J = 15.8 Hz, 1H), 3.70 (s, 3H), 3.52 (s, 2H), 3.11–2.92
m, 4H), 2.53 (s, 1H), 1.40 (s, 3H), 1.33 (s, 3H), 1.27 (s,
3
H), 1.19 (s, 3H), 1.16 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H).
1
3
3
C NMR (100 MHz, CDCl ) d 199.70, 177.08, 171.62,
1
5
4
3
2
70.03, 163.91, 128.71, 125.21, 120.55, 59.91, 53.23,
3.13, 51.93, 48.49, 45.67, 45.36, 45.15, 44.17, 43.50,
1.34, 37.98, 36.89, 36.25, 34.83, 31.99, 31.53, 31.45,
1.26, 28.72, 28.43, 26.69, 26.56, 25.66, 24.83, 24.77,
3.42, 23.39, 20.54, 18.69, 18.30, 15.62, 15.57. ESI-
+
HRMS (m/z) [M + H] calcd for C H N O4, 660.4740,
41 62 3
found 660.4731.
Flow cytometry assay
Biology
Apoptosis of mouse CT-26 colorectal cancer cells
induced by GA and 37 was also quantified by an
annexin V-FITC/PI (BD Bioscience, San Diego, CA, USA)
dual-staining assay (26). Briefly, cells were seeded into
6-well plates and were allowed to incubate for 24 h,
then compound GA or 37 was added. After treatment
for 24 h, cells were collected, centrifuged, and resus-
pended with PBS. After centrifugation, cells were resus-
pended with binding buffer and incubated with annexin
V and PI for 30 min in dark condition. Analysis was
performed by flow cytometry.
Cell lines and culture conditions
Cell lines used in this study were obtained from the Ameri-
can Type Culture Collection (ATCC). HCT-116, HCT-8,
HT-29, CT-26, PC-3, and DU-145 cell lines were cultured
in RPMI1640 medium. Mouse 4T1 breast cancer cells
were cultured in DMEM medium, and human MDA-MB-
2
31 breast cancer cells were cultured in MEM medium
supplemented with 1% sodium pyruvate and 1% non-
essential amino acids. Medium was supplemented with
4
Chem Biol Drug Des 2014

Novel GA Derivatives with Antitumor Activity
Transwell migration assay
were taken using an inverted microscope (Olympus), and
cells from six random areas per filter were counted.
The inhibitory effect on migration of mouse CT-26 colorec-
tal cancer cells was assessed by the modified Boyden’s
chamber (Millipore, Billerica, MA, USA) assay in 24-well cell
culture plate with 8.0-lm pore (27). After overnight starva-
tion, cells were collected, centrifuged, and resuspended
with serum-free medium. The top chambers were seeded
Results and Discussion
Synthesis of GA derivatives
4
with 10 9 10 mouse CT-26 colorectal cancer cells in
To determine which five-membered heterocyclic ring
contributed the most antitumor activity, a series of GA
derivatives with heterocyclic rings (pyrazole, 5-(trifluorom-
ethyl)-1H-pyrazole, ethyl 1H-pyrazole-5-carboxylate, 1,2,3-
thiadiazole, and 2-methyloxazole) fused at C-2 and C-3
positions were synthesized (Schemes 1 and 2) based on
our previous research (21,22). The synthesis of pyrazole
derivatives is outlined in Scheme 1. Oxidation of GA with
Jone’s reagent gave intermediate 1, which was then
converted to 6 or 7 by methyl or benzyl esterification with
MeI or BnBr, respectively. Intermediates 2, 3, 8, 9, 10,
and 11 were obtained by Claisen condensation of 1 with
100 lL serum-free RMPI 1640 medium containing different
concentrations of GA and 37. The bottom chambers were
filled with 700 lL medium supplemented with different
concentrations of GA and 37. Cells were allowed to
migrate for 12 h. 4% paraformaldehyde was added to the
wells with the chamber in order to fix the cells. After fixation
for 0.5 h, the non-migrated cells on the top surface of the
membrane were scraped with cotton swab. The cells were
washed with PBS for three times, and cells were stained
with 0.2% crystal violet. Then the chambers were washed
using water, and the membrane was left to dry. Images
COOR'
COOR'
COOR'
O
O
O
H
H
N
H
H
a
b
O
HON
O
O
O
6
7
R'= -Me
R'= -Bn
18 R'= -Me
19 R'= -Bn
1
6 R= -Me
7 R= -Bn
1
e
c
COOMe
f
COOMe
COOR'
O
O
O
H
H
H
O
H N
2
S
N
N
O
N
HN
N
23
24
20 R'= -Me
2
1 R'= -Bn
2 R'= -H
d
2
g
COOH
O
H
S
N
N
25
Scheme 2: Reagents and conditions: (a) isoamyl nitrite, t-BuOK, t-BuOH, rt, 5 h (65% for 16 and 56% for 17); (b) Zn, Ac
2
O, AcOH, rt,
1
3 2
2 h (68% for 18 and 81% for 19); (c) POCl , pyridine, rt, 12 h (93% for 20 and 87% for 21); (d) H (1 atm), Pd/C, MeOH, 12 h (94% for
2
2); (e) NH NHCONH
2
2
ÁHCl, AcONa, EtOH, reflux, 2 h; (f) SOCl
, CH Cl
2 2 2
, rt, 12 h (84% for 24 over two steps); (g) LiI, DMF, reflux, 48 h
(43% for 25).
Chem Biol Drug Des 2014
5

Gao et al.
corresponding esters in the presence of NaH. Then the
pyrazole derivatives 4, 5, 12, 13, 14, and 15 were synthe-
sized by condensation of corresponding intermediates with
hydrazine hydrate in AcOH.
3
because of the strong electron-withdrawing group CF ,
made the acylation of NH group in prazole unsuccessfully.
Antiproliferative activity
The synthesis of 1,2,3-thiadiazole and 2-methyloxazole
derivatives is outlined in Scheme 2. The 2-methyloxazole
derivatives 18 and 19 were obtained by treatment of 6
and 7 with isoamyl nitrite in the presence of t-BuOK in
t-BuOH, followed by reaction with zinc powder in HOAc/
To evaluate the anticancer potency of these synthesized
heterocyclic ring-fused GA derivatives, the antiproliferative
activity of 4–25 (Scheme 1 and 2) was first screened in an
SRB assay against eight cancer cell lines of different ori-
gin: HCT-116, HCT-8, HT-29, CT-26, PC-3, DU-145,
MDA-MB-231, 4T1 (Table 1). The results showed that
derivatives having a free carboxylic group in position C-30
(4 and 5) resulted in a lower inhibition of cell growth com-
pared with the corresponding methyl or benzyl esters (12,
14, and 13), and this was in accordance with previously
reports (13,14), thus they were excluded from further
investigations. The 2-methyloxazole was not a favorable
substituted group to improve antitumor activity, because
of both derivatives having a free carboxylic group in posi-
tion C-30 (22), and the corresponding methyl or benzyl
esters (20 or 21) gave IC50 values higher than 50 lM.
Compound 24 was found to be insoluble in dimethylsulf-
oxide (DMSO), therefore it was untested, and its corre-
sponding C-30 free carboxylic compound 25 displaying
expected poor activity. Most of the pyrazole derivatives
showed significantly improved IC50 values, such as 12 and
13, especially 14 (average IC50 = 13.94 lM) and 15 (aver-
age IC50 = 14.25 lM) exhibited approximately 6.5 times
more potent than GA (average IC50 = 90.86 lM). The praz-
Ac O, respectively. Compound 20 and 21 were synthe-
2
sized by cyclization of 18 and 19 in the presence of POCl
3
in pyridine. Hydrogenation of 21 with Pd/C under H affor-
2
ded 22. Intermediate 23 was obtained by reaction of 6
with semicarbazide hydrochloride. Reaction of 23 with
SOCl2 produced 24. The 1,2,3-thiadiazole derivative 25
was obtained by demethylation of C-30 methyl ester with
LiI in DMF.
The synthesis of piperidine derivatives 26–37 is outlined in
Scheme 3. Compounds 26 and 27 were produced by am-
2 2
idation of 12 with MeSO Cl and Ac O, respectively. Inter-
mediates 28, 29, 30, 31, and 32 were obtained by
amidation of 12 with corresponding piperidine caboxylic
acids in the presence of CDI and DMAP in CH Cl . Com-
2
2
pound 33 was produced by deprotection of Boc in the
presence of boron trifluoride etherate. Compounds 34, 35,
3
6, and 37 were prepared in a manner similar to that for
3. We also tried to amidation of 13 in a similar way, and
3
COOMe
COOMe
COOMe
O
O
O
H
H
H
O
a or b
R
N
N
HN
N
N
N
33
12
NH
2
6 R= -SO Me
2
2
7 R= -Ac
2
8 R=
c
n
O
O
n= 0
N
Boc
COOMe
29 R=
n
n
n
n
NBoc n= 0
NBoc n= 1
NBoc n= 2
NBoc n= 3
O
3
0 R=
HN
O
n
H
O
O
N
c
N
3
1 R=
2 R=
34 n= 0
3
5 n= 1
3
36 n= 2
37 n= 3
O
OH
BocN
n
O
Scheme 3: Reagents and conditions: (a) MeSO
2
Cl or Ac
2
O, DMAP, DCM, rt, 12 h (73% for 26 and 91% for 27); (b)
Cl
3 2
, Et O, rt, 0.5 h (80% for 33, 77% for 34, 68% for 35, 71% for 36, and 67% for 37).
(n = 0)
OH
orBocN
n
O
(n = 0, 1, 2, and 3), CDI (N,N’-carbonyldiimidazole), DMAP, CH
2
2
, rt, 3 h (76% for 28, 73% for 29, 78% for 30, 79% for 31,
and 82% for 32); (c) Et
2
OÁBF
6
Chem Biol Drug Des 2014

Novel GA Derivatives with Antitumor Activity
Chem Biol Drug Des 2014
7

Gao et al.
ole and ethyl 1H-pyrazole-5-carboxylate groups almost
contributed equally activity to corresponding derivatives
slightly more cytotoxic activity than compound 33
(substituted with 3-piperidinecarboxylic acid). Perhaps
more distance between the prazole to the NH group of
piperidine will afford more potency, and this speculation
was further confirmed by the results of 34 (n = 0), 35
(n = 1), 36 (n = 2), and 37 (n = 3). The free NH group in
piperidine was important for keeping the cytotoxic activity
against tumor cells, and if the group was protected with
the Boc (29), the activity would be decreased dramatically.
(
14 and 15). Prazole-substituted derivative with benzyl
esterification of the C-30 carboxylic group (14) exhibited
about twofold higher cytotoxic activity against tumor cells
than its corresponding methyl ester (12). Considering GA
itself has poor water solubility and exhibits low bioavailabil-
ity and if the hydrophobic benzyl ester group is formed at
the C-30, free carboxylic acid position will make the deriv-
ative more insoluble in water than corresponding methyl
ester, and therefore, the methyl ester 12 was selected as
a new lead for further modification. We also considered 13
as a further modifying lead and found the NH group of its
prazole ring was hard to substitute because of the strong
Apoptosis study using flow cytometry assay and
trypan blue staining
As previously reported (14), GA induces apoptosis on vari-
ous tumor cell lines. To examine whether this effect
remains or is lost on derivatization, we tested GA and 37
for the induction of apoptosis using flow cytometry and
trypan blue exclusion assays. As shown in Figures 1 and
2, we found that the ability of GA and 37 to trigger apop-
tosis is displayed obviously on mouse CT-26 colorectal
cancer cells.
3
electron-withdrawing group CF in presence.
We considered 12 as a new starting modifying structure
for more potent activity derivatives. Novel piperidine com-
pounds 26–37 were synthesized from 12 with amidation
and N-Boc deprotection reactions. Screening of these
derivatives (compounds 26–37) with the SRB assay
revealed that all of the piperidine compounds showed
more potent activity than their parent compound 12
(Table 2). The most potent compound 37 showed the
Migration assay
average IC50 8.02 lM, which was about 11-fold more
potent than the lead compound GA. Compound 34
Migration is a key step during the metastasis of cancer
(28), and to our best knowledge, there are no reports
describing inhibitory effect of GA and its analogs on
(substituted with 4-piperidinecarboxylic acid), possessed a
2
.31%
.65%
4.85%
12.29%
0
0.59%
1.77%
Control
GA (80 μM)
GA212 (8 μM)
Figure 1: GA and 37 induced apoptosis of mouse CT-26 colorectal cancer cells in flow cytometry assay compared with control group.
After treatment of GA or 37 for 24 h, CT-26 colorectal cancer cells with the supernatant were collected, centrifuged, and resuspended
with PBS. After centrifugation, cells were resuspended with binding buffer and stained with annexin V and PI for 0.5 h. Analysis was
performed by flow cytometry, and the percentage displayed the apoptosis cells of all cells.
Control
GA (80 μM)
37 (8 μM)
Figure 2: GA and 37 induced apoptosis of mouse CT-26 colorectal cancer cells in trypan blue exclusion assay compared with control
group. Mouse CT-26 colorectal cancer cells were seeded onto 6-well plates. After treatment of GA or 37 for 24 h, cells with the
supernatant were collected, centrifuged, and resuspended with serum-free medium. The cells were stained with trypan blue, and images
were taken using an inverted microscope. Arrows indicated the apoptotic or dead cells.
8
Chem Biol Drug Des 2014

Novel GA Derivatives with Antitumor Activity
Control
GA (80 μM)
Figure 3: GA and 37 suppressed
migration of mouse CT-26
colorectal cancer cells in transwell
migration assay compared with
control group. After starvation
overnight, the cells containing the
tested compounds were seeded
onto top side of the chambers.
Cells were allowed to migrate for
3
7 (4 μM)
37 (8 μM)
37 (16 μM)
120
00
1
1
2 h and fixed with 4%
paraformaldehyde. After fixation for
.5 h, the non-migrated cells on
8
0
*
**
*
**
0
60
the top side were scraped using
cotton swab. Migrated cells were
stained with 0.2% crystal violet,
and images were taken using an
inverted microscope. Cells from six
random areas per filter were
counted.
4
0
*
**
20
0
*
**
C
GA
4
8
16
3
7 (μM)
migration of tumor cell lines. To determine this effect, we
performed transwell migration assay. As shown in
Figure 3, we found that GA inhibited the migration of
mouse CT-26 colorectal cancer cells obviously, compared
with the control group. We also tested the antimetastatic
activity of 37 and other derivatives (such as 34 and the
data were not shown) and found that these derivatives,
especially 37, significantly inhibited the migration (IC50 was
about 4 lM) of mouse CT-26 colorectal cancer cells in a
dose-dependent manner. Surprisingly, 37 suppressed
migration completely at medium concentration (16 lM). In
addition, the results showed that the inhibitory effect on
migration of 37 at 4 lM was similar to that of GA at 80 lM,
it means that 37 was about 20-fold more potent than GA
on antimetastatic activity of mouse CT-26 colorectal can-
cer cells.
compared with the corresponding esters. Introduction of
prazole substituent was quite promising. Most of the
pyrazole derivatives showed significantly improved IC50
values, especially 14 and 15. The further modification of
prazole compound 12 with various piperidinecarboxylic
acids was efficiently afforded the most active compound
37. As demonstrated by flow cytometry and trypan blue
exclusion assays, tumor cell death trigged by the com-
pounds resulted from apoptotic processes. In addition,
we also described here for the first time that GA and the
synthetic compounds exhibited antimetastatic activity on
the tested cancer cell line.
In conclusion, we report GA heterocyclic derivatives as a
series of new chemical entities for the first time. Especially
37, which exhibited good antitumor and antimetastatic
activity, could be used as a promising lead for the devel-
opment of a new class of antitumor and antimetastatic
agents.
Conclusion
In this study, we synthesized a class of novel heterocyclic
ring-fused of GA analogs and screened them for antiprolif-
erative activity on cancer cell lines. Most of these com-
pounds exhibited an obvious increase in inhibitory
potency compared with GA. An unsubstituted carboxylic
group in C-30 resulted in a lower inhibition of cell growth
Acknowledgments
Shanghai Science and Technology Council (Grant
12ZR1408500), the Fundamental Research Funds for the
Central Universities, Innovation Program of Shanghai Munici-
Chem Biol Drug Des 2014
9

Gao et al.
pal Education Commission (13zz034), and National Natural
Science Foundation of China (81272463) are appreciated for
their financial support.
14. Csuk R., Schwarz S., Kluge R., Str o€ hl D. (2010)
Synthesis and biological activity of some antitumor
active derivatives from glycyrrhetinic acid. Eur J Med
Chem;45:5718–5823.
1
5. Gao Y., Guo X., Li X., Liu D., Song D., Xu Y., Sun M.,
Jing Y., Zhao L. (2010) The synthesis of glycyrrhetinic
acid derivatives containing a nitrogen heterocycle and
their antiproliferative effects in human leukemia cells.
Molecules;15:4439–4449.
References
1
. Newman D.J., Cragg G.M. (2007) Natural products as
sources of new drugs over the last 25 years. J Nat
Prod;70:461–477.
16. Lallemand B., Chaix F., Bury M., Bruy eꢀ re C., Ghostin
2
. Alqahtani A., Hamid K., Kam A., Wong K.H., Abdelhak
Z., Razmovski-Naumovski V., Chan K., Li K.M.,
Groundwater P.W., Li G.Q. (2013) The pentacyclic trit-
erpenoids in herbal medicines and their pharmacologi-
cal activities in diabetes and diabetic complications.
Curr Med Chem;20:908–931.
J., Becker J.P., Delporte C., Gelbcke M., Mathieu V.,
Dubois J., Pr eꢁ vost M., Jabin I., Kiss R. (2011) N-(2-{3-
[3,5-bis(trifluoromethyl)phenyl]ureido}ethyl)- glycyrrheti-
namide (6b): a novel anticancer glycyrrhetinic acid
derivative that targets the proteasome and displays
anti-kinase activity. J Med Chem;54:6501–6513.
3
. Salvador J.A., Moreira V.M., Goncßalves B.M., Leal
A.S., Jing Y. (2012) Ursane-type pentacyclic triterpe-
noids as useful platforms to discover anticancer drugs.
Nat Prod Rep;29:1463–1479.
17. Csuk R., Schwarz S., Siewert B., Kluge R., Str o€ hl D.
(2011) Synthesis and antitumor activity of ring A modi-
fied glycyrrhetinic acid derivatives. Eur
J
Med
Chem;46:5356–5369.
4
5
6
7
. Wang S.R., Fang W.S. (2009) Pentacyclic triterpenoids
and their saponins with apoptosis-inducing activity.
Curr Top Med Chem;9:1581–1596.
. James J.T., Dubery I.A. (2009) Pentacyclic triterpenoids
from the medicinal herb, Centella asiatica (L.) Urban.
Molecules;14:3922–3941.
. Asl M.N., Hosseinzadeh H. (2008) Review of pharma-
cological effects of Glycyrrhiza sp. and its bioactive
compounds. Phytother Res;22:709–724.
. Lauren D.R., Jensen D.J., Douglas J.A., Follett J.M. (2001)
Efficient method for determining the glycyrrhizin content of
fresh and dried roots, and root extracts, of Glycyrrhiza spe-
cies. Phytochem Anal;12:332–335.
18. Logashenko E.B., Salomatina O.V., Markov A.V.,
Korchagina D.V., Salakhutdinov N.F., Tolstikov G.A.,
Vlassov V.V., Zenkova M.A. (2011) Synthesis and
pro-apoptotic activity of novel glycyrrhetinic acid deriv-
atives. ChemBioChem;12:784–794.
19. Csuk R., Schwarz S., Kluge R., Str o€ hl D. (2012) Does
one keto group matter? Structure-activity relationships
of glycyrrhetinic acid derivatives modified at position
C-11. Arch Pharm Chem Life Sci;345:28–32.
20. Csuk R., Schwarz S., Siewert B., Kluge R., Str o€ hl D.
(2012) Conversions at C-30 of glycyrrhetinic acid and
their impact on antitumor activity. Arch Pharm Chem
Life Sci;345:223–230.
8
. Baltina L.A. (2003) Chemical modification of glycyrrhizic
acid as a route to new bioactive compounds for medi-
cine. Curr Med Chem;10:155–171.
21. Qiu W.W., Shen Q., Yang F., Wang B., Zou H., Li J.Y., Li
J., Tang J. (2009) Synthesis and biological evaluation of
heterocyclic ring-substituted maslinic acid derivatives as
novel inhibitors of protein tyrosine phosphatase 1B. Bio-
org Med Chem Lett;19:6618–6622.
9
. Hibasami H., Iwase H., Yoshioka K., Takahashi H.
(2006) Glycyrrhetic acid (a metabolic substance and
aglycon of glycyrrhizin) induces apoptosis in human
hepatoma, promyelotic leukemia and stomach cancer
cells. Int J Mol Med;17:215–219.
0. Jutooru I., Chadalapaka G., Chintharlapalli S., Papineni
S., Safe S. (2009) Induction of apoptosis and nonste-
roidal anti-inflammatory drug-activated gene 1 in pan-
creatic cancer cells by a glycyrrhetinic acid derivative.
Mol Carcinog;48:692–702.
1. Liu D., Song D., Guo G., Wang R., Lv J., Jing Y., Zhao
L. (2007) The synthesis of 18beta-glycyrrhetinic acid
derivatives which have increased antiproliferative and
apoptotic effects in leukemia cells. Bioorg Med
Chem;15:5432–5439.
22. Xu J., Li Z., Luo J., Yang F., Liu T., Liu M., Qiu W.W.,
Tang J. (2012) Synthesis and biological evaluation of
heterocyclic ring-fused betulinic acid derivatives as
novel inhibitors of osteoclast differentiation and bone
resorption. J Med Chem;55:3122–3134.
1
23. Santos R.C., Salvador J.A., Mar ꢁı n S., Cascante M.
(2009) Novel semisynthetic derivatives of betulin and
betulinic acid with cytotoxic activity. Bioorg Med
Chem;17:6241–6250.
1
24. Kumar V., Rani N., Aggarwal P., Sanna V.K., Singh
A.T., Jaggi M., Joshi N., Sharma P.K., Irchhaiya R.,
Burman A.C. (2008) Synthesis and cytotoxic activity of
heterocyclic ring-substituted betulinic acid derivatives.
Bioorg Med Chem Lett;18:5058–5062.
1
1
1
2. Lee C.S., Kim Y.J., Lee M.S., Han E.S., Lee S.J. (2008)
1
8beta-Glycyrrhetinic acid induces apoptotic cell death
25. Dong Y., Lu B., Zhang X., Zhang J., Lai L., Li D., Wu
Y., Song Y., Luo J., Pang X., Yi Z., Liu M. (2010)
Cucurbitacin E, a tetracyclic triterpenes compound
from Chinese medicine, inhibits tumor angiogenesis
through VEGFR2-mediated Jak2-STAT3 signaling
pathway. Carcinogenesis;31:2097–2104.
in SiHa cells and exhibits a synergistic effect against anti-
biotic anti-cancer drug toxicity. Life Sci;83:481–489.
3. Schwarz S., Csuk R. (2010) Synthesis and antitumour
activity of glycyrrhetinic acid derivatives. Bioorg Med
Chem;18:7458–7474.
0
Chem Biol Drug Des 2014

Novel GA Derivatives with Antitumor Activity
2
2
2
6. Dai F., Chen Y., Song Y., Huang L., Zhai D., Dong Y.,
Lai L., Zhang T., Li D., Pang X., Liu M., Yi Z. (2012) A
natural small molecule harmine inhibits angiogenesis
and suppresses tumour growth through activation of
p53 in endothelial cells. PLoS ONE;7:e52162.
7. Song Y., Dai F., Zhai D., Dong Y., Zhang J., Lu B.,
Luo J., Liu M., Yi Z. (2012) Usnic acid inhibits breast
tumor angiogenesis and growth by suppressing VEG-
FR2-mediated AKT and ERK1/2 signaling pathways.
Angiogenesis;15:421–432.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Appendix S1. Synthetic procedures for the preparation of
compounds 4, 5, 12, 13, 14, 15, 20, 21, 22, 22, 24, 25.
Appendix S2. Compound 34 suppressed migration of
mouse CT-26.
8. Zhang T., Li J., Dong Y., Lai L., Dai F., Deng H., Chen
Y., Liu M., Yi Z. (2012) Cucurbitacin E inhibits breast
tumor metastasis by suppressing cell migration and
invasion. Breast Cancer Res Treat;135:445–458.
Figure S1. Compounds 34 suppressed migration of
mouse CT-26 colorectal cancer cells in transwell migration
assay compared with control group.
Chem Biol Drug Des 2014
11