18β-Glycyrrhetinic acid derivatives induced mitochondrial-mediated apoptosis through reactive oxygen species-mediated p53 activation in NTUB1 cells

Article abstract of DOI:10.1016/j.bmc.2011.05.054

Twenty six 18β-glycyrrhetinic acid (GA) (1) derivatives 2-27 including twelve new GA derivatives 10, 11, 13-17, 21-25 were synthesized and evaluated for cytotoxicities against NTUB1 cells (human bladder cancer cell lines). seco-Compounds 9, 25, and 27 are the most potent compounds of this series, inhibiting cell growth of human NTUB1 cells with an IC₅₀ values of 2.34 ± 0.28, 4.76 ± 1.15, and 3.31 ± 0.61 μM, respectively. Exposure of NTUB1 to 25 for 24 h significantly increased the production of reactive oxygen species (ROS). Flow cytometric analysis exhibited that treatment of NTUB1 with 25 did not induce cell cycle arrest but accompanied by an increase of apoptotic cell death in a dose-dependant manner after 24 h. Mitochondrial membrane potential (MMP) decreased significantly in a dose-dependant manner when the NTUB1 cells were exposed to 25 for 24 h. Marked collapse of the MMP suggested that dysfunction of the mitochondria may be involved in the oxidative burst and apoptosis induced by 25. Western blot analysis shows that NTUB1 cells treated with 25 increased the level of p-p53 in a dose-dependant manner. Further, NAC treatment prevented p53 phosphorylation stimulated by 25. These results suggested that 25 induced a mitochondrial- mediated apoptosis in NTUB1 cells through activation of p53, which are mainly mediated ROS generated by 25.

Full text of DOI:10.1016/j.bmc.2011.05.054

Bioorganic & Medicinal Chemistry 19 (2011) 4274–4285
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
18b-Glycyrrhetinic acid derivatives induced mitochondrial-mediated apoptosis
through reactive oxygen species-mediated p53 activation in NTUB1 cells
Kai-Wei Lin a, A-Mei Huang b, Tzyh-Chyuan Hour b, Shyh-Chyun Yang a, , Yeong-Shiau Pu c,
⇑
Chun-Nan Lin d,e,
⇑
a School of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
b Institute of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
c Department of Urology, College of Medicine, National Taiwan University, Taipei 100, Taiwan
d Faculty of Fragrance and Cosmetics, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
e Department of Biological Science and Technology, School of Medicine, China Medical University, Taichung 404, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty six 18b-glycyrrhetinic acid (GA) (1) derivatives 2–27 including twelve new GA derivatives 10, 11,
13–17, 21–25 were synthesized and evaluated for cytotoxicities against NTUB1 cells (human bladder can-
cer cell lines). seco-Compounds 9, 25, and 27 are the most potent compounds of this series, inhibiting cell
Received 20 April 2011
Revised 24 May 2011
Accepted 25 May 2011
Available online 30 May 2011
growth of human NTUB1 cells with an IC50 values of 2.34 0.28, 4.76 1.15, and 3.31 0.61 lM, respec-
tively. Exposure of NTUB1 to 25 for 24 h significantly increased the production of reactive oxygen species
(ROS). Flow cytometric analysis exhibited that treatment of NTUB1 with 25 did not induce cell cycle
arrest but accompanied by an increase of apoptotic cell death in a dose-dependant manner after 24 h.
Mitochondrial membrane potential (MMP) decreased significantly in a dose-dependant manner when
the NTUB1 cells were exposed to 25 for 24 h. Marked collapse of the MMP suggested that dysfunction
of the mitochondria may be involved in the oxidative burst and apoptosis induced by 25. Western blot
analysis shows that NTUB1 cells treated with 25 increased the level of p-p53 in a dose-dependant man-
ner. Further, NAC treatment prevented p53 phosphorylation stimulated by 25. These results suggested
that 25 induced a mitochondrial-mediated apoptosis in NTUB1 cells through activation of p53, which
are mainly mediated ROS generated by 25.
Keywords:
Synthesis
18b-Glycyrrhetinic acid derivatives
Cytotoxicity
p53
Antioxidant
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
differently affected according to the types of cells examined and
the modes to generate ROS.5
Triterpenoids abundantly exist in plant kingdom. The natural
triterpenoid, erythrodiol, uvaol, and b-amyrin have been reported
to have cytotoxic effect against several human cancer cell lines.
These triterpenoids induced cycle cell arrest and promoted apopto-
sis in human cancer cells through a ROS-dependent mechanism.1–3
We also reported that a series of ursolic acid derivatives induced
cell cycle arrest in NTUB1 cells associated with ROS.4 Thus it is gen-
erally believed that natural or synthetic triterpenoids induce cell
death by oxidative stress.
Oxidative stress can induce cell death by apoptosis and apopto-
tic cell death is a process control by a specific signaling pathway.5
Several studies have demonstrated that the mitochondria stress
and caspase activation are the most typical events required for
apoptosis and the mode of cell death induced by oxidative stress
Recently, several 18b-glycyrrhetinic acid (18b-GA) derivatives
have been shown to possess cytotoxicity against several human
cancer cell lines.6–8 However, a series of structures and cytotoxic
relationships of GA derivatives did not appear in literature. Based
on the above reason, we synthesized a series of GA derivatives,
evaluated their cytotoxicities against human NTUB1 cells, and dis-
cussed their structure and cytotoxic relationships and mechanism
of action. In this study, we examined whether GA derivative
induces apoptosis using human bladder cancer cell lines, NTUB1.
We also investigated the cellular mechanism of GA derivative-
induced cell death. We have demonstrated that GA derivative-
stimulated ROS production leads to activation of p53 in the cells.
2. Results and discussion
2.1. Chemistry
⇑
Corresponding authors. Tel.: +886 7 3121101; fax: +886 7 5562365 (C.N.L.).
E-mail addresses: scyang@cc.kmu.edu.tw (S.-C. Yang), lincna@cc.kmu.edu.tw
Compounds 2–27 were synthesized as depicted in Schemes 1–3.
Starting material, 18b-GA (1) was oxidized to 3-keto compound (2)
(C.-N. Lin).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.054

K.-W. Lin et al. / Bioorg. Med. Chem. 19 (2011) 4274–4285
4275
30
29
19
COOR
d
COOR
COOH
21
20
H
H
H
12
26
O
O
22
28
O
13
18
25
17
a
11
1
4
14
2
16
9
8
10
5
15
O
3
27
7
O
O
HO
6
23
24
5 R = H
2 R= H
1
b
c
6 R = CH3
3 R= CH3
7 R = CH2C6H5
4 R= CH2C6H5
e
COOR
COOR1
H
O
H
O
f or g
HOOC
R2
8 R = CH3
10 R1 = CH3, R2 = COOCH(CH3)2
11 R1 = CH3, R2 = CONHCH(CH3)2
12 R1 = CH2C6H5, R2 = COOCH3
9 R = CH2C6H5
13 R1 = CH2C6H5, R2 = COOCH2CH3
14 R1 = CH2C6H5, R2 = COOCH(CH3)2
15 R1 = CH2C6H5, R2 = CONHCH2CH3
16 R1 = CH2C6H5, R2 = CONHCH2CH2CH3
17 R1 = CH2C6H5, R2 = CONHCH(CH3)2
Scheme 1. Reagents and conditions: (a) CrO3, DMF, rt, 12 h; (b) CH3OH, H2SO4, reflux, 48 h; (c) EDC, DMAP, CH2Cl2, ROH; (d) m-CPBA, CH2Cl2, rt, 12 h; (e) CH2Cl2, p-TSA, rt,
24 h; (f) various alkyl halides, K2CO3, (CH3)2CO; (g) EDC, DMAP, CH2Cl2, R0-NH2, rt, 24 h.
COOH
a
CONHCH(CH3)2
CONHCH(CH3)2
H
H
H
O
O
O
b
HOOC
O
O
O
O
19
5
18
c or d
CONHCH(CH3)2
H
O
R
20 R = COOCH3
21 R = COOCH2CH3
22 R = COOCH(CH3)2
23 R = CONHCH2CH3
24 R = CONHCH2CH2CH3
25 R = CONHCH(CH3)2
Scheme 2. Reagents and conditions: (a) EDC, DMAP, CH2Cl2, R0-NH2, rt, 24 h; (b) CH2Cl2, p-TSA, rt, 24 h; (c) various alkyl halides, K2CO3, (CH3)2CO; (d) EDC, DMAP, CH2Cl2, R0-
NH2, rt, 24 h.
using CrO3 in DMF.9 The treatment of 3-oxo-derivative 2 with ex-
cess of MeOH in the presence of H2SO4 as catalyst and benzyl alco-
hol in the presence of 1-ethyl-3-(3-dimethyl aminopropyl)
carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP) to
yield 3 and 4, respectively.9 The treatment of 3-oxo-derivatives 2,
3, and 4 with m-chloroperbenzoic acid (m-CPBA), respectively,

4276
K.-W. Lin et al. / Bioorg. Med. Chem. 19 (2011) 4274–4285
COOH
CONHC6H5
b
CONHC6H5
H
H
H
O
O
O
a
HOOC
O
O
O
O
26
Scheme 3. Reagents and conditions: (a) EDC, DMAP, CH2Cl2, R0-NH2, rt, 24 h; (b) CH2Cl2, p-TSA, rt, 24 h.
27
5
provided lactones 5, 6, and 7.9 The lactone ring of 6 or 7 was
cleaved by treatment of p-toluenesulfonic acid (p-TSA) in appropri-
ate solvent to give products 8 or 9. Treatment of seco-methyl ester
8 with isopropyl alcohol or isopropylamine in the presence of EDCI
as the activating agent and DMAP as the catalyst provided the cor-
responding 3-ester compound 10 or 3-amide compound 11. The
seco-benzylester 9 in acetone reacted with alkyl halide and
K2CO3 in acetone or treatment with various alkylamines in the
presence of EDCI and DMAP to afford seco-compounds 12–14 or
15–17 (Scheme 1).
Compound 5 in CH2Cl2 reacted with isopropylamine or aniline
in the presence of EDCI and DMAP to afford lactone 18 or 26. The
lactone 18 or 26 in CH2Cl2 reacted with p-TSA to give seco-com-
pound 19 or 27. The seco-compound 19 in acetone reacted with
various alkyl halides and K2CO3 or in CH2Cl2 reacted with alkyla-
mine in the presence of EDCI and DMAP to afford 20–22 or 23–
25 (Schemes 2 and 3).
longer than methyl halide decreased the cytotoxic effect against
NTUB1 cells. The amidation of C-3-carboxylic group of 19 with
increasing alkyl chain length of carbamoyl group decreased the
cytotoxic effect against NTUB1 cells while the amidation of C-3-
carboxylic acid of 19 with branched alkyl chain, such as 25, signif-
icantly increase the cytotoxic effect against NTUB1 cells (Table 4).
It indicated that amidation of the two carboxylic acid groups at C-3
and C-30 with isopropylamine significantly enhanced the cytotoxic
effect against NTUB1 cells.
The above results and discussion obtained from structure-cyto-
toxity relationships suggested that seco-compounds with a 3,30-
dioic acid 30-benzyl ester, 3,30-dioic acid 30-phenylcarbamate,
and 3,30-diisopropyl carbamate, such as 9, 25, and 27, obtained
from 18b-GA derivatives, exhibited the most potent cytotoxic ef-
fect against NTUB1 cells. These three compounds could be used
as lead compounds for further design and synthsis of novel anti-
cancer agents.
The known 18b-GA derivatives 2–9, 12, 18, 19, 20, 26, and 27
were identified by spectroscopic data and compared with those
of data reported in literature.9 The new 18b-GA derivatives were
characterized by various spectroscopic methods (Table 1 and
Experimental Section) and compared with data reported in
literature.9
For further evaluated the cytotoxic effect of 18b-GA derivatives
and mechanisms of induced cancer cell death in vitro, cytotoxici-
ties of selective compounds 21, 23, and 25 against PC3 (human
prostate cancer cell line), SV-HUC1 (immortalized normal human
urothelial cells), and NTUB1 were studied and compared with
those of cytotoxicities against NTUB1 cells (Fig. 1). As shown in
Figure 1, 21, 23, and 25 did not exhibited stronger cytotoxic effects
against PC3 than those of cytotoxicities against NTUB1 cells while
these compounds enhanced the cell growth of SV-HUC1 cells. It
suggested that these compounds did not show cytotoxicity against
human normal cells. It needs to verify the mechanism related to
why these compounds enhanced the cell growth in SV-HUC1 cells.
The MTT method is the most frequently used in the determina-
tion of the viable cell number but its inadequacy is evident in pres-
ence of compound that cause alterations in mitochondria
activity.10 It is necessary to use another assay to determine the
cytotoxicity for supporting the enhancement of cell growth of
SV-HUC1 cells induced by 21, 23, and 25.
The antioxidants, such as pyrrolidine dithiocarbamate, epigallo-
catechin gallate, genistein, and vitamin E, revealed synergistic
cytotoxicity to PC3 cells when combined with taxol.11 In addition,
recently we have reported that a low dose of xanthine oxidase
(XO) inhibitor, terpenoids, and antioxidant xanthones combined
with cisplatin enhanced the cytotoxic effect against NTUB1
cells.3,12,13
Cisplatin has been used as a chemotherapeutic agent against
malignant solid tumors in the head and neck region. However,
there have been several side effects, such as nephrotoxicity and cis-
platin resistance, when clinically used for treatment of cancers. For
continual evaluation of compounds in combination of cisplatin
would enhance the cytotoxicity induced by cisplatin, and attenuate
side effect and cisplatin resistance. The authors selected com-
pounds 12 and 20, previously reported as XO inhibitor and antiin-
flammatory agents9, to examine if 12 or 20 in combination of
cisplatin would enhance the cytotoxicity induced by cisplatin. As
2.2. Biological results and discussion
Cytotoxicity of 1 and its derivatives against NTUB1 cells were
studied and the cisplatin was used as positive control (Tables 2–
4). As shown in Table 2, several 18b-GA derivatives showed signif-
icant cytotoxic activities against NTUB1 cells. The oxidation of C-3
of 1 such as 2 significantly enhanced the cytotoxic activity against
NTUB1 cells while esterification of carboxylic acid group at C-30
decreased the cytotoxic activity against NTUB1 cells. The lactoniza-
tion of 2 and 4 at ring A significantly weakened the cytotoxic activ-
ity against NTUB1 cells while the lactonization of 3 at ring A
significantly increased the cytotoxic effect against NTUB1 cells.
The amidation of 5 with aniline such 26 also significantly increased
the cytotoxic effect against NTUB1 cells (Table 4). The cleavage of
the lactone ring in CH2Cl2 by treatment of p-TSA such as 6, 7, 18,
and 26 significantly enhanced the cytotoxicity against NTUB1 cells,
such as 8, 9, 19, and 27 (Tables 2–4). It clearly indicated that cleav-
ing the lactone ring of 18b-GA derivative in CH2Cl2 generally signif-
icantly enhanced the cytotoxicity against NTUB1 cells. The above
results also indicated that seco-compound with a 3,30-dioic acid
30-benzyl ester or 3,30-dioic acid 30-phenylcarbamate, obtained
from 18b-GA derivatives such as 9 and 27, displayed the most po-
tent cytotoxic effect against NTUB1 cells (Tables 3 and 4). As shown
in Table 2, the esterification or amidation of C-3 of 9 did not en-
hance the cytotoxocity against NTUB1 cells.
The esterification of C-3-carboxylic group with methyl halide of
19 enhanced the cytotoxic effect against NTUB1 cells such as 20
while the C-3–carboxylic group of 19 esterified with an alkyl halide

K.-W. Lin et al. / Bioorg. Med. Chem. 19 (2011) 4274–4285
4277
Table 1
13C NMR spectroscopic data for compounds 10, 11, 13–17 and 21–25
Position
10
23.5
11
23.8
13
23.8
14
23.8
15
23.8
16
23.8
17
23.8
21
23.8
22
23.8
23
23.8
24
23.8
25
23.8
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
OCH3
31.4
173.4
146.6
38.8
29.7
34.3
43.6
52.8
41.2
199.6
128.5
169.3
45.1
26.4
26.5
31.8
48.4
41.2
44.0
31.1
37.7
114.2
23.8
19.5
18.6
23.4
28.6
28.3
176.9
51.8
32.2
172.4
146.6
39.0
31.1
35.7
43.7
53.1
41.2
200.4
128.4
170.3
45.0
26.4
26.5
31.8
48.3
41.2
44.0
31.3
37.7
114.3
23.5
19.5
18.7
23.5
28.6
28.3
176.5
51.8
31.4
173.9
146.6
38.7
28.4
34.4
44.0
52.8
41.2
199.5
128.2
169.2
45.0
26.4
26.5
31.7
48.1
43.6
50.7
31.2
37.7
114.2
23.5
19.5
18.4
23.3
28.4
28.3
176.2
31.4
173.4
146.6
38.8
29.7
34.3
44.0
52.7
41.2
199.5
128.3
169.1
45.0
26.4
26.5
31.7
48.1
43.6
50.6
31.2
37.7
114.2
23.5
19.5
18.6
23.3
28.5
28.3
176.2
32.0
173.2
146.6
39.0
31.3
35.6
44.0
53.0
41.2
200.4
128.2
170.2
45.0
26.3
26.5
31.7
48.2
43.7
50.9
31.1
37.6
114.3
23.5
19.5
18.7
23.3
28.5
28.3
176.2
32.0
173.2
146.6
39.0
31.3
35.7
44.0
53.1
41.2
200.4
128.2
170.3
45.0
26.3
26.5
31.7
48.2
43.7
51.0
31.1
37.6
114.3
23.5
19.5
18.7
23.3
28.5
28.3
176.2
32.1
172.3
146.6
39.0
31.1
35.6
44.0
53.1
41.2
200.3
128.2
170.2
45.0
26.3
26.5
31.7
48.2
43.7
51.0
31.2
37.6
114.3
23.5
19.4
18.7
23.3
28.5
28.3
176.2
31.4
174.6
146.5
38.8
28.6
34.4
43.3
52.8
42.2
199.6
128.4
169.5
45.0
26.4
26.5
31.9
48.2
43.7
43.7
31.5
37.4
114.2
23.5
19.5
18.7
23.3
29.5
29.4
173.9
31.4
173.4
146.5
38.8
29.7
34.3
43.3
52.8
42.1
199.5
128.4
169.4
45.0
26.4
26.5
31.9
48.2
41.1
43.7
31.4
37.4
114.2
23.8
19.5
18.6
23.3
29.5
28.6
174.6
32.0
173.2
146.6
39.0
29.5
35.6
43.3
53.1
42.0
200.3
128.4
170.4
45.0
26.4
26.6
31.9
48.1
41.1
43.8
31.5
37.4
114.4
23.5
19.6
18.7
23.3
29.5
28.6
174.7
32.1
173.3
146.6
39.0
29.5
35.7
43.3
53.0
41.2
200.3
128.4
170.4
45.0
26.4
26.6
31.9
48.1
41.1
43.8
31.5
37.4
114.3
23.5
19.6
18.7
23.3
29.5
28.6
174.7
32.1
172.4
146.6
39.0
29.5
35.7
43.3
53.0
41.2
200.3
128.4
170.3
45.0
26.4
26.6
31.9
48.1
41.1
43.7
31.5
37.4
114.3
23.5
19.5
18.7
23.3
29.5
28.6
174.6
OCH2CH3
OCH2CH3
OCH2CH2CH3
OCH2CH2CH3
OCH2CH2CH3
OCH(CH3)2
OCH(CH3)(CH3)
OCH(CH3)(CH3)
OCH2
60.2
14.2
60.3
14.2
50.6
21.8
21.8
41.2
21.8
21.8
50.6
21.8
21.8
66.2
136.1
128.3
128.6
128.5
128.6
128.3
66.2
66.3
136.1
128.3
128.6
128.4
128.6
128.3
34.3
66.2
136.1
128.3
128.6
128.4
128.6
128.3
66.2
136.1
128.3
128.6
128.4
128.6
128.3
10
136.1
128.3
128.6
128.5
128.6
128.3
20
30
40
50
60
NH–CH2CH3
NH–CH2CH3
NH–CH2CH2CH3
NH–CH2CH2CH3
NH–CH2CH2CH3
NH–CH(CH3)2
NH–CH(CH3)2
NH–CH(CH3)(CH3)
NH–CH(CH3)(CH3)
NH–CH(CH3)(CH3)
NH–CH(CH3)(CH3)
44.9
14.8
14.7
41.3
22.8
11.4
42.1
31.3
11.4
50.9
50.9
51.0
50.8
50.6
51.0
50.9
50.9
22.7
22.7
22.8
22.9
22.7
22.7
22.7
22.7
22.8
23.0
22.8
23.0
22.8
22.9
22.8
22.9
shown in Table 5 and Figure 2, the combination of 12 or 20 and cis-
platin as the concentration used in Table 5 and Figure 2 signifi-
cantly enhanced cell death induced by 12 or 20 alone,
peutic efficacy of cisplatin, and reduce the side effect and resis-
tance of cisplatin. It is necessary to use several experimental data
for supporting the above results.
respectively, except for 20 (30
l
M) combined with 5 or 10
l
M cis-
ROS induce programed cell death or necrosis, induce or sup-
press the expression of many genes, and activate cell signaling cas-
cades.14 Based on the above result, the author selected 25 to
determine whether 25 stimulates ROS production in NTUB1 cells.
platin and 50 M combined with 10
l
lM cisplatin attenuated the
cell death induced by 20 alone, respectively. The above result sug-
gested an additive and possibly a more than additive effect of the
most concentration of 12 or 20 combined with cisplatin when
Exposure of cells to 20
lM cisplatin (positive control) and 25 (10
compared with 12 or 20 alone. While 50
ing weaker cytotoxic activity than that of 5
with 5 M cisplatin enhanced the cell death induced by 5
platin. It suggested that the combination of 50 M 12 or 20 and
M cisplatin or a low dose of cisplatin may enhance the thera-
l
M 12 or 20, each indicat-
M cisplatin, combined
M cis-
and 25 M), and 1 mM N-acetylcysteine (NAC) (negative control)
l
l
for 24 h, respectively, caused a significant increase, and decrease
in the intracellular generation of ROS, respectively, as determined
with the fluorescent dye, H2DCFDA, which preferentially detected
intracellular ROS (Fig. 3A–E). The generation of ROS by 25 was fur-
l
l
l
5
l

4278
K.-W. Lin et al. / Bioorg. Med. Chem. 19 (2011) 4274–4285
Table 2
Cytotoxic activity of 18b -glycyrrhetinic acid (1) and its derivatives 2–7
30
29
COOR
COOR1
COOH
21
19
18
20
H
H
H
12
26
O
O
22
28
O
13
14
25
17
11
1
4
2
16
9
8
10
5
15
O
3
27
7
O
O
HO
6
23
24
5-7
1
2-4
Compd
R
R1
IC50 SDa
NTUB1b
(lM)
Cisplatin
3.27 0.10
27.31 5.17
18.57 1.64
56.5 14.35
20.23 5.10
73.54 35.12
24.28 3.98
57.61 8.80
1
2
3
4
5
6
7
H
CH3
CH2C6H5
H
CH3
CH2C6H5
a
Data represent mean value (SD) of five independent determinations.
Human bladder cancer cell line.
b
Table 3
to apoptosis. Exposure of cells to NAC and H2O2 (positive control)
caused a attenuated and enhanced the population of sub-G1 phase,
respectively. These results also support that the induction of apop-
tosis by 25 mediated through ROS.
Cytotoxic activity of 18b -glycyrrhetinic acid derivatives 8–17
COR1
It has been suggested that ROS overproduction induced a reduc-
tion in the mitochondrial membrance potential (MMP) (Dwm) as
H
O
well as mitochondrial dysfunction.2 Thus, to detect the change of
MMP, the specific fluorescent probe JC-1 (5,50,6,60-tetrachloro-
1,10,3,30-tetraethyl benzimidazolocarbocyanine iodide) was used.
As shown in Figure 5A–E, positive control CCCP (carbonyl cyanide
3-chlorophenylhydrazone) and various concentrations of 25 signif-
icantly increased the number of JC-1 monomers (R2) and de-
creased that of JC-1 aggregates (R1). It suggested that 25
R
8-17
Compd
R
R1
IC50 SDa
NTUB1b
(
lM)
decreased
Dwm dramatically in the NTUB1 cells. Marked collapse
of the MMP suggested that dysfunction of mitochondria may be in-
volved in the oxidative burst and apoptosis induced by 25. Here,
we may demonstrate that 25 induce cell death of NTUB1 cells
mainly by apoptosis through mitochondria-mediated pathway.
p53 is an important regulator for apoptosis induced by ROS.5
There is a close relationship among ROS, DNA damage, and p53
activation.5 The most important p53 function is its ability to acti-
vate apoptosis, and disruption of this process can promote tumor
progression and chemoresistance.15 We investigated the effect of
25 on p53 phosphorylation in the cells. Western blot analysis dis-
plays that NTUB1 cells treated with 25 increased the level of p-p53
in a dose–dependent manner (Fig. 6A). Further, NAC treatment pre-
vented p53 phosphorylation stimulated by 25 (Fig. 6B). These data
strongly suggested that the generation of ROS played an important
role in p53 activation.
8
9
COOH
COOH
OCH3
OCH2C6H5
OCH3
20.72 4.97
2.34 0.28
25.30 2.28
31.35 0.55
9.41 2.72
27.76 6.48
18.20 1.58
42.74 11.15
49.39 11.52
30.12 2.41
10
11
12
13
14
15
16
17
COOCH(CH3)2
COOCH(CH3)2
COOCH3
COOCH2CH3
COOCH(CH3)2
CONHCH2CH3
CONHCH2CH2CH3
CONHCH(CH3)2
OCH3
OCH2C6H5
OCH2C6H5
OCH2C6H5
OCH2C6H5
OCH2C6H5
OCH2C6H5
a
Data represent mean value (SD) of five independent determinations.
Human bladder cancer cell line.
b
ther supported by the finding that cells treated with antioxidant,
NAC, attenuated the oxidation of H2DCFDA. These results indicate
the 25 is able to generate intracellular ROS and subsequently in-
duced cell death.
The effect of positive control cisplatin, negative control NAC,
and 25 on cell cycle progression was determined by using cell sort-
ing analysis in propidium iodide-stained NTUB1 cells. As shown in
Since the major enzyme involved in apoptosis is caspase-3, acti-
vation of caspase-3 in 25-treated cells was examined. NTUB1 cells
were treated with 10, 25, and 50 lM 25, respectively. After treat-
ment, cells were assessed for the occurrence of activated cas-
pase-3 by western blotting. There was no caspase-3 activity
induced by 25 (data not shown).
Figure 4A–G, treatment with 25, 50, and 75 lM 25 for 24 h did not
induce cell cycle arrest but induce the population of sub-G1 phase
in dose-dependent manner. Exposure of cells to 25 resulted in the
increase of sub-G1 phase cells which mean apoptotic cells. Expose
of cells to 25 also resulted in a notable reduction of cells in S phase.
It means that the cell exits from cell cycle and apoptosis occurs.
These results suggested that 25 induced cell death is mainly due
3. Conclusion
Here we report the design, synthesis, and biological evaluation
of 18b-GA derivatives which exhibited cytotoxic activity against

K.-W. Lin et al. / Bioorg. Med. Chem. 19 (2011) 4274–4285
4279
Table 4
Cytotoxic activity of 18b -glycyrrhetinic acid derivatives 18–27
CONHCH(CH3)2
COR
COR
H
H
O
H
O
O
R
HOOC
O
O
18, 26
19, 27
20-25
Compd
R
IC50 SDa
NTUB1b
(lM)
18
19
20
21
22
23
24
25
26
27
NHCH(CH3)2
NHCH(CH3)2
COOCH3
COOCH2CH3
COOCH (CH3)2
CONHCH2CH3
CONHCH2CH2CH3
CONHCH(CH3)2
NHC6H5
25.66 1.81
17.47 2.11
13.12 5.27
19.44 19.20
29.17 26.61
12.75 2.10
>50
4.76 1.15
15.95 5.30
3.31 0.61
NHC6H5
a
Data represent mean value (SD) of five independent determinations.
Human bladder cancer cell line
b
Figure 1. Cytotoxities of 21 (A), 23 (B), and 25 (C) against PC3, SV-HUC1, and NTUB1 cells. Cell viability was assessed by the MTT assay after treating with different
concentrations of compounds for 72 h. The data shown represent mean SD (n = 5) for one experiment performed in triplicate. P <0.05 (a), P <0.01 (b), and P <0.001 (c)
compared to the control value, respectively.

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K.-W. Lin et al. / Bioorg. Med. Chem. 19 (2011) 4274–4285
Table 5
4. Experimental
Percent cytotoxic inhibition (compared to untreated control cells) of different
concentrations of 12, 20, and 12 or 20 combined with
respectively, against NTUB1 cells.
5
or 10
l
M
cisplatin,
4.1. Chemistry
l
M
1
3
5
10
30
50
Reagents, starting material, and solvents were purchased from
commercial suppliers. Cisplatin was obtained from Pharmacia Up-
john, Milan, Italy. All culture reagents were obtained from Gibco
BRL. Optical rotations were recorded with a JASCO-370 polarimeter
using CHCl3 as solvent. IR spectra were determined with a Perkin–
Cisplatin
12
12 + 5
12 + 10
20
20 + 5
20 + 10
72
20
56
56
24
59
59
75
50
58
56
27
57
59
32
50
58
40
56
58
22
59
54
25
59
60
66
70
67
67
66
55
68
78
70
71
74
67
l
M Cisplatin
lm cisplatin
Elmer system 2000 FTIR spectrophotometer. 1H (400 MHz) and 13
C
l
M Cisplatin
lM Cisplatin
(100 MHz) NMR were recorded on a Varian UNITY-400 spectrom-
eter. Low-resolution mass spectra and high-resolution mass spec-
tra data were obtained on a JMX-HX 100 mass spectrometer.
Chromatography was performed using a flash-column technique
on Silica Gel 60 supplied by E. Merck.
120
100
80
60
40
20
0
A
B
Cisplatin 0 µM
Cisplatin 5 µM
Cisplatin 10 µM
c
4.2. General procedure for esterification and amidation of seco-
compounds
c
c
To a solution of seco-compound in dry acetone (10 mL) were
added an equivalent mole of potassium carbonate (2 mmol) fol-
lowed by alkyl halides (2 mmol). The reaction mixture was stirred
at room temperature or refluxed overnight and concentrated in the
vacuum. To the crude product was added 10% HCl solution, parti-
tioned with CH2Cl2 for three times, and concentrated. The residue
was purified by chromatography with ethyl acetate/n-hexane or
ethyl acetate/CH2Cl2 to yield pure product. To a solution of seco-
compound in dry CH2Cl2 (10 mL) were added EDCI (2 mmol) and
DMAP (catalytic amount) followed by various amines (2 mmol).
The reaction mixture was stirred at room temperature for 2–4 h
and concentrated in the vacuum. The residue was purified by chro-
matography with ethyl acetate/n-hexane or ethyl acetate/CH2Cl2 to
yield pure product.
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
0 µM 12
1 µM 12 3 µM 12
5 µM 12
50 µM 12
10 µM 12 30 µM 12
120
100
80
60
40
20
0
Cisplatin 0 µM
Cisplatin0 5 µM
Cisplatin0 10 µM
a
a
b
b
4.2.1. 3,4-seco-11-Oxo-18b-olean-4(23),12-dien-3,30-dioic acid
3-isopropyl 30-methyl, ester (10)
Compound 10 was prepared from 8 (80 mg, 0.16 mmol) follow-
ing the general procedure described for esterification using isopro-
pyl iodide as alcohol moiety. Compound 10 was obtained as white
c
c
c
c c
c
c
c
c
a c
c
c
b c
c
amorphous powder (86.2 mg, 0.16 mmol, 100%). ½a D25
ꢀ
8 (c 0.1,
CHCl3). IR (film on NaCl) cmꢁ1 1731, 1657. 1H NMR (CDCl3) d
0.82 (3H, s, Me-28), 1.15 (3H, s, Me-29), 1.16 (3H, s, Me-25), 1.17
(3H, s, Me-26), 1.21 (6H, d, J = 6.4 Hz, –CH(CH3)2), 1.38 (3H, s,
Me-27), 1.76 (3H, s, Me-24), 2.60 (1H, td, J = 13.4, 6.0 Hz, Hb -18),
3.70 (3H, s, OCH3), 4.70 (1H, br s, H-23), 4.90 (1H, br s, H-23),
4.95 (1H, m, –CH(CH3)2), 5.69 (1H, s, H-12). 13C NMR (CDCl3) see
Table 1. EI-MS (70 eV) m/z (% rel. int.), 540 [M]+ (63). HR-EI-MS
m/z calcd for C34H52O5, 540.3815; found: 540.3814.
0 µM 20
3 µM 20
1 µM 20
10 µM 20
50 µM 20
5 µM 20
30 µM 20
Figure 2. Cisplatin and combination of cisplatin with 12 (A) or 20 (B) induced cell
death. Cell viability was assessed by MTT assay for 72 h after treatment with
different concentrations of cisplatin (0, 5, and 10
cisplatin combined with different concentrations (0, 1, 3, 5, 10, 30 or 50
or 20. P <0.05 (a), P <0.01 (b), and P <0.001 (c) compared to the control value,
respectively.
l
M) and each concentration of
M ) of 12
l
4.2.2. 3-Isopropylcarbamoyl-11-oxo-18b-3,4-seco-olean-4(23),
12-diene 30-methyl ester (11)
Compound 11 was prepared from 8 (212 mg, 0.43 mmol)
following the general procedure described for amidation using
isopropyl amine as amine moiety. It was obtained as white
human NTUB1 cells. seco-Compounds 9, 25, and 27 are the most
potent compounds of this series of 18b-GA derivatives with an
IC50 values of 2.34 0.28, 4.76 1.15, and 3.31 0.61 lM, respec-
amorphous powder (201.6 mg, 0.37 mmol, 86.0%). ½a D25
ꢀ
10 (c
tively. Selective compound 25 induces cell death mainly by apop-
tosis through mitochondria-mediated and caspase-3-independent
pathway, where ROS-mediated p53 activation acted as upstream
signaling. It needs further to elucidate the detailed mechanism of
this series of compounds induced inhibition of tumor cell growth.
Several compounds were identified as potential p53 activity
modulators.16–18 The activities of these compounds may be due
to inhibition of p53-MDM2 (murin double minute-2) interaction
and subsequent p53 release and activation. Compound 25 exhib-
ited different mechanism of induced cell death from the above
reported compounds.
0.1, CHCl3). IR (film on NaCl) cmꢁ1 3299, 1727, 1661. 1H NMR
(CDCl3)
d
0.81 (3H, s, Me-28), 1.10 (3H, d, J = 6.8 Hz,
–CH(CH3)(CH3)), 1.11 (3H, d, J = 6.8 Hz, –CH(CH3)(CH3)), 1.14
(6H, s, Me-26 and 29), 1.17 (3H, s, Me-25), 1.37 (3H, s, Me-27),
1.77 (3H, s, Me-24), 2.47 (1H, td, J = 13.2, 6.0 Hz, Hb-18), 3.69
(3H, s, OCH3), 4.01 (1H, m, –CH(CH3)2), 4.73 (1H, br s, H-23),
4.90 (1H, br s, H-23), 5.48 (1H, d, J = 7.6 Hz, NH), 5.69 (1H, s,
H-12). 13C NMR (CDCl3) see Table 1. EI-MS (70 eV) m/z (% rel.
int.), 539 [M]+ (70). HR-EI-MS m/z calcd for C34H53NO4,
539.3974; found: 539.3956.

K.-W. Lin et al. / Bioorg. Med. Chem. 19 (2011) 4274–4285
4281
A
B
C
D
E
100
80
F
a
60
Control
Cisplatin, 20 µM
NAC, 1 mM
25, 10 µM
25, 25 µM
40
20
0
Figure 3. Effect of 25 on the production of ROS in NTUB1 cells: control (A), 20
lM cisplatin (B), 1 mM NAC (C), 10
lM 25 (D), and 25
lM 25 (E) for 24 h. The amount of ROS
was assayed by H2DCFDA staining. Each sampling measured the fluorescence intensity of region M1 of 3 ꢂ 105 cells/6 cm dish. The control cells were treated with medium.
Results were repeated by three independent experiments. Bar graph (F) shows percentage changes in intracellular ROS generation of region M1 of NTUB1 cells. Data as
mean SD, n = 3. ap <0.05 compared to the control value.
4.2.3. 3,4-seco-11-Oxo-18b-olean-4(23),12-dien-3,30. dioic acid
3-ethyl,30-benzyl ester (13)
1.22 (3H, t, J = 7.2 Hz, –CH2CH3), 1.37 (3H, s, Me-27), 1.75 (3H,
s, Me-24), 2.60 (1H, td, J = 13.2, 6.0 Hz, Hb-18), 4.10 (2H, q,
J = 7.2 Hz, –CH2CH3), 4.69 (1H, br s, H-23), 4.90 (1H, br s,
H-23), 5.09 (1H, d, J = 12.4 Hz, –OCHH–), 5.20 (1H, d,
J = 12.0 Hz, –OCHH–), 5.57 (1H, s, H-12), 7.37 (5H, m, aromatic
proton). 13C NMR (CDCl3) see Table 1. EI-MS (70 eV) m/z (% rel.
int.), 602 [M]+ (21). HR-EI-MS m/z calcd for C39H54O5,
602.3971; found: 602.3971.
Compound 13 was prepared from 9 (112 mg, 0.20 mmol) fol-
lowing the general procedure described for esterification using
ethyl iodide as alcohol moiety. It was obtained as white amor-
phous powder (81.6 mg, 0.13 mmol, 65.0%). ½a D25
ꢀ
3 (c 0.5, CHCl3).
IR (film on NaCl) cmꢁ1 1727, 1657, 1579. 1H NMR (CDCl3) d 0.74
(3H, s, Me-28), 1.15 (3H, s, Me-29), 1.16 (6H, s, Me-25 and 26),

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K.-W. Lin et al. / Bioorg. Med. Chem. 19 (2011) 4274–4285
Figure 4. Flow cytometry analysis of cisplatin and 25-treated NTUB1 cells. (8 ꢂ 105 Cell/10 cm dish) were treated with absence of cisplatin and 25 (control A), 20
lM cisplatin
(B), 25 lM 25 (C), 50 lM 25 (D), 75 lM 25 (E), 1 mM NAC (F), and 50 lM H2O2 (G), for 24 h. At the indicated time, cells were stained with propidium iodide (PI), DNA contents
were analyzed via flow cytometry and the amount of apoptosis was measured by accumulation of sub-G1 DNA contents in the cells. The control cells were treated with
medium. Results are representative of three independent experiments.
4.2.4. 3,4-seco-11-Oxo-18b-olean-4(23),12-dien-3,30. dioic acid
3-isopropyl,30-benzyl ester (14)
Compound 14 was prepared from 9 (100 mg, 0.17 mmol) fol-
lowing the general procedure described for esterification using iso-
propyl iodide as alcohol moiety. It was obtained as white
amine as amine moiety. It was obtained as white amorphous pow-
der (58.0 mg, 0.097 mmol, 57.1%). ½a D25
ꢀ
11 (c 0.35, CHCl3). IR (film
on NaCl) cmꢁ1 3299, 1727, 1657, 1553. 1H NMR (CDCl3) d 0.75
(3H, s, Me-28), 1.10 (3H, t, J = 7.2 Hz, –CH2CH3), 1.14 (3H, s, Me-
29), 1.15 (3H, s, Me-25), 1.16 (3H, s, Me-26), 1.36 (3H, s, Me-27),
1.77 (3H, s, Me-24), 2.49 (1H, td, J = 13.2, 6.0 Hz, Hb-18), 3.23
(2H, m, –CH2CH3), 4.74 (1H, br s, H-23), 4.90 (1H, br s, H-23),
5.09 (1H, d, J = 12.0 Hz, –OCHH–), 5.20 (1H, d, J = 12.0 Hz,
–OCHH–), 5.58 (1H, s, H-12), 5.67 (1H, br s, NH), 7.36 (5H, m, aro-
matic proton). 13C NMR (CDCl3) see Table 1. EI-MS (70 eV) m/z (%
rel. int.), 601 [M]+ (36). HR-EI-MS m/z calcd for C39H55NO4,
601.4131; found: 601.4131.
amorphous powder (49.4 mg, 0.08 mmol, 47.1%). ½a D25
ꢀ
16 (c 0.05,
CHCl3). IR (film on NaCl) cmꢁ1 1727, 1657, 1631. 1H NMR (CDCl3)
d 0.74 (3H, s, Me-28), 1.15 (3H, s, Me-29), 1.16 (6H, s, Me-25 and
26), 1.19 (6H, d, J = 6.4 Hz, –CH(CH3)2), 1.39 (3H, s, Me-27), 1.76
(3H, s, Me-24), 2.58 (1H, td, J = 13.2, 6.4 Hz, Hb-18), 4.69 (1H, br
s, H-23), 4.90 (1H, br s, H-23), 4.95 (1H, m, –CH(CH3)2), 5.09 (1H,
d, J = 12.4 Hz, –OCHH–), 5.20 (1H, d, J = 12.4 Hz, –OCHH–), 5.57
(1H, s, H-12), 7.38 (5H, m, aromatic proton). 13C NMR (CDCl3) see
Table 1. EI-MS (70 eV) m/z (% rel. int.), 616 [M]+ (33). HR-EI-MS
m/z calcd for C40H56O5, 616.4127; found: 616.4136.
4.2.6. 3-Propylcarbamoyl -11-oxo-18b-3,4-seco-olean-4(23),
12-diene 30-benzyl ester (16)
Compound 16 was prepared from 9 (125 mg, 0.22 mmol) fol-
lowing the general procedure described for amidation using propyl
amine as amine moiety. It was obtained as white amorphous pow-
4.2.5. 3-Ethylcarbamoyl -11-oxo-18b-3,4-seco-olean-4(23),12-
diene 30-benzyl ester (15)
Compound 15 was prepared from 9 (100 mg, 0.17 mmol) fol-
lowing the general procedure described for amidation using ethyl
der (105.2 mg, 0.17 mmol, 77.3%). ½a D25
ꢀ
15 (c 0.5, CHCl3). IR (film on
NaCl) cmꢁ1 3439, 1724, 1657, 1513. 1H NMR (CDCl3) d 0.75 (3H, s,

K.-W. Lin et al. / Bioorg. Med. Chem. 19 (2011) 4274–4285
4283
Figure 5. Collapse of MMP induced by CCCP and 25 in the NTUB1 cells were treated with absence CCCP and 25 (control A), 10
lM CCCP (B), 10
lM 25 (C), 25
l
M 25 (D),
50 lM 25 (E), for 24 h. Appropriate gates were made to define JC-1 aggregates (R2) and JC-1 monomers (R1).
Me-28), 0.90 (3H, t, J = 7.6 Hz, –CH2CH2CH3), 1.14 (3H, s, Me-29),
1.15 (3H, s, Me-25), 1.16 (3H, s, Me-26), 1.36 (3H, s, Me-27), 1.49
(2H, q, J = 7.2 Hz, –CH2CH2CH3), 1.77 (3H, s, Me-24), 2.49 (1H, td,
J = 13.2, 6.0 Hz, Hb-18), 3.16 (2H, dd, J = 13.6, 6.0 Hz, –CH2CH2CH3),
4.74 (1H, br s, H-23), 4.90 (1H, br s, H-23), 5.09 (1H, d, J = 12.4 Hz,
–OCHH–), 5.20 (1H, d, J = 12.4 Hz, –OCHH–), 5.73 (1H, br s, NH),
5.58 (1H, s, H-12), 7.36 (5H, m, aromatic proton). 13C NMR (CDCl3)
see Table 1. EI-MS (70 eV) m/z (% rel. int.), 615 [M]+ (36). HR-EI-MS
m/z calcd for C40H57NO4, 615.4287; found: 615.4283.
4.00 (1H, m, –CH(CH3)2), 4.74 (1H, br s, H-23), 4.91 (1H, br s,
H-23), 5.09 (1H, d, J = 12.4 Hz, –OCHH–), 5.20 (1H, d, J = 12.4 Hz,
–OCHH–), 5.56 (1H, br s, NH), 5.58 (1H, s, H-12), 7.36 (5H, m, aro-
matic proton). 13C NMR (CDCl3) see Table 1. EI-MS (70 eV) m/z (%
rel. int.), 615 [M]+ (42). HR-EI-MS m/z calcd for C40H57NO4,
615.4288; found: 615.4286.
4.2.8. 30-Isopropylcarbamoyl-11-oxo-18b-3,4-seco-olean-4(23),
12-diene 3-ethyl ester (21)
Compound 21 was prepared from 19 (100 mg, 0.19 mmol)
following the general procedure described for esterification using
ethyl iodide as alcohol moiety. It was obtained as white amorphous
4.2.7. 3-Isopropylcarbamoyl-11-oxo-18b-3,4-seco-olean-4(23),
12-diene 30-benzyl ester (17)
Compound 17 was prepared from 9 (100 mg, 0.17 mmol) fol-
lowing the general procedure described for amidation using iso-
propyl amine as amine moiety. It was obtained as white
powder (27.3 mg, 0.049 mmol, 25.8%). ½a D25
ꢀ
9 (c 0.25, CHCl3). IR
(film on NaCl) cmꢁ1 3365, 1735, 1650. 1H NMR (CDCl3) d 0.82
(3H, s, Me-28), 1.11 (3H, s, Me-26), 1.14 (3H, d, J = 6.4 Hz,
–CH(CH3)(CH3)), 1.16 (3H, s, Me-25),, 1.16 (3H, d, J = 6.4 Hz,
–CH(CH3)(CH3)), 1.17 (3H, s, Me-29), 1.23 (3H, t, J = 7.2 Hz, –CH2
CH3), 1.39 (3H, s, Me-27), 1.76 (3H, s, Me-24), 2.61 (1H, td,
J = 13.2, 6.4 Hz, Hb-18), 4.08 (2H, q, J = 7.2 Hz, –CH2CH3), 4.12
(1H, m, –CH(CH3)2), 4.70 (1H, br s, H-23), 4.90 (1H, br s, H-23),
5.33 (1H, d, J = 8.0 Hz, NH), 5.65 (1H, s, H-12). 13C NMR (CDCl3)
amorphous powder (92.9 mg, 0.15 mmol, 88.2%). ½a D25
ꢀ
7 (c 0.25,
CHCl3). IR (film on NaCl) cmꢁ1 3306, 1727, 1657, 1535. 1H NMR
(CDCl3)
d
0.75 (3H, s, Me-28), 1.10 (3H, d, J = 6.8 Hz,
–CH(CH3)(CH3)), 1.12 (3H, d, J = 6.8 Hz, –CH(CH3)(CH3)), 1.14 (3H,
s, Me-29), 1.15 (3H, s, Me-25), 1.16 (3H, s, Me-26), 1.36 (3H, s,
Me-27), 1.77 (3H, s, Me-24), 2.46 (1H, td, J = 13.2, 6.0 Hz, Hb-18),

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K.-W. Lin et al. / Bioorg. Med. Chem. 19 (2011) 4274–4285
–NHCH2CH3), 5.68 (1H, s, H-12). 13C NMR (CDCl3) see Table 1. EI-
MS (70 eV) m/z (% rel. int.), 552 [M]+ (47). HR-EI-MS m/z calcd
for C35H36N2O3, 552.4291; found: 552.4288.
4.2.11. 30-Isopropylcarbamoyl-11-oxo-18b-3,4-seco-olean-4(23),
12-diene 3-propyl carbamate (24)
Compound 24 was prepared from 19 (100 mg, 0.19 mmol) fol-
lowing the general procedure described for amidation using propyl
amine as amine moiety. It was obtained as white amorphous pow-
der (46.1 mg, 0.081 mmol, 42.6%). ½a D25
ꢀ
4 (c 0.25, CHCl3). IR (film on
NaCl) cmꢁ1 3424, 1735, 1653. 1H NMR (CDCl3) d 0.83 (3H, s, Me-
28), 0.91 (3H, t, J = 7.2 Hz, –CH2CH2CH3), 1.11 (3H, s, Me-26), 1.15
(3H, d, J = 6.8 Hz, –CH(CH3)(CH3)), 1.16 (3H, d, J = 6.8 Hz, –CH
(CH3)(CH3)), 1.16 (3H, s, Me-25), 1.17 (3H, s, Me-29), 1.38 (3H, s,
Me-27), 1.77 (3H, s, Me-24), 2.23 (2H, m, –CH2CH2CH3), 2.51 (1H,
td, J = 13.2, 6.0 Hz, Hb-18), 3.16 (2H, dd, J = 13.2, 6.4 Hz,
–
CH2CH2CH3), 4.12 (1H, m, –CH(CH3)2), 4.74 (1H, br s, H-23), 4.91
(1H, br s, H-23), 5.33 (1H, d, J = 8.4 Hz, NH), 5.67 (1H, s, H-12),
5.68 (1H, t, J = 4.0 Hz, –NHCH2CH2CH3) 13C NMR (CDCl3) see Table 1.
EI-MS (70 eV) m/z (% rel. int.), 566 [M]+ (95). HR-EI-MS m/z calcd
for C36H58N2O3, 566.4447; found: 566.4439.
4.2.12. 3,4-seco-11-Oxo-18b-olean-4(23),12-dien-3,30-diiso-
propyl carbamate (25)
Compound 25 was prepared from 19 (100 mg, 0.19 mmol) fol-
lowing the general procedure described for amidation using iso-
propyl amine as amine moiety. It was obtained as white
Figure 6. Compound 25 induced p53 phosphorylation in NTUB1 cell. NTUB1 cells
were incubated with 25 at different concentrations (0 – 50
(cisplatin), respectively, for 24 h (A) or the cells were exposed to 50
absence and presence of 1 mM NAC, and 50 M 25 combined with 1 mM NAC,
l
M) and 10
lM CDDP
amorphous powder (61.4 mg, 0.11 mmol, 58.0%). ½a D25
ꢀ
4 (c 0.25,
lM 25 in the
CHCl3). IR (film on NaCl) cmꢁ1 3439, 1727, 1650. 1H NMR (CDCl3)
d 0.80 (3H, s, Me-28), 1.11 (3H, s, Me-26), 1.12 (6H, d, J = 6.8 Hz,
–CH(CH3)2), 1.15 (3H, s, Me-29), 1.15 (6H, d, J = 6.8 Hz, –CH(CH3)2),
1.17 (3H, s, Me-25), 1.38 (3H, s, Me-27), 1.79 (3H, s, Me-24), 2.48
(1H, td, J = 13.2, 6.4 Hz, Hb-18), 4.02 (1H, m, –CH(CH3)2), 4.12
(1H, m, –CH(CH3)2), 4.74 (1H, br s, H-23), 4.90 (1H, br s, H-23),
5.34 (1H, d, J = 7.6 Hz, NH), 5.46 (1H, d, J = 7.6 Hz, NH), 5.65 (1H,
s, H-12). 13C NMR (CDCl3) see Table 1. EI-MS (70 eV) m/z (% rel.
int.), 566 [M]+ (89). HR-EI-MS m/z calcd for C36H58N2O3,
566.4447; found: 566.4444.
l
respectively, for 24 h (B) and then cellular proteins were analyzed by western
blotting. A representative result from triplicate experiments was shown.
see Table 1. EI-MS (70 eV) m/z (% rel. int.): 553 [M]+ (62). HR-EI-MS
m/z calcd for C35H55NO4, 553.4131; found: 553.4133.
4.2.9. 30-Isopropylcarbamoyl-11-oxo-18b-3,4-seco-olean-4(23),
12-diene 3-isopropyl ester (22)
Compound 22 was prepared from 19 (80 mg, 0.15 mmol) fol-
lowing the general procedure described for esterification using iso-
propyl iodide as alcohol moiety. It was obtained as white
4.3. Cell culture and MTT assay for cell viability
amorphous powder (73.3 mg, 0.13 mmol, 86.7%). ½a D25
ꢀ
1 (c 1.0,
NTUB1, a human bladder carcinoma cell and PC3 were main-
tained in RPMI 1640 medium supplemented with 10% fetal bovine
CHCl3). IR (film on NaCl) cmꢁ1 3373, 1727, 1661. 1H NMR (CDCl3)
d 0.82 (3H, s, Me-28), 1.11 (3H, s, Me-29), 1.14 (3H, d, J = 6.8 Hz,
–CH(CH3)(CH3)), 1.16 (3H, s, Me-25), 1.16 (3H, d, J = 6.8 Hz, –CH
(CH3)(CH3)), 1.17 (3H, s, Me-26), 1.20 (6H, d, J = 6.4 Hz, –CH(CH3)2),
1.39 (3H, s, Me-27), 1.76 (3H, s, Me-24), 2.59 (1H, td, J = 13.2,
6.4 Hz, Hb-18), 4.12 (1H, m, –CH(CH3)2), 4.69 (1H, br s, H-23),
4.90 (1H, br s, H-23), 4.95 (1H, m, –CH(CH3)2), 5.34 (1H, d,
J = 8.0 Hz, NH), 5.64 (1H, s, H-12). 13C NMR (CDCl3) see Table 1.
EI-MS (70 eV) m/z (% rel. int.), 567 [M]+ (64). HR-EI-MS m/z calcd
for C36H57NO4, 567.4288; found: 567.4269.
serum (FBS), 100 unit/mL penicillin-G, 100
lg/mL streptomycin,
and 2 mM -glutamine. SV-HUC1, immortalized normal human
L
urothelial cell line, was obtained from Amerrican type Culture Col-
lection (Rockville, USA) and maintained in F12 medium supple-
mented with 10% fetal bovine serum (FBS), 100 unit/mL
penicillin-G, 100 lg/mL streptomycin, and 2 mM L-glutamine. The
cells were cultured at 37 °C in a humidified atmosphere containing
5% CO2. For evaluating the cytotoxic effect of tested compounds, 12
and 20 combined with cisplatin, respectively, and positive control
cisplatin, a modified 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltet-
razolium bromide (MTT, Sigma Chemical Co.) assay was per-
formed.19 Briefly, the cells were plated at a density of 1800 cells/
well in 96-well plates and incubated at 37 °C overnight before drug
exposure. Cells were then cultured in the presence of graded con-
centrations of tested compounds, 12 and 20 combined with 5 and
4.2.10. 30-Isopropylcarbamoyl-11-oxo-18b-3,4-seco-olean-4(23),
12-diene 3-ethyl carbamate (23)
Compound 23 was prepared from 19 (100 mg, 0.19 mmol) fol-
lowing the general procedure described for amidation using ethyl
amine as amine moiety. It was obtained as white amorphous pow-
der (59.3 mg, 0.11 mmol, 58.0%). ½a D25
ꢀ
2 (c 0.25, CHCl3). IR (film on
10
lM cisplatin, (Pharmacia & Upjohn), respectively, 5 and 10 lM
NaCl) cmꢁ1 3292, 1731, 1653. 1H NMR (CDCl3) d 0.82 (3H, s,
Me-28), 1.10 (3H, t, J = 7.2 Hz, –CH2CH3), 1.11 (3H, s, Me-26),
1.15 (3H, d, J = 6.8 Hz, –CH(CH3)(CH3)), 1.16 (3H, d, J = 6.8 Hz,
–CH(CH3)(CH3)), 1.16 (3H, s, Me-25), 1.17 (3H, s, Me-29), 1.39
(3H, s, Me-27), 2.51 (1H, td, J = 13.2, 6.0 Hz, Hb-18), 3.23 (2H, m,
–CH2CH3), 4.12 (1H, m, –CH(CH3)2), 4.74 (1H, br s, H-23), 4.90
(1H, br s, H-23), 5.34 (1H, t, J = 8.4 Hz, NH), 5.68 (1H, t, J = 4.0 Hz,
cisplatin at 37 °C for 72 h. At the end of the culture period, 50 lL of
MTT (2 mg/mL in PB) was added to each well and allowed to react
for 3 h. Following centrifugation of plates at 1000g for 10 min,
media were removed and 150 lL DMSO were added to each well.
The proportions of surviving cells were determined by absorbance
spectrometry at 540 nm using MRX (DYNEXCO) microplate reader.
The cell viability was expressed as a percentage to the viable cells

K.-W. Lin et al. / Bioorg. Med. Chem. 19 (2011) 4274–4285
4285
of control culture condition. The IC50 values of each group were cal-
culated by the median-effect analysis and presented as
mean standard deviation (SD).
for another 1 h. Signals were detected by chemiluminescence ECL
reagent after TBST wash and visualized on Fuji SuperRX film.
Monoclonal antibody specific for b-acctin (NB600-501) was
purchased from Novus Biologicals (Littleton, CO, USA). p-p53 (Ser
15) (9284) polyclonal antibody was purchased from Cell Signaling
(Beverly, MA, USA). The monoclonal antibody specific to p53 (DO1)
was purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA).
4.4. Quantitative analysis of intracellular reactive oxygen
species (ROS)
Production of ROS was analyzed by flow cytometry as described
previously.20 Briefly, cells were plated and treated as indicated
conditions. 10
lM
2,7-dichlorodihydrofluorescein diacetate
4.8. Statistical analysis
(H2DCFHDA; Molecular Probes, Eugene, OR) was added to the trea-
ted cells 30 min prior harvest. The cells were collected by trypsin-
ization and washed with PBS. The green fluorescence of
intracellular DCF (20,70-dichlorofluorescein) was then analyzed
immediately by FACScan flow cytometer with a 525 nm band pass
filter (Becton Dickinson).
Data were expressed as mean SD. Statistical analysis were
performed using the Bonferroni t-test method after ANOVA for
multigroup comparison and the student’s t-test method for two
group comparison, with p <0.05 was considered to be statistically
significant.
4.5. Flow cytometry analysis
Acknowledgement
DNA content was determined following propidium iodide (PI)
This work was supported by a grand from the National Science
Council of the Republic of China (NSC 99-2320-B-037-022).
staining of cells as previously described.21 Briefly, 8 ꢂ 105 cells
were plated and treated with 10
lM cisplatin, various concentra-
M H2O2 for 24 h, respectively.
tions of 25, 1 mM NAC, and 50
l
References and notes
These cells were harvested by trypsinization, washed with 1ꢂ
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PBS, and fixed in ice-cold MeOH at – 20 °C. After overnight incuba-
tion, the cells were washed with PBS and incubated with 50
lg/mL
propidium iodide (Sigma, Co) and 50 g/mL RNase A (Sigma, Co) in
l
PBS at room temperature for 30 min. The fractions of cells in each
phase of cell cycle were analyzed using FACScan flow cytometer
and Cell Quest software (Becton Dickinson).
4.6. Measurement of mitochondrial membrane potential (MMP)
NTUB1 cells (3 ꢂ 105) treated with 10
l
M CCCP and different
concentrations of 25 (10, 25, and 50
lM) were incubated for
30 min with 1 l
M of 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzi-
midaolylcarbocyanine iodide (JC-1) (Moleecular Probes, Eugene,
OR, USA) in culture medium. The resulting cells were washed 3
times PBS and dislodged with trypsin-EDTA. Cells were collected
in PBS/2% BSA, washed twice by centrifugation (800g; 5 min),
and resuspended in 0.3 mL PBS/2% BSA for analysis by a FACSCali-
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of JC-1 aggregates (red fluorescence) to JC-1 monomers (green
fluorescence) was calculated to represent MMP.22
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Phytochemistry 2010, 71, 2140.
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4.7. Western blot analysis
Cells were harvested by trypsinization and resuspended with
suitable amount of PBS adjusted with the cell numbers. The cells
were mixed with equal volume of 2ꢂ sample buffer and boiled
for 10 min twice to denature the proteins. Cell extracts were sepa-
rated by SDS–PAGE. The proteins were transferred to nitrocellulose
membranes (Millipore, Billerica, MA, USA) using a semi-dry blotter.
The blotted membranes were treated with 5% (w/v) skimmed milk
in TBST buffer (100 mM Tris–HCl (pH 7.5), 150 mM NaCl and 0.1%
Tween-20). The membranes were incubated with specific antibod-
ies at 4 °C overnight. The membranes were washed with TBST buf-
fer and incubated with secondary antibody at room temperature
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