Synthesis and biological evaluation of furoxan-based nitric oxide-releasing derivatives of glycyrrhetinic acid as anti-hepatocellular carcinoma agents

Article abstract of DOI:10.1016/j.bmcl.2010.09.070

A series of novel furoxan-based nitric oxide (NO)-releasing derivatives of glycyrrhetinic acid (GA) were designed, synthesized, and evaluated for their in vitro cytotoxicity against human hepatocellular carcinoma (HCC) and non-tumor liver cells. Five furoxan/GA hybrids, 7b-d, 7f, and 7g, displayed potent cytotoxicity against HCC cells (IC₅₀: 0.25-1.10 μM against BEL-7402 cells and 1.32-6.78 μM against HepG2 cells), but had a little effect on the growth of LO2 cells, indicating that these compounds had selective cytotoxicity against HCC cells. Furthermore, these compounds produced high concentrations of NO in HCC cells, but low in LO2 cells and treatment with hemoglobin partially reduced the cytotoxicity of the hybrid in HCC cells. Apparently, the high concentrations of NO produced by NO donor moieties and the bioactivity of GA synergistically contribute to the cytotoxicity, but the NO is a major player against HCC cells in vitro. Potentially, our findings may aid in the design of new chemotherapeutic reagents for the intervention of human HCC at clinic.

Full text of DOI:10.1016/j.bmcl.2010.09.070

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6416–6420
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of furoxan-based nitric oxide-releasing
derivatives of glycyrrhetinic acid as anti-hepatocellular carcinoma agents
Yisheng Lai a, , Lihong Shen a,b, Zhenzhen Zhang c, Wenqing Liu a, Yihua Zhang a, Hui Ji c, , Jide Tian d
⇑
⇑
a Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
b Chemistry Department, Handan College, Handan 056005, PR China
c Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, PR China
d Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, CA 90095, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel furoxan-based nitric oxide (NO)-releasing derivatives of glycyrrhetinic acid (GA) were
designed, synthesized, and evaluated for their in vitro cytotoxicity against human hepatocellular carci-
noma (HCC) and non-tumor liver cells. Five furoxan/GA hybrids, 7b–d, 7f, and 7g, displayed potent cyto-
Received 18 August 2010
Revised 10 September 2010
Accepted 14 September 2010
Available online 17 September 2010
toxicity against HCC cells (IC50: 0.25–1.10 lM against BEL-7402 cells and 1.32–6.78 lM against HepG2
cells), but had a little effect on the growth of LO2 cells, indicating that these compounds had selective
cytotoxicity against HCC cells. Furthermore, these compounds produced high concentrations of NO in
HCC cells, but low in LO2 cells and treatment with hemoglobin partially reduced the cytotoxicity of
the hybrid in HCC cells. Apparently, the high concentrations of NO produced by NO donor moieties
and the bioactivity of GA synergistically contribute to the cytotoxicity, but the NO is a major player
against HCC cells in vitro. Potentially, our findings may aid in the design of new chemotherapeutic
reagents for the intervention of human HCC at clinic.
Keywords:
NO donor
Furoxan
Glycyrrhetinic acid
Hybrid compound
Anti-hepatocellular carcinoma agent
NO-releasing
Ó 2010 Elsevier Ltd. All rights reserved.
Nitric oxide (NO) is a critical regulator of the biological process
involved in many physiological functions.1 However, high concen-
trations of NO can induce cell cycle arrest and apoptosis, particu-
larly for some tumor cells.2–4 Indeed, the NO-based anti-cancer
reagents have been investigating for their potential application
for cancer therapy at clinic.5–7 Furoxans, an important class of
NO donors, can produce high concentrations of NO and exhibit
strong anti-cancer activity.8–11 However, due to their broad biolog-
ical effects, furoxan-based anti-cancer reagents may result in se-
vere adverse effect. Therefore, development and discovery of new
derivatives of furoxan that can selectively produce high concentra-
tions of NO in tumor cells will be of great significance in the inter-
vention of human tumors, such as hepatocellular carcinoma (HCC)
with little adverse effect.
Glycyrrhetinic acid (GA) is the pentacyclic triterpene aglycone
of glycyrrhizic acid. GA and glycyrrhizic acid have been shown to
possess anti-inflammatory, anti-oxidative, anti-ulcer, anti-aller-
genic, anti-cancer, and immunomodulatory activities.12 Further-
more, GA at a high concentration can induce apoptosis of HCC
cells.13 More importantly, GA can bind to the epidermal growth
factor receptor (EGFR)14 and steroid receptors15 with high affinity
and protect hepatocytes from the carbon tetrachloride and other
toxicant-induced hepatotoxicity in mice and rats.16–18 The high
affinity of GA binding to hepatocytes and its protective activity
make GA as an ideal leading compound for the design of novel
furoxan/GA hybrids, which may selectively produce high concen-
trations of NO in HCC cells, leading to potent cytotoxicity against
HCC cells, but little side effect on healthy hepatocytes.
To generate fruoxan/GA hybrids, the phenylsulfonylfuroxans
1a–h were synthesized from benzenethiol in a four-step sequence,
as previous reports.19 Simultaneously, GA solution of methanol
was refluxed in the presence of p-toluenesulfonic acid (p-TSA) to
yield glycyrrhetinic acid methyl ester 2,20 which was in turn esteri-
fied with succinic anhydride in the presence of DMAP to form
3-O-hemisuccinate GA methyl ester 3.21 Subsequently, compound
3 was further reacted with 1a–h in the presence of 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and
4-dimethylaminopyridine (DMAP) to generate the target com-
pounds 4a–h in 65–70% yields (Scheme 1).22
Given that coupling with an amino acid can help in delivering
the compounds to tumor cells,23–25 other target compounds were
designed using glycine as a linker and synthesized, as illustrated
in Scheme 2 and 3. First, compounds 1a–g were reacted with N-
Boc-glycine in the presence of dicyclohexylcarbodiimide (DCC)
and DMAP to form N-Boc-glycinates 5a–g in 55–80% yields. Subse-
quently, the Boc group was removed with trifluoroacetic acid to
generate the glycinates 6a–g, which were coupled with GA in the
presence of EDCI and DMAP to generate 7a–g in 45–55% yields
(Scheme 2).26,27
⇑
Corresponding authors. Tel.: +86 25 83271285; fax: +86 25 86635503 (Y.L.);
tel.: +86 25 86021369 (H.J.).
E-mail addresses: lcpu333@yahoo.com.cn (Y. Lai), huijicpu@163.com (H. Ji).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.070

Y. Lai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6416–6420
6417
CO2H
CO2CH3
CO2CH3
H
H
H
O
O
H
O
H
a
b
H
H
H
HO
HO2C(CH2)2CO2
HO
H
c
glycyrrhetinic acid (GA)
2
3
CO2CH3
a: R = (CH2)2O
b: R = (CH2)3O
c: R = (CH2)2CH(CH3)O
d: R = (CH2)4O
e: R = (CH2)2O(CH2)2O
f: R = CH2CH CHCH2O
g: R = CH2C CCH2O
h: R = CH2CH2NH
H
O
H
PhO2S
ORH
+
_
H
N
N
O
O
OROC(CH2)2CO2
PhO2S
+
_
1a-h
N
N
4a-h
O
O
Scheme 1. The synthetic pathway of 4a–h. Reagents and conditions: (a) CH3OH, p-TSA, reflux; (b) succinic anhydride, DMAP, dry CH2Cl2, reflux, 15 h; (c) 1a–h, DCC, DMAP,
dry CH2Cl2, rt, 24 h.
.
ORCOCH 2NH2 TFA
PhO2S
ORH
PhO2S
ORCOCH 2NH-Boc
PhO2S
a
b
+
_
+
+
_
_
N
N
N
N
N
O
N
O
O
O
O
O
1a-g
5a-g
6a-g
c
SO2Ph
CONHCH 2CORO
a: R = (CH2)2O
b: R = (CH2)3O
c: R = (CH2)2CH(CH3)O
d: R = (CH2)4O
+
_
H
N
N
O
O
O
e: R = (CH2)2O(CH2)2O
f: R = CH2CH CHCH2O
g: R = CH2C CCH2O
H
HO
H
7a-g
Scheme 2. The synthetic pathway of 7a–g. Reagents and conditions: (a) Boc-NHCH2CO2H, DCC, DMAP, dry CH2Cl2, rt, 24 h; (b) CF3CO2H, dry CH2Cl2, rt, 2 h; (c) GA, EDCI,
DMAP, DMF, rt, 24 h.
The condensation of GA with 1h in the presence of EDCI and
DMAP produced 8h in 48% yield. 3-O-Acetylation of GA with acetic
anhydride in the presence of DMAP furnished 9 in 98% yield,28
which was coupled with 1h, 6d, or 6e in the presence of EDCI
and DMAP to produce compounds 10h, 11d, and 11e, respectively.
Alternatively, 9 was treated with oxalyl chloride to form acyl chlo-
ride derivative 12, which was subsequently esterified with 1a, 1b,
1d, 1e, or 1g in the presence of triethylamine (Et3N) to form 13a,
13b, 13d, 13e, and 13g, respectively (Scheme 3).29
ever, other hybrids displayed various cytotoxicities against HCC
cells.
Next, we examined whether the new furoxan/GA hybrids
could selectively inhibit the proliferation of HCC cells by MTT as-
says.30 We found that the NO donor moieties 1b–d, 1f and 1g, like
adriamycin, inhibited the proliferation of both HCC BEL-7402 cells
and non-tumor human liver LO2 cells with a similar level of
inhibitory activity at the concentration of 10 lM (Fig. 1). In con-
trast, the corresponding hybrids 7b–d, 7f and 7g selectively inhib-
ited the proliferation of BEL-7402 cells, but with a little effect on
LO2 cells. Evidentially, treatment with 7b–d, 7f or 7g inhibited
the proliferation of BEL-7402 cells by 83.52–96.50%, respectively,
and the inhibitory effects of these compounds on LO2 cells were
less than 18.00%. These suggest that these new hybrids may have
little adverse effect and be safe for the intervention of human
HCC.
To understand their selectivity and diverse cytotoxicity against
HCC and LO2 cells, we examined the production of NO by individ-
ual compounds in HCC and LO2 cells by the Griess assay (Fig. 2).32
The furoxan moieties produced similar concentrations of NO in
HCC and LO2 cells, while treatment with hybrids 7b–d, 7f or 7g
generated high concentrations of NO in HCC cells, which were
3–5-fold higher than that in LO2 cells. The significantly higher
concentrations of NO produced in HCC cells may contribute to
the selective cytotoxicity of these new hybrids against HCC cells.
All of the final products were purified by column chromatogra-
phy and their structures were characterized by infrared (IR), mass
spectra (MS), 1H NMR, and elemental analyses.
The cytotoxicity of the target compounds and GA against
human HCC cells (HepG2, BEL-7402) was evaluated by MTT assay
using adriamycin as a positive control (Table 1).30 GA exhibited a
weak cytotoxicity against HCC cells tested, consistent with previ-
ous reports.13,31 In contrast, most of the new furoxan/GA hybrids
displayed potent anti-HCC activity, and some of them were stron-
ger than that of adriamycin. For example, the IC50 value of 7d
against HCC BEL-7402 cells was near fourfold less than that of adri-
amycin (0.25 vs 0.90 lM). More importantly, the hybrids 7b–d, 7f,
and 7g had stronger anti-proliferatory activity against BEL-7402
cells than that of their corresponding NO donor moieties 1b–d,
1f, and 1g (IC50 = 6.52–13.26
lM), suggesting a synergistic effect
of furoxan and GA on inhibition of HCC cells proliferation. How-

6418
Y. Lai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6416–6420
CO2H
CONHCH2CH2O
SO2Ph
H
+
_
H
O
H
N
N
O
O
O
a
H
H
HO
HO
CONHCH2CH2O
SO2Ph
H
8h
GA
+
_
H
N
N
O
O
O
b
CO2H
a
c
H
H
O
10h
CH3CO2
H
O
SO2Ph
CONHCH2CORO
H
+
_
CH3CO2
H
N
N
H
O
O
9
H
d: R = (CH2)4O
e: R = (CH2)2O(CH2)2O
CH3CO2
11d, 11e
H
d
COCl
SO2Ph
CORO
H
O
+
_
H
N
N
O
O
e
O
H
a: R =(CH2)2O
b: R = (CH2)3O
d: R = (CH2)4O
e: R = (CH2)2O(CH2)2O
g: R = CH2C CCH2O
CH3CO2
H
H
H
CH3CO2
13a,b,d,e,g
12
Scheme 3. The synthetic pathway of 8h, 10h, 11d, 11e, 13a, 13b, 13d, 13e, and 13g. Reagents and conditions: (a) 1h, EDCI, DMAP, DMF, rt, 24 h; (b) Ac2O, DMAP, reflux, 15 h;
(c) 6d or 6e, EDCI, DMAP, DMF, rt, 24 h; (d) oxalyl chloride, dry CH2Cl2, rt, 4 h; (e) 1a, 1b, 1d, 1e, or 1g, Et3N, dry THF, reflux, 12 h.
Table 1
BEL-7402 LO2
The inhibitory effects of the target compounds on the proliferation of human HCC
*
*
cells
100
90
80
70
60
50
40
30
20
10
0
*
*
a
a
*
Compound
IC50
HepG2
(
l
M)
Compound
IC50
HepG2
(lM)
BEL-7402
BEL-7402
ADMb
GAc
4a
4b
4c
4d
4e
4f
4g
4h
7a
7b
7c
2.03
>50
0.90
>50
7d
7e
7f
7g
6.78
5.18
1.32
3.39
7.40
23.72
8.39
6.40
14.71
11.52
9.83
31.45
7.50
0.25
3.78
0.32
0.84
2.47
14.16
4.84
9.47
3.84
15.35
5.04
18.18
13.41
26.03
36.52
15.67
7.90
11.55
2.90
9.06
7.85
9.22
6.03
8.20
19.92
7.37
13.41
2.94
1.38
0.43
1.10
8h
10h
11d
11e
13a
13b
13d
13e
13g
3.79
3.02
21.38
14.96
Figure 1. Cytotoxicity of the furoxan moieties and target compounds against HCC
BEL-7402 cells and non-tumor liver LO2 cells. BEL-7402 and LO2 cells were treated
with 10 lM of the indicated compounds and their cytotoxicities were determined
a
IC50 = compound at a concentration required to inhibit tumor cell proliferation
by 50%. Data are expressed as the mean IC50 from the dose–response curves of at
least three independent experiments.
by MTT. Data are expressed as mean% SD of the cytotoxicity of individual
*
compounds in both cells from three separate experiments. p <0.05, determined by
b
Adriamycin.
Glycyrrhetinic acid.
X2 tests.
c
ment with different concentrations of hemoglobin dramatically
reduced the concentrations of NO and inhibited the cytotoxicity
of 7d against the BEL-7402 cells. Furthermore, the inhibitory
effects of different concentrations of hemoglobin were dose-
dependent and negatively correlated with the concentrations of
NO (R2 = 0.87, p <0.01, determined by logistic regression analysis).
More importantly, the cytotoxicities of 7d were positively corre-
lated with the concentrations of NO (R2 = 0.79, p <0.01). Notably,
To further determine the role of NO in cytotoxicity of these
hybrids against HCC cells, HCC BEL-7402 cells were pre-treated
with varying concentrations of hemoglobin, a well-known NO
scavenger, and then treated with different concentrations of hybrid
compound 7d for the detection of their cytotoxicity and NO pro-
duction in Figure 3. As expected, 7d produced high concentration
of NO and displayed a strong cytotoxicity against the HCC cells
without pre-treatment with hemoglobin. In contrast, pre-treat-
treatment with 20 lM of hemoglobin only reduced the cytotoxicity

Y. Lai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6416–6420
6419
cytotoxicity.23–25 Thus, new hybrid compounds generated by cou-
pling NO donor moieties to 30-COOH of GA with a glycine as a lin-
ker should have potent cytotoxicity selectively against HCC cells.
In summary, a series of furoxan/GA hybrids were designed, syn-
thesized and evaluated for their cytotoxicity against human HCC
and non-tumor liver cells in vitro. Our data indicated that 7b–d,
7f and 7g displayed selective cytotoxicity against human HCC cells,
which was associated with higher concentrations of NO production
in HCC cells and potentially synergistic effect of NO donor moieties
and the bioactivity of GA. Potentially, our findings may aid in the
design of new chemotherapeutic reagents for the intervention of
human HCC at clinic.
20
15
10
5
1b
1c
1d
1f
1g
7b
7c
7d
7f
7g
GA
0
Acknowledgments
The authors thank the Center of Analysis & Measurement of
China Pharmaceutical University for the characterization of the
compounds and Ms. Yao Liu for her technical support. The authors
are also grateful to Drs. Xiaolei Ye, Zhangjian Huang and Lei Fang
for their helpful discussions.
Figure 2. Variable levels of NO produced by the compounds in human HCC cells
and non-tumor liver cells. Cells were treated in triplicate with each compound at
100 lM for 30–300 min. The cell lysates were prepared for determining the
contents of NO by Griess assay. Data are expressed as mean of individual
compounds in each type of cells after treatment with the compounds for
300 min. Similar patterns of NO production were detected at other time points
(data not shown).
References and notes
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D. G. Nitric Oxide 2008, 19, 192.
3. Wink, D. A.; Ridnour, L. A.; Hussain, S. P.; Harris, C. C. Nitric Oxide 2008, 19, 65.
4. Hirst, D.; Robson, T. Curr. Pharm. Des. 2010, 16, 411.
5. Yasuda, H.; Yamaya, M.; Nakayama, K.; Sasaki, T.; Ebihara, H.; Kanda, A.; Asada,
M.; Inoue, D.; Suzuki, T.; Okazaki, T.; Takahashi, H.; Yoshida, M.; Kaneta, T.;
Ishizama, K.; Yamanda, S.; Tomita, N.; Yamasaki, M.; Kikuchi, A.; Kubo, H.;
Sasaki, H. J. Clin. Oncol. 2006, 24, 688.
100%
80%
60%
40%
20%
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Cytotoxicity (control %)
Nitrate/nitrite (µM)
6. Seimens, D. R.; Heaton, J.; Adams, M.; Graham, C. Nitric Oxide 2007, 17, S15.
7. Siemens, D. R.; Heaton, J. P.; Adams, M. A.; Kawakami, J.; Graham, C. H. Urology
2009, 74, 878.
8. Cerecetto, H.; Porcal, W. Mini-Rev. Med. Chem. 2005, 5, 57.
9. Moharram, S.; Zhou, A.; Wiebe, L. I.; Knaus, E. E. J. Med. Chem. 2004, 47, 1840.
10. Maksimovic-Ivanic, D.; Mijatovic, S.; Harhaji, L.; Miljkovic, D.; Dabideen, D.;
Fan Cheng, K.; Mangano, K.; Malaponte, G.; Al-Abed, Y.; Libra, M.; Garotta, G.;
Nicoletti, F.; Stosic-Grujicic, S. Mol. Cancer Ther. 2008, 7, 510.
11. Boiani, M.; Cerecetto, H.; González, M.; Risso, M.; Olea-Azar, C.; Piro, O. E.;
Castellano, E. E.; López de Ceráin, A.; Ezpeleta, O.; Monge-Vega, A. Eur. J. Med.
Chem. 2001, 36, 771.
12. Asl, M. N.; Hosseinzadeh, H. Phytother. Res. 2008, 22, 709.
13. Hibasami, H.; Iwase, H.; Yoshioka, K.; Takahashi, H. Int. J. Mol. Med. 2006, 17,
215.
14. Kimura, M.; Inoue, H.; Hirabayashi, K.; Natsume, H.; Ogihara, M. Eur. J.
Pharmacol. 2001, 431, 151.
15. Armanini, D.; Karbowiak, I.; Funder, J. W. Clin. Endocrinol. 1983, 19, 609.
16. Nose, M.; Ito, M.; Kamimura, K.; Shimizu, M.; Ogihara, Y. Planta Med. 1994, 60,
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17. Lin, G.; Nnane, I. P.; Cheng, T. V. Toxicon 1999, 37, 1259.
18. Gumpricht, E.; Dahl, R.; Devereaux, M. W.; Sokol, R. J. J. Biol. Chem. 2005, 280,
10556.
0%
7d
10
0
10
20
10
10
10
5
10
1
5
0
5
5
5
5
5
1
Hemoglobin
20
10
Figure 3. The concentrations of NO produced by 7d were correlated with the
cytotoxicities against HCC cells. BEL-7402 cells were cultured in 24-well plates and
pre-treated in triplicate with the indicated concentrations (0, 1, 5, 10, or 20
hemoglobin for 1 h. The cells were treated with 5 or 10 M of 7d for 24 h, and the
cytotoxicity and NO production of 7d were determined by MTT and Griess assays,
respectively. Data are expressed as mean% of the cytotoxicity and mean concen-
trations of NO in individual experimental conditions. The intra-group and inter-
experimental variations were less than 10% and the indicated concentrations of
hemoglobin used did not affect the cell growth (<5%).
lM) of
l
19. Li, R. W.; Zhang, Y. H.; Ji, H.; Yu, X. L.; Peng, S. X. Acta Pharm. Sin. 2001, 36, 821.
20. Su, X.; Lawrence, H.; Ganeshapillai, D.; Cruttenden, A.; Purohit, A.; Reed, M. J.;
Vicker, N.; Potter, B. V. Bioorg. Med. Chem. 2004, 12, 4439.
21. Dean, P. D.; Halsall, T. G.; Whitehouse, M. W. J. Pharm. Pharmacol. 1967, 19, 682.
22. General procedure for the synthesis of the target compounds 4a–h: DMAP
(0.1 mmol) and appropriate phenylsulfonylfuroxans 1a–h (0.31 mmol) were
of 7d by near 66%. Apparently, the high concentrations of NO pro-
duced by NO donor moieties and the bioactivity of GA synergisti-
cally contribute to,33 but the NO is
a major player in the
added to
a mixture solution of 3-O-hemisuccinate GA methyl ester 3
(0.26 mmol) and DCC (0.33 mmol) in dry dichloromethane. The reaction
mixture was stirred at room temperature for 24 h. Filtration and removal of the
solvent in vacuo afforded the crude product, which was subsequently purified
by column chromatography using (PE/EtOAc = 3:1) to give pure 4a–h in 65–
70% yields.
cytotoxicity of these new hybrids against HCC cells in vitro.
Analysis of structure and activity relationship (SAR) revealed
that the hybrids with NO moieties coupling to 30-COOH of GA in
general had stronger cytotoxicity than that of 3-OH hybrids (e.g.,
7a or 13a vs 4a, 7b vs 4b, 7c vs 4c, 7d, 11d or 13d vs 4d, 7e or
11e vs 4e, 7f vs 4f, 7g vs 4g). Furthermore, the coupling model of
the NO donor moiety with GA also affected the cytotoxic activity
of these hybrids. For example, compound 4h containing an amide
bond was more active than its corresponding analog 4a containing
two ester bonds. Interestingly, the hybrids 7a–g with a glycine as a
linker were more cytotoxic to HCC cells than their corresponding
analogs without glycine residue (e.g., 7a vs 13a, 7b vs 13b, 7d vs
13d, 7e vs 13e, 7g vs 13g), supporting the notion that introduction
of a glycine residue into compounds can significantly enhance their
23. Song, X.; Lorenzi, P. L.; Landowski, C. P.; Vig, B. S.; Hilfinger, J. M.; Amidon, G. L.
Mol. Pharm. 2005, 2, 157.
24. Deshmukh, M.; Chao, P.; Kutscher, H. L.; Gao, D.; Sinko, P. J. J. Med. Chem. 2010,
53, 1038.
25. Ling, Y.; Ye, X.; Ji, H.; Zhang, Y.; Lai, Y.; Peng, S.; Tian, J. Bioorg. Med. Chem. 2010,
18, 3448.
26. General procedure for the synthesis of the target compounds 7a–g: A solution of
N-Boc-glycine (1.0 mmol), DCC (1.25 mmol), DMAP (0.125 mmol) and
appropriate phenylsulfonylfuroxans 1a–g (1.1 mmol) in dry CH2Cl2 was
stirred at room temperature for 24 h. After filtration, the filtrate was
evaporated in vacuo and the crude product was purified by column
chromatography (PE/EtOAc = 3:1) to afford N-Boc-glycinates 5a–g. A solution
of appropriate 5a–g (0.43 mmol) and trifluoroacetic acid (1 mL) in dry CH2Cl2

6420
Y. Lai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6416–6420
(4 mL) was stirred at room temperature for 2 h. The solvent was removed in
1H NMR (300 MHz, CDCl3): d 0.80–1.43 (m, 21H, 7 Â CH3), 2.33 (s, 1H, CHC@O),
2.78 (br s, 1H, OH), 3.20–3.25 (m, 1H, CHOH), 4.03–4.20 (m, 2H, NHCH2), 4.82
(s, 2H, CH2), 5.11 (s, 2H, CH2), 5.71(s, 1H, CH@), 6.13 (br s, 1H, NH), 7.61–7.66
(m, 2H, Ar-H), 7.75–7.80 (m, 1H, Ar-H), 8.07 (d, 2H, J = 7.5 Hz, Ar-H); MS (ESI)
m/z = 820 [M+1]+; Anal. Calcd for C44H57N3O10S: C, 64.45; H, 7.01; N, 5.12.
Found: C, 64.25; H, 7.06; N, 5.03.
vacuo. The crude residue was dissolved in the solution of DMF (15 mL) and
DMAP (1.60 mmol) with stirring at room temperature for 0.5 h, followed by the
addition of GA (0.43 mmol) and EDCI (1.30 mmol) to the reaction mixture for
another 24 h with stirring at room temperature. The mixture was diluted with
water (200 mL) and extracted with EtOAc (3 Â 50 mL). The combined organic
layer was washed with saturated NaCl solution, dried over anhydrous Na2SO4,
and concentrated under reduced pressure. The crude product was purified by
column chromatography (PE/EtOAc = 2:1) to yield 7a–g.
28. Sakano, K.; Ohshima, M. Agric. Biol. Chem. 1986, 50, 763.
29. General procedure for the synthesis of the target compounds 13a, 13b, 13d, 13e
and 13g: 3-Acetoxy-glycyrrhetinic acid
9 (6.0 mmol) dissolved in dry
27. Data for selected compounds: 7b: yield 50%, white solid, mp 87–89 °C; IR (KBr,
cmÀ1): 3442, 2958, 2867, 1751, 1652, 1616, 1550, 1525, 1452, 1384; 1H NMR
(300 MHz, CDCl3): d 0.80–1.43 (m, 21H, 7 Â CH3), 2.33 (s, 1H, CHC@O), 2.78 (br
s, 1H, OH), 3.20–3.25 (m, 1H, CHOH), 3.74–3.76 (m, 2H, NHCH2), 4.38 (t, 2H,
J = 6.0 Hz, OCH2), 4.53 (t, 2H, J = 6.0 Hz, OCH2), 5.72 (s, 1H, CH@), 6.17 (br s, 1H,
NH), 7.61–7.66 (m, 2H, Ar-H), 7.74–7.79 (m, 1H, Ar-H), 8.05 (d, 2H, J = 7.2 Hz,
Ar-H); MS (ESI) m/z = 810 [M+1]+; Anal. Calcd for C43H59N3O10S: C, 63.76; H,
7.34; N, 5.19. Found: C, 63.66; H, 7.38; N, 5.25. Compound 7c: yield 55%, white
solid, mp 96–98 °C; IR (KBr, cmÀ1): 3450, 2960, 2869, 1741, 1649, 1625, 1548,
1519, 1452, 1386; 1H NMR (300 MHz, CDCl3): d 0.80–1.45 (m, 21H, 7 Â CH3),
2.33 (s, 1H, CHC@O), 2.78 (br s, 1H, OH), 3.20–3.25 (m, 1H, CHOH), 4.07 (m, 2H,
NHCH2), 4.11 (t, 2H, J = 6.0 Hz, OCH2), 4.51 (t, 2H, J = 6.0 Hz, OCH2), 5.71 (s, 1H,
CH@), 6.17 (br s, 1H, NH), 7.62–7.65 (m, 2H, Ar-H), 7.72–7.77 (m, 1H, Ar-H),
8.05 (d, 2H, J = 7.4 Hz, Ar-H); MS (ESI) m/z = 824 [M+1]+; Anal. Calcd for
dichloromethane (20 mL), was added oxalyl chloride (3.0 mL) by slow dropwise.
The mixture was stirred at room temperature for 4 h, followed by concentration
under reduced pressure to 3-acetoxy-11-oxo-olean-12-en-30-oyl chloride 12. A
mixture of 12 (0.52 mmol) and appropriate phenylsulfonylfuroxans 1a–g
(0.53 mmol) in dry THF (20 mL) was refluxed in presence of triethylamine
(0.2 mL) for 12 h. The solution was cooled to room temperature. After filtration,
the filtratewasevaporated invacuoandthe crudeproductwas purified bycolumn
chromatography(PE/EtOAc = 5:1)toobtainpure13a, 13b, 13d, 13eand13gin80–
85% yield.
30. In vitro cell growth inhibition assay: The human hepatocellular carcinoma
(HepG2, BEL-7402) and non-tumor liver (LO2) cells (all from Shanghai
Institutes for Biological Science, Chinese Academy of Sciences) were cultured
in 10% heat inactivated fetal calf serum DMEM medium (Gibco, USA) at 37 °C,
5% CO2. The inhibitory effects of different compounds on the growth of these
cells were tested by MTT assay. Briefly, cells at 3 Â 105/well were cultured in
96-well plates overnight and treated in triplicate with different concentrations
of individual compounds for 24 h. Cells cultured in medium alone or treated
with vehicle were used as negative controls. During the last 4 h incubation, the
cells were exposed to MTT (5 mg/mL, sigma) and the resulting formazan
crystals were dissolved in 200 lM DMSO, followed by measuring at 570 nm on
a microplate reader (Thermo, USA).
31. Chintharlapalli, S.; Papineni, S.; Jutooru, I.; McAlees, A.; Safe, S. Mol. Cancer Ther.
2007, 6, 1588.
32. Nitrate/nitrite measurement in vitro: The human hepatocellular carcinoma
(HepG2, BEL-7402) and non-tumor liver (LO2) cells were cultured in the
C44H61N3O10S: C, 64.13; H, 7.46; N, 5.10. Found: C, 64.05; H, 7.66; N, 4.94.
Compound 7d: yield 50%, white solid, mp 80–82 °C; IR (KBr, cmÀ1): 3429, 2956,
2869, 1749, 1654, 1616, 1552, 1450, 1384; 1H NMR (300 MHz, CDCl3): d 0.80–
1.49 (m, 21H, 7 Â CH3), 2.33 (s, 1H, CHC = O), 2.78 (br s, 1H, OH), 3.20–3.25 (m,
1H, CHOH), 4.04–4.10 (m, 2H, NHCH2), 4.27 (t, 2H, J = 6.0 Hz, OCH2), 4.46 (t, 2H,
J = 6.0 Hz, OCH2), 5.72 (s, 1H, CH@), 6.18 (br s, 1H, NH), 7.60–7.65 (m, 2H, Ar-H),
7.74–7.79 (m, 1H, Ar-H), 8.05 (d, 2H, J = 7.2 Hz, Ar-H); MS (ESI) m/z = 822
[MÀ1]À; Anal. Calcd for C44H61N3O10S: C, 64.13; H, 7.46; N, 5.10. Found: C,
64.25; H, 7.55; N, 5.02. Compound 7f: yield 50%, white solid, mp 113–115 °C;
IR (KBr, cmÀ1): 3436, 2950, 2867, 1751, 1654, 1610, 1548, 1450, 1359; 1H NMR
(300 MHz, CDCl3): d 0.80–1.45 (m, 21H, 7 Â CH3), 2.35 (s, 1H, CHC@O), 2.78 (br
s, 1H, OH), 3.20–3.25 (m, 1H, CHOH), 4.05–4.20 (m, 2H, NHCH2), 4.74 (m, 2H,
OCH2), 5.05 (m, 2H, OCH2), 5.20–5.25 (m, 2H, 2 Â CH@), 5.70 (s, 1H, CH@), 7.60–
7.65 (m, 2H, Ar-H), 7.74–7.79 (m, 1H, Ar-H), 8.06 (d, 2H, J = 7.5 Hz, Ar-H); MS
(ESI) m/z = 822 [M+1]+; Anal. Calcd for C44H59N3O10S: C, 64.29; H, 7.23; N, 5.11.
Found: C, 63.93; H, 7.34; N, 5.22. Compound 7g: yield 55%, white solid, mp 91–
93 °C; IR (KBr, cmÀ1): 3436, 2956, 2867, 1757, 1652, 1620, 1544, 1454, 1359;
24-well plates for 24 h and treated in triplicate with 100 lM of individual
compounds for 30–300 min. The cells were harvested longitudinally and their
lysates were prepared. The levels of nitrate/nitrite in the lysates were
determined using the nitrate/nitrite colorimetric assay kit, according to
manufacturers’ instruction (Beyotime, China).
33. Lee, C. S.; Kim, Y. J.; Lee, M. S.; Han, E. S.; Lee, S. Life Sci. 2008, 83, 481.