Synthesis and bioactivity of novel nitric oxide-releasing ursolic acid derivatives

Article abstract of DOI:10.1016/j.cclet.2010.10.036

A series of furoxan-based novel nitric oxide-donating ursolic acid (UA) derivatives (7a-f) were synthesized, and their cytotoxic activities against HepG2 cells in vitro were evaluated by MTT method. It was found that 7a-d and 7f showed more potent cytotoxic activities than control 5-fluorouracil and UA.

Full text of DOI:10.1016/j.cclet.2010.10.036

Available online at www.sciencedirect.com
Chinese Chemical Letters 22 (2011) 413–416
www.elsevier.com/locate/cclet
Synthesis and bioactivity of novel nitric
oxide-releasing ursolic acid derivatives
a
a
b
c,
Li Chen a, , Wen Qiu , Jia Tang , Zhi Feng Wang , Shu Ying He
*
*
a Department of Phytochemistry, China Pharmaceutical University, Nanjing 210009, China
b Tianjin Hankang Medicinal & Biological Technology Co., Ltd., Tianjin 300203, China
c School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
Received 16 July 2010
Available online 15 January 2011
Abstract
A series of furoxan-based novel nitric oxide-donating ursolic acid (UA) derivatives (7a–f) were synthesized, and their cytotoxic
activities against HepG2 cells in vitro were evaluated by MTT method. It was found that 7a–d and 7f showed more potent cytotoxic
activities than control 5-fluorouracil and UA.
# 2010 Li Chen. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Ursolic acid; Nitric oxide; Furoxan; Cytotoxic activity
Ursolic acid (UA) is a well-known pentacyclic triterpene that is widely distributed throughout the plant kingdom. It
is of interest to pharmacists in the area of oncology because UA is capable of inducing apoptosis of tumor cells as well
as preventing malignant transformation of normal cells [1,2]. More important, it also showed the property of liver-
specific metabolism, which is associated with the distribution in liver and modulation of UA on cytochrome p450
(CYP) activity [3]. All these attract considerable attention from medicinal chemists to develop novel UA derivatives
with potent anti-hepatocellular carcinoma (HCC) effects using UA as lead compound.
OH
H
O
H
HO
Ursolic acid (UA)
* Corresponding authors.
E-mail addresses: chenliduo12@gmail.com (L. Chen), heshuying@yahoo.com.cn (S.Y. He).
1001-8417/$ – see front matter # 2010 Li Chen. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2010.10.036

[()TD$FIG]
414
L. Chen et al. / Chinese Chemical Letters 22 (2011) 413–416
SCH2COOH
SH
SO2CH2COOH
a
b
1
2
3
SO2Ph
PhO2S
HOR1
SO2Ph
5a
5b
5c
5d
R = (CH ) O
1 2 2
R = (CH ) O
1
2 3
d
c
N
N
N
N
R = CH(CH )(CH ) O
1
1
3
2 2
O
4
O
R = (CH ) O
O
2 4
O
5a-5d
6a
6b
R = -(CH ) -
2
2 2
OH
e
H
UA
R =
O
2
O
O
H
HO
R2
O
6a, 6b
OH
H
O
O
O
R1
H
PhO2S
f
5a~5d
O
R2
O
N
N
O
O
7a-f
R = O(CH ) , O(CH ) , O(CH ) (CH )CH, O(CH )
2 4
1
2 2
2 3
2 2
3
R = -(CH ) -,
2
2 2
Scheme 1. Reagents and conditions: (a) (i) NaOH (aq.), ClCH2COOH, rt, 3 h; (ii) 6 mol/L HCl; (b) 30% H2O2, AcOH, rt, 3 h; (c) fuming HNO3,
100 8C, 4 h; (d) HOR1H, THF, 25% NaOH, rt, 2 h; (e) anhydride, DMAP (cat), CH2Cl2, reflux 72 h; (f) DCC (cat), DMAP (cat), CH2Cl2, rt, 15 min.
Nitric oxide (NO), which is naturally generated from L-arginine under the catalysis of NO synthase (NOS) in vivo,
exerts a wide range of biological effects. NO can also be generated from synthetic NO-donors, such as furoxan, nitrate,
diazeniumdiolate, and others [4,5]. Recent studies have shown that NO appears to be critical for the tumoricidal activity
of the immune system. In general, researches demonstrated that high concentrations of NO are cytotoxic and can induce
the apoptosis of tumor cells, prevent tumors from metastasizing, and assistmacrophage to kill tumor cells[6,7]. Furoxans
are thermally stable compounds and represent one class of NO donors that can produce high levels of NO [8,9].
The above fact provides a solid biologic basis for the development of new types of NO-drug hybrids, whereby NO
donors are attached to currently known anti-cancer agent, provides additive anti-tumor effects by each compound
while minimizing their respective side effects [10]. Therefore, to obtain tissue-specific NO-related function, and based
on the fact that tumor cells are more sensitive to cell apoptosis induced by high intense of NO compared with normal
cells [6,11], a series of novel furoxan-based NO-releasing derivatives of UA (7a–f) were designed and synthesized by
coupling phenylsulfonyl-substituted furoxans to the 3-position of UA through various chemical linkers (Scheme 1).
By releasing NO and UA in liver, we hope to find novel and selective anticancer agents.
The starting material thiophenol 1 was firstly converted to 2-(phenylthio)acetic acid 2 by treatment with
chloroacetic acid in 90% yield. Then oxidation of 2 using 30% H2O2 in acetic acid afforded 2-(phenylsulfonyl)- acetic
acid 3, which was directly converted to bis(phenylsulfonyl)furoxan 4 by fuming nitric acid. The yield of these two
steps from 2 to 4 was 76%. Then compound 4 was converted to various mono-phenylsulfonylfuroxans 5a–d by
treatment with corresponding diols with the yields of 70%–80%.
Treatment of UA with succinic anhydride or phthalic anhydride respectively in CH2Cl2 in the presence of 4-
dimethylaminopyridine (DMAP) afforded intermediate 6a (53%) or 6b (67%). Finally, 6a or 6b were treated with
corresponding 5a–d in CH2Cl2 in the presence of DMAP and N, N0-dicyclohexylcarbodimide (DCC) to obtain target
compounds 7a–f in 37–48% yields. The structures of the target compounds were shown in Table 1 and the data of mp,
1
MS, IR, H NMR spectra and elemental analyses (EA) of selected compounds were shown in reference [12].
All target compounds were evaluated for cytotoxicity against HepG2 cells in vitro by MTT method, using 5-
fluorouracil as control. The results indicated that 7a–d and 7f showed significant cytotoxic activities which were

L. Chen et al. / Chinese Chemical Letters 22 (2011) 413–416
415
Table 1
The structures and cytotoxic activity (IC50 at mmol/L) of the target compounds against HepG2 cells (human hepatoma cells).
Compound
R1
R2
IC50 (mmol/L)
7a
7b
7c
7d
7e
O(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
3.20
10.74
5.42
O(CH2)3
O(CH2)2(CH3)CH
O(CH2)4
4.93
O(CH2)4
51.36
[DT$INLE]
7f
O(CH2)2(CH3)CH
9.76
[DT$INLE]
UA
21.10
15.96
5-Fluorouracil
stronger than that of 5-fluorouracil, especially 7a, 7c and 7d with IC50 values of 3.20, 5.42 and 4.93 mmol/L,
respectively (Table 1). The different activities of the tested compounds may be due to the different spacers, derivatives
with spacers having succinyl are more active than the compounds having phthaloyl.
In summary, a series of furoxan-based NO-donating ursolic acid derivatives were synthesized and evaluated for
their in vitro cytotoxicity against HepG2 cells. Preliminary bioassay indicated that five compounds (7a–d and 7f)
showed stronger cytotoxicity than UA and 5-fluorouracil, the mechanism of which may be high concentration of NO
released inducing cell apoptosis. Further biological evaluation and mechanism on this novel class of hybrids are
currently in progress.
Acknowledgments
This work was supported by a grant from the National Natural Science Foundation of China (No. 2097210),
research grants from the Natural Science Foundation of Jiangsu Province (No. BK2009302) and Tianjin Application
Bases and Advanced Technology Research Program Key Projects (No. JCZDJC21400).
References
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[8] S. Moharram, A. Zhou, L.I. Wiebe, et al. J. Med. Chem. 47 (2004) 1840.
[9] C. Medana, A. Di Stilo, S. Visentin, et al. Pharm. Res. 16 (1999) 956.
[10] (a) L. Fang, Y.H. Zhang, J. Lehmann, et al. Bioorg. Med. Chem. Lett. 17 (2007) 1062;
(b) L. Chen, Y.H. Zhang, X.W. Kong, et al. J. Med. Chem. 51 (2008) 4834.
[11] U.K. Mebmer, B. Brune, Arch. Biochem. Biophys. 1 (1) (1996) 1.
[12] Data. 7a: mp 180–182 8C. IR (KBr, cmꢀ1): 3326, 1733, 1169, 1362; MS (ESI, m/z): [MꢀH]ꢀ 823; 1H NMR (300 MHz, CDCl3): d 0.73 (s, 3H),
0.77 (s, 3H), 0.81 (s, 6H), 0.89 (d, 3H, J = 6.6 Hz), 0.95 (d, 3H, J = 6.4 Hz), 1.07 (s, 3H), 2.67 (s, 4H), 4.45 (dd, 1H, J = 10.2, 4.8 Hz), 4.51 (t,
2H, J = 6.0 Hz), 4.65 (t, 2H, J = 6.0 Hz), 5.26 (brs, 1H), 7.60–7.65 (m, 2H), 7.72–7.78 (m, 1H), 8.05 (m, 2H); Anal. Calcd. for C44H60N2O11S:
C 64.06, H 7.33, N 3.40; found: C 64.29, H 7.12, N 3.38. 7b: mp 172–174 8C. IR (KBr, cmꢀ1): 3335, 1731, 1169, 1380; MS (ESI, m/z): [MꢀH]ꢀ
837; 1H NMR (300 MHz, CDCl3): d 0.77 (s, 3H), 0.79 (s, 3H), 0.83 (s, 6H), 0.88 (d, 3H, J = 6.6 Hz), 0.94 (d, 3H, J = 6.4 Hz), 1.07 (s, 3H), 2.65
(s, 4H), 4.47 (t, 2H, J = 6.0 Hz), 4.50 (dd, 1H, J = 10.2, 4.8 Hz), 4.65 (t, 2H, J = 6.0 Hz), 5.26 (brs, 1H), 7.60–7.65 (m, 2H), 7.73–7.78 (m, 1H),
8.06 (m, 2H); Anal. Calcd. for C45H62N2O11S: C 64.42, H 7.45, N 3.34; found: C 64.34, H 7.56, N 3.30. 7c: mp 174–176 8C. IR (KBr, cmꢀ1):
3324, 1731, 1170, 1362; MS (ESI, m/z): [MꢀH]ꢀ 851; 1H NMR (300 MHz, CDCl3): d 0.76 (s, 3H), 0.77 (s, 3H), 0.85 (s, 6H), 0.87 (s, 3H), 0.93
(d, 3H, J = 6.6 Hz), 1.00 (d, 3H, J = 6.4 Hz), 2.65 (s, 4H), 2.20 (t, 2H, J = 4.5 Hz), 4.50 (dd, 1H, J = 10.2, 4.8 Hz), 4.50 (t, 2H, J = 6.0 Hz), 5.15
(m, 1H), 5.26 (brs, 1H), 7.60–7.66 (m, 2H), 7.72–7.77 (m, 1H), 8.06 (m, 2H); Anal. Calcd. for C46H64N2O11S: C 64.77, H 7.56, N 3.28; found:C
1
64.59, H 7.68, N 3.35. 7d: mp 163–165 8C. IR (KBr, cmꢀ1): 3324, 1735, 1169, 1380; MS (ESI, m/z): [MꢀH]ꢀ 851; H NMR (300 MHz,
CDCl3): d 0.76 (s, 3H), 0.77 (s, 3H), 0.83 (s, 6H), 0.88 (d, 3H, J = 6.6 Hz), 0.90 (d, 3H, J = 6.4 Hz), 1.00 (s, 3H), 2.65 (s, 4H), 2.20 (t, 2H,
J = 4.5 Hz), 4.55 (dd, 1H, J = 10.5, 5.1 Hz), 4.19 (t, 2H, J = 6.3 Hz), 4.45 (t, 2H, J = 6.3 Hz), 5.26 (brs, 1H), 7.60–7.66 (m, 2H), 7.74–7.79 (m,

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L. Chen et al. / Chinese Chemical Letters 22 (2011) 413–416
1H), 8.05 (m, 2H); Anal. Calcd. for C46H64N2O11S: C 64.77, H 7.56, N 3.28; found: C 64.66, H 7.70, N 3.17. 7e: mp 157–160 8C. IR (KBr,
cmꢀ1): 3420, 1787, 1723, 1165, 1367; MS (ESI, m/z): [MꢀH]ꢀ 899; 1H NMR (300 MHz, CDCl3): d 0.77 (s, 3H), 0.78 (s, 3H), 0.80 (s, 3H), 0.83
(s, 3H), 0.88 (d, 3H, J = 6.6 Hz), 0.90 (d, 3H, J = 6.4 Hz), 1.00 (s, 3H), 4.35 (m, 2H), 4.34 (dd, 1H, J = 10.5, 5.1 Hz), 4.70 (t, 2H, J = 6.3 Hz),
5.23 (brs, 1H), 7.50–7.80 (m, 5H), 7.70–8.10 (m, 4H); Anal. Calcd. for C50H64N2O11S: C 66.64, H 7.16, N 3.11; found: C 66.23, H 7.34, N 3.59.
7f: mp 178–180 8C. IR (KBr, cmꢀ1): 3388, 1793, 1723, 1161, 1367; MS (ESI, m/z): [MꢀH]ꢀ 899; 1H NMR (300 MHz, CDCl3): d 0.78 (3H, s),
0.81 (s, 3H), 0.86 (s, 3H), 0.92 (s, 3H), 0.95 (d, 3H, J = 6.6 Hz), 0.98 (d, 3H, J = 6.4 Hz), 1.15 (s, 3H), 4.40 (m, 2H), 4.45 (dd, 1H, J = 10.2,
4.8 Hz), 4.75 (m, 1H), 5.30 (brs, 1H), 7.50–7.80 (m, 5H), 7.70–8.10 (m, 4H); Anal. Calcd. for C50H64N2O11S: C 66.64, H 7.16, N 3.11; found: C
66.69, H 7.02, N 3.34